diff --git "a/test.tsv" "b/test.tsv"
deleted file mode 100644--- "a/test.tsv"
+++ /dev/null
@@ -1,31924 +0,0 @@
-words  labels
-The	O
-Bacteroidetes	O
-are	O
-dominant	O
-bacteria	O
-in	O
-the	O
-human	O
-gut	O
-that	O
-are	O
-responsible	O
-for	O
-the	O
-digestion	O
-of	O
-the	O
-complex	O
-polysaccharides	O
-that	O
-constitute	O
-“	O
-dietary	O
-fiber	O
-.”	O
-Although	O
-this	O
-symbiotic	O
-relationship	O
-has	O
-been	O
-appreciated	O
-for	O
-decades	O
-,	O
-little	O
-is	O
-currently	O
-known	O
-about	O
-how	O
-Bacteroidetes	O
-seek	O
-out	O
-and	O
-bind	O
-plant	O
-cell	O
-wall	O
-polysaccharides	O
-as	O
-a	O
-necessary	O
-first	O
-step	O
-in	O
-their	O
-metabolism	O
-.	O
-	O
-Here	O
-,	O
-we	O
-provide	O
-the	O
-first	O
-biochemical	O
-,	O
-crystallographic	O
-,	O
-and	O
-genetic	O
-insight	O
-into	O
-how	O
-two	O
-surface	O
-glycan	O
--	O
-binding	O
-proteins	O
-from	O
-the	O
-complex	O
-Bacteroides	O
-ovatus	O
-xyloglucan	O
-utilization	O
-locus	O
-(	O
-XyGUL	O
-)	O
-enable	O
-recognition	O
-and	O
-uptake	O
-of	O
-this	O
-ubiquitous	O
-vegetable	O
-polysaccharide	O
-.	O
-	O
-More	O
-importantly	O
-,	O
-this	O
-makes	O
-diet	O
-a	O
-tractable	O
-way	O
-to	O
-manipulate	O
-the	O
-abundance	O
-and	O
-metabolic	O
-output	O
-of	O
-the	O
-microbiota	O
-toward	O
-improved	O
-human	O
-health	O
-.	O
-	O
-The	O
-archetypal	O
-PUL	O
--	O
-encoded	O
-system	O
-is	O
-the	O
-starch	O
-utilization	O
-system	O
-(	O
-Sus	O
-)	O
-(	O
-Fig	O
-.	O
-1B	O
-)	O
-of	O
-Bacteroides	O
-thetaiotaomicron	O
-.	O
-	O
-The	O
-location	O
-of	O
-SGBP	O
--	O
-A	O
-/	O
-B	O
-is	O
-presented	O
-in	O
-this	O
-work	O
-;	O
-the	O
-location	O
-of	O
-GH5	O
-has	O
-been	O
-empirically	O
-determined	O
-,	O
-and	O
-the	O
-enzymes	O
-have	O
-been	O
-placed	O
-based	O
-upon	O
-their	O
-predicted	O
-cellular	O
-location	O
-.	O
-	O
-We	O
-recently	O
-reported	O
-the	O
-detailed	O
-molecular	O
-characterization	O
-of	O
-a	O
-PUL	O
-that	O
-confers	O
-the	O
-ability	O
-of	O
-the	O
-human	O
-gut	O
-commensal	O
-B	O
-.	O
-ovatus	O
-ATCC	O
-8483	O
-to	O
-grow	O
-on	O
-a	O
-prominent	O
-family	O
-of	O
-plant	O
-cell	O
-wall	O
-glycans	O
-,	O
-the	O
-xyloglucans	O
-(	O
-XyG	O
-).	O
-	O
-As	O
-the	O
-Sus	O
-SGBPs	O
-remain	O
-the	O
-only	O
-structurally	O
-characterized	O
-cohort	O
-to	O
-date	O
-,	O
-we	O
-therefore	O
-wondered	O
-whether	O
-such	O
-glycan	O
-binding	O
-and	O
-function	O
-are	O
-extended	O
-to	O
-other	O
-PUL	O
-that	O
-target	O
-more	O
-complex	O
-and	O
-heterogeneous	O
-polysaccharides	O
-,	O
-such	O
-as	O
-XyG	O
-.	O
-	O
-These	O
-data	O
-extend	O
-our	O
-current	O
-understanding	O
-of	O
-the	O
-Sus	O
--	O
-like	O
-glycan	O
-uptake	O
-paradigm	O
-within	O
-the	O
-Bacteroidetes	O
-and	O
-reveals	O
-how	O
-the	O
-complex	O
-dietary	O
-polysaccharide	O
-xyloglucan	O
-is	O
-recognized	O
-at	O
-the	O
-cell	O
-surface	O
-.	O
-	O
-Similarly	O
-,	O
-SGBP	O
--	O
-B	O
-also	O
-bound	O
-to	O
-XyG	O
-and	O
-XyGO2	O
-with	O
-approximately	O
-equal	O
-affinities	O
-,	O
-although	O
-in	O
-both	O
-cases	O
-,	O
-Ka	O
-values	O
-were	O
-nearly	O
-10	O
--	O
-fold	O
-lower	O
-than	O
-those	O
-for	O
-SGBP	O
--	O
-A	O
-.	O
-Also	O
-in	O
-contrast	O
-to	O
-SGBP	O
--	O
-A	O
-,	O
-SGBP	O
--	O
-B	O
-also	O
-bound	O
-to	O
-XyGO1	O
-,	O
-yet	O
-the	O
-affinity	O
-for	O
-this	O
-minimal	O
-repeating	O
-unit	O
-was	O
-poor	O
-,	O
-with	O
-a	O
-Ka	O
-value	O
-of	O
-ca	O
-.	O
-1	O
-order	O
-of	O
-magnitude	O
-lower	O
-than	O
-for	O
-XyG	O
-and	O
-XyGO2	O
-.	O
-	O
-As	O
-anticipated	O
-by	O
-sequence	O
-similarity	O
-,	O
-the	O
-high	O
--	O
-resolution	O
-tertiary	O
-structure	O
-of	O
-apo	O
--	O
-SGBP	O
--	O
-A	O
-(	O
-1	O
-.	O
-36	O
-Å	O
-,	O
-Rwork	O
-=	O
-14	O
-.	O
-7	O
-%,	O
-Rfree	O
-=	O
-17	O
-.	O
-4	O
-%,	O
-residues	O
-28	O
-to	O
-546	O
-)	O
-(	O
-Table	O
-2	O
-)	O
-displays	O
-the	O
-canonical	O
-“	O
-SusD	O
--	O
-like	O
-”	O
-protein	O
-fold	O
-dominated	O
-by	O
-four	O
-tetratrico	O
--	O
-peptide	O
-repeat	O
-(	O
-TPR	O
-)	O
-motifs	O
-that	O
-cradle	O
-the	O
-rest	O
-of	O
-the	O
-structure	O
-(	O
-Fig	O
-.	O
-4A	O
-).	O
-	O
-Cocrystallization	O
-of	O
-SGBP	O
--	O
-A	O
-with	O
-XyGO2	O
-generated	O
-a	O
-substrate	O
-complex	O
-structure	O
-(	O
-2	O
-.	O
-3	O
-Å	O
-,	O
-Rwork	O
-=	O
-21	O
-.	O
-8	O
-%,	O
-Rfree	O
-=	O
-24	O
-.	O
-8	O
-%,	O
-residues	O
-36	O
-to	O
-546	O
-)	O
-(	O
-Fig	O
-.	O
-4A	O
-and	O
-B	O
-;	O
-Table	O
-2	O
-)	O
-that	O
-revealed	O
-the	O
-distinct	O
-binding	O
--	O
-site	O
-architecture	O
-of	O
-the	O
-XyG	O
-binding	O
-protein	O
-.	O
-	O
-Seven	O
-of	O
-the	O
-eight	O
-backbone	O
-glucosyl	O
-residues	O
-of	O
-XyGO2	O
-could	O
-be	O
-convincingly	O
-modeled	O
-in	O
-the	O
-ligand	O
-electron	O
-density	O
-,	O
-and	O
-only	O
-two	O
-α	O
-(	O
-1	O
-→	O
-6	O
-)-	O
-linked	O
-xylosyl	O
-residues	O
-were	O
-observed	O
-(	O
-Fig	O
-.	O
-4B	O
-;	O
-cf	O
-.	O
-	O
-The	O
-functional	O
-importance	O
-of	O
-this	O
-platform	O
-is	O
-underscored	O
-by	O
-the	O
-observation	O
-that	O
-the	O
-W82A	B-mutant
-W283A	B-mutant
-W306A	B-mutant
-mutant	O
-of	O
-SGBP	O
--	O
-A	O
-,	O
-designated	O
-SGBP	B-mutant
--	I-mutant
-A	I-mutant
-*,	I-mutant
-is	O
-completely	O
-devoid	O
-of	O
-XyG	O
-affinity	O
-(	O
-Table	O
-3	O
-;	O
-see	O
-Fig	O
-.	O
-S4	O
-in	O
-the	O
-supplemental	O
-material	O
-).	O
-	O
-Protein	O
-name	O
-Ka	O
-ΔG	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-ΔH	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-TΔS	O
-(	O
-kcal	O
-⋅	O
-mol	O
-−	O
-1	O
-)	O
-Fold	O
-changeb	O
-M	O
-−	O
-1	O
-SGBP	O
--	O
-A	O
-(	O
-W82A	B-mutant
-W283A	B-mutant
-W306A	B-mutant
-)	O
-ND	O
-NB	O
-NB	O
-NB	O
-NB	O
-SGBP	O
--	O
-A	O
-(	O
-W82A	B-mutant
-)	O
-c	O
-4	O
-.	O
-9	O
-9	O
-.	O
-1	O
-×	O
-104	O
-−	O
-6	O
-.	O
-8	O
-−	O
-6	O
-.	O
-3	O
-0	O
-.	O
-5	O
-SGBP	O
--	O
-A	O
-(	O
-W306	O
-)	O
-ND	O
-NB	O
-NB	O
-NB	O
-NB	O
-SGBP	O
--	O
-B	O
-(	O
-230	O
-–	O
-489	O
-)	O
-0	O
-.	O
-7	O
-(	O
-8	O
-.	O
-6	O
-±	O
-0	O
-.	O
-20	O
-)	O
-×	O
-104	O
-−	O
-6	O
-.	O
-7	O
-−	O
-14	O
-.	O
-9	O
-±	O
-0	O
-.	O
-1	O
-−	O
-8	O
-.	O
-2	O
-SGBP	O
--	O
-B	O
-(	O
-Y363A	B-mutant
-)	O
-19	O
-.	O
-7	O
-(	O
-2	O
-.	O
-9	O
-±	O
-0	O
-.	O
-10	O
-)	O
-×	O
-103	O
-−	O
-4	O
-.	O
-7	O
-−	O
-18	O
-.	O
-1	O
-±	O
-0	O
-.	O
-1	O
-−	O
-13	O
-.	O
-3	O
-SGBP	O
--	O
-B	O
-(	O
-W364A	B-mutant
-)	O
-ND	O
-Weak	O
-Weak	O
-Weak	O
-Weak	O
-SGBP	O
--	O
-B	O
-(	O
-F414A	B-mutant
-)	O
-3	O
-.	O
-2	O
-(	O
-1	O
-.	O
-80	O
-±	O
-0	O
-.	O
-03	O
-)	O
-×	O
-104	O
-−	O
-5	O
-.	O
-8	O
-−	O
-11	O
-.	O
-4	O
-±	O
-0	O
-.	O
-1	O
-−	O
-5	O
-.	O
-6	O
-	O
-Binding	O
-thermodynamics	O
-are	O
-based	O
-on	O
-the	O
-concentration	O
-of	O
-the	O
-binding	O
-unit	O
-,	O
-XyGO2	O
-.	O
-	O
-Domains	O
-A	O
-,	O
-B	O
-,	O
-and	O
-C	O
-display	O
-similar	O
-β	O
--	O
-sandwich	O
-folds	O
-;	O
-domains	O
-B	O
-(	O
-residues	O
-134	O
-to	O
-230	O
-)	O
-and	O
-C	O
-(	O
-residues	O
-231	O
-to	O
-313	O
-)	O
-can	O
-be	O
-superimposed	O
-onto	O
-domain	O
-A	O
-(	O
-residues	O
-34	O
-to	O
-133	O
-)	O
-with	O
-RMSDs	O
-of	O
-1	O
-.	O
-1	O
-and	O
-1	O
-.	O
-2	O
-Å	O
-,	O
-respectively	O
-,	O
-for	O
-47	O
-atom	O
-pairs	O
-(	O
-23	O
-%	O
-and	O
-16	O
-%	O
-sequence	O
-identity	O
-,	O
-respectively	O
-).	O
-	O
-While	O
-there	O
-is	O
-no	O
-substrate	O
--	O
-complexed	O
-structure	O
-of	O
-Bacova_04391	O
-available	O
-,	O
-the	O
-binding	O
-site	O
-is	O
-predicted	O
-to	O
-include	O
-W241	O
-and	O
-Y404	O
-,	O
-which	O
-are	O
-proximal	O
-to	O
-the	O
-XyGO	O
-binding	O
-site	O
-in	O
-SGBP	O
--	O
-B	O
-.	O
-However	O
-,	O
-the	O
-opposing	O
-,	O
-clamp	O
--	O
-like	O
-arrangement	O
-of	O
-these	O
-residues	O
-in	O
-Bacova_04391	O
-is	O
-clearly	O
-distinct	O
-from	O
-the	O
-planar	O
-surface	O
-arrangement	O
-of	O
-the	O
-residues	O
-that	O
-interact	O
-with	O
-XyG	O
-in	O
-SGBP	O
--	O
-B	O
-(	O
-described	O
-below	O
-).	O
-	O
-Inspection	O
-of	O
-the	O
-tertiary	O
-structure	O
-indicates	O
-that	O
-domains	O
-C	O
-and	O
-D	O
-are	O
-effectively	O
-inseparable	O
-,	O
-with	O
-a	O
-contact	O
-interface	O
-of	O
-396	O
-Å2	O
-.	O
-	O
-Despite	O
-the	O
-lack	O
-of	O
-sequence	O
-and	O
-structural	O
-conservation	O
-,	O
-a	O
-similarly	O
-positioned	O
-proline	O
-joins	O
-the	O
-Ig	O
--	O
-like	O
-domains	O
-of	O
-the	O
-xylan	O
--	O
-binding	O
-Bacova_04391	O
-and	O
-the	O
-starch	O
--	O
-binding	O
-proteins	O
-SusE	O
-and	O
-SusF	O
-.	O
-We	O
-speculate	O
-that	O
-this	O
-is	O
-a	O
-biologically	O
-important	O
-adaptation	O
-that	O
-serves	O
-to	O
-project	O
-the	O
-glycan	O
-binding	O
-site	O
-of	O
-these	O
-proteins	O
-far	O
-from	O
-the	O
-membrane	O
-surface	O
-.	O
-	O
-In	O
-these	O
-growth	O
-experiments	O
-,	O
-overnight	O
-cultures	O
-of	O
-strains	O
-grown	O
-on	O
-minimal	O
-medium	O
-plus	O
-glucose	O
-were	O
-back	O
--	O
-diluted	O
-1	O
-:	O
-100	O
--	O
-fold	O
-into	O
-minimal	O
-medium	O
-containing	O
-5	O
-mg	O
-/	O
-ml	O
-of	O
-the	O
-reported	O
-carbohydrate	O
-.	O
-	O
-Complementation	O
-of	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-strain	O
-(	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-::	O
-SGBP	O
--	O
-A	O
-)	O
-restores	O
-growth	O
-to	O
-wild	O
--	O
-type	O
-rates	O
-on	O
-xyloglucan	O
-and	O
-XyGO1	O
-,	O
-yet	O
-the	O
-calculated	O
-rate	O
-of	O
-the	O
-complemented	O
-strain	O
-is	O
-~	O
-72	O
-%	O
-that	O
-of	O
-the	O
-WT	O
-Δtdk	B-mutant
-strain	O
-on	O
-XyGO2	O
-;	O
-similar	O
-results	O
-were	O
-obtained	O
-for	O
-the	O
-SGBP	O
--	O
-B	O
-complemented	O
-strain	O
-despite	O
-the	O
-fact	O
-that	O
-the	O
-growth	O
-curves	O
-do	O
-not	O
-appear	O
-much	O
-different	O
-(	O
-see	O
-Fig	O
-.	O
-S8C	O
-and	O
-F	O
-).	O
-	O
-Growth	O
-was	O
-measured	O
-over	O
-time	O
-in	O
-minimal	O
-medium	O
-containing	O
-(	O
-A	O
-)	O
-XyG	O
-,	O
-(	O
-B	O
-)	O
-XyGO2	O
-,	O
-(	O
-C	O
-)	O
-XyGO1	O
-,	O
-(	O
-D	O
-)	O
-glucose	O
-,	O
-and	O
-(	O
-E	O
-)	O
-xylose	O
-.	O
-	O
-In	O
-panel	O
-F	O
-,	O
-the	O
-growth	O
-rate	O
-of	O
-each	O
-strain	O
-on	O
-the	O
-five	O
-carbon	O
-sources	O
-is	O
-displayed	O
-,	O
-and	O
-in	O
-panel	O
-G	O
-,	O
-the	O
-normalized	O
-lag	O
-time	O
-of	O
-each	O
-culture	O
-,	O
-relative	O
-to	O
-its	O
-growth	O
-on	O
-glucose	O
-,	O
-is	O
-displayed	O
-.	O
-	O
-Intriguingly	O
-,	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-strain	O
-(	O
-ΔBacova_02650	B-mutant
-)	O
-(	O
-cf	O
-.	O
-	O
-Fig	O
-.	O
-1B	O
-)	O
-exhibited	O
-a	O
-minor	O
-growth	O
-defect	O
-on	O
-both	O
-XyG	O
-and	O
-XyGO2	O
-,	O
-with	O
-rates	O
-84	O
-.	O
-6	O
-%	O
-and	O
-93	O
-.	O
-9	O
-%	O
-that	O
-of	O
-the	O
-WT	O
-Δtdk	B-mutant
-strain	O
-.	O
-	O
-However	O
-,	O
-growth	O
-of	O
-the	O
-ΔSGBP	B-mutant
--	I-mutant
-B	I-mutant
-strain	O
-on	O
-XyGO1	O
-was	O
-54	O
-.	O
-2	O
-%	O
-the	O
-rate	O
-of	O
-the	O
-parental	O
-strain	O
-,	O
-despite	O
-the	O
-fact	O
-that	O
-SGBP	O
--	O
-B	O
-binds	O
-this	O
-substrate	O
-ca	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-the	O
-data	O
-indicate	O
-that	O
-SGBP	O
--	O
-A	O
-and	O
-SGBP	O
--	O
-B	O
-functionally	O
-complement	O
-each	O
-other	O
-in	O
-the	O
-capture	O
-of	O
-XyG	O
-polysaccharide	O
-,	O
-while	O
-SGBP	O
--	O
-B	O
-may	O
-allow	O
-B	O
-.	O
-ovatus	O
-to	O
-scavenge	O
-smaller	O
-XyGOs	O
-liberated	O
-by	O
-other	O
-gut	O
-commensals	O
-.	O
-	O
-It	O
-may	O
-then	O
-be	O
-that	O
-only	O
-after	O
-a	O
-sufficient	O
-amount	O
-of	O
-glycan	O
-is	O
-processed	O
-and	O
-imported	O
-by	O
-the	O
-cell	O
-is	O
-XyGUL	O
-upregulated	O
-and	O
-exponential	O
-growth	O
-on	O
-the	O
-glycan	O
-can	O
-begin	O
-.	O
-	O
-Likewise	O
-,	O
-such	O
-cognate	O
-interactions	O
-between	O
-homologous	O
-protein	O
-pairs	O
-such	O
-as	O
-SGBP	O
--	O
-A	O
-and	O
-its	O
-TBDT	O
-may	O
-underlie	O
-our	O
-observation	O
-that	O
-a	O
-ΔSGBP	B-mutant
--	I-mutant
-A	I-mutant
-mutant	O
-cannot	O
-grow	O
-on	O
-xyloglucan	O
-.	O
-	O
-Thus	O
-,	O
-understanding	O
-glycan	O
-capture	O
-at	O
-the	O
-cell	O
-surface	O
-is	O
-fundamental	O
-to	O
-explaining	O
-,	O
-and	O
-eventually	O
-predicting	O
-,	O
-how	O
-the	O
-carbohydrate	O
-content	O
-of	O
-the	O
-diet	O
-shapes	O
-the	O
-gut	O
-community	O
-structure	O
-as	O
-well	O
-as	O
-its	O
-causative	O
-health	O
-effects	O
-.	O
-	O
-PUL	O
--	O
-encoded	O
-TBDTs	O
-in	O
-Bacteroidetes	O
-are	O
-larger	O
-than	O
-the	O
-well	O
--	O
-characterized	O
-iron	O
--	O
-targeting	O
-TBDTs	O
-from	O
-many	O
-Proteobacteria	O
-and	O
-are	O
-further	O
-distinguished	O
-as	O
-the	O
-only	O
-known	O
-glycan	O
--	O
-importing	O
-TBDTs	O
-coexpressed	O
-with	O
-an	O
-SGBP	O
-.	O
-	O
-Our	O
-observation	O
-here	O
-that	O
-the	O
-physical	O
-presence	O
-of	O
-the	O
-SusD	O
-homolog	O
-SGBP	O
--	O
-A	O
-,	O
-independent	O
-of	O
-XyG	O
--	O
-binding	O
-ability	O
-,	O
-is	O
-both	O
-necessary	O
-and	O
-sufficient	O
-for	O
-XyG	O
-utilization	O
-further	O
-supports	O
-a	O
-model	O
-of	O
-glycan	O
-import	O
-whereby	O
-the	O
-SusC	O
--	O
-like	O
-TBDTs	O
-and	O
-the	O
-SusD	O
--	O
-like	O
-SGBPs	O
-must	O
-be	O
-intimately	O
-associated	O
-to	O
-support	O
-glycan	O
-uptake	O
-(	O
-Fig	O
-.	O
-1C	O
-).	O
-	O
-A	O
-molecular	O
-understanding	O
-of	O
-glycan	O
-uptake	O
-by	O
-human	O
-gut	O
-bacteria	O
-is	O
-therefore	O
-central	O
-to	O
-the	O
-development	O
-of	O
-strategies	O
-to	O
-improve	O
-human	O
-health	O
-through	O
-manipulation	O
-of	O
-the	O
-microbiota	O
-.	O
-	O
-A	O
-high	O
-affinity	O
-IL	O
--	O
-17A	O
-peptide	O
-antagonist	O
-(	O
-HAP	O
-)	O
-of	O
-15	O
-residues	O
-was	O
-identified	O
-through	O
-phage	O
--	O
-display	O
-screening	O
-followed	O
-by	O
-saturation	O
-mutagenesis	O
-optimization	O
-and	O
-amino	O
-acid	O
-substitutions	O
-.	O
-	O
-The	O
-family	O
-of	O
-IL	O
--	O
-17	O
-cytokines	O
-and	O
-receptors	O
-consists	O
-of	O
-six	O
-polypeptides	O
-,	O
-IL	O
--	O
-17A	O
--	O
-F	O
-,	O
-and	O
-five	O
-receptors	O
-,	O
-IL	O
--	O
-17RA	O
--	O
-E	O
-.	O
-IL	O
--	O
-17A	O
-is	O
-secreted	O
-from	O
-activated	O
-Th17	O
-cells	O
-,	O
-and	O
-several	O
-innate	O
-immune	O
-T	O
-cell	O
-types	O
-including	O
-macrophages	O
-,	O
-neutrophils	O
-,	O
-natural	O
-killer	O
-cells	O
-,	O
-and	O
-dendritic	O
-cells	O
-.	O
-	O
-There	O
-has	O
-been	O
-active	O
-research	O
-in	O
-identifying	O
-orally	O
-available	O
-chemical	O
-entities	O
-that	O
-would	O
-functionally	O
-antagonize	O
-IL	O
--	O
-17A	O
--	O
-mediated	O
-signaling	O
-.	O
-	O
-Since	O
-IL	O
--	O
-17RA	O
-is	O
-a	O
-shared	O
-receptor	O
-for	O
-at	O
-least	O
-IL	O
--	O
-17A	O
-,	O
-IL	O
--	O
-17F	O
-,	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17F	O
