diff --git "a/all.tsv" "b/all.tsv"
deleted file mode 100644--- "a/all.tsv"
+++ /dev/null
@@ -1,207260 +0,0 @@
-Molecular	O
-Dissection	O
-of	O
-Xyloglucan	O
-Recognition	O
-in	O
-a	O
-Prominent	O
-Human	O
-Gut	O
-Symbiont	O
-	O
-Polysaccharide	O
-utilization	O
-loci	O
-(	O
-PUL	O
-)	O
-within	O
-the	O
-genomes	O
-of	O
-resident	O
-human	O
-gut	O
-Bacteroidetes	O
-are	O
-central	O
-to	O
-the	O
-metabolism	O
-of	O
-the	O
-otherwise	O
-indigestible	O
-complex	O
-carbohydrates	O
-known	O
-as	O
-“	O
-dietary	O
-fiber	O
-.”	O
-However	O
-,	O
-functional	O
-characterization	O
-of	O
-PUL	O
-lags	O
-significantly	O
-behind	O
-sequencing	O
-efforts	O
-,	O
-which	O
-limits	O
-physiological	O
-understanding	O
-of	O
-the	O
-human	O
--	O
-bacterial	O
-symbiosis	O
-.	O
-	O
-In	O
-particular	O
-,	O
-the	O
-molecular	O
-basis	O
-of	O
-complex	O
-polysaccharide	O
-recognition	O
-,	O
-an	O
-essential	O
-prerequisite	O
-to	O
-hydrolysis	O
-by	O
-cell	O
-surface	O
-glycosidases	O
-and	O
-subsequent	O
-metabolism	O
-,	O
-is	O
-generally	O
-poorly	O
-understood	O
-.	O
-	O
-Here	O
-,	O
-we	O
-present	O
-the	O
-biochemical	O
-,	O
-structural	O
-,	O
-and	O
-reverse	O
-genetic	O
-characterization	O
-of	O
-two	O
-unique	O
-cell	O
-surface	O
-glycan	O
--	O
-binding	O
-proteins	O
-(	O
-SGBPs	O
-)	O
-encoded	O
-by	O
-a	O
-xyloglucan	O
-utilization	O
-locus	O
-(	O
-XyGUL	O
-)	O
-from	O
-Bacteroides	O
-ovatus	O
-,	O
-which	O
-are	O
-integral	O
-to	O
-growth	O
-on	O
-this	O
-key	O
-dietary	O
-vegetable	O
-polysaccharide	O
-.	O
-	O
-Biochemical	O
-analysis	O
-reveals	O
-that	O
-these	O
-outer	O
-membrane	O
--	O
-anchored	O
-proteins	O
-are	O
-in	O
-fact	O
-exquisitely	O
-specific	O
-for	O
-the	O
-highly	O
-branched	O
-xyloglucan	O
-(	O
-XyG	O
-)	O
-polysaccharide	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-SGBP	O
--	O
-A	O
-,	O
-a	O
-SusD	O
-homolog	O
-,	O
-with	O
-a	O
-bound	O
-XyG	O
-tetradecasaccharide	O
-reveals	O
-an	O
-extended	O
-carbohydrate	O
--	O
-binding	O
-platform	O
-that	O
-primarily	O
-relies	O
-on	O
-recognition	O
-of	O
-the	O
-β	O
--	O
-glucan	O
-backbone	O
-.	O
-	O
-The	O
-unique	O
-,	O
-tetra	O
--	O
-modular	O
-structure	O
-of	O
-SGBP	O
--	O
-B	O
-is	O
-comprised	O
-of	O
-tandem	O
-Ig	O
--	O
-like	O
-folds	O
-,	O
-with	O
-XyG	O
-binding	O
-mediated	O
-at	O
-the	O
-distal	O
-C	O
--	O
-terminal	O
-domain	O
-.	O
-	O
-Despite	O
-displaying	O
-similar	O
-affinities	O
-for	O
-XyG	O
-,	O
-reverse	O
--	O
-genetic	O
-analysis	O
-reveals	O
-that	O
-SGBP	O
--	O
-B	O
-is	O
-only	O
-required	O
-for	O
-the	O
-efficient	O
-capture	O
-of	O
-smaller	O
-oligosaccharides	O
-,	O
-whereas	O
-the	O
-presence	O
-of	O
-SGBP	O
--	O
-A	O
-is	O
-more	O
-critical	O
-than	O
-its	O
-carbohydrate	O
--	O
-binding	O
-ability	O
-for	O
-growth	O
-on	O
-XyG	O
-.	O
-Together	O
-,	O
-these	O
-data	O
-demonstrate	O
-that	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-play	O
-complementary	O
-,	O
-specialized	O
-roles	O
-in	O
-carbohydrate	O
-capture	O
-by	O
-B	O
-.	O
-ovatus	O
-and	O
-elaborate	O
-a	O
-model	O
-of	O
-how	O
-vegetable	O
-xyloglucans	O
-are	O
-accessed	O
-by	O
-the	O
-Bacteroidetes	O
-.	O
-	O
-The	O
-Bacteroidetes	O
-are	O
-dominant	O
-bacteria	O
-in	O
-the	O
-human	O
-gut	O
-that	O
-are	O
-responsible	O
-for	O
-the	O
-digestion	O
-of	O
-the	O
-complex	O
-polysaccharides	O
-that	O
-constitute	O
-“	O
-dietary	O
-fiber	O
-.”	O
-Although	O
-this	O
-symbiotic	O
-relationship	O
-has	O
-been	O
-appreciated	O
-for	O
-decades	O
-,	O
-little	O
-is	O
-currently	O
-known	O
-about	O
-how	O
-Bacteroidetes	O
-seek	O
-out	O
-and	O
-bind	O
-plant	O
-cell	O
-wall	O
-polysaccharides	O
-as	O
-a	O
-necessary	O
-first	O
-step	O
-in	O
-their	O
-metabolism	O
-.	O
-	O
-Here	O
-,	O
-we	O
-provide	O
-the	O
-first	O
-biochemical	O
-,	O
-crystallographic	O
-,	O
-and	O
-genetic	O
-insight	O
-into	O
-how	O
-two	O
-surface	O
-glycan	O
--	O
-binding	O
-proteins	O
-from	O
-the	O
-complex	O
-Bacteroides	O
-ovatus	O
-xyloglucan	O
-utilization	O
-locus	O
-(	O
-XyGUL	O
-)	O
-enable	O
-recognition	O
-and	O
-uptake	O
-of	O
-this	O
-ubiquitous	O
-vegetable	O
-polysaccharide	O
-.	O
-	O
-Our	O
-combined	O
-analysis	O
-illuminates	O
-new	O
-fundamental	O
-aspects	O
-of	O
-complex	O
-polysaccharide	O
-recognition	O
-,	O
-cleavage	O
-,	O
-and	O
-import	O
-at	O
-the	O
-Bacteroidetes	O
-cell	O
-surface	O
-that	O
-may	O
-facilitate	O
-the	O
-development	O
-of	O
-prebiotics	O
-to	O
-target	O
-this	O
-phylum	O
-of	O
-gut	O
-bacteria	O
-.	O
-	O
-The	O
-human	O
-gut	O
-microbiota	O
-influences	O
-the	O
-course	O
-of	O
-human	O
-development	O
-and	O
-health	O
-,	O
-playing	O
-key	O
-roles	O
-in	O
-immune	O
-stimulation	O
-,	O
-intestinal	O
-cell	O
-proliferation	O
-,	O
-and	O
-metabolic	O
-balance	O
-.	O
-	O
-This	O
-microbial	O
-community	O
-is	O
-largely	O
-bacterial	O
-,	O
-with	O
-the	O
-Bacteroidetes	O
-,	O
-Firmicutes	O
-,	O
-and	O
-Actinobacteria	O
-comprising	O
-the	O
-dominant	O
-phyla	O
-.	O
-	O
-The	O
-ability	O
-to	O
-acquire	O
-energy	O
-from	O
-carbohydrates	O
-of	O
-dietary	O
-or	O
-host	O
-origin	O
-is	O
-central	O
-to	O
-the	O
-adaptation	O
-of	O
-human	O
-gut	O
-bacterial	O
-species	O
-to	O
-their	O
-niche	O
-.	O
-	O
-More	O
-importantly	O
-,	O
-this	O
-makes	O
-diet	O
-a	O
-tractable	O
-way	O
-to	O
-manipulate	O
-the	O
-abundance	O
-and	O
-metabolic	O
-output	O
-of	O
-the	O
-microbiota	O
-toward	O
-improved	O
-human	O
-health	O
-.	O
-	O
-However	O
-,	O
-there	O
-is	O
-a	O
-paucity	O
-of	O
-data	O
-regarding	O
-how	O
-the	O
-vast	O
-array	O
-of	O
-complex	O
-carbohydrate	O
-structures	O
-are	O
-selectively	O
-recognized	O
-and	O
-imported	O
-by	O
-members	O
-of	O
-the	O
-microbiota	O
-,	O
-a	O
-critical	O
-process	O
-that	O
-enables	O
-these	O
-organisms	O
-to	O
-thrive	O
-in	O
-the	O
-competitive	O
-gut	O
-environment	O
-.	O
-	O
-The	O
-human	O
-gut	O
-bacteria	O
-Bacteroidetes	O
-share	O
-a	O
-profound	O
-capacity	O
-for	O
-dietary	O
-glycan	O
-degradation	O
-,	O
-with	O
-many	O
-species	O
-containing	O
->	O
-250	O
-predicted	O
-carbohydrate	O
--	O
-active	O
-enzymes	O
-(	O
-CAZymes	O
-),	O
-compared	O
-to	O
-50	O
-to	O
-100	O
-within	O
-many	O
-Firmicutes	O
-and	O
-only	O
-17	O
-in	O
-the	O
-human	O
-genome	O
-devoted	O
-toward	O
-carbohydrate	O
-utilization	O
-.	O
-	O
-A	O
-remarkable	O
-feature	O
-of	O
-the	O
-Bacteroidetes	O
-is	O
-the	O
-packaging	O
-of	O
-genes	O
-for	O
-carbohydrate	O
-catabolism	O
-into	O
-discrete	O
-polysaccharide	O
-utilization	O
-loci	O
-(	O
-PUL	O
-),	O
-which	O
-are	O
-transcriptionally	O
-regulated	O
-by	O
-specific	O
-substrate	O
-signatures	O
-.	O
-	O
-The	O
-archetypal	O
-PUL	O
--	O
-encoded	O
-system	O
-is	O
-the	O
-starch	O
-utilization	O
-system	O
-(	O
-Sus	O
-)	O
-(	O
-Fig	O
-.	O
-1B	O
-)	O
-of	O
-Bacteroides	O
-thetaiotaomicron	O
-.	O
-	O
-The	O
-Sus	O
-includes	O
-a	O
-lipid	O
--	O
-anchored	O
-,	O
-outer	O
-membrane	O
-endo	O
--	O
-amylase	O
-,	O
-SusG	O
-;	O
-a	O
-TonB	O
--	O
-dependent	O
-transporter	O
-(	O
-TBDT	O
-),	O
-SusC	O
-,	O
-which	O
-imports	O
-oligosaccharides	O
-with	O
-the	O
-help	O
-of	O
-an	O
-associated	O
-starch	O
--	O
-binding	O
-protein	O
-,	O
-SusD	O
-;	O
-two	O
-additional	O
-carbohydrate	O
--	O
-binding	O
-lipoproteins	O
-,	O
-SusE	O
-and	O
-SusF	O
-;	O
-and	O
-two	O
-periplasmic	O
-exo	O
--	O
-glucosidases	O
-,	O
-SusA	O
-and	O
-SusB	O
-,	O
-which	O
-generate	O
-glucose	O
-for	O
-transport	O
-into	O
-the	O
-cytoplasm	O
-.	O
-	O
-The	O
-importance	O
-of	O
-PUL	O
-as	O
-a	O
-successful	O
-evolutionary	O
-strategy	O
-is	O
-underscored	O
-by	O
-the	O
-observation	O
-that	O
-Bacteroidetes	O
-such	O
-as	O
-B	O
-.	O
-thetaiotaomicron	O
-and	O
-Bacteroides	O
-ovatus	O
-devote	O
-~	O
-18	O
-%	O
-of	O
-their	O
-genomes	O
-to	O
-these	O
-systems	O
-.	O
-	O
-Moving	O
-beyond	O
-seminal	O
-genomic	O
-and	O
-transcriptomic	O
-analyses	O
-,	O
-the	O
-current	O
-state	O
--	O
-of	O
--	O
-the	O
--	O
-art	O
-PUL	O
-characterization	O
-involves	O
-combined	O
-reverse	O
--	O
-genetic	O
-,	O
-biochemical	O
-,	O
-and	O
-structural	O
-studies	O
-to	O
-illuminate	O
-the	O
-molecular	O
-details	O
-of	O
-PUL	O
-function	O
-.	O
-	O
-Xyloglucan	O
-and	O
-the	O
-Bacteroides	O
-ovatus	O
-xyloglucan	O
-utilization	O
-locus	O
-(	O
-XyGUL	O
-).	O
-(	O
-A	O
-)	O
-Representative	O
-structures	O
-of	O
-common	O
-xyloglucans	O
-using	O
-the	O
-Consortium	O
-for	O
-Functional	O
-Glycomics	O
-Symbol	O
-Nomenclature	O
-(	O
-http	O
-://	O
-www	O
-.	O
-functionalglycomics	O
-.	O
-org	O
-/	O
-static	O
-/	O
-consortium	O
-/	O
-Nomenclature	O
-.	O
-shtml	O
-).	O
-	O
-Cleavage	O
-sites	O
-for	O
-BoXyGUL	O
-glycosidases	O
-(	O
-GHs	O
-)	O
-are	O
-indicated	O
-for	O
-solanaceous	O
-xyloglucan	O
-.	O
-(	O
-B	O
-)	O
-BtSus	O
-and	O
-BoXyGUL	O
-.	O
-(	O
-C	O
-)	O
-Localization	O
-of	O
-BoXyGUL	O
--	O
-encoded	O
-proteins	O
-in	O
-cellular	O
-membranes	O
-and	O
-concerted	O
-modes	O
-of	O
-action	O
-in	O
-the	O
-degradation	O
-of	O
-xyloglucans	O
-to	O
-monosaccharides	O
-.	O
-	O
-The	O
-location	O
-of	O
-SGBP	O
--	O
-A	O
-/	O
-B	O
-is	O
-presented	O
-in	O
-this	O
-work	O
-;	O
-the	O
-location	O
-of	O
-GH5	O
-has	O
-been	O
-empirically	O
-determined	O
-,	O
-and	O
-the	O
-enzymes	O
-have	O
-been	O
-placed	O
-based	O
-upon	O
-their	O
-predicted	O
-cellular	O
-location	O
-.	O
-	O
-We	O
-recently	O
-reported	O
-the	O
-detailed	O
-molecular	O
-characterization	O
-of	O
-a	O
-PUL	O
-that	O
-confers	O
-the	O
-ability	O
-of	O
-the	O
-human	O
-gut	O
-commensal	O
-B	O
-.	O
-ovatus	O
-ATCC	O
-8483	O
-to	O
-grow	O
-on	O
-a	O
-prominent	O
-family	O
-of	O
-plant	O
-cell	O
-wall	O
-glycans	O
-,	O
-the	O
-xyloglucans	O
-(	O
-XyG	O
-).	O
-	O
-XyG	O
-variants	O
-(	O
-Fig	O
-.	O
-1A	O
-)	O
-constitute	O
-up	O
-to	O
-25	O
-%	O
-of	O
-the	O
-dry	O
-weight	O
-of	O
-common	O
-vegetables	O
-.	O
-	O
-Analogous	O
-to	O
-the	O
-Sus	O
-locus	O
-,	O
-the	O
-xyloglucan	O
-utilization	O
-locus	O
-(	O
-XyGUL	O
-)	O
-encodes	O
-a	O
-cohort	O
-of	O
-carbohydrate	O
--	O
-binding	O
-,	O
--	O
-hydrolyzing	O
-,	O
-and	O
--	O
-importing	O
-proteins	O
-(	O
-Fig	O
-.	O
-1B	O
-and	O
-C	O
-).	O
-	O
-The	O
-number	O
-of	O
-glycoside	O
-hydrolases	O
-(	O
-GHs	O
-)	O
-encoded	O
-by	O
-the	O
-XyGUL	O
-is	O
-,	O
-however	O
-,	O
-more	O
-expansive	O
-than	O
-that	O
-by	O
-the	O
-Sus	O
-locus	O
-(	O
-Fig	O
-.	O
-1B	O
-),	O
-which	O
-reflects	O
-the	O
-greater	O
-complexity	O
-of	O
-glycosidic	O
-linkages	O
-found	O
-in	O
-XyG	O
-vis	O
--	O
-à	O
--	O
-vis	O
-starch	O
-.	O
-	O
-Whereas	O
-our	O
-previous	O
-study	O
-focused	O
-on	O
-the	O
-characterization	O
-of	O
-the	O
-linkage	O
-specificity	O
-of	O
-these	O
-GHs	O
-,	O
-a	O
-key	O
-outstanding	O
-question	O
-regarding	O
-this	O
-locus	O
-is	O
-how	O
-XyG	O
-recognition	O
-is	O
-mediated	O
-at	O
-the	O
-cell	O
-surface	O
-.	O
-	O
-In	O
-the	O
-archetypal	O
-starch	O
-utilization	O
-system	O
-of	O
-B	O
-.	O
-thetaiotaomicron	O
-,	O
-starch	O
-binding	O
-to	O
-the	O
-cell	O
-surface	O
-is	O
-mediated	O
-at	O
-eight	O
-distinct	O
-starch	O
--	O
-binding	O
-sites	O
-distributed	O
-among	O
-four	O
-surface	O
-glycan	O
--	O
-binding	O
-proteins	O
-(	O
-SGBPs	O
-):	O
-two	O
-within	O
-the	O
-amylase	O
-SusG	O
-,	O
-one	O
-within	O
-SusD	O
-,	O
-two	O
-within	O
-SusE	O
-,	O
-and	O
-three	O
-within	O
-SusF	O
-.	O
-The	O
-functional	O
-redundancy	O
-of	O
-many	O
-of	O
-these	O
-sites	O
-is	O
-high	O
-:	O
-whereas	O
-SusD	O
-is	O
-essential	O
-for	O
-growth	O
-on	O
-starch	O
-,	O
-combined	O
-mutations	O
-of	O
-the	O
-SusE	O
-,	O
-SusF	O
-,	O
-and	O
-SusG	O
-binding	O
-sites	O
-are	O
-required	O
-to	O
-impair	O
-growth	O
-on	O
-the	O
-polysaccharide	O
-.	O
-	O
-Bacteroidetes	O
-PUL	O
-ubiquitously	O
-encode	O
-homologs	O
-of	O
-SusC	O
-and	O
-SusD	O
-,	O
-as	O
-well	O
-as	O
-proteins	O
-whose	O
-genes	O
-are	O
-immediately	O
-downstream	O
-of	O
-susD	O
-,	O
-akin	O
-to	O
-susE	O
-/	O
-F	O
-,	O
-and	O
-these	O
-are	O
-typically	O
-annotated	O
-as	O
-“	O
-putative	O
-lipoproteins	O
-”.	O
-	O
-The	O
-genes	O
-coding	O
-for	O
-these	O
-proteins	O
-,	O
-sometimes	O
-referred	O
-to	O
-as	O
-“	O
-susE	O
-/	O
-F	O
-positioned	O
-,”	O
-display	O
-products	O
-with	O
-a	O
-wide	O
-variation	O
-in	O
-amino	O
-acid	O
-sequence	O
-and	O
-which	O
-have	O
-little	O
-or	O
-no	O
-homology	O
-to	O
-other	O
-PUL	O
--	O
-encoded	O
-proteins	O
-or	O
-known	O
-carbohydrate	O
--	O
-binding	O
-proteins	O
-.	O
-	O
-As	O
-the	O
-Sus	O
-SGBPs	O
-remain	O
-the	O
-only	O
-structurally	O
-characterized	O
-cohort	O
-to	O
-date	O
-,	O
-we	O
-therefore	O
-wondered	O
-whether	O
-such	O
-glycan	O
-binding	O
-and	O
-function	O
-are	O
-extended	O
-to	O
-other	O
-PUL	O
-that	O
-target	O
-more	O
-complex	O
-and	O
-heterogeneous	O
-polysaccharides	O
-,	O
-such	O
-as	O
-XyG	O
-.	O
-	O
-We	O
-describe	O
-here	O
-the	O
-detailed	O
-functional	O
-and	O
-structural	O
-characterization	O
-of	O
-the	O
-noncatalytic	O
-SGBPs	O
-encoded	O
-by	O
-Bacova_02651	O
-and	O
-Bacova_02650	O
-of	O
-the	O
-XyGUL	O
-,	O
-here	O
-referred	O
-to	O
-as	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-,	O
-to	O
-elucidate	O
-their	O
-molecular	O
-roles	O
-in	O
-carbohydrate	O
-acquisition	O
-in	O
-vivo	O
-.	O
-	O
-Combined	O
-biochemical	O
-,	O
-structural	O
-,	O
-and	O
-reverse	O
--	O
-genetic	O
-approaches	O
-clearly	O
-illuminate	O
-the	O
-distinct	O
-,	O
-yet	O
-complementary	O
-,	O
-functions	O
-that	O
-these	O
-two	O
-proteins	O
-play	O
-in	O
-XyG	O
-recognition	O
-as	O
-it	O
-impacts	O
-the	O
-physiology	O
-of	O
-B	O
-.	O
-ovatus	O
-.	O
-	O
-These	O
-data	O
-extend	O
-our	O
-current	O
-understanding	O
-of	O
-the	O
-Sus	O
--	O
-like	O
-glycan	O
-uptake	O
-paradigm	O
-within	O
-the	O
-Bacteroidetes	O
-and	O
-reveals	O
-how	O
-the	O
-complex	O
-dietary	O
-polysaccharide	O
-xyloglucan	O
-is	O
-recognized	O
-at	O
-the	O
-cell	O
-surface	O
-.	O
-	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-are	O
-cell	O
--	O
-surface	O
--	O
-localized	O
-,	O
-xyloglucan	O
--	O
-specific	O
-binding	O
-proteins	O
-.	O
-	O
-SGBP	O
--	O
-A	O
-,	O
-encoded	O
-by	O
-the	O
-XyGUL	O
-locus	O
-tag	O
-Bacova_02651	O
-(	O
-Fig	O
-.	O
-1B	O
-),	O
-shares	O
-26	O
-%	O
-amino	O
-acid	O
-sequence	O
-identity	O
-(	O
-40	O
-%	O
-similarity	O
-)	O
-with	O
-its	O
-homolog	O
-,	O
-B	O
-.	O
-thetaiotaomicron	O
-SusD	O
-,	O
-and	O
-similar	O
-homology	O
-with	O
-the	O
-SusD	O
--	O
-like	O
-proteins	O
-encoded	O
-within	O
-syntenic	O
-XyGUL	O
-identified	O
-in	O
-our	O
-earlier	O
-work	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-SGBP	O
--	O
-B	O
-,	O
-encoded	O
-by	O
-locus	O
-tag	O
-Bacova_02650	O
-,	O
-displays	O
-little	O
-sequence	O
-similarity	O
-to	O
-the	O
-products	O
-of	O
-similarly	O
-positioned	O
-genes	O
-in	O
-syntenic	O
-XyGUL	O
-nor	O
-to	O
-any	O
-other	O
-gene	O
-product	O
-among	O
-the	O
-diversity	O
-of	O
-Bacteroidetes	O
-PUL	O
-.	O
-	O
-Whereas	O
-sequence	O
-similarity	O
-among	O
-SusC	O
-/	O
-SusD	O
-homolog	O
-pairs	O
-often	O
-serves	O
-as	O
-a	O
-hallmark	O
-for	O
-PUL	O
-identification	O
-,	O
-the	O
-sequence	O
-similarities	O
-of	O
-downstream	O
-genes	O
-encoding	O
-SGBPs	O
-are	O
-generally	O
-too	O
-low	O
-to	O
-allow	O
-reliable	O
-bioinformatic	O
-classification	O
-of	O
-their	O
-products	O
-into	O
-protein	O
-families	O
-,	O
-let	O
-alone	O
-prediction	O
-of	O
-function	O
-.	O
-	O
-Hence	O
-,	O
-there	O
-is	O
-a	O
-critical	O
-need	O
-for	O
-the	O
-elucidation	O
-of	O
-detailed	O
-structure	O
--	O
-function	O
-relationships	O
-among	O
-PUL	O
-SGBPs	O
-,	O
-in	O
-light	O
-of	O
-the	O
-manifold	O
-glycan	O
-structures	O
-in	O
-nature	O
-.	O
-	O
-Immunofluorescence	O
-of	O
-formaldehyde	O
--	O
-fixed	O
-,	O
-nonpermeabilized	O
-cells	O
-grown	O
-in	O
-minimal	O
-medium	O
-with	O
-XyG	O
-as	O
-the	O
-sole	O
-carbon	O
-source	O
-to	O
-induce	O
-XyGUL	O
-expression	O
-,	O
-reveals	O
-that	O
-both	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-are	O
-presented	O
-on	O
-the	O
-cell	O
-surface	O
-by	O
-N	O
--	O
-terminal	O
-lipidation	O
-,	O
-as	O
-predicted	O
-by	O
-signal	O
-peptide	O
-analysis	O
-with	O
-SignalP	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Here	O
-,	O
-the	O
-SGBPs	O
-very	O
-likely	O
-work	O
-in	O
-concert	O
-with	O
-the	O
-cell	O
--	O
-surface	O
--	O
-localized	O
-endo	O
--	O
-xyloglucanase	O
-B	O
-.	O
-ovatus	O
-GH5	O
-(	O
-BoGH5	O
-)	O
-to	O
-recruit	O
-and	O
-cleave	O
-XyG	O
-for	O
-subsequent	O
-periplasmic	O
-import	O
-via	O
-the	O
-SusC	O
--	O
-like	O
-TBDT	O
-of	O
-the	O
-XyGUL	O
-(	O
-Fig	O
-.	O
-1B	O
-and	O
-C	O
-).	O
-	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-visualized	O
-by	O
-immunofluorescence	O
-.	O
-	O
-Formalin	O
--	O
-fixed	O
-,	O
-nonpermeabilized	O
-B	O
-.	O
-ovatus	O
-cells	O
-were	O
-grown	O
-in	O
-minimal	O
-medium	O
-plus	O
-XyG	O
-,	O
-probed	O
-with	O
-custom	O
-rabbit	O
-antibodies	O
-to	O
-SGBP	O
--	O
-A	O
-or	O
-SGBP	O
--	O
-B	O
-,	O
-and	O
-then	O
-stained	O
-with	O
-Alexa	O
-Fluor	O
-488	O
-goat	O
-anti	O
--	O
-rabbit	O
-IgG	O
-.	O
-(	O
-A	O
-)	O
-Overlay	O
-of	O
-bright	O
--	O
-field	O
-and	O
-FITC	O
-images	O
-of	O
-B	O
-.	O
-ovatus	O
-cells	O
-labeled	O
-with	O
-anti	O
--	O
-SGBP	O
--	O
-A	O
-.	O
-(	O
-B	O
-)	O
-Overlay	O
-of	O
-bright	O
--	O
-field	O
-and	O
-FITC	O
-images	O
-of	O
-B	O
-.	O
-ovatus	O
-cells	O
-labeled	O
-with	O
-anti	O
--	O
-SGBP	O
--	O
-B	O
-.	O
-(	O
-C	O
-)	O
-Bright	O
--	O
-field	O
-image	O
-of	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-cells	O
-labeled	O
-with	O
-anti	O
--	O
-SGBP	O
--	O
-B	O
-antibodies	O
-.	O
-	O
-(	O
-D	O
-)	O
-FITC	O
-images	O
-of	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-cells	O
-labeled	O
-with	O
-anti	O
--	O
-SGBP	O
--	O
-B	O
-antibodies	O
-.	O
-	O
-Cells	O
-lacking	O
-SGBP	O
--	O
-A	O
-(	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-)	O
-do	O
-not	O
-grow	O
-on	O
-XyG	O
-and	O
-therefore	O
-could	O
-not	O
-be	O
-tested	O
-in	O
-parallel	O
-.	O
-	O
-In	O
-our	O
-initial	O
-study	O
-focused	O
-on	O
-the	O
-functional	O
-characterization	O
-of	O
-the	O
-glycoside	O
-hydrolases	O
-of	O
-the	O
-XyGUL	O
-,	O
-we	O
-reported	O
-preliminary	O
-affinity	O
-PAGE	O
-and	O
-isothermal	O
-titration	O
-calorimetry	O
-(	O
-ITC	O
-)	O
-data	O
-indicating	O
-that	O
-both	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-are	O
-competent	O
-xyloglucan	O
--	O
-binding	O
-proteins	O
-(	O
-affinity	O
-constant	O
-[	O
-Ka	O
-]	O
-values	O
-of	O
-3	O
-.	O
-74	O
-×	O
-105	O
-M	O
-−	O
-1	O
-and	O
-4	O
-.	O
-98	O
-×	O
-104	O
-M	O
-−	O
-1	O
-,	O
-respectively	O
-[	O
-23	O
-]).	O
-	O
-Additional	O
-affinity	O
-PAGE	O
-analysis	O
-(	O
-Fig	O
-.	O
-3	O
-)	O
-demonstrates	O
-that	O
-SGBP	O
--	O
-A	O
-also	O
-has	O
-moderate	O
-affinity	O
-for	O
-the	O
-artificial	O
-soluble	O
-cellulose	O
-derivative	O
-hydroxyethyl	O
-cellulose	O
-[	O
-HEC	O
-;	O
-a	O
-β	O
-(	O
-1	O
-→	O
-4	O
-)-	O
-glucan	O
-]	O
-and	O
-limited	O
-affinity	O
-for	O
-mixed	O
--	O
-linkage	O
-β	O
-(	O
-1	O
-→	O
-3	O
-)/	O
-β	O
-(	O
-1	O
-→	O
-4	O
-)-	O
-glucan	O
-(	O
-MLG	O
-)	O
-and	O
-glucomannan	O
-(	O
-GM	O
-;	O
-mixed	O
-glucosyl	O
-and	O
-mannosyl	O
-backbone	O
-),	O
-which	O
-together	O
-indicate	O
-general	O
-binding	O
-to	O
-polysaccharide	O
-backbone	O
-residues	O
-and	O
-major	O
-contributions	O
-from	O
-side	O
--	O
-chain	O
-recognition	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-SGBP	O
--	O
-B	O
-bound	O
-to	O
-HEC	O
-more	O
-weakly	O
-than	O
-SGBP	O
--	O
-A	O
-and	O
-did	O
-not	O
-bind	O
-to	O
-MLG	O
-or	O
-GM	O
-.	O
-	O
-Neither	O
-SGBP	O
-recognized	O
-galactomannan	O
-(	O
-GGM	O
-),	O
-starch	O
-,	O
-carboxymethylcellulose	O
-,	O
-or	O
-mucin	O
-(	O
-see	O
-Fig	O
-.	O
-S1	O
-in	O
-the	O
-supplemental	O
-material	O
-).	O
-	O
-Together	O
-,	O
-these	O
-results	O
-highlight	O
-the	O
-high	O
-specificities	O
-of	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-for	O
-XyG	O
-,	O
-which	O
-is	O
-concordant	O
-with	O
-their	O
-association	O
-with	O
-XyG	O
--	O
-specific	O
-GHs	O
-in	O
-the	O
-XyGUL	O
-,	O
-as	O
-well	O
-as	O
-transcriptomic	O
-analysis	O
-indicating	O
-that	O
-B	O
-.	O
-ovatus	O
-has	O
-discrete	O
-PUL	O
-for	O
-MLG	O
-,	O
-GM	O
-,	O
-and	O
-GGM	O
-(	O
-11	O
-).	O
-	O
-Notably	O
-,	O
-the	O
-absence	O
-of	O
-carbohydrate	O
--	O
-binding	O
-modules	O
-in	O
-the	O
-GHs	O
-encoded	O
-by	O
-the	O
-XyGUL	O
-implies	O
-that	O
-noncatalytic	O
-recognition	O
-of	O
-xyloglucan	O
-is	O
-mediated	O
-entirely	O
-by	O
-SGBP	O
--	O
-A	O
-and	O
--	O
-B	O
-.	O
-	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-preferentially	O
-bind	O
-xyloglucan	O
-.	O
-	O
-Affinity	O
-electrophoresis	O
-(	O
-10	O
-%	O
-acrylamide	O
-)	O
-of	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-with	O
-BSA	O
-as	O
-a	O
-control	O
-protein	O
-.	O
-	O
-All	O
-samples	O
-were	O
-loaded	O
-on	O
-the	O
-same	O
-gel	O
-next	O
-to	O
-the	O
-BSA	O
-controls	O
-;	O
-thin	O
-black	O
-lines	O
-indicate	O
-where	O
-intervening	O
-lanes	O
-were	O
-removed	O
-from	O
-the	O
-final	O
-image	O
-for	O
-both	O
-space	O
-and	O
-clarity	O
-.	O
-	O
-The	O
-percentage	O
-of	O
-polysaccharide	O
-incorporated	O
-into	O
-each	O
-native	O
-gel	O
-is	O
-displayed	O
-.	O
-	O
-The	O
-vanguard	O
-endo	O
--	O
-xyloglucanase	O
-of	O
-the	O
-XyGUL	O
-,	O
-BoGH5	O
-,	O
-preferentially	O
-cleaves	O
-the	O
-polysaccharide	O
-at	O
-unbranched	O
-glucosyl	O
-residues	O
-to	O
-generate	O
-xylogluco	O
--	O
-oligosaccharides	O
-(	O
-XyGOs	O
-)	O
-comprising	O
-a	O
-Glc4	O
-backbone	O
-with	O
-variable	O
-side	O
--	O
-chain	O
-galactosylation	O
-(	O
-XyGO1	O
-)	O
-(	O
-Fig	O
-.	O
-1A	O
-;	O
-n	O
-=	O
-1	O
-)	O
-as	O
-the	O
-limit	O
-of	O
-digestion	O
-products	O
-in	O
-vitro	O
-;	O
-controlled	O
-digestion	O
-and	O
-fractionation	O
-by	O
-size	O
-exclusion	O
-chromatography	O
-allow	O
-the	O
-production	O
-of	O
-higher	O
--	O
-order	O
-oligosaccharides	O
-(	O
-e	O
-.	O
-g	O
-.,	O
-XyGO2	O
-)	O
-(	O
-Fig	O
-.	O
-1A	O
-;	O
-n	O
-=	O
-2	O
-).	O
-	O
-ITC	O
-demonstrates	O
-that	O
-SGBP	O
--	O
-A	O
-binds	O
-to	O
-XyG	O
-polysaccharide	O
-and	O
-XyGO2	O
-(	O
-based	O
-on	O
-a	O
-Glc8	O
-backbone	O
-)	O
-with	O
-essentially	O
-equal	O
-affinities	O
-,	O
-while	O
-no	O
-binding	O
-of	O
-XyGO1	O
-(	O
-Glc4	O
-backbone	O
-)	O
-was	O
-detectable	O
-(	O
-Table	O
-1	O
-;	O
-see	O
-Fig	O
-.	O
-S2	O
-and	O
-S3	O
-in	O
-the	O
-supplemental	O
-material	O
-).	O
-	O
-Similarly	O
-,	O
-SGBP	O
--	O
-B	O
-also	O
-bound	O
-to	O
-XyG	O
-and	O
-XyGO2	O
-with	O
-approximately	O
-equal	O
-affinities	O
-,	O
-although	O
-in	O
-both	O
-cases	O
-,	O
-Ka	O
-values	O
-were	O
-nearly	O
-10	O
--	O
-fold	O
-lower	O
-than	O
-those	O
-for	O
-SGBP	O
--	O
-A	O
-.	O
-Also	O
-in	O
-contrast	O
-to	O
-SGBP	O
--	O
-A	O
-,	O
-SGBP	O
--	O
-B	O
-also	O
-bound	O
-to	O
-XyGO1	O
-,	O
-yet	O
-the	O
-affinity	O
-for	O
-this	O
-minimal	O
-repeating	O
-unit	O
-was	O
-poor	O
-,	O
-with	O
-a	O
-Ka	O
-value	O
-of	O
-ca	O
-.	O
-1	O
-order	O
-of	O
-magnitude	O
-lower	O
-than	O
-for	O
-XyG	O
-and	O
-XyGO2	O
-.	O
-	O
-Together	O
-,	O
-these	O
-data	O
-clearly	O
-suggest	O
-that	O
-polysaccharide	O
-binding	O
-of	O
-both	O
-SGBPs	O
-is	O
-fulfilled	O
-by	O
-a	O
-dimer	O
-of	O
-the	O
-minimal	O
-repeat	O
-,	O
-corresponding	O
-to	O
-XyGO2	O
-(	O
-cf	O
-.	O
-	O
-The	O
-observation	O
-by	O
-affinity	O
-PAGE	O
-that	O
-these	O
-proteins	O
-specifically	O
-recognize	O
-XyG	O
-is	O
-further	O
-substantiated	O
-by	O
-their	O
-lack	O
-of	O
-binding	O
-for	O
-the	O
-undecorated	O
-oligosaccharide	O
-cellotetraose	O
-(	O
-Table	O
-1	O
-;	O
-see	O
-Fig	O
-.	O
-S3	O
-).	O
-	O
-Furthermore	O
-,	O
-SGBP	O
--	O
-A	O
-binds	O
-cellohexaose	O
-with	O
-~	O
-770	O
--	O
-fold	O
-weaker	O
-affinity	O
-than	O
-XyG	O
-,	O
-while	O
-SGBP	O
--	O
-B	O
-displays	O
-no	O
-detectable	O
-binding	O
-to	O
-this	O
-linear	O
-hexasaccharide	O
-.	O
-	O
-To	O
-provide	O
-molecular	O
--	O
-level	O
-insight	O
-into	O
-how	O
-the	O
-XyGUL	O
-SGBPs	O
-equip	O
-B	O
-.	O
-ovatus	O
-to	O
-specifically	O
-harvest	O
-XyG	O
-from	O
-the	O
-gut	O
-environment	O
-,	O
-we	O
-performed	O
-X	O
--	O
-ray	O
-crystallography	O
-analysis	O
-of	O
-both	O
-SGBP	O
--	O
-A	O
-and	O
-SGPB	O
--	O
-B	O
-in	O
-oligosaccharide	O
--	O
-complex	O
-forms	O
-.	O
-	O
-Summary	O
-of	O
-thermodynamic	O
-parameters	O
-for	O
-wild	O
--	O
-type	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-obtained	O
-by	O
-isothermal	O
-titration	O
-calorimetry	O
-at	O
-25	O
-°	O
-Ca	O
-	O
-Carbohydrate	O
-Ka	O
-(	O
-M	O
-−	O
-1	O
-)	O
-ΔG	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-ΔH	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-TΔS	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-SGBP	O
--	O
-A	O
-SGBP	O
--	O
-B	O
-SGBP	O
--	O
-A	O
-SGBP	O
--	O
-B	O
-SGBP	O
--	O
-A	O
-SGBP	O
--	O
-B	O
-SGBP	O
--	O
-A	O
-SGBP	O
--	O
-B	O
-XyGb	O
-(	O
-4	O
-.	O
-4	O
-±	O
-0	O
-.	O
-1	O
-)	O
-×	O
-105	O
-(	O
-5	O
-.	O
-7	O
-±	O
-0	O
-.	O
-2	O
-)	O
-×	O
-104	O
-−	O
-7	O
-.	O
-7	O
-−	O
-6	O
-.	O
-5	O
-−	O
-14	O
-±	O
-3	O
-−	O
-14	O
-±	O
-2	O
-−	O
-6	O
-.	O
-5	O
-−	O
-7	O
-.	O
-6	O
-XyGO2c	O
-3	O
-.	O
-0	O
-×	O
-105	O
-2	O
-.	O
-0	O
-×	O
-104	O
-−	O
-7	O
-.	O
-5	O
-−	O
-5	O
-.	O
-9	O
-−	O
-17	O
-.	O
-2	O
-−	O
-17	O
-.	O
-6	O
-−	O
-9	O
-.	O
-7	O
-−	O
-11	O
-.	O
-7	O
-XyGO1	O
-NBd	O
-(	O
-2	O
-.	O
-4	O
-±	O
-0	O
-.	O
-1	O
-)	O
-×	O
-103	O
-NB	O
-−	O
-4	O
-.	O
-6	O
-NB	O
-−	O
-4	O
-.	O
-4	O
-±	O
-0	O
-.	O
-2	O
-NB	O
-0	O
-.	O
-2	O
-Cellohexaose	O
-568	O
-.	O
-0	O
-±	O
-291	O
-.	O
-0	O
-NB	O
-−	O
-3	O
-.	O
-8	O
-NB	O
-−	O
-16	O
-±	O
-8	O
-NB	O
-−	O
-12	O
-.	O
-7	O
-NB	O
-Cellotetraose	O
-NB	O
-NB	O
-NB	O
-NB	O
-NB	O
-NB	O
-NB	O
-NB	O
-	O
-SGBP	O
--	O
-A	O
-is	O
-a	O
-SusD	O
-homolog	O
-with	O
-an	O
-extensive	O
-glycan	O
--	O
-binding	O
-platform	O
-.	O
-	O
-As	O
-anticipated	O
-by	O
-sequence	O
-similarity	O
-,	O
-the	O
-high	O
--	O
-resolution	O
-tertiary	O
-structure	O
-of	O
-apo	O
--	O
-SGBP	O
--	O
-A	O
-(	O
-1	O
-.	O
-36	O
-Å	O
-,	O
-Rwork	O
-=	O
-14	O
-.	O
-7	O
-%,	O
-Rfree	O
-=	O
-17	O
-.	O
-4	O
-%,	O
-residues	O
-28	O
-to	O
-546	O
-)	O
-(	O
-Table	O
-2	O
-)	O
-displays	O
-the	O
-canonical	O
-“	O
-SusD	O
--	O
-like	O
-”	O
-protein	O
-fold	O
-dominated	O
-by	O
-four	O
-tetratrico	O
--	O
-peptide	O
-repeat	O
-(	O
-TPR	O
-)	O
-motifs	O
-that	O
-cradle	O
-the	O
-rest	O
-of	O
-the	O
-structure	O
-(	O
-Fig	O
-.	O
-4A	O
-).	O
-	O
-Specifically	O
-,	O
-SGBP	O
--	O
-A	O
-overlays	O
-B	O
-.	O
-thetaiotaomicron	O
-SusD	O
-(	O
-BtSusD	O
-)	O
-with	O
-a	O
-root	O
-mean	O
-square	O
-deviation	O
-(	O
-RMSD	O
-)	O
-value	O
-of	O
-2	O
-.	O
-2	O
-Å	O
-for	O
-363	O
-Cα	O
-pairs	O
-,	O
-which	O
-is	O
-notable	O
-given	O
-the	O
-26	O
-%	O
-amino	O
-acid	O
-identity	O
-(	O
-40	O
-%	O
-similarity	O
-)	O
-between	O
-these	O
-homologs	O
-(	O
-Fig	O
-.	O
-4C	O
-).	O
-	O
-Cocrystallization	O
-of	O
-SGBP	O
--	O
-A	O
-with	O
-XyGO2	O
-generated	O
-a	O
-substrate	O
-complex	O
-structure	O
-(	O
-2	O
-.	O
-3	O
-Å	O
-,	O
-Rwork	O
-=	O
-21	O
-.	O
-8	O
-%,	O
-Rfree	O
-=	O
-24	O
-.	O
-8	O
-%,	O
-residues	O
-36	O
-to	O
-546	O
-)	O
-(	O
-Fig	O
-.	O
-4A	O
-and	O
-B	O
-;	O
-Table	O
-2	O
-)	O
-that	O
-revealed	O
-the	O
-distinct	O
-binding	O
--	O
-site	O
-architecture	O
-of	O
-the	O
-XyG	O
-binding	O
-protein	O
-.	O
-	O
-The	O
-SGBP	O
--	O
-A	O
-:	O
-XyGO2	O
-complex	O
-superimposes	O
-closely	O
-with	O
-the	O
-apo	O
-structure	O
-(	O
-RMSD	O
-of	O
-0	O
-.	O
-6	O
-Å	O
-)	O
-and	O
-demonstrates	O
-that	O
-no	O
-major	O
-conformational	O
-change	O
-occurs	O
-upon	O
-substrate	O
-binding	O
-;	O
-small	O
-deviations	O
-in	O
-the	O
-orientation	O
-of	O
-several	O
-surface	O
-loops	O
-are	O
-likely	O
-the	O
-result	O
-of	O
-differential	O
-crystal	O
-packing	O
-.	O
-	O
-It	O
-is	O
-particularly	O
-notable	O
-that	O
-although	O
-the	O
-location	O
-of	O
-the	O
-ligand	O
--	O
-binding	O
-site	O
-is	O
-conserved	O
-between	O
-SGBP	O
--	O
-A	O
-and	O
-SusD	O
-,	O
-that	O
-of	O
-SGBP	O
--	O
-A	O
-displays	O
-an	O
-~	O
-29	O
--	O
-Å	O
--	O
-long	O
-aromatic	O
-platform	O
-to	O
-accommodate	O
-the	O
-extended	O
-,	O
-linear	O
-XyG	O
-chain	O
-(	O
-see	O
-reference	O
-for	O
-a	O
-review	O
-of	O
-XyG	O
-secondary	O
-structure	O
-),	O
-versus	O
-the	O
-shorter	O
-,	O
-~	O
-18	O
--	O
-Å	O
--	O
-long	O
-,	O
-site	O
-within	O
-SusD	O
-that	O
-complements	O
-the	O
-helical	O
-conformation	O
-of	O
-amylose	O
-(	O
-Fig	O
-.	O
-4C	O
-and	O
-D	O
-).	O
-	O
-Molecular	O
-structure	O
-of	O
-SGBP	O
--	O
-A	O
-(	O
-Bacova_02651	O
-).	O
-(	O
-A	O
-)	O
-Overlay	O
-of	O
-SGBP	O
--	O
-A	O
-from	O
-the	O
-apo	O
-(	O
-rainbow	O
-)	O
-and	O
-XyGO2	O
-(	O
-gray	O
-)	O
-structures	O
-.	O
-	O
-The	O
-apo	O
-structure	O
-is	O
-color	O
-ramped	O
-from	O
-blue	O
-to	O
-red	O
-.	O
-	O
-An	O
-omit	O
-map	O
-(	O
-2σ	O
-)	O
-for	O
-XyGO2	O
-(	O
-orange	O
-and	O
-red	O
-sticks	O
-)	O
-is	O
-displayed	O
-.	O
-	O
-(	O
-B	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-omit	O
-map	O
-as	O
-in	O
-panel	O
-A	O
-,	O
-rotated	O
-90	O
-°	O
-clockwise	O
-.	O
-	O
-(	O
-C	O
-)	O
-Overlay	O
-of	O
-the	O
-Cα	O
-backbones	O
-of	O
-SGBP	O
--	O
-A	O
-(	O
-black	O
-)	O
-with	O
-XyGO2	O
-(	O
-orange	O
-and	O
-red	O
-spheres	O
-)	O
-and	O
-BtSusD	O
-(	O
-blue	O
-)	O
-with	O
-maltoheptaose	O
-(	O
-pink	O
-and	O
-red	O
-spheres	O
-),	O
-highlighting	O
-the	O
-conservation	O
-of	O
-the	O
-glycan	O
--	O
-binding	O
-site	O
-location	O
-.	O
-	O
-(	O
-D	O
-)	O
-Close	O
--	O
-up	O
-of	O
-the	O
-SGBP	O
--	O
-A	O
-(	O
-black	O
-and	O
-orange	O
-)	O
-and	O
-SusD	O
-(	O
-blue	O
-and	O
-pink	O
-)	O
-glycan	O
--	O
-binding	O
-sites	O
-.	O
-	O
-The	O
-approximate	O
-length	O
-of	O
-each	O
-glycan	O
--	O
-binding	O
-site	O
-is	O
-displayed	O
-,	O
-colored	O
-to	O
-match	O
-the	O
-protein	O
-structures	O
-.	O
-(	O
-E	O
-)	O
-Stereo	O
-view	O
-of	O
-the	O
-xyloglucan	O
--	O
-binding	O
-site	O
-of	O
-SGBP	O
--	O
-A	O
-,	O
-displaying	O
-all	O
-residues	O
-within	O
-4	O
-Å	O
-of	O
-the	O
-ligand	O
-.	O
-	O
-The	O
-backbone	O
-glucose	O
-residues	O
-are	O
-numbered	O
-from	O
-the	O
-nonreducing	O
-end	O
-;	O
-xylose	O
-residues	O
-are	O
-labeled	O
-X1	O
-and	O
-X2	O
-.	O
-	O
-Potential	O
-hydrogen	O
--	O
-bonding	O
-interactions	O
-are	O
-shown	O
-as	O
-dashed	O
-lines	O
-,	O
-and	O
-the	O
-distance	O
-is	O
-shown	O
-in	O
-angstroms	O
-.	O
-	O
-Seven	O
-of	O
-the	O
-eight	O
-backbone	O
-glucosyl	O
-residues	O
-of	O
-XyGO2	O
-could	O
-be	O
-convincingly	O
-modeled	O
-in	O
-the	O
-ligand	O
-electron	O
-density	O
-,	O
-and	O
-only	O
-two	O
-α	O
-(	O
-1	O
-→	O
-6	O
-)-	O
-linked	O
-xylosyl	O
-residues	O
-were	O
-observed	O
-(	O
-Fig	O
-.	O
-4B	O
-;	O
-cf	O
-.	O
-	O
-Indeed	O
-,	O
-the	O
-electron	O
-density	O
-for	O
-the	O
-ligand	O
-suggests	O
-some	O
-disorder	O
-,	O
-which	O
-may	O
-arise	O
-from	O
-multiple	O
-oligosaccharide	O
-orientations	O
-along	O
-the	O
-binding	O
-site	O
-.	O
-	O
-Three	O
-aromatic	O
-residues	O
-—	O
-W82	O
-,	O
-W283	O
-,	O
-W306	O
-—	O
-comprise	O
-the	O
-flat	O
-platform	O
-that	O
-stacks	O
-along	O
-the	O
-naturally	O
-twisted	O
-β	O
--	O
-glucan	O
-backbone	O
-(	O
-Fig	O
-.	O
-4E	O
-).	O
-	O
-The	O
-functional	O
-importance	O
-of	O
-this	O
-platform	O
-is	O
-underscored	O
-by	O
-the	O
-observation	O
-that	O
-the	O
-W82A	B-mutant
-W283A	B-mutant
-W306A	B-mutant
-mutant	O
-of	O
-SGBP	O
--	O
-A	O
-,	O
-designated	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*,	I-mutant
-is	O
-completely	O
-devoid	O
-of	O
-XyG	O
-affinity	O
-(	O
-Table	O
-3	O
-;	O
-see	O
-Fig	O
-.	O
-S4	O
-in	O
-the	O
-supplemental	O
-material	O
-).	O
-	O
-Dissection	O
-of	O
-the	O
-individual	O
-contribution	O
-of	O
-these	O
-residues	O
-reveals	O
-that	O
-the	O
-W82A	B-mutant
-mutant	O
-displays	O
-a	O
-significant	O
-4	O
-.	O
-9	O
--	O
-fold	O
-decrease	O
-in	O
-the	O
-Ka	O
-value	O
-for	O
-XyG	O
-,	O
-while	O
-the	O
-W306A	B-mutant
-substitution	O
-completely	O
-abolishes	O
-XyG	O
-binding	O
-.	O
-	O
-Contrasting	O
-with	O
-the	O
-clear	O
-importance	O
-of	O
-these	O
-hydrophobic	O
-interactions	O
-,	O
-there	O
-are	O
-remarkably	O
-few	O
-hydrogen	O
--	O
-bonding	O
-interactions	O
-with	O
-the	O
-ligand	O
-,	O
-which	O
-are	O
-provided	O
-by	O
-R65	O
-,	O
-N83	O
-,	O
-and	O
-S308	O
-,	O
-which	O
-are	O
-proximal	O
-to	O
-Glc5	O
-and	O
-Glc3	O
-.	O
-	O
-Most	O
-surprising	O
-in	O
-light	O
-of	O
-the	O
-saccharide	O
--	O
-binding	O
-data	O
-,	O
-however	O
-,	O
-was	O
-a	O
-lack	O
-of	O
-extensive	O
-recognition	O
-of	O
-the	O
-XyG	O
-side	O
-chains	O
-;	O
-only	O
-Y84	O
-appeared	O
-to	O
-provide	O
-a	O
-hydrophobic	O
-interface	O
-for	O
-a	O
-xylosyl	O
-residue	O
-(	O
-Xyl1	O
-).	O
-	O
-Summary	O
-of	O
-thermodynamic	O
-parameters	O
-for	O
-site	O
--	O
-directed	O
-mutants	O
-of	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-obtained	O
-by	O
-ITC	O
-with	O
-XyG	O
-at	O
-25	O
-°	O
-Ca	O
-	O
-Protein	O
-name	O
-Ka	O
-ΔG	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-ΔH	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-TΔS	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-Fold	O
-changeb	O
-M	O
-−	O
-1	O
-SGBP	O
--	O
-A	O
-(	O
-W82A	B-mutant
-W283A	B-mutant
-W306A	B-mutant
-)	O
-ND	O
-NB	O
-NB	O
-NB	O
-NB	O
-SGBP	O
--	O
-A	O
-(	O
-W82A	B-mutant
-)	O
-c	O
-4	O
-.	O
-9	O
-9	O
-.	O
-1	O
-×	O
-104	O
-−	O
-6	O
-.	O
-8	O
-−	O
-6	O
-.	O
-3	O
-0	O
-.	O
-5	O
-SGBP	O
--	O
-A	O
-(	O
-W306	O
-)	O
-ND	O
-NB	O
-NB	O
-NB	O
-NB	O
-SGBP	O
--	O
-B	O
-(	O
-230	O
-–	O
-489	O
-)	O
-0	O
-.	O
-7	O
-(	O
-8	O
-.	O
-6	O
-±	O
-0	O
-.	O
-20	O
-)	O
-×	O
-104	O
-−	O
-6	O
-.	O
-7	O
-−	O
-14	O
-.	O
-9	O
-±	O
-0	O
-.	O
-1	O
-−	O
-8	O
-.	O
-2	O
-SGBP	O
--	O
-B	O
-(	O
-Y363A	B-mutant
-)	O
-19	O
-.	O
-7	O
-(	O
-2	O
-.	O
-9	O
-±	O
-0	O
-.	O
-10	O
-)	O
-×	O
-103	O
-−	O
-4	O
-.	O
-7	O
-−	O
-18	O
-.	O
-1	O
-±	O
-0	O
-.	O
-1	O
-−	O
-13	O
-.	O
-3	O
-SGBP	O
--	O
-B	O
-(	O
-W364A	B-mutant
-)	O
-ND	O
-Weak	O
-Weak	O
-Weak	O
-Weak	O
-SGBP	O
--	O
-B	O
-(	O
-F414A	B-mutant
-)	O
-3	O
-.	O
-2	O
-(	O
-1	O
-.	O
-80	O
-±	O
-0	O
-.	O
-03	O
-)	O
-×	O
-104	O
-−	O
-5	O
-.	O
-8	O
-−	O
-11	O
-.	O
-4	O
-±	O
-0	O
-.	O
-1	O
-−	O
-5	O
-.	O
-6	O
-	O
-Binding	O
-thermodynamics	O
-are	O
-based	O
-on	O
-the	O
-concentration	O
-of	O
-the	O
-binding	O
-unit	O
-,	O
-XyGO2	O
-.	O
-	O
-Weak	O
-binding	O
-represents	O
-a	O
-Ka	O
-of	O
-<	O
-500	O
-M	O
-−	O
-1	O
-.	O
-	O
-Ka	O
-fold	O
-change	O
-=	O
-Ka	O
-of	O
-wild	O
--	O
-type	O
-protein	O
-/	O
-Ka	O
-of	O
-mutant	O
-protein	O
-for	O
-xyloglucan	O
-binding	O
-.	O
-	O
-SGBP	O
--	O
-B	O
-has	O
-a	O
-multimodular	O
-structure	O
-with	O
-a	O
-single	O
-,	O
-C	O
--	O
-terminal	O
-glycan	O
--	O
-binding	O
-domain	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-full	O
--	O
-length	O
-SGBP	O
--	O
-B	O
-in	O
-complex	O
-with	O
-XyGO2	O
-(	O
-2	O
-.	O
-37	O
-Å	O
-,	O
-Rwork	O
-=	O
-19	O
-.	O
-9	O
-%,	O
-Rfree	O
-=	O
-23	O
-.	O
-9	O
-%,	O
-residues	O
-34	O
-to	O
-489	O
-)	O
-(	O
-Table	O
-2	O
-)	O
-revealed	O
-an	O
-extended	O
-structure	O
-composed	O
-of	O
-three	O
-tandem	O
-immunoglobulin	O
-(	O
-Ig	O
-)-	O
-like	O
-domains	O
-(	O
-domains	O
-A	O
-,	O
-B	O
-,	O
-and	O
-C	O
-)	O
-followed	O
-at	O
-the	O
-C	O
-terminus	O
-by	O
-a	O
-novel	O
-xyloglucan	O
--	O
-binding	O
-domain	O
-(	O
-domain	O
-D	O
-)	O
-(	O
-Fig	O
-.	O
-5A	O
-).	O
-	O
-Domains	O
-A	O
-,	O
-B	O
-,	O
-and	O
-C	O
-display	O
-similar	O
-β	O
--	O
-sandwich	O
-folds	O
-;	O
-domains	O
-B	O
-(	O
-residues	O
-134	O
-to	O
-230	O
-)	O
-and	O
-C	O
-(	O
-residues	O
-231	O
-to	O
-313	O
-)	O
-can	O
-be	O
-superimposed	O
-onto	O
-domain	O
-A	O
-(	O
-residues	O
-34	O
-to	O
-133	O
-)	O
-with	O
-RMSDs	O
-of	O
-1	O
-.	O
-1	O
-and	O
-1	O
-.	O
-2	O
-Å	O
-,	O
-respectively	O
-,	O
-for	O
-47	O
-atom	O
-pairs	O
-(	O
-23	O
-%	O
-and	O
-16	O
-%	O
-sequence	O
-identity	O
-,	O
-respectively	O
-).	O
-	O
-These	O
-domains	O
-also	O
-display	O
-similarity	O
-to	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sandwich	O
-domains	O
-of	O
-many	O
-GH13	O
-enzymes	O
-,	O
-including	O
-the	O
-cyclodextrin	O
-glucanotransferase	O
-of	O
-Geobacillus	O
-stearothermophilus	O
-(	O
-Fig	O
-.	O
-5B	O
-).	O
-	O
-Such	O
-domains	O
-are	O
-not	O
-typically	O
-involved	O
-in	O
-carbohydrate	O
-binding	O
-.	O
-	O
-Indeed	O
-,	O
-visual	O
-inspection	O
-of	O
-the	O
-SGBP	O
--	O
-B	O
-structure	O
-,	O
-as	O
-well	O
-as	O
-individual	O
-production	O
-of	O
-the	O
-A	O
-and	O
-B	O
-domains	O
-and	O
-affinity	O
-PAGE	O
-analysis	O
-(	O
-see	O
-Fig	O
-.	O
-S5	O
-in	O
-the	O
-supplemental	O
-material	O
-),	O
-indicates	O
-that	O
-these	O
-domains	O
-do	O
-not	O
-contribute	O
-to	O
-XyG	O
-capture	O
-.	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-production	O
-of	O
-the	O
-fused	B-mutant
-domains	I-mutant
-C	I-mutant
-and	I-mutant
-D	I-mutant
-in	O
-tandem	O
-(	O
-SGBP	O
--	O
-B	O
-residues	O
-230	O
-to	O
-489	O
-)	O
-retains	O
-complete	O
-binding	O
-of	O
-xyloglucan	O
-in	O
-vitro	O
-,	O
-with	O
-the	O
-observed	O
-slight	O
-increase	O
-in	O
-affinity	O
-likely	O
-arising	O
-from	O
-a	O
-reduced	O
-potential	O
-for	O
-steric	O
-hindrance	O
-of	O
-the	O
-smaller	O
-protein	O
-construct	O
-during	O
-polysaccharide	O
-interactions	O
-(	O
-Table	O
-3	O
-).	O
-	O
-While	O
-neither	O
-the	O
-full	O
--	O
-length	O
-protein	O
-nor	O
-domain	O
-D	O
-displays	O
-structural	O
-homology	O
-to	O
-known	O
-XyG	O
--	O
-binding	O
-proteins	O
-,	O
-the	O
-topology	O
-of	O
-SGBP	O
--	O
-B	O
-resembles	O
-the	O
-xylan	O
--	O
-binding	O
-protein	O
-Bacova_04391	O
-(	O
-PDB	O
-3ORJ	O
-)	O
-encoded	O
-within	O
-a	O
-xylan	O
--	O
-targeting	O
-PUL	O
-of	O
-B	O
-.	O
-ovatus	O
-(	O
-Fig	O
-.	O
-5C	O
-).	O
-	O
-The	O
-structure	O
--	O
-based	O
-alignment	O
-of	O
-these	O
-proteins	O
-reveals	O
-17	O
-%	O
-sequence	O
-identity	O
-,	O
-with	O
-a	O
-core	O
-RMSD	O
-of	O
-3	O
-.	O
-6	O
-Å	O
-for	O
-253	O
-aligned	O
-residues	O
-.	O
-	O
-While	O
-there	O
-is	O
-no	O
-substrate	O
--	O
-complexed	O
-structure	O
-of	O
-Bacova_04391	O
-available	O
-,	O
-the	O
-binding	O
-site	O
-is	O
-predicted	O
-to	O
-include	O
-W241	O
-and	O
-Y404	O
-,	O
-which	O
-are	O
-proximal	O
-to	O
-the	O
-XyGO	O
-binding	O
-site	O
-in	O
-SGBP	O
--	O
-B	O
-.	O
-However	O
-,	O
-the	O
-opposing	O
-,	O
-clamp	O
--	O
-like	O
-arrangement	O
-of	O
-these	O
-residues	O
-in	O
-Bacova_04391	O
-is	O
-clearly	O
-distinct	O
-from	O
-the	O
-planar	O
-surface	O
-arrangement	O
-of	O
-the	O
-residues	O
-that	O
-interact	O
-with	O
-XyG	O
-in	O
-SGBP	O
--	O
-B	O
-(	O
-described	O
-below	O
-).	O
-	O
-Multimodular	O
-structure	O
-of	O
-SGBP	O
--	O
-B	O
-(	O
-Bacova_02650	O
-).	O
-(	O
-A	O
-)	O
-Full	O
--	O
-length	O
-structure	O
-of	O
-SGBP	O
--	O
-B	O
-,	O
-color	O
-coded	O
-by	O
-domain	O
-as	O
-indicated	O
-.	O
-	O
-Prolines	O
-between	O
-domains	O
-are	O
-indicated	O
-as	O
-spheres	O
-.	O
-	O
-An	O
-omit	O
-map	O
-(	O
-2σ	O
-)	O
-for	O
-XyGO2	O
-is	O
-displayed	O
-to	O
-highlight	O
-the	O
-location	O
-of	O
-the	O
-glycan	O
--	O
-binding	O
-site	O
-.	O
-	O
-(	O
-B	O
-)	O
-Overlay	O
-of	O
-SGBP	O
--	O
-B	O
-domains	O
-A	O
-,	O
-B	O
-,	O
-and	O
-C	O
-(	O
-colored	O
-as	O
-in	O
-panel	O
-A	O
-),	O
-with	O
-a	O
-C	O
--	O
-terminal	O
-Ig	O
--	O
-like	O
-domain	O
-of	O
-the	O
-G	O
-.	O
-stearothermophilus	O
-cyclodextrin	O
-glucanotransferase	O
-(	O
-PDB	O
-1CYG	O
-[	O
-residues	O
-375	O
-to	O
-493	O
-])	O
-in	O
-green	O
-.	O
-(	O
-C	O
-)	O
-Cα	O
-overlay	O
-of	O
-SGBP	O
--	O
-B	O
-(	O
-gray	O
-)	O
-and	O
-Bacova_04391	O
-(	O
-PDB	O
-3ORJ	O
-)	O
-(	O
-pink	O
-).	O
-	O
-(	O
-D	O
-)	O
-Close	O
--	O
-up	O
-omit	O
-map	O
-for	O
-the	O
-XyGO2	O
-ligand	O
-,	O
-contoured	O
-at	O
-2σ	O
-.	O
-(	O
-E	O
-)	O
-Stereo	O
-view	O
-of	O
-the	O
-xyloglucan	O
--	O
-binding	O
-site	O
-of	O
-SGBP	O
--	O
-B	O
-,	O
-displaying	O
-all	O
-residues	O
-within	O
-4	O
-Å	O
-of	O
-the	O
-ligand	O
-.	O
-	O
-The	O
-backbone	O
-glucose	O
-residues	O
-are	O
-numbered	O
-from	O
-the	O
-nonreducing	O
-end	O
-,	O
-xylose	O
-residues	O
-are	O
-shown	O
-as	O
-X1	O
-,	O
-X2	O
-,	O
-and	O
-X3	O
-,	O
-potential	O
-hydrogen	O
--	O
-bonding	O
-interactions	O
-are	O
-shown	O
-as	O
-dashed	O
-lines	O
-,	O
-and	O
-the	O
-distance	O
-is	O
-shown	O
-in	O
-angstroms	O
-.	O
-	O
-Inspection	O
-of	O
-the	O
-tertiary	O
-structure	O
-indicates	O
-that	O
-domains	O
-C	O
-and	O
-D	O
-are	O
-effectively	O
-inseparable	O
-,	O
-with	O
-a	O
-contact	O
-interface	O
-of	O
-396	O
-Å2	O
-.	O
-	O
-Domains	O
-A	O
-,	O
-B	O
-,	O
-and	O
-C	O
-do	O
-not	O
-pack	O
-against	O
-each	O
-other	O
-.	O
-	O
-Moreover	O
-,	O
-the	O
-five	O
--	O
-residue	O
-linkers	O
-between	O
-these	O
-first	O
-three	O
-domains	O
-all	O
-feature	O
-a	O
-proline	O
-as	O
-the	O
-middle	O
-residue	O
-,	O
-suggesting	O
-significant	O
-conformational	O
-rigidity	O
-(	O
-Fig	O
-.	O
-5A	O
-).	O
-	O
-Despite	O
-the	O
-lack	O
-of	O
-sequence	O
-and	O
-structural	O
-conservation	O
-,	O
-a	O
-similarly	O
-positioned	O
-proline	O
-joins	O
-the	O
-Ig	O
--	O
-like	O
-domains	O
-of	O
-the	O
-xylan	O
--	O
-binding	O
-Bacova_04391	O
-and	O
-the	O
-starch	O
--	O
-binding	O
-proteins	O
-SusE	O
-and	O
-SusF	O
-.	O
-We	O
-speculate	O
-that	O
-this	O
-is	O
-a	O
-biologically	O
-important	O
-adaptation	O
-that	O
-serves	O
-to	O
-project	O
-the	O
-glycan	O
-binding	O
-site	O
-of	O
-these	O
-proteins	O
-far	O
-from	O
-the	O
-membrane	O
-surface	O
-.	O
-	O
-Any	O
-mobility	O
-of	O
-SGBP	O
--	O
-B	O
-on	O
-the	O
-surface	O
-of	O
-the	O
-cell	O
-(	O
-beyond	O
-lateral	O
-diffusion	O
-within	O
-the	O
-membrane	O
-)	O
-is	O
-likely	O
-imparted	O
-by	O
-the	O
-eight	O
--	O
-residue	O
-linker	O
-that	O
-spans	O
-the	O
-predicted	O
-lipidated	O
-Cys	O
-(	O
-C28	O
-)	O
-and	O
-the	O
-first	O
-β	O
--	O
-strand	O
-of	O
-domain	O
-A	O
-.	O
-Other	O
-outer	O
-membrane	O
-proteins	O
-from	O
-various	O
-Sus	O
--	O
-like	O
-systems	O
-possess	O
-a	O
-similar	O
-10	O
--	O
-to	O
-20	O
--	O
-amino	O
--	O
-acid	O
-flexible	O
-linker	O
-between	O
-the	O
-lipidated	O
-Cys	O
-that	O
-tethers	O
-the	O
-protein	O
-to	O
-the	O
-outside	O
-the	O
-cell	O
-and	O
-the	O
-first	O
-secondary	O
-structure	O
-element	O
-.	O
-	O
-Analogously	O
-,	O
-the	O
-outer	O
-membrane	O
--	O
-anchored	O
-endo	O
--	O
-xyloglucanase	O
-BoGH5	O
-of	O
-the	O
-XyGUL	O
-contains	O
-a	O
-100	O
--	O
-amino	O
--	O
-acid	O
-,	O
-all	O
--	O
-β	O
--	O
-strand	O
-,	O
-N	O
--	O
-terminal	O
-module	O
-and	O
-flexible	O
-linker	O
-that	O
-imparts	O
-conformational	O
-flexibility	O
-and	O
-distances	O
-the	O
-catalytic	O
-module	O
-from	O
-the	O
-cell	O
-surface	O
-.	O
-	O
-XyG	O
-binds	O
-to	O
-domain	O
-D	O
-of	O
-SGBP	O
--	O
-B	O
-at	O
-the	O
-concave	O
-interface	O
-of	O
-the	O
-top	O
-β	O
--	O
-sheet	O
-,	O
-with	O
-binding	O
-mediated	O
-by	O
-loops	O
-connecting	O
-the	O
-β	O
--	O
-strands	O
-.	O
-	O
-Six	O
-glucosyl	O
-residues	O
-,	O
-comprising	O
-the	O
-main	O
-chain	O
-,	O
-and	O
-three	O
-branching	O
-xylosyl	O
-residues	O
-of	O
-XyGO2	O
-can	O
-be	O
-modeled	O
-in	O
-the	O
-density	O
-(	O
-Fig	O
-.	O
-5D	O
-;	O
-cf	O
-.	O
-	O
-The	O
-backbone	O
-is	O
-flat	O
-,	O
-with	O
-less	O
-of	O
-the	O
-“	O
-twisted	O
--	O
-ribbon	O
-”	O
-geometry	O
-observed	O
-in	O
-some	O
-cello	O
--	O
-and	O
-xylogluco	O
--	O
-oligosaccharides	O
-.	O
-	O
-The	O
-aromatic	O
-platform	O
-created	O
-by	O
-W330	O
-,	O
-W364	O
-,	O
-and	O
-Y363	O
-spans	O
-four	O
-glucosyl	O
-residues	O
-,	O
-compared	O
-to	O
-the	O
-longer	O
-platform	O
-of	O
-SGBP	O
--	O
-A	O
-,	O
-which	O
-supports	O
-six	O
-glucosyl	O
-residues	O
-(	O
-Fig	O
-.	O
-5E	O
-).	O
-	O
-The	O
-Y363A	B-mutant
-site	O
--	O
-directed	O
-mutant	O
-of	O
-SGBP	O
--	O
-B	O
-displays	O
-a	O
-20	O
--	O
-fold	O
-decrease	O
-in	O
-the	O
-Ka	O
-for	O
-XyG	O
-,	O
-while	O
-the	O
-W364A	B-mutant
-mutant	O
-lacks	O
-XyG	O
-binding	O
-(	O
-Table	O
-3	O
-;	O
-see	O
-Fig	O
-.	O
-S6	O
-in	O
-the	O
-supplemental	O
-material	O
-).	O
-	O
-There	O
-are	O
-no	O
-additional	O
-contacts	O
-between	O
-the	O
-protein	O
-and	O
-the	O
-β	O
--	O
-glucan	O
-backbone	O
-and	O
-surprisingly	O
-few	O
-interactions	O
-with	O
-the	O
-side	O
--	O
-chain	O
-xylosyl	O
-residues	O
-,	O
-despite	O
-that	O
-fact	O
-that	O
-ITC	O
-data	O
-demonstrate	O
-that	O
-SGBP	O
--	O
-B	O
-does	O
-not	O
-measurably	O
-bind	O
-the	O
-cellohexaose	O
-(	O
-Table	O
-1	O
-).	O
-	O
-F414	O
-stacks	O
-with	O
-the	O
-xylosyl	O
-residue	O
-of	O
-Glc3	O
-,	O
-while	O
-Q407	O
-is	O
-positioned	O
-for	O
-hydrogen	O
-bonding	O
-with	O
-the	O
-O4	O
-of	O
-xylosyl	O
-residue	O
-Xyl1	O
-.	O
-	O
-Surprisingly	O
-,	O
-an	O
-F414A	B-mutant
-mutant	O
-of	O
-SGBP	O
--	O
-B	O
-displays	O
-only	O
-a	O
-mild	O
-3	O
--	O
-fold	O
-decrease	O
-in	O
-the	O
-Ka	O
-value	O
-for	O
-XyG	O
-,	O
-again	O
-suggesting	O
-that	O
-glycan	O
-recognition	O
-is	O
-primarily	O
-mediated	O
-via	O
-contact	O
-with	O
-the	O
-β	O
--	O
-glucan	O
-backbone	O
-(	O
-Table	O
-3	O
-;	O
-see	O
-Fig	O
-.	O
-S6	O
-).	O
-	O
-Additional	O
-residues	O
-surrounding	O
-the	O
-binding	O
-site	O
-,	O
-including	O
-Y369	O
-and	O
-E412	O
-,	O
-may	O
-contribute	O
-to	O
-the	O
-recognition	O
-of	O
-more	O
-highly	O
-decorated	O
-XyG	O
-,	O
-but	O
-precisely	O
-how	O
-this	O
-is	O
-mediated	O
-is	O
-presently	O
-unclear	O
-.	O
-	O
-Hoping	O
-to	O
-achieve	O
-a	O
-higher	O
--	O
-resolution	O
-view	O
-of	O
-the	O
-SGBP	O
--	O
-B	O
-–	O
-xyloglucan	O
-interaction	O
-,	O
-we	O
-solved	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-fused	B-mutant
-CD	I-mutant
-domains	I-mutant
-in	O
-complex	O
-with	O
-XyGO2	O
-(	O
-1	O
-.	O
-57	O
-Å	O
-,	O
-Rwork	O
-=	O
-15	O
-.	O
-6	O
-%,	O
-Rfree	O
-=	O
-17	O
-.	O
-1	O
-%,	O
-residues	O
-230	O
-to	O
-489	O
-)	O
-(	O
-Table	O
-2	O
-).	O
-	O
-The	O
-CD	O
-domains	O
-of	O
-the	O
-truncated	O
-and	O
-full	O
--	O
-length	O
-proteins	O
-superimpose	O
-with	O
-a	O
-0	O
-.	O
-4	O
--	O
-Å	O
-RMSD	O
-of	O
-the	O
-Cα	O
-backbone	O
-,	O
-with	O
-no	O
-differences	O
-in	O
-the	O
-position	O
-of	O
-any	O
-of	O
-the	O
-glycan	O
--	O
-binding	O
-residues	O
-(	O
-see	O
-Fig	O
-.	O
-S7A	O
-in	O
-the	O
-supplemental	O
-material	O
-).	O
-	O
-While	O
-density	O
-is	O
-observed	O
-for	O
-XyGO2	O
-,	O
-the	O
-ligand	O
-could	O
-not	O
-be	O
-unambiguously	O
-modeled	O
-into	O
-this	O
-density	O
-to	O
-achieve	O
-a	O
-reasonable	O
-fit	O
-between	O
-the	O
-X	O
--	O
-ray	O
-data	O
-and	O
-the	O
-known	O
-stereochemistry	O
-of	O
-the	O
-sugar	O
-(	O
-see	O
-Fig	O
-.	O
-S7B	O
-and	O
-C	O
-).	O
-	O
-While	O
-this	O
-may	O
-occur	O
-for	O
-a	O
-number	O
-of	O
-reasons	O
-in	O
-crystal	O
-structures	O
-,	O
-it	O
-is	O
-likely	O
-that	O
-the	O
-poor	O
-ligand	O
-density	O
-even	O
-at	O
-higher	O
-resolution	O
-is	O
-due	O
-to	O
-movement	O
-or	O
-multiple	O
-orientations	O
-of	O
-the	O
-sugar	O
-averaged	O
-throughout	O
-the	O
-lattice	O
-.	O
-	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-have	O
-distinct	O
-,	O
-coordinated	O
-functions	O
-in	O
-vivo	O
-.	O
-	O
-The	O
-similarity	O
-of	O
-the	O
-glycan	O
-specificity	O
-of	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-presents	O
-an	O
-intriguing	O
-conundrum	O
-regarding	O
-their	O
-individual	O
-roles	O
-in	O
-XyG	O
-utilization	O
-by	O
-B	O
-.	O
-ovatus	O
-.	O
-	O
-To	O
-disentangle	O
-the	O
-functions	O
-of	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-in	O
-XyG	O
-recognition	O
-and	O
-uptake	O
-,	O
-we	O
-created	O
-individual	O
-in	O
--	O
-frame	O
-deletion	O
-and	O
-complementation	O
-mutant	O
-strains	O
-of	O
-B	O
-.	O
-ovatus	O
-.	O
-	O
-In	O
-these	O
-growth	O
-experiments	O
-,	O
-overnight	O
-cultures	O
-of	O
-strains	O
-grown	O
-on	O
-minimal	O
-medium	O
-plus	O
-glucose	O
-were	O
-back	O
--	O
-diluted	O
-1	O
-:	O
-100	O
--	O
-fold	O
-into	O
-minimal	O
-medium	O
-containing	O
-5	O
-mg	O
-/	O
-ml	O
-of	O
-the	O
-reported	O
-carbohydrate	O
-.	O
-	O
-Growth	O
-on	O
-glucose	O
-displayed	O
-the	O
-shortest	O
-lag	O
-time	O
-for	O
-each	O
-strain	O
-,	O
-and	O
-so	O
-lag	O
-times	O
-were	O
-normalized	O
-for	O
-each	O
-carbohydrate	O
-by	O
-subtracting	O
-the	O
-lag	O
-time	O
-of	O
-that	O
-strain	O
-in	O
-glucose	O
-(	O
-Fig	O
-.	O
-6	O
-;	O
-see	O
-Fig	O
-.	O
-S8	O
-in	O
-the	O
-supplemental	O
-material	O
-).	O
-	O
-A	O
-strain	O
-in	O
-which	O
-the	O
-entire	O
-XyGUL	O
-is	O
-deleted	O
-displays	O
-a	O
-lag	O
-of	O
-24	O
-.	O
-5	O
-h	O
-during	O
-growth	O
-on	O
-glucose	O
-compared	O
-to	O
-the	O
-isogenic	O
-parental	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-Δtdk	B-mutant
-strain	O
-,	O
-for	O
-which	O
-exponential	O
-growth	O
-lags	O
-for	O
-19	O
-.	O
-8	O
-h	O
-(	O
-see	O
-Fig	O
-.	O
-S8D	O
-).	O
-	O
-It	O
-is	O
-unknown	O
-whether	O
-this	O
-is	O
-because	O
-cultures	O
-were	O
-not	O
-normalized	O
-by	O
-the	O
-starting	O
-optical	O
-density	O
-(	O
-OD	O
-)	O
-or	O
-viable	O
-cells	O
-or	O
-reflects	O
-a	O
-minor	O
-defect	O
-for	O
-glucose	O
-utilization	O
-.	O
-	O
-The	O
-former	O
-seems	O
-more	O
-likely	O
-as	O
-the	O
-growth	O
-rates	O
-are	O
-nearly	O
-identical	O
-for	O
-these	O
-strains	O
-on	O
-glucose	O
-and	O
-xylose	O
-.	O
-	O
-The	O
-ΔXyGUL	B-mutant
-and	O
-WT	O
-Δtdk	B-mutant
-strains	O
-display	O
-normalized	O
-lag	O
-times	O
-on	O
-xylose	O
-within	O
-experimental	O
-error	O
-,	O
-and	O
-curiously	O
-some	O
-of	O
-the	O
-mutant	O
-and	O
-complemented	O
-strains	O
-display	O
-a	O
-nominally	O
-shorter	O
-lag	O
-time	O
-on	O
-xylose	O
-than	O
-the	O
-WT	O
-Δtdk	B-mutant
-strain	O
-.	O
-	O
-Complementation	O
-of	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-strain	O
-(	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-::	O
-SGBP	O
--	O
-A	O
-)	O
-restores	O
-growth	O
-to	O
-wild	O
--	O
-type	O
-rates	O
-on	O
-xyloglucan	O
-and	O
-XyGO1	O
-,	O
-yet	O
-the	O
-calculated	O
-rate	O
-of	O
-the	O
-complemented	O
-strain	O
-is	O
-~	O
-72	O
-%	O
-that	O
-of	O
-the	O
-WT	O
-Δtdk	B-mutant
-strain	O
-on	O
-XyGO2	O
-;	O
-similar	O
-results	O
-were	O
-obtained	O
-for	O
-the	O
-SGBP	O
--	O
-B	O
-complemented	O
-strain	O
-despite	O
-the	O
-fact	O
-that	O
-the	O
-growth	O
-curves	O
-do	O
-not	O
-appear	O
-much	O
-different	O
-(	O
-see	O
-Fig	O
-.	O
-S8C	O
-and	O
-F	O
-).	O
-	O
-The	O
-reason	O
-for	O
-this	O
-observation	O
-on	O
-XyGO2	O
-is	O
-unclear	O
-,	O
-as	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-mutant	O
-does	O
-not	O
-have	O
-a	O
-significantly	O
-different	O
-growth	O
-rate	O
-from	O
-the	O
-WT	O
-on	O
-XyGO2	O
-.	O
-	O
-Growth	O
-of	O
-select	O
-XyGUL	O
-mutants	O
-on	O
-xyloglucan	O
-and	O
-oligosaccharides	O
-.	O
-	O
-B	O
-.	O
-ovatus	O
-mutants	O
-were	O
-created	O
-in	O
-a	O
-thymidine	B-mutant
-kinase	I-mutant
-deletion	I-mutant
-(	O
-Δtdk	B-mutant
-)	O
-mutant	O
-as	O
-described	O
-previously	O
-.	O
-	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*	I-mutant
-denotes	O
-the	O
-Bacova_02651	O
-(	O
-W82A	B-mutant
-W283A	B-mutant
-W306A	B-mutant
-)	O
-allele	O
-,	O
-and	O
-the	O
-GH9	O
-gene	O
-is	O
-Bacova_02649	O
-.	O
-	O
-Growth	O
-was	O
-measured	O
-over	O
-time	O
-in	O
-minimal	O
-medium	O
-containing	O
-(	O
-A	O
-)	O
-XyG	O
-,	O
-(	O
-B	O
-)	O
-XyGO2	O
-,	O
-(	O
-C	O
-)	O
-XyGO1	O
-,	O
-(	O
-D	O
-)	O
-glucose	O
-,	O
-and	O
-(	O
-E	O
-)	O
-xylose	O
-.	O
-	O
-In	O
-panel	O
-F	O
-,	O
-the	O
-growth	O
-rate	O
-of	O
-each	O
-strain	O
-on	O
-the	O
-five	O
-carbon	O
-sources	O
-is	O
-displayed	O
-,	O
-and	O
-in	O
-panel	O
-G	O
-,	O
-the	O
-normalized	O
-lag	O
-time	O
-of	O
-each	O
-culture	O
-,	O
-relative	O
-to	O
-its	O
-growth	O
-on	O
-glucose	O
-,	O
-is	O
-displayed	O
-.	O
-	O
-Solid	O
-bars	O
-indicate	O
-conditions	O
-that	O
-are	O
-not	O
-statistically	O
-significant	O
-from	O
-the	O
-WT	O
-Δtdk	B-mutant
-cultures	O
-grown	O
-on	O
-the	O
-indicated	O
-carbohydrate	O
-,	O
-while	O
-open	O
-bars	O
-indicate	O
-a	O
-P	O
-value	O
-of	O
-<	O
-0	O
-.	O
-005	O
-compared	O
-to	O
-the	O
-WT	O
-Δtdk	B-mutant
-strain	O
-.	O
-	O
-Conditions	O
-denoted	O
-by	O
-the	O
-same	O
-letter	O
-(	O
-b	O
-,	O
-c	O
-,	O
-or	O
-d	O
-)	O
-are	O
-not	O
-statistically	O
-significant	O
-from	O
-each	O
-other	O
-but	O
-are	O
-significantly	O
-different	O
-from	O
-the	O
-condition	O
-labeled	O
-“	O
-a	O
-.”	O
-Complementation	O
-of	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-and	O
-ΔSBGP	B-mutant
--	I-mutant
-B	I-mutant
-was	O
-performed	O
-by	O
-allelic	O
-exchange	O
-of	O
-the	O
-wild	O
--	O
-type	O
-genes	O
-back	O
-into	O
-the	O
-genome	O
-for	O
-expression	O
-via	O
-the	O
-native	O
-promoter	O
-:	O
-these	O
-growth	O
-curves	O
-,	O
-quantified	O
-rates	O
-and	O
-lag	O
-times	O
-are	O
-displayed	O
-in	O
-Fig	O
-.	O
-S8	O
-in	O
-the	O
-supplemental	O
-material	O
-.	O
-	O
-The	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-(	O
-ΔBacova_02651	B-mutant
-)	O
-strain	O
-(	O
-cf	O
-.	O
-	O
-Fig	O
-.	O
-1B	O
-)	O
-was	O
-completely	O
-incapable	O
-of	O
-growth	O
-on	O
-XyG	O
-,	O
-XyGO1	O
-,	O
-and	O
-XyGO2	O
-,	O
-indicating	O
-that	O
-SGBP	O
--	O
-A	O
-is	O
-essential	O
-for	O
-XyG	O
-utilization	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-This	O
-result	O
-mirrors	O
-our	O
-previous	O
-data	O
-for	O
-the	O
-canonical	O
-Sus	O
-of	O
-B	O
-.	O
-thetaiotaomicron	O
-,	O
-which	O
-revealed	O
-that	O
-a	O
-homologous	O
-ΔsusD	B-mutant
-mutant	O
-is	O
-unable	O
-to	O
-grow	O
-on	O
-starch	O
-or	O
-malto	O
--	O
-oligosaccharides	O
-,	O
-despite	O
-normal	O
-cell	O
-surface	O
-expression	O
-of	O
-all	O
-other	O
-PUL	O
--	O
-encoded	O
-proteins	O
-.	O
-	O
-More	O
-recently	O
-,	O
-we	O
-demonstrated	O
-that	O
-this	O
-phenotype	O
-is	O
-due	O
-to	O
-the	O
-loss	O
-of	O
-the	O
-physical	O
-presence	O
-of	O
-SusD	O
-;	O
-complementation	O
-of	O
-ΔsusD	B-mutant
-with	O
-SusD	B-mutant
-*,	I-mutant
-a	O
-triple	O
-site	O
--	O
-directed	O
-mutant	O
-(	O
-W96A	B-mutant
-W320A	B-mutant
-Y296A	B-mutant
-)	O
-that	O
-ablates	O
-glycan	O
-binding	O
-,	O
-restores	O
-B	O
-.	O
-thetaiotaomicron	O
-growth	O
-on	O
-malto	O
--	O
-oligosaccharides	O
-and	O
-starch	O
-when	O
-sus	O
-transcription	O
-is	O
-induced	O
-by	O
-maltose	O
-addition	O
-.	O
-	O
-Similarly	O
-,	O
-the	O
-function	O
-of	O
-SGBP	O
--	O
-A	O
-extends	O
-beyond	O
-glycan	O
-binding	O
-.	O
-	O
-Complementation	O
-of	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-with	O
-the	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*	I-mutant
-(	O
-W82A	B-mutant
-W283A	B-mutant
-W306A	B-mutant
-)	O
-variant	O
-,	O
-which	O
-does	O
-not	O
-bind	O
-XyG	O
-,	O
-supports	O
-growth	O
-on	O
-XyG	O
-and	O
-XyGOs	O
-(	O
-Fig	O
-.	O
-6	O
-;	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-::	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*),	I-mutant
-with	O
-growth	O
-rates	O
-that	O
-are	O
-~	O
-70	O
-%	O
-that	O
-of	O
-the	O
-WT	O
-.	O
-	O
-In	O
-previous	O
-studies	O
-,	O
-we	O
-observed	O
-that	O
-carbohydrate	O
-binding	O
-by	O
-SusD	O
-enhanced	O
-the	O
-sensitivity	O
-of	O
-the	O
-cells	O
-to	O
-limiting	O
-concentrations	O
-of	O
-malto	O
--	O
-oligosaccharides	O
-by	O
-several	O
-orders	O
-of	O
-magnitude	O
-,	O
-such	O
-that	O
-the	O
-addition	O
-of	O
-0	O
-.	O
-5	O
-g	O
-/	O
-liter	O
-maltose	O
-was	O
-required	O
-to	O
-restore	O
-growth	O
-of	O
-the	O
-ΔsusD	B-mutant
-::	O
-SusD	B-mutant
-*	I-mutant
-strain	O
-on	O
-starch	O
-,	O
-which	O
-nonetheless	O
-occurred	O
-following	O
-an	O
-extended	O
-lag	O
-phase	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-::	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*	I-mutant
-strain	O
-does	O
-not	O
-display	O
-an	O
-extended	O
-lag	O
-time	O
-on	O
-any	O
-of	O
-the	O
-xyloglucan	O
-substrates	O
-compared	O
-to	O
-the	O
-WT	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-The	O
-specific	O
-glycan	O
-signal	O
-that	O
-upregulates	O
-BoXyGUL	O
-is	O
-currently	O
-unknown	O
-.	O
-	O
-From	O
-our	O
-present	O
-data	O
-,	O
-we	O
-cannot	O
-eliminate	O
-the	O
-possibility	O
-that	O
-the	O
-glycan	O
-binding	O
-by	O
-SGBP	O
--	O
-A	O
-enhances	O
-transcriptional	O
-activation	O
-of	O
-the	O
-XyGUL	O
-.	O
-	O
-However	O
-,	O
-the	O
-modest	O
-rate	O
-defect	O
-displayed	O
-by	O
-the	O
-SGBP	O
--	O
-A	O
-::	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*	I-mutant
-strain	O
-suggests	O
-that	O
-recognition	O
-of	O
-XyG	O
-and	O
-product	O
-import	O
-is	O
-somewhat	O
-less	O
-efficient	O
-in	O
-these	O
-cells	O
-.	O
-	O
-Intriguingly	O
-,	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-strain	O
-(	O
-ΔBacova_02650	B-mutant
-)	O
-(	O
-cf	O
-.	O
-	O
-Fig	O
-.	O
-1B	O
-)	O
-exhibited	O
-a	O
-minor	O
-growth	O
-defect	O
-on	O
-both	O
-XyG	O
-and	O
-XyGO2	O
-,	O
-with	O
-rates	O
-84	O
-.	O
-6	O
-%	O
-and	O
-93	O
-.	O
-9	O
-%	O
-that	O
-of	O
-the	O
-WT	O
-Δtdk	B-mutant
-strain	O
-.	O
-	O
-However	O
-,	O
-growth	O
-of	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-strain	O
-on	O
-XyGO1	O
-was	O
-54	O
-.	O
-2	O
-%	O
-the	O
-rate	O
-of	O
-the	O
-parental	O
-strain	O
-,	O
-despite	O
-the	O
-fact	O
-that	O
-SGBP	O
--	O
-B	O
-binds	O
-this	O
-substrate	O
-ca	O
-.	O
-	O
-10	O
--	O
-fold	O
-more	O
-weakly	O
-than	O
-XyGO2	O
-and	O
-XyG	O
-(	O
-Fig	O
-.	O
-6	O
-;	O
-Table	O
-1	O
-).	O
-	O
-As	O
-such	O
-,	O
-the	O
-data	O
-suggest	O
-that	O
-SGBP	O
--	O
-A	O
-can	O
-compensate	O
-for	O
-the	O
-loss	O
-of	O
-function	O
-of	O
-SGBP	O
--	O
-B	O
-on	O
-longer	O
-oligo	O
--	O
-and	O
-polysaccharides	O
-,	O
-while	O
-SGBP	O
--	O
-B	O
-may	O
-adapt	O
-the	O
-cell	O
-to	O
-recognize	O
-smaller	O
-oligosaccharides	O
-efficiently	O
-.	O
-	O
-Indeed	O
-,	O
-a	O
-double	O
-mutant	O
-,	O
-consisting	O
-of	O
-a	O
-crippled	O
-SGBP	O
--	O
-A	O
-and	O
-a	O
-deletion	O
-of	O
-SGBP	O
--	O
-B	O
-(	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-::	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*/	I-mutant
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-),	O
-exhibits	O
-an	O
-extended	O
-lag	O
-time	O
-on	O
-both	O
-XyG	O
-and	O
-XyGO2	O
-,	O
-as	O
-well	O
-as	O
-XyGO1	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-the	O
-data	O
-indicate	O
-that	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-functionally	O
-complement	O
-each	O
-other	O
-in	O
-the	O
-capture	O
-of	O
-XyG	O
-polysaccharide	O
-,	O
-while	O
-SGBP	O
--	O
-B	O
-may	O
-allow	O
-B	O
-.	O
-ovatus	O
-to	O
-scavenge	O
-smaller	O
-XyGOs	O
-liberated	O
-by	O
-other	O
-gut	O
-commensals	O
-.	O
-	O
-This	O
-additional	O
-role	O
-of	O
-SGBP	O
--	O
-B	O
-is	O
-especially	O
-notable	O
-in	O
-the	O
-context	O
-of	O
-studies	O
-on	O
-BtSusE	O
-and	O
-BtSusF	O
-(	O
-positioned	O
-similarly	O
-in	O
-the	O
-archetypal	O
-Sus	O
-locus	O
-)	O
-(	O
-Fig	O
-.	O
-1B	O
-),	O
-for	O
-which	O
-growth	O
-defects	O
-on	O
-starch	O
-or	O
-malto	O
--	O
-oligosaccharides	O
-have	O
-never	O
-been	O
-observed	O
-.	O
-	O
-Beyond	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-,	O
-we	O
-speculated	O
-that	O
-the	O
-catalytically	O
-feeble	O
-endo	O
--	O
-xyloglucanase	O
-GH9	O
-,	O
-which	O
-is	O
-expendable	O
-for	O
-growth	O
-in	O
-the	O
-presence	O
-of	O
-GH5	O
-,	O
-might	O
-also	O
-play	O
-a	O
-role	O
-in	O
-glycan	O
-binding	O
-to	O
-the	O
-cell	O
-surface	O
-.	O
-	O
-However	O
-,	O
-combined	O
-deletion	O
-of	O
-the	O
-genes	O
-encoding	O
-GH9	O
-(	O
-encoded	O
-by	O
-Bacova_02649	O
-)	O
-and	O
-SGBP	O
--	O
-B	O
-does	O
-not	O
-exacerbate	O
-the	O
-growth	O
-defect	O
-on	O
-XyGO1	O
-(	O
-Fig	O
-.	O
-6	O
-;	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-/	O
-ΔGH9	B-mutant
-).	O
-	O
-The	O
-necessity	O
-of	O
-SGBP	O
--	O
-B	O
-is	O
-elevated	O
-in	O
-the	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*	I-mutant
-strain	O
-,	O
-as	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-::	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*/	I-mutant
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-mutant	O
-displays	O
-an	O
-extended	O
-lag	O
-during	O
-growth	O
-on	O
-XyG	O
-and	O
-xylogluco	O
--	O
-oligosaccharides	O
-,	O
-while	O
-growth	O
-rate	O
-differences	O
-are	O
-more	O
-subtle	O
-.	O
-	O
-The	O
-precise	O
-reason	O
-for	O
-this	O
-lag	O
-is	O
-unclear	O
-,	O
-but	O
-recapitulating	O
-our	O
-findings	O
-on	O
-the	O
-role	O
-of	O
-SusD	O
-in	O
-malto	O
--	O
-oligosaccharide	O
-sensing	O
-in	O
-B	O
-.	O
-thetaiotaomicron	O
-,	O
-this	O
-extended	O
-lag	O
-may	O
-be	O
-due	O
-to	O
-inefficient	O
-import	O
-and	O
-thus	O
-sensing	O
-of	O
-xyloglucan	O
-in	O
-the	O
-environment	O
-in	O
-the	O
-absence	O
-of	O
-glycan	O
-binding	O
-by	O
-essential	O
-SGBPs	O
-.	O
-	O
-Our	O
-previous	O
-work	O
-demonstrates	O
-that	O
-B	O
-.	O
-ovatus	O
-cells	O
-grown	O
-in	O
-minimal	O
-medium	O
-plus	O
-glucose	O
-express	O
-low	O
-levels	O
-of	O
-the	O
-XyGUL	O
-transcript	O
-.	O
-	O
-Thus	O
-,	O
-in	O
-our	O
-experiments	O
-,	O
-we	O
-presume	O
-that	O
-each	O
-strain	O
-,	O
-initially	O
-grown	O
-in	O
-glucose	O
-,	O
-expresses	O
-low	O
-levels	O
-of	O
-the	O
-XyGUL	O
-transcript	O
-and	O
-thus	O
-low	O
-levels	O
-of	O
-the	O
-XyGUL	O
--	O
-encoded	O
-surface	O
-proteins	O
-,	O
-including	O
-the	O
-vanguard	O
-GH5	O
-.	O
-	O
-Presumably	O
-without	O
-glycan	O
-binding	O
-by	O
-the	O
-SGBPs	O
-,	O
-the	O
-GH5	O
-protein	O
-cannot	O
-efficiently	O
-process	O
-xyloglucan	O
-,	O
-and	O
-/	O
-or	O
-the	O
-lack	O
-of	O
-SGBP	O
-function	O
-prevents	O
-efficient	O
-capture	O
-and	O
-import	O
-of	O
-the	O
-processed	O
-oligosaccharides	O
-.	O
-	O
-It	O
-may	O
-then	O
-be	O
-that	O
-only	O
-after	O
-a	O
-sufficient	O
-amount	O
-of	O
-glycan	O
-is	O
-processed	O
-and	O
-imported	O
-by	O
-the	O
-cell	O
-is	O
-XyGUL	O
-upregulated	O
-and	O
-exponential	O
-growth	O
-on	O
-the	O
-glycan	O
-can	O
-begin	O
-.	O
-	O
-We	O
-hypothesize	O
-that	O
-during	O
-exponential	O
-growth	O
-the	O
-essential	O
-role	O
-of	O
-SGBP	O
--	O
-A	O
-extends	O
-beyond	O
-glycan	O
-recognition	O
-,	O
-perhaps	O
-due	O
-to	O
-a	O
-critical	O
-interaction	O
-with	O
-the	O
-TBDT	O
-.	O
-	O
-In	O
-the	O
-BtSus	O
-,	O
-SusD	O
-and	O
-the	O
-TBDT	O
-SusC	O
-interact	O
-,	O
-and	O
-we	O
-speculate	O
-that	O
-this	O
-interaction	O
-is	O
-necessary	O
-for	O
-glycan	O
-uptake	O
-,	O
-as	O
-suggested	O
-by	O
-the	O
-fact	O
-that	O
-a	O
-ΔsusD	B-mutant
-mutant	O
-cannot	O
-grow	O
-on	O
-starch	O
-,	O
-but	O
-a	O
-ΔsusD	B-mutant
-::	O
-SusD	B-mutant
-*	I-mutant
-strain	O
-regains	O
-this	O
-ability	O
-if	O
-a	O
-transcriptional	O
-activator	O
-of	O
-the	O
-sus	O
-operon	O
-is	O
-supplied	O
-.	O
-	O
-Likewise	O
-,	O
-such	O
-cognate	O
-interactions	O
-between	O
-homologous	O
-protein	O
-pairs	O
-such	O
-as	O
-SGBP	O
--	O
-A	O
-and	O
-its	O
-TBDT	O
-may	O
-underlie	O
-our	O
-observation	O
-that	O
-a	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-mutant	O
-cannot	O
-grow	O
-on	O
-xyloglucan	O
-.	O
-	O
-However	O
-,	O
-unlike	O
-the	O
-Sus	O
-,	O
-in	O
-which	O
-elimination	O
-of	O
-SusE	O
-and	O
-SusF	O
-does	O
-not	O
-affect	O
-growth	O
-on	O
-starch	O
-,	O
-SGBP	O
--	O
-B	O
-appears	O
-to	O
-have	O
-a	O
-dedicated	O
-role	O
-in	O
-growth	O
-on	O
-small	O
-xylogluco	O
--	O
-oligosaccharides	O
-.	O
-	O
-The	O
-ability	O
-of	O
-gut	O
--	O
-adapted	O
-microorganisms	O
-to	O
-thrive	O
-in	O
-the	O
-gastrointestinal	O
-tract	O
-is	O
-critically	O
-dependent	O
-upon	O
-their	O
-ability	O
-to	O
-efficiently	O
-recognize	O
-,	O
-cleave	O
-,	O
-and	O
-import	O
-glycans	O
-.	O
-	O
-The	O
-human	O
-gut	O
-,	O
-in	O
-particular	O
-,	O
-is	O
-a	O
-densely	O
-packed	O
-ecosystem	O
-with	O
-hundreds	O
-of	O
-species	O
-,	O
-in	O
-which	O
-there	O
-is	O
-potential	O
-for	O
-both	O
-competition	O
-and	O
-synergy	O
-in	O
-the	O
-utilization	O
-of	O
-different	O
-substrates	O
-.	O
-	O
-Recent	O
-work	O
-has	O
-elucidated	O
-that	O
-Bacteroidetes	O
-cross	O
--	O
-feed	O
-during	O
-growth	O
-on	O
-many	O
-glycans	O
-;	O
-the	O
-glycoside	O
-hydrolases	O
-expressed	O
-by	O
-one	O
-species	O
-liberate	O
-oligosaccharides	O
-for	O
-consumption	O
-by	O
-other	O
-members	O
-of	O
-the	O
-community	O
-.	O
-	O
-Thus	O
-,	O
-understanding	O
-glycan	O
-capture	O
-at	O
-the	O
-cell	O
-surface	O
-is	O
-fundamental	O
-to	O
-explaining	O
-,	O
-and	O
-eventually	O
-predicting	O
-,	O
-how	O
-the	O
-carbohydrate	O
-content	O
-of	O
-the	O
-diet	O
-shapes	O
-the	O
-gut	O
-community	O
-structure	O
-as	O
-well	O
-as	O
-its	O
-causative	O
-health	O
-effects	O
-.	O
-	O
-Here	O
-,	O
-we	O
-demonstrate	O
-that	O
-the	O
-surface	O
-glycan	O
-binding	O
-proteins	O
-encoded	O
-within	O
-the	O
-BoXyGUL	O
-play	O
-unique	O
-and	O
-essential	O
-roles	O
-in	O
-the	O
-acquisition	O
-of	O
-the	O
-ubiquitous	O
-and	O
-abundant	O
-vegetable	O
-polysaccharide	O
-xyloglucan	O
-.	O
-	O
-Yet	O
-,	O
-a	O
-number	O
-of	O
-questions	O
-remain	O
-regarding	O
-the	O
-molecular	O
-interplay	O
-of	O
-SGBPs	O
-with	O
-their	O
-cotranscribed	O
-cohort	O
-of	O
-glycoside	O
-hydrolases	O
-and	O
-TonB	O
--	O
-dependent	O
-transporters	O
-.	O
-	O
-A	O
-particularly	O
-understudied	O
-aspect	O
-of	O
-glycan	O
-utilization	O
-is	O
-the	O
-mechanism	O
-of	O
-import	O
-via	O
-TBDTs	O
-(	O
-SusC	O
-homologs	O
-)	O
-(	O
-Fig	O
-.	O
-1	O
-),	O
-which	O
-are	O
-ubiquitous	O
-and	O
-defining	O
-components	O
-of	O
-all	O
-PUL	O
-.	O
-	O
-PUL	O
--	O
-encoded	O
-TBDTs	O
-in	O
-Bacteroidetes	O
-are	O
-larger	O
-than	O
-the	O
-well	O
--	O
-characterized	O
-iron	O
--	O
-targeting	O
-TBDTs	O
-from	O
-many	O
-Proteobacteria	O
-and	O
-are	O
-further	O
-distinguished	O
-as	O
-the	O
-only	O
-known	O
-glycan	O
--	O
-importing	O
-TBDTs	O
-coexpressed	O
-with	O
-an	O
-SGBP	O
-.	O
-	O
-A	O
-direct	O
-interaction	O
-between	O
-the	O
-BtSusC	O
-TBDT	O
-and	O
-the	O
-SusD	O
-SGBP	O
-has	O
-been	O
-previously	O
-demonstrated	O
-,	O
-as	O
-has	O
-an	O
-interaction	O
-between	O
-the	O
-homologous	O
-components	O
-encoded	O
-by	O
-an	O
-N	O
--	O
-glycan	O
--	O
-scavenging	O
-PUL	O
-of	O
-Capnocytophaga	O
-canimorsus	O
-.	O
-	O
-Our	O
-observation	O
-here	O
-that	O
-the	O
-physical	O
-presence	O
-of	O
-the	O
-SusD	O
-homolog	O
-SGBP	O
--	O
-A	O
-,	O
-independent	O
-of	O
-XyG	O
--	O
-binding	O
-ability	O
-,	O
-is	O
-both	O
-necessary	O
-and	O
-sufficient	O
-for	O
-XyG	O
-utilization	O
-further	O
-supports	O
-a	O
-model	O
-of	O
-glycan	O
-import	O
-whereby	O
-the	O
-SusC	O
--	O
-like	O
-TBDTs	O
-and	O
-the	O
-SusD	O
--	O
-like	O
-SGBPs	O
-must	O
-be	O
-intimately	O
-associated	O
-to	O
-support	O
-glycan	O
-uptake	O
-(	O
-Fig	O
-.	O
-1C	O
-).	O
-	O
-It	O
-is	O
-yet	O
-presently	O
-unclear	O
-whether	O
-this	O
-interaction	O
-is	O
-static	O
-or	O
-dynamic	O
-and	O
-to	O
-what	O
-extent	O
-the	O
-association	O
-of	O
-cognate	O
-TBDT	O
-/	O
-SGBPs	O
-is	O
-dependent	O
-upon	O
-the	O
-structure	O
-of	O
-the	O
-carbohydrate	O
-to	O
-be	O
-imported	O
-.	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-there	O
-is	O
-clear	O
-evidence	O
-for	O
-independent	O
-TBDTs	O
-in	O
-Bacteroidetes	O
-that	O
-do	O
-not	O
-require	O
-SGBP	O
-association	O
-for	O
-activity	O
-.	O
-	O
-For	O
-example	O
-,	O
-it	O
-was	O
-recently	O
-demonstrated	O
-that	O
-expression	O
-of	O
-nanO	O
-,	O
-which	O
-encodes	O
-a	O
-SusC	O
--	O
-like	O
-TBDT	O
-as	O
-part	O
-of	O
-a	O
-sialic	O
--	O
-acid	O
--	O
-targeting	O
-PUL	O
-from	O
-B	O
-.	O
-fragilis	O
-,	O
-restored	O
-growth	O
-on	O
-this	O
-monosaccharide	O
-in	O
-a	O
-mutant	O
-strain	O
-of	O
-E	O
-.	O
-coli	O
-.	O
-	O
-In	O
-this	O
-instance	O
-,	O
-coexpression	O
-of	O
-the	O
-susD	O
--	O
-like	O
-gene	O
-nanU	O
-was	O
-not	O
-required	O
-,	O
-nor	O
-did	O
-the	O
-expression	O
-of	O
-the	O
-nanU	O
-gene	O
-enhance	O
-growth	O
-kinetics	O
-.	O
-	O
-Similarly	O
-,	O
-the	O
-deletion	O
-of	O
-BT1762	O
-encoding	O
-a	O
-fructan	O
--	O
-targeting	O
-SusD	O
--	O
-like	O
-protein	O
-in	O
-B	O
-.	O
-thetaiotaomicron	O
-did	O
-not	O
-result	O
-in	O
-a	O
-dramatic	O
-loss	O
-of	O
-growth	O
-on	O
-fructans	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-strict	O
-dependence	O
-on	O
-a	O
-SusD	O
--	O
-like	O
-SGBP	O
-for	O
-glycan	O
-uptake	O
-in	O
-the	O
-Bacteroidetes	O
-may	O
-be	O
-variable	O
-and	O
-substrate	O
-dependent	O
-.	O
-	O
-Furthermore	O
-,	O
-considering	O
-the	O
-broader	O
-distribution	O
-of	O
-TBDTs	O
-in	O
-PUL	O
-lacking	O
-SGBPs	O
-(	O
-sometimes	O
-known	O
-as	O
-carbohydrate	O
-utilization	O
-containing	O
-TBDT	O
-[	O
-CUT	O
-]	O
-loci	O
-;	O
-see	O
-reference	O
-and	O
-reviewed	O
-in	O
-reference	O
-)	O
-across	O
-bacterial	O
-phyla	O
-,	O
-it	O
-appears	O
-that	O
-the	O
-intimate	O
-biophysical	O
-association	O
-of	O
-these	O
-substrate	O
--	O
-transport	O
-and	O
--	O
-binding	O
-proteins	O
-is	O
-the	O
-result	O
-of	O
-specific	O
-evolution	O
-within	O
-the	O
-Bacteroidetes	O
-.	O
-	O
-Equally	O
-intriguing	O
-is	O
-the	O
-observation	O
-that	O
-while	O
-SusD	O
--	O
-like	O
-proteins	O
-such	O
-as	O
-SGBP	O
--	O
-A	O
-share	O
-moderate	O
-primary	O
-and	O
-high	O
-tertiary	O
-structural	O
-conservation	O
-,	O
-the	O
-genes	O
-for	O
-the	O
-SGBPs	O
-encoded	O
-immediately	O
-downstream	O
-(	O
-Fig	O
-.	O
-1B	O
-[	O
-sometimes	O
-referred	O
-to	O
-as	O
-“	O
-susE	O
-positioned	O
-”])	O
-encode	O
-glycan	O
--	O
-binding	O
-lipoproteins	O
-with	O
-little	O
-or	O
-no	O
-sequence	O
-or	O
-structural	O
-conservation	O
-,	O
-even	O
-among	O
-syntenic	O
-PUL	O
-that	O
-target	O
-the	O
-same	O
-polysaccharide	O
-.	O
-	O
-Such	O
-is	O
-the	O
-case	O
-for	O
-XyGUL	O
-from	O
-related	O
-Bacteroides	O
-species	O
-,	O
-which	O
-may	O
-encode	O
-either	O
-one	O
-or	O
-two	O
-of	O
-these	O
-predicted	O
-SGBPs	O
-,	O
-and	O
-these	O
-proteins	O
-vary	O
-considerably	O
-in	O
-length	O
-.	O
-	O
-The	O
-extremely	O
-low	O
-similarity	O
-of	O
-these	O
-SGBPs	O
-is	O
-striking	O
-in	O
-light	O
-of	O
-the	O
-moderate	O
-sequence	O
-conservation	O
-observed	O
-among	O
-homologous	O
-GHs	O
-in	O
-syntenic	O
-PUL	O
-.	O
-	O
-This	O
-,	O
-together	O
-with	O
-the	O
-observation	O
-that	O
-these	O
-SGBPs	O
-,	O
-as	O
-exemplified	O
-by	O
-BtSusE	O
-and	O
-BtSusF	O
-and	O
-the	O
-XyGUL	O
-SGBP	O
--	O
-B	O
-of	O
-the	O
-present	O
-study	O
-,	O
-are	O
-expendable	O
-for	O
-polysaccharide	O
-growth	O
-,	O
-implies	O
-a	O
-high	O
-degree	O
-of	O
-evolutionary	O
-flexibility	O
-to	O
-enhance	O
-glycan	O
-capture	O
-at	O
-the	O
-cell	O
-surface	O
-.	O
-	O
-Because	O
-the	O
-intestinal	O
-ecosystem	O
-is	O
-a	O
-dense	O
-consortium	O
-of	O
-bacteria	O
-that	O
-must	O
-compete	O
-for	O
-their	O
-nutrients	O
-,	O
-these	O
-multimodular	O
-SGBPs	O
-may	O
-reflect	O
-ongoing	O
-evolutionary	O
-experiments	O
-to	O
-enhance	O
-glycan	O
-uptake	O
-efficiency	O
-.	O
-	O
-Whether	O
-organisms	O
-that	O
-express	O
-longer	O
-SGBPs	O
-,	O
-extending	O
-further	O
-above	O
-the	O
-cell	O
-surface	O
-toward	O
-the	O
-extracellular	O
-environment	O
-,	O
-are	O
-better	O
-equipped	O
-to	O
-compete	O
-for	O
-available	O
-carbohydrates	O
-is	O
-presently	O
-unknown	O
-.	O
-	O
-However	O
-,	O
-the	O
-natural	O
-diversity	O
-of	O
-these	O
-proteins	O
-represents	O
-a	O
-rich	O
-source	O
-for	O
-the	O
-discovery	O
-of	O
-unique	O
-carbohydrate	O
--	O
-binding	O
-motifs	O
-to	O
-both	O
-inform	O
-gut	O
-microbiology	O
-and	O
-generate	O
-new	O
-,	O
-specific	O
-carbohydrate	O
-analytical	O
-reagents	O
-.	O
-	O
-In	O
-conclusion	O
-,	O
-the	O
-present	O
-study	O
-further	O
-illuminates	O
-the	O
-essential	O
-role	O
-that	O
-surface	O
--	O
-glycan	O
-binding	O
-proteins	O
-play	O
-in	O
-facilitating	O
-the	O
-catabolism	O
-of	O
-complex	O
-dietary	O
-carbohydrates	O
-by	O
-Bacteroidetes	O
-.	O
-	O
-The	O
-ability	O
-of	O
-our	O
-resident	O
-gut	O
-bacteria	O
-to	O
-recognize	O
-polysaccharides	O
-is	O
-the	O
-first	O
-committed	O
-step	O
-of	O
-glycan	O
-consumption	O
-by	O
-these	O
-organisms	O
-,	O
-a	O
-critical	O
-process	O
-that	O
-influences	O
-the	O
-community	O
-structure	O
-and	O
-thus	O
-the	O
-metabolic	O
-output	O
-(	O
-i	O
-.	O
-e	O
-.,	O
-short	O
--	O
-chain	O
-fatty	O
-acid	O
-and	O
-metabolite	O
-profile	O
-)	O
-of	O
-these	O
-organisms	O
-.	O
-	O
-A	O
-molecular	O
-understanding	O
-of	O
-glycan	O
-uptake	O
-by	O
-human	O
-gut	O
-bacteria	O
-is	O
-therefore	O
-central	O
-to	O
-the	O
-development	O
-of	O
-strategies	O
-to	O
-improve	O
-human	O
-health	O
-through	O
-manipulation	O
-of	O
-the	O
-microbiota	O
-.	O
-	O
-Inhibiting	O
-complex	O
-IL	O
--	O
-17A	O
-and	O
-IL	O
--	O
-17RA	O
-interactions	O
-with	O
-a	O
-linear	O
-peptide	O
-	O
-IL	O
--	O
-17A	O
-is	O
-a	O
-pro	O
--	O
-inflammatory	O
-cytokine	O
-that	O
-has	O
-been	O
-implicated	O
-in	O
-autoimmune	O
-and	O
-inflammatory	O
-diseases	O
-.	O
-	O
-Monoclonal	O
-antibodies	O
-inhibiting	O
-IL	O
--	O
-17A	O
-signaling	O
-have	O
-demonstrated	O
-remarkable	O
-efficacy	O
-,	O
-but	O
-an	O
-oral	O
-therapy	O
-is	O
-still	O
-lacking	O
-.	O
-	O
-A	O
-high	O
-affinity	O
-IL	O
--	O
-17A	O
-peptide	O
-antagonist	O
-(	O
-HAP	O
-)	O
-of	O
-15	O
-residues	O
-was	O
-identified	O
-through	O
-phage	O
--	O
-display	O
-screening	O
-followed	O
-by	O
-saturation	O
-mutagenesis	O
-optimization	O
-and	O
-amino	O
-acid	O
-substitutions	O
-.	O
-	O
-HAP	O
-binds	O
-specifically	O
-to	O
-IL	O
--	O
-17A	O
-and	O
-inhibits	O
-the	O
-interaction	O
-of	O
-the	O
-cytokine	O
-with	O
-its	O
-receptor	O
-,	O
-IL	O
--	O
-17RA	O
-.	O
-	O
-Tested	O
-in	O
-primary	O
-human	O
-cells	O
-,	O
-HAP	O
-blocked	O
-the	O
-production	O
-of	O
-multiple	O
-inflammatory	O
-cytokines	O
-.	O
-	O
-Crystal	O
-structure	O
-studies	O
-revealed	O
-that	O
-two	O
-HAP	O
-molecules	O
-bind	O
-to	O
-one	O
-IL	O
--	O
-17A	O
-dimer	O
-symmetrically	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-portions	O
-of	O
-HAP	O
-form	O
-a	O
-β	O
--	O
-strand	O
-that	O
-inserts	O
-between	O
-two	O
-IL	O
--	O
-17A	O
-monomers	O
-while	O
-the	O
-C	O
--	O
-terminal	O
-section	O
-forms	O
-an	O
-α	O
-helix	O
-that	O
-directly	O
-blocks	O
-IL	O
--	O
-17RA	O
-from	O
-binding	O
-to	O
-the	O
-same	O
-region	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-The	O
-family	O
-of	O
-IL	O
--	O
-17	O
-cytokines	O
-and	O
-receptors	O
-consists	O
-of	O
-six	O
-polypeptides	O
-,	O
-IL	O
--	O
-17A	O
--	O
-F	O
-,	O
-and	O
-five	O
-receptors	O
-,	O
-IL	O
--	O
-17RA	O
--	O
-E	O
-.	O
-IL	O
--	O
-17A	O
-is	O
-secreted	O
-from	O
-activated	O
-Th17	O
-cells	O
-,	O
-and	O
-several	O
-innate	O
-immune	O
-T	O
-cell	O
-types	O
-including	O
-macrophages	O
-,	O
-neutrophils	O
-,	O
-natural	O
-killer	O
-cells	O
-,	O
-and	O
-dendritic	O
-cells	O
-.	O
-	O
-IL	O
--	O
-17A	O
-signals	O
-through	O
-a	O
-specific	O
-cell	O
-surface	O
-receptor	O
-complex	O
-which	O
-consists	O
-of	O
-IL	O
--	O
-17RA	O
-and	O
-IL	O
--	O
-17RC	O
-.	O
-	O
-IL	O
--	O
-17A	O
-’	O
-s	O
-downstream	O
-signaling	O
-leads	O
-to	O
-increased	O
-production	O
-of	O
-inflammatory	O
-cytokines	O
-such	O
-as	O
-IL	O
--	O
-6	O
-,	O
-IL	O
--	O
-8	O
-,	O
-CCL	O
--	O
-20	O
-and	O
-CXCL1	O
-by	O
-various	O
-mechanisms	O
-including	O
-stimulation	O
-of	O
-transcription	O
-and	O
-stabilization	O
-of	O
-mRNA	O
-.	O
-	O
-Although	O
-various	O
-cell	O
-types	O
-have	O
-been	O
-reported	O
-to	O
-express	O
-IL	O
--	O
-17RA	O
-,	O
-the	O
-highest	O
-responses	O
-to	O
-IL	O
--	O
-17A	O
-come	O
-from	O
-epithelial	O
-cells	O
-,	O
-endothelial	O
-cells	O
-,	O
-keratinocytes	O
-and	O
-fibroblasts	O
-.	O
-	O
-IL	O
--	O
-17A	O
-and	O
-its	O
-signaling	O
-is	O
-important	O
-in	O
-host	O
-defense	O
-against	O
-certain	O
-fungal	O
-and	O
-bacterial	O
-infections	O
-as	O
-demonstrated	O
-by	O
-patients	O
-with	O
-autoantibodies	O
-against	O
-IL	O
--	O
-17A	O
-and	O
-IL	O
--	O
-17F	O
-,	O
-or	O
-with	O
-inborn	O
-errors	O
-of	O
-IL	O
--	O
-17	O
-immunity	O
-.	O
-	O
-In	O
-addition	O
-to	O
-its	O
-physiological	O
-role	O
-,	O
-IL	O
--	O
-17A	O
-is	O
-a	O
-key	O
-pathogenic	O
-factor	O
-in	O
-inflammatory	O
-and	O
-autoimmune	O
-diseases	O
-.	O
-	O
-In	O
-phase	O
-II	O
-and	O
-III	O
-clinical	O
-trials	O
-,	O
-neutralizing	O
-monoclonal	O
-antibodies	O
-against	O
-IL	O
--	O
-17A	O
-(	O
-secukinumab	O
-and	O
-ixekizumab	O
-)	O
-or	O
-its	O
-receptor	O
-IL	O
--	O
-17RA	O
-(	O
-brodalumab	O
-)	O
-are	O
-highly	O
-efficacious	O
-in	O
-treating	O
-moderate	O
-to	O
-severe	O
-plaque	O
-psoriasis	O
-and	O
-psoriatic	O
-arthritis	O
-.	O
-	O
-Secukinumab	O
-has	O
-been	O
-approved	O
-recently	O
-as	O
-a	O
-new	O
-psoriasis	O
-drug	O
-by	O
-the	O
-US	O
-Food	O
-and	O
-Drug	O
-Administration	O
-(	O
-Cosentyx	O
-™).	O
-	O
-In	O
-addition	O
-to	O
-psoriasis	O
-and	O
-psoriatic	O
-arthritis	O
-,	O
-IL	O
--	O
-17A	O
-blockade	O
-has	O
-also	O
-shown	O
-preclinical	O
-and	O
-clinical	O
-efficacies	O
-in	O
-ankylosing	O
-spondylitis	O
-and	O
-rheumatoid	O
-arthritis	O
-.	O
-	O
-Among	O
-IL	O
--	O
-17	O
-cytokines	O
-,	O
-IL	O
--	O
-17A	O
-and	O
-IL	O
--	O
-17F	O
-share	O
-the	O
-highest	O
-homology	O
-.	O
-	O
-These	O
-polypeptides	O
-form	O
-covalent	O
-homodimers	O
-,	O
-and	O
-IL	O
--	O
-17A	O
-and	O
-IL	O
--	O
-17F	O
-also	O
-form	O
-an	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17F	O
-hetereodimer	O
-.	O
-	O
-Structures	O
-are	O
-known	O
-for	O
-apo	O
-IL	O
--	O
-17F	O
-and	O
-its	O
-complex	O
-with	O
-IL	O
--	O
-17RA	O
-,	O
-for	O
-apo	O
-IL	O
--	O
-17A	O
-,	O
-its	O
-complex	O
-with	O
-an	O
-antibody	O
-Fab	O
-,	O
-and	O
-its	O
-complex	O
-with	O
-IL	O
--	O
-17RA	O
-.	O
-	O
-In	O
-these	O
-structures	O
-,	O
-both	O
-IL	O
--	O
-17A	O
-and	O
-IL	O
--	O
-17F	O
-adopt	O
-a	O
-cysteine	O
--	O
-knot	O
-fold	O
-with	O
-two	O
-intramolecular	O
-disulfides	O
-and	O
-two	O
-interchain	O
-disulfide	O
-bonds	O
-that	O
-covalently	O
-link	O
-two	O
-monomers	O
-.	O
-	O
-There	O
-has	O
-been	O
-active	O
-research	O
-in	O
-identifying	O
-orally	O
-available	O
-chemical	O
-entities	O
-that	O
-would	O
-functionally	O
-antagonize	O
-IL	O
--	O
-17A	O
--	O
-mediated	O
-signaling	O
-.	O
-	O
-Developing	O
-small	O
-molecules	O
-targeting	O
-protein	O
--	O
-protein	O
-interactions	O
-is	O
-difficult	O
-with	O
-particular	O
-challenges	O
-associated	O
-with	O
-the	O
-large	O
-,	O
-shallow	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-interfaces	O
-.	O
-	O
-Since	O
-IL	O
--	O
-17RA	O
-is	O
-a	O
-shared	O
-receptor	O
-for	O
-at	O
-least	O
-IL	O
--	O
-17A	O
-,	O
-IL	O
--	O
-17F	O
-,	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17F	O
-and	O
-IL	O
--	O
-17E	O
-,	O
-we	O
-chose	O
-to	O
-seek	O
-IL	O
--	O
-17A	O
--	O
-specific	O
-inhibitors	O
-that	O
-may	O
-have	O
-more	O
-defined	O
-pharmacological	O
-responses	O
-than	O
-IL	O
--	O
-17RA	O
-inhibitors	O
-.	O
-	O
-Our	O
-efforts	O
-resulted	O
-in	O
-discovery	O
-of	O
-a	O
-high	O
-affinity	O
-IL	O
--	O
-17A	O
-peptide	O
-antagonist	O
-(	O
-HAP	O
-),	O
-which	O
-we	O
-attempted	O
-to	O
-increase	O
-the	O
-functional	O
-production	O
-and	O
-pharmacokinetics	O
-after	O
-fusing	O
-HAP	O
-to	O
-antibodies	O
-for	O
-evaluation	O
-as	O
-a	O
-bispecific	O
-therapeutic	O
-in	O
-animal	O
-studies	O
-.	O
-	O
-Unfortunately	O
-,	O
-this	O
-past	O
-work	O
-revealed	O
-stability	O
-issues	O
-of	O
-the	O
-uncapped	O
-HAP	O
-in	O
-cell	O
-culture	O
-Here	O
-,	O
-we	O
-provide	O
-the	O
-details	O
-of	O
-the	O
-discovery	O
-and	O
-optimization	O
-that	O
-led	O
-to	O
-HAP	O
-and	O
-report	O
-the	O
-complex	O
-structure	O
-of	O
-IL	O
--	O
-17A	O
-with	O
-HAP	O
-,	O
-which	O
-provides	O
-structure	O
-based	O
-rationalization	O
-of	O
-peptide	O
-optimization	O
-and	O
-structure	O
-activity	O
-relationship	O
-(	O
-SAR	O
-).	O
-	O
-Identification	O
-of	O
-IL	O
--	O
-17A	O
-peptide	O
-inhibitors	O
-	O
-Peptides	O
-specifically	O
-binding	O
-to	O
-human	O
-IL	O
--	O
-17A	O
-were	O
-identified	O
-from	O
-phage	O
-panning	O
-using	O
-cyclic	O
-and	O
-linear	O
-peptide	O
-libraries	O
-(	O
-Supplementary	O
-Figure	O
-S1	O
-).	O
-	O
-Positive	O
-phage	O
-pools	O
-were	O
-then	O
-sub	O
--	O
-cloned	O
-into	O
-a	O
-maltose	O
--	O
-binding	O
-protein	O
-(	O
-MBP	O
-)	O
-fusion	O
-system	O
-.	O
-	O
-Single	O
-clones	O
-were	O
-isolated	O
-and	O
-sub	O
--	O
-cultured	O
-in	O
-growth	O
-medium	O
-,	O
-and	O
-culture	O
-supernatants	O
-were	O
-used	O
-in	O
-an	O
-enzyme	O
--	O
-linked	O
-immunosorbent	O
-assay	O
-(	O
-ELISA	O
-)	O
-to	O
-identify	O
-specific	O
-IL	O
--	O
-17A	O
--	O
-binding	O
-clones	O
-.	O
-	O
-The	O
-positive	O
-binding	O
-supernatants	O
-were	O
-tested	O
-for	O
-the	O
-ability	O
-to	O
-block	O
-biotinylated	O
-IL	O
--	O
-17A	O
-signaling	O
-through	O
-IL	O
--	O
-17RA	O
-in	O
-an	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-competition	O
-ELISA	O
-assay	O
-where	O
-unlabeled	O
-IL	O
--	O
-17A	O
-was	O
-used	O
-as	O
-positive	O
-control	O
-to	O
-inhibit	O
-biotinylated	O
-IL	O
--	O
-17A	O
-binding	O
-.	O
-	O
-Approximately	O
-10	O
-%	O
-of	O
-the	O
-clones	O
-that	O
-specifically	O
-bound	O
-to	O
-IL	O
--	O
-17A	O
-also	O
-prevented	O
-the	O
-cytokine	O
-from	O
-binding	O
-to	O
-IL	O
--	O
-17RA	O
-.	O
-	O
-Sequences	O
-identified	O
-from	O
-phage	O
-clones	O
-were	O
-chemically	O
-synthesized	O
-(	O
-Supplementary	O
-Table	O
-1	O
-)	O
-and	O
-tested	O
-for	O
-inhibition	O
-of	O
-IL	O
--	O
-17A	O
-binding	O
-to	O
-IL	O
--	O
-17RA	O
-(	O
-Table	O
-1	O
-).	O
-	O
-A	O
-15	O
--	O
-mer	O
-linear	O
-peptide	O
-1	O
-was	O
-shown	O
-to	O
-block	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-binding	O
-with	O
-an	O
-IC50	O
-of	O
-80	O
-nM	O
-in	O
-the	O
-competition	O
-ELISA	O
-assay	O
-(	O
-Table	O
-1	O
-).	O
-	O
-This	O
-peptide	O
-was	O
-then	O
-tested	O
-in	O
-a	O
-cell	O
--	O
-based	O
-functional	O
-assay	O
-wherein	O
-production	O
-of	O
-GRO	O
--	O
-α	O
-in	O
-BJ	O
-human	O
-fibroblast	O
-cells	O
-was	O
-measured	O
-as	O
-a	O
-function	O
-of	O
-IL	O
--	O
-17A	O
-stimulation	O
-using	O
-1	O
-ng	O
-/	O
-ml	O
-IL	O
--	O
-17A	O
-.	O
-	O
-Peptide	O
-1	O
-was	O
-found	O
-to	O
-be	O
-active	O
-in	O
-this	O
-functional	O
-assay	O
-with	O
-an	O
-IC50	O
-of	O
-370	O
-nM	O
-.	O
-	O
-Optimization	O
-of	O
-IL	O
--	O
-17A	O
-peptide	O
-inhibitors	O
-	O
-A	O
-SAR	O
-campaign	O
-was	O
-undertaken	O
-to	O
-improve	O
-the	O
-potency	O
-of	O
-peptide	O
-1	O
-.	O
-	O
-An	O
-alanine	O
-scan	O
-of	O
-peptide	O
-2	O
-,	O
-an	O
-analogue	O
-of	O
-1	O
-with	O
-a	O
-lysine	O
-to	O
-arginine	O
-substitution	O
-at	O
-position	O
-14	O
-,	O
-was	O
-initiated	O
-.	O
-	O
-When	O
-alanine	O
-was	O
-already	O
-present	O
-(	O
-positions	O
-7	O
-and	O
-15	O
-),	O
-substitution	O
-was	O
-made	O
-with	O
-lysine	O
-(	O
-Table	O
-1	O
-,	O
-peptides	O
-3	O
-–	O
-17	O
-).	O
-	O
-Positions	O
-1	O
-,	O
-2	O
-,	O
-4	O
-,	O
-5	O
-,	O
-7	O
-,	O
-14	O
-and	O
-15	O
-were	O
-shown	O
-to	O
-be	O
-amenable	O
-to	O
-substitution	O
-without	O
-significant	O
-loss	O
-(	O
-less	O
-than	O
-3	O
--	O
-fold	O
-)	O
-of	O
-binding	O
-affinity	O
-as	O
-measured	O
-by	O
-the	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-competition	O
-ELISA	O
-.	O
-	O
-In	O
-particular	O
-,	O
-at	O
-position	O
-5	O
-(	O
-13	O
-),	O
-substitution	O
-of	O
-methionine	O
-with	O
-alanine	O
-resulted	O
-in	O
-a	O
-seven	O
-fold	O
-improvement	O
-in	O
-potency	O
-(	O
-80	O
-nM	O
-versus	O
-11	O
-nM	O
-respectively	O
-).	O
-	O
-In	O
-order	O
-to	O
-rapidly	O
-evaluate	O
-the	O
-effects	O
-of	O
-substitution	O
-of	O
-natural	O
-amino	O
-acids	O
-at	O
-tolerant	O
-positions	O
-identified	O
-by	O
-the	O
-alanine	O
-scan	O
-,	O
-the	O
-lead	O
-sequence	O
-was	O
-subjected	O
-to	O
-site	O
--	O
-specific	O
-saturation	O
-mutagenesis	O
-using	O
-MBP	O
-.	O
-	O
-Each	O
-of	O
-the	O
-seven	O
-positions	O
-identified	O
-by	O
-the	O
-alanine	O
-scan	O
-was	O
-individually	O
-modified	O
-while	O
-keeping	O
-the	O
-rest	O
-of	O
-the	O
-sequence	O
-constant	O
-.	O
-	O
-Modifications	O
-at	O
-positions	O
-2	O
-and	O
-14	O
-were	O
-shown	O
-to	O
-display	O
-improvement	O
-in	O
-binding	O
-affinity	O
-(	O
-data	O
-not	O
-shown	O
-).	O
-	O
-Peptides	O
-with	O
-beneficial	O
-point	O
-mutations	O
-at	O
-positions	O
-2	O
-,	O
-5	O
-,	O
-and	O
-14	O
-were	O
-synthesized	O
-and	O
-evaluated	O
-in	O
-the	O
-competition	O
-ELISA	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Two	O
-of	O
-the	O
-changes	O
-,	O
-V2H	B-mutant
-(	O
-18	O
-)	O
-or	O
-V2T	B-mutant
-(	O
-21	O
-)	O
-displayed	O
-improved	O
-binding	O
-in	O
-the	O
-competition	O
-ELISA	O
-.	O
-	O
-Since	O
-the	O
-replacement	O
-of	O
-methionine	O
-at	O
-position	O
-5	O
-with	O
-alanine	O
-was	O
-beneficial	O
-,	O
-the	O
-additional	O
-hydrophobic	O
-amino	O
-acids	O
-isoleucine	O
-(	O
-24	O
-),	O
-leucine	O
-(	O
-25	O
-)	O
-and	O
-valine	O
-(	O
-26	O
-)	O
-were	O
-evaluated	O
-and	O
-an	O
-additional	O
-two	O
--	O
-three	O
-fold	O
-improvement	O
-in	O
-binding	O
-was	O
-observed	O
-for	O
-the	O
-valine	O
-and	O
-isoleucine	O
-replacements	O
-in	O
-comparison	O
-with	O
-alanine	O
-.	O
-	O
-Introduction	O
-of	O
-a	O
-methionine	O
-(	O
-27	O
-)	O
-or	O
-a	O
-carboxamide	O
-(	O
-28	O
-and	O
-29	O
-)	O
-at	O
-position	O
-14	O
-was	O
-shown	O
-to	O
-improve	O
-the	O
-binding	O
-affinity	O
-of	O
-the	O
-lead	O
-peptide	O
-.	O
-	O
-In	O
-general	O
-,	O
-there	O
-was	O
-good	O
-agreement	O
-between	O
-the	O
-respective	O
-binding	O
-affinities	O
-of	O
-the	O
-synthesized	O
-peptides	O
-and	O
-their	O
-MBP	O
-fusion	O
-counterparts	O
-,	O
-except	O
-for	O
-substitution	O
-of	O
-valine	O
-at	O
-position	O
-2	O
-to	O
-a	O
-tryptophan	O
-(	O
-22	O
-),	O
-which	O
-resulted	O
-in	O
-a	O
-fivefold	O
-loss	O
-of	O
-affinity	O
-,	O
-for	O
-the	O
-free	O
-peptide	O
-when	O
-compared	O
-with	O
-the	O
-MBP	O
-fusion	O
-.	O
-	O
-Combining	O
-the	O
-key	O
-amino	O
--	O
-acid	O
-residues	O
-identified	O
-by	O
-SAR	O
-into	O
-a	O
-single	O
-peptide	O
-sequence	O
-resulted	O
-in	O
-peptide	O
-30	O
-,	O
-named	O
-high	O
-affinity	O
-peptide	O
-(	O
-HAP	O
-),	O
-that	O
-was	O
-found	O
-to	O
-inhibit	O
-IL	O
--	O
-17A	O
-signaling	O
-in	O
-a	O
-BJ	O
-human	O
-fibroblast	O
-cell	O
-assay	O
-with	O
-an	O
-IC50	O
-of	O
-17	O
-nM	O
-,	O
-a	O
-more	O
-than	O
-20	O
--	O
-fold	O
-improvement	O
-over	O
-the	O
-phage	O
-peptide	O
-1	O
-(	O
-Table	O
-2	O
-and	O
-Supplementary	O
-Figure	O
-S2	O
-).	O
-	O
-We	O
-also	O
-examined	O
-the	O
-effect	O
-of	O
-removing	O
-the	O
-acetyl	O
-group	O
-at	O
-the	O
-N	O
--	O
-terminus	O
-of	O
-HAP	O
-(	O
-which	O
-is	O
-present	O
-in	O
-all	O
-the	O
-peptides	O
-made	O
-,	O
-see	O
-Supplementary	O
-Material	O
-).	O
-	O
-The	O
-un	O
--	O
-capped	O
-peptide	O
-(	O
-31	O
-)	O
-had	O
-an	O
-IC50	O
-of	O
-420	O
-nM	O
-in	O
-the	O
-cell	O
--	O
-based	O
-assay	O
-.	O
-	O
-The	O
-loss	O
-of	O
-cellular	O
-activity	O
-of	O
-31	O
-was	O
-most	O
-likely	O
-due	O
-to	O
-the	O
-degradation	O
-of	O
-the	O
-N	O
--	O
-terminus	O
-of	O
-31	O
-,	O
-since	O
-peptide	O
-31	O
-was	O
-shown	O
-to	O
-be	O
-able	O
-to	O
-bind	O
-to	O
-IL	O
--	O
-17A	O
-with	O
-similar	O
-affinity	O
-as	O
-HAP	O
-itself	O
-.	O
-	O
-Furthermore	O
-,	O
-our	O
-previous	O
-work	O
-had	O
-reported	O
-that	O
-in	O
-antibody	O
-fusions	O
-the	O
-uncapped	O
-peptide	O
-was	O
-degraded	O
-under	O
-cell	O
-assay	O
-conditions	O
-with	O
-removal	O
-of	O
-the	O
-first	O
-1	O
--	O
-3	O
-residues	O
-to	O
-inactive	O
-products	O
-with	O
-the	O
-same	O
-N	O
--	O
-terminal	O
-sequences	O
-as	O
-peptides	O
-32	O
-–	O
-34	O
-.	O
-	O
-In	O
-this	O
-work	O
-,	O
-32	O
-–	O
-34	O
-are	O
-capped	O
-by	O
-protective	O
-acetyl	O
-group	O
-and	O
-reflect	O
-the	O
-same	O
-inactivity	O
-as	O
-reported	O
-.	O
-	O
-C	O
--	O
-terminal	O
-truncations	O
-showed	O
-a	O
-more	O
-gradual	O
-reduction	O
-in	O
-activity	O
-(	O
-35	O
-–	O
-37	O
-;	O
-Table	O
-2	O
-).	O
-	O
-After	O
-deletion	O
-of	O
-three	O
-amino	O
-acids	O
-from	O
-the	O
-C	O
--	O
-terminal	O
-end	O
-(	O
-37	O
-),	O
-the	O
-peptide	O
-is	O
-no	O
-longer	O
-active	O
-.	O
-	O
-Dimerization	O
-of	O
-HAP	O
-can	O
-further	O
-increase	O
-its	O
-potency	O
-	O
-We	O
-reasoned	O
-that	O
-since	O
-the	O
-IL	O
--	O
-17A	O
-protein	O
-is	O
-almost	O
-exclusively	O
-present	O
-in	O
-a	O
-dimeric	O
-form	O
-,	O
-dimerizing	O
-the	O
-IL	O
--	O
-17A	O
-binding	O
-peptides	O
-could	O
-result	O
-in	O
-an	O
-improvement	O
-in	O
-binding	O
-affinity	O
-and	O
-inhibitory	O
-activity	O
-.	O
-	O
-Homodimers	O
-of	O
-HAP	O
-were	O
-made	O
-through	O
-attachment	O
-of	O
-polyethylene	O
-glycol	O
-(	O
-PEG	O
-)	O
-spacers	O
-of	O
-different	O
-lengths	O
-at	O
-amino	O
-acids	O
-4	O
-,	O
-7	O
-and	O
-14	O
-,	O
-as	O
-these	O
-positions	O
-were	O
-identified	O
-in	O
-the	O
-alanine	O
-scan	O
-analysis	O
-as	O
-not	O
-contributing	O
-significantly	O
-to	O
-the	O
-activity	O
-,	O
-and	O
-at	O
-each	O
-N	O
--	O
-terminus	O
-(	O
-Supplementary	O
-Table	O
-S2	O
-).	O
-	O
-Due	O
-to	O
-the	O
-high	O
-reactivity	O
-of	O
-the	O
-pentafluoroester	O
-(	O
-PFP	O
-)	O
-group	O
-used	O
-as	O
-the	O
-activating	O
-group	O
-in	O
-the	O
-PEG	O
-,	O
-the	O
-histidine	O
-at	O
-position	O
-2	O
-and	O
-the	O
-lysine	O
-at	O
-position	O
-15	O
-were	O
-replaced	O
-with	O
-threonine	O
-and	O
-dimethyllysine	O
-respectively	O
-to	O
-prevent	O
-formation	O
-of	O
-side	O
-products	O
-,	O
-which	O
-resulted	O
-in	O
-peptide	O
-38	O
-that	O
-was	O
-comparable	O
-in	O
-activity	O
-with	O
-HAP	O
-.	O
-	O
-This	O
-exercise	O
-revealed	O
-that	O
-several	O
-dimeric	O
-peptides	O
-with	O
-the	O
-longer	O
-PEG21	O
-spacer	O
-were	O
-significantly	O
-more	O
-potent	O
-than	O
-the	O
-monomer	O
-peptide	O
-in	O
-the	O
-cell	O
--	O
-based	O
-assay	O
-(	O
-Supplementary	O
-Table	O
-S2	O
-).	O
-	O
-Peptide	O
-45	O
-,	O
-dimerized	O
-via	O
-attachment	O
-of	O
-a	O
-PEG21	O
-spacer	O
-at	O
-position	O
-14	O
-(	O
-Supplementary	O
-Scheme	O
-S1	O
-and	O
-Figure	O
-S3	O
-),	O
-was	O
-the	O
-most	O
-potent	O
-with	O
-cellular	O
-IC50	O
-of	O
-0	O
-.	O
-1	O
-nM	O
-.	O
-This	O
-significant	O
-improvement	O
-in	O
-antagonism	O
-was	O
-not	O
-seen	O
-in	O
-the	O
-peptide	O
-monomer	O
-functionalized	O
-with	O
-a	O
-PEG21	O
-group	O
-at	O
-position	O
-14	O
-as	O
-peptide	O
-48	O
-had	O
-an	O
-IC50	O
-of	O
-21	O
-nM	O
-(	O
-Supplementary	O
-Scheme	O
-S2	O
-).	O
-	O
-The	O
-species	O
-cross	O
--	O
-reactivity	O
-of	O
-the	O
-dimeric	O
-peptide	O
-45	O
-and	O
-HAP	O
-were	O
-assessed	O
-in	O
-a	O
-murine	O
-functional	O
-cell	O
-assay	O
-using	O
-15	O
-ng	O
-/	O
-ml	O
-murine	O
-IL	O
--	O
-17A	O
-.	O
-	O
-Peptide	O
-45	O
-blocked	O
-the	O
-receptor	O
-binding	O
-of	O
-murine	O
-IL	O
--	O
-17A	O
-although	O
-with	O
-potency	O
-two	O
-orders	O
-of	O
-magnitude	O
-weaker	O
-than	O
-that	O
-observed	O
-against	O
-human	O
-IL	O
--	O
-17A	O
-(	O
-IC50	O
-=	O
-41	O
-nM	O
-vs	O
-IC50	O
-=	O
-0	O
-.	O
-1	O
-nM	O
-,	O
-respectively	O
-).	O
-	O
-The	O
-monomer	O
-HAP	O
-was	O
-much	O
-weaker	O
-(	O
-IC50	O
->	O
-1	O
-μM	O
-)	O
-in	O
-inhibiting	O
-murine	O
-IL	O
--	O
-17A	O
-signaling	O
-(	O
-Supplementary	O
-Figure	O
-S4	O
-).	O
-	O
-Although	O
-the	O
-dimeric	O
-peptide	O
-45	O
-is	O
-much	O
-more	O
-potent	O
-than	O
-HAP	O
-in	O
-the	O
-cell	O
--	O
-based	O
-assay	O
-,	O
-in	O
-subsequent	O
-studies	O
-we	O
-decided	O
-to	O
-focus	O
-our	O
-efforts	O
-solely	O
-on	O
-characterizations	O
-of	O
-the	O
-monomeric	O
-peptide	O
-HAP	O
-in	O
-hopes	O
-to	O
-identify	O
-smaller	O
-peptide	O
-inhibitors	O
-containing	O
-the	O
-best	O
-minimal	O
-functional	O
-group	O
-.	O
-	O
-Orthogonal	O
-assays	O
-to	O
-confirm	O
-HAP	O
-antagonism	O
-	O
-To	O
-further	O
-characterize	O
-the	O
-interaction	O
-of	O
-HAP	O
-with	O
-IL	O
--	O
-17A	O
-,	O
-we	O
-set	O
-out	O
-to	O
-determine	O
-its	O
-in	O
-vitro	O
-binding	O
-affinity	O
-,	O
-specificity	O
-and	O
-kinetic	O
-profile	O
-using	O
-Surface	O
-Plasmon	O
-Resonance	O
-(	O
-SPR	O
-)	O
-methods	O
-(	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-HAP	O
-binds	O
-to	O
-immobilized	O
-human	O
-IL	O
--	O
-17A	O
-homodimer	O
-tightly	O
-(	O
-Table	O
-3	O
-).	O
-	O
-It	O
-has	O
-slightly	O
-weaker	O
-affinity	O
-for	O
-human	O
-IL	O
--	O
-17A	O
-/	O
-F	O
-heterodimer	O
-and	O
->	O
-10	O
-fold	O
-weaker	O
-affinity	O
-for	O
-mouse	O
-IL	O
--	O
-17A	O
-(	O
-Table	O
-3	O
-).	O
-	O
-HAP	O
-does	O
-not	O
-show	O
-significant	O
-binding	O
-to	O
-immobilized	O
-human	O
-IL	O
--	O
-17F	O
-homodimer	O
-or	O
-IL	O
--	O
-17RA	O
-at	O
-concentrations	O
-up	O
-to	O
-100	O
-nM	O
-.	O
-	O
-Additionally	O
-,	O
-we	O
-investigated	O
-the	O
-antagonism	O
-of	O
-the	O
-human	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-interaction	O
-by	O
-HAP	O
-using	O
-orthogonal	O
-methods	O
-including	O
-SPR	O
-and	O
-Förster	O
-resonance	O
-energy	O
-transfer	O
-(	O
-FRET	O
-)	O
-competition	O
-assays	O
-(	O
-Fig	O
-.	O
-1B	O
-,	O
-C	O
-).	O
-	O
-In	O
-both	O
-assays	O
-,	O
-incubation	O
-of	O
-IL	O
--	O
-17A	O
-with	O
-HAP	O
-effectively	O
-blocks	O
-the	O
-binding	O
-of	O
-IL	O
--	O
-17A	O
-to	O
-immobilized	O
-IL	O
--	O
-17RA	O
-with	O
-similar	O
-sub	O
--	O
-nM	O
-IC50	O
-(	O
-Table	O
-3	O
-).	O
-	O
-HAP	O
-blocks	O
-IL	O
--	O
-17A	O
-signaling	O
-in	O
-a	O
-human	O
-primary	O
-cell	O
-assay	O
-	O
-While	O
-either	O
-IL	O
--	O
-17A	O
-or	O
-TNF	O
--	O
-α	O
-alone	O
-can	O
-stimulate	O
-the	O
-release	O
-of	O
-multiple	O
-inflammatory	O
-cytokines	O
-,	O
-when	O
-acting	O
-together	O
-they	O
-can	O
-synergistically	O
-enhance	O
-each	O
-other	O
-’	O
-s	O
-effects	O
-(	O
-Supplementary	O
-Figure	O
-S5	O
-).	O
-	O
-These	O
-integrative	O
-responses	O
-to	O
-IL	O
--	O
-17A	O
-and	O
-TNF	O
--	O
-α	O
-in	O
-human	O
-keratinocytes	O
-have	O
-been	O
-reported	O
-to	O
-account	O
-for	O
-key	O
-inflammatory	O
-pathogenic	O
-circuits	O
-in	O
-psoriasis	O
-.	O
-	O
-Thus	O
-,	O
-we	O
-chose	O
-to	O
-study	O
-HAP	O
-’	O
-s	O
-efficacy	O
-in	O
-blocking	O
-the	O
-production	O
-of	O
-IL	O
--	O
-8	O
-,	O
-IL	O
--	O
-6	O
-and	O
-CCL	O
--	O
-20	O
-by	O
-primary	O
-human	O
-keratinocytes	O
-stimulated	O
-by	O
-IL	O
--	O
-17A	O
-in	O
-the	O
-presence	O
-of	O
-TNF	O
--	O
-α	O
-,	O
-an	O
-assay	O
-which	O
-may	O
-be	O
-more	O
-disease	O
--	O
-relevant	O
-.	O
-	O
-HAP	O
-inhibits	O
-the	O
-production	O
-of	O
-all	O
-three	O
-cytokines	O
-in	O
-a	O
-dose	O
--	O
-dependent	O
-fashion	O
-(	O
-Fig	O
-.	O
-1D	O
-).	O
-	O
-Significantly	O
-,	O
-the	O
-baseline	O
-levels	O
-of	O
-IL	O
--	O
-8	O
-,	O
-IL	O
--	O
-6	O
-and	O
-CCL	O
--	O
-20	O
-stimulated	O
-by	O
-TNF	O
--	O
-α	O
-alone	O
-are	O
-not	O
-inhibited	O
-by	O
-HAP	O
-,	O
-further	O
-indicating	O
-the	O
-selectivity	O
-of	O
-HAP	O
-(	O
-Fig	O
-.	O
-1D	O
-).	O
-	O
-Such	O
-pharmacological	O
-selectivity	O
-may	O
-be	O
-important	O
-to	O
-suppress	O
-inflammatory	O
-pathogenic	O
-circuits	O
-in	O
-psoriasis	O
-,	O
-while	O
-sparing	O
-the	O
-anti	O
--	O
-infectious	O
-immune	O
-responses	O
-produced	O
-by	O
-TNF	O
--	O
-α	O
-.	O
-	O
-The	O
-relatively	O
-high	O
-IC50	O
-values	O
-in	O
-this	O
-assay	O
-(	O
-Table	O
-3	O
-)	O
-are	O
-probably	O
-due	O
-to	O
-the	O
-high	O
-IL	O
--	O
-17A	O
-concentration	O
-(	O
-100	O
-ng	O
-/	O
-ml	O
-)	O
-needed	O
-for	O
-detection	O
-of	O
-IL	O
--	O
-6	O
-.	O
-	O
-As	O
-a	O
-reference	O
-,	O
-a	O
-commercial	O
-anti	O
--	O
-IL	O
--	O
-17A	O
-antibody	O
-(	O
-R	O
-&	O
-D	O
-Systems	O
-)	O
-inhibits	O
-the	O
-production	O
-of	O
-IL	O
--	O
-8	O
-with	O
-an	O
-IC50	O
-of	O
-13	O
-(±	O
-6	O
-)	O
-nM	O
-(	O
-N	O
-=	O
-3	O
-).	O
-	O
-Indeed	O
-,	O
-the	O
-IC50	O
-was	O
-14	O
-(±	O
-9	O
-)	O
-nM	O
-(	O
-N	O
-=	O
-12	O
-)	O
-for	O
-HAP	O
-inhibition	O
-of	O
-IL	O
--	O
-8	O
-production	O
-when	O
-only	O
-5	O
-ng	O
-/	O
-ml	O
-IL	O
--	O
-17A	O
-was	O
-used	O
-in	O
-this	O
-assay	O
-.	O
-	O
-In	O
-patients	O
-,	O
-the	O
-concentration	O
-of	O
-IL	O
--	O
-17A	O
-in	O
-psoriatic	O
-lesions	O
-is	O
-reported	O
-to	O
-be	O
-0	O
-.	O
-01	O
-ng	O
-/	O
-ml	O
-,	O
-well	O
-below	O
-the	O
-EC50	O
-(	O
-5	O
-–	O
-10ng	O
-/	O
-ml	O
-)	O
-of	O
-IL	O
--	O
-17A	O
-induced	O
-IL	O
--	O
-8	O
-production	O
-in	O
-vitro	O
-.	O
-	O
-Similar	O
-to	O
-keratinocytes	O
-assay	O
-results	O
-,	O
-while	O
-HAP	O
-inhibits	O
-IL	O
--	O
-17A	O
-stimulated	O
-IL	O
--	O
-6	O
-production	O
-by	O
-BJ	O
-human	O
-fibroblast	O
-potently	O
-(	O
-IC50	O
-of	O
-17	O
-nM	O
-),	O
-it	O
-does	O
-not	O
-inhibit	O
-TNF	O
--	O
-α	O
-stimulated	O
-IL	O
--	O
-6	O
-production	O
-at	O
-concentrations	O
-up	O
-to	O
-10	O
-μM	O
-(	O
-Supplementary	O
-Figure	O
-S2	O
-).	O
-	O
-Crystallization	O
-and	O
-structure	O
-determination	O
-	O
-Extensive	O
-crystallization	O
-trials	O
-,	O
-either	O
-by	O
-co	O
--	O
-crystallization	O
-or	O
-by	O
-soaking	O
-HAP	O
-into	O
-preformed	O
-apo	O
-IL	O
--	O
-17A	O
-crystals	O
-,	O
-failed	O
-to	O
-lead	O
-to	O
-an	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-crystals	O
-.	O
-	O
-We	O
-theorized	O
-that	O
-HAP	O
-binding	O
-induced	O
-large	O
-conformational	O
-changes	O
-in	O
-IL	O
--	O
-17A	O
-that	O
-led	O
-to	O
-the	O
-difficulty	O
-of	O
-getting	O
-an	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-binary	O
-complex	O
-crystal	O
-.	O
-	O
-It	O
-is	O
-known	O
-that	O
-an	O
-antibody	O
-antigen	O
--	O
-binding	O
-fragment	O
-(	O
-Fab	O
-)	O
-can	O
-be	O
-used	O
-as	O
-crystallization	O
-chaperones	O
-in	O
-crystallizing	O
-difficult	O
-targets	O
-.	O
-	O
-We	O
-hypothesized	O
-that	O
-HAP	O
-may	O
-target	O
-the	O
-N	O
--	O
-terminal	O
-of	O
-IL	O
--	O
-17A	O
-which	O
-is	O
-known	O
-to	O
-be	O
-more	O
-flexible	O
-than	O
-its	O
-C	O
--	O
-terminal	O
-and	O
-conformational	O
-changes	O
-needed	O
-for	O
-HAP	O
-binding	O
-may	O
-be	O
-more	O
-likely	O
-there	O
-.	O
-	O
-We	O
-designed	O
-an	O
-antibody	O
-Fab	O
-known	O
-to	O
-target	O
-the	O
-C	O
--	O
-terminal	O
-half	O
-of	O
-IL	O
--	O
-17A	O
-based	O
-on	O
-a	O
-published	O
-IL	O
--	O
-17A	O
-/	O
-Fab	O
-complex	O
-crystal	O
-structure	O
-,	O
-and	O
-produced	O
-it	O
-in	O
-HEK293	O
-cells	O
-.	O
-	O
-In	O
-an	O
-SPR	O
-assay	O
-HAP	O
-and	O
-this	O
-Fab	O
-were	O
-able	O
-to	O
-co	O
--	O
-bind	O
-IL	O
--	O
-17A	O
-without	O
-large	O
-changes	O
-in	O
-their	O
-binding	O
-affinities	O
-and	O
-kinetics	O
-,	O
-confirming	O
-our	O
-hypothesis	O
-(	O
-Supplementary	O
-Figure	O
-S6	O
-).	O
-	O
-Furthermore	O
-,	O
-since	O
-it	O
-binds	O
-to	O
-an	O
-area	O
-far	O
-away	O
-from	O
-that	O
-of	O
-HAP	O
-(	O
-see	O
-below	O
-),	O
-this	O
-Fab	O
-should	O
-have	O
-minimum	O
-effects	O
-on	O
-HAP	O
-binding	O
-conformation	O
-.	O
-	O
-Crystals	O
-of	O
-Fab	O
-/	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-ternary	O
-complex	O
-were	O
-obtained	O
-readily	O
-in	O
-crystallization	O
-screens	O
-.	O
-	O
-Crystallization	O
-of	O
-IL	O
--	O
-17A	O
-and	O
-its	O
-binding	O
-partners	O
-was	O
-accomplished	O
-using	O
-two	O
-forms	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-These	O
-were	O
-,	O
-respectively	O
-,	O
-a	O
-presumably	O
-more	O
-homogeneous	O
-form	O
-of	O
-IL	O
--	O
-17A	O
-that	O
-lacked	O
-the	O
-disordered	O
-N	O
--	O
-terminal	O
-peptide	O
-and	O
-a	O
-full	O
--	O
-length	O
-form	O
-of	O
-the	O
-cytokine	O
-with	O
-a	O
-full	O
-complement	O
-of	O
-disulfide	O
-bonds	O
-.	O
-	O
-Crystals	O
-of	O
-the	O
-Fab	O
-/	O
-truncated	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-diffracted	O
-to	O
-2	O
-.	O
-2	O
-Å	O
-,	O
-and	O
-the	O
-Fab	O
-/	O
-full	O
-length	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-diffracted	O
-to	O
-3	O
-.	O
-0	O
-Å	O
-(	O
-Supplementary	O
-Table	O
-S3	O
-).	O
-	O
-Both	O
-structures	O
-were	O
-solved	O
-by	O
-molecular	O
-replacement	O
-.	O
-	O
-Both	O
-complexes	O
-crystallized	O
-in	O
-the	O
-space	O
-group	O
-of	O
-P321	O
-,	O
-with	O
-half	O
-the	O
-complex	O
-(	O
-1	O
-Fab	O
-/	O
-1	O
-IL	O
--	O
-17A	O
-monomer	O
-/	O
-1	O
-HAP	O
-)	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-The	O
-intact	O
-complex	O
-can	O
-be	O
-generated	O
-by	O
-applying	O
-crystallographic	O
-2	O
--	O
-fold	O
-symmetry	O
-.	O
-	O
-Electron	O
-densities	O
-for	O
-HAP	O
-residues	O
-Ile1	O
--	O
-Asn14	O
-were	O
-readily	O
-interpretable	O
-with	O
-the	O
-exception	O
-of	O
-Lys15	O
-,	O
-which	O
-is	O
-disordered	O
-.	O
-	O
-When	O
-considering	O
-the	O
-protein	O
-,	O
-the	O
-complex	O
-structure	O
-containing	O
-the	O
-full	O
-length	O
-IL	O
--	O
-17A	O
-is	O
-identical	O
-to	O
-that	O
-of	O
-the	O
-truncated	O
-IL	O
--	O
-17A	O
-,	O
-with	O
-the	O
-exception	O
-of	O
-Cys106	O
-(	O
-Ser106	O
-in	O
-the	O
-truncated	O
-IL	O
--	O
-17A	O
-),	O
-which	O
-is	O
-disordered	O
-.	O
-	O
-Cys106	O
-is	O
-covalently	O
-linked	O
-to	O
-Cys10	O
-that	O
-resides	O
-in	O
-the	O
-disordered	O
-N	O
--	O
-terminal	O
-peptide	O
-in	O
-the	O
-full	O
-length	O
-IL	O
--	O
-17A	O
-.	O
-	O
-Overall	O
-structure	O
-of	O
-Fab	O
-/	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-	O
-In	O
-a	O
-similar	O
-manner	O
-to	O
-the	O
-published	O
-structure	O
-of	O
-Fab	O
-/	O
-IL	O
--	O
-17A	O
-complex	O
-,	O
-two	O
-Fab	O
-molecules	O
-bind	O
-symmetrically	O
-to	O
-the	O
-C	O
--	O
-terminal	O
-of	O
-the	O
-cytokine	O
-dimer	O
-,	O
-interacting	O
-with	O
-epitopes	O
-from	O
-both	O
-monomers	O
-(	O
-Fig	O
-.	O
-2A	O
-).	O
-	O
-Two	O
-copies	O
-of	O
-HAP	O
-bind	O
-to	O
-the	O
-N	O
--	O
-terminal	O
-of	O
-the	O
-cytokine	O
-dimer	O
-,	O
-also	O
-symmetrically	O
-,	O
-and	O
-each	O
-HAP	O
-molecule	O
-also	O
-interacts	O
-with	O
-both	O
-IL	O
--	O
-17A	O
-monomers	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Based	O
-on	O
-disclosed	O
-epitopes	O
-of	O
-Secukinumab	O
-and	O
-Ixekizumab	O
-,	O
-HAP	O
-binds	O
-to	O
-IL	O
--	O
-17A	O
-at	O
-an	O
-area	O
-that	O
-is	O
-also	O
-different	O
-from	O
-those	O
-of	O
-those	O
-two	O
-antibodies	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-5	O
-residues	O
-of	O
-HAP	O
-,	O
-1IHVTI	O
-,	O
-form	O
-an	O
-amphipathic	O
-β	O
--	O
-strand	O
-that	O
-inserts	O
-between	O
-β	O
--	O
-strand	O
-4	O
-of	O
-one	O
-IL	O
--	O
-17A	O
-monomer	O
-and	O
-β	O
--	O
-strand	O
-0	O
-(	O
-the	O
-first	O
-ordered	O
-peptide	O
-of	O
-IL	O
--	O
-17A	O
-)	O
-of	O
-the	O
-second	O
-monomer	O
-.	O
-	O
-This	O
-β	O
--	O
-strand	O
-is	O
-parallel	O
-to	O
-both	O
-strands	O
-0	O
-and	O
-4	O
-(	O
-Fig	O
-.	O
-3B	O
-).	O
-	O
-Strands	O
-0	O
-of	O
-two	O
-IL	O
--	O
-17A	O
-monomer	O
-are	O
-antiparallel	O
-,	O
-as	O
-appeared	O
-in	O
-other	O
-IL	O
--	O
-17A	O
-structures	O
-.	O
-	O
-The	O
-C	O
--	O
-terminal	O
-8	O
-residues	O
-of	O
-the	O
-HAP	O
-that	O
-are	O
-ordered	O
-in	O
-the	O
-structure	O
-,	O
-7ADLWDWIN	O
-,	O
-form	O
-an	O
-amphipathic	O
-α	O
--	O
-helix	O
-interacting	O
-with	O
-the	O
-second	O
-IL	O
--	O
-17A	O
-monomer	O
-.	O
-	O
-Pro6	O
-of	O
-HAP	O
-makes	O
-a	O
-transition	O
-between	O
-the	O
-N	O
--	O
-terminal	O
-β	O
--	O
-strand	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-α	O
--	O
-helix	O
-of	O
-HAP	O
-.	O
-	O
-As	O
-a	O
-comparison	O
-,	O
-an	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-complex	O
-structure	O
-(	O
-PDB	O
-code	O
-4HSA	O
-)	O
-is	O
-also	O
-shown	O
-with	O
-IL	O
--	O
-17A	O
-in	O
-the	O
-same	O
-orientation	O
-(	O
-Fig	O
-.	O
-2C	O
-).	O
-	O
-Inhibition	O
-mechanism	O
-of	O
-IL	O
--	O
-17A	O
-signaling	O
-by	O
-HAP	O
-	O
-IL	O
--	O
-17RA	O
-binds	O
-IL	O
--	O
-17A	O
-at	O
-three	O
-regions	O
-on	O
-the	O
-IL	O
--	O
-17A	O
-homodimer	O
-.	O
-	O
-HAP	O
-binds	O
-IL	O
--	O
-17A	O
-at	O
-region	O
-I	O
-.	O
-Region	O
-I	O
-is	O
-formed	O
-by	O
-residues	O
-at	O
-the	O
-ends	O
-of	O
-β	O
-strands	O
-0	O
-and	O
-4	O
-,	O
-and	O
-from	O
-loops	O
-1	O
-–	O
-2	O
-and	O
-3	O
-–	O
-4	O
-of	O
-IL	O
--	O
-17A	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Conformational	O
-changes	O
-in	O
-region	O
-I	O
-induced	O
-by	O
-HAP	O
-binding	O
-alone	O
-may	O
-allosterically	O
-affect	O
-IL	O
--	O
-17RA	O
-binding	O
-,	O
-but	O
-more	O
-importantly	O
-,	O
-the	O
-α	O
--	O
-helix	O
-of	O
-HAP	O
-directly	O
-competes	O
-with	O
-IL	O
--	O
-17RA	O
-for	O
-binding	O
-to	O
-IL	O
--	O
-17A	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-The	O
-most	O
-significant	O
-interactions	O
-between	O
-the	O
-α	O
-helix	O
-of	O
-HAP	O
-and	O
-IL	O
--	O
-17A	O
-involve	O
-Trp12	O
-of	O
-HAP	O
-,	O
-which	O
-binds	O
-in	O
-a	O
-hydrophobic	O
-pocket	O
-in	O
-IL	O
--	O
-17A	O
-formed	O
-by	O
-the	O
-side	O
-chains	O
-of	O
-Phe110	O
-,	O
-Tyr62	O
-,	O
-Pro59	O
-and	O
-the	O
-hydrophobic	O
-portion	O
-of	O
-the	O
-Arg101	O
-side	O
-chain	O
-(	O
-Fig	O
-.	O
-3A	O
-).	O
-	O
-The	O
-Trp12	O
-side	O
-chain	O
-of	O
-HAP	O
-donates	O
-a	O
-hydrogen	O
-bond	O
-to	O
-the	O
-main	O
-chain	O
-oxygen	O
-of	O
-Pro69	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-The	O
-positively	O
-charged	O
-Arg101	O
-side	O
-chain	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-engages	O
-in	O
-a	O
-charge	O
--	O
-helix	O
-dipole	O
-interaction	O
-with	O
-the	O
-main	O
-chain	O
-oxygen	O
-of	O
-Trp12	O
-.	O
-	O
-Additionally	O
-,	O
-Leu9	O
-and	O
-Ile13	O
-of	O
-the	O
-HAP	O
-have	O
-hydrophobic	O
-interactions	O
-with	O
-IL	O
--	O
-17A	O
-,	O
-and	O
-the	O
-Asp8	O
-side	O
-chain	O
-has	O
-hydrogen	O
-bond	O
-and	O
-ion	O
-pair	O
-interactions	O
-with	O
-Tyr62	O
-and	O
-Lys114	O
-of	O
-IL	O
--	O
-17A	O
-,	O
-respectively	O
-.	O
-	O
-In	O
-region	O
-I	O
-,	O
-an	O
-IL	O
--	O
-17RA	O
-peptide	O
-interacts	O
-with	O
-IL	O
--	O
-17A	O
-in	O
-a	O
-very	O
-similar	O
-fashion	O
-to	O
-the	O
-α	O
--	O
-helix	O
-of	O
-HAP	O
-.	O
-	O
-The	O
-IL	O
--	O
-17RA	O
-peptide	O
-has	O
-sequences	O
-of	O
-27LDDSWI	O
-,	O
-and	O
-part	O
-of	O
-the	O
-peptide	O
-is	O
-also	O
-α	O
--	O
-helical	O
-(	O
-Fig	O
-.	O
-3B	O
-).	O
-	O
-Leu7	O
-,	O
-Trp31	O
-and	O
-Ile32	O
-of	O
-IL	O
--	O
-17RA	O
-interact	O
-very	O
-similarly	O
-with	O
-the	O
-same	O
-residues	O
-of	O
-IL	O
--	O
-17A	O
-as	O
-Leu9	O
-,	O
-Trp12	O
-and	O
-Ile13	O
-of	O
-HAP	O
-(	O
-Fig	O
-.	O
-3B	O
-).	O
-	O
-In	O
-this	O
-sense	O
-,	O
-the	O
-α	O
--	O
-helix	O
-of	O
-HAP	O
-with	O
-a	O
-sequence	O
-of	O
-9LWDWI	O
-is	O
-a	O
-good	O
-mimetic	O
-of	O
-the	O
-27LDDSWI	O
-peptide	O
-of	O
-IL	O
--	O
-17RA	O
-.	O
-	O
-The	O
-β	O
--	O
-strand	O
-of	O
-HAP	O
-has	O
-no	O
-equivalent	O
-in	O
-IL	O
--	O
-17RA	O
-.	O
-	O
-However	O
-,	O
-it	O
-mimics	O
-the	O
-β	O
--	O
-strand	O
-0	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-The	O
-amphipathic	O
-β	O
--	O
-strand	O
-of	O
-HAP	O
-orients	O
-the	O
-hydrophilic	O
-side	O
-chains	O
-of	O
-His2	O
-and	O
-Thr4	O
-outwards	O
-,	O
-and	O
-the	O
-hydrophobic	O
-side	O
-chains	O
-of	O
-Ile1	O
-,	O
-Val3	O
-and	O
-Ile5	O
-inward	O
-(	O
-Fig	O
-.	O
-3A	O
-).	O
-	O
-β	O
--	O
-strand	O
-0	O
-in	O
-IL	O
--	O
-17A	O
-is	O
-also	O
-amphipathic	O
-with	O
-the	O
-sequence	O
-of	O
-21TVMVNLNI	O
-.	O
-	O
-In	O
-all	O
-IL	O
--	O
-17A	O
-structures	O
-obtained	O
-to	O
-date	O
-,	O
-β	O
--	O
-strand	O
-0	O
-orients	O
-the	O
-hydrophilic	O
-side	O
-chains	O
-of	O
-Thr21	O
-,	O
-Asn25	O
-and	O
-Asn27	O
-outward	O
-,	O
-and	O
-the	O
-hydrophobic	O
-side	O
-chains	O
-of	O
-Val22	O
-,	O
-Val24	O
-,	O
-Leu26	O
-and	O
-Ile28	O
-inward	O
-.	O
-	O
-The	O
-binding	O
-pocket	O
-occupied	O
-by	O
-either	O
-Trp12	O
-of	O
-HAP	O
-or	O
-Trp31	O
-of	O
-IL	O
--	O
-17RA	O
-is	O
-not	O
-formed	O
-in	O
-the	O
-apo	O
-IL	O
--	O
-17A	O
-structure	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-Conformational	O
-changes	O
-of	O
-IL	O
--	O
-17A	O
-are	O
-needed	O
-for	O
-both	O
-HAP	O
-and	O
-IL	O
--	O
-17RA	O
-to	O
-bind	O
-to	O
-that	O
-region	O
-.	O
-	O
-Particularly	O
-for	O
-HAP	O
-,	O
-β	O
--	O
-strands	O
-0	O
-have	O
-to	O
-shift	O
-out	O
-of	O
-the	O
-hydrophobic	O
-cleft	O
-formed	O
-by	O
-the	O
-main	O
-body	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-by	O
-as	O
-much	O
-as	O
-10	O
-Å	O
-between	O
-Cα	O
-atoms	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-Disruptions	O
-of	O
-the	O
-apo	O
-IL	O
--	O
-17A	O
-structure	O
-by	O
-HAP	O
-binding	O
-are	O
-apparently	O
-compensated	O
-for	O
-by	O
-formation	O
-of	O
-the	O
-new	O
-interactions	O
-that	O
-involve	O
-almost	O
-the	O
-entire	O
-HAP	O
-molecule	O
-(	O
-Fig	O
-.	O
-3B	O
-).	O
-	O
-Structure	O
-basis	O
-for	O
-the	O
-observed	O
-SAR	O
-of	O
-peptides	O
-	O
-The	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-structure	O
-obtained	O
-is	O
-very	O
-consistent	O
-with	O
-the	O
-observed	O
-SAR	O
-of	O
-our	O
-identified	O
-peptide	O
-inhibitors	O
-,	O
-explaining	O
-well	O
-how	O
-the	O
-evolution	O
-of	O
-the	O
-initial	O
-phage	O
-peptide	O
-1	O
-to	O
-HAP	O
-and	O
-45	O
-improved	O
-its	O
-potency	O
-(	O
-Supplementary	O
-Figure	O
-S7	O
-).	O
-	O
-The	O
-important	O
-interactions	O
-involving	O
-Trp12	O
-of	O
-HAP	O
-explain	O
-the	O
->	O
-90	O
-times	O
-drop	O
-in	O
-potency	O
-of	O
-the	O
-W12A	B-mutant
-variant	O
-(	O
-6	O
-vs	O
-1	O
-,	O
-Table	O
-1	O
-).	O
-	O
-The	O
-amphipathic	O
-nature	O
-of	O
-the	O
-HAP	O
-β	O
--	O
-strand	O
-explains	O
-the	O
-preference	O
-of	O
-the	O
-hydrophilic	O
-residues	O
-at	O
-the	O
-2	O
-and	O
-4	O
-positions	O
-of	O
-peptides	O
-(	O
-14	O
-,	O
-18	O
-,	O
-19	O
-,	O
-21	O
-and	O
-23	O
-vs	O
-1	O
-and	O
-22	O
-,	O
-Table	O
-1	O
-).	O
-	O
-All	O
-N	O
--	O
-terminal	O
-residues	O
-of	O
-HAP	O
-are	O
-part	O
-of	O
-the	O
-β	O
--	O
-sheet	O
-with	O
-β	O
--	O
-stands	O
-0	O
-and	O
-4	O
-of	O
-IL	O
--	O
-17A	O
-,	O
-which	O
-explains	O
-why	O
-removal	O
-of	O
-the	O
-first	O
-1	O
-–	O
-3	O
-residues	O
-completely	O
-abolishes	O
-the	O
-ability	O
-of	O
-HAP	O
-to	O
-block	O
-IL	O
--	O
-17A	O
-cell	O
-signaling	O
-(	O
-31	O
-,	O
-32	O
-and	O
-33	O
-,	O
-Table	O
-2	O
-).	O
-	O
-The	O
-C	O
--	O
-terminal	O
-Asn14	O
-and	O
-Lys15	O
-of	O
-HAP	O
-are	O
-not	O
-directly	O
-involved	O
-in	O
-interactions	O
-with	O
-IL	O
--	O
-17A	O
-,	O
-and	O
-this	O
-is	O
-reflected	O
-in	O
-the	O
-gradual	O
-reduction	O
-in	O
-activity	O
-caused	O
-by	O
-C	O
--	O
-terminal	O
-truncations	O
-(	O
-35	O
-and	O
-36	O
-,	O
-Table	O
-2	O
-).	O
-	O
-Each	O
-peptide	O
-monomer	O
-in	O
-45	O
-may	O
-not	O
-necessarily	O
-be	O
-more	O
-potent	O
-than	O
-HAP	O
-,	O
-but	O
-two	O
-monomer	O
-peptides	O
-within	O
-the	O
-same	O
-molecule	O
-that	O
-can	O
-simultaneously	O
-bind	O
-to	O
-IL	O
--	O
-17A	O
-can	O
-greatly	O
-improve	O
-its	O
-potency	O
-due	O
-to	O
-avidity	O
-effects	O
-.	O
-	O
-HAP	O
-targets	O
-region	O
-I	O
-of	O
-IL	O
--	O
-17A	O
-,	O
-an	O
-area	O
-that	O
-has	O
-the	O
-least	O
-sequence	O
-conservation	O
-in	O
-IL	O
--	O
-17	O
-cytokines	O
-.	O
-	O
-This	O
-lack	O
-of	O
-sequence	O
-conservation	O
-in	O
-the	O
-HAP	O
-binding	O
-site	O
-explains	O
-the	O
-observed	O
-specificity	O
-of	O
-HAP	O
-binding	O
-to	O
-human	O
-IL	O
--	O
-17A	O
-.	O
-	O
-For	O
-example	O
-,	O
-inspection	O
-of	O
-the	O
-published	O
-IL	O
--	O
-17F	O
-crystal	O
-structure	O
-(	O
-PDB	O
-code	O
-1JPY	O
-)	O
-revealed	O
-a	O
-pocket	O
-of	O
-IL	O
--	O
-17F	O
-similar	O
-to	O
-that	O
-of	O
-IL	O
--	O
-17A	O
-for	O
-W12	O
-of	O
-HAP	O
-binding	O
-,	O
-but	O
-it	O
-is	O
-occupied	O
-by	O
-a	O
-Phe	O
--	O
-Phe	O
-motif	O
-at	O
-the	O
-N	O
--	O
-terminal	O
-peptide	O
-of	O
-IL	O
--	O
-17F	O
-.	O
-	O
-This	O
-Phe	O
--	O
-Phe	O
-motif	O
-is	O
-missing	O
-in	O
-IL	O
--	O
-17A	O
-.	O
-	O
-Sequence	O
-alignments	O
-between	O
-human	O
-and	O
-mouse	O
-IL	O
--	O
-17A	O
-indicated	O
-that	O
-among	O
-IL	O
--	O
-17A	O
-residues	O
-that	O
-interacting	O
-with	O
-HAP	O
-,	O
-majority	O
-differences	O
-occur	O
-in	O
-strand	O
-0	O
-of	O
-IL	O
--	O
-17A	O
-which	O
-interacts	O
-with	O
-the	O
-N	O
--	O
-terminal	O
-β	O
--	O
-strand	O
-of	O
-HAP	O
-.	O
-	O
-In	O
-human	O
-IL	O
--	O
-17A	O
-the	O
-sequences	O
-are	O
-21TVMVNLNI	O
-,	O
-and	O
-in	O
-mouse	O
-they	O
-are	O
-21NVKVNLKV	O
-.	O
-	O
-Using	O
-a	O
-combination	O
-of	O
-phage	O
-display	O
-and	O
-SAR	O
-we	O
-have	O
-discovered	O
-novel	O
-peptides	O
-that	O
-are	O
-IL	O
--	O
-17A	O
-antagonists	O
-.	O
-	O
-One	O
-of	O
-those	O
-peptides	O
-,	O
-HAP	O
-,	O
-also	O
-shows	O
-activity	O
-in	O
-inhibiting	O
-the	O
-production	O
-of	O
-multiple	O
-inflammatory	O
-cytokines	O
-by	O
-primary	O
-human	O
-keratinocytes	O
-stimulated	O
-by	O
-IL	O
--	O
-17A	O
-and	O
-TNF	O
--	O
-α	O
-,	O
-a	O
-disease	O
-relevant	O
--	O
-model	O
-.	O
-	O
-We	O
-have	O
-also	O
-determined	O
-the	O
-complex	O
-structure	O
-of	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-,	O
-which	O
-provides	O
-the	O
-structural	O
-basis	O
-for	O
-HAP	O
-’	O
-s	O
-antagonism	O
-to	O
-IL	O
--	O
-17A	O
-signaling	O
-.	O
-	O
-During	O
-IL	O
--	O
-17A	O
-signaling	O
-,	O
-IL	O
--	O
-17A	O
-binds	O
-to	O
-one	O
-copy	O
-of	O
-IL	O
--	O
-17RA	O
-and	O
-one	O
-copy	O
-of	O
-IL	O
--	O
-17RC	O
-.	O
-	O
-Since	O
-apo	O
-IL	O
--	O
-17A	O
-is	O
-a	O
-homodimer	O
-with	O
-2	O
-fold	O
-symmetry	O
-,	O
-IL	O
--	O
-17RA	O
-potentially	O
-can	O
-bind	O
-to	O
-either	O
-face	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-dimer	O
-.	O
-	O
-With	O
-two	O
-HAP	O
-molecules	O
-covering	O
-both	O
-faces	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-dimer	O
-,	O
-HAP	O
-can	O
-block	O
-IL	O
--	O
-17RA	O
-approaching	O
-from	O
-either	O
-face	O
-.	O
-	O
-To	O
-form	O
-the	O
-1	O
-:	O
-2	O
-complex	O
-observed	O
-in	O
-crystal	O
-structure	O
-,	O
-it	O
-is	O
-important	O
-that	O
-there	O
-is	O
-no	O
-strong	O
-negative	O
-cooperativity	O
-in	O
-the	O
-binding	O
-of	O
-two	O
-HAP	O
-molecules	O
-.	O
-	O
-In	O
-fact	O
-,	O
-in	O
-native	O
-electrospray	O
-ionization	O
-mass	O
-spectrometry	O
-analysis	O
-only	O
-1	O
-:	O
-2	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-was	O
-observed	O
-even	O
-when	O
-IL	O
--	O
-17A	O
-was	O
-in	O
-excess	O
-(	O
-Supplementary	O
-Figure	O
-S8	O
-),	O
-indicating	O
-a	O
-positive	O
-binding	O
-cooperativity	O
-that	O
-favors	O
-inhibition	O
-of	O
-IL	O
--	O
-17RA	O
-binding	O
-by	O
-HAP	O
-.	O
-	O
-HAP	O
-,	O
-with	O
-only	O
-15	O
-residues	O
-,	O
-can	O
-achieve	O
-almost	O
-the	O
-same	O
-binding	O
-affinity	O
-as	O
-the	O
-much	O
-larger	O
-IL	O
--	O
-17RA	O
-molecule	O
-,	O
-indicating	O
-a	O
-more	O
-efficient	O
-way	O
-of	O
-binding	O
-to	O
-IL	O
--	O
-17A	O
-.	O
-	O
-The	O
-interaction	O
-of	O
-IL	O
--	O
-17A	O
-with	O
-IL	O
--	O
-17RA	O
-has	O
-an	O
-extensive	O
-interface	O
-,	O
-covering	O
-~	O
-2	O
-,	O
-200	O
-Å2	O
-surface	O
-area	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-Due	O
-to	O
-the	O
-discontinuous	O
-nature	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-binding	O
-interface	O
-,	O
-it	O
-is	O
-classified	O
-as	O
-having	O
-tertiary	O
-structural	O
-epitopes	O
-on	O
-both	O
-binding	O
-partners	O
-,	O
-and	O
-is	O
-therefore	O
-hard	O
-to	O
-target	O
-using	O
-small	O
-molecules	O
-.	O
-	O
-Our	O
-studies	O
-of	O
-HAP	O
-demonstrated	O
-an	O
-uncommon	O
-mode	O
-of	O
-action	O
-for	O
-a	O
-peptide	O
-in	O
-inhibiting	O
-such	O
-a	O
-difficult	O
-protein	O
--	O
-protein	O
-interaction	O
-target	O
-,	O
-and	O
-suggest	O
-further	O
-possible	O
-improvements	O
-in	O
-its	O
-binding	O
-potency	O
-.	O
-	O
-One	O
-way	O
-of	O
-further	O
-improving	O
-HAP	O
-’	O
-s	O
-potency	O
-is	O
-by	O
-dimerization	O
-.	O
-	O
-Homo	O
--	O
-dimerization	O
-of	O
-HAP	O
-(	O
-45	O
-)	O
-achieved	O
-sub	O
--	O
-nanomolar	O
-potency	O
-against	O
-human	O
-IL	O
--	O
-17A	O
-in	O
-cell	O
-assay	O
-.	O
-	O
-In	O
-the	O
-crystal	O
-structure	O
-,	O
-the	O
-distance	O
-between	O
-the	O
-carbonyl	O
-of	O
-Asn14	O
-of	O
-one	O
-HAP	O
-molecule	O
-and	O
-the	O
-N	O
--	O
-terminus	O
-of	O
-the	O
-second	O
-is	O
-only	O
-15	O
-.	O
-7	O
-Å	O
-,	O
-suggesting	O
-the	O
-potential	O
-for	O
-more	O
-potent	O
-dimeric	O
-peptides	O
-to	O
-be	O
-designed	O
-by	O
-using	O
-linkers	O
-of	O
-different	O
-lengths	O
-at	O
-different	O
-positions	O
-.	O
-	O
-Another	O
-direction	O
-of	O
-improving	O
-HAP	O
-is	O
-by	O
-reducing	O
-its	O
-size	O
-.	O
-	O
-As	O
-demonstrated	O
-by	O
-the	O
-crystal	O
-structure	O
-,	O
-binding	O
-of	O
-the	O
-α	O
--	O
-helix	O
-of	O
-HAP	O
-should	O
-be	O
-sufficient	O
-for	O
-preventing	O
-IL	O
--	O
-17RA	O
-binding	O
-to	O
-IL	O
--	O
-17A	O
-.	O
-	O
-Theoretically	O
-,	O
-it	O
-is	O
-possible	O
-to	O
-design	O
-chemicals	O
-such	O
-as	O
-stapled	O
-α	O
--	O
-helical	O
-peptides	O
-to	O
-block	O
-α	O
--	O
-helix	O
--	O
-mediated	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-interactions	O
-.	O
-	O
-In	O
-summary	O
-,	O
-these	O
-peptide	O
--	O
-based	O
-anti	O
--	O
-IL	O
--	O
-17A	O
-modalities	O
-could	O
-be	O
-further	O
-developed	O
-as	O
-alternative	O
-therapeutic	O
-options	O
-to	O
-the	O
-reported	O
-monoclonal	O
-antibodies	O
-.	O
-	O
-We	O
-are	O
-also	O
-very	O
-interested	O
-in	O
-finding	O
-non	O
--	O
-peptidic	O
-small	O
-molecule	O
-IL	O
--	O
-17A	O
-antagonists	O
-,	O
-and	O
-HAP	O
-can	O
-be	O
-used	O
-as	O
-an	O
-excellent	O
-tool	O
-peptide	O
-.	O
-	O
-The	O
-strategy	O
-utilized	O
-in	O
-generating	O
-the	O
-complex	O
-structures	O
-of	O
-HAP	O
-may	O
-also	O
-be	O
-useful	O
-for	O
-enabling	O
-structure	O
-based	O
-design	O
-of	O
-some	O
-known	O
-small	O
-molecule	O
-IL	O
--	O
-17A	O
-antagonists	O
-.	O
-	O
-Binding	O
-of	O
-HAP	O
-to	O
-IL	O
--	O
-17A	O
-and	O
-inhibition	O
-of	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-are	O
-measured	O
-by	O
-SPR	O
-,	O
-FRET	O
-and	O
-cell	O
--	O
-based	O
-assays	O
-.	O
-	O
-(	O
-A	O
-)	O
-Typical	O
-SPR	O
-sensorgrams	O
-(	O
-black	O
-)	O
-of	O
-HAP	O
-at	O
-indicated	O
-concentrations	O
-binding	O
-to	O
-biotinylated	O
-human	O
-IL	O
--	O
-17A	O
-immobilized	O
-on	O
-a	O
-streptavidin	O
-chip	O
-surface	O
-,	O
-fitted	O
-with	O
-single	O
-site	O
-binding	O
-model	O
-curves	O
-(	O
-red	O
-).	O
-	O
-Kinetic	O
-parameters	O
-(	O
-ka	O
-,	O
-kd	O
-)	O
-were	O
-obtained	O
-by	O
-a	O
-global	O
-fit	O
-using	O
-three	O
-concentrations	O
-in	O
-triplicate	O
-.	O
-	O
-KD	O
-determined	O
-by	O
-the	O
-standard	O
-equation	O
-,	O
-KD	O
-=	O
-kd	O
-/	O
-ka	O
-.	O
-(	O
-B	O
-)	O
-HAP	O
-inhibits	O
-SPR	O
-signaling	O
-of	O
-IL	O
--	O
-17A	O
-binding	O
-to	O
-immobilized	O
-IL	O
--	O
-17RA	O
-.	O
-	O
-Data	O
-are	O
-mean	O
-and	O
-error	O
-bars	O
-of	O
-+/−	O
-standard	O
-deviation	O
-of	O
-three	O
-measurements	O
-.	O
-(	O
-C	O
-)	O
-Inhibition	O
-of	O
-IL	O
--	O
-17A	O
-and	O
-IL	O
--	O
-17RA	O
-binding	O
-by	O
-HAP	O
-measured	O
-by	O
-FRET	O
-assay	O
-.	O
-	O
-Data	O
-are	O
-mean	O
-and	O
-error	O
-bars	O
-of	O
-+/−	O
-standard	O
-deviation	O
-from	O
-299	O
-experiments	O
-,	O
-each	O
-performed	O
-in	O
-duplicate	O
-.	O
-(	O
-D	O
-)	O
-Example	O
-of	O
-HAP	O
-selective	O
-inhibition	O
-of	O
-the	O
-production	O
-of	O
-IL	O
--	O
-8	O
-(	O
-triangles	O
-),	O
-IL	O
--	O
-6	O
-(	O
-squares	O
-)	O
-and	O
-CCL	O
--	O
-20	O
-(	O
-circles	O
-)	O
-by	O
-primary	O
-human	O
-keratinocyte	O
-cells	O
-synergistically	O
-stimulated	O
-by	O
-100	O
-ng	O
-/	O
-ml	O
-IL	O
--	O
-17A	O
-and	O
-10	O
-ng	O
-/	O
-ml	O
-TNF	O
--	O
-α	O
-.	O
-	O
-HAP	O
-does	O
-not	O
-inhibit	O
-the	O
-baseline	O
-production	O
-of	O
-IL	O
--	O
-6	O
-,	O
-IL	O
--	O
-8	O
-and	O
-CCL	O
--	O
-20	O
-stimulated	O
-by	O
-10	O
-ng	O
-/	O
-ml	O
-TNF	O
--	O
-α	O
-alone	O
-(	O
-gray	O
-lines	O
-and	O
-symbols	O
-).	O
-	O
-Overall	O
-structure	O
-of	O
-the	O
-Fab	O
-/	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-in	O
-ribbon	O
-presentation	O
-.	O
-	O
-Two	O
-HAP	O
-molecules	O
-are	O
-colored	O
-blue	O
-and	O
-red	O
-,	O
-and	O
-IL	O
--	O
-17A	O
-monomers	O
-are	O
-colored	O
-ice	O
-blue	O
-and	O
-pink	O
-,	O
-respectively	O
-.	O
-	O
-(	O
-A	O
-)	O
-Overview	O
-of	O
-the	O
-distinct	O
-binding	O
-sites	O
-of	O
-Fab	O
-and	O
-HAP	O
-to	O
-IL	O
--	O
-17A	O
-.	O
-	O
-(	O
-B	O
-)	O
-Close	O
--	O
-in	O
-view	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-structure	O
-.	O
-	O
-IL	O
--	O
-17A	O
-β	O
--	O
-strands	O
-are	O
-labelled	O
-.	O
-	O
-Each	O
-of	O
-the	O
-two	O
-bound	O
-HAP	O
-interacts	O
-with	O
-both	O
-monomers	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-dimer	O
-.	O
-	O
-(	O
-C	O
-)	O
-As	O
-a	O
-comparison	O
-,	O
-the	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-complex	O
-was	O
-shown	O
-with	O
-IL	O
--	O
-17A	O
-in	O
-the	O
-same	O
-orientation	O
-.	O
-	O
-Three	O
-distinct	O
-areas	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-interface	O
-are	O
-labeled	O
-.	O
-	O
-Mechanism	O
-of	O
-the	O
-inhibition	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-interaction	O
-by	O
-HAP	O
-.	O
-	O
-(	O
-A	O
-)	O
-HAP	O
-binds	O
-at	O
-region	O
-I	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-IL	O
--	O
-17A	O
-dimer	O
-is	O
-in	O
-surface	O
-presentation	O
-(	O
-β	O
--	O
-strands	O
-0	O
-shown	O
-as	O
-ribbons	O
-for	O
-clarity	O
-).	O
-	O
-Polar	O
-interactions	O
-are	O
-shown	O
-in	O
-dashes	O
-.	O
-	O
-HAP	O
-residues	O
-as	O
-well	O
-as	O
-key	O
-IL	O
--	O
-17A	O
-residues	O
-are	O
-labeled	O
-.	O
-	O
-For	O
-clarity	O
-,	O
-a	O
-few	O
-HAP	O
-residues	O
-are	O
-also	O
-shown	O
-in	O
-stick	O
-model	O
-with	O
-carbon	O
-atoms	O
-colored	O
-green	O
-,	O
-oxygen	O
-in	O
-red	O
-and	O
-nitrogen	O
-in	O
-blue	O
-.	O
-	O
-(	O
-B	O
-)	O
-I	O
--	O
-17RA	O
-(	O
-ribbon	O
-in	O
-gold	O
-)	O
-peptide	O
-Leu27	O
--	O
-Ile32	O
-binds	O
-to	O
-the	O
-same	O
-area	O
-as	O
-the	O
-HAP	O
-α	O
--	O
-helix	O
-.	O
-	O
-Trp31	O
-of	O
-IL	O
--	O
-17RA	O
-binds	O
-to	O
-the	O
-same	O
-pocket	O
-in	O
-IL	O
--	O
-17A	O
-as	O
-Trp12	O
-of	O
-HAP	O
-.	O
-(	O
-C	O
-)	O
-As	O
-illustrated	O
-by	O
-overlay	O
-a	O
-single	O
-HAP	O
-molecule	O
-and	O
-β	O
--	O
-strands	O
-0	O
-(	O
-grey	O
-)	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-in	O
-the	O
-apo	O
-IL	O
--	O
-17A	O
-structure	O
-,	O
-conformational	O
-changes	O
-in	O
-region	O
-I	O
-of	O
-IL	O
--	O
-17A	O
-are	O
-needed	O
-for	O
-binding	O
-of	O
-both	O
-the	O
-β	O
--	O
-stand	O
-and	O
-α	O
--	O
-helix	O
-of	O
-the	O
-HAP	O
-.	O
-	O
-Notice	O
-that	O
-the	O
-Trp	O
-binding	O
-pocket	O
-for	O
-W12	O
-of	O
-HAP	O
-or	O
-W31	O
-of	O
-IL	O
--	O
-17RA	O
-is	O
-missing	O
-in	O
-the	O
-apo	O
-structure	O
-.	O
-	O
-ELISA	O
-competition	O
-activity	O
-of	O
-peptide	O
-analogues	O
-of	O
-1	O
-.	O
-	O
-Molecular	O
-Basis	O
-of	O
-Ligand	O
--	O
-Dependent	O
-Regulation	O
-of	O
-NadR	O
-,	O
-the	O
-Transcriptional	O
-Repressor	O
-of	O
-Meningococcal	O
-Virulence	O
-Factor	O
-NadA	O
-	O
-Neisseria	O
-adhesin	O
-A	O
-(	O
-NadA	O
-)	O
-is	O
-present	O
-on	O
-the	O
-meningococcal	O
-surface	O
-and	O
-contributes	O
-to	O
-adhesion	O
-to	O
-and	O
-invasion	O
-of	O
-human	O
-cells	O
-.	O
-	O
-NadA	O
-is	O
-also	O
-one	O
-of	O
-three	O
-recombinant	O
-antigens	O
-in	O
-the	O
-recently	O
--	O
-approved	O
-Bexsero	O
-vaccine	O
-,	O
-which	O
-protects	O
-against	O
-serogroup	O
-B	O
-meningococcus	O
-.	O
-	O
-The	O
-amount	O
-of	O
-NadA	O
-on	O
-the	O
-bacterial	O
-surface	O
-is	O
-of	O
-direct	O
-relevance	O
-in	O
-the	O
-constant	O
-battle	O
-of	O
-host	O
--	O
-pathogen	O
-interactions	O
-:	O
-it	O
-influences	O
-the	O
-ability	O
-of	O
-the	O
-pathogen	O
-to	O
-engage	O
-human	O
-cell	O
-surface	O
--	O
-exposed	O
-receptors	O
-and	O
-,	O
-conversely	O
-,	O
-the	O
-bacterial	O
-susceptibility	O
-to	O
-the	O
-antibody	O
--	O
-mediated	O
-immune	O
-response	O
-.	O
-	O
-It	O
-is	O
-therefore	O
-important	O
-to	O
-understand	O
-the	O
-mechanisms	O
-which	O
-regulate	O
-nadA	O
-expression	O
-levels	O
-,	O
-which	O
-are	O
-predominantly	O
-controlled	O
-by	O
-the	O
-transcriptional	O
-regulator	O
-NadR	O
-(	O
-Neisseria	O
-adhesin	O
-A	O
-Regulator	O
-)	O
-both	O
-in	O
-vitro	O
-and	O
-in	O
-vivo	O
-.	O
-	O
-NadR	O
-binds	O
-the	O
-nadA	O
-promoter	O
-and	O
-represses	O
-gene	O
-transcription	O
-.	O
-	O
-In	O
-the	O
-presence	O
-of	O
-4	O
--	O
-hydroxyphenylacetate	O
-(	O
-4	O
--	O
-HPA	O
-),	O
-a	O
-catabolite	O
-present	O
-in	O
-human	O
-saliva	O
-both	O
-under	O
-physiological	O
-conditions	O
-and	O
-during	O
-bacterial	O
-infection	O
-,	O
-the	O
-binding	O
-of	O
-NadR	O
-to	O
-the	O
-nadA	O
-promoter	O
-is	O
-attenuated	O
-and	O
-nadA	O
-expression	O
-is	O
-induced	O
-.	O
-	O
-NadR	O
-also	O
-mediates	O
-ligand	O
--	O
-dependent	O
-regulation	O
-of	O
-many	O
-other	O
-meningococcal	O
-genes	O
-,	O
-for	O
-example	O
-the	O
-highly	O
--	O
-conserved	O
-multiple	O
-adhesin	O
-family	O
-(	O
-maf	O
-)	O
-genes	O
-,	O
-which	O
-encode	O
-proteins	O
-emerging	O
-with	O
-important	O
-roles	O
-in	O
-host	O
--	O
-pathogen	O
-interactions	O
-,	O
-immune	O
-evasion	O
-and	O
-niche	O
-adaptation	O
-.	O
-	O
-To	O
-gain	O
-insights	O
-into	O
-the	O
-regulation	O
-of	O
-NadR	O
-mediated	O
-by	O
-4	O
--	O
-HPA	O
-,	O
-we	O
-combined	O
-structural	O
-,	O
-biochemical	O
-,	O
-and	O
-mutagenesis	O
-studies	O
-.	O
-	O
-In	O
-particular	O
-,	O
-two	O
-new	O
-crystal	O
-structures	O
-of	O
-ligand	O
--	O
-free	O
-and	O
-ligand	O
--	O
-bound	O
-NadR	O
-revealed	O
-(	O
-i	O
-)	O
-the	O
-molecular	O
-basis	O
-of	O
-‘	O
-conformational	O
-selection	O
-’	O
-by	O
-which	O
-a	O
-single	O
-molecule	O
-of	O
-4	O
--	O
-HPA	O
-binds	O
-and	O
-stabilizes	O
-dimeric	O
-NadR	O
-in	O
-a	O
-conformation	O
-unsuitable	O
-for	O
-DNA	O
--	O
-binding	O
-,	O
-(	O
-ii	O
-)	O
-molecular	O
-explanations	O
-for	O
-the	O
-binding	O
-specificities	O
-of	O
-different	O
-hydroxyphenylacetate	O
-ligands	O
-,	O
-including	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-which	O
-is	O
-produced	O
-during	O
-inflammation	O
-,	O
-(	O
-iii	O
-)	O
-the	O
-presence	O
-of	O
-a	O
-leucine	O
-residue	O
-essential	O
-for	O
-dimerization	O
-and	O
-conserved	O
-in	O
-many	O
-MarR	O
-family	O
-proteins	O
-,	O
-and	O
-(	O
-iv	O
-)	O
-four	O
-residues	O
-(	O
-His7	O
-,	O
-Ser9	O
-,	O
-Asn11	O
-and	O
-Phe25	O
-),	O
-which	O
-are	O
-involved	O
-in	O
-binding	O
-4	O
--	O
-HPA	O
-,	O
-and	O
-were	O
-confirmed	O
-in	O
-vitro	O
-to	O
-have	O
-key	O
-roles	O
-in	O
-the	O
-regulatory	O
-mechanism	O
-in	O
-bacteria	O
-.	O
-	O
-Overall	O
-,	O
-this	O
-study	O
-deepens	O
-our	O
-molecular	O
-understanding	O
-of	O
-the	O
-sophisticated	O
-regulatory	O
-mechanisms	O
-of	O
-the	O
-expression	O
-of	O
-nadA	O
-and	O
-other	O
-genes	O
-governed	O
-by	O
-NadR	O
-,	O
-dependent	O
-on	O
-interactions	O
-with	O
-niche	O
--	O
-specific	O
-signal	O
-molecules	O
-that	O
-may	O
-play	O
-important	O
-roles	O
-during	O
-meningococcal	O
-pathogenesis	O
-.	O
-	O
-Serogroup	O
-B	O
-meningococcus	O
-(	O
-MenB	O
-)	O
-causes	O
-fatal	O
-sepsis	O
-and	O
-invasive	O
-meningococcal	O
-disease	O
-,	O
-particularly	O
-in	O
-young	O
-children	O
-and	O
-adolescents	O
-,	O
-as	O
-highlighted	O
-by	O
-recent	O
-MenB	O
-outbreaks	O
-in	O
-universities	O
-of	O
-the	O
-United	O
-States	O
-and	O
-Canada	O
-.	O
-	O
-The	O
-Bexsero	O
-vaccine	O
-protects	O
-against	O
-MenB	O
-and	O
-has	O
-recently	O
-been	O
-approved	O
-in	O
->	O
-35	O
-countries	O
-worldwide	O
-.	O
-	O
-Neisseria	O
-adhesin	O
-A	O
-(	O
-NadA	O
-)	O
-present	O
-on	O
-the	O
-meningococcal	O
-surface	O
-can	O
-mediate	O
-binding	O
-to	O
-human	O
-cells	O
-and	O
-is	O
-one	O
-of	O
-the	O
-three	O
-MenB	O
-vaccine	O
-protein	O
-antigens	O
-.	O
-	O
-The	O
-amount	O
-of	O
-NadA	O
-exposed	O
-on	O
-the	O
-meningococcal	O
-surface	O
-also	O
-influences	O
-the	O
-antibody	O
--	O
-mediated	O
-serum	O
-bactericidal	O
-response	O
-measured	O
-in	O
-vitro	O
-.	O
-	O
-A	O
-deep	O
-understanding	O
-of	O
-nadA	O
-expression	O
-is	O
-therefore	O
-important	O
-,	O
-otherwise	O
-the	O
-contribution	O
-of	O
-NadA	O
-to	O
-vaccine	O
--	O
-induced	O
-protection	O
-against	O
-meningococcal	O
-meningitis	O
-may	O
-be	O
-underestimated	O
-.	O
-	O
-The	O
-abundance	O
-of	O
-surface	O
--	O
-exposed	O
-NadA	O
-is	O
-regulated	O
-by	O
-the	O
-ligand	O
--	O
-responsive	O
-transcriptional	O
-repressor	O
-NadR	O
-.	O
-Here	O
-,	O
-we	O
-present	O
-functional	O
-,	O
-biochemical	O
-and	O
-high	O
--	O
-resolution	O
-structural	O
-data	O
-on	O
-NadR	O
-.	O
-Our	O
-studies	O
-provide	O
-detailed	O
-insights	O
-into	O
-how	O
-small	O
-molecule	O
-ligands	O
-,	O
-such	O
-as	O
-hydroxyphenylacetate	O
-derivatives	O
-,	O
-found	O
-in	O
-relevant	O
-host	O
-niches	O
-,	O
-modulate	O
-the	O
-structure	O
-and	O
-activity	O
-of	O
-NadR	O
-,	O
-by	O
-‘	O
-conformational	O
-selection	O
-’	O
-of	O
-inactive	O
-forms	O
-.	O
-	O
-These	O
-findings	O
-shed	O
-light	O
-on	O
-the	O
-regulation	O
-of	O
-NadR	O
-,	O
-a	O
-key	O
-MarR	O
--	O
-family	O
-virulence	O
-factor	O
-of	O
-this	O
-important	O
-human	O
-pathogen	O
-.	O
-	O
-The	O
-‘	O
-Reverse	O
-Vaccinology	O
-’	O
-approach	O
-was	O
-pioneered	O
-to	O
-identify	O
-antigens	O
-for	O
-a	O
-protein	O
--	O
-based	O
-vaccine	O
-against	O
-serogroup	O
-B	O
-Neisseria	O
-meningitidis	O
-(	O
-MenB	O
-),	O
-a	O
-human	O
-pathogen	O
-causing	O
-potentially	O
--	O
-fatal	O
-sepsis	O
-and	O
-invasive	O
-meningococcal	O
-disease	O
-.	O
-	O
-Indeed	O
-,	O
-Reverse	O
-Vaccinology	O
-identified	O
-Neisseria	O
-adhesin	O
-A	O
-(	O
-NadA	O
-),	O
-a	O
-surface	O
--	O
-exposed	O
-protein	O
-involved	O
-in	O
-epithelial	O
-cell	O
-invasion	O
-and	O
-found	O
-in	O
-~	O
-30	O
-%	O
-of	O
-clinical	O
-isolates	O
-.	O
-	O
-Recently	O
-,	O
-we	O
-reported	O
-the	O
-crystal	O
-structure	O
-of	O
-NadA	O
-,	O
-providing	O
-insights	O
-into	O
-its	O
-biological	O
-and	O
-immunological	O
-functions	O
-.	O
-	O
-Recombinant	O
-NadA	O
-elicits	O
-a	O
-strong	O
-bactericidal	O
-immune	O
-response	O
-and	O
-is	O
-therefore	O
-included	O
-in	O
-the	O
-Bexsero	O
-vaccine	O
-that	O
-protects	O
-against	O
-MenB	O
-and	O
-which	O
-was	O
-recently	O
-approved	O
-in	O
-over	O
-35	O
-countries	O
-worldwide	O
-.	O
-	O
-Previous	O
-studies	O
-revealed	O
-that	O
-nadA	O
-expression	O
-levels	O
-are	O
-mainly	O
-regulated	O
-by	O
-the	O
-Neisseria	O
-adhesin	O
-A	O
-Regulator	O
-(	O
-NadR	O
-).	O
-	O
-Although	O
-additional	O
-factors	O
-influence	O
-nadA	O
-expression	O
-,	O
-we	O
-focused	O
-on	O
-its	O
-regulation	O
-by	O
-NadR	O
-,	O
-the	O
-major	O
-mediator	O
-of	O
-NadA	O
-phase	O
-variable	O
-expression	O
-.	O
-	O
-Studies	O
-of	O
-NadR	O
-also	O
-have	O
-broader	O
-implications	O
-,	O
-since	O
-a	O
-genome	O
--	O
-wide	O
-analysis	O
-of	O
-MenB	O
-wild	O
--	O
-type	O
-and	O
-nadR	O
-knock	O
--	O
-out	O
-strains	O
-revealed	O
-that	O
-NadR	O
-influences	O
-the	O
-regulation	O
-of	O
->	O
-30	O
-genes	O
-,	O
-including	O
-maf	O
-genes	O
-,	O
-from	O
-the	O
-multiple	O
-adhesin	O
-family	O
-.	O
-	O
-These	O
-genes	O
-encode	O
-a	O
-wide	O
-variety	O
-of	O
-proteins	O
-connected	O
-to	O
-many	O
-biological	O
-processes	O
-contributing	O
-to	O
-bacterial	O
-survival	O
-,	O
-adaptation	O
-in	O
-the	O
-host	O
-niche	O
-,	O
-colonization	O
-and	O
-invasion	O
-.	O
-	O
-NadR	O
-belongs	O
-to	O
-the	O
-MarR	O
-(	O
-Multiple	O
-Antibiotic	O
-Resistance	O
-Regulator	O
-)	O
-family	O
-,	O
-a	O
-group	O
-of	O
-ligand	O
--	O
-responsive	O
-transcriptional	O
-regulators	O
-ubiquitous	O
-in	O
-bacteria	O
-and	O
-archaea	O
-.	O
-	O
-MarR	O
-family	O
-proteins	O
-can	O
-promote	O
-bacterial	O
-survival	O
-in	O
-the	O
-presence	O
-of	O
-antibiotics	O
-,	O
-toxic	O
-chemicals	O
-,	O
-organic	O
-solvents	O
-or	O
-reactive	O
-oxygen	O
-species	O
-and	O
-can	O
-regulate	O
-virulence	O
-factor	O
-expression	O
-.	O
-	O
-MarR	O
-homologues	O
-can	O
-act	O
-either	O
-as	O
-transcriptional	O
-repressors	O
-or	O
-as	O
-activators	O
-.	O
-	O
-Although	O
->	O
-50	O
-MarR	O
-family	O
-structures	O
-are	O
-known	O
-,	O
-a	O
-molecular	O
-understanding	O
-of	O
-their	O
-ligand	O
--	O
-dependent	O
-regulatory	O
-mechanisms	O
-is	O
-still	O
-limited	O
-,	O
-often	O
-hampered	O
-by	O
-lack	O
-of	O
-identification	O
-of	O
-their	O
-ligands	O
-and	O
-/	O
-or	O
-DNA	O
-targets	O
-.	O
-	O
-A	O
-potentially	O
-interesting	O
-exception	O
-comes	O
-from	O
-the	O
-ligand	O
--	O
-free	O
-and	O
-salicylate	O
--	O
-bound	O
-forms	O
-of	O
-the	O
-Methanobacterium	O
-thermoautotrophicum	O
-protein	O
-MTH313	O
-which	O
-revealed	O
-that	O
-two	O
-salicylate	O
-molecules	O
-bind	O
-to	O
-one	O
-MTH313	O
-dimer	O
-and	O
-induce	O
-large	O
-conformational	O
-changes	O
-,	O
-apparently	O
-sufficient	O
-to	O
-prevent	O
-DNA	O
-binding	O
-.	O
-	O
-However	O
-,	O
-the	O
-homologous	O
-archeal	O
-Sulfolobus	O
-tokodaii	O
-protein	O
-ST1710	O
-presented	O
-essentially	O
-the	O
-same	O
-structure	O
-in	O
-ligand	O
--	O
-free	O
-and	O
-salicylate	O
--	O
-bound	O
-forms	O
-,	O
-apparently	O
-contrasting	O
-the	O
-mechanism	O
-proposed	O
-for	O
-MTH313	O
-.	O
-	O
-Despite	O
-these	O
-apparent	O
-differences	O
-,	O
-MTH313	O
-and	O
-ST1710	O
-bind	O
-salicylate	O
-in	O
-approximately	O
-the	O
-same	O
-site	O
-,	O
-between	O
-their	O
-dimerization	O
-and	O
-DNA	O
--	O
-binding	O
-domains	O
-.	O
-	O
-However	O
-,	O
-it	O
-is	O
-unknown	O
-whether	O
-salicylate	O
-is	O
-a	O
-relevant	O
-in	O
-vivo	O
-ligand	O
-of	O
-either	O
-of	O
-these	O
-two	O
-proteins	O
-,	O
-which	O
-share	O
-~	O
-20	O
-%	O
-sequence	O
-identity	O
-with	O
-NadR	O
-,	O
-rendering	O
-unclear	O
-the	O
-interpretation	O
-of	O
-these	O
-findings	O
-in	O
-relation	O
-to	O
-the	O
-regulatory	O
-mechanisms	O
-of	O
-NadR	O
-or	O
-other	O
-MarR	O
-family	O
-proteins	O
-.	O
-	O
-NadR	O
-binds	O
-nadA	O
-on	O
-three	O
-different	O
-operators	O
-(	O
-OpI	O
-,	O
-OpII	O
-and	O
-OpIII	O
-).	O
-	O
-The	O
-DNA	O
--	O
-binding	O
-activity	O
-of	O
-NadR	O
-is	O
-attenuated	O
-in	O
-vitro	O
-upon	O
-addition	O
-of	O
-various	O
-hydroxyphenylacetate	O
-(	O
-HPA	O
-)	O
-derivatives	O
-,	O
-including	O
-4	O
--	O
-HPA	O
-.	O
-	O
-4	O
--	O
-HPA	O
-is	O
-a	O
-small	O
-molecule	O
-derived	O
-from	O
-mammalian	O
-aromatic	O
-amino	O
-acid	O
-catabolism	O
-and	O
-is	O
-released	O
-in	O
-human	O
-saliva	O
-,	O
-where	O
-it	O
-has	O
-been	O
-detected	O
-at	O
-micromolar	O
-concentration	O
-.	O
-	O
-In	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-NadR	O
-is	O
-unable	O
-to	O
-bind	O
-the	O
-nadA	O
-promoter	O
-and	O
-nadA	O
-gene	O
-expression	O
-is	O
-induced	O
-.	O
-	O
-In	O
-vivo	O
-,	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-in	O
-the	O
-host	O
-niche	O
-of	O
-N	O
-.	O
-meningitidis	O
-serves	O
-as	O
-an	O
-inducer	O
-of	O
-NadA	O
-production	O
-,	O
-thereby	O
-promoting	O
-bacterial	O
-adhesion	O
-to	O
-host	O
-cells	O
-.	O
-	O
-Further	O
-,	O
-we	O
-recently	O
-reported	O
-that	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-,	O
-produced	O
-during	O
-inflammation	O
-,	O
-is	O
-another	O
-inducer	O
-of	O
-nadA	O
-expression	O
-.	O
-	O
-Extending	O
-our	O
-previous	O
-studies	O
-based	O
-on	O
-hydrogen	O
--	O
-deuterium	O
-exchange	O
-mass	O
-spectrometry	O
-(	O
-HDX	O
--	O
-MS	O
-),	O
-here	O
-we	O
-sought	O
-to	O
-reveal	O
-the	O
-molecular	O
-mechanisms	O
-and	O
-effects	O
-of	O
-NadR	O
-/	O
-HPA	O
-interactions	O
-via	O
-X	O
--	O
-ray	O
-crystallography	O
-,	O
-NMR	O
-spectroscopy	O
-and	O
-complementary	O
-biochemical	O
-and	O
-in	O
-vivo	O
-mutagenesis	O
-studies	O
-.	O
-	O
-We	O
-obtained	O
-detailed	O
-new	O
-insights	O
-into	O
-ligand	O
-specificity	O
-,	O
-how	O
-the	O
-ligand	O
-allosterically	O
-influences	O
-the	O
-DNA	O
--	O
-binding	O
-ability	O
-of	O
-NadR	O
-,	O
-and	O
-the	O
-regulation	O
-of	O
-nadA	O
-expression	O
-,	O
-thus	O
-also	O
-providing	O
-a	O
-deeper	O
-structural	O
-understanding	O
-of	O
-the	O
-ligand	O
--	O
-responsive	O
-MarR	O
-super	O
--	O
-family	O
-.	O
-	O
-Moreover	O
-,	O
-these	O
-findings	O
-are	O
-important	O
-because	O
-the	O
-activity	O
-of	O
-NadR	O
-impacts	O
-the	O
-potential	O
-coverage	O
-provided	O
-by	O
-anti	O
--	O
-NadA	O
-antibodies	O
-elicited	O
-by	O
-the	O
-Bexsero	O
-vaccine	O
-and	O
-influences	O
-host	O
--	O
-bacteria	O
-interactions	O
-that	O
-contribute	O
-to	O
-meningococcal	O
-pathogenesis	O
-.	O
-	O
-NadR	O
-is	O
-dimeric	O
-and	O
-is	O
-stabilized	O
-by	O
-specific	O
-hydroxyphenylacetate	O
-ligands	O
-	O
-Recombinant	O
-NadR	O
-was	O
-produced	O
-in	O
-E	O
-.	O
-coli	O
-using	O
-an	O
-expression	O
-construct	O
-prepared	O
-from	O
-N	O
-.	O
-meningitidis	O
-serogroup	O
-B	O
-strain	O
-MC58	O
-.	O
-	O
-Standard	O
-chromatographic	O
-techniques	O
-were	O
-used	O
-to	O
-obtain	O
-a	O
-highly	O
-purified	O
-sample	O
-of	O
-NadR	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-In	O
-analytical	O
-size	O
--	O
-exclusion	O
-high	O
--	O
-performance	O
-liquid	O
-chromatography	O
-(	O
-SE	O
--	O
-HPLC	O
-)	O
-experiments	O
-coupled	O
-with	O
-multi	O
--	O
-angle	O
-laser	O
-light	O
-scattering	O
-(	O
-MALLS	O
-),	O
-NadR	O
-presented	O
-a	O
-single	O
-species	O
-with	O
-an	O
-absolute	O
-molecular	O
-mass	O
-of	O
-35	O
-kDa	O
-(	O
-S1	O
-Fig	O
-).	O
-	O
-These	O
-data	O
-showed	O
-that	O
-NadR	O
-was	O
-dimeric	O
-in	O
-solution	O
-,	O
-since	O
-the	O
-theoretical	O
-molecular	O
-mass	O
-of	O
-the	O
-NadR	O
-dimer	O
-is	O
-33	O
-.	O
-73	O
-kDa	O
-;	O
-and	O
-,	O
-there	O
-was	O
-no	O
-change	O
-in	O
-oligomeric	O
-state	O
-on	O
-addition	O
-of	O
-4	O
--	O
-HPA	O
-.	O
-	O
-The	O
-thermal	O
-stability	O
-of	O
-NadR	O
-was	O
-examined	O
-using	O
-differential	O
-scanning	O
-calorimetry	O
-(	O
-DSC	O
-).	O
-	O
-Since	O
-ligand	O
--	O
-binding	O
-often	O
-increases	O
-protein	O
-stability	O
-,	O
-we	O
-also	O
-investigated	O
-the	O
-effect	O
-of	O
-various	O
-HPAs	O
-(	O
-Fig	O
-1A	O
-)	O
-on	O
-the	O
-melting	O
-temperature	O
-(	O
-Tm	O
-)	O
-of	O
-NadR	O
-.	O
-As	O
-a	O
-control	O
-of	O
-specificity	O
-,	O
-we	O
-also	O
-tested	O
-salicylate	O
-,	O
-a	O
-known	O
-ligand	O
-of	O
-some	O
-MarR	O
-proteins	O
-previously	O
-reported	O
-to	O
-increase	O
-the	O
-Tm	O
-of	O
-ST1710	O
-and	O
-MTH313	O
-.	O
-	O
-The	O
-Tm	O
-of	O
-NadR	O
-was	O
-67	O
-.	O
-4	O
-±	O
-0	O
-.	O
-1	O
-°	O
-C	O
-in	O
-the	O
-absence	O
-of	O
-ligand	O
-,	O
-and	O
-was	O
-unaffected	O
-by	O
-salicylate	O
-.	O
-	O
-However	O
-,	O
-an	O
-increased	O
-thermal	O
-stability	O
-was	O
-induced	O
-by	O
-4	O
--	O
-HPA	O
-and	O
-,	O
-to	O
-a	O
-lesser	O
-extent	O
-,	O
-by	O
-3	O
--	O
-HPA	O
-.	O
-	O
-Interestingly	O
-,	O
-NadR	O
-displayed	O
-the	O
-greatest	O
-Tm	O
-increase	O
-upon	O
-addition	O
-of	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-Table	O
-1	O
-and	O
-Fig	O
-1B	O
-).	O
-	O
-Stability	O
-of	O
-NadR	O
-is	O
-increased	O
-by	O
-small	O
-molecule	O
-ligands	O
-.	O
-	O
-(	O
-A	O
-)	O
-Molecular	O
-structures	O
-of	O
-3	O
--	O
-HPA	O
-(	O
-MW	O
-152	O
-.	O
-2	O
-),	O
-4	O
--	O
-HPA	O
-(	O
-MW	O
-152	O
-.	O
-2	O
-),	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-MW	O
-186	O
-.	O
-6	O
-)	O
-and	O
-salicylic	O
-acid	O
-(	O
-MW	O
-160	O
-.	O
-1	O
-).	O
-(	O
-B	O
-)	O
-DSC	O
-profiles	O
-,	O
-colored	O
-as	O
-follows	O
-:	O
-apo	O
--	O
-NadR	O
-(	O
-violet	O
-),	O
-NadR	O
-+	O
-salicylate	O
-(	O
-red	O
-),	O
-NadR	O
-+	O
-3	O
--	O
-HPA	O
-(	O
-green	O
-),	O
-NadR	O
-+	O
-4	O
--	O
-HPA	O
-(	O
-blue	O
-),	O
-NadR	O
-+	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-pink	O
-).	O
-	O
-All	O
-DSC	O
-profiles	O
-are	O
-representative	O
-of	O
-triplicate	O
-experiments	O
-.	O
-	O
-Melting	O
--	O
-point	O
-(	O
-Tm	O
-)	O
-and	O
-its	O
-ligand	O
--	O
-induced	O
-increase	O
-(	O
-ΔTm	O
-)	O
-derived	O
-from	O
-DSC	O
-thermostability	O
-experiments	O
-.	O
-	O
-Dissociation	O
-constants	O
-(	O
-KD	O
-)	O
-of	O
-the	O
-NadR	O
-/	O
-ligand	O
-interactions	O
-from	O
-SPR	O
-steady	O
--	O
-state	O
-binding	O
-experiments	O
-.	O
-	O
-Ligand	O
-Tm	O
-(°	O
-C	O
-)	O
-ΔTm	O
-(°	O
-C	O
-)	O
-KD	O
-(	O
-mM	O
-)	O
-No	O
-ligand	O
-67	O
-.	O
-4	O
-±	O
-0	O
-.	O
-1	O
-n	O
-.	O
-a	O
-.	O
-n	O
-.	O
-a	O
-.	O
-	O
-3	O
--	O
-HPA	O
-70	O
-.	O
-0	O
-±	O
-0	O
-.	O
-1	O
-2	O
-.	O
-7	O
-2	O
-.	O
-7	O
-±	O
-0	O
-.	O
-1	O
-4	O
--	O
-HPA	O
-70	O
-.	O
-7	O
-±	O
-0	O
-.	O
-1	O
-3	O
-.	O
-3	O
-1	O
-.	O
-5	O
-±	O
-0	O
-.	O
-1	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-71	O
-.	O
-3	O
-±	O
-0	O
-.	O
-2	O
-3	O
-.	O
-9	O
-1	O
-.	O
-1	O
-±	O
-0	O
-.	O
-1	O
-	O
-NadR	O
-displays	O
-distinct	O
-binding	O
-affinities	O
-for	O
-hydroxyphenylacetate	O
-ligands	O
-	O
-To	O
-further	O
-investigate	O
-the	O
-binding	O
-of	O
-HPAs	O
-to	O
-NadR	O
-,	O
-we	O
-used	O
-surface	O
-plasmon	O
-resonance	O
-(	O
-SPR	O
-).	O
-	O
-The	O
-SPR	O
-sensorgrams	O
-revealed	O
-very	O
-fast	O
-association	O
-and	O
-dissociation	O
-events	O
-,	O
-typical	O
-of	O
-small	O
-molecule	O
-ligands	O
-,	O
-thus	O
-prohibiting	O
-a	O
-detailed	O
-study	O
-of	O
-binding	O
-kinetics	O
-.	O
-	O
-However	O
-,	O
-steady	O
--	O
-state	O
-SPR	O
-analyses	O
-of	O
-the	O
-NadR	O
--	O
-HPA	O
-interactions	O
-allowed	O
-determination	O
-of	O
-the	O
-equilibrium	O
-dissociation	O
-constants	O
-(	O
-KD	O
-)	O
-(	O
-Table	O
-1	O
-and	O
-S2	O
-Fig	O
-).	O
-	O
-The	O
-interactions	O
-of	O
-4	O
--	O
-HPA	O
-and	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-with	O
-NadR	O
-exhibited	O
-KD	O
-values	O
-of	O
-1	O
-.	O
-5	O
-mM	O
-and	O
-1	O
-.	O
-1	O
-mM	O
-,	O
-respectively	O
-.	O
-	O
-3	O
--	O
-HPA	O
-showed	O
-a	O
-weaker	O
-interaction	O
-,	O
-with	O
-a	O
-KD	O
-of	O
-2	O
-.	O
-7	O
-mM	O
-,	O
-while	O
-salicylate	O
-showed	O
-only	O
-a	O
-very	O
-weak	O
-response	O
-that	O
-did	O
-not	O
-reach	O
-saturation	O
-,	O
-indicating	O
-a	O
-non	O
--	O
-specific	O
-interaction	O
-with	O
-NadR	O
-.	O
-A	O
-ranking	O
-of	O
-these	O
-KD	O
-values	O
-showed	O
-that	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-was	O
-the	O
-tightest	O
-binder	O
-,	O
-and	O
-thus	O
-matched	O
-the	O
-ranking	O
-of	O
-ligand	O
--	O
-induced	O
-Tm	O
-increases	O
-observed	O
-in	O
-the	O
-DSC	O
-experiments	O
-.	O
-	O
-Although	O
-these	O
-KD	O
-values	O
-indicate	O
-rather	O
-weak	O
-interactions	O
-,	O
-they	O
-are	O
-similar	O
-to	O
-the	O
-values	O
-reported	O
-previously	O
-for	O
-the	O
-MarR	O
-/	O
-salicylate	O
-interaction	O
-(	O
-KD	O
-~	O
-1	O
-mM	O
-)	O
-and	O
-the	O
-MTH313	O
-/	O
-salicylate	O
-interaction	O
-(	O
-KD	O
-2	O
-–	O
-3	O
-mM	O
-),	O
-and	O
-approximately	O
-20	O
--	O
-fold	O
-tighter	O
-than	O
-the	O
-ST1710	O
-/	O
-salicylate	O
-interaction	O
-(	O
-KD	O
-~	O
-20	O
-mM	O
-).	O
-	O
-Crystal	O
-structures	O
-of	O
-holo	O
--	O
-NadR	O
-and	O
-apo	O
--	O
-NadR	O
-	O
-To	O
-fully	O
-characterize	O
-the	O
-NadR	O
-/	O
-HPA	O
-interactions	O
-,	O
-we	O
-sought	O
-to	O
-determine	O
-crystal	O
-structures	O
-of	O
-NadR	O
-in	O
-ligand	O
--	O
-bound	O
-(	O
-holo	O
-)	O
-and	O
-ligand	O
--	O
-free	O
-(	O
-apo	O
-)	O
-forms	O
-.	O
-	O
-First	O
-,	O
-we	O
-crystallized	O
-NadR	O
-(	O
-a	O
-selenomethionine	O
--	O
-labelled	O
-derivative	O
-)	O
-in	O
-the	O
-presence	O
-of	O
-a	O
-200	O
--	O
-fold	O
-molar	O
-excess	O
-of	O
-4	O
--	O
-HPA	O
-.	O
-	O
-The	O
-structure	O
-of	O
-the	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-complex	O
-was	O
-determined	O
-at	O
-2	O
-.	O
-3	O
-Å	O
-resolution	O
-using	O
-a	O
-combination	O
-of	O
-the	O
-single	O
--	O
-wavelength	O
-anomalous	O
-dispersion	O
-(	O
-SAD	O
-)	O
-and	O
-molecular	O
-replacement	O
-(	O
-MR	O
-)	O
-methods	O
-,	O
-and	O
-was	O
-refined	O
-to	O
-R	O
-work	O
-/	O
-R	O
-free	O
-values	O
-of	O
-20	O
-.	O
-9	O
-/	O
-26	O
-.	O
-0	O
-%	O
-(	O
-Table	O
-2	O
-).	O
-	O
-Despite	O
-numerous	O
-attempts	O
-,	O
-we	O
-were	O
-unable	O
-to	O
-obtain	O
-high	O
--	O
-quality	O
-crystals	O
-of	O
-NadR	O
-complexed	O
-with	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-,	O
-3	O
-,	O
-4	O
--	O
-HPA	O
-,	O
-3	O
--	O
-HPA	O
-or	O
-DNA	O
-targets	O
-.	O
-	O
-However	O
-,	O
-it	O
-was	O
-eventually	O
-possible	O
-to	O
-crystallize	O
-apo	O
--	O
-NadR	O
-,	O
-and	O
-the	O
-structure	O
-was	O
-determined	O
-at	O
-2	O
-.	O
-7	O
-Å	O
-resolution	O
-by	O
-MR	O
-methods	O
-using	O
-the	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-complex	O
-as	O
-the	O
-search	O
-model	O
-.	O
-	O
-The	O
-apo	O
--	O
-NadR	O
-structure	O
-was	O
-refined	O
-to	O
-R	O
-work	O
-/	O
-R	O
-free	O
-values	O
-of	O
-19	O
-.	O
-1	O
-/	O
-26	O
-.	O
-8	O
-%	O
-(	O
-Table	O
-2	O
-).	O
-	O
-Data	O
-collection	O
-and	O
-refinement	O
-statistics	O
-for	O
-NadR	O
-structures	O
-.	O
-	O
-The	O
-asymmetric	O
-unit	O
-of	O
-the	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-crystals	O
-(	O
-holo	O
--	O
-NadR	O
-)	O
-contained	O
-one	O
-NadR	O
-homodimer	O
-,	O
-while	O
-the	O
-apo	O
--	O
-NadR	O
-crystals	O
-contained	O
-two	O
-homodimers	O
-.	O
-	O
-In	O
-the	O
-apo	O
--	O
-NadR	O
-crystals	O
-,	O
-the	O
-two	O
-homodimers	O
-were	O
-related	O
-by	O
-a	O
-rotation	O
-of	O
-~	O
-90	O
-°;	O
-the	O
-observed	O
-association	O
-of	O
-the	O
-two	O
-dimers	O
-was	O
-presumably	O
-merely	O
-an	O
-effect	O
-of	O
-crystal	O
-packing	O
-,	O
-since	O
-the	O
-interface	O
-between	O
-the	O
-two	O
-homodimers	O
-is	O
-small	O
-(<	O
-550	O
-Å2	O
-of	O
-buried	O
-surface	O
-area	O
-),	O
-and	O
-is	O
-not	O
-predicted	O
-to	O
-be	O
-physiologically	O
-relevant	O
-by	O
-the	O
-PISA	O
-software	O
-.	O
-	O
-Moreover	O
-,	O
-our	O
-SE	O
--	O
-HPLC	O
-/	O
-MALLS	O
-analyses	O
-(	O
-see	O
-above	O
-)	O
-revealed	O
-that	O
-in	O
-solution	O
-NadR	O
-is	O
-dimeric	O
-,	O
-and	O
-previous	O
-studies	O
-using	O
-native	O
-mass	O
-spectrometry	O
-(	O
-MS	O
-)	O
-revealed	O
-dimers	O
-,	O
-not	O
-tetramers	O
-.	O
-	O
-The	O
-NadR	O
-homodimer	O
-bound	O
-to	O
-4	O
--	O
-HPA	O
-has	O
-a	O
-dimerization	O
-interface	O
-mostly	O
-involving	O
-the	O
-top	O
-of	O
-its	O
-‘	O
-triangular	O
-’	O
-form	O
-,	O
-while	O
-the	O
-two	O
-DNA	O
--	O
-binding	O
-domains	O
-are	O
-located	O
-at	O
-the	O
-base	O
-(	O
-Fig	O
-2A	O
-).	O
-	O
-High	O
--	O
-quality	O
-electron	O
-density	O
-maps	O
-allowed	O
-clear	O
-identification	O
-of	O
-the	O
-bound	O
-ligand	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-Fig	O
-2B	O
-).	O
-	O
-The	O
-overall	O
-structure	O
-of	O
-NadR	O
-shows	O
-dimensions	O
-of	O
-~	O
-50	O
-×	O
-65	O
-×	O
-50	O
-Å	O
-and	O
-a	O
-large	O
-homodimer	O
-interface	O
-that	O
-buries	O
-a	O
-total	O
-surface	O
-area	O
-of	O
-~	O
-4800	O
-Å2	O
-.	O
-	O
-Each	O
-NadR	O
-monomer	O
-consists	O
-of	O
-six	O
-α	O
--	O
-helices	O
-and	O
-two	O
-short	O
-β	O
--	O
-strands	O
-,	O
-with	O
-helices	O
-α1	O
-,	O
-α5	O
-,	O
-and	O
-α6	O
-forming	O
-the	O
-dimer	O
-interface	O
-.	O
-	O
-Helices	O
-α3	O
-and	O
-α4	O
-form	O
-a	O
-helix	O
--	O
-turn	O
--	O
-helix	O
-motif	O
-,	O
-followed	O
-by	O
-the	O
-“	O
-wing	O
-motif	O
-”	O
-comprised	O
-of	O
-two	O
-short	O
-antiparallel	O
-β	O
--	O
-strands	O
-(	O
-β1	O
--	O
-β2	O
-)	O
-linked	O
-by	O
-a	O
-relatively	O
-long	O
-and	O
-flexible	O
-loop	O
-.	O
-	O
-Interestingly	O
-,	O
-in	O
-the	O
-α4	O
--	O
-β2	O
-region	O
-,	O
-the	O
-stretch	O
-of	O
-residues	O
-from	O
-R64	O
--	O
-R91	O
-presents	O
-seven	O
-positively	O
--	O
-charged	O
-side	O
-chains	O
-,	O
-all	O
-available	O
-for	O
-potential	O
-interactions	O
-with	O
-DNA	O
-.	O
-	O
-Together	O
-,	O
-these	O
-structural	O
-elements	O
-constitute	O
-the	O
-winged	O
-helix	O
--	O
-turn	O
--	O
-helix	O
-(	O
-wHTH	O
-)	O
-DNA	O
--	O
-binding	O
-domain	O
-and	O
-,	O
-together	O
-with	O
-the	O
-dimeric	O
-organization	O
-,	O
-are	O
-the	O
-hallmarks	O
-of	O
-MarR	O
-family	O
-structures	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-NadR	O
-in	O
-complex	O
-with	O
-4	O
--	O
-HPA	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-holo	O
--	O
-NadR	O
-homodimer	O
-is	O
-depicted	O
-in	O
-green	O
-and	O
-blue	O
-for	O
-chains	O
-A	O
-and	O
-B	O
-respectively	O
-,	O
-while	O
-yellow	O
-sticks	O
-depict	O
-the	O
-4	O
--	O
-HPA	O
-ligand	O
-(	O
-labelled	O
-).	O
-	O
-For	O
-simplicity	O
-,	O
-secondary	O
-structure	O
-elements	O
-are	O
-labelled	O
-for	O
-chain	O
-B	O
-only	O
-.	O
-	O
-Red	O
-dashes	O
-show	O
-hypothetical	O
-positions	O
-of	O
-chain	O
-B	O
-residues	O
-88	O
-–	O
-90	O
-that	O
-were	O
-not	O
-modeled	O
-due	O
-to	O
-lack	O
-of	O
-electron	O
-density	O
-.	O
-	O
-(	O
-B	O
-)	O
-A	O
-zoom	O
-into	O
-the	O
-pocket	O
-occupied	O
-by	O
-4	O
--	O
-HPA	O
-shows	O
-that	O
-the	O
-ligand	O
-contacts	O
-both	O
-chains	O
-A	O
-and	O
-B	O
-;	O
-blue	O
-mesh	O
-shows	O
-electron	O
-density	O
-around	O
-4	O
--	O
-HPA	O
-calculated	O
-from	O
-a	O
-composite	O
-omit	O
-map	O
-(	O
-omitting	O
-4	O
--	O
-HPA	O
-),	O
-using	O
-phenix	O
-.	O
-	O
-The	O
-map	O
-is	O
-contoured	O
-at	O
-1σ	O
-and	O
-the	O
-figure	O
-was	O
-prepared	O
-with	O
-a	O
-density	O
-mesh	O
-carve	O
-factor	O
-of	O
-1	O
-.	O
-7	O
-,	O
-using	O
-Pymol	O
-(	O
-www	O
-.	O
-pymol	O
-.	O
-org	O
-).	O
-	O
-A	O
-single	O
-conserved	O
-leucine	O
-residue	O
-(	O
-L130	O
-)	O
-is	O
-crucial	O
-for	O
-dimerization	O
-	O
-The	O
-NadR	O
-dimer	O
-interface	O
-is	O
-formed	O
-by	O
-at	O
-least	O
-32	O
-residues	O
-,	O
-which	O
-establish	O
-numerous	O
-inter	O
--	O
-chain	O
-salt	O
-bridges	O
-or	O
-hydrogen	O
-bonds	O
-,	O
-and	O
-many	O
-hydrophobic	O
-packing	O
-interactions	O
-(	O
-Fig	O
-3A	O
-and	O
-3B	O
-).	O
-	O
-To	O
-determine	O
-which	O
-residues	O
-were	O
-most	O
-important	O
-for	O
-dimerization	O
-,	O
-we	O
-studied	O
-the	O
-interface	O
-in	O
-silico	O
-and	O
-identified	O
-several	O
-residues	O
-as	O
-potential	O
-mediators	O
-of	O
-key	O
-stabilizing	O
-interactions	O
-.	O
-	O
-Using	O
-site	O
--	O
-directed	O
-mutagenesis	O
-,	O
-a	O
-panel	O
-of	O
-eight	O
-mutant	O
-NadR	O
-proteins	O
-was	O
-prepared	O
-(	O
-including	O
-mutations	O
-H7A	B-mutant
-,	O
-S9A	B-mutant
-,	O
-N11A	B-mutant
-,	O
-D112A	B-mutant
-,	O
-R114A	B-mutant
-,	O
-Y115A	B-mutant
-,	O
-K126A	B-mutant
-,	O
-L130K	B-mutant
-and	O
-L133K	B-mutant
-),	O
-sufficient	O
-to	O
-explore	O
-the	O
-entire	O
-dimer	O
-interface	O
-.	O
-	O
-Each	O
-mutant	O
-NadR	O
-protein	O
-was	O
-purified	O
-,	O
-and	O
-then	O
-its	O
-oligomeric	O
-state	O
-was	O
-examined	O
-by	O
-analytical	O
-SE	O
--	O
-HPLC	O
-.	O
-	O
-Almost	O
-all	O
-the	O
-mutants	O
-showed	O
-the	O
-same	O
-elution	O
-profile	O
-as	O
-the	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-NadR	O
-protein	O
-.	O
-	O
-Only	O
-the	O
-L130K	B-mutant
-mutation	O
-induced	O
-a	O
-notable	O
-change	O
-in	O
-the	O
-oligomeric	O
-state	O
-of	O
-NadR	O
-(	O
-Fig	O
-3C	O
-).	O
-	O
-Further	O
-,	O
-in	O
-SE	O
--	O
-MALLS	O
-analyses	O
-,	O
-the	O
-L130K	B-mutant
-mutant	O
-displayed	O
-two	O
-distinct	O
-species	O
-in	O
-solution	O
-,	O
-approximately	O
-80	O
-%	O
-being	O
-monomeric	O
-(	O
-a	O
-19	O
-kDa	O
-species	O
-),	O
-and	O
-only	O
-20	O
-%	O
-retaining	O
-the	O
-typical	O
-native	O
-dimeric	O
-state	O
-(	O
-a	O
-35	O
-kDa	O
-species	O
-)	O
-(	O
-Fig	O
-3D	O
-),	O
-demonstrating	O
-that	O
-Leu130	O
-is	O
-crucial	O
-for	O
-stable	O
-dimerization	O
-.	O
-	O
-It	O
-is	O
-notable	O
-that	O
-L130	O
-is	O
-usually	O
-present	O
-as	O
-Leu	O
-,	O
-or	O
-an	O
-alternative	O
-bulky	O
-hydrophobic	O
-amino	O
-acid	O
-(	O
-e	O
-.	O
-g	O
-.	O
-Phe	O
-,	O
-Val	O
-),	O
-in	O
-many	O
-MarR	O
-family	O
-proteins	O
-,	O
-suggesting	O
-a	O
-conserved	O
-role	O
-in	O
-stabilizing	O
-the	O
-dimer	O
-interface	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-most	O
-of	O
-the	O
-other	O
-residues	O
-identified	O
-in	O
-the	O
-NadR	O
-dimer	O
-interface	O
-were	O
-poorly	O
-conserved	O
-in	O
-the	O
-MarR	O
-family	O
-.	O
-	O
-Analysis	O
-of	O
-the	O
-NadR	O
-dimer	O
-interface	O
-.	O
-	O
-(	O
-A	O
-)	O
-Both	O
-orientations	O
-show	O
-chain	O
-A	O
-,	O
-green	O
-backbone	O
-ribbon	O
-,	O
-colored	O
-red	O
-to	O
-highlight	O
-all	O
-locations	O
-involved	O
-in	O
-dimerization	O
-;	O
-namely	O
-,	O
-inter	O
--	O
-chain	O
-salt	O
-bridges	O
-or	O
-hydrogen	O
-bonds	O
-involving	O
-Q4	O
-,	O
-S5	O
-,	O
-K6	O
-,	O
-H7	O
-,	O
-S9	O
-,	O
-I10	O
-,	O
-N11	O
-,	O
-I15	O
-,	O
-Q16	O
-,	O
-R18	O
-,	O
-D36	O
-,	O
-R43	O
-,	O
-A46	O
-,	O
-Q59	O
-,	O
-C61	O
-,	O
-Y104	O
-,	O
-D112	O
-,	O
-R114	O
-,	O
-Y115	O
-,	O
-D116	O
-,	O
-E119	O
-,	O
-K126	O
-,	O
-E136	O
-,	O
-E141	O
-,	O
-N145	O
-,	O
-and	O
-the	O
-hydrophobic	O
-packing	O
-interactions	O
-involving	O
-I10	O
-,	O
-I12	O
-,	O
-L14	O
-,	O
-I15	O
-,	O
-R18	O
-,	O
-Y115	O
-,	O
-I118	O
-,	O
-L130	O
-,	O
-L133	O
-,	O
-L134	O
-and	O
-L137	O
-.	O
-	O
-Chain	O
-B	O
-,	O
-grey	O
-surface	O
-,	O
-is	O
-marked	O
-blue	O
-to	O
-highlight	O
-residues	O
-probed	O
-by	O
-site	O
--	O
-directed	O
-mutagenesis	O
-(	O
-E136	O
-only	O
-makes	O
-a	O
-salt	O
-bridge	O
-with	O
-K126	O
-,	O
-therefore	O
-it	O
-was	O
-sufficient	O
-to	O
-make	O
-the	O
-K126A	B-mutant
-mutation	O
-to	O
-assess	O
-the	O
-importance	O
-of	O
-this	O
-ionic	O
-interaction	O
-;	O
-the	O
-H7	O
-position	O
-is	O
-labelled	O
-for	O
-monomer	O
-A	O
-,	O
-since	O
-electron	O
-density	O
-was	O
-lacking	O
-for	O
-monomer	O
-B	O
-).	O
-(	O
-B	O
-)	O
-A	O
-zoom	O
-into	O
-the	O
-environment	O
-of	O
-helix	O
-α6	O
-to	O
-show	O
-how	O
-residue	O
-L130	O
-chain	O
-B	O
-(	O
-blue	O
-side	O
-chain	O
-)	O
-is	O
-a	O
-focus	O
-of	O
-hydrophobic	O
-packing	O
-interactions	O
-with	O
-L130	O
-,	O
-L133	O
-,	O
-L134	O
-and	O
-L137	O
-of	O
-chain	O
-A	O
-(	O
-red	O
-side	O
-chains	O
-).	O
-	O
-(	O
-C	O
-)	O
-SE	O
--	O
-HPLC	O
-analyses	O
-of	O
-all	O
-mutant	O
-forms	O
-of	O
-NadR	O
-are	O
-compared	O
-with	O
-the	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-protein	O
-.	O
-	O
-The	O
-WT	O
-and	O
-most	O
-of	O
-the	O
-mutants	O
-show	O
-a	O
-single	O
-elution	O
-peak	O
-with	O
-an	O
-absorbance	O
-maximum	O
-at	O
-17	O
-.	O
-5	O
-min	O
-.	O
-	O
-Only	O
-the	O
-mutation	O
-L130K	B-mutant
-has	O
-a	O
-noteworthy	O
-effect	O
-on	O
-the	O
-oligomeric	O
-state	O
-,	O
-inducing	O
-a	O
-second	O
-peak	O
-with	O
-a	O
-longer	O
-retention	O
-time	O
-and	O
-a	O
-second	O
-peak	O
-maximum	O
-at	O
-18	O
-.	O
-6	O
-min	O
-.	O
-	O
-To	O
-a	O
-much	O
-lesser	O
-extent	O
-,	O
-the	O
-L133K	B-mutant
-mutation	O
-also	O
-appears	O
-to	O
-induce	O
-a	O
-‘	O
-shoulder	O
-’	O
-to	O
-the	O
-main	O
-peak	O
-,	O
-suggesting	O
-very	O
-weak	O
-ability	O
-to	O
-disrupt	O
-the	O
-dimer	O
-.	O
-(	O
-D	O
-)	O
-SE	O
--	O
-HPLC	O
-/	O
-MALLS	O
-analyses	O
-of	O
-the	O
-L130K	B-mutant
-mutant	O
-,	O
-shows	O
-20	O
-%	O
-dimer	O
-and	O
-80	O
-%	O
-monomer	O
-.	O
-	O
-The	O
-holo	O
--	O
-NadR	O
-structure	O
-presents	O
-only	O
-one	O
-occupied	O
-ligand	O
--	O
-binding	O
-pocket	O
-	O
-The	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-structure	O
-revealed	O
-the	O
-ligand	O
--	O
-binding	O
-site	O
-nestled	O
-between	O
-the	O
-dimerization	O
-and	O
-DNA	O
--	O
-binding	O
-domains	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-The	O
-ligand	O
-showed	O
-a	O
-different	O
-position	O
-and	O
-orientation	O
-compared	O
-to	O
-salicylate	O
-complexed	O
-with	O
-MTH313	O
-and	O
-ST1710	O
-(	O
-see	O
-Discussion	O
-).	O
-	O
-The	O
-binding	O
-pocket	O
-was	O
-almost	O
-entirely	O
-filled	O
-by	O
-4	O
--	O
-HPA	O
-and	O
-one	O
-water	O
-molecule	O
-,	O
-although	O
-there	O
-also	O
-remained	O
-a	O
-small	O
-tunnel	O
-2	O
--	O
-4Å	O
-in	O
-diameter	O
-and	O
-5	O
--	O
-6Å	O
-long	O
-leading	O
-from	O
-the	O
-pocket	O
-(	O
-proximal	O
-to	O
-the	O
-4	O
--	O
-hydroxyl	O
-position	O
-)	O
-to	O
-the	O
-protein	O
-surface	O
-.	O
-	O
-The	O
-tunnel	O
-was	O
-lined	O
-with	O
-rather	O
-hydrophobic	O
-amino	O
-acids	O
-,	O
-and	O
-did	O
-not	O
-contain	O
-water	O
-molecules	O
-.	O
-	O
-Unexpectedly	O
-,	O
-only	O
-one	O
-monomer	O
-of	O
-the	O
-holo	O
--	O
-NadR	O
-homodimer	O
-contained	O
-4	O
--	O
-HPA	O
-in	O
-the	O
-binding	O
-pocket	O
-,	O
-whereas	O
-the	O
-corresponding	O
-pocket	O
-of	O
-the	O
-other	O
-monomer	O
-was	O
-unoccupied	O
-by	O
-ligand	O
-,	O
-despite	O
-the	O
-large	O
-excess	O
-of	O
-4	O
--	O
-HPA	O
-used	O
-in	O
-the	O
-crystallization	O
-conditions	O
-.	O
-	O
-Inspection	O
-of	O
-the	O
-protein	O
--	O
-ligand	O
-interaction	O
-network	O
-revealed	O
-no	O
-bonds	O
-from	O
-NadR	O
-backbone	O
-groups	O
-to	O
-the	O
-ligand	O
-,	O
-but	O
-several	O
-key	O
-side	O
-chain	O
-mediated	O
-hydrogen	O
-(	O
-H	O
-)-	O
-bonds	O
-and	O
-ionic	O
-interactions	O
-,	O
-most	O
-notably	O
-between	O
-the	O
-carboxylate	O
-group	O
-of	O
-4	O
--	O
-HPA	O
-and	O
-Ser9	O
-of	O
-chain	O
-A	O
-(	O
-SerA9	O
-),	O
-and	O
-chain	O
-B	O
-residues	O
-TrpB39	O
-,	O
-ArgB43	O
-and	O
-TyrB115	O
-(	O
-Fig	O
-4A	O
-).	O
-	O
-At	O
-the	O
-other	O
-‘	O
-end	O
-’	O
-of	O
-the	O
-ligand	O
-,	O
-the	O
-4	O
--	O
-hydroxyl	O
-group	O
-was	O
-proximal	O
-to	O
-AspB36	O
-,	O
-with	O
-which	O
-it	O
-may	O
-establish	O
-an	O
-H	O
--	O
-bond	O
-(	O
-see	O
-bond	O
-distances	O
-in	O
-Table	O
-3	O
-).	O
-	O
-The	O
-water	O
-molecule	O
-observed	O
-in	O
-the	O
-pocket	O
-was	O
-bound	O
-by	O
-the	O
-carboxylate	O
-group	O
-and	O
-the	O
-side	O
-chains	O
-of	O
-SerA9	O
-and	O
-AsnA11	O
-.	O
-	O
-Atomic	O
-details	O
-of	O
-NadR	O
-/	O
-HPA	O
-interactions	O
-.	O
-	O
-A	O
-)	O
-A	O
-stereo	O
--	O
-view	O
-zoom	O
-into	O
-the	O
-binding	O
-pocket	O
-showing	O
-side	O
-chain	O
-sticks	O
-for	O
-all	O
-interactions	O
-between	O
-NadR	O
-and	O
-4	O
--	O
-HPA	O
-.	O
-	O
-Green	O
-and	O
-blue	O
-ribbons	O
-depict	O
-NadR	O
-chains	O
-A	O
-and	O
-B	O
-,	O
-respectively	O
-.	O
-	O
-4	O
--	O
-HPA	O
-is	O
-shown	O
-in	O
-yellow	O
-sticks	O
-,	O
-with	O
-oxygen	O
-atoms	O
-in	O
-red	O
-.	O
-	O
-A	O
-water	O
-molecule	O
-is	O
-shown	O
-by	O
-the	O
-red	O
-sphere	O
-.	O
-	O
-H	O
--	O
-bonds	O
-up	O
-to	O
-3	O
-.	O
-6Å	O
-are	O
-shown	O
-as	O
-dashed	O
-lines	O
-.	O
-	O
-The	O
-entire	O
-set	O
-of	O
-residues	O
-making	O
-H	O
--	O
-bonds	O
-or	O
-non	O
--	O
-bonded	O
-contacts	O
-with	O
-4	O
--	O
-HPA	O
-is	O
-as	O
-follows	O
-:	O
-SerA9	O
-,	O
-AsnA11	O
-,	O
-LeuB21	O
-,	O
-MetB22	O
-,	O
-PheB25	O
-,	O
-LeuB29	O
-,	O
-AspB36	O
-,	O
-TrpB39	O
-,	O
-ArgB43	O
-,	O
-ValB111	O
-and	O
-TyrB115	O
-(	O
-automated	O
-analysis	O
-performed	O
-using	O
-PDBsum	O
-and	O
-verified	O
-manually	O
-).	O
-	O
-Residues	O
-AsnA11	O
-and	O
-ArgB18	O
-likely	O
-make	O
-indirect	O
-yet	O
-local	O
-contributions	O
-to	O
-ligand	O
-binding	O
-,	O
-mainly	O
-by	O
-stabilizing	O
-the	O
-position	O
-of	O
-AspB36	O
-.	O
-	O
-Side	O
-chains	O
-mediating	O
-hydrophobic	O
-interactions	O
-are	O
-shown	O
-in	O
-orange	O
-.	O
-(	O
-B	O
-)	O
-A	O
-model	O
-was	O
-prepared	O
-to	O
-visualize	O
-putative	O
-interactions	O
-of	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-pink	O
-)	O
-with	O
-NadR	O
-,	O
-revealing	O
-the	O
-potential	O
-for	O
-additional	O
-contacts	O
-(	O
-dashed	O
-lines	O
-)	O
-of	O
-the	O
-chloro	O
-moiety	O
-(	O
-green	O
-stick	O
-)	O
-with	O
-LeuB29	O
-and	O
-AspB36	O
-.	O
-	O
-List	O
-of	O
-4	O
--	O
-HPA	O
-atoms	O
-bound	O
-to	O
-NadR	O
-via	O
-ionic	O
-interactions	O
-and	O
-/	O
-or	O
-H	O
--	O
-bonds	O
-.	O
-	O
-4	O
--	O
-HPA	O
-atom	O
-NadR	O
-residue	O
-/	O
-atom	O
-Distance	O
-(	O
-Å	O
-)	O
-O2	O
-TrpB39	O
-/	O
-NE1	O
-2	O
-.	O
-83	O
-O2	O
-ArgB43	O
-/	O
-NH1	O
-2	O
-.	O
-76	O
-O1	O
-ArgB43	O
-/	O
-NH1	O
-3	O
-.	O
-84	O
-O1	O
-SerA9	O
-/	O
-OG	O
-2	O
-.	O
-75	O
-O1	O
-TyrB115	O
-/	O
-OH	O
-2	O
-.	O
-50	O
-O2	O
-Water	O
-(*	O
-Ser9	O
-/	O
-Asn11	O
-)	O
-2	O
-.	O
-88	O
-OH	O
-AspB36	O
-/	O
-OD1	O
-/	O
-OD2	O
-3	O
-.	O
-6	O
-/	O
-3	O
-.	O
-7	O
-	O
-*	O
-Bond	O
-distance	O
-between	O
-the	O
-ligand	O
-carboxylate	O
-group	O
-and	O
-the	O
-water	O
-molecule	O
-,	O
-which	O
-in	O
-turn	O
-makes	O
-H	O
--	O
-bond	O
-to	O
-the	O
-SerA9	O
-and	O
-AsnA11	O
-side	O
-chains	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-H	O
--	O
-bonds	O
-involving	O
-the	O
-carboxylate	O
-and	O
-hydroxyl	O
-groups	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-binding	O
-of	O
-the	O
-phenyl	O
-moiety	O
-appeared	O
-to	O
-be	O
-stabilized	O
-by	O
-several	O
-van	O
-der	O
-Waals	O
-’	O
-contacts	O
-,	O
-particularly	O
-those	O
-involving	O
-the	O
-hydrophobic	O
-side	O
-chain	O
-atoms	O
-of	O
-LeuB21	O
-,	O
-MetB22	O
-,	O
-PheB25	O
-,	O
-LeuB29	O
-and	O
-ValB111	O
-(	O
-Fig	O
-4A	O
-).	O
-	O
-Notably	O
-,	O
-the	O
-phenyl	O
-ring	O
-of	O
-PheB25	O
-was	O
-positioned	O
-parallel	O
-to	O
-the	O
-phenyl	O
-ring	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-potentially	O
-forming	O
-π	O
--	O
-π	O
-parallel	O
--	O
-displaced	O
-stacking	O
-interactions	O
-.	O
-	O
-Consequently	O
-,	O
-residues	O
-in	O
-the	O
-4	O
--	O
-HPA	O
-binding	O
-pocket	O
-are	O
-mostly	O
-contributed	O
-by	O
-NadR	O
-chain	O
-B	O
-,	O
-and	O
-effectively	O
-created	O
-a	O
-polar	O
-‘	O
-floor	O
-’	O
-and	O
-a	O
-hydrophobic	O
-‘	O
-ceiling	O
-’,	O
-which	O
-house	O
-the	O
-ligand	O
-.	O
-	O
-Collectively	O
-,	O
-this	O
-mixed	O
-network	O
-of	O
-polar	O
-and	O
-hydrophobic	O
-interactions	O
-endows	O
-NadR	O
-with	O
-a	O
-strong	O
-recognition	O
-pattern	O
-for	O
-HPAs	O
-,	O
-with	O
-additional	O
-medium	O
--	O
-range	O
-interactions	O
-potentially	O
-established	O
-with	O
-the	O
-hydroxyl	O
-group	O
-at	O
-the	O
-4	O
--	O
-position	O
-.	O
-	O
-Structure	O
--	O
-activity	O
-relationships	O
-:	O
-molecular	O
-basis	O
-of	O
-enhanced	O
-stabilization	O
-by	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-	O
-We	O
-modelled	O
-the	O
-binding	O
-of	O
-other	O
-HPAs	O
-by	O
-in	O
-silico	O
-superposition	O
-onto	O
-4	O
--	O
-HPA	O
-in	O
-the	O
-holo	O
--	O
-NadR	O
-structure	O
-,	O
-and	O
-thereby	O
-obtained	O
-molecular	O
-explanations	O
-for	O
-the	O
-binding	O
-specificities	O
-of	O
-diverse	O
-ligands	O
-.	O
-	O
-For	O
-example	O
-,	O
-similar	O
-to	O
-4	O
--	O
-HPA	O
-,	O
-the	O
-binding	O
-of	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-could	O
-involve	O
-multiple	O
-bonds	O
-towards	O
-the	O
-carboxylate	O
-group	O
-of	O
-the	O
-ligand	O
-and	O
-some	O
-to	O
-the	O
-4	O
--	O
-hydroxyl	O
-group	O
-.	O
-	O
-Additionally	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-LeuB29	O
-and	O
-AspB36	O
-would	O
-be	O
-only	O
-2	O
-.	O
-6	O
-–	O
-3	O
-.	O
-5	O
-Å	O
-from	O
-the	O
-chlorine	O
-atom	O
-,	O
-thus	O
-providing	O
-van	O
-der	O
-Waals	O
-’	O
-interactions	O
-or	O
-H	O
--	O
-bonds	O
-to	O
-generate	O
-the	O
-additional	O
-binding	O
-affinity	O
-observed	O
-for	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-Fig	O
-4B	O
-).	O
-	O
-The	O
-presence	O
-of	O
-a	O
-single	O
-hydroxyl	O
-group	O
-at	O
-position	O
-2	O
-,	O
-as	O
-in	O
-2	O
--	O
-HPA	O
-,	O
-rather	O
-than	O
-at	O
-position	O
-4	O
-,	O
-would	O
-eliminate	O
-the	O
-possibility	O
-of	O
-favorable	O
-interactions	O
-with	O
-AspB36	O
-,	O
-resulting	O
-in	O
-the	O
-lack	O
-of	O
-NadR	O
-regulation	O
-by	O
-2	O
--	O
-HPA	O
-described	O
-previously	O
-.	O
-	O
-Finally	O
-,	O
-salicylate	O
-is	O
-presumably	O
-unable	O
-to	O
-specifically	O
-bind	O
-NadR	O
-due	O
-to	O
-the	O
-2	O
--	O
-hydroxyl	O
-substitution	O
-and	O
-the	O
-shorter	O
-aliphatic	O
-chain	O
-connecting	O
-its	O
-carboxylate	O
-group	O
-(	O
-Fig	O
-1A	O
-):	O
-the	O
-compound	O
-simply	O
-seems	O
-too	O
-small	O
-to	O
-simultaneously	O
-establish	O
-the	O
-network	O
-of	O
-beneficial	O
-bonds	O
-observed	O
-in	O
-the	O
-NadR	O
-/	O
-HPA	O
-interactions	O
-.	O
-	O
-Analysis	O
-of	O
-the	O
-pockets	O
-reveals	O
-the	O
-molecular	O
-basis	O
-for	O
-asymmetric	O
-binding	O
-and	O
-stoichiometry	O
-	O
-However	O
-,	O
-studies	O
-based	O
-on	O
-tryptophan	O
-fluorescence	O
-were	O
-confounded	O
-by	O
-the	O
-fluorescence	O
-of	O
-the	O
-HPA	O
-ligands	O
-,	O
-and	O
-isothermal	O
-titration	O
-calorimetry	O
-(	O
-ITC	O
-)	O
-was	O
-unfeasible	O
-due	O
-to	O
-the	O
-need	O
-for	O
-very	O
-high	O
-concentrations	O
-of	O
-NadR	O
-in	O
-the	O
-ITC	O
-chamber	O
-(	O
-due	O
-to	O
-the	O
-relatively	O
-low	O
-affinity	O
-),	O
-which	O
-exceeded	O
-the	O
-solubility	O
-limits	O
-of	O
-the	O
-protein	O
-.	O
-	O
-However	O
-,	O
-it	O
-was	O
-possible	O
-to	O
-calculate	O
-the	O
-binding	O
-stoichiometry	O
-of	O
-the	O
-NadR	O
--	O
-HPA	O
-interactions	O
-using	O
-an	O
-SPR	O
--	O
-based	O
-approach	O
-.	O
-	O
-In	O
-SPR	O
-,	O
-the	O
-signal	O
-measured	O
-is	O
-proportional	O
-to	O
-the	O
-total	O
-molecular	O
-mass	O
-proximal	O
-to	O
-the	O
-sensor	O
-surface	O
-;	O
-consequently	O
-,	O
-if	O
-the	O
-molecular	O
-weights	O
-of	O
-the	O
-interactors	O
-are	O
-known	O
-,	O
-then	O
-the	O
-stoichiometry	O
-of	O
-the	O
-resulting	O
-complex	O
-can	O
-be	O
-determined	O
-.	O
-	O
-This	O
-approach	O
-relies	O
-on	O
-the	O
-assumption	O
-that	O
-the	O
-captured	O
-protein	O
-(‘	O
-the	O
-ligand	O
-’,	O
-according	O
-to	O
-SPR	O
-conventions	O
-)	O
-is	O
-100	O
-%	O
-active	O
-and	O
-freely	O
--	O
-accessible	O
-to	O
-potential	O
-interactors	O
-(‘	O
-the	O
-analytes	O
-’).	O
-	O
-Firstly	O
-,	O
-NadR	O
-is	O
-expected	O
-to	O
-be	O
-covalently	O
-immobilized	O
-on	O
-the	O
-sensor	O
-chip	O
-as	O
-a	O
-dimer	O
-in	O
-random	O
-orientations	O
-,	O
-since	O
-it	O
-is	O
-a	O
-stable	O
-dimer	O
-in	O
-solution	O
-and	O
-has	O
-sixteen	O
-lysines	O
-well	O
--	O
-distributed	O
-around	O
-its	O
-surface	O
-,	O
-all	O
-able	O
-to	O
-act	O
-as	O
-potential	O
-sites	O
-for	O
-amine	O
-coupling	O
-to	O
-the	O
-chip	O
-,	O
-and	O
-none	O
-of	O
-which	O
-are	O
-close	O
-to	O
-the	O
-ligand	O
--	O
-binding	O
-pocket	O
-.	O
-	O
-Secondly	O
-,	O
-the	O
-HPA	O
-analytes	O
-are	O
-all	O
-very	O
-small	O
-(	O
-MW	O
-150	O
-–	O
-170	O
-,	O
-Fig	O
-1A	O
-)	O
-and	O
-therefore	O
-are	O
-expected	O
-to	O
-be	O
-able	O
-to	O
-diffuse	O
-readily	O
-into	O
-all	O
-potential	O
-binding	O
-sites	O
-,	O
-irrespective	O
-of	O
-the	O
-random	O
-orientations	O
-of	O
-the	O
-immobilized	O
-NadR	O
-dimers	O
-on	O
-the	O
-chip	O
-.	O
-	O
-The	O
-stoichiometry	O
-of	O
-the	O
-NadR	O
--	O
-HPA	O
-interactions	O
-was	O
-determined	O
-using	O
-Eq	O
-1	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-),	O
-and	O
-revealed	O
-stoichiometries	O
-of	O
-1	O
-.	O
-13	O
-for	O
-4	O
--	O
-HPA	O
-,	O
-1	O
-.	O
-02	O
-for	O
-3	O
--	O
-HPA	O
-,	O
-and	O
-1	O
-.	O
-21	O
-for	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-,	O
-strongly	O
-suggesting	O
-that	O
-one	O
-NadR	O
-dimer	O
-bound	O
-to	O
-1	O
-HPA	O
-analyte	O
-molecule	O
-.	O
-	O
-The	O
-crystallographic	O
-data	O
-,	O
-supported	O
-by	O
-the	O
-SPR	O
-studies	O
-of	O
-binding	O
-stoichiometry	O
-,	O
-revealed	O
-the	O
-lack	O
-of	O
-a	O
-second	O
-4	O
--	O
-HPA	O
-molecule	O
-in	O
-the	O
-homodimer	O
-,	O
-suggesting	O
-negative	O
-co	O
--	O
-operativity	O
-,	O
-a	O
-phenomenon	O
-previously	O
-described	O
-for	O
-the	O
-MTH313	O
-/	O
-salicylate	O
-interaction	O
-and	O
-for	O
-other	O
-MarR	O
-family	O
-proteins	O
-.	O
-	O
-To	O
-explore	O
-the	O
-molecular	O
-basis	O
-of	O
-asymmetry	O
-in	O
-holo	O
--	O
-NadR	O
-,	O
-we	O
-superposed	O
-its	O
-ligand	O
--	O
-free	O
-monomer	O
-(	O
-chain	O
-A	O
-)	O
-onto	O
-the	O
-ligand	O
--	O
-occupied	O
-monomer	O
-(	O
-chain	O
-B	O
-).	O
-	O
-Overall	O
-,	O
-the	O
-superposition	O
-revealed	O
-a	O
-high	O
-degree	O
-of	O
-structural	O
-similarity	O
-(	O
-Cα	O
-root	O
-mean	O
-square	O
-deviation	O
-(	O
-rmsd	O
-)	O
-of	O
-1	O
-.	O
-5Å	O
-),	O
-though	O
-on	O
-closer	O
-inspection	O
-a	O
-rotational	O
-difference	O
-of	O
-~	O
-9	O
-degrees	O
-along	O
-the	O
-long	O
-axis	O
-of	O
-helix	O
-α6	O
-was	O
-observed	O
-,	O
-suggesting	O
-that	O
-4	O
--	O
-HPA	O
-induced	O
-a	O
-slight	O
-conformational	O
-change	O
-(	O
-Fig	O
-5A	O
-).	O
-	O
-However	O
-,	O
-since	O
-residues	O
-of	O
-helix	O
-α6	O
-were	O
-not	O
-directly	O
-involved	O
-in	O
-ligand	O
-binding	O
-,	O
-an	O
-explanation	O
-for	O
-the	O
-lack	O
-of	O
-4	O
--	O
-HPA	O
-in	O
-monomer	O
-A	O
-did	O
-not	O
-emerge	O
-by	O
-analyzing	O
-only	O
-these	O
-backbone	O
-atom	O
-positions	O
-,	O
-suggesting	O
-that	O
-a	O
-more	O
-complex	O
-series	O
-of	O
-allosteric	O
-events	O
-may	O
-occur	O
-.	O
-	O
-Indeed	O
-,	O
-we	O
-noted	O
-interesting	O
-differences	O
-in	O
-the	O
-side	O
-chains	O
-of	O
-Met22	O
-,	O
-Phe25	O
-and	O
-Arg43	O
-,	O
-which	O
-in	O
-monomer	O
-B	O
-are	O
-used	O
-to	O
-contact	O
-the	O
-ligand	O
-while	O
-in	O
-monomer	O
-A	O
-they	O
-partially	O
-occupied	O
-the	O
-pocket	O
-and	O
-collectively	O
-reduced	O
-its	O
-volume	O
-significantly	O
-.	O
-	O
-Specifically	O
-,	O
-upon	O
-analysis	O
-with	O
-the	O
-CASTp	O
-software	O
-,	O
-the	O
-pocket	O
-in	O
-chain	O
-B	O
-containing	O
-the	O
-4	O
--	O
-HPA	O
-exhibited	O
-a	O
-total	O
-volume	O
-of	O
-approximately	O
-370	O
-Å3	O
-,	O
-while	O
-the	O
-pocket	O
-in	O
-chain	O
-A	O
-was	O
-occupied	O
-by	O
-these	O
-three	O
-side	O
-chains	O
-that	O
-adopted	O
-‘	O
-inward	O
-’	O
-positions	O
-and	O
-thereby	O
-divided	O
-the	O
-space	O
-into	O
-a	O
-few	O
-much	O
-smaller	O
-pockets	O
-,	O
-each	O
-with	O
-volume	O
-<	O
-50	O
-Å3	O
-,	O
-evidently	O
-rendering	O
-chain	O
-A	O
-unfavorable	O
-for	O
-ligand	O
-binding	O
-.	O
-	O
-Most	O
-notably	O
-,	O
-atomic	O
-clashes	O
-between	O
-the	O
-ligand	O
-and	O
-the	O
-side	O
-chains	O
-of	O
-MetA22	O
-,	O
-PheA25	O
-and	O
-ArgA43	O
-would	O
-occur	O
-if	O
-4	O
--	O
-HPA	O
-were	O
-present	O
-in	O
-the	O
-monomer	O
-A	O
-pocket	O
-(	O
-Fig	O
-5B	O
-).	O
-	O
-Subsequently	O
-,	O
-analyses	O
-of	O
-the	O
-pockets	O
-in	O
-apo	O
--	O
-NadR	O
-revealed	O
-that	O
-in	O
-the	O
-absence	O
-of	O
-ligand	O
-the	O
-long	O
-Arg43	O
-side	O
-chain	O
-was	O
-always	O
-in	O
-the	O
-open	O
-‘	O
-outward	O
-’	O
-position	O
-compatible	O
-with	O
-binding	O
-to	O
-the	O
-4	O
--	O
-HPA	O
-carboxylate	O
-group	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-apo	O
--	O
-form	O
-Met22	O
-and	O
-Phe25	O
-residues	O
-were	O
-still	O
-encroaching	O
-the	O
-spaces	O
-of	O
-the	O
-4	O
--	O
-hydroxyl	O
-group	O
-and	O
-the	O
-phenyl	O
-ring	O
-of	O
-the	O
-ligand	O
-,	O
-respectively	O
-(	O
-Fig	O
-5C	O
-).	O
-	O
-The	O
-‘	O
-outward	O
-’	O
-position	O
-of	O
-Arg43	O
-generated	O
-an	O
-open	O
-apo	O
--	O
-form	O
-pocket	O
-with	O
-volume	O
-approximately	O
-380Å3	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-these	O
-observations	O
-suggest	O
-that	O
-Arg43	O
-is	O
-a	O
-major	O
-determinant	O
-of	O
-ligand	O
-binding	O
-,	O
-and	O
-that	O
-its	O
-‘	O
-inward	O
-’	O
-position	O
-inhibits	O
-the	O
-binding	O
-of	O
-4	O
--	O
-HPA	O
-to	O
-the	O
-empty	O
-pocket	O
-of	O
-holo	O
--	O
-NadR	O
-.	O
-	O
-Structural	O
-differences	O
-of	O
-NadR	O
-in	O
-ligand	O
--	O
-bound	O
-or	O
-free	O
-forms	O
-.	O
-	O
-(	O
-A	O
-)	O
-Aligned	O
-monomers	O
-of	O
-holo	O
--	O
-NadR	O
-(	O
-chain	O
-A	O
-:	O
-green	O
-;	O
-chain	O
-B	O
-:	O
-blue	O
-),	O
-reveal	O
-major	O
-overall	O
-differences	O
-by	O
-the	O
-shift	O
-of	O
-helix	O
-α6	O
-.	O
-(	O
-B	O
-)	O
-Comparison	O
-of	O
-the	O
-two	O
-binding	O
-pockets	O
-in	O
-holo	O
--	O
-NadR	O
-shows	O
-that	O
-in	O
-the	O
-ligand	O
--	O
-free	O
-monomer	O
-A	O
-(	O
-green	O
-)	O
-residues	O
-Met22	O
-,	O
-Phe25	O
-and	O
-Arg43	O
-adopt	O
-‘	O
-inward	O
-’	O
-positions	O
-(	O
-highlighted	O
-by	O
-arrows	O
-)	O
-compared	O
-to	O
-the	O
-ligand	O
--	O
-occupied	O
-pocket	O
-(	O
-blue	O
-residues	O
-);	O
-these	O
-‘	O
-inward	O
-’	O
-conformations	O
-appear	O
-unfavorable	O
-for	O
-binding	O
-of	O
-4	O
--	O
-HPA	O
-due	O
-to	O
-clashes	O
-with	O
-the	O
-4	O
--	O
-hydroxyl	O
-group	O
-,	O
-the	O
-phenyl	O
-ring	O
-and	O
-the	O
-carboxylate	O
-group	O
-,	O
-respectively	O
-.	O
-	O
-In	O
-these	O
-crystals	O
-,	O
-the	O
-ArgA43	O
-side	O
-chain	O
-showed	O
-two	O
-alternate	O
-conformations	O
-,	O
-modelled	O
-with	O
-50	O
-%	O
-occupancy	O
-in	O
-each	O
-state	O
-,	O
-as	O
-indicated	O
-by	O
-the	O
-two	O
-‘	O
-mirrored	O
-’	O
-arrows	O
-.	O
-	O
-The	O
-inner	O
-conformer	O
-is	O
-the	O
-one	O
-that	O
-would	O
-display	O
-major	O
-clashes	O
-if	O
-4	O
--	O
-HPA	O
-were	O
-present	O
-.	O
-(	O
-C	O
-)	O
-Comparison	O
-of	O
-the	O
-empty	O
-pocket	O
-from	O
-holo	O
--	O
-NadR	O
-(	O
-green	O
-residues	O
-)	O
-with	O
-the	O
-four	O
-empty	O
-pockets	O
-of	O
-apo	O
--	O
-NadR	O
-(	O
-grey	O
-residues	O
-),	O
-shows	O
-that	O
-in	O
-the	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-the	O
-Arg43	O
-side	O
-chain	O
-is	O
-always	O
-observed	O
-in	O
-the	O
-‘	O
-outward	O
-’	O
-conformation	O
-.	O
-	O
-Finally	O
-,	O
-we	O
-applied	O
-15N	O
-heteronuclear	O
-solution	O
-NMR	O
-spectroscopy	O
-to	O
-examine	O
-the	O
-interaction	O
-of	O
-4	O
--	O
-HPA	O
-with	O
-apo	O
-NadR	O
-.	O
-We	O
-collected	O
-NMR	O
-spectra	O
-on	O
-NadR	O
-in	O
-the	O
-presence	O
-and	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-The	O
-1H	O
--	O
-15N	O
-TROSY	O
--	O
-HSQC	O
-spectrum	O
-of	O
-apo	O
--	O
-NadR	O
-,	O
-acquired	O
-at	O
-25	O
-°	O
-C	O
-,	O
-displayed	O
-approximately	O
-140	O
-distinct	O
-peaks	O
-(	O
-Fig	O
-6A	O
-),	O
-most	O
-of	O
-which	O
-correspond	O
-to	O
-backbone	O
-amide	O
-N	O
--	O
-H	O
-groups	O
-.	O
-	O
-The	O
-broad	O
-spectral	O
-dispersion	O
-and	O
-the	O
-number	O
-of	O
-peaks	O
-observed	O
-,	O
-which	O
-is	O
-close	O
-to	O
-the	O
-number	O
-of	O
-expected	O
-backbone	O
-amide	O
-N	O
--	O
-H	O
-groups	O
-for	O
-this	O
-polypeptide	O
-,	O
-confirmed	O
-that	O
-apo	O
--	O
-NadR	O
-is	O
-well	O
--	O
-folded	O
-under	O
-these	O
-conditions	O
-and	O
-exhibits	O
-one	O
-conformation	O
-appreciable	O
-on	O
-the	O
-NMR	O
-timescale	O
-,	O
-i	O
-.	O
-e	O
-.	O
-in	O
-the	O
-NMR	O
-experiments	O
-at	O
-25	O
-°	O
-C	O
-,	O
-two	O
-or	O
-more	O
-distinct	O
-conformations	O
-of	O
-apo	O
--	O
-NadR	O
-monomers	O
-were	O
-not	O
-readily	O
-apparent	O
-.	O
-	O
-Upon	O
-the	O
-addition	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-over	O
-45	O
-peaks	O
-showed	O
-chemical	O
-shift	O
-perturbations	O
-,	O
-i	O
-.	O
-e	O
-.	O
-changed	O
-position	O
-in	O
-the	O
-spectrum	O
-or	O
-disappeared	O
-,	O
-while	O
-the	O
-remaining	O
-peaks	O
-remained	O
-unchanged	O
-.	O
-	O
-This	O
-observation	O
-showed	O
-that	O
-4	O
--	O
-HPA	O
-was	O
-able	O
-to	O
-bind	O
-NadR	O
-and	O
-induce	O
-notable	O
-changes	O
-in	O
-specific	O
-regions	O
-of	O
-the	O
-protein	O
-.	O
-	O
-NMR	O
-spectra	O
-of	O
-NadR	O
-in	O
-the	O
-presence	O
-and	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-.	O
-	O
-(	O
-A	O
-)	O
-Superposition	O
-of	O
-two	O
-1H	O
--	O
-15N	O
-TROSY	O
--	O
-HSQC	O
-spectra	O
-recorded	O
-at	O
-25	O
-°	O
-C	O
-on	O
-apo	O
--	O
-NadR	O
-(	O
-cyan	O
-)	O
-and	O
-on	O
-NadR	O
-in	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-(	O
-red	O
-).	O
-	O
-(	O
-B	O
-,	O
-C	O
-)	O
-Overlay	O
-of	O
-selected	O
-regions	O
-of	O
-the	O
-1H	O
--	O
-15N	O
-TROSY	O
--	O
-HSQC	O
-spectra	O
-acquired	O
-at	O
-25	O
-°	O
-C	O
-of	O
-apo	O
--	O
-NadR	O
-(	O
-cyan	O
-)	O
-and	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-(	O
-red	O
-)	O
-superimposed	O
-with	O
-the	O
-spectra	O
-acquired	O
-at	O
-10	O
-°	O
-C	O
-of	O
-apo	O
--	O
-NadR	O
-(	O
-blue	O
-)	O
-and	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-(	O
-green	O
-).	O
-	O
-The	O
-spectra	O
-acquired	O
-at	O
-10	O
-°	O
-C	O
-are	O
-excluded	O
-from	O
-panel	O
-A	O
-for	O
-simplicity	O
-.	O
-	O
-However	O
-,	O
-in	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-the	O
-1H	O
--	O
-15N	O
-TROSY	O
--	O
-HSQC	O
-spectrum	O
-of	O
-NadR	O
-displayed	O
-approximately	O
-140	O
-peaks	O
-,	O
-as	O
-for	O
-apo	O
--	O
-NadR	O
-,	O
-i	O
-.	O
-e	O
-.	O
-two	O
-distinct	O
-stable	O
-conformations	O
-(	O
-that	O
-might	O
-have	O
-potentially	O
-revealed	O
-the	O
-molecular	O
-asymmetry	O
-observed	O
-crystallographically	O
-)	O
-were	O
-not	O
-notable	O
-.	O
-	O
-Considering	O
-the	O
-small	O
-size	O
-,	O
-fast	O
-diffusion	O
-and	O
-relatively	O
-low	O
-binding	O
-affinity	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-it	O
-would	O
-not	O
-be	O
-surprising	O
-if	O
-the	O
-ligand	O
-associates	O
-and	O
-dissociates	O
-rapidly	O
-on	O
-the	O
-NMR	O
-time	O
-scale	O
-,	O
-resulting	O
-in	O
-only	O
-one	O
-set	O
-of	O
-peaks	O
-whose	O
-chemical	O
-shifts	O
-represent	O
-the	O
-average	O
-environment	O
-of	O
-the	O
-bound	O
-and	O
-unbound	O
-states	O
-.	O
-	O
-Interestingly	O
-,	O
-by	O
-cooling	O
-the	O
-samples	O
-to	O
-10	O
-°	O
-C	O
-,	O
-we	O
-observed	O
-that	O
-a	O
-number	O
-of	O
-those	O
-peaks	O
-strongly	O
-affected	O
-by	O
-4	O
--	O
-HPA	O
-(	O
-and	O
-therefore	O
-likely	O
-to	O
-be	O
-in	O
-the	O
-ligand	O
--	O
-binding	O
-site	O
-)	O
-demonstrated	O
-evidence	O
-of	O
-peak	O
-splitting	O
-,	O
-i	O
-.	O
-e	O
-.	O
-a	O
-tendency	O
-to	O
-become	O
-two	O
-distinct	O
-peaks	O
-rather	O
-than	O
-one	O
-single	O
-peak	O
-(	O
-Fig	O
-6B	O
-and	O
-6C	O
-).	O
-	O
-These	O
-doubled	O
-peaks	O
-may	O
-therefore	O
-reveal	O
-that	O
-the	O
-cooler	O
-temperature	O
-partially	O
-trapped	O
-the	O
-existence	O
-in	O
-solution	O
-of	O
-two	O
-distinct	O
-states	O
-,	O
-in	O
-presence	O
-or	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-with	O
-minor	O
-conformational	O
-differences	O
-occurring	O
-at	O
-least	O
-in	O
-proximity	O
-to	O
-the	O
-binding	O
-pocket	O
-.	O
-	O
-Although	O
-more	O
-comprehensive	O
-NMR	O
-experiments	O
-and	O
-full	O
-chemical	O
-shift	O
-assignment	O
-of	O
-the	O
-spectra	O
-would	O
-be	O
-required	O
-to	O
-precisely	O
-define	O
-this	O
-multi	O
--	O
-state	O
-behavior	O
-,	O
-the	O
-NMR	O
-data	O
-clearly	O
-demonstrate	O
-that	O
-NadR	O
-exhibits	O
-conformational	O
-flexibility	O
-which	O
-is	O
-modulated	O
-by	O
-4	O
--	O
-HPA	O
-in	O
-solution	O
-.	O
-	O
-Apo	O
--	O
-NadR	O
-structures	O
-reveal	O
-intrinsic	O
-conformational	O
-flexibility	O
-	O
-The	O
-apo	O
--	O
-NadR	O
-crystal	O
-structure	O
-contained	O
-two	O
-homodimers	O
-in	O
-the	O
-asymmetric	O
-unit	O
-(	O
-chains	O
-A	O
-+	O
-B	O
-and	O
-chains	O
-C	O
-+	O
-D	O
-).	O
-	O
-Upon	O
-overall	O
-structural	O
-superposition	O
-,	O
-these	O
-dimers	O
-revealed	O
-a	O
-few	O
-minor	O
-differences	O
-in	O
-the	O
-α6	O
-helix	O
-(	O
-a	O
-major	O
-component	O
-of	O
-the	O
-dimer	O
-interface	O
-)	O
-and	O
-the	O
-helices	O
-α4	O
--	O
-α5	O
-(	O
-the	O
-DNA	O
-binding	O
-region	O
-),	O
-and	O
-an	O
-rmsd	O
-of	O
-1	O
-.	O
-55Å	O
-(	O
-Fig	O
-7A	O
-).	O
-	O
-Similarly	O
-,	O
-the	O
-entire	O
-holo	O
--	O
-homodimer	O
-could	O
-be	O
-closely	O
-superposed	O
-onto	O
-each	O
-of	O
-the	O
-apo	O
--	O
-homodimers	O
-,	O
-showing	O
-rmsd	O
-values	O
-of	O
-1	O
-.	O
-29Å	O
-and	O
-1	O
-.	O
-31Å	O
-,	O
-and	O
-with	O
-more	O
-notable	O
-differences	O
-in	O
-the	O
-α6	O
-helix	O
-positions	O
-(	O
-Fig	O
-7B	O
-).	O
-	O
-The	O
-slightly	O
-larger	O
-rmsd	O
-between	O
-the	O
-two	O
-apo	O
--	O
-homodimers	O
-,	O
-rather	O
-than	O
-between	O
-apo	O
--	O
-and	O
-holo	O
--	O
-homodimers	O
-,	O
-further	O
-indicate	O
-that	O
-apo	O
--	O
-NadR	O
-possesses	O
-a	O
-notable	O
-degree	O
-of	O
-intrinsic	O
-conformational	O
-flexibility	O
-.	O
-	O
-Overall	O
-apo	O
--	O
-and	O
-holo	O
--	O
-NadR	O
-structures	O
-are	O
-similar	O
-.	O
-	O
-(	O
-A	O
-)	O
-Pairwise	O
-alignment	O
-of	O
-the	O
-two	O
-distinct	O
-apo	O
--	O
-NadR	O
-homodimers	O
-(	O
-AB	O
-and	O
-CD	O
-)	O
-present	O
-in	O
-the	O
-apo	O
--	O
-NadR	O
-crystals	O
-.	O
-(	O
-B	O
-)	O
-Alignment	O
-of	O
-the	O
-holo	O
--	O
-NadR	O
-homodimer	O
-(	O
-green	O
-and	O
-blue	O
-chains	O
-)	O
-onto	O
-the	O
-apo	O
--	O
-NadR	O
-homodimers	O
-.	O
-	O
-Here	O
-,	O
-larger	O
-differences	O
-are	O
-observed	O
-in	O
-the	O
-α6	O
-helices	O
-(	O
-top	O
-).	O
-	O
-4	O
--	O
-HPA	O
-stabilizes	O
-concerted	O
-conformational	O
-changes	O
-in	O
-NadR	O
-that	O
-prevent	O
-DNA	O
--	O
-binding	O
-	O
-To	O
-further	O
-investigate	O
-the	O
-conformational	O
-rearrangements	O
-of	O
-NadR	O
-,	O
-we	O
-performed	O
-local	O
-structural	O
-alignments	O
-using	O
-only	O
-a	O
-subset	O
-of	O
-residues	O
-in	O
-the	O
-DNA	O
--	O
-binding	O
-helix	O
-(	O
-α4	O
-).	O
-	O
-By	O
-selecting	O
-and	O
-aligning	O
-residues	O
-Arg64	O
--	O
-Ala77	O
-of	O
-one	O
-α4	O
-helix	O
-per	O
-dimer	O
-,	O
-superposition	O
-of	O
-the	O
-holo	O
--	O
-homodimer	O
-onto	O
-the	O
-two	O
-apo	O
--	O
-homodimers	O
-revealed	O
-differences	O
-in	O
-the	O
-monomer	O
-conformations	O
-of	O
-each	O
-structure	O
-.	O
-	O
-While	O
-one	O
-monomer	O
-from	O
-each	O
-structure	O
-was	O
-closely	O
-superimposable	O
-(	O
-Fig	O
-8A	O
-,	O
-left	O
-side	O
-),	O
-the	O
-second	O
-monomer	O
-displayed	O
-quite	O
-large	O
-differences	O
-(	O
-Fig	O
-8A	O
-,	O
-right	O
-side	O
-).	O
-	O
-Most	O
-notably	O
-,	O
-the	O
-position	O
-of	O
-the	O
-DNA	O
--	O
-binding	O
-helix	O
-α4	O
-shifted	O
-by	O
-as	O
-much	O
-as	O
-6	O
-Å	O
-(	O
-Fig	O
-8B	O
-).	O
-	O
-Accordingly	O
-,	O
-helix	O
-α4	O
-was	O
-also	O
-found	O
-to	O
-be	O
-one	O
-of	O
-the	O
-most	O
-dynamic	O
-regions	O
-in	O
-previous	O
-HDX	O
--	O
-MS	O
-analyses	O
-of	O
-apo	O
--	O
-NadR	O
-in	O
-solution	O
-.	O
-	O
-Structural	O
-comparisons	O
-of	O
-NadR	O
-and	O
-modelling	O
-of	O
-interactions	O
-with	O
-DNA	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-holo	O
--	O
-homodimer	O
-structure	O
-is	O
-shown	O
-as	O
-green	O
-and	O
-blue	O
-cartoons	O
-,	O
-for	O
-chain	O
-A	O
-and	O
-B	O
-,	O
-respectively	O
-,	O
-while	O
-the	O
-two	O
-homodimers	O
-of	O
-apo	O
--	O
-NadR	O
-are	O
-both	O
-cyan	O
-and	O
-pale	O
-blue	O
-for	O
-chains	O
-A	O
-/	O
-C	O
-and	O
-B	O
-/	O
-D	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-three	O
-homodimers	O
-(	O
-chains	O
-AB	O
-holo	O
-,	O
-AB	O
-apo	O
-,	O
-and	O
-CD	O
-apo	O
-)	O
-were	O
-overlaid	O
-by	O
-structural	O
-alignment	O
-exclusively	O
-of	O
-all	O
-heavy	O
-atoms	O
-in	O
-residues	O
-R64	O
--	O
-A77	O
-(	O
-shown	O
-in	O
-red	O
-,	O
-with	O
-side	O
-chain	O
-sticks	O
-)	O
-of	O
-chains	O
-A	O
-holo	O
-,	O
-A	O
-apo	O
-,	O
-and	O
-C	O
-apo	O
-,	O
-belonging	O
-to	O
-helix	O
-α4	O
-(	O
-left	O
-).	O
-	O
-The	O
-α4	O
-helices	O
-aligned	O
-closely	O
-,	O
-Cα	O
-rmsd	O
-0	O
-.	O
-2Å	O
-for	O
-14	O
-residues	O
-.	O
-	O
-(	O
-B	O
-)	O
-The	O
-relative	O
-positions	O
-of	O
-the	O
-α4	O
-helices	O
-of	O
-the	O
-4	O
--	O
-HPA	O
--	O
-bound	O
-holo	O
-homodimer	O
-chain	O
-B	O
-(	O
-blue	O
-),	O
-and	O
-of	O
-apo	O
-homodimers	O
-AB	O
-and	O
-CD	O
-(	O
-showing	O
-chains	O
-B	O
-and	O
-D	O
-)	O
-in	O
-pale	O
-blue	O
-.	O
-	O
-Dashes	O
-indicate	O
-the	O
-Ala77	O
-Cα	O
-atoms	O
-,	O
-in	O
-the	O
-most	O
-highly	O
-shifted	O
-region	O
-of	O
-the	O
-‘	O
-non	O
--	O
-fixed	O
-’	O
-α4	O
-helix	O
-.	O
-	O
-(	O
-C	O
-)	O
-The	O
-double	O
--	O
-stranded	O
-DNA	O
-molecule	O
-(	O
-grey	O
-cartoon	O
-)	O
-from	O
-the	O
-OhrR	O
--	O
-ohrA	O
-complex	O
-is	O
-shown	O
-after	O
-superposition	O
-with	O
-NadR	O
-,	O
-to	O
-highlight	O
-the	O
-expected	O
-positions	O
-of	O
-the	O
-NadR	O
-α4	O
-helices	O
-in	O
-the	O
-DNA	O
-major	O
-grooves	O
-.	O
-	O
-For	O
-clarity	O
-,	O
-only	O
-the	O
-α4	O
-helices	O
-are	O
-shown	O
-in	O
-panels	O
-(	O
-B	O
-)	O
-and	O
-(	O
-C	O
-).	O
-(	O
-D	O
-)	O
-Upon	O
-comparison	O
-with	O
-the	O
-experimentally	O
--	O
-determined	O
-OhrR	O
-:	O
-ohrA	O
-structure	O
-(	O
-grey	O
-),	O
-the	O
-α4	O
-helix	O
-of	O
-holo	O
--	O
-NadR	O
-(	O
-blue	O
-)	O
-is	O
-shifted	O
-~	O
-8Å	O
-out	O
-of	O
-the	O
-major	O
-groove	O
-.	O
-	O
-However	O
-,	O
-structural	O
-comparisons	O
-revealed	O
-that	O
-the	O
-shift	O
-of	O
-holo	O
--	O
-NadR	O
-helix	O
-α4	O
-induced	O
-by	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-was	O
-also	O
-accompanied	O
-by	O
-several	O
-changes	O
-at	O
-the	O
-holo	O
-dimer	O
-interface	O
-,	O
-while	O
-such	O
-extensive	O
-structural	O
-differences	O
-were	O
-not	O
-observed	O
-in	O
-the	O
-apo	O
-dimer	O
-interfaces	O
-,	O
-particularly	O
-notable	O
-when	O
-comparing	O
-the	O
-α6	O
-helices	O
-(	O
-S3	O
-Fig	O
-).	O
-	O
-In	O
-summary	O
-,	O
-compared	O
-to	O
-ligand	O
--	O
-stabilized	O
-holo	O
--	O
-NadR	O
-,	O
-apo	O
--	O
-NadR	O
-displayed	O
-an	O
-intrinsic	O
-flexibility	O
-focused	O
-in	O
-the	O
-DNA	O
--	O
-binding	O
-region	O
-.	O
-	O
-This	O
-was	O
-also	O
-evident	O
-in	O
-the	O
-greater	O
-disorder	O
-(	O
-i	O
-.	O
-e	O
-.	O
-less	O
-well	O
--	O
-defined	O
-electron	O
-density	O
-)	O
-in	O
-the	O
-β1	O
--	O
-β2	O
-loops	O
-of	O
-the	O
-apo	O
-dimers	O
-(	O
-density	O
-for	O
-16	O
-residues	O
-per	O
-dimer	O
-was	O
-missing	O
-)	O
-compared	O
-to	O
-the	O
-holo	O
-dimer	O
-(	O
-density	O
-for	O
-only	O
-3	O
-residues	O
-was	O
-missing	O
-).	O
-	O
-In	O
-holo	O
--	O
-NadR	O
-,	O
-the	O
-distance	O
-separating	O
-the	O
-two	O
-DNA	O
--	O
-binding	O
-α4	O
-helices	O
-was	O
-32	O
-Å	O
-,	O
-while	O
-in	O
-apo	O
--	O
-NadR	O
-it	O
-was	O
-29	O
-Å	O
-for	O
-homodimer	O
-AB	O
-,	O
-and	O
-34	O
-Å	O
-for	O
-homodimer	O
-CD	O
-(	O
-Fig	O
-8C	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-apo	O
--	O
-homodimer	O
-AB	O
-presented	O
-the	O
-DNA	O
--	O
-binding	O
-helices	O
-in	O
-a	O
-conformation	O
-similar	O
-to	O
-that	O
-observed	O
-in	O
-the	O
-protein	O
-:	O
-DNA	O
-complex	O
-of	O
-OhrR	O
-:	O
-ohrA	O
-from	O
-Bacillus	O
-subtilis	O
-(	O
-Fig	O
-8C	O
-).	O
-	O
-Interestingly	O
-,	O
-OhrR	O
-contacts	O
-ohrA	O
-across	O
-22	O
-base	O
-pairs	O
-(	O
-bp	O
-),	O
-and	O
-similarly	O
-the	O
-main	O
-NadR	O
-target	O
-sites	O
-identified	O
-in	O
-the	O
-nadA	O
-promoter	O
-(	O
-the	O
-operators	O
-Op	O
-I	O
-and	O
-Op	O
-II	O
-)	O
-both	O
-span	O
-22	O
-bp	O
-.	O
-	O
-Pairwise	O
-superpositions	O
-showed	O
-that	O
-the	O
-NadR	O
-apo	O
--	O
-homodimer	O
-AB	O
-was	O
-the	O
-most	O
-similar	O
-to	O
-OhrR	O
-(	O
-rmsd	O
-2	O
-.	O
-6	O
-Å	O
-),	O
-while	O
-the	O
-holo	O
--	O
-homodimer	O
-was	O
-the	O
-most	O
-divergent	O
-(	O
-rmsd	O
-3	O
-.	O
-3	O
-Å	O
-)	O
-(	O
-Fig	O
-8C	O
-).	O
-	O
-Assuming	O
-the	O
-same	O
-DNA	O
--	O
-binding	O
-mechanism	O
-is	O
-used	O
-by	O
-OhrR	O
-and	O
-NadR	O
-,	O
-the	O
-apo	O
--	O
-homodimer	O
-AB	O
-seems	O
-ideally	O
-pre	O
--	O
-configured	O
-for	O
-DNA	O
-binding	O
-,	O
-while	O
-4	O
--	O
-HPA	O
-appeared	O
-to	O
-stabilize	O
-holo	O
--	O
-NadR	O
-in	O
-a	O
-conformation	O
-poorly	O
-suited	O
-for	O
-DNA	O
-binding	O
-.	O
-	O
-Specifically	O
-,	O
-in	O
-addition	O
-to	O
-the	O
-different	O
-inter	O
--	O
-helical	O
-translational	O
-distances	O
-,	O
-the	O
-α4	O
-helices	O
-in	O
-the	O
-holo	O
--	O
-NadR	O
-homodimer	O
-were	O
-also	O
-reoriented	O
-,	O
-resulting	O
-in	O
-movement	O
-of	O
-α4	O
-out	O
-of	O
-the	O
-major	O
-groove	O
-,	O
-by	O
-up	O
-to	O
-8Å	O
-,	O
-and	O
-presumably	O
-preventing	O
-efficient	O
-DNA	O
-binding	O
-in	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-(	O
-Fig	O
-8D	O
-).	O
-	O
-When	O
-aligned	O
-with	O
-OhrR	O
-,	O
-the	O
-apo	O
--	O
-homodimer	O
-CD	O
-presented	O
-yet	O
-another	O
-different	O
-intermediate	O
-conformation	O
-(	O
-rmsd	O
-2	O
-.	O
-9Å	O
-),	O
-apparently	O
-not	O
-ideally	O
-pre	O
--	O
-configured	O
-for	O
-DNA	O
-binding	O
-,	O
-but	O
-which	O
-in	O
-solution	O
-can	O
-presumably	O
-readily	O
-adopt	O
-the	O
-AB	O
-conformation	O
-due	O
-to	O
-the	O
-intrinsic	O
-flexibility	O
-described	O
-above	O
-.	O
-	O
-NadR	O
-residues	O
-His7	O
-,	O
-Ser9	O
-,	O
-Asn11	O
-and	O
-Phe25	O
-are	O
-essential	O
-for	O
-regulation	O
-of	O
-NadA	O
-expression	O
-in	O
-vivo	O
-	O
-While	O
-previous	O
-studies	O
-had	O
-correctly	O
-suggested	O
-the	O
-involvement	O
-of	O
-several	O
-NadR	O
-residues	O
-in	O
-ligand	O
-binding	O
-,	O
-the	O
-crystal	O
-structures	O
-presented	O
-here	O
-revealed	O
-additional	O
-residues	O
-with	O
-previously	O
-unknown	O
-roles	O
-in	O
-dimerization	O
-and	O
-/	O
-or	O
-binding	O
-to	O
-4	O
--	O
-HPA	O
-.	O
-	O
-To	O
-explore	O
-the	O
-functional	O
-involvement	O
-of	O
-these	O
-residues	O
-,	O
-we	O
-characterized	O
-the	O
-behavior	O
-of	O
-four	O
-new	O
-NadR	O
-mutants	O
-(	O
-H7A	B-mutant
-,	O
-S9A	B-mutant
-,	O
-N11A	B-mutant
-and	O
-F25A	B-mutant
-)	O
-in	O
-an	O
-in	O
-vivo	O
-assay	O
-using	O
-the	O
-previously	O
-described	O
-MC58	B-mutant
--	I-mutant
-Δ1843	I-mutant
-nadR	O
--	O
-null	O
-mutant	O
-strain	O
-,	O
-which	O
-was	O
-complemented	O
-either	O
-by	O
-wild	O
--	O
-type	O
-nadR	O
-or	O
-by	O
-the	O
-nadR	O
-mutants	O
-.	O
-	O
-NadA	O
-protein	O
-abundance	O
-levels	O
-were	O
-assessed	O
-by	O
-Western	O
-blotting	O
-to	O
-evaluate	O
-the	O
-ability	O
-of	O
-the	O
-NadR	O
-mutants	O
-to	O
-repress	O
-the	O
-nadA	O
-promoter	O
-,	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-.	O
-	O
-The	O
-nadR	O
-H7A	B-mutant
-,	O
-S9A	B-mutant
-and	O
-F25A	B-mutant
-complemented	O
-strains	O
-showed	O
-hyper	O
--	O
-repression	O
-of	O
-nadA	O
-expression	O
-in	O
-vivo	O
-,	O
-i	O
-.	O
-e	O
-.	O
-these	O
-mutants	O
-repressed	O
-nadA	O
-more	O
-efficiently	O
-than	O
-the	O
-NadR	O
-WT	O
-protein	O
-,	O
-either	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-while	O
-complementation	O
-with	O
-wild	O
--	O
-type	O
-nadR	O
-resulted	O
-in	O
-high	O
-production	O
-of	O
-NadA	O
-only	O
-in	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-(	O
-Fig	O
-9	O
-).	O
-	O
-Interestingly	O
-,	O
-and	O
-on	O
-the	O
-contrary	O
-,	O
-the	O
-nadR	O
-N11A	B-mutant
-complemented	O
-strain	O
-showed	O
-hypo	O
--	O
-repression	O
-(	O
-i	O
-.	O
-e	O
-.	O
-exhibited	O
-high	O
-expression	O
-of	O
-nadA	O
-both	O
-in	O
-absence	O
-and	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-).	O
-	O
-This	O
-mutagenesis	O
-data	O
-revealed	O
-that	O
-NadR	O
-residues	O
-His7	O
-,	O
-Ser9	O
-,	O
-Asn11	O
-and	O
-Phe25	O
-play	O
-key	O
-roles	O
-in	O
-the	O
-ligand	O
--	O
-mediated	O
-regulation	O
-of	O
-NadR	O
-;	O
-they	O
-are	O
-each	O
-involved	O
-in	O
-the	O
-controlled	O
-de	O
--	O
-repression	O
-of	O
-the	O
-nadA	O
-promoter	O
-and	O
-synthesis	O
-of	O
-NadA	O
-in	O
-response	O
-to	O
-4	O
--	O
-HPA	O
-in	O
-vivo	O
-.	O
-	O
-Structure	O
--	O
-based	O
-point	O
-mutations	O
-shed	O
-light	O
-on	O
-ligand	O
--	O
-induced	O
-regulation	O
-of	O
-NadR	O
-.	O
-	O
-Western	O
-blot	O
-analyses	O
-of	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-strain	O
-(	O
-lanes	O
-1	O
-–	O
-2	O
-)	O
-or	O
-isogenic	O
-nadR	O
-knockout	O
-strains	O
-(	O
-ΔNadR	B-mutant
-)	O
-complemented	O
-to	O
-express	O
-the	O
-indicated	O
-NadR	O
-WT	O
-or	O
-mutant	O
-proteins	O
-(	O
-lanes	O
-3	O
-–	O
-12	O
-)	O
-or	O
-not	O
-complemented	O
-(	O
-lanes	O
-13	O
-–	O
-14	O
-),	O
-grown	O
-in	O
-the	O
-presence	O
-(	O
-even	O
-lanes	O
-)	O
-or	O
-absence	O
-(	O
-odd	O
-lanes	O
-)	O
-of	O
-5mM	O
-4	O
--	O
-HPA	O
-,	O
-showing	O
-NadA	O
-and	O
-NadR	O
-expression	O
-.	O
-	O
-Complementation	O
-of	O
-ΔNadR	B-mutant
-with	O
-WT	O
-NadR	O
-enables	O
-induction	O
-of	O
-nadA	O
-expression	O
-by	O
-4	O
--	O
-HPA	O
-.	O
-	O
-The	O
-H7A	B-mutant
-,	O
-S9A	B-mutant
-and	O
-F25A	B-mutant
-mutants	O
-efficiently	O
-repress	O
-nadA	O
-expression	O
-but	O
-are	O
-less	O
-ligand	O
--	O
-responsive	O
-than	O
-WT	O
-NadR	O
-.	O
-The	O
-N11A	B-mutant
-mutant	O
-does	O
-not	O
-efficiently	O
-repress	O
-nadA	O
-expression	O
-either	O
-in	O
-presence	O
-or	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-.	O
-(	O
-The	O
-protein	O
-abundance	O
-levels	O
-of	O
-the	O
-meningococcal	O
-factor	O
-H	O
-binding	O
-protein	O
-(	O
-fHbp	O
-)	O
-were	O
-used	O
-as	O
-a	O
-gel	O
-loading	O
-control	O
-).	O
-	O
-NadA	O
-is	O
-a	O
-surface	O
--	O
-exposed	O
-meningococcal	O
-protein	O
-contributing	O
-to	O
-pathogenesis	O
-,	O
-and	O
-is	O
-one	O
-of	O
-three	O
-main	O
-antigens	O
-present	O
-in	O
-the	O
-vaccine	O
-Bexsero	O
-.	O
-	O
-A	O
-detailed	O
-understanding	O
-of	O
-the	O
-in	O
-vitro	O
-repression	O
-of	O
-nadA	O
-expression	O
-by	O
-the	O
-transcriptional	O
-regulator	O
-NadR	O
-is	O
-important	O
-,	O
-both	O
-because	O
-it	O
-is	O
-a	O
-relevant	O
-disease	O
--	O
-related	O
-model	O
-of	O
-how	O
-small	O
--	O
-molecule	O
-ligands	O
-can	O
-regulate	O
-MarR	O
-family	O
-proteins	O
-and	O
-thereby	O
-impact	O
-bacterial	O
-virulence	O
-,	O
-and	O
-because	O
-nadA	O
-expression	O
-levels	O
-are	O
-linked	O
-to	O
-the	O
-prediction	O
-of	O
-vaccine	O
-coverage	O
-.	O
-	O
-The	O
-repressive	O
-activity	O
-of	O
-NadR	O
-can	O
-be	O
-relieved	O
-by	O
-hydroxyphenylacetate	O
-(	O
-HPA	O
-)	O
-ligands	O
-,	O
-and	O
-HDX	O
--	O
-MS	O
-studies	O
-previously	O
-indicated	O
-that	O
-4	O
--	O
-HPA	O
-stabilizes	O
-dimeric	O
-NadR	O
-in	O
-a	O
-configuration	O
-incompatible	O
-with	O
-DNA	O
-binding	O
-.	O
-	O
-Despite	O
-these	O
-and	O
-other	O
-studies	O
-,	O
-the	O
-molecular	O
-mechanisms	O
-by	O
-which	O
-ligands	O
-regulate	O
-MarR	O
-family	O
-proteins	O
-are	O
-relatively	O
-poorly	O
-understood	O
-and	O
-likely	O
-differ	O
-depending	O
-on	O
-the	O
-specific	O
-ligand	O
-.	O
-	O
-Given	O
-the	O
-importance	O
-of	O
-NadR	O
--	O
-mediated	O
-regulation	O
-of	O
-NadA	O
-levels	O
-in	O
-the	O
-contexts	O
-of	O
-meningococcal	O
-pathogenesis	O
-,	O
-we	O
-sought	O
-to	O
-characterize	O
-NadR	O
-,	O
-and	O
-its	O
-interaction	O
-with	O
-ligands	O
-,	O
-at	O
-atomic	O
-resolution	O
-.	O
-	O
-Firstly	O
-,	O
-we	O
-confirmed	O
-that	O
-NadR	O
-is	O
-dimeric	O
-in	O
-solution	O
-and	O
-demonstrated	O
-that	O
-it	O
-retains	O
-its	O
-dimeric	O
-state	O
-in	O
-the	O
-presence	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-indicating	O
-that	O
-induction	O
-of	O
-a	O
-monomeric	O
-status	O
-is	O
-not	O
-the	O
-manner	O
-by	O
-which	O
-4	O
--	O
-HPA	O
-regulates	O
-NadR	O
-.	O
-These	O
-observations	O
-were	O
-in	O
-agreement	O
-with	O
-(	O
-i	O
-)	O
-a	O
-previous	O
-study	O
-of	O
-NadR	O
-performed	O
-using	O
-SEC	O
-and	O
-mass	O
-spectrometry	O
-,	O
-and	O
-(	O
-ii	O
-)	O
-crystallographic	O
-studies	O
-showing	O
-that	O
-several	O
-MarR	O
-homologues	O
-are	O
-dimeric	O
-.	O
-	O
-We	O
-also	O
-used	O
-structure	O
--	O
-guided	O
-site	O
--	O
-directed	O
-mutagenesis	O
-to	O
-identify	O
-an	O
-important	O
-conserved	O
-residue	O
-,	O
-Leu130	O
-,	O
-which	O
-stabilizes	O
-the	O
-NadR	O
-dimer	O
-interface	O
-,	O
-knowledge	O
-of	O
-which	O
-may	O
-also	O
-inform	O
-future	O
-studies	O
-to	O
-explore	O
-the	O
-regulatory	O
-mechanisms	O
-of	O
-other	O
-MarR	O
-family	O
-proteins	O
-.	O
-	O
-Secondly	O
-,	O
-we	O
-assessed	O
-the	O
-thermal	O
-stability	O
-and	O
-unfolding	O
-of	O
-NadR	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-ligands	O
-.	O
-	O
-All	O
-DSC	O
-profiles	O
-showed	O
-a	O
-single	O
-peak	O
-,	O
-suggesting	O
-that	O
-a	O
-single	O
-unfolding	O
-event	O
-simultaneously	O
-disrupted	O
-the	O
-dimer	O
-and	O
-the	O
-monomer	O
-.	O
-	O
-HPA	O
-ligands	O
-specifically	O
-increased	O
-the	O
-stability	O
-of	O
-NadR	O
-.	O
-The	O
-largest	O
-effects	O
-were	O
-induced	O
-by	O
-the	O
-naturally	O
--	O
-occurring	O
-compounds	O
-4	O
--	O
-HPA	O
-and	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-,	O
-which	O
-,	O
-in	O
-SPR	O
-assays	O
-,	O
-were	O
-found	O
-to	O
-bind	O
-NadR	O
-with	O
-KD	O
-values	O
-of	O
-1	O
-.	O
-5	O
-mM	O
-and	O
-1	O
-.	O
-1	O
-mM	O
-,	O
-respectively	O
-.	O
-	O
-Although	O
-these	O
-NadR	O
-/	O
-HPA	O
-interactions	O
-appeared	O
-rather	O
-weak	O
-,	O
-their	O
-distinct	O
-affinities	O
-and	O
-specificities	O
-matched	O
-their	O
-in	O
-vitro	O
-effects	O
-and	O
-their	O
-biological	O
-relevance	O
-appears	O
-similar	O
-to	O
-previous	O
-proposals	O
-that	O
-certain	O
-small	O
-molecules	O
-,	O
-including	O
-some	O
-antibiotics	O
-,	O
-in	O
-the	O
-millimolar	O
-concentration	O
-range	O
-may	O
-be	O
-broad	O
-inhibitors	O
-of	O
-MarR	O
-family	O
-proteins	O
-.	O
-	O
-Indeed	O
-,	O
-4	O
--	O
-HPA	O
-is	O
-found	O
-in	O
-human	O
-saliva	O
-and	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-is	O
-produced	O
-during	O
-inflammatory	O
-processes	O
-,	O
-suggesting	O
-that	O
-these	O
-natural	O
-ligands	O
-are	O
-encountered	O
-by	O
-N	O
-.	O
-meningitidis	O
-in	O
-the	O
-mucosa	O
-of	O
-the	O
-oropharynx	O
-during	O
-infections	O
-.	O
-	O
-It	O
-is	O
-also	O
-possible	O
-that	O
-NadR	O
-responds	O
-to	O
-currently	O
-unidentified	O
-HPA	O
-analogues	O
-.	O
-	O
-Indeed	O
-,	O
-in	O
-the	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-complex	O
-there	O
-was	O
-a	O
-water	O
-molecule	O
-close	O
-to	O
-the	O
-carboxylate	O
-group	O
-and	O
-also	O
-a	O
-small	O
-unfilled	O
-tunnel	O
-~	O
-5Å	O
-long	O
-,	O
-both	O
-factors	O
-suggesting	O
-that	O
-alternative	O
-larger	O
-ligands	O
-could	O
-occupy	O
-the	O
-pocket	O
-.	O
-	O
-The	O
-ability	O
-to	O
-respond	O
-to	O
-various	O
-ligands	O
-might	O
-enable	O
-NadR	O
-in	O
-vivo	O
-to	O
-orchestrate	O
-multiple	O
-response	O
-mechanisms	O
-and	O
-modulate	O
-expression	O
-of	O
-genes	O
-other	O
-than	O
-nadA	O
-.	O
-Ultimately	O
-,	O
-confirmation	O
-of	O
-the	O
-relevance	O
-of	O
-each	O
-ligand	O
-will	O
-require	O
-a	O
-deeper	O
-understanding	O
-of	O
-the	O
-available	O
-concentration	O
-in	O
-vivo	O
-in	O
-the	O
-host	O
-niche	O
-during	O
-bacterial	O
-colonization	O
-and	O
-inflammation	O
-.	O
-	O
-Here	O
-,	O
-we	O
-determined	O
-the	O
-first	O
-crystal	O
-structures	O
-of	O
-apo	O
--	O
-NadR	O
-and	O
-holo	O
--	O
-NadR	O
-.	O
-These	O
-experimentally	O
--	O
-determined	O
-structures	O
-enabled	O
-a	O
-new	O
-detailed	O
-characterization	O
-of	O
-the	O
-ligand	O
--	O
-binding	O
-pocket	O
-.	O
-	O
-In	O
-holo	O
--	O
-NadR	O
-,	O
-4	O
--	O
-HPA	O
-interacted	O
-directly	O
-with	O
-at	O
-least	O
-11	O
-polar	O
-and	O
-hydrophobic	O
-residues	O
-.	O
-	O
-Several	O
-,	O
-but	O
-not	O
-all	O
-,	O
-of	O
-these	O
-interactions	O
-were	O
-predicted	O
-previously	O
-by	O
-homology	O
-modelling	O
-combined	O
-with	O
-ligand	O
-docking	O
-in	O
-silico	O
-.	O
-	O
-Subsequently	O
-,	O
-we	O
-established	O
-the	O
-functional	O
-importance	O
-of	O
-His7	O
-,	O
-Ser9	O
-,	O
-Asn11	O
-and	O
-Phe25	O
-in	O
-the	O
-in	O
-vitro	O
-response	O
-of	O
-meningococcus	O
-to	O
-4	O
--	O
-HPA	O
-,	O
-via	O
-site	O
--	O
-directed	O
-mutagenesis	O
-.	O
-	O
-More	O
-unexpectedly	O
-,	O
-the	O
-crystal	O
-structure	O
-revealed	O
-that	O
-only	O
-one	O
-molecule	O
-of	O
-4	O
--	O
-HPA	O
-was	O
-bound	O
-per	O
-NadR	O
-dimer	O
-.	O
-	O
-We	O
-confirmed	O
-this	O
-stoichiometry	O
-in	O
-solution	O
-using	O
-SPR	O
-methods	O
-.	O
-	O
-We	O
-also	O
-used	O
-heteronuclear	O
-NMR	O
-spectroscopy	O
-to	O
-detect	O
-substantial	O
-conformational	O
-changes	O
-of	O
-NadR	O
-occurring	O
-in	O
-solution	O
-upon	O
-addition	O
-of	O
-4	O
--	O
-HPA	O
-.	O
-	O
-Moreover	O
-,	O
-NMR	O
-spectra	O
-at	O
-10	O
-°	O
-C	O
-suggested	O
-the	O
-existence	O
-of	O
-two	O
-distinct	O
-conformations	O
-of	O
-NadR	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-ligand	O
--	O
-binding	O
-pocket	O
-.	O
-	O
-More	O
-powerfully	O
-,	O
-our	O
-unique	O
-crystallographic	O
-observation	O
-of	O
-this	O
-‘	O
-occupied	O
-vs	O
-unoccupied	O
-site	O
-’	O
-asymmetry	O
-in	O
-the	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-interaction	O
-is	O
-,	O
-to	O
-our	O
-knowledge	O
-,	O
-the	O
-first	O
-example	O
-reported	O
-for	O
-a	O
-MarR	O
-family	O
-protein	O
-.	O
-	O
-Structural	O
-analyses	O
-suggested	O
-that	O
-‘	O
-inward	O
-’	O
-side	O
-chain	O
-positions	O
-of	O
-Met22	O
-,	O
-Phe25	O
-and	O
-especially	O
-Arg43	O
-precluded	O
-binding	O
-of	O
-a	O
-second	O
-ligand	O
-molecule	O
-.	O
-	O
-Such	O
-a	O
-mechanism	O
-indicates	O
-negative	O
-cooperativity	O
-,	O
-which	O
-may	O
-enhance	O
-the	O
-ligand	O
--	O
-responsiveness	O
-of	O
-NadR	O
-.	O
-	O
-Comparisons	O
-of	O
-the	O
-NadR	O
-/	O
-4	O
--	O
-HPA	O
-complex	O
-with	O
-available	O
-MarR	O
-family	O
-/	O
-salicylate	O
-complexes	O
-revealed	O
-that	O
-4	O
--	O
-HPA	O
-has	O
-a	O
-previously	O
-unobserved	O
-binding	O
-mode	O
-.	O
-	O
-Briefly	O
-,	O
-in	O
-the	O
-M	O
-.	O
-thermoautotrophicum	O
-MTH313	O
-dimer	O
-,	O
-one	O
-molecule	O
-of	O
-salicylate	O
-binds	O
-in	O
-the	O
-pocket	O
-of	O
-each	O
-monomer	O
-,	O
-though	O
-with	O
-two	O
-rather	O
-different	O
-positions	O
-and	O
-orientations	O
-,	O
-only	O
-one	O
-of	O
-which	O
-(	O
-site	O
--	O
-1	O
-)	O
-is	O
-thought	O
-to	O
-be	O
-biologically	O
-relevant	O
-(	O
-Fig	O
-10A	O
-).	O
-	O
-In	O
-the	O
-S	O
-.	O
-tokodaii	O
-protein	O
-ST1710	O
-,	O
-salicylate	O
-binds	O
-to	O
-the	O
-same	O
-position	O
-in	O
-each	O
-monomer	O
-of	O
-the	O
-dimer	O
-,	O
-in	O
-a	O
-site	O
-equivalent	O
-to	O
-the	O
-putative	O
-biologically	O
-relevant	O
-site	O
-of	O
-MTH313	O
-(	O
-Fig	O
-10B	O
-).	O
-	O
-Unlike	O
-other	O
-MarR	O
-family	O
-proteins	O
-which	O
-revealed	O
-multiple	O
-ligand	O
-binding	O
-interactions	O
-,	O
-we	O
-observed	O
-only	O
-1	O
-molecule	O
-of	O
-4	O
--	O
-HPA	O
-bound	O
-to	O
-NadR	O
-,	O
-suggesting	O
-a	O
-more	O
-specific	O
-and	O
-less	O
-promiscuous	O
-interaction	O
-.	O
-	O
-In	O
-NadR	O
-,	O
-the	O
-single	O
-molecule	O
-of	O
-4	O
--	O
-HPA	O
-binds	O
-in	O
-a	O
-position	O
-distinctly	O
-different	O
-from	O
-the	O
-salicylate	O
-binding	O
-site	O
-:	O
-translated	O
-by	O
->	O
-10	O
-Å	O
-and	O
-with	O
-a	O
-180	O
-°	O
-inverted	O
-orientation	O
-(	O
-Fig	O
-10C	O
-).	O
-	O
-NadR	O
-shows	O
-a	O
-ligand	O
-binding	O
-site	O
-distinct	O
-from	O
-other	O
-MarR	O
-homologues	O
-.	O
-	O
-(	O
-A	O
-)	O
-A	O
-structural	O
-alignment	O
-of	O
-MTH313	O
-chains	O
-A	O
-and	O
-B	O
-shows	O
-that	O
-salicylate	O
-is	O
-bound	O
-in	O
-distinct	O
-locations	O
-in	O
-each	O
-monomer	O
-;	O
-site	O
--	O
-1	O
-(	O
-thought	O
-to	O
-be	O
-the	O
-biologically	O
-relevant	O
-site	O
-)	O
-and	O
-site	O
--	O
-2	O
-differ	O
-by	O
-~	O
-7Å	O
-(	O
-indicated	O
-by	O
-black	O
-dotted	O
-line	O
-)	O
-and	O
-also	O
-by	O
-ligand	O
-orientation	O
-.	O
-	O
-(	O
-B	O
-)	O
-A	O
-structural	O
-alignment	O
-of	O
-MTH313	O
-chain	O
-A	O
-and	O
-ST1710	O
-(	O
-pink	O
-)	O
-(	O
-Cα	O
-rmsd	O
-2	O
-.	O
-3Å	O
-),	O
-shows	O
-that	O
-they	O
-bind	O
-salicylate	O
-in	O
-equivalent	O
-sites	O
-(	O
-differing	O
-by	O
-only	O
-~	O
-3Å	O
-)	O
-and	O
-with	O
-the	O
-same	O
-orientation	O
-.	O
-	O
-(	O
-C	O
-)	O
-Addition	O
-of	O
-holo	O
--	O
-NadR	O
-(	O
-chain	O
-B	O
-,	O
-blue	O
-)	O
-to	O
-the	O
-alignment	O
-reveals	O
-that	O
-bound	O
-4	O
--	O
-HPA	O
-differs	O
-in	O
-position	O
-by	O
->	O
-10	O
-Å	O
-compared	O
-to	O
-salicylate	O
-,	O
-and	O
-adopts	O
-a	O
-novel	O
-orientation	O
-.	O
-	O
-Interestingly	O
-,	O
-a	O
-crystal	O
-structure	O
-was	O
-previously	O
-reported	O
-for	O
-a	O
-functionally	O
--	O
-uncharacterized	O
-meningococcal	O
-homologue	O
-of	O
-NadR	O
-,	O
-termed	O
-NMB1585	O
-,	O
-which	O
-shares	O
-16	O
-%	O
-sequence	O
-identity	O
-with	O
-NadR	O
-.	O
-The	O
-two	O
-structures	O
-can	O
-be	O
-closely	O
-aligned	O
-(	O
-rmsd	O
-2	O
-.	O
-3	O
-Å	O
-),	O
-but	O
-NMB1585	O
-appears	O
-unsuited	O
-for	O
-binding	O
-HPAs	O
-,	O
-since	O
-its	O
-corresponding	O
-‘	O
-pocket	O
-’	O
-region	O
-is	O
-occupied	O
-by	O
-several	O
-bulky	O
-hydrophobic	O
-side	O
-chains	O
-.	O
-	O
-It	O
-can	O
-be	O
-speculated	O
-that	O
-MarR	O
-family	O
-members	O
-have	O
-evolved	O
-separately	O
-to	O
-engage	O
-distinct	O
-signaling	O
-molecules	O
-,	O
-thus	O
-enabling	O
-bacteria	O
-to	O
-use	O
-the	O
-overall	O
-conserved	O
-MarR	O
-scaffold	O
-to	O
-adapt	O
-and	O
-respond	O
-to	O
-diverse	O
-changing	O
-environmental	O
-stimuli	O
-experienced	O
-in	O
-their	O
-natural	O
-niches	O
-.	O
-	O
-Alternatively	O
-,	O
-it	O
-is	O
-possible	O
-that	O
-other	O
-MarR	O
-homologues	O
-(	O
-e	O
-.	O
-g	O
-.	O
-NMB1585	O
-)	O
-may	O
-have	O
-no	O
-extant	O
-functional	O
-binding	O
-pocket	O
-and	O
-thus	O
-may	O
-have	O
-lost	O
-the	O
-ability	O
-to	O
-respond	O
-to	O
-a	O
-ligand	O
-,	O
-acting	O
-instead	O
-as	O
-constitutive	O
-DNA	O
--	O
-binding	O
-regulatory	O
-proteins	O
-.	O
-	O
-The	O
-apo	O
--	O
-NadR	O
-crystal	O
-structures	O
-revealed	O
-two	O
-dimers	O
-with	O
-slightly	O
-different	O
-conformations	O
-,	O
-most	O
-divergent	O
-in	O
-the	O
-DNA	O
--	O
-binding	O
-domain	O
-.	O
-	O
-It	O
-is	O
-not	O
-unusual	O
-for	O
-a	O
-crystal	O
-structure	O
-to	O
-reveal	O
-multiple	O
-copies	O
-of	O
-the	O
-same	O
-protein	O
-in	O
-very	O
-slightly	O
-different	O
-conformations	O
-,	O
-which	O
-are	O
-likely	O
-representative	O
-of	O
-the	O
-lowest	O
--	O
-energy	O
-conformations	O
-sampled	O
-by	O
-the	O
-dynamic	O
-ensemble	O
-of	O
-molecular	O
-states	O
-occurring	O
-in	O
-solution	O
-,	O
-and	O
-which	O
-likely	O
-have	O
-only	O
-small	O
-energetic	O
-differences	O
-,	O
-as	O
-described	O
-previously	O
-for	O
-MexR	O
-(	O
-a	O
-MarR	O
-protein	O
-)	O
-or	O
-more	O
-recently	O
-for	O
-the	O
-solute	O
--	O
-binding	O
-protein	O
-FhuD2	O
-.	O
-	O
-Further	O
-,	O
-the	O
-holo	O
--	O
-NadR	O
-structure	O
-was	O
-overall	O
-more	O
-different	O
-from	O
-the	O
-two	O
-apo	O
--	O
-NadR	O
-structures	O
-(	O
-rmsd	O
-values	O
-~	O
-1	O
-.	O
-3Å	O
-),	O
-suggesting	O
-that	O
-the	O
-ligand	O
-selected	O
-and	O
-stabilized	O
-yet	O
-another	O
-conformation	O
-of	O
-NadR	O
-.	O
-These	O
-observations	O
-suggest	O
-that	O
-4	O
--	O
-HPA	O
-,	O
-and	O
-potentially	O
-other	O
-similar	O
-ligands	O
-,	O
-can	O
-shift	O
-the	O
-molecular	O
-equilibrium	O
-,	O
-changing	O
-the	O
-energy	O
-barriers	O
-that	O
-separate	O
-active	O
-and	O
-inactive	O
-states	O
-,	O
-and	O
-stabilizing	O
-the	O
-specific	O
-conformation	O
-of	O
-NadR	O
-poorly	O
-suited	O
-to	O
-bind	O
-DNA	O
-.	O
-	O
-Comparisons	O
-of	O
-the	O
-apo	O
--	O
-and	O
-holo	O
--	O
-NadR	O
-structures	O
-revealed	O
-that	O
-the	O
-largest	O
-differences	O
-occurred	O
-in	O
-the	O
-DNA	O
--	O
-binding	O
-helix	O
-α4	O
-.	O
-	O
-The	O
-shift	O
-of	O
-helix	O
-α4	O
-in	O
-holo	O
--	O
-NadR	O
-was	O
-also	O
-accompanied	O
-by	O
-rearrangements	O
-at	O
-the	O
-dimer	O
-interface	O
-,	O
-involving	O
-helices	O
-α1	O
-,	O
-α5	O
-,	O
-and	O
-α6	O
-,	O
-and	O
-this	O
-holo	O
--	O
-form	O
-appeared	O
-poorly	O
-suited	O
-for	O
-DNA	O
--	O
-binding	O
-when	O
-compared	O
-with	O
-the	O
-known	O
-OhrR	O
-:	O
-ohrA	O
-complex	O
-.	O
-	O
-While	O
-some	O
-flexibility	O
-of	O
-helix	O
-α4	O
-was	O
-also	O
-observed	O
-in	O
-the	O
-two	O
-apo	O
--	O
-structures	O
-,	O
-concomitant	O
-changes	O
-in	O
-the	O
-dimer	O
-interfaces	O
-were	O
-not	O
-observed	O
-,	O
-possibly	O
-due	O
-to	O
-the	O
-absence	O
-of	O
-ligand	O
-.	O
-	O
-One	O
-of	O
-the	O
-two	O
-conformations	O
-of	O
-apo	O
--	O
-NadR	O
-appeared	O
-ideally	O
-suited	O
-for	O
-DNA	O
--	O
-binding	O
-.	O
-	O
-Overall	O
-,	O
-these	O
-analyses	O
-suggest	O
-that	O
-the	O
-apo	O
--	O
-NadR	O
-dimer	O
-has	O
-a	O
-pre	O
--	O
-existing	O
-equilibrium	O
-that	O
-samples	O
-a	O
-variety	O
-of	O
-conformations	O
-,	O
-some	O
-of	O
-which	O
-are	O
-compatible	O
-with	O
-DNA	O
-binding	O
-.	O
-	O
-The	O
-noted	O
-flexibility	O
-may	O
-also	O
-explain	O
-how	O
-NadR	O
-can	O
-adapt	O
-to	O
-bind	O
-various	O
-DNA	O
-target	O
-sequences	O
-with	O
-slightly	O
-different	O
-structural	O
-features	O
-.	O
-	O
-Subsequently	O
-,	O
-upon	O
-ligand	O
-binding	O
-,	O
-holo	O
--	O
-NadR	O
-adopts	O
-a	O
-structure	O
-less	O
-suited	O
-for	O
-DNA	O
--	O
-binding	O
-and	O
-this	O
-conformation	O
-is	O
-selected	O
-and	O
-stabilized	O
-by	O
-a	O
-network	O
-of	O
-protein	O
--	O
-ligand	O
-interactions	O
-and	O
-concomitant	O
-rearrangements	O
-at	O
-the	O
-NadR	O
-holo	O
-dimer	O
-interface	O
-.	O
-	O
-In	O
-an	O
-alternative	O
-and	O
-less	O
-extensive	O
-manner	O
-,	O
-the	O
-binding	O
-of	O
-two	O
-salicylate	O
-molecules	O
-to	O
-the	O
-M	O
-.	O
-thermoautotrophicum	O
-protein	O
-MTH313	O
-appeared	O
-to	O
-induce	O
-large	O
-changes	O
-in	O
-the	O
-wHTH	O
-domain	O
-,	O
-which	O
-was	O
-associated	O
-with	O
-reduced	O
-DNA	O
--	O
-binding	O
-activity	O
-.	O
-	O
-Here	O
-we	O
-have	O
-presented	O
-two	O
-new	O
-crystal	O
-structures	O
-of	O
-the	O
-transcription	O
-factor	O
-,	O
-NadR	O
-,	O
-which	O
-regulates	O
-expression	O
-of	O
-the	O
-meningococcal	O
-surface	O
-protein	O
-,	O
-virulence	O
-factor	O
-and	O
-vaccine	O
-antigen	O
-NadA	O
-.	O
-Detailed	O
-structural	O
-analyses	O
-provided	O
-a	O
-molecular	O
-explanation	O
-for	O
-the	O
-ligand	O
--	O
-responsive	O
-regulation	O
-by	O
-NadR	O
-on	O
-the	O
-majority	O
-of	O
-the	O
-promoters	O
-of	O
-meningococcal	O
-genes	O
-regulated	O
-by	O
-NadR	O
-,	O
-including	O
-nadA	O
-.	O
-Intriguingly	O
-,	O
-NadR	O
-exhibits	O
-a	O
-reversed	O
-regulatory	O
-mechanism	O
-on	O
-a	O
-second	O
-class	O
-of	O
-promoters	O
-,	O
-including	O
-mafA	O
-of	O
-the	O
-multiple	O
-adhesin	O
-family	O
-–	O
-i	O
-.	O
-e	O
-.	O
-NadR	O
-represses	O
-these	O
-genes	O
-in	O
-the	O
-presence	O
-but	O
-not	O
-absence	O
-of	O
-4	O
--	O
-HPA	O
-.	O
-	O
-The	O
-latter	O
-may	O
-influence	O
-the	O
-surface	O
-abundance	O
-or	O
-secretion	O
-of	O
-maf	O
-proteins	O
-,	O
-an	O
-emerging	O
-class	O
-of	O
-highly	O
-conserved	O
-meningococcal	O
-putative	O
-adhesins	O
-and	O
-toxins	O
-with	O
-many	O
-important	O
-roles	O
-.	O
-	O
-Further	O
-work	O
-is	O
-required	O
-to	O
-investigate	O
-how	O
-the	O
-two	O
-different	O
-promoter	O
-types	O
-influence	O
-the	O
-ligand	O
--	O
-responsiveness	O
-of	O
-NadR	O
-during	O
-bacterial	O
-infection	O
-and	O
-may	O
-provide	O
-insights	O
-into	O
-the	O
-regulatory	O
-mechanisms	O
-occurring	O
-during	O
-these	O
-host	O
--	O
-pathogen	O
-interactions	O
-.	O
-	O
-Ultimately	O
-,	O
-knowledge	O
-of	O
-the	O
-ligand	O
--	O
-dependent	O
-activity	O
-of	O
-NadR	O
-will	O
-continue	O
-to	O
-deepen	O
-our	O
-understanding	O
-of	O
-nadA	O
-expression	O
-levels	O
-,	O
-which	O
-influence	O
-meningococcal	O
-pathogenesis	O
-.	O
-	O
-Structure	O
-of	O
-an	O
-OhrR	O
--	O
-ohrA	O
-operator	O
-complex	O
-reveals	O
-the	O
-DNA	O
-binding	O
-mechanism	O
-of	O
-the	O
-MarR	O
-family	O
-	O
-The	O
-structure	O
-of	O
-NMB1585	O
-,	O
-a	O
-MarR	O
--	O
-family	O
-regulator	O
-from	O
-Neisseria	O
-meningitidis	O
-	O
-Structural	O
-determinant	O
-for	O
-inducing	O
-RORgamma	O
-specific	O
-inverse	O
-agonism	O
-triggered	O
-by	O
-a	O
-synthetic	O
-benzoxazinone	O
-ligand	O
-	O
-The	O
-nuclear	O
-hormone	O
-receptor	O
-RORγ	O
-regulates	O
-transcriptional	O
-genes	O
-involved	O
-in	O
-the	O
-production	O
-of	O
-the	O
-pro	O
--	O
-inflammatory	O
-interleukin	O
-IL	O
--	O
-17	O
-which	O
-has	O
-been	O
-linked	O
-to	O
-autoimmune	O
-diseases	O
-such	O
-as	O
-rheumatoid	O
-arthritis	O
-,	O
-multiple	O
-sclerosis	O
-and	O
-inflammatory	O
-bowel	O
-disease	O
-.	O
-	O
-This	O
-transcriptional	O
-activity	O
-of	O
-RORγ	O
-is	O
-modulated	O
-through	O
-a	O
-protein	O
--	O
-protein	O
-interaction	O
-involving	O
-the	O
-activation	O
-function	O
-2	O
-(	O
-AF2	O
-)	O
-helix	O
-on	O
-the	O
-ligand	O
-binding	O
-domain	O
-of	O
-RORγ	O
-and	O
-a	O
-conserved	O
-LXXLL	O
-helix	O
-motif	O
-on	O
-coactivator	O
-proteins	O
-.	O
-	O
-Our	O
-goal	O
-was	O
-to	O
-develop	O
-a	O
-RORγ	O
-specific	O
-inverse	O
-agonist	O
-that	O
-would	O
-help	O
-down	O
-regulate	O
-pro	O
--	O
-inflammatory	O
-gene	O
-transcription	O
-by	O
-disrupting	O
-the	O
-protein	O
-protein	O
-interaction	O
-with	O
-coactivator	O
-proteins	O
-as	O
-a	O
-therapeutic	O
-agent	O
-.	O
-	O
-We	O
-identified	O
-a	O
-novel	O
-series	O
-of	O
-synthetic	O
-benzoxazinone	O
-ligands	O
-having	O
-an	O
-agonist	O
-(	O
-BIO592	O
-)	O
-and	O
-inverse	O
-agonist	O
-(	O
-BIO399	O
-)	O
-mode	O
-of	O
-action	O
-in	O
-a	O
-FRET	O
-based	O
-assay	O
-.	O
-	O
-We	O
-show	O
-that	O
-the	O
-AF2	O
-helix	O
-of	O
-RORγ	O
-is	O
-proteolytically	O
-sensitive	O
-when	O
-inverse	O
-agonist	O
-BIO399	O
-binds	O
-.	O
-	O
-Using	O
-x	O
--	O
-ray	O
-crystallography	O
-we	O
-show	O
-how	O
-small	O
-modifications	O
-on	O
-the	O
-benzoxazinone	O
-agonist	O
-BIO592	O
-trigger	O
-inverse	O
-agonism	O
-of	O
-RORγ	O
-.	O
-	O
-Using	O
-an	O
-in	O
-vivo	O
-reporter	O
-assay	O
-,	O
-we	O
-show	O
-that	O
-the	O
-inverse	O
-agonist	O
-BIO399	O
-displayed	O
-specificity	O
-for	O
-RORγ	O
-over	O
-ROR	O
-sub	O
--	O
-family	O
-members	O
-α	O
-and	O
-β	O
-.	O
-	O
-The	O
-synthetic	O
-benzoxazinone	O
-ligands	O
-identified	O
-in	O
-our	O
-FRET	O
-assay	O
-have	O
-an	O
-agonist	O
-(	O
-BIO592	O
-)	O
-or	O
-inverse	O
-agonist	O
-(	O
-BIO399	O
-)	O
-effect	O
-by	O
-stabilizing	O
-or	O
-destabilizing	O
-the	O
-agonist	O
-conformation	O
-of	O
-RORγ	O
-.	O
-	O
-The	O
-proteolytic	O
-sensitivity	O
-of	O
-the	O
-AF2	O
-helix	O
-of	O
-RORγ	O
-demonstrates	O
-that	O
-it	O
-destabilizes	O
-upon	O
-BIO399	O
-inverse	O
-agonist	O
-binding	O
-perturbing	O
-the	O
-coactivator	O
-protein	O
-binding	O
-site	O
-.	O
-	O
-Our	O
-structural	O
-investigation	O
-of	O
-the	O
-BIO592	O
-agonist	O
-and	O
-BIO399	O
-inverse	O
-agonist	O
-structures	O
-identified	O
-residue	O
-Met358	O
-on	O
-RORγ	O
-as	O
-the	O
-trigger	O
-for	O
-RORγ	O
-specific	O
-inverse	O
-agonism	O
-.	O
-	O
-Retinoid	O
--	O
-related	O
-orphan	O
-receptor	O
-gamma	O
-(	O
-RORγ	O
-)	O
-is	O
-a	O
-transcription	O
-factor	O
-belonging	O
-to	O
-a	O
-sub	O
--	O
-family	O
-of	O
-nuclear	O
-receptors	O
-that	O
-includes	O
-two	O
-closely	O
-related	O
-members	O
-RORα	O
-and	O
-RORβ	O
-.	O
-	O
-Even	O
-though	O
-a	O
-high	O
-degree	O
-of	O
-sequence	O
-similarity	O
-exists	O
-between	O
-the	O
-RORs	O
-,	O
-their	O
-functional	O
-roles	O
-in	O
-regulation	O
-for	O
-physiological	O
-processes	O
-involved	O
-in	O
-development	O
-and	O
-immunity	O
-are	O
-distinct	O
-.	O
-	O
-During	O
-development	O
-,	O
-RORγ	O
-regulates	O
-the	O
-transcriptional	O
-genes	O
-involved	O
-in	O
-the	O
-functioning	O
-of	O
-multiple	O
-pro	O
--	O
-inflammatory	O
-lymphocyte	O
-lineages	O
-including	O
-T	O
-helper	O
-cells	O
-(	O
-TH17cells	O
-)	O
-which	O
-are	O
-necessary	O
-for	O
-IL	O
--	O
-17	O
-production	O
-.	O
-	O
-IL	O
--	O
-17	O
-is	O
-a	O
-pro	O
--	O
-inflammatory	O
-interleukin	O
-linked	O
-to	O
-autoimmune	O
-diseases	O
-such	O
-as	O
-rheumatoid	O
-arthritis	O
-,	O
-multiple	O
-sclerosis	O
-and	O
-inflammatory	O
-bowel	O
-disease	O
-;	O
-making	O
-its	O
-transcriptional	O
-regulation	O
-through	O
-RORγ	O
-an	O
-attractive	O
-therapeutic	O
-target	O
-.	O
-	O
-RORγ	O
-consists	O
-of	O
-an	O
-N	O
--	O
-terminal	O
-DNA	O
-binding	O
-domain	O
-(	O
-DBD	O
-)	O
-connected	O
-to	O
-a	O
-C	O
--	O
-terminal	O
-ligand	O
-binding	O
-domain	O
-(	O
-LBD	O
-)	O
-via	O
-a	O
-flexible	O
-hinge	O
-region	O
-.	O
-	O
-The	O
-DBD	O
-is	O
-composed	O
-of	O
-two	O
-zinc	O
-fingers	O
-that	O
-allow	O
-it	O
-to	O
-interact	O
-with	O
-specifically	O
-encoded	O
-regions	O
-on	O
-the	O
-DNA	O
-called	O
-the	O
-nuclear	O
-receptor	O
-response	O
-elements	O
-.	O
-	O
-The	O
-LBD	O
-consists	O
-of	O
-a	O
-coactivator	O
-protein	O
-binding	O
-pocket	O
-and	O
-a	O
-hydrophobic	O
-ligand	O
-binding	O
-site	O
-(	O
-LBS	O
-)	O
-which	O
-are	O
-responsible	O
-for	O
-regulating	O
-transcription	O
-.	O
-	O
-The	O
-coactivator	O
-binding	O
-pocket	O
-of	O
-RORγ	O
-recognizes	O
-a	O
-conserved	O
-helix	O
-motif	O
-LXXLL	O
-(	O
-where	O
-X	O
-can	O
-be	O
-any	O
-amino	O
-acid	O
-)	O
-on	O
-transcriptional	O
-coactivator	O
-complexes	O
-and	O
-recruits	O
-it	O
-to	O
-activate	O
-transcription	O
-.	O
-	O
-Like	O
-other	O
-nuclear	O
-hormone	O
-receptors	O
-,	O
-RORγ	O
-’	O
-s	O
-helix12	O
-which	O
-makes	O
-up	O
-the	O
-C	O
--	O
-termini	O
-of	O
-the	O
-LBD	O
-is	O
-an	O
-essential	O
-part	O
-of	O
-the	O
-coactivator	O
-binding	O
-pocket	O
-and	O
-is	O
-commonly	O
-referred	O
-to	O
-as	O
-the	O
-activation	O
-function	O
-helix	O
-2	O
-(	O
-AF2	O
-).	O
-	O
-In	O
-RORγ	O
-,	O
-the	O
-conformation	O
-of	O
-the	O
-AF2	O
-helix	O
-required	O
-to	O
-form	O
-the	O
-coactivator	O
-binding	O
-pocket	O
-is	O
-mediated	O
-by	O
-a	O
-salt	O
-bridge	O
-between	O
-His479	O
-and	O
-Tyr502	O
-in	O
-addition	O
-to	O
-π	O
--	O
-π	O
-interactions	O
-between	O
-Tyr502	O
-and	O
-Phe506	O
-.	O
-	O
-The	O
-conformation	O
-of	O
-the	O
-AF2	O
-helix	O
-can	O
-be	O
-modulated	O
-through	O
-targeted	O
-ligands	O
-which	O
-bind	O
-the	O
-LBS	O
-and	O
-increase	O
-the	O
-binding	O
-of	O
-the	O
-coactivator	O
-protein	O
-(	O
-agonists	O
-)	O
-or	O
-disrupt	O
-binding	O
-(	O
-inverse	O
-agonists	O
-)	O
-thereby	O
-enhancing	O
-or	O
-inhibiting	O
-transcription	O
-.	O
-	O
-Since	O
-RORγ	O
-has	O
-been	O
-demonstrated	O
-to	O
-play	O
-an	O
-important	O
-role	O
-in	O
-pro	O
--	O
-inflammatory	O
-gene	O
-expression	O
-patterns	O
-implicated	O
-in	O
-several	O
-major	O
-autoimmune	O
-diseases	O
-,	O
-our	O
-aim	O
-was	O
-to	O
-develop	O
-RORγ	O
-inverse	O
-agonists	O
-that	O
-would	O
-help	O
-down	O
-regulate	O
-pro	O
--	O
-inflammatory	O
-gene	O
-transcription	O
-.	O
-	O
-FRET	O
-results	O
-for	O
-agonist	O
-BIO592	O
-(	O
-a	O
-)	O
-and	O
-Inverse	O
-Agonist	O
-BIO399	O
-(	O
-b	O
-)	O
-	O
-Here	O
-we	O
-present	O
-the	O
-identification	O
-of	O
-two	O
-synthetic	O
-benzoxazinone	O
-RORγ	O
-ligands	O
-,	O
-a	O
-weak	O
-agonist	O
-BIO592	O
-(	O
-Fig	O
-.	O
-1a	O
-)	O
-and	O
-an	O
-inverse	O
-agonist	O
-BIO399	O
-(	O
-Fig	O
-.	O
-1b	O
-)	O
-which	O
-were	O
-identified	O
-using	O
-a	O
-Fluorescence	O
-Resonance	O
-Energy	O
-transfer	O
-(	O
-FRET	O
-)	O
-based	O
-assay	O
-that	O
-monitored	O
-coactivator	O
-peptide	O
-recruitment	O
-.	O
-	O
-Using	O
-partial	O
-proteolysis	O
-in	O
-combination	O
-with	O
-mass	O
-spectrometry	O
-analysis	O
-we	O
-demonstrate	O
-that	O
-the	O
-AF2	O
-helix	O
-of	O
-RORγ	O
-destabilizes	O
-upon	O
-BIO399	O
-(	O
-inverse	O
-agonist	O
-)	O
-binding	O
-.	O
-	O
-Finally	O
-,	O
-comparing	O
-binding	O
-modes	O
-of	O
-our	O
-benzoxazinone	O
-RORγ	O
-crystal	O
-structures	O
-to	O
-other	O
-ROR	O
-structures	O
-,	O
-we	O
-hypothesize	O
-a	O
-new	O
-mode	O
-of	O
-action	O
-for	O
-achieving	O
-inverse	O
-agonism	O
-and	O
-selectivity	O
-.	O
-	O
-Using	O
-a	O
-FRET	O
-based	O
-assay	O
-we	O
-discovered	O
-agonist	O
-BIO592	O
-(	O
-Fig	O
-.	O
-1a	O
-)	O
-which	O
-increased	O
-the	O
-coactivator	O
-peptide	O
-TRAP220	O
-recruitment	O
-to	O
-RORγ	O
-(	O
-EC50	O
-0f	O
-58nM	O
-and	O
-Emax	O
-of	O
-130	O
-%)	O
-and	O
-a	O
-potent	O
-inverse	O
-agonist	O
-BIO399	O
-(	O
-Fig	O
-.	O
-1b	O
-)	O
-which	O
-inhibited	O
-coactivator	O
-recruitment	O
-(	O
-IC50	O
-:	O
-4	O
-.	O
-7nM	O
-).	O
-	O
-Interestingly	O
-,	O
-the	O
-structural	O
-difference	O
-between	O
-the	O
-agonist	O
-BIO592	O
-and	O
-inverse	O
-agonist	O
-BIO399	O
-was	O
-minor	O
-;	O
-with	O
-the	O
-2	O
-,	O
-3	O
--	O
-dihydrobenzo	O
-[	O
-1	O
-,	O
-4	O
-]	O
-oxazepin	O
--	O
-4	O
--	O
-one	O
-ring	O
-system	O
-of	O
-BIO399	O
-being	O
-3	O
-atoms	O
-larger	O
-than	O
-the	O
-benzo	O
-[	O
-1	O
-,	O
-4	O
-]	O
-oxazine	O
--	O
-3	O
--	O
-one	O
-ring	O
-system	O
-of	O
-BIO592	O
-.	O
-	O
-In	O
-order	O
-to	O
-understand	O
-how	O
-small	O
-changes	O
-in	O
-the	O
-core	O
-ring	O
-system	O
-leads	O
-to	O
-inverse	O
-agonism	O
-,	O
-we	O
-wanted	O
-to	O
-structurally	O
-determine	O
-the	O
-binding	O
-mode	O
-of	O
-both	O
-BIO592	O
-and	O
-BIO399	O
-in	O
-the	O
-LBS	O
-of	O
-RORγ	O
-using	O
-x	O
--	O
-ray	O
-crystallography	O
-.	O
-	O
-Structure	O
-of	O
-the	O
-RORγ518	O
--	O
-BIO592	O
--	O
-EBI96	O
-ternary	O
-complex	O
-is	O
-in	O
-a	O
-transcriptionally	O
-active	O
-conformation	O
-	O
-a	O
-The	O
-ternary	O
-structure	O
-of	O
-RORγ518	O
-BIO592	O
-and	O
-EBI96	O
-.	O
-	O
-b	O
-RORγ	O
-AF2	O
-helix	O
-in	O
-the	O
-agonist	O
-conformation	O
-.	O
-	O
-c	O
-EBI96	O
-coactivator	O
-peptide	O
-bound	O
-in	O
-the	O
-coactivator	O
-pocket	O
-of	O
-RORγ	O
-	O
-RORγ518	O
-bound	O
-to	O
-agonist	O
-BIO592	O
-was	O
-crystallized	O
-with	O
-a	O
-truncated	O
-form	O
-of	O
-the	O
-coactivator	O
-peptide	O
-EBI96	O
-to	O
-a	O
-resolution	O
-of	O
-2	O
-.	O
-6	O
-Å	O
-(	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-The	O
-structure	O
-of	O
-the	O
-ternary	O
-complex	O
-had	O
-features	O
-similar	O
-to	O
-other	O
-ROR	O
-agonist	O
-coactivator	O
-structures	O
-in	O
-a	O
-transcriptionally	O
-active	O
-canonical	O
-three	O
-layer	O
-helix	O
-fold	O
-with	O
-the	O
-AF2	O
-helix	O
-in	O
-the	O
-agonist	O
-conformation	O
-.	O
-	O
-The	O
-agonist	O
-conformation	O
-is	O
-stabilized	O
-by	O
-a	O
-hydrogen	O
-bond	O
-between	O
-His479	O
-and	O
-Tyr502	O
-,	O
-in	O
-addition	O
-to	O
-π	O
--	O
-π	O
-interactions	O
-between	O
