diff --git "a/dev.tsv" "b/dev.tsv"
deleted file mode 100644--- "a/dev.tsv"
+++ /dev/null
@@ -1,30805 +0,0 @@
-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
-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
-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
-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
-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
-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
-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
-The	O
-apo	O
-structure	O
-is	O
-color	O
-ramped	O
-from	O
-blue	O
-to	O
-red	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
-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
-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
-Prolines	O
-between	O
-domains	O
-are	O
-indicated	O
-as	O
-spheres	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
-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
-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
-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
-The	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-(	O
-ΔBacova_02651	B-mutant
-)	O
-strain	O
-(	O
-cf	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-blocks	O
-IL	O
--	O
-17A	O
-signaling	O
-in	O
-a	O
-human	O
-primary	O
-cell	O
-assay	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
-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
-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
-Both	O
-structures	O
-were	O
-solved	O
-by	O
-molecular	O
-replacement	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
-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
-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
-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
-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
-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
-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
-(	O
-A	O
-)	O
-HAP	O
-binds	O
-at	O
-region	O
-I	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-Polar	O
-interactions	O
-are	O
-shown	O
-in	O
-dashes	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Data	O
-collection	O
-and	O
-refinement	O
-statistics	O
-for	O
-NadR	O
-structures	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	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
-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
-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
-Structural	O
-comparisons	O
-of	O
-NadR	O
-and	O
-modelling	O
-of	O
-interactions	O
-with	O
-DNA	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
-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
-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
-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
-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
-We	O
-confirmed	O
-this	O
-stoichiometry	O
-in	O
-solution	O
-using	O
-SPR	O
-methods	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
-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
-NadR	O
-shows	O
-a	O
-ligand	O
-binding	O
-site	O
-distinct	O
-from	O
-other	O
-MarR	O
-homologues	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Oligomeric	O
-States	O
-of	O
-PduL	O
-Orthologs	O
-Are	O
-Influenced	O
-by	O
-the	O
-EP	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
-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
-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
-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
-Implications	O
-for	O
-Metabolosome	O
-Core	O
-Assembly	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
-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
-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
-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
-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
-Biochemical	O
-properties	O
-of	O
-the	O
-ectoine	O
-synthase	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
-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
-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
-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
-Dependency	O
-of	O
-the	O
-ectoine	O
-synthase	O
-activity	O
-on	O
-metals	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Each	O
-of	O
-these	O
-residues	O
-is	O
-evolutionarily	O
-highly	O
-conserved	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Dimer	O
-formation	O
-of	O
-the	O
-PIN	O
-domains	O
-	O
-Domain	O
--	O
-domain	O
-interaction	O
-between	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-	O
-Residues	O
-critical	O
-for	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	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
-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
-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
-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
-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
-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
-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
-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
-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
-Structural	O
-and	O
-functional	O
-analyses	O
-of	O
-Regnase	O
--	O
-1	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
-Domain	O
--	O
-domain	O
-interaction	O
-between	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	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
-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
-(	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
-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
-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
-Structure	O
-of	O
-PmC11	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
-The	O
-core	O
-caspase	O
--	O
-fold	O
-is	O
-highlighted	O
-in	O
-a	O
-box	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
-D	O
-,	O
-cysteine	O
-peptidase	O
-activity	O
-of	O
-PmC11	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
-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
-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
-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
-A	O
-,	O
-PmC11	O
-activity	O
-is	O
-inhibited	O
-by	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Phenotypic	O
-characterization	O
-of	O
-Δtsr3	B-mutant
-mutants	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-A	O
-notable	O
-exception	O
-is	O
-Trm10	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
-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
-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
-Hydrogen	O
-bonds	O
-are	O
-indicated	O
-by	O
-dashed	O
-lines	O
-.	O
-	O
-(	O
-E	O
-)	O
-Binding	O
-of	O
-14C	O
--	O
-labeled	O
-SAM	O
-to	O
-SsTsr3	O
-.	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
-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
-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
-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
-RNA	O
--	O
-binding	O
-of	O
-Tsr3	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Dodecamer	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
-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
-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
-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
-Annular	O
-Pore	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
-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
-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
-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
-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
-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
-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
-Annular	O
-Pores	O
-Formed	O
-by	O
-Aβ	O
-and	O
-Peptide	B-mutant
-2	I-mutant
-	O
-This	O
-mode	O
-of	O
-assembly	O
-is	O
-not	O
-unique	O
-to	O
-Aβ	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
-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
-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
-Branched	O
-causality	O
-model	O
-for	O
-ERα	O
-‐	O
-mediated	O
-cell	O
-proliferation	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
-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
-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
-Ligand	O
-‐	O
-specific	O
-signaling	O
-underlies	O
-ERα	O
-‐	O
-mediated	O
-cell	O
-proliferation	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-ERβ	O
-activity	O
-is	O
-not	O
-an	O
-independent	O
-predictor	O
-of	O
-E	O
-‐	O
-Luc	O
-activity	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Cdc42	O
--	O
-TOCA1	O
-Binding	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
-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
-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
-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
-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
-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
-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
-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
-The	O
-structure	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	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
-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
-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
-The	O
-overall	O
-CSP	O
-was	O
-calculated	O
-for	O
-each	O
-residue	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
-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
-Modeling	O
-the	O
-Cdc42	O
-·	O
-TOCA1	O
-HR1	O
-Complex	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
-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
-Competition	O
-between	O
-N	O
--	O
-WASP	O
-and	O
-TOCA1	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-The	O
-organization	O
-of	O
-the	O
-yeast	O
-ACC	O