-and	O
-IL	O
--	O
-17E	O
-,	O
-we	O
-chose	O
-to	O
-seek	O
-IL	O
--	O
-17A	O
--	O
-specific	O
-inhibitors	O
-that	O
-may	O
-have	O
-more	O
-defined	O
-pharmacological	O
-responses	O
-than	O
-IL	O
--	O
-17RA	O
-inhibitors	O
-.	O
-	O
-Positive	O
-phage	O
-pools	O
-were	O
-then	O
-sub	O
--	O
-cloned	O
-into	O
-a	O
-maltose	O
--	O
-binding	O
-protein	O
-(	O
-MBP	O
-)	O
-fusion	O
-system	O
-.	O
-	O
-Sequences	O
-identified	O
-from	O
-phage	O
-clones	O
-were	O
-chemically	O
-synthesized	O
-(	O
-Supplementary	O
-Table	O
-1	O
-)	O
-and	O
-tested	O
-for	O
-inhibition	O
-of	O
-IL	O
--	O
-17A	O
-binding	O
-to	O
-IL	O
--	O
-17RA	O
-(	O
-Table	O
-1	O
-).	O
-	O
-In	O
-particular	O
-,	O
-at	O
-position	O
-5	O
-(	O
-13	O
-),	O
-substitution	O
-of	O
-methionine	O
-with	O
-alanine	O
-resulted	O
-in	O
-a	O
-seven	O
-fold	O
-improvement	O
-in	O
-potency	O
-(	O
-80	O
-nM	O
-versus	O
-11	O
-nM	O
-respectively	O
-).	O
-	O
-Since	O
-the	O
-replacement	O
-of	O
-methionine	O
-at	O
-position	O
-5	O
-with	O
-alanine	O
-was	O
-beneficial	O
-,	O
-the	O
-additional	O
-hydrophobic	O
-amino	O
-acids	O
-isoleucine	O
-(	O
-24	O
-),	O
-leucine	O
-(	O
-25	O
-)	O
-and	O
-valine	O
-(	O
-26	O
-)	O
-were	O
-evaluated	O
-and	O
-an	O
-additional	O
-two	O
--	O
-three	O
-fold	O
-improvement	O
-in	O
-binding	O
-was	O
-observed	O
-for	O
-the	O
-valine	O
-and	O
-isoleucine	O
-replacements	O
-in	O
-comparison	O
-with	O
-alanine	O
-.	O
-	O
-Dimerization	O
-of	O
-HAP	O
-can	O
-further	O
-increase	O
-its	O
-potency	O
-	O
-Orthogonal	O
-assays	O
-to	O
-confirm	O
-HAP	O
-antagonism	O
-	O
-The	O
-relatively	O
-high	O
-IC50	O
-values	O
-in	O
-this	O
-assay	O
-(	O
-Table	O
-3	O
-)	O
-are	O
-probably	O
-due	O
-to	O
-the	O
-high	O
-IL	O
--	O
-17A	O
-concentration	O
-(	O
-100	O
-ng	O
-/	O
-ml	O
-)	O
-needed	O
-for	O
-detection	O
-of	O
-IL	O
--	O
-6	O
-.	O
-	O
-Crystallization	O
-and	O
-structure	O
-determination	O
-	O
-Crystals	O
-of	O
-the	O
-Fab	O
-/	O
-truncated	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-diffracted	O
-to	O
-2	O
-.	O
-2	O
-Å	O
-,	O
-and	O
-the	O
-Fab	O
-/	O
-full	O
-length	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-diffracted	O
-to	O
-3	O
-.	O
-0	O
-Å	O
-(	O
-Supplementary	O
-Table	O
-S3	O
-).	O
-	O
-Two	O
-copies	O
-of	O
-HAP	O
-bind	O
-to	O
-the	O
-N	O
--	O
-terminal	O
-of	O
-the	O
-cytokine	O
-dimer	O
-,	O
-also	O
-symmetrically	O
-,	O
-and	O
-each	O
-HAP	O
-molecule	O
-also	O
-interacts	O
-with	O
-both	O
-IL	O
--	O
-17A	O
-monomers	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Inhibition	O
-mechanism	O
-of	O
-IL	O
--	O
-17A	O
-signaling	O
-by	O
-HAP	O
-	O
-Structure	O
-basis	O
-for	O
-the	O
-observed	O
-SAR	O
-of	O
-peptides	O
-	O
-The	O
-C	O
--	O
-terminal	O
-Asn14	O
-and	O
-Lys15	O
-of	O
-HAP	O
-are	O
-not	O
-directly	O
-involved	O
-in	O
-interactions	O
-with	O
-IL	O
--	O
-17A	O
-,	O
-and	O
-this	O
-is	O
-reflected	O
-in	O
-the	O
-gradual	O
-reduction	O
-in	O
-activity	O
-caused	O
-by	O
-C	O
--	O
-terminal	O
-truncations	O
-(	O
-35	O
-and	O
-36	O
-,	O
-Table	O
-2	O
-).	O
-	O
-For	O
-example	O
-,	O
-inspection	O
-of	O
-the	O
-published	O
-IL	O
--	O
-17F	O
-crystal	O
-structure	O
-(	O
-PDB	O
-code	O
-1JPY	O
-)	O
-revealed	O
-a	O
-pocket	O
-of	O
-IL	O
--	O
-17F	O
-similar	O
-to	O
-that	O
-of	O
-IL	O
--	O
-17A	O
-for	O
-W12	O
-of	O
-HAP	O
-binding	O
-,	O
-but	O
-it	O
-is	O
-occupied	O
-by	O
-a	O
-Phe	O
--	O
-Phe	O
-motif	O
-at	O
-the	O
-N	O
--	O
-terminal	O
-peptide	O
-of	O
-IL	O
--	O
-17F	O
-.	O
-	O
-We	O
-have	O
-also	O
-determined	O
-the	O
-complex	O
-structure	O
-of	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-,	O
-which	O
-provides	O
-the	O
-structural	O
-basis	O
-for	O
-HAP	O
-’	O
-s	O
-antagonism	O
-to	O
-IL	O
--	O
-17A	O
-signaling	O
-.	O
-	O
-Since	O
-apo	O
-IL	O
--	O
-17A	O
-is	O
-a	O
-homodimer	O
-with	O
-2	O
-fold	O
-symmetry	O
-,	O
-IL	O
--	O
-17RA	O
-potentially	O
-can	O
-bind	O
-to	O
-either	O
-face	O
-of	O
-the	O
-IL	O
--	O
-17A	O
-dimer	O
-.	O
-	O
-The	O
-interaction	O
-of	O
-IL	O
--	O
-17A	O
-with	O
-IL	O
--	O
-17RA	O
-has	O
-an	O
-extensive	O
-interface	O
-,	O
-covering	O
-~	O
-2	O
-,	O
-200	O
-Å2	O
-surface	O
-area	O
-of	O
-IL	O
--	O
-17A	O
-.	O
-	O
-One	O
-way	O
-of	O
-further	O
-improving	O
-HAP	O
-’	O
-s	O
-potency	O
-is	O
-by	O
-dimerization	O
-.	O
-	O
-KD	O
-determined	O
-by	O
-the	O
-standard	O
-equation	O
-,	O
-KD	O
-=	O
-kd	O
-/	O
-ka	O
-.	O
-(	O
-B	O
-)	O
-HAP	O
-inhibits	O
-SPR	O
-signaling	O
-of	O
-IL	O
--	O
-17A	O
-binding	O
-to	O
-immobilized	O
-IL	O
--	O
-17RA	O
-.	O
-	O
-Overall	O
-structure	O
-of	O
-the	O
-Fab	O
-/	O
-IL	O
--	O
-17A	O
-/	O
-HAP	O
-complex	O
-in	O
-ribbon	O
-presentation	O
-.	O
-	O
-(	O
-C	O
-)	O
-As	O
-a	O
-comparison	O
-,	O
-the	O
-IL	O
--	O
-17A	O
-/	O
-IL	O
--	O
-17RA	O
-complex	O
-was	O
-shown	O
-with	O
-IL	O
--	O
-17A	O
-in	O
-the	O
-same	O
-orientation	O
-.	O
-	O
-ELISA	O
-competition	O
-activity	O
-of	O
-peptide	O
-analogues	O
-of	O
-1	O
-.	O
-	O
-The	O
-amount	O
-of	O
-NadA	O
-on	O
-the	O
-bacterial	O
-surface	O
-is	O
-of	O
-direct	O
-relevance	O
-in	O
-the	O
-constant	O
-battle	O
-of	O
-host	O
--	O
-pathogen	O
-interactions	O
-:	O
-it	O
-influences	O
-the	O
-ability	O
-of	O
-the	O
-pathogen	O
-to	O
-engage	O
-human	O
-cell	O
-surface	O
--	O
-exposed	O
-receptors	O
-and	O
-,	O
-conversely	O
-,	O
-the	O
-bacterial	O
-susceptibility	O
-to	O
-the	O
-antibody	O
--	O
-mediated	O
-immune	O
-response	O
-.	O
-	O
-NadR	O
-also	O
-mediates	O
-ligand	O
--	O
-dependent	O
-regulation	O
-of	O
-many	O
-other	O
-meningococcal	O
-genes	O
-,	O
-for	O
-example	O
-the	O
-highly	O
--	O
-conserved	O
-multiple	O
-adhesin	O
-family	O
-(	O
-maf	O
-)	O
-genes	O
-,	O
-which	O
-encode	O
-proteins	O
-emerging	O
-with	O
-important	O
-roles	O
-in	O
-host	O
--	O
-pathogen	O
-interactions	O
-,	O
-immune	O
-evasion	O
-and	O
-niche	O
-adaptation	O
-.	O
-	O
-The	O
-abundance	O
-of	O
-surface	O
--	O
-exposed	O
-NadA	O
-is	O
-regulated	O
-by	O
-the	O
-ligand	O
--	O
-responsive	O
-transcriptional	O
-repressor	O
-NadR	O
-.	O
-Here	O
-,	O
-we	O
-present	O
-functional	O
-,	O
-biochemical	O
-and	O
-high	O
--	O
-resolution	O
-structural	O
-data	O
-on	O
-NadR	O
-.	O
-Our	O
-studies	O
-provide	O
-detailed	O
-insights	O
-into	O
-how	O
-small	O
-molecule	O
-ligands	O
-,	O
-such	O
-as	O
-hydroxyphenylacetate	O
-derivatives	O
-,	O
-found	O
-in	O
-relevant	O
-host	O
-niches	O
-,	O
-modulate	O
-the	O
-structure	O
-and	O
-activity	O
-of	O
-NadR	O
-,	O
-by	O
-‘	O
-conformational	O
-selection	O
-’	O
-of	O
-inactive	O
-forms	O
-.	O
-	O
-The	O
-DNA	O
--	O
-binding	O
-activity	O
-of	O
-NadR	O
-is	O
-attenuated	O
-in	O
-vitro	O
-upon	O
-addition	O
-of	O
-various	O
-hydroxyphenylacetate	O
-(	O
-HPA	O
-)	O
-derivatives	O
-,	O
-including	O
-4	O
--	O
-HPA	O
-.	O
-	O
-Moreover	O
-,	O
-these	O
-findings	O
-are	O
-important	O
-because	O
-the	O
-activity	O
-of	O
-NadR	O
-impacts	O
-the	O
-potential	O
-coverage	O
-provided	O
-by	O
-anti	O
--	O
-NadA	O
-antibodies	O
-elicited	O
-by	O
-the	O
-Bexsero	O
-vaccine	O
-and	O
-influences	O
-host	O
--	O
-bacteria	O
-interactions	O
-that	O
-contribute	O
-to	O
-meningococcal	O
-pathogenesis	O
-.	O
-	O
-In	O
-analytical	O
-size	O
--	O
-exclusion	O
-high	O
--	O
-performance	O
-liquid	O
-chromatography	O
-(	O
-SE	O
--	O
-HPLC	O
-)	O
-experiments	O
-coupled	O
-with	O
-multi	O
--	O
-angle	O
-laser	O
-light	O
-scattering	O
-(	O
-MALLS	O
-),	O
-NadR	O
-presented	O
-a	O
-single	O
-species	O
-with	O
-an	O
-absolute	O
-molecular	O
-mass	O
-of	O
-35	O
-kDa	O
-(	O
-S1	O
-Fig	O
-).	O
-	O
-(	O
-A	O
-)	O
-Molecular	O
-structures	O
-of	O
-3	O
--	O
-HPA	O
-(	O
-MW	O
-152	O
-.	O
-2	O
-),	O
-4	O
--	O
-HPA	O
-(	O
-MW	O
-152	O
-.	O
-2	O
-),	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-MW	O
-186	O
-.	O
-6	O
-)	O
-and	O
-salicylic	O
-acid	O
-(	O
-MW	O
-160	O
-.	O
-1	O
-).	O
-(	O
-B	O
-)	O
-DSC	O
-profiles	O
-,	O
-colored	O
-as	O
-follows	O
-:	O
-apo	O
--	O
-NadR	O
-(	O
-violet	O
-),	O
-NadR	O
-+	O
-salicylate	O
-(	O
-red	O
-),	O
-NadR	O
-+	O
-3	O
--	O
-HPA	O
-(	O
-green	O
-),	O
-NadR	O
-+	O
-4	O
--	O
-HPA	O
-(	O
-blue	O
-),	O
-NadR	O
-+	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-(	O
-pink	O
-).	O
-	O
-All	O
-DSC	O
-profiles	O
-are	O
-representative	O
-of	O
-triplicate	O
-experiments	O
-.	O
-	O
-However	O
-,	O
-steady	O
--	O
-state	O
-SPR	O
-analyses	O
-of	O
-the	O
-NadR	O
--	O
-HPA	O
-interactions	O
-allowed	O
-determination	O
-of	O
-the	O
-equilibrium	O
-dissociation	O
-constants	O
-(	O
-KD	O
-)	O
-(	O
-Table	O
-1	O
-and	O
-S2	O
-Fig	O
-).	O
-	O
-The	O
-interactions	O
-of	O
-4	O
--	O
-HPA	O
-and	O
-3Cl	O
-,	O
-4	O
--	O
-HPA	O
-with	O
-NadR	O
-exhibited	O
-KD	O
-values	O
-of	O
-1	O
-.	O
-5	O
-mM	O
-and	O
-1	O
-.	O
-1	O
-mM	O
-,	O
-respectively	O
-.	O
-	O
-To	O
-fully	O
-characterize	O
-the	O
-NadR	O
-/	O
-HPA	O
-interactions	O
-,	O
-we	O
-sought	O
-to	O
-determine	O
-crystal	O
-structures	O
-of	O
-NadR	O
-in	O
-ligand	O
--	O
-bound	O
-(	O
-holo	O
-)	O
-and	O
-ligand	O
--	O
-free	O
-(	O
-apo	O
-)	O
-forms	O
-.	O
-	O
-The	O
-map	O
-is	O
-contoured	O
-at	O
-1σ	O
-and	O
-the	O
-figure	O
-was	O
-prepared	O
-with	O
-a	O
-density	O
-mesh	O
-carve	O
-factor	O
-of	O
-1	O
-.	O
-7	O
-,	O
-using	O
-Pymol	O
-(	O
-www	O
-.	O
-pymol	O
-.	O
-org	O
-).	O
-	O
-Only	O
-the	O
-mutation	O
-L130K	B-mutant
-has	O
-a	O
-noteworthy	O
-effect	O
-on	O
-the	O
-oligomeric	O
-state	O
-,	O
-inducing	O
-a	O
-second	O
-peak	O
-with	O
-a	O
-longer	O
-retention	O
-time	O
-and	O
-a	O
-second	O
-peak	O
-maximum	O
-at	O
-18	O
-.	O
-6	O
-min	O
-.	O
-	O
-To	O
-a	O
-much	O
-lesser	O
-extent	O
-,	O
-the	O
-L133K	B-mutant
-mutation	O
-also	O
-appears	O
-to	O
-induce	O
-a	O
-‘	O
-shoulder	O
-’	O
-to	O
-the	O
-main	O
-peak	O
-,	O
-suggesting	O
-very	O
-weak	O
-ability	O
-to	O
-disrupt	O
-the	O
-dimer	O
-.	O
-(	O
-D	O
-)	O
-SE	O
--	O
-HPLC	O
-/	O
-MALLS	O
-analyses	O
-of	O
-the	O
-L130K	B-mutant
-mutant	O
-,	O
-shows	O
-20	O
-%	O
-dimer	O
-and	O
-80	O
-%	O
-monomer	O
-.	O
-	O
-The	O
-ligand	O
-showed	O
-a	O
-different	O
-position	O
-and	O
-orientation	O
-compared	O
-to	O
-salicylate	O
-complexed	O
-with	O
-MTH313	O
-and	O
-ST1710	O
-(	O
-see	O
-Discussion	O
-).	O
-	O
-At	O
-the	O
-other	O
-‘	O
-end	O
-’	O
-of	O
-the	O
-ligand	O
-,	O
-the	O
-4	O
--	O
-hydroxyl	O
-group	O
-was	O
-proximal	O
-to	O
-AspB36	O
-,	O
-with	O
-which	O
-it	O
-may	O
-establish	O
-an	O
-H	O
--	O
-bond	O
-(	O
-see	O
-bond	O
-distances	O
-in	O
-Table	O
-3	O
-).	O
-	O
-The	O
-water	O
-molecule	O
-observed	O
-in	O
-the	O
-pocket	O
-was	O
-bound	O
-by	O
-the	O
-carboxylate	O
-group	O
-and	O
-the	O
-side	O
-chains	O
-of	O
-SerA9	O
-and	O
-AsnA11	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-H	O
--	O
-bonds	O
-involving	O
-the	O
-carboxylate	O
-and	O
-hydroxyl	O
-groups	O
-of	O
-4	O
--	O
-HPA	O
-,	O
-binding	O
-of	O
-the	O
-phenyl	O
-moiety	O
-appeared	O
-to	O
-be	O
-stabilized	O
-by	O
-several	O
-van	O
-der	O
-Waals	O
-’	O
-contacts	O
-,	O
-particularly	O
-those	O
-involving	O
-the	O
-hydrophobic	O
-side	O
-chain	O
-atoms	O
-of	O
-LeuB21	O
-,	O
-MetB22	O
-,	O
-PheB25	O
-,	O
-LeuB29	O
-and	O
-ValB111	O
-(	O
-Fig	O
-4A	O
-).	O
-	O
-The	O
-presence	O
-of	O
-a	O
-single	O
-hydroxyl	O
-group	O
-at	O
-position	O
-2	O
-,	O
-as	O
-in	O
-2	O
--	O
-HPA	O
-,	O
-rather	O
-than	O
-at	O
-position	O
-4	O
-,	O
-would	O
-eliminate	O
-the	O
-possibility	O
-of	O
-favorable	O
-interactions	O
-with	O
-AspB36	O
-,	O
-resulting	O
-in	O
-the	O
-lack	O
-of	O
-NadR	O
-regulation	O
-by	O
-2	O
--	O
-HPA	O
-described	O
-previously	O
-.	O
-	O
-Firstly	O
-,	O
-NadR	O
-is	O
-expected	O
-to	O
-be	O
-covalently	O
-immobilized	O
-on	O
-the	O
-sensor	O
-chip	O
-as	O
-a	O
-dimer	O
-in	O
-random	O
-orientations	O
-,	O
-since	O
-it	O
-is	O
-a	O
-stable	O
-dimer	O
-in	O
-solution	O
-and	O
-has	O
-sixteen	O
-lysines	O
-well	O
--	O
-distributed	O
-around	O
-its	O
-surface	O
-,	O
-all	O
-able	O
-to	O
-act	O
-as	O
-potential	O
-sites	O
-for	O
-amine	O
-coupling	O
-to	O
-the	O
-chip	O
-,	O
-and	O
-none	O
-of	O
-which	O
-are	O
-close	O
-to	O
-the	O
-ligand	O
--	O
-binding	O
-pocket	O
-.	O
-	O
-Secondly	O
-,	O
-the	O
-HPA	O
-analytes	O
-are	O
-all	O
-very	O
-small	O
-(	O
-MW	O
-150	O
-–	O
-170	O
-,	O
-Fig	O
-1A	O
-)	O
-and	O
-therefore	O
-are	O
-expected	O
-to	O
-be	O
-able	O
-to	O
-diffuse	O
-readily	O
-into	O
-all	O
-potential	O
-binding	O
-sites	O
-,	O
-irrespective	O
-of	O
-the	O
-random	O
-orientations	O
-of	O
-the	O
-immobilized	O
-NadR	O
-dimers	O
-on	O
-the	O
-chip	O
-.	O
-	O
-The	O
-crystallographic	O
-data	O
-,	O
-supported	O
-by	O
-the	O
-SPR	O
-studies	O
-of	O
-binding	O
-stoichiometry	O
-,	O
-revealed	O
-the	O
-lack	O
-of	O
-a	O
-second	O
-4	O
--	O
-HPA	O
-molecule	O
-in	O
-the	O
-homodimer	O
-,	O
-suggesting	O
-negative	O
-co	O
--	O
-operativity	O
-,	O
-a	O
-phenomenon	O
-previously	O
-described	O
-for	O
-the	O
-MTH313	O
-/	O
-salicylate	O
-interaction	O
-and	O
-for	O
-other	O
-MarR	O
-family	O
-proteins	O
-.	O
-	O
-To	O
-explore	O
-the	O
-molecular	O
-basis	O
-of	O
-asymmetry	O
-in	O
-holo	O
--	O
-NadR	O
-,	O
-we	O
-superposed	O
-its	O
-ligand	O
--	O
-free	O
-monomer	O
-(	O
-chain	O
-A	O
-)	O
-onto	O
-the	O
-ligand	O
--	O
-occupied	O
-monomer	O
-(	O
-chain	O
-B	O
-).	O
-	O
-However	O
-,	O
-since	O
-residues	O
-of	O
-helix	O
-α6	O
-were	O
-not	O
-directly	O
-involved	O
-in	O
-ligand	O
-binding	O
-,	O
-an	O
-explanation	O
-for	O
-the	O
-lack	O
-of	O
-4	O
--	O
-HPA	O
-in	O
-monomer	O
-A	O
-did	O
-not	O
-emerge	O
-by	O
-analyzing	O
-only	O
-these	O
-backbone	O
-atom	O
-positions	O
-,	O
-suggesting	O
-that	O
-a	O
-more	O
-complex	O
-series	O
-of	O
-allosteric	O
-events	O
-may	O
-occur	O
-.	O
-	O
-Specifically	O
-,	O
-upon	O
-analysis	O
-with	O
-the	O
-CASTp	O
-software	O
-,	O
-the	O
-pocket	O
-in	O
-chain	O
-B	O
-containing	O
-the	O
-4	O
--	O
-HPA	O
-exhibited	O
-a	O
-total	O
-volume	O
-of	O
-approximately	O
-370	O
-Å3	O
-,	O
-while	O
-the	O
-pocket	O
-in	O
-chain	O
-A	O
-was	O
-occupied	O
-by	O
-these	O
-three	O
-side	O
-chains	O
-that	O
-adopted	O
-‘	O
-inward	O
-’	O
-positions	O
-and	O
-thereby	O
-divided	O
-the	O
-space	O
-into	O
-a	O
-few	O
-much	O
-smaller	O
-pockets	O
-,	O
-each	O
-with	O
-volume	O
-<	O
-50	O
-Å3	O
-,	O
-evidently	O
-rendering	O
-chain	O
-A	O
-unfavorable	O
-for	O
-ligand	O
-binding	O
-.	O
-	O
-Although	O
-more	O
-comprehensive	O
-NMR	O
-experiments	O
-and	O
-full	O
-chemical	O
-shift	O
-assignment	O
-of	O
-the	O
-spectra	O
-would	O
-be	O
-required	O
-to	O
-precisely	O
-define	O
-this	O
-multi	O
--	O
-state	O
-behavior	O
-,	O
-the	O
-NMR	O
-data	O