-His479	O
-,	O
-Tyr502	O
-and	O
-Phe506	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-The	O
-hydrogen	O
-bond	O
-between	O
-His479	O
-and	O
-Tyr502	O
-has	O
-been	O
-reported	O
-to	O
-be	O
-critical	O
-for	O
-RORγ	O
-agonist	O
-activity	O
-.	O
-	O
-Disrupting	O
-this	O
-interaction	O
-through	O
-mutagenesis	O
-reduced	O
-transcriptional	O
-activity	O
-of	O
-RORγ	O
-.	O
-	O
-This	O
-reduced	O
-transcriptional	O
-activity	O
-has	O
-been	O
-attributed	O
-to	O
-the	O
-inability	O
-of	O
-the	O
-AF2	O
-helix	O
-to	O
-complete	O
-the	O
-formation	O
-of	O
-the	O
-coactivator	O
-binding	O
-pocket	O
-necessary	O
-for	O
-coactivator	O
-proteins	O
-to	O
-bind	O
-.	O
-	O
-Electron	O
-density	O
-for	O
-the	O
-coactivator	O
-peptide	O
-EBI96	O
-was	O
-observed	O
-for	O
-residues	O
-EFPYLLSLLG	O
-which	O
-formed	O
-a	O
-α	O
--	O
-helix	O
-stabilized	O
-through	O
-hydrophobic	O
-interactions	O
-with	O
-the	O
-coactivator	O
-binding	O
-pocket	O
-on	O
-RORγ	O
-(	O
-Fig	O
-.	O
-2c	O
-).	O
-	O
-This	O
-interaction	O
-is	O
-further	O
-stabilized	O
-through	O
-a	O
-conserved	O
-charged	O
-clamp	O
-wherein	O
-the	O
-backbone	O
-amide	O
-of	O
-Tyr7	O
-and	O
-carbonyl	O
-of	O
-Leu11	O
-of	O
-EBI96	O
-form	O
-hydrogen	O
-bonds	O
-with	O
-Glu504	O
-(	O
-helix12	O
-)	O
-and	O
-Lys336	O
-(	O
-helix3	O
-)	O
-of	O
-RORγ	O
-.	O
-	O
-Formation	O
-of	O
-this	O
-charged	O
-clamp	O
-is	O
-essential	O
-for	O
-RORγ	O
-’	O
-s	O
-function	O
-for	O
-playing	O
-a	O
-role	O
-in	O
-transcriptional	O
-activation	O
-and	O
-this	O
-has	O
-been	O
-corroborated	O
-through	O
-mutagenic	O
-studies	O
-in	O
-this	O
-region	O
-.	O
-	O
-BIO592	O
-binds	O
-in	O
-a	O
-collapsed	O
-conformation	O
-stabilizing	O
-the	O
-agonist	O
-conformation	O
-of	O
-RORγ	O
-	O
-a	O
-Collapsed	O
-binding	O
-mode	O
-of	O
-agonist	O
-BIO592	O
-in	O
-the	O
-hydrophobic	O
-LBS	O
-of	O
-RORγ	O
-.	O
-	O
-b	O
-Benzoxazinone	O
-ring	O
-system	O
-of	O
-agonist	O
-BIO592	O
-packing	O
-against	O
-His479	O
-of	O
-RORγ	O
-stabilizing	O
-agonist	O
-conformation	O
-of	O
-the	O
-AF2	O
-helix	O
-	O
-BIO592	O
-bound	O
-in	O
-a	O
-collapsed	O
-conformational	O
-state	O
-in	O
-the	O
-LBS	O
-of	O
-RORγ	O
-with	O
-the	O
-xylene	O
-ring	O
-positioned	O
-at	O
-the	O
-bottom	O
-of	O
-the	O
-pocket	O
-making	O
-hydrophobic	O
-interactions	O
-with	O
-Val376	O
-,	O
-Phe378	O
-,	O
-Phe388	O
-and	O
-Phe401	O
-,	O
-with	O
-the	O
-ethyl	O
--	O
-benzoxazinone	O
-ring	O
-making	O
-several	O
-hydrophobic	O
-interactions	O
-with	O
-Trp317	O
-,	O
-Leu324	O
-,	O
-Met358	O
-,	O
-Leu391	O
-,	O
-Ile	O
-400	O
-and	O
-His479	O
-(	O
-Fig	O
-.	O
-3a	O
-,	O
-Additional	O
-file	O
-3	O
-).	O
-	O
-The	O
-sulfonyl	O
-group	O
-faces	O
-the	O
-entrance	O
-of	O
-the	O
-pocket	O
-,	O
-while	O
-the	O
-CF3	O
-makes	O
-a	O
-hydrophobic	O
-contact	O
-with	O
-Ala327	O
-.	O
-	O
-Hydrophobic	O
-interaction	O
-between	O
-the	O
-ethyl	O
-group	O
-of	O
-the	O
-benzoxazinone	O
-and	O
-His479	O
-reinforce	O
-the	O
-His479	O
-sidechain	O
-position	O
-for	O
-making	O
-the	O
-hydrogen	O
-bond	O
-with	O
-Tyr502	O
-thereby	O
-stabilizing	O
-the	O
-agonist	O
-conformation	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-RORγ	O
-AF2	O
-helix	O
-is	O
-sensitive	O
-to	O
-proteolysis	O
-in	O
-the	O
-presence	O
-of	O
-Inverse	O
-Agonist	O
-BIO399	O
-	O
-Next	O
-,	O
-we	O
-attempted	O
-co	O
--	O
-crystallization	O
-with	O
-the	O
-inverse	O
-agonist	O
-BIO399	O
-.	O
-	O
-However	O
-,	O
-extensive	O
-crystallization	O
-efforts	O
-with	O
-BIO399	O
-and	O
-RORγ518	O
-or	O
-other	O
-AF2	O
-intact	O
-constructs	O
-did	O
-not	O
-produce	O
-crystals	O
-.	O
-	O
-We	O
-hypothesized	O
-that	O
-the	O
-RORγ518	O
-coactivator	O
-peptide	O
-interaction	O
-in	O
-the	O
-FRET	O
-assay	O
-was	O
-disrupted	O
-upon	O
-BIO399	O
-binding	O
-and	O
-that	O
-a	O
-conformational	O
-rearrangement	O
-of	O
-the	O
-AF2	O
-helix	O
-could	O
-have	O
-occurred	O
-,	O
-hindering	O
-crystallization	O
-.	O
-	O
-Specific	O
-proteolytic	O
-positions	O
-on	O
-RORγ518	O
-when	O
-treated	O
-with	O
-Actinase	O
-E	O
-alone	O
-(	O
-Green	O
-)	O
-or	O
-in	O
-the	O
-presence	O
-of	O
-BIO399	O
-(	O
-Red	O
-)	O
-and	O
-shared	O
-proteolytic	O
-sites	O
-(	O
-Yellow	O
-)	O
-	O
-The	O
-unfolding	O
-of	O
-the	O
-AF2	O
-helix	O
-has	O
-been	O
-observed	O
-for	O
-other	O
-nuclear	O
-hormone	O
-receptors	O
-when	O
-bound	O
-to	O
-an	O
-inverse	O
-agonist	O
-or	O
-antagonist	O
-.	O
-	O
-We	O
-used	O
-partial	O
-proteolysis	O
-in	O
-combination	O
-with	O
-mass	O
-spectrometry	O
-to	O
-determine	O
-if	O
-BIO399	O
-was	O
-causing	O
-the	O
-AF2	O
-helix	O
-to	O
-unfold	O
-.	O
-	O
-Results	O
-of	O
-the	O
-Actinase	O
-E	O
-proteolysis	O
-experiments	O
-on	O
-RORγ518	O
-,	O
-the	O
-ternary	O
-complex	O
-of	O
-RORγ518	O
-with	O
-agonist	O
-BIO592	O
-and	O
-coactivator	O
-EBI96	O
-,	O
-or	O
-in	O
-the	O
-presence	O
-of	O
-inverse	O
-agonist	O
-BIO399	O
-supported	O
-our	O
-hypothesis	O
-.	O
-	O
-Analysis	O
-of	O
-the	O
-fragmentation	O
-pattern	O
-showed	O
-minimal	O
-proteolytic	O
-removal	O
-of	O
-the	O
-AF2	O
-helix	O
-by	O
-Actinase	O
-E	O
-on	O
-RORγ518	O
-alone	O
-(	O
-ending	O
-at	O
-504	O
-to	O
-506	O
-)	O
-and	O
-the	O
-ternary	O
-complex	O
-remained	O
-primarily	O
-intact	O
-(	O
-ending	O
-at	O
-515	O
-/	O
-518	O
-)	O
-(	O
-Additional	O
-file	O
-4	O
-).	O
-	O
-However	O
-,	O
-in	O
-the	O
-presence	O
-of	O
-inverse	O
-agonist	O
-BIO399	O
-,	O
-the	O
-proteolytic	O
-pattern	O
-showed	O
-significantly	O
-less	O
-protection	O
-,	O
-albeit	O
-the	O
-products	O
-were	O
-more	O
-heterogeneous	O
-(	O
-majority	O
-ending	O
-at	O
-494	O
-/	O
-495	O
-),	O
-indicating	O
-the	O
-destabilization	O
-of	O
-the	O
-AF2	O
-helix	O
-compared	O
-to	O
-either	O
-the	O
-APO	O
-or	O
-ternary	O
-agonist	O
-complex	O
-(	O
-Fig	O
-.	O
-4	O
-,	O
-Additional	O
-file	O
-5	O
-).	O
-	O
-Several	O
-rounds	O
-of	O
-cocrystallization	O
-attempts	O
-with	O
-RORγ518	O
-or	O
-other	O
-RORγ	O
-AF2	O
-helix	O
-containing	O
-constructs	O
-complexed	O
-with	O
-BIO399	O
-had	O
-not	O
-produced	O
-crystals	O
-.	O
-	O
-We	O
-attributed	O
-the	O
-inability	O
-to	O
-form	O
-crystals	O
-to	O
-the	O
-unfolding	O
-of	O
-the	O
-AF2	O
-helix	O
-induced	O
-by	O
-BIO399	O
-.	O
-	O
-We	O
-reasoned	O
-that	O
-if	O
-we	O
-could	O
-remove	O
-the	O
-unfolded	O
-AF2	O
-helix	O
-using	O
-proteolysis	O
-we	O
-could	O
-produce	O
-a	O
-binary	O
-complex	O
-more	O
-amenable	O
-to	O
-crystallization	O
-.	O
-	O
-AF2	O
-truncated	O
-RORγ	O
-BIO399	O
-complex	O
-is	O
-more	O
-amenable	O
-to	O
-crystallization	O
-	O
-a	O
-The	O
-binary	O
-structure	O
-of	O
-AF2	O
--	O
-truncated	O
-RORγ	O
-and	O
-BIO399	O
-.	O
-	O
-b	O
-The	O
-superposition	O
-of	O
-inverse	O
-agonist	O
-BIO399	O
-(	O
-Cyan	O
-)	O
-and	O
-agonist	O
-BIO592	O
-(	O
-Green	O
-).	O
-	O
-c	O
-Movement	O
-of	O
-Met358	O
-and	O
-His479	O
-in	O
-the	O
-BIO399	O
-(	O
-Cyan	O
-)	O
-and	O
-BIO592	O
-(	O
-Green	O
-)	O
-structures	O
-	O
-The	O
-Actinase	O
-E	O
-treated	O
-RORγ518	O
-BIO399	O
-ternary	O
-complex	O
-(	O
-aeRORγ493	O
-/	O
-4	O
-)	O
-co	O
--	O
-crystallized	O
-readily	O
-in	O
-several	O
-PEG	O
-based	O
-conditions	O
-.	O
-	O
-The	O
-structure	O
-of	O
-aeRORγ493	O
-/	O
-4	O
-BIO399	O
-complex	O
-was	O
-solved	O
-to	O
-2	O
-.	O
-3	O
-Å	O
-and	O
-adopted	O
-a	O
-similar	O
-core	O
-fold	O
-to	O
-the	O
-BIO592	O
-agonist	O
-crystal	O
-structure	O
-(	O
-Fig	O
-.	O
-5a	O
-,	O
-Additional	O
-file	O
-3	O
-).	O
-	O
-The	O
-aeRORγ493	O
-/	O
-4	O
-BIO399	O
-structure	O
-diverged	O
-at	O
-the	O
-c	O
--	O
-terminal	O
-end	O
-of	O
-Helix	O
-11	O
-from	O
-the	O
-RORγ518	O
-BIO592	O
-EBI96	O
-structure	O
-,	O
-where	O
-helix	O
-11	O
-unwinds	O
-into	O
-a	O
-random	O
-coil	O
-after	O
-residue	O
-L475	O
-.	O
-	O
-Inverse	O
-agonist	O
-BIO399	O
-uses	O
-Met358	O
-as	O
-a	O
-trigger	O
-for	O
-inverse	O
-agonism	O
-	O
-BIO399	O
-binds	O
-to	O
-the	O
-ligand	O
-binding	O
-site	O
-of	O
-RORγ	O
-adopting	O
-a	O
-collapsed	O
-conformation	O
-as	O
-seen	O
-with	O
-BIO592	O
-where	O
-the	O
-two	O
-compounds	O
-superimpose	O
-with	O
-an	O
-RMSD	O
-of	O
-0	O
-.	O
-72	O
-Å	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-The	O
-majority	O
-of	O
-the	O
-side	O
-chains	O
-within	O
-4	O
-Å	O
-of	O
-BIO399	O
-and	O
-BIO592	O
-adopt	O
-similar	O
-rotomer	O
-conformations	O
-with	O
-the	O
-exceptions	O
-of	O
-Met358	O
-and	O
-His479	O
-(	O
-Fig	O
-.	O
-5c	O
-).	O
-	O
-The	O
-difference	O
-density	O
-map	O
-showed	O
-clear	O
-positive	O
-density	O
-for	O
-Met358	O
-in	O
-an	O
-alternate	O
-rotomer	O
-conformation	O
-compared	O
-to	O
-the	O
-one	O
-observed	O
-in	O
-the	O
-molecular	O
-replacement	O
-model	O
-or	O
-the	O
-other	O
-agonist	O
-containing	O
-models	O
-(	O
-Additional	O
-file	O
-6	O
-).	O
-	O
-We	O
-tried	O
-to	O
-refine	O
-Met358	O
-in	O
-the	O
-same	O
-conformation	O
-as	O
-the	O
-molecular	O
-replacement	O
-model	O
-or	O
-the	O
-other	O
-agonist	O
-containing	O
-models	O
-,	O
-but	O
-the	O
-results	O
-clearly	O
-indicated	O
-that	O
-this	O
-was	O
-not	O
-possible	O
-,	O
-thus	O
-confirming	O
-the	O
-new	O
-rotamer	O
-conformation	O
-for	O
-the	O
-Met358	O
-sidechain	O
-in	O
-the	O
-inverse	O
-agonist	O
-bound	O
-structure	O
-.	O
-	O
-The	O
-change	O
-in	O
-rotomer	O
-conformation	O
-of	O
-Met358	O
-between	O
-the	O
-agonist	O
-and	O
-inverse	O
-agonist	O
-structures	O
-is	O
-attributed	O
-to	O
-the	O
-gem	O
--	O
-dimethyl	O
-group	O
-on	O
-the	O
-larger	O
-7	O
-membered	O
-benzoxazinone	O
-ring	O
-system	O
-of	O
-BIO399	O
-.	O
-	O
-The	O
-comparison	O
-of	O
-the	O
-two	O
-structures	O
-shows	O
-that	O
-the	O
-agonist	O
-conformation	O
-observed	O
-in	O
-the	O
-BIO592	O
-structure	O
-would	O
-be	O
-perturbed	O
-by	O
-BIO399	O
-pushing	O
-Met358	O
-into	O
-Phe506	O
-of	O
-the	O
-AF2	O
-helix	O
-indicating	O
-that	O
-Met358	O
-is	O
-a	O
-trigger	O
-for	O
-inducing	O
-inverse	O
-agonism	O
-in	O
-RORγ	O
-(	O
-Fig	O
-.	O
-5c	O
-).	O
-	O
-BIO399	O
-and	O
-Inverse	O
-agonist	O
-T0901317	O
-bind	O
-in	O
-a	O
-collapsed	O
-conformation	O
-distinct	O
-from	O
-other	O
-RORγ	O
-Inverse	O
-Agonists	O
-Cocrystal	O
-structures	O
-	O
-a	O
-Overlay	O
-of	O
-RORγ	O
-structures	O
-bound	O
-to	O
-BIO596	O
-(	O
-Green	O
-),	O
-BIO399	O
-(	O
-Cyan	O
-)	O
-and	O
-T0901317	O
-(	O
-Pink	O
-).	O
-	O
-b	O
-Overlay	O
-of	O
-M358	O
-in	O
-RORγ	O
-structure	O
-BIO596	O
-(	O
-Green	O
-),	O
-BIO399	O
-(	O
-Cyan	O
-),	O
-Digoxin	O
-(	O
-Yellow	O
-),	O
-Compound	O
-2	O
-(	O
-Grey	O
-),	O
-Compound	O
-48	O
-(	O
-Salmon	O
-)	O
-and	O
-Compound	O
-4j	O
-(	O
-Orange	O
-)	O
-	O
-The	O
-co	O
--	O
-crystal	O
-structure	O
-of	O
-RORγ	O
-with	O
-T0901317	O
-(	O
-PDB	O
-code	O
-:	O
-4NB6	O
-),	O
-an	O
-inverse	O
-agonist	O
-of	O
-RORγ	O
-(	O
-IC50	O
-of	O
-54nM	O
-in	O
-an	O
-SRC1	O
-displacement	O
-FRET	O
-assay	O
-and	O
-an	O
-IC50	O
-of	O
-59nM	O
-in	O
-our	O
-FRET	O
-assay	O
-(	O
-Additional	O
-file	O
-7	O
-))	O
-shows	O
-that	O
-it	O
-adopts	O
-a	O
-collapsed	O
-conformation	O
-similar	O
-to	O
-the	O
-structure	O
-of	O
-BIO399	O
-described	O
-here	O
-.	O
-	O
-The	O
-two	O
-compounds	O
-superimpose	O
-with	O
-an	O
-RMSD	O
-of	O
-0	O
-.	O
-81	O
-Å	O
-(	O
-Fig	O
-.	O
-6a	O
-).	O
-	O
-The	O
-CF3	O
-group	O
-on	O
-the	O
-hexafluoropropanol	O
-group	O
-of	O
-T0901317	O
-was	O
-reported	O
-to	O
-fit	O
-the	O
-electron	O
-density	O
-in	O
-two	O
-conformations	O
-one	O
-of	O
-which	O
-pushes	O
-Met358	O
-into	O
-the	O
-vicinity	O
-of	O
-Phe506	O
-in	O
-the	O
-RORγ	O
-BIO592	O
-agonist	O
-structure	O
-.	O
-	O
-We	O
-hypothesize	O
-that	O
-since	O
-the	O
-Met358	O
-sidechain	O
-conformation	O
-in	O
-the	O
-T0901317	O
-RORγ	O
-structure	O
-is	O
-not	O
-in	O
-the	O
-BIO399	O
-conformation	O
-,	O
-this	O
-difference	O
-could	O
-account	O
-for	O
-the	O
-10	O
--	O
-fold	O
-reduction	O
-in	O
-the	O
-inverse	O
-agonism	O
-for	O
-T0901317	O
-compared	O
-to	O
-BIO399	O
-in	O
-the	O
-FRET	O
-assay	O
-.	O
-	O
-Co	O
--	O
-crystal	O
-structures	O
-of	O
-RORγ	O
-have	O
-been	O
-generated	O
-with	O
-several	O
-potent	O
-inverse	O
-agonists	O
-adopting	O
-a	O
-linear	O
-conformation	O
-distinct	O
-from	O
-the	O
-collapsed	O
-conformations	O
-seen	O
-for	O
-BIO399	O
-and	O
-T090131718	O
-.	O
-	O
-The	O
-inverse	O
-agonist	O
-activity	O
-for	O
-these	O
-compounds	O
-has	O
-been	O
-attributed	O
-to	O
-orientating	O
-Trp317	O
-to	O
-clash	O
-with	O
-Tyr502	O
-or	O
-a	O
-direct	O
-inverse	O
-agonist	O
-hydrogen	O
-bonding	O
-event	O
-with	O
-His479	O
-,	O
-both	O
-of	O
-which	O
-would	O
-perturb	O
-the	O
-agonist	O
-conformation	O
-of	O
-RORγ	O
-.	O
-	O
-BIO399	O
-neither	O
-orients	O
-the	O
-sidechain	O
-of	O
-Trp317	O
-toward	O
-Tyr502	O
-nor	O
-forms	O
-a	O
-hydrogen	O
-bond	O
-with	O
-His479	O
-suggesting	O
-its	O
-mode	O
-of	O
-action	O
-is	O
-distinct	O
-from	O
-linear	O
-inverse	O
-agonists	O
-(	O
-Additional	O
-file	O
-8	O
-).	O
-	O
-In	O
-the	O
-linear	O
-inverse	O
-agonist	O
-crystal	O
-structures	O
-the	O
-side	O
-chain	O
-of	O
-Met358	O
-resides	O
-in	O
-a	O
-similar	O
-position	O
-as	O
-the	O
-rotomer	O
-observed	O
-in	O
-RORγ	O
-agonist	O
-structures	O
-with	O
-BIO592	O
-described	O
-here	O
-or	O
-as	O
-observed	O
-in	O
-the	O
-hydroxycholesterol	O
-derivatives	O
-and	O
-therefore	O
-would	O
-not	O
-trigger	O
-inverse	O
-agonism	O
-with	O
-these	O
-ligands	O
-(	O
-Fig	O
-.	O
-6b	O
-).	O
-	O
-BIO399	O
-shows	O
-selectivity	O
-for	O
-RORγ	O
-over	O
-RORα	O
-and	O
-RORβ	O
-in	O
-a	O
-GAL4	O
-Cellular	O
-Reporter	O
-Assay	O
-	O
-GAL4	O
-cell	O
-assay	O
-selectivity	O
-profile	O
-for	O
-BIO399	O
-toward	O
-RORα	O
-and	O
-RORβ	O
-in	O
-GAL4	O
-	O
-a	O
-Overlay	O
-of	O
-RORα	O
-(	O
-yellow	O
-),	O
-β	O
-(	O
-pink	O
-)	O
-and	O
-γ	O
-(	O
-cyan	O
-)	O
-showing	O
-side	O
-chain	O
-differences	O
-at	O
-Met358	O
-inverse	O
-agonism	O
-trigger	O
-position	O
-and	O
-(	O
-b	O
-)	O
-around	O
-the	O
-benzoxazinone	O
-ring	O
-system	O
-of	O
-BIO399	O
-	O
-In	O
-order	O
-to	O
-assess	O
-the	O
-in	O
-vivo	O
-selectivity	O
-profile	O
-of	O
-BIO399	O
-a	O
-cellular	O
-reporter	O
-assay	O
-was	O
-implemented	O
-where	O
-the	O
-ligand	O
-binding	O
-domains	O
-of	O
-ROR	O
-α	O
-,	O
-β	O
-and	O
-γ	O
-were	O
-fused	O
-to	O
-the	O
-DNA	O
-binding	O
-domain	O
-of	O
-the	O
-transcriptional	O
-factor	O
-GAL4	O
-.	O
-	O
-The	O
-ROR	O
--	O
-GAL4	O
-fusion	O
-proteins	O
-were	O
-expressed	O
-in	O
-cells	O
-with	O
-the	O
-luciferase	O
-reporter	O
-gene	O
-under	O
-the	O
-control	O
-of	O
-a	O
-GAL4	O
-promoter	O
-.	O
-	O
-BIO399	O
-inhibited	O
-the	O
-luciferase	O
-activity	O
-when	O
-added	O
-to	O
-the	O
-cells	O
-expressing	O
-the	O
-RORγ	O
--	O
-GAL4	O
-fusion	O
-with	O
-an	O
-in	O
-vivo	O
-IC50	O
-of	O
-42	O
-.	O
-5nM	O
-while	O
-showing	O
->	O
-235	O
-and	O
-28	O
-fold	O
-selectivity	O
-over	O
-cells	O
-expressing	O
-GAL4	O
-fused	O
-to	O
-the	O
-LBD	O
-of	O
-ROR	O
-α	O
-or	O
-β	O
-,	O
-respectively	O
-(	O
-Table	O
-1	O
-).	O
-	O
-The	O
-LBS	O
-of	O
-RORs	O
-share	O
-a	O
-high	O
-degree	O
-of	O
-similarity	O
-.	O
-	O
-However	O
-,	O
-the	O
-inverse	O
-agonism	O
-trigger	O
-of	O
-BIO399	O
-,	O
-residue	O
-Met358	O
-,	O
-is	O
-a	O
-leucine	O
-in	O
-both	O
-RORα	O
-and	O
-β	O
-.	O
-	O
-This	O
-selectivity	O
-profile	O
-for	O
-BIO399	O
-is	O
-attributed	O
-to	O
-the	O
-shorter	O
-leucine	O
-side	O
-chain	O
-in	O
-RORα	O
-and	O
-β	O
-which	O
-would	O
-not	O
-reach	O
-the	O
-phenylalanine	O
-on	O
-the	O
-AF2	O
-helix	O
-further	O
-underscoring	O
-the	O
-role	O
-of	O
-Met358	O
-as	O
-a	O
-trigger	O
-for	O
-RORγ	O
-specific	O
-inverse	O
-agonism	O
-(	O
-Fig	O
-.	O
-7a	O
-).	O
-	O
-Furthermore	O
-,	O
-RORα	O
-contains	O
-two	O
-phenylalanine	O
-residues	O
-in	O
-its	O
-LBS	O
-whereas	O
-RORβ	O
-and	O
-γ	O
-have	O
-a	O
-leucine	O
-in	O
-the	O
-same	O
-position	O
-(	O
-Fig	O
-.	O
-6b	O
-).	O
-	O
-We	O
-hypothesize	O
-that	O
-the	O
-two	O
-phenylalanine	O
-residues	O
-in	O
-the	O
-LBS	O
-of	O
-RORα	O
-occlude	O
-the	O
-dihydrobenzoxazepinone	O
-ring	O
-system	O
-of	O
-BIO399	O
-from	O
-binding	O
-it	O
-and	O
-responsible	O
-for	O
-the	O
-increase	O
-in	O
-selectivity	O
-for	O
-RORα	O
-over	O
-β	O
-.	O
-	O
-We	O
-have	O
-identified	O
-a	O
-novel	O
-series	O
-of	O
-synthetic	O
-benzoxazinone	O
-ligands	O
-which	O
-modulate	O
-the	O
-transcriptional	O
-activity	O
-of	O
-RORγ	O
-in	O
-a	O
-FRET	O
-based	O
-assay	O
-.	O
-	O
-Using	O
-partial	O
-proteolysis	O
-we	O
-show	O
-a	O
-conformational	O
-change	O
-which	O
-destabilizes	O
-the	O
-AF2	O
-helix	O
-of	O
-RORγ	O
-when	O
-the	O
-inverse	O
-agonist	O
-BIO399	O
-binds	O
-.	O
-	O
-The	O
-two	O
-RORγ	O
-co	O
--	O
-crystal	O
-structures	O
-reported	O
-here	O
-show	O
-how	O
-a	O
-small	O
-change	O
-to	O
-the	O
-core	O
-ring	O
-system	O
-can	O
-modulate	O
-the	O
-mode	O
-of	O
-action	O
-from	O
-agonist	O
-(	O
-BIO592	O
-)	O
-to	O
-inverse	O
-agonism	O
-(	O
-BIO399	O
-).	O
-	O
-Finally	O
-,	O
-we	O
-are	O
-reporting	O
-a	O
-newly	O
-identified	O
-trigger	O
-for	O
-achieving	O
-RORγ	O
-specific	O
-inverse	O
-agonism	O
-in	O
-an	O
-in	O
-vivo	O
-setting	O
-through	O
-Met358	O
-which	O
-perturbs	O
-the	O
-agonist	O
-conformation	O
-of	O
-the	O
-AF2	O
-helix	O
-and	O
-prevents	O
-coactivator	O
-protein	O
-binding	O
-.	O
-	O
-The	O
-Structural	O
-Basis	O
-of	O
-Coenzyme	O
-A	O
-Recycling	O
-in	O
-a	O
-Bacterial	O
-Organelle	O
-	O
-Bacterial	O
-Microcompartments	O
-(	O
-BMCs	O
-)	O
-are	O
-proteinaceous	O
-organelles	O
-that	O
-encapsulate	O
-critical	O
-segments	O
-of	O
-autotrophic	O
-and	O
-heterotrophic	O
-metabolic	O
-pathways	O
-;	O
-they	O
-are	O
-functionally	O
-diverse	O
-and	O
-are	O
-found	O
-across	O
-23	O
-different	O
-phyla	O
-.	O
-	O
-The	O
-majority	O
-of	O
-catabolic	O
-BMCs	O
-(	O
-metabolosomes	O
-)	O
-compartmentalize	O
-a	O
-common	O
-core	O
-of	O
-enzymes	O
-to	O
-metabolize	O
-compounds	O
-via	O
-a	O
-toxic	O
-and	O
-/	O
-or	O
-volatile	O
-aldehyde	O
-intermediate	O
-.	O
-	O
-The	O
-core	O
-enzyme	O
-phosphotransacylase	O
-(	O
-PTAC	O
-)	O
-recycles	O
-Coenzyme	O
-A	O
-and	O
-generates	O
-an	O
-acyl	O
-phosphate	O
-that	O
-can	O
-serve	O
-as	O
-an	O
-energy	O
-source	O
-.	O
-	O
-The	O
-PTAC	O
-predominantly	O
-associated	O
-with	O
-metabolosomes	O
-(	O
-PduL	O
-)	O
-has	O
-no	O
-sequence	O
-homology	O
-to	O
-the	O
-PTAC	O
-ubiquitous	O
-among	O
-fermentative	O
-bacteria	O
-(	O
-Pta	O
-).	O
-	O
-Here	O
-,	O
-we	O
-report	O
-two	O
-high	O
--	O
-resolution	O
-PduL	O
-crystal	O
-structures	O
-with	O
-bound	O
-substrates	O
-.	O
-	O
-The	O
-PduL	O
-fold	O
-is	O
-unrelated	O
-to	O
-that	O
-of	O
-Pta	O
-;	O
-it	O
-contains	O
-a	O
-dimetal	O
-active	O
-site	O
-involved	O
-in	O
-a	O
-catalytic	O
-mechanism	O
-distinct	O
-from	O
-that	O
-of	O
-the	O
-housekeeping	O
-PTAC	O
-.	O
-	O
-Accordingly	O
-,	O
-PduL	O
-and	O
-Pta	O
-exemplify	O
-functional	O
-,	O
-but	O
-not	O
-structural	O
-,	O
-convergent	O
-evolution	O
-.	O
-	O
-The	O
-PduL	O
-structure	O
-,	O
-in	O
-the	O
-context	O
-of	O
-the	O
-catalytic	O
-core	O
-,	O
-completes	O
-our	O
-understanding	O
-of	O
-the	O
-structural	O
-basis	O
-of	O
-cofactor	O
-recycling	O
-in	O
-the	O
-metabolosome	O
-lumen	O
-.	O
-	O
-This	O
-study	O
-describes	O
-the	O
-structure	O
-of	O
-a	O
-novel	O
-phosphotransacylase	O
-enzyme	O
-that	O
-facilitates	O
-the	O
-recycling	O
-of	O
-the	O
-essential	O
-cofactor	O
-acetyl	O
--	O
-CoA	O
-within	O
-a	O
-bacterial	O
-organelle	O
-and	O
-discusses	O
-the	O
-properties	O
-of	O
-the	O
-enzyme	O
-'	O
-s	O
-active	O
-site	O
-and	O
-how	O
-it	O
-is	O
-packaged	O
-into	O
-the	O
-organelle	O
-.	O
-	O
-In	O
-metabolism	O
-,	O
-molecules	O
-with	O
-“	O
-high	O
--	O
-energy	O
-”	O
-bonds	O
-(	O
-e	O
-.	O
-g	O
-.,	O
-ATP	O
-and	O
-Acetyl	O
-~	O
-CoA	O
-)	O
-are	O
-critical	O
-for	O
-both	O
-catabolic	O
-and	O
-anabolic	O
-processes	O
-.	O
-	O
-The	O
-phosphotransacylase	O
-(	O
-Pta	O
-)	O
-enzyme	O
-catalyzes	O
-the	O
-conversion	O
-between	O
-acyl	O
--	O
-CoA	O
-and	O
-acyl	O
--	O
-phosphate	O
-.	O
-	O
-This	O
-reaction	O
-directly	O
-links	O
-an	O
-acyl	O
--	O
-CoA	O
-with	O
-ATP	O
-generation	O
-via	O
-substrate	O
--	O
-level	O
-phosphorylation	O
-,	O
-producing	O
-short	O
--	O
-chain	O
-fatty	O
-acids	O
-(	O
-e	O
-.	O
-g	O
-.,	O
-acetate	O
-),	O
-and	O
-also	O
-provides	O
-a	O
-path	O
-for	O
-short	O
--	O
-chain	O
-fatty	O
-acids	O
-to	O
-enter	O
-central	O
-metabolism	O
-.	O
-	O
-Due	O
-to	O
-this	O
-key	O
-function	O
-,	O
-Pta	O
-is	O
-conserved	O
-across	O
-the	O
-bacterial	O
-kingdom	O
-.	O
-	O
-Recently	O
-,	O
-a	O
-new	O
-type	O
-of	O
-phosphotransacylase	O
-was	O
-described	O
-that	O
-shares	O
-no	O
-evolutionary	O
-relation	O
-to	O
-Pta	O
-.	O
-	O
-This	O
-enzyme	O
-,	O
-PduL	O
-,	O
-is	O
-exclusively	O
-associated	O
-with	O
-organelles	O
-called	O
-bacterial	O
-microcompartments	O
-,	O
-which	O
-are	O
-used	O
-to	O
-catabolize	O
-various	O
-compounds	O
-.	O
-	O
-Not	O
-only	O
-does	O
-PduL	O
-facilitate	O
-substrate	O
-level	O
-phosphorylation	O
-,	O
-but	O
-it	O
-also	O
-is	O
-critical	O
-for	O
-cofactor	O
-recycling	O
-within	O
-,	O
-and	O
-product	O
-efflux	O
-from	O
-,	O
-the	O
-organelle	O
-.	O
-	O
-We	O
-solved	O
-the	O
-structure	O
-of	O
-this	O
-convergent	O
-phosphotransacylase	O
-and	O
-show	O
-that	O
-it	O
-is	O
-completely	O
-structurally	O
-different	O
-from	O
-Pta	O
-,	O
-including	O
-its	O
-active	O
-site	O
-architecture	O
-.	O
-	O
-Bacterial	O
-Microcompartments	O
-(	O
-BMCs	O
-)	O
-are	O
-organelles	O
-that	O
-encapsulate	O
-enzymes	O
-for	O
-sequential	O
-biochemical	O
-reactions	O
-within	O
-a	O
-protein	O
-shell	O
-.	O
-	O
-The	O
-shell	O
-is	O
-typically	O
-composed	O
-of	O
-three	O
-types	O
-of	O
-protein	O
-subunits	O
-,	O
-which	O
-form	O
-either	O
-hexagonal	O
-(	O
-BMC	O
--	O
-H	O
-and	O
-BMC	O
--	O
-T	O
-)	O
-or	O
-pentagonal	O
-(	O
-BMC	O
--	O
-P	O
-)	O
-tiles	O
-that	O
-assemble	O
-into	O
-a	O
-polyhedral	O
-shell	O
-.	O
-	O
-The	O
-facets	O
-of	O
-the	O
-shell	O
-are	O
-composed	O
-primarily	O
-of	O
-hexamers	O
-that	O
-are	O
-typically	O
-perforated	O
-by	O
-pores	O
-lined	O
-with	O
-highly	O
-conserved	O
-,	O
-polar	O
-residues	O
-that	O
-presumably	O
-function	O
-as	O
-the	O
-conduits	O
-for	O
-metabolites	O
-into	O
-and	O
-out	O
-of	O
-the	O
-shell	O
-.	O
-	O
-The	O
-vitamin	O
-B12	O
--	O
-dependent	O
-propanediol	O
--	O
-utilizing	O
-(	O
-PDU	O
-)	O
-BMC	O
-was	O
-one	O
-of	O
-the	O
-first	O
-functionally	O
-characterized	O
-catabolic	O
-BMCs	O
-;	O
-subsequently	O
-,	O
-other	O
-types	O
-have	O
-been	O
-implicated	O
-in	O
-the	O
-degradation	O
-of	O
-ethanolamine	O
-,	O
-choline	O
-,	O
-fucose	O
-,	O
-rhamnose	O
-,	O
-and	O
-ethanol	O
-,	O
-all	O
-of	O
-which	O
-produce	O
-different	O
-aldehyde	O
-intermediates	O
-(	O
-Table	O
-1	O
-).	O
-	O
-More	O
-recently	O
-,	O
-bioinformatic	O
-studies	O
-have	O
-demonstrated	O
-the	O
-widespread	O
-distribution	O
-of	O
-BMCs	O
-among	O
-diverse	O
-bacterial	O
-phyla	O
-and	O
-grouped	O
-them	O
-into	O
-23	O
-different	O
-functional	O
-types	O
-.	O
-	O
-The	O
-reactions	O
-carried	O
-out	O
-in	O
-the	O
-majority	O
-of	O
-catabolic	O
-BMCs	O
-(	O
-also	O
-known	O
-as	O
-metabolosomes	O
-)	O
-fit	O
-a	O
-generalized	O
-biochemical	O
-paradigm	O
-for	O
-the	O
-oxidation	O
-of	O
-aldehydes	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-This	O
-involves	O
-a	O
-BMC	O
--	O
-encapsulated	O
-signature	O
-enzyme	O
-that	O
-generates	O
-a	O
-toxic	O
-and	O
-/	O
-or	O
-volatile	O
-aldehyde	O
-that	O
-the	O
-BMC	O
-shell	O
-sequesters	O
-from	O
-the	O
-cytosol	O
-.	O
-	O
-The	O
-aldehyde	O
-is	O
-subsequently	O
-converted	O
-into	O
-an	O
-acyl	O
--	O
-CoA	O
-by	O
-aldehyde	O
-dehydrogenase	O
-,	O
-which	O
-uses	O
-NAD	O
-+	O
-and	O
-CoA	O
-as	O
-cofactors	O
-.	O
-	O
-These	O
-two	O
-cofactors	O
-are	O
-relatively	O
-large	O
-,	O
-and	O
-their	O
-diffusion	O
-across	O
-the	O
-protein	O
-shell	O
-is	O
-thought	O
-to	O
-be	O
-restricted	O
-,	O
-necessitating	O
-their	O
-regeneration	O
-within	O
-the	O
-BMC	O
-lumen	O
-.	O
-	O
-NAD	O
-+	O
-is	O
-recycled	O
-via	O
-alcohol	O
-dehydrogenase	O
-,	O
-and	O
-CoA	O
-is	O
-recycled	O
-via	O
-phosphotransacetylase	O
-(	O
-PTAC	O
-)	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-The	O
-final	O
-product	O
-of	O
-the	O
-BMC	O
-,	O
-an	O
-acyl	O
--	O
-phosphate	O
-,	O
-can	O
-then	O
-be	O
-used	O
-to	O
-generate	O
-ATP	O
-via	O
-acyl	O
-kinase	O
-,	O
-or	O
-revert	O
-back	O
-to	O
-acyl	O
--	O
-CoA	O
-by	O
-Pta	O
-for	O
-biosynthesis	O
-.	O
-	O
-Collectively	O
-,	O
-the	O
-aldehyde	O
-and	O
-alcohol	O
-dehydrogenases	O
-,	O
-as	O
-well	O
-as	O
-the	O
-PTAC	O
-,	O
-constitute	O
-the	O
-common	O
-metabolosome	O
-core	O
-.	O
-	O
-General	O
-biochemical	O
-model	O
-of	O
-aldehyde	O
--	O
-degrading	O
-BMCs	O
-(	O
-metabolosomes	O
-)	O
-illustrating	O
-the	O
-common	O
-metabolosome	O
-core	O
-enzymes	O
-and	O
-reactions	O
-.	O
-	O
-Substrates	O
-and	O
-cofactors	O
-involving	O
-the	O
-PTAC	O
-reaction	O
-are	O
-shown	O
-in	O
-red	O
-;	O
-other	O
-substrates	O
-and	O
-enzymes	O
-are	O
-shown	O
-in	O
-black	O
-,	O
-and	O
-other	O
-cofactors	O
-are	O
-shown	O
-in	O
-gray	O
-.	O
-	O
-Characterized	O
-and	O
-predicted	O
-catabolic	O
-BMC	O
-(	O
-metabolosome	O
-)	O
-types	O
-that	O
-represent	O
-the	O
-aldehyde	O
--	O
-degrading	O
-paradigm	O
-(	O
-for	O
-definition	O
-of	O
-types	O
-see	O
-Kerfeld	O
-and	O
-Erbilgin	O
-).	O
-	O
-Name	O
-PTAC	O
-Type	O
-Sequestered	O
-Aldehyde	O
-PDU	O
-*	O
-PduL	O
-propionaldehyde	O
-EUT1	O
-PTA_PTB	O
-acetaldehyde	O
-EUT2	O
-PduL	O
-acetaldehyde	O
-ETU	O
-None	O
-acetaldehyde	O
-GRM1	O
-/	O
-CUT	O
-PduL	O
-acetaldehyde	O
-GRM2	O
-PduL	O
-acetaldehyde	O
-GRM3	O
-*,	O
-4	O
-PduL	O
-propionaldehyde	O
-GRM5	O
-/	O
-GRP	O
-PduL	O
-propionaldehyde	O
-PVM	O
-*	O
-PduL	O
-lactaldehyde	O
-RMM1	O
-,	O
-2	O
-None	O
-unknown	O
-SPU	O
-PduL	O
-unknown	O
-	O
-*	O
-PduL	O
-from	O
-these	O
-functional	O
-types	O
-of	O
-metabolosomes	O
-were	O
-purified	O
-in	O
-this	O
-study	O
-.	O
-	O
-The	O
-activities	O
-of	O
-core	O
-enzymes	O
-are	O
-not	O
-confined	O
-to	O
-BMC	O
--	O
-associated	O
-functions	O
-:	O
-aldehyde	O
-and	O
-alcohol	O
-dehydrogenases	O
-are	O
-utilized	O
-in	O
-diverse	O
-metabolic	O
-reactions	O
-,	O
-and	O
-PTAC	O
-catalyzes	O
-a	O
-key	O
-biochemical	O
-reaction	O
-in	O
-the	O
-process	O
-of	O
-obtaining	O
-energy	O
-during	O
-fermentation	O
-.	O
-	O
-The	O
-concerted	O
-functioning	O
-of	O
-a	O
-PTAC	O
-and	O
-an	O
-acetate	O
-kinase	O
-(	O
-Ack	O
-)	O
-is	O
-crucial	O
-for	O
-ATP	O
-generation	O
-in	O
-the	O
-fermentation	O
-of	O
-pyruvate	O
-to	O
-acetate	O
-(	O
-see	O
-Reactions	O
-1	O
-and	O
-2	O
-).	O
-	O
-Both	O
-enzymes	O
-are	O
-,	O
-however	O
-,	O
-not	O
-restricted	O
-to	O
-fermentative	O
-organisms	O
-.	O
-	O
-They	O
-can	O
-also	O
-work	O
-in	O
-the	O
-reverse	O
-direction	O
-to	O
-activate	O
-acetate	O
-to	O
-the	O
-CoA	O
--	O
-thioester	O
-.	O
-	O
-This	O
-occurs	O
-,	O
-for	O
-example	O
-,	O
-during	O
-acetoclastic	O
-methanogenesis	O
-in	O
-the	O
-archaeal	O
-Methanosarcina	O
-species	O
-.	O
-	O
-Reaction	O
-1	O
-:	O
-acetyl	O
--	O
-S	O
--	O
-CoA	O
-+	O
-Pi	O
-←→	O
-acetyl	O
-phosphate	O
-+	O
-CoA	O
--	O
-SH	O
-(	O
-PTAC	O
-)	O
-	O
-Reaction	O
-2	O
-:	O
-acetyl	O
-phosphate	O
-+	O
-ADP	O
-←→	O
-acetate	O
-+	O
-ATP	O
-(	O
-Ack	O
-)	O
-	O
-The	O
-canonical	O
-PTAC	O
-,	O
-Pta	O
-,	O
-is	O
-an	O
-ancient	O
-enzyme	O
-found	O
-in	O
-some	O
-eukaryotes	O
-and	O
-archaea	O
-,	O
-and	O
-widespread	O
-among	O
-the	O
-bacteria	O
-;	O
-90	O
-%	O
-of	O
-the	O
-bacterial	O
-genomes	O
-in	O
-the	O
-Integrated	O
-Microbial	O
-Genomes	O
-database	O
-contain	O
-a	O
-gene	O
-encoding	O
-the	O
-PTA_PTB	O
-phosphotransacylase	O
-(	O
-Pfam	O
-domain	O
-PF01515	O
-).	O
-	O
-Pta	O
-has	O
-been	O
-extensively	O
-characterized	O
-due	O
-to	O
-its	O
-key	O
-role	O
-in	O
-fermentation	O
-.	O
-	O
-More	O
-recently	O
-,	O
-a	O
-second	O
-type	O
-of	O
-PTAC	O
-without	O
-any	O
-sequence	O
-homology	O
-to	O
-Pta	O
-was	O
-identified	O
-.	O
-	O
-This	O
-protein	O
-,	O
-PduL	O
-(	O
-Pfam	O
-domain	O
-PF06130	O
-),	O
-was	O
-shown	O
-to	O
-catalyze	O
-the	O
-conversion	O
-of	O
-propionyl	O
--	O
-CoA	O
-to	O
-propionyl	O
--	O
-phosphate	O
-and	O
-is	O
-associated	O
-with	O
-a	O
-BMC	O
-involved	O
-in	O
-propanediol	O
-utilization	O
-,	O
-the	O
-PDU	O
-BMC	O
-.	O
-	O
-Both	O
-pduL	O
-and	O
-pta	O
-genes	O
-can	O
-be	O
-found	O
-in	O
-genetic	O
-loci	O
-of	O
-functionally	O
-distinct	O
-BMCs	O
-,	O
-although	O
-the	O
-PduL	O
-type	O
-is	O
-much	O
-more	O
-prevalent	O
-,	O
-being	O
-found	O
-in	O
-all	O
-but	O
-one	O
-type	O
-of	O
-metabolosome	O
-locus	O
-:	O
-EUT1	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Furthermore	O
-,	O
-in	O
-the	O
-Integrated	O
-Microbial	O
-Genomes	O
-Database	O
-,	O
-91	O
-%	O
-of	O
-genomes	O
-that	O
-encode	O
-PF06130	O
-also	O
-encode	O
-genes	O
-for	O
-shell	O
-proteins	O
-.	O
-	O
-As	O
-a	O
-member	O
-of	O
-the	O
-core	O
-biochemical	O
-machinery	O
-of	O
-functionally	O
-diverse	O
-aldehyde	O
--	O
-oxidizing	O
-metabolosomes	O
-,	O
-PduL	O
-must	O
-have	O
-a	O
-certain	O
-level	O
-of	O
-substrate	O
-plasticity	O
-(	O
-see	O
-Table	O
-1	O
-)	O
-that	O
-is	O
-not	O
-required	O
-of	O
-Pta	O
-,	O
-which	O
-has	O
-generally	O
-been	O
-observed	O
-to	O
-prefer	O
-acetyl	O
--	O
-CoA	O
-.	O
-PduL	O
-from	O
-the	O
-PDU	O
-BMC	O
-of	O
-Salmonella	O
-enterica	O
-favors	O
-propionyl	O
--	O
-CoA	O
-over	O
-acetyl	O
--	O
-CoA	O
-,	O
-and	O
-it	O
-is	O
-likely	O
-that	O
-PduL	O
-orthologs	O
-in	O
-functionally	O
-diverse	O
-BMCs	O
-would	O
-have	O
-substrate	O
-preferences	O
-for	O
-other	O
-CoA	O
-derivatives	O
-.	O
-	O
-Another	O
-distinctive	O
-feature	O
-of	O
-BMC	O
--	O
-associated	O
-PduL	O
-homologs	O
-is	O
-an	O
-N	O
--	O
-terminal	O
-encapsulation	O
-peptide	O
-(	O
-EP	O
-)	O
-that	O
-is	O
-thought	O
-to	O
-“	O
-target	O
-”	O
-proteins	O
-for	O
-encapsulation	O
-by	O
-the	O
-BMC	O
-shell	O
-.	O
-	O
-EPs	O
-are	O
-frequently	O
-found	O
-on	O
-BMC	O
--	O
-associated	O
-proteins	O
-and	O
-have	O
-been	O
-shown	O
-to	O
-interact	O
-with	O
-shell	O
-proteins	O
-.	O
-	O
-EPs	O
-have	O
-also	O
-been	O
-observed	O
-to	O
-cause	O
-proteins	O
-to	O
-aggregate	O
-,	O
-and	O
-this	O
-has	O
-recently	O
-been	O
-suggested	O
-to	O
-be	O
-functionally	O
-relevant	O
-as	O
-an	O
-initial	O
-step	O
-in	O
-metabolosome	O
-assembly	O
-,	O
-in	O
-which	O
-a	O
-multifunctional	O
-protein	O
-core	O
-is	O
-formed	O
-,	O
-around	O
-which	O
-the	O
-shell	O
-assembles	O
-.	O
-	O
-Of	O
-the	O
-three	O
-common	O
-metabolosome	O
-core	O
-enzymes	O
-,	O
-crystal	O
-structures	O
-are	O
-available	O
-for	O
-both	O
-the	O
-alcohol	O
-and	O
-aldehyde	O
-dehydrogenases	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-structure	O
-of	O
-PduL	O
-,	O
-the	O
-PTAC	O
-found	O
-in	O
-the	O
-vast	O
-majority	O
-of	O
-catabolic	O
-BMCs	O
-,	O
-has	O
-not	O
-been	O
-determined	O
-.	O
-	O
-This	O
-is	O
-a	O
-major	O
-gap	O
-in	O
-our	O
-understanding	O
-of	O
-metabolosome	O
--	O
-encapsulated	O
-biochemistry	O
-and	O
-cofactor	O
-recycling	O
-.	O
-	O
-Moreover	O
-,	O
-it	O
-will	O
-be	O
-useful	O
-for	O
-guiding	O
-efforts	O
-to	O
-engineer	O
-novel	O
-BMC	O
-cores	O
-for	O
-biotechnological	O
-applications	O
-.	O
-	O
-The	O
-primary	O
-structure	O
-of	O
-PduL	O
-homologs	O
-is	O
-subdivided	O
-into	O
-two	O
-PF06130	O
-domains	O
-,	O
-each	O
-roughly	O
-80	O
-residues	O
-in	O
-length	O
-.	O
-	O
-No	O
-available	O
-protein	O
-structures	O
-contain	O
-the	O
-PF06130	O
-domain	O
-,	O
-and	O
-homology	O
-searches	O
-using	O
-the	O
-primary	O
-structure	O
-of	O
-PduL	O
-do	O
-not	O
-return	O
-any	O
-significant	O
-results	O
-that	O
-would	O
-allow	O
-prediction	O
-of	O
-the	O
-structure	O
-.	O
-	O
-Moreover	O
-,	O
-the	O
-evident	O
-novelty	O
-of	O
-PduL	O
-makes	O
-its	O
-structure	O
-interesting	O
-in	O
-the	O
-context	O
-of	O
-convergent	O
-evolution	O
-of	O
-PTAC	O
-function	O
-;	O
-to	O
--	O
-date	O
-,	O
-only	O
-the	O
-Pta	O
-active	O
-site	O
-and	O
-catalytic	O
-mechanism	O
-is	O
-known	O
-.	O
-	O
-Here	O
-we	O
-report	O
-high	O
--	O
-resolution	O
-crystal	O
-structures	O
-of	O
-a	O
-PduL	O
--	O
-type	O
-PTAC	O
-in	O
-both	O
-CoA	O
--	O
-and	O
-phosphate	O
--	O
-bound	O
-forms	O
-,	O
-completing	O
-our	O
-understanding	O
-of	O
-the	O
-structural	O
-basis	O
-of	O
-catalysis	O
-by	O
-the	O
-metabolosome	O
-common	O
-core	O
-enzymes	O
-.	O
-	O
-We	O
-propose	O
-a	O
-catalytic	O
-mechanism	O
-analogous	O
-but	O
-yet	O
-distinct	O
-from	O
-the	O
-ubiquitous	O
-Pta	O
-enzyme	O
-,	O
-highlighting	O
-the	O
-functional	O
-convergence	O
-of	O
-two	O
-enzymes	O
-with	O
-completely	O
-different	O
-structures	O
-and	O
-metal	O
-requirements	O
-.	O
-	O
-We	O
-also	O
-investigate	O
-the	O
-quaternary	O
-structures	O
-of	O
-three	O
-different	O
-PduL	O
-homologs	O
-and	O
-situate	O
-our	O
-findings	O
-in	O
-the	O
-context	O
-of	O
-organelle	O
-biogenesis	O
-in	O
-functionally	O
-diverse	O
-BMCs	O
-.	O
-	O
-Structure	O
-Determination	O
-of	O
-PduL	O
-	O
-We	O
-cloned	O
-,	O
-expressed	O
-,	O
-and	O
-purified	O
-three	O
-different	O
-PduL	O
-homologs	O
-from	O
-functionally	O
-distinct	O
-BMCs	O
-(	O
-Table	O
-1	O
-):	O
-from	O
-the	O
-well	O
--	O
-studied	O
-pdu	O
-locus	O
-in	O
-S	O
-.	O
-enterica	O
-Typhimurium	O
-LT2	O
-(	O
-sPduL	O
-),	O
-from	O
-the	O
-recently	O
-characterized	O
-pvm	O
-locus	O
-in	O
-Planctomyces	O
-limnophilus	O
-(	O
-pPduL	O
-),	O
-and	O
-from	O
-the	O
-grm3	O
-locus	O
-in	O
-Rhodopseudomonas	O
-palustris	O
-BisB18	O
-(	O
-rPduL	O
-).	O
-	O
-While	O
-purifying	O
-full	O
--	O
-length	O
-sPduL	O
-,	O
-we	O
-observed	O
-a	O
-tendency	O
-to	O
-aggregation	O
-as	O
-described	O
-previously	O
-,	O
-with	O
-a	O
-large	O
-fraction	O
-of	O
-the	O
-expressed	O
-protein	O
-found	O
-in	O
-the	O
-insoluble	O
-fraction	O
-in	O
-a	O
-white	O
-,	O
-cake	O
--	O
-like	O
-pellet	O
-.	O
-	O
-Remarkably	O
-,	O
-after	O
-removing	O
-the	O
-N	O
--	O
-terminal	O
-putative	O
-EP	O
-(	O
-27	O
-amino	O
-acids	O
-),	O
-most	O
-of	O
-the	O
-sPduLΔEP	B-mutant
-protein	O
-was	O
-in	O
-the	O
-soluble	O
-fraction	O
-upon	O
-cell	O
-lysis	O
-.	O
-	O
-Similar	O
-differences	O
-in	O
-solubility	O
-were	O
-observed	O
-for	O
-pPduL	O
-and	O
-rPduL	O
-when	O
-comparing	O
-EP	O
--	O
-truncated	O
-forms	O
-to	O
-the	O
-full	O
--	O
-length	O
-protein	O
-,	O
-but	O
-none	O
-were	O
-quite	O
-as	O
-dramatic	O
-as	O
-for	O
-sPduL	O
-.	O
-We	O
-confirmed	O
-that	O
-all	O
-homologs	O
-were	O
-active	O
-(	O
-S1a	O
-and	O
-S1b	O
-Fig	O
-).	O
-	O
-Among	O
-these	O
-,	O
-we	O
-were	O
-only	O
-able	O
-to	O
-obtain	O
-diffraction	O
--	O
-quality	O
-crystals	O
-of	O
-rPduL	O
-after	O
-removing	O
-the	O
-N	O
--	O
-terminal	O
-putative	O
-EP	O
-(	O
-33	O
-amino	O
-acids	O
-,	O
-also	O
-see	O
-Fig	O
-2a	O
-)	O
-(	O
-rPduLΔEP	B-mutant
-).	O
-	O
-Truncated	O
-rPduLΔEP	B-mutant
-had	O
-comparable	O
-enzymatic	O
-activity	O
-to	O
-the	O
-full	O
--	O
-length	O
-enzyme	O
-(	O
-S1a	O
-Fig	O
-).	O
-	O
-Structural	O
-overview	O
-of	O
-R	O
-.	O
-palustris	O
-PduL	O
-from	O
-the	O
-grm3	O
-locus	O
-.	O
-	O
-(	O
-a	O
-)	O
-Primary	O
-and	O
-secondary	O
-structure	O
-of	O
-rPduL	O
-(	O
-tubes	O
-represent	O
-α	O
--	O
-helices	O
-,	O
-arrows	O
-β	O
--	O
-sheets	O
-and	O
-dashed	O
-line	O
-residues	O
-disordered	O
-in	O
-the	O
-structure	O
-.	O
-	O
-The	O
-first	O
-33	O
-amino	O
-acids	O
-are	O
-present	O
-only	O
-in	O
-the	O
-wildtype	O
-construct	O
-and	O
-contains	O
-the	O
-predicted	O
-EP	O
-alpha	O
-helix	O
-,	O
-α0	O
-);	O
-the	O
-truncated	O
-rPduLΔEP	B-mutant
-that	O
-was	O
-crystallized	O
-begins	O
-with	O
-M	O
--	O
-G	O
--	O
-V	O
-.	O
-Coloring	O
-is	O
-according	O
-to	O
-structural	O
-domains	O
-(	O
-domain	O
-1	O
-D36	O
--	O
-N46	O
-/	O
-Q155	O
--	O
-C224	O
-,	O
-blue	O
-;	O
-loop	O
-insertion	O
-G61	O
--	O
-E81	O
-,	O
-grey	O
-;	O
-domain	O
-2	O
-R47	O
--	O
-F60	O
-/	O
-E82	O
--	O
-A154	O
-,	O
-red	O
-).	O
-	O
-Metal	O
-coordination	O
-residues	O
-are	O
-highlighted	O
-in	O
-light	O
-blue	O
-and	O
-CoA	O
-contacting	O
-residues	O
-in	O
-magenta	O
-,	O
-residues	O
-contacting	O
-the	O
-CoA	O
-of	O
-the	O
-other	O
-chain	O
-are	O
-also	O
-outlined	O
-.	O
-	O
-(	O
-b	O
-)	O
-Cartoon	O
-representation	O
-of	O
-the	O
-structure	O
-colored	O
-by	O
-domains	O
-and	O
-including	O
-secondary	O
-structure	O
-numbering	O
-.	O
-	O
-Coenzyme	O
-A	O
-is	O
-shown	O
-in	O
-magenta	O
-sticks	O
-and	O
-Zinc	O
-(	O
-grey	O
-)	O
-as	O
-spheres	O
-.	O
-	O
-We	O
-collected	O
-a	O
-native	O
-dataset	O
-from	O
-rPduLΔEP	B-mutant
-crystals	O
-diffracting	O
-to	O
-a	O
-resolution	O
-of	O
-1	O
-.	O
-54	O
-Å	O
-(	O
-Table	O
-2	O
-).	O
-	O
-Using	O
-a	O
-mercury	O
--	O
-derivative	O
-crystal	O
-form	O
-diffracting	O
-to	O
-1	O
-.	O
-99	O
-Å	O
-(	O
-Table	O
-2	O
-),	O
-we	O
-obtained	O
-high	O
-quality	O
-electron	O
-density	O
-for	O
-model	O
-building	O
-and	O
-used	O
-the	O
-initial	O
-model	O
-to	O
-refine	O
-against	O
-the	O
-native	O
-data	O
-to	O
-Rwork	O
-/	O
-Rfree	O
-values	O
-of	O
-18	O
-.	O
-9	O
-/	O
-22	O
-.	O
-1	O
-%.	O
-	O
-There	O
-are	O
-two	O
-PduL	O
-molecules	O
-in	O
-the	O
-asymmetric	O
-unit	O
-of	O
-the	O
-P212121	O
-unit	O
-cell	O
-.	O
-	O
-We	O
-were	O
-able	O
-to	O
-fit	O
-all	O
-of	O
-the	O
-primary	O
-structure	O
-of	O
-PduLΔEP	B-mutant
-into	O
-the	O
-electron	O
-density	O
-with	O
-the	O
-exception	O
-of	O
-three	O
-amino	O
-acids	O
-at	O
-the	O
-N	O
--	O
-terminus	O
-and	O
-two	O
-amino	O
-acids	O
-at	O
-the	O
-C	O
--	O
-terminus	O
-(	O
-Fig	O
-2a	O
-);	O
-the	O
-model	O
-is	O
-of	O
-excellent	O
-quality	O
-(	O
-Table	O
-2	O
-).	O
-	O
-A	O
-CoA	O
-cofactor	O
-as	O
-well	O
-as	O
-two	O
-metal	O
-ions	O
-are	O
-clearly	O
-resolved	O
-in	O
-the	O
-density	O
-(	O
-for	O
-omit	O
-maps	O
-of	O
-CoA	O
-see	O
-S2	O
-Fig	O
-).	O
-	O
-Structurally	O
-,	O
-PduL	O
-consists	O
-of	O
-two	O
-domains	O
-(	O
-Fig	O
-2	O
-,	O
-blue	O
-/	O
-red	O
-),	O
-each	O
-a	O
-beta	O
--	O
-barrel	O
-that	O
-is	O
-capped	O
-on	O
-both	O
-ends	O
-by	O
-short	O
-α	O
--	O
-helices	O
-.	O
-	O
-β	O
--	O
-Barrel	O
-1	O
-consists	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-β	O
-strand	O
-and	O
-β	O
-strands	O
-from	O
-the	O
-C	O
--	O
-terminal	O
-half	O
-of	O
-the	O
-polypeptide	O
-chain	O
-(	O
-β1	O
-,	O
-β10	O
--	O
-β14	O
-;	O
-residues	O
-37	O
-–	O
-46	O
-and	O
-155	O
-–	O
-224	O
-).	O
-	O
-β	O
--	O
-Barrel	O
-2	O
-consists	O
-mainly	O
-of	O
-the	O
-central	O
-segment	O
-of	O
-primary	O
-structure	O
-(	O
-β2	O
-,	O
-β5	O
-–	O
-β9	O
-;	O
-residues	O
-47	O
-–	O
-60	O
-and	O
-82	O
-–	O
-154	O
-)	O
-(	O
-Fig	O
-2	O
-,	O
-red	O
-),	O
-but	O
-is	O
-interrupted	O
-by	O
-a	O
-short	O
-two	O
--	O
-strand	O
-beta	O
-sheet	O
-(	O
-β3	O
--	O
-β4	O
-,	O
-residues	O
-61	O
-–	O
-81	O
-).	O
-	O
-This	O
-β	O
--	O
-sheet	O
-is	O
-involved	O
-in	O
-contacts	O
-between	O
-the	O
-two	O
-domains	O
-and	O
-forms	O
-a	O
-lid	O
-over	O
-the	O
-active	O
-site	O
-.	O
-	O
-Residues	O
-in	O
-this	O
-region	O
-(	O
-Gln42	O
-,	O
-Pro43	O
-,	O
-Gly44	O
-),	O
-covering	O
-the	O
-active	O
-site	O
-,	O
-are	O
-strongly	O
-conserved	O
-(	O
-Fig	O
-3	O
-).	O
-	O
-This	O
-structural	O
-arrangement	O
-is	O
-completely	O
-different	O
-from	O
-the	O
-functionally	O
-related	O
-Pta	O
-,	O
-which	O
-is	O
-composed	O
-of	O
-two	O
-domains	O
-,	O
-each	O
-consisting	O
-of	O
-a	O
-central	O
-flat	O
-beta	O
-sheet	O
-with	O
-alpha	O
--	O
-helices	O
-on	O
-the	O
-top	O
-and	O
-bottom	O
-.	O
-	O
-Primary	O
-structure	O
-conservation	O
-of	O
-the	O
-PduL	O
-protein	O
-family	O
-.	O
-	O
-Sequence	O
-logo	O
-calculated	O
-from	O
-the	O
-multiple	O
-sequence	O
-alignment	O
-of	O
-PduL	O
-homologs	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-),	O
-but	O
-not	O
-including	O
-putative	O
-EP	O
-sequences	O
-.	O
-	O
-Residues	O
-100	O
-%	O
-conserved	O
-across	O
-all	O
-PduL	O
-homologs	O
-in	O
-our	O
-dataset	O
-are	O
-noted	O
-with	O
-an	O
-asterisk	O
-,	O
-and	O
-residues	O
-conserved	O
-in	O
-over	O
-90	O
-%	O
-of	O
-sequences	O
-are	O
-noted	O
-with	O
-a	O
-colon	O
-.	O
-	O
-The	O
-sequences	O
-aligning	O
-to	O
-the	O
-PF06130	O
-domain	O
-(	O
-determined	O
-by	O
-BLAST	O
-)	O
-are	O
-highlighted	O
-in	O
-red	O
-and	O
-blue	O
-.	O
-	O
-The	O
-position	O
-numbers	O
-shown	O
-correspond	O
-to	O
-the	O
-residue	O
-numbering	O
-of	O
-rPduL	O
-;	O
-note	O
-that	O
-some	O
-positions	O
-in	O
-the	O
-logo	O
-represent	O
-gaps	O
-in	O
-the	O
-rPduL	O
-sequence	O
-.	O
-	O
-There	O
-are	O
-two	O
-PduL	O
-molecules	O
-in	O
-the	O
-asymmetric	O
-unit	O
-forming	O
-a	O
-butterfly	O
--	O
-shaped	O
-dimer	O
-(	O
-Fig	O
-4c	O
-).	O
-	O
-Consistent	O
-with	O
-this	O
-,	O
-results	O
-from	O
-size	O
-exclusion	O
-chromatography	O
-of	O
-rPduLΔEP	B-mutant
-suggest	O
-that	O
-it	O
-is	O
-a	O
-dimer	O
-in	O
-solution	O
-(	O
-Fig	O
-5e	O
-).	O
-	O
-The	O
-interface	O
-between	O
-the	O
-two	O
-chains	O
-buries	O
-882	O
-Å2	O
-per	O
-monomer	O
-and	O
-is	O
-mainly	O
-formed	O
-by	O
-α	O
--	O
-helices	O
-2	O
-and	O
-4	O
-and	O
-parts	O
-of	O
-β	O
--	O
-sheets	O
-12	O
-and	O
-14	O
-,	O
-as	O
-well	O
-as	O
-a	O
-π	O
-–	O
-π	O
-stacking	O
-of	O
-the	O
-adenine	O
-moiety	O
-of	O
-CoA	O
-with	O
-Phe116	O
-of	O
-the	O
-adjacent	O
-chain	O
-(	O
-Fig	O
-4c	O
-).	O
-	O
-The	O
-folds	O
-of	O
-the	O
-two	O
-chains	O
-in	O
-the	O
-asymmetric	O
-unit	O
-are	O
-very	O
-similar	O
-,	O
-superimposing	O
-with	O
-a	O
-rmsd	O
-of	O
-0	O
-.	O
-16	O
-Å	O
-over	O
-2	O
-,	O
-306	O
-aligned	O
-atom	O
-pairs	O
-.	O
-	O
-The	O
-peripheral	O
-helices	O
-and	O
-the	O
-short	O
-antiparallel	O
-β3	O
-–	O
-4	O
-sheet	O
-mediate	O
-most	O
-of	O
-the	O
-crystal	O
-contacts	O
-.	O
-	O
-Details	O
-of	O
-active	O
-site	O
-,	O
-dimeric	O
-assembly	O
-,	O
-and	O
-sequence	O
-conservation	O
-of	O
-PduL	O
-.	O
-	O
-(	O
-a	O
-,	O
-b	O
-)	O
-Proposed	O
-active	O
-site	O
-of	O
-PduL	O
-with	O
-relevant	O
-residues	O
-shown	O
-as	O
-sticks	O
-in	O
-atom	O
-coloring	O
-(	O
-nitrogen	O
-blue	O
-,	O
-oxygen	O
-red	O
-,	O
-sulfur	O
-yellow	O
-),	O
-zinc	O
-as	O
-grey	O
-colored	O
-spheres	O
-and	O
-coordinating	O
-ordered	O
-water	O
-molecules	O
-in	O
-red	O
-.	O
-	O
-Distances	O
-between	O
-atom	O
-centers	O
-are	O
-indicated	O
-in	O
-Å	O
-.	O
-(	O
-a	O
-)	O
-Coenzyme	O
-A	O
-containing	O
-,	O
-(	O
-b	O
-)	O
-phosphate	O
--	O
-bound	O
-structure	O
-.	O
-	O
-(	O
-c	O
-)	O
-View	O
-of	O
-the	O
-dimer	O
-in	O
-the	O
-asymmetric	O
-unit	O
-from	O
-the	O
-side	O
-,	O
-domains	O
-1	O
-and	O
-2	O
-colored	O
-as	O
-in	O
-Fig	O
-2	O
-and	O
-the	O
-two	O
-chains	O
-differentiated	O
-by	O
-blue	O
-/	O
-red	O
-versus	O
-slate	O
-/	O
-firebrick	O
-.	O
-	O
-The	O
-asterisk	O
-and	O
-double	O
-arrow	O
-marks	O
-the	O
-location	O
-of	O
-the	O
-π	O
-–	O
-π	O
-interaction	O
-between	O
-F116	O
-and	O
-the	O
-CoA	O
-base	O
-of	O
-the	O
-other	O
-dimer	O
-chain	O
-.	O
-	O
-(	O
-d	O
-)	O
-Surface	O
-representation	O
-of	O
-the	O
-structure	O
-with	O
-indicated	O
-conservation	O
-(	O
-red	O
-:	O
-high	O
-,	O
-white	O
-:	O
-intermediate	O
-,	O
-yellow	O
-:	O
-low	O
-).	O
-	O
-Size	O
-exclusion	O
-chromatography	O
-of	O
-PduL	O
-homologs	O
-.	O
-	O
-(	O
-a	O
-)–(	O
-c	O
-):	O
-Chromatograms	O
-of	O
-sPduL	O
-(	O
-a	O
-),	O
-rPduL	O
-(	O
-b	O
-),	O
-and	O
-pPduL	O
-(	O
-c	O
-)	O
-with	O
-(	O
-orange	O
-)	O
-or	O
-without	O
-(	O
-blue	O
-)	O
-the	O
-predicted	O
-EP	O
-,	O
-post	O
--	O
-nickel	O
-affinity	O
-purification	O
-,	O
-applied	O
-over	O
-a	O
-preparative	O
-size	O
-exclusion	O
-column	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-(	O
-d	O
-)–(	O
-f	O
-):	O
-Chromatograms	O
-of	O
-sPduL	O
-(	O
-d	O
-),	O
-rPduL	O
-(	O
-e	O
-),	O
-and	O
-pPduL	O
-(	O
-f	O
-)	O
-post	O
--	O
-preparative	O
-size	O
-exclusion	O
-chromatography	O
-with	O
-different	O
-size	O
-fractions	O
-separated	O
-,	O
-applied	O
-over	O
-an	O
-analytical	O
-size	O
-exclusion	O
-column	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-All	O
-chromatograms	O
-are	O
-cropped	O
-to	O
-show	O
-only	O
-the	O
-linear	O
-range	O
-of	O
-separation	O
-based	O
-on	O
-standard	O
-runs	O
-,	O
-shown	O
-in	O
-black	O
-squares	O
-with	O
-a	O
-dashed	O
-linear	O
-trend	O
-line	O
-.	O
-	O
-Active	O
-Site	O
-Properties	O
-	O
-CoA	O
-and	O
-the	O
-metal	O
-ions	O
-bind	O
-between	O
-the	O
-two	O
-domains	O
-,	O
-presumably	O
-in	O
-the	O
-active	O
-site	O
-(	O
-Figs	O
-2b	O
-and	O
-4a	O
-).	O
-	O
-To	O
-identify	O
-the	O
-bound	O
-metals	O
-,	O
-we	O
-performed	O
-an	O
-X	O
--	O
-ray	O
-fluorescence	O
-scan	O
-on	O
-the	O
-crystals	O
-at	O
-various	O
-wavelengths	O
-(	O
-corresponding	O
-to	O
-the	O
-K	O
--	O
-edges	O
-of	O
-Mn	O
-,	O
-Fe	O
-,	O
-Co	O
-,	O
-Ni	O
-,	O
-Cu	O
-,	O
-and	O
-Zn	O
-).	O
-	O
-There	O
-was	O
-a	O
-large	O
-signal	O
-at	O
-the	O
-zinc	O
-edge	O
-,	O
-and	O
-we	O
-tested	O
-for	O
-the	O
-presence	O
-of	O
-zinc	O
-by	O
-collecting	O
-full	O
-data	O
-sets	O
-before	O
-and	O
-after	O
-the	O
-Zn	O
-K	O
--	O
-edge	O
-(	O
-1	O
-.	O
-2861	O
-and	O
-1	O
-.	O
-2822	O
-Å	O
-,	O
-respectively	O
-).	O
-	O
-The	O
-large	O
-differences	O
-between	O
-the	O
-anomalous	O
-signals	O
-confirm	O
-the	O
-presence	O
-of	O
-zinc	O
-at	O
-both	O
-metal	O
-sites	O
-(	O
-S3	O
-Fig	O
-).	O
-	O
-The	O
-first	O
-zinc	O
-ion	O
-(	O
-Zn1	O
-)	O
-is	O
-in	O
-a	O
-tetrahedral	O
-coordination	O
-state	O
-with	O
-His48	O
-,	O
-His50	O
-,	O
-Glu109	O
-,	O
-and	O
-the	O
-CoA	O
-sulfur	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-The	O
-second	O
-(	O
-Zn2	O
-)	O
-is	O
-in	O
-octahedral	O
-coordination	O
-by	O
-three	O
-conserved	O
-histidine	O
-residues	O
-(	O
-His157	O
-,	O
-His159	O
-and	O
-His204	O
-)	O
-as	O
-well	O
-as	O
-three	O
-water	O
-molecules	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-The	O
-nitrogen	O
-atom	O
-coordinating	O
-the	O
-zinc	O
-is	O
-the	O
-Nε	O
-in	O
-each	O
-histidine	O
-residue	O
-,	O
-as	O
-is	O
-typical	O
-for	O
-this	O
-interaction	O
-.	O
-	O
-When	O
-the	O
-crystals	O
-were	O
-soaked	O
-in	O
-a	O
-sodium	O
-phosphate	O
-solution	O
-for	O
-2	O
-d	O
-prior	O
-to	O
-data	O
-collection	O
-,	O
-the	O
-CoA	O
-dissociates	O
-,	O
-and	O
-density	O
-for	O
-a	O
-phosphate	O
-molecule	O
-is	O
-visible	O
-at	O
-the	O
-active	O
-site	O
-(	O
-Table	O
-2	O
-,	O
-Fig	O
-4b	O
-).	O
-	O
-The	O
-phosphate	O
--	O
-bound	O
-structure	O
-aligns	O
-well	O
-with	O
-the	O
-CoA	O
--	O
-bound	O
-structure	O
-(	O
-0	O
-.	O
-43	O
-Å	O
-rmsd	O
-over	O
-2	O
-,	O
-361	O
-atoms	O
-for	O
-the	O
-monomer	O
-,	O
-0	O
-.	O
-83	O
-Å	O
-over	O
-5	O
-,	O
-259	O
-aligned	O
-atoms	O
-for	O
-the	O
-dimer	O
-).	O
-	O
-The	O
-phosphate	O
-contacts	O
-both	O
-zinc	O
-atoms	O
-(	O
-Fig	O
-4b	O
-)	O
-and	O
-replaces	O
-the	O
-coordination	O
-by	O
-CoA	O
-at	O
-Zn1	O
-;	O
-the	O
-coordination	O
-for	O
-Zn2	O
-changes	O
-from	O
-octahedral	O
-with	O
-three	O
-bound	O
-waters	O
-to	O
-tetrahedral	O
-with	O
-a	O
-phosphate	O
-ion	O
-as	O
-one	O
-of	O
-the	O
-ligands	O
-(	O
-Fig	O
-4b	O
-).	O
-	O
-Conserved	O
-Arg103	O
-seems	O
-to	O
-be	O
-involved	O
-in	O
-maintaining	O
-the	O
-phosphate	O
-in	O
-that	O
-position	O
-.	O
-	O
-The	O
-two	O
-zinc	O
-atoms	O
-are	O
-slightly	O
-closer	O
-together	O
-in	O
-the	O
-phosphate	O
--	O
-bound	O
-form	O
-(	O
-5	O
-.	O
-8	O
-Å	O
-vs	O
-6	O
-.	O
-3	O
-Å	O
-),	O
-possibly	O
-due	O
-to	O
-the	O
-bridging	O
-effect	O
-of	O
-the	O
-phosphate	O
-.	O
-	O
-An	O
-additional	O
-phosphate	O
-molecule	O
-is	O
-bound	O
-at	O
-a	O
-crystal	O
-contact	O
-interface	O
-,	O
-perhaps	O
-accounting	O
-for	O
-the	O
-14	O
-Å	O
-shorter	O
-c	O
--	O
-axis	O
-in	O
-the	O
-phosphate	O
--	O
-bound	O
-crystal	O
-form	O
-(	O
-Table	O
-2	O
-).	O
-	O
-Oligomeric	O
-States	O
-of	O
-PduL	O
-Orthologs	O
-Are	O
-Influenced	O
-by	O
-the	O
-EP	O
-	O
-Interestingly	O
-,	O
-some	O
-of	O
-the	O
-residues	O
-important	O
-for	O
-dimerization	O
-of	O
-rPduL	O
-,	O
-particularly	O
-Phe116	O
-,	O
-are	O
-poorly	O
-conserved	O
-across	O
-PduL	O
-homologs	O
-associated	O
-with	O
-functionally	O
-diverse	O
-BMCs	O
-(	O
-Figs	O
-4c	O
-and	O
-3	O
-),	O
-suggesting	O
-that	O
-they	O
-may	O
-have	O
-alternative	O
-oligomeric	O
-states	O
-.	O
-	O
-We	O
-tested	O
-this	O
-hypothesis	O
-by	O
-performing	O
-size	O
-exclusion	O
-chromatography	O
-on	O
-both	O
-full	O
--	O
-length	O
-and	O
-truncated	O
-variants	O
-(	O
-lacking	O
-the	O
-EP	O
-,	O
-ΔEP	B-mutant
-)	O
-of	O
-sPduL	O
-,	O
-rPduL	O
-,	O
-and	O
-pPduL	O
-.	O
-These	O
-three	O
-homologs	O
-are	O
-found	O
-in	O
-functionally	O
-distinct	O
-BMCs	O
-(	O
-Table	O
-1	O
-).	O
-	O
-It	O
-has	O
-been	O
-proposed	O
-that	O
-the	O
-catabolic	O
-BMCs	O
-may	O
-assemble	O
-in	O
-a	O
-core	O
--	O
-first	O
-manner	O
-,	O
-with	O
-the	O
-luminal	O
-enzymes	O
-(	O
-signature	O
-enzyme	O
-,	O
-aldehyde	O
-,	O
-and	O
-alcohol	O
-dehydrogenases	O
-and	O
-the	O
-BMC	O
-PTAC	O
-)	O
-forming	O
-an	O
-initial	O
-bolus	O
-,	O
-or	O
-prometabolosome	O
-,	O
-around	O
-which	O
-a	O
-shell	O
-assembles	O
-.	O
-	O
-Given	O
-the	O
-diversity	O
-of	O
-signature	O
-enzymes	O
-(	O
-Table	O
-1	O
-),	O
-it	O
-is	O
-plausible	O
-that	O
-PduL	O
-orthologs	O
-may	O
-adopt	O
-different	O
-oligomeric	O
-states	O
-that	O
-reflect	O
-the	O
-differences	O
-in	O
-the	O
-proteins	O
-being	O
-packaged	O
-with	O
-them	O
-in	O
-the	O
-organelle	O
-lumen	O
-.	O
-	O
-We	O
-found	O
-that	O
-not	O
-only	O
-did	O
-the	O
-different	O
-orthologs	O
-appear	O
-to	O
-assemble	O
-into	O
-different	O
-oligomeric	O
-states	O
-,	O
-but	O
-that	O
-quaternary	O
-structure	O
-was	O
-dependent	O
-on	O
-whether	O
-or	O
-not	O
-the	O
-EP	O
-was	O
-present	O
-.	O
-	O
-Full	O
--	O
-length	O
-sPduL	O
-was	O
-unstable	O
-in	O
-solution	O
-—	O
-precipitating	O
-over	O
-time	O
-—	O
-and	O
-eluted	O
-throughout	O
-the	O
-entire	O
-volume	O
-of	O
-a	O
-size	O
-exclusion	O
-column	O
-,	O
-indicating	O
-it	O
-was	O
-nonspecifically	O
-aggregating	O
-.	O
-	O
-However	O
-,	O
-when	O
-the	O
-putative	O
-EP	O
-(	O
-residues	O
-1	O
-–	O
-27	O
-)	O
-was	O
-removed	O
-(	O
-sPduL	B-mutant
-ΔEP	I-mutant
-),	O
-the	O
-truncated	O
-protein	O
-was	O
-stable	O
-and	O
-eluted	O
-as	O
-a	O
-single	O
-peak	O
-(	O
-Fig	O
-5a	O
-)	O
-consistent	O
-with	O
-the	O
-size	O
-of	O
-a	O
-monomer	O
-(	O
-Fig	O
-5d	O
-,	O
-blue	O
-curve	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-both	O
-full	O
--	O
-length	O
-rPduL	O
-and	O
-pPduL	O
-appeared	O
-to	O
-exist	O
-in	O
-two	O
-distinct	O
-oligomeric	O
-states	O
-(	O
-Fig	O
-5b	O
-and	O
-5c	O
-respectively	O
-,	O
-orange	O
-curves	O
-),	O
-one	O
-form	O
-of	O
-the	O
-approximate	O
-size	O
-of	O
-a	O
-dimer	O
-and	O
-the	O
-second	O
-,	O
-a	O
-higher	O
-molecular	O
-weight	O
-oligomer	O
-(~	O
-150	O
-kDa	O
-).	O
-	O
-Upon	O
-deletion	O
-of	O
-the	O
-putative	O
-EP	O
-(	O
-residues	O
-1	O
-–	O
-47	O
-for	O
-rPduL	O
-,	O
-and	O
-1	O
-–	O
-20	O
-for	O
-pPduL	O
-),	O
-there	O
-was	O
-a	O
-distinct	O
-change	O
-in	O
-the	O
-elution	O
-profiles	O
-(	O
-Fig	O
-5b	O
-and	O
-5c	O
-respectively	O
-,	O
-blue	O
-curves	O
-).	O
-	O
-pPduLΔEP	B-mutant
-eluted	O
-as	O
-two	O
-smaller	O
-forms	O
-,	O
-possibly	O
-corresponding	O
-to	O
-a	O
-trimer	O
-and	O
-a	O
-monomer	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-rPduLΔEP	B-mutant
-eluted	O
-as	O
-one	O
-smaller	O
-oligomer	O
-,	O
-possibly	O
-a	O
-dimer	O
-.	O
-	O
-We	O
-also	O
-analyzed	O
-purified	O
-rPduL	O
-and	O
-rPduLΔEP	B-mutant
-by	O
-size	O
-exclusion	O
-chromatography	O
-coupled	O
-with	O
-multiangle	O
-light	O
-scattering	O
-(	O
-SEC	O
--	O
-MALS	O
-)	O
-for	O
-a	O
-complementary	O
-approach	O
-to	O
-assessing	O
-oligomeric	O
-state	O
-.	O
-	O
-SEC	O
--	O
-MALS	O
-analysis	O
-of	O
-rPdulΔEP	B-mutant
-is	O
-consistent	O
-with	O
-a	O
-dimer	O
-(	O
-as	O
-observed	O
-in	O
-the	O
-crystal	O
-structure	O
-)	O
-with	O
-a	O
-weighted	O
-average	O
-(	O
-Mw	O
-)	O
-and	O
-number	O
-average	O
-(	O
-Mn	O
-)	O
-of	O
-the	O
-molar	O
-mass	O
-of	O
-58	O
-.	O
-4	O
-kDa	O
-+/−	O
-11	O
-.	O
-2	O
-%	O
-and	O
-58	O
-.	O
-8	O
-kDa	O
-+/−	O
-10	O
-.	O
-9	O
-%,	O
-respectively	O
-(	O
-S4a	O
-Fig	O
-).	O
-	O
-rPduL	O
-full	O
-length	O
-runs	O
-as	O
-Mw	O
-=	O
-140	O
-.	O
-3	O
-kDa	O
-+/−	O
-1	O
-.	O
-2	O
-%	O
-and	O
-Mn	O
-=	O
-140	O
-.	O
-5	O
-kDa	O
-+/−	O
-1	O
-.	O
-2	O
-%.	O
-	O
-This	O
-corresponds	O
-to	O
-an	O
-oligomeric	O
-state	O
-of	O
-six	O
-subunits	O
-(	O
-calculated	O
-molecular	O
-weight	O
-of	O
-144	O
-kDa	O
-).	O
-	O
-Collectively	O
-,	O
-these	O
-data	O
-strongly	O
-suggest	O
-that	O
-the	O
-N	O
--	O
-terminal	O
-EP	O
-of	O
-PduL	O
-plays	O
-a	O
-role	O
-in	O
-defining	O
-the	O
-quaternary	O
-structure	O
-of	O
-the	O
-protein	O
-.	O
-	O
-The	O
-BMC	O
-shell	O
-not	O
-only	O
-sequesters	O
-specific	O
-enzymes	O
-but	O
-also	O
-their	O
-cofactors	O
-,	O
-thereby	O
-establishing	O
-a	O
-private	O
-cofactor	O
-pool	O
-dedicated	O
-to	O
-the	O
-encapsulated	O
-reactions	O
-.	O
-	O
-In	O
-catabolic	O
-BMCs	O
-,	O
-CoA	O
-and	O
-NAD	O
-+	O
-must	O
-be	O
-continually	O
-recycled	O
-within	O
-the	O
-organelle	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-Homologs	O
-of	O
-the	O
-predominant	O
-cofactor	O
-utilizer	O
-(	O
-aldehyde	O
-dehydrogenase	O
-)	O
-and	O
-NAD	O
-+	O
-regenerator	O
-(	O
-alcohol	O
-dehydrogenase	O
-)	O
-have	O
-been	O
-structurally	O
-characterized	O
-,	O
-but	O
-until	O
-now	O
-structural	O
-information	O
-was	O
-lacking	O
-for	O
-PduL	O
-,	O
-which	O
-recycles	O
-CoA	O
-in	O
-the	O
-organelle	O
-lumen	O
-.	O
-	O
-Curiously	O
-,	O
-while	O
-the	O
-housekeeping	O
-Pta	O
-could	O
-provide	O
-this	O
-function	O
-,	O
-and	O
-indeed	O
-does	O
-so	O
-in	O
-the	O
-case	O
-of	O
-one	O
-type	O
-of	O
-ethanolamine	O
--	O
-utilizing	O
-(	O
-EUT	O
-)	O
-BMC	O
-,	O
-the	O
-evolutionarily	O
-unrelated	O
-PduL	O
-fulfills	O
-this	O
-function	O
-for	O
-the	O
-majority	O
-of	O
-metabolosomes	O
-using	O
-a	O
-novel	O
-structure	O
-and	O
-active	O
-site	O
-for	O
-convergent	O
-evolution	O
-of	O
-function	O
-.	O
-	O
-The	O
-Tertiary	O
-Structure	O
-of	O
-PduL	O
-Is	O
-Formed	O
-by	O
-Discontinuous	O
-Segments	O
-of	O
-Primary	O
-Structure	O
-	O
-The	O
-structure	O
-of	O
-PduL	O
-consists	O
-of	O
-two	O
-β	O
--	O
-barrel	O
-domains	O
-capped	O
-by	O
-short	O
-alpha	O
-helical	O
-segments	O
-(	O
-Fig	O
-2b	O
-).	O
-	O
-The	O
-two	O
-domains	O
-are	O
-structurally	O
-very	O
-similar	O
-(	O
-superimposing	O
-with	O
-a	O
-rmsd	O
-of	O
-1	O
-.	O
-34	O
-Å	O
-(	O
-over	O
-123	O
-out	O
-of	O
-320	O
-/	O
-348	O
-aligned	O
-backbone	O
-atoms	O
-,	O
-S5a	O
-Fig	O
-).	O
-	O
-However	O
-,	O
-the	O
-amino	O
-acid	O
-sequences	O
-of	O
-the	O
-two	O
-domains	O
-are	O
-only	O
-16	O
-%	O
-identical	O
-(	O
-mainly	O
-the	O
-RHxH	O
-motif	O
-,	O
-β2	O
-and	O
-β10	O
-),	O
-and	O
-34	O
-%	O
-similar	O
-.	O
-	O
-Our	O
-structure	O
-reveals	O
-that	O
-the	O
-two	O
-assigned	O
-PF06130	O
-domains	O
-(	O
-Fig	O
-3	O
-)	O
-do	O
-not	O
-form	O
-structurally	O
-discrete	O
-units	O
-;	O
-this	O
-reduces	O
-the	O
-apparent	O
-sequence	O
-conservation	O
-at	O
-the	O
-level	O
-of	O
-primary	O
-structure	O
-.	O
-	O
-One	O
-strand	O
-of	O
-the	O
-domain	O
-1	O
-beta	O
-barrel	O
-(	O
-shown	O
-in	O
-blue	O
-in	O
-Fig	O
-2	O
-)	O
-is	O
-contributed	O
-by	O
-the	O
-N	O
--	O
-terminus	O
-,	O
-while	O
-the	O
-rest	O
-of	O
-the	O
-domain	O
-is	O
-formed	O
-by	O
-the	O
-residues	O
-from	O
-the	O
-C	O
--	O
-terminal	O
-half	O
-of	O
-the	O
-protein	O
-.	O
-	O
-When	O
-aligned	O
-by	O
-structure	O
-,	O
-the	O
-β1	O
-strand	O
-of	O
-the	O
-first	O
-domain	O
-(	O
-Fig	O
-2a	O
-and	O
-2b	O
-,	O
-blue	O
-)	O
-corresponds	O
-to	O
-the	O
-final	O
-strand	O
-of	O
-the	O
-second	O
-domain	O
-(	O
-β9	O
-),	O
-effectively	O
-making	O
-the	O
-domains	O
-continuous	O
-if	O
-the	O
-first	O
-strand	O
-was	O
-transplanted	O
-to	O
-the	O
-C	O
--	O
-terminus	O
-.	O
-	O
-Refined	O
-domain	O
-assignment	O
-based	O
-on	O
-our	O
-structure	O
-should	O
-be	O
-able	O
-to	O
-predict	O
-domains	O
-of	O
-PF06130	O
-homologs	O
-much	O
-more	O
-accurately	O
-.	O
-	O
-The	O
-closest	O
-structural	O
-homolog	O
-of	O
-the	O
-PduL	O
-barrel	O
-domain	O
-is	O
-a	O
-subdomain	O
-of	O
-a	O
-multienzyme	O
-complex	O
-,	O
-the	O
-alpha	O
-subunit	O
-of	O
-ethylbenzene	O
-dehydrogenase	O
-(	O
-S5b	O
-Fig	O
-,	O
-rmsd	O
-of	O
-2	O
-.	O
-26	O
-Å	O
-over	O
-226	O
-aligned	O
-atoms	O
-consisting	O
-of	O
-one	O
-beta	O
-barrel	O
-and	O
-one	O
-capping	O
-helix	O
-).	O
-	O
-In	O
-contrast	O
-to	O
-PduL	O
-,	O
-there	O
-is	O
-only	O
-one	O
-barrel	O
-present	O
-in	O
-ethylbenzene	O
-dehydrogenase	O
-,	O
-and	O
-there	O
-is	O
-no	O
-comparable	O
-active	O
-site	O
-arrangement	O
-.	O
-	O
-The	O
-PduL	O
-signature	O
-primary	O
-structure	O
-,	O
-two	O
-PF06130	O
-domains	O
-,	O
-occurs	O
-in	O
-some	O
-multidomain	O
-proteins	O
-,	O
-most	O
-of	O
-them	O
-annotated	O
-as	O
-Acks	O
-,	O
-suggesting	O
-that	O
-PduL	O
-may	O
-also	O
-replace	O
-Pta	O
-in	O
-variants	O
-of	O
-the	O
-phosphotransacetylase	O
--	O
-Ack	O
-pathway	O
-.	O
-	O
-These	O
-PduL	O
-homologs	O
-lack	O
-EPs	O
-,	O
-and	O
-their	O
-fusion	O
-to	O
-Ack	O
-may	O
-have	O
-evolved	O
-as	O
-a	O
-way	O
-to	O
-facilitate	O
-substrate	O
-channeling	O
-between	O
-the	O
-two	O
-enzymes	O
-.	O
-	O
-Implications	O
-for	O
-Metabolosome	O
-Core	O
-Assembly	O
-	O
-For	O
-BMC	O
--	O
-encapsulated	O
-proteins	O
-to	O
-properly	O
-function	O
-together	O
-,	O
-they	O
-must	O
-be	O
-targeted	O
-to	O
-the	O
-lumen	O
-and	O
-assemble	O
-into	O
-an	O
-organization	O
-that	O
-facilitates	O
-substrate	O
-/	O
-product	O
-channeling	O
-among	O
-the	O
-different	O
-catalytic	O
-sites	O
-of	O
-the	O
-signature	O
-and	O
-core	O
-enzymes	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-extension	O
-on	O
-PduL	O
-homologs	O
-may	O
-serve	O
-both	O
-of	O
-these	O
-functions	O
-.	O
-	O
-The	O
-extension	O
-shares	O
-many	O
-features	O
-with	O
-previously	O
-characterized	O
-EPs	O
-:	O
-it	O
-is	O
-present	O
-only	O
-in	O
-homologs	O
-associated	O
-with	O
-BMC	O
-loci	O
-,	O
-and	O
-it	O
-is	O
-predicted	O
-to	O
-form	O
-an	O
-amphipathic	O
-α	O
--	O
-helix	O
-.	O
-	O
-Moreover	O
-,	O
-its	O
-removal	O
-affects	O
-the	O
-oligomeric	O
-state	O
-of	O
-the	O
-protein	O
-.	O
-	O
-EP	O
--	O
-mediated	O
-oligomerization	O
-has	O
-been	O
-observed	O
-for	O
-the	O
-signature	O
-and	O
-core	O
-BMC	O
-enzymes	O
-;	O
-for	O
-example	O
-,	O
-full	O
--	O
-length	O
-propanediol	O
-dehydratase	O
-and	O
-ethanolamine	O
-ammonia	O
--	O
-lyase	O
-(	O
-signature	O
-enzymes	O
-for	O
-PDU	O
-and	O
-EUT	O
-BMCs	O
-)	O
-subunits	O
-are	O
-also	O
-insoluble	O
-,	O
-but	O
-become	O
-soluble	O
-upon	O
-removal	O
-of	O
-the	O
-predicted	O
-EP	O
-.	O
-	O
-sPduL	O
-has	O
-also	O
-previously	O
-been	O
-reported	O
-to	O
-localize	O
-to	O
-inclusion	O
-bodies	O
-when	O
-overexpressed	O
-;	O
-we	O
-show	O
-here	O
-that	O
-this	O
-is	O
-dependent	O
-on	O
-the	O
-presence	O
-of	O
-the	O
-EP	O
-.	O
-	O
-This	O
-propensity	O
-of	O
-the	O
-EP	O
-to	O
-cause	O
-proteins	O
-to	O
-form	O
-complexes	O
-(	O
-Fig	O
-5	O
-)	O
-might	O
-not	O
-be	O
-a	O
-coincidence	O
-,	O
-but	O
-could	O
-be	O
-a	O
-necessary	O
-step	O
-in	O
-the	O
-assembly	O
-of	O
-BMCs	O
-.	O
-	O
-Structured	O
-aggregation	O
-of	O
-the	O
-core	O
-enzymes	O
-has	O
-been	O
-proposed	O
-to	O
-be	O
-the	O
-initial	O
-step	O
-in	O
-metabolosome	O
-assembly	O
-and	O
-is	O
-known	O
-to	O
-be	O
-the	O
-first	O
-step	O
-of	O
-β	O
--	O
-carboxysome	O
-biogenesis	O
-,	O
-where	O
-the	O
-core	O
-enzyme	O
-Ribulose	O
-Bisphosphate	O
-Carboxylase	O
-/	O
-Oxygenase	O
-(	O
-RuBisCO	O
-)	O
-is	O
-aggregated	O
-by	O
-the	O
-CcmM	O
-protein	O
-.	O
-	O
-Likewise	O
-,	O
-CsoS2	O
-,	O
-a	O
-protein	O
-in	O
-the	O
-α	O
--	O
-carboxysome	O
-core	O
-,	O
-also	O
-aggregates	O
-when	O
-purified	O
-and	O
-is	O
-proposed	O
-to	O
-facilitate	O
-the	O
-nucleation	O
-and	O
-encapsulation	O
-of	O
-RuBisCO	O
-molecules	O
-in	O
-the	O
-lumen	O
-of	O
-the	O
-organelle	O
-.	O
-	O
-This	O
-role	O
-for	O
-EPs	O
-in	O
-BMC	O
-assembly	O
-is	O
-in	O
-addition	O
-to	O
-their	O
-interaction	O
-with	O
-shell	O
-proteins	O
-.	O
-	O
-Moreover	O
-,	O
-the	O
-PduL	O
-crystal	O
-structures	O
-offer	O
-a	O
-clue	O
-as	O
-to	O
-how	O
-required	O
-cofactors	O
-enter	O
-the	O
-BMC	O
-lumen	O
-during	O
-assembly	O
-.	O
-	O
-Free	O
-CoA	O
-and	O
-NAD	O
-+/	O
-H	O
-could	O
-potentially	O
-be	O
-bound	O
-to	O
-the	O
-enzymes	O
-as	O
-the	O
-core	O
-assembles	O
-and	O
-is	O
-encapsulated	O
-.	O
-	O
-Our	O
-PduL	O
-crystals	O
-contained	O
-CoA	O
-that	O
-was	O
-captured	O
-from	O
-the	O
-Escherichia	O
-coli	O
-cytosol	O
-,	O
-indicating	O
-that	O
-the	O
-“	O
-ground	O
-state	O
-”	O
-of	O
-PduL	O
-is	O
-in	O
-the	O
-CoA	O
--	O
-bound	O
-form	O
-;	O
-this	O
-could	O
-provide	O
-an	O
-elegantly	O
-simple	O
-means	O
-of	O
-guaranteeing	O
-a	O
-1	O
-:	O
-1	O
-ratio	O
-of	O
-CoA	O
-:	O
-PduL	O
-within	O
-the	O
-metabolosome	O
-lumen	O
-.	O
-	O
-Active	O
-Site	O
-Identification	O
-and	O
-Structural	O
-Insights	O
-into	O
-Catalysis	O
-	O
-The	O
-active	O
-site	O
-of	O
-PduL	O
-is	O
-formed	O
-at	O
-the	O
-interface	O
-of	O
-the	O
-two	O
-structural	O
-domains	O
-(	O
-Fig	O
-2b	O
-).	O
-	O
-As	O
-expected	O
-,	O
-the	O
-amino	O
-acid	O
-sequence	O
-conservation	O
-is	O
-highest	O
-in	O
-the	O
-region	O
-around	O
-the	O
-proposed	O
-active	O
-site	O
-(	O
-Fig	O
-4d	O
-);	O
-highly	O
-conserved	O
-residues	O
-are	O
-also	O
-involved	O
-in	O
-CoA	O
-binding	O
-(	O
-Figs	O
-2a	O
-and	O
-3	O
-,	O
-residues	O
-Ser45	O
-,	O
-Lys70	O
-,	O
-Arg97	O
-,	O
-Leu99	O
-,	O
-His204	O
-,	O
-Asn211	O
-).	O
-	O
-All	O
-of	O
-the	O
-metal	O
--	O
-coordinating	O
-residues	O
-(	O
-Fig	O
-2a	O
-)	O
-are	O
-absolutely	O
-conserved	O
-,	O
-implicating	O
-them	O
-in	O
-catalysis	O
-or	O
-the	O
-correct	O
-spatial	O
-orientation	O
-of	O
-the	O
-substrates	O
-.	O
-	O
-Arg103	O
-,	O
-which	O
-contacts	O
-the	O
-phosphate	O
-(	O
-Fig	O
-4b	O
-),	O
-is	O
-present	O
-in	O
-all	O
-PduL	O
-homologs	O
-.	O
-	O
-The	O
-close	O
-resemblance	O
-between	O
-the	O
-structures	O
-binding	O
-CoA	O
-and	O
-phosphate	O
-likely	O
-indicates	O
-that	O
-no	O
-large	O
-changes	O
-in	O
-protein	O
-conformation	O
-are	O
-involved	O
-in	O
-catalysis	O
-,	O
-and	O
-that	O
-our	O
-crystal	O
-structures	O
-are	O
-representative	O
-of	O
-the	O
-active	O
-form	O
-.	O
-	O
-The	O
-native	O
-substrate	O
-for	O
-the	O
-forward	O
-reaction	O
-of	O
-rPduL	O
-and	O
-pPduL	O
-,	O
-propionyl	O
--	O
-CoA	O
-,	O
-most	O
-likely	O
-binds	O
-to	O
-the	O
-enzyme	O
-in	O
-the	O
-same	O
-way	O
-at	O
-the	O
-observed	O
-nucleotide	O
-and	O
-pantothenic	O
-acid	O
-moiety	O
-,	O
-but	O
-the	O
-propionyl	O
-group	O
-in	O
-the	O
-CoA	O
--	O
-thioester	O
-might	O
-point	O
-in	O
-a	O
-different	O
-direction	O
-.	O
-	O
-There	O
-is	O
-a	O
-pocket	O
-nearby	O
-the	O
-active	O
-site	O
-between	O
-the	O
-well	O
--	O
-conserved	O
-residues	O
-Ser45	O
-and	O
-Ala154	O
-,	O
-which	O
-could	O
-accommodate	O
-the	O
-propionyl	O
-group	O
-(	O
-S6	O
-Fig	O
-).	O
-	O
-A	O
-homology	O
-model	O
-of	O
-sPduL	O
-indicates	O
-that	O
-the	O
-residues	O
-making	O
-up	O
-this	O
-pocket	O
-and	O
-the	O
-surrounding	O
-active	O
-site	O
-region	O
-are	O
-identical	O
-to	O
-that	O
-of	O
-rPduL	O
-,	O
-which	O
-is	O
-not	O
-surprising	O
-,	O
-because	O
-these	O
-two	O
-homologs	O
-presumably	O
-have	O
-the	O
-same	O
-propionyl	O
--	O
-CoA	O
-substrate	O
-.	O
-	O
-The	O
-homology	O
-model	O
-of	O
-pPduL	O
-also	O
-has	O
-identical	O
-residues	O
-making	O
-up	O
-the	O
-pocket	O
-,	O
-but	O
-with	O
-a	O
-key	O
-difference	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-active	O
-site	O
-:	O
-Gln77	O
-of	O
-rPduL	O
-is	O
-replaced	O
-by	O
-a	O
-tyrosine	O
-(	O
-Tyr77	O
-)	O
-in	O
-pPduL	O
-.	O
-The	O
-physiological	O
-substrate	O
-of	O
-pPduL	O
-(	O
-Table	O
-1	O
-)	O
-is	O
-thought	O
-to	O
-be	O
-lactyl	O
--	O
-CoA	O
-,	O
-which	O
-contains	O
-an	O
-additional	O
-hydroxyl	O
-group	O
-relative	O
-to	O
-propionyl	O
--	O
-CoA	O
-.	O
-The	O
-presence	O
-of	O
-an	O
-aromatic	O
-residue	O
-at	O
-this	O
-position	O
-may	O
-underlie	O
-the	O
-substrate	O
-preference	O
-of	O
-the	O
-PduL	O
-enzyme	O
-from	O
-the	O
-pvm	O
-locus	O
-.	O
-	O
-Indeed	O
-,	O
-in	O
-the	O
-majority	O
-of	O
-PduLs	O
-encoded	O
-in	O
-pvm	O
-loci	O
-,	O
-Gln77	O
-is	O
-substituted	O
-by	O
-either	O
-a	O
-Tyr	O
-or	O
-Phe	O
-,	O
-whereas	O
-it	O
-is	O
-typically	O
-a	O
-Gln	O
-or	O
-Glu	O
-in	O
-PduLs	O
-in	O
-all	O
-other	O
-BMC	O
-types	O
-that	O
-degrade	O
-acetyl	O
--	O
-or	O
-propionyl	O
--	O
-CoA	O
-.	O
-A	O
-comparison	O
-of	O
-the	O
-PduL	O
-active	O
-site	O
-to	O
-that	O
-of	O
-the	O
-functionally	O
-identical	O
-Pta	O
-suggests	O
-that	O
-the	O
-two	O
-enzymes	O
-have	O
-distinctly	O
-different	O
-mechanisms	O
-.	O
-	O
-The	O
-catalytic	O
-mechanism	O
-of	O
-Pta	O
-involves	O
-the	O
-abstraction	O
-of	O
-a	O
-thiol	O
-hydrogen	O
-by	O
-an	O
-aspartate	O
-residue	O
-,	O
-resulting	O
-in	O
-the	O
-nucleophilic	O
-attack	O
-of	O
-thiolate	O
-upon	O
-the	O
-carbonyl	O
-carbon	O
-of	O
-acetyl	O
--	O
-phosphate	O
-,	O
-oriented	O
-by	O
-an	O
-arginine	O
-and	O
-stabilized	O
-by	O
-a	O
-serine	O
-—	O
-there	O
-are	O
-no	O
-metals	O
-involved	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-in	O
-the	O
-rPduL	O
-structure	O
-,	O
-there	O
-are	O
-no	O
-conserved	O
-aspartate	O
-residues	O
-in	O
-or	O
-around	O
-the	O
-active	O
-site	O
-,	O
-and	O
-the	O
-only	O
-well	O
--	O
-conserved	O
-glutamate	O
-residue	O
-in	O
-the	O
-active	O
-site	O
-is	O
-involved	O
-in	O
-coordinating	O
-one	O
-of	O
-the	O
-metal	O
-ions	O
-.	O
-	O
-These	O
-observations	O
-strongly	O
-suggest	O
-that	O
-an	O
-acidic	O
-residue	O
-is	O
-not	O
-directly	O
-involved	O
-in	O
-catalysis	O
-by	O
-PduL	O
-.	O
-Instead	O
-,	O
-the	O
-dimetal	O
-active	O
-site	O
-of	O
-PduL	O
-may	O
-create	O
-a	O
-nucleophile	O
-from	O
-one	O
-of	O
-the	O
-hydroxyl	O
-groups	O
-on	O
-free	O
-phosphate	O
-to	O
-attack	O
-the	O
-carbonyl	O
-carbon	O
-of	O
-the	O
-thioester	O
-bond	O
-of	O
-an	O
-acyl	O
--	O
-CoA	O
-.	O
-In	O
-the	O
-reverse	O
-direction	O
-,	O
-the	O
-metal	O
-ion	O
-(	O
-s	O
-)	O
-could	O
-stabilize	O
-the	O
-thiolate	O
-anion	O
-that	O
-would	O
-attack	O
-the	O
-carbonyl	O
-carbon	O
-of	O
-an	O
-acyl	O
--	O
-phosphate	O
-;	O
-a	O
-similar	O
-mechanism	O
-has	O
-been	O
-described	O
-for	O
-phosphatases	O
-where	O
-hydroxyl	O
-groups	O
-or	O
-hydroxide	O
-ions	O
-can	O
-act	O
-as	O
-a	O
-base	O
-when	O
-coordinated	O
-by	O
-a	O
-dimetal	O
-active	O
-site	O
-.	O
-	O
-Our	O
-structures	O
-provide	O
-the	O
-foundation	O
-for	O
-studies	O
-to	O
-elucidate	O
-the	O
-details	O
-of	O
-the	O
-catalytic	O
-mechanism	O
-of	O
-PduL	O
-.	O
-Conserved	O
-residues	O
-in	O
-the	O
-active	O
-site	O
-that	O
-may	O
-contribute	O
-to	O
-substrate	O
-binding	O
-and	O
-/	O
-or	O
-transition	O
-state	O
-stabilization	O
-include	O
-Ser127	O
-,	O
-Arg103	O
-,	O
-Arg194	O
-,	O
-Gln107	O
-,	O
-Gln74	O
-,	O
-and	O
-Gln	O
-/	O
-Glu77	O
-.	O
-	O
-In	O
-the	O
-phosphate	O
--	O
-bound	O
-crystal	O
-structure	O
-,	O
-Ser127	O
-and	O
-Arg103	O
-appear	O
-to	O
-position	O
-the	O
-phosphate	O
-(	O
-Fig	O
-4b	O
-).	O
-	O
-Alternatively	O
-,	O
-Arg103	O
-might	O
-act	O
-as	O
-a	O
-base	O
-to	O
-render	O
-the	O
-phosphate	O
-more	O
-nucleophilic	O
-.	O
-	O
-The	O
-functional	O
-groups	O
-of	O
-Gln74	O
-,	O
-Gln	O
-/	O
-Glu77	O
-,	O
-and	O
-Arg194	O
-are	O
-directed	O
-away	O
-from	O
-the	O
-active	O
-site	O
-in	O
-both	O
-CoA	O
-and	O
-phosphate	O
--	O
-bound	O
-crystal	O
-structures	O
-and	O
-do	O
-not	O
-appear	O
-to	O
-be	O
-involved	O
-in	O
-hydrogen	O
-bonding	O
-with	O
-these	O
-substrates	O
-,	O
-although	O
-they	O
-could	O
-be	O
-important	O
-for	O
-positioning	O
-an	O
-acyl	O
--	O
-phosphate	O
-.	O
-	O
-The	O
-free	O
-CoA	O
--	O
-bound	O
-form	O
-is	O
-presumably	O
-poised	O
-for	O
-attack	O
-upon	O
-an	O
-acyl	O
--	O
-phosphate	O
-,	O
-indicating	O
-that	O
-the	O
-enzyme	O
-initially	O
-binds	O
-CoA	O
-as	O
-opposed	O
-to	O
-acyl	O
--	O
-phosphate	O
-.	O
-	O
-This	O
-hypothesis	O
-is	O
-strengthened	O
-by	O
-the	O
-fact	O
-that	O
-the	O
-CoA	O
--	O
-bound	O
-crystals	O
-were	O
-obtained	O
-without	O
-added	O
-CoA	O
-,	O
-indicating	O
-that	O
-the	O
-protein	O
-bound	O
-CoA	O
-from	O
-the	O
-E	O
-.	O
-coli	O
-expression	O
-strain	O
-and	O
-retained	O
-it	O
-throughout	O
-purification	O
-and	O
-crystallization	O
-.	O
-	O
-The	O
-phosphate	O
--	O
-bound	O
-structure	O
-indicates	O
-that	O
-in	O
-the	O
-opposite	O
-reaction	O
-direction	O
-phosphate	O
-is	O
-bound	O
-first	O
-,	O
-and	O
-then	O
-an	O
-acyl	O
--	O
-CoA	O
-enters	O
-.	O
-	O
-The	O
-two	O
-high	O
--	O
-resolution	O
-crystal	O
-structures	O
-presented	O
-here	O
-will	O
-serve	O
-as	O
-the	O
-foundation	O
-for	O
-mechanistic	O
-studies	O
-on	O
-this	O
-noncanonical	O
-PTAC	O
-enzyme	O
-to	O
-determine	O
-how	O
-the	O
-dimetal	O
-active	O
-site	O
-functions	O
-to	O
-catalyze	O
-both	O
-forward	O
-and	O
-reverse	O
-reactions	O
-.	O
-	O
-Functional	O
-,	O
-but	O
-Not	O
-Structural	O
-,	O
-Convergence	O
-of	O
-PduL	O
-and	O
-Pta	O
-	O
-PduL	O
-and	O
-Pta	O
-are	O
-mechanistically	O
-and	O
-structurally	O
-distinct	O
-enzymes	O
-that	O
-catalyze	O
-the	O
-same	O
-reaction	O
-,	O
-a	O
-prime	O
-example	O
-of	O
-evolutionary	O
-convergence	O
-upon	O
-a	O
-function	O
-.	O
-	O
-There	O
-are	O
-several	O
-examples	O
-of	O
-such	O
-functional	O
-convergence	O
-of	O
-enzymes	O
-,	O
-although	O
-typically	O
-the	O
-enzymes	O
-have	O
-independently	O
-evolved	O
-similar	O
-,	O
-or	O
-even	O
-identical	O
-active	O
-sites	O
-;	O
-for	O
-example	O
-,	O
-the	O
-carbonic	O
-anhydrase	O
-family	O
-.	O
-	O
-However	O
-,	O
-apparently	O
-less	O
-frequent	O
-is	O
-functional	O
-convergence	O
-that	O
-is	O
-supported	O
-by	O
-distinctly	O
-different	O
-active	O
-sites	O
-and	O
-accordingly	O
-catalytic	O
-mechanism	O
-,	O
-as	O
-revealed	O
-by	O
-comparison	O
-of	O
-the	O
-structures	O
-of	O
-Pta	O
-and	O
-PduL	O
-.	O
-One	O
-well	O
--	O
-studied	O
-example	O
-of	O
-this	O
-is	O
-the	O
-β	O
--	O
-lactamase	O
-family	O
-of	O
-enzymes	O
-,	O
-in	O
-which	O
-the	O
-active	O
-site	O
-of	O
-Class	O
-A	O
-and	O
-Class	O
-C	O
-enzymes	O
-involve	O
-serine	O
--	O
-based	O
-catalysis	O
-,	O
-but	O
-Class	O
-B	O
-enzymes	O
-are	O
-metalloproteins	O
-.	O
-	O
-This	O
-is	O
-not	O
-surprising	O
-,	O
-as	O
-β	O
--	O
-lactamases	O
-are	O
-not	O
-so	O
-widespread	O
-among	O
-bacteria	O
-and	O
-therefore	O
-would	O
-be	O
-expected	O
-to	O
-have	O
-evolved	O
-independently	O
-several	O
-times	O
-as	O
-a	O
-defense	O
-mechanism	O
-against	O
-β	O
--	O
-lactam	O
-antibiotics	O
-.	O
-	O
-However	O
-,	O
-nearly	O
-all	O
-bacteria	O
-encode	O
-Pta	O
-,	O
-and	O
-it	O
-is	O
-not	O
-immediately	O
-clear	O
-why	O
-the	O
-Pta	O
-/	O
-PduL	O
-functional	O
-convergence	O
-should	O
-have	O
-evolved	O
-:	O
-it	O
-would	O
-seem	O
-to	O
-be	O
-evolutionarily	O
-more	O
-resourceful	O
-for	O
-the	O
-Pta	O
--	O
-encoding	O
-gene	O
-to	O
-be	O
-duplicated	O
-and	O
-repurposed	O
-for	O
-BMCs	O
-,	O
-as	O
-is	O
-apparently	O
-the	O
-case	O
-in	O
-one	O
-type	O
-of	O
-BMC	O
-—	O
-EUT1	O
-(	O
-Table	O
-1	O
-).	O
-	O
-There	O
-could	O
-be	O
-some	O
-intrinsic	O
-biochemical	O
-difference	O
-between	O
-the	O
-two	O
-enzymes	O
-that	O
-renders	O
-PduL	O
-a	O
-more	O
-attractive	O
-candidate	O
-for	O
-encapsulation	O
-in	O
-a	O
-BMC	O
-—	O
-for	O
-example	O
-,	O
-PduL	O
-might	O
-be	O
-more	O
-amenable	O
-to	O
-tight	O
-packaging	O
-,	O
-or	O
-is	O
-better	O
-suited	O
-for	O
-the	O
-chemical	O
-microenvironment	O
-formed	O
-within	O
-the	O
-lumen	O
-of	O
-the	O
-BMC	O
-,	O
-which	O
-can	O
-be	O
-quite	O
-different	O
-from	O
-the	O
-cytosol	O
-.	O
-	O
-Further	O
-biochemical	O
-comparison	O
-between	O
-the	O
-two	O
-PTACs	O
-will	O
-likely	O
-yield	O
-exciting	O
-results	O
-that	O
-could	O
-answer	O
-this	O
-evolutionary	O
-question	O
-.	O
-	O
-BMCs	O
-are	O
-now	O
-known	O
-to	O
-be	O
-widespread	O
-among	O
-the	O
-bacteria	O
-and	O
-are	O
-involved	O
-in	O
-critical	O
-segments	O
-of	O
-both	O
-autotrophic	O
-and	O
-heterotrophic	O
-biochemical	O
-pathways	O
-that	O
-confer	O
-to	O
-the	O
-host	O
-organism	O
-a	O
-competitive	O
-(	O
-metabolic	O
-)	O
-advantage	O
-in	O
-select	O
-niches	O
-.	O
-	O
-As	O
-one	O
-of	O
-the	O
-three	O
-common	O
-metabolosome	O
-core	O
-enzymes	O
-,	O
-the	O
-structure	O
-of	O
-PduL	O
-provides	O
-a	O
-key	O
-missing	O
-piece	O
-to	O
-our	O
-structural	O
-picture	O
-of	O
-the	O
-shared	O
-core	O
-biochemistry	O
-(	O
-Fig	O
-1	O
-)	O
-of	O
-functionally	O
-diverse	O
-catabolic	O
-BMCs	O
-.	O
-	O
-We	O
-have	O
-observed	O
-the	O
-oligomeric	O
-state	O
-differences	O
-of	O
-PduL	O
-to	O
-correlate	O
-with	O
-the	O
-presence	O
-of	O
-an	O
-EP	O
-,	O
-providing	O
-new	O
-insight	O
-into	O
-the	O
-function	O
-of	O
-this	O
-sequence	O
-extension	O
-in	O
-BMC	O
-assembly	O
-.	O
-	O
-Moreover	O
-,	O
-our	O
-results	O
-suggest	O
-a	O
-means	O
-for	O
-Coenzyme	O
-A	O
-incorporation	O
-during	O
-metabolosome	O
-biogenesis	O
-.	O
-	O
-A	O
-detailed	O
-understanding	O
-of	O
-the	O
-underlying	O
-principles	O
-governing	O
-the	O
-assembly	O
-and	O
-internal	O
-structural	O
-organization	O
-of	O
-BMCs	O
-is	O
-a	O
-requisite	O
-for	O
-synthetic	O
-biologists	O
-to	O
-design	O
-custom	O
-nanoreactors	O
-that	O
-use	O
-BMC	O
-architectures	O
-as	O
-a	O
-template	O
-.	O
-	O
-Furthermore	O
-,	O
-given	O
-the	O
-growing	O
-number	O
-of	O
-metabolosomes	O
-implicated	O
-in	O
-pathogenesis	O
-,	O
-the	O
-PduL	O
-structure	O
-will	O
-be	O
-useful	O
-in	O
-the	O
-development	O
-of	O
-therapeutics	O
-.	O
-	O
-The	O
-fact	O
-that	O
-PduL	O
-is	O
-confined	O
-almost	O
-exclusively	O
-to	O
-metabolosomes	O
-can	O
-be	O
-used	O
-to	O
-develop	O
-an	O
-inhibitor	O
-that	O
-blocks	O
-only	O
-PduL	O
-and	O
-not	O
-Pta	O
-as	O
-a	O
-way	O
-to	O
-selectively	O
-disrupt	O
-BMC	O
--	O
-based	O
-metabolism	O
-,	O
-while	O
-not	O
-affecting	O
-most	O
-commensal	O
-organisms	O
-that	O
-require	O
-PTAC	O
-activity	O
-.	O
-	O
-Biochemistry	O
-and	O
-Crystal	O
-Structure	O
-of	O
-Ectoine	O
-Synthase	O
-:	O
-A	O
-Metal	O
--	O
-Containing	O
-Member	O
-of	O
-the	O
-Cupin	O
-Superfamily	O
-	O
-Ectoine	O
-is	O
-a	O
-compatible	O
-solute	O
-and	O
-chemical	O
-chaperone	O
-widely	O
-used	O
-by	O
-members	O
-of	O
-the	O
-Bacteria	O
-and	O
-a	O
-few	O
-Archaea	O
-to	O
-fend	O
--	O
-off	O
-the	O
-detrimental	O
-effects	O
-of	O
-high	O
-external	O
-osmolarity	O
-on	O
-cellular	O
-physiology	O
-and	O
-growth	O
-.	O
-	O
-Ectoine	O
-synthase	O
-(	O
-EctC	O
-)	O
-catalyzes	O
-the	O
-last	O
-step	O
-in	O
-ectoine	O
-production	O
-and	O
-mediates	O
-the	O
-ring	O
-closure	O
-of	O
-the	O
-substrate	O
-N	O
--	O
-gamma	O
--	O
-acetyl	O
--	O
-L	O
--	O
-2	O
-,	O
-4	O
--	O
-diaminobutyric	O
-acid	O
-through	O
-a	O
-water	O
-elimination	O
-reaction	O
-.	O
-	O
-However	O
-,	O
-the	O
-crystal	O
-structure	O
-of	O
-ectoine	O
-synthase	O
-is	O
-not	O
-known	O
-and	O
-a	O
-clear	O
-understanding	O
-of	O
-how	O
-its	O
-fold	O
-contributes	O
-to	O
-enzyme	O
-activity	O
-is	O
-thus	O
-lacking	O
-.	O
-	O
-Using	O
-the	O
-ectoine	O
-synthase	O
-from	O
-the	O
-cold	O
--	O
-adapted	O
-marine	O
-bacterium	O
-Sphingopyxis	O
-alaskensis	O
-(	O
-Sa	O
-),	O
-we	O
-report	O
-here	O
-both	O
-a	O
-detailed	O
-biochemical	O
-characterization	O
-of	O
-the	O
-EctC	O
-enzyme	O
-and	O
-the	O
-high	O
--	O
-resolution	O
-crystal	O
-structure	O
-of	O
-its	O
-apo	O
--	O
-form	O
-.	O
-	O
-Structural	O
-analysis	O
-classified	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-as	O
-a	O
-member	O
-of	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-EctC	O
-forms	O
-a	O
-dimer	O
-with	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-arrangement	O
-,	O
-both	O
-in	O
-solution	O
-and	O
-in	O
-the	O
-crystal	O
-structure	O
-.	O
-	O
-The	O
-interface	O
-of	O
-the	O
-dimer	O
-assembly	O
-is	O
-shaped	O
-through	O
-backbone	O
--	O
-contacts	O
-and	O
-weak	O
-hydrophobic	O
-interactions	O
-mediated	O
-by	O
-two	O
-beta	O
--	O
-sheets	O
-within	O
-each	O
-monomer	O
-.	O
-	O
-We	O
-show	O
-for	O
-the	O
-first	O
-time	O
-that	O
-ectoine	O
-synthase	O
-harbors	O
-a	O
-catalytically	O
-important	O
-metal	O
-co	O
--	O
-factor	O
-;	O
-metal	O
-depletion	O
-and	O
-reconstitution	O
-experiments	O
-suggest	O
-that	O
-EctC	O
-is	O
-probably	O
-an	O
-iron	O
--	O
-dependent	O
-enzyme	O
-.	O
-	O
-We	O
-found	O
-that	O
-EctC	O
-not	O
-only	O
-effectively	O
-converts	O
-its	O
-natural	O
-substrate	O
-N	O
--	O
-gamma	O
--	O
-acetyl	O
--	O
-L	O
--	O
-2	O
-,	O
-4	O
--	O
-diaminobutyric	O
-acid	O
-into	O
-ectoine	O
-through	O
-a	O
-cyclocondensation	O
-reaction	O
-,	O
-but	O
-that	O
-it	O
-can	O
-also	O
-use	O
-the	O
-isomer	O
-N	O
--	O
-alpha	O
--	O
-acetyl	O
--	O
-L	O
--	O
-2	O
-,	O
-4	O
--	O
-diaminobutyric	O
-acid	O
-as	O
-its	O
-substrate	O
-,	O
-albeit	O
-with	O
-substantially	O
-reduced	O
-catalytic	O
-efficiency	O
-.	O
-	O
-Structure	O
--	O
-guided	O
-site	O
--	O
-directed	O
-mutagenesis	O
-experiments	O
-targeting	O
-amino	O
-acid	O
-residues	O
-that	O
-are	O
-evolutionarily	O
-highly	O
-conserved	O
-among	O
-the	O
-extended	O
-EctC	O
-protein	O
-family	O
-,	O
-including	O
-those	O
-forming	O
-the	O
-presumptive	O
-iron	O
--	O
-binding	O
-site	O
-,	O
-were	O
-conducted	O
-to	O
-functionally	O
-analyze	O
-the	O
-properties	O
-of	O
-the	O
-resulting	O
-EctC	O
-variants	O
-.	O
-	O
-An	O
-assessment	O
-of	O
-enzyme	O
-activity	O
-and	O
-iron	O
-content	O
-of	O
-these	O
-mutants	O
-give	O
-important	O
-clues	O
-for	O
-understanding	O
-the	O
-architecture	O
-of	O
-the	O
-active	O
-site	O
-positioned	O
-within	O
-the	O
-core	O
-of	O
-the	O
-EctC	O
-cupin	O
-barrel	O
-.	O
-	O
-Ectoine	O
-[(	O
-S	O
-)-	O
-2	O
--	O
-methyl	O
--	O
-1	O
-,	O
-4	O
-,	O
-5	O
-,	O
-6	O
--	O
-tetrahydropyrimidine	O
--	O
-4	O
--	O
-carboxylic	O
-acid	O
-]	O
-and	O
-its	O
-derivative	O
-5	O
--	O
-hydroxyectoine	O
-[(	O
-4S	O
-,	O
-5S	O
-)-	O
-5	O
--	O
-hydroxy	O
--	O
-2	O
--	O
-methyl	O
--	O
-1	O
-,	O
-4	O
-,	O
-5	O
-,	O
-6	O
--	O
-tetrahydropyrimidine	O
--	O
-4	O
--	O
-carboxylic	O
-acid	O
-]	O
-are	O
-such	O
-compatible	O
-solutes	O
-.	O
-	O
-Both	O
-marine	O
-and	O
-terrestrial	O
-microorganisms	O
-produce	O
-them	O
-widely	O
-in	O
-response	O
-to	O
-osmotic	O
-or	O
-temperature	O
-stress	O
-.	O
-	O
-Synthesis	O
-of	O
-ectoine	O
-occurs	O
-from	O
-the	O
-intermediate	O
-metabolite	O
-L	O
--	O
-aspartate	O
--	O
-ß	O
--	O
-semialdehyde	O
-and	O
-comprises	O
-the	O
-sequential	O
-activities	O
-of	O
-three	O
-enzymes	O
-:	O
-L	O
--	O
-2	O
-,	O
-4	O
--	O
-diaminobutyrate	O
-transaminase	O
-(	O
-EctB	O
-;	O
-EC	O
-2	O
-.	O
-6	O
-.	O
-1	O
-.	O
-76	O
-),	O
-2	O
-,	O
-4	O
--	O
-diaminobutyrate	O
-acetyltransferase	O
-(	O
-EctA	O
-;	O
-EC	O
-2	O
-.	O
-3	O
-.	O
-1	O
-.	O
-178	O
-),	O
-and	O
-ectoine	O
-synthase	O
-(	O
-EctC	O
-;	O
-EC	O
-4	O
-.	O
-2	O
-.	O
-1	O
-.	O
-108	O
-)	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-The	O
-ectoine	O
-derivative	O
-5	O
--	O
-hydroxyectoine	O
-,	O
-a	O
-highly	O
-effective	O
-stress	O
-protectant	O
-in	O
-its	O
-own	O
-right	O
-,	O
-is	O
-synthesized	O
-by	O
-a	O
-substantial	O
-subgroup	O
-of	O
-the	O
-ectoine	O
-producers	O
-.	O
-	O
-This	O
-stereospecific	O
-chemical	O
-modification	O
-of	O
-ectoine	O
-(	O
-Fig	O
-1	O
-)	O
-is	O
-catalyzed	O
-by	O
-the	O
-ectoine	O
-hydroxylase	O
-(	O
-EctD	O
-)	O
-(	O
-EC	O
-1	O
-.	O
-14	O
-.	O
-11	O
-),	O
-a	O
-member	O
-of	O
-the	O
-non	O
--	O
-heme	O
-containing	O
-iron	O
-(	O
-II	O
-)	O
-and	O
-2	O
--	O
-oxoglutarate	O
--	O
-dependent	O
-dioxygenase	O
-superfamily	O
-.	O
-	O
-The	O
-remarkable	O
-function	O
-preserving	O
-effects	O
-of	O
-ectoines	O
-for	O
-macromolecules	O
-and	O
-cells	O
-,	O
-frequently	O
-also	O
-addressed	O
-as	O
-chemical	O
-chaperones	O
-,	O
-led	O
-to	O
-a	O
-substantial	O
-interest	O
-in	O
-exploiting	O
-these	O
-compounds	O
-for	O
-biotechnological	O
-purposes	O
-and	O
-medical	O
-applications	O
-.	O
-	O
-Biosynthetic	O
-routes	O
-for	O
-ectoine	O
-and	O
-5	O
--	O
-hydroxyectoine	O
-.	O
-	O
-Scheme	O
-of	O
-the	O
-ectoine	O
-and	O
-5	O
--	O
-hydroxyectoine	O
-biosynthetic	O
-pathway	O
-.	O
-	O
-Here	O
-we	O
-focus	O
-on	O
-ectoine	O
-synthase	O
-(	O
-EctC	O
-),	O
-the	O
-key	O
-enzyme	O
-of	O
-the	O
-ectoine	O
-biosynthetic	O
-route	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-Biochemical	O
-characterizations	O
-of	O
-ectoine	O
-synthases	O
-from	O
-the	O
-extremophiles	O
-Halomonas	O
-elongata	O
-,	O
-Methylomicrobium	O
-alcaliphilum	O
-,	O
-and	O
-Acidiphilium	O
-cryptum	O
-,	O
-and	O
-from	O
-the	O
-nitrifying	O
-archaeon	O
-Nitrosopumilus	O
-maritimus	O
-have	O
-been	O
-carried	O
-out	O
-.	O
-	O
-Each	O
-of	O
-these	O
-enzymes	O
-catalyzes	O
-as	O
-their	O
-main	O
-activity	O
-the	O
-cyclization	O
-of	O
-N	O
--	O
-γ	O
--	O
-acetyl	O
--	O
-L	O
--	O
-2	O
-,	O
-4	O
--	O
-diaminobutyric	O
-acid	O
-(	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-),	O
-the	O
-reaction	O
-product	O
-of	O
-the	O
-2	O
-,	O
-4	O
--	O
-diaminobutyrate	O
-acetyltransferase	O
-(	O
-EctA	O
-),	O
-to	O
-ectoine	O
-with	O
-the	O
-concomitant	O
-release	O
-of	O
-a	O
-water	O
-molecule	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-In	O
-side	O
-reactions	O
-,	O
-EctC	O
-can	O
-promote	O
-the	O
-formation	O
-of	O
-the	O
-synthetic	O
-compatible	O
-solute	O
-5	O
--	O
-amino	O
--	O
-3	O
-,	O
-4	O
--	O
-dihydro	O
--	O
-2H	O
--	O
-pyrrole	O
--	O
-2	O
--	O
-carboxylate	O
-(	O
-ADPC	O
-)	O
-through	O
-the	O
-cyclic	O
-condensation	O
-of	O
-two	O
-glutamine	O
-molecules	O
-and	O
-it	O
-also	O
-possesses	O
-a	O
-minor	O
-hydrolytic	O
-activity	O
-for	O
-ectoine	O
-and	O
-synthetic	O
-ectoine	O
-derivatives	O
-with	O
-either	O
-reduced	O
-or	O
-expanded	O
-ring	O
-sizes	O
-.	O
-	O
-Although	O
-progress	O
-has	O
-been	O
-made	O
-with	O
-respect	O
-to	O
-the	O
-biochemical	O
-characterization	O
-of	O
-ectoine	O
-synthase	O
-,	O
-a	O
-clear	O
-understanding	O
-of	O
-how	O
-its	O
-structure	O
-contributes	O
-to	O
-its	O
-enzyme	O
-activity	O
-and	O
-reaction	O
-mechanism	O
-is	O
-still	O
-lacking	O
-.	O
-With	O
-this	O
-in	O
-mind	O
-,	O
-we	O
-have	O
-biochemically	O
-characterized	O
-the	O
-ectoine	O
-synthase	O
-from	O
-the	O
-cold	O
--	O
-adapted	O
-marine	O
-bacterium	O
-Sphingopyxis	O
-alaskensis	O
-(	O
-Sa	O
-).	O
-	O
-We	O
-demonstrate	O
-here	O
-for	O
-the	O
-first	O
-time	O
-that	O
-the	O
-ectoine	O
-synthase	O
-is	O
-a	O
-metal	O
--	O
-dependent	O
-enzyme	O
-,	O
-with	O
-iron	O
-as	O
-the	O
-most	O
-likely	O
-physiologically	O
-relevant	O
-co	O
--	O
-factor	O
-.	O
-	O
-The	O
-EctC	O
-protein	O
-forms	O
-a	O
-dimer	O
-in	O
-solution	O
-and	O
-our	O
-structural	O
-analysis	O
-identifies	O
-it	O
-as	O
-a	O
-member	O
-of	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-The	O
-two	O
-crystal	O
-structures	O
-that	O
-we	O
-report	O
-here	O
-for	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-with	O
-resolutions	O
-of	O
-1	O
-.	O
-2	O
-Å	O
-and	O
-2	O
-.	O
-0	O
-Å	O
-,	O
-respectively	O
-),	O
-and	O
-data	O
-derived	O
-from	O
-extensive	O
-site	O
--	O
-directed	O
-mutagenesis	O
-experiments	O
-targeting	O
-evolutionarily	O
-highly	O
-conserved	O
-residues	O
-within	O
-the	O
-extended	O
-EctC	O
-protein	O
-family	O
-,	O
-provide	O
-a	O
-first	O
-view	O
-into	O
-the	O
-architecture	O
-of	O
-the	O
-catalytic	O
-core	O
-of	O
-the	O
-ectoine	O
-synthase	O
-.	O
-	O
-Overproduction	O
-,	O
-purification	O
-and	O
-oligomeric	O
-state	O
-of	O
-the	O
-ectoine	O
-synthase	O
-in	O
-solution	O
-	O
-We	O
-focused	O
-our	O
-biochemical	O
-and	O
-structural	O
-studies	O
-on	O
-the	O
-ectoine	O
-synthase	O
-from	O
-S	O
-.	O
-alaskensis	O
-[(	O
-Sa	O
-)	O
-EctC	O
-],	O
-a	O
-cold	O
--	O
-adapted	O
-marine	O
-ultra	O
--	O
-microbacterium	O
-,	O
-from	O
-which	O
-we	O
-recently	O
-also	O
-determined	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-ectoine	O
-hydroxylase	O
-(	O
-EctD	O
-)	O
-in	O
-complex	O
-with	O
-either	O
-its	O
-substrate	O
-or	O
-its	O
-reaction	O
-product	O
-.	O
-	O
-We	O
-expressed	O
-a	O
-codon	O
--	O
-optimized	O
-version	O
-of	O
-the	O
-S	O
-.	O
-alaskensis	O
-ectC	O
-gene	O
-in	O
-E	O
-.	O
-coli	O
-to	O
-produce	O
-a	O
-recombinant	O
-protein	O
-with	O
-a	O
-carboxy	O
--	O
-terminally	O
-attached	O
-Strep	O
--	O
-tag	O
-II	O
-affinity	O
-peptide	O
-to	O
-allow	O
-purification	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
--	O
-Strep	O
--	O
-Tag	O
--	O
-II	O
-protein	O
-by	O
-affinity	O
-chromatography	O
-.	O
-	O
-The	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-was	O
-overproduced	O
-and	O
-isolated	O
-with	O
-good	O
-yields	O
-(	O
-30	O
-–	O
-40	O
-mg	O
-L	O
--	O
-1	O
-of	O
-culture	O
-)	O
-and	O
-purity	O
-(	O
-S2a	O
-Fig	O
-).	O
-	O
-Conventional	O
-size	O
--	O
-exclusion	O
-chromatography	O
-(	O
-SEC	O
-)	O
-has	O
-already	O
-shown	O
-that	O
-(	O
-Sa	O
-)	O
-EctC	O
-preparations	O
-produced	O
-in	O
-this	O
-fashion	O
-are	O
-homogeneous	O
-and	O
-that	O
-the	O
-protein	O
-forms	O
-dimers	O
-in	O
-solution	O
-.	O
-	O
-High	O
-performance	O
-liquid	O
-chromatography	O
-coupled	O
-with	O
-multi	O
--	O
-angle	O
-light	O
--	O
-scattering	O
-detection	O
-(	O
-HPLC	O
--	O
-MALS	O
-)	O
-experiments	O
-carried	O
-out	O
-here	O
-confirmed	O
-that	O
-the	O
-purified	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-was	O
-mono	O
--	O
-disperse	O
-and	O
-possessed	O
-a	O
-molecular	O
-mass	O
-of	O
-33	O
-.	O
-0	O
-±	O
-2	O
-.	O
-3	O
-kDa	O
-(	O
-S2b	O
-Fig	O
-).	O
-	O
-This	O
-value	O
-corresponds	O
-very	O
-well	O
-with	O
-the	O
-theoretically	O
-calculated	O
-molecular	O
-mass	O
-of	O
-an	O
-(	O
-Sa	O
-)	O
-EctC	O
-dimer	O
-(	O
-molecular	O
-mass	O
-of	O
-the	O
-monomer	O
-,	O
-including	O
-the	O
-Strep	O
--	O
-tag	O
-II	O
-affinity	O
-peptide	O
-:	O
-16	O
-.	O
-3	O
-kDa	O
-).	O
-	O
-Such	O
-a	O
-quaternary	O
-assembly	O
-as	O
-dimer	O
-has	O
-also	O
-been	O
-reported	O
-for	O
-the	O
-EctC	O
-proteins	O
-from	O
-H	O
-.	O
-elongata	O
-and	O
-N	O
-.	O
-maritimus	O
-.	O
-	O
-Biochemical	O
-properties	O
-of	O
-the	O
-ectoine	O
-synthase	O
-	O
-The	O
-EctA	O
--	O
-produced	O
-substrate	O
-of	O
-the	O
-ectoine	O
-synthase	O
-,	O
-N	O
--	O
-γ	O
--	O
-acetyl	O
--	O
-L	O
--	O
-2	O
-,	O
-4	O
--	O
-diaminobutyric	O
-acid	O
-(	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-)	O
-(	O
-Fig	O
-1	O
-),	O
-is	O
-commercially	O
-not	O
-available	O
-.	O
-	O
-We	O
-used	O
-alkaline	O
-hydrolysis	O
-of	O
-ectoine	O
-and	O
-subsequent	O
-chromatography	O
-on	O
-silica	O
-gel	O
-columns	O
-to	O
-obtain	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-in	O
-chemically	O
-highly	O
-purified	O
-form	O
-(	O
-S1a	O
-Fig	O
-).	O
-	O
-This	O
-procedure	O
-also	O
-yielded	O
-the	O
-isomer	O
-of	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-,	O
-N	O
--	O
-α	O
--	O
-acetyl	O
--	O
-L	O
--	O
-2	O
-,	O
-4	O
--	O
-diaminobutyric	O
-acid	O
-(	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-)	O
-(	O
-S1b	O
-Fig	O
-).	O
-	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-has	O
-so	O
-far	O
-not	O
-been	O
-considered	O
-as	O
-a	O
-substrate	O
-for	O
-EctC	O
-,	O
-but	O
-microorganisms	O
-that	O
-use	O
-ectoine	O
-as	O
-a	O
-nutrient	O
-produce	O
-it	O
-as	O
-an	O
-intermediate	O
-during	O
-catabolism	O
-.	O
-	O
-Using	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-as	O
-the	O
-substrate	O
-,	O
-we	O
-initially	O
-evaluated	O
-a	O
-set	O
-of	O
-biochemical	O
-parameters	O
-of	O
-the	O
-recombinant	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-S	O
-.	O
-alaskensis	O
-,	O
-from	O
-which	O
-the	O
-studied	O
-ectoine	O
-synthase	O
-was	O
-originally	O
-derived	O
-,	O
-is	O
-a	O
-microorganism	O
-that	O
-is	O
-well	O
--	O
-adapted	O
-to	O
-a	O
-life	O
-in	O
-permanently	O
-cold	O
-ocean	O
-waters	O
-.	O
-	O
-Consistent	O
-with	O
-the	O
-physicochemical	O
-attributes	O
-of	O
-this	O
-habitat	O
-,	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-was	O
-already	O
-enzymatically	O
-active	O
-at	O
-5	O
-°	O
-C	O
-,	O
-had	O
-a	O
-temperature	O
-optimum	O
-of	O
-15	O
-°	O
-C	O
-and	O
-was	O
-able	O
-to	O
-function	O
-over	O
-a	O
-broad	O
-range	O
-of	O
-temperatures	O
-(	O
-S3a	O
-Fig	O
-).	O
-	O
-It	O
-possessed	O
-an	O
-alkaline	O
-pH	O
-optimum	O
-of	O
-8	O
-.	O
-5	O
-(	O
-S3b	O
-Fig	O
-),	O
-a	O
-value	O
-similar	O
-to	O
-the	O
-ectoine	O
-synthases	O
-from	O
-the	O
-halo	O
--	O
-tolerant	O
-H	O
-.	O
-elongata	O
-(	O
-pH	O
-optimum	O
-of	O
-8	O
-.	O
-5	O
-to	O
-9	O
-.	O
-0	O
-),	O
-the	O
-alkaliphile	O
-M	O
-.	O
-alcaliphilum	O
-(	O
-pH	O
-optimum	O
-of	O
-9	O
-.	O
-0	O
-),	O
-and	O
-the	O
-acidophile	O
-Acidiphilium	O
-cryptum	O
-(	O
-pH	O
-optimum	O
-of	O
-8	O
-.	O
-5	O
-to	O
-9	O
-.	O
-0	O
-),	O
-whereas	O
-the	O
-EctC	O
-protein	O
-from	O
-N	O
-.	O
-maritimus	O
-has	O
-a	O
-neutral	O
-pH	O
-optimum	O
-(	O
-pH	O
-7	O
-.	O
-0	O
-).	O
-	O
-The	O
-salinity	O
-of	O
-the	O
-assay	O
-buffer	O
-had	O
-a	O
-significant	O
-influence	O
-on	O
-the	O
-maximal	O
-enzyme	O
-activity	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-An	O
-increase	O
-in	O
-either	O
-the	O
-NaCl	O
-or	O
-the	O
-KCl	O
-concentration	O
-led	O
-to	O
-an	O
-approximately	O
-5	O
--	O
-fold	O
-enhancement	O
-of	O
-the	O
-ectoine	O
-synthase	O
-activity	O
-.	O
-	O
-The	O
-maximum	O
-enzyme	O
-activity	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-occurred	O
-around	O
-250	O
-mM	O
-NaCl	O
-or	O
-KCl	O
-,	O
-respectively	O
-.	O
-	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-a	O
-highly	O
-salt	O
--	O
-tolerant	O
-enzyme	O
-since	O
-it	O
-exhibited	O
-substantial	O
-enzyme	O
-activity	O
-even	O
-at	O
-NaCl	O
-and	O
-KCl	O
-concentrations	O
-of	O
-1	O
-M	O
-in	O
-the	O
-assay	O
-buffer	O
-(	O
-S3c	O
-and	O
-S3d	O
-Fig	O
-).	O
-	O
-The	O
-stimulation	O
-of	O
-EctC	O
-enzyme	O
-activity	O
-by	O
-salts	O
-has	O
-previously	O
-also	O
-been	O
-observed	O
-for	O
-other	O
-ectoine	O
-synthases	O
-.	O
-	O
-The	O
-ectoine	O
-synthase	O
-is	O
-a	O
-metal	O
--	O
-containing	O
-protein	O
-	O
-Considerations	O
-based	O
-on	O
-bioinformatics	O
-suggests	O
-that	O
-EctC	O
-belongs	O
-to	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-Most	O
-of	O
-these	O
-proteins	O
-contain	O
-catalytically	O
-important	O
-transition	O
-state	O
-metals	O
-such	O
-as	O
-iron	O
-,	O
-copper	O
-,	O
-zinc	O
-,	O
-manganese	O
-,	O
-cobalt	O
-,	O
-or	O
-nickel	O
-.	O
-	O
-Cupins	O
-contain	O
-two	O
-conserved	O
-motifs	O
-:	O
-G	O
-(	O
-X	O
-)	O
-5HXH	O
-(	O
-X	O
-)	O
-3	O
-,	O
-4E	O
-(	O
-X	O
-)	O
-6G	O
-and	O
-G	O
-(	O
-X	O
-)	O
-5PXG	O
-(	O
-X	O
-)	O
-2H	O
-(	O
-X	O
-)	O
-3N	O
-(	O
-the	O
-letters	O
-in	O
-bold	O
-represent	O
-those	O
-residues	O
-that	O
-often	O
-coordinate	O
-the	O
-metal	O
-).	O
-	O
-Inspection	O
-of	O
-a	O
-previous	O
-alignment	O
-of	O
-the	O
-amino	O
-acid	O
-sequences	O
-of	O
-440	O
-EctC	O
--	O
-type	O
-proteins	O
-revealed	O
-that	O
-the	O
-canonical	O
-metal	O
--	O
-binding	O
-motif	O
-(	O
-s	O
-)	O
-of	O
-cupin	O
--	O
-type	O
-proteins	O
-is	O
-not	O
-conserved	O
-among	O
-members	O
-of	O
-the	O
-extended	O
-ectoine	O
-synthase	O
-protein	O
-family	O
-.	O
-	O
-An	O
-abbreviated	O
-alignment	O
-of	O
-the	O
-amino	O
-acid	O
-sequence	O
-of	O
-EctC	O
--	O
-type	O
-proteins	O
-is	O
-shown	O
-in	O
-Fig	O
-2	O
-.	O
-	O
-Abbreviated	O
-alignment	O
-of	O
-EctC	O
--	O
-type	O
-proteins	O
-.	O
-	O
-The	O
-amino	O
-acid	O
-sequences	O
-of	O
-20	O
-selected	O
-EctC	O
--	O
-type	O
-proteins	O
-are	O
-compared	O
-.	O
-	O
-Strictly	O
-conserved	O
-amino	O
-acid	O
-residues	O
-are	O
-shown	O
-in	O
-yellow	O
-.	O
-	O
-Dots	O
-shown	O
-above	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-sequence	O
-indicate	O
-residues	O
-likely	O
-to	O
-be	O
-involved	O
-in	O
-iron	O
--	O
-binding	O
-(	O
-red	O
-),	O
-ligand	O
--	O
-binding	O
-(	O
-green	O
-)	O
-and	O
-stabilization	O
-of	O
-the	O
-loop	O
--	O
-architecture	O
-(	O
-blue	O
-).	O
-	O
-The	O
-conserved	O
-residue	O
-Tyr	O
--	O
-52	O
-with	O
-so	O
--	O
-far	O
-undefined	O
-functions	O
-is	O
-indicated	O
-by	O
-a	O
-green	O
-dot	O
-circled	O
-in	O
-red	O
-.	O
-	O
-Secondary	O
-structural	O
-elements	O
-(	O
-α	O
--	O
-helices	O
-and	O
-β	O
--	O
-sheets	O
-)	O
-found	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structure	O
-are	O
-projected	O
-onto	O
-the	O
-amino	O
-acid	O
-sequences	O
-of	O
-EctC	O
--	O
-type	O
-proteins	O
-.	O
-	O
-Since	O
-variations	O
-of	O
-the	O
-above	O
--	O
-described	O
-metal	O
--	O
-binding	O
-motif	O
-occur	O
-frequently	O
-,	O
-we	O
-experimentally	O
-investigated	O
-the	O
-presence	O
-and	O
-nature	O
-of	O
-the	O
-metal	O
-that	O
-might	O
-be	O
-contained	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-by	O
-inductive	O
--	O
-coupled	O
-plasma	O
-mass	O
-spectrometry	O
-(	O
-ICP	O
--	O
-MS	O
-).	O
-	O
-For	O
-this	O
-analysis	O
-we	O
-used	O
-recombinant	O
-(	O
-Sa	O
-)	O
-EctC	O
-preparations	O
-from	O
-three	O
-independent	O
-protein	O
-overproduction	O
-and	O
-purification	O
-experiments	O
-.	O
-	O
-The	O
-ICP	O
--	O
-MS	O
-analyses	O
-yielded	O
-an	O
-iron	O
-content	O
-of	O
-0	O
-.	O
-66	O
-±	O
-0	O
-.	O
-06	O
-mol	O
-iron	O
-per	O
-mol	O
-of	O
-protein	O
-and	O
-the	O
-used	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-preparations	O
-also	O
-contained	O
-a	O
-minor	O
-amount	O
-of	O
-zinc	O
-(	O
-0	O
-.	O
-08	O
-mol	O
-zinc	O
-per	O
-mol	O
-of	O
-protein	O
-).	O
-	O
-All	O
-other	O
-assayed	O
-metals	O
-(	O
-copper	O
-and	O
-nickel	O
-)	O
-were	O
-only	O
-present	O
-in	O
-trace	O
-amounts	O
-(	O
-0	O
-.	O
-01	O
-mol	O
-metal	O
-per	O
-mol	O
-of	O
-protein	O
-,	O
-respectively	O
-).	O
-	O
-The	O
-presence	O
-of	O
-iron	O
-in	O
-these	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-preparations	O
-was	O
-further	O
-confirmed	O
-by	O
-a	O
-colorimetric	O
-method	O
-that	O
-is	O
-based	O
-on	O
-an	O
-iron	O
--	O
-complexing	O
-reagent	O
-;	O
-this	O
-procedure	O
-yielded	O
-an	O
-iron	O
--	O
-content	O
-of	O
-0	O
-.	O
-84	O
-±	O
-0	O
-.	O
-05	O
-mol	O
-per	O
-mol	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-Hence	O
-,	O
-both	O
-ICP	O
--	O
-MS	O
-and	O
-the	O
-colorimetric	O
-method	O
-clearly	O
-established	O
-that	O
-the	O
-recombinantly	O
-produced	O
-ectoine	O
-synthase	O
-from	O
-S	O
-.	O
-alaskensis	O
-is	O
-an	O
-iron	O
--	O
-containing	O
-protein	O
-.	O
-	O
-We	O
-note	O
-in	O
-this	O
-context	O
-,	O
-that	O
-the	O
-values	O
-obtained	O
-for	O
-the	O
-iron	O
-content	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-proteins	O
-varied	O
-by	O
-approximately	O
-10	O
-to	O
-20	O
-%	O
-between	O
-the	O
-two	O
-methods	O
-.	O
-	O
-The	O
-reason	O
-for	O
-this	O
-difference	O
-is	O
-not	O
-known	O
-,	O
-but	O
-indicates	O
-that	O
-the	O
-well	O
-established	O
-colorimetric	O
-assay	O
-probably	O
-overestimates	O
-the	O
-iron	O
-content	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-preparations	O
-to	O
-a	O
-certain	O
-degree	O
-.	O
-	O
-A	O
-metal	O
-cofactor	O
-is	O
-important	O
-for	O
-the	O
-catalytic	O
-activity	O
-of	O
-EctC	O
-	O
-The	O
-iron	O
-detected	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-preparations	O
-could	O
-serve	O
-a	O
-structural	O
-role	O
-,	O
-or	O
-most	O
-likely	O
-,	O
-could	O
-be	O
-critical	O
-for	O
-enzyme	O
-catalysis	O
-as	O
-is	O
-the	O
-case	O
-for	O
-many	O
-members	O
-of	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-To	O
-address	O
-these	O
-questions	O
-,	O
-we	O
-incubated	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-enzyme	O
-with	O
-increasing	O
-concentrations	O
-of	O
-the	O
-metal	O
-chelator	O
-ethylene	O
--	O
-diamine	O
--	O
-tetraacetic	O
--	O
-acid	O
-(	O
-EDTA	O
-)	O
-and	O
-subsequently	O
-assayed	O
-ectoine	O
-synthase	O
-activity	O
-.	O
-	O
-The	O
-addition	O
-of	O
-very	O
-low	O
-concentrations	O
-of	O
-EDTA	O
-(	O
-0	O
-.	O
-05	O
-mM	O
-)	O
-to	O
-the	O
-EctC	O
-enzyme	O
-already	O
-led	O
-to	O
-a	O
-noticeable	O
-inhibition	O
-of	O
-the	O
-ectoine	O
-synthase	O
-activity	O
-and	O
-the	O
-presence	O
-of	O
-1	O
-mM	O
-EDTA	O
-completely	O
-inhibited	O
-the	O
-enzyme	O
-(	O
-Fig	O
-3a	O
-).	O
-	O
-Dependency	O
-of	O
-the	O
-ectoine	O
-synthase	O
-activity	O
-on	O
-metals	O
-.	O
-	O
-(	O
-a	O
-)	O
-Impact	O
-of	O
-the	O
-iron	O
--	O
-chelator	O
-EDTA	O
-on	O
-the	O
-enzyme	O
-activity	O
-of	O
-the	O
-purified	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-Metal	O
-depletion	O
-and	O
-reconstitution	O
-experiments	O
-with	O
-(	O
-b	O
-)	O
-stoichiometric	O
-and	O
-(	O
-c	O
-)	O
-excess	O
-amounts	O
-of	O
-metals	O
-.	O
-	O
-The	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-was	O
-present	O
-at	O
-a	O
-concentration	O
-of	O
-10	O
-μM	O
-.	O
-The	O
-level	O
-of	O
-enzyme	O
-activity	O
-given	O
-in	O
-(	O
-b	O
-)	O
-is	O
-benchmarked	O
-relative	O
-to	O
-that	O
-of	O
-ectoine	O
-synthase	O
-enzyme	O
-assays	O
-in	O
-which	O
-1	O
-mM	O
-FeCl2	O
-was	O
-added	O
-.	O
-	O
-We	O
-then	O
-took	O
-such	O
-an	O
-inactivated	O
-enzyme	O
-preparation	O
-,	O
-removed	O
-the	O
-EDTA	O
-by	O
-dialysis	O
-,	O
-and	O
-added	O
-stoichiometric	O
-amounts	O
-(	O
-10	O
-μM	O
-)	O
-of	O
-various	O
-metals	O
-to	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-enzyme	O
-.	O
-	O
-The	O
-addition	O
-of	O
-FeCl2	O
-to	O
-the	O
-enzyme	O
-assay	O
-restored	O
-enzyme	O
-activity	O
-to	O
-about	O
-38	O
-%,	O
-whereas	O
-the	O
-addition	O
-of	O
-ZnCl2	O
-or	O
-CoCl2	O
-rescued	O
-(	O
-Sa	O
-)	O
-EctC	O
-enzyme	O
-activity	O
-only	O
-to	O
-5	O
-%	O
-and	O
-3	O
-%,	O
-respectively	O
-.	O
-	O
-All	O
-other	O
-tested	O
-metals	O
-,	O
-including	O
-Fe3	O
-+,	O
-were	O
-unable	O
-to	O
-restore	O
-activity	O
-(	O
-Fig	O
-3b	O
-).	O
-	O
-When	O
-the	O
-concentration	O
-of	O
-the	O
-various	O
-metals	O
-in	O
-the	O
-enzyme	O
-assay	O
-was	O
-increased	O
-100	O
--	O
-fold	O
-,	O
-Fe2	O
-+	O
-exhibited	O
-again	O
-the	O
-strongest	O
-stimulating	O
-effect	O
-on	O
-enzyme	O
-activity	O
-,	O
-and	O
-rescued	O
-enzyme	O
-activity	O
-to	O
-a	O
-degree	O
-similar	O
-to	O
-that	O
-exhibited	O
-by	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-preparations	O
-that	O
-had	O
-not	O
-been	O
-inactivated	O
-through	O
-EDTA	O
-treatment	O
-(	O
-Fig	O
-3c	O
-).	O
-	O
-However	O
-,	O
-a	O
-large	O
-molar	O
-excess	O
-of	O
-other	O
-transition	O
--	O
-state	O
-metals	O
-(	O
-zinc	O
-,	O
-cobalt	O
-,	O
-nickel	O
-,	O
-copper	O
-,	O
-and	O
-manganese	O
-)	O
-typically	O
-found	O
-in	O
-members	O
-of	O
-the	O
-cupin	O
-superfamily	O
-allowed	O
-the	O
-partial	O
-rescue	O
-of	O
-ectoine	O
-synthase	O
-activity	O
-as	O
-well	O
-(	O
-Fig	O
-3c	O
-).	O
-	O
-This	O
-is	O
-in	O
-line	O
-with	O
-literature	O
-data	O
-showing	O
-that	O
-cupin	O
--	O
-type	O
-enzymes	O
-are	O
-often	O
-promiscuous	O
-with	O
-respect	O
-to	O
-the	O
-use	O
-of	O
-the	O
-catalytically	O
-important	O
-metal	O
-.	O
-	O
-Kinetic	O
-parameters	O
-of	O
-EctC	O
-for	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-and	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-	O
-Based	O
-on	O
-the	O
-data	O
-presented	O
-in	O
-S3	O
-Fig	O
-,	O
-we	O
-formulated	O
-an	O
-optimized	O
-activity	O
-assay	O
-for	O
-the	O
-ectoine	O
-synthase	O
-of	O
-S	O
-.	O
-alaskensis	O
-and	O
-used	O
-it	O
-to	O
-determined	O
-the	O
-kinetic	O
-parameters	O
-for	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-enzyme	O
-for	O
-both	O
-its	O
-natural	O
-substrate	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-and	O
-the	O
-isomer	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-.	O
-	O
-The	O
-EctC	O
--	O
-catalyzed	O
-ring	O
--	O
-closure	O
-of	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-to	O
-form	O
-ectoine	O
-exhibited	O
-Michaelis	O
--	O
-Menten	O
--	O
-kinetics	O
-with	O
-an	O
-apparent	O
-Km	O
-of	O
-4	O
-.	O
-9	O
-±	O
-0	O
-.	O
-5	O
-mM	O
-,	O
-a	O
-vmax	O
-of	O
-25	O
-.	O
-0	O
-±	O
-0	O
-.	O
-8	O
-U	O
-/	O
-mg	O
-and	O
-a	O
-kcat	O
-of	O
-7	O
-.	O
-2	O
-s	O
--	O
-1	O
-(	O
-S4a	O
-Fig	O
-).	O
-	O
-Given	O
-the	O
-chemical	O
-relatedness	O
-of	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-to	O
-the	O
-natural	O
-substrate	O
-(	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-)	O
-of	O
-the	O
-ectoine	O
-synthase	O
-(	O
-S1a	O
-and	O
-S1b	O
-Fig	O
-),	O
-we	O
-wondered	O
-whether	O
-(	O
-Sa	O
-)	O
-EctC	O
-could	O
-also	O
-use	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-to	O
-produce	O
-ectoine	O
-.	O
-	O
-(	O
-Sa	O
-)	O
-EctC	O
-catalyzed	O
-this	O
-reaction	O
-with	O
-Michaelis	O
--	O
-Menten	O
--	O
-kinetics	O
-exhibiting	O
-an	O
-apparent	O
-Km	O
-of	O
-25	O
-.	O
-4	O
-±	O
-2	O
-.	O
-9	O
-mM	O
-,	O
-a	O
-vmax	O
-of	O
-24	O
-.	O
-6	O
-±	O
-1	O
-.	O
-0	O
-U	O
-/	O
-mg	O
-and	O
-a	O
-kcat	O
-0	O
-.	O
-6	O
-s	O
--	O
-1	O
-(	O
-S4b	O
-Fig	O
-).	O
-	O
-Hence	O
-,	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-is	O
-a	O
-newly	O
-recognized	O
-substrate	O
-for	O
-ectoine	O
-synthase	O
-.	O
-	O
-However	O
-,	O
-both	O
-the	O
-affinity	O
-(	O
-Km	O
-)	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-and	O
-its	O
-catalytic	O
-efficiency	O
-(	O
-kcat	O
-/	O
-Km	O
-)	O
-were	O
-strongly	O
-reduced	O
-in	O
-comparison	O
-with	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-.	O
-	O
-The	O
-Km	O
-dropped	O
-fife	O
--	O
-fold	O
-from	O
-4	O
-.	O
-9	O
-±	O
-0	O
-.	O
-5	O
-mM	O
-to	O
-25	O
-.	O
-4	O
-±	O
-2	O
-.	O
-9	O
-mM	O
-,	O
-and	O
-the	O
-catalytic	O
-efficiency	O
-was	O
-reduced	O
-from	O
-1	O
-.	O
-47	O
-mM	O
--	O
-1	O
-s	O
--	O
-1	O
-to	O
-0	O
-.	O
-02	O
-mM	O
--	O
-1	O
-s	O
--	O
-1	O
-,	O
-a	O
-73	O
--	O
-fold	O
-decrease	O
-.	O
-	O
-Both	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-and	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-are	O
-concomitantly	O
-formed	O
-during	O
-the	O
-enzymatic	O
-hydrolysis	O
-of	O
-the	O
-ectoine	O
-ring	O
-during	O
-catabolism	O
-.	O
-	O
-Our	O
-finding	O
-that	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-is	O
-a	O
-substrate	O
-for	O
-ectoine	O
-synthase	O
-has	O
-bearings	O
-for	O
-an	O
-understanding	O
-of	O
-the	O
-physiology	O
-of	O
-those	O
-microorganisms	O
-that	O
-can	O
-both	O
-synthesize	O
-and	O
-catabolize	O
-ectoine	O
-.	O
-	O
-However	O
-,	O
-these	O
-types	O
-of	O
-microorganisms	O
-should	O
-still	O
-be	O
-able	O
-to	O
-largely	O
-avoid	O
-a	O
-futile	O
-cycle	O
-since	O
-the	O
-affinity	O
-of	O
-ectoine	O
-synthase	O
-for	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-and	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-,	O
-and	O
-its	O
-catalytic	O
-efficiency	O
-for	O
-the	O
-two	O
-compounds	O
-,	O
-differs	O
-substantially	O
-(	O
-S4a	O
-and	O
-S4b	O
-Fig	O
-).	O
-	O
-Crystallization	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-	O
-Since	O
-no	O
-crystal	O
-structure	O
-of	O
-ectoine	O
-synthase	O
-has	O
-been	O
-reported	O
-,	O
-we	O
-set	O
-out	O
-to	O
-crystallize	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-Attempts	O
-to	O
-obtain	O
-crystals	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-in	O
-complex	O
-either	O
-with	O
-its	O
-substrate	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-or	O
-its	O
-reaction	O
-product	O
-ectoine	O
-were	O
-not	O
-successful	O
-.	O
-	O
-However	O
-,	O
-two	O
-crystal	O
-forms	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-in	O
-the	O
-absence	O
-of	O
-the	O
-substrate	O
-were	O
-obtained	O
-.	O
-	O
-Attempts	O
-to	O
-solve	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-by	O
-molecular	O
-replacement	O
-has	O
-previously	O
-failed	O
-.	O
-	O
-However	O
-,	O
-we	O
-were	O
-able	O
-to	O
-obtain	O
-crystals	O
-of	O
-form	O
-B	O
-that	O
-were	O
-derivatized	O
-with	O
-mercury	O
-and	O
-these	O
-diffracted	O
-up	O
-to	O
-2	O
-.	O
-8	O
-Å	O
-(	O
-S1	O
-Table	O
-).	O
-	O
-This	O
-dataset	O
-was	O
-used	O
-to	O
-derive	O
-an	O
-initial	O
-structural	O
-model	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-,	O
-which	O
-in	O
-turn	O
-was	O
-employed	O
-as	O
-a	O
-template	O
-for	O
-molecular	O
-replacement	O
-to	O
-phase	O
-the	O
-native	O
-dataset	O
-(	O
-2	O
-.	O
-0	O
-Å	O
-)	O
-of	O
-crystal	O
-form	O
-B	O
-.	O
-After	O
-several	O
-rounds	O
-of	O
-manual	O
-model	O
-building	O
-and	O
-refinement	O
-,	O
-four	O
-monomers	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-were	O
-identified	O
-and	O
-the	O
-crystal	O
-structure	O
-was	O
-refined	O
-to	O
-a	O
-final	O
-Rcryst	O
-of	O
-21	O
-.	O
-1	O
-%	O
-and	O
-an	O
-Rfree	O
-of	O
-24	O
-.	O
-8	O
-%	O
-(	O
-S1	O
-Table	O
-).	O
-	O
-Finally	O
-,	O
-a	O
-monomer	O
-of	O
-this	O
-structure	O
-was	O
-used	O
-as	O
-a	O
-template	O
-for	O
-molecular	O
-replacement	O
-to	O
-phase	O
-the	O
-high	O
--	O
-resolution	O
-(	O
-1	O
-.	O
-2	O
-Å	O
-)	O
-dataset	O
-of	O
-crystal	O
-form	O
-A	O
-,	O
-which	O
-was	O
-subsequently	O
-refined	O
-to	O
-a	O
-final	O
-Rcryst	O
-of	O
-12	O
-.	O
-4	O
-%	O
-and	O
-an	O
-Rfree	O
-of	O
-14	O
-.	O
-9	O
-%	O
-(	O
-S1	O
-Table	O
-).	O
-	O
-Overall	O
-fold	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-	O
-The	O
-two	O
-EctC	O
-structures	O
-that	O
-we	O
-determined	O
-revealed	O
-that	O
-the	O
-ectoine	O
-synthase	O
-belongs	O
-to	O
-the	O
-cupin	O
-superfamily	O
-with	O
-respect	O
-to	O
-its	O
-overall	O
-fold	O
-(	O
-Fig	O
-4a	O
-–	O
-4c	O
-).	O
-	O
-However	O
-,	O
-they	O
-represent	O
-two	O
-different	O
-states	O
-of	O
-the	O
-137	O
-amino	O
-acids	O
-comprising	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-First	O
-,	O
-the	O
-1	O
-.	O
-2	O
-Å	O
-structure	O
-reveals	O
-the	O
-spatial	O
-configuration	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-ranging	O
-from	O
-amino	O
-acid	O
-Met	O
--	O
-1	O
-to	O
-Glu	O
--	O
-115	O
-;	O
-hence	O
-,	O
-it	O
-lacks	O
-22	O
-amino	O
-acids	O
-at	O
-the	O
-carboxy	O
--	O
-terminus	O
-of	O
-the	O
-authentic	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-This	O
-structure	O
-adopts	O
-an	O
-open	O
-conformation	O
-with	O
-respect	O
-to	O
-the	O
-typical	O
-fold	O
-of	O
-cupin	O
-barrels	O
-and	O
-is	O
-therefore	O
-termed	O
-in	O
-the	O
-following	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-(	O
-Fig	O
-4b	O
-).	O
-	O
-In	O
-this	O
-structure	O
-no	O
-metal	O
-co	O
--	O
-factor	O
-was	O
-identified	O
-.	O
-	O
-The	O
-second	O
-crystal	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-was	O
-solved	O
-at	O
-a	O
-resolution	O
-of	O
-2	O
-.	O
-0	O
-Å	O
-and	O
-contained	O
-four	O
-molecules	O
-of	O
-the	O
-protein	O
-in	O
-the	O
-asymmetric	O
-unit	O
-of	O
-which	O
-protomer	O
-A	O
-comprised	O
-amino	O
-acid	O
-Met	O
--	O
-1	O
-to	O
-Gly	O
--	O
-121	O
-and	O
-adopts	O
-a	O
-closed	O
-conformation	O
-.	O
-	O
-Hence	O
-,	O
-it	O
-still	O
-lacks	O
-16	O
-amino	O
-acid	O
-residues	O
-of	O
-the	O
-carboxy	O
--	O
-terminus	O
-of	O
-the	O
-authentic	O
-137	O
-amino	O
-acids	O
-comprising	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-We	O
-therefore	O
-cannot	O
-exclude	O
-that	O
-this	O
-crystal	O
-structure	O
-does	O
-not	O
-represent	O
-the	O
-fully	O
-closed	O
-state	O
-of	O
-the	O
-ectoine	O
-synthase	O
-;	O
-consequently	O
-,	O
-we	O
-tentatively	O
-termed	O
-it	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-.	O
-	O
-Interestingly	O
-,	O
-the	O
-three	O
-other	O
-monomers	O
-present	O
-in	O
-the	O
-asymmetric	O
-unit	O
-all	O
-range	O
-from	O
-Met	O
--	O
-1	O
-to	O
-Glu	O
--	O
-115	O
-and	O
-adopt	O
-a	O
-conformation	O
-similar	O
-to	O
-the	O
-“	O
-open	O
-”	O
-EctC	O
-structure	O
-.	O
-	O
-Overall	O
-structure	O
-of	O
-the	O
-“	O
-open	O
-”	O
-and	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-crystal	O
-structures	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-.	O
-	O
-(	O
-a	O
-)	O
-The	O
-overall	O
-structure	O
-of	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-resolved	O
-at	O
-2	O
-.	O
-0	O
-Å	O
-is	O
-depicted	O
-in	O
-green	O
-in	O
-a	O
-cartoon	O
-(	O
-upper	O
-panel	O
-)	O
-and	O
-surface	O
-(	O
-lower	O
-panel	O
-)	O
-representation	O
-.	O
-	O
-The	O
-β	O
--	O
-strands	O
-are	O
-numbered	O
-β1	O
--	O
-β11	O
-and	O
-the	O
-helices	O
-α	O
--	O
-I	O
-to	O
-α	O
--	O
-II	O
-.	O
-	O
-(	O
-b	O
-)	O
-The	O
-overall	O
-structure	O
-of	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-was	O
-resolved	O
-at	O
-1	O
-.	O
-2	O
-Å	O
-and	O
-is	O
-depicted	O
-in	O
-yellow	O
-in	O
-a	O
-cartoon	O
-(	O
-upper	O
-panel	O
-)	O
-and	O
-surface	O
-(	O
-lower	O
-panel	O
-)	O
-representation	O
-.	O
-	O
-The	O
-entrance	O
-to	O
-the	O
-active	O
-site	O
-of	O
-the	O
-ectoine	O
-synthase	O
-is	O
-marked	O
-.	O
-	O
-(	O
-c	O
-)	O
-Overlay	O
-of	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-and	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structures	O
-.	O
-	O
-The	O
-overall	O
-structure	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-basically	O
-the	O
-same	O
-in	O
-both	O
-crystals	O
-except	O
-for	O
-the	O
-carboxy	O
--	O
-terminus	O
-,	O
-which	O
-covers	O
-the	O
-entry	O
-of	O
-one	O
-side	O
-of	O
-the	O
-cupin	O
-barrel	O
-from	O
-the	O
-surroundings	O
-in	O
-monomer	O
-A	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-.	O
-	O
-This	O
-is	O
-reflected	O
-by	O
-the	O
-calculated	O
-root	O
-mean	O
-square	O
-deviation	O
-(	O
-RMSD	O
-)	O
-of	O
-the	O
-Cα	O
-atoms	O
-that	O
-was	O
-about	O
-0	O
-.	O
-56	O
-Å	O
-(	O
-over	O
-117	O
-residues	O
-)	O
-when	O
-the	O
-four	O
-“	O
-open	O
-”	O
-monomers	O
-were	O
-compared	O
-with	O
-each	O
-other	O
-.	O
-	O
-However	O
-,	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-monomer	O
-has	O
-a	O
-slightly	O
-higher	O
-RMSD	O
-of	O
-1	O
-.	O
-4	O
-Å	O
-(	O
-over	O
-117	O
-residues	O
-)	O
-when	O
-compared	O
-with	O
-the	O
-“	O
-open	O
-”	O
-2	O
-.	O
-0	O
-Å	O
-structure	O
-.	O
-	O
-Therefore	O
-,	O
-we	O
-describe	O
-in	O
-the	O
-following	O
-the	O
-overall	O
-structure	O
-for	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-form	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-and	O
-subsequently	O
-highlight	O
-the	O
-structural	O
-differences	O
-between	O
-the	O
-“	O
-open	O
-”	O
-and	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-forms	O
-in	O
-more	O
-detail	O
-.	O
-	O
-The	O
-structure	O
-of	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-consists	O
-of	O
-11	O
-β	O
--	O
-strands	O
-(	O
-β1	O
--	O
-β11	O
-)	O
-and	O
-two	O
-α	O
--	O
-helices	O
-(	O
-α	O
--	O
-I	O
-and	O
-α	O
--	O
-II	O
-)	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-The	O
-β	O
--	O
-strands	O
-form	O
-two	O
-anti	O
--	O
-parallel	O
-β	O
--	O
-sheets	O
-:	O
-β2	O
-β3	O
-,	O
-β4	O
-,	O
-β11	O
-,	O
-β6	O
-,	O
-and	O
-β9	O
-,	O
-and	O
-a	O
-smaller	O
-three	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-(	O
-β7	O
-,	O
-β8	O
-,	O
-and	O
-β10	O
-),	O
-respectively	O
-.	O
-	O
-These	O
-two	O
-β	O
--	O
-sheets	O
-pack	O
-against	O
-each	O
-other	O
-,	O
-forming	O
-a	O
-cup	O
--	O
-shaped	O
-β	O
--	O
-sandwich	O
-with	O
-a	O
-topology	O
-characteristic	O
-for	O
-the	O
-cupin	O
--	O
-fold	O
-.	O
-	O
-Hence	O
-,	O
-(	O
-Sa	O
-)	O
-EctC	O
-adopts	O
-an	O
-overall	O
-bowl	O
-shape	O
-in	O
-which	O
-one	O
-side	O
-is	O
-opened	O
-towards	O
-the	O
-solvent	O
-(	O
-Fig	O
-4a	O
-to	O
-4c	O
-).	O
-	O
-In	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-,	O
-a	O
-longer	O
-carboxy	O
--	O
-terminal	O
-tail	O
-is	O
-visible	O
-in	O
-the	O
-electron	O
-density	O
-,	O
-folding	O
-into	O
-a	O
-small	O
-helix	O
-(	O
-α	O
--	O
-II	O
-)	O
-that	O
-closes	O
-the	O
-active	O
-site	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-The	O
-formation	O
-of	O
-this	O
-α	O
--	O
-II	O
-helix	O
-induces	O
-a	O
-reorientation	O
-and	O
-shift	O
-of	O
-a	O
-long	O
-unstructured	O
-loop	O
-(	O
-as	O
-observed	O
-in	O
-the	O
-“	O
-open	O
-”	O
-structure	O
-)	O
-connecting	O
-β4	O
-and	O
-β6	O
-,	O
-resulting	O
-in	O
-the	O
-formation	O
-of	O
-the	O
-stable	O
-β	O
--	O
-strand	O
-β5	O
-as	O
-observed	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-state	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-Structural	O
-comparison	O
-analyses	O
-using	O
-the	O
-DALI	O
-server	O
-revealed	O
-that	O
-(	O
-Sa	O
-)	O
-EctC	O
-adopts	O
-a	O
-fold	O
-similar	O
-to	O
-other	O
-members	O
-of	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-The	O
-highest	O
-structural	O
-similarities	O
-are	O
-observed	O
-for	O
-the	O
-Cupin	O
-2	O
-conserved	O
-barrel	O
-domain	O
-protein	O
-(	O
-YP_751781	O
-.	O
-1	O
-)	O
-from	O
-Shewanella	O
-frigidimarina	O
-(	O
-PDB	O
-accession	O
-code	O
-:	O
-2PFW	O
-)	O
-with	O
-a	O
-Z	O
--	O
-score	O
-of	O
-13	O
-.	O
-1	O
-and	O
-an	O
-RMSD	O
-of	O
-2	O
-.	O
-2	O
-Å	O
-over	O
-104	O
-Cα	O
--	O
-atoms	O
-(	O
-structural	O
-data	O
-for	O
-this	O
-protein	O
-have	O
-been	O
-deposited	O
-in	O
-the	O
-PDB	O
-but	O
-no	O
-publication	O
-connected	O
-to	O
-this	O
-structure	O
-is	O
-currently	O
-available	O
-),	O
-a	O
-manganese	O
--	O
-containing	O
-cupin	O
-(	O
-TM1459	O
-)	O
-from	O
-Thermotoga	O
-maritima	O
-(	O
-PDB	O
-accession	O
-code	O
-:	O
-1VJ2	O
-)	O
-with	O
-a	O
-Z	O
--	O
-score	O
-of	O
-12	O
-.	O
-8	O
-and	O
-an	O
-RMSD	O
-of	O
-2	O
-.	O
-0	O
-Å	O
-over	O
-103	O
-Cα	O
--	O
-atoms	O
-,	O
-the	O
-cyclase	O
-RemF	O
-from	O
-Streptomyces	O
-resistomycificus	O
-(	O
-PDB	O
-accession	O
-code	O
-:	O
-3HT1	O
-with	O
-a	O
-Z	O
--	O
-score	O
-of	O
-11	O
-.	O
-9	O
-and	O
-an	O
-RMSD	O
-of	O
-1	O
-.	O
-9	O
-Å	O
-over	O
-102	O
-Cα	O
--	O
-atoms	O
-),	O
-and	O
-an	O
-auxin	O
--	O
-binding	O
-protein	O
-1	O
-from	O
-Zea	O
-mays	O
-(	O
-PDB	O
-accession	O
-code	O
-:	O
-1LR5	O
-)	O
-with	O
-an	O
-Z	O
--	O
-score	O
-of	O
-11	O
-.	O
-8	O
-and	O
-an	O
-RMSD	O
-of	O
-2	O
-.	O
-8	O
-Å	O
-over	O
-104	O
-Cα	O
--	O
-atoms	O
-).	O
-	O
-Our	O
-data	O
-classify	O
-EctC	O
-,	O
-in	O
-addition	O
-to	O
-the	O
-polyketide	O
-cyclase	O
-RemF	O
-,	O
-as	O
-the	O
-second	O
-known	O
-cupin	O
--	O
-related	O
-enzyme	O
-that	O
-catalyze	O
-a	O
-cyclocondensation	O
-reaction	O
-.	O
-	O
-Next	O
-to	O
-RemF	O
-and	O
-the	O
-aldos	O
--	O
-2	O
--	O
-ulose	O
-dehydratase	O
-/	O
-isomerase	O
-,	O
-the	O
-ectoine	O
-synthase	O
-is	O
-only	O
-the	O
-third	O
-characterized	O
-dehydratase	O
-within	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-Analysis	O
-of	O
-the	O
-EctC	O
-dimer	O
-interface	O
-as	O
-observed	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structure	O
-	O
-Both	O
-the	O
-SEC	O
-analysis	O
-and	O
-the	O
-HPLC	O
--	O
-MALS	O
-experiments	O
-(	O
-S2b	O
-Fig	O
-)	O
-have	O
-shown	O
-that	O
-the	O
-ectoine	O
-synthase	O
-from	O
-S	O
-.	O
-alaskensis	O
-is	O
-a	O
-dimer	O
-in	O
-solution	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-this	O
-protein	O
-reflects	O
-this	O
-quaternary	O
-arrangement	O
-.	O
-	O
-In	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-crystal	O
-structure	O
-,	O
-(	O
-Sa	O
-)	O
-EctC	O
-has	O
-crystallized	O
-as	O
-a	O
-dimer	O
-of	O
-dimers	O
-within	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-This	O
-dimer	O
-(	O
-Fig	O
-5a	O
-and	O
-5b	O
-)	O
-is	O
-composed	O
-of	O
-two	O
-monomers	O
-arranged	O
-in	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-orientation	O
-and	O
-is	O
-stabilized	O
-via	O
-strong	O
-interactions	O
-mediated	O
-by	O
-two	O
-antiparallel	O
-β	O
--	O
-strands	O
-,	O
-β	O
--	O
-strand	O
-β1	O
-(	O
-sequence	O
-1MIVRN5	O
-)	O
-from	O
-monomer	O
-A	O
-and	O
-β	O
--	O
-strand	O
-β8	O
-from	O
-monomer	O
-B	O
-(	O
-sequence	O
-82GVMYAL87	O
-)	O
-(	O
-Fig	O
-5c	O
-).	O
-	O
-The	O
-strong	O
-interactions	O
-between	O
-these	O
-β	O
--	O
-strands	O
-rely	O
-primarily	O
-on	O
-backbone	O
-contacts	O
-.	O
-	O
-In	O
-addition	O
-to	O
-these	O
-interactions	O
-,	O
-some	O
-weaker	O
-hydrophobic	O
-interactions	O
-are	O
-also	O
-observed	O
-between	O
-the	O
-two	O
-monomers	O
-in	O
-some	O
-loops	O
-connecting	O
-the	O
-β	O
--	O
-strands	O
-.	O
-	O
-As	O
-calculated	O
-with	O
-PDBePISA	O
-,	O
-the	O
-surface	O
-area	O
-buried	O
-upon	O
-dimer	O
-formation	O
-is	O
-1462	O
-Å2	O
-,	O
-which	O
-is	O
-20	O
-.	O
-5	O
-%	O
-of	O
-the	O
-total	O
-accessible	O
-surface	O
-of	O
-a	O
-monomer	O
-of	O
-this	O
-protein	O
-.	O
-	O
-Both	O
-values	O
-fall	O
-within	O
-the	O
-range	O
-for	O
-known	O
-functional	O
-dimers	O
-.	O
-	O
-Crystal	O
-structure	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-.	O
-	O
-(	O
-a	O
-)	O
-Top	O
--	O
-view	O
-of	O
-the	O
-dimer	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-The	O
-position	O
-of	O
-the	O
-water	O
-molecule	O
-,	O
-described	O
-in	O
-detail	O
-in	O
-the	O
-text	O
-,	O
-is	O
-shown	O
-in	O
-one	O
-of	O
-the	O
-monomers	O
-as	O
-an	O
-orange	O
-sphere	O
-.	O
-(	O
-b	O
-)	O
-Side	O
--	O
-view	O
-of	O
-a	O
-(	O
-Sa	O
-)	O
-EctC	O
-dimer	O
-allowing	O
-an	O
-assessment	O
-of	O
-the	O
-dimer	O
-interface	O
-formed	O
-by	O
-two	O
-β	O
--	O
-strands	O
-of	O
-each	O
-monomer	O
-.	O
-	O
-(	O
-c	O
-)	O
-Close	O
--	O
-up	O
-representation	O
-of	O
-the	O
-dimer	O
-interface	O
-mediated	O
-by	O
-beta	O
--	O
-strand	O
-β1	O
-and	O
-β6	O
-.	O
-	O
-In	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-,	O
-one	O
-monomer	O
-is	O
-present	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-We	O
-therefore	O
-inspected	O
-the	O
-crystal	O
-packing	O
-and	O
-analyzed	O
-the	O
-monomer	O
--	O
-monomer	O
-interactions	O
-with	O
-symmetry	O
-related	O
-molecules	O
-to	O
-elucidate	O
-whether	O
-a	O
-physiologically	O
-relevant	O
-dimer	O
-could	O
-be	O
-deduced	O
-from	O
-this	O
-crystal	O
-form	O
-as	O
-well	O
-.	O
-	O
-Indeed	O
-,	O
-a	O
-similar	O
-dimer	O
-configuration	O
-to	O
-the	O
-one	O
-described	O
-for	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-is	O
-observed	O
-with	O
-the	O
-same	O
-monomer	O
--	O
-monomer	O
-interactions	O
-mediated	O
-by	O
-the	O
-two	O
-β	O
--	O
-sheets	O
-.	O
-	O
-The	O
-crystallographic	O
-two	O
--	O
-fold	O
-axis	O
-present	O
-within	O
-the	O
-crystal	O
-symmetry	O
-is	O
-located	O
-exactly	O
-in	O
-between	O
-the	O
-two	O
-monomers	O
-,	O
-resulting	O
-in	O
-a	O
-monomer	O
-within	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-Hence	O
-,	O
-the	O
-same	O
-dimer	O
-observed	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-can	O
-also	O
-be	O
-observed	O
-in	O
-the	O
-“	O
-open	O
-”	O
-structure	O
-.	O
-	O
-Interestingly	O
-,	O
-the	O
-proteins	O
-identified	O
-by	O
-the	O
-above	O
--	O
-described	O
-DALI	O
-search	O
-not	O
-only	O
-have	O
-folds	O
-similar	O
-to	O
-EctC	O
-,	O
-but	O
-are	O
-also	O
-functional	O
-dimers	O
-that	O
-adopt	O
-similar	O
-monomer	O
--	O
-monomer	O
-interactions	O
-within	O
-the	O
-dimer	O
-assembly	O
-as	O
-deduced	O
-from	O
-the	O
-inspection	O
-of	O
-the	O
-corresponding	O
-PDB	O
-files	O
-(	O
-2PFW	O
-,	O
-3HT1	O
-,	O
-1VJ2	O
-,	O
-1LR5	O
-).	O
-	O
-Structural	O
-rearrangements	O
-of	O
-the	O
-flexible	O
-(	O
-Sa	O
-)	O
-EctC	O
-carboxy	O
--	O
-terminus	O
-	O
-The	O
-cupin	O
-core	O
-represents	O
-the	O
-structural	O
-framework	O
-of	O
-ectoine	O
-synthase	O
-(	O
-Figs	O
-4	O
-and	O
-5	O
-).	O
-	O
-The	O
-major	O
-difference	O
-in	O
-the	O
-two	O
-crystal	O
-structures	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-reported	O
-here	O
-is	O
-the	O
-orientation	O
-of	O
-the	O
-carboxy	O
--	O
-terminus	O
-.	O
-	O
-Some	O
-amino	O
-acids	O
-located	O
-in	O
-the	O
-carboxy	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-137	O
-amino	O
-acids	O
-comprising	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-are	O
-highly	O
-conserved	O
-(	O
-Fig	O
-2	O
-)	O
-within	O
-the	O
-extended	O
-EctC	O
-protein	O
-family	O
-.	O
-	O
-At	O
-the	O
-end	O
-of	O
-β	O
--	O
-strand	O
-β11	O
-,	O
-two	O
-consecutive	O
-conserved	O
-proline	O
-residues	O
-(	O
-Pro	O
--	O
-109	O
-and	O
-Pro	O
--	O
-110	O
-)	O
-are	O
-present	O
-that	O
-are	O
-responsible	O
-for	O
-a	O
-turn	O
-in	O
-the	O
-main	O
-chain	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-In	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-,	O
-the	O
-visible	O
-electron	O
-density	O
-of	O
-the	O
-carboxy	O
--	O
-terminus	O
-is	O
-extended	O
-by	O
-7	O
-amino	O
-acid	O
-residues	O
-and	O
-ends	O
-at	O
-position	O
-Gly	O
--	O
-121	O
-.	O
-	O
-These	O
-additional	O
-amino	O
-acids	O
-fold	O
-into	O
-a	O
-small	O
-helix	O
-,	O
-which	O
-seals	O
-the	O
-open	O
-cavity	O
-of	O
-the	O
-cupin	O
--	O
-fold	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-Furthermore	O
-,	O
-this	O
-helix	O
-is	O
-stabilized	O
-via	O
-interactions	O
-with	O
-the	O
-loop	O
-region	O
-between	O
-β	O
--	O
-strands	O
-β4	O
-and	O
-β6	O
-,	O
-thereby	O
-inducing	O
-a	O
-structural	O
-rearrangement	O
-.	O
-	O
-This	O
-induces	O
-the	O
-formation	O
-of	O
-β	O
--	O
-strand	O
-β5	O
-,	O
-which	O
-is	O
-not	O
-present	O
-when	O
-the	O
-small	O
-C	O
--	O
-terminal	O
-helix	O
-is	O
-absent	O
-as	O
-observed	O
-in	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-.	O
-	O
-As	O
-a	O
-result	O
-,	O
-the	O
-newly	O
-formed	O
-β	O
--	O
-strand	O
-β5	O
-is	O
-reoriented	O
-and	O
-moved	O
-by	O
-2	O
-.	O
-4	O
-Å	O
-within	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-(	O
-Fig	O
-4a	O
-to	O
-4c	O
-).	O
-	O
-It	O
-is	O
-worth	O
-mentioning	O
-that	O
-β	O
--	O
-strand	O
-β5	O
-is	O
-located	O
-next	O
-to	O
-His	O
--	O
-93	O
-,	O
-which	O
-in	O
-all	O
-likelihood	O
-involved	O
-in	O
-metal	O
-binding	O
-(	O
-see	O
-below	O
-).	O
-	O
-The	O
-position	O
-of	O
-this	O
-His	O
-residue	O
-is	O
-slightly	O
-shifted	O
-in	O
-both	O
-(	O
-Sa	O
-)	O
-EctC	O
-structures	O
-,	O
-likely	O
-the	O
-result	O
-of	O
-the	O
-formation	O
-of	O
-β	O
--	O
-strand	O
-β5	O
-.	O
-	O
-Therefore	O
-the	O
-sealing	O
-of	O
-the	O
-cupin	O
-fold	O
-,	O
-as	O
-described	O
-above	O
-,	O
-seem	O
-to	O
-have	O
-an	O
-indirect	O
-influence	O
-on	O
-the	O
-architecture	O
-of	O
-the	O
-postulated	O
-iron	O
--	O
-binding	O
-site	O
-.	O
-	O
-The	O
-consecutive	O
-Pro	O
--	O
-109	O
-and	O
-Pro	O
--	O
-110	O
-residues	O
-found	O
-at	O
-the	O
-end	O
-of	O
-β	O
--	O
-strand	O
-β11are	O
-highly	O
-conserved	O
-in	O
-EctC	O
--	O
-type	O
-proteins	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-They	O
-are	O
-responsible	O
-for	O
-redirecting	O
-the	O
-main	O
-chain	O
-of	O
-the	O
-remaining	O
-carboxy	O
--	O
-terminus	O
-(	O
-27	O
-amino	O
-acid	O
-residues	O
-)	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-to	O
-close	O
-the	O
-cupin	O
-fold	O
-.	O
-	O
-In	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-this	O
-results	O
-in	O
-a	O
-complete	O
-closure	O
-of	O
-the	O
-entry	O
-of	O
-the	O
-cupin	O
-barrel	O
-(	O
-Fig	O
-4a	O
-to	O
-4c	O
-).	O
-	O
-In	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-,	O
-both	O
-proline	O
-residues	O
-are	O
-visible	O
-in	O
-the	O
-electron	O
-density	O
-;	O
-however	O
-,	O
-almost	O
-directly	O
-after	O
-Pro	O
--	O
-110	O
-,	O
-the	O
-electron	O
-density	O
-is	O
-drastically	O
-diminished	O
-caused	O
-by	O
-the	O
-flexibility	O
-of	O
-the	O
-carboxy	O
--	O
-terminus	O
-.	O
-	O
-A	O
-search	O
-for	O
-partners	O
-interacting	O
-with	O
-Pro	O
--	O
-109	O
-revealed	O
-that	O
-it	O
-interacts	O
-via	O
-its	O
-backbone	O
-oxygen	O
-with	O
-the	O
-side	O
-chain	O
-of	O
-His	O
--	O
-55	O
-as	O
-visible	O
-in	O
-both	O
-the	O
-“	O
-open	O
-”	O
-and	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structures	O
-.	O
-	O
-The	O
-Pro	O
--	O
-109	O
-/	O
-His	O
--	O
-55	O
-interaction	O
-ensures	O
-the	O
-stable	O
-orientation	O
-of	O
-both	O
-proline	O
-residues	O
-at	O
-the	O
-end	O
-of	O
-β	O
--	O
-strand	O
-β11	O
-.	O
-	O
-Since	O
-these	O
-proline	O
-residues	O
-are	O
-followed	O
-by	O
-the	O
-carboxy	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-,	O
-the	O
-interaction	O
-of	O
-His	O
--	O
-55	O
-with	O
-Pro	O
--	O
-109	O
-will	O
-likely	O
-play	O
-a	O
-substantial	O
-role	O
-in	O
-spatially	O
-orienting	O
-this	O
-very	O
-flexible	O
-part	O
-of	O
-the	O
-protein	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-interactions	O
-between	O
-Pro	O
--	O
-109	O
-and	O
-His	O
--	O
-55	O
-,	O
-the	O
-carboxy	O
--	O
-terminal	O
-region	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-held	O
-in	O
-position	O
-via	O
-an	O
-interaction	O
-of	O
-Glu	O
--	O
-115	O
-with	O
-His	O
--	O
-55	O
-,	O
-which	O
-stabilizes	O
-the	O
-conformation	O
-of	O
-the	O
-small	O
-helix	O
-in	O
-the	O
-carboxy	O
--	O
-terminus	O
-further	O
-.	O
-	O
-The	O
-interaction	O
-between	O
-Glu	O
--	O
-115	O
-and	O
-His	O
--	O
-55	O
-is	O
-only	O
-visible	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-where	O
-the	O
-partially	O
-extended	O
-carboxy	O
--	O
-terminus	O
-is	O
-resolved	O
-in	O
-the	O
-electron	O
-density	O
-.	O
-	O
-In	O
-the	O
-“	O
-open	O
-”	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-,	O
-this	O
-interaction	O
-does	O
-not	O
-occur	O
-since	O
-Glu	O
--	O
-115	O
-is	O
-rotated	O
-outwards	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-).	O
-	O
-Hence	O
-,	O
-one	O
-might	O
-speculate	O
-that	O
-this	O
-missing	O
-interaction	O
-might	O
-be	O
-responsible	O
-for	O
-the	O
-flexibility	O
-of	O
-the	O
-carboxy	O
--	O
-terminus	O
-in	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-and	O
-consequently	O
-results	O
-in	O
-less	O
-well	O
-defined	O
-electron	O
-density	O
-in	O
-this	O
-region	O
-.	O
-	O
-Architecture	O
-of	O
-the	O
-presumed	O
-metal	O
--	O
-binding	O
-site	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-and	O
-its	O
-flexible	O
-carboxy	O
--	O
-terminus	O
-.	O
-	O
-(	O
-a	O
-)	O
-The	O
-described	O
-water	O
-molecule	O
-(	O
-depicted	O
-as	O
-orange	O
-sphere	O
-)	O
-is	O
-bound	O
-via	O
-interactions	O
-with	O
-the	O
-side	O
-chains	O
-of	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-and	O
-His	O
--	O
-93	O
-.	O
-	O
-The	O
-position	O
-occupied	O
-by	O
-this	O
-water	O
-molecule	O
-represents	O
-probably	O
-the	O
-position	O
-of	O
-the	O
-Fe2	O
-+	O
-cofactor	O
-in	O
-the	O
-active	O
-side	O
-of	O
-the	O
-ectoine	O
-synthase	O
-.	O
-	O
-His	O
--	O
-55	O
-interacts	O
-with	O
-the	O
-double	O
-proline	O
-motif	O
-(	O
-Pro	O
--	O
-109	O
-and	O
-Pro	O
--	O
-110	O
-).	O
-	O
-It	O
-is	O
-further	O
-stabilized	O
-via	O
-an	O
-interaction	O
-with	O
-the	O
-side	O
-chain	O
-of	O
-Glu	O
--	O
-115	O
-which	O
-is	O
-localized	O
-in	O
-the	O
-flexible	O
-carboxy	O
--	O
-terminus	O
-(	O
-colored	O
-in	O
-orange	O
-)	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-that	O
-is	O
-visible	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-.	O
-	O
-(	O
-b	O
-)	O
-An	O
-overlay	O
-of	O
-the	O
-“	O
-open	O
-”	O
-(	O
-colored	O
-in	O
-light	O
-blue	O
-)	O
-and	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-colored	O
-in	O
-green	O
-)	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-The	O
-putative	O
-iron	O
-binding	O
-site	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-	O
-In	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-,	O
-each	O
-of	O
-the	O
-four	O
-monomers	O
-in	O
-the	O
-asymmetric	O
-unit	O
-contains	O
-a	O
-relative	O
-strong	O
-electron	O
-density	O
-positioned	O
-within	O
-the	O
-cupin	O
-barrel	O
-.	O
-	O
-Since	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-a	O
-metal	O
-containing	O
-protein	O
-(	O
-Fig	O
-3	O
-),	O
-we	O
-tried	O
-to	O
-fit	O
-either	O
-Fe2	O
-+,	O
-or	O
-Zn2	O
-+	O
-ions	O
-into	O
-this	O
-density	O
-and	O
-also	O
-refined	O
-occupancy	O
-.	O
-	O
-Only	O
-the	O
-refinement	O
-of	O
-Fe2	O
-+	O
-resulted	O
-in	O
-a	O
-visibly	O
-improved	O
-electron	O
-density	O
-,	O
-however	O
-with	O
-a	O
-low	O
-degree	O
-of	O
-occupancy	O
-.	O
-	O
-This	O
-possible	O
-iron	O
-molecule	O
-is	O
-bound	O
-via	O
-interactions	O
-with	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-and	O
-His	O
--	O
-93	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-).	O
-	O
-The	O
-distance	O
-between	O
-the	O
-side	O
-chains	O
-of	O
-these	O
-residues	O
-and	O
-the	O
-(	O
-putative	O
-)	O
-iron	O
-co	O
--	O
-factor	O
-is	O
-3	O
-.	O
-1	O
-Å	O
-for	O
-Glu	O
--	O
-57	O
-,	O
-2	O
-.	O
-9	O
-Å	O
-for	O
-Tyr	O
--	O
-85	O
-,	O
-and	O
-2	O
-.	O
-9	O
-Å	O
-for	O
-His	O
--	O
-93	O
-,	O
-respectively	O
-.	O
-	O
-These	O
-distances	O
-are	O
-to	O
-long	O
-when	O
-compared	O
-to	O
-other	O
-iron	O
-binding	O
-sites	O
-,	O
-a	O
-fact	O
-that	O
-might	O
-be	O
-caused	O
-by	O
-the	O
-absence	O
-of	O
-the	O
-proper	O
-substrate	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structure	O
-.	O
-	O
-Since	O
-both	O
-the	O
-refinement	O
-and	O
-the	O
-distance	O
-did	O
-not	O
-clearly	O
-identify	O
-an	O
-iron	O
-molecule	O
-,	O
-we	O
-decided	O
-to	O
-conservatively	O
-place	O
-a	O
-water	O
-molecule	O
-at	O
-this	O
-position	O
-.	O
-	O
-The	O
-position	O
-of	O
-this	O
-water	O
-molecule	O
-is	O
-described	O
-in	O
-more	O
-detail	O
-below	O
-and	O
-is	O
-highlighted	O
-in	O
-Figs	O
-5a	O
-and	O
-5b	O
-and	O
-6a	O
-and	O
-6b	O
-as	O
-a	O
-sphere	O
-.	O
-	O
-Interestingly	O
-,	O
-all	O
-three	O
-amino	O
-acids	O
-coordinating	O
-this	O
-water	O
-molecule	O
-are	O
-strictly	O
-conserved	O
-within	O
-an	O
-alignment	O
-of	O
-440	O
-members	O
-of	O
-the	O
-EctC	O
-protein	O
-family	O
-(	O
-for	O
-an	O
-abbreviated	O
-alignment	O
-of	O
-EctC	O
--	O
-type	O
-proteins	O
-see	O
-Fig	O
-2	O
-).	O
-	O
-In	O
-the	O
-“	O
-open	O
-”	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-,	O
-electron	O
-density	O
-is	O
-visible	O
-where	O
-the	O
-presumptive	O
-iron	O
-is	O
-positioned	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-.	O
-	O
-However	O
-,	O
-this	O
-electron	O
-density	O
-fits	O
-perfectly	O
-to	O
-a	O
-water	O
-molecule	O
-and	O
-not	O
-to	O
-an	O
-iron	O
-,	O
-and	O
-the	O
-water	O
-molecule	O
-was	O
-clearly	O
-visible	O
-after	O
-the	O
-refinement	O
-at	O
-this	O
-high	O
-resolution	O
-(	O
-1	O
-.	O
-2	O
-Å	O
-)	O
-of	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-.	O
-	O
-In	O
-a	O
-superimposition	O
-of	O
-both	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structures	O
-,	O
-the	O
-spatial	O
-arrangements	O
-of	O
-the	O
-side	O
-chains	O
-of	O
-the	O
-three	O
-amino	O
-acids	O
-(	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-and	O
-His	O
--	O
-93	O
-)	O
-likely	O
-to	O
-contact	O
-the	O
-iron	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-match	O
-nicely	O
-with	O
-those	O
-of	O
-the	O
-corresponding	O
-residues	O
-of	O
-the	O
-“	O
-iron	O
--	O
-free	O
-”	O
-“	O
-open	O
-”	O
-structure	O
-(	O
-Fig	O
-6b	O
-).	O
-	O
-Only	O
-His	O
--	O
-93	O
-is	O
-slightly	O
-rotated	O
-inwards	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-,	O
-most	O
-likely	O
-due	O
-to	O
-formation	O
-of	O
-β	O
--	O
-strand	O
-β5	O
-as	O
-described	O
-above	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-this	O
-observations	O
-indicate	O
-,	O
-that	O
-the	O
-architecture	O
-of	O
-the	O
-presumptive	O
-iron	O
--	O
-binding	O
-site	O
-is	O
-pre	O
--	O
-set	O
-for	O
-the	O
-binding	O
-of	O
-the	O
-catalytically	O
-important	O
-metal	O
-by	O
-the	O
-ectoine	O
-synthase	O
-.	O
-	O
-Of	O
-note	O
-is	O
-the	O
-different	O
-spatial	O
-arrangement	O
-of	O
-the	O
-side	O
--	O
-chain	O
-of	O
-Tyr	O
--	O
-52	O
-(	O
-located	O
-in	O
-a	O
-loop	O
-after	O
-the	O
-end	O
-of	O
-β	O
--	O
-strand	O
-β5	O
-)	O
-in	O
-the	O
-“	O
-open	O
-”	O
-and	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structures	O
-.	O
-	O
-In	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-,	O
-the	O
-hydroxyl	O
--	O
-group	O
-of	O
-the	O
-side	O
--	O
-chain	O
-of	O
-Tyr	O
--	O
-52	O
-points	O
-towards	O
-the	O
-iron	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-),	O
-but	O
-the	O
-corresponding	O
-distance	O
-(	O
-3	O
-.	O
-9	O
-Å	O
-)	O
-makes	O
-it	O
-highly	O
-unlikely	O
-that	O
-Tyr	O
--	O
-52	O
-is	O
-directly	O
-involved	O
-in	O
-metal	O
-binding	O
-.	O
-	O
-Nevertheless	O
-,	O
-its	O
-substitution	O
-by	O
-an	O
-Ala	O
-residue	O
-causes	O
-a	O
-strong	O
-decrease	O
-in	O
-iron	O
--	O
-content	O
-and	O
-enzyme	O
-activity	O
-of	O
-the	O
-mutant	O
-protein	O
-(	O
-Table	O
-1	O
-).	O
-	O
-It	O
-becomes	O
-apparent	O
-from	O
-an	O
-overlay	O
-of	O
-the	O
-“	O
-open	O
-”	O
-and	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structures	O
-that	O
-the	O
-side	O
--	O
-chain	O
-of	O
-Tyr	O
--	O
-52	O
-rotates	O
-away	O
-from	O
-the	O
-position	O
-of	O
-the	O
-presumptive	O
-iron	O
-,	O
-whereas	O
-the	O
-side	O
--	O
-chains	O
-of	O
-those	O
-residues	O
-that	O
-probably	O
-contacting	O
-the	O
-metal	O
-directly	O
-[	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-and	O
-His	O
--	O
-93	O
-],	O
-remain	O
-in	O
-place	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-).	O
-	O
-Since	O
-Tyr	O
--	O
-52	O
-is	O
-strictly	O
-conserved	O
-in	O
-an	O
-alignment	O
-of	O
-440	O
-EctC	O
--	O
-type	O
-proteins	O
-(	O
-Fig	O
-2	O
-),	O
-we	O
-speculate	O
-that	O
-it	O
-might	O
-be	O
-involved	O
-in	O
-contacting	O
-the	O
-substrate	O
-of	O
-the	O
-ectoine	O
-synthase	O
-and	O
-that	O
-the	O
-absence	O
-of	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-in	O
-our	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structures	O
-might	O
-endow	O
-the	O
-side	O
-chain	O
-of	O
-Tyr	O
--	O
-52	O
-with	O
-extra	O
-spatial	O
-flexibility	O
-.	O
-	O
-To	O
-further	O
-analyze	O
-the	O
-putative	O
-iron	O
-binding	O
-site	O
-(	O
-Fig	O
-6a	O
-),	O
-we	O
-performed	O
-structure	O
--	O
-guided	O
-site	O
--	O
-directed	O
-mutagenesis	O
-and	O
-assessed	O
-the	O
-resulting	O
-(	O
-Sa	O
-)	O
-EctC	O
-variants	O
-for	O
-their	O
-iron	O
-content	O
-and	O
-studied	O
-their	O
-enzyme	O
-activity	O
-.	O
-	O
-When	O
-those	O
-three	O
-residues	O
-(	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-His	O
--	O
-93	O
-)	O
-that	O
-likely	O
-form	O
-the	O
-mono	O
--	O
-nuclear	O
-iron	O
-center	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structure	O
-were	O
-individually	O
-replaced	O
-by	O
-an	O
-Ala	O
-residue	O
-,	O
-both	O
-the	O
-catalytic	O
-activity	O
-and	O
-the	O
-iron	O
-content	O
-of	O
-the	O
-mutant	O
-proteins	O
-was	O
-strongly	O
-reduced	O
-(	O
-Table	O
-1	O
-).	O
-	O
-For	O
-some	O
-of	O
-the	O
-presumptive	O
-iron	O
--	O
-coordinating	O
-residues	O
-,	O
-additional	O
-site	O
--	O
-directed	O
-mutagenesis	O
-experiments	O
-were	O
-carried	O
-out	O
-.	O
-	O
-To	O
-verify	O
-the	O
-importance	O
-of	O
-the	O
-negative	O
-charge	O
-in	O
-the	O
-position	O
-of	O
-Glu	O
--	O
-57	O
-,	O
-we	O
-created	O
-an	O
-Asp	O
-variant	O
-.	O
-	O
-This	O
-mutant	O
-protein	O
-rescued	O
-the	O
-enzyme	O
-activity	O
-and	O
-iron	O
-content	O
-of	O
-the	O
-Ala	O
-substitution	O
-substantially	O
-(	O
-Table	O
-1	O
-).	O
-	O
-We	O
-also	O
-replaced	O
-Tyr	O
--	O
-85	O
-with	O
-either	O
-a	O
-Phe	O
-or	O
-a	O
-Trp	O
-residue	O
-and	O
-both	O
-mutant	O
-proteins	O
-largely	O
-lost	O
-their	O
-catalytic	O
-activity	O
-and	O
-iron	O
-content	O
-(	O
-Table	O
-1	O
-)	O
-despite	O
-the	O
-fact	O
-that	O
-these	O
-substitutions	O
-were	O
-conservative	O
-.	O
-	O
-Collectively	O
-,	O
-these	O
-data	O
-suggest	O
-that	O
-the	O
-hydroxyl	O
-group	O
-of	O
-the	O
-Tyr	O
--	O
-85	O
-side	O
-chain	O
-is	O
-needed	O
-for	O
-the	O
-binding	O
-of	O
-the	O
-iron	O
-(	O
-Fig	O
-6a	O
-).	O
-	O
-We	O
-also	O
-replaced	O
-the	O
-presumptive	O
-iron	O
--	O
-binding	O
-residue	O
-His	O
--	O
-93	O
-by	O
-an	O
-Asn	O
-residue	O
-,	O
-yielding	O
-a	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-variant	O
-that	O
-possessed	O
-an	O
-enzyme	O
-activity	O
-of	O
-23	O
-%	O
-and	O
-iron	O
-content	O
-of	O
-only	O
-14	O
-%	O
-relative	O
-to	O
-that	O
-of	O
-the	O
-wild	O
--	O
-type	O
-protein	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Collectively	O
-,	O
-the	O
-data	O
-addressing	O
-the	O
-functionality	O
-of	O
-the	O
-putative	O
-iron	O
--	O
-coordinating	O
-residues	O
-(	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-His	O
--	O
-93	O
-)	O
-buttress	O
-our	O
-notion	O
-that	O
-the	O
-Fe2	O
-+	O
-present	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-is	O
-of	O
-catalytic	O
-importance	O
-.	O
-	O
-A	O
-chemically	O
-undefined	O
-ligand	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-provides	O
-clues	O
-for	O
-the	O
-binding	O
-of	O
-the	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-substrate	O
-	O
-Despite	O
-considerable	O
-efforts	O
-,	O
-either	O
-by	O
-trying	O
-co	O
--	O
-crystallization	O
-or	O
-soaking	O
-experiments	O
-,	O
-we	O
-were	O
-not	O
-able	O
-to	O
-obtain	O
-a	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structures	O
-that	O
-contained	O
-either	O
-the	O
-substrate	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-,	O
-or	O
-ectoine	O
-,	O
-the	O
-reaction	O
-product	O
-of	O
-ectoine	O
-synthase	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-However	O
-,	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-where	O
-the	O
-carboxy	O
--	O
-terminal	O
-loop	O
-is	O
-largely	O
-resolved	O
-,	O
-a	O
-long	O
-stretched	O
-electron	O
-density	O
-feature	O
-was	O
-detected	O
-in	O
-the	O
-predicted	O
-active	O
-site	O
-of	O
-the	O
-enzyme	O
-;	O
-it	O
-remained	O
-visible	O
-after	O
-crystallographic	O
-refinement	O
-.	O
-	O
-This	O
-is	O
-in	O
-contrast	O
-to	O
-the	O
-high	O
--	O
-resolution	O
-“	O
-open	O
-”	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-where	O
-no	O
-additional	O
-electron	O
-density	O
-was	O
-observed	O
-after	O
-refinement	O
-.	O
-	O
-We	O
-tried	O
-to	O
-fit	O
-all	O
-compounds	O
-used	O
-in	O
-the	O
-buffers	O
-during	O
-purification	O
-and	O
-crystallization	O
-into	O
-the	O
-observed	O
-electron	O
-density	O
-,	O
-but	O
-none	O
-matched	O
-.	O
-	O
-This	O
-observation	O
-indicates	O
-that	O
-the	O
-chemically	O
-undefined	O
-ligand	O
-was	O
-either	O
-trapped	O
-by	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-during	O
-its	O
-heterologous	O
-production	O
-in	O
-E	O
-.	O
-coli	O
-or	O
-during	O
-crystallization	O
-.	O
-	O
-Since	O
-we	O
-used	O
-PEG	O
-molecules	O
-in	O
-the	O
-crystallization	O
-conditions	O
-,	O
-the	O
-observed	O
-density	O
-might	O
-stem	O
-from	O
-an	O
-ordered	O
-part	O
-of	O
-a	O
-PEG	O
-molecule	O
-,	O
-or	O
-low	O
-molecular	O
-weight	O
-PEG	O
-species	O
-that	O
-might	O
-have	O
-been	O
-present	O
-in	O
-the	O
-PEG	O
-preparation	O
-used	O
-in	O
-our	O
-experiments	O
-.	O
-	O
-Estimating	O
-from	O
-the	O
-dimensions	O
-of	O
-the	O
-electron	O
-density	O
-feature	O
-,	O
-we	O
-modeled	O
-the	O
-chemically	O
-undefined	O
-compound	O
-trapped	O
-by	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-as	O
-a	O
-hexane	O
--	O
-1	O
-,	O
-6	O
--	O
-diol	O
-molecule	O
-(	O
-PDB	O
-identifier	O
-:	O
-HEZ	O
-)	O
-to	O
-best	O
-fit	O
-the	O
-observed	O
-electron	O
-density	O
-.	O
-	O
-However	O
-,	O
-to	O
-the	O
-best	O
-of	O
-our	O
-knowledge	O
-,	O
-hexane	O
--	O
-1	O
-,	O
-6	O
--	O
-diol	O
-is	O
-not	O
-part	O
-of	O
-the	O
-E	O
-.	O
-coli	O
-metabolome	O
-.	O
-	O
-Despite	O
-these	O
-notable	O
-limitations	O
-,	O
-we	O
-considered	O
-the	O
-serendipitously	O
-trapped	O
-compound	O
-as	O
-a	O
-mock	O
-ligand	O
-that	O
-might	O
-provide	O
-useful	O
-insights	O
-into	O
-the	O
-spatial	O
-positioning	O
-of	O
-the	O
-true	O
-EctC	O
-substrate	O
-and	O
-those	O
-residues	O
-that	O
-coordinate	O
-it	O
-within	O
-the	O
-ectoine	O
-synthase	O
-active	O
-site	O
-.	O
-	O
-We	O
-note	O
-that	O
-both	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-and	O
-hexane	O
--	O
-1	O
-,	O
-6	O
--	O
-diol	O
-are	O
-both	O
-C6	O
--	O
-compounds	O
-and	O
-display	O
-similar	O
-length	O
-(	O
-Fig	O
-7a	O
-).	O
-	O
-A	O
-chemically	O
-undefined	O
-ligand	O
-is	O
-captured	O
-in	O
-the	O
-active	O
-site	O
-of	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structure	O
-.	O
-	O
-(	O
-a	O
-)	O
-The	O
-observed	O
-electron	O
-density	O
-in	O
-the	O
-active	O
-site	O
-of	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-modeled	O
-as	O
-a	O
-hexane	O
--	O
-1	O
-,	O
-6	O
--	O
-diol	O
-molecule	O
-and	O
-compared	O
-with	O
-the	O
-electron	O
-density	O
-of	O
-the	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-substrate	O
-of	O
-the	O
-ectoine	O
-synthase	O
-to	O
-emphasize	O
-the	O
-similarity	O
-in	O
-size	O
-of	O
-these	O
-compounds	O
-.	O
-	O
-(	O
-b	O
-)	O
-The	O
-presumable	O
-binding	O
-site	O
-of	O
-the	O
-iron	O
-co	O
--	O
-factor	O
-and	O
-of	O
-the	O
-modeled	O
-hexane	O
--	O
-1	O
-,	O
-6	O
--	O
-diol	O
-molecule	O
-is	O
-depicted	O
-.	O
-	O
-The	O
-amino	O
-acid	O
-side	O
-chains	O
-involved	O
-in	O
-iron	O
--	O
-ligand	O
-binding	O
-are	O
-colored	O
-in	O
-blue	O
-and	O
-those	O
-involved	O
-in	O
-the	O
-binding	O
-of	O
-the	O
-chemically	O
-undefined	O
-ligand	O
-are	O
-colored	O
-in	O
-green	O
-using	O
-a	O
-ball	O
-and	O
-stick	O
-representation	O
-.	O
-	O
-The	O
-flexible	O
-carboxy	O
--	O
-terminal	O
-loop	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-highlighted	O
-in	O
-orange	O
-.	O
-	O
-The	O
-electron	O
-density	O
-was	O
-calculated	O
-as	O
-an	O
-omit	O
-map	O
-and	O
-contoured	O
-at	O
-1	O
-.	O
-0	O
-σ	O
-.	O
-	O
-We	O
-refined	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-with	O
-the	O
-trapped	O
-compound	O
-,	O
-and	O
-by	O
-doing	O
-so	O
-,	O
-the	O
-refinement	O
-parameters	O
-(	O
-especially	O
-R	O
--	O
-and	O
-Rfree	O
--	O
-factor	O
-)	O
-dropped	O
-by	O
-1	O
-.	O
-5	O
-%.	O
-	O
-We	O
-also	O
-calculated	O
-an	O
-omit	O
-map	O
-and	O
-the	O
-electron	O
-density	O
-reappeared	O
-(	O
-Fig	O
-7b	O
-).	O
-	O
-When	O
-analyzing	O
-the	O
-interactions	O
-of	O
-this	O
-compound	O
-within	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-,	O
-we	O
-found	O
-that	O
-it	O
-is	O
-bound	O
-via	O
-interactions	O
-with	O
-Trp	O
--	O
-21	O
-and	O
-Ser	O
--	O
-23	O
-of	O
-β	O
--	O
-sheet	O
-β3	O
-,	O
-Thr	O
--	O
-40	O
-located	O
-in	O
-β	O
--	O
-sheet	O
-β4	O
-,	O
-and	O
-Cys	O
--	O
-105	O
-and	O
-Phe	O
--	O
-107	O
-,	O
-which	O
-are	O
-both	O
-part	O
-of	O
-β	O
--	O
-sheet	O
-β11	O
-.	O
-	O
-Remarkably	O
-,	O
-all	O
-of	O
-these	O
-residues	O
-are	O
-highly	O
-conserved	O
-throughout	O
-the	O
-extended	O
-EctC	O
-protein	O
-family	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-Structure	O
--	O
-guided	O
-site	O
--	O
-directed	O
-mutagenesis	O
-of	O
-the	O
-catalytic	O
-core	O
-of	O
-the	O
-ectoine	O
-synthase	O
-	O
-In	O
-a	O
-previous	O
-alignment	O
-of	O
-the	O
-amino	O
-acid	O
-sequences	O
-of	O
-440	O
-EctC	O
--	O
-type	O
-proteins	O
-,	O
-13	O
-amino	O
-acids	O
-were	O
-identified	O
-as	O
-strictly	O
-conserved	O
-residues	O
-.	O
-	O
-These	O
-correspond	O
-to	O
-amino	O
-acids	O
-Thr	O
--	O
-40	O
-,	O
-Tyr	O
--	O
-52	O
-,	O
-His	O
--	O
-55	O
-,	O
-Glu	O
--	O
-57	O
-,	O
-Gly	O
--	O
-64	O
-,	O
-Tyr	O
--	O
-85	O
--	O
-Leu	O
--	O
-87	O
-,	O
-His	O
--	O
-93	O
-,	O
-Phe	O
--	O
-107	O
-,	O
-Pro	O
--	O
-109	O
-,	O
-Gly	O
--	O
-113	O
-,	O
-Glu	O
--	O
-115	O
-,	O
-and	O
-His	O
--	O
-117	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-Amino	O
-acid	O
-residues	O
-Gly	O
--	O
-64	O
-,	O
-Pro	O
--	O
-109	O
-,	O
-and	O
-Gly	O
--	O
-113	O
-likely	O
-fulfill	O
-structural	O
-roles	O
-since	O
-they	O
-are	O
-positioned	O
-either	O
-at	O
-the	O
-end	O
-or	O
-at	O
-the	O
-beginning	O
-of	O
-β	O
--	O
-strands	O
-and	O
-α	O
--	O
-helices	O
-.	O
-	O
-We	O
-considered	O
-the	O
-remaining	O
-ten	O
-residues	O
-as	O
-important	O
-either	O
-for	O
-ligand	O
-binding	O
-,	O
-for	O
-catalysis	O
-,	O
-or	O
-for	O
-the	O
-structurally	O
-correct	O
-orientation	O
-of	O
-the	O
-flexible	O
-carboxy	O
--	O
-terminus	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-.	O
-	O
-As	O
-described	O
-above	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-and	O
-His	O
--	O
-93	O
-are	O
-probably	O
-involved	O
-in	O
-iron	O
-binding	O
-(	O
-Table	O
-1	O
-and	O
-Fig	O
-6a	O
-).	O
-	O
-In	O
-view	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-with	O
-the	O
-serendipitously	O
-trapped	O
-compound	O
-(	O
-Fig	O
-7b	O
-),	O
-we	O
-probed	O
-the	O
-functional	O
-importance	O
-of	O
-the	O
-seven	O
-residues	O
-that	O
-contact	O
-this	O
-ligand	O
-by	O
-structure	O
--	O
-guided	O
-site	O
--	O
-directed	O
-mutagenesis	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Each	O
-of	O
-these	O
-mutant	O
-(	O
-Sa	O
-)	O
-EctC	O
-proteins	O
-was	O
-overproduced	O
-in	O
-E	O
-.	O
-coli	O
-and	O
-purified	O
-by	O
-affinity	O
-chromatography	O
-;	O
-they	O
-all	O
-yielded	O
-pure	O
-and	O
-stable	O
-protein	O
-preparations	O
-.	O
-	O
-We	O
-benchmarked	O
-the	O
-activity	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-variants	O
-in	O
-a	O
-single	O
-time	O
--	O
-point	O
-enzyme	O
-assay	O
-under	O
-conditions	O
-where	O
-10	O
-μM	O
-of	O
-the	O
-wild	O
--	O
-type	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-converted	O
-almost	O
-completely	O
-the	O
-supplied	O
-10	O
-mM	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-substrate	O
-to	O
-9	O
-.	O
-33	O
-mM	O
-ectoine	O
-within	O
-a	O
-time	O
-frame	O
-of	O
-20	O
-min	O
-.	O
-	O
-In	O
-addition	O
-,	O
-we	O
-determined	O
-the	O
-iron	O
-content	O
-of	O
-each	O
-of	O
-the	O
-mutant	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-by	O
-a	O
-colorimetric	O
-assay	O
-(	O
-Table	O
-1	O
-).	O
-	O
-The	O
-side	O
-chains	O
-of	O
-the	O
-evolutionarily	O
-conserved	O
-Trp	O
--	O
-21	O
-,	O
-Ser	O
--	O
-23	O
-,	O
-Thr	O
--	O
-40	O
-,	O
-Cys	O
--	O
-105	O
-,	O
-and	O
-Phe	O
--	O
-107	O
-residues	O
-(	O
-Fig	O
-2	O
-)	O
-make	O
-contacts	O
-with	O
-the	O
-chemically	O
-undefined	O
-ligand	O
-that	O
-we	O
-observed	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-(	O
-Fig	O
-7b	O
-).	O
-	O
-We	O
-replaced	O
-each	O
-of	O
-these	O
-residues	O
-with	O
-an	O
-Ala	O
-residue	O
-and	O
-found	O
-that	O
-none	O
-of	O
-them	O
-had	O
-an	O
-influence	O
-on	O
-the	O
-iron	O
-content	O
-of	O
-the	O
-mutant	O
-proteins	O
-.	O
-	O
-Thr	O
--	O
-40	O
-is	O
-positioned	O
-on	O
-β	O
--	O
-strand	O
-β5	O
-and	O
-its	O
-side	O
-chain	O
-protrudes	O
-into	O
-the	O
-lumen	O
-of	O
-the	O
-cupin	O
-barrel	O
-formed	O
-by	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-7b	O
-).	O
-	O
-We	O
-also	O
-replaced	O
-Phe	O
--	O
-107	O
-with	O
-either	O
-an	O
-Tyr	O
-or	O
-an	O
-Trp	O
-residue	O
-:	O
-the	O
-Phe	B-mutant
--	I-mutant
-107	I-mutant
-/	I-mutant
-Tyr	I-mutant
-substitution	O
-possessed	O
-near	O
-wild	O
--	O
-type	O
-enzyme	O
-activity	O
-(	O
-about	O
-95	O
-%)	O
-and	O
-the	O
-full	O
-iron	O
-content	O
-,	O
-but	O
-the	O
-Phe	B-mutant
--	I-mutant
-107	I-mutant
-/	I-mutant
-Trp	I-mutant
-substitution	O
-possessed	O
-only	O
-12	O
-%	O
-enzyme	O
-activity	O
-and	O
-72	O
-%	O
-iron	O
-content	O
-compared	O
-to	O
-the	O
-wild	O
--	O
-type	O
-protein	O
-.	O
-	O
-The	O
-properties	O
-of	O
-these	O
-mutant	O
-proteins	O
-indicate	O
-that	O
-the	O
-aromatic	O
-side	O
-chain	O
-at	O
-position	O
-107	O
-of	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-of	O
-importance	O
-but	O
-that	O
-a	O
-substitution	O
-with	O
-a	O
-bulky	O
-aromatic	O
-side	O
-chain	O
-is	O
-strongly	O
-detrimental	O
-to	O
-enzyme	O
-activity	O
-and	O
-concomitantly	O
-moderately	O
-impairs	O
-iron	O
-binding	O
-.	O
-	O
-Replacement	O
-of	O
-the	O
-only	O
-Cys	O
-residue	O
-in	O
-(	O
-Sa	O
-)	O
-EctC	O
-(	O
-Cys	O
--	O
-105	O
-;	O
-Fig	O
-2	O
-)	O
-by	O
-a	O
-Ser	O
-residue	O
-,	O
-a	O
-configuration	O
-that	O
-is	O
-naturally	O
-found	O
-in	O
-two	O
-EctC	O
-proteins	O
-among	O
-440	O
-inspected	O
-amino	O
-acid	O
-sequences	O
-,	O
-yielded	O
-a	O
-(	O
-Sa	O
-)	O
-EctC	O
-variant	O
-with	O
-84	O
-%	O
-wild	O
--	O
-type	O
-activity	O
-and	O
-an	O
-iron	O
-content	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-wild	O
--	O
-type	O
-protein	O
-.	O
-	O
-However	O
-,	O
-the	O
-Cys	B-mutant
--	I-mutant
-105	I-mutant
-/	I-mutant
-Ala	I-mutant
-variant	O
-was	O
-practically	O
-catalytically	O
-inactive	O
-while	O
-largely	O
-maintaining	O
-its	O
-iron	O
-content	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Since	O
-the	O
-side	O
--	O
-chains	O
-of	O
-Cys	O
-residues	O
-are	O
-chemically	O
-reactive	O
-and	O
-often	O
-participate	O
-in	O
-enzyme	O
-catalysis	O
-,	O
-Cys	O
--	O
-105	O
-(	O
-or	O
-Ser	O
--	O
-105	O
-)	O
-might	O
-serve	O
-such	O
-a	O
-role	O
-for	O
-ectoine	O
-synthase	O
-.	O
-	O
-We	O
-observed	O
-two	O
-amino	O
-acid	O
-substitutions	O
-that	O
-simultaneously	O
-strongly	O
-affected	O
-enzyme	O
-activity	O
-and	O
-iron	O
-content	O
-;	O
-these	O
-were	O
-the	O
-Tyr	B-mutant
--	I-mutant
-52	I-mutant
-/	I-mutant
-Ala	I-mutant
-and	O
-the	O
-His	B-mutant
--	I-mutant
-55	I-mutant
-/	I-mutant
-Ala	I-mutant
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-variants	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Based	O
-on	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structures	O
-that	O
-we	O
-present	O
-here	O
-,	O
-we	O
-can	O
-currently	O
-not	O
-firmly	O
-understand	O
-why	O
-the	O
-replacement	O
-of	O
-Tyr	O
--	O
-52	O
-by	O
-Ala	O
-impairs	O
-enzyme	O
-function	O
-and	O
-iron	O
-content	O
-so	O
-drastically	O
-(	O
-Table	O
-1	O
-).	O
-	O
-This	O
-is	O
-different	O
-for	O
-the	O
-His	B-mutant
--	I-mutant
-55	I-mutant
-/	I-mutant
-Ala	I-mutant
-substitution	O
-.	O
-	O
-The	O
-carboxy	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-is	O
-held	O
-in	O
-its	O
-position	O
-via	O
-an	O
-interaction	O
-of	O
-Glu	O
--	O
-115	O
-with	O
-His	O
--	O
-55	O
-,	O
-where	O
-His	O
--	O
-55	O
-in	O
-turn	O
-interacts	O
-with	O
-Pro	O
--	O
-110	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-).	O
-	O
-Each	O
-of	O
-these	O
-residues	O
-is	O
-evolutionarily	O
-highly	O
-conserved	O
-.	O
-	O
-The	O
-individual	O
-substitution	O
-of	O
-either	O
-Glu	O
--	O
-115	O
-or	O
-His	O
--	O
-55	O
-by	O
-an	O
-Ala	O
-residue	O
-is	O
-predicted	O
-to	O
-disrupt	O
-this	O
-interactive	O
-network	O
-and	O
-therefore	O
-should	O
-affect	O
-enzyme	O
-activity	O
-.	O
-	O
-Indeed	O
-,	O
-the	O
-Glu	B-mutant
--	I-mutant
-115	I-mutant
-/	I-mutant
-Ala	I-mutant
-and	O
-the	O
-His	B-mutant
--	I-mutant
-55	I-mutant
-/	I-mutant
-Ala	I-mutant
-substitutions	O
-possessed	O
-only	O
-21	O
-%	O
-and	O
-16	O
-%	O
-activity	O
-of	O
-the	O
-wild	O
--	O
-type	O
-protein	O
-,	O
-respectively	O
-(	O
-Table	O
-1	O
-).	O
-	O
-The	O
-Glu	B-mutant
--	I-mutant
-115	I-mutant
-/	I-mutant
-Ala	I-mutant
-mutant	O
-possessed	O
-wild	O
--	O
-type	O
-levels	O
-of	O
-iron	O
-,	O
-whereas	O
-the	O
-iron	O
-content	O
-of	O
-the	O
-His	B-mutant
--	I-mutant
-55	I-mutant
-/	I-mutant
-Ala	I-mutant
-substitutions	O
-dropped	O
-to	O
-15	O
-%	O
-of	O
-the	O
-wild	O
--	O
-type	O
-level	O
-(	O
-Table	O
-1	O
-).	O
-	O
-We	O
-also	O
-replaced	O
-Glu	O
--	O
-115	O
-with	O
-a	O
-negatively	O
-charged	O
-residue	O
-(	O
-Asp	O
-);	O
-this	O
-(	O
-Sa	O
-)	O
-EctC	O
-variant	O
-possessed	O
-wild	O
--	O
-type	O
-levels	O
-of	O
-iron	O
-and	O
-still	O
-exhibited	O
-77	O
-%	O
-of	O
-wild	O
--	O
-type	O
-enzyme	O
-activity	O
-.	O
-	O
-Collectively	O
-,	O
-these	O
-data	O
-suggest	O
-that	O
-the	O
-correct	O
-positioning	O
-of	O
-the	O
-carboxy	O
--	O
-terminus	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-is	O
-of	O
-structural	O
-and	O
-functional	O
-importance	O
-for	O
-the	O
-activity	O
-of	O
-the	O
-ectoine	O
-synthase	O
-.	O
-	O
-Residues	O
-Leu	O
--	O
-87	O
-and	O
-Asp	O
--	O
-91	O
-are	O
-highly	O
-conserved	O
-in	O
-the	O
-ectoine	O
-synthase	O
-protein	O
-family	O
-.	O
-	O
-The	O
-replacement	O
-of	O
-Leu	O
--	O
-87	O
-by	O
-Ala	O
-led	O
-to	O
-a	O
-substantial	O
-drop	O
-in	O
-enzyme	O
-activity	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Conversely	O
-,	O
-the	O
-replacement	O
-of	O
-Asp	O
--	O
-91	O
-by	O
-Ala	O
-and	O
-Glu	O
-,	O
-resulted	O
-in	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-variants	O
-with	O
-80	O
-%	O
-and	O
-98	O
-%	O
-enzyme	O
-activity	O
-,	O
-respectively	O
-(	O
-Table	O
-1	O
-).	O
-	O
-We	O
-currently	O
-cannot	O
-comment	O
-on	O
-possible	O
-functional	O
-role	O
-Asp	O
--	O
-91	O
-.	O
-	O
-However	O
-,	O
-Leu	O
--	O
-87	O
-is	O
-positioned	O
-at	O
-the	O
-end	O
-of	O
-one	O
-of	O
-the	O
-β	O
--	O
-sheets	O
-that	O
-form	O
-the	O
-dimer	O
-interface	O
-(	O
-Fig	O
-5c	O
-)	O
-and	O
-it	O
-might	O
-therefore	O
-possess	O
-a	O
-structural	O
-role	O
-.	O
-	O
-It	O
-is	O
-also	O
-located	O
-near	O
-Tyr	O
--	O
-85	O
-,	O
-one	O
-of	O
-the	O
-residues	O
-that	O
-probably	O
-coordinate	O
-the	O
-iron	O
-molecule	O
-with	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-active	O
-site	O
-(	O
-Fig	O
-6a	O
-)	O
-and	O
-therefore	O
-might	O
-exert	O
-indirect	O
-effects	O
-.	O
-	O
-His	O
--	O
-117	O
-is	O
-a	O
-strictly	O
-conserved	O
-residue	O
-and	O
-its	O
-substitution	O
-by	O
-an	O
-Ala	O
-residue	O
-results	O
-in	O
-a	O
-drop	O
-of	O
-enzyme	O
-activity	O
-(	O
-down	O
-to	O
-44	O
-%)	O
-and	O
-an	O
-iron	O
-content	O
-of	O
-83	O
-%	O
-(	O
-Table	O
-1	O
-).	O
-	O
-We	O
-note	O
-that	O
-His	O
--	O
-117	O
-is	O
-located	O
-close	O
-to	O
-the	O
-chemically	O
-undefined	O
-ligand	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-(	O
-Fig	O
-7b	O
-)	O
-and	O
-might	O
-thus	O
-play	O
-a	O
-role	O
-in	O
-contacting	O
-the	O
-natural	O
-substrate	O
-of	O
-the	O
-ectoine	O
-synthase	O
-.	O
-	O
-As	O
-an	O
-internal	O
-control	O
-for	O
-our	O
-mutagenesis	O
-experiments	O
-,	O
-we	O
-also	O
-substituted	O
-Thr	O
--	O
-41	O
-and	O
-His	O
--	O
-51	O
-,	O
-two	O
-residues	O
-that	O
-are	O
-not	O
-evolutionarily	O
-conserved	O
-in	O
-EctC	O
--	O
-type	O
-proteins	O
-with	O
-Ala	O
-residues	O
-.	O
-	O
-Both	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-variants	O
-exhibited	O
-wild	O
--	O
-type	O
-level	O
-enzyme	O
-activities	O
-and	O
-possessed	O
-a	O
-iron	O
-content	O
-matching	O
-that	O
-of	O
-the	O
-wild	O
--	O
-type	O
-(	O
-Table	O
-1	O
-).	O
-	O
-This	O
-illustrates	O
-that	O
-not	O
-every	O
-amino	O
-acid	O
-substitution	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-leads	O
-to	O
-an	O
-indiscriminate	O
-impairment	O
-of	O
-enzyme	O
-function	O
-and	O
-iron	O
-content	O
-.	O
-	O
-The	O
-crystallographic	O
-data	O
-presented	O
-here	O
-firmly	O
-identify	O
-ectoine	O
-synthase	O
-(	O
-EctC	O
-),	O
-an	O
-enzyme	O
-critical	O
-for	O
-the	O
-production	O
-of	O
-the	O
-microbial	O
-cytoprotectant	O
-and	O
-chemical	O
-chaperone	O
-ectoine	O
-,	O
-as	O
-a	O
-new	O
-member	O
-of	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-The	O
-overall	O
-fold	O
-and	O
-bowl	O
-shape	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Figs	O
-4	O
-and	O
-5	O
-)	O
-with	O
-its	O
-11	O
-β	O
--	O
-strands	O
-(	O
-β1	O
--	O
-β11	O
-)	O
-and	O
-two	O
-α	O
--	O
-helices	O
-(	O
-α	O
--	O
-I	O
-and	O
-α	O
--	O
-II	O
-)	O
-closely	O
-adheres	O
-to	O
-the	O
-design	O
-principles	O
-typically	O
-found	O
-in	O
-crystal	O
-structures	O
-of	O
-cupins	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-ectoine	O
-synthase	O
-,	O
-the	O
-polyketide	O
-cyclase	O
-RemF	O
-is	O
-the	O
-only	O
-other	O
-currently	O
-known	O
-cupin	O
--	O
-related	O
-enzyme	O
-that	O
-catalyze	O
-a	O
-cyclocondensation	O
-reaction	O
-although	O
-the	O
-substrates	O
-of	O
-EctC	O
-and	O
-RemF	O
-are	O
-rather	O
-different	O
-.	O
-	O
-As	O
-a	O
-consequence	O
-of	O
-the	O
-structural	O
-relatedness	O
-of	O
-EctC	O
-and	O
-RemF	O
-and	O
-the	O
-type	O
-of	O
-chemical	O
-reaction	O
-these	O
-two	O
-enzymes	O
-catalyze	O
-,	O
-is	O
-now	O
-understandable	O
-why	O
-bona	O
-fide	O
-EctC	O
--	O
-type	O
-proteins	O
-are	O
-frequently	O
-(	O
-mis	O
-)-	O
-annotated	O
-in	O
-microbial	O
-genome	O
-sequences	O
-as	O
-“	O
-RemF	O
--	O
-like	O
-”	O
-proteins	O
-.	O
-	O
-The	O
-pro	O
--	O
-and	O
-eukaryotic	O
-members	O
-of	O
-the	O
-cupin	O
-superfamily	O
-perform	O
-a	O
-variety	O
-of	O
-both	O
-enzymatic	O
-and	O
-non	O
--	O
-enzymatic	O
-functions	O
-that	O
-are	O
-built	O
-upon	O
-a	O
-common	O
-structural	O
-scaffold	O
-.	O
-	O
-Most	O
-cupins	O
-contain	O
-transition	O
-state	O
-metals	O
-that	O
-can	O
-promote	O
-different	O
-types	O
-of	O
-chemical	O
-reactions	O
-.	O
-	O
-Except	O
-for	O
-some	O
-cupin	O
--	O
-related	O
-proteins	O
-that	O
-seem	O
-to	O
-function	O
-as	O
-metallo	O
--	O
-chaperones	O
-,	O
-the	O
-bound	O
-metal	O
-is	O
-typically	O
-an	O
-essential	O
-part	O
-of	O
-the	O
-active	O
-sites	O
-.	O
-	O
-We	O
-report	O
-here	O
-for	O
-the	O
-first	O
-time	O
-that	O
-the	O
-ectoine	O
-synthase	O
-is	O
-a	O
-metal	O
--	O
-dependent	O
-enzyme	O
-.	O
-	O
-ICP	O
--	O
-MS	O
-,	O
-metal	O
--	O
-depletion	O
-and	O
-reconstitution	O
-experiments	O
-(	O
-Fig	O
-3	O
-)	O
-consistently	O
-identify	O
-iron	O
-as	O
-the	O
-biologically	O
-most	O
-relevant	O
-metal	O
-for	O
-the	O
-EctC	O
--	O
-catalyzed	O
-cyclocondensation	O
-reaction	O
-.	O
-	O
-However	O
-,	O
-as	O
-observed	O
-with	O
-other	O
-cupins	O
-,	O
-EctC	O
-is	O
-a	O
-somewhat	O
-promiscuous	O
-enzyme	O
-as	O
-far	O
-as	O
-the	O
-catalytically	O
-important	O
-metal	O
-is	O
-concerned	O
-when	O
-they	O
-are	O
-provided	O
-in	O
-large	O
-molar	O
-excess	O
-(	O
-Fig	O
-3c	O
-).	O
-	O
-Although	O
-some	O
-uncertainty	O
-remains	O
-with	O
-respect	O
-to	O
-the	O
-precise	O
-identity	O
-of	O
-amino	O
-acid	O
-residues	O
-that	O
-participate	O
-in	O
-metal	O
-binding	O
-by	O
-(	O
-Sa	O
-)	O
-EctC	O
-,	O
-our	O
-structure	O
--	O
-guided	O
-site	O
--	O
-directed	O
-mutagenesis	O
-experiments	O
-targeting	O
-the	O
-presumptive	O
-iron	O
--	O
-binding	O
-residues	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-)	O
-demonstrate	O
-that	O
-none	O
-of	O
-them	O
-can	O
-be	O
-spared	O
-(	O
-Table	O
-1	O
-).	O
-	O
-The	O
-architecture	O
-of	O
-the	O
-metal	O
-center	O
-of	O
-ectoine	O
-synthase	O
-seems	O
-to	O
-be	O
-subjected	O
-to	O
-considerable	O
-evolutionary	O
-constraints	O
-.	O
-	O
-The	O
-three	O
-residues	O
-(	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-His	O
--	O
-93	O
-)	O
-that	O
-we	O
-deem	O
-to	O
-form	O
-it	O
-(	O
-Figs	O
-6	O
-and	O
-7b	O
-)	O
-are	O
-strictly	O
-conserved	O
-in	O
-a	O
-large	O
-collection	O
-of	O
-EctC	O
--	O
-type	O
-proteins	O
-originating	O
-from	O
-16	O
-bacterial	O
-and	O
-three	O
-archaeal	O
-phyla	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-We	O
-also	O
-show	O
-here	O
-for	O
-the	O
-first	O
-time	O
-that	O
-,	O
-in	O
-addition	O
-to	O
-its	O
-natural	O
-substrate	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-,	O
-EctC	O
-also	O
-converts	O
-the	O
-isomer	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-into	O
-ectoine	O
-,	O
-albeit	O
-with	O
-a	O
-73	O
--	O
-fold	O
-reduced	O
-catalytic	O
-efficiency	O
-(	O
-S3a	O
-and	O
-S3b	O
-Fig	O
-).	O
-	O
-Hence	O
-,	O
-the	O
-active	O
-site	O
-of	O
-ectoine	O
-synthase	O
-must	O
-possess	O
-a	O
-certain	O
-degree	O
-of	O
-structural	O
-plasticity	O
-,	O
-a	O
-notion	O
-that	O
-is	O
-supported	O
-by	O
-the	O
-report	O
-on	O
-the	O
-EctC	O
--	O
-catalyzed	O
-formation	O
-of	O
-the	O
-synthetic	O
-compatible	O
-solute	O
-ADPC	O
-through	O
-the	O
-cyclic	O
-condensation	O
-of	O
-two	O
-glutamine	O
-molecules	O
-.	O
-	O
-Our	O
-finding	O
-that	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-serves	O
-as	O
-a	O
-substrate	O
-for	O
-ectoine	O
-synthase	O
-has	O
-physiologically	O
-relevant	O
-ramifications	O
-for	O
-those	O
-microorganisms	O
-that	O
-can	O
-both	O
-synthesize	O
-and	O
-catabolize	O
-ectoine	O
-,	O
-since	O
-they	O
-need	O
-to	O
-prevent	O
-a	O
-futile	O
-cycle	O
-of	O
-synthesis	O
-and	O
-degradation	O
-when	O
-N	O
--	O
-α	O
--	O
-ADABA	O
-is	O
-produced	O
-as	O
-an	O
-intermediate	O
-in	O
-the	O
-catabolic	O
-route	O
-.	O
-	O
-Although	O
-we	O
-cannot	O
-identify	O
-the	O
-true	O
-chemical	O
-nature	O
-of	O
-the	O
-C6	O
-compound	O
-that	O
-was	O
-trapped	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-nor	O
-its	O
-precise	O
-origin	O
-,	O
-we	O
-treated	O
-this	O
-compound	O
-as	O
-a	O
-proxy	O
-for	O
-the	O
-natural	O
-substrate	O
-of	O
-ectoine	O
-synthase	O
-,	O
-which	O
-is	O
-a	O
-C6	O
-compound	O
-as	O
-well	O
-(	O
-Fig	O
-7a	O
-).	O
-	O
-We	O
-assumed	O
-that	O
-its	O
-location	O
-and	O
-mode	O
-of	O
-binding	O
-gives	O
-,	O
-in	O
-all	O
-likelihood	O
-,	O
-clues	O
-as	O
-to	O
-the	O
-position	O
-of	O
-the	O
-true	O
-substrate	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-within	O
-the	O
-EctC	O
-active	O
-site	O
-.	O
-	O
-Indeed	O
-,	O
-site	O
--	O
-directed	O
-mutagenesis	O
-of	O
-those	O
-five	O
-residues	O
-that	O
-contact	O
-the	O
-unknown	O
-C6	O
-compound	O
-(	O
-Fig	O
-7b	O
-)	O
-yielded	O
-(	O
-Sa	O
-)	O
-EctC	O
-variants	O
-with	O
-strongly	O
-impaired	O
-enzyme	O
-function	O
-but	O
-near	O
-wild	O
--	O
-type	O
-levels	O
-of	O
-iron	O
-(	O
-Table	O
-1	O
-).	O
-	O
-This	O
-set	O
-of	O
-data	O
-and	O
-the	O
-fact	O
-that	O
-the	O
-targeted	O
-residues	O
-are	O
-strongly	O
-conserved	O
-among	O
-EctC	O
--	O
-type	O
-proteins	O
-(	O
-Fig	O
-2	O
-)	O
-is	O
-consistent	O
-with	O
-their	O
-potential	O
-role	O
-in	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-binding	O
-or	O
-enzyme	O
-catalysis	O
-.	O
-	O
-We	O
-therefore	O
-surmise	O
-that	O
-our	O
-crystallographic	O
-data	O
-and	O
-the	O
-site	O
--	O
-directed	O
-mutagenesis	O
-study	O
-reported	O
-here	O
-provide	O
-a	O
-structural	O
-and	O
-functional	O
-view	O
-into	O
-the	O
-architecture	O
-of	O
-the	O
-EctC	O
-active	O
-site	O
-(	O
-Fig	O
-7b	O
-).	O
-	O
-The	O
-ectoine	O
-synthase	O
-from	O
-the	O
-cold	O
--	O
-adapted	O
-marine	O
-bacterium	O
-S	O
-.	O
-alaskensis	O
-can	O
-be	O
-considered	O
-as	O
-a	O
-psychrophilic	O
-enzyme	O
-(	O
-S3a	O
-Fig	O
-),	O
-types	O
-of	O
-proteins	O
-with	O
-a	O
-considerable	O
-structural	O
-flexibility	O
-.	O
-	O
-This	O
-probably	O
-worked	O
-to	O
-the	O
-detriment	O
-of	O
-our	O
-efforts	O
-in	O
-solving	O
-crystal	O
-structures	O
-of	O
-the	O
-full	O
--	O
-length	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-in	O
-complex	O
-with	O
-either	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-or	O
-ectoine	O
-.	O
-	O
-Because	O
-microbial	O
-ectoine	O
-producers	O
-can	O
-colonize	O
-ecological	O
-niches	O
-with	O
-rather	O
-different	O
-physicochemical	O
-attributes	O
-,	O
-it	O
-seems	O
-promising	O
-to	O
-exploit	O
-this	O
-considerable	O
-biodiversity	O
-to	O
-identify	O
-EctC	O
-proteins	O
-with	O
-enhanced	O
-protein	O
-stability	O
-.	O
-	O
-It	O
-is	O
-hoped	O
-that	O
-these	O
-can	O
-be	O
-further	O
-employed	O
-to	O
-obtain	O
-EctC	O
-crystal	O
-structures	O
-with	O
-either	O
-the	O
-substrate	O
-or	O
-the	O
-reaction	O
-product	O
-.	O
-	O
-Together	O
-with	O
-our	O
-finding	O
-that	O
-ectoine	O
-synthase	O
-is	O
-metal	O
-dependent	O
-,	O
-these	O
-crystal	O
-structures	O
-should	O
-allow	O
-a	O
-more	O
-detailed	O
-understanding	O
-of	O
-the	O
-chemistry	O
-underlying	O
-the	O
-EctC	O
--	O
-catalyzed	O
-cyclocondensation	O
-reaction	O
-.	O
-	O
-Structural	O
-basis	O
-for	O
-the	O
-regulation	O
-of	O
-enzymatic	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-by	O
-domain	O
--	O
-domain	O
-interactions	O
-	O
-Regnase	O
--	O
-1	O
-is	O
-an	O
-RNase	O
-that	O
-directly	O
-cleaves	O
-mRNAs	O
-of	O
-inflammatory	O
-genes	O
-such	O
-as	O
-IL	O
--	O
-6	O
-and	O
-IL	O
--	O
-12p40	O
-,	O
-and	O
-negatively	O
-regulates	O
-cellular	O
-inflammatory	O
-responses	O
-.	O
-	O
-Here	O
-,	O
-we	O
-report	O
-the	O
-structures	O
-of	O
-four	O
-domains	O
-of	O
-Regnase	O
--	O
-1	O
-from	O
-Mus	O
-musculus	O
-—	O
-the	O
-N	O
--	O
-terminal	O
-domain	O
-(	O
-NTD	O
-),	O
-PilT	O
-N	O
--	O
-terminus	O
-like	O
-(	O
-PIN	O
-)	O
-domain	O
-,	O
-zinc	O
-finger	O
-(	O
-ZF	O
-)	O
-domain	O
-and	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-CTD	O
-).	O
-	O
-The	O
-PIN	O
-domain	O
-harbors	O
-the	O
-RNase	O
-catalytic	O
-center	O
-;	O
-however	O
-,	O
-it	O
-is	O
-insufficient	O
-for	O
-enzymatic	O
-activity	O
-.	O
-	O
-We	O
-found	O
-that	O
-the	O
-NTD	O
-associates	O
-with	O
-the	O
-PIN	O
-domain	O
-and	O
-significantly	O
-enhances	O
-its	O
-RNase	O
-activity	O
-.	O
-	O
-The	O
-PIN	O
-domain	O
-forms	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-oligomer	O
-and	O
-the	O
-dimer	O
-interface	O
-overlaps	O
-with	O
-the	O
-NTD	O
-binding	O
-site	O
-.	O
-	O
-Interestingly	O
-,	O
-mutations	O
-blocking	O
-PIN	O
-oligomerization	O
-had	O
-no	O
-RNase	O
-activity	O
-,	O
-indicating	O
-that	O
-both	O
-oligomerization	O
-and	O
-NTD	O
-binding	O
-are	O
-crucial	O
-for	O
-RNase	O
-activity	O
-in	O
-vitro	O
-.	O
-	O
-These	O
-results	O
-suggest	O
-that	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-is	O
-tightly	O
-controlled	O
-by	O
-both	O
-intramolecular	O
-(	O
-NTD	O
--	O
-PIN	O
-)	O
-and	O
-intermolecular	O
-(	O
-PIN	O
--	O
-PIN	O
-)	O
-interactions	O
-.	O
-	O
-The	O
-initial	O
-sensing	O
-of	O
-infection	O
-is	O
-mediated	O
-by	O
-a	O
-set	O
-of	O
-pattern	O
--	O
-recognition	O
-receptors	O
-(	O
-PRRs	O
-)	O
-such	O
-Toll	O
--	O
-like	O
-receptors	O
-(	O
-TLRs	O
-)	O
-and	O
-the	O
-intracellular	O
-signaling	O
-cascades	O
-triggered	O
-by	O
-TLRs	O
-evoke	O
-transcriptional	O
-expression	O
-of	O
-inflammatory	O
-mediators	O
-that	O
-coordinate	O
-the	O
-elimination	O
-of	O
-pathogens	O
-and	O
-infected	O
-cells	O
-.	O
-	O
-Regnase	O
--	O
-1	O
-(	O
-also	O
-known	O
-as	O
-Zc3h12a	O
-and	O
-MCPIP1	O
-)	O
-is	O
-an	O
-RNase	O
-whose	O
-expression	O
-level	O
-is	O
-stimulated	O
-by	O
-lipopolysaccharides	O
-and	O
-prevents	O
-autoimmune	O
-diseases	O
-by	O
-directly	O
-controlling	O
-the	O
-stability	O
-of	O
-mRNAs	O
-of	O
-inflammatory	O
-genes	O
-such	O
-as	O
-interleukin	O
-(	O
-IL	O
-)-	O
-6	O
-,	O
-IL	O
--	O
-1β	O
-,	O
-IL	O
--	O
-2	O
-,	O
-and	O
-IL	O
--	O
-12p40	O
-.	O
-	O
-Regnase	O
--	O
-1	O
-accelerates	O
-target	O
-mRNA	O
-degradation	O
-via	O
-their	O
-3	O
-′-	O
-terminal	O
-untranslated	O
-region	O
-(	O
-3	O
-′	O
-UTR	O
-),	O
-and	O
-also	O
-degrades	O
-its	O
-own	O
-mRNA	O
-.	O
-	O
-Regnase	O
--	O
-1	O
-is	O
-a	O
-member	O
-of	O
-Regnase	O
-family	O
-and	O
-is	O
-composed	O
-of	O
-a	O
-PilT	O
-N	O
--	O
-terminus	O
-like	O
-(	O
-PIN	O
-)	O
-domain	O
-followed	O
-by	O
-a	O
-CCCH	O
--	O
-type	O
-zinc	O
-–	O
-finger	O
-(	O
-ZF	O
-)	O
-domain	O
-,	O
-which	O
-are	O
-conserved	O
-among	O
-Regnase	O
-family	O
-members	O
-.	O
-	O
-Recently	O
-,	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-Regnase	O
--	O
-1	O
-PIN	O
-domain	O
-derived	O
-from	O
-Homo	O
-sapiens	O
-was	O
-reported	O
-.	O
-	O
-The	O
-structure	O
-combined	O
-with	O
-functional	O
-analyses	O
-revealed	O
-that	O
-four	O
-catalytically	O
-important	O
-Asp	O
-residues	O
-form	O
-the	O
-catalytic	O
-center	O
-and	O
-stabilize	O
-Mg2	O
-+	O
-binding	O
-that	O
-is	O
-crucial	O
-for	O
-RNase	O
-activity	O
-.	O
-	O
-Several	O
-CCCH	O
--	O
-type	O
-ZF	O
-motifs	O
-in	O
-RNA	O
--	O
-binding	O
-proteins	O
-have	O
-been	O
-reported	O
-to	O
-directly	O
-bind	O
-RNA	O
-.	O
-	O
-In	O
-addition	O
-,	O
-Regnase	O
--	O
-1	O
-has	O
-been	O
-predicted	O
-to	O
-possess	O
-other	O
-domains	O
-in	O
-the	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-regions	O
-.	O
-	O
-However	O
-,	O
-the	O
-structure	O
-and	O
-function	O
-of	O
-the	O
-ZF	O
-domain	O
-,	O
-N	O
--	O
-terminal	O
-domain	O
-(	O
-NTD	O
-)	O
-and	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-CTD	O
-)	O
-of	O
-Regnase	O
--	O
-1	O
-have	O
-not	O
-been	O
-solved	O
-.	O
-	O
-Here	O
-,	O
-we	O
-performed	O
-structural	O
-and	O
-functional	O
-analyses	O
-of	O
-individual	O
-domains	O
-of	O
-Regnase	O
--	O
-1	O
-derived	O
-from	O
-Mus	O
-musculus	O
-in	O
-order	O
-to	O
-understand	O
-the	O
-catalytic	O
-activity	O
-in	O
-vitro	O
-.	O
-	O
-Our	O
-data	O
-revealed	O
-that	O
-the	O
-catalytic	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-is	O
-regulated	O
-through	O
-both	O
-intra	O
-and	O
-intermolecular	O
-domain	O
-interactions	O
-in	O
-vitro	O
-.	O
-	O
-The	O
-NTD	O
-plays	O
-a	O
-crucial	O
-role	O
-in	O
-efficient	O
-cleavage	O
-of	O
-target	O
-mRNA	O
-,	O
-through	O
-intramolecular	O
-NTD	O
--	O
-PIN	O
-interactions	O
-.	O
-	O
-Moreover	O
-,	O
-Regnase	O
--	O
-1	O
-functions	O
-as	O
-a	O
-dimer	O
-through	O
-intermolecular	O
-PIN	O
--	O
-PIN	O
-interactions	O
-during	O
-cleavage	O
-of	O
-target	O
-mRNA	O
-.	O
-	O
-Our	O
-findings	O
-suggest	O
-that	O
-Regnase	O
--	O
-1	O
-cleaves	O
-its	O
-target	O
-mRNA	O
-by	O
-an	O
-NTD	O
--	O
-activated	O
-functional	O
-PIN	O
-dimer	O
-,	O
-while	O
-the	O
-ZF	O
-increases	O
-RNA	O
-affinity	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-PIN	O
-dimer	O
-.	O
-	O
-Domain	O
-structures	O
-of	O
-Regnase	O
--	O
-1	O
-	O
-We	O
-analyzed	O
-Rengase	O
--	O
-1	O
-derived	O
-from	O
-Mus	O
-musculus	O
-and	O
-solved	O
-the	O
-structures	O
-of	O
-the	O
-four	O
-domains	O
-;	O
-NTD	O
-,	O
-PIN	O
-,	O
-ZF	O
-,	O
-and	O
-CTD	O
-individually	O
-by	O
-X	O
--	O
-ray	O
-crystallography	O
-or	O
-NMR	O
-(	O
-Fig	O
-.	O
-1a	O
-–	O
-e	O
-).	O
-	O
-X	O
--	O
-ray	O
-crystallography	O
-was	O
-attempted	O
-for	O
-the	O
-fragment	O
-containing	O
-both	O
-the	O
-PIN	O
-and	O
-ZF	O
-domains	O
-,	O
-however	O
-,	O
-electron	O
-density	O
-was	O
-observed	O
-only	O
-for	O
-the	O
-PIN	O
-domain	O
-(	O
-Fig	O
-.	O
-1c	O
-),	O
-consistent	O
-with	O
-a	O
-previous	O
-report	O
-on	O
-Regnase	O
--	O
-1	O
-derived	O
-from	O
-Homo	O
-sapiens	O
-.	O
-	O
-This	O
-suggests	O
-that	O
-the	O
-PIN	O
-and	O
-ZF	O
-domains	O
-exist	O
-independently	O
-without	O
-interacting	O
-with	O
-each	O
-other	O
-.	O
-	O
-The	O
-domain	O
-structures	O
-of	O
-NTD	O
-,	O
-ZF	O
-,	O
-and	O
-CTD	O
-were	O
-determined	O
-by	O
-NMR	O
-(	O
-Fig	O
-.	O
-1b	O
-,	O
-d	O
-,	O
-e	O
-).	O
-	O
-The	O
-NTD	O
-and	O
-CTD	O
-are	O
-both	O
-composed	O
-of	O
-three	O
-α	O
-helices	O
-,	O
-and	O
-structurally	O
-resemble	O
-ubiquitin	O
-conjugating	O
-enzyme	O
-E2	O
-K	O
-(	O
-PDB	O
-ID	O
-:	O
-3K9O	O
-)	O
-and	O
-ubiquitin	O
-associated	O
-protein	O
-1	O
-(	O
-PDB	O
-ID	O
-:	O
-4AE4	O
-),	O
-respectively	O
-,	O
-according	O
-to	O
-the	O
-Dali	O
-server	O
-.	O
-	O
-Contribution	O
-of	O
-each	O
-domain	O
-of	O
-Regnase	O
--	O
-1	O
-to	O
-the	O
-mRNA	O
-binding	O
-activity	O
-	O
-Although	O
-the	O
-PIN	O
-domain	O
-is	O
-responsible	O
-for	O
-the	O
-catalytic	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-,	O
-the	O
-roles	O
-of	O
-the	O
-other	O
-domains	O
-are	O
-largely	O
-unknown	O
-.	O
-	O
-First	O
-,	O
-we	O
-evaluated	O
-a	O
-role	O
-of	O
-the	O
-NTD	O
-and	O
-ZF	O
-domains	O
-for	O
-mRNA	O
-binding	O
-by	O
-an	O
-in	O
-vitro	O
-gel	O
-shift	O
-assay	O
-(	O
-Fig	O
-.	O
-1f	O
-).	O
-	O
-Fluorescently	O
-5	O
-′-	O
-labeled	O
-RNA	O
-corresponding	O
-to	O
-nucleotides	O
-82	O
-–	O
-106	O
-of	O
-the	O
-IL	O
--	O
-6	O
-mRNA	O
-3	O
-′	O
-UTR	O
-and	O
-the	O
-catalytically	O
-inactive	O
-mutant	O
-(	O
-D226N	B-mutant
-and	O
-D244N	B-mutant
-)	O
-of	O
-Regnase	O
--	O
-1	O
-—	O
-hereafter	O
-referred	O
-to	O
-as	O
-the	O
-DDNN	B-mutant
-mutant	O
-—	O
-were	O
-utilized	O
-.	O
-	O
-Upon	O
-addition	O
-of	O
-a	O
-larger	O
-amount	O
-of	O
-Regnase	O
--	O
-1	O
-,	O
-the	O
-fluorescence	O
-of	O
-free	O
-RNA	O
-decreased	O
-,	O
-indicating	O
-that	O
-Regnase	O
--	O
-1	O
-bound	O
-to	O
-the	O
-RNA	O
-.	O
-	O
-Based	O
-on	O
-the	O
-decrease	O
-in	O
-the	O
-free	O
-RNA	O
-fluorescence	O
-band	O
-,	O
-we	O
-evaluated	O
-the	O
-contribution	O
-of	O
-each	O
-domain	O
-of	O
-Regnase	O
--	O
-1	O
-to	O
-RNA	O
-binding	O
-.	O
-	O
-While	O
-the	O
-RNA	O
-binding	O
-ability	O
-was	O
-not	O
-significantly	O
-changed	O
-in	O
-the	O
-presence	O
-of	O
-NTD	O
-,	O
-it	O
-increased	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-ZF	O
-domain	O
-(	O
-Fig	O
-.	O
-1f	O
-,	O
-g	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-Direct	O
-binding	O
-of	O
-the	O
-ZF	O
-domain	O
-and	O
-RNA	O
-were	O
-confirmed	O
-by	O
-NMR	O
-spectral	O
-changes	O
-.	O
-	O
-The	O
-fitting	O
-of	O
-the	O
-titration	O
-curve	O
-of	O
-Y314	O
-resulted	O
-in	O
-an	O
-apparent	O
-dissociation	O
-constant	O
-(	O
-Kd	O
-)	O
-of	O
-10	O
-±	O
-1	O
-.	O
-1	O
-μM	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-These	O
-results	O
-indicate	O
-that	O
-not	O
-only	O
-the	O
-PIN	O
-but	O
-also	O
-the	O
-ZF	O
-domain	O
-contribute	O
-to	O
-RNA	O
-binding	O
-,	O
-while	O
-the	O
-NTD	O
-is	O
-not	O
-likely	O
-to	O
-be	O
-involved	O
-in	O
-direct	O
-interaction	O
-with	O
-RNA	O
-.	O
-	O
-Contribution	O
-of	O
-each	O
-domain	O
-of	O
-Regnase	O
--	O
-1	O
-to	O
-RNase	O
-activity	O
-	O
-In	O
-order	O
-to	O
-characterize	O
-the	O
-role	O
-of	O
-each	O
-domain	O
-in	O
-the	O
-RNase	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-,	O
-we	O
-performed	O
-an	O
-in	O
-vitro	O
-cleavage	O
-assay	O
-using	O
-fluorescently	O
-5	O
-′-	O
-labeled	O
-RNA	O
-corresponding	O
-to	O
-nucleotides	O
-82	O
-–	O
-106	O
-of	O
-the	O
-IL	O
--	O
-6	O
-mRNA	O
-3	O
-′	O
-UTR	O
-(	O
-Fig	O
-.	O
-1g	O
-).	O
-	O
-Regnase	O
--	O
-1	O
-constructs	O
-consisting	O
-of	O
-NTD	B-mutant
--	I-mutant
-PIN	I-mutant
--	I-mutant
-ZF	I-mutant
-completely	O
-cleaved	O
-the	O
-target	O
-mRNA	O
-and	O
-generated	O
-the	O
-cleaved	O
-products	O
-.	O
-	O
-The	O
-apparent	O
-half	O
--	O
-life	O
-(	O
-T1	O
-/	O
-2	O
-)	O
-of	O
-the	O
-RNase	O
-activity	O
-was	O
-about	O
-20	O
-minutes	O
-.	O
-	O
-Regnase	O
--	O
-1	O
-lacking	O
-the	O
-ZF	O
-domain	O
-generated	O
-a	O
-smaller	O
-but	O
-appreciable	O
-amount	O
-of	O
-cleaved	O
-product	O
-(	O
-T1	O
-/	O
-2	O
-~	O
-70	O
-minutes	O
-),	O
-while	O
-those	O
-lacking	O
-the	O
-NTD	O
-did	O
-not	O
-generate	O
-cleaved	O
-products	O
-(	O
-T1	O
-/	O
-2	O
->	O
-90	O
-minutes	O
-).	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-NTD	B-mutant
--	I-mutant
-PIN	I-mutant
-(	I-mutant
-DDNN	I-mutant
-)-	I-mutant
-ZF	I-mutant
-,	O
-which	O
-possesses	O
-the	O
-NTD	O
-but	O
-lacks	O
-the	O
-catalytic	O
-residues	O
-in	O
-PIN	O
-,	O
-completely	O
-lost	O
-all	O
-RNase	O
-activity	O
-(	O
-Fig	O
-.	O
-1g	O
-,	O
-right	O
-panel	O
-),	O
-as	O
-expected	O
-,	O
-confirming	O
-that	O
-the	O
-RNase	O
-catalytic	O
-center	O
-is	O
-located	O
-in	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-Taken	O
-together	O
-with	O
-the	O
-results	O
-in	O
-the	O
-previous	O
-section	O
-,	O
-we	O
-conclude	O
-that	O
-the	O
-NTD	O
-is	O
-crucial	O
-for	O
-the	O
-RNase	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-in	O
-vitro	O
-,	O
-although	O
-it	O
-does	O
-not	O
-contribute	O
-to	O
-the	O
-direct	O
-mRNA	O
-binding	O
-.	O
-	O
-Dimer	O
-formation	O
-of	O
-the	O
-PIN	O
-domains	O
-	O
-During	O
-purification	O
-by	O
-gel	O
-filtration	O
-,	O
-the	O
-PIN	O
-domain	O
-exhibited	O
-extremely	O
-asymmetric	O
-elution	O
-peaks	O
-in	O
-a	O
-concentration	O
-dependent	O
-manner	O
-(	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-By	O
-comparison	O
-with	O
-the	O
-elution	O
-volume	O
-of	O
-standard	O
-marker	O
-proteins	O
-,	O
-the	O
-PIN	O
-domain	O
-was	O
-assumed	O
-to	O
-be	O
-in	O
-equilibrium	O
-between	O
-a	O
-monomer	O
-and	O
-a	O
-dimer	O
-in	O
-solution	O
-at	O
-concentrations	O
-in	O
-the	O
-20	O
-–	O
-200	O
-μM	O
-range	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-PIN	O
-domain	O
-has	O
-been	O
-determined	O
-in	O
-three	O
-distinct	O
-crystal	O
-forms	O
-with	O
-a	O
-space	O
-group	O
-of	O
-P3121	O
-(	O
-form	O
-I	O
-in	O
-this	O
-study	O
-and	O
-PDB	O
-ID	O
-3V33	O
-),	O
-P3221	O
-(	O
-form	O
-II	O
-in	O
-this	O
-study	O
-),	O
-and	O
-P41	O
-(	O
-PDB	O
-ID	O
-3V32	O
-and	O
-3V34	O
-),	O
-respectively	O
-.	O
-	O
-We	O
-found	O
-that	O
-the	O
-PIN	O
-domain	O
-formed	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-oligomer	O
-that	O
-was	O
-commonly	O
-observed	O
-in	O
-all	O
-three	O
-crystal	O
-forms	O
-in	O
-spite	O
-of	O
-the	O
-different	O
-crystallization	O
-conditions	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-Mutation	O
-of	O
-Arg215	O
-,	O
-whose	O
-side	O
-chain	O
-faces	O
-to	O
-the	O
-opposite	O
-side	O
-of	O
-the	O
-oligomeric	O
-surface	O
-,	O
-to	O
-Glu	O
-preserved	O
-the	O
-monomer	O
-/	O
-dimer	O
-equilibrium	O
-,	O
-similar	O
-to	O
-the	O
-wild	O
-type	O
-.	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-single	O
-mutations	O
-of	O
-side	O
-chains	O
-involved	O
-in	O
-the	O
-PIN	O
-–	O
-PIN	O
-oligomeric	O
-interaction	O
-resulted	O
-in	O
-monomer	O
-formation	O
-,	O
-judging	O
-from	O
-gel	O
-filtration	O
-(	O
-Fig	O
-.	O
-2a	O
-,	O
-b	O
-).	O
-	O
-Wild	O
-type	O
-and	O
-monomeric	O
-PIN	O
-mutants	O
-(	O
-P212A	B-mutant
-and	O
-D278R	B-mutant
-)	O
-were	O
-also	O
-analyzed	O
-by	O
-NMR	O
-.	O
-	O
-The	O
-spectra	O
-indicate	O
-that	O
-the	O
-dimer	O
-interface	O
-of	O
-the	O
-wild	O
-type	O
-PIN	O
-domain	O
-were	O
-significantly	O
-broadened	O
-compared	O
-to	O
-the	O
-monomeric	O
-mutants	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-These	O
-results	O
-indicate	O
-that	O
-the	O
-PIN	O
-domain	O
-forms	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-oligomer	O
-in	O
-solution	O
-similar	O
-to	O
-the	O
-crystal	O
-structure	O
-.	O
-	O
-Interestingly	O
-,	O
-the	O
-monomeric	O
-PIN	O
-mutants	O
-P212A	B-mutant
-,	O
-R214A	B-mutant
-,	O
-and	O
-D278R	B-mutant
-had	O
-no	O
-significant	O
-RNase	O
-activity	O
-for	O
-IL	O
--	O
-6	O
-mRNA	O
-in	O
-vitro	O
-(	O
-Fig	O
-.	O
-2c	O
-).	O
-	O
-The	O
-side	O
-chains	O
-of	O
-these	O
-residues	O
-point	O
-away	O
-from	O
-the	O
-catalytic	O
-center	O
-on	O
-the	O
-same	O
-molecule	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-Therefore	O
-,	O
-we	O
-concluded	O
-that	O
-head	O
--	O
-to	O
--	O
-tail	O
-PIN	O
-dimerization	O
-,	O
-together	O
-with	O
-the	O
-NTD	O
-,	O
-are	O
-required	O
-for	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-in	O
-vitro	O
-.	O
-	O
-Domain	O
--	O
-domain	O
-interaction	O
-between	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-	O
-While	O
-the	O
-NTD	O
-does	O
-not	O
-contribute	O
-to	O
-RNA	O
-binding	O
-(	O
-Fig	O
-.	O
-1f	O
-,	O
-g	O
-,	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-),	O
-it	O
-increases	O
-the	O
-RNase	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-(	O
-Fig	O
-.	O
-1h	O
-).	O
-	O
-In	O
-order	O
-to	O
-gain	O
-insight	O
-into	O
-the	O
-molecular	O
-mechanism	O
-of	O
-the	O
-NTD	O
--	O
-mediated	O
-enhancement	O
-of	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-,	O
-we	O
-further	O
-investigated	O
-the	O
-domain	O
--	O
-domain	O
-interaction	O
-between	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-using	O
-NMR	O
-.	O
-	O
-We	O
-used	O
-the	O
-catalytically	O
-inactive	O
-monomeric	O
-PIN	O
-mutant	O
-possessing	O
-both	O
-the	O
-DDNN	B-mutant
-and	O
-D278R	B-mutant
-mutations	O
-to	O
-avoid	O
-dimer	O
-formation	O
-of	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-The	O
-NMR	O
-signals	O
-from	O
-the	O
-PIN	O
-domain	O
-(	O
-residues	O
-V177	O
-,	O
-F210	O
--	O
-T211	O
-,	O
-R214	O
-,	O
-F228	O
--	O
-L232	O
-,	O
-and	O
-F234	O
--	O
-S236	O
-)	O
-exhibited	O
-significant	O
-chemical	O
-shift	O
-changes	O
-upon	O
-addition	O
-of	O
-the	O
-NTD	O
-(	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-Likewise	O
-,	O
-upon	O
-addition	O
-of	O
-the	O
-PIN	O
-domain	O
-,	O
-NMR	O
-signals	O
-derived	O
-from	O
-R56	O
-,	O
-L58	O
--	O
-G59	O
-,	O
-and	O
-V86	O
--	O
-H88	O
-in	O
-the	O
-NTD	O
-exhibited	O
-large	O
-chemical	O
-shift	O
-changes	O
-and	O
-residues	O
-D53	O
-,	O
-F55	O
-,	O
-K57	O
-,	O
-Y60	O
--	O
-S61	O
-,	O
-V68	O
-,	O
-T80	O
--	O
-G83	O
-,	O
-L85	O
-,	O
-and	O
-G89	O
-of	O
-the	O
-NTD	O
-as	O
-well	O
-as	O
-side	O
-chain	O
-amide	O
-signals	O
-of	O
-N79	O
-exhibited	O
-small	O
-but	O
-appreciable	O
-chemical	O
-shift	O
-changes	O
-(	O
-Fig	O
-.	O
-3b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-These	O
-results	O
-clearly	O
-indicate	O
-a	O
-direct	O
-interaction	O
-between	O
-the	O
-PIN	O
-domain	O
-and	O
-the	O
-NTD	O
-.	O
-	O
-Based	O
-on	O
-the	O
-titration	O
-curve	O
-for	O
-the	O
-chemical	O
-shift	O
-changes	O
-of	O
-L58	O
-,	O
-the	O
-apparent	O
-Kd	O
-between	O
-the	O
-isolated	O
-NTD	O
-and	O
-PIN	O
-was	O
-estimated	O
-to	O
-be	O
-110	O
-±	O
-5	O
-.	O
-8	O
-μM	O
-.	O
-Considering	O
-the	O
-fact	O
-that	O
-the	O
-NTD	O
-and	O
-PIN	O
-domains	O
-are	O
-attached	O
-by	O
-a	O
-linker	O
-,	O
-the	O
-actual	O
-binding	O
-affinity	O
-is	O
-expected	O
-much	O
-higher	O
-in	O
-the	O
-native	O
-protein	O
-.	O
-	O
-Mapping	O
-the	O
-residues	O
-with	O
-chemical	O
-shift	O
-changes	O
-reveals	O
-the	O
-putative	O
-PIN	O
-/	O
-NTD	O
-interface	O
-,	O
-which	O
-includes	O
-a	O
-helix	O
-that	O
-harbors	O
-catalytic	O
-residues	O
-D225	O
-and	O
-D226	O
-on	O
-the	O
-PIN	O
-domain	O
-(	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-Interestingly	O
-,	O
-the	O
-putative	O
-binding	O
-site	O
-for	O
-the	O
-NTD	O
-overlaps	O
-with	O
-the	O
-PIN	O
--	O
-PIN	O
-dimer	O
-interface	O
-,	O
-implying	O
-that	O
-NTD	O
-binding	O
-can	O
-“	O
-terminate	O
-”	O
-PIN	O
--	O
-PIN	O
-oligomerization	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-An	O
-in	O
-silico	O
-docking	O
-of	O
-the	O
-NTD	O
-and	O
-PIN	O
-domains	O
-using	O
-chemical	O
-shift	O
-restraints	O
-provided	O
-a	O
-model	O
-consistent	O
-with	O
-the	O
-NMR	O
-experiments	O
-(	O
-Fig	O
-.	O
-3c	O
-).	O
-	O
-Residues	O
-critical	O
-for	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-	O
-To	O
-gain	O
-insight	O
-into	O
-the	O
-residues	O
-critical	O
-for	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-,	O
-each	O
-basic	O
-or	O
-aromatic	O
-residue	O
-located	O
-around	O
-the	O
-catalytic	O
-site	O
-of	O
-the	O
-PIN	O
-oligomer	O
-was	O
-mutated	O
-to	O
-alanine	O
-,	O
-and	O
-the	O
-oligomerization	O
-and	O
-RNase	O
-activity	O
-were	O
-investigated	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-From	O
-the	O
-gel	O
-filtration	O
-assays	O
-,	O
-all	O
-mutants	O
-except	O
-R214A	B-mutant
-formed	O
-dimers	O
-,	O
-suggesting	O
-that	O
-any	O
-lack	O
-of	O
-RNase	O
-activity	O
-in	O
-the	O
-mutants	O
-,	O
-except	O
-R214A	B-mutant
-,	O
-was	O
-directly	O
-due	O
-to	O
-mutational	O
-effects	O
-of	O
-the	O
-specific	O
-residues	O
-and	O
-not	O
-to	O
-abrogation	O
-of	O
-dimer	O
-formation	O
-.	O
-	O
-The	O
-W182A	B-mutant
-,	O
-R183A	B-mutant
-,	O
-and	O
-R214A	B-mutant
-mutants	O
-markedly	O
-lost	O
-cleavage	O
-activity	O
-for	O
-IL	O
--	O
-6	O
-mRNA	O
-as	O
-well	O
-as	O
-for	O
-Regnase	O
--	O
-1	O
-mRNA	O
-.	O
-	O
-The	O
-K184A	B-mutant
-,	O
-R215A	B-mutant
-,	O
-and	O
-R220A	B-mutant
-mutants	O
-moderately	O
-but	O
-significantly	O
-decreased	O
-the	O
-cleavage	O
-activity	O
-for	O
-both	O
-target	O
-mRNAs	O
-.	O
-	O
-The	O
-importance	O
-of	O
-K219	O
-and	O
-R247	O
-was	O
-slightly	O
-different	O
-for	O
-IL	O
--	O
-6	O
-and	O
-Regnase	O
--	O
-1	O
-mRNA	O
-;	O
-both	O
-K219	O
-and	O
-R247	O
-were	O
-more	O
-important	O
-in	O
-the	O
-cleavage	O
-of	O
-IL	O
--	O
-6	O
-mRNA	O
-than	O
-for	O
-Regnase	O
--	O
-1	O
-mRNA	O
-.	O
-	O
-The	O
-other	O
-mutated	O
-residues	O
-—	O
-K152	O
-,	O
-R158	O
-,	O
-R188	O
-,	O
-R200	O
-,	O
-K204	O
-,	O
-K206	O
-,	O
-K257	O
-,	O
-and	O
-R258	O
-—	O
-were	O
-not	O
-critical	O
-for	O
-RNase	O
-activity	O
-.	O
-	O
-The	O
-importance	O
-of	O
-residues	O
-W182	O
-and	O
-R183	O
-can	O
-readily	O
-be	O
-understood	O
-in	O
-terms	O
-of	O
-the	O
-monomeric	O
-PIN	O
-structure	O
-as	O
-they	O
-are	O
-located	O
-near	O
-to	O
-the	O
-RNase	O
-catalytic	O
-site	O
-;	O
-however	O
-,	O
-the	O
-importance	O
-of	O
-residue	O
-K184	O
-,	O
-which	O
-points	O
-away	O
-from	O
-the	O
-active	O
-site	O
-is	O
-more	O
-easily	O
-rationalized	O
-in	O
-terms	O
-of	O
-the	O
-oligomeric	O
-structure	O
-,	O
-in	O
-which	O
-the	O
-“	O
-secondary	O
-”	O
-chain	O
-’	O
-s	O
-residue	O
-K184	O
-is	O
-positioned	O
-near	O
-the	O
-“	O
-primary	O
-”	O
-chain	O
-’	O
-s	O
-catalytic	O
-site	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-R214	O
-is	O
-important	O
-for	O
-oligomerization	O
-of	O
-the	O
-PIN	O
-domain	O
-and	O
-the	O
-“	O
-secondary	O
-”	O
-chain	O
-’	O
-s	O
-residue	O
-R214	O
-is	O
-also	O
-positioned	O
-near	O
-the	O
-“	O
-primary	O
-”	O
-chain	O
-’	O
-s	O
-active	O
-site	O
-within	O
-the	O
-dimer	O
-interface	O
-.	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-the	O
-putative	O
--	O
-RNA	O
-binding	O
-residues	O
-K184	O
-and	O
-R214	O
-are	O
-unique	O
-to	O
-Regnase	O
--	O
-1	O
-among	O
-PIN	O
-domains	O
-.	O
-	O
-Molecular	O
-mechanism	O
-of	O
-target	O
-mRNA	O
-cleavage	O
-by	O
-the	O
-PIN	O
-dimer	O
-	O
-Our	O
-mutational	O
-experiments	O
-indicated	O
-that	O
-the	O
-observed	O
-dimer	O
-is	O
-functional	O
-and	O
-that	O
-the	O
-role	O
-of	O
-the	O
-secondary	O
-PIN	O
-domain	O
-is	O
-to	O
-position	O
-Regnase	O
--	O
-1	O
--	O
-unique	O
-RNA	O
-binding	O
-residues	O
-near	O
-the	O
-active	O
-site	O
-of	O
-the	O
-primary	O
-PIN	O
-domain	O
-.	O
-	O
-If	O
-this	O
-model	O
-is	O
-correct	O
-,	O
-then	O
-we	O
-reasoned	O
-that	O
-a	O
-catalytically	O
-inactive	O
-PIN	O
-and	O
-a	O
-PIN	O
-lacking	O
-the	O
-putative	O
-RNA	O
--	O
-binding	O
-residues	O
-ought	O
-to	O
-be	O
-inactive	O
-in	O
-isolation	O
-but	O
-become	O
-active	O
-when	O
-mixed	O
-together	O
-.	O
-	O
-In	O
-order	O
-to	O
-test	O
-this	O
-hypothesis	O
-,	O
-we	O
-performed	O
-in	O
-vitro	O
-cleavage	O
-assays	O
-using	O
-combinations	O
-of	O
-Regnase	O
--	O
-1	O
-mutants	O
-that	O
-had	O
-no	O
-or	O
-decreased	O
-RNase	O
-activities	O
-by	O
-themselves	O
-(	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-One	O
-group	O
-consisted	O
-of	O
-catalytically	O
-active	O
-PIN	O
-domains	O
-with	O
-mutation	O
-of	O
-basic	O
-residues	O
-found	O
-in	O
-the	O
-previous	O
-section	O
-to	O
-confer	O
-decreased	O
-RNase	O
-activity	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-These	O
-were	O
-paired	O
-with	O
-a	O
-DDNN	B-mutant
-mutant	O
-that	O
-had	O
-no	O
-RNase	O
-activity	O
-by	O
-itself	O
-.	O
-	O
-When	O
-any	O
-members	O
-of	O
-the	O
-two	O
-groups	O
-are	O
-mixed	O
-,	O
-two	O
-kinds	O
-of	O
-heterodimers	O
-can	O
-be	O
-formed	O
-:	O
-one	O
-is	O
-composed	O
-of	O
-a	O
-DDNN	B-mutant
-primary	O
-PIN	O
-and	O
-a	O
-basic	O
-residue	O
-mutant	O
-secondary	O
-PIN	O
-and	O
-is	O
-expected	O
-to	O
-exhibit	O
-no	O
-RNase	O
-activity	O
-;	O
-the	O
-other	O
-is	O
-composed	O
-of	O
-a	O
-basic	O
-residue	O
-mutant	O
-primary	O
-PIN	O
-and	O
-a	O
-DDNN	B-mutant
-secondary	O
-PIN	O
-and	O
-is	O
-predicted	O
-to	O
-rescue	O
-RNase	O
-activity	O
-(	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-When	O
-we	O
-compared	O
-the	O
-fluorescence	O
-intensity	O
-of	O
-uncleaved	O
-IL	O
--	O
-6	O
-mRNA	O
-,	O
-basic	O
-residue	O
-mutants	O
-W182A	B-mutant
-,	O
-K184A	B-mutant
-,	O
-R214A	B-mutant
-,	O
-and	O
-R220A	B-mutant
-were	O
-rescued	O
-upon	O
-addition	O
-of	O
-the	O
-DDNN	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-Consistently	O
-,	O
-when	O
-we	O
-compared	O
-the	O
-fluorescence	O
-intensity	O
-of	O
-the	O
-uncleaved	O
-Regnase	O
--	O
-1	O
-mRNA	O
-,	O
-basic	O
-residue	O
-mutants	O
-K184A	B-mutant
-and	O
-R214A	B-mutant
-were	O
-rescued	O
-upon	O
-addition	O
-of	O
-the	O
-DDNN	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-5c	O
-).	O
-	O
-Rescue	O
-of	O
-K184A	B-mutant
-and	O
-R214A	B-mutant
-by	O
-the	O
-DDNN	B-mutant
-mutant	O
-was	O
-also	O
-confirmed	O
-by	O
-a	O
-significant	O
-increase	O
-in	O
-the	O
-cleaved	O
-products	O
-.	O
-	O
-This	O
-is	O
-particularly	O
-significant	O
-because	O
-the	O
-side	O
-chains	O
-of	O
-K184	O
-and	O
-R214	O
-in	O
-the	O
-primary	O
-PIN	O
-are	O
-oriented	O
-away	O
-from	O
-their	O
-own	O
-catalytic	O
-center	O
-,	O
-while	O
-those	O
-in	O
-the	O
-secondary	O
-PIN	O
-face	O
-toward	O
-the	O
-catalytic	O
-center	O
-of	O
-the	O
-primary	O
-PIN	O
-.	O
-	O
-R214	O
-is	O
-an	O
-important	O
-residue	O
-for	O
-dimer	O
-formation	O
-as	O
-shown	O
-in	O
-Fig	O
-.	O
-2	O
-,	O
-therefore	O
-,	O
-R214A	B-mutant
-in	O
-the	O
-secondary	O
-PIN	O
-cannot	O
-dimerize	O
-.	O
-	O
-According	O
-to	O
-the	O
-proposed	O
-model	O
-,	O
-an	O
-R214A	B-mutant
-PIN	O
-domain	O
-can	O
-only	O
-form	O
-a	O
-dimer	O
-when	O
-the	O
-DDNN	B-mutant
-PIN	O
-acts	O
-as	O
-the	O
-secondary	O
-PIN	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-the	O
-rescue	O
-experiments	O
-above	O
-support	O
-the	O
-proposed	O
-model	O
-in	O
-which	O
-the	O
-head	O
--	O
-to	O
--	O
-tail	O
-dimer	O
-is	O
-functional	O
-in	O
-vitro	O
-.	O
-	O
-We	O
-determined	O
-the	O
-individual	O
-domain	O
-structures	O
-of	O
-Regnase	O
--	O
-1	O
-by	O
-NMR	O
-and	O
-X	O
--	O
-ray	O
-crystallography	O
-.	O
-	O
-Although	O
-the	O
-function	O
-of	O
-the	O
-CTD	O
-remains	O
-elusive	O
-,	O
-we	O
-revealed	O
-the	O
-functions	O
-of	O
-the	O
-NTD	O
-,	O
-PIN	O
-,	O
-and	O
-ZF	O
-domains	O
-.	O
-	O
-A	O
-Regnase	O
--	O
-1	O
-construct	O
-consisting	O
-of	O
-PIN	O
-and	O
-ZF	O
-domains	O
-derived	O
-from	O
-Mus	O
-musculus	O
-was	O
-crystallized	O
-;	O
-however	O
-,	O
-the	O
-electron	O
-density	O
-of	O
-the	O
-ZF	O
-domain	O
-was	O
-low	O
-,	O
-indicating	O
-that	O
-the	O
-ZF	O
-domain	O
-is	O
-highly	O
-mobile	O
-in	O
-the	O
-absence	O
-of	O
-target	O
-mRNA	O
-or	O
-possibly	O
-other	O
-protein	O
--	O
-protein	O
-interactions	O
-.	O
-	O
-Our	O
-NMR	O
-experiments	O
-confirmed	O
-direct	O
-binding	O
-of	O
-the	O
-ZF	O
-domain	O
-to	O
-IL	O
--	O
-6	O
-mRNA	O
-with	O
-a	O
-Kd	O
-of	O
-10	O
-±	O
-1	O
-.	O
-1	O
-μM	O
-.	O
-Furthermore	O
-,	O
-an	O
-in	O
-vitro	O
-gel	O
-shift	O
-assay	O
-indicated	O
-that	O
-Regnase	O
--	O
-1	O
-containing	O
-the	O
-ZF	O
-domain	O
-enhanced	O
-target	O
-mRNA	O
--	O
-binding	O
-,	O
-but	O
-the	O
-protein	O
--	O
-RNA	O
-complex	O
-remained	O
-in	O
-the	O
-bottom	O
-of	O
-the	O
-well	O
-without	O
-entering	O
-into	O
-the	O
-polyacrylamide	O
-gel	O
-.	O
-	O
-These	O
-results	O
-indicate	O
-that	O
-Regnase	O
--	O
-1	O
-directly	O
-binds	O
-to	O
-RNA	O
-and	O
-precipitates	O
-under	O
-such	O
-experimental	O
-conditions	O
-.	O
-	O
-Due	O
-to	O
-this	O
-limitation	O
-,	O
-it	O
-is	O
-difficult	O
-to	O
-perform	O
-further	O
-structural	O
-analyses	O
-of	O
-mRNA	O
--	O
-Regnase	O
--	O
-1	O
-complexes	O
-by	O
-X	O
--	O
-ray	O
-crystallography	O
-or	O
-NMR	O
-.	O
-	O
-The	O
-previously	O
-reported	O
-crystal	O
-structure	O
-of	O
-the	O
-Regnase	O
--	O
-1	O
-PIN	O
-domain	O
-derived	O
-from	O
-Homo	O
-sapiens	O
-is	O
-nearly	O
-identical	O
-to	O
-the	O
-one	O
-derived	O
-from	O
-Mus	O
-musculus	O
-in	O
-this	O
-study	O
-,	O
-with	O
-a	O
-backbone	O
-RMSD	O
-of	O
-0	O
-.	O
-2	O
-Å	O
-.	O
-The	O
-amino	O
-acid	O
-sequences	O
-corresponding	O
-to	O
-PIN	O
-(	O
-residues	O
-134	O
-–	O
-295	O
-)	O
-are	O
-the	O
-two	O
-non	O
--	O
-identical	O
-residues	O
-are	O
-substituted	O
-with	O
-similar	O
-amino	O
-acids	O
-.	O
-	O
-Both	O
-the	O
-mouse	O
-and	O
-human	O
-PIN	O
-domains	O
-form	O
-head	O
--	O
-to	O
--	O
-tail	O
-oligomers	O
-in	O
-three	O
-distinct	O
-crystal	O
-forms	O
-.	O
-	O
-Rao	O
-and	O
-co	O
--	O
-workers	O
-previously	O
-argued	O
-that	O
-PIN	O
-dimerization	O
-is	O
-likely	O
-to	O
-be	O
-a	O
-crystallographic	O
-artifact	O
-with	O
-no	O
-physiological	O
-significance	O
-,	O
-since	O
-monomers	O
-were	O
-dominant	O
-in	O
-their	O
-analytical	O
-ultra	O
--	O
-centrifugation	O
-experiments	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-our	O
-gel	O
-filtration	O
-data	O
-,	O
-mutational	O
-analyses	O
-,	O
-and	O
-NMR	O
-spectra	O
-all	O
-indicate	O
-that	O
-the	O
-PIN	O
-domain	O
-forms	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-dimer	O
-in	O
-solution	O
-in	O
-a	O
-manner	O
-similar	O
-to	O
-the	O
-crystal	O
-structure	O
-.	O
-	O
-This	O
-inconsistency	O
-might	O
-be	O
-due	O
-to	O
-difference	O
-in	O
-the	O
-analytical	O
-methods	O
-and	O
-/	O
-or	O
-protein	O
-concentrations	O
-used	O
-in	O
-each	O
-experiment	O
-,	O
-since	O
-the	O
-oligomer	O
-formation	O
-of	O
-PIN	O
-was	O
-dependent	O
-on	O
-the	O
-protein	O
-concentration	O
-in	O
-our	O
-study	O
-.	O
-	O
-Single	O
-mutations	O
-to	O
-residues	O
-involved	O
-in	O
-the	O
-putative	O
-oligomeric	O
-interaction	O
-of	O
-PIN	O
-monomerized	O
-as	O
-expected	O
-and	O
-these	O
-mutants	O
-lost	O
-their	O
-RNase	O
-activity	O
-as	O
-well	O
-.	O
-	O
-Since	O
-the	O
-NMR	O
-spectra	O
-of	O
-monomeric	O
-mutants	O
-overlaps	O
-with	O
-those	O
-of	O
-the	O
-oligomeric	O
-forms	O
-,	O
-it	O
-is	O
-unlikely	O
-that	O
-the	O
-tertiary	O
-structure	O
-of	O
-the	O
-monomeric	O
-mutants	O
-were	O
-affected	O
-by	O
-the	O
-mutations	O
-.	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4b	O
-,	O
-c	O
-).	O
-	O
-Based	O
-on	O
-these	O
-observations	O
-,	O
-we	O
-concluded	O
-that	O
-PIN	O
--	O
-PIN	O
-dimer	O
-formation	O
-is	O
-critical	O
-for	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-in	O
-vitro	O
-.	O
-	O
-Within	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-PIN	O
-dimer	O
-,	O
-the	O
-Regnase	O
--	O
-1	O
-specific	O
-basic	O
-regions	O
-in	O
-both	O
-the	O
-“	O
-primary	O
-”	O
-and	O
-“	O
-secondary	O
-”	O
-PINs	O
-are	O
-located	O
-around	O
-the	O
-catalytic	O
-site	O
-of	O
-the	O
-primary	O
-PIN	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-Moreover	O
-,	O
-our	O
-structure	O
--	O
-based	O
-mutational	O
-analyses	O
-showed	O
-these	O
-two	O
-Regnase	O
--	O
-1	O
-specific	O
-basic	O
-regions	O
-were	O
-essential	O
-for	O
-target	O
-mRNA	O
-cleavage	O
-in	O
-vitro	O
-.	O
-	O
-The	O
-cleavage	O
-assay	O
-also	O
-showed	O
-that	O
-the	O
-NTD	O
-is	O
-crucial	O
-for	O
-efficient	O
-mRNA	O
-cleavage	O
-.	O
-	O
-Moreover	O
-,	O
-we	O
-found	O
-that	O
-the	O
-NTD	O
-associates	O
-with	O
-the	O
-oligomeric	O
-surface	O
-of	O
-the	O
-primary	O
-PIN	O
-,	O
-docking	O
-to	O
-a	O
-helix	O
-that	O
-harbors	O
-its	O
-catalytic	O
-residues	O
-(	O
-Figs	O
-2b	O
-and	O
-3a	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-this	O
-suggests	O
-that	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-compete	O
-for	O
-a	O
-common	O
-binding	O
-site	O
-.	O
-	O
-The	O
-affinity	O
-of	O
-the	O
-domain	O
--	O
-domain	O
-interaction	O
-between	O
-two	O
-PIN	O
-domains	O
-(	O
-Kd	O
-=	O
-~	O
-10	O
-−	O
-4	O
-M	O
-)	O
-is	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-NTD	O
--	O
-PIN	O
-(	O
-Kd	O
-=	O
-110	O
-±	O
-5	O
-.	O
-8	O
-μM	O
-)	O
-interactions	O
-;	O
-however	O
-,	O
-the	O
-covalent	O
-connection	O
-corresponding	O
-to	O
-residues	O
-90	O
-–	O
-133	O
-between	O
-the	O
-NTD	O
-and	O
-the	O
-primary	O
-PIN	O
-will	O
-greatly	O
-enhance	O
-the	O
-intramolecular	O
-domain	O
-interaction	O
-in	O
-the	O
-case	O
-of	O
-full	O
--	O
-length	O
-Regnase	O
--	O
-1	O
-.	O
-	O
-While	O
-further	O
-analyses	O
-are	O
-necessary	O
-to	O
-prove	O
-this	O
-point	O
-,	O
-our	O
-preliminary	O
-docking	O
-and	O
-molecular	O
-dynamics	O
-simulations	O
-indicate	O
-that	O
-NTD	O
--	O
-binding	O
-rearranges	O
-the	O
-catalytic	O
-residues	O
-of	O
-the	O
-PIN	O
-domain	O
-toward	O
-an	O
-active	O
-conformation	O
-suitable	O
-for	O
-binding	O
-Mg2	O
-+.	O
-	O
-In	O
-this	O
-context	O
-,	O
-it	O
-is	O
-interesting	O
-that	O
-,	O
-in	O
-response	O
-to	O
-TCR	O
-stimulation	O
-,	O
-Malt1	O
-cleaves	O
-Regnase	O
--	O
-1	O
-at	O
-R111	O
-to	O
-control	O
-immune	O
-responses	O
-in	O
-vivo	O
-.	O
-	O
-This	O
-result	O
-is	O
-consistent	O
-with	O
-a	O
-model	O
-in	O
-which	O
-the	O
-NTD	O
-acts	O
-as	O
-an	O
-enhancer	O
-,	O
-and	O
-cleavage	O
-of	O
-the	O
-linker	O
-lowers	O
-enzymatic	O
-activity	O
-dramatically	O
-.	O
-	O
-Based	O
-on	O
-these	O
-structural	O
-and	O
-functional	O
-analyses	O
-of	O
-Regnase	O
--	O
-1	O
-domain	O
--	O
-domain	O
-interactions	O
-,	O
-we	O
-performed	O
-docking	O
-simulations	O
-of	O
-the	O
-NTD	O
-,	O
-PIN	O
-dimer	O
-,	O
-and	O
-IL	O
--	O
-6	O
-mRNA	O
-.	O
-	O
-We	O
-incorporated	O
-information	O
-from	O
-the	O
-cleavage	O
-site	O
-of	O
-IL	O
--	O
-6	O
-mRNA	O
-in	O
-vitro	O
-is	O
-indicated	O
-by	O
-denaturing	O
-polyacrylamide	O
-gel	O
-electrophoresis	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7a	O
-,	O
-b	O
-).	O
-	O
-The	O
-docking	O
-result	O
-revealed	O
-multiple	O
-RNA	O
-binding	O
-modes	O
-that	O
-satisfied	O
-the	O
-experimental	O
-results	O
-in	O
-vitro	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7c	O
-,	O
-d	O
-),	O
-however	O
-,	O
-it	O
-should	O
-be	O
-noted	O
-that	O
-,	O
-in	O
-vivo	O
-,	O
-there	O
-would	O
-likely	O
-be	O
-many	O
-other	O
-RNA	O
--	O
-binding	O
-proteins	O
-that	O
-would	O
-protect	O
-loop	O
-regions	O
-from	O
-cleavage	O
-by	O
-Regnase	O
--	O
-1	O
-.	O
-	O
-The	O
-overall	O
-model	O
-of	O
-regulation	O
-of	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-through	O
-domain	O
--	O
-domain	O
-interactions	O
-in	O
-vitro	O
-is	O
-summarized	O
-in	O
-Fig	O
-.	O
-6	O
-.	O
-	O
-In	O
-the	O
-absence	O
-of	O
-target	O
-mRNA	O
-,	O
-the	O
-PIN	O
-domain	O
-forms	O
-head	O
--	O
-to	O
--	O
-tail	O
-oligomers	O
-at	O
-high	O
-concentration	O
-.	O
-	O
-A	O
-fully	O
-active	O
-catalytic	O
-center	O
-can	O
-be	O
-formed	O
-only	O
-when	O
-the	O
-NTD	O
-associates	O
-with	O
-the	O
-oligomer	O
-surface	O
-of	O
-the	O
-PIN	O
-domain	O
-,	O
-which	O
-terminates	O
-the	O
-head	O
--	O
-to	O
--	O
-tail	O
-oligomer	O
-formation	O
-in	O
-one	O
-direction	O
-(	O
-primary	O
-PIN	O
-),	O
-and	O
-forms	O
-a	O
-functional	O
-dimer	O
-together	O
-with	O
-the	O
-neighboring	O
-PIN	O
-(	O
-secondary	O
-PIN	O
-).	O
-	O
-While	O
-further	O
-investigations	O
-on	O
-the	O
-domain	O
--	O
-domain	O
-interactions	O
-of	O
-Regnase	O
--	O
-1	O
-in	O
-vivo	O
-are	O
-necessary	O
-,	O
-these	O
-intramolecular	O
-and	O
-intermolecular	O
-domain	O
-interactions	O
-of	O
-Regnase	O
--	O
-1	O
-appear	O
-to	O
-structurally	O
-constrain	O
-Regnase	O
--	O
-1activity	O
-,	O
-which	O
-,	O
-in	O
-turn	O
-,	O
-enables	O
-tight	O
-regulation	O
-of	O
-immune	O
-responses	O
-.	O
-	O
-Structural	O
-and	O
-functional	O
-analyses	O
-of	O
-Regnase	O
--	O
-1	O
-.	O
-	O
-(	O
-a	O
-)	O
-Domain	O
-architecture	O
-of	O
-Regnase	O
--	O
-1	O
-.	O
-(	O
-b	O
-)	O
-Solution	O
-structure	O
-of	O
-the	O
-NTD	O
-.	O
-(	O
-c	O
-)	O
-Crystal	O
-structure	O
-of	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-Catalytic	O
-Asp	O
-residues	O
-were	O
-shown	O
-in	O
-sticks	O
-.	O
-	O
-(	O
-d	O
-)	O
-Solution	O
-structure	O
-of	O
-the	O
-ZF	O
-domain	O
-.	O
-	O
-Three	O
-Cys	O
-residues	O
-and	O
-one	O
-His	O
-residue	O
-responsible	O
-for	O
-Zn2	O
-+-	O
-binding	O
-were	O
-shown	O
-in	O
-sticks	O
-.	O
-	O
-(	O
-e	O
-)	O
-Solution	O
-structure	O
-of	O
-the	O
-CTD	O
-.	O
-	O
-All	O
-the	O
-structures	O
-were	O
-colored	O
-in	O
-rainbow	O
-from	O
-N	O
--	O
-terminus	O
-(	O
-blue	O
-)	O
-to	O
-C	O
--	O
-terminus	O
-(	O
-red	O
-).	O
-	O
-(	O
-f	O
-)	O
-In	O
-vitro	O
-gel	O
-shift	O
-binding	O
-assay	O
-between	O
-Regnase	O
--	O
-1	O
-and	O
-IL	O
--	O
-6	O
-mRNA	O
-.	O
-	O
-Fluorescence	O
-intensity	O
-of	O
-the	O
-free	O
-IL	O
--	O
-6	O
-in	O
-each	O
-sample	O
-was	O
-indicated	O
-as	O
-the	O
-percentage	O
-against	O
-that	O
-in	O
-the	O
-absence	O
-of	O
-Regnase	O
--	O
-1	O
-.	O
-	O
-(	O
-g	O
-)	O
-Binding	O
-of	O
-Regnase	O
--	O
-1	O
-and	O
-IL	O
--	O
-6	O
-mRNA	O
-was	O
-plotted	O
-.	O
-	O
-The	O
-percentage	O
-of	O
-the	O
-bound	O
-IL	O
--	O
-6	O
-was	O
-calculated	O
-based	O
-on	O
-the	O
-fluorescence	O
-intensities	O
-of	O
-the	O
-free	O
-IL	O
--	O
-6	O
-quantified	O
-in	O
-(	O
-f	O
-).	O
-	O
-(	O
-h	O
-)	O
-In	O
-vitro	O
-cleavage	O
-assay	O
-of	O
-Regnase	O
--	O
-1	O
-to	O
-IL	O
--	O
-6	O
-mRNA	O
-.	O
-	O
-Fluorescence	O
-intensity	O
-of	O
-the	O
-uncleaved	O
-IL	O
--	O
-6	O
-mRNA	O
-was	O
-indicated	O
-as	O
-the	O
-percentage	O
-against	O
-that	O
-in	O
-the	O
-absence	O
-of	O
-Regnase	O
--	O
-1	O
-.	O
-	O
-Head	O
--	O
-to	O
--	O
-tail	O
-oligomer	O
-formation	O
-of	O
-the	O
-PIN	O
-domain	O
-is	O
-crucial	O
-for	O
-the	O
-RNase	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-.	O
-	O
-(	O
-a	O
-)	O
-Gel	O
-filtration	O
-analyses	O
-of	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-(	O
-b	O
-)	O
-Dimer	O
-structure	O
-of	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-Two	O
-PIN	O
-molecules	O
-in	O
-the	O
-crystal	O
-were	O
-colored	O
-white	O
-and	O
-green	O
-,	O
-respectively	O
-.	O
-	O
-Catalytic	O
-residues	O
-and	O
-mutated	O
-residues	O
-were	O
-shown	O
-in	O
-sticks	O
-.	O
-	O
-Residues	O
-important	O
-for	O
-the	O
-oligomeric	O
-interaction	O
-were	O
-colored	O
-red	O
-,	O
-while	O
-R215	O
-that	O
-was	O
-dispensable	O
-for	O
-the	O
-oligomeric	O
-interaction	O
-was	O
-colored	O
-blue	O
-.	O
-(	O
-c	O
-)	O
-RNase	O
-activity	O
-of	O
-monomeric	O
-mutants	O
-for	O
-IL	O
--	O
-6	O
-mRNA	O
-was	O
-analyzed	O
-.	O
-	O
-Domain	O
--	O
-domain	O
-interaction	O
-between	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-(	O
-a	O
-)	O
-NMR	O
-analyses	O
-of	O
-the	O
-NTD	O
--	O
-binding	O
-to	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-The	O
-residues	O
-with	O
-significant	O
-chemical	O
-shift	O
-changes	O
-were	O
-labeled	O
-in	O
-the	O
-overlaid	O
-spectra	O
-(	O
-left	O
-)	O
-and	O
-colored	O
-red	O
-on	O
-the	O
-surface	O
-and	O
-ribbon	O
-structure	O
-of	O
-the	O
-PIN	O
-domain	O
-(	O
-right	O
-).	O
-	O
-Pro	O
-and	O
-the	O
-residues	O
-without	O
-analysis	O
-were	O
-colored	O
-black	O
-and	O
-gray	O
-,	O
-respectively	O
-.	O
-	O
-(	O
-b	O
-)	O
-NMR	O
-analyses	O
-of	O
-the	O
-PIN	O
--	O
-binding	O
-to	O
-the	O
-NTD	O
-.	O
-	O
-The	O
-residues	O
-with	O
-significant	O
-chemical	O
-shift	O
-changes	O
-were	O
-labeled	O
-in	O
-the	O
-overlaid	O
-spectra	O
-(	O
-left	O
-)	O
-and	O
-colored	O
-red	O
-,	O
-yellow	O
-,	O
-or	O
-green	O
-on	O
-the	O
-surface	O
-and	O
-ribbon	O
-structure	O
-of	O
-the	O
-NTD	O
-.	O
-	O
-S62	O
-was	O
-colored	O
-gray	O
-and	O
-excluded	O
-from	O
-the	O
-analysis	O
-,	O
-due	O
-to	O
-low	O
-signal	O
-intensity	O
-.	O
-	O
-(	O
-c	O
-)	O
-Docking	O
-model	O
-of	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-The	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-are	O
-shown	O
-in	O
-cyan	O
-and	O
-white	O
-,	O
-respectively	O
-.	O
-	O
-Residues	O
-in	O
-close	O
-proximity	O
-(<	O
-5	O
-Å	O
-)	O
-to	O
-each	O
-other	O
-in	O
-the	O
-docking	O
-structure	O
-were	O
-colored	O
-yellow	O
-.	O
-	O
-Catalytic	O
-residues	O
-of	O
-the	O
-PIN	O
-domain	O
-are	O
-shown	O
-in	O
-sticks	O
-,	O
-and	O
-the	O
-residues	O
-that	O
-exhibited	O
-significant	O
-chemical	O
-shift	O
-changes	O
-in	O
-(	O
-a	O
-,	O
-b	O
-)	O
-were	O
-labeled	O
-.	O
-	O
-Critical	O
-residues	O
-in	O
-the	O
-PIN	O
-domain	O
-for	O
-the	O
-RNase	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-.	O
-	O
-(	O
-a	O
-)	O
-In	O
-vitro	O
-cleavage	O
-assay	O
-of	O
-basic	O
-residue	O
-mutants	O
-for	O
-IL	O
--	O
-6	O
-mRNA	O
-.	O
-	O
-(	O
-b	O
-)	O
-In	O
-vitro	O
-cleavage	O
-assay	O
-of	O
-basic	O
-residue	O
-mutants	O
-for	O
-Regnase	O
--	O
-1	O
-mRNA	O
-.	O
-	O
-The	O
-fluorescence	O
-intensity	O
-of	O
-the	O
-uncleaved	O
-mRNA	O
-was	O
-quantified	O
-and	O
-the	O
-results	O
-were	O
-mapped	O
-on	O
-the	O
-PIN	O
-dimer	O
-structure	O
-.	O
-	O
-Mutated	O
-basic	O
-residues	O
-were	O
-shown	O
-in	O
-sticks	O
-and	O
-those	O
-with	O
-significantly	O
-reduced	O
-RNase	O
-activities	O
-were	O
-colored	O
-red	O
-or	O
-yellow	O
-.	O
-	O
-Heterodimer	O
-formation	O
-by	O
-combination	O
-of	O
-the	O
-Regnase	O
--	O
-1	O
-basic	O
-residue	O
-mutants	O
-and	O
-the	O
-DDNN	B-mutant
-mutant	O
-restored	O
-the	O
-RNase	O
-activity	O
-.	O
-	O
-(	O
-a	O
-)	O
-Cartoon	O
-representation	O
-of	O
-the	O
-concept	O
-of	O
-the	O
-experiment	O
-.	O
-(	O
-b	O
-)	O
-In	O
-vitro	O
-cleavage	O
-assay	O
-of	O
-Regnase	O
--	O
-1	O
-for	O
-IL	O
--	O
-6	O
-mRNA	O
-.	O
-	O
-(	O
-c	O
-)	O
-In	O
-vitro	O
-cleavage	O
-assay	O
-of	O
-Regnase	O
--	O
-1	O
-for	O
-Regnase	O
--	O
-1	O
-mRNA	O
-.	O
-	O
-The	O
-fluorescence	O
-intensity	O
-of	O
-the	O
-uncleaved	O
-mRNA	O
-was	O
-quantified	O
-and	O
-the	O
-results	O
-were	O
-mapped	O
-on	O
-the	O
-PIN	O
-dimer	O
-.	O
-	O
-The	O
-mutations	O
-whose	O
-RNase	O
-activities	O
-were	O
-not	O
-increased	O
-in	O
-the	O
-presence	O
-of	O
-DDNN	B-mutant
-mutant	O
-were	O
-colored	O
-in	O
-blue	O
-on	O
-the	O
-primary	O
-PIN	O
-.	O
-	O
-The	O
-mutations	O
-whose	O
-RNase	O
-activities	O
-were	O
-restored	O
-in	O
-the	O
-presence	O
-of	O
-DDNN	B-mutant
-mutant	O
-were	O
-colored	O
-in	O
-red	O
-or	O
-yellow	O
-on	O
-the	O
-primary	O
-PIN	O
-.	O
-	O
-Schematic	O
-representation	O
-of	O
-regulation	O
-of	O
-the	O
-Regnase	O
--	O
-1	O
-catalytic	O
-activity	O
-through	O
-the	O
-domain	O
--	O
-domain	O
-interactions	O
-.	O
-	O
-Crystal	O
-Structure	O
-and	O
-Activity	O
-Studies	O
-of	O
-the	O
-C11	O
-Cysteine	O
-Peptidase	O
-from	O
-Parabacteroides	O
-merdae	O
-in	O
-the	O
-Human	O
-Gut	O
-Microbiome	O
-*	O
-	O
-Clan	O
-CD	O
-cysteine	O
-peptidases	O
-,	O
-a	O
-structurally	O
-related	O
-group	O
-of	O
-peptidases	O
-that	O
-include	O
-mammalian	O
-caspases	O
-,	O
-exhibit	O
-a	O
-wide	O
-range	O
-of	O
-important	O
-functions	O
-,	O
-along	O
-with	O
-a	O
-variety	O
-of	O
-specificities	O
-and	O
-activation	O
-mechanisms	O
-.	O
-	O
-However	O
-,	O
-for	O
-the	O
-clostripain	O
-family	O
-(	O
-denoted	O
-C11	O
-),	O
-little	O
-is	O
-currently	O
-known	O
-.	O
-	O
-Here	O
-,	O
-we	O
-describe	O
-the	O
-first	O
-crystal	O
-structure	O
-of	O
-a	O
-C11	O
-protein	O
-from	O
-the	O
-human	O
-gut	O
-bacterium	O
-,	O
-Parabacteroides	O
-merdae	O
-(	O
-PmC11	O
-),	O
-determined	O
-to	O
-1	O
-.	O
-7	O
--	O
-Å	O
-resolution	O
-.	O
-	O
-PmC11	O
-is	O
-a	O
-monomeric	O
-cysteine	O
-peptidase	O
-that	O
-comprises	O
-an	O
-extended	O
-caspase	O
--	O
-like	O
-α	O
-/	O
-β	O
-/	O
-α	O
-sandwich	O
-and	O
-an	O
-unusual	O
-C	O
--	O
-terminal	O
-domain	O
-.	O
-	O
-It	O
-shares	O
-core	O
-structural	O
-elements	O
-with	O
-clan	O
-CD	O
-cysteine	O
-peptidases	O
-but	O
-otherwise	O
-structurally	O
-differs	O
-from	O
-the	O
-other	O
-families	O
-in	O
-the	O
-clan	O
-.	O
-	O
-These	O
-studies	O
-also	O
-revealed	O
-a	O
-well	O
-ordered	O
-break	O
-in	O
-the	O
-polypeptide	O
-chain	O
-at	O
-Lys147	O
-,	O
-resulting	O
-in	O
-a	O
-large	O
-conformational	O
-rearrangement	O
-close	O
-to	O
-the	O
-active	O
-site	O
-.	O
-	O
-Biochemical	O
-and	O
-kinetic	O
-analysis	O
-revealed	O
-Lys147	O
-to	O
-be	O
-an	O
-intramolecular	O
-processing	O
-site	O
-at	O
-which	O
-cleavage	O
-is	O
-required	O
-for	O
-full	O
-activation	O
-of	O
-the	O
-enzyme	O
-,	O
-suggesting	O
-an	O
-autoinhibitory	O
-mechanism	O
-for	O
-self	O
--	O
-preservation	O
-.	O
-	O
-PmC11	O
-has	O
-an	O
-acidic	O
-binding	O
-pocket	O
-and	O
-a	O
-preference	O
-for	O
-basic	O
-substrates	O
-,	O
-and	O
-accepts	O
-substrates	O
-with	O
-Arg	O
-and	O
-Lys	O
-in	O
-P1	O
-and	O
-does	O
-not	O
-require	O
-Ca2	O
-+	O
-for	O
-activity	O
-.	O
-	O
-Collectively	O
-,	O
-these	O
-data	O
-provide	O
-insights	O
-into	O
-the	O
-mechanism	O
-and	O
-activity	O
-of	O
-PmC11	O
-and	O
-a	O
-detailed	O
-framework	O
-for	O
-studies	O
-on	O
-C11	O
-peptidases	O
-from	O
-other	O
-phylogenetic	O
-kingdoms	O
-.	O
-	O
-Cysteine	O
-peptidases	O
-play	O
-crucial	O
-roles	O
-in	O
-the	O
-virulence	O
-of	O
-bacterial	O
-and	O
-other	O
-eukaryotic	O
-pathogens	O
-.	O
-	O
-In	O
-the	O
-MEROPS	O
-peptidase	O
-database	O
-,	O
-clan	O
-CD	O
-contains	O
-groups	O
-(	O
-or	O
-families	O
-)	O
-of	O
-cysteine	O
-peptidases	O
-that	O
-share	O
-some	O
-highly	O
-conserved	O
-structural	O
-elements	O
-.	O
-	O
-Clan	O
-CD	O
-families	O
-are	O
-typically	O
-described	O
-using	O
-the	O
-name	O
-of	O
-their	O
-archetypal	O
-,	O
-or	O
-founding	O
-,	O
-member	O
-and	O
-also	O
-given	O
-an	O
-identification	O
-number	O
-preceded	O
-by	O
-a	O
-“	O
-C	O
-,”	O
-to	O
-denote	O
-cysteine	O
-peptidase	O
-.	O
-	O
-Although	O
-seven	O
-families	O
-(	O
-C14	O
-is	O
-additionally	O
-split	O
-into	O
-three	O
-subfamilies	O
-)	O
-have	O
-been	O
-described	O
-for	O
-this	O
-clan	O
-,	O
-crystal	O
-structures	O
-have	O
-only	O
-been	O
-determined	O
-from	O
-four	O
-:	O
-legumain	O
-(	O
-C13	O
-),	O
-caspase	O
-(	O
-C14a	O
-),	O
-paracaspase	O
-(	O
-C14b	O
-(	O
-P	O
-),	O
-metacaspase	O
-(	O
-C14b	O
-(	O
-M	O
-),	O
-gingipain	O
-(	O
-C25	O
-),	O
-and	O
-the	O
-cysteine	O
-peptidase	O
-domain	O
-(	O
-CPD	O
-)	O
-of	O
-various	O
-toxins	O
-(	O
-C80	O
-).	O
-	O
-No	O
-structural	O
-information	O
-is	O
-available	O
-for	O
-clostripain	O
-(	O
-C11	O
-),	O
-separase	O
-(	O
-C50	O
-),	O
-or	O
-PrtH	O
--	O
-peptidase	O
-(	O
-C85	O
-).	O
-	O
-Clan	O
-CD	O
-enzymes	O
-have	O
-a	O
-highly	O
-conserved	O
-His	O
-/	O
-Cys	O
-catalytic	O
-dyad	O
-and	O
-exhibit	O
-strict	O
-specificity	O
-for	O
-the	O
-P1	O
-residue	O
-of	O
-their	O
-substrates	O
-.	O
-	O
-However	O
-,	O
-despite	O
-these	O
-similarities	O
-,	O
-clan	O
-CD	O
-forms	O
-a	O
-functionally	O
-diverse	O
-group	O
-of	O
-enzymes	O
-:	O
-the	O
-overall	O
-structural	O
-diversity	O
-between	O
-(	O
-and	O
-at	O
-times	O
-within	O
-)	O
-the	O
-various	O
-families	O
-provides	O
-these	O
-peptidases	O
-with	O
-a	O
-wide	O
-variety	O
-of	O
-substrate	O
-specificities	O
-and	O
-activation	O
-mechanisms	O
-.	O
-	O
-The	O
-archetypal	O
-and	O
-arguably	O
-most	O
-notable	O
-family	O
-in	O
-the	O
-clan	O
-is	O
-that	O
-of	O
-the	O
-mammalian	O
-caspases	O
-(	O
-C14a	O
-),	O
-although	O
-clan	O
-CD	O
-members	O
-are	O
-distributed	O
-throughout	O
-the	O
-entire	O
-phylogenetic	O
-kingdom	O
-and	O
-are	O
-often	O
-required	O
-in	O
-fundamental	O
-biological	O
-processes	O
-.	O
-	O
-Interestingly	O
-,	O
-little	O
-is	O
-known	O
-about	O
-the	O
-structure	O
-or	O
-function	O
-of	O
-the	O
-C11	O
-proteins	O
-,	O
-despite	O
-their	O
-widespread	O
-distribution	O
-and	O
-its	O
-archetypal	O
-member	O
-,	O
-clostripain	O
-from	O
-Clostridium	O
-histolyticum	O
-,	O
-first	O
-reported	O
-in	O
-the	O
-literature	O
-in	O
-1938	O
-.	O
-	O
-Clostripain	O
-has	O
-been	O
-described	O
-as	O
-an	O
-arginine	O
--	O
-specific	O
-peptidase	O
-with	O
-a	O
-requirement	O
-for	O
-Ca2	O
-+	O
-and	O
-loss	O
-of	O
-an	O
-internal	O
-nonapeptide	O
-for	O
-full	O
-activation	O
-;	O
-lack	O
-of	O
-structural	O
-information	O
-on	O
-the	O
-family	O
-appears	O
-to	O
-have	O
-prohibited	O
-further	O
-investigation	O
-.	O
-	O
-As	O
-part	O
-of	O
-an	O
-ongoing	O
-project	O
-to	O
-characterize	O
-commensal	O
-bacteria	O
-in	O
-the	O
-microbiome	O
-that	O
-inhabit	O
-the	O
-human	O
-gut	O
-,	O
-the	O
-structure	O
-of	O
-C11	O
-peptidase	O
-,	O
-PmC11	O
-,	O
-from	O
-Parabacteroides	O
-merdae	O
-was	O
-determined	O
-using	O
-the	O
-Joint	O
-Center	O
-for	O
-Structural	O
-Genomics	O
-(	O
-JCSG	O
-)	O
-4	O
-HTP	O
-structural	O
-biology	O
-pipeline	O
-.	O
-	O
-The	O
-structure	O
-was	O
-analyzed	O
-,	O
-and	O
-the	O
-enzyme	O
-was	O
-biochemically	O
-characterized	O
-to	O
-provide	O
-the	O
-first	O
-structure	O
-/	O
-function	O
-correlation	O
-for	O
-a	O
-C11	O
-peptidase	O
-.	O
-	O
-Structure	O
-of	O
-PmC11	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-catalytically	O
-active	O
-form	O
-of	O
-PmC11	O
-revealed	O
-an	O
-extended	O
-caspase	O
--	O
-like	O
-α	O
-/	O
-β	O
-/	O
-α	O
-sandwich	O
-architecture	O
-comprised	O
-of	O
-a	O
-central	O
-nine	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-,	O
-with	O
-an	O
-unusual	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-CTD	O
-),	O
-starting	O
-at	O
-Lys250	O
-.	O
-	O
-A	O
-single	O
-cleavage	O
-was	O
-observed	O
-in	O
-the	O
-polypeptide	O
-chain	O
-at	O
-Lys147	O
-(	O
-Fig	O
-.	O
-1	O
-,	O
-A	O
-and	O
-B	O
-),	O
-where	O
-both	O
-ends	O
-of	O
-the	O
-cleavage	O
-site	O
-are	O
-fully	O
-visible	O
-and	O
-well	O
-ordered	O
-in	O
-the	O
-electron	O
-density	O
-.	O
-	O
-The	O
-central	O
-nine	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-(	O
-β1	O
-–	O
-β9	O
-)	O
-of	O
-PmC11	O
-consists	O
-of	O
-six	O
-parallel	O
-and	O
-three	O
-anti	O
--	O
-parallel	O
-β	O
--	O
-strands	O
-with	O
-4	O
-↑	O
-3	O
-↓	O
-2	O
-↑	O
-1	O
-↑	O
-5	O
-↑	O
-6	O
-↑	O
-7	O
-↓	O
-8	O
-↓	O
-9	O
-↑	O
-topology	O
-(	O
-Fig	O
-.	O
-1A	O
-)	O
-and	O
-the	O
-overall	O
-structure	O
-includes	O
-14	O
-α	O
--	O
-helices	O
-with	O
-six	O
-(	O
-α1	O
-–	O
-α2	O
-and	O
-α4	O
-–	O
-α7	O
-)	O
-closely	O
-surrounding	O
-the	O
-β	O
--	O
-sheet	O
-in	O
-an	O
-approximately	O
-parallel	O
-orientation	O
-.	O
-	O
-Helices	O
-α1	O
-,	O
-α7	O
-,	O
-and	O
-α6	O
-are	O
-located	O
-on	O
-one	O
-side	O
-of	O
-the	O
-β	O
--	O
-sheet	O
-with	O
-α2	O
-,	O
-α4	O
-,	O
-and	O
-α5	O
-on	O
-the	O
-opposite	O
-side	O
-(	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-Helix	O
-α3	O
-sits	O
-at	O
-the	O
-end	O
-of	O
-the	O
-loop	O
-following	O
-β5	O
-(	O
-L5	O
-),	O
-just	O
-preceding	O
-the	O
-Lys147	O
-cleavage	O
-site	O
-,	O
-with	O
-both	O
-L5	O
-and	O
-α3	O
-pointing	O
-away	O
-from	O
-the	O
-central	O
-β	O
--	O
-sheet	O
-and	O
-toward	O
-the	O
-CTD	O
-,	O
-which	O
-starts	O
-with	O
-α8	O
-.	O
-	O
-The	O
-structure	O
-also	O
-includes	O
-two	O
-short	O
-β	O
--	O
-hairpins	O
-(	O
-βA	O
-–	O
-βB	O
-and	O
-βD	O
-–	O
-βE	O
-)	O
-and	O
-a	O
-small	O
-β	O
--	O
-sheet	O
-(	O
-βC	O
-–	O
-βF	O
-),	O
-which	O
-is	O
-formed	O
-from	O
-two	O
-distinct	O
-regions	O
-of	O
-the	O
-sequence	O
-(	O
-βC	O
-precedes	O
-α11	O
-,	O
-α12	O
-and	O
-β9	O
-,	O
-whereas	O
-βF	O
-follows	O
-the	O
-βD	O
--	O
-βE	O
-hairpin	O
-)	O
-in	O
-the	O
-middle	O
-of	O
-the	O
-CTD	O
-(	O
-Fig	O
-.	O
-1B	O
-).	O
-	O
-Crystal	O
-structure	O
-of	O
-a	O
-C11	O
-peptidase	O
-from	O
-P	O
-.	O
-merdae	O
-.	O
-	O
-A	O
-,	O
-primary	O
-sequence	O
-alignment	O
-of	O
-PmC11	O
-(	O
-Uniprot	O
-ID	O
-A7A9N3	O
-)	O
-and	O
-clostripain	O
-(	O
-Uniprot	O
-ID	O
-P09870	O
-)	O
-from	O
-C	O
-.	O
-histolyticum	O
-with	O
-identical	O
-residues	O
-highlighted	O
-in	O
-gray	O
-shading	O
-.	O
-	O
-The	O
-secondary	O
-structure	O
-of	O
-PmC11	O
-from	O
-the	O
-crystal	O
-structure	O
-is	O
-mapped	O
-onto	O
-its	O
-sequence	O
-with	O
-the	O
-position	O
-of	O
-the	O
-PmC11	O
-catalytic	O
-dyad	O
-,	O
-autocatalytic	O
-cleavage	O
-site	O
-(	O
-Lys147	O
-),	O
-and	O
-S1	O
-binding	O
-pocket	O
-Asp	O
-(	O
-Asp177	O
-)	O
-highlighted	O
-by	O
-a	O
-red	O
-star	O
-,	O
-a	O
-red	O
-downturned	O
-triangle	O
-,	O
-and	O
-a	O
-red	O
-upturned	O
-triangle	O
-,	O
-respectively	O
-.	O
-	O
-Connecting	O
-loops	O
-are	O
-colored	O
-gray	O
-,	O
-the	O
-main	O
-β	O
--	O
-sheet	O
-is	O
-in	O
-orange	O
-,	O
-with	O
-other	O
-strands	O
-in	O
-olive	O
-,	O
-α	O
--	O
-helices	O
-are	O
-in	O
-blue	O
-,	O
-and	O
-the	O
-nonapeptide	O
-linker	O
-of	O
-clostripain	O
-that	O
-is	O
-excised	O
-upon	O
-autocleavage	O
-is	O
-underlined	O
-in	O
-red	O
-.	O
-	O
-Sequences	O
-around	O
-the	O
-catalytic	O
-site	O
-of	O
-clostripain	O
-and	O
-PmC11	O
-align	O
-well	O
-.	O
-	O
-B	O
-,	O
-topology	O
-diagram	O
-of	O
-PmC11	O
-colored	O
-as	O
-in	O
-A	O
-except	O
-that	O
-additional	O
-(	O
-non	O
--	O
-core	O
-)	O
-β	O
--	O
-strands	O
-are	O
-in	O
-yellow	O
-.	O
-	O
-Helices	O
-found	O
-on	O
-either	O
-side	O
-of	O
-the	O
-central	O
-β	O
--	O
-sheet	O
-are	O
-shown	O
-above	O
-and	O
-below	O
-the	O
-sheet	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-position	O
-of	O
-the	O
-catalytic	O
-dyad	O
-(	O
-H	O
-,	O
-C	O
-)	O
-and	O
-the	O
-processing	O
-site	O
-(	O
-Lys147	O
-)	O
-are	O
-highlighted	O
-.	O
-	O
-Helices	O
-(	O
-1	O
-–	O
-14	O
-)	O
-and	O
-β	O
--	O
-strands	O
-(	O
-1	O
-–	O
-9	O
-and	O
-A	O
--	O
-F	O
-)	O
-are	O
-numbered	O
-from	O
-the	O
-N	O
-terminus	O
-.	O
-	O
-The	O
-core	O
-caspase	O
--	O
-fold	O
-is	O
-highlighted	O
-in	O
-a	O
-box	O
-.	O
-	O
-C	O
-,	O
-tertiary	O
-structure	O
-of	O
-PmC11	O
-.	O
-	O
-The	O
-N	O
-and	O
-C	O
-termini	O
-(	O
-N	O
-and	O
-C	O
-)	O
-of	O
-PmC11	O
-along	O
-with	O
-the	O
-central	O
-β	O
--	O
-sheet	O
-(	O
-1	O
-–	O
-9	O
-),	O
-helix	O
-α5	O
-,	O
-and	O
-helices	O
-α8	O
-,	O
-α11	O
-,	O
-and	O
-α13	O
-from	O
-the	O
-C	O
--	O
-terminal	O
-domain	O
-,	O
-are	O
-all	O
-labeled	O
-.	O
-	O
-Loops	O
-are	O
-colored	O
-gray	O
-,	O
-the	O
-main	O
-β	O
--	O
-sheet	O
-is	O
-in	O
-orange	O
-,	O
-with	O
-other	O
-β	O
--	O
-strands	O
-in	O
-yellow	O
-,	O
-and	O
-α	O
--	O
-helices	O
-are	O
-in	O
-blue	O
-.	O
-	O
-The	O
-CTD	O
-of	O
-PmC11	O
-is	O
-composed	O
-of	O
-a	O
-tight	O
-helical	O
-bundle	O
-formed	O
-from	O
-helices	O
-α8	O
-–	O
-α14	O
-and	O
-includes	O
-strands	O
-βC	O
-and	O
-βF	O
-,	O
-and	O
-β	O
--	O
-hairpin	O
-βD	O
-–	O
-βE	O
-.	O
-The	O
-CTD	O
-sits	O
-entirely	O
-on	O
-one	O
-side	O
-of	O
-the	O
-enzyme	O
-interacting	O
-only	O
-with	O
-α3	O
-,	O
-α5	O
-,	O
-β9	O
-,	O
-and	O
-the	O
-loops	O
-surrounding	O
-β8	O
-.	O
-	O
-Of	O
-the	O
-interacting	O
-secondary	O
-structure	O
-elements	O
-,	O
-α5	O
-is	O
-perhaps	O
-the	O
-most	O
-interesting	O
-.	O
-	O
-This	O
-helix	O
-makes	O
-a	O
-total	O
-of	O
-eight	O
-hydrogen	O
-bonds	O
-with	O
-the	O
-CTD	O
-,	O
-including	O
-one	O
-salt	O
-bridge	O
-(	O
-Arg191	O
--	O
-Asp255	O
-)	O
-and	O
-is	O
-surrounded	O
-by	O
-the	O
-CTD	O
-on	O
-one	O
-side	O
-and	O
-the	O
-main	O
-core	O
-of	O
-the	O
-enzyme	O
-on	O
-the	O
-other	O
-,	O
-acting	O
-like	O
-a	O
-linchpin	O
-holding	O
-both	O
-components	O
-together	O
-(	O
-Fig	O
-.	O
-1C	O
-).	O
-	O
-PmC11	O
-is	O
-,	O
-as	O
-expected	O
-,	O
-most	O
-structurally	O
-similar	O
-to	O
-other	O
-members	O
-of	O
-clan	O
-CD	O
-with	O
-the	O
-top	O
-hits	O
-in	O
-a	O
-search	O
-of	O
-known	O
-structures	O
-being	O
-caspase	O
--	O
-7	O
-,	O
-gingipain	O
--	O
-K	O
-,	O
-and	O
-legumain	O
-(	O
-PBD	O
-codes	O
-4hq0	O
-,	O
-4tkx	O
-,	O
-and	O
-4aw9	O
-,	O
-respectively	O
-)	O
-(	O
-Table	O
-2	O
-).	O
-	O
-The	O
-C	O
--	O
-terminal	O
-domain	O
-is	O
-unique	O
-to	O
-PmC11	O
-within	O
-clan	O
-CD	O
-and	O
-structure	O
-comparisons	O
-for	O
-this	O
-domain	O
-alone	O
-does	O
-not	O
-produce	O
-any	O
-hits	O
-in	O
-the	O
-PDB	O
-(	O
-DaliLite	O
-,	O
-PDBeFold	O
-),	O
-suggesting	O
-a	O
-completely	O
-novel	O
-fold	O
-.	O
-	O
-As	O
-the	O
-archetypal	O
-and	O
-arguably	O
-most	O
-well	O
-studied	O
-member	O
-of	O
-clan	O
-CD	O
-,	O
-the	O
-caspases	O
-were	O
-used	O
-as	O
-the	O
-basis	O
-to	O
-investigate	O
-the	O
-structure	O
-/	O
-function	O
-relationships	O
-in	O
-PmC11	O
-,	O
-with	O
-caspase	O
--	O
-7	O
-as	O
-the	O
-representative	O
-member	O
-.	O
-	O
-Six	O
-of	O
-the	O
-central	O
-β	O
--	O
-strands	O
-in	O
-PmC11	O
-(	O
-β1	O
-–	O
-β2	O
-and	O
-β5	O
-–	O
-β8	O
-)	O
-share	O
-the	O
-same	O
-topology	O
-as	O
-the	O
-six	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-found	O
-in	O
-caspases	O
-,	O
-with	O
-strands	O
-β3	O
-,	O
-β4	O
-,	O
-and	O
-β9	O
-located	O
-on	O
-the	O
-outside	O
-of	O
-this	O
-core	O
-structure	O
-(	O
-Fig	O
-.	O
-1B	O
-,	O
-box	O
-).	O
-	O
-His133	O
-and	O
-Cys179	O
-were	O
-found	O
-at	O
-locations	O
-structurally	O
-homologous	O
-to	O
-the	O
-caspase	O
-catalytic	O
-dyad	O
-,	O
-and	O
-other	O
-clan	O
-CD	O
-structures	O
-,	O
-at	O
-the	O
-C	O
-termini	O
-of	O
-strands	O
-β5	O
-and	O
-β6	O
-,	O
-respectively	O
-(	O
-Figs	O
-.	O
-1	O
-,	O
-A	O
-and	O
-B	O
-,	O
-and	O
-2A	O
-).	O
-	O
-A	O
-multiple	O
-sequence	O
-alignment	O
-of	O
-C11	O
-proteins	O
-revealed	O
-that	O