-CD	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-A	O
-second	O
-hinge	O
-can	O
-be	O
-identified	O
-between	O
-CDC1	O
-/	O
-CDC2	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
-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
-Cartoon	O
-representation	O
-of	O
-crystal	O
-structures	O
-of	O
-multidomain	B-mutant
-constructs	I-mutant
-of	O
-CthACC	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
-The	O
-conformational	O
-dynamics	O
-of	O
-fungal	O
-ACC	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
-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
-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
-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
-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
-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
-2	O
-–	O
-228	O
-and	O
-aa	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
-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
-(	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
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-possess	O
-a	O
-similar	O
-ATP	O
-binding	O
-site	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-The	O
-active	O
-site	O
-is	O
-boxed	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
-Numbering	O
-as	O
-in	O
-E	O
-.	O
-coli	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
-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
-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
-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
-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
-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
-Unless	O
-specifically	O
-stated	O
-,	O
-the	O
-drawn	O
-conclusions	O
-also	O
-apply	O
-to	O
-ScMep2	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
-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
-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
-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
-Phosphorylation	O
-target	O
-site	O
-is	O
-at	O
-the	O
-periphery	O
-of	O
-Mep2	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	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
-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
-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
-Growth	O
-of	O
-ScMep2	B-mutant
-variants	I-mutant
-on	O
-low	O
-ammonium	O
-medium	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
-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
-Schematic	O
-model	O
-for	O
-phosphorylation	O
--	O
-based	O
-regulation	O
-of	O
-Mep2	O
-ammonium	O
-transporters	O
-.	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-CDRs	O
-are	O
-defined	O
-using	O
-the	O
-Dunbrack	O
-convention	O
-[	O
-12	O
-].	O
-	O
-CDR	O
-H2	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
-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
-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
-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
-CDR	O
-H3	O
-conformational	O
-diversity	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Some	O
-side	O
-chain	O
-atoms	O
-in	O
-CDR	O
-H3	O
-are	O
-missing	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
-Pairing	O
-of	O
-different	O
-germlines	O
-yields	O
-antibodies	O
-with	O
-various	O
-degrees	O
-of	O
-stability	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
-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
-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
-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
-Visualizing	O
-chaperone	O
--	O
-assisted	O
-protein	O
-folding	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
-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
-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
-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
-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
-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
-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
-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
-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
-Folding	O
-and	O
-interactions	O
-of	O
-Im7	O
-while	O
-bound	O
-to	O
-Spy	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	O
-c	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-Na	O
-+-	O
-binding	O
-sites	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
-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
-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
-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
-(	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
-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
-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
-(	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-U2AF65	O
-RRM	O
-extensions	O
-interact	O
-with	O
-the	O
-Py	O
-tract	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Structures	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-recognizing	O
-a	O
-contiguous	O
-Py	O
-tract	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
-(	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
-(	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
-RNA	O
-protects	O
-a	O
-nucleoprotein	O
-complex	O
-against	O
-radiation	O
-damage	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	O
-a	O
-)	O
-Domain	O
-organization	O
-of	O
-human	O
-MKP7	O
-and	O
-JNK1	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
-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
-Mutational	O
-analysis	O
-on	O
-interactions	O
-between	O
-MKP7	O
--	O
-CD	O
-and	O
-JNK1	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
-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
-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
-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
-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
-(	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
-(	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
-(	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
-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
-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
-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
-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
-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
-Cysteine	O
-residues	O
-engaged	O
-in	O
-disulphide	O
-bonds	O
-are	O
-depicted	O
-in	O
-green	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
-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
-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
-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
-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
-(	O
-B	O
-)	O
-Analytical	O
-size	O
--	O
-exclusion	O
-chromatography	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Crystal	O
-structure	O
-of	O
-a	O
-HAESA	O
-–	O
-IDA	O
-–	O
-SERK1	O
-signaling	O
-complex	O
-.	O
-	O
-Polar	O
-interactions	O
-are	O
-highlighted	O
-as	O
-dotted	O
-lines	O
-(	O
-in	O
-magenta	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
-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
-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
-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
-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
-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
-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
-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
-missing	O
-links	O
-in	O
-our	O
-understanding	O
-of	O
-tRNA	O
-translocation	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-The	O
-central	O
-protuberance	O
-(	O
-CP	O
-)	O
-is	O
-labeled	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
-IRES	O
-rearrangements	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
-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
-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
-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
-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
-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
-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
-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
-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
-Interactions	O
-of	O
-eEF2	O
-with	O
-the	O
-40S	O
-subunit	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Partitioned	O
-roles	O
-of	O
-40S	O
-subunit	O
-rearrangements	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	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
-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
-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
-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
-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
-Ser1	O
-lacks	O
-this	O
-stabilization	O
-and	O
-is	O
-therefore	O
-rotated	O
-by	O
-60	O
-°.	O
-	O
-Inhibition	O
-assays	O
-(	O
-left	O
-panel	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-Total	O
-H3	O
-was	O
-used	O
-as	O
-a	O
-loading	O
-control	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	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
-(	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
-(	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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-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
-(	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
-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
-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
-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
-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
-(	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
-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
-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