-clearly	O
-demonstrate	O
-that	O
-NadR	O
-exhibits	O
-conformational	O
-flexibility	O
-which	O
-is	O
-modulated	O
-by	O
-4	O
--	O
-HPA	O
-in	O
-solution	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-holo	O
--	O
-homodimer	O
-structure	O
-is	O
-shown	O
-as	O
-green	O
-and	O
-blue	O
-cartoons	O
-,	O
-for	O
-chain	O
-A	O
-and	O
-B	O
-,	O
-respectively	O
-,	O
-while	O
-the	O
-two	O
-homodimers	O
-of	O
-apo	O
--	O
-NadR	O
-are	O
-both	O
-cyan	O
-and	O
-pale	O
-blue	O
-for	O
-chains	O
-A	O
-/	O
-C	O
-and	O
-B	O
-/	O
-D	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-three	O
-homodimers	O
-(	O
-chains	O
-AB	O
-holo	O
-,	O
-AB	O
-apo	O
-,	O
-and	O
-CD	O
-apo	O
-)	O
-were	O
-overlaid	O
-by	O
-structural	O
-alignment	O
-exclusively	O
-of	O
-all	O
-heavy	O
-atoms	O
-in	O
-residues	O
-R64	O
--	O
-A77	O
-(	O
-shown	O
-in	O
-red	O
-,	O
-with	O
-side	O
-chain	O
-sticks	O
-)	O
-of	O
-chains	O
-A	O
-holo	O
-,	O
-A	O
-apo	O
-,	O
-and	O
-C	O
-apo	O
-,	O
-belonging	O
-to	O
-helix	O
-α4	O
-(	O
-left	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-apo	O
--	O
-homodimer	O
-AB	O
-presented	O
-the	O
-DNA	O
--	O
-binding	O
-helices	O
-in	O
-a	O
-conformation	O
-similar	O
-to	O
-that	O
-observed	O
-in	O
-the	O
-protein	O
-:	O
-DNA	O
-complex	O
-of	O
-OhrR	O
-:	O
-ohrA	O
-from	O
-Bacillus	O
-subtilis	O
-(	O
-Fig	O
-8C	O
-).	O
-	O
-This	O
-mutagenesis	O
-data	O
-revealed	O
-that	O
-NadR	O
-residues	O
-His7	O
-,	O
-Ser9	O
-,	O
-Asn11	O
-and	O
-Phe25	O
-play	O
-key	O
-roles	O
-in	O
-the	O
-ligand	O
--	O
-mediated	O
-regulation	O
-of	O
-NadR	O
-;	O
-they	O
-are	O
-each	O
-involved	O
-in	O
-the	O
-controlled	O
-de	O
--	O
-repression	O
-of	O
-the	O
-nadA	O
-promoter	O
-and	O
-synthesis	O
-of	O
-NadA	O
-in	O
-response	O
-to	O
-4	O
--	O
-HPA	O
-in	O
-vivo	O
-.	O
-	O
-Given	O
-the	O
-importance	O
-of	O
-NadR	O
--	O
-mediated	O
-regulation	O
-of	O
-NadA	O
-levels	O
-in	O
-the	O
-contexts	O
-of	O
-meningococcal	O
-pathogenesis	O
-,	O
-we	O
-sought	O
-to	O
-characterize	O
-NadR	O
-,	O
-and	O
-its	O
-interaction	O
-with	O
-ligands	O
-,	O
-at	O
-atomic	O
-resolution	O
-.	O
-	O
-(	O
-B	O
-)	O
-A	O
-structural	O
-alignment	O
-of	O
-MTH313	O
-chain	O
-A	O
-and	O
-ST1710	O
-(	O
-pink	O
-)	O
-(	O
-Cα	O
-rmsd	O
-2	O
-.	O
-3Å	O
-),	O
-shows	O
-that	O
-they	O
-bind	O
-salicylate	O
-in	O
-equivalent	O
-sites	O
-(	O
-differing	O
-by	O
-only	O
-~	O
-3Å	O
-)	O
-and	O
-with	O
-the	O
-same	O
-orientation	O
-.	O
-	O
-While	O
-some	O
-flexibility	O
-of	O
-helix	O
-α4	O
-was	O
-also	O
-observed	O
-in	O
-the	O
-two	O
-apo	O
--	O
-structures	O
-,	O
-concomitant	O
-changes	O
-in	O
-the	O
-dimer	O
-interfaces	O
-were	O
-not	O
-observed	O
-,	O
-possibly	O
-due	O
-to	O
-the	O
-absence	O
-of	O
-ligand	O
-.	O
-	O
-The	O
-latter	O
-may	O
-influence	O
-the	O
-surface	O
-abundance	O
-or	O
-secretion	O
-of	O
-maf	O
-proteins	O
-,	O
-an	O
-emerging	O
-class	O
-of	O
-highly	O
-conserved	O
-meningococcal	O
-putative	O
-adhesins	O
-and	O
-toxins	O
-with	O
-many	O
-important	O
-roles	O
-.	O
-	O
-Further	O
-work	O
-is	O
-required	O
-to	O
-investigate	O
-how	O
-the	O
-two	O
-different	O
-promoter	O
-types	O
-influence	O
-the	O
-ligand	O
--	O
-responsiveness	O
-of	O
-NadR	O
-during	O
-bacterial	O
-infection	O
-and	O
-may	O
-provide	O
-insights	O
-into	O
-the	O
-regulatory	O
-mechanisms	O
-occurring	O
-during	O
-these	O
-host	O
--	O
-pathogen	O
-interactions	O
-.	O
-	O
-Structure	O
-of	O
-an	O
-OhrR	O
--	O
-ohrA	O
-operator	O
-complex	O
-reveals	O
-the	O
-DNA	O
-binding	O
-mechanism	O
-of	O
-the	O
-MarR	O
-family	O
-	O
-Structural	O
-determinant	O
-for	O
-inducing	O
-RORgamma	O
-specific	O
-inverse	O
-agonism	O
-triggered	O
-by	O
-a	O
-synthetic	O
-benzoxazinone	O
-ligand	O
-	O
-Our	O
-goal	O
-was	O
-to	O
-develop	O
-a	O
-RORγ	O
-specific	O
-inverse	O
-agonist	O
-that	O
-would	O
-help	O
-down	O
-regulate	O
-pro	O
--	O
-inflammatory	O
-gene	O
-transcription	O
-by	O
-disrupting	O
-the	O
-protein	O
-protein	O
-interaction	O
-with	O
-coactivator	O
-proteins	O
-as	O
-a	O
-therapeutic	O
-agent	O
-.	O
-	O
-Using	O
-an	O
-in	O
-vivo	O
-reporter	O
-assay	O
-,	O
-we	O
-show	O
-that	O
-the	O
-inverse	O
-agonist	O
-BIO399	O
-displayed	O
-specificity	O
-for	O
-RORγ	O
-over	O
-ROR	O
-sub	O
--	O
-family	O
-members	O
-α	O
-and	O
-β	O
-.	O
-	O
-The	O
-synthetic	O
-benzoxazinone	O
-ligands	O
-identified	O
-in	O
-our	O
-FRET	O
-assay	O
-have	O
-an	O
-agonist	O
-(	O
-BIO592	O
-)	O
-or	O
-inverse	O
-agonist	O
-(	O
-BIO399	O
-)	O
-effect	O
-by	O
-stabilizing	O
-or	O
-destabilizing	O
-the	O
-agonist	O
-conformation	O
-of	O
-RORγ	O
-.	O
-	O
-Our	O
-structural	O
-investigation	O
-of	O
-the	O
-BIO592	O
-agonist	O
-and	O
-BIO399	O
-inverse	O
-agonist	O
-structures	O
-identified	O
-residue	O
-Met358	O
-on	O
-RORγ	O
-as	O
-the	O
-trigger	O
-for	O
-RORγ	O
-specific	O
-inverse	O
-agonism	O
-.	O
-	O
-Retinoid	O
--	O
-related	O
-orphan	O
-receptor	O
-gamma	O
-(	O
-RORγ	O
-)	O
-is	O
-a	O
-transcription	O
-factor	O
-belonging	O
-to	O
-a	O
-sub	O
--	O
-family	O
-of	O
-nuclear	O
-receptors	O
-that	O
-includes	O
-two	O
-closely	O
-related	O
-members	O
-RORα	O
-and	O
-RORβ	O
-.	O
-	O
-Here	O
-we	O
-present	O
-the	O
-identification	O
-of	O
-two	O
-synthetic	O
-benzoxazinone	O
-RORγ	O
-ligands	O
-,	O
-a	O
-weak	O
-agonist	O
-BIO592	O
-(	O
-Fig	O
-.	O
-1a	O
-)	O
-and	O
-an	O
-inverse	O
-agonist	O
-BIO399	O
-(	O
-Fig	O
-.	O
-1b	O
-)	O
-which	O
-were	O
-identified	O
-using	O
-a	O
-Fluorescence	O
-Resonance	O
-Energy	O
-transfer	O
-(	O
-FRET	O
-)	O
-based	O
-assay	O
-that	O
-monitored	O
-coactivator	O
-peptide	O
-recruitment	O
-.	O
-	O
-Using	O
-partial	O
-proteolysis	O
-in	O
-combination	O
-with	O
-mass	O
-spectrometry	O
-analysis	O
-we	O
-demonstrate	O
-that	O
-the	O
-AF2	O
-helix	O
-of	O
-RORγ	O
-destabilizes	O
-upon	O
-BIO399	O
-(	O
-inverse	O
-agonist	O
-)	O
-binding	O
-.	O
-	O
-Using	O
-a	O
-FRET	O
-based	O
-assay	O
-we	O
-discovered	O
-agonist	O
-BIO592	O
-(	O
-Fig	O
-.	O
-1a	O
-)	O
-which	O
-increased	O
-the	O
-coactivator	O
-peptide	O
-TRAP220	O
-recruitment	O
-to	O
-RORγ	O
-(	O
-EC50	O
-0f	O
-58nM	O
-and	O
-Emax	O
-of	O
-130	O
-%)	O
-and	O
-a	O
-potent	O
-inverse	O
-agonist	O
-BIO399	O
-(	O
-Fig	O
-.	O
-1b	O
-)	O
-which	O
-inhibited	O
-coactivator	O
-recruitment	O
-(	O
-IC50	O
-:	O
-4	O
-.	O
-7nM	O
-).	O
-	O
-c	O
-EBI96	O
-coactivator	O
-peptide	O
-bound	O
-in	O
-the	O
-coactivator	O
-pocket	O
-of	O
-RORγ	O
-	O
-Specific	O
-proteolytic	O
-positions	O
-on	O
-RORγ518	O
-when	O
-treated	O
-with	O
-Actinase	O
-E	O
-alone	O
-(	O
-Green	O
-)	O
-or	O
-in	O
-the	O
-presence	O
-of	O
-BIO399	O
-(	O
-Red	O
-)	O
-and	O
-shared	O
-proteolytic	O
-sites	O
-(	O
-Yellow	O
-)	O
-	O
-Several	O
-rounds	O
-of	O
-cocrystallization	O
-attempts	O
-with	O
-RORγ518	O
-or	O
-other	O
-RORγ	O
-AF2	O
-helix	O
-containing	O
-constructs	O
-complexed	O
-with	O
-BIO399	O
-had	O
-not	O
-produced	O
-crystals	O
-.	O
-	O
-We	O
-reasoned	O
-that	O
-if	O
-we	O
-could	O
-remove	O
-the	O
-unfolded	O
-AF2	O
-helix	O
-using	O
-proteolysis	O
-we	O
-could	O
-produce	O
-a	O
-binary	O
-complex	O
-more	O
-amenable	O
-to	O
-crystallization	O
-.	O
-	O
-The	O
-aeRORγ493	O
-/	O
-4	O
-BIO399	O
-structure	O
-diverged	O
-at	O
-the	O
-c	O
--	O
-terminal	O
-end	O
-of	O
-Helix	O
-11	O
-from	O
-the	O
-RORγ518	O
-BIO592	O
-EBI96	O
-structure	O
-,	O
-where	O
-helix	O
-11	O
-unwinds	O
-into	O
-a	O
-random	O
-coil	O
-after	O
-residue	O
-L475	O
-.	O
-	O
-BIO399	O
-binds	O
-to	O
-the	O
-ligand	O
-binding	O
-site	O
-of	O
-RORγ	O
-adopting	O
-a	O
-collapsed	O
-conformation	O
-as	O
-seen	O
-with	O
-BIO592	O
-where	O
-the	O
-two	O
-compounds	O
-superimpose	O
-with	O
-an	O
-RMSD	O
-of	O
-0	O
-.	O
-72	O
-Å	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-BIO399	O
-and	O
-Inverse	O
-agonist	O
-T0901317	O
-bind	O
-in	O
-a	O
-collapsed	O
-conformation	O
-distinct	O
-from	O
-other	O
-RORγ	O
-Inverse	O
-Agonists	O
-Cocrystal	O
-structures	O
-	O
-However	O
-,	O
-the	O
-inverse	O
-agonism	O
-trigger	O
-of	O
-BIO399	O
-,	O
-residue	O
-Met358	O
-,	O
-is	O
-a	O
-leucine	O
-in	O
-both	O
-RORα	O
-and	O
-β	O
-.	O
-	O
-The	O
-Structural	O
-Basis	O
-of	O
-Coenzyme	O
-A	O
-Recycling	O
-in	O
-a	O
-Bacterial	O
-Organelle	O
-	O
-The	O
-majority	O
-of	O
-catabolic	O
-BMCs	O
-(	O
-metabolosomes	O
-)	O
-compartmentalize	O
-a	O
-common	O
-core	O
-of	O
-enzymes	O
-to	O
-metabolize	O
-compounds	O
-via	O
-a	O
-toxic	O
-and	O
-/	O
-or	O
-volatile	O
-aldehyde	O
-intermediate	O
-.	O
-	O
-Accordingly	O
-,	O
-PduL	O
-and	O
-Pta	O
-exemplify	O
-functional	O
-,	O
-but	O
-not	O
-structural	O
-,	O
-convergent	O
-evolution	O
-.	O
-	O
-This	O
-enzyme	O
-,	O
-PduL	O
-,	O
-is	O
-exclusively	O
-associated	O
-with	O
-organelles	O
-called	O
-bacterial	O
-microcompartments	O
-,	O
-which	O
-are	O
-used	O
-to	O
-catabolize	O
-various	O
-compounds	O
-.	O
-	O
-The	O
-aldehyde	O
-is	O
-subsequently	O
-converted	O
-into	O
-an	O
-acyl	O
--	O
-CoA	O
-by	O
-aldehyde	O
-dehydrogenase	O
-,	O
-which	O
-uses	O
-NAD	O
-+	O
-and	O
-CoA	O
-as	O
-cofactors	O
-.	O
-	O
-NAD	O
-+	O
-is	O
-recycled	O
-via	O
-alcohol	O
-dehydrogenase	O
-,	O
-and	O
-CoA	O
-is	O
-recycled	O
-via	O
-phosphotransacetylase	O
-(	O
-PTAC	O
-)	O
-(	O
-Fig	O
-1	O
-).	O
-	O
-They	O
-can	O
-also	O
-work	O
-in	O
-the	O
-reverse	O
-direction	O
-to	O
-activate	O
-acetate	O
-to	O
-the	O
-CoA	O
--	O
-thioester	O
-.	O
-	O
-The	O
-canonical	O
-PTAC	O
-,	O
-Pta	O
-,	O
-is	O
-an	O
-ancient	O
-enzyme	O
-found	O
-in	O
-some	O
-eukaryotes	O
-and	O
-archaea	O
-,	O
-and	O
-widespread	O
-among	O
-the	O
-bacteria	O
-;	O
-90	O
-%	O
-of	O
-the	O
-bacterial	O
-genomes	O
-in	O
-the	O
-Integrated	O
-Microbial	O
-Genomes	O
-database	O
-contain	O
-a	O
-gene	O
-encoding	O
-the	O
-PTA_PTB	O
-phosphotransacylase	O
-(	O
-Pfam	O
-domain	O
-PF01515	O
-).	O
-	O
-The	O
-primary	O
-structure	O
-of	O
-PduL	O
-homologs	O
-is	O
-subdivided	O
-into	O
-two	O
-PF06130	O
-domains	O
-,	O
-each	O
-roughly	O
-80	O
-residues	O
-in	O
-length	O
-.	O
-	O
-Structure	O
-Determination	O
-of	O
-PduL	O
-	O
-Remarkably	O
-,	O
-after	O
-removing	O
-the	O
-N	O
--	O
-terminal	O
-putative	O
-EP	O
-(	O
-27	O
-amino	O
-acids	O
-),	O
-most	O
-of	O
-the	O
-sPduLΔEP	B-mutant
-protein	O
-was	O
-in	O
-the	O
-soluble	O
-fraction	O
-upon	O
-cell	O
-lysis	O
-.	O
-	O
-A	O
-CoA	O
-cofactor	O
-as	O
-well	O
-as	O
-two	O
-metal	O
-ions	O
-are	O
-clearly	O
-resolved	O
-in	O
-the	O
-density	O
-(	O
-for	O
-omit	O
-maps	O
-of	O
-CoA	O
-see	O
-S2	O
-Fig	O
-).	O
-	O
-The	O
-sequences	O
-aligning	O
-to	O
-the	O
-PF06130	O
-domain	O
-(	O
-determined	O
-by	O
-BLAST	O
-)	O
-are	O
-highlighted	O
-in	O
-red	O
-and	O
-blue	O
-.	O
-	O
-Distances	O
-between	O
-atom	O
-centers	O
-are	O
-indicated	O
-in	O
-Å	O
-.	O
-(	O
-a	O
-)	O
-Coenzyme	O
-A	O
-containing	O
-,	O
-(	O
-b	O
-)	O
-phosphate	O
--	O
-bound	O
-structure	O
-.	O
-	O
-(	O
-d	O
-)–(	O
-f	O
-):	O
-Chromatograms	O
-of	O
-sPduL	O
-(	O
-d	O
-),	O
-rPduL	O
-(	O
-e	O
-),	O
-and	O
-pPduL	O
-(	O
-f	O
-)	O
-post	O
--	O
-preparative	O
-size	O
-exclusion	O
-chromatography	O
-with	O
-different	O
-size	O
-fractions	O
-separated	O
-,	O
-applied	O
-over	O
-an	O
-analytical	O
-size	O
-exclusion	O
-column	O
-(	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-The	O
-phosphate	O
-contacts	O
-both	O
-zinc	O
-atoms	O
-(	O
-Fig	O
-4b	O
-)	O
-and	O
-replaces	O
-the	O
-coordination	O
-by	O
-CoA	O
-at	O
-Zn1	O
-;	O
-the	O
-coordination	O
-for	O
-Zn2	O
-changes	O
-from	O
-octahedral	O
-with	O
-three	O
-bound	O
-waters	O
-to	O
-tetrahedral	O
-with	O
-a	O
-phosphate	O
-ion	O
-as	O
-one	O
-of	O
-the	O
-ligands	O
-(	O
-Fig	O
-4b	O
-).	O
-	O
-The	O
-two	O
-zinc	O
-atoms	O
-are	O
-slightly	O
-closer	O
-together	O
-in	O
-the	O
-phosphate	O
--	O
-bound	O
-form	O
-(	O
-5	O
-.	O
-8	O
-Å	O
-vs	O
-6	O
-.	O
-3	O
-Å	O
-),	O
-possibly	O
-due	O
-to	O
-the	O
-bridging	O
-effect	O
-of	O
-the	O
-phosphate	O
-.	O
-	O
-rPduL	O
-full	O
-length	O
-runs	O
-as	O
-Mw	O
-=	O
-140	O
-.	O
-3	O
-kDa	O
-+/−	O
-1	O
-.	O
-2	O
-%	O
-and	O
-Mn	O
-=	O
-140	O
-.	O
-5	O
-kDa	O
-+/−	O
-1	O
-.	O
-2	O
-%.	O
-	O
-Moreover	O
-,	O
-the	O
-PduL	O
-crystal	O
-structures	O
-offer	O
-a	O
-clue	O
-as	O
-to	O
-how	O
-required	O
-cofactors	O
-enter	O
-the	O
-BMC	O
-lumen	O
-during	O
-assembly	O
-.	O
-	O
-The	O
-native	O
-substrate	O
-for	O
-the	O
-forward	O
-reaction	O
-of	O
-rPduL	O
-and	O
-pPduL	O
-,	O
-propionyl	O
--	O
-CoA	O
-,	O
-most	O
-likely	O
-binds	O
-to	O
-the	O
-enzyme	O
-in	O
-the	O
-same	O
-way	O
-at	O
-the	O
-observed	O
-nucleotide	O
-and	O
-pantothenic	O
-acid	O
-moiety	O
-,	O
-but	O
-the	O
-propionyl	O
-group	O
-in	O
-the	O
-CoA	O
--	O
-thioester	O
-might	O
-point	O
-in	O
-a	O
-different	O
-direction	O
-.	O
-	O
-Indeed	O
-,	O
-in	O
-the	O
-majority	O
-of	O
-PduLs	O
-encoded	O
-in	O
-pvm	O
-loci	O
-,	O
-Gln77	O
-is	O
-substituted	O
-by	O
-either	O
-a	O
-Tyr	O
-or	O
-Phe	O
-,	O
-whereas	O
-it	O
-is	O
-typically	O
-a	O
-Gln	O
-or	O
-Glu	O
-in	O
-PduLs	O
-in	O
-all	O
-other	O
-BMC	O
-types	O
-that	O
-degrade	O
-acetyl	O
--	O
-or	O
-propionyl	O
--	O
-CoA	O
-.	O
-A	O
-comparison	O
-of	O
-the	O
-PduL	O
-active	O
-site	O
-to	O
-that	O
-of	O
-the	O
-functionally	O
-identical	O
-Pta	O
-suggests	O
-that	O
-the	O
-two	O
-enzymes	O
-have	O
-distinctly	O
-different	O
-mechanisms	O
-.	O
-	O
-The	O
-two	O
-high	O
--	O
-resolution	O
-crystal	O
-structures	O
-presented	O
-here	O
-will	O
-serve	O
-as	O
-the	O
-foundation	O
-for	O
-mechanistic	O
-studies	O
-on	O
-this	O
-noncanonical	O
-PTAC	O
-enzyme	O
-to	O
-determine	O
-how	O
-the	O
-dimetal	O
-active	O
-site	O
-functions	O
-to	O
-catalyze	O
-both	O
-forward	O
-and	O
-reverse	O
-reactions	O
-.	O
-	O
-There	O
-could	O
-be	O
-some	O
-intrinsic	O
-biochemical	O
-difference	O
-between	O
-the	O
-two	O
-enzymes	O
-that	O
-renders	O
-PduL	O
-a	O
-more	O
-attractive	O
-candidate	O
-for	O
-encapsulation	O
-in	O
-a	O
-BMC	O
-—	O
-for	O
-example	O
-,	O
-PduL	O
-might	O
-be	O
-more	O
-amenable	O
-to	O
-tight	O
-packaging	O
-,	O
-or	O
-is	O
-better	O
-suited	O
-for	O
-the	O
-chemical	O
-microenvironment	O
-formed	O
-within	O
-the	O
-lumen	O
-of	O
-the	O
-BMC	O
-,	O
-which	O
-can	O
-be	O
-quite	O
-different	O
-from	O
-the	O
-cytosol	O
-.	O
-	O
-A	O
-detailed	O
-understanding	O
-of	O
-the	O
-underlying	O
-principles	O
-governing	O
-the	O
-assembly	O
-and	O
-internal	O
-structural	O
-organization	O
-of	O
-BMCs	O
-is	O
-a	O
-requisite	O
-for	O
-synthetic	O
-biologists	O
-to	O
-design	O
-custom	O
-nanoreactors	O
-that	O
-use	O
-BMC	O
-architectures	O
-as	O
-a	O
-template	O
-.	O
-	O
-Furthermore	O
-,	O
-given	O