-these	O
-residues	O
-are	O
-highly	O
-conserved	O
-(	O
-data	O
-not	O
-shown	O
-).	O
-	O
-Summary	O
-of	O
-PDBeFOLD	O
-superposition	O
-of	O
-structures	O
-found	O
-to	O
-be	O
-most	O
-similar	O
-to	O
-PmC11	O
-in	O
-the	O
-PBD	O
-based	O
-on	O
-DaliLite	O
-	O
-Biochemical	O
-and	O
-structural	O
-characterization	O
-of	O
-PmC11	O
-.	O
-	O
-A	O
-,	O
-ribbon	O
-representation	O
-of	O
-the	O
-overall	O
-structure	O
-of	O
-PmC11	O
-illustrating	O
-the	O
-catalytic	O
-site	O
-,	O
-cleavage	O
-site	O
-displacement	O
-,	O
-and	O
-potential	O
-S1	O
-binding	O
-site	O
-.	O
-	O
-The	O
-overall	O
-structure	O
-of	O
-PmC11	O
-is	O
-shown	O
-in	O
-gray	O
-,	O
-looking	O
-down	O
-into	O
-the	O
-catalytic	O
-site	O
-with	O
-the	O
-catalytic	O
-dyad	O
-in	O
-red	O
-.	O
-	O
-The	O
-two	O
-ends	O
-of	O
-the	O
-autolytic	O
-cleavage	O
-site	O
-(	O
-Lys147	O
-and	O
-Ala148	O
-,	O
-green	O
-)	O
-are	O
-displaced	O
-by	O
-19	O
-.	O
-5	O
-Å	O
-(	O
-thin	O
-black	O
-line	O
-)	O
-from	O
-one	O
-another	O
-and	O
-residues	O
-in	O
-the	O
-potential	O
-substrate	O
-binding	O
-pocket	O
-are	O
-highlighted	O
-in	O
-blue	O
-.	O
-	O
-B	O
-,	O
-size	O
-exclusion	O
-chromatography	O
-of	O
-PmC11	O
-.	O
-	O
-PmC11	O
-migrates	O
-as	O
-a	O
-monomer	O
-with	O
-a	O
-molecular	O
-mass	O
-around	O
-41	O
-kDa	O
-calculated	O
-from	O
-protein	O
-standards	O
-of	O
-known	O
-molecular	O
-weights	O
-.	O
-	O
-Elution	O
-fractions	O
-across	O
-the	O
-major	O
-peak	O
-(	O
-1	O
-–	O
-6	O
-)	O
-were	O
-analyzed	O
-by	O
-SDS	O
--	O
-PAGE	O
-on	O
-a	O
-4	O
-–	O
-12	O
-%	O
-gel	O
-in	O
-MES	O
-buffer	O
-.	O
-	O
-C	O
-,	O
-the	O
-active	O
-form	O
-of	O
-PmC11	O
-and	O
-two	O
-mutants	O
-,	O
-PmC11C179A	B-mutant
-(	O
-C	O
-)	O
-and	O
-PmC11K147A	B-mutant
-(	O
-K	O
-),	O
-were	O
-examined	O
-by	O
-SDS	O
--	O
-PAGE	O
-(	O
-lane	O
-1	O
-)	O
-and	O
-Western	O
-blot	O
-analysis	O
-using	O
-an	O
-anti	O
--	O
-His	O
-antibody	O
-(	O
-lane	O
-2	O
-)	O
-show	O
-that	O
-PmC11	O
-autoprocesses	O
-,	O
-whereas	O
-mutants	O
-,	O
-PmC11C179A	B-mutant
-and	O
-PmC11K147A	B-mutant
-,	O
-do	O
-not	O
-show	O
-autoprocessing	O
-in	O
-vitro	O
-.	O
-	O
-D	O
-,	O
-cysteine	O
-peptidase	O
-activity	O
-of	O
-PmC11	O
-.	O
-	O
-Km	O
-and	O
-Vmax	O
-of	O
-PmC11	O
-and	O
-K147A	B-mutant
-mutant	O
-were	O
-determined	O
-by	O
-monitoring	O
-change	O
-in	O
-the	O
-fluorescence	O
-corresponding	O
-to	O
-AMC	O
-release	O
-from	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-.	O
-	O
-E	O
-,	O
-intermolecular	O
-processing	O
-of	O
-PmC11C179A	B-mutant
-by	O
-PmC11	O
-.	O
-	O
-PmC11C179A	O
-(	O
-20	O
-μg	O
-)	O
-was	O
-incubated	O
-overnight	O
-at	O
-37	O
-°	O
-C	O
-with	O
-increasing	O
-amounts	O
-of	O
-processed	O
-PmC11	O
-and	O
-analyzed	O
-on	O
-a	O
-10	O
-%	O
-SDS	O
--	O
-PAGE	O
-gel	O
-.	O
-	O
-Inactive	O
-PmC11C179A	B-mutant
-was	O
-not	O
-processed	O
-to	O
-a	O
-major	O
-extent	O
-by	O
-active	O
-PmC11	O
-until	O
-around	O
-a	O
-ratio	O
-of	O
-1	O
-:	O
-4	O
-(	O
-5	O
-μg	O
-of	O
-active	O
-PmC11	O
-).	O
-	O
-A	O
-single	O
-lane	O
-of	O
-20	O
-μg	O
-of	O
-active	O
-PmC11	O
-(	O
-labeled	O
-20	O
-)	O
-is	O
-shown	O
-for	O
-comparison	O
-.	O
-	O
-F	O
-,	O
-activity	O
-of	O
-PmC11	O
-against	O
-basic	O
-substrates	O
-.	O
-	O
-G	O
-,	O
-electrostatic	O
-surface	O
-potential	O
-of	O
-PmC11	O
-shown	O
-in	O
-a	O
-similar	O
-orientation	O
-,	O
-where	O
-blue	O
-and	O
-red	O
-denote	O
-positively	O
-and	O
-negatively	O
-charged	O
-surface	O
-potential	O
-,	O
-respectively	O
-,	O
-contoured	O
-at	O
-±	O
-5	O
-kT	O
-/	O
-e	O
-.	O
-	O
-The	O
-position	O
-of	O
-the	O
-catalytic	O
-dyad	O
-,	O
-one	O
-potential	O
-key	O
-substrate	O
-binding	O
-residue	O
-Asp177	O
-,	O
-and	O
-the	O
-ends	O
-of	O
-the	O
-cleavage	O
-site	O
-Lys147	O
-and	O
-Ala148	O
-are	O
-indicated	O
-.	O
-	O
-Five	O
-of	O
-the	O
-α	O
--	O
-helices	O
-surrounding	O
-the	O
-β	O
--	O
-sheet	O
-of	O
-PmC11	O
-(	O
-α1	O
-,	O
-α2	O
-,	O
-α4	O
-,	O
-α6	O
-,	O
-and	O
-α7	O
-)	O
-are	O
-found	O
-in	O
-similar	O
-positions	O
-to	O
-the	O
-five	O
-structurally	O
-conserved	O
-helices	O
-in	O
-caspases	O
-and	O
-other	O
-members	O
-of	O
-clan	O
-CD	O
-,	O
-apart	O
-from	O
-family	O
-C80	O
-.	O
-	O
-Other	O
-than	O
-its	O
-more	O
-extended	O
-β	O
--	O
-sheet	O
-,	O
-PmC11	O
-differs	O
-most	O
-significantly	O
-from	O
-other	O
-clan	O
-CD	O
-members	O
-at	O
-its	O
-C	O
-terminus	O
-,	O
-where	O
-the	O
-CTD	O
-contains	O
-a	O
-further	O
-seven	O
-α	O
--	O
-helices	O
-and	O
-four	O
-β	O
--	O
-strands	O
-after	O
-β8	O
-.	O
-	O
-Autoprocessing	O
-of	O
-PmC11	O
-	O
-Purification	O
-of	O
-recombinant	O
-PmC11	O
-(	O
-molecular	O
-mass	O
-=	O
-42	O
-.	O
-6	O
-kDa	O
-)	O
-revealed	O
-partial	O
-processing	O
-into	O
-two	O
-cleavage	O
-products	O
-of	O
-26	O
-.	O
-4	O
-and	O
-16	O
-.	O
-2	O
-kDa	O
-,	O
-related	O
-to	O
-the	O
-observed	O
-cleavage	O
-at	O
-Lys147	O
-in	O
-the	O
-crystal	O
-structure	O
-(	O
-Fig	O
-.	O
-2A	O
-).	O
-	O
-Incubation	O
-of	O
-PmC11	O
-at	O
-37	O
-°	O
-C	O
-for	O
-16	O
-h	O
-,	O
-resulted	O
-in	O
-a	O
-fully	O
-processed	O
-enzyme	O
-that	O
-remained	O
-as	O
-an	O
-intact	O
-monomer	O
-when	O
-applied	O
-to	O
-a	O
-size	O
--	O
-exclusion	O
-column	O
-(	O
-Fig	O
-.	O
-2B	O
-).	O
-	O
-The	O
-single	O
-cleavage	O
-site	O
-of	O
-PmC11	O
-at	O
-Lys147	O
-is	O
-found	O
-immediately	O
-after	O
-α3	O
-,	O
-in	O
-loop	O
-L5	O
-within	O
-the	O
-central	O
-β	O
--	O
-sheet	O
-(	O
-Figs	O
-.	O
-1	O
-,	O
-A	O
-and	O
-B	O
-,	O
-and	O
-2A	O
-).	O
-	O
-The	O
-two	O
-ends	O
-of	O
-the	O
-cleavage	O
-site	O
-are	O
-remarkably	O
-well	O
-ordered	O
-in	O
-the	O
-crystal	O
-structure	O
-and	O
-displaced	O
-from	O
-one	O
-another	O
-by	O
-19	O
-.	O
-5	O
-Å	O
-(	O
-Fig	O
-.	O
-2A	O
-).	O
-	O
-Moreover	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-side	O
-of	O
-the	O
-cleavage	O
-site	O
-resides	O
-near	O
-the	O
-catalytic	O
-dyad	O
-with	O
-Ala148	O
-being	O
-4	O
-.	O
-5	O
-and	O
-5	O
-.	O
-7	O
-Å	O
-from	O
-His133	O
-and	O
-Cys179	O
-,	O
-respectively	O
-.	O
-	O
-Consequently	O
-,	O
-it	O
-appears	O
-feasible	O
-that	O
-the	O
-helix	O
-attached	O
-to	O
-Lys147	O
-(	O
-α3	O
-)	O
-could	O
-be	O
-responsible	O
-for	O
-steric	O
-autoinhibition	O
-of	O
-PmC11	O
-when	O
-Lys147	O
-is	O
-covalently	O
-bonded	O
-to	O
-Ala148	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-cleavage	O
-would	O
-be	O
-required	O
-for	O
-full	O
-activation	O
-of	O
-PmC11	O
-.	O
-	O
-To	O
-investigate	O
-this	O
-possibility	O
-,	O
-two	O
-mutant	O
-forms	O
-of	O
-the	O
-enzyme	O
-were	O
-created	O
-:	O
-PmC11C179A	B-mutant
-(	O
-a	O
-catalytically	O
-inactive	O
-mutant	O
-)	O
-and	O
-PmC11K147A	B-mutant
-(	O
-a	O
-cleavage	O
--	O
-site	O
-mutant	O
-).	O
-	O
-Initial	O
-SDS	O
--	O
-PAGE	O
-and	O
-Western	O
-blot	O
-analysis	O
-of	O
-both	O
-mutants	O
-revealed	O
-no	O
-discernible	O
-processing	O
-occurred	O
-as	O
-compared	O
-with	O
-active	O
-PmC11	O
-(	O
-Fig	O
-.	O
-2C	O
-).	O
-	O
-The	O
-PmC11K147A	B-mutant
-mutant	O
-enzyme	O
-had	O
-a	O
-markedly	O
-different	O
-reaction	O
-rate	O
-(	O
-Vmax	O
-)	O
-compared	O
-with	O
-WT	O
-,	O
-where	O
-the	O
-reaction	O
-velocity	O
-of	O
-PmC11	O
-was	O
-10	O
-times	O
-greater	O
-than	O
-that	O
-of	O
-PmC11K147A	B-mutant
-(	O
-Fig	O
-.	O
-2D	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-these	O
-data	O
-reveal	O
-that	O
-PmC11	O
-requires	O
-processing	O
-at	O
-Lys147	O
-for	O
-optimum	O
-activity	O
-.	O
-	O
-To	O
-investigate	O
-whether	O
-processing	O
-is	O
-a	O
-result	O
-of	O
-intra	O
--	O
-or	O
-intermolecular	O
-cleavage	O
-,	O
-the	O
-PmC11C179A	B-mutant
-mutant	O
-was	O
-incubated	O
-with	O
-increasing	O
-concentrations	O
-of	O
-processed	O
-and	O
-activated	O
-PmC11	O
-.	O
-	O
-These	O
-studies	O
-revealed	O
-that	O
-there	O
-was	O
-no	O
-apparent	O
-cleavage	O
-of	O
-PmC11C179A	B-mutant
-by	O
-the	O
-active	O
-enzyme	O
-at	O
-low	O
-concentrations	O
-of	O
-PmC11	O
-and	O
-that	O
-only	O
-limited	O
-cleavage	O
-was	O
-observed	O
-when	O
-the	O
-ratio	O
-of	O
-active	O
-enzyme	O
-(	O
-PmC11	O
-:	O
-PmC11C179A	B-mutant
-)	O
-was	O
-increased	O
-to	O
-∼	O
-1	O
-:	O
-10	O
-and	O
-1	O
-:	O
-4	O
-,	O
-with	O
-complete	O
-cleavage	O
-observed	O
-at	O
-a	O
-ratio	O
-of	O
-1	O
-:	O
-1	O
-(	O
-Fig	O
-.	O
-2E	O
-).	O
-	O
-This	O
-suggests	O
-that	O
-cleavage	O
-of	O
-PmC11C179A	B-mutant
-was	O
-most	O
-likely	O
-an	O
-effect	O
-of	O
-the	O
-increasing	O
-concentration	O
-of	O
-PmC11	O
-and	O
-intermolecular	O
-cleavage	O
-.	O
-	O
-Collectively	O
-,	O
-these	O
-data	O
-suggest	O
-that	O
-the	O
-pro	O
--	O
-form	O
-of	O
-PmC11	O
-is	O
-autoinhibited	O
-by	O
-a	O
-section	O
-of	O
-L5	O
-blocking	O
-access	O
-to	O
-the	O
-active	O
-site	O
-,	O
-prior	O
-to	O
-intramolecular	O
-cleavage	O
-at	O
-Lys147	O
-.	O
-	O
-This	O
-cleavage	O
-subsequently	O
-allows	O
-movement	O
-of	O
-the	O
-region	O
-containing	O
-Lys147	O
-and	O
-the	O
-active	O
-site	O
-to	O
-open	O
-up	O
-for	O
-substrate	O
-access	O
-.	O
-	O
-Substrate	O
-Specificity	O
-of	O
-PmC11	O
-	O
-The	O
-autocatalytic	O
-cleavage	O
-of	O
-PmC11	O
-at	O
-Lys147	O
-(	O
-sequence	O
-KLK	O
-∧	O
-A	O
-)	O
-demonstrates	O
-that	O
-the	O
-enzyme	O
-accepts	O
-substrates	O
-with	O
-Lys	O
-in	O
-the	O
-P1	O
-position	O
-.	O
-	O
-As	O
-expected	O
-,	O
-PmC11	O
-showed	O
-no	O
-activity	O
-against	O
-substrates	O
-with	O
-Pro	O
-or	O
-Asp	O
-in	O
-P1	O
-but	O
-was	O
-active	O
-toward	O
-substrates	O
-with	O
-a	O
-basic	O
-residue	O
-in	O
-P1	O
-such	O
-as	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-,	O
-Z	O
--	O
-GGR	O
--	O
-AMC	O
-,	O
-and	O
-BOC	O
--	O
-VLK	O
--	O
-AMC	O
-.	O
-	O
-The	O
-rate	O
-of	O
-cleavage	O
-was	O
-∼	O
-3	O
--	O
-fold	O
-greater	O
-toward	O
-the	O
-single	O
-Arg	O
-substrate	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-than	O
-for	O
-the	O
-other	O
-two	O
-(	O
-Fig	O
-.	O
-2F	O
-)	O
-and	O
-,	O
-unexpectedly	O
-,	O
-PmC11	O
-showed	O
-no	O
-activity	O
-toward	O
-BOC	O
--	O
-K	O
--	O
-AMC	O
-.	O
-	O
-These	O
-results	O
-confirm	O
-that	O
-PmC11	O
-accepts	O
-substrates	O
-containing	O
-Arg	O
-or	O
-Lys	O
-in	O
-P1	O
-with	O
-a	O
-possible	O
-preference	O
-for	O
-Arg	O
-.	O
-	O
-The	O
-catalytic	O
-dyad	O
-of	O
-PmC11	O
-sits	O
-near	O
-the	O
-bottom	O
-of	O
-an	O
-open	O
-pocket	O
-on	O
-the	O
-surface	O
-of	O
-the	O
-enzyme	O
-at	O
-a	O
-conserved	O
-location	O
-in	O
-the	O
-clan	O
-CD	O
-family	O
-.	O
-	O
-The	O
-PmC11	O
-structure	O
-reveals	O
-that	O
-the	O
-catalytic	O
-dyad	O
-forms	O
-part	O
-of	O
-a	O
-large	O
-acidic	O
-pocket	O
-(	O
-Fig	O
-.	O
-2G	O
-),	O
-consistent	O
-with	O
-a	O
-binding	O
-site	O
-for	O
-a	O
-basic	O
-substrate	O
-.	O
-	O
-This	O
-pocket	O
-is	O
-lined	O
-with	O
-the	O
-potential	O
-functional	O
-side	O
-chains	O
-of	O
-Asn50	O
-,	O
-Asp177	O
-,	O
-and	O
-Thr204	O
-with	O
-Gly134	O
-,	O
-Asp207	O
-,	O
-and	O
-Met205	O
-also	O
-contributing	O
-to	O
-the	O
-pocket	O
-(	O
-Fig	O
-.	O
-2A	O
-).	O
-	O
-Interestingly	O
-,	O
-these	O
-residues	O
-are	O
-in	O
-regions	O
-that	O
-are	O
-structurally	O
-similar	O
-to	O
-those	O
-involved	O
-in	O
-the	O
-S1	O
-binding	O
-pockets	O
-of	O
-other	O
-clan	O
-CD	O
-members	O
-(	O
-shown	O
-in	O
-Ref	O
-.).	O
-	O
-Because	O
-PmC11	O
-recognizes	O
-basic	O
-substrates	O
-,	O
-the	O
-tetrapeptide	O
-inhibitor	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-was	O
-tested	O
-as	O
-an	O
-enzyme	O
-inhibitor	O
-and	O
-was	O
-found	O
-to	O
-inhibit	O
-both	O
-the	O
-autoprocessing	O
-and	O
-activity	O
-of	O
-PmC11	O
-(	O
-Fig	O
-.	O
-3A	O
-).	O
-	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-was	O
-also	O
-shown	O
-to	O
-bind	O
-to	O
-the	O
-enzyme	O
-:	O
-a	O
-size	O
--	O
-shift	O
-was	O
-observed	O
-,	O
-by	O
-SDS	O
--	O
-PAGE	O
-analysis	O
-,	O
-in	O
-the	O
-larger	O
-processed	O
-product	O
-of	O
-PmC11	O
-suggesting	O
-that	O
-the	O
-inhibitor	O
-bound	O
-to	O
-the	O
-active	O
-site	O
-(	O
-Fig	O
-.	O
-3B	O
-).	O
-	O
-A	O
-structure	O
-overlay	O
-of	O
-PmC11	O
-with	O
-the	O
-MALT1	O
--	O
-paracacaspase	O
-(	O
-MALT1	O
--	O
-P	O
-),	O
-in	O
-complex	O
-with	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-,	O
-revealed	O
-that	O
-the	O
-PmC11	O
-dyad	O
-sits	O
-in	O
-a	O
-very	O
-similar	O
-position	O
-to	O
-that	O
-of	O
-active	O
-MALT1	O
--	O
-P	O
-and	O
-that	O
-Asn50	O
-,	O
-Asp177	O
-,	O
-and	O
-Asp207	O
-superimpose	O
-well	O
-with	O
-the	O
-principal	O
-MALT1	O
--	O
-P	O
-inhibitor	O
-binding	O
-residues	O
-(	O
-Asp365	O
-,	O
-Asp462	O
-,	O
-and	O
-Glu500	O
-,	O
-respectively	O
-(	O
-VRPR	O
--	O
-FMK	O
-from	O
-MALT1	O
--	O
-P	O
-with	O
-the	O
-corresponding	O
-PmC11	O
-residues	O
-from	O
-the	O
-structural	O
-overlay	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-1D	O
-),	O
-as	O
-described	O
-in	O
-Ref	O
-.).	O
-	O
-Asp177	O
-is	O
-located	O
-near	O
-the	O
-catalytic	O
-cysteine	O
-and	O
-is	O
-conserved	O
-throughout	O
-the	O
-C11	O
-family	O
-,	O
-suggesting	O
-it	O
-is	O
-the	O
-primary	O
-S1	O
-binding	O
-site	O
-residue	O
-.	O
-	O
-In	O
-the	O
-structure	O
-of	O
-PmC11	O
-,	O
-Asp207	O
-resides	O
-on	O
-a	O
-flexible	O
-loop	O
-pointing	O
-away	O
-from	O
-the	O
-S1	O
-binding	O
-pocket	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-However	O
-,	O
-this	O
-loop	O
-has	O
-been	O
-shown	O
-to	O
-be	O
-important	O
-for	O
-substrate	O
-binding	O
-in	O
-clan	O
-CD	O
-and	O
-this	O
-residue	O
-could	O
-easily	O
-rotate	O
-and	O
-be	O
-involved	O
-in	O
-substrate	O
-binding	O
-in	O
-PmC11	O
-.	O
-	O
-Thus	O
-,	O
-Asn50	O
-,	O
-Asp177	O
-,	O
-and	O
-Asp207	O
-are	O
-most	O
-likely	O
-responsible	O
-for	O
-the	O
-substrate	O
-specificity	O
-of	O
-PmC11	O
-.	O
-	O
-Asp177	O
-is	O
-highly	O
-conserved	O
-throughout	O
-the	O
-clan	O
-CD	O
-C11	O
-peptidases	O
-and	O
-is	O
-thought	O
-to	O
-be	O
-primarily	O
-responsible	O
-for	O
-substrate	O
-specificity	O
-of	O
-the	O
-clan	O
-CD	O
-enzymes	O
-,	O
-as	O
-also	O
-illustrated	O
-from	O
-the	O
-proximity	O
-of	O
-these	O
-residues	O
-relative	O
-to	O
-the	O
-inhibitor	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-when	O
-PmC11	O
-is	O
-overlaid	O
-on	O
-the	O
-MALT1	O
--	O
-P	O
-structure	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-PmC11	O
-binds	O
-and	O
-is	O
-inhibited	O
-by	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-and	O
-does	O
-not	O
-require	O
-Ca2	O
-+	O
-for	O
-activity	O
-.	O
-	O
-A	O
-,	O
-PmC11	O
-activity	O
-is	O
-inhibited	O
-by	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-.	O
-	O
-Cleavage	O
-of	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-by	O
-PmC11	O
-was	O
-measured	O
-in	O
-a	O
-fluorometric	O
-activity	O
-assay	O
-with	O
-(+,	O
-purple	O
-)	O
-and	O
-without	O
-(−,	O
-red	O
-)	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-.	O
-	O
-B	O
-,	O
-gel	O
--	O
-shift	O
-assay	O
-reveals	O
-that	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-binds	O
-to	O
-PmC11	O
-.	O
-	O
-PmC11	O
-was	O
-incubated	O
-with	O
-(+)	O
-or	O
-without	O
-(−)	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-and	O
-the	O
-samples	O
-analyzed	O
-on	O
-a	O
-10	O
-%	O
-SDS	O
--	O
-PAGE	O
-gel	O
-.	O
-	O
-A	O
-size	O
-shift	O
-can	O
-be	O
-observed	O
-in	O
-the	O
-larger	O
-processed	O
-product	O
-of	O
-PmC11	O
-(	O
-26	O
-.	O
-1	O
-kDa	O
-).	O
-	O
-C	O
-,	O
-PmC11	O
-with	O
-the	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-from	O
-the	O
-MALT1	O
--	O
-paracacaspase	O
-(	O
-MALT1	O
--	O
-P	O
-)	O
-superimposed	O
-.	O
-	O
-A	O
-three	O
--	O
-dimensional	O
-structural	O
-overlay	O
-of	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-from	O
-the	O
-MALT1	O
--	O
-P	O
-complex	O
-onto	O
-PmC11	O
-.	O
-	O
-The	O
-position	O
-and	O
-orientation	O
-of	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-was	O
-taken	O
-from	O
-superposition	O
-of	O
-the	O
-PmC11	O
-and	O
-MALTI_P	O
-structures	O
-and	O
-indicates	O
-the	O
-presumed	O
-active	O
-site	O
-of	O
-PmC11	O
-.	O
-	O
-Residues	O
-surrounding	O
-the	O
-inhibitor	O
-are	O
-labeled	O
-and	O
-represent	O
-potentially	O
-important	O
-binding	O
-site	O
-residues	O
-,	O
-labeled	O
-in	O
-black	O
-and	O
-shown	O
-in	O
-an	O
-atomic	O
-representation	O
-.	O
-	O
-C	O
-,	O
-divalent	O
-cations	O
-do	O
-not	O
-increase	O
-the	O
-activity	O
-of	O
-PmC11	O
-.	O
-	O
-The	O
-cleavage	O
-of	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-by	O
-PmC11	O
-was	O
-measured	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-cations	O
-Ca2	O
-+,	O
-Mn2	O
-+,	O
-Zn2	O
-+,	O
-Co2	O
-+,	O
-Cu2	O
-+,	O
-Mg2	O
-+,	O
-and	O
-Fe3	O
-+	O
-with	O
-EGTA	O
-as	O
-a	O
-negative	O
-control	O
-,	O
-and	O
-relative	O
-fluorescence	O
-measured	O
-against	O
-time	O
-(	O
-min	O
-).	O
-	O
-The	O
-addition	O
-of	O
-cations	O
-produced	O
-no	O
-improvement	O
-in	O
-activity	O
-of	O
-PmC11	O
-when	O
-compared	O
-in	O
-the	O
-presence	O
-of	O
-EGTA	O
-,	O
-suggesting	O
-that	O
-PmC11	O
-does	O
-not	O
-require	O
-metal	O
-ions	O
-for	O
-proteolytic	O
-activity	O
-.	O
-	O
-Furthermore	O
-,	O
-Cu2	O
-+,	O
-Fe2	O
-+,	O
-and	O
-Zn2	O
-+	O
-appear	O
-to	O
-inhibit	O
-PmC11	O
-.	O
-	O
-Comparison	O
-with	O
-Clostripain	O
-	O
-Clostripain	O
-from	O
-C	O
-.	O
-histolyticum	O
-is	O
-the	O
-founding	O
-member	O
-of	O
-the	O
-C11	O
-family	O
-of	O
-peptidases	O
-and	O
-contains	O
-an	O
-additional	O
-149	O
-residues	O
-compared	O
-with	O
-PmC11	O
-.	O
-	O
-A	O
-multiple	O
-sequence	O
-alignment	O
-revealed	O
-that	O
-most	O
-of	O
-the	O
-secondary	O
-structural	O
-elements	O
-are	O
-conserved	O
-between	O
-the	O
-two	O
-enzymes	O
-,	O
-although	O
-they	O
-are	O
-only	O
-∼	O
-23	O
-%	O
-identical	O
-(	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-Nevertheless	O
-,	O
-PmC11	O
-may	O
-be	O
-a	O
-good	O
-model	O
-for	O
-the	O
-core	O
-structure	O
-of	O
-clostripain	O
-.	O
-	O
-The	O
-primary	O
-structural	O
-alignment	O
-also	O
-shows	O
-that	O
-the	O
-catalytic	O
-dyad	O
-in	O
-PmC11	O
-is	O
-structurally	O
-conserved	O
-in	O
-clostripain	O
-(	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-Unlike	O
-PmC11	O
-,	O
-clostripain	O
-has	O
-two	O
-cleavage	O
-sites	O
-(	O
-Arg181	O
-and	O
-Arg190	O
-),	O
-which	O
-results	O
-in	O
-the	O
-removal	O
-of	O
-a	O
-nonapeptide	O
-,	O
-and	O
-is	O
-required	O
-for	O
-full	O
-activation	O
-of	O
-the	O
-enzyme	O
-(	O
-highlighted	O
-in	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-Interestingly	O
-,	O
-Arg190	O
-was	O
-found	O
-to	O
-align	O
-with	O
-Lys147	O
-in	O
-PmC11	O
-.	O
-	O
-In	O
-addition	O
-,	O
-the	O
-predicted	O
-primary	O
-S1	O
--	O
-binding	O
-residue	O
-in	O
-PmC11	O
-Asp177	O
-also	O
-overlays	O
-with	O
-the	O
-residue	O
-predicted	O
-to	O
-be	O
-the	O
-P1	O
-specificity	O
-determining	O
-residue	O
-in	O
-clostripain	O
-(	O
-Asp229	O
-,	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-As	O
-studies	O
-on	O
-clostripain	O
-revealed	O
-addition	O
-of	O
-Ca2	O
-+	O
-ions	O
-are	O
-required	O
-for	O
-full	O
-activation	O
-,	O
-the	O
-Ca2	O
-+	O
-dependence	O
-of	O
-PmC11	O
-was	O
-examined	O
-.	O
-	O
-Surprisingly	O
-,	O
-Ca2	O
-+	O
-did	O
-not	O
-enhance	O
-PmC11	O
-activity	O
-and	O
-,	O
-furthermore	O
-,	O
-other	O
-divalent	O
-cations	O
-,	O
-Mg2	O
-+,	O
-Mn2	O
-+,	O
-Co2	O
-+,	O
-Fe2	O
-+,	O
-Zn2	O
-+,	O
-and	O
-Cu2	O
-+,	O
-were	O
-not	O
-necessary	O
-for	O
-PmC11	O
-activity	O
-(	O
-Fig	O
-.	O
-3D	O
-).	O
-	O
-In	O
-support	O
-of	O
-these	O
-findings	O
-,	O
-EGTA	O
-did	O
-not	O
-inhibit	O
-PmC11	O
-suggesting	O
-that	O
-,	O
-unlike	O
-clostripain	O
-,	O
-PmC11	O
-does	O
-not	O
-require	O
-Ca2	O
-+	O
-or	O
-other	O
-divalent	O
-cations	O
-,	O
-for	O
-activity	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-PmC11	O
-now	O
-provides	O
-three	O
--	O
-dimensional	O
-information	O
-for	O
-a	O
-member	O
-of	O
-the	O
-clostripain	O
-C11	O
-family	O
-of	O
-cysteine	O
-peptidases	O
-.	O
-	O
-The	O
-enzyme	O
-exhibits	O
-all	O
-of	O
-the	O
-key	O
-structural	O
-elements	O
-of	O
-clan	O
-CD	O
-members	O
-,	O
-but	O
-is	O
-unusual	O
-in	O
-that	O
-it	O
-has	O
-a	O
-nine	O
--	O
-stranded	O
-central	O
-β	O
--	O
-sheet	O
-with	O
-a	O
-novel	O
-C	O
--	O
-terminal	O
-domain	O
-.	O
-	O
-The	O
-structural	O
-similarity	O
-of	O
-PmC11	O
-with	O
-its	O
-nearest	O
-structural	O
-neighbors	O
-in	O
-the	O
-PDB	O
-is	O
-decidedly	O
-low	O
-,	O
-overlaying	O
-better	O
-with	O
-six	O
--	O
-stranded	O
-caspase	O
--	O
-7	O
-than	O
-any	O
-of	O
-the	O
-other	O
-larger	O
-members	O
-of	O
-the	O
-clan	O
-(	O
-Table	O
-2	O
-).	O
-	O
-The	O
-substrate	O
-specificity	O
-of	O
-PmC11	O
-is	O
-Arg	O
-/	O
-Lys	O
-and	O
-the	O
-crystal	O
-structure	O
-revealed	O
-an	O
-acidic	O
-pocket	O
-for	O
-specific	O
-binding	O
-of	O
-such	O
-basic	O
-substrates	O
-.	O
-	O
-In	O
-addition	O
-,	O
-the	O
-structure	O
-suggested	O
-a	O
-mechanism	O
-of	O
-self	O
--	O
-inhibition	O
-in	O
-both	O
-PmC11	O
-and	O
-clostripain	O
-and	O
-an	O
-activation	O
-mechanism	O
-that	O
-requires	O
-autoprocessing	O
-.	O
-	O
-PmC11	O
-differs	O
-from	O
-clostripain	O
-in	O
-that	O
-is	O
-does	O
-not	O
-appear	O
-to	O
-require	O
-divalent	O
-cations	O
-for	O
-activation	O
-.	O
-	O
-Several	O
-other	O
-members	O
-of	O
-clan	O
-CD	O
-require	O
-processing	O
-for	O
-full	O
-activation	O
-including	O
-legumain	O
-,	O
-gingipain	O
--	O
-R	O
-,	O
-MARTX	O
--	O
-CPD	O
-,	O
-and	O
-the	O
-effector	O
-caspases	O
-,	O
-e	O
-.	O
-g	O
-.	O
-caspase	O
--	O
-7	O
-.	O
-	O
-To	O
-date	O
-,	O
-the	O
-effector	O
-caspases	O
-are	O
-the	O
-only	O
-group	O
-of	O
-enzymes	O
-that	O
-require	O
-cleavage	O
-of	O
-a	O
-loop	O
-within	O
-the	O
-central	O
-β	O
--	O
-sheet	O
-.	O
-	O
-This	O
-is	O
-also	O
-the	O
-case	O
-in	O
-PmC11	O
-,	O
-although	O
-the	O
-cleavage	O
-loop	O
-is	O
-structurally	O
-different	O
-to	O
-that	O
-found	O
-in	O
-the	O
-caspases	O
-and	O
-follows	O
-the	O
-catalytic	O
-His	O
-(	O
-Fig	O
-.	O
-1A	O
-),	O
-as	O
-opposed	O
-to	O
-the	O
-Cys	O
-in	O
-the	O
-caspases	O
-.	O
-	O
-All	O
-other	O
-clan	O
-CD	O
-members	O
-requiring	O
-cleavage	O
-for	O
-full	O
-activation	O
-do	O
-so	O
-at	O
-sites	O
-external	O
-to	O
-their	O
-central	O
-sheets	O
-.	O
-	O
-The	O
-caspases	O
-and	O
-gingipain	O
--	O
-R	O
-both	O
-undergo	O
-intermolecular	O
-(	O
-trans	O
-)	O
-cleavage	O
-and	O
-legumain	O
-and	O
-MARTX	O
--	O
-CPD	O
-are	O
-reported	O
-to	O
-perform	O
-intramolecular	O
-(	O
-cis	O
-)	O
-cleavage	O
-.	O
-	O
-In	O
-addition	O
-,	O
-several	O
-members	O
-of	O
-clan	O
-CD	O
-exhibit	O
-self	O
--	O
-inhibition	O
-,	O
-whereby	O
-regions	O
-of	O
-the	O
-enzyme	O
-block	O
-access	O
-to	O
-the	O
-active	O
-site	O
-.	O
-	O
-Like	O
-PmC11	O
-,	O
-these	O
-structures	O
-show	O
-preformed	O
-catalytic	O
-machinery	O
-and	O
-,	O
-for	O
-a	O
-substrate	O
-to	O
-gain	O
-access	O
-,	O
-movement	O
-and	O
-/	O
-or	O
-cleavage	O
-of	O
-the	O
-blocking	O
-region	O
-is	O
-required	O
-.	O
-	O
-The	O
-structure	O
-of	O
-PmC11	O
-gives	O
-the	O
-first	O
-insight	O
-into	O
-this	O
-class	O
-of	O
-relatively	O
-unexplored	O
-family	O
-of	O
-proteins	O
-and	O
-should	O
-allow	O
-important	O
-catalytic	O
-and	O
-substrate	O
-binding	O
-residues	O
-to	O
-be	O
-identified	O
-in	O
-a	O
-variety	O
-of	O
-orthologues	O
-.	O
-	O
-Indeed	O
-,	O
-insights	O
-gained	O
-from	O
-an	O
-analysis	O
-of	O
-the	O
-PmC11	O
-structure	O
-revealed	O
-the	O
-identity	O
-of	O
-the	O
-Trypanosoma	O
-brucei	O
-PNT1	O
-protein	O
-as	O
-a	O
-C11	O
-cysteine	O
-peptidase	O
-with	O
-an	O
-essential	O
-role	O
-in	O
-organelle	O
-replication	O
-.	O
-	O
-The	O
-PmC11	O
-structure	O
-should	O
-provide	O
-a	O
-good	O
-basis	O
-for	O
-structural	O
-modeling	O
-and	O
-,	O
-given	O
-the	O
-importance	O
-of	O
-other	O
-clan	O
-CD	O
-enzymes	O
-,	O
-this	O
-work	O
-should	O
-also	O
-advance	O
-the	O
-exploration	O
-of	O
-these	O
-peptidases	O
-and	O
-potentially	O
-identify	O
-new	O
-biologically	O
-important	O
-substrates	O
-.	O
-	O
-Ribosome	O
-biogenesis	O
-factor	O
-Tsr3	O
-is	O
-the	O
-aminocarboxypropyl	O
-transferase	O
-responsible	O
-for	O
-18S	O
-rRNA	O
-hypermodification	O
-in	O
-yeast	O
-and	O
-humans	O
-	O
-The	O
-chemically	O
-most	O
-complex	O
-modification	O
-in	O
-eukaryotic	O
-rRNA	O
-is	O
-the	O
-conserved	O
-hypermodified	O
-nucleotide	O
-N1	O
--	O
-methyl	O
--	O
-N3	O
--	O
-aminocarboxypropyl	O
--	O
-pseudouridine	O
-(	O
-m1acp3Ψ	O
-)	O
-located	O
-next	O
-to	O
-the	O
-P	O
--	O
-site	O
-tRNA	O
-on	O
-the	O
-small	O
-subunit	O
-18S	O
-rRNA	O
-.	O
-	O
-While	O
-S	O
--	O
-adenosylmethionine	O
-was	O
-identified	O
-as	O
-the	O
-source	O
-of	O
-the	O
-aminocarboxypropyl	O
-(	O
-acp	O
-)	O
-group	O
-more	O
-than	O
-40	O
-years	O
-ago	O
-the	O
-enzyme	O
-catalyzing	O
-the	O
-acp	O
-transfer	O
-remained	O
-elusive	O
-.	O
-	O
-Here	O
-we	O
-identify	O
-the	O
-cytoplasmic	O
-ribosome	O
-biogenesis	O
-protein	O
-Tsr3	O
-as	O
-the	O
-responsible	O
-enzyme	O
-in	O
-yeast	O
-and	O
-human	O
-cells	O
-.	O
-	O
-In	O
-functionally	O
-impaired	O
-Tsr3	O
--	O
-mutants	O
-,	O
-a	O
-reduced	O
-level	O
-of	O
-acp	O
-modification	O
-directly	O
-correlates	O
-with	O
-increased	O
-20S	O
-pre	O
--	O
-rRNA	O
-accumulation	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-archaeal	O
-Tsr3	O
-homologs	O
-revealed	O
-the	O
-same	O
-fold	O
-as	O
-in	O
-SPOUT	O
--	O
-class	O
-RNA	O
--	O
-methyltransferases	O
-but	O
-a	O
-distinct	O
-SAM	O
-binding	O
-mode	O
-.	O
-	O
-This	O
-unique	O
-SAM	O
-binding	O
-mode	O
-explains	O
-why	O
-Tsr3	O
-transfers	O
-the	O
-acp	O
-and	O
-not	O
-the	O
-methyl	O
-group	O
-of	O
-SAM	O
-to	O
-its	O
-substrate	O
-.	O
-	O
-Structurally	O
-,	O
-Tsr3	O
-therefore	O
-represents	O
-a	O
-novel	O
-class	O
-of	O
-acp	O
-transferase	O
-enzymes	O
-.	O
-	O
-Eukaryotic	O
-ribosome	O
-biogenesis	O
-is	O
-highly	O
-complex	O
-and	O
-requires	O
-a	O
-large	O
-number	O
-of	O
-non	O
--	O
-ribosomal	O
-proteins	O
-and	O
-small	O
-non	O
--	O
-coding	O
-RNAs	O
-in	O
-addition	O
-to	O
-ribosomal	O
-RNAs	O
-(	O
-rRNAs	O
-)	O
-and	O
-proteins	O
-.	O
-	O
-During	O
-eukaryotic	O
-ribosome	O
-biogenesis	O
-several	O
-dozens	O
-of	O
-rRNA	O
-nucleotides	O
-become	O
-chemically	O
-modified	O
-.	O
-	O
-The	O
-most	O
-abundant	O
-rRNA	O
-modifications	O
-are	O
-methylations	O
-at	O
-the	O
-2	O
-′-	O
-OH	O
-ribose	O
-moieties	O
-and	O
-isomerizations	O
-of	O
-uridine	O
-residues	O
-to	O
-pseudouridine	O
-,	O
-catalyzed	O
-by	O
-small	O
-nucleolar	O
-ribonucleoprotein	O
-particles	O
-(	O
-snoRNPs	O
-).	O
-	O
-In	O
-addition	O
-,	O
-18S	O
-and	O
-25S	O
-(	O
-yeast	O
-)/	O
-28S	O
-(	O
-humans	O
-)	O
-rRNAs	O
-contain	O
-several	O
-base	O
-modifications	O
-catalyzed	O
-by	O
-site	O
--	O
-specific	O
-and	O
-snoRNA	O
--	O
-independent	O
-enzymes	O
-.	O
-	O
-In	O
-Saccharomyces	O
-cerevisiae	O
-18S	O
-rRNA	O
-contains	O
-four	O
-base	O
-methylations	O
-,	O
-two	O
-acetylations	O
-and	O
-a	O
-single	O
-3	O
--	O
-amino	O
--	O
-3	O
--	O
-carboxypropyl	O
-(	O
-acp	O
-)	O
-modification	O
-,	O
-whereas	O
-six	O
-base	O
-methylations	O
-are	O
-present	O
-in	O
-the	O
-25S	O
-rRNA	O
-.	O
-	O
-While	O
-in	O
-humans	O
-the	O
-18S	O
-rRNA	O
-base	O
-modifications	O
-are	O
-highly	O
-conserved	O
-,	O
-only	O
-three	O
-of	O
-the	O
-yeast	O
-base	O
-modifications	O
-catalyzed	O
-by	O
-ScRrp8	O
-/	O
-HsNML	O
-,	O
-ScRcm1	O
-/	O
-HsNSUN5	O
-and	O
-ScNop2	O
-/	O
-HsNSUN1	O
-are	O
-preserved	O
-in	O
-the	O
-corresponding	O
-human	O
-28S	O
-rRNA	O
-.	O
-	O
-Ribosomal	O
-RNA	O
-modifications	O
-have	O
-been	O
-suggested	O
-to	O
-optimize	O
-ribosome	O
-function	O
-,	O
-although	O
-in	O
-most	O
-cases	O
-this	O
-remains	O
-to	O
-be	O
-clearly	O
-established	O
-.	O
-	O
-They	O
-might	O
-contribute	O
-to	O
-increased	O
-RNA	O
-stability	O
-by	O
-providing	O
-additional	O
-hydrogen	O
-bonds	O
-(	O
-pseudouridines	O
-),	O
-improved	O
-base	O
-stacking	O
-(	O
-pseudouridines	O
-and	O
-base	O
-methylations	O
-)	O
-or	O
-an	O
-increased	O
-resistance	O
-against	O
-hydrolysis	O
-(	O
-ribose	O
-methylations	O
-).	O
-	O
-Most	O
-modified	O
-rRNA	O
-nucleotides	O
-cluster	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-decoding	O
-or	O
-the	O
-peptidyl	O
-transferase	O
-center	O
-,	O
-suggesting	O
-an	O
-influence	O
-on	O
-ribosome	O
-functionality	O
-and	O
-stability	O
-.	O
-	O
-Defects	O
-of	O
-rRNA	O
-modification	O
-enzymes	O
-often	O
-lead	O
-to	O
-disturbed	O
-ribosome	O
-biogenesis	O
-or	O
-functionally	O
-impaired	O
-ribosomes	O
-,	O
-although	O
-the	O
-lack	O
-of	O
-individual	O
-rRNA	O
-modifications	O
-often	O
-has	O
-no	O
-or	O
-only	O
-a	O
-slight	O
-influence	O
-on	O
-the	O
-cell	O
-.	O
-	O
-The	O
-chemically	O
-most	O
-complex	O
-modification	O
-is	O
-located	O
-in	O
-the	O
-loop	O
-capping	O
-helix	O
-31	O
-of	O
-18S	O
-rRNA	O
-(	O
-Supplementary	O
-Figure	O
-S1B	O
-).	O
-	O
-There	O
-a	O
-uridine	O
-(	O
-U1191	O
-in	O
-yeast	O
-)	O
-is	O
-modified	O
-to	O
-1	O
--	O
-methyl	O
--	O
-3	O
--(	O
-3	O
--	O
-amino	O
--	O
-3	O
--	O
-carboxypropyl	O
-)-	O
-pseudouridine	O
-(	O
-m1acp3Ψ	O
-,	O
-Figure	O
-1A	O
-).	O
-	O
-This	O
-base	O
-modification	O
-was	O
-first	O
-described	O
-in	O
-1968	O
-for	O
-hamster	O
-cells	O
-and	O
-is	O
-conserved	O
-in	O
-eukaryotes	O
-.	O
-	O
-This	O
-hypermodified	O
-nucleotide	O
-,	O
-which	O
-is	O
-located	O
-at	O
-the	O
-P	O
--	O
-site	O
-tRNA	O
-,	O
-is	O
-synthesized	O
-in	O
-three	O
-steps	O
-beginning	O
-with	O
-the	O
-snR35	O
-H	O
-/	O
-ACA	O
-snoRNP	O
-guided	O
-conversion	O
-of	O
-uridine	O
-into	O
-pseudouridine	O
-.	O
-	O
-In	O
-a	O
-second	O
-step	O
-,	O
-the	O
-essential	O
-SPOUT	O
--	O
-class	O
-methyltransferase	O
-Nep1	O
-/	O
-Emg1	O
-modifies	O
-the	O
-pseudouridine	O
-to	O
-N1	O
--	O
-methylpseudouridine	O
-.	O
-	O
-Methylation	O
-can	O
-only	O
-occur	O
-once	O
-pseudouridylation	O
-has	O
-taken	O
-place	O
-,	O
-as	O
-the	O
-latter	O
-reaction	O
-generates	O
-the	O
-substrate	O
-for	O
-the	O
-former	O
-.	O
-	O
-The	O
-final	O
-acp	O
-modification	O
-leading	O
-to	O
-N1	O
--	O
-methyl	O
--	O
-N3	O
--	O
-aminocarboxypropyl	O
--	O
-pseudouridine	O
-occurs	O
-late	O
-during	O
-40S	O
-biogenesis	O
-in	O
-the	O
-cytoplasm	O
-,	O
-while	O
-the	O
-two	O
-former	O
-reactions	O
-are	O
-taking	O
-place	O
-in	O
-the	O
-nucleolus	O
-and	O
-nucleus	O
-,	O
-and	O
-is	O
-independent	O
-from	O
-pseudouridylation	O
-or	O
-methylation	O
-.	O
-	O
-Both	O
-the	O
-methyl	O
-and	O
-the	O
-acp	O
-group	O
-are	O
-derived	O
-from	O
-S	O
--	O
-adenosylmethionine	O
-(	O
-SAM	O
-),	O
-but	O
-the	O
-enzyme	O
-responsible	O
-for	O
-acp	O
-modification	O
-remained	O
-elusive	O
-for	O
-more	O
-than	O
-40	O
-years	O
-.	O
-	O
-Tsr3	O
-is	O
-necessary	O
-for	O
-acp	O
-modification	O
-of	O
-18S	O
-rRNA	O
-in	O
-yeast	O
-and	O
-human	O
-.	O
-(	O
-A	O
-)	O
-Hypermodified	O
-nucleotide	O
-m1acp3Ψ	O
-is	O
-synthesized	O
-in	O
-three	O
-steps	O
-:	O
-pseudouridylation	O
-catalyzed	O
-by	O
-snoRNP35	O
-,	O
-N1	O
--	O
-methylation	O
-catalyzed	O
-by	O
-methyltransferase	O
-Nep1	O
-and	O
-N3	O
--	O
-acp	O
-modification	O
-catalyzed	O
-by	O
-Tsr3	O
-.	O
-	O
-The	O
-asterisk	O
-indicates	O
-the	O
-C1	O
--	O
-atom	O
-labeled	O
-in	O
-the	O
-14C	O
--	O
-incorporation	O
-assay	O
-.	O
-	O
-(	O
-B	O
-)	O
-RP	O
--	O
-HPLC	O
-elution	O
-profile	O
-of	O
-yeast	O
-18S	O
-rRNA	O
-nucleosides	O
-.	O
-	O
-Hypermodified	O
-m1acp3Ψ	O
-elutes	O
-at	O
-7	O
-.	O
-4	O
-min	O
-(	O
-wild	O
-type	O
-,	O
-left	O
-profile	O
-)	O
-and	O
-is	O
-missing	O
-in	O
-Δtsr3	B-mutant
-(	O
-middle	O
-profile	O
-)	O
-and	O
-Δnep1	B-mutant
-Δnop6	I-mutant
-mutants	O
-(	O
-right	O
-profile	O
-).	O
-	O
-(	O
-C	O
-)	O
-14C	O
--	O
-acp	O
-labeling	O
-of	O
-18S	O
-rRNAs	O
-.	O
-	O
-Wild	O
-type	O
-(	O
-WT	O
-)	O
-and	O
-plasmid	O
-encoded	O
-18S	O
-rRNA	O
-(	O
-U1191U	B-mutant
-)	O
-show	O
-the	O
-14C	O
--	O
-acp	O
-signal	O
-,	O
-whereas	O
-the	O
-14C	O
--	O
-acp	O
-signal	O
-is	O
-missing	O
-in	O
-the	O
-U1191A	B-mutant
-mutant	O
-plasmid	O
-encoded	O
-18S	O
-rRNA	O
-(	O
-U1191A	B-mutant
-)	O
-and	O
-Δtsr3	B-mutant
-mutants	O
-(	O
-Δtsr3	B-mutant
-).	O
-	O
-Upper	O
-lanes	O
-show	O
-the	O
-ethidium	O
-bromide	O
-staining	O
-of	O
-the	O
-18S	O
-rRNAs	O
-for	O
-quantification	O
-.	O
-	O
-All	O
-samples	O
-were	O
-loaded	O
-on	O
-the	O
-gel	O
-with	O
-two	O
-different	O
-amounts	O
-of	O
-5	O
-and	O
-10	O
-μl	O
-.	O
-(	O
-D	O
-)	O
-Primer	O
-extension	O
-analysis	O
-of	O
-acp	O
-modification	O
-in	O
-yeast	O
-18S	O
-rRNA	O
-(	O
-right	O
-gel	O
-)	O
-including	O
-a	O
-sequencing	O
-ladder	O
-(	O
-left	O
-gel	O
-).	O
-	O
-The	O
-primer	O
-extension	O
-stop	O
-at	O
-nucleotide	O
-1191	O
-is	O
-missing	O
-exclusively	O
-in	O
-Δtsr3	B-mutant
-mutants	O
-and	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-recombinants	O
-.	O
-	O
-(	O
-E	O
-)	O
-Primer	O
-extension	O
-analysis	O
-of	O
-human	O
-18S	O
-rRNA	O
-after	O
-siRNA	O
-knockdown	O
-of	O
-HsNEP1	O
-/	O
-EMG1	O
-(	O
-541	O
-,	O
-542	O
-and	O
-543	O
-)	O
-and	O
-HsTSR3	O
-(	O
-544	O
-and	O
-545	O
-)	O
-(	O
-right	O
-gel	O
-),	O
-including	O
-a	O
-sequencing	O
-ladder	O
-(	O
-left	O
-gel	O
-).	O
-	O
-The	O
-primer	O
-extension	O
-arrest	O
-is	O
-reduced	O
-in	O
-HTC116	O
-cells	O
-transfected	O
-with	O
-siRNAs	O
-544	O
-and	O
-545	O
-.	O
-	O
-The	O
-efficiency	O
-of	O
-siRNA	O
-mediated	O
-HsTSR3	O
-repression	O
-correlates	O
-with	O
-the	O
-primer	O
-extension	O
-signals	O
-(	O
-see	O
-Supplementary	O
-Figure	O
-S2A	O
-).	O
-	O
-Only	O
-a	O
-few	O
-acp	O
-transferring	O
-enzymes	O
-have	O
-been	O
-characterized	O
-until	O
-now	O
-.	O
-	O
-During	O
-the	O
-biosynthesis	O
-of	O
-wybutosine	O
-,	O
-a	O
-tricyclic	O
-nucleoside	O
-present	O
-in	O
-eukaryotic	O
-and	O
-archaeal	O
-phenylalanine	O
-tRNA	O
-,	O
-Tyw2	O
-(	O
-Trm12	O
-in	O
-yeast	O
-)	O
-transfers	O
-an	O
-acp	O
-group	O
-from	O
-SAM	O
-to	O
-an	O
-acidic	O
-carbon	O
-atom	O
-.	O
-	O
-Archaeal	O
-Tyw2	O
-has	O
-a	O
-structure	O
-very	O
-similar	O
-to	O
-Rossmann	O
--	O
-fold	O
-(	O
-class	O
-I	O
-)	O
-RNA	O
--	O
-methyltransferases	O
-,	O
-but	O
-its	O
-distinctive	O
-SAM	O
--	O
-binding	O
-mode	O
-enables	O
-the	O
-transfer	O
-of	O
-the	O
-acp	O
-group	O
-instead	O
-of	O
-the	O
-methyl	O
-group	O
-of	O
-the	O
-cofactor	O
-.	O
-	O
-Another	O
-acp	O
-modification	O
-has	O
-been	O
-described	O
-in	O
-the	O
-diphtamide	O
-biosynthesis	O
-pathway	O
-,	O
-where	O
-an	O
-acp	O
-group	O
-is	O
-transferred	O
-from	O
-SAM	O
-to	O
-the	O
-carbon	O
-atom	O
-of	O
-a	O
-histidine	O
-residue	O
-of	O
-eukaryotic	O
-translation	O
-elongation	O
-factor	O
-2	O
-by	O
-use	O
-of	O
-a	O
-radical	O
-mechanism	O
-.	O
-	O
-In	O
-a	O
-recent	O
-bioinformatic	O
-study	O
-,	O
-the	O
-uncharacterized	O
-yeast	O
-gene	O
-YOR006c	O
-was	O
-predicted	O
-to	O
-be	O
-involved	O
-in	O
-ribosome	O
-biogenesis	O
-.	O
-	O
-It	O
-is	O
-highly	O
-conserved	O
-among	O
-eukaryotes	O
-and	O
-archaea	O
-(	O
-Supplementary	O
-Figure	O
-S1A	O
-)	O
-and	O
-its	O
-deletion	O
-leads	O
-to	O
-an	O
-accumulation	O
-of	O
-the	O
-20S	O
-pre	O
--	O
-rRNA	O
-precursor	O
-of	O
-18S	O
-rRNA	O
-,	O
-suggesting	O
-an	O
-influence	O
-on	O
-D	O
--	O
-site	O
-cleavage	O
-during	O
-the	O
-maturation	O
-of	O
-the	O
-small	O
-ribosomal	O
-subunit	O
-.	O
-	O
-On	O
-this	O
-basis	O
-,	O
-YOR006C	O
-was	O
-renamed	O
-‘	O
-Twenty	O
-S	O
-rRNA	O
-accumulation	O
-3	O
-′	O
-(	O
-TSR3	O
-).	O
-	O
-However	O
-,	O
-its	O
-function	O
-remained	O
-unclear	O
-although	O
-recently	O
-a	O
-putative	O
-nuclease	O
-function	O
-during	O
-18S	O
-rRNA	O
-maturation	O
-was	O
-predicted	O
-.	O
-	O
-Here	O
-,	O
-we	O
-identify	O
-Tsr3	O
-as	O
-the	O
-long	O
--	O
-sought	O
-acp	O
-transferase	O
-that	O
-catalyzes	O
-the	O
-last	O
-step	O
-in	O
-the	O
-biosynthesis	O
-of	O
-the	O
-hypermodified	O
-nucleotide	O
-m1acp3Ψ	O
-in	O
-yeast	O
-and	O
-human	O
-cells	O
-.	O
-	O
-Furthermore	O
-using	O
-catalytically	O
-defective	O
-mutants	O
-of	O
-yeast	O
-Tsr3	O
-we	O
-demonstrated	O
-that	O
-the	O
-acp	O
-modification	O
-is	O
-required	O
-for	O
-18S	O
-rRNA	O
-maturation	O
-.	O
-	O
-Surprisingly	O
-,	O
-the	O
-crystal	O
-structures	O
-of	O
-archaeal	O
-homologs	O
-revealed	O
-that	O
-Tsr3	O
-is	O
-structurally	O
-similar	O
-to	O
-the	O
-SPOUT	O
--	O
-class	O
-RNA	O
-methyltransferases	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-only	O
-other	O
-structurally	O
-characterized	O
-acp	O
-transferase	O
-enzyme	O
-Tyw2	O
-belongs	O
-to	O
-the	O
-Rossmann	O
--	O
-fold	O
-class	O
-of	O
-methyltransferase	O
-proteins	O
-.	O
-	O
-Interestingly	O
-,	O
-the	O
-two	O
-structurally	O
-very	O
-different	O
-enzymes	O
-use	O
-similar	O
-strategies	O
-in	O
-binding	O
-the	O
-SAM	O
--	O
-cofactor	O
-in	O
-order	O
-to	O
-ensure	O
-that	O
-in	O
-contrast	O
-to	O
-methyltransferases	O
-the	O
-acp	O
-and	O
-not	O
-the	O
-methyl	O
-group	O
-of	O
-SAM	O
-is	O
-transferred	O
-to	O
-the	O
-substrate	O
-.	O
-	O
-Tsr3	O
-is	O
-the	O
-enzyme	O
-responsible	O
-for	O
-18S	O
-rRNA	O
-acp	O
-modification	O
-in	O
-yeast	O
-and	O
-humans	O
-	O
-The	O
-S	O
-.	O
-cerevisiae	O
-18S	O
-rRNA	O
-acp	O
-transferase	O
-was	O
-identified	O
-in	O
-a	O
-systematic	O
-genetic	O
-screen	O
-where	O
-numerous	O
-deletion	O
-mutants	O
-from	O
-the	O
-EUROSCARF	O
-strain	O
-collection	O
-(	O
-www	O
-.	O
-euroscarf	O
-.	O
-de	O
-)	O
-were	O
-analyzed	O
-by	O
-HPLC	O
-for	O
-alterations	O
-in	O
-18S	O
-rRNA	O
-base	O
-modifications	O
-.	O
-	O
-For	O
-the	O
-Δtsr3	B-mutant
-deletion	O
-strain	O
-the	O
-HPLC	O
-elution	O
-profile	O
-of	O
-18S	O
-rRNA	O
-nucleosides	O
-(	O
-Figure	O
-1B	O
-)	O
-was	O
-very	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-pseudouridine	O
--	O
-N1	O
-methyltransferase	O
-mutant	O
-Δnep1	B-mutant
-,	O
-where	O
-a	O
-shoulder	O
-at	O
-∼	O
-7	O
-.	O
-4	O
-min	O
-elution	O
-time	O
-was	O
-missing	O
-in	O
-the	O
-elution	O
-profile	O
-.	O
-	O
-As	O
-previously	O
-reported	O
-this	O
-shoulder	O
-was	O
-identified	O
-by	O
-ESI	O
--	O
-MS	O
-as	O
-corresponding	O
-to	O
-m1acp3Ψ	O
-.	O
-	O
-In	O
-order	O
-to	O
-directly	O
-analyze	O
-the	O
-presence	O
-of	O
-the	O
-acp	O
-modification	O
-of	O
-nucleotide	O
-1191	O
-we	O
-used	O
-an	O
-in	O
-vivo14C	O
-incorporation	O
-assay	O
-with	O
-1	O
--	O
-14C	O
--	O
-methionine	O
-.	O
-	O
-Whereas	O
-the	O
-acp	O
-labeling	O
-of	O
-18S	O
-rRNA	O
-was	O
-clearly	O
-present	O
-in	O
-the	O
-wild	O
-type	O
-strain	O
-no	O
-radioactive	O
-labeling	O
-could	O
-be	O
-observed	O
-in	O
-a	O
-Δtsr3	B-mutant
-strain	O
-(	O
-Figure	O
-1C	O
-).	O
-	O
-No	O
-radioactive	O
-labeling	O
-was	O
-detected	O
-in	O
-the	O
-18S	B-mutant
-U1191A	I-mutant
-mutant	O
-which	O
-served	O
-as	O
-a	O
-control	O
-for	O
-the	O
-specificity	O
-of	O
-the	O
-14C	O
--	O
-aminocarboxypropyl	O
-incorporation	O
-.	O
-	O
-As	O
-previously	O
-shown	O
-,	O
-only	O
-the	O
-acp	O
-but	O
-none	O
-of	O
-the	O
-other	O
-modifications	O
-at	O
-U1191	O
-of	O
-yeast	O
-18S	O
-rRNA	O
-blocks	O
-reverse	O
-transcriptase	O
-activity	O
-.	O
-	O
-Therefore	O
-the	O
-presence	O
-of	O
-the	O
-acp	O
-modification	O
-can	O
-be	O
-directly	O
-assessed	O
-by	O
-primer	O
-extension	O
-.	O
-	O
-Indeed	O
-,	O
-in	O
-wild	O
--	O
-type	O
-yeast	O
-a	O
-strong	O
-primer	O
-extension	O
-stop	O
-signal	O
-occurred	O
-at	O
-position	O
-1192	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-in	O
-a	O
-Δtsr3	B-mutant
-mutant	O
-no	O
-primer	O
-extension	O
-stop	O
-signal	O
-was	O
-present	O
-at	O
-this	O
-position	O
-.	O
-	O
-As	O
-expected	O
-,	O
-in	O
-a	O
-Δsnr35	B-mutant
-deletion	O
-preventing	O
-pseudouridylation	O
-and	O
-N1	O
--	O
-methylation	O
-(	O
-resulting	O
-in	O
-acp3U	O
-)	O
-as	O
-well	O
-as	O
-in	O
-a	O
-Δnep1	B-mutant
-deletion	O
-strain	O
-where	O
-pseudouridine	O
-is	O
-not	O
-methylated	O
-(	O
-resulting	O
-in	O
-acp3Ψ	O
-)	O
-a	O
-primer	O
-extension	O
-stop	O
-signal	O
-of	O
-similar	O
-intensity	O
-as	O
-in	O
-the	O
-wild	O
-type	O
-was	O
-observed	O
-.	O
-	O
-In	O
-a	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-double	O
-deletion	O
-strain	O
-the	O
-18S	O
-rRNA	O
-contains	O
-an	O
-unmodified	O
-U	O
-and	O
-the	O
-primer	O
-extension	O
-stop	O
-signal	O
-was	O
-missing	O
-(	O
-Figure	O
-1D	O
-).	O
-	O
-The	O
-Tsr3	O
-protein	O
-is	O
-highly	O
-conserved	O
-in	O
-yeast	O
-and	O
-humans	O
-(	O
-50	O
-%	O
-identity	O
-).	O
-	O
-Human	O
-18S	O
-rRNA	O
-has	O
-also	O
-been	O
-shown	O
-to	O
-contain	O
-m1acp3Ψ	O
-in	O
-the	O
-18S	O
-rRNA	O
-at	O
-position	O
-1248	O
-.	O
-	O
-After	O
-siRNA	O
--	O
-mediated	O
-depletion	O
-of	O
-Tsr3	O
-in	O
-human	O
-colon	O
-carcinoma	O
-HCT116	O
-(+/+)	O
-cells	O
-the	O
-acp	O
-primer	O
-extension	O
-arrest	O
-was	O
-reduced	O
-in	O
-comparison	O
-to	O
-cells	O
-transfected	O
-with	O
-a	O
-non	O
--	O
-targeting	O
-scramble	O
-siRNA	O
-control	O
-(	O
-Figure	O
-1E	O
-,	O
-compare	O
-lanes	O
-544	O
-and	O
-scramble	O
-).	O
-	O
-The	O
-efficiency	O
-of	O
-siRNA	O
--	O
-mediated	O
-depletion	O
-was	O
-established	O
-by	O
-RT	O
--	O
-qPCR	O
-and	O
-found	O
-to	O
-be	O
-very	O
-high	O
-with	O
-siRNA	O
-544	O
-(	O
-Supplementary	O
-Figure	O
-S2A	O
-,	O
-remaining	O
-TSR3	O
-mRNA	O
-level	O
-of	O
-2	O
-%).	O
-	O
-By	O
-comparison	O
-,	O
-treating	O
-cells	O
-with	O
-siRNA	O
-545	O
-,	O
-which	O
-only	O
-reduced	O
-the	O
-TSR3	O
-mRNA	O
-to	O
-20	O
-%,	O
-did	O
-not	O
-markedly	O
-reduced	O
-the	O
-acp	O
-signal	O
-.	O
-	O
-This	O
-suggests	O
-that	O
-low	O
-residual	O
-levels	O
-of	O
-HsTsr3	O
-are	O
-sufficient	O
-to	O
-modify	O
-the	O
-RNA	O
-.	O
-	O
-Thus	O
-,	O
-HsTsr3	O
-is	O
-also	O
-responsible	O
-for	O
-the	O
-acp	O
-modification	O
-of	O
-18S	O
-rRNA	O
-nucleotide	O
-Ψ1248	O
-in	O
-helix	O
-31	O
-.	O
-	O
-Similar	O
-to	O
-yeast	O
-,	O
-siRNA	O
--	O
-mediated	O
-depletion	O
-of	O
-the	O
-Ψ1248	O
-N1	O
--	O
-methyltransferase	O
-Nep1	O
-/	O
-Emg1	O
-had	O
-no	O
-influence	O
-on	O
-the	O
-primer	O
-extension	O
-arrest	O
-(	O
-Figure	O
-1E	O
-).	O
-	O
-Phenotypic	O
-characterization	O
-of	O
-Δtsr3	B-mutant
-mutants	O
-	O
-Although	O
-the	O
-acp	O
-modification	O
-of	O
-18S	O
-rRNA	O
-is	O
-highly	O
-conserved	O
-in	O
-eukaryotes	O
-,	O
-yeast	O
-Δtsr3	B-mutant
-mutants	O
-showed	O
-only	O
-a	O
-minor	O
-growth	O
-defect	O
-.	O
-	O
-However	O
-,	O
-the	O
-Δtsr3	B-mutant
-deletion	O
-was	O
-synthetic	O
-sick	O
-with	O
-a	O
-Δsnr35	B-mutant
-deletion	O
-preventing	O
-pseudouridylation	O
-and	O
-Nep1	O
--	O
-catalyzed	O
-methylation	O
-of	O
-nucleotide	O
-1191	O
-(	O
-Figure	O
-2A	O
-).	O
-	O
-Interestingly	O
-,	O
-no	O
-increased	O
-growth	O
-defect	O
-could	O
-be	O
-observed	O
-for	O
-Δtsr3	B-mutant
-Δnep1	I-mutant
-recombinants	O
-containing	O
-the	O
-nep1	O
-suppressor	O
-mutation	O
-Δnop6	B-mutant
-as	O
-well	O
-as	O
-for	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-Δnep1	I-mutant
-recombinants	O
-with	O
-unmodified	O
-U1191	O
-(	O
-Supplementary	O
-Figure	O
-S2D	O
-and	O
-E	O
-).	O
-	O
-Phenotypic	O
-characterization	O
-of	O
-yeast	O
-TSR3	O
-deletion	O
-(	O
-Δtrs3	B-mutant
-)	O
-and	O
-human	O
-TSR3	O
-depletion	O
-(	O
-siRNAs	O
-544	O
-and	O
-545	O
-)	O
-and	O
-cellular	O
-localization	O
-of	O
-yeast	O
-Tsr3	O
-.	O
-(	O
-A	O
-)	O
-Growth	O
-of	O
-yeast	O
-wild	O
-type	O
-,	O
-Δtsr3	B-mutant
-,	O
-Δsnr35	B-mutant
-and	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-segregants	O
-after	O
-meiosis	O
-and	O
-tetrad	O
-dissection	O
-of	O
-Δtsr3	B-mutant
-/	O
-TSR3	O
-Δsnr35	B-mutant
-/	O
-SNR35	O
-heterozygous	O
-diploids	O
-.	O
-	O
-The	O
-Δtsr3	B-mutant
-deletion	O
-is	O
-synthetic	O
-sick	O
-with	O
-a	O
-Δsnr35	B-mutant
-deletion	O
-preventing	O
-U1191	O
-pseudouridylation	O
-.	O
-	O
-(	O
-B	O
-)	O
-In	O
-agar	O
-diffusion	O
-assays	O
-the	O
-yeast	O
-Δtsr3	B-mutant
-deletion	O
-mutant	O
-shows	O
-a	O
-hypersensitivity	O
-against	O
-paromomycin	O
-and	O
-hygromycin	O
-B	O
-which	O
-is	O
-further	O
-increased	O
-by	O
-recombination	O
-with	O
-Δsnr35	B-mutant
-.	O
-(	O
-C	O
-)	O
-Northern	O
-blot	O
-analysis	O
-with	O
-an	O
-ITS1	O
-hybridization	O
-probe	O
-after	O
-siRNA	O
-depletion	O
-of	O
-HsTSR3	O
-(	O
-siRNAs	O
-544	O
-and	O
-545	O
-)	O
-and	O
-a	O
-scrambled	O
-siRNA	O
-as	O
-control	O
-.	O
-	O
-The	O
-accumulation	O
-of	O
-18SE	O
-and	O
-47S	O
-and	O
-/	O
-or	O
-45S	O
-pre	O
--	O
-RNAs	O
-is	O
-enforced	O
-upon	O
-HsTSR3	O
-depletion	O
-.	O
-	O
-Right	O
-gel	O
-:	O
-Ethidium	O
-bromide	O
-staining	O
-showing	O
-18S	O
-and	O
-28S	O
-rRNAs	O
-.	O
-	O
-(	O
-D	O
-)	O
-Cytoplasmic	O
-localization	O
-of	O
-yeast	O
-Tsr3	O
-shown	O
-by	O
-fluorescence	O
-microscopy	O
-of	O
-GFP	B-mutant
--	I-mutant
-fused	I-mutant
-Tsr3	I-mutant
-.	O
-	O
-From	O
-left	O
-to	O
-right	O
-:	O
-differential	O
-interference	O
-contrast	O
-(	O
-DIC	O
-),	O
-green	O
-fluorescence	O
-of	O
-GFP	B-mutant
--	I-mutant
-Tsr3	I-mutant
-,	O
-red	O
-fluorescence	O
-of	O
-Nop56	B-mutant
--	I-mutant
-mRFP	I-mutant
-as	O
-nucleolar	O
-marker	O
-,	O
-and	O
-merge	O
-of	O
-GFP	B-mutant
--	I-mutant
-Tsr3	I-mutant
-/	O
-Nop56	B-mutant
--	I-mutant
-mRFP	I-mutant
-with	O
-DIC	O
-.	O
-(	O
-E	O
-)	O
-Elution	O
-profile	O
-(	O
-A254	O
-)	O
-after	O
-sucrose	O
-gradient	O
-separation	O
-of	O
-yeast	O
-ribosomal	O
-subunits	O
-and	O
-polysomes	O
-(	O
-upper	O
-part	O
-)	O
-and	O
-western	O
-blot	O
-analysis	O
-of	O
-3xHA	O
-tagged	O
-Tsr3	O
-(	O
-Tsr3	B-mutant
--	I-mutant
-3xHA	I-mutant
-)	O
-after	O
-SDS	O
--	O
-PAGE	O
-separation	O
-of	O
-polysome	O
-profile	O
-fractions	O
-taken	O
-every	O
-20	O
-s	O
-(	O
-lower	O
-part	O
-).	O
-	O
-The	O
-TSR3	O
-gene	O
-was	O
-genetically	O
-modified	O
-at	O
-its	O
-native	O
-locus	O
-,	O
-resulting	O
-in	O
-a	O
-C	O
--	O
-terminal	O
-fusion	O
-of	O
-Tsr3	O
-with	O
-a	O
-3xHA	O
-epitope	O
-expressed	O
-by	O
-the	O
-native	O
-promotor	O
-in	O
-yeast	O
-strain	O
-CEN	O
-.	O
-BM258	O
--	O
-5B	O
-.	O
-	O
-The	O
-influence	O
-of	O
-the	O
-acp	O
-modification	O
-of	O
-nucleotide	O
-1191	O
-on	O
-ribosome	O
-function	O
-was	O
-analyzed	O
-by	O
-treating	O
-Δtsr3	B-mutant
-mutants	O
-with	O
-protein	O
-synthesis	O
-inhibitors	O
-.	O
-	O
-Similar	O
-to	O
-a	O
-temperature	O
--	O
-sensitive	O
-nep1	O
-mutant	O
-,	O
-the	O
-Δtsr3	B-mutant
-deletion	O
-caused	O
-hypersensitivity	O
-to	O
-paromomycin	O
-and	O
-,	O
-to	O
-a	O
-lesser	O
-extent	O
-,	O
-to	O
-hygromycin	O
-B	O
-(	O
-Figure	O
-2B	O
-),	O
-but	O
-not	O
-to	O
-G418	O
-or	O
-cycloheximide	O
-(	O
-data	O
-not	O
-shown	O
-).	O
-	O
-In	O
-accordance	O
-with	O
-the	O
-synthetic	O
-sick	O
-growth	O
-phenotype	O
-the	O
-paromomycin	O
-and	O
-hygromycin	O
-B	O
-hypersensitivity	O
-further	O
-increased	O
-in	O
-a	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-recombination	O
-strain	O
-(	O
-Figure	O
-2B	O
-).	O
-	O
-In	O
-a	O
-yeast	O
-Δtsr3	B-mutant
-strain	O
-as	O
-well	O
-as	O
-in	O
-the	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-recombinant	O
-20S	O
-pre	O
--	O
-rRNA	O
-accumulated	O
-significantly	O
-and	O
-the	O
-level	O
-of	O
-mature	O
-18S	O
-rRNA	O
-was	O
-reduced	O
-(	O
-Supplementary	O
-Figures	O
-S2C	O
-and	O
-S3D	O
-),	O
-as	O
-reported	O
-previously	O
-.	O
-	O
-A	O
-minor	O
-effect	O
-on	O
-20S	O
-rRNA	O
-accumulation	O
-was	O
-also	O
-observed	O
-for	O
-Δsnr35	B-mutant
-,	O
-but	O
--	O
-probably	O
-due	O
-to	O
-different	O
-strain	O
-backgrounds	O
-–	O
-to	O
-a	O
-weaker	O
-extent	O
-than	O
-described	O
-earlier	O
-.	O
-	O
-In	O
-human	O
-cells	O
-,	O
-the	O
-depletion	O
-of	O
-HsTsr3	O
-in	O
-HCT116	O
-(+/+)	O
-cells	O
-caused	O
-an	O
-accumulation	O
-of	O
-the	O
-human	O
-20S	O
-pre	O
--	O
-rRNA	O
-equivalent	O
-18S	O
--	O
-E	O
-suggesting	O
-an	O
-evolutionary	O
-conserved	O
-role	O
-of	O
-Tsr3	O
-in	O
-the	O
-late	O
-steps	O
-of	O
-18S	O
-rRNA	O
-processing	O
-(	O
-Figure	O
-2C	O
-and	O
-Supplementary	O
-Figure	O
-S2B	O
-).	O
-	O
-Surprisingly	O
-,	O
-early	O
-nucleolar	O
-processing	O
-reactions	O
-were	O
-also	O
-inhibited	O
-,	O
-and	O
-this	O
-was	O
-observed	O
-in	O
-both	O
-yeast	O
-Δtsr3	B-mutant
-cells	O
-(	O
-see	O
-accumulation	O
-of	O
-35S	O
-in	O
-Supplementary	O
-Figure	O
-S2C	O
-)	O
-and	O
-Tsr3	O
-depleted	O
-human	O
-cells	O
-(	O
-see	O
-47S	O
-/	O
-45S	O
-accumulation	O
-in	O
-Figure	O
-2C	O
-and	O
-Northern	O
-blot	O
-quantification	O
-in	O
-Supplementary	O
-Figure	O
-S2B	O
-).	O
-	O
-Consistent	O
-with	O
-its	O
-role	O
-in	O
-late	O
-18S	O
-rRNA	O
-processing	O
-,	O
-TSR3	O
-deletion	O
-leads	O
-to	O
-a	O
-ribosomal	O
-subunit	O
-imbalance	O
-with	O
-a	O
-reduced	O
-40S	O
-to	O
-60S	O
-ratio	O
-of	O
-0	O
-.	O
-81	O
-(	O
-σ	O
-=	O
-0	O
-.	O
-024	O
-)	O
-which	O
-was	O
-further	O
-increased	O
-in	O
-a	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-recombinant	O
-to	O
-0	O
-.	O
-73	O
-(	O
-σ	O
-=	O
-0	O
-.	O
-023	O
-)	O
-(	O
-Supplementary	O
-Figure	O
-S2F	O
-).	O
-	O
-In	O
-polysome	O
-profiles	O
-,	O
-a	O
-reduced	O
-level	O
-of	O
-80S	O
-ribosomes	O
-and	O
-a	O
-strong	O
-signal	O
-for	O
-free	O
-60S	O
-subunits	O
-was	O
-observed	O
-in	O
-line	O
-with	O
-the	O
-40S	O
-subunit	O
-deficiency	O
-(	O
-Supplementary	O
-Figure	O
-S2G	O
-).	O
-	O
-Cellular	O
-localization	O
-of	O
-Tsr3	O
-in	O
-S	O
-.	O
-cerevisiae	O
-	O
-Fluorescence	O
-microscopy	O
-of	O
-GFP	O
--	O
-tagged	O
-Tsr3	O
-localized	O
-the	O
-fusion	O
-protein	O
-in	O
-the	O
-cytoplasm	O
-of	O
-yeast	O
-cells	O
-and	O
-no	O
-co	O
--	O
-localization	O
-with	O
-the	O
-nucleolar	O
-marker	O
-protein	O
-Nop56	O
-could	O
-be	O
-observed	O
-(	O
-Figure	O
-2D	O
-).	O
-	O
-This	O
-agrees	O
-with	O
-previous	O
-biochemical	O
-data	O
-suggesting	O
-that	O
-the	O
-acp	O
-modification	O
-of	O
-18S	O
-rRNA	O
-occurs	O
-late	O
-during	O
-40S	O
-subunit	O
-biogenesis	O
-in	O
-the	O
-cytoplasm	O
-,	O
-and	O
-makes	O
-an	O
-additional	O
-nuclear	O
-localization	O
-as	O
-reported	O
-in	O
-a	O
-previous	O
-large	O
--	O
-scale	O
-analysis	O
-unlikely	O
-.	O
-	O
-After	O
-polysome	O
-gradient	O
-separation	O
-C	O
--	O
-terminally	O
-epitope	O
--	O
-labeled	O
-Tsr3	B-mutant
--	I-mutant
-3xHA	I-mutant
-was	O
-exclusively	O
-detectable	O
-in	O
-the	O
-low	O
--	O
-density	O
-fraction	O
-(	O
-Figure	O
-2E	O
-).	O
-	O
-Such	O
-distribution	O
-on	O
-a	O
-density	O
-gradient	O
-suggests	O
-that	O
-Tsr3	O
-only	O
-interacts	O
-transiently	O
-with	O
-pre	O
--	O
-40S	O
-subunits	O
-,	O
-which	O
-presumably	O
-explains	O
-why	O
-it	O
-was	O
-not	O
-characterized	O
-in	O
-pre	O
--	O
-ribosome	O
-affinity	O
-purifications	O
-.	O
-	O
-Structure	O
-of	O
-Tsr3	O
-	O
-Searches	O
-for	O
-sequence	O
-homologs	O
-of	O
-S	O
-.	O
-cerevisiae	O
-Tsr3	O
-(	O
-ScTsr3	O
-)	O
-by	O
-us	O
-and	O
-others	O
-revealed	O
-that	O
-the	O
-genomes	O
-of	O
-many	O
-archaea	O
-contain	O
-genes	O
-encoding	O
-Tsr3	O
--	O
-like	O
-proteins	O
-.	O
-	O
-However	O
-,	O
-these	O
-archaeal	O
-homologs	O
-are	O
-significantly	O
-smaller	O
-than	O
-ScTsr3	O
-(∼	O
-190	O
-aa	O
-in	O
-archaea	O
-vs	O
-.	O
-313	O
-aa	O
-in	O
-yeast	O
-)	O
-due	O
-to	O
-shortened	O
-N	O
--	O
-and	O
-C	O
--	O
-termini	O
-(	O
-Supplementary	O
-Figure	O
-S1A	O
-).	O
-	O
-To	O
-locate	O
-the	O
-domains	O
-most	O
-important	O
-for	O
-Tsr3	O
-activity	O
-,	O
-ScTsr3	O
-fragments	O
-of	O
-different	O
-lengths	O
-containing	O
-the	O
-highly	O
-conserved	O
-central	O
-part	O
-were	O
-expressed	O
-in	O
-a	O
-Δtsr3	B-mutant
-mutant	O
-(	O
-Figure	O
-3A	O
-)	O
-and	O
-analyzed	O
-by	O
-primer	O
-extension	O
-(	O
-Figure	O
-3B	O
-)	O
-and	O
-Northern	O
-blotting	O
-(	O
-Figure	O
-3C	O
-).	O
-	O
-N	O
--	O
-terminal	O
-truncations	O
-of	O
-up	O
-to	O
-45	O
-aa	O
-and	O
-C	O
--	O
-terminal	O
-truncations	O
-of	O
-up	O
-to	O
-76	O
-aa	O
-mediated	O
-acp	O
-modification	O
-as	O
-efficiently	O
-as	O
-the	O
-full	O
--	O
-length	O
-protein	O
-and	O
-no	O
-significant	O
-increased	O
-levels	O
-of	O
-20S	O
-pre	O
--	O
-RNA	O
-were	O
-detected	O
-.	O
-	O
-Even	O
-a	O
-Tsr3	O
-fragment	O
-with	O
-a	O
-90	O
-aa	O
-C	O
--	O
-terminal	O
-truncation	O
-showed	O
-a	O
-residual	O
-primer	O
-extension	O
-stop	O
-,	O
-whereas	O
-N	O
--	O
-terminal	O
-truncations	O
-exceeding	O
-46	O
-aa	O
-almost	O
-completely	O
-abolished	O
-the	O
-primer	O
-extension	O
-arrest	O
-(	O
-Figure	O
-3B	O
-).	O
-	O
-Domain	O
-characterization	O
-of	O
-yeast	O
-Tsr3	O
-and	O
-correlation	O
-of	O
-acp	O
-modification	O
-with	O
-late	O
-18S	O
-rRNA	O
-processing	O
-steps	O
-.	O
-(	O
-A	O
-)	O
-Scheme	O
-of	O
-the	O
-TSR3	O
-gene	O
-with	O
-truncation	O
-positions	O
-in	O
-the	O
-open	O
-reading	O
-frame	O
-.	O
-	O
-TSR3	O
-fragments	O
-of	O
-different	O
-length	O
-were	O
-expressed	O
-under	O
-the	O
-native	O
-promotor	O
-from	O
-multicopy	O
-plasmids	O
-in	O
-a	O
-Δtsr3	B-mutant
-deletion	O
-strain	O
-.	O
-	O
-(	O
-B	O
-)	O
-Primer	O
-extension	O
-analysis	O
-of	O
-18S	O
-rRNA	O
-acp	O
-modification	O
-in	O
-yeast	O
-cells	O
-expressing	O
-the	O
-indicated	O
-TSR3	O
-fragments	O
-.	O
-	O
-N	O
--	O
-terminal	O
-deletions	O
-of	O
-36	O
-or	O
-45	O
-amino	O
-acids	O
-and	O
-C	O
--	O
-terminal	O
-deletions	O
-of	O
-43	O
-or	O
-76	O
-residues	O
-show	O
-a	O
-primer	O
-extension	O
-stop	O
-comparable	O
-to	O
-the	O
-wild	O
-type	O
-.	O
-	O
-Tsr3	O
-fragments	O
-37	O
-–	O
-223	O
-or	O
-46	O
-–	O
-223	O
-cause	O
-a	O
-nearly	O
-complete	O
-loss	O
-of	O
-the	O
-arrest	O
-signal	O
-.	O
-	O
-The	O
-box	O
-highlights	O
-the	O
-shortest	O
-Tsr3	O
-fragment	O
-(	O
-aa	O
-46	O
-–	O
-270	O
-)	O
-with	O
-wild	O
-type	O
-activity	O
-(	O
-strong	O
-primer	O
-extension	O
-block	O
-).	O
-(	O
-C	O
-)	O
-Northern	O
-blot	O
-analysis	O
-of	O
-20S	O
-pre	O
--	O
-rRNA	O
-accumulation	O
-.	O
-	O
-A	O
-weak	O
-20S	O
-rRNA	O
-signal	O
-,	O
-indicating	O
-normal	O
-processing	O
-,	O
-is	O
-observed	O
-for	O
-Tsr3	O
-fragment	O
-46	O
-–	O
-270	O
-(	O
-highlighted	O
-in	O
-a	O
-box	O
-)	O
-showing	O
-its	O
-functionality	O
-.	O
-	O
-Strong	O
-20S	O
-rRNA	O
-accumulation	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-Δtsr3	B-mutant
-deletion	O
-is	O
-observed	O
-for	O
-Tsr3	O
-fragments	O
-37	O
-–	O
-223	O
-or	O
-46	O
-–	O
-223	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-archaeal	O
-homologs	O
-correspond	O
-to	O
-the	O
-functional	O
-core	O
-of	O
-Tsr3	O
-.	O
-	O
-In	O
-order	O
-to	O
-define	O
-the	O
-structural	O
-basis	O
-for	O
-Tsr3	O
-function	O
-,	O
-homologs	O
-from	O
-thermophilic	O
-archaea	O
-were	O
-screened	O
-for	O
-crystallization	O
-.	O
-	O
-We	O
-focused	O
-on	O
-archaeal	O
-species	O
-containing	O
-a	O
-putative	O
-Nep1	O
-homolog	O
-suggesting	O
-that	O
-these	O
-species	O
-are	O
-in	O
-principle	O
-capable	O
-of	O
-synthesizing	O
-N1	O
--	O
-methyl	O
--	O
-N3	O
--	O
-acp	O
--	O
-pseudouridine	O
-.	O
-	O
-Well	O
-diffracting	O
-crystals	O
-were	O
-obtained	O
-for	O
-Tsr3	O
-homologs	O
-from	O
-the	O
-two	O
-crenarchaeal	O
-species	O
-Vulcanisaeta	O
-distributa	O
-(	O
-VdTsr3	O
-)	O
-and	O
-Sulfolobus	O
-solfataricus	O
-(	O
-SsTsr3	O
-)	O
-which	O
-share	O
-36	O
-%	O
-(	O
-VdTsr3	O
-)	O
-and	O
-38	O
-%	O
-(	O
-SsTsr3	O
-)	O
-identity	O
-with	O
-the	O
-ScTsr3	O
-core	O
-region	O
-(	O
-ScTsr3	O
-aa	O
-46	O
-–	O
-223	O
-).	O
-	O
-While	O
-for	O
-S	O
-.	O
-solfataricus	O
-the	O
-existence	O
-of	O
-a	O
-modified	O
-nucleotide	O
-of	O
-unknown	O
-chemical	O
-composition	O
-in	O
-the	O
-loop	O
-capping	O
-helix	O
-31	O
-of	O
-its	O
-16S	O
-rRNA	O
-has	O
-been	O
-demonstrated	O
-,	O
-no	O
-information	O
-regarding	O
-rRNA	O
-modifications	O
-is	O
-yet	O
-available	O
-for	O
-V	O
-.	O
-distributa	O
-.	O
-	O
-Crystals	O
-of	O
-VdTsr3	O
-diffracted	O
-to	O
-a	O
-resolution	O
-of	O
-1	O
-.	O
-6	O
-Å	O
-whereas	O
-crystals	O
-of	O
-SsTsr3	O
-diffracted	O
-to	O
-2	O
-.	O
-25	O
-Å	O
-.	O
-Serendipitously	O
-,	O
-VdTsr3	O
-was	O
-purified	O
-and	O
-crystallized	O
-in	O
-complex	O
-with	O
-endogenous	O
-(	O
-E	O
-.	O
-coli	O
-)	O
-SAM	O
-(	O
-Supplementary	O
-Figure	O
-S4	O
-)	O
-while	O
-SsTsr3	O
-crystals	O
-contained	O
-the	O
-protein	O
-in	O
-the	O
-apo	O
-state	O
-.	O
-	O
-The	O
-structure	O
-of	O
-VdTsr3	O
-was	O
-solved	O
-ab	O
-initio	O
-,	O
-by	O
-single	O
--	O
-wavelength	O
-anomalous	O
-diffraction	O
-phasing	O
-(	O
-Se	O
--	O
-SAD	O
-)	O
-with	O
-Se	O
-containing	O
-derivatives	O
-(	O
-selenomethionine	O
-and	O
-seleno	O
--	O
-substituted	O
-SAM	O
-).	O
-	O
-The	O
-structure	O
-of	O
-SsTsr3	O
-was	O
-solved	O
-by	O
-molecular	O
-replacement	O
-using	O
-VdTsr3	O
-as	O
-a	O
-search	O
-model	O
-(	O
-see	O
-Supplementary	O
-Table	O
-S1	O
-for	O
-data	O
-collection	O
-and	O
-refinement	O
-statistics	O
-).	O
-	O
-The	O
-structure	O
-of	O
-VdTsr3	O
-can	O
-be	O
-divided	O
-into	O
-two	O
-domains	O
-(	O
-Figure	O
-4A	O
-).	O
-	O
-The	O
-N	O
--	O
-terminal	O
-domain	O
-(	O
-aa	O
-1	O
-–	O
-92	O
-)	O
-has	O
-a	O
-mixed	O
-α	O
-/	O
-β	O
--	O
-structure	O
-centered	O
-around	O
-a	O
-five	O
--	O
-stranded	O
-all	O
--	O
-parallel	O
-β	O
--	O
-sheet	O
-(	O
-Figure	O
-4B	O
-)	O
-with	O
-the	O
-strand	O
-order	O
-β5	O
-↑-	O
-β3	O
-↑-	O
-β4	O
-↑-	O
-β1	O
-↑-	O
-β2	O
-↑.	O
-The	O
-loops	O
-connecting	O
-β1	O
-and	O
-β2	O
-,	O
-β3	O
-and	O
-β4	O
-and	O
-β4	O
-and	O
-β5	O
-include	O
-α	O
--	O
-helices	O
-α1	O
-,	O
-α2	O
-and	O
-α3	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-loop	O
-connecting	O
-β2	O
-and	O
-β3	O
-contains	O
-a	O
-single	O
-turn	O
-of	O
-a	O
-310	O
--	O
-helix	O
-.	O
-Helices	O
-α1	O
-and	O
-α2	O
-are	O
-located	O
-on	O
-one	O
-side	O
-of	O
-the	O
-five	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-while	O
-α3	O
-packs	O
-against	O
-the	O
-opposite	O
-β	O
--	O
-sheet	O
-surface	O
-.	O
-	O
-The	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-aa	O
-93	O
-–	O
-184	O
-)	O
-has	O
-a	O
-globular	O
-all	O
-α	O
--	O
-helical	O
-structure	O
-comprising	O
-α	O
--	O
-helices	O
-α4	O
-to	O
-α9	O
-.	O
-	O
-Remarkably	O
-,	O
-the	O
-entire	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-92	O
-aa	O
-)	O
-of	O
-the	O
-protein	O
-is	O
-threaded	O
-through	O
-the	O
-loop	O
-which	O
-connects	O
-β	O
--	O
-strand	O
-β3	O
-and	O
-α	O
--	O
-helix	O
-α2	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-domain	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-VdTsr3	O
-structure	O
-contains	O
-a	O
-deep	O
-trefoil	O
-knot	O
-.	O
-	O
-The	O
-structure	O
-of	O
-SsTsr3	O
-in	O
-the	O
-apo	O
-state	O
-is	O
-very	O
-similar	O
-to	O
-that	O
-of	O
-VdTsr3	O
-(	O
-Figure	O
-4C	O
-)	O
-with	O
-an	O
-RMSD	O
-for	O
-equivalent	O
-Cα	O
-atoms	O
-of	O
-1	O
-.	O
-1	O
-Å	O
-.	O
-The	O
-only	O
-significant	O
-difference	O
-in	O
-the	O
-global	O
-structure	O
-of	O
-the	O
-two	O
-proteins	O
-is	O
-the	O
-presence	O
-of	O
-an	O
-extended	O
-α	O
--	O
-helix	O
-α8	O
-and	O
-the	O
-absence	O
-of	O
-α	O
--	O
-helix	O
-α9	O
-in	O
-SsTsr3	O
-.	O
-	O
-Tsr3	O
-has	O
-a	O
-fold	O
-similar	O
-to	O
-SPOUT	O
--	O
-class	O
-RNA	O
-methyltransferases	O
-.	O
-(	O
-A	O
-)	O
-Cartoon	O
-representation	O
-of	O
-the	O
-X	O
--	O
-ray	O
-structure	O
-of	O
-VdTsr3	O
-in	O
-two	O
-orientations	O
-.	O
-	O
-β	O
--	O
-strands	O
-are	O
-colored	O
-in	O
-crimson	O
-whereas	O
-α	O
--	O
-helices	O
-in	O
-the	O
-N	O
--	O
-terminal	O
-domain	O
-are	O
-colored	O
-light	O
-blue	O
-and	O
-α	O
--	O
-helices	O
-in	O
-the	O
-C	O
--	O
-terminal	O
-domain	O
-are	O
-colored	O
-dark	O
-blue	O
-.	O
-	O
-The	O
-bound	O
-S	O
--	O
-adenosylmethionine	O
-is	O
-shown	O
-in	O
-a	O
-stick	O
-representation	O
-and	O
-colored	O
-by	O
-atom	O
-type	O
-.	O
-	O
-A	O
-red	O
-arrow	O
-marks	O
-the	O
-location	O
-of	O
-the	O
-topological	O
-knot	O
-in	O
-the	O
-structure	O
-.	O
-(	O
-B	O
-)	O
-Secondary	O
-structure	O
-representation	O
-of	O
-the	O
-VdTsr3	O
-structure	O
-.	O
-	O
-The	O
-color	O
-coding	O
-is	O
-the	O
-same	O
-as	O
-in	O
-(	O
-A	O
-).	O
-(	O
-C	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-X	O
--	O
-ray	O
-structures	O
-of	O
-VdTsr3	O
-in	O
-the	O
-SAM	O
--	O
-bound	O
-state	O
-(	O
-red	O
-)	O
-and	O
-SsTsr3	O
-(	O
-blue	O
-)	O
-in	O
-the	O
-apo	O
-state	O
-.	O
-	O
-The	O
-locations	O
-of	O
-the	O
-α	O
--	O
-helix	O
-α8	O
-which	O
-is	O
-longer	O
-in	O
-SsTsr3	O
-and	O
-of	O
-α	O
--	O
-helix	O
-α9	O
-which	O
-is	O
-only	O
-present	O
-in	O
-VdTsr3	O
-are	O
-indicated	O
-.	O
-(	O
-D	O
-)	O
-Secondary	O
-structure	O
-cartoon	O
-(	O
-left	O
-)	O
-of	O
-S	O
-.	O
-pombe	O
-Trm10	O
-(	O
-pdb4jwf	O
-)—	O
-the	O
-SPOUT	O
--	O
-class	O
-RNA	O
-methyltransferase	O
-structurally	O
-most	O
-similar	O
-to	O
-Tsr3	O
-and	O
-superposition	O
-of	O
-the	O
-VdTsr3	O
-and	O
-Trm10	O
-X	O
--	O
-ray	O
-structures	O
-(	O
-right	O
-).	O
-(	O
-E	O
-)	O
-Analytical	O
-gel	O
-filtration	O
-profiles	O
-for	O
-VdTsr3	O
-(	O
-red	O
-)	O
-and	O
-SsTsr3	O
-(	O
-blue	O
-)	O
-show	O
-that	O
-both	O
-proteins	O
-are	O
-monomeric	O
-in	O
-solution	O
-.	O
-	O
-Vd	O
-,	O
-Vulcanisaeta	O
-distributa	O
-;	O
-Ss	O
-,	O
-Sulfolobus	O
-solfataricus	O
-.	O
-	O
-Structure	O
-predictions	O
-suggested	O
-that	O
-Tsr3	O
-might	O
-contain	O
-a	O
-so	O
--	O
-called	O
-RLI	O
-domain	O
-which	O
-contains	O
-a	O
-‘	O
-bacterial	O
-like	O
-’	O
-ferredoxin	O
-fold	O
-and	O
-binds	O
-two	O
-iron	O
--	O
-sulfur	O
-clusters	O
-through	O
-eight	O
-conserved	O
-cysteine	O
-residues	O
-.	O
-	O
-However	O
-,	O
-no	O
-structural	O
-similarity	O
-to	O
-an	O
-RLI	O
--	O
-domain	O
-was	O
-detectable	O
-.	O
-	O
-This	O
-is	O
-in	O
-accordance	O
-with	O
-the	O
-functional	O
-analysis	O
-of	O
-alanine	O
-replacement	O
-mutations	O
-of	O
-cysteine	O
-residues	O
-in	O
-ScTsr3	O
-(	O
-Supplementary	O
-Figure	O
-S3	O
-).	O
-	O
-The	O
-β	O
--	O
-strand	O
-topology	O
-and	O
-the	O
-deep	O
-C	O
--	O
-terminal	O
-trefoil	O
-knot	O
-of	O
-archaeal	O
-Tsr3	O
-are	O
-the	O
-structural	O
-hallmarks	O
-of	O
-the	O
-SPOUT	O
--	O
-class	O
-RNA	O
--	O
-methyltransferase	O
-fold	O
-.	O
-	O
-The	O
-closest	O
-structural	O
-homolog	O
-identified	O
-in	O
-a	O
-DALI	O
-search	O
-is	O
-the	O
-tRNA	O
-methyltransferase	O
-Trm10	O
-(	O
-DALI	O
-Z	O
--	O
-score	O
-6	O
-.	O
-8	O
-)	O
-which	O
-methylates	O
-the	O
-N1	O
-nitrogen	O
-of	O
-G9	O
-/	O
-A9	O
-in	O
-many	O
-archaeal	O
-and	O
-eukaryotic	O
-tRNAs	O
-by	O
-using	O
-SAM	O
-as	O
-the	O
-methyl	O
-group	O
-donor	O
-.	O
-	O
-In	O
-comparison	O
-to	O
-Tsr3	O
-the	O
-central	O
-β	O
--	O
-sheet	O
-element	O
-of	O
-Trm10	O
-is	O
-extended	O
-by	O
-one	O
-additional	O
-β	O
--	O
-strand	O
-pairing	O
-to	O
-β2	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-trefoil	O
-knot	O
-of	O
-Trm10	O
-is	O
-not	O
-as	O
-deep	O
-as	O
-that	O
-of	O
-Tsr3	O
-(	O
-Figure	O
-4D	O
-).	O
-	O
-Interestingly	O
-,	O
-Nep1	O
-—	O
-the	O
-enzyme	O
-preceding	O
-Tsr3	O
-in	O
-the	O
-biosynthetic	O
-pathway	O
-for	O
-the	O
-synthesis	O
-of	O
-m1acp3Ψ	O
-—	O
-also	O
-belongs	O
-to	O
-the	O
-SPOUT	O
--	O
-class	O
-of	O
-RNA	O
-methyltransferases	O
-.	O
-	O
-However	O
-,	O
-the	O
-structural	O
-similarities	O
-between	O
-Nep1	O
-and	O
-Tsr3	O
-(	O
-DALI	O
-Z	O
--	O
-score	O
-4	O
-.	O
-4	O
-)	O
-are	O
-less	O
-pronounced	O
-than	O
-between	O
-Tsr3	O
-and	O
-Trm10	O
-.	O
-	O
-Most	O
-SPOUT	O
--	O
-class	O
-RNA	O
--	O
-methyltransferases	O
-are	O
-homodimers	O
-.	O
-	O
-A	O
-notable	O
-exception	O
-is	O
-Trm10	O
-.	O
-	O
-Gel	O
-filtration	O
-experiments	O
-with	O
-both	O
-VdTsr3	O
-and	O
-SsTsr3	O
-(	O
-Figure	O
-4E	O
-)	O
-showed	O
-that	O
-both	O
-proteins	O
-are	O
-monomeric	O
-in	O
-solution	O
-thereby	O
-extending	O
-the	O
-structural	O
-similarities	O
-to	O
-Trm10	O
-.	O
-	O
-So	O
-far	O
-,	O
-structural	O
-information	O
-is	O
-only	O
-available	O
-for	O
-one	O
-other	O
-enzyme	O
-that	O
-transfers	O
-the	O
-acp	O
-group	O
-from	O
-SAM	O
-to	O
-an	O
-RNA	O
-nucleotide	O
-.	O
-	O
-This	O
-enzyme	O
-,	O
-Tyw2	O
-,	O
-is	O
-part	O
-of	O
-the	O
-biosynthesis	O
-pathway	O
-of	O
-wybutosine	O
-nucleotides	O
-in	O
-tRNAs	O
-.	O
-	O
-However	O
-,	O
-there	O
-are	O
-no	O
-structural	O
-similarities	O
-between	O
-Tsr3	O
-and	O
-Tyw2	O
-,	O
-which	O
-contains	O
-an	O
-all	O
--	O
-parallel	O
-β	O
--	O
-sheet	O
-of	O
-a	O
-different	O
-topology	O
-and	O
-no	O
-knot	O
-structure	O
-.	O
-	O
-Instead	O
-,	O
-Tyw2	O
-has	O
-a	O
-fold	O
-typical	O
-for	O
-the	O
-class	O
--	O
-I	O
--	O
-or	O
-Rossmann	O
--	O
-fold	O
-class	O
-of	O
-methyltransferases	O
-(	O
-Supplementary	O
-Figure	O
-S5B	O
-).	O
-	O
-Cofactor	O
-binding	O
-of	O
-Tsr3	O
-	O
-The	O
-SAM	O
--	O
-binding	O
-site	O
-of	O
-Tsr3	O
-is	O
-located	O
-in	O
-a	O
-deep	O
-crevice	O
-between	O
-the	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-domains	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-trefoil	O
-knot	O
-as	O
-typical	O
-for	O
-SPOUT	O
--	O
-class	O
-RNA	O
--	O
-methyltransferases	O
-(	O
-Figure	O
-4A	O
-).	O
-	O
-The	O
-adenine	O
-base	O
-of	O
-the	O
-cofactor	O
-is	O
-recognized	O
-by	O
-hydrogen	O
-bonds	O
-between	O
-its	O
-N1	O
-nitrogen	O
-and	O
-the	O
-backbone	O
-amide	O
-of	O
-L93	O
-directly	O
-preceding	O
-β5	O
-as	O
-well	O
-as	O
-between	O
-its	O
-N6	O
--	O
-amino	O
-group	O
-and	O
-the	O
-backbone	O
-carbonyl	O
-group	O
-of	O
-Y108	O
-located	O
-in	O
-the	O
-loop	O
-connecting	O
-β5	O
-in	O
-the	O
-N	O
--	O
-terminal	O
-and	O
-α4	O
-in	O
-the	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-Figure	O
-5A	O
-).	O
-	O
-Furthermore	O
-,	O
-the	O
-adenine	O
-base	O
-of	O
-SAM	O
-is	O
-involved	O
-in	O
-hydrophobic	O
-packing	O
-interactions	O
-with	O
-the	O
-side	O
-chains	O
-of	O
-L45	O
-(	O
-β3	O
-),	O
-P47	O
-and	O
-W73	O
-(	O
-α3	O
-)	O
-in	O
-the	O
-N	O
--	O
-terminal	O
-domain	O
-as	O
-well	O
-as	O
-with	O
-L93	O
-,	O
-L110	O
-(	O
-both	O
-in	O
-the	O
-loop	O
-connecting	O
-β5	O
-and	O
-α4	O
-)	O
-and	O
-A115	O
-(	O
-α5	O
-)	O
-in	O
-the	O
-C	O
--	O
-terminal	O
-domain	O
-.	O
-	O
-The	O
-ribose	O
-2	O
-′	O
-and	O
-3	O
-′	O
-hydroxyl	O
-groups	O
-of	O
-SAM	O
-are	O
-hydrogen	O
-bonded	O
-to	O
-the	O
-backbone	O
-carbonyl	O
-group	O
-of	O
-I69	O
-.	O
-	O
-The	O
-acp	O
-side	O
-chain	O
-of	O
-SAM	O
-is	O
-fixed	O
-in	O
-position	O
-by	O
-hydrogen	O
-bonding	O
-of	O
-its	O
-carboxylate	O
-group	O
-to	O
-the	O
-backbone	O
-amide	O
-and	O
-the	O
-side	O
-chain	O
-hydroxyl	O
-group	O
-of	O
-T19	O
-in	O
-α1	O
-as	O
-well	O
-as	O
-the	O
-backbone	O
-amide	O
-group	O
-of	O
-T112	O
-in	O
-α4	O
-(	O
-C	O
--	O
-terminal	O
-domain	O
-).	O
-	O
-Most	O
-importantly	O
-,	O
-the	O
-methyl	O
-group	O
-of	O
-SAM	O
-is	O
-buried	O
-in	O
-a	O
-hydrophobic	O
-pocket	O
-formed	O
-by	O
-the	O
-sidechains	O
-of	O
-W73	O
-and	O
-A76	O
-both	O
-located	O
-in	O
-α3	O
-(	O
-Figure	O
-5A	O
-and	O
-B	O
-).	O
-	O
-W73	O
-is	O
-highly	O
-conserved	O
-in	O
-all	O
-known	O
-Tsr3	O
-proteins	O
-,	O
-whereas	O
-A76	O
-can	O
-be	O
-replaced	O
-by	O
-other	O
-hydrophobic	O
-amino	O
-acids	O
-.	O
-	O
-Consequently	O
-,	O
-the	O
-accessibility	O
-of	O
-this	O
-methyl	O
-group	O
-for	O
-a	O
-nucleophilic	O
-attack	O
-is	O
-strongly	O
-reduced	O
-in	O
-comparison	O
-with	O
-RNA	O
--	O
-methyltransferases	O
-such	O
-as	O
-Trm10	O
-(	O
-Figure	O
-5B	O
-,	O
-C	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-acp	O
-side	O
-chain	O
-of	O
-SAM	O
-is	O
-accessible	O
-for	O
-reactions	O
-in	O
-the	O
-Tsr3	O
--	O
-bound	O
-state	O
-(	O
-Figure	O
-5B	O
-).	O
-	O
-SAM	O
--	O
-binding	O
-by	O
-Tsr3	O
-.	O
-	O
-(	O
-A	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-SAM	O
--	O
-binding	O
-pocket	O
-of	O
-VdTsr3	O
-.	O
-	O
-Nitrogen	O
-atoms	O
-are	O
-dark	O
-blue	O
-,	O
-oxygen	O
-atoms	O
-red	O
-,	O
-sulfur	O
-atoms	O
-orange	O
-,	O
-carbon	O
-atoms	O
-of	O
-the	O
-protein	O
-light	O
-blue	O
-and	O
-carbon	O
-atoms	O
-of	O
-SAM	O
-yellow	O
-.	O
-	O
-Hydrogen	O
-bonds	O
-are	O
-indicated	O
-by	O
-dashed	O
-lines	O
-.	O
-	O
-(	O
-B	O
-)	O
-Solvent	O
-accessibility	O
-of	O
-the	O
-acp	O
-group	O
-of	O
-SAM	O
-bound	O
-to	O
-VdTsr3	O
-.	O
-	O
-The	O
-solvent	O
-accessible	O
-surface	O
-of	O
-the	O
-protein	O
-is	O
-shown	O
-in	O
-semitransparent	O
-gray	O
-whereas	O
-SAM	O
-is	O
-show	O
-in	O
-a	O
-stick	O
-representation	O
-.	O
-	O
-A	O
-red	O
-arrow	O
-indicates	O
-the	O
-reactive	O
-CH2	O
--	O
-moiety	O
-of	O
-the	O
-acp	O
-group	O
-.	O
-(	O
-C	O
-)	O
-Solvent	O
-accessibility	O
-of	O
-the	O
-SAM	O
-methyl	O
-group	O
-for	O
-SAM	O
-bound	O
-to	O
-the	O
-RNA	O
-methyltransferase	O
-Trm10	O
-.	O
-	O
-Bound	O
-SAM	O
-was	O
-modelled	O
-based	O
-on	O
-the	O
-X	O
--	O
-ray	O
-structure	O
-of	O
-the	O
-Trm10	O
-/	O
-SAH	O
--	O
-complex	O
-(	O
-pdb4jwf	O
-).	O
-	O
-A	O
-red	O
-arrow	O
-indicates	O
-the	O
-SAM	O
-methyl	O
-group	O
-.	O
-(	O
-D	O
-)	O
-Binding	O
-of	O
-SAM	O
-analogs	O
-to	O
-SsTsr3	O
-.	O
-	O
-Tryptophan	O
-fluorescence	O
-quenching	O
-curves	O
-upon	O
-addition	O
-of	O
-SAM	O
-(	O
-blue	O
-),	O
-5	O
-′-	O
-methyl	O
--	O
-thioadenosine	O
-(	O
-red	O
-)	O
-and	O
-SAH	O
-(	O
-black	O
-).	O
-	O
-(	O
-E	O
-)	O
-Binding	O
-of	O
-14C	O
--	O
-labeled	O
-SAM	O
-to	O
-SsTsr3	O
-.	O
-	O
-Radioactively	O
-labeled	O
-SAM	O
-is	O
-retained	O
-on	O
-a	O
-filter	O
-in	O
-the	O
-presence	O
-of	O
-SsTsr3	O
-.	O
-	O
-Addition	O
-of	O
-unlabeled	O
-SAM	O
-competes	O
-with	O
-the	O
-binding	O
-of	O
-labeled	O
-SAM	O
-.	O
-	O
-A	O
-W66A	B-mutant
--	O
-mutant	O
-of	O
-SsTsr3	O
-(	O
-W73	O
-in	O
-VdTsr3	O
-)	O
-does	O
-not	O
-bind	O
-SAM	O
-.	O
-	O
-(	O
-F	O
-)	O
-Primer	O
-extension	O
-(	O
-upper	O
-left	O
-)	O
-shows	O
-a	O
-strongly	O
-reduced	O
-acp	O
-modification	O
-of	O
-yeast	O
-18S	O
-rRNA	O
-in	O
-Δtsr3	B-mutant
-cells	O
-expressing	O
-Tsr3	B-mutant
--	I-mutant
-S62D	I-mutant
-,	O
--	B-mutant
-E111A	I-mutant
-or	O
-–	B-mutant
-W114A	I-mutant
-.	O
-	O
-This	O
-correlates	O
-with	O
-a	O
-20S	O
-pre	O
--	O
-rRNA	O
-accumulation	O
-comparable	O
-to	O
-the	O
-Δtsr3	B-mutant
-deletion	O
-(	O
-right	O
-:	O
-northern	O
-blot	O
-).	O
-	O
-3xHA	O
-tagged	O
-Tsr3	O
-mutants	O
-are	O
-expressed	O
-comparable	O
-to	O
-the	O
-wild	O
-type	O
-as	O
-shown	O
-by	O
-western	O
-blot	O
-(	O
-lower	O
-left	O
-).	O
-	O
-Binding	O
-affinities	O
-for	O
-SAM	O
-and	O
-its	O
-analogs	O
-5	O
-′-	O
-methylthioadenosin	O
-and	O
-SAH	O
-to	O
-SsTsr3	O
-were	O
-measured	O
-using	O
-tryptophan	O
-fluorescence	O
-quenching	O
-.	O
-	O
-VdTsr3	O
-could	O
-not	O
-be	O
-used	O
-in	O
-these	O
-experiments	O
-since	O
-we	O
-could	O
-not	O
-purify	O
-it	O
-in	O
-a	O
-stable	O
-SAM	O
--	O
-free	O
-form	O
-.	O
-	O
-SsTsr3	O
-bound	O
-SAM	O
-with	O
-a	O
-KD	O
-of	O
-6	O
-.	O
-5	O
-μM	O
-,	O
-which	O
-is	O
-similar	O
-to	O
-SAM	O
--	O
-KD	O
-'	O
-s	O
-reported	O
-for	O
-several	O
-SPOUT	O
--	O
-class	O
-methyltransferases	O
-.	O
-	O
-5	O
-′-	O
-methylthioadenosin	O
-—	O
-the	O
-reaction	O
-product	O
-after	O
-the	O
-acp	O
--	O
-transfer	O
-—	O
-binds	O
-only	O
-∼	O
-2	O
-.	O
-5	O
--	O
-fold	O
-weaker	O
-(	O
-KD	O
-=	O
-16	O
-.	O
-7	O
-μM	O
-)	O
-compared	O
-to	O
-SAM	O
-.	O
-	O
-S	O
--	O
-adenosylhomocysteine	O
-which	O
-lacks	O
-the	O
-methyl	O
-group	O
-of	O
-SAM	O
-binds	O
-with	O
-significantly	O
-lower	O
-affinity	O
-(	O
-KD	O
-=	O
-55	O
-.	O
-5	O
-μM	O
-)	O
-(	O
-Figure	O
-5D	O
-).	O
-	O
-This	O
-suggests	O
-that	O
-the	O
-hydrophobic	O
-interaction	O
-between	O
-SAM	O
-'	O
-s	O
-methyl	O
-group	O
-and	O
-the	O
-hydrophobic	O
-pocket	O
-of	O
-Tsr3	O
-is	O
-thermodynamically	O
-important	O
-for	O
-the	O
-interaction	O
-.	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-the	O
-loss	O
-of	O
-hydrogen	O
-bonds	O
-between	O
-the	O
-acp	O
-sidechain	O
-carboxylate	O
-group	O
-and	O
-the	O
-protein	O
-appears	O
-to	O
-be	O
-thermodynamically	O
-less	O
-important	O
-but	O
-these	O
-hydrogen	O
-bonds	O
-might	O
-play	O
-a	O
-crucial	O
-role	O
-for	O
-the	O
-proper	O
-orientation	O
-of	O
-the	O
-cofactor	O
-side	O
-chain	O
-in	O
-the	O
-substrate	O
-binding	O
-pocket	O
-.	O
-	O
-Accordingly	O
-,	O
-a	O
-W66A	B-mutant
--	O
-mutation	O
-(	O
-W73	O
-in	O
-VdTsr3	O
-)	O
-of	O
-SsTsr3	O
-significantly	O
-diminished	O
-SAM	O
--	O
-binding	O
-in	O
-a	O
-filter	O
-binding	O
-assay	O
-compared	O
-to	O
-the	O
-wild	O
-type	O
-(	O
-Figure	O
-5E	O
-).	O
-	O
-Furthermore	O
-,	O
-a	O
-W	O
-to	O
-A	O
-mutation	O
-at	O
-the	O
-equivalent	O
-position	O
-W114	O
-in	O
-ScTsr3	O
-strongly	O
-reduced	O
-the	O
-in	O
-vivo	O
-acp	O
-transferase	O
-activity	O
-(	O
-Figure	O
-5F	O
-).	O
-	O
-The	O
-side	O
-chain	O
-hydroxyl	O
-group	O
-of	O
-T19	O
-seems	O
-of	O
-minor	O
-importance	O
-for	O
-SAM	O
-binding	O
-since	O
-mutations	O
-of	O
-T17	O
-(	O
-T19	O
-in	O
-VdTsr3	O
-)	O
-to	O
-either	O
-A	O
-or	O
-D	O
-did	O
-not	O
-significantly	O
-influence	O
-the	O
-SAM	O
--	O
-binding	O
-affinity	O
-of	O
-SsTsr3	O
-(	O
-KD	O
-'	O
-s	O
-=	O
-3	O
-.	O
-9	O
-or	O
-11	O
-.	O
-2	O
-mM	O
-,	O
-respectively	O
-).	O
-	O
-Nevertheless	O
-,	O
-a	O
-mutation	O
-of	O
-the	O
-equivalent	O
-position	O
-S62	O
-of	O
-ScTsr3	O
-to	O
-D	O
-,	O
-but	O
-not	O
-to	O
-A	O
-,	O
-resulted	O
-in	O
-reduced	O
-acp	O
-modification	O
-in	O
-vivo	O
-,	O
-as	O
-shown	O
-by	O
-primer	O
-extension	O
-analysis	O
-(	O
-Figure	O
-5F	O
-).	O
-	O
-The	O
-acp	O
--	O
-transfer	O
-reaction	O
-catalyzed	O
-by	O
-Tsr3	O
-most	O
-likely	O
-requires	O
-the	O
-presence	O
-of	O
-a	O
-catalytic	O
-base	O
-in	O
-order	O
-to	O
-abstract	O
-a	O
-proton	O
-from	O
-the	O
-N3	O
-imino	O
-group	O
-of	O
-the	O
-modified	O
-pseudouridine	O
-.	O
-	O
-The	O
-side	O
-chain	O
-of	O
-D70	O
-(	O
-VdTsr3	O
-)	O
-located	O
-in	O
-β4	O
-is	O
-∼	O
-5	O
-Å	O
-away	O
-from	O
-the	O
-SAM	O
-sulfur	O
-atom	O
-.	O
-	O
-This	O
-residue	O
-is	O
-conserved	O
-as	O
-D	O
-or	O
-E	O
-both	O
-in	O
-archaeal	O
-and	O
-eukaryotic	O
-Tsr3	O
-homologs	O
-.	O
-	O
-Mutations	O
-of	O
-the	O
-corresponding	O
-residue	O
-in	O
-SsTsr3	O
-to	O
-A	O
-(	O
-D63	O
-)	O
-does	O
-not	O
-significantly	O
-alter	O
-the	O
-SAM	O
--	O
-binding	O
-affinity	O
-of	O
-the	O
-protein	O
-(	O
-KD	O
-=	O
-11	O
-.	O
-0	O
-μM	O
-).	O
-	O
-However	O
-,	O
-the	O
-mutation	O
-of	O
-the	O
-corresponding	O
-residue	O
-of	O
-ScTsr3	O
-(	O
-E111A	B-mutant
-)	O
-leads	O
-to	O
-a	O
-significant	O
-decrease	O
-of	O
-the	O
-acp	O
-transferase	O
-activity	O
-in	O
-vivo	O
-(	O
-Figure	O
-5F	O
-).	O
-	O
-RNA	O
--	O
-binding	O
-of	O
-Tsr3	O
-	O
-Analysis	O
-of	O
-the	O
-electrostatic	O
-surface	O
-properties	O
-of	O
-VdTsr3	O
-clearly	O
-identified	O
-positively	O
-charged	O
-surface	O
-patches	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-SAM	O
--	O
-binding	O
-site	O
-suggesting	O
-a	O
-putative	O
-RNA	O
--	O
-binding	O
-site	O
-(	O
-Figure	O
-6A	O
-).	O
-	O
-Furthermore	O
-,	O
-a	O
-negatively	O
-charged	O
-MES	O
--	O
-ion	O
-is	O
-found	O
-in	O
-the	O
-crystal	O
-structure	O
-of	O
-VdTsr3	O
-complexed	O
-to	O
-the	O
-side	O
-chain	O
-of	O
-K22	O
-in	O
-helix	O
-α1	O
-.	O
-	O
-Its	O
-negatively	O
-charged	O
-sulfate	O
-group	O
-might	O
-mimic	O
-an	O
-RNA	O
-backbone	O
-phosphate	O
-.	O
-	O
-Helix	O
-α1	O
-contains	O
-two	O
-more	O
-positively	O
-charged	O
-amino	O
-acids	O
-K17	O
-and	O
-R25	O
-as	O
-does	O
-the	O
-loop	O
-preceding	O
-it	O
-(	O
-R9	O
-).	O
-	O
-A	O
-second	O
-cluster	O
-of	O
-positively	O
-charged	O
-residues	O
-is	O
-found	O
-in	O
-or	O
-near	O
-helix	O
-α3	O
-(	O
-K74	O
-,	O
-R75	O
-,	O
-K82	O
-,	O
-R85	O
-and	O
-K87	O
-).	O
-	O
-Some	O
-of	O
-these	O
-amino	O
-acids	O
-are	O
-conserved	O
-between	O
-archaeal	O
-and	O
-eukaryotic	O
-Tsr3	O
-(	O
-Supplementary	O
-Figure	O
-S1A	O
-).	O
-	O
-In	O
-the	O
-C	O
--	O
-terminal	O
-domain	O
-,	O
-the	O
-surface	O
-exposed	O
-α	O
--	O
-helices	O
-α5	O
-and	O
-α7	O
-carry	O
-a	O
-significant	O
-amount	O
-of	O
-positively	O
-charged	O
-amino	O
-acids	O
-.	O
-	O
-A	O
-triple	O
-mutation	O
-of	O
-the	O
-conserved	O
-positively	O
-charged	O
-residues	O
-R60	O
-,	O
-K65	O
-and	O
-R131	O
-to	O
-A	O
-in	O
-ScTsr3	O
-resulted	O
-in	O
-a	O
-protein	O
-with	O
-a	O
-significantly	O
-impaired	O
-acp	O
-transferase	O
-activity	O
-in	O
-vivo	O
-(	O
-Figure	O
-6D	O
-)	O
-in	O
-line	O
-with	O
-an	O
-important	O
-functional	O
-role	O
-for	O
-these	O
-positively	O
-charged	O
-residues	O
-.	O
-	O
-RNA	O
--	O
-binding	O
-of	O
-Tsr3	O
-.	O
-	O
-(	O
-A	O
-)	O
-Electrostatic	O
-charge	O
-distribution	O
-on	O
-the	O
-surface	O
-of	O
-VdTsr3	O
-.	O
-	O
-SAM	O
-is	O
-shown	O
-in	O
-a	O
-stick	O
-representation	O
-.	O
-	O
-Also	O
-shown	O
-in	O
-stick	O
-representation	O
-is	O
-a	O
-negatively	O
-charged	O
-MES	O
-ion	O
-.	O
-	O
-Conserved	O
-basic	O
-amino	O
-acids	O
-are	O
-labeled	O
-.	O
-(	O
-B	O
-)	O
-Comparison	O
-of	O
-the	O
-secondary	O
-structures	O
-of	O
-helix	O
-31	O
-from	O
-the	O
-small	O
-ribosomal	O
-subunit	O
-rRNAs	O
-in	O
-S	O
-.	O
-cerevisiae	O
-and	O
-S	O
-.	O
-solfataricus	O
-with	O
-the	O
-location	O
-of	O
-the	O
-hypermodified	O
-nucleotide	O
-indicated	O
-in	O
-red	O
-.	O
-	O
-For	O
-S	O
-.	O
-solfataricus	O
-the	O
-chemical	O
-identity	O
-of	O
-the	O
-hypermodified	O
-nucleotide	O
-is	O
-not	O
-known	O
-but	O
-the	O
-existence	O
-of	O
-NEP1	O
-and	O
-TSR3	O
-homologs	O
-suggest	O
-that	O
-it	O
-is	O
-indeed	O
-N1	O
--	O
-methyl	O
--	O
-N3	O
--	O
-acp	O
--	O
-pseudouridine	O
-.	O
-	O
-(	O
-C	O
-)	O
-Binding	O
-of	O
-SsTsr3	O
-to	O
-RNA	O
-.	O
-	O
-5	O
-′-	O
-fluoresceine	O
-labeled	O
-RNA	O
-oligonucleotides	O
-corresponding	O
-either	O
-to	O
-the	O
-native	O
-(	O
-20mer	O
-–	O
-see	O
-inset	O
-)	O
-or	O
-a	O
-stabilized	O
-(	O
-20mer_GC	O
--	O
-inset	O
-)	O
-helix	O
-31	O
-of	O
-the	O
-small	O
-ribosomal	O
-subunit	O
-rRNA	O
-from	O
-S	O
-.	O
-solfataricus	O
-were	O
-titrated	O
-with	O
-increasing	O
-amounts	O
-of	O
-SsTsr3	O
-and	O
-the	O
-changes	O
-in	O
-the	O
-fluoresceine	O
-fluorescence	O
-anisotropy	O
-were	O
-measured	O
-and	O
-fitted	O
-to	O
-a	O
-binding	O
-curve	O
-(	O
-20mer	O
-–	O
-red	O
-,	O
-20mer_GC	O
-–	O
-blue	O
-).	O
-	O
-Oligo	O
--	O
-U9	O
--	O
-RNA	O
-was	O
-used	O
-for	O
-comparison	O
-(	O
-black	O
-).	O
-	O
-The	O
-20mer_GC	O
-RNA	O
-was	O
-also	O
-titrated	O
-with	O
-SsTsr3	O
-in	O
-the	O
-presence	O
-of	O
-2	O
-mM	O
-SAM	O
-(	O
-purple	O
-).	O
-(	O
-D	O
-)	O
-Mutants	O
-of	O
-ScTsr3	O
-R60	O
-,	O
-K65	O
-or	O
-R131	O
-(	O
-equivalent	O
-to	O
-K17	O
-,	O
-K22	O
-and	O
-R91	O
-in	O
-VdTsr3	O
-)	O
-expressed	O
-in	O
-Δtsr3	B-mutant
-yeast	O
-cells	O
-show	O
-a	O
-primer	O
-extension	O
-stop	O
-comparable	O
-to	O
-the	O
-wild	O
-type	O
-.	O
-	O
-Combination	O
-of	O
-the	O
-three	O
-point	O
-mutations	O
-(	O
-R60A	B-mutant
-/	O
-K65A	B-mutant
-/	O
-R131A	B-mutant
-)	O
-leads	O
-to	O
-a	O
-strongly	O
-reduced	O
-acp	O
-modification	O
-of	O
-18S	O
-rRNA	O
-.	O
-	O
-In	O
-order	O
-to	O
-explore	O
-the	O
-RNA	O
--	O
-ligand	O
-specificity	O
-of	O
-Tsr3	O
-we	O
-titrated	O
-SsTsr3	O
-prepared	O
-in	O
-RNase	O
--	O
-free	O
-form	O
-with	O
-5	O
-′-	O
-fluoresceine	O
--	O
-labeled	O
-RNA	O
-and	O
-determined	O
-the	O
-affinity	O
-by	O
-fluorescence	O
-anisotropy	O
-measurements	O
-.	O
-	O
-SsTsr3	O
-in	O
-the	O
-apo	O
-state	O
-bound	O
-a	O
-20mer	O
-RNA	O
-corresponding	O
-to	O
-helix	O
-31	O
-of	O
-S	O
-.	O
-solfataricus	O
-16S	O
-rRNA	O
-(	O
-Figure	O
-6B	O
-)	O
-with	O
-a	O
-KD	O
-of	O
-1	O
-.	O
-9	O
-μM	O
-and	O
-to	O
-a	O
-version	O
-of	O
-this	O
-hairpin	O
-stabilized	O
-by	O
-additional	O
-GC	O
-base	O
-pairs	O
-(	O
-20mer	O
--	O
-GC	O
-)	O
-with	O
-a	O
-KD	O
-of	O
-0	O
-.	O
-6	O
-μM	O
-(	O
-Figure	O
-6C	O
-).	O
-	O
-A	O
-single	O
-stranded	O
-oligoU	O
--	O
-RNA	O
-bound	O
-with	O
-a	O
-10	O
--	O
-fold	O
--	O
-reduced	O
-affinity	O
-(	O
-6	O
-.	O
-0	O
-μM	O
-).	O
-	O
-The	O
-presence	O
-of	O
-saturating	O
-amounts	O
-of	O
-SAM	O
-(	O
-2	O
-mM	O
-)	O
-did	O
-not	O
-have	O
-a	O
-significant	O
-influence	O
-on	O
-the	O
-RNA	O
--	O
-affinity	O
-of	O
-SsTsr3	O
-(	O
-KD	O
-of	O
-1	O
-.	O
-7	O
-μM	O
-for	O
-the	O
-20mer	O
--	O
-GC	O
--	O
-RNA	O
-)	O
-suggesting	O
-no	O
-cooperativity	O
-in	O
-substrate	O
-binding	O
-.	O
-	O
-U1191	O
-is	O
-the	O
-only	O
-hypermodified	O
-base	O
-in	O
-the	O
-yeast	O
-18S	O
-rRNA	O
-and	O
-is	O
-strongly	O
-conserved	O
-in	O
-eukaryotes	O
-.	O
-	O
-The	O
-formation	O
-of	O
-1	O
--	O
-methyl	O
--	O
-3	O
--(	O
-3	O
--	O
-amino	O
--	O
-3	O
--	O
-carboxypropyl	O
-)-	O
-pseudouridine	O
-(	O
-m1acp3Ψ	O
-)	O
-is	O
-very	O
-complex	O
-requiring	O
-three	O
-successive	O
-modification	O
-reactions	O
-involving	O
-one	O
-H	O
-/	O
-ACA	O
-snoRNP	O
-(	O
-snR35	O
-)	O
-and	O
-two	O
-protein	O
-enzymes	O
-(	O
-Nep1	O
-/	O
-Emg1	O
-and	O
-Tsr3	O
-).	O
-	O
-This	O
-makes	O
-it	O
-unique	O
-in	O
-eukaryotic	O
-rRNA	O
-modification	O
-.	O
-	O
-The	O
-m1acp3Ψ	O
-base	O
-is	O
-located	O
-at	O
-the	O
-tip	O
-of	O
-helix	O
-31	O
-on	O
-the	O
-18S	O
-rRNA	O
-(	O
-Supplementary	O
-Figure	O
-S1B	O
-)	O
-which	O
-,	O
-together	O
-with	O
-helices	O
-18	O
-,	O
-24	O
-,	O
-34	O
-and	O
-44	O
-,	O
-contribute	O
-to	O
-building	O
-the	O
-decoding	O
-center	O
-of	O
-the	O
-small	O
-ribosomal	O
-subunit	O
-.	O
-	O
-A	O
-similar	O
-modification	O
-(	O
-acp3U	O
-)	O
-was	O
-identified	O
-in	O
-Haloferax	O
-volcanii	O
-and	O
-corresponding	O
-modified	O
-nucleotides	O
-were	O
-also	O
-shown	O
-to	O
-occur	O
-in	O
-other	O
-archaea	O
-.	O
-	O
-As	O
-shown	O
-here	O
-TSR3	O
-encodes	O
-the	O
-transferase	O
-catalyzing	O
-the	O
-acp	O
-modification	O
-as	O
-the	O
-last	O
-step	O
-in	O
-the	O
-biosynthesis	O
-of	O
-m1acp3Ψ	O
-in	O
-yeast	O
-and	O
-human	O
-cells	O
-.	O
-	O
-Unexpectedly	O
-,	O
-archaeal	O
-Tsr3	O
-has	O
-a	O
-structure	O
-similar	O
-to	O
-SPOUT	O
--	O
-class	O
-RNA	O
-methyltransferases	O
-,	O
-and	O
-it	O
-is	O
-the	O
-first	O
-example	O
-for	O
-an	O
-enzyme	O
-of	O
-this	O
-class	O
-transferring	O
-an	O
-acp	O
-group	O
-,	O
-due	O
-to	O
-a	O
-modified	O
-SAM	O
--	O
-binding	O
-pocket	O
-that	O
-exposes	O
-the	O
-acp	O
-instead	O
-of	O
-the	O
-methyl	O
-group	O
-of	O
-SAM	O
-to	O
-its	O
-RNA	O
-substrate	O
-.	O
-	O
-Similar	O
-to	O
-the	O
-structurally	O
-unrelated	O
-Rossmann	O
--	O
-fold	O
-Tyw2	O
-acp	O
-transferase	O
-,	O
-the	O
-SAM	O
-methyl	O
-group	O
-of	O
-Tsr3	O
-is	O
-bound	O
-in	O
-an	O
-inaccessible	O
-hydrophobic	O
-pocket	O
-whereas	O
-the	O
-acp	O
-side	O
-chain	O
-becomes	O
-accessible	O
-for	O
-a	O
-nucleophilic	O
-attack	O
-by	O
-the	O
-N3	O
-of	O
-pseudouridine	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-in	O
-the	O
-structurally	O
-closely	O
-related	O
-RNA	O
-methyltransferase	O
-Trm10	O
-the	O
-methyl	O
-group	O
-of	O
-the	O
-cofactor	O
-SAM	O
-is	O
-accessible	O
-whereas	O
-its	O
-acp	O
-side	O
-chain	O
-is	O
-buried	O
-inside	O
-the	O
-protein	O
-.	O
-	O
-This	O
-suggests	O
-that	O
-enzymes	O
-with	O
-a	O
-SAM	O
--	O
-dependent	O
-acp	O
-transferase	O
-activity	O
-might	O
-have	O
-evolved	O
-from	O
-SAM	O
--	O
-dependent	O
-methyltransferases	O
-by	O
-slight	O
-modifications	O
-of	O
-the	O
-SAM	O
--	O
-binding	O
-pocket	O
-.	O
-	O
-Thus	O
-,	O
-additional	O
-examples	O
-for	O
-acp	O
-transferase	O
-enzymes	O
-might	O
-be	O
-found	O
-with	O
-similarities	O
-to	O
-other	O
-structural	O
-classes	O
-of	O
-methyltransferases	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-Nep1	O
-,	O
-the	O
-enzyme	O
-preceding	O
-Tsr3	O
-in	O
-the	O
-m1acp3Ψ	O
-biosynthesis	O
-pathway	O
-,	O
-Tsr3	O
-binds	O
-rather	O
-weakly	O
-and	O
-with	O
-little	O
-specificity	O
-to	O
-its	O
-isolated	O
-substrate	O
-RNA	O
-.	O
-	O
-This	O
-suggests	O
-that	O
-Tsr3	O
-is	O
-not	O
-stably	O
-incorporated	O
-into	O
-pre	O
--	O
-ribosomal	O
-particles	O
-and	O
-that	O
-its	O
-binding	O
-to	O
-the	O
-nascent	O
-ribosomal	O
-subunit	O
-possibly	O
-requires	O
-additional	O
-interactions	O
-with	O
-other	O
-pre	O
--	O
-ribosomal	O
-components	O
-.	O
-	O
-Consistently	O
-,	O
-in	O
-sucrose	O
-gradient	O
-analysis	O
-,	O
-Tsr3	O
-was	O
-found	O
-in	O
-low	O
--	O
-molecular	O
-weight	O
-fractions	O
-rather	O
-than	O
-with	O
-pre	O
--	O
-ribosome	O
-containing	O
-high	O
--	O
-molecular	O
-weight	O
-fractions	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-several	O
-enzymes	O
-that	O
-catalyze	O
-base	O
-specific	O
-modifications	O
-in	O
-rRNAs	O
-Tsr3	O
-is	O
-not	O
-an	O
-essential	O
-protein	O
-.	O
-	O
-Typically	O
-,	O
-other	O
-small	O
-subunit	O
-rRNA	O
-methyltransferases	O
-as	O
-Dim1	O
-,	O
-Bud23	O
-and	O
-Nep1	O
-/	O
-Emg1	O
-carry	O
-dual	O
-functions	O
-,	O
-in	O
-ribosome	O
-biogenesis	O
-and	O
-rRNA	O
-modification	O
-,	O
-and	O
-it	O
-is	O
-their	O
-involvement	O
-in	O
-pre	O
--	O
-RNA	O
-processing	O
-that	O
-is	O
-essential	O
-rather	O
-than	O
-their	O
-RNA	O
--	O
-methylating	O
-activity	O
-(,	O
-discussed	O
-in	O
-7	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-for	O
-several	O
-Tsr3	O
-mutants	O
-(	O
-SAM	O
--	O
-binding	O
-and	O
-cysteine	O
-mutations	O
-)	O
-we	O
-found	O
-a	O
-systematic	O
-correlation	O
-between	O
-the	O
-loss	O
-of	O
-acp	O
-modification	O
-and	O
-the	O
-efficiency	O
-of	O
-18S	O
-rRNA	O
-maturation	O
-.	O
-	O
-This	O
-demonstrates	O
-that	O
-,	O
-unlike	O
-the	O
-other	O
-small	O
-subunit	O
-rRNA	O
-base	O
-modifications	O
-,	O
-the	O
-acp	O
-modification	O
-is	O
-required	O
-for	O
-efficient	O
-pre	O
--	O
-rRNA	O
-processing	O
-.	O
-	O
-Recently	O
-,	O
-structural	O
-,	O
-functional	O
-,	O
-and	O
-CRAC	O
-(	O
-cross	O
--	O
-linking	O
-and	O
-cDNA	O
-analysis	O
-)	O
-experiments	O
-of	O
-late	O
-assembly	O
-factors	O
-involved	O
-in	O
-cytoplasmic	O
-processing	O
-of	O
-40S	O
-subunits	O
-,	O
-along	O
-with	O
-cryo	O
--	O
-EM	O
-studies	O
-of	O
-the	O
-late	O
-pre	O
--	O
-40S	O
-subunits	O
-have	O
-provided	O
-important	O
-insights	O
-into	O
-late	O
-pre	O
--	O
-40S	O
-processing	O
-.	O
-	O
-Apart	O
-from	O
-most	O
-of	O
-the	O
-ribosomal	O
-proteins	O
-,	O
-cytoplasmic	O
-pre	O
--	O
-40S	O
-particles	O
-contain	O
-20S	O
-rRNA	O
-and	O
-at	O
-least	O
-seven	O
-non	O
--	O
-ribosomal	O
-proteins	O
-including	O
-the	O
-D	O
--	O
-site	O
-endonuclease	O
-Nob1	O
-as	O
-well	O
-as	O
-Tsr1	O
-,	O
-a	O
-putative	O
-GTPase	O
-and	O
-Rio2	O
-which	O
-block	O
-the	O
-mRNA	O
-channel	O
-and	O
-the	O
-initiator	O
-tRNA	O
-binding	O
-site	O
-,	O
-respectively	O
-,	O
-thus	O
-preventing	O
-translation	O
-initiation	O
-.	O
-	O
-After	O
-structural	O
-changes	O
-,	O
-possibly	O
-driven	O
-by	O
-GTP	O
-hydrolysis	O
-,	O
-which	O
-go	O
-together	O
-with	O
-the	O
-formation	O
-of	O
-the	O
-decoding	O
-site	O
-,	O
-the	O
-20S	O
-pre	O
--	O
-rRNA	O
-becomes	O
-accessible	O
-for	O
-Nob1	O
-cleavage	O
-at	O
-site	O
-D	O
-.	O
-This	O
-also	O
-involves	O
-joining	O
-of	O
-pre	O
--	O
-40S	O
-and	O
-60S	O
-subunits	O
-to	O
-80S	O
--	O
-like	O
-particles	O
-in	O
-a	O
-translation	O
--	O
-like	O
-cycle	O
-promoted	O
-by	O
-eIF5B	O
-.	O
-	O
-The	O
-cleavage	O
-step	O
-most	O
-likely	O
-acts	O
-as	O
-a	O
-quality	O
-control	O
-check	O
-that	O
-ensures	O
-the	O
-proper	O
-40S	O
-subunit	O
-assembly	O
-with	O
-only	O
-completely	O
-processed	O
-precursors	O
-.	O
-	O
-Finally	O
-,	O
-termination	O
-factor	O
-Rli1	O
-,	O
-an	O
-ATPase	O
-,	O
-promotes	O
-the	O
-dissociation	O
-of	O
-assembly	O
-factors	O
-and	O
-the	O
-80S	O
--	O
-like	O
-complex	O
-dissociates	O
-and	O
-releases	O
-the	O
-mature	O
-40S	O
-subunit	O
-.	O
-	O
-Interestingly	O
-,	O
-differences	O
-in	O
-the	O
-level	O
-of	O
-acp	O
-modification	O
-were	O
-demonstrated	O
-for	O
-different	O
-steps	O
-of	O
-the	O
-cytoplasmic	O
-pre	O
--	O
-40S	O
-subunit	O
-maturation	O
-after	O
-analyzing	O
-purified	O
-20S	O
-pre	O
--	O
-rRNAs	O
-using	O
-different	O
-purification	O
-bait	O
-proteins	O
-.	O
-	O
-Early	O
-cytoplasmic	O
-pre	O
--	O
-40S	O
-subunits	O
-still	O
-containing	O
-the	O
-ribosome	O
-assembly	O
-factors	O
-Tsr1	O
-,	O
-Ltv1	O
-,	O
-Enp1	O
-and	O
-Rio2	O
-were	O
-not	O
-or	O
-only	O
-partially	O
-acp	O
-modified	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-late	O
-pre	O
--	O
-40S	O
-subunits	O
-containing	O
-Nob1	O
-and	O
-Rio1	O
-or	O
-already	O
-associated	O
-with	O
-60S	O
-subunits	O
-in	O
-80S	O
--	O
-like	O
-particles	O
-showed	O
-acp	O
-modification	O
-levels	O
-comparable	O
-to	O
-mature	O
-40S	O
-subunits	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-acp	O
-transfer	O
-to	O
-m1Ψ1191	O
-occurs	O
-during	O
-the	O
-step	O
-at	O
-which	O
-Rio2	O
-leaves	O
-the	O
-pre	O
--	O
-40S	O
-particle	O
-.	O
-	O
-These	O
-data	O
-and	O
-the	O
-finding	O
-that	O
-a	O
-missing	O
-acp	O
-modification	O
-hinders	O
-pre	O
--	O
-20S	O
-rRNA	O
-processing	O
-,	O
-suggest	O
-that	O
-the	O
-acp	O
-modification	O
-together	O
-with	O
-the	O
-release	O
-of	O
-Rio2	O
-promotes	O
-the	O
-formation	O
-of	O
-the	O
-decoding	O
-site	O
-and	O
-thus	O
-D	O
--	O
-site	O
-cleavage	O
-by	O
-Nob1	O
-.	O
-	O
-The	O
-interrelation	O
-between	O
-acp	O
-modification	O
-and	O
-Rio2	O
-release	O
-is	O
-also	O
-supported	O
-by	O
-CRAC	O
-analysis	O
-showing	O
-that	O
-Rio2	O
-binds	O
-to	O
-helix	O
-31	O
-next	O
-to	O
-the	O
-Ψ1191	O
-residue	O
-that	O
-receives	O
-the	O
-acp	O
-modification	O
-.	O
-	O
-Therefore	O
-,	O
-Rio2	O
-either	O
-blocks	O
-the	O
-access	O
-of	O
-Tsr3	O
-to	O
-helix	O
-31	O
-,	O
-and	O
-acp	O
-modification	O
-can	O
-only	O
-occur	O
-after	O
-Rio2	O
-is	O
-released	O
-,	O
-or	O
-the	O
-acp	O
-modification	O
-of	O
-m1Ψ1191	O
-and	O
-putative	O
-subsequent	O
-conformational	O
-changes	O
-of	O
-20S	O
-rRNA	O
-weaken	O
-the	O
-binding	O
-of	O
-Rio2	O
-to	O
-helix	O
-31	O
-and	O
-support	O
-its	O
-release	O
-from	O
-the	O
-pre	O
--	O
-rRNA	O
-.	O
-	O
-In	O
-summary	O
-,	O
-by	O
-identifying	O
-Tsr3	O
-as	O
-the	O
-enzyme	O
-responsible	O
-for	O
-introducing	O
-the	O
-acp	O
-group	O
-to	O
-the	O
-hypermodified	O
-m1acp3Ψ	O
-nucleotide	O
-at	O
-position	O
-1191	O
-(	O
-yeast	O
-)/	O
-1248	O
-(	O
-humans	O
-)	O
-of	O
-18S	O
-rRNA	O
-we	O
-added	O
-one	O
-of	O
-the	O
-last	O
-remaining	O
-pieces	O
-to	O
-the	O
-puzzle	O
-of	O
-eukaryotic	O
-small	O
-ribosomal	O
-subunit	O
-rRNA	O
-modifications	O
-.	O
-	O
-The	O
-current	O
-data	O
-together	O
-with	O
-the	O
-finding	O
-that	O
-acp	O
-modification	O
-takes	O
-place	O
-at	O
-the	O
-very	O
-last	O
-step	O
-in	O
-pre	O
--	O
-40S	O
-subunit	O
-maturation	O
-indicate	O
-that	O
-the	O
-acp	O
-modification	O
-probably	O
-supports	O
-the	O
-formation	O
-of	O
-the	O
-decoding	O
-site	O
-and	O
-efficient	O
-20S	O
-pre	O
--	O
-rRNA	O
-D	O
--	O
-site	O
-cleavage	O
-.	O
-	O
-Furthermore	O
-,	O
-our	O
-structural	O
-data	O
-unravelled	O
-how	O
-the	O
-regioselectivity	O
-of	O
-SAM	O
--	O
-dependent	O
-group	O
-transfer	O
-reactions	O
-can	O
-be	O
-tuned	O
-by	O
-distinct	O
-small	O
-evolutionary	O
-adaptions	O
-of	O
-the	O
-ligand	O
-binding	O
-pocket	O
-of	O
-SAM	O
--	O
-binding	O
-enzymes	O
-.	O
-	O
-Structural	O
-insights	O
-into	O
-the	O
-regulatory	O
-mechanism	O
-of	O
-the	O
-Pseudomonas	O
-aeruginosa	O
-YfiBNR	O
-system	O
-	O
-YfiBNR	O
-is	O
-a	O
-recently	O
-identified	O
-bis	O
--(	O
-3	O
-’-	O
-5	O
-’)-	O
-cyclic	O
-dimeric	O
-GMP	O
-(	O
-c	O
--	O
-di	O
--	O
-GMP	O
-)	O
-signaling	O
-system	O
-in	O
-opportunistic	O
-pathogens	O
-.	O
-	O
-In	O
-response	O
-to	O
-cell	O
-stress	O
-,	O
-YfiB	O
-in	O
-the	O
-outer	O
-membrane	O
-can	O
-sequester	O
-the	O
-periplasmic	O
-protein	O
-YfiR	O
-,	O
-releasing	O
-its	O
-inhibition	O
-of	O
-YfiN	O
-on	O
-the	O
-inner	O
-membrane	O
-and	O
-thus	O
-provoking	O
-the	O
-diguanylate	O
-cyclase	O
-activity	O
-of	O
-YfiN	O
-to	O
-induce	O
-c	O
--	O
-di	O
--	O
-GMP	O
-production	O
-.	O
-	O
-Here	O
-,	O
-we	O
-report	O
-the	O
-crystal	O
-structures	O
-of	O
-YfiB	O
-alone	O
-and	O
-of	O
-an	O
-active	O
-mutant	O
-YfiBL43P	B-mutant
-complexed	O
-with	O
-YfiR	O
-with	O
-2	O
-:	O
-2	O
-stoichiometry	O
-.	O
-	O
-Structural	O
-analyses	O
-revealed	O
-that	O
-in	O
-contrast	O
-to	O
-the	O
-compact	O
-conformation	O
-of	O
-the	O
-dimeric	O
-YfiB	O
-alone	O
-,	O
-YfiBL43P	B-mutant
-adopts	O
-a	O
-stretched	O
-conformation	O
-allowing	O
-activated	O
-YfiB	O
-to	O
-penetrate	O
-the	O
-peptidoglycan	O
-(	O
-PG	O
-)	O
-layer	O
-and	O
-access	O
-YfiR	O
-.	O
-YfiBL43P	B-mutant
-shows	O
-a	O
-more	O
-compact	O
-PG	O
--	O
-binding	O
-pocket	O
-and	O
-much	O
-higher	O
-PG	O
-binding	O
-affinity	O
-than	O
-wild	O
--	O
-type	O
-YfiB	O
-,	O
-suggesting	O
-a	O
-tight	O
-correlation	O
-between	O
-PG	O
-binding	O
-and	O
-YfiB	O
-activation	O
-.	O
-	O
-In	O
-addition	O
-,	O
-our	O
-crystallographic	O
-analyses	O
-revealed	O
-that	O
-YfiR	O
-binds	O
-Vitamin	O
-B6	O
-(	O
-VB6	O
-)	O
-or	O
-L	O
--	O
-Trp	O
-at	O
-a	O
-YfiB	O
--	O
-binding	O
-site	O
-and	O
-that	O
-both	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-are	O
-able	O
-to	O
-reduce	O
-YfiBL43P	B-mutant
--	O
-induced	O
-biofilm	O
-formation	O
-.	O
-	O
-Based	O
-on	O
-the	O
-structural	O
-and	O
-biochemical	O
-data	O
-,	O
-we	O
-propose	O
-an	O
-updated	O
-regulatory	O
-model	O
-of	O
-the	O
-YfiBNR	O
-system	O
-.	O
-	O
-Bis	O
--(	O
-3	O
-’-	O
-5	O
-’)-	O
-cyclic	O
-dimeric	O
-GMP	O
-(	O
-c	O
--	O
-di	O
--	O
-GMP	O
-)	O
-is	O
-a	O
-ubiquitous	O
-second	O
-messenger	O
-that	O
-bacteria	O
-use	O
-to	O
-facilitate	O
-behavioral	O
-adaptations	O
-to	O
-their	O
-ever	O
--	O
-changing	O
-environment	O
-.	O
-	O
-An	O
-increase	O
-in	O
-c	O
--	O
-di	O
--	O
-GMP	O
-promotes	O
-biofilm	O
-formation	O
-,	O
-and	O
-a	O
-decrease	O
-results	O
-in	O
-biofilm	O
-degradation	O
-(	O
-Boehm	O
-et	O
-al	O
-.,;	O
-Duerig	O
-et	O
-al	O
-.,;	O
-Hickman	O
-et	O
-al	O
-.,;	O
-Jenal	O
-,;	O
-Romling	O
-et	O
-al	O
-.,).	O
-	O
-The	O
-c	O
--	O
-di	O
--	O
-GMP	O
-level	O
-is	O
-regulated	O
-by	O
-two	O
-reciprocal	O
-enzyme	O
-systems	O
-,	O
-namely	O
-,	O
-diguanylate	O
-cyclases	O
-(	O
-DGCs	O
-)	O
-that	O
-synthesize	O
-c	O
--	O
-di	O
--	O
-GMP	O
-and	O
-phosphodiesterases	O
-(	O
-PDEs	O
-)	O
-that	O
-hydrolyze	O
-c	O
--	O
-di	O
--	O
-GMP	O
-(	O
-Kulasakara	O
-et	O
-al	O
-.,;	O
-Ross	O
-et	O
-al	O
-.,;	O
-Ross	O
-et	O
-al	O
-.,).	O
-Many	O
-of	O
-these	O
-enzymes	O
-are	O
-multiple	O
--	O
-domain	O
-proteins	O
-containing	O
-a	O
-variable	O
-N	O
--	O
-terminal	O
-domain	O
-that	O
-commonly	O
-acts	O
-as	O
-a	O
-signal	O
-sensor	O
-or	O
-transduction	O
-module	O
-,	O
-followed	O
-by	O
-the	O
-relatively	O
-conserved	O
-GGDEF	O
-motif	O
-in	O
-DGCs	O
-or	O
-EAL	O
-/	O
-HD	O
--	O
-GYP	O
-domains	O
-in	O
-PDEs	O
-(	O
-Hengge	O
-,;	O
-Navarro	O
-et	O
-al	O
-.,;	O
-Schirmer	O
-and	O
-Jenal	O
-,).	O
-	O
-Intriguingly	O
-,	O
-studies	O
-in	O
-diverse	O
-species	O
-have	O
-revealed	O
-that	O
-a	O
-single	O
-bacterium	O
-can	O
-have	O
-dozens	O
-of	O
-DGCs	O
-and	O
-PDEs	O
-(	O
-Hickman	O
-et	O
-al	O
-.,;	O
-Kirillina	O
-et	O
-al	O
-.,;	O
-Kulasakara	O
-et	O
-al	O
-.,;	O
-Tamayo	O
-et	O
-al	O
-.,).	O
-	O
-In	O
-Pseudomonas	O
-aeruginosa	O
-in	O
-particular	O
-,	O
-42	O
-genes	O
-containing	O
-putative	O
-DGCs	O
-and	O
-/	O
-or	O
-PDEs	O
-were	O
-identified	O
-(	O
-Kulasakara	O
-et	O
-al	O
-.,).	O
-	O
-The	O
-functional	O
-role	O
-of	O
-a	O
-number	O
-of	O
-downstream	O
-effectors	O
-of	O
-c	O
--	O
-di	O
--	O
-GMP	O
-has	O
-been	O
-characterized	O
-as	O
-affecting	O
-exopolysaccharide	O
-(	O
-EPS	O
-)	O
-production	O
-,	O
-transcription	O
-,	O
-motility	O
-,	O
-and	O
-surface	O
-attachment	O
-(	O
-Caly	O
-et	O
-al	O
-.,;	O
-Camilli	O
-and	O
-Bassler	O
-,;	O
-Ha	O
-and	O
-O	O
-’	O
-Toole	O
-,;	O
-Pesavento	O
-and	O
-Hengge	O
-,).	O
-	O
-However	O
-,	O
-due	O
-to	O
-the	O
-intricacy	O
-of	O
-c	O
--	O
-di	O
--	O
-GMP	O
-signaling	O
-networks	O
-and	O
-the	O
-diversity	O
-of	O
-experimental	O
-cues	O
-,	O
-the	O
-detailed	O
-mechanisms	O
-by	O
-which	O
-these	O
-signaling	O
-pathways	O
-specifically	O
-sense	O
-and	O
-integrate	O
-different	O
-inputs	O
-remain	O
-largely	O
-elusive	O
-.	O
-	O
-Biofilm	O
-formation	O
-protects	O
-pathogenic	O
-bacteria	O
-from	O
-antibiotic	O
-treatment	O
-,	O
-and	O
-c	O
--	O
-di	O
--	O
-GMP	O
--	O
-regulated	O
-biofilm	O
-formation	O
-has	O
-been	O
-extensively	O
-studied	O
-in	O
-P	O
-.	O
-aeruginosa	O
-(	O
-Evans	O
-,;	O
-Kirisits	O
-et	O
-al	O
-.,;	O
-Malone	O
-,;	O
-Reinhardt	O
-et	O
-al	O
-.,).	O
-	O
-In	O
-the	O
-lungs	O
-of	O
-cystic	O
-fibrosis	O
-(	O
-CF	O
-)	O
-patients	O
-,	O
-adherent	O
-biofilm	O
-formation	O
-and	O
-the	O
-appearance	O
-of	O
-small	O
-colony	O
-variant	O
-(	O
-SCV	O
-)	O
-morphologies	O
-of	O
-P	O
-.	O
-aeruginosa	O
-correlate	O
-with	O
-prolonged	O
-persistence	O
-of	O
-infection	O
-and	O
-poor	O
-lung	O
-function	O
-(	O
-Govan	O
-and	O
-Deretic	O
-,;	O
-Haussler	O
-et	O
-al	O
-.,;	O
-Haussler	O
-et	O
-al	O
-.,;	O
-Parsek	O
-and	O
-Singh	O
-,;	O
-Smith	O
-et	O
-al	O
-.,).	O
-	O
-Recently	O
-,	O
-Malone	O
-and	O
-coworkers	O
-identified	O
-the	O
-tripartite	O
-c	O
--	O
-di	O
--	O
-GMP	O
-signaling	O
-module	O
-system	O
-YfiBNR	O
-(	O
-also	O
-known	O
-as	O
-AwsXRO	O
-(	O
-Beaumont	O
-et	O
-al	O
-.,;	O
-Giddens	O
-et	O
-al	O
-.,)	O
-or	O
-Tbp	O
-(	O
-Ueda	O
-and	O
-Wood	O
-,))	O
-by	O
-genetic	O
-screening	O
-for	O
-mutants	O
-that	O
-displayed	O
-SCV	O
-phenotypes	O
-in	O
-P	O
-.	O
-aeruginosa	O
-PAO1	O
-(	O
-Malone	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-The	O
-YfiBNR	O
-system	O
-contains	O
-three	O
-protein	O
-members	O
-and	O
-modulates	O
-intracellular	O
-c	O
--	O
-di	O
--	O
-GMP	O
-levels	O
-in	O
-response	O
-to	O
-signals	O
-received	O
-in	O
-the	O
-periplasm	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-More	O
-recently	O
-,	O
-this	O
-system	O
-was	O
-also	O
-reported	O
-in	O
-other	O
-Gram	O
--	O
-negative	O
-bacteria	O
-,	O
-such	O
-as	O
-Escherichia	O
-coli	O
-(	O
-Hufnagel	O
-et	O
-al	O
-.,;	O
-Raterman	O
-et	O
-al	O
-.,;	O
-Sanchez	O
--	O
-Torres	O
-et	O
-al	O
-.,),	O
-Klebsiella	O
-pneumonia	O
-(	O
-Huertas	O
-et	O
-al	O
-.,)	O
-and	O
-Yersinia	O
-pestis	O
-(	O
-Ren	O
-et	O
-al	O
-.,).	O
-	O
-YfiN	O
-is	O
-an	O
-integral	O
-inner	O
--	O
-membrane	O
-protein	O
-with	O
-two	O
-potential	O
-transmembrane	O
-helices	O
-,	O
-a	O
-periplasmic	O
-Per	O
--	O
-Arnt	O
--	O
-Sim	O
-(	O
-PAS	O
-)	O
-domain	O
-,	O
-and	O
-cytosolic	O
-domains	O
-containing	O
-a	O
-HAMP	O
-domain	O
-(	O
-mediate	O
-input	O
--	O
-output	O
-signaling	O
-in	O
-histidine	O
-kinases	O
-,	O
-adenylyl	O
-cyclases	O
-,	O
-methyl	O
--	O
-accepting	O
-chemotaxis	O
-proteins	O
-,	O
-and	O
-phosphatases	O
-)	O
-and	O
-a	O
-C	O
--	O
-terminal	O
-GGDEF	O
-domain	O
-indicating	O
-a	O
-DGC	O
-’	O
-s	O
-function	O
-(	O
-Giardina	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-YfiN	O
-is	O
-repressed	O
-by	O
-specific	O
-interaction	O
-between	O
-its	O
-periplasmic	O
-PAS	O
-domain	O
-and	O
-the	O
-periplasmic	O
-protein	O
-YfiR	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-YfiB	O
-is	O
-an	O
-OmpA	O
-/	O
-Pal	O
--	O
-like	O
-outer	O
--	O
-membrane	O
-lipoprotein	O
-(	O
-Parsons	O
-et	O
-al	O
-.,)	O
-that	O
-can	O
-activate	O
-YfiN	O
-by	O
-sequestering	O
-YfiR	O
-(	O
-Malone	O
-et	O
-al	O
-.,)	O
-in	O
-an	O
-unknown	O
-manner	O
-.	O
-	O
-Whether	O
-YfiB	O
-directly	O
-recruits	O
-YfiR	O
-or	O
-recruits	O
-YfiR	O
-via	O
-a	O
-third	O
-partner	O
-is	O
-an	O
-open	O
-question	O
-.	O
-	O
-After	O
-the	O
-sequestration	O
-of	O
-YfiR	O
-by	O
-YfiB	O
-,	O
-the	O
-c	O
--	O
-di	O
--	O
-GMP	O
-produced	O
-by	O
-activated	O
-YfiN	O
-increases	O
-the	O
-biosynthesis	O
-of	O
-the	O
-Pel	O
-and	O
-Psl	O
-EPSs	O
-,	O
-resulting	O
-in	O
-the	O
-appearance	O
-of	O
-the	O
-SCV	O
-phenotype	O
-,	O
-which	O
-indicates	O
-enhanced	O
-biofilm	O
-formation	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-It	O
-has	O
-been	O
-reported	O
-that	O
-the	O
-activation	O
-of	O
-YfiN	O
-may	O
-be	O
-induced	O
-by	O
-redox	O
--	O
-driven	O
-misfolding	O
-of	O
-YfiR	O
-(	O
-Giardina	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-It	O
-is	O
-also	O
-proposed	O
-that	O
-the	O
-sequestration	O
-of	O
-YfiR	O
-by	O
-YfiB	O
-can	O
-be	O
-induced	O
-by	O
-certain	O
-YfiB	O
--	O
-mediated	O
-cell	O
-wall	O
-stress	O
-,	O
-and	O
-mutagenesis	O
-studies	O
-revealed	O
-a	O
-number	O
-of	O
-activation	O
-residues	O
-of	O
-YfiB	O
-that	O
-were	O
-located	O
-in	O
-close	O
-proximity	O
-to	O
-the	O
-predicted	O
-first	O
-helix	O
-of	O
-the	O
-periplasmic	O
-domain	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-In	O
-addition	O
-,	O
-quorum	O
-sensing	O
--	O
-related	O
-dephosphorylation	O
-of	O
-the	O
-PAS	O
-domain	O
-of	O
-YfiN	O
-may	O
-also	O
-be	O
-involved	O
-in	O
-the	O
-regulation	O
-(	O
-Ueda	O
-and	O
-Wood	O
-,;	O
-Xu	O
-et	O
-al	O
-.,).	O
-	O
-Recently	O
-,	O
-we	O
-solved	O
-the	O
-crystal	O
-structure	O
-of	O
-YfiR	O
-in	O
-both	O
-the	O
-non	O
--	O
-oxidized	O
-and	O
-the	O
-oxidized	O
-states	O
-,	O
-revealing	O
-breakage	O
-/	O
-formation	O
-of	O
-one	O
-disulfide	O
-bond	O
-(	O
-Cys71	O
--	O
-Cys110	O
-)	O
-and	O
-local	O
-conformational	O
-change	O
-around	O
-the	O
-other	O
-one	O
-(	O
-Cys145	O
--	O
-Cys152	O
-),	O
-indicating	O
-that	O
-Cys145	O
--	O
-Cys152	O
-plays	O
-an	O
-important	O
-role	O
-in	O
-maintaining	O
-the	O
-correct	O
-folding	O
-of	O
-YfiR	O
-(	O
-Yang	O
-et	O
-al	O
-.,).	O
-	O
-In	O
-the	O
-present	O
-study	O
-,	O
-we	O
-solved	O
-the	O
-crystal	O
-structures	O
-of	O
-an	O
-N	O
--	O
-terminal	O
-truncated	O
-form	O
-of	O
-YfiB	O
-(	O
-34	O
-–	O
-168	O
-)	O
-and	O
-YfiR	O
-in	O
-complex	O
-with	O
-an	O
-active	O
-mutant	O
-YfiBL43P	B-mutant
-.	O
-	O
-Most	O
-recently	O
-,	O
-Li	O
-and	O
-coworkers	O
-reported	O
-the	O
-crystal	O
-structures	O
-of	O
-YfiB	O
-(	O
-27	O
-–	O
-168	O
-)	O
-alone	O
-and	O
-YfiRC71S	B-mutant
-in	O
-complex	O
-with	O
-YfiB	O
-(	O
-59	O
-–	O
-168	O
-)	O
-(	O
-Li	O
-et	O
-al	O
-.,).	O
-	O
-Compared	O
-with	O
-the	O
-reported	O
-complex	O
-structure	O
-,	O
-YfiBL43P	B-mutant
-in	O
-our	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-structure	O
-has	O
-additional	O
-visible	O
-N	O
--	O
-terminal	O
-residues	O
-44	O
-–	O
-58	O
-that	O
-are	O
-shown	O
-to	O
-play	O
-essential	O
-roles	O
-in	O
-YfiB	O
-activation	O
-and	O
-biofilm	O
-formation	O
-.	O
-	O
-Therefore	O
-,	O
-we	O
-are	O
-able	O
-to	O
-visualize	O
-the	O
-detailed	O
-allosteric	O
-arrangement	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-structure	O
-of	O
-YfiB	O
-and	O
-its	O
-important	O
-role	O
-in	O
-YfiB	O
--	O
-YfiR	O
-interaction	O
-.	O
-	O
-In	O
-addition	O
-,	O
-we	O
-found	O
-that	O
-the	O
-YfiBL43P	B-mutant
-shows	O
-a	O
-much	O
-higher	O
-PG	O
--	O
-binding	O
-affinity	O
-than	O
-wild	O
--	O
-type	O
-YfiB	O
-,	O
-most	O
-likely	O
-due	O
-to	O
-its	O
-more	O
-compact	O
-PG	O
--	O
-binding	O
-pocket	O
-.	O
-	O
-Moreover	O
-,	O
-we	O
-found	O
-that	O
-Vitamin	O
-B6	O
-(	O
-VB6	O
-)	O
-or	O
-L	O
--	O
-Trp	O
-can	O
-bind	O
-YfiR	O
-with	O
-an	O
-affinity	O
-in	O
-the	O
-ten	O
-millimolar	O
-range	O
-.	O
-	O
-Together	O
-with	O
-functional	O
-data	O
-,	O
-these	O
-results	O
-provide	O
-new	O
-mechanistic	O
-insights	O
-into	O
-how	O
-activated	O
-YfiB	O
-sequesters	O
-YfiR	O
-and	O
-releases	O
-the	O
-suppression	O
-of	O
-YfiN	O
-.	O
-These	O
-findings	O
-may	O
-facilitate	O
-the	O
-development	O
-and	O
-optimization	O
-of	O
-anti	O
--	O
-biofilm	O
-drugs	O
-for	O
-the	O
-treatment	O
-of	O
-chronic	O
-infections	O
-.	O
-	O
-Overall	O
-structure	O
-of	O
-YfiB	O
-	O
-We	O
-obtained	O
-two	O
-crystal	O
-forms	O
-of	O
-YfiB	O
-(	O
-residues	O
-34	O
-–	O
-168	O
-,	O
-lacking	O
-the	O
-signal	O
-peptide	O
-from	O
-residues	O
-1	O
-–	O
-26	O
-and	O
-periplasmic	O
-residues	O
-27	O
-–	O
-33	O
-),	O
-crystal	O
-forms	O
-I	O
-and	O
-II	O
-,	O
-belonging	O
-to	O
-space	O
-groups	O
-P21	O
-and	O
-P41	O
-,	O
-respectively	O
-.	O
-	O
-Overall	O
-structure	O
-of	O
-YfiB	O
-.	O
-(	O
-A	O
-)	O
-The	O
-overall	O
-structure	O
-of	O
-the	O
-YfiB	O
-monomer	O
-.	O
-(	O
-B	O
-)	O
-A	O
-topology	O
-diagram	O
-of	O
-the	O
-YfiB	O
-monomer	O
-.	O
-(	O
-C	O
-and	O
-D	O
-)	O
-The	O
-analytical	O
-ultracentrifugation	O
-experiment	O
-results	O
-for	O
-the	O
-wild	O
--	O
-type	O
-YfiB	O
-and	O
-YfiBL43P	B-mutant
-	O
-Two	O
-dimeric	O
-types	O
-of	O
-YfiB	O
-dimer	O
-.	O
-(	O
-A	O
-–	O
-C	O
-)	O
-The	O
-“	O
-head	O
-to	O
-head	O
-”	O
-dimer	O
-.	O
-	O
-The	O
-“	O
-back	O
-to	O
-back	O
-”	O
-dimer	O
-.	O
-	O
-(	O
-A	O
-)	O
-and	O
-(	O
-E	O
-)	O
-indicate	O
-the	O
-front	O
-views	O
-of	O
-the	O
-two	O
-dimers	O
-,	O
-(	O
-B	O
-)	O
-and	O
-(	O
-F	O
-)	O
-indicate	O
-the	O
-top	O
-views	O
-of	O
-the	O
-two	O
-dimers	O
-,	O
-and	O
-(	O
-C	O
-)	O
-and	O
-(	O
-D	O
-)	O
-indicate	O
-the	O
-details	O
-of	O
-the	O
-two	O
-dimeric	O
-interfaces	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-YfiB	O
-monomer	O
-consists	O
-of	O
-a	O
-five	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-(	O
-β1	O
--	O
-2	O
--	O
-5	O
--	O
-3	O
--	O
-4	O
-)	O
-flanked	O
-by	O
-five	O
-α	O
--	O
-helices	O
-(	O
-α1	O
-–	O
-5	O
-)	O
-on	O
-one	O
-side	O
-.	O
-	O
-In	O
-addition	O
-,	O
-there	O
-is	O
-a	O
-short	O
-helix	O
-turn	O
-connecting	O
-the	O
-β4	O
-strand	O
-and	O
-α4	O
-helix	O
-(	O
-Fig	O
-.	O
-1A	O
-and	O
-1B	O
-).	O
-	O
-Each	O
-crystal	O
-form	O
-contains	O
-three	O
-different	O
-dimeric	O
-types	O
-of	O
-YfiB	O
-,	O
-two	O
-of	O
-which	O
-are	O
-present	O
-in	O
-both	O
-,	O
-suggesting	O
-that	O
-the	O
-rest	O
-of	O
-the	O
-dimeric	O
-types	O
-may	O
-result	O
-from	O
-crystal	O
-packing	O
-.	O
-	O
-Here	O
-,	O
-we	O
-refer	O
-to	O
-the	O
-two	O
-dimeric	O
-types	O
-as	O
-“	O
-head	O
-to	O
-head	O
-”	O
-and	O
-“	O
-back	O
-to	O
-back	O
-”	O
-according	O
-to	O
-the	O
-interacting	O
-mode	O
-(	O
-Fig	O
-.	O
-2A	O
-and	O
-2E	O
-),	O
-with	O
-the	O
-total	O
-buried	O
-surface	O
-areas	O
-being	O
-316	O
-.	O
-8	O
-Å2	O
-and	O
-554	O
-.	O
-3	O
-Å2	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-“	O
-head	O
-to	O
-head	O
-”	O
-dimer	O
-exhibits	O
-a	O
-clamp	O
-shape	O
-.	O
-	O
-The	O
-dimerization	O
-occurs	O
-mainly	O
-via	O
-hydrophobic	O
-interactions	O
-formed	O
-by	O
-A37	O
-and	O
-I40	O
-on	O
-the	O
-α1	O
-helices	O
-,	O
-L50	O
-on	O
-the	O
-β1	O
-strands	O
-,	O
-and	O
-W55	O
-on	O
-the	O
-β2	O
-strands	O
-of	O
-both	O
-molecules	O
-,	O
-making	O
-a	O
-hydrophobic	O
-interacting	O
-core	O
-(	O
-Fig	O
-.	O
-2A	O
-–	O
-C	O
-).	O
-	O
-The	O
-“	O
-back	O
-to	O
-back	O
-”	O
-dimer	O
-presents	O
-a	O
-Y	O
-shape	O
-.	O
-	O
-The	O
-dimeric	O
-interaction	O
-is	O
-mainly	O
-hydrophilic	O
-,	O
-occurring	O
-among	O
-the	O
-main	O
--	O
-chain	O
-and	O
-side	O
--	O
-chain	O
-atoms	O
-of	O
-N68	O
-,	O
-L69	O
-,	O
-D70	O
-and	O
-R71	O
-on	O
-the	O
-α2	O
--	O
-α3	O
-loops	O
-and	O
-R116	O
-and	O
-S120	O
-on	O
-the	O
-α4	O
-helices	O
-of	O
-both	O
-molecules	O
-,	O
-resulting	O
-in	O
-a	O
-complex	O
-hydrogen	O
-bond	O
-network	O
-(	O
-Fig	O
-.	O
-2D	O
-–	O
-F	O
-).	O
-	O
-The	O
-YfiB	O
--	O
-YfiR	O
-interaction	O
-	O
-Overall	O
-structure	O
-of	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-and	O
-the	O
-conserved	O
-surface	O
-in	O
-YfiR	O
-.	O
-(	O
-A	O
-)	O
-The	O
-overall	O
-structure	O
-of	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-.	O
-	O
-The	O
-YfiBL43P	B-mutant
-molecules	O
-are	O
-shown	O
-in	O
-cyan	O
-and	O
-yellow	O
-.	O
-	O
-The	O
-YfiR	O
-molecules	O
-are	O
-shown	O
-in	O
-green	O
-and	O
-magenta	O
-.	O
-	O
-Two	O
-interacting	O
-regions	O
-are	O
-highlighted	O
-by	O
-red	O
-rectangles	O
-.	O
-(	O
-B	O
-)	O
-Structural	O
-superposition	O
-of	O
-apo	O
-YfiB	O
-and	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-.	O
-	O
-To	O
-illustrate	O
-the	O
-differences	O
-between	O
-apo	O
-YfiB	O
-and	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-,	O
-the	O
-apo	O
-YfiB	O
-is	O
-shown	O
-in	O
-pink	O
-,	O
-except	O
-residues	O
-34	O
-–	O
-70	O
-are	O
-shown	O
-in	O
-red	O
-,	O
-whereas	O
-the	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-is	O
-shown	O
-in	O
-cyan	O
-,	O
-except	O
-residues	O
-44	O
-–	O
-70	O
-are	O
-shown	O
-in	O
-blue	O
-.	O
-(	O
-C	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-differences	O
-between	O
-apo	O
-YfiB	O
-and	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-.	O
-	O
-The	O
-residues	O
-proposed	O
-to	O
-contribute	O
-to	O
-YfiB	O
-activation	O
-are	O
-illustrated	O
-in	O
-sticks	O
-.	O
-	O
-The	O
-key	O
-residues	O
-in	O
-apo	O
-YfiB	O
-are	O
-shown	O
-in	O
-red	O
-and	O
-those	O
-in	O
-YfiBL43P	B-mutant
-are	O
-shown	O
-in	O
-blue	O
-.	O
-(	O
-D	O
-)	O
-Close	O
--	O
-up	O
-views	O
-showing	O
-interactions	O
-in	O
-regions	O
-I	O
-and	O
-II	O
-.	O
-	O
-YfiBL43P	B-mutant
-and	O
-YfiR	O
-are	O
-shown	O
-in	O
-cyan	O
-and	O
-green	O
-,	O
-respectively	O
-.	O
-(	O
-E	O
-and	O
-F	O
-)	O
-The	O
-conserved	O
-surface	O
-in	O
-YfiR	O
-contributes	O
-to	O
-the	O
-interaction	O
-with	O
-YfiB	O
-.	O
-(	O
-G	O
-)	O
-The	O
-residues	O
-of	O
-YfiR	O
-responsible	O
-for	O
-interacting	O
-with	O
-YfiB	O
-are	O
-shown	O
-in	O
-green	O
-sticks	O
-,	O
-and	O
-the	O
-proposed	O
-YfiN	O
--	O
-interacting	O
-residues	O
-are	O
-shown	O
-in	O
-yellow	O
-sticks	O
-.	O
-	O
-The	O
-red	O
-sticks	O
-,	O
-which	O
-represent	O
-the	O
-YfiB	O
--	O
-interacting	O
-residues	O
-,	O
-are	O
-also	O
-responsible	O
-for	O
-the	O
-proposed	O
-interactions	O
-with	O
-YfiN	O
-	O
-To	O
-gain	O
-structural	O
-insights	O
-into	O
-the	O
-YfiB	O
--	O
-YfiR	O
-interaction	O
-,	O
-we	O
-co	O
--	O
-expressed	O
-YfiB	O
-(	O
-residues	O
-34	O
-–	O
-168	O
-)	O
-and	O
-YfiR	O
-(	O
-residues	O
-35	O
-–	O
-190	O
-,	O
-lacking	O
-the	O
-signal	O
-peptide	O
-),	O
-but	O
-failed	O
-to	O
-obtain	O
-the	O
-complex	O
-,	O
-in	O
-accordance	O
-with	O
-a	O
-previous	O
-report	O
-in	O
-which	O
-no	O
-stable	O
-complex	O
-of	O
-YfiB	O
--	O
-YfiR	O
-was	O
-observed	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-It	O
-has	O
-been	O
-reported	O
-that	O
-single	O
-mutants	O
-of	O
-Q39	O
-,	O
-L43	O
-,	O
-F48	O
-and	O
-W55	O
-contribute	O
-to	O
-YfiB	O
-activation	O
-leading	O
-to	O
-the	O
-induction	O
-of	O
-the	O
-SCV	O
-phenotype	O
-in	O
-P	O
-.	O
-aeruginosa	O
-PAO1	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-It	O
-is	O
-likely	O
-that	O
-these	O
-residues	O
-may	O
-be	O
-involved	O
-in	O
-the	O
-conformational	O
-changes	O
-of	O
-YfiB	O
-that	O
-are	O
-related	O
-to	O
-YfiR	O
-sequestration	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-Therefore	O
-,	O
-we	O
-constructed	O
-two	O
-such	O
-single	O
-mutants	O
-of	O
-YfiB	O
-(	O
-YfiBL43P	B-mutant
-and	O
-YfiBF48S	B-mutant
-).	O
-	O
-As	O
-expected	O
-,	O
-both	O
-mutants	O
-form	O
-a	O
-stable	O
-complex	O
-with	O
-YfiR	O
-.	O
-Finally	O
-,	O
-we	O
-crystalized	O
-YfiR	O
-in	O
-complex	O
-with	O
-the	O
-YfiBL43P	B-mutant
-mutant	O
-and	O
-solved	O
-the	O
-structure	O
-at	O
-1	O
-.	O
-78	O
-Å	O
-resolution	O
-by	O
-molecular	O
-replacement	O
-using	O
-YfiR	O
-and	O
-YfiB	O
-as	O
-models	O
-.	O
-	O
-The	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-is	O
-a	O
-2	O
-:	O
-2	O
-heterotetramer	O
-(	O
-Fig	O
-.	O
-3A	O
-)	O
-in	O
-which	O
-the	O
-YfiR	O
-dimer	O
-is	O
-clamped	O
-by	O
-two	O
-separated	O
-YfiBL43P	B-mutant
-molecules	O
-with	O
-a	O
-total	O
-buried	O
-surface	O
-area	O
-of	O
-3161	O
-.	O
-2	O
-Å2	O
-.	O
-	O
-The	O
-YfiR	O
-dimer	O
-in	O
-the	O
-complex	O
-is	O
-identical	O
-to	O
-the	O
-non	O
--	O
-oxidized	O
-YfiR	O
-dimer	O
-alone	O
-(	O
-Yang	O
-et	O
-al	O
-.,),	O
-with	O
-only	O
-Cys145	O
--	O
-Cys152	O
-of	O
-the	O
-two	O
-disulfide	O
-bonds	O
-well	O
-formed	O
-,	O
-suggesting	O
-Cys71	O
--	O
-Cys110	O
-disulfide	O
-bond	O
-formation	O
-is	O
-not	O
-essential	O
-for	O
-forming	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-structural	O
-conformation	O
-of	O
-YfiBL43P	B-mutant
-,	O
-from	O
-the	O
-foremost	O
-N	O
--	O
-terminus	O
-to	O
-residue	O
-D70	O
-,	O
-is	O
-significantly	O
-altered	O
-compared	O
-with	O
-that	O
-of	O
-the	O
-apo	O
-YfiB	O
-.	O
-The	O
-majority	O
-of	O
-the	O
-α1	O
-helix	O
-(	O
-residues	O
-34	O
-–	O
-43	O
-)	O
-is	O
-invisible	O
-on	O
-the	O
-electron	O
-density	O
-map	O
-,	O
-and	O
-the	O
-α2	O
-helix	O
-and	O
-β1	O
-and	O
-β2	O
-strands	O
-are	O
-rearranged	O
-to	O
-form	O
-a	O
-long	O
-loop	O
-containing	O
-two	O
-short	O
-α	O
--	O
-helix	O
-turns	O
-(	O
-Fig	O
-.	O
-3B	O
-and	O
-3C	O
-),	O
-thus	O
-embracing	O
-the	O
-YfiR	O
-dimer	O
-.	O
-	O
-The	O
-observed	O
-changes	O
-in	O
-conformation	O
-of	O
-YfiB	O
-and	O
-the	O
-results	O
-of	O
-mutagenesis	O
-suggest	O
-a	O
-mechanism	O
-by	O
-which	O
-YfiB	O
-sequesters	O
-YfiR	O
-.	O
-	O
-The	O
-YfiB	O
--	O
-YfiR	O
-interface	O
-can	O
-be	O
-divided	O
-into	O
-two	O
-regions	O
-(	O
-Fig	O
-.	O
-3A	O
-and	O
-3D	O
-).	O
-	O
-Region	O
-I	O
-is	O
-formed	O
-by	O
-numerous	O
-main	O
--	O
-chain	O
-and	O
-side	O
--	O
-chain	O
-hydrophilic	O
-interactions	O
-between	O
-residues	O
-E45	O
-,	O
-G47	O
-and	O
-E53	O
-from	O
-the	O
-N	O
--	O
-terminal	O
-extended	O
-loop	O
-of	O
-YfiB	O
-and	O
-residues	O
-S57	O
-,	O
-R60	O
-,	O
-A89	O
-and	O
-H177	O
-from	O
-YfiR	O
-(	O
-Fig	O
-.	O
-3D	O
--	O
-I	O
-(	O
-i	O
-)).	O
-	O
-Additionally	O
-,	O
-three	O
-hydrophobic	O
-anchoring	O
-sites	O
-exist	O
-in	O
-region	O
-I	O
-.	O
-The	O
-residues	O
-F48	O
-and	O
-W55	O
-of	O
-YfiB	O
-are	O
-inserted	O
-into	O
-the	O
-hydrophobic	O
-cores	O
-mainly	O
-formed	O
-by	O
-the	O
-main	O
-chain	O
-and	O
-side	O
-chain	O
-carbon	O
-atoms	O
-of	O
-residues	O
-S57	O
-/	O
-Q88	O
-/	O
-A89	O
-/	O
-N90	O
-and	O
-R60	O
-/	O
-R175	O
-/	O
-H177	O
-of	O
-YfiR	O
-,	O
-respectively	O
-;	O
-and	O
-F57	O
-of	O
-YfiB	O
-is	O
-inserted	O
-into	O
-the	O
-hydrophobic	O
-pocket	O
-formed	O
-by	O
-L166	O
-/	O
-I169	O
-/	O
-V176	O
-/	O
-P178	O
-/	O
-L181	O
-of	O
-YfiR	O
-(	O
-Fig	O
-.	O
-3D	O
--	O
-I	O
-(	O
-ii	O
-)).	O
-	O
-In	O
-region	O
-II	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-R96	O
-,	O
-E98	O
-and	O
-E157	O
-from	O
-YfiB	O
-interact	O
-with	O
-the	O
-side	O
-chains	O
-of	O
-E163	O
-,	O
-S146	O
-and	O
-R171	O
-from	O
-YfiR	O
-,	O
-respectively	O
-.	O
-	O
-Additionally	O
-,	O
-the	O
-main	O
-chains	O
-of	O
-I163	O
-and	O
-V165	O
-from	O
-YfiB	O
-form	O
-hydrogen	O
-bonds	O
-with	O
-the	O
-main	O
-chains	O
-of	O
-L166	O
-and	O
-A164	O
-from	O
-YfiR	O
-,	O
-respectively	O
-,	O
-and	O
-the	O
-main	O
-chain	O
-of	O
-P166	O
-from	O
-YfiB	O
-interacts	O
-with	O
-the	O
-side	O
-chain	O
-of	O
-R185	O
-from	O
-YfiR	O
-(	O
-Fig	O
-.	O
-3D	O
--	O
-II	O
-).	O
-	O
-These	O
-two	O
-regions	O
-contribute	O
-a	O
-robust	O
-hydrogen	O
--	O
-bonding	O
-network	O
-to	O
-the	O
-YfiB	O
--	O
-YfiR	O
-interface	O
-,	O
-resulting	O
-in	O
-a	O
-tightly	O
-bound	O
-complex	O
-.	O
-	O
-Based	O
-on	O
-the	O
-observations	O
-that	O
-two	O
-separated	O
-YfiBL43P	B-mutant
-molecules	O
-form	O
-a	O
-2	O
-:	O
-2	O
-complex	O
-structure	O
-with	O
-YfiR	O
-dimer	O
-,	O
-we	O
-performed	O
-an	O
-analytical	O
-ultracentrifugation	O
-experiment	O
-to	O
-check	O
-the	O
-oligomeric	O
-states	O
-of	O
-wild	O
--	O
-type	O
-YfiB	O
-and	O
-YfiBL43P	B-mutant
-.	O
-	O
-The	O
-results	O
-showed	O
-that	O
-wild	O
--	O
-type	O
-YfiB	O
-exists	O
-in	O
-both	O
-monomeric	O
-and	O
-dimeric	O
-states	O
-in	O
-solution	O
-,	O
-while	O
-YfiBL43P	B-mutant
-primarily	O
-adopts	O
-the	O
-monomer	O
-state	O
-in	O
-solution	O
-(	O
-Fig	O
-.	O
-1C	O
-–	O
-D	O
-).	O
-	O
-This	O
-suggests	O
-that	O
-the	O
-N	O
--	O
-terminus	O
-of	O
-YfiB	O
-plays	O
-an	O
-important	O
-role	O
-in	O
-forming	O
-the	O
-dimeric	O
-YfiB	O
-in	O
-solution	O
-and	O
-that	O
-the	O
-conformational	O
-change	O
-of	O
-residue	O
-L43	O
-is	O
-associated	O
-with	O
-the	O
-stretch	O
-of	O
-the	O
-N	O
--	O
-terminus	O
-and	O
-opening	O
-of	O
-the	O
-dimer	O
-.	O
-	O
-Therefore	O
-,	O
-it	O
-is	O
-possible	O
-that	O
-both	O
-dimeric	O
-types	O
-might	O
-exist	O
-in	O
-solution	O
-.	O
-	O
-For	O
-simplicity	O
-,	O
-we	O
-only	O
-discuss	O
-the	O
-“	O
-head	O
-to	O
-head	O
-”	O
-dimer	O
-in	O
-the	O
-following	O
-text	O
-.	O
-	O
-The	O
-PG	O
--	O
-binding	O
-site	O
-of	O
-YfiB	O
-	O
-The	O
-PG	O
--	O
-binding	O
-site	O
-in	O
-YfiB	O
-.	O
-(	O
-A	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-PG	O
--	O
-binding	O
-sites	O
-of	O
-the	O
-H	O
-.	O
-influenzae	O
-Pal	O
-/	O
-PG	O
--	O
-P	O
-complex	O
-and	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-complexed	O
-with	O
-sulfate	O
-ions	O
-.	O
-	O
-(	O
-B	O
-)	O
-Close	O
--	O
-up	O
-view	O
-showing	O
-the	O
-key	O
-residues	O
-of	O
-Pal	O
-interacting	O
-with	O
-the	O
-m	O
--	O
-Dap5	O
-ε	O
--	O
-carboxylate	O
-group	O
-of	O
-PG	O
--	O
-P	O
-.	O
-Pal	O
-is	O
-shown	O
-in	O
-wheat	O
-and	O
-PG	O
--	O
-P	O
-is	O
-in	O
-magenta	O
-.	O
-	O
-(	O
-C	O
-)	O
-Close	O
--	O
-up	O
-view	O
-showing	O
-the	O
-key	O
-residues	O
-of	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-interacting	O
-with	O
-a	O
-sulfate	O
-ion	O
-.	O
-	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-is	O
-shown	O
-in	O
-cyan	O
-;	O
-the	O
-sulfate	O
-ion	O
-,	O
-in	O
-green	O
-;	O
-and	O
-the	O
-water	O
-molecule	O
-,	O
-in	O
-yellow	O
-.	O
-(	O
-D	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-PG	O
--	O
-binding	O
-sites	O
-of	O
-apo	O
-YfiB	O
-and	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-,	O
-the	O
-key	O
-residues	O
-are	O
-shown	O
-in	O
-stick	O
-.	O
-	O
-Apo	O
-YfiB	O
-is	O
-shown	O
-in	O
-yellow	O
-and	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-in	O
-cyan	O
-.	O
-(	O
-E	O
-and	O
-F	O
-)	O
-MST	O
-data	O
-and	O
-analysis	O
-for	O
-binding	O
-affinities	O
-of	O
-(	O
-E	O
-)	O
-YfiB	O
-wild	O
--	O
-type	O
-and	O
-(	O
-F	O
-)	O
-YfiBL43P	B-mutant
-with	O
-PG	O
-.	O
-(	O
-G	O
-)	O
-The	O
-sequence	O
-alignment	O
-of	O
-P	O
-.	O
-aeruginosa	O
-and	O
-E	O
-.	O
-coli	O
-sources	O
-of	O
-YfiB	O
-,	O
-Pal	O
-and	O
-the	O
-periplasmic	O
-domain	O
-of	O
-OmpA	O
-	O
-PG	O
--	O
-associated	O
-lipoprotein	O
-(	O
-Pal	O
-)	O
-is	O
-highly	O
-conserved	O
-in	O
-Gram	O
--	O
-negative	O
-bacteria	O
-and	O
-anchors	O
-to	O
-the	O
-outer	O
-membrane	O
-through	O
-an	O
-N	O
--	O
-terminal	O
-lipid	O
-attachment	O
-and	O
-to	O
-PG	O
-layer	O
-through	O
-its	O
-periplasmic	O
-domain	O
-,	O
-which	O
-is	O
-implicated	O
-in	O
-maintaining	O
-outer	O
-membrane	O
-integrity	O
-.	O
-	O
-Previous	O
-homology	O
-modeling	O
-studies	O
-suggested	O
-that	O
-YfiB	O
-contains	O
-a	O
-Pal	O
--	O
-like	O
-PG	O
--	O
-binding	O
-site	O
-(	O
-Parsons	O
-et	O
-al	O
-.,),	O
-and	O
-the	O
-mutation	O
-of	O
-two	O
-residues	O
-at	O
-this	O
-site	O
-,	O
-D102	O
-and	O
-G105	O
-,	O
-reduces	O
-the	O
-ability	O
-for	O
-biofilm	O
-formation	O
-and	O
-surface	O
-attachment	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-In	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-,	O
-one	O
-sulfate	O
-ion	O
-is	O
-found	O
-at	O
-the	O
-bottom	O
-of	O
-each	O
-YfiBL43P	B-mutant
-molecule	O
-(	O
-Fig	O
-.	O
-3A	O
-)	O
-and	O
-forms	O
-a	O
-strong	O
-hydrogen	O
-bond	O
-with	O
-D102	O
-of	O
-YfiBL43P	B-mutant
-(	O
-Fig	O
-.	O
-4A	O
-and	O
-4C	O
-).	O
-	O
-Structural	O
-superposition	O
-between	O
-YfiBL43P	B-mutant
-and	O
-Haemophilus	O
-influenzae	O
-Pal	O
-complexed	O
-with	O
-biosynthetic	O
-peptidoglycan	O
-precursor	O
-(	O
-PG	O
--	O
-P	O
-),	O
-UDP	O
--	O
-N	O
--	O
-acetylmuramyl	O
--	O
-L	O
--	O
-Ala	O
--	O
-α	O
--	O
-D	O
--	O
-Glu	O
--	O
-m	O
--	O
-Dap	O
--	O
-D	O
--	O
-Ala	O
--	O
-D	O
--	O
-Ala	O
-(	O
-m	O
--	O
-Dap	O
-is	O
-meso	O
--	O
-diaminopimelate	O
-)	O
-(	O
-PDB	O
-code	O
-:	O
-2aiz	O
-)	O
-(	O
-Parsons	O
-et	O
-al	O
-.,),	O
-revealed	O
-that	O
-the	O
-sulfate	O
-ion	O
-is	O
-located	O
-at	O
-the	O
-position	O
-of	O
-the	O
-m	O
--	O
-Dap5	O
-ϵ	O
--	O
-carboxylate	O
-group	O
-in	O
-the	O
-Pal	O
-/	O
-PG	O
--	O
-P	O
-complex	O
-(	O
-Fig	O
-.	O
-4A	O
-).	O
-	O
-In	O
-the	O
-Pal	O
-/	O
-PG	O
--	O
-P	O
-complex	O
-structure	O
-,	O
-the	O
-m	O
--	O
-Dap5	O
-ϵ	O
--	O
-carboxylate	O
-group	O
-interacts	O
-with	O
-the	O
-side	O
--	O
-chain	O
-atoms	O
-of	O
-D71	O
-and	O
-the	O
-main	O
--	O
-chain	O
-amide	O
-of	O
-D37	O
-(	O
-Fig	O
-.	O
-4B	O
-).	O
-	O
-Similarly	O
-,	O
-in	O
-the	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-structure	O
-,	O
-the	O
-sulfate	O
-ion	O
-interacts	O
-with	O
-the	O
-side	O
--	O
-chain	O
-atoms	O
-of	O
-D102	O
-(	O
-corresponding	O
-to	O
-D71	O
-in	O
-Pal	O
-)	O
-and	O
-R117	O
-(	O
-corresponding	O
-to	O
-R86	O
-in	O
-Pal	O
-)	O
-and	O
-the	O
-main	O
--	O
-chain	O
-amide	O
-of	O
-N68	O
-(	O
-corresponding	O
-to	O
-D37	O
-in	O
-Pal	O
-).	O
-	O
-Moreover	O
-,	O
-a	O
-water	O
-molecule	O
-was	O
-found	O
-to	O
-bridge	O
-the	O
-sulfate	O
-ion	O
-and	O
-the	O
-side	O
-chains	O
-of	O
-N67	O
-and	O
-D102	O
-,	O
-strengthening	O
-the	O
-hydrogen	O
-bond	O
-network	O
-(	O
-Fig	O
-.	O
-4C	O
-).	O
-	O
-In	O
-addition	O
-,	O
-sequence	O
-alignment	O
-of	O
-YfiB	O
-with	O
-Pal	O
-and	O
-the	O
-periplasmic	O
-domain	O
-of	O
-OmpA	O
-(	O
-proteins	O
-containing	O
-PG	O
--	O
-binding	O
-site	O
-)	O
-showed	O
-that	O
-N68	O
-and	O
-D102	O
-are	O
-highly	O
-conserved	O
-(	O
-Fig	O
-.	O
-4G	O
-,	O
-blue	O
-stars	O
-),	O
-suggesting	O
-that	O
-these	O
-residues	O
-contribute	O
-to	O
-the	O
-PG	O
--	O
-binding	O
-ability	O
-of	O
-YfiB	O
-.	O
-	O
-Interestingly	O
-,	O
-superposition	O
-of	O
-apo	O
-YfiB	O
-with	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-revealed	O
-that	O
-the	O
-PG	O
--	O
-binding	O
-region	O
-is	O
-largely	O
-altered	O
-mainly	O
-due	O
-to	O
-different	O
-conformation	O
-of	O
-the	O
-N68	O
-containing	O
-loop	O
-.	O
-	O
-Compared	O
-to	O
-YfiBL43P	B-mutant
-,	O
-the	O
-N68	O
--	O
-containing	O
-loop	O
-of	O
-the	O
-apo	O
-YfiB	O
-flips	O
-away	O
-about	O
-7	O
-Å	O
-,	O
-and	O
-D102	O
-and	O
-R117	O
-swing	O
-slightly	O
-outward	O
-;	O
-thus	O
-,	O
-the	O
-PG	O
--	O
-binding	O
-pocket	O
-is	O
-enlarged	O
-with	O
-no	O
-sulfate	O
-ion	O
-or	O
-water	O
-bound	O
-(	O
-Fig	O
-.	O
-4D	O
-).	O
-	O
-Therefore	O
-,	O
-we	O
-proposed	O
-that	O
-the	O
-PG	O
--	O
-binding	O
-ability	O
-of	O
-inactive	O
-YfiB	O
-might	O
-be	O
-weaker	O
-than	O
-that	O
-of	O
-active	O
-YfiB	O
-.	O
-To	O
-validate	O
-this	O
-,	O
-we	O
-performed	O
-a	O
-microscale	O
-thermophoresis	O
-(	O
-MST	O
-)	O
-assay	O
-to	O
-measure	O
-the	O
-binding	O
-affinities	O
-of	O
-PG	O
-to	O
-wild	O
--	O
-type	O
-YfiB	O
-and	O
-YfiBL43P	B-mutant
-,	O
-respectively	O
-.	O
-	O
-The	O
-results	O
-indicated	O
-that	O
-the	O
-PG	O
--	O
-binding	O
-affinity	O
-of	O
-YfiBL43P	B-mutant
-is	O
-65	O
-.	O
-5	O
-μmol	O
-/	O
-L	O
-,	O
-which	O
-is	O
-about	O
-16	O
--	O
-fold	O
-stronger	O
-than	O
-that	O
-of	O
-wild	O
--	O
-type	O
-YfiB	O
-(	O
-Kd	O
-=	O
-1	O
-.	O
-1	O
-mmol	O
-/	O
-L	O
-)	O
-(	O
-Fig	O
-.	O
-4E	O
-–	O
-F	O
-).	O
-	O
-As	O
-the	O
-experiment	O
-is	O
-performed	O
-in	O
-the	O
-absence	O
-of	O
-YfiR	O
-,	O
-it	O
-suggests	O
-that	O
-an	O
-increase	O
-in	O
-the	O
-PG	O
--	O
-binding	O
-affinity	O
-of	O
-YfiB	O
-is	O
-not	O
-a	O
-result	O
-of	O
-YfiB	O
--	O
-YfiR	O
-interaction	O
-and	O
-is	O
-highly	O
-coupled	O
-to	O
-the	O
-activation	O
-of	O
-YfiB	O
-characterized	O
-by	O
-a	O
-stretched	O
-N	O
--	O
-terminal	O
-conformation	O
-.	O
-	O
-The	O
-conserved	O
-surface	O
-in	O
-YfiR	O
-is	O
-functional	O
-for	O
-binding	O
-YfiB	O
-and	O
-YfiN	O
-	O
-Calculation	O
-using	O
-the	O
-ConSurf	O
-Server	O
-(	O
-http	O
-://	O
-consurf	O
-.	O
-tau	O
-.	O
-ac	O
-.	O
-il	O
-/),	O
-which	O
-estimates	O
-the	O
-evolutionary	O
-conservation	O
-of	O
-amino	O
-acid	O
-positions	O
-and	O
-visualizes	O
-information	O
-on	O
-the	O
-structure	O
-surface	O
-,	O
-revealed	O
-a	O
-conserved	O
-surface	O
-on	O
-YfiR	O
-that	O
-contributes	O
-to	O
-the	O
-interaction	O
-with	O
-YfiB	O
-(	O
-Fig	O
-.	O
-3E	O
-and	O
-3F	O
-).	O
-	O
-Interestingly	O
-,	O
-the	O
-majority	O
-of	O
-this	O
-conserved	O
-surface	O
-contributes	O
-to	O
-the	O
-interaction	O
-with	O
-YfiB	O
-(	O
-Fig	O
-.	O
-3E	O
-and	O
-3F	O
-).	O
-	O
-Malone	O
-JG	O
-et	O
-al	O
-.	O
-have	O
-reported	O
-that	O
-F151	O
-,	O
-E163	O
-,	O
-I169	O
-and	O
-Q187	O
-,	O
-located	O
-near	O
-the	O
-C	O
--	O
-terminus	O
-of	O
-YfiR	O
-,	O
-comprise	O
-a	O
-putative	O
-YfiN	O
-binding	O
-site	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-Interestingly	O
-,	O
-these	O
-residues	O
-are	O
-part	O
-of	O
-the	O
-conserved	O
-surface	O
-of	O
-YfiR	O
-(	O
-Fig	O
-.	O
-3G	O
-).	O
-	O
-F151	O
-,	O
-E163	O
-and	O
-I169	O
-form	O
-a	O
-hydrophobic	O
-core	O
-while	O
-,	O
-Q187	O
-is	O
-located	O
-at	O
-the	O
-end	O
-of	O
-the	O
-α6	O
-helix	O
-.	O
-	O
-E163	O
-and	O
-I169	O
-are	O
-YfiB	O
--	O
-interacting	O
-residues	O
-of	O
-YfiR	O
-,	O
-in	O
-which	O
-E163	O
-forms	O
-a	O
-hydrogen	O
-bond	O
-with	O
-R96	O
-of	O
-YfiB	O
-(	O
-Fig	O
-.	O
-3D	O
--	O
-II	O
-)	O
-and	O
-I169	O
-is	O
-involved	O
-in	O
-forming	O
-the	O
-L166	O
-/	O
-I169	O
-/	O
-V176	O
-/	O
-P178	O
-/	O
-L181	O
-hydrophobic	O
-core	O
-for	O
-anchoring	O
-F57	O
-of	O
-YfiB	O
-(	O
-Fig	O
-.	O
-3D	O
--	O
-I	O
-(	O
-ii	O
-)).	O
-	O
-Collectively	O
-,	O
-a	O
-part	O
-of	O
-the	O
-YfiB	O
--	O
-YfiR	O
-interface	O
-overlaps	O
-with	O
-the	O
-proposed	O
-YfiR	O
--	O
-YfiN	O
-interface	O
-,	O
-suggesting	O
-alteration	O
-in	O
-the	O
-association	O
--	O
-disassociation	O
-equilibrium	O
-of	O
-YfiR	O
--	O
-YfiN	O
-and	O
-hence	O
-the	O
-ability	O
-of	O
-YfiB	O
-to	O
-sequester	O
-YfiR	O
-.	O
-	O
-YfiR	O
-binds	O
-small	O
-molecules	O
-	O
-Previous	O
-studies	O
-indicated	O
-that	O
-YfiR	O
-constitutes	O
-a	O
-YfiB	O
--	O
-independent	O
-sensing	O
-device	O
-that	O
-can	O
-activate	O
-YfiN	O
-in	O
-response	O
-to	O
-the	O
-redox	O
-status	O
-of	O
-the	O
-periplasm	O
-,	O
-and	O
-we	O
-have	O
-reported	O
-YfiR	O
-structures	O
-in	O
-both	O
-the	O
-non	O
--	O
-oxidized	O
-and	O
-the	O
-oxidized	O
-states	O
-earlier	O
-,	O
-revealing	O
-that	O
-the	O
-Cys145	O
--	O
-Cys152	O
-disulfide	O
-bond	O
-plays	O
-an	O
-essential	O
-role	O
-in	O
-maintaining	O
-the	O
-correct	O
-folding	O
-of	O
-YfiR	O
-(	O
-Yang	O
-et	O
-al	O
-.,).	O
-	O
-However	O
-,	O
-whether	O
-YfiR	O
-is	O
-involved	O
-in	O
-other	O
-regulatory	O
-mechanisms	O
-is	O
-still	O
-an	O
-open	O
-question	O
-.	O
-	O
-Overall	O
-Structures	O
-of	O
-VB6	O
--	O
-bound	O
-and	O
-Trp	O
--	O
-bound	O
-YfiR	O
-.	O
-(	O
-A	O
-)	O
-Superposition	O
-of	O
-the	O
-overall	O
-structures	O
-of	O
-VB6	O
--	O
-bound	O
-and	O
-Trp	O
--	O
-bound	O
-YfiR	O
-.	O
-(	O
-B	O
-)	O
-Close	O
--	O
-up	O
-views	O
-showing	O
-the	O
-key	O
-residues	O
-of	O
-YfiR	O
-that	O
-bind	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-.	O
-	O
-The	O
-electron	O
-densities	O
-of	O
-VB6	O
-and	O
-Trp	O
-are	O
-countered	O
-at	O
-3	O
-.	O
-0σ	O
-and	O
-2	O
-.	O
-3σ	O
-,	O
-respectively	O
-,	O
-in	O
-|	O
-Fo	O
-|-|	O
-Fc	O
-|	O
-maps	O
-.	O
-(	O
-C	O
-)	O
-Superposition	O
-of	O
-the	O
-hydrophobic	O
-pocket	O
-of	O
-YfiR	O
-with	O
-VB6	O
-,	O
-L	O
--	O
-Trp	O
-and	O
-F57	O
-of	O
-YfiB	O
-	O
-Intriguingly	O
-,	O
-a	O
-Dali	O
-search	O
-(	O
-Holm	O
-and	O
-Rosenstrom	O
-,)	O
-indicated	O
-that	O
-the	O
-closest	O
-homologs	O
-of	O
-YfiR	O
-shared	O
-the	O
-characteristic	O
-of	O
-being	O
-able	O
-to	O
-bind	O
-several	O
-structurally	O
-similar	O
-small	O
-molecules	O
-,	O
-such	O
-as	O
-L	O
--	O
-Trp	O
-,	O
-L	O
--	O
-Phe	O
-,	O
-B	O
--	O
-group	O
-vitamins	O
-and	O
-their	O
-analogs	O
-,	O
-encouraging	O
-us	O
-to	O
-test	O
-whether	O
-YfiR	O
-can	O
-recognize	O
-these	O
-molecules	O
-.	O
-	O
-For	O
-this	O
-purpose	O
-,	O
-we	O
-co	O
--	O
-crystallized	O
-YfiR	O
-or	O
-soaked	O
-YfiR	O
-crystals	O
-with	O
-different	O
-small	O
-molecules	O
-,	O
-including	O
-L	O
--	O
-Trp	O
-and	O
-B	O
--	O
-group	O
-vitamins	O
-.	O
-	O
-Fortunately	O
-,	O
-we	O
-found	O
-obvious	O
-small	O
--	O
-molecule	O
-density	O
-in	O
-the	O
-VB6	O
--	O
-bound	O
-and	O
-Trp	O
--	O
-bound	O
-YfiR	O
-crystal	O
-structures	O
-(	O
-Fig	O
-.	O
-5A	O
-and	O
-5B	O
-),	O
-and	O
-in	O
-both	O
-structures	O
-,	O
-the	O
-YfiR	O
-dimers	O
-resemble	O
-the	O
-oxidized	O
-YfiR	O
-structure	O
-in	O
-which	O
-both	O
-two	O
-disulfide	O
-bonds	O
-are	O
-well	O
-formed	O
-(	O
-Yang	O
-et	O
-al	O
-.,).	O
-	O
-Functional	O
-analysis	O
-of	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-.	O
-(	O
-A	O
-and	O
-B	O
-)	O
-The	O
-effect	O
-of	O
-increasing	O
-concentrations	O
-of	O
-VB6	O
-or	O
-L	O
--	O
-Trp	O
-on	O
-YfiBL43P	B-mutant
--	O
-induced	O
-attachment	O
-(	O
-bars	O
-).	O
-	O
-The	O
-relative	O
-optical	O
-density	O
-is	O
-represented	O
-as	O
-curves	O
-.	O
-	O
-Wild	O
--	O
-type	O
-YfiB	O
-is	O
-used	O
-as	O
-negative	O
-control	O
-.	O
-	O
-(	O
-C	O
-and	O
-D	O
-)	O
-BIAcore	O
-data	O
-and	O
-analysis	O
-for	O
-binding	O
-affinities	O
-of	O
-(	O
-C	O
-)	O
-VB6	O
-and	O
-(	O
-D	O
-)	O
-L	O
--	O
-Trp	O
-with	O
-YfiR	O
-.	O
-(	O
-E	O
-–	O
-G	O
-)	O
-ITC	O
-data	O
-and	O
-analysis	O
-for	O
-titration	O
-of	O
-(	O
-E	O
-)	O
-YfiB	O
-wild	O
--	O
-type	O
-,	O
-(	O
-F	O
-)	O
-YfiBL43P	O
-,	O
-and	O
-(	O
-G	O
-)	O
-YfiBL43P	B-mutant
-/	O
-F57A	B-mutant
-into	O
-YfiR	O
-	O
-Structural	O
-analyses	O
-revealed	O
-that	O
-the	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-molecules	O
-are	O
-bound	O
-at	O
-the	O
-periphery	O
-of	O
-the	O
-YfiR	O
-dimer	O
-,	O
-but	O
-not	O
-at	O
-the	O
-dimer	O
-interface	O
-.	O
-	O
-Interestingly	O
-,	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-were	O
-found	O
-to	O
-occupy	O
-the	O
-same	O
-hydrophobic	O
-pocket	O
-,	O
-formed	O
-by	O
-L166	O
-/	O
-I169	O
-/	O
-V176	O
-/	O
-P178	O
-/	O
-L181	O
-of	O
-YfiR	O
-,	O
-which	O
-is	O
-also	O
-a	O
-binding	O
-pocket	O
-for	O
-F57	O
-of	O
-YfiB	O
-,	O
-as	O
-observed	O
-in	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-(	O
-Fig	O
-.	O
-5C	O
-).	O
-	O
-To	O
-evaluate	O
-the	O
-importance	O
-of	O
-F57	O
-in	O
-YfiBL43P	O
--	O
-YfiR	O
-interaction	O
-,	O
-the	O
-binding	O
-affinities	O
-of	O
-YfiBL43P	B-mutant
-and	O
-YfiBL43P	B-mutant
-/	O
-F57A	B-mutant
-for	O
-YfiR	O
-were	O
-measured	O
-by	O
-isothermal	O
-titration	O
-calorimetry	O
-(	O
-ITC	O
-).	O
-	O
-The	O
-results	O
-showed	O
-Kd	O
-values	O
-of	O
-1	O
-.	O
-4	O
-×	O
-10	O
-−	O
-7	O
-mol	O
-/	O
-L	O
-and	O
-5	O
-.	O
-3	O
-×	O
-10	O
-−	O
-7	O
-mol	O
-/	O
-L	O
-for	O
-YfiBL43P	B-mutant
-and	O
-YfiBL43P	B-mutant
-/	O
-F57A	B-mutant
-,	O
-respectively	O
-,	O
-revealing	O
-that	O
-the	O
-YfiBL43P	B-mutant
-/	O
-F57A	B-mutant
-mutant	O
-caused	O
-a	O
-3	O
-.	O
-8	O
--	O
-fold	O
-reduction	O
-in	O
-the	O
-binding	O
-affinity	O
-compared	O
-with	O
-the	O
-YfiBL43P	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-6F	O
-and	O
-6G	O
-).	O
-	O
-In	O
-parallel	O
-,	O
-to	O
-better	O
-understand	O
-the	O
-putative	O
-functional	O
-role	O
-of	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-,	O
-yfiB	O
-was	O
-deleted	O
-in	O
-a	O
-PAO1	O
-wild	O
--	O
-type	O
-strain	O
-,	O
-and	O
-a	O
-construct	O
-expressing	O
-the	O
-YfiBL43P	B-mutant
-mutant	O
-was	O
-transformed	O
-into	O
-the	O
-PAO1	O
-ΔyfiB	B-mutant
-strain	O
-to	O
-trigger	O
-YfiBL43P	B-mutant
--	O
-induced	O
-biofilm	O
-formation	O
-.	O
-	O
-Growth	O
-and	O
-surface	O
-attachment	O
-assays	O
-were	O
-carried	O
-out	O
-for	O
-the	O
-yfiB	B-mutant
--	I-mutant
-L43P	I-mutant
-strain	O
-in	O
-the	O
-presence	O
-of	O
-increasing	O
-concentrations	O
-of	O
-VB6	O
-or	O
-L	O
--	O
-Trp	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-.	O
-6A	O
-and	O
-6B	O
-,	O
-the	O
-over	O
--	O
-expression	O
-of	O
-YfiBL43P	B-mutant
-induced	O
-strong	O
-surface	O
-attachment	O
-and	O
-much	O
-slower	O
-growth	O
-of	O
-the	O
-yfiB	B-mutant
--	I-mutant
-L43P	I-mutant
-strain	O
-,	O
-and	O
-as	O
-expected	O
-,	O
-a	O
-certain	O
-amount	O
-of	O
-VB6	O
-or	O
-L	O
--	O
-Trp	O
-(	O
-4	O
-–	O
-6	O
-mmol	O
-/	O
-L	O
-for	O
-VB6	O
-and	O
-6	O
-–	O
-10	O
-mmol	O
-/	O
-L	O
-for	O
-L	O
--	O
-Trp	O
-)	O
-could	O
-reduce	O
-the	O
-surface	O
-attachment	O
-.	O
-	O
-Interestingly	O
-,	O
-at	O
-a	O
-concentration	O
-higher	O
-than	O
-8	O
-mmol	O
-/	O
-L	O
-,	O
-VB6	O
-lost	O
-its	O
-ability	O
-to	O
-inhibit	O
-biofilm	O
-formation	O
-,	O
-implying	O
-that	O
-the	O
-VB6	O
--	O
-involving	O
-regulatory	O
-mechanism	O
-is	O
-highly	O
-complicated	O
-and	O
-remains	O
-to	O
-be	O
-further	O
-investigated	O
-.	O
-	O
-Of	O
-note	O
-,	O
-both	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-have	O
-been	O
-reported	O
-to	O
-correlate	O
-with	O
-biofilm	O
-formation	O
-in	O
-certain	O
-Gram	O
--	O
-negative	O
-bacteria	O
-(	O
-Grubman	O
-et	O
-al	O
-.,;	O
-Shimazaki	O
-et	O
-al	O
-.,).	O
-	O
-In	O
-Helicobacter	O
-pylori	O
-in	O
-particular	O
-,	O
-VB6	O
-biosynthetic	O
-enzymes	O
-act	O
-as	O
-novel	O
-virulence	O
-factors	O
-,	O
-and	O
-VB6	O
-is	O
-required	O
-for	O
-full	O
-motility	O
-and	O
-virulence	O
-(	O
-Grubman	O
-et	O
-al	O
-.,).	O
-	O
-In	O
-E	O
-.	O
-coli	O
-,	O
-mutants	O
-with	O
-decreased	O
-tryptophan	O
-synthesis	O
-show	O
-greater	O
-biofilm	O
-formation	O
-,	O
-and	O
-matured	O
-biofilm	O
-is	O
-degraded	O
-by	O
-L	O
--	O
-tryptophan	O
-addition	O
-(	O
-Shimazaki	O
-et	O
-al	O
-.,).	O
-	O
-To	O
-answer	O
-the	O
-question	O
-whether	O
-competition	O
-of	O
-VB6	O
-or	O
-L	O
--	O
-Trp	O
-for	O
-the	O
-YfiB	O
-F57	O
--	O
-binding	O
-pocket	O
-of	O
-YfiR	O
-plays	O
-an	O
-essential	O
-role	O
-in	O
-inhibiting	O
-biofilm	O
-formation	O
-,	O
-we	O
-measured	O
-the	O
-binding	O
-affinities	O
-of	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-for	O
-YfiR	O
-via	O
-BIAcore	O
-experiments	O
-.	O
-	O
-The	O
-results	O
-showed	O
-relatively	O
-weak	O
-Kd	O
-values	O
-of	O
-35	O
-.	O
-2	O
-mmol	O
-/	O
-L	O
-and	O
-76	O
-.	O
-9	O
-mmol	O
-/	O
-L	O
-for	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-,	O
-respectively	O
-(	O
-Fig	O
-.	O
-6C	O
-and	O
-6D	O
-).	O
-	O
-Based	O
-on	O
-our	O
-results	O
-,	O
-we	O
-concluded	O
-that	O
-VB6	O
-or	O
-L	O
--	O
-Trp	O
-can	O
-bind	O
-to	O
-YfiR	O
-,	O
-however	O
-,	O
-VB6	O
-or	O
-L	O
--	O
-Trp	O
-alone	O
-may	O
-have	O
-little	O
-effects	O
-in	O
-interrupting	O
-the	O
-YfiB	O
--	O
-YfiR	O
-interaction	O
-,	O
-the	O
-mechanism	O
-by	O
-which	O
-VB6	O
-or	O
-L	O
--	O
-Trp	O
-inhibits	O
-biofilm	O
-formation	O
-remains	O
-unclear	O
-and	O
-requires	O
-further	O
-investigation	O
-.	O
-	O
-Previous	O
-studies	O
-suggested	O
-that	O
-in	O
-response	O
-to	O
-cell	O
-stress	O
-,	O
-YfiB	O
-in	O
-the	O
-outer	O
-membrane	O
-sequesters	O
-the	O
-periplasmic	O
-protein	O
-YfiR	O
-,	O
-releasing	O
-its	O
-inhibition	O
-of	O
-YfiN	O
-on	O
-the	O
-inner	O
-membrane	O
-and	O
-thus	O
-inducing	O
-the	O
-diguanylate	O
-cyclase	O
-activity	O
-of	O
-YfiN	O
-to	O
-allow	O
-c	O
--	O
-di	O
--	O
-GMP	O
-production	O
-(	O
-Giardina	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-Here	O
-,	O
-we	O
-report	O
-the	O
-crystal	O
-structures	O
-of	O
-YfiB	O
-alone	O
-and	O
-an	O
-active	O
-mutant	O
-YfiBL43P	B-mutant
-in	O
-complex	O
-with	O
-YfiR	O
-,	O
-indicating	O
-that	O
-YfiR	O
-forms	O
-a	O
-2	O
-:	O
-2	O
-complex	O
-with	O
-YfiB	O
-via	O
-a	O
-region	O
-composed	O
-of	O
-conserved	O
-residues	O
-.	O
-	O
-Our	O
-structural	O
-data	O
-analysis	O
-shows	O
-that	O
-the	O
-activated	O
-YfiB	O
-has	O
-an	O
-N	O
--	O
-terminal	O
-portion	O
-that	O
-is	O
-largely	O
-altered	O
-,	O
-adopting	O
-a	O
-stretched	O
-conformation	O
-compared	O
-with	O
-the	O
-compact	O
-conformation	O
-of	O
-the	O
-apo	O
-YfiB	O
-.	O
-The	O
-apo	O
-YfiB	O
-structure	O
-constructed	O
-beginning	O
-at	O
-residue	O
-34	O
-has	O
-a	O
-compact	O
-conformation	O
-of	O
-approximately	O
-45	O
-Å	O
-in	O
-length	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-preceding	O
-8	O
-aa	O
-loop	O
-(	O
-from	O
-the	O
-lipid	O
-acceptor	O
-Cys26	O
-to	O
-Gly34	O
-),	O
-the	O
-full	O
-length	O
-of	O
-the	O
-periplasmic	O
-portion	O
-of	O
-apo	O
-YfiB	O
-can	O
-reach	O
-approximately	O
-60	O
-Å	O
-.	O
-It	O
-was	O
-reported	O
-that	O
-the	O
-distance	O
-between	O
-the	O
-outer	O
-membrane	O
-and	O
-the	O
-cell	O
-wall	O
-is	O
-approximately	O
-50	O
-Å	O
-and	O
-that	O
-the	O
-thickness	O
-of	O
-the	O
-PG	O
-layer	O
-is	O
-approximately	O
-70	O
-Å	O
-(	O
-Matias	O
-et	O
-al	O
-.,).	O
-	O
-Thus	O
-,	O
-YfiB	O
-alone	O
-represents	O
-an	O
-inactive	O
-form	O
-that	O
-may	O
-only	O
-partially	O
-insert	O
-into	O
-the	O
-PG	O
-matrix	O
-.	O
-	O
-By	O
-contrast	O
-,	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-(	O
-residues	O
-44	O
-–	O
-168	O
-)	O
-has	O
-a	O
-stretched	O
-conformation	O
-of	O
-approximately	O
-55	O
-Å	O
-in	O
-length	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-17	O
-preceding	O
-intracellular	O
-residues	O
-(	O
-from	O
-the	O
-lipid	O
-acceptor	O
-Cys26	O
-to	O
-Leu43	O
-),	O
-the	O
-length	O
-of	O
-the	O
-intracellular	O
-portion	O
-of	O
-active	O
-YfiB	O
-may	O
-extend	O
-over	O
-100	O
-Å	O
-,	O
-assuming	O
-a	O
-fully	O
-stretched	O
-conformation	O
-.	O
-	O
-Provided	O
-that	O
-the	O
-diameter	O
-of	O
-the	O
-widest	O
-part	O
-of	O
-the	O
-YfiB	O
-dimer	O
-is	O
-approximately	O
-64	O
-Å	O
-,	O
-which	O
-is	O
-slightly	O
-smaller	O
-than	O
-the	O
-smallest	O
-diameter	O
-of	O
-the	O
-PG	O
-pore	O
-(	O
-70	O
-Å	O
-)	O
-(	O
-Meroueh	O
-et	O
-al	O
-.,),	O
-the	O
-YfiB	O
-dimer	O
-should	O
-be	O
-able	O
-to	O
-penetrate	O
-the	O
-PG	O
-layer	O
-.	O
-	O
-Regulatory	O
-model	O
-of	O
-the	O
-YfiBNR	O
-tripartite	O
-system	O
-.	O
-	O
-The	O
-periplasmic	O
-domain	O
-of	O
-YfiB	O
-and	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-are	O
-depicted	O
-according	O
-to	O
-the	O
-crystal	O
-structures	O
-.	O
-	O
-The	O
-lipid	O
-acceptor	O
-Cys26	O
-is	O
-indicated	O
-as	O
-blue	O
-ball	O
-.	O
-	O
-The	O
-loop	O
-connecting	O
-Cys26	O
-and	O
-Gly34	O
-of	O
-YfiB	O
-is	O
-modeled	O
-.	O
-	O
-The	O
-PAS	O
-domain	O
-of	O
-YfiN	O
-is	O
-shown	O
-as	O
-pink	O
-oval	O
-.	O
-	O
-Once	O
-activated	O
-by	O
-certain	O
-cell	O
-stress	O
-,	O
-the	O
-dimeric	O
-YfiB	O
-transforms	O
-from	O
-a	O
-compact	O
-conformation	O
-to	O
-a	O
-stretched	O
-conformation	O
-,	O
-allowing	O
-the	O
-periplasmic	O
-domain	O
-of	O
-the	O
-membrane	O
--	O
-anchored	O
-YfiB	O
-to	O
-penetrate	O
-the	O
-cell	O
-wall	O
-and	O
-sequester	O
-the	O
-YfiR	O
-dimer	O
-,	O
-thus	O
-relieving	O
-the	O
-repression	O
-of	O
-YfiN	O
-	O
-These	O
-results	O
-,	O
-together	O
-with	O
-our	O
-observation	O
-that	O
-activated	O
-YfiB	O
-has	O
-a	O
-much	O
-higher	O
-cell	O
-wall	O
-binding	O
-affinity	O
-,	O
-and	O
-previous	O
-mutagenesis	O
-data	O
-showing	O
-that	O
-(	O
-1	O
-)	O
-both	O
-PG	O
-binding	O
-and	O
-membrane	O
-anchoring	O
-are	O
-required	O
-for	O
-YfiB	O
-activity	O
-and	O
-(	O
-2	O
-)	O
-activating	O
-mutations	O
-possessing	O
-an	O
-altered	O
-N	O
--	O
-terminal	O
-loop	O
-length	O
-are	O
-dominant	O
-over	O
-the	O
-loss	O
-of	O
-PG	O
-binding	O
-(	O
-Malone	O
-et	O
-al	O
-.,),	O
-suggest	O
-an	O
-updated	O
-regulatory	O
-model	O
-of	O
-the	O
-YfiBNR	O
-system	O
-(	O
-Fig	O
-.	O
-7	O
-).	O
-	O
-In	O
-this	O
-model	O
-,	O
-in	O
-response	O
-to	O
-a	O
-particular	O
-cell	O
-stress	O
-that	O
-is	O
-yet	O
-to	O
-be	O
-identified	O
-,	O
-the	O
-dimeric	O
-YfiB	O
-is	O
-activated	O
-from	O
-a	O
-compact	O
-,	O
-inactive	O
-conformation	O
-to	O
-a	O
-stretched	O
-conformation	O
-,	O
-which	O
-possesses	O
-increased	O
-PG	O
-binding	O
-affinity	O
-.	O
-	O
-This	O
-allows	O
-the	O
-C	O
--	O
-terminal	O
-portion	O
-of	O
-the	O
-membrane	O
--	O
-anchored	O
-YfiB	O
-to	O
-reach	O
-,	O
-bind	O
-and	O
-penetrate	O
-the	O
-cell	O
-wall	O
-and	O
-sequester	O
-the	O
-YfiR	O
-dimer	O
-.	O
-	O
-The	O
-YfiBNR	O
-system	O
-provides	O
-a	O
-good	O
-example	O
-of	O
-a	O
-delicate	O
-homeostatic	O
-system	O
-that	O
-integrates	O
-multiple	O
-signals	O
-to	O
-regulate	O
-the	O
-c	O
--	O
-di	O
--	O
-GMP	O
-level	O
-.	O
-	O
-Homologs	O
-of	O
-the	O
-YfiBNR	O
-system	O
-are	O
-functionally	O
-conserved	O
-in	O
-P	O
-.	O
-aeruginosa	O
-(	O
-Malone	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,),	O
-E	O
-.	O
-coli	O
-(	O
-Hufnagel	O
-et	O
-al	O
-.,;	O
-Raterman	O
-et	O
-al	O
-.,;	O
-Sanchez	O
--	O
-Torres	O
-et	O
-al	O
-.,),	O
-K	O
-.	O
-pneumonia	O
-(	O
-Huertas	O
-et	O
-al	O
-.,)	O
-and	O
-Y	O
-.	O
-pestis	O
-(	O
-Ren	O
-et	O
-al	O
-.,),	O
-where	O
-they	O
-affect	O
-c	O
--	O
-di	O
--	O
-GMP	O
-production	O
-and	O
-biofilm	O
-formation	O
-.	O
-	O
-The	O
-mechanism	O
-by	O
-which	O
-activated	O
-YfiB	O
-relieves	O
-the	O
-repression	O
-of	O
-YfiN	O
-may	O
-be	O
-applicable	O
-to	O
-the	O
-YfiBNR	O
-system	O
-in	O
-other	O
-bacteria	O
-and	O
-to	O
-analogous	O
-outside	O
--	O
-in	O
-signaling	O
-for	O
-c	O
--	O
-di	O
--	O
-GMP	O
-production	O
-,	O
-which	O
-in	O
-turn	O
-may	O
-be	O
-relevant	O
-to	O
-the	O
-development	O
-of	O
-drugs	O
-that	O
-can	O
-circumvent	O
-complicated	O
-antibiotic	O
-resistance	O
-.	O
-	O
-X	O
--	O
-ray	O
-Crystallographic	O
-Structures	O
-of	O
-a	O
-Trimer	O
-,	O
-Dodecamer	O
-,	O
-and	O
-Annular	O
-Pore	O
-Formed	O
-by	O
-an	O
-Aβ17	O
-–	O
-36	O
-β	O
--	O
-Hairpin	O
-	O
-High	O
--	O
-resolution	O
-structures	O
-of	O
-oligomers	O
-formed	O
-by	O
-the	O
-β	O
--	O
-amyloid	O
-peptide	O
-Aβ	O
-are	O
-needed	O
-to	O
-understand	O
-the	O
-molecular	O
-basis	O
-of	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-and	O
-develop	O
-therapies	O
-.	O
-	O
-This	O
-paper	O
-presents	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structures	O
-of	O
-oligomers	O
-formed	O
-by	O
-a	O
-20	O
--	O
-residue	O
-peptide	O
-segment	O
-derived	O
-from	O
-Aβ	O
-.	O
-	O
-The	O
-development	O
-of	O
-a	O
-peptide	O
-in	O
-which	O
-Aβ17	O
-–	O
-36	O
-is	O
-stabilized	O
-as	O
-a	O
-β	O
--	O
-hairpin	O
-is	O
-described	O
-,	O
-and	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structures	O
-of	O
-oligomers	O
-it	O
-forms	O
-are	O
-reported	O
-.	O
-	O
-Two	O
-covalent	O
-constraints	O
-act	O
-in	O
-tandem	O
-to	O
-stabilize	O
-the	O
-Aβ17	O
-–	O
-36	O
-peptide	O
-in	O
-a	O
-hairpin	O
-conformation	O
-:	O
-a	O
-δ	O
--	O
-linked	O
-ornithine	O
-turn	O
-connecting	O
-positions	O
-17	O
-and	O
-36	O
-to	O
-create	O
-a	O
-macrocycle	O
-and	O
-an	O
-intramolecular	O
-disulfide	O
-linkage	O
-between	O
-positions	O
-24	O
-and	O
-29	O
-.	O
-	O
-An	O
-N	O
--	O
-methyl	O
-group	O
-at	O
-position	O
-33	O
-blocks	O
-uncontrolled	O
-aggregation	O
-.	O
-	O
-The	O
-peptide	O
-readily	O
-crystallizes	O
-as	O
-a	O
-folded	O
-β	O
--	O
-hairpin	O
-,	O
-which	O
-assembles	O
-hierarchically	O
-in	O
-the	O
-crystal	O
-lattice	O
-.	O
-	O
-Three	O
-β	O
--	O
-hairpin	O
-monomers	O
-assemble	O
-to	O
-form	O
-a	O
-triangular	O
-trimer	O
-,	O
-four	O
-trimers	O
-assemble	O
-in	O
-a	O
-tetrahedral	O
-arrangement	O
-to	O
-form	O
-a	O
-dodecamer	O
-,	O
-and	O
-five	O
-dodecamers	O
-pack	O
-together	O
-to	O
-form	O
-an	O
-annular	O
-pore	O
-.	O
-	O
-This	O
-hierarchical	O
-assembly	O
-provides	O
-a	O
-model	O
-,	O
-in	O
-which	O
-full	O
--	O
-length	O
-Aβ	O
-transitions	O
-from	O
-an	O
-unfolded	O
-monomer	O
-to	O
-a	O
-folded	O
-β	O
--	O
-hairpin	O
-,	O
-which	O
-assembles	O
-to	O
-form	O
-oligomers	O
-that	O
-further	O
-pack	O
-to	O
-form	O
-an	O
-annular	O
-pore	O
-.	O
-	O
-High	O
--	O
-resolution	O
-structures	O
-of	O
-oligomers	O
-formed	O
-by	O
-the	O
-β	O
--	O
-amyloid	O
-peptide	O
-Aβ	O
-are	O
-desperately	O
-needed	O
-to	O
-understand	O
-the	O
-molecular	O
-basis	O
-of	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-and	O
-ultimately	O
-develop	O
-preventions	O
-or	O
-treatments	O
-.	O
-	O
-In	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-,	O
-monomeric	O
-Aβ	O
-aggregates	O
-to	O
-form	O
-soluble	O
-low	O
-molecular	O
-weight	O
-oligomers	O
-,	O
-such	O
-as	O
-dimers	O
-,	O
-trimers	O
-,	O
-tetramers	O
-,	O
-hexamers	O
-,	O
-nonamers	O
-,	O
-and	O
-dodecamers	O
-,	O
-as	O
-well	O
-as	O
-high	O
-molecular	O
-weight	O
-aggregates	O
-,	O
-such	O
-as	O
-annular	O
-protofibrils	O
-.	O
-	O
-Over	O
-the	O
-last	O
-two	O
-decades	O
-the	O
-role	O
-of	O
-Aβ	O
-oligomers	O
-in	O
-the	O
-pathophysiology	O
-of	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-has	O
-begun	O
-to	O
-unfold	O
-.	O
-	O
-Mouse	O
-models	O
-for	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-have	O
-helped	O
-shape	O
-our	O
-current	O
-understanding	O
-about	O
-the	O
-Aβ	O
-oligomerization	O
-that	O
-precedes	O
-neurodegeneration	O
-.	O
-	O
-Aβ	O
-isolated	O
-from	O
-the	O
-brains	O
-of	O
-young	O
-plaque	O
--	O
-free	O
-Tg2576	O
-mice	O
-forms	O
-a	O
-mixture	O
-of	O
-low	O
-molecular	O
-weight	O
-oligomers	O
-.	O
-	O
-A	O
-56	O
-kDa	O
-soluble	O
-oligomer	O
-identified	O
-by	O
-SDS	O
--	O
-PAGE	O
-was	O
-found	O
-to	O
-be	O
-especially	O
-important	O
-within	O
-this	O
-mixture	O
-.	O
-	O
-This	O
-oligomer	O
-was	O
-termed	O
-Aβ	O
-*	O
-56	O
-and	O
-appears	O
-to	O
-be	O
-a	O
-dodecamer	O
-of	O
-Aβ	O
-.	O
-	O
-Purified	O
-Aβ	O
-*	O
-56	O
-injected	O
-intercranially	O
-into	O
-healthy	O
-rats	O
-was	O
-found	O
-to	O
-impair	O
-memory	O
-,	O
-providing	O
-evidence	O
-that	O
-this	O
-Aβ	O
-oligomer	O
-may	O
-cause	O
-memory	O
-loss	O
-in	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-.	O
-	O
-Smaller	O
-oligomers	O
-with	O
-molecular	O
-weights	O
-consistent	O
-with	O
-trimers	O
-,	O
-hexamers	O
-,	O
-and	O
-nonamers	O
-were	O
-also	O
-identified	O
-within	O
-the	O
-mixture	O
-of	O
-low	O
-molecular	O
-weight	O
-oligomers	O
-.	O
-	O
-Treatment	O
-of	O
-the	O
-mixture	O
-of	O
-low	O
-molecular	O
-weight	O
-oligomers	O
-with	O
-hexafluoroisopropanol	O
-resulted	O
-in	O
-the	O
-dissociation	O
-of	O
-the	O
-putative	O
-dodecamers	O
-,	O
-nonamers	O
-,	O
-and	O
-hexamers	O
-into	O
-trimers	O
-and	O
-monomers	O
-,	O
-suggesting	O
-that	O
-trimers	O
-may	O
-be	O
-the	O
-building	O
-block	O
-of	O
-the	O
-dodecamers	O
-,	O
-nonamers	O
-,	O
-and	O
-hexamers	O
-.	O
-	O
-Recently	O
-,	O
-Aβ	O
-trimers	O
-and	O
-Aβ	O
-*	O
-56	O
-were	O
-identified	O
-in	O
-the	O
-brains	O
-of	O
-cognitively	O
-normal	O
-humans	O
-and	O
-were	O
-found	O
-to	O
-increase	O
-with	O
-age	O
-.	O
-	O
-A	O
-type	O
-of	O
-large	O
-oligomers	O
-called	O
-annular	O
-protofibrils	O
-(	O
-APFs	O
-)	O
-have	O
-also	O
-been	O
-observed	O
-in	O
-the	O
-brains	O
-of	O
-transgenic	O
-mice	O
-and	O
-isolated	O
-from	O
-the	O
-brains	O
-of	O
-Alzheimer	O
-’	O
-s	O
-patients	O
-.	O
-	O
-APFs	O
-were	O
-first	O
-discovered	O
-in	O
-vitro	O
-using	O
-chemically	O
-synthesized	O
-Aβ	O
-that	O
-aggregated	O
-into	O
-porelike	O
-structures	O
-that	O
-could	O
-be	O
-observed	O
-by	O
-atomic	O
-force	O
-microscopy	O
-(	O
-AFM	O
-)	O
-and	O
-transmission	O
-electron	O
-microscopy	O
-(	O
-TEM	O
-).	O
-	O
-The	O
-sizes	O
-of	O
-APFs	O
-prepared	O
-in	O
-vitro	O
-vary	O
-among	O
-different	O
-studies	O
-.	O
-	O
-Lashuel	O
-et	O
-al	O
-.	O
-observed	O
-APFs	O
-with	O
-an	O
-outer	O
-diameter	O
-that	O
-ranged	O
-from	O
-7	O
-–	O
-10	O
-nm	O
-and	O
-an	O
-inner	O
-diameter	O
-that	O
-ranged	O
-from	O
-1	O
-.	O
-5	O
-–	O
-2	O
-nm	O
-,	O
-consistent	O
-with	O
-molecular	O
-weights	O
-of	O
-150	O
-–	O
-250	O
-kDa	O
-.	O
-	O
-Quist	O
-et	O
-al	O
-.	O
-observed	O
-APFs	O
-with	O
-an	O
-outer	O
-diameter	O
-of	O
-16	O
-nm	O
-embedded	O
-in	O
-a	O
-lipid	O
-bilayer	O
-.	O
-	O
-Kayed	O
-et	O
-al	O
-.	O
-observed	O
-APFs	O
-with	O
-an	O
-outer	O
-diameter	O
-that	O
-ranged	O
-from	O
-8	O
-–	O
-25	O
-nm	O
-,	O
-which	O
-were	O
-composed	O
-of	O
-small	O
-spherical	O
-Aβ	O
-oligomers	O
-,	O
-3	O
-–	O
-5	O
-nm	O
-in	O
-diameter	O
-.	O
-	O
-Although	O
-the	O
-APFs	O
-in	O
-these	O
-studies	O
-differ	O
-in	O
-size	O
-,	O
-they	O
-share	O
-a	O
-similar	O
-annular	O
-morphology	O
-and	O
-appear	O
-to	O
-be	O
-composed	O
-of	O
-smaller	O
-oligomers	O
-.	O
-	O
-APFs	O
-have	O
-also	O
-been	O
-observed	O
-in	O
-the	O
-brains	O
-of	O
-APP23	O
-transgenic	O
-mice	O
-by	O
-immunofluorescence	O
-with	O
-an	O
-anti	O
--	O
-APF	O
-antibody	O
-and	O
-were	O
-found	O
-to	O
-accumulate	O
-in	O
-neuronal	O
-processes	O
-and	O
-synapses	O
-.	O
-	O
-In	O
-a	O
-subsequent	O
-study	O
-,	O
-APFs	O
-were	O
-isolated	O
-from	O
-the	O
-brains	O
-of	O
-Alzheimer	O
-’	O
-s	O
-patients	O
-by	O
-immunoprecipitation	O
-with	O
-an	O
-anti	O
--	O
-APF	O
-antibody	O
-.	O
-	O
-These	O
-APFs	O
-had	O
-an	O
-outer	O
-diameter	O
-that	O
-ranged	O
-from	O
-11	O
-–	O
-14	O
-nm	O
-and	O
-an	O
-inner	O
-diameter	O
-that	O
-ranged	O
-from	O
-2	O
-.	O
-5	O
-–	O
-4	O
-nm	O
-.	O
-	O
-Dimers	O
-of	O
-Aβ	O
-have	O
-also	O
-been	O
-isolated	O
-from	O
-the	O
-brains	O
-of	O
-Alzheimer	O
-’	O
-s	O
-patients	O
-.−	O
-Aβ	O
-dimers	O
-inhibit	O
-long	O
--	O
-term	O
-potentiation	O
-in	O
-mice	O
-and	O
-promote	O
-hyperphosphorylation	O
-of	O
-the	O
-microtubule	O
--	O
-associated	O
-protein	O
-tau	O
-,	O
-leading	O
-to	O
-neuritic	O
-damage	O
-.	O
-	O
-Aβ	O
-dimers	O
-have	O
-only	O
-been	O
-isolated	O
-from	O
-human	O
-or	O
-transgenic	O
-mouse	O
-brains	O
-that	O
-contain	O
-the	O
-pathognomonic	O
-fibrillar	O
-Aβ	O
-plaques	O
-associated	O
-with	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-endogenous	O
-rise	O
-of	O
-Aβ	O
-dimers	O
-in	O
-the	O
-brains	O
-of	O
-Tg2576	O
-and	O
-J20	O
-transgenic	O
-mice	O
-coincides	O
-with	O
-the	O
-deposition	O
-of	O
-Aβ	O
-plaques	O
-.	O
-	O
-These	O
-observations	O
-suggest	O
-that	O
-the	O
-Aβ	O
-trimers	O
-,	O
-hexamers	O
-,	O
-dodecamers	O
-,	O
-and	O
-related	O
-assemblies	O
-may	O
-be	O
-associated	O
-with	O
-presymptomatic	O
-neurodegeneration	O
-,	O
-while	O
-Aβ	O
-dimers	O
-are	O
-more	O
-closely	O
-associated	O
-with	O
-fibril	O
-formation	O
-and	O
-plaque	O
-deposition	O
-during	O
-symptomatic	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-.−	O
-	O
-The	O
-approach	O
-of	O
-isolating	O
-and	O
-characterizing	O
-Aβ	O
-oligomers	O
-has	O
-not	O
-provided	O
-any	O
-high	O
--	O
-resolution	O
-structures	O
-of	O
-Aβ	O
-oligomers	O
-.	O
-	O
-Techniques	O
-such	O
-as	O
-SDS	O
--	O
-PAGE	O
-,	O
-TEM	O
-,	O
-and	O
-AFM	O
-have	O
-only	O
-provided	O
-information	O
-about	O
-the	O
-molecular	O
-weights	O
-,	O
-sizes	O
-,	O
-morphologies	O
-,	O
-and	O
-stoichiometry	O
-of	O
-Aβ	O
-oligomers	O
-.	O
-	O
-High	O
--	O
-resolution	O
-structural	O
-studies	O
-of	O
-Aβ	O
-have	O
-primarily	O
-focused	O
-on	O
-Aβ	O
-fibrils	O
-and	O
-Aβ	O
-monomers	O
-.	O
-	O
-Solid	O
--	O
-state	O
-NMR	O
-spectroscopy	O
-studies	O
-of	O
-Aβ	O
-fibrils	O
-revealed	O
-that	O
-Aβ	O
-fibrils	O
-are	O
-generally	O
-composed	O
-of	O
-extended	O
-networks	O
-of	O
-in	O
--	O
-register	O
-parallel	O
-β	O
--	O
-sheets	O
-.−	O
-X	O
--	O
-ray	O
-crystallographic	O
-studies	O
-using	O
-fragments	O
-of	O
-Aβ	O
-have	O
-provided	O
-additional	O
-information	O
-about	O
-how	O
-Aβ	O
-fibrils	O
-pack	O
-.	O
-	O
-Solution	O
--	O
-phase	O
-NMR	O
-and	O
-solid	O
--	O
-state	O
-NMR	O
-have	O
-been	O
-used	O
-to	O
-study	O
-the	O
-structures	O
-of	O
-the	O
-Aβ	O
-monomers	O
-within	O
-oligomeric	O
-assemblies	O
-.−	O
-A	O
-major	O
-finding	O
-from	O
-these	O
-studies	O
-is	O
-that	O
-oligomeric	O
-assemblies	O
-of	O
-Aβ	O
-are	O
-primarily	O
-composed	O
-of	O
-antiparallel	O
-β	O
--	O
-sheets	O
-.	O
-	O
-Many	O
-of	O
-these	O
-studies	O
-have	O
-reported	O
-the	O
-monomer	O
-subunit	O
-as	O
-adopting	O
-a	O
-β	O
--	O
-hairpin	O
-conformation	O
-,	O
-in	O
-which	O
-the	O
-hydrophobic	O
-central	O
-and	O
-C	O
--	O
-terminal	O
-regions	O
-form	O
-an	O
-antiparallel	O
-β	O
--	O
-sheet	O
-.	O
-	O
-In	O
-2008	O
-,	O
-Hoyer	O
-et	O
-al	O
-.	O
-reported	O
-the	O
-NMR	O
-structure	O
-of	O
-an	O
-Aβ	O
-monomer	O
-bound	O
-to	O
-an	O
-artificial	O
-binding	O
-protein	O
-called	O
-an	O
-affibody	O
-(	O
-PDB	O
-2OTK	O
-).	O
-	O
-The	O
-structure	O
-revealed	O
-that	O
-monomeric	O
-Aβ	O
-forms	O
-a	O
-β	O
--	O
-hairpin	O
-when	O
-bound	O
-to	O
-the	O
-affibody	O
-.	O
-	O
-This	O
-Aβ	O
-β	O
--	O
-hairpin	O
-encompasses	O
-residues	O
-17	O
-–	O
-37	O
-and	O
-contains	O
-two	O
-β	O
--	O
-strands	O
-comprising	O
-Aβ17	O
-–	O
-24	O
-and	O
-Aβ30	O
-–	O
-37	O
-connected	O
-by	O
-an	O
-Aβ25	O
-–	O
-29	O
-loop	O
-.	O
-	O
-Sequestering	O
-Aβ	O
-within	O
-the	O
-affibody	O
-prevents	O
-its	O
-fibrilization	O
-and	O
-reduces	O
-its	O
-neurotoxicity	O
-,	O
-providing	O
-evidence	O
-that	O
-the	O
-β	O
--	O
-hairpin	O
-structure	O
-may	O
-contribute	O
-to	O
-the	O
-ability	O
-of	O
-Aβ	O
-to	O
-form	O
-neurotoxic	O
-oligomers	O
-.	O
-	O
-In	O
-a	O
-related	O
-study	O
-,	O
-Sandberg	O
-et	O
-al	O
-.	O
-constrained	O
-Aβ	O
-in	O
-a	O
-β	O
--	O
-hairpin	O
-conformation	O
-by	O
-mutating	O
-residues	O
-A21	O
-and	O
-A30	O
-to	O
-cysteine	O
-and	O
-forming	O
-an	O
-intramolecular	O
-disulfide	O
-bond	O
-.	O
-	O
-Locking	O
-Aβ	O
-into	O
-a	O
-β	O
--	O
-hairpin	O
-structure	O
-resulted	O
-in	O
-the	O
-formation	O
-Aβ	O
-oligomers	O
-,	O
-which	O
-were	O
-observed	O
-by	O
-size	O
-exclusion	O
-chromatography	O
-(	O
-SEC	O
-)	O
-and	O
-SDS	O
--	O
-PAGE	O
-.	O
-	O
-The	O
-oligomers	O
-with	O
-a	O
-molecular	O
-weight	O
-of	O
-∼	O
-100	O
-kDa	O
-that	O
-were	O
-isolated	O
-by	O
-SEC	O
-were	O
-toxic	O
-toward	O
-neuronally	O
-derived	O
-SH	O
--	O
-SY5Y	O
-cells	O
-.	O
-	O
-This	O
-study	O
-provides	O
-evidence	O
-for	O
-the	O
-role	O
-of	O
-β	O
--	O
-hairpin	O
-structure	O
-in	O
-Aβ	O
-oligomerization	O
-and	O
-neurotoxicity	O
-.	O
-	O
-Inspired	O
-by	O
-these	O
-β	O
--	O
-hairpin	O
-structures	O
-,	O
-our	O
-laboratory	O
-developed	O
-a	O
-macrocyclic	O
-β	O
--	O
-sheet	O
-peptide	O
-derived	O
-from	O
-Aβ17	O
-–	O
-36	O
-designed	O
-to	O
-mimic	O
-an	O
-Aβ	O
-β	O
--	O
-hairpin	O
-and	O
-reported	O
-its	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-.	O
-	O
-This	O
-peptide	O
-(	O
-peptide	B-mutant
-1	I-mutant
-)	O
-consists	O
-of	O
-two	O
-β	O
--	O
-strands	O
-comprising	O
-Aβ17	O
-–	O
-23	O
-and	O
-Aβ30	O
-–	O
-36	O
-covalently	O
-linked	O
-by	O
-two	O
-δ	O
--	O
-linked	O
-ornithine	O
-(	O
-δOrn	O
-)	O
-β	O
--	O
-turn	O
-mimics	O
-.	O
-	O
-The	O
-δOrn	O
-that	O
-connects	O
-residues	O
-D23	O
-and	O
-A30	O
-replaces	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-.	O
-	O
-The	O
-δOrn	O
-that	O
-connects	O
-residues	O
-L17	O
-and	O
-V36	O
-enforces	O
-β	O
--	O
-hairpin	O
-structure	O
-.	O
-	O
-We	O
-incorporated	O
-an	O
-N	O
--	O
-methyl	O
-group	O
-at	O
-position	O
-G33	O
-to	O
-prevent	O
-uncontrolled	O
-aggregation	O
-and	O
-precipitation	O
-of	O
-the	O
-peptide	O
-.	O
-	O
-To	O
-improve	O
-the	O
-solubility	O
-of	O
-the	O
-peptide	O
-we	O
-replaced	O
-M35	O
-with	O
-the	O
-hydrophilic	O
-isostere	O
-of	O
-methionine	O
-,	O
-ornithine	O
-(	O
-α	O
--	O
-linked	O
-)	O
-(	O
-Figure	O
-1B	O
-).	O
-	O
-The	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-1	I-mutant
-reveals	O
-that	O
-it	O
-folds	O
-to	O
-form	O
-a	O
-β	O
--	O
-hairpin	O
-that	O
-assembles	O
-to	O
-form	O
-trimers	O
-and	O
-that	O
-the	O
-trimers	O
-further	O
-assemble	O
-to	O
-form	O
-hexamers	O
-and	O
-dodecamers	O
-.	O
-	O
-(	O
-A	O
-)	O
-Cartoon	O
-illustrating	O
-the	O
-design	O
-of	O
-peptides	O
-1	O
-and	O
-2	O
-and	O
-their	O
-relationship	O
-to	O
-an	O
-Aβ17	O
-–	O
-36	O
-β	O
--	O
-hairpin	O
-.	O
-	O
-(	O
-B	O
-)	O
-Chemical	O
-structure	O
-of	O
-peptide	B-mutant
-1	I-mutant
-illustrating	O
-Aβ17	O
-–	O
-23	O
-and	O
-Aβ30	O
-–	O
-36	O
-,	O
-M35Orn	O
-,	O
-the	O
-N	O
--	O
-methyl	O
-group	O
-,	O
-and	O
-the	O
-δ	O
--	O
-linked	O
-ornithine	O
-turns	O
-.	O
-(	O
-C	O
-)	O
-Chemical	O
-structure	O
-of	O
-peptide	B-mutant
-2	I-mutant
-illustrating	O
-Aβ17	O
-–	O
-36	O
-,	O
-the	O
-N	O
--	O
-methyl	O
-group	O
-,	O
-the	O
-disulfide	O
-bond	O
-across	O
-positions	O
-24	O
-and	O
-29	O
-,	O
-and	O
-the	O
-δ	O
--	O
-linked	O
-ornithine	O
-turn	O
-.	O
-	O
-Our	O
-design	O
-of	O
-peptide	B-mutant
-1	I-mutant
-omitted	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-.	O
-	O
-To	O
-visualize	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-,	O
-we	O
-performed	O
-replica	O
--	O
-exchange	O
-molecular	O
-dynamics	O
-(	O
-REMD	O
-)	O
-simulations	O
-on	O
-Aβ17	O
-–	O
-36	O
-using	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-coordinates	O
-of	O
-Aβ17	O
-–	O
-23	O
-and	O
-Aβ30	O
-–	O
-36	O
-from	O
-peptide	B-mutant
-1	I-mutant
-.	O
-	O
-These	O
-studies	O
-provided	O
-a	O
-working	O
-model	O
-for	O
-a	O
-trimer	O
-of	O
-Aβ17	O
-–	O
-36	O
-β	O
--	O
-hairpins	O
-and	O
-demonstrated	O
-that	O
-the	O
-trimer	O
-should	O
-be	O
-capable	O
-of	O
-accommodating	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-.	O
-	O
-In	O
-the	O
-current	O
-study	O
-we	O
-set	O
-out	O
-to	O
-restore	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-,	O
-reintroduce	O
-the	O
-methionine	O
-residue	O
-at	O
-position	O
-35	O
-,	O
-and	O
-determine	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structures	O
-of	O
-oligomers	O
-that	O
-form	O
-.	O
-	O
-We	O
-designed	O
-peptide	B-mutant
-2	I-mutant
-as	O
-a	O
-homologue	O
-of	O
-peptide	B-mutant
-1	I-mutant
-that	O
-embodies	O
-these	O
-ideas	O
-.	O
-	O
-Peptide	B-mutant
-2	I-mutant
-contains	O
-a	O
-methionine	O
-residue	O
-at	O
-position	O
-35	O
-and	O
-an	O
-Aβ24	O
-–	O
-29	O
-loop	O
-with	O
-residues	O
-24	O
-and	O
-29	O
-(	O
-Val	O
-and	O
-Gly	O
-)	O
-mutated	O
-to	O
-cysteine	O
-and	O
-linked	O
-by	O
-a	O
-disulfide	O
-bond	O
-(	O
-Figure	O
-1C	O
-).	O
-	O
-Here	O
-,	O
-we	O
-describe	O
-the	O
-development	O
-of	O
-peptide	B-mutant
-2	I-mutant
-and	O
-report	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structures	O
-of	O
-the	O
-trimer	O
-,	O
-dodecamer	O
-,	O
-and	O
-annular	O
-pore	O
-observed	O
-within	O
-the	O
-crystal	O
-structure	O
-.	O
-	O
-Development	O
-of	O
-Peptide	B-mutant
-2	I-mutant
-	O
-We	O
-developed	O
-peptide	B-mutant
-2	I-mutant
-from	O
-peptide	B-mutant
-1	I-mutant
-by	O
-an	O
-iterative	O
-process	O
-,	O
-in	O
-which	O
-we	O
-first	O
-attempted	O
-to	O
-restore	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-without	O
-a	O
-disulfide	O
-linkage	O
-.	O
-	O
-We	O
-envisioned	O
-peptide	B-mutant
-3	I-mutant
-as	O
-a	O
-homologue	O
-of	O
-peptide	B-mutant
-1	I-mutant
-with	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-in	O
-place	O
-of	O
-the	O
-δOrn	O
-that	O
-connects	O
-D23	O
-and	O
-A30	O
-and	O
-p	O
--	O
-iodophenylalanine	O
-(	O
-FI	O
-)	O
-in	O
-place	O
-of	O
-F19	O
-.	O
-	O
-We	O
-routinely	O
-use	O
-p	O
--	O
-iodophenylalanine	O
-to	O
-determine	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-phases	O
-.	O
-	O
-After	O
-determining	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-the	O
-p	O
--	O
-iodophenylalanine	O
-variant	O
-we	O
-attempt	O
-to	O
-determine	O
-the	O
-structure	O
-of	O
-the	O
-native	O
-phenylalanine	O
-compound	O
-by	O
-isomorphous	O
-replacement	O
-.	O
-	O
-Upon	O
-synthesizing	O
-peptide	B-mutant
-3	I-mutant
-,	O
-we	O
-found	O
-that	O
-it	O
-formed	O
-an	O
-amorphous	O
-precipitate	O
-in	O
-most	O
-crystallization	O
-conditions	O
-screened	O
-and	O
-failed	O
-to	O
-afford	O
-crystals	O
-in	O
-any	O
-condition	O
-.	O
-	O
-We	O
-postulate	O
-that	O
-the	O
-loss	O
-of	O
-the	O
-δOrn	O
-constraint	O
-leads	O
-to	O
-conformational	O
-heterogeneity	O
-that	O
-prevents	O
-peptide	B-mutant
-3	I-mutant
-from	O
-crystallizing	O
-.	O
-	O
-To	O
-address	O
-this	O
-issue	O
-,	O
-we	O
-next	O
-incorporated	O
-a	O
-disulfide	O
-bond	O
-between	O
-residues	O
-24	O
-and	O
-29	O
-as	O
-a	O
-conformational	O
-constraint	O
-that	O
-serves	O
-as	O
-a	O
-surrogate	O
-for	O
-δOrn	O
-.	O
-	O
-We	O
-designed	O
-peptide	B-mutant
-4	I-mutant
-to	O
-embody	O
-this	O
-idea	O
-,	O
-mutating	O
-Val24	O
-and	O
-Gly29	O
-to	O
-cysteine	O
-and	O
-forming	O
-an	O
-interstrand	O
-disulfide	O
-linkage	O
-.	O
-	O
-We	O
-mutated	O
-these	O
-residues	O
-because	O
-they	O
-occupy	O
-the	O
-same	O
-position	O
-as	O
-the	O
-δOrn	O
-that	O
-connects	O
-D23	O
-and	O
-A30	O
-in	O
-peptide	B-mutant
-1	I-mutant
-.	O
-	O
-Residues	O
-V24	O
-and	O
-G29	O
-form	O
-a	O
-non	O
--	O
-hydrogen	O
--	O
-bonded	O
-pair	O
-,	O
-which	O
-can	O
-readily	O
-accommodate	O
-disulfide	O
-linkages	O
-in	O
-antiparallel	O
-β	O
--	O
-sheets	O
-.	O
-	O
-Disulfide	O
-bonds	O
-across	O
-non	O
--	O
-hydrogen	O
--	O
-bonded	O
-pairs	O
-stabilize	O
-β	O
--	O
-hairpins	O
-,	O
-while	O
-disulfide	O
-bonds	O
-across	O
-hydrogen	O
--	O
-bonded	O
-pairs	O
-do	O
-not	O
-.	O
-	O
-Although	O
-the	O
-disulfide	O
-bond	O
-between	O
-positions	O
-24	O
-and	O
-29	O
-helps	O
-stabilize	O
-the	O
-β	O
--	O
-hairpin	O
-,	O
-it	O
-does	O
-not	O
-alter	O
-the	O
-charge	O
-or	O
-substantially	O
-change	O
-the	O
-hydrophobicity	O
-of	O
-the	O
-Aβ17	O
-–	O
-36	O
-β	O
--	O
-hairpin	O
-.	O
-	O
-We	O
-were	O
-gratified	O
-to	O
-find	O
-that	O
-peptide	B-mutant
-4	I-mutant
-afforded	O
-crystals	O
-suitable	O
-for	O
-X	O
--	O
-ray	O
-crystallography	O
-.	O
-	O
-As	O
-the	O
-next	O
-step	O
-in	O
-the	O
-iterative	O
-process	O
-,	O
-we	O
-determined	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-this	O
-peptide	O
-(	O
-PDB	O
-5HOW	O
-).	O
-	O
-After	O
-determining	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-4	I-mutant
-we	O
-reintroduced	O
-the	O
-native	O
-phenylalanine	O
-at	O
-position	O
-19	O
-and	O
-the	O
-methionine	O
-at	O
-position	O
-35	O
-to	O
-afford	O
-peptide	B-mutant
-2	I-mutant
-.	O
-	O
-We	O
-completed	O
-the	O
-iterative	O
-process	O
-—	O
-from	O
-1	O
-to	O
-3	O
-to	O
-4	O
-to	O
-2	O
-—	O
-by	O
-successfully	O
-determining	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-2	I-mutant
-(	O
-PDB	O
-5HOX	O
-and	O
-5HOY	O
-).	O
-	O
-The	O
-following	O
-sections	O
-describe	O
-the	O
-synthesis	O
-of	O
-peptides	B-mutant
-2	I-mutant
-–	I-mutant
-4	I-mutant
-and	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-2	I-mutant
-.	O
-	O
-Synthesis	O
-of	O
-Peptides	B-mutant
-2	I-mutant
-–	I-mutant
-4	I-mutant
-	O
-We	O
-synthesized	O
-peptides	B-mutant
-2	I-mutant
-–	I-mutant
-4	I-mutant
-by	O
-similar	O
-procedures	O
-to	O
-those	O
-we	O
-have	O
-developed	O
-for	O
-other	O
-macrocyclic	O
-peptides	O
-.	O
-	O
-In	O
-synthesizing	O
-peptides	B-mutant
-2	I-mutant
-and	I-mutant
-4	I-mutant
-we	O
-formed	O
-the	O
-disulfide	O
-linkage	O
-after	O
-macrolactamization	O
-and	O
-deprotection	O
-of	O
-the	O
-acid	O
--	O
-labile	O
-side	O
-chain	O
-protecting	O
-groups	O
-.	O
-	O
-We	O
-used	O
-acid	O
--	O
-stable	O
-Acm	O
--	O
-protected	O
-cysteine	O
-residues	O
-at	O
-positions	O
-24	O
-and	O
-29	O
-and	O
-removed	O
-the	O
-Acm	O
-groups	O
-by	O
-oxidation	O
-with	O
-I2	O
-in	O
-aqueous	O
-acetic	O
-acid	O
-to	O
-afford	O
-the	O
-disulfide	O
-linkage	O
-.	O
-	O
-Peptides	B-mutant
-2	I-mutant
-–	I-mutant
-4	I-mutant
-were	O
-purified	O
-by	O
-RP	O
--	O
-HPLC	O
-.	O
-	O
-Crystallization	O
-,	O
-X	O
--	O
-ray	O
-Crystallographic	O
-Data	O
-Collection	O
-,	O
-Data	O
-Processing	O
-,	O
-and	O
-Structure	O
-Determination	O
-of	O
-Peptides	B-mutant
-2	I-mutant
-and	I-mutant
-4	I-mutant
-	O
-We	O
-screened	O
-crystallization	O
-conditions	O
-for	O
-peptide	B-mutant
-4	I-mutant
-in	O
-a	O
-96	O
--	O
-well	O
--	O
-plate	O
-format	O
-using	O
-three	O
-different	O
-Hampton	O
-Research	O
-crystallization	O
-kits	O
-(	O
-Crystal	O
-Screen	O
-,	O
-Index	O
-,	O
-and	O
-PEG	O
-/	O
-Ion	O
-)	O
-with	O
-three	O
-ratios	O
-of	O
-peptide	O
-and	O
-mother	O
-liquor	O
-per	O
-condition	O
-(	O
-864	O
-experiments	O
-).	O
-	O
-Peptide	B-mutant
-4	I-mutant
-afforded	O
-crystals	O
-in	O
-a	O
-single	O
-set	O
-of	O
-conditions	O
-containing	O
-HEPES	O
-buffer	O
-and	O
-Jeffamine	O
-M	O
--	O
-600	O
-—	O
-the	O
-same	O
-crystallization	O
-conditions	O
-that	O
-afforded	O
-crystals	O
-of	O
-peptide	B-mutant
-1	I-mutant
-.	O
-	O
-Peptide	B-mutant
-2	I-mutant
-also	O
-afforded	O
-crystals	O
-in	O
-these	O
-conditions	O
-.	O
-	O
-We	O
-further	O
-optimized	O
-these	O
-conditions	O
-to	O
-rapidly	O
-(∼	O
-72	O
-h	O
-)	O
-yield	O
-crystals	O
-suitable	O
-for	O
-X	O
--	O
-ray	O
-crystallography	O
-.	O
-	O
-The	O
-optimized	O
-conditions	O
-consist	O
-of	O
-0	O
-.	O
-1	O
-M	O
-HEPES	O
-at	O
-pH	O
-6	O
-.	O
-4	O
-with	O
-31	O
-%	O
-Jeffamine	O
-M	O
--	O
-600	O
-for	O
-peptide	B-mutant
-4	I-mutant
-and	O
-0	O
-.	O
-1	O
-M	O
-HEPES	O
-pH	O
-7	O
-.	O
-1	O
-with	O
-29	O
-%	O
-Jeffamine	O
-M	O
--	O
-600	O
-for	O
-peptide	B-mutant
-2	I-mutant
-.	O
-	O
-Crystal	O
-diffraction	O
-data	O
-for	O
-peptides	B-mutant
-4	I-mutant
-and	I-mutant
-2	I-mutant
-were	O
-collected	O
-in	O
--	O
-house	O
-with	O
-a	O
-Rigaku	O
-MicroMax	O
-007HF	O
-X	O
--	O
-ray	O
-diffractometer	O
-at	O
-1	O
-.	O
-54	O
-Å	O
-wavelength	O
-.	O
-	O
-Crystal	O
-diffraction	O
-data	O
-for	O
-peptide	B-mutant
-2	I-mutant
-were	O
-also	O
-collected	O
-at	O
-the	O
-Advanced	O
-Light	O
-Source	O
-at	O
-Lawrence	O
-Berkeley	O
-National	O
-Laboratory	O
-with	O
-a	O
-synchrotron	O
-source	O
-at	O
-1	O
-.	O
-00	O
-Å	O
-wavelength	O
-to	O
-achieve	O
-higher	O
-resolution	O
-.	O
-	O
-Data	O
-from	O
-peptides	B-mutant
-4	I-mutant
-and	I-mutant
-2	I-mutant
-suitable	O
-for	O
-refinement	O
-at	O
-2	O
-.	O
-30	O
-Å	O
-were	O
-obtained	O
-from	O
-the	O
-diffractometer	O
-;	O
-data	O
-from	O
-peptide	B-mutant
-2	I-mutant
-suitable	O
-for	O
-refinement	O
-at	O
-1	O
-.	O
-90	O
-Å	O
-were	O
-obtained	O
-from	O
-the	O
-synchrotron	O
-.	O
-	O
-Data	O
-for	O
-peptides	B-mutant
-4	I-mutant
-and	I-mutant
-2	I-mutant
-were	O
-scaled	O
-and	O
-merged	O
-using	O
-XDS	O
-.	O
-	O
-Phases	O
-for	O
-peptide	B-mutant
-4	I-mutant
-were	O
-determined	O
-by	O
-single	O
--	O
-wavelength	O
-anomalous	O
-diffraction	O
-(	O
-SAD	O
-)	O
-phasing	O
-by	O
-using	O
-the	O
-coordinates	O
-of	O
-the	O
-iodine	O
-anomalous	O
-signal	O
-from	O
-p	O
--	O
-iodophenylalanine	O
-.	O
-	O
-Phases	O
-for	O
-peptide	B-mutant
-2	I-mutant
-were	O
-determined	O
-by	O
-isomorphous	O
-replacement	O
-of	O
-peptide	B-mutant
-4	I-mutant
-.	O
-	O
-The	O
-structures	O
-of	O
-peptides	B-mutant
-2	I-mutant
-and	I-mutant
-4	I-mutant
-were	O
-solved	O
-and	O
-refined	O
-in	O
-the	O
-P6122	O
-space	O
-group	O
-.	O
-	O
-The	O
-asymmetric	O
-unit	O
-of	O
-each	O
-peptide	O
-consists	O
-of	O
-six	O
-monomers	O
-,	O
-arranged	O
-as	O
-two	O
-trimers	O
-.	O
-	O
-Peptides	B-mutant
-2	I-mutant
-and	I-mutant
-4	I-mutant
-form	O
-morphologically	O
-identical	O
-structures	O
-and	O
-assemblies	O
-in	O
-the	O
-crystal	O
-lattice	O
-.	O
-	O
-X	O
--	O
-ray	O
-Crystallographic	O
-Structure	O
-of	O
-Peptide	B-mutant
-2	I-mutant
-and	O
-the	O
-Oligomers	O
-It	O
-Forms	O
-	O
-The	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-2	I-mutant
-reveals	O
-that	O
-it	O
-folds	O
-to	O
-form	O
-a	O
-twisted	O
-β	O
--	O
-hairpin	O
-comprising	O
-two	O
-β	O
--	O
-strands	O
-connected	O
-by	O
-a	O
-loop	O
-(	O
-Figure	O
-2A	O
-).	O
-	O
-Eight	O
-residues	O
-make	O
-up	O
-each	O
-surface	O
-of	O
-the	O
-β	O
--	O
-hairpin	O
-:	O
-L17	O
-,	O
-F19	O
-,	O
-A21	O
-,	O
-D23	O
-,	O
-A30	O
-,	O
-I32	O
-,	O
-L34	O
-,	O
-and	O
-V36	O
-make	O
-up	O
-one	O
-surface	O
-;	O
-V18	O
-,	O
-F20	O
-,	O
-E22	O
-,	O
-C24	O
-,	O
-C29	O
-,	O
-I31	O
-,	O
-G33	O
-,	O
-and	O
-M35	O
-make	O
-up	O
-the	O
-other	O
-surface	O
-.	O
-	O
-The	O
-β	O
--	O
-strands	O
-of	O
-the	O
-monomers	O
-in	O
-the	O
-asymmetric	O
-unit	O
-are	O
-virtually	O
-identical	O
-,	O
-differing	O
-primarily	O
-in	O
-rotamers	O
-of	O
-F20	O
-,	O
-E22	O
-,	O
-C24	O
-,	O
-C29	O
-,	O
-I31	O
-,	O
-and	O
-M35	O
-(	O
-Figure	O
-S1	O
-).	O
-	O
-The	O
-disulfide	O
-linkages	O
-suffered	O
-radiation	O
-damage	O
-under	O
-synchrotron	O
-radiation	O
-.	O
-	O
-We	O
-refined	O
-three	O
-of	O
-the	O
-β	O
--	O
-hairpins	O
-with	O
-intact	O
-disulfide	O
-linkages	O
-and	O
-three	O
-with	O
-thiols	O
-to	O
-represent	O
-cleaved	O
-disulfide	O
-linkages	O
-in	O
-the	O
-synchrotron	O
-data	O
-set	O
-(	O
-PDB	O
-5HOX	O
-).	O
-	O
-No	O
-evidence	O
-for	O
-cleavage	O
-of	O
-the	O
-disulfides	O
-was	O
-observed	O
-in	O
-the	O
-refinement	O
-of	O
-the	O
-data	O
-set	O
-collected	O
-on	O
-the	O
-X	O
--	O
-ray	O
-diffractometer	O
-,	O
-and	O
-we	O
-refined	O
-all	O
-disulfide	O
-linkages	O
-as	O
-intact	O
-(	O
-PDB	O
-5HOY	O
-).	O
-	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-2	I-mutant
-(	O
-PDB	O
-5HOX	O
-,	O
-synchrotron	O
-data	O
-set	O
-).	O
-(	O
-A	O
-)	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-a	O
-representative	O
-β	O
--	O
-hairpin	O
-monomer	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-.	O
-(	O
-B	O
-)	O
-Overlay	O
-of	O
-the	O
-six	O
-β	O
--	O
-hairpin	O
-monomers	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-The	O
-β	O
--	O
-hairpins	O
-are	O
-shown	O
-as	O
-cartoons	O
-to	O
-illustrate	O
-the	O
-differences	O
-in	O
-the	O
-Aβ25	O
-–	O
-28	O
-loops	O
-.	O
-	O
-The	O
-Aβ25	O
-–	O
-28	O
-loops	O
-of	O
-the	O
-six	O
-monomers	O
-within	O
-the	O
-asymmetric	O
-unit	O
-vary	O
-substantially	O
-in	O
-backbone	O
-geometry	O
-and	O
-side	O
-chain	O
-rotamers	O
-(	O
-Figures	O
-2B	O
-and	O
-S1	O
-).	O
-	O
-The	O
-electron	O
-density	O
-for	O
-the	O
-loops	O
-is	O
-weak	O
-and	O
-diffuse	O
-compared	O
-to	O
-the	O
-electron	O
-density	O
-for	O
-the	O
-β	O
--	O
-strands	O
-.	O
-	O
-The	O
-B	O
-values	O
-for	O
-the	O
-loops	O
-are	O
-large	O
-,	O
-indicating	O
-that	O
-the	O
-loops	O
-are	O
-dynamic	O
-and	O
-not	O
-well	O
-ordered	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-differences	O
-in	O
-backbone	O
-geometry	O
-and	O
-side	O
-chain	O
-rotamers	O
-among	O
-the	O
-loops	O
-are	O
-likely	O
-of	O
-little	O
-significance	O
-and	O
-should	O
-be	O
-interpreted	O
-with	O
-caution	O
-.	O
-	O
-Peptide	B-mutant
-2	I-mutant
-assembles	O
-into	O
-oligomers	O
-similar	O
-in	O
-morphology	O
-to	O
-those	O
-formed	O
-by	O
-peptide	B-mutant
-1	I-mutant
-.	O
-	O
-Like	O
-peptide	B-mutant
-1	I-mutant
-,	O
-peptide	B-mutant
-2	I-mutant
-forms	O
-a	O
-triangular	O
-trimer	O
-,	O
-and	O
-four	O
-trimers	O
-assemble	O
-to	O
-form	O
-a	O
-dodecamer	O
-.	O
-	O
-In	O
-the	O
-higher	O
--	O
-order	O
-assembly	O
-of	O
-the	O
-dodecamers	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-a	O
-new	O
-structure	O
-emerges	O
-,	O
-not	O
-seen	O
-in	O
-peptide	B-mutant
-1	I-mutant
-,	O
-an	O
-annular	O
-pore	O
-consisting	O
-of	O
-five	O
-dodecamers	O
-.	O
-	O
-Trimer	O
-	O
-Peptide	B-mutant
-2	I-mutant
-forms	O
-a	O
-trimer	O
-,	O
-much	O
-like	O
-that	O
-which	O
-we	O
-observed	O
-previously	O
-for	O
-peptide	B-mutant
-1	I-mutant
-,	O
-in	O
-which	O
-three	O
-β	O
--	O
-hairpins	O
-assemble	O
-to	O
-form	O
-an	O
-equilateral	O
-triangle	O
-(	O
-Figure	O
-3A	O
-).	O
-	O
-The	O
-trimer	O
-maintains	O
-all	O
-of	O
-the	O
-same	O
-stabilizing	O
-contacts	O
-as	O
-those	O
-of	O
-peptide	B-mutant
-1	I-mutant
-.	O
-	O
-Hydrogen	O
-bonding	O
-and	O
-hydrophobic	O
-interactions	O
-between	O
-residues	O
-on	O
-the	O
-β	O
--	O
-strands	O
-comprising	O
-Aβ17	O
-–	O
-23	O
-and	O
-Aβ30	O
-–	O
-36	O
-stabilize	O
-the	O
-core	O
-of	O
-the	O
-trimer	O
-.	O
-	O
-The	O
-disulfide	O
-bonds	O
-between	O
-residues	O
-24	O
-and	O
-29	O
-are	O
-adjacent	O
-to	O
-the	O
-structural	O
-core	O
-of	O
-the	O
-trimer	O
-and	O
-do	O
-not	O
-make	O
-any	O
-substantial	O
-intermolecular	O
-contacts	O
-.	O
-	O
-Two	O
-crystallographically	O
-distinct	O
-trimers	O
-comprise	O
-the	O
-peptide	O
-portion	O
-of	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-The	O
-two	O
-trimers	O
-are	O
-almost	O
-identical	O
-in	O
-structure	O
-,	O
-differing	O
-slightly	O
-among	O
-side	O
-chain	O
-rotamers	O
-and	O
-loop	O
-conformations	O
-.	O
-	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-the	O
-trimer	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-.	O
-(	O
-A	O
-)	O
-Triangular	O
-trimer	O
-.	O
-	O
-The	O
-three	O
-water	O
-molecules	O
-in	O
-the	O
-center	O
-hole	O
-of	O
-the	O
-trimer	O
-are	O
-shown	O
-as	O
-spheres	O
-.	O
-(	O
-B	O
-)	O
-Detailed	O
-view	O
-of	O
-the	O
-intermolecular	O
-hydrogen	O
-bonds	O
-between	O
-the	O
-main	O
-chains	O
-of	O
-V18	O
-and	O
-E22	O
-and	O
-δOrn	O
-and	O
-C24	O
-,	O
-at	O
-the	O
-three	O
-corners	O
-of	O
-the	O
-triangular	O
-trimer	O
-.	O
-(	O
-C	O
-)	O
-The	O
-F19	O
-face	O
-of	O
-the	O
-trimer	O
-,	O
-with	O
-key	O
-side	O
-chains	O
-shown	O
-as	O
-spheres	O
-.	O
-(	O
-D	O
-)	O
-The	O
-F20	O
-face	O
-of	O
-the	O
-trimer	O
-,	O
-with	O
-key	O
-side	O
-chains	O
-as	O
-spheres	O
-.	O
-	O
-A	O
-network	O
-of	O
-18	O
-intermolecular	O
-hydrogen	O
-bonds	O
-helps	O
-stabilize	O
-the	O
-trimer	O
-.	O
-	O
-At	O
-the	O
-corners	O
-of	O
-the	O
-trimer	O
-,	O
-the	O
-pairs	O
-of	O
-β	O
--	O
-hairpin	O
-monomers	O
-form	O
-four	O
-hydrogen	O
-bonds	O
-:	O
-two	O
-between	O
-the	O
-main	O
-chains	O
-of	O
-V18	O
-and	O
-E22	O
-and	O
-two	O
-between	O
-δOrn	O
-and	O
-the	O
-main	O
-chain	O
-of	O
-C24	O
-(	O
-Figure	O
-3B	O
-).	O
-	O
-Three	O
-ordered	O
-water	O
-molecules	O
-fill	O
-the	O
-hole	O
-in	O
-the	O
-center	O
-of	O
-the	O
-trimer	O
-,	O
-hydrogen	O
-bonding	O
-to	O
-each	O
-other	O
-and	O
-to	O
-the	O
-main	O
-chain	O
-of	O
-F20	O
-(	O
-Figure	O
-3A	O
-).	O
-	O
-Hydrophobic	O
-contacts	O
-between	O
-residues	O
-at	O
-the	O
-three	O
-corners	O
-of	O
-the	O
-trimer	O
-,	O
-where	O
-the	O
-β	O
--	O
-hairpins	O
-meet	O
-,	O
-further	O
-stabilize	O
-the	O
-trimer	O
-.	O
-	O
-At	O
-each	O
-corner	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-residues	O
-L17	O
-,	O
-F19	O
-,	O
-and	O
-V36	O
-of	O
-one	O
-β	O
--	O
-hairpin	O
-pack	O
-against	O
-the	O
-side	O
-chains	O
-of	O
-residues	O
-A21	O
-,	O
-I32	O
-,	O
-L34	O
-,	O
-and	O
-also	O
-D23	O
-of	O
-the	O
-adjacent	O
-β	O
--	O
-hairpin	O
-to	O
-create	O
-a	O
-hydrophobic	O
-cluster	O
-(	O
-Figure	O
-3C	O
-).	O
-The	O
-three	O
-hydrophobic	O
-clusters	O
-create	O
-a	O
-large	O
-hydrophobic	O
-surface	O
-on	O
-one	O
-face	O
-of	O
-the	O
-trimer	O
-.	O
-	O
-The	O
-other	O
-face	O
-of	O
-the	O
-trimer	O
-displays	O
-a	O
-smaller	O
-hydrophobic	O
-surface	O
-,	O
-which	O
-includes	O
-the	O
-side	O
-chains	O
-of	O
-residues	O
-V18	O
-,	O
-F20	O
-,	O
-and	O
-I31	O
-of	O
-the	O
-three	O
-β	O
--	O
-hairpins	O
-(	O
-Figure	O
-3D	O
-).	O
-	O
-In	O
-subsequent	O
-discussion	O
-,	O
-we	O
-designate	O
-the	O
-former	O
-surface	O
-the	O
-“	O
-F19	O
-face	O
-”	O
-and	O
-the	O
-latter	O
-surface	O
-the	O
-“	O
-F20	O
-face	O
-”.	O
-	O
-Dodecamer	O
-	O
-Four	O
-trimers	O
-assemble	O
-to	O
-form	O
-a	O
-dodecamer	O
-.	O
-	O
-The	O
-four	O
-trimers	O
-arrange	O
-in	O
-a	O
-tetrahedral	O
-fashion	O
-,	O
-creating	O
-a	O
-central	O
-cavity	O
-inside	O
-the	O
-dodecamer	O
-.	O
-Because	O
-each	O
-trimer	O
-is	O
-triangular	O
-,	O
-the	O
-resulting	O
-arrangement	O
-resembles	O
-an	O
-octahedron	O
-.	O
-	O
-Each	O
-of	O
-the	O
-12	O
-β	O
--	O
-hairpins	O
-constitutes	O
-an	O
-edge	O
-of	O
-the	O
-octahedron	O
-,	O
-and	O
-the	O
-triangular	O
-trimers	O
-occupy	O
-four	O
-of	O
-the	O
-eight	O
-faces	O
-of	O
-the	O
-octahedron	O
-.	O
-	O
-Figure	O
-4A	O
-illustrates	O
-the	O
-octahedral	O
-shape	O
-of	O
-the	O
-dodecamer	O
-.	O
-	O
-Figure	O
-4B	O
-illustrates	O
-the	O
-tetrahedral	O
-arrangement	O
-of	O
-the	O
-four	O
-trimers	O
-.	O
-	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-the	O
-dodecamer	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-.	O
-(	O
-A	O
-)	O
-View	O
-of	O
-the	O
-dodecamer	O
-that	O
-illustrates	O
-the	O
-octahedral	O
-shape	O
-.	O
-(	O
-B	O
-)	O
-View	O
-of	O
-the	O
-dodecamer	O
-that	O
-illustrates	O
-the	O
-tetrahedral	O
-arrangement	O
-of	O
-the	O
-four	O
-trimers	O
-that	O
-comprise	O
-the	O
-dodecamer	O
-.	O
-(	O
-C	O
-)	O
-View	O
-of	O
-two	O
-trimer	O
-subunits	O
-from	O
-inside	O
-the	O
-cavity	O
-of	O
-the	O
-dodecamer	O
-.	O
-	O
-Residues	O
-L17	O
-,	O
-L34	O
-,	O
-and	O
-V36	O
-are	O
-shown	O
-as	O
-spheres	O
-,	O
-illustrating	O
-the	O
-hydrophobic	O
-packing	O
-that	O
-occurs	O
-at	O
-the	O
-six	O
-vertices	O
-of	O
-the	O
-dodecamer	O
-.	O
-(	O
-D	O
-)	O
-Detailed	O
-view	O
-of	O
-one	O
-of	O
-the	O
-six	O
-vertices	O
-of	O
-the	O
-dodecamer	O
-.	O
-	O
-The	O
-F19	O
-faces	O
-of	O
-the	O
-trimers	O
-line	O
-the	O
-interior	O
-of	O
-the	O
-dodecamer	O
-.	O
-	O
-At	O
-the	O
-six	O
-vertices	O
-,	O
-hydrophobic	O
-packing	O
-between	O
-the	O
-side	O
-chains	O
-of	O
-L17	O
-,	O
-L34	O
-,	O
-and	O
-V36	O
-helps	O
-stabilize	O
-the	O
-dodecamer	O
-(	O
-Figures	O
-4C	O
-and	O
-D	O
-).	O
-	O
-Salt	O
-bridges	O
-between	O
-the	O
-side	O
-chains	O
-of	O
-D23	O
-and	O
-δOrn	O
-at	O
-the	O
-vertices	O
-further	O
-stabilize	O
-the	O
-dodecamer	O
-.	O
-	O
-Each	O
-of	O
-the	O
-six	O
-vertices	O
-includes	O
-two	O
-Aβ25	O
-–	O
-28	O
-loops	O
-that	O
-extend	O
-past	O
-the	O
-core	O
-of	O
-the	O
-dodecamer	O
-without	O
-making	O
-any	O
-substantial	O
-intermolecular	O
-contacts	O
-.	O
-	O
-The	O
-exterior	O
-of	O
-the	O
-dodecamer	O
-displays	O
-four	O
-F20	O
-faces	O
-(	O
-Figure	O
-S3	O
-).	O
-	O
-In	O
-the	O
-crystal	O
-lattice	O
-,	O
-each	O
-F20	O
-face	O
-of	O
-one	O
-dodecamer	O
-packs	O
-against	O
-an	O
-F20	O
-face	O
-of	O
-another	O
-dodecamer	O
-.	O
-	O
-Although	O
-the	O
-asymmetric	O
-unit	O
-comprises	O
-half	O
-a	O
-dodecamer	O
-,	O
-the	O
-crystal	O
-lattice	O
-may	O
-be	O
-thought	O
-of	O
-as	O
-being	O
-built	O
-of	O
-dodecamers	O
-.	O
-	O
-The	O
-electron	O
-density	O
-map	O
-for	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-2	I-mutant
-has	O
-long	O
-tubes	O
-of	O
-electron	O
-density	O
-inside	O
-the	O
-central	O
-cavity	O
-of	O
-the	O
-dodecamer	O
-.	O
-	O
-The	O
-shape	O
-and	O
-length	O
-of	O
-the	O
-electron	O
-density	O
-is	O
-consistent	O
-with	O
-the	O
-structure	O
-of	O
-Jeffamine	O
-M	O
--	O
-600	O
-,	O
-which	O
-is	O
-an	O
-essential	O
-component	O
-of	O
-the	O
-crystallization	O
-conditions	O
-.	O
-	O
-Jeffamine	O
-M	O
--	O
-600	O
-is	O
-a	O
-polypropylene	O
-glycol	O
-derivative	O
-with	O
-a	O
-2	O
--	O
-methoxyethoxy	O
-unit	O
-at	O
-one	O
-end	O
-and	O
-a	O
-2	O
--	O
-aminopropyl	O
-unit	O
-at	O
-the	O
-other	O
-end	O
-.	O
-	O
-Although	O
-Jeffamine	O
-M	O
--	O
-600	O
-is	O
-a	O
-heterogeneous	O
-mixture	O
-with	O
-varying	O
-chain	O
-lengths	O
-and	O
-stereochemistry	O
-,	O
-we	O
-modeled	O
-a	O
-single	O
-stereoisomer	O
-with	O
-nine	O
-propylene	O
-glycol	O
-units	O
-(	O
-n	O
-=	O
-9	O
-)	O
-to	O
-fit	O
-the	O
-electron	O
-density	O
-.	O
-	O
-The	O
-Jeffamine	O
-M	O
--	O
-600	O
-appears	O
-to	O
-stabilize	O
-the	O
-dodecamer	O
-by	O
-occupying	O
-the	O
-central	O
-cavity	O
-and	O
-making	O
-hydrophobic	O
-contacts	O
-with	O
-residues	O
-lining	O
-the	O
-cavity	O
-(	O
-Figure	O
-S3	O
-).	O
-	O
-In	O
-a	O
-dodecamer	O
-formed	O
-by	O
-full	O
--	O
-length	O
-Aβ	O
-,	O
-the	O
-hydrophobic	O
-C	O
--	O
-terminal	O
-residues	O
-(	O
-Aβ37	O
-–	O
-40	O
-or	O
-Aβ37	O
-–	O
-42	O
-)	O
-might	O
-play	O
-a	O
-similar	O
-role	O
-in	O
-filling	O
-the	O
-dodecamer	O
-and	O
-thus	O
-create	O
-a	O
-packed	O
-hydrophobic	O
-core	O
-within	O
-the	O
-central	O
-cavity	O
-of	O
-the	O
-dodecamer	O
-.	O
-	O
-Annular	O
-Pore	O
-	O
-Five	O
-dodecamers	O
-assemble	O
-to	O
-form	O
-an	O
-annular	O
-porelike	O
-structure	O
-(	O
-Figure	O
-5A	O
-).	O
-	O
-Hydrophobic	O
-packing	O
-between	O
-the	O
-F20	O
-faces	O
-of	O
-trimers	O
-displayed	O
-on	O
-the	O
-outer	O
-surface	O
-of	O
-each	O
-dodecamer	O
-stabilizes	O
-the	O
-porelike	O
-assembly	O
-.	O
-	O
-Two	O
-morphologically	O
-distinct	O
-interactions	O
-between	O
-trimers	O
-occur	O
-at	O
-the	O
-interfaces	O
-of	O
-the	O
-five	O
-dodecamers	O
-:	O
-one	O
-in	O
-which	O
-the	O
-trimers	O
-are	O
-eclipsed	O
-(	O
-Figure	O
-5B	O
-),	O
-and	O
-one	O
-in	O
-which	O
-the	O
-trimers	O
-are	O
-staggered	O
-(	O
-Figure	O
-5C	O
-).	O
-	O
-Hydrophobic	O
-packing	O
-between	O
-the	O
-side	O
-chains	O
-of	O
-F20	O
-,	O
-I31	O
-,	O
-and	O
-E22	O
-stabilizes	O
-these	O
-interfaces	O
-(	O
-Figure	O
-5D	O
-and	O
-E	O
-).	O
-	O
-The	O
-annular	O
-pore	O
-contains	O
-three	O
-eclipsed	O
-interfaces	O
-and	O
-two	O
-staggered	O
-interfaces	O
-.	O
-	O
-The	O
-eclipsed	O
-interfaces	O
-occur	O
-between	O
-dodecamers	O
-1	O
-and	O
-2	O
-,	O
-1	O
-and	O
-5	O
-,	O
-and	O
-3	O
-and	O
-4	O
-,	O
-as	O
-shown	O
-in	O
-Figure	O
-5A	O
-.	O
-	O
-The	O
-staggered	O
-interfaces	O
-occur	O
-between	O
-dodecamers	O
-2	O
-and	O
-3	O
-and	O
-4	O
-and	O
-5	O
-.	O
-	O
-The	O
-annular	O
-pore	O
-is	O
-not	O
-completely	O
-flat	O
-,	O
-instead	O
-,	O
-adopting	O
-a	O
-slightly	O
-puckered	O
-shape	O
-,	O
-which	O
-accommodates	O
-the	O
-eclipsed	O
-and	O
-staggered	O
-interfaces	O
-.	O
-	O
-Ten	O
-Aβ25	O
-–	O
-28	O
-loops	O
-from	O
-the	O
-vertices	O
-of	O
-the	O
-five	O
-dodecamers	O
-line	O
-the	O
-hole	O
-in	O
-the	O
-center	O
-of	O
-the	O
-pore	O
-.	O
-	O
-The	O
-hydrophilic	O
-side	O
-chains	O
-of	O
-S26	O
-,	O
-N27	O
-,	O
-and	O
-K28	O
-decorate	O
-the	O
-hole	O
-.	O
-	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-the	O
-annular	O
-pore	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-.	O
-(	O
-A	O
-)	O
-Annular	O
-porelike	O
-structure	O
-illustrating	O
-the	O
-relationship	O
-of	O
-the	O
-five	O
-dodecamers	O
-that	O
-form	O
-the	O
-pore	O
-(	O
-top	O
-view	O
-).	O
-	O
-(	O
-B	O
-)	O
-Eclipsed	O
-interface	O
-between	O
-dodecamers	O
-1	O
-and	O
-2	O
-(	O
-side	O
-view	O
-).	O
-	O
-The	O
-same	O
-eclipsed	O
-interface	O
-also	O
-occurs	O
-between	O
-dodecamers	O
-1	O
-and	O
-5	O
-and	O
-3	O
-and	O
-4	O
-.	O
-(	O
-C	O
-)	O
-Staggered	O
-interface	O
-between	O
-dodecamers	O
-2	O
-and	O
-3	O
-(	O
-side	O
-view	O
-).	O
-	O
-The	O
-same	O
-staggered	O
-interface	O
-also	O
-occurs	O
-between	O
-dodecamers	O
-4	O
-and	O
-5	O
-.	O
-(	O
-D	O
-)	O
-Eclipsed	O
-interface	O
-between	O
-dodecamers	O
-1	O
-and	O
-5	O
-(	O
-top	O
-view	O
-).	O
-	O
-The	O
-annular	O
-pore	O
-is	O
-comparable	O
-in	O
-size	O
-to	O
-other	O
-large	O
-protein	O
-assemblies	O
-.	O
-	O
-The	O
-diameter	O
-of	O
-the	O
-hole	O
-in	O
-the	O
-center	O
-of	O
-the	O
-pore	O
-is	O
-∼	O
-2	O
-nm	O
-.	O
-	O
-The	O
-thickness	O
-of	O
-the	O
-pore	O
-is	O
-∼	O
-5	O
-nm	O
-,	O
-which	O
-is	O
-comparable	O
-to	O
-that	O
-of	O
-a	O
-lipid	O
-bilayer	O
-membrane	O
-.	O
-	O
-It	O
-is	O
-important	O
-to	O
-note	O
-that	O
-the	O
-annular	O
-pore	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-is	O
-not	O
-a	O
-discrete	O
-unit	O
-in	O
-the	O
-crystal	O
-lattice	O
-.	O
-	O
-Rather	O
-,	O
-the	O
-crystal	O
-lattice	O
-is	O
-composed	O
-of	O
-conjoined	O
-annular	O
-pores	O
-in	O
-which	O
-all	O
-four	O
-F20	O
-faces	O
-on	O
-the	O
-surface	O
-of	O
-each	O
-dodecamer	O
-contact	O
-F20	O
-faces	O
-on	O
-other	O
-dodecamers	O
-(	O
-Figure	O
-S4	O
-).	O
-	O
-The	O
-crystal	O
-lattice	O
-shows	O
-how	O
-the	O
-dodecamers	O
-can	O
-further	O
-assemble	O
-to	O
-form	O
-larger	O
-structures	O
-.	O
-	O
-Each	O
-dodecamer	O
-may	O
-be	O
-thought	O
-of	O
-as	O
-a	O
-tetravalent	O
-building	O
-block	O
-with	O
-the	O
-potential	O
-to	O
-assemble	O
-on	O
-all	O
-four	O
-faces	O
-to	O
-form	O
-higher	O
--	O
-order	O
-supramolecular	O
-assemblies	O
-.	O
-	O
-The	O
-X	O
--	O
-ray	O
-crystallographic	O
-study	O
-of	O
-peptide	B-mutant
-2	I-mutant
-described	O
-here	O
-provides	O
-high	O
--	O
-resolution	O
-structures	O
-of	O
-oligomers	O
-formed	O
-by	O
-an	O
-Aβ17	O
-–	O
-36	O
-β	O
--	O
-hairpin	O
-.	O
-	O
-The	O
-crystallographic	O
-assembly	O
-of	O
-peptide	B-mutant
-2	I-mutant
-into	O
-a	O
-trimer	O
-,	O
-dodecamer	O
-,	O
-and	O
-annular	O
-pore	O
-provides	O
-a	O
-model	O
-for	O
-the	O
-assembly	O
-of	O
-the	O
-full	O
--	O
-length	O
-Aβ	O
-peptide	O
-to	O
-form	O
-oligomers	O
-.	O
-	O
-In	O
-this	O
-model	O
-Aβ	O
-folds	O
-to	O
-form	O
-a	O
-β	O
--	O
-hairpin	O
-comprising	O
-the	O
-hydrophobic	O
-central	O
-and	O
-C	O
--	O
-terminal	O
-regions	O
-.	O
-	O
-Three	O
-β	O
--	O
-hairpins	O
-assemble	O
-to	O
-form	O
-a	O
-trimer	O
-,	O
-and	O
-four	O
-trimers	O
-assemble	O
-to	O
-form	O
-a	O
-dodecamer	O
-.	O
-	O
-The	O
-dodecamers	O
-further	O
-assemble	O
-to	O
-form	O
-an	O
-annular	O
-pore	O
-(	O
-Figure	O
-6	O
-).	O
-	O
-Model	O
-for	O
-the	O
-hierarchical	O
-assembly	O
-of	O
-an	O
-Aβ	O
-β	O
--	O
-hairpin	O
-into	O
-a	O
-trimer	O
-,	O
-dodecamer	O
-,	O
-and	O
-annular	O
-pore	O
-based	O
-on	O
-the	O
-crystallographic	O
-assembly	O
-of	O
-peptide	B-mutant
-2	I-mutant
-.	O
-	O
-Monomeric	O
-Aβ	O
-folds	O
-to	O
-form	O
-a	O
-β	O
--	O
-hairpin	O
-in	O
-which	O
-the	O
-hydrophobic	O
-central	O
-and	O
-C	O
--	O
-terminal	O
-regions	O
-form	O
-an	O
-antiparallel	O
-β	O
--	O
-sheet	O
-.	O
-	O
-Three	O
-β	O
--	O
-hairpin	O
-monomers	O
-assemble	O
-to	O
-form	O
-a	O
-triangular	O
-trimer	O
-.	O
-	O
-Four	O
-triangular	O
-trimers	O
-assemble	O
-to	O
-form	O
-a	O
-dodecamer	O
-.	O
-	O
-Five	O
-dodecamers	O
-assemble	O
-to	O
-form	O
-an	O
-annular	O
-pore	O
-.	O
-	O
-The	O
-molecular	O
-weights	O
-shown	O
-correspond	O
-to	O
-an	O
-Aβ42	O
-monomer	O
-(∼	O
-4	O
-.	O
-5	O
-kDa	O
-),	O
-an	O
-Aβ42	O
-trimer	O
-(∼	O
-13	O
-.	O
-5	O
-kDa	O
-),	O
-an	O
-Aβ42	O
-dodecamer	O
-(∼	O
-54	O
-kDa	O
-),	O
-and	O
-an	O
-Aβ42	O
-annular	O
-pore	O
-composed	O
-of	O
-five	O
-dodecamers	O
-(∼	O
-270	O
-kDa	O
-).	O
-	O
-The	O
-model	O
-put	O
-forth	O
-in	O
-Figure	O
-6	O
-is	O
-consistent	O
-with	O
-the	O
-current	O
-understanding	O
-of	O
-endogenous	O
-Aβ	O
-oligomerization	O
-and	O
-explains	O
-at	O
-atomic	O
-resolution	O
-many	O
-key	O
-observations	O
-about	O
-Aβ	O
-oligomers	O
-.	O
-	O
-Two	O
-general	O
-types	O
-of	O
-endogenous	O
-Aβ	O
-oligomers	O
-have	O
-been	O
-observed	O
-:	O
-Aβ	O
-oligomers	O
-that	O
-occur	O
-on	O
-a	O
-pathway	O
-to	O
-fibrils	O
-,	O
-or	O
-“	O
-fibrillar	O
-oligomers	O
-”,	O
-and	O
-Aβ	O
-oligomers	O
-that	O
-evade	O
-a	O
-fibrillar	O
-fate	O
-,	O
-or	O
-“	O
-nonfibrillar	O
-oligomers	O
-”.−	O
-Fibrillar	O
-oligomers	O
-accumulate	O
-in	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-later	O
-than	O
-nonfibrillar	O
-oligomers	O
-and	O
-coincide	O
-with	O
-the	O
-deposition	O
-of	O
-plaques	O
-.	O
-	O
-Nonfibrillar	O
-oligomers	O
-accumulate	O
-early	O
-in	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-before	O
-plaque	O
-deposition	O
-.	O
-	O
-Fibrillar	O
-and	O
-nonfibrillar	O
-oligomers	O
-have	O
-structurally	O
-distinct	O
-characteristics	O
-,	O
-which	O
-are	O
-reflected	O
-in	O
-their	O
-reactivity	O
-with	O
-the	O
-fibril	O
--	O
-specific	O
-OC	O
-antibody	O
-and	O
-the	O
-oligomer	O
--	O
-specific	O
-A11	O
-antibody	O
-.	O
-	O
-Fibrillar	O
-oligomers	O
-are	O
-recognized	O
-by	O
-the	O
-OC	O
-antibody	O
-but	O
-not	O
-the	O
-A11	O
-antibody	O
-,	O
-whereas	O
-nonfibrillar	O
-oligomers	O
-are	O
-recognized	O
-by	O
-the	O
-A11	O
-antibody	O
-but	O
-not	O
-the	O
-OC	O
-antibody	O
-.	O
-	O
-These	O
-criteria	O
-have	O
-been	O
-used	O
-to	O
-classify	O
-the	O
-Aβ	O
-oligomers	O
-that	O
-accumulate	O
-in	O
-vivo	O
-.	O
-	O
-Aβ	O
-dimers	O
-have	O
-been	O
-classified	O
-as	O
-fibrillar	O
-oligomers	O
-,	O
-whereas	O
-Aβ	O
-trimers	O
-,	O
-Aβ	O
-*	O
-56	O
-,	O
-and	O
-APFs	O
-have	O
-been	O
-classified	O
-as	O
-nonfibrillar	O
-oligomers	O
-.	O
-	O
-Larson	O
-and	O
-Lesné	O
-proposed	O
-a	O
-model	O
-for	O
-the	O
-endogenous	O
-production	O
-of	O
-nonfibrillar	O
-oligomers	O
-that	O
-explains	O
-these	O
-observations	O
-.	O
-	O
-In	O
-this	O
-model	O
-,	O
-folded	O
-Aβ	O
-monomer	O
-assembles	O
-into	O
-a	O
-trimer	O
-,	O
-the	O
-trimer	O
-further	O
-assembles	O
-into	O
-hexamers	O
-and	O
-dodecamers	O
-,	O
-and	O
-the	O
-dodecamers	O
-further	O
-assemble	O
-to	O
-form	O
-annular	O
-protofibrils	O
-.	O
-	O
-The	O
-hierarchical	O
-assembly	O
-of	O
-peptide	B-mutant
-2	I-mutant
-is	O
-consistent	O
-with	O
-this	O
-model	O
-;	O
-and	O
-the	O
-trimer	O
-,	O
-dodecamer	O
-,	O
-and	O
-annular	O
-pore	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-may	O
-share	O
-similarities	O
-to	O
-the	O
-trimers	O
-,	O
-Aβ	O
-*	O
-56	O
-,	O
-and	O
-APFs	O
-observed	O
-in	O
-vivo	O
-.	O
-	O
-At	O
-this	O
-point	O
-,	O
-we	O
-can	O
-only	O
-speculate	O
-whether	O
-the	O
-trimer	O
-and	O
-dodecamer	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-share	O
-structural	O
-similarities	O
-to	O
-Aβ	O
-trimers	O
-and	O
-Aβ	O
-*	O
-56	O
-,	O
-as	O
-little	O
-is	O
-known	O
-about	O
-the	O
-structure	O
-of	O
-Aβ	O
-trimers	O
-and	O
-Aβ	O
-*	O
-56	O
-.	O
-	O
-The	O
-crystallographically	O
-observed	O
-annular	O
-pore	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-is	O
-morphologically	O
-similar	O
-to	O
-the	O
-APFs	O
-formed	O
-by	O
-full	O
--	O
-length	O
-Aβ	O
-.	O
-	O
-The	O
-annular	O
-pore	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-is	O
-comparable	O
-in	O
-size	O
-to	O
-the	O
-APFs	O
-prepared	O
-in	O
-vitro	O
-or	O
-isolated	O
-from	O
-Alzheimer	O
-’	O
-s	O
-brains	O
-(	O
-Figure	O
-7	O
-and	O
-Table	O
-1	O
-).	O
-	O
-The	O
-varying	O
-sizes	O
-of	O
-APFs	O
-formed	O
-by	O
-full	O
--	O
-length	O
-Aβ	O
-might	O
-result	O
-from	O
-differences	O
-in	O
-the	O
-number	O
-of	O
-oligomer	O
-subunits	O
-comprising	O
-each	O
-APF	O
-.	O
-	O
-Although	O
-the	O
-annular	O
-pore	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-contains	O
-five	O
-dodecamer	O
-subunits	O
-,	O
-pores	O
-containing	O
-fewer	O
-or	O
-more	O
-subunits	O
-can	O
-easily	O
-be	O
-envisioned	O
-.	O
-	O
-The	O
-dodecamers	O
-that	O
-comprise	O
-the	O
-annular	O
-pore	O
-exhibit	O
-two	O
-modes	O
-of	O
-assembly	O
-—	O
-eclipsed	O
-interactions	O
-and	O
-staggered	O
-interactions	O
-between	O
-the	O
-F20	O
-faces	O
-of	O
-trimers	O
-within	O
-dodecamers	O
-.	O
-	O
-These	O
-two	O
-modes	O
-of	O
-assembly	O
-might	O
-reflect	O
-a	O
-dynamic	O
-interaction	O
-between	O
-dodecamers	O
-,	O
-which	O
-could	O
-permit	O
-assemblies	O
-of	O
-more	O
-dodecamers	O
-into	O
-larger	O
-annular	O
-pores	O
-.	O
-	O
-Surface	O
-views	O
-of	O
-the	O
-annular	O
-pore	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-.	O
-(	O
-A	O
-)	O
-Top	O
-view	O
-.	O
-	O
-Annular	O
-Pores	O
-Formed	O
-by	O
-Aβ	O
-and	O
-Peptide	B-mutant
-2	I-mutant
-	O
-annular	O
-pore	O
-source	O
-outer	O
-diameter	O
-inner	O
-diameter	O
-observation	O
-method	O
-peptide	O
-2	O
-∼	O
-11	O
-–	O
-12	O
-nm	O
-∼	O
-2	O
-nm	O
-X	O
--	O
-ray	O
-crystallography	O
-synthetic	O
-Aβ	O
-7	O
-–	O
-10	O
-nm	O
-1	O
-.	O
-5	O
-–	O
-2	O
-nm	O
-TEM	O
-synthetic	O
-Aβ	O
-16	O
-nm	O
-not	O
-reported	O
-AFM	O
-synthetic	O
-Aβ	O
-8	O
-–	O
-25	O
-nm	O
-not	O
-reported	O
-TEM	O
-Alzheimer	O
-’	O
-s	O
-brain	O
-11	O
-–	O
-14	O
-nm	O
-2	O
-.	O
-5	O
-–	O
-4	O
-nm	O
-TEM	O
-	O
-Dot	O
-blot	O
-analysis	O
-shows	O
-that	O
-peptide	B-mutant
-2	I-mutant
-is	O
-reactive	O
-toward	O
-the	O
-A11	O
-antibody	O
-(	O
-Figure	O
-S5	O
-).	O
-	O
-This	O
-reactivity	O
-suggests	O
-that	O
-peptide	B-mutant
-2	I-mutant
-forms	O
-oligomers	O
-in	O
-solution	O
-that	O
-share	O
-structural	O
-similarities	O
-to	O
-the	O
-nonfibrillar	O
-oligomers	O
-formed	O
-by	O
-full	O
--	O
-length	O
-Aβ	O
-.	O
-	O
-Further	O
-studies	O
-are	O
-needed	O
-to	O
-elucidate	O
-the	O
-species	O
-that	O
-peptide	B-mutant
-2	I-mutant
-forms	O
-in	O
-solution	O
-and	O
-to	O
-study	O
-their	O
-biological	O
-properties	O
-.	O
-	O
-Preliminary	O
-attempts	O
-to	O
-study	O
-these	O
-species	O
-by	O
-SEC	O
-and	O
-SDS	O
--	O
-PAGE	O
-have	O
-not	O
-provided	O
-a	O
-clear	O
-measure	O
-of	O
-the	O
-structures	O
-formed	O
-in	O
-solution	O
-.	O
-	O
-The	O
-difficulty	O
-in	O
-studying	O
-the	O
-oligomers	O
-formed	O
-in	O
-solution	O
-may	O
-reflect	O
-the	O
-propensity	O
-of	O
-the	O
-dodecamer	O
-to	O
-assemble	O
-on	O
-all	O
-four	O
-F20	O
-faces	O
-.	O
-	O
-The	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-and	O
-A11	O
-reactivity	O
-of	O
-peptide	B-mutant
-2	I-mutant
-support	O
-the	O
-model	O
-proposed	O
-by	O
-Larsen	O
-and	O
-Lesné	O
-and	O
-suggest	O
-that	O
-β	O
--	O
-hairpins	O
-constitute	O
-a	O
-fundamental	O
-building	O
-block	O
-for	O
-nonfibrillar	O
-oligomers	O
-.	O
-	O
-What	O
-makes	O
-β	O
--	O
-hairpins	O
-special	O
-is	O
-that	O
-three	O
-β	O
--	O
-hairpins	O
-can	O
-nestle	O
-together	O
-to	O
-form	O
-trimers	O
-,	O
-stabilized	O
-by	O
-a	O
-network	O
-of	O
-hydrogen	O
-bonds	O
-and	O
-hydrophobic	O
-interactions	O
-.	O
-	O
-This	O
-mode	O
-of	O
-assembly	O
-is	O
-not	O
-unique	O
-to	O
-Aβ	O
-.	O
-	O
-The	O
-foldon	O
-domain	O
-of	O
-bacteriophage	O
-T4	O
-fibritin	O
-is	O
-composed	O
-of	O
-three	O
-β	O
--	O
-hairpins	O
-that	O
-assemble	O
-into	O
-a	O
-triangular	O
-trimer	O
-similar	O
-to	O
-the	O
-triangular	O
-trimer	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-.	O
-	O
-Additionally	O
-,	O
-our	O
-research	O
-group	O
-has	O
-observed	O
-a	O
-similar	O
-assembly	O
-of	O
-a	O
-β	O
--	O
-hairpin	O
-peptide	O
-derived	O
-from	O
-β2	O
--	O
-microglobulin	O
-.	O
-	O
-Although	O
-we	O
-began	O
-these	O
-studies	O
-with	O
-a	O
-relatively	O
-simple	O
-hypothesis	O
-—	O
-that	O
-the	O
-trimers	O
-and	O
-dodecamers	O
-formed	O
-by	O
-peptide	B-mutant
-1	I-mutant
-could	O
-accommodate	O
-the	O
-Aβ24	O
-–	O
-29	O
-loop	O
-—	O
-an	O
-even	O
-more	O
-exciting	O
-finding	O
-has	O
-emerged	O
-—	O
-that	O
-the	O
-dodecamers	O
-can	O
-assemble	O
-to	O
-form	O
-annular	O
-pores	O
-.	O
-	O
-This	O
-finding	O
-could	O
-not	O
-have	O
-been	O
-anticipated	O
-from	O
-the	O
-X	O
--	O
-ray	O
-crystallographic	O
-structure	O
-of	O
-peptide	B-mutant
-1	I-mutant
-and	O
-reveals	O
-a	O
-new	O
-level	O
-of	O
-hierarchical	O
-assembly	O
-that	O
-recapitulates	O
-micrographic	O
-observations	O
-of	O
-annular	O
-protofibrils	O
-.	O
-	O
-The	O
-crystallographically	O
-observed	O
-dodecamer	O
-,	O
-in	O
-turn	O
-,	O
-recapitulates	O
-the	O
-observation	O
-of	O
-Aβ	O
-*	O
-56	O
-,	O
-which	O
-appears	O
-to	O
-be	O
-a	O
-dodecamer	O
-of	O
-Aβ	O
-.	O
-	O
-The	O
-crystallographically	O
-observed	O
-trimer	O
-recapitulates	O
-the	O
-Aβ	O
-trimers	O
-that	O
-are	O
-observed	O
-even	O
-before	O
-the	O
-onset	O
-of	O
-symptoms	O
-in	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-.	O
-	O
-Our	O
-approach	O
-of	O
-constraining	O
-Aβ17	O
-–	O
-36	O
-into	O
-a	O
-β	O
--	O
-hairpin	O
-conformation	O
-and	O
-blocking	O
-aggregation	O
-with	O
-an	O
-N	O
--	O
-methyl	O
-group	O
-has	O
-allowed	O
-us	O
-to	O
-crystallize	O
-a	O
-large	O
-fragment	O
-of	O
-what	O
-is	O
-generally	O
-considered	O
-to	O
-be	O
-an	O
-uncrystallizable	O
-peptide	O
-.	O
-	O
-We	O
-believe	O
-this	O
-iterative	O
-,	O
-“	O
-bottom	O
-up	O
-”	O
-approach	O
-of	O
-identifying	O
-the	O
-minimal	O
-modification	O
-required	O
-to	O
-crystallize	O
-Aβ	O
-peptides	O
-will	O
-ultimately	O
-allow	O
-larger	O
-fragments	O
-of	O
-Aβ	O
-to	O
-be	O
-crystallized	O
-,	O
-thus	O
-providing	O
-greater	O
-insights	O
-into	O
-the	O
-structures	O
-of	O
-Aβ	O
-oligomers	O
-.	O
-	O
-Predictive	O
-features	O
-of	O
-ligand	O
-‐	O
-specific	O
-signaling	O
-through	O
-the	O
-estrogen	O
-receptor	O
-	O
-Some	O
-estrogen	O
-receptor	O
-‐	O
-α	O
-(	O
-ERα	O
-)‐	O
-targeted	O
-breast	O
-cancer	O
-therapies	O
-such	O
-as	O
-tamoxifen	O
-have	O
-tissue	O
-‐	O
-selective	O
-or	O
-cell	O
-‐	O
-specific	O
-activities	O
-,	O
-while	O
-others	O
-have	O
-similar	O
-activities	O
-in	O
-different	O
-cell	O
-types	O
-.	O
-	O
-To	O
-identify	O
-biophysical	O
-determinants	O
-of	O
-cell	O
-‐	O
-specific	O
-signaling	O
-and	O
-breast	O
-cancer	O
-cell	O
-proliferation	O
-,	O
-we	O
-synthesized	O
-241	O
-ERα	O
-ligands	O
-based	O
-on	O
-19	O
-chemical	O
-scaffolds	O
-,	O
-and	O
-compared	O
-ligand	O
-response	O
-using	O
-quantitative	O
-bioassays	O
-for	O
-canonical	O
-ERα	O
-activities	O
-and	O
-X	O
-‐	O
-ray	O
-crystallography	O
-.	O
-	O
-Ligands	O
-that	O
-regulate	O
-the	O
-dynamics	O
-and	O
-stability	O
-of	O
-the	O
-coactivator	O
-‐	O
-binding	O
-site	O
-in	O
-the	O
-C	O
-‐	O
-terminal	O
-ligand	O
-‐	O
-binding	O
-domain	O
-,	O
-called	O
-activation	O
-function	O
-‐	O
-2	O
-(	O
-AF	O
-‐	O
-2	O
-),	O
-showed	O
-similar	O
-activity	O
-profiles	O
-in	O
-different	O
-cell	O
-types	O
-.	O
-	O
-Such	O
-ligands	O
-induced	O
-breast	O
-cancer	O
-cell	O
-proliferation	O
-in	O
-a	O
-manner	O
-that	O
-was	O
-predicted	O
-by	O
-the	O
-canonical	O
-recruitment	O
-of	O
-the	O
-coactivators	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-and	O
-induction	O
-of	O
-the	O
-GREB1	O
-proliferative	O
-gene	O
-.	O
-	O
-For	O
-some	O
-ligand	O
-series	O
-,	O
-a	O
-single	O
-inter	O
-‐	O
-atomic	O
-distance	O
-in	O
-the	O
-ligand	O
-‐	O
-binding	O
-domain	O
-predicted	O
-their	O
-proliferative	O
-effects	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-N	O
-‐	O
-terminal	O
-coactivator	O
-‐	O
-binding	O
-site	O
-,	O
-activation	O
-function	O
-‐	O
-1	O
-(	O
-AF	O
-‐	O
-1	O
-),	O
-determined	O
-cell	O
-‐	O
-specific	O
-signaling	O
-induced	O
-by	O
-ligands	O
-that	O
-used	O
-alternate	O
-mechanisms	O
-to	O
-control	O
-cell	O
-proliferation	O
-.	O
-	O
-Thus	O
-,	O
-incorporating	O
-systems	O
-structural	O
-analyses	O
-with	O
-quantitative	O
-chemical	O
-biology	O
-reveals	O
-how	O
-ligands	O
-can	O
-achieve	O
-distinct	O
-allosteric	O
-signaling	O
-outcomes	O
-through	O
-ERα	O
-.	O
-	O
-Many	O
-drugs	O
-are	O
-small	O
-‐	O
-molecule	O
-ligands	O
-of	O
-allosteric	O
-signaling	O
-proteins	O
-,	O
-including	O
-G	O
-protein	O
-‐	O
-coupled	O
-receptors	O
-(	O
-GPCRs	O
-)	O
-and	O
-nuclear	O
-receptors	O
-such	O
-as	O
-ERα	O
-.	O
-	O
-Small	O
-‐	O
-molecule	O
-ligands	O
-control	O
-receptor	O
-activity	O
-by	O
-modulating	O
-recruitment	O
-of	O
-effector	O
-enzymes	O
-to	O
-distal	O
-regions	O
-of	O
-the	O
-receptor	O
-,	O
-relative	O
-to	O
-the	O
-ligand	O
-‐	O
-binding	O
-site	O
-.	O
-	O
-For	O
-example	O
-,	O
-selective	O
-estrogen	O
-receptor	O
-modulators	O
-(	O
-SERMs	O
-)	O
-such	O
-as	O
-tamoxifen	O
-(	O
-Nolvadex	O
-®;	O
-AstraZeneca	O
-)	O
-or	O
-raloxifene	O
-(	O
-Evista	O
-®;	O
-Eli	O
-Lilly	O
-)	O
-(	O
-Fig	O
-1A	O
-)	O
-block	O
-the	O
-ERα	O
-‐	O
-mediated	O
-proliferative	O
-effects	O
-of	O
-the	O
-native	O
-estrogen	O
-,	O
-17β	O
-‐	O
-estradiol	O
-(	O
-E2	O
-),	O
-on	O
-breast	O
-cancer	O
-cells	O
-,	O
-but	O
-promote	O
-beneficial	O
-estrogenic	O
-effects	O
-on	O
-bone	O
-mineral	O
-density	O
-and	O
-adverse	O
-estrogenic	O
-effects	O
-such	O
-as	O
-uterine	O
-proliferation	O
-,	O
-fatty	O
-liver	O
-,	O
-or	O
-stroke	O
-(	O
-Frolik	O
-et	O
-al	O
-,	O
-1996	O
-;	O
-Fisher	O
-et	O
-al	O
-,	O
-1998	O
-;	O
-McDonnell	O
-et	O
-al	O
-,	O
-2002	O
-;	O
-Jordan	O
-,	O
-2003	O
-).	O
-	O
-Allosteric	O
-control	O
-of	O
-ERα	O
-activity	O
-	O
-Chemical	O
-structures	O
-of	O
-some	O
-common	O
-ERα	O
-ligands	O
-.	O
-	O
-E2	O
-‐	O
-rings	O
-are	O
-numbered	O
-A	O
-‐	O
-D	O
-.	O
-The	O
-E	O
-‐	O
-ring	O
-is	O
-the	O
-common	O
-site	O
-of	O
-attachment	O
-for	O
-BSC	O
-found	O
-in	O
-many	O
-SERMS	O
-.	O
-	O
-ERα	O
-domain	O
-organization	O
-lettered	O
-,	O
-A	O
-‐	O
-F	O
-.	O
-DBD	O
-,	O
-DNA	O
-‐	O
-binding	O
-domain	O
-;	O
-LBD	O
-,	O
-ligand	O
-‐	O
-binding	O
-domain	O
-;	O
-AF	O
-,	O
-activation	O
-function	O
-	O
-Schematic	O
-illustration	O
-of	O
-the	O
-canonical	O
-ERα	O
-signaling	O
-pathway	O
-.	O
-	O
-Linear	O
-causality	O
-model	O
-for	O
-ERα	O
-‐	O
-mediated	O
-cell	O
-proliferation	O
-.	O
-	O
-Branched	O
-causality	O
-model	O
-for	O
-ERα	O
-‐	O
-mediated	O
-cell	O
-proliferation	O
-.	O
-	O
-ERα	O
-contains	O
-structurally	O
-conserved	O
-globular	O
-domains	O
-of	O
-the	O
-nuclear	O
-receptor	O
-superfamily	O
-,	O
-including	O
-a	O
-DNA	O
-‐	O
-binding	O
-domain	O
-(	O
-DBD	O
-)	O
-that	O
-is	O
-connected	O
-by	O
-a	O
-flexible	O
-hinge	O
-region	O
-to	O
-the	O
-ligand	O
-‐	O
-binding	O
-domain	O
-(	O
-LBD	O
-),	O
-as	O
-well	O
-as	O
-unstructured	O
-AB	O
-and	O
-F	O
-domains	O
-at	O
-its	O
-amino	O
-and	O
-carboxyl	O
-termini	O
-,	O
-respectively	O
-(	O
-Fig	O
-1B	O
-).	O
-	O
-The	O
-LBD	O
-contains	O
-a	O
-ligand	O
-‐	O
-dependent	O
-coactivator	O
-‐	O
-binding	O
-site	O
-called	O
-activation	O
-function	O
-‐	O
-2	O
-(	O
-AF	O
-‐	O
-2	O
-).	O
-	O
-However	O
-,	O
-the	O
-agonist	O
-activity	O
-of	O
-SERMs	O
-derives	O
-from	O
-activation	O
-function	O
-‐	O
-1	O
-(	O
-AF	O
-‐	O
-1	O
-)—	O
-a	O
-coactivator	O
-recruitment	O
-site	O
-located	O
-in	O
-the	O
-AB	O
-domain	O
-(	O
-Berry	O
-et	O
-al	O
-,	O
-1990	O
-;	O
-Shang	O
-&	O
-Brown	O
-,	O
-2002	O
-;	O
-Abot	O
-et	O
-al	O
-,	O
-2013	O
-).	O
-	O
-AF	O
-‐	O
-1	O
-and	O
-AF	O
-‐	O
-2	O
-bind	O
-distinct	O
-but	O
-overlapping	O
-sets	O
-of	O
-coregulators	O
-(	O
-Webb	O
-et	O
-al	O
-,	O
-1998	O
-;	O
-Endoh	O
-et	O
-al	O
-,	O
-1999	O
-;	O
-Delage	O
-‐	O
-Mourroux	O
-et	O
-al	O
-,	O
-2000	O
-;	O
-Yi	O
-et	O
-al	O
-,	O
-2015	O
-).	O
-	O
-AF	O
-‐	O
-2	O
-binds	O
-the	O
-signature	O
-LxxLL	O
-motif	O
-peptides	O
-of	O
-coactivators	O
-such	O
-as	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-(	O
-also	O
-known	O
-as	O
-SRC	O
-‐	O
-1	O
-/	O
-2	O
-/	O
-3	O
-).	O
-	O
-AF	O
-‐	O
-1	O
-binds	O
-a	O
-separate	O
-surface	O
-on	O
-these	O
-coactivators	O
-(	O
-Webb	O
-et	O
-al	O
-,	O
-1998	O
-;	O
-Yi	O
-et	O
-al	O
-,	O
-2015	O
-).	O
-	O
-Yet	O
-,	O
-it	O
-is	O
-unknown	O
-how	O
-different	O
-ERα	O
-ligands	O
-control	O
-AF	O
-‐	O
-1	O
-through	O
-the	O
-LBD	O
-,	O
-and	O
-whether	O
-this	O
-inter	O
-‐	O
-domain	O
-communication	O
-is	O
-required	O
-for	O
-cell	O
-‐	O
-specific	O
-signaling	O
-or	O
-anti	O
-‐	O
-proliferative	O
-responses	O
-.	O
-	O
-In	O
-the	O
-canonical	O
-model	O
-of	O
-the	O
-ERα	O
-signaling	O
-pathway	O
-(	O
-Fig	O
-1C	O
-),	O
-E2	O
-‐	O
-bound	O
-ERα	O
-forms	O
-a	O
-homodimer	O
-that	O
-binds	O
-DNA	O
-at	O
-estrogen	O
-‐	O
-response	O
-elements	O
-(	O
-EREs	O
-),	O
-recruits	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-(	O
-Metivier	O
-et	O
-al	O
-,	O
-2003	O
-;	O
-Johnson	O
-&	O
-O	O
-'	O
-Malley	O
-,	O
-2012	O
-),	O
-and	O
-activates	O
-the	O
-GREB1	O
-gene	O
-,	O
-which	O
-is	O
-required	O
-for	O
-proliferation	O
-of	O
-ERα	O
-‐	O
-positive	O
-breast	O
-cancer	O
-cells	O
-(	O
-Ghosh	O
-et	O
-al	O
-,	O
-2000	O
-;	O
-Rae	O
-et	O
-al	O
-,	O
-2005	O
-;	O
-Deschenes	O
-et	O
-al	O
-,	O
-2007	O
-;	O
-Liu	O
-et	O
-al	O
-,	O
-2012	O
-;	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-).	O
-	O
-However	O
-,	O
-ERα	O
-‐	O
-mediated	O
-proliferative	O
-responses	O
-vary	O
-in	O
-a	O
-ligand	O
-‐	O
-dependent	O
-manner	O
-(	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-);	O
-thus	O
-,	O
-it	O
-is	O
-not	O
-known	O
-whether	O
-this	O
-canonical	O
-model	O
-is	O
-widely	O
-applicable	O
-across	O
-diverse	O
-ERα	O
-ligands	O
-.	O
-	O
-Our	O
-long	O
-‐	O
-term	O
-goal	O
-is	O
-to	O
-be	O
-able	O
-to	O
-predict	O
-proliferative	O
-or	O
-anti	O
-‐	O
-proliferative	O
-activity	O
-of	O
-a	O
-ligand	O
-in	O
-different	O
-tissues	O
-from	O
-its	O
-crystal	O
-structure	O
-by	O
-identifying	O
-different	O
-structural	O
-perturbations	O
-that	O
-lead	O
-to	O
-specific	O
-signaling	O
-outcomes	O
-.	O
-	O
-The	O
-simplest	O
-response	O
-model	O
-for	O
-ligand	O
-‐	O
-specific	O
-proliferative	O
-effects	O
-is	O
-a	O
-linear	O
-causality	O
-model	O
-,	O
-where	O
-the	O
-degree	O
-of	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-determines	O
-GREB1	O
-expression	O
-,	O
-which	O
-in	O
-turn	O
-drives	O
-ligand	O
-‐	O
-specific	O
-cell	O
-proliferation	O
-(	O
-Fig	O
-1D	O
-).	O
-	O
-In	O
-this	O
-signaling	O
-model	O
-,	O
-multiple	O
-coregulator	O
-binding	O
-events	O
-and	O
-target	O
-genes	O
-(	O
-Won	O
-Jeong	O
-et	O
-al	O
-,	O
-2012	O
-;	O
-Nwachukwu	O
-et	O
-al	O
-,	O
-2014	O
-),	O
-LBD	O
-conformation	O
-,	O
-nucleocytoplasmic	O
-shuttling	O
-,	O
-the	O
-occupancy	O
-and	O
-dynamics	O
-of	O
-DNA	O
-binding	O
-,	O
-and	O
-other	O
-biophysical	O
-features	O
-could	O
-contribute	O
-independently	O
-to	O
-cell	O
-proliferation	O
-(	O
-Lickwar	O
-et	O
-al	O
-,	O
-2012	O
-).	O
-	O
-To	O
-test	O
-these	O
-signaling	O
-models	O
-,	O
-we	O
-profiled	O
-a	O
-diverse	O
-library	O
-of	O
-ERα	O
-ligands	O
-using	O
-systems	O
-biology	O
-approaches	O
-to	O
-X	O
-‐	O
-ray	O
-crystallography	O
-and	O
-chemical	O
-biology	O
-(	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-),	O
-including	O
-a	O
-series	O
-of	O
-quantitative	O
-bioassays	O
-for	O
-ERα	O
-function	O
-that	O
-were	O
-statistically	O
-robust	O
-and	O
-reproducible	O
-,	O
-based	O
-on	O
-the	O
-Z	O
-’‐	O
-statistic	O
-(	O
-Fig	O
-EV1A	O
-and	O
-B	O
-;	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-We	O
-also	O
-determined	O
-the	O
-structures	O
-of	O
-76	O
-distinct	O
-ERα	O
-LBD	O
-complexes	O
-bound	O
-to	O
-different	O
-ligand	O
-types	O
-,	O
-which	O
-allowed	O
-us	O
-to	O
-understand	O
-how	O
-diverse	O
-ligand	O
-scaffolds	O
-distort	O
-the	O
-active	O
-conformation	O
-of	O
-the	O
-ERα	O
-LBD	O
-.	O
-	O
-Our	O
-findings	O
-here	O
-indicate	O
-that	O
-specific	O
-structural	O
-perturbations	O
-can	O
-be	O
-tied	O
-to	O
-ligand	O
-‐	O
-selective	O
-domain	O
-usage	O
-and	O
-signaling	O
-patterns	O
-,	O
-thus	O
-providing	O
-a	O
-framework	O
-for	O
-structure	O
-‐	O
-based	O
-design	O
-of	O
-improved	O
-breast	O
-cancer	O
-therapeutics	O
-,	O
-and	O
-understanding	O
-the	O
-different	O
-phenotypic	O
-effects	O
-of	O
-environmental	O
-estrogens	O
-.	O
-	O
-High	O
-‐	O
-throughput	O
-screens	O
-for	O
-ERα	O
-ligand	O
-profiling	O
-	O
-Summary	O
-of	O
-ligand	O
-screening	O
-assays	O
-used	O
-to	O
-measure	O
-ER	O
-‐	O
-mediated	O
-activities	O
-.	O
-	O
-ERE	O
-,	O
-estrogen	O
-‐	O
-response	O
-element	O
-;	O
-Luc	O
-,	O
-luciferase	O
-reporter	O
-gene	O
-;	O
-M2H	O
-,	O
-mammalian	O
-2	O
-‐	O
-hybrid	O
-;	O
-UAS	O
-,	O
-upstream	O
-‐	O
-activating	O
-sequence	O
-.	O
-	O
-Strength	O
-of	O
-AF	O
-‐	O
-1	O
-signaling	O
-does	O
-not	O
-determine	O
-cell	O
-‐	O
-specific	O
-signaling	O
-	O
-To	O
-compare	O
-ERα	O
-signaling	O
-induced	O
-by	O
-diverse	O
-ligand	O
-types	O
-,	O
-we	O
-synthesized	O
-and	O
-assayed	O
-a	O
-library	O
-of	O
-241	O
-ERα	O
-ligands	O
-containing	O
-19	O
-distinct	O
-molecular	O
-scaffolds	O
-.	O
-	O
-These	O
-include	O
-15	O
-indirect	O
-modulator	O
-series	O
-,	O
-which	O
-lack	O
-a	O
-SERM	O
-‐	O
-like	O
-side	O
-chain	O
-and	O
-modulate	O
-coactivator	O
-binding	O
-indirectly	O
-from	O
-the	O
-ligand	O
-‐	O
-binding	O
-pocket	O
-(	O
-Fig	O
-2A	O
-–	O
-E	O
-;	O
-Dataset	O
-EV1	O
-)	O
-(	O
-Zheng	O
-et	O
-al	O
-,	O
-2012	O
-)	O
-(	O
-Zhu	O
-et	O
-al	O
-,	O
-2012	O
-)	O
-(	O
-Muthyala	O
-et	O
-al	O
-,	O
-2003	O
-;	O
-Seo	O
-et	O
-al	O
-,	O
-2006	O
-)	O
-(	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-)	O
-(	O
-Wang	O
-et	O
-al	O
-,	O
-2012	O
-)	O
-(	O
-Liao	O
-et	O
-al	O
-,	O
-2014	O
-)	O
-(	O
-Min	O
-et	O
-al	O
-,	O
-2013	O
-).	O
-	O
-We	O
-also	O
-generated	O
-four	O
-direct	O
-modulator	O
-series	O
-with	O
-side	O
-chains	O
-designed	O
-to	O
-directly	O
-dislocate	O
-h12	O
-and	O
-thereby	O
-completely	O
-occlude	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-(	O
-Fig	O
-2C	O
-and	O
-E	O
-;	O
-Dataset	O
-EV1	O
-)	O
-(	O
-Kieser	O
-et	O
-al	O
-,	O
-2010	O
-).	O
-	O
-Ligand	O
-profiling	O
-using	O
-our	O
-quantitative	O
-bioassays	O
-revealed	O
-a	O
-wide	O
-range	O
-of	O
-ligand	O
-‐	O
-induced	O
-GREB1	O
-expression	O
-,	O
-reporter	O
-gene	O
-activities	O
-,	O
-ERα	O
-‐	O
-coactivator	O
-interactions	O
-,	O
-and	O
-proliferative	O
-effects	O
-on	O
-MCF	O
-‐	O
-7	O
-breast	O
-cancer	O
-cells	O
-(	O
-Figs	O
-EV1	O
-and	O
-EV2A	O
-–	O
-J	O
-).	O
-	O
-This	O
-wide	O
-variance	O
-enabled	O
-us	O
-to	O
-probe	O
-specific	O
-features	O
-of	O
-ERα	O
-signaling	O
-using	O
-ligand	O
-class	O
-analyses	O
-,	O
-and	O
-identify	O
-signaling	O
-patterns	O
-shared	O
-by	O
-specific	O
-ligand	O
-series	O
-or	O
-scaffolds	O
-.	O
-	O
-Classes	O
-of	O
-compounds	O
-in	O
-the	O
-ERα	O
-ligand	O
-library	O
-	O
-Structure	O
-of	O
-the	O
-E2	O
-‐	O
-bound	O
-ERα	O
-LBD	O
-in	O
-complex	O
-with	O
-an	O
-NCOA2	O
-peptide	O
-of	O
-(	O
-PDB	O
-1GWR	O
-).	O
-	O
-Structural	O
-details	O
-of	O
-the	O
-ERα	O
-LBD	O
-bound	O
-to	O
-the	O
-indicated	O
-ligands	O
-.	O
-	O
-Unlike	O
-E2	O
-(	O
-PDB	O
-1GWR	O
-),	O
-TAM	O
-is	O
-a	O
-direct	O
-modulator	O
-with	O
-a	O
-BSC	O
-that	O
-dislocates	O
-h12	O
-to	O
-block	O
-the	O
-NCOA2	O
-‐	O
-binding	O
-site	O
-(	O
-PDB	O
-3ERT	O
-).	O
-	O
-OBHS	O
-is	O
-an	O
-indirect	O
-modulator	O
-that	O
-dislocates	O
-the	O
-h11	O
-C	O
-‐	O
-terminus	O
-to	O
-destabilize	O
-the	O
-h11	O
-–	O
-h12	O
-interface	O
-(	O
-PDB	O
-4ZN9	O
-).	O
-	O
-The	O
-ERα	O
-ligand	O
-library	O
-contains	O
-241	O
-ligands	O
-representing	O
-15	O
-indirect	O
-modulator	O
-scaffolds	O
-,	O
-plus	O
-4	O
-direct	O
-modulator	O
-scaffolds	O
-.	O
-	O
-ERα	O
-ligands	O
-induced	O
-a	O
-range	O
-of	O
-agonist	O
-activity	O
-profiles	O
-	O
-To	O
-this	O
-end	O
-,	O
-we	O
-compared	O
-the	O
-average	O
-ligand	O
-‐	O
-induced	O
-GREB1	O
-mRNA	O
-levels	O
-in	O
-MCF	O
-‐	O
-7	O
-cells	O
-and	O
-3	O
-×	O
-ERE	O
-‐	O
-Luc	O
-reporter	O
-gene	O
-activity	O
-in	O
-Ishikawa	O
-endometrial	O
-cancer	O
-cells	O
-(	O
-E	O
-‐	O
-Luc	O
-)	O
-or	O
-in	O
-HepG2	O
-cells	O
-transfected	O
-with	O
-wild	O
-‐	O
-type	O
-ERα	O
-(	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-)	O
-(	O
-Figs	O
-3A	O
-and	O
-EV2A	O
-–	O
-C	O
-).	O
-	O
-Direct	O
-modulators	O
-showed	O
-significant	O
-differences	O
-in	O
-average	O
-activity	O
-between	O
-cell	O
-types	O
-except	O
-OBHS	O
-‐	O
-ASC	O
-analogs	O
-,	O
-which	O
-had	O
-similar	O
-low	O
-agonist	O
-activities	O
-in	O
-the	O
-three	O
-cell	O
-types	O
-.	O
-	O
-While	O
-it	O
-was	O
-known	O
-that	O
-direct	O
-modulators	O
-such	O
-as	O
-tamoxifen	O
-drive	O
-cell	O
-‐	O
-specific	O
-signaling	O
-,	O
-these	O
-experiments	O
-reveal	O
-that	O
-indirect	O
-modulators	O
-also	O
-drive	O
-cell	O
-‐	O
-specific	O
-signaling	O
-,	O
-since	O
-eight	O
-of	O
-fourteen	O
-classes	O
-showed	O
-significant	O
-differences	O
-in	O
-average	O
-activity	O
-(	O
-Figs	O
-3A	O
-and	O
-EV2A	O
-–	O
-C	O
-).	O
-	O
-Ligand	O
-‐	O
-specific	O
-signaling	O
-underlies	O
-ERα	O
-‐	O
-mediated	O
-cell	O
-proliferation	O
-	O
-(	O
-A	O
-)	O
-Ligand	O
-‐	O
-specific	O
-ERα	O
-activities	O
-in	O
-HepG2	O
-,	O
-Ishikawa	O
-and	O
-MCF	O
-‐	O
-7	O
-cells	O
-.	O
-	O
-The	O
-ligand	O
-‐	O
-induced	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-and	O
-E	O
-‐	O
-Luc	O
-activities	O
-and	O
-GREB1	O
-mRNA	O
-levels	O
-are	O
-shown	O
-by	O
-scaffold	O
-(	O
-mean	O
-+	O
-SD	O
-).	O
-	O
-(	O
-B	O
-)	O
-Ligand	O
-class	O
-analysis	O
-of	O
-the	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-and	O
-ERα	B-mutant
-‐	I-mutant
-ΔAB	I-mutant
-activities	O
-in	O
-HepG2	O
-cells	O
-.	O
-	O
-Significant	O
-sensitivity	O
-to	O
-AB	O
-domain	O
-deletion	O
-was	O
-determined	O
-by	O
-Student	O
-'	O
-s	O
-t	O
-‐	O
-test	O
-(	O
-n	O
-=	O
-number	O
-of	O
-ligands	O
-per	O
-scaffold	O
-in	O
-Fig	O
-2	O
-).	O
-	O
-Correlation	O
-and	O
-regression	O
-analyses	O
-in	O
-a	O
-large	O
-test	O
-set	O
-.	O
-	O
-In	O
-cluster	O
-1	O
-,	O
-the	O
-first	O
-three	O
-comparisons	O
-(	O
-rows	O
-)	O
-showed	O
-significant	O
-positive	O
-correlations	O
-(	O
-F	O
-‐	O
-test	O
-for	O
-nonzero	O
-slope	O
-,	O
-P	O
-≤	O
-0	O
-.	O
-05	O
-).	O
-	O
-In	O
-cluster	O
-2	O
-,	O
-only	O
-one	O
-of	O
-these	O
-comparisons	O
-revealed	O
-a	O
-significant	O
-positive	O
-correlation	O
-,	O
-while	O
-none	O
-was	O
-significant	O
-in	O
-cluster	O
-3	O
-.	O
-+,	O
-statistically	O
-significant	O
-correlations	O
-gained	O
-by	O
-deletion	O
-of	O
-the	O
-AB	O
-or	O
-F	O
-domains	O
-.	O
-	O
-−,	O
-significant	O
-correlations	O
-lost	O
-upon	O
-deletion	O
-of	O
-AB	O
-or	O
-F	O
-domains	O
-.	O
-	O
-Tamoxifen	O
-depends	O
-on	O
-AF	O
-‐	O
-1	O
-for	O
-its	O
-cell	O
-‐	O
-specific	O
-activity	O
-(	O
-Sakamoto	O
-et	O
-al	O
-,	O
-2002	O
-);	O
-therefore	O
-,	O
-we	O
-asked	O
-whether	O
-cell	O
-‐	O
-specific	O
-signaling	O
-observed	O
-here	O
-is	O
-due	O
-to	O
-a	O
-similar	O
-dependence	O
-on	O
-AF	O
-‐	O
-1	O
-for	O
-activity	O
-(	O
-Fig	O
-EV1	O
-).	O
-	O
-To	O
-test	O
-this	O
-idea	O
-,	O
-we	O
-compared	O
-the	O
-average	O
-L	O
-‐	O
-Luc	O
-activities	O
-of	O
-each	O
-scaffold	O
-in	O
-HepG2	O
-cells	O
-co	O
-‐	O
-transfected	O
-with	O
-wild	O
-‐	O
-type	O
-ERα	O
-or	O
-with	O
-ERα	O
-lacking	O
-the	O
-AB	O
-domain	O
-(	O
-Figs	O
-1B	O
-and	O
-EV1	O
-).	O
-	O
-While	O
-E2	O
-showed	O
-similar	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-and	O
-ERα	B-mutant
-‐	I-mutant
-ΔAB	I-mutant
-activities	O
-,	O
-tamoxifen	O
-showed	O
-complete	O
-loss	O
-of	O
-activity	O
-without	O
-the	O
-AB	O
-domain	O
-(	O
-Fig	O
-EV1B	O
-).	O
-	O
-Deletion	O
-of	O
-the	O
-AB	O
-domain	O
-significantly	O
-reduced	O
-the	O
-average	O
-L	O
-‐	O
-Luc	O
-activities	O
-of	O
-14	O
-scaffolds	O
-(	O
-Student	O
-'	O
-s	O
-t	O
-‐	O
-test	O
-,	O
-P	O
-≤	O
-0	O
-.	O
-05	O
-)	O
-(	O
-Fig	O
-3B	O
-).	O
-	O
-These	O
-“	O
-AF	O
-‐	O
-1	O
-‐	O
-sensitive	O
-”	O
-activities	O
-were	O
-exhibited	O
-by	O
-both	O
-direct	O
-and	O
-indirect	O
-modulators	O
-,	O
-and	O
-were	O
-not	O
-limited	O
-to	O
-scaffolds	O
-that	O
-showed	O
-cell	O
-‐	O
-specific	O
-signaling	O
-(	O
-Fig	O
-3A	O
-and	O
-B	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-strength	O
-of	O
-AF	O
-‐	O
-1	O
-signaling	O
-does	O
-not	O
-determine	O
-cell	O
-‐	O
-specific	O
-signaling	O
-.	O
-	O
-Identifying	O
-cell	O
-‐	O
-specific	O
-signaling	O
-clusters	O
-in	O
-ERα	O
-ligand	O
-classes	O
-	O
-For	O
-each	O
-ligand	O
-class	O
-or	O
-scaffold	O
-,	O
-we	O
-calculated	O
-the	O
-Pearson	O
-'	O
-s	O
-correlation	O
-coefficient	O
-,	O
-r	O
-,	O
-for	O
-pairwise	O
-comparison	O
-of	O
-activity	O
-profiles	O
-in	O
-breast	O
-(	O
-GREB1	O
-),	O
-liver	O
-(	O
-L	O
-‐	O
-Luc	O
-),	O
-and	O
-endometrial	O
-cells	O
-(	O
-E	O
-‐	O
-Luc	O
-).	O
-	O
-The	O
-value	O
-of	O
-r	O
-ranges	O
-from	O
-−	O
-1	O
-to	O
-1	O
-,	O
-and	O
-it	O
-defines	O
-the	O
-extent	O
-to	O
-which	O
-the	O
-data	O
-fit	O
-a	O
-straight	O
-line	O
-when	O
-compounds	O
-show	O
-similar	O
-agonist	O
-/	O
-antagonist	O
-activity	O
-profiles	O
-between	O
-cell	O
-types	O
-(	O
-Fig	O
-EV3A	O
-).	O
-	O
-We	O
-also	O
-calculated	O
-the	O
-coefficient	O
-of	O
-determination	O
-,	O
-r	O
-2	O
-,	O
-which	O
-describes	O
-the	O
-percentage	O
-of	O
-variance	O
-in	O
-a	O
-dependent	O
-variable	O
-such	O
-as	O
-proliferation	O
-that	O
-can	O
-be	O
-predicted	O
-by	O
-an	O
-independent	O
-variable	O
-such	O
-as	O
-GREB1	O
-expression	O
-.	O
-	O
-We	O
-present	O
-both	O
-calculations	O
-as	O
-r	O
-2	O
-to	O
-readily	O
-compare	O
-signaling	O
-specificities	O
-using	O
-a	O
-heat	O
-map	O
-on	O
-which	O
-the	O
-red	O
-–	O
-yellow	O
-palette	O
-indicates	O
-significant	O
-positive	O
-correlations	O
-(	O
-P	O
-≤	O
-0	O
-.	O
-05	O
-,	O
-F	O
-‐	O
-test	O
-for	O
-nonzero	O
-slope	O
-),	O
-while	O
-the	O
-blue	O
-palette	O
-denotes	O
-negative	O
-correlations	O
-(	O
-Fig	O
-3C	O
-–	O
-F	O
-).	O
-	O
-The	O
-side	O
-chain	O
-of	O
-OBHS	O
-‐	O
-BSC	O
-analogs	O
-induces	O
-cell	O
-‐	O
-specific	O
-signaling	O
-	O
-Correlation	O
-analysis	O
-of	O
-OBHS	O
-versus	O
-OBHS	O
-‐	O
-BSC	O
-activity	O
-across	O
-cell	O
-types	O
-.	O
-	O
-Correlation	O
-analysis	O
-of	O
-L	O
-‐	O
-Luc	O
-ERα	B-mutant
-‐	I-mutant
-ΔAB	I-mutant
-activity	O
-versus	O
-endogenous	O
-ERα	O
-activity	O
-of	O
-OBHS	O
-analogs	O
-.	O
-	O
-In	O
-panel	O
-(	O
-D	O
-),	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-activity	O
-from	O
-panel	O
-(	O
-B	O
-)	O
-is	O
-shown	O
-for	O
-comparison	O
-.	O
-	O
-Correlation	O
-analysis	O
-of	O
-L	O
-‐	O
-Luc	O
-ERα	B-mutant
-‐	I-mutant
-ΔF	I-mutant
-activity	O
-versus	O
-endogenous	O
-ERα	O
-activities	O
-of	O
-OBHS	O
-analogs	O
-.	O
-	O
-Correlation	O
-analysis	O
-of	O
-MCF	O
-‐	O
-7	O
-cell	O
-proliferation	O
-versus	O
-NCOA2	O
-/	O
-3	O
-recruitment	O
-or	O
-GREB1	O
-levels	O
-observed	O
-in	O
-response	O
-to	O
-(	O
-G	O
-)	O
-OBHS	O
-‐	O
-N	O
-and	O
-(	O
-H	O
-)	O
-OBHS	O
-‐	O
-BSC	O
-analogs	O
-.	O
-	O
-Scaffolds	O
-in	O
-cluster	O
-1	O
-exhibited	O
-strongly	O
-correlated	O
-GREB1	O
-levels	O
-,	O
-E	O
-‐	O
-Luc	O
-and	O
-L	O
-‐	O
-Luc	O
-activity	O
-profiles	O
-across	O
-the	O
-three	O
-cell	O
-types	O
-(	O
-Fig	O
-3C	O
-lanes	O
-1	O
-–	O
-4	O
-),	O
-suggesting	O
-these	O
-ligands	O
-use	O
-similar	O
-ERα	O
-signaling	O
-pathways	O
-in	O
-the	O
-breast	O
-,	O
-endometrial	O
-,	O
-and	O
-liver	O
-cell	O
-types	O
-.	O
-	O
-This	O
-cluster	O
-includes	O
-WAY	O
-‐	O
-C	O
-,	O
-OBHS	O
-,	O
-OBHS	O
-‐	O
-N	O
-,	O
-and	O
-triaryl	O
-‐	O
-ethylene	O
-analogs	O
-,	O
-all	O
-of	O
-which	O
-are	O
-indirect	O
-modulators	O
-.	O
-	O
-This	O
-cluster	O
-includes	O
-two	O
-classes	O
-of	O
-direct	O
-modulators	O
-(	O
-cyclofenil	O
-‐	O
-ASC	O
-and	O
-WAY	O
-dimer	O
-),	O
-and	O
-six	O
-classes	O
-of	O
-indirect	O
-modulators	O
-(	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-,	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-,	O
-S	O
-‐	O
-OBHS	O
-‐	O
-2	O
-and	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-,	O
-furan	O
-,	O
-and	O
-WAY	O
-‐	O
-D	O
-).	O
-	O
-For	O
-example	O
-,	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-,	O
-furan	O
-,	O
-and	O
-S	O
-‐	O
-OBHS	O
-‐	O
-2	O
-drove	O
-positively	O
-correlated	O
-GREB1	O
-levels	O
-and	O
-E	O
-‐	O
-Luc	O
-but	O
-not	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-activity	O
-(	O
-Fig	O
-3C	O
-lanes	O
-5	O
-–	O
-7	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-WAY	O
-dimer	O
-and	O
-WAY	O
-‐	O
-D	O
-analogs	O
-drove	O
-positively	O
-correlated	O
-GREB1	O
-levels	O
-and	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-but	O
-not	O
-E	O
-‐	O
-Luc	O
-activity	O
-(	O
-Fig	O
-3C	O
-lanes	O
-8	O
-and	O
-9	O
-).	O
-	O
-This	O
-cluster	O
-includes	O
-two	O
-direct	O
-modulator	O
-scaffolds	O
-(	O
-OBHS	O
-‐	O
-ASC	O
-and	O
-OBHS	O
-‐	O
-BSC	O
-),	O
-and	O
-five	O
-indirect	O
-modulator	O
-scaffolds	O
-(	O
-A	O
-‐	O
-CD	O
-,	O
-cyclofenil	O
-,	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-,	O
-imine	O
-,	O
-and	O
-imidazopyridine	O
-).	O
-	O
-These	O
-results	O
-suggest	O
-that	O
-addition	O
-of	O
-an	O
-extended	O
-side	O
-chain	O
-to	O
-an	O
-ERα	O
-ligand	O
-scaffold	O
-is	O
-sufficient	O
-to	O
-induce	O
-cell	O
-‐	O
-specific	O
-signaling	O
-,	O
-where	O
-the	O
-relative	O
-activity	O
-profiles	O
-of	O
-the	O
-individual	O
-ligands	O
-change	O
-between	O
-cell	O
-types	O
-.	O
-	O
-This	O
-is	O
-demonstrated	O
-by	O
-directly	O
-comparing	O
-the	O
-signaling	O
-specificities	O
-of	O
-matched	O
-OBHS	O
-(	O
-indirect	O
-modulator	O
-,	O
-cluster	O
-1	O
-)	O
-and	O
-OBHS	O
-‐	O
-BSC	O
-analogs	O
-(	O
-direct	O
-modulator	O
-,	O
-cluster	O
-3	O
-),	O
-which	O
-differ	O
-only	O
-in	O
-the	O
-basic	O
-side	O
-chain	O
-(	O
-Fig	O
-2E	O
-).	O
-	O
-The	O
-activities	O
-of	O
-OBHS	O
-analogs	O
-were	O
-positively	O
-correlated	O
-across	O
-the	O
-three	O
-cell	O
-types	O
-,	O
-but	O
-the	O
-side	O
-chain	O
-of	O
-OBHS	O
-‐	O
-BSC	O
-analogs	O
-was	O
-sufficient	O
-to	O
-abolish	O
-these	O
-correlations	O
-(	O
-Figs	O
-3C	O
-lanes	O
-1	O
-and	O
-19	O
-,	O
-and	O
-EV3A	O
-–	O
-C	O
-).	O
-	O
-Thus	O
-,	O
-examining	O
-the	O
-correlated	O
-patterns	O
-of	O
-ERα	O
-activity	O
-within	O
-each	O
-scaffold	O
-demonstrates	O
-that	O
-an	O
-extended	O
-side	O
-chain	O
-is	O
-not	O
-required	O
-for	O
-cell	O
-‐	O
-specific	O
-signaling	O
-.	O
-	O
-Modulation	O
-of	O
-signaling	O
-specificity	O
-by	O
-AF	O
-‐	O
-1	O
-	O
-To	O
-evaluate	O
-the	O
-role	O
-of	O
-AF	O
-‐	O
-1	O
-and	O
-the	O
-F	O
-domain	O
-in	O
-ERα	O
-signaling	O
-specificity	O
-,	O
-we	O
-compared	O
-activity	O
-of	O
-truncated	O
-ERα	O
-constructs	O
-in	O
-HepG2	O
-liver	O
-cells	O
-with	O
-endogenous	O
-ERα	O
-activity	O
-in	O
-the	O
-other	O
-cell	O
-types	O
-.	O
-	O
-The	O
-positive	O
-correlation	O
-between	O
-the	O
-L	O
-‐	O
-Luc	O
-and	O
-E	O
-‐	O
-Luc	O
-activities	O
-or	O
-GREB1	O
-levels	O
-induced	O
-by	O
-scaffolds	O
-in	O
-cluster	O
-1	O
-was	O
-generally	O
-retained	O
-without	O
-the	O
-AB	O
-domain	O
-,	O
-or	O
-the	O
-F	O
-domain	O
-(	O
-Fig	O
-3D	O
-lanes	O
-1	O
-–	O
-4	O
-).	O
-	O
-This	O
-demonstrates	O
-that	O
-the	O
-signaling	O
-specificities	O
-underlying	O
-these	O
-positive	O
-correlations	O
-are	O
-not	O
-modified	O
-by	O
-AF	O
-‐	O
-1	O
-.	O
-	O
-OBHS	O
-analogs	O
-showed	O
-an	O
-average	O
-L	O
-‐	O
-Luc	O
-ERα	B-mutant
-‐	I-mutant
-ΔAB	I-mutant
-activity	O
-of	O
-3	O
-.	O
-2	O
-%	O
-±	O
-3	O
-(	O
-mean	O
-+	O
-SEM	O
-)	O
-relative	O
-to	O
-E2	O
-.	O
-	O
-Despite	O
-this	O
-nearly	O
-complete	O
-lack	O
-of	O
-activity	O
-,	O
-the	O
-pattern	O
-of	O
-L	O
-‐	O
-Luc	O
-ERα	B-mutant
-‐	I-mutant
-ΔAB	I-mutant
-activity	O
-was	O
-still	O
-highly	O
-correlated	O
-with	O
-the	O
-E	O
-‐	O
-Luc	O
-activity	O
-and	O
-GREB1	O
-expression	O
-(	O
-Fig	O
-EV3D	O
-and	O
-E	O
-),	O
-demonstrating	O
-that	O
-very	O
-small	O
-AF	O
-‐	O
-2	O
-activities	O
-can	O
-be	O
-amplified	O
-by	O
-AF	O
-‐	O
-1	O
-to	O
-produce	O
-robust	O
-signals	O
-.	O
-	O
-Similarly	O
-,	O
-deletion	O
-of	O
-the	O
-F	O
-domain	O
-did	O
-not	O
-abolish	O
-correlations	O
-between	O
-the	O
-L	O
-‐	O
-Luc	O
-and	O
-E	O
-‐	O
-Luc	O
-or	O
-GREB1	O
-levels	O
-induced	O
-by	O
-OBHS	O
-analogs	O
-(	O
-Fig	O
-EV3F	O
-).	O
-	O
-These	O
-similar	O
-patterns	O
-of	O
-ligand	O
-activity	O
-in	O
-the	O
-wild	O
-‐	O
-type	O
-and	O
-deletion	O
-mutants	O
-suggest	O
-that	O
-AF	O
-‐	O
-1	O
-and	O
-the	O
-F	O
-domain	O
-purely	O
-amplify	O
-the	O
-AF	O
-‐	O
-2	O
-activities	O
-of	O
-ligands	O
-in	O
-cluster	O
-1	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-AF	O
-‐	O
-1	O
-was	O
-a	O
-determinant	O
-of	O
-signaling	O
-specificity	O
-for	O
-scaffolds	O
-in	O
-cluster	O
-2	O
-.	O
-	O
-Deletion	O
-of	O
-the	O
-AB	O
-or	O
-F	O
-domain	O
-altered	O
-correlations	O
-for	O
-six	O
-of	O
-the	O
-eight	O
-scaffolds	O
-in	O
-this	O
-cluster	O
-(	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-,	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-,	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-,	O
-WAY	O
-‐	O
-D	O
-,	O
-WAY	O
-dimer	O
-,	O
-and	O
-cyclofenil	O
-‐	O
-ASC	O
-)	O
-(	O
-Fig	O
-3D	O
-lanes	O
-5	O
-–	O
-12	O
-).	O
-	O
-Comparing	O
-Fig	O
-3C	O
-and	O
-D	O
-,	O
-the	O
-+	O
-and	O
-−	O
-signs	O
-indicate	O
-where	O
-the	O
-deletion	O
-mutant	O
-assays	O
-led	O
-to	O
-a	O
-gain	O
-or	O
-loss	O
-of	O
-statically	O
-significant	O
-correlation	O
-,	O
-respectively	O
-.	O
-	O
-Thus	O
-,	O
-in	O
-cluster	O
-2	O
-,	O
-AF	O
-‐	O
-1	O
-substantially	O
-modulated	O
-the	O
-specificity	O
-of	O
-ligands	O
-with	O
-cell	O
-‐	O
-specific	O
-activity	O
-(	O
-Fig	O
-3D	O
-lanes	O
-5	O
-–	O
-12	O
-).	O
-	O
-For	O
-ligands	O
-in	O
-cluster	O
-3	O
-,	O
-we	O
-could	O
-not	O
-eliminate	O
-a	O
-role	O
-for	O
-AF	O
-‐	O
-1	O
-in	O
-determining	O
-signaling	O
-specificity	O
-,	O
-since	O
-this	O
-cluster	O
-lacked	O
-positively	O
-correlated	O
-activity	O
-profiles	O
-(	O
-Fig	O
-3C	O
-),	O
-and	O
-deletion	O
-of	O
-the	O
-AB	O
-or	O
-F	O
-domain	O
-rarely	O
-induced	O
-such	O
-correlations	O
-(	O
-Fig	O
-3D	O
-),	O
-except	O
-for	O
-A	O
-‐	O
-CD	O
-and	O
-OBHS	O
-‐	O
-ASC	O
-analogs	O
-,	O
-where	O
-deletion	O
-of	O
-the	O
-AB	O
-domain	O
-or	O
-F	O
-domain	O
-led	O
-to	O
-positive	O
-correlations	O
-with	O
-E	O
-‐	O
-Luc	O
-activity	O
-and	O
-/	O
-or	O
-GREB1	O
-levels	O
-(	O
-Fig	O
-3D	O
-lanes	O
-13	O
-and	O
-18	O
-).	O
-	O
-Thus	O
-,	O
-ligands	O
-in	O
-cluster	O
-2	O
-rely	O
-on	O
-AF	O
-‐	O
-1	O
-for	O
-both	O
-activity	O
-(	O
-Fig	O
-3B	O
-)	O
-and	O
-signaling	O
-specificity	O
-(	O
-Fig	O
-3D	O
-).	O
-	O
-Ligand	O
-‐	O
-specific	O
-control	O
-of	O
-GREB1	O
-expression	O
-	O
-To	O
-determine	O
-whether	O
-ligand	O
-classes	O
-control	O
-expression	O
-of	O
-native	O
-ERα	O
-target	O
-genes	O
-through	O
-the	O
-canonical	O
-linear	O
-signaling	O
-pathway	O
-,	O
-we	O
-performed	O
-pairwise	O
-linear	O
-regression	O
-analyses	O
-using	O
-ERα	O
-–	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-interactions	O
-in	O
-M2H	O
-assay	O
-as	O
-independent	O
-predictors	O
-of	O
-GREB1	O
-expression	O
-(	O
-the	O
-dependent	O
-variable	O
-)	O
-(	O
-Figs	O
-EV1	O
-and	O
-EV2A	O
-,	O
-F	O
-–	O
-H	O
-).	O
-	O
-In	O
-cluster	O
-1	O
-,	O
-the	O
-recruitment	O
-of	O
-NCOA1	O
-and	O
-NCOA2	O
-was	O
-highest	O
-for	O
-WAY	O
-‐	O
-C	O
-,	O
-followed	O
-by	O
-triaryl	O
-‐	O
-ethylene	O
-,	O
-OBHS	O
-‐	O
-N	O
-,	O
-and	O
-OBHS	O
-series	O
-,	O
-while	O
-for	O
-NCOA3	O
-,	O
-OBHS	O
-‐	O
-N	O
-compounds	O
-induced	O
-the	O
-most	O
-recruitment	O
-and	O
-OBHS	O
-ligands	O
-were	O
-inverse	O
-agonists	O
-(	O
-Fig	O
-EV2F	O
-–	O
-H	O
-).	O
-	O
-The	O
-average	O
-induction	O
-of	O
-GREB1	O
-by	O
-cluster	O
-1	O
-ligands	O
-showed	O
-greater	O
-variance	O
-,	O
-with	O
-a	O
-range	O
-between	O
-~	O
-25	O
-and	O
-~	O
-75	O
-%	O
-for	O
-OBHS	O
-and	O
-a	O
-range	O
-from	O
-full	O
-agonist	O
-to	O
-inverse	O
-agonist	O
-for	O
-the	O
-others	O
-in	O
-cluster	O
-1	O
-(	O
-Fig	O
-EV2A	O
-).	O
-	O
-GREB1	O
-levels	O
-induced	O
-by	O
-OBHS	O
-analogs	O
-were	O
-determined	O
-by	O
-recruitment	O
-of	O
-NCOA1	O
-but	O
-not	O
-NCOA2	O
-/	O
-3	O
-(	O
-Fig	O
-3E	O
-lane	O
-1	O
-),	O
-suggesting	O
-that	O
-there	O
-may	O
-be	O
-alternate	O
-or	O
-preferential	O
-use	O
-of	O
-these	O
-coactivators	O
-by	O
-different	O
-classes	O
-.	O
-	O
-However	O
-,	O
-in	O
-cluster	O
-1	O
-,	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-generally	O
-predicted	O
-GREB1	O
-levels	O
-(	O
-Fig	O
-3E	O
-lanes	O
-1	O
-–	O
-4	O
-),	O
-consistent	O
-with	O
-the	O
-canonical	O
-signaling	O
-model	O
-(	O
-Fig	O
-1D	O
-).	O
-	O
-For	O
-clusters	O
-2	O
-and	O
-3	O
-,	O
-GREB1	O
-activity	O
-was	O
-generally	O
-not	O
-predicted	O
-by	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-.	O
-	O
-Direct	O
-modulators	O
-showed	O
-low	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-(	O
-Fig	O
-EV2F	O
-–	O
-H	O
-),	O
-but	O
-only	O
-OBHS	O
-‐	O
-ASC	O
-analogs	O
-had	O
-NCOA2	O
-recruitment	O
-profiles	O
-that	O
-predicted	O
-a	O
-full	O
-range	O
-of	O
-effects	O
-on	O
-GREB1	O
-levels	O
-(	O
-Figs	O
-3E	O
-lanes	O
-9	O
-,	O
-11	O
-,	O
-18	O
-–	O
-19	O
-,	O
-and	O
-EV2A	O
-).	O
-	O
-The	O
-indirect	O
-modulators	O
-in	O
-clusters	O
-2	O
-and	O
-3	O
-stimulated	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-and	O
-GREB1	O
-expression	O
-with	O
-substantial	O
-variance	O
-(	O
-Figs	O
-3A	O
-and	O
-EV2F	O
-–	O
-H	O
-).	O
-	O
-However	O
-,	O
-ligand	O
-‐	O
-induced	O
-GREB1	O
-levels	O
-were	O
-generally	O
-not	O
-determined	O
-by	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-(	O
-Fig	O
-3E	O
-lanes	O
-5	O
-–	O
-19	O
-),	O
-consistent	O
-with	O
-an	O
-alternate	O
-causality	O
-model	O
-(	O
-Fig	O
-1E	O
-).	O
-	O
-Out	O
-of	O
-11	O
-indirect	O
-modulator	O
-series	O
-in	O
-cluster	O
-2	O
-or	O
-3	O
-,	O
-only	O
-the	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-class	O
-had	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-profiles	O
-that	O
-predicted	O
-GREB1	O
-levels	O
-(	O
-Fig	O
-3E	O
-lane	O
-12	O
-).	O
-	O
-These	O
-results	O
-suggest	O
-that	O
-compounds	O
-that	O
-show	O
-cell	O
-‐	O
-specific	O
-signaling	O
-do	O
-not	O
-activate	O
-GREB1	O
-,	O
-or	O
-use	O
-coactivators	O
-other	O
-than	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-to	O
-control	O
-GREB1	O
-expression	O
-(	O
-Fig	O
-1E	O
-).	O
-	O
-To	O
-determine	O
-mechanisms	O
-for	O
-ligand	O
-‐	O
-dependent	O
-control	O
-of	O
-breast	O
-cancer	O
-cell	O
-proliferation	O
-,	O
-we	O
-performed	O
-linear	O
-regression	O
-analyses	O
-across	O
-the	O
-19	O
-scaffolds	O
-using	O
-MCF	O
-‐	O
-7	O
-cell	O
-proliferation	O
-as	O
-the	O
-dependent	O
-variable	O
-,	O
-and	O
-the	O
-other	O
-activities	O
-as	O
-independent	O
-variables	O
-(	O
-Fig	O
-3F	O
-).	O
-	O
-In	O
-cluster	O
-1	O
-,	O
-E	O
-‐	O
-Luc	O
-and	O
-L	O
-‐	O
-Luc	O
-activities	O
-,	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-,	O
-and	O
-GREB1	O
-levels	O
-generally	O
-predicted	O
-the	O
-proliferative	O
-response	O
-(	O
-Fig	O
-3F	O
-lanes	O
-2	O
-–	O
-4	O
-).	O
-	O
-With	O
-the	O
-OBHS	O
-‐	O
-N	O
-compounds	O
-,	O
-NCOA3	O
-and	O
-GREB1	O
-showed	O
-near	O
-perfect	O
-prediction	O
-of	O
-proliferation	O
-(	O
-Fig	O
-EV3G	O
-),	O
-with	O
-unexplained	O
-variance	O
-similar	O
-to	O
-the	O
-noise	O
-in	O
-the	O
-assays	O
-.	O
-	O
-The	O
-lack	O
-of	O
-significant	O
-predictors	O
-for	O
-OBHS	O
-analogs	O
-(	O
-Fig	O
-3F	O
-lane	O
-1	O
-)	O
-reflects	O
-their	O
-small	O
-range	O
-of	O
-proliferative	O
-effects	O
-on	O
-MCF	O
-‐	O
-7	O
-cells	O
-(	O
-Fig	O
-EV2I	O
-).	O
-	O
-The	O
-significant	O
-correlations	O
-with	O
-GREB1	O
-expression	O
-and	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-observed	O
-in	O
-this	O
-cluster	O
-are	O
-consistent	O
-with	O
-the	O
-canonical	O
-signaling	O
-model	O
-(	O
-Fig	O
-1D	O
-),	O
-where	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-determines	O
-GREB1	O
-expression	O
-,	O
-which	O
-then	O
-drives	O
-proliferation	O
-.	O
-	O
-Despite	O
-this	O
-phenotypic	O
-variance	O
-,	O
-proliferation	O
-was	O
-not	O
-generally	O
-predicted	O
-by	O
-correlated	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-and	O
-GREB1	O
-induction	O
-(	O
-Figs	O
-3F	O
-lanes	O
-5	O
-–	O
-19	O
-,	O
-and	O
-EV3H	O
-).	O
-	O
-Out	O
-of	O
-15	O
-ligand	O
-series	O
-in	O
-these	O
-clusters	O
-,	O
-only	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-induced	O
-a	O
-proliferative	O
-response	O
-that	O
-was	O
-predicted	O
-by	O
-GREB1	O
-levels	O
-,	O
-which	O
-were	O
-not	O
-determined	O
-by	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-(	O
-Fig	O
-3E	O
-and	O
-F	O
-lane	O
-10	O
-).	O
-	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-,	O
-cyclofenil	O
-,	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-,	O
-and	O
-imidazopyridine	O
-analogs	O
-had	O
-NCOA1	O
-/	O
-3	O
-recruitment	O
-profiles	O
-that	O
-predicted	O
-their	O
-proliferative	O
-effects	O
-,	O
-without	O
-determining	O
-GREB1	O
-levels	O
-(	O
-Fig	O
-3E	O
-and	O
-F	O
-,	O
-lanes	O
-5	O
-and	O
-14	O
-–	O
-16	O
-).	O
-	O
-Similarly	O
-,	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-,	O
-cyclofenil	O
-‐	O
-ASC	O
-,	O
-and	O
-OBHS	O
-‐	O
-ASC	O
-had	O
-positively	O
-correlated	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-and	O
-GREB1	O
-levels	O
-,	O
-but	O
-none	O
-of	O
-these	O
-activities	O
-determined	O
-their	O
-proliferative	O
-effects	O
-(	O
-Fig	O
-3E	O
-and	O
-F	O
-lanes	O
-11	O
-–	O
-12	O
-and	O
-18	O
-).	O
-	O
-For	O
-ligands	O
-that	O
-show	O
-cell	O
-‐	O
-specific	O
-signaling	O
-,	O
-ERα	O
-‐	O
-mediated	O
-recruitment	O
-of	O
-other	O
-coregulators	O
-and	O
-activation	O
-of	O
-other	O
-target	O
-genes	O
-likely	O
-determine	O
-their	O
-proliferative	O
-effects	O
-on	O
-MCF	O
-‐	O
-7	O
-cells	O
-.	O
-	O
-NCOA3	O
-occupancy	O
-at	O
-GREB1	O
-did	O
-not	O
-predict	O
-the	O
-proliferative	O
-response	O
-	O
-We	O
-also	O
-questioned	O
-whether	O
-promoter	O
-occupancy	O
-by	O
-coactivators	O
-is	O
-statistically	O
-robust	O
-and	O
-reproducible	O
-for	O
-ligand	O
-class	O
-analysis	O
-using	O
-a	O
-chromatin	O
-immunoprecipitation	O
-(	O
-ChIP	O
-)‐	O
-based	O
-quantitative	O
-assay	O
-,	O
-and	O
-whether	O
-it	O
-has	O
-a	O
-better	O
-predictive	O
-power	O
-than	O
-the	O
-M2H	O
-assay	O
-.	O
-	O
-ERα	O
-and	O
-NCOA3	O
-cycle	O
-on	O
-and	O
-off	O
-the	O
-GREB1	O
-promoter	O
-(	O
-Nwachukwu	O
-et	O
-al	O
-,	O
-2014	O
-).	O
-	O
-Therefore	O
-,	O
-we	O
-first	O
-performed	O
-a	O
-time	O
-‐	O
-course	O
-study	O
-,	O
-and	O
-found	O
-that	O
-E2	O
-and	O
-the	O
-WAY	O
-‐	O
-C	O
-analog	O
-,	O
-AAPII	O
-‐	O
-151	O
-‐	O
-4	O
-,	O
-induced	O
-recruitment	O
-of	O
-NCOA3	O
-to	O
-the	O
-GREB1	O
-promoter	O
-in	O
-a	O
-temporal	O
-cycle	O
-that	O
-peaked	O
-after	O
-45	O
-min	O
-in	O
-MCF	O
-‐	O
-7	O
-cells	O
-(	O
-Fig	O
-4A	O
-).	O
-	O
-At	O
-this	O
-time	O
-point	O
-,	O
-other	O
-WAY	O
-‐	O
-C	O
-analogs	O
-also	O
-induced	O
-recruitment	O
-of	O
-NCOA3	O
-at	O
-this	O
-site	O
-to	O
-varying	O
-degrees	O
-(	O
-Fig	O
-4B	O
-).	O
-	O
-The	O
-Z	O
-’	O
-for	O
-this	O
-assay	O
-was	O
-0	O
-.	O
-6	O
-,	O
-showing	O
-statistical	O
-robustness	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-We	O
-prepared	O
-biological	O
-replicates	O
-with	O
-different	O
-cell	O
-passage	O
-numbers	O
-and	O
-separately	O
-prepared	O
-samples	O
-,	O
-which	O
-showed	O
-r	O
-2	O
-of	O
-0	O
-.	O
-81	O
-,	O
-demonstrating	O
-high	O
-reproducibility	O
-(	O
-Fig	O
-4C	O
-).	O
-	O
-NCOA3	O
-occupancy	O
-at	O
-GREB1	O
-is	O
-statistically	O
-robust	O
-but	O
-does	O
-not	O
-predict	O
-transcriptional	O
-activity	O
-	O
-Kinetic	O
-ChIP	O
-assay	O
-examining	O
-recruitment	O
-of	O
-NCOA3	O
-to	O
-the	O
-GREB1	O
-gene	O
-in	O
-MCF	O
-‐	O
-7	O
-cells	O
-stimulated	O
-with	O
-E2	O
-or	O
-the	O
-indicated	O
-WAY	O
-‐	O
-C	O
-analog	O
-.	O
-	O
-NCOA3	O
-occupancy	O
-at	O
-GREB1	O
-was	O
-compared	O
-by	O
-ChIP	O
-assay	O
-45	O
-min	O
-after	O
-stimulation	O
-with	O
-vehicle	O
-,	O
-E2	O
-,	O
-or	O
-the	O
-WAY	O
-‐	O
-C	O
-analogs	O
-.	O
-	O
-In	O
-panel	O
-(	O
-B	O
-),	O
-the	O
-average	O
-recruitment	O
-of	O
-two	O
-biological	O
-replicates	O
-are	O
-shown	O
-as	O
-mean	O
-+	O
-SEM	O
-,	O
-and	O
-the	O
-Z	O
-‐	O
-score	O
-is	O
-indicated	O
-.	O
-	O
-In	O
-panel	O
-(	O
-C	O
-),	O
-correlation	O
-analysis	O
-was	O
-performed	O
-for	O
-two	O
-biological	O
-replicates	O
-.	O
-	O
-Linear	O
-regression	O
-analyses	O
-comparing	O
-the	O
-ability	O
-of	O
-NCOA3	O
-recruitment	O
-,	O
-measured	O
-by	O
-ChIP	O
-or	O
-M2H	O
-,	O
-to	O
-predict	O
-other	O
-agonist	O
-activities	O
-of	O
-WAY	O
-‐	O
-C	O
-analogs	O
-.	O
-*	O
-Significant	O
-positive	O
-correlation	O
-(	O
-F	O
-‐	O
-test	O
-for	O
-nonzero	O
-slope	O
-,	O
-P	O
-‐	O
-value	O
-).	O
-	O
-The	O
-M2H	O
-assay	O
-for	O
-NCOA3	O
-recruitment	O
-broadly	O
-correlated	O
-with	O
-the	O
-other	O
-assays	O
-,	O
-and	O
-was	O
-predictive	O
-for	O
-GREB1	O
-expression	O
-and	O
-cell	O
-proliferation	O
-(	O
-Fig	O
-3E	O
-).	O
-	O
-However	O
-,	O
-the	O
-ChIP	O
-assays	O
-for	O
-WAY	O
-‐	O
-C	O
-‐	O
-induced	O
-recruitment	O
-of	O
-NCOA3	O
-to	O
-the	O
-GREB1	O
-promoter	O
-did	O
-not	O
-correlate	O
-with	O
-any	O
-of	O
-the	O
-other	O
-WAY	O
-‐	O
-C	O
-activity	O
-profiles	O
-(	O
-Fig	O
-4D	O
-),	O
-although	O
-the	O
-positive	O
-correlation	O
-between	O
-ChIP	O
-assays	O
-and	O
-NCOA3	O
-recruitment	O
-via	O
-M2H	O
-assay	O
-showed	O
-a	O
-trend	O
-toward	O
-significance	O
-with	O
-r	O
-2	O
-=	O
-0	O
-.	O
-36	O
-and	O
-P	O
-=	O
-0	O
-.	O
-09	O
-(	O
-F	O
-‐	O
-test	O
-for	O
-nonzero	O
-slope	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-simplified	O
-coactivator	O
-‐	O
-binding	O
-assay	O
-showed	O
-much	O
-greater	O
-predictive	O
-power	O
-than	O
-the	O
-ChIP	O
-assay	O
-for	O
-ligand	O
-‐	O
-specific	O
-effects	O
-on	O
-GREB1	O
-expression	O
-and	O
-cell	O
-proliferation	O
-.	O
-	O
-ERβ	O
-activity	O
-is	O
-not	O
-an	O
-independent	O
-predictor	O
-of	O
-cell	O
-‐	O
-specific	O
-activity	O
-	O
-One	O
-difference	O
-between	O
-MCF	O
-‐	O
-7	O
-breast	O
-cancer	O
-cells	O
-and	O
-Ishikawa	O
-endometrial	O
-cancer	O
-cells	O
-is	O
-the	O
-contribution	O
-of	O
-ERβ	O
-to	O
-estrogenic	O
-response	O
-,	O
-as	O
-Ishikawa	O
-cells	O
-may	O
-express	O
-ERβ	O
-(	O
-Bhat	O
-&	O
-Pezzuto	O
-,	O
-2001	O
-).	O
-	O
-When	O
-overexpressed	O
-in	O
-MCF	O
-‐	O
-7	O
-cells	O
-,	O
-ERβ	O
-alters	O
-E2	O
-‐	O
-induced	O
-expression	O
-of	O
-only	O
-a	O
-subset	O
-of	O
-ERα	O
-‐	O
-target	O
-genes	O
-(	O
-Wu	O
-et	O
-al	O
-,	O
-2011	O
-),	O
-raising	O
-the	O
-possibility	O
-that	O
-ligand	O
-‐	O
-induced	O
-ERβ	O
-activity	O
-may	O
-contribute	O
-to	O
-E	O
-‐	O
-Luc	O
-activities	O
-,	O
-and	O
-thus	O
-underlie	O
-the	O
-lack	O
-of	O
-correlation	O
-between	O
-the	O
-E	O
-‐	O
-Luc	O
-and	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-activities	O
-or	O
-GREB1	O
-levels	O
-induced	O
-by	O
-cell	O
-‐	O
-specific	O
-modulators	O
-in	O
-cluster	O
-2	O
-and	O
-cluster	O
-3	O
-(	O
-Fig	O
-3C	O
-).	O
-	O
-To	O
-test	O
-this	O
-idea	O
-,	O
-we	O
-determined	O
-the	O
-L	O
-‐	O
-Luc	O
-ERβ	O
-activity	O
-profiles	O
-of	O
-the	O
-ligands	O
-(	O
-Fig	O
-EV1	O
-).	O
-	O
-All	O
-direct	O
-modulator	O
-and	O
-two	O
-indirect	O
-modulator	O
-scaffolds	O
-(	O
-OBHS	O
-and	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-)	O
-lacked	O
-ERβ	O
-agonist	O
-activity	O
-.	O
-	O
-For	O
-most	O
-scaffolds	O
-,	O
-L	O
-‐	O
-Luc	O
-ERβ	O
-and	O
-E	O
-‐	O
-Luc	O
-activities	O
-were	O
-not	O
-correlated	O
-,	O
-except	O
-for	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-and	O
-cyclofenil	O
-analogs	O
-,	O
-which	O
-showed	O
-moderate	O
-but	O
-significant	O
-correlations	O
-(	O
-Fig	O
-EV4A	O
-).	O
-	O
-Nevertheless	O
-,	O
-the	O
-E	O
-‐	O
-Luc	O
-activities	O
-of	O
-both	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-and	O
-cyclofenil	O
-analogs	O
-were	O
-better	O
-predicted	O
-by	O
-their	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-than	O
-L	O
-‐	O
-Luc	O
-ERβ	O
-activities	O
-(	O
-Fig	O
-EV4A	O
-and	O
-B	O
-).	O
-	O
-ERβ	O
-activity	O
-is	O
-not	O
-an	O
-independent	O
-predictor	O
-of	O
-E	O
-‐	O
-Luc	O
-activity	O
-	O
-ERβ	O
-activity	O
-in	O
-HepG2	O
-cells	O
-rarely	O
-correlates	O
-with	O
-E	O
-‐	O
-Luc	O
-activity	O
-.	O
-	O
-ERα	O
-activity	O
-of	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-and	O
-cyclofenil	O
-analogs	O
-correlates	O
-with	O
-E	O
-‐	O
-Luc	O
-activity	O
-.	O
-	O
-Data	O
-information	O
-:	O
-The	O
-r	O
-2	O
-and	O
-P	O
-values	O
-for	O
-the	O
-indicated	O
-correlations	O
-are	O
-shown	O
-in	O
-both	O
-panels	O
-.	O
-*	O
-Significant	O
-positive	O
-correlation	O
-(	O
-F	O
-‐	O
-test	O
-for	O
-nonzero	O
-slope	O
-,	O
-P	O
-‐	O
-value	O
-)	O
-	O
-To	O
-overcome	O
-barriers	O
-to	O
-crystallization	O
-of	O
-ERα	O
-LBD	O
-complexes	O
-,	O
-we	O
-developed	O
-a	O
-conformation	O
-‐	O
-trapping	O
-X	O
-‐	O
-ray	O
-crystallography	O
-approach	O
-using	O
-the	O
-ERα	B-mutant
-‐	I-mutant
-Y537S	I-mutant
-mutation	O
-(	O
-Nettles	O
-et	O
-al	O
-,	O
-2008	O
-;	O
-Bruning	O
-et	O
-al	O
-,	O
-2010	O
-;	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-).	O
-	O
-To	O
-further	O
-validate	O
-this	O
-approach	O
-,	O
-we	O
-solved	O
-the	O
-structure	O
-of	O
-the	O
-ERα	B-mutant
-‐	I-mutant
-Y537S	I-mutant
-LBD	O
-in	O
-complex	O
-with	O
-diethylstilbestrol	O
-(	O
-DES	O
-),	O
-which	O
-bound	O
-identically	O
-in	O
-the	O
-wild	O
-‐	O
-type	O
-and	O
-ERα	B-mutant
-‐	I-mutant
-Y537S	I-mutant
-LBDs	O
-,	O
-demonstrating	O
-again	O
-that	O
-this	O
-surface	O
-mutation	O
-stabilizes	O
-h12	O
-dynamics	O
-to	O
-facilitate	O
-crystallization	O
-without	O
-changing	O
-ligand	O
-binding	O
-(	O
-Appendix	O
-Fig	O
-S1A	O
-and	O
-B	O
-)	O
-(	O
-Nettles	O
-et	O
-al	O
-,	O
-2008	O
-;	O
-Bruning	O
-et	O
-al	O
-,	O
-2010	O
-;	O
-Delfosse	O
-et	O
-al	O
-,	O
-2012	O
-).	O
-	O
-Using	O
-this	O
-approach	O
-,	O
-we	O
-solved	O
-76	O
-ERα	O
-LBD	O
-structures	O
-in	O
-the	O
-active	O
-conformation	O
-and	O
-bound	O
-to	O
-ligands	O
-studied	O
-here	O
-(	O
-Appendix	O
-Fig	O
-S1C	O
-).	O
-	O
-Eleven	O
-of	O
-these	O
-structures	O
-have	O
-been	O
-published	O
-,	O
-while	O
-65	O
-are	O
-new	O
-,	O
-including	O
-the	O
-DES	O
-‐	O
-bound	O
-ERα	B-mutant
-‐	I-mutant
-Y537S	I-mutant
-LBD	O
-.	O
-	O
-We	O
-present	O
-57	O
-of	O
-these	O
-new	O
-structures	O
-here	O
-(	O
-Dataset	O
-EV2	O
-),	O
-while	O
-the	O
-remaining	O
-eight	O
-new	O
-structures	O
-bound	O
-to	O
-OBHS	O
-‐	O
-N	O
-analogs	O
-will	O
-be	O
-published	O
-elsewhere	O
-(	O
-S	O
-.	O
-Srinivasan	O
-et	O
-al	O
-,	O
-in	O
-preparation	O
-).	O
-	O
-Examining	O
-many	O
-closely	O
-related	O
-structures	O
-allows	O
-us	O
-to	O
-visualize	O
-subtle	O
-structural	O
-differences	O
-,	O
-in	O
-effect	O
-using	O
-X	O
-‐	O
-ray	O
-crystallography	O
-as	O
-a	O
-systems	O
-biology	O
-tool	O
-.	O
-	O
-The	O
-indirect	O
-modulator	O
-scaffolds	O
-in	O
-cluster	O
-1	O
-did	O
-not	O
-show	O
-cell	O
-‐	O
-specific	O
-signaling	O
-(	O
-Fig	O
-3C	O
-),	O
-but	O
-shared	O
-common	O
-structural	O
-perturbations	O
-that	O
-we	O
-designed	O
-to	O
-modulate	O
-h12	O
-dynamics	O
-.	O
-	O
-Based	O
-on	O
-our	O
-original	O
-OBHS	O
-structure	O
-,	O
-the	O
-OBHS	O
-,	O
-OBHS	O
-‐	O
-N	O
-,	O
-and	O
-triaryl	O
-‐	O
-ethylene	O
-compounds	O
-were	O
-modified	O
-with	O
-h11	O
-‐	O
-directed	O
-pendant	O
-groups	O
-(	O
-Zheng	O
-et	O
-al	O
-,	O
-2012	O
-;	O
-Zhu	O
-et	O
-al	O
-,	O
-2012	O
-;	O
-Liao	O
-et	O
-al	O
-,	O
-2014	O
-).	O
-	O
-Superposing	O
-the	O
-LBDs	O
-based	O
-on	O
-the	O
-class	O
-of	O
-bound	O
-ligands	O
-provides	O
-an	O
-ensemble	O
-view	O
-of	O
-the	O
-structural	O
-variance	O
-and	O
-clarifies	O
-what	O
-part	O
-of	O
-the	O
-ligand	O
-‐	O
-binding	O
-pocket	O
-is	O
-differentially	O
-perturbed	O
-or	O
-targeted	O
-.	O
-	O
-The	O
-24	O
-structures	O
-containing	O
-OBHS	O
-,	O
-OBHS	O
-‐	O
-N	O
-,	O
-or	O
-triaryl	O
-‐	O
-ethylene	O
-analogs	O
-showed	O
-structural	O
-diversity	O
-in	O
-the	O
-same	O
-part	O
-of	O
-the	O
-scaffolds	O
-(	O
-Figs	O
-5A	O
-and	O
-EV5A	O
-),	O
-and	O
-the	O
-same	O
-region	O
-of	O
-the	O
-LBD	O
-—	O
-the	O
-C	O
-‐	O
-terminal	O
-end	O
-of	O
-h11	O
-(	O
-Figs	O
-5B	O
-and	O
-C	O
-,	O
-and	O
-EV5B	O
-),	O
-which	O
-in	O
-turn	O
-nudges	O
-h12	O
-(	O
-Fig	O
-5C	O
-and	O
-D	O
-).	O
-	O
-We	O
-observed	O
-that	O
-the	O
-OBHS	O
-‐	O
-N	O
-analogs	O
-displaced	O
-h11	O
-along	O
-a	O
-vector	O
-away	O
-from	O
-Leu354	O
-in	O
-a	O
-region	O
-of	O
-h3	O
-that	O
-is	O
-unaffected	O
-by	O
-the	O
-ligands	O
-,	O
-and	O
-toward	O
-the	O
-dimer	O
-interface	O
-.	O
-	O
-For	O
-the	O
-triaryl	O
-‐	O
-ethylene	O
-analogs	O
-,	O
-the	O
-displacement	O
-of	O
-h11	O
-was	O
-in	O
-a	O
-perpendicular	O
-direction	O
-,	O
-away	O
-from	O
-Ile424	O
-in	O
-h8	O
-and	O
-toward	O
-h12	O
-.	O
-	O
-Remarkably	O
-,	O
-these	O
-individual	O
-inter	O
-‐	O
-atomic	O
-distances	O
-showed	O
-a	O
-ligand	O
-class	O
-‐	O
-specific	O
-ability	O
-to	O
-significantly	O
-predict	O
-proliferative	O
-effects	O
-(	O
-Fig	O
-5E	O
-and	O
-F	O
-),	O
-demonstrating	O
-the	O
-feasibility	O
-of	O
-developing	O
-a	O
-minimal	O
-set	O
-of	O
-activity	O
-predictors	O
-from	O
-crystal	O
-structures	O
-.	O
-	O
-Structure	O
-‐	O
-class	O
-analysis	O
-of	O
-triaryl	O
-‐	O
-ethylene	O
-analogs	O
-.	O
-	O
-Triaryl	O
-‐	O
-ethylene	O
-analogs	O
-bound	O
-to	O
-the	O
-superposed	O
-crystal	O
-structures	O
-of	O
-the	O
-ERα	O
-LBD	O
-are	O
-shown	O
-.	O
-	O
-Arrows	O
-indicate	O
-chemical	O
-variance	O
-in	O
-the	O
-orientation	O
-of	O
-the	O
-different	O
-h11	O
-‐	O
-directed	O
-ligand	O
-side	O
-groups	O
-(	O
-PDB	O
-5DK9	O
-,	O
-5DKB	O
-,	O
-5DKE	O
-,	O
-5DKG	O
-,	O
-5DKS	O
-,	O
-5DL4	O
-,	O
-5DLR	O
-,	O
-5DMC	O
-,	O
-5DMF	O
-and	O
-5DP0	O
-).	O
-	O
-Triaryl	O
-‐	O
-ethylene	O
-analogs	O
-induce	O
-variance	O
-of	O
-ERα	O
-conformations	O
-at	O
-the	O
-C	O
-‐	O
-terminal	O
-region	O
-of	O
-h11	O
-.	O
-	O
-Panel	O
-(	O
-B	O
-)	O
-shows	O
-the	O
-crystal	O
-structure	O
-of	O
-a	O
-triaryl	O
-‐	O
-ethylene	O
-analog	O
-‐	O
-bound	O
-ERα	O
-LBD	O
-(	O
-PDB	O
-5DLR	O
-).	O
-	O
-The	O
-h11	O
-–	O
-h12	O
-interface	O
-(	O
-circled	O
-)	O
-includes	O
-the	O
-C	O
-‐	O
-terminal	O
-part	O
-of	O
-h11	O
-.	O
-	O
-This	O
-region	O
-was	O
-expanded	O
-in	O
-panel	O
-(	O
-C	O
-),	O
-where	O
-the	O
-10	O
-triaryl	O
-‐	O
-ethylene	O
-analog	O
-‐	O
-bound	O
-ERα	O
-LBD	O
-structures	O
-(	O
-see	O
-Datasets	O
-EV1	O
-and	O
-EV2	O
-)	O
-were	O
-superposed	O
-to	O
-show	O
-variations	O
-in	O
-the	O
-h11	O
-C	O
-‐	O
-terminus	O
-(	O
-PDB	O
-5DK9	O
-,	O
-5DKB	O
-,	O
-5DKE	O
-,	O
-5DKG	O
-,	O
-5DKS	O
-,	O
-5DL4	O
-,	O
-5DLR	O
-,	O
-5DMC	O
-,	O
-5DMF	O
-,	O
-and	O
-5DP0	O
-).	O
-	O
-ERα	O
-LBDs	O
-in	O
-complex	O
-with	O
-diethylstilbestrol	O
-(	O
-DES	O
-)	O
-or	O
-a	O
-triaryl	O
-‐	O
-ethylene	O
-analog	O
-were	O
-superposed	O
-to	O
-show	O
-that	O
-the	O
-ligand	O
-‐	O
-induced	O
-difference	O
-in	O
-h11	O
-conformation	O
-is	O
-transmitted	O
-to	O
-the	O
-C	O
-‐	O
-terminus	O
-of	O
-h12	O
-(	O
-PDB	O
-4ZN7	O
-,	O
-5DMC	O
-).	O
-	O
-Inter	O
-‐	O
-atomic	O
-distances	O
-predict	O
-the	O
-proliferative	O
-effects	O
-of	O
-specific	O
-ligand	O
-series	O
-.	O
-	O
-Ile424	O
-–	O
-His524	O
-distance	O
-measured	O
-in	O
-the	O
-crystal	O
-structures	O
-correlates	O
-with	O
-the	O
-proliferative	O
-effect	O
-of	O
-triaryl	O
-‐	O
-ethylene	O
-analogs	O
-in	O
-MCF	O
-‐	O
-7	O
-cells	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-Leu354	O
-–	O
-Leu525	O
-distance	O
-correlates	O
-with	O
-the	O
-proliferative	O
-effects	O
-of	O
-OBHS	O
-‐	O
-N	O
-analogs	O
-in	O
-MCF	O
-‐	O
-7	O
-cells	O
-.	O
-	O
-Structure	O
-‐	O
-class	O
-analysis	O
-of	O
-WAY	O
-‐	O
-C	O
-analogs	O
-.	O
-	O
-WAY	O
-‐	O
-C	O
-side	O
-groups	O
-subtly	O
-nudge	O
-h12	O
-Leu540	O
-.	O
-	O
-ERα	O
-LBD	O
-structures	O
-bound	O
-to	O
-4	O
-distinct	O
-WAY	O
-‐	O
-C	O
-analogs	O
-were	O
-superposed	O
-(	O
-PDB	O
-4	O
-IU7	O
-,	O
-4IV4	O
-,	O
-4IVW	O
-,	O
-4IW6	O
-)	O
-(	O
-see	O
-Datasets	O
-EV1	O
-and	O
-EV2	O
-).	O
-	O
-Structure	O
-‐	O
-class	O
-analysis	O
-of	O
-indirect	O
-modulators	O
-	O
-Structure	O
-‐	O
-class	O
-analysis	O
-of	O
-indirect	O
-modulators	O
-in	O
-cluster	O
-1	O
-.	O
-	O
-Crystal	O
-structures	O
-of	O
-the	O
-ERα	O
-LBD	O
-bound	O
-to	O
-OBHS	O
-and	O
-OBHS	O
-‐	O
-N	O
-analogs	O
-were	O
-superposed	O
-.	O
-	O
-Arrows	O
-indicate	O
-chemical	O
-variance	O
-in	O
-the	O
-orientation	O
-of	O
-the	O
-different	O
-h11	O
-‐	O
-directed	O
-ligand	O
-side	O
-groups	O
-.	O
-	O
-Panel	O
-(	O
-B	O
-)	O
-shows	O
-the	O
-ligand	O
-‐	O
-induced	O
-conformational	O
-variation	O
-at	O
-the	O
-C	O
-‐	O
-terminal	O
-region	O
-of	O
-h11	O
-(	O
-OBHS	O
-:	O
-PDB	O
-4ZN9	O
-,	O
-4ZNH	O
-,	O
-4ZNS	O
-,	O
-4ZNT	O
-,	O
-4ZNU	O
-,	O
-4ZNV	O
-,	O
-and	O
-4ZNW	O
-;	O
-OBHS	O
-‐	O
-N	O
-:	O
-PDB	O
-4ZUB	O
-,	O
-4ZUC	O
-,	O
-4ZWH	O
-,	O
-4ZWK	O
-,	O
-5BNU	O
-,	O
-5BP6	O
-,	O
-5BPR	O
-,	O
-and	O
-5BQ4	O
-).	O
-	O
-Structure	O
-‐	O
-class	O
-analysis	O
-of	O
-indirect	O
-modulators	O
-in	O
-clusters	O
-2	O
-and	O
-3	O
-.	O
-	O
-Crystal	O
-structures	O
-of	O
-the	O
-ERα	O
-LBD	O
-bound	O
-to	O
-ligands	O
-with	O
-cell	O
-‐	O
-specific	O
-activities	O
-were	O
-superposed	O
-.	O
-	O
-The	O
-bound	O
-ligands	O
-are	O
-shown	O
-,	O
-and	O
-arrows	O
-indicate	O
-considerable	O
-variation	O
-in	O
-the	O
-orientation	O
-of	O
-the	O
-different	O
-h3	O
-‐,	O
-h8	O
-‐,	O
-h11	O
-‐,	O
-or	O
-h12	O
-‐	O
-directed	O
-ligand	O
-side	O
-groups	O
-.	O
-	O
-As	O
-visualized	O
-in	O
-four	O
-LBD	O
-structures	O
-(	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-),	O
-WAY	O
-‐	O
-C	O
-analogs	O
-were	O
-designed	O
-with	O
-small	O
-substitutions	O
-that	O
-slightly	O
-nudge	O
-h12	O
-Leu540	O
-,	O
-without	O
-exiting	O
-the	O
-ligand	O
-‐	O
-binding	O
-pocket	O
-(	O
-Fig	O
-5G	O
-and	O
-H	O
-).	O
-	O
-Therefore	O
-,	O
-changing	O
-h12	O
-dynamics	O
-maintains	O
-the	O
-canonical	O
-signaling	O
-pathway	O
-defined	O
-by	O
-E2	O
-(	O
-Fig	O
-1D	O
-)	O
-to	O
-support	O
-AF	O
-‐	O
-2	O
-‐	O
-driven	O
-signaling	O
-and	O
-recruit	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-for	O
-GREB1	O
-‐	O
-stimulated	O
-proliferation	O
-.	O
-	O
-Ligands	O
-with	O
-cell	O
-‐	O
-specific	O
-activity	O
-alter	O
-the	O
-shape	O
-of	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-	O
-Direct	O
-modulators	O
-like	O
-tamoxifen	O
-drive	O
-AF	O
-‐	O
-1	O
-‐	O
-dependent	O
-cell	O
-‐	O
-specific	O
-activity	O
-by	O
-completely	O
-occluding	O
-AF	O
-‐	O
-2	O
-,	O
-but	O
-it	O
-is	O
-not	O
-known	O
-how	O
-indirect	O
-modulators	O
-produce	O
-cell	O
-‐	O
-specific	O
-ERα	O
-activity	O
-.	O
-	O
-Therefore	O
-,	O
-we	O
-examined	O
-another	O
-50	O
-LBD	O
-structures	O
-containing	O
-ligands	O
-in	O
-clusters	O
-2	O
-and	O
-3	O
-.	O
-	O
-These	O
-structures	O
-demonstrated	O
-that	O
-cell	O
-‐	O
-specific	O
-activity	O
-derived	O
-from	O
-altering	O
-the	O
-shape	O
-of	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-without	O
-an	O
-extended	O
-side	O
-chain	O
-.	O
-	O
-Ligands	O
-in	O
-cluster	O
-2	O
-and	O
-cluster	O
-3	O
-showed	O
-conformational	O
-heterogeneity	O
-in	O
-parts	O
-of	O
-the	O
-scaffold	O
-that	O
-were	O
-directed	O
-toward	O
-multiple	O
-regions	O
-of	O
-the	O
-receptor	O
-including	O
-h3	O
-,	O
-h8	O
-,	O
-h11	O
-,	O
-h12	O
-,	O
-and	O
-/	O
-or	O
-the	O
-β	O
-‐	O
-sheets	O
-(	O
-Fig	O
-EV5C	O
-–	O
-G	O
-).	O
-	O
-For	O
-instance	O
-,	O
-S	O
-‐	O
-OBHS	O
-‐	O
-2	O
-and	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-analogs	O
-(	O
-Fig	O
-2	O
-)	O
-had	O
-similar	O
-ERα	O
-activity	O
-profiles	O
-in	O
-the	O
-different	O
-cell	O
-types	O
-(	O
-Fig	O
-EV2A	O
-–	O
-C	O
-),	O
-but	O
-the	O
-2	O
-‐	O
-versus	O
-3	O
-‐	O
-methyl	O
-substituted	O
-phenol	O
-rings	O
-altered	O
-the	O
-correlated	O
-signaling	O
-patterns	O
-in	O
-different	O
-cell	O
-types	O
-(	O
-Fig	O
-3B	O
-lanes	O
-7	O
-and	O
-12	O
-).	O
-	O
-This	O
-difference	O
-in	O
-ligand	O
-positioning	O
-altered	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-via	O
-a	O
-shift	O
-in	O
-the	O
-N	O
-‐	O
-terminus	O
-of	O
-h12	O
-,	O
-which	O
-directly	O
-contacts	O
-the	O
-coactivator	O
-.	O
-	O
-This	O
-effect	O
-is	O
-evident	O
-in	O
-a	O
-single	O
-structure	O
-due	O
-to	O
-its	O
-1	O
-Å	O
-magnitude	O
-(	O
-Fig	O
-6A	O
-and	O
-B	O
-).	O
-	O
-The	O
-shifts	O
-in	O
-h12	O
-residues	O
-Asp538	O
-and	O
-Leu539	O
-led	O
-to	O
-rotation	O
-of	O
-the	O
-coactivator	O
-peptide	O
-(	O
-Fig	O
-6C	O
-).	O
-	O
-Thus	O
-,	O
-cell	O
-‐	O
-specific	O
-activity	O
-can	O
-stem	O
-from	O
-perturbation	O
-of	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-without	O
-an	O
-extended	O
-side	O
-chain	O
-,	O
-which	O
-presumably	O
-alters	O
-the	O
-receptor	O
-–	O
-coregulator	O
-interaction	O
-profile	O
-.	O
-	O
-S	O
-‐	O
-OBHS	O
-‐	O
-2	O
-/	O
-3	O
-analogs	O
-subtly	O
-distort	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-.	O
-	O
-Panel	O
-(	O
-A	O
-)	O
-shows	O
-the	O
-crystal	O
-structure	O
-of	O
-an	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-‐	O
-bound	O
-ERα	O
-LBD	O
-(	O
-PDB	O
-5DUH	O
-).	O
-	O
-The	O
-h3	O
-–	O
-h12	O
-interface	O
-(	O
-circled	O
-)	O
-at	O
-AF	O
-‐	O
-2	O
-(	O
-pink	O
-)	O
-was	O
-expanded	O
-in	O
-panels	O
-(	O
-B	O
-,	O
-C	O
-).	O
-	O
-The	O
-S	O
-‐	O
-OBHS	O
-‐	O
-2	O
-/	O
-3	O
-‐	O
-bound	O
-ERα	O
-LBDs	O
-were	O
-superposed	O
-to	O
-show	O
-shifts	O
-in	O
-h3	O
-(	O
-panel	O
-B	O
-)	O
-and	O
-the	O
-NCOA2	O
-peptide	O
-docked	O
-at	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-(	O
-panel	O
-C	O
-).	O
-	O
-Crystal	O
-structures	O
-show	O
-that	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-shift	O
-h3	O
-and	O
-h11	O
-further	O
-apart	O
-compared	O
-to	O
-an	O
-A	O
-‐	O
-CD	O
-‐	O
-ring	O
-estrogen	O
-(	O
-PDB	O
-4PPS	O
-,	O
-5DRM	O
-,	O
-5DRJ	O
-).	O
-	O
-The	O
-2F	O
-o	O
-‐	O
-F	O
-c	O
-electron	O
-density	O
-map	O
-and	O
-F	O
-o	O
-‐	O
-F	O
-c	O
-difference	O
-map	O
-of	O
-a	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-‐	O
-bound	O
-structure	O
-(	O
-PDB	O
-5DRJ	O
-)	O
-were	O
-contoured	O
-at	O
-1	O
-.	O
-0	O
-sigma	O
-and	O
-±	O
-3	O
-.	O
-0	O
-sigma	O
-,	O
-respectively	O
-.	O
-	O
-Average	O
-(	O
-mean	O
-+	O
-SEM	O
-)	O
-α	O
-‐	O
-carbon	O
-distance	O
-measured	O
-from	O
-h3	O
-Thr347	O
-to	O
-h11	O
-Leu525	O
-of	O
-A	O
-‐	O
-CD	O
-‐,	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-‐,	O
-and	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-‐	O
-bound	O
-ERα	O
-LBDs	O
-.	O
-	O
-*	O
-Two	O
-‐	O
-tailed	O
-Student	O
-'	O
-s	O
-t	O
-‐	O
-test	O
-,	O
-P	O
-=	O
-0	O
-.	O
-002	O
-(	O
-PDB	O
-A	O
-‐	O
-CD	O
-:	O
-5DI7	O
-,	O
-5DID	O
-,	O
-5DIE	O
-,	O
-5DIG	O
-,	O
-and	O
-4PPS	O
-;	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-:	O
-4IWC	O
-,	O
-5DRM	O
-,	O
-and	O
-5DRJ	O
-;	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-:	O
-5DTV	O
-and	O
-5DU5	O
-).	O
-	O
-Crystal	O
-structures	O
-show	O
-that	O
-a	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-analog	O
-shifts	O
-h3	O
-(	O
-F	O
-)	O
-and	O
-the	O
-NCOA2	O
-(	O
-G	O
-)	O
-peptide	O
-compared	O
-to	O
-an	O
-A	O
-‐	O
-CD	O
-‐	O
-ring	O
-estrogen	O
-(	O
-PDB	O
-4PPS	O
-,	O
-5DTV	O
-).	O
-	O
-Hierarchical	O
-clustering	O
-of	O
-ligand	O
-‐	O
-specific	O
-binding	O
-of	O
-154	O
-interacting	O
-peptides	O
-to	O
-the	O
-ERα	O
-LBD	O
-was	O
-performed	O
-in	O
-triplicate	O
-by	O
-MARCoNI	O
-analysis	O
-.	O
-	O
-The	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-showed	O
-perturbation	O
-of	O
-h11	O
-,	O
-as	O
-well	O
-as	O
-h3	O
-,	O
-which	O
-forms	O
-part	O
-of	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-.	O
-	O
-These	O
-compounds	O
-bind	O
-the	O
-LBD	O
-in	O
-an	O
-unusual	O
-fashion	O
-because	O
-they	O
-have	O
-a	O
-phenol	O
-‐	O
-to	O
-‐	O
-phenol	O
-length	O
-of	O
-~	O
-12	O
-Å	O
-,	O
-which	O
-is	O
-longer	O
-than	O
-steroids	O
-and	O
-other	O
-prototypical	O
-ERα	O
-agonists	O
-that	O
-are	O
-~	O
-10	O
-Å	O
-in	O
-length	O
-.	O
-	O
-One	O
-phenol	O
-pushed	O
-further	O
-toward	O
-h3	O
-(	O
-Fig	O
-6D	O
-),	O
-while	O
-the	O
-other	O
-phenol	O
-pushed	O
-toward	O
-the	O
-C	O
-‐	O
-terminus	O
-of	O
-h11	O
-to	O
-a	O
-greater	O
-extent	O
-than	O
-A	O
-‐	O
-CD	O
-‐	O
-ring	O
-estrogens	O
-(	O
-Nwachukwu	O
-et	O
-al	O
-,	O
-2014	O
-),	O
-which	O
-are	O
-close	O
-structural	O
-analogs	O
-of	O
-E2	O
-that	O
-lack	O
-a	O
-B	O
-‐	O
-ring	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-To	O
-quantify	O
-this	O
-difference	O
-,	O
-we	O
-compared	O
-the	O
-distance	O
-between	O
-α	O
-‐	O
-carbons	O
-at	O
-h3	O
-Thr347	O
-and	O
-h11	O
-Leu525	O
-in	O
-the	O
-set	O
-of	O
-structures	O
-containing	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-(	O
-n	O
-=	O
-3	O
-)	O
-or	O
-A	O
-‐	O
-CD	O
-‐	O
-ring	O
-analogs	O
-(	O
-n	O
-=	O
-5	O
-)	O
-(	O
-Fig	O
-6E	O
-).	O
-	O
-We	O
-observed	O
-a	O
-difference	O
-of	O
-0	O
-.	O
-4	O
-Å	O
-that	O
-was	O
-significant	O
-(	O
-two	O
-‐	O
-tailed	O
-Student	O
-'	O
-s	O
-t	O
-‐	O
-test	O
-,	O
-P	O
-=	O
-0	O
-.	O
-002	O
-)	O
-due	O
-to	O
-the	O
-very	O
-tight	O
-clustering	O
-of	O
-the	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-‐	O
-induced	O
-LBD	O
-conformation	O
-.	O
-	O
-The	O
-shifts	O
-in	O
-h3	O
-suggest	O
-these	O
-compounds	O
-are	O
-positioned	O
-to	O
-alter	O
-coregulator	O
-preferences	O
-.	O
-	O
-The	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-and	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-scaffolds	O
-are	O
-isomeric	O
-,	O
-but	O
-with	O
-aryl	O
-groups	O
-at	O
-obtuse	O
-and	O
-acute	O
-angles	O
-,	O
-respectively	O
-(	O
-Fig	O
-2	O
-).	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-ERα	O
-in	O
-complex	O
-with	O
-a	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-is	O
-unknown	O
-;	O
-however	O
-,	O
-we	O
-solved	O
-two	O
-crystal	O
-structures	O
-of	O
-ERα	O
-bound	O
-to	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-analogs	O
-and	O
-one	O
-structure	O
-containing	O
-a	O
-furan	O
-ligand	O
-—	O
-all	O
-of	O
-which	O
-have	O
-a	O
-3	O
-,	O
-4	O
-‐	O
-diaryl	O
-configuration	O
-(	O
-Fig	O
-2	O
-;	O
-Datasets	O
-EV1	O
-and	O
-EV2	O
-).	O
-	O
-In	O
-these	O
-structures	O
-,	O
-the	O
-A	O
-‐	O
-ring	O
-mimetic	O
-of	O
-the	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-scaffold	O
-bound	O
-h3	O
-Glu353	O
-as	O
-expected	O
-,	O
-but	O
-the	O
-other	O
-phenol	O
-wrapped	O
-around	O
-h3	O
-to	O
-form	O
-a	O
-hydrogen	O
-bond	O
-with	O
-Thr347	O
-,	O
-indicating	O
-a	O
-change	O
-in	O
-binding	O
-epitopes	O
-in	O
-the	O
-ERα	O
-ligand	O
-‐	O
-binding	O
-pocket	O
-(	O
-Fig	O
-6F	O
-).	O
-	O
-The	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-analogs	O
-also	O
-induced	O
-a	O
-shift	O
-in	O
-h3	O
-positioning	O
-,	O
-which	O
-translated	O
-again	O
-into	O
-a	O
-shift	O
-in	O
-the	O
-bound	O
-coactivator	O
-peptide	O
-(	O
-Fig	O
-6F	O
-).	O
-	O
-Therefore	O
-,	O
-these	O
-indirect	O
-modulators	O
-,	O
-including	O
-S	O
-‐	O
-OBHS	O
-‐	O
-2	O
-,	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-,	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-,	O
-and	O
-3	O
-,	O
-4	O
-‐	O
-DTPD	O
-analogs	O
-—	O
-all	O
-of	O
-which	O
-show	O
-cell	O
-‐	O
-specific	O
-activity	O
-profiles	O
-—	O
-induced	O
-shifts	O
-in	O
-h3	O
-and	O
-h12	O
-that	O
-were	O
-transmitted	O
-to	O
-the	O
-coactivator	O
-peptide	O
-via	O
-an	O
-altered	O
-AF	O
-‐	O
-2	O
-surface	O
-.	O
-	O
-To	O
-test	O
-whether	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-shows	O
-changes	O
-in	O
-shape	O
-in	O
-solution	O
-,	O
-we	O
-used	O
-the	O
-microarray	O
-assay	O
-for	O
-real	O
-‐	O
-time	O
-coregulator	O
-–	O
-nuclear	O
-receptor	O
-interaction	O
-(	O
-MARCoNI	O
-)	O
-analysis	O
-(	O
-Aarts	O
-et	O
-al	O
-,	O
-2013	O
-).	O
-	O
-Here	O
-,	O
-the	O
-ligand	O
-‐	O
-dependent	O
-interactions	O
-of	O
-the	O
-ERα	O
-LBD	O
-with	O
-over	O
-150	O
-distinct	O
-LxxLL	O
-motif	O
-peptides	O
-were	O
-assayed	O
-to	O
-define	O
-structural	O
-fingerprints	O
-for	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-,	O
-in	O
-a	O
-manner	O
-similar	O
-to	O
-the	O
-use	O
-of	O
-phage	O
-display	O
-peptides	O
-as	O
-structural	O
-probes	O
-(	O
-Connor	O
-et	O
-al	O
-,	O
-2001	O
-).	O
-	O
-Despite	O
-the	O
-similar	O
-average	O
-activities	O
-of	O
-these	O
-ligand	O
-classes	O
-(	O
-Fig	O
-3A	O
-and	O
-B	O
-),	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-and	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-analogs	O
-displayed	O
-remarkably	O
-different	O
-peptide	O
-recruitment	O
-patterns	O
-(	O
-Fig	O
-6H	O
-),	O
-consistent	O
-with	O
-the	O
-structural	O
-analyses	O
-.	O
-	O
-Hierarchical	O
-clustering	O
-revealed	O
-that	O
-many	O
-of	O
-the	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-recapitulated	O
-most	O
-of	O
-the	O
-peptide	O
-recruitment	O
-and	O
-dismissal	O
-patterns	O
-observed	O
-with	O
-E2	O
-(	O
-Fig	O
-6H	O
-).	O
-	O
-However	O
-,	O
-there	O
-was	O
-a	O
-unique	O
-cluster	O
-of	O
-peptides	O
-that	O
-were	O
-recruited	O
-by	O
-E2	O
-but	O
-not	O
-the	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-analogs	O
-dismissed	O
-most	O
-of	O
-the	O
-peptides	O
-from	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-(	O
-Fig	O
-6H	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-isomeric	O
-attachment	O
-of	O
-diaryl	O
-groups	O
-to	O
-the	O
-thiophene	O
-core	O
-changed	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-from	O
-inside	O
-the	O
-ligand	O
-‐	O
-binding	O
-pocket	O
-,	O
-as	O
-predicted	O
-by	O
-the	O
-crystal	O
-structures	O
-.	O
-	O
-Together	O
-,	O
-these	O
-findings	O
-suggest	O
-that	O
-without	O
-an	O
-extended	O
-side	O
-chain	O
-,	O
-cell	O
-‐	O
-specific	O
-activity	O
-stems	O
-from	O
-different	O
-coregulator	O
-recruitment	O
-profiles	O
-,	O
-due	O
-to	O
-unique	O
-ligand	O
-‐	O
-induced	O
-conformations	O
-of	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-,	O
-in	O
-addition	O
-to	O
-differential	O
-usage	O
-of	O
-AF	O
-‐	O
-1	O
-.	O
-	O
-Indirect	O
-modulators	O
-in	O
-cluster	O
-1	O
-avoid	O
-this	O
-by	O
-perturbing	O
-the	O
-h11	O
-–	O
-h12	O
-interface	O
-,	O
-and	O
-modulating	O
-the	O
-dynamics	O
-of	O
-h12	O
-without	O
-changing	O
-the	O
-shape	O
-of	O
-AF	O
-‐	O
-2	O
-when	O
-stabilized	O
-.	O
-	O
-Our	O
-goal	O
-was	O
-to	O
-identify	O
-a	O
-minimal	O
-set	O
-of	O
-predictors	O
-that	O
-would	O
-link	O
-specific	O
-structural	O
-perturbations	O
-to	O
-ERα	O
-signaling	O
-pathways	O
-that	O
-control	O
-cell	O
-‐	O
-specific	O
-signaling	O
-and	O
-proliferation	O
-.	O
-	O
-We	O
-found	O
-a	O
-very	O
-strong	O
-set	O
-of	O
-predictors	O
-,	O
-where	O
-ligands	O
-in	O
-cluster	O
-1	O
-,	O
-defined	O
-by	O
-similar	O
-signaling	O
-across	O
-cell	O
-types	O
-,	O
-showed	O
-indirect	O
-modulation	O
-of	O
-h12	O
-dynamics	O
-via	O
-the	O
-h11	O
-–	O
-12	O
-interface	O
-or	O
-slight	O
-contact	O
-with	O
-h12	O
-.	O
-	O
-This	O
-perturbation	O
-determined	O
-proliferation	O
-that	O
-correlated	O
-strongly	O
-with	O
-AF	O
-‐	O
-2	O
-activity	O
-,	O
-recruitment	O
-of	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-family	O
-members	O
-,	O
-and	O
-induction	O
-of	O
-the	O
-GREB1	O
-gene	O
-,	O
-consistent	O
-with	O
-the	O
-canonical	O
-ERα	O
-signaling	O
-pathway	O
-(	O
-Fig	O
-1D	O
-).	O
-	O
-For	O
-ligands	O
-in	O
-cluster	O
-1	O
-,	O
-deletion	O
-of	O
-AF	O
-‐	O
-1	O
-reduced	O
-activity	O
-to	O
-varying	O
-degrees	O
-,	O
-but	O
-did	O
-not	O
-change	O
-the	O
-underlying	O
-signaling	O
-patterns	O
-established	O
-through	O
-AF	O
-‐	O
-2	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-an	O
-extended	O
-side	O
-chain	O
-designed	O
-to	O
-directly	O
-reposition	O
-h12	O
-and	O
-completely	O
-disrupt	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-results	O
-in	O
-cell	O
-‐	O
-specific	O
-signaling	O
-.	O
-	O
-Compared	O
-to	O
-cluster	O
-1	O
-,	O
-the	O
-structural	O
-rules	O
-are	O
-less	O
-clear	O
-in	O
-clusters	O
-2	O
-and	O
-3	O
-,	O
-but	O
-a	O
-number	O
-of	O
-indirect	O
-modulator	O
-classes	O
-perturbed	O
-the	O
-LBD	O
-conformation	O
-at	O
-the	O
-intersection	O
-of	O
-h3	O
-,	O
-the	O
-h12	O
-N	O
-‐	O
-terminus	O
-,	O
-and	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-.	O
-	O
-Ligands	O
-in	O
-these	O
-classes	O
-altered	O
-the	O
-shape	O
-of	O
-AF	O
-‐	O
-2	O
-to	O
-affect	O
-coregulator	O
-preferences	O
-.	O
-	O
-For	O
-direct	O
-and	O
-indirect	O
-modulators	O
-in	O
-cluster	O
-2	O
-or	O
-3	O
-,	O
-the	O
-canonical	O
-ERα	O
-signaling	O
-pathway	O
-involving	O
-recruitment	O
-of	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-and	O
-induction	O
-of	O
-GREB1	O
-did	O
-not	O
-generally	O
-predict	O
-their	O
-proliferative	O
-effects	O
-,	O
-indicating	O
-an	O
-alternate	O
-causal	O
-model	O
-(	O
-Fig	O
-1E	O
-).	O
-	O
-These	O
-principles	O
-outlined	O
-above	O
-provide	O
-a	O
-structural	O
-basis	O
-for	O
-how	O
-the	O
-ligand	O
-–	O
-receptor	O
-interface	O
-leads	O
-to	O
-different	O
-signaling	O
-specificities	O
-through	O
-AF	O
-‐	O
-1	O
-and	O
-AF	O
-‐	O
-2	O
-.	O
-	O
-It	O
-is	O
-noteworthy	O
-that	O
-regulation	O
-of	O
-h12	O
-dynamics	O
-indirectly	O
-through	O
-h11	O
-can	O
-virtually	O
-abolish	O
-AF	O
-‐	O
-2	O
-activity	O
-,	O
-and	O
-yet	O
-still	O
-drive	O
-robust	O
-transcriptional	O
-activity	O
-through	O
-AF	O
-‐	O
-1	O
-,	O
-as	O
-demonstrated	O
-with	O
-the	O
-OBHS	O
-series	O
-.	O
-	O
-This	O
-finding	O
-can	O
-be	O
-explained	O
-by	O
-the	O
-fact	O
-that	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-contain	O
-distinct	O
-binding	O
-sites	O
-for	O
-interaction	O
-with	O
-AF	O
-‐	O
-1	O
-and	O
-AF	O
-‐	O
-2	O
-(	O
-McInerney	O
-et	O
-al	O
-,	O
-1996	O
-;	O
-Webb	O
-et	O
-al	O
-,	O
-1998	O
-),	O
-which	O
-allows	O
-ligands	O
-to	O
-nucleate	O
-ERα	O
-–	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-interaction	O
-through	O
-AF	O
-‐	O
-2	O
-,	O
-and	O
-reinforce	O
-this	O
-interaction	O
-with	O
-additional	O
-binding	O
-to	O
-AF	O
-‐	O
-1	O
-.	O
-	O
-Completely	O
-blocking	O
-AF	O
-‐	O
-2	O
-with	O
-an	O
-extended	O
-side	O
-chain	O
-or	O
-altering	O
-the	O
-shape	O
-of	O
-AF	O
-‐	O
-2	O
-changes	O
-the	O
-preference	O
-away	O
-from	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-for	O
-determining	O
-GREB1	O
-levels	O
-and	O
-proliferation	O
-of	O
-breast	O
-cancer	O
-cells	O
-.	O
-	O
-AF	O
-‐	O
-2	O
-blockade	O
-also	O
-allows	O
-AF	O
-‐	O
-1	O
-to	O
-function	O
-independently	O
-,	O
-which	O
-is	O
-important	O
-since	O
-AF	O
-‐	O
-1	O
-drives	O
-tissue	O
-‐	O
-selective	O
-effects	O
-in	O
-vivo	O
-.	O
-	O
-This	O
-was	O
-demonstrated	O
-with	O
-AF	O
-‐	O
-1	O
-knockout	O
-mice	O
-that	O
-show	O
-E2	O
-‐	O
-dependent	O
-vascular	O
-protection	O
-,	O
-but	O
-not	O
-uterine	O
-proliferation	O
-,	O
-thus	O
-highlighting	O
-the	O
-role	O
-of	O
-AF	O
-‐	O
-1	O
-in	O
-tissue	O
-‐	O
-selective	O
-or	O
-cell	O
-‐	O
-specific	O
-signaling	O
-(	O
-Billon	O
-‐	O
-Gales	O
-et	O
-al	O
-,	O
-2009	O
-;	O
-Abot	O
-et	O
-al	O
-,	O
-2013	O
-).	O
-	O
-Here	O
-,	O
-we	O
-examined	O
-many	O
-LBD	O
-structures	O
-and	O
-tested	O
-several	O
-variables	O
-that	O
-were	O
-not	O
-predictive	O
-,	O
-including	O
-ERβ	O
-activity	O
-,	O
-the	O
-strength	O
-of	O
-AF	O
-‐	O
-1	O
-signaling	O
-,	O
-and	O
-NCOA3	O
-occupancy	O
-at	O
-the	O
-GREB1	O
-gene	O
-.	O
-	O
-Similarly	O
-,	O
-we	O
-visualized	O
-structures	O
-to	O
-identify	O
-patterns	O
-.	O
-	O
-For	O
-example	O
-,	O
-phage	O
-display	O
-was	O
-used	O
-to	O
-identify	O
-the	O
-androgen	O
-receptor	O
-interactome	O
-,	O
-which	O
-was	O
-cloned	O
-into	O
-an	O
-M2H	O
-library	O
-and	O
-used	O
-to	O
-identify	O
-clusters	O
-of	O
-ligand	O
-‐	O
-selective	O
-interactions	O
-(	O
-Norris	O
-et	O
-al	O
-,	O
-2009	O
-).	O
-	O
-Also	O
-,	O
-we	O
-have	O
-used	O
-siRNA	O
-screening	O
-to	O
-identify	O
-a	O
-number	O
-of	O
-coregulators	O
-required	O
-for	O
-ERα	O
-‐	O
-mediated	O
-repression	O
-of	O
-the	O
-IL	O
-‐	O
-6	O
-gene	O
-(	O
-Nwachukwu	O
-et	O
-al	O
-,	O
-2014	O
-).	O
-	O
-If	O
-we	O
-calculated	O
-inter	O
-‐	O
-atomic	O
-distance	O
-matrices	O
-containing	O
-4	O
-,	O
-000	O
-atoms	O
-per	O
-structure	O
-×	O
-76	O
-ligand	O
-–	O
-receptor	O
-complexes	O
-,	O
-we	O
-would	O
-have	O
-3	O
-×	O
-105	O
-predictions	O
-.	O
-	O
-We	O
-have	O
-identified	O
-atomic	O
-vectors	O
-for	O
-the	O
-OBHS	O
-‐	O
-N	O
-and	O
-triaryl	O
-‐	O
-ethylene	O
-classes	O
-that	O
-predict	O
-ligand	O
-response	O
-(	O
-Fig	O
-5E	O
-and	O
-F	O
-).	O
-	O
-Indeed	O
-,	O
-the	O
-most	O
-anti	O
-‐	O
-proliferative	O
-compound	O
-in	O
-the	O
-OBHS	O
-‐	O
-N	O
-series	O
-had	O
-a	O
-fulvestrant	O
-‐	O
-like	O
-profile	O
-across	O
-a	O
-battery	O
-of	O
-assays	O
-(	O
-S	O
-.	O
-Srinivasan	O
-et	O
-al	O
-,	O
-in	O
-preparation	O
-).	O
-	O
-Secondly	O
-,	O
-our	O
-finding	O
-that	O
-WAY	O
-‐	O
-C	O
-compounds	O
-do	O
-not	O
-rely	O
-of	O
-AF	O
-‐	O
-1	O
-for	O
-signaling	O
-efficacy	O
-may	O
-derive	O
-from	O
-the	O
-slight	O
-contacts	O
-with	O
-h12	O
-observed	O
-in	O
-crystal	O
-structures	O
-(	O
-Figs	O
-3B	O
-and	O
-5H	O
-),	O
-unlike	O
-other	O
-compounds	O
-in	O
-cluster	O
-1	O
-that	O
-dislocate	O
-h11	O
-and	O
-rely	O
-on	O
-AF	O
-‐	O
-1	O
-for	O
-signaling	O
-efficacy	O
-(	O
-Figs	O
-3B	O
-and	O
-5C	O
-,	O
-and	O
-EV5B	O
-).	O
-	O
-Some	O
-of	O
-these	O
-ligands	O
-altered	O
-the	O
-shape	O
-of	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-by	O
-perturbing	O
-the	O
-h3	O
-–	O
-h12	O
-interface	O
-,	O
-thus	O
-providing	O
-a	O
-route	O
-to	O
-new	O
-SERM	O
-‐	O
-like	O
-activity	O
-profiles	O
-by	O
-combining	O
-indirect	O
-and	O
-direct	O
-modulation	O
-of	O
-receptor	O
-structure	O
-.	O
-	O
-Incorporation	O
-of	O
-statistical	O
-approaches	O
-to	O
-understand	O
-relationships	O
-between	O
-structure	O
-and	O
-signaling	O
-variables	O
-moves	O
-us	O
-toward	O
-predictive	O
-models	O
-for	O
-complex	O
-ERα	O
-‐	O
-mediated	O
-responses	O
-such	O
-as	O
-in	O
-vivo	O
-uterine	O
-proliferation	O
-or	O
-tumor	O
-growth	O
-,	O
-and	O
-more	O
-generally	O
-toward	O
-structure	O
-‐	O
-based	O
-design	O
-for	O
-other	O
-allosteric	O
-drug	O
-targets	O
-including	O
-GPCRs	O
-and	O
-other	O
-nuclear	O
-receptors	O
-.	O
-	O
-Investigation	O
-of	O
-the	O
-Interaction	O
-between	O
-Cdc42	O
-and	O
-Its	O
-Effector	O
-TOCA1	O
-	O
-Transducer	O
-of	O
-Cdc42	O
--	O
-dependent	O
-actin	O
-assembly	O
-protein	O
-1	O
-(	O
-TOCA1	O
-)	O
-is	O
-an	O
-effector	O
-of	O
-the	O
-Rho	O
-family	O
-small	O
-G	O
-protein	O
-Cdc42	O
-.	O
-	O
-It	O
-contains	O
-a	O
-membrane	O
--	O
-deforming	O
-F	O
--	O
-BAR	O
-domain	O
-as	O
-well	O
-as	O
-a	O
-Src	O
-homology	O
-3	O
-(	O
-SH3	O
-)	O
-domain	O
-and	O
-a	O
-G	O
-protein	O
--	O
-binding	O
-homology	O
-region	O
-1	O
-(	O
-HR1	O
-)	O
-domain	O
-.	O
-	O
-TOCA1	O
-binding	O
-to	O
-Cdc42	O
-leads	O
-to	O
-actin	O
-rearrangements	O
-,	O
-which	O
-are	O
-thought	O
-to	O
-be	O
-involved	O
-in	O
-processes	O
-such	O
-as	O
-endocytosis	O
-,	O
-filopodia	O
-formation	O
-,	O
-and	O
-cell	O
-migration	O
-.	O
-	O
-We	O
-have	O
-solved	O
-the	O
-structure	O
-of	O
-the	O
-HR1	O
-domain	O
-of	O
-TOCA1	O
-,	O
-providing	O
-the	O
-first	O
-structural	O
-data	O
-for	O
-this	O
-protein	O
-.	O
-	O
-We	O
-have	O
-found	O
-that	O
-the	O
-TOCA1	O
-HR1	O
-,	O
-like	O
-the	O
-closely	O
-related	O
-CIP4	O
-HR1	O
-,	O
-has	O
-interesting	O
-structural	O
-features	O
-that	O
-are	O
-not	O
-observed	O
-in	O
-other	O
-HR1	O
-domains	O
-.	O
-	O
-We	O
-have	O
-also	O
-investigated	O
-the	O
-binding	O
-of	O
-the	O
-TOCA	O
-HR1	O
-domain	O
-to	O
-Cdc42	O
-and	O
-the	O
-potential	O
-ternary	O
-complex	O
-between	O
-Cdc42	O
-and	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-regions	O
-of	O
-TOCA1	O
-and	O
-a	O
-member	O
-of	O
-the	O
-Wiskott	O
--	O
-Aldrich	O
-syndrome	O
-protein	O
-family	O
-,	O
-N	O
--	O
-WASP	O
-.	O
-	O
-TOCA1	O
-binds	O
-Cdc42	O
-with	O
-micromolar	O
-affinity	O
-,	O
-in	O
-contrast	O
-to	O
-the	O
-nanomolar	O
-affinity	O
-of	O
-the	O
-N	O
--	O
-WASP	O
-G	O
-protein	O
--	O
-binding	O
-region	O
-for	O
-Cdc42	O
-.	O
-	O
-NMR	O
-experiments	O
-show	O
-that	O
-the	O
-Cdc42	O
--	O
-binding	O
-domain	O
-from	O
-N	O
--	O
-WASP	O
-is	O
-able	O
-to	O
-displace	O
-TOCA1	O
-HR1	O
-from	O
-Cdc42	O
-,	O
-whereas	O
-the	O
-N	O
--	O
-WASP	O
-domain	O
-but	O
-not	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-inhibits	O
-actin	O
-polymerization	O
-.	O
-	O
-This	O
-suggests	O
-that	O
-TOCA1	O
-binding	O
-to	O
-Cdc42	O
-is	O
-an	O
-early	O
-step	O
-in	O
-the	O
-Cdc42	O
--	O
-dependent	O
-pathways	O
-that	O
-govern	O
-actin	O
-dynamics	O
-,	O
-and	O
-the	O
-differential	O
-binding	O
-affinities	O
-of	O
-the	O
-effectors	O
-facilitate	O
-a	O
-handover	O
-from	O
-TOCA1	O
-to	O
-N	O
--	O
-WASP	O
-,	O
-which	O
-can	O
-then	O
-drive	O
-recruitment	O
-of	O
-the	O
-actin	O
--	O
-modifying	O
-machinery	O
-.	O
-	O
-The	O
-Ras	O
-superfamily	O
-of	O
-small	O
-GTPases	O
-comprises	O
-over	O
-150	O
-members	O
-that	O
-regulate	O
-a	O
-multitude	O
-of	O
-cellular	O
-processes	O
-in	O
-eukaryotes	O
-.	O
-	O
-The	O
-superfamily	O
-can	O
-be	O
-divided	O
-into	O
-five	O
-families	O
-based	O
-on	O
-structural	O
-and	O
-functional	O
-similarities	O
-:	O
-Ras	O
-,	O
-Rho	O
-,	O
-Rab	O
-,	O
-Arf	O
-,	O
-and	O
-Ran	O
-.	O
-	O
-All	O
-members	O
-share	O
-a	O
-well	O
-defined	O
-core	O
-structure	O
-of	O
-∼	O
-20	O
-kDa	O
-known	O
-as	O
-the	O
-G	O
-domain	O
-,	O
-which	O
-is	O
-responsible	O
-for	O
-guanine	O
-nucleotide	O
-binding	O
-.	O
-	O
-These	O
-molecular	O
-switches	O
-cycle	O
-between	O
-active	O
-,	O
-GTP	O
--	O
-bound	O
-,	O
-and	O
-inactive	O
-,	O
-GDP	O
--	O
-bound	O
-,	O
-states	O
-with	O
-the	O
-help	O
-of	O
-auxiliary	O
-proteins	O
-.	O
-	O
-The	O
-guanine	O
-nucleotide	O
-exchange	O
-factors	O
-mediate	O
-formation	O
-of	O
-the	O
-active	O
-state	O
-by	O
-promoting	O
-the	O
-dissociation	O
-of	O
-GDP	O
-,	O
-allowing	O
-GTP	O
-to	O
-bind	O
-.	O
-	O
-The	O
-GTPase	O
--	O
-activating	O
-proteins	O
-stimulate	O
-the	O
-rate	O
-of	O
-intrinsic	O
-GTP	O
-hydrolysis	O
-,	O
-mediating	O
-the	O
-return	O
-to	O
-the	O
-inactive	O
-state	O
-(	O
-reviewed	O
-in	O
-Ref	O
-.).	O
-	O
-The	O
-overall	O
-conformation	O
-of	O
-small	O
-G	O
-proteins	O
-in	O
-the	O
-active	O
-and	O
-inactive	O
-states	O
-is	O
-similar	O
-,	O
-but	O
-they	O
-differ	O
-significantly	O
-in	O
-two	O
-main	O
-regions	O
-known	O
-as	O
-switch	O
-I	O
-and	O
-switch	O
-II	O
-.	O
-	O
-These	O
-regions	O
-are	O
-responsible	O
-for	O
-“	O
-sensing	O
-”	O
-the	O
-nucleotide	O
-state	O
-,	O
-with	O
-the	O
-GTP	O
--	O
-bound	O
-state	O
-showing	O
-greater	O
-rigidity	O
-and	O
-the	O
-GDP	O
--	O
-bound	O
-state	O
-adopting	O
-a	O
-more	O
-relaxed	O
-conformation	O
-(	O
-reviewed	O
-in	O
-Ref	O
-.).	O
-	O
-In	O
-the	O
-active	O
-state	O
-,	O
-G	O
-proteins	O
-bind	O
-to	O
-an	O
-array	O
-of	O
-downstream	O
-effectors	O
-,	O
-through	O
-which	O
-they	O
-exert	O
-their	O
-extensive	O
-roles	O
-within	O
-the	O
-cell	O
-.	O
-	O
-The	O
-structures	O
-of	O
-more	O
-than	O
-60	O
-small	O
-G	O
-protein	O
-·	O
-effector	O
-complexes	O
-have	O
-been	O
-solved	O
-,	O
-and	O
-,	O
-not	O
-surprisingly	O
-,	O
-the	O
-switch	O
-regions	O
-have	O
-been	O
-implicated	O
-in	O
-a	O
-large	O
-proportion	O
-of	O
-the	O
-G	O
-protein	O
--	O
-effector	O
-interactions	O
-(	O
-reviewed	O
-in	O
-Ref	O
-.).	O
-	O
-However	O
-,	O
-because	O
-each	O
-of	O
-the	O
-150	O
-members	O
-of	O
-the	O
-superfamily	O
-interacts	O
-with	O
-multiple	O
-effectors	O
-,	O
-there	O
-are	O
-still	O
-a	O
-huge	O
-number	O
-of	O
-known	O
-G	O
-protein	O
--	O
-effector	O
-interactions	O
-that	O
-have	O
-not	O
-yet	O
-been	O
-studied	O
-structurally	O
-.	O
-	O
-The	O
-Rho	O
-family	O
-comprises	O
-20	O
-members	O
-,	O
-of	O
-which	O
-three	O
-,	O
-RhoA	O
-,	O
-Rac1	O
-,	O
-and	O
-Cdc42	O
-,	O
-have	O
-been	O
-relatively	O
-well	O
-studied	O
-.	O
-	O
-RhoA	O
-acts	O
-to	O
-rearrange	O
-existing	O
-actin	O
-structures	O
-to	O
-form	O
-stress	O
-fibers	O
-,	O
-whereas	O
-Rac1	O
-and	O
-Cdc42	O
-promote	O
-de	O
-novo	O
-actin	O
-polymerization	O
-to	O
-form	O
-lamellipodia	O
-and	O
-filopodia	O
-,	O
-respectively	O
-.	O
-	O
-A	O
-number	O
-of	O
-RhoA	O
-and	O
-Rac1	O
-effector	O
-proteins	O
-,	O
-including	O
-the	O
-formins	O
-and	O
-members	O
-of	O
-the	O
-protein	O
-kinase	O
-C	O
--	O
-related	O
-kinase	O
-(	O
-PRK	O
-)	O
-6	O
-family	O
-,	O
-along	O
-with	O
-Cdc42	O
-effectors	O
-,	O
-including	O
-the	O
-Wiskott	O
--	O
-Aldrich	O
-syndrome	O
-(	O
-WASP	O
-)	O
-family	O
-and	O
-the	O
-transducer	O
-of	O
-Cdc42	O
--	O
-dependent	O
-actin	O
-assembly	O
-(	O
-TOCA	O
-)	O
-family	O
-,	O
-have	O
-also	O
-been	O
-linked	O
-to	O
-the	O
-pathways	O
-that	O
-govern	O
-cytoskeletal	O
-dynamics	O
-.	O
-	O
-Cdc42	O
-effectors	O
-,	O
-TOCA1	O
-and	O
-the	O
-ubiquitously	O
-expressed	O
-member	O
-of	O
-the	O
-WASP	O
-family	O
-,	O
-N	O
--	O
-WASP	O
-,	O
-have	O
-been	O
-implicated	O
-in	O
-the	O
-regulation	O
-of	O
-actin	O
-polymerization	O
-downstream	O
-of	O
-Cdc42	O
-and	O
-phosphatidylinositol	O
-4	O
-,	O
-5	O
--	O
-bisphosphate	O
-(	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-).	O
-	O
-N	O
--	O
-WASP	O
-exists	O
-in	O
-an	O
-autoinhibited	O
-conformation	O
-,	O
-which	O
-is	O
-released	O
-upon	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-and	O
-Cdc42	O
-binding	O
-or	O
-by	O
-other	O
-factors	O
-,	O
-such	O
-as	O
-phosphorylation	O
-.	O
-	O
-Following	O
-their	O
-release	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-regions	O
-of	O
-N	O
--	O
-WASP	O
-are	O
-free	O
-to	O
-interact	O
-with	O
-G	O
--	O
-actin	O
-and	O
-a	O
-known	O
-nucleator	O
-of	O
-actin	O
-assembly	O
-,	O
-the	O
-Arp2	O
-/	O
-3	O
-complex	O
-.	O
-	O
-The	O
-importance	O
-of	O
-TOCA1	O
-in	O
-actin	O
-polymerization	O
-has	O
-been	O
-demonstrated	O
-in	O
-a	O
-range	O
-of	O
-in	O
-vitro	O
-and	O
-in	O
-vivo	O
-studies	O
-,	O
-but	O
-the	O
-exact	O
-role	O
-of	O
-TOCA1	O
-in	O
-the	O
-many	O
-pathways	O
-involving	O
-actin	O
-assembly	O
-remains	O
-unclear	O
-.	O
-	O
-The	O
-most	O
-widely	O
-studied	O
-role	O
-of	O
-TOCA1	O
-is	O
-in	O
-membrane	O
-invagination	O
-and	O
-endocytosis	O
-,	O
-although	O
-it	O
-has	O
-also	O
-been	O
-implicated	O
-in	O
-filopodia	O
-formation	O
-,	O
-neurite	O
-elongation	O
-,	O
-transcriptional	O
-reprogramming	O
-via	O
-nuclear	O
-actin	O
-,	O
-and	O
-interaction	O
-with	O
-ZO	O
--	O
-1	O
-at	O
-tight	O
-junctions	O
-.	O
-	O
-TOCA1	O
-comprises	O
-an	O
-N	O
--	O
-terminal	O
-F	O
--	O
-BAR	O
-domain	O
-,	O
-a	O
-central	O
-homology	O
-region	O
-1	O
-(	O
-HR1	O
-)	O
-domain	O
-,	O
-and	O
-a	O
-C	O
--	O
-terminal	O
-SH3	O
-domain	O
-.	O
-	O
-The	O
-F	O
--	O
-BAR	O
-domain	O
-is	O
-a	O
-known	O
-dimerization	O
-,	O
-membrane	O
--	O
-binding	O
-,	O
-and	O
-membrane	O
--	O
-deforming	O
-module	O
-found	O
-in	O
-a	O
-number	O
-of	O
-cell	O
-signaling	O
-proteins	O
-.	O
-	O
-The	O
-TOCA1	O
-SH3	O
-domain	O
-has	O
-many	O
-known	O
-binding	O
-partners	O
-,	O
-including	O
-N	O
--	O
-WASP	O
-and	O
-dynamin	O
-.	O
-	O
-The	O
-HR1	O
-domain	O
-has	O
-been	O
-directly	O
-implicated	O
-in	O
-the	O
-interaction	O
-between	O
-TOCA1	O
-and	O
-Cdc42	O
-,	O
-representing	O
-the	O
-first	O
-Cdc42	O
--	O
-HR1	O
-domain	O
-interaction	O
-to	O
-be	O
-identified	O
-.	O
-	O
-Other	O
-HR1	O
-domains	O
-studied	O
-so	O
-far	O
-,	O
-including	O
-those	O
-from	O
-the	O
-PRK	O
-family	O
-,	O
-have	O
-been	O
-found	O
-to	O
-bind	O
-their	O
-cognate	O
-Rho	O
-family	O
-G	O
-protein	O
--	O
-binding	O
-partner	O
-with	O
-high	O
-specificity	O
-and	O
-affinities	O
-in	O
-the	O
-nanomolar	O
-range	O
-.	O
-	O
-The	O
-structures	O
-of	O
-the	O
-PRK1	O
-HR1a	O
-domain	O
-in	O
-complex	O
-with	O
-RhoA	O
-and	O
-the	O
-HR1b	O
-domain	O
-in	O
-complex	O
-with	O
-Rac1	O
-show	O
-that	O
-the	O
-HR1	O
-domain	O
-comprises	O
-an	O
-anti	O
--	O
-parallel	O
-coiled	O
--	O
-coil	O
-that	O
-interacts	O
-with	O
-its	O
-G	O
-protein	O
-binding	O
-partner	O
-via	O
-both	O
-helices	O
-.	O
-	O
-Both	O
-of	O
-the	O
-G	O
-protein	O
-switch	O
-regions	O
-are	O
-involved	O
-in	O
-the	O
-interaction	O
-.	O
-	O
-The	O
-coiled	O
--	O
-coil	O
-fold	O
-is	O
-shared	O
-by	O
-the	O
-HR1	O
-domain	O
-of	O
-the	O
-TOCA	O
-family	O
-protein	O
-,	O
-CIP4	O
-,	O
-and	O
-,	O
-based	O
-on	O
-sequence	O
-homology	O
-,	O
-by	O
-TOCA1	O
-itself	O
-.	O
-	O
-These	O
-HR1	O
-domains	O
-,	O
-however	O
-,	O
-show	O
-specificity	O
-for	O
-Cdc42	O
-,	O
-rather	O
-than	O
-RhoA	O
-or	O
-Rac1	O
-.	O
-	O
-How	O
-different	O
-HR1	O
-domain	O
-proteins	O
-distinguish	O
-their	O
-specific	O
-G	O
-protein	O
-partners	O
-remains	O
-only	O
-partially	O
-understood	O
-,	O
-and	O
-structural	O
-characterization	O
-of	O
-a	O
-novel	O
-G	O
-protein	O
--	O
-HR1	O
-domain	O
-interaction	O
-would	O
-add	O
-to	O
-the	O
-growing	O
-body	O
-of	O
-information	O
-pertaining	O
-to	O
-these	O
-protein	O
-complexes	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-biological	O
-function	O
-of	O
-the	O
-interaction	O
-between	O
-TOCA1	O
-and	O
-Cdc42	O
-remains	O
-poorly	O
-understood	O
-,	O
-and	O
-so	O
-far	O
-there	O
-has	O
-been	O
-no	O
-biophysical	O
-or	O
-structural	O
-insight	O
-.	O
-	O
-The	O
-interactions	O
-of	O
-TOCA1	O
-and	O
-N	O
--	O
-WASP	O
-with	O
-Cdc42	O
-as	O
-well	O
-as	O
-with	O
-each	O
-other	O
-have	O
-raised	O
-questions	O
-as	O
-to	O
-whether	O
-the	O
-two	O
-Cdc42	O
-effectors	O
-can	O
-interact	O
-with	O
-a	O
-single	O
-molecule	O
-of	O
-Cdc42	O
-simultaneously	O
-.	O
-	O
-There	O
-is	O
-some	O
-evidence	O
-for	O
-a	O
-ternary	O
-complex	O
-between	O
-Cdc42	O
-,	O
-N	O
--	O
-WASP	O
-,	O
-and	O
-TOCA1	O
-,	O
-but	O
-there	O
-was	O
-no	O
-direct	O
-demonstration	O
-of	O
-simultaneous	O
-contacts	O
-between	O
-the	O
-two	O
-effectors	O
-and	O
-a	O
-single	O
-molecule	O
-of	O
-Cdc42	O
-.	O
-	O
-Nonetheless	O
-,	O
-the	O
-substantial	O
-difference	O
-between	O
-the	O
-structures	O
-of	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-regions	O
-of	O
-the	O
-two	O
-effectors	O
-is	O
-intriguing	O
-and	O
-implies	O
-that	O
-they	O
-bind	O
-to	O
-Cdc42	O
-quite	O
-differently	O
-,	O
-providing	O
-motivation	O
-for	O
-investigating	O
-the	O
-possibility	O
-that	O
-Cdc42	O
-can	O
-bind	O
-both	O
-effectors	O
-concurrently	O
-.	O
-	O
-WASP	O
-interacts	O
-with	O
-Cdc42	O
-via	O
-a	O
-conserved	O
-,	O
-unstructured	O
-binding	O
-motif	O
-known	O
-as	O
-the	O
-Cdc42	O
--	O
-and	O
-Rac	O
--	O
-interactive	O
-binding	O
-region	O
-(	O
-CRIB	O
-),	O
-which	O
-forms	O
-an	O
-intermolecular	O
-β	O
--	O
-sheet	O
-,	O
-expanding	O
-the	O
-anti	O
--	O
-parallel	O
-β2	O
-and	O
-β3	O
-strands	O
-of	O
-Cdc42	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-TOCA	O
-family	O
-proteins	O
-are	O
-thought	O
-to	O
-interact	O
-via	O
-the	O
-HR1	O
-domain	O
-,	O
-which	O
-may	O
-form	O
-a	O
-triple	O
-coiled	O
--	O
-coil	O
-with	O
-switch	O
-II	O
-of	O
-Rac1	O
-,	O
-like	O
-the	O
-HR1b	O
-domain	O
-of	O
-PRK1	O
-.	O
-	O
-Here	O
-,	O
-we	O
-present	O
-the	O
-solution	O
-NMR	O
-structure	O
-of	O
-the	O
-HR1	O
-domain	O
-of	O
-TOCA1	O
-,	O
-providing	O
-the	O
-first	O
-structural	O
-data	O
-for	O
-this	O
-protein	O
-.	O
-	O
-We	O
-also	O
-present	O
-data	O
-pertaining	O
-to	O
-binding	O
-of	O
-the	O
-TOCA	O
-HR1	O
-domain	O
-to	O
-Cdc42	O
-,	O
-which	O
-is	O
-the	O
-first	O
-biophysical	O
-description	O
-of	O
-an	O
-HR1	O
-domain	O
-binding	O
-this	O
-particular	O
-Rho	O
-family	O
-small	O
-G	O
-protein	O
-.	O
-	O
-Finally	O
-,	O
-we	O
-investigate	O
-the	O
-potential	O
-ternary	O
-complex	O
-between	O
-Cdc42	O
-and	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-regions	O
-of	O
-TOCA1	O
-and	O
-N	O
--	O
-WASP	O
-,	O
-contributing	O
-to	O
-our	O
-understanding	O
-of	O
-G	O
-protein	O
--	O
-effector	O
-interactions	O
-as	O
-well	O
-as	O
-the	O
-roles	O
-of	O
-Cdc42	O
-,	O
-N	O
--	O
-WASP	O
-,	O
-and	O
-TOCA1	O
-in	O
-the	O
-pathways	O
-that	O
-govern	O
-actin	O
-dynamics	O
-.	O
-	O
-Cdc42	O
--	O
-TOCA1	O
-Binding	O
-	O
-TOCA1	O
-was	O
-identified	O
-in	O
-Xenopus	O
-extracts	O
-as	O
-a	O
-protein	O
-necessary	O
-for	O
-Cdc42	O
--	O
-dependent	O
-actin	O
-assembly	O
-and	O
-was	O
-shown	O
-to	O
-bind	O
-to	O
-Cdc42	O
-·	O
-GTPγS	O
-but	O
-not	O
-to	O
-Cdc42	O
-·	O
-GDP	O
-or	O
-to	O
-Rac1	O
-and	O
-RhoA	O
-.	O
-Given	O
-its	O
-homology	O
-to	O
-other	O
-Rho	O
-family	O
-binding	O
-modules	O
-,	O
-it	O
-is	O
-likely	O
-that	O
-the	O
-HR1	O
-domain	O
-of	O
-TOCA1	O
-is	O
-sufficient	O
-to	O
-bind	O
-Cdc42	O
-.	O
-	O
-The	O
-C	O
-.	O
-elegans	O
-TOCA1	O
-orthologues	O
-also	O
-bind	O
-to	O
-Cdc42	O
-via	O
-their	O
-consensus	O
-HR1	O
-domain	O
-.	O
-	O
-The	O
-HR1	O
-domains	O
-from	O
-the	O
-PRK	O
-family	O
-bind	O
-their	O
-G	O
-protein	O
-partners	O
-with	O
-a	O
-high	O
-affinity	O
-,	O
-exhibiting	O
-a	O
-range	O
-of	O
-submicromolar	O
-dissociation	O
-constants	O
-(	O
-Kd	O
-)	O
-as	O
-low	O
-as	O
-26	O
-nm	O
-.	O
-	O
-A	O
-Kd	O
-in	O
-the	O
-nanomolar	O
-range	O
-was	O
-therefore	O
-expected	O
-for	O
-the	O
-interaction	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-with	O
-Cdc42	O
-.	O
-	O
-We	O
-generated	O
-an	O
-X	O
-.	O
-tropicalis	O
-TOCA1	O
-HR1	O
-domain	O
-construct	O
-encompassing	O
-residues	O
-330	O
-–	O
-426	O
-.	O
-	O
-This	O
-region	O
-comprises	O
-the	O
-complete	O
-HR1	O
-domain	O
-based	O
-on	O
-secondary	O
-structure	O
-predictions	O
-and	O
-sequence	O
-alignments	O
-with	O
-another	O
-TOCA	O
-family	O
-member	O
-,	O
-CIP4	O
-,	O
-whose	O
-structure	O
-has	O
-been	O
-determined	O
-.	O
-	O
-The	O
-interaction	O
-between	O
-[	O
-3H	O
-]	O
-GTP	O
-·	O
-Cdc42	O
-and	O
-a	O
-C	O
--	O
-terminally	O
-His	O
--	O
-tagged	O
-TOCA1	O
-HR1	O
-domain	O
-construct	O
-was	O
-investigated	O
-using	O
-SPA	O
-.	O
-	O
-The	O
-binding	O
-isotherm	O
-for	O
-the	O
-interaction	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-1A	O
-,	O
-together	O
-with	O
-the	O
-Cdc42	O
--	O
-PAK	O
-interaction	O
-as	O
-a	O
-positive	O
-control	O
-.	O
-	O
-The	O
-binding	O
-of	O
-TOCA1	O
-HR1	O
-to	O
-Cdc42	O
-was	O
-unexpectedly	O
-weak	O
-,	O
-with	O
-a	O
-Kd	O
-of	O
->	O
-1	O
-μm	O
-.	O
-	O
-It	O
-was	O
-not	O
-possible	O
-to	O
-estimate	O
-the	O
-Kd	O
-more	O
-accurately	O
-using	O
-direct	O
-SPA	O
-experiments	O
-,	O
-because	O
-saturation	O
-could	O
-not	O
-be	O
-reached	O
-due	O
-to	O
-nonspecific	O
-signal	O
-at	O
-higher	O
-protein	O
-concentrations	O
-.	O
-	O
-The	O
-TOCA1	O
-HR1	O
--	O
-Cdc42	O
-interaction	O
-is	O
-low	O
-affinity	O
-.	O
-	O
-A	O
-,	O
-curves	O
-derived	O
-from	O
-direct	O
-binding	O
-assays	O
-in	O
-which	O
-the	O
-indicated	O
-concentrations	O
-of	O
-Cdc42Δ7Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-were	O
-incubated	O
-with	O
-30	O
-nm	O
-GST	B-mutant
--	I-mutant
-PAK	I-mutant
-or	O
-HR1	B-mutant
--	I-mutant
-His6	I-mutant
-in	O
-SPAs	O
-.	O
-	O
-The	O
-SPA	O
-signal	O
-was	O
-corrected	O
-by	O
-subtraction	O
-of	O
-control	O
-data	O
-with	O
-no	O
-GST	B-mutant
--	I-mutant
-PAK	I-mutant
-or	O
-HR1	B-mutant
--	I-mutant
-His6	I-mutant
-.	O
-	O
-The	O
-data	O
-were	O
-fitted	O
-to	O
-a	O
-binding	O
-isotherm	O
-to	O
-give	O
-an	O
-apparent	O
-Kd	O
-and	O
-are	O
-expressed	O
-as	O
-a	O
-percentage	O
-of	O
-the	O
-maximum	O
-signal	O
-;	O
-B	O
-and	O
-C	O
-,	O
-competition	O
-SPA	O
-experiments	O
-were	O
-carried	O
-out	O
-with	O
-the	O
-indicated	O
-concentrations	O
-of	O
-ACK	O
-GBD	O
-(	O
-B	O
-)	O
-or	O
-HR1	O
-domain	O
-(	O
-C	O
-)	O
-titrated	O
-into	O
-30	O
-nm	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-and	O
-either	O
-30	O
-nm	O
-Cdc42Δ7Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-or	O
-full	O
--	O
-length	O
-Cdc42Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-.	O
-	O
-The	O
-Kd	O
-values	O
-derived	O
-for	O
-the	O
-ACK	O
-GBD	O
-with	O
-Cdc42Δ7	B-mutant
-and	O
-full	O
--	O
-length	O
-Cdc42	O
-were	O
-0	O
-.	O
-032	O
-±	O
-0	O
-.	O
-01	O
-and	O
-0	O
-.	O
-011	O
-±	O
-0	O
-.	O
-01	O
-μm	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-Kd	O
-values	O
-derived	O
-for	O
-the	O
-TOCA1	O
-HR1	O
-with	O
-Cdc42Δ7	B-mutant
-and	O
-full	O
--	O
-length	O
-Cdc42	O
-were	O
-6	O
-.	O
-05	O
-±	O
-1	O
-.	O
-96	O
-and	O
-5	O
-.	O
-39	O
-±	O
-1	O
-.	O
-69	O
-μm	O
-,	O
-respectively	O
-.	O
-	O
-It	O
-was	O
-possible	O
-that	O
-the	O
-low	O
-affinity	O
-observed	O
-was	O
-due	O
-to	O
-negative	O
-effects	O
-of	O
-immobilization	O
-of	O
-the	O
-HR1	O
-domain	O
-,	O
-so	O
-other	O
-methods	O
-were	O
-employed	O
-,	O
-which	O
-utilized	O
-untagged	O
-proteins	O
-.	O
-	O
-Isothermal	O
-titration	O
-calorimetry	O
-was	O
-carried	O
-out	O
-,	O
-but	O
-no	O
-heat	O
-changes	O
-were	O
-observed	O
-at	O
-a	O
-range	O
-of	O
-concentrations	O
-and	O
-temperatures	O
-(	O
-data	O
-not	O
-shown	O
-),	O
-suggesting	O
-that	O
-the	O
-interaction	O
-is	O
-predominantly	O
-entropically	O
-driven	O
-.	O
-	O
-Other	O
-G	O
-protein	O
--	O
-HR1	O
-domain	O
-interactions	O
-have	O
-also	O
-failed	O
-to	O
-show	O
-heat	O
-changes	O
-in	O
-our	O
-hands	O
-.	O
-7	O
-Infrared	O
-interferometry	O
-with	O
-immobilized	O
-Cdc42	O
-was	O
-also	O
-attempted	O
-but	O
-was	O
-unsuccessful	O
-for	O
-both	O
-TOCA1	O
-HR1	O
-and	O
-for	O
-the	O
-positive	O
-control	O
-,	O
-ACK	O
-.	O
-	O
-The	O
-affinity	O
-was	O
-therefore	O
-determined	O
-using	O
-competition	O
-SPAs	O
-.	O
-	O
-A	O
-complex	O
-of	O
-a	O
-GST	O
-fusion	O
-of	O
-the	O
-GBD	O
-of	O
-ACK	O
-,	O
-which	O
-binds	O
-with	O
-a	O
-high	O
-affinity	O
-to	O
-Cdc42	O
-,	O
-with	O
-radiolabeled	O
-[	O
-3H	O
-]	O
-GTP	O
-·	O
-Cdc42	O
-was	O
-preformed	O
-,	O
-and	O
-the	O
-effect	O
-of	O
-increasing	O
-concentrations	O
-of	O
-untagged	O
-TOCA1	O
-HR1	O
-domain	O
-was	O
-examined	O
-.	O
-	O
-Competition	O
-of	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-GBD	O
-bound	O
-to	O
-[	O
-3H	O
-]	O
-GTP	O
-·	O
-Cdc42	O
-by	O
-free	O
-ACK	O
-GBD	O
-was	O
-used	O
-as	O
-a	O
-control	O
-and	O
-to	O
-establish	O
-the	O
-value	O
-of	O
-background	O
-counts	O
-when	O
-Cdc42	O
-is	O
-fully	O
-displaced	O
-.	O
-	O
-The	O
-data	O
-were	O
-fitted	O
-to	O
-a	O
-binding	O
-isotherm	O
-describing	O
-competition	O
-.	O
-	O
-Free	O
-ACK	O
-competed	O
-with	O
-itself	O
-with	O
-an	O
-affinity	O
-of	O
-32	O
-nm	O
-,	O
-similar	O
-to	O
-the	O
-value	O
-obtained	O
-by	O
-direct	O
-binding	O
-of	O
-23	O
-nm	O
-.	O
-	O
-The	O
-TOCA1	O
-HR1	O
-domain	O
-also	O
-fully	O
-competed	O
-with	O
-the	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-but	O
-bound	O
-with	O
-an	O
-affinity	O
-of	O
-6	O
-μm	O
-(	O
-Fig	O
-.	O
-1	O
-,	O
-B	O
-and	O
-C	O
-),	O
-in	O
-agreement	O
-with	O
-the	O
-low	O
-affinity	O
-observed	O
-in	O
-the	O
-direct	O
-binding	O
-experiments	O
-.	O
-	O
-The	O
-Cdc42	O
-construct	O
-used	O
-in	O
-the	O
-binding	O
-assays	O
-has	O
-seven	O
-residues	O
-deleted	O
-from	O
-the	O
-C	O
-terminus	O
-to	O
-facilitate	O
-purification	O
-.	O
-	O
-These	O
-residues	O
-are	O
-not	O
-generally	O
-required	O
-for	O
-G	O
-protein	O
--	O
-effector	O
-interactions	O
-,	O
-including	O
-the	O
-interaction	O
-between	O
-RhoA	O
-and	O
-the	O
-PRK1	O
-HR1a	O
-domain	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-C	O
-terminus	O
-of	O
-Rac1	O
-contains	O
-a	O
-polybasic	O
-sequence	O
-,	O
-which	O
-is	O
-crucial	O
-for	O
-Rac1	O
-binding	O
-to	O
-the	O
-HR1b	O
-domain	O
-from	O
-PRK1	O
-.	O
-	O
-As	O
-the	O
-observed	O
-affinity	O
-between	O
-TOCA1	O
-HR1	O
-and	O
-Cdc42	O
-was	O
-much	O
-lower	O
-than	O
-expected	O
-,	O
-we	O
-reasoned	O
-that	O
-the	O
-C	O
-terminus	O
-of	O
-Cdc42	O
-might	O
-be	O
-necessary	O
-for	O
-a	O
-high	O
-affinity	O
-interaction	O
-.	O
-	O
-The	O
-binding	O
-experiments	O
-were	O
-repeated	O
-with	O
-full	O
--	O
-length	O
-[	O
-3H	O
-]	O
-GTP	O
-·	O
-Cdc42	O
-,	O
-but	O
-the	O
-affinity	O
-of	O
-the	O
-HR1	O
-domain	O
-for	O
-full	O
--	O
-length	O
-Cdc42	O
-was	O
-similar	O
-to	O
-its	O
-affinity	O
-for	O
-truncated	O
-Cdc42	O
-(	O
-Kd	O
-≈	O
-5	O
-μm	O
-;	O
-Fig	O
-.	O
-1C	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-Cdc42	O
-is	O
-not	O
-required	O
-for	O
-maximal	O
-binding	O
-of	O
-TOCA1	O
-HR1	O
-.	O
-	O
-Another	O
-possible	O
-explanation	O
-for	O
-the	O
-low	O
-affinities	O
-observed	O
-was	O
-that	O
-the	O
-HR1	O
-domain	O
-alone	O
-is	O
-not	O
-sufficient	O
-for	O
-maximal	O
-binding	O
-of	O
-the	O
-TOCA	O
-proteins	O
-to	O
-Cdc42	O
-and	O
-that	O
-the	O
-other	O
-domains	O
-are	O
-required	O
-.	O
-	O
-Indeed	O
-,	O
-GST	O
-pull	O
--	O
-downs	O
-performed	O
-with	O
-in	O
-vitro	O
-translated	O
-human	O
-TOCA1	O
-fragments	O
-had	O
-suggested	O
-that	O
-residues	O
-N	O
--	O
-terminal	O
-to	O
-the	O
-HR1	O
-domain	O
-may	O
-be	O
-required	O
-to	O
-stabilize	O
-the	O
-HR1	O
-domain	O
-structure	O
-.	O
-	O
-Furthermore	O
-,	O
-both	O
-BAR	O
-and	O
-SH3	O
-domains	O
-have	O
-been	O
-implicated	O
-in	O
-interactions	O
-with	O
-small	O
-G	O
-proteins	O
-(	O
-e	O
-.	O
-g	O
-.	O
-the	O
-BAR	O
-domain	O
-of	O
-Arfaptin2	O
-binds	O
-to	O
-Rac1	O
-and	O
-Arl1	O
-),	O
-while	O
-an	O
-SH3	O
-domain	O
-mediates	O
-the	O
-interaction	O
-between	O
-Rac1	O
-and	O
-the	O
-guanine	O
-nucleotide	O
-exchange	O
-factor	O
-,	O
-β	O
--	O
-PIX	O
-.	O
-	O
-TOCA1	O
-dimerizes	O
-via	O
-its	O
-F	O
--	O
-BAR	O
-domain	O
-,	O
-which	O
-could	O
-also	O
-affect	O
-Cdc42	O
-binding	O
-,	O
-for	O
-example	O
-by	O
-presenting	O
-two	O
-HR1	O
-domains	O
-for	O
-Cdc42	O
-interactions	O
-.	O
-	O
-Various	O
-TOCA1	O
-fragments	O
-(	O
-Fig	O
-.	O
-2A	O
-)	O
-were	O
-therefore	O
-assessed	O
-for	O
-binding	O
-to	O
-full	O
--	O
-length	O
-Cdc42	O
-by	O
-direct	O
-SPA	O
-.	O
-	O
-The	O
-isolated	O
-F	O
--	O
-BAR	O
-domain	O
-showed	O
-no	O
-binding	O
-to	O
-full	O
--	O
-length	O
-Cdc42	O
-(	O
-Fig	O
-.	O
-2B	O
-).	O
-	O
-Full	O
--	O
-length	O
-TOCA1	O
-and	O
-ΔSH3	B-mutant
-TOCA1	O
-bound	O
-with	O
-micromolar	O
-affinity	O
-(	O
-Fig	O
-.	O
-2B	O
-),	O
-in	O
-a	O
-similar	O
-manner	O
-to	O
-the	O
-isolated	O
-HR1	O
-domain	O
-(	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-The	O
-HR1	B-mutant
--	I-mutant
-SH3	I-mutant
-protein	O
-could	O
-not	O
-be	O
-purified	O
-to	O
-homogeneity	O
-as	O
-a	O
-fusion	O
-protein	O
-,	O
-so	O
-it	O
-was	O
-assayed	O
-in	O
-competition	O
-assays	O
-after	O
-cleavage	O
-of	O
-the	O
-His	O
-tag	O
-.	O
-	O
-This	O
-construct	O
-competed	O
-with	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-GBD	O
-to	O
-give	O
-a	O
-similar	O
-affinity	O
-to	O
-the	O
-HR1	O
-domain	O
-alone	O
-(	O
-Kd	O
-=	O
-4	O
-.	O
-6	O
-±	O
-4	O
-μm	O
-;	O
-Fig	O
-.	O
-2C	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-these	O
-data	O
-suggest	O
-that	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-is	O
-sufficient	O
-for	O
-maximal	O
-binding	O
-and	O
-that	O
-this	O
-binding	O
-is	O
-of	O
-a	O
-relatively	O
-low	O
-affinity	O
-compared	O
-with	O
-many	O
-other	O
-Cdc42	O
-·	O
-effector	O
-complexes	O
-.	O
-	O
-The	O
-Cdc42	O
--	O
-HR1	O
-interaction	O
-is	O
-of	O
-low	O
-affinity	O
-in	O
-the	O
-context	O
-of	O
-full	O
--	O
-length	O
-protein	O
-and	O
-in	O
-TOCA1	O
-paralogues	O
-.	O
-	O
-A	O
-,	O
-diagram	O
-illustrating	O
-the	O
-TOCA1	O
-constructs	O
-assayed	O
-for	O
-Cdc42	O
-binding	O
-.	O
-	O
-Domain	O
-boundaries	O
-are	O
-derived	O
-from	O
-secondary	O
-structure	O
-predictions	O
-;	O
-B	O
-,	O
-binding	O
-curves	O
-derived	O
-from	O
-direct	O
-binding	O
-assays	O
-,	O
-in	O
-which	O
-the	O
-indicated	O
-concentrations	O
-of	O
-Cdc42Δ7Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-were	O
-incubated	O
-with	O
-30	O
-nm	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-or	O
-His	O
--	O
-tagged	O
-TOCA1	O
-constructs	O
-,	O
-as	O
-indicated	O
-,	O
-in	O
-SPAs	O
-.	O
-	O
-The	O
-SPA	O
-signal	O
-was	O
-corrected	O
-by	O
-subtraction	O
-of	O
-control	O
-data	O
-with	O
-no	O
-fusion	O
-protein	O
-.	O
-	O
-The	O
-data	O
-were	O
-fitted	O
-to	O
-a	O
-binding	O
-isotherm	O
-to	O
-give	O
-an	O
-apparent	O
-Kd	O
-and	O
-are	O
-expressed	O
-as	O
-a	O
-percentage	O
-of	O
-the	O
-maximum	O
-signal	O
-.	O
-	O
-C	O
-–	O
-E	O
-,	O
-representative	O
-examples	O
-of	O
-competition	O
-SPA	O
-experiments	O
-carried	O
-out	O
-with	O
-the	O
-indicated	O
-concentrations	O
-of	O
-the	O
-TOCA1	O
-HR1	B-mutant
--	I-mutant
-SH3	I-mutant
-construct	O
-titrated	O
-into	O
-30	O
-nm	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-and	O
-30	O
-nm	O
-Cdc42Δ7Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-(	O
-C	O
-)	O
-or	O
-HR1CIP4	O
-(	O
-D	O
-)	O
-or	O
-HR1FBP17	O
-(	O
-E	O
-)	O
-titrated	O
-into	O
-30	O
-nm	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-and	O
-30	O
-nm	O
-Cdc42FLQ61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-.	O
-	O
-The	O
-low	O
-affinity	O
-of	O
-the	O
-TOCA1	O
-HR1	O
--	O
-Cdc42	O
-interaction	O
-raised	O
-the	O
-question	O
-of	O
-whether	O
-the	O
-other	O
-known	O
-Cdc42	O
--	O
-binding	O
-TOCA	O
-family	O
-proteins	O
-,	O
-FBP17	O
-and	O
-CIP4	O
-,	O
-also	O
-bind	O
-weakly	O
-.	O
-	O
-The	O
-HR1	O
-domains	O
-from	O
-FBP17	O
-and	O
-CIP4	O
-were	O
-purified	O
-and	O
-assayed	O
-for	O
-Cdc42	O
-binding	O
-in	O
-competition	O
-SPAs	O
-,	O
-analogous	O
-to	O
-those	O
-carried	O
-out	O
-with	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-.	O
-	O
-The	O
-affinities	O
-of	O
-both	O
-the	O
-FBP17	O
-and	O
-CIP4	O
-HR1	O
-domains	O
-were	O
-also	O
-in	O
-the	O
-low	O
-micromolar	O
-range	O
-(	O
-10	O
-and	O
-5	O
-μm	O
-,	O
-respectively	O
-)	O
-(	O
-Fig	O
-.	O
-2	O
-,	O
-D	O
-and	O
-E	O
-),	O
-suggesting	O
-that	O
-low	O
-affinity	O
-interactions	O
-with	O
-Cdc42	O
-are	O
-a	O
-common	O
-feature	O
-within	O
-the	O
-TOCA	O
-family	O
-.	O
-	O
-Structure	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-Domain	O
-	O
-Because	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-was	O
-sufficient	O
-for	O
-maximal	O
-Cdc42	O
--	O
-binding	O
-,	O
-we	O
-used	O
-this	O
-construct	O
-for	O
-structural	O
-studies	O
-.	O
-	O
-Initial	O
-experiments	O
-were	O
-performed	O
-with	O
-TOCA1	O
-residues	O
-324	O
-–	O
-426	O
-,	O
-but	O
-we	O
-observed	O
-that	O
-the	O
-N	O
-terminus	O
-was	O
-cleaved	O
-during	O
-purification	O
-to	O
-yield	O
-a	O
-new	O
-N	O
-terminus	O
-at	O
-residue	O
-330	O
-(	O
-data	O
-not	O
-shown	O
-).	O
-	O
-We	O
-therefore	O
-engineered	O
-a	O
-construct	O
-comprising	O
-residues	O
-330	O
-–	O
-426	O
-to	O
-produce	O
-the	O
-minimal	O
-,	O
-stable	O
-HR1	O
-domain	O
-.	O
-	O
-2	O
-,	O
-778	O
-non	O
--	O
-degenerate	O
-NOE	O
-restraints	O
-were	O
-used	O
-in	O
-initial	O
-structure	O
-calculations	O
-(	O
-1	O
-,	O
-791	O
-unambiguous	O
-and	O
-987	O
-ambiguous	O
-),	O
-derived	O
-from	O
-three	O
--	O
-dimensional	O
-15N	O
--	O
-separated	O
-NOESY	O
-and	O
-13C	O
--	O
-separated	O
-NOESY	O
-experiments	O
-.	O
-	O
-There	O
-were	O
-1	O
-,	O
-845	O
-unambiguous	O
-NOEs	O
-and	O
-757	O
-ambiguous	O
-NOEs	O
-after	O
-eight	O
-iterations	O
-.	O
-	O
-100	O
-structures	O
-were	O
-calculated	O
-in	O
-the	O
-final	O
-iteration	O
-;	O
-the	O
-50	O
-lowest	O
-energy	O
-structures	O
-were	O
-water	O
--	O
-refined	O
-;	O
-and	O
-of	O
-these	O
-,	O
-the	O
-35	O
-lowest	O
-energy	O
-structures	O
-were	O
-analyzed	O
-.	O
-	O
-Table	O
-1	O
-indicates	O
-that	O
-the	O
-HR1	O
-domain	O
-structure	O
-is	O
-well	O
-defined	O
-by	O
-the	O
-NMR	O
-data	O
-.	O
-	O
-a	O
-<	O
-SA	O
->,	O
-the	O
-average	O
-root	O
-mean	O
-square	O
-deviations	O
-for	O
-the	O
-ensemble	O
-±	O
-S	O
-.	O
-D	O
-.	O
-	O
-b	O
-<	O
-SA	O
->	O
-c	O
-,	O
-values	O
-for	O
-the	O
-structure	O
-that	O
-is	O
-closest	O
-to	O
-the	O
-mean	O
-.	O
-	O
-The	O
-structure	O
-closest	O
-to	O
-the	O
-mean	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-3A	O
-.	O
-	O
-The	O
-two	O
-α	O
--	O
-helices	O
-of	O
-the	O
-HR1	O
-domain	O
-interact	O
-to	O
-form	O
-an	O
-anti	O
--	O
-parallel	O
-coiled	O
--	O
-coil	O
-with	O
-a	O
-slight	O
-left	O
--	O
-handed	O
-twist	O
-,	O
-reminiscent	O
-of	O
-the	O
-HR1	O
-domains	O
-of	O
-CIP4	O
-(	O
-PDB	O
-code	O
-2KE4	O
-)	O
-and	O
-PRK1	O
-(	O
-PDB	O
-codes	O
-1CXZ	O
-and	O
-1URF	O
-).	O
-	O
-A	O
-sequence	O
-alignment	O
-illustrating	O
-the	O
-secondary	O
-structure	O
-elements	O
-of	O
-the	O
-TOCA1	O
-and	O
-CIP4	O
-HR1	O
-domains	O
-and	O
-the	O
-HR1a	O
-and	O
-HR1b	O
-domains	O
-from	O
-PRK1	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-3B	O
-.	O
-	O
-The	O
-structure	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-.	O
-	O
-A	O
-,	O
-the	O
-backbone	O
-trace	O
-of	O
-the	O
-35	O
-lowest	O
-energy	O
-structures	O
-of	O
-the	O
-HR1	O
-domain	O
-overlaid	O
-with	O
-the	O
-structure	O
-closest	O
-to	O
-the	O
-mean	O
-is	O
-shown	O
-alongside	O
-a	O
-schematic	O
-representation	O
-of	O
-the	O
-structure	O
-closest	O
-to	O
-the	O
-mean	O
-.	O
-	O
-Flexible	O
-regions	O
-at	O
-the	O
-N	O
-and	O
-C	O
-termini	O
-(	O
-residues	O
-330	O
-–	O
-333	O
-and	O
-421	O
-–	O
-426	O
-)	O
-are	O
-omitted	O
-for	O
-clarity	O
-.	O
-	O
-B	O
-,	O
-a	O
-sequence	O
-alignment	O
-of	O
-the	O
-HR1	O
-domains	O
-from	O
-TOCA1	O
-,	O
-CIP4	O
-,	O
-and	O
-PRK1	O
-.	O
-	O
-The	O
-secondary	O
-structure	O
-was	O
-deduced	O
-using	O
-Stride	O
-,	O
-based	O
-on	O
-the	O
-Ramachandran	O
-angles	O
-,	O
-and	O
-is	O
-indicated	O
-as	O
-follows	O
-:	O
-gray	O
-,	O
-turn	O
-;	O
-yellow	O
-,	O
-α	O
--	O
-helix	O
-;	O
-blue	O
-,	O
-310	O
-helix	O
-;	O
-white	O
-,	O
-coil	O
-.	O
-	O
-C	O
-,	O
-a	O
-close	O
--	O
-up	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-region	O
-of	O
-TOCA1	O
-HR1	O
-,	O
-indicating	O
-some	O
-of	O
-the	O
-NOEs	O
-defining	O
-its	O
-position	O
-with	O
-respect	O
-to	O
-the	O
-two	O
-α	O
--	O
-helices	O
-.	O
-	O
-Dotted	O
-lines	O
-,	O
-NOE	O
-restraints	O
-.	O
-	O
-D	O
-,	O
-a	O
-close	O
--	O
-up	O
-of	O
-the	O
-interhelix	O
-loop	O
-region	O
-showing	O
-some	O
-of	O
-the	O
-contacts	O
-between	O
-the	O
-loop	O
-and	O
-helix	O
-1	O
-.	O
-	O
-In	O
-the	O
-HR1a	O
-domain	O
-of	O
-PRK1	O
-,	O
-a	O
-region	O
-N	O
--	O
-terminal	O
-to	O
-helix	O
-1	O
-forms	O
-a	O
-short	O
-α	O
--	O
-helix	O
-,	O
-which	O
-packs	O
-against	O
-both	O
-helices	O
-of	O
-the	O
-HR1	O
-domain	O
-.	O
-	O
-This	O
-region	O
-of	O
-TOCA1	O
-HR1	O
-(	O
-residues	O
-334	O
-–	O
-340	O
-)	O
-is	O
-well	O
-defined	O
-in	O
-the	O
-family	O
-of	O
-structures	O
-(	O
-Fig	O
-.	O
-3A	O
-)	O
-but	O
-does	O
-not	O
-form	O
-an	O
-α	O
--	O
-helix	O
-.	O
-	O
-It	O
-instead	O
-forms	O
-a	O
-series	O
-of	O
-turns	O
-,	O
-defined	O
-by	O
-NOE	O
-restraints	O
-observed	O
-between	O
-residues	O
-separated	O
-by	O
-one	O
-(	O
-residues	O
-332	O
-–	O
-334	O
-,	O
-333	O
-–	O
-335	O
-,	O
-etc	O
-.)	O
-or	O
-two	O
-(	O
-residues	O
-337	O
-–	O
-340	O
-)	O
-residues	O
-in	O
-the	O
-sequence	O
-and	O
-the	O
-φ	O
-and	O
-ψ	O
-angles	O
-,	O
-assessed	O
-using	O
-Stride	O
-.	O
-	O
-These	O
-turns	O
-cause	O
-the	O
-chain	O
-to	O
-reverse	O
-direction	O
-,	O
-allowing	O
-the	O
-N	O
--	O
-terminal	O
-segment	O
-(	O
-residues	O
-334	O
-–	O
-340	O
-)	O
-to	O
-contact	O
-both	O
-helices	O
-of	O
-the	O
-HR1	O
-domain	O
-.	O
-	O
-Long	O
-range	O
-NOEs	O
-were	O
-observed	O
-linking	O
-Leu	O
--	O
-334	O
-,	O
-Glu	O
--	O
-335	O
-,	O
-and	O
-Asp	O
--	O
-336	O
-with	O
-Trp	O
--	O
-413	O
-of	O
-helix	O
-2	O
-,	O
-Leu	O
--	O
-334	O
-with	O
-Lys	O
--	O
-409	O
-of	O
-helix	O
-2	O
-,	O
-and	O
-Phe	O
--	O
-337	O
-and	O
-Ser	O
--	O
-338	O
-with	O
-Arg	O
--	O
-345	O
-,	O
-Arg	O
--	O
-348	O
-,	O
-and	O
-Leu	O
--	O
-349	O
-of	O
-helix	O
-1	O
-.	O
-	O
-The	O
-two	O
-α	O
--	O
-helices	O
-of	O
-TOCA1	O
-HR1	O
-are	O
-separated	O
-by	O
-a	O
-long	O
-loop	O
-of	O
-10	O
-residues	O
-(	O
-residues	O
-380	O
-–	O
-389	O
-)	O
-that	O
-contains	O
-two	O
-short	O
-310	O
-helices	O
-(	O
-residues	O
-381	O
-–	O
-383	O
-and	O
-386	O
-–	O
-389	O
-).	O
-	O
-Interestingly	O
-,	O
-side	O
-chains	O
-of	O
-residues	O
-within	O
-the	O
-loop	O
-region	O
-point	O
-back	O
-toward	O
-helix	O
-1	O
-;	O
-for	O
-example	O
-,	O
-there	O
-are	O
-numerous	O
-distinct	O
-NOEs	O
-between	O
-the	O
-side	O
-chains	O
-of	O
-Asn	O
--	O
-380	O
-and	O
-Met	O
--	O
-383	O
-of	O
-the	O
-loop	O
-region	O
-and	O
-Tyr	O
--	O
-377	O
-and	O
-Val	O
--	O
-376	O
-of	O
-helix	O
-1	O
-(	O
-Fig	O
-.	O
-3D	O
-).	O
-	O
-The	O
-backbone	O
-NH	O
-and	O
-CHα	O
-groups	O
-of	O
-Gly	O
--	O
-384	O
-and	O
-Asp	O
--	O
-385	O
-also	O
-show	O
-NOEs	O
-with	O
-the	O
-side	O
-chain	O
-of	O
-Tyr	O
--	O
-377	O
-.	O
-	O
-Mapping	O
-the	O
-TOCA1	O
-and	O
-Cdc42	O
-Binding	O
-Interfaces	O
-	O
-The	O
-HR1TOCA1	O
--	O
-Cdc42	O
-interface	O
-was	O
-investigated	O
-using	O
-NMR	O
-spectroscopy	O
-.	O
-	O
-A	O
-series	O
-of	O
-15N	O
-HSQC	O
-experiments	O
-was	O
-recorded	O
-on	O
-15N	O
--	O
-labeled	O
-TOCA1	O
-HR1	O
-domain	O
-in	O
-the	O
-presence	O
-of	O
-increasing	O
-concentrations	O
-of	O
-unlabeled	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-to	O
-map	O
-the	O
-Cdc42	O
--	O
-binding	O
-surface	O
-.	O
-	O
-A	O
-comparison	O
-of	O
-the	O
-15N	O
-HSQC	O
-spectra	O
-of	O
-free	O
-HR1	O
-and	O
-HR1	O
-in	O
-the	O
-presence	O
-of	O
-excess	O
-Cdc42	O
-shows	O
-that	O
-although	O
-some	O
-peaks	O
-were	O
-shifted	O
-,	O
-several	O
-were	O
-much	O
-broader	O
-in	O
-the	O
-complex	O
-,	O
-and	O
-a	O
-considerable	O
-subset	O
-had	O
-disappeared	O
-(	O
-Fig	O
-.	O
-4A	O
-).	O
-	O
-This	O
-behavior	O
-cannot	O
-be	O
-explained	O
-by	O
-the	O
-increase	O
-in	O
-molecular	O
-mass	O
-(	O
-from	O
-12	O
-to	O
-33	O
-kDa	O
-)	O
-when	O
-Cdc42	O
-binds	O
-and	O
-is	O
-more	O
-likely	O
-to	O
-be	O
-due	O
-to	O
-conformational	O
-exchange	O
-.	O
-	O
-Overall	O
-chemical	O
-shift	O
-perturbations	O
-(	O
-CSPs	O
-)	O
-were	O
-calculated	O
-for	O
-each	O
-residue	O
-,	O
-whereas	O
-those	O
-that	O
-had	O
-disappeared	O
-were	O
-assigned	O
-a	O
-shift	O
-change	O
-of	O
-0	O
-.	O
-2	O
-(	O
-Fig	O
-.	O
-4B	O
-).	O
-	O
-A	O
-peak	O
-that	O
-disappeared	O
-or	O
-had	O
-a	O
-CSP	O
-above	O
-the	O
-mean	O
-CSP	O
-for	O
-the	O
-spectrum	O
-was	O
-considered	O
-to	O
-be	O
-significantly	O
-affected	O
-.	O
-	O
-Mapping	O
-the	O
-binding	O
-surface	O
-of	O
-Cdc42	O
-onto	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-.	O
-	O
-A	O
-,	O
-the	O
-15N	O
-HSQC	O
-of	O
-200	O
-μm	O
-TOCA1	O
-HR1	O
-domain	O
-is	O
-shown	O
-in	O
-the	O
-free	O
-form	O
-(	O
-black	O
-)	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-a	O
-4	O
--	O
-fold	O
-molar	O
-excess	O
-of	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-(	O
-red	O
-).	O
-	O
-B	O
-,	O
-CSPs	O
-were	O
-calculated	O
-as	O
-described	O
-under	O
-“	O
-Experimental	O
-Procedures	O
-”	O
-and	O
-are	O
-shown	O
-for	O
-backbone	O
-and	O
-side	O
-chain	O
-NH	O
-groups	O
-.	O
-	O
-The	O
-mean	O
-CSP	O
-is	O
-marked	O
-with	O
-a	O
-red	O
-line	O
-.	O
-	O
-Residues	O
-that	O
-disappeared	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-were	O
-assigned	O
-a	O
-CSP	O
-of	O
-0	O
-.	O
-2	O
-but	O
-were	O
-excluded	O
-when	O
-calculating	O
-the	O
-mean	O
-CSP	O
-and	O
-are	O
-indicated	O
-with	O
-open	O
-bars	O
-.	O
-	O
-Those	O
-that	O
-were	O
-not	O
-traceable	O
-due	O
-to	O
-spectral	O
-overlap	O
-were	O
-assigned	O
-a	O
-CSP	O
-of	O
-zero	O
-and	O
-are	O
-marked	O
-with	O
-an	O
-asterisk	O
-below	O
-the	O
-bar	O
-.	O
-	O
-Residues	O
-with	O
-affected	O
-side	O
-chain	O
-CSPs	O
-derived	O
-from	O
-13C	O
-HSQCs	O
-are	O
-marked	O
-with	O
-green	O
-asterisks	O
-above	O
-the	O
-bars	O
-.	O
-	O
-C	O
-,	O
-a	O
-schematic	O
-representation	O
-of	O
-the	O
-HR1	O
-domain	O
-.	O
-	O
-Residues	O
-with	O
-significantly	O
-affected	O
-backbone	O
-or	O
-side	O
-chain	O
-chemical	O
-shifts	O
-when	O
-Cdc42	O
-bound	O
-and	O
-that	O
-are	O
-buried	O
-are	O
-colored	O
-dark	O
-blue	O
-,	O
-whereas	O
-those	O
-that	O
-are	O
-solvent	O
--	O
-accessible	O
-are	O
-colored	O
-yellow	O
-.	O
-	O
-Residues	O
-with	O
-significantly	O
-affected	O
-backbone	O
-and	O
-side	O
-chain	O
-groups	O
-that	O
-are	O
-solvent	O
--	O
-accessible	O
-are	O
-colored	O
-red	O
-.	O
-	O
-A	O
-close	O
--	O
-up	O
-of	O
-the	O
-binding	O
-region	O
-is	O
-shown	O
-,	O
-with	O
-affected	O
-side	O
-chain	O
-heavy	O
-atoms	O
-shown	O
-as	O
-sticks	O
-.	O
-	O
-D	O
-,	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-region	O
-is	O
-marked	O
-in	O
-red	O
-onto	O
-structures	O
-of	O
-the	O
-HR1	O
-domains	O
-as	O
-indicated	O
-.	O
-	O
-15N	O
-HSQC	O
-shift	O
-mapping	O
-experiments	O
-report	O
-on	O
-changes	O
-to	O
-amide	O
-groups	O
-,	O
-which	O
-are	O
-mainly	O
-inaccessible	O
-because	O
-they	O
-are	O
-buried	O
-inside	O
-the	O
-helices	O
-and	O
-are	O
-involved	O
-in	O
-hydrogen	O
-bonds	O
-.	O
-	O
-Therefore	O
-,	O
-13C	O
-HSQC	O
-and	O
-methyl	O
--	O
-selective	O
-SOFAST	O
--	O
-HMQC	O
-experiments	O
-were	O
-also	O
-recorded	O
-on	O
-15N	O
-,	O
-13C	O
--	O
-labeled	O
-TOCA1	O
-HR1	O
-to	O
-yield	O
-more	O
-information	O
-on	O
-side	O
-chain	O
-involvement	O
-.	O
-	O
-Side	O
-chains	O
-whose	O
-CH	O
-groups	O
-disappeared	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-are	O
-marked	O
-on	O
-the	O
-graph	O
-in	O
-Fig	O
-.	O
-4B	O
-with	O
-green	O
-asterisks	O
-.	O
-	O
-TOCA1	O
-residues	O
-whose	O
-signals	O
-were	O
-affected	O
-by	O
-Cdc42	O
-binding	O
-were	O
-mapped	O
-onto	O
-the	O
-structure	O
-of	O
-TOCA1	O
-HR1	O
-(	O
-Fig	O
-.	O
-4C	O
-).	O
-	O
-The	O
-changes	O
-were	O
-localized	O
-to	O
-one	O
-end	O
-of	O
-the	O
-coiled	O
--	O
-coil	O
-,	O
-and	O
-the	O
-binding	O
-site	O
-appeared	O
-to	O
-include	O
-residues	O
-from	O
-both	O
-α	O
--	O
-helices	O
-and	O
-the	O
-loop	O
-region	O
-that	O
-joins	O
-them	O
-.	O
-	O
-The	O
-residues	O
-in	O
-the	O
-interhelical	O
-loop	O
-and	O
-helix	O
-1	O
-that	O
-contact	O
-each	O
-other	O
-(	O
-Fig	O
-.	O
-3D	O
-)	O
-show	O
-shift	O
-changes	O
-in	O
-their	O
-backbone	O
-NH	O
-and	O
-side	O
-chains	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-.	O
-	O
-For	O
-example	O
-,	O
-the	O
-side	O
-chain	O
-of	O
-Asn	O
--	O
-380	O
-and	O
-the	O
-backbones	O
-of	O
-Val	O
--	O
-376	O
-and	O
-Tyr	O
--	O
-377	O
-were	O
-significantly	O
-affected	O
-but	O
-are	O
-all	O
-buried	O
-in	O
-the	O
-free	O
-TOCA1	O
-HR1	O
-structure	O
-,	O
-indicating	O
-that	O
-local	O
-conformational	O
-changes	O
-in	O
-the	O
-loop	O
-may	O
-facilitate	O
-complex	O
-formation	O
-.	O
-	O
-The	O
-chemical	O
-shift	O
-mapping	O
-data	O
-indicate	O
-that	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-region	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-is	O
-broadly	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-CIP4	O
-and	O
-PRK1	O
-HR1	O
-domains	O
-(	O
-Figs	O
-.	O
-3B	O
-and	O
-4D	O
-).	O
-	O
-The	O
-corresponding	O
-15N	O
-and	O
-13C	O
-NMR	O
-experiments	O
-were	O
-also	O
-recorded	O
-on	O
-15N	O
--	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-or	O
-15N	O
-/	O
-13C	O
--	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-in	O
-the	O
-presence	O
-of	O
-unlabeled	O
-HR1	O
-domain	O
-.	O
-	O
-The	O
-overall	O
-CSP	O
-was	O
-calculated	O
-for	O
-each	O
-residue	O
-.	O
-	O
-As	O
-was	O
-the	O
-case	O
-when	O
-labeled	O
-HR1	O
-was	O
-observed	O
-,	O
-several	O
-peaks	O
-were	O
-shifted	O
-in	O
-the	O
-complex	O
-,	O
-but	O
-many	O
-disappeared	O
-,	O
-indicating	O
-exchange	O
-on	O
-an	O
-unfavorable	O
-,	O
-millisecond	O
-time	O
-scale	O
-(	O
-Fig	O
-.	O
-5A	O
-).	O
-	O
-Detailed	O
-side	O
-chain	O
-data	O
-could	O
-not	O
-be	O
-obtained	O
-for	O
-all	O
-residues	O
-due	O
-to	O
-spectral	O
-overlap	O
-,	O
-but	O
-constant	O
-time	O
-13C	O
-HSQC	O
-and	O
-methyl	O
--	O
-selective	O
-SOFAST	O
--	O
-HMQC	O
-experiments	O
-provided	O
-further	O
-information	O
-on	O
-certain	O
-well	O
-resolved	O
-side	O
-chains	O
-(	O
-marked	O
-with	O
-green	O
-asterisks	O
-in	O
-Fig	O
-.	O
-5B	O
-).	O
-	O
-Mapping	O
-the	O
-binding	O
-surface	O
-of	O
-the	O
-HR1	O
-domain	O
-onto	O
-Cdc42	O
-.	O
-	O
-A	O
-,	O
-the	O
-15N	O
-HSQC	O
-of	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-is	O
-shown	O
-in	O
-its	O
-free	O
-form	O
-(	O
-black	O
-)	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-excess	O
-TOCA1	O
-HR1	O
-domain	O
-(	O
-1	O
-:	O
-2	O
-.	O
-2	O
-,	O
-red	O
-).	O
-	O
-B	O
-,	O
-CSPs	O
-are	O
-shown	O
-for	O
-backbone	O
-NH	O
-groups	O
-.	O
-	O
-The	O
-red	O
-line	O
-indicates	O
-the	O
-mean	O
-CSP	O
-,	O
-plus	O
-one	O
-S	O
-.	O
-D	O
-.	O
-Residues	O
-that	O
-disappeared	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-were	O
-assigned	O
-a	O
-CSP	O
-of	O
-0	O
-.	O
-1	O
-and	O
-are	O
-indicated	O
-with	O
-open	O
-bars	O
-.	O
-	O
-Residues	O
-with	O
-disappeared	O
-peaks	O
-in	O
-13C	O
-HSQC	O
-experiments	O
-are	O
-marked	O
-on	O
-the	O
-chart	O
-with	O
-green	O
-asterisks	O
-.	O
-	O
-C	O
-,	O
-the	O
-residues	O
-with	O
-significantly	O
-affected	O
-backbone	O
-and	O
-side	O
-chain	O
-groups	O
-are	O
-highlighted	O
-on	O
-an	O
-NMR	O
-structure	O
-of	O
-free	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-;	O
-those	O
-that	O
-are	O
-buried	O
-are	O
-colored	O
-dark	O
-blue	O
-,	O
-whereas	O
-those	O
-that	O
-are	O
-solvent	O
--	O
-accessible	O
-are	O
-colored	O
-red	O
-.	O
-	O
-Residues	O
-with	O
-either	O
-side	O
-chain	O
-or	O
-backbone	O
-groups	O
-affected	O
-are	O
-colored	O
-blue	O
-if	O
-buried	O
-and	O
-yellow	O
-if	O
-solvent	O
--	O
-accessible	O
-.	O
-	O
-Residues	O
-without	O
-information	O
-from	O
-shift	O
-mapping	O
-are	O
-colored	O
-gray	O
-.	O
-	O
-The	O
-flexible	O
-switch	O
-regions	O
-are	O
-circled	O
-.	O
-	O
-As	O
-many	O
-of	O
-the	O
-peaks	O
-disappeared	O
-,	O
-the	O
-mean	O
-chemical	O
-shift	O
-change	O
-was	O
-relatively	O
-low	O
-,	O
-so	O
-a	O
-threshold	O
-of	O
-the	O
-mean	O
-plus	O
-one	O
-S	O
-.	O
-D	O
-.	O
-value	O
-was	O
-used	O
-to	O
-define	O
-a	O
-significant	O
-CSP	O
-.	O
-	O
-Parts	O
-of	O
-the	O
-switch	O
-regions	O
-(	O
-Fig	O
-.	O
-5	O
-,	O
-B	O
-and	O
-C	O
-)	O
-are	O
-invisible	O
-in	O
-NMR	O
-spectra	O
-recorded	O
-on	O
-free	O
-Cdc42	O
-due	O
-to	O
-conformational	O
-exchange	O
-.	O
-	O
-These	O
-switch	O
-regions	O
-become	O
-visible	O
-in	O
-Cdc42	O
-and	O
-other	O
-small	O
-G	O
-protein	O
-·	O
-effector	O
-complexes	O
-due	O
-to	O
-a	O
-decrease	O
-in	O
-conformational	O
-freedom	O
-upon	O
-complex	O
-formation	O
-.	O
-	O
-The	O
-switch	O
-regions	O
-of	O
-Cdc42	O
-did	O
-not	O
-,	O
-however	O
-,	O
-become	O
-visible	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-.	O
-	O
-Indeed	O
-,	O
-Ser	O
--	O
-30	O
-of	O
-switch	O
-I	O
-and	O
-Arg	O
--	O
-66	O
-,	O
-Arg	O
--	O
-68	O
-,	O
-Leu	O
--	O
-70	O
-,	O
-and	O
-Ser	O
--	O
-71	O
-of	O
-switch	O
-II	O
-are	O
-visible	O
-in	O
-free	O
-Cdc42	O
-but	O
-disappear	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-HR1	O
-domain	O
-.	O
-	O
-This	O
-suggests	O
-that	O
-the	O
-switch	O
-regions	O
-are	O
-not	O
-rigidified	O
-in	O
-the	O
-HR1	O
-complex	O
-and	O
-are	O
-still	O
-in	O
-conformational	O
-exchange	O
-.	O
-	O
-Nevertheless	O
-,	O
-mapping	O
-of	O
-the	O
-affected	O
-residues	O
-onto	O
-the	O
-NMR	O
-structure	O
-of	O
-free	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-(	O
-Fig	O
-.	O
-5C	O
-)	O
-8	O
-shows	O
-that	O
-,	O
-although	O
-they	O
-are	O
-relatively	O
-widespread	O
-compared	O
-with	O
-changes	O
-in	O
-the	O
-HR1	O
-domain	O
-,	O
-in	O
-general	O
-,	O
-they	O
-are	O
-on	O
-the	O
-face	O
-of	O
-the	O
-protein	O
-that	O
-includes	O
-the	O
-switches	O
-.	O
-	O
-Although	O
-the	O
-binding	O
-interface	O
-may	O
-be	O
-overestimated	O
-,	O
-this	O
-suggests	O
-that	O
-the	O
-switch	O
-regions	O
-are	O
-involved	O
-in	O
-binding	O
-to	O
-TOCA1	O
-.	O
-	O
-Modeling	O
-the	O
-Cdc42	O
-·	O
-TOCA1	O
-HR1	O
-Complex	O
-	O
-The	O
-Cdc42	O
-·	O
-HR1TOCA1	O
-complex	O
-was	O
-not	O
-amenable	O
-to	O
-full	O
-structural	O
-analysis	O
-due	O
-to	O
-the	O
-weak	O
-interaction	O
-and	O
-the	O
-extensive	O
-exchange	O
-broadening	O
-seen	O
-in	O
-the	O
-NMR	O
-experiments	O
-.	O
-	O
-HADDOCK	O
-was	O
-therefore	O
-used	O
-to	O
-perform	O
-rigid	O
-body	O
-docking	O
-based	O
-on	O
-the	O
-structures	O
-of	O
-free	O
-HR1	O
-domain	O
-and	O
-Cdc42	O
-and	O
-ambiguous	O
-interaction	O
-restraints	O
-derived	O
-from	O
-the	O
-titration	O
-experiments	O
-described	O
-above	O
-.	O
-	O
-The	O
-orientation	O
-of	O
-the	O
-HR1	O
-domain	O
-with	O
-respect	O
-to	O
-Cdc42	O
-cannot	O
-be	O
-definitively	O
-concluded	O
-in	O
-the	O
-absence	O
-of	O
-unambiguous	O
-distance	O
-restraints	O
-;	O
-hence	O
-,	O
-HADDOCK	O
-produced	O
-a	O
-set	O
-of	O
-models	O
-in	O
-which	O
-the	O
-HR1	O
-domain	O
-contacts	O
-the	O
-same	O
-surface	O
-on	O
-Cdc42	O
-but	O
-is	O
-in	O
-various	O
-orientations	O
-with	O
-respect	O
-to	O
-Cdc42	O
-.	O
-	O
-The	O
-cluster	O
-with	O
-the	O
-lowest	O
-root	O
-mean	O
-square	O
-deviation	O
-from	O
-the	O
-lowest	O
-energy	O
-structure	O
-is	O
-assumed	O
-to	O
-be	O
-the	O
-best	O
-model	O
-.	O
-	O
-By	O
-these	O
-criteria	O
-,	O
-in	O
-the	O
-best	O
-model	O
-,	O
-the	O
-HR1	O
-domain	O
-is	O
-in	O
-a	O
-similar	O
-orientation	O
-to	O
-the	O
-HR1a	O
-domain	O
-of	O
-PRK1	O
-bound	O
-to	O
-RhoA	O
-and	O
-the	O
-HR1b	O
-domain	O
-bound	O
-to	O
-Rac1	O
-.	O
-	O
-A	O
-representative	O
-model	O
-from	O
-this	O
-cluster	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-6A	O
-alongside	O
-the	O
-Rac1	O
--	O
-HR1b	O
-structure	O
-(	O
-PDB	O
-code	O
-2RMK	O
-)	O
-in	O
-Fig	O
-.	O
-6B	O
-.	O
-	O
-Model	O
-of	O
-Cdc42	O
-·	O
-HR1	O
-complex	O
-.	O
-	O
-A	O
-,	O
-a	O
-representative	O
-model	O
-of	O
-the	O
-Cdc42	O
-·	O
-HR1	O
-complex	O
-from	O
-the	O
-cluster	O
-closest	O
-to	O
-the	O
-lowest	O
-energy	O
-model	O
-produced	O
-using	O
-HADDOCK	O
-.	O
-	O
-Residues	O
-of	O
-Cdc42	O
-that	O
-are	O
-affected	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-HR1	O
-domain	O
-but	O
-are	O
-not	O
-in	O
-close	O
-proximity	O
-to	O
-it	O
-are	O
-colored	O
-in	O
-red	O
-and	O
-labeled	O
-.	O
-	O
-B	O
-,	O
-structure	O
-of	O
-Rac1	O
-in	O
-complex	O
-with	O
-the	O
-HR1b	O
-domain	O
-of	O
-PRK1	O
-(	O
-PDB	O
-code	O
-2RMK	O
-).	O
-	O
-C	O
-,	O
-sequence	O
-alignment	O
-of	O
-RhoA	O
-,	O
-Cdc42	O
-and	O
-Rac1	O
-.	O
-	O
-Contact	O
-residues	O
-of	O
-RhoA	O
-and	O
-Rac1	O
-to	O
-PRK1	O
-HR1a	O
-and	O
-HR1b	O
-,	O
-respectively	O
-,	O
-are	O
-colored	O
-cyan	O
-.	O
-	O
-Residues	O
-of	O
-Cdc42	O
-that	O
-disappear	O
-or	O
-show	O
-chemical	O
-shift	O
-changes	O
-in	O
-the	O
-presence	O
-of	O
-TOCA1	O
-are	O
-colored	O
-cyan	O
-if	O
-also	O
-identified	O
-as	O
-contacts	O
-in	O
-RhoA	O
-and	O
-Rac1	O
-and	O
-yellow	O
-if	O
-they	O
-are	O
-not	O
-.	O
-	O
-Residues	O
-equivalent	O
-to	O
-Rac1	O
-and	O
-RhoA	O
-contact	O
-sites	O
-but	O
-that	O
-are	O
-invisible	O
-in	O
-free	O
-Cdc42	O
-are	O
-gray	O
-.	O
-	O
-D	O
-,	O
-regions	O
-of	O
-interest	O
-of	O
-the	O
-Cdc42	O
-·	O
-HR1	O
-domain	O
-model	O
-.	O
-	O
-The	O
-four	O
-lowest	O
-energy	O
-structures	O
-in	O
-the	O
-chosen	O
-HADDOCK	O
-cluster	O
-are	O
-shown	O
-overlaid	O
-,	O
-with	O
-the	O
-residues	O
-of	O
-interest	O
-shown	O
-as	O
-sticks	O
-and	O
-labeled	O
-.	O
-	O
-Cdc42	O
-is	O
-shown	O
-in	O
-cyan	O
-,	O
-and	O
-TOCA1	O
-is	O
-shown	O
-in	O
-purple	O
-.	O
-	O
-A	O
-sequence	O
-alignment	O
-of	O
-RhoA	O
-,	O
-Cdc42	O
-,	O
-and	O
-Rac1	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-6C	O
-.	O
-	O
-The	O
-RhoA	O
-and	O
-Rac1	O
-contact	O
-residues	O
-in	O
-the	O
-switch	O
-regions	O
-are	O
-invisible	O
-in	O
-the	O
-spectra	O
-of	O
-Cdc42	O
-,	O
-but	O
-they	O
-are	O
-generally	O
-conserved	O
-between	O
-all	O
-three	O
-G	O
-proteins	O
-.	O
-	O
-Several	O
-Cdc42	O
-residues	O
-identified	O
-by	O
-chemical	O
-shift	O
-mapping	O
-are	O
-not	O
-in	O
-close	O
-contact	O
-in	O
-the	O
-Cdc42	O
-·	O
-TOCA1	O
-model	O
-(	O
-Fig	O
-.	O
-6A	O
-).	O
-	O
-Some	O
-of	O
-these	O
-can	O
-be	O
-rationalized	O
-;	O
-for	O
-example	O
-,	O
-Thr	O
--	O
-24Cdc42	O
-,	O
-Leu	O
--	O
-160Cdc42	O
-,	O
-and	O
-Lys	O
--	O
-163Cdc42	O
-all	O
-pack	O
-behind	O
-switch	O
-I	O
-and	O
-are	O
-likely	O
-to	O
-be	O
-affected	O
-by	O
-conformational	O
-changes	O
-within	O
-the	O
-switch	O
-,	O
-while	O
-Glu	O
--	O
-95Cdc42	O
-and	O
-Lys	O
--	O
-96Cdc42	O
-are	O
-in	O
-the	O
-helix	O
-behind	O
-switch	O
-II	O
-.	O
-	O
-Other	O
-residues	O
-that	O
-are	O
-affected	O
-in	O
-the	O
-Cdc42	O
-·	O
-TOCA1	O
-complex	O
-but	O
-that	O
-do	O
-not	O
-correspond	O
-to	O
-contact	O
-residues	O
-of	O
-RhoA	O
-or	O
-Rac1	O
-(	O
-Fig	O
-.	O
-6C	O
-)	O
-include	O
-Gln	O
--	O
-2Cdc42	O
-,	O
-Lys	O
--	O
-16Cdc42	O
-,	O
-Thr	O
--	O
-52Cdc42	O
-,	O
-and	O
-Arg	O
--	O
-68Cdc42	O
-.	O
-	O
-Lys	O
--	O
-16Cdc42	O
-is	O
-unlikely	O
-to	O
-be	O
-a	O
-contact	O
-residue	O
-because	O
-it	O
-is	O
-involved	O
-in	O
-nucleotide	O
-binding	O
-,	O
-but	O
-the	O
-others	O
-may	O
-represent	O
-specific	O
-Cdc42	O
--	O
-TOCA1	O
-contacts	O
-.	O
-	O
-Competition	O
-between	O
-N	O
--	O
-WASP	O
-and	O
-TOCA1	O
-	O
-From	O
-the	O
-known	O
-interactions	O
-and	O
-effects	O
-of	O
-the	O
-proteins	O
-in	O
-biological	O
-systems	O
-,	O
-it	O
-has	O
-been	O
-suggested	O
-that	O
-TOCA1	O
-and	O
-N	O
--	O
-WASP	O
-could	O
-bind	O
-Cdc42	O
-simultaneously	O
-.	O
-	O
-Studies	O
-in	O
-CHO	O
-cells	O
-indicated	O
-that	O
-a	O
-Cdc42	O
-·	O
-N	O
--	O
-WASP	O
-·	O
-TOCA1	O
-complex	O
-existed	O
-because	O
-FRET	O
-was	O
-observed	O
-between	O
-RFP	O
--	O
-TOCA1	O
-and	O
-GFP	O
--	O
-N	O
--	O
-WASP	O
-,	O
-and	O
-the	O
-efficiency	O
-was	O
-decreased	O
-when	O
-an	O
-N	O
--	O
-WASP	O
-mutant	O
-was	O
-used	O
-that	O
-no	O
-longer	O
-binds	O
-Cdc42	O
-.	O
-	O
-An	O
-overlay	O
-of	O
-the	O
-HADDOCK	O
-model	O
-of	O
-the	O
-Cdc42	O
-·	O
-HR1TOCA1	O
-complex	O
-and	O
-the	O
-structure	O
-of	O
-Cdc42	O
-in	O
-complex	O
-with	O
-the	O
-GBD	O
-of	O
-the	O
-N	O
--	O
-WASP	O
-homologue	O
-,	O
-WASP	O
-(	O
-PDB	O
-code	O
-1CEE	O
-),	O
-shows	O
-that	O
-the	O
-HR1	O
-and	O
-GBD	O
-binding	O
-sites	O
-only	O
-partly	O
-overlap	O
-,	O
-and	O
-,	O
-therefore	O
-,	O
-a	O
-ternary	O
-complex	O
-remained	O
-possible	O
-(	O
-Fig	O
-.	O
-7A	O
-).	O
-	O
-Interestingly	O
-,	O
-the	O
-presence	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-would	O
-not	O
-prevent	O
-the	O
-core	O
-CRIB	O
-of	O
-WASP	O
-from	O
-binding	O
-to	O
-Cdc42	O
-,	O
-although	O
-the	O
-regions	O
-C	O
--	O
-terminal	O
-to	O
-the	O
-CRIB	O
-that	O
-are	O
-required	O
-for	O
-high	O
-affinity	O
-binding	O
-of	O
-WASP	O
-would	O
-interfere	O
-sterically	O
-with	O
-the	O
-TOCA1	O
-HR1	O
-.	O
-	O
-A	O
-basic	O
-region	O
-in	O
-WASP	O
-including	O
-three	O
-lysines	O
-(	O
-residues	O
-230	O
-–	O
-232	O
-),	O
-N	O
--	O
-terminal	O
-to	O
-the	O
-core	O
-CRIB	O
-,	O
-has	O
-been	O
-implicated	O
-in	O
-an	O
-electrostatic	O
-steering	O
-mechanism	O
-,	O
-and	O
-these	O
-residues	O
-would	O
-be	O
-free	O
-to	O
-bind	O
-in	O
-the	O
-presence	O
-of	O
-TOCA1	O
-HR1	O
-(	O
-Fig	O
-.	O
-7A	O
-).	O
-	O
-The	O
-N	O
--	O
-WASP	O
-GBD	O
-displaces	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-.	O
-	O
-A	O
-,	O
-the	O
-model	O
-of	O
-the	O
-Cdc42	O
-·	O
-TOCA1	O
-HR1	O
-domain	O
-complex	O
-overlaid	O
-with	O
-the	O
-Cdc42	O
--	O
-WASP	O
-structure	O
-.	O
-	O
-Cdc42	O
-is	O
-shown	O
-in	O
-green	O
-,	O
-and	O
-TOCA1	O
-is	O
-shown	O
-in	O
-purple	O
-.	O
-	O
-The	O
-core	O
-CRIB	O
-region	O
-of	O
-WASP	O
-is	O
-shown	O
-in	O
-red	O
-,	O
-whereas	O
-its	O
-basic	O
-region	O
-is	O
-shown	O
-in	O
-orange	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-required	O
-for	O
-maximal	O
-affinity	O
-is	O
-shown	O
-in	O
-cyan	O
-.	O
-	O
-A	O
-semitransparent	O
-surface	O
-representation	O
-of	O
-Cdc42	O
-and	O
-WASP	O
-is	O
-shown	O
-overlaid	O
-with	O
-the	O
-schematic	O
-.	O
-	O
-B	O
-,	O
-competition	O
-SPA	O
-experiments	O
-carried	O
-out	O
-with	O
-indicated	O
-concentrations	O
-of	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-construct	O
-titrated	O
-into	O
-30	O
-nm	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-or	O
-GST	B-mutant
--	I-mutant
-WASP	I-mutant
-GBD	O
-and	O
-30	O
-nm	O
-Cdc42Δ7Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-.	O
-	O
-C	O
-,	O
-Selected	O
-regions	O
-of	O
-the	O
-15N	O
-HSQC	O
-of	O
-145	O
-μm	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-with	O
-the	O
-indicated	O
-ratios	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-,	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-,	O
-or	O
-both	O
-,	O
-showing	O
-that	O
-the	O
-TOCA	O
-HR1	O
-domain	O
-does	O
-not	O
-displace	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-.	O
-	O
-D	O
-,	O
-selected	O
-regions	O
-of	O
-the	O
-15N	O
-HSQC	O
-of	O
-600	O
-μm	O
-TOCA1	O
-HR1	O
-domain	O
-in	O
-complex	O
-with	O
-Cdc42	O
-in	O
-the	O
-absence	O
-and	O
-presence	O
-of	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-,	O
-showing	O
-displacement	O
-of	O
-Cdc42	O
-from	O
-the	O
-HR1	O
-domain	O
-by	O
-N	O
--	O
-WASP	O
-.	O
-	O
-An	O
-N	O
--	O
-WASP	O
-GBD	O
-construct	O
-was	O
-produced	O
-,	O
-and	O
-its	O
-affinity	O
-for	O
-Cdc42	O
-was	O
-measured	O
-by	O
-competition	O
-SPA	O
-(	O
-Fig	O
-.	O
-7B	O
-).	O
-	O
-The	O
-Kd	O
-that	O
-was	O
-determined	O
-(	O
-37	O
-nm	O
-)	O
-is	O
-consistent	O
-with	O
-the	O
-previously	O
-reported	O
-affinity	O
-.	O
-	O
-Unlabeled	O
-N	O
--	O
-WASP	O
-GBD	O
-was	O
-titrated	O
-into	O
-15N	O
--	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-,	O
-and	O
-the	O
-backbone	O
-NH	O
-groups	O
-were	O
-monitored	O
-using	O
-HSQCs	O
-(	O
-Fig	O
-.	O
-7C	O
-).	O
-	O
-Unlabeled	O
-HR1TOCA1	O
-was	O
-then	O
-added	O
-to	O
-the	O
-Cdc42	O
-·	O
-N	O
--	O
-WASP	O
-complex	O
-,	O
-and	O
-no	O
-changes	O
-were	O
-seen	O
-,	O
-suggesting	O
-that	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-was	O
-not	O
-displaced	O
-even	O
-in	O
-the	O
-presence	O
-of	O
-a	O
-5	O
--	O
-fold	O
-excess	O
-of	O
-HR1TOCA1	O
-.	O
-	O
-These	O
-experiments	O
-were	O
-recorded	O
-at	O
-sufficiently	O
-high	O
-protein	O
-concentrations	O
-(	O
-145	O
-μm	O
-Cdc42	O
-,	O
-145	O
-μm	O
-N	O
--	O
-WASP	O
-GBD	O
-,	O
-725	O
-μm	O
-TOCA1	O
-HR1	O
-domain	O
-)	O
-to	O
-be	O
-far	O
-in	O
-excess	O
-of	O
-the	O
-Kd	O
-values	O
-of	O
-the	O
-individual	O
-interactions	O
-(	O
-TOCA1	O
-Kd	O
-≈	O
-5	O
-μm	O
-,	O
-N	O
--	O
-WASP	O
-Kd	O
-=	O
-37	O
-nm	O
-).	O
-	O
-A	O
-comparison	O
-of	O
-the	O
-HSQC	O
-experiments	O
-recorded	O
-on	O
-15N	O
--	O
-Cdc42	O
-alone	O
-,	O
-in	O
-the	O
-presence	O
-of	O
-TOCA1	O
-HR1	O
-,	O
-N	O
--	O
-WASP	O
-GBD	O
-,	O
-or	O
-both	O
-,	O
-shows	O
-that	O
-the	O
-spectra	O
-in	O
-the	O
-presence	O
-of	O
-N	O
--	O
-WASP	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-both	O
-N	O
--	O
-WASP	O
-and	O
-TOCA1	O
-HR1	O
-are	O
-identical	O
-(	O
-Fig	O
-.	O
-7C	O
-).	O
-	O
-Furthermore	O
-,	O
-15N	O
--	O
-TOCA1	O
-HR1	O
-was	O
-monitored	O
-in	O
-the	O
-presence	O
-of	O
-unlabeled	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-(	O
-1	O
-:	O
-1	O
-)	O
-before	O
-and	O
-after	O
-the	O
-addition	O
-of	O
-0	O
-.	O
-25	O
-and	O
-1	O
-.	O
-0	O
-eq	O
-of	O
-unlabeled	O
-N	O
--	O
-WASP	O
-GBD	O
-.	O
-	O
-The	O
-spectrum	O
-when	O
-N	O
--	O
-WASP	O
-and	O
-TOCA1	O
-were	O
-equimolar	O
-was	O
-identical	O
-to	O
-that	O
-of	O
-the	O
-free	O
-HR1	O
-domain	O
-,	O
-whereas	O
-the	O
-spectrum	O
-in	O
-the	O
-presence	O
-of	O
-0	O
-.	O
-25	O
-eq	O
-of	O
-N	O
--	O
-WASP	O
-was	O
-intermediate	O
-between	O
-the	O
-TOCA1	O
-HR1	O
-free	O
-and	O
-complex	O
-spectra	O
-(	O
-Fig	O
-.	O
-7D	O
-).	O
-	O
-When	O
-in	O
-fast	O
-exchange	O
-,	O
-the	O
-NMR	O
-signal	O
-represents	O
-a	O
-population	O
--	O
-weighted	O
-average	O
-between	O
-free	O
-and	O
-bound	O
-states	O
-,	O
-so	O
-the	O
-intermediate	O
-spectrum	O
-indicates	O
-that	O
-the	O
-population	O
-comprises	O
-a	O
-mixture	O
-of	O
-free	O
-and	O
-bound	O
-HR1	O
-domain	O
-.	O
-	O
-Again	O
-,	O
-the	O
-experiments	O
-were	O
-recorded	O
-on	O
-protein	O
-samples	O
-far	O
-in	O
-excess	O
-of	O
-the	O
-individual	O
-Kd	O
-values	O
-(	O
-600	O
-μm	O
-each	O
-protein	O
-).	O
-	O
-These	O
-data	O
-indicate	O
-that	O
-the	O
-HR1	O
-domain	O
-is	O
-displaced	O
-from	O
-Cdc42	O
-by	O
-N	O
--	O
-WASP	O
-and	O
-that	O
-a	O
-ternary	O
-complex	O
-comprising	O
-TOCA1	O
-HR1	O
-,	O
-N	O
--	O
-WASP	O
-GBD	O
-,	O
-and	O
-Cdc42	O
-is	O
-not	O
-formed	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-the	O
-data	O
-in	O
-Fig	O
-.	O
-7	O
-,	O
-C	O
-and	O
-D	O
-,	O
-indicate	O
-unidirectional	O
-competition	O
-for	O
-Cdc42	O
-binding	O
-in	O
-which	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-displaces	O
-TOCA1	O
-HR1	O
-but	O
-not	O
-vice	O
-versa	O
-.	O
-	O
-To	O
-extend	O
-these	O
-studies	O
-to	O
-a	O
-more	O
-complex	O
-system	O
-and	O
-to	O
-assess	O
-the	O
-ability	O
-of	O
-TOCA1	O
-HR1	O
-to	O
-compete	O
-with	O
-full	O
--	O
-length	O
-N	O
--	O
-WASP	O
-,	O
-pyrene	O
-actin	O
-assays	O
-were	O
-employed	O
-.	O
-	O
-These	O
-assays	O
-,	O
-described	O
-in	O
-detail	O
-elsewhere	O
-,	O
-were	O
-carried	O
-out	O
-using	O
-pyrene	O
-actin	O
--	O
-supplemented	O
-Xenopus	O
-extracts	O
-into	O
-which	O
-exogenous	O
-TOCA1	O
-HR1	O
-domain	O
-or	O
-N	O
--	O
-WASP	O
-GBD	O
-was	O
-added	O
-,	O
-to	O
-assess	O
-their	O
-effects	O
-on	O
-actin	O
-polymerization	O
-.	O
-	O
-Actin	O
-polymerization	O
-in	O
-all	O
-cases	O
-was	O
-initiated	O
-by	O
-the	O
-addition	O
-of	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
--	O
-containing	O
-liposomes	O
-.	O
-	O
-Actin	O
-polymerization	O
-triggered	O
-by	O
-the	O
-addition	O
-of	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
--	O
-containing	O
-liposomes	O
-has	O
-previously	O
-been	O
-shown	O
-to	O
-depend	O
-on	O
-TOCA1	O
-and	O
-N	O
--	O
-WASP	O
-.	O
-	O
-Endogenous	O
-N	O
--	O
-WASP	O
-is	O
-present	O
-at	O
-∼	O
-100	O
-nm	O
-in	O
-Xenopus	O
-extracts	O
-,	O
-whereas	O
-TOCA1	O
-is	O
-present	O
-at	O
-a	O
-10	O
--	O
-fold	O
-lower	O
-concentration	O
-than	O
-N	O
--	O
-WASP	O
-.	O
-	O
-The	O
-addition	O
-of	O
-the	O
-isolated	O
-N	O
--	O
-WASP	O
-GBD	O
-significantly	O
-inhibited	O
-the	O
-polymerization	O
-of	O
-actin	O
-at	O
-concentrations	O
-as	O
-low	O
-as	O
-100	O
-nm	O
-and	O
-completely	O
-abolished	O
-polymerization	O
-at	O
-higher	O
-concentrations	O
-(	O
-Fig	O
-.	O
-8	O
-).	O
-	O
-The	O
-GBD	O
-presumably	O
-acts	O
-as	O
-a	O
-dominant	O
-negative	O
-,	O
-sequestering	O
-endogenous	O
-Cdc42	O
-and	O
-preventing	O
-endogenous	O
-full	O
--	O
-length	O
-N	O
--	O
-WASP	O
-from	O
-binding	O
-and	O
-becoming	O
-activated	O
-.	O
-	O
-The	O
-addition	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-to	O
-100	O
-μm	O
-had	O
-no	O
-significant	O
-effect	O
-on	O
-the	O
-rate	O
-of	O
-actin	O
-polymerization	O
-or	O
-maximum	O
-fluorescence	O
-.	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-endogenous	O
-N	O
--	O
-WASP	O
-,	O
-activated	O
-by	O
-other	O
-components	O
-of	O
-the	O
-assay	O
-,	O
-outcompeting	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-for	O
-Cdc42	O
-binding	O
-.	O
-	O
-Actin	O
-polymerization	O
-downstream	O
-of	O
-Cdc42	O
-·	O
-N	O
--	O
-WASP	O
-·	O
-TOCA1	O
-is	O
-inhibited	O
-by	O
-excess	O
-N	O
--	O
-WASP	O
-GBD	O
-but	O
-not	O
-by	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-.	O
-	O
-Fluorescence	O
-curves	O
-show	O
-actin	O
-polymerization	O
-in	O
-the	O
-presence	O
-of	O
-increasing	O
-concentrations	O
-of	O
-N	O
--	O
-WASP	O
-GBD	O
-or	O
-TOCA1	O
-HR1	O
-domain	O
-as	O
-indicated	O
-.	O
-	O
-The	O
-Cdc42	O
--	O
-TOCA1	O
-Interaction	O
-	O
-The	O
-TOCA1	O
-HR1	O
-domain	O
-alone	O
-is	O
-sufficient	O
-for	O
-Cdc42	O
-binding	O
-in	O
-vitro	O
-,	O
-yet	O
-the	O
-affinity	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-for	O
-Cdc42	O
-is	O
-remarkably	O
-low	O
-(	O
-Kd	O
-≈	O
-5	O
-μm	O
-).	O
-	O
-This	O
-is	O
-over	O
-100	O
-times	O
-lower	O
-than	O
-that	O
-of	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-(	O
-Kd	O
-=	O
-37	O
-nm	O
-)	O
-and	O
-considerably	O
-lower	O
-than	O
-other	O
-known	O
-G	O
-protein	O
--	O
-HR1	O
-domain	O
-interactions	O
-.	O
-	O
-The	O
-polybasic	O
-tract	O
-within	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-Cdc42	O
-does	O
-not	O
-appear	O
-to	O
-be	O
-required	O
-for	O
-binding	O
-to	O
-TOCA1	O
-,	O
-which	O
-is	O
-in	O
-contrast	O
-to	O
-the	O
-interaction	O
-between	O
-Rac1	O
-and	O
-the	O
-HR1b	O
-domain	O
-of	O
-PRK1	O
-but	O
-more	O
-similar	O
-to	O
-the	O
-PRK1	O
-HR1a	O
--	O
-RhoA	O
-interaction	O
-.	O
-	O
-A	O
-single	O
-binding	O
-interface	O
-on	O
-both	O
-the	O
-HR1	O
-domain	O
-and	O
-Cdc42	O
-can	O
-be	O
-concluded	O
-from	O
-the	O
-data	O
-presented	O
-here	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-interfaces	O
-are	O
-comparable	O
-with	O
-those	O
-of	O
-other	O
-G	O
-protein	O
--	O
-HR1	O
-interactions	O
-(	O
-Fig	O
-.	O
-4	O
-),	O
-and	O
-the	O
-lowest	O
-energy	O
-model	O
-produced	O
-in	O
-rigid	O
-body	O
-docking	O
-resembles	O
-previously	O
-studied	O
-G	O
-protein	O
-·	O
-HR1	O
-complexes	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-It	O
-seems	O
-,	O
-therefore	O
-,	O
-that	O
-the	O
-interaction	O
-,	O
-despite	O
-its	O
-relatively	O
-low	O
-affinity	O
-,	O
-is	O
-specific	O
-and	O
-sterically	O
-similar	O
-to	O
-other	O
-HR1	O
-domain	O
--	O
-G	O
-protein	O
-interactions	O
-.	O
-	O
-The	O
-TOCA1	O
-HR1	O
-domain	O
-is	O
-a	O
-left	O
--	O
-handed	O
-coiled	O
--	O
-coil	O
-comparable	O
-with	O
-other	O
-known	O
-HR1	O
-domains	O
-.	O
-	O
-A	O
-short	O
-region	O
-N	O
--	O
-terminal	O
-to	O
-the	O
-coiled	O
--	O
-coil	O
-exhibits	O
-a	O
-series	O
-of	O
-turns	O
-and	O
-contacts	O
-residues	O
-of	O
-both	O
-helices	O
-of	O
-the	O
-coiled	O
--	O
-coil	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-The	O
-corresponding	O
-sequence	O
-in	O
-CIP4	O
-also	O
-includes	O
-a	O
-series	O
-of	O
-turns	O
-but	O
-is	O
-flexible	O
-,	O
-whereas	O
-in	O
-the	O
-HR1a	O
-domain	O
-of	O
-PRK1	O
-,	O
-the	O
-equivalent	O
-region	O
-adopts	O
-an	O
-α	O
--	O
-helical	O
-structure	O
-that	O
-packs	O
-against	O
-the	O
-coiled	O
--	O
-coil	O
-.	O
-	O
-The	O
-contacts	O
-between	O
-the	O
-N	O
--	O
-terminal	O
-region	O
-and	O
-the	O
-coiled	O
--	O
-coil	O
-are	O
-predominantly	O
-hydrophobic	O
-in	O
-both	O
-cases	O
-,	O
-but	O
-sequence	O
--	O
-specific	O
-contacts	O
-do	O
-not	O
-appear	O
-to	O
-be	O
-conserved	O
-.	O
-	O
-This	O
-region	O
-is	O
-distant	O
-from	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-interface	O
-of	O
-the	O
-HR1	O
-domains	O
-,	O
-so	O
-the	O
-structural	O
-differences	O
-may	O
-relate	O
-to	O
-the	O
-structure	O
-and	O
-regulation	O
-of	O
-these	O
-domains	O
-rather	O
-than	O
-their	O
-G	O
-protein	O
-interactions	O
-.	O
-	O
-The	O
-interhelical	O
-loops	O
-of	O
-TOCA1	O
-and	O
-CIP4	O
-differ	O
-from	O
-the	O
-same	O
-region	O
-in	O
-the	O
-HR1	O
-domains	O
-of	O
-PRK1	O
-in	O
-that	O
-they	O
-are	O
-longer	O
-and	O
-contain	O
-two	O
-short	O
-stretches	O
-of	O
-310	O
--	O
-helix	O
-.	O
-	O
-This	O
-region	O
-lies	O
-within	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-surface	O
-of	O
-all	O
-of	O
-the	O
-HR1	O
-domains	O
-(	O
-Fig	O
-.	O
-4D	O
-).	O
-	O
-TOCA1	O
-and	O
-CIP4	O
-both	O
-bind	O
-weakly	O
-to	O
-Cdc42	O
-,	O
-whereas	O
-the	O
-HR1a	O
-domain	O
-of	O
-PRK1	O
-binds	O
-tightly	O
-to	O
-RhoA	O
-and	O
-Rac1	O
-,	O
-and	O
-the	O
-HR1b	O
-domain	O
-binds	O
-to	O
-Rac1	O
-.	O
-	O
-The	O
-structural	O
-features	O
-shared	O
-by	O
-TOCA1	O
-and	O
-CIP4	O
-may	O
-therefore	O
-be	O
-related	O
-to	O
-Cdc42	O
-binding	O
-specificity	O
-and	O
-the	O
-low	O
-affinities	O
-.	O
-	O
-In	O
-free	O
-TOCA1	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-the	O
-interhelical	O
-region	O
-make	O
-extensive	O
-contacts	O
-with	O
-residues	O
-in	O
-helix	O
-1	O
-.	O
-	O
-Many	O
-of	O
-these	O
-residues	O
-are	O
-significantly	O
-affected	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-,	O
-so	O
-it	O
-is	O
-likely	O
-that	O
-the	O
-conformation	O
-of	O
-this	O
-loop	O
-is	O
-altered	O
-in	O
-the	O
-Cdc42	O
-complex	O
-.	O
-	O
-These	O
-observations	O
-therefore	O
-provide	O
-a	O
-molecular	O
-mechanism	O
-whereby	O
-mutation	O
-of	O
-Met383	O
--	O
-Gly384	O
--	O
-Asp385	O
-to	O
-Ile383	O
--	O
-Ser384	O
--	O
-Thr385	O
-abolishes	O
-TOCA1	O
-binding	O
-to	O
-Cdc42	O
-.	O
-	O
-The	O
-lowest	O
-energy	O
-model	O
-produced	O
-by	O
-HADDOCK	O
-using	O
-ambiguous	O
-interaction	O
-restraints	O
-from	O
-the	O
-titration	O
-data	O
-resembled	O
-the	O
-NMR	O
-structures	O
-of	O
-RhoA	O
-and	O
-Rac1	O
-in	O
-complex	O
-with	O
-their	O
-HR1	O
-domain	O
-partners	O
-.	O
-	O
-For	O
-example	O
-,	O
-Phe	O
--	O
-56Cdc42	O
-,	O
-which	O
-is	O
-not	O
-visible	O
-in	O
-free	O
-Cdc42	O
-or	O
-Cdc42	O
-·	O
-HR1TOCA1	O
-,	O
-is	O
-close	O
-to	O
-the	O
-TOCA1	O
-HR1	O
-(	O
-Fig	O
-.	O
-6A	O
-).	O
-	O
-Phe	O
--	O
-56Cdc42	O
-,	O
-which	O
-is	O
-a	O
-Trp	O
-in	O
-both	O
-Rac1	O
-and	O
-RhoA	O
-(	O
-Fig	O
-.	O
-6C	O
-),	O
-is	O
-thought	O
-to	O
-pack	O
-behind	O
-switch	O
-I	O
-when	O
-Cdc42	O
-interacts	O
-with	O
-ACK	O
-,	O
-maintaining	O
-the	O
-switch	O
-in	O
-a	O
-binding	O
--	O
-competent	O
-orientation	O
-.	O
-	O
-This	O
-residue	O
-has	O
-also	O
-been	O
-identified	O
-as	O
-important	O
-for	O
-Cdc42	O
--	O
-WASP	O
-binding	O
-.	O
-	O
-Phe	O
--	O
-56Cdc42	O
-is	O
-therefore	O
-likely	O
-to	O
-be	O
-involved	O
-in	O
-the	O
-Cdc42	O
--	O
-TOCA1	O
-interaction	O
-,	O
-probably	O
-by	O
-stabilizing	O
-the	O
-position	O
-of	O
-switch	O
-I	O
-.	O
-	O
-Some	O
-residues	O
-that	O
-are	O
-affected	O
-in	O
-the	O
-Cdc42	O
-·	O
-HR1TOCA1	O
-complex	O
-but	O
-do	O
-not	O
-correspond	O
-to	O
-contact	O
-residues	O
-of	O
-RhoA	O
-or	O
-Rac1	O
-(	O
-Fig	O
-.	O
-6C	O
-)	O
-may	O
-contact	O
-HR1TOCA1	O
-directly	O
-(	O
-Fig	O
-.	O
-6D	O
-).	O
-	O
-Gln	O
--	O
-2Cdc42	O
-,	O
-which	O
-has	O
-also	O
-been	O
-identified	O
-as	O
-a	O
-contact	O
-residue	O
-in	O
-the	O
-Cdc42	O
-·	O
-ACK	O
-complex	O
-,	O
-contacts	O
-Val	O
--	O
-376TOCA1	O
-and	O
-Asn	O
--	O
-380TOCA1	O
-in	O
-the	O
-model	O
-and	O
-disrupts	O
-the	O
-contacts	O
-between	O
-the	O
-interhelical	O
-loop	O
-and	O
-the	O
-first	O
-helix	O
-of	O
-the	O
-TOCA1	O
-coiled	O
--	O
-coil	O
-.	O
-	O
-Thr	O
--	O
-52Cdc42	O
-,	O
-which	O
-has	O
-also	O
-been	O
-identified	O
-as	O
-making	O
-minor	O
-contacts	O
-with	O
-ACK	O
-,	O
-falls	O
-near	O
-the	O
-side	O
-chains	O
-of	O
-HR1TOCA1	O
-helix	O
-1	O
-,	O
-particularly	O
-Lys	O
--	O
-372TOCA1	O
-,	O
-whereas	O
-the	O
-equivalent	O
-position	O
-in	O
-Rac1	O
-is	O
-Asn	O
--	O
-52Rac1	O
-.	O
-	O
-N52T	B-mutant
-is	O
-one	O
-of	O
-a	O
-combination	O
-of	O
-seven	O
-residues	O
-found	O
-to	O
-confer	O
-ACK	O
-binding	O
-on	O
-Rac1	O
-and	O
-so	O
-may	O
-represent	O
-a	O
-specific	O
-Cdc42	O
--	O
-effector	O
-contact	O
-residue	O
-.	O
-	O
-The	O
-position	O
-equivalent	O
-to	O
-Lys	O
--	O
-372TOCA1	O
-in	O
-PRK1	O
-is	O
-Glu	O
--	O
-58HR1a	O
-or	O
-Gln	O
--	O
-151HR1b	O
-.	O
-	O
-Thr	O
--	O
-52Cdc42	O
--	O
-Lys	O
--	O
-372TOCA1	O
-may	O
-therefore	O
-represent	O
-a	O
-specific	O
-Cdc42	O
--	O
-HR1TOCA1	O
-contact	O
-.	O
-	O
-Arg	O
--	O
-68Cdc42	O
-of	O
-switch	O
-II	O
-is	O
-positioned	O
-close	O
-to	O
-Glu	O
--	O
-395TOCA1	O
-(	O
-Fig	O
-.	O
-6D	O
-),	O
-suggesting	O
-a	O
-direct	O
-electrostatic	O
-contact	O
-between	O
-switch	O
-II	O
-of	O
-Cdc42	O
-and	O
-helix	O
-2	O
-of	O
-the	O
-HR1	O
-domain	O
-.	O
-	O
-The	O
-equivalent	O
-Arg	O
-in	O
-Rac1	O
-and	O
-RhoA	O
-is	O
-pointing	O
-away	O
-from	O
-the	O
-HR1	O
-domains	O
-of	O
-PRK1	O
-.	O
-	O
-The	O
-importance	O
-of	O
-this	O
-residue	O
-in	O
-the	O
-Cdc42	O
--	O
-TOCA1	O
-interaction	O
-remains	O
-unclear	O
-,	O
-although	O
-its	O
-mutation	O
-reduces	O
-binding	O
-to	O
-RhoGAP	O
-,	O
-suggesting	O
-that	O
-it	O
-can	O
-be	O
-involved	O
-in	O
-Cdc42	O
-interactions	O
-.	O
-	O
-The	O
-solution	O
-structure	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-presented	O
-here	O
-,	O
-along	O
-with	O
-the	O
-model	O
-of	O
-the	O
-HR1TOCA1	O
-·	O
-Cdc42	O
-complex	O
-is	O
-consistent	O
-with	O
-a	O
-conserved	O
-mode	O
-of	O
-binding	O
-across	O
-the	O
-known	O
-HR1	O
-domain	O
--	O
-Rho	O
-family	O
-interactions	O
-,	O
-despite	O
-their	O
-differing	O
-affinities	O
-.	O
-	O
-The	O
-weak	O
-binding	O
-prevented	O
-detailed	O
-structural	O
-and	O
-thermodynamic	O
-studies	O
-of	O
-the	O
-complex	O
-.	O
-	O
-Nonetheless	O
-,	O
-structural	O
-studies	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-,	O
-combined	O
-with	O
-chemical	O
-shift	O
-mapping	O
-,	O
-have	O
-highlighted	O
-some	O
-potentially	O
-interesting	O
-differences	O
-between	O
-Cdc42	O
--	O
-HR1TOCA1	O
-and	O
-RhoA	O
-/	O
-Rac1	O
--	O
-HR1PRK1	O
-binding	O
-.	O
-	O
-We	O
-have	O
-previously	O
-postulated	O
-that	O
-the	O
-inherent	O
-flexibility	O
-of	O
-HR1	O
-domains	O
-contributes	O
-to	O
-their	O
-ability	O
-to	O
-bind	O
-to	O
-different	O
-Rho	O
-family	O
-G	O
-proteins	O
-,	O
-with	O
-Rho	O
--	O
-binding	O
-HR1	O
-domains	O
-displaying	O
-increased	O
-flexibility	O
-,	O
-reflected	O
-in	O
-their	O
-lower	O
-melting	O
-temperatures	O
-(	O
-Tm	O
-)	O
-and	O
-Rac	O
-binders	O
-being	O
-more	O
-rigid	O
-.	O
-	O
-The	O
-Tm	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-is	O
-61	O
-.	O
-9	O
-°	O
-C	O
-(	O
-data	O
-not	O
-shown	O
-),	O
-which	O
-is	O
-the	O
-highest	O
-Tm	O
-that	O
-we	O
-have	O
-measured	O
-for	O
-an	O
-HR1	O
-domain	O
-thus	O
-far	O
-.	O
-	O
-As	O
-such	O
-,	O
-the	O
-ability	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-to	O
-bind	O
-to	O
-Cdc42	O
-(	O
-a	O
-close	O
-relative	O
-of	O
-Rac1	O
-rather	O
-than	O
-RhoA	O
-)	O
-fits	O
-this	O
-trend	O
-.	O
-	O
-An	O
-investigation	O
-into	O
-the	O
-local	O
-motions	O
-,	O
-particularly	O
-in	O
-the	O
-G	O
-protein	O
--	O
-binding	O
-regions	O
-,	O
-may	O
-offer	O
-further	O
-insight	O
-into	O
-the	O
-differential	O
-specificities	O
-and	O
-affinities	O
-of	O
-G	O
-protein	O
--	O
-HR1	O
-domain	O
-interactions	O
-.	O
-	O
-The	O
-low	O
-affinity	O
-of	O
-the	O
-Cdc42	O
--	O
-HR1TOCA1	O
-interaction	O
-is	O
-consistent	O
-with	O
-a	O
-tightly	O
-spatially	O
-and	O
-temporally	O
-regulated	O
-pathway	O
-,	O
-requiring	O
-combinatorial	O
-signals	O
-leading	O
-to	O
-a	O
-series	O
-of	O
-coincident	O
-weak	O
-interactions	O
-that	O
-elicit	O
-full	O
-activation	O
-.	O
-	O
-The	O
-HR1	O
-domains	O
-from	O
-other	O
-TOCA	O
-family	O
-members	O
-,	O
-CIP4	O
-and	O
-FBP17	O
-,	O
-also	O
-bind	O
-at	O
-low	O
-micromolar	O
-affinities	O
-to	O
-Cdc42	O
-,	O
-so	O
-the	O
-low	O
-affinity	O
-interaction	O
-appears	O
-to	O
-be	O
-commonplace	O
-among	O
-this	O
-family	O
-of	O
-HR1	O
-domain	O
-proteins	O
-,	O
-in	O
-contrast	O
-to	O
-the	O
-PRK	O
-family	O
-.	O
-	O
-The	O
-low	O
-affinity	O
-of	O
-the	O
-HR1TOCA1	O
--	O
-Cdc42	O
-interaction	O
-in	O
-the	O
-context	O
-of	O
-the	O
-physiological	O
-concentration	O
-of	O
-TOCA1	O
-in	O
-Xenopus	O
-extracts	O
-(∼	O
-10	O
-nm	O
-)	O
-suggests	O
-that	O
-binding	O
-between	O
-TOCA1	O
-and	O
-Cdc42	O
-is	O
-likely	O
-to	O
-occur	O
-in	O
-vivo	O
-only	O
-when	O
-TOCA1	O
-is	O
-at	O
-high	O
-local	O
-concentrations	O
-and	O
-membrane	O
--	O
-localized	O
-and	O
-therefore	O
-in	O
-close	O
-proximity	O
-to	O
-activated	O
-Cdc42	O
-.	O
-	O
-Evidence	O
-suggests	O
-that	O
-the	O
-TOCA	O
-family	O
-of	O
-proteins	O
-are	O
-recruited	O
-to	O
-the	O
-membrane	O
-via	O
-an	O
-interaction	O
-between	O
-their	O
-F	O
--	O
-BAR	O
-domain	O
-and	O
-specific	O
-signaling	O
-lipids	O
-.	O
-	O
-For	O
-example	O
-,	O
-electrostatic	O
-interactions	O
-between	O
-the	O
-F	O
--	O
-BAR	O
-domain	O
-and	O
-the	O
-membrane	O
-are	O
-required	O
-for	O
-TOCA1	O
-recruitment	O
-to	O
-membrane	O
-vesicles	O
-and	O
-tubules	O
-,	O
-and	O
-TOCA1	O
--	O
-dependent	O
-actin	O
-polymerization	O
-is	O
-known	O
-to	O
-depend	O
-specifically	O
-on	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-isolated	O
-F	O
--	O
-BAR	O
-domain	O
-of	O
-FBP17	O
-has	O
-been	O
-shown	O
-to	O
-induce	O
-membrane	O
-tubulation	O
-of	O
-brain	O
-liposomes	O
-and	O
-BAR	O
-domain	O
-proteins	O
-that	O
-promote	O
-tubulation	O
-cluster	O
-on	O
-membranes	O
-at	O
-high	O
-densities	O
-.	O
-	O
-Once	O
-at	O
-the	O
-membrane	O
-,	O
-high	O
-local	O
-concentrations	O
-of	O
-TOCA1	O
-could	O
-exceed	O
-the	O
-Kd	O
-of	O
-F	O
--	O
-BAR	O
-dimerization	O
-(	O
-likely	O
-to	O
-be	O
-comparable	O
-with	O
-that	O
-of	O
-the	O
-FCHo2	O
-F	O
--	O
-BAR	O
-domain	O
-(	O
-2	O
-.	O
-5	O
-μm	O
-))	O
-and	O
-that	O
-of	O
-the	O
-Cdc42	O
--	O
-HR1TOCA1	O
-interaction	O
-.	O
-	O
-Cdc42	O
--	O
-HR1TOCA1	O
-binding	O
-would	O
-then	O
-be	O
-favorable	O
-,	O
-as	O
-long	O
-as	O
-coincident	O
-activation	O
-of	O
-Cdc42	O
-had	O
-occurred	O
-,	O
-leading	O
-to	O
-stabilization	O
-of	O
-TOCA1	O
-at	O
-the	O
-membrane	O
-and	O
-downstream	O
-activation	O
-of	O
-N	O
--	O
-WASP	O
-.	O
-	O
-It	O
-has	O
-been	O
-postulated	O
-that	O
-WASP	O
-and	O
-N	O
--	O
-WASP	O
-exist	O
-in	O
-equilibrium	O
-between	O
-folded	O
-(	O
-inactive	O
-)	O
-and	O
-unfolded	O
-(	O
-active	O
-)	O
-forms	O
-,	O
-and	O
-the	O
-affinity	O
-of	O
-Cdc42	O
-for	O
-the	O
-unfolded	O
-WASP	O
-proteins	O
-is	O
-significantly	O
-enhanced	O
-.	O
-	O
-The	O
-unfolded	O
-,	O
-high	O
-affinity	O
-state	O
-of	O
-WASP	O
-is	O
-represented	O
-by	O
-a	O
-short	O
-peptide	O
-,	O
-the	O
-GBD	O
-,	O
-which	O
-binds	O
-with	O
-a	O
-low	O
-nanomolar	O
-affinity	O
-to	O
-Cdc42	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-the	O
-best	O
-estimate	O
-of	O
-the	O
-affinity	O
-of	O
-full	O
--	O
-length	O
-WASP	O
-for	O
-Cdc42	O
-is	O
-low	O
-micromolar	O
-.	O
-	O
-In	O
-the	O
-inactive	O
-state	O
-of	O
-WASP	O
-,	O
-the	O
-actin	O
--	O
-and	O
-Arp2	O
-/	O
-3	O
--	O
-binding	O
-VCA	O
-domain	O
-contacts	O
-the	O
-GBD	O
-,	O
-competing	O
-for	O
-Cdc42	O
-binding	O
-.	O
-	O
-The	O
-high	O
-affinity	O
-of	O
-Cdc42	O
-for	O
-the	O
-unfolded	O
-,	O
-active	O
-form	O
-pushes	O
-the	O
-equilibrium	O
-in	O
-favor	O
-of	O
-(	O
-N	O
--)	O
-WASP	O
-activation	O
-.	O
-	O
-Binding	O
-of	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-to	O
-the	O
-basic	O
-region	O
-just	O
-N	O
--	O
-terminal	O
-to	O
-the	O
-GBD	O
-further	O
-favors	O
-the	O
-active	O
-conformation	O
-.	O
-	O
-A	O
-substantial	O
-body	O
-of	O
-data	O
-has	O
-illuminated	O
-the	O
-complex	O
-regulation	O
-of	O
-WASP	O
-/	O
-N	O
--	O
-WASP	O
-proteins	O
-,	O
-and	O
-current	O
-evidence	O
-suggests	O
-that	O
-these	O
-allosteric	O
-activation	O
-mechanisms	O
-and	O
-oligomerization	O
-combine	O
-to	O
-regulate	O
-WASP	O
-activity	O
-,	O
-allowing	O
-the	O
-synchronization	O
-and	O
-integration	O
-of	O
-multiple	O
-potential	O
-activation	O
-signals	O
-(	O
-reviewed	O
-in	O
-Ref	O
-.).	O
-	O
-We	O
-envisage	O
-that	O
-TOCA1	O
-is	O
-first	O
-recruited	O
-to	O
-the	O
-appropriate	O
-membrane	O
-in	O
-response	O
-to	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-via	O
-its	O
-F	O
--	O
-BAR	O
-domain	O
-,	O
-where	O
-the	O
-local	O
-increase	O
-in	O
-concentration	O
-favors	O
-F	O
--	O
-BAR	O
--	O
-mediated	O
-dimerization	O
-of	O
-TOCA1	O
-.	O
-	O
-Cdc42	O
-is	O
-activated	O
-in	O
-response	O
-to	O
-co	O
--	O
-incident	O
-signals	O
-and	O
-can	O
-then	O
-bind	O
-to	O
-TOCA1	O
-,	O
-further	O
-stabilizing	O
-TOCA1	O
-at	O
-the	O
-membrane	O
-.	O
-	O
-TOCA1	O
-can	O
-then	O
-recruit	O
-N	O
--	O
-WASP	O
-via	O
-an	O
-interaction	O
-between	O
-its	O
-SH3	O
-domain	O
-and	O
-the	O
-N	O
--	O
-WASP	O
-proline	O
--	O
-rich	O
-region	O
-.	O
-	O
-The	O
-recruitment	O
-of	O
-N	O
--	O
-WASP	O
-alone	O
-and	O
-of	O
-the	O
-N	O
--	O
-WASP	O
-·	O
-WIP	O
-complex	O
-by	O
-TOCA1	O
-and	O
-FBP17	O
-has	O
-been	O
-demonstrated	O
-.	O
-	O
-WIP	O
-inhibits	O
-the	O
-activation	O
-of	O
-N	O
--	O
-WASP	O
-by	O
-Cdc42	O
-,	O
-an	O
-effect	O
-that	O
-is	O
-reversed	O
-by	O
-TOCA1	O
-.	O
-	O
-It	O
-may	O
-therefore	O
-be	O
-envisaged	O
-that	O
-WIP	O
-and	O
-TOCA1	O
-exert	O
-opposing	O
-allosteric	O
-effects	O
-on	O
-N	O
--	O
-WASP	O
-,	O
-with	O
-TOCA1	O
-favoring	O
-the	O
-unfolded	O
-,	O
-active	O
-conformation	O
-of	O
-N	O
--	O
-WASP	O
-and	O
-increasing	O
-its	O
-affinity	O
-for	O
-Cdc42	O
-.	O
-	O
-TOCA1	O
-may	O
-also	O
-activate	O
-N	O
--	O
-WASP	O
-by	O
-effective	O
-oligomerization	O
-because	O
-clustering	O
-of	O
-TOCA1	O
-at	O
-the	O
-membrane	O
-following	O
-coincident	O
-interactions	O
-with	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-and	O
-Cdc42	O
-would	O
-in	O
-turn	O
-lead	O
-to	O
-clustering	O
-of	O
-N	O
--	O
-WASP	O
-,	O
-in	O
-addition	O
-to	O
-pushing	O
-the	O
-equilibrium	O
-toward	O
-the	O
-unfolded	O
-,	O
-active	O
-state	O
-.	O
-	O
-In	O
-a	O
-cellular	O
-context	O
-,	O
-full	O
--	O
-length	O
-TOCA1	O
-and	O
-N	O
--	O
-WASP	O
-are	O
-likely	O
-to	O
-have	O
-similar	O
-affinities	O
-for	O
-active	O
-Cdc42	O
-,	O
-but	O
-in	O
-the	O
-unfolded	O
-,	O
-active	O
-conformation	O
-,	O
-the	O
-affinity	O
-of	O
-N	O
--	O
-WASP	O
-for	O
-Cdc42	O
-dramatically	O
-increases	O
-.	O
-	O
-Our	O
-binding	O
-data	O
-suggest	O
-that	O
-TOCA1	O
-HR1	O
-binding	O
-is	O
-not	O
-allosterically	O
-regulated	O
-,	O
-and	O
-our	O
-NMR	O
-data	O
-,	O
-along	O
-with	O
-the	O
-high	O
-stability	O
-of	O
-TOCA1	O
-HR1	O
-,	O
-suggest	O
-that	O
-there	O
-is	O
-no	O
-widespread	O
-conformational	O
-change	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-.	O
-	O
-As	O
-full	O
--	O
-length	O
-TOCA1	O
-and	O
-the	O
-isolated	O
-HR1	O
-domain	O
-bind	O
-Cdc42	O
-with	O
-similar	O
-affinities	O
-,	O
-the	O
-N	O
--	O
-WASP	O
--	O
-Cdc42	O
-interaction	O
-will	O
-be	O
-favored	O
-because	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-can	O
-easily	O
-outcompete	O
-the	O
-TOCA1	O
-HR1	O
-for	O
-Cdc42	O
-.	O
-	O
-A	O
-combination	O
-of	O
-allosteric	O
-activation	O
-by	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-,	O
-activated	O
-Cdc42	O
-and	O
-TOCA1	O
-,	O
-and	O
-oligomeric	O
-activation	O
-implemented	O
-by	O
-TOCA1	O
-would	O
-lead	O
-to	O
-full	O
-activation	O
-of	O
-N	O
--	O
-WASP	O
-and	O
-downstream	O
-actin	O
-polymerization	O
-.	O
-	O
-In	O
-such	O
-an	O
-array	O
-of	O
-molecules	O
-localized	O
-to	O
-a	O
-discrete	O
-region	O
-of	O
-the	O
-membrane	O
-,	O
-it	O
-is	O
-plausible	O
-that	O
-WASP	O
-could	O
-bind	O
-to	O
-a	O
-second	O
-Cdc42	O
-molecule	O
-rather	O
-than	O
-displacing	O
-TOCA1	O
-from	O
-its	O
-cognate	O
-Cdc42	O
-.	O
-	O
-Our	O
-NMR	O
-and	O
-affinity	O
-data	O
-,	O
-however	O
-,	O
-are	O
-consistent	O
-with	O
-displacement	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-by	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-.	O
-	O
-Furthermore	O
-,	O
-TOCA1	O
-is	O
-required	O
-for	O
-Cdc42	O
--	O
-mediated	O
-activation	O
-of	O
-N	O
--	O
-WASP	O
-·	O
-WIP	O
-,	O
-implying	O
-that	O
-it	O
-may	O
-not	O
-be	O
-possible	O
-for	O
-Cdc42	O
-to	O
-bind	O
-and	O
-activate	O
-N	O
--	O
-WASP	O
-prior	O
-to	O
-TOCA1	O
--	O
-Cdc42	O
-binding	O
-.	O
-	O
-The	O
-commonly	O
-used	O
-MGD	B-mutant
-→	I-mutant
-IST	I-mutant
-(	O
-Cdc42	O
--	O
-binding	O
-deficient	O
-)	O
-mutant	O
-of	O
-TOCA1	O
-has	O
-a	O
-reduced	O
-ability	O
-to	O
-activate	O
-the	O
-N	O
--	O
-WASP	O
-·	O
-WIP	O
-complex	O
-,	O
-further	O
-indicating	O
-the	O
-importance	O
-of	O
-the	O
-Cdc42	O
--	O
-HR1TOCA1	O
-interaction	O
-prior	O
-to	O
-downstream	O
-activation	O
-of	O
-N	O
--	O
-WASP	O
-.	O
-	O
-In	O
-light	O
-of	O
-this	O
-,	O
-we	O
-favor	O
-an	O
-“	O
-effector	O
-handover	O
-”	O
-scheme	O
-whereby	O
-TOCA1	O
-interacts	O
-with	O
-Cdc42	O
-prior	O
-to	O
-N	O
--	O
-WASP	O
-activation	O
-,	O
-after	O
-which	O
-N	O
--	O
-WASP	O
-displaces	O
-TOCA1	O
-from	O
-its	O
-bound	O
-Cdc42	O
-in	O
-order	O
-to	O
-be	O
-fully	O
-activated	O
-rather	O
-than	O
-binding	O
-a	O
-second	O
-Cdc42	O
-molecule	O
-.	O
-	O
-Potentially	O
-,	O
-the	O
-TOCA1	O
--	O
-Cdc42	O
-interaction	O
-functions	O
-to	O
-position	O
-N	O
--	O
-WASP	O
-and	O
-Cdc42	O
-such	O
-that	O
-they	O
-are	O
-poised	O
-to	O
-interact	O
-with	O
-high	O
-affinity	O
-.	O
-	O
-The	O
-concomitant	O
-release	O
-of	O
-TOCA1	O
-from	O
-Cdc42	O
-while	O
-still	O
-bound	O
-to	O
-N	O
--	O
-WASP	O
-presumably	O
-enhances	O
-the	O
-ability	O
-of	O
-TOCA1	O
-to	O
-further	O
-activate	O
-N	O
--	O
-WASP	O
-·	O
-WIP	O
--	O
-induced	O
-actin	O
-polymerization	O
-.	O
-	O
-There	O
-is	O
-an	O
-advantage	O
-to	O
-such	O
-an	O
-effector	O
-handover	O
-,	O
-in	O
-that	O
-N	O
--	O
-WASP	O
-would	O
-only	O
-be	O
-robustly	O
-recruited	O
-when	O
-F	O
--	O
-BAR	O
-domains	O
-are	O
-already	O
-present	O
-.	O
-	O
-Hence	O
-,	O
-actin	O
-polymerization	O
-cannot	O
-occur	O
-until	O
-F	O
--	O
-BAR	O
-domains	O
-are	O
-poised	O
-for	O
-membrane	O
-distortion	O
-.	O
-	O
-Our	O
-model	O
-of	O
-the	O
-Cdc42	O
-·	O
-HR1TOCA1	O
-complex	O
-indicates	O
-a	O
-mechanism	O
-by	O
-which	O
-such	O
-a	O
-handover	O
-could	O
-take	O
-place	O
-(	O
-Fig	O
-.	O
-9	O
-)	O
-because	O
-it	O
-shows	O
-that	O
-the	O
-effector	O
-binding	O
-sites	O
-only	O
-partially	O
-overlap	O
-on	O
-Cdc42	O
-.	O
-	O
-The	O
-lysine	O
-residues	O
-thought	O
-to	O
-be	O
-involved	O
-in	O
-an	O
-electrostatic	O
-steering	O
-mechanism	O
-in	O
-WASP	O
--	O
-Cdc42	O
-binding	O
-are	O
-conserved	O
-in	O
-N	O
--	O
-WASP	O
-and	O
-would	O
-be	O
-able	O
-to	O
-interact	O
-with	O
-Cdc42	O
-even	O
-when	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-is	O
-already	O
-bound	O
-.	O
-	O
-It	O
-has	O
-been	O
-postulated	O
-that	O
-the	O
-initial	O
-interactions	O
-between	O
-this	O
-basic	O
-region	O
-and	O
-Cdc42	O
-could	O
-stabilize	O
-the	O
-active	O
-conformation	O
-of	O
-WASP	O
-,	O
-leading	O
-to	O
-high	O
-affinity	O
-binding	O
-between	O
-the	O
-core	O
-CRIB	O
-and	O
-Cdc42	O
-.	O
-	O
-The	O
-region	O
-C	O
--	O
-terminal	O
-to	O
-the	O
-core	O
-CRIB	O
-,	O
-required	O
-for	O
-maximal	O
-affinity	O
-binding	O
-,	O
-would	O
-then	O
-fully	O
-displace	O
-the	O
-TOCA1	O
-HR1	O
-.	O
-	O
-A	O
-simplified	O
-model	O
-of	O
-the	O
-early	O
-stages	O
-of	O
-Cdc42	O
-·	O
-N	O
--	O
-WASP	O
-·	O
-TOCA1	O
--	O
-dependent	O
-actin	O
-polymerization	O
-.	O
-	O
-Step	O
-1	O
-,	O
-TOCA1	O
-is	O
-recruited	O
-to	O
-the	O
-membrane	O
-via	O
-its	O
-F	O
--	O
-BAR	O
-domain	O
-and	O
-/	O
-or	O
-Cdc42	O
-interactions	O
-.	O
-	O
-F	O
--	O
-BAR	O
-oligomerization	O
-is	O
-expected	O
-to	O
-occur	O
-following	O
-membrane	O
-binding	O
-,	O
-but	O
-a	O
-single	O
-monomer	O
-is	O
-shown	O
-for	O
-clarity	O
-.	O
-	O
-Step	O
-2	O
-,	O
-N	O
--	O
-WASP	O
-exists	O
-in	O
-an	O
-inactive	O
-,	O
-folded	O
-conformation	O
-.	O
-	O
-The	O
-TOCA1	O
-SH3	O
-domain	O
-interacts	O
-with	O
-N	O
--	O
-WASP	O
-,	O
-causing	O
-an	O
-activatory	O
-allosteric	O
-effect	O
-.	O
-	O
-The	O
-HR1TOCA1	O
--	O
-Cdc42	O
-and	O
-SH3TOCA1	O
--	O
-N	O
--	O
-WASP	O
-interactions	O
-position	O
-Cdc42	O
-and	O
-N	O
--	O
-WASP	O
-for	O
-binding	O
-.	O
-	O
-Step	O
-3	O
-,	O
-electrostatic	O
-interactions	O
-between	O
-Cdc42	O
-and	O
-the	O
-basic	O
-region	O
-upstream	O
-of	O
-the	O
-CRIB	O
-initiate	O
-Cdc42	O
-·	O
-N	O
--	O
-WASP	O
-binding	O
-.	O
-	O
-Step	O
-4	O
-,	O
-the	O
-core	O
-CRIB	O
-binds	O
-with	O
-high	O
-affinity	O
-while	O
-the	O
-region	O
-C	O
--	O
-terminal	O
-to	O
-the	O
-CRIB	O
-displaces	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-and	O
-increases	O
-the	O
-affinity	O
-of	O
-the	O
-N	O
--	O
-WASP	O
--	O
-Cdc42	O
-interaction	O
-further	O
-.	O
-	O
-The	O
-VCA	O
-domain	O
-is	O
-released	O
-for	O
-downstream	O
-interactions	O
-,	O
-and	O
-actin	O
-polymerization	O
-proceeds	O
-.	O
-	O
-WH1	O
-,	O
-WASP	O
-homology	O
-1	O
-domain	O
-;	O
-PP	O
-,	O
-proline	O
--	O
-rich	O
-region	O
-;	O
-VCA	O
-,	O
-verprolin	O
-homology	O
-,	O
-cofilin	O
-homology	O
-,	O
-acidic	O
-region	O
-.	O
-	O
-In	O
-conclusion	O
-,	O
-the	O
-data	O
-presented	O
-here	O
-show	O
-that	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-is	O
-sufficient	O
-for	O
-Cdc42	O
-binding	O
-in	O
-vitro	O
-and	O
-that	O
-the	O
-interaction	O
-is	O
-of	O
-micromolar	O
-affinity	O
-,	O
-lower	O
-than	O
-that	O
-of	O
-other	O
-G	O
-protein	O
--	O
-HR1	O
-domain	O
-interactions	O
-.	O
-	O
-The	O
-analogous	O
-HR1	O
-domains	O
-from	O
-other	O
-TOCA1	O
-family	O
-members	O
-,	O
-FBP17	O
-and	O
-CIP4	O
-,	O
-also	O
-exhibit	O
-micromolar	O
-affinity	O
-for	O
-Cdc42	O
-.	O
-	O
-A	O
-role	O
-for	O
-the	O
-TOCA1	O
--,	O
-FBP17	O
--,	O
-and	O
-CIP4	O
--	O
-Cdc42	O
-interactions	O
-in	O
-the	O
-recruitment	O
-of	O
-these	O
-proteins	O
-to	O
-the	O
-membrane	O
-therefore	O
-appears	O
-unlikely	O
-.	O
-	O
-Instead	O
-,	O
-our	O
-findings	O
-agree	O
-with	O
-earlier	O
-suggestions	O
-that	O
-the	O
-F	O
--	O
-BAR	O
-domain	O
-is	O
-responsible	O
-for	O
-membrane	O
-recruitment	O
-.	O
-	O
-The	O
-role	O
-of	O
-the	O
-Cdc42	O
--	O
-TOCA1	O
-interaction	O
-remains	O
-somewhat	O
-elusive	O
-,	O
-but	O
-it	O
-may	O
-serve	O
-to	O
-position	O
-activated	O
-Cdc42	O
-and	O
-N	O
--	O
-WASP	O
-to	O
-allow	O
-full	O
-activation	O
-of	O
-N	O
--	O
-WASP	O
-and	O
-as	O
-such	O
-serve	O
-to	O
-couple	O
-F	O
--	O
-BAR	O
--	O
-mediated	O
-membrane	O
-deformation	O
-with	O
-N	O
--	O
-WASP	O
-activation	O
-.	O
-	O
-We	O
-envisage	O
-a	O
-complex	O
-interplay	O
-of	O
-equilibria	O
-between	O
-free	O
-and	O
-bound	O
-,	O
-active	O
-and	O
-inactive	O
-Cdc42	O
-,	O
-TOCA	O
-family	O
-,	O
-and	O
-WASP	O
-family	O
-proteins	O
-,	O
-facilitating	O
-a	O
-tightly	O
-spatially	O
-and	O
-temporally	O
-regulated	O
-pathway	O
-requiring	O
-numerous	O
-simultaneous	O
-events	O
-in	O
-order	O
-to	O
-achieve	O
-appropriate	O
-and	O
-robust	O
-activation	O
-of	O
-the	O
-downstream	O
-pathway	O
-.	O
-	O
-Our	O
-data	O
-are	O
-therefore	O
-easily	O
-reconciled	O
-with	O
-the	O
-dynamic	O
-instability	O
-models	O
-described	O
-in	O
-relation	O
-to	O
-the	O
-formation	O
-of	O
-endocytic	O
-vesicles	O
-and	O
-with	O
-the	O
-current	O
-data	O
-pertaining	O
-to	O
-the	O
-complex	O
-activation	O
-of	O
-WASP	O
-/	O
-N	O
--	O
-WASP	O
-pathways	O
-by	O
-allosteric	O
-and	O
-oligomeric	O
-effects	O
-.	O
-	O
-It	O
-is	O
-clear	O
-from	O
-the	O
-data	O
-presented	O
-here	O
-that	O
-TOCA1	O
-and	O
-N	O
--	O
-WASP	O
-do	O
-not	O
-bind	O
-Cdc42	O
-simultaneously	O
-and	O
-that	O
-N	O
--	O
-WASP	O
-is	O
-likely	O
-to	O
-outcompete	O
-TOCA1	O
-for	O
-Cdc42	O
-binding	O
-.	O
-	O
-We	O
-therefore	O
-postulate	O
-an	O
-effector	O
-handover	O
-mechanism	O
-based	O
-on	O
-current	O
-evidence	O
-surrounding	O
-WASP	O
-/	O
-N	O
--	O
-WASP	O
-activation	O
-and	O
-our	O
-model	O
-of	O
-the	O
-Cdc42	O
-·	O
-HR1TOCA1	O
-complex	O
-.	O
-	O
-The	O
-displacement	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-from	O
-Cdc42	O
-by	O
-N	O
--	O
-WASP	O
-may	O
-represent	O
-a	O
-unidirectional	O
-step	O
-in	O
-the	O
-pathway	O
-of	O
-Cdc42	O
-·	O
-N	O
--	O
-WASP	O
-·	O
-TOCA1	O
--	O
-dependent	O
-actin	O
-assembly	O
-.	O
-	O
-The	O
-dynamic	O
-organization	O
-of	O
-fungal	O
-acetyl	O
--	O
-CoA	O
-carboxylase	O
-	O
-Acetyl	O
--	O
-CoA	O
-carboxylases	O
-(	O
-ACCs	O
-)	O
-catalyse	O
-the	O
-committed	O
-step	O
-in	O
-fatty	O
--	O
-acid	O
-biosynthesis	O
-:	O
-the	O
-ATP	O
--	O
-dependent	O
-carboxylation	O
-of	O
-acetyl	O
--	O
-CoA	O
-to	O
-malonyl	O
--	O
-CoA	O
-.	O
-They	O
-are	O
-important	O
-regulatory	O
-hubs	O
-for	O
-metabolic	O
-control	O
-and	O
-relevant	O
-drug	O
-targets	O
-for	O
-the	O
-treatment	O
-of	O
-the	O
-metabolic	O
-syndrome	O
-and	O
-cancer	O
-.	O
-	O
-Eukaryotic	O
-ACCs	O
-are	O
-single	O
--	O
-chain	O
-multienzymes	O
-characterized	O
-by	O
-a	O
-large	O
-,	O
-non	O
--	O
-catalytic	O
-central	O
-domain	O
-(	O
-CD	O
-),	O
-whose	O
-role	O
-in	O
-ACC	O
-regulation	O
-remains	O
-poorly	O
-characterized	O
-.	O
-	O
-Here	O
-we	O
-report	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-yeast	O
-ACC	O
-CD	O
-,	O
-revealing	O
-a	O
-unique	O
-four	O
--	O
-domain	O
-organization	O
-.	O
-	O
-A	O
-regulatory	O
-loop	O
-,	O
-which	O
-is	O
-phosphorylated	O
-at	O
-the	O
-key	O
-functional	O
-phosphorylation	O
-site	O
-of	O
-fungal	O
-ACC	O
-,	O
-wedges	O
-into	O
-a	O
-crevice	O
-between	O
-two	O
-domains	O
-of	O
-CD	O
-.	O
-	O
-Combining	O
-the	O
-yeast	O
-CD	O
-structure	O
-with	O
-intermediate	O
-and	O
-low	O
--	O
-resolution	O
-data	O
-of	O
-larger	B-mutant
-fragments	I-mutant
-up	O
-to	O
-intact	O
-ACCs	O
-provides	O
-a	O
-comprehensive	O
-characterization	O
-of	O
-the	O
-dynamic	O
-fungal	O
-ACC	O
-architecture	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-related	O
-carboxylases	O
-,	O
-large	O
--	O
-scale	O
-conformational	O
-changes	O
-are	O
-required	O
-for	O
-substrate	O
-turnover	O
-,	O
-and	O
-are	O
-mediated	O
-by	O
-the	O
-CD	O
-under	O
-phosphorylation	O
-control	O
-.	O
-	O
-Acetyl	O
--	O
-CoA	O
-carboxylases	O
-are	O
-central	O
-regulatory	O
-hubs	O
-of	O
-fatty	O
-acid	O
-metabolism	O
-and	O
-are	O
-important	O
-targets	O
-for	O
-drug	O
-development	O
-in	O
-obesity	O
-and	O
-cancer	O
-.	O
-	O
-Here	O
-,	O
-the	O
-authors	O
-demonstrate	O
-that	O
-the	O
-regulation	O
-of	O
-these	O
-highly	O
-dynamic	O
-enzymes	O
-in	O
-fungi	O
-is	O
-governed	O
-by	O
-a	O
-mechanism	O
-based	O
-on	O
-phosphorylation	O
--	O
-dependent	O
-conformational	O
-variability	O
-.	O
-	O
-Biotin	O
--	O
-dependent	O
-acetyl	O
--	O
-CoA	O
-carboxylases	O
-(	O
-ACCs	O
-)	O
-are	O
-essential	O
-enzymes	O
-that	O
-catalyse	O
-the	O
-ATP	O
--	O
-dependent	O
-carboxylation	O
-of	O
-acetyl	O
--	O
-CoA	O
-to	O
-malonyl	O
--	O
-CoA	O
-.	O
-This	O
-reaction	O
-provides	O
-the	O
-committed	O
-activated	O
-substrate	O
-for	O
-the	O
-biosynthesis	O
-of	O
-fatty	O
-acids	O
-via	O
-fatty	O
--	O
-acid	O
-synthase	O
-.	O
-	O
-By	O
-catalysing	O
-this	O
-rate	O
--	O
-limiting	O
-step	O
-in	O
-fatty	O
--	O
-acid	O
-biosynthesis	O
-,	O
-ACC	O
-plays	O
-a	O
-key	O
-role	O
-in	O
-anabolic	O
-metabolism	O
-.	O
-	O
-ACC	O
-inhibition	O
-and	O
-knock	O
--	O
-out	O
-studies	O
-show	O
-the	O
-potential	O
-of	O
-targeting	O
-ACC	O
-for	O
-treatment	O
-of	O
-the	O
-metabolic	O
-syndrome	O
-.	O
-	O
-Furthermore	O
-,	O
-elevated	O
-ACC	O
-activity	O
-is	O
-observed	O
-in	O
-malignant	O
-tumours	O
-.	O
-	O
-A	O
-direct	O
-link	O
-between	O
-ACC	O
-and	O
-cancer	O
-is	O
-provided	O
-by	O
-cancer	O
--	O
-associated	O
-mutations	B-mutant
-in	O
-the	O
-breast	O
-cancer	O
-susceptibility	O
-gene	O
-1	O
-(	O
-BRCA1	O
-),	O
-which	O
-relieve	O
-inhibitory	O
-interactions	O
-of	O
-BRCA1	O
-with	O
-ACC	O
-.	O
-	O
-Thus	O
-,	O
-ACC	O
-is	O
-a	O
-relevant	O
-drug	O
-target	O
-for	O
-type	O
-2	O
-diabetes	O
-and	O
-cancer	O
-.	O
-	O
-Microbial	O
-ACCs	O
-are	O
-also	O
-the	O
-principal	O
-target	O
-of	O
-antifungal	O
-and	O
-antibiotic	O
-compounds	O
-,	O
-such	O
-as	O
-Soraphen	O
-A	O
-.	O
-	O
-The	O
-principal	O
-functional	O
-protein	O
-components	O
-of	O
-ACCs	O
-have	O
-been	O
-described	O
-already	O
-in	O
-the	O
-late	O
-1960s	O
-for	O
-Escherichia	O
-coli	O
-(	O
-E	O
-.	O
-coli	O
-)	O
-ACC	O
-:	O
-Biotin	O
-carboxylase	O
-(	O
-BC	O
-)	O
-catalyses	O
-the	O
-ATP	O
--	O
-dependent	O
-carboxylation	O
-of	O
-a	O
-biotin	O
-moiety	O
-,	O
-which	O
-is	O
-covalently	O
-linked	O
-to	O
-the	O
-biotin	O
-carboxyl	O
-carrier	O
-protein	O
-(	O
-BCCP	O
-).	O
-	O
-Carboxyltransferase	O
-(	O
-CT	O
-)	O
-transfers	O
-the	O
-activated	O
-carboxyl	O
-group	O
-from	O
-carboxybiotin	O
-to	O
-acetyl	O
--	O
-CoA	O
-to	O
-yield	O
-malonyl	O
--	O
-CoA	O
-.	O
-Prokaryotic	O
-ACCs	O
-are	O
-transient	O
-assemblies	O
-of	O
-individual	O
-BC	O
-,	O
-CT	O
-and	O
-BCCP	O
-subunits	O
-.	O
-	O
-Eukaryotic	O
-ACCs	O
-,	O
-instead	O
-,	O
-are	O
-multienzymes	O
-,	O
-which	O
-integrate	O
-all	O
-functional	O
-components	O
-into	O
-a	O
-single	O
-polypeptide	O
-chain	O
-of	O
-∼	O
-2	O
-,	O
-300	O
-amino	O
-acids	O
-.	O
-	O
-Human	O
-ACC	O
-occurs	O
-in	O
-two	O
-closely	O
-related	O
-isoforms	O
-,	O
-ACC1	O
-and	O
-2	O
-,	O
-located	O
-in	O
-the	O
-cytosol	O
-and	O
-at	O
-the	O
-outer	O
-mitochondrial	O
-membrane	O
-,	O
-respectively	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-canonical	O
-ACC	O
-components	O
-,	O
-eukaryotic	O
-ACCs	O
-contain	O
-two	O
-non	O
--	O
-catalytic	O
-regions	O
-,	O
-the	O
-large	O
-central	O
-domain	O
-(	O
-CD	O
-)	O
-and	O
-the	O
-BC	O
-–	O
-CT	O
-interaction	O
-domain	O
-(	O
-BT	O
-).	O
-	O
-The	O
-CD	O
-comprises	O
-one	O
--	O
-third	O
-of	O
-the	O
-protein	O
-and	O
-is	O
-a	O
-unique	O
-feature	O
-of	O
-eukaryotic	O
-ACCs	O
-without	O
-homologues	O
-in	O
-other	O
-proteins	O
-.	O
-	O
-The	O
-function	O
-of	O
-this	O
-domain	O
-remains	O
-poorly	O
-characterized	O
-,	O
-although	O
-phosphorylation	O
-of	O
-several	O
-serine	O
-residues	O
-in	O
-the	O
-CD	O
-regulates	O
-ACC	O
-activity	O
-.	O
-	O
-The	O
-BT	O
-domain	O
-has	O
-been	O
-visualized	O
-in	O
-bacterial	O
-carboxylases	O
-,	O
-where	O
-it	O
-mediates	O
-contacts	O
-between	O
-α	O
--	O
-and	O
-β	O
--	O
-subunits	O
-.	O
-	O
-Structural	O
-studies	O
-on	O
-the	O
-functional	O
-architecture	O
-of	O
-intact	O
-ACCs	O
-have	O
-been	O
-hindered	O
-by	O
-their	O
-huge	O
-size	O
-and	O
-pronounced	O
-dynamics	O
-,	O
-as	O
-well	O
-as	O
-the	O
-transient	O
-assembly	O
-mode	O
-of	O
-bacterial	O
-ACCs	O
-.	O
-	O
-However	O
-,	O
-crystal	O
-structures	O
-of	O
-individual	O
-components	O
-or	O
-domains	O
-from	O
-prokaryotic	O
-and	O
-eukaryotic	O
-ACCs	O
-,	O
-respectively	O
-,	O
-have	O
-been	O
-solved	O
-.	O
-	O
-The	O
-structure	O
-determination	O
-of	O
-the	O
-holoenzymes	O
-of	O
-bacterial	O
-biotin	O
--	O
-dependent	O
-carboxylases	O
-,	O
-which	O
-lack	O
-the	O
-characteristic	O
-CD	O
-,	O
-such	O
-as	O
-the	O
-pyruvate	O
-carboxylase	O
-(	O
-PC	O
-),	O
-propionyl	O
--	O
-CoA	O
-carboxylase	O
-,	O
-3	O
--	O
-methyl	O
--	O
-crotonyl	O
--	O
-CoA	O
-carboxylase	O
-and	O
-a	O
-long	O
--	O
-chain	O
-acyl	O
--	O
-CoA	O
-carboxylase	O
-revealed	O
-strikingly	O
-divergent	O
-architectures	O
-despite	O
-a	O
-general	O
-conservation	O
-of	O
-all	O
-functional	O
-components	O
-.	O
-	O
-In	O
-these	O
-structures	O
-,	O
-the	O
-BC	O
-and	O
-CT	O
-active	O
-sites	O
-are	O
-at	O
-distances	O
-between	O
-40	O
-and	O
-80	O
-Å	O
-,	O
-such	O
-that	O
-substrate	O
-transfer	O
-could	O
-be	O
-mediated	O
-solely	O
-by	O
-the	O
-mobility	O
-of	O
-the	O
-flexibly	O
-tethered	O
-BCCP	O
-.	O
-	O
-Human	O
-ACC1	O
-is	O
-regulated	O
-allosterically	O
-,	O
-via	O
-specific	O
-protein	O
-–	O
-protein	O
-interactions	O
-,	O
-and	O
-by	O
-reversible	O
-phosphorylation	O
-.	O
-	O
-Dynamic	O
-polymerization	O
-of	O
-human	O
-ACC1	O
-is	O
-linked	O
-to	O
-increased	O
-activity	O
-and	O
-is	O
-regulated	O
-allosterically	O
-by	O
-the	O
-activator	O
-citrate	O
-and	O
-the	O
-inhibitor	O
-palmitate	O
-,	O
-or	O
-by	O
-binding	O
-of	O
-the	O
-small	O
-protein	O
-MIG	O
--	O
-12	O
-(	O
-ref	O
-.).	O
-	O
-Human	O
-ACC1	O
-is	O
-further	O
-regulated	O
-by	O
-specific	O
-phosphorylation	O
--	O
-dependent	O
-binding	O
-of	O
-BRCA1	O
-to	O
-Ser1263	O
-in	O
-the	O
-CD	O
-.	O
-	O
-BRCA1	O
-binds	O
-only	O
-to	O
-the	O
-phosphorylated	O
-form	O
-of	O
-ACC1	O
-and	O
-prevents	O
-ACC	O
-activation	O
-by	O
-phosphatase	O
--	O
-mediated	O
-dephosphorylation	O
-.	O
-	O
-Furthermore	O
-,	O
-phosphorylation	O
-by	O
-AMP	O
--	O
-activated	O
-protein	O
-kinase	O
-(	O
-AMPK	O
-)	O
-and	O
-cAMP	O
--	O
-dependent	O
-protein	O
-kinase	O
-(	O
-PKA	O
-)	O
-leads	O
-to	O
-a	O
-decrease	O
-in	O
-ACC1	O
-activity	O
-.	O
-	O
-AMPK	O
-phosphorylates	O
-ACC1	O
-in	O
-vitro	O
-at	O
-Ser80	O
-,	O
-Ser1201	O
-and	O
-Ser1216	O
-and	O
-PKA	O
-at	O
-Ser78	O
-and	O
-Ser1201	O
-.	O
-	O
-However	O
-,	O
-regulatory	O
-effects	O
-on	O
-ACC1	O
-activity	O
-are	O
-mainly	O
-mediated	O
-by	O
-phosphorylation	O
-of	O
-Ser80	O
-and	O
-Ser1201	O
-(	O
-refs	O
-).	O
-	O
-Phosphorylated	O
-Ser80	O
-,	O
-which	O
-is	O
-highly	O
-conserved	O
-only	O
-in	O
-higher	O
-eukaryotes	O
-,	O
-presumably	O
-binds	O
-into	O
-the	O
-Soraphen	O
-A	O
--	O
-binding	O
-pocket	O
-.	O
-	O
-The	O
-regulatory	O
-Ser1201	O
-shows	O
-only	O
-moderate	O
-conservation	O
-across	O
-higher	O
-eukaryotes	O
-,	O
-while	O
-the	O
-phosphorylated	O
-Ser1216	O
-is	O
-highly	O
-conserved	O
-across	O
-all	O
-eukaryotes	O
-.	O
-	O
-However	O
-,	O
-no	O
-effect	O
-of	O
-Ser1216	O
-phosphorylation	O
-on	O
-ACC	O
-activity	O
-has	O
-been	O
-reported	O
-in	O
-higher	O
-eukaryotes	O
-.	O
-	O
-For	O
-fungal	O
-ACC	O
-,	O
-neither	O
-spontaneous	O
-nor	O
-inducible	O
-polymerization	O
-has	O
-been	O
-detected	O
-despite	O
-considerable	O
-sequence	O
-conservation	O
-to	O
-human	O
-ACC1	O
-.	O
-	O
-The	O
-BRCA1	O
--	O
-interacting	O
-phosphoserine	O
-position	O
-is	O
-not	O
-conserved	O
-in	O
-fungal	O
-ACC	O
-,	O
-and	O
-no	O
-other	O
-phospho	O
--	O
-dependent	O
-protein	O
-–	O
-protein	O
-interactions	O
-of	O
-fungal	O
-ACC	O
-have	O
-been	O
-described	O
-.	O
-	O
-In	O
-yeast	O
-ACC	O
-,	O
-phosphorylation	O
-sites	O
-have	O
-been	O
-identified	O
-at	O
-Ser2	O
-,	O
-Ser735	O
-,	O
-Ser1148	O
-,	O
-Ser1157	O
-and	O
-Ser1162	O
-(	O
-ref	O
-.).	O
-	O
-Of	O
-these	O
-,	O
-only	O
-Ser1157	O
-is	O
-highly	O
-conserved	O
-in	O
-fungal	O
-ACC	O
-and	O
-aligns	O
-to	O
-Ser1216	O
-in	O
-human	O
-ACC1	O
-.	O
-	O
-Its	O
-phosphorylation	O
-by	O
-the	O
-AMPK	O
-homologue	O
-SNF1	O
-results	O
-in	O
-strongly	O
-reduced	O
-ACC	O
-activity	O
-.	O
-	O
-Despite	O
-the	O
-outstanding	O
-relevance	O
-of	O
-ACC	O
-in	O
-primary	O
-metabolism	O
-and	O
-disease	O
-,	O
-the	O
-dynamic	O
-organization	O
-and	O
-regulation	O
-of	O
-the	O
-giant	O
-eukaryotic	O
-,	O
-and	O
-in	O
-particular	O
-fungal	O
-ACC	O
-,	O
-remain	O
-poorly	O
-characterized	O
-.	O
-	O
-Here	O
-we	O
-provide	O
-the	O
-structure	O
-of	O
-Saccharomyces	O
-cerevisiae	O
-(	O
-Sce	O
-)	O
-ACC	O
-CD	O
-,	O
-intermediate	O
--	O
-and	O
-low	O
--	O
-resolution	O
-structures	O
-of	O
-human	O
-(	O
-Hsa	O
-)	O
-ACC	O
-CD	O
-and	O
-larger	B-mutant
-fragments	I-mutant
-of	O
-fungal	O
-ACC	O
-from	O
-Chaetomium	O
-thermophilum	O
-(	O
-Cth	O
-;	O
-Fig	O
-.	O
-1a	O
-).	O
-	O
-Integrating	O
-these	O
-data	O
-with	O
-small	O
--	O
-angle	O
-X	O
--	O
-ray	O
-scattering	O
-(	O
-SAXS	O
-)	O
-and	O
-electron	O
-microscopy	O
-(	O
-EM	O
-)	O
-observations	O
-yield	O
-a	O
-comprehensive	O
-representation	O
-of	O
-the	O
-dynamic	O
-structure	O
-and	O
-regulation	O
-of	O
-fungal	O
-ACC	O
-.	O
-	O
-The	O
-organization	O
-of	O
-the	O
-yeast	O
-ACC	O
-CD	O
-	O
-First	O
-,	O
-we	O
-focused	O
-on	O
-structure	O
-determination	O
-of	O
-the	O
-82	O
--	O
-kDa	O
-CD	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-CD	O
-of	O
-SceACC	O
-(	O
-SceCD	O
-)	O
-was	O
-determined	O
-at	O
-3	O
-.	O
-0	O
-Å	O
-resolution	O
-by	O
-experimental	O
-phasing	O
-and	O
-refined	O
-to	O
-Rwork	O
-/	O
-Rfree	O
-=	O
-0	O
-.	O
-20	O
-/	O
-0	O
-.	O
-24	O
-(	O
-Table	O
-1	O
-).	O
-	O
-The	O
-overall	O
-extent	O
-of	O
-the	O
-SceCD	O
-is	O
-70	O
-by	O
-75	O
-Å	O
-(	O
-Fig	O
-.	O
-1b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1a	O
-,	O
-b	O
-),	O
-and	O
-the	O
-attachment	O
-points	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-26	O
--	O
-residue	O
-linker	O
-to	O
-the	O
-BCCP	O
-domain	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-CT	O
-domain	O
-are	O
-separated	O
-by	O
-46	O
-Å	O
-(	O
-the	O
-N	O
--	O
-and	O
-C	O
-termini	O
-are	O
-indicated	O
-with	O
-spheres	O
-in	O
-Fig	O
-.	O
-1b	O
-).	O
-	O
-SceCD	O
-comprises	O
-four	O
-distinct	O
-domains	O
-,	O
-an	O
-N	O
--	O
-terminal	O
-α	O
--	O
-helical	O
-domain	O
-(	O
-CDN	O
-),	O
-and	O
-a	O
-central	O
-four	O
--	O
-helix	O
-bundle	O
-linker	O
-domain	O
-(	O
-CDL	O
-),	O
-followed	O
-by	O
-two	O
-α	O
-–	O
-β	O
--	O
-fold	O
-C	O
--	O
-terminal	O
-domains	O
-(	O
-CDC1	O
-/	O
-CDC2	O
-).	O
-	O
-CDN	O
-adopts	O
-a	O
-letter	O
-C	O
-shape	O
-,	O
-where	O
-one	O
-of	O
-the	O
-ends	O
-is	O
-a	O
-regular	O
-four	O
--	O
-helix	O
-bundle	O
-(	O
-Nα3	O
--	O
-6	O
-),	O
-the	O
-other	O
-end	O
-is	O
-a	O
-helical	O
-hairpin	O
-(	O
-Nα8	O
-,	O
-9	O
-)	O
-and	O
-the	O
-bridging	O
-region	O
-comprises	O
-six	O
-helices	O
-(	O
-Nα1	O
-,	O
-2	O
-,	O
-7	O
-,	O
-10	O
-–	O
-12	O
-).	O
-	O
-CDL	O
-is	O
-composed	O
-of	O
-a	O
-small	O
-,	O
-irregular	O
-four	O
--	O
-helix	O
-bundle	O
-(	O
-Lα1	O
-–	O
-4	O
-)	O
-and	O
-tightly	O
-interacts	O
-with	O
-the	O
-open	O
-face	O
-of	O
-CDC1	O
-via	O
-an	O
-interface	O
-of	O
-1	O
-,	O
-300	O
-Å2	O
-involving	O
-helices	O
-Lα3	O
-and	O
-Lα4	O
-.	O
-	O
-CDL	O
-does	O
-not	O
-interact	O
-with	O
-CDN	O
-apart	O
-from	O
-the	O
-covalent	O
-linkage	O
-and	O
-forms	O
-only	O
-a	O
-small	O
-contact	O
-to	O
-CDC2	O
-via	O
-a	O
-loop	O
-between	O
-Lα2	O
-/	O
-α3	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-end	O
-of	O
-Lα1	O
-,	O
-with	O
-an	O
-interface	O
-area	O
-of	O
-400	O
-Å2	O
-.	O
-	O
-CDC1	O
-/	O
-CDC2	O
-share	O
-a	O
-common	O
-fold	O
-;	O
-they	O
-are	O
-composed	O
-of	O
-six	O
--	O
-stranded	O
-β	O
--	O
-sheets	O
-flanked	O
-on	O
-one	O
-side	O
-by	O
-two	O
-long	O
-,	O
-bent	O
-helices	O
-inserted	O
-between	O
-strands	O
-β3	O
-/	O
-β4	O
-and	O
-β4	O
-/	O
-β5	O
-.	O
-	O
-CDC2	O
-is	O
-extended	O
-at	O
-its	O
-C	O
-terminus	O
-by	O
-an	O
-additional	O
-β	O
--	O
-strand	O
-and	O
-an	O
-irregular	O
-β	O
--	O
-hairpin	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-a	O
-root	O
-mean	O
-square	O
-deviation	O
-of	O
-main	O
-chain	O
-atom	O
-positions	O
-of	O
-2	O
-.	O
-2	O
-Å	O
-,	O
-CDC1	O
-/	O
-CDC2	O
-are	O
-structurally	O
-more	O
-closely	O
-related	O
-to	O
-each	O
-other	O
-than	O
-to	O
-any	O
-other	O
-protein	O
-(	O
-Fig	O
-.	O
-1c	O
-);	O
-they	O
-may	O
-thus	O
-have	O
-evolved	O
-by	O
-duplication	O
-.	O
-	O
-Close	O
-structural	O
-homologues	O
-could	O
-not	O
-be	O
-found	O
-for	O
-the	O
-CDN	O
-or	O
-the	O
-CDC	O
-domains	O
-.	O
-	O
-A	O
-regulatory	O
-loop	O
-mediates	O
-interdomain	O
-interactions	O
-	O
-To	O
-define	O
-the	O
-functional	O
-state	O
-of	O
-insect	O
--	O
-cell	O
--	O
-expressed	O
-ACC	O
-variants	O
-,	O
-we	O
-employed	O
-mass	O
-spectrometry	O
-(	O
-MS	O
-)	O
-for	O
-phosphorylation	O
-site	O
-detection	O
-.	O
-	O
-In	O
-insect	O
--	O
-cell	O
--	O
-expressed	O
-full	O
--	O
-length	O
-SceACC	O
-,	O
-the	O
-highly	O
-conserved	O
-Ser1157	O
-is	O
-the	O
-only	O
-fully	O
-occupied	O
-phosphorylation	O
-site	O
-with	O
-functional	O
-relevance	O
-in	O
-S	O
-.	O
-cerevisiae	O
-.	O
-	O
-Additional	O
-phosphorylation	O
-was	O
-detected	O
-for	O
-Ser2101	O
-and	O
-Tyr2179	O
-;	O
-however	O
-,	O
-these	O
-sites	O
-are	O
-neither	O
-conserved	O
-across	O
-fungal	O
-ACC	O
-nor	O
-natively	O
-phosphorylated	O
-in	O
-yeast	O
-.	O
-	O
-MS	O
-analysis	O
-of	O
-dissolved	O
-crystals	O
-confirmed	O
-the	O
-phosphorylated	O
-state	O
-of	O
-Ser1157	O
-also	O
-in	O
-SceCD	O
-crystals	O
-.	O
-	O
-The	O
-SceCD	O
-structure	O
-thus	O
-authentically	O
-represents	O
-the	O
-state	O
-of	O
-SceACC	O
-,	O
-where	O
-the	O
-enzyme	O
-is	O
-inhibited	O
-by	O
-SNF1	O
--	O
-dependent	O
-phosphorylation	O
-.	O
-	O
-In	O
-the	O
-SceCD	O
-crystal	O
-structure	O
-,	O
-the	O
-phosphorylated	O
-Ser1157	O
-resides	O
-in	O
-a	O
-regulatory	O
-36	O
--	O
-amino	O
--	O
-acid	O
-loop	O
-between	O
-strands	O
-β2	O
-and	O
-β3	O
-of	O
-CDC1	O
-(	O
-Fig	O
-.	O
-1b	O
-,	O
-d	O
-),	O
-which	O
-contains	O
-two	O
-additional	O
-less	O
--	O
-conserved	O
-phosphorylation	O
-sites	O
-(	O
-Ser1148	O
-and	O
-Ser1162	O
-)	O
-confirmed	O
-in	O
-yeast	O
-,	O
-but	O
-not	O
-occupied	O
-here	O
-.	O
-	O
-This	O
-regulatory	O
-loop	O
-wedges	O
-between	O
-the	O
-CDC1	O
-and	O
-CDC2	O
-domains	O
-and	O
-provides	O
-the	O
-largest	O
-contribution	O
-to	O
-the	O
-interdomain	O
-interface	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-regulatory	O
-loop	O
-also	O
-directly	O
-contacts	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-CDC2	O
-leading	O
-into	O
-CT	O
-.	O
-	O
-Phosphoserine	O
-1157	O
-is	O
-tightly	O
-bound	O
-by	O
-two	O
-highly	O
-conserved	O
-arginines	O
-(	O
-Arg1173	O
-and	O
-Arg1260	O
-)	O
-of	O
-CDC1	O
-(	O
-Fig	O
-.	O
-1d	O
-).	O
-	O
-Already	O
-the	O
-binding	O
-of	O
-phosphorylated	O
-Ser1157	O
-apparently	O
-stabilizes	O
-the	O
-regulatory	O
-loop	O
-conformation	O
-;	O
-the	O
-accessory	O
-phosphorylation	O
-sites	O
-Ser1148	O
-and	O
-Ser1162	O
-in	O
-the	O
-same	O
-loop	O
-may	O
-further	O
-modulate	O
-the	O
-strength	O
-of	O
-interaction	O
-between	O
-the	O
-regulatory	O
-loop	O
-and	O
-the	O
-CDC1	O
-and	O
-CDC2	O
-domains	O
-.	O
-	O
-Phosphorylation	O
-of	O
-the	O
-regulatory	O
-loop	O
-thus	O
-determines	O
-interdomain	O
-interactions	O
-of	O
-CDC1	O
-and	O
-CDC2	O
-,	O
-suggesting	O
-that	O
-it	O
-may	O
-exert	O
-its	O
-regulatory	O
-function	O
-by	O
-modifying	O
-the	O
-overall	O
-structure	O
-and	O
-dynamics	O
-of	O
-the	O
-CD	O
-.	O
-	O
-The	O
-functional	O
-role	O
-of	O
-Ser1157	O
-was	O
-confirmed	O
-by	O
-an	O
-activity	O
-assay	O
-based	O
-on	O
-the	O
-incorporation	O
-of	O
-radioactive	O
-carbonate	O
-into	O
-acid	O
-non	O
--	O
-volatile	O
-material	O
-.	O
-	O
-Phosphorylated	O
-SceACC	O
-shows	O
-only	O
-residual	O
-activity	O
-(	O
-kcat	O
-=	O
-0	O
-.	O
-4	O
-±	O
-0	O
-.	O
-2	O
-s	O
-−	O
-1	O
-,	O
-s	O
-.	O
-d	O
-.	O
-based	O
-on	O
-five	O
-replicate	O
-measurements	O
-),	O
-which	O
-increases	O
-16	O
--	O
-fold	O
-(	O
-kcat	O
-=	O
-6	O
-.	O
-5	O
-±	O
-0	O
-.	O
-3	O
-s	O
-−	O
-1	O
-)	O
-after	O
-dephosphorylation	O
-with	O
-λ	O
-protein	O
-phosphatase	O
-.	O
-	O
-The	O
-values	O
-obtained	O
-for	O
-dephosphorylated	O
-SceACC	O
-are	O
-comparable	O
-to	O
-earlier	O
-measurements	O
-of	O
-non	O
--	O
-phosphorylated	O
-yeast	O
-ACC	O
-expressed	O
-in	O
-E	O
-.	O
-coli	O
-.	O
-	O
-The	O
-variable	O
-CD	O
-is	O
-conserved	O
-between	O
-yeast	O
-and	O
-human	O
-	O
-To	O
-compare	O
-the	O
-organization	O
-of	O
-fungal	O
-and	O
-human	O
-ACC	O
-CD	O
-,	O
-we	O
-determined	O
-the	O
-structure	O
-of	O
-a	O
-human	O
-ACC1	B-mutant
-fragment	I-mutant
-that	O
-comprises	O
-the	O
-BT	O
-and	O
-CD	O
-domains	O
-(	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-),	O
-but	O
-lacks	O
-the	O
-mobile	O
-BCCP	O
-in	O
-between	O
-(	O
-Fig	O
-.	O
-1a	O
-).	O
-	O
-An	O
-experimentally	O
-phased	O
-map	O
-was	O
-obtained	O
-at	O
-3	O
-.	O
-7	O
-Å	O
-resolution	O
-for	O
-a	O
-cadmium	O
--	O
-derivatized	O
-crystal	O
-and	O
-was	O
-interpreted	O
-by	O
-a	O
-poly	O
--	O
-alanine	O
-model	O
-(	O
-Fig	O
-.	O
-1e	O
-and	O
-Table	O
-1	O
-).	O
-	O
-Each	O
-of	O
-the	O
-four	O
-CD	O
-domains	O
-in	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-individually	O
-resembles	O
-the	O
-corresponding	O
-SceCD	O
-domain	O
-;	O
-however	O
-,	O
-human	O
-and	O
-yeast	O
-CDs	O
-exhibit	O
-distinct	O
-overall	O
-structures	O
-.	O
-	O
-In	O
-agreement	O
-with	O
-their	O
-tight	O
-interaction	O
-in	O
-SceCD	O
-,	O
-the	O
-relative	O
-spatial	O
-arrangement	O
-of	O
-CDL	O
-and	O
-CDC1	O
-is	O
-preserved	O
-in	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-,	O
-but	O
-the	O
-human	O
-CDL	O
-/	O
-CDC1	O
-didomain	O
-is	O
-tilted	O
-by	O
-30	O
-°	O
-based	O
-on	O
-a	O
-superposition	O
-of	O
-human	O
-and	O
-yeast	O
-CDC2	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-As	O
-a	O
-result	O
-,	O
-the	O
-N	O
-terminus	O
-of	O
-CDL	O
-at	O
-helix	O
-Lα1	O
-,	O
-which	O
-connects	O
-to	O
-CDN	O
-,	O
-is	O
-shifted	O
-by	O
-12	O
-Å	O
-.	O
-Remarkably	O
-,	O
-CDN	O
-of	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-adopts	O
-a	O
-completely	O
-different	O
-orientation	O
-compared	O
-with	O
-SceCD	O
-.	O
-	O
-With	O
-CDL	O
-/	O
-CDC1	O
-superposed	O
-,	O
-CDN	O
-in	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-is	O
-rotated	O
-by	O
-160	O
-°	O
-around	O
-a	O
-hinge	O
-at	O
-the	O
-connection	O
-of	O
-CDN	O
-/	O
-CDL	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1d	O
-).	O
-	O
-This	O
-rotation	O
-displaces	O
-the	O
-N	O
-terminus	O
-of	O
-CDN	O
-in	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-by	O
-51	O
-Å	O
-compared	O
-with	O
-SceCD	O
-,	O
-resulting	O
-in	O
-a	O
-separation	O
-of	O
-the	O
-attachment	O
-points	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-linker	O
-to	O
-the	O
-BCCP	O
-domain	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-CT	O
-domain	O
-by	O
-67	O
-Å	O
-(	O
-the	O
-attachment	O
-points	O
-are	O
-indicated	O
-with	O
-spheres	O
-in	O
-Fig	O
-.	O
-1e	O
-).	O
-	O
-The	O
-BT	O
-domain	O
-of	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-consists	O
-of	O
-a	O
-helix	O
-that	O
-is	O
-surrounded	O
-at	O
-its	O
-N	O
-terminus	O
-by	O
-an	O
-antiparallel	O
-eight	O
--	O
-stranded	O
-β	O
--	O
-barrel	O
-.	O
-	O
-It	O
-resembles	O
-the	O
-BT	O
-of	O
-propionyl	O
--	O
-CoA	O
-carboxylase	O
-;	O
-only	O
-the	O
-four	O
-C	O
--	O
-terminal	O
-strands	O
-of	O
-the	O
-β	O
--	O
-barrel	O
-are	O
-slightly	O
-tilted	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-MS	O
-analysis	O
-of	O
-insect	O
--	O
-cell	O
--	O
-expressed	O
-human	O
-full	O
--	O
-length	O
-ACC	O
-,	O
-Ser80	O
-shows	O
-the	O
-highest	O
-degree	O
-of	O
-phosphorylation	O
-(	O
-90	O
-%).	O
-	O
-Ser29	O
-and	O
-Ser1263	O
-,	O
-implicated	O
-in	O
-insulin	O
--	O
-dependent	O
-phosphorylation	O
-and	O
-BRCA1	O
-binding	O
-,	O
-respectively	O
-,	O
-are	O
-phosphorylated	O
-at	O
-intermediate	O
-levels	O
-(	O
-40	O
-%).	O
-	O
-The	O
-highly	O
-conserved	O
-Ser1216	O
-(	O
-corresponding	O
-to	O
-S	O
-.	O
-cerevisiae	O
-Ser1157	O
-),	O
-as	O
-well	O
-as	O
-Ser1201	O
-,	O
-both	O
-in	O
-the	O
-regulatory	O
-loop	O
-discussed	O
-above	O
-,	O
-are	O
-not	O
-phosphorylated	O
-.	O
-	O
-However	O
-,	O
-residual	O
-phosphorylation	O
-levels	O
-were	O
-detected	O
-for	O
-Ser1204	O
-(	O
-7	O
-%)	O
-and	O
-Ser1218	O
-(	O
-7	O
-%)	O
-in	O
-the	O
-same	O
-loop	O
-.	O
-	O
-MS	O
-analysis	O
-of	O
-the	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-crystallization	O
-sample	O
-reveals	O
-partial	O
-proteolytic	O
-digestion	O
-of	O
-the	O
-regulatory	O
-loop	O
-.	O
-	O
-Accordingly	O
-,	O
-most	O
-of	O
-this	O
-loop	O
-is	O
-not	O
-represented	O
-in	O
-the	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-crystal	O
-structure	O
-.	O
-	O
-The	O
-absence	O
-of	O
-the	O
-regulatory	O
-loop	O
-might	O
-be	O
-linked	O
-to	O
-the	O
-less	O
--	O
-restrained	O
-interface	O
-of	O
-CDL	O
-/	O
-CDC1	O
-and	O
-CDC2	O
-and	O
-altered	O
-relative	O
-orientations	O
-of	O
-these	O
-domains	O
-.	O
-	O
-Besides	O
-the	O
-regulatory	O
-loop	O
-,	O
-also	O
-the	O
-phosphopeptide	O
-target	O
-region	O
-for	O
-BRCA1	O
-interaction	O
-is	O
-not	O
-resolved	O
-presumably	O
-because	O
-of	O
-pronounced	O
-flexibility	O
-.	O
-	O
-At	O
-the	O
-level	O
-of	O
-isolated	O
-yeast	O
-and	O
-human	O
-CD	O
-,	O
-the	O
-structural	O
-analysis	O
-indicates	O
-the	O
-presence	O
-of	O
-at	O
-least	O
-two	O
-hinges	O
-,	O
-one	O
-with	O
-large	O
--	O
-scale	O
-flexibility	O
-at	O
-the	O
-CDN	O
-/	O
-CDL	O
-connection	O
-,	O
-and	O
-one	O
-with	O
-tunable	O
-plasticity	O
-between	O
-CDL	O
-/	O
-CDC1	O
-and	O
-CDC2	O
-,	O
-plausibly	O
-affected	O
-by	O
-phosphorylation	O
-in	O
-the	O
-regulatory	O
-loop	O
-region	O
-.	O
-	O
-The	O
-integration	O
-of	O
-CD	O
-into	O
-the	O
-fungal	O
-ACC	O
-multienzyme	O
-	O
-To	O
-further	O
-obtain	O
-insights	O
-into	O
-the	O
-functional	O
-architecture	O
-of	O
-fungal	O
-ACC	O
-,	O
-we	O
-characterized	O
-larger	B-mutant
-multidomain	I-mutant
-fragments	I-mutant
-up	O
-to	O
-the	O
-intact	O
-enzymes	O
-.	O
-	O
-Using	O
-molecular	O
-replacement	O
-based	O
-on	O
-fungal	O
-ACC	O
-CD	O
-and	O
-CT	O
-models	O
-,	O
-we	O
-obtained	O
-structures	O
-of	O
-a	O
-variant	B-mutant
-comprising	O
-CthCT	O
-and	O
-CDC1	O
-/	O
-CDC2	O
-in	O
-two	O
-crystal	O
-forms	O
-at	O
-resolutions	O
-of	O
-3	O
-.	O
-6	O
-and	O
-4	O
-.	O
-5	O
-Å	O
-(	O
-CthCD	B-mutant
--	I-mutant
-CTCter1	I-mutant
-/	I-mutant
-2	I-mutant
-),	O
-respectively	O
-,	O
-as	O
-well	O
-as	O
-of	O
-a	O
-CthCT	O
-linked	O
-to	O
-the	O
-entire	O
-CD	O
-at	O
-7	O
-.	O
-2	O
-Å	O
-resolution	O
-(	O
-CthCD	B-mutant
--	I-mutant
-CT	I-mutant
-;	O
-Figs	O
-1a	O
-and	O
-2	O
-,	O
-Table	O
-1	O
-).	O
-	O
-No	O
-crystals	O
-diffracting	O
-to	O
-sufficient	O
-resolution	O
-were	O
-obtained	O
-for	O
-larger	B-mutant
-BC	I-mutant
--	I-mutant
-containing	I-mutant
-fragments	I-mutant
-,	O
-or	O
-for	O
-full	O
--	O
-length	O
-Cth	O
-or	O
-SceACC	O
-.	O
-	O
-To	O
-improve	O
-crystallizability	O
-,	O
-we	O
-generated	O
-ΔBCCP	B-mutant
-variants	I-mutant
-of	O
-full	O
--	O
-length	O
-ACC	O
-,	O
-which	O
-,	O
-based	O
-on	O
-SAXS	O
-analysis	O
-,	O
-preserve	O
-properties	O
-of	O
-intact	O
-ACC	O
-(	O
-Supplementary	O
-Table	O
-1	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2a	O
-–	O
-c	O
-).	O
-	O
-For	O
-CthΔBCCP	B-mutant
-,	O
-crystals	O
-diffracting	O
-to	O
-8	O
-.	O
-4	O
-Å	O
-resolution	O
-were	O
-obtained	O
-.	O
-	O
-However	O
-,	O
-molecular	O
-replacement	O
-did	O
-not	O
-reveal	O
-a	O
-unique	O
-positioning	O
-of	O
-the	O
-BC	O
-domain	O
-.	O
-	O
-Owing	O
-to	O
-the	O
-limited	O
-resolution	O
-the	O
-discussion	O
-of	O
-structures	O
-of	O
-CthCD	B-mutant
--	I-mutant
-CT	I-mutant
-and	O
-CthΔBCCP	B-mutant
-is	O
-restricted	O
-to	O
-the	O
-analysis	O
-of	O
-domain	O
-localization	O
-.	O
-	O
-Still	O
-,	O
-these	O
-structures	O
-contribute	O
-considerably	O
-to	O
-the	O
-visualization	O
-of	O
-an	O
-intrinsically	O
-dynamic	O
-fungal	O
-ACC	O
-.	O
-	O
-In	O
-all	O
-these	O
-crystal	O
-structures	O
-,	O
-the	O
-CT	O
-domains	O
-build	O
-a	O
-canonical	O
-head	O
--	O
-to	O
--	O
-tail	O
-dimer	O
-,	O
-with	O
-active	O
-sites	O
-formed	O
-by	O
-contributions	O
-from	O
-both	O
-protomers	O
-(	O
-Fig	O
-.	O
-2	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-The	O
-connection	O
-of	O
-CD	O
-and	O
-CT	O
-is	O
-provided	O
-by	O
-a	O
-10	O
--	O
-residue	O
-peptide	O
-stretch	O
-,	O
-which	O
-links	O
-the	O
-N	O
-terminus	O
-of	O
-CT	O
-to	O
-the	O
-irregular	O
-β	O
--	O
-hairpin	O
-/	O
-β	O
--	O
-strand	O
-extension	O
-of	O
-CDC2	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-The	O
-connecting	O
-region	O
-is	O
-remarkably	O
-similar	O
-in	O
-isolated	O
-CD	O
-and	O
-CthCD	B-mutant
--	I-mutant
-CTCter	I-mutant
-structures	O
-,	O
-indicating	O
-inherent	O
-conformational	O
-stability	O
-.	O
-	O
-CD	O
-/	O
-CT	O
-contacts	O
-are	O
-only	O
-formed	O
-in	O
-direct	O
-vicinity	O
-of	O
-the	O
-covalent	O
-linkage	O
-and	O
-involve	O
-the	O
-β	O
--	O
-hairpin	O
-extension	O
-of	O
-CDC2	O
-as	O
-well	O
-as	O
-the	O
-loop	O
-between	O
-strands	O
-β2	O
-/	O
-β3	O
-of	O
-the	O
-CT	O
-N	O
--	O
-lobe	O
-,	O
-which	O
-contains	O
-a	O
-conserved	O
-RxxGxN	O
-motif	O
-.	O
-	O
-The	O
-neighbouring	O
-loop	O
-on	O
-the	O
-CT	O
-side	O
-(	O
-between	O
-CT	O
-β1	O
-/	O
-β2	O
-)	O
-is	O
-displaced	O
-by	O
-2	O
-.	O
-5	O
-Å	O
-compared	O
-to	O
-isolated	O
-CT	O
-structures	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3c	O
-).	O
-	O
-On	O
-the	O
-basis	O
-of	O
-an	O
-interface	O
-area	O
-of	O
-∼	O
-600	O
-Å2	O
-and	O
-its	O
-edge	O
--	O
-to	O
--	O
-edge	O
-connection	O
-characteristics	O
-,	O
-the	O
-interface	O
-between	O
-CT	O
-and	O
-CD	O
-might	O
-be	O
-classified	O
-as	O
-conformationally	O
-variable	O
-.	O
-	O
-Indeed	O
-,	O
-the	O
-comparison	O
-of	O
-the	O
-positioning	O
-of	O
-eight	O
-instances	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-part	O
-of	O
-CD	O
-relative	O
-to	O
-CT	O
-in	O
-crystal	O
-structures	O
-determined	O
-here	O
-,	O
-reveals	O
-flexible	O
-interdomain	O
-linking	O
-(	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-The	O
-CDC2	O
-/	O
-CT	O
-interface	O
-acts	O
-as	O
-a	O
-true	O
-hinge	O
-with	O
-observed	O
-rotation	O
-up	O
-to	O
-16	O
-°,	O
-which	O
-results	O
-in	O
-a	O
-translocation	O
-of	O
-the	O
-distal	O
-end	O
-of	O
-CDC2	O
-by	O
-8	O
-Å	O
-.	O
-	O
-The	O
-interface	O
-between	O
-CDC2	O
-and	O
-CDL	O
-/	O
-CDC1	O
-,	O
-which	O
-is	O
-mediated	O
-by	O
-the	O
-phosphorylated	O
-regulatory	O
-loop	O
-in	O
-the	O
-SceCD	O
-structure	O
-,	O
-is	O
-less	O
-variable	O
-than	O
-the	O
-CD	O
-–	O
-CT	O
-junction	O
-,	O
-and	O
-permits	O
-only	O
-limited	O
-rotation	O
-and	O
-tilting	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-Analysis	O
-of	O
-the	O
-impact	O
-of	O
-phosphorylation	O
-on	O
-the	O
-interface	O
-between	O
-CDC2	O
-and	O
-CDL	O
-/	O
-CDC1	O
-in	O
-CthACC	B-mutant
-variant	I-mutant
-structures	O
-is	O
-precluded	O
-by	O
-the	O
-limited	O
-crystallographic	O
-resolution	O
-.	O
-	O
-However	O
-,	O
-MS	O
-analysis	O
-of	O
-CthCD	B-mutant
--	I-mutant
-CT	I-mutant
-and	O
-CthΔBCCP	B-mutant
-constructs	O
-revealed	O
-between	O
-60	O
-and	O
-70	O
-%	O
-phosphorylation	O
-of	O
-Ser1170	O
-(	O
-corresponding	O
-to	O
-SceACC	O
-Ser1157	O
-).	O
-	O
-The	O
-CDN	O
-domain	O
-positioning	O
-relative	O
-to	O
-CDL	O
-/	O
-CDC1	O
-is	O
-highly	O
-variable	O
-with	O
-three	O
-main	O
-orientations	O
-observed	O
-in	O
-the	O
-structures	O
-of	O
-SceCD	O
-and	O
-the	O
-larger	B-mutant
-CthACC	I-mutant
-fragments	I-mutant
-:	O
-CDN	O
-tilts	O
-,	O
-resulting	O
-in	O
-a	O
-displacement	O
-of	O
-its	O
-N	O
-terminus	O
-by	O
-23	O
-Å	O
-(	O
-Fig	O
-.	O
-4a	O
-,	O
-observed	O
-in	O
-both	O
-protomers	O
-of	O
-CthCD	B-mutant
--	I-mutant
-CT	I-mutant
-and	O
-one	O
-protomer	O
-of	O
-CthΔBCCP	B-mutant
-,	O
-denoted	O
-as	O
-CthCD	B-mutant
--	I-mutant
-CT1	I-mutant
-/	I-mutant
-2	I-mutant
-and	O
-CthΔBCCP1	B-mutant
-,	O
-respectively	O
-).	O
-	O
-In	O
-addition	O
-,	O
-CDN	O
-can	O
-rotate	O
-around	O
-hinges	O
-in	O
-the	O
-connection	O
-between	O
-CDN	O
-/	O
-CDL	O
-by	O
-70	O
-°	O
-(	O
-Fig	O
-.	O
-4b	O
-,	O
-observed	O
-in	O
-the	O
-second	O
-protomer	O
-of	O
-CthΔBCCP	B-mutant
-,	O
-denoted	O
-as	O
-CthΔBCCP2	B-mutant
-)	O
-and	O
-160	O
-°	O
-(	O
-Fig	O
-.	O
-4c	O
-,	O
-observed	O
-in	O
-SceCD	O
-)	O
-leading	O
-to	O
-displacement	O
-of	O
-the	O
-anchor	O
-site	O
-for	O
-the	O
-BCCP	O
-linker	O
-by	O
-up	O
-to	O
-33	O
-and	O
-40	O
-Å	O
-,	O
-respectively	O
-.	O
-	O
-Conformational	O
-variability	O
-in	O
-the	O
-CD	O
-thus	O
-contributes	O
-considerably	O
-to	O
-variations	O
-in	O
-the	O
-spacing	O
-between	O
-the	O
-BC	O
-and	O
-CT	O
-domains	O
-,	O
-and	O
-may	O
-extend	O
-to	O
-distance	O
-variations	O
-beyond	O
-the	O
-mobility	O
-range	O
-of	O
-the	O
-flexibly	O
-tethered	O
-BCCP	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-the	O
-occurrence	O
-of	O
-related	O
-conformational	O
-changes	O
-between	O
-fungal	O
-and	O
-human	O
-ACC	B-mutant
-fragments	I-mutant
-,	O
-the	O
-observed	O
-set	O
-of	O
-conformations	O
-may	O
-well	O
-represent	O
-general	O
-states	O
-present	O
-in	O
-all	O
-eukaryotic	O
-ACCs	O
-.	O
-	O
-Large	O
--	O
-scale	O
-conformational	O
-variability	O
-of	O
-fungal	O
-ACC	O
-	O
-To	O
-obtain	O
-a	O
-comprehensive	O
-view	O
-of	O
-fungal	O
-ACC	O
-dynamics	O
-in	O
-solution	O
-,	O
-we	O
-employed	O
-SAXS	O
-and	O
-EM	O
-.	O
-	O
-SAXS	O
-analysis	O
-of	O
-CthACC	O
-agrees	O
-with	O
-a	O
-dimeric	O
-state	O
-and	O
-an	O
-elongated	O
-shape	O
-with	O
-a	O
-maximum	O
-extent	O
-of	O
-350	O
-Å	O
-(	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-The	O
-smooth	O
-appearance	O
-of	O
-scattering	O
-curves	O
-and	O
-derived	O
-distance	O
-distributions	O
-might	O
-indicate	O
-substantial	O
-interdomain	O
-flexibility	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2a	O
-–	O
-c	O
-).	O
-	O
-Direct	O
-observation	O
-of	O
-individual	O
-full	O
--	O
-length	O
-CthACC	O
-particles	O
-,	O
-according	O
-to	O
-MS	O
-results	O
-predominantly	O
-in	O
-a	O
-phosphorylated	O
-low	O
--	O
-activity	O
-state	O
-,	O
-in	O
-negative	O
-stain	O
-EM	O
-reveals	O
-a	O
-large	O
-set	O
-of	O
-conformations	O
-from	O
-rod	O
--	O
-like	O
-extended	O
-to	O
-U	O
--	O
-shaped	O
-particles	O
-.	O
-	O
-Class	O
-averages	O
-,	O
-obtained	O
-by	O
-maximum	O
--	O
-likelihood	O
--	O
-based	O
-two	O
--	O
-dimensional	O
-(	O
-2D	O
-)	O
-classification	O
-,	O
-are	O
-focused	O
-on	O
-the	O
-dimeric	O
-CT	O
-domain	O
-and	O
-the	O
-full	O
-BC	B-mutant
-–	I-mutant
-BCCP	I-mutant
-–	I-mutant
-CD	I-mutant
-domain	O
-of	O
-only	O
-one	O
-protomer	O
-,	O
-due	O
-to	O
-the	O
-non	O
--	O
-coordinated	O
-motions	O
-of	O
-the	O
-lateral	O
-BC	O
-/	O
-CD	O
-regions	O
-relative	O
-to	O
-the	O
-CT	O
-dimer	O
-.	O
-	O
-They	O
-identify	O
-the	O
-connections	O
-between	O
-CDN	O
-/	O
-CDL	O
-and	O
-between	O
-CDC2	O
-/	O
-CT	O
-as	O
-major	O
-contributors	O
-to	O
-conformational	O
-heterogeneity	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4a	O
-,	O
-b	O
-).	O
-	O
-The	O
-flexibility	O
-in	O
-the	O
-CDC2	O
-/	O
-CT	O
-hinge	O
-appears	O
-substantially	O
-larger	O
-than	O
-the	O
-variations	O
-observed	O
-in	O
-the	O
-set	O
-of	O
-crystal	O
-structures	O
-.	O
-	O
-The	O
-BC	O
-domain	O
-is	O
-not	O
-completely	O
-disordered	O
-,	O
-but	O
-laterally	O
-attached	O
-to	O
-BT	O
-/	O
-CDN	O
-in	O
-a	O
-generally	O
-conserved	O
-position	O
-,	O
-albeit	O
-with	O
-increased	O
-flexibility	O
-.	O
-	O
-Surprisingly	O
-,	O
-in	O
-both	O
-the	O
-linear	O
-and	O
-U	O
--	O
-shaped	O
-conformations	O
-,	O
-the	O
-approximate	O
-distances	O
-between	O
-the	O
-BC	O
-and	O
-CT	O
-active	O
-sites	O
-would	O
-remain	O
-larger	O
-than	O
-110	O
-Å	O
-.	O
-These	O
-observed	O
-distances	O
-are	O
-considerably	O
-larger	O
-than	O
-in	O
-static	O
-structures	O
-of	O
-any	O
-other	O
-related	O
-biotin	O
--	O
-dependent	O
-carboxylase	O
-.	O
-	O
-Furthermore	O
-,	O
-based	O
-on	O
-an	O
-average	O
-length	O
-of	O
-the	O
-BCCP	O
-–	O
-CD	O
-linker	O
-in	O
-fungal	O
-ACC	O
-of	O
-26	O
-amino	O
-acids	O
-,	O
-mobility	O
-of	O
-the	O
-BCCP	O
-alone	O
-would	O
-not	O
-be	O
-sufficient	O
-to	O
-bridge	O
-the	O
-active	O
-sites	O
-of	O
-BC	O
-and	O
-CT	O
-.	O
-	O
-The	O
-most	O
-relevant	O
-candidate	O
-site	O
-for	O
-mediating	O
-such	O
-additional	O
-flexibility	O
-and	O
-permitting	O
-an	O
-extended	O
-set	O
-of	O
-conformations	O
-is	O
-the	O
-CDC1	O
-/	O
-CDC2	O
-interface	O
-,	O
-which	O
-is	O
-rigidified	O
-by	O
-the	O
-Ser1157	O
--	O
-phosphorylated	O
-regulatory	O
-loop	O
-,	O
-as	O
-depicted	O
-in	O
-the	O
-SceCD	O
-crystal	O
-structure	O
-.	O
-	O
-Altogether	O
-,	O
-the	O
-architecture	O
-of	O
-fungal	O
-ACC	O
-is	O
-based	O
-on	O
-the	O
-central	O
-dimeric	O
-CT	O
-domain	O
-(	O
-Fig	O
-.	O
-4d	O
-).	O
-	O
-The	O
-CD	O
-consists	O
-of	O
-four	O
-distinct	O
-subdomains	O
-and	O
-acts	O
-as	O
-a	O
-tether	O
-from	O
-the	O
-CT	O
-to	O
-the	O
-mobile	O
-BCCP	O
-and	O
-an	O
-oriented	O
-BC	O
-domain	O
-.	O
-	O
-The	O
-CD	O
-has	O
-no	O
-direct	O
-role	O
-in	O
-substrate	O
-recognition	O
-or	O
-catalysis	O
-but	O
-contributes	O
-to	O
-the	O
-regulation	O
-of	O
-all	O
-eukaryotic	O
-ACCs	O
-.	O
-	O
-In	O
-higher	O
-eukaryotic	O
-ACCs	O
-,	O
-regulation	O
-via	O
-phosphorylation	O
-is	O
-achieved	O
-by	O
-combining	O
-the	O
-effects	O
-of	O
-phosphorylation	O
-at	O
-Ser80	O
-,	O
-Ser1201	O
-and	O
-Ser1263	O
-.	O
-	O
-In	O
-fungal	O
-ACC	O
-,	O
-however	O
-,	O
-Ser1157	O
-in	O
-the	O
-regulatory	O
-loop	O
-of	O
-the	O
-CD	O
-is	O
-the	O
-only	O
-phosphorylation	O
-site	O
-that	O
-has	O
-been	O
-demonstrated	O
-to	O
-be	O
-both	O
-phosphorylated	O
-in	O
-vivo	O
-and	O
-involved	O
-in	O
-the	O
-regulation	O
-of	O
-ACC	O
-activity	O
-.	O
-	O
-In	O
-its	O
-phosphorylated	O
-state	O
-,	O
-the	O
-regulatory	O
-loop	O
-containing	O
-Ser1157	O
-wedges	O
-between	O
-CDC1	O
-/	O
-CDC2	O
-and	O
-presumably	O
-limits	O
-the	O
-conformational	O
-freedom	O
-at	O
-this	O
-interdomain	O
-interface	O
-.	O
-	O
-However	O
-,	O
-flexibility	O
-at	O
-this	O
-hinge	O
-may	O
-be	O
-required	O
-for	O
-full	O
-ACC	O
-activity	O
-,	O
-as	O
-the	O
-distances	O
-between	O
-the	O
-BCCP	O
-anchor	O
-points	O
-and	O
-the	O
-active	O
-sites	O
-of	O
-BC	O
-and	O
-CT	O
-observed	O
-here	O
-are	O
-such	O
-large	O
-that	O
-mobility	O
-of	O
-the	O
-BCCP	O
-alone	O
-is	O
-not	O
-sufficient	O
-for	O
-substrate	O
-transfer	O
-.	O
-	O
-The	O
-current	O
-data	O
-thus	O
-suggest	O
-that	O
-regulation	O
-of	O
-fungal	O
-ACC	O
-is	O
-mediated	O
-by	O
-controlling	O
-the	O
-dynamics	O
-of	O
-the	O
-unique	O
-CD	O
-,	O
-rather	O
-than	O
-directly	O
-affecting	O
-catalytic	O
-turnover	O
-at	O
-the	O
-active	O
-sites	O
-of	O
-BC	O
-and	O
-CT	O
-.	O
-	O
-A	O
-comparison	O
-between	O
-fungal	O
-and	O
-human	O
-ACC	O
-will	O
-help	O
-to	O
-further	O
-discriminate	O
-mechanistic	O
-differences	O
-that	O
-contribute	O
-to	O
-the	O
-extended	O
-control	O
-and	O
-polymerization	O
-of	O
-human	O
-ACC	O
-.	O
-	O
-Most	O
-recently	O
-,	O
-a	O
-crystal	O
-structure	O
-of	O
-near	O
-full	O
--	O
-length	O
-non	O
--	O
-phosphorylated	O
-ACC	O
-from	O
-S	O
-.	O
-cerevisae	O
-(	O
-lacking	O
-only	O
-21	O
-N	O
--	O
-terminal	O
-amino	O
-acids	O
-,	O
-here	O
-denoted	O
-as	O
-flACC	O
-)	O
-was	O
-published	O
-by	O
-Wei	O
-and	O
-Tong	O
-.	O
-	O
-In	O
-flACC	O
-,	O
-the	O
-ACC	O
-dimer	O
-obeys	O
-twofold	O
-symmetry	O
-and	O
-assembles	O
-in	O
-a	O
-triangular	O
-architecture	O
-with	O
-dimeric	O
-BC	O
-domains	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-In	O
-their	O
-study	O
-,	O
-mutational	O
-data	O
-indicate	O
-a	O
-requirement	O
-for	O
-BC	O
-dimerization	O
-for	O
-catalytic	O
-activity	O
-.	O
-	O
-The	O
-transition	O
-from	O
-the	O
-elongated	O
-open	O
-shape	O
-,	O
-observed	O
-in	O
-our	O
-experiments	O
-,	O
-towards	O
-a	O
-compact	O
-triangular	O
-shape	O
-is	O
-based	O
-on	O
-an	O
-intricate	O
-interplay	O
-of	O
-several	O
-hinge	O
--	O
-bending	O
-motions	O
-in	O
-the	O
-CD	O
-(	O
-Fig	O
-.	O
-4d	O
-).	O
-	O
-Comparison	O
-of	O
-flACC	O
-with	O
-our	O
-CthΔBCCP	B-mutant
-structure	O
-reveals	O
-the	O
-CDC2	O
-/	O
-CT	O
-hinge	O
-as	O
-a	O
-major	O
-contributor	O
-to	O
-conformational	O
-flexibility	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5b	O
-,	O
-c	O
-).	O
-	O
-In	O
-flACC	O
-,	O
-CDC2	O
-rotates	O
-∼	O
-120	O
-°	O
-with	O
-respect	O
-to	O
-the	O
-CT	O
-domain	O
-.	O
-	O
-A	O
-second	O
-hinge	O
-can	O
-be	O
-identified	O
-between	O
-CDC1	O
-/	O
-CDC2	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-a	O
-superposition	O
-of	O
-CDC2	O
-,	O
-CDC1	O
-of	O
-the	O
-phosphorylated	O
-SceCD	O
-is	O
-rotated	O
-by	O
-30	O
-°	O
-relative	O
-to	O
-CDC1	O
-of	O
-the	O
-non	O
--	O
-phosphorylated	O
-flACC	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5d	O
-),	O
-similar	O
-to	O
-what	O
-we	O
-have	O
-observed	O
-for	O
-the	O
-non	O
--	O
-phosphorylated	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-(	O
-Supplementary	O
-Fig	O
-.	O
-1d	O
-).	O
-	O
-When	O
-inspecting	O
-all	O
-individual	O
-protomer	O
-and	O
-fragment	B-mutant
-structures	O
-in	O
-their	O
-study	O
-,	O
-Wei	O
-and	O
-Tong	O
-also	O
-identify	O
-the	O
-CDN	O
-/	O
-CDC1	O
-connection	O
-as	O
-a	O
-highly	O
-flexible	O
-hinge	O
-,	O
-in	O
-agreement	O
-with	O
-our	O
-observations	O
-.	O
-	O
-The	O
-only	O
-bona	O
-fide	O
-regulatory	O
-phophorylation	O
-site	O
-of	O
-fungal	O
-ACC	O
-in	O
-the	O
-regulatory	O
-loop	O
-is	O
-directly	O
-participating	O
-in	O
-CDC1	O
-/	O
-CDC2	O
-domain	O
-interactions	O
-and	O
-thus	O
-stabilizes	O
-the	O
-hinge	O
-conformation	O
-.	O
-	O
-In	O
-flACC	O
-,	O
-the	O
-regulatory	O
-loop	O
-is	O
-mostly	O
-disordered	O
-,	O
-illustrating	O
-the	O
-increased	O
-flexibility	O
-due	O
-to	O
-the	O
-absence	O
-of	O
-the	O
-phosphoryl	O
-group	O
-.	O
-	O
-Only	O
-in	O
-three	O
-out	O
-of	O
-eight	O
-observed	O
-protomers	O
-a	O
-short	O
-peptide	O
-stretch	O
-(	O
-including	O
-Ser1157	O
-)	O
-was	O
-modelled	O
-.	O
-	O
-In	O
-those	O
-instances	O
-the	O
-Ser1157	O
-residue	O
-is	O
-located	O
-at	O
-a	O
-distance	O
-of	O
-14	O
-–	O
-20	O
-Å	O
-away	O
-from	O
-the	O
-location	O
-of	O
-the	O
-phosphorylated	O
-serine	O
-observed	O
-here	O
-,	O
-based	O
-on	O
-superposition	O
-of	O
-either	O
-CDC1	O
-or	O
-CDC2	O
-.	O
-	O
-Applying	O
-the	O
-conformation	O
-of	O
-the	O
-CDC1	O
-/	O
-CDC2	O
-hinge	O
-observed	O
-in	O
-SceCD	O
-on	O
-flACC	O
-leads	O
-to	O
-CDN	O
-sterically	O
-clashing	O
-with	O
-CDC2	O
-and	O
-BT	O
-/	O
-CDN	O
-clashing	O
-with	O
-CT	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-,	O
-b	O
-).	O
-	O
-Thus	O
-,	O
-in	O
-accordance	O
-with	O
-the	O
-results	O
-presented	O
-here	O
-,	O
-phosphorylation	O
-of	O
-Ser1157	O
-in	O
-SceACC	O
-most	O
-likely	O
-limits	O
-flexibility	O
-in	O
-the	O
-CDC1	O
-/	O
-CDC2	O
-hinge	O
-such	O
-that	O
-activation	O
-through	O
-BC	O
-dimerization	O
-is	O
-not	O
-possible	O
-(	O
-Fig	O
-.	O
-4d	O
-),	O
-which	O
-however	O
-does	O
-not	O
-exclude	O
-intermolecular	O
-dimerization	O
-.	O
-	O
-In	O
-addition	O
-,	O
-EM	O
-micrographs	O
-of	O
-phosphorylated	O
-and	O
-dephosphorylated	O
-SceACC	O
-display	O
-for	O
-both	O
-samples	O
-mainly	O
-elongated	O
-and	O
-U	O
--	O
-shaped	O
-conformations	O
-and	O
-reveal	O
-no	O
-apparent	O
-differences	O
-in	O
-particle	O
-shape	O
-distributions	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7	O
-).	O
-	O
-This	O
-implicates	O
-that	O
-the	O
-triangular	O
-shape	O
-with	O
-dimeric	O
-BC	O
-domains	O
-has	O
-a	O
-low	O
-population	O
-also	O
-in	O
-the	O
-active	O
-form	O
-,	O
-even	O
-though	O
-a	O
-biasing	O
-influence	O
-of	O
-grid	O
-preparation	O
-cannot	O
-be	O
-excluded	O
-completely	O
-.	O
-	O
-Large	O
--	O
-scale	O
-conformational	O
-variability	O
-has	O
-also	O
-been	O
-observed	O
-in	O
-most	O
-other	O
-carrier	O
-protein	O
--	O
-based	O
-multienzymes	O
-,	O
-including	O
-polyketide	O
-and	O
-fatty	O
--	O
-acid	O
-synthases	O
-(	O
-with	O
-the	O
-exception	O
-of	O
-fungal	O
--	O
-type	O
-fatty	O
--	O
-acid	O
-synthases	O
-),	O
-non	O
--	O
-ribosomal	O
-peptide	O
-synthetases	O
-and	O
-the	O
-pyruvate	O
-dehydrogenase	O
-complexes	O
-,	O
-although	O
-based	O
-on	O
-completely	O
-different	O
-architectures	O
-.	O
-	O
-Together	O
-,	O
-this	O
-structural	O
-information	O
-suggests	O
-that	O
-variable	O
-carrier	O
-protein	O
-tethering	O
-is	O
-not	O
-sufficient	O
-for	O
-efficient	O
-substrate	O
-transfer	O
-and	O
-catalysis	O
-in	O
-any	O
-of	O
-these	O
-systems	O
-.	O
-	O
-The	O
-determination	O
-of	O
-a	O
-set	O
-of	O
-crystal	O
-structures	O
-of	O
-SceACC	O
-in	O
-two	O
-states	O
-,	O
-unphosphorylated	O
-and	O
-phosphorylated	O
-at	O
-the	O
-major	O
-regulatory	O
-site	O
-Ser1157	O
-,	O
-provides	O
-a	O
-unique	O
-depiction	O
-of	O
-multienzyme	O
-regulation	O
-by	O
-post	O
--	O
-translational	O
-modification	O
-(	O
-Fig	O
-.	O
-4d	O
-).	O
-	O
-The	O
-phosphorylated	O
-regulatory	O
-loop	O
-binds	O
-to	O
-an	O
-allosteric	O
-site	O
-at	O
-the	O
-interface	O
-of	O
-two	O
-non	O
--	O
-catalytic	O
-domains	O
-and	O
-restricts	O
-conformational	O
-freedom	O
-at	O
-several	O
-hinges	O
-in	O
-the	O
-dynamic	O
-ACC	O
-.	O
-	O
-It	O
-disfavours	O
-the	O
-adoption	O
-of	O
-a	O
-rare	O
-,	O
-compact	O
-conformation	O
-,	O
-in	O
-which	O
-intramolecular	O
-dimerization	O
-of	O
-the	O
-BC	O
-domains	O
-results	O
-in	O
-catalytic	O
-turnover	O
-.	O
-	O
-The	O
-regulation	O
-of	O
-activity	O
-thus	O
-results	O
-from	O
-restrained	O
-large	O
--	O
-scale	O
-conformational	O
-dynamics	O
-rather	O
-than	O
-a	O
-direct	O
-or	O
-indirect	O
-influence	O
-on	O
-active	O
-site	O
-structure	O
-.	O
-	O
-To	O
-our	O
-best	O
-knowledge	O
-,	O
-ACC	O
-is	O
-the	O
-first	O
-multienzyme	O
-for	O
-which	O
-such	O
-a	O
-phosphorylation	O
--	O
-dependent	O
-mechanical	O
-control	O
-mechanism	O
-has	O
-been	O
-visualized	O
-.	O
-	O
-However	O
-,	O
-the	O
-example	O
-of	O
-ACC	O
-now	O
-demonstrates	O
-the	O
-possibility	O
-of	O
-regulating	O
-activity	O
-by	O
-controlled	O
-dynamics	O
-of	O
-non	O
--	O
-enzymatic	O
-linker	O
-regions	O
-also	O
-in	O
-other	O
-families	O
-of	O
-carrier	O
--	O
-dependent	O
-multienzymes	O
-.	O
-	O
-The	O
-phosphorylated	O
-central	O
-domain	O
-of	O
-yeast	O
-ACC	O
-.	O
-	O
-(	O
-a	O
-)	O
-Schematic	O
-overview	O
-of	O
-the	O
-domain	O
-organization	O
-of	O
-eukaryotic	O
-ACCs	O
-.	O
-	O
-Crystallized	O
-constructs	O
-are	O
-indicated	O
-.	O
-	O
-(	O
-b	O
-)	O
-Cartoon	O
-representation	O
-of	O
-the	O
-SceCD	O
-crystal	O
-structure	O
-.	O
-	O
-CDN	O
-is	O
-linked	O
-by	O
-a	O
-four	O
--	O
-helix	O
-bundle	O
-(	O
-CDL	O
-)	O
-to	O
-two	O
-α	O
-–	O
-β	O
--	O
-fold	O
-domains	O
-(	O
-CDC1	O
-and	O
-CDC2	O
-).	O
-	O
-The	O
-regulatory	O
-loop	O
-is	O
-shown	O
-as	O
-bold	O
-cartoon	O
-,	O
-and	O
-the	O
-phosphorylated	O
-Ser1157	O
-is	O
-marked	O
-by	O
-a	O
-red	O
-triangle	O
-.	O
-	O
-(	O
-c	O
-)	O
-Superposition	O
-of	O
-CDC1	O
-and	O
-CDC2	O
-reveals	O
-highly	O
-conserved	O
-folds	O
-.	O
-(	O
-d	O
-)	O
-The	O
-regulatory	O
-loop	O
-with	O
-the	O
-phosphorylated	O
-Ser1157	O
-is	O
-bound	O
-into	O
-a	O
-crevice	O
-between	O
-CDC1	O
-and	O
-CDC2	O
-,	O
-the	O
-conserved	O
-residues	O
-Arg1173	O
-and	O
-Arg1260	O
-coordinate	O
-the	O
-phosphoryl	O
--	O
-group	O
-.	O
-	O
-(	O
-e	O
-)	O
-Structural	O
-overview	O
-of	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-.	O
-	O
-The	O
-attachment	O
-points	O
-to	O
-the	O
-N	O
--	O
-terminal	O
-BCCP	O
-domain	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-CT	O
-domain	O
-are	O
-indicated	O
-with	O
-spheres	O
-.	O
-	O
-Architecture	O
-of	O
-the	O
-CD	O
-–	O
-CT	O
-core	O
-of	O
-fungal	O
-ACC	O
-.	O
-	O
-Cartoon	O
-representation	O
-of	O
-crystal	O
-structures	O
-of	O
-multidomain	B-mutant
-constructs	I-mutant
-of	O
-CthACC	O
-.	O
-	O
-One	O
-protomer	O
-is	O
-shown	O
-in	O
-colour	O
-and	O
-one	O
-in	O
-grey	O
-.	O
-	O
-Individual	O
-domains	O
-are	O
-labelled	O
-;	O
-the	O
-active	O
-site	O
-of	O
-CT	O
-and	O
-the	O
-position	O
-of	O
-the	O
-conserved	O
-regulatory	O
-phosphoserine	O
-site	O
-based	O
-on	O
-SceCD	O
-are	O
-indicated	O
-by	O
-an	O
-asterisk	O
-and	O
-a	O
-triangle	O
-,	O
-respectively	O
-.	O
-	O
-Variability	O
-of	O
-the	O
-connections	O
-of	O
-CDC2	O
-to	O
-CT	O
-and	O
-CDC1	O
-in	O
-fungal	O
-ACC	O
-.	O
-	O
-(	O
-a	O
-)	O
-Hinge	O
-properties	O
-of	O
-the	O
-CDC2	O
-–	O
-CT	O
-connection	O
-analysed	O
-by	O
-a	O
-CT	O
--	O
-based	O
-superposition	O
-of	O
-eight	O
-instances	O
-of	O
-the	O
-CDC2	B-mutant
--	I-mutant
-CT	I-mutant
-segment	I-mutant
-.	O
-	O
-For	O
-clarity	O
-,	O
-only	O
-one	O
-protomer	O
-of	O
-CthCD	B-mutant
--	I-mutant
-CTCter1	I-mutant
-is	O
-shown	O
-in	O
-full	O
-colour	O
-as	O
-reference	O
-.	O
-	O
-For	O
-other	O
-instances	O
-,	O
-CDC2	O
-domains	O
-are	O
-shown	O
-in	O
-transparent	O
-tube	O
-representation	O
-with	O
-only	O
-one	O
-helix	O
-each	O
-highlighted	O
-.	O
-	O
-The	O
-range	O
-of	O
-hinge	O
-bending	O
-is	O
-indicated	O
-and	O
-the	O
-connection	O
-points	O
-between	O
-CDC2	O
-and	O
-CT	O
-(	O
-blue	O
-)	O
-as	O
-well	O
-as	O
-between	O
-CDC1	O
-and	O
-CDC2	O
-(	O
-green	O
-and	O
-grey	O
-)	O
-are	O
-marked	O
-as	O
-spheres	O
-.	O
-	O
-(	O
-b	O
-)	O
-The	O
-interdomain	O
-interface	O
-of	O
-CDC1	O
-and	O
-CDC2	O
-exhibits	O
-only	O
-limited	O
-plasticity	O
-.	O
-	O
-Representation	O
-as	O
-in	O
-a	O
-,	O
-but	O
-the	O
-CDC1	O
-and	O
-CDC2	O
-are	O
-superposed	O
-based	O
-on	O
-CDC2	O
-.	O
-	O
-One	O
-protomer	O
-of	O
-CthΔBCCP	B-mutant
-is	O
-shown	O
-in	O
-colour	O
-,	O
-the	O
-CDL	O
-domains	O
-are	O
-omitted	O
-for	O
-clarity	O
-and	O
-the	O
-position	O
-of	O
-the	O
-phosphorylated	O
-serine	O
-based	O
-on	O
-SceCD	O
-is	O
-indicated	O
-with	O
-a	O
-red	O
-triangle	O
-.	O
-	O
-The	O
-connection	O
-points	O
-from	O
-CDC1	O
-to	O
-CDC2	O
-and	O
-to	O
-CDL	O
-are	O
-represented	O
-by	O
-green	O
-spheres	O
-.	O
-	O
-The	O
-conformational	O
-dynamics	O
-of	O
-fungal	O
-ACC	O
-.	O
-	O
-(	O
-a	O
-–	O
-c	O
-)	O
-Large	O
--	O
-scale	O
-conformational	O
-variability	O
-of	O
-the	O
-CDN	O
-domain	O
-relative	O
-to	O
-the	O
-CDL	O
-/	O
-CDC1	O
-domain	O
-.	O
-	O
-CthCD	B-mutant
--	I-mutant
-CT1	I-mutant
-(	O
-in	O
-colour	O
-)	O
-serves	O
-as	O
-reference	O
-,	O
-the	O
-compared	O
-structures	O
-(	O
-as	O
-indicated	O
-,	O
-numbers	O
-after	O
-construct	O
-name	O
-differentiate	O
-between	O
-individual	O
-protomers	O
-)	O
-are	O
-shown	O
-in	O
-grey	O
-.	O
-	O
-Domains	O
-other	O
-than	O
-CDN	O
-and	O
-CDL	O
-/	O
-CDC1	O
-are	O
-omitted	O
-for	O
-clarity	O
-.	O
-	O
-The	O
-domains	O
-are	O
-labelled	O
-and	O
-the	O
-distances	O
-between	O
-the	O
-N	O
-termini	O
-of	O
-CDN	O
-(	O
-spheres	O
-)	O
-in	O
-the	O
-compared	O
-structures	O
-are	O
-indicated	O
-.	O
-	O
-(	O
-d	O
-)	O
-Schematic	O
-model	O
-of	O
-fungal	O
-ACC	O
-showing	O
-the	O
-intrinsic	O
-,	O
-regulated	O
-flexibility	O
-of	O
-CD	O
-in	O
-the	O
-phosphorylated	O
-inhibited	O
-or	O
-the	O
-non	O
--	O
-phosphorylated	O
-activated	O
-state	O
-.	O
-	O
-Flexibility	O
-of	O
-the	O
-CDC2	O
-/	O
-CT	O
-and	O
-CDN	O
-/	O
-CDL	O
-hinges	O
-is	O
-illustrated	O
-by	O
-arrows	O
-.	O
-	O
-The	O
-Ser1157	O
-phosphorylation	O
-site	O
-and	O
-the	O
-regulatory	O
-loop	O
-are	O
-schematically	O
-indicated	O
-in	O
-magenta	O
-.	O
-	O
-Crystal	O
-Structures	O
-of	O
-Putative	O
-Sugar	O
-Kinases	O
-from	O
-Synechococcus	O
-Elongatus	O
-PCC	O
-7942	O
-and	O
-Arabidopsis	O
-Thaliana	O
-	O
-The	O
-genome	O
-of	O
-the	O
-Synechococcus	O
-elongatus	O
-strain	O
-PCC	O
-7942	O
-encodes	O
-a	O
-putative	O
-sugar	O
-kinase	O
-(	O
-SePSK	O
-),	O
-which	O
-shares	O
-44	O
-.	O
-9	O
-%	O
-sequence	O
-identity	O
-with	O
-the	O
-xylulose	O
-kinase	O
--	O
-1	O
-(	O
-AtXK	O
--	O
-1	O
-)	O
-from	O
-Arabidopsis	O
-thaliana	O
-.	O
-	O
-Sequence	O
-alignment	O
-suggests	O
-that	O
-both	O
-kinases	O
-belong	O
-to	O
-the	O
-ribulokinase	O
--	O
-like	O
-carbohydrate	O
-kinases	O
-,	O
-a	O
-sub	O
--	O
-family	O
-of	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-.	O
-	O
-Here	O
-we	O
-solved	O
-the	O
-structures	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-in	O
-both	O
-their	O
-apo	O
-forms	O
-and	O
-in	O
-complex	O
-with	O
-nucleotide	O
-substrates	O
-.	O
-	O
-The	O
-two	O
-kinases	O
-exhibit	O
-nearly	O
-identical	O
-overall	O
-architecture	O
-,	O
-with	O
-both	O
-kinases	O
-possessing	O
-ATP	O
-hydrolysis	O
-activity	O
-in	O
-the	O
-absence	O
-of	O
-substrates	O
-.	O
-	O
-In	O
-addition	O
-,	O
-our	O
-enzymatic	O
-assays	O
-suggested	O
-that	O
-SePSK	O
-has	O
-the	O
-capability	O
-to	O
-phosphorylate	O
-D	O
--	O
-ribulose	O
-.	O
-	O
-In	O
-order	O
-to	O
-understand	O
-the	O
-catalytic	O
-mechanism	O
-of	O
-SePSK	O
-,	O
-we	O
-solved	O
-the	O
-structure	O
-of	O
-SePSK	O
-in	O
-complex	O
-with	O
-D	O
--	O
-ribulose	O
-and	O
-found	O
-two	O
-potential	O
-substrate	O
-binding	O
-pockets	O
-in	O
-SePSK	O
-.	O
-	O
-Using	O
-mutation	O
-and	O
-activity	O
-analysis	O
-,	O
-we	O
-further	O
-verified	O
-the	O
-key	O
-residues	O
-important	O
-for	O
-its	O
-catalytic	O
-activity	O
-.	O
-	O
-Moreover	O
-,	O
-our	O
-structural	O
-comparison	O
-with	O
-other	O
-family	O
-members	O
-suggests	O
-that	O
-there	O
-are	O
-major	O
-conformational	O
-changes	O
-in	O
-SePSK	O
-upon	O
-substrate	O
-binding	O
-,	O
-facilitating	O
-the	O
-catalytic	O
-process	O
-.	O
-	O
-Together	O
-,	O
-these	O
-results	O
-provide	O
-important	O
-information	O
-for	O
-a	O
-more	O
-detailed	O
-understanding	O
-of	O
-the	O
-cofactor	O
-and	O
-substrate	O
-binding	O
-mode	O
-as	O
-well	O
-as	O
-the	O
-catalytic	O
-mechanism	O
-of	O
-SePSK	O
-,	O
-and	O
-possible	O
-similarities	O
-with	O
-its	O
-plant	O
-homologue	O
-AtXK	O
--	O
-1	O
-.	O
-	O
-Carbohydrates	O
-are	O
-essential	O
-cellular	O
-compounds	O
-involved	O
-in	O
-the	O
-metabolic	O
-processes	O
-present	O
-in	O
-all	O
-organisms	O
-.	O
-	O
-Phosphorylation	O
-is	O
-one	O
-of	O
-the	O
-various	O
-pivotal	O
-modifications	O
-of	O
-carbohydrates	O
-,	O
-and	O
-is	O
-catalyzed	O
-by	O
-specific	O
-sugar	O
-kinases	O
-.	O
-	O
-These	O
-kinases	O
-exhibit	O
-considerable	O
-differences	O
-in	O
-their	O
-folding	O
-pattern	O
-and	O
-substrate	O
-specificity	O
-.	O
-	O
-Based	O
-on	O
-sequence	O
-analysis	O
-,	O
-they	O
-can	O
-be	O
-divided	O
-into	O
-four	O
-families	O
-,	O
-namely	O
-HSP	O
-70_NBD	O
-family	O
-,	O
-FGGY	O
-family	O
-,	O
-Mer_B	O
-like	O
-family	O
-and	O
-Parm_like	O
-family	O
-.	O
-	O
-The	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-contain	O
-different	O
-types	O
-of	O
-sugar	O
-kinases	O
-,	O
-all	O
-of	O
-which	O
-possess	O
-different	O
-catalytic	O
-substrates	O
-with	O
-preferences	O
-for	O
-short	O
--	O
-chained	O
-sugar	O
-substrates	O
-,	O
-ranging	O
-from	O
-triose	O
-to	O
-heptose	O
-.	O
-	O
-These	O
-sugar	O
-substrates	O
-include	O
-L	O
--	O
-ribulose	O
-,	O
-erythritol	O
-,	O
-L	O
--	O
-fuculose	O
-,	O
-D	O
--	O
-glycerol	O
-,	O
-D	O
--	O
-gluconate	O
-,	O
-L	O
--	O
-xylulose	O
-,	O
-D	O
--	O
-ribulose	O
-,	O
-L	O
--	O
-rhamnulose	O
-and	O
-D	O
--	O
-xylulose	O
-.	O
-	O
-Structures	O
-reported	O
-in	O
-the	O
-Protein	O
-Data	O
-Bank	O
-of	O
-the	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-exhibit	O
-a	O
-similar	O
-overall	O
-architecture	O
-containing	O
-two	O
-protein	O
-domains	O
-,	O
-one	O
-of	O
-which	O
-is	O
-responsible	O
-for	O
-the	O
-binding	O
-of	O
-substrate	O
-,	O
-while	O
-the	O
-second	O
-is	O
-used	O
-for	O
-binding	O
-cofactor	O
-ATP	O
-.	O
-	O
-While	O
-the	O
-binding	O
-pockets	O
-for	O
-substrates	O
-are	O
-at	O
-the	O
-same	O
-position	O
-,	O
-each	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-uses	O
-different	O
-substrate	O
--	O
-binding	O
-residues	O
-,	O
-resulting	O
-in	O
-high	O
-substrate	O
-specificity	O
-.	O
-	O
-Synpcc7942_2462	O
-from	O
-the	O
-cyanobacteria	O
-Synechococcus	O
-elongatus	O
-PCC	O
-7942	O
-encodes	O
-a	O
-putative	O
-sugar	O
-kinase	O
-(	O
-SePSK	O
-),	O
-and	O
-this	O
-kinase	O
-contains	O
-426	O
-amino	O
-acids	O
-.	O
-	O
-The	O
-At2g21370	O
-gene	O
-product	O
-from	O
-Arabidopsis	O
-thaliana	O
-,	O
-xylulose	O
-kinase	O
--	O
-1	O
-(	O
-AtXK	O
--	O
-1	O
-),	O
-whose	O
-mature	O
-form	O
-contains	O
-436	O
-amino	O
-acids	O
-,	O
-is	O
-located	O
-in	O
-the	O
-chloroplast	O
-(	O
-ChloroP	O
-1	O
-.	O
-1	O
-Server	O
-).	O
-	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-display	O
-a	O
-sequence	O
-identity	O
-of	O
-44	O
-.	O
-9	O
-%,	O
-and	O
-belong	O
-to	O
-the	O
-ribulokinase	O
--	O
-like	O
-carbohydrate	O
-kinases	O
-,	O
-a	O
-sub	O
--	O
-family	O
-of	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-.	O
-	O
-Members	O
-of	O
-this	O
-sub	O
--	O
-family	O
-are	O
-responsible	O
-for	O
-the	O
-phosphorylation	O
-of	O
-sugars	O
-similar	O
-to	O
-L	O
--	O
-ribulose	O
-and	O
-D	O
--	O
-ribulose	O
-.	O
-	O
-The	O
-sequence	O
-and	O
-the	O
-substrate	O
-specificity	O
-of	O
-ribulokinase	O
--	O
-like	O
-carbohydrate	O
-kinases	O
-are	O
-different	O
-,	O
-but	O
-they	O
-share	O
-the	O
-common	O
-folding	O
-feature	O
-with	O
-two	O
-domains	O
-.	O
-	O
-Domain	O
-I	O
-exhibits	O
-a	O
-ribonuclease	O
-H	O
--	O
-like	O
-folding	O
-pattern	O
-,	O
-and	O
-is	O
-responsible	O
-for	O
-the	O
-substrate	O
-binding	O
-,	O
-while	O
-domain	O
-II	O
-possesses	O
-an	O
-actin	O
--	O
-like	O
-ATPase	O
-domain	O
-that	O
-binds	O
-cofactor	O
-ATP	O
-.	O
-	O
-Two	O
-possible	O
-xylulose	O
-kinases	O
-(	O
-xylulose	O
-kinase	O
--	O
-1	O
-:	O
-XK	O
--	O
-1	O
-and	O
-xylulose	O
-kinase	O
--	O
-2	O
-:	O
-XK	O
--	O
-2	O
-)	O
-from	O
-Arabidopsis	O
-thaliana	O
-were	O
-previously	O
-proposed	O
-.	O
-	O
-It	O
-was	O
-shown	O
-that	O
-XK	O
--	O
-2	O
-(	O
-At5g49650	O
-)	O
-located	O
-in	O
-the	O
-cytosol	O
-is	O
-indeed	O
-xylulose	O
-kinase	O
-.	O
-	O
-However	O
-,	O
-the	O
-function	O
-of	O
-XK	O
--	O
-1	O
-(	O
-At2g21370	O
-)	O
-inside	O
-the	O
-chloroplast	O
-stroma	O
-has	O
-remained	O
-unknown	O
-.	O
-	O
-SePSK	O
-from	O
-Synechococcus	O
-elongatus	O
-strain	O
-PCC	O
-7942	O
-is	O
-the	O
-homolog	O
-of	O
-AtXK	O
--	O
-1	O
-,	O
-though	O
-its	O
-physiological	O
-function	O
-and	O
-substrates	O
-remain	O
-unclear	O
-.	O
-	O
-In	O
-order	O
-to	O
-obtain	O
-functional	O
-and	O
-structural	O
-information	O
-about	O
-these	O
-two	O
-proteins	O
-,	O
-here	O
-we	O
-reported	O
-the	O
-crystal	O
-structures	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-.	O
-	O
-Our	O
-findings	O
-provide	O
-new	O
-details	O
-of	O
-the	O
-catalytic	O
-mechanism	O
-of	O
-SePSK	O
-and	O
-lay	O
-the	O
-foundation	O
-for	O
-future	O
-studies	O
-into	O
-its	O
-homologs	O
-in	O
-eukaryotes	O
-.	O
-	O
-Overall	O
-structures	O
-of	O
-apo	O
--	O
-SePSK	O
-and	O
-apo	O
--	O
-AtXK	O
--	O
-1	O
-	O
-The	O
-attempt	O
-to	O
-solve	O
-the	O
-SePSK	O
-structure	O
-by	O
-molecular	O
-replacement	O
-method	O
-failed	O
-with	O
-ribulokinase	O
-from	O
-Bacillus	O
-halodurans	O
-(	O
-PDB	O
-code	O
-:	O
-3QDK	O
-,	O
-15	O
-.	O
-7	O
-%	O
-sequence	O
-identity	O
-)	O
-as	O
-an	O
-initial	O
-model	O
-.	O
-	O
-We	O
-therefore	O
-used	O
-single	O
-isomorphous	O
-replacement	O
-anomalous	O
-scattering	O
-method	O
-(	O
-SIRAS	O
-)	O
-for	O
-successful	O
-solution	O
-of	O
-the	O
-apo	O
--	O
-SePSK	O
-structure	O
-at	O
-a	O
-resolution	O
-of	O
-2	O
-.	O
-3	O
-Å	O
-.	O
-Subsequently	O
-,	O
-the	O
-apo	O
--	O
-SePSK	O
-structure	O
-was	O
-used	O
-as	O
-molecular	O
-replacement	O
-model	O
-to	O
-solve	O
-all	O
-other	O
-structures	O
-identified	O
-in	O
-this	O
-study	O
-.	O
-	O
-Our	O
-structural	O
-analysis	O
-showed	O
-that	O
-apo	O
--	O
-SePSK	O
-consists	O
-of	O
-one	O
-SePSK	O
-protein	O
-molecule	O
-in	O
-an	O
-asymmetric	O
-unit	O
-.	O
-	O
-The	O
-amino	O
--	O
-acid	O
-residues	O
-were	O
-traced	O
-from	O
-Val2	O
-to	O
-His419	O
-,	O
-except	O
-for	O
-the	O
-Met1	O
-residue	O
-and	O
-the	O
-seven	O
-residues	O
-at	O
-the	O
-C	O
--	O
-termini	O
-.	O
-	O
-Apo	O
--	O
-SePSK	O
-contains	O
-two	O
-domains	O
-referred	O
-to	O
-further	O
-on	O
-as	O
-domain	O
-I	O
-and	O
-domain	O
-II	O
-(	O
-Fig	O
-1A	O
-).	O
-	O
-Domain	O
-I	O
-consists	O
-of	O
-non	O
--	O
-contiguous	O
-portions	O
-of	O
-the	O
-polypeptide	O
-chains	O
-(	O
-aa	O
-.	O
-	O
-2	O
-–	O
-228	O
-and	O
-aa	O
-.	O
-	O
-402	O
-–	O
-419	O
-),	O
-exhibiting	O
-11	O
-α	O
--	O
-helices	O
-and	O
-11	O
-β	O
--	O
-sheets	O
-.	O
-	O
-Among	O
-all	O
-these	O
-structural	O
-elements	O
-,	O
-α4	O
-/	O
-α5	O
-/	O
-α11	O
-/	O
-α18	O
-,	O
-β3	O
-/	O
-β2	O
-/	O
-β1	O
-/	O
-β6	O
-/	O
-β19	O
-/	O
-β20	O
-/	O
-β17	O
-and	O
-α21	O
-/	O
-α32	O
-form	O
-three	O
-patches	O
-,	O
-referred	O
-to	O
-as	O
-A1	O
-,	O
-B1	O
-and	O
-A2	O
-,	O
-exhibiting	O
-the	O
-core	O
-region	O
-.	O
-	O
-In	O
-addition	O
-,	O
-four	O
-β	O
--	O
-sheets	O
-(	O
-β7	O
-,	O
-β10	O
-,	O
-β12	O
-and	O
-β16	O
-)	O
-and	O
-five	O
-α	O
--	O
-helices	O
-(	O
-α8	O
-,	O
-α9	O
-,	O
-α13	O
-,	O
-α14	O
-and	O
-α15	O
-)	O
-flank	O
-the	O
-left	O
-side	O
-of	O
-the	O
-core	O
-region	O
-.	O
-	O
-Domain	O
-II	O
-is	O
-comprised	O
-of	O
-aa	O
-.	O
-	O
-229	O
-–	O
-401	O
-and	O
-classified	O
-into	O
-B2	O
-(	O
-β31	O
-/	O
-β29	O
-/	O
-β22	O
-/	O
-β23	O
-/	O
-β25	O
-/	O
-β24	O
-)	O
-and	O
-A3	O
-(	O
-α26	O
-/	O
-α27	O
-/	O
-α28	O
-/	O
-α30	O
-)	O
-(	O
-Fig	O
-1A	O
-and	O
-S1	O
-Fig	O
-).	O
-	O
-In	O
-the	O
-SePSK	O
-structure	O
-,	O
-B1	O
-and	O
-B2	O
-are	O
-sandwiched	O
-by	O
-A1	O
-,	O
-A2	O
-and	O
-A3	O
-,	O
-and	O
-the	O
-whole	O
-structure	O
-shows	O
-the	O
-A1	O
-/	O
-B1	O
-/	O
-A2	O
-/	O
-B2	O
-/	O
-A3	O
-(	O
-α	O
-/	O
-β	O
-/	O
-α	O
-/	O
-β	O
-/	O
-α	O
-)	O
-folding	O
-pattern	O
-,	O
-which	O
-is	O
-in	O
-common	O
-with	O
-other	O
-members	O
-of	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-(	O
-S2	O
-Fig	O
-).	O
-	O
-The	O
-overall	O
-folding	O
-of	O
-SePSK	O
-resembles	O
-a	O
-clip	O
-,	O
-with	O
-A2	O
-of	O
-domain	O
-I	O
-acting	O
-as	O
-a	O
-hinge	O
-region	O
-.	O
-	O
-Overall	O
-structures	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-.	O
-	O
-(	O
-A	O
-)	O
-Three	O
--	O
-dimensional	O
-structure	O
-of	O
-apo	O
--	O
-SePSK	O
-.	O
-	O
-The	O
-secondary	O
-structural	O
-elements	O
-are	O
-indicated	O
-(	O
-α	O
--	O
-helix	O
-:	O
-cyan	O
-,	O
-β	O
--	O
-sheet	O
-:	O
-yellow	O
-).	O
-	O
-(	O
-B	O
-)	O
-Three	O
--	O
-dimensional	O
-structure	O
-of	O
-apo	O
--	O
-AtXK	O
--	O
-1	O
-.	O
-	O
-The	O
-secondary	O
-structural	O
-elements	O
-are	O
-indicated	O
-(	O
-α	O
--	O
-helix	O
-:	O
-green	O
-,	O
-β	O
--	O
-sheet	O
-:	O
-wheat	O
-).	O
-	O
-Apo	O
--	O
-AtXK	O
--	O
-1	O
-exhibits	O
-a	O
-folding	O
-pattern	O
-similar	O
-to	O
-that	O
-of	O
-SePSK	O
-in	O
-line	O
-with	O
-their	O
-high	O
-sequence	O
-identity	O
-(	O
-Fig	O
-1B	O
-and	O
-S1	O
-Fig	O
-).	O
-	O
-However	O
-,	O
-superposition	O
-of	O
-structures	O
-of	O
-AtXK	O
--	O
-1	O
-and	O
-SePSK	O
-shows	O
-some	O
-differences	O
-,	O
-especially	O
-at	O
-the	O
-loop	O
-regions	O
-.	O
-	O
-A	O
-considerable	O
-difference	O
-is	O
-found	O
-in	O
-the	O
-loop3	O
-linking	O
-β3	O
-and	O
-α4	O
-,	O
-which	O
-is	O
-stretched	O
-out	O
-in	O
-the	O
-AtXK	O
--	O
-1	O
-structure	O
-,	O
-while	O
-in	O
-the	O
-SePSK	O
-structure	O
-,	O
-it	O
-is	O
-bent	O
-back	O
-towards	O
-the	O
-inner	O
-part	O
-.	O
-	O
-The	O
-corresponding	O
-residues	O
-between	O
-these	O
-two	O
-structures	O
-(	O
-SePSK	O
--	O
-Lys35	O
-and	O
-AtXK	O
--	O
-1	O
--	O
-Lys48	O
-)	O
-have	O
-a	O
-distance	O
-of	O
-15	O
-.	O
-4	O
-Å	O
-(	O
-S3	O
-Fig	O
-).	O
-	O
-Activity	O
-assays	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-	O
-In	O
-order	O
-to	O
-understand	O
-the	O
-function	O
-of	O
-these	O
-two	O
-kinases	O
-,	O
-we	O
-performed	O
-structural	O
-comparison	O
-using	O
-Dali	O
-server	O
-.	O
-	O
-The	O
-structures	O
-most	O
-closely	O
-related	O
-to	O
-SePSK	O
-are	O
-xylulose	O
-kinase	O
-,	O
-glycerol	O
-kinase	O
-and	O
-ribulose	O
-kinase	O
-,	O
-implying	O
-that	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-might	O
-function	O
-similarly	O
-to	O
-these	O
-kinases	O
-.	O
-	O
-We	O
-first	O
-tested	O
-whether	O
-both	O
-enzymes	O
-possessed	O
-ATP	O
-hydrolysis	O
-activity	O
-in	O
-the	O
-absence	O
-of	O
-substrates	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-2A	O
-,	O
-both	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-exhibited	O
-ATP	O
-hydrolysis	O
-activity	O
-.	O
-	O
-This	O
-finding	O
-is	O
-in	O
-agreement	O
-with	O
-a	O
-previous	O
-result	O
-showing	O
-that	O
-xylulose	O
-kinase	O
-(	O
-PDB	O
-code	O
-:	O
-2ITM	O
-)	O
-possessed	O
-ATP	O
-hydrolysis	O
-activity	O
-without	O
-adding	O
-substrate	O
-.	O
-	O
-To	O
-further	O
-identify	O
-the	O
-actual	O
-substrate	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-,	O
-five	O
-different	O
-sugar	O
-molecules	O
-,	O
-including	O
-D	O
--	O
-ribulose	O
-,	O
-L	O
--	O
-ribulose	O
-,	O
-D	O
--	O
-xylulose	O
-,	O
-L	O
--	O
-xylulose	O
-and	O
-Glycerol	O
-,	O
-were	O
-used	O
-in	O
-enzymatic	O
-activity	O
-assays	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-2B	O
-,	O
-the	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-SePSK	O
-greatly	O
-increased	O
-upon	O
-adding	O
-D	O
--	O
-ribulose	O
-than	O
-adding	O
-other	O
-potential	O
-substrates	O
-,	O
-suggesting	O
-that	O
-it	O
-has	O
-D	O
--	O
-ribulose	O
-kinase	O
-activity	O
-.	O
-	O
-In	O
-contrary	O
-,	O
-limited	O
-increasing	O
-of	O
-ATP	O
-hydrolysis	O
-activity	O
-was	O
-detected	O
-for	O
-AtXK	O
--	O
-1	O
-upon	O
-addition	O
-of	O
-D	O
--	O
-ribulose	O
-(	O
-Fig	O
-2C	O
-),	O
-despite	O
-its	O
-structural	O
-similarity	O
-with	O
-SePSK	O
-.	O
-	O
-The	O
-enzymatic	O
-activity	O
-assays	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-.	O
-	O
-Both	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-showed	O
-ATP	O
-hydrolysis	O
-activity	O
-in	O
-the	O
-absence	O
-of	O
-substrate	O
-.	O
-	O
-While	O
-the	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-SePSK	O
-greatly	O
-increases	O
-upon	O
-addition	O
-of	O
-D	O
--	O
-ribulose	O
-(	O
-DR	O
-).	O
-	O
-(	O
-B	O
-)	O
-The	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-SePSK	O
-with	O
-addition	O
-of	O
-five	O
-different	O
-substrates	O
-.	O
-	O
-The	O
-substrates	O
-are	O
-DR	O
-(	O
-D	O
--	O
-ribulose	O
-),	O
-LR	O
-(	O
-L	O
--	O
-ribulose	O
-),	O
-DX	O
-(	O
-D	O
--	O
-xylulose	O
-),	O
-LX	O
-(	O
-L	O
--	O
-xylulose	O
-)	O
-and	O
-GLY	O
-(	O
-Glycerol	O
-).	O
-(	O
-C	O
-)	O
-The	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-with	O
-or	O
-without	O
-D	O
--	O
-ribulose	O
-.	O
-(	O
-D	O
-)	O
-The	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-and	O
-single	O
--	O
-site	O
-mutants	O
-of	O
-SePSK	O
-.	O
-	O
-Three	O
-single	O
--	O
-site	O
-mutants	O
-of	O
-SePSK	O
-are	O
-D8A	B-mutant
--	O
-SePSK	O
-,	O
-T11A	B-mutant
--	O
-SePSK	O
-and	O
-D221A	B-mutant
--	O
-SePSK	O
-.	O
-	O
-The	O
-ATP	O
-hydrolysis	O
-activity	O
-measured	O
-via	O
-luminescent	O
-ADP	O
--	O
-Glo	O
-assay	O
-(	O
-Promega	O
-).	O
-	O
-To	O
-understand	O
-the	O
-catalytic	O
-mechanism	O
-of	O
-SePSK	O
-,	O
-we	O
-performed	O
-structural	O
-comparisons	O
-among	O
-xylulose	O
-kinase	O
-,	O
-glycerol	O
-kinase	O
-,	O
-ribulose	O
-kinase	O
-and	O
-SePSK	O
-.	O
-	O
-Our	O
-results	O
-suggested	O
-that	O
-three	O
-conserved	O
-residues	O
-(	O
-D8	O
-,	O
-T11	O
-and	O
-D221	O
-of	O
-SePSK	O
-)	O
-play	O
-an	O
-important	O
-role	O
-in	O
-SePSK	O
-function	O
-.	O
-	O
-Mutations	O
-of	O
-the	O
-corresponding	O
-residue	O
-in	O
-xylulose	O
-kinase	O
-and	O
-glycerol	O
-kinase	O
-from	O
-Escherichia	O
-coli	O
-greatly	O
-reduced	O
-their	O
-activity	O
-.	O
-	O
-To	O
-identify	O
-the	O
-function	O
-of	O
-these	O
-three	O
-residues	O
-of	O
-SePSK	O
-,	O
-we	O
-constructed	O
-D8A	B-mutant
-,	O
-T11A	B-mutant
-and	O
-D221A	B-mutant
-mutants	O
-.	O
-	O
-Using	O
-enzymatic	O
-activity	O
-assays	O
-,	O
-we	O
-found	O
-that	O
-all	O
-of	O
-these	O
-mutants	O
-exhibit	O
-much	O
-lower	O
-activity	O
-of	O
-ATP	O
-hydrolysis	O
-after	O
-adding	O
-D	O
--	O
-ribulose	O
-than	O
-that	O
-of	O
-wild	O
-type	O
-,	O
-indicating	O
-the	O
-possibility	O
-that	O
-these	O
-three	O
-residues	O
-are	O
-involved	O
-in	O
-the	O
-catalytic	O
-process	O
-of	O
-phosphorylation	O
-D	O
--	O
-ribulose	O
-and	O
-are	O
-vital	O
-for	O
-the	O
-function	O
-of	O
-SePSK	O
-(	O
-Fig	O
-2D	O
-).	O
-	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-possess	O
-a	O
-similar	O
-ATP	O
-binding	O
-site	O
-	O
-To	O
-obtain	O
-more	O
-detailed	O
-information	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-in	O
-complex	O
-with	O
-ATP	O
-,	O
-we	O
-soaked	O
-the	O
-apo	O
--	O
-crystals	O
-in	O
-the	O
-reservoir	O
-adding	O
-cofactor	O
-ATP	O
-,	O
-and	O
-obtained	O
-the	O
-structures	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-bound	O
-with	O
-ATP	O
-at	O
-the	O
-resolution	O
-of	O
-2	O
-.	O
-3	O
-Å	O
-and	O
-1	O
-.	O
-8	O
-Å	O
-,	O
-respectively	O
-.	O
-	O
-In	O
-both	O
-structures	O
-,	O
-a	O
-strong	O
-electron	O
-density	O
-was	O
-found	O
-in	O
-the	O
-conserved	O
-ATP	O
-binding	O
-pocket	O
-,	O
-but	O
-can	O
-only	O
-be	O
-fitted	O
-with	O
-an	O
-ADP	O
-molecule	O
-(	O
-S4	O
-Fig	O
-).	O
-	O
-Thus	O
-the	O
-two	O
-structures	O
-were	O
-named	O
-ADP	O
--	O
-SePSK	O
-and	O
-ADP	O
--	O
-AtXK	O
--	O
-1	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-extremely	O
-weak	O
-electron	O
-densities	O
-of	O
-ATP	O
-γ	O
--	O
-phosphate	O
-in	O
-both	O
-structures	O
-suggest	O
-that	O
-the	O
-γ	O
--	O
-phosphate	O
-group	O
-of	O
-ATP	O
-is	O
-either	O
-flexible	O
-or	O
-hydrolyzed	O
-by	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-.	O
-	O
-This	O
-result	O
-was	O
-consistent	O
-with	O
-our	O
-enzymatic	O
-activity	O
-assays	O
-where	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-showed	O
-ATP	O
-hydrolysis	O
-activity	O
-without	O
-adding	O
-any	O
-substrates	O
-(	O
-Fig	O
-2A	O
-and	O
-2C	O
-).	O
-	O
-To	O
-avoid	O
-hydrolysis	O
-of	O
-ATP	O
-,	O
-we	O
-soaked	O
-the	O
-crystals	O
-of	O
-apo	O
--	O
-SePSK	O
-and	O
-apo	O
--	O
-AtXK	O
--	O
-1	O
-into	O
-the	O
-reservoir	O
-adding	O
-AMP	O
--	O
-PNP	O
-.	O
-	O
-However	O
-,	O
-we	O
-found	O
-that	O
-the	O
-electron	O
-densities	O
-of	O
-γ	O
--	O
-phosphate	O
-group	O
-of	O
-AMP	O
--	O
-PNP	O
-(	O
-AMP	O
--	O
-PNP	O
-γ	O
--	O
-phosphate	O
-)	O
-are	O
-still	O
-weak	O
-in	O
-the	O
-AMP	O
--	O
-PNP	O
--	O
-SePSK	O
-and	O
-AMP	O
--	O
-PNP	O
--	O
-AtXK	O
--	O
-1	O
-structures	O
-,	O
-suggesting	O
-high	O
-flexibility	O
-of	O
-ATP	O
--	O
-γ	O
--	O
-phosphate	O
-.	O
-	O
-The	O
-γ	O
--	O
-phosphate	O
-group	O
-of	O
-ATP	O
-is	O
-transferred	O
-to	O
-the	O
-sugar	O
-substrate	O
-during	O
-the	O
-reaction	O
-process	O
-,	O
-so	O
-this	O
-flexibility	O
-might	O
-be	O
-important	O
-for	O
-the	O
-ability	O
-of	O
-these	O
-kinases	O
-.	O
-	O
-The	O
-overall	O
-structures	O
-as	O
-well	O
-as	O
-the	O
-coordination	O
-modes	O
-of	O
-ADP	O
-and	O
-AMP	O
--	O
-PNP	O
-in	O
-the	O
-AMP	O
--	O
-PNP	O
--	O
-AtXK	O
--	O
-1	O
-,	O
-ADP	O
--	O
-AtXK	O
--	O
-1	O
-,	O
-ADP	O
--	O
-SePSK	O
-and	O
-AMP	O
--	O
-PNP	O
--	O
-SePSK	O
-structures	O
-are	O
-nearly	O
-identical	O
-(	O
-S5	O
-Fig	O
-),	O
-therefore	O
-the	O
-structure	O
-of	O
-AMP	O
--	O
-PNP	O
--	O
-SePSK	O
-is	O
-used	O
-here	O
-to	O
-describe	O
-the	O
-structural	O
-details	O
-and	O
-to	O
-compare	O
-with	O
-those	O
-of	O
-other	O
-family	O
-members	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-3A	O
-,	O
-one	O
-SePSK	O
-protein	O
-molecule	O
-is	O
-in	O
-an	O
-asymmetric	O
-unit	O
-with	O
-one	O
-AMP	O
--	O
-PNP	O
-molecule	O
-.	O
-	O
-The	O
-AMP	O
--	O
-PNP	O
-is	O
-bound	O
-at	O
-the	O
-domain	O
-II	O
-,	O
-where	O
-it	O
-fits	O
-well	O
-inside	O
-a	O
-positively	O
-charged	O
-groove	O
-.	O
-	O
-The	O
-AMP	O
--	O
-PNP	O
-binding	O
-pocket	O
-consists	O
-of	O
-four	O
-α	O
--	O
-helices	O
-(	O
-α26	O
-,	O
-α28	O
-,	O
-α27	O
-and	O
-α30	O
-)	O
-and	O
-forms	O
-a	O
-shape	O
-resembling	O
-a	O
-half	O
--	O
-fist	O
-(	O
-Fig	O
-3A	O
-and	O
-3B	O
-).	O
-	O
-The	O
-head	O
-group	O
-of	O
-the	O
-AMP	O
--	O
-PNP	O
-is	O
-embedded	O
-in	O
-a	O
-pocket	O
-surrounded	O
-by	O
-Trp383	O
-,	O
-Asn380	O
-,	O
-Gly376	O
-and	O
-Gly377	O
-.	O
-	O
-The	O
-purine	O
-ring	O
-of	O
-AMP	O
--	O
-PNP	O
-is	O
-positioned	O
-in	O
-parallel	O
-to	O
-the	O
-indole	O
-ring	O
-of	O
-Trp383	O
-.	O
-	O
-In	O
-addition	O
-,	O
-it	O
-is	O
-hydrogen	O
--	O
-bonded	O
-with	O
-the	O
-side	O
-chain	O
-amide	O
-of	O
-Asn380	O
-(	O
-Fig	O
-3B	O
-).	O
-	O
-The	O
-tail	O
-of	O
-AMP	O
--	O
-PNP	O
-points	O
-to	O
-the	O
-hinge	O
-region	O
-of	O
-SePSK	O
-,	O
-and	O
-its	O
-α	O
--	O
-phosphate	O
-and	O
-β	O
--	O
-phosphate	O
-groups	O
-are	O
-stabilized	O
-by	O
-Gly376	O
-and	O
-Ser243	O
-,	O
-respectively	O
-.	O
-	O
-Together	O
-,	O
-this	O
-structure	O
-clearly	O
-shows	O
-that	O
-the	O
-AMP	O
--	O
-PNP	O
--	O
-β	O
--	O
-phosphate	O
-is	O
-sticking	O
-out	O
-of	O
-the	O
-ATP	O
-binding	O
-pocket	O
-,	O
-thus	O
-the	O
-γ	O
--	O
-phosphate	O
-group	O
-is	O
-at	O
-the	O
-empty	O
-space	O
-between	O
-domain	O
-I	O
-and	O
-domain	O
-II	O
-and	O
-is	O
-unconstrained	O
-in	O
-its	O
-movement	O
-by	O
-the	O
-protein	O
-.	O
-	O
-Structure	O
-of	O
-SePSK	O
-in	O
-complex	O
-with	O
-AMP	O
--	O
-PNP	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-electron	O
-density	O
-of	O
-AMP	O
--	O
-PNP	O
-.	O
-	O
-The	O
-SePSK	O
-structure	O
-is	O
-shown	O
-in	O
-the	O
-electrostatic	O
-potential	O
-surface	O
-mode	O
-.	O
-	O
-The	O
-AMP	O
--	O
-PNP	O
-is	O
-depicted	O
-as	O
-sticks	O
-with	O
-its	O
-ǀFoǀ	O
--	O
-ǀFcǀ	O
-map	O
-contoured	O
-at	O
-3	O
-σ	O
-shown	O
-as	O
-cyan	O
-mesh	O
-.	O
-	O
-(	O
-B	O
-)	O
-The	O
-AMP	O
--	O
-PNP	O
-binding	O
-pocket	O
-.	O
-	O
-The	O
-head	O
-of	O
-AMP	O
--	O
-PNP	O
-is	O
-sandwiched	O
-by	O
-four	O
-residues	O
-(	O
-Leu293	O
-,	O
-Gly376	O
-,	O
-Gly377	O
-and	O
-Trp383	O
-).	O
-	O
-The	O
-four	O
-α	O
--	O
-helices	O
-(	O
-α26	O
-,	O
-α28	O
-,	O
-α27	O
-and	O
-α30	O
-)	O
-are	O
-labeled	O
-in	O
-red	O
-.	O
-	O
-The	O
-AMP	O
--	O
-PNP	O
-and	O
-coordinated	O
-residues	O
-are	O
-shown	O
-as	O
-sticks	O
-.	O
-	O
-The	O
-potential	O
-substrate	O
-binding	O
-site	O
-in	O
-SePSK	O
-	O
-The	O
-results	O
-from	O
-our	O
-activity	O
-assays	O
-suggested	O
-that	O
-SePSK	O
-has	O
-D	O
--	O
-ribulose	O
-kinase	O
-activity	O
-.	O
-	O
-To	O
-better	O
-understand	O
-the	O
-interaction	O
-pattern	O
-between	O
-SePSK	O
-and	O
-D	O
--	O
-ribulose	O
-,	O
-the	O
-apo	O
--	O
-SePSK	O
-crystals	O
-were	O
-soaked	O
-into	O
-the	O
-reservoir	O
-with	O
-10	O
-mM	O
-D	O
--	O
-ribulose	O
-(	O
-RBL	O
-)	O
-and	O
-the	O
-RBL	O
--	O
-SePSK	O
-structure	O
-was	O
-solved	O
-.	O
-	O
-As	O
-shown	O
-in	O
-S6	O
-Fig	O
-,	O
-two	O
-residual	O
-electron	O
-densities	O
-are	O
-visible	O
-in	O
-domain	O
-I	O
-,	O
-which	O
-can	O
-be	O
-interpreted	O
-as	O
-two	O
-D	O
--	O
-ribulose	O
-molecules	O
-with	O
-reasonable	O
-fit	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-4A	O
-,	O
-the	O
-nearest	O
-distance	O
-between	O
-the	O
-carbon	O
-skeleton	O
-of	O
-two	O
-D	O
--	O
-ribulose	O
-molecules	O
-are	O
-approx	O
-.	O
-	O
-7	O
-.	O
-1	O
-Å	O
-(	O
-RBL1	O
--	O
-C4	O
-and	O
-RBL2	O
--	O
-C1	O
-).	O
-	O
-RBL1	O
-is	O
-located	O
-in	O
-the	O
-pocket	O
-consisting	O
-of	O
-α21	O
-and	O
-the	O
-loop	O
-between	O
-β6	O
-and	O
-β7	O
-.	O
-	O
-The	O
-O4	O
-and	O
-O5	O
-of	O
-RBL1	O
-are	O
-coordinated	O
-with	O
-the	O
-side	O
-chain	O
-carboxyl	O
-group	O
-of	O
-Asp221	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-O2	O
-of	O
-RBL1	O
-interacts	O
-with	O
-the	O
-main	O
-chain	O
-amide	O
-nitrogen	O
-of	O
-Ser72	O
-(	O
-Fig	O
-4B	O
-).	O
-	O
-This	O
-pocket	O
-is	O
-at	O
-a	O
-similar	O
-position	O
-of	O
-substrate	O
-binding	O
-site	O
-of	O
-other	O
-sugar	O
-kinase	O
-,	O
-such	O
-as	O
-L	O
--	O
-ribulokinase	O
-(	O
-PDB	O
-code	O
-:	O
-3QDK	O
-)	O
-(	O
-S7	O
-Fig	O
-).	O
-	O
-However	O
-,	O
-structural	O
-comparison	O
-shows	O
-that	O
-the	O
-substrate	O
-ligating	O
-residues	O
-between	O
-the	O
-two	O
-structures	O
-are	O
-not	O
-strictly	O
-conserved	O
-.	O
-	O
-Based	O
-on	O
-the	O
-structures	O
-,	O
-the	O
-ligating	O
-residues	O
-of	O
-RBL1	O
-in	O
-RBL	O
--	O
-SePSK	O
-structure	O
-are	O
-Ser72	O
-,	O
-Asp221	O
-and	O
-Ser222	O
-,	O
-and	O
-the	O
-interacting	O
-residues	O
-of	O
-L	O
--	O
-ribulose	O
-with	O
-L	O
--	O
-ribulokinase	O
-are	O
-Ala96	O
-,	O
-Lys208	O
-,	O
-Asp274	O
-and	O
-Glu329	O
-(	O
-S7	O
-Fig	O
-).	O
-	O
-Glu329	O
-in	O
-3QDK	O
-has	O
-no	O
-counterpart	O
-in	O
-RBL	O
--	O
-SePSK	O
-structure	O
-.	O
-	O
-In	O
-addition	O
-,	O
-although	O
-Lys208	O
-of	O
-L	O
--	O
-ribulokinase	O
-has	O
-the	O
-corresponding	O
-residue	O
-(	O
-Lys163	O
-)	O
-in	O
-RBL	O
--	O
-SePSK	O
-structure	O
-,	O
-the	O
-hydrogen	O
-bond	O
-of	O
-Lys163	O
-is	O
-broken	O
-because	O
-of	O
-the	O
-conformational	O
-change	O
-of	O
-two	O
-α	O
--	O
-helices	O
-(	O
-α9	O
-and	O
-α13	O
-)	O
-of	O
-SePSK	O
-.	O
-	O
-The	O
-binding	O
-of	O
-D	O
--	O
-ribulose	O
-(	O
-RBL	O
-)	O
-with	O
-SePSK	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-electrostatic	O
-potential	O
-surface	O
-map	O
-of	O
-RBL	O
--	O
-SePSK	O
-and	O
-a	O
-zoom	O
--	O
-in	O
-view	O
-of	O
-RBL	O
-binding	O
-site	O
-.	O
-	O
-The	O
-RBL1	O
-and	O
-RBL2	O
-are	O
-depicted	O
-as	O
-sticks	O
-.	O
-(	O
-B	O
-)	O
-Interaction	O
-of	O
-two	O
-D	O
--	O
-ribulose	O
-molecules	O
-(	O
-RBL1	O
-and	O
-RBL2	O
-)	O
-with	O
-SePSK	O
-.	O
-	O
-The	O
-RBL	O
-molecules	O
-(	O
-carbon	O
-atoms	O
-colored	O
-yellow	O
-)	O
-and	O
-amino	O
-acid	O
-residues	O
-of	O
-SePSK	O
-(	O
-carbon	O
-atoms	O
-colored	O
-green	O
-)	O
-involved	O
-in	O
-RBL	O
-interaction	O
-are	O
-shown	O
-as	O
-sticks	O
-.	O
-	O
-The	O
-hydrogen	O
-bonds	O
-are	O
-indicated	O
-by	O
-the	O
-black	O
-dashed	O
-lines	O
-and	O
-the	O
-numbers	O
-near	O
-the	O
-dashed	O
-lines	O
-are	O
-the	O
-distances	O
-(	O
-Å	O
-).	O
-(	O
-C	O
-)	O
-The	O
-binding	O
-affinity	O
-assays	O
-of	O
-SePSK	O
-with	O
-D	O
--	O
-ribulose	O
-.	O
-	O
-Single	O
--	O
-cycle	O
-kinetic	O
-data	O
-are	O
-reflecting	O
-the	O
-interaction	O
-of	O
-SePSK	O
-and	O
-D8A	B-mutant
--	O
-SePSK	O
-with	O
-D	O
--	O
-ribulose	O
-.	O
-	O
-It	O
-shows	O
-two	O
-experimental	O
-sensorgrams	O
-after	O
-minus	O
-the	O
-empty	O
-sensorgrams	O
-.	O
-	O
-The	O
-original	O
-data	O
-is	O
-shown	O
-as	O
-black	O
-curve	O
-,	O
-and	O
-the	O
-fitted	O
-data	O
-is	O
-shown	O
-as	O
-different	O
-color	O
-(	O
-wild	O
-type	O
-SePSK	O
-:	O
-red	O
-curve	O
-,	O
-D8A	B-mutant
--	O
-SePSK	O
-:	O
-green	O
-curve	O
-).	O
-	O
-Dissociation	O
-rate	O
-constant	O
-of	O
-wild	O
-type	O
-and	O
-D8A	B-mutant
--	O
-SePSK	O
-are	O
-3	O
-ms	O
--	O
-1	O
-and	O
-9	O
-ms	O
--	O
-1	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-binding	O
-pocket	O
-of	O
-RBL2	O
-with	O
-relatively	O
-weak	O
-electron	O
-density	O
-is	O
-near	O
-the	O
-N	O
--	O
-terminal	O
-region	O
-of	O
-SePSK	O
-and	O
-is	O
-negatively	O
-charged	O
-.	O
-	O
-The	O
-side	O
-chain	O
-of	O
-Asp8	O
-interacts	O
-strongly	O
-with	O
-O3	O
-and	O
-O4	O
-of	O
-RBL2	O
-.	O
-	O
-The	O
-hydroxyl	O
-group	O
-of	O
-Ser12	O
-coordinates	O
-with	O
-O2	O
-of	O
-RBL2	O
-.	O
-	O
-The	O
-backbone	O
-amide	O
-nitrogens	O
-of	O
-Gly13	O
-and	O
-Arg15	O
-also	O
-keep	O
-hydrogen	O
-bonds	O
-with	O
-RBL2	O
-(	O
-Fig	O
-4B	O
-).	O
-	O
-Structural	O
-comparison	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-showed	O
-that	O
-while	O
-the	O
-RBL1	O
-binding	O
-pocket	O
-is	O
-conserved	O
-,	O
-the	O
-RBL2	O
-pocket	O
-is	O
-disrupted	O
-in	O
-AtXK	O
--	O
-1	O
-structure	O
-,	O
-despite	O
-the	O
-fact	O
-that	O
-the	O
-residues	O
-interacting	O
-with	O
-RBL2	O
-are	O
-highly	O
-conserved	O
-between	O
-the	O
-two	O
-proteins	O
-.	O
-	O
-In	O
-the	O
-RBL	O
--	O
-SePSK	O
-structure	O
-,	O
-a	O
-2	O
-.	O
-6	O
-Å	O
-hydrogen	O
-bond	O
-is	O
-present	O
-between	O
-RBL2	O
-and	O
-Ser12	O
-(	O
-Fig	O
-4B	O
-),	O
-while	O
-in	O
-the	O
-AtXK	O
--	O
-1	O
-structure	O
-this	O
-hydrogen	O
-bond	O
-with	O
-the	O
-corresponding	O
-residue	O
-(	O
-Ser22	O
-)	O
-is	O
-broken	O
-.	O
-	O
-This	O
-break	O
-is	O
-probably	O
-induced	O
-by	O
-the	O
-conformational	O
-change	O
-of	O
-the	O
-two	O
-β	O
--	O
-sheets	O
-(	O
-β1	O
-and	O
-β2	O
-),	O
-with	O
-the	O
-result	O
-that	O
-the	O
-linking	O
-loop	O
-(	O
-loop	O
-1	O
-)	O
-is	O
-located	O
-further	O
-away	O
-from	O
-the	O
-RBL2	O
-binding	O
-site	O
-.	O
-	O
-This	O
-change	O
-might	O
-be	O
-the	O
-reason	O
-that	O
-AtXK	O
--	O
-1	O
-only	O
-shows	O
-limited	O
-increasing	O
-in	O
-its	O
-ATP	O
-hydrolysis	O
-ability	O
-upon	O
-adding	O
-D	O
--	O
-ribulose	O
-as	O
-a	O
-substrate	O
-after	O
-comparing	O
-with	O
-SePSK	O
-(	O
-Fig	O
-2C	O
-).	O
-	O
-Our	O
-SePSK	O
-structure	O
-shows	O
-that	O
-the	O
-Asp8	O
-residue	O
-forms	O
-strong	O
-hydrogen	O
-bond	O
-with	O
-RBL2	O
-(	O
-Fig	O
-4B	O
-).	O
-	O
-In	O
-addition	O
-,	O
-our	O
-enzymatic	O
-assays	O
-indicated	O
-that	O
-Asp8	O
-is	O
-important	O
-for	O
-the	O
-activity	O
-of	O
-SePSK	O
-(	O
-Fig	O
-2D	O
-).	O
-	O
-To	O
-further	O
-verified	O
-this	O
-result	O
-,	O
-we	O
-measured	O
-the	O
-binding	O
-affinity	O
-for	O
-D	O
--	O
-ribulose	O
-of	O
-both	O
-wild	O
-type	O
-(	O
-WT	O
-)	O
-and	O
-D8A	B-mutant
-mutant	O
-of	O
-SePSK	O
-using	O
-a	O
-surface	O
-plasmon	O
-resonance	O
-method	O
-.	O
-	O
-The	O
-results	O
-showed	O
-that	O
-the	O
-affinity	O
-of	O
-D8A	B-mutant
--	O
-SePSK	O
-with	O
-D	O
--	O
-ribulose	O
-is	O
-weaker	O
-than	O
-that	O
-of	O
-WT	O
-with	O
-a	O
-reduction	O
-of	O
-approx	O
-.	O
-	O
-Dissociation	O
-rate	O
-constant	O
-(	O
-Kd	O
-)	O
-of	O
-wild	O
-type	O
-and	O
-D8A	B-mutant
--	O
-SePSK	O
-are	O
-3	O
-ms	O
--	O
-1	O
-and	O
-9	O
-ms	O
--	O
-1	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-results	O
-implied	O
-that	O
-the	O
-second	O
-RBL	O
-binding	O
-site	O
-plays	O
-a	O
-role	O
-in	O
-the	O
-D	O
--	O
-ribulose	O
-kinase	O
-function	O
-of	O
-SePSK	O
-.	O
-	O
-However	O
-,	O
-considering	O
-the	O
-high	O
-concentration	O
-of	O
-D	O
--	O
-ribulose	O
-used	O
-for	O
-crystal	O
-soaking	O
-,	O
-as	O
-well	O
-as	O
-the	O
-relatively	O
-weak	O
-electron	O
-density	O
-of	O
-RBL2	O
-,	O
-it	O
-is	O
-also	O
-possible	O
-that	O
-the	O
-second	O
-binding	O
-site	O
-of	O
-D	O
--	O
-ribulose	O
-in	O
-SePSK	O
-is	O
-an	O
-artifact	O
-.	O
-	O
-Simulated	O
-conformational	O
-change	O
-of	O
-SePSK	O
-during	O
-the	O
-catalytic	O
-process	O
-	O
-It	O
-was	O
-reported	O
-earlier	O
-that	O
-the	O
-crossing	O
-angle	O
-between	O
-the	O
-domain	O
-I	O
-and	O
-domain	O
-II	O
-in	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-is	O
-different	O
-.	O
-	O
-In	O
-addition	O
-,	O
-this	O
-difference	O
-may	O
-be	O
-caused	O
-by	O
-the	O
-binding	O
-of	O
-substrates	O
-and	O
-/	O
-or	O
-ATP	O
-.	O
-	O
-As	O
-reported	O
-previously	O
-,	O
-members	O
-of	O
-the	O
-sugar	O
-kinase	O
-family	O
-undergo	O
-a	O
-conformational	O
-change	O
-to	O
-narrow	O
-the	O
-crossing	O
-angle	O
-between	O
-two	O
-domains	O
-and	O
-reduce	O
-the	O
-distance	O
-between	O
-substrate	O
-and	O
-ATP	O
-in	O
-order	O
-to	O
-facilitate	O
-the	O
-catalytic	O
-reaction	O
-of	O
-phosphorylation	O
-of	O
-sugar	O
-substrates	O
-.	O
-	O
-After	O
-comparing	O
-structures	O
-of	O
-apo	O
--	O
-SePSK	O
-,	O
-RBL	O
--	O
-SePSK	O
-and	O
-AMP	O
--	O
-PNP	O
--	O
-SePSK	O
-,	O
-we	O
-noticed	O
-that	O
-these	O
-structures	O
-presented	O
-here	O
-are	O
-similar	O
-.	O
-	O
-Superposing	O
-the	O
-structures	O
-of	O
-RBL	O
--	O
-SePSK	O
-and	O
-AMP	O
--	O
-PNP	O
--	O
-SePSK	O
-,	O
-the	O
-results	O
-show	O
-that	O
-the	O
-nearest	O
-distance	O
-between	O
-AMP	O
--	O
-PNP	O
-γ	O
--	O
-phosphate	O
-and	O
-RBL1	O
-/	O
-RBL2	O
-is	O
-7	O
-.	O
-5	O
-Å	O
-(	O
-RBL1	O
--	O
-O5	O
-)/	O
-6	O
-.	O
-7	O
-Å	O
-(	O
-RBL2	O
--	O
-O1	O
-)	O
-(	O
-S8	O
-Fig	O
-).	O
-	O
-This	O
-distance	O
-is	O
-too	O
-long	O
-to	O
-transfer	O
-the	O
-γ	O
--	O
-phosphate	O
-group	O
-from	O
-ATP	O
-to	O
-the	O
-substrate	O
-.	O
-	O
-Since	O
-the	O
-two	O
-domains	O
-of	O
-SePSK	O
-are	O
-widely	O
-separated	O
-in	O
-this	O
-structure	O
-,	O
-we	O
-hypothesize	O
-that	O
-our	O
-structures	O
-of	O
-SePSK	O
-represent	O
-its	O
-open	O
-form	O
-,	O
-and	O
-that	O
-a	O
-conformational	O
-rearrangement	O
-must	O
-occur	O
-to	O
-switch	O
-to	O
-the	O
-closed	O
-state	O
-in	O
-order	O
-to	O
-facilitate	O
-the	O
-catalytic	O
-process	O
-of	O
-phosphorylation	O
-of	O
-sugar	O
-substrates	O
-.	O
-	O
-For	O
-studying	O
-such	O
-potential	O
-conformational	O
-change	O
-,	O