-the	O
-growing	O
-number	O
-of	O
-metabolosomes	O
-implicated	O
-in	O
-pathogenesis	O
-,	O
-the	O
-PduL	O
-structure	O
-will	O
-be	O
-useful	O
-in	O
-the	O
-development	O
-of	O
-therapeutics	O
-.	O
-	O
-EctC	O
-forms	O
-a	O
-dimer	O
-with	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-arrangement	O
-,	O
-both	O
-in	O
-solution	O
-and	O
-in	O
-the	O
-crystal	O
-structure	O
-.	O
-	O
-We	O
-show	O
-for	O
-the	O
-first	O
-time	O
-that	O
-ectoine	O
-synthase	O
-harbors	O
-a	O
-catalytically	O
-important	O
-metal	O
-co	O
--	O
-factor	O
-;	O
-metal	O
-depletion	O
-and	O
-reconstitution	O
-experiments	O
-suggest	O
-that	O
-EctC	O
-is	O
-probably	O
-an	O
-iron	O
--	O
-dependent	O
-enzyme	O
-.	O
-	O
-Structure	O
--	O
-guided	O
-site	O
--	O
-directed	O
-mutagenesis	O
-experiments	O
-targeting	O
-amino	O
-acid	O
-residues	O
-that	O
-are	O
-evolutionarily	O
-highly	O
-conserved	O
-among	O
-the	O
-extended	O
-EctC	O
-protein	O
-family	O
-,	O
-including	O
-those	O
-forming	O
-the	O
-presumptive	O
-iron	O
--	O
-binding	O
-site	O
-,	O
-were	O
-conducted	O
-to	O
-functionally	O
-analyze	O
-the	O
-properties	O
-of	O
-the	O
-resulting	O
-EctC	O
-variants	O
-.	O
-	O
-This	O
-stereospecific	O
-chemical	O
-modification	O
-of	O
-ectoine	O
-(	O
-Fig	O
-1	O
-)	O
-is	O
-catalyzed	O
-by	O
-the	O
-ectoine	O
-hydroxylase	O
-(	O
-EctD	O
-)	O
-(	O
-EC	O
-1	O
-.	O
-14	O
-.	O
-11	O
-),	O
-a	O
-member	O
-of	O
-the	O
-non	O
--	O
-heme	O
-containing	O
-iron	O
-(	O
-II	O
-)	O
-and	O
-2	O
--	O
-oxoglutarate	O
--	O
-dependent	O
-dioxygenase	O
-superfamily	O
-.	O
-	O
-Scheme	O
-of	O
-the	O
-ectoine	O
-and	O
-5	O
--	O
-hydroxyectoine	O
-biosynthetic	O
-pathway	O
-.	O
-	O
-The	O
-EctC	O
-protein	O
-forms	O
-a	O
-dimer	O
-in	O
-solution	O
-and	O
-our	O
-structural	O
-analysis	O
-identifies	O
-it	O
-as	O
-a	O
-member	O
-of	O
-the	O
-cupin	O
-superfamily	O
-.	O
-	O
-(	O
-Sa	O
-)	O
-EctC	O
-is	O
-a	O
-highly	O
-salt	O
--	O
-tolerant	O
-enzyme	O
-since	O
-it	O
-exhibited	O
-substantial	O
-enzyme	O
-activity	O
-even	O
-at	O
-NaCl	O
-and	O
-KCl	O
-concentrations	O
-of	O
-1	O
-M	O
-in	O
-the	O
-assay	O
-buffer	O
-(	O
-S3c	O
-and	O
-S3d	O
-Fig	O
-).	O
-	O
-The	O
-ectoine	O
-synthase	O
-is	O
-a	O
-metal	O
--	O
-containing	O
-protein	O
-	O
-The	O
-amino	O
-acid	O
-sequences	O
-of	O
-20	O
-selected	O
-EctC	O
--	O
-type	O
-proteins	O
-are	O
-compared	O
-.	O
-	O
-A	O
-metal	O
-cofactor	O
-is	O
-important	O
-for	O
-the	O
-catalytic	O
-activity	O
-of	O
-EctC	O
-	O
-To	O
-address	O
-these	O
-questions	O
-,	O
-we	O
-incubated	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-enzyme	O
-with	O
-increasing	O
-concentrations	O
-of	O
-the	O
-metal	O
-chelator	O
-ethylene	O
--	O
-diamine	O
--	O
-tetraacetic	O
--	O
-acid	O
-(	O
-EDTA	O
-)	O
-and	O
-subsequently	O
-assayed	O
-ectoine	O
-synthase	O
-activity	O
-.	O
-	O
-The	O
-EctC	O
--	O
-catalyzed	O
-ring	O
--	O
-closure	O
-of	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-to	O
-form	O
-ectoine	O
-exhibited	O
-Michaelis	O
--	O
-Menten	O
--	O
-kinetics	O
-with	O
-an	O
-apparent	O
-Km	O
-of	O
-4	O
-.	O
-9	O
-±	O
-0	O
-.	O
-5	O
-mM	O
-,	O
-a	O
-vmax	O
-of	O
-25	O
-.	O
-0	O
-±	O
-0	O
-.	O
-8	O
-U	O
-/	O
-mg	O
-and	O
-a	O
-kcat	O
-of	O
-7	O
-.	O
-2	O
-s	O
--	O
-1	O
-(	O
-S4a	O
-Fig	O
-).	O
-	O
-(	O
-Sa	O
-)	O
-EctC	O
-catalyzed	O
-this	O
-reaction	O
-with	O
-Michaelis	O
--	O
-Menten	O
--	O
-kinetics	O
-exhibiting	O
-an	O
-apparent	O
-Km	O
-of	O
-25	O
-.	O
-4	O
-±	O
-2	O
-.	O
-9	O
-mM	O
-,	O
-a	O
-vmax	O
-of	O
-24	O
-.	O
-6	O
-±	O
-1	O
-.	O
-0	O
-U	O
-/	O
-mg	O
-and	O
-a	O
-kcat	O
-0	O
-.	O
-6	O
-s	O
--	O
-1	O
-(	O
-S4b	O
-Fig	O
-).	O
-	O
-However	O
-,	O
-two	O
-crystal	O
-forms	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-in	O
-the	O
-absence	O
-of	O
-the	O
-substrate	O
-were	O
-obtained	O
-.	O
-	O
-Overall	O
-fold	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-	O
-The	O
-β	O
--	O
-strands	O
-are	O
-numbered	O
-β1	O
--	O
-β11	O
-and	O
-the	O
-helices	O
-α	O
--	O
-I	O
-to	O
-α	O
--	O
-II	O
-.	O
-	O
-The	O
-entrance	O
-to	O
-the	O
-active	O
-site	O
-of	O
-the	O
-ectoine	O
-synthase	O
-is	O
-marked	O
-.	O
-	O
-(	O
-c	O
-)	O
-Overlay	O
-of	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-and	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structures	O
-.	O
-	O
-Hence	O
-,	O
-(	O
-Sa	O
-)	O
-EctC	O
-adopts	O
-an	O
-overall	O
-bowl	O
-shape	O
-in	O
-which	O
-one	O
-side	O
-is	O
-opened	O
-towards	O
-the	O
-solvent	O
-(	O
-Fig	O
-4a	O
-to	O
-4c	O
-).	O
-	O
-The	O
-formation	O
-of	O
-this	O
-α	O
--	O
-II	O
-helix	O
-induces	O
-a	O
-reorientation	O
-and	O
-shift	O
-of	O
-a	O
-long	O
-unstructured	O
-loop	O
-(	O
-as	O
-observed	O
-in	O
-the	O
-“	O
-open	O
-”	O
-structure	O
-)	O
-connecting	O
-β4	O
-and	O
-β6	O
-,	O
-resulting	O
-in	O
-the	O
-formation	O
-of	O
-the	O
-stable	O
-β	O
--	O
-strand	O
-β5	O
-as	O
-observed	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-state	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-Both	O
-the	O
-SEC	O
-analysis	O
-and	O
-the	O
-HPLC	O
--	O
-MALS	O
-experiments	O
-(	O
-S2b	O
-Fig	O
-)	O
-have	O
-shown	O
-that	O
-the	O
-ectoine	O
-synthase	O
-from	O
-S	O
-.	O
-alaskensis	O
-is	O
-a	O
-dimer	O
-in	O
-solution	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-this	O
-protein	O
-reflects	O
-this	O
-quaternary	O
-arrangement	O
-.	O
-	O
-As	O
-calculated	O
-with	O
-PDBePISA	O
-,	O
-the	O
-surface	O
-area	O
-buried	O
-upon	O
-dimer	O
-formation	O
-is	O
-1462	O
-Å2	O
-,	O
-which	O
-is	O
-20	O
-.	O
-5	O
-%	O
-of	O
-the	O
-total	O
-accessible	O
-surface	O
-of	O
-a	O
-monomer	O
-of	O
-this	O
-protein	O
-.	O
-	O
-In	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-,	O
-one	O
-monomer	O
-is	O
-present	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-We	O
-therefore	O
-inspected	O
-the	O
-crystal	O
-packing	O
-and	O
-analyzed	O
-the	O
-monomer	O
--	O
-monomer	O
-interactions	O
-with	O
-symmetry	O
-related	O
-molecules	O
-to	O
-elucidate	O
-whether	O
-a	O
-physiologically	O
-relevant	O
-dimer	O
-could	O
-be	O
-deduced	O
-from	O
-this	O
-crystal	O
-form	O
-as	O
-well	O
-.	O
-	O
-These	O
-additional	O
-amino	O
-acids	O
-fold	O
-into	O
-a	O
-small	O
-helix	O
-,	O
-which	O
-seals	O
-the	O
-open	O
-cavity	O
-of	O
-the	O
-cupin	O
--	O
-fold	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-(	O
-Fig	O
-4a	O
-).	O
-	O
-As	O
-a	O
-result	O
-,	O
-the	O
-newly	O
-formed	O
-β	O
--	O
-strand	O
-β5	O
-is	O
-reoriented	O
-and	O
-moved	O
-by	O
-2	O
-.	O
-4	O
-Å	O
-within	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-(	O
-Fig	O
-4a	O
-to	O
-4c	O
-).	O
-	O
-Therefore	O
-the	O
-sealing	O
-of	O
-the	O
-cupin	O
-fold	O
-,	O
-as	O
-described	O
-above	O
-,	O
-seem	O
-to	O
-have	O
-an	O
-indirect	O
-influence	O
-on	O
-the	O
-architecture	O
-of	O
-the	O
-postulated	O
-iron	O
--	O
-binding	O
-site	O
-.	O
-	O
-In	O
-the	O
-“	O
-open	O
-”	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-,	O
-this	O
-interaction	O
-does	O
-not	O
-occur	O
-since	O
-Glu	O
--	O
-115	O
-is	O
-rotated	O
-outwards	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-).	O
-	O
-Hence	O
-,	O
-one	O
-might	O
-speculate	O
-that	O
-this	O
-missing	O
-interaction	O
-might	O
-be	O
-responsible	O
-for	O
-the	O
-flexibility	O
-of	O
-the	O
-carboxy	O
--	O
-terminus	O
-in	O
-the	O
-“	O
-open	O
-”	O
-(	O
-Sa	O
-)	O
-EctC	O
-structure	O
-and	O
-consequently	O
-results	O
-in	O
-less	O
-well	O
-defined	O
-electron	O
-density	O
-in	O
-this	O
-region	O
-.	O
-	O
-These	O
-distances	O
-are	O
-to	O
-long	O
-when	O
-compared	O
-to	O
-other	O
-iron	O
-binding	O
-sites	O
-,	O
-a	O
-fact	O
-that	O
-might	O
-be	O
-caused	O
-by	O
-the	O
-absence	O
-of	O
-the	O
-proper	O
-substrate	O
-in	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-crystal	O
-structure	O
-.	O
-	O
-Since	O
-both	O
-the	O
-refinement	O
-and	O
-the	O
-distance	O
-did	O
-not	O
-clearly	O
-identify	O
-an	O
-iron	O
-molecule	O
-,	O
-we	O
-decided	O
-to	O
-conservatively	O
-place	O
-a	O
-water	O
-molecule	O
-at	O
-this	O
-position	O
-.	O
-	O
-Only	O
-His	O
--	O
-93	O
-is	O
-slightly	O
-rotated	O
-inwards	O
-in	O
-the	O
-“	O
-semi	O
--	O
-closed	O
-”	O
-structure	O
-,	O
-most	O
-likely	O
-due	O
-to	O
-formation	O
-of	O
-β	O
--	O
-strand	O
-β5	O
-as	O
-described	O
-above	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-this	O
-observations	O
-indicate	O
-,	O
-that	O
-the	O
-architecture	O
-of	O
-the	O
-presumptive	O
-iron	O
--	O
-binding	O
-site	O
-is	O
-pre	O
--	O
-set	O
-for	O
-the	O
-binding	O
-of	O
-the	O
-catalytically	O
-important	O
-metal	O
-by	O
-the	O
-ectoine	O
-synthase	O
-.	O
-	O
-This	O
-is	O
-in	O
-contrast	O
-to	O
-the	O
-high	O
--	O
-resolution	O
-“	O
-open	O
-”	O
-structure	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-where	O
-no	O
-additional	O
-electron	O
-density	O
-was	O
-observed	O
-after	O
-refinement	O
-.	O
-	O
-When	O
-analyzing	O
-the	O
-interactions	O
-of	O
-this	O
-compound	O
-within	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-,	O
-we	O
-found	O
-that	O
-it	O
-is	O
-bound	O
-via	O
-interactions	O
-with	O
-Trp	O
--	O
-21	O
-and	O
-Ser	O
--	O
-23	O
-of	O
-β	O
--	O
-sheet	O
-β3	O
-,	O
-Thr	O
--	O
-40	O
-located	O
-in	O
-β	O
--	O
-sheet	O
-β4	O
-,	O
-and	O
-Cys	O
--	O
-105	O
-and	O
-Phe	O
--	O
-107	O
-,	O
-which	O
-are	O
-both	O
-part	O
-of	O
-β	O
--	O
-sheet	O
-β11	O
-.	O
-	O
-As	O
-described	O
-above	O
-,	O
-the	O
-side	O
-chains	O
-of	O
-Glu	O
--	O
-57	O
-,	O
-Tyr	O
--	O
-85	O
-,	O
-and	O
-His	O
--	O
-93	O
-are	O
-probably	O
-involved	O
-in	O
-iron	O
-binding	O
-(	O
-Table	O
-1	O
-and	O
-Fig	O
-6a	O
-).	O
-	O
-However	O
-,	O
-the	O
-Cys	B-mutant
--	I-mutant
-105	I-mutant
-/	I-mutant
-Ala	I-mutant
-variant	O
-was	O
-practically	O
-catalytically	O
-inactive	O
-while	O
-largely	O
-maintaining	O
-its	O
-iron	O
-content	O
-(	O
-Table	O
-1	O
-).	O
-	O
-We	O
-observed	O
-two	O
-amino	O
-acid	O
-substitutions	O
-that	O
-simultaneously	O
-strongly	O
-affected	O
-enzyme	O
-activity	O
-and	O
-iron	O
-content	O
-;	O
-these	O
-were	O
-the	O
-Tyr	B-mutant
--	I-mutant
-52	I-mutant
-/	I-mutant
-Ala	I-mutant
-and	O
-the	O
-His	B-mutant
--	I-mutant
-55	I-mutant
-/	I-mutant
-Ala	I-mutant
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-variants	O
-(	O
-Table	O
-1	O
-).	O
-	O
-The	O
-carboxy	O
--	O
-terminal	O
-region	O
-of	O
-the	O
-(	O
-Sa	O
-)	O
-EctC	O
-protein	O
-is	O
-held	O
-in	O
-its	O
-position	O
-via	O
-an	O
-interaction	O
-of	O
-Glu	O
--	O
-115	O
-with	O
-His	O
--	O
-55	O
-,	O
-where	O
-His	O
--	O
-55	O
-in	O
-turn	O
-interacts	O
-with	O
-Pro	O
--	O
-110	O
-(	O
-Fig	O
-6a	O
-and	O
-6b	O
-).	O
-	O
-The	O
-Glu	B-mutant
--	I-mutant
-115	I-mutant
-/	I-mutant
-Ala	I-mutant
-mutant	O
-possessed	O
-wild	O
--	O
-type	O
-levels	O
-of	O
-iron	O
-,	O
-whereas	O
-the	O
-iron	O
-content	O
-of	O
-the	O
-His	B-mutant
--	I-mutant
-55	I-mutant
-/	I-mutant
-Ala	I-mutant
-substitutions	O
-dropped	O
-to	O
-15	O
-%	O
-of	O
-the	O
-wild	O
--	O
-type	O
-level	O
-(	O
-Table	O
-1	O
-).	O
-	O
-As	O
-a	O
-consequence	O
-of	O
-the	O
-structural	O
-relatedness	O
-of	O
-EctC	O
-and	O
-RemF	O
-and	O
-the	O
-type	O
-of	O
-chemical	O
-reaction	O
-these	O
-two	O
-enzymes	O
-catalyze	O
-,	O
-is	O
-now	O
-understandable	O
-why	O
-bona	O
-fide	O
-EctC	O
--	O
-type	O
-proteins	O
-are	O
-frequently	O
-(	O
-mis	O
-)-	O
-annotated	O
-in	O
-microbial	O
-genome	O
-sequences	O
-as	O
-“	O
-RemF	O
--	O
-like	O
-”	O
-proteins	O
-.	O
-	O
-Except	O
-for	O
-some	O
-cupin	O
--	O
-related	O
-proteins	O
-that	O
-seem	O
-to	O
-function	O
-as	O
-metallo	O
--	O
-chaperones	O
-,	O
-the	O
-bound	O
-metal	O
-is	O
-typically	O
-an	O
-essential	O
-part	O
-of	O
-the	O
-active	O
-sites	O
-.	O
-	O
-The	O
-architecture	O
-of	O
-the	O
-metal	O
-center	O
-of	O
-ectoine	O
-synthase	O
-seems	O
-to	O
-be	O
-subjected	O
-to	O
-considerable	O
-evolutionary	O
-constraints	O
-.	O
-	O
-This	O
-set	O
-of	O
-data	O
-and	O
-the	O
-fact	O
-that	O
-the	O
-targeted	O
-residues	O
-are	O
-strongly	O
-conserved	O
-among	O
-EctC	O
--	O
-type	O
-proteins	O
-(	O
-Fig	O
-2	O
-)	O
-is	O
-consistent	O
-with	O
-their	O
-potential	O
-role	O
-in	O
-N	O
--	O
-γ	O
--	O
-ADABA	O
-binding	O
-or	O
-enzyme	O
-catalysis	O
-.	O
-	O
-Because	O
-microbial	O
-ectoine	O
-producers	O
-can	O
-colonize	O
-ecological	O
-niches	O
-with	O
-rather	O
-different	O
-physicochemical	O
-attributes	O
-,	O
-it	O
-seems	O
-promising	O
-to	O
-exploit	O
-this	O
-considerable	O
-biodiversity	O
-to	O
-identify	O
-EctC	O
-proteins	O
-with	O
-enhanced	O
-protein	O
-stability	O
-.	O
-	O
-Structural	O
-basis	O
-for	O
-the	O
-regulation	O
-of	O
-enzymatic	O
-activity	O
-of	O
-Regnase	O
--	O
-1	O
-by	O
-domain	O
--	O
-domain	O
-interactions	O
-	O
-Domain	O
-structures	O
-of	O
-Regnase	O
--	O
-1	O
-	O
-The	O
-domain	O
-structures	O
-of	O
-NTD	O
-,	O
-ZF	O
-,	O
-and	O
-CTD	O
-were	O
-determined	O
-by	O
-NMR	O
-(	O
-Fig	O
-.	O
-1b	O
-,	O
-d	O
-,	O
-e	O
-).	O
-	O
-Based	O
-on	O
-the	O
-decrease	O
-in	O
-the	O
-free	O
-RNA	O
-fluorescence	O
-band	O
-,	O
-we	O
-evaluated	O
-the	O
-contribution	O
-of	O
-each	O
-domain	O
-of	O
-Regnase	O
--	O
-1	O
-to	O
-RNA	O
-binding	O
-.	O
-	O
-Contribution	O
-of	O
-each	O
-domain	O
-of	O
-Regnase	O
--	O
-1	O
-to	O
-RNase	O
-activity	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-NTD	B-mutant
--	I-mutant
-PIN	I-mutant
-(	I-mutant
-DDNN	I-mutant
-)-	I-mutant
-ZF	I-mutant
-,	O
-which	O
-possesses	O
-the	O
-NTD	O
-but	O
-lacks	O
-the	O
-catalytic	O
-residues	O
-in	O
-PIN	O
-,	O
-completely	O
-lost	O
-all	O
-RNase	O
-activity	O
-(	O
-Fig	O
-.	O
-1g	O
-,	O
-right	O
-panel	O
-),	O
-as	O
-expected	O
-,	O
-confirming	O
-that	O
-the	O
-RNase	O
-catalytic	O
-center	O
-is	O
-located	O
-in	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-By	O
-comparison	O
-with	O
-the	O
-elution	O
-volume	O
-of	O
-standard	O
-marker	O
-proteins	O
-,	O
-the	O
-PIN	O
-domain	O
-was	O
-assumed	O
-to	O
-be	O
-in	O
-equilibrium	O
-between	O
-a	O
-monomer	O
-and	O
-a	O
-dimer	O
-in	O
-solution	O
-at	O
-concentrations	O
-in	O
-the	O
-20	O
-–	O
-200	O
-μM	O
-range	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-PIN	O
-domain	O
-has	O
-been	O
-determined	O
-in	O
-three	O
-distinct	O
-crystal	O
-forms	O
-with	O
-a	O
-space	O
-group	O
-of	O
-P3121	O
-(	O
-form	O
-I	O
-in	O
-this	O
-study	O
-and	O
-PDB	O
-ID	O
-3V33	O
-),	O
-P3221	O
-(	O
-form	O
-II	O
-in	O
-this	O
-study	O
-),	O
-and	O
-P41	O
-(	O
-PDB	O
-ID	O
-3V32	O
-and	O
-3V34	O
-),	O
-respectively	O
-.	O
-	O
-Mutation	O
-of	O
-Arg215	O
-,	O
-whose	O