-a	O
-simulation	O
-on	O
-the	O
-Hingeprot	O
-Server	O
-was	O
-performed	O
-to	O
-predict	O
-the	O
-movement	O
-of	O
-different	O
-SePSK	O
-domains	O
-.	O
-	O
-The	O
-results	O
-showed	O
-that	O
-domain	O
-I	O
-and	O
-domain	O
-II	O
-are	O
-closer	O
-to	O
-each	O
-other	O
-with	O
-Ala228	O
-and	O
-Thr401	O
-in	O
-A2	O
-as	O
-Hinge	O
--	O
-residues	O
-.	O
-	O
-Based	O
-on	O
-the	O
-above	O
-results	O
-,	O
-SePSK	O
-is	O
-divided	O
-into	O
-two	O
-rigid	O
-parts	O
-.	O
-	O
-The	O
-domain	O
-I	O
-of	O
-RBL	O
--	O
-SePSK	O
-(	O
-aa	O
-.	O
-1	O
-–	O
-228	O
-,	O
-aa	O
-.	O
-402	O
-–	O
-421	O
-)	O
-and	O
-the	O
-domain	O
-II	O
-of	O
-AMP	O
--	O
-PNP	O
--	O
-SePSK	O
-(	O
-aa	O
-.	O
-229	O
-–	O
-401	O
-)	O
-were	O
-superposed	O
-with	O
-structures	O
-,	O
-including	O
-apo	O
--	O
-AtXK	O
--	O
-1	O
-,	O
-apo	O
--	O
-SePSK	O
-,	O
-xylulose	O
-kinase	O
-from	O
-Lactobacillus	O
-acidophilus	O
-(	O
-PDB	O
-code	O
-:	O
-3LL3	O
-)	O
-and	O
-the	O
-S58W	B-mutant
-mutant	O
-form	O
-of	O
-glycerol	O
-kinase	O
-from	O
-Escherichia	O
-coli	O
-(	O
-PDB	O
-code	O
-:	O
-1GLJ	O
-).	O
-	O
-The	O
-results	O
-of	O
-superposition	O
-displayed	O
-different	O
-crossing	O
-angle	O
-between	O
-these	O
-two	O
-domains	O
-.	O
-	O
-After	O
-superposition	O
-,	O
-the	O
-distances	O
-of	O
-AMP	O
--	O
-PNP	O
-γ	O
--	O
-phosphate	O
-and	O
-the	O
-fifth	O
-hydroxyl	O
-group	O
-of	O
-RBL1	O
-are	O
-7	O
-.	O
-9	O
-Å	O
-(	O
-superposed	O
-with	O
-AtXK	O
--	O
-1	O
-),	O
-7	O
-.	O
-4	O
-Å	O
-(	O
-superposed	O
-with	O
-SePSK	O
-),	O
-6	O
-.	O
-6	O
-Å	O
-(	O
-superposed	O
-with	O
-3LL3	O
-)	O
-and	O
-6	O
-.	O
-1	O
-Å	O
-(	O
-superposed	O
-with	O
-1GLJ	O
-).	O
-	O
-Meanwhile	O
-,	O
-the	O
-distances	O
-of	O
-AMP	O
--	O
-PNP	O
-γ	O
--	O
-phosphate	O
-and	O
-the	O
-first	O
-hydroxyl	O
-group	O
-of	O
-RBL2	O
-are	O
-7	O
-.	O
-2	O
-Å	O
-(	O
-superposed	O
-with	O
-AtXK	O
--	O
-1	O
-),	O
-6	O
-.	O
-7	O
-Å	O
-(	O
-superposed	O
-with	O
-SePSK	O
-),	O
-3	O
-.	O
-7	O
-Å	O
-(	O
-superposed	O
-with	O
-3LL3	O
-),	O
-until	O
-AMP	O
--	O
-PNP	O
-γ	O
--	O
-phosphate	O
-fully	O
-contacts	O
-RBL2	O
-after	O
-superposition	O
-with	O
-1GLJ	O
-(	O
-Fig	O
-5	O
-).	O
-	O
-This	O
-distance	O
-between	O
-RBL2	O
-and	O
-AMP	O
--	O
-PNP	O
--	O
-γ	O
--	O
-phosphate	O
-is	O
-close	O
-enough	O
-to	O
-facilitate	O
-phosphate	O
-transferring	O
-.	O
-	O
-Together	O
-,	O
-our	O
-superposition	O
-results	O
-provided	O
-snapshots	O
-of	O
-the	O
-conformational	O
-changes	O
-at	O
-different	O
-catalytic	O
-stages	O
-of	O
-SePSK	O
-and	O
-potentially	O
-revealed	O
-the	O
-closed	O
-form	O
-of	O
-SePSK	O
-.	O
-	O
-Simulated	O
-conformational	O
-change	O
-of	O
-SePSK	O
-during	O
-the	O
-catalytic	O
-process	O
-.	O
-	O
-The	O
-structures	O
-are	O
-shown	O
-as	O
-cartoon	O
-and	O
-the	O
-ligands	O
-are	O
-shown	O
-as	O
-sticks	O
-.	O
-	O
-Domain	O
-I	O
-from	O
-D	O
--	O
-ribulose	O
--	O
-SePSK	O
-(	O
-green	O
-)	O
-and	O
-Domain	O
-II	O
-from	O
-AMP	O
--	O
-PNP	O
--	O
-SePSK	O
-(	O
-cyan	O
-)	O
-are	O
-superposed	O
-with	O
-apo	O
--	O
-AtXK	O
--	O
-1	O
-(	O
-1st	O
-),	O
-apo	O
--	O
-SePSK	O
-(	O
-2nd	O
-),	O
-3LL3	O
-(	O
-3rd	O
-)	O
-and	O
-1GLJ	O
-(	O
-4th	O
-),	O
-respectively	O
-.	O
-	O
-The	O
-numbers	O
-near	O
-the	O
-black	O
-dashed	O
-lines	O
-show	O
-the	O
-distances	O
-(	O
-Å	O
-)	O
-between	O
-two	O
-nearest	O
-atoms	O
-of	O
-RBL	O
-and	O
-AMP	O
--	O
-PNP	O
-.	O
-	O
-In	O
-summary	O
-,	O
-our	O
-structural	O
-and	O
-enzymatic	O
-analyses	O
-provide	O
-evidence	O
-that	O
-SePSK	O
-shows	O
-D	O
--	O
-ribulose	O
-kinase	O
-activity	O
-,	O
-and	O
-exhibits	O
-the	O
-conserved	O
-features	O
-of	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-.	O
-	O
-Three	O
-conserved	O
-residues	O
-in	O
-SePSK	O
-were	O
-identified	O
-to	O
-be	O
-essential	O
-for	O
-this	O
-function	O
-.	O
-	O
-Our	O
-results	O
-provide	O
-the	O
-detailed	O
-information	O
-about	O
-the	O
-interaction	O
-of	O
-SePSK	O
-with	O
-ATP	O
-and	O
-substrates	O
-.	O
-	O
-Moreover	O
-,	O
-structural	O
-superposition	O
-results	O
-enable	O
-us	O
-to	O
-visualize	O
-the	O
-conformational	O
-change	O
-of	O
-SePSK	O
-during	O
-the	O
-catalytic	O
-process	O
-.	O
-	O
-In	O
-conclusion	O
-,	O
-our	O
-results	O
-provide	O
-important	O
-information	O
-for	O
-a	O
-more	O
-detailed	O
-understanding	O
-of	O
-the	O
-mechanisms	O
-of	O
-SePSK	O
-and	O
-other	O
-members	O
-of	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-.	O
-	O
-Structural	O
-insights	O
-into	O
-the	O
-Escherichia	O
-coli	O
-lysine	O
-decarboxylases	O
-and	O
-molecular	O
-determinants	O
-of	O
-interaction	O
-with	O
-the	O
-AAA	O
-+	O
-ATPase	O
-RavA	O
-	O
-The	O
-inducible	O
-lysine	O
-decarboxylase	O
-LdcI	O
-is	O
-an	O
-important	O
-enterobacterial	O
-acid	O
-stress	O
-response	O
-enzyme	O
-whereas	O
-LdcC	O
-is	O
-its	O
-close	O
-paralogue	O
-thought	O
-to	O
-play	O
-mainly	O
-a	O
-metabolic	O
-role	O
-.	O
-	O
-A	O
-unique	O
-macromolecular	O
-cage	O
-formed	O
-by	O
-two	O
-decamers	O
-of	O
-the	O
-Escherichia	O
-coli	O
-LdcI	O
-and	O
-five	O
-hexamers	O
-of	O
-the	O
-AAA	O
-+	O
-ATPase	O
-RavA	O
-was	O
-shown	O
-to	O
-counteract	O
-acid	O
-stress	O
-under	O
-starvation	O
-.	O
-	O
-Previously	O
-,	O
-we	O
-proposed	O
-a	O
-pseudoatomic	O
-model	O
-of	O
-the	O
-LdcI	O
--	O
-RavA	O
-cage	O
-based	O
-on	O
-its	O
-cryo	O
--	O
-electron	O
-microscopy	O
-map	O
-and	O
-crystal	O
-structures	O
-of	O
-an	O
-inactive	O
-LdcI	O
-decamer	O
-and	O
-a	O
-RavA	O
-monomer	O
-.	O
-	O
-We	O
-now	O
-present	O
-cryo	O
--	O
-electron	O
-microscopy	O
-3D	O
-reconstructions	O
-of	O
-the	O
-E	O
-.	O
-coli	O
-LdcI	O
-and	O
-LdcC	O
-,	O
-and	O
-an	O
-improved	O
-map	O
-of	O
-the	O
-LdcI	O
-bound	O
-to	O
-the	O
-LARA	O
-domain	O
-of	O
-RavA	O
-,	O
-at	O
-pH	O
-optimal	O
-for	O
-their	O
-enzymatic	O
-activity	O
-.	O
-	O
-Comparison	O
-with	O
-each	O
-other	O
-and	O
-with	O
-available	O
-structures	O
-uncovers	O
-differences	O
-between	O
-LdcI	O
-and	O
-LdcC	O
-explaining	O
-why	O
-only	O
-the	O
-acid	O
-stress	O
-response	O
-enzyme	O
-is	O
-capable	O
-of	O
-binding	O
-RavA	O
-.	O
-We	O
-identify	O
-interdomain	O
-movements	O
-associated	O
-with	O
-the	O
-pH	O
--	O
-dependent	O
-enzyme	O
-activation	O
-and	O
-with	O
-the	O
-RavA	O
-binding	O
-.	O
-	O
-Multiple	O
-sequence	O
-alignment	O
-coupled	O
-to	O
-a	O
-phylogenetic	O
-analysis	O
-reveals	O
-that	O
-certain	O
-enterobacteria	O
-exert	O
-evolutionary	O
-pressure	O
-on	O
-the	O
-lysine	O
-decarboxylase	O
-towards	O
-the	O
-cage	O
--	O
-like	O
-assembly	O
-with	O
-RavA	O
-,	O
-implying	O
-that	O
-this	O
-complex	O
-may	O
-have	O
-an	O
-important	O
-function	O
-under	O
-particular	O
-stress	O
-conditions	O
-.	O
-	O
-Enterobacterial	O
-inducible	O
-decarboxylases	O
-of	O
-basic	O
-amino	O
-acids	O
-lysine	O
-,	O
-arginine	O
-and	O
-ornithine	O
-have	O
-a	O
-common	O
-evolutionary	O
-origin	O
-and	O
-belong	O
-to	O
-the	O
-α	O
--	O
-family	O
-of	O
-pyridoxal	O
--	O
-5	O
-′-	O
-phosphate	O
-(	O
-PLP	O
-)-	O
-dependent	O
-enzymes	O
-.	O
-	O
-They	O
-counteract	O
-acid	O
-stress	O
-experienced	O
-by	O
-the	O
-bacterium	O
-in	O
-the	O
-host	O
-digestive	O
-and	O
-urinary	O
-tract	O
-,	O
-and	O
-in	O
-particular	O
-in	O
-the	O
-extremely	O
-acidic	O
-stomach	O
-.	O
-	O
-Each	O
-decarboxylase	O
-is	O
-induced	O
-by	O
-an	O
-excess	O
-of	O
-the	O
-target	O
-amino	O
-acid	O
-and	O
-a	O
-specific	O
-range	O
-of	O
-extracellular	O
-pH	O
-,	O
-and	O
-works	O
-in	O
-conjunction	O
-with	O
-a	O
-cognate	O
-inner	O
-membrane	O
-antiporter	O
-.	O
-	O
-Decarboxylation	O
-of	O
-the	O
-amino	O
-acid	O
-into	O
-a	O
-polyamine	O
-is	O
-catalysed	O
-by	O
-a	O
-PLP	O
-cofactor	O
-in	O
-a	O
-multistep	O
-reaction	O
-that	O
-consumes	O
-a	O
-cytoplasmic	O
-proton	O
-and	O
-produces	O
-a	O
-CO2	O
-molecule	O
-passively	O
-diffusing	O
-out	O
-of	O
-the	O
-cell	O
-,	O
-while	O
-the	O
-polyamine	O
-is	O
-excreted	O
-by	O
-the	O
-antiporter	O
-in	O
-exchange	O
-for	O
-a	O
-new	O
-amino	O
-acid	O
-substrate	O
-.	O
-	O
-Consequently	O
-,	O
-these	O
-enzymes	O
-buffer	O
-both	O
-the	O
-bacterial	O
-cytoplasm	O
-and	O
-the	O
-local	O
-extracellular	O
-environment	O
-.	O
-	O
-These	O
-amino	O
-acid	O
-decarboxylases	O
-are	O
-therefore	O
-called	O
-acid	O
-stress	O
-inducible	O
-or	O
-biodegradative	O
-to	O
-distinguish	O
-them	O
-from	O
-their	O
-biosynthetic	O
-lysine	O
-and	O
-ornithine	O
-decarboxylase	O
-paralogs	O
-catalysing	O
-the	O
-same	O
-reaction	O
-but	O
-responsible	O
-for	O
-the	O
-polyamine	O
-production	O
-at	O
-neutral	O
-pH	O
-.	O
-	O
-Inducible	O
-enterobacterial	O
-amino	O
-acid	O
-decarboxylases	O
-have	O
-been	O
-intensively	O
-studied	O
-since	O
-the	O
-early	O
-1940	O
-because	O
-the	O
-ability	O
-of	O
-bacteria	O
-to	O
-withstand	O
-acid	O
-stress	O
-can	O
-be	O
-linked	O
-to	O
-their	O
-pathogenicity	O
-in	O
-humans	O
-.	O
-	O
-In	O
-particular	O
-,	O
-the	O
-inducible	O
-lysine	O
-decarboxylase	O
-LdcI	O
-(	O
-or	O
-CadA	O
-)	O
-attracts	O
-attention	O
-due	O
-to	O
-its	O
-broad	O
-pH	O
-range	O
-of	O
-activity	O
-and	O
-its	O
-capacity	O
-to	O
-promote	O
-survival	O
-and	O
-growth	O
-of	O
-pathogenic	O
-enterobacteria	O
-such	O
-as	O
-Salmonella	O
-enterica	O
-serovar	O
-Typhimurium	O
-,	O
-Vibrio	O
-cholerae	O
-and	O
-Vibrio	O
-vulnificus	O
-under	O
-acidic	O
-conditions	O
-.	O
-	O
-Furthermore	O
-,	O
-both	O
-LdcI	O
-and	O
-the	O
-biosynthetic	O
-lysine	O
-decarboxylase	O
-LdcC	O
-of	O
-uropathogenic	O
-Escherichia	O
-coli	O
-(	O
-UPEC	O
-)	O
-appear	O
-to	O
-play	O
-an	O
-important	O
-role	O
-in	O
-increased	O
-resistance	O
-of	O
-this	O
-pathogen	O
-to	O
-nitrosative	O
-stress	O
-produced	O
-by	O
-nitric	O
-oxide	O
-and	O
-other	O
-damaging	O
-reactive	O
-nitrogen	O
-intermediates	O
-accumulating	O
-during	O
-the	O
-course	O
-of	O
-urinary	O
-tract	O
-infections	O
-(	O
-UTI	O
-).	O
-	O
-This	O
-effect	O
-is	O
-attributed	O
-to	O
-cadaverine	O
-,	O
-the	O
-diamine	O
-produced	O
-by	O
-decarboxylation	O
-of	O
-lysine	O
-by	O
-LdcI	O
-and	O
-LdcC	O
-,	O
-that	O
-was	O
-shown	O
-to	O
-enhance	O
-UPEC	O
-colonisation	O
-of	O
-the	O
-bladder	O
-.	O
-	O
-In	O
-addition	O
-,	O
-the	O
-biosynthetic	O
-E	O
-.	O
-coli	O
-lysine	O
-decarboxylase	O
-LdcC	O
-,	O
-long	O
-thought	O
-to	O
-be	O
-constitutively	O
-expressed	O
-in	O
-low	O
-amounts	O
-,	O
-was	O
-demonstrated	O
-to	O
-be	O
-strongly	O
-upregulated	O
-by	O
-fluoroquinolones	O
-via	O
-their	O
-induction	O
-of	O
-RpoS	O
-.	O
-A	O
-direct	O
-correlation	O
-between	O
-the	O
-level	O
-of	O
-cadaverine	O
-and	O
-the	O
-resistance	O
-of	O
-E	O
-.	O
-coli	O
-to	O
-these	O
-antibiotics	O
-commonly	O
-used	O
-as	O
-a	O
-first	O
--	O
-line	O
-treatment	O
-of	O
-UTI	O
-could	O
-be	O
-established	O
-.	O
-	O
-Both	O
-acid	O
-pH	O
-and	O
-cadaverine	O
-induce	O
-closure	O
-of	O
-outer	O
-membrane	O
-porins	O
-thereby	O
-contributing	O
-to	O
-bacterial	O
-protection	O
-from	O
-acid	O
-stress	O
-,	O
-but	O
-also	O
-from	O
-certain	O
-antibiotics	O
-,	O
-by	O
-reduction	O
-in	O
-membrane	O
-permeability	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-E	O
-.	O
-coli	O
-LdcI	O
-as	O
-well	O
-as	O
-its	O
-low	O
-resolution	O
-characterisation	O
-by	O
-electron	O
-microscopy	O
-(	O
-EM	O
-)	O
-showed	O
-that	O
-it	O
-is	O
-a	O
-decamer	O
-made	O
-of	O
-two	O
-pentameric	O
-rings	O
-.	O
-	O
-Each	O
-monomer	O
-is	O
-composed	O
-of	O
-three	O
-domains	O
-–	O
-an	O
-N	O
--	O
-terminal	O
-wing	O
-domain	O
-(	O
-residues	O
-1	O
-–	O
-129	O
-),	O
-a	O
-PLP	O
--	O
-binding	O
-core	O
-domain	O
-(	O
-residues	O
-130	O
-–	O
-563	O
-),	O
-and	O
-a	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-CTD	O
-,	O
-residues	O
-564	O
-–	O
-715	O
-).	O
-	O
-Monomers	O
-tightly	O
-associate	O
-via	O
-their	O
-core	O
-domains	O
-into	O
-2	O
--	O
-fold	O
-symmetrical	O
-dimers	O
-with	O
-two	O
-complete	O
-active	O
-sites	O
-,	O
-and	O
-further	O
-build	O
-a	O
-toroidal	O
-D5	O
--	O
-symmetrical	O
-structure	O
-held	O
-by	O
-the	O
-wing	O
-and	O
-core	O
-domain	O
-interactions	O
-around	O
-the	O
-central	O
-pore	O
-,	O
-with	O
-the	O
-CTDs	O
-at	O
-the	O
-periphery	O
-.	O
-	O
-Ten	O
-years	O
-ago	O
-we	O
-showed	O
-that	O
-the	O
-E	O
-.	O
-coli	O
-AAA	O
-+	O
-ATPase	O
-RavA	O
-,	O
-involved	O
-in	O
-multiple	O
-stress	O
-response	O
-pathways	O
-,	O
-tightly	O
-interacted	O
-with	O
-LdcI	O
-but	O
-was	O
-not	O
-capable	O
-of	O
-binding	O
-to	O
-LdcC	O
-.	O
-We	O
-described	O
-how	O
-two	O
-double	O
-pentameric	O
-rings	O
-of	O
-the	O
-LdcI	O
-tightly	O
-associate	O
-with	O
-five	O
-hexameric	O
-rings	O
-of	O
-RavA	O
-to	O
-form	O
-a	O
-unique	O
-cage	O
--	O
-like	O
-architecture	O
-that	O
-enables	O
-the	O
-bacterium	O
-to	O
-withstand	O
-acid	O
-stress	O
-even	O
-under	O
-conditions	O
-of	O
-nutrient	O
-deprivation	O
-eliciting	O
-stringent	O
-response	O
-.	O
-	O
-Furthermore	O
-,	O
-we	O
-recently	O
-solved	O
-the	O
-structure	O
-of	O
-the	O
-E	O
-.	O
-coli	O
-LdcI	O
--	O
-RavA	O
-complex	O
-by	O
-cryo	O
--	O
-electron	O
-microscopy	O
-(	O
-cryoEM	O
-)	O
-and	O
-combined	O
-it	O
-with	O
-the	O
-crystal	O
-structures	O
-of	O
-the	O
-individual	O
-proteins	O
-.	O
-	O
-This	O
-allowed	O
-us	O
-to	O
-make	O
-a	O
-pseudoatomic	O
-model	O
-of	O
-the	O
-whole	O
-assembly	O
-,	O
-underpinned	O
-by	O
-a	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcI	O
--	O
-LARA	O
-complex	O
-(	O
-with	O
-LARA	O
-standing	O
-for	O
-LdcI	O
-associating	O
-domain	O
-of	O
-RavA	O
-),	O
-and	O
-to	O
-identify	O
-conformational	O
-rearrangements	O
-and	O
-specific	O
-elements	O
-essential	O
-for	O
-complex	O
-formation	O
-.	O
-	O
-The	O
-main	O
-determinants	O
-of	O
-the	O
-LdcI	O
--	O
-RavA	O
-cage	O
-assembly	O
-appeared	O
-to	O
-be	O
-the	O
-N	O
--	O
-terminal	O
-loop	O
-of	O
-the	O
-LARA	O
-domain	O
-of	O
-RavA	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-LdcI	O
-.	O
-	O
-In	O
-spite	O
-of	O
-this	O
-wealth	O
-of	O
-structural	O
-information	O
-,	O
-the	O
-fact	O
-that	O
-LdcC	O
-does	O
-not	O
-interact	O
-with	O
-RavA	O
-,	O
-although	O
-the	O
-two	O
-lysine	O
-decarboxylases	O
-are	O
-69	O
-%	O
-identical	O
-and	O
-84	O
-%	O
-similar	O
-,	O
-and	O
-the	O
-physiological	O
-significance	O
-of	O
-the	O
-absence	O
-of	O
-this	O
-interaction	O
-remained	O
-unexplored	O
-.	O
-	O
-To	O
-solve	O
-this	O
-discrepancy	O
-,	O
-in	O
-the	O
-present	O
-work	O
-we	O
-provided	O
-a	O
-three	O
--	O
-dimensional	O
-(	O
-3D	O
-)	O
-cryoEM	O
-reconstruction	O
-of	O
-LdcC	O
-and	O
-compared	O
-it	O
-with	O
-the	O
-available	O
-LdcI	O
-and	O
-LdcI	O
--	O
-RavA	O
-structures	O
-.	O
-	O
-Given	O
-that	O
-the	O
-LdcI	O
-crystal	O
-structures	O
-were	O
-obtained	O
-at	O
-high	O
-pH	O
-where	O
-the	O
-enzyme	O
-is	O
-inactive	O
-(	O
-LdcIi	O
-,	O
-pH	O
-8	O
-.	O
-5	O
-),	O
-whereas	O
-the	O
-cryoEM	O
-reconstructions	O
-of	O
-LdcI	O
--	O
-RavA	O
-and	O
-LdcI	O
--	O
-LARA	O
-were	O
-done	O
-at	O
-acidic	O
-pH	O
-optimal	O
-for	O
-the	O
-enzymatic	O
-activity	O
-,	O
-for	O
-a	O
-meaningful	O
-comparison	O
-,	O
-we	O
-also	O
-produced	O
-a	O
-3D	O
-reconstruction	O
-of	O
-the	O
-LdcI	O
-at	O
-active	O
-pH	O
-(	O
-LdcIa	O
-,	O
-pH	O
-6	O
-.	O
-2	O
-).	O
-	O
-This	O
-comparison	O
-pinpointed	O
-differences	O
-between	O
-the	O
-biodegradative	O
-and	O
-the	O
-biosynthetic	O
-lysine	O
-decarboxylases	O
-and	O
-brought	O
-to	O
-light	O
-interdomain	O
-movements	O
-associated	O
-to	O
-pH	O
--	O
-dependent	O
-enzyme	O
-activation	O
-and	O
-RavA	O
-binding	O
-,	O
-notably	O
-at	O
-the	O
-predicted	O
-RavA	O
-binding	O
-site	O
-at	O
-the	O
-level	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-LdcI	O
-.	O
-Consequently	O
-,	O
-we	O
-tested	O
-the	O
-capacity	O
-of	O
-cage	O
-formation	O
-by	O
-LdcI	B-mutant
--	I-mutant
-LdcC	I-mutant
-chimeras	I-mutant
-where	O
-we	O
-interchanged	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheets	O
-in	O
-question	O
-.	O
-	O
-Finally	O
-,	O
-we	O
-performed	O
-multiple	O
-sequence	O
-alignment	O
-of	O
-22	O
-lysine	O
-decarboxylases	O
-from	O
-Enterobacteriaceae	O
-containing	O
-the	O
-ravA	O
--	O
-viaA	O
-operon	O
-in	O
-their	O
-genome	O
-.	O
-	O
-Remarkably	O
-,	O
-this	O
-analysis	O
-revealed	O
-that	O
-several	O
-specific	O
-residues	O
-in	O
-the	O
-above	O
--	O
-mentioned	O
-β	O
--	O
-sheet	O
-,	O
-independently	O
-of	O
-the	O
-rest	O
-of	O
-the	O
-protein	O
-sequence	O
-,	O
-are	O
-sufficient	O
-to	O
-define	O
-if	O
-a	O
-particular	O
-lysine	O
-decarboxylase	O
-should	O
-be	O
-classified	O
-as	O
-an	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-or	O
-an	O
-“	O
-LdcI	O
--	O
-like	O
-”.	O
-	O
-This	O
-fascinating	O
-parallelism	O
-between	O
-the	O
-propensity	O
-for	O
-RavA	O
-binding	O
-and	O
-the	O
-genetic	O
-environment	O
-of	O
-an	O
-enterobacterial	O
-lysine	O
-decarboxylase	O
-,	O
-as	O
-well	O
-as	O
-the	O
-high	O
-degree	O
-of	O
-conservation	O
-of	O
-this	O
-small	O
-structural	O
-motif	O
-,	O
-emphasize	O
-the	O
-functional	O
-importance	O
-of	O
-the	O
-interaction	O
-between	O
-biodegradative	O
-enterobacterial	O
-lysine	O
-decarboxylases	O
-and	O
-the	O
-AAA	O
-+	O
-ATPase	O
-RavA	O
-.	O
-	O
-CryoEM	O
-3D	O
-reconstructions	O
-of	O
-LdcC	O
-,	O
-LdcIa	O
-and	O
-LdcI	O
--	O
-LARA	O
-	O
-In	O
-the	O
-frame	O
-of	O
-this	O
-work	O
-,	O
-we	O
-produced	O
-two	O
-novel	O
-subnanometer	O
-resolution	O
-cryoEM	O
-reconstructions	O
-of	O
-the	O
-E	O
-.	O
-coli	O
-lysine	O
-decarboxylases	O
-at	O
-pH	O
-optimal	O
-for	O
-their	O
-enzymatic	O
-activity	O
-–	O
-a	O
-5	O
-.	O
-5	O
-Å	O
-resolution	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcC	O
-(	O
-pH	O
-7	O
-.	O
-5	O
-)	O
-for	O
-which	O
-no	O
-3D	O
-structural	O
-information	O
-has	O
-been	O
-previously	O
-available	O
-(	O
-Figs	O
-1A	O
-,	O
-B	O
-and	O
-S1	O
-),	O
-and	O
-a	O
-6	O
-.	O
-1	O
-Å	O
-resolution	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcIa	O
-,	O
-(	O
-pH	O
-6	O
-.	O
-2	O
-)	O
-(	O
-Figs	O
-1C	O
-,	O
-D	O
-and	O
-S2	O
-).	O
-	O
-In	O
-addition	O
-,	O
-we	O
-improved	O
-our	O
-earlier	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcI	O
--	O
-LARA	O
-complex	O
-from	O
-7	O
-.	O
-5	O
-Å	O
-to	O
-6	O
-.	O
-2	O
-Å	O
-resolution	O
-(	O
-Figs	O
-1E	O
-,	O
-F	O
-and	O
-S3	O
-).	O
-	O
-Based	O
-on	O
-these	O
-reconstructions	O
-,	O
-reliable	O
-pseudoatomic	O
-models	O
-of	O
-the	O
-three	O
-assemblies	O
-were	O
-obtained	O
-by	O
-flexible	O
-fitting	O
-of	O
-either	O
-the	O
-crystal	O
-structure	O
-of	O
-LdcIi	O
-or	O
-a	O
-derived	O
-structural	O
-homology	O
-model	O
-of	O
-LdcC	O
-(	O
-Table	O
-S1	O
-).	O
-	O
-Significant	O
-differences	O
-between	O
-these	O
-pseudoatomic	O
-models	O
-can	O
-be	O
-interpreted	O
-as	O
-movements	O
-between	O
-specific	O
-biological	O
-states	O
-of	O
-the	O
-proteins	O
-as	O
-described	O
-below	O
-.	O
-	O
-The	O
-wing	O
-domains	O
-as	O
-a	O
-stable	O
-anchor	O
-at	O
-the	O
-center	O
-of	O
-the	O
-double	O
--	O
-ring	O
-	O
-As	O
-a	O
-first	O
-step	O
-of	O
-a	O
-comparative	O
-analysis	O
-,	O
-we	O
-superimposed	O
-the	O
-three	O
-cryoEM	O
-reconstructions	O
-(	O
-LdcIa	O
-,	O
-LdcI	O
--	O
-LARA	O
-and	O
-LdcC	O
-)	O
-and	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-LdcIi	O
-decamer	O
-(	O
-Fig	O
-.	O
-2	O
-and	O
-Movie	O
-S1	O
-).	O
-	O
-This	O
-superposition	O
-reveals	O
-that	O
-the	O
-densities	O
-lining	O
-the	O
-central	O
-hole	O
-of	O
-the	O
-toroid	O
-are	O
-roughly	O
-at	O
-the	O
-same	O
-location	O
-,	O
-while	O
-the	O
-rest	O
-of	O
-the	O
-structure	O
-exhibits	O
-noticeable	O
-changes	O
-.	O
-	O
-Specifically	O
-,	O
-at	O
-the	O
-center	O
-of	O
-the	O
-double	O
--	O
-ring	O
-the	O
-wing	O
-domains	O
-of	O
-the	O
-subunits	O
-provide	O
-the	O
-conserved	O
-basis	O
-for	O
-the	O
-assembly	O
-with	O
-the	O
-lowest	O
-root	O
-mean	O
-square	O
-deviation	O
-(	O
-RMSD	O
-)	O
-(	O
-between	O
-1	O
-.	O
-4	O
-and	O
-2	O
-Å	O
-for	O
-the	O
-Cα	O
-atoms	O
-only	O
-),	O
-whereas	O
-the	O
-peripheral	O
-CTDs	O
-containing	O
-the	O
-RavA	O
-binding	O
-interface	O
-manifest	O
-the	O
-highest	O
-RMSD	O
-(	O
-up	O
-to	O
-4	O
-.	O
-2	O
-Å	O
-)	O
-(	O
-Table	O
-S2	O
-).	O
-	O
-In	O
-addition	O
-,	O
-the	O
-wing	O
-domains	O
-of	O
-all	O
-structures	O
-are	O
-very	O
-similar	O
-,	O
-with	O
-the	O
-RMSD	O
-after	O
-optimal	O
-rigid	O
-body	O
-alignment	O
-(	O
-RMSDmin	O
-)	O
-less	O
-than	O
-1	O
-.	O
-1	O
-Å	O
-.	O
-Thus	O
-,	O
-taking	O
-the	O
-limited	O
-resolution	O
-of	O
-the	O
-cryoEM	O
-maps	O
-into	O
-account	O
-,	O
-we	O
-consider	O
-that	O
-the	O
-wing	O
-domains	O
-of	O
-all	O
-the	O
-four	O
-structures	O
-are	O
-essentially	O
-identical	O
-and	O
-that	O
-in	O
-the	O
-present	O
-study	O
-the	O
-RMSD	O
-of	O
-less	O
-than	O
-2	O
-Å	O
-can	O
-serve	O
-as	O
-a	O
-baseline	O
-below	O
-which	O
-differences	O
-may	O
-be	O
-assumed	O
-as	O
-insignificant	O
-.	O
-	O
-This	O
-preservation	O
-of	O
-the	O
-central	O
-part	O
-of	O
-the	O
-double	O
--	O
-ring	O
-assembly	O
-may	O
-help	O
-the	O
-enzymes	O
-to	O
-maintain	O
-their	O
-decameric	O
-state	O
-upon	O
-activation	O
-and	O
-incorporation	O
-into	O
-the	O
-LdcI	O
--	O
-RavA	O
-cage	O
-.	O
-	O
-The	O
-core	O
-domain	O
-and	O
-the	O
-active	O
-site	O
-rearrangements	O
-upon	O
-pH	O
--	O
-dependent	O
-enzyme	O
-activation	O
-and	O
-LARA	O
-binding	O
-	O
-Both	O
-visual	O
-inspection	O
-(	O
-Fig	O
-.	O
-2	O
-)	O
-and	O
-RMSD	O
-calculations	O
-(	O
-Table	O
-S2	O
-)	O
-show	O
-that	O
-globally	O
-the	O
-three	O
-structures	O
-at	O
-active	O
-pH	O
-(	O
-LdcIa	O
-,	O
-LdcI	O
--	O
-LARA	O
-and	O
-LdcC	O
-)	O
-are	O
-more	O
-similar	O
-to	O
-each	O
-other	O
-than	O
-to	O
-the	O
-structure	O
-determined	O
-at	O
-high	O
-pH	O
-conditions	O
-(	O
-LdcIi	O
-).	O
-	O
-The	O
-decameric	O
-enzyme	O
-is	O
-built	O
-of	O
-five	O
-dimers	O
-associating	O
-into	O
-a	O
-5	O
--	O
-fold	O
-symmetrical	O
-double	O
--	O
-ring	O
-(	O
-two	O
-monomers	O
-making	O
-a	O
-dimer	O
-are	O
-delineated	O
-in	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-As	O
-common	O
-for	O
-the	O
-α	O
-family	O
-of	O
-the	O
-PLP	O
--	O
-dependent	O
-decarboxylases	O
-,	O
-dimerization	O
-is	O
-required	O
-for	O
-the	O
-enzymatic	O
-activity	O
-because	O
-the	O
-active	O
-site	O
-is	O
-buried	O
-in	O
-the	O
-dimer	O
-interface	O
-(	O
-Fig	O
-.	O
-3A	O
-,	O
-B	O
-).	O
-	O
-This	O
-interface	O
-is	O
-formed	O
-essentially	O
-by	O
-the	O
-core	O
-domains	O
-with	O
-some	O
-contribution	O
-of	O
-the	O
-CTDs	O
-.	O
-	O
-The	O
-core	O
-domain	O
-is	O
-built	O
-by	O
-the	O
-PLP	O
--	O
-binding	O
-subdomain	O
-(	O
-PLP	O
--	O
-SD	O
-,	O
-residues	O
-184	O
-–	O
-417	O
-)	O
-flanked	O
-by	O
-two	O
-smaller	O
-subdomains	O
-rich	O
-in	O
-partly	O
-disordered	O
-loops	O
-–	O
-the	O
-linker	O
-region	O
-(	O
-residues	O
-130	O
-–	O
-183	O
-)	O
-and	O
-the	O
-subdomain	O
-4	O
-(	O
-residues	O
-418	O
-–	O
-563	O
-).	O
-	O
-Zooming	O
-in	O
-the	O
-variations	O
-in	O
-the	O
-PLP	O
--	O
-SD	O
-shows	O
-that	O
-most	O
-of	O
-the	O
-structural	O
-changes	O
-concern	O
-displacements	O
-in	O
-the	O
-active	O
-site	O
-(	O
-Fig	O
-.	O
-3C	O
-–	O
-F	O
-).	O
-	O
-The	O
-most	O
-conspicuous	O
-differences	O
-between	O
-the	O
-PLP	O
--	O
-SDs	O
-can	O
-be	O
-linked	O
-to	O
-the	O
-pH	O
--	O
-dependent	O
-activation	O
-of	O
-the	O
-enzymes	O
-.	O
-	O
-The	O
-resolution	O
-of	O
-the	O
-cryoEM	O
-maps	O
-does	O
-not	O
-allow	O
-modeling	O
-the	O
-position	O
-of	O
-the	O
-PLP	O
-moiety	O
-and	O
-calls	O
-for	O
-caution	O
-in	O
-detailed	O
-mechanistic	O
-interpretations	O
-in	O
-terms	O
-of	O
-individual	O
-amino	O
-acids	O
-.	O
-	O
-In	O
-particular	O
-,	O
-transition	O
-from	O
-LdcIi	O
-to	O
-LdcI	O
--	O
-LARA	O
-involves	O
-~	O
-3	O
-.	O
-5	O
-Å	O
-and	O
-~	O
-4	O
-.	O
-5	O
-Å	O
-shifts	O
-away	O
-from	O
-the	O
-5	O
--	O
-fold	O
-axis	O
-in	O
-the	O
-active	O
-site	O
-α	O
--	O
-helices	O
-spanning	O
-residues	O
-218	O
-–	O
-232	O
-and	O
-246	O
-–	O
-254	O
-respectively	O
-(	O
-Fig	O
-.	O
-3C	O
-–	O
-E	O
-).	O
-	O
-Between	O
-these	O
-two	O
-extremes	O
-,	O
-the	O
-PLP	O
--	O
-SDs	O
-of	O
-LdcIa	O
-and	O
-LdcC	O
-are	O
-similar	O
-both	O
-in	O
-the	O
-context	O
-of	O
-the	O
-decamer	O
-(	O
-Fig	O
-.	O
-3F	O
-)	O
-and	O
-in	O
-terms	O
-of	O
-RMSDmin	O
-=	O
-0	O
-.	O
-9	O
-Å	O
-,	O
-which	O
-probably	O
-reflects	O
-the	O
-fact	O
-that	O
-,	O
-at	O
-the	O
-optimal	O
-pH	O
-,	O
-these	O
-lysine	O
-decarboxylases	O
-have	O
-a	O
-similar	O
-enzymatic	O
-activity	O
-.	O
-	O
-In	O
-addition	O
-,	O
-our	O
-earlier	O
-biochemical	O
-observation	O
-that	O
-the	O
-enzymatic	O
-activity	O
-of	O
-LdcIa	O
-is	O
-unaffected	O
-by	O
-RavA	O
-binding	O
-is	O
-consistent	O
-with	O
-the	O
-relatively	O
-small	O
-changes	O
-undergone	O
-by	O
-the	O
-active	O
-site	O
-upon	O
-transition	O
-from	O
-LdcIa	O
-to	O
-LdcI	O
--	O
-LARA	O
-.	O
-	O
-Worthy	O
-of	O
-note	O
-,	O
-our	O
-previous	O
-comparison	O
-of	O
-the	O
-crystal	O
-structure	O
-of	O
-LdcIi	O
-with	O
-that	O
-of	O
-the	O
-inducible	O
-arginine	O
-decarboxylase	O
-AdiA	O
-revealed	O
-high	O
-conservation	O
-of	O
-the	O
-PLP	O
--	O
-coordinating	O
-residues	O
-and	O
-identified	O
-a	O
-patch	O
-of	O
-negatively	O
-charged	O
-residues	O
-lining	O
-the	O
-active	O
-site	O
-channel	O
-as	O
-a	O
-potential	O
-binding	O
-site	O
-for	O
-the	O
-target	O
-amino	O
-acid	O
-substrate	O
-(	O
-Figs	O
-S3	O
-and	O
-S4	O
-in	O
-ref	O
-.).	O
-	O
-Rearrangements	O
-of	O
-the	O
-ppGpp	O
-binding	O
-pocket	O
-upon	O
-pH	O
--	O
-dependent	O
-enzyme	O
-activation	O
-and	O
-LARA	O
-binding	O
-	O
-An	O
-inhibitor	O
-of	O
-the	O
-LdcI	O
-and	O
-LdcC	O
-activity	O
-,	O
-the	O
-stringent	O
-response	O
-alarmone	O
-ppGpp	O
-,	O
-is	O
-known	O
-to	O
-bind	O
-at	O
-the	O
-interface	O
-between	O
-neighboring	O
-monomers	O
-within	O
-each	O
-ring	O
-(	O
-Fig	O
-.	O
-S4	O
-).	O
-	O
-The	O
-ppGpp	O
-binding	O
-pocket	O
-is	O
-made	O
-up	O
-by	O
-residues	O
-from	O
-all	O
-domains	O
-and	O
-is	O
-located	O
-approximately	O
-30	O
-Å	O
-away	O
-from	O
-the	O
-PLP	O
-moiety	O
-.	O
-	O
-Whereas	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-ppGpp	O
--	O
-LdcIi	O
-was	O
-solved	O
-to	O
-2	O
-Å	O
-resolution	O
-,	O
-only	O
-a	O
-4	O
-.	O
-1	O
-Å	O
-resolution	O
-structure	O
-of	O
-the	O
-ppGpp	O
--	O
-free	O
-LdcIi	O
-could	O
-be	O
-obtained	O
-.	O
-	O
-At	O
-this	O
-resolution	O
-,	O
-the	O
-apo	O
--	O
-LdcIi	O
-and	O
-ppGpp	O
--	O
-LdcIi	O
-structures	O
-(	O
-both	O
-solved	O
-at	O
-pH	O
-8	O
-.	O
-5	O
-)	O
-appeared	O
-indistinguishable	O
-except	O
-for	O
-the	O
-presence	O
-of	O
-ppGpp	O
-(	O
-Fig	O
-.	O
-S11	O
-in	O
-ref	O
-.	O
-).	O
-	O
-Thus	O
-,	O
-we	O
-speculated	O
-that	O
-inhibition	O
-of	O
-LdcI	O
-by	O
-ppGpp	O
-would	O
-be	O
-accompanied	O
-by	O
-a	O
-transduction	O
-of	O
-subtle	O
-structural	O
-changes	O
-at	O
-the	O
-level	O
-of	O
-individual	O
-amino	O
-acid	O
-side	O
-chains	O
-between	O
-the	O
-ppGpp	O
-binding	O
-pocket	O
-and	O
-the	O
-active	O
-site	O
-of	O
-the	O
-enzyme	O
-.	O
-	O
-All	O
-our	O
-current	O
-cryoEM	O
-reconstructions	O
-of	O
-the	O
-lysine	O
-decarboxylases	O
-were	O
-obtained	O
-in	O
-the	O
-absence	O
-of	O
-ppGpp	O
-in	O
-order	O
-to	O
-be	O
-closer	O
-to	O
-the	O
-active	O
-state	O
-of	O
-the	O
-enzymes	O
-under	O
-study	O
-.	O
-	O
-While	O
-differences	O
-in	O
-the	O
-ppGpp	O
-binding	O
-site	O
-could	O
-indeed	O
-be	O
-visualized	O
-(	O
-Fig	O
-.	O
-S4	O
-),	O
-the	O
-level	O
-of	O
-resolution	O
-warns	O
-against	O
-speculations	O
-about	O
-their	O
-significance	O
-.	O
-	O
-The	O
-fact	O
-that	O
-interaction	O
-with	O
-RavA	O
-reduces	O
-the	O
-ppGpp	O
-affinity	O
-for	O
-LdcI	O
-despite	O
-the	O
-long	O
-distance	O
-of	O
-~	O
-30	O
-Å	O
-between	O
-the	O
-LARA	O
-domain	O
-binding	O
-site	O
-and	O
-the	O
-closest	O
-ppGpp	O
-binding	O
-pocket	O
-(	O
-Fig	O
-.	O
-S5	O
-)	O
-seems	O
-to	O
-favor	O
-an	O
-allosteric	O
-regulation	O
-mechanism	O
-.	O
-	O
-Interestingly	O
-,	O
-although	O
-a	O
-number	O
-of	O
-ppGpp	O
-binding	O
-residues	O
-are	O
-strictly	O
-conserved	O
-between	O
-LdcI	O
-and	O
-AdiA	O
-that	O
-also	O
-forms	O
-decamers	O
-at	O
-low	O
-pH	O
-optimal	O
-for	O
-its	O
-arginine	O
-decarboxylase	O
-activity	O
-,	O
-no	O
-ppGpp	O
-regulation	O
-of	O
-AdiA	O
-could	O
-be	O
-demonstrated	O
-.	O
-	O
-Swinging	O
-and	O
-stretching	O
-of	O
-the	O
-CTDs	O
-upon	O
-pH	O
--	O
-dependent	O
-LdcI	O
-activation	O
-and	O
-LARA	O
-binding	O
-	O
-Inspection	O
-of	O
-the	O
-superimposed	O
-decameric	O
-structures	O
-(	O
-Figs	O
-2	O
-and	O
-S6	O
-)	O
-suggests	O
-a	O
-depiction	O
-of	O
-the	O
-wing	O
-domains	O
-as	O
-an	O
-anchor	O
-around	O
-which	O
-the	O
-peripheral	O
-CTDs	O
-swing	O
-.	O
-	O
-This	O
-swinging	O
-movement	O
-seems	O
-to	O
-be	O
-mediated	O
-by	O
-the	O
-core	O
-domains	O
-and	O
-is	O
-accompanied	O
-by	O
-a	O
-stretching	O
-of	O
-the	O
-whole	O
-LdcI	O
-subunits	O
-attracted	O
-by	O
-the	O
-RavA	O
-magnets	O
-.	O
-	O
-Indeed	O
-,	O
-all	O
-CTDs	O
-have	O
-very	O
-similar	O
-structures	O
-(	O
-RMSDmin	O
-<	O
-1	O
-Å	O
-).	O
-	O
-Yet	O
-the	O
-superposition	O
-of	O
-the	O
-decamers	O
-lays	O
-bare	O
-a	O
-progressive	O
-movement	O
-of	O
-the	O
-CTD	O
-as	O
-a	O
-whole	O
-upon	O
-enzyme	O
-activation	O
-by	O
-pH	O
-and	O
-the	O
-binding	O
-of	O
-LARA	O
-.	O
-	O
-The	O
-LdcIi	O
-monomer	O
-is	O
-the	O
-most	O
-compact	O
-,	O
-whereas	O
-LdcIa	O
-and	O
-especially	O
-LdcI	O
--	O
-LARA	O
-gradually	O
-extend	O
-their	O
-CTDs	O
-towards	O
-the	O
-LARA	O
-domain	O
-of	O
-RavA	O
-(	O
-Figs	O
-2	O
-and	O
-4	O
-).	O
-	O
-These	O
-small	O
-but	O
-noticeable	O
-swinging	O
-and	O
-stretching	O
-(	O
-up	O
-to	O
-~	O
-4	O
-Å	O
-)	O
-may	O
-be	O
-related	O
-to	O
-the	O
-incorporation	O
-of	O
-the	O
-LdcI	O
-decamer	O
-into	O
-the	O
-LdcI	O
--	O
-RavA	O
-cage	O
-.	O
-	O
-The	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-a	O
-lysine	O
-decarboxylase	O
-as	O
-a	O
-major	O
-determinant	O
-of	O
-the	O
-interaction	O
-with	O
-RavA	O
-	O
-In	O
-our	O
-previous	O
-contribution	O
-,	O
-based	O
-on	O
-the	O
-fit	O
-of	O
-the	O
-LdcIi	O
-and	O
-the	O
-LARA	O
-crystal	O
-structures	O
-into	O
-the	O
-LdcI	O
--	O
-LARA	O
-cryoEM	O
-density	O
-,	O
-we	O
-predicted	O
-that	O
-the	O
-LdcI	O
--	O
-RavA	O
-interaction	O
-should	O
-involve	O
-the	O
-C	O
--	O
-terminal	O
-two	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-of	O
-the	O
-LdcI	O
-.	O
-Our	O
-present	O
-cryoEM	O
-maps	O
-and	O
-pseudoatomic	O
-models	O
-provide	O
-first	O
-structure	O
--	O
-based	O
-insights	O
-into	O
-the	O
-differences	O
-between	O
-the	O
-inducible	O
-and	O
-the	O
-constitutive	O
-lysine	O
-decarboxylases	O
-.	O
-	O
-Therefore	O
-,	O
-we	O
-wanted	O
-to	O
-check	O
-the	O
-influence	O
-of	O
-the	O
-primary	O
-sequence	O
-of	O
-the	O
-two	O
-proteins	O
-in	O
-this	O
-region	O
-on	O
-their	O
-ability	O
-to	O
-interact	O
-with	O
-RavA	O
-.	O
-To	O
-this	O
-end	O
-,	O
-we	O
-swapped	O
-the	O
-relevant	O
-β	O
--	O
-sheets	O
-of	O
-the	O
-two	O
-proteins	O
-and	O
-produced	O
-their	O
-chimeras	B-mutant
-,	O
-namely	O
-LdcIC	B-mutant
-(	O
-i	O
-.	O
-e	O
-.	O
-LdcI	O
-with	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-LdcC	O
-)	O
-and	O
-LdcCI	B-mutant
-(	O
-i	O
-.	O
-e	O
-.	O
-LdcC	O
-with	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-LdcI	O
-)	O
-(	O
-Fig	O
-.	O
-5A	O
-–	O
-C	O
-).	O
-	O
-Both	B-mutant
-constructs	I-mutant
-could	O
-be	O
-purified	O
-and	O
-could	O
-form	O
-decamers	O
-visually	O
-indistinguishable	O
-from	O
-the	O
-wild	O
--	O
-type	O
-proteins	O
-.	O
-	O
-As	O
-expected	O
-,	O
-binding	O
-of	O
-LdcI	O
-to	O
-RavA	O
-was	O
-completely	O
-abolished	O
-by	O
-this	O
-procedure	O
-and	O
-no	O
-LdcIC	O
--	O
-RavA	O
-complex	O
-could	O
-be	O
-detected	O
-.	O
-	O
-On	O
-the	O
-contrary	O
-,	O
-introduction	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-LdcI	O
-into	O
-LdcC	O
-led	O
-to	O
-an	O
-assembly	O
-of	O
-the	O
-LdcCI	O
--	O
-RavA	O
-complex	O
-.	O
-	O
-On	O
-the	O
-negative	O
-stain	O
-EM	O
-grid	O
-,	O
-the	O
-chimeric	O
-cages	O
-appeared	O
-less	O
-rigid	O
-than	O
-the	O
-native	O
-LdcI	O
--	O
-RavA	O
-,	O
-which	O
-probably	O
-means	O
-that	O
-the	O
-environment	O
-of	O
-the	O
-β	O
--	O
-sheet	O
-contributes	O
-to	O
-the	O
-efficiency	O
-of	O
-the	O
-interaction	O
-and	O
-the	O
-stability	O
-of	O
-the	O
-entire	O
-architecture	O
-(	O
-Fig	O
-.	O
-5D	O
-–	O
-F	O
-).	O
-	O
-The	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-a	O
-lysine	O
-decarboxylase	O
-is	O
-a	O
-highly	O
-conserved	O
-signature	O
-allowing	O
-to	O
-distinguish	O
-between	O
-LdcI	O
-and	O
-LdcC	O
-	O
-Alignment	O
-of	O
-the	O
-primary	O
-sequences	O
-of	O
-the	O
-E	O
-.	O
-coli	O
-LdcI	O
-and	O
-LdcC	O
-shows	O
-that	O
-some	O
-amino	O
-acid	O
-residues	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-are	O
-the	O
-same	O
-in	O
-the	O
-two	O
-proteins	O
-,	O
-whereas	O
-others	O
-are	O
-notably	O
-different	O
-in	O
-chemical	O
-nature	O
-.	O
-	O
-Importantly	O
-,	O
-most	O
-of	O
-the	O
-amino	O
-acid	O
-differences	O
-between	O
-the	O
-two	O
-enzymes	O
-are	O
-located	O
-in	O
-this	O
-very	O
-region	O
-.	O
-	O
-Thus	O
-,	O
-to	O
-advance	O
-beyond	O
-our	O
-experimental	O
-confirmation	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-as	O
-a	O
-major	O
-determinant	O
-of	O
-the	O
-capacity	O
-of	O
-a	O
-particular	O
-lysine	O
-decarboxylase	O
-to	O
-form	O
-a	O
-cage	O
-with	O
-RavA	O
-,	O
-we	O
-set	O
-out	O
-to	O
-investigate	O
-whether	O
-certain	O
-residues	O
-in	O
-this	O
-β	O
--	O
-sheet	O
-are	O
-conserved	O
-in	O
-lysine	O
-decarboxylases	O
-of	O
-different	O
-enterobacteria	O
-that	O
-have	O
-the	O
-ravA	O
--	O
-viaA	O
-operon	O
-in	O
-their	O
-genome	O
-.	O
-	O
-We	O
-inspected	O
-the	O
-genetic	O
-environment	O
-of	O
-lysine	O
-decarboxylases	O
-from	O
-22	O
-enterobacterial	O
-species	O
-referenced	O
-in	O
-the	O
-NCBI	O
-database	O
-,	O
-corrected	O
-the	O
-gene	O
-annotation	O
-where	O
-necessary	O
-(	O
-Tables	O
-S3	O
-and	O
-S4	O
-),	O
-and	O
-performed	O
-multiple	O
-sequence	O
-alignment	O
-coupled	O
-to	O
-a	O
-phylogenetic	O
-analysis	O
-(	O
-see	O
-Methods	O
-).	O
-	O
-First	O
-of	O
-all	O
-,	O
-consensus	O
-sequence	O
-for	O
-the	O
-entire	O
-lysine	O
-decarboxylase	O
-family	O
-was	O
-derived	O
-.	O
-	O
-Second	O
-,	O
-the	O
-phylogenetic	O
-analysis	O
-clearly	O
-split	O
-the	O
-lysine	O
-decarboxylases	O
-into	O
-two	O
-groups	O
-(	O
-Fig	O
-.	O
-6A	O
-).	O
-	O
-All	O
-lysine	O
-decarboxylases	O
-predicted	O
-to	O
-be	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-or	O
-biodegradable	O
-based	O
-on	O
-their	O
-genetic	O
-environment	O
-,	O
-as	O
-for	O
-example	O
-their	O
-organization	O
-in	O
-an	O
-operon	O
-with	O
-a	O
-gene	O
-encoding	O
-the	O
-CadB	O
-antiporter	O
-(	O
-see	O
-Methods	O
-),	O
-were	O
-found	O
-in	O
-one	O
-group	O
-,	O
-whereas	O
-all	O
-enzymes	O
-predicted	O
-as	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-or	O
-biosynthetic	O
-partitioned	O
-into	O
-another	O
-group	O
-.	O
-	O
-Thus	O
-,	O
-consensus	O
-sequences	O
-could	O
-also	O
-be	O
-determined	O
-for	O
-each	O
-of	O
-the	O
-two	O
-groups	O
-(	O
-Figs	O
-6B	O
-,	O
-C	O
-and	O
-S7	O
-).	O
-	O
-Inspection	O
-of	O
-these	O
-consensus	O
-sequences	O
-revealed	O
-important	O
-differences	O
-between	O
-the	O
-groups	O
-regarding	O
-charge	O
-,	O
-size	O
-and	O
-hydrophobicity	O
-of	O
-several	O
-residues	O
-precisely	O
-at	O
-the	O
-level	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-that	O
-is	O
-responsible	O
-for	O
-the	O
-interaction	O
-with	O
-RavA	O
-(	O
-Fig	O
-.	O
-6B	O
-–	O
-D	O
-).	O
-	O
-For	O
-example	O
-,	O
-in	O
-our	O
-previous	O
-study	O
-,	O
-site	O
--	O
-directed	O
-mutations	O
-identified	O
-Y697	O
-as	O
-critically	O
-required	O
-for	O
-the	O
-RavA	O
-binding	O
-.	O
-	O
-Our	O
-current	O
-analysis	O
-shows	O
-that	O
-Y697	O
-is	O
-strictly	O
-conserved	O
-in	O
-the	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-group	O
-whereas	O
-the	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-enzymes	O
-always	O
-have	O
-a	O
-lysine	O
-in	O
-this	O
-position	O
-;	O
-it	O
-also	O
-uncovers	O
-several	O
-other	O
-residues	O
-potentially	O
-essential	O
-for	O
-the	O
-interaction	O
-with	O
-RavA	O
-which	O
-can	O
-now	O
-be	O
-addressed	O
-by	O
-site	O
--	O
-directed	O
-mutagenesis	O
-.	O
-	O
-The	O
-third	O
-and	O
-most	O
-remarkable	O
-finding	O
-was	O
-that	O
-exactly	O
-the	O
-same	O
-separation	O
-into	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-and	O
-“	O
-LdcC	O
-”-	O
-like	O
-groups	O
-can	O
-be	O
-obtained	O
-based	O
-on	O
-a	O
-comparison	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheets	O
-only	O
-,	O
-without	O
-taking	O
-the	O
-rest	O
-of	O
-the	O
-primary	O
-sequence	O
-into	O
-account	O
-.	O
-	O
-Therefore	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-emerges	O
-as	O
-being	O
-a	O
-highly	O
-conserved	O
-signature	O
-sequence	O
-,	O
-sufficient	O
-to	O
-unambiguously	O
-discriminate	O
-between	O
-the	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-and	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-enterobacterial	O
-lysine	O
-decarboxylases	O
-independently	O
-of	O
-any	O
-other	O
-information	O
-(	O
-Figs	O
-6	O
-and	O
-S7	O
-).	O
-	O
-Our	O
-structures	O
-show	O
-that	O
-this	O
-motif	O
-is	O
-not	O
-involved	O
-in	O
-the	O
-enzymatic	O
-activity	O
-or	O
-the	O
-oligomeric	O
-state	O
-of	O
-the	O
-proteins	O
-.	O
-	O
-Thus	O
-,	O
-enterobacteria	O
-identified	O
-here	O
-(	O
-Fig	O
-.	O
-6	O
-,	O
-Table	O
-S4	O
-)	O
-appear	O
-to	O
-exert	O
-evolutionary	O
-pressure	O
-on	O
-the	O
-biodegradative	O
-lysine	O
-decarboxylase	O
-towards	O
-the	O
-RavA	O
-binding	O
-.	O
-	O
-One	O
-of	O
-the	O
-elucidated	O
-roles	O
-of	O
-the	O
-LdcI	O
--	O
-RavA	O
-cage	O
-is	O
-to	O
-maintain	O
-LdcI	O
-activity	O
-under	O
-conditions	O
-of	O
-enterobacterial	O
-starvation	O
-by	O
-preventing	O
-LdcI	O
-inhibition	O
-by	O
-the	O
-stringent	O
-response	O
-alarmone	O
-ppGpp	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-recently	O
-documented	O
-interaction	O
-of	O
-both	O
-LdcI	O
-and	O
-RavA	O
-with	O
-specific	O
-subunits	O
-of	O
-the	O
-respiratory	O
-complex	O
-I	O
-,	O
-together	O
-with	O
-the	O
-unanticipated	O
-link	O
-between	O
-RavA	O
-and	O
-maturation	O
-of	O
-numerous	O
-iron	O
--	O
-sulfur	O
-proteins	O
-,	O
-tend	O
-to	O
-suggest	O
-an	O
-additional	O
-intriguing	O
-function	O
-for	O
-this	O
-3	O
-.	O
-5	O
-MDa	O
-assembly	O
-.	O
-	O
-The	O
-conformational	O
-rearrangements	O
-of	O
-LdcI	O
-upon	O
-enzyme	O
-activation	O
-and	O
-RavA	O
-binding	O
-revealed	O
-in	O
-this	O
-work	O
-,	O
-and	O
-our	O
-amazing	O
-finding	O
-that	O
-the	O
-molecular	O
-determinant	O
-of	O
-the	O
-LdcI	O
--	O
-RavA	O
-interaction	O
-is	O
-the	O
-one	O
-that	O
-straightforwardly	O
-determines	O
-if	O
-a	O
-particular	O
-enterobacterial	O
-lysine	O
-decarboxylase	O
-belongs	O
-to	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-or	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-proteins	O
-,	O
-should	O
-give	O
-a	O
-new	O
-impetus	O
-to	O
-functional	O
-studies	O
-of	O
-the	O
-unique	O
-LdcI	O
--	O
-RavA	O
-cage	O
-.	O
-	O
-Besides	O
-,	O
-the	O
-structures	O
-and	O
-the	O
-pseudoatomic	O
-models	O
-of	O
-the	O
-active	O
-ppGpp	O
--	O
-free	O
-states	O
-of	O
-both	O
-the	O
-biodegradative	O
-and	O
-the	O
-biosynthetic	O
-E	O
-.	O
-coli	O
-lysine	O
-decarboxylases	O
-offer	O
-an	O
-additional	O
-tool	O
-for	O
-analysis	O
-of	O
-their	O
-role	O
-in	O
-UPEC	O
-infectivity	O
-.	O
-	O
-Together	O
-with	O
-the	O
-apo	O
--	O
-LdcI	O
-and	O
-ppGpp	O
--	O
-LdcIi	O
-crystal	O
-structures	O
-,	O
-our	O
-cryoEM	O
-reconstructions	O
-provide	O
-a	O
-structural	O
-framework	O
-for	O
-future	O
-studies	O
-of	O
-structure	O
--	O
-function	O
-relationships	O
-of	O
-lysine	O
-decarboxylases	O
-from	O
-other	O
-enterobacteria	O
-and	O
-even	O
-of	O
-their	O
-homologues	O
-outside	O
-Enterobacteriaceae	O
-.	O
-For	O
-example	O
-,	O
-the	O
-lysine	O
-decarboxylase	O
-of	O
-Eikenella	O
-corrodens	O
-is	O
-thought	O
-to	O
-play	O
-a	O
-major	O
-role	O
-in	O
-the	O
-periodontal	O
-disease	O
-and	O
-its	O
-inhibitors	O
-were	O
-shown	O
-to	O
-retard	O
-gingivitis	O
-development	O
-.	O
-	O
-Finally	O
-,	O
-cadaverine	O
-being	O
-an	O
-important	O
-platform	O
-chemical	O
-for	O
-the	O
-production	O
-of	O
-industrial	O
-polymers	O
-such	O
-as	O
-nylon	O
-,	O
-structural	O
-information	O
-is	O
-valuable	O
-for	O
-optimisation	O
-of	O
-bacterial	O
-lysine	O
-decarboxylases	O
-used	O
-for	O
-its	O
-production	O
-in	O
-biotechnology	O
-.	O
-	O
-3D	O
-cryoEM	O
-reconstructions	O
-of	O
-LdcC	O
-,	O
-LdcI	O
--	O
-LARA	O
-and	O
-LdcIa	O
-.	O
-	O
-(	O
-A	O
-,	O
-C	O
-,	O
-E	O
-)	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcC	O
-(	O
-A	O
-),	O
-LdcIa	O
-(	O
-C	O
-)	O
-and	O
-LdcI	O
--	O
-LARA	O
-(	O
-E	O
-)	O
-decamers	O
-with	O
-one	O
-protomer	O
-in	O
-light	O
-grey	O
-.	O
-	O
-In	O
-the	O
-rest	O
-of	O
-the	O
-protomers	O
-,	O
-the	O
-wing	O
-,	O
-core	O
-and	O
-C	O
--	O
-terminal	O
-domains	O
-are	O
-colored	O
-from	O
-light	O
-to	O
-dark	O
-in	O
-shades	O
-of	O
-green	O
-for	O
-LdcC	O
-(	O
-A	O
-),	O
-pink	O
-for	O
-LdcIa	O
-(	O
-C	O
-)	O
-and	O
-blue	O
-for	O
-LdcI	O
-in	O
-LdcI	O
--	O
-LARA	O
-(	O
-E	O
-).	O
-	O
-In	O
-(	O
-E	O
-),	O
-the	O
-LARA	O
-domain	O
-density	O
-is	O
-shown	O
-in	O
-dark	O
-grey	O
-.	O
-	O
-Two	O
-monomers	O
-making	O
-a	O
-dimer	O
-are	O
-delineated	O
-.	O
-	O
-Scale	O
-bar	O
-50	O
-Å	O
-.	O
-(	O
-B	O
-,	O
-D	O
-,	O
-F	O
-)	O
-One	O
-protomer	O
-from	O
-the	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcC	O
-(	O
-B	O
-),	O
-LdcIa	O
-(	O
-D	O
-)	O
-and	O
-LdcI	O
--	O
-LARA	O
-(	O
-F	O
-)	O
-in	O
-light	O
-grey	O
-with	O
-the	O
-pseudoatomic	O
-model	O
-represented	O
-as	O
-cartoons	O
-and	O
-colored	O
-as	O
-the	O
-densities	O
-in	O
-(	O
-A	O
-,	O
-C	O
-,	O
-E	O
-).	O
-	O
-Superposition	O
-of	O
-the	O
-pseudoatomic	O
-models	O
-of	O
-LdcC	O
-,	O
-LdcI	O
-from	O
-LdcI	O
--	O
-LARA	O
-and	O
-LdcIa	O
-colored	O
-as	O
-in	O
-Fig	O
-.	O
-1	O
-,	O
-and	O
-the	O
-crystal	O
-structure	O
-of	O
-LdcIi	O
-in	O
-shades	O
-of	O
-yellow	O
-.	O
-	O
-Only	O
-one	O
-of	O
-the	O
-two	O
-rings	O
-of	O
-the	O
-double	O
-toroid	O
-is	O
-shown	O
-for	O
-clarity	O
-.	O
-	O
-The	O
-dashed	O
-circle	O
-indicates	O
-the	O
-central	O
-region	O
-that	O
-remains	O
-virtually	O
-unchanged	O
-between	O
-all	O
-the	O
-structures	O
-,	O
-while	O
-the	O
-periphery	O
-undergoes	O
-visible	O
-movements	O
-.	O
-	O
-Conformational	O
-rearrangements	O
-in	O
-the	O
-enzyme	O
-active	O
-site	O
-.	O
-	O
-(	O
-A	O
-)	O
-LdcIi	O
-crystal	O
-structure	O
-,	O
-with	O
-one	O
-ring	O
-represented	O
-as	O
-a	O
-grey	O
-surface	O
-and	O
-the	O
-second	O
-as	O
-a	O
-cartoon	O
-.	O
-	O
-A	O
-monomer	O
-with	O
-its	O
-PLP	O
-cofactor	O
-is	O
-delineated	O
-.	O
-	O
-The	O
-PLP	O
-moieties	O
-of	O
-the	O
-cartoon	O
-ring	O
-are	O
-shown	O
-in	O
-red	O
-.	O
-	O
-(	O
-B	O
-)	O
-The	O
-LdcIi	O
-dimer	O
-extracted	O
-from	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-decamer	O
-.	O
-	O
-One	O
-monomer	O
-is	O
-colored	O
-in	O
-shades	O
-of	O
-yellow	O
-as	O
-in	O
-Figs	O
-1	O
-and	O
-2	O
-,	O
-while	O
-the	O
-monomer	O
-related	O
-by	O
-C2	O
-symmetry	O
-is	O
-grey	O
-.	O
-	O
-The	O
-PLP	O
-is	O
-red	O
-.	O
-	O
-The	O
-active	O
-site	O
-is	O
-boxed	O
-.	O
-	O
-Stretching	O
-of	O
-the	O
-LdcI	O
-monomer	O
-upon	O
-pH	O
--	O
-dependent	O
-enzyme	O
-activation	O
-and	O
-LARA	O
-binding	O
-.	O
-	O
-(	O
-A	O
-–	O
-C	O
-)	O
-A	O
-slice	O
-through	O
-the	O
-pseudoatomic	O
-models	O
-of	O
-the	O
-LdcI	O
-monomers	O
-extracted	O
-from	O
-the	O
-superimposed	O
-decamers	O
-(	O
-Fig	O
-.	O
-2	O
-)	O
-The	O
-rectangle	O
-indicates	O
-the	O
-regions	O
-enlarged	O
-in	O
-(	O
-D	O
-–	O
-F	O
-).	O
-	O
-(	O
-A	O
-)	O
-compares	O
-LdcIi	O
-(	O
-yellow	O
-)	O
-and	O
-LdcIa	O
-(	O
-pink	O
-),	O
-(	O
-B	O
-)	O
-compares	O
-LdcIa	O
-(	O
-pink	O
-)	O
-and	O
-LdcI	O
--	O
-LARA	O
-(	O
-blue	O
-),	O
-and	O
-(	O
-C	O
-)	O
-compares	O
-LdcIi	O
-(	O
-yellow	O
-),	O
-LdcIa	O
-(	O
-pink	O
-)	O
-and	O
-LdcI	O
--	O
-LARA	O
-(	O
-blue	O
-)	O
-simultaneously	O
-in	O
-order	O
-to	O
-show	O
-the	O
-progressive	O
-stretching	O
-described	O
-in	O
-the	O
-text	O
-.	O
-	O
-The	O
-cryoEM	O
-density	O
-of	O
-the	O
-LARA	O
-domain	O
-is	O
-represented	O
-as	O
-a	O
-grey	O
-surface	O
-to	O
-show	O
-the	O
-position	O
-of	O
-the	O
-binding	O
-site	O
-and	O
-the	O
-direction	O
-of	O
-the	O
-movement	O
-.	O
-	O
-(	O
-D	O
-–	O
-F	O
-)	O
-Inserts	O
-zooming	O
-at	O
-the	O
-CTD	O
-part	O
-in	O
-proximity	O
-of	O
-the	O
-LARA	O
-binding	O
-site	O
-.	O
-	O
-Analysis	O
-of	O
-the	O
-LdcIC	B-mutant
-and	O
-LdcCI	B-mutant
-chimeras	B-mutant
-.	O
-	O
-(	O
-A	O
-)	O
-A	O
-slice	O
-through	O
-the	O
-pseudoatomic	O
-models	O
-of	O
-the	O
-LdcIa	O
-(	O
-purple	O
-)	O
-and	O
-LdcC	O
-(	O
-green	O
-)	O
-monomers	O
-extracted	O
-from	O
-the	O
-superimposed	O
-decamers	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-(	O
-B	O
-)	O
-The	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-in	O
-LdcIa	O
-and	O
-LdcC	O
-enlarged	O
-from	O
-(	O
-A	O
-,	O
-C	O
-)	O
-Exchanged	O
-primary	O
-sequences	O
-(	O
-capital	O
-letters	O
-)	O
-and	O
-their	O
-immediate	O
-vicinity	O
-(	O
-lower	O
-case	O
-letters	O
-)	O
-colored	O
-as	O
-in	O
-(	O
-A	O
-,	O
-B	O
-),	O
-with	O
-the	O
-corresponding	O
-secondary	O
-structure	O
-elements	O
-and	O
-the	O
-amino	O
-acid	O
-numbering	O
-shown	O
-.	O
-	O
-(	O
-D	O
-,	O
-E	O
-)	O
-A	O
-gallery	O
-of	O
-negative	O
-stain	O
-EM	O
-images	O
-of	O
-(	O
-D	O
-)	O
-the	O
-wild	O
-type	O
-LdcI	O
--	O
-RavA	O
-cage	O
-and	O
-(	O
-E	O
-)	O
-the	O
-LdcCI	B-mutant
--	I-mutant
-RavA	I-mutant
-cage	I-mutant
--	I-mutant
-like	I-mutant
-particles	I-mutant
-.	O
-(	O
-F	O
-)	O
-Some	O
-representative	O
-class	O
-averages	O
-of	O
-the	O
-LdcCI	B-mutant
--	I-mutant
-RavA	I-mutant
-cage	I-mutant
--	I-mutant
-like	I-mutant
-particles	I-mutant
-.	O
-	O
-Sequence	O
-analysis	O
-of	O
-enterobacterial	O
-lysine	O
-decarboxylases	O
-.	O
-	O
-(	O
-A	O
-)	O
-Maximum	O
-likelihood	O
-tree	O
-with	O
-the	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-and	O
-the	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-groups	O
-highlighted	O
-in	O
-green	O
-and	O
-pink	O
-,	O
-respectively	O
-.	O
-	O
-(	O
-B	O
-)	O
-Analysis	O
-of	O
-consensus	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-and	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-sequences	O
-around	O
-the	O
-first	O
-and	O
-second	O
-C	O
--	O
-terminal	O
-β	O
--	O
-strands	O
-.	O
-	O
-Numbering	O
-as	O
-in	O
-E	O
-.	O
-coli	O
-.	O
-	O
-(	O
-C	O
-)	O
-Signature	O
-sequences	O
-of	O
-LdcI	O
-and	O
-LdcC	O
-in	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-.	O
-	O
-Polarity	O
-differences	O
-are	O
-highlighted	O
-.	O
-(	O
-D	O
-)	O
-Position	O
-and	O
-nature	O
-of	O
-these	O
-differences	O
-at	O
-the	O
-surface	O
-of	O
-the	O
-respective	O
-cryoEM	O
-maps	O
-with	O
-the	O
-color	O
-code	O
-as	O
-in	O
-B	O
-.	O
-See	O
-also	O
-Fig	O
-.	O
-S7	O
-and	O
-Tables	O
-S3	O
-and	O
-S4	O
-.	O
-	O
-Structural	O
-basis	O
-for	O
-Mep2	O
-ammonium	O
-transceptor	O
-activation	O
-by	O
-phosphorylation	O
-	O
-Mep2	O
-proteins	O
-are	O
-fungal	O
-transceptors	O
-that	O
-play	O
-an	O
-important	O
-role	O
-as	O
-ammonium	O
-sensors	O
-in	O
-fungal	O
-development	O
-.	O
-	O
-Mep2	O
-activity	O
-is	O
-tightly	O
-regulated	O
-by	O
-phosphorylation	O
-,	O
-but	O
-how	O
-this	O
-is	O
-achieved	O
-at	O
-the	O
-molecular	O
-level	O
-is	O
-not	O
-clear	O
-.	O
-	O
-Here	O
-we	O
-report	O
-X	O
--	O
-ray	O
-crystal	O
-structures	O
-of	O
-the	O
-Mep2	O
-orthologues	O
-from	O
-Saccharomyces	O
-cerevisiae	O
-and	O
-Candida	O
-albicans	O
-and	O
-show	O
-that	O
-under	O
-nitrogen	O
--	O
-sufficient	O
-conditions	O
-the	O
-transporters	O
-are	O
-not	O
-phosphorylated	O
-and	O
-present	O
-in	O
-closed	O
-,	O
-inactive	O
-conformations	O
-.	O
-	O
-Relative	O
-to	O
-the	O
-open	O
-bacterial	O
-ammonium	O
-transporters	O
-,	O
-non	O
--	O
-phosphorylated	O
-Mep2	O
-exhibits	O
-shifts	O
-in	O
-cytoplasmic	O
-loops	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-(	O
-CTR	O
-)	O
-to	O
-occlude	O
-the	O
-cytoplasmic	O
-exit	O
-of	O
-the	O
-channel	O
-and	O
-to	O
-interact	O
-with	O
-His2	O
-of	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-.	O
-	O
-The	O
-phosphorylation	O
-site	O
-in	O
-the	O
-CTR	O
-is	O
-solvent	O
-accessible	O
-and	O
-located	O
-in	O
-a	O
-negatively	O
-charged	O
-pocket	O
-∼	O
-30	O
-Å	O
-away	O
-from	O
-the	O
-channel	O
-exit	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-phosphorylation	O
--	O
-mimicking	O
-Mep2	B-mutant
-variants	I-mutant
-from	O
-C	O
-.	O
-albicans	O
-show	O
-large	O
-conformational	O
-changes	O
-in	O
-a	O
-conserved	O
-and	O
-functionally	O
-important	O
-region	O
-of	O
-the	O
-CTR	O
-.	O
-	O
-The	O
-results	O
-allow	O
-us	O
-to	O
-propose	O
-a	O
-model	O
-for	O
-regulation	O
-of	O
-eukaryotic	O
-ammonium	O
-transport	O
-by	O
-phosphorylation	O
-.	O
-	O
-Mep2	O
-proteins	O
-are	O
-tightly	O
-regulated	O
-fungal	O
-ammonium	O
-transporters	O
-.	O
-	O
-Here	O
-,	O
-the	O
-authors	O
-report	O
-the	O
-crystal	O
-structures	O
-of	O
-closed	O
-states	O
-of	O
-Mep2	O
-proteins	O
-and	O
-propose	O
-a	O
-model	O
-for	O
-their	O
-regulation	O
-by	O
-comparing	O
-them	O
-with	O
-the	O
-open	O
-ammonium	O
-transporters	O
-of	O
-bacteria	O
-.	O
-	O
-Transceptors	O
-are	O
-membrane	O
-proteins	O
-that	O
-function	O
-not	O
-only	O
-as	O
-transporters	O
-but	O
-also	O
-as	O
-receptors	O
-/	O
-sensors	O
-during	O
-nutrient	O
-sensing	O
-to	O
-activate	O
-downstream	O
-signalling	O
-pathways	O
-.	O
-	O
-A	O
-common	O
-feature	O
-of	O
-transceptors	O
-is	O
-that	O
-they	O
-are	O
-induced	O
-when	O
-cells	O
-are	O
-starved	O
-for	O
-their	O
-substrate	O
-.	O
-	O
-While	O
-most	O
-studies	O
-have	O
-focused	O
-on	O
-the	O
-Saccharomyces	O
-cerevisiae	O
-transceptors	O
-for	O
-phosphate	O
-(	O
-Pho84	O
-),	O
-amino	O
-acids	O
-(	O
-Gap1	O
-)	O
-and	O
-ammonium	O
-(	O
-Mep2	O
-),	O
-transceptors	O
-are	O
-found	O
-in	O
-higher	O
-eukaryotes	O
-as	O
-well	O
-(	O
-for	O
-example	O
-,	O
-the	O
-mammalian	O
-SNAT2	O
-amino	O
--	O
-acid	O
-transporter	O
-and	O
-the	O
-GLUT2	O
-glucose	O
-transporter	O
-).	O
-	O
-One	O
-of	O
-the	O
-most	O
-important	O
-unresolved	O
-questions	O
-in	O
-the	O
-field	O
-is	O
-how	O
-the	O
-transceptors	O
-couple	O
-to	O
-downstream	O
-signalling	O
-pathways	O
-.	O
-	O
-One	O
-hypothesis	O
-is	O
-that	O
-downstream	O
-signalling	O
-is	O
-dependent	O
-on	O
-a	O
-specific	O
-conformation	O
-of	O
-the	O
-transporter	O
-.	O
-	O
-Mep2	O
-(	O
-methylammonium	O
-(	O
-MA	O
-)	O
-permease	O
-)	O
-proteins	O
-are	O
-ammonium	O
-transceptors	O
-that	O
-are	O
-ubiquitous	O
-in	O
-fungi	O
-.	O
-	O
-They	O
-belong	O
-to	O
-the	O
-Amt	O
-/	O
-Mep	O
-/	O
-Rh	O
-family	O
-of	O
-transporters	O
-that	O
-are	O
-present	O
-in	O
-all	O
-kingdoms	O
-of	O
-life	O
-and	O
-they	O
-take	O
-up	O
-ammonium	O
-from	O
-the	O
-extracellular	O
-environment	O
-.	O
-	O
-Fungi	O
-typically	O
-have	O
-more	O
-than	O
-one	O
-Mep	O
-paralogue	O
-,	O
-for	O
-example	O
-,	O
-Mep1	O
--	O
-3	O
-in	O
-S	O
-.	O
-cerevisiae	O
-.	O
-	O
-Of	O
-these	O
-,	O
-only	O
-Mep2	O
-proteins	O
-function	O
-as	O
-ammonium	O
-receptors	O
-/	O
-sensors	O
-in	O
-fungal	O
-development	O
-.	O
-	O
-Under	O
-conditions	O
-of	O
-nitrogen	O
-limitation	O
-,	O
-Mep2	O
-initiates	O
-a	O
-signalling	O
-cascade	O
-that	O
-results	O
-in	O
-a	O
-switch	O
-from	O
-the	O
-yeast	O
-form	O
-to	O
-filamentous	O
-(	O
-pseudohyphal	O
-)	O
-growth	O
-that	O
-may	O
-be	O
-required	O
-for	O
-fungal	O
-pathogenicity	O
-.	O
-	O
-As	O
-is	O
-the	O
-case	O
-for	O
-other	O
-transceptors	O
-,	O
-it	O
-is	O
-not	O
-clear	O
-how	O
-Mep2	O
-interacts	O
-with	O
-downstream	O
-signalling	O
-partners	O
-,	O
-but	O
-the	O
-protein	O
-kinase	O
-A	O
-and	O
-mitogen	O
--	O
-activated	O
-protein	O
-kinase	O
-pathways	O
-have	O
-been	O
-proposed	O
-as	O
-downstream	O
-effectors	O
-of	O
-Mep2	O
-(	O
-refs	O
-).	O
-	O
-Compared	O
-with	O
-Mep1	O
-and	O
-Mep3	O
-,	O
-Mep2	O
-is	O
-highly	O
-expressed	O
-and	O
-functions	O
-as	O
-a	O
-low	O
--	O
-capacity	O
-,	O
-high	O
--	O
-affinity	O
-transporter	O
-in	O
-the	O
-uptake	O
-of	O
-MA	O
-.	O
-	O
-In	O
-addition	O
-,	O
-Mep2	O
-is	O
-also	O
-important	O
-for	O
-uptake	O
-of	O
-ammonium	O
-produced	O
-by	O
-growth	O
-on	O
-other	O
-nitrogen	O
-sources	O
-.	O
-	O
-With	O
-the	O
-exception	O
-of	O
-the	O
-human	O
-RhCG	O
-structure	O
-,	O
-no	O
-structural	O
-information	O
-is	O
-available	O
-for	O
-eukaryotic	O
-ammonium	O
-transporters	O
-.	O
-	O
-By	O
-contrast	O
-,	O
-several	O
-bacterial	O
-Amt	O
-orthologues	O
-have	O
-been	O
-characterized	O
-in	O
-detail	O
-via	O
-high	O
--	O
-resolution	O
-crystal	O
-structures	O
-and	O
-a	O
-number	O
-of	O
-molecular	O
-dynamics	O
-(	O
-MD	O
-)	O
-studies	O
-.	O
-	O
-All	O
-the	O
-solved	O
-structures	O
-including	O
-that	O
-of	O
-RhCG	O
-are	O
-very	O
-similar	O
-,	O
-establishing	O
-the	O
-basic	O
-architecture	O
-of	O
-ammonium	O
-transporters	O
-.	O
-	O
-The	O
-proteins	O
-form	O
-stable	O
-trimers	O
-,	O
-with	O
-each	O
-monomer	O
-having	O
-11	O
-transmembrane	O
-(	O
-TM	O
-)	O
-helices	O
-and	O
-a	O
-central	O
-channel	O
-for	O
-the	O
-transport	O
-of	O
-ammonium	O
-.	O
-	O
-All	O
-structures	O
-show	O
-the	O
-transporters	O
-in	O
-open	O
-conformations	O
-.	O
-	O
-Where	O
-earlier	O
-studies	O
-favoured	O
-the	O
-transport	O
-of	O
-ammonia	O
-gas	O
-,	O
-recent	O
-data	O
-and	O
-theoretical	O
-considerations	O
-suggest	O
-that	O
-Amt	O
-/	O
-Mep	O
-proteins	O
-are	O
-instead	O
-active	O
-,	O
-electrogenic	O
-transporters	O
-of	O
-either	O
-NH4	O
-+	O
-(	O
-uniport	O
-)	O
-or	O
-NH3	O
-/	O
-H	O
-+	O
-(	O
-symport	O
-).	O
-	O
-A	O
-highly	O
-conserved	O
-pair	O
-of	O
-channel	O
--	O
-lining	O
-histidine	O
-residues	O
-dubbed	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-may	O
-serve	O
-as	O
-a	O
-proton	O
-relay	O
-system	O
-while	O
-NH3	O
-moves	O
-through	O
-the	O
-channel	O
-during	O
-NH3	O
-/	O
-H	O
-+	O
-symport	O
-.	O
-	O
-Ammonium	O
-transport	O
-is	O
-tightly	O
-regulated	O
-.	O
-	O
-In	O
-animals	O
-,	O
-this	O
-is	O
-due	O
-to	O
-toxicity	O
-of	O
-elevated	O
-intracellular	O
-ammonium	O
-levels	O
-,	O
-whereas	O
-for	O
-microorganisms	O
-ammonium	O
-is	O
-a	O
-preferred	O
-nitrogen	O
-source	O
-.	O
-	O
-In	O
-bacteria	O
-,	O
-amt	O
-genes	O
-are	O
-present	O
-in	O
-an	O
-operon	O
-with	O
-glnK	O
-,	O
-encoding	O
-a	O
-PII	O
--	O
-like	O
-signal	O
-transduction	O
-class	O
-protein	O
-.	O
-	O
-By	O
-binding	O
-tightly	O
-to	O
-Amt	O
-proteins	O
-without	O
-inducing	O
-a	O
-conformational	O
-change	O
-in	O
-the	O
-transporter	O
-,	O
-GlnK	O
-sterically	O
-blocks	O
-ammonium	O
-conductance	O
-when	O
-nitrogen	O
-levels	O
-are	O
-sufficient	O
-.	O
-	O
-Under	O
-conditions	O
-of	O
-nitrogen	O
-limitation	O
-,	O
-GlnK	O
-becomes	O
-uridylated	O
-,	O
-blocking	O
-its	O
-ability	O
-to	O
-bind	O
-and	O
-inhibit	O
-Amt	O
-proteins	O
-.	O
-	O
-Importantly	O
-,	O
-eukaryotes	O
-do	O
-not	O
-have	O
-GlnK	O
-orthologues	O
-and	O
-have	O
-a	O
-different	O
-mechanism	O
-for	O
-regulation	O
-of	O
-ammonium	O
-transport	O
-activity	O
-.	O
-	O
-In	O
-plants	O
-,	O
-transporter	O
-phosphorylation	O
-and	O
-dephosphorylation	O
-are	O
-known	O
-to	O
-regulate	O
-activity	O
-.	O
-	O
-In	O
-S	O
-.	O
-cerevisiae	O
-,	O
-phosphorylation	O
-of	O
-Ser457	O
-within	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-(	O
-CTR	O
-)	O
-in	O
-the	O
-cytoplasm	O
-was	O
-recently	O
-proposed	O
-to	O
-cause	O
-Mep2	O
-opening	O
-,	O
-possibly	O
-via	O
-inducing	O
-a	O
-conformational	O
-change	O
-.	O
-	O
-To	O
-elucidate	O
-the	O
-mechanism	O
-of	O
-Mep2	O
-transport	O
-regulation	O
-,	O
-we	O
-present	O
-here	O
-X	O
--	O
-ray	O
-crystal	O
-structures	O
-of	O
-the	O
-Mep2	O
-transceptors	O
-from	O
-S	O
-.	O
-cerevisiae	O
-and	O
-C	O
-.	O
-albicans	O
-.	O
-	O
-The	O
-structures	O
-are	O
-similar	O
-to	O
-each	O
-other	O
-but	O
-show	O
-considerable	O
-differences	O
-to	O
-all	O
-other	O
-ammonium	O
-transporter	O
-structures	O
-.	O
-	O
-The	O
-most	O
-striking	O
-difference	O
-is	O
-the	O
-fact	O
-that	O
-the	O
-Mep2	O
-proteins	O
-have	O
-closed	O
-conformations	O
-.	O
-	O
-The	O
-putative	O
-phosphorylation	O
-site	O
-is	O
-solvent	O
-accessible	O
-and	O
-located	O
-in	O
-a	O
-negatively	O
-charged	O
-pocket	O
-∼	O
-30	O
-Å	O
-away	O
-from	O
-the	O
-channel	O
-exit	O
-.	O
-	O
-The	O
-channels	O
-of	O
-phosphorylation	O
--	O
-mimicking	O
-mutants	O
-of	O
-C	O
-.	O
-albicans	O
-Mep2	O
-are	O
-still	O
-closed	O
-but	O
-show	O
-large	O
-conformational	O
-changes	O
-within	O
-a	O
-conserved	O
-part	O
-of	O
-the	O
-CTR	O
-.	O
-	O
-Together	O
-with	O
-a	O
-structure	O
-of	O
-a	O
-C	O
--	O
-terminal	O
-Mep2	B-mutant
-variant	I-mutant
-lacking	O
-the	O
-segment	O
-containing	O
-the	O
-phosphorylation	O
-site	O
-,	O
-the	O
-results	O
-allow	O
-us	O
-to	O
-propose	O
-a	O
-structural	O
-model	O
-for	O
-phosphorylation	O
--	O
-based	O
-regulation	O
-of	O
-eukaryotic	O
-ammonium	O
-transport	O
-.	O
-	O
-General	O
-architecture	O
-of	O
-Mep2	O
-ammonium	O
-transceptors	O
-	O
-The	O
-Mep2	O
-protein	O
-of	O
-S	O
-.	O
-cerevisiae	O
-(	O
-ScMep2	O
-)	O
-was	O
-overexpressed	O
-in	O
-S	O
-.	O
-cerevisiae	O
-in	O
-high	O
-yields	O
-,	O
-enabling	O
-structure	O
-determination	O
-by	O
-X	O
--	O
-ray	O
-crystallography	O
-using	O
-data	O
-to	O
-3	O
-.	O
-2	O
-Å	O
-resolution	O
-by	O
-molecular	O
-replacement	O
-(	O
-MR	O
-)	O
-with	O
-the	O
-archaebacterial	O
-Amt	O
--	O
-1	O
-structure	O
-(	O
-see	O
-Methods	O
-section	O
-).	O
-	O
-Given	O
-that	O
-the	O
-modest	O
-resolution	O
-of	O
-the	O
-structure	O
-and	O
-the	O
-limited	O
-detergent	O
-stability	O
-of	O
-ScMep2	O
-would	O
-likely	O
-complicate	O
-structure	O
-–	O
-function	O
-studies	O
-,	O
-several	O
-other	O
-fungal	O
-Mep2	O
-orthologues	O
-were	O
-subsequently	O
-overexpressed	O
-and	O
-screened	O
-for	O
-diffraction	O
--	O
-quality	O
-crystals	O
-.	O
-	O
-Of	O
-these	O
-,	O
-Mep2	O
-from	O
-C	O
-.	O
-albicans	O
-(	O
-CaMep2	O
-)	O
-showed	O
-superior	O
-stability	O
-in	O
-relatively	O
-harsh	O
-detergents	O
-such	O
-as	O
-nonyl	O
--	O
-glucoside	O
-,	O
-allowing	O
-structure	O
-determination	O
-in	O
-two	O
-different	O
-crystal	O
-forms	O
-to	O
-high	O
-resolution	O
-(	O
-up	O
-to	O
-1	O
-.	O
-5	O
-Å	O
-).	O
-	O
-Despite	O
-different	O
-crystal	O
-packing	O
-(	O
-Supplementary	O
-Table	O
-1	O
-),	O
-the	O
-two	O
-CaMep2	O
-structures	O
-are	O
-identical	O
-to	O
-each	O
-other	O
-and	O
-very	O
-similar	O
-to	O
-ScMep2	O
-(	O
-Cα	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.	O
-	O
-(	O
-root	O
-mean	O
-square	O
-deviation	O
-)=	O
-0	O
-.	O
-7	O
-Å	O
-for	O
-434	O
-residues	O
-),	O
-with	O
-the	O
-main	O
-differences	O
-confined	O
-to	O
-the	O
-N	O
-terminus	O
-and	O
-the	O
-CTR	O
-(	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-Electron	O
-density	O
-is	O
-visible	O
-for	O
-the	O
-entire	O
-polypeptide	O
-chains	O
-,	O
-with	O
-the	O
-exception	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-43	O
-(	O
-ScMep2	O
-)	O
-and	O
-25	O
-residues	O
-(	O
-CaMep2	O
-),	O
-which	O
-are	O
-poorly	O
-conserved	O
-and	O
-presumably	O
-disordered	O
-.	O
-	O
-Both	O
-Mep2	O
-proteins	O
-show	O
-the	O
-archetypal	O
-trimeric	O
-assemblies	O
-in	O
-which	O
-each	O
-monomer	O
-consists	O
-of	O
-11	O
-TM	O
-helices	O
-surrounding	O
-a	O
-central	O
-pore	O
-.	O
-	O
-Important	O
-functional	O
-features	O
-such	O
-as	O
-the	O
-extracellular	O
-ammonium	O
-binding	O
-site	O
-,	O
-the	O
-Phe	O
-gate	O
-and	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-within	O
-the	O
-hydrophobic	O
-channel	O
-are	O
-all	O
-very	O
-similar	O
-to	O
-those	O
-present	O
-in	O
-the	O
-bacterial	O
-transporters	O
-and	O
-RhCG	O
-.	O
-	O
-In	O
-the	O
-remainder	O
-of	O
-the	O
-manuscript	O
-,	O
-we	O
-will	O
-specifically	O
-discuss	O
-CaMep2	O
-due	O
-to	O
-the	O
-superior	O
-resolution	O
-of	O
-the	O
-structure	O
-.	O
-	O
-Unless	O
-specifically	O
-stated	O
-,	O
-the	O
-drawn	O
-conclusions	O
-also	O
-apply	O
-to	O
-ScMep2	O
-.	O
-	O
-While	O
-the	O
-overall	O
-architecture	O
-of	O
-Mep2	O
-is	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-prokaryotic	O
-transporters	O
-(	O
-Cα	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.	O
-with	O
-Amt	O
--	O
-1	O
-=	O
-1	O
-.	O
-4	O
-Å	O
-for	O
-361	O
-residues	O
-),	O
-there	O
-are	O
-large	O
-differences	O
-within	O
-the	O
-N	O
-terminus	O
-,	O
-intracellular	O
-loops	O
-(	O
-ICLs	O
-)	O
-ICL1	O
-and	O
-ICL3	O
-,	O
-and	O
-the	O
-CTR	O
-.	O
-	O
-The	O
-N	O
-termini	O
-of	O
-the	O
-Mep2	O
-proteins	O
-are	O
-∼	O
-20	O
-–	O
-25	O
-residues	O
-longer	O
-compared	O
-with	O
-their	O
-bacterial	O
-counterparts	O
-(	O
-Figs	O
-1	O
-and	O
-2	O
-),	O
-substantially	O
-increasing	O
-the	O
-size	O
-of	O
-the	O
-extracellular	O
-domain	O
-.	O
-	O
-Moreover	O
-,	O
-the	O
-N	O
-terminus	O
-of	O
-one	O
-monomer	O
-interacts	O
-with	O
-the	O
-extended	O
-extracellular	O
-loop	O
-ECL5	O
-of	O
-a	O
-neighbouring	O
-monomer	O
-.	O
-	O
-Together	O
-with	O
-additional	O
-,	O
-smaller	O
-differences	O
-in	O
-other	O
-extracellular	O
-loops	O
-,	O
-these	O
-changes	O
-generate	O
-a	O
-distinct	O
-vestibule	O
-leading	O
-to	O
-the	O
-ammonium	O
-binding	O
-site	O
-that	O
-is	O
-much	O
-more	O
-pronounced	O
-than	O
-in	O
-the	O
-bacterial	O
-proteins	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-vestibule	O
-and	O
-the	O
-resulting	O
-inter	O
--	O
-monomer	O
-interactions	O
-likely	O
-increase	O
-the	O
-stability	O
-of	O
-the	O
-Mep2	O
-trimer	O
-,	O
-in	O
-support	O
-of	O
-data	O
-for	O
-plant	O
-AMT	O
-proteins	O
-.	O
-	O
-However	O
-,	O
-given	O
-that	O
-an	O
-N	O
--	O
-terminal	O
-deletion	O
-mutant	O
-(	O
-2	B-mutant
--	I-mutant
-27Δ	I-mutant
-)	O
-grows	O
-as	O
-well	O
-as	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-Mep2	O
-on	O
-minimal	O
-ammonium	O
-medium	O
-(	O
-Fig	O
-.	O
-3	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-),	O
-the	O
-importance	O
-of	O
-the	O
-N	O
-terminus	O
-for	O
-Mep2	O
-activity	O
-is	O
-not	O
-clear	O
-.	O
-	O
-Mep2	O
-channels	O
-are	O
-closed	O
-by	O
-a	O
-two	O
--	O
-tier	O
-channel	O
-block	O
-	O
-The	O
-largest	O
-differences	O
-between	O
-the	O
-Mep2	O
-structures	O
-and	O
-the	O
-other	O
-known	O
-ammonium	O
-transporter	O
-structures	O
-are	O
-located	O
-on	O
-the	O
-intracellular	O
-side	O
-of	O
-the	O
-membrane	O
-.	O
-	O
-In	O
-the	O
-vicinity	O
-of	O
-the	O
-Mep2	O
-channel	O
-exit	O
-,	O
-the	O
-cytoplasmic	O
-end	O
-of	O
-TM2	O
-has	O
-unwound	O
-,	O
-generating	O
-a	O
-longer	O
-ICL1	O
-even	O
-though	O
-there	O
-are	O
-no	O
-insertions	O
-in	O
-this	O
-region	O
-compared	O
-to	O
-the	O
-bacterial	O
-proteins	O
-(	O
-Figs	O
-2	O
-and	O
-4	O
-).	O
-	O
-ICL1	O
-has	O
-also	O
-moved	O
-inwards	O
-relative	O
-to	O
-its	O
-position	O
-in	O
-the	O
-bacterial	O
-Amts	O
-.	O
-	O
-The	O
-largest	O
-backbone	O
-movements	O
-of	O
-equivalent	O
-residues	O
-within	O
-ICL1	O
-are	O
-∼	O
-10	O
-Å	O
-,	O
-markedly	O
-affecting	O
-the	O
-conserved	O
-basic	O
-RxK	O
-motif	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-The	O
-head	O
-group	O
-of	O
-Arg54	O
-has	O
-moved	O
-∼	O
-11	O
-Å	O
-relative	O
-to	O
-that	O
-in	O
-Amt	O
--	O
-1	O
-,	O
-whereas	O
-the	O
-shift	O
-of	O
-the	O
-head	O
-group	O
-of	O
-the	O
-variable	O
-Lys55	O
-residue	O
-is	O
-almost	O
-20	O
-Å	O
-.	O
-The	O
-side	O
-chain	O
-of	O
-Lys56	O
-in	O
-the	O
-basic	O
-motif	O
-points	O
-in	O
-an	O
-opposite	O
-direction	O
-in	O
-the	O
-Mep2	O
-structures	O
-compared	O
-with	O
-that	O
-of	O
-,	O
-for	O
-example	O
-,	O
-Amt	O
--	O
-1	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-In	O
-addition	O
-to	O
-changing	O
-the	O
-RxK	O
-motif	O
-,	O
-the	O
-movement	O
-of	O
-ICL1	O
-has	O
-another	O
-,	O
-crucial	O
-functional	O
-consequence	O
-.	O
-	O
-At	O
-the	O
-C	O
--	O
-terminal	O
-end	O
-of	O
-TM1	O
-,	O
-the	O
-side	O
--	O
-chain	O
-hydroxyl	O
-group	O
-of	O
-the	O
-relatively	O
-conserved	O
-Tyr49	O
-(	O
-Tyr53	O
-in	O
-ScMep2	O
-)	O
-makes	O
-a	O
-strong	O
-hydrogen	O
-bond	O
-with	O
-the	O
-ɛ2	O
-nitrogen	O
-atom	O
-of	O
-the	O
-absolutely	O
-conserved	O
-His342	O
-of	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-(	O
-His348	O
-in	O
-ScMep2	O
-),	O
-closing	O
-the	O
-channel	O
-(	O
-Figs	O
-4	O
-and	O
-5	O
-).	O
-	O
-In	O
-bacterial	O
-Amt	O
-proteins	O
-,	O
-this	O
-Tyr	O
-side	O
-chain	O
-is	O
-rotated	O
-∼	O
-4	O
-Å	O
-away	O
-as	O
-a	O
-result	O
-of	O
-the	O
-different	O
-conformation	O
-of	O
-TM1	O
-,	O
-leaving	O
-the	O
-channel	O
-open	O
-and	O
-the	O
-histidine	O
-available	O
-for	O
-its	O
-putative	O
-role	O
-in	O
-substrate	O
-transport	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Compared	O
-with	O
-ICL1	O
-,	O
-the	O
-backbone	O
-conformational	O
-changes	O
-observed	O
-for	O
-the	O
-neighbouring	O
-ICL2	O
-are	O
-smaller	O
-,	O
-but	O
-large	O
-shifts	O
-are	O
-nevertheless	O
-observed	O
-for	O
-the	O
-conserved	O
-residues	O
-Glu140	O
-and	O
-Arg141	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-Finally	O
-,	O
-the	O
-important	O
-ICL3	O
-linking	O
-the	O
-pseudo	O
--	O
-symmetrical	O
-halves	O
-(	O
-TM1	O
--	O
-5	O
-and	O
-TM6	O
--	O
-10	O
-)	O
-of	O
-the	O
-transporter	O
-is	O
-also	O
-shifted	O
-up	O
-to	O
-∼	O
-10	O
-Å	O
-and	O
-forms	O
-an	O
-additional	O
-barrier	O
-that	O
-closes	O
-the	O
-channel	O
-on	O
-the	O
-cytoplasmic	O
-side	O
-(	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-This	O
-two	O
--	O
-tier	O
-channel	O
-block	O
-likely	O
-ensures	O
-that	O
-very	O
-little	O
-ammonium	O
-transport	O
-will	O
-take	O
-place	O
-under	O
-nitrogen	O
--	O
-sufficient	O
-conditions	O
-.	O
-	O
-The	O
-closed	O
-state	O
-of	O
-the	O
-channel	O
-might	O
-also	O
-explain	O
-why	O
-no	O
-density	O
-,	O
-which	O
-could	O
-correspond	O
-to	O
-ammonium	O
-(	O
-or	O
-water	O
-),	O
-is	O
-observed	O
-in	O
-the	O
-hydrophobic	O
-part	O
-of	O
-the	O
-Mep2	O
-channel	O
-close	O
-to	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-.	O
-	O
-Significantly	O
-,	O
-this	O
-is	O
-also	O
-true	O
-for	O
-ScMep2	O
-,	O
-which	O
-was	O
-crystallized	O
-in	O
-the	O
-presence	O
-of	O
-0	O
-.	O
-2	O
-M	O
-ammonium	O
-ions	O
-(	O
-see	O
-Methods	O
-section	O
-).	O
-	O
-The	O
-final	O
-region	O
-in	O
-Mep2	O
-that	O
-shows	O
-large	O
-differences	O
-compared	O
-with	O
-the	O
-bacterial	O
-transporters	O
-is	O
-the	O
-CTR	O
-.	O
-	O
-In	O
-Mep2	O
-,	O
-the	O
-CTR	O
-has	O
-moved	O
-away	O
-and	O
-makes	O
-relatively	O
-few	O
-contacts	O
-with	O
-the	O
-main	O
-body	O
-of	O
-the	O
-transporter	O
-,	O
-generating	O
-a	O
-more	O
-elongated	O
-protein	O
-(	O
-Figs	O
-1	O
-and	O
-4	O
-).	O
-	O
-By	O
-contrast	O
-,	O
-in	O
-the	O
-structures	O
-of	O
-bacterial	O
-proteins	O
-,	O
-the	O
-CTR	O
-is	O
-docked	O
-tightly	O
-onto	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-the	O
-transporters	O
-(	O
-corresponding	O
-to	O
-TM1	O
--	O
-5	O
-),	O
-resulting	O
-in	O
-a	O
-more	O
-compact	O
-structure	O
-.	O
-	O
-This	O
-is	O
-illustrated	O
-by	O
-the	O
-positions	O
-of	O
-the	O
-five	O
-universally	O
-conserved	O
-residues	O
-within	O
-the	O
-CTR	O
-,	O
-that	O
-is	O
-,	O
-Arg415	O
-(	O
-370	O
-),	O
-Glu421	O
-(	O
-376	O
-),	O
-Gly424	O
-(	O
-379	O
-),	O
-Asp426	O
-(	O
-381	O
-)	O
-and	O
-Tyr	O
-435	O
-(	O
-390	O
-)	O
-in	O
-CaMep2	O
-(	O
-Amt	O
--	O
-1	O
-)	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-These	O
-residues	O
-include	O
-those	O
-of	O
-the	O
-‘	O
-ExxGxD	O
-'	O
-motif	O
-,	O
-which	O
-when	O
-mutated	O
-generate	O
-inactive	O
-transporters	O
-.	O
-	O
-In	O
-Amt	O
--	O
-1	O
-and	O
-other	O
-bacterial	O
-ammonium	O
-transporters	O
-,	O
-these	O
-CTR	O
-residues	O
-interact	O
-with	O
-residues	O
-within	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-the	O
-protein	O
-.	O
-	O
-On	O
-one	O
-side	O
-,	O
-the	O
-Tyr390	O
-hydroxyl	O
-in	O
-Amt	O
--	O
-1	O
-is	O
-hydrogen	O
-bonded	O
-with	O
-the	O
-side	O
-chain	O
-of	O
-the	O
-conserved	O
-His185	O
-at	O
-the	O
-C	O
--	O
-terminal	O
-end	O
-of	O
-loop	O
-ICL3	O
-.	O
-	O
-At	O
-the	O
-other	O
-end	O
-of	O
-ICL3	O
-,	O
-the	O
-backbone	O
-carbonyl	O
-groups	O
-of	O
-Gly172	O
-and	O
-Lys173	O
-are	O
-hydrogen	O
-bonded	O
-to	O
-the	O
-side	O
-chain	O
-of	O
-Arg370	O
-.	O
-	O
-Similar	O
-interactions	O
-were	O
-also	O
-modelled	O
-in	O
-the	O
-active	O
-,	O
-non	O
--	O
-phosphorylated	O
-plant	O
-AtAmt	O
--	O
-1	O
-;	O
-1	O
-structure	O
-(	O
-for	O
-example	O
-,	O
-Y467	O
--	O
-H239	O
-and	O
-D458	O
--	O
-K71	O
-).	O
-	O
-The	O
-result	O
-of	O
-these	O
-interactions	O
-is	O
-that	O
-the	O
-CTR	O
-‘	O
-hugs	O
-'	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-the	O
-transporters	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-Also	O
-noteworthy	O
-is	O
-Asp381	O
-,	O
-the	O
-side	O
-chain	O
-of	O
-which	O
-interacts	O
-strongly	O
-with	O
-the	O
-positive	O
-dipole	O
-on	O
-the	O
-N	O
--	O
-terminal	O
-end	O
-of	O
-TM2	O
-.	O
-	O
-This	O
-interaction	O
-in	O
-the	O
-centre	O
-of	O
-the	O
-protein	O
-may	O
-be	O
-particularly	O
-important	O
-to	O
-stabilize	O
-the	O
-open	O
-conformations	O
-of	O
-ammonium	O
-transporters	O
-.	O
-	O
-In	O
-the	O
-Mep2	O
-structures	O
-,	O
-none	O
-of	O
-the	O
-interactions	O
-mentioned	O
-above	O
-are	O
-present	O
-.	O
-	O
-Phosphorylation	O
-target	O
-site	O
-is	O
-at	O
-the	O
-periphery	O
-of	O
-Mep2	O
-	O
-Recently	O
-Boeckstaens	O
-et	O
-al	O
-.	O
-provided	O
-evidence	O
-that	O
-Ser457	O
-in	O
-ScMep2	O
-(	O
-corresponding	O
-to	O
-Ser453	O
-in	O
-CaMep2	O
-)	O
-is	O
-phosphorylated	O
-by	O
-the	O
-TORC1	O
-effector	O
-kinase	O
-Npr1	O
-under	O
-nitrogen	O
--	O
-limiting	O
-conditions	O
-.	O
-	O
-In	O
-the	O
-absence	O
-of	O
-Npr1	O
-,	O
-plasmid	O
--	O
-encoded	O
-WT	O
-Mep2	O
-in	O
-a	O
-S	O
-.	O
-cerevisiae	O
-mep1	B-mutant
--	I-mutant
-3Δ	I-mutant
-strain	O
-(	O
-triple	B-mutant
-mepΔ	I-mutant
-)	O
-does	O
-not	O
-allow	O
-growth	O
-on	O
-low	O
-concentrations	O
-of	O
-ammonium	O
-,	O
-suggesting	O
-that	O
-the	O
-transporter	O
-is	O
-inactive	O
-(	O
-Fig	O
-.	O
-3	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-Conversely	O
-,	O
-the	O
-phosphorylation	O
--	O
-mimicking	O
-S457D	B-mutant
-variant	O
-is	O
-active	O
-both	O
-in	O
-the	O
-triple	B-mutant
-mepΔ	I-mutant
-background	O
-and	O
-in	O
-a	O
-triple	B-mutant
-mepΔ	I-mutant
-npr1Δ	I-mutant
-strain	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-Mutation	O
-of	O
-other	O
-potential	O
-phosphorylation	O
-sites	O
-in	O
-the	O
-CTR	O
-did	O
-not	O
-support	O
-growth	O
-in	O
-the	O
-npr1Δ	B-mutant
-background	O
-.	O
-	O
-Collectively	O
-,	O
-these	O
-data	O
-suggest	O
-that	O
-phosphorylation	O
-of	O
-Ser457	O
-opens	O
-the	O
-Mep2	O
-channel	O
-to	O
-allow	O
-ammonium	O
-uptake	O
-.	O
-	O
-Ser457	O
-is	O
-located	O
-in	O
-a	O
-part	O
-of	O
-the	O
-CTR	O
-that	O
-is	O
-conserved	O
-in	O
-a	O
-subgroup	O
-of	O
-Mep2	O
-proteins	O
-,	O
-but	O
-which	O
-is	O
-not	O
-present	O
-in	O
-bacterial	O
-proteins	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-This	O
-segment	O
-(	O
-residues	O
-450	O
-–	O
-457	O
-in	O
-ScMep2	O
-and	O
-446	O
-–	O
-453	O
-in	O
-CaMep2	O
-)	O
-was	O
-dubbed	O
-an	O
-autoinhibitory	O
-(	O
-AI	O
-)	O
-region	O
-based	O
-on	O
-the	O
-fact	O
-that	O
-its	O
-removal	O
-generates	O
-an	O
-active	O
-transporter	O
-in	O
-the	O
-absence	O
-of	O
-Npr1	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-Where	O
-is	O
-the	O
-AI	O
-region	O
-and	O
-the	O
-Npr1	O
-phosphorylation	O
-site	O
-located	O
-?	O
-Our	O
-structures	O
-reveal	O
-that	O
-surprisingly	O
-,	O
-the	O
-AI	O
-region	O
-is	O
-folded	O
-back	O
-onto	O
-the	O
-CTR	O
-and	O
-is	O
-not	O
-located	O
-near	O
-the	O
-centre	O
-of	O
-the	O
-trimer	O
-as	O
-expected	O
-from	O
-the	O
-bacterial	O
-structures	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-The	O
-AI	O
-region	O
-packs	O
-against	O
-the	O
-cytoplasmic	O
-ends	O
-of	O
-TM2	O
-and	O
-TM4	O
-,	O
-physically	O
-linking	O
-the	O
-main	O
-body	O
-of	O
-the	O
-transporter	O
-with	O
-the	O
-CTR	O
-via	O
-main	O
-chain	O
-interactions	O
-and	O
-side	O
--	O
-chain	O
-interactions	O
-of	O
-Val447	O
-,	O
-Asp449	O
-,	O
-Pro450	O
-and	O
-Arg452	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-The	O
-AI	O
-regions	O
-have	O
-very	O
-similar	O
-conformations	O
-in	O
-CaMep2	O
-and	O
-ScMep2	O
-,	O
-despite	O
-considerable	O
-differences	O
-in	O
-the	O
-rest	O
-of	O
-the	O
-CTR	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-Strikingly	O
-,	O
-the	O
-Npr1	O
-target	O
-serine	O
-residue	O
-is	O
-located	O
-at	O
-the	O
-periphery	O
-of	O
-the	O
-trimer	O
-,	O
-far	O
-away	O
-(∼	O
-30	O
-Å	O
-)	O
-from	O
-any	O
-channel	O
-exit	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-Despite	O
-its	O
-location	O
-at	O
-the	O
-periphery	O
-of	O
-the	O
-trimer	O
-,	O
-the	O
-electron	O
-density	O
-for	O
-the	O
-serine	O
-is	O
-well	O
-defined	O
-in	O
-both	O
-Mep2	O
-structures	O
-and	O
-corresponds	O
-to	O
-the	O
-non	O
--	O
-phosphorylated	O
-state	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-This	O
-makes	O
-sense	O
-since	O
-the	O
-proteins	O
-were	O
-expressed	O
-in	O
-rich	O
-medium	O
-and	O
-confirms	O
-the	O
-recent	O
-suggestion	O
-by	O
-Boeckstaens	O
-et	O
-al	O
-.	O
-that	O
-the	O
-non	O
--	O
-phosphorylated	O
-form	O
-of	O
-Mep2	O
-corresponds	O
-to	O
-the	O
-inactive	O
-state	O
-.	O
-	O
-For	O
-ScMep2	O
-,	O
-Ser457	O
-is	O
-the	O
-most	O
-C	O
--	O
-terminal	O
-residue	O
-for	O
-which	O
-electron	O
-density	O
-is	O
-visible	O
-,	O
-indicating	O
-that	O
-the	O
-region	O
-beyond	O
-Ser457	O
-is	O
-disordered	O
-.	O
-	O
-In	O
-CaMep2	O
-,	O
-the	O
-visible	O
-part	O
-of	O
-the	O
-sequence	O
-extends	O
-for	O
-two	O
-residues	O
-beyond	O
-Ser453	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-The	O
-peripheral	O
-location	O
-and	O
-disorder	O
-of	O
-the	O
-CTR	O
-beyond	O
-the	O
-kinase	O
-target	O
-site	O
-should	O
-facilitate	O
-the	O
-phosphorylation	O
-by	O
-Npr1	O
-.	O
-	O
-The	O
-disordered	O
-part	O
-of	O
-the	O
-CTR	O
-is	O
-not	O
-conserved	O
-in	O
-ammonium	O
-transporters	O
-(	O
-Fig	O
-.	O
-2	O
-),	O
-suggesting	O
-that	O
-it	O
-is	O
-not	O
-important	O
-for	O
-transport	O
-.	O
-	O
-Interestingly	O
-,	O
-a	O
-ScMep2	O
-457Δ	B-mutant
-truncation	O
-mutant	O
-in	O
-which	O
-a	O
-His	O
--	O
-tag	O
-directly	O
-follows	O
-Ser457	O
-is	O
-highly	O
-expressed	O
-but	O
-has	O
-low	O
-activity	O
-(	O
-Fig	O
-.	O
-3	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1b	O
-),	O
-suggesting	O
-that	O
-the	O
-His	O
--	O
-tag	O
-interferes	O
-with	O
-phosphorylation	O
-by	O
-Npr1	O
-.	O
-	O
-The	O
-same	O
-mutant	B-mutant
-lacking	O
-the	O
-His	O
--	O
-tag	O
-has	O
-WT	O
-properties	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1b	O
-),	O
-confirming	O
-that	O
-the	O
-region	O
-following	O
-the	O
-phosphorylation	O
-site	O
-is	O
-dispensable	O
-for	O
-function	O
-.	O
-	O
-Mep2	O
-lacking	O
-the	O
-AI	O
-region	O
-is	O
-conformationally	O
-heterogeneous	O
-	O
-Given	O
-that	O
-Ser457	O
-/	O
-453	O
-is	O
-far	O
-from	O
-any	O
-channel	O
-exit	O
-(	O
-Fig	O
-.	O
-6	O
-),	O
-the	O
-crucial	O
-question	O
-is	O
-how	O
-phosphorylation	O
-opens	O
-the	O
-Mep2	O
-channel	O
-to	O
-generate	O
-an	O
-active	O
-transporter	O
-.	O
-	O
-Boeckstaens	O
-et	O
-al	O
-.	O
-proposed	O
-that	O
-phosphorylation	O
-does	O
-not	O
-affect	O
-channel	O
-activity	O
-directly	O
-,	O
-but	O
-instead	O
-relieves	O
-inhibition	O
-by	O
-the	O
-AI	O
-region	O
-.	O
-	O
-The	O
-data	O
-behind	O
-this	O
-hypothesis	O
-is	O
-the	O
-observation	O
-that	O
-a	O
-ScMep2	O
-449	B-mutant
--	I-mutant
-485Δ	I-mutant
-deletion	O
-mutant	O
-lacking	O
-the	O
-AI	O
-region	O
-is	O
-highly	O
-active	O
-in	O
-MA	O
-uptake	O
-both	O
-in	O
-the	O
-triple	B-mutant
-mepΔ	I-mutant
-and	O
-triple	B-mutant
-mepΔ	I-mutant
-npr1Δ	I-mutant
-backgrounds	O
-,	O
-implying	O
-that	O
-this	O
-Mep2	B-mutant
-variant	I-mutant
-has	O
-a	O
-constitutively	O
-open	O
-channel	O
-.	O
-	O
-We	O
-obtained	O
-a	O
-similar	O
-result	O
-for	O
-ammonium	O
-uptake	O
-by	O
-the	O
-446Δ	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-3	O
-),	O
-supporting	O
-the	O
-data	O
-from	O
-Marini	O
-et	O
-al	O
-.	O
-We	O
-then	O
-constructed	O
-and	O
-purified	O
-the	O
-analogous	O
-CaMep2	O
-442Δ	B-mutant
-truncation	O
-mutant	O
-and	O
-determined	O
-the	O
-crystal	O
-structure	O
-using	O
-data	O
-to	O
-3	O
-.	O
-4	O
-Å	O
-resolution	O
-.	O
-	O
-The	O
-structure	O
-shows	O
-that	O
-removal	O
-of	O
-the	O
-AI	O
-region	O
-markedly	O
-increases	O
-the	O
-dynamics	O
-of	O
-the	O
-cytoplasmic	O
-parts	O
-of	O
-the	O
-transporter	O
-.	O
-	O
-This	O
-is	O
-not	O
-unexpected	O
-given	O
-the	O
-fact	O
-that	O
-the	O
-AI	O
-region	O
-bridges	O
-the	O
-CTR	O
-and	O
-the	O
-main	O
-body	O
-of	O
-Mep2	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-Density	O
-for	O
-ICL3	O
-and	O
-the	O
-CTR	O
-beyond	O
-residue	O
-Arg415	O
-is	O
-missing	O
-in	O
-the	O
-442Δ	B-mutant
-mutant	O
-,	O
-and	O
-the	O
-density	O
-for	O
-the	O
-other	O
-ICLs	O
-including	O
-ICL1	O
-is	O
-generally	O
-poor	O
-with	O
-visible	O
-parts	O
-of	O
-the	O
-structure	O
-having	O
-high	O
-B	O
--	O
-factors	O
-(	O
-Fig	O
-.	O
-7	O
-).	O
-	O
-Interestingly	O
-,	O
-however	O
-,	O
-the	O
-Tyr49	O
--	O
-His342	O
-hydrogen	O
-bond	O
-that	O
-closes	O
-the	O
-channel	O
-in	O
-the	O
-WT	O
-protein	O
-is	O
-still	O
-present	O
-(	O
-Fig	O
-.	O
-7	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Why	O
-then	O
-does	O
-this	O
-mutant	O
-appear	O
-to	O
-be	O
-constitutively	O
-active	O
-?	O
-We	O
-propose	O
-two	O
-possibilities	O
-.	O
-	O
-The	O
-first	O
-one	O
-is	O
-that	O
-the	O
-open	O
-state	O
-is	O
-disfavoured	O
-by	O
-crystallization	O
-because	O
-of	O
-lower	O
-stability	O
-or	O
-due	O
-to	O
-crystal	O
-packing	O
-constraints	O
-.	O
-	O
-The	O
-second	O
-possibility	O
-is	O
-that	O
-the	O
-Tyr	O
-–	O
-His	O
-hydrogen	O
-bond	O
-has	O
-to	O
-be	O
-disrupted	O
-by	O
-the	O
-incoming	O
-substrate	O
-to	O
-open	O
-the	O
-channel	O
-.	O
-	O
-The	O
-latter	O
-model	O
-would	O
-fit	O
-well	O
-with	O
-the	O
-NH3	O
-/	O
-H	O
-+	O
-symport	O
-model	O
-in	O
-which	O
-the	O
-proton	O
-is	O
-relayed	O
-by	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-.	O
-	O
-The	O
-importance	O
-of	O
-the	O
-Tyr	O
-–	O
-His	O
-hydrogen	O
-bond	O
-is	O
-underscored	O
-by	O
-the	O
-fact	O
-that	O
-its	O
-removal	O
-in	O
-the	O
-ScMep2	O
-Y53A	B-mutant
-mutant	O
-results	O
-in	O
-a	O
-constitutively	O
-active	O
-transporter	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-Phosphorylation	O
-causes	O
-a	O
-conformational	O
-change	O
-in	O
-the	O
-CTR	O
-	O
-Do	O
-the	O
-Mep2	O
-structures	O
-provide	O
-any	O
-clues	O
-regarding	O
-the	O
-potential	O
-effect	O
-of	O
-phosphorylation	O
-?	O
-	O
-The	O
-side	O
--	O
-chain	O
-hydroxyl	O
-of	O
-Ser457	O
-/	O
-453	O
-is	O
-located	O
-in	O
-a	O
-well	O
--	O
-defined	O
-electronegative	O
-pocket	O
-that	O
-is	O
-solvent	O
-accessible	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-The	O
-closest	O
-atoms	O
-to	O
-the	O
-serine	O
-hydroxyl	O
-group	O
-are	O
-the	O
-backbone	O
-carbonyl	O
-atoms	O
-of	O
-Asp419	O
-,	O
-Glu420	O
-and	O
-Glu421	O
-,	O
-which	O
-are	O
-3	O
-–	O
-4	O
-Å	O
-away	O
-.	O
-	O
-We	O
-therefore	O
-predict	O
-that	O
-phosphorylation	O
-of	O
-Ser453	O
-will	O
-result	O
-in	O
-steric	O
-clashes	O
-as	O
-well	O
-as	O
-electrostatic	O
-repulsion	O
-,	O
-which	O
-in	O
-turn	O
-might	O
-cause	O
-substantial	O
-conformational	O
-changes	O
-within	O
-the	O
-CTR	O
-.	O
-	O
-To	O
-test	O
-this	O
-hypothesis	O
-,	O
-we	O
-determined	O
-the	O
-structure	O
-of	O
-the	O
-phosphorylation	O
--	O
-mimicking	O
-R452D	B-mutant
-/	I-mutant
-S453D	I-mutant
-protein	O
-(	O
-hereafter	O
-termed	O
-‘	O
-DD	B-mutant
-mutant	I-mutant
-'),	O
-using	O
-data	O
-to	O
-a	O
-resolution	O
-of	O
-2	O
-.	O
-4	O
-Å	O
-.	O
-The	O
-additional	O
-mutation	O
-of	O
-the	O
-arginine	O
-preceding	O
-the	O
-phosphorylation	O
-site	O
-was	O
-introduced	O
-(	O
-i	O
-)	O
-to	O
-increase	O
-the	O
-negative	O
-charge	O
-density	O
-and	O
-make	O
-it	O
-more	O
-comparable	O
-to	O
-a	O
-phosphate	O
-at	O
-neutral	O
-pH	O
-,	O
-and	O
-(	O
-ii	O
-)	O
-to	O
-further	O
-destabilize	O
-the	O
-interactions	O
-of	O
-the	O
-AI	O
-region	O
-with	O
-the	O
-main	O
-body	O
-of	O
-the	O
-transporter	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-The	O
-ammonium	O
-uptake	O
-activity	O
-of	O
-the	O
-S	O
-.	O
-cerevisiae	O
-version	O
-of	O
-the	O
-DD	B-mutant
-mutant	I-mutant
-is	O
-the	O
-same	O
-as	O
-that	O
-of	O
-WT	O
-Mep2	O
-and	O
-the	O
-S453D	B-mutant
-mutant	O
-,	O
-indicating	O
-that	O
-the	O
-mutations	O
-do	O
-not	O
-affect	O
-transporter	O
-functionality	O
-in	O
-the	O
-triple	B-mutant
-mepΔ	I-mutant
-background	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-Unexpectedly	O
-,	O
-the	O
-AI	O
-segment	O
-containing	O
-the	O
-mutated	O
-residues	O
-has	O
-only	O
-undergone	O
-a	O
-slight	O
-shift	O
-compared	O
-with	O
-the	O
-WT	O
-protein	O
-(	O
-Fig	O
-.	O
-8	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-By	O
-contrast	O
-,	O
-the	O
-conserved	O
-part	O
-of	O
-the	O
-CTR	O
-has	O
-undergone	O
-a	O
-large	O
-conformational	O
-change	O
-involving	O
-formation	O
-of	O
-a	O
-12	O
--	O
-residue	O
--	O
-long	O
-α	O
--	O
-helix	O
-from	O
-Leu427	O
-to	O
-Asp438	O
-.	O
-	O
-In	O
-addition	O
-,	O
-residues	O
-Glu420	O
--	O
-Leu423	O
-including	O
-Glu421	O
-of	O
-the	O
-ExxGxD	O
-motif	O
-are	O
-now	O
-disordered	O
-(	O
-Fig	O
-.	O
-8	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-This	O
-is	O
-the	O
-first	O
-time	O
-a	O
-large	O
-conformational	O
-change	O
-has	O
-been	O
-observed	O
-in	O
-an	O
-ammonium	O
-transporter	O
-as	O
-a	O
-result	O
-of	O
-a	O
-mutation	O
-,	O
-and	O
-confirms	O
-previous	O
-hypotheses	O
-that	O
-phosphorylation	O
-causes	O
-structural	O
-changes	O
-in	O
-the	O
-CTR	O
-.	O
-	O
-To	O
-exclude	O
-the	O
-possibility	O
-that	O
-the	O
-additional	O
-R452D	B-mutant
-mutation	O
-is	O
-responsible	O
-for	O
-the	O
-observed	O
-changes	O
-,	O
-we	O
-also	O
-determined	O
-the	O
-structure	O
-of	O
-the	O
-‘	O
-single	B-mutant
-D	I-mutant
-'	O
-S453D	B-mutant
-mutant	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-4	O
-,	O
-the	O
-consequence	O
-of	O
-the	O
-single	B-mutant
-D	I-mutant
-mutation	O
-is	O
-very	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-DD	B-mutant
-substitution	I-mutant
-,	O
-with	O
-conformational	O
-changes	O
-and	O
-increased	O
-dynamics	O
-confined	O
-to	O
-the	O
-conserved	O
-part	O
-of	O
-the	O
-CTR	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-To	O
-supplement	O
-the	O
-crystal	O
-structures	O
-,	O
-we	O
-also	O
-performed	O
-modelling	O
-and	O
-MD	O
-studies	O
-of	O
-WT	O
-CaMep2	O
-,	O
-the	O
-DD	B-mutant
-mutant	I-mutant
-and	O
-phosphorylated	O
-protein	O
-(	O
-S453J	B-mutant
-).	O
-	O
-In	O
-the	O
-WT	O
-structure	O
-,	O
-the	O
-acidic	O
-residues	O
-Asp419	O
-,	O
-Glu420	O
-and	O
-Glu421	O
-are	O
-within	O
-hydrogen	O
-bonding	O
-distance	O
-of	O
-Ser453	O
-.	O
-	O
-After	O
-200	O
-ns	O
-of	O
-MD	O
-simulation	O
-,	O
-the	O
-interactions	O
-between	O
-these	O
-residues	O
-and	O
-Ser453	O
-remain	O
-intact	O
-.	O
-	O
-The	O
-protein	O
-backbone	O
-has	O
-an	O
-average	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.	O
-of	O
-only	O
-∼	O
-3	O
-Å	O
-during	O
-the	O
-200	O
--	O
-ns	O
-simulation	O
-,	O
-indicating	O
-that	O
-the	O
-protein	O
-is	O
-stable	O
-.	O
-	O
-There	O
-is	O
-flexibility	O
-in	O
-the	O
-side	O
-chains	O
-of	O
-the	O
-acidic	O
-residues	O
-so	O
-that	O
-they	O
-are	O
-able	O
-to	O
-form	O
-stable	O
-hydrogen	O
-bonds	O
-with	O
-Ser453	O
-.	O
-	O
-In	O
-particular	O
-,	O
-persistent	O
-hydrogen	O
-bonds	O
-are	O
-observed	O
-between	O
-the	O
-Ser453	O
-hydroxyl	O
-group	O
-and	O
-the	O
-acidic	O
-group	O
-of	O
-Glu420	O
-,	O
-and	O
-also	O
-between	O
-the	O
-amine	O
-group	O
-of	O
-Ser453	O
-and	O
-the	O
-backbone	O
-carbonyl	O
-of	O
-Glu420	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-The	O
-DD	B-mutant
-mutant	I-mutant
-is	O
-also	O
-stable	O
-during	O
-the	O
-simulations	O
-,	O
-but	O
-the	O
-average	O
-backbone	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-of	O
-∼	O
-3	O
-.	O
-6	O
-Å	O
-suggests	O
-slightly	O
-more	O
-conformational	O
-flexibility	O
-than	O
-WT	O
-.	O
-	O
-As	O
-the	O
-simulation	O
-proceeds	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-the	O
-acidic	O
-residues	O
-move	O
-away	O
-from	O
-Asp452	O
-and	O
-Asp453	O
-,	O
-presumably	O
-to	O
-avoid	O
-electrostatic	O
-repulsion	O
-.	O
-	O
-For	O
-example	O
-,	O
-the	O
-distance	O
-between	O
-the	O
-Asp453	O
-acidic	O
-oxygens	O
-and	O
-the	O
-Glu420	O
-acidic	O
-oxygens	O
-increases	O
-from	O
-∼	O
-7	O
-to	O
->	O
-22	O
-Å	O
-after	O
-200	O
-ns	O
-simulations	O
-,	O
-and	O
-thus	O
-these	O
-residues	O
-are	O
-not	O
-interacting	O
-.	O
-	O
-The	O
-protein	O
-is	O
-structurally	O
-stable	O
-throughout	O
-the	O
-simulation	O
-with	O
-little	O
-deviation	O
-in	O
-the	O
-other	O
-parts	O
-of	O
-the	O
-protein	O
-.	O
-	O
-Finally	O
-,	O
-the	O
-S453J	B-mutant
-mutant	O
-is	O
-also	O
-stable	O
-throughout	O
-the	O
-200	O
--	O
-ns	O
-simulation	O
-and	O
-has	O
-an	O
-average	O
-backbone	O
-deviation	O
-of	O
-∼	O
-3	O
-.	O
-8	O
-Å	O
-,	O
-which	O
-is	O
-similar	O
-to	O
-the	O
-DD	B-mutant
-mutant	I-mutant
-.	O
-	O
-The	O
-movement	O
-of	O
-the	O
-acidic	O
-residues	O
-away	O
-from	O
-Arg452	O
-and	O
-Sep453	O
-is	O
-more	O
-pronounced	O
-in	O
-this	O
-simulation	O
-in	O
-comparison	O
-with	O
-the	O
-movement	O
-away	O
-from	O
-Asp452	O
-and	O
-Asp453	O
-in	O
-the	O
-DD	B-mutant
-mutant	I-mutant
-.	O
-	O
-The	O
-distance	O
-between	O
-the	O
-phosphate	O
-of	O
-Sep453	O
-and	O
-the	O
-acidic	O
-oxygen	O
-atoms	O
-of	O
-Glu420	O
-is	O
-initially	O
-∼	O
-11	O
-Å	O
-,	O
-but	O
-increases	O
-to	O
->	O
-30	O
-Å	O
-after	O
-200	O
-ns	O
-.	O
-	O
-The	O
-short	O
-helix	O
-formed	O
-by	O
-residues	O
-Leu427	O
-to	O
-Asp438	O
-unravels	O
-during	O
-the	O
-simulations	O
-to	O
-a	O
-disordered	O
-state	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-MD	O
-simulations	O
-support	O
-the	O
-notion	O
-from	O
-the	O
-crystal	O
-structures	O
-that	O
-phosphorylation	O
-generates	O
-conformational	O
-changes	O
-in	O
-the	O
-conserved	O
-part	O
-of	O
-the	O
-CTR	O
-.	O
-	O
-However	O
-,	O
-the	O
-conformational	O
-changes	O
-for	O
-the	O
-phosphomimetic	B-mutant
-mutants	I-mutant
-in	O
-the	O
-crystals	O
-are	O
-confined	O
-to	O
-the	O
-CTR	O
-(	O
-Fig	O
-.	O
-8	O
-),	O
-and	O
-the	O
-channels	O
-are	O
-still	O
-closed	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-One	O
-possible	O
-explanation	O
-is	O
-that	O
-the	O
-mutants	B-mutant
-do	O
-not	O
-accurately	O
-mimic	O
-a	O
-phosphoserine	O
-,	O
-but	O
-the	O
-observation	O
-that	O
-the	O
-S453D	B-mutant
-and	O
-DD	B-mutant
-mutants	I-mutant
-are	O
-fully	O
-active	O
-in	O
-the	O
-absence	O
-of	O
-Npr1	O
-suggests	O
-that	O
-the	O
-mutations	O
-do	O
-mimic	O
-the	O
-effect	O
-of	O
-phosphorylation	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-The	O
-fact	O
-that	O
-the	O
-S453D	B-mutant
-structure	O
-was	O
-obtained	O
-in	O
-the	O
-presence	O
-of	O
-10	O
-mM	O
-ammonium	O
-ions	O
-suggests	O
-that	O
-the	O
-crystallization	O
-process	O
-favours	O
-closed	O
-states	O
-of	O
-the	O
-Mep2	O
-channels	O
-.	O
-	O
-Knowledge	O
-about	O
-ammonium	O
-transporter	O
-structure	O
-has	O
-been	O
-obtained	O
-from	O
-experimental	O
-and	O
-theoretical	O
-studies	O
-on	O
-bacterial	O
-family	O
-members	O
-.	O
-	O
-In	O
-addition	O
-,	O
-a	O
-number	O
-of	O
-biochemical	O
-and	O
-genetic	O
-studies	O
-are	O
-available	O
-for	O
-bacterial	O
-,	O
-fungal	O
-and	O
-plant	O
-proteins	O
-.	O
-	O
-These	O
-efforts	O
-have	O
-advanced	O
-our	O
-knowledge	O
-considerably	O
-but	O
-have	O
-not	O
-yet	O
-yielded	O
-atomic	O
--	O
-level	O
-answers	O
-to	O
-several	O
-important	O
-mechanistic	O
-questions	O
-,	O
-including	O
-how	O
-ammonium	O
-transport	O
-is	O
-regulated	O
-in	O
-eukaryotes	O
-and	O
-the	O
-mechanism	O
-of	O
-ammonium	O
-signalling	O
-.	O
-	O
-In	O
-Arabidopsis	O
-thaliana	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-,	O
-phosphorylation	O
-of	O
-the	O
-CTR	O
-residue	O
-T460	O
-under	O
-conditions	O
-of	O
-high	O
-ammonium	O
-inhibits	O
-transport	O
-activity	O
-,	O
-that	O
-is	O
-,	O
-the	O
-default	O
-(	O
-non	O
--	O
-phosphorylated	O
-)	O
-state	O
-of	O
-the	O
-plant	O
-transporter	O
-is	O
-open	O
-.	O
-	O
-Interestingly	O
-,	O
-phosphomimetic	B-mutant
-mutations	I-mutant
-introduced	O
-into	O
-one	O
-monomer	O
-inactivate	O
-the	O
-entire	O
-trimer	O
-,	O
-indicating	O
-that	O
-(	O
-i	O
-)	O
-heterotrimerization	O
-occurs	O
-and	O
-(	O
-ii	O
-)	O
-the	O
-CTR	O
-mediates	O
-allosteric	O
-regulation	O
-of	O
-ammonium	O
-transport	O
-activity	O
-via	O
-phosphorylation	O
-.	O
-	O
-Owing	O
-to	O
-the	O
-lack	O
-of	O
-structural	O
-information	O
-for	O
-plant	O
-AMTs	O
-,	O
-the	O
-details	O
-of	O
-channel	O
-closure	O
-and	O
-inter	O
--	O
-monomer	O
-crosstalk	O
-are	O
-not	O
-yet	O
-clear	O
-.	O
-	O
-Contrasting	O
-with	O
-the	O
-plant	O
-transporters	O
-,	O
-the	O
-inactive	O
-states	O
-of	O
-Mep2	O
-proteins	O
-under	O
-conditions	O
-of	O
-high	O
-ammonium	O
-are	O
-non	O
--	O
-phosphorylated	O
-,	O
-with	O
-channels	O
-that	O
-are	O
-closed	O
-on	O
-the	O
-cytoplasmic	O
-side	O
-.	O
-	O
-The	O
-reason	O
-why	O
-similar	O
-transporters	O
-such	O
-as	O
-A	O
-.	O
-thaliana	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-and	O
-Mep2	O
-are	O
-regulated	O
-in	O
-opposite	O
-ways	O
-by	O
-phosphorylation	O
-(	O
-inactivation	O
-in	O
-plants	O
-and	O
-activation	O
-in	O
-fungi	O
-)	O
-is	O
-not	O
-known	O
-.	O
-	O
-In	O
-fungi	O
-,	O
-preventing	O
-ammonium	O
-entry	O
-via	O
-channel	O
-closure	O
-in	O
-ammonium	O
-transporters	O
-would	O
-be	O
-one	O
-way	O
-to	O
-alleviate	O
-ammonium	O
-toxicity	O
-,	O
-in	O
-addition	O
-to	O
-ammonium	O
-excretion	O
-via	O
-Ato	O
-transporters	O
-and	O
-amino	O
--	O
-acid	O
-secretion	O
-.	O
-	O
-By	O
-determining	O
-the	O
-first	O
-structures	O
-of	O
-closed	O
-ammonium	O
-transporters	O
-and	O
-comparing	O
-those	O
-structures	O
-with	O
-the	O
-permanently	O
-open	O
-bacterial	O
-proteins	O
-,	O
-we	O
-demonstrate	O
-that	O
-Mep2	O
-channel	O
-closure	O
-is	O
-likely	O
-due	O
-to	O
-movements	O
-of	O
-the	O
-CTR	O
-and	O
-ICL1	O
-and	O
-ICL3	O
-.	O
-	O
-More	O
-specifically	O
-,	O
-the	O
-close	O
-interactions	O
-between	O
-the	O
-CTR	O
-and	O
-ICL1	O
-/	O
-ICL3	O
-present	O
-in	O
-open	O
-transporters	O
-are	O
-disrupted	O
-,	O
-causing	O
-ICL3	O
-to	O
-move	O
-outwards	O
-and	O
-block	O
-the	O
-channel	O
-(	O
-Figs	O
-4	O
-and	O
-9a	O
-).	O
-	O
-In	O
-addition	O
-,	O
-ICL1	O
-has	O
-shifted	O
-inwards	O
-to	O
-contribute	O
-to	O
-the	O
-channel	O
-closure	O
-by	O
-engaging	O
-His2	O
-from	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-via	O
-hydrogen	O
-bonding	O
-with	O
-a	O
-highly	O
-conserved	O
-tyrosine	O
-hydroxyl	O
-group	O
-.	O
-	O
-Upon	O
-phosphorylation	O
-by	O
-the	O
-Npr1	O
-kinase	O
-in	O
-response	O
-to	O
-nitrogen	O
-limitation	O
-,	O
-the	O
-region	O
-around	O
-the	O
-conserved	O
-ExxGxD	O
-motif	O
-undergoes	O
-a	O
-conformational	O
-change	O
-that	O
-opens	O
-the	O
-channel	O
-(	O
-Fig	O
-.	O
-9	O
-).	O
-	O
-Importantly	O
-,	O
-the	O
-structural	O
-similarities	O
-in	O
-the	O
-TM	O
-parts	O
-of	O
-Mep2	O
-and	O
-AfAmt	O
--	O
-1	O
-(	O
-Fig	O
-.	O
-5a	O
-)	O
-suggest	O
-that	O
-channel	O
-opening	O
-/	O
-closure	O
-does	O
-not	O
-require	O
-substantial	O
-changes	O
-in	O
-the	O
-residues	O
-lining	O
-the	O
-channel	O
-.	O
-	O
-How	O
-exactly	O
-the	O
-channel	O
-opens	O
-and	O
-whether	O
-opening	O
-is	O
-intra	O
--	O
-monomeric	O
-are	O
-still	O
-open	O
-questions	O
-;	O
-it	O
-is	O
-possible	O
-that	O
-the	O
-change	O
-in	O
-the	O
-CTR	O
-may	O
-disrupt	O
-its	O
-interactions	O
-with	O
-ICL3	O
-of	O
-the	O
-neighbouring	O
-monomer	O
-(	O
-Fig	O
-.	O
-9b	O
-),	O
-which	O
-could	O
-result	O
-in	O
-opening	O
-of	O
-the	O
-neighbouring	O
-channel	O
-via	O
-inward	O
-movement	O
-of	O
-its	O
-ICL3	O
-.	O
-	O
-Owing	O
-to	O
-the	O
-crosstalk	O
-between	O
-monomers	O
-,	O
-a	O
-single	O
-phosphorylation	O
-event	O
-might	O
-lead	O
-to	O
-opening	O
-of	O
-the	O
-entire	O
-trimer	O
-,	O
-although	O
-this	O
-has	O
-not	O
-yet	O
-been	O
-tested	O
-(	O
-Fig	O
-.	O
-9b	O
-).	O
-	O
-Whether	O
-or	O
-not	O
-Mep2	O
-channel	O
-opening	O
-requires	O
-,	O
-in	O
-addition	O
-to	O
-phosphorylation	O
-,	O
-disruption	O
-of	O
-the	O
-Tyr	O
-–	O
-His2	O
-interaction	O
-by	O
-the	O
-ammonium	O
-substrate	O
-is	O
-not	O
-yet	O
-clear	O
-.	O
-	O
-Is	O
-our	O
-model	O
-for	O
-opening	O
-and	O
-closing	O
-of	O
-Mep2	O
-channels	O
-valid	O
-for	O
-other	O
-eukaryotic	O
-ammonium	O
-transporters	O
-?	O
-Our	O
-structural	O
-data	O
-support	O
-previous	O
-studies	O
-and	O
-clarify	O
-the	O
-central	O
-role	O
-of	O
-the	O
-CTR	O
-and	O
-cytoplasmic	O
-loops	O
-in	O
-the	O
-transition	O
-between	O
-closed	O
-and	O
-open	O
-states	O
-.	O
-	O
-However	O
-,	O
-even	O
-the	O
-otherwise	O
-highly	O
-similar	O
-Mep2	O
-proteins	O
-of	O
-S	O
-.	O
-cerevisiae	O
-and	O
-C	O
-.	O
-albicans	O
-have	O
-different	O
-structures	O
-for	O
-their	O
-CTRs	O
-(	O
-Fig	O
-.	O
-1	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-In	O
-addition	O
-,	O
-the	O
-AI	O
-region	O
-of	O
-the	O
-CTR	O
-containing	O
-the	O
-Npr1	O
-kinase	O
-site	O
-is	O
-conserved	O
-in	O
-only	O
-a	O
-subset	O
-of	O
-fungal	O
-transporters	O
-,	O
-suggesting	O
-that	O
-the	O
-details	O
-of	O
-the	O
-structural	O
-changes	O
-underpinning	O
-regulation	O
-vary	O
-.	O
-	O
-Nevertheless	O
-,	O
-given	O
-the	O
-central	O
-role	O
-of	O
-absolutely	O
-conserved	O
-residues	O
-within	O
-the	O
-ICL1	O
--	O
-ICL3	O
--	O
-CTR	O
-interaction	O
-network	O
-(	O
-Fig	O
-.	O
-4	O
-),	O
-we	O
-propose	O
-that	O
-the	O
-structural	O
-basics	O
-of	O
-fungal	O
-ammonium	O
-transporter	O
-activation	O
-are	O
-conserved	O
-.	O
-	O
-The	O
-fact	O
-that	O
-Mep2	O
-orthologues	O
-of	O
-distantly	O
-related	O
-fungi	O
-are	O
-fully	O
-functional	O
-in	O
-ammonium	O
-transport	O
-and	O
-signalling	O
-in	O
-S	O
-.	O
-cerevisiae	O
-supports	O
-this	O
-notion	O
-.	O
-	O
-It	O
-should	O
-also	O
-be	O
-noted	O
-that	O
-the	O
-tyrosine	O
-residue	O
-interacting	O
-with	O
-His2	O
-is	O
-highly	O
-conserved	O
-in	O
-fungal	O
-Mep2	O
-orthologues	O
-,	O
-suggesting	O
-that	O
-the	O
-Tyr	O
-–	O
-His2	O
-hydrogen	O
-bond	O
-might	O
-be	O
-a	O
-general	O
-way	O
-to	O
-close	O
-Mep2	O
-proteins	O
-.	O
-	O
-With	O
-regards	O
-to	O
-plant	O
-AMTs	O
-,	O
-it	O
-has	O
-been	O
-proposed	O
-that	O
-phosphorylation	O
-at	O
-T460	O
-generates	O
-conformational	O
-changes	O
-that	O
-would	O
-close	O
-the	O
-neighbouring	O
-pore	O
-via	O
-the	O
-C	O
-terminus	O
-.	O
-	O
-This	O
-assumption	O
-was	O
-based	O
-partly	O
-on	O
-a	O
-homology	O
-model	O
-for	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-based	O
-on	O
-the	O
-(	O
-open	O
-)	O
-archaebacterial	O
-AfAmt	O
--	O
-1	O
-structure	O
-,	O
-which	O
-suggested	O
-that	O
-the	O
-C	O
-terminus	O
-of	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-would	O
-extend	O
-further	O
-to	O
-the	O
-neighbouring	O
-monomer	O
-.	O
-	O
-Our	O
-Mep2	O
-structures	O
-show	O
-that	O
-this	O
-assumption	O
-may	O
-not	O
-be	O
-correct	O
-(	O
-Fig	O
-.	O
-4	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-In	O
-addition	O
-,	O
-the	O
-considerable	O
-differences	O
-between	O
-structurally	O
-resolved	O
-CTR	O
-domains	O
-means	O
-that	O
-the	O
-exact	O
-environment	O
-of	O
-T460	O
-in	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-is	O
-also	O
-not	O
-known	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-Based	O
-on	O
-the	O
-available	O
-structural	O
-information	O
-,	O
-we	O
-consider	O
-it	O
-more	O
-likely	O
-that	O
-phosphorylation	O
--	O
-mediated	O
-pore	O
-closure	O
-in	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-is	O
-intra	O
--	O
-monomeric	O
-,	O
-via	O
-disruption	O
-of	O
-the	O
-interactions	O
-between	O
-the	O
-CTR	O
-and	O
-ICL1	O
-/	O
-ICL3	O
-(	O
-for	O
-example	O
-,	O
-Y467	O
--	O
-H239	O
-and	O
-D458	O
--	O
-K71	O
-).	O
-	O
-There	O
-is	O
-generally	O
-no	O
-equivalent	O
-for	O
-CaMep2	O
-Tyr49	O
-in	O
-plant	O
-AMTs	O
-,	O
-indicating	O
-that	O
-a	O
-Tyr	O
-–	O
-His2	O
-hydrogen	O
-bond	O
-as	O
-observed	O
-in	O
-Mep2	O
-may	O
-not	O
-contribute	O
-to	O
-the	O
-closed	O
-state	O
-in	O
-plant	O
-transporters	O
-.	O
-	O
-We	O
-propose	O
-that	O
-intra	O
--	O
-monomeric	O
-CTR	O
--	O
-ICL1	O
-/	O
-ICL3	O
-interactions	O
-lie	O
-at	O
-the	O
-basis	O
-of	O
-regulation	O
-of	O
-both	O
-fungal	O
-and	O
-plant	O
-ammonium	O
-transporters	O
-;	O
-close	O
-interactions	O
-generate	O
-open	O
-channels	O
-,	O
-whereas	O
-the	O
-lack	O
-of	O
-‘	O
-intra	O
--'	O
-interactions	O
-leads	O
-to	O
-inactive	O
-states	O
-.	O
-	O
-The	O
-need	O
-to	O
-regulate	O
-in	O
-opposite	O
-ways	O
-may	O
-be	O
-the	O
-reason	O
-why	O
-the	O
-phosphorylation	O
-sites	O
-are	O
-in	O
-different	O
-parts	O
-of	O
-the	O
-CTR	O
-,	O
-that	O
-is	O
-,	O
-centrally	O
-located	O
-close	O
-to	O
-the	O
-ExxGxD	O
-motif	O
-in	O
-AMTs	O
-and	O
-peripherally	O
-in	O
-Mep2	O
-.	O
-	O
-In	O
-this	O
-way	O
-,	O
-phosphorylation	O
-can	O
-either	O
-lead	O
-to	O
-channel	O
-closing	O
-(	O
-in	O
-the	O
-case	O
-of	O
-AMTs	O
-)	O
-or	O
-channel	O
-opening	O
-in	O
-the	O
-case	O
-of	O
-Mep2	O
-.	O
-	O
-Our	O
-model	O
-also	O
-provides	O
-an	O
-explanation	O
-for	O
-the	O
-observation	O
-that	O
-certain	B-mutant
-mutations	I-mutant
-within	O
-the	O
-CTR	O
-completely	O
-abolish	O
-transport	O
-activity	O
-.	O
-	O
-An	O
-example	O
-of	O
-an	O
-inactivating	O
-residue	O
-is	O
-the	O
-glycine	O
-of	O
-the	O
-ExxGxD	O
-motif	O
-of	O
-the	O
-CTR	O
-.	O
-	O
-Mutation	O
-of	O
-this	O
-residue	O
-(	O
-G393	O
-in	O
-EcAmtB	O
-;	O
-G456	O
-in	O
-AtAmt	O
--	O
-1	O
-;	O
-1	O
-)	O
-inactivates	O
-transporters	O
-as	O
-diverse	O
-as	O
-Escherichia	O
-coli	O
-AmtB	O
-and	O
-A	O
-.	O
-thaliana	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-(	O
-refs	O
-).	O
-	O
-Such	O
-mutations	O
-likely	O
-cause	O
-structural	O
-changes	O
-in	O
-the	O
-CTR	O
-that	O
-prevent	O
-close	O
-contacts	O
-between	O
-the	O
-CTR	O
-and	O
-ICL1	O
-/	O
-ICL3	O
-,	O
-thereby	O
-stabilizing	O
-a	O
-closed	O
-state	O
-that	O
-may	O
-be	O
-similar	O
-to	O
-that	O
-observed	O
-in	O
-Mep2	O
-.	O
-	O
-Regulation	O
-and	O
-modulation	O
-of	O
-membrane	O
-transport	O
-by	O
-phosphorylation	O
-is	O
-known	O
-to	O
-occur	O
-in	O
-,	O
-for	O
-example	O
-,	O
-aquaporins	O
-and	O
-urea	O
-transporters	O
-,	O
-and	O
-is	O
-likely	O
-to	O
-be	O
-a	O
-common	O
-theme	O
-for	O
-eukaryotic	O
-channels	O
-and	O
-transporters	O
-.	O
-	O
-Recently	O
-,	O
-phosphorylation	O
-was	O
-also	O
-shown	O
-to	O
-modulate	O
-substrate	O
-affinity	O
-in	O
-nitrate	O
-transporters	O
-.	O
-	O
-With	O
-respect	O
-to	O
-ammonium	O
-transport	O
-,	O
-phosphorylation	O
-has	O
-thus	O
-far	O
-only	O
-been	O
-shown	O
-for	O
-A	O
-.	O
-thaliana	O
-AMTs	O
-and	O
-for	O
-S	O
-.	O
-cerevisiae	O
-Mep2	O
-(	O
-refs	O
-).	O
-	O
-However	O
-,	O
-the	O
-absence	O
-of	O
-GlnK	O
-proteins	O
-in	O
-eukaryotes	O
-suggests	O
-that	O
-phosphorylation	O
--	O
-based	O
-regulation	O
-of	O
-ammonium	O
-transport	O
-may	O
-be	O
-widespread	O
-.	O
-	O
-With	O
-respect	O
-to	O
-Mep2	O
--	O
-mediated	O
-signalling	O
-to	O
-induce	O
-pseudohyphal	O
-growth	O
-,	O
-two	O
-models	O
-have	O
-been	O
-put	O
-forward	O
-as	O
-to	O
-how	O
-this	O
-occurs	O
-and	O
-why	O
-it	O
-is	O
-specific	O
-to	O
-Mep2	O
-proteins	O
-.	O
-	O
-In	O
-one	O
-model	O
-,	O
-signalling	O
-is	O
-proposed	O
-to	O
-depend	O
-on	O
-the	O
-nature	O
-of	O
-the	O
-transported	O
-substrate	O
-,	O
-which	O
-might	O
-be	O
-different	O
-in	O
-certain	O
-subfamilies	O
-of	O
-ammonium	O
-transporters	O
-(	O
-for	O
-example	O
-,	O
-Mep1	O
-/	O
-Mep3	O
-versus	O
-Mep2	O
-).	O
-	O
-For	O
-example	O
-,	O
-NH3	O
-uniport	O
-or	O
-symport	O
-of	O
-NH3	O
-/	O
-H	O
-+	O
-might	O
-result	O
-in	O
-changes	O
-in	O
-local	O
-pH	O
-,	O
-but	O
-NH4	O
-+	O
-uniport	O
-might	O
-not	O
-,	O
-and	O
-this	O
-difference	O
-might	O
-determine	O
-signalling	O
-.	O
-	O
-In	O
-the	O
-other	O
-model	O
-,	O
-signalling	O
-is	O
-thought	O
-to	O
-require	O
-a	O
-distinct	O
-conformation	O
-of	O
-the	O
-Mep2	O
-transporter	O
-occurring	O
-during	O
-the	O
-transport	O
-cycle	O
-.	O
-	O
-While	O
-the	O
-current	O
-study	O
-does	O
-not	O
-specifically	O
-address	O
-the	O
-mechanism	O
-of	O
-signalling	O
-underlying	O
-pseudohyphal	O
-growth	O
-,	O
-our	O
-structures	O
-do	O
-show	O
-that	O
-Mep2	O
-proteins	O
-can	O
-assume	O
-different	O
-conformations	O
-.	O
-	O
-It	O
-is	O
-clear	O
-that	O
-ammonium	O
-transport	O
-across	O
-biomembranes	O
-remains	O
-a	O
-fascinating	O
-and	O
-challenging	O
-field	O
-in	O
-large	O
-part	O
-due	O
-to	O
-the	O
-unique	O
-properties	O
-of	O
-the	O
-substrate	O
-.	O
-	O
-Our	O
-Mep2	O
-structural	O
-work	O
-now	O
-provides	O
-a	O
-foundation	O
-for	O
-future	O
-studies	O
-to	O
-uncover	O
-the	O
-details	O
-of	O
-the	O
-structural	O
-changes	O
-that	O
-occur	O
-during	O
-eukaryotic	O
-ammonium	O
-transport	O
-and	O
-signaling	O
-,	O
-and	O
-to	O
-assess	O
-the	O
-possibility	O
-to	O
-utilize	O
-small	O
-molecules	O
-to	O
-shut	O
-down	O
-ammonium	O
-sensing	O
-and	O
-downstream	O
-signalling	O
-pathways	O
-in	O
-pathogenic	O
-fungi	O
-.	O
-	O
-X	O
--	O
-ray	O
-crystal	O
-structures	O
-of	O
-Mep2	O
-transceptors	O
-.	O
-	O
-(	O
-a	O
-)	O
-Monomer	O
-cartoon	O
-models	O
-viewed	O
-from	O
-the	O
-side	O
-for	O
-(	O
-left	O
-)	O
-A	O
-.	O
-fulgidus	O
-Amt	O
--	O
-1	O
-(	O
-PDB	O
-ID	O
-2B2H	O
-),	O
-S	O
-.	O
-cerevisiae	O
-Mep2	O
-(	O
-middle	O
-)	O
-and	O
-C	O
-.	O
-albicans	O
-Mep2	O
-(	O
-right	O
-).	O
-	O
-The	O
-region	O
-showing	O
-ICL1	O
-(	O
-blue	O
-),	O
-ICL3	O
-(	O
-green	O
-)	O
-and	O
-the	O
-CTR	O
-(	O
-red	O
-)	O
-is	O
-boxed	O
-for	O
-comparison	O
-.	O
-	O
-(	O
-b	O
-)	O
-CaMep2	O
-trimer	O
-viewed	O
-from	O
-the	O
-intracellular	O
-side	O
-(	O
-right	O
-).	O
-	O
-One	O
-monomer	O
-is	O
-coloured	O
-as	O
-in	O
-a	O
-and	O
-one	O
-monomer	O
-is	O
-coloured	O
-by	O
-B	O
--	O
-factor	O
-(	O
-blue	O
-,	O
-low	O
-;	O
-red	O
-;	O
-high	O
-).	O
-	O
-The	O
-CTR	O
-is	O
-boxed	O
-.	O
-	O
-(	O
-c	O
-)	O
-Overlay	O
-of	O
-ScMep2	O
-(	O
-grey	O
-)	O
-and	O
-CaMep2	O
-(	O
-rainbow	O
-),	O
-illustrating	O
-the	O
-differences	O
-in	O
-the	O
-CTRs	O
-.	O
-	O
-Sequence	O
-conservation	O
-in	O
-ammonium	O
-transporters	O
-.	O
-	O
-ClustalW	O
-alignment	O
-of	O
-CaMep2	O
-,	O
-ScMep2	O
-,	O
-A	O
-.	O
-fulgidus	O
-Amt	O
--	O
-1	O
-,	O
-E	O
-.	O
-coli	O
-AmtB	O
-and	O
-A	O
-.	O
-thaliana	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-.	O
-	O
-The	O
-secondary	O
-structure	O
-elements	O
-observed	O
-for	O
-CaMep2	O
-are	O
-indicated	O
-,	O
-with	O
-the	O
-numbers	O
-corresponding	O
-to	O
-the	O
-centre	O
-of	O
-the	O
-TM	O
-segment	O
-.	O
-	O
-The	O
-conserved	O
-RxK	O
-motif	O
-in	O
-ICL1	O
-is	O
-boxed	O
-in	O
-blue	O
-,	O
-the	O
-ER	O
-motif	O
-in	O
-ICL2	O
-in	O
-cyan	O
-,	O
-the	O
-conserved	O
-ExxGxD	O
-motif	O
-of	O
-the	O
-CTR	O
-in	O
-red	O
-and	O
-the	O
-AI	O
-region	O
-in	O
-yellow	O
-.	O
-	O
-Coloured	O
-residues	O
-are	O
-functionally	O
-important	O
-and	O
-correspond	O
-to	O
-those	O
-of	O
-the	O
-Phe	O
-gate	O
-(	O
-blue	O
-),	O
-the	O
-binding	O
-site	O
-Trp	O
-residue	O
-(	O
-magenta	O
-)	O
-and	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-(	O
-red	O
-).	O
-	O
-The	O
-Npr1	O
-kinase	O
-site	O
-in	O
-the	O
-AI	O
-region	O
-is	O
-highlighted	O
-pink	O
-.	O
-	O
-The	O
-grey	O
-sequences	O
-at	O
-the	O
-C	O
-termini	O
-of	O
-CaMep2	O
-and	O
-ScMep2	O
-are	O
-not	O
-visible	O
-in	O
-the	O
-structures	O
-and	O
-are	O
-likely	O
-disordered	O
-.	O
-	O
-Growth	O
-of	O
-ScMep2	B-mutant
-variants	I-mutant
-on	O
-low	O
-ammonium	O
-medium	O
-.	O
-	O
-(	O
-a	O
-)	O
-The	O
-triple	B-mutant
-mepΔ	I-mutant
-strain	O
-(	O
-black	O
-)	O
-and	O
-triple	O
-mepΔ	O
-npr1Δ	O
-strain	O
-(	O
-grey	O
-)	O
-containing	O
-plasmids	O
-expressing	O
-WT	O
-and	O
-variant	B-mutant
-ScMep2	I-mutant
-were	O
-grown	O
-on	O
-minimal	O
-medium	O
-containing	O
-1	O
-mM	O
-ammonium	O
-sulphate	O
-.	O
-	O
-The	O
-quantified	O
-cell	O
-density	O
-reflects	O
-logarithmic	O
-growth	O
-after	O
-24	O
-h	O
-.	O
-Error	O
-bars	O
-are	O
-the	O
-s	O
-.	O
-d	O
-.	O
-for	O
-three	O
-replicates	O
-of	O
-each	O
-strain	O
-(	O
-b	O
-)	O
-The	O
-strains	O
-used	O
-in	O
-a	O
-were	O
-also	O
-serially	O
-diluted	O
-and	O
-spotted	O
-onto	O
-minimal	O
-agar	O
-plates	O
-containing	O
-glutamate	O
-(	O
-0	O
-.	O
-1	O
-%)	O
-or	O
-ammonium	O
-sulphate	O
-(	O
-1	O
-mM	O
-),	O
-and	O
-grown	O
-for	O
-3	O
-days	O
-at	O
-30	O
-°	O
-C	O
-.	O
-	O
-Structural	O
-differences	O
-between	O
-Mep2	O
-and	O
-bacterial	O
-ammonium	O
-transporters	O
-.	O
-	O
-(	O
-a	O
-)	O
-ICL1	O
-in	O
-AfAmt	O
--	O
-1	O
-(	O
-light	O
-blue	O
-)	O
-and	O
-CaMep2	O
-(	O
-dark	O
-blue	O
-),	O
-showing	O
-unwinding	O
-and	O
-inward	O
-movement	O
-in	O
-the	O
-fungal	O
-protein	O
-.	O
-(	O
-b	O
-)	O
-Stereo	O
-diagram	O
-viewed	O
-from	O
-the	O
-cytosol	O
-of	O
-ICL1	O
-,	O
-ICL3	O
-(	O
-green	O
-)	O
-and	O
-the	O
-CTR	O
-(	O
-red	O
-)	O
-in	O
-AfAmt	O
--	O
-1	O
-(	O
-light	O
-colours	O
-)	O
-and	O
-CaMep2	O
-(	O
-dark	O
-colours	O
-).	O
-	O
-The	O
-side	O
-chains	O
-of	O
-residues	O
-in	O
-the	O
-RxK	O
-motif	O
-as	O
-well	O
-as	O
-those	O
-of	O
-Tyr49	O
-and	O
-His342	O
-are	O
-labelled	O
-.	O
-	O
-The	O
-numbering	O
-is	O
-for	O
-CaMep2	O
-.	O
-	O
-(	O
-c	O
-)	O
-Conserved	O
-residues	O
-in	O
-ICL1	O
--	O
-3	O
-and	O
-the	O
-CTR	O
-.	O
-	O
-Views	O
-from	O
-the	O
-cytosol	O
-for	O
-CaMep2	O
-(	O
-left	O
-)	O
-and	O
-AfAmt	O
--	O
-1	O
-,	O
-highlighting	O
-the	O
-large	O
-differences	O
-in	O
-conformation	O
-of	O
-the	O
-conserved	O
-residues	O
-in	O
-ICL1	O
-(	O
-RxK	O
-motif	O
-;	O
-blue	O
-),	O
-ICL2	O
-(	O
-ER	O
-motif	O
-;	O
-cyan	O
-),	O
-ICL3	O
-(	O
-green	O
-)	O
-and	O
-the	O
-CTR	O
-(	O
-red	O
-).	O
-	O
-The	O
-labelled	O
-residues	O
-are	O
-analogous	O
-within	O
-both	O
-structures	O
-.	O
-	O
-In	O
-b	O
-and	O
-c	O
-,	O
-the	O
-centre	O
-of	O
-the	O
-trimer	O
-is	O
-at	O
-top	O
-.	O
-	O
-Channel	O
-closures	O
-in	O
-Mep2	O
-.	O
-	O
-(	O
-a	O
-)	O
-Stereo	O
-superposition	O
-of	O
-AfAmt	O
--	O
-1	O
-and	O
-CaMep2	O
-showing	O
-the	O
-residues	O
-of	O
-the	O
-Phe	O
-gate	O
-,	O
-His2	O
-of	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-and	O
-the	O
-tyrosine	O
-residue	O
-Y49	O
-in	O
-TM1	O
-that	O
-forms	O
-a	O
-hydrogen	O
-bond	O
-with	O
-His2	O
-in	O
-CaMep2	O
-.	O
-(	O
-b	O
-)	O
-Surface	O
-views	O
-from	O
-the	O
-side	O
-in	O
-rainbow	O
-colouring	O
-,	O
-showing	O
-the	O
-two	O
--	O
-tier	O
-channel	O
-block	O
-(	O
-indicated	O
-by	O
-the	O
-arrows	O
-)	O
-in	O
-CaMep2	O
-.	O
-	O
-The	O
-Npr1	O
-kinase	O
-target	O
-Ser453	O
-is	O
-dephosphorylated	O
-and	O
-located	O
-in	O
-an	O
-electronegative	O
-pocket	O
-.	O
-	O
-(	O
-a	O
-)	O
-Stereoviews	O
-of	O
-CaMep2	O
-showing	O
-2Fo	O
-–	O
-Fc	O
-electron	O
-density	O
-(	O
-contoured	O
-at	O
-1	O
-.	O
-0	O
-σ	O
-)	O
-for	O
-CTR	O
-residues	O
-Asp419	O
--	O
-Met422	O
-and	O
-for	O
-Tyr446	O
--	O
-Thr455	O
-of	O
-the	O
-AI	O
-region	O
-.	O
-	O
-The	O
-phosphorylation	O
-target	O
-residue	O
-Ser453	O
-is	O
-labelled	O
-in	O
-bold	O
-.	O
-	O
-(	O
-b	O
-)	O
-Overlay	O
-of	O
-the	O
-CTRs	O
-of	O
-ScMep2	O
-(	O
-grey	O
-)	O
-and	O
-CaMep2	O
-(	O
-green	O
-),	O
-showing	O
-the	O
-similar	O
-electronegative	O
-environment	O
-surrounding	O
-the	O
-phosphorylation	O
-site	O
-(	O
-P	O
-).	O
-	O
-The	O
-AI	O
-regions	O
-are	O
-coloured	O
-magenta	O
-.	O
-	O
-(	O
-c	O
-)	O
-Cytoplasmic	O
-view	O
-of	O
-the	O
-Mep2	O
-trimer	O
-indicating	O
-the	O
-large	O
-distance	O
-between	O
-Ser453	O
-and	O
-the	O
-channel	O
-exits	O
-(	O
-circles	O
-;	O
-Ile52	O
-lining	O
-the	O
-channel	O
-exit	O
-is	O
-shown	O
-).	O
-	O
-Effect	O
-of	O
-removal	O
-of	O
-the	O
-AI	O
-region	O
-on	O
-Mep2	O
-structure	O
-.	O
-	O
-(	O
-a	O
-)	O
-Side	O
-views	O
-for	O
-WT	O
-CaMep2	O
-(	O
-left	O
-)	O
-and	O
-the	O
-truncation	O
-mutant	O
-442Δ	B-mutant
-(	O
-right	O
-).	O
-	O
-The	O
-latter	O
-is	O
-shown	O
-as	O
-a	O
-putty	O
-model	O
-according	O
-to	O
-B	O
--	O
-factors	O
-to	O
-illustrate	O
-the	O
-disorder	O
-in	O
-the	O
-protein	O
-on	O
-the	O
-cytoplasmic	O
-side	O
-.	O
-	O
-Missing	O
-regions	O
-are	O
-labelled	O
-.	O
-(	O
-b	O
-)	O
-Stereo	O
-superpositions	O
-of	O
-WT	O
-CaMep2	O
-and	O
-the	O
-truncation	O
-mutant	O
-.	O
-	O
-2Fo	O
-–	O
-Fc	O
-electron	O
-density	O
-(	O
-contoured	O
-at	O
-1	O
-.	O
-0	O
-σ	O
-)	O
-for	O
-residues	O
-Tyr49	O
-and	O
-His342	O
-is	O
-shown	O
-for	O
-the	O
-truncation	O
-mutant	O
-.	O
-	O
-Phosphorylation	O
-causes	O
-conformational	O
-changes	O
-in	O
-the	O
-CTR	O
-.	O
-	O
-(	O
-a	O
-)	O
-Cytoplasmic	O
-view	O
-of	O
-the	O
-DD	B-mutant
-mutant	I-mutant
-trimer	O
-,	O
-with	O
-WT	O
-CaMep2	O
-superposed	O
-in	O
-grey	O
-for	O
-one	O
-of	O
-the	O
-monomers	O
-.	O
-	O
-The	O
-arrow	O
-indicates	O
-the	O
-phosphorylation	O
-site	O
-.	O
-	O
-The	O
-AI	O
-region	O
-is	O
-coloured	O
-magenta	O
-.	O
-	O
-(	O
-b	O
-)	O
-Monomer	O
-side	O
--	O
-view	O
-superposition	O
-of	O
-WT	O
-CaMep2	O
-and	O
-the	O
-DD	B-mutant
-mutant	I-mutant
-,	O
-showing	O
-the	O
-conformational	O
-change	O
-and	O
-disorder	O
-around	O
-the	O
-ExxGxD	O
-motif	O
-.	O
-	O
-Side	O
-chains	O
-for	O
-residues	O
-452	O
-and	O
-453	O
-are	O
-shown	O
-as	O
-stick	O
-models	O
-.	O
-	O
-Schematic	O
-model	O
-for	O
-phosphorylation	O
--	O
-based	O
-regulation	O
-of	O
-Mep2	O
-ammonium	O
-transporters	O
-.	O
-	O
-(	O
-a	O
-)	O
-In	O
-the	O
-closed	O
-,	O
-non	O
--	O
-phosphorylated	O
-state	O
-(	O
-i	O
-),	O
-the	O
-CTR	O
-(	O
-magenta	O
-)	O
-and	O
-ICL3	O
-(	O
-green	O
-)	O
-are	O
-far	O
-apart	O
-with	O
-the	O
-latter	O
-blocking	O
-the	O
-intracellular	O
-channel	O
-exit	O
-(	O
-indicated	O
-with	O
-a	O
-hatched	O
-circle	O
-).	O
-	O
-Upon	O
-phosphorylation	O
-and	O
-mimicked	O
-by	O
-the	O
-CaMep2	O
-S453D	B-mutant
-and	O
-DD	B-mutant
-mutants	I-mutant
-(	O
-ii	O
-),	O
-the	O
-region	O
-around	O
-the	O
-ExxGxD	O
-motif	O
-undergoes	O
-a	O
-conformational	O
-change	O
-that	O
-results	O
-in	O
-the	O
-CTR	O
-interacting	O
-with	O
-the	O
-inward	O
--	O
-moving	O
-ICL3	O
-,	O
-opening	O
-the	O
-channel	O
-(	O
-full	O
-circle	O
-)	O
-(	O
-iii	O
-).	O
-	O
-The	O
-open	O
--	O
-channel	O
-Mep2	O
-structure	O
-is	O
-represented	O
-by	O
-archaebacterial	O
-Amt	O
--	O
-1	O
-and	O
-shown	O
-in	O
-lighter	O
-colours	O
-consistent	O
-with	O
-Fig	O
-.	O
-4	O
-.	O
-	O
-As	O
-discussed	O
-in	O
-the	O
-text	O
-,	O
-similar	O
-structural	O
-arrangements	O
-may	O
-occur	O
-in	O
-plant	O
-AMTs	O
-.	O
-	O
-In	O
-this	O
-case	O
-however	O
-,	O
-the	O
-open	O
-channel	O
-corresponds	O
-to	O
-the	O
-non	O
--	O
-phosphorylated	O
-state	O
-;	O
-phosphorylation	O
-breaks	O
-the	O
-CTR	O
-–	O
-ICL3	O
-interactions	O
-leading	O
-to	O
-channel	O
-closure	O
-.	O
-(	O
-b	O
-)	O
-Model	O
-based	O
-on	O
-AMT	O
-transporter	O
-analogy	O
-showing	O
-how	O
-phosphorylation	O
-of	O
-a	O
-Mep2	O
-monomer	O
-might	O
-allosterically	O
-open	O
-channels	O
-in	O
-the	O
-entire	O
-trimer	O
-via	O
-disruption	O
-of	O
-the	O
-interactions	O
-between	O
-the	O
-CTR	O
-and	O
-ICL3	O
-of	O
-a	O
-neighbouring	O
-monomer	O
-(	O
-arrow	O
-).	O
-	O
-Structural	O
-diversity	O
-in	O
-a	O
-human	O
-antibody	O
-germline	O
-library	O
-	O
-To	O
-support	O
-antibody	O
-therapeutic	O
-development	O
-,	O
-the	O
-crystal	O
-structures	O
-of	O
-a	O
-set	O
-of	O
-16	O
-germline	O
-variants	O
-composed	O
-of	O
-4	O
-different	O
-kappa	O
-light	O
-chains	O
-paired	O
-with	O
-4	O
-different	O
-heavy	O
-chains	O
-have	O
-been	O
-determined	O
-.	O
-	O
-All	O
-four	O
-heavy	O
-chains	O
-of	O
-the	O
-antigen	O
--	O
-binding	O
-fragments	O
-(	O
-Fabs	O
-)	O
-have	O
-the	O
-same	O
-complementarity	O
--	O
-determining	O
-region	O
-(	O
-CDR	O
-)	O
-H3	O
-that	O
-was	O
-reported	O
-in	O
-an	O
-earlier	O
-Fab	O
-structure	O
-.	O
-	O
-The	O
-structure	O
-analyses	O
-include	O
-comparisons	O
-of	O
-the	O
-overall	O
-structures	O
-,	O
-canonical	O
-structures	O
-of	O
-the	O
-CDRs	O
-and	O
-the	O
-VH	O
-:	O
-VL	O
-packing	O
-interactions	O
-.	O
-	O
-The	O
-CDR	O
-conformations	O
-for	O
-the	O
-most	O
-part	O
-are	O
-tightly	O
-clustered	O
-,	O
-especially	O
-for	O
-the	O
-ones	O
-with	O
-shorter	O
-lengths	O
-.	O
-	O
-The	O
-longer	O
-CDRs	O
-with	O
-tandem	O
-glycines	O
-or	O
-serines	O
-have	O
-more	O
-conformational	O
-diversity	O
-than	O
-the	O
-others	O
-.	O
-	O
-CDR	O
-H3	O
-,	O
-despite	O
-having	O
-the	O
-same	O
-amino	O
-acid	O
-sequence	O
-,	O
-exhibits	O
-the	O
-largest	O
-conformational	O
-diversity	O
-.	O
-	O
-About	O
-half	O
-of	O
-the	O
-structures	O
-have	O
-CDR	O
-H3	O
-conformations	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-parent	O
-;	O
-the	O
-others	O
-diverge	O
-significantly	O
-.	O
-	O
-One	O
-conclusion	O
-is	O
-that	O
-the	O
-CDR	O
-H3	O
-conformations	O
-are	O
-influenced	O
-by	O
-both	O
-their	O
-amino	O
-acid	O
-sequence	O
-and	O
-their	O
-structural	O
-environment	O
-determined	O
-by	O
-the	O
-heavy	O
-and	O
-light	O
-chain	O
-pairing	O
-.	O
-	O
-The	O
-stem	O
-regions	O
-of	O
-14	O
-of	O
-the	O
-variant	O
-pairs	O
-are	O
-in	O
-the	O
-‘	O
-kinked	O
-’	O
-conformation	O
-,	O
-and	O
-only	O
-2	O
-are	O
-in	O
-the	O
-extended	O
-conformation	O
-.	O
-	O
-The	O
-packing	O
-of	O
-the	O
-VH	O
-and	O
-VL	O
-domains	O
-is	O
-consistent	O
-with	O
-our	O
-knowledge	O
-of	O
-antibody	O
-structure	O
-,	O
-and	O
-the	O
-tilt	O
-angles	O
-between	O
-these	O
-domains	O
-cover	O
-a	O
-range	O
-of	O
-11	O
-degrees	O
-.	O
-	O
-Two	O
-of	O
-16	O
-structures	O
-showed	O
-particularly	O
-large	O
-variations	O
-in	O
-the	O
-tilt	O
-angles	O
-when	O
-compared	O
-with	O
-the	O
-other	O
-pairings	O
-.	O
-	O
-The	O
-structures	O
-and	O
-their	O
-analyses	O
-provide	O
-a	O
-rich	O
-foundation	O
-for	O
-future	O
-antibody	O
-modeling	O
-and	O
-engineering	O
-efforts	O
-.	O
-	O
-At	O
-present	O
-,	O
-therapeutic	O
-antibodies	O
-are	O
-the	O
-largest	O
-class	O
-of	O
-biotherapeutic	O
-proteins	O
-that	O
-are	O
-in	O
-clinical	O
-trials	O
-.	O
-	O
-The	O
-use	O
-of	O
-monoclonal	O
-antibodies	O
-as	O
-therapeutics	O
-began	O
-in	O
-the	O
-early	O
-1980s	O
-,	O
-and	O
-their	O
-composition	O
-has	O
-transitioned	O
-from	O
-murine	O
-antibodies	O
-to	O
-generally	O
-less	O
-immunogenic	O
-humanized	O
-and	O
-human	O
-antibodies	O
-.	O
-	O
-The	O
-technologies	O
-currently	O
-used	O
-to	O
-obtain	O
-human	O
-antibodies	O
-include	O
-transgenic	O
-mice	O
-containing	O
-human	O
-antibody	O
-repertoires	O
-,	O
-cloning	O
-directly	O
-from	O
-human	O
-B	O
-cells	O
-,	O
-and	O
-in	O
-vitro	O
-selection	O
-from	O
-antibody	O
-libraries	O
-using	O
-various	O
-display	O
-technologies	O
-.	O
-	O
-Once	O
-a	O
-candidate	O
-antibody	O
-is	O
-identified	O
-,	O
-protein	O
-engineering	O
-is	O
-usually	O
-required	O
-to	O
-produce	O
-a	O
-molecule	O
-with	O
-the	O
-right	O
-biophysical	O
-and	O
-functional	O
-properties	O
-.	O
-	O
-All	O
-engineering	O
-efforts	O
-are	O
-guided	O
-by	O
-our	O
-understanding	O
-of	O
-the	O
-atomic	O
-structures	O
-of	O
-antibodies	O
-.	O
-	O
-In	O
-such	O
-efforts	O
-,	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-specific	O
-antibody	O
-may	O
-not	O
-be	O
-available	O
-,	O
-but	O
-modeling	O
-can	O
-be	O
-used	O
-to	O
-guide	O
-the	O
-engineering	O
-efforts	O
-.	O
-	O
-Today	O
-'	O
-s	O
-antibody	O
-modeling	O
-approaches	O
-,	O
-which	O
-normally	O
-focus	O
-on	O
-the	O
-variable	O
-region	O
-,	O
-are	O
-being	O
-developed	O
-by	O
-the	O
-application	O
-of	O
-structural	O
-principles	O
-and	O
-insights	O
-that	O
-are	O
-evolving	O
-as	O
-our	O
-knowledge	O
-of	O
-antibody	O
-structures	O
-continues	O
-to	O
-expand	O
-.	O
-	O
-Our	O
-current	O
-structural	O
-knowledge	O
-of	O
-antibodies	O
-is	O
-based	O
-on	O
-a	O
-multitude	O
-of	O
-studies	O
-that	O
-used	O
-many	O
-techniques	O
-to	O
-gain	O
-insight	O
-into	O
-the	O
-functional	O
-and	O
-structural	O
-properties	O
-of	O
-this	O
-class	O
-of	O
-macromolecule	O
-.	O
-	O
-Five	O
-different	O
-antibody	O
-isotypes	O
-occur	O
-,	O
-IgG	O
-,	O
-IgD	O
-,	O
-IgE	O
-,	O
-IgA	O
-and	O
-IgM	O
-,	O
-and	O
-each	O
-isotype	O
-has	O
-a	O
-unique	O
-role	O
-in	O
-the	O
-adaptive	O
-immune	O
-system	O
-.	O
-	O
-IgG	O
-,	O
-IgD	O
-and	O
-IgE	O
-isotypes	O
-are	O
-composed	O
-of	O
-2	O
-heavy	O
-chains	O
-(	O
-HCs	O
-)	O
-and	O
-2	O
-light	O
-chains	O
-(	O
-LCs	O
-)	O
-linked	O
-through	O
-disulfide	O
-bonds	O
-,	O
-while	O
-IgA	O
-and	O
-IgM	O
-are	O
-double	O
-and	O
-quintuple	O
-versions	O
-of	O
-antibodies	O
-,	O
-respectively	O
-.	O
-	O
-Isotypes	O
-IgG	O
-,	O
-IgD	O
-and	O
-IgA	O
-each	O
-have	O
-4	O
-domains	O
-,	O
-one	O
-variable	O
-(	O
-V	O
-)	O
-and	O
-3	O
-constant	O
-(	O
-C	O
-)	O
-domains	O
-,	O
-while	O
-IgE	O
-and	O
-IgM	O
-each	O
-have	O
-the	O
-same	O
-4	O
-domains	O
-along	O
-with	O
-an	O
-additional	O
-C	O
-domain	O
-.	O
-	O
-These	O
-multimeric	O
-forms	O
-are	O
-linked	O
-with	O
-an	O
-additional	O
-J	O
-chain	O
-.	O
-	O
-The	O
-LCs	O
-that	O
-associate	O
-with	O
-the	O
-HCs	O
-are	O
-divided	O
-into	O
-2	O
-functionally	O
-indistinguishable	O
-classes	O
-,	O
-κ	O
-and	O
-λ	O
-.	O
-	O
-Both	O
-κ	O
-and	O
-λ	O
-polypeptide	O
-chains	O
-are	O
-composed	O
-of	O
-a	O
-single	O
-V	O
-domain	O
-and	O
-a	O
-single	O
-C	O
-domain	O
-.	O
-	O
-The	O
-heavy	O
-and	O
-light	O
-chains	O
-are	O
-composed	O
-of	O
-structural	O
-domains	O
-that	O
-have	O
-∼	O
-110	O
-amino	O
-acid	O
-residues	O
-.	O
-	O
-These	O
-domains	O
-have	O
-a	O
-common	O
-folding	O
-pattern	O
-often	O
-referred	O
-to	O
-as	O
-the	O
-“	O
-immunoglobulin	O
-fold	O
-,”	O
-formed	O
-by	O
-the	O
-packing	O
-together	O
-of	O
-2	O
-anti	O
--	O
-parallel	O
-β	O
--	O
-sheets	O
-.	O
-	O
-All	O
-immunoglobulin	O
-chains	O
-have	O
-an	O
-N	O
--	O
-terminal	O
-V	O
-domain	O
-followed	O
-by	O
-1	O
-to	O
-4	O
-C	O
-domains	O
-,	O
-depending	O
-upon	O
-the	O
-chain	O
-type	O
-.	O
-	O
-In	O
-antibodies	O
-,	O
-the	O
-heavy	O
-and	O
-light	O
-chain	O
-V	O
-domains	O
-pack	O
-together	O
-forming	O
-the	O
-antigen	O
-combining	O
-site	O
-.	O
-	O
-This	O
-site	O
-,	O
-which	O
-interacts	O
-with	O
-the	O
-antigen	O
-(	O
-or	O
-target	O
-),	O
-is	O
-the	O
-focus	O
-of	O
-current	O
-antibody	O
-modeling	O
-efforts	O
-.	O
-	O
-This	O
-interaction	O
-site	O
-is	O
-composed	O
-of	O
-6	O
-complementarity	O
--	O
-determining	O
-regions	O
-(	O
-CDRs	O
-)	O
-that	O
-were	O
-identified	O
-in	O
-early	O
-antibody	O
-amino	O
-acid	O
-sequence	O
-analyses	O
-to	O
-be	O
-hypervariable	O
-in	O
-nature	O
-,	O
-and	O
-thus	O
-are	O
-responsible	O
-for	O
-the	O
-sequence	O
-and	O
-structural	O
-diversity	O
-of	O
-our	O
-antibody	O
-repertoire	O
-.	O
-	O
-The	O
-sequence	O
-diversity	O
-of	O
-the	O
-CDR	O
-regions	O
-presents	O
-a	O
-substantial	O
-challenge	O
-to	O
-antibody	O
-modeling	O
-.	O
-	O
-However	O
-,	O
-an	O
-initial	O
-structural	O
-analysis	O
-of	O
-the	O
-combining	O
-sites	O
-of	O
-the	O
-small	O
-set	O
-of	O
-structures	O
-of	O
-immunoglobulin	O
-fragments	O
-available	O
-in	O
-the	O
-1980s	O
-found	O
-that	O
-5	O
-of	O
-the	O
-6	O
-hypervariable	O
-loops	O
-or	O
-CDRs	O
-had	O
-canonical	O
-structures	O
-(	O
-a	O
-limited	O
-set	O
-of	O
-main	O
--	O
-chain	O
-conformations	O
-).	O
-	O
-A	O
-CDR	O
-canonical	O
-structure	O
-is	O
-defined	O
-by	O
-its	O
-length	O
-and	O
-conserved	O
-residues	O
-located	O
-in	O
-the	O
-hypervariable	O
-loop	O
-and	O
-framework	O
-residues	O
-(	O
-V	O
--	O
-region	O
-residues	O
-that	O
-are	O
-not	O
-part	O
-of	O
-the	O
-CDRs	O
-).	O
-	O
-Furthermore	O
-,	O
-studies	O
-of	O
-antibody	O
-sequences	O
-revealed	O
-that	O
-the	O
-total	O
-number	O
-of	O
-canonical	O
-structures	O
-are	O
-limited	O
-for	O
-each	O
-CDR	O
-,	O
-indicating	O
-possibly	O
-that	O
-antigen	O
-recognition	O
-may	O
-be	O
-affected	O
-by	O
-structural	O
-restrictions	O
-at	O
-the	O
-antigen	O
--	O
-binding	O
-site	O
-.	O
-	O
-Later	O
-studies	O
-found	O
-that	O
-the	O
-CDR	O
-loop	O
-length	O
-is	O
-the	O
-primary	O
-determining	O
-factor	O
-of	O
-antigen	O
--	O
-binding	O
-site	O
-topography	O
-because	O
-it	O
-is	O
-the	O
-primary	O
-factor	O
-for	O
-determining	O
-a	O
-canonical	O
-structure	O
-.	O
-	O
-Additional	O
-efforts	O
-have	O
-led	O
-to	O
-our	O
-current	O
-understanding	O
-that	O
-the	O
-LC	O
-CDRs	O
-L1	O
-,	O
-L2	O
-,	O
-and	O
-L3	O
-have	O
-preferred	O
-sets	O
-of	O
-canonical	O
-structures	O
-based	O
-on	O
-length	O
-and	O
-amino	O
-acid	O
-sequence	O
-composition	O
-.	O
-	O
-This	O
-was	O
-also	O
-found	O
-to	O
-be	O
-the	O
-case	O
-for	O
-the	O
-H1	O
-and	O
-H2	O
-CDRs	O
-.	O
-	O
-Classification	O
-schemes	O
-for	O
-the	O
-canonical	O
-structures	O
-of	O
-these	O
-5	O
-CDRs	O
-have	O
-emerged	O
-and	O
-evolved	O
-as	O
-the	O
-number	O
-of	O
-depositions	O
-in	O
-the	O
-Protein	O
-Data	O
-Bank	O
-of	O
-Fab	O
-fragments	O
-of	O
-antibodies	O
-grow	O
-.	O
-	O
-Recently	O
-,	O
-a	O
-comprehensive	O
-CDR	O
-classification	O
-scheme	O
-was	O
-reported	O
-identifying	O
-72	O
-clusters	O
-of	O
-conformations	O
-observed	O
-in	O
-antibody	O
-structures	O
-.	O
-	O
-The	O
-knowledge	O
-and	O
-predictability	O
-of	O
-these	O
-CDR	O
-canonical	O
-structures	O
-have	O
-greatly	O
-advanced	O
-antibody	O
-modeling	O
-efforts	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-CDRs	O
-L1	O
-,	O
-L2	O
-,	O
-L3	O
-,	O
-H1	O
-and	O
-H2	O
-,	O
-no	O
-canonical	O
-structures	O
-have	O
-been	O
-observed	O
-for	O
-CDR	O
-H3	O
-,	O
-which	O
-is	O
-the	O
-most	O
-variable	O
-in	O
-length	O
-and	O
-amino	O
-acid	O
-sequence	O
-.	O
-	O
-Some	O
-clustering	O
-of	O
-conformations	O
-was	O
-observed	O
-for	O
-the	O
-shortest	O
-lengths	O
-;	O
-however	O
-,	O
-for	O
-the	O
-longer	O
-loops	O
-,	O
-only	O
-the	O
-portions	O
-nearest	O
-the	O
-framework	O
-(	O
-torso	O
-,	O
-stem	O
-or	O
-anchor	O
-region	O
-)	O
-were	O
-found	O
-to	O
-have	O
-defined	O
-conformations	O
-.	O
-	O
-In	O
-the	O
-torso	O
-region	O
-,	O
-2	O
-primary	O
-groups	O
-could	O
-be	O
-identified	O
-,	O
-which	O
-led	O
-to	O
-sequence	O
--	O
-based	O
-rules	O
-that	O
-can	O
-predict	O
-with	O
-some	O
-degree	O
-of	O
-reliability	O
-the	O
-conformation	O
-of	O
-the	O
-stem	O
-region	O
-.	O
-	O
-The	O
-“	O
-kinked	O
-”	O
-or	O
-“	O
-bulged	O
-”	O
-conformation	O
-is	O
-the	O
-most	O
-prevalent	O
-,	O
-but	O
-an	O
-“	O
-extended	O
-”	O
-or	O
-“	O
-non	O
--	O
-bulged	O
-”	O
-conformation	O
-is	O
-also	O
-,	O
-but	O
-less	O
-frequently	O
-,	O
-observed	O
-.	O
-	O
-The	O
-cataloging	O
-and	O
-development	O
-of	O
-the	O
-rules	O
-for	O
-predicting	O
-the	O
-conformation	O
-of	O
-the	O
-anchor	O
-region	O
-of	O
-CDR	O
-H3	O
-continue	O
-to	O
-be	O
-refined	O
-,	O
-producing	O
-new	O
-insight	O
-into	O
-the	O
-CDR	O
-H3	O
-conformations	O
-and	O
-new	O
-tools	O
-for	O
-antibody	O
-engineering	O
-.	O
-	O
-Current	O
-antibody	O
-modeling	O
-approaches	O
-take	O
-advantage	O
-of	O
-the	O
-most	O
-recent	O
-advances	O
-in	O
-homology	O
-modeling	O
-,	O
-the	O
-evolving	O
-understanding	O
-of	O
-the	O
-CDR	O
-canonical	O
-structures	O
-,	O
-the	O
-emerging	O
-rules	O
-for	O
-CDR	O
-H3	O
-modeling	O
-and	O
-the	O
-growing	O
-body	O
-of	O
-antibody	O
-structural	O
-data	O
-available	O
-from	O
-the	O
-PDB	O
-.	O
-	O
-Recent	O
-antibody	O
-modeling	O
-assessments	O
-show	O
-continued	O
-improvement	O
-in	O
-the	O
-quality	O
-of	O
-the	O
-models	O
-being	O
-generated	O
-by	O
-a	O
-variety	O
-of	O
-modeling	O
-methods	O
-.	O
-	O
-Although	O
-antibody	O
-modeling	O
-is	O
-improving	O
-,	O
-the	O
-latest	O
-assessment	O
-revealed	O
-a	O
-number	O
-of	O
-challenges	O
-that	O
-need	O
-to	O
-be	O
-overcome	O
-to	O
-provide	O
-accurate	O
-3	O
--	O
-dimensional	O
-models	O
-of	O
-antibody	O
-V	O
-regions	O
-,	O
-including	O
-accuracies	O
-in	O
-the	O
-modeling	O
-of	O
-CDR	O
-H3	O
-.	O
-	O
-The	O
-need	O
-for	O
-improvement	O
-in	O
-this	O
-area	O
-was	O
-also	O
-highlighted	O
-in	O
-a	O
-recent	O
-study	O
-reporting	O
-an	O
-approach	O
-and	O
-results	O
-that	O
-may	O
-influence	O
-future	O
-antibody	O
-modeling	O
-efforts	O
-.	O
-	O
-One	O
-important	O
-finding	O
-of	O
-the	O
-antibody	O
-modeling	O
-assessments	O
-was	O
-that	O
-errors	O
-in	O
-the	O
-structural	O
-templates	O
-that	O
-are	O
-used	O
-as	O
-the	O
-basis	O
-for	O
-homology	O
-models	O
-can	O
-propagate	O
-into	O
-the	O
-final	O
-models	O
-,	O
-producing	O
-inaccuracies	O
-that	O
-may	O
-negatively	O
-influence	O
-the	O
-predictive	O
-nature	O
-of	O
-the	O
-V	O
-region	O
-model	O
-.	O
-	O
-To	O
-support	O
-antibody	O
-engineering	O
-and	O
-therapeutic	O
-development	O
-efforts	O
-,	O
-a	O
-phage	O
-library	O
-was	O
-designed	O
-and	O
-constructed	O
-based	O
-on	O
-a	O
-limited	O
-number	O
-of	O
-scaffolds	O
-built	O
-with	O
-frequently	O
-used	O
-human	O
-germ	O
--	O
-line	O
-IGV	O
-and	O
-IGJ	O
-gene	O
-segments	O
-that	O
-encode	O
-antigen	O
-combining	O
-sites	O
-suitable	O
-for	O
-recognition	O
-of	O
-peptides	O
-and	O
-proteins	O
-.	O
-	O
-This	O
-Fab	O
-library	O
-is	O
-composed	O
-of	O
-3	O
-HC	O
-germlines	O
-,	O
-IGHV1	B-mutant
--	I-mutant
-69	I-mutant
-(	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-),	O
-IGHV3	B-mutant
--	I-mutant
-23	I-mutant
-(	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-)	O
-and	O
-IGHV5	B-mutant
--	I-mutant
-51	I-mutant
-(	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-),	O
-and	O
-4	O
-LC	O
-germlines	O
-(	O
-all	O
-κ	O
-),	O
-IGKV1	B-mutant
--	I-mutant
-39	I-mutant
-(	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-),	O
-IGKV3	B-mutant
--	I-mutant
-11	I-mutant
-(	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-),	O
-IGKV3	B-mutant
--	I-mutant
-20	I-mutant
-(	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-)	O
-and	O
-IGKV4	B-mutant
--	I-mutant
-1	I-mutant
-(	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-).	O
-	O
-Selection	O
-of	O
-these	O
-genes	O
-was	O
-based	O
-on	O
-the	O
-high	O
-frequency	O
-of	O
-their	O
-use	O
-and	O
-their	O
-cognate	O
-canonical	O
-structures	O
-that	O
-were	O
-found	O
-binding	O
-to	O
-peptides	O
-and	O
-proteins	O
-,	O
-as	O
-well	O
-as	O
-their	O
-ability	O
-to	O
-be	O
-expressed	O
-in	O
-bacteria	O
-and	O
-displayed	O
-on	O
-filamentous	O
-phage	O
-.	O
-	O
-The	O
-implementation	O
-of	O
-the	O
-library	O
-involves	O
-the	O
-diversification	O
-of	O
-the	O
-human	O
-germline	O
-genes	O
-to	O
-mimic	O
-that	O
-found	O
-in	O
-natural	O
-human	O
-libraries	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-determinations	O
-and	O
-structural	O
-analyses	O
-of	O
-all	O
-germline	O
-Fabs	O
-in	O
-the	O
-library	O
-described	O
-above	O
-along	O
-with	O
-the	O
-structures	O
-of	O
-a	O
-fourth	O
-HC	O
-germline	O
-,	O
-IGHV3	B-mutant
--	I-mutant
-53	I-mutant
-(	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-),	O
-paired	O
-with	O
-the	O
-4	O
-LCs	O
-of	O
-the	O
-library	O
-have	O
-been	O
-carried	O
-out	O
-to	O
-support	O
-antibody	O
-therapeutic	O
-development	O
-.	O
-	O
-All	O
-16	O
-HCs	O
-of	O
-the	O
-Fabs	O
-have	O
-the	O
-same	O
-CDR	O
-H3	O
-that	O
-was	O
-reported	O
-in	O
-an	O
-earlier	O
-Fab	O
-structure	O
-.	O
-	O
-This	O
-is	O
-the	O
-first	O
-systematic	O
-study	O
-of	O
-the	O
-same	O
-VH	O
-and	O
-VL	O
-structures	O
-in	O
-the	O
-context	O
-of	O
-different	O
-pairings	O
-.	O
-	O
-The	O
-structure	O
-analyses	O
-include	O
-comparisons	O
-of	O
-the	O
-overall	O
-structures	O
-,	O
-canonical	O
-structures	O
-of	O
-the	O
-L1	O
-,	O
-L2	O
-,	O
-L3	O
-,	O
-H1	O
-and	O
-H2	O
-CDRs	O
-,	O
-the	O
-structures	O
-of	O
-all	O
-CDR	O
-H3s	O
-,	O
-and	O
-the	O
-VH	O
-:	O
-VL	O
-packing	O
-interactions	O
-.	O
-	O
-The	O
-structures	O
-and	O
-their	O
-analyses	O
-provide	O
-a	O
-foundation	O
-for	O
-future	O
-antibody	O
-engineering	O
-and	O
-structure	O
-determination	O
-efforts	O
-.	O
-	O
-Crystal	O
-structures	O
-	O
-Crystal	O
-data	O
-,	O
-X	O
--	O
-ray	O
-data	O
-,	O
-and	O
-refinement	O
-statistics	O
-.	O
-	O
-(	O
-Continued	O
-)	O
-Crystal	O
-data	O
-,	O
-X	O
--	O
-ray	O
-data	O
-,	O
-and	O
-refinement	O
-statistics	O
-.	O
-	O
-The	O
-crystal	O
-structures	O
-of	O
-a	O
-germline	O
-library	O
-composed	O
-of	O
-16	O
-Fabs	O
-generated	O
-by	O
-combining	O
-4	O
-HCs	O
-(	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-)	O
-and	O
-4	O
-LCs	O
-(	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-,	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-,	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-and	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-)	O
-have	O
-been	O
-determined	O
-.	O
-	O
-The	O
-Fab	O
-heavy	O
-and	O
-light	O
-chain	O
-sequences	O
-for	O
-the	O
-variants	O
-numbered	O
-according	O
-to	O
-Chothia	O
-are	O
-shown	O
-in	O
-Fig	O
-.	O
-S1	O
-.	O
-	O
-The	O
-four	O
-different	O
-HCs	O
-all	O
-have	O
-the	O
-same	O
-CDR	O
-H3	O
-sequence	O
-,	O
-ARYDGIYGELDF	O
-.	O
-	O
-Crystallization	O
-of	O
-the	O
-16	O
-Fabs	O
-was	O
-previously	O
-reported	O
-.	O
-	O
-Three	O
-sets	O
-of	O
-the	O
-crystals	O
-were	O
-isomorphous	O
-with	O
-nearly	O
-identical	O
-unit	O
-cells	O
-(	O
-Table	O
-1	O
-).	O
-	O
-These	O
-include	O
-(	O
-1	O
-)	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-11	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L4	O
--	O
-1	O
-in	O
-P212121	O
-,	O
-(	O
-2	O
-)	O
-H3	O
--	O
-53	O
-:	O
-L1	O
--	O
-39	O
-,	O
-H3	O
--	O
-53	O
-:	O
-L3	O
--	O
-11	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L3	O
--	O
-20	O
-in	O
-P6522	O
-,	O
-and	O
-(	O
-3	O
-)	O
-H5	O
--	O
-51	O
-:	O
-L1	O
--	O
-39	O
-,	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-11	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-in	O
-P212121	O
-.	O
-	O
-Variations	O
-occur	O
-in	O
-the	O
-pH	O
-(	O
-buffer	O
-)	O
-and	O
-the	O
-additives	O
-,	O
-and	O
-,	O
-in	O
-group	O
-3	O
-,	O
-PEG	O
-3350	O
-is	O
-the	O
-precipitant	O
-for	O
-one	O
-variants	O
-while	O
-ammonium	O
-sulfate	O
-is	O
-the	O
-precipitant	O
-for	O
-the	O
-other	O
-two	O
-.	O
-	O
-The	O
-similarity	O
-in	O
-the	O
-crystal	O
-forms	O
-is	O
-attributed	O
-in	O
-part	O
-to	O
-cross	O
--	O
-seeding	O
-using	O
-the	O
-microseed	O
-matrix	O
-screening	O
-for	O
-groups	O
-2	O
-and	O
-3	O
-.	O
-	O
-The	O
-crystal	O
-structures	O
-of	O
-the	O
-16	O
-Fabs	O
-have	O
-been	O
-determined	O
-at	O
-resolutions	O
-ranging	O
-from	O
-3	O
-.	O
-3	O
-Å	O
-to	O
-1	O
-.	O
-65	O
-Å	O
-(	O
-Table	O
-1	O
-).	O
-	O
-The	O
-number	O
-of	O
-Fab	O
-molecules	O
-in	O
-the	O
-crystallographic	O
-asymmetric	O
-unit	O
-varies	O
-from	O
-1	O
-(	O
-for	O
-12	O
-Fabs	O
-)	O
-to	O
-2	O
-(	O
-for	O
-4	O
-Fabs	O
-).	O
-	O
-Overall	O
-the	O
-structures	O
-are	O
-fairly	O
-complete	O
-,	O
-and	O
-,	O
-as	O
-can	O
-be	O
-expected	O
-,	O
-the	O
-models	O
-for	O
-the	O
-higher	O
-resolution	O
-structures	O
-are	O
-more	O
-complete	O
-than	O
-those	O
-for	O
-the	O
-lower	O
-resolution	O
-structures	O
-(	O
-Table	O
-S1	O
-).	O
-	O
-Invariably	O
-,	O
-the	O
-HCs	O
-have	O
-more	O
-disorder	O
-than	O
-the	O
-LCs	O
-.	O
-	O
-For	O
-the	O
-LC	O
-,	O
-the	O
-disorder	O
-is	O
-observed	O
-at	O
-2	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-residues	O
-with	O
-few	O
-exceptions	O
-.	O
-	O
-Apart	O
-from	O
-the	O
-C	O
--	O
-terminus	O
-,	O
-only	O
-a	O
-few	O
-surface	O
-residues	O
-in	O
-LC	O
-are	O
-disordered	O
-.	O
-	O
-The	O
-HCs	O
-feature	O
-the	O
-largest	O
-number	O
-of	O
-disordered	O
-residues	O
-,	O
-with	O
-the	O
-lower	O
-resolution	O
-structures	O
-having	O
-the	O
-most	O
-.	O
-	O
-The	O
-C	O
--	O
-terminal	O
-residues	O
-including	O
-the	O
-6xHis	O
-tags	O
-are	O
-disordered	O
-in	O
-all	O
-16	O
-structures	O
-.	O
-	O
-In	O
-addition	O
-to	O
-these	O
-,	O
-2	O
-primary	O
-disordered	O
-stretches	O
-of	O
-residues	O
-are	O
-observed	O
-in	O
-a	O
-number	O
-of	O
-structures	O
-(	O
-Table	O
-S1	O
-).	O
-	O
-One	O
-involves	O
-the	O
-loop	O
-connecting	O
-the	O
-first	O
-2	O
-β	O
--	O
-strands	O
-of	O
-the	O
-constant	O
-domain	O
-(	O
-in	O
-all	O
-Fabs	O
-except	O
-H3	O
--	O
-23	O
-:	O
-L1	O
--	O
-39	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-11	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L1	O
--	O
-39	O
-).	O
-	O
-The	O
-other	O
-is	O
-located	O
-in	O
-CDR	O
-H3	O
-(	O
-in	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-11	O
-,	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-and	O
-in	O
-one	O
-of	O
-2	O
-copies	O
-of	O
-H3	O
--	O
-23	O
-:	O
-L4	O
--	O
-1	O
-).	O
-	O
-CDR	O
-H1	O
-and	O
-CDR	O
-H2	O
-also	O
-show	O
-some	O
-degree	O
-of	O
-disorder	O
-,	O
-but	O
-to	O
-a	O
-lesser	O
-extent	O
-.	O
-	O
-CDR	O
-canonical	O
-structures	O
-	O
-Several	O
-CDR	O
-definitions	O
-have	O
-evolved	O
-over	O
-decades	O
-of	O
-antibody	O
-research	O
-.	O
-	O
-Depending	O
-on	O
-the	O
-focus	O
-of	O
-the	O
-study	O
-,	O
-the	O
-CDR	O
-boundaries	O
-differ	O
-slightly	O
-between	O
-various	O
-definitions	O
-.	O
-	O
-In	O
-this	O
-work	O
-,	O
-we	O
-use	O
-the	O
-CDR	O
-definition	O
-of	O
-North	O
-et	O
-al	O
-.,	O
-which	O
-is	O
-similar	O
-to	O
-that	O
-of	O
-Martin	O
-with	O
-the	O
-following	O
-exceptions	O
-:	O
-1	O
-)	O
-CDRs	O
-H1	O
-and	O
-H3	O
-begin	O
-immediately	O
-after	O
-the	O
-Cys	O
-;	O
-and	O
-2	O
-)	O
-CDR	O
-L2	O
-includes	O
-an	O
-additional	O
-residue	O
-at	O
-the	O
-N	O
--	O
-terminal	O
-side	O
-,	O
-typically	O
-Tyr	O
-.	O
-	O
-CDR	O
-H1	O
-	O
-The	O
-superposition	O
-of	O
-CDR	O
-H1	O
-backbones	O
-for	O
-all	O
-HC	O
-:	O
-LC	O
-pairs	O
-with	O
-heavy	O
-chains	O
-:	O
-(	O
-A	O
-)	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-,	O
-(	O
-B	O
-)	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-,	O
-(	O
-C	O
-)	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-and	O
-(	O
-D	O
-)	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-.	O
-	O
-CDRs	O
-are	O
-defined	O
-using	O
-the	O
-Dunbrack	O
-convention	O
-[	O
-12	O
-].	O
-	O
-Assignments	O
-for	O
-2	O
-copies	O
-of	O
-the	O
-Fab	O
-in	O
-the	O
-asymmetric	O
-unit	O
-are	O
-given	O
-for	O
-5	O
-structures	O
-.	O
-	O
-No	O
-assignment	O
-(	O
-NA	O
-)	O
-for	O
-CDRs	O
-with	O
-missing	O
-residues	O
-.	O
-	O
-The	O
-four	O
-HCs	O
-feature	O
-CDR	O
-H1	O
-of	O
-the	O
-same	O
-length	O
-,	O
-and	O
-their	O
-sequences	O
-are	O
-highly	O
-similar	O
-(	O
-Table	O
-2	O
-).	O
-	O
-The	O
-CDR	O
-H1	O
-backbone	O
-conformations	O
-for	O
-all	O
-variants	O
-for	O
-each	O
-of	O
-the	O
-HCs	O
-are	O
-shown	O
-in	O
-Fig	O
-.	O
-1	O
-.	O
-	O
-Three	O
-of	O
-the	O
-HCs	O
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-,	O
-have	O
-the	O
-same	O
-canonical	O
-structure	O
-,	O
-H1	B-mutant
--	I-mutant
-13	I-mutant
--	I-mutant
-1	I-mutant
-,	O
-and	O
-the	O
-backbone	O
-conformations	O
-are	O
-tightly	O
-clustered	O
-for	O
-each	O
-set	O
-of	O
-Fab	O
-structures	O
-as	O
-reflected	O
-in	O
-the	O
-rmsd	O
-values	O
-(	O
-Fig	O
-.	O
-1B	O
--	O
-D	O
-).	O
-	O
-Some	O
-deviation	O
-is	O
-observed	O
-for	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-,	O
-mostly	O
-due	O
-to	O
-H3	O
--	O
-53	O
-:	O
-L4	O
--	O
-1	O
-,	O
-which	O
-exhibits	O
-a	O
-significant	O
-degree	O
-of	O
-disorder	O
-in	O
-CDR	O
-H1	O
-.	O
-	O
-The	O
-electron	O
-density	O
-for	O
-the	O
-backbone	O
-is	O
-weak	O
-and	O
-discontinuous	O
-,	O
-and	O
-completely	O
-missing	O
-for	O
-several	O
-side	O
-chains	O
-.	O
-	O
-The	O
-CDR	O
-H1	O
-structures	O
-with	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-shown	O
-in	O
-Fig	O
-.	O
-1A	O
-are	O
-quite	O
-variable	O
-,	O
-both	O
-for	O
-the	O
-structures	O
-with	O
-different	O
-LCs	O
-and	O
-for	O
-the	O
-copies	O
-of	O
-the	O
-same	O
-Fab	O
-in	O
-the	O
-asymmetric	O
-unit	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-11	O
-and	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-.	O
-	O
-In	O
-total	O
-,	O
-6	O
-independent	O
-Fab	O
-structures	O
-produce	O
-5	O
-different	O
-canonical	O
-structures	O
-,	O
-namely	O
-H1	B-mutant
--	I-mutant
-13	I-mutant
--	I-mutant
-1	I-mutant
-,	O
-H1	B-mutant
--	I-mutant
-13	I-mutant
--	I-mutant
-3	I-mutant
-,	O
-H1	B-mutant
--	I-mutant
-13	I-mutant
--	I-mutant
-4	I-mutant
-,	O
-H1	B-mutant
--	I-mutant
-13	I-mutant
--	I-mutant
-6	I-mutant
-and	O
-H1	B-mutant
--	I-mutant
-13	I-mutant
--	I-mutant
-10	I-mutant
-.	O
-	O
-A	O
-major	O
-difference	O
-of	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-from	O
-the	O
-other	O
-germlines	O
-in	O
-the	O
-experimental	O
-data	O
-set	O
-is	O
-the	O
-presence	O
-of	O
-Gly	O
-instead	O
-of	O
-Phe	O
-or	O
-Tyr	O
-at	O
-position	O
-27	O
-(	O
-residue	O
-5	O
-of	O
-13	O
-in	O
-CDR	O
-H1	O
-).	O
-	O
-Glycine	O
-introduces	O
-the	O
-possibility	O
-of	O
-a	O
-higher	O
-degree	O
-of	O
-conformational	O
-flexibility	O
-that	O
-undoubtedly	O
-translates	O
-to	O
-the	O
-differences	O
-observed	O
-,	O
-and	O
-contributes	O
-to	O
-the	O
-elevated	O
-thermal	O
-parameters	O
-for	O
-the	O
-atoms	O
-in	O
-the	O
-amino	O
-acid	O
-residues	O
-in	O
-this	O
-region	O
-.	O
-	O
-CDR	O
-H2	O
-	O
-The	O
-superposition	O
-of	O
-CDR	O
-H2	O
-backbones	O
-for	O
-all	O
-HC	O
-:	O
-LC	O
-pairs	O
-with	O
-heavy	O
-chains	O
-:	O
-(	O
-A	O
-)	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-,	O
-(	O
-B	O
-)	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-,	O
-(	O
-C	O
-)	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-and	O
-(	O
-D	O
-)	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-.	O
-	O
-The	O
-canonical	O
-structures	O
-of	O
-CDR	O
-H2	O
-have	O
-fairly	O
-consistent	O
-conformations	O
-(	O
-Table	O
-2	O
-,	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Each	O
-of	O
-the	O
-4	O
-HCs	O
-adopts	O
-only	O
-one	O
-canonical	O
-structure	O
-regardless	O
-of	O
-the	O
-pairing	O
-LC	O
-.	O
-	O
-Germlines	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-have	O
-the	O
-same	O
-canonical	O
-structure	O
-assignment	O
-H2	B-mutant
--	I-mutant
-10	I-mutant
--	I-mutant
-1	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-has	O
-H2	B-mutant
--	I-mutant
-10	I-mutant
--	I-mutant
-2	I-mutant
-,	O
-and	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-has	O
-H2	B-mutant
--	I-mutant
-9	I-mutant
--	I-mutant
-3	I-mutant
-.	O
-	O
-The	O
-conformations	O
-for	O
-all	O
-of	O
-these	O
-CDR	O
-H2s	O
-are	O
-tightly	O
-clustered	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-In	O
-one	O
-case	O
-,	O
-in	O
-the	O
-second	O
-Fab	O
-of	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-CDR	O
-H2	O
-is	O
-partially	O
-disordered	O
-(	O
-Δ55	B-mutant
--	I-mutant
-60	I-mutant
-).	O
-	O
-Although	O
-three	O
-of	O
-the	O
-germlines	O
-have	O
-CDR	O
-H2	O
-of	O
-the	O
-same	O
-length	O
-,	O
-10	O
-residues	O
-,	O
-they	O
-adopt	O
-2	O
-distinctively	O
-different	O
-conformations	O
-depending	O
-mostly	O
-on	O
-the	O
-residue	O
-at	O
-position	O
-71	O
-from	O
-the	O
-so	O
--	O
-called	O
-CDR	O
-H4	O
-.	O
-	O
-Arg71	O
-in	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-fills	O
-the	O
-space	O
-between	O
-CDRs	O
-H2	O
-and	O
-H4	O
-,	O
-and	O
-defines	O
-the	O
-conformation	O
-of	O
-the	O
-tip	O
-of	O
-CDR	O
-H2	O
-so	O
-that	O
-residue	O
-54	O
-points	O
-away	O
-from	O
-the	O
-antigen	O
-binding	O
-site	O
-.	O
-	O
-Germlines	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-are	O
-unique	O
-in	O
-the	O
-human	O
-repertoire	O
-in	O
-having	O
-an	O
-Ala	O
-at	O
-position	O
-71	O
-that	O
-leaves	O
-enough	O
-space	O
-for	O
-H	O
--	O
-Pro52a	O
-to	O
-pack	O
-deeper	O
-against	O
-CDR	O
-H4	O
-so	O
-that	O
-the	O
-following	O
-residues	O
-53	O
-and	O
-54	O
-point	O
-toward	O
-the	O
-putative	O
-antigen	O
-.	O
-	O
-Conformations	O
-of	O
-CDR	O
-H2	O
-in	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-,	O
-both	O
-of	O
-which	O
-have	O
-canonical	O
-structure	O
-H2	B-mutant
--	I-mutant
-10	I-mutant
--	I-mutant
-1	I-mutant
-,	O
-show	O
-little	O
-deviation	O
-within	O
-each	O
-set	O
-of	O
-4	O
-structures	O
-.	O
-	O
-However	O
-,	O
-there	O
-is	O
-a	O
-significant	O
-shift	O
-of	O
-the	O
-CDR	O
-as	O
-a	O
-rigid	O
-body	O
-when	O
-the	O
-2	O
-sets	O
-are	O
-superimposed	O
-.	O
-	O
-Most	O
-likely	O
-this	O
-is	O
-the	O
-result	O
-of	O
-interaction	O
-of	O
-CDR	O
-H2	O
-with	O
-CDR	O
-H1	O
-,	O
-namely	O
-with	O
-the	O
-residue	O
-at	O
-position	O
-33	O
-(	O
-residue	O
-11	O
-of	O
-13	O
-in	O
-CDR	O
-H1	O
-).	O
-	O
-Germline	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-has	O
-Ala	O
-at	O
-position	O
-33	O
-whereas	O
-in	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-position	O
-33	O
-is	O
-occupied	O
-by	O
-a	O
-bulky	O
-Trp	O
-,	O
-which	O
-stacks	O
-against	O
-H	O
--	O
-Tyr52	O
-and	O
-drives	O
-CDR	O
-H2	O
-away	O
-from	O
-the	O
-center	O
-.	O
-	O
-CDR	O
-L1	O
-	O
-The	O
-superposition	O
-of	O
-CDR	O
-L1	O
-backbones	O
-for	O
-all	O
-HC	O
-:	O
-LC	O
-pairs	O
-with	O
-light	O
-chains	O
-:	O
-(	O
-A	O
-)	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-,	O
-(	O
-B	O
-)	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-,	O
-(	O
-C	O
-)	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-and	O
-(	O
-D	O
-)	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-.	O
-	O
-The	O
-four	O
-LC	O
-CDRs	O
-L1	O
-feature	O
-3	O
-different	O
-lengths	O
-(	O
-11	O
-,	O
-12	O
-and	O
-17	O
-residues	O
-)	O
-having	O
-a	O
-total	O
-of	O
-4	O
-different	O
-canonical	O
-structure	O
-assignments	O
-.	O
-	O
-Of	O
-these	O
-LCs	O
-,	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-and	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-have	O
-the	O
-same	O
-canonical	O
-structure	O
-,	O
-L1	B-mutant
--	I-mutant
-11	I-mutant
--	I-mutant
-1	I-mutant
-,	O
-and	O
-superimpose	O
-very	O
-well	O
-(	O
-Fig	O
-.	O
-3A	O
-,	O
-B	O
-).	O
-	O
-For	O
-the	O
-remaining	O
-2	O
-,	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-has	O
-2	O
-different	O
-assignments	O
-,	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-1	I-mutant
-and	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-2	I-mutant
-,	O
-while	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-has	O
-a	O
-single	O
-assignment	O
-,	O
-L1	B-mutant
--	I-mutant
-17	I-mutant
--	I-mutant
-1	I-mutant
-.	O
-	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-has	O
-the	O
-longest	O
-CDR	O
-L1	O
-,	O
-composed	O
-of	O
-17	O
-amino	O
-acid	O
-residues	O
-(	O
-Fig	O
-.	O
-3D	O
-).	O
-	O
-Despite	O
-this	O
-,	O
-the	O
-conformations	O
-are	O
-tightly	O
-clustered	O
-(	O
-rmsd	O
-is	O
-0	O
-.	O
-20	O
-Å	O
-).	O
-	O
-The	O
-backbone	O
-conformations	O
-of	O
-the	O
-stem	O
-regions	O
-superimpose	O
-well	O
-.	O
-	O
-Some	O
-changes	O
-in	O
-conformation	O
-occur	O
-between	O
-residues	O
-30a	O
-and	O
-30f	O
-(	O
-residues	O
-8	O
-and	O
-13	O
-of	O
-17	O
-in	O
-CDR	O
-L1	O
-).	O
-	O
-This	O
-is	O
-the	O
-tip	O
-of	O
-the	O
-loop	O
-region	O
-,	O
-which	O
-appears	O
-to	O
-have	O
-similar	O
-conformations	O
-that	O
-fan	O
-out	O
-the	O
-structures	O
-because	O
-of	O
-the	O
-slight	O
-differences	O
-in	O
-torsion	O
-angles	O
-in	O
-the	O
-backbone	O
-near	O
-Tyr30a	O
-and	O
-Lys30f	O
-.	O
-	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-is	O
-the	O
-most	O
-variable	O
-in	O
-CDR	O
-L1	O
-among	O
-the	O
-4	O
-germlines	O
-as	O
-indicated	O
-by	O
-an	O
-rmsd	O
-of	O
-0	O
-.	O
-54	O
-Å	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-Two	O
-structures	O
-,	O
-H3	O
--	O
-53	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-are	O
-assigned	O
-to	O
-canonical	O
-structure	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-1	I-mutant
-with	O
-virtually	O
-identical	O
-backbone	O
-conformations	O
-.	O
-	O
-The	O
-third	O
-structure	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-has	O
-CDR	O
-L1	O
-as	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-2	I-mutant
-,	O
-which	O
-deviates	O
-from	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-1	I-mutant
-at	O
-residues	O
-29	O
--	O
-32	O
-,	O
-i	O
-.	O
-e	O
-.,	O
-at	O
-the	O
-site	O
-of	O
-insertion	O
-with	O
-respect	O
-to	O
-the	O
-11	O
--	O
-residue	O
-CDR	O
-.	O
-	O
-The	O
-fourth	O
-member	O
-of	O
-the	O
-set	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-was	O
-crystallized	O
-with	O
-2	O
-Fabs	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-The	O
-conformation	O
-of	O
-CDR	O
-L1	O
-in	O
-these	O
-2	O
-Fabs	O
-is	O
-slightly	O
-different	O
-,	O
-and	O
-both	O
-conformations	O
-fall	O
-somewhere	O
-between	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-1	I-mutant
-and	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-2	I-mutant
-.	O
-	O
-This	O
-reflects	O
-the	O
-lack	O
-of	O
-accuracy	O
-in	O
-the	O
-structure	O
-due	O
-to	O
-low	O
-resolution	O
-of	O
-the	O
-X	O
--	O
-ray	O
-data	O
-(	O
-3	O
-.	O
-3	O
-Å	O
-).	O
-	O
-CDR	O
-L2	O
-	O
-The	O
-superposition	O
-of	O
-CDR	O
-L2	O
-backbones	O
-for	O
-all	O
-HC	O
-:	O
-LC	O
-pairs	O
-with	O
-light	O
-chains	O
-:	O
-(	O
-A	O
-)	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-,	O
-(	O
-B	O
-)	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-,	O
-(	O
-C	O
-)	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-and	O
-(	O
-D	O
-)	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-.	O
-	O
-All	O
-four	O
-LCs	O
-have	O
-CDR	O
-L2	O
-of	O
-the	O
-same	O
-length	O
-and	O
-canonical	O
-structure	O
-,	O
-L2	B-mutant
--	I-mutant
-8	I-mutant
--	I-mutant
-1	I-mutant
-(	O
-Table	O
-2	O
-).	O
-	O
-The	O
-CDR	O
-L2	O
-conformations	O
-for	O
-each	O
-of	O
-the	O
-LCs	O
-paired	O
-with	O
-the	O
-4	O
-HCs	O
-are	O
-clustered	O
-more	O
-tightly	O
-than	O
-any	O
-of	O
-the	O
-other	O
-CDRs	O
-(	O
-rmsd	O
-values	O
-are	O
-in	O
-the	O
-range	O
-0	O
-.	O
-09	O
--	O
-0	O
-.	O
-16	O
-Å	O
-),	O
-and	O
-all	O
-4	O
-sets	O
-have	O
-virtually	O
-the	O
-same	O
-conformation	O
-despite	O
-the	O
-sequence	O
-diversity	O
-of	O
-the	O
-loop	O
-.	O
-	O
-CDR	O
-L3	O
-	O
-The	O
-superposition	O
-of	O
-CDR	O
-L3	O
-backbones	O
-for	O
-all	O
-HC	O
-:	O
-LC	O
-pairs	O
-with	O
-light	O
-chains	O
-:	O
-(	O
-A	O
-)	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-,	O
-(	O
-B	O
-)	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-,	O
-(	O
-C	O
-)	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-and	O
-(	O
-D	O
-)	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-.	O
-	O
-As	O
-with	O
-CDR	O
-L2	O
-,	O
-all	O
-4	O
-LCs	O
-have	O
-CDR	O
-L3	O
-of	O
-the	O
-same	O
-length	O
-and	O
-canonical	O
-structure	O
-,	O
-L3	B-mutant
--	I-mutant
-9	I-mutant
--	I-mutant
-cis7	I-mutant
--	I-mutant
-1	I-mutant
-(	O
-Table	O
-2	O
-).	O
-	O
-The	O
-conformations	O
-of	O
-CDR	O
-L3	O
-for	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-,	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-,	O
-and	O
-particularly	O
-for	O
-L320	O
-,	O
-are	O
-not	O
-as	O
-tightly	O
-clustered	O
-as	O
-those	O
-of	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-(	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-The	O
-slight	O
-conformational	O
-variability	O
-occurs	O
-in	O
-the	O
-region	O
-of	O
-amino	O
-acid	O
-residues	O
-90	O
--	O
-92	O
-,	O
-which	O
-is	O
-in	O
-contact	O
-with	O
-CDR	O
-H3	O
-.	O
-	O
-CDR	O
-H3	O
-conformational	O
-diversity	O
-	O
-As	O
-mentioned	O
-earlier	O
-,	O
-all	O
-16	O
-Fabs	O
-have	O
-the	O
-same	O
-CDR	O
-H3	O
-,	O
-for	O
-which	O
-the	O
-amino	O
-acid	O
-sequence	O
-is	O
-derived	O
-from	O
-the	O
-anti	O
--	O
-CCL2	O
-antibody	O
-CNTO	O
-888	O
-.	O
-	O
-The	O
-loop	O
-and	O
-the	O
-2	O
-β	O
--	O
-strands	O
-of	O
-the	O
-CDR	O
-H3	O
-in	O
-this	O
-‘	O
-parent	O
-’	O
-structure	O
-are	O
-stabilized	O
-by	O
-H	O
--	O
-bonds	O
-between	O
-the	O
-carbonyl	O
-oxygen	O
-and	O
-peptide	O
-nitrogen	O
-atoms	O
-in	O
-the	O
-2	O
-strands	O
-.	O
-	O
-An	O
-interesting	O
-feature	O
-of	O
-these	O
-CDR	O
-H3	O
-structures	O
-is	O
-the	O
-presence	O
-of	O
-a	O
-water	O
-molecule	O
-that	O
-interacts	O
-with	O
-the	O
-peptide	O
-nitrogens	O
-and	O
-carbonyl	O
-oxygens	O
-near	O
-the	O
-bridging	O
-loop	O
-connecting	O
-the	O
-2	O
-β	O
--	O
-strands	O
-.	O
-	O
-This	O
-water	O
-is	O
-present	O
-in	O
-both	O
-the	O
-bound	O
-(	O
-4DN4	O
-)	O
-and	O
-unbound	O
-(	O
-4DN3	O
-)	O
-forms	O
-of	O
-CNTO	O
-888	O
-.	O
-	O
-The	O
-stem	O
-region	O
-of	O
-CDR	O
-H3	O
-in	O
-the	O
-parental	O
-Fab	O
-is	O
-in	O
-a	O
-‘	O
-kinked	O
-’	O
-conformation	O
-,	O
-in	O
-which	O
-the	O
-indole	O
-nitrogen	O
-of	O
-Trp103	O
-forms	O
-a	O
-hydrogen	O
-bond	O
-with	O
-the	O
-carbonyl	O
-oxygen	O
-of	O
-Leu100b	O
-.	O
-	O
-The	O
-carboxyl	O
-group	O
-of	O
-Asp101	O
-forms	O
-a	O
-salt	O
-bridge	O
-with	O
-Arg94	O
-.	O
-	O
-Ribbon	O
-representations	O
-of	O
-(	O
-A	O
-)	O
-the	O
-superposition	O
-of	O
-all	O
-CDR	O
-H3s	O
-of	O
-the	O
-structures	O
-with	O
-complete	O
-backbone	O
-traces	O
-.	O
-(	O
-B	O
-)	O
-The	O
-CDR	O
-H3s	O
-rotated	O
-90	O
-°	O
-about	O
-the	O
-y	O
-axis	O
-of	O
-the	O
-page	O
-.	O
-	O
-The	O
-structure	O
-of	O
-each	O
-CDR	O
-H3	O
-is	O
-represented	O
-with	O
-a	O
-different	O
-color	O
-.	O
-	O
-Despite	O
-having	O
-the	O
-same	O
-amino	O
-acid	O
-sequence	O
-in	O
-all	O
-variants	O
-,	O
-CDR	O
-H3	O
-has	O
-the	O
-highest	O
-degree	O
-of	O
-structural	O
-diversity	O
-and	O
-disorder	O
-of	O
-all	O
-of	O
-the	O
-CDRs	O
-in	O
-the	O
-experimental	O
-set	O
-.	O
-	O
-Three	O
-of	O
-the	O
-21	O
-Fab	O
-structures	O
-(	O
-including	O
-multiple	O
-copies	O
-in	O
-the	O
-asymmetric	O
-unit	O
-),	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-11	O
-,	O
-H551	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L4	O
--	O
-1	O
-(	O
-one	O
-of	O
-the	O
-2	O
-Fabs	O
-),	O
-have	O
-missing	O
-(	O
-disordered	O
-)	O
-residues	O
-at	O
-the	O
-apex	O
-of	O
-the	O
-CDR	O
-loop	O
-.	O
-	O
-Another	O
-four	O
-of	O
-the	O
-Fabs	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L1	O
--	O
-39	O
-,	O
-H3	O
--	O
-53	O
-:	O
-L1	O
--	O
-39	O
-,	O
-H3	O
--	O
-53	O
-:	O
-L3	O
--	O
-11	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L4	O
--	O
-1	O
-have	O
-missing	O
-side	O
--	O
-chain	O
-atoms	O
-.	O
-	O
-The	O
-variations	O
-in	O
-CDR	O
-H3	O
-conformation	O
-are	O
-illustrated	O
-in	O
-Fig	O
-.	O
-6	O
-for	O
-the	O
-18	O
-Fab	O
-structures	O
-that	O
-have	O
-ordered	O
-backbone	O
-atoms	O
-.	O
-	O
-A	O
-comparison	O
-of	O
-representatives	O
-of	O
-the	O
-“	O
-kinked	O
-”	O
-and	O
-“	O
-extended	O
-”	O
-structures	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-“	O
-kinked	O
-”	O
-CDR	O
-H3	O
-of	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-11	O
-with	O
-purple	O
-carbon	O
-atoms	O
-and	O
-yellow	O
-dashed	O
-lines	O
-connecting	O
-the	O
-H	O
--	O
-bond	O
-pairs	O
-for	O
-Leu100b	O
-O	O
-and	O
-Trp103	O
-NE1	O
-,	O
-Arg94	O
-NE	O
-and	O
-Asp101	O
-OD1	O
-,	O
-and	O
-Arg94	O
-NH2	O
-and	O
-Asp101	O
-OD2	O
-.	O
-	O
-(	O
-B	O
-)	O
-The	O
-“	O
-extended	O
-”	O
-CDR	O
-H3	O
-of	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-with	O
-green	O
-carbon	O
-atoms	O
-and	O
-yellow	O
-dashed	O
-lines	O
-connecting	O
-the	O
-H	O
--	O
-bond	O
-pairs	O
-for	O
-Asp101	O
-OD1	O
-and	O
-OD2	O
-and	O
-Trp103	O
-NE1	O
-.	O
-	O
-In	O
-10	O
-of	O
-the	O
-18	O
-Fab	O
-structures	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L1	O
--	O
-39	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-11	O
-(	O
-2	O
-Fabs	O
-),	O
-H1	O
--	O
-69	O
-:	O
-L4	O
--	O
-1	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-11	O
-(	O
-2	O
-Fabs	O
-),	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-H3	O
--	O
-53	O
-:	O
-L3	O
--	O
-11	O
-,	O
-H3	O
--	O
-53	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L1	O
--	O
-39	O
-,	O
-the	O
-CDRs	O
-have	O
-similar	O
-conformations	O
-to	O
-that	O
-found	O
-in	O
-4DN3	O
-.	O
-	O
-The	O
-bases	O
-of	O
-these	O
-structures	O
-have	O
-the	O
-‘	O
-kinked	O
-’	O
-conformation	O
-with	O
-the	O
-H	O
--	O
-bond	O
-between	O
-Trp103	O
-and	O
-Leu100b	O
-.	O
-	O
-A	O
-representative	O
-CDR	O
-H3	O
-structure	O
-for	O
-H1	O
--	O
-69	O
-:	O
-L1	O
--	O
-39	O
-illustrating	O
-this	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-7A	O
-.	O
-	O
-The	O
-largest	O
-backbone	O
-conformational	O
-deviation	O
-for	O
-the	O
-set	O
-is	O
-at	O
-Tyr99	O
-,	O
-where	O
-the	O
-C	O
-=	O
-O	O
-is	O
-rotated	O
-by	O
-90	O
-°	O
-relative	O
-to	O
-that	O
-observed	O
-in	O
-4DN3	O
-.	O
-	O
-Also	O
-,	O
-it	O
-is	O
-worth	O
-noting	O
-that	O
-only	O
-one	O
-of	O
-these	O
-structures	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L4	O
--	O
-1	O
-,	O
-has	O
-the	O
-conserved	O
-water	O
-molecule	O
-in	O
-CDR	O
-H3	O
-observed	O
-in	O
-the	O
-4DN3	O
-and	O
-4DN4	O
-structures	O
-.	O
-	O
-In	O
-fact	O
-,	O
-it	O
-is	O
-the	O
-only	O
-Fab	O
-in	O
-the	O
-set	O
-that	O
-has	O
-a	O
-water	O
-molecule	O
-present	O
-at	O
-this	O
-site	O
-.	O
-	O
-The	O
-CDR	O
-H3	O
-for	O
-this	O
-structure	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-S3	O
-.	O
-	O
-The	O
-remaining	O
-8	O
-Fabs	O
-can	O
-be	O
-grouped	O
-into	O
-5	O
-different	O
-conformational	O
-classes	O
-.	O
-	O
-Three	O
-of	O
-the	O
-Fabs	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L1	O
--	O
-39	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L4	O
--	O
-1	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L1	O
--	O
-39	O
-,	O
-have	O
-distinctive	O
-conformations	O
-.	O
-	O
-The	O
-stem	O
-regions	O
-in	O
-these	O
-3	O
-cases	O
-are	O
-in	O
-the	O
-‘	O
-kinked	O
-’	O
-conformation	O
-consistent	O
-with	O
-that	O
-observed	O
-for	O
-4DN3	O
-.	O
-	O
-The	O
-five	O
-remaining	O
-Fabs	O
-,	O
-H5	O
--	O
-51	O
-:	O
-L4	O
--	O
-1	O
-(	O
-2	O
-copies	O
-),	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-(	O
-2	O
-copies	O
-)	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L4	O
--	O
-1	O
-,	O
-have	O
-3	O
-different	O
-CDR	O
-H3	O
-conformations	O
-(	O
-Fig	O
-.	O
-S4	O
-).	O
-	O
-The	O
-stem	O
-regions	O
-of	O
-CDR	O
-H3	O
-for	O
-the	O
-H5	O
--	O
-51	O
-:	O
-L4	O
--	O
-1	O
-Fabs	O
-are	O
-in	O
-the	O
-‘	O
-kinked	O
-’	O
-conformation	O
-while	O
-,	O
-surprisingly	O
-,	O
-those	O
-of	O
-the	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-pair	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L4	O
--	O
-1	O
-are	O
-in	O
-the	O
-‘	O
-extended	O
-’	O
-conformation	O
-(	O
-Fig	O
-.	O
-7B	O
-).	O
-	O
-VH	O
-:	O
-VL	O
-domain	O
-packing	O
-	O
-The	O
-VH	O
-and	O
-VL	O
-domains	O
-have	O
-a	O
-β	O
--	O
-sandwich	O
-structure	O
-(	O
-also	O
-often	O
-referred	O
-as	O
-a	O
-Greek	O
-key	O
-motif	O
-)	O
-and	O
-each	O
-is	O
-composed	O
-of	O
-a	O
-4	O
--	O
-stranded	O
-and	O
-a	O
-5	O
--	O
-stranded	O
-antiparallel	O
-β	O
--	O
-sheets	O
-.	O
-	O
-The	O
-two	O
-domains	O
-pack	O
-together	O
-such	O
-that	O
-the	O
-5	O
--	O
-stranded	O
-β	O
--	O
-sheets	O
-,	O
-which	O
-have	O
-hydrophobic	O
-surfaces	O
-,	O
-interact	O
-with	O
-each	O
-other	O
-bringing	O
-the	O
-CDRs	O
-from	O
-both	O
-the	O
-VH	O
-and	O
-VL	O
-domains	O
-into	O
-close	O
-proximity	O
-.	O
-	O
-The	O
-domain	O
-packing	O
-of	O
-the	O
-variants	O
-was	O
-assessed	O
-by	O
-computing	O
-the	O
-domain	O
-interface	O
-interactions	O
-,	O
-the	O
-VH	O
-:	O
-VL	O
-tilt	O
-angles	O
-,	O
-the	O
-buried	O
-surface	O
-area	O
-and	O
-surface	O
-complementarity	O
-.	O
-	O
-VH	O
-:	O
-VL	O
-interface	O
-amino	O
-acid	O
-residue	O
-interactions	O
-	O
-The	O
-conserved	O
-VH	O
-:	O
-VL	O
-interactions	O
-as	O
-viewed	O
-along	O
-the	O
-VH	O
-/	O
-VL	O
-axis	O
-.	O
-	O
-The	O
-VH	O
-residues	O
-are	O
-in	O
-blue	O
-,	O
-the	O
-VL	O
-residues	O
-are	O
-in	O
-orange	O
-.	O
-	O
-The	O
-VH	O
-:	O
-VL	O
-interface	O
-is	O
-pseudosymmetric	O
-,	O
-and	O
-involves	O
-2	O
-stretches	O
-of	O
-the	O
-polypeptide	O
-chain	O
-from	O
-each	O
-domain	O
-,	O
-namely	O
-CDR3	O
-and	O
-the	O
-framework	O
-region	O
-between	O
-CDRs	O
-1	O
-and	O
-2	O
-.	O
-	O
-These	O
-stretches	O
-form	O
-antiparallel	O
-β	O
--	O
-hairpins	O
-within	O
-the	O
-internal	O
-5	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-.	O
-	O
-There	O
-are	O
-a	O
-few	O
-principal	O
-inter	O
--	O
-domain	O
-interactions	O
-that	O
-are	O
-conserved	O
-not	O
-only	O
-in	O
-the	O
-experimental	O
-set	O
-of	O
-16	O
-Fabs	O
-,	O
-but	O
-in	O
-all	O
-human	O
-antibodies	O
-.	O
-	O
-They	O
-include	O
-:	O
-1	O
-)	O
-a	O
-bidentate	O
-hydrogen	O
-bond	O
-between	O
-L	O
--	O
-Gln38	O
-and	O
-H	O
--	O
-Gln39	O
-;	O
-2	O
-)	O
-H	O
--	O
-Leu45	O
-in	O
-a	O
-hydrophobic	O
-pocket	O
-between	O
-L	O
--	O
-Phe98	O
-,	O
-L	O
--	O
-Tyr87	O
-and	O
-L	O
--	O
-Pro44	O
-;	O
-3	O
-)	O
-L	O
--	O
-Pro44	O
-stacked	O
-against	O
-H	O
--	O
-Trp103	O
-;	O
-and	O
-4	O
-)	O
-L	O
--	O
-Ala43	O
-opposite	O
-the	O
-face	O
-of	O
-H	O
--	O
-Tyr91	O
-(	O
-Fig	O
-.	O
-8	O
-).	O
-	O
-With	O
-the	O
-exception	O
-of	O
-L	O
--	O
-Ala43	O
-,	O
-all	O
-other	O
-residues	O
-are	O
-conserved	O
-in	O
-human	O
-germlines	O
-.	O
-	O
-Position	O
-43	O
-may	O
-be	O
-alternatively	O
-occupied	O
-by	O
-Ser	O
-,	O
-Val	O
-or	O
-Pro	O
-(	O
-as	O
-in	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-),	O
-but	O
-the	O
-hydrophobic	O
-interaction	O
-with	O
-H	O
--	O
-Tyr91	O
-is	O
-preserved	O
-.	O
-	O
-These	O
-core	O
-interactions	O
-provide	O
-enough	O
-stability	O
-to	O
-the	O
-VH	O
-:	O
-VL	O
-dimer	O
-so	O
-that	O
-additional	O
-VH	O
--	O
-VL	O
-contacts	O
-can	O
-tolerate	O
-amino	O
-acid	O
-sequence	O
-variations	O
-in	O
-CDRs	O
-H3	O
-and	O
-L3	O
-that	O
-form	O
-part	O
-of	O
-the	O
-VH	O
-:	O
-VL	O
-interface	O
-.	O
-	O
-In	O
-total	O
-,	O
-about	O
-20	O
-residues	O
-are	O
-involved	O
-in	O
-the	O
-VH	O
-:	O
-VL	O
-interactions	O
-on	O
-each	O
-side	O
-(	O
-Fig	O
-.	O
-S5	O
-).	O
-	O
-Half	O
-of	O
-them	O
-are	O
-in	O
-the	O
-framework	O
-regions	O
-and	O
-those	O
-residues	O
-(	O
-except	O
-residue	O
-61	O
-in	O
-HC	O
-,	O
-which	O
-is	O
-actually	O
-in	O
-CDR2	O
-in	O
-Kabat	O
-'	O
-s	O
-definition	O
-)	O
-are	O
-conserved	O
-in	O
-the	O
-set	O
-of	O
-16	O
-Fabs	O
-.	O
-	O
-One	O
-notable	O
-exception	O
-is	O
-H	O
--	O
-Trp47	O
-,	O
-which	O
-exhibits	O
-2	O
-conformations	O
-of	O
-the	O
-indole	O
-ring	O
-.	O
-	O
-In	O
-most	O
-of	O
-the	O
-structures	O
-,	O
-it	O
-has	O
-the	O
-χ2	O
-angle	O
-of	O
-∼	O
-80	O
-°,	O
-while	O
-the	O
-ring	O
-is	O
-flipped	O
-over	O
-(	O
-χ2	O
-=	O
-−	O
-100	O
-°)	O
-in	O
-H5	O
--	O
-51	O
-:	O
-L3	O
-:	O
-11	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-.	O
-	O
-Interestingly	O
-,	O
-these	O
-are	O
-the	O
-only	O
-2	O
-structures	O
-with	O
-residues	O
-missing	O
-in	O
-CDR	O
-H3	O
-because	O
-of	O
-disorder	O
-,	O
-although	O
-both	O
-structures	O
-are	O
-determined	O
-at	O
-high	O
-resolution	O
-and	O
-the	O
-rest	O
-of	O
-the	O
-structure	O
-is	O
-well	O
-defined	O
-.	O
-	O
-Apparently	O
-,	O
-residues	O
-flanking	O
-CDR	O
-H3	O
-in	O
-the	O
-2	O
-VH	O
-:	O
-VL	O
-pairings	O
-are	O
-inconsistent	O
-with	O
-any	O
-stable	O
-conformation	O
-of	O
-CDR	O
-H3	O
-,	O
-which	O
-translates	O
-into	O
-a	O
-less	O
-restricted	O
-conformational	O
-space	O
-for	O
-some	O
-of	O
-them	O
-,	O
-including	O
-H	O
--	O
-Trp47	O
-.	O
-	O
-VH	O
-:	O
-VL	O
-tilt	O
-angles	O
-	O
-The	O
-relative	O
-orientation	O
-of	O
-VH	O
-and	O
-VL	O
-has	O
-been	O
-measured	O
-in	O
-a	O
-number	O
-of	O
-different	O
-ways	O
-.	O
-	O
-The	O
-first	O
-approach	O
-uses	O
-ABangles	O
-,	O
-the	O
-results	O
-of	O
-which	O
-are	O
-shown	O
-in	O
-Table	O
-S2	O
-.	O
-	O
-The	O
-four	O
-LCs	O
-all	O
-are	O
-classified	O
-as	O
-Type	O
-A	O
-because	O
-they	O
-have	O
-a	O
-proline	O
-at	O
-position	O
-44	O
-,	O
-and	O
-the	O
-results	O
-for	O
-each	O
-orientation	O
-parameter	O
-are	O
-within	O
-the	O
-range	O
-of	O
-values	O
-of	O
-this	O
-type	O
-reported	O
-by	O
-Dunbar	O
-and	O
-co	O
--	O
-workers	O
-.	O
-	O
-In	O
-fact	O
-,	O
-the	O
-parameter	O
-values	O
-for	O
-the	O
-set	O
-of	O
-16	O
-Fabs	O
-are	O
-in	O
-the	O
-middle	O
-of	O
-the	O
-distribution	O
-observed	O
-for	O
-351	O
-non	O
--	O
-redundant	O
-antibody	O
-structures	O
-determined	O
-at	O
-3	O
-.	O
-0	O
-Å	O
-resolution	O
-or	O
-better	O
-.	O
-	O
-The	O
-only	O
-exception	O
-is	O
-HC1	O
-,	O
-which	O
-is	O
-shifted	O
-toward	O
-smaller	O
-angles	O
-with	O
-the	O
-mean	O
-value	O
-of	O
-70	O
-.	O
-8	O
-°	O
-as	O
-compared	O
-to	O
-the	O
-distribution	O
-centered	O
-at	O
-72	O
-°	O
-for	O
-the	O
-entire	O
-PDB	O
-.	O
-	O
-This	O
-probably	O
-reflects	O
-the	O
-invariance	O
-of	O
-CDR	O
-H3	O
-in	O
-the	O
-current	O
-set	O
-as	O
-opposed	O
-to	O
-the	O
-CDR	O
-H3	O
-diversity	O
-in	O
-the	O
-PDB	O
-.	O
-	O
-The	O
-second	O
-approach	O
-used	O
-for	O
-comparing	O
-tilt	O
-angles	O
-involved	O
-computing	O
-the	O
-difference	O
-in	O
-the	O
-tilt	O
-angles	O
-between	O
-all	O
-pairs	O
-of	O
-structures	O
-.	O
-	O
-For	O
-structures	O
-with	O
-2	O
-copies	O
-of	O
-the	O
-Fab	O
-in	O
-the	O
-asymmetric	O
-unit	O
-,	O
-only	O
-one	O
-structure	O
-was	O
-used	O
-.	O
-	O
-The	O
-differences	O
-between	O
-independent	O
-Fabs	O
-in	O
-the	O
-same	O
-structure	O
-are	O
-4	O
-.	O
-9	O
-°	O
-for	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-1	O
-.	O
-6	O
-°	O
-for	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-11	O
-,	O
-1	O
-.	O
-4	O
-°	O
-for	O
-H3	O
--	O
-23	O
-:	O
-L4	O
--	O
-1	O
-,	O
-3	O
-.	O
-3	O
-°	O
-for	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-11	O
-,	O
-and	O
-2	O
-.	O
-5	O
-°	O
-for	O
-H5	O
--	O
-51	O
-:	O
-L4	O
--	O
-1	O
-.	O
-	O
-With	O
-the	O
-exception	O
-of	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-the	O
-angles	O
-are	O
-within	O
-the	O
-range	O
-of	O
-2	O
--	O
-3	O
-°	O
-as	O
-are	O
-observed	O
-in	O
-the	O
-identical	O
-structures	O
-in	O
-the	O
-PDB	O
-.	O
-	O
-In	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-one	O
-of	O
-the	O
-Fabs	O
-is	O
-substantially	O
-disordered	O
-so	O
-that	O
-part	O
-of	O
-CDR	O
-H2	O
-(	O
-the	O
-outer	O
-β	O
--	O
-strand	O
-,	O
-residues	O
-55	O
--	O
-60	O
-)	O
-is	O
-completely	O
-missing	O
-.	O
-	O
-This	O
-kind	O
-of	O
-disorder	O
-may	O
-compromise	O
-the	O
-integrity	O
-of	O
-the	O
-VH	O
-domain	O
-and	O
-its	O
-interaction	O
-with	O
-the	O
-VL	O
-.	O
-	O
-Indeed	O
-,	O
-this	O
-Fab	O
-has	O
-the	O
-largest	O
-twist	O
-angle	O
-HC2	O
-within	O
-the	O
-experimental	O
-set	O
-that	O
-exceeds	O
-the	O
-mean	O
-value	O
-by	O
-2	O
-.	O
-5	O
-standard	O
-deviations	O
-(	O
-Table	O
-S2	O
-).	O
-	O
-An	O
-illustration	O
-of	O
-the	O
-difference	O
-in	O
-tilt	O
-angle	O
-for	O
-2	O
-pairs	O
-of	O
-variants	O
-by	O
-the	O
-superposition	O
-of	O
-the	O
-VH	O
-domains	O
-of	O
-(	O
-A	O
-)	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-on	O
-that	O
-of	O
-H5	O
--	O
-51	O
-:	O
-L1	O
--	O
-39	O
-(	O
-the	O
-VL	O
-domain	O
-is	O
-off	O
-by	O
-a	O
-rigid	O
--	O
-body	O
-roatation	O
-of	O
-10	O
-.	O
-5	O
-°)	O
-and	O
-(	O
-B	O
-)	O
-H1	O
--	O
-69	O
-:	O
-L4	O
--	O
-1	O
-on	O
-that	O
-of	O
-H5	O
--	O
-51	O
-:	O
-L1	O
--	O
-39	O
-(	O
-the	O
-VL	O
-domain	O
-is	O
-off	O
-by	O
-a	O
-rigid	O
--	O
-body	O
-roatation	O
-of	O
-1	O
-.	O
-6	O
-°).	O
-	O
-Differences	O
-in	O
-VH	O
-:	O
-VL	O
-tilt	O
-angles	O
-.	O
-	O
-The	O
-differences	O
-in	O
-the	O
-tilt	O
-angle	O
-are	O
-shown	O
-for	O
-all	O
-pairs	O
-of	O
-V	O
-regions	O
-in	O
-Table	O
-3	O
-.	O
-	O
-The	O
-smallest	O
-differences	O
-in	O
-the	O
-tilt	O
-angle	O
-are	O
-between	O
-the	O
-Fabs	O
-in	O
-isomorphous	O
-crystal	O
-forms	O
-.	O
-	O
-The	O
-largest	O
-deviations	O
-in	O
-the	O
-tilt	O
-angle	O
-,	O
-up	O
-to	O
-11	O
-.	O
-0	O
-°,	O
-are	O
-found	O
-for	O
-2	O
-structures	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-that	O
-stand	O
-out	O
-from	O
-the	O
-other	O
-Fabs	O
-.	O
-	O
-One	O
-of	O
-the	O
-2	O
-structures	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-has	O
-its	O
-CDR	O
-H3	O
-in	O
-the	O
-‘	O
-extended	O
-’	O
-conformation	O
-;	O
-the	O
-other	O
-structure	O
-has	O
-it	O
-in	O
-the	O
-‘	O
-kinked	O
-’	O
-conformation	O
-.	O
-	O
-Two	O
-examples	O
-illustrating	O
-large	O
-(	O
-10	O
-.	O
-5	O
-°)	O
-and	O
-small	O
-(	O
-1	O
-.	O
-6	O
-°)	O
-differences	O
-in	O
-the	O
-tilt	O
-angles	O
-are	O
-shown	O
-in	O
-Fig	O
-.	O
-9	O
-.	O
-	O
-VH	O
-:	O
-VL	O
-buried	O
-surface	O
-area	O
-and	O
-complementarity	O
-	O
-VH	O
-:	O
-VL	O
-surface	O
-areas	O
-and	O
-surface	O
-complementarity	O
-.	O
-	O
-Some	O
-side	O
-chain	O
-atoms	O
-in	O
-CDR	O
-H3	O
-are	O
-missing	O
-.	O
-	O
-Residues	O
-in	O
-CDR	O
-H3	O
-are	O
-missing	O
-:	O
-YGE	O
-in	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-11	O
-,	O
-GIY	O
-in	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-.	O
-	O
-The	O
-results	O
-of	O
-the	O
-PISA	O
-contact	O
-surface	O
-calculation	O
-and	O
-surface	O
-complementarity	O
-calculation	O
-are	O
-shown	O
-in	O
-Table	O
-4	O
-.	O
-	O
-The	O
-interface	O
-areas	O
-are	O
-calculated	O
-as	O
-the	O
-average	O
-of	O
-the	O
-VH	O
-and	O
-VL	O
-contact	O
-surfaces	O
-.	O
-	O
-Six	O
-of	O
-the	O
-16	O
-structures	O
-have	O
-CDR	O
-H3	O
-side	O
-chains	O
-or	O
-complete	O
-residues	O
-missing	O
-,	O
-and	O
-therefore	O
-their	O
-interfaces	O
-are	O
-much	O
-smaller	O
-than	O
-in	O
-the	O
-other	O
-10	O
-structures	O
-with	O
-complete	O
-CDRs	O
-(	O
-the	O
-results	O
-are	O
-provided	O
-for	O
-all	O
-Fabs	O
-for	O
-completeness	O
-).	O
-	O
-Among	O
-the	O
-complete	O
-structures	O
-,	O
-the	O
-interface	O
-areas	O
-range	O
-from	O
-684	O
-to	O
-836	O
-Å2	O
-.	O
-	O
-Interestingly	O
-,	O
-the	O
-2	O
-structures	O
-that	O
-have	O
-the	O
-largest	O
-tilt	O
-angle	O
-differences	O
-with	O
-the	O
-other	O
-variants	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-have	O
-the	O
-smallest	O
-VH	O
-:	O
-VL	O
-interfaces	O
-,	O
-684	O
-and	O
-725	O
-Å2	O
-,	O
-respectively	O
-.	O
-	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-is	O
-also	O
-unique	O
-in	O
-that	O
-it	O
-has	O
-the	O
-lowest	O
-value	O
-(	O
-0	O
-.	O
-676	O
-)	O
-of	O
-surface	O
-complementarity	O
-.	O
-	O
-Melting	O
-temperatures	O
-for	O
-the	O
-16	O
-Fabs	O
-.	O
-	O
-Colors	O
-:	O
-blue	O
-(	O
-Tm	O
-<	O
-70	O
-°	O
-C	O
-),	O
-green	O
-(	O
-70	O
-°	O
-C	O
-<	O
-Tm	O
-<	O
-73	O
-°	O
-C	O
-),	O
-yellow	O
-(	O
-73	O
-°	O
-C	O
-<	O
-Tm	O
-<	O
-78	O
-°	O
-C	O
-),	O
-orange	O
-(	O
-Tm	O
->	O
-78	O
-°	O
-C	O
-).	O
-	O
-Melting	O
-temperatures	O
-(	O
-Tm	O
-)	O
-were	O
-measured	O
-for	O
-all	O
-Fabs	O
-using	O
-differential	O
-scanning	O
-calorimetry	O
-(	O
-Table	O
-5	O
-).	O
-	O
-It	O
-appears	O
-that	O
-for	O
-each	O
-given	O
-LC	O
-,	O
-the	O
-Fabs	O
-with	O
-germlines	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-are	O
-substantially	O
-more	O
-stable	O
-than	O
-those	O
-with	O
-germlines	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-.	O
-	O
-In	O
-addition	O
-,	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-provides	O
-a	O
-much	O
-higher	O
-degree	O
-of	O
-stabilization	O
-than	O
-the	O
-other	O
-3	O
-LC	O
-germlines	O
-when	O
-combined	O
-with	O
-any	O
-of	O
-the	O
-HCs	O
-.	O
-	O
-As	O
-a	O
-result	O
-,	O
-the	O
-Tm	O
-for	O
-pairs	O
-H1	O
--	O
-69	O
-:	O
-L1	O
--	O
-39	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L1	O
--	O
-39	O
-is	O
-12	O
--	O
-13	O
-°	O
-higher	O
-than	O
-for	O
-pairs	O
-H3	O
--	O
-53	O
-:	O
-L3	O
--	O
-20	O
-,	O
-H3	O
--	O
-53	O
-:	O
-L4	O
--	O
-1	O
-,	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L4	O
--	O
-1	O
-.	O
-	O
-These	O
-findings	O
-correlate	O
-well	O
-with	O
-the	O
-degree	O
-of	O
-conformational	O
-disorder	O
-observed	O
-in	O
-the	O
-crystal	O
-structures	O
-.	O
-	O
-Parts	O
-of	O
-CDR	O
-H3	O
-main	O
-chain	O
-are	O
-completely	O
-disordered	O
-,	O
-and	O
-were	O
-not	O
-modeled	O
-in	O
-Fabs	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-11	O
-that	O
-have	O
-the	O
-lowest	O
-Tms	O
-in	O
-the	O
-set	O
-.	O
-	O
-No	O
-electron	O
-density	O
-is	O
-observed	O
-for	O
-a	O
-number	O
-of	O
-side	O
-chains	O
-in	O
-CDRs	O
-H3	O
-and	O
-L3	O
-in	O
-all	O
-Fabs	O
-with	O
-germline	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-,	O
-which	O
-indicates	O
-loose	O
-packing	O
-of	O
-the	O
-variable	O
-domains	O
-.	O
-	O
-All	O
-those	O
-molecules	O
-are	O
-relatively	O
-unstable	O
-,	O
-as	O
-is	O
-reflected	O
-in	O
-their	O
-low	O
-Tms	O
-.	O
-	O
-This	O
-is	O
-the	O
-first	O
-report	O
-of	O
-a	O
-systematic	O
-structural	O
-investigation	O
-of	O
-a	O
-phage	O
-germline	O
-library	O
-.	O
-	O
-The	O
-16	O
-Fab	O
-structures	O
-offer	O
-a	O
-unique	O
-look	O
-at	O
-all	O
-pairings	O
-of	O
-4	O
-different	O
-HCs	O
-(	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-,	O
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-)	O
-and	O
-4	O
-different	O
-LCs	O
-(	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-,	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-,	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-and	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-),	O
-all	O
-with	O
-the	O
-same	O
-CDR	O
-H3	O
-.	O
-	O
-The	O
-structural	O
-data	O
-set	O
-taken	O
-as	O
-a	O
-whole	O
-provides	O
-insight	O
-into	O
-how	O
-the	O
-backbone	O
-conformations	O
-of	O
-the	O
-CDRs	O
-of	O
-a	O
-specific	O
-heavy	O
-or	O
-light	O
-chain	O
-vary	O
-when	O
-it	O
-is	O
-paired	O
-with	O
-4	O
-different	O
-light	O
-or	O
-heavy	O
-chains	O
-,	O
-respectively	O
-.	O
-	O
-A	O
-large	O
-variability	O
-in	O
-the	O
-CDR	O
-conformations	O
-for	O
-the	O
-sets	O
-of	O
-HCs	O
-and	O
-LCs	O
-is	O
-observed	O
-.	O
-	O
-In	O
-some	O
-cases	O
-the	O
-CDR	O
-conformations	O
-for	O
-all	O
-members	O
-of	O
-a	O
-set	O
-are	O
-virtually	O
-identical	O
-,	O
-for	O
-others	O
-subtle	O
-changes	O
-occur	O
-in	O
-a	O
-few	O
-members	O
-of	O
-a	O
-set	O
-,	O
-and	O
-in	O
-some	O
-cases	O
-larger	O
-deviations	O
-are	O
-observed	O
-within	O
-a	O
-set	O
-.	O
-	O
-The	O
-five	O
-variants	O
-that	O
-crystallized	O
-with	O
-2	O
-copies	O
-of	O
-the	O
-Fab	O
-in	O
-the	O
-asymmetric	O
-unit	O
-serve	O
-somewhat	O
-as	O
-controls	O
-for	O
-the	O
-influence	O
-of	O
-crystal	O
-packing	O
-on	O
-the	O
-conformations	O
-of	O
-the	O
-CDRs	O
-.	O
-	O
-In	O
-four	O
-of	O
-the	O
-5	O
-structures	O
-the	O
-CDR	O
-conformations	O
-are	O
-consistent	O
-.	O
-	O
-In	O
-only	O
-one	O
-case	O
-,	O
-that	O
-of	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-(	O
-the	O
-lowest	O
-resolution	O
-structure	O
-),	O
-do	O
-we	O
-see	O
-differences	O
-in	O
-the	O
-conformations	O
-of	O
-the	O
-2	O
-copies	O
-of	O
-CDRs	O
-H1	O
-and	O
-L1	O
-.	O
-	O
-This	O
-variability	O
-is	O
-likely	O
-a	O
-result	O
-of	O
-2	O
-factors	O
-,	O
-crystal	O
-packing	O
-interactions	O
-and	O
-internal	O
-instability	O
-of	O
-the	O
-variable	O
-domain	O
-.	O
-	O
-For	O
-the	O
-CDRs	O
-with	O
-canonical	O
-structures	O
-,	O
-the	O
-largest	O
-changes	O
-in	O
-conformation	O
-occur	O
-for	O
-CDR	O
-H1	O
-of	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-.	O
-	O
-The	O
-other	O
-2	O
-HCs	O
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-,	O
-have	O
-canonical	O
-structures	O
-that	O
-are	O
-remarkably	O
-well	O
-conserved	O
-(	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-Of	O
-the	O
-4	O
-HCs	O
-,	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-has	O
-the	O
-greatest	O
-number	O
-of	O
-canonical	O
-structure	O
-assignments	O
-(	O
-Table	O
-2	O
-).	O
-	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-is	O
-unique	O
-in	O
-having	O
-a	O
-pair	O
-of	O
-glycine	O
-residues	O
-at	O
-positions	O
-26	O
-and	O
-27	O
-,	O
-which	O
-provide	O
-more	O
-conformational	O
-freedom	O
-in	O
-CDR	O
-H1	O
-.	O
-	O
-Besides	O
-IGHV1	B-mutant
--	I-mutant
-69	I-mutant
-,	O
-only	O
-the	O
-germlines	O
-of	O
-the	O
-VH4	O
-family	O
-possess	O
-double	O
-glycines	O
-in	O
-CDR	O
-H1	O
-,	O
-and	O
-it	O
-will	O
-be	O
-interesting	O
-to	O
-see	O
-if	O
-they	O
-are	O
-also	O
-conformationally	O
-unstable	O
-.	O
-	O
-Having	O
-all	O
-16	O
-VH	O
-:	O
-VL	O
-pairs	O
-with	O
-the	O
-same	O
-CDR	O
-H3	O
-provides	O
-some	O
-insights	O
-into	O
-why	O
-molecular	O
-modeling	O
-efforts	O
-of	O
-CDR	O
-H3	O
-have	O
-proven	O
-so	O
-difficult	O
-.	O
-	O
-As	O
-mentioned	O
-in	O
-the	O
-Results	O
-section	O
-,	O
-this	O
-data	O
-set	O
-is	O
-composed	O
-of	O
-21	O
-Fabs	O
-,	O
-since	O
-5	O
-of	O
-the	O
-16	O
-variants	O
-have	O
-2	O
-Fab	O
-copies	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-For	O
-the	O
-18	O
-Fabs	O
-with	O
-complete	O
-backbone	O
-atoms	O
-for	O
-CDR	O
-H3	O
-,	O
-10	O
-have	O
-conformations	O
-similar	O
-to	O
-that	O
-of	O
-the	O
-parent	O
-,	O
-while	O
-the	O
-others	O
-have	O
-significantly	O
-different	O
-conformations	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-Thus	O
-,	O
-it	O
-is	O
-likely	O
-that	O
-the	O
-CDR	O
-H3	O
-conformation	O
-is	O
-dependent	O
-upon	O
-2	O
-dominating	O
-factors	O
-:	O
-1	O
-)	O
-amino	O
-acid	O
-sequence	O
-;	O
-and	O
-2	O
-)	O
-VH	O
-and	O
-VL	O
-context	O
-.	O
-	O
-More	O
-than	O
-half	O
-of	O
-the	O
-variants	O
-retain	O
-the	O
-conformation	O
-of	O
-the	O
-parent	O
-despite	O
-having	O
-differences	O
-in	O
-the	O
-VH	O
-:	O
-VL	O
-pairing	O
-.	O
-	O
-This	O
-subset	O
-includes	O
-2	O
-structures	O
-with	O
-2	O
-copies	O
-of	O
-the	O
-Fab	O
-in	O
-the	O
-asymmetric	O
-unit	O
-,	O
-all	O
-of	O
-which	O
-are	O
-nearly	O
-identical	O
-in	O
-conformation	O
-.	O
-	O
-The	O
-remaining	O
-8	O
-structures	O
-exhibit	O
-“	O
-non	O
--	O
-parental	O
-”	O
-conformations	O
-,	O
-indicating	O
-that	O
-the	O
-VH	O
-and	O
-VL	O
-context	O
-can	O
-also	O
-be	O
-a	O
-dominating	O
-factor	O
-influencing	O
-CDR	O
-H3	O
-.	O
-	O
-This	O
-subset	O
-also	O
-has	O
-2	O
-structures	O
-with	O
-2	O
-Fab	O
-copies	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-Interestingly	O
-,	O
-as	O
-described	O
-earlier	O
-,	O
-these	O
-2	O
-pairs	O
-differ	O
-in	O
-the	O
-stem	O
-regions	O
-with	O
-the	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-pair	O
-in	O
-the	O
-‘	O
-extended	O
-’	O
-conformation	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L4	O
--	O
-1	O
-pair	O
-in	O
-the	O
-‘	O
-kinked	O
-’	O
-conformation	O
-.	O
-	O
-The	O
-CDR	O
-H3	O
-conformational	O
-analysis	O
-shows	O
-that	O
-,	O
-for	O
-each	O
-set	O
-of	O
-variants	O
-of	O
-one	O
-HC	O
-paired	O
-with	O
-the	O
-4	O
-different	O
-LCs	O
-,	O
-both	O
-“	O
-parental	O
-”	O
-and	O
-“	O
-non	O
--	O
-parental	O
-”	O
-conformations	O
-are	O
-observed	O
-.	O
-	O
-The	O
-same	O
-variability	O
-is	O
-observed	O
-for	O
-the	O
-sets	O
-of	O
-variants	O
-composed	O
-of	O
-one	O
-LC	O
-paired	O
-with	O
-each	O
-of	O
-the	O
-4	O
-HCs	O
-.	O
-	O
-Thus	O
-,	O
-no	O
-patterns	O
-of	O
-conformational	O
-preference	O
-for	O
-a	O
-particular	O
-HC	O
-or	O
-LC	O
-emerge	O
-to	O
-shed	O
-any	O
-direct	O
-light	O
-on	O
-what	O
-drives	O
-the	O
-conformational	O
-differences	O
-.	O
-	O
-This	O
-finding	O
-supports	O
-the	O
-hypothesis	O
-of	O
-Weitzner	O
-et	O
-al	O
-.	O
-that	O
-the	O
-H3	O
-conformation	O
-is	O
-controlled	O
-both	O
-by	O
-its	O
-sequence	O
-and	O
-its	O
-environment	O
-.	O
-	O
-In	O
-looking	O
-at	O
-a	O
-possible	O
-correlation	O
-between	O
-the	O
-tilt	O
-angle	O
-and	O
-the	O
-conformation	O
-of	O
-CDR	O
-H3	O
-,	O
-no	O
-clear	O
-trends	O
-are	O
-observed	O
-.	O
-	O
-Two	O
-variants	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-have	O
-the	O
-largest	O
-differences	O
-in	O
-the	O
-tilt	O
-angles	O
-compared	O
-to	O
-other	O
-variants	O
-as	O
-seen	O
-in	O
-Table	O
-3	O
-.	O
-	O
-The	O
-absolute	O
-VH	O
-:	O
-VL	O
-orientation	O
-parameters	O
-for	O
-the	O
-2	O
-Fabs	O
-(	O
-Table	O
-S2	O
-)	O
-show	O
-significant	O
-deviation	O
-in	O
-HL	O
-,	O
-LC1	O
-and	O
-HC2	O
-values	O
-(	O
-2	O
--	O
-3	O
-standard	O
-deviations	O
-from	O
-the	O
-mean	O
-).	O
-	O
-One	O
-of	O
-the	O
-variants	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-has	O
-the	O
-CDR	O
-H3	O
-conformation	O
-similar	O
-to	O
-the	O
-parent	O
-,	O
-but	O
-the	O
-other	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-is	O
-different	O
-.	O
-	O
-As	O
-noted	O
-in	O
-the	O
-Results	O
-section	O
-,	O
-the	O
-2	O
-variants	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-are	O
-outliers	O
-in	O
-terms	O
-of	O
-the	O
-tilt	O
-angle	O
-;	O
-at	O
-the	O
-same	O
-time	O
-,	O
-both	O
-have	O
-the	O
-smallest	O
-VH	O
-:	O
-VL	O
-interface	O
-.	O
-	O
-These	O
-smaller	O
-interfaces	O
-may	O
-perhaps	O
-translate	O
-to	O
-a	O
-significant	O
-deviation	O
-in	O
-how	O
-VH	O
-is	O
-oriented	O
-relative	O
-to	O
-VL	O
-than	O
-the	O
-other	O
-variants	O
-.	O
-	O
-These	O
-deviations	O
-from	O
-the	O
-other	O
-variants	O
-can	O
-also	O
-be	O
-seen	O
-to	O
-some	O
-extent	O
-in	O
-VH	O
-:	O
-VL	O
-orientation	O
-parameters	O
-in	O
-Table	O
-S2	O
-,	O
-as	O
-well	O
-as	O
-in	O
-the	O
-smaller	O
-number	O
-of	O
-residues	O
-involved	O
-in	O
-the	O
-VH	O
-:	O
-VL	O
-interfaces	O
-of	O
-these	O
-2	O
-variants	O
-(	O
-Fig	O
-.	O
-S5	O
-).	O
-	O
-These	O
-differences	O
-undoubtedly	O
-influence	O
-the	O
-conformation	O
-of	O
-the	O
-CDRs	O
-,	O
-in	O
-particular	O
-CDR	O
-H1	O
-(	O
-Fig	O
-.	O
-1A	O
-)	O
-and	O
-CDR	O
-L1	O
-(	O
-Fig	O
-.	O
-3C	O
-),	O
-especially	O
-with	O
-the	O
-tandem	O
-glycines	O
-and	O
-multiple	O
-serines	O
-present	O
-,	O
-respectively	O
-.	O
-	O
-Pairing	O
-of	O
-different	O
-germlines	O
-yields	O
-antibodies	O
-with	O
-various	O
-degrees	O
-of	O
-stability	O
-.	O
-	O
-As	O
-indicated	O
-by	O
-the	O
-melting	O
-temperatures	O
-,	O
-germlines	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-for	O
-HC	O
-and	O
-germline	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-for	O
-LC	O
-produce	O
-more	O
-stable	O
-Fabs	O
-compared	O
-to	O
-the	O
-other	O
-germlines	O
-in	O
-the	O
-experimental	O
-set	O
-.	O
-	O
-One	O
-possible	O
-explanation	O
-of	O
-the	O
-clear	O
-preference	O
-of	O
-LC	O
-germline	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-is	O
-that	O
-CDR	O
-L3	O
-has	O
-smaller	O
-residues	O
-at	O
-positions	O
-91	O
-and	O
-94	O
-,	O
-allowing	O
-for	O
-more	O
-room	O
-to	O
-accommodate	O
-CDR	O
-H3	O
-.	O
-	O
-Other	O
-germlines	O
-have	O
-bulky	O
-residues	O
-,	O
-Tyr	O
-,	O
-Arg	O
-and	O
-Trp	O
-,	O
-at	O
-these	O
-positions	O
-,	O
-whereas	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-has	O
-Ser	O
-and	O
-Thr	O
-.	O
-	O
-Various	O
-combinations	O
-of	O
-germline	O
-sequences	O
-for	O
-VL	O
-and	O
-VH	O
-impose	O
-certain	O
-constraints	O
-on	O
-CDR	O
-H3	O
-,	O
-which	O
-has	O
-to	O
-adapt	O
-to	O
-the	O
-environment	O
-.	O
-	O
-A	O
-more	O
-compact	O
-CDR	O
-L3	O
-may	O
-be	O
-beneficial	O
-in	O
-this	O
-situation	O
-.	O
-	O
-At	O
-the	O
-other	O
-end	O
-of	O
-the	O
-stability	O
-range	O
-is	O
-LC	O
-germline	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-,	O
-which	O
-yields	O
-antibodies	O
-with	O
-the	O
-lowest	O
-Tms	O
-.	O
-	O
-While	O
-pairings	O
-with	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-may	O
-be	O
-safely	O
-called	O
-a	O
-mismatch	O
-,	O
-those	O
-with	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-have	O
-Tms	O
-about	O
-5	O
--	O
-6	O
-°	O
-higher	O
-.	O
-	O
-Curiously	O
-,	O
-the	O
-2	O
-Fabs	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-deviate	O
-markedly	O
-in	O
-their	O
-tilt	O
-angles	O
-from	O
-the	O
-rest	O
-of	O
-the	O
-panel	O
-.	O
-	O
-It	O
-is	O
-possible	O
-that	O
-by	O
-adopting	O
-extreme	O
-tilt	O
-angles	O
-the	O
-structure	O
-modulates	O
-CDR	O
-H3	O
-and	O
-its	O
-environment	O
-,	O
-which	O
-apparently	O
-cannot	O
-be	O
-achieved	O
-solely	O
-by	O
-conformational	O
-rearrangement	O
-of	O
-the	O
-CDR	O
-.	O
-	O
-Note	O
-that	O
-most	O
-of	O
-the	O
-VH	O
-:	O
-VL	O
-interface	O
-residues	O
-are	O
-invariant	O
-;	O
-therefore	O
-,	O
-significant	O
-change	O
-of	O
-the	O
-tilt	O
-angle	O
-must	O
-come	O
-with	O
-a	O
-penalty	O
-in	O
-free	O
-energy	O
-.	O
-	O
-Yet	O
-,	O
-for	O
-the	O
-2	O
-antibodies	O
-,	O
-the	O
-total	O
-gain	O
-in	O
-stability	O
-merits	O
-the	O
-domain	O
-repacking	O
-.	O
-	O
-Overall	O
-,	O
-the	O
-stability	O
-of	O
-the	O
-Fab	O
-,	O
-as	O
-measured	O
-by	O
-Tm	O
-,	O
-is	O
-a	O
-result	O
-of	O
-the	O
-mutual	O
-adjustment	O
-of	O
-the	O
-HC	O
-and	O
-LC	O
-variable	O
-domains	O
-and	O
-adjustment	O
-of	O
-CDR	O
-H3	O
-to	O
-the	O
-VH	O
-:	O
-VL	O
-cleft	O
-.	O
-	O
-The	O
-final	O
-conformation	O
-represents	O
-an	O
-energetic	O
-minimum	O
-;	O
-however	O
-,	O
-in	O
-most	O
-cases	O
-it	O
-is	O
-very	O
-shallow	O
-,	O
-so	O
-that	O
-a	O
-single	O
-mutation	O
-can	O
-cause	O
-a	O
-dramatic	O
-rearrangement	O
-of	O
-the	O
-structure	O
-.	O
-	O
-In	O
-summary	O
-,	O
-the	O
-analysis	O
-of	O
-this	O
-structural	O
-library	O
-of	O
-germline	O
-variants	O
-composed	O
-of	O
-all	O
-pairs	O
-of	O
-4	O
-HCs	O
-and	O
-4LCs	O
-,	O
-all	O
-with	O
-the	O
-same	O
-CDR	O
-H3	O
-,	O
-offers	O
-some	O
-unique	O
-insights	O
-into	O
-antibody	O
-structure	O
-and	O
-how	O
-pairing	O
-and	O
-sequence	O
-may	O
-influence	O
-,	O
-or	O
-not	O
-,	O
-the	O
-canonical	O
-structures	O
-of	O
-the	O
-L1	O
-,	O
-L2	O
-,	O
-L3	O
-,	O
-H1	O
-and	O
-H2	O
-CDRs	O
-.	O
-	O
-Comparison	O
-of	O
-the	O
-CDR	O
-H3s	O
-reveals	O
-a	O
-large	O
-set	O
-of	O
-variants	O
-with	O
-conformations	O
-similar	O
-to	O
-the	O
-parent	O
-,	O
-while	O
-a	O
-second	O
-set	O
-has	O
-significant	O
-conformational	O
-variability	O
-,	O
-indicating	O
-that	O
-both	O
-the	O
-sequence	O
-and	O
-the	O
-structural	O
-context	O
-define	O
-the	O
-CDR	O
-H3	O
-conformation	O
-.	O
-	O
-Quite	O
-unexpectedly	O
-,	O
-2	O
-of	O
-the	O
-variants	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L4	O
--	O
-1	O
-,	O
-have	O
-the	O
-‘	O
-extended	O
-’	O
-stem	O
-region	O
-differing	O
-from	O
-the	O
-other	O
-14	O
-that	O
-have	O
-a	O
-‘	O
-kinked	O
-’	O
-stem	O
-region	O
-.	O
-	O
-These	O
-data	O
-reveal	O
-the	O
-difficulty	O
-of	O
-modeling	O
-CDR	O
-H3	O
-accurately	O
-,	O
-as	O
-shown	O
-again	O
-in	O
-Antibody	O
-Modeling	O
-Assessment	O
-II	O
-.	O
-	O
-Furthermore	O
-,	O
-antibody	O
-CDRs	O
-,	O
-H3	O
-in	O
-particular	O
-,	O
-may	O
-go	O
-through	O
-conformational	O
-changes	O
-upon	O
-binding	O
-their	O
-targets	O
-,	O
-making	O
-structural	O
-prediction	O
-for	O
-docking	O
-purposes	O
-an	O
-even	O
-more	O
-difficult	O
-task	O
-.	O
-	O
-Fortunately	O
-,	O
-for	O
-most	O
-applications	O
-of	O
-antibody	O
-modeling	O
-,	O
-such	O
-as	O
-engineering	O
-affinity	O
-and	O
-biophysical	O
-properties	O
-,	O
-an	O
-accurate	O
-CDR	O
-H3	O
-structure	O
-is	O
-not	O
-always	O
-necessary	O
-.	O
-	O
-For	O
-those	O
-applications	O
-where	O
-accurate	O
-CDR	O
-structures	O
-are	O
-essential	O
-,	O
-such	O
-as	O
-docking	O
-,	O
-the	O
-results	O
-in	O
-this	O
-work	O
-demonstrate	O
-the	O
-importance	O
-of	O
-experimental	O
-structures	O
-.	O
-	O
-With	O
-the	O
-recent	O
-advances	O
-in	O
-expression	O
-and	O
-crystallization	O
-methods	O
-,	O
-Fab	O
-structures	O
-can	O
-be	O
-obtained	O
-rapidly	O
-.	O
-	O
-The	O
-set	O
-of	O
-16	O
-germline	O
-Fab	O
-structures	O
-offers	O
-a	O
-unique	O
-dataset	O
-to	O
-facilitate	O
-software	O
-development	O
-for	O
-antibody	O
-modeling	O
-.	O
-	O
-The	O
-results	O
-essentially	O
-support	O
-the	O
-underlying	O
-idea	O
-of	O
-canonical	O
-structures	O
-,	O
-indicating	O
-that	O
-most	O
-CDRs	O
-with	O
-germline	O
-sequences	O
-tend	O
-to	O
-adopt	O
-predefined	O
-conformations	O
-.	O
-	O
-From	O
-this	O
-point	O
-of	O
-view	O
-,	O
-a	O
-novel	O
-approach	O
-to	O
-design	O
-combinatorial	O
-antibody	O
-libraries	O
-would	O
-be	O
-to	O
-cover	O
-the	O
-range	O
-of	O
-CDR	O
-conformations	O
-that	O
-may	O
-not	O
-necessarily	O
-coincide	O
-with	O
-the	O
-germline	O
-usage	O
-in	O
-the	O
-human	O
-repertoire	O
-.	O
-	O
-This	O
-would	O
-insure	O
-more	O
-structural	O
-diversity	O
-,	O
-leading	O
-to	O
-a	O
-more	O
-diverse	O
-panel	O
-of	O
-antibodies	O
-that	O
-would	O
-bind	O
-to	O
-a	O
-broad	O
-spectrum	O
-of	O
-targets	O
-.	O
-	O
-Visualizing	O
-chaperone	O
--	O
-assisted	O
-protein	O
-folding	O
-	O
-Challenges	O
-in	O
-determining	O
-the	O
-structures	O
-of	O
-heterogeneous	O
-and	O
-dynamic	O
-protein	O
-complexes	O
-have	O
-greatly	O
-hampered	O
-past	O
-efforts	O
-to	O
-obtain	O
-a	O
-mechanistic	O
-understanding	O
-of	O
-many	O
-important	O
-biological	O
-processes	O
-.	O
-	O
-One	O
-such	O
-process	O
-is	O
-chaperone	O
--	O
-assisted	O
-protein	O
-folding	O
-,	O
-where	O
-obtaining	O
-structural	O
-ensembles	O
-of	O
-chaperone	O
-:	O
-substrate	O
-complexes	O
-would	O
-ultimately	O
-reveal	O
-how	O
-chaperones	O
-help	O
-proteins	O
-fold	O
-into	O
-their	O
-native	O
-state	O
-.	O
-	O
-To	O
-address	O
-this	O
-problem	O
-,	O
-we	O
-devised	O
-a	O
-novel	O
-structural	O
-biology	O
-approach	O
-based	O
-on	O
-X	O
--	O
-ray	O
-crystallography	O
-,	O
-termed	O
-Residual	O
-Electron	O
-and	O
-Anomalous	O
-Density	O
-(	O
-READ	O
-).	O
-	O
-READ	O
-enabled	O
-us	O
-to	O
-visualize	O
-even	O
-sparsely	O
-populated	O
-conformations	O
-of	O
-the	O
-substrate	O
-protein	O
-immunity	O
-protein	O
-7	O
-(	O
-Im7	O
-)	O
-in	O
-complex	O
-with	O
-the	O
-E	O
-.	O
-coli	O
-chaperone	O
-Spy	O
-.	O
-	O
-This	O
-study	O
-resulted	O
-in	O
-a	O
-series	O
-of	O
-snapshots	O
-depicting	O
-the	O
-various	O
-folding	O
-states	O
-of	O
-Im7	O
-while	O
-bound	O
-to	O
-Spy	O
-.	O
-	O
-The	O
-ensemble	O
-shows	O
-that	O
-Spy	O
--	O
-associated	O
-Im7	O
-samples	O
-conformations	O
-ranging	O
-from	O
-unfolded	O
-to	O
-partially	O
-folded	O
-and	O
-native	O
--	O
-like	O
-states	O
-,	O
-and	O
-reveals	O
-how	O
-a	O
-substrate	O
-can	O
-explore	O
-its	O
-folding	O
-landscape	O
-while	O
-bound	O
-to	O
-a	O
-chaperone	O
-.	O
-	O
-High	O
--	O
-resolution	O
-structural	O
-models	O
-of	O
-protein	O
--	O
-protein	O
-interactions	O
-are	O
-critical	O
-for	O
-obtaining	O
-mechanistic	O
-insights	O
-into	O
-biological	O
-processes	O
-.	O
-	O
-However	O
-,	O
-many	O
-protein	O
--	O
-protein	O
-interactions	O
-are	O
-highly	O
-dynamic	O
-,	O
-making	O
-it	O
-difficult	O
-to	O
-obtain	O
-high	O
--	O
-resolution	O
-data	O
-.	O
-	O
-Particularly	O
-challenging	O
-are	O
-interactions	O
-of	O
-intrinsically	O
-or	O
-conditionally	O
-disordered	O
-sections	O
-of	O
-proteins	O
-with	O
-their	O
-partner	O
-proteins	O
-.	O
-	O
-Recent	O
-advances	O
-in	O
-X	O
--	O
-ray	O
-crystallography	O
-and	O
-NMR	O
-spectroscopy	O
-continue	O
-to	O
-improve	O
-our	O
-ability	O
-to	O
-analyze	O
-biomolecules	O
-that	O
-exist	O
-in	O
-multiple	O
-conformations	O
-.	O
-	O
-X	O
--	O
-ray	O
-crystallography	O
-has	O
-historically	O
-provided	O
-valuable	O
-information	O
-on	O
-small	O
--	O
-scale	O
-conformational	O
-changes	O
-,	O
-but	O
-observing	O
-large	O
--	O
-amplitude	O
-heterogeneous	O
-conformational	O
-changes	O
-often	O
-falls	O
-beyond	O
-the	O
-reach	O
-of	O
-current	O
-crystallographic	O
-techniques	O
-.	O
-	O
-NMR	O
-can	O
-theoretically	O
-be	O
-used	O
-to	O
-determine	O
-heterogeneous	O
-ensembles	O
-,	O
-but	O
-in	O
-practice	O
-,	O
-this	O
-proves	O
-to	O
-be	O
-very	O
-challenging	O
-.	O
-	O
-It	O
-is	O
-clear	O
-that	O
-molecular	O
-chaperones	O
-aid	O
-in	O
-protein	O
-folding	O
-.	O
-	O
-Structural	O
-characterization	O
-of	O
-chaperone	O
--	O
-assisted	O
-protein	O
-folding	O
-likely	O
-would	O
-help	O
-bring	O
-clarity	O
-to	O
-this	O
-question	O
-.	O
-	O
-Structural	O
-models	O
-of	O
-chaperone	O
--	O
-substrate	O
-complexes	O
-have	O
-recently	O
-begun	O
-to	O
-provide	O
-information	O
-as	O
-to	O
-how	O
-a	O
-chaperone	O
-can	O
-recognize	O
-its	O
-substrate	O
-.	O
-	O
-However	O
-,	O
-the	O
-impact	O
-that	O
-chaperones	O
-have	O
-on	O
-their	O
-substrates	O
-,	O
-and	O
-how	O
-these	O
-interactions	O
-affect	O
-the	O
-folding	O
-process	O
-remain	O
-largely	O
-unknown	O
-.	O
-	O
-For	O
-most	O
-chaperones	O
-,	O
-it	O
-is	O
-still	O
-unclear	O
-whether	O
-the	O
-chaperone	O
-actively	O
-participates	O
-in	O
-and	O
-affects	O
-the	O
-folding	O
-of	O
-the	O
-substrate	O
-proteins	O
-,	O
-or	O
-merely	O
-provides	O
-a	O
-suitable	O
-microenvironment	O
-enabling	O
-the	O
-substrate	O
-to	O
-fold	O
-on	O
-its	O
-own	O
-.	O
-	O
-This	O
-is	O
-a	O
-truly	O
-fundamental	O
-question	O
-in	O
-the	O
-chaperone	O
-field	O
-,	O
-and	O
-one	O
-that	O
-has	O
-eluded	O
-the	O
-community	O
-largely	O
-because	O
-of	O
-the	O
-highly	O
-dynamic	O
-nature	O
-of	O
-the	O
-chaperone	O
--	O
-substrate	O
-complexes	O
-.	O
-	O
-To	O
-address	O
-this	O
-question	O
-,	O
-we	O
-investigated	O
-the	O
-ATP	O
--	O
-independent	O
-Escherichia	O
-coli	O
-periplasmic	O
-chaperone	O
-Spy	O
-.	O
-	O
-Spy	O
-prevents	O
-protein	O
-aggregation	O
-and	O
-aids	O
-in	O
-protein	O
-folding	O
-under	O
-various	O
-stress	O
-conditions	O
-,	O
-including	O
-treatment	O
-with	O
-tannin	O
-and	O
-butanol	O
-.	O
-	O
-We	O
-originally	O
-discovered	O
-Spy	O
-by	O
-its	O
-ability	O
-to	O
-stabilize	O
-the	O
-protein	O
--	O
-folding	O
-model	O
-Im7	O
-in	O
-vivo	O
-and	O
-recently	O
-demonstrated	O
-that	O
-Im7	O
-folds	O
-while	O
-associated	O
-with	O
-Spy	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-Spy	O
-revealed	O
-that	O
-it	O
-forms	O
-a	O
-thin	O
-α	O
--	O
-helical	O
-homodimeric	O
-cradle	O
-.	O
-	O
-Crosslinking	O
-and	O
-genetic	O
-experiments	O
-suggested	O
-that	O
-Spy	O
-interacts	O
-with	O
-substrates	O
-somewhere	O
-on	O
-its	O
-concave	O
-side	O
-.	O
-	O
-By	O
-using	O
-a	O
-novel	O
-X	O
--	O
-ray	O
-crystallography	O
--	O
-based	O
-approach	O
-to	O
-model	O
-disorder	O
-in	O
-crystal	O
-structures	O
-,	O
-we	O
-have	O
-now	O
-determined	O
-the	O
-high	O
--	O
-resolution	O
-ensemble	O
-of	O
-the	O
-dynamic	O
-Spy	O
-:	O
-Im7	O
-complex	O
-.	O
-	O
-This	O
-work	O
-provides	O
-a	O
-detailed	O
-view	O
-of	O
-chaperone	O
--	O
-mediated	O
-protein	O
-folding	O
-and	O
-shows	O
-how	O
-substrates	O
-like	O
-Im7	O
-find	O
-their	O
-native	O
-fold	O
-while	O
-bound	O
-to	O
-their	O
-chaperones	O
-.	O
-	O
-Crystallizing	O
-the	O
-Spy	O
-:	O
-Im7	O
-complex	O
-	O
-We	O
-reasoned	O
-that	O
-to	O
-obtain	O
-crystals	O
-of	O
-complexes	O
-between	O
-Spy	O
-(	O
-domain	O
-boundaries	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-)	O
-and	O
-its	O
-substrate	O
-proteins	O
-,	O
-our	O
-best	O
-approach	O
-was	O
-to	O
-identify	O
-crystallization	O
-conditions	O
-that	O
-yielded	O
-Spy	O
-crystals	O
-in	O
-the	O
-presence	O
-of	O
-protein	O
-substrates	O
-but	O
-not	O
-in	O
-their	O
-absence	O
-.	O
-	O
-We	O
-therefore	O
-screened	O
-crystallization	O
-conditions	O
-for	O
-Spy	O
-with	O
-four	O
-different	O
-substrate	O
-proteins	O
-:	O
-a	O
-fragment	O
-of	O
-the	O
-largely	O
-unfolded	O
-bovine	O
-α	O
--	O
-casein	O
-protein	O
-,	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-E	O
-.	O
-coli	O
-Im7	O
-,	O
-an	O
-unfolded	O
-variant	O
-of	O
-Im7	O
-(	O
-L18A	B-mutant
-L19A	B-mutant
-L37A	B-mutant
-),	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-Im7	O
-(	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-),	O
-which	O
-encompasses	O
-the	O
-entire	O
-Spy	O
--	O
-binding	O
-portion	O
-of	O
-Im7	O
-.	O
-	O
-We	O
-found	O
-conditions	O
-in	O
-which	O
-all	O
-four	O
-substrates	O
-co	O
--	O
-crystallized	O
-with	O
-Spy	O
-,	O
-but	O
-in	O
-which	O
-Spy	O
-alone	O
-did	O
-not	O
-yield	O
-crystals	O
-.	O
-	O
-Subsequent	O
-crystal	O
-washing	O
-and	O
-dissolution	O
-experiments	O
-confirmed	O
-the	O
-presence	O
-of	O
-the	O
-substrates	O
-in	O
-the	O
-co	O
--	O
-crystals	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-The	O
-crystals	O
-diffracted	O
-to	O
-~	O
-1	O
-.	O
-8	O
-Å	O
-resolution	O
-.	O
-	O
-We	O
-used	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-selenomethionine	O
-crystals	O
-for	O
-phasing	O
-with	O
-single	O
--	O
-wavelength	O
-anomalous	O
-diffraction	O
-(	O
-SAD	O
-)	O
-experiments	O
-,	O
-and	O
-used	O
-this	O
-solution	O
-to	O
-build	O
-the	O
-well	O
--	O
-ordered	O
-Spy	O
-portions	O
-of	O
-all	O
-four	O
-complexes	O
-.	O
-	O
-However	O
-,	O
-modeling	O
-of	O
-the	O
-substrate	O
-in	O
-the	O
-complex	O
-proved	O
-to	O
-be	O
-a	O
-substantial	O
-challenge	O
-,	O
-as	O
-the	O
-electron	O
-density	O
-of	O
-the	O
-substrate	O
-was	O
-discontinuous	O
-and	O
-fragmented	O
-.	O
-	O
-Even	O
-the	O
-minimal	O
-binding	O
-portion	O
-of	O
-Im7	O
-(	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-)	O
-showed	O
-highly	O
-dispersed	O
-electron	O
-density	O
-(	O
-Fig	O
-.	O
-1a	O
-).	O
-	O
-We	O
-hypothesized	O
-that	O
-the	O
-fragmented	O
-density	O
-was	O
-due	O
-to	O
-multiple	O
-,	O
-partially	O
-occupied	O
-conformations	O
-of	O
-the	O
-substrate	O
-bound	O
-within	O
-the	O
-crystal	O
-.	O
-	O
-Such	O
-residual	O
-density	O
-is	O
-typically	O
-not	O
-considered	O
-usable	O
-by	O
-traditional	O
-X	O
--	O
-ray	O
-crystallography	O
-methods	O
-.	O
-	O
-Thus	O
-,	O
-we	O
-developed	O
-a	O
-new	O
-approach	O
-to	O
-interpret	O
-the	O
-chaperone	O
--	O
-bound	O
-substrate	O
-in	O
-multiple	O
-conformations	O
-.	O
-	O
-READ	O
-:	O
-a	O
-strategy	O
-to	O
-visualize	O
-heterogeneous	O
-and	O
-dynamic	O
-biomolecules	O
-	O
-To	O
-determine	O
-the	O
-structure	O
-of	O
-the	O
-substrate	O
-portion	O
-of	O
-these	O
-Spy	O
-:	O
-substrate	O
-complexes	O
-,	O
-we	O
-conceived	O
-of	O
-an	O
-approach	O
-that	O
-we	O
-term	O
-READ	O
-,	O
-for	O
-Residual	O
-Electron	O
-and	O
-Anomalous	O
-Density	O
-.	O
-	O
-We	O
-split	O
-this	O
-approach	O
-into	O
-five	O
-steps	O
-:	O
-(	O
-1	O
-)	O
-By	O
-using	O
-a	O
-well	O
--	O
-diffracting	O
-Spy	O
-:	O
-substrate	O
-co	O
--	O
-crystal	O
-,	O
-we	O
-first	O
-determined	O
-the	O
-structure	O
-of	O
-the	O
-folded	O
-domain	O
-of	O
-Spy	O
-and	O
-obtained	O
-high	O
-quality	O
-residual	O
-electron	O
-density	O
-within	O
-the	O
-dynamic	O
-regions	O
-of	O
-the	O
-substrate	O
-.	O
-	O
-(	O
-2	O
-)	O
-We	O
-then	O
-labeled	O
-individual	O
-residues	O
-in	O
-the	O
-flexible	O
-regions	O
-of	O
-the	O
-substrate	O
-with	O
-the	O
-strong	O
-anomalous	O
-scatterer	O
-iodine	O
-,	O
-which	O
-serves	O
-to	O
-locate	O
-these	O
-residues	O
-in	O
-three	O
--	O
-dimensional	O
-space	O
-using	O
-their	O
-anomalous	O
-density	O
-.	O
-	O
-(	O
-3	O
-)	O
-We	O
-performed	O
-molecular	O
-dynamics	O
-(	O
-MD	O
-)	O
-simulations	O
-to	O
-generate	O
-a	O
-pool	O
-of	O
-energetically	O
-reasonable	O
-conformations	O
-of	O
-the	O
-dynamic	O
-complex	O
-and	O
-(	O
-4	O
-)	O
-applied	O
-a	O
-sample	O
--	O
-and	O
--	O
-select	O
-algorithm	O
-to	O
-determine	O
-the	O
-minimal	O
-set	O
-of	O
-substrate	O
-conformations	O
-that	O
-fit	O
-both	O
-the	O
-residual	O
-and	O
-anomalous	O
-density	O
-.	O
-	O
-Importantly	O
-,	O
-even	O
-though	O
-we	O
-only	O
-labeled	O
-a	O
-subset	O
-of	O
-the	O
-residues	O
-in	O
-the	O
-flexible	O
-regions	O
-of	O
-the	O
-substrate	O
-with	O
-iodine	O
-,	O
-the	O
-residual	O
-electron	O
-density	O
-can	O
-provide	O
-spatial	O
-information	O
-on	O
-many	O
-of	O
-the	O
-other	O
-flexible	O
-residues	O
-.	O
-	O
-The	O
-electron	O
-density	O
-then	O
-allowed	O
-us	O
-to	O
-connect	O
-the	O
-labeled	O
-residues	O
-of	O
-the	O
-substrate	O
-by	O
-confining	O
-the	O
-protein	O
-chain	O
-within	O
-regions	O
-of	O
-detectable	O
-density	O
-.	O
-	O
-In	O
-this	O
-way	O
-,	O
-the	O
-two	O
-forms	O
-of	O
-data	O
-together	O
-were	O
-able	O
-to	O
-describe	O
-multiple	O
-conformations	O
-of	O
-the	O
-substrate	O
-within	O
-the	O
-crystal	O
-.	O
-	O
-As	O
-described	O
-in	O
-detail	O
-below	O
-,	O
-we	O
-developed	O
-the	O
-READ	O
-method	O
-to	O
-uncover	O
-the	O
-ensemble	O
-of	O
-conformations	O
-that	O
-the	O
-Spy	O
--	O
-binding	O
-domain	O
-of	O
-Im7	O
-(	O
-i	O
-.	O
-e	O
-.,	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-)	O
-adopts	O
-while	O
-bound	O
-to	O
-Spy	O
-.	O
-	O
-However	O
-,	O
-we	O
-believe	O
-that	O
-READ	O
-will	O
-prove	O
-generally	O
-applicable	O
-to	O
-visualizing	O
-heterogeneous	O
-and	O
-dynamic	O
-complexes	O
-that	O
-have	O
-previously	O
-escaped	O
-detailed	O
-structural	O
-analysis	O
-.	O
-	O
-Collecting	O
-READ	O
-data	O
-for	O
-the	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-complex	O
-	O
-To	O
-apply	O
-the	O
-READ	O
-technique	O
-to	O
-the	O
-folding	O
-mechanism	O
-employed	O
-by	O
-the	O
-chaperone	O
-Spy	O
-,	O
-we	O
-selected	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-for	O
-further	O
-investigation	O
-because	O
-NMR	O
-data	O
-suggested	O
-that	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-could	O
-recapitulate	O
-unfolded	O
-,	O
-partially	O
-folded	O
-,	O
-and	O
-native	O
--	O
-like	O
-states	O
-of	O
-Im7	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-Moreover	O
-,	O
-binding	O
-experiments	O
-indicated	O
-that	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-comprises	O
-the	O
-entire	O
-Spy	O
--	O
-binding	O
-region	O
-.	O
-	O
-To	O
-introduce	O
-the	O
-anomalous	O
-scatterer	O
-iodine	O
-,	O
-we	O
-replaced	O
-eight	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-residues	O
-with	O
-the	O
-non	O
--	O
-canonical	O
-amino	O
-acid	O
-4	O
--	O
-iodophenylalanine	O
-(	O
-pI	O
--	O
-Phe	O
-).	O
-	O
-Its	O
-strong	O
-anomalous	O
-scattering	O
-allowed	O
-us	O
-to	O
-track	O
-the	O
-positions	O
-of	O
-these	O
-individual	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-residues	O
-one	O
-at	O
-a	O
-time	O
-,	O
-potentially	O
-even	O
-if	O
-the	O
-residue	O
-was	O
-found	O
-in	O
-several	O
-locations	O
-in	O
-the	O
-same	O
-crystal	O
-.	O
-	O
-We	O
-then	O
-co	O
--	O
-crystallized	O
-Spy	O
-and	O
-the	O
-eight	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-peptides	O
-,	O
-each	O
-of	O
-which	O
-harbored	O
-an	O
-individual	O
-pI	O
--	O
-Phe	O
-substitution	O
-at	O
-one	O
-distinct	O
-position	O
-,	O
-and	O
-collected	O
-anomalous	O
-data	O
-for	O
-all	O
-eight	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-complexes	O
-(	O
-Fig	O
-.	O
-1B	O
-,	O
-Supplementary	O
-Table	O
-1	O
-Supplementary	O
-Dataset	O
-1	O
-,	O
-and	O
-Supplementary	O
-Table	O
-2	O
-).	O
-	O
-Consistent	O
-with	O
-our	O
-electron	O
-density	O
-map	O
-,	O
-we	O
-found	O
-that	O
-the	O
-majority	O
-of	O
-anomalous	O
-signals	O
-emerged	O
-in	O
-the	O
-cradle	O
-of	O
-Spy	O
-,	O
-implying	O
-that	O
-this	O
-is	O
-the	O
-likely	O
-Im7	O
-substrate	O
-binding	O
-site	O
-.	O
-	O
-Consistent	O
-with	O
-the	O
-fragmented	O
-density	O
-,	O
-however	O
-,	O
-we	O
-observed	O
-multiple	O
-iodine	O
-positions	O
-for	O
-seven	O
-of	O
-the	O
-eight	O
-substituted	O
-residues	O
-.	O
-	O
-Together	O
-,	O
-these	O
-results	O
-indicated	O
-that	O
-the	O
-Im7	O
-substrate	O
-binds	O
-Spy	O
-in	O
-multiple	O
-conformations	O
-.	O
-	O
-READ	O
-sample	O
--	O
-and	O
--	O
-select	O
-procedure	O
-	O
-To	O
-determine	O
-the	O
-structural	O
-ensemble	O
-that	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-adopts	O
-while	O
-bound	O
-to	O
-Spy	O
-,	O
-we	O
-combined	O
-the	O
-residual	O
-electron	O
-density	O
-and	O
-the	O
-anomalous	O
-signals	O
-from	O
-our	O
-pI	O
--	O
-Phe	O
-substituted	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-complexes	O
-.	O
-	O
-To	O
-generate	O
-an	O
-accurate	O
-depiction	O
-of	O
-the	O
-chaperone	O
--	O
-substrate	O
-interactions	O
-,	O
-we	O
-devised	O
-a	O
-selection	O
-protocol	O
-based	O
-on	O
-a	O
-sample	O
--	O
-and	O
--	O
-select	O
-procedure	O
-employed	O
-in	O
-NMR	O
-spectroscopy	O
-.	O
-	O
-During	O
-each	O
-round	O
-of	O
-the	O
-selection	O
-,	O
-a	O
-genetic	O
-algorithm	O
-alters	O
-the	O
-ensemble	O
-and	O
-its	O
-agreement	O
-to	O
-the	O
-experimental	O
-data	O
-is	O
-re	O
--	O
-evaluated	O
-.	O
-	O
-If	O
-successful	O
-,	O
-the	O
-selection	O
-identifies	O
-the	O
-smallest	O
-group	O
-of	O
-specific	O
-conformations	O
-that	O
-best	O
-fits	O
-the	O
-residual	O
-electron	O
-density	O
-and	O
-anomalous	O
-signals	O
-.	O
-	O
-The	O
-READ	O
-sample	O
--	O
-and	O
--	O
-select	O
-algorithm	O
-is	O
-diagrammed	O
-in	O
-Fig	O
-.	O
-2	O
-.	O
-	O
-Prior	O
-to	O
-performing	O
-the	O
-selection	O
-,	O
-we	O
-generated	O
-a	O
-large	O
-and	O
-diverse	O
-pool	O
-of	O
-chaperone	O
--	O
-substrate	O
-complexes	O
-using	O
-coarse	O
--	O
-grained	O
-MD	O
-simulations	O
-in	O
-a	O
-pseudo	O
--	O
-crystal	O
-environment	O
-(	O
-Fig	O
-.	O
-2	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-The	O
-coarse	O
--	O
-grained	O
-simulations	O
-are	O
-based	O
-on	O
-a	O
-single	O
--	O
-residue	O
-resolution	O
-model	O
-for	O
-protein	O
-folding	O
-and	O
-were	O
-extended	O
-here	O
-to	O
-describe	O
-Spy	O
--	O
-Im76	O
--	O
-45	O
-binding	O
-events	O
-(	O
-Online	O
-Methods	O
-).	O
-	O
-The	O
-initial	O
-conditions	O
-of	O
-the	O
-binding	O
-simulations	O
-are	O
-not	O
-biased	O
-toward	O
-a	O
-particular	O
-conformation	O
-of	O
-the	O
-substrate	O
-or	O
-any	O
-specific	O
-chaperone	O
--	O
-substrate	O
-interaction	O
-(	O
-Online	O
-Methods	O
-).	O
-	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-binds	O
-and	O
-unbinds	O
-to	O
-Spy	O
-throughout	O
-the	O
-simulations	O
-.	O
-	O
-This	O
-strategy	O
-allows	O
-a	O
-wide	O
-range	O
-of	O
-substrate	O
-conformations	O
-to	O
-interact	O
-with	O
-the	O
-chaperone	O
-.	O
-	O
-From	O
-the	O
-MD	O
-simulations	O
-,	O
-we	O
-extracted	O
-~	O
-10	O
-,	O
-000	O
-diverse	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-complexes	O
-to	O
-be	O
-used	O
-by	O
-the	O
-ensuing	O
-selection	O
-.	O
-	O
-Each	O
-complex	O
-within	O
-this	O
-pool	O
-comprises	O
-one	O
-Spy	O
-dimer	O
-bound	O
-to	O
-a	O
-single	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-substrate	O
-.	O
-	O
-This	O
-pool	O
-was	O
-then	O
-used	O
-by	O
-the	O
-selection	O
-algorithm	O
-to	O
-identify	O
-the	O
-minimal	O
-ensemble	O
-that	O
-best	O
-satisfies	O
-both	O
-the	O
-residual	O
-electron	O
-and	O
-anomalous	O
-crystallographic	O
-data	O
-.	O
-	O
-The	O
-anomalous	O
-scattering	O
-portion	O
-of	O
-the	O
-selection	O
-uses	O
-our	O
-basic	O
-knowledge	O
-of	O
-pI	O
--	O
-Phe	O
-geometry	O
-:	O
-the	O
-iodine	O
-is	O
-separated	O
-from	O
-its	O
-respective	O
-Cα	O
-atom	O
-in	O
-each	O
-coarse	O
--	O
-grained	O
-conformer	O
-by	O
-6	O
-.	O
-5	O
-Å	O
-.	O
-The	O
-selection	O
-then	O
-picks	O
-ensembles	O
-that	O
-best	O
-reproduce	O
-the	O
-collection	O
-of	O
-iodine	O
-anomalous	O
-signals	O
-.	O
-	O
-Simultaneously	O
-,	O
-it	O
-uses	O
-the	O
-residual	O
-electron	O
-density	O
-to	O
-help	O
-choose	O
-ensembles	O
-.	O
-	O
-To	O
-make	O
-the	O
-electron	O
-density	O
-selection	O
-practical	O
-,	O
-we	O
-needed	O
-to	O
-develop	O
-a	O
-method	O
-to	O
-rapidly	O
-evaluate	O
-the	O
-agreement	O
-between	O
-the	O
-selected	O
-sub	O
--	O
-ensembles	O
-and	O
-the	O
-experimental	O
-electron	O
-density	O
-on	O
--	O
-the	O
--	O
-fly	O
-during	O
-the	O
-selection	O
-procedure	O
-.	O
-	O
-To	O
-accomplish	O
-this	O
-task	O
-,	O
-we	O
-generated	O
-a	O
-compressed	O
-version	O
-of	O
-the	O
-experimental	O
-2mFo	O
-−	O
-DFc	O
-electron	O
-density	O
-map	O
-for	O
-use	O
-in	O
-the	O
-selection	O
-.	O
-	O
-This	O
-process	O
-provided	O
-us	O
-with	O
-a	O
-target	O
-map	O
-that	O
-the	O
-ensuing	O
-selection	O
-tried	O
-to	O
-recapitulate	O
-.	O
-	O
-To	O
-reduce	O
-the	O
-extent	O
-of	O
-3D	O
-space	O
-to	O
-be	O
-explored	O
-,	O
-this	O
-compressed	O
-map	O
-was	O
-created	O
-by	O
-only	O
-using	O
-density	O
-from	O
-regions	O
-of	O
-space	O
-significantly	O
-sampled	O
-by	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-in	O
-the	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-MD	O
-simulations	O
-.	O
-	O
-For	O
-each	O
-of	O
-the	O
-~	O
-10	O
-,	O
-000	O
-complexes	O
-in	O
-the	O
-coarse	O
--	O
-grained	O
-MD	O
-pool	O
-,	O
-the	O
-electron	O
-density	O
-at	O
-the	O
-Cα	O
-positions	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-was	O
-extracted	O
-and	O
-used	O
-to	O
-construct	O
-an	O
-electron	O
-density	O
-map	O
-(	O
-Online	O
-Methods	O
-).	O
-	O
-These	O
-individual	O
-electron	O
-density	O
-maps	O
-from	O
-the	O
-separate	O
-conformers	O
-could	O
-then	O
-be	O
-combined	O
-(	O
-Fig	O
-.	O
-2	O
-)	O
-and	O
-compared	O
-to	O
-the	O
-averaged	O
-experimental	O
-electron	O
-density	O
-map	O
-as	O
-part	O
-of	O
-the	O
-selection	O
-algorithm	O
-.	O
-	O
-This	O
-approach	O
-allowed	O
-us	O
-to	O
-simultaneously	O
-use	O
-both	O
-the	O
-iodine	O
-anomalous	O
-signals	O
-and	O
-the	O
-residual	O
-electron	O
-density	O
-in	O
-the	O
-selection	O
-procedure	O
-.	O
-	O
-The	O
-selection	O
-resulted	O
-in	O
-small	O
-ensembles	O
-from	O
-the	O
-MD	O
-pool	O
-that	O
-best	O
-fit	O
-the	O
-READ	O
-data	O
-(	O
-Fig	O
-.	O
-1c	O
-,	O
-d	O
-).	O
-	O
-Before	O
-analyzing	O
-the	O
-details	O
-of	O
-the	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-complex	O
-,	O
-we	O
-first	O
-engaged	O
-in	O
-a	O
-series	O
-of	O
-validation	O
-tests	O
-to	O
-verify	O
-the	O
-ensemble	O
-and	O
-selection	O
-procedure	O
-(	O
-Supplementary	O
-Note	O
-1	O
-,	O
-Figures	O
-1c	O
-,	O
-d	O
-,	O
-Supplemental	O
-Figures	O
-5	O
--	O
-7	O
-).	O
-	O
-Of	O
-note	O
-,	O
-the	O
-final	O
-six	O
--	O
-membered	O
-ensemble	O
-was	O
-the	O
-largest	O
-ensemble	O
-that	O
-could	O
-simultaneously	O
-decrease	O
-the	O
-RFree	O
-and	O
-pass	O
-the	O
-10	O
--	O
-fold	O
-cross	O
--	O
-validation	O
-test	O
-.	O
-	O
-This	O
-ensemble	O
-depicts	O
-the	O
-conformations	O
-that	O
-the	O
-substrate	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-adopts	O
-while	O
-bound	O
-to	O
-the	O
-chaperone	O
-Spy	O
-(	O
-Fig	O
-.	O
-3	O
-Supplementary	O
-Movie	O
-1	O
-,	O
-and	O
-Table	O
-1	O
-).	O
-	O
-Folding	O
-and	O
-interactions	O
-of	O
-Im7	O
-while	O
-bound	O
-to	O
-Spy	O
-	O
-Our	O
-results	O
-showed	O
-that	O
-by	O
-using	O
-this	O
-novel	O
-READ	O
-approach	O
-,	O
-we	O
-were	O
-able	O
-to	O
-obtain	O
-structural	O
-information	O
-about	O
-the	O
-dynamic	O
-interaction	O
-of	O
-a	O
-chaperone	O
-with	O
-its	O
-substrate	O
-protein	O
-.	O
-	O
-We	O
-were	O
-particularly	O
-interested	O
-in	O
-finding	O
-answers	O
-to	O
-one	O
-of	O
-the	O
-most	O
-fundamental	O
-questions	O
-in	O
-chaperone	O
-biology	O
-—	O
-how	O
-does	O
-chaperone	O
-binding	O
-affect	O
-substrate	O
-structure	O
-and	O
-vice	O
-versa	O
-.	O
-	O
-By	O
-analyzing	O
-the	O
-individual	O
-structures	O
-of	O
-the	O
-six	O
--	O
-member	O
-ensemble	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-bound	O
-to	O
-Spy	O
-,	O
-we	O
-observed	O
-that	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-takes	O
-on	O
-several	O
-different	O
-conformations	O
-while	O
-bound	O
-.	O
-	O
-We	O
-found	O
-these	O
-conformations	O
-to	O
-be	O
-highly	O
-heterogeneous	O
-and	O
-to	O
-include	O
-unfolded	O
-,	O
-partially	O
-folded	O
-,	O
-and	O
-native	O
--	O
-like	O
-states	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-The	O
-ensemble	O
-primarily	O
-encompasses	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-laying	O
-diagonally	O
-within	O
-the	O
-Spy	O
-cradle	O
-in	O
-several	O
-different	O
-orientations	O
-,	O
-but	O
-some	O
-conformations	O
-traverse	O
-as	O
-far	O
-as	O
-the	O
-tips	O
-or	O
-even	O
-extend	O
-over	O
-the	O
-side	O
-of	O
-the	O
-cradle	O
-(	O
-Figs	O
-.	O
-3	O
-,	O
-4a	O
-).	O
-	O
-We	O
-constructed	O
-a	O
-contact	O
-map	O
-of	O
-the	O
-complex	O
-,	O
-which	O
-shows	O
-the	O
-frequency	O
-of	O
-interactions	O
-for	O
-chaperone	O
--	O
-substrate	O
-residue	O
-pairs	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-We	O
-found	O
-that	O
-the	O
-primary	O
-interaction	O
-sites	O
-on	O
-Spy	O
-reside	O
-at	O
-the	O
-N	O
-and	O
-C	O
-termini	O
-(	O
-Arg122	O
-,	O
-Thr124	O
-,	O
-and	O
-Phe29	O
-)	O
-as	O
-well	O
-as	O
-on	O
-the	O
-concave	O
-face	O
-of	O
-the	O
-chaperone	O
-(	O
-Arg61	O
-,	O
-Arg43	O
-,	O
-Lys47	O
-,	O
-His96	O
-,	O
-and	O
-Met46	O
-).	O
-	O
-The	O
-Spy	O
--	O
-contacting	O
-residues	O
-comprise	O
-a	O
-mixture	O
-of	O
-charged	O
-,	O
-polar	O
-,	O
-and	O
-hydrophobic	O
-residues	O
-.	O
-	O
-Surprisingly	O
-,	O
-we	O
-noted	O
-that	O
-in	O
-the	O
-ensemble	O
-,	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-interacts	O
-with	O
-only	O
-38	O
-%	O
-of	O
-the	O
-hydrophobic	O
-residues	O
-in	O
-the	O
-Spy	O
-cradle	O
-,	O
-but	O
-interacts	O
-with	O
-61	O
-%	O
-of	O
-the	O
-hydrophilic	O
-residues	O
-in	O
-the	O
-cradle	O
-.	O
-	O
-This	O
-mixture	O
-suggests	O
-the	O
-importance	O
-of	O
-both	O
-electrostatic	O
-and	O
-hydrophobic	O
-components	O
-in	O
-binding	O
-the	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-ensemble	O
-.	O
-	O
-With	O
-respect	O
-to	O
-the	O
-substrate	O
-,	O
-we	O
-observed	O
-that	O
-nearly	O
-every	O
-residue	O
-in	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-is	O
-in	O
-contact	O
-with	O
-Spy	O
-(	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-However	O
-,	O
-we	O
-did	O
-notice	O
-that	O
-despite	O
-this	O
-uniformity	O
-,	O
-regions	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-preferentially	O
-interact	O
-with	O
-different	O
-regions	O
-in	O
-Spy	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-For	O
-example	O
-,	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-binds	O
-more	O
-consistently	O
-in	O
-the	O
-Spy	O
-cradle	O
-,	O
-whereas	O
-the	O
-C	O
--	O
-terminal	O
-half	O
-predominantly	O
-binds	O
-to	O
-the	O
-outer	O
-edges	O
-of	O
-Spy	O
-’	O
-s	O
-concave	O
-surface	O
-.	O
-	O
-Not	O
-unexpectedly	O
-,	O
-we	O
-found	O
-that	O
-as	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-progresses	O
-from	O
-the	O
-unfolded	O
-to	O
-the	O
-native	O
-state	O
-,	O
-its	O
-interactions	O
-with	O
-Spy	O
-shift	O
-accordingly	O
-.	O
-	O
-Whereas	O
-the	O
-least	O
--	O
-folded	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-pose	O
-in	O
-the	O
-ensemble	O
-forms	O
-the	O
-most	O
-hydrophobic	O
-contacts	O
-with	O
-Spy	O
-(	O
-Fig	O
-.	O
-3	O
-),	O
-the	O
-two	O
-most	O
--	O
-folded	O
-conformations	O
-form	O
-the	O
-fewest	O
-hydrophobic	O
-contacts	O
-(	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-This	O
-shift	O
-in	O
-contacts	O
-is	O
-likely	O
-due	O
-to	O
-hydrophobic	O
-residues	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-preferentially	O
-forming	O
-intra	O
--	O
-molecular	O
-contacts	O
-upon	O
-folding	O
-(	O
-i	O
-.	O
-e	O
-.,	O
-hydrophobic	O
-collapse	O
-),	O
-effectively	O
-removing	O
-themselves	O
-from	O
-the	O
-interaction	O
-sites	O
-.	O
-	O
-The	O
-diversity	O
-of	O
-conformations	O
-and	O
-binding	O
-sites	O
-observed	O
-here	O
-emphasizes	O
-the	O
-dynamic	O
-and	O
-heterogeneous	O
-nature	O
-of	O
-the	O
-chaperone	O
--	O
-substrate	O
-ensemble	O
-.	O
-	O
-Although	O
-we	O
-do	O
-not	O
-yet	O
-have	O
-time	O
-resolution	O
-data	O
-of	O
-these	O
-various	O
-snapshots	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-,	O
-this	O
-ensemble	O
-illustrates	O
-how	O
-a	O
-substrate	O
-samples	O
-its	O
-folding	O
-landscape	O
-while	O
-bound	O
-to	O
-a	O
-chaperone	O
-.	O
-	O
-Spy	O
-changes	O
-conformation	O
-upon	O
-substrate	O
-binding	O
-	O
-Comparing	O
-the	O
-structure	O
-of	O
-Spy	O
-in	O
-its	O
-substrate	O
--	O
-bound	O
-and	O
-apo	O
-states	O
-revealed	O
-that	O
-the	O
-Spy	O
-dimer	O
-also	O
-undergoes	O
-significant	O
-conformational	O
-changes	O
-upon	O
-substrate	O
-binding	O
-(	O
-Fig	O
-.	O
-5a	O
-and	O
-Supplementary	O
-Movie	O
-2	O
-).	O
-	O
-Upon	O
-substrate	O
-binding	O
-,	O
-the	O
-Spy	O
-dimer	O
-twists	O
-9	O
-°	O
-about	O
-its	O
-center	O
-relative	O
-to	O
-its	O
-apo	O
-form	O
-.	O
-	O
-This	O
-twist	O
-yields	O
-asymmetry	O
-and	O
-results	O
-in	O
-substantially	O
-different	O
-interaction	O
-patterns	O
-in	O
-the	O
-two	O
-Spy	O
-monomers	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-It	O
-is	O
-possible	O
-that	O
-this	O
-twist	O
-serves	O
-to	O
-increase	O
-heterogeneity	O
-in	O
-Spy	O
-by	O
-providing	O
-more	O
-binding	O
-poses	O
-.	O
-	O
-Additionally	O
-,	O
-we	O
-observed	O
-that	O
-the	O
-linker	O
-region	O
-(	O
-residues	O
-47	O
-–	O
-57	O
-)	O
-of	O
-Spy	O
-,	O
-which	O
-participates	O
-in	O
-substrate	O
-interaction	O
-,	O
-becomes	O
-mostly	O
-disordered	O
-upon	O
-binding	O
-the	O
-substrate	O
-.	O
-	O
-This	O
-increased	O
-disorder	O
-might	O
-explain	O
-how	O
-Spy	O
-is	O
-able	O
-to	O
-recognize	O
-and	O
-bind	O
-different	O
-substrates	O
-and	O
-/	O
-or	O
-differing	O
-conformations	O
-of	O
-the	O
-same	O
-substrate	O
-.	O
-	O
-Importantly	O
-,	O
-we	O
-observed	O
-the	O
-same	O
-structural	O
-changes	O
-in	O
-Spy	O
-regardless	O
-of	O
-which	O
-of	O
-the	O
-four	O
-substrates	O
-was	O
-bound	O
-(	O
-Fig	O
-.	O
-5b	O
-,	O
-Table	O
-1	O
-).	O
-	O
-The	O
-RMSD	O
-between	O
-the	O
-well	O
--	O
-folded	O
-sections	O
-of	O
-Spy	O
-in	O
-the	O
-four	O
-chaperone	O
--	O
-substrate	O
-complexes	O
-was	O
-very	O
-small	O
-,	O
-less	O
-than	O
-0	O
-.	O
-3	O
-Å	O
-.	O
-Combined	O
-with	O
-competition	O
-experiments	O
-showing	O
-that	O
-the	O
-substrates	O
-compete	O
-in	O
-solution	O
-for	O
-Spy	O
-binding	O
-(	O
-Fig	O
-.	O
-5c	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-8	O
-),	O
-we	O
-conclude	O
-that	O
-all	O
-the	O
-tested	O
-substrates	O
-share	O
-the	O
-same	O
-overall	O
-Spy	O
-binding	O
-site	O
-.	O
-	O
-To	O
-shed	O
-light	O
-on	O
-how	O
-chaperones	O
-interact	O
-with	O
-their	O
-substrates	O
-,	O
-we	O
-developed	O
-a	O
-novel	O
-structural	O
-biology	O
-method	O
-(	O
-READ	O
-)	O
-and	O
-applied	O
-it	O
-to	O
-determine	O
-a	O
-conformational	O
-ensemble	O
-of	O
-the	O
-chaperone	O
-Spy	O
-bound	O
-to	O
-substrate	O
-.	O
-	O
-As	O
-a	O
-substrate	O
-,	O
-we	O
-used	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-,	O
-the	O
-chaperone	O
--	O
-interacting	O
-portion	O
-of	O
-the	O
-protein	O
--	O
-folding	O
-model	O
-protein	O
-Im7	O
-.	O
-	O
-In	O
-the	O
-chaperone	O
--	O
-bound	O
-ensemble	O
-,	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-samples	O
-unfolded	O
-,	O
-partially	O
-folded	O
-,	O
-and	O
-native	O
--	O
-like	O
-states	O
-.	O
-	O
-The	O
-ensemble	O
-provides	O
-an	O
-unprecedented	O
-description	O
-of	O
-the	O
-conformations	O
-that	O
-a	O
-substrate	O
-assumes	O
-while	O
-exploring	O
-its	O
-chaperone	O
--	O
-associated	O
-folding	O
-landscape	O
-.	O
-	O
-This	O
-substrate	O
--	O
-chaperone	O
-ensemble	O
-helps	O
-accomplish	O
-the	O
-longstanding	O
-goal	O
-of	O
-obtaining	O
-a	O
-detailed	O
-view	O
-of	O
-how	O
-a	O
-chaperone	O
-aids	O
-protein	O
-folding	O
-.	O
-	O
-We	O
-recently	O
-showed	O
-that	O
-Im7	O
-can	O
-fold	O
-while	O
-remaining	O
-continuously	O
-bound	O
-to	O
-Spy	O
-.	O
-	O
-The	O
-high	O
--	O
-resolution	O
-ensemble	O
-obtained	O
-here	O
-now	O
-provides	O
-insight	O
-into	O
-exactly	O
-how	O
-this	O
-occurs	O
-.	O
-	O
-The	O
-structures	O
-of	O
-our	O
-ensemble	O
-agree	O
-well	O
-with	O
-lower	O
--	O
-resolution	O
-crosslinking	O
-data	O
-,	O
-which	O
-indicate	O
-that	O
-chaperone	O
--	O
-substrate	O
-interactions	O
-primarily	O
-occur	O
-on	O
-the	O
-concave	O
-surface	O
-of	O
-Spy	O
-.	O
-	O
-The	O
-ensemble	O
-suggests	O
-a	O
-model	O
-in	O
-which	O
-Spy	O
-provides	O
-an	O
-amphipathic	O
-surface	O
-that	O
-allows	O
-substrate	O
-proteins	O
-to	O
-assume	O
-different	O
-conformations	O
-while	O
-bound	O
-to	O
-the	O
-chaperone	O
-.	O
-	O
-This	O
-model	O
-is	O
-consistent	O
-with	O
-previous	O
-studies	O
-postulating	O
-that	O
-the	O
-flexible	O
-binding	O
-of	O
-chaperones	O
-allows	O
-for	O
-substrate	O
-protein	O
-folding	O
-.	O
-	O
-The	O
-amphipathic	O
-concave	O
-surface	O
-of	O
-Spy	O
-likely	O
-facilitates	O
-this	O
-flexible	O
-binding	O
-and	O
-may	O
-be	O
-a	O
-crucial	O
-feature	O
-for	O
-Spy	O
-and	O
-potentially	O
-other	O
-chaperones	O
-,	O
-allowing	O
-them	O
-to	O
-bind	O
-multiple	O
-conformations	O
-of	O
-many	O
-different	O
-substrates	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-Spy	O
-’	O
-s	O
-binding	O
-hotspots	O
-,	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-displays	O
-substantially	O
-less	O
-specificity	O
-in	O
-its	O
-binding	O
-sites	O
-.	O
-	O
-Nearly	O
-all	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-residues	O
-come	O
-in	O
-contact	O
-with	O
-Spy	O
-.	O
-	O
-Unfolded	O
-substrate	O
-conformers	O
-interact	O
-with	O
-Spy	O
-through	O
-both	O
-hydrophobic	O
-and	O
-hydrophilic	O
-interactions	O
-,	O
-whereas	O
-the	O
-binding	O
-of	O
-native	O
--	O
-like	O
-states	O
-is	O
-mainly	O
-hydrophilic	O
-.	O
-	O
-This	O
-trend	O
-suggests	O
-that	O
-complex	O
-formation	O
-between	O
-an	O
-ATP	O
--	O
-independent	O
-chaperone	O
-and	O
-its	O
-unfolded	O
-substrate	O
-may	O
-initially	O
-involve	O
-hydrophobic	O
-interactions	O
-,	O
-effectively	O
-shielding	O
-the	O
-exposed	O
-aggregation	O
--	O
-sensitive	O
-hydrophobic	O
-regions	O
-in	O
-the	O
-substrate	O
-.	O
-	O
-Once	O
-the	O
-substrate	O
-begins	O
-to	O
-fold	O
-within	O
-this	O
-protected	O
-environment	O
-,	O
-it	O
-progressively	O
-buries	O
-its	O
-own	O
-hydrophobic	O
-residues	O
-,	O
-and	O
-its	O
-interactions	O
-with	O
-the	O
-chaperone	O
-shift	O
-towards	O
-becoming	O
-more	O
-electrostatic	O
-.	O
-	O
-Notably	O
-,	O
-the	O
-most	O
-frequent	O
-contacts	O
-between	O
-Spy	O
-and	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-are	O
-charge	O
--	O
-charge	O
-interactions	O
-.	O
-	O
-The	O
-negatively	O
-charged	O
-Im7	O
-residues	O
-Glu21	O
-,	O
-Asp32	O
-,	O
-and	O
-Asp35	O
-reside	O
-on	O
-the	O
-surface	O
-of	O
-Im7	O
-and	O
-form	O
-interactions	O
-with	O
-Spy	O
-’	O
-s	O
-positively	O
-charged	O
-cradle	O
-in	O
-both	O
-the	O
-unfolded	O
-and	O
-native	O
--	O
-like	O
-states	O
-.	O
-	O
-Residues	O
-Asp32	O
-and	O
-Asp35	O
-are	O
-close	O
-to	O
-each	O
-other	O
-in	O
-the	O
-folded	O
-state	O
-of	O
-Im7	O
-.	O
-	O
-This	O
-proximity	O
-likely	O
-causes	O
-electrostatic	O
-repulsion	O
-that	O
-destabilizes	O
-Im7	O
-’	O
-s	O
-native	O
-state	O
-.	O
-	O
-Interaction	O
-with	O
-Spy	O
-’	O
-s	O
-positively	O
--	O
-charged	O
-residues	O
-likely	O
-relieves	O
-the	O
-charge	O
-repulsion	O
-between	O
-Asp32	O
-and	O
-Asp35	O
-,	O
-promoting	O
-their	O
-compaction	O
-into	O
-a	O
-helical	O
-conformation	O
-.	O
-	O
-As	O
-inter	O
--	O
-molecular	O
-hydrophobic	O
-interactions	O
-between	O
-Spy	O
-and	O
-the	O
-substrate	O
-become	O
-progressively	O
-replaced	O
-by	O
-intra	O
--	O
-molecular	O
-interactions	O
-within	O
-the	O
-substrate	O
-,	O
-the	O
-affinity	O
-between	O
-chaperone	O
-and	O
-substrates	O
-could	O
-decrease	O
-,	O
-eventually	O
-leading	O
-to	O
-release	O
-of	O
-the	O
-folded	O
-client	O
-protein	O
-.	O
-	O
-Recently	O
-,	O
-we	O
-employed	O
-a	O
-genetic	O
-selection	O
-system	O
-to	O
-improve	O
-the	O
-chaperone	O
-activity	O
-of	O
-Spy	O
-.	O
-	O
-This	O
-selection	O
-resulted	O
-in	O
-“	O
-Super	O
-Spy	O
-”	O
-variants	O
-that	O
-were	O
-more	O
-effective	O
-at	O
-both	O
-preventing	O
-aggregation	O
-and	O
-promoting	O
-protein	O
-folding	O
-.	O
-	O
-In	O
-conjunction	O
-with	O
-our	O
-bound	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-ensemble	O
-,	O
-these	O
-mutants	O
-now	O
-allowed	O
-us	O
-to	O
-investigate	O
-structural	O
-features	O
-important	O
-to	O
-chaperone	O
-function	O
-.	O
-	O
-Previous	O
-analysis	O
-revealed	O
-that	O
-the	O
-Super	O
-Spy	O
-variants	O
-either	O
-bound	O
-Im7	O
-tighter	O
-than	O
-WT	O
-Spy	O
-,	O
-increased	O
-chaperone	O
-flexibility	O
-as	O
-measured	O
-via	O
-H	O
-/	O
-D	O
-exchange	O
-,	O
-or	O
-both	O
-.	O
-	O
-Our	O
-ensemble	O
-revealed	O
-that	O
-two	O
-of	O
-the	O
-Super	O
-Spy	O
-mutations	O
-(	O
-H96L	B-mutant
-and	O
-Q100L	B-mutant
-)	O
-form	O
-part	O
-of	O
-the	O
-chaperone	O
-contact	O
-surface	O
-that	O
-binds	O
-to	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-(	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-Moreover	O
-,	O
-our	O
-co	O
--	O
-structure	O
-suggests	O
-that	O
-the	O
-L32P	B-mutant
-substitution	O
-,	O
-which	O
-increases	O
-Spy	O
-’	O
-s	O
-flexibility	O
-,	O
-could	O
-operate	O
-by	O
-unhinging	O
-the	O
-N	O
--	O
-terminal	O
-helix	O
-and	O
-effectively	O
-expanding	O
-the	O
-size	O
-of	O
-the	O
-disordered	O
-linker	O
-.	O
-	O
-This	O
-possibility	O
-is	O
-supported	O
-by	O
-the	O
-Spy	O
-:	O
-substrate	O
-structures	O
-,	O
-in	O
-which	O
-the	O
-linker	O
-region	O
-becomes	O
-more	O
-flexible	O
-compared	O
-to	O
-the	O
-apo	O
-state	O
-(	O
-Fig	O
-.	O
-6a	O
-).	O
-	O
-By	O
-sampling	O
-multiple	O
-conformations	O
-,	O
-this	O
-linker	O
-region	O
-may	O
-allow	O
-diverse	O
-substrate	O
-conformations	O
-to	O
-be	O
-accommodated	O
-.	O
-	O
-Other	O
-Super	O
-Spy	O
-mutations	O
-(	O
-F115I	B-mutant
-and	O
-F115L	B-mutant
-)	O
-caused	O
-increased	O
-flexibility	O
-but	O
-not	O
-tighter	O
-substrate	O
-binding	O
-.	O
-	O
-This	O
-residue	O
-does	O
-not	O
-directly	O
-contact	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-in	O
-our	O
-READ	O
--	O
-derived	O
-ensemble	O
-.	O
-	O
-Instead	O
-,	O
-when	O
-Spy	O
-is	O
-bound	O
-to	O
-substrate	O
-,	O
-F115	O
-engages	O
-in	O
-close	O
-CH	O
-⋯	O
-π	O
-hydrogen	O
-bonds	O
-with	O
-Tyr104	O
-(	O
-Fig	O
-.	O
-6b	O
-).	O
-	O
-This	O
-interaction	O
-presumably	O
-reduces	O
-the	O
-mobility	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-helix	O
-.	O
-	O
-The	O
-F115I	B-mutant
-/	O
-L	B-mutant
-substitutions	O
-would	O
-replace	O
-these	O
-hydrogen	O
-bonds	O
-with	O
-hydrophobic	O
-interactions	O
-that	O
-have	O
-little	O
-angular	O
-dependence	O
-.	O
-	O
-As	O
-a	O
-result	O
-,	O
-the	O
-C	O
--	O
-terminus	O
-,	O
-and	O
-possibly	O
-also	O
-the	O
-flexible	O
-linker	O
-,	O
-is	O
-likely	O
-to	O
-become	O
-more	O
-flexible	O
-and	O
-thus	O
-more	O
-accommodating	O
-of	O
-different	O
-conformations	O
-of	O
-substrates	O
-.	O
-	O
-Overall	O
-,	O
-comparison	O
-of	O
-our	O
-ensemble	O
-to	O
-the	O
-Super	O
-Spy	O
-variants	O
-provides	O
-specific	O
-examples	O
-to	O
-corroborate	O
-the	O
-importance	O
-of	O
-conformational	O
-flexibility	O
-in	O
-chaperone	O
--	O
-substrate	O
-interactions	O
-.	O
-	O
-Despite	O
-extensive	O
-studies	O
-,	O
-exactly	O
-how	O
-complex	O
-chaperone	O
-machines	O
-help	O
-proteins	O
-fold	O
-remains	O
-controversial	O
-.	O
-	O
-Our	O
-study	O
-indicates	O
-that	O
-the	O
-chaperone	O
-Spy	O
-employs	O
-a	O
-simple	O
-surface	O
-binding	O
-approach	O
-that	O
-allows	O
-the	O
-substrate	O
-to	O
-explore	O
-various	O
-conformations	O
-and	O
-form	O
-transiently	O
-favorable	O
-interactions	O
-while	O
-being	O
-protected	O
-from	O
-aggregation	O
-.	O
-	O
-We	O
-speculate	O
-that	O
-many	O
-other	O
-chaperones	O
-could	O
-utilize	O
-a	O
-similar	O
-strategy	O
-.	O
-	O
-ATP	O
-and	O
-co	O
--	O
-chaperone	O
-dependencies	O
-may	O
-have	O
-emerged	O
-later	O
-through	O
-evolution	O
-to	O
-better	O
-modulate	O
-and	O
-control	O
-chaperone	O
-action	O
-.	O
-	O
-In	O
-addition	O
-to	O
-insights	O
-into	O
-chaperone	O
-function	O
-,	O
-this	O
-work	O
-presents	O
-a	O
-new	O
-method	O
-for	O
-determining	O
-heterogeneous	O
-structural	O
-ensembles	O
-via	O
-a	O
-hybrid	O
-methodology	O
-of	O
-X	O
--	O
-ray	O
-crystallography	O
-and	O
-computational	O
-modeling	O
-.	O
-	O
-Heterogeneous	O
-dynamic	O
-complexes	O
-or	O
-disordered	O
-regions	O
-of	O
-single	O
-proteins	O
-,	O
-once	O
-considered	O
-solely	O
-approachable	O
-by	O
-NMR	O
-spectroscopy	O
-,	O
-can	O
-now	O
-be	O
-visualized	O
-through	O
-X	O
--	O
-ray	O
-crystallography	O
-.	O
-	O
-Crystallographic	O
-data	O
-and	O
-ensemble	O
-selection	O
-.	O
-(	O
-a	O
-)	O
-2mFo	O
-−	O
-DFc	O
-omit	O
-map	O
-of	O
-residual	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-and	O
-flexible	O
-linker	O
-electron	O
-density	O
-contoured	O
-at	O
-0	O
-.	O
-5	O
-σ	O
-.	O
-	O
-This	O
-is	O
-the	O
-residual	O
-density	O
-that	O
-is	O
-used	O
-in	O
-the	O
-READ	O
-selection	O
-.	O
-	O
-(	O
-b	O
-)	O
-Composites	O
-of	O
-iodine	O
-positions	O
-detected	O
-from	O
-anomalous	O
-signals	O
-using	O
-pI	O
--	O
-Phe	O
-substitutions	O
-,	O
-colored	O
-and	O
-numbered	O
-by	O
-sequence	O
-.	O
-	O
-Multiple	O
-iodine	O
-positions	O
-were	O
-detected	O
-for	O
-most	O
-residues	O
-.	O
-	O
-Agreement	O
-to	O
-the	O
-residual	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-electron	O
-density	O
-(	O
-c	O
-)	O
-and	O
-anomalous	O
-iodine	O
-signals	O
-(	O
-d	O
-)	O
-for	O
-ensembles	O
-of	O
-varying	O
-size	O
-generated	O
-by	O
-randomly	O
-choosing	O
-from	O
-the	O
-MD	O
-pool	O
-(	O
-blue	O
-)	O
-and	O
-from	O
-the	O
-selection	O
-procedure	O
-(	O
-black	O
-).	O
-	O
-The	O
-cost	O
-function	O
-,	O
-χ2	O
-,	O
-decreases	O
-as	O
-the	O
-agreement	O
-to	O
-the	O
-experimental	O
-data	O
-increases	O
-and	O
-is	O
-defined	O
-in	O
-the	O
-Online	O
-Methods	O
-.	O
-	O
-Flowchart	O
-of	O
-the	O
-READ	O
-sample	O
--	O
-and	O
--	O
-select	O
-process	O
-.	O
-	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-ensemble	O
-,	O
-arranged	O
-by	O
-RMSD	O
-to	O
-native	O
-state	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-.	O
-Although	O
-the	O
-six	O
--	O
-membered	O
-ensemble	O
-from	O
-the	O
-READ	O
-selection	O
-should	O
-be	O
-considered	O
-only	O
-as	O
-an	O
-ensemble	O
-,	O
-for	O
-clarity	O
-,	O
-the	O
-individual	O
-conformers	O
-are	O
-shown	O
-separately	O
-here	O
-.	O
-	O
-Spy	O
-is	O
-depicted	O
-as	O
-a	O
-gray	O
-surface	O
-and	O
-the	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-conformer	O
-is	O
-shown	O
-as	O
-orange	O
-balls	O
-.	O
-	O
-Atoms	O
-that	O
-were	O
-either	O
-not	O
-directly	O
-selected	O
-in	O
-the	O
-READ	O
-procedure	O
-,	O
-or	O
-whose	O
-position	O
-could	O
-not	O
-be	O
-justified	O
-based	O
-on	O
-agreement	O
-with	O
-the	O
-residual	O
-electron	O
-density	O
-were	O
-removed	O
-,	O
-leading	O
-to	O
-non	O
--	O
-contiguous	O
-sections	O
-.	O
-	O
-Dashed	O
-lines	O
-connect	O
-non	O
--	O
-contiguous	O
-segments	O
-of	O
-the	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-substrate	O
-.	O
-	O
-Residues	O
-of	O
-the	O
-Spy	O
-flexible	O
-linker	O
-region	O
-that	O
-fit	O
-the	O
-residual	O
-electron	O
-density	O
-are	O
-shown	O
-as	O
-larger	O
-gray	O
-spheres	O
-.	O
-	O
-Shown	O
-below	O
-each	O
-ensemble	O
-member	O
-is	O
-the	O
-RMSD	O
-of	O
-each	O
-conformer	O
-to	O
-the	O
-native	O
-state	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-,	O
-as	O
-well	O
-as	O
-the	O
-percentage	O
-of	O
-contacts	O
-between	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-and	O
-Spy	O
-that	O
-are	O
-hydrophobic	O
-.	O
-	O
-Contact	O
-maps	O
-of	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-complex	O
-.	O
-	O
-(	O
-a	O
-)	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-contact	O
-map	O
-projected	O
-onto	O
-the	O
-bound	O
-Spy	O
-dimer	O
-(	O
-above	O
-)	O
-and	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-(	O
-below	O
-)	O
-structures	O
-.	O
-	O
-For	O
-clarity	O
-,	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-is	O
-represented	O
-with	O
-a	O
-single	O
-conformation	O
-.	O
-	O
-The	O
-frequency	O
-plotted	O
-is	O
-calculated	O
-as	O
-the	O
-average	O
-contact	O
-frequency	O
-from	O
-Spy	O
-to	O
-every	O
-residue	O
-of	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-and	O
-vice	O
--	O
-versa	O
-.	O
-	O
-As	O
-the	O
-residues	O
-involved	O
-in	O
-contacts	O
-are	O
-more	O
-evenly	O
-distributed	O
-in	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-compared	O
-to	O
-Spy	O
-,	O
-its	O
-contact	O
-map	O
-was	O
-amplified	O
-.	O
-(	O
-b	O
-)	O
-Detailed	O
-contact	O
-maps	O
-of	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-.	O
-	O
-Contacts	O
-to	O
-the	O
-two	O
-Spy	O
-monomers	O
-are	O
-depicted	O
-separately	O
-.	O
-	O
-Note	O
-that	O
-the	O
-flexible	O
-linker	O
-region	O
-of	O
-Spy	O
-(	O
-residues	O
-47	O
-–	O
-57	O
-)	O
-is	O
-not	O
-represented	O
-in	O
-the	O
-2D	O
-contact	O
-maps	O
-.	O
-	O
-Spy	O
-conformation	O
-changes	O
-upon	O
-substrate	O
-binding	O
-.	O
-	O
-(	O
-a	O
-)	O
-Overlay	O
-of	O
-apo	O
-Spy	O
-(	O
-PDB	O
-ID	O
-:	O
-3O39	O
-,	O
-gray	O
-)	O
-and	O
-bound	O
-Spy	O
-(	O
-green	O
-).	O
-(	O
-b	O
-)	O
-Overlay	O
-of	O
-WT	O
-Spy	O
-bound	O
-to	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-(	O
-green	O
-),	O
-H96L	B-mutant
-Spy	O
-bound	O
-to	O
-Im7	O
-L18A	B-mutant
-L19	B-mutant
-AL13A	I-mutant
-(	O
-blue	O
-),	O
-H96L	B-mutant
-Spy	O
-bound	O
-to	O
-WT	O
-Im7	O
-(	O
-yellow	O
-),	O
-and	O
-WT	O
-Spy	O
-bound	O
-to	O
-casein	O
-(	O
-salmon	O
-).	O
-(	O
-c	O
-)	O
-Competition	O
-assay	O
-showing	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-competes	O
-with	O
-Im7	O
-L18A	B-mutant
-L19A	B-mutant
-L37A	B-mutant
-H40W	B-mutant
-for	O
-the	O
-same	O
-binding	O
-site	O
-on	O
-Spy	O
-(	O
-further	O
-substrate	O
-competition	O
-assays	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-8	O
-).	O
-	O
-Flexibility	O
-of	O
-Spy	O
-linker	O
-region	O
-and	O
-effect	O
-of	O
-Super	O
-Spy	O
-mutants	O
-.	O
-(	O
-a	O
-)	O
-The	O
-Spy	O
-linker	O
-region	O
-adopts	O
-one	O
-dominant	O
-conformation	O
-in	O
-its	O
-apo	O
-state	O
-(	O
-PDB	O
-ID	O
-3039	O
-,	O
-gray	O
-),	O
-but	O
-expands	O
-and	O
-adopts	O
-multiple	O
-conformations	O
-in	O
-bound	O
-states	O
-(	O
-green	O
-).	O
-	O
-(	O
-b	O
-)	O
-F115	O
-and	O
-L32	O
-tether	O
-Spy	O
-’	O
-s	O
-linker	O
-region	O
-to	O
-its	O
-cradle	O
-,	O
-decreasing	O
-Spy	O
-activity	O
-by	O
-limiting	O
-linker	O
-region	O
-flexibility	O
-.	O
-	O
-The	O
-Super	O
-Spy	O
-mutants	O
-F115L	B-mutant
-,	O
-F115I	B-mutant
-,	O
-and	O
-L32P	B-mutant
-are	O
-proposed	O
-to	O
-gain	O
-activity	O
-by	O
-increasing	O
-the	O
-flexibility	O
-or	O
-size	O
-of	O
-this	O
-linker	O
-region	O
-.	O
-	O
-L32	O
-,	O
-F115	O
-,	O
-and	O
-Y104	O
-are	O
-rendered	O
-in	O
-purple	O
-to	O
-illustrate	O
-residues	O
-that	O
-are	O
-most	O
-affected	O
-by	O
-Super	O
-Spy	O
-mutations	O
-;	O
-CH	O
-⋯	O
-π	O
-hydrogen	O
-bonds	O
-are	O
-depicted	O
-by	O
-orange	O
-dashes	O
-.	O
-	O
-Mechanism	O
-of	O
-extracellular	O
-ion	O
-exchange	O
-and	O
-binding	O
--	O
-site	O
-occlusion	O
-in	O
-the	O
-sodium	O
--	O
-calcium	O
-exchanger	O
-	O
-Na	O
-+/	O
-Ca2	O
-+	O
-exchangers	O
-utilize	O
-the	O
-Na	O
-+	O
-electrochemical	O
-gradient	O
-across	O
-the	O
-plasma	O
-membrane	O
-to	O
-extrude	O
-intracellular	O
-Ca2	O
-+,	O
-and	O
-play	O
-a	O
-central	O
-role	O
-in	O
-Ca2	O
-+	O
-homeostasis	O
-.	O
-	O
-Here	O
-,	O
-we	O
-elucidate	O
-their	O
-mechanisms	O
-of	O
-extracellular	O
-ion	O
-recognition	O
-and	O
-exchange	O
-through	O
-a	O
-structural	O
-analysis	O
-of	O
-the	O
-exchanger	O
-from	O
-Methanococcus	O
-jannaschii	O
-(	O
-NCX_Mj	O
-)	O
-bound	O
-to	O
-Na	O
-+,	O
-Ca2	O
-+	O
-or	O
-Sr2	O
-+	O
-in	O
-various	O
-occupancies	O
-and	O
-in	O
-an	O
-apo	O
-state	O
-.	O
-	O
-This	O
-analysis	O
-defines	O
-the	O
-binding	O
-mode	O
-and	O
-relative	O
-affinity	O
-of	O
-these	O
-ions	O
-,	O
-establishes	O
-the	O
-structural	O
-basis	O
-for	O
-the	O
-anticipated	O
-3Na	O
-+:	O
-1Ca2	O
-+	O
-exchange	O
-stoichiometry	O
-,	O
-and	O
-reveals	O
-the	O
-conformational	O
-changes	O
-at	O
-the	O
-onset	O
-of	O
-the	O
-alternating	O
--	O
-access	O
-transport	O
-mechanism	O
-.	O
-	O
-An	O
-independent	O
-analysis	O
-of	O
-the	O
-dynamics	O
-and	O
-conformational	O
-free	O
--	O
-energy	O
-landscape	O
-of	O
-NCX_Mj	O
-in	O
-different	O
-ion	O
--	O
-occupancy	O
-states	O
-,	O
-based	O
-on	O
-enhanced	O
--	O
-sampling	O
-molecular	O
--	O
-dynamics	O
-simulations	O
-,	O
-demonstrates	O
-that	O
-the	O
-crystal	O
-structures	O
-reflect	O
-mechanistically	O
-relevant	O
-,	O
-interconverting	O
-conformations	O
-.	O
-	O
-These	O
-calculations	O
-also	O
-reveal	O
-the	O
-mechanism	O
-by	O
-which	O
-the	O
-outward	O
--	O
-to	O
--	O
-inward	O
-transition	O
-is	O
-controlled	O
-by	O
-the	O
-ion	O
--	O
-occupancy	O
-state	O
-,	O
-thereby	O
-explaining	O
-the	O
-emergence	O
-of	O
-strictly	O
--	O
-coupled	O
-Na	O
-+/	O
-Ca2	O
-+	O
-antiport	O
-.	O
-	O
-Na	O
-+/	O
-Ca2	O
-+	O
-exchangers	O
-(	O
-NCX	O
-)	O
-play	O
-physiologically	O
-essential	O
-roles	O
-in	O
-Ca2	O
-+	O
-signaling	O
-and	O
-homeostasis	O
-.	O
-	O
-NCX	O
-catalyzes	O
-the	O
-uphill	O
-extrusion	O
-of	O
-intracellular	O
-Ca2	O
-+	O
-across	O
-the	O
-cell	O
-membrane	O
-,	O
-by	O
-coupling	O
-this	O
-process	O
-to	O
-the	O
-downhill	O
-permeation	O
-of	O
-Na	O
-+	O
-into	O
-the	O
-cell	O
-,	O
-with	O
-a	O
-3	O
-Na	O
-+	O
-to	O
-1	O
-Ca2	O
-+	O
-stoichiometry	O
-.	O
-	O
-The	O
-mechanism	O
-of	O
-NCX	O
-proteins	O
-is	O
-therefore	O
-highly	O
-likely	O
-to	O
-be	O
-consistent	O
-with	O
-the	O
-alternating	O
--	O
-access	O
-model	O
-of	O
-secondary	O
--	O
-active	O
-transport	O
-.	O
-	O
-The	O
-basic	O
-functional	O
-unit	O
-for	O
-ion	O
-transport	O
-in	O
-NCX	O
-consists	O
-of	O
-ten	O
-membrane	O
--	O
-spanning	O
-segments	O
-,	O
-comprising	O
-two	O
-homologous	O
-halves	O
-.	O
-	O
-Each	O
-of	O
-these	O
-halves	O
-contains	O
-a	O
-highly	O
-conserved	O
-region	O
-,	O
-referred	O
-to	O
-as	O
-α	O
--	O
-repeat	O
-,	O
-known	O
-to	O
-be	O
-important	O
-for	O
-ion	O
-binding	O
-and	O
-translocation	O
-;	O
-in	O
-eukaryotic	O
-NCX	O
-,	O
-the	O
-two	O
-halves	O
-are	O
-connected	O
-by	O
-a	O
-large	O
-intracellular	O
-regulatory	O
-domain	O
-,	O
-which	O
-is	O
-absent	O
-in	O
-microbial	O
-NCX	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-Despite	O
-a	O
-long	O
-history	O
-of	O
-physiological	O
-and	O
-functional	O
-studies	O
-,	O
-the	O
-molecular	O
-mechanism	O
-of	O
-NCX	O
-has	O
-been	O
-elusive	O
-,	O
-owing	O
-to	O
-the	O
-lack	O
-of	O
-structural	O
-information	O
-.	O
-	O
-Our	O
-recent	O
-atomic	O
--	O
-resolution	O
-structure	O
-of	O
-NCX_Mj	O
-from	O
-Methanococcus	O
-jannaschii	O
-provided	O
-the	O
-first	O
-view	O
-of	O
-the	O
-basic	O
-functional	O
-unit	O
-of	O
-an	O
-NCX	O
-protein	O
-.	O
-	O
-This	O
-structure	O
-shows	O
-the	O
-exchanger	O
-in	O
-an	O
-outward	O
--	O
-facing	O
-conformation	O
-and	O
-reveals	O
-four	O
-putative	O
-ion	O
--	O
-binding	O
-sites	O
-,	O
-denominated	O
-internal	O
-(	O
-Sint	O
-),	O
-external	O
-(	O
-Sext	O
-),	O
-Ca2	O
-+-	O
-binding	O
-(	O
-SCa	O
-)	O
-and	O
-middle	O
-(	O
-Smid	O
-),	O
-clustered	O
-in	O
-the	O
-center	O
-of	O
-the	O
-protein	O
-and	O
-occluded	O
-from	O
-the	O
-solvent	O
-(	O
-Fig	O
-.	O
-1a	O
--	O
-b	O
-).	O
-	O
-With	O
-similar	O
-ion	O
-exchange	O
-properties	O
-to	O
-those	O
-of	O
-its	O
-eukaryotic	O
-counterparts	O
-,	O
-NCX_Mj	O
-provides	O
-a	O
-compelling	O
-model	O
-system	O
-to	O
-investigate	O
-the	O
-structural	O
-basis	O
-for	O
-the	O
-specificity	O
-,	O
-stoichiometry	O
-and	O
-mechanism	O
-of	O
-the	O
-ion	O
--	O
-exchange	O
-reaction	O
-catalyzed	O
-by	O
-NCX	O
-.	O
-	O
-In	O
-this	O
-study	O
-,	O
-we	O
-set	O
-out	O
-to	O
-determine	O
-the	O
-structures	O
-of	O
-outward	O
--	O
-facing	O
-wild	O
--	O
-type	O
-NCX_Mj	O
-in	O
-complex	O
-with	O
-Na	O
-+,	O
-Ca2	O
-+	O
-and	O
-Sr2	O
-+,	O
-at	O
-various	O
-concentrations	O
-.	O
-	O
-These	O
-structures	O
-reveal	O
-the	O
-mode	O
-of	O
-recognition	O
-of	O
-these	O
-ions	O
-,	O
-their	O
-relative	O
-affinities	O
-,	O
-and	O
-the	O
-mechanism	O
-of	O
-extracellular	O
-ion	O
-exchange	O
-,	O
-for	O
-a	O
-well	O
--	O
-defined	O
-,	O
-functional	O
-conformation	O
-in	O
-a	O
-membrane	O
--	O
-like	O
-environment	O
-.	O
-	O
-An	O
-independent	O
-analysis	O
-based	O
-on	O
-molecular	O
--	O
-dynamics	O
-simulations	O
-demonstrates	O
-that	O
-the	O
-structures	O
-capture	O
-mechanistically	O
-relevant	O
-states	O
-.	O
-	O
-These	O
-calculations	O
-also	O
-reveal	O
-how	O
-the	O
-ion	O
-occupancy	O
-state	O
-of	O
-the	O
-outward	O
--	O
-facing	O
-exchanger	O
-determines	O
-the	O
-feasibility	O
-of	O
-the	O
-transition	O
-to	O
-the	O
-inward	O
--	O
-facing	O
-conformation	O
-,	O
-thereby	O
-addressing	O
-a	O
-key	O
-outstanding	O
-question	O
-in	O
-secondary	O
--	O
-active	O
-transport	O
-,	O
-namely	O
-how	O
-the	O
-transported	O
-substrates	O
-control	O
-the	O
-alternating	O
--	O
-access	O
-mechanism	O
-.	O
-	O
-Extracellular	O
-Na	O
-+	O
-binding	O
-	O
-The	O
-assignment	O
-of	O
-the	O
-four	O
-central	O
-binding	O
-sites	O
-identified	O
-in	O
-the	O
-previously	O
-reported	O
-NCX_Mj	O
-structure	O
-was	O
-hampered	O
-by	O
-the	O
-presence	O
-of	O
-both	O
-Na	O
-+	O
-and	O
-Ca2	O
-+	O
-in	O
-the	O
-protein	O
-crystals	O
-.	O
-	O
-To	O
-conclusively	O
-clarify	O
-this	O
-assignment	O
-,	O
-we	O
-first	O
-set	O
-out	O
-to	O
-examine	O
-the	O
-Na	O
-+	O
-occupancy	O
-of	O
-these	O
-sites	O
-without	O
-Ca2	O
-+.	O
-	O
-Crystals	O
-were	O
-grown	O
-in	O
-150	O
-mM	O
-NaCl	O
-using	O
-the	O
-lipidic	O
-cubic	O
-phase	O
-(	O
-LCP	O
-)	O
-technique	O
-.	O
-	O
-The	O
-crystallization	O
-solutions	O
-around	O
-the	O
-LCP	O
-droplets	O
-were	O
-then	O
-slowly	O
-replaced	O
-by	O
-solutions	O
-containing	O
-different	O
-concentrations	O
-of	O
-NaCl	O
-and	O
-EGTA	O
-(	O
-Methods	O
-).	O
-	O
-X	O
--	O
-ray	O
-diffraction	O
-of	O
-these	O
-soaked	O
-crystals	O
-revealed	O
-a	O
-Na	O
-+-	O
-dependent	O
-variation	O
-in	O
-the	O
-electron	O
--	O
-density	O
-distribution	O
-at	O
-sites	O
-Sext	O
-,	O
-SCa	O
-and	O
-Sint	O
-,	O
-indicating	O
-a	O
-Na	O
-+	O
-occupancy	O
-change	O
-(	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-Occupancy	O
-refinement	O
-indicated	O
-two	O
-Na	O
-+	O
-ions	O
-bind	O
-to	O
-Sint	O
-and	O
-SCa	O
-at	O
-low	O
-Na	O
-+	O
-concentrations	O
-(	O
-Fig	O
-.	O
-1c	O
-),	O
-with	O
-a	O
-slight	O
-preference	O
-for	O
-Sint	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Binding	O
-of	O
-a	O
-third	O
-Na	O
-+	O
-to	O
-Sext	O
-occurs	O
-at	O
-higher	O
-concentrations	O
-,	O
-as	O
-no	O
-density	O
-was	O
-observed	O
-there	O
-at	O
-10	O
-mM	O
-Na	O
-+	O
-or	O
-lower	O
-(	O
-Fig	O
-.	O
-1c	O
-);	O
-Sext	O
-is	O
-however	O
-partially	O
-occupied	O
-at	O
-20	O
-mM	O
-Na	O
-+,	O
-and	O
-fully	O
-occupied	O
-at	O
-150	O
-mM	O
-(	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-The	O
-Na	O
-+	O
-occupation	O
-at	O
-SCa	O
-,	O
-compounded	O
-with	O
-the	O
-expected	O
-3Na	O
-+:	O
-1Ca2	O
-+	O
-stoichiometry	O
-,	O
-implies	O
-our	O
-previous	O
-assignment	O
-of	O
-the	O
-Smid	O
-site	O
-must	O
-be	O
-re	O
--	O
-evaluated	O
-.	O
-	O
-Indeed	O
-,	O
-two	O
-observations	O
-indicate	O
-that	O
-a	O
-water	O
-molecule	O
-rather	O
-than	O
-a	O
-Na	O
-+	O
-ion	O
-occupies	O
-Smid	O
-,	O
-as	O
-was	O
-predicted	O
-in	O
-a	O
-recent	O
-simulation	O
-study	O
-.	O
-	O
-First	O
-,	O
-the	O
-electron	O
-density	O
-at	O
-Smid	O
-does	O
-not	O
-depend	O
-significantly	O
-on	O
-the	O
-Na	O
-+	O
-concentration	O
-.	O
-	O
-Second	O
-,	O
-the	O
-protein	O
-coordination	O
-geometry	O
-at	O
-Smid	O
-is	O
-clearly	O
-suboptimal	O
-for	O
-Na	O
-+	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1d	O
-).	O
-	O
-The	O
-water	O
-molecule	O
-at	O
-Smid	O
-forms	O
-hydrogen	O
--	O
-bonds	O
-with	O
-the	O
-highly	O
-conserved	O
-Glu54	O
-and	O
-Glu213	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1d	O
-),	O
-stabilizing	O
-their	O
-orientation	O
-to	O
-properly	O
-coordinate	O
-multiple	O
-Na	O
-+	O
-ions	O
-at	O
-Sext	O
-,	O
-SCa	O
-and	O
-Sint	O
-.	O
-	O
-It	O
-can	O
-be	O
-inferred	O
-from	O
-this	O
-assignment	O
-that	O
-Glu54	O
-and	O
-Glu213	O
-are	O
-ionized	O
-,	O
-while	O
-Asp240	O
-,	O
-which	O
-flanks	O
-Smid	O
-(	O
-and	O
-is	O
-replaced	O
-by	O
-Asn	O
-in	O
-eukaryotic	O
-NCX	O
-)	O
-would	O
-be	O
-protonated	O
-,	O
-as	O
-indicated	O
-by	O
-the	O
-abovementioned	O
-simulation	O
-study	O
-.	O
-	O
-Na	O
-+-	O
-dependent	O
-conformational	O
-change	O
-	O
-The	O
-NCX_Mj	O
-structures	O
-in	O
-various	O
-Na	O
-+	O
-concentrations	O
-also	O
-reveal	O
-that	O
-Na	O
-+	O
-binding	O
-to	O
-Sext	O
-is	O
-coupled	O
-to	O
-a	O
-subtle	O
-but	O
-important	O
-conformational	O
-change	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-When	O
-Na	O
-+	O
-binds	O
-to	O
-Sext	O
-at	O
-high	O
-concentrations	O
-,	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-TM7	O
-is	O
-bent	O
-into	O
-two	O
-short	O
-helices	O
-,	O
-TM7a	O
-and	O
-TM7b	O
-(	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-TM7b	O
-occludes	O
-the	O
-four	O
-central	O
-binding	O
-sites	O
-from	O
-the	O
-external	O
-solution	O
-,	O
-with	O
-the	O
-backbone	O
-carbonyl	O
-of	O
-Ala206	O
-coordinating	O
-the	O
-Na	O
-+	O
-ion	O
-(	O
-Fig	O
-.	O
-2b	O
--	O
-d	O
-).	O
-	O
-However	O
-,	O
-when	O
-Sext	O
-becomes	O
-empty	O
-at	O
-low	O
-Na	O
-+	O
-concentrations	O
-,	O
-TM7a	O
-and	O
-TM7b	O
-become	O
-a	O
-continuous	O
-straight	O
-helix	O
-(	O
-Fig	O
-.	O
-2a	O
-),	O
-and	O
-the	O
-carbonyl	O
-group	O
-of	O
-Ala206	O
-retracts	O
-away	O
-(	O
-Fig	O
-.	O
-2b	O
--	O
-d	O
-).	O
-	O
-TM7a	O
-also	O
-forms	O
-hydrophobic	O
-contacts	O
-with	O
-the	O
-C	O
--	O
-terminal	O
-half	O
-of	O
-TM6	O
-.	O
-	O
-These	O
-contacts	O
-are	O
-absent	O
-in	O
-the	O
-structure	O
-with	O
-Na	O
-+	O
-at	O
-Sext	O
-,	O
-in	O
-which	O
-there	O
-is	O
-an	O
-open	O
-gap	O
-between	O
-the	O
-two	O
-helices	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-This	O
-difference	O
-is	O
-noteworthy	O
-because	O
-TM6	O
-and	O
-TM1	O
-are	O
-believed	O
-to	O
-undergo	O
-a	O
-sliding	O
-motion	O
-,	O
-relative	O
-to	O
-the	O
-rest	O
-of	O
-the	O
-protein	O
-,	O
-when	O
-the	O
-transporter	O
-switches	O
-to	O
-the	O
-inward	O
--	O
-facing	O
-conformation	O
-.	O
-	O
-The	O
-straightening	O
-of	O
-TM7ab	O
-also	O
-opens	O
-up	O
-a	O
-passageway	O
-from	O
-the	O
-external	O
-solution	O
-to	O
-Sext	O
-and	O
-Smid	O
-,	O
-while	O
-SCa	O
-and	O
-Sint	O
-remain	O
-occluded	O
-(	O
-Fig	O
-.	O
-2d	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-structures	O
-at	O
-high	O
-and	O
-low	O
-Na	O
-+	O
-concentrations	O
-represent	O
-the	O
-outward	O
--	O
-facing	O
-occluded	O
-and	O
-partially	O
-open	O
-states	O
-,	O
-respectively	O
-.	O
-	O
-This	O
-conformational	O
-change	O
-is	O
-dependent	O
-on	O
-the	O
-Na	O
-+	O
-occupancy	O
-of	O
-Sext	O
-and	O
-occurs	O
-when	O
-Na	O
-+	O
-already	O
-occupies	O
-Sint	O
-and	O
-SCa	O
-.	O
-	O
-Our	O
-crystallographic	O
-titration	O
-experiment	O
-indicates	O
-that	O
-the	O
-K1	O
-/	O
-2	O
-of	O
-this	O
-Na	O
-+-	O
-driven	O
-conformational	O
-transition	O
-is	O
-~	O
-20	O
-mM	O
-.	O
-At	O
-this	O
-concentration	O
-,	O
-Sext	O
-is	O
-partially	O
-occupied	O
-and	O
-the	O
-NCX_Mj	O
-crystal	O
-is	O
-a	O
-mixture	O
-of	O
-both	O
-the	O
-occluded	O
-and	O
-partially	O
-open	O
-conformations	O
-.	O
-	O
-This	O
-structurally	O
--	O
-derived	O
-Na	O
-+	O
-affinity	O
-agrees	O
-well	O
-with	O
-the	O
-external	O
-Na	O
-+	O
-concentration	O
-required	O
-for	O
-NCX	O
-activation	O
-in	O
-eukaryotes	O
-.	O
-	O
-The	O
-finding	O
-that	O
-the	O
-Na	O
-+	O
-occupancy	O
-change	O
-from	O
-2	O
-to	O
-3	O
-ions	O
-coincides	O
-with	O
-a	O
-conformational	O
-change	O
-of	O
-the	O
-transporter	O
-also	O
-provides	O
-a	O
-rationale	O
-to	O
-the	O
-Hill	O
-coefficient	O
-of	O
-the	O
-Na	O
-+-	O
-dependent	O
-activation	O
-process	O
-in	O
-eukaryotic	O
-NCX	O
-.	O
-	O
-Extracellular	O
-Ca2	O
-+	O
-and	O
-Sr2	O
-+	O
-binding	O
-and	O
-their	O
-competition	O
-with	O
-Na	O
-+	O
-	O
-To	O
-determine	O
-how	O
-Ca2	O
-+	O
-binds	O
-to	O
-NCX_Mj	O
-and	O
-competes	O
-with	O
-Na	O
-+,	O
-we	O
-first	O
-titrated	O
-the	O
-crystals	O
-with	O
-Sr2	O
-+	O
-(	O
-Methods	O
-).	O
-	O
-Sr2	O
-+	O
-is	O
-transported	O
-by	O
-NCX	O
-similarly	O
-to	O
-Ca2	O
-+	O
-,	O
-and	O
-is	O
-distinguishable	O
-from	O
-Na	O
-+	O
-by	O
-its	O
-greater	O
-electron	O
--	O
-density	O
-intensity	O
-.	O
-	O
-Protein	O
-crystals	O
-soaked	O
-with	O
-10	O
-mM	O
-Sr2	O
-+	O
-and	O
-2	O
-.	O
-5	O
-mM	O
-Na	O
-+	O
-revealed	O
-a	O
-strong	O
-electron	O
--	O
-density	O
-peak	O
-at	O
-site	O
-SCa	O
-,	O
-indicating	O
-binding	O
-of	O
-a	O
-single	O
-Sr2	O
-+	O
-ion	O
-(	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-The	O
-Sr2	O
-+-	O
-loaded	O
-NCX_Mj	O
-structure	O
-adopts	O
-the	O
-partially	O
-open	O
-conformation	O
-observed	O
-at	O
-low	O
-Na	O
-+	O
-concentrations	O
-.	O
-	O
-Binding	O
-of	O
-Sr2	O
-+,	O
-however	O
-,	O
-excludes	O
-Na	O
-+	O
-entirely	O
-.	O
-	O
-Crystal	O
-titrations	O
-with	O
-decreasing	O
-Sr2	O
-+	O
-or	O
-increasing	O
-Na	O
-+	O
-demonstrated	O
-that	O
-Sr2	O
-+	O
-binds	O
-to	O
-the	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-with	O
-low	O
-affinity	O
-,	O
-and	O
-that	O
-it	O
-can	O
-be	O
-out	O
--	O
-competed	O
-by	O
-Na	O
-+	O
-even	O
-at	O
-low	O
-concentrations	O
-(	O
-Supplementary	O
-Note	O
-1	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2a	O
--	O
-b	O
-).	O
-	O
-Thus	O
-,	O
-in	O
-100	O
-mM	O
-Na	O
-+	O
-and	O
-10	O
-mM	O
-Sr2	O
-+,	O
-Na	O
-+	O
-completely	O
-replaced	O
-Sr2	O
-+	O
-(	O
-Fig	O
-.	O
-3a	O
-)	O
-and	O
-reverted	O
-NCX_Mj	O
-to	O
-the	O
-Na	O
-+-	O
-loaded	O
-,	O
-fully	O
-occluded	O
-state	O
-.	O
-	O
-Similar	O
-titration	O
-experiments	O
-showed	O
-that	O
-Ca2	O
-+	O
-and	O
-Sr2	O
-+	O
-binding	O
-to	O
-NCX_Mj	O
-are	O
-not	O
-exactly	O
-alike	O
-The	O
-electron	O
-density	O
-distribution	O
-from	O
-crystals	O
-soaked	O
-in	O
-high	O
-Ca2	O
-+	O
-and	O
-low	O
-Na	O
-+,	O
-indicates	O
-that	O
-Ca2	O
-+	O
-can	O
-bind	O
-to	O
-Smid	O
-as	O
-well	O
-as	O
-SCa	O
-,	O
-with	O
-a	O
-preference	O
-for	O
-SCa	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-Binding	O
-of	O
-Ca2	O
-+	O
-to	O
-both	O
-sites	O
-simultaneously	O
-is	O
-highly	O
-improbable	O
-due	O
-to	O
-their	O
-close	O
-proximity	O
-,	O
-and	O
-at	O
-least	O
-one	O
-water	O
-molecule	O
-can	O
-be	O
-discerned	O
-coordinating	O
-the	O
-ion	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-The	O
-partial	O
-Ca2	O
-+	O
-occupancy	O
-at	O
-Smid	O
-is	O
-likely	O
-caused	O
-by	O
-Asp240	O
-,	O
-which	O
-flanks	O
-this	O
-site	O
-and	O
-can	O
-in	O
-principle	O
-coordinate	O
-Ca2	O
-+.	O
-	O
-Previous	O
-functional	O
-and	O
-computational	O
-studies	O
-,	O
-however	O
-,	O
-indicate	O
-Asp240	O
-becomes	O
-protonated	O
-during	O
-transport	O
-.	O
-	O
-Indeed	O
-,	O
-in	O
-most	O
-NCX	O
-proteins	O
-Asp240	O
-is	O
-substituted	O
-by	O
-Asn	O
-,	O
-which	O
-would	O
-likely	O
-weaken	O
-or	O
-abrogate	O
-Ca2	O
-+	O
-binding	O
-to	O
-Smid	O
-.	O
-	O
-SCa	O
-is	O
-therefore	O
-the	O
-functional	O
-Ca2	O
-+	O
-site	O
-.	O
-	O
-Similarly	O
-to	O
-Sr2	O
-+,	O
-Ca2	O
-+	O
-binds	O
-with	O
-low	O
-affinity	O
-to	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-and	O
-can	O
-be	O
-readily	O
-displaced	O
-by	O
-Na	O
-+	O
-(	O
-Supplementary	O
-Note	O
-1	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2c	O
-).	O
-	O
-This	O
-finding	O
-is	O
-consistent	O
-with	O
-physiological	O
-and	O
-biochemical	O
-data	O
-for	O
-both	O
-eukaryotic	O
-NCX	O
-and	O
-NCX_Mj	O
-indicating	O
-that	O
-the	O
-apparent	O
-Ca2	O
-+	O
-affinity	O
-is	O
-much	O
-lower	O
-on	O
-the	O
-extracellular	O
-than	O
-the	O
-cytoplasmic	O
-side	O
-.	O
-	O
-Specifically	O
-,	O
-our	O
-crystallographic	O
-titration	O
-assay	O
-indicates	O
-Ca2	O
-+	O
-binds	O
-with	O
-sub	O
--	O
-millimolar	O
-affinity	O
-,	O
-in	O
-good	O
-agreement	O
-with	O
-the	O
-external	O
-apparent	O
-Ca2	O
-+	O
-affinities	O
-deduced	O
-functionally	O
-for	O
-cardiac	O
-NCX	O
-(	O
-Km	O
-~	O
-0	O
-.	O
-32	O
-mM	O
-)	O
-and	O
-NCX_Mj	O
-(	O
-Km	O
-~	O
-0	O
-.	O
-175	O
-mM	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-these	O
-crystal	O
-titration	O
-experiments	O
-demonstrate	O
-that	O
-the	O
-four	O
-binding	O
-sites	O
-in	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-exhibit	O
-different	O
-specificity	O
-:	O
-Sint	O
-and	O
-Sext	O
-are	O
-Na	O
-+	O
-specific	O
-whereas	O
-SCa	O
-,	O
-previously	O
-hypothesized	O
-to	O
-be	O
-Ca2	O
-+	O
-specific	O
-,	O
-can	O
-also	O
-bind	O
-Na	O
-+,	O
-confirming	O
-our	O
-earlier	O
-simulation	O
-study	O
-,	O
-as	O
-well	O
-as	O
-Sr2	O
-+;	O
-Smid	O
-can	O
-also	O
-transiently	O
-accommodate	O
-Ca2	O
-+	O
-but	O
-during	O
-transport	O
-Smid	O
-is	O
-most	O
-likely	O
-occupied	O
-by	O
-water	O
-.	O
-	O
-The	O
-ion	O
--	O
-binding	O
-sites	O
-in	O
-NCX_Mj	O
-can	O
-therefore	O
-accommodate	O
-up	O
-to	O
-three	O
-Na	O
-+	O
-ions	O
-or	O
-a	O
-single	O
-divalent	O
-ion	O
-,	O
-and	O
-occupancy	O
-by	O
-Na	O
-+	O
-and	O
-Ca2	O
-+	O
-(	O
-or	O
-Sr2	O
-+)	O
-are	O
-mutually	O
-exclusive	O
-,	O
-as	O
-was	O
-deduced	O
-for	O
-eukaryotic	O
-exchangers	O
-.	O
-	O
-A	O
-structure	O
-of	O
-NCX_Mj	O
-without	O
-Na	O
-+	O
-or	O
-Ca2	O
-+	O
-bound	O
-	O
-An	O
-apo	O
-state	O
-of	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-is	O
-likely	O
-to	O
-exist	O
-transiently	O
-in	O
-physiological	O
-conditions	O
-,	O
-despite	O
-the	O
-high	O
-amounts	O
-of	O
-extracellular	O
-Na	O
-+	O
-(~	O
-150	O
-mM	O
-)	O
-and	O
-Ca2	O
-+	O
-(~	O
-2	O
-mM	O
-).	O
-	O
-We	O
-were	O
-able	O
-to	O
-determine	O
-an	O
-apo	O
--	O
-state	O
-structure	O
-of	O
-NCX_Mj	O
-,	O
-by	O
-crystallizing	O
-the	O
-protein	O
-at	O
-lower	O
-pH	O
-and	O
-in	O
-the	O
-absence	O
-of	O
-Na	O
-+	O
-(	O
-Methods	O
-).	O
-	O
-This	O
-structure	O
-is	O
-similar	O
-to	O
-the	O
-partially	O
-open	O
-structure	O
-with	O
-two	O
-Na	O
-+	O
-or	O
-either	O
-one	O
-Ca2	O
-+	O
-or	O
-one	O
-Sr2	O
-+	O
-ion	O
-,	O
-with	O
-two	O
-noticeable	O
-differences	O
-.	O
-	O
-First	O
-,	O
-TM7ab	O
-along	O
-with	O
-the	O
-extracellular	O
-half	O
-of	O
-the	O
-TM6	O
-and	O
-TM1	O
-swing	O
-further	O
-away	O
-from	O
-the	O
-protein	O
-core	O
-(	O
-Fig	O
-.	O
-3c	O
-),	O
-resulting	O
-in	O
-a	O
-slightly	O
-wider	O
-passageway	O
-into	O
-the	O
-binding	O
-sites	O
-.	O
-	O
-Second	O
-,	O
-Glu54	O
-and	O
-Glu213	O
-side	O
-chains	O
-rotate	O
-away	O
-from	O
-the	O
-binding	O
-sites	O
-and	O
-appear	O
-to	O
-form	O
-hydrogen	O
--	O
-bonds	O
-with	O
-residues	O
-involved	O
-in	O
-ion	O
-coordination	O
-in	O
-the	O
-fully	O
-Na	O
-+-	O
-loaded	O
-structure	O
-(	O
-Fig	O
-.	O
-3d	O
-).	O
-	O
-Although	O
-the	O
-binding	O
-sites	O
-are	O
-thus	O
-fully	O
-accessible	O
-to	O
-the	O
-external	O
-solution	O
-(	O
-Fig	O
-.	O
-3e	O
-),	O
-the	O
-lack	O
-of	O
-electron	O
-density	O
-therein	O
-indicates	O
-no	O
-ions	O
-or	O
-ordered	O
-solvent	O
-molecules	O
-.	O
-	O
-This	O
-apo	O
-structure	O
-might	O
-therefore	O
-represent	O
-the	O
-unloaded	O
-,	O
-open	O
-state	O
-of	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-.	O
-	O
-Alternatively	O
-,	O
-this	O
-structure	O
-might	O
-capture	O
-a	O
-fully	O
-protonated	O
-state	O
-of	O
-the	O
-transporter	O
-,	O
-to	O
-which	O
-Na	O
-+	O
-and	O
-Ca2	O
-+	O
-cannot	O
-bind	O
-.	O
-	O
-Such	O
-interpretation	O
-would	O
-be	O
-consistent	O
-with	O
-the	O
-computer	O
-simulations	O
-reported	O
-below	O
-.	O
-	O
-Indeed	O
-,	O
-transport	O
-assays	O
-of	O
-NCX_Mj	O
-have	O
-shown	O
-that	O
-even	O
-in	O
-the	O
-presence	O
-of	O
-Na	O
-+	O
-or	O
-Ca2	O
-+,	O
-low	O
-pH	O
-inactivates	O
-the	O
-transport	O
-cycle	O
-.	O
-	O
-Ion	O
-occupancy	O
-determines	O
-the	O
-free	O
--	O
-energy	O
-landscape	O
-of	O
-NCX_Mj	O
-	O
-That	O
-secondary	O
--	O
-active	O
-transporters	O
-are	O
-able	O
-to	O
-harness	O
-an	O
-electrochemical	O
-gradient	O
-of	O
-one	O
-substrate	O
-to	O
-power	O
-the	O
-uphill	O
-transport	O
-of	O
-another	O
-relies	O
-on	O
-a	O
-seemingly	O
-simple	O
-principle	O
-:	O
-they	O
-must	O
-not	O
-transition	O
-between	O
-outward	O
--	O
-and	O
-inward	O
--	O
-open	O
-conformations	O
-unless	O
-in	O
-two	O
-precise	O
-substrate	O
-occupancy	O
-states	O
-.	O
-	O
-NCX	O
-must	O
-be	O
-loaded	O
-either	O
-with	O
-3	O
-Na	O
-+	O
-or	O
-1	O
-Ca2	O
-+,	O
-and	O
-therefore	O
-functions	O
-as	O
-an	O
-antiporter	O
-;	O
-symporters	O
-,	O
-by	O
-contrast	O
-,	O
-undergo	O
-the	O
-alternating	O
--	O
-access	O
-transition	O
-only	O
-when	O
-all	O
-substrates	O
-and	O
-coupling	O
-ions	O
-are	O
-concurrently	O
-bound	O
-,	O
-or	O
-in	O
-the	O
-apo	O
-state	O
-.	O
-	O
-To	O
-examine	O
-this	O
-central	O
-question	O
-,	O
-we	O
-sought	O
-to	O
-characterize	O
-the	O
-conformational	O
-free	O
--	O
-energy	O
-landscape	O
-of	O
-NCX_Mj	O
-and	O
-to	O
-examine	O
-its	O
-dependence	O
-on	O
-the	O
-ion	O
--	O
-occupancy	O
-state	O
-,	O
-using	O
-molecular	O
-dynamics	O
-(	O
-MD	O
-)	O
-simulations	O
-.	O
-	O
-This	O
-computational	O
-analysis	O
-was	O
-based	O
-solely	O
-on	O
-the	O
-published	O
-structure	O
-of	O
-NCX_Mj	O
-,	O
-independently	O
-of	O
-the	O
-crystallographic	O
-studies	O
-described	O
-above	O
-.	O
-	O
-As	O
-it	O
-happens	O
-,	O
-the	O
-results	O
-confirm	O
-that	O
-the	O
-structures	O
-now	O
-available	O
-are	O
-representing	O
-interconverting	O
-states	O
-of	O
-the	O
-functional	O
-cycle	O
-of	O
-NCX_Mj	O
-,	O
-while	O
-revealing	O
-how	O
-the	O
-alternating	O
--	O
-access	O
-mechanism	O
-is	O
-controlled	O
-by	O
-the	O
-ion	O
--	O
-occupancy	O
-state	O
-.	O
-	O
-A	O
-series	O
-of	O
-exploratory	O
-MD	O
-simulations	O
-was	O
-initially	O
-carried	O
-out	O
-to	O
-examine	O
-what	O
-features	O
-of	O
-the	O
-NCX_Mj	O
-structure	O
-might	O
-depend	O
-on	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-occupancy	O
-.	O
-	O
-Specifically	O
-,	O
-we	O
-first	O
-simulated	O
-the	O
-outward	O
--	O
-occluded	O
-form	O
-,	O
-in	O
-the	O
-ion	O
-configuration	O
-we	O
-previously	O
-predicted	O
-,	O
-now	O
-confirmed	O
-by	O
-the	O
-high	O
--	O
-Na	O
-+	O
-crystal	O
-structure	O
-described	O
-above	O
-(	O
-Fig	O
-.	O
-1b	O
-).	O
-	O
-That	O
-is	O
-,	O
-Na	O
-+	O
-ions	O
-occupy	O
-Sext	O
-,	O
-SCa	O
-,	O
-and	O
-Sint	O
-,	O
-while	O
-D240	O
-is	O
-protonated	O
-and	O
-a	O
-water	O
-molecule	O
-occupies	O
-Smid	O
-.	O
-	O
-The	O
-Na	O
-+	O
-ion	O
-at	O
-Sext	O
-was	O
-then	O
-relocated	O
-from	O
-the	O
-site	O
-to	O
-the	O
-bulk	O
-solution	O
-(	O
-Methods	O
-),	O
-and	O
-this	O
-system	O
-was	O
-then	O
-allowed	O
-to	O
-evolve	O
-freely	O
-in	O
-time	O
-.	O
-	O
-The	O
-Na	O
-+	O
-ions	O
-at	O
-SCa	O
-and	O
-Sint	O
-were	O
-displaced	O
-subsequently	O
-,	O
-and	O
-an	O
-analogous	O
-simulation	O
-was	O
-then	O
-carried	O
-out	O
-.	O
-	O
-These	O
-initial	O
-simulations	O
-revealed	O
-noticeable	O
-changes	O
-in	O
-the	O
-transporter	O
-,	O
-consistent	O
-with	O
-those	O
-observed	O
-in	O
-the	O
-new	O
-crystal	O
-structures	O
-.	O
-	O
-The	O
-most	O
-notable	O
-change	O
-upon	O
-displacement	O
-of	O
-Na	O
-+	O
-from	O
-Sext	O
-was	O
-the	O
-straightening	O
-of	O
-TM7ab	O
-(	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-When	O
-3	O
-Na	O
-+	O
-ions	O
-are	O
-bound	O
-,	O
-TM7ab	O
-primarily	O
-folds	O
-as	O
-two	O
-distinct	O
-,	O
-non	O
--	O
-collinear	O
-α	O
--	O
-helical	O
-fragments	O
-,	O
-owing	O
-to	O
-the	O
-loss	O
-of	O
-the	O
-backbone	O
-carbonyl	O
--	O
-amide	O
-hydrogen	O
--	O
-bonds	O
-between	O
-F202	O
-and	O
-A206	O
-,	O
-and	O
-T203	O
-and	O
-F207	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-This	O
-distortion	O
-occludes	O
-Sext	O
-from	O
-the	O
-exterior	O
-(	O
-Fig	O
-.	O
-4d	O
-,	O
-4h	O
--	O
-i	O
-)	O
-and	O
-appears	O
-to	O
-be	O
-induced	O
-by	O
-the	O
-Na	O
-+	O
-ion	O
-itself	O
-,	O
-which	O
-pulls	O
-the	O
-carbonyl	O
-group	O
-of	O
-A206	O
-into	O
-its	O
-coordination	O
-sphere	O
-(	O
-Fig	O
-.	O
-4g	O
-).	O
-	O
-With	O
-Sext	O
-empty	O
-,	O
-however	O
-,	O
-TM7ab	O
-forms	O
-a	O
-canonical	O
-α	O
--	O
-helix	O
-(	O
-Fig	O
-.	O
-4a	O
--	O
-b	O
-,	O
-4g	O
-),	O
-thereby	O
-creating	O
-an	O
-opening	O
-between	O
-TM3	O
-and	O
-TM7	O
-,	O
-which	O
-in	O
-turn	O
-allows	O
-water	O
-molecules	O
-from	O
-the	O
-external	O
-solution	O
-to	O
-reach	O
-into	O
-Sext	O
-(	O
-Fig	O
-.	O
-4e	O
-,	O
-4h	O
--	O
-i	O
-),	O
-i	O
-.	O
-e	O
-.	O
-the	O
-transporter	O
-is	O
-no	O
-longer	O
-occluded	O
-.	O
-	O
-Displacement	O
-of	O
-Na	O
-+	O
-from	O
-SCa	O
-and	O
-Sint	O
-induces	O
-further	O
-changes	O
-(	O
-Fig	O
-.	O
-4c	O
-).	O
-	O
-The	O
-most	O
-noticeable	O
-is	O
-an	O
-increased	O
-separation	O
-between	O
-TM7	O
-and	O
-TM2	O
-(	O
-Fig	O
-.	O
-4f	O
-),	O
-previously	O
-brought	O
-together	O
-by	O
-concurrent	O
-backbone	O
-interactions	O
-with	O
-the	O
-Na	O
-+	O
-ion	O
-at	O
-SCa	O
-(	O
-Fig	O
-.	O
-4d	O
--	O
-e	O
-).	O
-	O
-TM1	O
-and	O
-TM6	O
-also	O
-slide	O
-further	O
-towards	O
-the	O
-membrane	O
-center	O
-,	O
-relative	O
-to	O
-the	O
-outward	O
--	O
-occluded	O
-state	O
-(	O
-Fig	O
-.	O
-4c	O
-).	O
-	O
-Together	O
-,	O
-these	O
-changes	O
-open	O
-a	O
-second	O
-aqueous	O
-channel	O
-leading	O
-directly	O
-into	O
-SCa	O
-and	O
-Sint	O
-(	O
-Fig	O
-.	O
-4f	O
-,	O
-Fig	O
-.	O
-4h	O
--	O
-i	O
-).	O
-	O
-The	O
-transporter	O
-thus	O
-becomes	O
-fully	O
-outward	O
--	O
-open	O
-.	O
-	O
-To	O
-more	O
-rigorously	O
-characterize	O
-the	O
-influence	O
-of	O
-the	O
-ion	O
--	O
-occupancy	O
-state	O
-on	O
-the	O
-conformational	O
-dynamics	O
-of	O
-the	O
-exchanger	O
-,	O
-we	O
-carried	O
-out	O
-a	O
-series	O
-of	O
-enhanced	O
--	O
-sampling	O
-MD	O
-calculations	O
-designed	O
-to	O
-reversibly	O
-simulate	O
-the	O
-transition	O
-between	O
-the	O
-outward	O
--	O
-occluded	O
-and	O
-fully	O
-outward	O
--	O
-open	O
-states	O
-,	O
-and	O
-thus	O
-quantify	O
-the	O
-free	O
--	O
-energy	O
-landscape	O
-encompassing	O
-these	O
-states	O
-(	O
-Methods	O
-).	O
-	O
-As	O
-above	O
-,	O
-we	O
-initially	O
-examined	O
-three	O
-occupancy	O
-states	O
-,	O
-namely	O
-with	O
-Na	O
-+	O
-in	O
-Sext	O
-,	O
-SCa	O
-and	O
-Sint	O
-,	O
-with	O
-Na	O
-+	O
-only	O
-at	O
-SCa	O
-and	O
-Sint	O
-,	O
-and	O
-without	O
-Na	O
-+.	O
-	O
-These	O
-calculations	O
-demonstrate	O
-that	O
-the	O
-Na	O
-+	O
-occupancy	O
-state	O
-of	O
-the	O
-transporter	O
-has	O
-a	O
-profound	O
-effect	O
-on	O
-its	O
-conformational	O
-free	O
--	O
-energy	O
-landscape	O
-.	O
-	O
-When	O
-all	O
-Na	O
-+	O
-sites	O
-are	O
-occupied	O
-,	O
-the	O
-global	O
-free	O
--	O
-energy	O
-minimum	O
-corresponds	O
-to	O
-a	O
-conformation	O
-in	O
-which	O
-the	O
-ions	O
-are	O
-maximally	O
-coordinated	O
-by	O
-the	O
-protein	O
-(	O
-Fig	O
-.	O
-5a	O
-,	O
-5c	O
-);	O
-TM7ab	O
-is	O
-bent	O
-and	O
-packs	O
-closely	O
-with	O
-TM2	O
-and	O
-TM3	O
-,	O
-and	O
-so	O
-the	O
-binding	O
-sites	O
-are	O
-occluded	O
-from	O
-the	O
-solvent	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-At	O
-a	O
-small	O
-energetic	O
-cost	O
-,	O
-however	O
-,	O
-the	O
-transporter	O
-can	O
-adopt	O
-a	O
-metastable	O
-‘	O
-half	O
--	O
-open	O
-’	O
-conformation	O
-in	O
-which	O
-TM7ab	O
-is	O
-completely	O
-straight	O
-and	O
-Sext	O
-is	O
-open	O
-to	O
-the	O
-exterior	O
-(	O
-Fig	O
-.	O
-5a	O
-,	O
-5b	O
-).	O
-	O
-The	O
-Na	O
-+	O
-ion	O
-at	O
-Sext	O
-remains	O
-fully	O
-coordinated	O
-,	O
-but	O
-an	O
-ordered	O
-water	O
-molecule	O
-now	O
-mediates	O
-its	O
-interaction	O
-with	O
-A206	O
-:	O
-O	O
-,	O
-relieving	O
-the	O
-strain	O
-on	O
-the	O
-F202	O
-:	O
-O	O
-–	O
-A206	O
-:	O
-N	O
-hydrogen	O
--	O
-bond	O
-(	O
-Fig	O
-.	O
-5c	O
-).	O
-	O
-This	O
-semi	O
--	O
-open	O
-conformation	O
-is	O
-nearly	O
-identical	O
-to	O
-that	O
-found	O
-to	O
-be	O
-the	O
-most	O
-probable	O
-when	O
-Na	O
-+	O
-occupies	O
-only	O
-SCa	O
-and	O
-Sint	O
-(	O
-2	O
-×	O
-Na	O
-+,	O
-Fig	O
-.	O
-5a	O
-),	O
-demonstrating	O
-that	O
-binding	O
-(	O
-or	O
-release	O
-)	O
-of	O
-Na	O
-+	O
-to	O
-Sext	O
-occurs	O
-in	O
-this	O
-metastable	O
-conformation	O
-.	O
-	O
-Interestingly	O
-,	O
-this	O
-doubly	O
-occupied	O
-state	O
-can	O
-also	O
-access	O
-conformations	O
-in	O
-which	O
-the	O
-second	O
-aqueous	O
-channel	O
-mentioned	O
-above	O
-,	O
-i	O
-.	O
-e	O
-.	O
-leading	O
-to	O
-SCa	O
-between	O
-TM7	O
-and	O
-TM2	O
-and	O
-over	O
-the	O
-gating	O
-helices	O
-TM1	O
-and	O
-TM6	O
-,	O
-also	O
-becomes	O
-open	O
-(	O
-Fig	O
-.	O
-5b	O
--	O
-c	O
-).	O
-	O
-Crucially	O
-,	O
-though	O
-,	O
-the	O
-free	O
--	O
-energy	O
-landscape	O
-for	O
-this	O
-partially	O
-occupied	O
-state	O
-demonstrates	O
-that	O
-the	O
-occluded	O
-conformation	O
-is	O
-no	O
-longer	O
-energetically	O
-feasible	O
-(	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-Displacement	O
-of	O
-the	O
-two	O
-remaining	O
-Na	O
-+	O
-ions	O
-from	O
-SCa	O
-and	O
-Sint	O
-further	O
-reshapes	O
-the	O
-free	O
--	O
-energy	O
-landscape	O
-of	O
-the	O
-transporter	O
-(	O
-No	O
-ions	O
-,	O
-Fig	O
-.	O
-5a	O
-),	O
-which	O
-now	O
-can	O
-only	O
-adopt	O
-a	O
-fully	O
-open	O
-state	O
-featuring	O
-the	O
-two	O
-aqueous	O
-channels	O
-(	O
-Fig	O
-.	O
-5b	O
--	O
-c	O
-).	O
-	O
-The	O
-transition	O
-to	O
-the	O
-occluded	O
-state	O
-in	O
-this	O
-apo	O
-state	O
-is	O
-again	O
-energetically	O
-unfeasible	O
-.	O
-	O
-From	O
-a	O
-mechanistic	O
-standpoint	O
-,	O
-it	O
-is	O
-satisfying	O
-to	O
-observe	O
-how	O
-the	O
-open	O
-and	O
-semi	O
--	O
-open	O
-states	O
-are	O
-each	O
-compatible	O
-with	O
-two	O
-different	O
-Na	O
-+	O
-occupancies	O
-,	O
-explaining	O
-how	O
-sequential	O
-Na	O
-+	O
-binding	O
-to	O
-energetically	O
-accessible	O
-conformations	O
-(	O
-prior	O
-to	O
-those	O
-binding	O
-events	O
-)	O
-progressively	O
-reshape	O
-the	O
-free	O
--	O
-energy	O
-landscape	O
-of	O
-the	O
-transporter	O
-;	O
-by	O
-contrast	O
-,	O
-the	O
-occluded	O
-conformation	O
-is	O
-forbidden	O
-unless	O
-the	O
-Na	O
-+	O
-occupancy	O
-is	O
-complete	O
-.	O
-	O
-This	O
-processivity	O
-is	O
-logical	O
-since	O
-three	O
-Na	O
-+	O
-ions	O
-are	O
-involved	O
-,	O
-but	O
-also	O
-implies	O
-that	O
-in	O
-the	O
-Ca2	O
-+-	O
-bound	O
-state	O
-,	O
-which	O
-includes	O
-a	O
-single	O
-ion	O
-,	O
-the	O
-transporter	O
-ought	O
-to	O
-be	O
-able	O
-to	O
-access	O
-all	O
-three	O
-major	O
-conformations	O
-,	O
-i	O
-.	O
-e	O
-.	O
-the	O
-outward	O
--	O
-open	O
-state	O
-,	O
-in	O
-order	O
-to	O
-release	O
-(	O
-or	O
-re	O
--	O
-bind	O
-)	O
-Ca2	O
-+,	O
-but	O
-also	O
-the	O
-occluded	O
-conformation	O
-,	O
-and	O
-thus	O
-the	O
-semi	O
--	O
-open	O
-intermediate	O
-,	O
-in	O
-order	O
-to	O
-transition	O
-to	O
-the	O
-inward	O
--	O
-open	O
-state	O
-.	O
-	O
-By	O
-contrast	O
-,	O
-occupancy	O
-by	O
-H	O
-+,	O
-which	O
-as	O
-mentioned	O
-are	O
-not	O
-transported	O
-,	O
-might	O
-be	O
-compatible	O
-with	O
-a	O
-semi	O
--	O
-open	O
-state	O
-as	O
-well	O
-as	O
-with	O
-the	O
-fully	O
-open	O
-conformation	O
-,	O
-but	O
-should	O
-not	O
-be	O
-conducive	O
-to	O
-occlusion	O
-.	O
-	O
-To	O
-assess	O
-this	O
-hypothesis	O
-,	O
-we	O
-carried	O
-out	O
-enhanced	O
--	O
-sampling	O
-simulations	O
-for	O
-the	O
-Ca2	O
-+	O
-and	O
-H	O
-+-	O
-bound	O
-states	O
-of	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-analogous	O
-to	O
-those	O
-described	O
-above	O
-for	O
-Na	O
-+	O
-(	O
-see	O
-Supplementary	O
-Note	O
-2	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3	O
--	O
-4	O
-for	O
-details	O
-on	O
-how	O
-the	O
-structures	O
-of	O
-the	O
-Ca2	O
-+-	O
-bound	O
-state	O
-was	O
-predicted	O
-).	O
-	O
-The	O
-calculated	O
-free	O
--	O
-energy	O
-landscape	O
-for	O
-Ca2	O
-+-	O
-bound	O
-NCX_Mj	O
-confirms	O
-the	O
-hypothesis	O
-outlined	O
-above	O
-(	O
-1	O
-×	O
-Ca2	O
-+,	O
-Fig	O
-.	O
-6a	O
-):	O
-consistent	O
-with	O
-the	O
-fact	O
-that	O
-NCX_Mj	O
-transports	O
-a	O
-single	O
-Ca2	O
-+,	O
-the	O
-occluded	O
-,	O
-dehydrated	O
-conformation	O
-is	O
-one	O
-of	O
-the	O
-major	O
-energetic	O
-minima	O
-,	O
-but	O
-clearly	O
-the	O
-exchanger	O
-can	O
-also	O
-adopt	O
-the	O
-semi	O
--	O
-open	O
-and	O
-open	O
-states	O
-that	O
-would	O
-be	O
-required	O
-for	O
-Ca2	O
-+	O
-release	O
-and	O
-Na	O
-+	O
-entry	O
-,	O
-via	O
-either	O
-of	O
-the	O
-aqueous	O
-access	O
-channels	O
-that	O
-lead	O
-to	O
-Sext	O
-and	O
-SCa	O
-(	O
-Fig	O
-.	O
-6b	O
--	O
-c	O
-).	O
-	O
-By	O
-contrast	O
-,	O
-protonation	O
-of	O
-Glu54	O
-and	O
-Glu213	O
-makes	O
-the	O
-occluded	O
-conformation	O
-energetically	O
-unfeasible	O
-,	O
-consistent	O
-with	O
-the	O
-fact	O
-that	O
-NCX_Mj	O
-does	O
-not	O
-transport	O
-protons	O
-;	O
-in	O
-this	O
-H	O
-+-	O
-bound	O
-state	O
-,	O
-though	O
-,	O
-the	O
-exchanger	O
-can	O
-adopt	O
-the	O
-semi	O
--	O
-open	O
-conformation	O
-captured	O
-in	O
-the	O
-low	O
-pH	O
-,	O
-apo	O
-crystal	O
-structure	O
-(	O
-2	O
-×	O
-H	O
-+,	O
-Fig	O
-.	O
-6a	O
--	O
-c	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-this	O
-systematic	O
-computational	O
-analysis	O
-of	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-clearly	O
-demonstrates	O
-that	O
-the	O
-alternating	O
--	O
-access	O
-and	O
-ion	O
--	O
-recognition	O
-mechanisms	O
-in	O
-this	O
-Na	O
-+/	O
-Ca2	O
-+	O
-exchanger	O
-are	O
-coupled	O
-through	O
-the	O
-influence	O
-that	O
-the	O
-bound	O
-ions	O
-have	O
-on	O
-the	O
-free	O
--	O
-energy	O
-landscape	O
-of	O
-the	O
-protein	O
-,	O
-which	O
-in	O
-turn	O
-determines	O
-whether	O
-or	O
-not	O
-the	O
-occluded	O
-conformation	O
-is	O
-energetically	O
-feasible	O
-.	O
-	O
-This	O
-occluded	O
-conformation	O
-,	O
-which	O
-is	O
-a	O
-necessary	O
-intermediate	O
-between	O
-the	O
-outward	O
-and	O
-inward	O
--	O
-open	O
-states	O
-,	O
-and	O
-which	O
-entails	O
-the	O
-internal	O
-dehydration	O
-of	O
-the	O
-protein	O
-,	O
-is	O
-only	O
-attainable	O
-upon	O
-complete	O
-occupancy	O
-of	O
-the	O
-binding	O
-sites	O
-.	O
-	O
-The	O
-alternating	O
--	O
-access	O
-hypothesis	O
-implicitly	O
-dictates	O
-that	O
-the	O
-switch	O
-between	O
-outward	O
--	O
-and	O
-inward	O
--	O
-open	O
-conformations	O
-of	O
-a	O
-given	O
-secondary	O
--	O
-active	O
-transporter	O
-must	O
-not	O
-occur	O
-unless	O
-the	O
-appropriate	O
-type	O
-and	O
-number	O
-of	O
-substrates	O
-are	O
-recognized	O
-.	O
-	O
-It	O
-is	O
-however	O
-also	O
-non	O
--	O
-trivial	O
-:	O
-antiporters	O
-,	O
-for	O
-example	O
-,	O
-do	O
-not	O
-undergo	O
-the	O
-alternating	O
--	O
-access	O
-transition	O
-without	O
-a	O
-cargo	O
-,	O
-but	O
-this	O
-is	O
-precisely	O
-how	O
-membrane	O
-symporters	O
-reset	O
-their	O
-transport	O
-cycles	O
-.	O
-	O
-Similarly	O
-puzzling	O
-is	O
-that	O
-a	O
-given	O
-antiporter	O
-will	O
-undergo	O
-this	O
-transition	O
-upon	O
-recognition	O
-of	O
-substrates	O
-of	O
-different	O
-charge	O
-,	O
-size	O
-and	O
-number	O
-.	O
-	O
-Yet	O
-,	O
-when	O
-multiple	O
-species	O
-are	O
-to	O
-be	O
-co	O
--	O
-translocated	O
-,	O
-by	O
-either	O
-an	O
-antiporter	O
-or	O
-a	O
-symporter	O
-,	O
-partial	O
-occupancies	O
-must	O
-not	O
-be	O
-conducive	O
-to	O
-the	O
-alternating	O
--	O
-access	O
-switch	O
-.	O
-	O
-Here	O
-,	O
-we	O
-have	O
-provided	O
-novel	O
-insights	O
-into	O
-this	O
-intriguing	O
-mechanism	O
-of	O
-conformational	O
-control	O
-through	O
-structural	O
-studies	O
-and	O
-quantitative	O
-molecular	O
-simulations	O
-of	O
-a	O
-Na	O
-+/	O
-Ca2	O
-+	O
-exchanger	O
-.	O
-	O
-Specifically	O
-,	O
-our	O
-studies	O
-of	O
-NCX_Mj	O
-reveal	O
-the	O
-mechanism	O
-of	O
-forward	O
-ion	O
-exchange	O
-(	O
-Fig	O
-.	O
-7	O
-).	O
-	O
-The	O
-internal	O
-symmetry	O
-of	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-and	O
-the	O
-inward	O
--	O
-facing	O
-crystal	O
-structures	O
-of	O
-several	O
-Ca2	O
-+/	O
-H	O
-+	O
-exchangers	O
-indicate	O
-that	O
-the	O
-alternating	O
--	O
-access	O
-mechanism	O
-of	O
-NCX	O
-proteins	O
-entails	O
-a	O
-sliding	O
-motion	O
-of	O
-TM1	O
-and	O
-TM6	O
-relative	O
-to	O
-the	O
-rest	O
-of	O
-the	O
-transporter	O
-.	O
-	O
-Here	O
-,	O
-we	O
-demonstrate	O
-that	O
-conformational	O
-changes	O
-in	O
-the	O
-extracellular	O
-region	O
-of	O
-the	O
-TM2	O
--	O
-TM3	O
-and	O
-TM7	O
--	O
-TM8	O
-bundle	O
-precede	O
-and	O
-are	O
-necessary	O
-for	O
-the	O
-transition	O
-,	O
-and	O
-are	O
-associated	O
-with	O
-ion	O
-recognition	O
-and	O
-/	O
-or	O
-release	O
-.	O
-	O
-The	O
-most	O
-apparent	O
-of	O
-these	O
-changes	O
-involves	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-TM7	O
-(	O
-TM7ab	O
-);	O
-together	O
-with	O
-more	O
-subtle	O
-displacements	O
-in	O
-TM2	O
-and	O
-TM3	O
-,	O
-this	O
-change	O
-in	O
-TM7ab	O
-correlates	O
-with	O
-the	O
-opening	O
-and	O
-closing	O
-of	O
-two	O
-distinct	O
-aqueous	O
-channels	O
-leading	O
-into	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-from	O
-the	O
-extracellular	O
-solution	O
-.	O
-	O
-Interestingly	O
-,	O
-the	O
-bending	O
-of	O
-TM7	O
-associated	O
-with	O
-the	O
-occlusion	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-also	O
-unlocks	O
-its	O
-interaction	O
-with	O
-TM6	O
-,	O
-and	O
-thus	O
-enables	O
-TM6	O
-and	O
-TM1	O
-to	O
-freely	O
-slide	O
-to	O
-the	O
-inward	O
--	O
-facing	O
-conformation	O
-.	O
-	O
-The	O
-crystal	O
-structures	O
-of	O
-NCX_Mj	O
-reported	O
-here	O
-,	O
-with	O
-either	O
-Na	O
-+,	O
-Ca2	O
-+,	O
-Sr2	O
-+	O
-or	O
-H	O
-+	O
-bound	O
-,	O
-capture	O
-the	O
-exchanger	O
-in	O
-different	O
-conformational	O
-states	O
-.	O
-	O
-These	O
-states	O
-can	O
-only	O
-represent	O
-a	O
-subset	O
-among	O
-all	O
-possible	O
-,	O
-but	O
-they	O
-ought	O
-to	O
-reflect	O
-inherent	O
-preferences	O
-of	O
-the	O
-transporter	O
-,	O
-modulated	O
-by	O
-the	O
-experimental	O
-conditions	O
-.	O
-	O
-For	O
-example	O
-,	O
-in	O
-the	O
-crystal	O
-of	O
-NCX_Mj	O
-in	O
-LCP	O
-,	O
-the	O
-extracellular	O
-half	O
-of	O
-the	O
-gating	O
-helices	O
-(	O
-TM6	O
-and	O
-TM1	O
-)	O
-form	O
-a	O
-lattice	O
-contact	O
-,	O
-which	O
-might	O
-ultimately	O
-restrict	O
-the	O
-degree	O
-of	O
-opening	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-in	O
-some	O
-cases	O
-(	O
-e	O
-.	O
-g	O
-.	O
-in	O
-the	O
-apo	O
-,	O
-low	O
-pH	O
-structure	O
-).	O
-	O
-Nonetheless	O
-,	O
-the	O
-calculated	O
-free	O
--	O
-energy	O
-landscapes	O
-,	O
-derived	O
-without	O
-knowledge	O
-of	O
-the	O
-experimental	O
-data	O
-,	O
-reassuringly	O
-confirm	O
-that	O
-the	O
-crystallized	O
-structures	O
-correspond	O
-to	O
-mechanistically	O
-relevant	O
-,	O
-interconverting	O
-states	O
-.	O
-	O
-The	O
-simulations	O
-also	O
-demonstrate	O
-how	O
-this	O
-landscape	O
-is	O
-drastically	O
-re	O
--	O
-shaped	O
-upon	O
-each	O
-ion	O
--	O
-binding	O
-event	O
-.	O
-	O
-Indeed	O
-,	O
-we	O
-show	O
-that	O
-it	O
-is	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-the	O
-occluded	O
-state	O
-in	O
-this	O
-landscape	O
-that	O
-explains	O
-the	O
-antiport	O
-function	O
-of	O
-NCX_Mj	O
-and	O
-its	O
-3Na	O
-+:	O
-1Ca2	O
-+	O
-stoichiometry	O
-.	O
-	O
-We	O
-posit	O
-that	O
-a	O
-similar	O
-principle	O
-might	O
-govern	O
-the	O
-alternating	O
--	O
-access	O
-mechanism	O
-in	O
-other	O
-transporters	O
-;	O
-that	O
-is	O
-,	O
-we	O
-anticipate	O
-that	O
-for	O
-both	O
-symporters	O
-and	O
-antiporters	O
-,	O
-it	O
-is	O
-the	O
-feasibility	O
-of	O
-the	O
-occluded	O
-state	O
-,	O
-encoded	O
-in	O
-the	O
-protein	O
-conformational	O
-free	O
--	O
-energy	O
-landscape	O
-and	O
-its	O
-dependence	O
-on	O
-substrate	O
-binding	O
-,	O
-that	O
-ultimately	O
-explains	O
-their	O
-specific	O
-coupling	O
-mechanisms	O
-.	O
-	O
-In	O
-multiple	O
-ways	O
-,	O
-our	O
-findings	O
-provide	O
-an	O
-explanation	O
-for	O
-,	O
-existing	O
-functional	O
-,	O
-biochemical	O
-and	O
-biophysical	O
-data	O
-for	O
-both	O
-NCX_Mj	O
-and	O
-its	O
-eukaryotic	O
-homologues	O
-.	O
-	O
-The	O
-striking	O
-quantitative	O
-agreement	O
-between	O
-the	O
-ion	O
--	O
-binding	O
-affinities	O
-inferred	O
-from	O
-our	O
-crystallographic	O
-titrations	O
-and	O
-the	O
-Km	O
-and	O
-K1	O
-/	O
-2	O
-values	O
-previously	O
-deduced	O
-from	O
-functional	O
-assays	O
-has	O
-been	O
-discussed	O
-above	O
-.	O
-	O
-Consistent	O
-with	O
-that	O
-finding	O
-,	O
-mutations	O
-that	O
-have	O
-been	O
-shown	O
-to	O
-inactivate	O
-or	O
-diminish	O
-the	O
-transport	O
-activity	O
-of	O
-NCX_Mj	O
-and	O
-cardiac	O
-NCX	O
-perfectly	O
-map	O
-to	O
-the	O
-first	O
-ion	O
--	O
-coordination	O
-shell	O
-in	O
-our	O
-NCX_Mj	O
-structures	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4c	O
--	O
-d	O
-).	O
-	O
-The	O
-crystallographic	O
-data	O
-also	O
-provides	O
-the	O
-long	O
--	O
-sought	O
-structural	O
-basis	O
-for	O
-the	O
-‘	O
-two	O
--	O
-site	O
-’	O
-model	O
-proposed	O
-to	O
-describe	O
-competitive	O
-cation	O
-binding	O
-in	O
-eukaryotic	O
-NCX	O
-,	O
-underscoring	O
-the	O
-relevance	O
-of	O
-these	O
-studies	O
-of	O
-NCX_Mj	O
-as	O
-a	O
-prototypical	O
-Na	O
-+/	O
-Ca2	O
-+	O
-exchanger	O
-.	O
-	O
-Specifically	O
-,	O
-our	O
-crystal	O
-titrations	O
-suggest	O
-that	O
-,	O
-during	O
-forward	O
-Na	O
-+/	O
-Ca2	O
-+	O
-exchange	O
-,	O
-sites	O
-Sint	O
-and	O
-SCa	O
-,	O
-which	O
-Ca2	O
-+	O
-and	O
-Na	O
-+	O
-compete	O
-for	O
-,	O
-can	O
-be	O
-grouped	O
-into	O
-one	O
-;	O
-Na	O
-+	O
-binding	O
-to	O
-these	O
-sites	O
-does	O
-not	O
-require	O
-high	O
-Na	O
-+	O
-concentrations	O
-,	O
-and	O
-two	O
-Na	O
-+	O
-ions	O
-along	O
-with	O
-a	O
-water	O
-molecule	O
-(	O
-at	O
-Smid	O
-)	O
-are	O
-sufficient	O
-to	O
-displace	O
-Ca2	O
-+,	O
-explaining	O
-the	O
-Hill	O
-coefficient	O
-of	O
-~	O
-2	O
-for	O
-Na	O
-+-	O
-dependent	O
-inhibition	O
-of	O
-Ca2	O
-+	O
-fluxes	O
-.	O
-	O
-The	O
-Sext	O
-site	O
-,	O
-by	O
-contrast	O
-,	O
-might	O
-be	O
-thought	O
-as	O
-an	O
-activation	O
-site	O
-for	O
-inward	O
-Na	O
-+	O
-translocation	O
-,	O
-since	O
-this	O
-is	O
-where	O
-the	O
-third	O
-Na	O
-+	O
-ion	O
-binds	O
-at	O
-high	O
-Na	O
-+	O
-concentration	O
-,	O
-enabling	O
-the	O
-transition	O
-to	O
-the	O
-occluded	O
-state	O
-.	O
-	O
-Interestingly	O
-,	O
-binding	O
-of	O
-Ca2	O
-+	O
-to	O
-Smid	O
-appears	O
-to	O
-be	O
-also	O
-possible	O
-,	O
-but	O
-available	O
-evidence	O
-indicates	O
-that	O
-this	O
-event	O
-transiently	O
-blocks	O
-the	O
-exchange	O
-cycle	O
-.	O
-	O
-Indeed	O
-,	O
-structures	O
-of	O
-NCX_Mj	O
-bound	O
-to	O
-Cd2	O
-+	O
-or	O
-Mn2	O
-+,	O
-both	O
-of	O
-which	O
-inhibit	O
-transport	O
-,	O
-show	O
-these	O
-ions	O
-at	O
-Smid	O
-;	O
-by	O
-contrast	O
-,	O
-Sr2	O
-+	O
-binds	O
-only	O
-to	O
-SCa	O
-,	O
-and	O
-accordingly	O
-,	O
-is	O
-transported	O
-by	O
-NCX	O
-similarly	O
-to	O
-calcium	O
-.	O
-	O
-Lastly	O
-,	O
-our	O
-theory	O
-that	O
-occlusion	O
-of	O
-NCX_Mj	O
-is	O
-selectively	O
-induced	O
-upon	O
-Ca2	O
-+	O
-or	O
-Na	O
-+	O
-recognition	O
-is	O
-consonant	O
-with	O
-a	O
-recent	O
-analysis	O
-of	O
-the	O
-rate	O
-of	O
-hydrogen	O
--	O
-deuterium	O
-exchange	O
-(	O
-HDX	O
-)	O
-in	O
-NCX_Mj	O
-,	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-these	O
-ions	O
-,	O
-in	O
-conditions	O
-that	O
-favor	O
-outward	O
--	O
-facing	O
-conformations	O
-.	O
-	O
-Specifically	O
-,	O
-saturating	O
-amounts	O
-of	O
-Ca2	O
-+	O
-or	O
-Na	O
-+	O
-resulted	O
-in	O
-a	O
-noticeable	O
-slowdown	O
-in	O
-the	O
-HDX	O
-rate	O
-for	O
-extracellular	O
-portions	O
-of	O
-the	O
-α	O
--	O
-repeat	O
-helices	O
-.	O
-	O
-We	O
-interpret	O
-these	O
-observations	O
-as	O
-reflecting	O
-that	O
-the	O
-solvent	O
-accessibility	O
-of	O
-the	O
-protein	O
-interior	O
-is	O
-diminished	O
-upon	O
-ion	O
-recognition	O
-,	O
-consistent	O
-with	O
-our	O
-finding	O
-that	O
-opening	O
-and	O
-closing	O
-of	O
-extracellular	O
-aqueous	O
-pathways	O
-to	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-depend	O
-on	O
-ion	O
-occupancy	O
-state	O
-.	O
-	O
-In	O
-addition	O
-,	O
-the	O
-increased	O
-compactness	O
-of	O
-the	O
-protein	O
-tertiary	O
-structure	O
-in	O
-the	O
-occluded	O
-state	O
-would	O
-also	O
-slow	O
-down	O
-the	O
-dynamics	O
-of	O
-the	O
-secondary	O
--	O
-structure	O
-elements	O
-,	O
-and	O
-thus	O
-further	O
-reduce	O
-the	O
-HDX	O
-rate	O
-.	O
-	O
-Our	O
-data	O
-would	O
-also	O
-explain	O
-the	O
-observation	O
-that	O
-the	O
-reduction	O
-in	O
-the	O
-HDX	O
-rate	O
-is	O
-comparable	O
-for	O
-Na	O
-+	O
-and	O
-Ca2	O
-+,	O
-as	O
-well	O
-as	O
-the	O
-finding	O
-that	O
-the	O
-degree	O
-of	O
-deuterium	O
-incorporation	O
-remains	O
-non	O
--	O
-negligible	O
-even	O
-under	O
-saturating	O
-ion	O
-concentrations	O
-.	O
-	O
-As	O
-the	O
-calculated	O
-free	O
--	O
-energy	O
-landscapes	O
-show	O
-,	O
-Na	O
-+	O
-and	O
-Ca2	O
-+	O
-induce	O
-the	O
-occlusion	O
-of	O
-the	O
-transporter	O
-in	O
-a	O
-comparable	O
-manner	O
-,	O
-and	O
-yet	O
-the	O
-ion	O
--	O
-bound	O
-states	O
-retain	O
-the	O
-ability	O
-to	O
-explore	O
-conformations	O
-that	O
-are	O
-partially	O
-or	O
-fully	O
-open	O
-to	O
-the	O
-extracellular	O
-solution	O
-,	O
-precisely	O
-so	O
-as	O
-to	O
-be	O
-able	O
-to	O
-unload	O
-and	O
-re	O
--	O
-load	O
-the	O
-substrates	O
-.	O
-	O
-Na	O
-+	O
-binding	O
-to	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-.	O
-	O
-(	O
-a	O
-)	O
-Overall	O
-structure	O
-of	O
-native	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-from	O
-crystals	O
-grown	O
-in	O
-150	O
-mM	O
-Na	O
-+.	O
-	O
-Colored	O
-spheres	O
-represent	O
-the	O
-bound	O
-Na	O
-+	O
-(	O
-green	O
-)	O
-and	O
-water	O
-(	O
-red	O
-).	O
-	O
-(	O
-b	O
-)	O
-Structural	O
-details	O
-and	O
-definition	O
-of	O
-the	O
-four	O
-central	O
-binding	O
-sites	O
-.	O
-	O
-The	O
-electron	O
-density	O
-(	O
-grey	O
-mesh	O
-,	O
-1	O
-.	O
-9	O
-Å	O
-Fo	O
--	O
-Fc	O
-ion	O
-omit	O
-map	O
-contoured	O
-at	O
-4σ	O
-)	O
-at	O
-Smid	O
-was	O
-modeled	O
-as	O
-water	O
-(	O
-red	O
-sphere	O
-)	O
-and	O
-those	O
-at	O
-Sext	O
-,	O
-SCa	O
-and	O
-Sint	O
-as	O
-Na	O
-+	O
-ions	O
-(	O
-green	O
-spheres	O
-).	O
-	O
-Further	O
-details	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-.	O
-(	O
-c	O
-)	O
-Concentration	O
--	O
-dependent	O
-change	O
-in	O
-Na	O
-+	O
-occupancy	O
-(	O
-see	O
-also	O
-Table	O
-1	O
-).	O
-	O
-All	O
-Fo	O
-–	O
-Fc	O
-ion	O
--	O
-omit	O
-maps	O
-are	O
-calculated	O
-to	O
-2	O
-.	O
-4	O
-Å	O
-and	O
-contoured	O
-at	O
-3σ	O
-for	O
-comparison	O
-.	O
-	O
-The	O
-displacement	O
-of	O
-A206	O
-reflects	O
-the	O
-[	O
-Na	O
-+]-	O
-dependent	O
-conformational	O
-change	O
-from	O
-the	O
-partially	O
-open	O
-to	O
-the	O
-occluded	O
-state	O
-(	O
-observed	O
-at	O
-low	O
-and	O
-high	O
-Na	O
-+	O
-concentrations	O
-,	O
-respectively	O
-).	O
-	O
-At	O
-20	O
-mM	O
-Na	O
-+,	O
-both	O
-conformations	O
-co	O
--	O
-exist	O
-.	O
-	O
-No	O
-significant	O
-changes	O
-were	O
-observed	O
-in	O
-the	O
-side	O
--	O
-chains	O
-involved	O
-in	O
-ion	O
-or	O
-water	O
-coordination	O
-at	O
-the	O
-SCa	O
-,	O
-Sint	O
-and	O
-Smid	O
-sites	O
-.	O
-	O
-Na	O
-+-	O
-occupancy	O
-dependent	O
-conformational	O
-change	O
-in	O
-NCX_Mj	O
-.	O
-	O
-(	O
-a	O
-)	O
-Superimposition	O
-of	O
-the	O
-NCX_Mj	O
-crystal	O
-structures	O
-obtained	O
-in	O
-high	O
-(	O
-100	O
-mM	O
-,	O
-cyan	O
-cylinders	O
-)	O
-and	O
-low	O
-(	O
-10	O
-mM	O
-,	O
-brown	O
-cylinders	O
-)	O
-Na	O
-+	O
-concentrations	O
-.	O
-	O
-(	O
-b	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-interface	O
-between	O
-TM6	O
-and	O
-TM7ab	O
-in	O
-the	O
-NCX_Mj	O
-structures	O
-obtained	O
-at	O
-high	O
-and	O
-low	O
-Na	O
-+	O
-concentrations	O
-(	O
-top	O
-and	O
-lower	O
-panels	O
-,	O
-respectively	O
-).	O
-	O
-Residues	O
-forming	O
-van	O
--	O
-der	O
--	O
-Waals	O
-contacts	O
-in	O
-the	O
-structure	O
-at	O
-low	O
-Na	O
-+	O
-concentration	O
-are	O
-shown	O
-in	O
-detail	O
-.	O
-	O
-(	O
-c	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-Na	O
-+-	O
-binding	O
-sites	O
-.	O
-	O
-The	O
-vacant	O
-Sext	O
-site	O
-in	O
-the	O
-structure	O
-at	O
-low	O
-Na	O
-+	O
-concentration	O
-is	O
-indicated	O
-with	O
-a	O
-white	O
-sphere	O
-.	O
-	O
-Residues	O
-surrounding	O
-this	O
-site	O
-are	O
-also	O
-indicated	O
-;	O
-note	O
-A206	O
-(	O
-labeled	O
-in	O
-red	O
-)	O
-coordinates	O
-Na	O
-+	O
-at	O
-Sext	O
-via	O
-its	O
-backbone	O
-carbonyl	O
-oxygen	O
-.	O
-	O
-(	O
-d	O
-)	O
-Extracellular	O
-solvent	O
-accessibility	O
-of	O
-the	O
-ion	O
-binding	O
-sites	O
-in	O
-the	O
-structures	O
-at	O
-high	O
-and	O
-low	O
-[	O
-Na	O
-+].	O
-	O
-Putative	O
-solvent	O
-channels	O
-are	O
-represented	O
-as	O
-light	O
--	O
-purple	O
-surfaces	O
-.	O
-	O
-Divalent	O
-cation	O
-binding	O
-and	O
-apo	O
-structure	O
-of	O
-NCX_Mj	O
-.	O
-(	O
-a	O
-)	O
-A	O
-single	O
-Sr2	O
-+	O
-(	O
-dark	O
-blue	O
-sphere	O
-)	O
-binds	O
-at	O
-SCa	O
-in	O
-crystals	O
-titrated	O
-with	O
-10	O
-mM	O
-Sr2	O
-+	O
-and	O
-2	O
-.	O
-5	O
-mM	O
-Na	O
-+	O
-(	O
-see	O
-also	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Residues	O
-involved	O
-in	O
-Sr2	O
-+	O
-coordination	O
-are	O
-labeled	O
-.	O
-	O
-There	O
-are	O
-no	O
-significant	O
-changes	O
-in	O
-the	O
-side	O
--	O
-chains	O
-involved	O
-in	O
-ion	O
-coordination	O
-,	O
-relative	O
-to	O
-the	O
-Na	O
-+-	O
-bound	O
-state	O
-.	O
-	O
-T50	O
-and	O
-T209	O
-(	O
-labeled	O
-in	O
-red	O
-)	O
-coordinate	O
-Sr2	O
-+	O
-through	O
-their	O
-backbone	O
-carbonyl	O
--	O
-oxygen	O
-atoms	O
-.	O
-	O
-High	O
-Na	O
-+	O
-concentration	O
-(	O
-100	O
-mM	O
-)	O
-completely	O
-displaces	O
-Sr2	O
-+	O
-and	O
-reverts	O
-NCX_Mj	O
-to	O
-the	O
-occluded	O
-state	O
-(	O
-right	O
-panel	O
-).	O
-	O
-The	O
-contour	O
-level	O
-of	O
-the	O
-Fo	O
-–	O
-Fc	O
-omit	O
-map	O
-of	O
-the	O
-structure	O
-at	O
-high	O
-Na	O
-+	O
-concentration	O
-was	O
-lowered	O
-(	O
-to	O
-4σ	O
-)	O
-so	O
-as	O
-to	O
-visualize	O
-the	O
-density	O
-from	O
-Na	O
-+	O
-ions	O
-and	O
-H2O	O
-.	O
-	O
-(	O
-b	O
-)	O
-Ca2	O
-+	O
-(	O
-tanned	O
-spheres	O
-)	O
-binds	O
-either	O
-to	O
-SCa	O
-or	O
-Smid	O
-in	O
-crystals	O
-titrated	O
-with	O
-10	O
-mM	O
-Ca2	O
-+	O
-and	O
-2	O
-.	O
-5	O
-mM	O
-Na	O
-+	O
-(	O
-see	O
-also	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-The	O
-relative	O
-occupancies	O
-are	O
-55	O
-%	O
-and	O
-45	O
-%,	O
-respectively	O
-.	O
-(	O
-c	O
-)	O
-Superimposition	O
-of	O
-NCX_Mj	O
-structures	O
-obtained	O
-at	O
-low	O
-Na	O
-+	O
-concentration	O
-(	O
-10	O
-mM	O
-)	O
-and	O
-pH	O
-6	O
-.	O
-5	O
-(	O
-brown	O
-)	O
-and	O
-in	O
-the	O
-absence	O
-of	O
-Na	O
-+	O
-and	O
-pH	O
-4	O
-(	O
-light	O
-green	O
-),	O
-referred	O
-to	O
-as	O
-apo	O
-state	O
-.	O
-(	O
-d	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-in	O
-the	O
-apo	O
-(	O
-or	O
-high	O
-H	O
-+)	O
-state	O
-.	O
-	O
-The	O
-side	O
-chains	O
-of	O
-E54	O
-and	O
-E213	O
-from	O
-the	O
-low	O
-Na	O
-+	O
-structure	O
-are	O
-also	O
-shown	O
-(	O
-light	O
-brown	O
-)	O
-for	O
-comparison	O
-.	O
-	O
-White	O
-spheres	O
-indicate	O
-the	O
-location	O
-Sint	O
-,	O
-Smid	O
-SCa	O
-.	O
-(	O
-e	O
-)	O
-Extracellular	O
-solvent	O
-accessibility	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-in	O
-apo	O
-NCX_Mj	O
-.	O
-	O
-Spontaneous	O
-changes	O
-in	O
-the	O
-structure	O
-of	O
-outward	O
--	O
-occluded	O
-,	O
-fully	O
-Na	O
-+-	O
-occupied	O
-NCX_Mj	O
-(	O
-PDB	O
-code	O
-3V5U	O
-)	O
-upon	O
-sequential	O
-displacement	O
-of	O
-Na	O
-+.	O
-	O
-(	O
-a	O
-)	O
-Representative	O
-simulation	O
-snapshots	O
-of	O
-NCX_Mj	O
-(	O
-Methods	O
-)	O
-with	O
-Na	O
-+	O
-bound	O
-at	O
-Sext	O
-,	O
-SCa	O
-and	O
-Sint	O
-(	O
-orange	O
-cartoons	O
-,	O
-green	O
-spheres	O
-)	O
-and	O
-with	O
-Na	O
-+	O
-bound	O
-only	O
-at	O
-SCa	O
-and	O
-Sint	O
-(	O
-marine	O
-cartoons	O
-,	O
-yellow	O
-spheres	O
-)	O
-(	O
-b	O
-)	O
-Close	O
--	O
-up	O
-of	O
-the	O
-backbone	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-TM7	O
-(	O
-TM7ab	O
-),	O
-in	O
-the	O
-same	O
-Na	O
-+	O
-occupancy	O
-states	O
-depicted	O
-in	O
-(	O
-a	O
-).	O
-	O
-(	O
-c	O
-)	O
-Representative	O
-simulation	O
-snapshots	O
-(	O
-same	O
-as	O
-above	O
-)	O
-with	O
-Na	O
-+	O
-bound	O
-at	O
-SCa	O
-and	O
-Sint	O
-(	O
-marine	O
-cartoons	O
-,	O
-yellow	O
-spheres	O
-)	O
-and	O
-without	O
-any	O
-Na	O
-+	O
-bound	O
-(	O
-grey	O
-cartoons	O
-).	O
-	O
-(	O
-d	O
-)	O
-Close	O
--	O
-up	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-region	O
-in	O
-the	O
-fully	O
-Na	O
-+-	O
-occupied	O
-state	O
-.	O
-	O
-Approximate	O
-distances	O
-between	O
-TM2	O
-,	O
-TM3	O
-and	O
-TM7	O
-are	O
-indicated	O
-in	O
-Å	O
-.	O
-(	O
-e	O
-)	O
-Close	O
--	O
-up	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-region	O
-in	O
-the	O
-partially	O
-Na	O
-+-	O
-occupied	O
-state	O
-.	O
-	O
-(	O
-f	O
-)	O
-Close	O
--	O
-up	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-region	O
-in	O
-the	O
-Na	O
-+-	O
-free	O
-state	O
-.	O
-(	O
-g	O
--	O
-i	O
-)	O
-Analytical	O
-descriptors	O
-of	O
-the	O
-changes	O
-just	O
-described	O
-,	O
-calculated	O
-from	O
-the	O
-simulations	O
-of	O
-each	O
-Na	O
-+-	O
-occupancy	O
-state	O
-depicted	O
-in	O
-panels	O
-(	O
-a	O
--	O
-f	O
-).	O
-	O
-These	O
-descriptors	O
-were	O
-employed	O
-as	O
-collective	O
-variables	O
-in	O
-the	O
-Bias	O
--	O
-Exchange	O
-Metadynamics	O
-simulations	O
-(	O
-Methods	O
-).	O
-	O
-(	O
-g	O
-)	O
-Probability	O
-distributions	O
-of	O
-an	O
-analytical	O
-descriptor	O
-of	O
-the	O
-backbone	O
-hydrogen	O
--	O
-bonding	O
-pattern	O
-in	O
-TM7ab	O
-(	O
-Eq	O
-.	O
-2	O
-).	O
-(	O
-h	O
-)	O
-Mean	O
-value	O
-(	O
-with	O
-standard	O
-deviation	O
-)	O
-of	O
-a	O
-quantitative	O
-descriptor	O
-of	O
-the	O
-solvent	O
-accessibility	O
-of	O
-the	O
-Sext	O
-site	O
-(	O
-Eq	O
-.	O
-1	O
-).	O
-(	O
-i	O
-)	O
-Mean	O
-value	O
-(	O
-with	O
-standard	O
-deviation	O
-)	O
-of	O
-a	O
-quantitative	O
-descriptor	O
-of	O
-the	O
-solvent	O
-accessibility	O
-of	O
-the	O
-SCa	O
-site	O
-(	O
-Eq	O
-.	O
-1	O
-).	O
-	O
-Thermodynamic	O
-basis	O
-for	O
-the	O
-proposed	O
-mechanism	O
-of	O
-substrate	O
-control	O
-of	O
-the	O
-alternating	O
--	O
-access	O
-transition	O
-of	O
-NCX	O
-.	O
-(	O
-a	O
-)	O
-Calculated	O
-conformational	O
-free	O
--	O
-energy	O
-landscapes	O
-for	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-,	O
-for	O
-two	O
-different	O
-Na	O
-+-	O
-occupancy	O
-states	O
-,	O
-and	O
-for	O
-a	O
-state	O
-with	O
-no	O
-ions	O
-bound	O
-.	O
-	O
-The	O
-free	O
-energy	O
-is	O
-plotted	O
-as	O
-a	O
-function	O
-of	O
-two	O
-coordinates	O
-,	O
-each	O
-describing	O
-the	O
-degree	O
-of	O
-opening	O
-of	O
-the	O
-aqueous	O
-channels	O
-leading	O
-to	O
-the	O
-Sext	O
-and	O
-SCa	O
-sites	O
-,	O
-respectively	O
-(	O
-see	O
-Methods	O
-).	O
-	O
-Black	O
-circles	O
-map	O
-the	O
-X	O
--	O
-ray	O
-structures	O
-of	O
-NCX_Mj	O
-obtained	O
-at	O
-high	O
-and	O
-low	O
-Na	O
-+	O
-concentration	O
-,	O
-as	O
-well	O
-as	O
-that	O
-at	O
-low	O
-pH	O
-,	O
-reported	O
-in	O
-this	O
-study	O
-.	O
-	O
-(	O
-b	O
-)	O
-Density	O
-isosurfaces	O
-for	O
-water	O
-molecules	O
-within	O
-12	O
-Å	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-region	O
-(	O
-grey	O
-volumes	O
-),	O
-for	O
-each	O
-of	O
-the	O
-major	O
-conformational	O
-free	O
--	O
-energy	O
-minima	O
-in	O
-each	O
-ion	O
--	O
-occupancy	O
-state	O
-.	O
-	O
-Na	O
-+	O
-ions	O
-are	O
-shown	O
-as	O
-green	O
-spheres	O
-.	O
-	O
-The	O
-two	O
-inverted	O
--	O
-topology	O
-repeats	O
-in	O
-the	O
-transporter	O
-structure	O
-(	O
-transparent	O
-cartoons	O
-)	O
-are	O
-colored	O
-differently	O
-(	O
-TM1	O
--	O
-5	O
-,	O
-orange	O
-;	O
-TM6	O
--	O
-10	O
-,	O
-marine	O
-).	O
-	O
-(	O
-c	O
-)	O
-Close	O
--	O
-up	O
-views	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-region	O
-in	O
-the	O
-same	O
-conformational	O
-free	O
--	O
-energy	O
-minima	O
-.	O
-	O
-Key	O
-residues	O
-involved	O
-in	O
-Na	O
-+	O
-and	O
-water	O
-coordination	O
-(	O
-W	O
-)	O
-are	O
-highlighted	O
-(	O
-sticks	O
-,	O
-black	O
-lines	O
-).	O
-	O
-The	O
-water	O
--	O
-density	O
-maps	O
-in	O
-(	O
-b	O
-)	O
-is	O
-shown	O
-here	O
-as	O
-a	O
-grey	O
-mesh	O
-.	O
-	O
-Note	O
-D240	O
-is	O
-protonated	O
-,	O
-while	O
-E54	O
-and	O
-E213	O
-are	O
-ionized	O
-.	O
-	O
-Thermodynamic	O
-basis	O
-for	O
-the	O
-proposed	O
-mechanism	O
-of	O
-substrate	O
-control	O
-of	O
-the	O
-alternating	O
--	O
-access	O
-transition	O
-of	O
-NCX	O
-.	O
-(	O
-a	O
-)	O
-Calculated	O
-free	O
--	O
-energy	O
-landscapes	O
-for	O
-outward	O
--	O
-facing	O
-NCX_Mj	O
-,	O
-for	O
-the	O
-Ca2	O
-+	O
-and	O
-the	O
-fully	O
-protonated	O
-state	O
-.	O
-	O
-The	O
-free	O
-energy	O
-is	O
-plotted	O
-as	O
-in	O
-Fig	O
-.	O
-5	O
-.	O
-	O
-For	O
-Ca2	O
-+,	O
-a	O
-map	O
-is	O
-shown	O
-in	O
-which	O
-a	O
-correction	O
-for	O
-the	O
-charge	O
--	O
-transfer	O
-between	O
-the	O
-ion	O
-and	O
-the	O
-protein	O
-is	O
-introduced	O
-,	O
-alongside	O
-the	O
-uncorrected	O
-map	O
-(	O
-see	O
-Supplementary	O
-Notes	O
-3	O
--	O
-4	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-5	O
--	O
-6	O
-).	O
-	O
-The	O
-uncorrected	O
-map	O
-overstabilizes	O
-the	O
-open	O
-state	O
-relative	O
-to	O
-the	O
-semi	O
--	O
-open	O
-and	O
-occluded	O
-because	O
-it	O
-also	O
-overestimates	O
-the	O
-cost	O
-of	O
-dehydration	O
-of	O
-the	O
-ion	O
-,	O
-once	O
-it	O
-is	O
-bound	O
-to	O
-the	O
-protein	O
-(	O
-this	O
-effect	O
-is	O
-negligible	O
-for	O
-Na	O
-+).	O
-	O
-Black	O
-circles	O
-map	O
-the	O
-crystal	O
-structures	O
-obtained	O
-at	O
-high	O
-Ca2	O
-+	O
-concentration	O
-and	O
-at	O
-low	O
-pH	O
-(	O
-or	O
-high	O
-H	O
-+)	O
-reported	O
-in	O
-this	O
-study	O
-.	O
-	O
-(	O
-b	O
-)	O
-Water	O
--	O
-density	O
-isosurfaces	O
-analogous	O
-to	O
-those	O
-in	O
-Fig	O
-.	O
-5	O
-are	O
-shown	O
-for	O
-each	O
-of	O
-the	O
-major	O
-conformational	O
-free	O
--	O
-energy	O
-minima	O
-in	O
-the	O
-free	O
--	O
-energy	O
-maps	O
-.	O
-	O
-The	O
-Ca2	O
-+	O
-ion	O
-is	O
-shown	O
-as	O
-a	O
-red	O
-sphere	O
-;	O
-the	O
-protein	O
-is	O
-shown	O
-as	O
-in	O
-Fig	O
-.	O
-5	O
-.	O
-(	O
-c	O
-)	O
-Close	O
--	O
-up	O
-views	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-region	O
-in	O
-the	O
-same	O
-conformational	O
-free	O
--	O
-energy	O
-minima	O
-.	O
-	O
-Key	O
-residues	O
-involved	O
-in	O
-Ca2	O
-+	O
-and	O
-water	O
-coordination	O
-(	O
-W	O
-)	O
-are	O
-highlighted	O
-(	O
-sticks	O
-,	O
-black	O
-lines	O
-).	O
-	O
-The	O
-water	O
--	O
-density	O
-maps	O
-in	O
-(	O
-b	O
-)	O
-are	O
-shown	O
-here	O
-as	O
-a	O
-grey	O
-mesh	O
-.	O
-	O
-In	O
-the	O
-occluded	O
-state	O
-with	O
-Ca2	O
-+	O
-bound	O
-,	O
-helix	O
-TM7ab	O
-bends	O
-in	O
-the	O
-same	O
-way	O
-as	O
-in	O
-the	O
-fully	O
-occupied	O
-Na	O
-+	O
-state	O
-,	O
-as	O
-the	O
-carbonyl	O
-of	O
-Ala206	O
-forms	O
-a	O
-hydrogen	O
--	O
-bonding	O
-interaction	O
-with	O
-Ser210	O
-.	O
-	O
-Structural	O
-mechanism	O
-of	O
-extracellular	O
-forward	O
-ion	O
-exchange	O
-in	O
-NCX	O
-.	O
-	O
-The	O
-carbonyl	O
-groups	O
-of	O
-Ala47	O
-(	O
-on	O
-TM2b	O
-)	O
-and	O
-Ala206	O
-(	O
-on	O
-TM7b	O
-),	O
-and	O
-the	O
-side	O
-chains	O
-of	O
-Glu54	O
-(	O
-on	O
-TM2c	O
-)	O
-and	O
-Glu213	O
-(	O
-on	O
-TM7c	O
-)	O
-are	O
-highlighted	O
-;	O
-these	O
-are	O
-four	O
-of	O
-the	O
-key	O
-residues	O
-for	O
-ion	O
-chelation	O
-and	O
-conformational	O
-changes	O
-.	O
-	O
-The	O
-green	O
-open	O
-cylinders	O
-represent	O
-the	O
-gating	O
-helices	O
-TM1	O
-and	O
-TM6	O
-.	O
-	O
-Asterisks	O
-mark	O
-the	O
-states	O
-whose	O
-crystal	O
-structures	O
-have	O
-been	O
-determined	O
-in	O
-this	O
-study	O
-.	O
-	O
-These	O
-states	O
-and	O
-their	O
-connectivity	O
-can	O
-also	O
-be	O
-deduced	O
-from	O
-the	O
-calculated	O
-free	O
--	O
-energy	O
-landscapes	O
-,	O
-which	O
-also	O
-reveal	O
-a	O
-Ca2	O
-+-	O
-loaded	O
-outward	O
--	O
-facing	O
-occluded	O
-state	O
-,	O
-and	O
-an	O
-unloaded	O
-,	O
-fully	O
-open	O
-state	O
-.	O
-	O
-An	O
-extended	O
-U2AF65	O
-–	O
-RNA	O
--	O
-binding	O
-domain	O
-recognizes	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-signal	O
-	O
-How	O
-the	O
-essential	O
-pre	O
--	O
-mRNA	O
-splicing	O
-factor	O
-U2AF65	O
-recognizes	O
-the	O
-polypyrimidine	O
-(	O
-Py	O
-)	O
-signals	O
-of	O
-the	O
-major	O
-class	O
-of	O
-3	O
-′	O
-splice	O
-sites	O
-in	O
-human	O
-gene	O
-transcripts	O
-remains	O
-incompletely	O
-understood	O
-.	O
-	O
-We	O
-determined	O
-four	O
-structures	O
-of	O
-an	O
-extended	O
-U2AF65	O
-–	O
-RNA	O
--	O
-binding	O
-domain	O
-bound	O
-to	O
-Py	O
--	O
-tract	O
-oligonucleotides	O
-at	O
-resolutions	O
-between	O
-2	O
-.	O
-0	O
-and	O
-1	O
-.	O
-5	O
-Å	O
-.	O
-These	O
-structures	O
-together	O
-with	O
-RNA	O
-binding	O
-and	O
-splicing	O
-assays	O
-reveal	O
-unforeseen	O
-roles	O
-for	O
-U2AF65	O
-inter	O
--	O
-domain	O
-residues	O
-in	O
-recognizing	O
-a	O
-contiguous	O
-,	O
-nine	O
--	O
-nucleotide	O
-Py	O
-tract	O
-.	O
-	O
-The	O
-U2AF65	O
-linker	O
-residues	O
-between	O
-the	O
-dual	O
-RNA	O
-recognition	O
-motifs	O
-(	O
-RRMs	O
-)	O
-recognize	O
-the	O
-central	O
-nucleotide	O
-,	O
-whereas	O
-the	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-RRM	O
-extensions	O
-recognize	O
-the	O
-3	O
-′	O
-terminus	O
-and	O
-third	O
-nucleotide	O
-.	O
-	O
-Single	O
--	O
-molecule	O
-FRET	O
-experiments	O
-suggest	O
-that	O
-conformational	O
-selection	O
-and	O
-induced	O
-fit	O
-of	O
-the	O
-U2AF65	O
-RRMs	O
-are	O
-complementary	O
-mechanisms	O
-for	O
-Py	O
--	O
-tract	O
-association	O
-.	O
-	O
-Altogether	O
-,	O
-these	O
-results	O
-advance	O
-the	O
-mechanistic	O
-understanding	O
-of	O
-molecular	O
-recognition	O
-for	O
-a	O
-major	O
-class	O
-of	O
-splice	O
-site	O
-signals	O
-.	O
-	O
-The	O
-pre	O
--	O
-mRNA	O
-splicing	O
-factor	O
-U2AF65	O
-recognizes	O
-3	O
-′	O
-splice	O
-sites	O
-in	O
-human	O
-gene	O
-transcripts	O
-,	O
-but	O
-the	O
-details	O
-are	O
-not	O
-fully	O
-understood	O
-.	O
-	O
-Here	O
-,	O
-the	O
-authors	O
-report	O
-U2AF65	O
-structures	O
-and	O
-single	O
-molecule	O
-FRET	O
-that	O
-reveal	O
-mechanistic	O
-insights	O
-into	O
-splice	O
-site	O
-recognition	O
-.	O
-	O
-The	O
-differential	O
-skipping	O
-or	O
-inclusion	O
-of	O
-alternatively	O
-spliced	O
-pre	O
--	O
-mRNA	O
-regions	O
-is	O
-a	O
-major	O
-source	O
-of	O
-diversity	O
-for	O
-nearly	O
-all	O
-human	O
-gene	O
-transcripts	O
-.	O
-	O
-The	O
-splice	O
-sites	O
-are	O
-marked	O
-by	O
-relatively	O
-short	O
-consensus	O
-sequences	O
-and	O
-are	O
-regulated	O
-by	O
-additional	O
-pre	O
--	O
-mRNA	O
-motifs	O
-(	O
-reviewed	O
-in	O
-ref	O
-.).	O
-	O
-At	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-of	O
-the	O
-major	O
-intron	O
-class	O
-,	O
-these	O
-include	O
-a	O
-polypyrimidine	O
-(	O
-Py	O
-)	O
-tract	O
-comprising	O
-primarily	O
-Us	O
-or	O
-Cs	O
-,	O
-which	O
-is	O
-preceded	O
-by	O
-a	O
-branch	O
-point	O
-sequence	O
-(	O
-BPS	O
-)	O
-that	O
-ultimately	O
-serves	O
-as	O
-the	O
-nucleophile	O
-in	O
-the	O
-splicing	O
-reaction	O
-and	O
-an	O
-AG	O
--	O
-dinucleotide	O
-at	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-junction	O
-.	O
-	O
-Disease	O
--	O
-causing	O
-mutations	O
-often	O
-compromise	O
-pre	O
--	O
-mRNA	O
-splicing	O
-(	O
-reviewed	O
-in	O
-refs	O
-),	O
-yet	O
-a	O
-priori	O
-predictions	O
-of	O
-splice	O
-sites	O
-and	O
-the	O
-consequences	O
-of	O
-their	O
-mutations	O
-are	O
-challenged	O
-by	O
-the	O
-brevity	O
-and	O
-degeneracy	O
-of	O
-known	O
-splice	O
-site	O
-sequences	O
-.	O
-	O
-High	O
--	O
-resolution	O
-structures	O
-of	O
-intact	O
-splicing	O
-factor	O
-–	O
-RNA	O
-complexes	O
-would	O
-offer	O
-key	O
-insights	O
-regarding	O
-the	O
-juxtaposition	O
-of	O
-the	O
-distinct	O
-splice	O
-site	O
-consensus	O
-sequences	O
-and	O
-their	O
-relationship	O
-to	O
-disease	O
--	O
-causing	O
-point	O
-mutations	O
-.	O
-	O
-The	O
-early	O
--	O
-stage	O
-pre	O
--	O
-mRNA	O
-splicing	O
-factor	O
-U2AF65	O
-is	O
-essential	O
-for	O
-viability	O
-in	O
-vertebrates	O
-and	O
-other	O
-model	O
-organisms	O
-(	O
-for	O
-example	O
-,	O
-ref	O
-.).	O
-	O
-A	O
-tightly	O
-controlled	O
-assembly	O
-among	O
-U2AF65	O
-,	O
-the	O
-pre	O
--	O
-mRNA	O
-,	O
-and	O
-partner	O
-proteins	O
-sequentially	O
-identifies	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-and	O
-promotes	O
-association	O
-of	O
-the	O
-spliceosome	O
-,	O
-which	O
-ultimately	O
-accomplishes	O
-the	O
-task	O
-of	O
-splicing	O
-.	O
-	O
-Initially	O
-U2AF65	O
-recognizes	O
-the	O
-Py	O
--	O
-tract	O
-splice	O
-site	O
-signal	O
-.	O
-	O
-In	O
-turn	O
-,	O
-the	O
-ternary	O
-complex	O
-of	O
-U2AF65	O
-with	O
-SF1	O
-and	O
-U2AF35	O
-identifies	O
-the	O
-surrounding	O
-BPS	O
-and	O
-3	O
-′	O
-splice	O
-site	O
-junctions	O
-.	O
-	O
-Subsequently	O
-U2AF65	O
-recruits	O
-the	O
-U2	O
-small	O
-nuclear	O
-ribonucleoprotein	O
-particle	O
-(	O
-snRNP	O
-)	O
-and	O
-ultimately	O
-dissociates	O
-from	O
-the	O
-active	O
-spliceosome	O
-.	O
-	O
-Biochemical	O
-characterizations	O
-of	O
-U2AF65	O
-demonstrated	O
-that	O
-tandem	O
-RNA	O
-recognition	O
-motifs	O
-(	O
-RRM1	O
-and	O
-RRM2	O
-)	O
-recognize	O
-the	O
-Py	O
-tract	O
-(	O
-Fig	O
-.	O
-1a	O
-).	O
-	O
-Milestone	O
-crystal	O
-structures	O
-of	O
-the	O
-core	O
-U2AF65	O
-RRM1	O
-and	O
-RRM2	O
-connected	O
-by	O
-a	O
-shortened	O
-inter	O
--	O
-RRM	O
-linker	O
-(	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-)	O
-detailed	O
-a	O
-subset	O
-of	O
-nucleotide	O
-interactions	O
-with	O
-the	O
-individual	O
-U2AF65	O
-RRMs	O
-.	O
-	O
-A	O
-subsequent	O
-NMR	O
-structure	O
-characterized	O
-the	O
-side	O
--	O
-by	O
--	O
-side	O
-arrangement	O
-of	O
-the	O
-minimal	O
-U2AF65	O
-RRM1	O
-and	O
-RRM2	O
-connected	O
-by	O
-a	O
-linker	O
-of	O
-natural	O
-length	O
-(	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-),	O
-yet	O
-depended	O
-on	O
-the	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-crystal	O
-structures	O
-for	O
-RNA	O
-interactions	O
-and	O
-an	O
-ab	O
-initio	O
-model	O
-for	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-conformation	O
-.	O
-	O
-As	O
-such	O
-,	O
-the	O
-molecular	O
-mechanisms	O
-for	O
-Py	O
--	O
-tract	O
-recognition	O
-by	O
-the	O
-intact	O
-U2AF65	O
-–	O
-RNA	O
--	O
-binding	O
-domain	O
-remained	O
-unknown	O
-.	O
-	O
-Here	O
-,	O
-we	O
-use	O
-X	O
--	O
-ray	O
-crystallography	O
-and	O
-biochemical	O
-studies	O
-to	O
-reveal	O
-new	O
-roles	O
-in	O
-Py	O
--	O
-tract	O
-recognition	O
-for	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-and	O
-key	O
-residues	O
-surrounding	O
-the	O
-core	O
-U2AF65	O
-RRMs	O
-.	O
-	O
-We	O
-use	O
-single	O
--	O
-molecule	O
-Förster	O
-resonance	O
-energy	O
-transfer	O
-(	O
-smFRET	O
-)	O
-to	O
-characterize	O
-the	O
-conformational	O
-dynamics	O
-of	O
-this	O
-extended	O
-U2AF65	O
-–	O
-RNA	O
--	O
-binding	O
-domain	O
-during	O
-Py	O
--	O
-tract	O
-recognition	O
-.	O
-	O
-Cognate	O
-U2AF65	O
-–	O
-Py	O
--	O
-tract	O
-recognition	O
-requires	O
-RRM	O
-extensions	O
-	O
-The	O
-RNA	O
-affinity	O
-of	O
-the	O
-minimal	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-domain	O
-comprising	O
-the	O
-core	O
-RRM1	O
-–	O
-RRM2	O
-folds	O
-(	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-,	O
-residues	O
-148	O
-–	O
-336	O
-)	O
-is	O
-relatively	O
-weak	O
-compared	O
-with	O
-full	O
--	O
-length	O
-U2AF65	O
-(	O
-Fig	O
-.	O
-1a	O
-,	O
-b	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-Historically	O
-,	O
-this	O
-difference	O
-was	O
-attributed	O
-to	O
-the	O
-U2AF65	O
-arginine	O
-–	O
-serine	O
-rich	O
-domain	O
-,	O
-which	O
-contacts	O
-pre	O
--	O
-mRNA	O
-–	O
-U2	O
-snRNA	O
-duplexes	O
-outside	O
-of	O
-the	O
-Py	O
-tract	O
-.	O
-	O
-We	O
-noticed	O
-that	O
-the	O
-RNA	O
--	O
-binding	O
-affinity	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-domain	O
-was	O
-greatly	O
-enhanced	O
-by	O
-the	O
-addition	O
-of	O
-seven	O
-and	O
-six	O
-residues	O
-at	O
-the	O
-respective	O
-N	O
-and	O
-C	O
-termini	O
-of	O
-the	O
-minimal	O
-RRM1	O
-and	O
-RRM2	O
-(	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-residues	O
-141	O
-–	O
-342	O
-;	O
-Fig	O
-.	O
-1a	O
-).	O
-	O
-In	O
-a	O
-fluorescence	O
-anisotropy	O
-assay	O
-for	O
-binding	O
-a	O
-representative	O
-Py	O
-tract	O
-derived	O
-from	O
-the	O
-well	O
--	O
-characterized	O
-splice	O
-site	O
-of	O
-the	O
-adenovirus	O
-major	O
-late	O
-promoter	O
-(	O
-AdML	O
-),	O
-the	O
-RNA	O
-affinity	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-increased	O
-by	O
-100	O
--	O
-fold	O
-relative	O
-to	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-to	O
-comparable	O
-levels	O
-as	O
-full	O
--	O
-length	O
-U2AF65	O
-(	O
-Fig	O
-.	O
-1b	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-1a	O
-–	O
-d	O
-).	O
-	O
-Likewise	O
-,	O
-both	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-and	O
-full	O
--	O
-length	O
-U2AF65	O
-showed	O
-similar	O
-sequence	O
-specificity	O
-for	O
-U	O
--	O
-rich	O
-stretches	O
-in	O
-the	O
-5	O
-′-	O
-region	O
-of	O
-the	O
-Py	O
-tract	O
-and	O
-promiscuity	O
-for	O
-C	O
--	O
-rich	O
-regions	O
-in	O
-the	O
-3	O
-′-	O
-region	O
-(	O
-Fig	O
-.	O
-1c	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-1e	O
-–	O
-h	O
-).	O
-	O
-U2AF65	O
--	O
-bound	O
-Py	O
-tract	O
-comprises	O
-nine	O
-contiguous	O
-nucleotides	O
-	O
-To	O
-investigate	O
-the	O
-structural	O
-basis	O
-for	O
-cognate	O
-U2AF65	O
-recognition	O
-of	O
-a	O
-contiguous	O
-Py	O
-tract	O
-,	O
-we	O
-determined	O
-four	O
-crystal	O
-structures	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-bound	O
-to	O
-Py	O
--	O
-tract	O
-oligonucleotides	O
-(	O
-Fig	O
-.	O
-2a	O
-;	O
-Table	O
-1	O
-).	O
-	O
-By	O
-sequential	O
-boot	O
-strapping	O
-(	O
-Methods	O
-),	O
-we	O
-optimized	O
-the	O
-oligonucleotide	O
-length	O
-,	O
-the	O
-position	O
-of	O
-a	O
-Br	O
--	O
-dU	O
-,	O
-and	O
-the	O
-identity	O
-of	O
-the	O
-terminal	O
-nucleotide	O
-(	O
-rU	O
-,	O
-dU	O
-and	O
-rC	O
-)	O
-to	O
-achieve	O
-full	O
-views	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-bound	O
-to	O
-contiguous	O
-Py	O
-tracts	O
-at	O
-up	O
-to	O
-1	O
-.	O
-5	O
-Å	O
-resolution	O
-.	O
-	O
-The	O
-protein	O
-and	O
-oligonucleotide	O
-conformations	O
-are	O
-nearly	O
-identical	O
-among	O
-the	O
-four	O
-new	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-RRM1	O
-and	O
-RRM2	O
-associate	O
-with	O
-the	O
-Py	O
-tract	O
-in	O
-a	O
-parallel	O
-,	O
-side	O
--	O
-by	O
--	O
-side	O
-arrangement	O
-(	O
-shown	O
-for	O
-representative	O
-structure	O
-iv	O
-in	O
-Fig	O
-.	O
-2b	O
-,	O
-c	O
-;	O
-Supplementary	O
-Movie	O
-1	O
-).	O
-	O
-An	O
-extended	O
-conformation	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-traverses	O
-across	O
-the	O
-α	O
--	O
-helical	O
-surface	O
-of	O
-RRM1	O
-and	O
-the	O
-central	O
-β	O
--	O
-strands	O
-of	O
-RRM2	O
-and	O
-is	O
-well	O
-defined	O
-in	O
-the	O
-electron	O
-density	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-The	O
-extensions	O
-at	O
-the	O
-N	O
-terminus	O
-of	O
-RRM1	O
-and	O
-C	O
-terminus	O
-of	O
-RRM2	O
-adopt	O
-well	O
--	O
-ordered	O
-α	O
--	O
-helices	O
-.	O
-	O
-Both	O
-RRM1	O
-/	O
-RRM2	O
-extensions	O
-and	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-directly	O
-recognize	O
-the	O
-bound	O
-oligonucleotide	O
-.	O
-	O
-We	O
-compare	O
-the	O
-global	O
-conformation	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-with	O
-the	O
-prior	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-crystal	O
-structure	O
-and	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-NMR	O
-structure	O
-in	O
-the	O
-Supplementary	O
-Discussion	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-.	O
-	O
-The	O
-discovery	O
-of	O
-nine	O
-U2AF65	O
--	O
-binding	O
-sites	O
-for	O
-contiguous	O
-Py	O
--	O
-tract	O
-nucleotides	O
-was	O
-unexpected	O
-.	O
-	O
-Based	O
-on	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structures	O
-,	O
-we	O
-originally	O
-hypothesized	O
-that	O
-the	O
-U2AF65	O
-RRMs	O
-would	O
-bind	O
-the	O
-minimal	O
-seven	O
-nucleotides	O
-observed	O
-in	O
-these	O
-structures	O
-.	O
-	O
-Surprisingly	O
-,	O
-the	O
-RRM2	O
-extension	O
-/	O
-inter	O
--	O
-RRM	O
-linker	O
-contribute	O
-new	O
-central	O
-nucleotide	O
--	O
-binding	O
-sites	O
-near	O
-the	O
-RRM1	O
-/	O
-RRM2	O
-junction	O
-and	O
-the	O
-RRM1	O
-extension	O
-recognizes	O
-the	O
-3	O
-′-	O
-terminal	O
-nucleotide	O
-(	O
-Fig	O
-.	O
-2c	O
-;	O
-Supplementary	O
-Movie	O
-1	O
-).	O
-	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-characterize	O
-ribose	O
-(	O
-r	O
-)	O
-nucleotides	O
-at	O
-all	O
-of	O
-the	O
-binding	O
-sites	O
-except	O
-the	O
-seventh	O
-and	O
-eighth	O
-deoxy	O
--(	O
-d	O
-)	O
-U	O
-,	O
-which	O
-are	O
-likely	O
-to	O
-lack	O
-2	O
-′-	O
-hydroxyl	O
-contacts	O
-based	O
-on	O
-the	O
-RNA	O
--	O
-bound	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structure	O
-.	O
-	O
-Qualitatively	O
-,	O
-a	O
-subset	O
-of	O
-the	O
-U2AF651	O
-,	O
-2L	O
--	O
-nucleotide	O
--	O
-binding	O
-sites	O
-(	O
-sites	O
-1	O
-–	O
-3	O
-and	O
-7	O
-–	O
-9	O
-)	O
-share	O
-similar	O
-locations	O
-to	O
-those	O
-of	O
-the	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structures	O
-(	O
-Supplementary	O
-Figs	O
-2c	O
-,	O
-d	O
-and	O
-3	O
-).	O
-	O
-Yet	O
-,	O
-only	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-interactions	O
-at	O
-sites	O
-1	O
-and	O
-7	O
-are	O
-nearly	O
-identical	O
-to	O
-those	O
-of	O
-the	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structures	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-,	O
-f	O
-).	O
-	O
-In	O
-striking	O
-departures	O
-from	O
-prior	O
-partial	O
-views	O
-,	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-reveal	O
-three	O
-unanticipated	O
-nucleotide	O
--	O
-binding	O
-sites	O
-at	O
-the	O
-centre	O
-of	O
-the	O
-Py	O
-tract	O
-,	O
-as	O
-well	O
-as	O
-numerous	O
-new	O
-interactions	O
-that	O
-underlie	O
-cognate	O
-recognition	O
-of	O
-the	O
-Py	O
-tract	O
-(	O
-Fig	O
-.	O
-3a	O
-–	O
-h	O
-).	O
-	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-interacts	O
-with	O
-the	O
-Py	O
-tract	O
-	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-RRM2	O
-,	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-and	O
-RRM1	O
-concomitantly	O
-recognize	O
-the	O
-three	O
-central	O
-nucleotides	O
-of	O
-the	O
-Py	O
-tract	O
-,	O
-which	O
-are	O
-likely	O
-to	O
-coordinate	O
-the	O
-conformational	O
-arrangement	O
-of	O
-these	O
-disparate	O
-portions	O
-of	O
-the	O
-protein	O
-.	O
-	O
-Residues	O
-in	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-comprise	O
-a	O
-centrally	O
-located	O
-binding	O
-site	O
-for	O
-the	O
-fifth	O
-nucleotide	O
-on	O
-the	O
-RRM2	O
-surface	O
-and	O
-abutting	O
-the	O
-RRM1	O
-/	O
-RRM2	O
-interface	O
-(	O
-Fig	O
-.	O
-3d	O
-).	O
-	O
-The	O
-backbone	O
-amide	O
-of	O
-the	O
-linker	O
-V254	O
-and	O
-the	O
-carbonyl	O
-of	O
-T252	O
-engage	O
-in	O
-hydrogen	O
-bonds	O
-with	O
-the	O
-rU5	O
--	O
-O4	O
-and	O
--	O
-N3H	O
-atoms	O
-.	O
-	O
-In	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-strand	O
-of	O
-RRM1	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-K225	O
-and	O
-R227	O
-donate	O
-additional	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-rU5	O
--	O
-O2	O
-lone	O
-pair	O
-electrons	O
-.	O
-	O
-The	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-also	O
-participates	O
-in	O
-the	O
-preceding	O
-rU4	O
--	O
-binding	O
-site	O
-,	O
-where	O
-the	O
-V254	O
-backbone	O
-carbonyl	O
-and	O
-D256	O
-carboxylate	O
-position	O
-the	O
-K260	O
-side	O
-chain	O
-to	O
-hydrogen	O
-bond	O
-with	O
-the	O
-rU4	O
--	O
-O4	O
-(	O
-Fig	O
-.	O
-3c	O
-).	O
-	O
-Otherwise	O
-,	O
-the	O
-rU4	O
-nucleotide	O
-packs	O
-against	O
-F304	O
-in	O
-the	O
-signature	O
-ribonucleoprotein	O
-consensus	O
-motif	O
-(	O
-RNP	O
-)-	O
-2	O
-of	O
-RRM2	O
-.	O
-	O
-At	O
-the	O
-opposite	O
-side	O
-of	O
-the	O
-central	O
-fifth	O
-nucleotide	O
-,	O
-the	O
-sixth	O
-rU6	O
-nucleotide	O
-is	O
-located	O
-at	O
-the	O
-inter	O
--	O
-RRM1	O
-/	O
-RRM2	O
-interface	O
-(	O
-Fig	O
-.	O
-3e	O
-;	O
-Supplementary	O
-Movie	O
-1	O
-).	O
-	O
-This	O
-nucleotide	O
-twists	O
-to	O
-face	O
-away	O
-from	O
-the	O
-U2AF65	O
-linker	O
-and	O
-instead	O
-inserts	O
-the	O
-rU6	O
--	O
-uracil	O
-into	O
-a	O
-sandwich	O
-between	O
-the	O
-β2	O
-/	O
-β3	O
-loops	O
-of	O
-RRM1	O
-and	O
-RRM2	O
-.	O
-	O
-The	O
-rU6	O
-base	O
-edge	O
-is	O
-relatively	O
-solvent	O
-exposed	O
-;	O
-accordingly	O
-,	O
-the	O
-rU6	O
-hydrogen	O
-bonds	O
-with	O
-U2AF65	O
-are	O
-water	O
-mediated	O
-apart	O
-from	O
-a	O
-single	O
-direct	O
-interaction	O
-by	O
-the	O
-RRM1	O
--	O
-N196	O
-side	O
-chain	O
-.	O
-	O
-We	O
-tested	O
-the	O
-contribution	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-interactions	O
-with	O
-the	O
-new	O
-central	O
-nucleotide	O
-to	O
-Py	O
--	O
-tract	O
-affinity	O
-(	O
-Fig	O
-.	O
-3i	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-4a	O
-,	O
-b	O
-).	O
-	O
-Mutagenesis	O
-of	O
-either	O
-V254	O
-in	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-to	O
-proline	O
-or	O
-RRM1	O
-–	O
-R227	O
-to	O
-alanine	O
-,	O
-which	O
-remove	O
-the	O
-hydrogen	O
-bond	O
-with	O
-the	O
-fifth	O
-uracil	O
--	O
-O4	O
-or	O
--	O
-O2	O
-,	O
-reduced	O
-the	O
-affinities	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-for	O
-the	O
-representative	O
-AdML	O
-Py	O
-tract	O
-by	O
-four	O
--	O
-or	O
-five	O
--	O
-fold	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-energetic	O
-penalties	O
-due	O
-to	O
-these	O
-mutations	O
-(	O
-ΔΔG	O
-0	O
-.	O
-8	O
-–	O
-0	O
-.	O
-9	O
-kcal	O
-mol	O
-−	O
-1	O
-)	O
-are	O
-consistent	O
-with	O
-the	O
-loss	O
-of	O
-each	O
-hydrogen	O
-bond	O
-with	O
-the	O
-rU5	O
-base	O
-and	O
-support	O
-the	O
-relevance	O
-of	O
-the	O
-central	O
-nucleotide	O
-interactions	O
-observed	O
-in	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-.	O
-	O
-U2AF65	O
-RRM	O
-extensions	O
-interact	O
-with	O
-the	O
-Py	O
-tract	O
-	O
-The	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-extensions	O
-of	O
-the	O
-U2AF65	O
-RRM1	O
-and	O
-RRM2	O
-directly	O
-contact	O
-the	O
-bound	O
-Py	O
-tract	O
-.	O
-	O
-Rather	O
-than	O
-interacting	O
-with	O
-a	O
-new	O
-5	O
-′-	O
-terminal	O
-nucleotide	O
-as	O
-we	O
-had	O
-hypothesized	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-α	O
--	O
-helix	O
-of	O
-RRM2	O
-instead	O
-folds	O
-across	O
-one	O
-surface	O
-of	O
-rU3	O
-in	O
-the	O
-third	O
-binding	O
-site	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-There	O
-,	O
-a	O
-salt	O
-bridge	O
-between	O
-the	O
-K340	O
-side	O
-chain	O
-and	O
-nucleotide	O
-phosphate	O
-,	O
-as	O
-well	O
-as	O
-G338	O
--	O
-base	O
-stacking	O
-and	O
-a	O
-hydrogen	O
-bond	O
-between	O
-the	O
-backbone	O
-amide	O
-of	O
-G338	O
-and	O
-the	O
-rU3	O
--	O
-O4	O
-,	O
-secure	O
-the	O
-RRM2	O
-extension	O
-.	O
-	O
-Indirectly	O
-,	O
-the	O
-additional	O
-contacts	O
-with	O
-the	O
-third	O
-nucleotide	O
-shift	O
-the	O
-rU2	O
-nucleotide	O
-in	O
-the	O
-second	O
-binding	O
-site	O
-closer	O
-to	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-strand	O
-of	O
-RRM2	O
-.	O
-	O
-Consequently	O
-,	O
-the	O
-U2AF651	O
-,	O
-2L	O
--	O
-bound	O
-rU2	O
--	O
-O4	O
-and	O
--	O
-N3H	O
-form	O
-dual	O
-hydrogen	O
-bonds	O
-with	O
-the	O
-K329	O
-backbone	O
-atoms	O
-(	O
-Fig	O
-.	O
-3a	O
-),	O
-rather	O
-than	O
-a	O
-single	O
-hydrogen	O
-bond	O
-with	O
-the	O
-K329	O
-side	O
-chain	O
-as	O
-in	O
-the	O
-prior	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structure	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-At	O
-the	O
-N	O
-terminus	O
-,	O
-the	O
-α	O
--	O
-helical	O
-extension	O
-of	O
-U2AF65	O
-RRM1	O
-positions	O
-the	O
-Q147	O
-side	O
-chain	O
-to	O
-bridge	O
-the	O
-eighth	O
-and	O
-ninth	O
-nucleotides	O
-at	O
-the	O
-3	O
-′	O
-terminus	O
-of	O
-the	O
-Py	O
-tract	O
-(	O
-Fig	O
-.	O
-3f	O
-–	O
-h	O
-).	O
-	O
-The	O
-Q147	O
-residue	O
-participates	O
-in	O
-hydrogen	O
-bonds	O
-with	O
-the	O
--	O
-N3H	O
-of	O
-the	O
-eighth	O
-uracil	O
-and	O
--	O
-O2	O
-of	O
-the	O
-ninth	O
-pyrimidine	O
-.	O
-	O
-The	O
-adjacent	O
-R146	O
-guanidinium	O
-group	O
-donates	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-3	O
-′-	O
-terminal	O
-ribose	O
--	O
-O2	O
-′	O
-and	O
-O3	O
-′	O
-atoms	O
-,	O
-where	O
-it	O
-could	O
-form	O
-a	O
-salt	O
-bridge	O
-with	O
-a	O
-phospho	O
--	O
-diester	O
-group	O
-in	O
-the	O
-context	O
-of	O
-a	O
-longer	O
-pre	O
--	O
-mRNA	O
-.	O
-	O
-Consistent	O
-with	O
-loss	O
-of	O
-a	O
-hydrogen	O
-bond	O
-with	O
-the	O
-ninth	O
-pyrimidine	O
--	O
-O2	O
-(	O
-ΔΔG	O
-1	O
-.	O
-0	O
-kcal	O
-mol	O
-−	O
-1	O
-),	O
-mutation	O
-of	O
-the	O
-Q147	O
-to	O
-an	O
-alanine	O
-reduced	O
-U2AF651	O
-,	O
-2L	O
-affinity	O
-for	O
-the	O
-AdML	O
-Py	O
-tract	O
-by	O
-five	O
--	O
-fold	O
-(	O
-Fig	O
-.	O
-3i	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-4c	O
-).	O
-	O
-We	O
-compare	O
-U2AF65	O
-interactions	O
-with	O
-uracil	O
-relative	O
-to	O
-cytosine	O
-pyrimidines	O
-at	O
-the	O
-ninth	O
-binding	O
-site	O
-in	O
-Fig	O
-.	O
-3g	O
-,	O
-h	O
-and	O
-the	O
-Supplementary	O
-Discussion	O
-.	O
-	O
-Versatile	O
-primary	O
-sequence	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-reveal	O
-that	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-mediates	O
-an	O
-extensive	O
-interface	O
-with	O
-the	O
-second	O
-α	O
--	O
-helix	O
-of	O
-RRM1	O
-,	O
-the	O
-β2	O
-/	O
-β3	O
-strands	O
-of	O
-RRM2	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-α	O
--	O
-helical	O
-extension	O
-of	O
-RRM1	O
-.	O
-	O
-Altogether	O
-,	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-residues	O
-(	O
-R228	O
-–	O
-K260	O
-)	O
-bury	O
-2	O
-,	O
-800	O
-Å2	O
-of	O
-surface	O
-area	O
-in	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-holo	O
--	O
-protein	O
-,	O
-suggestive	O
-of	O
-a	O
-cognate	O
-interface	O
-compared	O
-with	O
-1	O
-,	O
-900	O
-Å2	O
-for	O
-a	O
-typical	O
-protein	O
-–	O
-protein	O
-complex	O
-.	O
-	O
-The	O
-path	O
-of	O
-the	O
-linker	O
-initiates	O
-at	O
-P229	O
-following	O
-the	O
-core	O
-RRM1	O
-β	O
--	O
-strand	O
-,	O
-in	O
-a	O
-kink	O
-that	O
-is	O
-positioned	O
-by	O
-intra	O
--	O
-molecular	O
-stacking	O
-among	O
-the	O
-consecutive	O
-R228	O
-,	O
-Y232	O
-and	O
-P234	O
-side	O
-chains	O
-(	O
-Fig	O
-.	O
-4a	O
-,	O
-lower	O
-right	O
-).	O
-	O
-A	O
-second	O
-kink	O
-at	O
-P236	O
-,	O
-coupled	O
-with	O
-respective	O
-packing	O
-of	O
-the	O
-L235	O
-and	O
-M238	O
-side	O
-chains	O
-on	O
-the	O
-N	O
--	O
-terminal	O
-α	O
--	O
-helical	O
-RRM1	O
-extension	O
-and	O
-the	O
-core	O
-RRM1	O
-α2	O
--	O
-helix	O
-,	O
-reverses	O
-the	O
-direction	O
-of	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-towards	O
-the	O
-RRM1	O
-/	O
-RRM2	O
-interface	O
-and	O
-away	O
-from	O
-the	O
-RNA	O
--	O
-binding	O
-site	O
-.	O
-	O
-In	O
-the	O
-neighbouring	O
-apical	O
-region	O
-of	O
-the	O
-linker	O
-,	O
-the	O
-V244	O
-and	O
-V246	O
-side	O
-chains	O
-pack	O
-in	O
-a	O
-hydrophobic	O
-pocket	O
-between	O
-two	O
-α	O
--	O
-helices	O
-of	O
-the	O
-core	O
-RRM1	O
-.	O
-	O
-The	O
-adjacent	O
-V249	O
-and	O
-V250	O
-are	O
-notable	O
-for	O
-their	O
-respective	O
-interactions	O
-that	O
-connect	O
-RRM1	O
-and	O
-RRM2	O
-at	O
-this	O
-distal	O
-interface	O
-from	O
-the	O
-RNA	O
--	O
-binding	O
-site	O
-(	O
-Fig	O
-.	O
-4a	O
-,	O
-top	O
-).	O
-	O
-A	O
-third	O
-kink	O
-stacks	O
-P247	O
-and	O
-G248	O
-with	O
-Y245	O
-and	O
-re	O
--	O
-orients	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-linker	O
-towards	O
-the	O
-RRM2	O
-and	O
-bound	O
-RNA	O
-.	O
-	O
-At	O
-the	O
-RNA	O
-surface	O
-,	O
-the	O
-key	O
-V254	O
-that	O
-recognizes	O
-the	O
-fifth	O
-uracil	O
-is	O
-secured	O
-via	O
-hydrophobic	O
-contacts	O
-between	O
-its	O
-side	O
-chain	O
-and	O
-the	O
-β	O
--	O
-sheet	O
-surface	O
-of	O
-RRM2	O
-,	O
-chiefly	O
-the	O
-consensus	O
-RNP1	O
--	O
-F304	O
-residue	O
-that	O
-stacks	O
-with	O
-the	O
-fourth	O
-uracil	O
-(	O
-Fig	O
-.	O
-4a	O
-,	O
-lower	O
-left	O
-).	O
-	O
-Few	O
-direct	O
-contacts	O
-are	O
-made	O
-between	O
-the	O
-remaining	O
-residues	O
-of	O
-the	O
-linker	O
-and	O
-the	O
-U2AF65	O
-RRM2	O
-;	O
-instead	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-conformation	O
-of	O
-the	O
-linker	O
-appears	O
-primarily	O
-RNA	O
-mediated	O
-(	O
-Fig	O
-.	O
-3c	O
-,	O
-d	O
-).	O
-	O
-We	O
-investigated	O
-whether	O
-the	O
-observed	O
-contacts	O
-between	O
-the	O
-RRMs	O
-and	O
-linker	O
-were	O
-critical	O
-for	O
-RNA	O
-binding	O
-by	O
-structure	O
--	O
-guided	O
-mutagenesis	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-We	O
-titrated	O
-these	O
-mutant	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-proteins	O
-into	O
-fluorescein	O
--	O
-labelled	O
-AdML	O
-Py	O
--	O
-tract	O
-RNA	O
-and	O
-fit	O
-the	O
-fluorescence	O
-anisotropy	O
-changes	O
-to	O
-obtain	O
-the	O
-apparent	O
-equilibrium	O
-affinities	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4d	O
-–	O
-h	O
-).	O
-	O
-We	O
-introduced	O
-glycine	O
-substitutions	O
-to	O
-maximally	O
-reduce	O
-the	O
-buried	O
-surface	O
-area	O
-without	O
-directly	O
-interfering	O
-with	O
-its	O
-hydrogen	O
-bonds	O
-between	O
-backbone	O
-atoms	O
-and	O
-the	O
-base	O
-.	O
-	O
-First	O
-,	O
-we	O
-replaced	O
-V249	O
-and	O
-V250	O
-at	O
-the	O
-RRM1	O
-/	O
-RRM2	O
-interface	O
-and	O
-V254	O
-at	O
-the	O
-bound	O
-RNA	O
-site	O
-with	O
-glycine	O
-(	O
-3Gly	B-mutant
-).	O
-	O
-However	O
-,	O
-the	O
-resulting	O
-decrease	O
-in	O
-the	O
-AdML	O
-RNA	O
-affinity	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
--	I-mutant
-3Gly	I-mutant
-mutant	O
-relative	O
-to	O
-wild	O
--	O
-type	O
-protein	O
-was	O
-not	O
-significant	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-In	O
-parallel	O
-,	O
-we	O
-replaced	O
-five	O
-linker	O
-residues	O
-(	O
-S251	O
-,	O
-T252	O
-,	O
-V253	O
-,	O
-V254	O
-and	O
-P255	O
-)	O
-at	O
-the	O
-fifth	O
-nucleotide	O
--	O
-binding	O
-site	O
-with	O
-glycines	O
-(	O
-5Gly	B-mutant
-)	O
-and	O
-also	O
-found	O
-that	O
-the	O
-RNA	O
-affinity	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
--	I-mutant
-5Gly	I-mutant
-mutant	O
-likewise	O
-decreased	O
-only	O
-slightly	O
-relative	O
-to	O
-wild	O
--	O
-type	O
-protein	O
-.	O
-	O
-A	O
-more	O
-conservative	O
-substitution	O
-of	O
-these	O
-five	O
-residues	O
-(	O
-251	O
-–	O
-255	O
-)	O
-with	O
-an	O
-unrelated	O
-sequence	O
-capable	O
-of	O
-backbone	O
--	O
-mediated	O
-hydrogen	O
-bonds	O
-(	O
-STVVP	B-mutant
->	I-mutant
-NLALA	I-mutant
-)	O
-confirmed	O
-the	O
-subtle	O
-impact	O
-of	O
-this	O
-versatile	O
-inter	O
--	O
-RRM	O
-sequence	O
-on	O
-affinity	O
-for	O
-the	O
-AdML	O
-Py	O
-tract	O
-.	O
-	O
-Finally	O
-,	O
-to	O
-ensure	O
-that	O
-these	O
-selective	O
-mutations	O
-were	O
-sufficient	O
-to	O
-disrupt	O
-the	O
-linker	O
-/	O
-RRM	O
-contacts	O
-,	O
-we	O
-substituted	O
-glycine	O
-for	O
-the	O
-majority	O
-of	O
-buried	O
-hydrophobic	O
-residues	O
-in	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-(	O
-including	O
-M144	O
-,	O
-L235	O
-,	O
-M238	O
-,	O
-V244	O
-,	O
-V246	O
-,	O
-V249	O
-,	O
-V250	O
-,	O
-S251	O
-,	O
-T252	O
-,	O
-V253	O
-,	O
-V254	O
-,	O
-P255	O
-;	O
-called	O
-12Gly	B-mutant
-).	O
-	O
-Despite	O
-12	O
-concurrent	O
-mutations	O
-,	O
-the	O
-AdML	O
-RNA	O
-affinity	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
--	I-mutant
-12Gly	I-mutant
-variant	O
-was	O
-reduced	O
-by	O
-only	O
-three	O
--	O
-fold	O
-relative	O
-to	O
-the	O
-unmodified	O
-protein	O
-(	O
-Fig	O
-.	O
-4b	O
-),	O
-which	O
-is	O
-less	O
-than	O
-the	O
-penalty	O
-of	O
-the	O
-V254P	B-mutant
-mutation	O
-that	O
-disrupts	O
-the	O
-rU5	O
-hydrogen	O
-bond	O
-(	O
-Fig	O
-.	O
-3d	O
-,	O
-i	O
-).	O
-	O
-To	O
-test	O
-the	O
-interplay	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-with	O
-its	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-RRM	O
-extensions	O
-,	O
-we	O
-constructed	O
-an	O
-internal	O
-linker	O
-deletion	O
-of	O
-20	O
--	O
-residues	O
-within	O
-the	O
-extended	O
-RNA	O
--	O
-binding	O
-domain	O
-(	O
-dU2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-).	O
-	O
-We	O
-found	O
-that	O
-the	O
-affinity	O
-of	O
-dU2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-for	O
-the	O
-AdML	O
-RNA	O
-was	O
-significantly	O
-reduced	O
-relative	O
-to	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-(	O
-four	O
--	O
-fold	O
-,	O
-Figs	O
-1b	O
-and	O
-4b	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-4i	O
-).	O
-	O
-Yet	O
-,	O
-it	O
-is	O
-well	O
-known	O
-that	O
-the	O
-linker	O
-deletion	O
-in	O
-the	O
-context	O
-of	O
-the	O
-minimal	O
-RRM1	O
-–	O
-RRM2	O
-boundaries	O
-has	O
-no	O
-detectable	O
-effect	O
-on	O
-the	O
-RNA	O
-affinities	O
-of	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-compared	O
-with	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-(	O
-refs	O
-;	O
-Figs	O
-1b	O
-and	O
-4b	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-4j	O
-).	O
-	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-suggest	O
-that	O
-an	O
-extended	O
-conformation	O
-of	O
-the	O
-truncated	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-inter	O
--	O
-RRM	O
-linker	O
-would	O
-suffice	O
-to	O
-connect	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-RRM1	O
-C	O
-terminus	O
-to	O
-the	O
-N	O
-terminus	O
-of	O
-RRM2	O
-(	O
-24	O
-Å	O
-distance	O
-between	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-R227	O
--	O
-Cα	O
-–	O
-H259	O
--	O
-Cα	O
-atoms	O
-),	O
-which	O
-agrees	O
-with	O
-the	O
-greater	O
-RNA	O
-affinities	O
-of	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-and	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-dual	O
-RRMs	O
-compared	O
-with	O
-the	O
-individual	O
-U2AF65	O
-RRMs	O
-.	O
-	O
-However	O
-,	O
-stretching	O
-of	O
-the	O
-truncated	O
-dU2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-linker	O
-to	O
-connect	O
-the	O
-RRM	O
-termini	O
-is	O
-expected	O
-to	O
-disrupt	O
-its	O
-nucleotide	O
-interactions	O
-.	O
-	O
-Likewise	O
-,	O
-deletion	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-RRM1	O
-extension	O
-in	O
-the	O
-shortened	O
-constructs	O
-would	O
-remove	O
-packing	O
-interactions	O
-that	O
-position	O
-the	O
-linker	O
-in	O
-a	O
-kinked	O
-turn	O
-following	O
-P229	O
-(	O
-Fig	O
-.	O
-4a	O
-),	O
-consistent	O
-with	O
-the	O
-lower	O
-RNA	O
-affinities	O
-of	O
-dU2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-and	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-compared	O
-with	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-.	O
-	O
-To	O
-further	O
-test	O
-cooperation	O
-among	O
-the	O
-U2AF65	O
-RRM	O
-extensions	O
-and	O
-inter	O
--	O
-RRM	O
-linker	O
-for	O
-RNA	O
-recognition	O
-,	O
-we	O
-tested	O
-the	O
-impact	O
-of	O
-a	O
-triple	O
-Q147A	B-mutant
-/	O
-V254P	B-mutant
-/	O
-R227A	B-mutant
-mutation	O
-(	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
--	I-mutant
-3Mut	I-mutant
-)	O
-for	O
-RNA	O
-binding	O
-(	O
-Fig	O
-.	O
-4b	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-4d	O
-).	O
-	O
-Notably	O
-,	O
-the	O
-Q147A	B-mutant
-/	O
-V254P	B-mutant
-/	O
-R227A	B-mutant
-mutation	O
-reduced	O
-the	O
-RNA	O
-affinity	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
--	I-mutant
-3Mut	I-mutant
-protein	O
-by	O
-30	O
--	O
-fold	O
-more	O
-than	O
-would	O
-be	O
-expected	O
-based	O
-on	O
-simple	O
-addition	O
-of	O
-the	O
-ΔΔG	O
-'	O
-s	O
-for	O
-the	O
-single	O
-mutations	O
-.	O
-	O
-This	O
-difference	O
-indicates	O
-that	O
-the	O
-linearly	O
-distant	O
-regions	O
-of	O
-the	O
-U2AF65	O
-primary	O
-sequence	O
-,	O
-including	O
-Q147	O
-in	O
-the	O
-N	O
--	O
-terminal	O
-RRM1	O
-extension	O
-and	O
-R227	O
-/	O
-V254	O
-in	O
-the	O
-N	O
--/	O
-C	O
--	O
-terminal	O
-linker	O
-regions	O
-at	O
-the	O
-fifth	O
-nucleotide	O
-site	O
-,	O
-cooperatively	O
-recognize	O
-the	O
-Py	O
-tract	O
-.	O
-	O
-Altogether	O
-,	O
-we	O
-conclude	O
-that	O
-the	O
-conformation	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-is	O
-key	O
-for	O
-recognizing	O
-RNA	O
-and	O
-is	O
-positioned	O
-by	O
-the	O
-RRM	O
-extension	O
-but	O
-otherwise	O
-relatively	O
-independent	O
-of	O
-the	O
-side	O
-chain	O
-composition	O
-.	O
-	O
-The	O
-non	O
--	O
-additive	O
-effects	O
-of	O
-the	O
-Q147A	B-mutant
-/	O
-V254P	B-mutant
-/	O
-R227A	B-mutant
-triple	O
-mutation	O
-,	O
-coupled	O
-with	O
-the	O
-context	O
--	O
-dependent	O
-penalties	O
-of	O
-an	O
-internal	O
-U2AF65	O
-linker	O
-deletion	O
-,	O
-highlights	O
-the	O
-importance	O
-of	O
-the	O
-structural	O
-interplay	O
-among	O
-the	O
-U2AF65	O
-linker	O
-and	O
-the	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-extensions	O
-flanking	O
-the	O
-core	O
-RRMs	O
-.	O
-	O
-Importance	O
-of	O
-U2AF65	O
-–	O
-RNA	O
-contacts	O
-for	O
-pre	O
--	O
-mRNA	O
-splicing	O
-	O
-We	O
-proceeded	O
-to	O
-test	O
-the	O
-importance	O
-of	O
-new	O
-U2AF65	O
-–	O
-Py	O
--	O
-tract	O
-interactions	O
-for	O
-splicing	O
-of	O
-a	O
-model	O
-pre	O
--	O
-mRNA	O
-substrate	O
-in	O
-a	O
-human	O
-cell	O
-line	O
-(	O
-Fig	O
-.	O
-5	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-As	O
-a	O
-representative	O
-splicing	O
-substrate	O
-,	O
-we	O
-utilized	O
-a	O
-well	O
--	O
-characterized	O
-minigene	O
-splicing	O
-reporter	O
-(	O
-called	O
-pyPY	O
-)	O
-comprising	O
-a	O
-weak	O
-(	O
-that	O
-is	O
-,	O
-degenerate	O
-,	O
-py	O
-)	O
-and	O
-strong	O
-(	O
-that	O
-is	O
-,	O
-U	O
--	O
-rich	O
-,	O
-PY	O
-)	O
-polypyrimidine	O
-tracts	O
-preceding	O
-two	O
-alternative	O
-splice	O
-sites	O
-(	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-When	O
-transfected	O
-into	O
-HEK293T	O
-cells	O
-containing	O
-only	O
-endogenous	O
-U2AF65	O
-,	O
-the	O
-PY	O
-splice	O
-site	O
-is	O
-used	O
-and	O
-the	O
-remaining	O
-transcript	O
-remains	O
-unspliced	O
-.	O
-	O
-When	O
-co	O
--	O
-transfected	O
-with	O
-an	O
-expression	O
-plasmid	O
-for	O
-wild	O
--	O
-type	O
-U2AF65	O
-,	O
-use	O
-of	O
-the	O
-py	O
-splice	O
-site	O
-significantly	O
-increases	O
-(	O
-by	O
-more	O
-than	O
-five	O
--	O
-fold	O
-)	O
-and	O
-as	O
-documented	O
-converts	O
-a	O
-fraction	O
-of	O
-the	O
-unspliced	O
-to	O
-spliced	O
-transcript	O
-.	O
-	O
-The	O
-strong	O
-PY	O
-splice	O
-site	O
-is	O
-insensitive	O
-to	O
-added	O
-U2AF65	O
-,	O
-suggesting	O
-that	O
-endogenous	O
-U2AF65	O
-levels	O
-are	O
-sufficient	O
-to	O
-saturate	O
-this	O
-site	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-We	O
-introduced	O
-the	O
-triple	O
-mutation	O
-(	O
-V254P	B-mutant
-/	O
-R227A	B-mutant
-/	O
-Q147A	B-mutant
-)	O
-that	O
-significantly	O
-reduced	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-association	O
-with	O
-the	O
-Py	O
-tract	O
-(	O
-Fig	O
-.	O
-4b	O
-)	O
-in	O
-the	O
-context	O
-of	O
-full	O
--	O
-length	O
-U2AF65	O
-(	O
-U2AF65	B-mutant
--	I-mutant
-3Mut	I-mutant
-).	O
-	O
-Co	O
--	O
-transfection	O
-of	O
-the	O
-U2AF65	B-mutant
--	I-mutant
-3Mut	I-mutant
-with	O
-the	O
-pyPY	O
-splicing	O
-substrate	O
-significantly	O
-reduced	O
-splicing	O
-of	O
-the	O
-weak	O
-‘	O
-py	O
-'	O
-splice	O
-site	O
-relative	O
-to	O
-wild	O
--	O
-type	O
-U2AF65	O
-(	O
-Fig	O
-.	O
-5b	O
-,	O
-c	O
-).	O
-	O
-We	O
-conclude	O
-that	O
-the	O
-Py	O
--	O
-tract	O
-interactions	O
-with	O
-these	O
-residues	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-and	O
-RRM	O
-extensions	O
-are	O
-important	O
-for	O
-splicing	O
-as	O
-well	O
-as	O
-for	O
-binding	O
-a	O
-representative	O
-of	O
-the	O
-major	O
-U2	O
--	O
-class	O
-of	O
-splice	O
-sites	O
-.	O
-	O
-Sparse	O
-inter	O
--	O
-RRM	O
-contacts	O
-underlie	O
-apo	O
--	O
-U2AF65	O
-dynamics	O
-	O
-The	O
-direct	O
-interface	O
-between	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-RRM1	O
-and	O
-RRM2	O
-is	O
-minor	O
-,	O
-burying	O
-265	O
-Å2	O
-of	O
-solvent	O
-accessible	O
-surface	O
-area	O
-compared	O
-with	O
-570	O
-Å2	O
-on	O
-average	O
-for	O
-a	O
-crystal	O
-packing	O
-interface	O
-.	O
-	O
-A	O
-handful	O
-of	O
-inter	O
--	O
-RRM	O
-hydrogen	O
-bonds	O
-are	O
-apparent	O
-between	O
-the	O
-side	O
-chains	O
-of	O
-RRM1	O
--	O
-N155	O
-and	O
-RRM2	O
--	O
-K292	O
-,	O
-RRM1	O
--	O
-N155	O
-and	O
-RRM2	O
--	O
-D272	O
-as	O
-well	O
-as	O
-the	O
-backbone	O
-atoms	O
-of	O
-RRM1	O
--	O
-G221	O
-and	O
-RRM2	O
--	O
-D273	O
-(	O
-Fig	O
-.	O
-4c	O
-).	O
-	O
-This	O
-minor	O
-U2AF65	O
-RRM1	O
-/	O
-RRM2	O
-interface	O
-,	O
-coupled	O
-with	O
-the	O
-versatile	O
-sequence	O
-of	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-,	O
-highlighted	O
-the	O
-potential	O
-role	O
-for	O
-inter	O
--	O
-RRM	O
-conformational	O
-dynamics	O
-in	O
-U2AF65	O
--	O
-splice	O
-site	O
-recognition	O
-.	O
-	O
-Paramagnetic	O
-resonance	O
-enhancement	O
-(	O
-PRE	O
-)	O
-measurements	O
-previously	O
-had	O
-suggested	O
-a	O
-predominant	O
-back	O
--	O
-to	O
--	O
-back	O
-,	O
-or	O
-‘	O
-closed	O
-'	O
-conformation	O
-of	O
-the	O
-apo	O
--	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-RRM1	O
-and	O
-RRM2	O
-in	O
-equilibrium	O
-with	O
-a	O
-minor	O
-‘	O
-open	O
-'	O
-conformation	O
-resembling	O
-the	O
-RNA	O
--	O
-bound	O
-inter	O
--	O
-RRM	O
-arrangement	O
-.	O
-	O
-Yet	O
-,	O
-small	O
--	O
-angle	O
-X	O
--	O
-ray	O
-scattering	O
-(	O
-SAXS	O
-)	O
-data	O
-indicated	O
-that	O
-both	O
-the	O
-minimal	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-and	O
-longer	O
-constructs	O
-comprise	O
-a	O
-highly	O
-diverse	O
-continuum	O
-of	O
-conformations	O
-in	O
-the	O
-absence	O
-of	O
-RNA	O
-that	O
-includes	O
-the	O
-‘	O
-closed	O
-'	O
-and	O
-‘	O
-open	O
-'	O
-conformations	O
-.	O
-	O
-To	O
-complement	O
-the	O
-static	O
-portraits	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structure	O
-that	O
-we	O
-had	O
-determined	O
-by	O
-X	O
--	O
-ray	O
-crystallography	O
-,	O
-we	O
-used	O
-smFRET	O
-to	O
-characterize	O
-the	O
-probability	O
-distribution	O
-functions	O
-and	O
-time	O
-dependence	O
-of	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-conformational	O
-dynamics	O
-in	O
-solution	O
-.	O
-	O
-The	O
-inter	O
--	O
-RRM	O
-dynamics	O
-of	O
-U2AF65	O
-were	O
-followed	O
-using	O
-FRET	O
-between	O
-fluorophores	O
-attached	O
-to	O
-RRM1	O
-and	O
-RRM2	O
-(	O
-Fig	O
-.	O
-6a	O
-,	O
-b	O
-,	O
-Methods	O
-).	O
-	O
-The	O
-positions	O
-of	O
-single	O
-cysteine	O
-mutations	O
-for	O
-fluorophore	O
-attachment	O
-(	O
-A181C	B-mutant
-in	O
-RRM1	O
-and	O
-Q324C	B-mutant
-in	O
-RRM2	O
-)	O
-were	O
-chosen	O
-based	O
-on	O
-inspection	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-and	O
-the	O
-‘	O
-closed	O
-'	O
-model	O
-of	O
-apo	O
--	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-.	O
-	O
-Criteria	O
-included	O
-(	O
-i	O
-)	O
-residue	O
-locations	O
-that	O
-are	O
-distant	O
-from	O
-and	O
-hence	O
-not	O
-expected	O
-to	O
-interfere	O
-with	O
-the	O
-RRM	O
-/	O
-RNA	O
-or	O
-inter	O
--	O
-RRM	O
-interfaces	O
-,	O
-(	O
-ii	O
-)	O
-inter	O
--	O
-dye	O
-distances	O
-(	O
-50	O
-Å	O
-for	O
-U2AF651	O
-,	O
-2L	O
-–	O
-Py	O
-tract	O
-and	O
-30	O
-Å	O
-for	O
-the	O
-closed	O
-apo	O
--	O
-model	O
-)	O
-that	O
-are	O
-expected	O
-to	O
-be	O
-near	O
-the	O
-Förster	O
-radius	O
-(	O
-Ro	O
-)	O
-for	O
-the	O
-Cy3	O
-/	O
-Cy5	O
-pair	O
-(	O
-56	O
-Å	O
-),	O
-where	O
-changes	O
-in	O
-the	O
-efficiency	O
-of	O
-energy	O
-transfer	O
-are	O
-most	O
-sensitive	O
-to	O
-distance	O
-,	O
-and	O
-(	O
-iii	O
-)	O
-FRET	O
-efficiencies	O
-that	O
-are	O
-calculated	O
-to	O
-be	O
-significantly	O
-greater	O
-for	O
-the	O
-‘	O
-closed	O
-'	O
-apo	O
--	O
-model	O
-as	O
-opposed	O
-to	O
-the	O
-‘	O
-open	O
-'	O
-RNA	O
--	O
-bound	O
-structures	O
-(	O
-by	O
-∼	O
-30	O
-%).	O
-	O
-The	O
-FRET	O
-efficiencies	O
-of	O
-either	O
-of	O
-these	O
-structurally	O
-characterized	O
-conformations	O
-also	O
-are	O
-expected	O
-to	O
-be	O
-significantly	O
-greater	O
-than	O
-elongated	O
-U2AF65	O
-conformations	O
-that	O
-lack	O
-inter	O
--	O
-RRM	O
-contacts	O
-.	O
-	O
-Double	O
--	O
-cysteine	O
-variant	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-was	O
-modified	O
-with	O
-equimolar	O
-amount	O
-of	O
-Cy3	O
-and	O
-Cy5	O
-.	O
-	O
-Only	O
-traces	O
-that	O
-showed	O
-single	O
-photobleaching	O
-events	O
-for	O
-both	O
-donor	O
-and	O
-acceptor	O
-dyes	O
-and	O
-anti	O
--	O
-correlated	O
-changes	O
-in	O
-acceptor	O
-and	O
-donor	O
-fluorescence	O
-were	O
-included	O
-in	O
-smFRET	O
-data	O
-analysis	O
-.	O
-	O
-We	O
-first	O
-characterized	O
-the	O
-conformational	O
-dynamics	O
-spectrum	O
-of	O
-U2AF65	O
-in	O
-the	O
-absence	O
-of	O
-RNA	O
-(	O
-Fig	O
-.	O
-6c	O
-,	O
-d	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-7a	O
-,	O
-b	O
-).	O
-	O
-The	O
-double	O
--	O
-labelled	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-protein	O
-was	O
-tethered	O
-to	O
-a	O
-slide	O
-via	O
-biotin	O
--	O
-NTA	O
-/	O
-Ni	O
-+	O
-2	O
-resin	O
-.	O
-	O
-Virtually	O
-no	O
-fluorescent	O
-molecules	O
-were	O
-detected	O
-in	O
-the	O
-absence	O
-of	O
-biotin	O
--	O
-NTA	O
-/	O
-Ni	O
-+	O
-2	O
-,	O
-which	O
-demonstrates	O
-the	O
-absence	O
-of	O
-detectable	O
-non	O
--	O
-specific	O
-binding	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-to	O
-the	O
-slide	O
-.	O
-	O
-The	O
-FRET	O
-distribution	O
-histogram	O
-built	O
-from	O
-more	O
-than	O
-a	O
-thousand	O
-traces	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-in	O
-the	O
-absence	O
-of	O
-ligand	O
-showed	O
-an	O
-extremely	O
-broad	O
-distribution	O
-centred	O
-at	O
-a	O
-FRET	O
-efficiency	O
-of	O
-∼	O
-0	O
-.	O
-4	O
-(	O
-Fig	O
-.	O
-6d	O
-).	O
-	O
-Approximately	O
-40	O
-%	O
-of	O
-the	O
-smFRET	O
-traces	O
-showed	O
-apparent	O
-transitions	O
-between	O
-multiple	O
-FRET	O
-values	O
-(	O
-for	O
-example	O
-,	O
-Fig	O
-.	O
-6c	O
-).	O
-	O
-Despite	O
-the	O
-large	O
-width	O
-of	O
-the	O
-FRET	O
--	O
-distribution	O
-histogram	O
-,	O
-the	O
-majority	O
-(	O
-80	O
-%)	O
-of	O
-traces	O
-that	O
-showed	O
-fluctuations	O
-sampled	O
-only	O
-two	O
-distinct	O
-FRET	O
-states	O
-(	O
-for	O
-example	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-7a	O
-).	O
-	O
-Approximately	O
-70	O
-%	O
-of	O
-observed	O
-fluctuations	O
-were	O
-interchanges	O
-between	O
-the	O
-∼	O
-0	O
-.	O
-65	O
-and	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-values	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7b	O
-).	O
-	O
-We	O
-cannot	O
-exclude	O
-a	O
-possibility	O
-that	O
-tethering	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-to	O
-the	O
-microscope	O
-slide	O
-introduces	O
-structural	O
-heterogeneity	O
-into	O
-the	O
-protein	O
-and	O
-,	O
-thus	O
-,	O
-contributes	O
-to	O
-the	O
-breadth	O
-of	O
-the	O
-FRET	O
-distribution	O
-histogram	O
-.	O
-	O
-However	O
-,	O
-the	O
-presence	O
-of	O
-repetitive	O
-fluctuations	O
-between	O
-particular	O
-FRET	O
-values	O
-supports	O
-the	O
-hypothesis	O
-that	O
-RNA	O
--	O
-free	O
-U2AF65	O
-samples	O
-several	O
-distinct	O
-conformations	O
-.	O
-	O
-This	O
-result	O
-is	O
-consistent	O
-with	O
-the	O
-broad	O
-ensembles	O
-of	O
-extended	O
-solution	O
-conformations	O
-that	O
-best	O
-fit	O
-the	O
-SAXS	O
-data	O
-collected	O
-for	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-as	O
-well	O
-as	O
-for	O
-a	O
-longer	O
-construct	O
-(	O
-residues	O
-136	O
-–	O
-347	O
-).	O
-	O
-We	O
-conclude	O
-that	O
-weak	O
-contacts	O
-between	O
-the	O
-U2AF65	O
-RRM1	O
-and	O
-RRM2	O
-permit	O
-dissociation	O
-of	O
-these	O
-RRMs	O
-in	O
-the	O
-absence	O
-of	O
-RNA	O
-.	O
-	O
-U2AF65	O
-conformational	O
-selection	O
-and	O
-induced	O
-fit	O
-by	O
-bound	O
-RNA	O
-	O
-We	O
-next	O
-used	O
-smFRET	O
-to	O
-probe	O
-the	O
-conformational	O
-selection	O
-of	O
-distinct	O
-inter	O
--	O
-RRM	O
-arrangements	O
-following	O
-association	O
-of	O
-U2AF65	O
-with	O
-the	O
-AdML	O
-Py	O
--	O
-tract	O
-prototype	O
-.	O
-	O
-Addition	O
-of	O
-the	O
-AdML	O
-RNA	O
-to	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-selectively	O
-increases	O
-a	O
-fraction	O
-of	O
-molecules	O
-showing	O
-an	O
-∼	O
-0	O
-.	O
-45	O
-apparent	O
-FRET	O
-efficiency	O
-,	O
-suggesting	O
-that	O
-RNA	O
-binding	O
-stabilizes	O
-a	O
-single	O
-conformation	O
-,	O
-which	O
-corresponds	O
-to	O
-the	O
-0	O
-.	O
-45	O
-FRET	O
-state	O
-(	O
-Fig	O
-.	O
-6e	O
-,	O
-f	O
-).	O
-	O
-To	O
-assess	O
-the	O
-possible	O
-contributions	O
-of	O
-RNA	O
--	O
-free	O
-conformations	O
-of	O
-U2AF65	O
-and	O
-/	O
-or	O
-structural	O
-heterogeneity	O
-introduced	O
-by	O
-tethering	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-to	O
-the	O
-slide	O
-to	O
-the	O
-observed	O
-distribution	O
-of	O
-FRET	O
-values	O
-,	O
-we	O
-reversed	O
-the	O
-immobilization	O
-scheme	O
-.	O
-	O
-We	O
-tethered	O
-the	O
-AdML	O
-RNA	O
-to	O
-the	O
-slide	O
-via	O
-a	O
-biotinylated	O
-oligonucleotide	O
-DNA	O
-handle	O
-and	O
-added	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-in	O
-the	O
-absence	O
-of	O
-biotin	O
--	O
-NTA	O
-resin	O
-(	O
-Fig	O
-.	O
-6g	O
-,	O
-h	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-7c	O
-–	O
-g	O
-).	O
-	O
-A	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-was	O
-again	O
-predominant	O
-,	O
-indicating	O
-a	O
-similar	O
-RNA	O
--	O
-bound	O
-conformation	O
-and	O
-structural	O
-dynamics	O
-for	O
-the	O
-untethered	O
-and	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-).	O
-	O
-We	O
-examined	O
-the	O
-effect	O
-on	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-conformations	O
-of	O
-purine	O
-interruptions	O
-that	O
-often	O
-occur	O
-in	O
-relatively	O
-degenerate	O
-human	O
-Py	O
-tracts	O
-.	O
-	O
-We	O
-introduced	O
-an	O
-rArA	O
-purine	O
-dinucleotide	O
-within	O
-a	O
-variant	O
-of	O
-the	O
-AdML	O
-Py	O
-tract	O
-(	O
-detailed	O
-in	O
-Methods	O
-).	O
-	O
-Insertion	O
-of	O
-adenine	O
-nucleotides	O
-decreased	O
-binding	O
-affinity	O
-of	O
-U2AF65	O
-to	O
-RNA	O
-by	O
-approximately	O
-five	O
--	O
-fold	O
-.	O
-	O
-Nevertheless	O
-,	O
-in	O
-the	O
-presence	O
-of	O
-saturating	O
-concentrations	O
-of	O
-rArA	O
--	O
-interrupted	O
-RNA	O
-slide	O
--	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-showed	O
-a	O
-prevalent	O
-∼	O
-0	O
-.	O
-45	O
-apparent	O
-FRET	O
-value	O
-(	O
-Fig	O
-.	O
-6i	O
-,	O
-j	O
-),	O
-which	O
-was	O
-also	O
-predominant	O
-in	O
-the	O
-presence	O
-of	O
-continuous	O
-Py	O
-tract	O
-.	O
-	O
-Therefore	O
-,	O
-RRM1	O
--	O
-to	O
--	O
-RRM2	O
-distance	O
-remains	O
-similar	O
-regardless	O
-of	O
-whether	O
-U2AF65	O
-is	O
-bound	O
-to	O
-interrupted	O
-or	O
-continuous	O
-Py	O
-tract	O
-.	O
-	O
-The	O
-inter	O
--	O
-fluorophore	O
-distances	O
-derived	O
-from	O
-the	O
-observed	O
-0	O
-.	O
-45	O
-FRET	O
-state	O
-agree	O
-with	O
-the	O
-distances	O
-between	O
-the	O
-α	O
--	O
-carbon	O
-atoms	O
-of	O
-the	O
-respective	O
-residues	O
-in	O
-the	O
-crystal	O
-structures	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-bound	O
-to	O
-Py	O
--	O
-tract	O
-oligonucleotides	O
-.	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-inferring	O
-distances	O
-from	O
-FRET	O
-values	O
-is	O
-prone	O
-to	O
-significant	O
-error	O
-because	O
-of	O
-uncertainties	O
-in	O
-the	O
-determination	O
-of	O
-fluorophore	O
-orientation	O
-factor	O
-κ2	O
-and	O
-Förster	O
-radius	O
-R0	O
-,	O
-the	O
-parameters	O
-used	O
-in	O
-distance	O
-calculations	O
-.	O
-	O
-Nevertheless	O
-,	O
-the	O
-predominant	O
-0	O
-.	O
-45	O
-FRET	O
-state	O
-in	O
-the	O
-presence	O
-of	O
-RNA	O
-agrees	O
-with	O
-the	O
-Py	O
--	O
-tract	O
--	O
-bound	O
-crystal	O
-structure	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-.	O
-	O
-Importantly	O
-,	O
-the	O
-majority	O
-of	O
-traces	O
-(∼	O
-70	O
-%)	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-bound	O
-to	O
-the	O
-slide	O
--	O
-tethered	O
-RNA	O
-lacked	O
-FRET	O
-fluctuations	O
-and	O
-predominately	O
-exhibited	O
-a	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-(	O
-for	O
-example	O
-,	O
-Fig	O
-.	O
-6g	O
-).	O
-	O
-The	O
-remaining	O
-∼	O
-30	O
-%	O
-of	O
-traces	O
-for	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-bound	O
-to	O
-the	O
-slide	O
--	O
-tethered	O
-RNA	O
-showed	O
-fluctuations	O
-between	O
-distinct	O
-FRET	O
-values	O
-.	O
-	O
-The	O
-majority	O
-of	O
-traces	O
-that	O
-show	O
-fluctuations	O
-began	O
-at	O
-high	O
-(	O
-0	O
-.	O
-65	O
-–	O
-0	O
-.	O
-8	O
-)	O
-FRET	O
-value	O
-and	O
-transitioned	O
-to	O
-a	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7c	O
-–	O
-g	O
-).	O
-	O
-Hidden	O
-Markov	O
-modelling	O
-analysis	O
-of	O
-smFRET	O
-traces	O
-suggests	O
-that	O
-RNA	O
--	O
-bound	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-can	O
-sample	O
-at	O
-least	O
-two	O
-other	O
-conformations	O
-corresponding	O
-to	O
-∼	O
-0	O
-.	O
-7	O
-–	O
-0	O
-.	O
-8	O
-and	O
-∼	O
-0	O
-.	O
-3	O
-FRET	O
-values	O
-in	O
-addition	O
-to	O
-the	O
-predominant	O
-conformation	O
-corresponding	O
-to	O
-the	O
-0	O
-.	O
-45	O
-FRET	O
-state	O
-.	O
-	O
-Although	O
-a	O
-compact	O
-conformation	O
-(	O
-or	O
-multiple	O
-conformations	O
-)	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-corresponding	O
-to	O
-∼	O
-0	O
-.	O
-7	O
-–	O
-0	O
-.	O
-8	O
-FRET	O
-values	O
-can	O
-bind	O
-RNA	O
-,	O
-on	O
-RNA	O
-binding	O
-,	O
-these	O
-compact	O
-conformations	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-transition	O
-into	O
-a	O
-more	O
-stable	O
-structural	O
-state	O
-that	O
-corresponds	O
-to	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-and	O
-is	O
-likely	O
-similar	O
-to	O
-the	O
-side	O
--	O
-by	O
--	O
-side	O
-inter	O
--	O
-RRM	O
--	O
-arrangement	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-crystal	O
-structures	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-sequence	O
-of	O
-structural	O
-rearrangements	O
-of	O
-U2AF65	O
-observed	O
-in	O
-smFRET	O
-traces	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7c	O
-–	O
-g	O
-)	O
-suggests	O
-that	O
-a	O
-‘	O
-conformational	O
-selection	O
-'	O
-mechanism	O
-of	O
-Py	O
--	O
-tract	O
-recognition	O
-(	O
-that	O
-is	O
-,	O
-RNA	O
-ligand	O
-stabilization	O
-of	O
-a	O
-pre	O
--	O
-configured	O
-U2AF65	O
-conformation	O
-)	O
-is	O
-complemented	O
-by	O
-‘	O
-induced	O
-fit	O
-'	O
-(	O
-that	O
-is	O
-,	O
-RNA	O
--	O
-induced	O
-rearrangement	O
-of	O
-the	O
-U2AF65	O
-RRMs	O
-to	O
-achieve	O
-the	O
-final	O
-‘	O
-side	O
--	O
-by	O
--	O
-side	O
-'	O
-conformation	O
-),	O
-as	O
-discussed	O
-below	O
-.	O
-	O
-The	O
-U2AF65	O
-structures	O
-and	O
-analyses	O
-presented	O
-here	O
-represent	O
-a	O
-successful	O
-step	O
-towards	O
-defining	O
-a	O
-molecular	O
-map	O
-of	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-.	O
-	O
-Several	O
-observations	O
-indicate	O
-that	O
-the	O
-numerous	O
-intramolecular	O
-contacts	O
-,	O
-here	O
-revealed	O
-among	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-and	O
-RRM1	O
-,	O
-RRM2	O
-,	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-RRM1	O
-extension	O
-,	O
-synergistically	O
-coordinate	O
-U2AF65	O
-–	O
-Py	O
--	O
-tract	O
-recognition	O
-.	O
-	O
-Truncation	O
-of	O
-U2AF65	O
-to	O
-the	O
-core	O
-RRM1	O
-–	O
-RRM2	O
-region	O
-reduces	O
-its	O
-RNA	O
-affinity	O
-by	O
-100	O
--	O
-fold	O
-.	O
-	O
-Likewise	O
-,	O
-deletion	O
-of	O
-20	O
-inter	O
--	O
-RRM	O
-linker	O
-residues	O
-significantly	O
-reduces	O
-U2AF65	O
-–	O
-RNA	O
-binding	O
-only	O
-when	O
-introduced	O
-in	O
-the	O
-context	O
-of	O
-the	O
-longer	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-construct	O
-comprising	O
-the	O
-RRM	O
-extensions	O
-,	O
-which	O
-in	O
-turn	O
-position	O
-the	O
-linker	O
-for	O
-RNA	O
-interactions	O
-.	O
-	O
-Notably	O
-,	O
-a	O
-triple	O
-mutation	O
-of	O
-three	O
-residues	O
-(	O
-V254P	B-mutant
-,	O
-Q147A	B-mutant
-and	O
-R227A	B-mutant
-)	O
-in	O
-the	O
-respective	O
-inter	O
--	O
-RRM	O
-linker	O
-,	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-extensions	O
-non	O
--	O
-additively	O
-reduce	O
-RNA	O
-binding	O
-by	O
-150	O
--	O
-fold	O
-.	O
-	O
-Altogether	O
-,	O
-these	O
-data	O
-indicate	O
-that	O
-interactions	O
-among	O
-the	O
-U2AF65	O
-RRM1	O
-/	O
-RRM2	O
-,	O
-inter	O
--	O
-RRM	O
-linker	O
-,	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-extensions	O
-are	O
-mutually	O
-inter	O
--	O
-dependent	O
-for	O
-cognate	O
-Py	O
--	O
-tract	O
-recognition	O
-.	O
-	O
-The	O
-implications	O
-of	O
-this	O
-finding	O
-for	O
-U2AF65	O
-conservation	O
-and	O
-Py	O
--	O
-tract	O
-recognition	O
-are	O
-detailed	O
-in	O
-the	O
-Supplementary	O
-Discussion	O
-.	O
-	O
-Recently	O
-,	O
-high	O
--	O
-throughput	O
-sequencing	O
-studies	O
-have	O
-shown	O
-that	O
-somatic	O
-mutations	O
-in	O
-pre	O
--	O
-mRNA	O
-splicing	O
-factors	O
-occur	O
-in	O
-the	O
-majority	O
-of	O
-patients	O
-with	O
-myelodysplastic	O
-syndrome	O
-(	O
-MDS	O
-).	O
-	O
-MDS	O
--	O
-relevant	O
-mutations	O
-are	O
-common	O
-in	O
-the	O
-small	O
-U2AF	O
-subunit	O
-(	O
-U2AF35	O
-,	O
-or	O
-U2AF1	O
-),	O
-yet	O
-such	O
-mutations	O
-are	O
-rare	O
-in	O
-the	O
-large	O
-U2AF65	O
-subunit	O
-(	O
-also	O
-called	O
-U2AF2	O
-)—	O
-possibly	O
-due	O
-to	O
-the	O
-selective	O
-versus	O
-nearly	O
-universal	O
-requirements	O
-of	O
-these	O
-factors	O
-for	O
-splicing	O
-.	O
-	O
-A	O
-confirmed	O
-somatic	O
-mutation	O
-of	O
-U2AF65	O
-in	O
-patients	O
-with	O
-MDS	O
-,	O
-L187V	B-mutant
-,	O
-is	O
-located	O
-on	O
-a	O
-solvent	O
--	O
-exposed	O
-surface	O
-of	O
-RRM1	O
-that	O
-is	O
-distinct	O
-from	O
-the	O
-RNA	O
-interface	O
-(	O
-Fig	O
-.	O
-7a	O
-).	O
-	O
-This	O
-L187	O
-surface	O
-is	O
-oriented	O
-towards	O
-the	O
-N	O
-terminus	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-construct	O
-,	O
-where	O
-it	O
-is	O
-expected	O
-to	O
-abut	O
-the	O
-U2AF35	O
--	O
-binding	O
-site	O
-in	O
-the	O
-context	O
-of	O
-the	O
-full	O
--	O
-length	O
-U2AF	O
-heterodimer	O
-.	O
-	O
-Likewise	O
-,	O
-an	O
-unconfirmed	O
-M144I	B-mutant
-mutation	O
-reported	O
-by	O
-the	O
-same	O
-group	O
-corresponds	O
-to	O
-the	O
-N	O
--	O
-terminal	O
-residue	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-which	O
-is	O
-separated	O
-by	O
-only	O
-∼	O
-20	O
-residues	O
-from	O
-the	O
-U2AF35	O
--	O
-binding	O
-site	O
-.	O
-	O
-As	O
-such	O
-,	O
-we	O
-suggest	O
-that	O
-the	O
-MDS	O
--	O
-relevant	O
-U2AF65	O
-mutations	O
-contribute	O
-to	O
-MDS	O
-progression	O
-indirectly	O
-,	O
-by	O
-destabilizing	O
-a	O
-relevant	O
-conformation	O
-of	O
-the	O
-conjoined	O
-U2AF35	O
-subunit	O
-rather	O
-than	O
-affecting	O
-U2AF65	O
-functions	O
-in	O
-RNA	O
-binding	O
-or	O
-spliceosome	O
-recruitment	O
-per	O
-se	O
-.	O
-	O
-Our	O
-smFRET	O
-results	O
-agree	O
-with	O
-prior	O
-NMR	O
-/	O
-PRE	O
-evidence	O
-for	O
-multi	O
--	O
-domain	O
-conformational	O
-selection	O
-as	O
-one	O
-mechanistic	O
-basis	O
-for	O
-U2AF65	O
-–	O
-RNA	O
-association	O
-(	O
-Fig	O
-.	O
-7b	O
-).	O
-	O
-An	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-is	O
-likely	O
-to	O
-correspond	O
-to	O
-the	O
-U2AF65	O
-conformation	O
-visualized	O
-in	O
-our	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-crystal	O
-structures	O
-,	O
-in	O
-which	O
-the	O
-RRM1	O
-and	O
-RRM2	O
-bind	O
-side	O
--	O
-by	O
--	O
-side	O
-to	O
-the	O
-Py	O
--	O
-tract	O
-oligonucleotide	O
-.	O
-	O
-The	O
-lesser	O
-0	O
-.	O
-65	O
-–	O
-0	O
-.	O
-8	O
-and	O
-0	O
-.	O
-2	O
-–	O
-0	O
-.	O
-3	O
-FRET	O
-values	O
-in	O
-the	O
-untethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-experiment	O
-could	O
-correspond	O
-to	O
-respective	O
-variants	O
-of	O
-the	O
-‘	O
-closed	O
-',	O
-back	O
--	O
-to	O
--	O
-back	O
-U2AF65	O
-conformations	O
-characterized	O
-by	O
-NMR	O
-/	O
-PRE	O
-data	O
-,	O
-or	O
-to	O
-extended	O
-U2AF65	O
-conformations	O
-,	O
-in	O
-which	O
-the	O
-intramolecular	O
-RRM1	O
-/	O
-RRM2	O
-interactions	O
-have	O
-dissociated	O
-the	O
-protein	O
-is	O
-bound	O
-to	O
-RNA	O
-via	O
-single	O
-RRMs	O
-.	O
-	O
-An	O
-increased	O
-prevalence	O
-of	O
-the	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-following	O
-U2AF65	O
-–	O
-RNA	O
-binding	O
-,	O
-coupled	O
-with	O
-the	O
-apparent	O
-absence	O
-of	O
-transitions	O
-in	O
-many	O
-∼	O
-0	O
-.	O
-45	O
--	O
-value	O
-single	O
-molecule	O
-traces	O
-(	O
-for	O
-example	O
-,	O
-Fig	O
-.	O
-6e	O
-),	O
-suggests	O
-a	O
-population	O
-shift	O
-in	O
-which	O
-RNA	O
-binds	O
-to	O
-(	O
-and	O
-draws	O
-the	O
-equilibrium	O
-towards	O
-)	O
-a	O
-pre	O
--	O
-configured	O
-inter	O
--	O
-RRM	O
-proximity	O
-that	O
-most	O
-often	O
-corresponds	O
-to	O
-the	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-.	O
-	O
-Notably	O
-,	O
-our	O
-smFRET	O
-results	O
-reveal	O
-that	O
-U2AF65	O
-–	O
-Py	O
--	O
-tract	O
-recognition	O
-can	O
-be	O
-characterized	O
-by	O
-an	O
-‘	O
-extended	O
-conformational	O
-selection	O
-'	O
-model	O
-(	O
-Fig	O
-.	O
-7b	O
-).	O
-	O
-Examples	O
-of	O
-‘	O
-extended	O
-conformational	O
-selection	O
-'	O
-during	O
-ligand	O
-binding	O
-have	O
-been	O
-characterized	O
-for	O
-a	O
-growing	O
-number	O
-of	O
-macromolecules	O
-(	O
-for	O
-example	O
-,	O
-adenylate	O
-kinase	O
-,	O
-LAO	O
--	O
-binding	O
-protein	O
-,	O
-poly	O
--	O
-ubiquitin	O
-,	O
-maltose	O
--	O
-binding	O
-protein	O
-and	O
-the	O
-preQ1	O
-riboswitch	O
-,	O
-among	O
-others	O
-).	O
-	O
-Here	O
-,	O
-the	O
-majority	O
-of	O
-changes	O
-in	O
-smFRET	O
-traces	O
-for	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-bound	O
-to	O
-slide	O
--	O
-tethered	O
-RNA	O
-began	O
-at	O
-high	O
-(	O
-0	O
-.	O
-65	O
-–	O
-0	O
-.	O
-8	O
-)	O
-FRET	O
-value	O
-and	O
-transition	O
-to	O
-the	O
-predominant	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7c	O
-–	O
-g	O
-).	O
-	O
-These	O
-transitions	O
-could	O
-correspond	O
-to	O
-rearrangement	O
-from	O
-the	O
-‘	O
-closed	O
-'	O
-NMR	O
-/	O
-PRE	O
--	O
-based	O
-U2AF65	O
-conformation	O
-in	O
-which	O
-the	O
-RNA	O
--	O
-binding	O
-surface	O
-of	O
-only	O
-a	O
-single	O
-RRM	O
-is	O
-exposed	O
-and	O
-available	O
-for	O
-RNA	O
-binding	O
-,	O
-to	O
-the	O
-structural	O
-state	O
-seen	O
-in	O
-the	O
-side	O
--	O
-by	O
--	O
-side	O
-,	O
-RNA	O
--	O
-bound	O
-crystal	O
-structure	O
-.	O
-	O
-As	O
-such	O
-,	O
-the	O
-smFRET	O
-approach	O
-reconciles	O
-prior	O
-inconsistencies	O
-between	O
-two	O
-major	O
-conformations	O
-that	O
-were	O
-detected	O
-by	O
-NMR	O
-/	O
-PRE	O
-experiments	O
-and	O
-a	O
-broad	O
-ensemble	O
-of	O
-diverse	O
-inter	O
--	O
-RRM	O
-arrangements	O
-that	O
-fit	O
-the	O
-SAXS	O
-data	O
-for	O
-the	O
-apo	O
--	O
-protein	O
-.	O
-	O
-Similar	O
-interdisciplinary	O
-structural	O
-approaches	O
-are	O
-likely	O
-to	O
-illuminate	O
-whether	O
-similar	O
-mechanistic	O
-bases	O
-for	O
-RNA	O
-binding	O
-are	O
-widespread	O
-among	O
-other	O
-members	O
-of	O
-the	O
-vast	O
-multi	O
--	O
-RRM	O
-family	O
-.	O
-	O
-The	O
-finding	O
-that	O
-U2AF65	O
-recognizes	O
-a	O
-nine	O
-base	O
-pair	O
-Py	O
-tract	O
-contributes	O
-to	O
-an	O
-elusive	O
-‘	O
-code	O
-'	O
-for	O
-predicting	O
-splicing	O
-patterns	O
-from	O
-primary	O
-sequences	O
-in	O
-the	O
-post	O
--	O
-genomic	O
-era	O
-(	O
-reviewed	O
-in	O
-ref	O
-.).	O
-	O
-Based	O
-on	O
-(	O
-i	O
-)	O
-similar	O
-RNA	O
-affinities	O
-of	O
-U2AF65	O
-and	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-(	O
-ii	O
-)	O
-indistinguishable	O
-conformations	O
-among	O
-four	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-in	O
-two	O
-different	O
-crystal	O
-packing	O
-arrangements	O
-and	O
-(	O
-iii	O
-)	O
-penalties	O
-of	O
-structure	O
--	O
-guided	O
-mutations	O
-in	O
-RNA	O
-binding	O
-and	O
-splicing	O
-assays	O
-,	O
-we	O
-suggest	O
-that	O
-the	O
-extended	O
-inter	O
--	O
-RRM	O
-regions	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-underlie	O
-cognate	O
-Py	O
--	O
-tract	O
-recognition	O
-by	O
-the	O
-full	O
--	O
-length	O
-U2AF65	O
-protein	O
-.	O
-	O
-Further	O
-research	O
-will	O
-be	O
-needed	O
-to	O
-understand	O
-the	O
-roles	O
-of	O
-SF1	O
-and	O
-U2AF35	O
-subunits	O
-in	O
-the	O
-conformational	O
-equilibria	O
-underlying	O
-U2AF65	O
-association	O
-with	O
-Py	O
-tracts	O
-.	O
-	O
-Moreover	O
-,	O
-structural	O
-differences	O
-among	O
-U2AF65	O
-homologues	O
-and	O
-paralogues	O
-may	O
-regulate	O
-splice	O
-site	O
-selection	O
-.	O
-	O
-Ultimately	O
-,	O
-these	O
-guidelines	O
-will	O
-assist	O
-the	O
-identification	O
-of	O
-3	O
-′	O
-splice	O
-sites	O
-and	O
-the	O
-relationship	O
-of	O
-disease	O
--	O
-causing	O
-mutations	O
-to	O
-penalties	O
-for	O
-U2AF65	O
-association	O
-.	O
-	O
-The	O
-intact	O
-U2AF65	O
-RRM1	O
-/	O
-RRM2	O
--	O
-containing	O
-domain	O
-and	O
-flanking	O
-residues	O
-are	O
-required	O
-for	O
-binding	O
-contiguous	O
-Py	O
-tracts	O
-.	O
-	O
-(	O
-a	O
-)	O
-Domain	O
-organization	O
-of	O
-full	O
--	O
-length	O
-(	O
-fl	O
-)	O
-U2AF65	O
-and	O
-constructs	O
-used	O
-for	O
-RNA	O
-binding	O
-and	O
-structural	O
-experiments	O
-.	O
-	O
-An	O
-internal	O
-deletion	O
-(	O
-d	B-mutant
-,	O
-Δ	B-mutant
-)	O
-of	O
-residues	O
-238	O
-–	O
-257	O
-removes	O
-a	O
-portion	O
-of	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-from	O
-the	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-and	O
-dU2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-constructs	O
-.	O
-	O
-(	O
-b	O
-)	O
-Comparison	O
-of	O
-the	O
-apparent	O
-equilibrium	O
-affinities	O
-of	O
-various	O
-U2AF65	O
-constructs	O
-for	O
-binding	O
-the	O
-prototypical	O
-AdML	O
-Py	O
-tract	O
-(	O
-5	O
-′-	O
-CCCUUUUUUUUCC	O
--	O
-3	O
-′).	O
-	O
-The	O
-flU2AF65	O
-protein	O
-includes	O
-a	O
-heterodimerization	O
-domain	O
-of	O
-the	O
-U2AF35	O
-subunit	O
-to	O
-promote	O
-solubility	O
-and	O
-folding	O
-.	O
-	O
-The	O
-apparent	O
-equilibrium	O
-dissociation	O
-constants	O
-(	O
-KD	O
-)	O
-for	O
-binding	O
-the	O
-AdML	O
-13mer	O
-are	O
-as	O
-follows	O
-:	O
-flU2AF65	O
-,	O
-30	O
-±	O
-3	O
-nM	O
-;	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-35	O
-±	O
-6	O
-nM	O
-;	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-,	O
-3	O
-,	O
-600	O
-±	O
-300	O
-nM	O
-.	O
-(	O
-c	O
-)	O
-Comparison	O
-of	O
-the	O
-RNA	O
-sequence	O
-specificities	O
-of	O
-flU2AF65	O
-and	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-constructs	O
-binding	O
-C	O
--	O
-rich	O
-Py	O
-tracts	O
-with	O
-4U	O
-'	O
-s	O
-embedded	O
-in	O
-either	O
-the	O
-5	O
-′-	O
-(	O
-light	O
-grey	O
-fill	O
-)	O
-or	O
-3	O
-′-	O
-(	O
-dark	O
-grey	O
-fill	O
-)	O
-regions	O
-.	O
-	O
-The	O
-KD	O
-'	O
-s	O
-for	O
-binding	O
-5	O
-′-	O
-CCUUUUCCCCCCC	O
--	O
-3	O
-′	O
-are	O
-:	O
-flU2AF65	O
-,	O
-41	O
-±	O
-2	O
-nM	O
-;	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-31	O
-±	O
-3	O
-nM	O
-.	O
-The	O
-KD	O
-'	O
-s	O
-for	O
-binding	O
-5	O
-′-	O
-CCCCCCCUUUUCC	O
--	O
-3	O
-′	O
-are	O
-:	O
-flU2AF65	O
-,	O
-414	O
-±	O
-12	O
-nM	O
-;	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-417	O
-±	O
-10	O
-nM	O
-.	O
-Bar	O
-graphs	O
-are	O
-hatched	O
-to	O
-match	O
-the	O
-constructs	O
-shown	O
-in	O
-a	O
-.	O
-The	O
-average	O
-apparent	O
-equilibrium	O
-affinity	O
-(	O
-KA	O
-)	O
-and	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.	O
-for	O
-three	O
-independent	O
-titrations	O
-are	O
-plotted	O
-.	O
-	O
-The	O
-purified	O
-protein	O
-and	O
-average	O
-fitted	O
-fluorescence	O
-anisotropy	O
-RNA	O
--	O
-binding	O
-curves	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-.	O
-	O
-RRM	O
-,	O
-RNA	O
-recognition	O
-motif	O
-;	O
-RS	O
-,	O
-arginine	O
--	O
-serine	O
-rich	O
-;	O
-UHM	O
-,	O
-U2AF	O
-homology	O
-motif	O
-;	O
-ULM	O
-,	O
-U2AF	O
-ligand	O
-motif	O
-.	O
-	O
-Structures	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-recognizing	O
-a	O
-contiguous	O
-Py	O
-tract	O
-.	O
-	O
-(	O
-a	O
-)	O
-Alignment	O
-of	O
-oligonucleotide	O
-sequences	O
-that	O
-were	O
-co	O
--	O
-crystallized	O
-in	O
-the	O
-indicated	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-.	O
-	O
-The	O
-regions	O
-of	O
-RRM1	O
-,	O
-RRM2	O
-and	O
-linker	O
-contacts	O
-are	O
-indicated	O
-above	O
-by	O
-respective	O
-black	O
-and	O
-blue	O
-arrows	O
-from	O
-N	O
--	O
-to	O
-C	O
--	O
-terminus	O
-.	O
-	O
-For	O
-clarity	O
-,	O
-we	O
-consistently	O
-number	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-nucleotide	O
--	O
-binding	O
-sites	O
-from	O
-one	O
-to	O
-nine	O
-,	O
-although	O
-in	O
-some	O
-cases	O
-the	O
-co	O
--	O
-crystallized	O
-oligonucleotide	O
-comprises	O
-eight	O
-nucleotides	O
-and	O
-as	O
-such	O
-leaves	O
-the	O
-first	O
-binding	O
-site	O
-empty	O
-.	O
-	O
-The	O
-prior	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-nucleotide	O
--	O
-binding	O
-sites	O
-are	O
-given	O
-in	O
-parentheses	O
-(	O
-site	O
-4	O
-'	O
-interacts	O
-with	O
-dU2AF65	B-mutant
-RRM1	O
-and	O
-RRM2	O
-by	O
-crystallographic	O
-symmetry	O
-).	O
-	O
-(	O
-b	O
-)	O
-Stereo	O
-views	O
-of	O
-a	O
-‘	O
-kicked	O
-'	O
-2	O
-|	O
-Fo	O
-|−|	O
-Fc	O
-|	O
-electron	O
-density	O
-map	O
-contoured	O
-at	O
-1σ	O
-for	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-,	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-residues	O
-(	O
-blue	O
-)	O
-or	O
-bound	O
-oligonucleotide	O
-of	O
-a	O
-representative	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structure	O
-(	O
-structure	O
-iv	O
-,	O
-bound	O
-to	O
-5	O
-′-(	O
-P	O
-)	O
-rUrUrUdUrUrU	O
-(	O
-BrdU	O
-)	O
-dUrC	O
-)	O
-(	O
-magenta	O
-).	O
-	O
-Crystallographic	O
-statistics	O
-are	O
-given	O
-in	O
-Table	O
-1	O
-and	O
-the	O
-overall	O
-conformations	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-and	O
-prior	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-/	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structures	O
-are	O
-compared	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-.	O
-	O
-BrdU	O
-,	O
-5	O
--	O
-bromo	O
--	O
-deoxy	O
--	O
-uridine	O
-;	O
-d	O
-,	O
-deoxy	O
--	O
-ribose	O
-;	O
-P	O
--,	O
-5	O
-′-	O
-phosphorylation	O
-;	O
-r	O
-,	O
-ribose	O
-.	O
-	O
-Representative	O
-views	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-interactions	O
-with	O
-each	O
-new	O
-nucleotide	O
-of	O
-the	O
-bound	O
-Py	O
-tract	O
-.	O
-	O
-New	O
-residues	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-are	O
-coloured	O
-a	O
-darker	O
-shade	O
-of	O
-blue	O
-,	O
-apart	O
-from	O
-residues	O
-that	O
-were	O
-tested	O
-by	O
-site	O
--	O
-directed	O
-mutagenesis	O
-,	O
-which	O
-are	O
-coloured	O
-yellow	O
-.	O
-	O
-The	O
-nucleotide	O
--	O
-binding	O
-sites	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-and	O
-prior	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structure	O
-are	O
-compared	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-–	O
-h	O
-.	O
-The	O
-first	O
-and	O
-seventh	O
-U2AF651	O
-,	O
-2L	O
--	O
-binding	O
-sites	O
-are	O
-unchanged	O
-from	O
-the	O
-prior	O
-dU2AF651	O
-,	O
-2	O
-–	O
-RNA	O
-structure	O
-and	O
-are	O
-portrayed	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-,	O
-f	O
-.	O
-The	O
-four	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structures	O
-are	O
-similar	O
-with	O
-the	O
-exception	O
-of	O
-pH	O
--	O
-dependent	O
-variations	O
-at	O
-the	O
-ninth	O
-site	O
-that	O
-are	O
-detailed	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-3i	O
-,	O
-j	O
-.	O
-The	O
-representative	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structure	O
-shown	O
-has	O
-the	O
-highest	O
-resolution	O
-and	O
-/	O
-or	O
-ribose	O
-nucleotide	O
-at	O
-the	O
-given	O
-site	O
-:	O
-(	O
-a	O
-)	O
-rU2	O
-of	O
-structure	O
-iv	O
-;	O
-(	O
-b	O
-)	O
-rU3	O
-of	O
-structure	O
-iii	O
-;	O
-(	O
-c	O
-)	O
-rU4	O
-of	O
-structure	O
-i	O
-;	O
-(	O
-d	O
-)	O
-rU5	O
-of	O
-structure	O
-iii	O
-;	O
-(	O
-e	O
-)	O
-rU6	O
-of	O
-structure	O
-ii	O
-;	O
-(	O
-f	O
-)	O
-dU8	O
-of	O
-structure	O
-iii	O
-;	O
-(	O
-g	O
-)	O
-dU9	O
-of	O
-structure	O
-iii	O
-;	O
-(	O
-h	O
-)	O
-rC9	O
-of	O
-structure	O
-iv	O
-.	O
-	O
-(	O
-i	O
-)	O
-Bar	O
-graph	O
-of	O
-apparent	O
-equilibrium	O
-affinities	O
-(	O
-KA	O
-)	O
-of	O
-the	O
-wild	O
-type	O
-(	O
-blue	O
-)	O
-and	O
-the	O
-indicated	O
-mutant	O
-(	O
-yellow	O
-)	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-proteins	O
-binding	O
-the	O
-AdML	O
-Py	O
-tract	O
-(	O
-5	O
-′-	O
-CCCUUUUUUUUCC	O
--	O
-3	O
-′).	O
-	O
-The	O
-apparent	O
-equilibrium	O
-dissociation	O
-constants	O
-(	O
-KD	O
-)	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-mutant	O
-proteins	O
-are	O
-:	O
-wild	O
-type	O
-(	O
-WT	O
-),	O
-35	O
-±	O
-6	O
-nM	O
-;	O
-R227A	B-mutant
-,	O
-166	O
-±	O
-2	O
-nM	O
-;	O
-V254P	B-mutant
-,	O
-137	O
-±	O
-10	O
-nM	O
-;	O
-Q147A	B-mutant
-,	O
-171	O
-±	O
-21	O
-nM	O
-.	O
-The	O
-average	O
-KA	O
-and	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.	O
-for	O
-three	O
-independent	O
-titrations	O
-are	O
-plotted	O
-.	O
-	O
-The	O
-average	O
-fitted	O
-fluorescence	O
-anisotropy	O
-RNA	O
--	O
-binding	O
-curves	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-4a	O
-–	O
-c	O
-.	O
-	O
-The	O
-U2AF65	O
-linker	O
-/	O
-RRM	O
-and	O
-inter	O
--	O
-RRM	O
-interactions	O
-.	O
-	O
-(	O
-a	O
-)	O
-Contacts	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-with	O
-the	O
-RRMs	O
-.	O
-	O
-A	O
-semi	O
--	O
-transparent	O
-space	O
--	O
-filling	O
-surface	O
-is	O
-shown	O
-for	O
-the	O
-RRM1	O
-(	O
-green	O
-)	O
-and	O
-RRM2	O
-(	O
-light	O
-blue	O
-).	O
-	O
-Residues	O
-V249	O
-,	O
-V250	O
-,	O
-V254	O
-(	O
-yellow	O
-)	O
-are	O
-mutated	O
-to	O
-V249G	B-mutant
-/	O
-V250G	B-mutant
-/	O
-V254G	B-mutant
-in	O
-the	O
-3Gly	B-mutant
-mutant	I-mutant
-;	O
-residues	O
-S251	O
-,	O
-T252	O
-,	O
-V253	O
-,	O
-P255	O
-(	O
-red	O
-)	O
-along	O
-with	O
-V254	O
-are	O
-mutated	O
-to	O
-S251G	B-mutant
-/	O
-T252G	B-mutant
-/	O
-V253G	B-mutant
-/	O
-V254G	B-mutant
-/	O
-P255G	B-mutant
-in	O
-the	O
-5Gly	B-mutant
-mutant	I-mutant
-or	O
-to	O
-S251N	B-mutant
-/	O
-T252L	B-mutant
-/	O
-V253A	B-mutant
-/	O
-V254L	B-mutant
-/	O
-P255A	B-mutant
-in	O
-the	O
-NLALA	B-mutant
-mutant	I-mutant
-;	O
-residues	O
-M144	O
-,	O
-L235	O
-,	O
-M238	O
-,	O
-V244	O
-,	O
-V246	O
-(	O
-orange	O
-)	O
-along	O
-with	O
-V249	O
-,	O
-V250	O
-,	O
-S251	O
-,	O
-T252	O
-,	O
-V253	O
-,	O
-V254	O
-,	O
-P255	O
-are	O
-mutated	O
-to	O
-M144G	B-mutant
-/	O
-L235G	B-mutant
-/	O
-M238G	B-mutant
-/	O
-V244G	B-mutant
-/	O
-V246G	B-mutant
-/	O
-V249G	B-mutant
-/	O
-V250G	B-mutant
-/	O
-S251G	B-mutant
-/	O
-T252G	B-mutant
-/	O
-V253G	B-mutant
-/	O
-V254G	B-mutant
-/	O
-P255G	B-mutant
-in	O
-the	O
-12Gly	B-mutant
-mutant	I-mutant
-.	O
-	O
-Other	O
-linker	O
-residues	O
-are	O
-coloured	O
-either	O
-dark	O
-blue	O
-for	O
-new	O
-residues	O
-in	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structure	O
-or	O
-light	O
-blue	O
-for	O
-the	O
-remaining	O
-inter	O
--	O
-RRM	O
-residues	O
-.	O
-	O
-The	O
-central	O
-panel	O
-shows	O
-an	O
-overall	O
-view	O
-with	O
-stick	O
-diagrams	O
-for	O
-mutated	O
-residues	O
-;	O
-boxed	O
-regions	O
-are	O
-expanded	O
-to	O
-show	O
-the	O
-C	O
--	O
-terminal	O
-(	O
-bottom	O
-left	O
-)	O
-and	O
-central	O
-linker	O
-regions	O
-(	O
-top	O
-)	O
-at	O
-the	O
-inter	O
--	O
-RRM	O
-interfaces	O
-,	O
-and	O
-N	O
--	O
-terminal	O
-linker	O
-region	O
-contacts	O
-with	O
-RRM1	O
-(	O
-bottom	O
-right	O
-).	O
-	O
-(	O
-b	O
-)	O
-Bar	O
-graph	O
-of	O
-apparent	O
-equilibrium	O
-affinities	O
-(	O
-KA	O
-)	O
-for	O
-the	O
-AdML	O
-Py	O
-tract	O
-(	O
-5	O
-′-	O
-CCCUUUUUUUUCC	O
--	O
-3	O
-′)	O
-of	O
-the	O
-wild	O
--	O
-type	O
-(	O
-blue	O
-)	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-protein	O
-compared	O
-with	O
-mutations	O
-of	O
-the	O
-residues	O
-shown	O
-in	O
-a	O
-:	O
-3Gly	B-mutant
-(	O
-yellow	O
-),	O
-5Gly	B-mutant
-(	O
-red	O
-),	O
-NLALA	B-mutant
-(	O
-hatched	O
-red	O
-),	O
-12Gly	B-mutant
-(	O
-orange	O
-)	O
-and	O
-the	O
-linker	O
-deletions	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-in	O
-the	O
-minimal	O
-RRM1	O
-–	O
-RRM2	O
-region	O
-(	O
-residues	O
-148	O
-–	O
-237	O
-,	O
-258	O
-–	O
-336	O
-)	O
-or	O
-dU2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-(	O
-residues	O
-141	O
-–	O
-237	O
-,	O
-258	O
-–	O
-342	O
-).	O
-	O
-The	O
-apparent	O
-equilibrium	O
-dissociation	O
-constants	O
-(	O
-KD	O
-)	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-mutant	O
-proteins	O
-are	O
-:	O
-wild	O
-type	O
-(	O
-WT	O
-),	O
-35	O
-±	O
-6	O
-nM	O
-;	O
-3Gly	B-mutant
-,	O
-47	O
-±	O
-4	O
-nM	O
-;	O
-5Gly	B-mutant
-,	O
-61	O
-±	O
-3	O
-nM	O
-;	O
-12Gly	B-mutant
-,	O
-88	O
-±	O
-21	O
-nM	O
-;	O
-NLALA	B-mutant
-,	O
-45	O
-±	O
-3	O
-nM	O
-;	O
-dU2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-123	O
-±	O
-5	O
-nM	O
-;	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-,	O
-5000	O
-±	O
-100	O
-nM	O
-;	O
-3Mut	B-mutant
-,	O
-5630	O
-±	O
-70	O
-nM	O
-.	O
-The	O
-average	O
-KA	O
-and	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.	O
-for	O
-three	O
-independent	O
-titrations	O
-are	O
-plotted	O
-.	O
-	O
-The	O
-fitted	O
-fluorescence	O
-anisotropy	O
-RNA	O
--	O
-binding	O
-curves	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-4d	O
-–	O
-j	O
-.	O
-(	O
-c	O
-)	O
-Close	O
-view	O
-of	O
-the	O
-U2AF65	O
-RRM1	O
-/	O
-RRM2	O
-interface	O
-following	O
-a	O
-two	O
--	O
-fold	O
-rotation	O
-about	O
-the	O
-x	O
--	O
-axis	O
-relative	O
-to	O
-a	O
-.	O
-	O
-U2AF65	O
-inter	O
--	O
-domain	O
-residues	O
-are	O
-important	O
-for	O
-splicing	O
-a	O
-representative	O
-pre	O
--	O
-mRNA	O
-substrate	O
-in	O
-human	O
-cells	O
-.	O
-	O
-(	O
-a	O
-)	O
-Schematic	O
-diagram	O
-of	O
-the	O
-pyPY	O
-reporter	O
-minigene	O
-construct	O
-comprising	O
-two	O
-alternative	O
-splice	O
-sites	O
-preceded	O
-by	O
-either	O
-the	O
-weak	O
-IgM	O
-Py	O
-tract	O
-(	O
-py	O
-)	O
-or	O
-the	O
-strong	O
-AdML	O
-Py	O
-tract	O
-(	O
-PY	O
-)	O
-(	O
-sequences	O
-inset	O
-).	O
-	O
-(	O
-b	O
-)	O
-Representative	O
-RT	O
--	O
-PCR	O
-of	O
-pyPY	O
-transcripts	O
-from	O
-HEK293T	O
-cells	O
-co	O
--	O
-transfected	O
-with	O
-constructs	O
-encoding	O
-the	O
-pyPY	O
-minigene	O
-and	O
-either	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-U2AF65	O
-or	O
-a	O
-triple	O
-U2AF65	O
-mutant	O
-(	O
-3Mut	B-mutant
-)	O
-of	O
-Q147A	B-mutant
-,	O
-R227A	B-mutant
-and	O
-V254P	B-mutant
-residues	O
-.	O
-(	O
-c	O
-)	O
-A	O
-bar	O
-graph	O
-of	O
-the	O
-average	O
-percentage	O
-of	O
-the	O
-py	O
--	O
-spliced	O
-mRNA	O
-relative	O
-to	O
-total	O
-detected	O
-pyPY	O
-transcripts	O
-(	O
-spliced	O
-and	O
-unspliced	O
-)	O
-for	O
-the	O
-corresponding	O
-gel	O
-lanes	O
-(	O
-black	O
-,	O
-no	O
-U2AF65	O
-added	O
-;	O
-white	O
-,	O
-WT	O
-U2AF65	O
-;	O
-grey	O
-,	O
-3Mut	B-mutant
-U2AF65	O
-).	O
-	O
-Protein	O
-overexpression	O
-and	O
-qRT	O
--	O
-PCR	O
-results	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-5	O
-.	O
-	O
-RNA	O
-binding	O
-stabilizes	O
-the	O
-side	O
--	O
-by	O
--	O
-side	O
-conformation	O
-of	O
-U2AF65	O
-RRMs	O
-.	O
-	O
-(	O
-a	O
-,	O
-b	O
-)	O
-Views	O
-of	O
-FRET	O
-pairs	O
-chosen	O
-to	O
-follow	O
-the	O
-relative	O
-movement	O
-of	O
-RRM1	O
-and	O
-RRM2	O
-on	O
-the	O
-crystal	O
-structure	O
-of	O
-‘	O
-side	O
--	O
-by	O
--	O
-side	O
-'	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-RRMs	O
-bound	O
-to	O
-a	O
-Py	O
--	O
-tract	O
-oligonucleotide	O
-(	O
-a	O
-,	O
-representative	O
-structure	O
-iv	O
-)	O
-or	O
-‘	O
-closed	O
-'	O
-NMR	O
-/	O
-PRE	O
--	O
-based	O
-model	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-(	O
-b	O
-,	O
-PDB	O
-ID	O
-2YH0	O
-)	O
-in	O
-identical	O
-orientations	O
-of	O
-RRM2	O
-.	O
-	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-proteins	O
-were	O
-doubly	O
-labelled	O
-at	O
-A181C	B-mutant
-/	O
-Q324C	B-mutant
-such	O
-that	O
-a	O
-mixture	O
-of	O
-Cy3	O
-/	O
-Cy5	O
-fluorophores	O
-are	O
-expected	O
-to	O
-be	O
-present	O
-at	O
-each	O
-site	O
-.	O
-	O
-(	O
-c	O
-–	O
-f	O
-,	O
-i	O
-,	O
-j	O
-)	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-protein	O
-was	O
-immobilized	O
-on	O
-the	O
-microscope	O
-slide	O
-via	O
-biotin	O
--	O
-NTA	O
-/	O
-Ni	O
-+	O
-2	O
-(	O
-orange	O
-line	O
-)	O
-on	O
-a	O
-neutravidin	O
-(	O
-black	O
-X	O
-)-	O
-biotin	O
--	O
-PEG	O
-(	O
-orange	O
-triangle	O
-)-	O
-treated	O
-surface	O
-and	O
-imaged	O
-either	O
-in	O
-the	O
-absence	O
-of	O
-ligands	O
-(	O
-c	O
-,	O
-d	O
-),	O
-in	O
-the	O
-presence	O
-of	O
-5	O
-μM	O
-AdML	O
-Py	O
--	O
-tract	O
-RNA	O
-(	O
-5	O
-′-	O
-CCUUUUUUUUCC	O
--	O
-3	O
-′)	O
-(	O
-e	O
-,	O
-f	O
-),	O
-or	O
-in	O
-the	O
-presence	O
-of	O
-10	O
-μM	O
-adenosine	O
--	O
-interrupted	O
-variant	O
-RNA	O
-(	O
-5	O
-′-	O
-CUUUUUAAUUUCCA	O
--	O
-3	O
-′)	O
-(	O
-i	O
-,	O
-j	O
-).	O
-	O
-The	O
-untethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-protein	O
-(	O
-1	O
-nM	O
-)	O
-was	O
-added	O
-to	O
-AdML	O
-RNA	O
-–	O
-polyethylene	O
--	O
-glycol	O
--	O
-linker	O
-–	O
-DNA	O
-oligonucleotide	O
-(	O
-10	O
-nM	O
-),	O
-which	O
-was	O
-immobilized	O
-on	O
-the	O
-microscope	O
-slide	O
-by	O
-annealing	O
-with	O
-a	O
-complementary	O
-biotinyl	O
--	O
-DNA	O
-oligonucleotide	O
-(	O
-black	O
-vertical	O
-line	O
-).	O
-	O
-Typical	O
-single	O
--	O
-molecule	O
-FRET	O
-traces	O
-(	O
-c	O
-,	O
-e	O
-,	O
-g	O
-,	O
-i	O
-)	O
-show	O
-fluorescence	O
-intensities	O
-from	O
-Cy3	O
-(	O
-green	O
-)	O
-and	O
-Cy5	O
-(	O
-red	O
-)	O
-and	O
-the	O
-calculated	O
-apparent	O
-FRET	O
-efficiency	O
-(	O
-blue	O
-).	O
-	O
-Additional	O
-traces	O
-for	O
-untethered	O
-,	O
-RNA	O
--	O
-bound	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-7c	O
-,	O
-d	O
-.	O
-Histograms	O
-(	O
-d	O
-,	O
-f	O
-,	O
-h	O
-,	O
-j	O
-)	O
-show	O
-the	O
-distribution	O
-of	O
-FRET	O
-values	O
-in	O
-RNA	O
--	O
-free	O
-,	O
-slide	O
--	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-d	O
-);	O
-AdML	O
-RNA	O
--	O
-bound	O
-,	O
-slide	O
--	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-f	O
-);	O
-AdML	O
-RNA	O
--	O
-bound	O
-,	O
-untethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-h	O
-)	O
-and	O
-adenosine	O
--	O
-interrupted	O
-RNA	O
--	O
-bound	O
-,	O
-slide	O
--	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-j	O
-).	O
-	O
-N	O
-is	O
-the	O
-number	O
-of	O
-single	O
--	O
-molecule	O
-traces	O
-compiled	O
-for	O
-each	O
-histogram	O
-.	O
-	O
-Schematic	O
-models	O
-of	O
-U2AF65	O
-recognizing	O
-the	O
-Py	O
-tract	O
-.	O
-	O
-(	O
-a	O
-)	O
-Diagram	O
-of	O
-the	O
-U2AF65	O
-,	O
-SF1	O
-and	O
-U2AF35	O
-splicing	O
-factors	O
-bound	O
-to	O
-the	O
-consensus	O
-elements	O
-of	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-.	O
-	O
-A	O
-surface	O
-representation	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-is	O
-shown	O
-bound	O
-to	O
-nine	O
-nucleotides	O
-(	O
-nt	O
-);	O
-the	O
-relative	O
-distances	O
-and	O
-juxtaposition	O
-of	O
-the	O
-branch	O
-point	O
-sequence	O
-(	O
-BPS	O
-)	O
-and	O
-consensus	O
-AG	O
-dinucleotide	O
-at	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-are	O
-unknown	O
-.	O
-	O
-MDS	O
--	O
-relevant	O
-mutated	O
-residues	O
-of	O
-U2AF65	O
-are	O
-shown	O
-as	O
-yellow	O
-spheres	O
-(	O
-L187	O
-and	O
-M144	O
-).	O
-	O
-(	O
-b	O
-)	O
-Following	O
-binding	O
-to	O
-the	O
-Py	O
--	O
-tract	O
-RNA	O
-,	O
-a	O
-conformation	O
-corresponding	O
-to	O
-high	O
-FRET	O
-and	O
-consistent	O
-with	O
-the	O
-‘	O
-closed	O
-',	O
-back	O
--	O
-to	O
--	O
-back	O
-apo	O
--	O
-U2AF65	O
-model	O
-resulting	O
-from	O
-PRE	O
-/	O
-NMR	O
-characterization	O
-(	O
-PDB	O
-ID	O
-2YH0	O
-)	O
-often	O
-transitions	O
-to	O
-a	O
-conformation	O
-corresponding	O
-to	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-,	O
-which	O
-is	O
-consistent	O
-with	O
-‘	O
-open	O
-',	O
-side	O
--	O
-by	O
--	O
-side	O
-RRMs	O
-such	O
-as	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-crystal	O
-structures	O
-.	O
-	O
-Alternatively	O
-,	O
-a	O
-conformation	O
-of	O
-U2AF65	O
-corresponding	O
-to	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-can	O
-directly	O
-bind	O
-to	O
-RNA	O
-;	O
-RNA	O
-binding	O
-stabilizes	O
-the	O
-‘	O
-open	O
-',	O
-side	O
--	O
-by	O
--	O
-side	O
-conformation	O
-and	O
-thus	O
-shifts	O
-the	O
-U2AF65	O
-population	O
-towards	O
-the	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-.	O
-	O
-RRM1	O
-,	O
-green	O
-;	O
-RRM2	O
-,	O
-pale	O
-blue	O
-;	O
-RRM	O
-extensions	O
-/	O
-linker	O
-,	O
-blue	O
-.	O
-	O
-RNA	O
-protects	O
-a	O
-nucleoprotein	O
-complex	O
-against	O
-radiation	O
-damage	O
-	O
-Systematic	O
-analysis	O
-of	O
-radiation	O
-damage	O
-within	O
-a	O
-protein	O
-–	O
-RNA	O
-complex	O
-over	O
-a	O
-large	O
-dose	O
-range	O
-(	O
-1	O
-.	O
-3	O
-–	O
-25	O
-MGy	O
-)	O
-reveals	O
-significant	O
-differential	O
-susceptibility	O
-of	O
-RNA	O
-and	O
-protein	O
-.	O
-	O
-A	O
-new	O
-method	O
-of	O
-difference	O
-electron	O
--	O
-density	O
-quantification	O
-is	O
-presented	O
-.	O
-	O
-Radiation	O
-damage	O
-during	O
-macromolecular	O
-X	O
--	O
-ray	O
-crystallographic	O
-data	O
-collection	O
-is	O
-still	O
-the	O
-main	O
-impediment	O
-for	O
-many	O
-macromolecular	O
-structure	O
-determinations	O
-.	O
-	O
-Although	O
-this	O
-has	O
-been	O
-well	O
-characterized	O
-within	O
-protein	O
-crystals	O
-,	O
-far	O
-less	O
-is	O
-known	O
-about	O
-specific	O
-damage	O
-effects	O
-within	O
-the	O
-larger	O
-class	O
-of	O
-nucleoprotein	O
-complexes	O
-.	O
-	O
-Here	O
-,	O
-a	O
-methodology	O
-has	O
-been	O
-developed	O
-whereby	O
-per	O
--	O
-atom	O
-density	O
-changes	O
-could	O
-be	O
-quantified	O
-with	O
-increasing	O
-dose	O
-over	O
-a	O
-wide	O
-(	O
-1	O
-.	O
-3	O
-–	O
-25	O
-.	O
-0	O
-MGy	O
-)	O
-range	O
-and	O
-at	O
-higher	O
-resolution	O
-(	O
-1	O
-.	O
-98	O
-Å	O
-)	O
-than	O
-the	O
-previous	O
-systematic	O
-specific	O
-damage	O
-study	O
-on	O
-a	O
-protein	O
-–	O
-DNA	O
-complex	O
-.	O
-	O
-Specific	O
-damage	O
-manifestations	O
-were	O
-determined	O
-within	O
-the	O
-large	O
-trp	O
-RNA	O
--	O
-binding	O
-attenuation	O
-protein	O
-(	O
-TRAP	O
-)	O
-bound	O
-to	O
-a	O
-single	O
--	O
-stranded	O
-RNA	O
-that	O
-forms	O
-a	O
-belt	O
-around	O
-the	O
-protein	O
-.	O
-	O
-Over	O
-a	O
-large	O
-dose	O
-range	O
-,	O
-the	O
-RNA	O
-was	O
-found	O
-to	O
-be	O
-far	O
-less	O
-susceptible	O
-to	O
-radiation	O
--	O
-induced	O
-chemical	O
-changes	O
-than	O
-the	O
-protein	O
-.	O
-	O
-The	O
-availability	O
-of	O
-two	O
-TRAP	O
-molecules	O
-in	O
-the	O
-asymmetric	O
-unit	O
-,	O
-of	O
-which	O
-only	O
-one	O
-contained	O
-bound	O
-RNA	O
-,	O
-allowed	O
-a	O
-controlled	O
-investigation	O
-into	O
-the	O
-exact	O
-role	O
-of	O
-RNA	O
-binding	O
-in	O
-protein	O
-specific	O
-damage	O
-susceptibility	O
-.	O
-	O
-The	O
-11	O
--	O
-fold	O
-symmetry	O
-within	O
-each	O
-TRAP	O
-ring	O
-permitted	O
-statistically	O
-significant	O
-analysis	O
-of	O
-the	O
-Glu	O
-and	O
-Asp	O
-damage	O
-patterns	O
-,	O
-with	O
-RNA	O
-binding	O
-unexpectedly	O
-being	O
-observed	O
-to	O
-protect	O
-these	O
-otherwise	O
-highly	O
-sensitive	O
-residues	O
-within	O
-the	O
-11	O
-RNA	O
--	O
-binding	O
-pockets	O
-distributed	O
-around	O
-the	O
-outside	O
-of	O
-the	O
-protein	O
-molecule	O
-.	O
-	O
-Additionally	O
-,	O
-the	O
-method	O
-enabled	O
-a	O
-quantification	O
-of	O
-the	O
-reduction	O
-in	O
-radiation	O
--	O
-induced	O
-Lys	O
-and	O
-Phe	O
-disordering	O
-upon	O
-RNA	O
-binding	O
-directly	O
-from	O
-the	O
-electron	O
-density	O
-.	O
-	O
-With	O
-the	O
-wide	O
-use	O
-of	O
-high	O
--	O
-flux	O
-third	O
--	O
-generation	O
-synchrotron	O
-sources	O
-,	O
-radiation	O
-damage	O
-(	O
-RD	O
-)	O
-has	O
-once	O
-again	O
-become	O
-a	O
-dominant	O
-reason	O
-for	O
-the	O
-failure	O
-of	O
-structure	O
-determination	O
-using	O
-macromolecular	O
-crystallography	O
-(	O
-MX	O
-)	O
-in	O
-experiments	O
-conducted	O
-both	O
-at	O
-room	O
-temperature	O
-and	O
-under	O
-cryocooled	O
-conditions	O
-(	O
-100	O
-K	O
-).	O
-	O
-Significant	O
-progress	O
-has	O
-been	O
-made	O
-in	O
-recent	O
-years	O
-in	O
-understanding	O
-the	O
-inevitable	O
-manifestations	O
-of	O
-X	O
--	O
-ray	O
--	O
-induced	O
-RD	O
-within	O
-protein	O
-crystals	O
-,	O
-and	O
-there	O
-is	O
-now	O
-a	O
-body	O
-of	O
-literature	O
-on	O
-possible	O
-strategies	O
-to	O
-mitigate	O
-the	O
-effects	O
-of	O
-RD	O
-(	O
-e	O
-.	O
-g	O
-.	O
-Zeldin	O
-,	O
-Brockhauser	O
-et	O
-al	O
-.,	O
-2013	O
-;	O
-Bourenkov	O
-&	O
-Popov	O
-,	O
-2010	O
-).	O
-	O
-However	O
-,	O
-there	O
-is	O
-still	O
-no	O
-general	O
-consensus	O
-within	O
-the	O
-field	O
-on	O
-how	O
-to	O
-minimize	O
-RD	O
-during	O
-MX	O
-data	O
-collection	O
-,	O
-and	O
-debates	O
-on	O
-the	O
-dependence	O
-of	O
-RD	O
-progression	O
-on	O
-incident	O
-X	O
--	O
-ray	O
-energy	O
-(	O
-Shimizu	O
-et	O
-al	O
-.,	O
-2007	O
-;	O
-Liebschner	O
-et	O
-al	O
-.,	O
-2015	O
-)	O
-and	O
-the	O
-efficacy	O
-of	O
-radical	O
-scavengers	O
-(	O
-Allan	O
-et	O
-al	O
-.,	O
-2013	O
-)	O
-have	O
-yet	O
-to	O
-be	O
-resolved	O
-.	O
-	O
-Global	O
-radiation	O
-damage	O
-is	O
-observed	O
-within	O
-reciprocal	O
-space	O
-as	O
-the	O
-overall	O
-decay	O
-of	O
-the	O
-summed	O
-intensity	O
-of	O
-reflections	O
-detected	O
-within	O
-the	O
-diffraction	O
-pattern	O
-as	O
-dose	O
-increases	O
-(	O
-Garman	O
-,	O
-2010	O
-;	O
-Murray	O
-&	O
-Garman	O
-,	O
-2002	O
-).	O
-	O
-Dose	O
-is	O
-defined	O
-as	O
-the	O
-absorbed	O
-energy	O
-per	O
-unit	O
-mass	O
-of	O
-crystal	O
-in	O
-grays	O
-(	O
-Gy	O
-;	O
-1	O
-Gy	O
-=	O
-1	O
-J	O
-kg	O
-−	O
-1	O
-),	O
-and	O
-is	O
-the	O
-metric	O
-against	O
-which	O
-damage	O
-progression	O
-should	O
-be	O
-monitored	O
-during	O
-MX	O
-data	O
-collection	O
-,	O
-as	O
-opposed	O
-to	O
-time	O
-.	O
-	O
-At	O
-100	O
-K	O
-,	O
-an	O
-experimental	O
-dose	O
-limit	O
-of	O
-30	O
-MGy	O
-has	O
-been	O
-recommended	O
-as	O
-an	O
-upper	O
-limit	O
-beyond	O
-which	O
-the	O
-biological	O
-information	O
-derived	O
-from	O
-any	O
-macromolecular	O
-crystal	O
-may	O
-be	O
-compromised	O
-(	O
-Owen	O
-et	O
-al	O
-.,	O
-2006	O
-).	O
-	O
-Specific	O
-radiation	O
-damage	O
-(	O
-SRD	O
-)	O
-is	O
-observed	O
-in	O
-the	O
-real	O
--	O
-space	O
-electron	O
-density	O
-,	O
-and	O
-has	O
-been	O
-detected	O
-at	O
-much	O
-lower	O
-doses	O
-than	O
-any	O
-observable	O
-decay	O
-in	O
-the	O
-intensity	O
-of	O
-reflections	O
-.	O
-	O
-Indeed	O
-,	O
-the	O
-C	O
-—	O
-Se	O
-bond	O
-in	O
-selenomethionine	O
-,	O
-the	O
-stability	O
-of	O
-which	O
-is	O
-key	O
-for	O
-the	O
-success	O
-of	O
-experimental	O
-phasing	O
-methods	O
-,	O
-can	O
-be	O
-cleaved	O
-at	O
-a	O
-dose	O
-as	O
-low	O
-as	O
-2	O
-MGy	O
-for	O
-a	O
-crystal	O
-maintained	O
-at	O
-100	O
-K	O
-(	O
-Holton	O
-,	O
-2007	O
-).	O
-	O
-SRD	O
-has	O
-been	O
-well	O
-characterized	O
-in	O
-a	O
-large	O
-range	O
-of	O
-proteins	O
-,	O
-and	O
-is	O
-seen	O
-to	O
-follow	O
-a	O
-reproducible	O
-order	O
-:	O
-metallo	O
--	O
-centre	O
-reduction	O
-,	O
-disulfide	O
--	O
-bond	O
-cleavage	O
-,	O
-acidic	O
-residue	O
-decarboxylation	O
-and	O
-methionine	O
-methylthio	O
-cleavage	O
-(	O
-Ravelli	O
-&	O
-McSweeney	O
-,	O
-2000	O
-;	O
-Burmeister	O
-,	O
-2000	O
-;	O
-Weik	O
-et	O
-al	O
-.,	O
-2000	O
-;	O
-Yano	O
-et	O
-al	O
-.,	O
-2005	O
-).	O
-	O
-There	O
-are	O
-a	O
-number	O
-of	O
-cases	O
-where	O
-SRD	O
-manifestations	O
-have	O
-compromised	O
-the	O
-biological	O
-information	O
-extracted	O
-from	O
-MX	O
--	O
-determined	O
-structures	O
-at	O
-much	O
-lower	O
-doses	O
-than	O
-the	O
-recommended	O
-30	O
-MGy	O
-limit	O
-,	O
-leading	O
-to	O
-false	O
-structural	O
-interpretations	O
-of	O
-protein	O
-mechanisms	O
-.	O
-	O
-Active	O
--	O
-site	O
-residues	O
-appear	O
-to	O
-be	O
-particularly	O
-susceptible	O
-,	O
-particularly	O
-for	O
-photosensitive	O
-proteins	O
-and	O
-in	O
-instances	O
-where	O
-chemical	O
-strain	O
-is	O
-an	O
-intrinsic	O
-feature	O
-of	O
-the	O
-reaction	O
-mechanism	O
-.	O
-	O
-For	O
-instance	O
-,	O
-structure	O
-determination	O
-of	O
-the	O
-purple	O
-membrane	O
-protein	O
-bacterio	O
-­	O
-rhodopsin	O
-required	O
-careful	O
-corrections	O
-for	O
-radiation	O
--	O
-induced	O
-structural	O
-changes	O
-before	O
-the	O
-correct	O
-photosensitive	O
-intermediate	O
-states	O
-could	O
-be	O
-isolated	O
-(	O
-Matsui	O
-et	O
-al	O
-.,	O
-2002	O
-).	O
-	O
-The	O
-significant	O
-chemical	O
-strain	O
-required	O
-for	O
-catalysis	O
-within	O
-the	O
-active	O
-site	O
-of	O
-phosphoserine	O
-aminotransferase	O
-has	O
-been	O
-observed	O
-to	O
-diminish	O
-during	O
-X	O
--	O
-ray	O
-exposure	O
-(	O
-Dubnovitsky	O
-et	O
-al	O
-.,	O
-2005	O
-).	O
-	O
-Since	O
-the	O
-majority	O
-of	O
-SRD	O
-studies	O
-to	O
-date	O
-have	O
-focused	O
-on	O
-proteins	O
-,	O
-much	O
-less	O
-is	O
-known	O
-about	O
-the	O
-effects	O
-of	O
-X	O
--	O
-ray	O
-irradiation	O
-on	O
-the	O
-wider	O
-class	O
-of	O
-crystalline	O
-nucleoprotein	O
-complexes	O
-or	O
-how	O
-to	O
-correct	O
-for	O
-such	O
-radiation	O
--	O
-induced	O
-structural	O
-changes	O
-.	O
-	O
-Understanding	O
-RD	O
-to	O
-such	O
-complexes	O
-is	O
-crucial	O
-,	O
-since	O
-DNA	O
-is	O
-rarely	O
-naked	O
-within	O
-a	O
-cell	O
-,	O
-instead	O
-dynamically	O
-interacting	O
-with	O
-proteins	O
-,	O
-facilitating	O
-replication	O
-,	O
-transcription	O
-,	O
-modification	O
-and	O
-DNA	O
-repair	O
-.	O
-	O
-As	O
-of	O
-early	O
-2016	O
-,	O
->	O
-5400	O
-nucleoprotein	O
-complex	O
-structures	O
-have	O
-been	O
-deposited	O
-within	O
-the	O
-PDB	O
-,	O
-with	O
-91	O
-%	O
-solved	O
-by	O
-MX	O
-.	O
-	O
-It	O
-is	O
-essential	O
-to	O
-understand	O
-how	O
-these	O
-increasingly	O
-complex	O
-macromolecular	O
-structures	O
-are	O
-affected	O
-by	O
-the	O
-radiation	O
-used	O
-to	O
-solve	O
-them	O
-.	O
-	O
-Nucleoproteins	O
-also	O
-represent	O
-one	O
-of	O
-the	O
-main	O
-targets	O
-of	O
-radiotherapy	O
-,	O
-and	O
-an	O
-insight	O
-into	O
-the	O
-damage	O
-mechanisms	O
-induced	O
-by	O
-X	O
--	O
-ray	O
-irradiation	O
-could	O
-inform	O
-innovative	O
-treatments	O
-.	O
-	O
-Investigations	O
-on	O
-sub	O
--	O
-ionization	O
--	O
-level	O
-LEEs	O
-(	O
-0	O
-–	O
-15	O
-eV	O
-)	O
-interacting	O
-with	O
-both	O
-dried	O
-and	O
-aqueous	O
-oligonucleotides	O
-(	O
-Alizadeh	O
-&	O
-Sanche	O
-,	O
-2014	O
-;	O
-Simons	O
-,	O
-2006	O
-)	O
-concluded	O
-that	O
-resonant	O
-electron	O
-attachment	O
-to	O
-DNA	O
-bases	O
-and	O
-the	O
-sugar	O
--	O
-phosphate	O
-backbone	O
-could	O
-lead	O
-to	O
-the	O
-preferential	O
-cleavage	O
-of	O
-strong	O
-(∼	O
-4	O
-eV	O
-,	O
-385	O
-kJ	O
-mol	O
-−	O
-1	O
-)	O
-sugar	O
--	O
-phosphate	O
-C	O
-—	O
-O	O
-covalent	O
-bonds	O
-within	O
-the	O
-DNA	O
-backbone	O
-and	O
-then	O
-base	O
--	O
-sugar	O
-N1	O
-—	O
-C	O
-bonds	O
-,	O
-eventually	O
-leading	O
-to	O
-single	O
--	O
-strand	O
-breakages	O
-(	O
-SSBs	O
-;	O
-Ptasińska	O
-&	O
-Sanche	O
-,	O
-2007	O
-).	O
-	O
-Electrons	O
-have	O
-been	O
-shown	O
-to	O
-be	O
-mobile	O
-at	O
-77	O
-K	O
-by	O
-electron	O
-spin	O
-resonance	O
-spectroscopy	O
-studies	O
-(	O
-Symons	O
-,	O
-1997	O
-;	O
-Jones	O
-et	O
-al	O
-.,	O
-1987	O
-),	O
-with	O
-rapid	O
-electron	O
-quantum	O
-tunnelling	O
-and	O
-positive	O
-hole	O
-migration	O
-along	O
-the	O
-protein	O
-backbone	O
-and	O
-through	O
-stacked	O
-DNA	O
-bases	O
-indicated	O
-as	O
-a	O
-dominant	O
-mechanism	O
-by	O
-which	O
-oxidative	O
-and	O
-reductive	O
-damage	O
-localizes	O
-at	O
-distances	O
-from	O
-initial	O
-ionization	O
-sites	O
-at	O
-100	O
-K	O
-(	O
-O	O
-’	O
-Neill	O
-et	O
-al	O
-.,	O
-2002	O
-).	O
-	O
-The	O
-investigation	O
-of	O
-naturally	O
-forming	O
-nucleoprotein	O
-complexes	O
-circumvents	O
-the	O
-inherent	O
-challenges	O
-in	O
-making	O
-controlled	O
-comparisons	O
-of	O
-damage	O
-mechanisms	O
-between	O
-protein	O
-and	O
-nucleic	O
-acids	O
-crystallized	O
-separately	O
-.	O
-Recently	O
-,	O
-for	O
-a	O
-well	O
-characterized	O
-bacterial	O
-protein	O
-–	O
-DNA	O
-complex	O
-(	O
-C	O
-.	O
-Esp1396I	O
-;	O
-PDB	O
-entry	O
-3clc	O
-;	O
-resolution	O
-2	O
-.	O
-8	O
-Å	O
-;	O
-McGeehan	O
-et	O
-al	O
-.,	O
-2008	O
-)	O
-it	O
-was	O
-concluded	O
-that	O
-over	O
-a	O
-wide	O
-dose	O
-range	O
-(	O
-2	O
-.	O
-1	O
-–	O
-44	O
-.	O
-6	O
-MGy	O
-)	O
-the	O
-protein	O
-was	O
-far	O
-more	O
-susceptible	O
-to	O
-SRD	O
-than	O
-the	O
-DNA	O
-within	O
-the	O
-crystal	O
-(	O
-Bury	O
-et	O
-al	O
-.,	O
-2015	O
-).	O
-	O
-Only	O
-at	O
-doses	O
-above	O
-20	O
-MGy	O
-were	O
-precursors	O
-of	O
-phosphodiester	O
--	O
-bond	O
-cleavage	O
-observed	O
-within	O
-AT	O
--	O
-rich	O
-regions	O
-of	O
-the	O
-35	O
--	O
-mer	O
-DNA	O
-.	O
-	O
-For	O
-crystalline	O
-complexes	O
-such	O
-as	O
-C	O
-.	O
-Esp1396I	O
-,	O
-whether	O
-the	O
-protein	O
-is	O
-intrinsically	O
-more	O
-susceptible	O
-to	O
-X	O
--	O
-ray	O
--	O
-induced	O
-damage	O
-or	O
-whether	O
-the	O
-protein	O
-scavenges	O
-electrons	O
-to	O
-protect	O
-the	O
-DNA	O
-remains	O
-unclear	O
-in	O
-the	O
-absence	O
-of	O
-a	O
-non	O
--	O
-nucleic	O
-acid	O
--	O
-bound	O
-protein	O
-control	O
-obtained	O
-under	O
-exactly	O
-the	O
-same	O
-crystallization	O
-and	O
-data	O
--	O
-collection	O
-conditions	O
-.	O
-	O
-To	O
-monitor	O
-the	O
-effects	O
-of	O
-nucleic	O
-acid	O
-binding	O
-on	O
-protein	O
-damage	O
-susceptibility	O
-,	O
-a	O
-crystal	O
-containing	O
-two	O
-protein	O
-molecules	O
-per	O
-asymmetric	O
-unit	O
-,	O
-only	O
-one	O
-of	O
-which	O
-was	O
-bound	O
-to	O
-RNA	O
-,	O
-is	O
-reported	O
-here	O
-(	O
-Fig	O
-.	O
-1	O
-▸).	O
-	O
-Using	O
-newly	O
-developed	O
-methodology	O
-,	O
-we	O
-present	O
-a	O
-controlled	O
-SRD	O
-investigation	O
-at	O
-1	O
-.	O
-98	O
-Å	O
-resolution	O
-using	O
-a	O
-large	O
-(∼	O
-91	O
-kDa	O
-)	O
-crystalline	O
-protein	O
-–	O
-RNA	O
-complex	O
-:	O
-trp	O
-RNA	O
--	O
-binding	O
-attenuation	O
-protein	O
-(	O
-TRAP	O
-)	O
-bound	O
-to	O
-a	O
-53	O
-bp	O
-RNA	O
-sequence	O
-(	O
-GAGUU	O
-)	O
-10GAG	O
-(	O
-PDB	O
-entry	O
-1gtf	O
-;	O
-Hopcroft	O
-et	O
-al	O
-.,	O
-2002	O
-).	O
-	O
-TRAP	O
-consists	O
-of	O
-11	O
-identical	O
-subunits	O
-assembled	O
-into	O
-a	O
-ring	O
-with	O
-11	O
--	O
-fold	O
-rotational	O
-symmetry	O
-.	O
-	O
-It	O
-binds	O
-with	O
-high	O
-affinity	O
-(	O
-K	O
-d	O
-≃	O
-1	O
-.	O
-0	O
-nM	O
-)	O
-to	O
-RNA	O
-segments	O
-containing	O
-11	O
-GAG	O
-/	O
-UAG	O
-triplets	O
-separated	O
-by	O
-two	O
-or	O
-three	O
-spacer	O
-nucleotides	O
-(	O
-Elliott	O
-et	O
-al	O
-.,	O
-2001	O
-)	O
-to	O
-regulate	O
-the	O
-transcription	O
-of	O
-tryptophan	O
-biosynthetic	O
-genes	O
-in	O
-Bacillus	O
-subtilis	O
-(	O
-Antson	O
-et	O
-al	O
-.,	O
-1999	O
-).	O
-	O
-In	O
-this	O
-structure	O
-,	O
-the	O
-bases	O
-of	O
-the	O
-G1	O
--	O
-A2	O
--	O
-G3	O
-nucleotides	O
-form	O
-direct	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-protein	O
-,	O
-unlike	O
-the	O
-U4	O
--	O
-U5	O
-nucleotides	O
-,	O
-which	O
-appear	O
-to	O
-be	O
-more	O
-flexible	O
-.	O
-	O
-Ten	O
-successive	O
-1	O
-.	O
-98	O
-Å	O
-resolution	O
-MX	O
-data	O
-sets	O
-were	O
-collected	O
-from	O
-the	O
-same	O
-TRAP	O
-–	O
-RNA	O
-crystal	O
-to	O
-analyse	O
-X	O
--	O
-ray	O
--	O
-induced	O
-structural	O
-changes	O
-over	O
-a	O
-large	O
-dose	O
-range	O
-(	O
-d	O
-1	O
-=	O
-1	O
-.	O
-3	O
-MGy	O
-to	O
-d	O
-10	O
-=	O
-25	O
-.	O
-0	O
-MGy	O
-).	O
-	O
-To	O
-avoid	O
-the	O
-previous	O
-necessity	O
-for	O
-visual	O
-inspection	O
-of	O
-electron	O
--	O
-density	O
-maps	O
-to	O
-detect	O
-SRD	O
-sites	O
-,	O
-a	O
-computational	O
-approach	O
-was	O
-designed	O
-to	O
-quantify	O
-the	O
-electron	O
--	O
-density	O
-change	O
-for	O
-each	O
-refined	O
-atom	O
-with	O
-increasing	O
-dose	O
-,	O
-thus	O
-providing	O
-a	O
-rapid	O
-systematic	O
-method	O
-for	O
-SRD	O
-study	O
-on	O
-such	O
-large	O
-multimeric	O
-complexes	O
-.	O
-	O
-By	O
-employing	O
-the	O
-high	O
-11	O
--	O
-fold	O
-structural	O
-symmetry	O
-within	O
-each	O
-TRAP	O
-macromolecule	O
-,	O
-this	O
-approach	O
-permitted	O
-a	O
-thorough	O
-statistical	O
-quantification	O
-of	O
-the	O
-RD	O
-effects	O
-of	O
-RNA	O
-binding	O
-to	O
-TRAP	O
-.	O
-	O
-Per	O
--	O
-atom	O
-quantification	O
-of	O
-electron	O
-density	O
-	O
-To	O
-quantify	O
-the	O
-exact	O
-effects	O
-of	O
-nucleic	O
-acid	O
-binding	O
-to	O
-a	O
-protein	O
-on	O
-SRD	O
-susceptibility	O
-,	O
-a	O
-high	O
--	O
-throughput	O
-and	O
-automated	O
-pipeline	O
-was	O
-created	O
-to	O
-systematically	O
-calculate	O
-the	O
-electron	O
--	O
-density	O
-change	O
-for	O
-every	O
-refined	O
-atom	O
-within	O
-the	O
-TRAP	O
-–	O
-RNA	O
-structure	O
-as	O
-a	O
-function	O
-of	O
-dose	O
-.	O
-	O
-This	O
-provides	O
-an	O
-atom	O
--	O
-specific	O
-quantification	O
-of	O
-density	O
-–	O
-dose	O
-dynamics	O
-,	O
-which	O
-was	O
-previously	O
-lacking	O
-within	O
-the	O
-field	O
-.	O
-	O
-Previous	O
-studies	O
-have	O
-characterized	O
-SRD	O
-sites	O
-by	O
-reporting	O
-magnitudes	O
-of	O
-F	O
-obs	O
-(	O
-d	O
-n	O
-)	O
-−	O
-F	O
-obs	O
-(	O
-d	O
-1	O
-)	O
-Fourier	O
-difference	O
-map	O
-peaks	O
-in	O
-terms	O
-of	O
-the	O
-sigma	O
-(	O
-σ	O
-)	O
-contour	O
-level	O
-(	O
-the	O
-number	O
-of	O
-standard	O
-deviations	O
-from	O
-the	O
-mean	O
-map	O
-electron	O
--	O
-density	O
-value	O
-)	O
-at	O
-which	O
-peaks	O
-become	O
-visible	O
-.	O
-	O
-However	O
-,	O
-these	O
-σ	O
-levels	O
-depend	O
-on	O
-the	O
-standard	O
-deviation	O
-values	O
-of	O
-the	O
-map	O
-,	O
-which	O
-can	O
-deviate	O
-between	O
-data	O
-sets	O
-,	O
-and	O
-are	O
-thus	O
-unsuitable	O
-for	O
-quantitative	O
-comparison	O
-of	O
-density	O
-between	O
-different	O
-dose	O
-data	O
-sets	O
-.	O
-	O
-Instead	O
-,	O
-we	O
-use	O
-here	O
-a	O
-maximum	O
-density	O
--	O
-loss	O
-metric	O
-(	O
-D	O
-loss	O
-),	O
-which	O
-is	O
-the	O
-per	O
--	O
-atom	O
-equivalent	O
-of	O
-the	O
-magnitude	O
-of	O
-these	O
-negative	O
-Fourier	O
-difference	O
-map	O
-peaks	O
-in	O
-units	O
-of	O
-e	O
-Å	O
-−	O
-3	O
-.	O
-	O
-Large	O
-positive	O
-D	O
-loss	O
-values	O
-indicate	O
-radiation	O
--	O
-induced	O
-atomic	O
-disordering	O
-reproducibly	O
-throughout	O
-the	O
-unit	O
-cells	O
-with	O
-respect	O
-to	O
-the	O
-initial	O
-low	O
--	O
-dose	O
-data	O
-set	O
-.	O
-	O
-For	O
-each	O
-TRAP	O
-–	O
-RNA	O
-data	O
-set	O
-,	O
-the	O
-D	O
-loss	O
-metric	O
-successfully	O
-identified	O
-the	O
-recognized	O
-forms	O
-of	O
-protein	O
-SRD	O
-(	O
-Fig	O
-.	O
-2	O
-▸	O
-a	O
-),	O
-with	O
-clear	O
-Glu	O
-and	O
-Asp	O
-side	O
--	O
-chain	O
-decarboxylation	O
-even	O
-in	O
-the	O
-first	O
-difference	O
-map	O
-calculated	O
-(	O
-3	O
-.	O
-9	O
-MGy	O
-;	O
-Fig	O
-.	O
-3	O
-▸	O
-a	O
-).	O
-	O
-The	O
-main	O
-sequence	O
-of	O
-TRAP	O
-does	O
-not	O
-contain	O
-any	O
-Trp	O
-and	O
-Cys	O
-residues	O
-(	O
-and	O
-thus	O
-contains	O
-no	O
-disulfide	O
-bonds	O
-).	O
-	O
-The	O
-substrate	O
-Trp	O
-amino	O
--	O
-acid	O
-ligands	O
-also	O
-exhibited	O
-disordering	O
-of	O
-the	O
-free	O
-terminal	O
-carboxyl	O
-groups	O
-at	O
-higher	O
-doses	O
-(	O
-Fig	O
-.	O
-2	O
-▸	O
-a	O
-);	O
-however	O
-,	O
-no	O
-clear	O
-Fourier	O
-difference	O
-peaks	O
-could	O
-be	O
-observed	O
-visually	O
-.	O
-	O
-Even	O
-for	O
-radiation	O
--	O
-insensitive	O
-residues	O
-(	O
-e	O
-.	O
-g	O
-.	O
-Gly	O
-)	O
-the	O
-average	O
-D	O
-loss	O
-increases	O
-with	O
-dose	O
-:	O
-this	O
-is	O
-the	O
-effect	O
-of	O
-global	O
-radiation	O
-damage	O
-,	O
-since	O
-as	O
-dose	O
-increases	O
-the	O
-electron	O
-density	O
-associated	O
-with	O
-each	O
-refined	O
-atom	O
-becomes	O
-weaker	O
-as	O
-the	O
-atomic	O
-occupancy	O
-decreases	O
-(	O
-Fig	O
-.	O
-2	O
-▸	O
-b	O
-).	O
-	O
-Only	O
-Glu	O
-and	O
-Asp	O
-residues	O
-exhibit	O
-a	O
-rate	O
-of	O
-D	O
-loss	O
-increase	O
-that	O
-consistently	O
-exceeds	O
-the	O
-average	O
-decay	O
-(	O
-Fig	O
-.	O
-2	O
-▸	O
-b	O
-,	O
-dashed	O
-line	O
-)	O
-at	O
-each	O
-dose	O
-.	O
-	O
-The	O
-rate	O
-of	O
-D	O
-loss	O
-(	O
-attributed	O
-to	O
-side	O
--	O
-chain	O
-decarboxylation	O
-)	O
-was	O
-consistently	O
-larger	O
-for	O
-Glu	O
-compared	O
-with	O
-Asp	O
-residues	O
-over	O
-the	O
-large	O
-dose	O
-range	O
-(	O
-Fig	O
-.	O
-2	O
-▸	O
-b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-S3	O
-);	O
-this	O
-observation	O
-is	O
-consistent	O
-with	O
-our	O
-calculations	O
-on	O
-model	O
-systems	O
-(	O
-see	O
-above	O
-)	O
-that	O
-suggest	O
-that	O
-,	O
-without	O
-considering	O
-differential	O
-hydrogen	O
--	O
-bonding	O
-environments	O
-,	O
-CO2	O
-loss	O
-is	O
-more	O
-exothermic	O
-by	O
-around	O
-8	O
-kJ	O
-mol	O
-−	O
-1	O
-from	O
-oxidized	O
-Glu	O
-residues	O
-than	O
-from	O
-their	O
-Asp	O
-counterparts	O
-.	O
-	O
-RNA	O
-is	O
-less	O
-susceptible	O
-to	O
-electron	O
--	O
-density	O
-loss	O
-than	O
-protein	O
-within	O
-the	O
-TRAP	O
-–	O
-RNA	O
-complex	O
-	O
-Visual	O
-inspection	O
-of	O
-Fourier	O
-difference	O
-maps	O
-illustrated	O
-the	O
-clear	O
-lack	O
-of	O
-RNA	O
-electron	O
--	O
-density	O
-degradation	O
-with	O
-increasing	O
-dose	O
-compared	O
-with	O
-the	O
-obvious	O
-protein	O
-damage	O
-manifestations	O
-(	O
-Figs	O
-.	O
-3	O
-▸	O
-b	O
-and	O
-3	O
-▸	O
-c	O
-).	O
-	O
-Only	O
-at	O
-the	O
-highest	O
-doses	O
-investigated	O
-(>	O
-20	O
-MGy	O
-)	O
-was	O
-density	O
-loss	O
-observed	O
-at	O
-the	O
-RNA	O
-phosphate	O
-and	O
-C	O
-—	O
-O	O
-bonds	O
-of	O
-the	O
-phosphodiester	O
-backbone	O
-.	O
-	O
-However	O
-,	O
-the	O
-median	O
-D	O
-loss	O
-was	O
-lower	O
-by	O
-a	O
-factor	O
-of	O
->	O
-2	O
-for	O
-RNA	O
-P	O
-atoms	O
-than	O
-for	O
-Glu	O
-and	O
-Asp	O
-side	O
--	O
-chain	O
-groups	O
-at	O
-25	O
-.	O
-0	O
-MGy	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-S4	O
-),	O
-and	O
-furthermore	O
-could	O
-not	O
-be	O
-numerically	O
-distinguished	O
-from	O
-Gly	O
-Cα	O
-atoms	O
-within	O
-TRAP	O
-,	O
-which	O
-are	O
-not	O
-radiation	O
--	O
-sensitive	O
-at	O
-the	O
-doses	O
-tested	O
-here	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-S3	O
-).	O
-	O
-RNA	O
-binding	O
-protects	O
-radiation	O
--	O
-sensitive	O
-residues	O
-	O
-For	O
-the	O
-large	O
-number	O
-of	O
-acidic	O
-residues	O
-per	O
-TRAP	O
-ring	O
-(	O
-four	O
-Asp	O
-and	O
-six	O
-Glu	O
-residues	O
-per	O
-protein	O
-monomer	O
-),	O
-a	O
-strong	O
-dependence	O
-of	O
-decarboxylation	O
-susceptibility	O
-on	O
-local	O
-environment	O
-was	O
-observed	O
-(	O
-Fig	O
-.	O
-4	O
-▸).	O
-	O
-For	O
-each	O
-Glu	O
-Cδ	O
-or	O
-Asp	O
-Cγ	O
-atom	O
-,	O
-D	O
-loss	O
-provided	O
-a	O
-direct	O
-measure	O
-of	O
-the	O
-rate	O
-of	O
-side	O
--	O
-chain	O
-carboxyl	O
--	O
-group	O
-disordering	O
-and	O
-subsequent	O
-decarboxylation	O
-.	O
-	O
-For	O
-acidic	O
-residues	O
-with	O
-no	O
-differing	O
-interactions	O
-between	O
-nonbound	O
-and	O
-bound	O
-TRAP	O
-(	O
-Fig	O
-.	O
-4	O
-▸	O
-a	O
-),	O
-similar	O
-damage	O
-was	O
-apparent	O
-between	O
-the	O
-two	O
-rings	O
-within	O
-the	O
-asymmetric	O
-unit	O
-,	O
-as	O
-expected	O
-.	O
-	O
-However	O
-,	O
-TRAP	O
-residues	O
-directly	O
-on	O
-the	O
-RNA	O
--	O
-binding	O
-interfaces	O
-exhibited	O
-greater	O
-damage	O
-accumulation	O
-in	O
-nonbound	O
-TRAP	O
-(	O
-Fig	O
-.	O
-4	O
-▸	O
-b	O
-),	O
-and	O
-for	O
-residues	O
-at	O
-the	O
-ring	O
-–	O
-ring	O
-interfaces	O
-(	O
-where	O
-crystal	O
-contacts	O
-were	O
-detected	O
-)	O
-bound	O
-TRAP	O
-exhibited	O
-enhanced	O
-SRD	O
-accumulation	O
-(	O
-Fig	O
-.	O
-4	O
-▸	O
-c	O
-).	O
-	O
-Three	O
-acidic	O
-residues	O
-(	O
-Glu36	O
-,	O
-Asp39	O
-and	O
-Glu42	O
-)	O
-are	O
-involved	O
-in	O
-RNA	O
-interactions	O
-within	O
-each	O
-of	O
-the	O
-11	O
-TRAP	O
-ring	O
-subunits	O
-,	O
-and	O
-Fig	O
-.	O
-5	O
-▸	O
-shows	O
-their	O
-density	O
-changes	O
-with	O
-increasing	O
-dose	O
-.	O
-	O
-Hotelling	O
-’	O
-s	O
-T	O
--	O
-squared	O
-test	O
-(	O
-the	O
-multivariate	O
-counterpart	O
-of	O
-Student	O
-’	O
-s	O
-t	O
--	O
-test	O
-)	O
-was	O
-used	O
-to	O
-reject	O
-the	O
-null	O
-hypothesis	O
-that	O
-the	O
-means	O
-of	O
-the	O
-D	O
-loss	O
-metric	O
-were	O
-equal	O
-for	O
-the	O
-bound	O
-and	O
-nonbound	O
-groups	O
-in	O
-Fig	O
-.	O
-5	O
-▸.	O
-	O
-A	O
-significant	O
-reduction	O
-in	O
-D	O
-loss	O
-is	O
-seen	O
-for	O
-Glu36	O
-in	O
-RNA	O
--	O
-bound	O
-compared	O
-with	O
-nonbound	O
-TRAP	O
-,	O
-indicative	O
-of	O
-a	O
-lower	O
-rate	O
-of	O
-side	O
--	O
-chain	O
-decarboxylation	O
-(	O
-Fig	O
-.	O
-5	O
-▸	O
-a	O
-;	O
-p	O
-=	O
-6	O
-.	O
-06	O
-×	O
-10	O
-−	O
-5	O
-).	O
-	O
-For	O
-each	O
-TRAP	O
-ring	O
-subunit	O
-,	O
-the	O
-Glu36	O
-side	O
--	O
-chain	O
-carboxyl	O
-group	O
-accepts	O
-a	O
-pair	O
-of	O
-hydrogen	O
-bonds	O
-from	O
-the	O
-two	O
-N	O
-atoms	O
-of	O
-the	O
-G3	O
-RNA	O
-base	O
-.	O
-	O
-In	O
-our	O
-analysis	O
-,	O
-Asp39	O
-in	O
-the	O
-TRAP	O
-–(	O
-GAGUU	O
-)	O
-10GAG	O
-structure	O
-appears	O
-to	O
-exhibit	O
-two	O
-distinct	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-G1	O
-base	O
-within	O
-each	O
-of	O
-the	O
-11	O
-TRAP	O
-–	O
-RNA	O
-interfaces	O
-,	O
-as	O
-does	O
-Glu36	O
-to	O
-G3	O
-;	O
-however	O
-,	O
-the	O
-reduction	O
-in	O
-density	O
-disordering	O
-upon	O
-RNA	O
-binding	O
-is	O
-far	O
-less	O
-significant	O
-for	O
-Asp39	O
-than	O
-for	O
-Glu36	O
-(	O
-Fig	O
-.	O
-5	O
-▸	O
-b	O
-,	O
-p	O
-=	O
-0	O
-.	O
-0925	O
-).	O
-	O
-RNA	O
-binding	O
-reduces	O
-radiation	O
--	O
-induced	O
-disorder	O
-on	O
-the	O
-atomic	O
-scale	O
-	O
-One	O
-oxygen	O
-(	O
-O	O
-∊	O
-1	O
-)	O
-of	O
-Glu42	O
-appears	O
-to	O
-form	O
-a	O
-hydrogen	O
-bond	O
-to	O
-a	O
-nearby	O
-water	O
-within	O
-each	O
-TRAP	O
-RNA	O
--	O
-binding	O
-pocket	O
-,	O
-with	O
-the	O
-other	O
-(	O
-O	O
-∊	O
-2	O
-)	O
-being	O
-involved	O
-in	O
-a	O
-salt	O
--	O
-bridge	O
-interaction	O
-with	O
-Arg58	O
-(	O
-Hopcroft	O
-et	O
-al	O
-.,	O
-2002	O
-;	O
-Antson	O
-et	O
-al	O
-.,	O
-1999	O
-).	O
-	O
-Salt	O
--	O
-bridge	O
-interactions	O
-have	O
-previously	O
-been	O
-suggested	O
-to	O
-reduce	O
-the	O
-glutamate	O
-decarboxylation	O
-rate	O
-within	O
-the	O
-large	O
-(∼	O
-62	O
-.	O
-4	O
-kDa	O
-)	O
-myrosinase	O
-protein	O
-structure	O
-(	O
-Burmeister	O
-,	O
-2000	O
-).	O
-	O
-A	O
-significant	O
-difference	O
-was	O
-observed	O
-between	O
-the	O
-D	O
-loss	O
-dynamics	O
-for	O
-the	O
-nonbound	O
-/	O
-bound	O
-Glu42	O
-O	O
-∊	O
-1	O
-atoms	O
-(	O
-Fig	O
-.	O
-5	O
-▸	O
-c	O
-;	O
-p	O
-=	O
-0	O
-.	O
-007	O
-)	O
-but	O
-not	O
-for	O
-the	O
-Glu42	O
-O	O
-∊	O
-2	O
-atoms	O
-(	O
-Fig	O
-.	O
-5	O
-▸	O
-d	O
-;	O
-p	O
-=	O
-0	O
-.	O
-239	O
-),	O
-indicating	O
-that	O
-the	O
-stabilizing	O
-strength	O
-of	O
-this	O
-salt	O
--	O
-bridge	O
-interaction	O
-was	O
-conserved	O
-upon	O
-RNA	O
-binding	O
-and	O
-that	O
-the	O
-water	O
--	O
-mediated	O
-hydrogen	O
-bond	O
-had	O
-a	O
-greater	O
-relative	O
-susceptibility	O
-to	O
-atomic	O
-disordering	O
-in	O
-the	O
-absence	O
-of	O
-RNA	O
-.	O
-	O
-The	O
-density	O
--	O
-change	O
-dynamics	O
-were	O
-statistically	O
-indistinguishable	O
-between	O
-bound	O
-and	O
-nonbound	O
-TRAP	O
-for	O
-each	O
-Glu42	O
-carboxyl	O
-group	O
-Cδ	O
-atom	O
-(	O
-p	O
-=	O
-0	O
-.	O
-435	O
-),	O
-indicating	O
-that	O
-upon	O
-RNA	O
-binding	O
-the	O
-conserved	O
-salt	O
--	O
-bridge	O
-interaction	O
-ultimately	O
-dictated	O
-the	O
-overall	O
-Glu42	O
-decarboxylation	O
-rate	O
-.	O
-	O
-The	O
-RNA	O
--	O
-stabilizing	O
-effect	O
-was	O
-not	O
-restricted	O
-to	O
-radiation	O
--	O
-sensitive	O
-acidic	O
-residues	O
-.	O
-	O
-The	O
-side	O
-chain	O
-of	O
-Phe32	O
-stacks	O
-against	O
-the	O
-G3	O
-base	O
-within	O
-the	O
-11	O
-TRAP	O
-RNA	O
--	O
-binding	O
-interfaces	O
-(	O
-Antson	O
-et	O
-al	O
-.,	O
-1999	O
-).	O
-	O
-With	O
-increasing	O
-dose	O
-,	O
-the	O
-D	O
-loss	O
-associated	O
-with	O
-the	O
-Phe32	O
-side	O
-chain	O
-was	O
-significantly	O
-reduced	O
-upon	O
-RNA	O
-binding	O
-(	O
-Fig	O
-.	O
-5	O
-▸	O
-e	O
-;	O
-Phe32	O
-Cζ	O
-;	O
-p	O
-=	O
-0	O
-.	O
-0014	O
-),	O
-an	O
-indication	O
-that	O
-radiation	O
--	O
-induced	O
-conformation	O
-disordering	O
-of	O
-Phe32	O
-had	O
-been	O
-reduced	O
-.	O
-	O
-The	O
-extended	O
-aliphatic	O
-Lys37	O
-side	O
-chain	O
-stacks	O
-against	O
-the	O
-nearby	O
-G1	O
-base	O
-,	O
-making	O
-a	O
-series	O
-of	O
-nonpolar	O
-contacts	O
-within	O
-each	O
-RNA	O
--	O
-binding	O
-interface	O
-.	O
-	O
-The	O
-D	O
-loss	O
-for	O
-Lys37	O
-side	O
--	O
-chain	O
-atoms	O
-was	O
-also	O
-reduced	O
-when	O
-stacked	O
-against	O
-the	O
-G1	O
-base	O
-(	O
-Fig	O
-.	O
-5	O
-▸	O
-f	O
-;	O
-p	O
-=	O
-0	O
-.	O
-0243	O
-for	O
-Lys37	O
-C	O
-∊	O
-atoms	O
-).	O
-	O
-Representative	O
-Phe32	O
-and	O
-Lys37	O
-atoms	O
-were	O
-selected	O
-to	O
-illustrate	O
-these	O
-trends	O
-.	O
-	O
-Here	O
-,	O
-MX	O
-radiation	O
--	O
-induced	O
-specific	O
-structural	O
-changes	O
-within	O
-the	O
-large	O
-TRAP	O
-–	O
-RNA	O
-assembly	O
-over	O
-a	O
-large	O
-dose	O
-range	O
-(	O
-1	O
-.	O
-3	O
-–	O
-25	O
-.	O
-0	O
-MGy	O
-)	O
-have	O
-been	O
-analysed	O
-using	O
-a	O
-high	O
--	O
-throughput	O
-quantitative	O
-approach	O
-,	O
-providing	O
-a	O
-measure	O
-of	O
-the	O
-electron	O
--	O
-density	O
-distribution	O
-for	O
-each	O
-refined	O
-atom	O
-with	O
-increasing	O
-dose	O
-,	O
-D	O
-loss	O
-.	O
-	O
-Compared	O
-with	O
-previous	O
-studies	O
-,	O
-the	O
-results	O
-provide	O
-a	O
-further	O
-step	O
-in	O
-the	O
-detailed	O
-characterization	O
-of	O
-SRD	O
-effects	O
-in	O
-MX	O
-.	O
-	O
-Our	O
-method	O
-­	O
-ology	O
-,	O
-which	O
-eliminated	O
-tedious	O
-and	O
-error	O
--	O
-prone	O
-visual	O
-inspection	O
-,	O
-permitted	O
-the	O
-determination	O
-on	O
-a	O
-per	O
--	O
-atom	O
-basis	O
-of	O
-the	O
-most	O
-damaged	O
-sites	O
-,	O
-as	O
-characterized	O
-by	O
-F	O
-obs	O
-(	O
-d	O
-n	O
-)	O
-−	O
-F	O
-obs	O
-(	O
-d	O
-1	O
-)	O
-Fourier	O
-difference	O
-map	O
-peaks	O
-between	O
-successive	O
-data	O
-sets	O
-collected	O
-from	O
-the	O
-same	O
-crystal	O
-.	O
-	O
-Here	O
-,	O
-it	O
-provided	O
-the	O
-precision	O
-required	O
-to	O
-quantify	O
-the	O
-role	O
-of	O
-RNA	O
-in	O
-the	O
-damage	O
-susceptibilities	O
-of	O
-equivalent	O
-atoms	O
-between	O
-RNA	O
--	O
-bound	O
-and	O
-nonbound	O
-TRAP	O
-,	O
-but	O
-it	O
-is	O
-applicable	O
-to	O
-any	O
-MX	O
-SRD	O
-study	O
-.	O
-	O
-The	O
-RNA	O
-was	O
-found	O
-to	O
-be	O
-substantially	O
-more	O
-radiation	O
--	O
-resistant	O
-than	O
-the	O
-protein	O
-,	O
-even	O
-at	O
-the	O
-highest	O
-doses	O
-investigated	O
-(∼	O
-25	O
-.	O
-0	O
-MGy	O
-),	O
-which	O
-is	O
-in	O
-strong	O
-concurrence	O
-with	O
-our	O
-previous	O
-SRD	O
-investigation	O
-of	O
-the	O
-C	O
-.	O
-Esp1396I	O
-protein	O
-–	O
-DNA	O
-complex	O
-(	O
-Bury	O
-et	O
-al	O
-.,	O
-2015	O
-).	O
-	O
-Consistent	O
-with	O
-that	O
-study	O
-,	O
-at	O
-high	O
-doses	O
-of	O
-above	O
-∼	O
-20	O
-MGy	O
-,	O
-F	O
-obs	O
-(	O
-d	O
-n	O
-)	O
-−	O
-F	O
-obs	O
-(	O
-d	O
-1	O
-)	O
-map	O
-density	O
-was	O
-detected	O
-around	O
-P	O
-,	O
-O3	O
-′	O
-and	O
-O5	O
-′	O
-atoms	O
-of	O
-the	O
-RNA	O
-backbone	O
-,	O
-with	O
-no	O
-significant	O
-difference	O
-density	O
-localized	O
-to	O
-RNA	O
-ribose	O
-and	O
-basic	O
-subunits	O
-.	O
-	O
-RNA	O
-backbone	O
-disordering	O
-thus	O
-appears	O
-to	O
-be	O
-the	O
-main	O
-radiation	O
--	O
-induced	O
-effect	O
-in	O
-RNA	O
-,	O
-with	O
-the	O
-protein	O
-–	O
-base	O
-interactions	O
-maintained	O
-even	O
-at	O
-high	O
-doses	O
-(>	O
-20	O
-MGy	O
-).	O
-	O
-The	O
-U4	O
-phosphate	O
-exhibited	O
-marginally	O
-larger	O
-D	O
-loss	O
-values	O
-above	O
-20	O
-MGy	O
-than	O
-G1	O
-,	O
-A2	O
-and	O
-G3	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-S4	O
-).	O
-	O
-Since	O
-U4	O
-is	O
-the	O
-only	O
-refined	O
-nucleotide	O
-not	O
-to	O
-exhibit	O
-significant	O
-base	O
-–	O
-protein	O
-interactions	O
-around	O
-TRAP	O
-(	O
-with	O
-a	O
-water	O
--	O
-mediated	O
-hydrogen	O
-bond	O
-detected	O
-in	O
-only	O
-three	O
-of	O
-the	O
-11	O
-subunits	O
-and	O
-a	O
-single	O
-Arg58	O
-hydrogen	O
-bond	O
-suggested	O
-in	O
-a	O
-further	O
-four	O
-subunits	O
-),	O
-this	O
-increased	O
-U4	O
-D	O
-loss	O
-can	O
-be	O
-explained	O
-owing	O
-to	O
-its	O
-greater	O
-flexibility	O
-.	O
-	O
-At	O
-25	O
-.	O
-0	O
-MGy	O
-,	O
-the	O
-magnitude	O
-of	O
-the	O
-RNA	O
-backbone	O
-D	O
-loss	O
-was	O
-of	O
-the	O
-same	O
-order	O
-as	O
-for	O
-the	O
-radiation	O
--	O
-insensitive	O
-Gly	O
-Cα	O
-atoms	O
-and	O
-on	O
-average	O
-less	O
-than	O
-half	O
-that	O
-of	O
-the	O
-acidic	O
-residues	O
-of	O
-the	O
-protein	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-S3	O
-).	O
-	O
-Consequently	O
-,	O
-no	O
-clear	O
-single	O
--	O
-strand	O
-breaks	O
-could	O
-be	O
-located	O
-,	O
-and	O
-since	O
-RNA	O
--	O
-binding	O
-within	O
-the	O
-current	O
-TRAP	O
-–(	O
-GAGUU	O
-)	O
-10GAG	O
-complex	O
-is	O
-mediated	O
-predominantly	O
-through	O
-base	O
-–	O
-protein	O
-interactions	O
-,	O
-the	O
-biological	O
-integrity	O
-of	O
-the	O
-RNA	O
-complex	O
-was	O
-dictated	O
-by	O
-the	O
-rate	O
-at	O
-which	O
-protein	O
-decarboxylation	O
-occurred	O
-.	O
-	O
-RNA	O
-interacting	O
-with	O
-TRAP	O
-was	O
-shown	O
-to	O
-offer	O
-significant	O
-protection	O
-against	O
-radiation	O
--	O
-induced	O
-structural	O
-changes	O
-.	O
-	O
-Both	O
-Glu36	O
-and	O
-Asp39	O
-bind	O
-directly	O
-to	O
-RNA	O
-,	O
-each	O
-through	O
-two	O
-hydrogen	O
-bonds	O
-to	O
-guanine	O
-bases	O
-(	O
-G3	O
-and	O
-G1	O
-,	O
-respectively	O
-).	O
-	O
-However	O
-,	O
-compared	O
-with	O
-Asp39	O
-,	O
-Glu36	O
-is	O
-strikingly	O
-less	O
-decarboxylated	O
-when	O
-bound	O
-to	O
-RNA	O
-(	O
-Fig	O
-.	O
-4	O
-▸).	O
-	O
-This	O
-is	O
-in	O
-good	O
-agreement	O
-with	O
-previous	O
-mutagenesis	O
-and	O
-nucleoside	O
-analogue	O
-studies	O
-(	O
-Elliott	O
-et	O
-al	O
-.,	O
-2001	O
-),	O
-which	O
-indicated	O
-that	O
-the	O
-G1	O
-nucleotide	O
-does	O
-not	O
-bind	O
-to	O
-TRAP	O
-as	O
-strongly	O
-as	O
-do	O
-A2	O
-and	O
-G3	O
-,	O
-and	O
-plays	O
-little	O
-role	O
-in	O
-the	O
-high	O
-RNA	O
--	O
-binding	O
-affinity	O
-of	O
-TRAP	O
-(	O
-K	O
-d	O
-≃	O
-1	O
-.	O
-1	O
-±	O
-0	O
-.	O
-4	O
-nM	O
-).	O
-	O
-For	O
-Glu36	O
-and	O
-Asp39	O
-,	O
-no	O
-direct	O
-quantitative	O
-correlation	O
-could	O
-be	O
-established	O
-between	O
-hydrogen	O
--	O
-bond	O
-length	O
-and	O
-D	O
-loss	O
-(	O
-linear	O
-R	O
-2	O
-of	O
-<	O
-0	O
-.	O
-23	O
-for	O
-all	O
-doses	O
-;	O
-Supplementary	O
-Fig	O
-.	O
-S5	O
-).	O
-	O
-Thus	O
-,	O
-another	O
-factor	O
-must	O
-be	O
-responsible	O
-for	O
-this	O
-clear	O
-reduction	O
-in	O
-Glu36	O
-CO2	O
-decarboxyl	O
-­	O
-ation	O
-in	O
-RNA	O
--	O
-bound	O
-TRAP	O
-.	O
-	O
-The	O
-Glu36	O
-carboxyl	O
-side	O
-chain	O
-also	O
-potentially	O
-forms	O
-hydrogen	O
-bonds	O
-to	O
-His34	O
-and	O
-Lys56	O
-,	O
-but	O
-since	O
-these	O
-interactions	O
-are	O
-conserved	O
-irrespective	O
-of	O
-G3	O
-nucleotide	O
-binding	O
-,	O
-this	O
-cannot	O
-directly	O
-account	O
-for	O
-the	O
-stabilization	O
-effect	O
-on	O
-Glu36	O
-in	O
-RNA	O
--	O
-bound	O
-TRAP	O
-.	O
-	O
-When	O
-bound	O
-to	O
-RNA	O
-,	O
-the	O
-average	O
-solvent	O
--	O
-accessible	O
-area	O
-of	O
-the	O
-Glu36	O
-side	O
--	O
-chain	O
-O	O
-atoms	O
-is	O
-reduced	O
-from	O
-∼	O
-15	O
-to	O
-0	O
-Å2	O
-.	O
-	O
-We	O
-propose	O
-that	O
-with	O
-no	O
-solvent	O
-accessibility	O
-Glu36	O
-decarboxylation	O
-is	O
-inhibited	O
-,	O
-since	O
-the	O
-CO2	O
--	O
-formation	O
-rate	O
-K	O
-2	O
-is	O
-greatly	O
-reduced	O
-,	O
-and	O
-suggest	O
-that	O
-steric	O
-hindrance	O
-prevents	O
-each	O
-radicalized	O
-Glu36	O
-CO2	O
-group	O
-from	O
-achieving	O
-the	O
-planar	O
-conformation	O
-required	O
-for	O
-complete	O
-dissociation	O
-from	O
-TRAP	O
-.	O
-	O
-The	O
-electron	O
--	O
-recombination	O
-rate	O
-K	O
-−	O
-1	O
-remains	O
-high	O
-,	O
-however	O
-,	O
-owing	O
-to	O
-rapid	O
-electron	O
-migration	O
-through	O
-the	O
-protein	O
-–	O
-RNA	O
-complex	O
-to	O
-refill	O
-the	O
-Glu36	O
-positive	O
-hole	O
-(	O
-the	O
-precursor	O
-for	O
-Glu	O
-decarboxylation	O
-).	O
-	O
-Upon	O
-RNA	O
-binding	O
-,	O
-the	O
-Asp39	O
-side	O
--	O
-chain	O
-carboxyl	O
-group	O
-solvent	O
--	O
-accessible	O
-area	O
-changes	O
-from	O
-∼	O
-75	O
-to	O
-35	O
-Å2	O
-,	O
-still	O
-allowing	O
-a	O
-high	O
-CO2	O
--	O
-formation	O
-rate	O
-K	O
-2	O
-.	O
-	O
-The	O
-prevalence	O
-of	O
-radical	O
-attack	O
-from	O
-solvent	O
-channels	O
-surrounding	O
-the	O
-protein	O
-in	O
-the	O
-crystal	O
-is	O
-a	O
-questionable	O
-cause	O
-,	O
-considering	O
-previous	O
-observations	O
-indicating	O
-that	O
-the	O
-strongly	O
-oxidizing	O
-hydroxyl	O
-radical	O
-is	O
-immobile	O
-at	O
-100	O
-K	O
-(	O
-Allan	O
-et	O
-al	O
-.,	O
-2013	O
-;	O
-Owen	O
-et	O
-al	O
-.,	O
-2012	O
-).	O
-	O
-By	O
-comparing	O
-equivalent	O
-acidic	O
-residues	O
-with	O
-and	O
-without	O
-RNA	O
-,	O
-we	O
-have	O
-now	O
-deconvoluted	O
-the	O
-role	O
-of	O
-solvent	O
-accessibility	O
-from	O
-other	O
-local	O
-protein	O
-environment	O
-factors	O
-,	O
-and	O
-thus	O
-propose	O
-a	O
-suitable	O
-mechanism	O
-by	O
-which	O
-exceptionally	O
-low	O
-solvent	O
-accessibility	O
-can	O
-reduce	O
-the	O
-rate	O
-of	O
-decarboxylation	O
-.	O
-	O
-Apart	O
-from	O
-these	O
-RNA	O
--	O
-binding	O
-interfaces	O
-,	O
-RNA	O
-binding	O
-was	O
-seen	O
-to	O
-enhance	O
-decarboxylation	O
-for	O
-residues	O
-Glu50	O
-,	O
-Glu71	O
-and	O
-Glu73	O
-,	O
-all	O
-of	O
-which	O
-are	O
-involved	O
-in	O
-crystal	O
-contacts	O
-between	O
-TRAP	O
-rings	O
-(	O
-Fig	O
-.	O
-4	O
-▸	O
-c	O
-).	O
-	O
-However	O
-,	O
-for	O
-each	O
-of	O
-these	O
-residues	O
-the	O
-exact	O
-crystal	O
-contacts	O
-are	O
-not	O
-preserved	O
-between	O
-bound	O
-and	O
-nonbound	O
-TRAP	O
-or	O
-even	O
-between	O
-monomers	O
-within	O
-one	O
-TRAP	O
-ring	O
-.	O
-	O
-For	O
-example	O
-,	O
-in	O
-bound	O
-TRAP	O
-,	O
-Glu73	O
-hydrogen	O
--	O
-bonds	O
-to	O
-a	O
-nearby	O
-lysine	O
-on	O
-each	O
-of	O
-the	O
-11	O
-subunits	O
-,	O
-whereas	O
-in	O
-nonbound	O
-TRAP	O
-no	O
-such	O
-interaction	O
-exists	O
-and	O
-Glu73	O
-interacts	O
-with	O
-a	O
-variable	O
-number	O
-of	O
-refined	O
-waters	O
-in	O
-each	O
-subunit	O
-.	O
-	O
-Radiation	O
--	O
-induced	O
-side	O
--	O
-chain	O
-conformational	O
-changes	O
-have	O
-been	O
-poorly	O
-characterized	O
-in	O
-previous	O
-SRD	O
-investigations	O
-owing	O
-to	O
-their	O
-strong	O
-dependence	O
-on	O
-packing	O
-density	O
-and	O
-geometric	O
-strain	O
-.	O
-	O
-Such	O
-structural	O
-changes	O
-are	O
-known	O
-to	O
-have	O
-significant	O
-roles	O
-within	O
-enzymatic	O
-pathways	O
-,	O
-and	O
-experimenters	O
-must	O
-be	O
-aware	O
-of	O
-these	O
-possible	O
-confounding	O
-factors	O
-when	O
-assigning	O
-true	O
-functional	O
-mechanisms	O
-using	O
-MX	O
-.	O
-	O
-Our	O
-results	O
-show	O
-that	O
-RNA	O
-binding	O
-to	O
-TRAP	O
-physically	O
-stabilizes	O
-non	O
--	O
-acidic	O
-residues	O
-within	O
-the	O
-TRAP	O
-macromolecule	O
-,	O
-most	O
-notably	O
-Lys37	O
-and	O
-Phe32	O
-,	O
-which	O
-stack	O
-against	O
-the	O
-G1	O
-and	O
-G3	O
-bases	O
-,	O
-respectively	O
-.	O
-	O
-It	O
-has	O
-been	O
-suggested	O
-(	O
-Burmeister	O
-,	O
-2000	O
-)	O
-that	O
-Tyr	O
-residues	O
-can	O
-lose	O
-their	O
-aromatic	O
-–	O
-OH	O
-group	O
-owing	O
-to	O
-radiation	O
--	O
-induced	O
-effects	O
-;	O
-however	O
-,	O
-no	O
-energetically	O
-favourable	O
-pathway	O
-for	O
-–	O
-OH	O
-cleavage	O
-exists	O
-and	O
-this	O
-has	O
-not	O
-been	O
-detected	O
-in	O
-aqueous	O
-radiation	O
--	O
-chemistry	O
-studies	O
-.	O
-	O
-In	O
-TRAP	O
-,	O
-D	O
-loss	O
-increased	O
-at	O
-a	O
-similar	O
-rate	O
-for	O
-both	O
-the	O
-Tyr	O
-O	O
-atoms	O
-and	O
-aromatic	O
-ring	O
-atoms	O
-,	O
-suggesting	O
-that	O
-full	O
-ring	O
-conformational	O
-disordering	O
-is	O
-more	O
-likely	O
-.	O
-	O
-Indeed	O
-,	O
-no	O
-convincing	O
-reproducible	O
-Fourier	O
-difference	O
-peaks	O
-above	O
-the	O
-background	O
-map	O
-noise	O
-were	O
-observed	O
-around	O
-any	O
-Tyr	O
-terminal	O
-–	O
-OH	O
-groups	O
-.	O
-	O
-The	O
-RNA	O
--	O
-stabilization	O
-effects	O
-on	O
-protein	O
-are	O
-observed	O
-at	O
-short	O
-ranges	O
-and	O
-are	O
-restricted	O
-to	O
-within	O
-the	O
-RNA	O
--	O
-binding	O
-interfaces	O
-around	O
-the	O
-TRAP	O
-ring	O
-.	O
-	O
-For	O
-example	O
-,	O
-Asp17	O
-is	O
-located	O
-∼	O
-6	O
-.	O
-8	O
-Å	O
-from	O
-the	O
-G1	O
-base	O
-,	O
-outside	O
-the	O
-RNA	O
--	O
-binding	O
-interfaces	O
-,	O
-and	O
-has	O
-indistinguishable	O
-Cγ	O
-atom	O
-D	O
-loss	O
-dose	O
--	O
-dynamics	O
-between	O
-RNA	O
--	O
-bound	O
-and	O
-nonbound	O
-TRAP	O
-(	O
-p	O
->	O
-0	O
-.	O
-9	O
-).	O
-	O
-An	O
-increase	O
-in	O
-the	O
-dose	O
-at	O
-which	O
-functionally	O
-important	O
-residues	O
-remain	O
-intact	O
-has	O
-biological	O
-ramifications	O
-for	O
-understanding	O
-the	O
-mechanisms	O
-at	O
-which	O
-ionizing	O
-radiation	O
-damage	O
-is	O
-mitigated	O
-within	O
-naturally	O
-forming	O
-DNA	O
-–	O
-protein	O
-and	O
-RNA	O
-–	O
-protein	O
-complexes	O
-.	O
-	O
-Observations	O
-of	O
-lower	O
-protein	O
-radiation	O
--	O
-sensitivity	O
-in	O
-DNA	O
--	O
-bound	O
-forms	O
-have	O
-been	O
-recorded	O
-in	O
-solution	O
-at	O
-RT	O
-at	O
-much	O
-lower	O
-doses	O
-(∼	O
-1	O
-kGy	O
-)	O
-than	O
-those	O
-used	O
-for	O
-typical	O
-MX	O
-experiments	O
-[	O
-e	O
-.	O
-g	O
-.	O
-an	O
-oestrogen	O
-response	O
-element	O
-–	O
-receptor	O
-complex	O
-(	O
-Stísová	O
-et	O
-al	O
-.,	O
-2006	O
-)	O
-and	O
-a	O
-DNA	O
-glycosylase	O
-and	O
-its	O
-abasic	O
-DNA	O
-target	O
-site	O
-(	O
-Gillard	O
-et	O
-al	O
-.,	O
-2004	O
-)].	O
-	O
-In	O
-these	O
-studies	O
-,	O
-the	O
-main	O
-damaging	O
-species	O
-is	O
-predicted	O
-to	O
-be	O
-the	O
-oxidizing	O
-hydroxyl	O
-radical	O
-produced	O
-through	O
-solvent	O
-irradiation	O
-,	O
-which	O
-is	O
-known	O
-to	O
-add	O
-to	O
-double	O
-covalent	O
-bonds	O
-within	O
-both	O
-DNA	O
-and	O
-RNA	O
-bases	O
-to	O
-induce	O
-strand	O
-breaks	O
-and	O
-base	O
-modification	O
-(	O
-Spotheim	O
--	O
-Maurizot	O
-&	O
-Davídková	O
-,	O
-2011	O
-;	O
-Chance	O
-et	O
-al	O
-.,	O
-1997	O
-).	O
-	O
-It	O
-was	O
-suggested	O
-that	O
-physical	O
-screening	O
-of	O
-DNA	O
-by	O
-protein	O
-shielded	O
-the	O
-DNA	O
-–	O
-protein	O
-interaction	O
-sites	O
-from	O
-radical	O
-damage	O
-,	O
-yielding	O
-an	O
-extended	O
-life	O
--	O
-dose	O
-for	O
-the	O
-nucleoprotein	O
-complex	O
-compared	O
-with	O
-separate	O
-protein	O
-and	O
-DNA	O
-constituents	O
-at	O
-RT	O
-.	O
-	O
-However	O
-,	O
-in	O
-the	O
-current	O
-MX	O
-study	O
-at	O
-100	O
-K	O
-,	O
-the	O
-main	O
-damaging	O
-species	O
-are	O
-believed	O
-to	O
-be	O
-migrating	O
-LEEs	O
-and	O
-holes	O
-produced	O
-directly	O
-within	O
-the	O
-protein	O
-–	O
-RNA	O
-components	O
-or	O
-in	O
-closely	O
-associated	O
-solvent	O
-.	O
-	O
-The	O
-results	O
-presented	O
-here	O
-suggest	O
-that	O
-biologically	O
-relevant	O
-nucleoprotein	O
-complexes	O
-also	O
-exhibit	O
-prolonged	O
-life	O
--	O
-doses	O
-under	O
-the	O
-effect	O
-of	O
-LEE	O
--	O
-induced	O
-structural	O
-changes	O
-,	O
-involving	O
-direct	O
-physical	O
-protection	O
-of	O
-key	O
-RNA	O
--	O
-binding	O
-residues	O
-.	O
-	O
-Such	O
-reduced	O
-radiation	O
--	O
-sensitivity	O
-in	O
-this	O
-case	O
-ensures	O
-that	O
-the	O
-interacting	O
-protein	O
-remains	O
-bound	O
-long	O
-enough	O
-to	O
-the	O
-RNA	O
-to	O
-complete	O
-its	O
-function	O
-,	O
-even	O
-whilst	O
-exposed	O
-to	O
-ionizing	O
-radiation	O
-.	O
-	O
-Within	O
-the	O
-nonbound	O
-TRAP	O
-macromolecule	O
-,	O
-the	O
-acidic	O
-residues	O
-within	O
-the	O
-unoccupied	O
-RNA	O
--	O
-binding	O
-interfaces	O
-(	O
-Asp39	O
-,	O
-Glu36	O
-,	O
-Glu42	O
-)	O
-are	O
-notably	O
-amongst	O
-the	O
-most	O
-susceptible	O
-residues	O
-within	O
-the	O
-asymmetric	O
-unit	O
-(	O
-Fig	O
-.	O
-4	O
-▸).	O
-	O
-When	O
-exposed	O
-to	O
-X	O
--	O
-rays	O
-,	O
-these	O
-residues	O
-will	O
-be	O
-preferentially	O
-damaged	O
-by	O
-X	O
--	O
-rays	O
-and	O
-subsequently	O
-reduce	O
-the	O
-affinity	O
-with	O
-which	O
-TRAP	O
-binds	O
-to	O
-RNA	O
-.	O
-	O
-Within	O
-the	O
-cellular	O
-environment	O
-,	O
-this	O
-mechanism	O
-could	O
-reduce	O
-the	O
-risk	O
-that	O
-radiation	O
--	O
-damaged	O
-proteins	O
-might	O
-bind	O
-to	O
-RNA	O
-,	O
-thus	O
-avoiding	O
-the	O
-detrimental	O
-introduction	O
-of	O
-incorrect	O
-DNA	O
--	O
-repair	O
-,	O
-transcriptional	O
-and	O
-base	O
--	O
-modification	O
-pathways	O
-.	O
-	O
-The	O
-TRAP	O
-–(	O
-GAGUU	O
-)	O
-10GAG	O
-complex	O
-asymmetric	O
-unit	O
-(	O
-PDB	O
-entry	O
-1gtf	O
-;	O
-Hopcroft	O
-et	O
-al	O
-.,	O
-2002	O
-).	O
-	O
-Bound	O
-tryptophan	O
-ligands	O
-are	O
-represented	O
-as	O
-coloured	O
-spheres	O
-.	O
-	O
-RNA	O
-is	O
-shown	O
-is	O
-yellow	O
-.	O
-	O
-(	O
-a	O
-)	O
-Electron	O
--	O
-density	O
-loss	O
-sites	O
-as	O
-indicated	O
-by	O
-D	O
-loss	O
-in	O
-the	O
-TRAP	O
-–	O
-RNA	O
-complex	O
-crystal	O
-by	O
-residue	O
-/	O
-nucleotide	O
-type	O
-for	O
-five	O
-doses	O
-[	O
-sites	O
-determined	O
-above	O
-the	O
-4	O
-×	O
-average	O
-D	O
-loss	O
-threshold	O
-,	O
-calculated	O
-over	O
-the	O
-TRAP	O
-–	O
-RNA	O
-structure	O
-for	O
-the	O
-first	O
-difference	O
-map	O
-:	O
-F	O
-obs	O
-(	O
-d	O
-2	O
-)	O
-−	O
-F	O
-obs	O
-(	O
-d	O
-1	O
-)].	O
-	O
-(	O
-b	O
-)	O
-Average	O
-D	O
-loss	O
-for	O
-each	O
-residue	O
-/	O
-nucleotide	O
-type	O
-with	O
-respect	O
-to	O
-the	O
-DWD	O
-(	O
-diffraction	O
--	O
-weighted	O
-dose	O
-;	O
-Zeldin	O
-,	O
-Brock	O
-­	O
-hauser	O
-et	O
-al	O
-.,	O
-2013	O
-).	O
-	O
-Only	O
-a	O
-subset	O
-of	O
-key	O
-TRAP	O
-residue	O
-types	O
-are	O
-included	O
-.	O
-	O
-The	O
-average	O
-D	O
-loss	O
-(	O
-calculated	O
-over	O
-the	O
-whole	O
-TRAP	O
-asymmetric	O
-unit	O
-)	O
-is	O
-shown	O
-at	O
-each	O
-dose	O
-(	O
-dashed	O
-line	O
-).	O
-	O
-In	O
-(	O
-a	O
-)	O
-clear	O
-difference	O
-density	O
-is	O
-observed	O
-around	O
-the	O
-Glu42	O
-carboxyl	O
-side	O
-chain	O
-in	O
-chain	O
-H	O
-,	O
-within	O
-the	O
-lowest	O
-dose	O
-difference	O
-map	O
-at	O
-d	O
-2	O
-=	O
-3	O
-.	O
-9	O
-MGy	O
-.	O
-	O
-Radiation	O
--	O
-induced	O
-protein	O
-disordering	O
-is	O
-evident	O
-across	O
-the	O
-large	O
-dose	O
-range	O
-(	O
-b	O
-,	O
-c	O
-);	O
-in	O
-comparison	O
-,	O
-no	O
-clear	O
-deterioration	O
-of	O
-the	O
-RNA	O
-density	O
-was	O
-observed	O
-.	O
-	O
-D	O
-loss	O
-calculated	O
-for	O
-all	O
-side	O
--	O
-chain	O
-carboxyl	O
-group	O
-Glu	O
-Cδ	O
-and	O
-Asp	O
-Cγ	O
-atoms	O
-within	O
-the	O
-TRAP	O
-–	O
-RNA	O
-complex	O
-for	O
-a	O
-dose	O
-of	O
-19	O
-.	O
-3	O
-MGy	O
-(	O
-d	O
-8	O
-).	O
-	O
-Residues	O
-have	O
-been	O
-grouped	O
-by	O
-amino	O
--	O
-acid	O
-number	O
-,	O
-and	O
-split	O
-into	O
-bound	O
-and	O
-nonbound	O
-groupings	O
-,	O
-with	O
-each	O
-bar	O
-representing	O
-the	O
-mean	O
-calculated	O
-over	O
-11	O
-equivalent	O
-atoms	O
-around	O
-a	O
-TRAP	O
-ring	O
-.	O
-	O
-D	O
-loss	O
-against	O
-dose	O
-for	O
-(	O
-a	O
-)	O
-Glu36	O
-Cδ	O
-,	O
-(	O
-b	O
-)	O
-Asp39	O
-Cγ	O
-,	O
-(	O
-c	O
-)	O
-Glu42	O
-O	O
-∊	O
-1	O
-,	O
-(	O
-d	O
-)	O
-Glu42	O
-O	O
-∊	O
-2	O
-,	O
-(	O
-e	O
-)	O
-Phe32	O
-Cζ	O
-and	O
-(	O
-f	O
-)	O
-Lys37	O
-C	O
-∊	O
-atoms	O
-.	O
-	O
-95	O
-%	O
-CI	O
-are	O
-included	O
-for	O
-each	O
-set	O
-of	O
-11	O
-equivalent	O
-atoms	O
-grouped	O
-as	O
-bound	O
-/	O
-nonbound	O
-.	O
-	O
-RNA	O
--	O
-binding	O
-interface	O
-interactions	O
-are	O
-shown	O
-for	O
-TRAP	O
-chain	O
-N	O
-,	O
-with	O
-the	O
-F	O
-obs	O
-(	O
-d	O
-7	O
-)	O
-−	O
-F	O
-obs	O
-(	O
-d	O
-1	O
-)	O
-Fourier	O
-difference	O
-map	O
-(	O
-dose	O
-16	O
-.	O
-7	O
-MGy	O
-)	O
-overlaid	O
-and	O
-contoured	O
-at	O
-a	O
-±	O
-4σ	O
-level	O
-.	O
-	O
-A	O
-conserved	O
-motif	O
-in	O
-JNK	O
-/	O
-p38	O
--	O
-specific	O
-MAPK	O
-phosphatases	O
-as	O
-a	O
-determinant	O
-for	O
-JNK1	O
-recognition	O
-and	O
-inactivation	O
-	O
-Mitogen	O
--	O
-activated	O
-protein	O
-kinases	O
-(	O
-MAPKs	O
-),	O
-important	O
-in	O
-a	O
-large	O
-array	O
-of	O
-signalling	O
-pathways	O
-,	O
-are	O
-tightly	O
-controlled	O
-by	O
-a	O
-cascade	O
-of	O
-protein	O
-kinases	O
-and	O
-by	O
-MAPK	O
-phosphatases	O
-(	O
-MKPs	O
-).	O
-	O
-MAPK	O
-signalling	O
-efficiency	O
-and	O
-specificity	O
-is	O
-modulated	O
-by	O
-protein	O
-–	O
-protein	O
-interactions	O
-between	O
-individual	O
-MAPKs	O
-and	O
-the	O
-docking	O
-motifs	O
-in	O
-cognate	O
-binding	O
-partners	O
-.	O
-	O
-Two	O
-types	O
-of	O
-docking	O
-interactions	O
-have	O
-been	O
-identified	O
-:	O
-D	O
--	O
-motif	O
--	O
-mediated	O
-interaction	O
-and	O
-FXF	O
--	O
-docking	O
-interaction	O
-.	O
-	O
-Here	O
-we	O
-report	O
-the	O
-crystal	O
-structure	O
-of	O
-JNK1	O
-bound	O
-to	O
-the	O
-catalytic	O
-domain	O
-of	O
-MKP7	O
-at	O
-2	O
-.	O
-4	O
--	O
-Å	O
-resolution	O
-,	O
-providing	O
-high	O
--	O
-resolution	O
-structural	O
-insight	O
-into	O
-the	O
-FXF	O
--	O
-docking	O
-interaction	O
-.	O
-	O
-The	O
-285FNFL288	O
-segment	O
-in	O
-MKP7	O
-directly	O
-binds	O
-to	O
-a	O
-hydrophobic	O
-site	O
-on	O
-JNK1	O
-that	O
-is	O
-near	O
-the	O
-MAPK	O
-insertion	O
-and	O
-helix	O
-αG	O
-.	O
-Biochemical	O
-studies	O
-further	O
-reveal	O
-that	O
-this	O
-highly	O
-conserved	O
-structural	O
-motif	O
-is	O
-present	O
-in	O
-all	O
-members	O
-of	O
-the	O
-MKP	O
-family	O
-,	O
-and	O
-the	O
-interaction	O
-mode	O
-is	O
-universal	O
-and	O
-critical	O
-for	O
-the	O
-MKP	O
--	O
-MAPK	O
-recognition	O
-and	O
-biological	O
-function	O
-.	O
-	O
-The	O
-important	O
-MAPK	O
-family	O
-of	O
-signalling	O
-proteins	O
-is	O
-controlled	O
-by	O
-MAPK	O
-phosphatases	O
-(	O
-MKPs	O
-).	O
-	O
-Here	O
-,	O
-the	O
-authors	O
-report	O
-the	O
-structure	O
-of	O
-MKP7	O
-bound	O
-to	O
-JNK1	O
-and	O
-characterise	O
-the	O
-conserved	O
-MKP	O
--	O
-MAPK	O
-interaction	O
-.	O
-	O
-The	O
-mitogen	O
--	O
-activated	O
-protein	O
-kinases	O
-(	O
-MAPKs	O
-)	O
-are	O
-central	O
-components	O
-of	O
-the	O
-signal	O
--	O
-transduction	O
-pathways	O
-,	O
-which	O
-mediate	O
-the	O
-cellular	O
-response	O
-to	O
-a	O
-variety	O
-of	O
-extracellular	O
-stimuli	O
-,	O
-ranging	O
-from	O
-growth	O
-factors	O
-to	O
-environmental	O
-stresses	O
-.	O
-	O
-The	O
-MAPK	O
-signalling	O
-pathways	O
-are	O
-evolutionally	O
-highly	O
-conserved	O
-.	O
-	O
-The	O
-basic	O
-assembly	O
-of	O
-MAPK	O
-pathways	O
-is	O
-a	O
-three	O
--	O
-tier	O
-kinase	O
-module	O
-that	O
-establishes	O
-a	O
-sequential	O
-activation	O
-cascade	O
-:	O
-a	O
-MAPK	O
-kinase	O
-kinase	O
-activates	O
-a	O
-MAPK	O
-kinase	O
-,	O
-which	O
-in	O
-turn	O
-activates	O
-a	O
-MAPK	O
-.	O
-	O
-The	O
-three	O
-best	O
--	O
-characterized	O
-MAPK	O
-signalling	O
-pathways	O
-are	O
-mediated	O
-by	O
-the	O
-kinases	O
-extracellular	O
-signal	O
--	O
-regulated	O
-kinase	O
-(	O
-ERK	O
-),	O
-c	O
--	O
-Jun	O
-N	O
--	O
-terminal	O
-kinase	O
-(	O
-JNK	O
-)	O
-and	O
-p38	O
-.	O
-	O
-The	O
-ERK	O
-pathway	O
-is	O
-activated	O
-by	O
-various	O
-mitogens	O
-and	O
-phorbol	O
-esters	O
-,	O
-whereas	O
-the	O
-JNK	O
-and	O
-p38	O
-pathways	O
-are	O
-stimulated	O
-mainly	O
-by	O
-environmental	O
-stress	O
-and	O
-inflammatory	O
-cytokines	O
-.	O
-	O
-The	O
-MAPKs	O
-are	O
-activated	O
-by	O
-MAPK	O
-kinases	O
-that	O
-phosphorylate	O
-the	O
-MAPKs	O
-at	O
-conserved	O
-threonine	O
-and	O
-tyrosine	O
-residues	O
-within	O
-their	O
-activation	O
-loop	O
-.	O
-	O
-After	O
-activation	O
-,	O
-each	O
-MAPK	O
-phosphorylates	O
-a	O
-distinct	O
-set	O
-of	O
-protein	O
-substrates	O
-,	O
-which	O
-act	O
-as	O
-the	O
-critical	O
-effectors	O
-that	O
-enable	O
-cells	O
-to	O
-mount	O
-the	O
-appropriate	O
-responses	O
-to	O
-varied	O
-stimuli	O
-.	O
-	O
-MAPKs	O
-lie	O
-at	O
-the	O
-bottom	O
-of	O
-conserved	O
-three	O
--	O
-component	O
-phosphorylation	O
-cascades	O
-and	O
-utilize	O
-docking	O
-interactions	O
-to	O
-link	O
-module	O
-components	O
-and	O
-bind	O
-substrates	O
-.	O
-	O
-Two	O
-types	O
-of	O
-docking	O
-motifs	O
-have	O
-been	O
-identified	O
-in	O
-MAPK	O
-substrates	O
-and	O
-cognate	O
-proteins	O
-:	O
-kinase	O
--	O
-interacting	O
-motif	O
-(	O
-D	O
--	O
-motif	O
-)	O
-and	O
-FXF	O
--	O
-motif	O
-(	O
-also	O
-called	O
-DEF	O
-motif	O
-,	O
-docking	O
-site	O
-for	O
-ERK	O
-FXF	O
-).	O
-	O
-The	O
-best	O
--	O
-studied	O
-docking	O
-interactions	O
-are	O
-those	O
-between	O
-MAP	O
-kinases	O
-and	O
-‘	O
-D	O
--	O
-motifs	O
-',	O
-which	O
-consists	O
-of	O
-two	O
-or	O
-more	O
-basic	O
-residues	O
-followed	O
-by	O
-a	O
-short	O
-linker	O
-and	O
-a	O
-cluster	O
-of	O
-hydrophobic	O
-residues	O
-.	O
-	O
-The	O
-D	O
--	O
-motif	O
--	O
-docking	O
-site	O
-(	O
-D	O
--	O
-site	O
-)	O
-in	O
-MAPKs	O
-is	O
-situated	O
-in	O
-a	O
-noncatalytic	O
-region	O
-opposite	O
-of	O
-the	O
-kinase	O
-catalytic	O
-pocket	O
-and	O
-is	O
-comprised	O
-of	O
-a	O
-highly	O
-acidic	O
-patch	O
-and	O
-a	O
-hydrophobic	O
-groove	O
-.	O
-	O
-D	O
--	O
-motifs	O
-are	O
-found	O
-in	O
-many	O
-MAPK	O
--	O
-interacting	O
-proteins	O
-,	O
-including	O
-substrates	O
-,	O
-activating	O
-kinases	O
-and	O
-inactivating	O
-phosphatases	O
-,	O
-as	O
-well	O
-as	O
-scaffolding	O
-proteins	O
-.	O
-	O
-A	O
-second	O
-docking	O
-motif	O
-for	O
-MAPKs	O
-consists	O
-of	O
-two	O
-Phe	O
-residues	O
-separated	O
-by	O
-one	O
-residue	O
-(	O
-FXF	O
--	O
-motif	O
-).	O
-	O
-This	O
-motif	O
-has	O
-been	O
-observed	O
-in	O
-several	O
-MAPK	O
-substrates	O
-.	O
-	O
-The	O
-FXF	O
--	O
-motif	O
--	O
-binding	O
-site	O
-of	O
-ERK2	O
-has	O
-been	O
-mapped	O
-to	O
-a	O
-hydrophobic	O
-pocket	O
-formed	O
-between	O
-the	O
-P	O
-+	O
-1	O
-site	O
-,	O
-αG	O
-helix	O
-and	O
-the	O
-MAPK	O
-insert	O
-.	O
-	O
-However	O
-,	O
-the	O
-generality	O
-and	O
-mechanism	O
-of	O
-the	O
-FXF	O
--	O
-mediated	O
-interaction	O
-is	O
-unclear	O
-.	O
-	O
-The	O
-physiological	O
-outcome	O
-of	O
-MAPK	O
-signalling	O
-depends	O
-on	O
-both	O
-the	O
-magnitude	O
-and	O
-the	O
-duration	O
-of	O
-kinase	O
-activation	O
-.	O
-	O
-Downregulation	O
-of	O
-MAPK	O
-activity	O
-can	O
-be	O
-achieved	O
-through	O
-direct	O
-dephosphorylation	O
-of	O
-the	O
-phospho	O
--	O
-threonine	O
-and	O
-/	O
-or	O
-tyrosine	O
-residues	O
-by	O
-various	O
-serine	O
-/	O
-threonine	O
-phosphatases	O
-,	O
-tyrosine	O
-phosphatases	O
-and	O
-dual	O
--	O
-specificity	O
-phosphatases	O
-(	O
-DUSPs	O
-)	O
-termed	O
-MKPs	O
-.	O
-	O
-MKPs	O
-constitute	O
-a	O
-group	O
-of	O
-DUSPs	O
-that	O
-are	O
-characterized	O
-by	O
-their	O
-ability	O
-to	O
-dephosphorylate	O
-both	O
-phosphotyrosine	O
-and	O
-phosphoserine	O
-/	O
-phospho	O
--	O
-threonine	O
-residues	O
-within	O
-a	O
-substrate	O
-.	O
-	O
-Dysregulated	O
-expression	O
-of	O
-MKPs	O
-has	O
-been	O
-associated	O
-with	O
-pathogenesis	O
-of	O
-various	O
-diseases	O
-,	O
-and	O
-understanding	O
-their	O
-precise	O
-recognition	O
-mechanism	O
-presents	O
-an	O
-important	O
-challenge	O
-and	O
-opportunity	O
-for	O
-drug	O
-development	O
-.	O
-	O
-Here	O
-,	O
-we	O
-present	O
-the	O
-crystal	O
-structure	O
-of	O
-JNK1	O
-in	O
-complex	O
-with	O
-the	O
-catalytic	O
-domain	O
-of	O
-MKP7	O
-.	O
-	O
-This	O
-structure	O
-reveals	O
-the	O
-molecular	O
-mechanism	O
-underlying	O
-the	O
-docking	O
-interaction	O
-between	O
-MKP7	O
-and	O
-JNK1	O
-.	O
-	O
-In	O
-the	O
-JNK1	O
-–	O
-MKP7	O
-complex	O
-,	O
-a	O
-hydrophobic	O
-motif	O
-(	O
-285FNFL288	O
-)	O
-that	O
-initiates	O
-the	O
-helix	O
-α5	O
-in	O
-the	O
-MKP7	O
-catalytic	O
-domain	O
-directly	O
-binds	O
-to	O
-the	O
-FXF	O
--	O
-motif	O
--	O
-binding	O
-site	O
-on	O
-JNK1	O
-,	O
-providing	O
-the	O
-structural	O
-insight	O
-into	O
-the	O
-classic	O
-FXF	O
--	O
-type	O
-docking	O
-interaction	O
-.	O
-	O
-Biochemical	O
-and	O
-modelling	O
-studies	O
-further	O
-demonstrate	O
-that	O
-the	O
-molecular	O
-interactions	O
-mediate	O
-this	O
-key	O
-element	O
-for	O
-substrate	O
-recognition	O
-are	O
-highly	O
-conserved	O
-among	O
-all	O
-MKP	O
--	O
-family	O
-members	O
-.	O
-	O
-Thus	O
-,	O
-our	O
-study	O
-reveals	O
-a	O
-hitherto	O
-unrecognized	O
-interaction	O
-mode	O
-for	O
-encoding	O
-complex	O
-target	O
-specificity	O
-among	O
-MAPK	O
-isoforms	O
-.	O
-	O
-Interaction	O
-of	O
-JNK1	O
-with	O
-the	O
-MKP7	O
-catalytic	O
-domain	O
-	O
-DUSPs	O
-belong	O
-to	O
-the	O
-protein	O
--	O
-tyrosine	O
-phosphatases	O
-(	O
-PTPase	O
-)	O
-superfamily	O
-,	O
-which	O
-is	O
-defined	O
-by	O
-the	O
-PTPase	O
--	O
-signature	O
-motif	O
-CXXGXXR	O
-.	O
-	O
-MKPs	O
-represent	O
-a	O
-distinct	O
-subfamily	O
-within	O
-a	O
-larger	O
-group	O
-of	O
-DUSPs	O
-.	O
-	O
-In	O
-mammalian	O
-cells	O
-,	O
-the	O
-MKP	O
-subfamily	O
-includes	O
-10	O
-distinct	O
-catalytically	O
-active	O
-MKPs	O
-.	O
-	O
-All	O
-MKPs	O
-contain	O
-a	O
-highly	O
-conserved	O
-C	O
--	O
-terminal	O
-catalytic	O
-domain	O
-(	O
-CD	O
-)	O
-and	O
-an	O
-N	O
--	O
-terminal	O
-kinase	O
--	O
-binding	O
-domain	O
-(	O
-KBD	O
-).	O
-	O
-The	O
-KBD	O
-is	O
-homologous	O
-to	O
-the	O
-rhodanese	O
-family	O
-and	O
-contains	O
-an	O
-intervening	O
-cluster	O
-of	O
-basic	O
-amino	O
-acids	O
-,	O
-which	O
-has	O
-been	O
-suggested	O
-to	O
-be	O
-important	O
-for	O
-interacting	O
-with	O
-the	O
-target	O
-MAPKs	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-sequence	O
-similarity	O
-,	O
-substrate	O
-specificity	O
-and	O
-predominant	O
-subcellular	O
-localization	O
-,	O
-the	O
-MKP	O
-family	O
-can	O
-be	O
-further	O
-divided	O
-into	O
-three	O
-groups	O
-(	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-Biochemical	O
-and	O
-structural	O
-studies	O
-have	O
-revealed	O
-that	O
-the	O
-KBD	O
-of	O
-MKPs	O
-is	O
-critical	O
-for	O
-MKP3	O
-docking	O
-to	O
-ERK2	O
-,	O
-and	O
-MKP5	O
-binding	O
-to	O
-p38α	O
-,	O
-although	O
-their	O
-binding	O
-mechanisms	O
-are	O
-completely	O
-different	O
-.	O
-	O
-However	O
-,	O
-it	O
-is	O
-unknown	O
-if	O
-other	O
-MAPKs	O
-can	O
-interact	O
-with	O
-the	O
-KBD	O
-of	O
-their	O
-cognate	O
-phosphatases	O
-in	O
-the	O
-same	O
-manner	O
-as	O
-observed	O
-for	O
-recognition	O
-of	O
-ERK2	O
-and	O
-p38α	O
-by	O
-their	O
-MKPs	O
-,	O
-or	O
-whether	O
-they	O
-recognize	O
-distinct	O
-docking	O
-motifs	O
-of	O
-MKPs	O
-.	O
-	O
-MKP7	O
-,	O
-the	O
-biggest	O
-molecule	O
-in	O
-the	O
-MKP	O
-family	O
-,	O
-selectively	O
-inactivates	O
-JNK	O
-and	O
-p38	O
-following	O
-stress	O
-activation	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-CD	O
-and	O
-KBD	O
-,	O
-MKP7	O
-has	O
-a	O
-long	O
-C	O
--	O
-terminal	O
-region	O
-that	O
-contains	O
-both	O
-nuclear	O
-localization	O
-and	O
-export	O
-sequences	O
-by	O
-which	O
-MKP7	O
-shuttles	O
-between	O
-the	O
-nucleus	O
-and	O
-the	O
-cytoplasm	O
-(	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-To	O
-quantitatively	O
-assess	O
-the	O
-contribution	O
-of	O
-the	O
-N	O
--	O
-terminal	O
-domain	O
-to	O
-the	O
-MKP7	O
--	O
-catalysed	O
-JNK1	O
-dephosphorylation	O
-,	O
-we	O
-first	O
-measured	O
-the	O
-kinetic	O
-parameters	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-truncation	O
-of	O
-MKP7	O
-(	O
-MKP7ΔC304	B-mutant
-,	O
-residues	O
-5	O
-–	O
-303	O
-)	O
-and	O
-MKP7	O
--	O
-CD	O
-(	O
-residues	O
-156	O
-–	O
-301	O
-)	O
-towards	O
-phosphorylated	O
-JNK1	O
-(	O
-pJNK1	O
-).	O
-	O
-Figure	O
-2b	O
-shows	O
-the	O
-variation	O
-of	O
-initial	O
-rates	O
-of	O
-the	O
-MKP7ΔC304	B-mutant
-and	O
-MKP7	O
--	O
-CD	O
--	O
-catalysed	O
-reaction	O
-with	O
-the	O
-concentration	O
-of	O
-phospho	O
--	O
-JNK1	O
-.	O
-Because	O
-the	O
-concentrations	O
-of	O
-MKP7	O
-and	O
-pJNK1	O
-were	O
-comparable	O
-in	O
-the	O
-reaction	O
-,	O
-the	O
-assumption	O
-that	O
-the	O
-free	O
--	O
-substrate	O
-concentration	O
-is	O
-equal	O
-to	O
-the	O
-total	O
-substrate	O
-concentration	O
-is	O
-not	O
-valid	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-kinetic	O
-data	O
-were	O
-analysed	O
-using	O
-the	O
-general	O
-initial	O
-velocity	O
-equation	O
-,	O
-taking	O
-substrate	O
-depletion	O
-into	O
-account	O
-:	O
-	O
-The	O
-kcat	O
-and	O
-Km	O
-of	O
-the	O
-MKP7	O
--	O
-CD	O
-(	O
-0	O
-.	O
-028	O
-s	O
-−	O
-1	O
-and	O
-0	O
-.	O
-26	O
-μM	O
-)	O
-so	O
-determined	O
-were	O
-nearly	O
-identical	O
-to	O
-those	O
-of	O
-MKP7ΔC304	B-mutant
-(	O
-0	O
-.	O
-029	O
-s	O
-−	O
-1	O
-and	O
-0	O
-.	O
-27	O
-μM	O
-),	O
-indicating	O
-that	O
-the	O
-MKP7	O
--	O
-KBD	O
-has	O
-no	O
-effect	O
-on	O
-enzyme	O
-catalysis	O
-.	O
-	O
-We	O
-next	O
-examined	O
-the	O
-interaction	O
-of	O
-JNK1	O
-with	O
-the	O
-CD	O
-and	O
-KBD	O
-of	O
-MKP7	O
-by	O
-gel	O
-filtration	O
-analysis	O
-.	O
-	O
-When	O
-3	O
-molar	O
-equivalents	O
-of	O
-CD	O
-were	O
-mixed	O
-with	O
-1	O
-molar	O
-equivalent	O
-of	O
-JNK1	O
-,	O
-a	O
-significant	O
-amount	O
-of	O
-CD	O
-co	O
--	O
-migrated	O
-with	O
-JNK1	O
-to	O
-earlier	O
-fractions	O
-,	O
-and	O
-the	O
-excess	O
-amount	O
-of	O
-CD	O
-was	O
-eluted	O
-from	O
-the	O
-size	O
-exclusion	O
-column	O
-as	O
-a	O
-monomer	O
-,	O
-indicating	O
-stable	O
-complex	O
-formation	O
-(	O
-Fig	O
-.	O
-2c	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-no	O
-KBD	O
-–	O
-JNK1	O
-complex	O
-was	O
-detected	O
-when	O
-3	O
-molar	O
-equivalents	O
-of	O
-KBD	O
-were	O
-mixed	O
-with	O
-1	O
-molar	O
-equivalent	O
-of	O
-JNK1	O
-.	O
-	O
-To	O
-further	O
-confirm	O
-the	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-interaction	O
-,	O
-we	O
-performed	O
-a	O
-pull	O
--	O
-down	O
-assay	O
-using	O
-the	O
-purified	O
-proteins	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-.	O
-2d	O
-,	O
-the	O
-CD	O
-of	O
-MKP7	O
-can	O
-be	O
-pulled	O
-down	O
-by	O
-JNK1	O
-,	O
-while	O
-the	O
-KBD	O
-failed	O
-to	O
-bind	O
-to	O
-the	O
-counterpart	O
-protein	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-our	O
-data	O
-indicate	O
-that	O
-the	O
-CD	O
-of	O
-MKP7	O
-,	O
-but	O
-not	O
-the	O
-KBD	O
-domain	O
-,	O
-is	O
-responsible	O
-for	O
-JNK	O
-substrate	O
--	O
-binding	O
-and	O
-enzymatic	O
-specificity	O
-.	O
-	O
-Crystal	O
-structure	O
-of	O
-JNK1	O
-in	O
-complex	O
-with	O
-the	O
-MKP7	O
--	O
-CD	O
-	O
-To	O
-understand	O
-the	O
-molecular	O
-basis	O
-of	O
-JNK1	O
-recognition	O
-by	O
-MKP7	O
-,	O
-we	O
-determined	O
-the	O
-crystal	O
-structure	O
-of	O
-unphosphorylated	O
-JNK1	O
-in	O
-complex	O
-with	O
-the	O
-MKP7	O
--	O
-CD	O
-(	O
-Fig	O
-.	O
-3a	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-1a	O
-and	O
-Table	O
-1	O
-).	O
-	O
-In	O
-the	O
-complex	O
-,	O
-JNK1	O
-has	O
-its	O
-characteristic	O
-bilobal	O
-structure	O
-comprising	O
-an	O
-N	O
--	O
-terminal	O
-lobe	O
-rich	O
-in	O
-β	O
--	O
-sheet	O
-and	O
-a	O
-C	O
--	O
-terminal	O
-lobe	O
-that	O
-is	O
-mostly	O
-α	O
--	O
-helical	O
-.	O
-	O
-The	O
-overall	O
-folding	O
-of	O
-MKP7	O
--	O
-CD	O
-is	O
-typical	O
-of	O
-DUSPs	O
-,	O
-with	O
-a	O
-central	O
-twisted	O
-five	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-surrounded	O
-by	O
-six	O
-α	O
--	O
-helices	O
-.	O
-	O
-One	O
-side	O
-of	O
-the	O
-β	O
--	O
-sheet	O
-is	O
-covered	O
-with	O
-two	O
-α	O
--	O
-helices	O
-and	O
-the	O
-other	O
-is	O
-covered	O
-with	O
-four	O
-α	O
--	O
-helices	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-The	O
-catalytic	O
-domain	O
-of	O
-MKP7	O
-interacts	O
-with	O
-JNK1	O
-through	O
-a	O
-contiguous	O
-surface	O
-area	O
-that	O
-is	O
-remote	O
-from	O
-the	O
-active	O
-site	O
-.	O
-	O
-MKP7	O
--	O
-CD	O
-is	O
-positioned	O
-onto	O
-the	O
-JNK1	O
-molecule	O
-so	O
-that	O
-the	O
-active	O
-site	O
-of	O
-the	O
-phosphatase	O
-faces	O
-towards	O
-the	O
-activation	O
-segment	O
-.	O
-	O
-In	O
-an	O
-alignment	O
-of	O
-the	O
-structure	O
-of	O
-MKP7	O
--	O
-CD	O
-with	O
-that	O
-of	O
-VHR	O
-,	O
-an	O
-atypical	O
-‘	O
-MKP	O
-'	O
-consisting	O
-of	O
-only	O
-a	O
-catalytic	O
-domain	O
-,	O
-119	O
-of	O
-147	O
-MKP7	O
--	O
-CD	O
-residues	O
-could	O
-be	O
-superimposed	O
-with	O
-a	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.	O
-(	O
-root	O
-mean	O
-squared	O
-deviation	O
-)	O
-of	O
-1	O
-.	O
-05	O
-Å	O
-(	O
-Fig	O
-.	O
-3c	O
-).	O
-	O
-The	O
-most	O
-striking	O
-difference	O
-is	O
-that	O
-helix	O
-α0	O
-and	O
-loop	O
-α0	O
-–	O
-β1	O
-of	O
-VHR	O
-are	O
-absent	O
-in	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-Another	O
-region	O
-that	O
-cannot	O
-be	O
-aligned	O
-with	O
-VHR	O
-is	O
-found	O
-in	O
-loop	O
-β3	O
-–	O
-β4	O
-.	O
-	O
-This	O
-loop	O
-is	O
-shortened	O
-by	O
-nine	O
-residues	O
-in	O
-MKP7	O
--	O
-CD	O
-compared	O
-with	O
-that	O
-in	O
-VHR	O
-.	O
-	O
-Since	O
-helix	O
-α0	O
-and	O
-the	O
-following	O
-loop	O
-α0	O
-–	O
-β1	O
-are	O
-known	O
-for	O
-a	O
-substrate	O
--	O
-recognition	O
-motif	O
-of	O
-VHR	O
-and	O
-other	O
-phosphatases	O
-,	O
-the	O
-absence	O
-of	O
-these	O
-moieties	O
-implicates	O
-a	O
-different	O
-substrate	O
--	O
-binding	O
-mode	O
-of	O
-MKP7	O
-.	O
-	O
-The	O
-active	O
-site	O
-of	O
-MKP7	O
-consists	O
-of	O
-the	O
-phosphate	O
--	O
-binding	O
-loop	O
-(	O
-P	O
--	O
-loop	O
-,	O
-Cys244	O
--	O
-Leu245	O
--	O
-Ala246	O
--	O
-Gly247	O
--	O
-Ile248	O
--	O
-Ser249	O
--	O
-Arg250	O
-),	O
-and	O
-Asp213	O
-in	O
-the	O
-general	O
-acid	O
-loop	O
-(	O
-Fig	O
-.	O
-3b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1b	O
-).	O
-	O
-The	O
-MKP7	O
--	O
-CD	O
-structure	O
-near	O
-the	O
-active	O
-site	O
-exhibits	O
-a	O
-typical	O
-active	O
-conformation	O
-as	O
-found	O
-in	O
-VHR	O
-and	O
-other	O
-PTPs	O
-.	O
-	O
-The	O
-catalytic	O
-residue	O
-,	O
-Cys244	O
-,	O
-is	O
-located	O
-just	O
-after	O
-strand	O
-β5	O
-and	O
-optimally	O
-positioned	O
-for	O
-nucleophilic	O
-attack	O
-.	O
-	O
-Asp213	O
-in	O
-MKP7	O
-also	O
-adopts	O
-a	O
-position	O
-similar	O
-to	O
-that	O
-of	O
-Asp92	O
-in	O
-VHR	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1c	O
-),	O
-indicating	O
-that	O
-Asp213	O
-is	O
-likely	O
-to	O
-function	O
-as	O
-the	O
-general	O
-acid	O
-in	O
-MKP7	O
-.	O
-	O
-We	O
-also	O
-observed	O
-the	O
-binding	O
-of	O
-a	O
-chloride	O
-ion	O
-in	O
-the	O
-active	O
-site	O
-of	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-It	O
-is	O
-located	O
-3	O
-.	O
-36	O
-Å	O
-from	O
-the	O
-Cys244	O
-side	O
-chain	O
-and	O
-makes	O
-electrostatic	O
-interactions	O
-with	O
-the	O
-dipole	O
-moment	O
-of	O
-helix	O
-α3	O
-and	O
-with	O
-several	O
-main	O
--	O
-chain	O
-amide	O
-groups	O
-.	O
-	O
-The	O
-side	O
-chain	O
-of	O
-strictly	O
-conserved	O
-Arg250	O
-is	O
-oriented	O
-towards	O
-the	O
-negatively	O
-charged	O
-chloride	O
-,	O
-similar	O
-to	O
-the	O
-canonical	O
-phosphate	O
--	O
-coordinating	O
-conformation	O
-.	O
-	O
-Thus	O
-this	O
-chloride	O
-ion	O
-is	O
-a	O
-mimic	O
-for	O
-the	O
-phosphate	O
-group	O
-of	O
-the	O
-substrate	O
-,	O
-as	O
-revealed	O
-by	O
-a	O
-comparison	O
-with	O
-the	O
-structure	O
-of	O
-PTP1B	O
-in	O
-complex	O
-with	O
-phosphotyrosine	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1d	O
-).	O
-	O
-Although	O
-the	O
-catalytically	O
-important	O
-residues	O
-in	O
-MKP7	O
--	O
-CD	O
-are	O
-well	O
-aligned	O
-with	O
-those	O
-in	O
-VHR	O
-,	O
-the	O
-residues	O
-in	O
-the	O
-P	O
--	O
-loop	O
-of	O
-MKP7	O
-are	O
-smaller	O
-and	O
-have	O
-a	O
-more	O
-hydrophobic	O
-character	O
-than	O
-those	O
-of	O
-VHR	O
-(	O
-Cys124	O
--	O
-Arg125	O
--	O
-Glu126	O
--	O
-Gly127	O
--	O
-Tyr128	O
--	O
-Gly129	O
--	O
-Arg130	O
-;	O
-Fig	O
-.	O
-3b	O
-,	O
-c	O
-).	O
-	O
-The	O
-difference	O
-in	O
-the	O
-polarity	O
-/	O
-hydrophobicity	O
-of	O
-the	O
-surface	O
-may	O
-also	O
-point	O
-to	O
-the	O
-origin	O
-of	O
-the	O
-differences	O
-in	O
-the	O
-substrate	O
--	O
-recognition	O
-mechanism	O
-for	O
-these	O
-two	O
-phosphatases	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1e	O
-,	O
-f	O
-).	O
-	O