-side	O
-chain	O
-faces	O
-to	O
-the	O
-opposite	O
-side	O
-of	O
-the	O
-oligomeric	O
-surface	O
-,	O
-to	O
-Glu	O
-preserved	O
-the	O
-monomer	O
-/	O
-dimer	O
-equilibrium	O
-,	O
-similar	O
-to	O
-the	O
-wild	O
-type	O
-.	O
-	O
-Therefore	O
-,	O
-we	O
-concluded	O
-that	O
-head	O
--	O
-to	O
--	O
-tail	O
-PIN	O
-dimerization	O
-,	O
-together	O
-with	O
-the	O
-NTD	O
-,	O
-are	O
-required	O
-for	O
-Regnase	O
--	O
-1	O
-RNase	O
-activity	O
-in	O
-vitro	O
-.	O
-	O
-Likewise	O
-,	O
-upon	O
-addition	O
-of	O
-the	O
-PIN	O
-domain	O
-,	O
-NMR	O
-signals	O
-derived	O
-from	O
-R56	O
-,	O
-L58	O
--	O
-G59	O
-,	O
-and	O
-V86	O
--	O
-H88	O
-in	O
-the	O
-NTD	O
-exhibited	O
-large	O
-chemical	O
-shift	O
-changes	O
-and	O
-residues	O
-D53	O
-,	O
-F55	O
-,	O
-K57	O
-,	O
-Y60	O
--	O
-S61	O
-,	O
-V68	O
-,	O
-T80	O
--	O
-G83	O
-,	O
-L85	O
-,	O
-and	O
-G89	O
-of	O
-the	O
-NTD	O
-as	O
-well	O
-as	O
-side	O
-chain	O
-amide	O
-signals	O
-of	O
-N79	O
-exhibited	O
-small	O
-but	O
-appreciable	O
-chemical	O
-shift	O
-changes	O
-(	O
-Fig	O
-.	O
-3b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-5	O
-).	O
-	O
-The	O
-importance	O
-of	O
-residues	O
-W182	O
-and	O
-R183	O
-can	O
-readily	O
-be	O
-understood	O
-in	O
-terms	O
-of	O
-the	O
-monomeric	O
-PIN	O
-structure	O
-as	O
-they	O
-are	O
-located	O
-near	O
-to	O
-the	O
-RNase	O
-catalytic	O
-site	O
-;	O
-however	O
-,	O
-the	O
-importance	O
-of	O
-residue	O
-K184	O
-,	O
-which	O
-points	O
-away	O
-from	O
-the	O
-active	O
-site	O
-is	O
-more	O
-easily	O
-rationalized	O
-in	O
-terms	O
-of	O
-the	O
-oligomeric	O
-structure	O
-,	O
-in	O
-which	O
-the	O
-“	O
-secondary	O
-”	O
-chain	O
-’	O
-s	O
-residue	O
-K184	O
-is	O
-positioned	O
-near	O
-the	O
-“	O
-primary	O
-”	O
-chain	O
-’	O
-s	O
-catalytic	O
-site	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-the	O
-putative	O
--	O
-RNA	O
-binding	O
-residues	O
-K184	O
-and	O
-R214	O
-are	O
-unique	O
-to	O
-Regnase	O
--	O
-1	O
-among	O
-PIN	O
-domains	O
-.	O
-	O
-Molecular	O
-mechanism	O
-of	O
-target	O
-mRNA	O
-cleavage	O
-by	O
-the	O
-PIN	O
-dimer	O
-	O
-Our	O
-mutational	O
-experiments	O
-indicated	O
-that	O
-the	O
-observed	O
-dimer	O
-is	O
-functional	O
-and	O
-that	O
-the	O
-role	O
-of	O
-the	O
-secondary	O
-PIN	O
-domain	O
-is	O
-to	O
-position	O
-Regnase	O
--	O
-1	O
--	O
-unique	O
-RNA	O
-binding	O
-residues	O
-near	O
-the	O
-active	O
-site	O
-of	O
-the	O
-primary	O
-PIN	O
-domain	O
-.	O
-	O
-We	O
-determined	O
-the	O
-individual	O
-domain	O
-structures	O
-of	O
-Regnase	O
--	O
-1	O
-by	O
-NMR	O
-and	O
-X	O
--	O
-ray	O
-crystallography	O
-.	O
-	O
-Both	O
-the	O
-mouse	O
-and	O
-human	O
-PIN	O
-domains	O
-form	O
-head	O
--	O
-to	O
--	O
-tail	O
-oligomers	O
-in	O
-three	O
-distinct	O
-crystal	O
-forms	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-our	O
-gel	O
-filtration	O
-data	O
-,	O
-mutational	O
-analyses	O
-,	O
-and	O
-NMR	O
-spectra	O
-all	O
-indicate	O
-that	O
-the	O
-PIN	O
-domain	O
-forms	O
-a	O
-head	O
--	O
-to	O
--	O
-tail	O
-dimer	O
-in	O
-solution	O
-in	O
-a	O
-manner	O
-similar	O
-to	O
-the	O
-crystal	O
-structure	O
-.	O
-	O
-Taken	O
-together	O
-,	O
-this	O
-suggests	O
-that	O
-the	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-compete	O
-for	O
-a	O
-common	O
-binding	O
-site	O
-.	O
-	O
-While	O
-further	O
-investigations	O
-on	O
-the	O
-domain	O
--	O
-domain	O
-interactions	O
-of	O
-Regnase	O
--	O
-1	O
-in	O
-vivo	O
-are	O
-necessary	O
-,	O
-these	O
-intramolecular	O
-and	O
-intermolecular	O
-domain	O
-interactions	O
-of	O
-Regnase	O
--	O
-1	O
-appear	O
-to	O
-structurally	O
-constrain	O
-Regnase	O
--	O
-1activity	O
-,	O
-which	O
-,	O
-in	O
-turn	O
-,	O
-enables	O
-tight	O
-regulation	O
-of	O
-immune	O
-responses	O
-.	O
-	O
-The	O
-percentage	O
-of	O
-the	O
-bound	O
-IL	O
--	O
-6	O
-was	O
-calculated	O
-based	O
-on	O
-the	O
-fluorescence	O
-intensities	O
-of	O
-the	O
-free	O
-IL	O
--	O
-6	O
-quantified	O
-in	O
-(	O
-f	O
-).	O
-	O
-(	O
-b	O
-)	O
-Dimer	O
-structure	O
-of	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-Two	O
-PIN	O
-molecules	O
-in	O
-the	O
-crystal	O
-were	O
-colored	O
-white	O
-and	O
-green	O
-,	O
-respectively	O
-.	O
-	O
-(	O
-a	O
-)	O
-NMR	O
-analyses	O
-of	O
-the	O
-NTD	O
--	O
-binding	O
-to	O
-the	O
-PIN	O
-domain	O
-.	O
-	O
-The	O
-residues	O
-with	O
-significant	O
-chemical	O
-shift	O
-changes	O
-were	O
-labeled	O
-in	O
-the	O
-overlaid	O
-spectra	O
-(	O
-left	O
-)	O
-and	O
-colored	O
-red	O
-on	O
-the	O
-surface	O
-and	O
-ribbon	O
-structure	O
-of	O
-the	O
-PIN	O
-domain	O
-(	O
-right	O
-).	O
-	O
-The	O
-NTD	O
-and	O
-the	O
-PIN	O
-domain	O
-are	O
-shown	O
-in	O
-cyan	O
-and	O
-white	O
-,	O
-respectively	O
-.	O
-	O
-Catalytic	O
-residues	O
-of	O
-the	O
-PIN	O
-domain	O
-are	O
-shown	O
-in	O
-sticks	O
-,	O
-and	O
-the	O
-residues	O
-that	O
-exhibited	O
-significant	O
-chemical	O
-shift	O
-changes	O
-in	O
-(	O
-a	O
-,	O
-b	O
-)	O
-were	O
-labeled	O
-.	O
-	O
-(	O
-b	O
-)	O
-In	O
-vitro	O
-cleavage	O
-assay	O
-of	O
-basic	O
-residue	O
-mutants	O
-for	O
-Regnase	O
--	O
-1	O
-mRNA	O
-.	O
-	O
-The	O
-mutations	O
-whose	O
-RNase	O
-activities	O
-were	O
-not	O
-increased	O
-in	O
-the	O
-presence	O
-of	O
-DDNN	B-mutant
-mutant	O
-were	O
-colored	O
-in	O
-blue	O
-on	O
-the	O
-primary	O
-PIN	O
-.	O
-	O
-In	O
-the	O
-MEROPS	O
-peptidase	O
-database	O
-,	O
-clan	O
-CD	O
-contains	O
-groups	O
-(	O
-or	O
-families	O
-)	O
-of	O
-cysteine	O
-peptidases	O
-that	O
-share	O
-some	O
-highly	O
-conserved	O
-structural	O
-elements	O
-.	O
-	O
-The	O
-structure	O
-was	O
-analyzed	O
-,	O
-and	O
-the	O
-enzyme	O
-was	O
-biochemically	O
-characterized	O
-to	O
-provide	O
-the	O
-first	O
-structure	O
-/	O
-function	O
-correlation	O
-for	O
-a	O
-C11	O
-peptidase	O
-.	O
-	O
-The	O
-crystal	O
-structure	O
-of	O
-the	O
-catalytically	O
-active	O
-form	O
-of	O
-PmC11	O
-revealed	O
-an	O
-extended	O
-caspase	O
--	O
-like	O
-α	O
-/	O
-β	O
-/	O
-α	O
-sandwich	O
-architecture	O
-comprised	O
-of	O
-a	O
-central	O
-nine	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-,	O
-with	O
-an	O
-unusual	O
-C	O
--	O
-terminal	O
-domain	O
-(	O
-CTD	O
-),	O
-starting	O
-at	O
-Lys250	O
-.	O
-	O
-The	O
-structure	O
-also	O
-includes	O
-two	O
-short	O
-β	O
--	O
-hairpins	O
-(	O
-βA	O
-–	O
-βB	O
-and	O
-βD	O
-–	O
-βE	O
-)	O
-and	O
-a	O
-small	O
-β	O
--	O
-sheet	O
-(	O
-βC	O
-–	O
-βF	O
-),	O
-which	O
-is	O
-formed	O
-from	O
-two	O
-distinct	O
-regions	O
-of	O
-the	O
-sequence	O
-(	O
-βC	O
-precedes	O
-α11	O
-,	O
-α12	O
-and	O
-β9	O
-,	O
-whereas	O
-βF	O
-follows	O
-the	O
-βD	O
--	O
-βE	O
-hairpin	O
-)	O
-in	O
-the	O
-middle	O
-of	O
-the	O
-CTD	O
-(	O
-Fig	O
-.	O
-1B	O
-).	O
-	O
-His133	O
-and	O
-Cys179	O
-were	O
-found	O
-at	O
-locations	O
-structurally	O
-homologous	O
-to	O
-the	O
-caspase	O
-catalytic	O
-dyad	O
-,	O
-and	O
-other	O
-clan	O
-CD	O
-structures	O
-,	O
-at	O
-the	O
-C	O
-termini	O
-of	O
-strands	O
-β5	O
-and	O
-β6	O
-,	O
-respectively	O
-(	O
-Figs	O
-.	O
-1	O
-,	O
-A	O
-and	O
-B	O
-,	O
-and	O
-2A	O
-).	O
-	O
-A	O
-multiple	O
-sequence	O
-alignment	O
-of	O
-C11	O
-proteins	O
-revealed	O
-that	O
-these	O
-residues	O
-are	O
-highly	O
-conserved	O
-(	O
-data	O
-not	O
-shown	O
-).	O
-	O
-B	O
-,	O
-size	O
-exclusion	O
-chromatography	O
-of	O
-PmC11	O
-.	O
-	O
-Incubation	O
-of	O
-PmC11	O
-at	O
-37	O
-°	O
-C	O
-for	O
-16	O
-h	O
-,	O
-resulted	O
-in	O
-a	O
-fully	O
-processed	O
-enzyme	O
-that	O
-remained	O
-as	O
-an	O
-intact	O
-monomer	O
-when	O
-applied	O
-to	O
-a	O
-size	O
--	O
-exclusion	O
-column	O
-(	O
-Fig	O
-.	O
-2B	O
-).	O
-	O
-As	O
-expected	O
-,	O
-PmC11	O
-showed	O
-no	O
-activity	O
-against	O
-substrates	O
-with	O
-Pro	O
-or	O
-Asp	O
-in	O
-P1	O
-but	O
-was	O
-active	O
-toward	O
-substrates	O
-with	O
-a	O
-basic	O
-residue	O
-in	O
-P1	O
-such	O
-as	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-,	O
-Z	O
--	O
-GGR	O
--	O
-AMC	O
-,	O
-and	O
-BOC	O
--	O
-VLK	O
--	O
-AMC	O
-.	O
-	O
-The	O
-rate	O
-of	O
-cleavage	O
-was	O
-∼	O
-3	O
--	O
-fold	O
-greater	O
-toward	O
-the	O
-single	O
-Arg	O
-substrate	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-than	O
-for	O
-the	O
-other	O
-two	O
-(	O
-Fig	O
-.	O
-2F	O
-)	O
-and	O
-,	O
-unexpectedly	O
-,	O
-PmC11	O
-showed	O
-no	O
-activity	O
-toward	O
-BOC	O
--	O
-K	O
--	O
-AMC	O
-.	O
-	O
-These	O
-results	O
-confirm	O
-that	O
-PmC11	O
-accepts	O
-substrates	O
-containing	O
-Arg	O
-or	O
-Lys	O
-in	O
-P1	O
-with	O
-a	O
-possible	O
-preference	O
-for	O
-Arg	O
-.	O
-	O
-Because	O
-PmC11	O
-recognizes	O
-basic	O
-substrates	O
-,	O
-the	O
-tetrapeptide	O
-inhibitor	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-was	O
-tested	O
-as	O
-an	O
-enzyme	O
-inhibitor	O
-and	O
-was	O
-found	O
-to	O
-inhibit	O
-both	O
-the	O
-autoprocessing	O
-and	O
-activity	O
-of	O
-PmC11	O
-(	O
-Fig	O
-.	O
-3A	O
-).	O
-	O
-In	O
-the	O
-structure	O
-of	O
-PmC11	O
-,	O
-Asp207	O
-resides	O
-on	O
-a	O
-flexible	O
-loop	O
-pointing	O
-away	O
-from	O
-the	O
-S1	O
-binding	O
-pocket	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-The	O
-position	O
-and	O
-orientation	O
-of	O
-Z	O
--	O
-VRPR	O
--	O
-FMK	O
-was	O
-taken	O
-from	O
-superposition	O
-of	O
-the	O
-PmC11	O
-and	O
-MALTI_P	O
-structures	O
-and	O
-indicates	O
-the	O
-presumed	O
-active	O
-site	O
-of	O
-PmC11	O
-.	O
-	O
-C	O
-,	O
-divalent	O
-cations	O
-do	O
-not	O
-increase	O
-the	O
-activity	O
-of	O
-PmC11	O
-.	O
-	O
-The	O
-cleavage	O
-of	O
-Bz	O
--	O
-R	O
--	O
-AMC	O
-by	O
-PmC11	O
-was	O
-measured	O
-in	O
-the	O
-presence	O
-of	O
-the	O
-cations	O
-Ca2	O
-+,	O
-Mn2	O
-+,	O
-Zn2	O
-+,	O
-Co2	O
-+,	O
-Cu2	O
-+,	O
-Mg2	O
-+,	O
-and	O
-Fe3	O
-+	O
-with	O
-EGTA	O
-as	O
-a	O
-negative	O
-control	O
-,	O
-and	O
-relative	O
-fluorescence	O
-measured	O
-against	O
-time	O
-(	O
-min	O
-).	O
-	O
-The	O
-addition	O
-of	O
-cations	O
-produced	O
-no	O
-improvement	O
-in	O
-activity	O
-of	O
-PmC11	O
-when	O
-compared	O
-in	O
-the	O
-presence	O
-of	O
-EGTA	O
-,	O
-suggesting	O
-that	O
-PmC11	O
-does	O
-not	O
-require	O
-metal	O
-ions	O
-for	O
-proteolytic	O
-activity	O
-.	O
-	O
-Several	O
-other	O
-members	O
-of	O
-clan	O
-CD	O
-require	O
-processing	O
-for	O
-full	O
-activation	O
-including	O
-legumain	O
-,	O
-gingipain	O
--	O
-R	O
-,	O
-MARTX	O
--	O
-CPD	O
-,	O
-and	O
-the	O
-effector	O
-caspases	O
-,	O
-e	O
-.	O
-g	O
-.	O
-caspase	O
--	O
-7	O
-.	O
-	O
-The	O
-caspases	O
-and	O
-gingipain	O
--	O
-R	O
-both	O
-undergo	O
-intermolecular	O
-(	O
-trans	O
-)	O
-cleavage	O
-and	O
-legumain	O
-and	O
-MARTX	O
--	O
-CPD	O
-are	O
-reported	O
-to	O
-perform	O
-intramolecular	O
-(	O
-cis	O
-)	O
-cleavage	O
-.	O
-	O
-The	O
-PmC11	O
-structure	O
-should	O
-provide	O
-a	O
-good	O
-basis	O
-for	O
-structural	O
-modeling	O
-and	O
-,	O
-given	O
-the	O
-importance	O
-of	O
-other	O
-clan	O
-CD	O
-enzymes	O
-,	O
-this	O
-work	O
-should	O
-also	O
-advance	O
-the	O
-exploration	O
-of	O
-these	O
-peptidases	O
-and	O
-potentially	O
-identify	O
-new	O
-biologically	O
-important	O
-substrates	O
-.	O
-	O
-The	O
-chemically	O
-most	O
-complex	O
-modification	O
-in	O
-eukaryotic	O
-rRNA	O
-is	O
-the	O
-conserved	O
-hypermodified	O
-nucleotide	O
-N1	O
--	O
-methyl	O
--	O
-N3	O
--	O
-aminocarboxypropyl	O
--	O
-pseudouridine	O
-(	O
-m1acp3Ψ	O
-)	O
-located	O
-next	O
-to	O
-the	O
-P	O
--	O
-site	O
-tRNA	O
-on	O
-the	O
-small	O
-subunit	O
-18S	O
-rRNA	O
-.	O
-	O
-While	O
-S	O
--	O
-adenosylmethionine	O
-was	O
-identified	O
-as	O
-the	O
-source	O
-of	O
-the	O
-aminocarboxypropyl	O
-(	O
-acp	O
-)	O
-group	O
-more	O
-than	O
-40	O
-years	O
-ago	O
-the	O
-enzyme	O
-catalyzing	O
-the	O
-acp	O
-transfer	O
-remained	O
-elusive	O
-.	O
-	O
-In	O
-Saccharomyces	O
-cerevisiae	O
-18S	O
-rRNA	O
-contains	O
-four	O
-base	O
-methylations	O
-,	O
-two	O
-acetylations	O
-and	O
-a	O
-single	O
-3	O
--	O
-amino	O
--	O
-3	O
--	O
-carboxypropyl	O
-(	O
-acp	O
-)	O
-modification	O
-,	O
-whereas	O
-six	O
-base	O
-methylations	O
-are	O
-present	O
-in	O
-the	O
-25S	O
-rRNA	O
-.	O
-	O
-While	O
-in	O
-humans	O
-the	O
-18S	O
-rRNA	O
-base	O
-modifications	O
-are	O
-highly	O
-conserved	O
-,	O
-only	O
-three	O
-of	O
-the	O
-yeast	O
-base	O
-modifications	O
-catalyzed	O
-by	O
-ScRrp8	O
-/	O
-HsNML	O
-,	O
-ScRcm1	O
-/	O
-HsNSUN5	O
-and	O
-ScNop2	O
-/	O
-HsNSUN1	O
-are	O
-preserved	O
-in	O
-the	O
-corresponding	O
-human	O
-28S	O
-rRNA	O
-.	O
-	O
-They	O
-might	O
-contribute	O
-to	O
-increased	O
-RNA	O
-stability	O
-by	O
-providing	O
-additional	O
-hydrogen	O
-bonds	O
-(	O
-pseudouridines	O
-),	O
-improved	O
-base	O
-stacking	O
-(	O
-pseudouridines	O
-and	O
-base	O
-methylations	O
-)	O
-or	O
-an	O
-increased	O
-resistance	O
-against	O
-hydrolysis	O
-(	O
-ribose	O
-methylations	O
-).	O
-	O
-Defects	O
-of	O
-rRNA	O
-modification	O
-enzymes	O
-often	O
-lead	O
-to	O
-disturbed	O
-ribosome	O
-biogenesis	O
-or	O
-functionally	O
-impaired	O
-ribosomes	O
-,	O
-although	O
-the	O
-lack	O
-of	O
-individual	O
-rRNA	O
-modifications	O
-often	O
-has	O
-no	O
-or	O
-only	O
-a	O
-slight	O
-influence	O
-on	O
-the	O
-cell	O
-.	O
-	O
-The	O
-asterisk	O
-indicates	O
-the	O
-C1	O
--	O
-atom	O
-labeled	O
-in	O
-the	O
-14C	O
--	O
-incorporation	O
-assay	O
-.	O
-	O
-(	O
-C	O
-)	O
-14C	O
--	O
-acp	O
-labeling	O
-of	O
-18S	O
-rRNAs	O
-.	O
-	O
-The	O
-primer	O
-extension	O
-arrest	O
-is	O
-reduced	O
-in	O
-HTC116	O
-cells	O
-transfected	O
-with	O
-siRNAs	O
-544	O
-and	O
-545	O
-.	O
-	O
-As	O
-previously	O
-reported	O
-this	O
-shoulder	O
-was	O
-identified	O
-by	O
-ESI	O
--	O
-MS	O
-as	O
-corresponding	O
-to	O
-m1acp3Ψ	O
-.	O
-	O
-Whereas	O
-the	O
-acp	O
-labeling	O
-of	O
-18S	O
-rRNA	O
-was	O
-clearly	O
-present	O
-in	O
-the	O
-wild	O
-type	O
-strain	O
-no	O
-radioactive	O
-labeling	O
-could	O
-be	O
-observed	O
-in	O
-a	O
-Δtsr3	B-mutant
-strain	O
-(	O
-Figure	O
-1C	O
-).	O
-	O
-Human	O
-18S	O
-rRNA	O
-has	O
-also	O
-been	O
-shown	O
-to	O
-contain	O
-m1acp3Ψ	O
-in	O
-the	O
-18S	O
-rRNA	O
-at	O
-position	O
-1248	O
-.	O
-	O
-By	O
-comparison	O
-,	O
-treating	O
-cells	O
-with	O
-siRNA	O
-545	O
-,	O
-which	O
-only	O
-reduced	O
-the	O
-TSR3	O
-mRNA	O
-to	O
-20	O
-%,	O
-did	O
-not	O
-markedly	O
-reduced	O
-the	O
-acp	O
-signal	O
-.	O
-	O
-The	O
-TSR3	O
-gene	O
-was	O
-genetically	O
-modified	O
-at	O
-its	O
-native	O
-locus	O
-,	O
-resulting	O
-in	O
-a	O
-C	O
--	O
-terminal	O
-fusion	O
-of	O
-Tsr3	O
-with	O
-a	O
-3xHA	O
-epitope	O
-expressed	O
-by	O
-the	O
-native	O
-promotor	O
-in	O
-yeast	O
-strain	O
-CEN	O
-.	O
-BM258	O
--	O
-5B	O
-.	O
-	O
-In	O
-accordance	O
-with	O
-the	O
-synthetic	O
-sick	O
-growth	O
-phenotype	O
-the	O
-paromomycin	O
-and	O
-hygromycin	O
-B	O
-hypersensitivity	O
-further	O
-increased	O
-in	O
-a	O
-Δtsr3	B-mutant
-Δsnr35	I-mutant
-recombination	O
-strain	O
-(	O
-Figure	O
-2B	O
-).	O
-	O
-Domain	O
-characterization	O
-of	O
-yeast	O
-Tsr3	O
-and	O
-correlation	O
-of	O
-acp	O
-modification	O
-with	O
-late	O
-18S	O
-rRNA	O
-processing	O
-steps	O
-.	O
-(	O
-A	O
-)	O
-Scheme	O
-of	O
-the	O
-TSR3	O
-gene	O
-with	O
-truncation	O
-positions	O
-in	O
-the	O
-open	O
-reading	O
-frame	O
-.	O
-	O
-The	O
-loop	O
-connecting	O
-β2	O
-and	O
-β3	O
-contains	O
-a	O
-single	O
-turn	O
-of	O
-a	O
-310	O
--	O
-helix	O
-.	O
-Helices	O
-α1	O
-and	O
-α2	O
-are	O
-located	O
-on	O
-one	O
-side	O
-of	O
-the	O
-five	O
--	O
-stranded	O
-β	O
--	O
-sheet	O
-while	O
-α3	O
-packs	O
-against	O
-the	O
-opposite	O
-β	O
--	O
-sheet	O
-surface	O
-.	O
-	O
-The	O
-bound	O
-S	O
--	O
-adenosylmethionine	O
-is	O
-shown	O
-in	O
-a	O
-stick	O
-representation	O
-and	O
-colored	O
-by	O
-atom	O
-type	O
-.	O
-	O
-The	O
-color	O
-coding	O
-is	O
-the	O
-same	O
-as	O
-in	O
-(	O
-A	O
-).	O
-(	O
-C	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-X	O
--	O
-ray	O
-structures	O
-of	O
-VdTsr3	O
-in	O
-the	O
-SAM	O
--	O
-bound	O
-state	O
-(	O
-red	O
-)	O
-and	O
-SsTsr3	O
-(	O
-blue	O
-)	O
-in	O
-the	O
-apo	O
-state	O
-.	O
-	O
-In	O
-comparison	O
-to	O
-Tsr3	O
-the	O
-central	O
-β	O
--	O
-sheet	O
-element	O
-of	O
-Trm10	O
-is	O
-extended	O
-by	O
-one	O
-additional	O
-β	O
--	O
-strand	O
-pairing	O
-to	O
-β2	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-trefoil	O
-knot	O
-of	O
-Trm10	O
-is	O
-not	O
-as	O
-deep	O
-as	O
-that	O
-of	O
-Tsr3	O
-(	O
-Figure	O
-4D	O
-).	O
-	O
-W73	O
-is	O
-highly	O
-conserved	O
-in	O
-all	O
-known	O
-Tsr3	O
-proteins	O
-,	O
-whereas	O
-A76	O
-can	O
-be	O
-replaced	O
-by	O
-other	O
-hydrophobic	O
-amino	O
-acids	O
-.	O
-	O
-(	O
-A	O
-)	O
-Close	O
--	O
-up	O
-view	O
-of	O
-the	O
-SAM	O
--	O
-binding	O
-pocket	O
-of	O
-VdTsr3	O
-.	O
-	O
-Bound	O
-SAM	O
-was	O
-modelled	O
-based	O
-on	O
-the	O
-X	O
--	O
-ray	O
-structure	O
-of	O
-the	O
-Trm10	O
-/	O
-SAH	O
--	O
-complex	O
-(	O
-pdb4jwf	O
-).	O
-	O
-A	O
-red	O
-arrow	O
-indicates	O
-the	O
-SAM	O
-methyl	O
-group	O
-.	O
-(	O
-D	O
-)	O
-Binding	O
-of	O
-SAM	O
-analogs	O
-to	O
-SsTsr3	O
-.	O
-	O
-SsTsr3	O
-bound	O
-SAM	O
-with	O
-a	O
-KD	O
-of	O
-6	O
-.	O
-5	O
-μM	O
-,	O
-which	O
-is	O
-similar	O
-to	O
-SAM	O
--	O
-KD	O
-'	O
-s	O
-reported	O
-for	O
-several	O
-SPOUT	O
--	O
-class	O
-methyltransferases	O
-.	O
-	O
-5	O
-′-	O
-methylthioadenosin	O
-—	O
-the	O
-reaction	O
-product	O
-after	O
-the	O
-acp	O
--	O
-transfer	O
-—	O
-binds	O
-only	O
-∼	O
-2	O
-.	O
-5	O
--	O
-fold	O
-weaker	O
-(	O
-KD	O
-=	O
-16	O
-.	O
-7	O
-μM	O
-)	O
-compared	O
-to	O
-SAM	O
-.	O
-	O
-Mutations	O
-of	O
-the	O
-corresponding	O
-residue	O
-in	O
-SsTsr3	O
-to	O
-A	O
-(	O
-D63	O
-)	O
-does	O
-not	O
-significantly	O
-alter	O
-the	O
-SAM	O
--	O
-binding	O
-affinity	O
-of	O
-the	O
-protein	O
-(	O
-KD	O
-=	O
-11	O
-.	O
-0	O
-μM	O
-).	O
-	O
-Analysis	O
-of	O
-the	O
-electrostatic	O
-surface	O
-properties	O
-of	O
-VdTsr3	O
-clearly	O
-identified	O
-positively	O
-charged	O
-surface	O
-patches	O
-in	O
-the	O
-vicinity	O
-of	O
-the	O
-SAM	O
--	O
-binding	O
-site	O
-suggesting	O
-a	O
-putative	O
-RNA	O
--	O
-binding	O
-site	O
-(	O
-Figure	O
-6A	O
-).	O
-	O
-Its	O
-negatively	O
-charged	O
-sulfate	O
-group	O
-might	O
-mimic	O
-an	O
-RNA	O
-backbone	O
-phosphate	O
-.	O
-	O
-In	O
-order	O
-to	O
-explore	O
-the	O
-RNA	O
--	O
-ligand	O
-specificity	O
-of	O
-Tsr3	O
-we	O
-titrated	O
-SsTsr3	O
-prepared	O
-in	O
-RNase	O
--	O
-free	O
-form	O
-with	O
-5	O
-′-	O
-fluoresceine	O
--	O
-labeled	O
-RNA	O
-and	O
-determined	O
-the	O
-affinity	O
-by	O
-fluorescence	O
-anisotropy	O
-measurements	O
-.	O
-	O
-A	O
-single	O
-stranded	O
-oligoU	O
--	O
-RNA	O
-bound	O
-with	O
-a	O
-10	O
--	O
-fold	O
--	O
-reduced	O
-affinity	O
-(	O
-6	O
-.	O
-0	O
-μM	O
-).	O
-	O
-This	O
-makes	O
-it	O
-unique	O
-in	O
-eukaryotic	O
-rRNA	O
-modification	O
-.	O
-	O
-A	O
-similar	O
-modification	O
-(	O
-acp3U	O
-)	O
-was	O
-identified	O
-in	O
-Haloferax	O
-volcanii	O
-and	O
-corresponding	O
-modified	O
-nucleotides	O
-were	O
-also	O
-shown	O
-to	O
-occur	O
-in	O
-other	O
-archaea	O
-.	O
-	O
-This	O
-demonstrates	O
-that	O
-,	O
-unlike	O
-the	O
-other	O
-small	O
-subunit	O
-rRNA	O
-base	O
-modifications	O
-,	O
-the	O
-acp	O
-modification	O
-is	O
-required	O
-for	O
-efficient	O
-pre	O
--	O
-rRNA	O
-processing	O
-.	O
-	O
-After	O
-structural	O
-changes	O
-,	O
-possibly	O
-driven	O
-by	O
-GTP	O
-hydrolysis	O
-,	O
-which	O
-go	O
-together	O
-with	O
-the	O
-formation	O
-of	O
-the	O
-decoding	O
-site	O
-,	O
-the	O
-20S	O
-pre	O
--	O
-rRNA	O
-becomes	O
-accessible	O
-for	O
-Nob1	O
-cleavage	O
-at	O
-site	O
-D	O
-.	O
-This	O
-also	O
-involves	O
-joining	O
-of	O
-pre	O
--	O
-40S	O
-and	O
-60S	O
-subunits	O
-to	O
-80S	O
--	O
-like	O
-particles	O
-in	O
-a	O
-translation	O
--	O
-like	O
-cycle	O
-promoted	O
-by	O
-eIF5B	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-acp	O
-transfer	O
-to	O
-m1Ψ1191	O
-occurs	O
-during	O
-the	O
-step	O
-at	O
-which	O
-Rio2	O
-leaves	O
-the	O
-pre	O
--	O
-40S	O
-particle	O
-.	O
-	O
-The	O
-current	O
-data	O
-together	O
-with	O
-the	O
-finding	O
-that	O
-acp	O
-modification	O
-takes	O
-place	O
-at	O
-the	O
-very	O
-last	O
-step	O
-in	O
-pre	O
--	O
-40S	O
-subunit	O
-maturation	O
-indicate	O
-that	O
-the	O
-acp	O
-modification	O
-probably	O
-supports	O
-the	O
-formation	O
-of	O
-the	O
-decoding	O
-site	O
-and	O
-efficient	O
-20S	O
-pre	O
--	O
-rRNA	O
-D	O
--	O
-site	O
-cleavage	O
-.	O
-	O
-Furthermore	O
-,	O
-our	O
-structural	O
-data	O
-unravelled	O
-how	O
-the	O
-regioselectivity	O
-of	O
-SAM	O
--	O
-dependent	O
-group	O
-transfer	O
-reactions	O
-can	O
-be	O
-tuned	O
-by	O
-distinct	O
-small	O
-evolutionary	O
-adaptions	O
-of	O
-the	O
-ligand	O
-binding	O
-pocket	O
-of	O
-SAM	O
--	O
-binding	O
-enzymes	O
-.	O
-	O
-In	O
-addition	O
-,	O
-our	O
-crystallographic	O
-analyses	O
-revealed	O
-that	O
-YfiR	O
-binds	O
-Vitamin	O
-B6	O
-(	O
-VB6	O
-)	O
-or	O
-L	O
--	O
-Trp	O
-at	O
-a	O
-YfiB	O
--	O
-binding	O
-site	O
-and	O
-that	O
-both	O
-VB6	O
-and	O
-L	O
--	O
-Trp	O
-are	O
-able	O
-to	O
-reduce	O
-YfiBL43P	B-mutant
--	O
-induced	O
-biofilm	O
-formation	O
-.	O
-	O
-An	O
-increase	O
-in	O
-c	O
--	O
-di	O
--	O
-GMP	O
-promotes	O
-biofilm	O
-formation	O
-,	O
-and	O
-a	O
-decrease	O
-results	O
-in	O
-biofilm	O
-degradation	O
-(	O
-Boehm	O
-et	O
-al	O
-.,;	O
-Duerig	O
-et	O
-al	O
-.,;	O
-Hickman	O
-et	O
-al	O
-.,;	O
-Jenal	O
-,;	O
-Romling	O
-et	O
-al	O
-.,).	O
-	O
-The	O
-c	O
--	O
-di	O
--	O
-GMP	O
-level	O
-is	O
-regulated	O
-by	O
-two	O
-reciprocal	O
-enzyme	O
-systems	O
-,	O
-namely	O
-,	O
-diguanylate	O
-cyclases	O
-(	O
-DGCs	O
-)	O
-that	O
-synthesize	O
-c	O
--	O
-di	O
--	O
-GMP	O
-and	O
-phosphodiesterases	O
-(	O
-PDEs	O
-)	O
-that	O
-hydrolyze	O
-c	O
--	O
-di	O
--	O
-GMP	O
-(	O
-Kulasakara	O
-et	O
-al	O
-.,;	O
-Ross	O
-et	O
-al	O
-.,;	O
-Ross	O
-et	O
-al	O
-.,).	O
-Many	O
-of	O
-these	O
-enzymes	O
-are	O
-multiple	O
--	O
-domain	O
-proteins	O
-containing	O
-a	O
-variable	O
-N	O
--	O
-terminal	O
-domain	O
-that	O
-commonly	O
-acts	O
-as	O
-a	O
-signal	O
-sensor	O
-or	O
-transduction	O
-module	O
-,	O
-followed	O
-by	O
-the	O
-relatively	O
-conserved	O
-GGDEF	O
-motif	O
-in	O
-DGCs	O
-or	O
-EAL	O
-/	O
-HD	O
--	O
-GYP	O
-domains	O
-in	O
-PDEs	O
-(	O
-Hengge	O
-,;	O
-Navarro	O
-et	O
-al	O
-.,;	O
-Schirmer	O
-and	O
-Jenal	O
-,).	O
-	O
-YfiN	O
-is	O
-an	O
-integral	O
-inner	O
--	O
-membrane	O
-protein	O
-with	O
-two	O
-potential	O
-transmembrane	O
-helices	O
-,	O
-a	O
-periplasmic	O
-Per	O
--	O
-Arnt	O
--	O
-Sim	O
-(	O
-PAS	O
-)	O
-domain	O
-,	O
-and	O
-cytosolic	O
-domains	O
-containing	O
-a	O
-HAMP	O
-domain	O
-(	O
-mediate	O
-input	O
--	O
-output	O
-signaling	O
-in	O
-histidine	O
-kinases	O
-,	O
-adenylyl	O
-cyclases	O
-,	O
-methyl	O
--	O
-accepting	O
-chemotaxis	O
-proteins	O
-,	O
-and	O
-phosphatases	O
-)	O
-and	O
-a	O
-C	O
--	O
-terminal	O
-GGDEF	O
-domain	O
-indicating	O
-a	O
-DGC	O
-’	O
-s	O
-function	O
-(	O
-Giardina	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-YfiN	O
-is	O
-repressed	O
-by	O
-specific	O
-interaction	O
-between	O
-its	O
-periplasmic	O
-PAS	O
-domain	O
-and	O
-the	O
-periplasmic	O
-protein	O
-YfiR	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-After	O
-the	O
-sequestration	O
-of	O
-YfiR	O
-by	O
-YfiB	O
-,	O
-the	O
-c	O
--	O
-di	O
--	O
-GMP	O
-produced	O
-by	O
-activated	O
-YfiN	O
-increases	O
-the	O
-biosynthesis	O
-of	O
-the	O
-Pel	O
-and	O
-Psl	O
-EPSs	O
-,	O
-resulting	O
-in	O
-the	O
-appearance	O
-of	O
-the	O
-SCV	O
-phenotype	O
-,	O
-which	O
-indicates	O
-enhanced	O
-biofilm	O
-formation	O
-(	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-Recently	O
-,	O
-we	O
-solved	O
-the	O
-crystal	O
-structure	O
-of	O
-YfiR	O
-in	O
-both	O
-the	O
-non	O
--	O
-oxidized	O
-and	O
-the	O
-oxidized	O
-states	O
-,	O
-revealing	O
-breakage	O
-/	O
-formation	O
-of	O
-one	O
-disulfide	O
-bond	O
-(	O
-Cys71	O
--	O
-Cys110	O
-)	O
-and	O
-local	O
-conformational	O
-change	O
-around	O
-the	O
-other	O
-one	O
-(	O
-Cys145	O
--	O
-Cys152	O
-),	O
-indicating	O
-that	O
-Cys145	O
--	O
-Cys152	O
-plays	O
-an	O
-important	O
-role	O
-in	O
-maintaining	O
-the	O
-correct	O
-folding	O
-of	O
-YfiR	O
-(	O
-Yang	O
-et	O
-al	O
-.,).	O
-	O
-The	O
-“	O
-back	O
-to	O
-back	O
-”	O
-dimer	O
-.	O
-	O
-The	O
-dimerization	O
-occurs	O
-mainly	O
-via	O
-hydrophobic	O
-interactions	O
-formed	O
-by	O
-A37	O
-and	O
-I40	O
-on	O
-the	O
-α1	O
-helices	O
-,	O
-L50	O
-on	O
-the	O
-β1	O
-strands	O
-,	O
-and	O
-W55	O
-on	O
-the	O
-β2	O
-strands	O
-of	O
-both	O
-molecules	O
-,	O
-making	O
-a	O
-hydrophobic	O
-interacting	O
-core	O
-(	O
-Fig	O
-.	O
-2A	O
-–	O
-C	O
-).	O
-	O
-The	O
-“	O
-back	O
-to	O
-back	O
-”	O
-dimer	O
-presents	O
-a	O
-Y	O
-shape	O
-.	O
-	O
-Overall	O
-structure	O
-of	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-and	O
-the	O
-conserved	O
-surface	O
-in	O
-YfiR	O
-.	O
-(	O
-A	O
-)	O
-The	O
-overall	O
-structure	O
-of	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-.	O
-	O
-Two	O
-interacting	O
-regions	O
-are	O
-highlighted	O
-by	O
-red	O
-rectangles	O
-.	O
-(	O
-B	O
-)	O
-Structural	O
-superposition	O
-of	O
-apo	O
-YfiB	O
-and	O
-YfiR	O
--	O
-bound	O
-YfiBL43P	B-mutant
-.	O
-	O
-The	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-is	O
-a	O
-2	O
-:	O
-2	O
-heterotetramer	O
-(	O
-Fig	O
-.	O
-3A	O
-)	O
-in	O
-which	O
-the	O
-YfiR	O
-dimer	O
-is	O
-clamped	O
-by	O
-two	O
-separated	O
-YfiBL43P	B-mutant
-molecules	O
-with	O
-a	O
-total	O
-buried	O
-surface	O
-area	O
-of	O
-3161	O
-.	O
-2	O
-Å2	O
-.	O
-	O
-Additionally	O
-,	O
-three	O
-hydrophobic	O
-anchoring	O
-sites	O
-exist	O
-in	O
-region	O
-I	O
-.	O
-The	O
-residues	O
-F48	O
-and	O
-W55	O
-of	O
-YfiB	O
-are	O
-inserted	O
-into	O
-the	O
-hydrophobic	O
-cores	O
-mainly	O
-formed	O
-by	O
-the	O
-main	O
-chain	O
-and	O
-side	O
-chain	O
-carbon	O
-atoms	O
-of	O
-residues	O
-S57	O
-/	O
-Q88	O
-/	O
-A89	O
-/	O
-N90	O
-and	O
-R60	O
-/	O
-R175	O
-/	O
-H177	O
-of	O
-YfiR	O
-,	O
-respectively	O
-;	O
-and	O
-F57	O
-of	O
-YfiB	O
-is	O
-inserted	O
-into	O
-the	O
-hydrophobic	O
-pocket	O
-formed	O
-by	O
-L166	O
-/	O
-I169	O
-/	O
-V176	O
-/	O
-P178	O
-/	O
-L181	O
-of	O
-YfiR	O
-(	O
-Fig	O
-.	O
-3D	O
--	O
-I	O
-(	O
-ii	O
-)).	O
-	O
-This	O
-suggests	O
-that	O
-the	O
-N	O
--	O
-terminus	O
-of	O
-YfiB	O
-plays	O
-an	O
-important	O
-role	O
-in	O
-forming	O
-the	O
-dimeric	O
-YfiB	O
-in	O
-solution	O
-and	O
-that	O
-the	O
-conformational	O
-change	O
-of	O
-residue	O
-L43	O
-is	O
-associated	O
-with	O
-the	O
-stretch	O
-of	O
-the	O
-N	O
--	O
-terminus	O
-and	O
-opening	O
-of	O
-the	O
-dimer	O
-.	O
-	O
-The	O
-PG	O
--	O
-binding	O
-site	O
-of	O
-YfiB	O
-	O
-In	O
-the	O
-YfiB	O
--	O
-YfiR	O
-complex	O
-,	O
-one	O
-sulfate	O
-ion	O
-is	O
-found	O
-at	O
-the	O
-bottom	O
-of	O
-each	O
-YfiBL43P	B-mutant
-molecule	O
-(	O
-Fig	O
-.	O
-3A	O
-)	O
-and	O
-forms	O
-a	O
-strong	O
-hydrogen	O
-bond	O
-with	O
-D102	O
-of	O
-YfiBL43P	B-mutant
-(	O
-Fig	O
-.	O
-4A	O
-and	O
-4C	O
-).	O
-	O
-Moreover	O
-,	O
-a	O
-water	O
-molecule	O
-was	O
-found	O
-to	O
-bridge	O
-the	O
-sulfate	O
-ion	O
-and	O
-the	O
-side	O
-chains	O
-of	O
-N67	O
-and	O
-D102	O
-,	O
-strengthening	O
-the	O
-hydrogen	O
-bond	O
-network	O
-(	O
-Fig	O
-.	O
-4C	O
-).	O
-	O
-The	O
-results	O
-indicated	O
-that	O
-the	O
-PG	O
--	O
-binding	O
-affinity	O
-of	O
-YfiBL43P	B-mutant
-is	O
-65	O
-.	O
-5	O
-μmol	O
-/	O
-L	O
-,	O
-which	O
-is	O
-about	O
-16	O
--	O
-fold	O
-stronger	O
-than	O
-that	O
-of	O
-wild	O
--	O
-type	O
-YfiB	O
-(	O
-Kd	O
-=	O
-1	O
-.	O
-1	O
-mmol	O
-/	O
-L	O
-)	O
-(	O
-Fig	O
-.	O
-4E	O
-–	O
-F	O
-).	O
-	O
-The	O
-relative	O
-optical	O
-density	O
-is	O
-represented	O
-as	O
-curves	O
-.	O
-	O
-Wild	O
--	O
-type	O
-YfiB	O
-is	O
-used	O
-as	O
-negative	O
-control	O
-.	O
-	O
-Previous	O
-studies	O
-suggested	O
-that	O
-in	O
-response	O
-to	O
-cell	O
-stress	O
-,	O
-YfiB	O
-in	O
-the	O
-outer	O
-membrane	O
-sequesters	O
-the	O
-periplasmic	O
-protein	O
-YfiR	O
-,	O
-releasing	O
-its	O
-inhibition	O
-of	O
-YfiN	O
-on	O
-the	O
-inner	O
-membrane	O
-and	O
-thus	O
-inducing	O
-the	O
-diguanylate	O
-cyclase	O
-activity	O
-of	O
-YfiN	O
-to	O
-allow	O
-c	O
--	O
-di	O
--	O
-GMP	O
-production	O
-(	O
-Giardina	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,).	O
-	O
-Here	O
-,	O
-we	O
-report	O
-the	O
-crystal	O
-structures	O
-of	O
-YfiB	O
-alone	O
-and	O
-an	O
-active	O
-mutant	O
-YfiBL43P	B-mutant
-in	O
-complex	O
-with	O
-YfiR	O
-,	O
-indicating	O
-that	O
-YfiR	O
-forms	O
-a	O
-2	O
-:	O
-2	O
-complex	O
-with	O
-YfiB	O
-via	O
-a	O
-region	O
-composed	O
-of	O
-conserved	O
-residues	O
-.	O
-	O
-Our	O
-structural	O
-data	O
-analysis	O
-shows	O
-that	O
-the	O
-activated	O
-YfiB	O
-has	O
-an	O
-N	O
--	O
-terminal	O
-portion	O
-that	O
-is	O
-largely	O
-altered	O
-,	O
-adopting	O
-a	O
-stretched	O
-conformation	O
-compared	O
-with	O
-the	O
-compact	O
-conformation	O
-of	O
-the	O
-apo	O
-YfiB	O
-.	O
-The	O
-apo	O
-YfiB	O
-structure	O
-constructed	O
-beginning	O
-at	O
-residue	O
-34	O
-has	O
-a	O
-compact	O
-conformation	O
-of	O
-approximately	O
-45	O
-Å	O
-in	O
-length	O
-.	O
-	O
-In	O
-this	O
-model	O
-,	O
-in	O
-response	O
-to	O
-a	O
-particular	O
-cell	O
-stress	O
-that	O
-is	O
-yet	O
-to	O
-be	O
-identified	O
-,	O
-the	O
-dimeric	O
-YfiB	O
-is	O
-activated	O
-from	O
-a	O
-compact	O
-,	O
-inactive	O
-conformation	O
-to	O
-a	O
-stretched	O