-In	O
-the	O
-complex	O
-,	O
-MKP7	O
--	O
-CD	O
-and	O
-JNK1	O
-form	O
-extensive	O
-protein	O
-–	O
-protein	O
-interactions	O
-involving	O
-the	O
-C	O
--	O
-terminal	O
-helices	O
-of	O
-MKP7	O
--	O
-CD	O
-and	O
-C	O
--	O
-lobe	O
-of	O
-JNK1	O
-(	O
-Fig	O
-.	O
-3d	O
-,	O
-e	O
-).	O
-	O
-As	O
-a	O
-result	O
-,	O
-the	O
-buried	O
-solvent	O
--	O
-accessible	O
-surface	O
-area	O
-is	O
-∼	O
-1	O
-,	O
-315	O
-Å	O
-.	O
-In	O
-the	O
-C	O
--	O
-terminal	O
-domain	O
-,	O
-JNK1	O
-has	O
-an	O
-insertion	O
-after	O
-the	O
-helix	O
-αG	O
-.	O
-This	O
-insertion	O
-consists	O
-of	O
-two	O
-helices	O
-(	O
-α1L14	O
-and	O
-α2L14	O
-)	O
-that	O
-are	O
-common	O
-to	O
-all	O
-members	O
-of	O
-the	O
-MAPK	O
-family	O
-.	O
-	O
-The	O
-interactive	O
-surface	O
-in	O
-JNK1	O
-,	O
-formed	O
-by	O
-the	O
-helices	O
-αG	O
-and	O
-α2L14	O
-,	O
-displays	O
-a	O
-hydrophobic	O
-region	O
-,	O
-centred	O
-at	O
-Trp234	O
-(	O
-Fig	O
-.	O
-3d	O
-).	O
-	O
-The	O
-MKP7	O
--	O
-docking	O
-region	O
-includes	O
-two	O
-helices	O
-,	O
-α4	O
-and	O
-α5	O
-,	O
-and	O
-the	O
-general	O
-acid	O
-loop	O
-.	O
-	O
-The	O
-aromatic	O
-ring	O
-of	O
-Phe285	O
-on	O
-MKP7	O
-α5	O
--	O
-helix	O
-is	O
-nestled	O
-in	O
-a	O
-hydrophobic	O
-pocket	O
-on	O
-JNK1	O
-,	O
-formed	O
-by	O
-side	O
-chains	O
-of	O
-Ile197	O
-,	O
-Leu198	O
-,	O
-Ile231	O
-,	O
-Trp234	O
-,	O
-Val256	O
-,	O
-Tyr259	O
-,	O
-Val260	O
-and	O
-the	O
-aliphatic	O
-portion	O
-of	O
-His230	O
-(	O
-Fig	O
-.	O
-3d	O
-,	O
-f	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1g	O
-).	O
-	O
-In	O
-addition	O
-,	O
-there	O
-are	O
-hydrogen	O
-bonds	O
-between	O
-Ser282	O
-and	O
-Asn286	O
-of	O
-MKP7	O
-and	O
-His230	O
-and	O
-Thr255	O
-of	O
-JNK1	O
-,	O
-and	O
-the	O
-main	O
-chain	O
-of	O
-Phe215	O
-in	O
-the	O
-general	O
-acid	O
-loop	O
-of	O
-MKP7	O
-is	O
-hydrogen	O
--	O
-bonded	O
-to	O
-the	O
-side	O
-chain	O
-of	O
-Gln253	O
-in	O
-JNK1	O
-.	O
-	O
-The	O
-second	O
-interactive	O
-area	O
-involves	O
-the	O
-α4	O
-helix	O
-of	O
-MKP7	O
-and	O
-charged	O
-/	O
-polar	O
-residues	O
-of	O
-JNK1	O
-(	O
-Fig	O
-.	O
-3e	O
-).	O
-	O
-The	O
-carboxylate	O
-of	O
-Asp268	O
-in	O
-MKP7	O
-forms	O
-a	O
-salt	O
-bridge	O
-with	O
-side	O
-chain	O
-of	O
-Arg263	O
-in	O
-JNK1	O
-,	O
-and	O
-Lys275	O
-of	O
-MKP7	O
-forms	O
-a	O
-hydrogen	O
-bond	O
-and	O
-a	O
-salt	O
-bridge	O
-with	O
-Thr228	O
-and	O
-Asp229	O
-of	O
-JNK1	O
-,	O
-respectively	O
-.	O
-	O
-Mutational	O
-analysis	O
-of	O
-the	O
-JNK1	O
-–	O
-MKP7	O
-docking	O
-interface	O
-	O
-To	O
-assess	O
-the	O
-importance	O
-of	O
-the	O
-aforementioned	O
-interactions	O
-,	O
-we	O
-generated	O
-a	O
-series	O
-of	O
-point	O
-mutations	O
-on	O
-the	O
-MKP7	O
--	O
-CD	O
-and	O
-examined	O
-their	O
-effect	O
-on	O
-the	O
-MKP7	O
--	O
-catalysed	O
-JNK1	O
-dephosphorylation	O
-(	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-When	O
-the	O
-hydrophobic	O
-residues	O
-Phe285	O
-and	O
-Phe287	O
-on	O
-the	O
-α5	O
-of	O
-MKP7	O
--	O
-CD	O
-were	O
-replaced	O
-by	O
-Asp	O
-or	O
-Ala	O
-,	O
-their	O
-phosphatase	O
-activities	O
-for	O
-JNK1	O
-dephosphorylation	O
-decreased	O
-∼	O
-10	O
--	O
-fold	O
-.	O
-	O
-In	O
-comparison	O
-,	O
-replacement	O
-of	O
-the	O
-other	O
-residues	O
-(	O
-Phe215	O
-,	O
-Asp268	O
-,	O
-Lys275	O
-,	O
-Ser282	O
-,	O
-Asn286	O
-and	O
-Leu292	O
-)	O
-with	O
-an	O
-Ala	O
-or	O
-Asp	O
-individually	O
-led	O
-to	O
-a	O
-modest	O
-decrease	O
-in	O
-catalytic	O
-efficiencies	O
-,	O
-suggesting	O
-that	O
-this	O
-position	O
-may	O
-only	O
-affect	O
-some	O
-selectivity	O
-of	O
-MKP	O
-.	O
-	O
-Mutation	O
-of	O
-Leu288	O
-markedly	O
-reduced	O
-its	O
-solubility	O
-when	O
-expressed	O
-in	O
-Escherichia	O
-coli	O
-,	O
-resulting	O
-in	O
-the	O
-insoluble	O
-aggregation	O
-of	O
-the	O
-mutant	O
-protein	O
-.	O
-	O
-Gel	O
-filtration	O
-analysis	O
-further	O
-confirmed	O
-the	O
-key	O
-role	O
-of	O
-Phe285	O
-in	O
-the	O
-MKP7	O
-–	O
-JNK1	O
-interaction	O
-:	O
-no	O
-F285D	O
-–	O
-JNK1	O
-complex	O
-was	O
-detected	O
-when	O
-3	O
-molar	O
-equivalents	O
-of	O
-MKP7	O
--	O
-CD	O
-(	O
-F285D	B-mutant
-)	O
-were	O
-mixed	O
-with	O
-1	O
-molar	O
-equivalent	O
-of	O
-JNK1	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-Interestingly	O
-,	O
-mutation	O
-of	O
-Phe287	O
-results	O
-in	O
-a	O
-considerable	O
-loss	O
-of	O
-activity	O
-against	O
-pJNK1	O
-without	O
-altering	O
-the	O
-affinity	O
-of	O
-MKP7	O
--	O
-CD	O
-for	O
-JNK1	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-We	O
-also	O
-generated	O
-a	O
-series	O
-of	O
-point	O
-mutations	O
-in	O
-the	O
-JNK1	O
-and	O
-assessed	O
-the	O
-effect	O
-on	O
-JNK1	O
-binding	O
-using	O
-the	O
-GST	O
-pull	O
--	O
-down	O
-assay	O
-(	O
-Fig	O
-.	O
-4c	O
-).	O
-	O
-Substitution	O
-at	O
-Asp229	O
-,	O
-Trp234	O
-,	O
-Thr255	O
-,	O
-Val256	O
-,	O
-Tyr259	O
-and	O
-Val260	O
-significantly	O
-reduced	O
-the	O
-binding	O
-affinity	O
-of	O
-MKP7	O
--	O
-CD	O
-for	O
-JNK	O
-.	O
-	O
-To	O
-determine	O
-whether	O
-the	O
-deficiencies	O
-in	O
-their	O
-abilities	O
-to	O
-bind	O
-partner	O
-proteins	O
-or	O
-carry	O
-out	O
-catalytic	O
-function	O
-are	O
-owing	O
-to	O
-misfolding	O
-of	O
-the	O
-purified	O
-mutant	O
-proteins	O
-,	O
-we	O
-also	O
-examined	O
-the	O
-folding	O
-properties	O
-of	O
-the	O
-JNK1	O
-and	O
-MKP7	O
-mutants	O
-with	O
-circular	O
-dichroism	O
-.	O
-	O
-The	O
-spectra	O
-of	O
-these	O
-mutants	O
-are	O
-similar	O
-to	O
-the	O
-wild	O
--	O
-type	O
-proteins	O
-,	O
-indicating	O
-that	O
-these	O
-mutants	O
-fold	O
-as	O
-well	O
-as	O
-the	O
-wild	O
--	O
-type	O
-proteins	O
-(	O
-Fig	O
-.	O
-4d	O
-,	O
-e	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-these	O
-results	O
-are	O
-consistent	O
-with	O
-the	O
-present	O
-crystallographic	O
-model	O
-,	O
-which	O
-reveal	O
-the	O
-hydrophobic	O
-contacts	O
-between	O
-the	O
-MKP7	O
-catalytic	O
-domain	O
-and	O
-JNK1	O
-have	O
-a	O
-predominant	O
-role	O
-in	O
-the	O
-enzyme	O
-–	O
-substrate	O
-interaction	O
-,	O
-and	O
-hydrophobic	O
-residue	O
-Phe285	O
-in	O
-the	O
-MKP7	O
--	O
-CD	O
-is	O
-a	O
-key	O
-residue	O
-for	O
-its	O
-high	O
--	O
-affinity	O
-binding	O
-to	O
-JNK1	O
-.	O
-	O
-It	O
-has	O
-previously	O
-been	O
-reported	O
-that	O
-several	O
-cytosolic	O
-and	O
-inducible	O
-nuclear	O
-MKPs	O
-undergo	O
-catalytic	O
-activation	O
-upon	O
-interaction	O
-with	O
-the	O
-MAPK	O
-substrates	O
-.	O
-	O
-This	O
-allosteric	O
-activation	O
-of	O
-MKP3	O
-has	O
-been	O
-well	O
--	O
-documented	O
-in	O
-vitro	O
-using	O
-pNPP	O
-,	O
-a	O
-small	O
--	O
-molecule	O
-phosphotyrosine	O
-analogue	O
-of	O
-its	O
-normal	O
-substrate	O
-.	O
-	O
-We	O
-then	O
-assayed	O
-pNPPase	O
-activities	O
-of	O
-MKP7ΔC304	B-mutant
-and	O
-MKP7	O
--	O
-CD	O
-in	O
-the	O
-presence	O
-of	O
-JNK1	O
-.	O
-	O
-Incubation	O
-of	O
-MKP7	O
-with	O
-JNK1	O
-did	O
-not	O
-markedly	O
-stimulate	O
-the	O
-phosphatase	O
-activity	O
-,	O
-which	O
-is	O
-consistent	O
-with	O
-previous	O
-results	O
-that	O
-MKP7	O
-solely	O
-possesses	O
-the	O
-intrinsic	O
-activity	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-The	O
-small	O
-pNPP	O
-molecule	O
-binds	O
-directly	O
-at	O
-the	O
-enzyme	O
-active	O
-site	O
-and	O
-can	O
-be	O
-used	O
-to	O
-probe	O
-the	O
-reaction	O
-mechanism	O
-of	O
-protein	O
-phosphatases	O
-.	O
-	O
-We	O
-therefore	O
-examined	O
-the	O
-effects	O
-of	O
-the	O
-MKP7	O
--	O
-CD	O
-mutants	O
-on	O
-their	O
-pNPPase	O
-activities	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-.	O
-4f	O
-,	O
-all	O
-the	O
-mutants	O
-,	O
-except	O
-F287D	B-mutant
-/	I-mutant
-A	I-mutant
-,	O
-showed	O
-little	O
-or	O
-no	O
-activity	O
-change	O
-compared	O
-with	O
-the	O
-wild	O
--	O
-type	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-In	O
-the	O
-JNK1	O
-/	O
-MKP7	O
--	O
-CD	O
-complex	O
-structure	O
-,	O
-Phe287	O
-of	O
-MKP7	O
-does	O
-not	O
-make	O
-contacts	O
-with	O
-JNK1	O
-substrate	O
-.	O
-	O
-It	O
-penetrates	O
-into	O
-a	O
-pocket	O
-formed	O
-by	O
-residues	O
-from	O
-the	O
-P	O
--	O
-loop	O
-and	O
-general	O
-acid	O
-loop	O
-and	O
-forms	O
-hydrophobic	O
-contacts	O
-with	O
-the	O
-aliphatic	O
-portions	O
-of	O
-side	O
-chains	O
-of	O
-Arg250	O
-,	O
-Glu217	O
-and	O
-Ile219	O
-,	O
-suggesting	O
-that	O
-Phe287	O
-in	O
-MKP7	O
-would	O
-play	O
-a	O
-similar	O
-role	O
-to	O
-that	O
-of	O
-its	O
-structural	O
-counterpart	O
-in	O
-the	O
-PTPs	O
-(	O
-Gln266	O
-in	O
-PTP1B	O
-)	O
-and	O
-VHR	O
-(	O
-Phe166	O
-in	O
-VHR	O
-)	O
-in	O
-the	O
-precise	O
-alignment	O
-of	O
-active	O
--	O
-site	O
-residues	O
-in	O
-MKP7	O
-with	O
-respect	O
-to	O
-the	O
-substrate	O
-for	O
-efficient	O
-catalysis	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2c	O
-).	O
-	O
-Kinase	O
--	O
-associated	O
-phosphatase	O
-(	O
-KAP	O
-),	O
-a	O
-member	O
-of	O
-the	O
-DUSP	O
-family	O
-,	O
-plays	O
-a	O
-crucial	O
-role	O
-in	O
-cell	O
-cycle	O
-regulation	O
-by	O
-dephosphorylating	O
-the	O
-pThr160	O
-residue	O
-of	O
-CDK2	O
-(	O
-cyclin	O
--	O
-dependent	O
-kinase	O
-2	O
-).	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-CDK2	O
-/	O
-KAP	O
-complex	O
-has	O
-been	O
-determined	O
-at	O
-3	O
-.	O
-0	O
-Å	O
-(	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-The	O
-interface	O
-between	O
-these	O
-two	O
-proteins	O
-consists	O
-of	O
-three	O
-discontinuous	O
-contact	O
-regions	O
-.	O
-	O
-Biochemical	O
-results	O
-suggested	O
-that	O
-the	O
-affinity	O
-and	O
-specificity	O
-between	O
-KAP	O
-and	O
-CDK2	O
-results	O
-from	O
-the	O
-recognition	O
-site	O
-comprising	O
-CDK2	O
-residues	O
-from	O
-the	O
-αG	O
-helix	O
-and	O
-L14	O
-loop	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-helical	O
-region	O
-of	O
-KAP	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-There	O
-is	O
-a	O
-hydrogen	O
-bond	O
-between	O
-the	O
-main	O
--	O
-chain	O
-nitrogen	O
-of	O
-Ile183	O
-(	O
-KAP	O
-)	O
-and	O
-side	O
-chain	O
-oxygen	O
-of	O
-Glu208	O
-(	O
-CDK2	O
-),	O
-and	O
-salt	O
-bridges	O
-between	O
-Lys184	O
-of	O
-KAP	O
-and	O
-Asp235	O
-of	O
-CDK2	O
-.	O
-	O
-Structural	O
-analysis	O
-and	O
-sequence	O
-alignment	O
-reveal	O
-that	O
-one	O
-of	O
-the	O
-few	O
-differences	O
-between	O
-MKP7	O
--	O
-CD	O
-and	O
-KAP	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-region	O
-is	O
-the	O
-presence	O
-of	O
-the	O
-motif	O
-FNFL	O
-in	O
-MKP7	O
--	O
-CD	O
-,	O
-which	O
-corresponds	O
-to	O
-IKQY	O
-in	O
-KAP	O
-(	O
-Fig	O
-.	O
-5c	O
-).	O
-	O
-The	O
-substitution	O
-of	O
-the	O
-two	O
-hydrophobic	O
-residues	O
-with	O
-charged	O
-/	O
-polar	O
-residues	O
-(	O
-F285I	B-mutant
-/	O
-N286K	B-mutant
-)	O
-seriously	O
-disrupts	O
-the	O
-hydrophobic	O
-interaction	O
-required	O
-for	O
-MKP7	O
-binding	O
-on	O
-JNK1	O
-(	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-In	O
-addition	O
-,	O
-His230	O
-and	O
-Val256	O
-in	O
-JNK1	O
-are	O
-replaced	O
-by	O
-the	O
-negatively	O
-charged	O
-residues	O
-Glu208	O
-and	O
-Asp235	O
-in	O
-CDK2	O
-(	O
-Fig	O
-.	O
-5d	O
-),	O
-and	O
-the	O
-charge	O
-distribution	O
-on	O
-the	O
-CDK2	O
-interactive	O
-surface	O
-is	O
-quite	O
-different	O
-from	O
-that	O
-of	O
-JNK	O
-.	O
-	O
-These	O
-data	O
-indicated	O
-that	O
-a	O
-unique	O
-hydrophobic	O
-pocket	O
-formed	O
-between	O
-the	O
-MAPK	O
-insert	O
-and	O
-αG	O
-helix	O
-plays	O
-a	O
-major	O
-role	O
-in	O
-the	O
-substrate	O
-recognition	O
-by	O
-MKPs	O
-.	O
-	O
-F	O
--	O
-site	O
-interaction	O
-is	O
-crucial	O
-for	O
-JNK1	O
-inactivation	O
-in	O
-vivo	O
-	O
-JNK	O
-is	O
-activated	O
-following	O
-cellular	O
-exposure	O
-to	O
-a	O
-number	O
-of	O
-acute	O
-stimuli	O
-such	O
-as	O
-anisomycin	O
-,	O
-H2O2	O
-,	O
-ultraviolet	O
-light	O
-,	O
-sorbitol	O
-,	O
-DNA	O
--	O
-damaging	O
-agents	O
-and	O
-several	O
-strong	O
-apoptosis	O
-inducers	O
-(	O
-etoposide	O
-,	O
-cisplatin	O
-and	O
-taxol	O
-).	O
-	O
-To	O
-assess	O
-the	O
-effects	O
-of	O
-MKP7	O
-and	O
-its	O
-mutants	O
-on	O
-the	O
-activation	O
-of	O
-endogenous	O
-JNK	O
-in	O
-vivo	O
-,	O
-HEK293T	O
-cells	O
-were	O
-transfected	O
-with	O
-blank	O
-vector	O
-or	O
-with	O
-HA	O
--	O
-tagged	O
-constructs	O
-for	O
-full	O
--	O
-length	O
-MKP7	O
-,	O
-MKP7ΔC304	B-mutant
-and	O
-MKP7	O
--	O
-CD	O
-or	O
-MKP7	O
-mutants	O
-,	O
-and	O
-stimulated	O
-with	O
-ultraviolet	O
-or	O
-etoposide	O
-treatment	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-.	O
-6a	O
-–	O
-c	O
-,	O
-immunobloting	O
-showed	O
-similar	O
-expression	O
-levels	O
-for	O
-the	O
-different	O
-MKP7	O
-constructs	O
-in	O
-all	O
-the	O
-cells	O
-.	O
-	O
-Overexpressed	O
-full	O
--	O
-length	O
-MKP7	O
-,	O
-MKP7ΔC304	B-mutant
-and	O
-MKP7	O
--	O
-CD	O
-significantly	O
-reduced	O
-the	O
-endogenous	O
-level	O
-of	O
-phosphorylated	O
-JNK	O
-compared	O
-with	O
-vector	O
--	O
-transfected	O
-cells	O
-.	O
-	O
-Parallel	O
-experiments	O
-showed	O
-clearly	O
-that	O
-the	O
-D	O
--	O
-motif	O
-mutants	O
-(	O
-R56A	B-mutant
-/	O
-R57A	B-mutant
-and	O
-V63A	B-mutant
-/	O
-I65A	B-mutant
-)	O
-dephosphorylated	O
-JNK	O
-as	O
-did	O
-the	O
-wild	O
-type	O
-under	O
-the	O
-same	O
-conditions	O
-,	O
-further	O
-confirming	O
-that	O
-the	O
-MKP7	O
--	O
-KBD	O
-is	O
-not	O
-required	O
-for	O
-the	O
-JNK	O
-inactivation	O
-in	O
-vivo	O
-.	O
-	O
-Consistent	O
-with	O
-the	O
-in	O
-vitro	O
-data	O
-,	O
-the	O
-level	O
-of	O
-phosphorylated	O
-JNK	O
-was	O
-not	O
-or	O
-little	O
-altered	O
-in	O
-MKP7	O
-FXF	O
--	O
-motif	O
-mutants	O
-(	O
-F285D	B-mutant
-,	O
-F287D	B-mutant
-and	O
-L288D	B-mutant
-)-	O
-transfected	O
-cells	O
-,	O
-and	O
-the	O
-MKP7	O
-D268A	B-mutant
-and	O
-N286A	B-mutant
-mutants	O
-retained	O
-the	O
-ability	O
-to	O
-reduce	O
-the	O
-phosphorylation	O
-levels	O
-of	O
-JNK	O
-.	O
-	O
-We	O
-next	O
-tested	O
-in	O
-vivo	O
-interactions	O
-between	O
-JNK1	O
-mutants	O
-and	O
-full	O
--	O
-length	O
-MKP7	O
-by	O
-coimmunoprecipitation	O
-experiments	O
-under	O
-unstimulated	O
-conditions	O
-.	O
-	O
-When	O
-co	O
--	O
-expressed	O
-in	O
-HEK293T	O
-cells	O
-,	O
-wild	O
--	O
-type	O
-(	O
-HA	O
-)-	O
-JNK1	O
-was	O
-readily	O
-precipitated	O
-with	O
-(	O
-Myc	O
-)-	O
-MKP7	O
-(	O
-Fig	O
-.	O
-6d	O
-),	O
-indicating	O
-that	O
-MKP7	O
-binds	O
-dephosphorylated	O
-JNK1	O
-protein	O
-in	O
-vivo	O
-.	O
-	O
-In	O
-agreement	O
-with	O
-the	O
-in	O
-vitro	O
-pull	O
--	O
-down	O
-results	O
-,	O
-the	O
-mutants	O
-D229A	B-mutant
-,	O
-W234D	B-mutant
-and	O
-Y259D	B-mutant
-were	O
-not	O
-co	O
--	O
-precipitated	O
-with	O
-MKP7	O
-,	O
-and	O
-the	O
-I231D	B-mutant
-mutant	O
-had	O
-only	O
-little	O
-effect	O
-on	O
-the	O
-JNK1	O
-–	O
-MKP7	O
-interaction	O
-(	O
-Fig	O
-.	O
-6d	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-Activation	O
-of	O
-the	O
-JNK	O
-signalling	O
-pathway	O
-is	O
-frequently	O
-associated	O
-with	O
-apoptotic	O
-cell	O
-death	O
-,	O
-and	O
-inhibition	O
-of	O
-JNK	O
-can	O
-prevent	O
-apoptotic	O
-death	O
-of	O
-multiple	O
-cells	O
-.	O
-	O
-To	O
-examine	O
-whether	O
-the	O
-inhibition	O
-of	O
-JNK	O
-activity	O
-by	O
-MKP7	O
-would	O
-provide	O
-protections	O
-against	O
-the	O
-apoptosis	O
-,	O
-we	O
-analysed	O
-the	O
-rate	O
-of	O
-apoptosis	O
-in	O
-ultraviolet	O
--	O
-irradiated	O
-cells	O
-transfected	O
-with	O
-MKP7	O
-(	O
-wild	O
-type	O
-or	O
-mutants	O
-)	O
-by	O
-flow	O
-cytometry	O
-.	O
-	O
-The	O
-results	O
-showed	O
-similar	O
-apoptotic	O
-rates	O
-between	O
-cells	O
-transfected	O
-with	O
-blank	O
-vector	O
-or	O
-with	O
-MKP7	O
-(	O
-wild	O
-type	O
-or	O
-mutants	O
-)	O
-under	O
-unstimulated	O
-conditions	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3b	O
-),	O
-while	O
-ultraviolet	O
--	O
-irradiation	O
-significantly	O
-increased	O
-apoptotic	O
-rate	O
-in	O
-cells	O
-transfected	O
-with	O
-blank	O
-vector	O
-(	O
-Fig	O
-.	O
-6e	O
-).	O
-	O
-Expressions	O
-of	O
-wild	O
--	O
-type	O
-MKP7	O
-,	O
-MKP7ΔC304	B-mutant
-and	O
-MKP7	O
--	O
-CD	O
-significantly	O
-decreased	O
-the	O
-proportion	O
-of	O
-apoptotic	O
-cells	O
-after	O
-ultraviolet	O
-treatment	O
-.	O
-	O
-Moreover	O
-,	O
-treatment	O
-of	O
-cells	O
-expressing	O
-MKP7	O
--	O
-KBD	O
-mutants	O
-(	O
-R56A	B-mutant
-/	O
-R57A	B-mutant
-and	O
-V63A	B-mutant
-/	O
-I65A	B-mutant
-)	O
-decreased	O
-the	O
-apoptosis	O
-rates	O
-to	O
-a	O
-similar	O
-extent	O
-as	O
-MKP7	O
-wild	O
-type	O
-did	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-cells	O
-transfected	O
-with	O
-the	O
-MKP7	O
-FXF	O
--	O
-motif	O
-mutants	O
-(	O
-F285D	B-mutant
-,	O
-F287D	B-mutant
-and	O
-L288D	B-mutant
-)	O
-showed	O
-little	O
-protective	O
-effect	O
-after	O
-ultraviolet	O
-treatment	O
-and	O
-similar	O
-levels	O
-of	O
-apoptosis	O
-rates	O
-were	O
-detected	O
-to	O
-cells	O
-transfected	O
-with	O
-control	O
-vectors	O
-(	O
-Fig	O
-.	O
-6e	O
-,	O
-f	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-our	O
-results	O
-suggested	O
-that	O
-FXF	O
--	O
-motif	O
--	O
-mediated	O
-,	O
-rather	O
-than	O
-KBD	O
--	O
-mediated	O
-,	O
-interaction	O
-is	O
-essential	O
-for	O
-MKP7	O
-to	O
-block	O
-ultraviolet	O
--	O
-induced	O
-apoptosis	O
-.	O
-	O
-A	O
-similar	O
-docking	O
-mechanism	O
-for	O
-JNK1	O
-recognition	O
-by	O
-MKP5	O
-	O
-MKP5	O
-belongs	O
-to	O
-the	O
-same	O
-subfamily	O
-as	O
-MKP7	O
-.	O
-	O
-MKP5	O
-is	O
-unique	O
-among	O
-the	O
-MKPs	O
-in	O
-possessing	O
-an	O
-additional	O
-domain	O
-of	O
-unknown	O
-function	O
-at	O
-the	O
-N	O
--	O
-terminus	O
-(	O
-Fig	O
-.	O
-7a	O
-).	O
-	O
-The	O
-KBD	O
-of	O
-MKP5	O
-interacts	O
-with	O
-the	O
-D	O
--	O
-site	O
-of	O
-p38α	O
-to	O
-mediate	O
-the	O
-enzyme	O
-–	O
-substrate	O
-interaction	O
-.	O
-	O
-Deletion	O
-of	O
-the	O
-KBD	O
-in	O
-MKP5	O
-leads	O
-to	O
-a	O
-280	O
--	O
-fold	O
-increase	O
-in	O
-Km	O
-for	O
-p38α	O
-substrate	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-p38α	O
-substrate	O
-,	O
-deletion	O
-of	O
-the	O
-MKP5	O
--	O
-KBD	O
-had	O
-little	O
-effects	O
-on	O
-the	O
-kinetic	O
-parameters	O
-for	O
-the	O
-JNK1	O
-dephosphorylation	O
-,	O
-indicating	O
-that	O
-the	O
-KBD	O
-of	O
-MKP5	O
-is	O
-not	O
-required	O
-for	O
-the	O
-JNK1	O
-dephosphorylation	O
-(	O
-Fig	O
-.	O
-7b	O
-).	O
-	O
-The	O
-substrate	O
-specificity	O
-constant	O
-kcat	O
-/	O
-Km	O
-value	O
-for	O
-MKP5	O
--	O
-CD	O
-was	O
-calculated	O
-as	O
-1	O
-.	O
-0	O
-×	O
-105	O
-M	O
-−	O
-1	O
-s	O
-−	O
-1	O
-,	O
-which	O
-is	O
-very	O
-close	O
-to	O
-that	O
-of	O
-MKP7	O
--	O
-CD	O
-(	O
-1	O
-.	O
-07	O
-×	O
-105	O
-M	O
-−	O
-1	O
-s	O
-−	O
-1	O
-).	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-human	O
-MKP5	O
--	O
-CD	O
-has	O
-been	O
-determined	O
-.	O
-	O
-Comparisons	O
-between	O
-catalytic	O
-domains	O
-structures	O
-of	O
-MKP5	O
-and	O
-MKP7	O
-reveal	O
-that	O
-the	O
-overall	O
-folds	O
-of	O
-the	O
-two	O
-proteins	O
-are	O
-highly	O
-similar	O
-,	O
-with	O
-only	O
-a	O
-few	O
-regions	O
-exhibiting	O
-small	O
-deviations	O
-(	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.	O
-of	O
-0	O
-.	O
-79	O
-Å	O
-;	O
-Fig	O
-.	O
-7c	O
-).	O
-	O
-Given	O
-the	O
-distinct	O
-interaction	O
-mode	O
-revealed	O
-by	O
-the	O
-crystal	O
-structure	O
-of	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-,	O
-one	O
-obvious	O
-question	O
-is	O
-whether	O
-this	O
-is	O
-a	O
-general	O
-mechanism	O
-used	O
-by	O
-all	O
-members	O
-of	O
-the	O
-JNK	O
--	O
-specific	O
-MKPs	O
-.	O
-	O
-To	O
-address	O
-this	O
-issue	O
-,	O
-we	O
-first	O
-examined	O
-the	O
-docking	O
-ability	O
-of	O
-JNK1	O
-to	O
-the	O
-KBD	O
-and	O
-CD	O
-of	O
-MKP5	O
-using	O
-gel	O
-filtration	O
-analysis	O
-and	O
-pull	O
--	O
-down	O
-assays	O
-.	O
-	O
-It	O
-can	O
-be	O
-seen	O
-from	O
-gel	O
-filtration	O
-experiments	O
-that	O
-JNK1	O
-can	O
-forms	O
-a	O
-stable	O
-heterodimer	O
-with	O
-MKP5	O
--	O
-CD	O
-in	O
-solution	O
-,	O
-but	O
-no	O
-detectable	O
-interaction	O
-was	O
-found	O
-with	O
-the	O
-KBD	O
-domain	O
-(	O
-Fig	O
-.	O
-7d	O
-).	O
-	O
-Pull	O
--	O
-down	O
-assays	O
-also	O
-confirmed	O
-the	O
-protein	O
-–	O
-protein	O
-interactions	O
-observed	O
-above	O
-.	O
-	O
-The	O
-catalytic	O
-domain	O
-of	O
-MKP5	O
-,	O
-but	O
-not	O
-its	O
-KBD	O
-,	O
-was	O
-able	O
-to	O
-pull	O
--	O
-down	O
-a	O
-detectable	O
-amount	O
-of	O
-JNK1	O
-(	O
-Fig	O
-.	O
-7e	O
-),	O
-implicating	O
-a	O
-different	O
-substrate	O
--	O
-recognition	O
-mechanisms	O
-for	O
-p38	O
-and	O
-JNK	O
-MAPKs	O
-.	O
-	O
-To	O
-further	O
-test	O
-our	O
-hypothesis	O
-,	O
-we	O
-generated	O
-forms	O
-of	O
-MKP5	O
--	O
-CD	O
-bearing	O
-mutations	O
-corresponding	O
-to	O
-the	O
-changes	O
-we	O
-made	O
-on	O
-MKP7	O
--	O
-CD	O
-on	O
-the	O
-basis	O
-of	O
-sequence	O
-and	O
-structural	O
-alignment	O
-and	O
-examined	O
-their	O
-effects	O
-on	O
-the	O
-phosphatase	O
-activity	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-.	O
-7f	O
-,	O
-the	O
-T432A	B-mutant
-and	O
-L449F	B-mutant
-MKP5	O
-mutant	O
-showed	O
-little	O
-or	O
-no	O
-difference	O
-in	O
-phosphatase	O
-activity	O
-,	O
-whereas	O
-the	O
-other	O
-mutants	O
-showed	O
-reduced	O
-specific	O
-activities	O
-of	O
-MKP5	O
-.	O
-	O
-As	O
-in	O
-the	O
-case	O
-of	O
-MKP7	O
-,	O
-all	O
-the	O
-mutants	O
-,	O
-except	O
-F451D	B-mutant
-/	I-mutant
-A	I-mutant
-,	O
-showed	O
-no	O
-pNPPase	O
-activity	O
-changes	O
-compared	O
-with	O
-the	O
-wild	O
--	O
-type	O
-MKP5	O
--	O
-CD	O
-(	O
-Fig	O
-.	O
-7g	O
-),	O
-and	O
-the	O
-point	O
-mutations	O
-in	O
-JNK1	O
-also	O
-reduced	O
-the	O
-binding	O
-affinity	O
-of	O
-MKP5	O
--	O
-CD	O
-for	O
-JNK1	O
-(	O
-Fig	O
-.	O
-7h	O
-).	O
-	O
-In	O
-addition	O
-,	O
-there	O
-were	O
-no	O
-significant	O
-differences	O
-in	O
-the	O
-CD	O
-spectra	O
-between	O
-wild	O
--	O
-type	O
-and	O
-mutant	O
-proteins	O
-,	O
-indicating	O
-that	O
-the	O
-overall	O
-structures	O
-of	O
-these	O
-mutants	O
-did	O
-not	O
-change	O
-significantly	O
-from	O
-that	O
-of	O
-wild	O
--	O
-type	O
-MKP5	O
-protein	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-our	O
-results	O
-suggest	O
-that	O
-MKP5	O
-binds	O
-JNK1	O
-in	O
-a	O
-docking	O
-mode	O
-similar	O
-to	O
-that	O
-in	O
-the	O
-JNK1	O
-–	O
-MKP7	O
-complex	O
-,	O
-and	O
-the	O
-detailed	O
-interaction	O
-model	O
-can	O
-be	O
-generated	O
-using	O
-molecular	O
-dynamics	O
-simulation	O
-based	O
-on	O
-the	O
-structure	O
-of	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-complex	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4b	O
-,	O
-c	O
-).	O
-	O
-In	O
-this	O
-model	O
-,	O
-the	O
-MKP5	O
--	O
-CD	O
-adopts	O
-a	O
-conformation	O
-nearly	O
-identical	O
-to	O
-that	O
-in	O
-its	O
-unbound	O
-form	O
-,	O
-suggesting	O
-that	O
-the	O
-conformation	O
-of	O
-the	O
-catalytic	O
-domain	O
-undergoes	O
-little	O
-change	O
-,	O
-if	O
-any	O
-at	O
-all	O
-,	O
-upon	O
-JNK1	O
-binding	O
-.	O
-	O
-In	O
-particular	O
-,	O
-Leu449	O
-of	O
-MKP5	O
-,	O
-which	O
-is	O
-equivalent	O
-to	O
-the	O
-key	O
-residue	O
-Phe285	O
-of	O
-MKP7	O
-,	O
-buried	O
-deeply	O
-within	O
-the	O
-hydrophobic	O
-pocket	O
-of	O
-JNK1	O
-in	O
-the	O
-same	O
-way	O
-as	O
-Phe285	O
-in	O
-the	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-complex	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4d	O
-).	O
-	O
-Despite	O
-the	O
-strong	O
-similarities	O
-between	O
-JNK1	O
-–	O
-MKP5	O
--	O
-CD	O
-and	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-,	O
-however	O
-,	O
-there	O
-are	O
-differences	O
-.	O
-	O
-The	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-interaction	O
-is	O
-better	O
-and	O
-more	O
-extensive	O
-.	O
-	O
-Asp268	O
-of	O
-MKP7	O
--	O
-CD	O
-forms	O
-salt	O
-bridge	O
-with	O
-JNK1	O
-Arg263	O
-,	O
-whereas	O
-the	O
-corresponding	O
-residue	O
-Thr432	O
-in	O
-MKP5	O
--	O
-CD	O
-may	O
-not	O
-interact	O
-with	O
-JNK1	O
-.	O
-	O
-In	O
-addition	O
-,	O
-the	O
-key	O
-interacting	O
-residues	O
-of	O
-MKP7	O
--	O
-CD	O
-,	O
-Phe215	O
-,	O
-Leu267	O
-and	O
-Leu288	O
-,	O
-are	O
-replaced	O
-by	O
-less	O
-hydrophobic	O
-residues	O
-,	O
-Asn379	O
-,	O
-Met431	O
-and	O
-Met452	O
-in	O
-MKP5	O
--	O
-CD	O
-(	O
-Fig	O
-.	O
-5c	O
-),	O
-respectively	O
-,	O
-which	O
-may	O
-result	O
-in	O
-weaker	O
-hydrophobic	O
-interactions	O
-between	O
-MKP5	O
--	O
-CD	O
-and	O
-JNK1	O
-.	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-the	O
-experimental	O
-observation	O
-showing	O
-that	O
-JNK1	O
-binds	O
-to	O
-MKP7	O
--	O
-CD	O
-much	O
-more	O
-tightly	O
-than	O
-MKP5	O
--	O
-CD	O
-(	O
-Km	O
-value	O
-of	O
-MKP5	O
--	O
-CD	O
-for	O
-pJNK1	O
-substrate	O
-is	O
-∼	O
-20	O
--	O
-fold	O
-higher	O
-than	O
-that	O
-of	O
-MKP7	O
--	O
-CD	O
-).	O
-	O
-The	O
-MAPKs	O
-p38	O
-,	O
-ERK	O
-and	O
-JNK	O
-,	O
-are	O
-central	O
-to	O
-evolutionarily	O
-conserved	O
-signalling	O
-pathways	O
-that	O
-are	O
-present	O
-in	O
-all	O
-eukaryotic	O
-cells	O
-.	O
-	O
-Each	O
-MAPK	O
-cascade	O
-is	O
-activated	O
-in	O
-response	O
-to	O
-a	O
-diverse	O
-array	O
-of	O
-extracellular	O
-signals	O
-and	O
-culminates	O
-in	O
-the	O
-dual	O
--	O
-phosphorylation	O
-of	O
-a	O
-threonine	O
-and	O
-a	O
-tyrosine	O
-residue	O
-in	O
-the	O
-MAPK	O
--	O
-activation	O
-loop	O
-.	O
-	O
-The	O
-propagation	O
-of	O
-MAPK	O
-signals	O
-is	O
-attenuated	O
-through	O
-the	O
-actions	O
-of	O
-the	O
-MKPs	O
-.	O
-	O
-Most	O
-studies	O
-have	O
-focused	O
-on	O
-the	O
-dephosphorylation	O
-of	O
-MAPKs	O
-by	O
-phosphatases	O
-containing	O
-the	O
-‘	O
-kinase	O
--	O
-interaction	O
-motif	O
-'	O
-(	O
-D	O
--	O
-motif	O
-),	O
-including	O
-a	O
-group	O
-of	O
-DUSPs	O
-(	O
-MKPs	O
-)	O
-and	O
-a	O
-distinct	O
-subfamily	O
-of	O
-tyrosine	O
-phosphatases	O
-(	O
-HePTP	O
-,	O
-STEP	O
-and	O
-PTP	O
--	O
-SL	O
-).	O
-	O
-Crystal	O
-structures	O
-of	O
-ERK2	O
-bound	O
-with	O
-the	O
-D	O
--	O
-motif	O
-sequences	O
-derived	O
-from	O
-MKP3	O
-and	O
-HePTP	O
-have	O
-been	O
-reported	O
-.	O
-	O
-These	O
-structures	O
-revealed	O
-that	O
-linear	O
-docking	O
-motifs	O
-in	O
-interacting	O
-proteins	O
-bind	O
-to	O
-a	O
-common	O
-docking	O
-site	O
-on	O
-MAPKs	O
-outside	O
-the	O
-kinase	O
-active	O
-site	O
-.	O
-	O
-The	O
-particular	O
-amino	O
-acids	O
-and	O
-their	O
-spacing	O
-within	O
-D	O
--	O
-motif	O
-sequences	O
-and	O
-amino	O
-acid	O
-composition	O
-of	O
-the	O
-docking	O
-sites	O
-on	O
-MAPKs	O
-appear	O
-to	O
-determine	O
-the	O
-specificity	O
-of	O
-D	O
--	O
-motifs	O
-for	O
-individual	O
-MAPKs	O
-.	O
-	O
-Recently	O
-,	O
-the	O
-crystal	O
-structure	O
-of	O
-a	O
-complex	O
-between	O
-the	O
-KBD	O
-of	O
-MKP5	O
-and	O
-p38α	O
-has	O
-been	O
-obtained	O
-.	O
-	O
-This	O
-complex	O
-has	O
-revealed	O
-a	O
-distinct	O
-interaction	O
-mode	O
-for	O
-MKP5	O
-.	O
-	O
-The	O
-KBD	O
-of	O
-MKP5	O
-binds	O
-to	O
-p38α	O
-in	O
-the	O
-opposite	O
-polypeptide	O
-direction	O
-compared	O
-with	O
-how	O
-the	O
-D	O
--	O
-motif	O
-of	O
-MKP3	O
-binds	O
-to	O
-ERK2	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-the	O
-canonical	O
-D	O
--	O
-motif	O
--	O
-binding	O
-mode	O
-,	O
-separate	O
-helices	O
-,	O
-α2	O
-and	O
-α3	O
-′,	O
-in	O
-the	O
-KBD	O
-of	O
-MKP5	O
-engage	O
-the	O
-p38α	O
--	O
-docking	O
-site	O
-.	O
-	O
-Further	O
-structural	O
-and	O
-biochemical	O
-studies	O
-indicate	O
-that	O
-KBD	O
-of	O
-MKP7	O
-may	O
-interact	O
-with	O
-p38α	O
-in	O
-a	O
-similar	O
-manner	O
-to	O
-that	O
-of	O
-MKP5	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-MKP5	O
-,	O
-removal	O
-of	O
-the	O
-KBD	O
-domain	O
-from	O
-MKP7	O
-does	O
-not	O
-drastically	O
-affect	O
-enzyme	O
-catalysis	O
-,	O
-and	O
-the	O
-kinetic	O
-parameters	O
-of	O
-MKP7	O
--	O
-CD	O
-for	O
-p38α	O
-substrate	O
-are	O
-very	O
-similar	O
-to	O
-those	O
-for	O
-JNK1	O
-substrate	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-these	O
-results	O
-suggest	O
-that	O
-MKP7	O
-utilizes	O
-a	O
-bipartite	O
-recognition	O
-mechanism	O
-to	O
-achieve	O
-the	O
-efficiency	O
-and	O
-fidelity	O
-of	O
-p38α	O
-signalling	O
-.	O
-	O
-The	O
-MKP7	O
--	O
-KBD	O
-docks	O
-to	O
-the	O
-D	O
--	O
-site	O
-located	O
-on	O
-the	O
-back	O
-side	O
-of	O
-the	O
-p38α	O
-catalytic	O
-pocket	O
-for	O
-high	O
--	O
-affinity	O
-association	O
-,	O
-whereas	O
-the	O
-interaction	O
-of	O
-the	O
-MKP7	O
--	O
-CD	O
-with	O
-another	O
-p38α	O
-structural	O
-region	O
-,	O
-which	O
-is	O
-close	O
-to	O
-the	O
-activation	O
-loop	O
-,	O
-may	O
-not	O
-only	O
-stabilize	O
-binding	O
-but	O
-also	O
-provide	O
-contacts	O
-crucial	O
-for	O
-organizing	O
-the	O
-MKP7	O
-active	O
-site	O
-with	O
-respect	O
-to	O
-the	O
-phosphoreceptor	O
-in	O
-the	O
-p38α	O
-substrate	O
-for	O
-efficient	O
-dephosphorylation	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-canonical	O
-D	O
--	O
-site	O
-,	O
-the	O
-MAPK	O
-ERK2	O
-contains	O
-a	O
-second	O
-binding	O
-site	O
-utilized	O
-by	O
-transcription	O
-factor	O
-substrates	O
-and	O
-phosphatases	O
-,	O
-the	O
-FXF	O
--	O
-motif	O
--	O
-binding	O
-site	O
-(	O
-also	O
-called	O
-F	O
--	O
-site	O
-),	O
-that	O
-is	O
-exposed	O
-in	O
-active	O
-ERK2	O
-and	O
-the	O
-D	O
--	O
-motif	O
-peptide	O
--	O
-induced	O
-conformation	O
-of	O
-MAPKs	O
-.	O
-	O
-This	O
-hydrophobic	O
-site	O
-was	O
-first	O
-identified	O
-by	O
-changes	O
-in	O
-deuterium	O
-exchange	O
-profiles	O
-,	O
-and	O
-is	O
-near	O
-the	O
-MAPK	O
-insertion	O
-and	O
-helix	O
-αG	O
-.	O
-Interestingly	O
-,	O
-many	O
-of	O
-the	O
-equivalent	O
-residues	O
-in	O
-JNK1	O
-,	O
-important	O
-for	O
-MKP7	O
--	O
-CD	O
-recognition	O
-,	O
-are	O
-also	O
-used	O
-for	O
-substrate	O
-binding	O
-by	O
-ERK2	O
-(	O
-ref	O
-.),	O
-indicating	O
-that	O
-this	O
-site	O
-is	O
-overlapped	O
-with	O
-the	O
-DEF	O
--	O
-site	O
-previously	O
-identified	O
-in	O
-ERK2	O
-(	O
-Fig	O
-.	O
-5d	O
-).	O
-	O
-MKP3	O
-is	O
-highly	O
-specific	O
-in	O
-dephosphorylating	O
-and	O
-inactivating	O
-ERK2	O
-,	O
-and	O
-the	O
-phosphatase	O
-activity	O
-of	O
-the	O
-MKP3	O
--	O
-catalysed	O
-pNPP	O
-reaction	O
-can	O
-be	O
-markedly	O
-increased	O
-in	O
-the	O
-presence	O
-of	O
-ERK2	O
-(	O
-refs	O
-).	O
-	O
-Sequence	O
-alignment	O
-of	O
-all	O
-MKPs	O
-reveals	O
-a	O
-high	O
-degree	O
-of	O
-conservation	O
-of	O
-residues	O
-surrounding	O
-the	O
-interacting	O
-region	O
-observed	O
-in	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-complex	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-Therefore	O
-,	O
-it	O
-is	O
-tempting	O
-to	O
-speculate	O
-that	O
-the	O
-catalytic	O
-domain	O
-of	O
-MKP3	O
-may	O
-bind	O
-to	O
-ERK2	O
-in	O
-a	O
-manner	O
-analogous	O
-to	O
-the	O
-way	O
-by	O
-which	O
-MKP7	O
--	O
-CD	O
-binds	O
-to	O
-JNK1	O
-.	O
-	O
-A	O
-comprehensive	O
-examination	O
-of	O
-the	O
-molecular	O
-basis	O
-of	O
-the	O
-specific	O
-ERK2	O
-recognition	O
-by	O
-MKP3	O
-is	O
-underway	O
-.	O
-	O
-The	O
-ongoing	O
-work	O
-demonstrates	O
-that	O
-although	O
-the	O
-overall	O
-interaction	O
-modes	O
-are	O
-similar	O
-between	O
-the	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-and	O
-ERK2	O
-–	O
-MKP3	O
--	O
-CD	O
-complexes	O
-,	O
-the	O
-ERK2	O
-–	O
-MKP3	O
--	O
-CD	O
-interaction	O
-is	O
-less	O
-extensive	O
-and	O
-helix	O
-α4	O
-from	O
-MKP3	O
--	O
-CD	O
-does	O
-not	O
-interact	O
-directly	O
-with	O
-ERK2	O
-.	O
-	O
-The	O
-FXF	O
--	O
-motif	O
--	O
-mediated	O
-interaction	O
-is	O
-critical	O
-for	O
-both	O
-pERK2	O
-inactivation	O
-and	O
-ERK2	O
--	O
-induced	O
-MKP3	O
-activation	O
-(	O
-manuscript	O
-in	O
-preparation	O
-).	O
-	O
-In	O
-summary	O
-,	O
-we	O
-have	O
-resolved	O
-the	O
-structure	O
-of	O
-JNK1	O
-in	O
-complex	O
-with	O
-the	O
-catalytic	O
-domain	O
-of	O
-MKP7	O
-.	O
-	O
-This	O
-structure	O
-reveals	O
-an	O
-FXF	O
--	O
-docking	O
-interaction	O
-mode	O
-between	O
-MAPK	O
-and	O
-MKP	O
-.	O
-	O
-Results	O
-from	O
-biochemical	O
-characterization	O
-of	O
-the	O
-Phe285	O
-and	O
-Phe287	O
-MKP7	O
-mutants	O
-combined	O
-with	O
-structural	O
-information	O
-support	O
-the	O
-conclusion	O
-that	O
-the	O
-two	O
-Phe	O
-residues	O
-serve	O
-different	O
-roles	O
-in	O
-the	O
-catalytic	O
-reaction	O
-.	O
-	O
-Phe285	O
-is	O
-essential	O
-for	O
-JNK1	O
-substrate	O
-binding	O
-,	O
-whereas	O
-Phe287	O
-plays	O
-a	O
-role	O
-for	O
-the	O
-precise	O
-alignment	O
-of	O
-active	O
--	O
-site	O
-residues	O
-,	O
-which	O
-are	O
-important	O
-for	O
-transition	O
--	O
-state	O
-stabilization	O
-.	O
-	O
-This	O
-newly	O
-identified	O
-FXF	O
--	O
-type	O
-motif	O
-is	O
-present	O
-in	O
-all	O
-MKPs	O
-,	O
-except	O
-that	O
-the	O
-residue	O
-at	O
-the	O
-first	O
-position	O
-in	O
-MKP5	O
-is	O
-an	O
-equivalent	O
-hydrophobic	O
-leucine	O
-residue	O
-(	O
-see	O
-also	O
-Fig	O
-.	O
-7f	O
-,	O
-g	O
-),	O
-suggesting	O
-that	O
-these	O
-two	O
-Phe	O
-residues	O
-would	O
-play	O
-a	O
-similar	O
-role	O
-in	O
-facilitating	O
-substrate	O
-recognition	O
-and	O
-catalysis	O
-,	O
-respectively	O
-.	O
-	O
-An	O
-important	O
-feature	O
-of	O
-MKP	O
-–	O
-JNK1	O
-interactions	O
-is	O
-that	O
-MKP7	O
-or	O
-MKP5	O
-only	O
-interact	O
-with	O
-the	O
-F	O
--	O
-site	O
-of	O
-JNK1	O
-.	O
-	O
-One	O
-possible	O
-explanation	O
-is	O
-that	O
-JNK1	O
-needs	O
-to	O
-use	O
-the	O
-D	O
--	O
-site	O
-to	O
-interact	O
-with	O
-JIP	O
--	O
-1	O
-,	O
-a	O
-scaffold	O
-protein	O
-for	O
-JNK	O
-signalling	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-JNK	O
--	O
-binding	O
-domain	O
-of	O
-JIP	O
--	O
-1	O
-interacts	O
-with	O
-the	O
-D	O
--	O
-site	O
-on	O
-JNK	O
-and	O
-this	O
-interaction	O
-is	O
-required	O
-for	O
-JIP	O
--	O
-1	O
--	O
-mediated	O
-enhancement	O
-of	O
-JNK	O
-activation	O
-.	O
-	O
-In	O
-addition	O
-,	O
-JIP	O
--	O
-1	O
-can	O
-also	O
-associate	O
-with	O
-MKP7	O
-via	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-MKP7	O
-(	O
-ref	O
-.).	O
-	O
-When	O
-MKP7	O
-is	O
-bound	O
-to	O
-JIP	O
--	O
-1	O
-,	O
-it	O
-reduces	O
-JNK	O
-activation	O
-,	O
-leading	O
-to	O
-reduced	O
-phosphorylation	O
-of	O
-the	O
-JNK	O
-target	O
-c	O
--	O
-Jun	O
-.	O
-	O
-Thus	O
-,	O
-our	O
-biochemical	O
-and	O
-structural	O
-data	O
-allow	O
-us	O
-to	O
-present	O
-a	O
-model	O
-for	O
-the	O
-JNK1	O
-–	O
-JIP	O
--	O
-1	O
-–	O
-MKP7	O
-ternary	O
-complex	O
-and	O
-provide	O
-an	O
-important	O
-insight	O
-into	O
-the	O
-assembly	O
-and	O
-function	O
-of	O
-JNK	O
-signalling	O
-modules	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-The	O
-cDNAs	O
-of	O
-human	O
-MKP7	O
-and	O
-MKP5	O
-were	O
-kindly	O
-provided	O
-by	O
-Dr	O
-Mathijs	O
-Baens	O
-(	O
-University	O
-of	O
-Leuven	O
-)	O
-and	O
-Dr	O
-Eisuke	O
-Nishida	O
-(	O
-Kyoto	O
-University	O
-),	O
-respectively	O
-.	O
-	O
-The	O
-cDNAs	O
-of	O
-human	O
-ASK1	O
-,	O
-MKK4	O
-,	O
-MKK7	O
-and	O
-JNK1	O
-were	O
-kindly	O
-provided	O
-by	O
-Dr	O
-Zhenguo	O
-Wu	O
-(	O
-Hong	O
-Kong	O
-University	O
-of	O
-Science	O
-and	O
-Technology	O
-).	O
-	O
-The	O
-human	O
-full	O
--	O
-length	O
-JNK1	O
-,	O
-MKK4	O
-,	O
-MKK7	O
-and	O
-the	O
-kinase	O
-domain	O
-of	O
-ASK1	O
-(	O
-659	O
-–	O
-951	O
-)	O
-were	O
-cloned	O
-into	O
-pGEX4T	O
--	O
-2	O
-,	O
-pET15b	O
-and	O
-/	O
-or	O
-pET21b	O
-vectors	O
-to	O
-produce	O
-a	O
-GST	O
--	O
-or	O
-His	O
--	O
-tagged	O
-protein	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-unphosphorylated	O
-JNK1	O
-in	O
-complex	O
-with	O
-the	O
-catalytic	O
-domain	O
-of	O
-MKP7	O
-was	O
-refined	O
-to	O
-2	O
-.	O
-4	O
-Å	O
-resolution	O
-.	O
-	O
-Domain	O
-structures	O
-of	O
-ten	O
-human	O
-MKPs	O
-and	O
-the	O
-atypical	O
-VHR	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-sequence	O
-similarity	O
-,	O
-protein	O
-structure	O
-,	O
-substrate	O
-specificity	O
-and	O
-subcellular	O
-localization	O
-,	O
-the	O
-ten	O
-members	O
-of	O
-MKP	O
-family	O
-can	O
-be	O
-divided	O
-into	O
-three	O
-groups	O
-.	O
-	O
-The	O
-first	O
-subfamily	O
-comprises	O
-MKP1	O
-,	O
-MKP2	O
-,	O
-PAC1	O
-and	O
-hVH3	O
-,	O
-which	O
-are	O
-inducible	O
-nuclear	O
-phosphatases	O
-and	O
-can	O
-dephosphorylate	O
-ERK	O
-(	O
-and	O
-JNK	O
-,	O
-p38	O
-)	O
-MAPKs	O
-.	O
-	O
-The	O
-second	O
-subfamily	O
-contains	O
-MKP3	O
-,	O
-MKP4	O
-and	O
-MKPX	O
-,	O
-which	O
-are	O
-cytoplasmic	O
-ERK	O
--	O
-specific	O
-MKPs	O
-.	O
-	O
-The	O
-third	O
-subfamily	O
-comprises	O
-MKP5	O
-,	O
-MKP7	O
-and	O
-hVH5	O
-,	O
-which	O
-were	O
-located	O
-in	O
-both	O
-nucleus	O
-and	O
-cytoplasm	O
-,	O
-and	O
-selectively	O
-inactivate	O
-JNK	O
-and	O
-p38	O
-.	O
-	O
-All	O
-MKPs	O
-contain	O
-both	O
-the	O
-CD	O
-and	O
-KBD	O
-domains	O
-,	O
-whereas	O
-VHR	O
-,	O
-an	O
-atypical	O
-MKP	O
-,	O
-only	O
-contains	O
-a	O
-highly	O
-conserved	O
-catalytic	O
-domain	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-CD	O
-and	O
-KBD	O
-,	O
-MKP7	O
-contains	O
-a	O
-unique	O
-long	O
-C	O
--	O
-terminal	O
-region	O
-that	O
-contains	O
-NES	O
-,	O
-NLS	O
-and	O
-PEST	O
-motifs	O
-,	O
-which	O
-has	O
-no	O
-effect	O
-on	O
-the	O
-binding	O
-ability	O
-and	O
-phosphatase	O
-activity	O
-of	O
-MKP7	O
-toward	O
-MAPKs	O
-.	O
-	O
-NES	O
-,	O
-nuclear	O
-export	O
-signal	O
-;	O
-NLS	O
-,	O
-nuclear	O
-localization	O
-signal	O
-;	O
-PEST	O
-,	O
-C	O
--	O
-terminal	O
-sequence	O
-rich	O
-in	O
-prolines	O
-,	O
-glutamates	O
-,	O
-serines	O
-and	O
-threonines	O
-.	O
-	O
-MKP7	O
--	O
-CD	O
-is	O
-crucial	O
-for	O
-JNK1	O
-binding	O
-and	O
-enzyme	O
-catalysis	O
-.	O
-	O
-(	O
-a	O
-)	O
-Domain	O
-organization	O
-of	O
-human	O
-MKP7	O
-and	O
-JNK1	O
-.	O
-	O
-The	O
-KBD	O
-and	O
-CD	O
-of	O
-MKP7	O
-are	O
-shown	O
-in	O
-green	O
-and	O
-blue	O
-,	O
-and	O
-the	O
-N	O
--	O
-lobe	O
-and	O
-C	O
--	O
-lobe	O
-of	O
-JNK1	O
-are	O
-coloured	O
-in	O
-lemon	O
-and	O
-yellow	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-colour	O
-scheme	O
-is	O
-the	O
-same	O
-in	O
-the	O
-following	O
-figures	O
-unless	O
-indicated	O
-otherwise	O
-.	O
-(	O
-b	O
-)	O
-Plots	O
-of	O
-initial	O
-velocity	O
-of	O
-the	O
-MKP7	O
--	O
-catalysed	O
-reaction	O
-versus	O
-phospho	O
--	O
-JNK1	O
-concentration	O
-.	O
-	O
-The	O
-error	O
-bars	O
-represent	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.	O
-(	O
-c	O
-)	O
-Gel	O
-filtration	O
-analysis	O
-for	O
-interaction	O
-of	O
-JNK1	O
-with	O
-MKP7	O
--	O
-CD	O
-and	O
-MKP7	O
--	O
-KBD	O
-.	O
-	O
-(	O
-d	O
-)	O
-GST	O
--	O
-mediated	O
-pull	O
--	O
-down	O
-assay	O
-for	O
-interaction	O
-of	O
-JNK1	O
-with	O
-MKP7	O
--	O
-CD	O
-and	O
-MKP7	O
--	O
-KBD	O
-.	O
-	O
-The	O
-top	O
-panel	O
-shows	O
-the	O
-relative	O
-affinities	O
-of	O
-MKP7	O
--	O
-CD	O
-and	O
-MKP7	O
--	O
-KBD	O
-to	O
-JNK1	O
-,	O
-with	O
-the	O
-affinity	O
-of	O
-MKP7	O
--	O
-CD	O
-defined	O
-as	O
-100	O
-%;	O
-the	O
-middle	O
-panel	O
-is	O
-the	O
-electrophoretic	O
-pattern	O
-of	O
-MKP7	O
-and	O
-JNK1	O
-after	O
-GST	O
-pull	O
--	O
-down	O
-assays	O
-.	O
-	O
-The	O
-protein	O
-amounts	O
-of	O
-MKP7	O
-used	O
-are	O
-shown	O
-at	O
-the	O
-bottom	O
-.	O
-	O
-Structure	O
-of	O
-JNK1	O
-in	O
-complex	O
-with	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-(	O
-a	O
-)	O
-Ribbon	O
-diagram	O
-of	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-complex	O
-in	O
-two	O
-views	O
-related	O
-by	O
-a	O
-45	O
-°	O
-rotation	O
-around	O
-a	O
-vertical	O
-axis	O
-.	O
-(	O
-b	O
-)	O
-Structure	O
-of	O
-MKP7	O
--	O
-CD	O
-with	O
-its	O
-active	O
-site	O
-highlight	O
-in	O
-cyan	O
-.	O
-	O
-The	O
-2Fo	O
-−	O
-Fc	O
-omit	O
-map	O
-(	O
-contoured	O
-at	O
-1	O
-.	O
-5σ	O
-)	O
-for	O
-the	O
-P	O
--	O
-loop	O
-of	O
-MKP7	O
--	O
-CD	O
-is	O
-shown	O
-at	O
-inset	O
-of	O
-b	O
-.	O
-(	O
-c	O
-)	O
-Structure	O
-of	O
-VHR	O
-with	O
-its	O
-active	O
-site	O
-highlighted	O
-in	O
-marine	O
-blue	O
-.	O
-(	O
-d	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-JNK1	O
-–	O
-MKP7	O
-interface	O
-showing	O
-interacting	O
-amino	O
-acids	O
-of	O
-JNK1	O
-(	O
-orange	O
-)	O
-and	O
-MKP7	O
--	O
-CD	O
-(	O
-cyan	O
-).	O
-	O
-The	O
-JNK1	O
-is	O
-shown	O
-in	O
-surface	O
-representation	O
-coloured	O
-according	O
-to	O
-electrostatic	O
-potential	O
-(	O
-positive	O
-,	O
-blue	O
-;	O
-negative	O
-,	O
-red	O
-).	O
-	O
-(	O
-e	O
-)	O
-Interaction	O
-networks	O
-mainly	O
-involving	O
-helices	O
-α4	O
-and	O
-α5	O
-from	O
-MKP7	O
--	O
-CD	O
-,	O
-and	O
-αG	O
-and	O
-α2L14	O
-of	O
-JNK1	O
-.	O
-	O
-MKP7	O
--	O
-CD	O
-is	O
-shown	O
-in	O
-surface	O
-representation	O
-coloured	O
-according	O
-to	O
-electrostatic	O
-potential	O
-(	O
-positive	O
-,	O
-blue	O
-;	O
-negative	O
-,	O
-red	O
-).	O
-	O
-Blue	O
-dashed	O
-lines	O
-represent	O
-polar	O
-interactions	O
-.	O
-	O
-(	O
-f	O
-)	O
-The	O
-2Fo	O
-−	O
-Fc	O
-omit	O
-map	O
-(	O
-contoured	O
-at	O
-1	O
-.	O
-5σ	O
-)	O
-clearly	O
-shows	O
-electron	O
-density	O
-for	O
-the	O
-285FNFL288	O
-segment	O
-of	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-Mutational	O
-analysis	O
-on	O
-interactions	O
-between	O
-MKP7	O
--	O
-CD	O
-and	O
-JNK1	O
-.	O
-	O
-(	O
-a	O
-)	O
-Effects	O
-of	O
-mutations	O
-in	O
-MKP7	O
--	O
-CD	O
-on	O
-the	O
-JNK1	O
-dephosphorylation	O
-(	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.,	O
-n	O
-=	O
-3	O
-).	O
-	O
-Residues	O
-involved	O
-in	O
-hydrophobic	O
-and	O
-hydrophilic	O
-contacts	O
-are	O
-coloured	O
-in	O
-red	O
-and	O
-blue	O
-,	O
-respectively	O
-.	O
-(	O
-b	O
-)	O
-Gel	O
-filtration	O
-analysis	O
-for	O
-interaction	O
-of	O
-JNK1	O
-with	O
-MKP7	O
--	O
-CD	O
-mutant	O
-F285D	B-mutant
-.	O
-	O
-Mutant	O
-F285D	B-mutant
-and	O
-JNK1	O
-were	O
-eluted	O
-as	O
-monomers	O
-,	O
-with	O
-the	O
-molecular	O
-masses	O
-of	O
-∼	O
-17	O
-and	O
-44	O
-kDa	O
-,	O
-respectively	O
-.	O
-	O
-However	O
-,	O
-in	O
-contrast	O
-to	O
-the	O
-wild	O
--	O
-type	O
-MKP7	O
--	O
-CD	O
-,	O
-mutant	O
-F285D	B-mutant
-did	O
-not	O
-co	O
--	O
-migrate	O
-with	O
-JNK1	O
-.	O
-	O
-(	O
-c	O
-)	O
-Pull	O
--	O
-down	O
-assays	O
-of	O
-MKP7	O
--	O
-CD	O
-by	O
-GST	O
--	O
-tagged	O
-JNK1	O
-mutants	O
-.	O
-	O
-The	O
-top	O
-panel	O
-shows	O
-the	O
-relative	O
-affinities	O
-of	O
-MKP7	O
--	O
-CD	O
-to	O
-JNK1	O
-mutants	O
-,	O
-with	O
-the	O
-affinity	O
-of	O
-wild	O
--	O
-type	O
-JNK1	O
-defined	O
-as	O
-100	O
-%,	O
-the	O
-middle	O
-panel	O
-is	O
-the	O
-electrophoretic	O
-pattern	O
-of	O
-MKP7	O
--	O
-CD	O
-and	O
-JNK1	O
-mutants	O
-after	O
-GST	O
-pull	O
--	O
-down	O
-assays	O
-.	O
-	O
-The	O
-protein	O
-amounts	O
-of	O
-MKP7	O
--	O
-CD	O
-used	O
-are	O
-shown	O
-at	O
-the	O
-bottom	O
-.	O
-(	O
-d	O
-)	O
-Circular	O
-dichroism	O
-spectra	O
-for	O
-MKP7	O
--	O
-CD	O
-wild	O
-type	O
-and	O
-mutants	O
-.	O
-	O
-Measurements	O
-were	O
-averaged	O
-for	O
-three	O
-scans	O
-.	O
-(	O
-e	O
-)	O
-Circular	O
-dichroism	O
-spectra	O
-for	O
-JNK1	O
-wild	O
-type	O
-and	O
-mutants	O
-.	O
-	O
-(	O
-f	O
-)	O
-Effects	O
-of	O
-mutations	O
-in	O
-MKP7	O
--	O
-CD	O
-on	O
-the	O
-pNPP	O
-hydrolysis	O
-reaction	O
-(	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.,	O
-n	O
-=	O
-3	O
-).	O
-	O
-Comparison	O
-of	O
-CDK2	O
--	O
-KAP	O
-and	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-(	O
-a	O
-)	O
-Superposition	O
-of	O
-the	O
-complex	O
-structures	O
-of	O
-CDK2	O
--	O
-KAP	O
-(	O
-PDB	O
-1FQ1	O
-)	O
-and	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-The	O
-N	O
--	O
-lobe	O
-and	O
-C	O
--	O
-lobe	O
-of	O
-CDK2	O
-are	O
-coloured	O
-in	O
-grey	O
-and	O
-pink	O
-,	O
-respectively	O
-,	O
-and	O
-KAP	O
-is	O
-coloured	O
-in	O
-green	O
-.	O
-	O
-The	O
-interactions	O
-between	O
-these	O
-two	O
-proteins	O
-consist	O
-of	O
-three	O
-discontinuous	O
-contact	O
-regions	O
-,	O
-centred	O
-at	O
-the	O
-multiple	O
-hydrogen	O
-bonds	O
-between	O
-the	O
-pThr160	O
-of	O
-CDK2	O
-and	O
-the	O
-active	O
-site	O
-of	O
-KAP	O
-(	O
-region	O
-I	O
-).	O
-	O
-Interestingly	O
-,	O
-the	O
-recognition	O
-of	O
-CDK2	O
-by	O
-KAP	O
-is	O
-augmented	O
-by	O
-a	O
-similar	O
-interface	O
-as	O
-that	O
-observed	O
-in	O
-the	O
-complex	O
-of	O
-JNK1	O
-and	O
-MKP7	O
--	O
-CD	O
-(	O
-region	O
-II	O
-).	O
-	O
-(	O
-b	O
-)	O
-Interactions	O
-networks	O
-at	O
-the	O
-auxiliary	O
-region	O
-II	O
-mainly	O
-involving	O
-helix	O
-α7	O
-from	O
-KAP	O
-and	O
-the	O
-αG	O
-helix	O
-and	O
-following	O
-L14	O
-loop	O
-of	O
-CDK2	O
-.	O
-	O
-The	O
-CDK2	O
-is	O
-shown	O
-in	O
-surface	O
-representation	O
-coloured	O
-according	O
-to	O
-the	O
-electrostatic	O
-potential	O
-(	O
-positive	O
-,	O
-blue	O
-;	O
-negative	O
-,	O
-red	O
-).	O
-	O
-Residues	O
-of	O
-KAP	O
-and	O
-CDK2	O
-are	O
-highlighted	O
-as	O
-green	O
-and	O
-red	O
-sticks	O
-,	O
-respectively	O
-.	O
-	O
-One	O
-remarkable	O
-difference	O
-between	O
-these	O
-two	O
-kinase	O
--	O
-phosphatase	O
-complexes	O
-is	O
-that	O
-helix	O
-α6	O
-of	O
-KAP	O
-(	O
-corresponding	O
-to	O
-helix	O
-α4	O
-of	O
-MKP7	O
--	O
-CD	O
-)	O
-plays	O
-little	O
-,	O
-if	O
-any	O
-,	O
-role	O
-in	O
-the	O
-formation	O
-of	O
-a	O
-stable	O
-heterodimer	O
-of	O
-CDK2	O
-and	O
-KAP	O
-.	O
-(	O
-c	O
-)	O
-Sequence	O
-alignment	O
-of	O
-the	O
-JNK	O
--	O
-interacting	O
-regions	O
-on	O
-MKPs	O
-.	O
-	O
-Residues	O
-of	O
-MKP7	O
--	O
-CD	O
-involved	O
-in	O
-JNK1	O
-recognition	O
-are	O
-indicated	O
-by	O
-cyan	O
-asterisks	O
-,	O
-and	O
-the	O
-conserved	O
-FXF	O
--	O
-motif	O
-is	O
-highlighted	O
-in	O
-cyan	O
-.	O
-	O
-The	O
-secondary	O
-structure	O
-assignments	O
-of	O
-MKP7	O
--	O
-CD	O
-and	O
-KAP	O
-are	O
-shown	O
-above	O
-and	O
-below	O
-each	O
-sequence	O
-.	O
-	O
-(	O
-d	O
-)	O
-Sequence	O
-alignment	O
-of	O
-the	O
-F	O
--	O
-site	O
-regions	O
-on	O
-MAPKs	O
-.	O
-	O
-Residues	O
-of	O
-JNK1	O
-involved	O
-in	O
-recognition	O
-of	O
-MKP7	O
-are	O
-indicated	O
-by	O
-orange	O
-asterisks	O
-,	O
-and	O
-those	O
-forming	O
-the	O
-F	O
--	O
-site	O
-are	O
-highlighted	O
-in	O
-yellow	O
-.	O
-	O
-FXF	O
--	O
-motif	O
-is	O
-critical	O
-for	O
-controlling	O
-the	O
-phosphorylation	O
-of	O
-JNK	O
-and	O
-ultraviolet	O
--	O
-induced	O
-apoptosis	O
-.	O
-	O
-(	O
-a	O
-–	O
-c	O
-)	O
-FXF	O
--	O
-motif	O
-is	O
-essential	O
-for	O
-the	O
-dephosphorylation	O
-of	O
-JNK	O
-by	O
-MKP7	O
-.	O
-	O
-HEK293T	O
-cells	O
-were	O
-infected	O
-with	O
-lentiviruses	O
-expressing	O
-MKP7	O
-and	O
-its	O
-mutants	O
-(	O
-1	O
-.	O
-0	O
-μg	O
-).	O
-	O
-After	O
-36	O
-h	O
-infection	O
-,	O
-cells	O
-were	O
-untreated	O
-in	O
-a	O
-,	O
-stimulated	O
-with	O
-30	O
-μM	O
-etoposide	O
-for	O
-3	O
-h	O
-in	O
-b	O
-or	O
-irradiated	O
-with	O
-25	O
-J	O
-m	O
-−	O
-2	O
-ultraviolet	O
-light	O
-at	O
-30	O
-min	O
-before	O
-lysis	O
-in	O
-c	O
-.	O
-Whole	O
--	O
-cell	O
-extracts	O
-were	O
-then	O
-immunoblotted	O
-with	O
-antibody	O
-indicated	O
-.	O
-	O
-Shown	O
-is	O
-a	O
-typical	O
-immunoblot	O
-for	O
-phosphorylated	O
-JNK	O
-from	O
-three	O
-independent	O
-experiments	O
-.	O
-	O
-(	O
-d	O
-)	O
-F	O
--	O
-site	O
-is	O
-required	O
-for	O
-JNK1	O
-to	O
-interact	O
-with	O
-MKP7	O
-.	O
-	O
-HEK293T	O
-cells	O
-were	O
-co	O
--	O
-transfected	O
-with	O
-MKP7	O
-full	O
--	O
-length	O
-(	O
-1	O
-.	O
-0	O
-μg	O
-)	O
-and	O
-JNK1	O
-(	O
-wild	O
-type	O
-or	O
-mutants	O
-as	O
-indicated	O
-,	O
-1	O
-.	O
-0	O
-μg	O
-).	O
-	O
-Whole	O
--	O
-cell	O
-extracts	O
-were	O
-then	O
-immunoprecipitated	O
-with	O
-antibody	O
-against	O
-Myc	O
-for	O
-MKP7	O
-;	O
-immunobloting	O
-was	O
-carried	O
-out	O
-with	O
-antibodies	O
-indicated	O
-.	O
-	O
-IP	O
-,	O
-immunoprecipitation	O
-;	O
-TCL	O
-,	O
-total	O
-cell	O
-lysate	O
-.	O
-	O
-(	O
-e	O
-)	O
-Effect	O
-of	O
-MKP7	O
-(	O
-wild	O
-type	O
-or	O
-mutants	O
-)	O
-expression	O
-on	O
-ultraviolet	O
--	O
-induced	O
-apoptosis	O
-.	O
-	O
-HeLa	O
-cells	O
-were	O
-infected	O
-with	O
-lentiviruses	O
-expressing	O
-MKP7	O
-full	O
--	O
-length	O
-and	O
-its	O
-mutants	O
-.	O
-	O
-Cells	O
-were	O
-then	O
-subjected	O
-to	O
-flow	O
-cytometry	O
-analysis	O
-.	O
-	O
-Apoptotic	O
-cells	O
-were	O
-determined	O
-by	O
-Annexin	O
--	O
-V	O
--	O
-APC	O
-/	O
-PI	O
-staining	O
-.	O
-	O
-The	O
-results	O
-using	O
-Annexin	O
--	O
-V	O
-stain	O
-for	O
-membrane	O
-phosphatidylserine	O
-eversion	O
-,	O
-combined	O
-with	O
-propidium	O
-iodide	O
-(	O
-PI	O
-)	O
-uptake	O
-to	O
-evaluate	O
-cells	O
-whose	O
-membranes	O
-had	O
-been	O
-compromised	O
-.	O
-	O
-Staining	O
-with	O
-both	O
-Annexin	O
--	O
-V	O
-and	O
-PI	O
-indicate	O
-apoptosis	O
-(	O
-upper	O
-right	O
-quadrant	O
-).	O
-	O
-(	O
-f	O
-)	O
-Statistical	O
-analysis	O
-of	O
-apoptotic	O
-cells	O
-(	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.,	O
-n	O
-=	O
-3	O
-),	O
-*	O
-P	O
-<	O
-0	O
-.	O
-05	O
-,	O
-***	O
-P	O
-<	O
-0	O
-.	O
-001	O
-(	O
-ANOVA	O
-followed	O
-by	O
-Tukey	O
-'	O
-s	O
-test	O
-).	O
-	O
-MKP5	O
--	O
-CD	O
-is	O
-crucial	O
-for	O
-JNK1	O
-binding	O
-and	O
-enzyme	O
-catalysis	O
-.	O
-	O
-(	O
-a	O
-)	O
-Domain	O
-organization	O
-of	O
-human	O
-MKP5	O
-.	O
-	O
-The	O
-KBD	O
-and	O
-CD	O
-of	O
-MKP5	O
-are	O
-shown	O
-in	O
-brown	O
-and	O
-grey	O
-,	O
-respectively	O
-.	O
-(	O
-b	O
-)	O
-Plots	O
-of	O
-initial	O
-velocity	O
-of	O
-the	O
-MKP5	O
--	O
-catalysed	O
-reaction	O
-versus	O
-phospho	O
--	O
-JNK1	O
-concentration	O
-.	O
-	O
-The	O
-solid	O
-lines	O
-are	O
-best	O
--	O
-fitting	O
-results	O
-according	O
-to	O
-the	O
-Michaelis	O
-–	O
-Menten	O
-equation	O
-with	O
-Km	O
-and	O
-kcat	O
-values	O
-indicated	O
-.	O
-	O
-The	O
-error	O
-bars	O
-represent	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.	O
-(	O
-c	O
-)	O
-Structural	O
-comparison	O
-of	O
-the	O
-JNK	O
--	O
-interacting	O
-residues	O
-on	O
-MKP5	O
--	O
-CD	O
-(	O
-PDB	O
-1ZZW	O
-)	O
-and	O
-MKP7	O
--	O
-CD	O
-.	O
-	O
-The	O
-corresponding	O
-residues	O
-on	O
-MKP5	O
-are	O
-depicted	O
-as	O
-orange	O
-sticks	O
-,	O
-and	O
-MKP5	O
-residues	O
-numbers	O
-are	O
-in	O
-parentheses	O
-.	O
-	O
-(	O
-d	O
-)	O
-Gel	O
-filtration	O
-analysis	O
-for	O
-interaction	O
-of	O
-JNK1	O
-with	O
-MKP5	O
--	O
-CD	O
-and	O
-MKP5	O
--	O
-KBD	O
-.	O
-(	O
-e	O
-)	O
-GST	O
--	O
-mediated	O
-pull	O
--	O
-down	O
-assays	O
-for	O
-interaction	O
-of	O
-JNK1	O
-with	O
-MKP5	O
--	O
-CD	O
-and	O
-MKP5	O
--	O
-KBD	O
-.	O
-	O
-The	O
-panels	O
-are	O
-arranged	O
-the	O
-same	O
-as	O
-in	O
-Fig	O
-.	O
-2d	O
-.	O
-(	O
-f	O
-)	O
-Effects	O
-of	O
-mutations	O
-in	O
-MKP5	O
--	O
-CD	O
-on	O
-the	O
-JNK1	O
-dephosphorylation	O
-(	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.,	O
-n	O
-=	O
-3	O
-).	O
-	O
-(	O
-g	O
-)	O
-Effects	O
-of	O
-mutations	O
-in	O
-MKP5	O
--	O
-CD	O
-on	O
-the	O
-pNPP	O
-hydrolysis	O
-reaction	O
-(	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.,	O
-n	O
-=	O
-3	O
-).	O
-	O
-(	O
-h	O
-)	O
-Pull	O
--	O
-down	O
-assays	O
-of	O
-MKP5	O
--	O
-CD	O
-by	O
-GST	O
--	O
-tagged	O
-JNK1	O
-mutants	O
-.	O
-	O
-Mechanistic	O
-insight	O
-into	O
-a	O
-peptide	O
-hormone	O
-signaling	O
-complex	O
-mediating	O
-floral	O
-organ	O
-abscission	O
-	O
-Plants	O
-constantly	O
-renew	O
-during	O
-their	O
-life	O
-cycle	O
-and	O
-thus	O
-require	O
-to	O
-shed	O
-senescent	O
-and	O
-damaged	O
-organs	O
-.	O
-	O
-Floral	O
-abscission	O
-is	O
-controlled	O
-by	O
-the	O
-leucine	O
--	O
-rich	O
-repeat	O
-receptor	O
-kinase	O
-(	O
-LRR	O
--	O
-RK	O
-)	O
-HAESA	O
-and	O
-the	O
-peptide	O
-hormone	O
-IDA	O
-.	O
-	O
-It	O
-is	O
-unknown	O
-how	O
-expression	O
-of	O
-IDA	O
-in	O
-the	O
-abscission	O
-zone	O
-leads	O
-to	O
-HAESA	O
-activation	O
-.	O
-	O
-Here	O
-we	O
-show	O
-that	O
-IDA	O
-is	O
-sensed	O
-directly	O
-by	O
-the	O
-HAESA	O
-ectodomain	O
-.	O
-	O
-Crystal	O
-structures	O
-of	O
-HAESA	O
-in	O
-complex	O
-with	O
-IDA	O
-reveal	O
-a	O
-hormone	O
-binding	O
-pocket	O
-that	O
-accommodates	O
-an	O
-active	O
-dodecamer	O
-peptide	O
-.	O
-	O
-A	O
-central	O
-hydroxyproline	O
-residue	O
-anchors	O
-IDA	O
-to	O
-the	O
-receptor	O
-.	O
-	O
-The	O
-HAESA	O
-co	O
--	O
-receptor	O
-SERK1	O
-,	O
-a	O
-positive	O
-regulator	O
-of	O
-the	O
-floral	O
-abscission	O
-pathway	O
-,	O
-allows	O
-for	O
-high	O
--	O
-affinity	O
-sensing	O
-of	O
-the	O
-peptide	O
-hormone	O
-by	O
-binding	O
-to	O
-an	O
-Arg	O
--	O
-His	O
--	O
-Asn	O
-motif	O
-in	O
-IDA	O
-.	O
-	O
-This	O
-sequence	O
-pattern	O
-is	O
-conserved	O
-among	O
-diverse	O
-plant	O
-peptides	O
-,	O
-suggesting	O
-that	O
-plant	O
-peptide	O
-hormone	O
-receptors	O
-may	O
-share	O
-a	O
-common	O
-ligand	O
-binding	O
-mode	O
-and	O
-activation	O
-mechanism	O
-.	O
-	O
-Plants	O
-can	O
-shed	O
-their	O
-leaves	O
-,	O
-flowers	O
-or	O
-other	O
-organs	O
-when	O
-they	O
-no	O
-longer	O
-need	O
-them	O
-.	O
-But	O
-how	O
-does	O
-a	O
-leaf	O
-or	O
-a	O
-flower	O
-know	O
-when	O
-to	O
-let	O
-go	O
-?	O
-A	O
-receptor	O
-protein	O
-called	O
-HAESA	O
-is	O
-found	O
-on	O
-the	O
-surface	O
-of	O
-the	O
-cells	O
-that	O
-surround	O
-a	O
-future	O
-break	O
-point	O
-on	O
-the	O
-plant	O
-.	O
-When	O
-its	O
-time	O
-to	O
-shed	O
-an	O
-organ	O
-,	O
-a	O
-hormone	O
-called	O
-IDA	O
-instructs	O
-HAESA	O
-to	O
-trigger	O
-the	O
-shedding	O
-process	O
-.	O
-	O
-However	O
-,	O
-the	O
-molecular	O
-details	O
-of	O
-how	O
-IDA	O
-triggers	O
-organ	O
-shedding	O
-are	O
-not	O
-clear	O
-.	O
-	O
-The	O
-shedding	O
-of	O
-floral	O
-organs	O
-(	O
-or	O
-leaves	O
-)	O
-can	O
-be	O
-easily	O
-studied	O
-in	O
-a	O
-model	O
-plant	O
-called	O
-Arabidopsis	O
-.	O
-	O
-Santiago	O
-et	O
-al	O
-.	O
-used	O
-protein	O
-biochemistry	O
-,	O
-structural	O
-biology	O
-and	O
-genetics	O
-to	O
-uncover	O
-how	O
-the	O
-IDA	O
-hormone	O
-activates	O
-HAESA	O
-.	O
-	O
-The	O
-experiments	O
-show	O
-that	O
-IDA	O
-binds	O
-directly	O
-to	O
-a	O
-canyon	O
-shaped	O
-pocket	O
-in	O
-HAESA	O
-that	O
-extends	O
-out	O
-from	O
-the	O
-surface	O
-of	O
-the	O
-cell	O
-.	O
-	O
-IDA	O
-binding	O
-to	O
-HAESA	O
-allows	O
-another	O
-receptor	O
-protein	O
-called	O
-SERK1	O
-to	O
-bind	O
-to	O
-HAESA	O
-,	O
-which	O
-results	O
-in	O
-the	O
-release	O
-of	O
-signals	O
-inside	O
-the	O
-cell	O
-that	O
-trigger	O
-the	O
-shedding	O
-of	O
-organs	O
-.	O
-	O
-The	O
-next	O
-step	O
-following	O
-on	O
-from	O
-this	O
-work	O
-is	O
-to	O
-understand	O
-what	O
-signals	O
-are	O
-produced	O
-when	O
-IDA	O
-activates	O
-HAESA	O
-.	O
-	O
-Another	O
-challenge	O
-will	O
-be	O
-to	O
-find	O
-out	O
-where	O
-IDA	O
-is	O
-produced	O
-in	O
-the	O
-plant	O
-and	O
-what	O
-causes	O
-it	O
-to	O
-accumulate	O
-in	O
-specific	O
-places	O
-in	O
-preparation	O
-for	O
-organ	O
-shedding	O
-.	O
-	O
-The	O
-HAESA	O
-ectodomain	O
-folds	O
-into	O
-a	O
-superhelical	O
-assembly	O
-of	O
-21	O
-leucine	O
--	O
-rich	O
-repeats	O
-.	O
-	O
-(	O
-A	O
-)	O
-SDS	O
-PAGE	O
-analysis	O
-of	O
-the	O
-purified	O
-Arabidopsis	O
-thaliana	O
-HAESA	O
-ectodomain	O
-(	O
-residues	O
-20	O
-–	O
-620	O
-)	O
-obtained	O
-by	O
-secreted	O
-expression	O
-in	O
-insect	O
-cells	O
-.	O
-	O
-The	O
-calculated	O
-molecular	O
-mass	O
-is	O
-65	O
-.	O
-7	O
-kDa	O
-,	O
-the	O
-actual	O
-molecular	O
-mass	O
-obtained	O
-by	O
-mass	O
-spectrometry	O
-is	O
-74	O
-,	O
-896	O
-Da	O
-,	O
-accounting	O
-for	O
-the	O
-N	O
--	O
-glycans	O
-.	O
-(	O
-B	O
-)	O
-Ribbon	O
-diagrams	O
-showing	O
-front	O
-(	O
-left	O
-panel	O
-)	O
-and	O
-side	O
-views	O
-(	O
-right	O
-panel	O
-)	O
-of	O
-the	O
-isolated	O
-HAESA	O
-LRR	O
-domain	O
-.	O
-	O
-The	O
-N	O
--	O
-(	O
-residues	O
-20	O
-–	O
-88	O
-)	O
-and	O
-C	O
--	O
-terminal	O
-(	O
-residues	O
-593	O
-–	O
-615	O
-)	O
-capping	O
-domains	O
-are	O
-shown	O
-in	O
-yellow	O
-,	O
-the	O
-central	O
-21	O
-LRR	O
-motifs	O
-are	O
-in	O
-blue	O
-and	O
-disulphide	O
-bonds	O
-are	O
-highlighted	O
-in	O
-green	O
-(	O
-in	O
-bonds	O
-representation	O
-).	O
-(	O
-C	O
-)	O
-Structure	O
-based	O
-sequence	O
-alignment	O
-of	O
-the	O
-21	O
-leucine	O
--	O
-rich	O
-repeats	O
-in	O
-HAESA	O
-with	O
-the	O
-plant	O
-LRR	O
-consensus	O
-sequence	O
-shown	O
-for	O
-comparison	O
-.	O
-	O
-Conserved	O
-hydrophobic	O
-residues	O
-are	O
-shaded	O
-in	O
-gray	O
-,	O
-N	O
--	O
-glycosylation	O
-sites	O
-visible	O
-in	O
-our	O
-structures	O
-are	O
-highlighted	O
-in	O
-blue	O
-,	O
-cysteine	O
-residues	O
-involved	O
-in	O
-disulphide	O
-bridge	O
-formation	O
-in	O
-green	O
-.	O
-(	O
-D	O
-)	O
-Asn	O
--	O
-linked	O
-glycans	O
-mask	O
-the	O
-N	O
--	O
-terminal	O
-portion	O
-of	O
-the	O
-HAESA	O
-ectodomain	O
-.	O
-	O
-Oligomannose	O
-core	O
-structures	O
-(	O
-containing	O
-two	O
-N	O
--	O
-actylglucosamines	O
-and	O
-three	O
-terminal	O
-mannose	O
-units	O
-)	O
-as	O
-found	O
-in	O
-Trichoplusia	O
-ni	O
-cells	O
-and	O
-in	O
-plants	O
-were	O
-modeled	O
-onto	O
-the	O
-seven	O
-glycosylation	O
-sites	O
-observed	O
-in	O
-our	O
-HAESA	O
-structures	O
-,	O
-to	O
-visualize	O
-the	O
-surface	O
-areas	O
-potentially	O
-not	O
-masked	O
-by	O
-carbohydrate	O
-.	O
-	O
-The	O
-HAESA	O
-ectodomain	O
-is	O
-shown	O
-in	O
-blue	O
-(	O
-in	O
-surface	O
-representation	O
-),	O
-the	O
-glycan	O
-structures	O
-are	O
-shown	O
-in	O
-yellow	O
-.	O
-	O
-Hydrophobic	O
-contacts	O
-and	O
-a	O
-hydrogen	O
--	O
-bond	O
-network	O
-mediate	O
-the	O
-interaction	O
-between	O
-HAESA	O
-and	O
-the	O
-peptide	O
-hormone	O
-IDA	O
-.	O
-	O
-(	O
-A	O
-)	O
-Details	O
-of	O
-the	O
-IDA	O
-binding	O
-pocket	O
-.	O
-	O
-HAESA	O
-is	O
-shown	O
-in	O
-blue	O
-(	O
-ribbon	O
-diagram	O
-),	O
-the	O
-C	O
--	O
-terminal	O
-Arg	O
--	O
-His	O
--	O
-Asn	O
-motif	O
-(	O
-left	O
-panel	O
-),	O
-the	O
-central	O
-Hyp	O
-anchor	O
-(	O
-center	O
-)	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-Pro	O
--	O
-rich	O
-motif	O
-in	O
-IDA	O
-(	O
-right	O
-panel	O
-)	O
-are	O
-shown	O
-in	O
-yellow	O
-(	O
-in	O
-bonds	O
-representation	O
-).	O
-	O
-HAESA	O
-interface	O
-residues	O
-are	O
-shown	O
-as	O
-sticks	O
-,	O
-selected	O
-hydrogen	O
-bond	O
-interactions	O
-are	O
-denoted	O
-as	O
-dotted	O
-lines	O
-(	O
-in	O
-magenta	O
-).	O
-(	O
-B	O
-)	O
-View	O
-of	O
-the	O
-complete	O
-IDA	O
-(	O
-in	O
-bonds	O
-representation	O
-,	O
-in	O
-yellow	O
-)	O
-binding	O
-pocket	O
-in	O
-HAESA	O
-(	O
-surface	O
-view	O
-,	O
-in	O
-blue	O
-).	O
-	O
-Orientation	O
-as	O
-in	O
-(	O
-A	O
-).	O
-(	O
-C	O
-)	O
-Structure	O
-based	O
-sequence	O
-alignment	O
-of	O
-leucine	O
--	O
-rich	O
-repeats	O
-in	O
-HAESA	O
-with	O
-the	O
-plant	O
-LRR	O
-consensus	O
-sequence	O
-shown	O
-for	O
-comparison	O
-.	O
-	O
-Residues	O
-mediating	O
-hydrophobic	O
-interactions	O
-with	O
-the	O
-IDA	O
-peptide	O
-are	O
-highlighted	O
-in	O
-blue	O
-,	O
-residues	O
-contributing	O
-to	O
-hydrogen	O
-bond	O
-interactions	O
-and	O
-/	O
-or	O
-salt	O
-bridges	O
-are	O
-shown	O
-in	O
-red	O
-.	O
-	O
-The	O
-IDA	O
-binding	O
-pocket	O
-covers	O
-LRRs	O
-2	O
-–	O
-14	O
-and	O
-all	O
-residues	O
-originate	O
-from	O
-the	O
-inner	O
-surface	O
-of	O
-the	O
-HAESA	O
-superhelix	O
-.	O
-	O
-The	O
-IDA	O
--	O
-HAESA	O
-and	O
-SERK1	O
--	O
-HAESA	O
-complex	O
-interfaces	O
-are	O
-conserved	O
-among	O
-HAESA	O
-and	O
-HAESA	O
--	O
-like	O
-proteins	O
-from	O
-different	O
-plant	O
-species	O
-.	O
-	O
-Structure	O
--	O
-based	O
-sequence	O
-alignment	O
-of	O
-the	O
-HAESA	O
-family	O
-members	O
-:	O
-Arabidopsis	O
-thaliana	O
-HAESA	O
-(	O
-Uniprot	O
-(	O
-http	O
-://	O
-www	O
-.	O
-uniprot	O
-.	O
-org	O
-)	O
-ID	O
-P47735	O
-),	O
-Arabidopsis	O
-thaliana	O
-HSL2	O
-(	O
-Uniprot	O
-ID	O
-C0LGX3	O
-),	O
-Capsella	O
-rubella	O
-HAESA	O
-(	O
-Uniprot	O
-ID	O
-R0F2U6	O
-),	O
-Citrus	O
-clementina	O
-HSL2	O
-(	O
-Uniprot	O
-ID	O
-V4U227	O
-),	O
-Vitis	O
-vinifera	O
-HAESA	O
-(	O
-Uniprot	O
-ID	O
-F6HM39	O
-).	O
-	O
-The	O
-alignment	O
-includes	O
-a	O
-secondary	O
-structure	O
-assignment	O
-calculated	O
-with	O
-the	O
-program	O
-DSSP	O
-and	O
-colored	O
-according	O
-to	O
-Figure	O
-1	O
-,	O
-with	O
-the	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-caps	O
-and	O
-the	O
-21	O
-LRR	O
-motifs	O
-indicated	O
-in	O
-orange	O
-and	O
-blue	O
-,	O
-respectively	O
-.	O
-	O
-Cysteine	O
-residues	O
-engaged	O
-in	O
-disulphide	O
-bonds	O
-are	O
-depicted	O
-in	O
-green	O
-.	O
-	O
-HAESA	O
-residues	O
-interacting	O
-with	O
-the	O
-IDA	O
-peptide	O
-and	O
-/	O
-or	O
-the	O
-SERK1	O
-co	O
--	O
-receptor	O
-kinase	O
-ectodomain	O
-are	O
-highlighted	O
-in	O
-blue	O
-and	O
-orange	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-peptide	O
-hormone	O
-IDA	O
-binds	O
-to	O
-the	O
-HAESA	O
-LRR	O
-ectodomain	O
-.	O
-	O
-(	O
-A	O
-)	O
-Multiple	O
-sequence	O
-alignment	O
-of	O
-selected	O
-IDA	O
-family	O
-members	O
-.	O
-	O
-The	O
-conserved	O
-PIP	O
-motif	O
-is	O
-highlighted	O
-in	O
-yellow	O
-,	O
-the	O
-central	O
-Hyp	O
-in	O
-blue	O
-.	O
-	O
-The	O
-PKGV	O
-motif	O
-present	O
-in	O
-our	O
-N	O
--	O
-terminally	O
-extended	O
-IDA	O
-peptide	O
-is	O
-highlighted	O
-in	O
-red	O
-.	O
-(	O
-B	O
-)	O
-Isothermal	O
-titration	O
-calorimetry	O
-of	O
-the	O
-HAESA	O
-ectodomain	O
-vs	O
-.	O
-IDA	O
-and	O
-including	O
-the	O
-synthetic	O
-peptide	O
-sequence	O
-.	O
-	O
-(	O
-C	O
-)	O
-Structure	O
-of	O
-the	O
-HAESA	O
-–	O
-IDA	O
-complex	O
-with	O
-HAESA	O
-shown	O
-in	O
-blue	O
-(	O
-ribbon	O
-diagram	O
-).	O
-	O
-IDA	O
-(	O
-in	O
-bonds	O
-representation	O
-,	O
-surface	O
-view	O
-included	O
-)	O
-is	O
-depicted	O
-in	O
-yellow	O
-.	O
-	O
-The	O
-peptide	O
-binding	O
-pocket	O
-covers	O
-HAESA	O
-LRRs	O
-2	O
-–	O
-14	O
-.	O
-(	O
-D	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-entire	O
-IDA	O
-(	O
-in	O
-yellow	O
-)	O
-peptide	O
-binding	O
-site	O
-in	O
-HAESA	O
-(	O
-in	O
-blue	O
-).	O
-	O
-Details	O
-of	O
-the	O
-interactions	O
-between	O
-the	O
-central	O
-Hyp	O
-anchor	O
-in	O
-IDA	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-Arg	O
--	O
-His	O
--	O
-Asn	O
-motif	O
-with	O
-HAESA	O
-are	O
-highlighted	O
-in	O
-(	O
-E	O
-)	O
-and	O
-(	O
-F	O
-),	O
-respectively	O
-.	O
-	O
-Hydrogren	O
-bonds	O
-are	O
-depicted	O
-as	O
-dotted	O
-lines	O
-(	O
-in	O
-magenta	O
-),	O
-a	O
-water	O
-molecule	O
-is	O
-shown	O
-as	O
-a	O
-red	O
-sphere	O
-.	O
-	O
-During	O
-their	O
-growth	O
-,	O
-development	O
-and	O
-reproduction	O
-plants	O
-use	O
-cell	O
-separation	O
-processes	O
-to	O
-detach	O
-no	O
--	O
-longer	O
-required	O
-,	O
-damaged	O
-or	O
-senescent	O
-organs	O
-.	O
-	O
-Abscission	O
-of	O
-floral	O
-organs	O
-in	O
-Arabidopsis	O
-is	O
-a	O
-model	O
-system	O
-to	O
-study	O
-these	O
-cell	O
-separation	O
-processes	O
-in	O
-molecular	O
-detail	O
-.	O
-	O
-The	O
-LRR	O
--	O
-RKs	O
-HAESA	O
-(	O
-greek	O
-:	O
-to	O
-adhere	O
-to	O
-)	O
-and	O
-HAESA	O
--	O
-LIKE	O
-2	O
-(	O
-HSL2	O
-)	O
-redundantly	O
-control	O
-floral	O
-abscission	O
-.	O
-	O
-Loss	O
--	O
-of	O
--	O
-function	O
-of	O
-the	O
-secreted	O
-small	O
-protein	O
-INFLORESCENCE	O
-DEFICIENT	O
-IN	O
-ABSCISSION	O
-(	O
-IDA	O
-)	O
-causes	O
-floral	O
-organs	O
-to	O
-remain	O
-attached	O
-while	O
-its	O
-over	O
--	O
-expression	O
-leads	O
-to	O
-premature	O
-shedding	O
-.	O
-	O
-Full	O
--	O
-length	O
-IDA	O
-is	O
-proteolytically	O
-processed	O
-and	O
-a	O
-conserved	O
-stretch	O
-of	O
-20	O
-amino	O
--	O
-acids	O
-(	O
-termed	O
-EPIP	O
-)	O
-can	O
-rescue	O
-the	O
-IDA	O
-loss	O
--	O
-of	O
--	O
-function	O
-phenotype	O
-(	O
-Figure	O
-1A	O
-).	O
-	O
-It	O
-has	O
-been	O
-demonstrated	O
-that	O
-a	O
-dodecamer	O
-peptide	O
-within	O
-EPIP	O
-is	O
-able	O
-to	O
-activate	O
-HAESA	O
-and	O
-HSL2	O
-in	O
-transient	O
-assays	O
-in	O
-tobacco	O
-cells	O
-.	O
-	O
-This	O
-sequence	O
-motif	O
-is	O
-highly	O
-conserved	O
-among	O
-IDA	O
-family	O
-members	O
-(	O
-IDA	O
--	O
-LIKE	O
-PROTEINS	O
-,	O
-IDLs	O
-)	O
-and	O
-contains	O
-a	O
-central	O
-Pro	O
-residue	O
-,	O
-presumed	O
-to	O
-be	O
-post	O
--	O
-translationally	O
-modified	O
-to	O
-hydroxyproline	O
-(	O
-Hyp	O
-;	O
-Figure	O
-1A	O
-).	O
-	O
-The	O
-available	O
-genetic	O
-and	O
-biochemical	O
-evidence	O
-suggests	O
-that	O
-IDA	O
-and	O
-HAESA	O
-together	O
-control	O
-floral	O
-abscission	O
-,	O
-but	O
-it	O
-is	O
-poorly	O
-understood	O
-if	O
-IDA	O
-is	O
-directly	O
-sensed	O
-by	O
-the	O
-receptor	O
-kinase	O
-HAESA	O
-and	O
-how	O
-IDA	O
-binding	O
-at	O
-the	O
-cell	O
-surface	O
-would	O
-activate	O
-the	O
-receptor	O
-.	O
-	O
-IDA	O
-directly	O
-binds	O
-to	O
-the	O
-LRR	O
-domain	O
-of	O
-HAESA	O
-	O
-Active	O
-IDA	O
--	O
-family	O
-peptide	O
-hormones	O
-are	O
-hydroxyprolinated	O
-dodecamers	O
-.	O
-	O
-Close	O
--	O
-up	O
-views	O
-of	O
-(	O
-A	O
-)	O
-IDA	O
-,	O
-(	O
-B	O
-)	O
-the	O
-N	O
--	O
-terminally	O
-extended	O
-PKGV	B-mutant
--	I-mutant
-IDA	I-mutant
-and	O
-(	O
-C	O
-)	O
-IDL1	O
-bound	O
-to	O
-the	O
-HAESA	O
-hormone	O
-binding	O
-pocket	O
-(	O
-in	O
-bonds	O
-representation	O
-,	O
-in	O
-yellow	O
-)	O
-and	O
-including	O
-simulated	O
-annealing	O
-2Fo	O
-–	O
-Fc	O
-omit	O
-electron	O
-density	O
-maps	O
-contoured	O
-at	O
-1	O
-.	O
-0	O
-σ	O
-.	O
-	O
-Note	O
-that	O
-Pro58IDA	O
-and	O
-Leu67IDA	O
-are	O
-the	O
-first	O
-residues	O
-defined	O
-by	O
-electron	O
-density	O
-when	O
-bound	O
-to	O
-the	O
-HAESA	O
-ectodomain	O
-.	O
-(	O
-D	O
-)	O
-Table	O
-summaries	O
-for	O
-equilibrium	O
-dissociation	O
-constants	O
-(	O
-Kd	O
-),	O
-binding	O
-enthalpies	O
-(	O
-ΔH	O
-),	O
-binding	O
-entropies	O
-(	O
-ΔS	O
-)	O
-and	O
-stoichoimetries	O
-(	O
-N	O
-)	O
-for	O
-different	O
-IDA	O
-peptides	O
-binding	O
-to	O
-the	O
-HAESA	O
-ectodomain	O
-(	O
-±	O
-fitting	O
-errors	O
-;	O
-n	O
-.	O
-d	O
-.	O
-	O
-no	O
-detectable	O
-binding	O
-).	O
-(	O
-E	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-active	O
-IDA	O
-(	O
-in	O
-bonds	O
-representation	O
-,	O
-in	O
-gray	O
-)	O
-and	O
-IDL1	O
-peptide	O
-(	O
-in	O
-yellow	O
-)	O
-hormones	O
-bound	O
-to	O
-the	O
-HAESA	O
-ectodomain	O
-.	O
-	O
-Root	O
-mean	O
-square	O
-deviation	O
-(	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.)	O
-is	O
-1	O
-.	O
-0	O
-Å	O
-comparing	O
-100	O
-corresponding	O
-atoms	O
-.	O
-	O
-The	O
-receptor	O
-kinase	O
-SERK1	O
-acts	O
-as	O
-a	O
-HAESA	O
-co	O
--	O
-receptor	O
-and	O
-promotes	O
-high	O
--	O
-affinity	O
-IDA	O
-sensing	O
-.	O
-	O
-(	O
-A	O
-)	O
-Petal	O
-break	O
--	O
-strength	O
-assays	O
-measure	O
-the	O
-force	O
-(	O
-expressed	O
-in	O
-gram	O
-equivalents	O
-)	O
-required	O
-to	O
-remove	O
-the	O
-petals	O
-from	O
-the	O
-flower	O
-of	O
-serk	O
-mutant	O
-plants	O
-compared	O
-to	O
-haesa	O
-/	O
-hsl2	O
-mutant	O
-and	O
-Col	O
--	O
-0	O
-wild	O
--	O
-type	O
-flowers	O
-.	O
-	O
-Petal	O
-break	O
--	O
-strength	O
-was	O
-found	O
-significantly	O
-increased	O
-in	O
-almost	O
-all	O
-positions	O
-(	O
-indicated	O
-with	O
-a	O
-*)	O
-for	O
-haesa	O
-/	O
-hsl2	O
-and	O
-serk1	O
--	O
-1	O
-mutant	O
-plants	O
-with	O
-respect	O
-to	O
-the	O
-Col	O
--	O
-0	O
-control	O
-.	O
-	O
-(	O
-B	O
-)	O
-Analytical	O
-size	O
--	O
-exclusion	O
-chromatography	O
-.	O
-	O
-The	O
-HAESA	O
-LRR	O
-domain	O
-elutes	O
-as	O
-a	O
-monomer	O
-(	O
-black	O
-dotted	O
-line	O
-),	O
-as	O
-does	O
-the	O
-isolated	O
-SERK1	O
-ectodomain	O
-(	O
-blue	O
-dotted	O
-line	O
-).	O
-	O
-A	O
-HAESA	O
-–	O
-IDA	O
-–	O
-SERK1	O
-complex	O
-elutes	O
-as	O
-an	O
-apparent	O
-heterodimer	O
-(	O
-red	O
-line	O
-),	O
-while	O
-a	O
-mixture	O
-of	O
-HAESA	O
-and	O
-SERK1	O
-yields	O
-two	O
-isolated	O
-peaks	O
-that	O
-correspond	O
-to	O
-monomeric	O
-HAESA	O
-and	O
-SERK1	O
-,	O
-respectively	O
-(	O
-black	O
-line	O
-).	O
-	O
-Void	O
-(	O
-V0	O
-)	O
-volume	O
-and	O
-total	O
-volume	O
-(	O
-Vt	O
-)	O
-are	O
-shown	O
-,	O
-together	O
-with	O
-elution	O
-volumes	O
-for	O
-molecular	O
-mass	O
-standards	O
-(	O
-A	O
-,	O
-Thyroglobulin	O
-,	O
-669	O
-,	O
-000	O
-Da	O
-;	O
-B	O
-,	O
-Ferritin	O
-,	O
-440	O
-,	O
-00	O
-Da	O
-,	O
-C	O
-,	O
-Aldolase	O
-,	O
-158	O
-,	O
-000	O
-Da	O
-;	O
-D	O
-,	O
-Conalbumin	O
-,	O
-75	O
-,	O
-000	O
-Da	O
-;	O
-E	O
-,	O
-Ovalbumin	O
-,	O
-44	O
-,	O
-000	O
-Da	O
-;	O
-F	O
-,	O
-Carbonic	O
-anhydrase	O
-,	O
-29	O
-,	O
-000	O
-Da	O
-).	O
-	O
-A	O
-SDS	O
-PAGE	O
-of	O
-the	O
-peak	O
-fractions	O
-is	O
-shown	O
-alongside	O
-.	O
-	O
-Purified	O
-HAESA	O
-and	O
-SERK1	O
-are	O
-~	O
-75	O
-and	O
-~	O
-28	O
-kDa	O
-,	O
-respectively	O
-.	O
-(	O
-C	O
-)	O
-Isothermal	O
-titration	O
-calorimetry	O
-of	O
-wild	O
--	O
-type	O
-and	O
-Hyp64	O
-→	O
-Pro	O
-IDA	O
-versus	O
-the	O
-HAESA	O
-and	O
-SERK1	O
-ectodomains	O
-.	O
-	O
-The	O
-titration	O
-of	O
-IDA	O
-wild	O
--	O
-type	O
-versus	O
-the	O
-isolated	O
-HAESA	O
-ectodomain	O
-from	O
-Figure	O
-1B	O
-is	O
-shown	O
-for	O
-comparison	O
-(	O
-red	O
-line	O
-;	O
-n	O
-.	O
-d	O
-.	O
-	O
-no	O
-detectable	O
-binding	O
-)	O
-(	O
-D	O
-)	O
-Analytical	O
-size	O
--	O
-exclusion	O
-chromatography	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-IDA	O
-Hyp64	O
-→	O
-Pro	O
-mutant	O
-peptide	O
-reveals	O
-no	O
-complex	O
-formation	O
-between	O
-HAESA	O
-and	O
-SERK1	O
-ectodomains	O
-.	O
-	O
-(	O
-E	O
-)	O
-In	O
-vitro	O
-kinase	O
-assays	O
-of	O
-the	O
-HAESA	O
-and	O
-SERK1	O
-kinase	O
-domains	O
-.	O
-	O
-Wild	O
--	O
-type	O
-HAESA	O
-and	O
-SERK1	O
-kinase	O
-domains	O
-(	O
-KDs	O
-)	O
-exhibit	O
-auto	O
--	O
-phosphorylation	O
-activities	O
-(	O
-lanes	O
-1	O
-+	O
-3	O
-).	O
-	O
-Mutant	O
-(	O
-m	O
-)	O
-versions	O
-,	O
-which	O
-carry	O
-point	O
-mutations	O
-in	O
-their	O
-active	O
-sites	O
-(	O
-Asp837HAESA	B-mutant
-→	I-mutant
-Asn	I-mutant
-,	O
-Asp447SERK1	B-mutant
-→	I-mutant
-Asn	I-mutant
-)	O
-possess	O
-no	O
-autophosphorylation	O
-activity	O
-(	O
-lanes	O
-2	O
-+	O
-4	O
-).	O
-	O
-Transphosphorylation	O
-activity	O
-from	O
-the	O
-active	O
-kinase	O
-to	O
-the	O
-mutated	O
-form	O
-can	O
-be	O
-observed	O
-in	O
-both	O
-directions	O
-(	O
-lanes	O
-5	O
-+	O
-6	O
-).	O
-	O
-We	O
-purified	O
-the	O
-HAESA	O
-ectodomain	O
-(	O
-residues	O
-20	O
-–	O
-620	O
-)	O
-from	O
-baculovirus	O
--	O
-infected	O
-insect	O
-cells	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1A	O
-,	O
-see	O
-Materials	O
-and	O
-methods	O
-)	O
-and	O
-quantified	O
-the	O
-interaction	O
-of	O
-the	O
-~	O
-75	O
-kDa	O
-glycoprotein	O
-with	O
-synthetic	O
-IDA	O
-peptides	O
-using	O
-isothermal	O
-titration	O
-calorimetry	O
-(	O
-ITC	O
-).	O
-	O
-A	O
-Hyp	O
--	O
-modified	O
-dodecamer	O
-comprising	O
-the	O
-highly	O
-conserved	O
-PIP	O
-motif	O
-in	O
-IDA	O
-(	O
-Figure	O
-1A	O
-)	O
-interacts	O
-with	O
-HAESA	O
-with	O
-1	O
-:	O
-1	O
-stoichiometry	O
-(	O
-N	O
-)	O
-and	O
-with	O
-a	O
-dissociation	O
-constant	O
-(	O
-Kd	O
-)	O
-of	O
-~	O
-20	O
-μM	O
-(	O
-Figure	O
-1B	O
-).	O
-	O
-We	O
-next	O
-determined	O
-crystal	O
-structures	O
-of	O
-the	O
-apo	O
-HAESA	O
-ectodomain	O
-and	O
-of	O
-a	O
-HAESA	O
--	O
-IDA	O
-complex	O
-,	O
-at	O
-1	O
-.	O
-74	O
-and	O
-1	O
-.	O
-86	O
-Å	O
-resolution	O
-,	O
-respectively	O
-(	O
-Figure	O
-1C	O
-;	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1B	O
-–	O
-D	O
-;	O
-Tables	O
-1	O
-,	O
-2	O
-).	O
-	O
-IDA	O
-binds	O
-in	O
-a	O
-completely	O
-extended	O
-conformation	O
-along	O
-the	O
-inner	O
-surface	O
-of	O
-the	O
-HAESA	O
-ectodomain	O
-,	O
-covering	O
-LRRs	O
-2	O
-–	O
-14	O
-(	O
-Figure	O
-1C	O
-,	O
-D	O
-,	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2	O
-).	O
-	O
-The	O
-central	O
-Hyp64IDA	O
-is	O
-buried	O
-in	O
-a	O
-specific	O
-pocket	O
-formed	O
-by	O
-HAESA	O
-LRRs	O
-8	O
-–	O
-10	O
-,	O
-with	O
-its	O
-hydroxyl	O
-group	O
-establishing	O
-hydrogen	O
-bonds	O
-with	O
-the	O
-strictly	O
-conserved	O
-Glu266HAESA	O
-and	O
-with	O
-a	O
-water	O
-molecule	O
-,	O
-which	O
-in	O
-turn	O
-is	O
-coordinated	O
-by	O
-the	O
-main	O
-chain	O
-oxygens	O
-of	O
-Phe289HAESA	O
-and	O
-Ser311HAESA	O
-(	O
-Figure	O
-1E	O
-;	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-3	O
-).	O
-	O
-The	O
-restricted	O
-size	O
-of	O
-the	O
-Hyp	O
-pocket	O
-suggests	O
-that	O
-IDA	O
-does	O
-not	O
-require	O
-arabinosylation	O
-of	O
-Hyp64IDA	O
-for	O
-activity	O
-in	O
-vivo	O
-,	O
-a	O
-modification	O
-that	O
-has	O
-been	O
-reported	O
-for	O
-Hyp	O
-residues	O
-in	O
-plant	O
-CLE	O
-peptide	O
-hormones	O
-.	O
-	O
-The	O
-C	O
--	O
-terminal	O
-Arg	O
--	O
-His	O
--	O
-Asn	O
-motif	O
-in	O
-IDA	O
-maps	O
-to	O
-a	O
-cavity	O
-formed	O
-by	O
-HAESA	O
-LRRs	O
-11	O
-–	O
-14	O
-(	O
-Figure	O
-1D	O
-,	O
-F	O
-).	O
-	O
-The	O
-COO	O
--	O
-group	O
-of	O
-Asn69IDA	O
-is	O
-in	O
-direct	O
-contact	O
-with	O
-Arg407HAESA	O
-and	O
-Arg409HAESA	O
-and	O
-HAESA	O
-cannot	O
-bind	O
-a	O
-C	O
--	O
-terminally	O
-extended	O
-IDA	B-mutant
--	I-mutant
-SFVN	I-mutant
-peptide	O
-(	O
-Figures	O
-1D	O
-,	O
-F	O
-,	O
-2D	O
-).	O
-	O
-This	O
-suggests	O
-that	O
-the	O
-conserved	O
-Asn69IDA	O
-may	O
-constitute	O
-the	O
-very	O
-C	O
--	O
-terminus	O
-of	O
-the	O
-mature	O
-IDA	O
-peptide	O
-in	O
-planta	O
-and	O
-that	O
-active	O
-IDA	O
-is	O
-generated	O
-by	O
-proteolytic	O
-processing	O
-from	O
-a	O
-longer	O
-pre	O
--	O
-protein	O
-.	O
-	O
-Mutation	O
-of	O
-Arg417HSL2	O
-(	O
-which	O
-corresponds	O
-to	O
-Arg409HAESA	O
-)	O
-causes	O
-a	O
-loss	O
--	O
-of	O
--	O
-function	O
-phenotype	O
-in	O
-HSL2	O
-,	O
-which	O
-indicates	O
-that	O
-the	O
-peptide	O
-binding	O
-pockets	O
-in	O
-different	O
-HAESA	O
-receptors	O
-have	O
-common	O
-structural	O
-and	O
-sequence	O
-features	O
-.	O
-	O
-Indeed	O
-,	O
-we	O
-find	O
-many	O
-of	O
-the	O
-residues	O
-contributing	O
-to	O
-the	O
-formation	O
-of	O
-the	O
-IDA	O
-binding	O
-surface	O
-in	O
-HAESA	O
-to	O
-be	O
-conserved	O
-in	O
-HSL2	O
-and	O
-in	O
-other	O
-HAESA	O
--	O
-type	O
-receptors	O
-in	O
-different	O
-plant	O
-species	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-3	O
-).	O
-	O
-A	O
-N	O
--	O
-terminal	O
-Pro	O
--	O
-rich	O
-motif	O
-in	O
-IDA	O
-makes	O
-contacts	O
-with	O
-LRRs	O
-2	O
-–	O
-6	O
-of	O
-the	O
-receptor	O
-(	O
-Figure	O
-1D	O
-,	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2A	O
-–	O
-C	O
-).	O
-	O
-Other	O
-hydrophobic	O
-and	O
-polar	O
-interactions	O
-are	O
-mediated	O
-by	O
-Ser62IDA	O
-,	O
-Ser65IDA	O
-and	O
-by	O
-backbone	O
-atoms	O
-along	O
-the	O
-IDA	O
-peptide	O
-(	O
-Figure	O
-1D	O
-,	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2A	O
-–	O
-C	O
-).	O
-	O
-HAESA	O
-specifically	O
-senses	O
-IDA	O
--	O
-family	O
-dodecamer	O
-peptides	O
-	O
-We	O
-next	O
-investigated	O
-whether	O
-HAESA	O
-binds	O
-N	O
--	O
-terminally	O
-extended	O
-versions	O
-of	O
-IDA	O
-.	O
-	O
-We	O
-obtained	O
-a	O
-structure	O
-of	O
-HAESA	O
-in	O
-complex	O
-with	O
-a	O
-PKGV	B-mutant
--	I-mutant
-IDA	I-mutant
-peptide	O
-at	O
-1	O
-.	O
-94	O
-Å	O
-resolution	O
-(	O
-Table	O
-2	O
-).	O
-	O
-In	O
-this	O
-structure	O
-,	O
-no	O
-additional	O
-electron	O
-density	O
-accounts	O
-for	O
-the	O
-PKGV	O
-motif	O
-at	O
-the	O
-IDA	O
-N	O
--	O
-terminus	O
-(	O
-Figure	O
-2A	O
-,	O
-B	O
-).	O
-	O
-Consistently	O
-,	O
-PKGV	B-mutant
--	I-mutant
-IDA	I-mutant
-and	O
-IDA	O
-have	O
-similar	O
-binding	O
-affinities	O
-in	O
-our	O
-ITC	O
-assays	O
-,	O
-further	O
-indicating	O
-that	O
-HAESA	O
-senses	O
-a	O
-dodecamer	O
-peptide	O
-comprising	O
-residues	O
-58	O
--	O
-69IDA	O
-(	O
-Figure	O
-2D	O
-).	O
-	O
-We	O
-next	O
-tested	O
-if	O
-HAESA	O
-binds	O
-other	O
-IDA	O
-peptide	O
-family	O
-members	O
-.	O
-	O
-IDL1	O
-,	O
-which	O
-can	O
-rescue	O
-IDA	O
-loss	O
--	O
-of	O
--	O
-function	O
-mutants	O
-when	O
-introduced	O
-in	O
-abscission	O
-zone	O
-cells	O
-,	O
-can	O
-also	O
-be	O
-sensed	O
-by	O
-HAESA	O
-,	O
-albeit	O
-with	O
-lower	O
-affinity	O
-(	O
-Figure	O
-2D	O
-).	O
-	O
-A	O
-2	O
-.	O
-56	O
-Å	O
-co	O
--	O
-crystal	O
-structure	O
-with	O
-IDL1	O
-reveals	O
-that	O
-different	O
-IDA	O
-family	O
-members	O
-use	O
-a	O
-common	O
-binding	O
-mode	O
-to	O
-interact	O
-with	O
-HAESA	O
--	O
-type	O
-receptors	O
-(	O
-Figure	O
-2A	O
-–	O
-C	O
-,	O
-E	O
-,	O
-Table	O
-2	O
-).	O
-	O
-We	O
-do	O
-not	O
-detect	O
-interaction	O
-between	O
-HAESA	O
-and	O
-a	O
-synthetic	O
-peptide	O
-missing	O
-the	O
-C	O
--	O
-terminal	O
-Asn69IDA	O
-(	O
-ΔN69	B-mutant
-),	O
-highlighting	O
-the	O
-importance	O
-of	O
-the	O
-polar	O
-interactions	O
-between	O
-the	O
-IDA	O
-carboxy	O
--	O
-terminus	O
-and	O
-Arg407HAESA	O
-/	O
-Arg409HAESA	O
-(	O
-Figures	O
-1F	O
-,	O
-2D	O
-).	O
-	O
-Replacing	O
-Hyp64IDA	O
-,	O
-which	O
-is	O
-common	O
-to	O
-all	O
-IDLs	O
-,	O
-with	O
-proline	O
-impairs	O
-the	O
-interaction	O
-with	O
-the	O
-receptor	O
-,	O
-as	O
-does	O
-the	O
-Lys66IDA	B-mutant
-/	I-mutant
-Arg67IDA	I-mutant
-→	I-mutant
-Ala	I-mutant
-double	O
--	O
-mutant	O
-discussed	O
-below	O
-(	O
-Figure	O
-1A	O
-,	O
-2D	O
-).	O
-	O
-Notably	O
-,	O
-HAESA	O
-can	O
-discriminate	O
-between	O
-IDLs	O
-and	O
-functionally	O
-unrelated	O
-dodecamer	O
-peptides	O
-with	O
-Hyp	O
-modifications	O
-,	O
-such	O
-as	O
-CLV3	O
-(	O
-Figures	O
-2D	O
-,	O
-7	O
-).	O
-	O
-The	O
-co	O
--	O
-receptor	O
-kinase	O
-SERK1	O
-allows	O
-for	O
-high	O
--	O
-affinity	O
-IDA	O
-sensing	O
-	O
-Our	O
-binding	O
-assays	O
-reveal	O
-that	O
-IDA	O
-family	O
-peptides	O
-are	O
-sensed	O
-by	O
-the	O
-isolated	O
-HAESA	O
-ectodomain	O
-with	O
-relatively	O
-weak	O
-binding	O
-affinities	O
-(	O
-Figures	O
-1B	O
-,	O
-2A	O
-–	O
-D	O
-).	O
-	O
-It	O
-has	O
-been	O
-recently	O
-reported	O
-that	O
-SOMATIC	O
-EMBRYOGENESIS	O
-RECEPTOR	O
-KINASES	O
-(	O
-SERKs	O
-)	O
-are	O
-positive	O
-regulators	O
-of	O
-floral	O
-abscission	O
-and	O
-can	O
-interact	O
-with	O
-HAESA	O
-and	O
-HSL2	O
-in	O
-an	O
-IDA	O
--	O
-dependent	O
-manner	O
-.	O
-	O
-As	O
-all	O
-five	O
-SERK	O
-family	O
-members	O
-appear	O
-to	O
-be	O
-expressed	O
-in	O
-the	O
-Arabidopsis	O
-abscission	O
-zone	O
-,	O
-we	O
-quantified	O
-their	O
-relative	O
-contribution	O
-to	O
-floral	O
-abscission	O
-in	O
-Arabidopsis	O
-using	O
-a	O
-petal	O
-break	O
--	O
-strength	O
-assay	O
-.	O
-	O
-Our	O
-experiments	O
-suggest	O
-that	O
-among	O
-the	O
-SERK	O
-family	O
-members	O
-,	O
-SERK1	O
-is	O
-a	O
-positive	O
-regulator	O
-of	O
-floral	O
-abscission	O
-.	O
-	O
-We	O
-found	O
-that	O
-the	O
-force	O
-required	O
-to	O
-remove	O
-the	O
-petals	O
-of	O
-serk1	O
--	O
-1	O
-mutants	O
-is	O
-significantly	O
-higher	O
-than	O
-that	O
-needed	O
-for	O
-wild	O
--	O
-type	O
-plants	O
-,	O
-as	O
-previously	O
-observed	O
-for	O
-haesa	O
-/	O
-hsl2	O
-mutants	O
-,	O
-and	O
-that	O
-floral	O
-abscission	O
-is	O
-delayed	O
-in	O
-serk1	O
--	O
-1	O
-(	O
-Figure	O
-3A	O
-).	O
-	O
-The	O
-serk2	O
--	O
-2	O
-,	O
-serk3	O
--	O
-1	O
-,	O
-serk4	O
--	O
-1	O
-and	O
-serk5	O
--	O
-1	O
-mutant	O
-lines	O
-showed	O
-a	O
-petal	O
-break	O
--	O
-strength	O
-profile	O
-not	O
-significantly	O
-different	O
-from	O
-wild	O
--	O
-type	O
-plants	O
-.	O
-	O
-Possibly	O
-because	O
-SERKs	O
-have	O
-additional	O
-roles	O
-in	O
-plant	O
-development	O
-such	O
-as	O
-in	O
-pollen	O
-formation	O
-and	O
-brassinosteroid	O
-signaling	O
-,	O
-we	O
-found	O
-that	O
-higher	O
--	O
-order	O
-SERK	O
-mutants	O
-exhibit	O
-pleiotropic	O
-phenotypes	O
-in	O
-the	O
-flower	O
-,	O
-rendering	O
-their	O
-analysis	O
-and	O
-comparison	O
-by	O
-quantitative	O
-petal	O
-break	O
--	O
-strength	O
-assays	O
-difficult	O
-.	O
-	O
-We	O
-thus	O
-focused	O
-on	O
-analyzing	O
-the	O
-contribution	O
-of	O
-SERK1	O
-to	O
-HAESA	O
-ligand	O
-sensing	O
-and	O
-receptor	O
-activation	O
-.	O
-	O
-In	O
-vitro	O
-,	O
-the	O
-LRR	O
-ectodomain	O
-of	O
-SERK1	O
-(	O
-residues	O
-24	O
-–	O
-213	O
-)	O
-forms	O
-stable	O
-,	O
-IDA	O
--	O
-dependent	O
-heterodimeric	O
-complexes	O
-with	O
-HAESA	O
-in	O
-size	O
-exclusion	O
-chromatography	O
-experiments	O
-(	O
-Figure	O
-3B	O
-).	O
-	O
-We	O
-next	O
-quantified	O
-the	O
-contribution	O
-of	O
-SERK1	O
-to	O
-IDA	O
-recognition	O
-by	O
-HAESA	O
-.	O
-	O
-We	O
-found	O
-that	O
-HAESA	O
-senses	O
-IDA	O
-with	O
-a	O
-~	O
-60	O
-fold	O
-higher	O
-binding	O
-affinity	O
-in	O
-the	O
-presence	O
-of	O
-SERK1	O
-,	O
-suggesting	O
-that	O
-SERK1	O
-is	O
-involved	O
-in	O
-the	O
-specific	O
-recognition	O
-of	O
-the	O
-peptide	O
-hormone	O
-(	O
-Figure	O
-3C	O
-).	O
-	O
-We	O
-next	O
-titrated	O
-SERK1	O
-into	O
-a	O
-solution	O
-containing	O
-only	O
-the	O
-HAESA	O
-ectodomain	O
-.	O
-	O
-In	O
-this	O
-case	O
-,	O
-there	O
-was	O
-no	O
-detectable	O
-interaction	O
-between	O
-receptor	O
-and	O
-co	O
--	O
-receptor	O
-,	O
-while	O
-in	O
-the	O
-presence	O
-of	O
-IDA	O
-,	O
-SERK1	O
-strongly	O
-binds	O
-HAESA	O
-with	O
-a	O
-dissociation	O
-constant	O
-in	O
-the	O
-mid	O
--	O
-nanomolar	O
-range	O
-(	O
-Figure	O
-3C	O
-).	O
-	O
-This	O
-suggests	O
-that	O
-IDA	O
-itself	O
-promotes	O
-receptor	O
-–	O
-co	O
--	O
-receptor	O
-association	O
-,	O
-as	O
-previously	O
-described	O
-for	O
-the	O
-steroid	O
-hormone	O
-brassinolide	O
-and	O
-for	O
-other	O
-LRR	O
--	O
-RK	O
-complexes	O
-.	O
-	O
-Importantly	O
-,	O
-hydroxyprolination	O
-of	O
-IDA	O
-is	O
-critical	O
-for	O
-HAESA	O
--	O
-IDA	O
--	O
-SERK1	O
-complex	O
-formation	O
-(	O
-Figure	O
-3C	O
-,	O
-D	O
-).	O
-	O
-Our	O
-calorimetry	O
-experiments	O
-now	O
-reveal	O
-that	O
-SERKs	O
-may	O
-render	O
-HAESA	O
-,	O
-and	O
-potentially	O
-other	O
-receptor	O
-kinases	O
-,	O
-competent	O
-for	O
-high	O
--	O
-affinity	O
-sensing	O
-of	O
-their	O
-cognate	O
-ligands	O
-.	O
-	O
-Upon	O
-IDA	O
-binding	O
-at	O
-the	O
-cell	O
-surface	O
-,	O
-the	O
-kinase	O
-domains	O
-of	O
-HAESA	O
-and	O
-SERK1	O
-,	O
-which	O
-have	O
-been	O
-shown	O
-to	O
-be	O
-active	O
-protein	O
-kinases	O
-,	O
-may	O
-interact	O
-in	O
-the	O
-cytoplasm	O
-to	O
-activate	O
-each	O
-other	O
-.	O
-	O
-Consistently	O
-,	O
-the	O
-HAESA	O
-kinase	O
-domain	O
-can	O
-transphosphorylate	O
-SERK1	O
-and	O
-vice	O
-versa	O
-in	O
-in	O
-vitro	O
-transphosphorylation	O
-assays	O
-(	O
-Figure	O
-3E	O
-).	O
-	O
-Together	O
-,	O
-our	O
-genetic	O
-and	O
-biochemical	O
-experiments	O
-implicate	O
-SERK1	O
-as	O
-a	O
-HAESA	O
-co	O
--	O
-receptor	O
-in	O
-the	O
-Arabidopsis	O
-abscission	O
-zone	O
-.	O
-	O
-SERK1	O
-senses	O
-a	O
-conserved	O
-motif	O
-in	O
-IDA	O
-family	O
-peptides	O
-	O
-Crystal	O
-structure	O
-of	O
-a	O
-HAESA	O
-–	O
-IDA	O
-–	O
-SERK1	O
-signaling	O
-complex	O
-.	O
-	O
-(	O
-A	O
-)	O
-Overview	O
-of	O
-the	O
-ternary	O
-complex	O
-with	O
-HAESA	O
-in	O
-blue	O
-(	O
-surface	O
-representation	O
-),	O
-IDA	O
-in	O
-yellow	O
-(	O
-bonds	O
-representation	O
-)	O
-and	O
-SERK1	O
-in	O
-orange	O
-(	O
-surface	O
-view	O
-).	O
-(	O
-B	O
-)	O
-The	O
-HAESA	O
-ectodomain	O
-undergoes	O
-a	O
-conformational	O
-change	O
-upon	O
-SERK1	O
-co	O
--	O
-receptor	O
-binding	O
-.	O
-	O
-Shown	O
-are	O
-Cα	O
-traces	O
-of	O
-a	O
-structural	O
-superposition	O
-of	O
-the	O
-unbound	O
-(	O
-yellow	O
-)	O
-and	O
-SERK1	O
--	O
-bound	O
-(	O
-blue	O
-)	O
-HAESA	O
-ectodomains	O
-(	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.	O
-is	O
-1	O
-.	O
-5	O
-Å	O
-between	O
-572	O
-corresponding	O
-Cα	O
-atoms	O
-).	O
-	O
-SERK1	O
-(	O
-in	O
-orange	O
-)	O
-and	O
-IDA	O
-(	O
-in	O
-red	O
-)	O
-are	O
-shown	O
-alongside	O
-.	O
-	O
-The	O
-conformational	O
-change	O
-in	O
-the	O
-C	O
--	O
-terminal	O
-LRRs	O
-and	O
-capping	O
-domain	O
-is	O
-indicated	O
-by	O
-an	O
-arrow	O
-.	O
-(	O
-C	O
-)	O
-SERK1	O
-forms	O
-an	O
-integral	O
-part	O
-of	O
-the	O
-receptor	O
-'	O
-s	O
-peptide	O
-binding	O
-pocket	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-capping	O
-domain	O
-of	O
-SERK1	O
-(	O
-in	O
-orange	O
-)	O
-directly	O
-contacts	O
-the	O
-C	O
--	O
-terminal	O
-part	O
-of	O
-IDA	O
-(	O
-in	O
-yellow	O
-,	O
-in	O
-bonds	O
-representation	O
-)	O
-and	O
-the	O
-receptor	O
-HAESA	O
-(	O
-in	O
-blue	O
-).	O
-	O
-Polar	O
-contacts	O
-of	O
-SERK1	O
-with	O
-IDA	O
-are	O
-shown	O
-in	O
-magenta	O
-,	O
-with	O
-the	O
-HAESA	O
-LRR	O
-domain	O
-in	O
-gray	O
-.	O
-(	O
-D	O
-)	O
-Details	O
-of	O
-the	O
-zipper	O
--	O
-like	O
-SERK1	O
--	O
-HAESA	O
-interface	O
-.	O
-	O
-Ribbon	O
-diagrams	O
-of	O
-HAESA	O
-(	O
-in	O
-blue	O
-)	O
-and	O
-SERK1	O
-(	O
-in	O
-orange	O
-)	O
-are	O
-shown	O
-with	O
-selected	O
-interface	O
-residues	O
-(	O
-in	O
-bonds	O
-representation	O
-).	O
-	O
-Polar	O
-interactions	O
-are	O
-highlighted	O
-as	O
-dotted	O
-lines	O
-(	O
-in	O
-magenta	O
-).	O
-	O
-To	O
-understand	O
-in	O
-molecular	O
-terms	O
-how	O
-SERK1	O
-contributes	O
-to	O
-high	O
--	O
-affinity	O
-IDA	O
-recognition	O
-,	O
-we	O
-solved	O
-a	O
-2	O
-.	O
-43	O
-Å	O
-crystal	O
-structure	O
-of	O
-the	O
-ternary	O
-HAESA	O
-–	O
-IDA	O
-–	O
-SERK1	O
-complex	O
-(	O
-Figure	O
-4A	O
-,	O
-Table	O
-2	O
-).	O
-	O
-HAESA	O
-LRRs	O
-16	O
-–	O
-21	O
-and	O
-its	O
-C	O
--	O
-terminal	O
-capping	O
-domain	O
-undergo	O
-a	O
-conformational	O
-change	O
-upon	O
-SERK1	O
-binding	O
-(	O
-Figure	O
-4B	O
-).	O
-	O
-The	O
-SERK1	O
-ectodomain	O
-interacts	O
-with	O
-the	O
-IDA	O
-peptide	O
-binding	O
-site	O
-using	O
-a	O
-loop	O
-region	O
-(	O
-residues	O
-51	O
--	O
-59SERK1	O
-)	O
-from	O
-its	O
-N	O
--	O
-terminal	O
-cap	O
-(	O
-Figure	O
-4A	O
-,	O
-C	O
-).	O
-	O
-SERK1	O
-loop	O
-residues	O
-establish	O
-multiple	O
-hydrophobic	O
-and	O
-polar	O
-contacts	O
-with	O
-Lys66IDA	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-Arg	O
--	O
-His	O
--	O
-Asn	O
-motif	O
-in	O
-IDA	O
-(	O
-Figure	O
-4C	O
-).	O
-	O
-SERK1	O
-LRRs	O
-1	O
-–	O
-5	O
-and	O
-its	O
-C	O
--	O
-terminal	O
-capping	O
-domain	O
-form	O
-an	O
-additional	O
-zipper	O
--	O
-like	O
-interface	O
-with	O
-residues	O
-originating	O
-from	O
-HAESA	O
-LRRs	O
-15	O
-–	O
-21	O
-and	O
-from	O
-the	O
-HAESA	O
-C	O
--	O
-terminal	O
-cap	O
-(	O
-Figure	O
-4D	O
-).	O
-	O
-SERK1	O
-binds	O
-HAESA	O
-using	O
-these	O
-two	O
-distinct	O
-interaction	O
-surfaces	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-3	O
-),	O
-with	O
-the	O
-N	O
--	O
-cap	O
-of	O
-the	O
-SERK1	O
-LRR	O
-domain	O
-partially	O
-covering	O
-the	O
-IDA	O
-peptide	O
-binding	O
-cleft	O
-.	O
-	O
-The	O
-IDA	O
-C	O
--	O
-terminal	O
-motif	O
-is	O
-required	O
-for	O
-HAESA	O
--	O
-SERK1	O
-complex	O
-formation	O
-and	O
-for	O
-IDA	O
-bioactivity	O
-.	O
-	O
-(	O
-A	O
-)	O
-Size	O
-exclusion	O
-chromatography	O
-experiments	O
-similar	O
-to	O
-Figure	O
-3B	O
-,	O
-D	O
-reveal	O
-that	O
-IDA	O
-mutant	O
-peptides	O
-targeting	O
-the	O
-C	O
--	O
-terminal	O
-motif	O
-do	O
-not	O
-form	O
-biochemically	O
-stable	O
-HAESA	O
--	O
-IDA	O
--	O
-SERK1	O
-complexes	O
-.	O
-	O
-Deletion	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-Asn69IDA	O
-completely	O
-inhibits	O
-complex	O
-formation	O
-.	O
-	O
-Purified	O
-HAESA	O
-and	O
-SERK1	O
-are	O
-~	O
-75	O
-and	O
-~	O
-28	O
-kDa	O
-,	O
-respectively	O
-.	O
-	O
-Left	O
-panel	O
-:	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R67A	I-mutant
-;	O
-center	O
-:	O
-IDA	B-mutant
-ΔN69	I-mutant
-,	O
-right	O
-panel	O
-:	O
-SDS	O
--	O
-PAGE	O
-of	O
-peak	O
-fractions	O
-.	O
-	O
-Note	O
-that	O
-the	O
-HAESA	O
-and	O
-SERK1	O
-input	O
-lanes	O
-have	O
-already	O
-been	O
-shown	O
-in	O
-Figure	O
-3D	O
-.	O
-(	O
-B	O
-)	O
-Isothermal	O
-titration	O
-thermographs	O
-of	O
-wild	O
--	O
-type	O
-and	O
-mutant	O
-IDA	O
-peptides	O
-titrated	O
-into	O
-a	O
-HAESA	O
--	O
-SERK1	O
-mixture	O
-in	O
-the	O
-cell	O
-.	O
-	O
-Table	O
-summaries	O
-for	O
-calorimetric	O
-binding	O
-constants	O
-and	O
-stoichoimetries	O
-for	O
-different	O
-IDA	O
-peptides	O
-binding	O
-to	O
-the	O
-HAESA	O
-–	O
-SERK1	O
-ectodomain	O
-mixture	O
-(	O
-±	O
-fitting	O
-errors	O
-;	O
-n	O
-.	O
-d	O
-.	O
-	O
-(	O
-C	O
-)	O
-Quantitative	O
-petal	O
-break	O
--	O
-strength	O
-assay	O
-for	O
-Col	O
--	O
-0	O
-wild	O
--	O
-type	O
-flowers	O
-and	O
-35S	O
-::	O
-IDA	O
-wild	O
--	O
-type	O
-and	O
-35S	O
-::	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R67A	I-mutant
-mutant	O
-flowers	O
-.	O
-	O
-35S	O
-::	O
-IDA	O
-plants	O
-showed	O
-significantly	O
-increased	O
-abscission	O
-compared	O
-to	O
-Col	O
--	O
-0	O
-controls	O
-in	O
-inflorescence	O
-positions	O
-2	O
-and	O
-3	O
-(	O
-a	O
-).	O
-	O
-Up	O
-to	O
-inflorescence	O
-position	O
-4	O
-,	O
-petal	O
-break	O
-in	O
-35S	O
-::	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R67A	I-mutant
-mutant	O
-plants	O
-was	O
-significantly	O
-increased	O
-compared	O
-to	O
-both	O
-Col	O
--	O
-0	O
-control	O
-plants	O
-(	O
-b	O
-)	O
-and	O
-35S	O
-::	O
-IDA	O
-plants	O
-(	O
-c	O
-)	O
-(	O
-D	O
-)	O
-Normalized	O
-expression	O
-levels	O
-(	O
-relative	O
-expression	O
-±	O
-standard	O
-error	O
-;	O
-ida	O
-:	O
--	O
-0	O
-.	O
-02	O
-±	O
-0	O
-.	O
-001	O
-;	O
-Col	O
--	O
-0	O
-:	O
-1	O
-±	O
-0	O
-.	O
-11	O
-;	O
-35S	O
-::	O
-IDA	O
-124	O
-±	O
-0	O
-.	O
-75	O
-;	O
-35S	O
-::	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R67A	I-mutant
-:	O
-159	O
-±	O
-0	O
-.	O
-58	O
-)	O
-of	O
-IDA	O
-wild	O
--	O
-type	O
-and	O
-mutant	O
-transcripts	O
-in	O
-the	O
-35S	O
-promoter	O
-over	O
--	O
-expression	O
-lines	O
-analyzed	O
-in	O
-(	O
-C	O
-).	O
-(	O
-E	O
-)	O
-Magnified	O
-view	O
-of	O
-representative	O
-abscission	O
-zones	O
-from	O
-35S	O
-::	O
-IDA	O
-,	O
-Col	O
--	O
-0	O
-wild	O
--	O
-type	O
-and	O
-35S	O
-::	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R67A	I-mutant
-double	O
--	O
-mutant	O
-T3	O
-transgenic	O
-lines	O
-.	O
-	O
-15	O
-out	O
-of	O
-15	O
-35S	O
-::	O
-IDA	O
-plants	O
-,	O
-0	O
-out	O
-of	O
-15	O
-Col	O
--	O
-0	O
-plants	O
-and	O
-0	O
-out	O
-of	O
-15	O
-35S	O
-::	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R67A	I-mutant
-double	O
--	O
-mutant	O
-plants	O
-,	O
-showed	O
-an	O
-enlarged	O
-abscission	O
-zone	O
-,	O
-respectively	O
-(	O
-3	O
-independent	O
-lines	O
-were	O
-analyzed	O
-).	O
-	O
-The	O
-four	O
-C	O
--	O
-terminal	O
-residues	O
-in	O
-IDA	O
-(	O
-Lys66IDA	O
--	O
-Asn69IDA	O
-)	O
-are	O
-conserved	O
-among	O
-IDA	O
-family	O
-members	O
-and	O
-are	O
-in	O
-direct	O
-contact	O
-with	O
-SERK1	O
-(	O
-Figures	O
-1A	O
-,	O
-4C	O
-).	O
-	O
-We	O
-thus	O
-assessed	O
-their	O
-contribution	O
-to	O
-HAESA	O
-–	O
-SERK1	O
-complex	O
-formation	O
-.	O
-	O
-Deletion	O
-of	O
-the	O
-buried	O
-Asn69IDA	O
-completely	O
-inhibits	O
-receptor	O
-–	O
-co	O
--	O
-receptor	O
-complex	O
-formation	O
-and	O
-HSL2	O
-activation	O
-(	O
-Figure	O
-5A	O
-,	O
-B	O
-).	O
-	O
-A	O
-synthetic	O
-Lys66IDA	B-mutant
-/	I-mutant
-Arg67IDA	I-mutant
-→	I-mutant
-Ala	I-mutant
-mutant	O
-peptide	O
-(	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R66A	I-mutant
-)	O
-showed	O
-a	O
-10	O
-fold	O
-reduced	O
-binding	O
-affinity	O
-when	O
-titrated	O
-in	O
-a	O
-HAESA	O
-/	O
-SERK1	O
-protein	O
-solution	O
-(	O
-Figures	O
-5A	O
-,	O
-B	O
-,	O
-2D	O
-).	O
-	O
-We	O
-over	O
--	O
-expressed	O
-full	O
--	O
-length	O
-wild	O
--	O
-type	O
-IDA	O
-or	O
-this	O
-Lys66IDA	B-mutant
-/	I-mutant
-Arg67IDA	I-mutant
-→	I-mutant
-Ala	I-mutant
-double	O
--	O
-mutant	O
-to	O
-similar	O
-levels	O
-in	O
-Col	O
--	O
-0	O
-Arabidopsis	O
-plants	O
-(	O
-Figure	O
-5D	O
-).	O
-	O
-We	O
-found	O
-that	O
-over	O
--	O
-expression	O
-of	O
-wild	O
--	O
-type	O
-IDA	O
-leads	O
-to	O
-early	O
-floral	O
-abscission	O
-and	O
-an	O
-enlargement	O
-of	O
-the	O
-abscission	O
-zone	O
-(	O
-Figure	O
-5C	O
-–	O
-E	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-over	O
--	O
-expression	O
-of	O
-the	O
-IDA	B-mutant
-Lys66IDA	I-mutant
-/	I-mutant
-Arg67IDA	I-mutant
-→	I-mutant
-Ala	I-mutant
-double	O
-mutant	O
-significantly	O
-delays	O
-floral	O
-abscission	O
-when	O
-compared	O
-to	O
-wild	O
--	O
-type	O
-control	O
-plants	O
-,	O
-suggesting	O
-that	O
-the	O
-mutant	O
-IDA	O
-peptide	O
-has	O
-reduced	O
-activity	O
-in	O
-planta	O
-(	O
-Figure	O
-5C	O
-–	O
-E	O
-).	O
-	O
-Comparison	O
-of	O
-35S	O
-::	O
-IDA	O
-wild	O
--	O
-type	O
-and	O
-mutant	O
-plants	O
-further	O
-indicates	O
-that	O
-mutation	O
-of	O
-Lys66IDA	B-mutant
-/	I-mutant
-Arg67IDA	I-mutant
-→	I-mutant
-Ala	I-mutant
-may	O
-cause	O
-a	O
-weak	O
-dominant	O
-negative	O
-effect	O
-(	O
-Figure	O
-5C	O
-–	O
-E	O
-).	O
-	O
-In	O
-agreement	O
-with	O
-our	O
-structures	O
-and	O
-biochemical	O
-assays	O
-,	O
-this	O
-experiment	O
-suggests	O
-a	O
-role	O
-of	O
-the	O
-conserved	O
-IDA	O
-C	O
--	O
-terminus	O
-in	O
-the	O
-control	O
-of	O
-floral	O
-abscission	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-animal	O
-LRR	O
-receptors	O
-,	O
-plant	O
-LRR	O
--	O
-RKs	O
-harbor	O
-spiral	O
--	O
-shaped	O
-ectodomains	O
-and	O
-thus	O
-they	O
-require	O
-shape	O
--	O
-complementary	O
-co	O
--	O
-receptor	O
-proteins	O
-for	O
-receptor	O
-activation	O
-.	O
-	O
-For	O
-a	O
-rapidly	O
-growing	O
-number	O
-of	O
-plant	O
-signaling	O
-pathways	O
-,	O
-SERK	O
-proteins	O
-act	O
-as	O
-these	O
-essential	O
-co	O
--	O
-receptors	O
-(;	O
-).	O
-	O
-SERK1	O
-has	O
-been	O
-previously	O
-reported	O
-as	O
-a	O
-positive	O
-regulator	O
-in	O
-plant	O
-embryogenesis	O
-,	O
-male	O
-sporogenesis	O
-,	O
-brassinosteroid	O
-signaling	O
-and	O
-in	O
-phytosulfokine	O
-perception	O
-.	O
-	O
-Recent	O
-findings	O
-by	O
-and	O
-our	O
-mechanistic	O
-studies	O
-now	O
-also	O
-support	O
-a	O
-positive	O
-role	O
-for	O
-SERK1	O
-in	O
-floral	O
-abscission	O
-.	O
-	O
-As	O
-serk1	O
--	O
-1	O
-mutant	O
-plants	O
-show	O
-intermediate	O
-abscission	O
-phenotypes	O
-when	O
-compared	O
-to	O
-haesa	O
-/	O
-hsl2	O
-mutants	O
-,	O
-SERK1	O
-likely	O
-acts	O
-redundantly	O
-with	O
-other	O
-SERKs	O
-in	O
-the	O
-abscission	O
-zone	O
-(	O
-Figure	O
-3A	O
-).	O
-	O
-It	O
-has	O
-been	O
-previously	O
-suggested	O
-that	O
-SERK1	O
-can	O
-inhibit	O
-cell	O
-separation	O
-.	O
-	O
-However	O
-our	O
-results	O
-show	O
-that	O
-SERK1	O
-also	O
-can	O
-activate	O
-this	O
-process	O
-upon	O
-IDA	O
-sensing	O
-,	O
-indicating	O
-that	O
-SERKs	O
-may	O
-fulfill	O
-several	O
-different	O
-functions	O
-in	O
-the	O
-course	O
-of	O
-the	O
-abscission	O
-process	O
-.	O
-	O
-While	O
-the	O
-sequence	O
-of	O
-the	O
-mature	O
-IDA	O
-peptide	O
-has	O
-not	O
-been	O
-experimentally	O
-determined	O
-in	O
-planta	O
-,	O
-our	O
-HAESA	O
--	O
-IDA	O
-complex	O
-structures	O
-and	O
-calorimetry	O
-assays	O
-suggest	O
-that	O
-active	O
-IDLs	O
-are	O
-hydroxyprolinated	O
-dodecamers	O
-.	O
-	O
-It	O
-will	O
-be	O
-thus	O
-interesting	O
-to	O
-see	O
-if	O
-proteolytic	O
-processing	O
-of	O
-full	O
--	O
-length	O
-IDA	O
-in	O
-vivo	O
-is	O
-regulated	O
-in	O
-a	O
-cell	O
--	O
-type	O
-or	O
-tissue	O
--	O
-specific	O
-manner	O
-.	O
-	O
-The	O
-central	O
-Hyp	O
-residue	O
-in	O
-IDA	O
-is	O
-found	O
-buried	O
-in	O
-the	O
-HAESA	O
-peptide	O
-binding	O
-surface	O
-and	O
-thus	O
-this	O
-post	O
--	O
-translational	O
-modification	O
-may	O
-regulate	O
-IDA	O
-bioactivity	O
-.	O
-	O
-Our	O
-comparative	O
-structural	O
-and	O
-biochemical	O
-analysis	O
-further	O
-suggests	O
-that	O
-IDLs	O
-share	O
-a	O
-common	O
-receptor	O
-binding	O
-mode	O
-,	O
-but	O
-may	O
-preferably	O
-bind	O
-to	O
-HAESA	O
-,	O
-HSL1	O
-or	O
-HSL2	O
-in	O
-different	O
-plant	O
-tissues	O
-and	O
-organs	O
-.	O
-	O
-In	O
-our	O
-quantitative	O
-biochemical	O
-assays	O
-,	O
-the	O
-presence	O
-of	O
-SERK1	O
-dramatically	O
-increases	O
-the	O
-HAESA	O
-binding	O
-specificity	O
-and	O
-affinity	O
-for	O
-IDA	O
-.	O
-	O
-This	O
-observation	O
-is	O
-consistent	O
-with	O
-our	O
-complex	O
-structure	O
-in	O
-which	O
-receptor	O
-and	O
-co	O
--	O
-receptor	O
-together	O
-form	O
-the	O
-IDA	O
-binding	O
-pocket	O
-.	O
-	O
-The	O
-fact	O
-that	O
-SERK1	O
-specifically	O
-interacts	O
-with	O
-the	O
-very	O
-C	O
--	O
-terminus	O
-of	O
-IDLs	O
-may	O
-allow	O
-for	O
-the	O
-rational	O
-design	O
-of	O
-peptide	O
-hormone	O
-antagonists	O
-,	O
-as	O
-previously	O
-demonstrated	O
-for	O
-the	O
-brassinosteroid	O
-pathway	O
-.	O
-	O
-Importantly	O
-,	O
-our	O
-calorimetry	O
-assays	O
-reveal	O
-that	O
-the	O
-SERK1	O
-ectodomain	O
-binds	O
-HAESA	O
-with	O
-nanomolar	O
-affinity	O
-,	O
-but	O
-only	O
-in	O
-the	O
-presence	O
-of	O
-IDA	O
-(	O
-Figure	O
-3C	O
-).	O
-	O
-This	O
-ligand	O
--	O
-induced	O
-formation	O
-of	O
-a	O
-receptor	O
-–	O
-co	O
--	O
-receptor	O
-complex	O
-may	O
-allow	O
-the	O
-HAESA	O
-and	O
-SERK1	O
-kinase	O
-domains	O
-to	O
-efficiently	O
-trans	O
--	O
-phosphorylate	O
-and	O
-activate	O
-each	O
-other	O
-in	O
-the	O
-cytoplasm	O
-.	O
-	O
-It	O
-is	O
-of	O
-note	O
-that	O
-our	O
-reported	O
-binding	O
-affinities	O
-for	O
-IDA	O
-and	O
-SERK1	O
-have	O
-been	O
-measured	O
-using	O
-synthetic	O
-peptides	O
-and	O
-the	O
-isolated	O
-HAESA	O
-and	O
-SERK1	O
-ectodomains	O
-,	O
-and	O
-thus	O
-might	O
-differ	O
-in	O
-the	O
-context	O
-of	O
-the	O
-full	O
--	O
-length	O
-,	O
-membrane	O
--	O
-embedded	O
-signaling	O
-complex	O
-.	O
-	O
-SERK1	O
-uses	O
-partially	O
-overlapping	O
-surface	O
-areas	O
-to	O
-activate	O
-different	O
-plant	O
-signaling	O
-receptors	O
-.	O
-	O
-(	O
-A	O
-)	O
-Structural	O
-comparison	O
-of	O
-plant	O
-steroid	O
-and	O
-peptide	O
-hormone	O
-membrane	O
-signaling	O
-complexes	O
-.	O
-	O
-Left	O
-panel	O
-:	O
-Ribbon	O
-diagram	O
-of	O
-HAESA	O
-(	O
-in	O
-blue	O
-),	O
-SERK1	O
-(	O
-in	O
-orange	O
-)	O
-and	O
-IDA	O
-(	O
-in	O
-bonds	O
-and	O
-surface	O
-represention	O
-).	O
-	O
-Right	O
-panel	O
-:	O
-Ribbon	O
-diagram	O
-of	O
-the	O
-plant	O
-steroid	O
-receptor	O
-BRI1	O
-(	O
-in	O
-blue	O
-)	O
-bound	O
-to	O
-brassinolide	O
-(	O
-in	O
-gray	O
-,	O
-in	O
-bonds	O
-representation	O
-)	O
-and	O
-to	O
-SERK1	O
-,	O
-shown	O
-in	O
-the	O
-same	O
-orientation	O
-(	O
-PDB	O
--	O
-ID	O
-.	O
-4lsx	O
-).	O
-	O
-(	O
-B	O
-)	O
-View	O
-of	O
-the	O
-inner	O
-surface	O
-of	O
-the	O
-SERK1	O
-LRR	O
-domain	O
-(	O
-PDB	O
--	O
-ID	O
-4lsc	O
-,	O
-surface	O
-representation	O
-,	O
-in	O
-gray	O
-).	O
-	O
-A	O
-ribbon	O
-diagram	O
-of	O
-SERK1	O
-in	O
-the	O
-same	O
-orientation	O
-is	O
-shown	O
-alongside	O
-.	O
-	O
-Residues	O
-interacting	O
-with	O
-the	O
-HAESA	O
-or	O
-BRI1	O
-LRR	O
-domains	O
-are	O
-shown	O
-in	O
-orange	O
-or	O
-magenta	O
-,	O
-respectively	O
-.	O
-	O
-Comparison	O
-of	O
-our	O
-HAESA	O
-–	O
-IDA	O
-–	O
-SERK1	O
-structure	O
-with	O
-the	O
-brassinosteroid	O
-receptor	O
-signaling	O
-complex	O
-,	O
-where	O
-SERK1	O
-also	O
-acts	O
-as	O
-co	O
--	O
-receptor	O
-,	O
-reveals	O
-an	O
-overall	O
-conserved	O
-mode	O
-of	O
-SERK1	O
-binding	O
-,	O
-while	O
-the	O
-ligand	O
-binding	O
-pockets	O
-map	O
-to	O
-very	O
-different	O
-areas	O
-in	O
-the	O
-corresponding	O
-receptors	O
-(	O
-LRRs	O
-2	O
-–	O
-14	O
-;	O
-HAESA	O
-;	O
-LRRs	O
-21	O
-–	O
-25	O
-,	O
-BRI1	O
-)	O
-and	O
-may	O
-involve	O
-an	O
-island	O
-domain	O
-(	O
-BRI1	O
-)	O
-or	O
-not	O
-(	O
-HAESA	O
-)	O
-(	O
-Figure	O
-6A	O
-).	O
-	O
-Several	O
-residues	O
-in	O
-the	O
-SERK1	O
-N	O
--	O
-terminal	O
-capping	O
-domain	O
-(	O
-Thr59SERK1	O
-,	O
-Phe61SERK1	O
-)	O
-and	O
-the	O
-LRR	O
-inner	O
-surface	O
-(	O
-Asp75SERK1	O
-,	O
-Tyr101SERK1	O
-,	O
-SER121SERK1	O
-,	O
-Phe145SERK1	O
-)	O
-contribute	O
-to	O
-the	O
-formation	O
-of	O
-both	O
-complexes	O
-(	O
-Figures	O
-4C	O
-,	O
-D	O
-,	O
-6B	O
-).	O
-	O
-In	O
-addition	O
-,	O
-residues	O
-53	O
--	O
-55SERK1	O
-from	O
-the	O
-SERK1	O
-N	O
--	O
-terminal	O
-cap	O
-mediate	O
-specific	O
-interactions	O
-with	O
-the	O
-IDA	O
-peptide	O
-(	O
-Figures	O
-4C	O
-,	O
-6B	O
-).	O
-	O
-These	O
-residues	O
-are	O
-not	O
-involved	O
-in	O
-the	O
-sensing	O
-of	O
-the	O
-steroid	O
-hormone	O
-brassinolide	O
-.	O
-	O
-In	O
-both	O
-cases	O
-however	O
-,	O
-the	O
-co	O
--	O
-receptor	O
-completes	O
-the	O
-hormone	O
-binding	O
-pocket	O
-.	O
-	O
-This	O
-fact	O
-together	O
-with	O
-the	O
-largely	O
-overlapping	O
-SERK1	O
-binding	O
-surfaces	O
-in	O
-HAESA	O
-and	O
-BRI1	O
-allows	O
-us	O
-to	O
-speculate	O
-that	O
-SERK1	O
-may	O
-promote	O
-high	O
--	O
-affinity	O
-peptide	O
-hormone	O
-and	O
-brassinosteroid	O
-sensing	O
-by	O
-simply	O
-slowing	O
-down	O
-dissociation	O
-of	O
-the	O
-ligand	O
-from	O
-its	O
-cognate	O
-receptor	O
-.	O
-	O
-Different	O
-plant	O
-peptide	O
-hormone	O
-families	O
-contain	O
-a	O
-C	O
--	O
-terminal	O
-(	O
-Arg	O
-)-	O
-His	O
--	O
-Asn	O
-motif	O
-,	O
-which	O
-in	O
-IDA	O
-represents	O
-the	O
-co	O
--	O
-receptor	O
-recognition	O
-site	O
-.	O
-	O
-Structure	O
--	O
-guided	O
-multiple	O
-sequence	O
-alignment	O
-of	O
-IDA	O
-and	O
-IDA	O
--	O
-like	O
-peptides	O
-with	O
-other	O
-plant	O
-peptide	O
-hormone	O
-families	O
-,	O
-including	O
-CLAVATA3	O
-–	O
-EMBRYO	O
-SURROUNDING	O
-REGION	O
--	O
-RELATED	O
-(	O
-CLV3	O
-/	O
-CLE	O
-),	O
-ROOT	O
-GROWTH	O
-FACTOR	O
-–	O
-GOLVEN	O
-(	O
-RGF	O
-/	O
-GLV	O
-),	O
-PRECURSOR	O
-GENE	O
-PROPEP1	O
-(	O
-PEP1	O
-)	O
-from	O
-Arabidopsis	O
-thaliana	O
-.	O
-	O
-The	O
-conserved	O
-(	O
-Arg	O
-)-	O
-His	O
--	O
-Asn	O
-motif	O
-is	O
-highlighted	O
-in	O
-red	O
-,	O
-the	O
-central	O
-Hyp	O
-residue	O
-in	O
-IDLs	O
-and	O
-CLEs	O
-is	O
-marked	O
-in	O
-blue	O
-.	O
-	O
-Our	O
-experiments	O
-reveal	O
-that	O
-SERK1	O
-recognizes	O
-a	O
-C	O
--	O
-terminal	O
-Arg	O
--	O
-His	O
--	O
-Asn	O
-motif	O
-in	O
-IDA	O
-.	O
-	O
-Importantly	O
-,	O
-this	O
-motif	O
-can	O
-also	O
-be	O
-found	O
-in	O
-other	O
-peptide	O
-hormone	O
-families	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-Among	O
-these	O
-are	O
-the	O
-CLE	O
-peptides	O
-regulating	O
-stem	O
-cell	O
-maintenance	O
-in	O
-the	O
-shoot	O
-and	O
-the	O
-root	O
-.	O
-	O
-It	O
-is	O
-interesting	O
-to	O
-note	O
-,	O
-that	O
-CLEs	O
-in	O
-their	O
-mature	O
-form	O
-are	O
-also	O
-hydroxyprolinated	O
-dodecamers	O
-,	O
-which	O
-bind	O
-to	O
-a	O
-surface	O
-area	O
-in	O
-the	O
-BARELY	O
-ANY	O
-MERISTEM	O
-1	O
-receptor	O
-that	O
-would	O
-correspond	O
-to	O
-part	O
-of	O
-the	O
-IDA	O
-binding	O
-cleft	O
-in	O
-HAESA	O
-.	O
-	O
-Diverse	O
-plant	O
-peptide	O
-hormones	O
-may	O
-thus	O
-also	O
-bind	O
-their	O
-LRR	O
--	O
-RK	O
-receptors	O
-in	O
-an	O
-extended	O
-conformation	O
-along	O
-the	O
-inner	O
-surface	O
-of	O
-the	O
-LRR	O
-domain	O
-and	O
-may	O
-also	O
-use	O
-small	O
-,	O
-shape	O
--	O
-complementary	O
-co	O
--	O
-receptors	O
-for	O
-high	O
--	O
-affinity	O
-ligand	O
-binding	O
-and	O
-receptor	O
-activation	O
-.	O
-	O
-Ensemble	O
-cryo	O
--	O
-EM	O
-uncovers	O
-inchworm	O
--	O
-like	O
-translocation	O
-of	O
-a	O
-viral	O
-IRES	O
-through	O
-the	O
-ribosome	O
-	O
-Internal	O
-ribosome	O
-entry	O
-sites	O
-(	O
-IRESs	O
-)	O
-mediate	O
-cap	O
--	O
-independent	O
-translation	O
-of	O
-viral	O
-mRNAs	O
-.	O
-	O
-Using	O
-electron	O
-cryo	O
--	O
-microscopy	O
-of	O
-a	O
-single	O
-specimen	O
-,	O
-we	O
-present	O
-five	O
-ribosome	O
-structures	O
-formed	O
-with	O
-the	O
-Taura	O
-syndrome	O
-virus	O
-IRES	O
-and	O
-translocase	O
-eEF2	O
-•	O
-GTP	O
-bound	O
-with	O
-sordarin	O
-.	O
-	O
-The	O
-structures	O
-suggest	O
-a	O
-trajectory	O
-of	O
-IRES	O
-translocation	O
-,	O
-required	O
-for	O
-translation	O
-initiation	O
-,	O
-and	O
-provide	O
-an	O
-unprecedented	O
-view	O
-of	O
-eEF2	O
-dynamics	O
-.	O
-	O
-The	O
-IRES	O
-rearranges	O
-from	O
-extended	O
-to	O
-bent	O
-to	O
-extended	O
-conformations	O
-.	O
-	O
-This	O
-inchworm	O
--	O
-like	O
-movement	O
-is	O
-coupled	O
-with	O
-ribosomal	O
-inter	O
--	O
-subunit	O
-rotation	O
-and	O
-40S	O
-head	O
-swivel	O
-.	O
-	O
-eEF2	O
-,	O
-attached	O
-to	O
-the	O
-60S	O
-subunit	O
-,	O
-slides	O
-along	O
-the	O
-rotating	O
-40S	O
-subunit	O
-to	O
-enter	O
-the	O
-A	O
-site	O
-.	O
-	O
-Its	O
-diphthamide	O
--	O
-bearing	O
-tip	O
-at	O
-domain	O
-IV	O
-separates	O
-the	O
-tRNA	O
--	O
-mRNA	O
--	O
-like	O
-pseudoknot	O
-I	O
-(	O
-PKI	O
-)	O
-of	O
-the	O
-IRES	O
-from	O
-the	O
-decoding	O
-center	O
-.	O
-	O
-This	O
-unlocks	O
-40S	O
-domains	O
-,	O
-facilitating	O
-head	O
-swivel	O
-and	O
-biasing	O
-IRES	O
-translocation	O
-via	O
-hitherto	O
--	O
-elusive	O
-intermediates	O
-with	O
-PKI	O
-captured	O
-between	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-.	O
-	O
-The	O
-structures	O
-suggest	O
-missing	O
-links	O
-in	O
-our	O
-understanding	O
-of	O
-tRNA	O
-translocation	O
-.	O
-	O
-Virus	O
-propagation	O
-relies	O
-on	O
-the	O
-host	O
-translational	O
-apparatus	O
-.	O
-	O
-To	O
-efficiently	O
-compete	O
-with	O
-host	O
-mRNAs	O
-and	O
-engage	O
-in	O
-translation	O
-under	O
-stress	O
-,	O
-some	O
-viral	O
-mRNAs	O
-undergo	O
-cap	O
--	O
-independent	O
-translation	O
-.	O
-	O
-To	O
-this	O
-end	O
-,	O
-internal	O
-ribosome	O
-entry	O
-site	O
-(	O
-IRES	O
-)	O
-RNAs	O
-are	O
-employed	O
-(	O
-reviewed	O
-in	O
-.	O
-	O
-An	O
-IRES	O
-is	O
-located	O
-at	O
-the	O
-5	O
-’	O
-untranslated	O
-region	O
-of	O
-the	O
-viral	O
-mRNA	O
-,	O
-preceding	O
-an	O
-open	O
-reading	O
-frame	O
-(	O
-ORF	O
-).	O
-	O
-To	O
-initiate	O
-translation	O
-,	O
-a	O
-structured	O
-IRES	O
-RNA	O
-interacts	O
-with	O
-the	O
-40S	O
-subunit	O
-or	O
-the	O
-80S	O
-ribosome	O
-,	O
-resulting	O
-in	O
-precise	O
-positioning	O
-of	O
-the	O
-downstream	O
-start	O
-codon	O
-in	O
-the	O
-small	O
-40S	O
-subunit	O
-.	O
-	O
-The	O
-canonical	O
-scenario	O
-of	O
-cap	O
--	O
-dependent	O
-and	O
-IRES	O
--	O
-dependent	O
-initiation	O
-involves	O
-positioning	O
-of	O
-the	O
-AUG	O
-start	O
-codon	O
-and	O
-the	O
-initiator	O
-tRNAMet	O
-in	O
-the	O
-ribosomal	O
-peptidyl	O
--	O
-tRNA	O
-(	O
-P	O
-)	O
-site	O
-,	O
-facilitated	O
-by	O
-interaction	O
-with	O
-initiation	O
-factors	O
-.	O
-	O
-Subsequent	O
-binding	O
-of	O
-an	O
-elongator	O
-aminoacyl	O
--	O
-tRNA	O
-to	O
-the	O
-ribosomal	O
-A	O
-site	O
-transitions	O
-the	O
-initiation	O
-complex	O
-into	O
-the	O
-elongation	O
-cycle	O
-of	O
-translation	O
-.	O
-	O
-Upon	O
-peptide	O
-bond	O
-formation	O
-,	O
-the	O
-two	O
-tRNAs	O
-and	O
-their	O
-respective	O
-mRNA	O
-codons	O
-translocate	O
-from	O
-the	O
-A	O
-and	O
-P	O
-to	O
-P	O
-and	O
-E	O
-(	O
-exit	O
-)	O
-sites	O
-,	O
-freeing	O
-the	O
-A	O
-site	O
-for	O
-the	O
-next	O
-elongator	O
-tRNA	O
-.	O
-	O
-An	O
-unusual	O
-strategy	O
-of	O
-initiation	O
-is	O
-used	O
-by	O
-intergenic	O
--	O
-region	O
-(	O
-IGR	O
-)	O
-IRESs	O
-found	O
-in	O
-Dicistroviridae	O
-arthropod	O
--	O
-infecting	O
-viruses	O
-.	O
-	O
-These	O
-include	O
-shrimp	O
--	O
-infecting	O
-Taura	O
-syndrome	O
-virus	O
-(	O
-TSV	O
-),	O
-and	O
-insect	O
-viruses	O
-Plautia	O
-stali	O
-intestine	O
-virus	O
-(	O
-PSIV	O
-)	O
-and	O
-Cricket	O
-paralysis	O
-virus	O
-(	O
-CrPV	O
-).	O
-	O
-The	O
-IGR	O
-IRES	O
-mRNAs	O
-do	O
-not	O
-contain	O
-an	O
-AUG	O
-start	O
-codon	O
-.	O
-	O
-The	O
-IGR	O
--	O
-IRES	O
--	O
-driven	O
-initiation	O
-does	O
-not	O
-involve	O
-initiator	O
-tRNAMet	O
-and	O
-initiation	O
-factors	O
-.	O
-	O
-As	O
-such	O
-,	O
-this	O
-group	O
-of	O
-IRESs	O
-represents	O
-the	O
-most	O
-streamlined	O
-mechanism	O
-of	O
-eukaryotic	O
-translation	O
-initiation	O
-.	O
-	O
-A	O
-recent	O
-demonstration	O
-of	O
-bacterial	O
-translation	O
-initiation	O
-by	O
-an	O
-IGR	O
-IRES	O
-indicates	O
-that	O
-the	O
-IRESs	O
-take	O
-advantage	O
-of	O
-conserved	O
-structural	O
-and	O
-dynamic	O
-properties	O
-of	O
-the	O
-ribosome	O
-.	O
-	O
-Early	O
-electron	O
-cryo	O
--	O
-microscopy	O
-(	O
-cryo	O
--	O
-EM	O
-)	O
-studies	O
-have	O
-found	O
-that	O
-the	O
-CrPV	O
-IRES	O
-packs	O
-in	O
-the	O
-ribosome	O
-intersubunit	O
-space	O
-.	O
-	O
-Recent	O
-cryo	O
--	O
-EM	O
-structures	O
-of	O
-ribosome	O
--	O
-bound	O
-TSV	O
-IRES	O
-and	O
-CrPV	O
-IRES	O
-revealed	O
-that	O
-IGR	O
-IRESs	O
-position	O
-the	O
-ORF	O
-by	O
-mimicking	O
-a	O
-translating	O
-ribosome	O
-bound	O
-with	O
-tRNA	O
-and	O
-mRNA	O
-.	O
-	O
-The	O
-~	O
-200	O
--	O
-nt	O
-IRES	O
-RNAs	O
-span	O
-from	O
-the	O
-A	O
-site	O
-beyond	O
-the	O
-E	O
-site	O
-.	O
-	O
-A	O
-conserved	O
-tRNA	O
--	O
-mRNA	O
-–	O
-like	O
-structural	O
-element	O
-of	O
-pseudoknot	O
-I	O
-(	O
-PKI	O
-)	O
-interacts	O
-with	O
-the	O
-decoding	O
-center	O
-in	O
-the	O
-A	O
-site	O
-of	O
-the	O
-40S	O
-subunit	O
-.	O
-	O
-The	O
-codon	O
--	O
-anticodon	O
--	O
-like	O
-helix	O
-of	O
-PKI	O
-is	O
-stabilized	O
-by	O
-interactions	O
-with	O
-the	O
-universally	O
-conserved	O
-decoding	O
--	O
-center	O
-nucleotides	O
-G577	O
-,	O
-A1755	O
-and	O
-A1756	O
-(	O
-G530	O
-,	O
-A1492	O
-and	O
-A1493	O
-in	O
-E	O
-.	O
-coli	O
-16S	O
-ribosomal	O
-RNA	O
-,	O
-or	O
-rRNA	O
-).	O
-	O
-The	O
-downstream	O
-initiation	O
-codon	O
-—	O
-coding	O
-for	O
-alanine	O
-—	O
-is	O
-placed	O
-in	O
-the	O
-mRNA	O
-tunnel	O
-,	O
-preceding	O
-the	O
-decoding	O
-center	O
-.	O
-	O
-PKI	O
-of	O
-IGR	O
-IRESs	O
-therefore	O
-mimics	O
-an	O
-A	O
--	O
-site	O
-elongator	O
-tRNA	O
-interacting	O
-with	O
-an	O
-mRNA	O
-sense	O
-codon	O
-,	O
-but	O
-not	O
-a	O
-P	O
--	O
-site	O
-initiator	O
-tRNAMet	O
-and	O
-the	O
-AUG	O
-start	O
-codon	O
-.	O
-	O
-How	O
-this	O
-non	O
--	O
-canonical	O
-initiation	O
-complex	O
-transitions	O
-to	O
-the	O
-elongation	O
-step	O
-is	O
-not	O
-fully	O
-understood	O
-.	O
-	O
-For	O
-a	O
-cognate	O
-aminoacyl	O
--	O
-tRNA	O
-to	O
-bind	O
-the	O
-first	O
-viral	O
-mRNA	O
-codon	O
-,	O
-PKI	O
-has	O
-to	O
-be	O
-translocated	O
-from	O
-the	O
-A	O
-site	O
-,	O
-so	O
-that	O
-the	O
-first	O
-codon	O
-can	O
-be	O
-presented	O
-in	O
-the	O
-A	O
-site	O
-.	O
-	O
-A	O
-cryo	O
--	O
-EM	O
-structure	O
-of	O
-the	O
-ribosome	O
-bound	O
-with	O
-a	O
-CrPV	O
-IRES	O
-and	O
-release	O
-factor	O
-eRF1	O
-occupying	O
-the	O
-A	O
-site	O
-provided	O
-insight	O
-into	O
-the	O
-post	O
--	O
-translocation	O
-state	O
-.	O
-	O
-In	O
-this	O
-structure	O
-,	O
-PKI	O
-is	O
-positioned	O
-in	O
-the	O
-P	O
-site	O
-and	O
-the	O
-first	O
-mRNA	O
-codon	O
-is	O
-located	O
-in	O
-the	O
-A	O
-site	O
-.	O
-	O
-How	O
-the	O
-large	O
-IRES	O
-RNA	O
-translocates	O
-within	O
-the	O
-ribosome	O
-,	O
-allowing	O
-PKI	O
-translocation	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
-is	O
-not	O
-known	O
-.	O
-	O
-The	O
-structural	O
-similarity	O
-of	O
-PKI	O
-and	O
-the	O
-tRNA	O
-anticodon	O
-stem	O
-loop	O
-(	O
-ASL	O
-)	O
-bound	O
-to	O
-a	O
-codon	O
-suggests	O
-that	O
-their	O
-mechanisms	O
-of	O
-translocation	O
-are	O
-similar	O
-to	O
-some	O
-extent	O
-.	O
-	O
-Translocation	O
-of	O
-the	O
-IRES	O
-or	O
-tRNA	O
--	O
-mRNA	O
-requires	O
-eukaryotic	O
-elongation	O
-factor	O
-2	O
-(	O
-eEF2	O
-),	O
-a	O
-structural	O
-and	O
-functional	O
-homolog	O
-of	O
-the	O
-well	O
--	O
-studied	O
-bacterial	O
-EF	O
--	O
-G	O
-.	O
-Pre	O
--	O
-translocation	O
-tRNA	O
--	O
-bound	O
-ribosomes	O
-contain	O
-a	O
-peptidyl	O
--	O
-and	O
-deacyl	O
--	O
-tRNA	O
-,	O
-both	O
-base	O
--	O
-paired	O
-to	O
-mRNA	O
-codons	O
-in	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-(	O
-termed	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-).	O
-	O
-Translocation	O
-of	O
-2tRNA	O
-•	O
-mRNA	O
-involves	O
-two	O
-major	O
-large	O
--	O
-scale	O
-ribosome	O
-rearrangements	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1	O
-)	O
-(	O
-reviewed	O
-in	O
-).	O
-	O
-First	O
-,	O
-studies	O
-of	O
-bacterial	O
-ribosomes	O
-showed	O
-that	O
-a	O
-~	O
-10	O
-°	O
-rotation	O
-of	O
-the	O
-small	O
-subunit	O
-relative	O
-to	O
-the	O
-large	O
-subunit	O
-,	O
-known	O
-as	O
-intersubunit	O
-rotation	O
-,	O
-or	O
-ratcheting	O
-,	O
-is	O
-required	O
-for	O
-translocation	O
-.	O
-	O
-Intersubunit	O
-rotation	O
-occurs	O
-spontaneously	O
-upon	O
-peptidyl	O
-transfer	O
-,	O
-and	O
-is	O
-coupled	O
-with	O
-formation	O
-of	O
-hybrid	O
-tRNA	O
-states	O
-.	O
-	O
-In	O
-the	O
-rotated	O
-pre	O
--	O
-translocation	O
-ribosome	O
-,	O
-the	O
-peptidyl	O
--	O
-tRNA	O
-binds	O
-the	O
-A	O
-site	O
-of	O
-the	O
-small	O
-subunit	O
-with	O
-its	O
-ASL	O
-and	O
-the	O
-P	O
-site	O
-of	O
-the	O
-large	O
-subunit	O
-with	O
-the	O
-CCA	O
-3	O
-’	O
-end	O
-(	O
-A	O
-/	O
-P	O
-hybrid	O
-state	O
-).	O
-	O
-Concurrently	O
-,	O
-the	O
-deacyl	O
--	O
-tRNA	O
-interacts	O
-with	O
-the	O
-P	O
-site	O
-of	O
-the	O
-small	O
-subunit	O
-and	O
-the	O
-E	O
-site	O
-of	O
-the	O
-large	O
-subunit	O
-(	O
-P	O
-/	O
-E	O
-hybrid	O
-state	O
-).	O
-	O
-The	O
-ribosome	O
-can	O
-undergo	O
-spontaneous	O
-,	O
-thermally	O
--	O
-driven	O
-forward	O
--	O
-reverse	O
-rotation	O
-that	O
-shifts	O
-the	O
-two	O
-tRNAs	O
-between	O
-the	O
-hybrid	O
-and	O
-'	O
-classical	O
-'	O
-states	O
-while	O
-the	O
-anticodon	O
-stem	O
-loops	O
-remain	O
-non	O
--	O
-translocated	O
-.	O
-	O
-Binding	O
-of	O
-EF	O
--	O
-G	O
-next	O
-to	O
-the	O
-A	O
-site	O
-and	O
-reverse	O
-rotation	O
-of	O
-the	O
-small	O
-subunit	O
-results	O
-in	O
-translocation	O
-of	O
-both	O
-ASLs	O
-on	O
-the	O
-small	O
-subunit	O
-.	O
-	O
-EF	O
--	O
-G	O
-is	O
-thought	O
-to	O
-'	O
-unlock	O
-'	O
-the	O
-pre	O
--	O
-translocation	O
-ribosome	O
-,	O
-allowing	O
-movement	O
-of	O
-the	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-,	O
-however	O
-the	O
-structural	O
-details	O
-of	O
-this	O
-unlocking	O
-are	O
-not	O
-known	O
-.	O
-	O
-The	O
-second	O
-large	O
--	O
-scale	O
-rearrangement	O
-involves	O
-rotation	O
-,	O
-or	O
-swiveling	O
-,	O
-of	O
-the	O
-head	O
-of	O
-the	O
-small	O
-subunit	O
-relative	O
-to	O
-the	O
-body	O
-.	O
-	O
-The	O
-head	O
-can	O
-rotate	O
-by	O
-up	O
-to	O
-~	O
-20	O
-°	O
-around	O
-the	O
-axis	O
-nearly	O
-orthogonal	O
-to	O
-that	O
-of	O
-intersubunit	O
-rotation	O
-,	O
-in	O
-the	O
-absence	O
-of	O
-tRNA	O
-or	O
-in	O
-the	O
-presence	O
-of	O
-a	O
-single	O
-P	O
-/	O
-E	O
-tRNA	O
-and	O
-eEF2	O
-or	O
-EF	O
--	O
-G	O
-.	O
-Förster	O
-resonance	O
-energy	O
-transfer	O
-(	O
-FRET	O
-)	O
-data	O
-suggest	O
-that	O
-head	O
-swivel	O
-of	O
-the	O
-rotated	O
-small	O
-subunit	O
-facilitates	O
-EF	O
--	O
-G	O
--	O
-mediated	O
-movement	O
-of	O
-2tRNA	O
-•	O
-mRNA	O
-.	O
-	O
-Structures	O
-of	O
-the	O
-70S	O
-•	O
-EF	O
--	O
-G	O
-complex	O
-bound	O
-with	O
-two	O
-nearly	O
-translocated	O
-tRNAs	O
-,	O
-exhibit	O
-a	O
-large	O
-18	O
-°	O
-to	O
-21	O
-°	O
-head	O
-swivel	O
-in	O
-a	O
-mid	O
--	O
-rotated	O
-subunit	O
-,	O
-whereas	O
-no	O
-head	O
-swivel	O
-is	O
-observed	O
-in	O
-the	O
-fully	O
-rotated	O
-pre	O
--	O
-translocation	O
-or	O
-in	O
-the	O
-non	O
--	O
-rotated	O
-post	O
--	O
-translocation	O
-70S	O
-•	O
-2tRNA	O
-•	O
-EF	O
--	O
-G	O
-structures	O
-.	O
-	O
-The	O
-structural	O
-role	O
-of	O
-head	O
-swivel	O
-is	O
-not	O
-fully	O
-understood	O
-.	O
-	O
-The	O
-head	O
-swivel	O
-was	O
-proposed	O
-to	O
-facilitate	O
-transition	O
-of	O
-the	O
-tRNA	O
-from	O
-the	O
-P	O
-to	O
-E	O
-site	O
-by	O
-widening	O
-a	O
-constriction	O
-between	O
-these	O
-sites	O
-on	O
-the	O
-30S	O
-subunit	O
-.	O
-	O
-This	O
-widening	O
-allows	O
-the	O
-ASL	O
-to	O
-sample	O
-positions	O
-between	O
-the	O
-P	O
-and	O
-E	O
-sites	O
-.	O
-	O
-Whether	O
-and	O
-how	O
-the	O
-head	O
-swivel	O
-mediates	O
-tRNA	O
-transition	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
-remains	O
-unknown	O
-.	O
-	O
-Comparison	O
-of	O
-70S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-and	O
-80S	O
-•	O
-IRES	O
-translocation	O
-complexes	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structures	O
-of	O
-bacterial	O
-70S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-translocation	O
-complexes	O
-,	O
-ordered	O
-according	O
-to	O
-the	O
-position	O
-of	O
-the	O
-translocating	O
-A	O
-->	O
-P	O
-tRNA	O
-(	O
-orange	O
-).	O
-	O
-The	O
-large	O
-ribosomal	O
-subunit	O
-is	O
-shown	O
-in	O
-cyan	O
-;	O
-the	O
-small	O
-subunit	O
-in	O
-light	O
-yellow	O
-(	O
-head	O
-)	O
-and	O
-wheat	O
--	O
-yellow	O
-(	O
-body	O
-),	O
-elongation	O
-factor	O
-G	O
-(	O
-EF	O
--	O
-G	O
-)	O
-is	O
-shown	O
-in	O
-green	O
-.	O
-	O
-Nucleotides	O
-C1054	O
-,	O
-G966	O
-and	O
-G693	O
-of	O
-16S	O
-rRNA	O
-are	O
-shown	O
-in	O
-black	O
-to	O
-denote	O
-the	O
-A	O
-,	O
-P	O
-and	O
-E	O
-sites	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-extents	O
-of	O
-the	O
-30S	O
-subunit	O
-rotation	O
-and	O
-head	O
-swivel	O
-relative	O
-to	O
-their	O
-positions	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-structure	O
-are	O
-shown	O
-with	O
-arrows	O
-.	O
-	O
-References	O
-and	O
-PDB	O
-codes	O
-of	O
-the	O
-structures	O
-are	O
-shown	O
-.	O
-	O
-(	O
-b	O
-)	O
-Structures	O
-of	O
-the	O
-80S	O
-•	O
-IRES	O
-complexes	O
-in	O
-the	O
-absence	O
-and	O
-presence	O
-of	O
-eEF2	O
-(	O
-this	O
-work	O
-).	O
-	O
-The	O
-large	O
-ribosomal	O
-subunit	O
-is	O
-shown	O
-in	O
-cyan	O
-;	O
-the	O
-small	O
-subunit	O
-in	O
-light	O
-yellow	O
-(	O
-head	O
-)	O
-and	O
-wheat	O
--	O
-yellow	O
-(	O
-body	O
-);	O
-the	O
-TSV	O
-IRES	O
-in	O
-red	O
-,	O
-eEF2	O
-in	O
-green	O
-.	O
-	O
-Nucleotides	O
-C1274	O
-,	O
-U1191	O
-of	O
-the	O
-40S	O
-head	O
-and	O
-G904	O
-of	O
-the	O
-platform	O
-(	O
-corresponding	O
-to	O
-C1054	O
-,	O
-G966	O
-and	O
-G693	O
-in	O
-E	O
-.	O
-coli	O
-16S	O
-rRNA	O
-)	O
-are	O
-shown	O
-in	O
-black	O
-to	O
-denote	O
-the	O
-A	O
-,	O
-P	O
-and	O
-E	O
-sites	O
-,	O
-respectively	O
-.	O
-	O
-Unresolved	O
-regions	O
-of	O
-the	O
-IRES	O
-in	O
-densities	O
-for	O
-Structures	O
-III	O
-and	O
-V	O
-are	O
-shown	O
-in	O
-gray	O
-.	O
-	O
-The	O
-extents	O
-of	O
-the	O
-40S	O
-subunit	O
-rotation	O
-and	O
-head	O
-swivel	O
-relative	O
-to	O
-their	O
-positions	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-structure	O
-are	O
-shown	O
-with	O
-arrows	O
-.	O
-	O
-Schematic	O
-of	O
-cryo	O
--	O
-EM	O
-refinement	O
-and	O
-classification	O
-procedures	O
-.	O
-	O
-All	O
-particles	O
-were	O
-initially	O
-aligned	O
-to	O
-a	O
-single	O
-model	O
-.	O
-	O
-3D	O
-classification	O
-using	O
-a	O
-3D	O
-mask	O
-around	O
-the	O
-40S	O
-head	O
-,	O
-TSV	O
-IRES	O
-and	O
-eEF2	O
-,	O
-of	O
-the	O
-4x	O
-binned	O
-stack	O
-was	O
-used	O
-to	O
-identify	O
-particles	O
-containing	O
-both	O
-the	O
-IRES	O
-and	O
-eEF2	O
-.	O
-	O
-Subsequent	O
-3D	O
-classification	O
-using	O
-a	O
-2D	O
-mask	O
-comprising	O
-PKI	O
-and	O
-domain	O
-IV	O
-of	O
-eEF2	O
-yielded	O
-5	O
-'	O
-purified	O
-'	O
-classes	O
-representing	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-Sub	O
--	O
-classification	O
-of	O
-each	O
-class	O
-did	O
-not	O
-yield	O
-additional	O
-classes	O
-,	O
-but	O
-helped	O
-improve	O
-density	O
-in	O
-the	O
-PKI	O
-region	O
-of	O
-class	O
-III	O
-(	O
-estimated	O
-resolution	O
-and	O
-percentage	O
-of	O
-particles	O
-in	O
-the	O
-sub	O
--	O
-classified	O
-reconstruction	O
-are	O
-shown	O
-in	O
-parentheses	O
-).	O
-	O
-Cryo	O
--	O
-EM	O
-density	O
-of	O
-Structures	O
-I	O
--	O
-V	O
-.	O
-	O
-In	O
-panels	O
-(	O
-a	O
--	O
-e	O
-),	O
-the	O
-maps	O
-are	O
-segmented	O
-and	O
-colored	O
-as	O
-in	O
-Figure	O
-1	O
-.	O
-	O
-The	O
-maps	O
-in	O
-all	O
-panels	O
-were	O
-B	O
--	O
-softened	O
-by	O
-applying	O
-a	O
-B	O
--	O
-factor	O
-of	O
-30	O
-Å2	O
-.	O
-	O
-(	O
-a	O
--	O
-e	O
-)	O
-Cryo	O
--	O
-EM	O
-map	O
-of	O
-Structures	O
-I	O
-,	O
-II	O
-,	O
-III	O
-,	O
-IV	O
-and	O
-V	O
-.	O
-(	O
-f	O
--	O
-j	O
-)	O
-Local	O
-resolution	O
-of	O
-unfiltered	O
-and	O
-unmasked	O
-cryo	O
--	O
-EM	O
-reconstructions	O
-,	O
-assessed	O
-using	O
-Blocres	O
-from	O
-the	O
-BSoft	O
-package	O
-,	O
-for	O
-Structures	O
-I	O
-,	O
-II	O
-,	O
-III	O
-,	O
-IV	O
-and	O
-V	O
-.	O
-(	O
-k	O
--	O
-o	O
-)	O
-Cryo	O
--	O
-EM	O
-density	O
-for	O
-the	O
-TSV	O
-IRES	O
-(	O
-red	O
-model	O
-)	O
-and	O
-eEF2	O
-(	O
-green	O
-model	O
-)	O
-in	O
-Structures	O
-I	O
-,	O
-II	O
-,	O
-III	O
-,	O
-IV	O
-and	O
-V	O
-.	O
-(	O
-p	O
-)	O
-Fourier	O
-shell	O
-correlation	O
-(	O
-FSC	O
-)	O
-curves	O
-for	O
-Structures	O
-I	O
--	O
-V	O
-.	O
-The	O
-horizontal	O
-axis	O
-is	O
-labeled	O
-with	O
-spatial	O
-frequency	O
-Å	O
--	O
-1	O
-and	O
-with	O
-Å	O
-.	O
-The	O
-resolutions	O
-stated	O
-in	O
-the	O
-text	O
-correspond	O
-to	O
-an	O
-FSC	O
-threshold	O
-value	O
-of	O
-0	O
-.	O
-143	O
-,	O
-shown	O
-as	O
-a	O
-dotted	O
-line	O
-,	O
-for	O
-the	O
-FREALIGN	O
--	O
-derived	O
-FSC	O
-('	O
-Part_FSC	O
-').	O
-	O
-Cryo	O
--	O
-EM	O
-structures	O
-of	O
-the	O
-80S	O
-•	O
-TSV	O
-IRES	O
-bound	O
-with	O
-eEF2	O
-•	O
-GDP	O
-•	O
-sordarin	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-In	O
-all	O
-panels	O
-,	O
-the	O
-large	O
-ribosomal	O
-subunit	O
-is	O
-shown	O
-in	O
-cyan	O
-;	O
-the	O
-small	O
-subunit	O
-in	O
-light	O
-yellow	O
-(	O
-head	O
-)	O
-and	O
-wheat	O
--	O
-yellow	O
-(	O
-body	O
-);	O
-the	O
-TSV	O
-IRES	O
-in	O
-red	O
-,	O
-eEF2	O
-in	O
-green	O
-.	O
-	O
-Nucleotides	O
-C1274	O
-,	O
-U1191	O
-of	O
-the	O
-40S	O
-head	O
-and	O
-G904	O
-of	O
-the	O
-platform	O
-(	O
-C1054	O
-,	O
-G966	O
-and	O
-G693	O
-in	O
-E	O
-.	O
-coli	O
-16S	O
-rRNA	O
-)	O
-are	O
-shown	O
-in	O
-black	O
-to	O
-denote	O
-the	O
-A	O
-,	O
-P	O
-and	O
-E	O
-sites	O
-,	O
-respectively	O
-.	O
-	O
-(	O
-b	O
-)	O
-Schematic	O
-representation	O
-of	O
-the	O
-structures	O
-shown	O
-in	O
-panel	O
-a	O
-,	O
-denoting	O
-the	O
-conformations	O
-of	O
-the	O
-small	O
-subunit	O
-relative	O
-to	O
-the	O
-large	O
-subunit	O
-.	O
-	O
-A	O
-,	O
-P	O
-and	O
-E	O
-sites	O
-are	O
-shown	O
-as	O
-rectangles	O
-.	O
-	O
-All	O
-measurements	O
-are	O
-relative	O
-to	O
-the	O
-non	O
--	O
-rotated	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-structure	O
-.	O
-	O
-We	O
-sought	O
-to	O
-address	O
-the	O
-following	O
-questions	O
-by	O
-structural	O
-visualization	O
-of	O
-80S	O
-•	O
-IRES	O
-•	O
-eEF2	O
-translocation	O
-complexes	O
-:	O
-(	O
-1	O
-)	O
-How	O
-does	O
-a	O
-large	O
-IRES	O
-RNA	O
-move	O
-through	O
-the	O
-restricted	O
-intersubunit	O
-space	O
-,	O
-bringing	O
-PKI	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
-of	O
-the	O
-small	O
-subunit	O
-?	O
-(	O
-2	O
-)	O
-How	O
-does	O
-eEF2	O
-mediate	O
-IRES	O
-translocation	O
-?	O
-(	O
-3	O
-)	O
-Does	O
-IRES	O
-translocation	O
-involve	O
-large	O
-rearrangements	O
-in	O
-the	O
-ribosome	O
-,	O
-similar	O
-to	O
-tRNA	O
-translocation	O
-?	O
-(	O
-4	O
-)	O
-What	O
-,	O
-if	O
-any	O
-,	O
-is	O
-the	O
-mechanistic	O
-role	O
-of	O
-40S	O
-head	O
-rotation	O
-in	O
-IRES	O
-translocation	O
-?	O
-	O
-We	O
-used	O
-cryo	O
--	O
-EM	O
-to	O
-visualize	O
-80S	O
-•	O
-TSV	O
-IRES	O
-complexes	O
-formed	O
-in	O
-the	O
-presence	O
-of	O
-eEF2	O
-•	O
-GTP	O
-and	O
-the	O
-translation	O
-inhibitor	O
-sordarin	O
-,	O
-which	O
-stabilizes	O
-eEF2	O
-on	O
-the	O
-ribosome	O
-.	O
-	O
-Although	O
-the	O
-mechanism	O
-of	O
-sordarin	O
-action	O
-is	O
-not	O
-fully	O
-understood	O
-,	O
-the	O
-inhibitor	O
-does	O
-not	O
-affect	O
-the	O
-conformation	O
-of	O
-eEF2	O
-•	O
-GDPNP	O
-on	O
-the	O
-ribosome	O
-,	O
-rendering	O
-it	O
-an	O
-excellent	O
-tool	O
-in	O
-translocation	O
-studies	O
-.	O
-	O
-Maximum	O
--	O
-likelihood	O
-classification	O
-using	O
-FREALIGN	O
-identified	O
-five	O
-IRES	O
--	O
-eEF2	O
--	O
-bound	O
-ribosome	O
-structures	O
-within	O
-a	O
-single	O
-sample	O
-(	O
-Figures	O
-1	O
-and	O
-2	O
-).	O
-	O
-The	O
-structures	O
-differ	O
-in	O
-the	O
-positions	O
-and	O
-conformations	O
-of	O
-ribosomal	O
-subunits	O
-(	O
-Figures	O
-1b	O
-and	O
-2	O
-),	O
-IRES	O
-RNA	O
-(	O
-Figures	O
-3	O
-and	O
-4	O
-)	O
-and	O
-eEF2	O
-(	O
-Figures	O
-5	O
-and	O
-6	O
-).	O
-	O
-This	O
-ensemble	O
-of	O
-structures	O
-allowed	O
-us	O
-to	O
-reconstruct	O
-a	O
-sequence	O
-of	O
-steps	O
-in	O
-IRES	O
-translocation	O
-induced	O
-by	O
-eEF2	O
-.	O
-	O
-We	O
-used	O
-single	O
--	O
-particle	O
-cryo	O
--	O
-EM	O
-and	O
-maximum	O
--	O
-likelihood	O
-image	O
-classification	O
-in	O
-FREALIGN	O
-to	O
-obtain	O
-three	O
--	O
-dimensional	O
-density	O
-maps	O
-from	O
-a	O
-single	O
-specimen	O
-.	O
-	O
-The	O
-translocation	O
-complex	O
-was	O
-formed	O
-using	O
-S	O
-.	O
-cerevisiae	O
-80S	O
-ribosomes	O
-,	O
-Taura	O
-syndrome	O
-virus	O
-IRES	O
-,	O
-and	O
-S	O
-.	O
-cerevisiae	O
-eEF2	O
-in	O
-the	O
-presence	O
-of	O
-GTP	O
-and	O
-the	O
-eEF2	O
--	O
-binding	O
-translation	O
-inhibitor	O
-sordarin	O
-.	O
-	O
-Unsupervised	O
-cryo	O
--	O
-EM	O
-data	O
-classification	O
-was	O
-combined	O
-with	O
-the	O
-use	O
-of	O
-three	O
--	O
-dimensional	O
-and	O
-two	O
--	O
-dimensional	O
-masking	O
-around	O
-the	O
-ribosomal	O
-A	O
-site	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2	O
-).	O
-	O
-This	O
-approach	O
-revealed	O
-five	O
-80S	O
-•	O
-IRES	O
-•	O
-eEF2	O
-•	O
-GDP	O
-structures	O
-at	O
-average	O
-resolutions	O
-of	O
-3	O
-.	O
-5	O
-to	O
-4	O
-.	O
-2	O
-Å	O
-,	O
-sufficient	O
-to	O
-locate	O
-IRES	O
-domains	O
-and	O
-to	O
-resolve	O
-individual	O
-residues	O
-in	O
-the	O
-core	O
-regions	O
-of	O
-the	O
-ribosome	O
-and	O
-eEF2	O
-(	O
-Figures	O
-3c	O
-,	O
-d	O
-,	O
-and	O
-5f	O
-,	O
-h	O
-;	O
-see	O
-also	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2	O
-and	O
-Figure	O
-5	O
-—	O
-figure	O
-supplement	O
-2	O
-),	O
-including	O
-the	O
-post	O
--	O
-translational	O
-modification	O
-diphthamide	O
-699	O
-(	O
-Figure	O
-3c	O
-).	O
-	O
-Large	O
--	O
-scale	O
-rearrangements	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-,	O
-coupled	O
-with	O
-the	O
-movement	O
-of	O
-PKI	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
-and	O
-eEF2	O
-entry	O
-into	O
-the	O
-A	O
-site	O
-.	O
-	O
-(	O
-a	O
-)	O
-Rotational	O
-states	O
-of	O
-the	O
-40S	O
-subunit	O
-in	O
-the	O
-80S	O
-•	O
-IRES	O
-structure	O
-(	O
-INIT	O
-;	O
-PDB	O
-3J6Y	O
-)	O
-and	O
-in	O
-80S	O
-•	O
-IRES	O
-•	O
-eEF2	O
-Structures	O
-I	O
-,	O
-II	O
-,	O
-III	O
-,	O
-IV	O
-and	O
-V	O
-(	O
-this	O
-work	O
-).	O
-	O
-For	O
-each	O
-structure	O
-,	O
-the	O
-triangle	O
-outlines	O
-the	O
-contours	O
-of	O
-the	O
-40S	O
-body	O
-;	O
-the	O
-lower	O
-angle	O
-illustrates	O
-the	O
-extent	O
-of	O
-intersubunit	O
-(	O
-body	O
-)	O
-rotation	O
-.	O
-	O
-The	O
-sizes	O
-of	O
-the	O
-arrows	O
-correspond	O
-to	O
-the	O
-extent	O
-of	O
-the	O
-head	O
-swivel	O
-(	O
-yellow	O
-)	O
-and	O
-subunit	O
-rotation	O
-(	O
-black	O
-).	O
-	O
-The	O
-views	O
-were	O
-obtained	O
-by	O
-structural	O
-alignment	O
-of	O
-the	O
-25S	O
-rRNAs	O
-;	O
-the	O
-sarcin	O
--	O
-ricin	O
-loop	O
-(	O
-SRL	O
-)	O
-of	O
-25S	O
-rRNA	O
-is	O
-shown	O
-in	O
-gray	O
-for	O
-reference	O
-.	O
-	O
-(	O
-b	O
-)	O
-Solvent	O
-view	O
-(	O
-opposite	O
-from	O
-that	O
-shown	O
-in	O
-(	O
-a	O
-))	O
-of	O
-the	O
-40S	O
-subunit	O
-in	O
-the	O
-80S	O
-•	O
-IRES	O
-structure	O
-(	O
-INIT	O
-;	O
-PDB	O
-3J6Y	O
-)	O
-and	O
-in	O
-80S	O
-•	O
-IRES	O
-•	O
-eEF2	O
-Structures	O
-I	O
-,	O
-II	O
-,	O
-III	O
-,	O
-IV	O
-and	O
-V	O
-(	O
-this	O
-work	O
-).	O
-	O
-The	O
-structures	O
-are	O
-colored	O
-as	O
-in	O
-Figure	O
-1	O
-.	O
-	O
-(	O
-a	O
-)	O
-Comparison	O
-of	O
-the	O
-40S	O
--	O
-subunit	O
-rotational	O
-states	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-,	O
-sampling	O
-a	O
-~	O
-10	O
-°	O
-range	O
-between	O
-Structure	O
-I	O
-(	O
-fully	O
-rotated	O
-)	O
-and	O
-Structure	O
-V	O
-(	O
-non	O
--	O
-rotated	O
-).	O
-	O
-18S	O
-ribosomal	O
-RNA	O
-is	O
-shown	O
-and	O
-ribosomal	O
-proteins	O
-are	O
-omitted	O
-for	O
-clarity	O
-.	O
-	O
-The	O
-superpositions	O
-of	O
-Structures	O
-I	O
--	O
-V	O
-were	O
-performed	O
-by	O
-structural	O
-alignments	O
-of	O
-the	O
-25S	O
-ribosomal	O
-RNAs	O
-.	O
-	O
-(	O
-b	O
-)	O
-Bar	O
-graph	O
-of	O
-the	O
-angles	O
-characterizing	O
-the	O
-40S	O
-rotational	O
-and	O
-40S	O
-head	O
-swiveling	O
-states	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-Measurements	O
-for	O
-the	O
-two	O
-80S	O
-•	O
-IRES	O
-(	O
-INIT	O
-)	O
-structures	O
-are	O
-included	O
-for	O
-comparison	O
-.	O
-	O
-(	O
-c	O
-)	O
-Comparison	O
-of	O
-the	O
-40S	O
-conformations	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-shows	O
-distinct	O
-positions	O
-of	O
-the	O
-head	O
-relative	O
-to	O
-the	O
-body	O
-of	O
-the	O
-40S	O
-subunit	O
-(	O
-head	O
-swivel	O
-).	O
-	O
-Conformation	O
-of	O
-the	O
-non	O
--	O
-swiveled	O
-40S	O
-subunit	O
-in	O
-the	O
-S	O
-.	O
-cerevisiae	O
-80S	O
-ribosome	O
-bound	O
-with	O
-two	O
-tRNAs	O
-is	O
-shown	O
-for	O
-reference	O
-(	O
-blue	O
-).	O
-	O
-(	O
-d	O
-)	O
-Comparison	O
-of	O
-conformations	O
-of	O
-the	O
-L1	O
-and	O
-P	O
-stalks	O
-of	O
-the	O
-large	O
-subunit	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-with	O
-those	O
-in	O
-the	O
-80S	O
-•	O
-IRES	O
-and	O
-tRNA	O
--	O
-bound	O
-80S	O
-structures	O
-.	O
-	O
-Superpositions	O
-were	O
-performed	O
-by	O
-structural	O
-alignments	O
-of	O
-25S	O
-ribosomal	O
-RNAs	O
-.	O
-	O
-The	O
-central	O
-protuberance	O
-(	O
-CP	O
-)	O
-is	O
-labeled	O
-.	O
-	O
-(	O
-e	O
-)	O
-Bar	O
-graph	O
-of	O
-the	O
-positions	O
-of	O
-PKI	O
-and	O
-domain	O
-IV	O
-of	O
-eEF2	O
-relative	O
-to	O
-the	O
-P	O
-site	O
-residues	O
-of	O
-the	O
-head	O
-(	O
-U1191	O
-)	O
-and	O
-body	O
-(	O
-C1637	O
-)	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-(	O
-f	O
-and	O
-g	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-rearrangements	O
-in	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-from	O
-the	O
-initiation	O
-state	O
-(	O
-INIT	O
-:	O
-PDB	O
-ID	O
-3J6Y	O
-)	O
-to	O
-the	O
-post	O
--	O
-translocation	O
-Structure	O
-V	O
-.	O
-The	O
-fragment	O
-shown	O
-within	O
-a	O
-rectangle	O
-in	O
-panel	O
-f	O
-is	O
-magnified	O
-in	O
-panel	O
-g	O
-.	O
-Nucleotides	O
-of	O
-the	O
-40S	O
-body	O
-are	O
-shown	O
-in	O
-orange	O
-,	O
-40S	O
-head	O
-in	O
-yellow	O
-.	O
-	O
-The	O
-superpositions	O
-of	O
-structures	O
-were	O
-performed	O
-by	O
-structural	O
-alignments	O
-of	O
-the	O
-18S	O
-ribosomal	O
-RNAs	O
-excluding	O
-the	O
-head	O
-region	O
-(	O
-nt	O
-1150	O
-–	O
-1620	O
-).	O
-	O
-Our	O
-structures	O
-represent	O
-hitherto	O
-uncharacterized	O
-translocation	O
-complexes	O
-of	O
-the	O
-TSV	O
-IRES	O
-captured	O
-within	O
-globally	O
-distinct	O
-80S	O
-conformations	O
-(	O
-Figures	O
-1b	O
-and	O
-2	O
-).	O
-	O
-We	O
-numbered	O
-the	O
-structures	O
-from	O
-I	O
-to	O
-V	O
-,	O
-according	O
-to	O
-the	O
-position	O
-of	O
-the	O
-tRNA	O
--	O
-mRNA	O
--	O
-like	O
-PKI	O
-on	O
-the	O
-40S	O
-subunit	O
-(	O
-Figure	O
-2	O
-—	O
-source	O
-data	O
-1	O
-).	O
-	O
-Specifically	O
-,	O
-PKI	O
-is	O
-partially	O
-withdrawn	O
-from	O
-the	O
-A	O
-site	O
-in	O
-Structure	O
-I	O
-,	O
-and	O
-fully	O
-translocated	O
-to	O
-the	O
-P	O
-site	O
-in	O
-Structure	O
-V	O
-(	O
-Figure	O
-4	O
-;	O
-see	O
-also	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Thus	O
-Structures	O
-I	O
-to	O
-IV	O
-represent	O
-different	O
-positions	O
-of	O
-PKI	O
-between	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-(	O
-Figure	O
-2	O
-—	O
-source	O
-data	O
-1	O
-),	O
-suggesting	O
-that	O
-these	O
-structures	O
-describe	O
-intermediate	O
-states	O
-of	O
-translocation	O
-.	O
-	O
-Structure	O
-V	O
-corresponds	O
-to	O
-the	O
-post	O
--	O
-translocation	O
-state	O
-.	O
-	O
-Changes	O
-in	O
-ribosome	O
-conformation	O
-and	O
-eEF2	O
-positions	O
-are	O
-coupled	O
-with	O
-IRES	O
-movement	O
-through	O
-the	O
-ribosome	O
-	O
-Using	O
-the	O
-post	O
--	O
-translocation	O
-S	O
-.	O
-cerevisiae	O
-80S	O
-ribosome	O
-bound	O
-with	O
-the	O
-P	O
-and	O
-E	O
-site	O
-tRNAs	O
-as	O
-a	O
-reference	O
-(	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-),	O
-in	O
-which	O
-both	O
-the	O
-subunit	O
-rotation	O
-and	O
-the	O
-head	O
--	O
-body	O
-swivel	O
-are	O
-0	O
-°,	O
-we	O
-found	O
-that	O
-the	O
-ribosome	O
-adopts	O
-four	O
-globally	O
-distinct	O
-conformations	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-(	O
-Figure	O
-1b	O
-;	O
-see	O
-also	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1	O
-and	O
-Figure	O
-2	O
-—	O
-source	O
-data	O
-1	O
-).	O
-	O
-Structure	O
-I	O
-comprises	O
-the	O
-most	O
-rotated	O
-ribosome	O
-conformation	O
-(~	O
-10	O
-°),	O
-characteristic	O
-of	O
-pre	O
--	O
-translocation	O
-hybrid	O
--	O
-tRNA	O
-states	O
-.	O
-	O
-From	O
-Structure	O
-I	O
-to	O
-V	O
-,	O
-the	O
-body	O
-of	O
-the	O
-small	O
-subunit	O
-undergoes	O
-backward	O
-(	O
-reverse	O
-)	O
-rotation	O
-(	O
-Figure	O
-2b	O
-;	O
-see	O
-also	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2	O
-and	O
-Figure	O
-2	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Structures	O
-II	O
-and	O
-III	O
-are	O
-in	O
-mid	O
--	O
-rotation	O
-conformations	O
-(~	O
-5	O
-°).	O
-	O
-Structure	O
-IV	O
-adopts	O
-a	O
-slightly	O
-rotated	O
-conformation	O
-(~	O
-1	O
-°).	O
-	O
-Structure	O
-V	O
-is	O
-in	O
-a	O
-nearly	O
-non	O
--	O
-rotated	O
-conformation	O
-(	O
-0	O
-.	O
-5	O
-°),	O
-very	O
-similar	O
-to	O
-that	O
-of	O
-post	O
--	O
-translocation	O
-ribosome	O
--	O
-tRNA	O
-complexes	O
-.	O
-	O
-Thus	O
-,	O
-intersubunit	O
-rotation	O
-of	O
-~	O
-9	O
-°	O
-from	O
-Structure	O
-I	O
-to	O
-V	O
-covers	O
-a	O
-nearly	O
-complete	O
-range	O
-of	O
-relative	O
-subunit	O
-positions	O
-,	O
-similar	O
-to	O
-what	O
-was	O
-reported	O
-for	O
-tRNA	O
--	O
-bound	O
-yeast	O
-,	O
-bacterial	O
-and	O
-mammalian	O
-ribosomes	O
-.	O
-	O
-40S	O
-head	O
-swivel	O
-	O
-The	O
-pattern	O
-of	O
-40S	O
-head	O
-swivel	O
-between	O
-the	O
-structures	O
-is	O
-different	O
-from	O
-that	O
-of	O
-intersubunit	O
-rotation	O
-(	O
-Figures	O
-2c	O
-and	O
-d	O
-;	O
-see	O
-also	O
-Figure	O
-2	O
-—	O
-source	O
-data	O
-1	O
-).	O
-	O
-As	O
-with	O
-the	O
-intersubunit	O
-rotation	O
-,	O
-the	O
-small	O
-head	O
-swivel	O
-(~	O
-1	O
-°)	O
-in	O
-the	O
-non	O
--	O
-rotated	O
-Structure	O
-V	O
-is	O
-closest	O
-to	O
-that	O
-in	O
-the	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-post	O
--	O
-translocation	O
-ribosome	O
-.	O
-	O
-However	O
-in	O
-the	O
-pre	O
--	O
-translocation	O
-intermediates	O
-(	O
-from	O
-Structure	O
-I	O
-to	O
-IV	O
-),	O
-the	O
-beak	O
-of	O
-the	O
-head	O
-domain	O
-first	O
-turns	O
-toward	O
-the	O
-large	O
-subunit	O
-and	O
-then	O
-backs	O
-off	O
-(	O
-Figure	O
-2	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-The	O
-head	O
-samples	O
-a	O
-mid	O
--	O
-swiveled	O
-position	O
-in	O
-Structure	O
-I	O
-(	O
-12	O
-°),	O
-then	O
-a	O
-highly	O
--	O
-swiveled	O
-position	O
-in	O
-Structures	O
-II	O
-and	O
-III	O
-(	O
-17	O
-°)	O
-and	O
-a	O
-less	O
-swiveled	O
-position	O
-in	O
-Structure	O
-IV	O
-(	O
-14	O
-°).	O
-	O
-The	O
-maximum	O
-head	O
-swivel	O
-is	O
-observed	O
-in	O
-the	O
-mid	O
--	O
-rotated	O
-complexes	O
-II	O
-and	O
-III	O
-,	O
-in	O
-which	O
-PKI	O
-transitions	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
-,	O
-while	O
-eEF2	O
-occupies	O
-the	O
-A	O
-site	O
-partially	O
-.	O
-	O
-By	O
-comparison	O
-,	O
-the	O
-similarly	O
-mid	O
--	O
-rotated	O
-(	O
-4	O
-°)	O
-80S	O
-•	O
-TSV	O
-IRES	O
-initiation	O
-complex	O
-,	O
-in	O
-the	O
-absence	O
-of	O
-eEF2	O
-,	O
-adopts	O
-a	O
-mid	O
--	O
-swiveled	O
-position	O
-(~	O
-10	O
-°)	O
-(	O
-Figure	O
-2c	O
-).	O
-	O
-These	O
-observations	O
-suggest	O
-that	O
-eEF2	O
-is	O
-necessary	O
-for	O
-inducing	O
-or	O
-stabilizing	O
-the	O
-large	O
-head	O
-swivel	O
-of	O
-the	O
-40S	O
-subunit	O
-characteristic	O
-for	O
-IRES	O
-translocation	O
-intermediates	O
-.	O
-	O
-IRES	O
-rearrangements	O
-	O
-Comparison	O
-of	O
-the	O
-TSV	O
-IRES	O
-and	O
-eEF2	O
-positions	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-	O
-(	O
-a	O
-)	O
-Positions	O
-of	O
-the	O
-IRES	O
-and	O
-eEF2	O
-in	O
-the	O
-initiation	O
-,	O
-pre	O
--	O
-translocation	O
-(	O
-I	O
-)	O
-and	O
-post	O
--	O
-translocation	O
-(	O
-V	O
-)	O
-states	O
-,	O
-relative	O
-to	O
-the	O
-body	O
-of	O
-the	O
-40S	O
-subunit	O
-(	O
-not	O
-shown	O
-)	O
-(	O
-b	O
-)	O
-Positions	O
-of	O
-the	O
-IRES	O
-and	O
-eEF2	O
-in	O
-the	O
-initiation	O
-state	O
-(	O
-INIT	O
-)	O
-and	O
-intermediate	O
-steps	O
-of	O
-translocation	O
-(	O
-II	O
-,	O
-III	O
-and	O
-IV	O
-),	O
-relative	O
-to	O
-the	O
-body	O
-of	O
-the	O
-40S	O
-subunit	O
-(	O
-not	O
-shown	O
-).	O
-	O
-Superpositions	O
-were	O
-obtained	O
-by	O
-structural	O
-alignments	O
-of	O
-the	O
-18S	O
-rRNAs	O
-excluding	O
-the	O
-head	O
-domains	O
-(	O
-nt	O
-1150	O
-–	O
-1620	O
-).	O
-	O
-Positions	O
-of	O
-the	O
-IRES	O
-relative	O
-to	O
-proteins	O
-uS7	O
-,	O
-uS11	O
-and	O
-eS25	O
-.	O
-	O
-(	O
-a	O
-)	O
-Intra	O
--	O
-IRES	O
-rearrangements	O
-from	O
-the	O
-80S	O
-*	O
-IRES	O
-initiation	O
-structure	O
-(	O
-INIT	O
-;	O
-PDB	O
-3J6Y	O
-,)	O
-to	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-For	O
-each	O
-structure	O
-(	O
-shown	O
-in	O
-red	O
-),	O
-the	O
-conformation	O
-from	O
-a	O
-preceding	O
-structure	O
-is	O
-shown	O
-in	O
-light	O
-red	O
-for	O
-comparison	O
-.	O
-	O
-Superpositions	O
-were	O
-obtained	O
-by	O
-structural	O
-alignments	O
-of	O
-18S	O
-rRNA	O
-.	O
-	O
-(	O
-b	O
-)	O
-Positions	O
-of	O
-the	O
-IRES	O
-and	O
-eEF2	O
-relative	O
-to	O
-those	O
-of	O
-classical	O
-P	O
--	O
-and	O
-E	O
--	O
-site	O
-tRNAs	O
-in	O
-the	O
-80S	O
-•	O
-tRNA	O
-complex	O
-.	O
-(	O
-c	O
-)	O
-Positions	O
-of	O
-the	O
-IRES	O
-relative	O
-to	O
-proteins	O
-uS11	O
-(	O
-40S	O
-platform	O
-)	O
-and	O
-uS7	O
-and	O
-eS25	O
-(	O
-40S	O
-head	O
-),	O
-which	O
-interact	O
-with	O
-the	O
-5	O
-′	O
-domain	O
-of	O
-the	O
-IRES	O
-in	O
-the	O
-initiation	O
-state	O
-(	O
-left	O
-panel	O
-).	O
-	O
-In	O
-all	O
-panels	O
-,	O
-superpositions	O
-were	O
-obtained	O
-by	O
-structural	O
-alignments	O
-of	O
-the	O
-18S	O
-rRNAs	O
-.	O
-	O
-Ribosomal	O
-proteins	O
-of	O
-the	O
-initiation	O
-state	O
-are	O
-shown	O
-in	O
-gray	O
-for	O
-comparison	O
-.	O
-	O
-Positions	O
-of	O
-the	O
-L1stalk	O
-,	O
-tRNA	O
-and	O
-TSV	O
-IRES	O
-relative	O
-to	O
-proteins	O
-uS7	O
-and	O
-eS25	O
-,	O
-in	O
-80S	O
-•	O
-tRNA	O
-structures	O
-and	O
-80S	O
-•	O
-IRES	O
-structures	O
-I	O
-and	O
-V	O
-(	O
-this	O
-work	O
-).	O
-	O
-The	O
-view	O
-shows	O
-the	O
-vicinity	O
-of	O
-the	O
-ribosomal	O
-E	O
-site	O
-.	O
-	O
-Loop	O
-1	O
-.	O
-1	O
-and	O
-stem	O
-loops	O
-4	O
-and	O
-5	O
-of	O
-the	O
-IRES	O
-are	O
-labeled	O
-.	O
-	O
-Interactions	O
-of	O
-the	O
-stem	O
-loops	O
-4	O
-and	O
-5	O
-of	O
-the	O
-TSV	O
-with	O
-proteins	O
-uS7	O
-and	O
-eS25	O
-.	O
-	O
-Position	O
-and	O
-interactions	O
-of	O
-loop	O
-3	O
-(	O
-variable	O
-loop	O
-region	O
-)	O
-of	O
-the	O
-PKI	O
-domain	O
-in	O
-Structure	O
-V	O
-(	O
-this	O
-work	O
-)	O
-resembles	O
-those	O
-of	O
-the	O
-anticodon	O
-stem	O
-loop	O
-of	O
-the	O
-E	O
--	O
-site	O
-tRNA	O
-(	O
-blue	O
-)	O
-in	O
-the	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-.	O
-	O
-Positions	O
-of	O
-tRNAs	O
-and	O
-the	O
-TSV	O
-IRES	O
-relative	O
-to	O
-the	O
-A	O
--	O
-site	O
-finger	O
-(	O
-blue	O
-,	O
-nt	O
-1008	O
-–	O
-1043	O
-of	O
-25S	O
-rRNA	O
-)	O
-and	O
-the	O
-P	O
-site	O
-of	O
-the	O
-large	O
-subunit	O
-,	O
-comprising	O
-helix	O
-84	O
-of	O
-25S	O
-rRNA	O
-(	O
-nt	O
-.	O
-	O
-2668	O
-–	O
-2687	O
-)	O
-and	O
-protein	O
-uL5	O
-(	O
-collectively	O
-labeled	O
-as	O
-central	O
-protuberance	O
-,	O
-CP	O
-,	O
-in	O
-the	O
-upper	O
--	O
-row	O
-first	O
-figure	O
-,	O
-and	O
-individually	O
-labeled	O
-in	O
-the	O
-lower	O
--	O
-row	O
-first	O
-figure	O
-).	O
-	O
-Structures	O
-of	O
-translocation	O
-complexes	O
-of	O
-the	O
-bacterial	O
-70S	O
-ribosome	O
-bound	O
-with	O
-two	O
-tRNAs	O
-and	O
-yeast	O
-80S	O
-complexes	O
-with	O
-tRNAs	O
-are	O
-shown	O
-in	O
-the	O
-upper	O
-row	O
-and	O
-labeled	O
-.	O
-	O
-Structures	O
-of	O
-80S	O
-•	O
-IRES	O
-complexes	O
-in	O
-the	O
-absence	O
-of	O
-eEF2	O
-(	O
-INIT	O
-;	O
-PDB	O
-3J6Y	O
-,)	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-eEF2	O
-(	O
-this	O
-work	O
-)	O
-are	O
-shown	O
-in	O
-the	O
-lower	O
-row	O
-and	O
-labeled	O
-.	O
-	O
-Interactions	O
-of	O
-the	O
-TSV	O
-IRES	O
-with	O
-uL5	O
-and	O
-eL42	O
-.	O
-	O
-Structures	O
-of	O
-80S	O
-•	O
-IRES	O
-complexes	O
-in	O
-the	O
-absence	O
-of	O
-eEF2	O
-(	O
-INIT	O
-;	O
-PDB	O
-3J6Y	O
-,)	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-eEF2	O
-(	O
-this	O
-work	O
-)	O
-are	O
-shown	O
-in	O
-the	O
-upper	O
-row	O
-and	O
-labeled	O
-.	O
-	O
-Structures	O
-of	O
-the	O
-80S	O
-complexes	O
-with	O
-tRNAs	O
-are	O
-shown	O
-in	O
-the	O
-lower	O
-row	O
-in	O
-a	O
-view	O
-similar	O
-to	O
-that	O
-for	O
-the	O
-80S	O
-•	O
-IRES	O
-complex	O
-.	O
-	O
-Positions	O
-of	O
-the	O
-IRES	O
-relative	O
-to	O
-eEF2	O
-and	O
-elements	O
-of	O
-the	O
-ribosome	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-	O
-(	O
-a	O
-)	O
-Secondary	O
-structure	O
-of	O
-the	O
-TSV	O
-IRES	O
-.	O
-	O
-The	O
-TSV	O
-IRES	O
-comprises	O
-two	O
-domains	O
-:	O
-the	O
-5	O
-'	O
-domain	O
-(	O
-blue	O
-)	O
-and	O
-the	O
-PKI	O
-domain	O
-(	O
-red	O
-).	O
-	O
-The	O
-open	O
-reading	O
-frame	O
-(	O
-gray	O
-)	O
-is	O
-immediately	O
-following	O
-pseudoknot	O
-I	O
-(	O
-PKI	O
-).	O
-	O
-(	O
-b	O
-)	O
-Three	O
--	O
-dimensional	O
-structure	O
-of	O
-the	O
-TSV	O
-IRES	O
-(	O
-Structure	O
-II	O
-).	O
-	O
-Pseudoknots	O
-and	O
-stem	O
-loops	O
-are	O
-labeled	O
-and	O
-colored	O
-as	O
-in	O
-(	O
-a	O
-).	O
-	O
-(	O
-c	O
-)	O
-Positions	O
-of	O
-the	O
-IRES	O
-and	O
-eEF2	O
-on	O
-the	O
-small	O
-subunit	O
-in	O
-Structures	O
-I	O
-to	O
-V	O
-.	O
-The	O
-initiation	O
--	O
-state	O
-IRES	O
-is	O
-shown	O
-in	O
-gray	O
-.	O
-	O
-The	O
-insert	O
-shows	O
-density	O
-for	O
-interaction	O
-of	O
-diphthamide	O
-699	O
-(	O
-eEF2	O
-;	O
-green	O
-)	O
-with	O
-the	O
-codon	O
--	O
-anticodon	O
--	O
-like	O
-helix	O
-(	O
-PKI	O
-;	O
-red	O
-)	O
-in	O
-Structure	O
-V	O
-.	O
-(	O
-d	O
-and	O
-e	O
-)	O
-Density	O
-of	O
-the	O
-P	O
-site	O
-in	O
-Structure	O
-V	O
-shows	O
-that	O
-interactions	O
-of	O
-PKI	O
-with	O
-the	O
-18S	O
-rRNA	O
-nucleotides	O
-(	O
-c	O
-)	O
-are	O
-nearly	O
-identical	O
-to	O
-those	O
-in	O
-the	O
-P	O
-site	O
-of	O
-the	O
-2tRNA	O
-•	O
-mRNA	O
--	O
-bound	O
-70S	O
-ribosome	O
-(	O
-d	O
-).	O
-	O
-In	O
-each	O
-structure	O
-,	O
-the	O
-TSV	O
-IRES	O
-adopts	O
-a	O
-distinct	O
-conformation	O
-in	O
-the	O
-intersubunit	O
-space	O
-of	O
-the	O
-ribosome	O
-(	O
-Figures	O
-3	O
-and	O
-4	O
-).	O
-	O
-The	O
-IRES	O
-(	O
-nt	O
-6758	O
-–	O
-6952	O
-)	O
-consists	O
-of	O
-two	O
-globular	O
-parts	O
-(	O
-Figure	O
-3a	O
-):	O
-the	O
-5	O
-’-	O
-region	O
-(	O
-domains	O
-I	O
-and	O
-II	O
-,	O
-nt	O
-6758	O
-–	O
-6888	O
-)	O
-and	O
-the	O
-PKI	O
-domain	O
-(	O
-domain	O
-III	O
-,	O
-nt	O
-6889	O
-–	O
-6952	O
-).	O
-	O
-We	O
-collectively	O
-term	O
-domains	O
-I	O
-and	O
-II	O
-the	O
-5	O
-’	O
-domain	O
-.	O
-	O
-The	O
-PKI	O
-domain	O
-comprises	O
-PKI	O
-and	O
-stem	O
-loop	O
-3	O
-(	O
-SL3	O
-),	O
-which	O
-stacks	O
-on	O
-top	O
-of	O
-the	O
-stem	O
-of	O
-PKI	O
-.	O
-	O
-The	O
-6953GCU	O
-triplet	O
-immediately	O
-following	O
-the	O
-PKI	O
-domain	O
-is	O
-the	O
-first	O
-codon	O
-of	O
-the	O
-open	O
-reading	O
-frame	O
-.	O
-	O
-In	O
-the	O
-eEF2	O
--	O
-free	O
-80S	O
-•	O
-IRES	O
-initiation	O
-complex	O
-(	O
-INIT	O
-),	O
-the	O
-bulk	O
-of	O
-the	O
-5	O
-’-	O
-domain	O
-(	O
-nt	O
-.	O
-	O
-6758	O
-–	O
-6888	O
-)	O
-binds	O
-near	O
-the	O
-E	O
-site	O
-,	O
-contacting	O
-the	O
-ribosome	O
-mostly	O
-by	O
-means	O
-of	O
-three	O
-protruding	O
-structural	O
-elements	O
-:	O
-the	O
-L1	O
-.	O
-1	O
-region	O
-and	O
-stem	O
-loops	O
-4	O
-and	O
-5	O
-(	O
-SL4	O
-and	O
-SL5	O
-).	O
-	O
-In	O
-Structures	O
-I	O
-to	O
-IV	O
-,	O
-these	O
-contacts	O
-remain	O
-as	O
-in	O
-the	O
-initiation	O
-complex	O
-(	O
-Figure	O
-1a	O
-).	O
-	O
-Specifically	O
-,	O
-the	O
-L1	O
-.	O
-1	O
-region	O
-interacts	O
-with	O
-the	O
-L1	O
-stalk	O
-of	O
-the	O
-large	O
-subunit	O
-,	O
-while	O
-SL4	O
-and	O
-SL5	O
-bind	O
-at	O
-the	O
-side	O
-of	O
-the	O
-40S	O
-head	O
-and	O
-interact	O
-with	O
-proteins	O
-uS7	O
-,	O
-uS11	O
-and	O
-eS25	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-2	O
-and	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-3	O
-;	O
-ribosomal	O
-proteins	O
-are	O
-termed	O
-according	O
-to	O
-).	O
-	O
-In	O
-Structures	O
-I	O
--	O
-IV	O
-,	O
-the	O
-minor	O
-groove	O
-of	O
-SL4	O
-(	O
-at	O
-nt	O
-6840	O
-–	O
-6846	O
-)	O
-binds	O
-next	O
-to	O
-an	O
-α	O
--	O
-helix	O
-of	O
-uS7	O
-,	O
-which	O
-is	O
-rich	O
-in	O
-positively	O
-charged	O
-residues	O
-(	O
-K212	O
-,	O
-K213	O
-,	O
-R219	O
-and	O
-K222	O
-).	O
-	O
-The	O
-tip	O
-of	O
-SL4	O
-binds	O
-in	O
-the	O
-vicinity	O
-of	O
-R157	O
-in	O
-the	O
-β	O
--	O
-hairpin	O
-of	O
-uS7	O
-and	O
-of	O
-Y58	O
-in	O
-uS11	O
-.	O
-	O
-The	O
-minor	O
-groove	O
-of	O
-SL5	O
-(	O
-at	O
-nt	O
-6862	O
-–	O
-6868	O
-)	O
-contacts	O
-the	O
-positively	O
-charged	O
-region	O
-of	O
-eS25	O
-(	O
-R49	O
-,	O
-R58	O
-and	O
-R68	O
-)	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-4	O
-).	O
-	O
-In	O
-Structure	O
-V	O
-,	O
-however	O
-,	O
-the	O
-density	O
-for	O
-SL5	O
-is	O
-missing	O
-suggesting	O
-that	O
-SL5	O
-is	O
-mobile	O
-,	O
-while	O
-weak	O
-SL4	O
-density	O
-suggests	O
-that	O
-SL4	O
-is	O
-shifted	O
-along	O
-the	O
-surface	O
-of	O
-uS7	O
-,	O
-~	O
-20	O
-Å	O
-away	O
-from	O
-its	O
-initial	O
-position	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-2c	O
-).	O
-	O
-The	O
-L1	O
-.	O
-1	O
-region	O
-remains	O
-in	O
-contact	O
-with	O
-the	O
-L1	O
-stalk	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-3	O
-).	O
-	O
-Inchworm	O
--	O
-like	O
-translocation	O
-of	O
-the	O
-TSV	O
-IRES	O
-.	O
-	O
-Conformations	O
-and	O
-positions	O
-of	O
-the	O
-IRES	O
-in	O
-the	O
-initiation	O
-state	O
-and	O
-in	O
-Structures	O
-I	O
--	O
-V	O
-are	O
-shown	O
-relative	O
-to	O
-those	O
-of	O
-the	O
-A	O
--,	O
-P	O
--	O
-and	O
-E	O
--	O
-site	O
-tRNAs	O
-.	O
-	O
-The	O
-view	O
-was	O
-obtained	O
-by	O
-structural	O
-alignment	O
-of	O
-the	O
-body	O
-domains	O
-of	O
-18S	O
-rRNAs	O
-of	O
-the	O
-corresponding	O
-80S	O
-structures	O
-.	O
-	O
-Distances	O
-between	O
-nucleotides	O
-6848	O
-and	O
-6913	O
-in	O
-SL4	O
-and	O
-PKI	O
-,	O
-respectively	O
-,	O
-are	O
-shown	O
-(	O
-see	O
-also	O
-Figure	O
-2	O
-—	O
-source	O
-data	O
-1	O
-).	O
-	O
-The	O
-shape	O
-of	O
-the	O
-IRES	O
-changes	O
-considerably	O
-from	O
-the	O
-initiation	O
-state	O
-to	O
-Structures	O
-I	O
-through	O
-V	O
-,	O
-from	O
-an	O
-extended	O
-to	O
-compact	O
-to	O
-extended	O
-conformation	O
-(	O
-Figure	O
-4	O
-;	O
-see	O
-also	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-2a	O
-).	O
-	O
-Because	O
-in	O
-Structures	O
-I	O
-to	O
-IV	O
-the	O
-PKI	O
-domain	O
-shifts	O
-toward	O
-the	O
-P	O
-site	O
-,	O
-while	O
-the	O
-5	O
-’	O
-remains	O
-unchanged	O
-near	O
-the	O
-E	O
-site	O
-,	O
-the	O
-distance	O
-between	O
-the	O
-domains	O
-shortens	O
-(	O
-Figure	O
-4	O
-).	O
-	O
-In	O
-the	O
-80S	O
-•	O
-IRES	O
-initiation	O
-state	O
-,	O
-the	O
-A	O
--	O
-site	O
--	O
-bound	O
-PKI	O
-is	O
-separated	O
-from	O
-SL4	O
-by	O
-almost	O
-50	O
-Å	O
-(	O
-Figure	O
-4	O
-).	O
-	O
-In	O
-Structures	O
-I	O
-and	O
-II	O
-,	O
-the	O
-PKI	O
-is	O
-partially	O
-retracted	O
-from	O
-the	O
-A	O
-site	O
-and	O
-the	O
-distance	O
-from	O
-SL4	O
-shortens	O
-to	O
-~	O
-35	O
-Å	O
-.	O
-As	O
-PKI	O
-moves	O
-toward	O
-the	O
-P	O
-site	O
-in	O
-Structures	O
-III	O
-and	O
-IV	O
-,	O
-the	O
-PKI	O
-domain	O
-approaches	O
-to	O
-within	O
-~	O
-25	O
-Å	O
-of	O
-SL4	O
-.	O
-	O
-Because	O
-the	O
-5	O
-’-	O
-domain	O
-in	O
-the	O
-following	O
-structure	O
-(	O
-V	O
-)	O
-moves	O
-by	O
-~	O
-20	O
-Å	O
-along	O
-the	O
-40S	O
-head	O
-,	O
-the	O
-IRES	O
-returns	O
-to	O
-an	O
-extended	O
-conformation	O
-(~	O
-45	O
-Å	O
-)	O
-that	O
-is	O
-similar	O
-to	O
-that	O
-in	O
-the	O
-80S	O
-•	O
-IRES	O
-initiation	O
-complex	O
-.	O
-	O
-Rearrangements	O
-of	O
-the	O
-IRES	O
-involve	O
-restructuring	O
-of	O
-several	O
-interactions	O
-with	O
-the	O
-ribosome	O
-.	O
-	O
-In	O
-Structure	O
-I	O
-,	O
-SL3	O
-of	O
-the	O
-PKI	O
-domain	O
-is	O
-positioned	O
-between	O
-the	O
-A	O
--	O
-site	O
-finger	O
-(	O
-nt	O
-1008	O
-–	O
-1043	O
-of	O
-25S	O
-rRNA	O
-)	O
-and	O
-the	O
-P	O
-site	O
-of	O
-the	O
-60S	O
-subunit	O
-,	O
-comprising	O
-helix	O
-84	O
-of	O
-25S	O
-rRNA	O
-(	O
-nt	O
-.	O
-	O
-2668	O
-–	O
-2687	O
-)	O
-and	O
-protein	O
-uL5	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-6	O
-).	O
-	O
-This	O
-position	O
-of	O
-SL3	O
-is	O
-~	O
-25	O
-Å	O
-away	O
-from	O
-that	O
-in	O
-the	O
-80S	O
-•	O
-IRES	O
-initiation	O
-state	O
-,	O
-in	O
-which	O
-PKI	O
-and	O
-SL3	O
-closely	O
-mimic	O
-the	O
-ASL	O
-and	O
-elbow	O
-of	O
-the	O
-A	O
--	O
-site	O
-tRNA	O
-,	O
-respectively	O
-.	O
-	O
-As	O
-such	O
-,	O
-the	O
-transition	O
-from	O
-the	O
-initiation	O
-state	O
-to	O
-Structure	O
-I	O
-involves	O
-repositioning	O
-of	O
-SL3	O
-around	O
-the	O
-A	O
--	O
-site	O
-finger	O
-,	O
-resembling	O
-the	O
-transition	O
-between	O
-the	O
-pre	O
--	O
-translocation	O
-A	O
-/	O
-P	O
-and	O
-A	O
-/	O
-P	O
-*	O
-tRNA	O
-.	O
-	O
-The	O
-second	O
-set	O
-of	O
-major	O
-structural	O
-changes	O
-involves	O
-interaction	O
-of	O
-the	O
-P	O
-site	O
-region	O
-of	O
-the	O
-large	O
-subunit	O
-with	O
-the	O
-hinge	O
-point	O
-of	O
-the	O
-IRES	O
-bending	O
-between	O
-the	O
-5	O
-´	O
-domain	O
-and	O
-the	O
-PKI	O
-domain	O
-(	O
-nt	O
-.	O
-6886	O
-–	O
-6890	O
-).	O
-	O
-In	O
-the	O
-highly	O
-bent	O
-Structures	O
-III	O
-and	O
-IV	O
-,	O
-the	O
-hinge	O
-region	O
-interacts	O
-with	O
-the	O
-universally	O
-conserved	O
-uL5	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-tail	O
-of	O
-eL42	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-7	O
-).	O
-	O
-However	O
-,	O
-in	O
-the	O
-extended	O
-conformations	O
-,	O
-these	O
-parts	O
-of	O
-the	O
-IRES	O
-and	O
-the	O
-60S	O
-subunit	O
-are	O
-separated	O
-by	O
-more	O
-than	O
-10	O
-Å	O
-,	O
-suggesting	O
-that	O
-an	O
-interaction	O
-between	O
-them	O
-stabilizes	O
-the	O
-bent	O
-conformations	O
-but	O
-not	O
-the	O
-extended	O
-ones	O
-.	O
-	O
-Another	O
-local	O
-rearrangement	O
-concerns	O
-loop	O
-3	O
-,	O
-also	O
-known	O
-as	O
-the	O
-variable	O
-loop	O
-region	O
-,	O
-which	O
-connects	O
-the	O
-ASL	O
--	O
-and	O
-mRNA	O
--	O
-like	O
-parts	O
-of	O
-PKI	O
-.	O
-	O
-This	O
-loop	O
-is	O
-poorly	O
-resolved	O
-in	O
-Structures	O
-I	O
-through	O
-IV	O
-,	O
-suggesting	O
-conformational	O
-flexibility	O
-in	O
-agreement	O
-with	O
-structural	O
-studies	O
-of	O
-the	O
-isolated	O
-PKI	O
-and	O
-biochemical	O
-studies	O
-of	O
-unbound	O
-IRESs	O
-.	O
-	O
-In	O
-Structure	O
-V	O
-,	O
-loop	O
-3	O
-is	O
-bound	O
-in	O
-the	O
-40S	O
-E	O
-site	O
-and	O
-the	O
-backbone	O
-of	O
-loop	O
-3	O
-near	O
-the	O
-codon	O
--	O
-like	O
-part	O
-of	O
-PKI	O
-(	O
-at	O
-nt	O
-.	O
-	O
-6945	O
-–	O
-6946	O
-)	O
-interacts	O
-with	O
-R148	O
-and	O
-R157	O
-in	O
-β	O
--	O
-hairpin	O
-of	O
-uS7	O
-.	O
-	O
-The	O
-interaction	O
-of	O
-loop	O
-3	O
-backbone	O
-with	O
-uS7	O
-resembles	O
-that	O
-of	O
-the	O
-anticodon	O
--	O
-stem	O
-loop	O
-of	O
-E	O
--	O
-site	O
-tRNA	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-structure	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-5	O
-).	O
-	O
-Ordering	O
-of	O
-loop	O
-3	O
-suggests	O
-that	O
-this	O
-flexible	O
-region	O
-contributes	O
-to	O
-the	O
-stabilization	O
-of	O
-the	O
-PKI	O
-domain	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-state	O
-.	O
-	O
-This	O
-interpretation	O
-is	O
-consistent	O
-with	O
-the	O
-recent	O
-observation	O
-that	O
-alterations	O
-in	O
-loop	O
-3	O
-of	O
-the	O
-CrPV	O
-IRES	O
-result	O
-in	O
-decreased	O
-efficiency	O
-of	O
-translocation	O
-.	O
-	O
-eEF2	O
-structures	O
-	O
-Elements	O
-of	O
-the	O
-80S	O
-ribosome	O
-that	O
-contact	O
-eEF2	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-	O
-The	O
-view	O
-and	O
-colors	O
-are	O
-as	O
-in	O
-Figure	O
-5b	O
-:	O
-eEF2	O
-is	O
-shown	O
-in	O
-green	O
-,	O
-IRES	O
-RNA	O
-in	O
-red	O
-,	O
-40S	O
-subunit	O
-elements	O
-in	O
-orange	O
-,	O
-60S	O
-in	O
-cyan	O
-/	O
-teal	O
-.	O
-	O
-Cryo	O
--	O
-EM	O
-density	O
-of	O
-the	O
-GTPase	O
-region	O
-in	O
-Structures	O
-I	O
-and	O
-II	O
-.	O
-	O
-The	O
-switch	O
-loop	O
-I	O
-in	O
-Structure	O
-I	O
-is	O
-shown	O
-in	O
-blue	O
-.	O
-	O
-The	O
-putative	O
-position	O
-of	O
-the	O
-switch	O
-loop	O
-I	O
-,	O
-unresolved	O
-in	O
-the	O
-density	O
-of	O
-Structure	O
-II	O
-,	O
-is	O
-shown	O
-with	O
-a	O
-dashed	O
-line	O
-.	O
-	O
-Colors	O
-for	O
-the	O
-ribosome	O
-and	O
-eEF2	O
-are	O
-as	O
-in	O
-Figure	O
-1	O
-.	O
-	O
-Conformations	O
-and	O
-interactions	O
-of	O
-eEF2	O
-.	O
-	O
-(	O
-a	O
-)	O
-Conformations	O
-of	O
-eEF2	O
-in	O
-Structures	O
-I	O
--	O
-V	O
-and	O
-domain	O
-organization	O
-of	O
-eEF2	O
-are	O
-shown	O
-.	O
-	O
-Roman	O
-numerals	O
-denote	O
-eEF2	O
-domains	O
-.	O
-	O
-Superposition	O
-was	O
-obtained	O
-by	O
-structural	O
-alignment	O
-of	O
-domains	O
-I	O
-and	O
-II	O
-.	O
-	O
-(	O
-b	O
-)	O
-Elements	O
-of	O
-the	O
-80S	O
-ribosome	O
-in	O
-Structures	O
-I	O
-and	O
-V	O
-that	O
-contact	O
-eEF2	O
-.	O
-	O
-eEF2	O
-is	O
-shown	O
-in	O
-green	O
-,	O
-IRES	O
-RNA	O
-in	O
-red	O
-,	O
-40S	O
-subunit	O
-elements	O
-in	O
-orange	O
-,	O
-60S	O
-in	O
-cyan	O
-/	O
-teal	O
-.	O
-	O
-(	O
-c	O
-)	O
-Comparison	O
-of	O
-conformations	O
-of	O
-eEF2	O
-•	O
-sordarin	O
-in	O
-Structure	O
-I	O
-(	O
-light	O
-green	O
-)	O
-with	O
-those	O
-of	O
-free	O
-apo	O
--	O
-eEF2	O
-(	O
-magenta	O
-)	O
-and	O
-eEF2	O
-•	O
-sordarin	O
-(	O
-teal	O
-).	O
-	O
-(	O
-d	O
-)	O
-Interactions	O
-of	O
-the	O
-GTPase	O
-domains	O
-with	O
-the	O
-40S	O
-and	O
-60S	O
-subunits	O
-in	O
-Structure	O
-I	O
-(	O
-colored	O
-in	O
-green	O
-/	O
-blue	O
-,	O
-eEF2	O
-;	O
-orange	O
-,	O
-40S	O
-;	O
-cyan	O
-/	O
-teal	O
-,	O
-60S	O
-)	O
-and	O
-in	O
-Structure	O
-II	O
-(	O
-gray	O
-).	O
-	O
-Switch	O
-loop	O
-I	O
-(	O
-SWI	O
-)	O
-in	O
-Structure	O
-I	O
-is	O
-in	O
-blue	O
-;	O
-dashed	O
-line	O
-shows	O
-the	O
-putative	O
-location	O
-of	O
-unresolved	O
-switch	O
-loop	O
-I	O
-in	O
-Structure	O
-II	O
-.	O
-	O
-Superposition	O
-was	O
-obtained	O
-by	O
-structural	O
-alignment	O
-of	O
-the	O
-25S	O
-rRNAs	O
-.	O
-	O
-(	O
-e	O
-)	O
-Comparison	O
-of	O
-the	O
-GTP	O
--	O
-like	O
-conformation	O
-of	O
-eEF2	O
-•	O
-GDP	O
-in	O
-Structure	O
-I	O
-(	O
-light	O
-green	O
-)	O
-with	O
-those	O
-of	O
-70S	O
--	O
-bound	O
-elongation	O
-factors	O
-EF	O
--	O
-Tu	O
-•	O
-GDPCP	O
-(	O
-teal	O
-)	O
-and	O
-EF	O
--	O
-G	O
-•	O
-GDP	O
-•	O
-fusidic	O
-acid	O
-(	O
-magenta	O
-;	O
-fusidic	O
-acid	O
-not	O
-shown	O
-).	O
-(	O
-f	O
-)	O
-Cryo	O
--	O
-EM	O
-density	O
-showing	O
-guanosine	O
-diphosphate	O
-bound	O
-in	O
-the	O
-GTPase	O
-center	O
-(	O
-green	O
-)	O
-next	O
-to	O
-the	O
-sarcin	O
--	O
-ricin	O
-loop	O
-of	O
-25S	O
-rRNA	O
-(	O
-cyan	O
-)	O
-of	O
-Structure	O
-II	O
-.	O
-(	O
-g	O
-)	O
-Comparison	O
-of	O
-the	O
-sordarin	O
--	O
-binding	O
-sites	O
-in	O
-the	O
-ribosome	O
--	O
-bound	O
-(	O
-light	O
-green	O
-;	O
-Structure	O
-II	O
-)	O
-and	O
-isolated	O
-eEF2	O
-(	O
-teal	O
-).	O
-	O
-(	O
-h	O
-)	O
-Cryo	O
--	O
-EM	O
-density	O
-showing	O
-the	O
-sordarin	O
--	O
-binding	O
-pocket	O
-of	O
-eEF2	O
-(	O
-Structure	O
-II	O
-).	O
-	O
-Sordarin	O
-is	O
-shown	O
-in	O
-pink	O
-with	O
-oxygen	O
-atoms	O
-in	O
-red	O
-.	O
-	O
-Elongation	O
-factor	O
-eEF2	O
-in	O
-all	O
-five	O
-structures	O
-is	O
-bound	O
-with	O
-GDP	O
-and	O
-sordarin	O
-(	O
-Figure	O
-5	O
-).	O
-	O
-The	O
-elongation	O
-factor	O
-consists	O
-of	O
-three	O
-dynamic	O
-superdomains	O
-:	O
-an	O
-N	O
--	O
-terminal	O
-globular	O
-superdomain	O
-formed	O
-by	O
-the	O
-G	O
-(	O
-GTPase	O
-)	O
-domain	O
-(	O
-domain	O
-I	O
-)	O
-and	O
-domain	O
-II	O
-;	O
-a	O
-linker	O
-domain	O
-III	O
-;	O
-and	O
-a	O
-C	O
--	O
-terminal	O
-superdomain	O
-comprising	O
-domains	O
-IV	O
-and	O
-V	O
-(	O
-Figure	O
-5a	O
-).	O
-	O
-Domain	O
-IV	O
-extends	O
-from	O
-the	O
-main	O
-body	O
-and	O
-is	O
-critical	O
-for	O
-translocation	O
-catalyzed	O
-by	O
-eEF2	O
-or	O
-EF	O
--	O
-G	O
-.	O
-ADP	O
--	O
-ribosylation	O
-of	O
-eEF2	O
-at	O
-the	O
-tip	O
-of	O
-domain	O
-IV	O
-or	O
-deletion	O
-of	O
-domain	O
-IV	O
-from	O
-EF	O
--	O
-G	O
-abrogate	O
-translocation	O
-.	O
-	O
-In	O
-post	O
--	O
-translocation	O
--	O
-like	O
-80S	O
-•	O
-tRNA	O
-•	O
-eEF2	O
-complexes	O
-,	O
-domain	O
-IV	O
-binds	O
-in	O
-the	O
-40S	O
-A	O
-site	O
-,	O
-suggesting	O
-direct	O
-involvement	O
-of	O
-domain	O
-IV	O
-in	O
-translocation	O
-of	O
-tRNA	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
-.	O
-	O
-GDP	O
-in	O
-our	O
-structures	O
-is	O
-bound	O
-in	O
-the	O
-GTPase	O
-center	O
-(	O
-Figures	O
-5d	O
-,	O
-e	O
-and	O
-f	O
-)	O
-and	O
-sordarin	O
-is	O
-sandwiched	O
-between	O
-the	O
-β	O
--	O
-platforms	O
-of	O
-domains	O
-III	O
-and	O
-V	O
-(	O
-Figures	O
-5g	O
-and	O
-h	O
-),	O
-as	O
-in	O
-the	O
-structure	O
-of	O
-free	O
-eEF2	O
-•	O
-sordarin	O
-complex	O
-.	O
-	O
-The	O
-global	O
-conformations	O
-of	O
-eEF2	O
-(	O
-Figure	O
-5a	O
-)	O
-are	O
-similar	O
-in	O
-these	O
-structures	O
-(	O
-all	O
--	O
-atom	O
-RMSD	O
-≤	O
-2	O
-Å	O
-),	O
-but	O
-the	O
-positions	O
-of	O
-eEF2	O
-relative	O
-to	O
-the	O
-40S	O
-subunit	O
-differ	O
-substantially	O
-as	O
-a	O
-result	O
-of	O
-40S	O
-subunit	O
-rotation	O
-(	O
-Figure	O
-2	O
-—	O
-source	O
-data	O
-1	O
-).	O
-	O
-From	O
-Structure	O
-I	O
-to	O
-V	O
-,	O
-eEF2	O
-is	O
-rigidly	O
-attached	O
-to	O
-the	O
-GTPase	O
--	O
-associated	O
-center	O
-of	O
-the	O
-60S	O
-subunit	O
-.	O
-	O
-The	O
-GTPase	O
--	O
-associated	O
-center	O
-comprises	O
-the	O
-P	O
-stalk	O
-(	O
-L11	O
-and	O
-L7	O
-/	O
-L12	O
-stalk	O
-in	O
-bacteria	O
-)	O
-and	O
-the	O
-sarcin	O
--	O
-ricin	O
-loop	O
-(	O
-SRL	O
-,	O
-nt	O
-3012	O
-–	O
-3042	O
-).	O
-	O
-The	O
-tips	O
-of	O
-25S	O
-rRNA	O
-helices	O
-43	O
-and	O
-44	O
-of	O
-the	O
-P	O
-stalk	O
-(	O
-nucleotides	O
-G1242	O
-and	O
-A1270	O
-,	O
-respectively	O
-)	O
-stack	O
-on	O
-V754	O
-and	O
-Y744	O
-of	O
-domain	O
-V	O
-.	O
-An	O
-αββ	O
-motif	O
-of	O
-the	O
-eukaryote	O
--	O
-specific	O
-protein	O
-P0	O
-(	O
-aa	O
-126	O
-–	O
-154	O
-)	O
-packs	O
-in	O
-the	O
-crevice	O
-between	O
-the	O
-long	O
-α	O
--	O
-helix	O
-D	O
-(	O
-aa	O
-172	O
-–	O
-188	O
-)	O
-of	O
-the	O
-GTPase	O
-domain	O
-and	O
-the	O
-β	O
--	O
-sheet	O
-region	O
-(	O
-aa	O
-246	O
-–	O
-263	O
-)	O
-of	O
-the	O
-GTPase	O
-domain	O
-insert	O
-(	O
-or	O
-G	O
-’	O
-insert	O
-)	O
-of	O
-eEF2	O
-(	O
-secondary	O
--	O
-structure	O
-nomenclatures	O
-for	O
-eEF2	O
-and	O
-EF	O
--	O
-G	O
-are	O
-the	O
-same	O
-).	O
-	O
-Although	O
-the	O
-P	O
-/	O
-L11	O
-stalk	O
-is	O
-known	O
-to	O
-be	O
-dynamic	O
-,	O
-its	O
-position	O
-remains	O
-unchanged	O
-from	O
-Structure	O
-I	O
-to	O
-V	O
-:	O
-all	O
--	O
-atom	O
-root	O
--	O
-mean	O
--	O
-square	O
-differences	O
-for	O
-the	O
-25S	O
-rRNA	O
-of	O
-the	O
-P	O
-stalk	O
-(	O
-nt	O
-1223	O
-–	O
-1286	O
-)	O
-are	O
-within	O
-2	O
-.	O
-5	O
-Å	O
-.	O
-However	O
-,	O
-with	O
-respect	O
-to	O
-its	O
-position	O
-in	O
-the	O
-80S	O
-•	O
-IRES	O
-complex	O
-in	O
-the	O
-absence	O
-of	O
-eEF2	O
-and	O
-in	O
-the	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-,	O
-the	O
-P	O
-stalk	O
-is	O
-shifted	O
-by	O
-~	O
-13	O
-Å	O
-toward	O
-the	O
-A	O
-site	O
-(	O
-Figure	O
-2d	O
-).	O
-	O
-The	O
-sarcin	O
--	O
-ricin	O
-loop	O
-interacts	O
-with	O
-the	O
-GTP	O
--	O
-binding	O
-site	O
-of	O
-eEF2	O
-(	O
-Figures	O
-5d	O
-and	O
-f	O
-).	O
-	O
-While	O
-the	O
-overall	O
-mode	O
-of	O
-this	O
-interaction	O
-is	O
-similar	O
-to	O
-that	O
-seen	O
-in	O
-70S	O
-•	O
-EF	O
--	O
-G	O
-crystal	O
-structures	O
-,	O
-there	O
-is	O
-an	O
-important	O
-local	O
-difference	O
-between	O
-Structure	O
-I	O
-and	O
-Structures	O
-II	O
--	O
-V	O
-in	O
-switch	O
-loop	O
-I	O
-,	O
-as	O
-discussed	O
-below	O
-.	O
-	O
-Repositioning	O
-(	O
-sliding	O
-)	O
-of	O
-the	O
-positively	O
--	O
-charged	O
-cluster	O
-of	O
-domain	O
-IV	O
-of	O
-eEF2	O
-over	O
-the	O
-phosphate	O
-backbone	O
-(	O
-red	O
-)	O
-of	O
-the	O
-18S	O
-helices	O
-33	O
-and	O
-34	O
-.	O
-	O
-Structures	O
-I	O
-through	O
-V	O
-are	O
-shown	O
-.	O
-	O
-Electrostatic	O
-surface	O
-of	O
-eEF2	O
-is	O
-shown	O
-;	O
-negatively	O
-and	O
-positively	O
-charged	O
-regions	O
-are	O
-shown	O
-in	O
-red	O
-and	O
-blue	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-view	O
-was	O
-obtained	O
-by	O
-structural	O
-alignment	O
-of	O
-the	O
-18S	O
-rRNAs	O
-.	O
-	O
-Interactions	O
-of	O
-eEF2	O
-with	O
-the	O
-40S	O
-subunit	O
-.	O
-	O
-(	O
-a	O
-)	O
-eEF2	O
-(	O
-green	O
-)	O
-interacts	O
-only	O
-with	O
-the	O
-body	O
-in	O
-Structure	O
-I	O
-(	O
-eEF2	O
-domains	O
-are	O
-labeled	O
-with	O
-roman	O
-numerals	O
-in	O
-white	O
-),	O
-and	O
-with	O
-both	O
-the	O
-head	O
-and	O
-body	O
-in	O
-Structures	O
-II	O
-through	O
-V	O
-.	O
-Colors	O
-are	O
-as	O
-in	O
-Figure	O
-1	O
-,	O
-except	O
-for	O
-the	O
-40S	O
-structural	O
-elements	O
-that	O
-contact	O
-eEF2	O
-,	O
-which	O
-are	O
-labeled	O
-and	O
-shown	O
-in	O
-purple	O
-.	O
-(	O
-b	O
-)	O
-Entry	O
-of	O
-eEF2	O
-into	O
-the	O
-40S	O
-A	O
-site	O
-,	O
-from	O
-Structure	O
-I	O
-through	O
-V	O
-.	O
-Distances	O
-to	O
-the	O
-A	O
--	O
-site	O
-accommodated	O
-eEF2	O
-(	O
-Structure	O
-V	O
-)	O
-are	O
-shown	O
-.	O
-	O
-The	O
-view	O
-was	O
-obtained	O
-by	O
-superpositions	O
-of	O
-the	O
-body	O
-domains	O
-of	O
-18S	O
-rRNAs	O
-.	O
-	O
-(	O
-c	O
-)	O
-Rearrangements	O
-,	O
-from	O
-Structure	O
-I	O
-through	O
-V	O
-,	O
-of	O
-a	O
-positively	O
-charged	O
-cluster	O
-of	O
-eEF2	O
-(	O
-K613	O
-,	O
-R617	O
-and	O
-R631	O
-)	O
-positioned	O
-over	O
-the	O
-phosphate	O
-backbone	O
-of	O
-18S	O
-helices	O
-33	O
-and	O
-34	O
-,	O
-suggesting	O
-a	O
-role	O
-of	O
-electrostatic	O
-interactions	O
-in	O
-eEF2	O
-diffusion	O
-over	O
-the	O
-40S	O
-surface	O
-.	O
-	O
-(	O
-d	O
-)	O
-Shift	O
-of	O
-the	O
-tip	O
-of	O
-domain	O
-III	O
-of	O
-eEF2	O
-,	O
-interacting	O
-with	O
-uS12	O
-upon	O
-reverse	O
-subunit	O
-rotation	O
-from	O
-Structure	O
-I	O
-to	O
-Structure	O
-V	O
-.	O
-Structure	O
-I	O
-colored	O
-as	O
-in	O
-Figure	O
-1	O
-,	O
-except	O
-uS12	O
-,	O
-which	O
-is	O
-in	O
-purple	O
-;	O
-Structure	O
-V	O
-is	O
-in	O
-gray	O
-.	O
-	O
-There	O
-are	O
-two	O
-modest	O
-but	O
-noticeable	O
-domain	O
-rearrangements	O
-between	O
-Structures	O
-I	O
-and	O
-V	O
-.	O
-Unlike	O
-in	O
-free	O
-eEF2	O
-,	O
-which	O
-can	O
-sample	O
-large	O
-movements	O
-of	O
-at	O
-least	O
-50	O
-Å	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-superdomain	O
-relative	O
-to	O
-the	O
-N	O
--	O
-terminal	O
-superdomain	O
-(	O
-Figure	O
-5c	O
-),	O
-eEF2	O
-undergoes	O
-moderate	O
-repositioning	O
-of	O
-domain	O
-IV	O
-(~	O
-3	O
-Å	O
-;	O
-Figure	O
-5a	O
-)	O
-and	O
-domain	O
-III	O
-(~	O
-5	O
-Å	O
-;	O
-Figure	O
-6d	O
-).	O
-	O
-This	O
-limited	O
-flexibility	O
-of	O
-the	O
-ribosome	O
--	O
-bound	O
-eEF2	O
-is	O
-likely	O
-the	O
-result	O
-of	O
-simultaneous	O
-fixation	O
-of	O
-eEF2	O
-superdomains	O
-,	O
-via	O
-domains	O
-I	O
-and	O
-V	O
-,	O
-by	O
-the	O
-GTPase	O
--	O
-associated	O
-center	O
-of	O
-the	O
-large	O
-subunit	O
-.	O
-	O
-Domain	O
-IV	O
-of	O
-eEF2	O
-binds	O
-at	O
-the	O
-40S	O
-A	O
-site	O
-in	O
-Structures	O
-I	O
-to	O
-V	O
-but	O
-the	O
-mode	O
-of	O
-interaction	O
-differs	O
-in	O
-each	O
-complex	O
-(	O
-Figure	O
-6	O
-).	O
-	O
-Because	O
-eEF2	O
-is	O
-rigidly	O
-attached	O
-to	O
-the	O
-60S	O
-subunit	O
-and	O
-does	O
-not	O
-undergo	O
-large	O
-inter	O
--	O
-subunit	O
-rearrangements	O
-,	O
-gradual	O
-entry	O
-of	O
-domain	O
-IV	O
-into	O
-the	O
-A	O
-site	O
-between	O
-Structures	O
-I	O
-and	O
-V	O
-is	O
-due	O
-to	O
-40S	O
-subunit	O
-rotation	O
-and	O
-head	O
-swivel	O
-.	O
-	O
-eEF2	O
-settles	O
-into	O
-the	O
-A	O
-site	O
-from	O
-Structure	O
-I	O
-to	O
-V	O
-,	O
-as	O
-the	O
-tip	O
-of	O
-domain	O
-IV	O
-shifts	O
-by	O
-~	O
-10	O
-Å	O
-relative	O
-to	O
-the	O
-body	O
-and	O
-by	O
-~	O
-20	O
-Å	O
-relative	O
-to	O
-the	O
-swiveling	O
-head	O
-.	O
-	O
-Modest	O
-intra	O
--	O
-eEF2	O
-shifts	O
-of	O
-domain	O
-IV	O
-between	O
-Structures	O
-I	O
-to	O
-V	O
-outline	O
-a	O
-stochastic	O
-trajectory	O
-(	O
-Figure	O
-5a	O
-),	O
-consistent	O
-with	O
-local	O
-adjustments	O
-of	O
-the	O
-domain	O
-in	O
-the	O
-A	O
-site	O
-.	O
-	O
-At	O
-the	O
-central	O
-region	O
-of	O
-eEF2	O
-,	O
-domains	O
-II	O
-and	O
-III	O
-contact	O
-the	O
-40S	O
-body	O
-(	O
-mainly	O
-at	O
-nucleotides	O
-48	O
-–	O
-52	O
-and	O
-429	O
-–	O
-432	O
-of	O
-18S	O
-rRNA	O
-helix	O
-5	O
-and	O
-uS12	O
-).	O
-	O
-From	O
-Structure	O
-I	O
-to	O
-V	O
-,	O
-these	O
-central	O
-domains	O
-migrate	O
-by	O
-~	O
-10	O
-Å	O
-along	O
-the	O
-40S	O
-surface	O
-(	O
-Figure	O
-6c	O
-).	O
-	O
-Comparison	O
-of	O
-eEF2	O
-conformations	O
-reveals	O
-that	O
-in	O
-Structure	O
-V	O
-,	O
-domain	O
-III	O
-is	O
-displaced	O
-as	O
-a	O
-result	O
-of	O
-interaction	O
-with	O
-uS12	O
-,	O
-as	O
-discussed	O
-below	O
-.	O
-	O
-In	O
-summary	O
-,	O
-between	O
-Structures	O
-I	O
-and	O
-V	O
-,	O
-a	O
-step	O
--	O
-wise	O
-translocation	O
-of	O
-PKI	O
-by	O
-~	O
-15	O
-Å	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
--	O
-within	O
-the	O
-40S	O
-subunit	O
-–	O
-occurs	O
-simultaneously	O
-with	O
-the	O
-~	O
-11	O
-Å	O
-side	O
--	O
-way	O
-entry	O
-of	O
-domain	O
-IV	O
-into	O
-the	O
-A	O
-site	O
-coupled	O
-with	O
-~	O
-3	O
-to	O
-5	O
-Å	O
-inter	O
--	O
-domain	O
-rearrangements	O
-in	O
-eEF2	O
-.	O
-	O
-These	O
-shifts	O
-occur	O
-during	O
-the	O
-reverse	O
-rotation	O
-of	O
-the	O
-40S	O
-body	O
-coupled	O
-with	O
-the	O
-forward	O
--	O
-then	O
--	O
-reverse	O
-head	O
-swivel	O
-.	O
-	O
-To	O
-elucidate	O
-the	O
-detailed	O
-structural	O
-mechanism	O
-of	O
-IRES	O
-translocation	O
-and	O
-the	O
-roles	O
-of	O
-eEF2	O
-and	O
-ribosome	O
-rearrangements	O
-,	O
-we	O
-describe	O
-in	O
-the	O
-following	O
-sections	O
-the	O
-interactions	O
-of	O
-PKI	O
-and	O
-eEF2	O
-with	O
-the	O
-ribosomal	O
-A	O
-and	O
-P	O
-sites	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-(	O
-Figure	O
-2g	O
-;	O
-see	O
-also	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Structure	O
-I	O
-represents	O
-a	O
-pre	O
--	O
-translocation	O
-IRES	O
-and	O
-initial	O
-entry	O
-of	O
-eEF2	O
-in	O
-a	O
-GTP	O
--	O
-like	O
-state	O
-	O
-In	O
-the	O
-fully	O
-rotated	O
-Structure	O
-I	O
-,	O
-PKI	O
-is	O
-shifted	O
-toward	O
-the	O
-P	O
-site	O
-by	O
-~	O
-3	O
-Å	O
-relative	O
-to	O
-its	O
-position	O
-in	O
-the	O
-initiation	O
-complex	O
-but	O
-maintains	O
-interactions	O
-with	O
-the	O
-partially	O
-swiveled	O
-head	O
-.	O
-	O
-At	O
-the	O
-head	O
-,	O
-C1274	O
-of	O
-the	O
-18S	O
-rRNA	O
-(	O
-C1054	O
-in	O
-E	O
-.	O
-coli	O
-)	O
-base	O
-pairs	O
-with	O
-the	O
-first	O
-nucleotide	O
-of	O
-the	O
-ORF	O
-immediately	O
-downstream	O
-of	O
-PKI	O
-.	O
-	O
-The	O
-C1274	O
-:	O
-G6953	O
-base	O
-pair	O
-provides	O
-a	O
-stacking	O
-platform	O
-for	O
-the	O
-codon	O
--	O
-anticodon	O
-–	O
-like	O
-helix	O
-of	O
-PKI	O
-.	O
-	O
-We	O
-therefore	O
-define	O
-C1274	O
-as	O
-the	O
-foundation	O
-of	O
-the	O
-'	O
-head	O
-A	O
-site	O
-'.	O
-	O
-Accordingly	O
-,	O
-we	O
-use	O
-U1191	O
-(	O
-G966	O
-in	O
-E	O
-.	O
-coli	O
-)	O
-and	O
-C1637	O
-(	O
-C1400	O
-in	O
-E	O
-.	O
-coli	O
-)	O
-as	O
-the	O
-reference	O
-points	O
-of	O
-the	O
-'	O
-head	O
-P	O
-site	O
-'	O
-and	O
-'	O
-body	O
-P	O
-site	O
-'	O
-(	O
-Figure	O
-2g	O
-),	O
-respectively	O
-,	O
-because	O
-these	O
-nucleotides	O
-form	O
-a	O
-stacking	O
-foundation	O
-for	O
-the	O
-fully	O
-translocated	O
-mRNA	O
--	O
-tRNA	O
-helix	O
-in	O
-tRNA	O
--	O
-bound	O
-structures	O
-and	O
-in	O
-our	O
-post	O
--	O
-translocation	O
-Structure	O
-V	O
-discussed	O
-below	O
-.	O
-	O
-Interactions	O
-of	O
-the	O
-residues	O
-at	O
-the	O
-eEF2	O
-tip	O
-with	O
-the	O
-decoding	O
-center	O
-of	O
-the	O
-IRES	O
--	O
-bound	O
-ribosome	O
-.	O
-	O
-Key	O
-elements	O
-of	O
-the	O
-decoding	O
-center	O
-of	O
-the	O
-'	O
-locked	O
-'	O
-initiation	O
-structure	O
-,	O
-'	O
-unlocked	O
-'	O
-Structure	O
-I	O
-,	O
-and	O
-post	O
--	O
-translocation	O
-Structure	O
-V	O
-(	O
-this	O
-work	O
-)	O
-are	O
-shown	O
-.	O
-	O
-The	O
-histidine	O
--	O
-diphthamide	O
-tip	O
-of	O
-eEF2	O
-is	O
-shown	O
-in	O
-green	O
-.	O
-	O
-The	O
-codon	O
--	O
-anticodon	O
--	O
-like	O
-helix	O
-of	O
-PKI	O
-is	O
-shown	O
-in	O
-red	O
-,	O
-the	O
-downstream	O
-first	O
-codon	O
-of	O
-the	O
-ORF	O
-in	O
-magenta	O
-.	O
-	O
-Nucleotides	O
-of	O
-the	O
-18S	O
-rRNA	O
-body	O
-are	O
-in	O
-orange	O
-and	O
-head	O
-in	O
-yellow	O
-;	O
-25S	O
-rRNA	O
-nucleotide	O
-A2256	O
-is	O
-blue	O
-.	O
-	O
-A	O
-and	O
-P	O
-sites	O
-are	O
-schematically	O
-demarcated	O
-by	O
-dotted	O
-lines	O
-.	O
-	O
-The	O
-interaction	O
-of	O
-PKI	O
-with	O
-the	O
-40S	O
-body	O
-is	O
-substantially	O
-rearranged	O
-relative	O
-to	O
-that	O
-in	O
-the	O
-initiation	O
-state	O
-.	O
-	O
-In	O
-the	O
-latter	O
-,	O
-PKI	O
-is	O
-stabilized	O
-by	O
-interactions	O
-with	O
-the	O
-universally	O
-conserved	O
-decoding	O
--	O
-center	O
-nucleotides	O
-G577	O
-,	O
-A1755	O
-and	O
-A1756	O
-('	O
-body	O
-A	O
-site	O
-'),	O
-as	O
-in	O
-the	O
-A	O
--	O
-site	O
-tRNA	O
-bound	O
-complexes	O
-.	O
-	O
-In	O
-Structure	O
-I	O
-,	O
-PKI	O
-does	O
-not	O
-contact	O
-these	O
-nucleotides	O
-(	O
-Figures	O
-2g	O
-and	O
-7	O
-).	O
-	O
-The	O
-position	O
-of	O
-eEF2	O
-on	O
-the	O
-40S	O
-subunit	O
-of	O
-Structure	O
-I	O
-is	O
-markedly	O
-distinct	O
-from	O
-those	O
-in	O
-Structures	O
-II	O
-to	O
-V	O
-.	O
-The	O
-translocase	O
-interacts	O
-with	O
-the	O
-40S	O
-body	O
-but	O
-does	O
-not	O
-contact	O
-the	O
-head	O
-(	O
-Figures	O
-5b	O
-and	O
-6a	O
-;	O
-Figure	O
-5	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Domain	O
-IV	O
-is	O
-partially	O
-engaged	O
-with	O
-the	O
-body	O
-A	O
-site	O
-.	O
-	O
-The	O
-tip	O
-of	O
-domain	O
-IV	O
-is	O
-wedged	O
-between	O
-PKI	O
-and	O
-decoding	O
--	O
-center	O
-nucleotides	O
-A1755	O
-and	O
-A1756	O
-,	O
-which	O
-are	O
-bulged	O
-out	O
-of	O
-h44	O
-.	O
-	O
-This	O
-tip	O
-contains	O
-the	O
-histidine	O
--	O
-diphthamide	O
-triad	O
-(	O
-H583	O
-,	O
-H694	O
-and	O
-Diph699	O
-),	O
-which	O
-interacts	O
-with	O
-the	O
-codon	O
--	O
-anticodon	O
--	O
-like	O
-helix	O
-of	O
-PKI	O
-and	O
-A1756	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-Histidines	O
-583	O
-and	O
-694	O
-interact	O
-with	O
-the	O
-phosphate	O
-backbone	O
-of	O
-the	O
-anticodon	O
--	O
-like	O
-strand	O
-(	O
-at	O
-G6907	O
-and	O
-C6908	O
-).	O
-	O
-Diphthamide	O
-is	O
-a	O
-unique	O
-posttranslational	O
-modification	O
-conserved	O
-in	O
-archaeal	O
-and	O
-eukaryotic	O
-EF2	O
-(	O
-at	O
-residue	O
-699	O
-in	O
-S	O
-.	O
-cerevisiae	O
-)	O
-and	O
-involves	O
-addition	O
-of	O
-a	O
-~	O
-7	O
--	O
-Å	O
-long	O
-3	O
--	O
-carboxyamido	O
--	O
-3	O
--(	O
-trimethylamino	O
-)-	O
-propyl	O
-moiety	O
-to	O
-the	O
-histidine	O
-imidazole	O
-ring	O
-at	O
-CE1	O
-.	O
-	O
-The	O
-trimethylamino	O
-end	O
-of	O
-Diph699	O
-packs	O
-over	O
-A1756	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-The	O
-opposite	O
-surface	O
-of	O
-the	O
-tail	O
-is	O
-oriented	O
-toward	O
-the	O
-minor	O
--	O
-groove	O
-side	O
-of	O
-the	O
-second	O
-base	O
-pair	O
-of	O
-the	O
-codon	O
--	O
-anticodon	O
-helix	O
-(	O
-G6906	O
-:	O
-C6951	O
-).	O
-	O
-Thus	O
-,	O
-in	O
-comparison	O
-with	O
-the	O
-initiation	O
-state	O
-,	O
-the	O
-histidine	O
--	O
-diphthamide	O
-tip	O
-of	O
-eEF2	O
-replaces	O
-the	O
-codon	O
--	O
-anticodon	O
-–	O
-like	O
-helix	O
-of	O
-PKI	O
-.	O
-	O
-The	O
-splitting	O
-of	O
-the	O
-interaction	O
-of	O
-A1755	O
--	O
-A1756	O
-and	O
-PKI	O
-is	O
-achieved	O
-by	O
-providing	O
-the	O
-histidine	O
--	O
-diphthamine	O
-tip	O
-as	O
-a	O
-binding	O
-partner	O
-for	O
-both	O
-A1756	O
-and	O
-the	O
-minor	O
-groove	O
-of	O
-the	O
-codon	O
--	O
-anticodon	O
-helix	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-Unlike	O
-in	O
-Structures	O
-II	O
-to	O
-V	O
-,	O
-the	O
-conformation	O
-of	O
-the	O
-eEF2	O
-GTPase	O
-center	O
-in	O
-Structure	O
-I	O
-resembles	O
-that	O
-of	O
-a	O
-GTP	O
--	O
-bound	O
-translocase	O
-(	O
-Figure	O
-5e	O
-).	O
-	O
-In	O
-translational	O
-GTPases	O
-,	O
-switch	O
-loops	O
-I	O
-and	O
-II	O
-are	O
-involved	O
-in	O
-the	O
-GTPase	O
-activity	O
-(	O
-reviewed	O
-in	O
-).	O
-	O
-Switch	O
-loop	O
-II	O
-(	O
-aa	O
-105	O
-–	O
-110	O
-),	O
-which	O
-carries	O
-the	O
-catalytic	O
-H108	O
-(	O
-H92	O
-in	O
-E	O
-.	O
-coli	O
-EF	O
--	O
-G	O
-;	O
-is	O
-well	O
-resolved	O
-in	O
-all	O
-five	O
-structures	O
-.	O
-	O
-The	O
-histidine	O
-resides	O
-next	O
-to	O
-the	O
-backbone	O
-of	O
-G3028	O
-of	O
-the	O
-sarcin	O
--	O
-ricin	O
-loop	O
-and	O
-near	O
-the	O
-diphosphate	O
-of	O
-GDP	O
-(	O
-Figure	O
-5e	O
-).	O
-	O
-By	O
-contrast	O
-,	O
-switch	O
-loop	O
-I	O
-(	O
-aa	O
-50	O
-–	O
-70	O
-in	O
-S	O
-.	O
-cerevisiae	O
-eEF2	O
-)	O
-is	O
-resolved	O
-only	O
-in	O
-Structure	O
-I	O
-(	O
-Figure	O
-5	O
-—	O
-figure	O
-supplement	O
-2	O
-).	O
-	O
-The	O
-N	O
--	O
-terminal	O
-part	O
-of	O
-the	O
-loop	O
-(	O
-aa	O
-50	O
-–	O
-60	O
-)	O
-is	O
-sandwiched	O
-between	O
-the	O
-tip	O
-of	O
-helix	O
-14	O
-(	O
-415CAAA418	O
-)	O
-of	O
-the	O
-18S	O
-rRNA	O
-of	O
-the	O
-40S	O
-subunit	O
-and	O
-helix	O
-A	O
-(	O
-aa	O
-32	O
-–	O
-42	O
-)	O
-of	O
-eEF2	O
-(	O
-Figure	O
-5d	O
-).	O
-	O
-Bulged	O
-A416	O
-interacts	O
-with	O
-the	O
-switch	O
-loop	O
-in	O
-the	O
-vicinity	O
-of	O
-D53	O
-.	O
-	O
-Next	O
-to	O
-GDP	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-part	O
-of	O
-the	O
-switch	O
-loop	O
-(	O
-aa	O
-61	O
-–	O
-67	O
-)	O
-adopts	O
-a	O
-helical	O
-fold	O
-.	O
-	O
-As	O
-such	O
-,	O
-the	O
-conformations	O
-of	O
-SWI	O
-and	O
-the	O
-GTPase	O
-center	O
-in	O
-general	O
-are	O
-similar	O
-to	O
-those	O
-observed	O
-in	O
-ribosome	O
--	O
-bound	O
-EF	O
--	O
-Tu	O
-and	O
-EF	O
--	O
-G	O
-in	O
-the	O
-presence	O
-of	O
-GTP	O
-analogs	O
-.	O
-	O
-Structure	O
-II	O
-reveals	O
-PKI	O
-between	O
-the	O
-body	O
-A	O
-and	O
-P	O
-sites	O
-and	O
-eEF2	O
-partially	O
-advanced	O
-into	O
-the	O
-A	O
-site	O
-	O
-In	O
-Structure	O
-II	O
-,	O
-relative	O
-to	O
-Structure	O
-I	O
-,	O
-PKI	O
-is	O
-further	O
-shifted	O
-along	O
-the	O
-40S	O
-body	O
-,	O
-traversing	O
-~	O
-4	O
-Å	O
-toward	O
-the	O
-P	O
-site	O
-(	O
-Figures	O
-2e	O
-,	O
-f	O
-,	O
-and	O
-g	O
-),	O
-while	O
-stacking	O
-on	O
-C1274	O
-at	O
-the	O
-head	O
-A	O
-site	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-intermediate	O
-position	O
-of	O
-PKI	O
-is	O
-possible	O
-due	O
-to	O
-a	O
-large	O
-swivel	O
-of	O
-the	O
-head	O
-relative	O
-to	O
-the	O
-body	O
-,	O
-which	O
-brings	O
-the	O
-head	O
-A	O
-site	O
-close	O
-to	O
-the	O
-body	O
-P	O
-site	O
-.	O
-	O
-Domain	O
-IV	O
-of	O
-eEF2	O
-is	O
-further	O
-entrenched	O
-in	O
-the	O
-A	O
-site	O
-by	O
-~	O
-3	O
-Å	O
-relative	O
-to	O
-the	O
-body	O
-and	O
-~	O
-8	O
-Å	O
-relative	O
-to	O
-the	O
-head	O
-,	O
-preserving	O
-its	O
-interactions	O
-with	O
-PKI	O
-.	O
-	O
-The	O
-decoding	O
-center	O
-residues	O
-A1755	O
-and	O
-A1756	O
-are	O
-rearranged	O
-to	O
-pack	O
-inside	O
-helix	O
-44	O
-,	O
-making	O
-room	O
-for	O
-eEF2	O
-.	O
-	O
-This	O
-conformation	O
-of	O
-decoding	O
-center	O
-residues	O
-is	O
-also	O
-observed	O
-in	O
-the	O
-absence	O
-of	O
-A	O
--	O
-site	O
-ligands	O
-.	O
-	O
-The	O
-head	O
-interface	O
-of	O
-domain	O
-IV	O
-interacts	O
-with	O
-the	O
-40S	O
-head	O
-(	O
-Figure	O
-6a	O
-).	O
-	O
-Here	O
-,	O
-a	O
-positively	O
-charged	O
-surface	O
-of	O
-eEF2	O
-,	O
-formed	O
-by	O
-K613	O
-,	O
-R617	O
-and	O
-R631	O
-contacts	O
-the	O
-phosphate	O
-backbone	O
-of	O
-helix	O
-33	O
-(	O
-Figures	O
-6c	O
-;	O
-see	O
-also	O
-Figure	O
-6	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Structure	O
-III	O
-represents	O
-a	O
-highly	O
-bent	O
-IRES	O
-with	O
-PKI	O
-captured	O
-between	O
-the	O
-head	O
-A	O
-and	O
-P	O
-sites	O
-	O
-Consistent	O
-with	O
-the	O
-similar	O
-head	O
-swivels	O
-in	O
-Structure	O
-III	O
-and	O
-Structure	O
-II	O
-,	O
-relative	O
-positions	O
-of	O
-the	O
-40S	O
-head	O
-A	O
-site	O
-and	O
-body	O
-P	O
-site	O
-remain	O
-as	O
-in	O
-Structure	O
-II	O
-.	O
-	O
-Among	O
-the	O
-five	O
-structures	O
-,	O
-the	O
-PKI	O
-domain	O
-is	O
-least	O
-ordered	O
-in	O
-Structure	O
-III	O
-and	O
-lacks	O
-density	O
-for	O
-SL3	O
-.	O
-	O
-The	O
-map	O
-allows	O
-placement	O
-of	O
-PKI	O
-at	O
-the	O
-body	O
-P	O
-site	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-3	O
-).	O
-	O
-Thus	O
-,	O
-in	O
-Structure	O
-III	O
-,	O
-PKI	O
-has	O
-translocated	O
-along	O
-the	O
-40S	O
-body	O
-,	O
-but	O
-the	O
-head	O
-remains	O
-fully	O
-swiveled	O
-so	O
-that	O
-PKI	O
-is	O
-between	O
-the	O
-head	O
-A	O
-and	O
-P	O
-sites	O
-.	O
-	O
-Lower	O
-resolution	O
-of	O
-the	O
-map	O
-in	O
-this	O
-region	O
-suggests	O
-that	O
-PKI	O
-is	O
-somewhat	O
-destabilized	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-body	O
-P	O
-site	O
-in	O
-the	O
-absence	O
-of	O
-stacking	O
-with	O
-the	O
-foundations	O
-of	O
-the	O
-head	O
-A	O
-site	O
-(	O
-C1274	O
-)	O
-or	O
-P	O
-site	O
-(	O
-U1191	O
-).	O
-	O
-The	O
-position	O
-of	O
-eEF2	O
-is	O
-similar	O
-to	O
-that	O
-in	O
-Structure	O
-II	O
-.	O
-	O
-Structure	O
-IV	O
-represents	O
-a	O
-highly	O
-bent	O
-IRES	O
-with	O
-PKI	O
-partially	O
-accommodated	O
-in	O
-the	O
-P	O
-site	O
-	O
-In	O
-Structure	O
-IV	O
-,	O
-the	O
-40S	O
-subunit	O
-is	O
-almost	O
-non	O
--	O
-rotated	O
-relative	O
-to	O
-the	O
-60S	O
-subunit	O
-,	O
-and	O
-the	O
-40S	O
-head	O
-is	O
-mid	O
--	O
-swiveled	O
-.	O
-	O
-Unwinding	O
-of	O
-the	O
-head	O
-moves	O
-the	O
-head	O
-P	O
--	O
-site	O
-residue	O
-U1191	O
-and	O
-body	O
-P	O
--	O
-site	O
-residue	O
-C1637	O
-closer	O
-together	O
-,	O
-resulting	O
-in	O
-a	O
-partially	O
-restored	O
-40S	O
-P	O
-site	O
-.	O
-	O
-Whereas	O
-C1637	O
-forms	O
-a	O
-stacking	O
-platform	O
-for	O
-the	O
-last	O
-base	O
-pair	O
-of	O
-PKI	O
-,	O
-U1191	O
-does	O
-not	O
-yet	O
-stack	O
-on	O
-PKI	O
-because	O
-the	O
-head	O
-remains	O
-partially	O
-swiveled	O
-.	O
-	O
-This	O
-renders	O
-PKI	O
-partially	O
-accommodated	O
-in	O
-the	O
-P	O
-site	O
-(	O
-Figure	O
-2g	O
-).	O
-	O
-Unwinding	O
-of	O
-the	O
-40S	O
-head	O
-also	O
-positions	O
-the	O
-head	O
-A	O
-site	O
-closer	O
-to	O
-the	O
-body	O
-A	O
-site	O
-.	O
-	O
-This	O
-results	O
-in	O
-rearrangements	O
-of	O
-eEF2	O
-interactions	O
-with	O
-the	O
-head	O
-,	O
-allowing	O
-eEF2	O
-to	O
-advance	O
-further	O
-into	O
-the	O
-A	O
-site	O
-.	O
-	O
-To	O
-this	O
-end	O
-,	O
-the	O
-head	O
--	O
-interacting	O
-interface	O
-of	O
-domain	O
-IV	O
-slides	O
-along	O
-the	O
-surface	O
-of	O
-the	O
-head	O
-by	O
-5	O
-Å	O
-.	O
-Helix	O
-A	O
-of	O
-domain	O
-IV	O
-is	O
-positioned	O
-next	O
-to	O
-the	O
-backbone	O
-of	O
-h34	O
-,	O
-with	O
-positively	O
-charged	O
-residues	O
-K613	O
-,	O
-R617	O
-and	O
-R631	O
-rearranged	O
-from	O
-the	O
-backbone	O
-of	O
-h33	O
-(	O
-Figure	O
-6c	O
-;	O
-see	O
-also	O
-Figure	O
-6	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Structure	O
-V	O
-represents	O
-an	O
-extended	O
-IRES	O
-with	O
-PKI	O
-fully	O
-accommodated	O
-in	O
-the	O
-P	O
-site	O
-and	O
-domain	O
-IV	O
-of	O
-eEF2	O
-in	O
-the	O
-A	O
-site	O
-	O
-In	O
-the	O
-nearly	O
-non	O
--	O
-rotated	O
-and	O
-non	O
--	O
-swiveled	O
-ribosome	O
-conformation	O
-in	O
-Structure	O
-V	O
-closely	O
-resembling	O
-that	O
-of	O
-the	O
-post	O
--	O
-translocation	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-,	O
-PKI	O
-is	O
-fully	O
-accommodated	O
-in	O
-the	O
-P	O
-site	O
-.	O
-	O
-The	O
-codon	O
--	O
-anticodon	O
-–	O
-like	O
-helix	O
-is	O
-stacked	O
-on	O
-P	O
--	O
-site	O
-residues	O
-U1191	O
-and	O
-C1637	O
-(	O
-Figure	O
-3d	O
-),	O
-analogous	O
-to	O
-stacking	O
-of	O
-the	O
-tRNA	O
--	O
-mRNA	O
-helix	O
-(	O
-Figure	O
-3e	O
-).	O
-	O
-A	O
-notable	O
-conformational	O
-change	O
-in	O
-eEF2	O
-from	O
-that	O
-in	O
-the	O
-preceding	O
-Structures	O
-is	O
-visible	O
-in	O
-the	O
-position	O
-of	O
-domain	O
-III	O
-,	O
-which	O
-contacts	O
-uS12	O
-(	O
-Figure	O
-6d	O
-).	O
-	O
-In	O
-Structure	O
-V	O
-,	O
-protein	O
-uS12	O
-is	O
-shifted	O
-along	O
-with	O
-the	O
-40S	O
-body	O
-as	O
-a	O
-result	O
-of	O
-intersubunit	O
-rotation	O
-.	O
-	O
-In	O
-this	O
-position	O
-,	O
-uS12	O
-forms	O
-extensive	O
-interactions	O
-with	O
-eEF2	O
-domains	O
-II	O
-and	O
-III	O
-.	O
-	O
-Specifically	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-tail	O
-of	O
-uS12	O
-packs	O
-against	O
-the	O
-β	O
--	O
-barrel	O
-of	O
-domain	O
-II	O
-,	O
-while	O
-the	O
-β	O
--	O
-barrel	O
-of	O
-uS12	O
-packs	O
-against	O
-helix	O
-A	O
-of	O
-domain	O
-III	O
-.	O
-	O
-This	O
-shifts	O
-the	O
-tip	O
-of	O
-helix	O
-A	O
-of	O
-domain	O
-III	O
-(	O
-at	O
-aa	O
-500	O
-)	O
-by	O
-~	O
-5	O
-Å	O
-(	O
-relative	O
-to	O
-that	O
-in	O
-Structure	O
-I	O
-)	O
-toward	O
-domain	O
-I	O
-.	O
-Although	O
-domain	O
-III	O
-remains	O
-in	O
-contact	O
-with	O
-domain	O
-V	O
-,	O
-the	O
-shift	O
-occurs	O
-in	O
-the	O
-direction	O
-that	O
-could	O
-eventually	O
-disconnect	O
-the	O
-β	O
--	O
-platforms	O
-of	O
-these	O
-domains	O
-.	O
-	O
-Domain	O
-IV	O
-of	O
-eEF2	O
-is	O
-fully	O
-accommodated	O
-in	O
-the	O
-A	O
-site	O
-.	O
-	O
-The	O
-first	O
-codon	O
-of	O
-the	O
-open	O
-reading	O
-frame	O
-is	O
-also	O
-positioned	O
-in	O
-the	O
-A	O
-site	O
-,	O
-with	O
-bases	O
-exposed	O
-toward	O
-eEF2	O
-(	O
-Figure	O
-7	O
-),	O
-resembling	O
-the	O
-conformations	O
-of	O
-the	O
-A	O
--	O
-site	O
-codons	O
-in	O
-EF	O
--	O
-G	O
--	O
-bound	O
-70S	O
-complexes	O
-.	O
-	O
-As	O
-in	O
-the	O
-preceding	O
-Structures	O
-,	O
-the	O
-histidine	O
--	O
-diphthamide	O
-tip	O
-is	O
-bound	O
-in	O
-the	O
-minor	O
-groove	O
-of	O
-the	O
-P	O
--	O
-site	O
-codon	O
--	O
-anticodon	O
-helix	O
-.	O
-	O
-Diph699	O
-slightly	O
-rearranges	O
-,	O
-relative	O
-to	O
-that	O
-in	O
-Structure	O
-I	O
-(	O
-Figure	O
-7	O
-),	O
-and	O
-interacts	O
-with	O
-four	O
-out	O
-of	O
-six	O
-codon	O
--	O
-anticodon	O
-nucleotides	O
-.	O
-	O
-The	O
-imidazole	O
-moiety	O
-stacks	O
-on	O
-G6907	O
-(	O
-corresponding	O
-to	O
-nt	O
-36	O
-in	O
-the	O
-tRNA	O
-anticodon	O
-)	O
-and	O
-hydrogen	O
-bonds	O
-with	O
-O2	O
-’	O
-of	O
-G6906	O
-(	O
-nt	O
-35	O
-of	O
-tRNA	O
-).	O
-	O
-The	O
-amide	O
-at	O
-the	O
-diphthamide	O
-end	O
-interacts	O
-with	O
-N2	O
-of	O
-G6906	O
-and	O
-O2	O
-and	O
-O2	O
-’	O
-of	O
-C6951	O
-(	O
-corresponding	O
-to	O
-nt	O
-2	O
-of	O
-the	O
-codon	O
-).	O
-	O
-The	O
-trimethylamino	O
--	O
-group	O
-is	O
-positioned	O
-over	O
-the	O
-ribose	O
-of	O
-C6952	O
-(	O
-codon	O
-nt	O
-3	O
-).	O
-	O
-IRES	O
-translocation	O
-mechanism	O
-	O
-Animation	O
-showing	O
-the	O
-transition	O
-from	O
-the	O
-initiation	O
-80S	O
-•	O
-TSV	O
-IRES	O
-structures	O
-(	O
-Koh	O
-et	O
-al	O
-.,	O
-2014	O
-)	O
-to	O
-eEF2	O
--	O
-bound	O
-Structures	O
-I	O
-through	O
-V	O
-(	O
-this	O
-work	O
-).	O
-	O
-Four	O
-views	O
-(	O
-scenes	O
-)	O
-are	O
-shown	O
-:	O
-(	O
-1	O
-)	O
-A	O
-view	O
-down	O
-the	O
-intersubunit	O
-space	O
-,	O
-with	O
-the	O
-head	O
-of	O
-the	O
-40S	O
-subunit	O
-oriented	O
-toward	O
-a	O
-viewer	O
-,	O
-as	O
-in	O
-Figure	O
-1a	O
-;	O
-(	O
-2	O
-)	O
-A	O
-view	O
-at	O
-the	O
-solvent	O
-side	O
-of	O
-the	O
-40S	O
-subunit	O
-,	O
-with	O
-the	O
-40S	O
-head	O
-shown	O
-at	O
-the	O
-top	O
-,	O
-as	O
-in	O
-Figure	O
-2	O
-—	O
-figure	O
-supplement	O
-1	O
-;	O
-(	O
-3	O
-)	O
-A	O
-view	O
-down	O
-at	O
-the	O
-subunit	O
-interface	O
-of	O
-the	O
-40S	O
-subunit	O
-;	O
-(	O
-4	O
-)	O
-A	O
-close	O
--	O
-up	O
-view	O
-of	O
-the	O
-decoding	O
-center	O
-(	O
-A	O
-site	O
-)	O
-and	O
-the	O
-P	O
-site	O
-,	O
-as	O
-in	O
-Figure	O
-2g	O
-.	O
-Each	O
-scene	O
-is	O
-shown	O
-twice	O
-.	O
-	O
-In	O
-scenes	O
-1	O
-,	O
-2	O
-and	O
-3	O
-,	O
-nucleotides	O
-C1274	O
-,	O
-U1191	O
-of	O
-the	O
-40S	O
-head	O
-and	O
-G904	O
-of	O
-the	O
-40S	O
-platform	O
-are	O
-shown	O
-in	O
-black	O
-to	O
-denote	O
-the	O
-A	O
-,	O
-P	O
-and	O
-E	O
-sites	O
-,	O
-respectively	O
-.	O
-	O
-In	O
-scene	O
-4	O
-,	O
-C1274	O
-and	O
-U1191	O
-are	O
-labeled	O
-and	O
-shown	O
-in	O
-yellow	O
-;	O
-G577	O
-,	O
-A1755	O
-and	O
-A1756	O
-of	O
-the	O
-40S	O
-body	O
-A	O
-site	O
-and	O
-C1637	O
-of	O
-the	O
-body	O
-P	O
-site	O
-are	O
-labeled	O
-and	O
-shown	O
-in	O
-orange	O
-.	O
-	O
-In	O
-this	O
-work	O
-we	O
-have	O
-captured	O
-the	O
-structures	O
-of	O
-the	O
-TSV	O
-IRES	O
-,	O
-whose	O
-PKI	O
-samples	O
-positions	O
-between	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-(	O
-Structures	O
-I	O
-–	O
-IV	O
-),	O
-as	O
-well	O
-as	O
-in	O
-the	O
-P	O
-site	O
-(	O
-Structure	O
-V	O
-).	O
-	O
-We	O
-propose	O
-that	O
-together	O
-with	O
-the	O
-previously	O
-reported	O
-initiation	O
-state	O
-,	O
-these	O
-structures	O
-represent	O
-the	O
-trajectory	O
-of	O
-eEF2	O
--	O
-induced	O
-IRES	O
-translocation	O
-(	O
-shown	O
-as	O
-an	O
-animation	O
-in	O
-http	O
-://	O
-labs	O
-.	O
-umassmed	O
-.	O
-edu	O
-/	O
-korostelevlab	O
-/	O
-msc	O
-/	O
-iresmovie	O
-.	O
-gif	O
-and	O
-Video	O
-1	O
-).	O
-	O
-Our	O
-structures	O
-reveal	O
-previously	O
-unseen	O
-intermediate	O
-states	O
-of	O
-eEF2	O
-or	O
-EF	O
--	O
-G	O
-engagement	O
-with	O
-the	O
-A	O
-site	O
-,	O
-providing	O
-the	O
-structural	O
-basis	O
-for	O
-the	O
-mechanism	O
-of	O
-translocase	O
-action	O
-.	O
-	O
-Furthermore	O
-,	O
-they	O
-provide	O
-insight	O
-into	O
-the	O
-mechanism	O
-of	O
-eEF2	O
-•	O
-GTP	O
-association	O
-with	O
-the	O
-pre	O
--	O
-translocation	O
-ribosome	O
-and	O
-eEF2	O
-•	O
-GDP	O
-dissociation	O
-from	O
-the	O
-post	O
--	O
-translocation	O
-ribosome	O
-,	O
-also	O
-delineating	O
-the	O
-mechanism	O
-of	O
-translation	O
-inhibition	O
-by	O
-the	O
-antifungal	O
-drug	O
-sordarin	O
-.	O
-	O
-In	O
-summary	O
-,	O
-the	O
-reported	O
-ensemble	O
-of	O
-structures	O
-substantially	O
-enhances	O
-our	O
-understanding	O
-of	O
-the	O
-translocation	O
-mechanism	O
-,	O
-including	O
-that	O
-of	O
-tRNAs	O
-as	O
-discussed	O
-below	O
-.	O
-	O
-Translocation	O
-of	O
-the	O
-TSV	O
-IRES	O
-on	O
-the	O
-40S	O
-subunit	O
-globally	O
-resembles	O
-a	O
-step	O
-of	O
-an	O
-inchworm	O
-(	O
-Figure	O
-4	O
-;	O
-see	O
-also	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-2	O
-).	O
-	O
-At	O
-the	O
-start	O
-(	O
-initiation	O
-state	O
-),	O
-the	O
-IRES	O
-adopts	O
-an	O
-extended	O
-conformation	O
-(	O
-extended	O
-inchworm	O
-).	O
-	O
-The	O
-front	O
-'	O
-legs	O
-'	O
-(	O
-SL4	O
-and	O
-SL5	O
-)	O
-of	O
-the	O
-5	O
-’-	O
-domain	O
-(	O
-front	O
-end	O
-)	O
-are	O
-attached	O
-to	O
-the	O
-40S	O
-head	O
-proteins	O
-uS7	O
-,	O
-uS11	O
-and	O
-eS25	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-2	O
-).	O
-	O
-PKI	O
-,	O
-representing	O
-the	O
-hind	O
-end	O
-,	O
-is	O
-bound	O
-in	O
-the	O
-A	O
-site	O
-.	O
-	O
-In	O
-the	O
-first	O
-sub	O
--	O
-step	O
-(	O
-Structures	O
-I	O
-to	O
-IV	O
-),	O
-the	O
-hind	O
-end	O
-advances	O
-from	O
-the	O
-A	O
-to	O
-the	O
-P	O
-site	O
-and	O
-approaches	O
-the	O
-front	O
-end	O
-,	O
-which	O
-remains	O
-attached	O
-to	O
-the	O
-40S	O
-surface	O
-.	O
-	O
-This	O
-shortens	O
-the	O
-distance	O
-between	O
-PKI	O
-and	O
-SL4	O
-by	O
-up	O
-to	O
-20	O
-Å	O
-relative	O
-to	O
-the	O
-initiating	O
-IRES	O
-structure	O
-,	O
-resulting	O
-in	O
-a	O
-bent	O
-IRES	O
-conformation	O
-(	O
-bent	O
-inchworm	O
-).	O
-	O
-Finally	O
-(	O
-Structures	O
-IV	O
-to	O
-V	O
-),	O
-as	O
-the	O
-hind	O
-end	O
-is	O
-accommodated	O
-in	O
-the	O
-P	O
-site	O
-,	O
-the	O
-front	O
-'	O
-legs	O
-'	O
-advance	O
-by	O
-departing	O
-from	O
-their	O
-initial	O
-binding	O
-sites	O
-.	O
-	O
-This	O
-converts	O
-the	O
-IRES	O
-into	O
-an	O
-extended	O
-conformation	O
-,	O
-rendering	O
-the	O
-inchworm	O
-prepared	O
-for	O
-the	O
-next	O
-translocation	O
-step	O
-.	O
-	O
-Notably	O
-,	O
-at	O
-all	O
-steps	O
-,	O
-the	O
-head	O
-of	O
-the	O
-IRES	O
-inchworm	O
-(	O
-L1	O
-.	O
-1	O
-region	O
-)	O
-is	O
-supported	O
-by	O
-the	O
-mobile	O
-L1	O
-stalk	O
-.	O
-	O
-In	O
-the	O
-post	O
--	O
-translocation	O
-CrPV	O
-IRES	O
-structure	O
-,	O
-the	O
-5	O
-’-	O
-domain	O
-similarly	O
-protrudes	O
-between	O
-the	O
-subunits	O
-and	O
-interacts	O
-with	O
-the	O
-L1	O
-stalk	O
-,	O
-as	O
-in	O
-the	O
-initiation	O
-state	O
-for	O
-this	O
-IRES	O
-.	O
-	O
-This	O
-underlines	O
-structural	O
-similarity	O
-for	O
-the	O
-TSV	O
-and	O
-CrPV	O
-IRES	O
-translocation	O
-mechanisms	O
-.	O
-	O
-Upon	O
-translocation	O
-,	O
-the	O
-GCU	O
-start	O
-codon	O
-is	O
-positioned	O
-in	O
-the	O
-A	O
-site	O
-(	O
-Structure	O
-V	O
-),	O
-ready	O
-for	O
-interaction	O
-with	O
-Ala	O
--	O
-tRNAAla	O
-upon	O
-eEF2	O
-departure	O
-.	O
-	O
-Recent	O
-studies	O
-have	O
-shown	O
-that	O
-in	O
-some	O
-cases	O
-a	O
-fraction	O
-of	O
-IGR	O
-IRES	O
--	O
-driven	O
-translation	O
-results	O
-from	O
-an	O
-alternative	O
-reading	O
-frame	O
-,	O
-which	O
-is	O
-shifted	O
-by	O
-one	O
-nucleotide	O
-relative	O
-to	O
-the	O
-normal	O
-ORF	O
-.	O
-	O
-One	O
-of	O
-the	O
-mechanistic	O
-scenarios	O
-(	O
-discussed	O
-in	O
-)	O
-involves	O
-binding	O
-of	O
-the	O
-first	O
-aminoacyl	O
--	O
-tRNA	O
-to	O
-the	O
-post	O
--	O
-translocated	O
-IRES	O
-mRNA	O
-frame	O
-shifted	O
-by	O
-one	O
-nucleotide	O
-(	O
-predominantly	O
-a	O
-+	O
-1	O
-frame	O
-shift	O
-).	O
-	O
-In	O
-our	O
-structures	O
-,	O
-the	O
-IRES	O
-presents	O
-to	O
-the	O
-decoding	O
-center	O
-a	O
-pre	O
--	O
-translocated	O
-or	O
-fully	O
-translocated	O
-ORF	O
-,	O
-rather	O
-than	O
-a	O
-+	O
-1	O
-(	O
-more	O
-translocated	O
-)	O
-ORF	O
-,	O
-suggesting	O
-that	O
-eEF2	O
-does	O
-not	O
-induce	O
-a	O
-highly	O
-populated	O
-fraction	O
-of	O
-+	O
-1	O
-shifted	O
-IRES	O
-mRNAs	O
-.	O
-	O
-It	O
-is	O
-likely	O
-that	O
-alternative	O
-frame	O
-setting	O
-occurs	O
-following	O
-eEF2	O
-release	O
-and	O
-that	O
-this	O
-depends	O
-on	O
-transient	O
-displacement	O
-of	O
-the	O
-start	O
-codon	O
-in	O
-the	O
-decoding	O
-center	O
-,	O
-allowing	O
-binding	O
-of	O
-the	O
-corresponding	O
-amino	O
-acyl	O
--	O
-tRNA	O
-to	O
-an	O
-off	O
--	O
-frame	O
-codon	O
-.	O
-	O
-Further	O
-structural	O
-studies	O
-involving	O
-80S	O
-•	O
-IRES	O
-•	O
-tRNA	O
-complexes	O
-are	O
-necessary	O
-to	O
-understand	O
-the	O
-mechanisms	O
-underlying	O
-alternative	O
-reading	O
-frame	O
-selection	O
-.	O
-	O
-The	O
-presence	O
-of	O
-several	O
-translocation	O
-complexes	O
-in	O
-a	O
-single	O
-sample	O
-suggests	O
-that	O
-the	O
-structures	O
-represent	O
-equilibrium	O
-states	O
-of	O
-forward	O
-and	O
-reverse	O
-translocation	O
-of	O
-the	O
-IRES	O
-,	O
-which	O
-interconvert	O
-among	O
-each	O
-other	O
-.	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-the	O
-observations	O
-that	O
-the	O
-intergenic	O
-IRESs	O
-are	O
-prone	O
-to	O
-reverse	O
-translocation	O
-.	O
-	O
-Specifically	O
-,	O
-biochemical	O
-toe	O
--	O
-printing	O
-studies	O
-in	O
-the	O
-presence	O
-of	O
-eEF2	O
-•	O
-GTP	O
-identified	O
-IRES	O
-in	O
-a	O
-non	O
--	O
-translocated	O
-position	O
-unless	O
-eEF1a	O
-•	O
-aa	O
--	O
-tRNA	O
-is	O
-also	O
-present	O
-.	O
-	O
-These	O
-findings	O
-indicate	O
-that	O
-IRES	O
-translocation	O
-by	O
-eEF2	O
-is	O
-futile	O
-:	O
-the	O
-IRES	O
-returns	O
-to	O
-the	O
-A	O
-site	O
-upon	O
-releasing	O
-eEF2	O
-•	O
-GDP	O
-unless	O
-an	O
-amino	O
--	O
-acyl	O
-tRNA	O
-enters	O
-the	O
-A	O
-site	O
-and	O
-blocks	O
-IRES	O
-back	O
--	O
-translocation	O
-.	O
-	O
-This	O
-contrasts	O
-with	O
-the	O
-post	O
--	O
-translocated	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-,	O
-in	O
-which	O
-the	O
-classical	O
-P	O
-and	O
-E	O
--	O
-site	O
-tRNAs	O
-are	O
-stabilized	O
-in	O
-the	O
-non	O
--	O
-rotated	O
-ribosome	O
-after	O
-translocase	O
-release	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-meta	O
--	O
-stability	O
-of	O
-the	O
-post	O
--	O
-translocation	O
-IRES	O
-is	O
-likely	O
-due	O
-to	O
-the	O
-absence	O
-of	O
-stabilizing	O
-structural	O
-features	O
-present	O
-in	O
-the	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-.	O
-	O
-In	O
-the	O
-initiation	O
-state	O
-,	O
-the	O
-IRES	O
-resembles	O
-a	O
-pre	O
--	O
-translocation	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-reduced	O
-to	O
-the	O
-A	O
-/	O
-P	O
--	O
-tRNA	O
-anticodon	O
--	O
-stem	O
-loop	O
-and	O
-elbow	O
-in	O
-the	O
-A	O
-site	O
-and	O
-the	O
-P	O
-/	O
-E	O
--	O
-tRNA	O
-elbow	O
-contacting	O
-the	O
-L1	O
-stalk	O
-.	O
-	O
-Because	O
-the	O
-anticodon	O
--	O
-stem	O
-loop	O
-of	O
-the	O
-A	O
--	O
-tRNA	O
-is	O
-sufficient	O
-for	O
-translocation	O
-completion	O
-,	O
-we	O
-ascribe	O
-the	O
-meta	O
--	O
-stability	O
-of	O
-the	O
-post	O
--	O
-translocation	O
-IRES	O
-to	O
-the	O
-absence	O
-of	O
-the	O
-P	O
-/	O
-E	O
--	O
-tRNA	O
-elements	O
-,	O
-either	O
-the	O
-ASL	O
-or	O
-the	O
-acceptor	O
-arm	O
-,	O
-or	O
-both	O
-.	O
-	O
-Furthermore	O
-,	O
-interactions	O
-of	O
-SL4	O
-and	O
-SL5	O
-with	O
-the	O
-40S	O
-subunit	O
-likely	O
-contribute	O
-to	O
-stabilization	O
-of	O
-pre	O
--	O
-translocation	O
-structures	O
-.	O
-	O
-Partitioned	O
-roles	O
-of	O
-40S	O
-subunit	O
-rearrangements	O
-	O
-Our	O
-structures	O
-delineate	O
-the	O
-mechanistic	O
-functions	O
-for	O
-intersubunit	O
-rotation	O
-and	O
-head	O
-swivel	O
-in	O
-translocation	O
-.	O
-	O
-Specifically	O
-,	O
-intersubunit	O
-rotation	O
-allows	O
-eEF2	O
-entry	O
-into	O
-the	O
-A	O
-site	O
-,	O
-while	O
-the	O
-head	O
-swivel	O
-mediates	O
-PKI	O
-translocation	O
-.	O
-	O
-Various	O
-degrees	O
-of	O
-intersubunit	O
-rotation	O
-have	O
-been	O
-observed	O
-in	O
-cryo	O
--	O
-EM	O
-studies	O
-of	O
-the	O
-80S	O
-•	O
-IRES	O
-initiation	O
-complexes	O
-.	O
-	O
-This	O
-suggests	O
-that	O
-the	O
-subunits	O
-are	O
-capable	O
-of	O
-spontaneous	O
-rotation	O
-,	O
-as	O
-is	O
-the	O
-case	O
-for	O
-tRNA	O
--	O
-bound	O
-pre	O
--	O
-translocation	O
-complexes	O
-.	O
-	O
-The	O
-pre	O
--	O
-translocation	O
-Structure	O
-I	O
-with	O
-eEF2	O
-least	O
-advanced	O
-into	O
-the	O
-A	O
-site	O
-adopts	O
-a	O
-fully	O
-rotated	O
-conformation	O
-.	O
-	O
-Reverse	O
-intersubunit	O
-rotation	O
-from	O
-Structure	O
-I	O
-to	O
-V	O
-shifts	O
-the	O
-translocation	O
-tunnel	O
-(	O
-the	O
-tunnel	O
-between	O
-the	O
-A	O
-,	O
-P	O
-and	O
-E	O
-sites	O
-)	O
-toward	O
-eEF2	O
-,	O
-which	O
-is	O
-rigidly	O
-attached	O
-to	O
-the	O
-60S	O
-subunit	O
-.	O
-	O
-This	O
-allows	O
-eEF2	O
-to	O
-move	O
-into	O
-the	O
-A	O
-site	O
-.	O
-	O
-As	O
-such	O
-,	O
-reverse	O
-intersubunit	O
-rotation	O
-facilitates	O
-full	O
-docking	O
-of	O
-eEF2	O
-in	O
-the	O
-A	O
-site	O
-.	O
-	O
-Because	O
-the	O
-histidine	O
--	O
-diphthamide	O
-tip	O
-of	O
-eEF2	O
-(	O
-H583	O
-,	O
-H694	O
-and	O
-Diph699	O
-)	O
-attaches	O
-to	O
-the	O
-codon	O
--	O
-anticodon	O
--	O
-like	O
-helix	O
-of	O
-PKI	O
-,	O
-eEF2	O
-appears	O
-to	O
-directly	O
-force	O
-PKI	O
-out	O
-of	O
-the	O
-A	O
-site	O
-.	O
-	O
-The	O
-head	O
-swivel	O
-allows	O
-gradual	O
-translocation	O
-of	O
-PKI	O
-to	O
-the	O
-P	O
-site	O
-,	O
-first	O
-with	O
-respect	O
-to	O
-the	O
-body	O
-and	O
-then	O
-to	O
-the	O
-head	O
-.	O
-	O
-The	O
-fully	O
-swiveled	O
-conformations	O
-of	O
-Structures	O
-II	O
-and	O
-III	O
-represent	O
-the	O
-mid	O
--	O
-point	O
-of	O
-translocation	O
-,	O
-in	O
-which	O
-PKI	O
-relocates	O
-between	O
-the	O
-head	O
-A	O
-site	O
-and	O
-body	O
-P	O
-site	O
-.	O
-	O
-We	O
-note	O
-that	O
-such	O
-mid	O
--	O
-states	O
-have	O
-not	O
-been	O
-observed	O
-for	O
-2tRNA	O
-•	O
-mRNA	O
-,	O
-but	O
-their	O
-formation	O
-can	O
-explain	O
-the	O
-formation	O
-of	O
-subsequent	O
-pe	O
-/	O
-E	O
-hybrid	O
-and	O
-ap	O
-/	O
-P	O
-chimeric	O
-structures	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Reverse	O
-swivel	O
-from	O
-Structure	O
-III	O
-to	O
-V	O
-brings	O
-the	O
-head	O
-to	O
-the	O
-non	O
--	O
-swiveled	O
-position	O
-,	O
-restoring	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-on	O
-the	O
-small	O
-subunit	O
-.	O
-	O
-The	O
-functions	O
-of	O
-eEF2	O
-in	O
-translocation	O
-	O
-To	O
-our	O
-knowledge	O
-,	O
-our	O
-work	O
-provides	O
-the	O
-first	O
-high	O
--	O
-resolution	O
-view	O
-of	O
-the	O
-dynamics	O
-of	O
-a	O
-ribosomal	O
-translocase	O
-that	O
-is	O
-inferred	O
-from	O
-an	O
-ensemble	O
-of	O
-structures	O
-sampled	O
-under	O
-uniform	O
-conditions	O
-.	O
-	O
-The	O
-structures	O
-,	O
-therefore	O
-,	O
-offer	O
-a	O
-unique	O
-opportunity	O
-to	O
-address	O
-the	O
-role	O
-of	O
-the	O
-elongation	O
-factors	O
-during	O
-translocation	O
-.	O
-	O
-Translocases	O
-are	O
-efficient	O
-enzymes	O
-.	O
-	O
-While	O
-the	O
-ribosome	O
-itself	O
-has	O
-the	O
-capacity	O
-to	O
-translocate	O
-in	O
-the	O
-absence	O
-of	O
-the	O
-translocase	O
-,	O
-spontaneous	O
-translocation	O
-is	O
-slow	O
-.	O
-	O
-EF	O
--	O
-G	O
-enhances	O
-the	O
-translocation	O
-rate	O
-by	O
-several	O
-orders	O
-of	O
-magnitude	O
-,	O
-aided	O
-by	O
-an	O
-additional	O
-2	O
--	O
-to	O
-50	O
--	O
-fold	O
-boost	O
-from	O
-GTP	O
-hydrolysis	O
-.	O
-	O
-Due	O
-to	O
-the	O
-lack	O
-of	O
-structures	O
-of	O
-translocation	O
-intermediates	O
-,	O
-the	O
-mechanistic	O
-role	O
-of	O
-eEF2	O
-/	O
-EF	O
--	O
-G	O
-is	O
-not	O
-fully	O
-understood	O
-.	O
-	O
-The	O
-80S	O
-•	O
-IRES	O
-•	O
-eEF2	O
-structures	O
-reported	O
-here	O
-suggest	O
-two	O
-main	O
-roles	O
-for	O
-eEF2	O
-in	O
-translocation	O
-.	O
-	O
-As	O
-discussed	O
-above	O
-,	O
-the	O
-first	O
-role	O
-is	O
-to	O
-directly	O
-shift	O
-PKI	O
-out	O
-of	O
-the	O
-A	O
-site	O
-upon	O
-spontaneous	O
-reverse	O
-intersubunit	O
-rotation	O
-.	O
-	O
-In	O
-our	O
-structures	O
-,	O
-the	O
-tip	O
-of	O
-domain	O
-IV	O
-docks	O
-next	O
-to	O
-PKI	O
-,	O
-with	O
-diphthamide	O
-699	O
-fit	O
-into	O
-the	O
-minor	O
-groove	O
-of	O
-the	O
-codon	O
--	O
-anticodon	O
--	O
-like	O
-helix	O
-of	O
-PKI	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-This	O
-arrangement	O
-rationalizes	O
-inactivation	O
-of	O
-eEF2	O
-by	O
-diphtheria	O
-toxin	O
-,	O
-which	O
-catalyzes	O
-ADP	O
--	O
-ribosylation	O
-of	O
-the	O
-diphthamide	O
-(	O
-reviewed	O
-in	O
-).	O
-	O
-The	O
-enzyme	O
-ADP	O
--	O
-ribosylates	O
-the	O
-NE2	O
-atom	O
-of	O
-the	O
-imidazole	O
-ring	O
-,	O
-which	O
-in	O
-our	O
-structures	O
-interacts	O
-with	O
-the	O
-first	O
-two	O
-residues	O
-of	O
-the	O
-anticodon	O
--	O
-like	O
-strand	O
-of	O
-PKI	O
-.	O
-	O
-The	O
-bulky	O
-ADP	O
--	O
-ribosyl	O
-moiety	O
-at	O
-this	O
-position	O
-would	O
-disrupt	O
-the	O
-interaction	O
-,	O
-rendering	O
-eEF2	O
-unable	O
-to	O
-bind	O
-to	O
-the	O
-A	O
-site	O
-and	O
-/	O
-or	O
-stalled	O
-on	O
-ribosomes	O
-in	O
-a	O
-non	O
--	O
-productive	O
-conformation	O
-.	O
-	O
-As	O
-eEF2	O
-shifts	O
-PKI	O
-toward	O
-the	O
-P	O
-site	O
-in	O
-the	O
-course	O
-of	O
-reverse	O
-intersubunit	O
-rotation	O
-,	O
-the	O
-60S	O
--	O
-attached	O
-translocase	O
-migrates	O
-along	O
-the	O
-surface	O
-of	O
-the	O
-40S	O
-subunit	O
-,	O
-guided	O
-by	O
-electrostatic	O
-interactions	O
-.	O
-	O
-Positively	O
--	O
-charged	O
-patches	O
-of	O
-domains	O
-II	O
-and	O
-III	O
-(	O
-R391	O
-,	O
-K394	O
-,	O
-R433	O
-,	O
-R510	O
-)	O
-and	O
-IV	O
-(	O
-K613	O
-,	O
-R617	O
-,	O
-R609	O
-,	O
-R631	O
-,	O
-K651	O
-)	O
-slide	O
-over	O
-rRNA	O
-of	O
-the	O
-40S	O
-body	O
-(	O
-h5	O
-)	O
-and	O
-head	O
-(	O
-h18	O
-and	O
-h33	O
-/	O
-h34	O
-),	O
-respectively	O
-.	O
-	O
-The	O
-Structures	O
-reveal	O
-hopping	O
-of	O
-the	O
-positive	O
-clusters	O
-over	O
-rRNA	O
-helices	O
-.	O
-	O
-For	O
-example	O
-,	O
-between	O
-Structures	O
-II	O
-and	O
-V	O
-,	O
-the	O
-K613	O
-/	O
-R617	O
-/	O
-R631	O
-cluster	O
-of	O
-domain	O
-IV	O
-hops	O
-by	O
-~	O
-19	O
-Å	O
-(	O
-for	O
-Cα	O
-of	O
-R617	O
-)	O
-from	O
-the	O
-phosphate	O
-backbone	O
-of	O
-h33	O
-(	O
-at	O
-nt	O
-1261	O
-–	O
-1264	O
-)	O
-to	O
-that	O
-of	O
-the	O
-neighboring	O
-h34	O
-(	O
-at	O
-nt	O
-1442	O
-–	O
-1445	O
-).	O
-	O
-Thus	O
-,	O
-sliding	O
-of	O
-eEF2	O
-involves	O
-reorganization	O
-of	O
-electrostatic	O
-,	O
-perhaps	O
-isoenergetic	O
-interactions	O
-,	O
-echoing	O
-those	O
-implied	O
-in	O
-extraordinarily	O
-fast	O
-ribosome	O
-inactivation	O
-rates	O
-by	O
-the	O
-small	O
--	O
-protein	O
-ribotoxins	O
-and	O
-in	O
-fast	O
-protein	O
-association	O
-and	O
-diffusion	O
-along	O
-DNA	O
-.	O
-	O
-Comparison	O
-of	O
-our	O
-structures	O
-with	O
-the	O
-80S	O
-•	O
-IRES	O
-initiation	O
-structure	O
-reveals	O
-the	O
-structural	O
-basis	O
-for	O
-the	O
-second	O
-key	O
-function	O
-of	O
-the	O
-translocase	O
-:	O
-'	O
-unlocking	O
-'	O
-of	O
-intrasubunit	O
-rearrangements	O
-that	O
-are	O
-required	O
-for	O
-step	O
--	O
-wise	O
-translocation	O
-of	O
-PKI	O
-on	O
-the	O
-small	O
-subunit	O
-.	O
-	O
-The	O
-unlocking	O
-model	O
-of	O
-the	O
-ribosome	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-pre	O
--	O
-translocation	O
-complex	O
-has	O
-been	O
-proposed	O
-decades	O
-ago	O
-and	O
-functional	O
-requirement	O
-of	O
-the	O
-translocase	O
-in	O
-this	O
-process	O
-has	O
-been	O
-implicated	O
-.	O
-	O
-However	O
-,	O
-the	O
-structural	O
-and	O
-mechanistic	O
-definitions	O
-of	O
-the	O
-locked	O
-and	O
-unlocked	O
-states	O
-have	O
-remained	O
-unclear	O
-,	O
-ranging	O
-from	O
-the	O
-globally	O
-distinct	O
-ribosome	O
-conformations	O
-to	O
-unknown	O
-local	O
-rearrangements	O
-,	O
-e	O
-.	O
-g	O
-.	O
-those	O
-in	O
-the	O
-decoding	O
-center	O
-.	O
-	O
-FRET	O
-data	O
-indicate	O
-that	O
-translocation	O
-of	O
-2tRNA	O
-•	O
-mRNA	O
-on	O
-the	O
-70S	O
-ribosome	O
-requires	O
-a	O
-forward	O
--	O
-and	O
--	O
-reverse	O
-head	O
-swivel	O
-,	O
-which	O
-may	O
-be	O
-related	O
-to	O
-the	O
-unlocking	O
-phenomenon	O
-.	O
-	O
-Whereas	O
-intersubunit	O
-rotation	O
-of	O
-the	O
-pre	O
--	O
-translocation	O
-complex	O
-occurs	O
-spontaneously	O
-,	O
-the	O
-head	O
-swivel	O
-is	O
-induced	O
-by	O
-the	O
-eEF2	O
-/	O
-EF	O
--	O
-G	O
-translocase	O
-,	O
-consistent	O
-with	O
-requirement	O
-of	O
-eEF2	O
-for	O
-unlocking	O
-.	O
-	O
-Structural	O
-studies	O
-revealed	O
-large	O
-head	O
-swivels	O
-in	O
-various	O
-70S	O
-•	O
-tRNA	O
-•	O
-EF	O
--	O
-G	O
-and	O
-80S	O
-•	O
-tRNA	O
-•	O
-eEF2	O
-complexes	O
-,	O
-but	O
-not	O
-in	O
-'	O
-locked	O
-'	O
-complexes	O
-with	O
-the	O
-A	O
-site	O
-occupied	O
-by	O
-the	O
-tRNA	O
-in	O
-the	O
-absence	O
-of	O
-the	O
-translocase	O
-.	O
-	O
-Our	O
-structures	O
-suggest	O
-that	O
-eEF2	O
-induces	O
-head	O
-swivel	O
-by	O
-'	O
-unlocking	O
-'	O
-the	O
-head	O
--	O
-body	O
-interactions	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-Binding	O
-of	O
-the	O
-ASL	O
-to	O
-the	O
-A	O
-site	O
-is	O
-known	O
-from	O
-structural	O
-studies	O
-of	O
-bacterial	O
-ribosomes	O
-to	O
-result	O
-in	O
-'	O
-domain	O
-closure	O
-'	O
-of	O
-the	O
-small	O
-subunit	O
-,	O
-i	O
-.	O
-e	O
-.	O
-closer	O
-association	O
-of	O
-the	O
-head	O
-,	O
-shoulder	O
-and	O
-body	O
-domains	O
-.	O
-	O
-The	O
-domain	O
-closure	O
-'	O
-locks	O
-'	O
-cognate	O
-tRNA	O
-in	O
-the	O
-A	O
-site	O
-via	O
-stacking	O
-on	O
-the	O
-head	O
-A	O
-site	O
-(	O
-C1274	O
-in	O
-S	O
-.	O
-cerevisiae	O
-or	O
-C1054	O
-in	O
-E	O
-.	O
-coli	O
-)	O
-and	O
-interactions	O
-with	O
-the	O
-body	O
-A	O
--	O
-site	O
-nucleotides	O
-A1755	O
-and	O
-A1756	O
-(	O
-A1492	O
-and	O
-A1493	O
-in	O
-E	O
-.	O
-coli	O
-).	O
-	O
-This	O
-'	O
-locked	O
-'	O
-state	O
-is	O
-identical	O
-to	O
-that	O
-observed	O
-for	O
-PKI	O
-in	O
-the	O
-80S	O
-•	O
-IRES	O
-initiation	O
-structures	O
-in	O
-the	O
-absence	O
-of	O
-eEF2	O
-.	O
-	O
-Structure	O
-I	O
-demonstrates	O
-that	O
-at	O
-an	O
-early	O
-pre	O
--	O
-translocation	O
-step	O
-,	O
-the	O
-histidine	O
--	O
-diphthamide	O
-tip	O
-of	O
-eEF2	O
-is	O
-wedged	O
-between	O
-A1755	O
-and	O
-A1756	O
-and	O
-PKI	O
-.	O
-	O
-This	O
-destabilization	O
-allows	O
-PKI	O
-to	O
-detach	O
-from	O
-the	O
-body	O
-A	O
-site	O
-upon	O
-spontaneous	O
-reverse	O
-40S	O
-body	O
-rotation	O
-,	O
-while	O
-maintaining	O
-interactions	O
-with	O
-the	O
-head	O
-A	O
-site	O
-.	O
-	O
-Destabilization	O
-of	O
-the	O
-head	O
--	O
-bound	O
-PKI	O
-at	O
-the	O
-body	O
-A	O
-site	O
-thus	O
-allows	O
-mobility	O
-of	O
-the	O
-head	O
-relative	O
-to	O
-the	O
-body	O
-.	O
-	O
-The	O
-histidine	O
--	O
-diphthamide	O
--	O
-induced	O
-disengagement	O
-of	O
-PKI	O
-from	O
-A1755	O
-and	O
-A1756	O
-therefore	O
-provides	O
-the	O
-structural	O
-definition	O
-for	O
-the	O
-'	O
-unlocking	O
-'	O
-mode	O
-of	O
-eEF2	O
-action	O
-.	O
-	O
-In	O
-summary	O
-,	O
-our	O
-structures	O
-are	O
-consistent	O
-with	O
-a	O
-model	O
-of	O
-eEF2	O
--	O
-induced	O
-translocation	O
-in	O
-which	O
-both	O
-PKI	O
-and	O
-eEF2	O
-passively	O
-migrate	O
-into	O
-the	O
-P	O
-and	O
-A	O
-site	O
-,	O
-respectively	O
-,	O
-during	O
-spontaneous	O
-40S	O
-body	O
-rotation	O
-and	O
-head	O
-swivel	O
-,	O
-the	O
-latter	O
-being	O
-allowed	O
-by	O
-'	O
-unlocking	O
-'	O
-of	O
-the	O
-A	O
-site	O
-by	O
-eEF2	O
-.	O
-	O
-Observation	O
-of	O
-different	O
-PKI	O
-conformations	O
-sampling	O
-a	O
-range	O
-of	O
-positions	O
-between	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-in	O
-the	O
-presence	O
-of	O
-eEF2	O
-•	O
-GDP	O
-implies	O
-that	O
-thermal	O
-fluctuations	O
-of	O
-the	O
-40S	O
-head	O
-domain	O
-are	O
-sufficient	O
-for	O
-translocation	O
-along	O
-the	O
-energetically	O
-flat	O
-trajectory	O
-.	O
-	O
-Insights	O
-into	O
-eEF2	O
-association	O
-with	O
-and	O
-dissociation	O
-from	O
-the	O
-ribosome	O
-	O
-The	O
-conformational	O
-rearrangements	O
-in	O
-eEF2	O
-from	O
-Structure	O
-I	O
-through	O
-Structure	O
-V	O
-provide	O
-insights	O
-into	O
-the	O
-mechanisms	O
-of	O
-eEF2	O
-association	O
-with	O
-the	O
-pre	O
--	O
-translocation	O
-ribosome	O
-and	O
-dissociation	O
-from	O
-the	O
-post	O
--	O
-translocation	O
-ribosome	O
-.	O
-	O
-In	O
-all	O
-five	O
-structures	O
-,	O
-the	O
-GTPase	O
-domain	O
-is	O
-attached	O
-to	O
-the	O
-P	O
-stalk	O
-and	O
-the	O
-sarcin	O
--	O
-ricin	O
-loop	O
-.	O
-	O
-In	O
-the	O
-fully	O
--	O
-rotated	O
-pre	O
--	O
-translocation	O
--	O
-like	O
-Structure	O
-I	O
-,	O
-an	O
-additional	O
-interaction	O
-exists	O
-.	O
-	O
-Here	O
-,	O
-switch	O
-loop	O
-I	O
-interacts	O
-with	O
-helix	O
-14	O
-(	O
-415CAAA418	O
-)	O
-of	O
-the	O
-18S	O
-rRNA	O
-.	O
-	O
-This	O
-stabilization	O
-renders	O
-the	O
-GTPase	O
-center	O
-to	O
-adopt	O
-a	O
-GTP	O
--	O
-bound	O
-conformation	O
-,	O
-similar	O
-to	O
-those	O
-observed	O
-in	O
-other	O
-translational	O
-GTPases	O
-in	O
-the	O
-presence	O
-of	O
-GTP	O
-analogs	O
-and	O
-in	O
-the	O
-80S	O
-•	O
-eEF2	O
-complex	O
-bound	O
-with	O
-a	O
-transition	O
--	O
-state	O
-mimic	O
-GDP	O
-•	O
-AlF4	O
-–.	O
-The	O
-switch	O
-loop	O
-contacts	O
-the	O
-base	O
-of	O
-A416	O
-(	O
-invariable	O
-A344	O
-in	O
-E	O
-.	O
-coli	O
-and	O
-A463	O
-in	O
-H	O
-.	O
-sapiens	O
-).	O
-	O
-Mutations	O
-of	O
-residues	O
-flanking	O
-A344	O
-in	O
-E	O
-.	O
-coli	O
-16S	O
-rRNA	O
-modestly	O
-inhibit	O
-translation	O
-but	O
-do	O
-not	O
-specifically	O
-affect	O
-EF	O
--	O
-G	O
--	O
-mediated	O
-translocation	O
-.	O
-	O
-However	O
-,	O
-the	O
-effect	O
-of	O
-A344	O
-mutation	O
-on	O
-translation	O
-was	O
-not	O
-addressed	O
-in	O
-that	O
-study	O
-,	O
-leaving	O
-the	O
-question	O
-open	O
-whether	O
-this	O
-residue	O
-is	O
-critical	O
-for	O
-eEF2	O
-/	O
-EF	O
--	O
-G	O
-function	O
-.	O
-	O
-The	O
-interaction	O
-between	O
-h14	O
-and	O
-switch	O
-loop	O
-I	O
-is	O
-not	O
-resolved	O
-in	O
-Structures	O
-II	O
-to	O
-V	O
-,	O
-in	O
-all	O
-of	O
-which	O
-the	O
-small	O
-subunit	O
-is	O
-partially	O
-rotated	O
-or	O
-non	O
--	O
-rotated	O
-,	O
-so	O
-that	O
-helix	O
-14	O
-is	O
-placed	O
-at	O
-least	O
-6	O
-Å	O
-farther	O
-from	O
-eEF2	O
-(	O
-Figure	O
-5d	O
-).	O
-	O
-We	O
-conclude	O
-that	O
-unlike	O
-other	O
-conformations	O
-of	O
-the	O
-ribosome	O
-,	O
-the	O
-fully	O
-rotated	O
-40S	O
-subunit	O
-of	O
-the	O
-pre	O
--	O
-translocation	O
-ribosome	O
-provides	O
-an	O
-interaction	O
-surface	O
-,	O
-complementing	O
-the	O
-P	O
-stalk	O
-and	O
-SRL	O
-,	O
-for	O
-binding	O
-of	O
-the	O
-GTP	O
--	O
-bound	O
-translocase	O
-.	O
-	O
-This	O
-structural	O
-basis	O
-rationalizes	O
-the	O
-observation	O
-of	O
-transient	O
-stabilization	O
-of	O
-the	O
-rotated	O
-70S	O
-ribosome	O
-upon	O
-EF	O
--	O
-G	O
-•	O
-GTP	O
-binding	O
-and	O
-prior	O
-to	O
-translocation	O
-.	O
-	O
-The	O
-least	O
-rotated	O
-conformation	O
-of	O
-the	O
-post	O
--	O
-translocation	O
-Structure	O
-V	O
-suggests	O
-conformational	O
-changes	O
-that	O
-may	O
-trigger	O
-eEF2	O
-release	O
-from	O
-the	O
-ribosome	O
-at	O
-the	O
-end	O
-of	O
-translocation	O
-.	O
-	O
-The	O
-most	O
-pronounced	O
-inter	O
--	O
-domain	O
-rearrangement	O
-in	O
-eEF2	O
-involves	O
-movement	O
-of	O
-domain	O
-III	O
-.	O
-	O
-In	O
-the	O
-rotated	O
-or	O
-mid	O
--	O
-rotated	O
-Structures	O
-I	O
-through	O
-III	O
-,	O
-this	O
-domain	O
-remains	O
-rigidly	O
-associated	O
-with	O
-domain	O
-V	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-superdomain	O
-and	O
-does	O
-not	O
-undergo	O
-noticeable	O
-rearrangements	O
-.	O
-	O
-In	O
-Structure	O
-V	O
-,	O
-however	O
-,	O
-the	O
-tip	O
-of	O
-helix	O
-A	O
-of	O
-domain	O
-III	O
-is	O
-displaced	O
-toward	O
-domain	O
-I	O
-by	O
-~	O
-5	O
-Å	O
-relative	O
-to	O
-that	O
-in	O
-mid	O
--	O
-rotated	O
-or	O
-fully	O
-rotated	O
-structures	O
-.	O
-	O
-This	O
-displacement	O
-is	O
-caused	O
-by	O
-the	O
-8	O
-Å	O
-movement	O
-of	O
-the	O
-40S	O
-body	O
-protein	O
-uS12	O
-upon	O
-reverse	O
-intersubunit	O
-rotation	O
-from	O
-Structure	O
-I	O
-to	O
-V	O
-(	O
-Figure	O
-6d	O
-).	O
-	O
-We	O
-propose	O
-that	O
-the	O
-shift	O
-of	O
-domain	O
-III	O
-by	O
-uS12	O
-initiates	O
-intra	O
--	O
-domain	O
-rearrangements	O
-in	O
-eEF2	O
-,	O
-which	O
-unstack	O
-the	O
-β	O
--	O
-platform	O
-of	O
-domain	O
-III	O
-from	O
-that	O
-of	O
-domain	O
-V	O
-.	O
-This	O
-would	O
-result	O
-in	O
-a	O
-conformation	O
-characteristic	O
-of	O
-free	O
-eEF2	O
-and	O
-EF	O
--	O
-G	O
-in	O
-which	O
-the	O
-β	O
--	O
-platforms	O
-are	O
-nearly	O
-perpendicular	O
-.	O
-	O
-As	O
-we	O
-discuss	O
-below	O
-,	O
-Structure	O
-V	O
-captures	O
-a	O
-'	O
-pre	O
--	O
-unstacking	O
-'	O
-state	O
-due	O
-to	O
-stabilization	O
-of	O
-the	O
-interface	O
-between	O
-domains	O
-III	O
-and	O
-V	O
-by	O
-sordarin	O
-.	O
-	O
-Sordarin	O
-stabilizes	O
-GDP	O
--	O
-bound	O
-eEF2	O
-on	O
-the	O
-ribosome	O
-	O
-Sordarin	O
-is	O
-a	O
-potent	O
-antifungal	O
-antibiotic	O
-that	O
-inhibits	O
-translation	O
-.	O
-	O
-Based	O
-on	O
-biochemical	O
-experiments	O
-,	O
-two	O
-alternative	O
-mechanisms	O
-of	O
-action	O
-were	O
-proposed	O
-:	O
-sordarin	O
-either	O
-prevents	O
-eEF2	O
-departure	O
-by	O
-inhibiting	O
-GTP	O
-hydrolysis	O
-or	O
-acts	O
-after	O
-GTP	O
-hydrolysis	O
-.	O
-	O
-Although	O
-our	O
-complex	O
-was	O
-assembled	O
-using	O
-eEF2	O
-•	O
-GTP	O
-,	O
-density	O
-maps	O
-clearly	O
-show	O
-GDP	O
-and	O
-Mg2	O
-+	O
-in	O
-each	O
-structure	O
-(	O
-Figure	O
-5g	O
-).	O
-	O
-Our	O
-structures	O
-therefore	O
-indicate	O
-that	O
-sordarin	O
-stalls	O
-eEF2	O
-on	O
-the	O
-ribosome	O
-in	O
-the	O
-GDP	O
--	O
-bound	O
-form	O
-,	O
-i	O
-.	O
-e	O
-.	O
-following	O
-GTP	O
-hydrolysis	O
-and	O
-phosphate	O
-release	O
-.	O
-	O
-The	O
-mechanism	O
-of	O
-stalling	O
-is	O
-suggested	O
-by	O
-comparison	O
-of	O
-pre	O
--	O
-translocation	O
-and	O
-post	O
--	O
-translocation	O
-structures	O
-in	O
-our	O
-ensemble	O
-.	O
-	O
-In	O
-all	O
-five	O
-structures	O
-,	O
-sordarin	O
-is	O
-bound	O
-between	O
-domains	O
-III	O
-and	O
-V	O
-of	O
-eEF2	O
-,	O
-stabilized	O
-by	O
-hydrophobic	O
-interactions	O
-identical	O
-to	O
-those	O
-in	O
-the	O
-isolated	O
-eEF2	O
-•	O
-sordarin	O
-complex	O
-(	O
-Figures	O
-5g	O
-and	O
-h	O
-).	O
-	O
-In	O
-the	O
-nearly	O
-non	O
--	O
-rotated	O
-post	O
--	O
-translocation	O
-Structure	O
-V	O
-,	O
-the	O
-tip	O
-of	O
-domain	O
-III	O
-is	O
-shifted	O
-,	O
-however	O
-the	O
-interface	O
-between	O
-domains	O
-III	O
-and	O
-V	O
-remains	O
-unchanged	O
-,	O
-suggesting	O
-strong	O
-stabilization	O
-of	O
-this	O
-interface	O
-by	O
-sordarin	O
-.	O
-	O
-We	O
-note	O
-that	O
-Structure	O
-V	O
-is	O
-slightly	O
-more	O
-rotated	O
-than	O
-the	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-in	O
-the	O
-absence	O
-of	O
-eEF2	O
-•	O
-sordarin	O
-,	O
-implying	O
-that	O
-sordarin	O
-interferes	O
-with	O
-the	O
-final	O
-stages	O
-of	O
-reverse	O
-rotation	O
-of	O
-the	O
-post	O
--	O
-translocation	O
-ribosome	O
-.	O
-	O
-We	O
-propose	O
-that	O
-sordarin	O
-acts	O
-to	O
-prevent	O
-full	O
-reverse	O
-rotation	O
-and	O
-release	O
-of	O
-eEF2	O
-•	O
-GDP	O
-by	O
-stabilizing	O
-the	O
-interdomain	O
-interface	O
-and	O
-thus	O
-blocking	O
-uS12	O
--	O
-induced	O
-disengagement	O
-of	O
-domain	O
-III	O
-from	O
-domain	O
-V	O
-.	O
-	O
-Implications	O
-for	O
-tRNA	O
-and	O
-mRNA	O
-translocation	O
-during	O
-translation	O
-	O
-Because	O
-translocation	O
-of	O
-tRNA	O
-must	O
-involve	O
-large	O
--	O
-scale	O
-dynamics	O
-,	O
-this	O
-step	O
-has	O
-long	O
-been	O
-regarded	O
-as	O
-the	O
-most	O
-puzzling	O
-step	O
-of	O
-translation	O
-.	O
-	O
-Intersubunit	O
-rearrangements	O
-and	O
-tRNA	O
-hybrid	O
-states	O
-have	O
-been	O
-proposed	O
-to	O
-play	O
-key	O
-roles	O
-half	O
-a	O
-century	O
-ago	O
-.	O
-	O
-Despite	O
-an	O
-impressive	O
-body	O
-of	O
-biochemical	O
-,	O
-fluorescence	O
-and	O
-structural	O
-data	O
-accumulated	O
-since	O
-then	O
-,	O
-translocation	O
-remains	O
-the	O
-least	O
-understood	O
-step	O
-of	O
-elongation	O
-.	O
-	O
-The	O
-structural	O
-understanding	O
-of	O
-ribosome	O
-and	O
-tRNA	O
-dynamics	O
-has	O
-been	O
-greatly	O
-aided	O
-by	O
-a	O
-wealth	O
-of	O
-X	O
--	O
-ray	O
-and	O
-cryo	O
--	O
-EM	O
-structures	O
-(	O
-reviewed	O
-in	O
-).	O
-	O
-However	O
-,	O
-visualization	O
-of	O
-the	O
-eEF2	O
-/	O
-EF	O
--	O
-G	O
--	O
-induced	O
-translocation	O
-is	O
-confined	O
-to	O
-very	O
-early	O
-pre	O
--	O
-EF	O
--	O
-G	O
--	O
-entry	O
-states	O
-and	O
-late	O
-(	O
-almost	O
-translocated	O
-or	O
-fully	O
-translocated	O
-)	O
-states	O
-,	O
-leaving	O
-most	O
-of	O
-the	O
-path	O
-from	O
-the	O
-A	O
-to	O
-the	O
-P	O
-site	O
-uncharacterized	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1	O
-).	O
-	O
-Our	O
-study	O
-provides	O
-new	O
-insights	O
-into	O
-the	O
-structural	O
-understanding	O
-of	O
-tRNA	O
-translocation	O
-.	O
-	O
-First	O
-,	O
-we	O
-propose	O
-that	O
-tRNA	O
-and	O
-IRES	O
-translocations	O
-occur	O
-via	O
-the	O
-same	O
-general	O
-trajectory	O
-.	O
-	O
-This	O
-is	O
-evident	O
-from	O
-the	O
-fact	O
-that	O
-ribosome	O
-rearrangements	O
-in	O
-translocation	O
-are	O
-inherent	O
-to	O
-the	O
-ribosome	O
-and	O
-likely	O
-occur	O
-in	O
-similar	O
-ways	O
-in	O
-both	O
-cases	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-step	O
--	O
-wise	O
-coupling	O
-of	O
-ribosome	O
-dynamics	O
-with	O
-IRES	O
-translocation	O
-is	O
-overall	O
-consistent	O
-with	O
-that	O
-observed	O
-for	O
-2tRNA	O
-•	O
-mRNA	O
-translocation	O
-in	O
-solution	O
-.	O
-	O
-For	O
-example	O
-,	O
-fluorescence	O
-and	O
-biochemical	O
-studies	O
-revealed	O
-that	O
-the	O
-early	O
-pre	O
--	O
-translocation	O
-EF	O
--	O
-G	O
--	O
-bound	O
-ribosomes	O
-are	O
-fully	O
-rotated	O
-and	O
-translocation	O
-of	O
-the	O
-tRNA	O
--	O
-mRNA	O
-complex	O
-occurs	O
-during	O
-reverse	O
-rotation	O
-of	O
-the	O
-small	O
-subunit	O
-,	O
-coupled	O
-with	O
-head	O
-swivel	O
-.	O
-	O
-The	O
-sequence	O
-of	O
-ribosome	O
-rearrangements	O
-during	O
-IRES	O
-translocation	O
-also	O
-agrees	O
-with	O
-that	O
-inferred	O
-from	O
-70S	O
-•	O
-EF	O
--	O
-G	O
-structures	O
-,	O
-including	O
-those	O
-in	O
-which	O
-the	O
-A	O
--	O
-to	O
--	O
-P	O
--	O
-site	O
-translocating	O
-tRNA	O
-was	O
-not	O
-present	O
-.	O
-	O
-Specifically	O
-,	O
-an	O
-earlier	O
-translocation	O
-intermediate	O
-ribosome	O
-(	O
-TIpre	O
-)	O
-was	O
-proposed	O
-to	O
-adopt	O
-a	O
-rotated	O
-(	O
-7	O
-–	O
-9	O
-°)	O
-body	O
-and	O
-a	O
-partly	O
-rotated	O
-head	O
-(	O
-5	O
-–	O
-7	O
-.	O
-5	O
-°),	O
-in	O
-agreement	O
-with	O
-the	O
-conformation	O
-of	O
-our	O
-Structure	O
-I	O
-.	O
-The	O
-most	O
-swiveled	O
-head	O
-(	O
-18	O
-–	O
-21	O
-°)	O
-was	O
-observed	O
-in	O
-a	O
-mid	O
--	O
-rotated	O
-ribosome	O
-(	O
-3	O
-–	O
-5	O
-°)	O
-of	O
-a	O
-later	O
-translocation	O
-intermediate	O
-TIpost	O
-,	O
-similar	O
-to	O
-the	O
-conformation	O
-of	O
-our	O
-Structure	O
-III	O
-.	O
-	O
-Overall	O
-,	O
-these	O
-correlations	O
-suggest	O
-that	O
-the	O
-intermediate	O
-locations	O
-of	O
-the	O
-elusive	O
-A	O
--	O
-to	O
--	O
-P	O
--	O
-site	O
-translocating	O
-tRNA	O
-are	O
-similar	O
-to	O
-those	O
-of	O
-PKI	O
-in	O
-our	O
-structures	O
-.	O
-	O
-Second	O
-,	O
-the	O
-structures	O
-clarify	O
-the	O
-structural	O
-basis	O
-of	O
-the	O
-often	O
--	O
-used	O
-but	O
-structurally	O
-undefined	O
-terms	O
-'	O
-locking	O
-'	O
-and	O
-'	O
-unlocking	O
-'	O
-with	O
-respect	O
-to	O
-the	O
-pre	O
--	O
-translocation	O
-complex	O
-(	O
-Figure	O
-6f	O
-).	O
-	O
-We	O
-deem	O
-the	O
-pre	O
--	O
-translocation	O
-complex	O
-locked	O
-,	O
-because	O
-the	O
-A	O
--	O
-site	O
-bound	O
-ASL	O
--	O
-mRNA	O
-is	O
-stabilized	O
-by	O
-interactions	O
-with	O
-the	O
-decoding	O
-center	O
-.	O
-	O
-These	O
-interactions	O
-are	O
-maintained	O
-for	O
-the	O
-classical	O
--	O
-and	O
-hybrid	O
--	O
-state	O
-tRNAs	O
-in	O
-the	O
-spontaneously	O
-sampled	O
-non	O
--	O
-rotated	O
-and	O
-rotated	O
-ribosomes	O
-,	O
-respectively	O
-.	O
-	O
-Unlocking	O
-involves	O
-separation	O
-of	O
-the	O
-codon	O
--	O
-anticodon	O
-helix	O
-from	O
-the	O
-decoding	O
-center	O
-residues	O
-by	O
-the	O
-protruding	O
-tip	O
-of	O
-eEF2	O
-/	O
-EF	O
--	O
-G	O
-(	O
-Figure	O
-7	O
-),	O
-occurring	O
-in	O
-the	O
-fully	O
-rotated	O
-ribosome	O
-at	O
-an	O
-early	O
-pre	O
--	O
-translocation	O
-step	O
-.	O
-	O
-This	O
-unlatches	O
-the	O
-head	O
-,	O
-allowing	O
-creation	O
-of	O
-hitherto	O
-elusive	O
-intermediate	O
-tRNA	O
-positions	O
-during	O
-spontaneous	O
-reverse	O
-body	O
-rotation	O
-.	O
-	O
-Third	O
-,	O
-our	O
-findings	O
-uncover	O
-a	O
-new	O
-role	O
-of	O
-the	O
-head	O
-swivel	O
-.	O
-	O
-Previous	O
-studies	O
-showed	O
-that	O
-this	O
-movement	O
-widens	O
-the	O
-constriction	O
-('	O
-gate	O
-')	O
-between	O
-the	O
-P	O
-and	O
-E	O
-sites	O
-,	O
-thus	O
-allowing	O
-the	O
-P	O
--	O
-tRNA	O
-passage	O
-to	O
-the	O
-E	O
-site	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-'	O
-gate	O
--	O
-opening	O
-'	O
-role	O
-,	O
-we	O
-now	O
-show	O
-that	O
-the	O
-head	O
-swivel	O
-brings	O
-the	O
-head	O
-A	O
-site	O
-to	O
-the	O
-body	O
-P	O
-site	O
-,	O
-allowing	O
-a	O
-step	O
--	O
-wise	O
-conveying	O
-of	O
-the	O
-codon	O
--	O
-anticodon	O
-helix	O
-between	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-.	O
-	O
-Finally	O
-,	O
-the	O
-similar	O
-populations	O
-of	O
-particles	O
-(	O
-within	O
-a	O
-2X	O
-range	O
-)	O
-in	O
-our	O
-80S	O
-•	O
-IRES	O
-•	O
-eEF2	O
-reconstructions	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2	O
-)	O
-suggest	O
-that	O
-the	O
-intermediate	O
-translocation	O
-states	O
-sample	O
-several	O
-energetically	O
-similar	O
-and	O
-interconverting	O
-conformations	O
-.	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-the	O
-idea	O
-of	O
-a	O
-rather	O
-flat	O
-energy	O
-landscape	O
-of	O
-translocation	O
-,	O
-suggested	O
-by	O
-recent	O
-work	O
-that	O
-measured	O
-mechanical	O
-work	O
-produced	O
-by	O
-the	O
-ribosome	O
-during	O
-translocation	O
-.	O
-	O
-Our	O
-findings	O
-implicate	O
-,	O
-however	O
-,	O
-that	O
-the	O
-energy	O
-landscape	O
-is	O
-not	O
-completely	O
-flat	O
-and	O
-contains	O
-local	O
-minima	O
-for	O
-transient	O
-positions	O
-of	O
-the	O
-codon	O
--	O
-anticodon	O
-helix	O
-between	O
-the	O
-A	O
-and	O
-P	O
-sites	O
-.	O
-	O
-The	O
-shift	O
-of	O
-the	O
-PKI	O
-with	O
-respect	O
-to	O
-the	O
-body	O
-occurs	O
-during	O
-forward	O
-head	O
-swivel	O
-in	O
-two	O
-major	O
-sub	O
--	O
-steps	O
-of	O
-~	O
-4	O
-Å	O
-each	O
-(	O
-initiation	O
-complex	O
-to	O
-I	O
-,	O
-and	O
-I	O
-to	O
-II	O
-),	O
-after	O
-which	O
-PKI	O
-undergoes	O
-small	O
-shifts	O
-to	O
-settle	O
-in	O
-the	O
-body	O
-P	O
-site	O
-in	O
-Structures	O
-III	O
-,	O
-IV	O
-and	O
-V	O
-(	O
-Figure	O
-2	O
-—	O
-source	O
-data	O
-1	O
-).	O
-	O
-Movement	O
-of	O
-PKI	O
-relative	O
-to	O
-the	O
-head	O
-occurs	O
-during	O
-the	O
-subsequent	O
-reverse	O
-swivel	O
-in	O
-three	O
-3	O
-–	O
-7	O
-Å	O
-sub	O
--	O
-steps	O
-(	O
-II	O
-to	O
-III	O
-to	O
-IV	O
-to	O
-V	O
-).	O
-	O
-We	O
-note	O
-that	O
-four	O
-of	O
-our	O
-near	O
--	O
-atomic	O
-resolution	O
-maps	O
-comprised	O
-~	O
-30	O
-,	O
-000	O
-particles	O
-each	O
-,	O
-the	O
-minimum	O
-number	O
-required	O
-for	O
-a	O
-near	O
--	O
-atomic	O
--	O
-resolution	O
-reconstruction	O
-of	O
-the	O
-ribosome	O
-.	O
-	O
-Translation	O
-of	O
-viral	O
-mRNA	O
-	O
-Our	O
-work	O
-sheds	O
-light	O
-on	O
-the	O
-dynamic	O
-mechanism	O
-of	O
-cap	O
--	O
-independent	O
-translation	O
-by	O
-IGR	O
-IRESs	O
-,	O
-tightly	O
-coupled	O
-with	O
-the	O
-universally	O
-conserved	O
-dynamic	O
-properties	O
-of	O
-the	O
-ribosome	O
-.	O
-	O
-The	O
-cryo	O
--	O
-EM	O
-structures	O
-demonstrate	O
-that	O
-the	O
-TSV	O
-IRES	O
-structurally	O
-and	O
-dynamically	O
-represents	O
-a	O
-chimera	O
-of	O
-the	O
-2tRNA	O
-•	O
-mRNA	O
-translocating	O
-complex	O
-(	O
-A	O
-/	O
-P	O
--	O
-tRNA	O
-•	O
-P	O
-/	O
-E	O
--	O
-tRNA	O
-•	O
-mRNA	O
-).	O
-	O
-Like	O
-in	O
-the	O
-2tRNA	O
-•	O
-mRNA	O
-translocating	O
-complex	O
-in	O
-which	O
-the	O
-two	O
-tRNAs	O
-move	O
-independently	O
-of	O
-each	O
-other	O
-,	O
-the	O
-PKI	O
-domain	O
-moves	O
-relative	O
-to	O
-the	O
-5	O
-´-	O
-domain	O
-,	O
-causing	O
-the	O
-IRES	O
-to	O
-undergo	O
-an	O
-inchworm	O
--	O
-walk	O
-translocation	O
-.	O
-	O
-A	O
-large	O
-structural	O
-difference	O
-between	O
-the	O
-IRES	O
-and	O
-the	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-exists	O
-,	O
-however	O
-,	O
-in	O
-that	O
-the	O
-IRES	O
-lacks	O
-three	O
-out	O
-of	O
-six	O
-tRNA	O
--	O
-like	O
-domains	O
-involved	O
-in	O
-tRNA	O
-translocation	O
-.	O
-	O
-This	O
-difference	O
-likely	O
-accounts	O
-for	O
-the	O
-inefficient	O
-translocation	O
-of	O
-the	O
-IRES	O
-,	O
-which	O
-is	O
-difficult	O
-to	O
-stabilize	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-state	O
-and	O
-therefore	O
-is	O
-prone	O
-to	O
-reverse	O
-translocation	O
-.	O
-	O
-Although	O
-structurally	O
-handicapped	O
-,	O
-the	O
-TSV	O
-IRES	O
-manages	O
-to	O
-translocate	O
-by	O
-employing	O
-ribosome	O
-dynamics	O
-that	O
-are	O
-remarkably	O
-similar	O
-to	O
-that	O
-in	O
-2tRNA	O
-•	O
-mRNA	O
-translocation	O
-.	O
-	O
-The	O
-uniformity	O
-of	O
-ribosome	O
-dynamics	O
-underscores	O
-the	O
-idea	O
-that	O
-translocation	O
-is	O
-an	O
-inherent	O
-and	O
-structurally	O
--	O
-optimized	O
-property	O
-of	O
-the	O
-ribosome	O
-,	O
-supported	O
-also	O
-by	O
-translocation	O
-activity	O
-in	O
-the	O
-absence	O
-of	O
-the	O
-elongation	O
-factor	O
-.	O
-	O
-This	O
-property	O
-is	O
-rendered	O
-by	O
-the	O
-relative	O
-mobility	O
-of	O
-the	O
-three	O
-major	O
-building	O
-blocks	O
-,	O
-the	O
-60S	O
-subunit	O
-and	O
-the	O
-40S	O
-head	O
-and	O
-body	O
-,	O
-assisted	O
-by	O
-ligand	O
--	O
-interacting	O
-extensions	O
-including	O
-the	O
-L1	O
-stalk	O
-and	O
-the	O
-P	O
-stalk	O
-.	O
-	O
-Intergenic	O
-IRESs	O
-,	O
-in	O
-turn	O
-,	O
-represent	O
-a	O
-striking	O
-example	O
-of	O
-convergent	O
-molecular	O
-evolution	O
-.	O
-	O
-Viral	O
-mRNAs	O
-have	O
-evolved	O
-to	O
-adopt	O
-an	O
-atypical	O
-structure	O
-to	O
-employ	O
-the	O
-inherent	O
-ribosome	O
-dynamics	O
-,	O
-to	O
-be	O
-able	O
-to	O
-hijack	O
-the	O
-host	O
-translational	O
-machinery	O
-in	O
-a	O
-simple	O
-fashion	O
-.	O
-	O
-Ensemble	O
-cryo	O
--	O
-EM	O
-	O
-Our	O
-current	O
-understanding	O
-of	O
-macromolecular	O
-machines	O
-,	O
-such	O
-as	O
-the	O
-ribosome	O
-,	O
-is	O
-often	O
-limited	O
-by	O
-a	O
-gap	O
-between	O
-biophysical	O
-/	O
-biochemical	O
-studies	O
-and	O
-structural	O
-studies	O
-.	O
-	O
-For	O
-example	O
-,	O
-Förster	O
-resonance	O
-energy	O
-transfer	O
-can	O
-provide	O
-insight	O
-into	O
-the	O
-macromolecular	O
-dynamics	O
-of	O
-an	O
-assembly	O
-at	O
-the	O
-single	O
--	O
-molecule	O
-level	O
-but	O
-is	O
-limited	O
-to	O
-specifically	O
-labeled	O
-locations	O
-within	O
-the	O
-assembly	O
-.	O
-	O
-High	O
--	O
-resolution	O
-crystal	O
-structures	O
-,	O
-on	O
-the	O
-other	O
-hand	O
-,	O
-can	O
-provide	O
-static	O
-images	O
-of	O
-an	O
-assembly	O
-,	O
-and	O
-the	O
-structural	O
-dynamics	O
-can	O
-only	O
-be	O
-inferred	O
-by	O
-comparing	O
-structures	O
-that	O
-are	O
-usually	O
-obtained	O
-in	O
-different	O
-experiments	O
-and	O
-under	O
-different	O
-,	O
-often	O
-non	O
--	O
-native	O
-,	O
-conditions	O
-.	O
-	O
-Cryo	O
--	O
-EM	O
-offers	O
-the	O
-possibility	O
-of	O
-obtaining	O
-integrated	O
-information	O
-of	O
-both	O
-structure	O
-and	O
-dynamics	O
-as	O
-demonstrated	O
-in	O
-lower	O
--	O
-resolution	O
-studies	O
-of	O
-bacterial	O
-ribosome	O
-complexes	O
-.	O
-	O
-This	O
-is	O
-presumably	O
-one	O
-of	O
-the	O
-reasons	O
-why	O
-most	O
-recent	O
-studies	O
-of	O
-ribosome	O
-complexes	O
-have	O
-focused	O
-on	O
-a	O
-single	O
-high	O
--	O
-resolution	O
-structure	O
-despite	O
-the	O
-non	O
--	O
-uniform	O
-local	O
-resolution	O
-of	O
-the	O
-maps	O
-that	O
-likely	O
-reflects	O
-structural	O
-heterogeneity	O
-.	O
-	O
-The	O
-computational	O
-efficiency	O
-of	O
-FREALIGN	O
-has	O
-allowed	O
-us	O
-to	O
-classify	O
-a	O
-relatively	O
-large	O
-dataset	O
-(	O
-1	O
-.	O
-1	O
-million	O
-particles	O
-)	O
-into	O
-15	O
-classes	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-2	O
-)	O
-and	O
-obtain	O
-eight	O
-near	O
--	O
-atomic	O
--	O
-resolution	O
-structures	O
-from	O
-it	O
-.	O
-	O
-The	O
-classification	O
-,	O
-which	O
-followed	O
-an	O
-initial	O
-alignment	O
-of	O
-all	O
-particles	O
-to	O
-a	O
-single	O
-reference	O
-,	O
-required	O
-about	O
-130	O
-,	O
-000	O
-CPU	O
-hours	O
-or	O
-about	O
-five	O
-to	O
-six	O
-full	O
-days	O
-on	O
-a	O
-1000	O
--	O
-CPU	O
-cluster	O
-.	O
-	O
-Therefore	O
-,	O
-cryo	O
--	O
-EM	O
-has	O
-the	O
-potential	O
-to	O
-become	O
-a	O
-standard	O
-tool	O
-for	O
-uncovering	O
-detailed	O
-dynamic	O
-pathways	O
-of	O
-complex	O
-macromolecular	O
-machines	O
-.	O
-	O
-A	O
-unified	O
-mechanism	O
-for	O
-proteolysis	O
-and	O
-autocatalytic	O
-activation	O
-in	O
-the	O
-20S	O
-proteasome	O
-	O
-Biogenesis	O
-of	O
-the	O
-20S	O
-proteasome	O
-is	O
-tightly	O
-regulated	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-propeptides	O
-protecting	O
-the	O
-active	O
--	O
-site	O
-threonines	O
-are	O
-autocatalytically	O
-released	O
-only	O
-on	O
-completion	O
-of	O
-assembly	O
-.	O
-	O
-However	O
-,	O
-the	O
-trigger	O
-for	O
-the	O
-self	O
--	O
-activation	O
-and	O
-the	O
-reason	O
-for	O
-the	O
-strict	O
-conservation	O
-of	O
-threonine	O
-as	O
-the	O
-active	O
-site	O
-nucleophile	O
-remain	O
-enigmatic	O
-.	O
-	O
-Here	O
-we	O
-use	O
-mutagenesis	O
-,	O
-X	O
--	O
-ray	O
-crystallography	O
-and	O
-biochemical	O
-assays	O
-to	O
-suggest	O
-that	O
-Lys33	O
-initiates	O
-nucleophilic	O
-attack	O
-of	O
-the	O
-propeptide	O
-by	O
-deprotonating	O
-the	O
-Thr1	O
-hydroxyl	O
-group	O
-and	O
-that	O
-both	O
-residues	O
-together	O
-with	O
-Asp17	O
-are	O
-part	O
-of	O
-a	O
-catalytic	O
-triad	O
-.	O
-	O
-Substitution	O
-of	O
-Thr1	O
-by	O
-Cys	O
-disrupts	O
-the	O
-interaction	O
-with	O
-Lys33	O
-and	O
-inactivates	O
-the	O
-proteasome	O
-.	O
-	O
-Although	O
-a	O
-Thr1Ser	B-mutant
-mutant	O
-is	O
-active	O
-,	O
-it	O
-is	O
-less	O
-efficient	O
-compared	O
-with	O
-wild	O
-type	O
-because	O
-of	O
-the	O
-unfavourable	O
-orientation	O
-of	O
-Ser1	O
-towards	O
-incoming	O
-substrates	O
-.	O
-	O
-This	O
-work	O
-provides	O
-insights	O
-into	O
-the	O
-basic	O
-mechanism	O
-of	O
-proteolysis	O
-and	O
-propeptide	O
-autolysis	O
-,	O
-as	O
-well	O
-as	O
-the	O
-evolutionary	O
-pressures	O
-that	O
-drove	O
-the	O
-proteasome	O
-to	O
-become	O
-a	O
-threonine	O
-protease	O
-.	O
-	O
-The	O
-proteasome	O
-,	O
-an	O
-essential	O
-molecular	O
-machine	O
-,	O
-is	O
-a	O
-threonine	O
-protease	O
-,	O
-but	O
-the	O
-evolution	O
-and	O
-the	O
-components	O
-of	O
-its	O
-proteolytic	O
-centre	O
-are	O
-unclear	O
-.	O
-	O
-Here	O
-,	O
-the	O
-authors	O
-use	O
-structural	O
-biology	O
-and	O
-biochemistry	O
-to	O
-investigate	O
-the	O
-role	O
-of	O
-proteasome	O
-active	O
-site	O
-residues	O
-on	O
-maturation	O
-and	O
-activity	O
-.	O
-	O
-The	O
-20S	O
-proteasome	O
-core	O
-particle	O
-(	O
-CP	O
-)	O
-is	O
-the	O
-key	O
-non	O
--	O
-lysosomal	O
-protease	O
-of	O
-eukaryotic	O
-cells	O
-.	O
-	O
-Its	O
-seven	O
-different	O
-α	O
-and	O
-seven	O
-different	O
-β	O
-subunits	O
-assemble	O
-into	O
-four	O
-heptameric	O
-rings	O
-that	O
-are	O
-stacked	O
-on	O
-each	O
-other	O
-to	O
-form	O
-a	O
-hollow	O
-cylinder	O
-.	O
-	O
-While	O
-the	O
-inactive	O
-α	O
-subunits	O
-build	O
-the	O
-two	O
-outer	O
-rings	O
-,	O
-the	O
-β	O
-subunits	O
-form	O
-the	O
-inner	O
-rings	O
-.	O
-	O
-Only	O
-three	O
-out	O
-of	O
-the	O
-seven	O
-different	O
-β	O
-subunits	O
-,	O
-namely	O
-β1	O
-,	O
-β2	O
-and	O
-β5	O
-,	O
-bear	O
-N	O
--	O
-terminal	O
-proteolytic	O
-active	O
-centres	O
-,	O
-and	O
-before	O
-CP	O
-maturation	O
-these	O
-are	O
-protected	O
-by	O
-propeptides	O
-.	O
-	O
-In	O
-the	O
-last	O
-stage	O
-of	O
-CP	O
-biogenesis	O
-,	O
-the	O
-prosegments	O
-are	O
-autocatalytically	O
-removed	O
-through	O
-nucleophilic	O
-attack	O
-by	O
-the	O
-active	O
-site	O
-residue	O
-Thr1	O
-on	O
-the	O
-preceding	O
-peptide	O
-bond	O
-involving	O
-Gly	O
-(-	O
-1	O
-).	O
-	O
-Release	O
-of	O
-the	O
-propeptides	O
-creates	O
-a	O
-functionally	O
-active	O
-CP	O
-that	O
-cleaves	O
-proteins	O
-into	O
-short	O
-peptides	O
-.	O
-	O
-Although	O
-the	O
-chemical	O
-nature	O
-of	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-and	O
-hence	O
-substrate	O
-preferences	O
-are	O
-unique	O
-to	O
-each	O
-of	O
-the	O
-distinct	O
-active	O
-β	O
-subunits	O
-,	O
-all	O
-active	O
-sites	O
-employ	O
-an	O
-identical	O
-reaction	O
-mechanism	O
-to	O
-hydrolyse	O
-peptide	O
-bonds	O
-.	O
-	O
-Nucleophilic	O
-attack	O
-of	O
-Thr1Oγ	O
-on	O
-the	O
-carbonyl	O
-carbon	O
-atom	O
-of	O
-the	O
-scissile	O
-peptide	O
-bond	O
-creates	O
-a	O
-first	O
-cleavage	O
-product	O
-and	O
-a	O
-covalent	O
-acyl	O
--	O
-enzyme	O
-intermediate	O
-.	O
-	O
-Hydrolysis	O
-of	O
-this	O
-complex	O
-by	O
-the	O
-addition	O
-of	O
-a	O
-nucleophilic	O
-water	O
-molecule	O
-regenerates	O
-the	O
-enzyme	O
-and	O
-releases	O
-the	O
-second	O
-peptide	O
-fragment	O
-.	O
-	O
-The	O
-proteasome	O
-belongs	O
-to	O
-the	O
-family	O
-of	O
-N	O
--	O
-terminal	O
-nucleophilic	O
-(	O
-Ntn	O
-)	O
-hydrolases	O
-,	O
-and	O
-the	O
-free	O
-N	O
--	O
-terminal	O
-amine	O
-group	O
-of	O
-Thr1	O
-was	O
-proposed	O
-to	O
-deprotonate	O
-the	O
-Thr1	O
-hydroxyl	O
-group	O
-to	O
-generate	O
-a	O
-nucleophilic	O
-Thr1Oγ	O
-for	O
-peptide	O
--	O
-bond	O
-cleavage	O
-.	O
-	O
-This	O
-mechanism	O
-,	O
-however	O
-,	O
-cannot	O
-explain	O
-autocatalytic	O
-precursor	O
-processing	O
-because	O
-in	O
-the	O
-immature	O
-active	O
-sites	O
-,	O
-Thr1N	O
-is	O
-part	O
-of	O
-the	O
-peptide	O
-bond	O
-with	O
-Gly	O
-(-	O
-1	O
-),	O
-the	O
-bond	O
-that	O
-needs	O
-to	O
-be	O
-hydrolysed	O
-.	O
-	O
-An	O
-alternative	O
-candidate	O
-for	O
-deprotonating	O
-the	O
-Thr1	O
-hydroxyl	O
-group	O
-is	O
-the	O
-side	O
-chain	O
-of	O
-Lys33	O
-as	O
-it	O
-is	O
-within	O
-hydrogen	O
--	O
-bonding	O
-distance	O
-to	O
-Thr1OH	O
-(	O
-2	O
-.	O
-7	O
-Å	O
-).	O
-	O
-In	O
-principle	O
-it	O
-could	O
-function	O
-as	O
-the	O
-general	O
-base	O
-during	O
-both	O
-autocatalytic	O
-removal	O
-of	O
-the	O
-propeptide	O
-and	O
-protein	O
-substrate	O
-cleavage	O
-.	O
-	O
-Here	O
-we	O
-provide	O
-experimental	O
-evidences	O
-for	O
-this	O
-distinct	O
-view	O
-of	O
-the	O
-proteasome	O
-active	O
--	O
-site	O
-mechanism	O
-.	O
-	O
-Data	O
-from	O
-biochemical	O
-and	O
-structural	O
-analyses	O
-of	O
-proteasome	O
-variants	O
-with	O
-mutations	O
-in	O
-the	O
-β5	O
-propeptide	O
-and	O
-the	O
-active	O
-site	O
-strongly	O
-support	O
-the	O
-model	O
-and	O
-deliver	O
-novel	O
-insights	O
-into	O
-the	O
-structural	O
-constraints	O
-required	O
-for	O
-the	O
-autocatalytic	O
-activation	O
-of	O
-the	O
-proteasome	O
-.	O
-	O
-Furthermore	O
-,	O
-we	O
-determine	O
-the	O
-advantages	O
-of	O
-Thr	O
-over	O
-Cys	O
-or	O
-Ser	O
-as	O
-the	O
-active	O
--	O
-site	O
-nucleophile	O
-using	O
-X	O
--	O
-ray	O
-crystallography	O
-together	O
-with	O
-activity	O
-and	O
-inhibition	O
-assays	O
-.	O
-	O
-Inactivation	O
-of	O
-proteasome	O
-subunits	O
-by	O
-T1A	B-mutant
-mutations	O
-	O
-Proteasome	O
--	O
-mediated	O
-degradation	O
-of	O
-cell	O
--	O
-cycle	O
-regulators	O
-and	O
-potentially	O
-toxic	O
-misfolded	O
-proteins	O
-is	O
-required	O
-for	O
-the	O
-viability	O
-of	O
-eukaryotic	O
-cells	O
-.	O
-	O
-Inactivation	O
-of	O
-the	O
-active	O
-site	O
-Thr1	O
-by	O
-mutation	O
-to	O
-Ala	O
-has	O
-been	O
-used	O
-to	O
-study	O
-substrate	O
-specificity	O
-and	O
-the	O
-hierarchy	O
-of	O
-the	O
-proteasome	O
-active	O
-sites	O
-.	O
-	O
-Yeast	O
-strains	O
-carrying	O
-the	O
-single	O
-mutations	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-or	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-,	O
-or	O
-both	O
-,	O
-are	O
-viable	O
-,	O
-even	O
-though	O
-one	O
-or	O
-two	O
-of	O
-the	O
-three	O
-distinct	O
-catalytic	O
-β	O
-subunits	O
-are	O
-disabled	O
-and	O
-carry	O
-remnants	O
-of	O
-their	O
-N	O
--	O
-terminal	O
-propeptides	O
-(	O
-Table	O
-1	O
-).	O
-	O
-These	O
-results	O
-indicate	O
-that	O
-the	O
-β1	O
-and	O
-β2	O
-proteolytic	O
-activities	O
-are	O
-not	O
-essential	O
-for	O
-cell	O
-survival	O
-.	O
-	O
-By	O
-contrast	O
-,	O
-the	O
-T1A	B-mutant
-mutation	O
-in	O
-subunit	O
-β5	O
-has	O
-been	O
-reported	O
-to	O
-be	O
-lethal	O
-or	O
-nearly	O
-so	O
-.	O
-	O
-Viability	O
-is	O
-restored	O
-if	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-subunit	O
-has	O
-its	O
-propeptide	O
-(	O
-pp	O
-)	O
-deleted	O
-but	O
-expressed	O
-separately	O
-in	O
-trans	O
-(	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-pp	O
-trans	O
-),	O
-although	O
-substantial	O
-phenotypic	O
-impairment	O
-remains	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Our	O
-present	O
-crystallographic	O
-analysis	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-pp	O
-trans	O
-mutant	O
-demonstrates	O
-that	O
-the	O
-mutation	O
-per	O
-se	O
-does	O
-not	O
-structurally	O
-alter	O
-the	O
-catalytic	O
-active	O
-site	O
-and	O
-that	O
-the	O
-trans	O
--	O
-expressed	O
-β5	O
-propeptide	O
-is	O
-not	O
-bound	O
-in	O
-the	O
-β5	O
-substrate	O
--	O
-binding	O
-channel	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1a	O
-).	O
-	O
-The	O
-extremely	O
-weak	O
-growth	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-pp	O
-cis	O
-described	O
-by	O
-Chen	O
-and	O
-Hochstrasser	O
-compared	O
-with	O
-the	O
-inviability	O
-reported	O
-by	O
-Heinemeyer	O
-et	O
-al	O
-.	O
-prompted	O
-us	O
-to	O
-analyse	O
-this	O
-discrepancy	O
-.	O
-	O
-Sequencing	O
-of	O
-the	O
-plasmids	O
-,	O
-testing	O
-them	O
-in	O
-both	O
-published	O
-yeast	O
-strain	O
-backgrounds	O
-and	O
-site	O
--	O
-directed	O
-mutagenesis	O
-revealed	O
-that	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-pp	O
-cis	O
-is	O
-viable	O
-,	O
-but	O
-suffers	O
-from	O
-a	O
-marked	O
-growth	O
-defect	O
-that	O
-requires	O
-extended	O
-incubation	O
-of	O
-4	O
-–	O
-5	O
-days	O
-for	O
-initial	O
-colony	O
-formation	O
-(	O
-Table	O
-1	O
-and	O
-Supplementary	O
-Methods	O
-).	O
-	O
-We	O
-also	O
-identified	O
-an	O
-additional	O
-point	O
-mutation	O
-K81R	B-mutant
-in	O
-subunit	O
-β5	O
-that	O
-was	O
-present	O
-in	O
-the	O
-allele	O
-used	O
-in	O
-ref	O
-..	O
-This	O
-single	O
-amino	O
--	O
-acid	O
-exchange	O
-is	O
-located	O
-at	O
-the	O
-interface	O
-of	O
-the	O
-subunits	O
-α4	O
-,	O
-β4	O
-and	O
-β5	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1b	O
-)	O
-and	O
-might	O
-weakly	O
-promote	O
-CP	O
-assembly	O
-by	O
-enhancing	O
-inter	O
--	O
-subunit	O
-contacts	O
-.	O
-	O
-The	O
-slightly	O
-better	O
-growth	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-mutant	O
-allowed	O
-us	O
-to	O
-solve	O
-the	O
-crystal	O
-structure	O
-of	O
-a	O
-yeast	O
-proteasome	O
-(	O
-yCP	O
-)	O
-with	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-mutation	O
-,	O
-which	O
-is	O
-discussed	O
-in	O
-the	O
-following	O
-section	O
-(	O
-for	O
-details	O
-see	O
-Supplementary	O
-Note	O
-1	O
-).	O
-	O
-Propeptide	O
-conformation	O
-and	O
-triggering	O
-of	O
-autolysis	O
-	O
-In	O
-the	O
-final	O
-steps	O
-of	O
-proteasome	O
-biogenesis	O
-,	O
-the	O
-propeptides	O
-are	O
-autocatalytically	O
-cleaved	O
-from	O
-the	O
-mature	O
-β	O
--	O
-subunit	O
-domains	O
-.	O
-	O
-For	O
-subunit	O
-β1	O
-,	O
-this	O
-process	O
-was	O
-previously	O
-inferred	O
-to	O
-require	O
-that	O
-the	O
-propeptide	O
-residue	O
-at	O
-position	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-subunit	O
-precursor	O
-occupies	O
-the	O
-S1	O
-specificity	O
-pocket	O
-of	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-formed	O
-by	O
-amino	O
-acid	O
-45	O
-(	O
-for	O
-details	O
-see	O
-Supplementary	O
-Note	O
-2	O
-).	O
-	O
-Furthermore	O
-,	O
-it	O
-was	O
-observed	O
-that	O
-the	O
-prosegment	O
-forms	O
-an	O
-antiparallel	O
-β	O
--	O
-sheet	O
-in	O
-the	O
-active	O
-site	O
-,	O
-and	O
-that	O
-Gly	O
-(-	O
-1	O
-)	O
-adopts	O
-a	O
-γ	O
--	O
-turn	O
-conformation	O
-,	O
-which	O
-by	O
-definition	O
-is	O
-characterized	O
-by	O
-a	O
-hydrogen	O
-bond	O
-between	O
-Leu	O
-(-	O
-2	O
-)	O
-O	O
-and	O
-Thr1NH	O
-(	O
-ref	O
-.).	O
-	O
-Here	O
-we	O
-again	O
-analysed	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-crystallographically	O
-but	O
-in	O
-addition	O
-determined	O
-the	O
-structures	O
-of	O
-the	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-single	O
-and	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-β2	I-mutant
--	I-mutant
-T1A	I-mutant
-double	O
-mutants	O
-(	O
-Protein	O
-Data	O
-Bank	O
-(	O
-PDB	O
-)	O
-entry	O
-codes	O
-are	O
-provided	O
-in	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-In	O
-subunit	O
-β1	O
-,	O
-we	O
-found	O
-that	O
-Gly	O
-(-	O
-1	O
-)	O
-indeed	O
-forms	O
-a	O
-sharp	O
-turn	O
-,	O
-which	O
-relaxes	O
-on	O
-prosegment	O
-cleavage	O
-(	O
-Fig	O
-.	O
-1a	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-However	O
-,	O
-the	O
-γ	O
--	O
-turn	O
-conformation	O
-and	O
-the	O
-associated	O
-hydrogen	O
-bond	O
-initially	O
-proposed	O
-is	O
-for	O
-geometric	O
-and	O
-chemical	O
-reasons	O
-inappropriate	O
-and	O
-would	O
-not	O
-perfectly	O
-position	O
-the	O
-carbonyl	O
-carbon	O
-atom	O
-of	O
-Gly	O
-(-	O
-1	O
-)	O
-for	O
-nucleophilic	O
-attack	O
-by	O
-Thr1	O
-.	O
-	O
-Regarding	O
-the	O
-β2	O
-propeptide	O
-,	O
-Thr	O
-(-	O
-2	O
-)	O
-occupies	O
-the	O
-S1	O
-pocket	O
-but	O
-is	O
-less	O
-deeply	O
-anchored	O
-compared	O
-with	O
-Leu	O
-(-	O
-2	O
-)	O
-in	O
-β1	O
-,	O
-which	O
-might	O
-be	O
-due	O
-to	O
-the	O
-rather	O
-large	O
-β2	O
--	O
-S1	O
-pocket	O
-created	O
-by	O
-Gly45	O
-.	O
-	O
-Thr	O
-(-	O
-2	O
-)	O
-positions	O
-Gly	O
-(-	O
-1	O
-)	O
-O	O
-via	O
-hydrogen	O
-bonding	O
-(∼	O
-2	O
-.	O
-8	O
-Å	O
-)	O
-in	O
-a	O
-perfect	O
-trajectory	O
-for	O
-the	O
-nucleophilic	O
-attack	O
-by	O
-Thr1Oγ	O
-(	O
-Fig	O
-.	O
-1b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-Next	O
-,	O
-we	O
-examined	O
-the	O
-position	O
-of	O
-the	O
-β5	O
-propeptide	O
-in	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-mutant	O
-.	O
-	O
-Surprisingly	O
-,	O
-Gly	O
-(-	O
-1	O
-)	O
-is	O
-completely	O
-extended	O
-and	O
-forces	O
-the	O
-histidine	O
-side	O
-chain	O
-at	O
-position	O
-(-	O
-2	O
-)	O
-to	O
-occupy	O
-the	O
-S2	O
-instead	O
-of	O
-the	O
-S1	O
-pocket	O
-,	O
-thereby	O
-disrupting	O
-the	O
-antiparallel	O
-β	O
--	O
-sheet	O
-.	O
-	O
-Nonetheless	O
-,	O
-the	O
-carbonyl	O
-carbon	O
-of	O
-Gly	O
-(-	O
-1	O
-)	O
-would	O
-be	O
-ideally	O
-placed	O
-for	O
-nucleophilic	O
-attack	O
-by	O
-Thr1Oγ	O
-(	O
-Fig	O
-.	O
-1c	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-2c	O
-,	O
-d	O
-).	O
-	O
-As	O
-the	O
-K81R	B-mutant
-mutation	O
-is	O
-located	O
-far	O
-from	O
-the	O
-active	O
-site	O
-(	O
-Thr1Cα	O
-–	O
-Arg81Cα	O
-:	O
-24	O
-Å	O
-),	O
-any	O
-influence	O
-on	O
-propeptide	O
-conformation	O
-can	O
-be	O
-excluded	O
-.	O
-	O
-Instead	O
-,	O
-the	O
-plasticity	O
-of	O
-the	O
-β5	O
-S1	O
-pocket	O
-caused	O
-by	O
-the	O
-rotational	O
-flexibility	O
-of	O
-Met45	O
-might	O
-prevent	O
-stable	O
-accommodation	O
-of	O
-His	O
-(-	O
-2	O
-)	O
-in	O
-the	O
-S1	O
-site	O
-and	O
-thus	O
-also	O
-promote	O
-its	O
-immediate	O
-release	O
-after	O
-autolysis	O
-.	O
-	O
-Processing	O
-of	O
-β	O
--	O
-subunit	O
-precursors	O
-requires	O
-deprotonation	O
-of	O
-Thr1OH	O
-;	O
-however	O
-,	O
-the	O
-general	O
-base	O
-initiating	O
-autolysis	O
-is	O
-unknown	O
-.	O
-	O
-Remarkably	O
-,	O
-eukaryotic	O
-proteasomal	O
-β5	O
-subunits	O
-bear	O
-a	O
-His	O
-residue	O
-in	O
-position	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-propeptide	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-As	O
-histidine	O
-commonly	O
-functions	O
-as	O
-a	O
-proton	O
-shuttle	O
-in	O
-the	O
-catalytic	O
-triads	O
-of	O
-serine	O
-proteases	O
-,	O
-we	O
-investigated	O
-the	O
-role	O
-of	O
-His	O
-(-	O
-2	O
-)	O
-in	O
-processing	O
-of	O
-the	O
-β5	O
-propeptide	O
-by	O
-exchanging	O
-it	O
-for	O
-Asn	O
-,	O
-Lys	O
-,	O
-Phe	O
-and	O
-Ala	O
-.	O
-All	O
-yeast	O
-mutants	O
-were	O
-viable	O
-at	O
-30	O
-°	O
-C	O
-,	O
-but	O
-suffered	O
-from	O
-growth	O
-defects	O
-at	O
-37	O
-°	O
-C	O
-with	O
-the	O
-H	B-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-N	I-mutant
-and	O
-H	B-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-F	I-mutant
-mutants	O
-being	O
-most	O
-affected	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3b	O
-and	O
-Table	O
-1	O
-).	O
-	O
-In	O
-agreement	O
-,	O
-the	O
-chymotrypsin	O
--	O
-like	O
-(	O
-ChT	O
--	O
-L	O
-)	O
-activity	O
-of	O
-H	B-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-N	I-mutant
-and	O
-H	B-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-F	I-mutant
-mutant	O
-yCPs	O
-was	O
-impaired	O
-in	O
-situ	O
-and	O
-in	O
-vitro	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3c	O
-).	O
-	O
-Structural	O
-analyses	O
-revealed	O
-that	O
-the	O
-propeptides	O
-of	O
-all	O
-mutant	O
-yCPs	O
-shared	O
-residual	O
-2FO	O
-–	O
-FC	O
-electron	O
-densities	O
-.	O
-	O
-Gly	O
-(-	O
-1	O
-)	O
-and	O
-Phe	O
-/	O
-Lys	O
-(-	O
-2	O
-)	O
-were	O
-visualized	O
-at	O
-low	O
-occupancy	O
-,	O
-while	O
-Ala	O
-/	O
-Asn	O
-(-	O
-2	O
-)	O
-could	O
-not	O
-be	O
-assigned	O
-.	O
-	O
-This	O
-observation	O
-indicates	O
-a	O
-mixture	O
-of	O
-processed	O
-and	O
-unprocessed	O
-β5	O
-subunits	O
-and	O
-partially	O
-impaired	O
-autolysis	O
-,	O
-thereby	O
-excluding	O
-any	O
-essential	O
-role	O
-of	O
-residue	O
-(-	O
-2	O
-)	O
-as	O
-the	O
-general	O
-base	O
-.	O
-	O
-Next	O
-,	O
-we	O
-examined	O
-the	O
-effect	O
-of	O
-residue	O
-(-	O
-2	O
-)	O
-on	O
-the	O
-orientation	O
-of	O
-the	O
-propeptide	O
-by	O
-creating	O
-mutants	O
-that	O
-combine	O
-the	O
-T1A	B-mutant
-(	O
-K81R	B-mutant
-)	O
-mutation	O
-(	O
-s	O
-)	O
-with	O
-H	B-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-L	I-mutant
-,	O
-H	B-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-T	I-mutant
-or	O
-H	B-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-A	I-mutant
-substitutions	O
-.	O
-	O
-Leu	O
-(-	O
-2	O
-)	O
-is	O
-encoded	O
-in	O
-the	O
-yeast	O
-β1	O
-subunit	O
-precursor	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-);	O
-Thr	O
-(-	O
-2	O
-)	O
-is	O
-generally	O
-part	O
-of	O
-β2	O
--	O
-propeptides	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-);	O
-and	O
-Ala	O
-(-	O
-2	O
-)	O
-was	O
-expected	O
-to	O
-fit	O
-the	O
-β5	O
--	O
-S1	O
-pocket	O
-without	O
-inducing	O
-conformational	O
-changes	O
-of	O
-Met45	O
-,	O
-allowing	O
-it	O
-to	O
-accommodate	O
-‘	O
-β1	O
--	O
-like	O
-'	O
-propeptide	O
-positioning	O
-.	O
-	O
-As	O
-expected	O
-from	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-mutants	O
-,	O
-the	O
-yeasts	O
-show	O
-severe	O
-growth	O
-phenotypes	O
-,	O
-with	O
-minor	O
-variations	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4a	O
-and	O
-Table	O
-1	O
-).	O
-	O
-We	O
-determined	O
-crystal	O
-structures	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-L	I-mutant
--	I-mutant
-T1A	I-mutant
-,	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-T	I-mutant
--	I-mutant
-T1A	I-mutant
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-A	I-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-mutants	O
-(	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-For	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-A	I-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-variant	O
-,	O
-only	O
-the	O
-residues	O
-Gly	O
-(-	O
-1	O
-)	O
-and	O
-Ala	O
-(-	O
-2	O
-)	O
-could	O
-be	O
-visualized	O
-,	O
-indicating	O
-that	O
-Ala	O
-(-	O
-2	O
-)	O
-leads	O
-to	O
-insufficient	O
-stabilization	O
-of	O
-the	O
-propeptide	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4d	O
-).	O
-	O
-By	O
-contrast	O
-,	O
-the	O
-prosegments	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-L	I-mutant
--	I-mutant
-T1A	I-mutant
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-T	I-mutant
--	I-mutant
-T1A	I-mutant
-mutants	O
-were	O
-significantly	O
-better	O
-resolved	O
-in	O
-the	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-maps	O
-yet	O
-not	O
-at	O
-full	O
-occupancy	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4b	O
-,	O
-c	O
-and	O
-Supplementary	O
-Table	O
-1	O
-),	O
-suggesting	O
-that	O
-the	O
-natural	O
-propeptide	O
-bearing	O
-His	O
-(-	O
-2	O
-)	O
-is	O
-most	O
-favourable	O
-.	O
-	O
-Nevertheless	O
-,	O
-both	O
-Leu	O
-(-	O
-2	O
-)	O
-and	O
-Thr	O
-(-	O
-2	O
-)	O
-were	O
-found	O
-to	O
-occupy	O
-the	O
-S1	O
-specificity	O
-pocket	O
-formed	O
-by	O
-Met45	O
-(	O
-Fig	O
-.	O
-2a	O
-,	O
-b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-4f	O
-–	O
-h	O
-).	O
-	O
-This	O
-result	O
-proves	O
-that	O
-the	O
-naturally	O
-occurring	O
-His	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-β5	O
-propeptide	O
-does	O
-not	O
-stably	O
-fit	O
-into	O
-the	O
-S1	O
-site	O
-.	O
-	O
-Since	O
-Gly	O
-(-	O
-1	O
-)	O
-adopts	O
-the	O
-same	O
-position	O
-in	O
-both	O
-wild	O
--	O
-type	O
-(	O
-WT	O
-)	O
-and	O
-mutant	O
-β5	O
-propeptides	O
-,	O
-and	O
-since	O
-in	O
-all	O
-cases	O
-its	O
-carbonyl	O
-carbon	O
-is	O
-perfectly	O
-placed	O
-for	O
-nucleophilic	O
-attack	O
-by	O
-Thr1Oγ	O
-(	O
-Fig	O
-.	O
-2b	O
-),	O
-we	O
-propose	O
-that	O
-neither	O
-binding	O
-of	O
-residue	O
-(-	O
-2	O
-)	O
-to	O
-the	O
-S1	O
-pocket	O
-nor	O
-formation	O
-of	O
-the	O
-antiparallel	O
-β	O
--	O
-sheet	O
-is	O
-essential	O
-for	O
-autolysis	O
-of	O
-the	O
-propeptide	O
-.	O
-	O
-Next	O
-,	O
-we	O
-determined	O
-the	O
-crystal	O
-structure	O
-of	O
-a	O
-chimeric	O
-yCP	O
-having	O
-the	O
-yeast	O
-β1	O
--	O
-propeptide	O
-replaced	O
-by	O
-its	O
-β5	O
-counterpart	O
-.	O
-	O
-Although	O
-we	O
-observed	O
-fragments	O
-of	O
-2FO	O
-–	O
-FC	O
-electron	O
-density	O
-in	O
-the	O
-β1	O
-active	O
-site	O
-,	O
-the	O
-data	O
-were	O
-not	O
-interpretable	O
-.	O
-	O
-Bearing	O
-in	O
-mind	O
-that	O
-in	O
-contrast	O
-to	O
-Thr	O
-(-	O
-2	O
-)	O
-in	O
-β2	O
-,	O
-Leu	O
-(-	O
-2	O
-)	O
-in	O
-subunit	O
-β1	O
-is	O
-not	O
-conserved	O
-among	O
-species	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-),	O
-we	O
-created	O
-a	O
-β2	B-mutant
--	I-mutant
-T	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-V	I-mutant
-proteasome	O
-mutant	O
-.	O
-	O
-As	O
-proven	O
-by	O
-the	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-crystal	O
-structures	O
-,	O
-Thr	O
-(-	O
-2	O
-)	O
-hydrogen	O
-bonds	O
-to	O
-Gly	O
-(-	O
-1	O
-)	O
-O	O
-.	O
-Although	O
-this	O
-interaction	O
-was	O
-not	O
-observed	O
-for	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-T	I-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-2c	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-4c	O
-,	O
-i	O
-),	O
-exchange	O
-of	O
-Thr	O
-(-	O
-2	O
-)	O
-by	O
-Val	O
-in	O
-β2	O
-,	O
-a	O
-conservative	O
-mutation	O
-regarding	O
-size	O
-but	O
-drastic	O
-with	O
-respect	O
-to	O
-polarity	O
-,	O
-was	O
-found	O
-to	O
-inhibit	O
-maturation	O
-of	O
-this	O
-subunit	O
-(	O
-Fig	O
-.	O
-2d	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-4e	O
-,	O
-j	O
-).	O
-	O
-Notably	O
-,	O
-the	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-map	O
-displays	O
-a	O
-different	O
-orientation	O
-for	O
-the	O
-β2	O
-propeptide	O
-than	O
-has	O
-been	O
-observed	O
-for	O
-the	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-proteasome	O
-.	O
-	O
-In	O
-particular	O
-,	O
-Val	O
-(-	O
-2	O
-)	O
-is	O
-displaced	O
-from	O
-the	O
-S1	O
-site	O
-and	O
-Gly	O
-(-	O
-1	O
-)	O
-is	O
-severely	O
-shifted	O
-(	O
-movement	O
-of	O
-the	O
-carbonyl	O
-oxygen	O
-atom	O
-of	O
-3	O
-.	O
-8	O
-Å	O
-),	O
-thereby	O
-preventing	O
-nucleophilic	O
-attack	O
-of	O
-Thr1	O
-(	O
-Fig	O
-.	O
-2d	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-4j	O
-,	O
-k	O
-).	O
-	O
-These	O
-results	O
-further	O
-confirm	O
-that	O
-correct	O
-positioning	O
-of	O
-the	O
-active	O
--	O
-site	O
-residues	O
-and	O
-Gly	O
-(-	O
-1	O
-)	O
-is	O
-decisive	O
-for	O
-the	O
-maturation	O
-of	O
-the	O
-proteasome	O
-.	O
-	O
-The	O
-active	O
-site	O
-of	O
-the	O
-proteasome	O
-	O
-Proton	O
-shuttling	O
-from	O
-the	O
-proteasomal	O
-active	O
-site	O
-Thr1OH	O
-to	O
-Thr1NH2	O
-via	O
-a	O
-nucleophilic	O
-water	O
-molecule	O
-was	O
-suggested	O
-to	O
-initiate	O
-peptide	O
--	O
-bond	O
-hydrolysis	O
-.	O
-	O
-However	O
-,	O
-in	O
-the	O
-immature	O
-particle	O
-Thr1NH2	O
-is	O
-blocked	O
-by	O
-the	O
-propeptide	O
-and	O
-cannot	O
-activate	O
-Thr1Oγ	O
-.	O
-	O
-Instead	O
-,	O
-Lys33NH2	O
-,	O
-which	O
-is	O
-in	O
-hydrogen	O
--	O
-bonding	O
-distance	O
-to	O
-Thr1Oγ	O
-(	O
-2	O
-.	O
-7	O
-Å	O
-)	O
-in	O
-all	O
-catalytically	O
-active	O
-β	O
-subunits	O
-(	O
-Fig	O
-.	O
-3a	O
-,	O
-b	O
-),	O
-was	O
-proposed	O
-to	O
-serve	O
-as	O
-the	O
-proton	O
-acceptor	O
-.	O
-	O
-A	O
-proposed	O
-catalytic	O
-tetrad	O
-model	O
-involving	O
-Thr1OH	O
-,	O
-Thr1NH2	O
-,	O
-Lys33NH2	O
-and	O
-Asp17Oδ	O
-,	O
-as	O
-well	O
-as	O
-a	O
-nucleophilic	O
-water	O
-molecule	O
-as	O
-the	O
-proton	O
-shuttle	O
-appeared	O
-to	O
-accommodate	O
-all	O
-possible	O
-views	O
-of	O
-the	O
-proteasomal	O
-active	O
-site	O
-.	O
-	O
-Twenty	O
-years	O
-later	O
-,	O
-with	O
-a	O
-plethora	O
-of	O
-yCP	O
-X	O
--	O
-ray	O
-structures	O
-in	O
-hand	O
-,	O
-we	O
-decided	O
-to	O
-re	O
--	O
-analyse	O
-the	O
-active	O
-site	O
-of	O
-the	O
-proteasome	O
-and	O
-to	O
-resolve	O
-the	O
-uncertainty	O
-regarding	O
-the	O
-nature	O
-of	O
-the	O
-general	O
-base	O
-.	O
-	O
-Mutation	O
-of	O
-β5	O
--	O
-Lys33	O
-to	O
-Ala	O
-causes	O
-a	O
-strongly	O
-deleterious	O
-phenotype	O
-,	O
-and	O
-previous	O
-structural	O
-and	O
-biochemical	O
-analyses	O
-confirmed	O
-that	O
-this	O
-is	O
-caused	O
-by	O
-failure	O
-of	O
-propeptide	O
-cleavage	O
-,	O
-and	O
-consequently	O
-,	O
-lack	O
-of	O
-ChT	O
--	O
-L	O
-activity	O
-(	O
-Fig	O
-.	O
-4a	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-3b	O
-and	O
-Table	O
-1	O
-;	O
-for	O
-details	O
-see	O
-Supplementary	O
-Note	O
-1	O
-).	O
-	O
-The	O
-phenotype	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-K33A	I-mutant
-mutant	O
-was	O
-however	O
-less	O
-pronounced	O
-than	O
-for	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-yeast	O
-(	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-This	O
-discrepancy	O
-in	O
-growth	O
-was	O
-traced	O
-to	O
-an	O
-additional	O
-point	O
-mutation	O
-L	B-mutant
-(-	I-mutant
-49	I-mutant
-)	I-mutant
-S	I-mutant
-in	O
-the	O
-β5	O
--	O
-propeptide	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-K33A	I-mutant
-mutant	O
-(	O
-see	O
-also	O
-Supplementary	O
-Note	O
-1	O
-).	O
-	O
-Structural	O
-comparison	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-L	I-mutant
-(-	I-mutant
-49	I-mutant
-)	I-mutant
-S	I-mutant
--	I-mutant
-K33A	I-mutant
-and	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-active	O
-sites	O
-revealed	O
-that	O
-mutation	O
-of	O
-Lys33	O
-to	O
-Ala	O
-creates	O
-a	O
-cavity	O
-that	O
-is	O
-filled	O
-with	O
-Thr1	O
-and	O
-the	O
-remnant	O
-propeptide	O
-.	O
-	O
-This	O
-structural	O
-alteration	O
-destroys	O
-active	O
--	O
-site	O
-integrity	O
-and	O
-abolishes	O
-catalytic	O
-activity	O
-of	O
-the	O
-β5	O
-active	O
-site	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-Additional	O
-proof	O
-for	O
-the	O
-key	O
-function	O
-of	O
-Lys33	O
-was	O
-obtained	O
-from	O
-the	O
-β5	B-mutant
--	I-mutant
-K33A	I-mutant
-mutant	O
-,	O
-with	O
-the	O
-propeptide	O
-expressed	O
-separately	O
-from	O
-the	O
-main	O
-subunit	O
-(	O
-pp	O
-trans	O
-).	O
-	O
-The	O
-Thr1	O
-N	O
-terminus	O
-of	O
-this	O
-mutant	O
-is	O
-not	O
-blocked	O
-by	O
-the	O
-propeptide	O
-,	O
-yet	O
-its	O
-catalytic	O
-activity	O
-is	O
-reduced	O
-by	O
-∼	O
-83	O
-%	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6b	O
-).	O
-	O
-Consistent	O
-with	O
-this	O
-,	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-K33A	I-mutant
-pp	O
-trans	O
-mutant	O
-in	O
-complex	O
-with	O
-carfilzomib	O
-only	O
-showed	O
-partial	O
-occupancy	O
-of	O
-the	O
-ligand	O
-at	O
-the	O
-β5	O
-active	O
-sites	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5b	O
-and	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-Since	O
-no	O
-acetylation	O
-of	O
-the	O
-Thr1	O
-N	O
-terminus	O
-was	O
-observed	O
-for	O
-the	O
-β5	B-mutant
--	I-mutant
-K33A	I-mutant
-pp	O
-trans	O
-apo	O
-crystal	O
-structure	O
-,	O
-the	O
-reduced	O
-reactivity	O
-towards	O
-substrates	O
-and	O
-inhibitors	O
-indicates	O
-that	O
-Lys33NH2	O
-,	O
-rather	O
-than	O
-Thr1NH2	O
-,	O
-deprotonates	O
-and	O
-activates	O
-Thr1OH	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-K33A	I-mutant
-pp	O
-trans	O
-mutant	O
-without	O
-inhibitor	O
-revealed	O
-that	O
-Thr1Oγ	O
-strongly	O
-coordinates	O
-a	O
-well	O
--	O
-defined	O
-water	O
-molecule	O
-(∼	O
-2	O
-Å	O
-;	O
-Fig	O
-.	O
-3c	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-5c	O
-,	O
-d	O
-).	O
-	O
-This	O
-water	O
-hydrogen	O
-bonds	O
-also	O
-to	O
-Arg19O	O
-(∼	O
-3	O
-.	O
-0	O
-Å	O
-)	O
-and	O
-Asp17Oδ	O
-(∼	O
-3	O
-.	O
-0	O
-Å	O
-),	O
-and	O
-thereby	O
-presumably	O
-enables	O
-residual	O
-activity	O
-of	O
-the	O
-mutant	O
-.	O
-	O
-Remarkably	O
-,	O
-the	O
-solvent	O
-molecule	O
-occupies	O
-the	O
-position	O
-normally	O
-taken	O
-by	O
-Lys33NH2	O
-in	O
-the	O
-WT	O
-proteasome	O
-structure	O
-(	O
-Fig	O
-.	O
-3c	O
-),	O
-further	O
-corroborating	O
-the	O
-essential	O
-role	O
-of	O
-Lys33	O
-as	O
-the	O
-general	O
-base	O
-for	O
-autolysis	O
-and	O
-proteolysis	O
-.	O
-	O
-Conservative	O
-substitution	O
-of	O
-Lys33	O
-by	O
-Arg	O
-delays	O
-autolysis	O
-of	O
-the	O
-β5	O
-precursor	O
-and	O
-impairs	O
-yeast	O
-growth	O
-(	O
-for	O
-details	O
-see	O
-Supplementary	O
-Note	O
-1	O
-).	O
-	O
-While	O
-Thr1	O
-occupies	O
-the	O
-same	O
-position	O
-as	O
-in	O
-WT	O
-yCPs	O
-,	O
-Arg33	O
-is	O
-unable	O
-to	O
-hydrogen	O
-bond	O
-to	O
-Asp17	O
-,	O
-thereby	O
-inactivating	O
-the	O
-β5	O
-active	O
-site	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5e	O
-).	O
-	O
-The	O
-conservative	O
-mutation	O
-of	O
-Asp17	O
-to	O
-Asn	O
-in	O
-subunit	O
-β5	O
-of	O
-the	O
-yCP	O
-also	O
-provokes	O
-a	O
-severe	O
-growth	O
-defect	O
-(	O
-Supplementary	O
-Note	O
-1	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-and	O
-Table	O
-1	O
-).	O
-	O
-Notably	O
-,	O
-only	O
-with	O
-the	O
-additional	O
-point	O
-mutation	O
-L	B-mutant
-(-	I-mutant
-49	I-mutant
-)	I-mutant
-S	I-mutant
-present	O
-in	O
-the	O
-β5	O
-propeptide	O
-could	O
-we	O
-purify	O
-a	O
-small	O
-amount	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-mutant	O
-yCP	O
-.	O
-	O
-As	O
-determined	O
-by	O
-crystallographic	O
-analysis	O
-,	O
-this	O
-mutant	O
-β5	O
-subunit	O
-was	O
-partially	O
-processed	O
-(	O
-Table	O
-1	O
-)	O
-but	O
-displayed	O
-impaired	O
-reactivity	O
-towards	O
-the	O
-proteasome	O
-inhibitor	O
-carfilzomib	O
-compared	O
-with	O
-the	O
-subunits	O
-β1	O
-and	O
-β2	O
-,	O
-and	O
-with	O
-WT	O
-β5	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7a	O
-).	O
-	O
-In	O
-contrast	O
-to	O
-the	O
-cis	O
--	O
-construct	O
-,	O
-expression	O
-of	O
-the	O
-β5	O
-propeptide	O
-in	O
-trans	O
-allowed	O
-straightforward	O
-isolation	O
-and	O
-crystallization	O
-of	O
-the	O
-D17N	B-mutant
-mutant	O
-proteasome	O
-.	O
-	O
-The	O
-ChT	O
--	O
-L	O
-activity	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-pp	O
-in	O
-trans	O
-CP	O
-towards	O
-the	O
-canonical	O
-β5	O
-model	O
-substrates	O
-N	O
--	O
-succinyl	O
--	O
-Leu	O
--	O
-Leu	O
--	O
-Val	O
--	O
-Tyr	O
--	O
-7	O
--	O
-amino	O
--	O
-4	O
--	O
-methylcoumarin	O
-(	O
-Suc	O
--	O
-LLVY	O
--	O
-AMC	O
-)	O
-and	O
-carboxybenzyl	O
--	O
-Gly	O
--	O
-Gly	O
--	O
-Leu	O
--	O
-para	O
--	O
-nitroanilide	O
-(	O
-Z	O
--	O
-GGL	O
--	O
-pNA	O
-)	O
-was	O
-severely	O
-reduced	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6b	O
-),	O
-confirming	O
-that	O
-Asp17	O
-is	O
-of	O
-fundamental	O
-importance	O
-for	O
-the	O
-catalytic	O
-activity	O
-of	O
-the	O
-mature	O
-proteasome	O
-.	O
-	O
-Even	O
-though	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-pp	O
-trans	O
-yCP	O
-crystal	O
-structure	O
-appeared	O
-identical	O
-to	O
-the	O
-WT	O
-yCP	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7b	O
-),	O
-the	O
-co	O
--	O
-crystal	O
-structure	O
-with	O
-the	O
-α	O
-′,	O
-β	O
-′	O
-epoxyketone	O
-inhibitor	O
-carfilzomib	O
-visualized	O
-only	O
-partial	O
-occupancy	O
-of	O
-the	O
-ligand	O
-in	O
-the	O
-β5	O
-active	O
-site	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7a	O
-).	O
-	O
-This	O
-observation	O
-is	O
-consistent	O
-with	O
-a	O
-strongly	O
-reduced	O
-reactivity	O
-of	O
-β5	O
--	O
-Thr1	O
-and	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-pp	O
-cis	O
-mutant	O
-in	O
-complex	O
-with	O
-carfilzomib	O
-.	O
-	O
-Autolysis	O
-and	O
-residual	O
-catalytic	O
-activity	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-mutants	O
-may	O
-originate	O
-from	O
-the	O
-carbonyl	O
-group	O
-of	O
-Asn17	O
-,	O
-which	O
-albeit	O
-to	O
-a	O
-lower	O
-degree	O
-still	O
-can	O
-polarize	O
-Lys33	O
-for	O
-the	O
-activation	O
-of	O
-Thr1	O
-.	O
-	O
-In	O
-agreement	O
-,	O
-an	O
-E17A	B-mutant
-mutant	O
-in	O
-the	O
-proteasomal	O
-β	O
--	O
-subunit	O
-of	O
-the	O
-archaeon	O
-Thermoplasma	O
-acidophilum	O
-prevents	O
-autolysis	O
-and	O
-catalysis	O
-.	O
-	O
-Strikingly	O
-,	O
-although	O
-the	O
-X	O
--	O
-ray	O
-data	O
-on	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-mutant	O
-with	O
-the	O
-propeptide	O
-expressed	O
-in	O
-cis	O
-and	O
-in	O
-trans	O
-looked	O
-similar	O
-,	O
-there	O
-was	O
-a	O
-pronounced	O
-difference	O
-in	O
-their	O
-growth	O
-phenotypes	O
-observed	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-7b	O
-).	O
-	O
-On	O
-the	O
-basis	O
-of	O
-these	O
-results	O
-,	O
-we	O
-propose	O
-that	O
-CPs	O
-from	O
-all	O
-domains	O
-of	O
-life	O
-use	O
-a	O
-catalytic	O
-triad	O
-consisting	O
-of	O
-Thr1	O
-,	O
-Lys33	O
-and	O
-Asp	O
-/	O
-Glu17	O
-for	O
-both	O
-autocatalytic	O
-precursor	O
-processing	O
-and	O
-proteolysis	O
-(	O
-Fig	O
-.	O
-3d	O
-).	O
-	O
-This	O
-model	O
-is	O
-also	O
-consistent	O
-with	O
-the	O
-fact	O
-that	O
-no	O
-defined	O
-water	O
-molecule	O
-is	O
-observed	O
-in	O
-the	O
-mature	O
-WT	O
-proteasomal	O
-active	O
-site	O
-that	O
-could	O
-shuttle	O
-the	O
-proton	O
-from	O
-Thr1Oγ	O
-to	O
-Thr1NH2	O
-.	O
-	O
-To	O
-explore	O
-this	O
-active	O
--	O
-site	O
-model	O
-further	O
-,	O
-we	O
-exchanged	O
-the	O
-conserved	O
-Asp166	O
-residue	O
-for	O
-Asn	O
-in	O
-the	O
-yeast	O
-β5	O
-subunit	O
-.	O
-	O
-Asp166Oδ	O
-is	O
-hydrogen	O
--	O
-bonded	O
-to	O
-Thr1NH2	O
-via	O
-Ser129OH	O
-and	O
-Ser169OH	O
-,	O
-and	O
-therefore	O
-was	O
-proposed	O
-to	O
-be	O
-involved	O
-in	O
-catalysis	O
-.	O
-	O
-The	O
-β5	B-mutant
--	I-mutant
-D166N	I-mutant
-pp	O
-cis	O
-yeast	O
-mutant	O
-is	O
-significantly	O
-impaired	O
-in	O
-growth	O
-and	O
-its	O
-ChT	O
--	O
-L	O
-activity	O
-is	O
-drastically	O
-reduced	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-,	O
-b	O
-and	O
-Table	O
-1	O
-).	O
-	O
-X	O
--	O
-ray	O
-data	O
-on	O
-the	O
-β5	B-mutant
--	I-mutant
-D166N	I-mutant
-mutant	O
-indicate	O
-that	O
-the	O
-β5	O
-propeptide	O
-is	O
-hydrolysed	O
-,	O
-but	O
-due	O
-to	O
-reorientation	O
-of	O
-Ser129OH	O
-,	O
-the	O
-interaction	O
-with	O
-Asn166Oδ	O
-is	O
-disrupted	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-8a	O
-).	O
-	O
-Instead	O
-,	O
-a	O
-water	O
-molecule	O
-is	O
-bound	O
-to	O
-Ser129OH	O
-and	O
-Thr1NH2	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-8b	O
-),	O
-which	O
-may	O
-enable	O
-precursor	O
-processing	O
-.	O
-	O
-The	O
-hydrogen	O
-bonds	O
-involving	O
-Ser169OH	O
-are	O
-intact	O
-and	O
-may	O
-account	O
-for	O
-residual	O
-substrate	O
-turnover	O
-.	O
-	O
-Soaking	O
-the	O
-β5	B-mutant
--	I-mutant
-D166N	I-mutant
-crystals	O
-with	O
-carfilzomib	O
-and	O
-MG132	O
-resulted	O
-in	O
-covalent	O
-modification	O
-of	O
-Thr1	O
-at	O
-high	O
-occupancy	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-8c	O
-).	O
-	O
-In	O
-the	O
-carfilzomib	O
-complex	O
-structure	O
-,	O
-Thr1Oγ	O
-and	O
-Thr1N	O
-incorporate	O
-into	O
-a	O
-morpholine	O
-ring	O
-structure	O
-and	O
-Ser129	O
-adopts	O
-its	O
-WT	O
--	O
-like	O
-orientation	O
-.	O
-	O
-In	O
-the	O
-MG132	O
--	O
-bound	O
-state	O
-,	O
-Thr1N	O
-is	O
-unmodified	O
-,	O
-and	O
-we	O
-again	O
-observe	O
-that	O
-Ser129	O
-is	O
-hydrogen	O
--	O
-bonded	O
-to	O
-a	O
-water	O
-molecule	O
-instead	O
-of	O
-Asn166	O
-.	O
-	O
-Whereas	O
-Asn	O
-can	O
-to	O
-some	O
-degree	O
-replace	O
-Asp166	O
-due	O
-to	O
-its	O
-carbonyl	O
-group	O
-in	O
-the	O
-side	O
-chain	O
-,	O
-Ala	O
-at	O
-this	O
-position	O
-was	O
-found	O
-to	O
-prevent	O
-both	O
-autolysis	O
-and	O
-catalysis	O
-.	O
-	O
-These	O
-results	O
-suggest	O
-that	O
-Asp166	O
-and	O
-Ser129	O
-function	O
-as	O
-a	O
-proton	O
-shuttle	O
-and	O
-affect	O
-the	O
-protonation	O
-state	O
-of	O
-Thr1N	O
-during	O
-autolysis	O
-and	O
-catalysis	O
-.	O
-	O
-Substitution	O
-of	O
-the	O
-active	O
--	O
-site	O
-Thr1	O
-by	O
-Cys	O
-	O
-Mutation	O
-of	O
-Thr1	O
-to	O
-Cys	O
-inactivates	O
-the	O
-20S	O
-proteasome	O
-from	O
-the	O
-archaeon	O
-T	O
-.	O
-acidophilum	O
-.	O
-	O
-In	O
-yeast	O
-,	O
-this	O
-mutation	O
-causes	O
-a	O
-strong	O
-growth	O
-defect	O
-(	O
-Fig	O
-.	O
-4a	O
-and	O
-Table	O
-1	O
-),	O
-although	O
-the	O
-propeptide	O
-is	O
-hydrolysed	O
-,	O
-as	O
-shown	O
-here	O
-by	O
-its	O
-X	O
--	O
-ray	O
-structure	O
-.	O
-	O
-In	O
-one	O
-of	O
-the	O
-two	O
-β5	O
-subunits	O
-,	O
-however	O
-,	O
-we	O
-found	O
-the	O
-cleaved	O
-propeptide	O
-still	O
-bound	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-(	O
-Fig	O
-.	O
-4c	O
-).	O
-	O
-His	O
-(-	O
-2	O
-)	O
-occupies	O
-the	O
-S2	O
-pocket	O
-like	O
-observed	O
-for	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-mutant	O
-,	O
-but	O
-in	O
-contrast	O
-to	O
-the	O
-latter	O
-,	O
-the	O
-propeptide	O
-in	O
-the	O
-T1C	B-mutant
-mutant	O
-adopts	O
-an	O
-antiparallel	O
-β	O
--	O
-sheet	O
-conformation	O
-as	O
-known	O
-from	O
-inhibitors	O
-like	O
-MG132	O
-(	O
-Fig	O
-.	O
-4c	O
-–	O
-e	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-9b	O
-).	O
-	O
-On	O
-the	O
-basis	O
-of	O
-the	O
-phenotype	O
-of	O
-the	O
-T1C	B-mutant
-mutant	O
-and	O
-the	O
-propeptide	O
-remnant	O
-identified	O
-in	O
-its	O
-active	O
-site	O
-,	O
-we	O
-suppose	O
-that	O
-autolysis	O
-is	O
-retarded	O
-and	O
-may	O
-not	O
-have	O
-been	O
-completed	O
-before	O
-crystallization	O
-.	O
-	O
-Owing	O
-to	O
-the	O
-unequal	O
-positions	O
-of	O
-the	O
-two	O
-β5	O
-subunits	O
-within	O
-the	O
-CP	O
-in	O
-the	O
-crystal	O
-lattice	O
-,	O
-maturation	O
-and	O
-propeptide	O
-displacement	O
-may	O
-occur	O
-at	O
-different	O
-timescales	O
-in	O
-the	O
-two	O
-subunits	O
-.	O
-	O
-Despite	O
-propeptide	O
-hydrolysis	O
-,	O
-the	O
-β5	B-mutant
--	I-mutant
-T1C	I-mutant
-active	O
-site	O
-is	O
-catalytically	O
-inactive	O
-(	O
-Fig	O
-.	O
-4b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-9a	O
-).	O
-	O
-In	O
-agreement	O
-,	O
-soaking	O
-crystals	O
-with	O
-the	O
-CP	O
-inhibitors	O
-bortezomib	O
-or	O
-carfilzomib	O
-modifies	O
-only	O
-the	O
-β1	O
-and	O
-β2	O
-active	O
-sites	O
-,	O
-while	O
-leaving	O
-the	O
-β5	B-mutant
--	I-mutant
-T1C	I-mutant
-proteolytic	O
-centres	O
-unmodified	O
-even	O
-though	O
-they	O
-are	O
-only	O
-partially	O
-occupied	O
-by	O
-the	O
-cleaved	O
-propeptide	O
-remnant	O
-.	O
-	O
-Moreover	O
-,	O
-the	O
-structural	O
-data	O
-reveal	O
-that	O
-the	O
-thiol	O
-group	O
-of	O
-Cys1	O
-is	O
-rotated	O
-by	O
-74	O
-°	O
-with	O
-respect	O
-to	O
-the	O
-hydroxyl	O
-side	O
-chain	O
-of	O
-Thr1	O
-(	O
-Fig	O
-.	O
-4f	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-9b	O
-).	O
-	O
-Consequently	O
-,	O
-the	O
-hydrogen	O
-bond	O
-bridging	O
-the	O
-active	O
--	O
-site	O
-nucleophile	O
-and	O
-Lys33	O
-in	O
-WT	O
-CPs	O
-is	O
-broken	O
-with	O
-Cys1	O
-.	O
-	O
-Notably	O
-,	O
-the	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-map	O
-of	O
-the	O
-T1C	B-mutant
-mutant	O
-also	O
-indicates	O
-that	O
-Lys33NH2	O
-is	O
-disordered	O
-.	O
-	O
-Together	O
-,	O
-these	O
-observations	O
-suggest	O
-that	O
-efficient	O
-peptide	O
--	O
-bond	O
-hydrolysis	O
-requires	O
-that	O
-Lys33NH2	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-active	O
-site	O
-nucleophile	O
-.	O
-	O
-The	O
-benefit	O
-of	O
-Thr	O
-over	O
-Ser	O
-as	O
-the	O
-active	O
--	O
-site	O
-nucleophile	O
-	O
-All	O
-proteasomes	O
-strictly	O
-employ	O
-threonine	O
-as	O
-the	O
-active	O
--	O
-site	O
-residue	O
-instead	O
-of	O
-serine	O
-.	O
-	O
-To	O
-investigate	O
-the	O
-reason	O
-for	O
-this	O
-singularity	O
-,	O
-we	O
-analysed	O
-a	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-mutant	O
-,	O
-which	O
-is	O
-viable	O
-but	O
-suffers	O
-from	O
-growth	O
-defects	O
-(	O
-Fig	O
-.	O
-4a	O
-and	O
-Table	O
-1	O
-).	O
-	O
-Activity	O
-assays	O
-with	O
-the	O
-β5	O
--	O
-specific	O
-substrate	O
-Suc	O
--	O
-LLVY	O
--	O
-AMC	O
-demonstrated	O
-that	O
-the	O
-ChT	O
--	O
-L	O
-activity	O
-of	O
-the	O
-T1S	B-mutant
-mutant	O
-is	O
-reduced	O
-by	O
-40	O
-–	O
-45	O
-%	O
-compared	O
-with	O
-WT	O
-proteasomes	O
-depending	O
-on	O
-the	O
-incubation	O
-temperature	O
-(	O
-Fig	O
-.	O
-4b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-9c	O
-).	O
-	O
-By	O
-contrast	O
-,	O
-turnover	O
-of	O
-the	O
-substrate	O
-Z	O
--	O
-GGL	O
--	O
-pNA	O
-,	O
-used	O
-to	O
-monitor	O
-ChT	O
--	O
-L	O
-activity	O
-in	O
-situ	O
-but	O
-in	O
-a	O
-less	O
-quantitative	O
-fashion	O
-,	O
-is	O
-not	O
-detectably	O
-impaired	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-9a	O
-).	O
-	O
-Crystal	O
-structure	O
-analysis	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-mutant	O
-confirmed	O
-precursor	O
-processing	O
-(	O
-Fig	O
-.	O
-4g	O
-),	O
-and	O
-ligand	O
--	O
-complex	O
-structures	O
-with	O
-bortezomib	O
-and	O
-carfilzomib	O
-unambiguously	O
-corroborated	O
-the	O
-reactivity	O
-of	O
-Ser1	O
-(	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-However	O
-,	O
-the	O
-apo	O
-crystal	O
-structure	O
-revealed	O
-that	O
-Ser1Oγ	O
-is	O
-turned	O
-away	O
-from	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-(	O
-Fig	O
-.	O
-4g	O
-).	O
-	O
-Compared	O
-with	O
-Thr1Oγ	O
-in	O
-WT	O
-CP	O
-structures	O
-,	O
-Ser1Oγ	O
-is	O
-rotated	O
-by	O
-60	O
-°.	O
-	O
-Because	O
-both	O
-conformations	O
-of	O
-Ser1Oγ	O
-are	O
-hydrogen	O
--	O
-bonded	O
-to	O
-Lys33NH2	O
-(	O
-Fig	O
-.	O
-4h	O
-),	O
-the	O
-relay	O
-system	O
-is	O
-capable	O
-of	O
-hydrolysing	O
-peptide	O
-substrates	O
-,	O
-albeit	O
-at	O
-lower	O
-rates	O
-compared	O
-with	O
-Thr1	O
-.	O
-	O
-The	O
-active	O
--	O
-site	O
-residue	O
-Thr1	O
-is	O
-fixed	O
-in	O
-its	O
-position	O
-,	O
-as	O
-its	O
-methyl	O
-group	O
-is	O
-engaged	O
-in	O
-hydrophobic	O
-interactions	O
-with	O
-Thr3	O
-and	O
-Ala46	O
-(	O
-Fig	O
-.	O
-4h	O
-).	O
-	O
-Consequently	O
-,	O
-the	O
-hydroxyl	O
-group	O
-of	O
-Thr1	O
-requires	O
-no	O
-reorientation	O
-before	O
-substrate	O
-cleavage	O
-and	O
-is	O
-thus	O
-more	O
-catalytically	O
-efficient	O
-than	O
-Ser1	O
-.	O
-	O
-In	O
-agreement	O
-,	O
-at	O
-an	O
-elevated	O
-growing	O
-temperature	O
-of	O
-37	O
-°	O
-C	O
-the	O
-T1S	B-mutant
-mutant	O
-is	O
-unable	O
-to	O
-grow	O
-(	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-In	O
-vitro	O
-,	O
-the	O
-mutant	O
-proteasome	O
-is	O
-less	O
-susceptible	O
-to	O
-proteasome	O
-inhibition	O
-by	O
-bortezomib	O
-(	O
-3	O
-.	O
-7	O
--	O
-fold	O
-)	O
-and	O
-carfilzomib	O
-(	O
-1	O
-.	O
-8	O
--	O
-fold	O
-;	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-Nevertheless	O
-,	O
-inhibitor	O
-complex	O
-structures	O
-indicate	O
-identical	O
-binding	O
-modes	O
-compared	O
-with	O
-the	O
-WT	O
-yCP	O
-structures	O
-,	O
-with	O
-the	O
-same	O
-inhibitors	O
-.	O
-	O
-Notably	O
-,	O
-the	O
-affinity	O
-of	O
-the	O
-tetrapeptide	O
-carfilzomib	O
-is	O
-less	O
-impaired	O
-,	O
-as	O
-it	O
-is	O
-better	O
-stabilized	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-than	O
-the	O
-dipeptide	O
-bortezomib	O
-,	O
-which	O
-lacks	O
-a	O
-defined	O
-P3	O
-site	O
-and	O
-has	O
-only	O
-a	O
-few	O
-interactions	O
-with	O
-the	O
-surrounding	O
-protein	O
-.	O
-	O
-Hence	O
-,	O
-the	O
-mean	O
-residence	O
-time	O
-of	O
-carfilzomib	O
-at	O
-the	O
-active	O
-site	O
-is	O
-prolonged	O
-and	O
-the	O
-probability	O
-to	O
-covalently	O
-react	O
-with	O
-Ser1	O
-is	O
-increased	O
-.	O
-	O
-Considered	O
-together	O
-,	O
-these	O
-results	O
-provide	O
-a	O
-plausible	O
-explanation	O
-for	O
-the	O
-invariance	O
-of	O
-threonine	O
-as	O
-the	O
-active	O
--	O
-site	O
-nucleophile	O
-in	O
-proteasomes	O
-in	O
-all	O
-three	O
-domains	O
-of	O
-life	O
-,	O
-as	O
-well	O
-as	O
-in	O
-proteasome	O
--	O
-like	O
-proteases	O
-such	O
-as	O
-HslV	O
-(	O
-ref	O
-.).	O
-	O
-The	O
-20S	O
-proteasome	O
-CP	O
-is	O
-the	O
-major	O
-non	O
--	O
-lysosomal	O
-protease	O
-in	O
-eukaryotic	O
-cells	O
-,	O
-and	O
-its	O
-assembly	O
-is	O
-highly	O
-organized	O
-.	O
-	O
-The	O
-β	O
--	O
-subunit	O
-propeptides	O
-,	O
-particularly	O
-that	O
-of	O
-β5	O
-,	O
-are	O
-key	O
-factors	O
-that	O
-help	O
-drive	O
-proper	O
-assembly	O
-of	O
-the	O
-CP	O
-complex	O
-.	O
-	O
-In	O
-addition	O
-,	O
-they	O
-prevent	O
-irreversible	O
-inactivation	O
-of	O
-the	O
-Thr1	O
-N	O
-terminus	O
-by	O
-N	O
--	O
-acetylation	O
-.	O
-	O
-By	O
-contrast	O
-,	O
-the	O
-prosegments	O
-of	O
-β	O
-subunits	O
-are	O
-dispensable	O
-for	O
-archaeal	O
-proteasome	O
-assembly	O
-,	O
-at	O
-least	O
-when	O
-heterologously	O
-expressed	O
-in	O
-Escherichia	O
-coli	O
-.	O
-	O
-In	O
-eukaryotes	O
-,	O
-deletion	O
-of	O
-or	O
-failure	O
-to	O
-cleave	O
-the	O
-β1	O
-and	O
-β2	O
-propeptides	O
-is	O
-well	O
-tolerated	O
-.	O
-	O
-However	O
-,	O
-removal	O
-of	O
-the	O
-β5	O
-prosegment	O
-or	O
-any	O
-interference	O
-with	O
-its	O
-cleavage	O
-causes	O
-severe	O
-phenotypic	O
-defects	O
-.	O
-	O
-These	O
-observations	O
-highlight	O
-the	O
-unique	O
-function	O
-and	O
-importance	O
-of	O
-the	O
-β5	O
-propeptide	O
-as	O
-well	O
-as	O
-the	O
-β5	O
-active	O
-site	O
-for	O
-maturation	O
-and	O
-function	O
-of	O
-the	O
-eukaryotic	O
-CP	O
-.	O
-	O
-Here	O
-we	O
-have	O
-described	O
-the	O
-atomic	O
-structures	O
-of	O
-various	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-mutants	O
-,	O
-which	O
-allowed	O
-for	O
-the	O
-first	O
-time	O
-visualization	O
-of	O
-the	O
-residual	O
-β5	O
-propeptide	O
-.	O
-	O
-Depending	O
-on	O
-the	O
-(-	O
-2	O
-)	O
-residue	O
-we	O
-observed	O
-various	O
-propeptide	O
-conformations	O
-,	O
-but	O
-Gly	O
-(-	O
-1	O
-)	O
-is	O
-in	O
-all	O
-structures	O
-perfectly	O
-located	O
-for	O
-the	O
-nucleophilic	O
-attack	O
-by	O
-Thr1Oγ	O
-,	O
-although	O
-it	O
-does	O
-not	O
-adopt	O
-the	O
-tight	O
-turn	O
-observed	O
-for	O
-the	O
-prosegment	O
-of	O
-subunit	O
-β1	O
-.	O
-	O
-From	O
-these	O
-data	O
-we	O
-conclude	O
-that	O
-only	O
-the	O
-positioning	O
-of	O
-Gly	O
-(-	O
-1	O
-)	O
-and	O
-Thr1	O
-as	O
-well	O
-as	O
-the	O
-integrity	O
-of	O
-the	O
-proteasomal	O
-active	O
-site	O
-are	O
-required	O
-for	O
-autolysis	O
-.	O
-	O
-In	O
-this	O
-regard	O
-,	O
-inappropriate	O
-N	O
--	O
-acetylation	O
-of	O
-the	O
-Thr1	O
-N	O
-terminus	O
-cannot	O
-be	O
-removed	O
-by	O
-Thr1Oγ	O
-due	O
-to	O
-the	O
-rotational	O
-freedom	O
-and	O
-flexibility	O
-of	O
-the	O
-acetyl	O
-group	O
-.	O
-	O
-The	O
-propeptide	O
-needs	O
-some	O
-anchoring	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-to	O
-properly	O
-position	O
-Gly	O
-(-	O
-1	O
-),	O
-but	O
-this	O
-seems	O
-to	O
-be	O
-independent	O
-of	O
-the	O
-orientation	O
-of	O
-residue	O
-(-	O
-2	O
-).	O
-	O
-Autolytic	O
-activation	O
-of	O
-the	O
-CP	O
-constitutes	O
-one	O
-of	O
-the	O
-final	O
-steps	O
-of	O
-proteasome	O
-biogenesis	O
-,	O
-but	O
-the	O
-trigger	O
-for	O
-propeptide	O
-cleavage	O
-had	O
-remained	O
-enigmatic	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-the	O
-numerous	O
-CP	O
-:	O
-ligand	O
-complexes	O
-solved	O
-during	O
-the	O
-past	O
-18	O
-years	O
-and	O
-in	O
-the	O
-current	O
-study	O
-,	O
-we	O
-provide	O
-a	O
-revised	O
-interpretation	O
-of	O
-proteasome	O
-active	O
--	O
-site	O
-architecture	O
-.	O
-	O
-We	O
-propose	O
-a	O
-catalytic	O
-triad	O
-for	O
-the	O
-active	O
-site	O
-of	O
-the	O
-CP	O
-consisting	O
-of	O
-residues	O
-Thr1	O
-,	O
-Lys33	O
-and	O
-Asp	O
-/	O
-Glu17	O
-,	O
-which	O
-are	O
-conserved	O
-among	O
-all	O
-proteolytically	O
-active	O
-eukaryotic	O
-,	O
-bacterial	O
-and	O
-archaeal	O
-proteasome	O
-subunits	O
-.	O
-	O
-Lys33NH2	O
-is	O
-expected	O
-to	O
-act	O
-as	O
-the	O
-proton	O
-acceptor	O
-during	O
-autocatalytic	O
-removal	O
-of	O
-the	O
-propeptides	O
-,	O
-as	O
-well	O
-as	O
-during	O
-substrate	O
-proteolysis	O
-,	O
-while	O
-Asp17Oδ	O
-orients	O
-Lys33NH2	O
-and	O
-makes	O
-it	O
-more	O
-prone	O
-to	O
-protonation	O
-by	O
-raising	O
-its	O
-pKa	O
-(	O
-hydrogen	O
-bond	O
-distance	O
-:	O
-Lys33NH3	O
-+–	O
-Asp17Oδ	O
-:	O
-2	O
-.	O
-9	O
-Å	O
-).	O
-	O
-Analogously	O
-to	O
-the	O
-proteasome	O
-,	O
-a	O
-Thr	O
-–	O
-Lys	O
-–	O
-Asp	O
-triad	O
-is	O
-also	O
-found	O
-in	O
-L	O
--	O
-asparaginase	O
-.	O
-	O
-Thus	O
-,	O
-specific	O
-protein	O
-surroundings	O
-can	O
-significantly	O
-alter	O
-the	O
-chemical	O
-properties	O
-of	O
-amino	O
-acids	O
-such	O
-as	O
-Lys	O
-to	O
-function	O
-as	O
-an	O
-acid	O
-–	O
-base	O
-catalyst	O
-.	O
-	O
-In	O
-this	O
-new	O
-view	O
-of	O
-the	O
-proteasomal	O
-active	O
-site	O
-,	O
-the	O
-positively	O
-charged	O
-Thr1NH3	O
-+-	O
-terminus	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-amide	O
-nitrogen	O
-of	O
-incoming	O
-peptide	O
-substrates	O
-and	O
-stabilizes	O
-as	O
-well	O
-as	O
-activates	O
-them	O
-for	O
-the	O
-endoproteolytic	O
-cleavage	O
-by	O
-Thr1Oγ	O
-(	O
-Fig	O
-.	O
-3d	O
-).	O
-	O
-Consistent	O
-with	O
-this	O
-model	O
-,	O
-the	O
-positively	O
-charged	O
-Thr1	O
-N	O
-terminus	O
-is	O
-engaged	O
-in	O
-hydrogen	O
-bonds	O
-with	O
-inhibitory	O
-compounds	O
-like	O
-fellutamide	O
-B	O
-(	O
-ref	O
-.),	O
-α	O
--	O
-ketoamides	O
-,	O
-homobelactosin	O
-C	O
-(	O
-ref	O
-.)	O
-and	O
-salinosporamide	O
-A	O
-(	O
-ref	O
-.).	O
-	O
-Furthermore	O
-,	O
-opening	O
-of	O
-the	O
-β	O
--	O
-lactone	O
-compound	O
-omuralide	O
-by	O
-Thr1	O
-creates	O
-a	O
-C3	O
--	O
-hydroxyl	O
-group	O
-,	O
-whose	O
-proton	O
-originates	O
-from	O
-Thr1NH3	O
-+.	O
-	O
-The	O
-resulting	O
-uncharged	O
-Thr1NH2	O
-is	O
-hydrogen	O
--	O
-bridged	O
-to	O
-the	O
-C3	O
--	O
-OH	O
-group	O
-.	O
-	O
-In	O
-agreement	O
-,	O
-acetylation	O
-of	O
-the	O
-Thr1	O
-N	O
-terminus	O
-irreversibly	O
-blocks	O
-hydrolytic	O
-activity	O
-,	O
-and	O
-binding	O
-of	O
-substrates	O
-is	O
-prevented	O
-for	O
-steric	O
-reasons	O
-.	O
-	O
-By	O
-acting	O
-as	O
-a	O
-proton	O
-donor	O
-during	O
-catalysis	O
-,	O
-the	O
-Thr1	O
-N	O
-terminus	O
-may	O
-also	O
-favour	O
-cleavage	O
-of	O
-substrate	O
-peptide	O
-bonds	O
-(	O
-Fig	O
-.	O
-3d	O
-).	O
-	O
-Cleavage	O
-of	O
-the	O
-scissile	O
-peptide	O
-bond	O
-requires	O
-protonation	O
-of	O
-the	O
-emerging	O
-free	O
-amine	O
-,	O
-and	O
-in	O
-the	O
-proteasome	O
-,	O
-the	O
-Thr1	O
-amine	O
-group	O
-is	O
-likely	O
-to	O
-assume	O
-this	O
-function	O
-.	O
-	O
-Analogously	O
-,	O
-Thr1NH3	O
-+	O
-might	O
-promote	O
-the	O
-bivalent	O
-reaction	O
-mode	O
-of	O
-epoxyketone	O
-inhibitors	O
-by	O
-protonating	O
-the	O
-epoxide	O
-moiety	O
-to	O
-create	O
-a	O
-positively	O
-charged	O
-trivalent	O
-oxygen	O
-atom	O
-that	O
-is	O
-subsequently	O
-nucleophilically	O
-attacked	O
-by	O
-Thr1NH2	O
-.	O
-	O
-During	O
-autolysis	O
-the	O
-Thr1	O
-N	O
-terminus	O
-is	O
-engaged	O
-in	O
-a	O
-hydroxyoxazolidine	O
-ring	O
-intermediate	O
-(	O
-Fig	O
-.	O
-3d	O
-),	O
-which	O
-is	O
-unstable	O
-and	O
-short	O
--	O
-lived	O
-.	O
-	O
-Breakdown	O
-of	O
-this	O
-tetrahedral	O
-transition	O
-state	O
-releases	O
-the	O
-Thr1	O
-N	O
-terminus	O
-that	O
-is	O
-protonated	O
-by	O
-aspartic	O
-acid	O
-166	O
-via	O
-Ser129OH	O
-to	O
-yield	O
-Thr1NH3	O
-+.	O
-	O
-The	O
-residues	O
-Ser129	O
-and	O
-Asp166	O
-are	O
-expected	O
-to	O
-increase	O
-the	O
-pKa	O
-value	O
-of	O
-Thr1N	O
-,	O
-thereby	O
-favouring	O
-its	O
-charged	O
-state	O
-.	O
-	O
-Consistent	O
-with	O
-playing	O
-an	O
-essential	O
-role	O
-in	O
-proton	O
-shuttling	O
-,	O
-the	O
-mutation	O
-D166A	B-mutant
-prevents	O
-autolysis	O
-of	O
-the	O
-archaeal	O
-CP	O
-and	O
-the	O
-exchange	O
-D166N	B-mutant
-impairs	O
-catalytic	O
-activity	O
-of	O
-the	O
-yeast	O
-CP	O
-about	O
-60	O
-%.	O
-	O
-The	O
-mutation	O
-D166N	B-mutant
-lowers	O
-the	O
-pKa	O
-of	O
-Thr1N	O
-,	O
-which	O
-is	O
-thus	O
-more	O
-likely	O
-to	O
-exist	O
-in	O
-the	O
-uncharged	O
-deprotonated	O
-state	O
-(	O
-Thr1NH2	O
-).	O
-	O
-This	O
-interpretation	O
-agrees	O
-with	O
-the	O
-strongly	O
-reduced	O
-catalytic	O
-activity	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-D166N	I-mutant
-mutant	O
-on	O
-the	O
-one	O
-hand	O
-,	O
-and	O
-the	O
-ability	O
-to	O
-react	O
-readily	O
-with	O
-carfilzomib	O
-on	O
-the	O
-other	O
-.	O
-	O
-Hence	O
-,	O
-the	O
-proteasome	O
-can	O
-be	O
-viewed	O
-as	O
-having	O
-a	O
-second	O
-triad	O
-that	O
-is	O
-essential	O
-for	O
-efficient	O
-proteolysis	O
-.	O
-	O
-While	O
-Lys33NH2	O
-and	O
-Asp17Oδ	O
-are	O
-required	O
-to	O
-deprotonate	O
-the	O
-Thr1	O
-hydroxyl	O
-side	O
-chain	O
-,	O
-Ser129OH	O
-and	O
-Asp166OH	O
-serve	O
-to	O
-protonate	O
-the	O
-N	O
--	O
-terminal	O
-amine	O
-group	O
-of	O
-Thr1	O
-.	O
-	O
-In	O
-accord	O
-with	O
-the	O
-proposed	O
-Thr1	O
-–	O
-Lys33	O
-–	O
-Asp17	O
-catalytic	O
-triad	O
-,	O
-crystallographic	O
-data	O
-on	O
-the	O
-proteolytically	O
-inactive	O
-β5	B-mutant
--	I-mutant
-T1C	I-mutant
-mutant	O
-demonstrate	O
-that	O
-the	O
-interaction	O
-of	O
-Lys33NH2	O
-and	O
-Cys1	O
-is	O
-broken	O
-.	O
-	O
-However	O
-,	O
-owing	O
-to	O
-Cys	O
-being	O
-a	O
-strong	O
-nucleophile	O
-,	O
-the	O
-propeptide	O
-can	O
-still	O
-be	O
-cleaved	O
-off	O
-over	O
-time	O
-.	O
-	O
-While	O
-only	O
-one	O
-single	O
-turnover	O
-is	O
-necessary	O
-for	O
-autolysis	O
-,	O
-continuous	O
-enzymatic	O
-activity	O
-is	O
-required	O
-for	O
-significant	O
-and	O
-detectable	O
-substrate	O
-hydrolysis	O
-.	O
-	O
-Notably	O
-,	O
-in	O
-the	O
-Ntn	O
-hydrolase	O
-penicillin	O
-acylase	O
-,	O
-substitution	O
-of	O
-the	O
-catalytic	O
-N	O
--	O
-terminal	O
-Ser	O
-residue	O
-by	O
-Cys	O
-also	O
-inactivates	O
-the	O
-enzyme	O
-but	O
-still	O
-enables	O
-precursor	O
-processing	O
-.	O
-	O
-To	O
-investigate	O
-why	O
-the	O
-CP	O
-specifically	O
-employs	O
-threonine	O
-as	O
-its	O
-active	O
--	O
-site	O
-residue	O
-,	O
-we	O
-used	O
-a	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-mutant	O
-of	O
-the	O
-yCP	O
-and	O
-characterized	O
-it	O
-biochemically	O
-and	O
-structurally	O
-.	O
-	O
-Activity	O
-assays	O
-with	O
-the	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-mutant	O
-revealed	O
-reduced	O
-turnover	O
-of	O
-Suc	O
--	O
-LLVY	O
--	O
-AMC	O
-.	O
-	O
-We	O
-also	O
-observed	O
-slightly	O
-lower	O
-affinity	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-mutant	O
-yCP	O
-for	O
-the	O
-Food	O
-and	O
-Drug	O
-Administration	O
--	O
-approved	O
-proteasome	O
-inhibitors	O
-bortezomib	O
-and	O
-carfilzomib	O
-.	O
-	O
-Structural	O
-analyses	O
-support	O
-these	O
-findings	O
-with	O
-the	O
-T1S	B-mutant
-mutant	O
-and	O
-provide	O
-an	O
-explanation	O
-for	O
-the	O
-strict	O
-use	O
-of	O
-Thr	O
-residues	O
-in	O
-proteasomes	O
-.	O
-	O
-Thr1	O
-is	O
-well	O
-anchored	O
-in	O
-the	O
-active	O
-site	O
-by	O
-hydrophobic	O
-interactions	O
-of	O
-its	O
-Cγ	O
-methyl	O
-group	O
-with	O
-Ala46	O
-(	O
-Cβ	O
-),	O
-Lys33	O
-(	O
-carbon	O
-side	O
-chain	O
-)	O
-and	O
-Thr3	O
-(	O
-Cγ	O
-).	O
-	O
-Notably	O
-,	O
-proteolytically	O
-active	O
-proteasome	O
-subunits	O
-from	O
-archaea	O
-,	O
-yeast	O
-and	O
-mammals	O
-,	O
-including	O
-constitutive	O
-,	O
-immuno	O
--	O
-and	O
-thymoproteasome	O
-subunits	O
-,	O
-either	O
-encode	O
-Thr	O
-or	O
-Ile	O
-at	O
-position	O
-3	O
-,	O
-indicating	O
-the	O
-importance	O
-of	O
-the	O
-Cγ	O
-for	O
-fixing	O
-the	O
-position	O
-of	O
-the	O
-nucleophilic	O
-Thr1	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-Thr1	O
-,	O
-the	O
-hydroxyl	O
-group	O
-of	O
-Ser1	O
-occupies	O
-the	O
-position	O
-of	O
-the	O
-Thr1	O
-methyl	O
-side	O
-chain	O
-in	O
-the	O
-WT	O
-enzyme	O
-,	O
-which	O
-requires	O
-its	O
-reorientation	O
-relative	O
-to	O
-the	O
-substrate	O
-to	O
-allow	O
-cleavage	O
-(	O
-Fig	O
-.	O
-4g	O
-,	O
-h	O
-).	O
-	O
-Notably	O
-,	O
-in	O
-the	O
-threonine	O
-aspartase	O
-Taspase1	O
-,	O
-mutation	O
-of	O
-the	O
-active	O
--	O
-site	O
-Thr234	O
-to	O
-Ser	O
-also	O
-places	O
-the	O
-side	O
-chain	O
-in	O
-the	O
-position	O
-of	O
-the	O
-methyl	O
-group	O
-of	O
-Thr234	O
-in	O
-the	O
-WT	O
-,	O
-thereby	O
-reducing	O
-catalytic	O
-activity	O
-.	O
-	O
-Similarly	O
-,	O
-although	O
-the	O
-serine	O
-mutant	O
-is	O
-active	O
-,	O
-threonine	O
-is	O
-more	O
-efficient	O
-in	O
-the	O
-context	O
-of	O
-the	O
-proteasome	O
-active	O
-site	O
-.	O
-	O
-The	O
-greater	O
-suitability	O
-of	O
-threonine	O
-for	O
-the	O
-proteasome	O
-active	O
-site	O
-,	O
-which	O
-has	O
-been	O
-noted	O
-in	O
-biochemical	O
-as	O
-well	O
-as	O
-in	O
-kinetic	O
-studies	O
-,	O
-constitutes	O
-a	O
-likely	O
-reason	O
-for	O
-the	O
-conservation	O
-of	O
-the	O
-Thr1	O
-residue	O
-in	O
-all	O
-proteasomes	O
-from	O
-bacteria	O
-to	O
-eukaryotes	O
-.	O
-	O
-Conformation	O
-of	O
-proteasomal	O
-propeptides	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-and	O
-the	O
-matured	O
-WT	O
-β1	O
-active	O
--	O
-site	O
-Thr1	O
-.	O
-	O
-Only	O
-the	O
-residues	O
-(-	O
-5	O
-)	O
-to	O
-(-	O
-1	O
-)	O
-of	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-are	O
-displayed	O
-.	O
-	O
-The	O
-major	O
-determinant	O
-of	O
-the	O
-S1	O
-specificity	O
-pocket	O
-,	O
-residue	O
-45	O
-,	O
-is	O
-depicted	O
-.	O
-	O
-Note	O
-the	O
-tight	O
-conformation	O
-of	O
-Gly	O
-(-	O
-1	O
-)	O
-and	O
-Ala1	O
-before	O
-propeptide	O
-removal	O
-(	O
-G	O
-(-	O
-1	O
-)	O
-turn	O
-;	O
-cyan	O
-double	O
-arrow	O
-)	O
-compared	O
-with	O
-the	O
-relaxed	O
-,	O
-processed	O
-WT	O
-active	O
--	O
-site	O
-Thr1	O
-(	O
-red	O
-double	O
-arrow	O
-).	O
-	O
-The	O
-black	O
-arrow	O
-indicates	O
-the	O
-attack	O
-of	O
-Thr1Oγ	O
-onto	O
-the	O
-carbonyl	O
-carbon	O
-atom	O
-of	O
-Gly	O
-(-	O
-1	O
-).	O
-	O
-(	O
-b	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-and	O
-the	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-highlights	O
-subtle	O
-differences	O
-in	O
-their	O
-conformations	O
-,	O
-but	O
-illustrates	O
-that	O
-Ala1	O
-and	O
-Gly	O
-(-	O
-1	O
-)	O
-match	O
-well	O
-.	O
-	O
-Thr	O
-(-	O
-2	O
-)	O
-OH	O
-is	O
-hydrogen	O
--	O
-bonded	O
-to	O
-Gly	O
-(-	O
-1	O
-)	O
-O	O
-(∼	O
-2	O
-.	O
-8	O
-Å	O
-;	O
-black	O
-dashed	O
-line	O
-).	O
-	O
-(	O
-c	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-,	O
-the	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-propeptide	O
-remnants	O
-depict	O
-their	O
-differences	O
-in	O
-conformation	O
-.	O
-	O
-While	O
-residue	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-β1	O
-and	O
-β2	O
-prosegments	O
-fit	O
-the	O
-S1	O
-pocket	O
-,	O
-His	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-β5	O
-propeptide	O
-occupies	O
-the	O
-S2	O
-pocket	O
-.	O
-	O
-Nonetheless	O
-,	O
-in	O
-all	O
-mutants	O
-the	O
-carbonyl	O
-carbon	O
-atom	O
-of	O
-Gly	O
-(-	O
-1	O
-)	O
-is	O
-ideally	O
-placed	O
-for	O
-the	O
-nucleophilic	O
-attack	O
-by	O
-Thr1Oγ	O
-.	O
-	O
-The	O
-hydrogen	O
-bond	O
-between	O
-Thr	O
-(-	O
-2	O
-)	O
-OH	O
-and	O
-Gly	O
-(-	O
-1	O
-)	O
-O	O
-(∼	O
-2	O
-.	O
-8	O
-Å	O
-)	O
-is	O
-indicated	O
-by	O
-a	O
-black	O
-dashed	O
-line	O
-.	O
-	O
-Mutations	O
-of	O
-residue	O
-(-	O
-2	O
-)	O
-and	O
-their	O
-influence	O
-on	O
-propeptide	O
-conformation	O
-and	O
-autolysis	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-L	I-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-propeptide	O
-.	O
-	O
-The	O
-(-	O
-2	O
-)	O
-residues	O
-of	O
-both	O
-prosegments	O
-point	O
-into	O
-the	O
-S1	O
-pocket	O
-.	O
-	O
-(	O
-b	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β5	O
-propeptides	O
-in	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-L	I-mutant
--	I-mutant
-T1A	I-mutant
-,	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-T	I-mutant
--	I-mutant
-T1A	I-mutant
-,	O
-β5	B-mutant
--(	I-mutant
-H	I-mutant
--	I-mutant
-2	I-mutant
-)	I-mutant
-A	I-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-and	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-mutant	O
-proteasomes	O
-.	O
-	O
-While	O
-the	O
-residues	O
-(-	O
-2	O
-)	O
-to	O
-(-	O
-4	O
-)	O
-vary	O
-in	O
-their	O
-conformation	O
-,	O
-Gly	O
-(-	O
-1	O
-)	O
-and	O
-Ala1	O
-are	O
-located	O
-in	O
-all	O
-structures	O
-at	O
-the	O
-same	O
-positions	O
-.	O
-	O
-(	O
-c	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-T	I-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-propeptide	O
-.	O
-	O
-The	O
-(-	O
-2	O
-)	O
-residues	O
-of	O
-both	O
-prosegments	O
-point	O
-into	O
-the	O
-S1	O
-pocket	O
-,	O
-but	O
-only	O
-Thr	O
-(-	O
-2	O
-)	O
-OH	O
-of	O
-β2	O
-forms	O
-a	O
-hydrogen	O
-bridge	O
-to	O
-Gly	O
-(-	O
-1	O
-)	O
-O	O
-(	O
-black	O
-dashed	O
-line	O
-).	O
-	O
-(	O
-d	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-matured	O
-β2	O
-active	O
-site	O
-,	O
-the	O
-WT	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-and	O
-the	O
-β2	B-mutant
--	I-mutant
-T	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-V	I-mutant
-mutant	O
-propeptide	O
-.	O
-	O
-Notably	O
-,	O
-Val	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-latter	O
-does	O
-not	O
-occupy	O
-the	O
-S1	O
-pocket	O
-,	O
-thereby	O
-changing	O
-the	O
-orientation	O
-of	O
-Gly	O
-(-	O
-1	O
-)	O
-and	O
-preventing	O
-nucleophilic	O
-attack	O
-of	O
-Thr1Oγ	O
-on	O
-the	O
-carbonyl	O
-carbon	O
-atom	O
-of	O
-Gly	O
-(-	O
-1	O
-).	O
-	O
-Architecture	O
-and	O
-proposed	O
-reaction	O
-mechanism	O
-of	O
-the	O
-proteasomal	O
-active	O
-site	O
-.	O
-	O
-(	O
-a	O
-)	O
-Hydrogen	O
--	O
-bonding	O
-network	O
-at	O
-the	O
-mature	O
-WT	O
-β5	O
-proteasomal	O
-active	O
-site	O
-(	O
-dotted	O
-lines	O
-).	O
-	O
-Thr1OH	O
-is	O
-hydrogen	O
--	O
-bonded	O
-to	O
-Lys33NH2	O
-(	O
-2	O
-.	O
-7	O
-Å	O
-),	O
-which	O
-in	O
-turn	O
-interacts	O
-with	O
-Asp17Oδ	O
-.	O
-	O
-The	O
-Thr1	O
-N	O
-terminus	O
-is	O
-engaged	O
-in	O
-hydrogen	O
-bonds	O
-with	O
-Ser129Oγ	O
-,	O
-the	O
-carbonyl	O
-oxygen	O
-of	O
-residue	O
-168	O
-,	O
-Ser169Oγ	O
-and	O
-Asp166Oδ	O
-.	O
-(	O
-b	O
-)	O
-The	O
-orientations	O
-of	O
-the	O
-active	O
--	O
-site	O
-residues	O
-involved	O
-in	O
-hydrogen	O
-bonding	O
-are	O
-strictly	O
-conserved	O
-in	O
-each	O
-proteolytic	O
-centre	O
-,	O
-as	O
-shown	O
-by	O
-superposition	O
-of	O
-the	O
-β	O
-subunits	O
-.	O
-	O
-(	O
-c	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-WT	O
-β5	O
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-K33A	I-mutant
-pp	O
-trans	O
-mutant	O
-active	O
-site	O
-.	O
-	O
-In	O
-the	O
-latter	O
-,	O
-a	O
-water	O
-molecule	O
-(	O
-red	O
-sphere	O
-)	O
-is	O
-found	O
-at	O
-the	O
-position	O
-where	O
-in	O
-the	O
-WT	O
-structure	O
-the	O
-side	O
-chain	O
-amine	O
-group	O
-of	O
-Lys33	O
-is	O
-located	O
-.	O
-	O
-Similarly	O
-to	O
-Lys33	O
-,	O
-the	O
-water	O
-molecule	O
-hydrogen	O
-bonds	O
-to	O
-Arg19O	O
-,	O
-Asp17Oδ	O
-and	O
-Thr1OH	O
-.	O
-	O
-Note	O
-,	O
-the	O
-strong	O
-interaction	O
-with	O
-the	O
-water	O
-molecule	O
-causes	O
-a	O
-minor	O
-shift	O
-of	O
-Thr1	O
-,	O
-while	O
-all	O
-other	O
-active	O
--	O
-site	O
-residues	O
-remain	O
-in	O
-place	O
-.	O
-	O
-(	O
-d	O
-)	O
-Proposed	O
-chemical	O
-reaction	O
-mechanism	O
-for	O
-autocatalytic	O
-precursor	O
-processing	O
-and	O
-proteolysis	O
-in	O
-the	O
-proteasome	O
-.	O
-	O
-The	O
-active	O
--	O
-site	O
-Thr1	O
-is	O
-depicted	O
-in	O
-blue	O
-,	O
-the	O
-propeptide	O
-segment	O
-and	O
-the	O
-peptide	O
-substrate	O
-are	O
-coloured	O
-in	O
-green	O
-,	O
-whereas	O
-the	O
-scissile	O
-peptide	O
-bond	O
-is	O
-highlighted	O
-in	O
-red	O
-.	O
-	O
-Autolysis	O
-(	O
-left	O
-set	O
-of	O
-structures	O
-)	O
-is	O
-initiated	O
-by	O
-deprotonation	O
-of	O
-Thr1OH	O
-via	O
-Lys33NH2	O
-and	O
-the	O
-formation	O
-of	O
-a	O
-tetrahedral	O
-transition	O
-state	O
-.	O
-	O
-The	O
-strictly	O
-conserved	O
-oxyanion	O
-hole	O
-Gly47NH	O
-stabilizing	O
-the	O
-negatively	O
-charged	O
-intermediate	O
-is	O
-illustrated	O
-as	O
-a	O
-semicircle	O
-.	O
-	O
-Collapse	O
-of	O
-the	O
-transition	O
-state	O
-frees	O
-the	O
-Thr1	O
-N	O
-terminus	O
-(	O
-by	O
-completing	O
-an	O
-N	O
--	O
-to	O
--	O
-O	O
-acyl	O
-shift	O
-of	O
-the	O
-propeptide	O
-),	O
-which	O
-is	O
-subsequently	O
-protonated	O
-by	O
-Asp166OH	O
-via	O
-Ser129OH	O
-.	O
-	O
-Next	O
-,	O
-Thr1NH2	O
-polarizes	O
-a	O
-water	O
-molecule	O
-for	O
-the	O
-nucleophilic	O
-attack	O
-of	O
-the	O
-acyl	O
--	O
-enzyme	O
-intermediate	O
-.	O
-	O
-On	O
-hydrolysis	O
-of	O
-the	O
-latter	O
-,	O
-the	O
-active	O
--	O
-site	O
-Thr1	O
-is	O
-ready	O
-for	O
-catalysis	O
-(	O
-right	O
-set	O
-of	O
-structures	O
-).	O
-	O
-The	O
-charged	O
-Thr1	O
-N	O
-terminus	O
-may	O
-engage	O
-in	O
-the	O
-orientation	O
-of	O
-the	O
-amide	O
-moiety	O
-and	O
-donate	O
-a	O
-proton	O
-to	O
-the	O
-emerging	O
-N	O
-terminus	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-cleavage	O
-product	O
-.	O
-	O
-The	O
-resulting	O
-deprotonated	O
-Thr1NH2	O
-finally	O
-activates	O
-a	O
-water	O
-molecule	O
-for	O
-hydrolysis	O
-of	O
-the	O
-acyl	O
--	O
-enzyme	O
-.	O
-	O
-The	O
-proteasome	O
-favours	O
-threonine	O
-as	O
-the	O
-active	O
--	O
-site	O
-nucleophile	O
-.	O
-	O
-(	O
-a	O
-)	O
-Growth	O
-tests	O
-by	O
-serial	O
-dilution	O
-of	O
-WT	O
-and	O
-pre2	O
-(	O
-β5	O
-)	O
-mutant	O
-yeast	O
-cultures	O
-reveal	O
-growth	O
-defects	O
-of	O
-the	O
-active	O
--	O
-site	O
-mutants	O
-under	O
-the	O
-indicated	O
-conditions	O
-after	O
-2	O
-days	O
-(	O
-2	O
-d	O
-)	O
-of	O
-incubation	O
-.	O
-	O
-(	O
-b	O
-)	O
-Purified	O
-WT	O
-and	O
-mutant	O
-proteasomes	O
-were	O
-tested	O
-for	O
-their	O
-chymotrypsin	O
--	O
-like	O
-activity	O
-(	O
-β5	O
-)	O
-using	O
-the	O
-substrate	O
-Suc	O
--	O
-LLVY	O
--	O
-AMC	O
-.	O
-	O
-(	O
-c	O
-)	O
-Illustration	O
-of	O
-the	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-map	O
-(	O
-blue	O
-mesh	O
-contoured	O
-at	O
-1σ	O
-)	O
-for	O
-the	O
-β5	B-mutant
--	I-mutant
-T1C	I-mutant
-propeptide	O
-fragment	O
-.	O
-	O
-The	O
-prosegment	O
-is	O
-cleaved	O
-but	O
-still	O
-bound	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-.	O
-	O
-Notably	O
-,	O
-His	O
-(-	O
-2	O
-)	O
-does	O
-not	O
-occupy	O
-the	O
-S1	O
-pocket	O
-formed	O
-by	O
-Met45	O
-,	O
-similar	O
-to	O
-what	O
-was	O
-observed	O
-for	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-mutant	O
-.	O
-	O
-(	O
-d	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-K81R	I-mutant
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-T1C	I-mutant
-mutant	O
-subunits	O
-onto	O
-the	O
-WT	O
-β5	O
-subunit	O
-.	O
-(	O
-e	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1C	I-mutant
-propeptide	O
-onto	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-active	O
-site	O
-(	O
-blue	O
-)	O
-and	O
-the	O
-WT	O
-β5	O
-active	O
-site	O
-in	O
-complex	O
-with	O
-the	O
-proteasome	O
-inhibitor	O
-MG132	O
-(	O
-ref	O
-.).	O
-	O
-The	O
-inhibitor	O
-as	O
-well	O
-as	O
-the	O
-propeptides	O
-adopt	O
-similar	O
-conformations	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-.	O
-	O
-(	O
-f	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-WT	O
-β5	O
-and	O
-β5	B-mutant
--	I-mutant
-T1C	I-mutant
-mutant	O
-active	O
-sites	O
-illustrates	O
-the	O
-different	O
-orientations	O
-of	O
-the	O
-hydroxyl	O
-group	O
-of	O
-Thr1	O
-and	O
-the	O
-thiol	O
-side	O
-chain	O
-of	O
-Cys1	O
-.	O
-	O
-(	O
-g	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-WT	O
-β5	O
-and	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-mutant	O
-active	O
-sites	O
-reveals	O
-different	O
-orientations	O
-of	O
-the	O
-hydroxyl	O
-groups	O
-of	O
-Thr1	O
-and	O
-Ser1	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-map	O
-for	O
-Ser1	O
-(	O
-blue	O
-mesh	O
-contoured	O
-at	O
-1σ	O
-)	O
-is	O
-illustrated	O
-.	O
-	O
-(	O
-h	O
-)	O
-The	O
-methyl	O
-group	O
-of	O
-Thr1	O
-is	O
-anchored	O
-by	O
-hydrophobic	O
-interactions	O
-with	O
-Ala46Cβ	O
-and	O
-Thr3Cγ	O
-.	O
-	O
-Ser1	O
-lacks	O
-this	O
-stabilization	O
-and	O
-is	O
-therefore	O
-rotated	O
-by	O
-60	O
-°.	O
-	O
-Inhibition	O
-of	O
-WT	O
-and	O
-mutant	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-proteasomes	O
-by	O
-bortezomib	O
-and	O
-carfilzomib	O
-.	O
-	O
-Inhibition	O
-assays	O
-(	O
-left	O
-panel	O
-).	O
-	O
-Purified	O
-yeast	O
-proteasomes	O
-were	O
-tested	O
-for	O
-the	O
-susceptibility	O
-of	O
-their	O
-ChT	O
--	O
-L	O
-(	O
-β5	O
-)	O
-activity	O
-to	O
-inhibition	O
-by	O
-bortezomib	O
-and	O
-carfilzomib	O
-using	O
-the	O
-substrate	O
-Suc	O
--	O
-LLVY	O
--	O
-AMC	O
-.	O
-	O
-IC50	O
-values	O
-were	O
-determined	O
-in	O
-triplicate	O
-;	O
-s	O
-.	O
-d	O
-.'	O
-s	O
-are	O
-indicated	O
-by	O
-error	O
-bars	O
-.	O
-	O
-Note	O
-that	O
-IC50	O
-values	O
-depend	O
-on	O
-time	O
-and	O
-enzyme	O
-concentration	O
-.	O
-	O
-Proteasomes	O
-(	O
-final	O
-concentration	O
-:	O
-66	O
-nM	O
-)	O
-were	O
-incubated	O
-with	O
-inhibitor	O
-for	O
-45	O
-min	O
-before	O
-substrate	O
-addition	O
-(	O
-final	O
-concentration	O
-:	O
-200	O
-μM	O
-).	O
-	O
-Structures	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-T1S	I-mutant
-mutant	O
-in	O
-complex	O
-with	O
-both	O
-ligands	O
-(	O
-green	O
-)	O
-prove	O
-the	O
-reactivity	O
-of	O
-Ser1	O
-(	O
-right	O
-panel	O
-).	O
-	O
-The	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-maps	O
-(	O
-blue	O
-mesh	O
-)	O
-for	O
-Ser1	O
-(	O
-brown	O
-)	O
-and	O
-the	O
-covalently	O
-bound	O
-ligands	O
-(	O
-green	O
-;	O
-only	O
-the	O
-P1	O
-site	O
-(	O
-Leu1	O
-)	O
-is	O
-shown	O
-)	O
-are	O
-contoured	O
-at	O
-1σ	O
-.	O
-	O
-The	O
-WT	O
-proteasome	O
-:	O
-inhibitor	O
-complex	O
-structures	O
-(	O
-inhibitor	O
-in	O
-grey	O
-;	O
-Thr1	O
-in	O
-black	O
-)	O
-are	O
-superimposed	O
-and	O
-demonstrate	O
-that	O
-mutation	O
-of	O
-Thr1	O
-to	O
-Ser	O
-does	O
-not	O
-affect	O
-the	O
-binding	O
-mode	O
-of	O
-bortezomib	O
-or	O
-carfilzomib	O
-.	O
-	O
-The	O
-Taf14	O
-YEATS	O
-domain	O
-is	O
-a	O
-reader	O
-of	O
-histone	O
-crotonylation	O
-	O
-The	O
-discovery	O
-of	O
-new	O
-histone	O
-modifications	O
-is	O
-unfolding	O
-at	O
-startling	O
-rates	O
-,	O
-however	O
-,	O
-the	O
-identification	O
-of	O
-effectors	O
-capable	O
-of	O
-interpreting	O
-these	O
-modifications	O
-has	O
-lagged	O
-behind	O
-.	O
-	O
-Here	O
-we	O
-report	O
-the	O
-YEATS	O
-domain	O
-as	O
-an	O
-effective	O
-reader	O
-of	O
-histone	O
-lysine	O
-crotonylation	O
-–	O
-an	O
-epigenetic	O
-signature	O
-associated	O
-with	O
-active	O
-transcription	O
-.	O
-	O
-We	O
-show	O
-that	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-engages	O
-crotonyllysine	O
-via	O
-a	O
-unique	O
-π	O
--	O
-π	O
--	O
-π	O
--	O
-stacking	O
-mechanism	O
-and	O
-that	O
-other	O
-YEATS	O
-domains	O
-have	O
-crotonyllysine	O
-binding	O
-activity	O
-.	O
-	O
-Crotonylation	O
-of	O
-lysine	O
-residues	O
-(	O
-crotonyllysine	O
-,	O
-Kcr	O
-)	O
-has	O
-emerged	O
-as	O
-one	O
-of	O
-the	O
-fundamental	O
-histone	O
-post	O
--	O
-translational	O
-modifications	O
-(	O
-PTMs	O
-)	O
-found	O
-in	O
-mammalian	O
-chromatin	O
-.	O
-	O
-The	O
-crotonyllysine	O
-mark	O
-on	O
-histone	O
-H3K18	O
-is	O
-produced	O
-by	O
-p300	O
-,	O
-a	O
-histone	O
-acetyltransferase	O
-also	O
-responsible	O
-for	O
-acetylation	O
-of	O
-histones	O
-.	O
-	O
-Owing	O
-to	O
-some	O
-differences	O
-in	O
-their	O
-genomic	O
-distribution	O
-,	O
-the	O
-crotonyllysine	O
-and	O
-acetyllysine	O
-(	O
-Kac	O
-)	O
-modifications	O
-have	O
-been	O
-linked	O
-to	O
-distinct	O
-functional	O
-outcomes	O
-.	O
-	O
-p300	O
--	O
-catalyzed	O
-histone	O
-crotonylation	O
-,	O
-which	O
-is	O
-likely	O
-metabolically	O
-regulated	O
-,	O
-stimulates	O
-transcription	O
-to	O
-a	O
-greater	O
-degree	O
-than	O
-p300	O
--	O
-catalyzed	O
-acetylation	O
-.	O
-	O
-The	O
-discovery	O
-of	O
-individual	O
-biological	O
-roles	O
-for	O
-the	O
-crotonyllysine	O
-and	O
-acetyllysine	O
-marks	O
-suggests	O
-that	O
-these	O
-PTMs	O
-can	O
-be	O
-read	O
-by	O
-distinct	O
-readers	O
-.	O
-	O
-While	O
-a	O
-number	O
-of	O
-acetyllysine	O
-readers	O
-have	O
-been	O
-identified	O
-and	O
-characterized	O
-,	O
-a	O
-specific	O
-reader	O
-of	O
-the	O
-crotonyllysine	O
-mark	O
-remains	O
-unknown	O
-(	O
-reviewed	O
-in	O
-).	O
-	O
-A	O
-recent	O
-survey	O
-of	O
-bromodomains	O
-(	O
-BDs	O
-)	O
-demonstrates	O
-that	O
-only	O
-one	O
-BD	O
-associates	O
-very	O
-weakly	O
-with	O
-a	O
-crotonylated	O
-peptide	O
-,	O
-however	O
-it	O
-binds	O
-more	O
-tightly	O
-to	O
-acetylated	O
-peptides	O
-,	O
-inferring	O
-that	O
-bromodomains	O
-do	O
-not	O
-possess	O
-physiologically	O
-relevant	O
-crotonyllysine	O
-binding	O
-activity	O
-.	O
-	O
-The	O
-family	O
-of	O
-acetyllysine	O
-readers	O
-has	O
-been	O
-expanded	O
-with	O
-the	O
-discovery	O
-that	O
-the	O
-YEATS	O
-(	O
-Yaf9	O
-,	O
-ENL	O
-,	O
-AF9	O
-,	O
-Taf14	O
-,	O
-Sas5	O
-)	O
-domains	O
-of	O
-human	O
-AF9	O
-and	O
-yeast	O
-Taf14	O
-are	O
-capable	O
-of	O
-recognizing	O
-the	O
-histone	O
-mark	O
-H3K9ac	O
-.	O
-	O
-The	O
-acetyllysine	O
-binding	O
-function	O
-of	O
-the	O
-AF9	O
-YEATS	O
-domain	O
-is	O
-essential	O
-for	O
-the	O
-recruitment	O
-of	O
-the	O
-histone	O
-methyltransferase	O
-DOT1L	O
-to	O
-H3K9ac	O
--	O
-containing	O
-chromatin	O
-and	O
-for	O
-DOT1L	O
--	O
-mediated	O
-H3K79	O
-methylation	O
-and	O
-transcription	O
-.	O
-	O
-Similarly	O
-,	O
-activation	O
-of	O
-a	O
-subset	O
-of	O
-genes	O
-and	O
-DNA	O
-damage	O
-repair	O
-in	O
-yeast	O
-require	O
-the	O
-acetyllysine	O
-binding	O
-activity	O
-of	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-.	O
-	O
-Consistent	O
-with	O
-its	O
-role	O
-in	O
-gene	O
-regulation	O
-,	O
-Taf14	O
-was	O
-identified	O
-as	O
-a	O
-core	O
-component	O
-of	O
-the	O
-transcription	O
-factor	O
-complexes	O
-TFIID	O
-and	O
-TFIIF	O
-.	O
-	O
-However	O
-,	O
-Taf14	O
-is	O
-also	O
-found	O
-in	O
-a	O
-number	O
-of	O
-chromatin	O
--	O
-remodeling	O
-complexes	O
-(	O
-i	O
-.	O
-e	O
-.,	O
-INO80	O
-,	O
-SWI	O
-/	O
-SNF	O
-and	O
-RSC	O
-)	O
-and	O
-the	O
-histone	O
-acetyltransferase	O
-complex	O
-NuA3	O
-,	O
-indicating	O
-a	O
-multifaceted	O
-role	O
-of	O
-Taf14	O
-in	O
-transcriptional	O
-regulation	O
-and	O
-chromatin	O
-biology	O
-.	O
-	O
-In	O
-this	O
-study	O
-,	O
-we	O
-identified	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-as	O
-a	O
-reader	O
-of	O
-crotonyllysine	O
-that	O
-binds	O
-to	O
-histone	O
-H3	O
-crotonylated	O
-at	O
-lysine	O
-9	O
-(	O
-H3K9cr	O
-)	O
-via	O
-a	O
-distinctive	O
-binding	O
-mechanism	O
-.	O
-	O
-We	O
-found	O
-that	O
-H3K9cr	O
-is	O
-present	O
-in	O
-yeast	O
-and	O
-is	O
-dynamically	O
-regulated	O
-.	O
-	O
-To	O
-elucidate	O
-the	O
-molecular	O
-basis	O
-for	O
-recognition	O
-of	O
-the	O
-H3K9cr	O
-mark	O
-,	O
-we	O
-obtained	O
-a	O
-crystal	O
-structure	O
-of	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-in	O
-complex	O
-with	O
-H3K9cr5	O
--	O
-13	O
-(	O
-residues	O
-5	O
-–	O
-13	O
-of	O
-H3	O
-)	O
-peptide	O
-(	O
-Fig	O
-.	O
-1	O
-,	O
-Supplementary	O
-Results	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-and	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-The	O
-Taf14	O
-YEATS	O
-domain	O
-adopts	O
-an	O
-immunoglobin	O
--	O
-like	O
-β	O
-sandwich	O
-fold	O
-containing	O
-eight	O
-anti	O
--	O
-parallel	O
-β	O
-strands	O
-linked	O
-by	O
-short	O
-loops	O
-that	O
-form	O
-a	O
-binding	O
-site	O
-for	O
-H3K9cr	O
-(	O
-Fig	O
-.	O
-1b	O
-).	O
-	O
-The	O
-H3K9cr	O
-peptide	O
-lays	O
-in	O
-an	O
-extended	O
-conformation	O
-in	O
-an	O
-orientation	O
-orthogonal	O
-to	O
-the	O
-β	O
-strands	O
-and	O
-is	O
-stabilized	O
-through	O
-an	O
-extensive	O
-network	O
-of	O
-direct	O
-and	O
-water	O
--	O
-mediated	O
-hydrogen	O
-bonds	O
-and	O
-a	O
-salt	O
-bridge	O
-(	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-The	O
-most	O
-striking	O
-feature	O
-of	O
-the	O
-crotonyllysine	O
-recognition	O
-mechanism	O
-is	O
-the	O
-unique	O
-coordination	O
-of	O
-crotonylated	O
-lysine	O
-residue	O
-.	O
-	O
-The	O
-fully	O
-extended	O
-side	O
-chain	O
-of	O
-K9cr	O
-transverses	O
-the	O
-narrow	O
-tunnel	O
-,	O
-crossing	O
-the	O
-β	O
-sandwich	O
-at	O
-right	O
-angle	O
-in	O
-a	O
-corkscrew	O
--	O
-like	O
-manner	O
-(	O
-Fig	O
-.	O
-1b	O
-and	O
-Supplementary	O
-Figure	O
-1b	O
-).	O
-	O
-The	O
-planar	O
-crotonyl	O
-group	O
-is	O
-inserted	O
-between	O
-Trp81	O
-and	O
-Phe62	O
-of	O
-the	O
-protein	O
-,	O
-the	O
-aromatic	O
-rings	O
-of	O
-which	O
-are	O
-positioned	O
-strictly	O
-parallel	O
-to	O
-each	O
-other	O
-and	O
-at	O
-equal	O
-distance	O
-from	O
-the	O
-crotonyl	O
-group	O
-,	O
-yielding	O
-a	O
-novel	O
-aromatic	O
--	O
-amide	O
-/	O
-aliphatic	O
--	O
-aromatic	O
-π	O
--	O
-π	O
--	O
-π	O
--	O
-stacking	O
-system	O
-that	O
-,	O
-to	O
-our	O
-knowledge	O
-,	O
-has	O
-not	O
-been	O
-reported	O
-previously	O
-for	O
-any	O
-protein	O
--	O
-protein	O
-interaction	O
-(	O
-Fig	O
-.	O
-1d	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-The	O
-side	O
-chain	O
-of	O
-Trp81	O
-appears	O
-to	O
-adopt	O
-two	O
-conformations	O
-,	O
-one	O
-of	O
-which	O
-provides	O
-maximum	O
-π	O
--	O
-stacking	O
-with	O
-the	O
-alkene	O
-functional	O
-group	O
-while	O
-the	O
-other	O
-rotamer	O
-affords	O
-maximum	O
-π	O
--	O
-stacking	O
-with	O
-the	O
-amide	O
-π	O
-electrons	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-The	O
-dual	O
-conformation	O
-of	O
-Trp81	O
-is	O
-likely	O
-due	O
-to	O
-the	O
-conjugated	O
-nature	O
-of	O
-the	O
-C	O
-=	O
-C	O
-and	O
-C	O
-=	O
-O	O
-π	O
--	O
-orbitals	O
-within	O
-the	O
-crotonyl	O
-functional	O
-group	O
-.	O
-	O
-In	O
-addition	O
-to	O
-π	O
--	O
-π	O
--	O
-π	O
-stacking	O
-,	O
-the	O
-crotonyl	O
-group	O
-is	O
-stabilized	O
-by	O
-a	O
-set	O
-of	O
-hydrogen	O
-bonds	O
-and	O
-electrostatic	O
-interactions	O
-.	O
-	O
-The	O
-π	O
-bond	O
-conjugation	O
-of	O
-the	O
-crotonyl	O
-group	O
-gives	O
-rise	O
-to	O
-a	O
-dipole	O
-moment	O
-of	O
-the	O
-alkene	O
-moiety	O
-,	O
-resulting	O
-in	O
-a	O
-partial	O
-positive	O
-charge	O
-on	O
-the	O
-β	O
--	O
-carbon	O
-(	O
-Cβ	O
-)	O
-and	O
-a	O
-partial	O
-negative	O
-charge	O
-on	O
-the	O
-α	O
--	O
-carbon	O
-(	O
-Cα	O
-).	O
-	O
-This	O
-provides	O
-the	O
-capability	O
-for	O
-the	O
-alkene	O
-moiety	O
-to	O
-form	O
-electrostatic	O
-contacts	O
-,	O
-as	O
-Cα	O
-and	O
-Cβ	O
-lay	O
-within	O
-electrostatic	O
-interaction	O
-distances	O
-of	O
-the	O
-carbonyl	O
-oxygen	O
-of	O
-Gln79	O
-and	O
-of	O
-the	O
-hydroxyl	O
-group	O
-of	O
-Thr61	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-hydroxyl	O
-group	O
-of	O
-Thr61	O
-also	O
-participates	O
-in	O
-a	O
-hydrogen	O
-bond	O
-with	O
-the	O
-amide	O
-nitrogen	O
-of	O
-the	O
-K9cr	O
-side	O
-chain	O
-(	O
-Fig	O
-.	O
-1d	O
-).	O
-	O
-The	O
-fixed	O
-position	O
-of	O
-the	O
-Thr61	O
-hydroxyl	O
-group	O
-,	O
-which	O
-facilitates	O
-interactions	O
-with	O
-both	O
-the	O
-amide	O
-and	O
-Cα	O
-of	O
-K9cr	O
-,	O
-is	O
-achieved	O
-through	O
-a	O
-hydrogen	O
-bond	O
-with	O
-imidazole	O
-ring	O
-of	O
-His59	O
-.	O
-	O
-Extra	O
-stabilization	O
-of	O
-K9cr	O
-is	O
-attained	O
-by	O
-a	O
-hydrogen	O
-bond	O
-formed	O
-between	O
-its	O
-carbonyl	O
-oxygen	O
-and	O
-the	O
-backbone	O
-nitrogen	O
-of	O
-Trp81	O
-,	O
-as	O
-well	O
-as	O
-a	O
-water	O
--	O
-mediated	O
-hydrogen	O
-bond	O
-with	O
-the	O
-backbone	O
-carbonyl	O
-group	O
-of	O
-Gly82	O
-(	O
-Fig	O
-1d	O
-).	O
-	O
-This	O
-distinctive	O
-mechanism	O
-was	O
-corroborated	O
-through	O
-mapping	O
-the	O
-Taf14	O
-YEATS	O
--	O
-H3K9cr	O
-binding	O
-interface	O
-in	O
-solution	O
-using	O
-NMR	O
-chemical	O
-shift	O
-perturbation	O
-analysis	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2a	O
-,	O
-b	O
-).	O
-	O
-Binding	O
-of	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-to	O
-H3K9cr	O
-is	O
-robust	O
-.	O
-	O
-The	O
-dissociation	O
-constant	O
-(	O
-Kd	O
-)	O
-for	O
-the	O
-Taf14	O
-YEATS	O
--	O
-H3K9cr5	O
--	O
-13	O
-complex	O
-was	O
-found	O
-to	O
-be	O
-9	O
-.	O
-5	O
-μM	O
-,	O
-as	O
-measured	O
-by	O
-fluorescence	O
-spectroscopy	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2c	O
-).	O
-	O
-This	O
-value	O
-is	O
-in	O
-the	O
-range	O
-of	O
-binding	O
-affinities	O
-exhibited	O
-by	O
-the	O
-majority	O
-of	O
-histone	O
-readers	O
-,	O
-thus	O
-attesting	O
-to	O
-the	O
-physiological	O
-relevance	O
-of	O
-the	O
-H3K9cr	O
-recognition	O
-by	O
-Taf14	O
-.	O
-	O
-To	O
-determine	O
-whether	O
-H3K9cr	O
-is	O
-present	O
-in	O
-yeast	O
-,	O
-we	O
-generated	O
-whole	O
-cell	O
-extracts	O
-from	O
-logarithmically	O
-growing	O
-yeast	O
-cells	O
-and	O
-subjected	O
-them	O
-to	O
-Western	O
-blot	O
-analysis	O
-using	O
-antibodies	O
-directed	O
-towards	O
-H3K9cr	O
-,	O
-H3K9ac	O
-and	O
-H3	O
-(	O
-Fig	O
-.	O
-2a	O
-,	O
-b	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-and	O
-Supplementary	O
-Table	O
-2	O
-).	O
-	O
-Both	O
-H3K9cr	O
-and	O
-H3K9ac	O
-were	O
-detected	O
-in	O
-yeast	O
-histones	O
-;	O
-to	O
-our	O
-knowledge	O
-,	O
-this	O
-is	O
-the	O
-first	O
-report	O
-of	O
-H3K9cr	O
-occurring	O
-in	O
-yeast	O
-.	O
-	O
-We	O
-next	O
-asked	O
-if	O
-H3K9cr	O
-is	O
-regulated	O
-by	O
-the	O
-actions	O
-of	O
-histone	O
-acetyltransferases	O
-(	O
-HATs	O
-)	O
-and	O
-histone	O
-deacetylases	O
-(	O
-HDACs	O
-).	O
-	O
-Towards	O
-this	O
-end	O
-,	O
-we	O
-probed	O
-extracts	O
-derived	O
-from	O
-yeast	O
-cells	O
-in	O
-which	O
-major	O
-yeast	O
-HATs	O
-(	O
-HAT1	O
-,	O
-Gcn5	O
-,	O
-and	O
-Rtt109	O
-)	O
-or	O
-HDACs	O
-(	O
-Rpd3	O
-,	O
-Hos1	O
-,	O
-and	O
-Hos2	O
-)	O
-were	O
-deleted	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Figure	O
-2a	O
-,	O
-b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3e	O
-,	O
-H3K9cr	O
-levels	O
-were	O
-abolished	O
-or	O
-reduced	O
-considerably	O
-in	O
-the	O
-HAT	O
-deletion	O
-strains	O
-,	O
-whereas	O
-they	O
-were	O
-dramatically	O
-increased	O
-in	O
-the	O
-HDAC	O
-deletion	O
-strains	O
-.	O
-	O
-Furthermore	O
-,	O
-fluctuations	O
-in	O
-the	O
-H3K9cr	O
-levels	O
-were	O
-more	O
-substantial	O
-than	O
-fluctuations	O
-in	O
-the	O
-corresponding	O
-H3K9ac	O
-levels	O
-.	O
-	O
-Together	O
-,	O
-these	O
-results	O
-reveal	O
-that	O
-H3K9cr	O
-is	O
-a	O
-dynamic	O
-mark	O
-of	O
-chromatin	O
-in	O
-yeast	O
-and	O
-suggest	O
-an	O
-important	O
-role	O
-for	O
-this	O
-modification	O
-in	O
-transcription	O
-as	O
-it	O
-is	O
-regulated	O
-by	O
-HATs	O
-and	O
-HDACs	O
-.	O
-	O
-We	O
-have	O
-previously	O
-shown	O
-that	O
-among	O
-acetylated	O
-histone	O
-marks	O
-,	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-prefers	O
-acetylated	O
-H3K9	O
-(	O
-also	O
-see	O
-Supplementary	O
-Fig	O
-.	O
-3b	O
-),	O
-however	O
-it	O
-binds	O
-to	O
-H3K9cr	O
-tighter	O
-.	O
-	O
-The	O
-selectivity	O
-of	O
-Taf14	O
-towards	O
-crotonyllysine	O
-was	O
-substantiated	O
-by	O
-1H	O
-,	O
-15N	O
-HSQC	O
-experiments	O
-,	O
-in	O
-which	O
-either	O
-H3K9cr5	O
--	O
-13	O
-or	O
-H3K9ac5	O
--	O
-13	O
-peptide	O
-was	O
-titrated	O
-into	O
-the	O
-15N	O
--	O
-labeled	O
-Taf14	O
-YEATS	O
-domain	O
-(	O
-Fig	O
-.	O
-2c	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-4a	O
-,	O
-b	O
-).	O
-	O
-Binding	O
-of	O
-H3K9cr	O
-induced	O
-resonance	O
-changes	O
-in	O
-slow	O
-exchange	O
-regime	O
-on	O
-the	O
-NMR	O
-time	O
-scale	O
-,	O
-indicative	O
-of	O
-strong	O
-interaction	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-binding	O
-of	O
-H3K9ac	O
-resulted	O
-in	O
-an	O
-intermediate	O
-exchange	O
-,	O
-which	O
-is	O
-characteristic	O
-of	O
-a	O
-weaker	O
-association	O
-.	O
-	O
-Furthermore	O
-,	O
-crosspeaks	O
-of	O
-Gly80	O
-and	O
-Trp81	O
-of	O
-the	O
-YEATS	O
-domain	O
-were	O
-uniquely	O
-perturbed	O
-by	O
-H3K9cr	O
-and	O
-H3K9ac	O
-,	O
-indicating	O
-a	O
-different	O
-chemical	O
-environment	O
-in	O
-the	O
-respective	O
-crotonyllysine	O
-and	O
-acetyllysine	O
-binding	O
-pockets	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4a	O
-).	O
-	O
-These	O
-differences	O
-support	O
-our	O
-model	O
-that	O
-Trp81	O
-adopts	O
-two	O
-conformations	O
-upon	O
-complex	O
-formation	O
-with	O
-the	O
-H3K9cr	O
-mark	O
-as	O
-compared	O
-to	O
-H3K9ac	O
-(	O
-Supplementary	O
-Figs	O
-.	O
-1c	O
-,	O
-d	O
-and	O
-4c	O
-).	O
-	O
-One	O
-of	O
-the	O
-conformations	O
-,	O
-characterized	O
-by	O
-the	O
-π	O
-stacking	O
-involving	O
-two	O
-aromatic	O
-residues	O
-and	O
-the	O
-alkene	O
-group	O
-,	O
-is	O
-observed	O
-only	O
-in	O
-the	O
-YEATS	O
--	O
-H3K9cr	O
-complex	O
-.	O
-	O
-To	O
-establish	O
-whether	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-is	O
-able	O
-to	O
-recognize	O
-other	O
-recently	O
-identified	O
-acyllysine	O
-marks	O
-,	O
-we	O
-performed	O
-solution	O
-pull	O
--	O
-down	O
-assays	O
-using	O
-H3	O
-peptides	O
-acetylated	O
-,	O
-propionylated	O
-,	O
-butyrylated	O
-,	O
-and	O
-crotonylated	O
-at	O
-lysine	O
-9	O
-(	O
-residues	O
-1	O
-–	O
-20	O
-of	O
-H3	O
-).	O
-	O
-As	O
-shown	O
-in	O
-Figure	O
-2d	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-5a	O
-,	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-binds	O
-more	O
-strongly	O
-to	O
-H3K9cr1	O
--	O
-20	O
-,	O
-as	O
-compared	O
-to	O
-other	O
-acylated	O
-histone	O
-peptides	O
-.	O
-	O
-The	O
-preference	O
-for	O
-H3K9cr	O
-over	O
-H3K9ac	O
-,	O
-H3K9pr	O
-and	O
-H3K9bu	O
-was	O
-supported	O
-by	O
-1H	O
-,	O
-15N	O
-HSQC	O
-titration	O
-experiments	O
-.	O
-	O
-Addition	O
-of	O
-H3K9ac1	O
--	O
-20	O
-,	O
-H3K9pr1	O
--	O
-20	O
-,	O
-and	O
-H3K9bu1	O
--	O
-20	O
-peptides	O
-caused	O
-chemical	O
-shift	O
-perturbations	O
-in	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-in	O
-intermediate	O
-exchange	O
-regime	O
-,	O
-implying	O
-that	O
-these	O
-interactions	O
-are	O
-weaker	O
-compared	O
-to	O
-the	O
-interaction	O
-with	O
-the	O
-H3K9cr1	O
--	O
-20	O
-peptide	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-We	O
-concluded	O
-that	O
-H3K9cr	O
-is	O
-the	O
-preferred	O
-target	O
-of	O
-this	O
-domain	O
-.	O
-	O
-From	O
-comparative	O
-structural	O
-analysis	O
-of	O
-the	O
-YEATS	O
-complexes	O
-,	O
-Gly80	O
-emerged	O
-as	O
-candidate	O
-residue	O
-potentially	O
-responsible	O
-for	O
-the	O
-preference	O
-for	O
-crotonyllysine	O
-.	O
-	O
-In	O
-attempt	O
-to	O
-generate	O
-a	O
-mutant	O
-capable	O
-of	O
-accommodating	O
-a	O
-short	O
-acetyl	O
-moiety	O
-but	O
-discriminating	O
-against	O
-a	O
-longer	O
-,	O
-planar	O
-crotonyl	O
-moiety	O
-,	O
-we	O
-mutated	O
-Gly80	O
-to	O
-more	O
-bulky	O
-residues	O
-,	O
-however	O
-all	O
-mutants	O
-of	O
-Gly80	O
-lost	O
-their	O
-binding	O
-activities	O
-towards	O
-either	O
-acylated	O
-peptide	O
-,	O
-suggesting	O
-that	O
-Gly80	O
-is	O
-absolutely	O
-required	O
-for	O
-the	O
-interaction	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-mutation	O
-of	O
-Val24	O
-,	O
-a	O
-residue	O
-located	O
-on	O
-another	O
-side	O
-of	O
-Trp81	O
-,	O
-had	O
-no	O
-effect	O
-on	O
-binding	O
-(	O
-Fig	O
-.	O
-2d	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-5a	O
-,	O
-c	O
-).	O
-	O
-To	O
-determine	O
-if	O
-the	O
-binding	O
-to	O
-crotonyllysine	O
-is	O
-conserved	O
-,	O
-we	O
-tested	O
-human	O
-YEATS	O
-domains	O
-by	O
-pull	O
--	O
-down	O
-experiments	O
-using	O
-singly	O
-and	O
-multiply	O
-acetylated	O
-,	O
-propionylated	O
-,	O
-butyrylated	O
-,	O
-and	O
-crotonylated	O
-histone	O
-peptides	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-We	O
-found	O
-that	O
-all	O
-YEATS	O
-domains	O
-tested	O
-are	O
-capable	O
-of	O
-binding	O
-to	O
-crotonyllysine	O
-peptides	O
-,	O
-though	O
-they	O
-display	O
-variable	O
-preferences	O
-for	O
-the	O
-acyl	O
-moieties	O
-.	O
-	O
-While	O
-YEATS2	O
-and	O
-ENL	O
-showed	O
-selectivity	O
-for	O
-the	O
-crotonylated	O
-peptides	O
-,	O
-GAS41	O
-and	O
-AF9	O
-bound	O
-acylated	O
-peptides	O
-almost	O
-equally	O
-well	O
-.	O
-	O
-Unlike	O
-the	O
-YEATS	O
-domain	O
-,	O
-a	O
-known	O
-acetyllysine	O
-reader	O
-,	O
-bromodomain	O
-,	O
-does	O
-not	O
-recognize	O
-crotonyllysine	O
-.	O
-	O
-We	O
-assayed	O
-a	O
-large	O
-set	O
-of	O
-BDs	O
-in	O
-pull	O
--	O
-down	O
-experiments	O
-and	O
-found	O
-that	O
-this	O
-module	O
-is	O
-highly	O
-specific	O
-for	O
-acetyllysine	O
-and	O
-propionyllysine	O
-containing	O
-peptides	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-7	O
-).	O
-	O
-However	O
-,	O
-bromodomains	O
-did	O
-not	O
-interact	O
-(	O
-or	O
-associated	O
-very	O
-weakly	O
-)	O
-with	O
-longer	O
-acyl	O
-modifications	O
-,	O
-including	O
-crotonyllysine	O
-,	O
-as	O
-in	O
-the	O
-case	O
-of	O
-BDs	O
-of	O
-TAF1	O
-and	O
-BRD2	O
-,	O
-supporting	O
-recent	O
-reports	O
-.	O
-	O
-These	O
-results	O
-demonstrate	O
-that	O
-the	O
-YEATS	O
-domain	O
-is	O
-currently	O
-the	O
-sole	O
-reader	O
-of	O
-crotonyllysine	O
-.	O
-	O
-In	O
-conclusion	O
-,	O
-we	O
-have	O
-identified	O
-the	O
-YEATS	O
-domain	O
-of	O
-Taf14	O
-as	O
-the	O
-first	O
-reader	O
-of	O
-histone	O
-crotonylation	O
-.	O
-	O
-The	O
-unique	O
-and	O
-previously	O
-unobserved	O
-aromatic	O
--	O
-amide	O
-/	O
-aliphatic	O
--	O
-aromatic	O
-π	O
--	O
-π	O
--	O
-π	O
--	O
-stacking	O
-mechanism	O
-facilitates	O
-the	O
-specific	O
-recognition	O
-of	O
-the	O
-crotonyl	O
-moiety	O
-.	O
-	O
-We	O
-further	O
-demonstrate	O
-that	O
-H3K9cr	O
-exists	O
-in	O
-yeast	O
-and	O
-is	O
-dynamically	O
-regulated	O
-by	O
-HATs	O
-and	O
-HDACs	O
-.	O
-	O
-As	O
-we	O
-previously	O
-showed	O
-the	O
-importance	O
-of	O
-acyllysine	O
-binding	O
-by	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-for	O
-the	O
-DNA	O
-damage	O
-response	O
-and	O
-gene	O
-transcription	O
-,	O
-it	O
-will	O
-be	O
-essential	O
-in	O
-the	O
-future	O
-to	O
-define	O
-the	O
-physiological	O
-role	O
-of	O
-crotonyllysine	O
-recognition	O
-and	O
-to	O
-differentiate	O
-the	O
-activities	O
-of	O
-Taf14	O
-that	O
-are	O
-due	O
-to	O
-binding	O
-to	O
-crotonyllysine	O
-and	O
-acetyllysine	O
-modifications	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-functional	O
-significance	O
-of	O
-crotonyllysine	O
-recognition	O
-by	O
-other	O
-YEATS	O
-proteins	O
-will	O
-be	O
-of	O
-great	O
-importance	O
-to	O
-elucidate	O
-and	O
-compare	O
-.	O
-	O
-The	O
-structural	O
-mechanism	O
-for	O
-the	O
-recognition	O
-of	O
-H3K9cr	O
-	O
-(	O
-a	O
-)	O
-Chemical	O
-structure	O
-of	O
-crotonyllysine	O
-.	O
-(	O
-b	O
-)	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-(	O
-wheat	O
-)	O
-in	O
-complex	O
-with	O
-the	O
-H3K9cr5	O
--	O
-13	O
-peptide	O
-(	O
-green	O
-).	O
-(	O
-c	O
-)	O
-H3K9cr	O
-is	O
-stabilized	O
-via	O
-an	O
-extensive	O
-network	O
-of	O
-intermolecular	O
-electrostatic	O
-and	O
-polar	O
-interactions	O
-with	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-.	O
-	O
-(	O
-d	O
-)	O
-The	O
-π	O
--	O
-π	O
--	O
-π	O
-stacking	O
-mechanism	O
-involving	O
-the	O
-alkene	O
-moiety	O
-of	O
-crotonyllysine	O
-.	O
-	O
-H3K9cr	O
-is	O
-a	O
-selective	O
-target	O
-of	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-	O
-(	O
-a	O
-,	O
-b	O
-)	O
-Western	O
-blot	O
-analysis	O
-comparing	O
-the	O
-levels	O
-of	O
-H3K9cr	O
-and	O
-H3K9ac	O
-in	O
-wild	O
-type	O
-(	O
-WT	O
-),	O
-HAT	O
-deletion	O
-,	O
-or	O
-HDAC	O
-deletion	O
-yeast	O
-strains	O
-.	O
-	O
-Total	O
-H3	O
-was	O
-used	O
-as	O
-a	O
-loading	O
-control	O
-.	O
-	O
-(	O
-c	O
-)	O
-Superimposed	O
-1H	O
-,	O
-15N	O
-HSQC	O
-spectra	O
-of	O
-Taf14	O
-YEATS	O
-recorded	O
-as	O
-H3K9cr5	O
--	O
-13	O
-and	O
-H3K9ac5	O
--	O
-13	O
-peptides	O
-were	O
-titrated	O
-in	O
-.	O
-	O
-Spectra	O
-are	O
-color	O
-coded	O
-according	O
-to	O
-the	O
-protein	O
-:	O
-peptide	O
-molar	O
-ratio	O
-.	O
-	O
-(	O
-d	O
-)	O
-Western	O
-blot	O
-analyses	O
-of	O
-peptide	O
-pull	O
--	O
-down	O
-assays	O
-using	O
-wild	O
--	O
-type	O
-and	O
-mutated	O
-Taf14	O
-YEATS	O
-domains	O
-and	O
-indicated	O
-peptides	O
-.	O
-	O
-Structure	O
-of	O
-the	O
-GAT	O
-domain	O
-of	O
-the	O
-endosomal	O
-adapter	O
-protein	O
-Tom1	O
-	O
-Cellular	O
-homeostasis	O
-requires	O
-correct	O
-delivery	O
-of	O
-cell	O
--	O
-surface	O
-receptor	O
-proteins	O
-(	O
-cargo	O
-)	O
-to	O
-their	O
-target	O
-subcellular	O
-compartments	O
-.	O
-	O
-The	O
-adapter	O
-proteins	O
-Tom1	O
-and	O
-Tollip	O
-are	O
-involved	O
-in	O
-sorting	O
-of	O
-ubiquitinated	O
-cargo	O
-in	O
-endosomal	O
-compartments	O
-.	O
-	O
-Recruitment	O
-of	O
-Tom1	O
-to	O
-the	O
-endosomal	O
-compartments	O
-is	O
-mediated	O
-by	O
-its	O
-GAT	O
-domain	O
-’	O
-s	O
-association	O
-to	O
-Tollip	O
-’	O
-s	O
-Tom1	O
--	O
-binding	O
-domain	O
-(	O
-TBD	O
-).	O
-	O
-In	O
-this	O
-data	O
-article	O
-,	O
-we	O
-report	O
-the	O
-solution	O
-NMR	O
--	O
-derived	O
-structure	O
-of	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-.	O
-	O
-The	O
-estimated	O
-protein	O
-structure	O
-exhibits	O
-a	O
-bundle	O
-of	O
-three	O
-helical	O
-elements	O
-.	O
-	O
-We	O
-compare	O
-the	O
-Tom1	O
-GAT	O
-structure	O
-with	O
-those	O
-structures	O
-corresponding	O
-to	O
-the	O
-Tollip	O
-TBD	O
--	O
-and	O
-ubiquitin	O
--	O
-bound	O
-states	O
-.	O
-	O
-Subject	O
-area	O
-Biology	O
-More	O
-specific	O
-subject	O
-area	O
-Structural	O
-biology	O
-Type	O
-of	O
-data	O
-Table	O
-,	O
-text	O
-file	O
-,	O
-graph	O
-,	O
-figures	O
-How	O
-data	O
-was	O
-acquired	O
-Circular	O
-dichroism	O
-and	O
-NMR	O
-.	O
-	O
-NMR	O
-data	O
-was	O
-recorded	O
-using	O
-a	O
-Bruker	O
-800	O
-MHz	O
-Data	O
-format	O
-PDB	O
-format	O
-text	O
-file	O
-.	O
-	O
-Analyzed	O
-by	O
-CS	O
--	O
-Rosetta	O
-,	O
-Protein	O
-Structure	O
-Validation	O
-Server	O
-(	O
-PSVS	O
-),	O
-NMRPipe	O
-,	O
-NMRDraw	O
-,	O
-and	O
-PyMol	O
-Experimental	O
-factors	O
-Recombinant	O
-human	O
-Tom1	O
-GAT	O
-domain	O
-was	O
-purified	O
-to	O
-homogeneity	O
-before	O
-use	O
-Experimental	O
-features	O
-Solution	O
-structure	O
-of	O
-Tom1	O
-GAT	O
-was	O
-determined	O
-from	O
-NMR	O
-chemical	O
-shift	O
-data	O
-Data	O
-source	O
-location	O
-Virginia	O
-and	O
-Colorado	O
-,	O
-United	O
-States	O
-.	O
-	O
-Tom1	O
-GAT	O
-structural	O
-data	O
-is	O
-publicly	O
-available	O
-in	O
-the	O
-RCSB	O
-Protein	O
-Data	O
-Bank	O
-(	O
-http	O
-://	O
-www	O
-.	O
-rscb	O
-.	O
-org	O
-/)	O
-under	O
-the	O
-accession	O
-number	O
-PDB	O
-:	O
-2n9d	O
-	O
-The	O
-Tom1	O
-GAT	O
-domain	O
-solution	O
-structure	O
-will	O
-provide	O
-additional	O
-tools	O
-for	O
-modulating	O
-its	O
-biological	O
-function	O
-.	O
-	O
-Tom1	O
-GAT	O
-can	O
-adopt	O
-distinct	O
-conformations	O
-upon	O
-ligand	O
-binding	O
-.	O
-	O
-A	O
-conformational	O
-response	O
-of	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-upon	O
-Tollip	O
-TBD	O
-binding	O
-can	O
-serve	O
-as	O
-an	O
-example	O
-to	O
-explain	O
-mutually	O
-exclusive	O
-ligand	O
-binding	O
-events	O
-.	O
-	O
-Analysis	O
-of	O
-the	O
-far	O
--	O
-UV	O
-circular	O
-dichroism	O
-(	O
-CD	O
-)	O
-spectrum	O
-of	O
-the	O
-Tom	O
-1	O
-GAT	O
-domain	O
-(	O
-Fig	O
-.	O
-1	O
-)	O
-predicts	O
-58	O
-.	O
-7	O
-%	O
-α	O
--	O
-helix	O
-,	O
-3	O
-%	O
-β	O
--	O
-strand	O
-,	O
-15	O
-.	O
-5	O
-%	O
-turn	O
-,	O
-and	O
-22	O
-.	O
-8	O
-%	O
-disordered	O
-regions	O
-.	O
-	O
-The	O
-Tom1	O
-GAT	O
-structural	O
-restraints	O
-yielded	O
-ten	O
-helical	O
-structures	O
-(	O
-Fig	O
-.	O
-2A	O
-,	O
-B	O
-)	O
-with	O
-a	O
-root	O
-mean	O
-square	O
-deviation	O
-(	O
-RMSD	O
-)	O
-of	O
-0	O
-.	O
-9	O
-Å	O
-for	O
-backbone	O
-and	O
-1	O
-.	O
-3	O
-Å	O
-for	O
-all	O
-heavy	O
-atoms	O
-(	O
-Table	O
-1	O
-)	O
-and	O
-estimated	O
-the	O
-presence	O
-of	O
-three	O
-helices	O
-spanning	O
-residues	O
-Q216	O
--	O
-E240	O
-(	O
-α	O
--	O
-helix	O
-1	O
-),	O
-P248	O
--	O
-Q274	O
-(	O
-α	O
--	O
-helix	O
-2	O
-),	O
-and	O
-E278	O
--	O
-T306	O
-(	O
-α	O
--	O
-helix	O
-3	O
-).	O
-	O
-Unlike	O
-ubiquitin	O
-binding	O
-,	O
-data	O
-suggest	O
-that	O
-conformational	O
-changes	O
-of	O
-the	O
-Tom1	O
-GAT	O
-α	O
--	O
-helices	O
-1	O
-and	O
-2	O
-occur	O
-upon	O
-Tollip	O
-TBD	O
-binding	O
-(	O
-Fig	O
-.	O
-3A	O
-,	O
-B	O
-).	O
-	O
-Representative	O
-far	O
--	O
-UV	O
-CD	O
-spectrum	O
-of	O
-the	O
-His	O
--	O
-Tom1	O
-GAT	O
-domain	O
-.	O
-	O
-(	O
-A	O
-)	O
-Stereo	O
-view	O
-displaying	O
-the	O
-best	O
--	O
-fit	O
-backbone	O
-superposition	O
-of	O
-the	O
-refined	O
-structures	O
-for	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-.	O
-	O
-Helices	O
-are	O
-shown	O
-in	O
-orange	O
-,	O
-whereas	O
-loops	O
-are	O
-colored	O
-in	O
-green	O
-.	O
-(	O
-B	O
-)	O
-Ribbon	O
-illustration	O
-of	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-.	O
-	O
-(	O
-A	O
-)	O
-Two	O
-views	O
-of	O
-the	O
-superimposed	O
-structures	O
-of	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-in	O
-the	O
-free	O
-state	O
-(	O
-gray	O
-)	O
-with	O
-that	O
-in	O
-the	O
-Tollip	O
-TBD	O
--	O
-bound	O
-state	O
-(	O
-red	O
-).	O
-(	O
-B	O
-)	O
-Two	O
-views	O
-of	O
-the	O
-superimposed	O
-structures	O
-of	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-(	O
-gray	O
-)	O
-with	O
-that	O
-in	O
-the	O
-Ub	O
--	O
-bound	O
-state	O
-(	O
-green	O
-).	O
-	O
-NMR	O
-and	O
-refinement	O
-statistics	O
-for	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-.	O
-	O
-NMR	O
-structural	O
-statistics	O
-for	O
-lowest	O
-energy	O
-conformers	O
-of	O
-Tom1	O
-GAT	O
-using	O
-PSVS	O
-.	O
-	O
-deviations	O
-were	O
-obtained	O
-by	O
-superimposing	O
-residues	O
-215	O
-–	O
-309	O
-of	O
-Tom1	O
-GAT	O
-among	O
-10	O
-lowest	O
-energy	O
-refined	O
-structures	O
-.	O
-	O
-Haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-/	O
-Sigma	O
--	O
-2	O
-receptor	O
-facilitates	O
-cancer	O
-proliferation	O
-and	O
-chemoresistance	O
-	O
-Progesterone	O
--	O
-receptor	O
-membrane	O
-component	O
-1	O
-(	O
-PGRMC1	O
-/	O
-Sigma	O
--	O
-2	O
-receptor	O
-)	O
-is	O
-a	O
-haem	O
--	O
-containing	O
-protein	O
-that	O
-interacts	O
-with	O
-epidermal	O
-growth	O
-factor	O
-receptor	O
-(	O
-EGFR	O
-)	O
-and	O
-cytochromes	O
-P450	O
-to	O
-regulate	O
-cancer	O
-proliferation	O
-and	O
-chemoresistance	O
-;	O
-its	O
-structural	O
-basis	O
-remains	O
-unknown	O
-.	O
-	O
-Here	O
-crystallographic	O
-analyses	O
-of	O
-the	O
-PGRMC1	O
-cytosolic	O
-domain	O
-at	O
-1	O
-.	O
-95	O
-Å	O
-resolution	O
-reveal	O
-that	O
-it	O
-forms	O
-a	O
-stable	O
-dimer	O
-through	O
-stacking	O
-interactions	O
-of	O
-two	O
-protruding	O
-haem	O
-molecules	O
-.	O
-	O
-The	O
-haem	O
-iron	O
-is	O
-five	O
--	O
-coordinated	O
-by	O
-Tyr113	O
-,	O
-and	O
-the	O
-open	O
-surface	O
-of	O
-the	O
-haem	O
-mediates	O
-dimerization	O
-.	O
-	O
-Carbon	O
-monoxide	O
-(	O
-CO	O
-)	O
-interferes	O
-with	O
-PGRMC1	O
-dimerization	O
-by	O
-binding	O
-to	O
-the	O
-sixth	O
-coordination	O
-site	O
-of	O
-the	O
-haem	O
-.	O
-	O
-Haem	O
--	O
-mediated	O
-PGRMC1	O
-dimerization	O
-is	O
-required	O
-for	O
-interactions	O
-with	O
-EGFR	O
-and	O
-cytochromes	O
-P450	O
-,	O
-cancer	O
-proliferation	O
-and	O
-chemoresistance	O
-against	O
-anti	O
--	O
-cancer	O
-drugs	O
-;	O
-these	O
-events	O
-are	O
-attenuated	O
-by	O
-either	O
-CO	O
-or	O
-haem	O
-deprivation	O
-in	O
-cancer	O
-cells	O
-.	O
-	O
-This	O
-study	O
-demonstrates	O
-protein	O
-dimerization	O
-via	O
-haem	O
-–	O
-haem	O
-stacking	O
-,	O
-which	O
-has	O
-not	O
-been	O
-seen	O
-in	O
-eukaryotes	O
-,	O
-and	O
-provides	O
-insights	O
-into	O
-its	O
-functional	O
-significance	O
-in	O
-cancer	O
-.	O
-	O
-PGRMC1	O
-binds	O
-to	O
-EGFR	O
-and	O
-cytochromes	O
-P450	O
-,	O
-and	O
-is	O
-known	O
-to	O
-be	O
-involved	O
-in	O
-cancer	O
-proliferation	O
-and	O
-in	O
-drug	O
-resistance	O
-.	O
-	O
-Here	O
-,	O
-the	O
-authors	O
-determine	O
-the	O
-structure	O
-of	O
-the	O
-cytosolic	O
-domain	O
-of	O
-PGRMC1	O
-,	O
-which	O
-forms	O
-a	O
-dimer	O
-via	O
-haem	O
-–	O
-haem	O
-stacking	O
-,	O
-and	O
-propose	O
-how	O
-this	O
-interaction	O
-could	O
-be	O
-involved	O
-in	O
-its	O
-function	O
-.	O
-	O
-Much	O
-attention	O
-has	O
-been	O
-paid	O
-to	O
-the	O
-roles	O
-of	O
-haem	O
--	O
-iron	O
-in	O
-cancer	O
-development	O
-.	O
-	O
-Increased	O
-dietary	O
-intake	O
-of	O
-haem	O
-is	O
-a	O
-risk	O
-factor	O
-for	O
-several	O
-types	O
-of	O
-cancer	O
-.	O
-	O
-Previous	O
-studies	O
-showed	O
-that	O
-deprivation	O
-of	O
-iron	O
-or	O
-haem	O
-suppresses	O
-tumourigenesis	O
-.	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-carbon	O
-monoxide	O
-(	O
-CO	O
-),	O
-the	O
-gaseous	O
-mediator	O
-generated	O
-by	O
-oxidative	O
-degradation	O
-of	O
-haem	O
-via	O
-haem	O
-oxygenase	O
-(	O
-HO	O
-),	O
-inhibits	O
-tumour	O
-growth	O
-.	O
-	O
-Thus	O
-,	O
-a	O
-tenuous	O
-balance	O
-between	O
-free	O
-haem	O
-and	O
-CO	O
-plays	O
-key	O
-roles	O
-in	O
-cancer	O
-development	O
-and	O
-chemoresistance	O
-,	O
-although	O
-the	O
-underlying	O
-mechanisms	O
-are	O
-not	O
-fully	O
-understood	O
-.	O
-	O
-To	O
-gain	O
-insight	O
-into	O
-the	O
-underlying	O
-mechanisms	O
-,	O
-we	O
-took	O
-chemical	O
-biological	O
-approaches	O
-using	O
-affinity	O
-nanobeads	O
-carrying	O
-haem	O
-and	O
-identified	O
-progesterone	O
--	O
-receptor	O
-membrane	O
-component	O
-1	O
-(	O
-PGRMC1	O
-)	O
-as	O
-a	O
-haem	O
--	O
-binding	O
-protein	O
-from	O
-mouse	O
-liver	O
-extracts	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-PGRMC1	O
-is	O
-a	O
-member	O
-of	O
-the	O
-membrane	O
--	O
-associated	O
-progesterone	O
-receptor	O
-(	O
-MAPR	O
-)	O
-family	O
-with	O
-a	O
-cytochrome	O
-b5	O
--	O
-like	O
-haem	O
--	O
-binding	O
-region	O
-,	O
-and	O
-is	O
-known	O
-to	O
-be	O
-highly	O
-expressed	O
-in	O
-various	O
-types	O
-of	O
-cancers	O
-.	O
-	O
-PGRMC1	O
-is	O
-anchored	O
-to	O
-the	O
-cell	O
-membrane	O
-through	O
-the	O
-N	O
--	O
-terminal	O
-transmembrane	O
-helix	O
-and	O
-interacts	O
-with	O
-epidermal	O
-growth	O
-factor	O
-receptor	O
-(	O
-EGFR	O
-)	O
-and	O
-cytochromes	O
-P450	O
-(	O
-ref	O
-).	O
-	O
-While	O
-PGRMC1	O
-is	O
-implicated	O
-in	O
-cell	O
-proliferation	O
-and	O
-cholesterol	O
-biosynthesis	O
-,	O
-the	O
-structural	O
-basis	O
-on	O
-which	O
-PGRMC1	O
-exerts	O
-its	O
-function	O
-remains	O
-largely	O
-unknown	O
-.	O
-	O
-Here	O
-we	O
-show	O
-that	O
-PGRMC1	O
-exhibits	O
-a	O
-unique	O
-haem	O
--	O
-dependent	O
-dimerization	O
-.	O
-	O
-The	O
-dimer	O
-binds	O
-to	O
-EGFR	O
-and	O
-cytochromes	O
-P450	O
-to	O
-enhance	O
-tumour	O
-cell	O
-proliferation	O
-and	O
-chemoresistance	O
-.	O
-	O
-The	O
-dimer	O
-is	O
-dissociated	O
-to	O
-monomers	O
-by	O
-physiological	O
-levels	O
-of	O
-CO	O
-,	O
-suggesting	O
-that	O
-PGRMC1	O
-serves	O
-as	O
-a	O
-CO	O
--	O
-sensitive	O
-molecular	O
-switch	O
-regulating	O
-cancer	O
-cell	O
-proliferation	O
-.	O
-	O
-X	O
--	O
-ray	O
-crystal	O
-structure	O
-of	O
-PGRMC1	O
-	O
-We	O
-solved	O
-the	O
-crystal	O
-structure	O
-of	O
-the	O
-haem	O
--	O
-bound	O
-PGRMC1	O
-cytosolic	O
-domain	O
-(	O
-a	O
-.	O
-a	O
-.	O
-72	O
-–	O
-195	O
-)	O
-at	O
-1	O
-.	O
-95	O
-Å	O
-resolution	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-In	O
-the	O
-presence	O
-of	O
-haem	O
-,	O
-PGRMC1	O
-forms	O
-a	O
-dimeric	O
-structure	O
-largely	O
-through	O
-hydrophobic	O
-interactions	O
-between	O
-the	O
-haem	O
-moieties	O
-of	O
-two	O
-monomers	O
-(	O
-Fig	O
-.	O
-1a	O
-,	O
-Table	O
-1	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-;	O
-a	O
-stereo	O
--	O
-structural	O
-image	O
-is	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-4	O
-).	O
-	O
-While	O
-the	O
-overall	O
-fold	O
-of	O
-PGRMC1	O
-is	O
-similar	O
-to	O
-that	O
-of	O
-canonical	O
-cytochrome	O
-b5	O
-,	O
-their	O
-haem	O
-irons	O
-are	O
-coordinated	O
-differently	O
-.	O
-	O
-In	O
-cytochrome	O
-b5	O
-,	O
-the	O
-haem	O
-iron	O
-is	O
-six	O
--	O
-coordinated	O
-by	O
-two	O
-axial	O
-histidine	O
-residues	O
-.	O
-	O
-These	O
-histidines	O
-are	O
-missing	O
-in	O
-PGRMC1	O
-,	O
-and	O
-the	O
-haem	O
-iron	O
-is	O
-five	O
--	O
-coordinated	O
-by	O
-Tyr113	O
-(	O
-Y113	O
-)	O
-alone	O
-(	O
-Fig	O
-.	O
-1b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-A	O
-homologous	O
-helix	O
-that	O
-holds	O
-haem	O
-in	O
-cytochrome	O
-b5	O
-is	O
-longer	O
-,	O
-shifts	O
-away	O
-from	O
-haem	O
-,	O
-and	O
-does	O
-not	O
-form	O
-a	O
-coordinate	O
-bond	O
-in	O
-PGRMC1	O
-(	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-Consequently	O
-,	O
-the	O
-five	O
--	O
-coordinated	O
-haem	O
-of	O
-PGRMC1	O
-has	O
-an	O
-open	O
-surface	O
-that	O
-allows	O
-its	O
-dimerization	O
-through	O
-hydrophobic	O
-haem	O
-–	O
-haem	O
-stacking	O
-.	O
-	O
-Contrary	O
-to	O
-our	O
-finding	O
-,	O
-Kaluka	O
-et	O
-al	O
-.	O
-recently	O
-reported	O
-that	O
-Tyr164	O
-of	O
-PGRMC1	O
-is	O
-the	O
-axial	O
-ligand	O
-of	O
-haem	O
-because	O
-mutation	O
-of	O
-this	O
-residue	O
-impairs	O
-haem	O
-binding	O
-.	O
-	O
-Our	O
-structural	O
-data	O
-revealed	O
-that	O
-Tyr164	O
-and	O
-a	O
-few	O
-other	O
-residues	O
-such	O
-as	O
-Tyr107	O
-and	O
-Lys163	O
-are	O
-in	O
-fact	O
-hydrogen	O
--	O
-bonded	O
-to	O
-haem	O
-propionates	O
-.	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-observations	O
-by	O
-Min	O
-et	O
-al	O
-.	O
-that	O
-Tyr	O
-107	O
-and	O
-Tyr113	O
-of	O
-PGRMC1	O
-are	O
-involved	O
-in	O
-binding	O
-with	O
-haem	O
-.	O
-	O
-These	O
-amino	O
-acid	O
-residues	O
-are	O
-conserved	O
-among	O
-MAPR	O
-family	O
-members	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5a	O
-),	O
-suggesting	O
-that	O
-these	O
-proteins	O
-share	O
-the	O
-ability	O
-to	O
-exhibit	O
-haem	O
--	O
-dependent	O
-dimerization	O
-.	O
-	O
-PGRMC1	O
-exhibits	O
-haem	O
--	O
-dependent	O
-dimerization	O
-in	O
-solution	O
-	O
-In	O
-the	O
-PGRMC1	O
-crystal	O
-,	O
-two	O
-different	O
-types	O
-of	O
-crystal	O
-contacts	O
-(	O
-chain	O
-A	O
-–	O
-A	O
-″	O
-and	O
-A	O
-–	O
-B	O
-)	O
-were	O
-observed	O
-in	O
-addition	O
-to	O
-the	O
-haem	O
--	O
-mediated	O
-dimer	O
-(	O
-chain	O
-A	O
-–	O
-A	O
-′)	O
-(	O
-Supplementary	O
-Figs	O
-3	O
-and	O
-6a	O
-).	O
-	O
-To	O
-confirm	O
-that	O
-haem	O
--	O
-assisted	O
-dimerization	O
-of	O
-PGRMC1	O
-occurs	O
-in	O
-solution	O
-,	O
-we	O
-analysed	O
-the	O
-structure	O
-of	O
-apo	O
--	O
-and	O
-haem	O
--	O
-bound	O
-PGMRC1	O
-by	O
-two	O
--	O
-dimensional	O
-nuclear	O
-magnetic	O
-resonance	O
-(	O
-NMR	O
-)	O
-using	O
-heteronuclear	O
-single	O
--	O
-quantum	O
-coherence	O
-and	O
-transverse	O
-relaxation	O
--	O
-optimized	O
-spectroscopy	O
-(	O
-Supplementary	O
-Figs	O
-6b	O
-and	O
-7	O
-).	O
-	O
-NMR	O
-signals	O
-from	O
-some	O
-amino	O
-acid	O
-residues	O
-of	O
-PGRMC1	O
-disappeared	O
-due	O
-to	O
-the	O
-paramagnetic	O
-relaxation	O
-effect	O
-of	O
-haem	O
-(	O
-Supplementary	O
-Figs	O
-6b	O
-);	O
-these	O
-residues	O
-were	O
-located	O
-in	O
-the	O
-haem	O
--	O
-binding	O
-region	O
-.	O
-	O
-When	O
-chemical	O
-shifts	O
-of	O
-apo	O
--	O
-and	O
-haem	O
--	O
-bound	O
-forms	O
-of	O
-PGMRC1	O
-were	O
-compared	O
-,	O
-some	O
-amino	O
-acid	O
-residues	O
-close	O
-to	O
-those	O
-which	O
-disappeared	O
-because	O
-of	O
-the	O
-paramagnetic	O
-relaxation	O
-effect	O
-of	O
-haem	O
-exhibit	O
-notable	O
-chemical	O
-shifts	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-,	O
-b	O
-;	O
-dark	O
-yellow	O
-).	O
-	O
-However	O
-,	O
-at	O
-the	O
-interfaces	O
-of	O
-the	O
-other	O
-possible	O
-dimeric	O
-structures	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-,	O
-chain	O
-A	O
-–	O
-A	O
-″;	O
-cyan	O
-and	O
-chain	O
-A	O
-–	O
-B	O
-;	O
-violet	O
-),	O
-no	O
-significant	O
-difference	O
-was	O
-observed	O
-.	O
-	O
-Furthermore	O
-,	O
-free	O
-energy	O
-of	O
-dissociation	O
-predicted	O
-by	O
-PISA	O
-suggested	O
-that	O
-the	O
-haem	O
--	O
-mediated	O
-dimer	O
-is	O
-stable	O
-in	O
-solution	O
-while	O
-the	O
-other	O
-potential	O
-interactions	O
-are	O
-not	O
-.	O
-	O
-We	O
-also	O
-attempted	O
-to	O
-predict	O
-the	O
-secondary	O
-structure	O
-of	O
-PGRMC1	O
-through	O
-NMR	O
-data	O
-by	O
-calculating	O
-with	O
-TALOS	O
-+	O
-program	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-8	O
-);	O
-the	O
-prediction	O
-suggested	O
-that	O
-the	O
-overall	O
-secondary	O
-structure	O
-is	O
-comparable	O
-between	O
-apo	O
--	O
-and	O
-haem	O
--	O
-bound	O
-forms	O
-of	O
-PGRMC1	O
-in	O
-solution	O
-.	O
-	O
-We	O
-analysed	O
-the	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-the	O
-PGRMC1	O
-cytosolic	O
-domain	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-in	O
-solution	O
-(	O
-Fig	O
-.	O
-2	O
-and	O
-Table	O
-2	O
-).	O
-	O
-Mass	O
-spectrometry	O
-(	O
-MS	O
-)	O
-analyses	O
-under	O
-non	O
--	O
-denaturing	O
-condition	O
-demonstrated	O
-that	O
-the	O
-apo	O
--	O
-monomer	O
-PGRMC1	O
-resulted	O
-in	O
-dimerization	O
-by	O
-binding	O
-with	O
-haem	O
-(	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-a	O
-disulfide	O
-bond	O
-between	O
-two	O
-Cys129	O
-residues	O
-is	O
-observed	O
-in	O
-the	O
-crystal	O
-of	O
-PGRMC1	O
-(	O
-Fig	O
-.	O
-1a	O
-),	O
-while	O
-Cys129	O
-is	O
-not	O
-conserved	O
-among	O
-the	O
-MAPR	O
-family	O
-proteins	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-This	O
-observation	O
-led	O
-us	O
-to	O
-examine	O
-whether	O
-or	O
-not	O
-the	O
-disulfide	O
-bond	O
-contributes	O
-to	O
-PGRMC1	O
-dimerization	O
-.	O
-	O
-MS	O
-analyses	O
-under	O
-non	O
--	O
-denaturing	O
-conditions	O
-clearly	O
-showed	O
-that	O
-the	O
-Cys129Ser	B-mutant
-(	O
-C129S	B-mutant
-)	O
-mutant	O
-is	O
-dimerized	O
-in	O
-the	O
-presence	O
-of	O
-haem	O
-,	O
-indicating	O
-that	O
-the	O
-haem	O
--	O
-mediated	O
-dimerization	O
-of	O
-PGRMC1	O
-occurs	O
-independently	O
-of	O
-the	O
-disulfide	O
-bond	O
-formation	O
-via	O
-Cys129	O
-(	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-Supporting	O
-this	O
-,	O
-MS	O
-analyses	O
-under	O
-denaturing	O
-conditions	O
-showed	O
-that	O
-haem	O
--	O
-mediated	O
-PGRMC1	O
-dimer	O
-is	O
-completely	O
-dissociated	O
-into	O
-monomer	O
-,	O
-indicating	O
-that	O
-dimerization	O
-of	O
-this	O
-kind	O
-is	O
-not	O
-mediated	O
-by	O
-any	O
-covalent	O
-bond	O
-such	O
-as	O
-disulfide	O
-bond	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-9	O
-).	O
-	O
-We	O
-also	O
-analysed	O
-the	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-by	O
-diffusion	O
--	O
-ordered	O
-NMR	O
-spectroscopy	O
-(	O
-DOSY	O
-)	O
-analyses	O
-(	O
-Table	O
-2	O
-,	O
-Supplementary	O
-Fig	O
-.	O
-10	O
-).	O
-	O
-The	O
-results	O
-suggested	O
-that	O
-the	O
-hydrodynamic	O
-radius	O
-of	O
-haem	O
--	O
-bound	O
-PGRMC1	O
-is	O
-larger	O
-than	O
-that	O
-of	O
-apo	O
--	O
-PGRMC1	O
-.	O
-	O
-To	O
-further	O
-evaluate	O
-changes	O
-in	O
-molecular	O
-weights	O
-in	O
-dimerization	O
-of	O
-PGRMC1	O
-,	O
-sedimentation	O
-velocity	O
-analytical	O
-ultracentrifugation	O
-(	O
-SV	O
--	O
-AUC	O
-)	O
-analysis	O
-was	O
-carried	O
-out	O
-.	O
-	O
-Whereas	O
-the	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-)	O
-apo	O
--	O
-PGRMC1	O
-appeared	O
-at	O
-a	O
-1	O
-.	O
-9	O
-S	O
-peak	O
-as	O
-monomer	O
-,	O
-the	O
-haem	O
--	O
-binding	O
-PGRMC1	O
-was	O
-converted	O
-into	O
-dimer	O
-at	O
-a	O
-3	O
-.	O
-1	O
-S	O
-peak	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-Similarly	O
-,	O
-the	O
-C129S	B-mutant
-mutant	O
-of	O
-PGRMC1	O
-converted	O
-from	O
-monomer	O
-to	O
-dimer	O
-by	O
-binding	O
-to	O
-haem	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-SV	O
--	O
-AUC	O
-analyses	O
-also	O
-allowed	O
-us	O
-to	O
-examine	O
-the	O
-stability	O
-of	O
-haem	O
-/	O
-PGRMC1	O
-dimer	O
-.	O
-	O
-To	O
-this	O
-end	O
-,	O
-we	O
-used	O
-different	O
-concentrations	O
-(	O
-3	O
-.	O
-5	O
-–	O
-147	O
-μmol	O
-l	O
-−	O
-1	O
-)	O
-of	O
-haem	O
--	O
-bound	O
-PGRMC1	O
-protein	O
-(	O
-a	O
-.	O
-a	O
-.	O
-72	O
-–	O
-195	O
-),	O
-which	O
-were	O
-identical	O
-to	O
-that	O
-used	O
-in	O
-the	O
-crystallographic	O
-analysis	O
-.	O
-	O
-The	O
-sedimentation	O
-coefficients	O
-calculated	O
-on	O
-the	O
-basis	O
-of	O
-the	O
-crystal	O
-structure	O
-were	O
-1	O
-.	O
-71	O
-S	O
-for	O
-monomer	O
-and	O
-2	O
-.	O
-56	O
-S	O
-for	O
-dimer	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-11	O
-,	O
-upper	O
-panel	O
-).	O
-	O
-The	O
-results	O
-showed	O
-that	O
-the	O
-PGRMC1	O
-dimer	O
-is	O
-not	O
-dissociated	O
-into	O
-monomer	O
-at	O
-all	O
-concentrations	O
-examined	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-11	O
-,	O
-lower	O
-panel	O
-),	O
-suggesting	O
-that	O
-the	O
-Kd	O
-value	O
-of	O
-haem	O
--	O
-mediated	O
-dimer	O
-of	O
-PGRMC1	O
-is	O
-under	O
-3	O
-.	O
-5	O
-μmol	O
-l	O
-−	O
-1	O
-.	O
-	O
-A	O
-value	O
-of	O
-this	O
-kind	O
-implies	O
-that	O
-the	O
-PGRMC1	O
-dimer	O
-is	O
-more	O
-stable	O
-than	O
-other	O
-dimers	O
-of	O
-extracellular	O
-domain	O
-of	O
-membrane	O
-proteins	O
-such	O
-as	O
-Toll	O
-like	O
-receptor	O
-9	O
-(	O
-dimerization	O
-Kd	O
-of	O
-20	O
-μmol	O
-l	O
-−	O
-1	O
-)	O
-(	O
-ref	O
-.)	O
-and	O
-plexin	O
-A2	O
-receptor	O
-(	O
-dimerization	O
-Kd	O
-higher	O
-than	O
-300	O
-μmol	O
-l	O
-−	O
-1	O
-)	O
-(	O
-ref	O
-.).	O
-	O
-The	O
-current	O
-analytical	O
-data	O
-confirmed	O
-that	O
-apo	O
--	O
-PGRMC1	O
-monomer	O
-converts	O
-into	O
-dimer	O
-by	O
-binding	O
-to	O
-haem	O
-in	O
-solution	O
-(	O
-Table	O
-2	O
-).	O
-	O
-We	O
-also	O
-showed	O
-by	O
-haem	O
-titration	O
-experiments	O
-that	O
-haem	O
-binding	O
-to	O
-PGRMC1	O
-was	O
-of	O
-low	O
-affinity	O
-with	O
-a	O
-Kd	O
-value	O
-of	O
-50	O
-nmol	O
-l	O
-−	O
-1	O
-;	O
-this	O
-is	O
-comparable	O
-with	O
-that	O
-of	O
-iron	O
-regulatory	O
-protein	O
-2	O
-,	O
-which	O
-is	O
-known	O
-to	O
-be	O
-regulated	O
-by	O
-intracellular	O
-levels	O
-of	O
-haem	O
-(	O
-Fig	O
-.	O
-2c	O
-and	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-These	O
-results	O
-raised	O
-the	O
-possibility	O
-that	O
-the	O
-function	O
-of	O
-PGRMC1	O
-is	O
-regulated	O
-by	O
-intracellular	O
-haem	O
-concentrations	O
-.	O
-	O
-CO	O
-inhibits	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-	O
-Crystallographic	O
-analyses	O
-revealed	O
-that	O
-Tyr113	O
-of	O
-PGRMC1	O
-is	O
-an	O
-axial	O
-ligand	O
-for	O
-haem	O
-and	O
-contributes	O
-to	O
-haem	O
--	O
-dependent	O
-dimerization	O
-(	O
-Fig	O
-.	O
-1a	O
-).	O
-	O
-Analysis	O
-of	O
-UV	O
--	O
-visible	O
-spectra	O
-revealed	O
-that	O
-the	O
-heme	O
-of	O
-PGRMC1	O
-is	O
-reducible	O
-from	O
-ferric	O
-to	O
-ferrous	O
-state	O
-,	O
-thus	O
-allowing	O
-CO	O
-binding	O
-(	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-Furthermore	O
-,	O
-the	O
-UV	O
--	O
-visible	O
-spectrum	O
-of	O
-the	O
-wild	O
-type	O
-PGRMC1	O
-was	O
-the	O
-same	O
-as	O
-that	O
-of	O
-the	O
-C129S	B-mutant
-mutant	O
-of	O
-PGRMC1	O
-,	O
-and	O
-the	O
-R	O
-/	O
-Z	O
-ratio	O
-determined	O
-by	O
-the	O
-intensities	O
-between	O
-the	O
-Soret	O
-band	O
-(	O
-394	O
-nm	O
-)	O
-peak	O
-and	O
-the	O
-274	O
--	O
-nm	O
-peak	O
-showed	O
-that	O
-these	O
-proteins	O
-were	O
-fully	O
-loaded	O
-with	O
-haem	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-12	O
-).	O
-	O
-Analysis	O
-of	O
-the	O
-ferric	O
-form	O
-of	O
-PGRMC1	O
-using	O
-resonance	O
-Raman	O
-spectroscopy	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-13	O
-)	O
-showed	O
-that	O
-the	O
-relative	O
-intensity	O
-of	O
-oxidation	O
-and	O
-spin	O
-state	O
-marker	O
-bands	O
-(	O
-ν4	O
-and	O
-ν3	O
-)	O
-is	O
-close	O
-to	O
-1	O
-.	O
-0	O
-,	O
-which	O
-is	O
-consistent	O
-with	O
-it	O
-being	O
-a	O
-haem	O
-protein	O
-with	O
-a	O
-proximal	O
-Tyr	O
-coordination	O
-.	O
-	O
-A	O
-specific	O
-Raman	O
-shift	O
-peaking	O
-at	O
-vFe	O
-–	O
-CO	O
-=	O
-500	O
-cm	O
-−	O
-1	O
-demonstrated	O
-that	O
-the	O
-CO	O
--	O
-bound	O
-haem	O
-of	O
-PGRMC1	O
-is	O
-six	O
--	O
-coordinated	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-13	O
-).	O
-	O
-Since	O
-PGRMC1	O
-dimerization	O
-involves	O
-the	O
-open	O
-surface	O
-of	O
-haem	O
-on	O
-the	O
-opposite	O
-side	O
-of	O
-the	O
-axial	O
-Tyr113	O
-,	O
-no	O
-space	O
-for	O
-CO	O
-binding	O
-is	O
-available	O
-in	O
-the	O
-dimeric	O
-structure	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-This	O
-prompted	O
-us	O
-to	O
-ask	O
-if	O
-CO	O
-binding	O
-to	O
-haem	O
-causes	O
-dissociation	O
-of	O
-the	O
-PGRMC1	O
-dimer	O
-.	O
-	O
-Analysis	O
-by	O
-gel	O
-filtration	O
-chromatography	O
-revealed	O
-that	O
-the	O
-relative	O
-molecular	O
-sizes	O
-of	O
-the	O
-wild	O
--	O
-type	O
-and	O
-the	O
-C129S	B-mutant
-mutant	O
-of	O
-PGRMC1	O
-are	O
-increased	O
-by	O
-adding	O
-haem	O
-to	O
-apo	O
--	O
-PGRMC1	O
-regardless	O
-of	O
-the	O
-oxidation	O
-state	O
-of	O
-the	O
-iron	O
-(	O
-Fig	O
-.	O
-3c	O
-),	O
-which	O
-is	O
-in	O
-agreement	O
-with	O
-the	O
-results	O
-in	O
-Table	O
-1	O
-.	O
-	O
-CO	O
-application	O
-to	O
-ferrous	O
-PGRMC1	O
-abolished	O
-the	O
-haem	O
--	O
-dependent	O
-increase	O
-in	O
-its	O
-molecular	O
-size	O
-.	O
-	O
-Under	O
-this	O
-reducing	O
-condition	O
-in	O
-the	O
-presence	O
-of	O
-dithionite	O
-,	O
-analyses	O
-of	O
-UV	O
--	O
-visible	O
-spectra	O
-indicated	O
-that	O
-CO	O
--	O
-binding	O
-with	O
-haem	O
--	O
-PGRMC1	O
-is	O
-stable	O
-,	O
-showing	O
-only	O
-20	O
-%	O
-reduction	O
-of	O
-the	O
-absorbance	O
-at	O
-412	O
-nm	O
-within	O
-2	O
-h	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-14	O
-).	O
-	O
-Furthermore	O
-,	O
-the	O
-Tyr113Phe	B-mutant
-(	O
-Y113F	B-mutant
-)	O
-mutant	O
-of	O
-PGRMC1	O
-was	O
-not	O
-responsive	O
-to	O
-haem	O
-.	O
-	O
-These	O
-results	O
-suggest	O
-that	O
-CO	O
-favours	O
-the	O
-six	O
--	O
-coordinate	O
-form	O
-of	O
-haem	O
-and	O
-interferes	O
-with	O
-the	O
-haem	O
--	O
-mediated	O
-dimerization	O
-of	O
-PGRMC1	O
-.	O
-	O
-To	O
-examine	O
-the	O
-inhibitory	O
-effects	O
-of	O
-CO	O
-on	O
-haem	O
--	O
-mediated	O
-PGRMC1	O
-dimerization	O
-,	O
-SV	O
--	O
-AUC	O
-analysis	O
-was	O
-carried	O
-out	O
-.	O
-	O
-The	O
-peak	O
-corresponding	O
-to	O
-the	O
-haem	O
-/	O
-PGRMC1	O
-dimer	O
-was	O
-detected	O
-under	O
-reducing	O
-conditions	O
-in	O
-the	O
-presence	O
-of	O
-dithionite	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-15	O
-,	O
-middle	O
-panel	O
-).	O
-	O
-Under	O
-these	O
-circumstances	O
-,	O
-CO	O
-application	O
-induced	O
-dissociation	O
-of	O
-the	O
-haem	O
--	O
-mediated	O
-dimers	O
-of	O
-PGRMC1	O
-to	O
-generate	O
-a	O
-peak	O
-of	O
-monomers	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-15	O
-,	O
-lower	O
-panel	O
-).	O
-	O
-These	O
-observations	O
-raised	O
-the	O
-transition	O
-model	O
-for	O
-structural	O
-regulation	O
-of	O
-PGRMC1	O
-in	O
-response	O
-to	O
-haem	O
-(	O
-Fig	O
-.	O
-3d	O
-).	O
-	O
-As	O
-mentioned	O
-above	O
-,	O
-apo	O
--	O
-PGRMC1	O
-exists	O
-as	O
-monomer	O
-.	O
-	O
-By	O
-binding	O
-with	O
-haem	O
-(	O
-binding	O
-Kd	O
-=	O
-50	O
-nmol	O
-l	O
-−	O
-1	O
-),	O
-PGRMC1	O
-forms	O
-a	O
-stable	O
-dimer	O
-(	O
-dimerization	O
-Kd	O
-<<	O
-3	O
-.	O
-5	O
-μmol	O
-l	O
-−	O
-1	O
-)	O
-through	O
-stacking	O
-of	O
-the	O
-two	O
-open	O
-surfaces	O
-of	O
-the	O
-five	O
--	O
-coordinated	O
-haem	O
-molecules	O
-in	O
-each	O
-monomer	O
-.	O
-	O
-CO	O
-induces	O
-the	O
-dissociation	O
-of	O
-the	O
-haem	O
--	O
-mediated	O
-dimer	O
-of	O
-PGRMC1	O
-by	O
-interfering	O
-with	O
-the	O
-haem	O
--	O
-stacking	O
-interface	O
-via	O
-formation	O
-of	O
-the	O
-six	O
--	O
-coordinated	O
-CO	O
--	O
-haem	O
--	O
-PGRMC1	O
-complex	O
-.	O
-	O
-Such	O
-a	O
-dynamic	O
-structural	O
-regulation	O
-led	O
-us	O
-to	O
-further	O
-examine	O
-the	O
-regulation	O
-of	O
-PGRMC1	O
-functions	O
-in	O
-cancer	O
-cells	O
-.	O
-	O
-PGRMC1	O
-dimerization	O
-is	O
-required	O
-for	O
-binding	O
-to	O
-EGFR	O
-	O
-Because	O
-PGRMC1	O
-is	O
-known	O
-to	O
-interact	O
-with	O
-EGFR	O
-and	O
-to	O
-accelerate	O
-tumour	O
-progression	O
-,	O
-we	O
-examined	O
-the	O
-effect	O
-of	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-on	O
-its	O
-interaction	O
-with	O
-EGFR	O
-by	O
-using	O
-purified	O
-proteins	O
-.	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-.	O
-4a	O
-,	O
-the	O
-cytosolic	O
-domain	O
-of	O
-wild	O
--	O
-type	O
-PGRMC1	O
-,	O
-but	O
-not	O
-the	O
-Y113F	B-mutant
-mutant	O
-,	O
-interacted	O
-with	O
-purified	O
-EGFR	O
-in	O
-a	O
-haem	O
--	O
-dependent	O
-manner	O
-.	O
-	O
-This	O
-interaction	O
-was	O
-disrupted	O
-by	O
-the	O
-ruthenium	O
--	O
-based	O
-CO	O
--	O
-releasing	O
-molecule	O
-,	O
-CORM3	O
-,	O
-but	O
-not	O
-by	O
-RuCl3	O
-as	O
-a	O
-control	O
-reagent	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-We	O
-further	O
-analysed	O
-the	O
-intracellular	O
-interaction	O
-between	O
-PGRMC1	O
-and	O
-EGFR	O
-.	O
-	O
-FLAG	O
--	O
-tagged	O
-PGRMC1	O
-ectopically	O
-expressed	O
-in	O
-human	O
-colon	O
-cancer	O
-HCT116	O
-cells	O
-was	O
-immunoprecipitated	O
-with	O
-anti	O
--	O
-FLAG	O
-antibody	O
-,	O
-and	O
-co	O
--	O
-immunoprecipitated	O
-EGFR	O
-and	O
-endogenous	O
-PGRMC1	O
-binding	O
-to	O
-FLAG	O
--	O
-PGRMC1	O
-were	O
-detected	O
-by	O
-Western	O
-blotting	O
-(	O
-Fig	O
-.	O
-4c	O
-).	O
-	O
-The	O
-C129S	B-mutant
-mutant	O
-of	O
-PGRMC1	O
-also	O
-interacted	O
-with	O
-endogenous	O
-PGRMC1	O
-and	O
-EGFR	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-16	O
-).	O
-	O
-Whereas	O
-FLAG	O
--	O
-tagged	O
-wild	O
--	O
-type	O
-PGRMC1	O
-interacted	O
-with	O
-endogenous	O
-PGRMC1	O
-and	O
-EGFR	O
-,	O
-the	O
-Y113F	B-mutant
-mutant	O
-did	O
-not	O
-.	O
-	O
-We	O
-also	O
-examined	O
-the	O
-effect	O
-of	O
-succinylacetone	O
-(	O
-SA	O
-),	O
-an	O
-inhibitor	O
-of	O
-haem	O
-biosynthesis	O
-(	O
-Fig	O
-.	O
-4d	O
-).	O
-	O
-As	O
-expected	O
-,	O
-SA	O
-significantly	O
-reduced	O
-PGRMC1	O
-dimerization	O
-and	O
-its	O
-interaction	O
-with	O
-EGFR	O
-(	O
-Fig	O
-.	O
-4e	O
-),	O
-indicating	O
-that	O
-haem	O
--	O
-mediated	O
-dimerization	O
-of	O
-PGMRC1	O
-is	O
-critical	O
-for	O
-its	O
-binding	O
-to	O
-EGFR	O
-.	O
-	O
-PGRMC1	O
-dimer	O
-facilitates	O
-EGFR	O
--	O
-mediated	O
-cancer	O
-growth	O
-	O
-Next	O
-,	O
-we	O
-investigated	O
-the	O
-functional	O
-significance	O
-of	O
-PGRMC1	O
-dimerization	O
-in	O
-EGFR	O
-signaling	O
-.	O
-	O
-EGF	O
--	O
-induced	O
-phosphorylations	O
-of	O
-EGFR	O
-and	O
-its	O
-downstream	O
-targets	O
-AKT	O
-and	O
-ERK	O
-were	O
-decreased	O
-by	O
-PGRMC1	O
-knockdown	O
-(	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-)	O
-(	O
-Fig	O
-.	O
-4f	O
-).	O
-	O
-Similarly	O
-,	O
-EGFR	O
-signaling	O
-was	O
-suppressed	O
-by	O
-treatment	O
-of	O
-HCT116	O
-cells	O
-with	O
-SA	O
-(	O
-Fig	O
-.	O
-4g	O
-)	O
-or	O
-CORM3	O
-(	O
-Fig	O
-.	O
-4h	O
-).	O
-	O
-These	O
-results	O
-suggested	O
-that	O
-haem	O
--	O
-mediated	O
-dimerization	O
-of	O
-PGRMC1	O
-is	O
-critical	O
-for	O
-EGFR	O
-signaling	O
-.	O
-	O
-To	O
-further	O
-investigate	O
-the	O
-role	O
-of	O
-the	O
-dimerized	O
-form	O
-of	O
-PGRMC1	O
-in	O
-cancer	O
-proliferation	O
-,	O
-we	O
-performed	O
-PGRMC1	O
-knockdown	O
--	O
-rescue	O
-experiments	O
-using	O
-FLAG	O
--	O
-tagged	O
-wild	O
--	O
-type	O
-and	O
-Y113F	B-mutant
-PGRMC1	O
-expression	O
-vectors	O
-,	O
-in	O
-which	O
-silent	O
-mutations	O
-were	O
-introduced	O
-into	O
-the	O
-nucleotide	O
-sequence	O
-targeted	O
-by	O
-shRNA	O
-(	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-While	O
-proliferation	O
-of	O
-HCT116	O
-cells	O
-was	O
-not	O
-affected	O
-by	O
-knocking	O
-down	O
-PGRMC1	O
-,	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-were	O
-more	O
-sensitive	O
-to	O
-the	O
-EGFR	O
-inhibitor	O
-erlotinib	O
-than	O
-control	O
-HCT116	O
-cells	O
-,	O
-and	O
-the	O
-knockdown	O
-effect	O
-was	O
-reversed	O
-by	O
-co	O
--	O
-expression	O
-of	O
-shRNA	O
--	O
-resistant	O
-wild	O
--	O
-type	O
-PGRMC1	O
-but	O
-not	O
-of	O
-the	O
-Y113F	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-Chemosensitivity	O
-enhancement	O
-by	O
-two	O
-different	O
-shRNAs	O
-to	O
-PGRMC1	O
-was	O
-seen	O
-also	O
-in	O
-HCT116	O
-cells	O
-and	O
-human	O
-hepatoma	O
-HuH7	O
-cells	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-17	O
-).	O
-	O
-Furthermore	O
-,	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-inhibited	O
-spheroid	O
-formation	O
-of	O
-HCT116	O
-cells	O
-in	O
-culture	O
-,	O
-and	O
-this	O
-inhibition	O
-was	O
-reversed	O
-by	O
-co	O
--	O
-expression	O
-of	O
-wild	O
--	O
-type	O
-PGRMC1	O
-but	O
-not	O
-of	O
-the	O
-Y113F	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-5c	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-18	O
-).	O
-	O
-Thus	O
-,	O
-PGRMC1	O
-dimerization	O
-is	O
-important	O
-for	O
-cancer	O
-cell	O
-proliferation	O
-and	O
-chemoresistance	O
-.	O
-	O
-We	O
-examined	O
-the	O
-role	O
-of	O
-PGRMC1	O
-in	O
-metastatic	O
-progression	O
-by	O
-xenograft	O
-transplantation	O
-assays	O
-using	O
-super	O
--	O
-immunodeficient	O
-NOD	O
-/	O
-scid	O
-/	O
-γnull	O
-(	O
-NOG	O
-)	O
-mice	O
-.	O
-	O
-Ten	O
-days	O
-after	O
-intra	O
--	O
-splenic	O
-implantation	O
-of	O
-HCT116	O
-cells	O
-that	O
-were	O
-genetically	O
-tagged	O
-with	O
-a	O
-fluorescent	O
-protein	O
-Venus	O
-,	O
-the	O
-group	O
-implanted	O
-with	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-showed	O
-a	O
-significant	O
-decrease	O
-of	O
-liver	O
-metastasis	O
-in	O
-comparison	O
-with	O
-the	O
-control	O
-group	O
-(	O
-Fig	O
-.	O
-5d	O
-).	O
-	O
-Interaction	O
-of	O
-PGRMC1	O
-dimer	O
-with	O
-cytochromes	O
-P450	O
-	O
-Since	O
-PGRMC1	O
-has	O
-been	O
-shown	O
-to	O
-interact	O
-with	O
-cytochromes	O
-P450	O
-(	O
-ref	O
-),	O
-we	O
-investigated	O
-whether	O
-the	O
-haem	O
--	O
-mediated	O
-dimerization	O
-of	O
-PGRMC1	O
-is	O
-necessary	O
-for	O
-their	O
-interactions	O
-.	O
-	O
-Recombinant	O
-CYP1A2	O
-and	O
-CYP3A4	O
-including	O
-a	O
-microsomal	O
-formulation	O
-containing	O
-cytochrome	O
-b5	O
-and	O
-cytochrome	O
-P450	O
-reductase	O
-,	O
-drug	O
--	O
-metabolizing	O
-cytochromes	O
-P450	O
-,	O
-interacted	O
-with	O
-wild	O
--	O
-type	O
-PGRMC1	O
-,	O
-but	O
-not	O
-with	O
-the	O
-Y113F	B-mutant
-mutant	O
-,	O
-in	O
-a	O
-haem	O
--	O
-dependent	O
-manner	O
-(	O
-Fig	O
-.	O
-6a	O
-,	O
-b	O
-).	O
-	O
-Moreover	O
-,	O
-the	O
-interaction	O
-of	O
-PGRMC1	O
-with	O
-CYP1A2	O
-was	O
-blocked	O
-by	O
-CORM3	O
-under	O
-reducing	O
-conditions	O
-(	O
-Fig	O
-.	O
-6c	O
-),	O
-indicating	O
-that	O
-PGRMC1	O
-dimerization	O
-is	O
-necessary	O
-for	O
-its	O
-interaction	O
-with	O
-cytochromes	O
-P450	O
-.	O
-	O
-Doxorubicin	O
-is	O
-an	O
-anti	O
--	O
-cancer	O
-reagent	O
-that	O
-is	O
-metabolized	O
-into	O
-inactive	O
-doxorubicinol	O
-by	O
-CYP2D6	O
-and	O
-CYP3A4	O
-(	O
-Fig	O
-.	O
-6d	O
-).	O
-	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-significantly	O
-suppressed	O
-the	O
-conversion	O
-of	O
-doxorubicin	O
-to	O
-doxorubicinol	O
-(	O
-Fig	O
-.	O
-6d	O
-)	O
-and	O
-increased	O
-sensitivity	O
-to	O
-doxorubicin	O
-(	O
-Fig	O
-.	O
-6e	O
-).	O
-	O
-Enhanced	O
-doxorubicin	O
-sensitivity	O
-was	O
-modestly	O
-but	O
-significantly	O
-induced	O
-by	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-.	O
-	O
-This	O
-effect	O
-was	O
-reversed	O
-by	O
-co	O
--	O
-expression	O
-of	O
-the	O
-wild	O
--	O
-type	O
-PGRMC1	O
-but	O
-not	O
-of	O
-the	O
-Y113F	B-mutant
-mutant	O
-,	O
-suggesting	O
-that	O
-PGRMC1	O
-enhances	O
-doxorubicin	O
-resistance	O
-of	O
-cancer	O
-cells	O
-by	O
-facilitating	O
-its	O
-degradation	O
-via	O
-cytochromes	O
-P450	O
-.	O
-	O
-To	O
-gain	O
-further	O
-insight	O
-into	O
-the	O
-interaction	O
-between	O
-PGRMC1	O
-and	O
-cytochromes	O
-P450	O
-,	O
-surface	O
-plasmon	O
-resonance	O
-analyses	O
-were	O
-conducted	O
-using	O
-recombinant	O
-CYP51	O
-and	O
-PGRMC1	O
-.	O
-	O
-This	O
-was	O
-based	O
-on	O
-a	O
-previous	O
-study	O
-showing	O
-that	O
-PGRMC1	O
-binds	O
-to	O
-CYP51	O
-and	O
-enhances	O
-cholesterol	O
-biosynthesis	O
-by	O
-CYP51	O
-(	O
-refs	O
-).	O
-	O
-CYP51	O
-interacted	O
-with	O
-PGRMC1	O
-in	O
-a	O
-concentration	O
--	O
-dependent	O
-manner	O
-in	O
-the	O
-presence	O
-of	O
-haem	O
-,	O
-but	O
-not	O
-in	O
-its	O
-absence	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-19	O
-),	O
-suggesting	O
-the	O
-requirement	O
-for	O
-the	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-.	O
-	O
-The	O
-Kd	O
-value	O
-of	O
-PGRMC1	O
-binding	O
-to	O
-CYP51	O
-was	O
-in	O
-a	O
-micromolar	O
-range	O
-and	O
-comparable	O
-with	O
-those	O
-of	O
-other	O
-haem	O
-proteins	O
-,	O
-such	O
-as	O
-cytochrome	O
-P450	O
-reductase	O
-and	O
-neuroglobin	O
-/	O
-Gαi1	O
-(	O
-ref	O
-.),	O
-suggesting	O
-that	O
-haem	O
--	O
-dependent	O
-PGRMC1	O
-interaction	O
-with	O
-CYP51	O
-is	O
-biologically	O
-relevant	O
-.	O
-	O
-In	O
-this	O
-study	O
-,	O
-we	O
-showed	O
-that	O
-PGRMC1	O
-dimerizes	O
-by	O
-stacking	O
-interactions	O
-of	O
-haem	O
-molecules	O
-from	O
-each	O
-monomer	O
-.	O
-	O
-Recently	O
-,	O
-Lucas	O
-et	O
-al	O
-.	O
-reported	O
-that	O
-translationally	O
--	O
-controlled	O
-tumour	O
-protein	O
-was	O
-dimerized	O
-by	O
-binding	O
-with	O
-haem	O
-,	O
-but	O
-its	O
-structural	O
-basis	O
-remains	O
-unclear	O
-.	O
-	O
-This	O
-is	O
-the	O
-report	O
-showing	O
-crystallographic	O
-evidence	O
-that	O
-indicates	O
-roles	O
-of	O
-the	O
-direct	O
-haem	O
-–	O
-haem	O
-stacking	O
-in	O
-haem	O
--	O
-mediated	O
-dimerization	O
-in	O
-eukaryotes	O
-,	O
-although	O
-a	O
-few	O
-examples	O
-are	O
-known	O
-in	O
-bacteria	O
-.	O
-	O
-Sequence	O
-alignments	O
-show	O
-that	O
-haem	O
--	O
-binding	O
-residues	O
-(	O
-Tyr113	O
-,	O
-Tyr107	O
-,	O
-Lys163	O
-and	O
-Tyr164	O
-)	O
-in	O
-PGRMC1	O
-are	O
-conserved	O
-among	O
-MAPR	O
-proteins	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-In	O
-the	O
-current	O
-study	O
-,	O
-the	O
-Y113	O
-residue	O
-plays	O
-a	O
-crucial	O
-role	O
-for	O
-the	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-and	O
-resultant	O
-regulation	O
-of	O
-cancer	O
-proliferation	O
-and	O
-chemoresistance	O
-(	O
-Figs	O
-5c	O
-and	O
-6e	O
-).	O
-	O
-Since	O
-the	O
-Y113	O
-residue	O
-is	O
-involved	O
-in	O
-the	O
-putative	O
-consensus	O
-motif	O
-of	O
-phosphorylation	O
-by	O
-tyrosine	O
-kinases	O
-such	O
-as	O
-Abl	O
-and	O
-Lck	O
-,	O
-we	O
-investigated	O
-whether	O
-phosphorylated	O
-Y113	O
-is	O
-present	O
-in	O
-HCT116	O
-cells	O
-by	O
-ESI	O
--	O
-MS	O
-analysis	O
-.	O
-	O
-Recently	O
-,	O
-Peluso	O
-et	O
-al	O
-.	O
-reported	O
-that	O
-PGRMC1	O
-binds	O
-to	O
-PGRMC2	O
-,	O
-suggesting	O
-that	O
-MAPR	O
-family	O
-members	O
-may	O
-also	O
-undergo	O
-haem	O
--	O
-mediated	O
-heterodimerization	O
-.	O
-	O
-We	O
-showed	O
-that	O
-the	O
-haem	O
--	O
-mediated	O
-dimer	O
-of	O
-PGRMC1	O
-enables	O
-interaction	O
-with	O
-different	O
-subclasses	O
-of	O
-cytochromes	O
-P450	O
-(	O
-CYP	O
-)	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-While	O
-the	O
-effects	O
-of	O
-PGRMC1	O
-on	O
-cholesterol	O
-synthesis	O
-mediated	O
-by	O
-CYP51	O
-have	O
-been	O
-well	O
-documented	O
-in	O
-yeast	O
-and	O
-human	O
-cells	O
-,	O
-it	O
-has	O
-not	O
-been	O
-clear	O
-whether	O
-drug	O
--	O
-metabolizing	O
-CYP	O
-activities	O
-are	O
-regulated	O
-by	O
-PGRMC1	O
-.	O
-	O
-Szczesna	O
--	O
-Skorupa	O
-and	O
-Kemper	O
-reported	O
-that	O
-PGRMC1	O
-exhibited	O
-an	O
-inhibitory	O
-effect	O
-on	O
-CYP3A4	O
-drug	O
-metabolizing	O
-activity	O
-by	O
-competitively	O
-binding	O
-with	O
-cytochrome	O
-P450	O
-reductase	O
-(	O
-CPR	O
-)	O
-in	O
-HEK293	O
-or	O
-HepG2	O
-cells	O
-.	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-Oda	O
-et	O
-al	O
-.	O
-reported	O
-that	O
-PGRMC1	O
-had	O
-no	O
-effect	O
-to	O
-CYP2E1	O
-and	O
-CYP3A4	O
-activities	O
-in	O
-HepG2	O
-cell	O
-.	O
-	O
-Several	O
-other	O
-groups	O
-showed	O
-that	O
-PGRMC1	O
-enhanced	O
-chemoresistance	O
-in	O
-several	O
-cancer	O
-cells	O
-such	O
-as	O
-uterine	O
-sarcoma	O
-,	O
-breast	O
-cancer	O
-,	O
-endometrial	O
-tumour	O
-and	O
-ovarian	O
-cancer	O
-;	O
-however	O
-,	O
-no	O
-evidence	O
-of	O
-PGRMC1	O
--	O
-dependent	O
-regulation	O
-of	O
-CYP	O
-activity	O
-was	O
-provided	O
-.	O
-	O
-Our	O
-results	O
-showed	O
-that	O
-PGRMC1	O
-contributes	O
-to	O
-enhancement	O
-of	O
-the	O
-doxorubicin	O
-metabolism	O
-,	O
-which	O
-is	O
-mediated	O
-by	O
-CYP2D6	O
-or	O
-CYP3A4	O
-in	O
-human	O
-colon	O
-cancer	O
-HCT116	O
-cells	O
-(	O
-Fig	O
-.	O
-6d	O
-).	O
-	O
-While	O
-the	O
-effects	O
-of	O
-structural	O
-diversity	O
-of	O
-CYP	O
-family	O
-proteins	O
-and	O
-interactions	O
-with	O
-different	O
-xenobiotic	O
-substrates	O
-should	O
-further	O
-be	O
-examined	O
-,	O
-the	O
-current	O
-results	O
-suggest	O
-that	O
-the	O
-interaction	O
-of	O
-drug	O
--	O
-metabolizing	O
-CYPs	O
-with	O
-the	O
-haem	O
--	O
-mediated	O
-dimer	O
-of	O
-PGRMC1	O
-plays	O
-a	O
-crucial	O
-role	O
-in	O
-regulating	O
-their	O
-activities	O
-.	O
-	O
-We	O
-showed	O
-that	O
-haem	O
--	O
-mediated	O
-dimerization	O
-of	O
-PGRMC1	O
-enhances	O
-proliferation	O
-and	O
-chemoresistance	O
-of	O
-cancer	O
-cells	O
-through	O
-binding	O
-to	O
-and	O
-regulating	O
-EGFR	O
-and	O
-cytochromes	O
-P450	O
-(	O
-illustrated	O
-in	O
-Fig	O
-.	O
-7	O
-).	O
-	O
-Since	O
-the	O
-haem	O
--	O
-binding	O
-affinity	O
-of	O
-PGRMC1	O
-is	O
-lower	O
-than	O
-those	O
-of	O
-constitutive	O
-haem	O
--	O
-binding	O
-proteins	O
-such	O
-as	O
-myoglobin	O
-,	O
-PGMRC1	O
-is	O
-probably	O
-interconverted	O
-between	O
-apo	O
--	O
-monomer	O
-and	O
-haem	O
--	O
-bound	O
-dimer	O
-forms	O
-in	O
-response	O
-to	O
-changes	O
-in	O
-the	O
-intracellular	O
-haem	O
-concentration	O
-.	O
-	O
-Considering	O
-microenvironments	O
-in	O
-and	O
-around	O
-malignant	O
-tumours	O
-,	O
-the	O
-haem	O
-concentration	O
-in	O
-cancer	O
-cells	O
-is	O
-likely	O
-to	O
-be	O
-elevated	O
-through	O
-multiple	O
-mechanisms	O
-,	O
-such	O
-as	O
-(	O
-i	O
-)	O
-an	O
-increased	O
-intake	O
-of	O
-haem	O
-,	O
-(	O
-ii	O
-)	O
-mutation	O
-of	O
-enzymes	O
-in	O
-TCA	O
-cycle	O
-(	O
-for	O
-example	O
-,	O
-fumarate	O
-hydratase	O
-)	O
-that	O
-increases	O
-the	O
-level	O
-of	O
-succinyl	O
-CoA	O
-,	O
-a	O
-substrate	O
-for	O
-haem	O
-biosynthesis	O
-and	O
-(	O
-iii	O
-)	O
-metastasis	O
-to	O
-haem	O
--	O
-rich	O
-organs	O
-such	O
-as	O
-liver	O
-,	O
-brain	O
-and	O
-bone	O
-marrow	O
-.	O
-	O
-Moreover	O
-,	O
-exposure	O
-of	O
-cancer	O
-cells	O
-to	O
-stimuli	O
-such	O
-as	O
-hypoxia	O
-,	O
-radiation	O
-and	O
-chemotherapy	O
-causes	O
-cell	O
-damages	O
-and	O
-leads	O
-to	O
-protein	O
-degradation	O
-,	O
-resulting	O
-in	O
-increased	O
-levels	O
-of	O
-TCA	O
-cycle	O
-intermediates	O
-and	O
-in	O
-an	O
-enhanced	O
-haem	O
-biosynthesis	O
-.	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-excessive	O
-haem	O
-induces	O
-HO	O
--	O
-1	O
-,	O
-the	O
-enzyme	O
-that	O
-oxidatively	O
-degrades	O
-haem	O
-and	O
-generates	O
-CO	O
-.	O
-	O
-Thus	O
-,	O
-HO	O
--	O
-1	O
-induction	O
-in	O
-cancer	O
-cells	O
-may	O
-inhibit	O
-the	O
-haem	O
--	O
-mediated	O
-dimerization	O
-of	O
-PGRMC1	O
-through	O
-the	O
-production	O
-of	O
-CO	O
-and	O
-thereby	O
-suppress	O
-tumour	O
-progression	O
-.	O
-	O
-This	O
-idea	O
-is	O
-consistent	O
-with	O
-the	O
-observation	O
-that	O
-HO	O
--	O
-1	O
-induction	O
-or	O
-CO	O
-inhibits	O
-tumour	O
-growth	O
-.	O
-	O
-Besides	O
-the	O
-regulatory	O
-roles	O
-of	O
-PGRMC1	O
-/	O
-Sigma	O
--	O
-2	O
-receptor	O
-in	O
-proliferation	O
-and	O
-chemoresistance	O
-in	O
-cancer	O
-cells	O
-(	O
-ref	O
-.),	O
-recent	O
-reports	O
-show	O
-that	O
-PGRMC1	O
-is	O
-able	O
-to	O
-bind	O
-to	O
-amyloid	O
-beta	O
-oligomer	O
-to	O
-enhance	O
-its	O
-neurotoxicity	O
-.	O
-	O
-Furthermore	O
-,	O
-Sigma	O
--	O
-2	O
-ligand	O
--	O
-binding	O
-is	O
-decreased	O
-in	O
-transgenic	O
-amyloid	O
-beta	O
-deposition	O
-model	O
-APP	O
-/	O
-PS1	O
-female	O
-mice	O
-.	O
-	O
-These	O
-results	O
-suggest	O
-a	O
-possible	O
-involvement	O
-of	O
-PGRMC1	O
-in	O
-Alzheimer	O
-'	O
-s	O
-disease	O
-.	O
-	O
-The	O
-roles	O
-of	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-in	O
-the	O
-functional	O
-regulation	O
-of	O
-its	O
-target	O
-proteins	O
-deserve	O
-further	O
-studies	O
-to	O
-find	O
-evidence	O
-that	O
-therapeutic	O
-interventions	O
-to	O
-interfere	O
-with	O
-the	O
-function	O
-of	O
-the	O
-dimer	O
-may	O
-control	O
-varied	O
-disease	O
-conditions	O
-.	O
-	O
-Alzheimer	O
-'	O
-s	O
-therapeutics	O
-targeting	O
-amyloid	O
-beta	O
-1	O
--	O
-42	O
-oligomers	O
-II	O
-:	O
-Sigma	O
--	O
-2	O
-/	O
-PGRMC1	O
-receptors	O
-mediate	O
-Abeta	O
-42	O
-oligomer	O
-binding	O
-and	O
-synaptotoxicity	O
-	O
-X	O
--	O
-ray	O
-crystal	O
-structure	O
-of	O
-PGRMC1	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structure	O
-of	O
-the	O
-PGRMC1	O
-dimer	O
-formed	O
-through	O
-stacked	O
-haems	O
-.	O
-	O
-Two	O
-PGRMC1	O
-subunits	O
-(	O
-blue	O
-and	O
-green	O
-ribbons	O
-)	O
-dimerize	O
-via	O
-stacking	O
-of	O
-the	O
-haem	O
-molecules	O
-.	O
-	O
-(	O
-b	O
-)	O
-Haem	O
-coordination	O
-of	O
-PGRMC1	O
-with	O
-Tyr113	O
-.	O
-	O
-Comparison	O
-of	O
-PGRMC1	O
-(	O
-blue	O
-)	O
-and	O
-cytochrome	O
-b5	O
-(	O
-yellow	O
-,	O
-ID	O
-:	O
-3NER	O
-).	O
-(	O
-c	O
-)	O
-PGRMC1	O
-has	O
-a	O
-longer	O
-helix	O
-(	O
-a	O
-.	O
-a	O
-.	O
-147	O
-–	O
-163	O
-),	O
-which	O
-is	O
-shifted	O
-away	O
-from	O
-the	O
-haem	O
-(	O
-arrow	O
-).	O
-	O
-PGRCM1	O
-is	O
-dimerized	O
-by	O
-binding	O
-with	O
-haem	O
-.	O
-	O
-(	O
-a	O
-)	O
-Mass	O
-spectrometric	O
-analyses	O
-of	O
-the	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-)	O
-PGRMC1	O
-or	O
-the	O
-C129S	B-mutant
-mutant	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-haem	O
-under	O
-non	O
--	O
-denaturing	O
-condition	O
-.	O
-	O
-Both	O
-proteins	O
-had	O
-identical	O
-lengths	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-).	O
-	O
-(	O
-b	O
-)	O
-SV	O
--	O
-AUC	O
-analyses	O
-of	O
-the	O
-wt	O
--	O
-PGRMC1	O
-and	O
-the	O
-C129S	B-mutant
-mutant	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-haem	O
-.	O
-	O
-SV	O
--	O
-AUC	O
-experiments	O
-were	O
-performed	O
-with	O
-1	O
-.	O
-5	O
-mg	O
-ml	O
-−	O
-1	O
-of	O
-PGRMC1	O
-proteins	O
-.	O
-	O
-The	O
-major	O
-peak	O
-with	O
-sedimentation	O
-coefficient	O
-S20	O
-,	O
-w	O
-of	O
-1	O
-.	O
-9	O
-∼	O
-2	O
-.	O
-0	O
-S	O
-(	O
-monomer	O
-)	O
-or	O
-3	O
-.	O
-1	O
-S	O
-(	O
-dimer	O
-)	O
-was	O
-detected	O
-.	O
-	O
-(	O
-c	O
-)	O
-Difference	O
-absorption	O
-spectra	O
-of	O
-PGRMC1	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-titrated	O
-with	O
-haem	O
-(	O
-left	O
-panel	O
-).	O
-	O
-The	O
-titration	O
-curve	O
-of	O
-haem	O
-to	O
-PGRMC1	O
-(	O
-right	O
-panel	O
-).	O
-	O
-The	O
-absorbance	O
-difference	O
-at	O
-400	O
-nm	O
-is	O
-plotted	O
-against	O
-the	O
-haem	O
-concentration	O
-.	O
-	O
-Carbon	O
-monoxide	O
-inhibits	O
-haem	O
--	O
-dependent	O
-PGRMC1	O
-dimerization	O
-.	O
-	O
-(	O
-a	O
-)	O
-UV	O
--	O
-visible	O
-absorption	O
-spectra	O
-of	O
-PGRMC1	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-).	O
-	O
-Measurements	O
-were	O
-performed	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-oxidized	O
-form	O
-of	O
-haem	O
-(	O
-ferric	O
-),	O
-the	O
-reduced	O
-form	O
-of	O
-haem	O
-(	O
-ferrous	O
-)	O
-and	O
-the	O
-reduced	O
-form	O
-of	O
-haem	O
-plus	O
-CO	O
-gas	O
-(	O
-ferrous	O
-+	O
-CO	O
-).	O
-	O
-(	O
-b	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-haem	O
-stacking	O
-.	O
-	O
-(	O
-c	O
-)	O
-Gel	O
--	O
-filtration	O
-chromatography	O
-analyses	O
-of	O
-PGRMC1	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-)	O
-and	O
-the	O
-Y113F	B-mutant
-or	O
-C129S	B-mutant
-mutant	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-haem	O
-,	O
-dithionite	O
-and	O
-/	O
-or	O
-CO	O
-.	O
-(	O
-d	O
-)	O
-Transition	O
-model	O
-for	O
-structural	O
-regulation	O
-of	O
-PGRMC1	O
-in	O
-response	O
-to	O
-haem	O
-and	O
-CO	O
-.	O
-	O
-Haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-is	O
-necessary	O
-for	O
-tumour	O
-proliferation	O
-mediated	O
-by	O
-EGFR	O
-signalling	O
-.	O
-	O
-(	O
-a	O
-)	O
-FLAG	O
--	O
-PGRMC1	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-)	O
-and	O
-Y113F	B-mutant
-mutant	O
-proteins	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-),	O
-in	O
-either	O
-apo	O
--	O
-or	O
-haem	O
--	O
-bound	O
-form	O
-,	O
-were	O
-incubated	O
-with	O
-purified	O
-EGFR	O
-and	O
-co	O
--	O
-immunoprecipitated	O
-with	O
-anti	O
--	O
-FLAG	O
-antibody	O
--	O
-conjugated	O
-beads	O
-.	O
-	O
-Input	O
-and	O
-bound	O
-proteins	O
-were	O
-detected	O
-by	O
-Western	O
-blotting	O
-.	O
-	O
-(	O
-b	O
-)	O
-In	O
-vitro	O
-binding	O
-assay	O
-was	O
-performed	O
-as	O
-in	O
-(	O
-a	O
-)	O
-using	O
-haem	O
--	O
-bound	O
-FLAG	O
--	O
-PGRMC1	O
-wt	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-and	O
-purified	O
-EGFR	O
-with	O
-or	O
-without	O
-treatment	O
-of	O
-RuCl3	O
-and	O
-CORM3	O
-.	O
-	O
-(	O
-c	O
-)	O
-FLAG	O
--	O
-PGRMC1	O
-wt	O
-or	O
-Y113F	B-mutant
-(	O
-full	O
-length	O
-)	O
-was	O
-over	O
--	O
-expressed	O
-in	O
-HCT116	O
-cells	O
-and	O
-immunoprecipitated	O
-with	O
-anti	O
--	O
-FLAG	O
-antibody	O
--	O
-conjugated	O
-beads	O
-.	O
-	O
-Co	O
--	O
-immunoprecipitated	O
-proteins	O
-(	O
-FLAG	O
--	O
-PGRMC1	O
-,	O
-endogenous	O
-PGRMC1	O
-and	O
-EGFR	O
-)	O
-were	O
-detected	O
-with	O
-Western	O
-blotting	O
-by	O
-using	O
-anti	O
--	O
-PGRMC1	O
-or	O
-anti	O
--	O
-EGFR	O
-antibody	O
-.	O
-	O
-(	O
-d	O
-)	O
-HCT116	O
-cells	O
-were	O
-treated	O
-with	O
-or	O
-without	O
-250	O
-μmol	O
-l	O
-−	O
-1	O
-of	O
-succinylacetone	O
-(	O
-SA	O
-)	O
-for	O
-48	O
-h	O
-.	O
-The	O
-intracellular	O
-haem	O
-was	O
-extracted	O
-and	O
-quantified	O
-by	O
-reverse	O
--	O
-phase	O
-HPLC	O
-.	O
-	O
-of	O
-four	O
-separate	O
-experiments	O
-.	O
-**	O
-P	O
-<	O
-0	O
-.	O
-01	O
-using	O
-unpaired	O
-Student	O
-'	O
-s	O
-t	O
--	O
-test	O
-.	O
-(	O
-e	O
-)	O
-Co	O
--	O
-immunoprecipitation	O
-assay	O
-was	O
-performed	O
-as	O
-in	O
-(	O
-c	O
-)	O
-with	O
-or	O
-without	O
-SA	O
-treatment	O
-in	O
-HCT116	O
-cells	O
-.	O
-	O
-(	O
-f	O
-)	O
-HCT116	O
-cells	O
-expressing	O
-control	O
-shRNA	O
-or	O
-those	O
-knocking	O
-down	O
-PGRMC1	O
-(	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-)	O
-were	O
-treated	O
-with	O
-EGF	O
-or	O
-left	O
-untreated	O
-,	O
-and	O
-components	O
-of	O
-the	O
-EGFR	O
-signaling	O
-pathway	O
-were	O
-detected	O
-by	O
-Western	O
-blotting	O
-.	O
-	O
-(	O
-g	O
-,	O
-h	O
-)	O
-HCT116	O
-cells	O
-were	O
-treated	O
-with	O
-or	O
-without	O
-EGF	O
-,	O
-SA	O
-,	O
-RuCl3	O
-and	O
-CORM3	O
-as	O
-indicated	O
-,	O
-and	O
-components	O
-of	O
-the	O
-EGFR	O
-signaling	O
-pathway	O
-were	O
-detected	O
-by	O
-Western	O
-blotting	O
-.	O
-	O
-Haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-accelerates	O
-tumour	O
-growth	O
-through	O
-the	O
-EGFR	O
-signaling	O
-pathway	O
-.	O
-	O
-(	O
-a	O
-)	O
-Nucleotide	O
-sequences	O
-of	O
-PGRMC1	O
-targeted	O
-by	O
-shRNA	O
-and	O
-of	O
-the	O
-shRNA	O
--	O
-resistant	O
-full	O
-length	O
-PGRMC1	O
-expression	O
-vector	O
-.	O
-	O
-Stable	O
-PGRMC1	B-mutant
--	I-mutant
-knockdown	I-mutant
-(	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-)	O
-HCT116	O
-cells	O
-were	O
-transiently	O
-transfected	O
-with	O
-the	O
-shRNA	O
--	O
-resistant	O
-expression	O
-vector	O
-of	O
-wild	O
--	O
-type	O
-PGRMC1	O
-(	O
-wt	O
-)	O
-or	O
-the	O
-Y113F	B-mutant
-mutant	O
-(	O
-Y113F	B-mutant
-).	O
-	O
-(	O
-b	O
-)	O
-Erlotinib	O
-was	O
-added	O
-to	O
-HCT116	O
-(	O
-control	O
-)	O
-cells	O
-,	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-or	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-expressing	O
-shRNA	O
--	O
-resistant	O
-PGRMC1	O
-wt	O
-or	O
-Y113F	B-mutant
-,	O
-and	O
-cell	O
-viability	O
-was	O
-examined	O
-by	O
-MTT	O
-assay	O
-.	O
-	O
-of	O
-four	O
-separate	O
-experiments	O
-.	O
-*	O
-P	O
-<	O
-0	O
-.	O
-01	O
-using	O
-ANOVA	O
-with	O
-Fischer	O
-'	O
-s	O
-LSD	O
-test	O
-.	O
-	O
-(	O
-c	O
-)	O
-Spheroid	O
-formation	O
-in	O
-control	O
-and	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-HCT116	O
-cells	O
-.	O
-	O
-The	O
-graph	O
-represents	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-of	O
-each	O
-spheroid	O
-size	O
-.	O
-*	O
-P	O
-<	O
-0	O
-.	O
-01	O
-using	O
-ANOVA	O
-with	O
-Fischer	O
-'	O
-s	O
-LSD	O
-test	O
-.	O
-	O
-Scale	O
-bar	O
-:	O
-0	O
-.	O
-1	O
-mm	O
-.	O
-(	O
-d	O
-)	O
-Tumour	O
--	O
-bearing	O
-livers	O
-of	O
-NOG	O
-mice	O
-at	O
-10	O
-days	O
-after	O
-intrasplenic	O
-injection	O
-of	O
-HCT116	O
-(	O
-control	O
-)	O
-or	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-.	O
-	O
-of	O
-10	O
-separate	O
-experiments	O
-.	O
-*	O
-P	O
-<	O
-0	O
-.	O
-05	O
-using	O
-unpaired	O
-Student	O
-'	O
-s	O
-t	O
--	O
-test	O
-.	O
-	O
-Haem	O
--	O
-dependent	O
-PGRMC1	O
-dimerization	O
-enhances	O
-tumour	O
-chemoresistance	O
-through	O
-interaction	O
-with	O
-cytochromes	O
-P450	O
-.	O
-	O
-(	O
-a	O
-,	O
-b	O
-)	O
-FLAG	O
--	O
-PGRMC1	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-)	O
-and	O
-Y113F	B-mutant
-mutant	O
-proteins	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-),	O
-in	O
-either	O
-apo	O
-or	O
-haem	O
--	O
-bound	O
-form	O
-,	O
-were	O
-incubated	O
-with	O
-CYP1A2	O
-(	O
-a	O
-)	O
-or	O
-CYP3A4	O
-(	O
-b	O
-)	O
-and	O
-immunoprecipitated	O
-with	O
-anti	O
--	O
-FLAG	O
-antibody	O
--	O
-conjugated	O
-beads	O
-.	O
-	O
-(	O
-c	O
-)	O
-Binding	O
-assay	O
-was	O
-performed	O
-as	O
-in	O
-(	O
-a	O
-)	O
-using	O
-haem	O
--	O
-bound	O
-FLAG	O
--	O
-PGRMC1	O
-wt	O
-and	O
-CYP1A2	O
-with	O
-or	O
-without	O
-RuCl3	O
-and	O
-CORM3	O
-.	O
-	O
-(	O
-d	O
-)	O
-Schematic	O
-illustration	O
-of	O
-doxorubicin	O
-metabolism	O
-is	O
-shown	O
-on	O
-the	O
-left	O
-.	O
-	O
-Doxorubicin	O
-was	O
-incubated	O
-with	O
-HCT116	O
-cells	O
-expressing	O
-control	O
-shRNA	O
-or	O
-shPGRMC1	O
-(	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-),	O
-and	O
-the	O
-doxorubicinol	O
-/	O
-doxorubicin	O
-ratios	O
-in	O
-cell	O
-pellets	O
-were	O
-determined	O
-using	O
-LC	O
--	O
-MS	O
-.	O
-	O
-of	O
-four	O
-separate	O
-experiments	O
-.	O
-**	O
-P	O
-<	O
-0	O
-.	O
-01	O
-versus	O
-control	O
-using	O
-unpaired	O
-Student	O
-'	O
-s	O
-t	O
--	O
-test	O
-.	O
-(	O
-e	O
-)	O
-Indicated	O
-amounts	O
-of	O
-doxorubicin	O
-were	O
-added	O
-to	O
-HCT116	O
-(	O
-control	O
-)	O
-cells	O
-,	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-,	O
-or	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-expressing	O
-shRNA	O
--	O
-resistant	O
-full	O
--	O
-length	O
-PGRMC1	O
-wt	O
-or	O
-Y113F	B-mutant
-,	O
-and	O
-cell	O
-viability	O
-was	O
-examined	O
-by	O
-MTT	O
-assay	O
-.	O
-	O
-Schematic	O
-diagram	O
-for	O
-the	O
-regulation	O
-of	O
-PGRMC1	O
-functions	O
-.	O
-	O
-Apo	O
--	O
-PGRMC1	O
-exists	O
-as	O
-an	O
-inactive	O
-monomer	O
-.	O
-	O
-On	O
-binding	O
-to	O
-haem	O
-,	O
-PGRMC1	O
-forms	O
-a	O
-dimer	O
-through	O
-stacking	O
-interactions	O
-between	O
-the	O
-haem	O
-moieties	O
-,	O
-which	O
-enables	O
-PGRMC1	O
-to	O
-interact	O
-with	O
-EGFR	O
-and	O
-cytochromes	O
-P450	O
-,	O
-leading	O
-to	O
-an	O
-enhanced	O
-proliferation	O
-and	O
-chemoresistance	O
-of	O
-cancer	O
-cells	O
-.	O
-	O
-CO	O
-interferes	O
-with	O
-the	O
-stacking	O
-interactions	O
-of	O
-the	O
-haems	O
-and	O
-thereby	O
-inhibits	O
-PGRMC1	O
-functions	O
-.	O
-	O
-PGRMC1	O
-proteins	O
-exhibit	O
-haem	O
--	O
-dependent	O
-dimerization	O
-in	O
-solution	O
-.	O
-	O
-Apo	O
-form	O
-Haem	O
--	O
-bound	O
-form	O
-Mass	O
-(	O
-Da	O
-)	O
-Mass	O
-(	O
-Da	O
-)	O
-aPGRMC1	O
-wt	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-ESI	O
--	O
-MS	O
-—	O
-17	O
-,	O
-844	O
-.	O
-14	O
-—	O
-36	O
-,	O
-920	O
-.	O
-19	O
-Theoretical	O
-17	O
-,	O
-843	O
-.	O
-65	O
-36	O
-,	O
-918	O
-.	O
-06	O
-Hydrodynamic	O
-radius	O
-10	O
-−	O
-9	O
-(	O
-m	O
-)	O
-MW	O
-(	O
-kDa	O
-)	O
-Hydrodynamic	O
-radius	O
-10	O
-−	O
-9	O
-(	O
-m	O
-)	O
-MW	O
-(	O
-kDa	O
-)	O
-DOSY	O
-2	O
-.	O
-04	O
-–	O
-2	O
-.	O
-15	O
-20	O
-2	O
-.	O
-94	O
-–	O
-3	O
-.	O
-02	O
-42	O
-S20	O
-,	O
-w	O
-(	O
-S	O
-)	O
-MW	O
-(	O
-kDa	O
-)	O
-S20	O
-,	O
-w	O
-(	O
-S	O
-)	O
-MW	O
-(	O
-kDa	O
-)	O
-SV	O
--	O
-AUC	O
-1	O
-.	O
-9	O
-17	O
-.	O
-6	O
-3	O
-.	O
-1	O
-35	O
-.	O
-5	O
-bPGRMC1	O
-C129S	B-mutant
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-ESI	O
--	O
-MS	O
-—	O
-17	O
-,	O
-827	O
-.	O
-91	O
-—	O
-36	O
-,	O
-887	O
-.	O
-07	O
-Theoretical	O
-17	O
-,	O
-827	O
-.	O
-59	O
-36	O
-,	O
-885	O
-.	O
-6	O
-S20	O
-,	O
-w	O
-(	O
-S	O
-)	O
-MW	O
-(	O
-kDa	O
-)	O
-S20	O
-,	O
-w	O
-(	O
-S	O
-)	O
-MW	O
-(	O
-kDa	O
-)	O
-SV	O
--	O
-AUC	O
-2	O
-.	O
-0	O
-18	O
-.	O
-1	O
-3	O
-.	O
-1	O
-35	O
-.	O
-8	O
-	O
-Differences	O
-in	O
-molecular	O
-weights	O
-of	O
-the	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-;	O
-a	O
-)	O
-and	O
-the	O
-C129S	B-mutant
-mutant	O
-(	O
-b	O
-)	O
-PGRMC1	O
-proteins	O
-in	O
-the	O
-absence	O
-(	O
-apo	O
-form	O
-)	O
-or	O
-the	O
-presence	O
-of	O
-haem	O
-(	O
-haem	O
--	O
-bound	O
-form	O
-).	O
-	O
-The	O
-protein	O
-sizes	O
-of	O
-the	O
-wt	O
-and	O
-C129S	B-mutant
-PGRMC1	O
-cytosolic	O
-domains	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-haem	O
-were	O
-estimated	O
-by	O
-ESI	O
--	O
-MS	O
-,	O
-DOSY	O
-and	O
-SV	O
--	O
-AUC	O
-.	O
-	O
-Hotspot	O
-autoimmune	O
-T	O
-cell	O
-receptor	O
-binding	O
-underlies	O
-pathogen	O
-and	O
-insulin	O
-peptide	O
-cross	O
--	O
-reactivity	O
-	O
-However	O
-,	O
-the	O
-mechanisms	O
-that	O
-allow	O
-the	O
-clonal	O
-T	O
-cell	O
-antigen	O
-receptor	O
-(	O
-TCR	O
-)	O
-to	O
-functionally	O
-engage	O
-multiple	O
-peptide	O
-–	O
-major	O
-histocompatibility	O
-complexes	O
-(	O
-pMHC	O
-)	O
-are	O
-unclear	O
-.	O
-	O
-Here	O
-,	O
-we	O
-studied	O
-multiligand	O
-discrimination	O
-by	O
-a	O
-human	O
-,	O
-preproinsulin	O
-reactive	O
-,	O
-MHC	O
-class	O
--	O
-I	O
-–	O
-restricted	O
-CD8	O
-+	O
-T	O
-cell	O
-clone	O
-(	O
-1E6	O
-)	O
-that	O
-can	O
-recognize	O
-over	O
-1	O
-million	O
-different	O
-peptides	O
-.	O
-	O
-We	O
-generated	O
-high	O
--	O
-resolution	O
-structures	O
-of	O
-the	O
-1E6	O
-TCR	O
-bound	O
-to	O
-7	O
-altered	O
-peptide	O
-ligands	O
-,	O
-including	O
-a	O
-pathogen	O
--	O
-derived	O
-peptide	O
-that	O
-was	O
-an	O
-order	O
-of	O
-magnitude	O
-more	O
-potent	O
-than	O
-the	O
-natural	O
-self	O
--	O
-peptide	O
-.	O
-	O
-Evaluation	O
-of	O
-these	O
-structures	O
-demonstrated	O
-that	O
-binding	O
-was	O
-stabilized	O
-through	O
-a	O
-conserved	O
-lock	O
--	O
-and	O
--	O
-key	O
-–	O
-like	O
-minimal	O
-binding	O
-footprint	O
-that	O
-enables	O
-1E6	O
-TCR	O
-to	O
-tolerate	O
-vast	O
-numbers	O
-of	O
-substitutions	O
-outside	O
-of	O
-this	O
-so	O
--	O
-called	O
-hotspot	O
-.	O
-	O
-Highly	O
-potent	O
-antigens	O
-of	O
-the	O
-1E6	O
-TCR	O
-engaged	O
-with	O
-a	O
-strong	O
-antipathogen	O
--	O
-like	O
-binding	O
-affinity	O
-;	O
-this	O
-engagement	O
-was	O
-governed	O
-though	O
-an	O
-energetic	O
-switch	O
-from	O
-an	O
-enthalpically	O
-to	O
-entropically	O
-driven	O
-interaction	O
-compared	O
-with	O
-the	O
-natural	O
-autoimmune	O
-ligand	O
-.	O
-	O
-T	O
-cells	O
-perform	O
-an	O
-essential	O
-role	O
-in	O
-adaptive	O
-immunity	O
-by	O
-interrogating	O
-the	O
-host	O
-proteome	O
-for	O
-anomalies	O
-,	O
-classically	O
-by	O
-recognizing	O
-peptides	O
-bound	O
-in	O
-major	O
-histocompatibility	O
-(	O
-MHC	O
-)	O
-molecules	O
-at	O
-the	O
-cell	O
-surface	O
-.	O
-	O
-Recent	O
-data	O
-supports	O
-the	O
-notion	O
-that	O
-,	O
-to	O
-perform	O
-this	O
-role	O
-,	O
-the	O
-highly	O
-variable	O
-αβ	O
-T	O
-cell	O
-antigen	O
-receptor	O
-(	O
-TCR	O
-)	O
-must	O
-be	O
-able	O
-to	O
-recognize	O
-thousands	O
-,	O
-if	O
-not	O
-millions	O
-,	O
-of	O
-different	O
-peptide	O
-ligands	O
-.	O
-	O
-This	O
-ability	O
-is	O
-required	O
-to	O
-enable	O
-the	O
-estimated	O
-25	O
-million	O
-distinct	O
-TCRs	O
-expressed	O
-in	O
-humans	O
-to	O
-provide	O
-effective	O
-immune	O
-coverage	O
-against	O
-all	O
-possible	O
-foreign	O
-peptide	O
-antigens	O
-.	O
-	O
-Several	O
-mechanisms	O
-,	O
-by	O
-which	O
-TCRs	O
-could	O
-bind	O
-to	O
-a	O
-large	O
-number	O
-of	O
-different	O
-peptide	O
--	O
-MHC	O
-(	O
-pMHC	O
-),	O
-have	O
-been	O
-proposed	O
-.	O
-	O
-Structures	O
-of	O
-unligated	O
-and	O
-ligated	O
-TCRs	O
-have	O
-shown	O
-that	O
-the	O
-TCR	O
-complementarity	O
-determining	O
-region	O
-(	O
-CDR	O
-)	O
-loops	O
-can	O
-be	O
-flexible	O
-,	O
-perhaps	O
-enabling	O
-peptide	O
-binding	O
-using	O
-different	O
-loop	O
-conformations	O
-.	O
-	O
-Both	O
-MHC	O
-and	O
-peptide	O
-have	O
-also	O
-been	O
-shown	O
-to	O
-undergo	O
-structural	O
-changes	O
-upon	O
-TCR	O
-binding	O
-,	O
-mediating	O
-an	O
-induced	O
-fit	O
-between	O
-the	O
-TCR	O
-and	O
-pMHC	O
-.	O
-	O
-Other	O
-studies	O
-,	O
-mainly	O
-in	O
-the	O
-murine	O
-system	O
-,	O
-have	O
-demonstrated	O
-that	O
-the	O
-same	O
-TCR	O
-can	O
-interact	O
-with	O
-different	O
-pMHCs	O
-using	O
-a	O
-common	O
-or	O
-divergent	O
-modality	O
-.	O
-	O
-Recent	O
-studies	O
-in	O
-model	O
-murine	O
-systems	O
-demonstrate	O
-that	O
-TCR	O
-cross	O
--	O
-reactivity	O
-can	O
-be	O
-governed	O
-by	O
-recognition	O
-of	O
-a	O
-conserved	O
-region	O
-in	O
-the	O
-peptide	O
-that	O
-allows	O
-tolerance	O
-of	O
-peptide	O
-sequence	O
-variation	O
-outside	O
-of	O
-this	O
-hotspot	O
-.	O
-	O
-We	O
-recently	O
-reported	O
-that	O
-the	O
-1E6	O
-human	O
-CD8	O
-+	O
-T	O
-cell	O
-clone	O
-—	O
-which	O
-mediates	O
-the	O
-destruction	O
-of	O
-β	O
-cells	O
-through	O
-the	O
-recognition	O
-of	O
-a	O
-major	O
-,	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-–	O
-restricted	O
-,	O
-preproinsulin	O
-signal	O
-peptide	O
-(	O
-ALWGPDPAAA15	O
-–	O
-24	O
-)	O
-—	O
-can	O
-recognize	O
-upwards	O
-of	O
-1	O
-million	O
-different	O
-peptides	O
-.	O
-	O
-CD8	O
-+	O
-T	O
-cells	O
-that	O
-recognize	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-–	O
-ALWGPDPAAA	O
-have	O
-been	O
-shown	O
-to	O
-populate	O
-insulitic	O
-lesions	O
-in	O
-patients	O
-with	O
-type	O
-1	O
-diabetes	O
-(	O
-T1D	O
-).	O
-	O
-We	O
-demonstrated	O
-that	O
-the	O
-TCR	O
-from	O
-the	O
-1E6	O
-T	O
-cell	O
-clone	O
-bound	O
-to	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-–	O
-ALWGPDPAAA	O
-using	O
-a	O
-limited	O
-footprint	O
-and	O
-very	O
-weak	O
-binding	O
-affinity	O
-.	O
-	O
-This	O
-first	O
-experimental	O
-evidence	O
-of	O
-a	O
-high	O
-level	O
-of	O
-CD8	O
-+	O
-T	O
-cell	O
-cross	O
--	O
-reactivity	O
-in	O
-a	O
-human	O
-autoimmune	O
-disease	O
-system	O
-hinted	O
-toward	O
-molecular	O
-mimicry	O
-by	O
-a	O
-more	O
-potent	O
-pathogenic	O
-peptide	O
-as	O
-a	O
-potential	O
-mechanism	O
-leading	O
-to	O
-β	O
-cell	O
-destruction	O
-.	O
-	O
-Here	O
-,	O
-we	O
-solved	O
-the	O
-structure	O
-of	O
-the	O
-1E6	O
-TCR	O
-with	O
-7	O
-altered	O
-peptide	O
-ligands	O
-(	O
-APLs	O
-)	O
-determined	O
-by	O
-our	O
-previously	O
-published	O
-combinatorial	O
-peptide	O
-library	O
-(	O
-CPL	O
-)	O
-screening	O
-,	O
-2	O
-of	O
-which	O
-mapped	O
-within	O
-human	O
-pathogens	O
-.	O
-	O
-These	O
-APLs	O
-differed	O
-from	O
-the	O
-natural	O
-preproinsulin	O
-peptide	O
-by	O
-up	O
-to	O
-7	O
-of	O
-10	O
-residues	O
-.	O
-	O
-We	O
-also	O
-solved	O
-the	O
-structure	O
-of	O
-each	O
-unligated	O
-APL	O
-to	O
-investigate	O
-whether	O
-structural	O
-changes	O
-occurred	O
-before	O
-or	O
-after	O
-binding	O
-—	O
-which	O
-,	O
-combined	O
-with	O
-an	O
-in	O
--	O
-depth	O
-cellular	O
-and	O
-biophysical	O
-analysis	O
-of	O
-the	O
-1E6	O
-interaction	O
-with	O
-each	O
-APL	O
-,	O
-demonstrated	O
-the	O
-molecular	O
-mechanism	O
-mediating	O
-the	O
-high	O
-level	O
-of	O
-cross	O
--	O
-reactivity	O
-exhibited	O
-by	O
-this	O
-preproinsulin	O
--	O
-reactive	O
-human	O
-CD8	O
-+	O
-T	O
-cell	O
-clone	O
-.	O
-	O
-The	O
-1E6	O
-T	O
-cell	O
-clone	O
-recognizes	O
-APLs	O
-across	O
-a	O
-large	O
-dynamic	O
-range	O
-.	O
-	O
-We	O
-have	O
-previously	O
-demonstrated	O
-that	O
-the	O
-1E6	O
-T	O
-cell	O
-clone	O
-can	O
-recognize	O
-over	O
-1	O
-million	O
-different	O
-peptides	O
-with	O
-a	O
-potency	O
-comparable	O
-with	O
-,	O
-or	O
-better	O
-than	O
-,	O
-the	O
-cognate	O
-preproinsulin	O
-peptide	O
-ALWGPDPAAA	O
-.	O
-	O
-From	O
-this	O
-large	O
-functional	O
-scan	O
-,	O
-we	O
-selected	O
-7	O
-different	O
-APLs	O
-that	O
-activated	O
-the	O
-1E6	O
-T	O
-cell	O
-clone	O
-across	O
-a	O
-wide	O
-(	O
-4	O
--	O
-log	O
-)	O
-functional	O
-range	O
-(	O
-Table	O
-1	O
-).	O
-	O
-Two	O
-of	O
-these	O
-peptides	O
-,	O
-MVWGPDPLYV	O
-and	O
-RQFGPDWIVA	O
-(	O
-bold	O
-text	O
-signifies	O
-amino	O
-acids	O
-that	O
-are	O
-different	O
-from	O
-the	O
-index	O
-preproinsulin	O
-–	O
-derived	O
-sequence	O
-),	O
-are	O
-contained	O
-within	O
-the	O
-proteomes	O
-of	O
-the	O
-human	O
-pathogens	O
-Bacteroides	O
-fragilis	O
-/	O
-thetaiotaomicron	O
-and	O
-Clostridium	O
-asparagiforme	O
-,	O
-respectively	O
-.	O
-	O
-Competitive	O
-functional	O
-testing	O
-revealed	O
-that	O
-the	O
-preproinsulin	O
--	O
-derived	O
-sequence	O
-ALWGPDPAAA	O
-was	O
-one	O
-of	O
-the	O
-least	O
-potent	O
-targets	O
-for	O
-1E6	O
-,	O
-with	O
-only	O
-the	O
-MVWGPDPLYV	O
-and	O
-YLGGPDFPTI	O
-demonstrating	O
-a	O
-similar	O
-low	O
--	O
-activity	O
-profile	O
-in	O
-MIP	O
--	O
-1β	O
-secretion	O
-and	O
-target	O
-killing	O
-assays	O
-(	O
-Figure	O
-1	O
-,	O
-A	O
-and	O
-B	O
-).	O
-	O
-The	O
-RQFGPDWIVA	O
-sequence	O
-(	O
-present	O
-in	O
-C	O
-.	O
-asparagiforme	O
-)	O
-activated	O
-the	O
-1E6	O
-T	O
-cell	O
-with	O
-around	O
-1	O
-log	O
-–	O
-greater	O
-potency	O
-compared	O
-with	O
-ALWGPDPAAA	O
-.	O
-	O
-At	O
-the	O
-other	O
-end	O
-of	O
-the	O
-spectrum	O
-,	O
-the	O
-RQFGPDFPTI	O
-peptide	O
-stimulated	O
-MIP	O
--	O
-1β	O
-release	O
-and	O
-killing	O
-by	O
-1E6	O
-at	O
-an	O
-exogenous	O
-peptide	O
-concentration	O
-2	O
-–	O
-3	O
-logs	O
-lower	O
-compared	O
-with	O
-ALWGPDPAAA	O
-.	O
-	O
-The	O
-pattern	O
-of	O
-peptide	O
-potency	O
-was	O
-closely	O
-mirrored	O
-by	O
-pMHC	O
-tetramer	O
-staining	O
-experiments	O
-(	O
-Figure	O
-1C	O
-and	O
-plots	O
-shown	O
-in	O
-Supplemental	O
-Figure	O
-1	O
-;	O
-supplemental	O
-material	O
-available	O
-online	O
-with	O
-this	O
-article	O
-;	O
-doi	O
-:	O
-10	O
-.	O
-1172	O
-/	O
-JCI85679DS1	O
-).	O
-	O
-Here	O
-,	O
-the	O
-A2	O
--	O
-RQFGPDFPTI	O
-tetramer	O
-stained	O
-1E6	O
-with	O
-the	O
-greatest	O
-MFI	O
-,	O
-gradually	O
-decreasing	O
-to	O
-the	O
-weakest	O
-tetramers	O
-:	O
-A2	O
--	O
-MVWGPDPLYV	O
-and	O
--	O
-YLGGPDFPTI	O
-.	O
-	O
-To	O
-parallel	O
-the	O
-functional	O
-analysis	O
-,	O
-we	O
-also	O
-performed	O
-thermal	O
-melt	O
-(	O
-Tm	O
-)	O
-experiments	O
-using	O
-synchrotron	O
-radiation	O
-circular	O
-dichroism	O
-(	O
-SRCD	O
-)	O
-to	O
-investigate	O
-the	O
-stability	O
-of	O
-each	O
-APL	O
-(	O
-Figure	O
-1D	O
-).	O
-	O
-The	O
-range	O
-of	O
-Tm	O
-was	O
-between	O
-49	O
-.	O
-4	O
-°	O
-C	O
-(	O
-RQFGPDWIVA	O
-)	O
-and	O
-60	O
-.	O
-3	O
-°	O
-C	O
-(	O
-YQFGPDFPIA	O
-),	O
-with	O
-an	O
-average	O
-approximately	O
-55	O
-°	O
-C	O
-,	O
-similar	O
-to	O
-our	O
-previous	O
-findings	O
-.	O
-	O
-This	O
-pattern	O
-of	O
-stability	O
-did	O
-not	O
-correlate	O
-with	O
-the	O
-T	O
-cell	O
-activation	O
-or	O
-tetramer	O
-staining	O
-experiments	O
-,	O
-indicating	O
-that	O
-peptide	O
-binding	O
-to	O
-the	O
-MHC	O
-do	O
-not	O
-explain	O
-ligand	O
-potency	O
-.	O
-	O
-The	O
-1E6	O
-TCR	O
-can	O
-bind	O
-peptides	O
-with	O
-strong	O
-antipathogen	O
--	O
-like	O
-affinities	O
-.	O
-	O
-We	O
-,	O
-and	O
-others	O
-,	O
-have	O
-previously	O
-demonstrated	O
-that	O
-antipathogenic	O
-TCRs	O
-tend	O
-to	O
-bind	O
-with	O
-stronger	O
-affinity	O
-compared	O
-with	O
-self	O
--	O
-reactive	O
-TCRs	O
-,	O
-likely	O
-a	O
-consequence	O
-of	O
-the	O
-deletion	O
-of	O
-T	O
-cells	O
-with	O
-high	O
--	O
-affinity	O
-self	O
--	O
-reactive	O
-TCR	O
-during	O
-thymic	O
-selection	O
-.	O
-	O
-In	O
-accordance	O
-with	O
-this	O
-trend	O
-,	O
-the	O
-1E6	O
-TCR	O
-bound	O
-the	O
-natural	O
-preproinsulin	O
-peptide	O
-,	O
-ALWGPDPAAA	O
-,	O
-with	O
-the	O
-weakest	O
-affinity	O
-currently	O
-published	O
-for	O
-a	O
-human	O
-CD8	O
-+	O
-T	O
-cell	O
-–	O
-derived	O
-TCR	O
-with	O
-a	O
-biologically	O
-relevant	O
-ligand	O
-(	O
-KD	O
->	O
-200	O
-μM	O
-;	O
-KD	O
-,	O
-equilibrium	O
-binding	O
-constant	O
-).	O
-	O
-Surface	O
-plasmon	O
-resonance	O
-(	O
-SPR	O
-)	O
-analysis	O
-of	O
-the	O
-1E6	O
-TCR	O
-–	O
-pMHC	O
-interaction	O
-for	O
-all	O
-7	O
-APLs	O
-(	O
-Figure	O
-2	O
-,	O
-A	O
-–	O
-H	O
-)	O
-demonstrated	O
-that	O
-stronger	O
-binding	O
-affinity	O
-(	O
-represented	O
-as	O
-ΔG	O
-°,	O
-kcal	O
-/	O
-mol	O
-)	O
-correlated	O
-well	O
-with	O
-the	O
-EC50	O
-values	O
-(	O
-peptide	O
-concentration	O
-required	O
-to	O
-reach	O
-half	O
--	O
-maximal	O
-1E6	O
-T	O
-cell	O
-killing	O
-)	O
-for	O
-each	O
-ligand	O
-,	O
-demonstrated	O
-by	O
-a	O
-Pearson	O
-’	O
-s	O
-correlation	O
-analysis	O
-value	O
-of	O
-0	O
-.	O
-8	O
-(	O
-P	O
-=	O
-0	O
-.	O
-01	O
-)	O
-(	O
-Figure	O
-2I	O
-).	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-this	O
-correlation	O
-,	O
-although	O
-consistent	O
-with	O
-the	O
-T	O
-cell	O
-killing	O
-experiments	O
-,	O
-uses	O
-only	O
-approximate	O
-affinities	O
-calculated	O
-for	O
-the	O
-2	O
-weakest	O
-ligands	O
-.	O
-	O
-First	O
-,	O
-the	O
-1E6	O
-T	O
-cell	O
-could	O
-still	O
-functionally	O
-respond	O
-to	O
-peptide	O
-when	O
-the	O
-TCR	O
-binding	O
-affinity	O
-was	O
-extremely	O
-weak	O
-,	O
-e	O
-.	O
-g	O
-.,	O
-the	O
-1E6	O
-TCR	O
-binding	O
-affinity	O
-for	O
-the	O
-A2	O
--	O
-MVWGPDPLYV	O
-peptide	O
-was	O
-KD	O
-=	O
-~	O
-600	O
-μM	O
-.	O
-Second	O
-,	O
-the	O
-1E6	O
-TCR	O
-bound	O
-to	O
-A2	O
--	O
-RQFGPDFPTI	O
-with	O
-KD	O
-=	O
-0	O
-.	O
-5	O
-μM	O
-,	O
-equivalent	O
-to	O
-the	O
-binding	O
-affinity	O
-of	O
-the	O
-very	O
-strongest	O
-antipathogen	O
-TCRs	O
-.	O
-	O
-Third	O
-,	O
-the	O
-1E6	O
-TCR	O
-bound	O
-to	O
-A2	O
--	O
-RQFGPDWIVA	O
-peptide	O
-,	O
-within	O
-the	O
-C	O
-.	O
-asparagiforme	O
-proteome	O
-,	O
-with	O
-approximately	O
-4	O
--	O
-fold	O
-stronger	O
-affinity	O
-than	O
-A2	O
--	O
-ALWGPDPAAA	O
-,	O
-demonstrating	O
-the	O
-potential	O
-for	O
-a	O
-pathogen	O
--	O
-derived	O
-antigen	O
-to	O
-initiate	O
-a	O
-response	O
-to	O
-the	O
-self	O
--	O
-derived	O
-sequence	O
-.	O
-	O
-Finally	O
-,	O
-these	O
-data	O
-demonstrate	O
-the	O
-largest	O
-range	O
-of	O
-binding	O
-affinities	O
-reported	O
-for	O
-a	O
-natural	O
-,	O
-endogenous	O
-human	O
-TCR	O
-of	O
-more	O
-than	O
-3	O
-logs	O
-of	O
-magnitude	O
-(	O
-A2	O
--	O
-MVWGPDPLYV	O
-vs	O
-.	O
-A2	O
--	O
-RQFGPDFPTI	O
-).	O
-	O
-To	O
-confirm	O
-the	O
-affinity	O
-spread	O
-detected	O
-by	O
-SPR	O
-,	O
-and	O
-to	O
-evaluate	O
-whether	O
-experiments	O
-performed	O
-using	O
-soluble	O
-molecules	O
-were	O
-biologically	O
-relevant	O
-to	O
-events	O
-at	O
-the	O
-T	O
-cell	O
-surface	O
-,	O
-we	O
-determined	O
-the	O
-effective	O
-2D	O
-affinity	O
-of	O
-each	O
-APL	O
-using	O
-an	O
-adhesion	O
-frequency	O
-assay	O
-in	O
-which	O
-a	O
-human	O
-rbc	O
-coated	O
-in	O
-pMHC	O
-acted	O
-as	O
-an	O
-adhesion	O
-sensor	O
-.	O
-	O
-In	O
-agreement	O
-with	O
-SPR	O
-experiments	O
-,	O
-the	O
-range	O
-of	O
-2D	O
-affinities	O
-we	O
-detected	O
-differed	O
-by	O
-around	O
-3	O
-logs	O
-,	O
-with	O
-the	O
-A2	O
--	O
-MVWGPDPLYV	O
-generating	O
-the	O
-weakest	O
-2D	O
-affinity	O
-(	O
-2	O
-.	O
-6	O
-×	O
-10	O
-–	O
-5	O
-AcKa	O
-μm4	O
-)	O
-and	O
-A2	O
--	O
-RQFGPDFPTI	O
-the	O
-strongest	O
-(	O
-4	O
-.	O
-5	O
-��	O
-10	O
-–	O
-2	O
-AcKa	O
-μm4	O
-)	O
-(	O
-Figure	O
-2J	O
-).	O
-	O
-As	O
-with	O
-the	O
-3D	O
-affinity	O
-measurements	O
-,	O
-the	O
-2D	O
-affinity	O
-measurements	O
-correlated	O
-well	O
-with	O
-the	O
-EC50	O
-values	O
-for	O
-each	O
-ligand	O
-(	O
-Figure	O
-2K	O
-)	O
-demonstrating	O
-a	O
-strong	O
-correlation	O
-(	O
-Pearson	O
-’	O
-s	O
-correlation	O
-=	O
-0	O
-.	O
-8	O
-,	O
-P	O
-=	O
-0	O
-.	O
-01	O
-)	O
-between	O
-T	O
-cell	O
-antigen	O
-sensitivity	O
-and	O
-TCR	O
-binding	O
-affinity	O
-.	O
-	O
-Of	O
-note	O
-,	O
-these	O
-data	O
-demonstrate	O
-a	O
-close	O
-agreement	O
-between	O
-the	O
-3D	O
-affinity	O
-values	O
-generated	O
-using	O
-SPR	O
-and	O
-2D	O
-affinity	O
-values	O
-generated	O
-using	O
-adhesion	O
-frequency	O
-assays	O
-.	O
-	O
-The	O
-1E6	O
-TCR	O
-uses	O
-a	O
-consensus	O
-binding	O
-mode	O
-to	O
-engage	O
-multiple	O
-APLs	O
-.	O
-	O
-Our	O
-previous	O
-structure	O
-of	O
-the	O
-1E6	O
--	O
-A2	O
--	O
-ALWGPDPAAA	O
-complex	O
-demonstrated	O
-a	O
-limited	O
-binding	O
-footprint	O
-between	O
-the	O
-TCR	O
-and	O
-pMHC	O
-.	O
-	O
-The	O
-low	O
-number	O
-of	O
-contacts	O
-between	O
-the	O
-2	O
-molecules	O
-most	O
-likely	O
-contributed	O
-to	O
-the	O
-weak	O
-binding	O
-affinity	O
-of	O
-the	O
-interaction	O
-.	O
-	O
-In	O
-order	O
-to	O
-examine	O
-the	O
-mechanism	O
-by	O
-which	O
-the	O
-1E6	O
-TCR	O
-engaged	O
-a	O
-wide	O
-range	O
-of	O
-peptides	O
-with	O
-divergent	O
-binding	O
-affinities	O
-,	O
-we	O
-solved	O
-the	O
-structure	O
-of	O
-the	O
-1E6	O
-TCR	O
-in	O
-complex	O
-with	O
-all	O
-7	O
-APLs	O
-used	O
-in	O
-Figure	O
-2	O
-.	O
-	O
-All	O
-structures	O
-were	O
-solved	O
-in	O
-space	O
-group	O
-P1	O
-to	O
-2	O
-–	O
-3	O
-Å	O
-resolution	O
-with	O
-crystallographic	O
-Rwork	O
-/	O
-Rfree	O
-ratios	O
-within	O
-accepted	O
-limits	O
-as	O
-shown	O
-in	O
-the	O
-theoretically	O
-expected	O
-distribution	O
-(	O
-ref	O
-.	O
-and	O
-Supplemental	O
-Table	O
-1	O
-).	O
-	O
-The	O
-1E6	O
-TCR	O
-used	O
-a	O
-very	O
-similar	O
-overall	O
-binding	O
-modality	O
-to	O
-engage	O
-all	O
-of	O
-the	O
-APLs	O
-,	O
-with	O
-root	O
-mean	O
-square	O
-deviation	O
-ranging	O
-between	O
-0	O
-.	O
-81	O
-and	O
-1	O
-.	O
-12	O
-Å2	O
-(	O
-compared	O
-with	O
-1E6	O
--	O
-A2	O
--	O
-ALWGPDPAAA	O
-).	O
-	O
-The	O
-relatively	O
-broad	O
-range	O
-of	O
-buried	O
-surface	O
-areas	O
-(	O
-1	O
-,	O
-670	O
-–	O
-1	O
-,	O
-920	O
-Å2	O
-)	O
-did	O
-not	O
-correlate	O
-well	O
-with	O
-TCR	O
-binding	O
-affinity	O
-(	O
-Pearson	O
-’	O
-s	O
-correlation	O
-=	O
-0	O
-.	O
-45	O
-,	O
-P	O
-=	O
-0	O
-.	O
-2	O
-).	O
-	O
-The	O
-surface	O
-complementarity	O
-values	O
-(	O
-0	O
-.	O
-52	O
-–	O
-0	O
-.	O
-7	O
-)	O
-correlated	O
-slightly	O
-with	O
-affinity	O
-(	O
-Pearson	O
-’	O
-s	O
-correlation	O
-=	O
-0	O
-.	O
-7	O
-,	O
-P	O
-=	O
-0	O
-.	O
-05	O
-)	O
-but	O
-could	O
-not	O
-explain	O
-all	O
-differences	O
-in	O
-binding	O
-(	O
-Figure	O
-3A	O
-and	O
-Table	O
-2	O
-).	O
-	O
-The	O
-TCR	O
-CDR	O
-loops	O
-were	O
-in	O
-a	O
-very	O
-similar	O
-position	O
-in	O
-all	O
-complexes	O
-,	O
-apart	O
-from	O
-some	O
-slight	O
-deviations	O
-in	O
-the	O
-TCR	O
-β	O
--	O
-chain	O
-(	O
-Figure	O
-3B	O
-);	O
-the	O
-peptides	O
-were	O
-all	O
-presented	O
-in	O
-a	O
-similar	O
-conformation	O
-(	O
-Figure	O
-3C	O
-);	O
-and	O
-there	O
-was	O
-minimal	O
-variation	O
-in	O
-crossing	O
-angles	O
-of	O
-the	O
-TCR	O
-(	O
-42	O
-.	O
-3	O
-°–	O
-45	O
-.	O
-6	O
-°)	O
-(	O
-Figure	O
-3D	O
-).	O
-	O
-Overall	O
-,	O
-the	O
-1E6	O
-TCR	O
-used	O
-a	O
-canonical	O
-binding	O
-mode	O
-to	O
-engage	O
-each	O
-APL	O
-with	O
-the	O
-TCR	O
-α	O
--	O
-chain	O
-positioned	O
-over	O
-the	O
-MHC	O
-class	O
-I	O
-(	O
-MHCI	O
-)	O
-α2	O
--	O
-helix	O
-and	O
-the	O
-TCR	O
-β	O
--	O
-chain	O
-over	O
-the	O
-MHCI	O
-α	O
--	O
-1	O
-helix	O
-,	O
-straddling	O
-the	O
-peptide	O
-cargo	O
-.	O
-	O
-However	O
-,	O
-subtle	O
-differences	O
-in	O
-the	O
-respective	O
-interfaces	O
-were	O
-apparent	O
-(	O
-discussed	O
-below	O
-)	O
-and	O
-resulted	O
-in	O
-altered	O
-binding	O
-affinities	O
-of	O
-the	O
-respective	O
-complexes	O
-.	O
-	O
-Interactions	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-different	O
-APLs	O
-are	O
-focused	O
-around	O
-a	O
-conserved	O
-GPD	O
-peptide	O
-motif	O
-.	O
-	O
-We	O
-next	O
-performed	O
-an	O
-in	O
--	O
-depth	O
-atomic	O
-analysis	O
-of	O
-the	O
-contacts	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-each	O
-APL	O
-to	O
-determine	O
-the	O
-structural	O
-basis	O
-for	O
-the	O
-altered	O
-T	O
-cell	O
-peptide	O
-sensitivities	O
-and	O
-TCR	O
-binding	O
-affinities	O
-(	O
-Table	O
-2	O
-).	O
-	O
-Concomitant	O
-with	O
-our	O
-global	O
-analysis	O
-of	O
-1E6	O
-TCR	O
-binding	O
-to	O
-the	O
-APLs	O
-,	O
-we	O
-observed	O
-a	O
-common	O
-interaction	O
-element	O
-,	O
-consistent	O
-with	O
-our	O
-previous	O
-findings	O
-,	O
-that	O
-utilized	O
-TCR	O
-residues	O
-Tyr97α	O
-and	O
-Trp97β	O
-,	O
-forming	O
-an	O
-aromatic	O
-cap	O
-over	O
-a	O
-central	O
-GPD	O
-motif	O
-that	O
-was	O
-present	O
-in	O
-all	O
-of	O
-the	O
-APLs	O
-(	O
-Figure	O
-4	O
-).	O
-	O
-Interactions	O
-between	O
-these	O
-2	O
-TCR	O
-and	O
-3	O
-peptide	O
-residues	O
-accounted	O
-for	O
-41	O
-%–	O
-50	O
-%	O
-of	O
-the	O
-total	O
-contacts	O
-across	O
-all	O
-complexes	O
-(	O
-Table	O
-2	O
-),	O
-demonstrating	O
-the	O
-conserved	O
-peptide	O
-centric	O
-binding	O
-mode	O
-utilized	O
-by	O
-the	O
-1E6	O
-TCR	O
-.	O
-	O
-This	O
-fixed	O
-anchoring	O
-between	O
-the	O
-2	O
-molecules	O
-was	O
-important	O
-for	O
-stabilization	O
-of	O
-the	O
-TCR	O
--	O
-pMHC	O
-complex	O
-,	O
-as	O
-—	O
-although	O
-other	O
-peptides	O
-without	O
-the	O
-‘	O
-GDP	O
-’	O
-motif	O
-were	O
-tested	O
-and	O
-shown	O
-to	O
-activate	O
-the	O
-1E6	O
-T	O
-cell	O
-clone	O
-—	O
-we	O
-were	O
-unable	O
-to	O
-measure	O
-robust	O
-affinities	O
-using	O
-SPR	O
-(	O
-data	O
-not	O
-shown	O
-).	O
-	O
-These	O
-data	O
-support	O
-the	O
-requirement	O
-for	O
-a	O
-conserved	O
-interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-the	O
-GPD	O
-motif	O
-,	O
-as	O
-we	O
-observed	O
-in	O
-our	O
-previously	O
-published	O
-1E6	O
--	O
-A2	O
--	O
-ALWGPDPAAA	O
-structure	O
-.	O
-	O
-Focused	O
-hotspot	O
-binding	O
-around	O
-a	O
-conserved	O
-GPD	O
-motif	O
-enables	O
-the	O
-1E6	O
-TCR	O
-to	O
-tolerate	O
-peptide	O
-degeneracy	O
-.	O
-	O
-Although	O
-the	O
-1E6	O
-TCR	O
-formed	O
-a	O
-similar	O
-overall	O
-interaction	O
-with	O
-each	O
-APL	O
-,	O
-the	O
-stabilization	O
-between	O
-the	O
-TCR	O
-and	O
-the	O
-GPD	O
-motif	O
-enabled	O
-fine	O
-differences	O
-in	O
-the	O
-contact	O
-network	O
-with	O
-both	O
-the	O
-peptide	O
-and	O
-MHC	O
-surface	O
-that	O
-allowed	O
-discrimination	O
-between	O
-each	O
-ligand	O
-(	O
-Figure	O
-5	O
-).	O
-	O
-For	O
-example	O
-,	O
-the	O
-1E6	O
-TCR	O
-made	O
-only	O
-47	O
-peptide	O
-contacts	O
-with	O
-A2	O
--	O
-MVWGPDPLYV	O
-(	O
-KD	O
-=	O
-~	O
-600	O
-μM	O
-)	O
-compared	O
-with	O
-63	O
-and	O
-57	O
-contacts	O
-with	O
-A2	O
--	O
-YQFGPDFPIA	O
-(	O
-KD	O
-=	O
-7	O
-.	O
-4	O
-μM	O
-)	O
-and	O
-A2	O
--	O
-RQFGPDFPTI	O
-(	O
-KD	O
-=	O
-0	O
-.	O
-5	O
-μM	O
-),	O
-respectively	O
-.	O
-	O
-Although	O
-the	O
-number	O
-of	O
-peptide	O
-contacts	O
-was	O
-a	O
-good	O
-predictor	O
-of	O
-TCR	O
-binding	O
-affinity	O
-for	O
-some	O
-of	O
-the	O
-APLs	O
-,	O
-for	O
-others	O
-,	O
-the	O
-correlation	O
-was	O
-poor	O
-(	O
-Pearson	O
-’	O
-s	O
-correlation	O
-=	O
-0	O
-.	O
-045	O
-,	O
-P	O
-=	O
-0	O
-.	O
-92	O
-),	O
-possibly	O
-because	O
-of	O
-different	O
-resolutions	O
-for	O
-each	O
-complex	O
-structure	O
-.	O
-	O
-For	O
-example	O
-,	O
-the	O
-1E6	O
-TCR	O
-made	O
-64	O
-peptide	O
-contacts	O
-with	O
-A2	O
--	O
-YLGGPDFPTI	O
-(	O
-KD	O
-=	O
-~	O
-400	O
-μM	O
-)	O
-compared	O
-with	O
-43	O
-contacts	O
-with	O
-A2	O
--	O
-RQWGPDPAAV	O
-(	O
-KD	O
-=	O
-7	O
-.	O
-8	O
-μM	O
-).	O
-	O
-The	O
-most	O
-important	O
-peptide	O
-modification	O
-in	O
-terms	O
-of	O
-generating	O
-new	O
-contacts	O
-was	O
-peptide	O
-position	O
-1	O
-.	O
-	O
-The	O
-stronger	O
-ligands	O
-all	O
-encoded	O
-larger	O
-side	O
-chains	O
-(	O
-Arg	O
-or	O
-Tyr	O
-)	O
-at	O
-peptide	O
-position	O
-1	O
-(	O
-Figure	O
-5	O
-,	O
-E	O
-–	O
-H	O
-),	O
-enabling	O
-interactions	O
-with	O
-1E6	O
-that	O
-were	O
-not	O
-present	O
-in	O
-the	O
-weaker	O
-APLs	O
-that	O
-lacked	O
-large	O
-side	O
-chains	O
-in	O
-this	O
-position	O
-(	O
-Figure	O
-5	O
-,	O
-A	O
-,	O
-C	O
-,	O
-and	O
-D	O
-).	O
-	O
-We	O
-have	O
-previously	O
-shown	O
-that	O
-the	O
-1E6	O
-TCR	O
-uses	O
-a	O
-rigid	O
-lock	O
--	O
-and	O
--	O
-key	O
-mechanism	O
-during	O
-binding	O
-to	O
-A2	O
--	O
-ALWGPDPAAA	O
-.	O
-	O
-These	O
-data	O
-demonstrated	O
-that	O
-the	O
-unligated	O
-structure	O
-of	O
-the	O
-1E6	O
-TCR	O
-was	O
-virtually	O
-identical	O
-to	O
-its	O
-ligated	O
-counterparts	O
-.	O
-	O
-In	O
-order	O
-to	O
-determine	O
-whether	O
-any	O
-of	O
-the	O
-APLs	O
-required	O
-an	O
-induced	O
-fit	O
-mechanism	O
-during	O
-binding	O
-that	O
-could	O
-explain	O
-the	O
-difference	O
-in	O
-free	O
-binding	O
-energy	O
-(	O
-ΔG	O
-)	O
-between	O
-each	O
-complex	O
-(	O
-Table	O
-2	O
-),	O
-we	O
-solved	O
-the	O
-unligated	O
-structures	O
-of	O
-all	O
-7	O
-APLs	O
-(	O
-the	O
-A2	O
--	O
-ALWGPDPAAA	O
-structure	O
-has	O
-been	O
-previously	O
-published	O
-and	O
-was	O
-used	O
-in	O
-this	O
-comparison	O
-,	O
-ref	O
-.)	O
-(	O
-Figure	O
-6	O
-and	O
-Supplemental	O
-Table	O
-2	O
-).	O
-	O
-The	O
-unligated	O
-A2	O
--	O
-MVWGPDPLYV	O
-(	O
-KD	O
-=	O
-~	O
-600	O
-μM	O
-)	O
-structure	O
-revealed	O
-that	O
-the	O
-side	O
-chain	O
-Tyr9	O
-swung	O
-around	O
-8	O
-Å	O
-in	O
-the	O
-complex	O
-structure	O
-,	O
-subsequently	O
-making	O
-contacts	O
-with	O
-TCR	O
-residues	O
-Asp30β	O
-and	O
-Asn51β	O
-(	O
-Figure	O
-6A	O
-and	O
-Figure	O
-5A	O
-,	O
-respectively	O
-).	O
-	O
-This	O
-movement	O
-could	O
-result	O
-in	O
-an	O
-entropic	O
-penalty	O
-contributing	O
-to	O
-the	O
-weak	O
-TCR	O
-binding	O
-affinity	O
-we	O
-observed	O
-for	O
-this	O
-ligand	O
-.	O
-	O
-Additional	O
-small	O
-movements	O
-in	O
-the	O
-Cα	O
-backbone	O
-of	O
-the	O
-peptide	O
-around	O
-peptide	O
-residue	O
-Asp6	O
-were	O
-apparent	O
-in	O
-the	O
-A2	O
--	O
-YLGGPDFPTI	O
-(	O
-KD	O
-=	O
-~	O
-400	O
-μM	O
-),	O
-A2	O
--	O
-ALWGPDPAAA	O
-(	O
-KD	O
-=	O
-~	O
-208	O
-μM	O
-),	O
-and	O
-A2	O
--	O
-RQFGPDWIVA	O
-(	O
-KD	O
-=	O
-44	O
-.	O
-4	O
-μM	O
-)	O
-structures	O
-(	O
-Figure	O
-6	O
-,	O
-B	O
-,	O
-C	O
-,	O
-and	O
-E	O
-).	O
-	O
-The	O
-unligated	O
-structures	O
-of	O
-A2	O
--	O
-AQWGPDAAA	O
-,	O
-A2	O
--	O
-RQWGPDPAAV	O
-,	O
-A2	O
--	O
-YQFGPDFPIA	O
-,	O
-and	O
-A2	O
--	O
-RQFGPDFPTI	O
-were	O
-virtually	O
-identical	O
-when	O
-in	O
-complex	O
-with	O
-1E6	O
-(	O
-Figure	O
-6	O
-,	O
-D	O
-and	O
-F	O
-–	O
-H	O
-).	O
-	O
-Apart	O
-from	O
-the	O
-case	O
-of	O
-A2	O
--	O
-AQWGPDAAA	O
-(	O
-KD	O
-=	O
-61	O
-.	O
-9	O
-μM	O
-),	O
-these	O
-observations	O
-support	O
-the	O
-conclusion	O
-that	O
-the	O
-higher	O
--	O
-affinity	O
-ligands	O
-required	O
-less	O
-conformational	O
-melding	O
-during	O
-binding	O
-,	O
-which	O
-could	O
-be	O
-energetically	O
-beneficial	O
-(	O
-lower	O
-entopic	O
-cost	O
-)	O
-during	O
-ligation	O
-with	O
-the	O
-1E6	O
-TCR	O
-.	O
-	O
-Peptide	O
-modifications	O
-alter	O
-the	O
-interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-the	O
-MHC	O
-surface	O
-.	O
-	O
-In	O
-addition	O
-to	O
-changes	O
-between	O
-the	O
-TCR	O
-and	O
-peptide	O
-component	O
-,	O
-we	O
-also	O
-observed	O
-that	O
-different	O
-APLs	O
-had	O
-different	O
-knock	O
--	O
-on	O
-effects	O
-between	O
-the	O
-TCR	O
-and	O
-MHC	O
-.	O
-	O
-MHC	O
-residue	O
-Arg65	O
-that	O
-forms	O
-part	O
-of	O
-the	O
-MHC	O
-restriction	O
-triad	O
-(	O
-Arg65	O
-,	O
-Ala69	O
-,	O
-and	O
-Gln155	O
-)	O
-played	O
-a	O
-central	O
-role	O
-in	O
-TCR	O
--	O
-MHC	O
-contacts	O
-,	O
-with	O
-Gln155	O
-playing	O
-a	O
-less	O
-important	O
-role	O
-and	O
-Ala69	O
-playing	O
-no	O
-role	O
-in	O
-binding	O
-at	O
-the	O
-interface	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-Generally	O
-,	O
-the	O
-weaker	O
--	O
-affinity	O
-APLs	O
-made	O
-fewer	O
-contacts	O
-with	O
-the	O
-MHC	O
-surface	O
-(	O
-27	O
-–	O
-29	O
-interactions	O
-)	O
-compared	O
-with	O
-the	O
-stronger	O
--	O
-affinity	O
-APLs	O
-(	O
-29	O
-–	O
-35	O
-contacts	O
-),	O
-consistent	O
-with	O
-a	O
-better	O
-Pearson	O
-’	O
-s	O
-correlation	O
-value	O
-(	O
-0	O
-.	O
-55	O
-)	O
-compared	O
-with	O
-TCR	O
--	O
-peptide	O
-interactions	O
-versus	O
-affinity	O
-(	O
-0	O
-.	O
-045	O
-).	O
-	O
-For	O
-instance	O
-,	O
-contacts	O
-were	O
-made	O
-between	O
-TCR	O
-residue	O
-Val53β	O
-and	O
-MHC	O
-residue	O
-Gln72	O
-in	O
-all	O
-APLs	O
-except	O
-for	O
-in	O
-the	O
-weakest	O
-affinity	O
-ligand	O
-pair	O
-,	O
-1E6	O
--	O
-A2	O
--	O
-MVWGPDPLYV	O
-,	O
-in	O
-which	O
-a	O
-subtle	O
-change	O
-in	O
-TCR	O
-conformation	O
-—	O
-probably	O
-mediated	O
-by	O
-different	O
-peptide	O
-contacts	O
-—	O
-abrogated	O
-this	O
-interaction	O
-(	O
-Figure	O
-7A	O
-).	O
-	O
-An	O
-energetic	O
-switch	O
-from	O
-unfavorable	O
-to	O
-favorable	O
-entropy	O
-(	O
-order	O
--	O
-to	O
--	O
-disorder	O
-)	O
-correlates	O
-with	O
-antigen	O
-potency	O
-.	O
-	O
-Our	O
-analysis	O
-of	O
-the	O
-contact	O
-network	O
-provided	O
-some	O
-clues	O
-that	O
-could	O
-explain	O
-the	O
-different	O
-antigen	O
-potencies	O
-and	O
-binding	O
-affinities	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-the	O
-different	O
-APLs	O
-.	O
-	O
-For	O
-example	O
-,	O
-the	O
-1E6	O
-TCR	O
-bound	O
-to	O
-A2	O
--	O
-RQWGPDPAAV	O
-with	O
-the	O
-third	O
-strongest	O
-affinity	O
-(	O
-KD	O
-=	O
-7	O
-.	O
-8	O
-μM	O
-)	O
-but	O
-made	O
-fewer	O
-contacts	O
-than	O
-with	O
-A2	O
--	O
-ALWGPDPAAA	O
-(	O
-KD	O
-=	O
-~	O
-208	O
-μM	O
-)	O
-(	O
-Table	O
-2	O
-).	O
-	O
-Thus	O
-,	O
-we	O
-performed	O
-an	O
-in	O
--	O
-depth	O
-thermodynamic	O
-analysis	O
-of	O
-6	O
-of	O
-the	O
-ligands	O
-under	O
-investigation	O
-(	O
-Figure	O
-8	O
-and	O
-Supplemental	O
-Table	O
-3	O
-).	O
-	O
-The	O
-weak	O
-binding	O
-affinity	O
-between	O
-1E6	O
-and	O
-A2	O
--	O
-MVWGPDPLYV	O
-and	O
-A2	O
--	O
-YLGGPDFPTI	O
-generated	O
-thermodynamic	O
-data	O
-that	O
-were	O
-not	O
-robust	O
-enough	O
-to	O
-gain	O
-insight	O
-into	O
-the	O
-enthalpic	O
-(	O
-ΔH	O
-°)	O
-and	O
-entropic	O
-(	O
-TΔS	O
-°)	O
-changes	O
-that	O
-contributed	O
-to	O
-the	O
-different	O
-binding	O
-affinities	O
-/	O
-potencies	O
-for	O
-each	O
-APL	O
-.	O
-	O
-The	O
-overall	O
-free	O
-binding	O
-energies	O
-(	O
-ΔG	O
-°)	O
-were	O
-between	O
-–	O
-4	O
-.	O
-4	O
-and	O
-–	O
-8	O
-.	O
-6	O
-kcal	O
-/	O
-mol	O
-,	O
-reflecting	O
-the	O
-wide	O
-range	O
-of	O
-TCR	O
-binding	O
-affinities	O
-we	O
-observed	O
-for	O
-the	O
-different	O
-APLs	O
-.	O
-	O
-The	O
-enthalpic	O
-contribution	O
-in	O
-each	O
-complex	O
-did	O
-not	O
-follow	O
-a	O
-clear	O
-trend	O
-with	O
-affinity	O
-,	O
-with	O
-all	O
-but	O
-the	O
-1E6	O
--	O
-A2	O
--	O
-RQFGPDFPTI	O
-interaction	O
-(	O
-ΔH	O
-°	O
-=	O
-6	O
-.	O
-3	O
-kcal	O
-/	O
-mol	O
-)	O
-generating	O
-an	O
-energetically	O
-favorable	O
-enthalpy	O
-value	O
-(	O
-ΔH	O
-°	O
-=	O
-–	O
-3	O
-.	O
-7	O
-to	O
-–	O
-11	O
-.	O
-4	O
-kcal	O
-/	O
-mol	O
-);	O
-this	O
-indicated	O
-a	O
-net	O
-gain	O
-in	O
-electrostatic	O
-interactions	O
-during	O
-complex	O
-formation	O
-.	O
-	O
-However	O
-,	O
-there	O
-was	O
-a	O
-clear	O
-switch	O
-in	O
-entropy	O
-between	O
-the	O
-weaker	O
--	O
-affinity	O
-and	O
-stronger	O
--	O
-affinity	O
-ligands	O
-,	O
-indicated	O
-by	O
-a	O
-strong	O
-Pearson	O
-’	O
-s	O
-correlation	O
-value	O
-between	O
-entropy	O
-and	O
-affinity	O
-(	O
-Pearson	O
-’	O
-s	O
-correlation	O
-value	O
-0	O
-.	O
-93	O
-,	O
-P	O
-=	O
-0	O
-.	O
-007	O
-).	O
-	O
-For	O
-instance	O
-,	O
-the	O
-A2	O
--	O
-ALWGPDPAAA	O
-,	O
-A2	O
--	O
-AQWGPDAAA	O
-,	O
-and	O
-A2	O
--	O
-RQFGPDWIVA	O
-(	O
-KD	O
-=	O
-~	O
-208	O
-μM	O
-,	O
-KD	O
-=	O
-61	O
-.	O
-9	O
-μM	O
-,	O
-and	O
-KD	O
-=	O
-44	O
-.	O
-4	O
-μM	O
-,	O
-respectively	O
-)	O
-were	O
-all	O
-entropically	O
-unfavorable	O
-(	O
-TΔS	O
-°	O
-=	O
-–	O
-2	O
-.	O
-9	O
-to	O
-–	O
-5	O
-.	O
-6	O
-kcal	O
-/	O
-mol	O
-),	O
-indicating	O
-a	O
-net	O
-change	O
-from	O
-disorder	O
-to	O
-order	O
-.	O
-	O
-Conversely	O
-,	O
-the	O
-stronger	O
--	O
-affinity	O
-ligands	O
-A2	O
--	O
-RQWGPDPAAV	O
-(	O
-KD	O
-=	O
-7	O
-.	O
-8	O
-μM	O
-),	O
-A2	O
--	O
-YQFGPDFPIA	O
-(	O
-KD	O
-=	O
-7	O
-.	O
-4	O
-μM	O
-),	O
-and	O
-A2	O
--	O
-RQFGPDFPTI	O
-(	O
-KD	O
-=	O
-0	O
-.	O
-5	O
-μM	O
-)	O
-exhibited	O
-favorable	O
-entropy	O
-(	O
-TΔS	O
-°	O
-=	O
-2	O
-.	O
-2	O
-to	O
-14	O
-.	O
-9	O
-kcal	O
-/	O
-mol	O
-),	O
-indicating	O
-an	O
-order	O
--	O
-to	O
--	O
-disorder	O
-change	O
-during	O
-binding	O
-,	O
-possibly	O
-through	O
-the	O
-expulsion	O
-of	O
-ordered	O
-water	O
-molecules	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-structures	O
-of	O
-the	O
-unligated	O
-pMHCs	O
-demonstrated	O
-that	O
-,	O
-for	O
-these	O
-stronger	O
--	O
-affinity	O
-ligands	O
-,	O
-there	O
-was	O
-less	O
-conformational	O
-difference	O
-between	O
-the	O
-TCR	O
-ligated	O
-pMHCs	O
-compared	O
-with	O
-the	O
-weaker	O
--	O
-affinity	O
-ligands	O
-(	O
-Figure	O
-6	O
-).	O
-	O
-The	O
-potential	O
-requirement	O
-for	O
-a	O
-larger	O
-degree	O
-of	O
-induced	O
-fit	O
-during	O
-binding	O
-to	O
-these	O
-weaker	O
--	O
-affinity	O
-ligands	O
-is	O
-consistent	O
-with	O
-the	O
-larger	O
-entropic	O
-penalties	O
-observed	O
-for	O
-these	O
-interactions	O
-.	O
-	O
-Potential	O
-epitopes	O
-for	O
-1E6	O
-TCR	O
-occur	O
-commonly	O
-in	O
-the	O
-viral	O
-proteome	O
-.	O
-	O
-We	O
-searched	O
-a	O
-database	O
-of	O
-over	O
-1	O
-,	O
-924	O
-,	O
-572	O
-unique	O
-decamer	O
-peptides	O
-from	O
-the	O
-proteome	O
-of	O
-viral	O
-pathogens	O
-that	O
-are	O
-known	O
-,	O
-or	O
-strongly	O
-suspected	O
-,	O
-to	O
-infect	O
-humans	O
-.	O
-	O
-Three	O
-hundred	O
-forty	O
--	O
-two	O
-of	O
-these	O
-decamers	O
-conformed	O
-to	O
-the	O
-motif	O
-xxxGPDxxxx	O
-.	O
-	O
-Of	O
-these	O
-,	O
-53	O
-peptides	O
-contained	O
-the	O
-motif	O
-xOxGPDxxxO	O
-,	O
-where	O
-O	O
-is	O
-one	O
-of	O
-the	O
-hydrophobic	O
-amino	O
-acid	O
-residues	O
-A	O
-,	O
-V	O
-,	O
-I	O
-,	O
-L	O
-,	O
-M	O
-,	O
-Y	O
-,	O
-F	O
-,	O
-and	O
-W	O
-that	O
-might	O
-allow	O
-binding	O
-to	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-(	O
-Supplemental	O
-Table	O
-4	O
-).	O
-	O
-Thus	O
-,	O
-there	O
-are	O
-many	O
-pathogen	O
--	O
-encoded	O
-peptides	O
-that	O
-could	O
-act	O
-as	O
-agonists	O
-for	O
-the	O
-1E6	O
-T	O
-cell	O
-beyond	O
-the	O
-MVWGPDPLYV	O
-and	O
-RQFGPDWIVA	O
-sequences	O
-studied	O
-here	O
-.	O
-	O
-Extension	O
-of	O
-these	O
-analyses	O
-to	O
-include	O
-the	O
-larger	O
-genomes	O
-of	O
-bacterial	O
-pathogens	O
-would	O
-be	O
-expected	O
-to	O
-considerably	O
-increase	O
-these	O
-numbers	O
-.	O
-	O
-The	O
-binding	O
-affinity	O
-of	O
-the	O
-1E6	O
-TCR	O
-interaction	O
-with	O
-A2	O
--	O
-RQFGPDWIVA	O
-is	O
-considerably	O
-higher	O
-than	O
-with	O
-the	O
-disease	O
--	O
-implicated	O
-A2	O
--	O
-ALWGPDPAAA	O
-sequence	O
-(	O
-KD	O
-=	O
-44	O
-.	O
-4	O
-μM	O
-and	O
-KD	O
->	O
-200	O
-μM	O
-,	O
-respectively	O
-),	O
-highlighting	O
-how	O
-a	O
-pathogen	O
--	O
-derived	O
-sequence	O
-might	O
-be	O
-capable	O
-of	O
-priming	O
-a	O
-1E6	O
--	O
-like	O
-T	O
-cell	O
-.	O
-	O
-T	O
-cell	O
-antigen	O
-discrimination	O
-is	O
-governed	O
-by	O
-an	O
-interaction	O
-between	O
-the	O
-clonally	O
-expressed	O
-TCR	O
-and	O
-pMHC	O
-,	O
-mediated	O
-by	O
-the	O
-chemical	O
-characteristics	O
-of	O
-the	O
-interacting	O
-molecules	O
-.	O
-	O
-It	O
-has	O
-recently	O
-become	O
-clear	O
-that	O
-TCR	O
-cross	O
--	O
-reactivity	O
-with	O
-large	O
-numbers	O
-of	O
-different	O
-pMHC	O
-ligands	O
-is	O
-essential	O
-to	O
-plug	O
-holes	O
-in	O
-T	O
-cell	O
-immune	O
-coverage	O
-that	O
-pathogens	O
-could	O
-exploit	O
-.	O
-	O
-Flexibility	O
-at	O
-the	O
-interface	O
-between	O
-the	O
-TCR	O
-and	O
-pMHC	O
-,	O
-demonstrated	O
-in	O
-various	O
-studies	O
-,	O
-has	O
-been	O
-suggested	O
-as	O
-a	O
-mechanism	O
-mediating	O
-T	O
-cell	O
-cross	O
--	O
-reactivity	O
-with	O
-multiple	O
-distinct	O
-epitopes	O
-.	O
-	O
-This	O
-notion	O
-is	O
-attractive	O
-because	O
-the	O
-CDR	O
-loops	O
-,	O
-which	O
-form	O
-the	O
-TCR	O
-antigen	O
--	O
-binding	O
-site	O
-,	O
-are	O
-usually	O
-the	O
-most	O
-flexible	O
-part	O
-of	O
-the	O
-TCR	O
-and	O
-have	O
-the	O
-ability	O
-to	O
-mold	O
-around	O
-differently	O
-shaped	O
-ligands	O
-.	O
-	O
-Focused	O
-binding	O
-around	O
-a	O
-minimal	O
-peptide	O
-motif	O
-has	O
-also	O
-been	O
-implicated	O
-as	O
-an	O
-alternative	O
-mechanism	O
-enabling	O
-TCR	O
-cross	O
--	O
-reactivity	O
-.	O
-	O
-Notably	O
-among	O
-these	O
-studies	O
-,	O
-Garcia	O
-and	O
-colleagues	O
-recently	O
-used	O
-the	O
-alloreactive	O
-murine	O
-TCR	O
--	O
-MHC	O
-pair	O
-of	O
-the	O
-42F3	O
-TCR	O
-and	O
-H2	O
--	O
-Ld	O
-to	O
-demonstrate	O
-recognition	O
-of	O
-a	O
-large	O
-number	O
-of	O
-different	O
-peptides	O
-via	O
-conserved	O
-hotspot	O
-contacts	O
-with	O
-prominent	O
-up	O
--	O
-facing	O
-peptide	O
-residues	O
-.	O
-	O
-Sethi	O
-and	O
-colleagues	O
-recently	O
-demonstrated	O
-that	O
-the	O
-MHCII	O
--	O
-restricted	O
-Hy	O
-.	O
-1B11	O
-TCR	O
-,	O
-which	O
-was	O
-isolated	O
-from	O
-a	O
-patient	O
-with	O
-multiple	O
-sclerosis	O
-,	O
-could	O
-anchor	O
-into	O
-a	O
-deep	O
-pocket	O
-formed	O
-from	O
-peptide	O
-residues	O
-2	O
-,	O
-3	O
-,	O
-and	O
-5	O
-(	O
-from	O
-MBP85	O
-–	O
-99	O
-bound	O
-to	O
-HLA	O
--	O
-DQ1	O
-).	O
-	O
-This	O
-motif	O
-was	O
-conserved	O
-in	O
-at	O
-least	O
-2	O
-potential	O
-foreign	O
-peptides	O
-,	O
-originating	O
-from	O
-Herpes	O
-simplex	O
-virus	O
-and	O
-Pseudomonas	O
-aeruginosa	O
-,	O
-enabling	O
-TCR	O
-recognition	O
-of	O
-foreign	O
-epitopes	O
-.	O
-	O
-First	O
-,	O
-we	O
-currently	O
-know	O
-nothing	O
-about	O
-how	O
-human	O
-MHCI	O
-–	O
-restricted	O
-TCRs	O
-mediate	O
-cross	O
--	O
-reactivity	O
-in	O
-the	O
-context	O
-of	O
-a	O
-clinically	O
-relevant	O
-model	O
-of	O
-autoimmunity	O
-,	O
-thought	O
-to	O
-be	O
-a	O
-major	O
-pathway	O
-of	O
-disease	O
-initiation	O
-in	O
-several	O
-autoimmune	O
-diseases	O
-.	O
-	O
-Second	O
-,	O
-molecular	O
-studies	O
-have	O
-not	O
-yet	O
-revealed	O
-a	O
-broad	O
-set	O
-of	O
-rules	O
-that	O
-determine	O
-TCR	O
-cross	O
--	O
-reactivity	O
-because	O
-,	O
-with	O
-the	O
-exception	O
-of	O
-the	O
-allo	O
-–	O
-TCR	O
--	O
-MHC	O
-pair	O
-of	O
-the	O
-42F3	O
-TCR	O
-and	O
-H2	O
--	O
-Ld	O
-that	O
-did	O
-not	O
-encounter	O
-each	O
-other	O
-during	O
-T	O
-cell	O
-development	O
-,	O
-studies	O
-have	O
-been	O
-limited	O
-to	O
-structures	O
-of	O
-a	O
-TCR	O
-with	O
-only	O
-2	O
-or	O
-3	O
-different	O
-ligands	O
-.	O
-	O
-Here	O
-,	O
-we	O
-investigated	O
-a	O
-highly	O
-cross	O
--	O
-reactive	O
-MHCI	O
--	O
-restricted	O
-TCR	O
-isolated	O
-from	O
-a	O
-patient	O
-with	O
-T1D	O
-that	O
-recognizes	O
-an	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-–	O
-restricted	O
-preproinsulin	O
-signal	O
-peptide	O
-(	O
-ALWGPDPAAA15	O
-–	O
-24	O
-).	O
-	O
-Human	O
-CD8	O
-+	O
-T	O
-cell	O
-clones	O
-expressing	O
-TCRs	O
-with	O
-this	O
-specificity	O
-mediate	O
-the	O
-destruction	O
-of	O
-β	O
-cells	O
-,	O
-have	O
-been	O
-found	O
-in	O
-islets	O
-early	O
-in	O
-infection	O
-,	O
-and	O
-are	O
-proposed	O
-to	O
-be	O
-a	O
-major	O
-driver	O
-of	O
-disease	O
-.	O
-	O
-We	O
-solved	O
-the	O
-structure	O
-of	O
-the	O
-1E6	O
-TCR	O
-with	O
-7	O
-APLs	O
-to	O
-enable	O
-a	O
-comprehensive	O
-analysis	O
-of	O
-the	O
-molecular	O
-basis	O
-of	O
-TCR	O
-degeneracy	O
-.	O
-	O
-Overall	O
-,	O
-the	O
-difference	O
-in	O
-antigen	O
-potency	O
-correlated	O
-well	O
-with	O
-the	O
-binding	O
-energy	O
-(	O
-ΔG	O
-°	O
-kcal	O
-/	O
-mol	O
-)	O
-of	O
-the	O
-1E6	O
-TCR	O
-for	O
-the	O
-different	O
-epitopes	O
-,	O
-which	O
-ranged	O
-from	O
-values	O
-of	O
-ΔG	O
-°	O
-=	O
-~–	O
-4	O
-.	O
-4	O
-to	O
-–	O
-8	O
-.	O
-6	O
-kcal	O
-/	O
-mol	O
-(	O
-calculated	O
-from	O
-3D	O
-affinity	O
-data	O
-)	O
-or	O
-2D	O
-affinity	O
-values	O
-of	O
-AcKa	O
-=	O
-2	O
-.	O
-5	O
-×	O
-10	O
-–	O
-5	O
-to	O
-4	O
-.	O
-4	O
-×	O
-10	O
-–	O
-2	O
-μm4	O
-.	O
-	O
-The	O
-weaker	O
-end	O
-of	O
-this	O
-spectrum	O
-extends	O
-our	O
-understanding	O
-of	O
-the	O
-limits	O
-in	O
-which	O
-T	O
-cells	O
-can	O
-functionally	O
-operate	O
-in	O
-terms	O
-of	O
-TCR	O
-3D	O
-binding	O
-affinity	O
-and	O
-is	O
-in	O
-line	O
-with	O
-the	O
-types	O
-of	O
-very	O
-low	O
-affinity	O
-,	O
-yet	O
-fully	O
-functional	O
-self	O
--	O
-reactive	O
-CD8	O
-+	O
-T	O
-cells	O
-we	O
-have	O
-observed	O
-in	O
-tumor	O
--	O
-infiltrating	O
-lymphocytes	O
-.	O
-	O
-Previous	O
-studies	O
-of	O
-autoreactive	O
-TCRs	O
-have	O
-shown	O
-that	O
-their	O
-binding	O
-mode	O
-is	O
-generally	O
-atypical	O
-,	O
-either	O
-due	O
-to	O
-an	O
-unusual	O
-binding	O
-manner	O
-,	O
-weak	O
-TCR	O
-binding	O
-affinity	O
-,	O
-an	O
-unstable	O
-pMHC	O
-,	O
-or	O
-a	O
-combination	O
-of	O
-these	O
-factors	O
-.	O
-	O
-Our	O
-data	O
-demonstrate	O
-the	O
-potential	O
-for	O
-an	O
-autoreactive	O
-TCR	O
-to	O
-bind	O
-with	O
-a	O
-conventional	O
-binding	O
-mode	O
-to	O
-a	O
-stable	O
-pMHC	O
-with	O
-antipathogen	O
--	O
-like	O
-affinity	O
-(	O
-KD	O
-=	O
-0	O
-.	O
-5	O
-μM	O
-)	O
-depending	O
-on	O
-the	O
-peptide	O
-sequence	O
-.	O
-	O
-Our	O
-structural	O
-analysis	O
-revealed	O
-that	O
-the	O
-1E6	O
-TCR	O
-bound	O
-with	O
-a	O
-conserved	O
-conformation	O
-across	O
-all	O
-APLs	O
-investigated	O
-.	O
-	O
-This	O
-binding	O
-orientation	O
-was	O
-mediated	O
-through	O
-a	O
-focused	O
-interaction	O
-with	O
-TCR	O
-residues	O
-Tyr97α	O
-and	O
-Trp97β	O
-that	O
-formed	O
-an	O
-aromatic	O
-cap	O
-over	O
-a	O
-central	O
-‘	O
-GDP	O
-’	O
-motif	O
-that	O
-was	O
-common	O
-to	O
-all	O
-APLs	O
-.	O
-	O
-We	O
-have	O
-previously	O
-demonstrated	O
-the	O
-importance	O
-of	O
-the	O
-GPD	O
-motif	O
-using	O
-a	O
-peptide	O
-library	O
-scan	O
-,	O
-as	O
-well	O
-as	O
-a	O
-CPL	O
-scan	O
-approach	O
-.	O
-	O
-Although	O
-the	O
-1E6	O
-T	O
-cell	O
-was	O
-able	O
-to	O
-activate	O
-weakly	O
-with	O
-peptides	O
-that	O
-lacked	O
-this	O
-motif	O
-,	O
-we	O
-were	O
-unable	O
-to	O
-robustly	O
-measure	O
-binding	O
-affinities	O
-or	O
-generate	O
-complex	O
-structures	O
-with	O
-these	O
-ligands	O
-,	O
-highlighting	O
-the	O
-central	O
-role	O
-of	O
-this	O
-interaction	O
-during	O
-1E6	O
-T	O
-cell	O
-antigen	O
-recognition	O
-.	O
-	O
-This	O
-hotspot	O
-binding	O
-,	O
-defined	O
-as	O
-a	O
-localized	O
-cluster	O
-of	O
-interactions	O
-that	O
-dominate	O
-binding	O
-energy	O
-during	O
-protein	O
--	O
-protein	O
-interactions	O
-,	O
-has	O
-been	O
-previously	O
-shown	O
-to	O
-contribute	O
-to	O
-TCR	O
-recognition	O
-of	O
-MHC	O
-as	O
-a	O
-mechanism	O
-that	O
-tunes	O
-T	O
-cell	O
-cross	O
--	O
-reactivity	O
-by	O
-providing	O
-fixed	O
-anchor	O
-points	O
-that	O
-enable	O
-TCRs	O
-to	O
-tolerate	O
-a	O
-variable	O
-peptide	O
-cargo	O
-.	O
-	O
-Alternatively	O
-,	O
-interactions	O
-between	O
-the	O
-TCR	O
-and	O
-peptide	O
-have	O
-been	O
-shown	O
-to	O
-dominate	O
-the	O
-energetic	O
-landscape	O
-during	O
-ligand	O
-engagement	O
-,	O
-ensuring	O
-that	O
-T	O
-cells	O
-retain	O
-peptide	O
-specificity	O
-.	O
-	O
-The	O
-binding	O
-mechanism	O
-utilized	O
-by	O
-the	O
-1E6	O
-TCR	O
-during	O
-pMHC	O
-recognition	O
-is	O
-consistent	O
-with	O
-both	O
-of	O
-these	O
-models	O
-.	O
-	O
-Ligand	O
-engagement	O
-is	O
-dominated	O
-by	O
-peptide	O
-interactions	O
-,	O
-but	O
-hotspot	O
--	O
-like	O
-interactions	O
-with	O
-the	O
-central	O
-GPD	O
-motif	O
-enable	O
-the	O
-1E6	O
-TCR	O
-to	O
-tolerate	O
-peptide	O
-residues	O
-that	O
-vary	O
-outside	O
-of	O
-this	O
-region	O
-,	O
-explaining	O
-how	O
-T	O
-cells	O
-expressing	O
-this	O
-TCR	O
-may	O
-cross	O
--	O
-react	O
-with	O
-a	O
-large	O
-number	O
-of	O
-different	O
-peptides	O
-.	O
-	O
-These	O
-findings	O
-are	O
-also	O
-analogous	O
-to	O
-the	O
-observed	O
-binding	O
-mode	O
-of	O
-the	O
-Hy	O
-.	O
-1B11	O
-TCR	O
-,	O
-in	O
-which	O
-one	O
-aromatic	O
-residue	O
-of	O
-the	O
-TCR	O
-CDR3α	O
-loop	O
-anchored	O
-into	O
-a	O
-pocket	O
-created	O
-by	O
-a	O
-conserved	O
-peptide	O
-motif	O
-.	O
-	O
-In	O
-both	O
-of	O
-these	O
-examples	O
-,	O
-self	O
--	O
-recognition	O
-is	O
-mediated	O
-by	O
-TCR	O
-residues	O
-with	O
-aromatic	O
-side	O
-chains	O
-.	O
-	O
-Combined	O
-with	O
-evidence	O
-demonstrating	O
-that	O
-aromatic	O
-side	O
-chains	O
-are	O
-conserved	O
-in	O
-the	O
-CDR2	O
-loops	O
-of	O
-TCRs	O
-from	O
-many	O
-species	O
-,	O
-we	O
-speculate	O
-that	O
-these	O
-aromatic	O
-residues	O
-could	O
-impart	O
-a	O
-level	O
-of	O
-“	O
-stickiness	O
-”	O
-to	O
-TCRs	O
-,	O
-which	O
-might	O
-be	O
-enriched	O
-in	O
-an	O
-autoimmune	O
-setting	O
-when	O
-the	O
-TCR	O
-often	O
-binds	O
-in	O
-a	O
-nonoptimal	O
-fashion	O
-.	O
-	O
-Despite	O
-some	O
-weak	O
-statistical	O
-correlation	O
-between	O
-the	O
-surface	O
-complementarity	O
-(	O
-SC	O
-)	O
-and	O
-affinity	O
-,	O
-closer	O
-inspection	O
-of	O
-the	O
-interface	O
-revealed	O
-no	O
-obvious	O
-structural	O
-signature	O
-that	O
-could	O
-definitively	O
-explain	O
-the	O
-differences	O
-in	O
-antigen	O
-potency	O
-and	O
-TCR	O
-binding	O
-strength	O
-between	O
-the	O
-different	O
-ligands	O
-.	O
-	O
-However	O
-,	O
-similar	O
-to	O
-our	O
-findings	O
-in	O
-other	O
-systems	O
-,	O
-modifications	O
-to	O
-residues	O
-outside	O
-of	O
-the	O
-canonical	O
-central	O
-peptide	O
-bulge	O
-were	O
-important	O
-for	O
-generating	O
-new	O
-interactions	O
-.	O
-	O
-For	O
-example	O
-,	O
-all	O
-of	O
-the	O
-stronger	O
-ligands	O
-encoded	O
-larger	O
-side	O
-chains	O
-(	O
-Arg	O
-or	O
-Tyr	O
-)	O
-at	O
-peptide	O
-position	O
-1	O
-that	O
-enabled	O
-new	O
-interactions	O
-with	O
-1E6	O
-not	O
-present	O
-with	O
-the	O
-Ala	O
-at	O
-this	O
-position	O
-in	O
-the	O
-natural	O
-preproinsulin	O
-peptide	O
-.	O
-	O
-These	O
-data	O
-also	O
-explain	O
-our	O
-previous	O
-findings	O
-that	O
-alteration	O
-of	O
-the	O
-anchor	O
-residue	O
-at	O
-peptide	O
-position	O
-2	O
-(	O
-Leu	B-mutant
--	I-mutant
-Gln	I-mutant
-)	O
-has	O
-a	O
-direct	O
-effect	O
-on	O
-1E6	O
-TCR	O
-binding	O
-affinity	O
-because	O
-our	O
-structural	O
-analysis	O
-demonstrated	O
-that	O
-1E6	O
-made	O
-3	O
-additional	O
-bonds	O
-with	O
-A2	O
--	O
-AQWGPDPAAA	O
-compared	O
-with	O
-A2	O
--	O
-ALWGPDPAAA	O
-,	O
-consistent	O
-with	O
-the	O
->	O
-3	O
--	O
-fold	O
-stronger	O
-binding	O
-affinity	O
-.	O
-	O
-We	O
-have	O
-recently	O
-demonstrated	O
-how	O
-a	O
-suboptimal	O
-position	O
-2	O
-anchor	O
-in	O
-a	O
-melanoma	O
--	O
-derived	O
-antigen	O
-can	O
-improve	O
-TCR	O
-binding	O
-through	O
-a	O
-similar	O
-mechanism	O
-.	O
-	O
-These	O
-results	O
-challenge	O
-the	O
-notion	O
-that	O
-the	O
-most	O
-potent	O
-peptide	O
-antigens	O
-exhibit	O
-the	O
-greatest	O
-pMHC	O
-stability	O
-and	O
-have	O
-implications	O
-for	O
-the	O
-design	O
-of	O
-anchor	O
-residue	O
-–	O
-modified	O
-heteroclitic	O
-peptides	O
-for	O
-vaccination	O
-.	O
-	O
-Early	O
-thermodynamic	O
-analysis	O
-of	O
-TCR	O
--	O
-pMHC	O
-interactions	O
-suggested	O
-a	O
-common	O
-energetic	O
-signature	O
-,	O
-driven	O
-by	O
-favorable	O
-enthalpy	O
-(	O
-generally	O
-mediated	O
-through	O
-an	O
-increase	O
-in	O
-electrostatic	O
-interactions	O
-)	O
-and	O
-unfavorable	O
-entropy	O
-(	O
-changes	O
-from	O
-disorder	O
-to	O
-order	O
-).	O
-	O
-These	O
-parameters	O
-aligned	O
-well	O
-with	O
-structural	O
-data	O
-,	O
-demonstrating	O
-that	O
-TCRs	O
-engaged	O
-pMHC	O
-using	O
-an	O
-induced	O
-fit	O
-binding	O
-mode	O
-.	O
-	O
-However	O
-,	O
-more	O
-recent	O
-data	O
-have	O
-shown	O
-that	O
-TCRs	O
-can	O
-utilize	O
-a	O
-range	O
-of	O
-energetic	O
-strategies	O
-during	O
-pMHC	O
-binding	O
-,	O
-currently	O
-with	O
-no	O
-obvious	O
-pattern	O
-in	O
-terms	O
-of	O
-TCR	O
-affinity	O
-,	O
-binding	O
-mechanism	O
-,	O
-or	O
-specificity	O
-(	O
-pathogen	O
-,	O
-cancer	O
-,	O
-or	O
-self	O
--	O
-ligands	O
-).	O
-	O
-Although	O
-no	O
-energetic	O
-signature	O
-appears	O
-to	O
-exist	O
-for	O
-different	O
-TCRs	O
-,	O
-we	O
-used	O
-thermodynamic	O
-analysis	O
-here	O
-to	O
-explore	O
-whether	O
-changes	O
-in	O
-energetics	O
-could	O
-help	O
-explain	O
-ligand	O
-discrimination	O
-by	O
-a	O
-single	O
-TCR	O
-.	O
-	O
-This	O
-analysis	O
-demonstrated	O
-a	O
-strong	O
-relationship	O
-(	O
-according	O
-to	O
-the	O
-Pearson	O
-’	O
-s	O
-correlation	O
-analysis	O
-)	O
-between	O
-the	O
-energetic	O
-signature	O
-used	O
-by	O
-the	O
-1E6	O
-TCR	O
-and	O
-the	O
-sensitivity	O
-of	O
-the	O
-1E6	O
-T	O
-cell	O
-clone	O
-to	O
-different	O
-APLs	O
-.	O
-	O
-The	O
-weaker	O
-APL	O
-ligands	O
-were	O
-characterized	O
-by	O
-favorable	O
-enthalpy	O
-and	O
-unfavorable	O
-entropy	O
-,	O
-whereas	O
-the	O
-stronger	O
-ligands	O
-progressively	O
-shifted	O
-to	O
-favorable	O
-entropy	O
-.	O
-	O
-These	O
-differences	O
-were	O
-consistent	O
-with	O
-a	O
-greater	O
-degree	O
-of	O
-movement	O
-between	O
-the	O
-unligated	O
-and	O
-ligated	O
-pMHCs	O
-for	O
-the	O
-weaker	O
-ligands	O
-,	O
-suggesting	O
-a	O
-greater	O
-requirement	O
-for	O
-disorder	O
--	O
-to	O
--	O
-order	O
-changes	O
-during	O
-TCR	O
-binding	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-enhanced	O
-antigen	O
-potency	O
-was	O
-probably	O
-mediated	O
-through	O
-a	O
-shift	O
-from	O
-an	O
-induced	O
-fit	O
-to	O
-a	O
-lock	O
--	O
-and	O
--	O
-key	O
-interaction	O
-between	O
-the	O
-stronger	O
-ligands	O
-(	O
-less	O
-requirement	O
-for	O
-energetically	O
-unfavorable	O
-disorder	O
--	O
-to	O
--	O
-order	O
-changes	O
-),	O
-resulting	O
-in	O
-a	O
-more	O
-energetically	O
-favorable	O
-ΔG	O
-value	O
-.	O
-	O
-Importantly	O
-,	O
-the	O
-preproinsulin	O
--	O
-derived	O
-epitope	O
-was	O
-one	O
-of	O
-the	O
-least	O
-potent	O
-peptides	O
-,	O
-demonstrating	O
-that	O
-the	O
-1E6	O
-T	O
-cell	O
-clone	O
-had	O
-the	O
-ability	O
-to	O
-respond	O
-to	O
-different	O
-peptide	O
-sequences	O
-with	O
-far	O
-greater	O
-potency	O
-.	O
-	O
-The	O
-RQFGPDWIVA	O
-peptide	O
-,	O
-which	O
-was	O
-substantially	O
-more	O
-potent	O
-than	O
-the	O
-preproinsulin	O
-peptide	O
-,	O
-is	O
-within	O
-the	O
-proteome	O
-of	O
-a	O
-common	O
-human	O
-pathogen	O
-(	O
-C	O
-.	O
-asparagiforme	O
-),	O
-demonstrating	O
-the	O
-potential	O
-for	O
-an	O
-encounter	O
-between	O
-a	O
-naive	O
-1E6	O
--	O
-like	O
-T	O
-cell	O
-and	O
-a	O
-foreign	O
-peptide	O
-with	O
-a	O
-more	O
-potent	O
-ligand	O
-that	O
-might	O
-then	O
-break	O
-self	O
--	O
-tolerance	O
-.	O
-	O
-Indeed	O
-,	O
-we	O
-found	O
-over	O
-50	O
-decamer	O
-peptides	O
-from	O
-the	O
-proteome	O
-of	O
-likely	O
-,	O
-or	O
-known	O
-,	O
-human	O
-viral	O
-pathogens	O
-alone	O
-that	O
-contained	O
-both	O
-the	O
-conserved	O
-central	O
-GPD	O
-motif	O
-and	O
-anchor	O
-residues	O
-at	O
-positions	O
-2	O
-and	O
-10	O
-that	O
-would	O
-enable	O
-binding	O
-to	O
-HLA	O
--	O
-A	O
-*	O
-02	O
-:	O
-01	O
-.	O
-	O
-Further	O
-experiments	O
-will	O
-be	O
-required	O
-to	O
-determine	O
-whether	O
-any	O
-naturally	O
-presented	O
-,	O
-human	O
-pathogen	O
-–	O
-derived	O
-peptides	O
-act	O
-as	O
-active	O
-ligands	O
-for	O
-1E6	O
-,	O
-but	O
-our	O
-work	O
-presented	O
-here	O
-demonstrates	O
-that	O
-it	O
-is	O
-at	O
-least	O
-feasible	O
-for	O
-an	O
-autoimmune	O
-TCR	O
-to	O
-bind	O
-to	O
-a	O
-different	O
-peptide	O
-sequence	O
-that	O
-could	O
-be	O
-present	O
-in	O
-a	O
-pathogen	O
-proteome	O
-with	O
-substantially	O
-higher	O
-affinity	O
-and	O
-potency	O
-than	O
-the	O
-interaction	O
-it	O
-might	O
-use	O
-to	O
-attack	O
-self	O
--	O
-tissue	O
-.	O
-	O
-In	O
-summary	O
-,	O
-this	O
-investigation	O
-into	O
-the	O
-molecular	O
-basis	O
-of	O
-T	O
-cell	O
-cross	O
--	O
-reactivity	O
-using	O
-a	O
-clinically	O
-relevant	O
-cytotoxic	O
-CD8	O
-+	O
-T	O
-cell	O
-clone	O
-that	O
-kills	O
-human	O
-pancreatic	O
-β	O
-cells	O
-provides	O
-answers	O
-to	O
-a	O
-number	O
-of	O
-previously	O
-outstanding	O
-questions	O
-.	O
-	O
-First	O
-,	O
-our	O
-data	O
-shows	O
-that	O
-a	O
-single	O
-TCR	O
-has	O
-the	O
-potential	O
-to	O
-functionally	O
-(	O
-assessed	O
-through	O
-T	O
-cell	O
-activation	O
-)	O
-bind	O
-to	O
-different	O
-ligands	O
-with	O
-affinities	O
-ranging	O
-across	O
-3	O
-orders	O
-of	O
-magnitude	O
-.	O
-	O
-Second	O
-,	O
-this	O
-is	O
-the	O
-first	O
-example	O
-in	O
-which	O
-ligands	O
-have	O
-been	O
-identified	O
-and	O
-characterized	O
-for	O
-a	O
-human	O
-autoreactive	O
-TCR	O
-that	O
-are	O
-substantially	O
-more	O
-potent	O
-than	O
-the	O
-natural	O
-self	O
--	O
-ligand	O
-,	O
-demonstrating	O
-the	O
-potential	O
-for	O
-a	O
-pathogenic	O
-ligand	O
-to	O
-break	O
-self	O
--	O
-tolerance	O
-and	O
-prime	O
-self	O
--	O
-reactive	O
-T	O
-cells	O
-.	O
-	O
-Third	O
-,	O
-this	O
-first	O
-structural	O
-analysis	O
-of	O
-a	O
-cross	O
--	O
-reactive	O
-human	O
-MHCI	O
-–	O
-restricted	O
-autoimmune	O
-TCR	O
-showed	O
-that	O
-degeneracy	O
-was	O
-mediated	O
-through	O
-TCR	O
--	O
-pMHC	O
-anchoring	O
-by	O
-a	O
-conserved	O
-minimal	O
-binding	O
-peptide	O
-motif	O
-.	O
-	O
-Finally	O
-,	O
-TCR	O
-ligand	O
-discrimination	O
-was	O
-characterized	O
-by	O
-an	O
-energetic	O
-shift	O
-from	O
-an	O
-enthalpically	O
-to	O
-entropically	O
-driven	O
-interaction	O
-.	O
-	O
-Our	O
-demonstration	O
-of	O
-the	O
-molecular	O
-mechanism	O
-governing	O
-cross	O
--	O
-reactivity	O
-by	O
-this	O
-preproinsulin	O
-reactive	O
-human	O
-CD8	O
-+	O
-T	O
-cell	O
-clone	O
-supports	O
-the	O
-notion	O
-first	O
-put	O
-forward	O
-by	O
-Wucherpfennig	O
-and	O
-Strominger	O
-that	O
-molecular	O
-mimicry	O
-could	O
-mediate	O
-autoimmunity	O
-and	O
-has	O
-far	O
--	O
-reaching	O
-implications	O
-for	O
-the	O
-complex	O
-nature	O
-of	O
-T	O
-cell	O
-antigen	O
-discrimination	O
-.	O
-	O
-The	O
-1E6	O
-T	O
-cell	O
-clone	O
-reacts	O
-with	O
-a	O
-broad	O
-sensitivity	O
-range	O
-to	O
-APLs	O
-.	O
-	O
-(	O
-A	O
-and	O
-B	O
-)	O
-The	O
-1E6	O
-T	O
-cell	O
-clone	O
-was	O
-tested	O
-in	O
-a	O
-peptide	O
-dilution	O
-assay	O
-,	O
-in	O
-triplicate	O
-,	O
-with	O
-MVWGPDPLYV	O
-(	O
-gray	O
-),	O
-YLGGPDFPTI	O
-(	O
-red	O
-),	O
-ALWGPDPAAA	O
-(	O
-blue	O
-),	O
-AQWGPDPAAA	O
-(	O
-green	O
-),	O
-RQFGPDWIVA	O
-(	O
-dark	O
-blue	O
-),	O
-RQWGPDPAAV	O
-(	O
-purple	O
-),	O
-YQFGPDFPTA	O
-(	O
-yellow	O
-),	O
-and	O
-RQFGPDFPTI	O
-(	O
-cyan	O
-)	O
-peptides	O
-presented	O
-by	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-–	O
-expressing	O
-C1R	O
-cells	O
-for	O
-release	O
-of	O
-MIP	O
--	O
-1β	O
-(	O
-A	O
-)	O
-and	O
-killing	O
-(	O
-B	O
-).	O
-	O
-(	O
-C	O
-)	O
-The	O
-1E6	O
-T	O
-cell	O
-clone	O
-was	O
-stained	O
-,	O
-in	O
-duplicate	O
-,	O
-with	O
-tetramers	O
-composed	O
-of	O
-each	O
-APL	O
-(	O
-colored	O
-as	O
-above	O
-)	O
-presented	O
-by	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-.	O
-(	O
-D	O
-)	O
-The	O
-stability	O
-of	O
-each	O
-APL	O
-(	O
-colored	O
-as	O
-above	O
-)	O
-was	O
-tested	O
-,	O
-in	O
-duplicate	O
-,	O
-using	O
-CD	O
-by	O
-recording	O
-the	O
-peak	O
-at	O
-218	O
-nm	O
-absorbance	O
-from	O
-5	O
-°	O
-C	O
-–	O
-90	O
-°	O
-C	O
-.	O
-	O
-Tm	O
-values	O
-were	O
-calculated	O
-using	O
-a	O
-Boltzmann	O
-fit	O
-to	O
-each	O
-set	O
-of	O
-data	O
-.	O
-	O
-3D	O
-and	O
-2D	O
-binding	O
-analysis	O
-of	O
-the	O
-1E6	O
-TCR	O
-with	O
-A2	O
--	O
-ALW	O
-and	O
-the	O
-APLs	O
-.	O
-	O
-(	O
-A	O
-–	O
-H	O
-)	O
-Binding	O
-affinity	O
-of	O
-the	O
-1E6	O
-TCR	O
-interaction	O
-at	O
-25	O
-°	O
-C	O
-using	O
-SPR	O
-.	O
-	O
-Eight	O
-serial	O
-dilutions	O
-of	O
-the	O
-1E6	O
-TCR	O
-were	O
-measured	O
-(	O
-shown	O
-in	O
-the	O
-inset	O
-);	O
-representative	O
-data	O
-from	O
-3	O
-independent	O
-experiments	O
-are	O
-plotted	O
-.	O
-	O
-The	O
-equilibrium	O
-binding	O
-constant	O
-(	O
-KD	O
-)	O
-values	O
-were	O
-calculated	O
-using	O
-a	O
-nonlinear	O
-curve	O
-fit	O
-(	O
-y	O
-=	O
-[	O
-P1x	O
-]/[	O
-P2	O
-+	O
-X	O
-]).	O
-	O
-In	O
-order	O
-to	O
-calculate	O
-each	O
-response	O
-,	O
-the	O
-1E6	O
-TCR	O
-was	O
-also	O
-injected	O
-over	O
-a	O
-control	O
-sample	O
-(	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-–	O
-ILAKFLHWL	O
-)	O
-that	O
-was	O
-deducted	O
-from	O
-the	O
-experimental	O
-data	O
-.	O
-	O
-(	O
-A	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-MVWGPDPLYV	O
-(	O
-approximate	O
-value	O
-);	O
-(	O
-B	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-YLGGPDFPTI	O
-(	O
-approximate	O
-value	O
-);	O
-(	O
-C	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-ALWGPDPAAA	O
-;	O
-(	O
-D	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-AQWGPDPAAA	O
-;	O
-(	O
-E	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-RQFGPDWIVA	O
-;	O
-(	O
-F	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-RQWGPDPAAV	O
-;	O
-(	O
-G	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-YQFGPDFPTA	O
-;	O
-and	O
-(	O
-H	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-RQFGPDFPTI	O
-.	O
-(	O
-I	O
-)	O
-ΔG	O
-values	O
-,	O
-calculated	O
-from	O
-SPR	O
-experiments	O
-,	O
-plotted	O
-against	O
-1	O
-/	O
-EC50	O
-(	O
-the	O
-reciprocal	O
-peptide	O
-concentration	O
-required	O
-to	O
-reach	O
-half	O
--	O
-maximal	O
-1E6	O
-T	O
-cell	O
-killing	O
-)	O
-showing	O
-Pearson	O
-’	O
-s	O
-coefficient	O
-analysis	O
-(	O
-r	O
-)	O
-and	O
-P	O
-value	O
-(	O
-including	O
-approximate	O
-values	O
-from	O
-A	O
-and	O
-B	O
-).	O
-	O
-(	O
-J	O
-)	O
-Effective	O
-2D	O
-affinity	O
-(	O
-AcKa	O
-)	O
-calculated	O
-using	O
-adhesion	O
-frequency	O
-assays	O
-,	O
-using	O
-at	O
-least	O
-5	O
-cell	O
-pairs	O
-,	O
-and	O
-calculated	O
-as	O
-an	O
-average	O
-of	O
-100	O
-cell	O
-cell	O
-contacts	O
-.	O
-	O
-(	O
-K	O
-)	O
-Effective	O
-2D	O
-affinity	O
-plotted	O
-against	O
-1	O
-/	O
-EC50	O
-showing	O
-Pearson	O
-’	O
-s	O
-coefficient	O
-analysis	O
-(	O
-r	O
-)	O
-and	O
-P	O
-value	O
-.	O
-	O
-The	O
-1E6	O
-TCR	O
-uses	O
-a	O
-conserved	O
-binding	O
-mode	O
-to	O
-engage	O
-A2	O
--	O
-ALWGPDPAAA	O
-and	O
-the	O
-APLs	O
-.	O
-	O
-(	O
-A	O
-)	O
-Superposition	O
-of	O
-the	O
-1E6	O
-TCR	O
-(	O
-multicolored	O
-illustration	O
-)	O
-in	O
-complex	O
-with	O
-all	O
-7	O
-APLs	O
-(	O
-multicolored	O
-sticks	O
-)	O
-and	O
-the	O
-A2	O
--	O
-ALWGPDPAAA	O
-ligand	O
-using	O
-the	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-(	O
-gray	O
-illustration	O
-)	O
-molecule	O
-to	O
-align	O
-all	O
-of	O
-the	O
-structures	O
-.	O
-	O
-The	O
-1E6	O
-TCR	O
-and	O
-each	O
-peptide	O
-are	O
-colored	O
-according	O
-to	O
-the	O
-APL	O
-used	O
-in	O
-the	O
-complex	O
-as	O
-in	O
-Figure	O
-1	O
-.	O
-(	O
-B	O
-)	O
-Position	O
-of	O
-the	O
-1E6	O
-TCR	O
-CDR	O
-loops	O
-(	O
-multicolored	O
-lines	O
-)	O
-in	O
-each	O
-complex	O
-.	O
-	O
-The	O
-ALWGPDPAAA	O
-peptide	O
-(	O
-green	O
-sticks	O
-)	O
-is	O
-shown	O
-in	O
-the	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-binding	O
-groove	O
-(	O
-gray	O
-surface	O
-).	O
-(	O
-C	O
-)	O
-The	O
-Cα	O
-backbone	O
-conformation	O
-of	O
-each	O
-APL	O
-(	O
-multicolored	O
-illustration	O
-)	O
-in	O
-the	O
-context	O
-of	O
-the	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-α1	O
-helices	O
-(	O
-gray	O
-illustration	O
-).	O
-(	O
-D	O
-)	O
-Crossing	O
-angle	O
-of	O
-the	O
-1E6	O
-TCR	O
-(	O
-multicolored	O
-lines	O
-)	O
-calculated	O
-using	O
-previously	O
-published	O
-parameters	O
-in	O
-the	O
-context	O
-of	O
-the	O
-ALWGPDPAAA	O
-peptide	O
-(	O
-green	O
-sticks	O
-)	O
-bound	O
-in	O
-the	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-binding	O
-groove	O
-(	O
-gray	O
-surface	O
-).	O
-	O
-A	O
-conserved	O
-interaction	O
-with	O
-a	O
-GPD	O
-motif	O
-underpins	O
-the	O
-1E6	O
-TCR	O
-interaction	O
-with	O
-the	O
-APLs	O
-.	O
-	O
-Interaction	O
-between	O
-1E6	O
-TCR	O
-(	O
-gray	O
-illustration	O
-)	O
-residues	O
-Tyr97α	O
-and	O
-Tyr97β	O
-(	O
-the	O
-position	O
-of	O
-these	O
-side	O
-chains	O
-in	O
-the	O
-TCR	O
-in	O
-complex	O
-with	O
-all	O
-7	O
-APLs	O
-,	O
-and	O
-the	O
-previously	O
-reported	O
-A2	O
--	O
-ALWGPDPAAA	O
-epitope	O
-,	O
-is	O
-shown	O
-in	O
-multicolored	O
-sticks	O
-;	O
-ref	O
-.)	O
-and	O
-the	O
-GPD	O
-peptide	O
-motif	O
-(	O
-the	O
-position	O
-of	O
-these	O
-side	O
-chains	O
-in	O
-all	O
-7	O
-APLs	O
-and	O
-A2	O
--	O
-ALWGPDPAAA	O
-in	O
-complex	O
-with	O
-the	O
-1E6	O
-TCR	O
-is	O
-shown	O
-in	O
-multicolored	O
-sticks	O
-).	O
-	O
-The	O
-rest	O
-of	O
-the	O
-peptide	O
-,	O
-and	O
-the	O
-MHCα1	O
-helix	O
-,	O
-are	O
-shown	O
-as	O
-a	O
-gray	O
-illustration	O
-.	O
-	O
-The	O
-1E6	O
-TCR	O
-makes	O
-distinct	O
-peptide	O
-contacts	O
-with	O
-peripheral	O
-APL	O
-residues	O
-.	O
-	O
-Interactions	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-peptide	O
-residues	O
-outside	O
-of	O
-the	O
-conserved	O
-GPD	O
-motif	O
-.	O
-	O
-The	O
-MHCα1	O
-helix	O
-is	O
-shown	O
-in	O
-gray	O
-illustrations	O
-.	O
-	O
-Hydrogen	O
-bonds	O
-are	O
-shown	O
-as	O
-red	O
-dotted	O
-lines	O
-;	O
-van	O
-der	O
-Waals	O
-(	O
-vdW	O
-)	O
-contacts	O
-are	O
-shown	O
-as	O
-black	O
-dotted	O
-lines	O
-.	O
-	O
-Boxes	O
-show	O
-total	O
-contacts	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-each	O
-peptide	O
-ligand	O
-.	O
-	O
-(	O
-A	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-black	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-MVWGPDPLYV	O
-(	O
-black	O
-illustration	O
-and	O
-sticks	O
-).	O
-(	O
-B	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-red	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-YLGGPDFPTI	O
-(	O
-red	O
-illustration	O
-and	O
-sticks	O
-).	O
-(	O
-C	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-blue	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-ALWGPDPAAA	O
-(	O
-blue	O
-illustration	O
-and	O
-sticks	O
-)	O
-reproduced	O
-from	O
-previous	O
-published	O
-data	O
-.	O
-	O
-(	O
-D	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-green	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-AQWGPDPAAA	O
-(	O
-green	O
-illustration	O
-and	O
-sticks	O
-).	O
-(	O
-E	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-dark	O
-blue	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-RQFGPDWIVA	O
-(	O
-dark	O
-blue	O
-illustration	O
-and	O
-sticks	O
-).	O
-(	O
-F	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-purple	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-RQWGPDPAAV	O
-(	O
-purple	O
-illustration	O
-and	O
-sticks	O
-).	O
-(	O
-G	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-yellow	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-YQFGPDFPTA	O
-(	O
-yellow	O
-illustration	O
-and	O
-sticks	O
-).	O
-(	O
-H	O
-)	O
-Interaction	O
-between	O
-the	O
-1E6	O
-TCR	O
-(	O
-cyan	O
-illustration	O
-and	O
-sticks	O
-)	O
-and	O
-A2	O
--	O
-RQFGPDFPTI	O
-(	O
-cyan	O
-illustration	O
-and	O
-sticks	O
-).	O
-	O
-Comparison	O
-of	O
-ligated	O
-and	O
-unligated	O
-APLs	O
-.	O
-	O
-Superposition	O
-of	O
-each	O
-APL	O
-in	O
-unligated	O
-form	O
-and	O
-ligated	O
-to	O
-the	O
-1E6	O
-TCR	O
-.	O
-	O
-All	O
-unligated	O
-pMHCs	O
-are	O
-shown	O
-as	O
-light	O
-green	O
-illustrations	O
-.	O
-	O
-Peptide	O
-sequences	O
-are	O
-shown	O
-underneath	O
-each	O
-structure	O
-aligned	O
-with	O
-the	O
-peptide	O
-structure	O
-.	O
-	O
-(	O
-A	O
-)	O
-A2	O
--	O
-MVWGPDPLYV	O
-(	O
-black	O
-sticks	O
-).	O
-	O
-A	O
-large	O
-conformational	O
-shift	O
-was	O
-observed	O
-for	O
-Tyr8	O
-in	O
-the	O
-ligated	O
-versus	O
-unligated	O
-states	O
-(	O
-black	O
-circle	O
-).	O
-(	O
-B	O
-)	O
-A2	O
--	O
-YLGGPDFPTI	O
-(	O
-red	O
-sticks	O
-).	O
-(	O
-C	O
-)	O
-A2	O
--	O
-ALWGPDPAAA	O
-(	O
-blue	O
-sticks	O
-)	O
-reproduced	O
-from	O
-previous	O
-published	O
-data	O
-.	O
-(	O
-D	O
-)	O
-A2	O
--	O
-AQWGPDPAAA	O
-(	O
-green	O
-sticks	O
-).	O
-(	O
-E	O
-)	O
-A2	O
--	O
-RQFGPDWIVA	O
-(	O
-dark	O
-blue	O
-sticks	O
-).	O
-(	O
-F	O
-)	O
-A2	O
--	O
-RQWGPDPAAV	O
-(	O
-purple	O
-sticks	O
-).	O
-(	O
-G	O
-)	O
-A2	O
--	O
-YQFGPDFPTA	O
-(	O
-yellow	O
-sticks	O
-).	O
-(	O
-H	O
-)	O
-A2	O
--	O
-RQFGPDFPTI	O
-(	O
-cyan	O
-sticks	O
-).	O
-	O
-The	O
-1E6	O
-TCR	O
-makes	O
-distinct	O
-peptide	O
-contacts	O
-with	O
-the	O
-MHC	O
-surface	O
-depending	O
-on	O
-the	O
-peptide	O
-cargo	O
-.	O
-	O
-Interactions	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-the	O
-MHC	O
-α1	O
-helix	O
-residues	O
-Arg65	O
-,	O
-Lys66	O
-,	O
-and	O
-Gln72	O
-.	O
-	O
-Hydrogen	O
-bonds	O
-are	O
-shown	O
-as	O
-red	O
-dotted	O
-lines	O
-;	O
-vdW	O
-contacts	O
-are	O
-shown	O
-as	O
-black	O
-dotted	O
-lines	O
-.	O
-	O
-MHCα1	O
-helix	O
-are	O
-shown	O
-in	O
-gray	O
-illustrations	O
-.	O
-	O
-Boxes	O
-show	O
-total	O
-contacts	O
-between	O
-the	O
-1E6	O
-TCR	O
-and	O
-these	O
-key	O
-residues	O
-(	O
-green	O
-boxes	O
-are	O
-MHC	O
-residues	O
-;	O
-white	O
-boxes	O
-are	O
-TCR	O
-residues	O
-).	O
-	O
-Thermodynamic	O
-analysis	O
-of	O
-the	O
-1E6	O
-TCR	O
-with	O
-A2	O
--	O
-ALWGPDPAAA	O
-and	O
-the	O
-APLs	O
-.	O
-	O
-Eight	O
-serial	O
-dilutions	O
-of	O
-the	O
-1E6	O
-TCR	O
-were	O
-injected	O
-,	O
-in	O
-duplicate	O
-,	O
-over	O
-each	O
-immobilized	O
-APL	O
-and	O
-A2	O
--	O
-ALW	O
-at	O
-5	O
-°	O
-C	O
-,	O
-13	O
-°	O
-C	O
-,	O
-18	O
-°	O
-C	O
-,	O
-25	O
-°	O
-C	O
-,	O
-30	O
-°	O
-C	O
-,	O
-and	O
-37	O
-°	O
-C	O
-.	O
-	O
-The	O
-equilibrium	O
-binding	O
-constant	O
-(	O
-KD	O
-)	O
-values	O
-were	O
-calculated	O
-using	O
-a	O
-nonlinear	O
-curve	O
-fit	O
-(	O
-y	O
-=	O
-[	O
-P1x	O
-]/[	O
-P2	O
-+	O
-X	O
-]),	O
-and	O
-thermodynamic	O
-parameters	O
-were	O
-calculated	O
-according	O
-to	O
-the	O
-Gibbs	O
--	O
-Helmholtz	O
-equation	O
-(	O
-ΔG	O
-°	O
-=	O
-ΔH	O
-−	O
-TΔS	O
-°).	O
-	O
-The	O
-binding	O
-free	O
-energies	O
-,	O
-ΔG	O
-°	O
-(	O
-ΔG	O
-°	O
-=	O
-RTlnKD	O
-),	O
-were	O
-plotted	O
-against	O
-temperature	O
-(	O
-K	O
-)	O
-using	O
-nonlinear	O
-regression	O
-to	O
-fit	O
-the	O
-3	O
--	O
-parameters	O
-van	O
-’	O
-t	O
-Hoff	O
-equation	O
-(	O
-RT	O
-ln	O
-KD	O
-=	O
-ΔH	O
-°	O
-–	O
-TΔS	O
-°	O
-+	O
-ΔCp	O
-°[	O
-T	O
--	O
-T0	O
-]	O
-–	O
-TΔCp	O
-°	O
-ln	O
-[	O
-T	O
-/	O
-T0	O
-]	O
-with	O
-T0	O
-=	O
-298	O
-K	O
-).	O
-	O
-(	O
-A	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-ALWGPDPAAA	O
-;	O
-(	O
-B	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-AQWGPDPAAA	O
-;	O
-(	O
-C	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-RQFGPDWIVA	O
-;	O
-(	O
-D	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-RQWGPDPAAV	O
-,	O
-(	O
-E	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-YQFGPDFPTA	O
-;	O
-and	O
-(	O
-F	O
-)	O
-1E6	O
--	O
-A2	O
--	O
-RQFGPDFPTI	O
-.	O
-	O
-1E6	O
-TCR	O
--	O
-pMHC	O
-contacts	O
-,	O
-affinity	O
-measurements	O
-and	O
-thermodynamics	O
-	O