-conformation	O
-,	O
-which	O
-possesses	O
-increased	O
-PG	O
-binding	O
-affinity	O
-.	O
-	O
-Homologs	O
-of	O
-the	O
-YfiBNR	O
-system	O
-are	O
-functionally	O
-conserved	O
-in	O
-P	O
-.	O
-aeruginosa	O
-(	O
-Malone	O
-et	O
-al	O
-.,;	O
-Malone	O
-et	O
-al	O
-.,),	O
-E	O
-.	O
-coli	O
-(	O
-Hufnagel	O
-et	O
-al	O
-.,;	O
-Raterman	O
-et	O
-al	O
-.,;	O
-Sanchez	O
--	O
-Torres	O
-et	O
-al	O
-.,),	O
-K	O
-.	O
-pneumonia	O
-(	O
-Huertas	O
-et	O
-al	O
-.,)	O
-and	O
-Y	O
-.	O
-pestis	O
-(	O
-Ren	O
-et	O
-al	O
-.,),	O
-where	O
-they	O
-affect	O
-c	O
--	O
-di	O
--	O
-GMP	O
-production	O
-and	O
-biofilm	O
-formation	O
-.	O
-	O
-High	O
--	O
-resolution	O
-structures	O
-of	O
-oligomers	O
-formed	O
-by	O
-the	O
-β	O
--	O
-amyloid	O
-peptide	O
-Aβ	O
-are	O
-needed	O
-to	O
-understand	O
-the	O
-molecular	O
-basis	O
-of	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-and	O
-develop	O
-therapies	O
-.	O
-	O
-Over	O
-the	O
-last	O
-two	O
-decades	O
-the	O
-role	O
-of	O
-Aβ	O
-oligomers	O
-in	O
-the	O
-pathophysiology	O
-of	O
-Alzheimer	O
-’	O
-s	O
-disease	O
-has	O
-begun	O
-to	O
-unfold	O
-.	O
-	O
-Aβ	O
-isolated	O
-from	O
-the	O
-brains	O
-of	O
-young	O
-plaque	O
--	O
-free	O
-Tg2576	O
-mice	O
-forms	O
-a	O
-mixture	O
-of	O
-low	O
-molecular	O
-weight	O
-oligomers	O
-.	O
-	O
-Smaller	O
-oligomers	O
-with	O
-molecular	O
-weights	O
-consistent	O
-with	O
-trimers	O
-,	O
-hexamers	O
-,	O
-and	O
-nonamers	O
-were	O
-also	O
-identified	O
-within	O
-the	O
-mixture	O
-of	O
-low	O
-molecular	O
-weight	O
-oligomers	O
-.	O
-	O
-A	O
-type	O
-of	O
-large	O
-oligomers	O
-called	O
-annular	O
-protofibrils	O
-(	O
-APFs	O
-)	O
-have	O
-also	O
-been	O
-observed	O
-in	O
-the	O
-brains	O
-of	O
-transgenic	O
-mice	O
-and	O
-isolated	O
-from	O
-the	O
-brains	O
-of	O
-Alzheimer	O
-’	O
-s	O
-patients	O
-.	O
-	O
-Lashuel	O
-et	O
-al	O
-.	O
-observed	O
-APFs	O
-with	O
-an	O
-outer	O
-diameter	O
-that	O
-ranged	O
-from	O
-7	O
-–	O
-10	O
-nm	O
-and	O
-an	O
-inner	O
-diameter	O
-that	O
-ranged	O
-from	O
-1	O
-.	O
-5	O
-–	O
-2	O
-nm	O
-,	O
-consistent	O
-with	O
-molecular	O
-weights	O
-of	O
-150	O
-–	O
-250	O
-kDa	O
-.	O
-	O
-Kayed	O
-et	O
-al	O
-.	O
-observed	O
-APFs	O
-with	O
-an	O
-outer	O
-diameter	O
-that	O
-ranged	O
-from	O
-8	O
-–	O
-25	O
-nm	O
-,	O
-which	O
-were	O
-composed	O
-of	O
-small	O
-spherical	O
-Aβ	O
-oligomers	O
-,	O
-3	O
-–	O
-5	O
-nm	O
-in	O
-diameter	O
-.	O
-	O
-Sequestering	O
-Aβ	O
-within	O
-the	O
-affibody	O
-prevents	O
-its	O
-fibrilization	O
-and	O
-reduces	O
-its	O
-neurotoxicity	O
-,	O
-providing	O
-evidence	O
-that	O
-the	O
-β	O
--	O
-hairpin	O
-structure	O
-may	O
-contribute	O
-to	O
-the	O
-ability	O
-of	O
-Aβ	O
-to	O
-form	O
-neurotoxic	O
-oligomers	O
-.	O
-	O
-(	O
-A	O
-)	O
-Cartoon	O
-illustrating	O
-the	O
-design	O
-of	O
-peptides	O
-1	O
-and	O
-2	O
-and	O
-their	O
-relationship	O
-to	O
-an	O
-Aβ17	O
-–	O
-36	O
-β	O
--	O
-hairpin	O
-.	O
-	O
-Peptide	B-mutant
-2	I-mutant
-contains	O
-a	O
-methionine	O
-residue	O
-at	O
-position	O
-35	O
-and	O
-an	O
-Aβ24	O
-–	O
-29	O
-loop	O
-with	O
-residues	O
-24	O
-and	O
-29	O
-(	O
-Val	O
-and	O
-Gly	O
-)	O
-mutated	O
-to	O
-cysteine	O
-and	O
-linked	O
-by	O
-a	O
-disulfide	O
-bond	O
-(	O
-Figure	O
-1C	O
-).	O
-	O
-To	O
-address	O
-this	O
-issue	O
-,	O
-we	O
-next	O
-incorporated	O
-a	O
-disulfide	O
-bond	O
-between	O
-residues	O
-24	O
-and	O
-29	O
-as	O
-a	O
-conformational	O
-constraint	O
-that	O
-serves	O
-as	O
-a	O
-surrogate	O
-for	O
-δOrn	O
-.	O
-	O
-We	O
-mutated	O
-these	O
-residues	O
-because	O
-they	O
-occupy	O
-the	O
-same	O
-position	O
-as	O
-the	O
-δOrn	O
-that	O
-connects	O
-D23	O
-and	O
-A30	O
-in	O
-peptide	B-mutant
-1	I-mutant
-.	O
-	O
-In	O
-synthesizing	O
-peptides	B-mutant
-2	I-mutant
-and	I-mutant
-4	I-mutant
-we	O
-formed	O
-the	O
-disulfide	O
-linkage	O
-after	O
-macrolactamization	O
-and	O
-deprotection	O
-of	O
-the	O
-acid	O
--	O
-labile	O
-side	O
-chain	O
-protecting	O
-groups	O
-.	O
-	O
-Crystal	O
-diffraction	O
-data	O
-for	O
-peptides	B-mutant
-4	I-mutant
-and	I-mutant
-2	I-mutant
-were	O
-collected	O
-in	O
--	O
-house	O
-with	O
-a	O
-Rigaku	O
-MicroMax	O
-007HF	O
-X	O
--	O
-ray	O
-diffractometer	O
-at	O
-1	O
-.	O
-54	O
-Å	O
-wavelength	O
-.	O
-	O
-Data	O
-for	O
-peptides	B-mutant
-4	I-mutant
-and	I-mutant
-2	I-mutant
-were	O
-scaled	O
-and	O
-merged	O
-using	O
-XDS	O
-.	O
-	O
-X	O
--	O
-ray	O
-Crystallographic	O
-Structure	O
-of	O
-Peptide	B-mutant
-2	I-mutant
-and	O
-the	O
-Oligomers	O
-It	O
-Forms	O
-	O
-The	O
-B	O
-values	O
-for	O
-the	O
-loops	O
-are	O
-large	O
-,	O
-indicating	O
-that	O
-the	O
-loops	O
-are	O
-dynamic	O
-and	O
-not	O
-well	O
-ordered	O
-.	O
-	O
-Thus	O
-,	O
-the	O
-differences	O
-in	O
-backbone	O
-geometry	O
-and	O
-side	O
-chain	O
-rotamers	O
-among	O
-the	O
-loops	O
-are	O
-likely	O
-of	O
-little	O
-significance	O
-and	O
-should	O
-be	O
-interpreted	O
-with	O
-caution	O
-.	O
-	O
-Like	O
-peptide	B-mutant
-1	I-mutant
-,	O
-peptide	B-mutant
-2	I-mutant
-forms	O
-a	O
-triangular	O
-trimer	O
-,	O
-and	O
-four	O
-trimers	O
-assemble	O
-to	O
-form	O
-a	O
-dodecamer	O
-.	O
-	O
-In	O
-the	O
-higher	O
--	O
-order	O
-assembly	O
-of	O
-the	O
-dodecamers	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-a	O
-new	O
-structure	O
-emerges	O
-,	O
-not	O
-seen	O
-in	O
-peptide	B-mutant
-1	I-mutant
-,	O
-an	O
-annular	O
-pore	O
-consisting	O
-of	O
-five	O
-dodecamers	O
-.	O
-	O
-The	O
-trimer	O
-maintains	O
-all	O
-of	O
-the	O
-same	O
-stabilizing	O
-contacts	O
-as	O
-those	O
-of	O
-peptide	B-mutant
-1	I-mutant
-.	O
-	O
-In	O
-the	O
-crystal	O
-lattice	O
-,	O
-each	O
-F20	O
-face	O
-of	O
-one	O
-dodecamer	O
-packs	O
-against	O
-an	O
-F20	O
-face	O
-of	O
-another	O
-dodecamer	O
-.	O
-	O
-Jeffamine	O
-M	O
--	O
-600	O
-is	O
-a	O
-polypropylene	O
-glycol	O
-derivative	O
-with	O
-a	O
-2	O
--	O
-methoxyethoxy	O
-unit	O
-at	O
-one	O
-end	O
-and	O
-a	O
-2	O
--	O
-aminopropyl	O
-unit	O
-at	O
-the	O
-other	O
-end	O
-.	O
-	O
-Hydrophobic	O
-packing	O
-between	O
-the	O
-F20	O
-faces	O
-of	O
-trimers	O
-displayed	O
-on	O
-the	O
-outer	O
-surface	O
-of	O
-each	O
-dodecamer	O
-stabilizes	O
-the	O
-porelike	O
-assembly	O
-.	O
-	O
-The	O
-eclipsed	O
-interfaces	O
-occur	O
-between	O
-dodecamers	O
-1	O
-and	O
-2	O
-,	O
-1	O
-and	O
-5	O
-,	O
-and	O
-3	O
-and	O
-4	O
-,	O
-as	O
-shown	O
-in	O
-Figure	O
-5A	O
-.	O
-	O
-The	O
-crystallographic	O
-assembly	O
-of	O
-peptide	B-mutant
-2	I-mutant
-into	O
-a	O
-trimer	O
-,	O
-dodecamer	O
-,	O
-and	O
-annular	O
-pore	O
-provides	O
-a	O
-model	O
-for	O
-the	O
-assembly	O
-of	O
-the	O
-full	O
--	O
-length	O
-Aβ	O
-peptide	O
-to	O
-form	O
-oligomers	O
-.	O
-	O
-In	O
-this	O
-model	O
-Aβ	O
-folds	O
-to	O
-form	O
-a	O
-β	O
--	O
-hairpin	O
-comprising	O
-the	O
-hydrophobic	O
-central	O
-and	O
-C	O
--	O
-terminal	O
-regions	O
-.	O
-	O
-Three	O
-β	O
--	O
-hairpins	O
-assemble	O
-to	O
-form	O
-a	O
-trimer	O
-,	O
-and	O
-four	O
-trimers	O
-assemble	O
-to	O
-form	O
-a	O
-dodecamer	O
-.	O
-	O
-The	O
-model	O
-put	O
-forth	O
-in	O
-Figure	O
-6	O
-is	O
-consistent	O
-with	O
-the	O
-current	O
-understanding	O
-of	O
-endogenous	O
-Aβ	O
-oligomerization	O
-and	O
-explains	O
-at	O
-atomic	O
-resolution	O
-many	O
-key	O
-observations	O
-about	O
-Aβ	O
-oligomers	O
-.	O
-	O
-Fibrillar	O
-and	O
-nonfibrillar	O
-oligomers	O
-have	O
-structurally	O
-distinct	O
-characteristics	O
-,	O
-which	O
-are	O
-reflected	O
-in	O
-their	O
-reactivity	O
-with	O
-the	O
-fibril	O
--	O
-specific	O
-OC	O
-antibody	O
-and	O
-the	O
-oligomer	O
--	O
-specific	O
-A11	O
-antibody	O
-.	O
-	O
-At	O
-this	O
-point	O
-,	O
-we	O
-can	O
-only	O
-speculate	O
-whether	O
-the	O
-trimer	O
-and	O
-dodecamer	O
-formed	O
-by	O
-peptide	B-mutant
-2	I-mutant
-share	O
-structural	O
-similarities	O
-to	O
-Aβ	O
-trimers	O
-and	O
-Aβ	O
-*	O
-56	O
-,	O
-as	O
-little	O
-is	O
-known	O
-about	O
-the	O
-structure	O
-of	O
-Aβ	O
-trimers	O
-and	O
-Aβ	O
-*	O
-56	O
-.	O
-	O
-These	O
-two	O
-modes	O
-of	O
-assembly	O
-might	O
-reflect	O
-a	O
-dynamic	O
-interaction	O
-between	O
-dodecamers	O
-,	O
-which	O
-could	O
-permit	O
-assemblies	O
-of	O
-more	O
-dodecamers	O
-into	O
-larger	O
-annular	O
-pores	O
-.	O
-	O
-Preliminary	O
-attempts	O
-to	O
-study	O
-these	O
-species	O
-by	O
-SEC	O
-and	O
-SDS	O
--	O
-PAGE	O
-have	O
-not	O
-provided	O
-a	O
-clear	O
-measure	O
-of	O
-the	O
-structures	O
-formed	O
-in	O
-solution	O
-.	O
-	O
-Our	O
-approach	O
-of	O
-constraining	O
-Aβ17	O
-–	O
-36	O
-into	O
-a	O
-β	O
--	O
-hairpin	O
-conformation	O
-and	O
-blocking	O
-aggregation	O
-with	O
-an	O
-N	O
--	O
-methyl	O
-group	O
-has	O
-allowed	O
-us	O
-to	O
-crystallize	O
-a	O
-large	O
-fragment	O
-of	O
-what	O
-is	O
-generally	O
-considered	O
-to	O
-be	O
-an	O
-uncrystallizable	O
-peptide	O
-.	O
-	O
-Ligands	O
-that	O
-regulate	O
-the	O
-dynamics	O
-and	O
-stability	O
-of	O
-the	O
-coactivator	O
-‐	O
-binding	O
-site	O
-in	O
-the	O
-C	O
-‐	O
-terminal	O
-ligand	O
-‐	O
-binding	O
-domain	O
-,	O
-called	O
-activation	O
-function	O
-‐	O
-2	O
-(	O
-AF	O
-‐	O
-2	O
-),	O
-showed	O
-similar	O
-activity	O
-profiles	O
-in	O
-different	O
-cell	O
-types	O
-.	O
-	O
-Such	O
-ligands	O
-induced	O
-breast	O
-cancer	O
-cell	O
-proliferation	O
-in	O
-a	O
-manner	O
-that	O
-was	O
-predicted	O
-by	O
-the	O
-canonical	O
-recruitment	O
-of	O
-the	O
-coactivators	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-and	O
-induction	O
-of	O
-the	O
-GREB1	O
-proliferative	O
-gene	O
-.	O
-	O
-For	O
-example	O
-,	O
-selective	O
-estrogen	O
-receptor	O
-modulators	O
-(	O
-SERMs	O
-)	O
-such	O
-as	O
-tamoxifen	O
-(	O
-Nolvadex	O
-®;	O
-AstraZeneca	O
-)	O
-or	O
-raloxifene	O
-(	O
-Evista	O
-®;	O
-Eli	O
-Lilly	O
-)	O
-(	O
-Fig	O
-1A	O
-)	O
-block	O
-the	O
-ERα	O
-‐	O
-mediated	O
-proliferative	O
-effects	O
-of	O
-the	O
-native	O
-estrogen	O
-,	O
-17β	O
-‐	O
-estradiol	O
-(	O
-E2	O
-),	O
-on	O
-breast	O
-cancer	O
-cells	O
-,	O
-but	O
-promote	O
-beneficial	O
-estrogenic	O
-effects	O
-on	O
-bone	O
-mineral	O
-density	O
-and	O
-adverse	O
-estrogenic	O
-effects	O
-such	O
-as	O
-uterine	O
-proliferation	O
-,	O
-fatty	O
-liver	O
-,	O
-or	O
-stroke	O
-(	O
-Frolik	O
-et	O
-al	O
-,	O
-1996	O
-;	O
-Fisher	O
-et	O
-al	O
-,	O
-1998	O
-;	O
-McDonnell	O
-et	O
-al	O
-,	O
-2002	O
-;	O
-Jordan	O
-,	O
-2003	O
-).	O
-	O
-E2	O
-‐	O
-rings	O
-are	O
-numbered	O
-A	O
-‐	O
-D	O
-.	O
-The	O
-E	O
-‐	O
-ring	O
-is	O
-the	O
-common	O
-site	O
-of	O
-attachment	O
-for	O
-BSC	O
-found	O
-in	O
-many	O
-SERMS	O
-.	O
-	O
-Linear	O
-causality	O
-model	O
-for	O
-ERα	O
-‐	O
-mediated	O
-cell	O
-proliferation	O
-.	O
-	O
-AF	O
-‐	O
-1	O
-binds	O
-a	O
-separate	O
-surface	O
-on	O
-these	O
-coactivators	O
-(	O
-Webb	O
-et	O
-al	O
-,	O
-1998	O
-;	O
-Yi	O
-et	O
-al	O
-,	O
-2015	O
-).	O
-	O
-However	O
-,	O
-ERα	O
-‐	O
-mediated	O
-proliferative	O
-responses	O
-vary	O
-in	O
-a	O
-ligand	O
-‐	O
-dependent	O
-manner	O
-(	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-);	O
-thus	O
-,	O
-it	O
-is	O
-not	O
-known	O
-whether	O
-this	O
-canonical	O
-model	O
-is	O
-widely	O
-applicable	O
-across	O
-diverse	O
-ERα	O
-ligands	O
-.	O
-	O
-In	O
-this	O
-signaling	O
-model	O
-,	O
-multiple	O
-coregulator	O
-binding	O
-events	O
-and	O
-target	O
-genes	O
-(	O
-Won	O
-Jeong	O
-et	O
-al	O
-,	O
-2012	O
-;	O
-Nwachukwu	O
-et	O
-al	O
-,	O
-2014	O
-),	O
-LBD	O
-conformation	O
-,	O
-nucleocytoplasmic	O
-shuttling	O
-,	O
-the	O
-occupancy	O
-and	O
-dynamics	O
-of	O
-DNA	O
-binding	O
-,	O
-and	O
-other	O
-biophysical	O
-features	O
-could	O
-contribute	O
-independently	O
-to	O
-cell	O
-proliferation	O
-(	O
-Lickwar	O
-et	O
-al	O
-,	O
-2012	O
-).	O
-	O
-To	O
-test	O
-these	O
-signaling	O
-models	O
-,	O
-we	O
-profiled	O
-a	O
-diverse	O
-library	O
-of	O
-ERα	O
-ligands	O
-using	O
-systems	O
-biology	O
-approaches	O
-to	O
-X	O
-‐	O
-ray	O
-crystallography	O
-and	O
-chemical	O
-biology	O
-(	O
-Srinivasan	O
-et	O
-al	O
-,	O
-2013	O
-),	O
-including	O
-a	O
-series	O
-of	O
-quantitative	O
-bioassays	O
-for	O
-ERα	O
-function	O
-that	O
-were	O
-statistically	O
-robust	O
-and	O
-reproducible	O
-,	O
-based	O
-on	O
-the	O
-Z	O
-’‐	O
-statistic	O
-(	O
-Fig	O
-EV1A	O
-and	O
-B	O
-;	O
-see	O
-Materials	O
-and	O
-Methods	O
-).	O
-	O
-Structure	O
-of	O
-the	O
-E2	O
-‐	O
-bound	O
-ERα	O
-LBD	O
-in	O
-complex	O
-with	O
-an	O
-NCOA2	O
-peptide	O
-of	O
-(	O
-PDB	O
-1GWR	O
-).	O
-	O
-In	O
-cluster	O
-1	O
-,	O
-the	O
-first	O
-three	O
-comparisons	O
-(	O
-rows	O
-)	O
-showed	O
-significant	O
-positive	O
-correlations	O
-(	O
-F	O
-‐	O
-test	O
-for	O
-nonzero	O
-slope	O
-,	O
-P	O
-≤	O
-0	O
-.	O
-05	O
-).	O
-	O
-−,	O
-significant	O
-correlations	O
-lost	O
-upon	O
-deletion	O
-of	O
-AB	O
-or	O
-F	O
-domains	O
-.	O
-	O
-Tamoxifen	O
-depends	O
-on	O
-AF	O
-‐	O
-1	O
-for	O
-its	O
-cell	O
-‐	O
-specific	O
-activity	O
-(	O
-Sakamoto	O
-et	O
-al	O
-,	O
-2002	O
-);	O
-therefore	O
-,	O
-we	O
-asked	O
-whether	O
-cell	O
-‐	O
-specific	O
-signaling	O
-observed	O
-here	O
-is	O
-due	O
-to	O
-a	O
-similar	O
-dependence	O
-on	O
-AF	O
-‐	O
-1	O
-for	O
-activity	O
-(	O
-Fig	O
-EV1	O
-).	O
-	O
-Thus	O
-,	O
-the	O
-strength	O
-of	O
-AF	O
-‐	O
-1	O
-signaling	O
-does	O
-not	O
-determine	O
-cell	O
-‐	O
-specific	O
-signaling	O
-.	O
-	O
-Identifying	O
-cell	O
-‐	O
-specific	O
-signaling	O
-clusters	O
-in	O
-ERα	O
-ligand	O
-classes	O
-	O
-The	O
-side	O
-chain	O
-of	O
-OBHS	O
-‐	O
-BSC	O
-analogs	O
-induces	O
-cell	O
-‐	O
-specific	O
-signaling	O
-	O
-In	O
-panel	O
-(	O
-D	O
-),	O
-L	O
-‐	O
-Luc	O
-ERα	O
-‐	O
-WT	O
-activity	O
-from	O
-panel	O
-(	O
-B	O
-)	O
-is	O
-shown	O
-for	O
-comparison	O
-.	O
-	O
-Thus	O
-,	O
-examining	O
-the	O
-correlated	O
-patterns	O
-of	O
-ERα	O
-activity	O
-within	O
-each	O
-scaffold	O
-demonstrates	O
-that	O
-an	O
-extended	O
-side	O
-chain	O
-is	O
-not	O
-required	O
-for	O
-cell	O
-‐	O
-specific	O
-signaling	O
-.	O
-	O
-Deletion	O
-of	O
-the	O
-AB	O
-or	O
-F	O
-domain	O
-altered	O
-correlations	O
-for	O
-six	O
-of	O
-the	O
-eight	O
-scaffolds	O
-in	O
-this	O
-cluster	O
-(	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-,	O
-3	O
-,	O
-4	O
-‐	O
-DTP	O
-,	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-,	O
-WAY	O
-‐	O
-D	O
-,	O
-WAY	O
-dimer	O
-,	O
-and	O
-cyclofenil	O
-‐	O
-ASC	O
-)	O
-(	O
-Fig	O
-3D	O
-lanes	O
-5	O
-–	O
-12	O
-).	O
-	O
-Thus	O
-,	O
-in	O
-cluster	O
-2	O
-,	O
-AF	O
-‐	O
-1	O
-substantially	O
-modulated	O
-the	O
-specificity	O
-of	O
-ligands	O
-with	O
-cell	O
-‐	O
-specific	O
-activity	O
-(	O
-Fig	O
-3D	O
-lanes	O
-5	O
-–	O
-12	O
-).	O
-	O
-To	O
-determine	O
-whether	O
-ligand	O
-classes	O
-control	O
-expression	O
-of	O
-native	O
-ERα	O
-target	O
-genes	O
-through	O
-the	O
-canonical	O
-linear	O
-signaling	O
-pathway	O
-,	O
-we	O
-performed	O
-pairwise	O
-linear	O
-regression	O
-analyses	O
-using	O
-ERα	O
-–	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-interactions	O
-in	O
-M2H	O
-assay	O
-as	O
-independent	O
-predictors	O
-of	O
-GREB1	O
-expression	O
-(	O
-the	O
-dependent	O
-variable	O
-)	O
-(	O
-Figs	O
-EV1	O
-and	O
-EV2A	O
-,	O
-F	O
-–	O
-H	O
-).	O
-	O
-For	O
-clusters	O
-2	O
-and	O
-3	O
-,	O
-GREB1	O
-activity	O
-was	O
-generally	O
-not	O
-predicted	O
-by	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-.	O
-	O
-However	O
-,	O
-ligand	O
-‐	O
-induced	O
-GREB1	O
-levels	O
-were	O
-generally	O
-not	O
-determined	O
-by	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-(	O
-Fig	O
-3E	O
-lanes	O
-5	O
-–	O
-19	O
-),	O
-consistent	O
-with	O
-an	O
-alternate	O
-causality	O
-model	O
-(	O
-Fig	O
-1E	O
-).	O
-	O
-Out	O
-of	O
-11	O
-indirect	O
-modulator	O
-series	O
-in	O
-cluster	O
-2	O
-or	O
-3	O
-,	O
-only	O
-the	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-class	O
-had	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-profiles	O
-that	O
-predicted	O
-GREB1	O
-levels	O
-(	O
-Fig	O
-3E	O
-lane	O
-12	O
-).	O
-	O
-With	O
-the	O
-OBHS	O
-‐	O
-N	O
-compounds	O
-,	O
-NCOA3	O
-and	O
-GREB1	O
-showed	O
-near	O
-perfect	O
-prediction	O
-of	O
-proliferation	O
-(	O
-Fig	O
-EV3G	O
-),	O
-with	O
-unexplained	O
-variance	O
-similar	O
-to	O
-the	O
-noise	O
-in	O
-the	O
-assays	O
-.	O
-	O
-Out	O
-of	O
-15	O
-ligand	O
-series	O
-in	O
-these	O
-clusters	O
-,	O
-only	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-induced	O
-a	O
-proliferative	O
-response	O
-that	O
-was	O
-predicted	O
-by	O
-GREB1	O
-levels	O
-,	O
-which	O
-were	O
-not	O
-determined	O
-by	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-(	O
-Fig	O
-3E	O
-and	O
-F	O
-lane	O
-10	O
-).	O
-	O
-Similarly	O
-,	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-,	O
-cyclofenil	O
-‐	O
-ASC	O
-,	O
-and	O
-OBHS	O
-‐	O
-ASC	O
-had	O
-positively	O
-correlated	O
-NCOA1	O
-/	O
-2	O
-/	O
-3	O
-recruitment	O
-and	O
-GREB1	O
-levels	O
-,	O
-but	O
-none	O
-of	O
-these	O
-activities	O
-determined	O
-their	O
-proliferative	O
-effects	O
-(	O
-Fig	O
-3E	O
-and	O
-F	O
-lanes	O
-11	O
-–	O
-12	O
-and	O
-18	O
-).	O
-	O
-NCOA3	O
-occupancy	O
-at	O
-GREB1	O
-is	O
-statistically	O
-robust	O
-but	O
-does	O
-not	O
-predict	O
-transcriptional	O
-activity	O
-	O
-All	O
-direct	O
-modulator	O
-and	O
-two	O
-indirect	O
-modulator	O
-scaffolds	O
-(	O
-OBHS	O
-and	O
-S	O
-‐	O
-OBHS	O
-‐	O
-3	O
-)	O
-lacked	O
-ERβ	O
-agonist	O
-activity	O
-.	O
-	O
-ERα	O
-activity	O
-of	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-and	O
-cyclofenil	O
-analogs	O
-correlates	O
-with	O
-E	O
-‐	O
-Luc	O
-activity	O
-.	O
-	O
-Therefore	O
-,	O
-we	O
-examined	O
-another	O
-50	O
-LBD	O
-structures	O
-containing	O
-ligands	O
-in	O
-clusters	O
-2	O
-and	O
-3	O
-.	O
-	O
-Ligands	O
-in	O
-cluster	O
-2	O
-and	O
-cluster	O
-3	O
-showed	O
-conformational	O
-heterogeneity	O
-in	O
-parts	O
-of	O
-the	O
-scaffold	O
-that	O
-were	O
-directed	O
-toward	O
-multiple	O
-regions	O
-of	O
-the	O
-receptor	O
-including	O
-h3	O
-,	O
-h8	O
-,	O
-h11	O
-,	O
-h12	O
-,	O
-and	O
-/	O
-or	O
-the	O
-β	O
-‐	O
-sheets	O
-(	O
-Fig	O
-EV5C	O
-–	O
-G	O
-).	O
-	O
-Hierarchical	O
-clustering	O
-revealed	O
-that	O
-many	O
-of	O
-the	O
-2	O
-,	O
-5	O
-‐	O
-DTP	O
-analogs	O
-recapitulated	O
-most	O
-of	O
-the	O
-peptide	O
-recruitment	O
-and	O
-dismissal	O
-patterns	O
-observed	O
-with	O
-E2	O
-(	O
-Fig	O
-6H	O
-).	O
-	O
-Also	O
-,	O
-we	O
-have	O
-used	O
-siRNA	O
-screening	O
-to	O
-identify	O
-a	O
-number	O
-of	O
-coregulators	O
-required	O
-for	O
-ERα	O
-‐	O
-mediated	O
-repression	O
-of	O
-the	O
-IL	O
-‐	O
-6	O
-gene	O
-(	O
-Nwachukwu	O
-et	O
-al	O
-,	O
-2014	O
-).	O
-	O
-Some	O
-of	O
-these	O
-ligands	O
-altered	O
-the	O
-shape	O
-of	O
-the	O
-AF	O
-‐	O
-2	O
-surface	O
-by	O
-perturbing	O
-the	O
-h3	O
-–	O
-h12	O
-interface	O
-,	O
-thus	O
-providing	O
-a	O
-route	O
-to	O
-new	O
-SERM	O
-‐	O
-like	O
-activity	O
-profiles	O
-by	O
-combining	O
-indirect	O
-and	O
-direct	O
-modulation	O
-of	O
-receptor	O
-structure	O
-.	O
-	O
-Incorporation	O
-of	O
-statistical	O
-approaches	O
-to	O
-understand	O
-relationships	O
-between	O
-structure	O
-and	O
-signaling	O
-variables	O
-moves	O
-us	O
-toward	O
-predictive	O
-models	O
-for	O
-complex	O
-ERα	O
-‐	O
-mediated	O
-responses	O
-such	O
-as	O
-in	O
-vivo	O
-uterine	O
-proliferation	O
-or	O
-tumor	O
-growth	O
-,	O
-and	O
-more	O
-generally	O
-toward	O
-structure	O
-‐	O
-based	O
-design	O
-for	O
-other	O
-allosteric	O
-drug	O
-targets	O
-including	O
-GPCRs	O
-and	O
-other	O
-nuclear	O
-receptors	O
-.	O
-	O
-We	O
-have	O
-solved	O
-the	O
-structure	O
-of	O
-the	O
-HR1	O
-domain	O
-of	O
-TOCA1	O
-,	O
-providing	O
-the	O
-first	O
-structural	O
-data	O
-for	O
-this	O
-protein	O
-.	O
-	O
-The	O
-superfamily	O
-can	O
-be	O
-divided	O
-into	O
-five	O
-families	O
-based	O
-on	O
-structural	O
-and	O
-functional	O
-similarities	O
-:	O
-Ras	O
-,	O
-Rho	O
-,	O
-Rab	O
-,	O
-Arf	O
-,	O
-and	O
-Ran	O
-.	O
-	O
-These	O
-regions	O
-are	O
-responsible	O
-for	O
-“	O
-sensing	O
-”	O
-the	O
-nucleotide	O
-state	O
-,	O
-with	O
-the	O
-GTP	O
--	O
-bound	O
-state	O
-showing	O
-greater	O
-rigidity	O
-and	O
-the	O
-GDP	O
--	O
-bound	O
-state	O
-adopting	O
-a	O
-more	O
-relaxed	O
-conformation	O
-(	O
-reviewed	O
-in	O
-Ref	O
-.).	O
-	O
-A	O
-number	O
-of	O
-RhoA	O
-and	O
-Rac1	O
-effector	O
-proteins	O
-,	O
-including	O
-the	O
-formins	O
-and	O
-members	O
-of	O
-the	O
-protein	O
-kinase	O
-C	O
--	O
-related	O
-kinase	O
-(	O
-PRK	O
-)	O
-6	O
-family	O
-,	O
-along	O
-with	O
-Cdc42	O
-effectors	O
-,	O
-including	O
-the	O
-Wiskott	O
--	O
-Aldrich	O
-syndrome	O
-(	O
-WASP	O
-)	O
-family	O
-and	O
-the	O
-transducer	O
-of	O
-Cdc42	O
--	O
-dependent	O
-actin	O
-assembly	O
-(	O
-TOCA	O
-)	O
-family	O
-,	O
-have	O
-also	O
-been	O
-linked	O
-to	O
-the	O
-pathways	O
-that	O
-govern	O
-cytoskeletal	O
-dynamics	O
-.	O
-	O
-Cdc42	O
-effectors	O
-,	O
-TOCA1	O
-and	O
-the	O
-ubiquitously	O
-expressed	O
-member	O
-of	O
-the	O
-WASP	O
-family	O
-,	O
-N	O
--	O
-WASP	O
-,	O
-have	O
-been	O
-implicated	O
-in	O
-the	O
-regulation	O
-of	O
-actin	O
-polymerization	O
-downstream	O
-of	O
-Cdc42	O
-and	O
-phosphatidylinositol	O
-4	O
-,	O
-5	O
--	O
-bisphosphate	O
-(	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-).	O
-	O
-The	O
-data	O
-were	O
-fitted	O
-to	O
-a	O
-binding	O
-isotherm	O
-to	O
-give	O
-an	O
-apparent	O
-Kd	O
-and	O
-are	O
-expressed	O
-as	O
-a	O
-percentage	O
-of	O
-the	O
-maximum	O
-signal	O
-;	O
-B	O
-and	O
-C	O
-,	O
-competition	O
-SPA	O
-experiments	O
-were	O
-carried	O
-out	O
-with	O
-the	O
-indicated	O
-concentrations	O
-of	O
-ACK	O
-GBD	O
-(	O
-B	O
-)	O
-or	O
-HR1	O
-domain	O
-(	O
-C	O
-)	O
-titrated	O
-into	O
-30	O
-nm	O
-GST	B-mutant
--	I-mutant
-ACK	I-mutant
-and	O
-either	O
-30	O
-nm	O
-Cdc42Δ7Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-or	O
-full	O
--	O
-length	O
-Cdc42Q61L	O
-·[	O
-3H	O
-]	O
-GTP	O
-.	O
-	O
-The	O
-binding	O
-experiments	O
-were	O
-repeated	O
-with	O
-full	O
--	O
-length	O
-[	O
-3H	O
-]	O
-GTP	O
-·	O
-Cdc42	O
-,	O
-but	O
-the	O
-affinity	O
-of	O
-the	O
-HR1	O
-domain	O
-for	O
-full	O
--	O
-length	O
-Cdc42	O
-was	O
-similar	O
-to	O
-its	O
-affinity	O
-for	O
-truncated	O
-Cdc42	O
-(	O
-Kd	O
-≈	O
-5	O
-μm	O
-;	O
-Fig	O
-.	O
-1C	O
-).	O
-	O
-Another	O
-possible	O
-explanation	O
-for	O
-the	O
-low	O
-affinities	O
-observed	O
-was	O
-that	O
-the	O
-HR1	O
-domain	O
-alone	O
-is	O
-not	O
-sufficient	O
-for	O
-maximal	O
-binding	O
-of	O
-the	O
-TOCA	O
-proteins	O
-to	O
-Cdc42	O
-and	O
-that	O
-the	O
-other	O
-domains	O
-are	O
-required	O
-.	O
-	O
-Furthermore	O
-,	O
-both	O
-BAR	O
-and	O
-SH3	O
-domains	O
-have	O
-been	O
-implicated	O
-in	O
-interactions	O
-with	O
-small	O
-G	O
-proteins	O
-(	O
-e	O
-.	O
-g	O
-.	O
-the	O
-BAR	O
-domain	O
-of	O
-Arfaptin2	O
-binds	O
-to	O
-Rac1	O
-and	O
-Arl1	O
-),	O
-while	O
-an	O
-SH3	O
-domain	O
-mediates	O
-the	O
-interaction	O
-between	O
-Rac1	O
-and	O
-the	O
-guanine	O
-nucleotide	O
-exchange	O
-factor	O
-,	O
-β	O
--	O
-PIX	O
-.	O
-	O
-Full	O
--	O
-length	O
-TOCA1	O
-and	O
-ΔSH3	B-mutant
-TOCA1	O
-bound	O
-with	O
-micromolar	O
-affinity	O
-(	O
-Fig	O
-.	O
-2B	O
-),	O
-in	O
-a	O
-similar	O
-manner	O
-to	O
-the	O
-isolated	O
-HR1	O
-domain	O
-(	O
-Fig	O
-.	O
-1A	O
-).	O
-	O
-There	O
-were	O
-1	O
-,	O
-845	O
-unambiguous	O
-NOEs	O
-and	O
-757	O
-ambiguous	O
-NOEs	O
-after	O
-eight	O
-iterations	O
-.	O
-	O
-A	O
-sequence	O
-alignment	O
-illustrating	O
-the	O
-secondary	O
-structure	O
-elements	O
-of	O
-the	O
-TOCA1	O
-and	O
-CIP4	O
-HR1	O
-domains	O
-and	O
-the	O
-HR1a	O
-and	O
-HR1b	O
-domains	O
-from	O
-PRK1	O
-is	O
-shown	O
-in	O
-Fig	O
-.	O
-3B	O
-.	O
-	O
-A	O
-series	O
-of	O
-15N	O
-HSQC	O
-experiments	O
-was	O
-recorded	O
-on	O
-15N	O
--	O
-labeled	O
-TOCA1	O
-HR1	O
-domain	O
-in	O
-the	O
-presence	O
-of	O
-increasing	O
-concentrations	O
-of	O
-unlabeled	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-to	O
-map	O
-the	O
-Cdc42	O
--	O
-binding	O
-surface	O
-.	O
-	O
-B	O
-,	O
-CSPs	O
-were	O
-calculated	O
-as	O
-described	O
-under	O
-“	O
-Experimental	O
-Procedures	O
-”	O
-and	O
-are	O
-shown	O
-for	O
-backbone	O
-and	O
-side	O
-chain	O
-NH	O
-groups	O
-.	O
-	O
-Residues	O
-that	O
-disappeared	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-were	O
-assigned	O
-a	O
-CSP	O
-of	O
-0	O
-.	O
-2	O
-but	O
-were	O
-excluded	O
-when	O
-calculating	O
-the	O
-mean	O
-CSP	O
-and	O
-are	O
-indicated	O
-with	O
-open	O
-bars	O
-.	O
-	O
-Residues	O
-with	O
-affected	O
-side	O
-chain	O
-CSPs	O
-derived	O
-from	O
-13C	O
-HSQCs	O
-are	O
-marked	O
-with	O
-green	O
-asterisks	O
-above	O
-the	O
-bars	O
-.	O
-	O
-The	O
-corresponding	O
-15N	O
-and	O
-13C	O
-NMR	O
-experiments	O
-were	O
-also	O
-recorded	O
-on	O
-15N	O
--	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-or	O
-15N	O
-/	O
-13C	O
--	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-in	O
-the	O
-presence	O
-of	O
-unlabeled	O
-HR1	O
-domain	O
-.	O
-	O
-A	O
-,	O
-the	O
-15N	O
-HSQC	O
-of	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-is	O
-shown	O
-in	O
-its	O
-free	O
-form	O
-(	O
-black	O
-)	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-excess	O
-TOCA1	O
-HR1	O
-domain	O
-(	O
-1	O
-:	O
-2	O
-.	O
-2	O
-,	O
-red	O
-).	O
-	O
-C	O
-,	O
-the	O
-residues	O
-with	O
-significantly	O
-affected	O
-backbone	O
-and	O
-side	O
-chain	O
-groups	O
-are	O
-highlighted	O
-on	O
-an	O
-NMR	O
-structure	O
-of	O
-free	O
-Cdc42Δ7Q61L	O
-·	O
-GMPPNP	O
-;	O
-those	O
-that	O
-are	O
-buried	O
-are	O
-colored	O
-dark	O
-blue	O
-,	O
-whereas	O
-those	O
-that	O
-are	O
-solvent	O
--	O
-accessible	O
-are	O
-colored	O
-red	O
-.	O
-	O
-Residues	O
-without	O
-information	O
-from	O
-shift	O
-mapping	O
-are	O
-colored	O
-gray	O
-.	O
-	O
-HADDOCK	O
-was	O
-therefore	O
-used	O
-to	O
-perform	O
-rigid	O
-body	O
-docking	O
-based	O
-on	O
-the	O
-structures	O
-of	O
-free	O
-HR1	O
-domain	O
-and	O
-Cdc42	O
-and	O
-ambiguous	O
-interaction	O
-restraints	O
-derived	O
-from	O
-the	O
-titration	O
-experiments	O
-described	O
-above	O
-.	O
-	O
-Residues	O
-equivalent	O
-to	O
-Rac1	O
-and	O
-RhoA	O
-contact	O
-sites	O
-but	O
-that	O
-are	O
-invisible	O
-in	O
-free	O
-Cdc42	O
-are	O
-gray	O
-.	O
-	O
-D	O
-,	O
-regions	O
-of	O
-interest	O
-of	O
-the	O
-Cdc42	O
-·	O
-HR1	O
-domain	O
-model	O
-.	O
-	O
-The	O
-four	O
-lowest	O
-energy	O
-structures	O
-in	O
-the	O
-chosen	O
-HADDOCK	O
-cluster	O
-are	O
-shown	O
-overlaid	O
-,	O
-with	O
-the	O
-residues	O
-of	O
-interest	O
-shown	O
-as	O
-sticks	O
-and	O
-labeled	O
-.	O
-	O
-Lys	O
--	O
-16Cdc42	O
-is	O
-unlikely	O
-to	O
-be	O
-a	O
-contact	O
-residue	O
-because	O
-it	O
-is	O
-involved	O
-in	O
-nucleotide	O
-binding	O
-,	O
-but	O
-the	O
-others	O
-may	O
-represent	O
-specific	O
-Cdc42	O
--	O
-TOCA1	O
-contacts	O
-.	O
-	O
-Cdc42	O
-is	O
-shown	O
-in	O
-green	O
-,	O
-and	O
-TOCA1	O
-is	O
-shown	O
-in	O
-purple	O
-.	O
-	O
-A	O
-comparison	O
-of	O
-the	O
-HSQC	O
-experiments	O
-recorded	O
-on	O
-15N	O
--	O
-Cdc42	O
-alone	O
-,	O
-in	O
-the	O
-presence	O
-of	O
-TOCA1	O
-HR1	O
-,	O
-N	O
--	O
-WASP	O
-GBD	O
-,	O
-or	O
-both	O
-,	O
-shows	O
-that	O
-the	O
-spectra	O
-in	O
-the	O
-presence	O
-of	O
-N	O
--	O
-WASP	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-both	O
-N	O
--	O
-WASP	O
-and	O
-TOCA1	O
-HR1	O
-are	O
-identical	O
-(	O
-Fig	O
-.	O
-7C	O
-).	O
-	O
-The	O
-spectrum	O
-when	O
-N	O
--	O
-WASP	O
-and	O
-TOCA1	O
-were	O
-equimolar	O
-was	O
-identical	O
-to	O
-that	O
-of	O
-the	O
-free	O
-HR1	O
-domain	O
-,	O
-whereas	O
-the	O
-spectrum	O
-in	O
-the	O
-presence	O
-of	O
-0	O
-.	O
-25	O
-eq	O
-of	O
-N	O
--	O
-WASP	O
-was	O
-intermediate	O
-between	O
-the	O
-TOCA1	O
-HR1	O
-free	O
-and	O
-complex	O
-spectra	O
-(	O
-Fig	O
-.	O
-7D	O
-).	O
-	O
-Taken	O
-together	O
-,	O
-the	O
-data	O
-in	O
-Fig	O
-.	O
-7	O
-,	O
-C	O
-and	O
-D	O
-,	O
-indicate	O
-unidirectional	O
-competition	O
-for	O
-Cdc42	O
-binding	O
-in	O
-which	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-displaces	O
-TOCA1	O
-HR1	O
-but	O
-not	O
-vice	O
-versa	O
-.	O
-	O
-The	O
-GBD	O
-presumably	O
-acts	O
-as	O
-a	O
-dominant	O
-negative	O
-,	O
-sequestering	O
-endogenous	O
-Cdc42	O
-and	O
-preventing	O
-endogenous	O
-full	O
--	O
-length	O
-N	O
--	O
-WASP	O
-from	O
-binding	O
-and	O
-becoming	O
-activated	O
-.	O
-	O
-The	O
-TOCA1	O
-HR1	O
-domain	O
-alone	O
-is	O
-sufficient	O
-for	O
-Cdc42	O
-binding	O
-in	O
-vitro	O
-,	O
-yet	O
-the	O
-affinity	O
-of	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-for	O
-Cdc42	O
-is	O
-remarkably	O
-low	O
-(	O
-Kd	O
-≈	O
-5	O
-μm	O
-).	O
-	O
-The	O
-polybasic	O
-tract	O
-within	O
-the	O
-C	O
--	O
-terminal	O
-region	O
-of	O
-Cdc42	O
-does	O
-not	O
-appear	O
-to	O
-be	O
-required	O
-for	O
-binding	O
-to	O
-TOCA1	O
-,	O
-which	O
-is	O
-in	O
-contrast	O
-to	O
-the	O
-interaction	O
-between	O
-Rac1	O
-and	O
-the	O
-HR1b	O
-domain	O
-of	O
-PRK1	O
-but	O
-more	O
-similar	O
-to	O
-the	O
-PRK1	O
-HR1a	O
--	O
-RhoA	O
-interaction	O
-.	O
-	O
-The	O
-equivalent	O
-Arg	O
-in	O
-Rac1	O
-and	O
-RhoA	O
-is	O
-pointing	O
-away	O
-from	O
-the	O
-HR1	O
-domains	O
-of	O
-PRK1	O
-.	O
-	O
-Furthermore	O
-,	O
-the	O
-isolated	O
-F	O
--	O
-BAR	O
-domain	O
-of	O
-FBP17	O
-has	O
-been	O
-shown	O
-to	O
-induce	O
-membrane	O
-tubulation	O
-of	O
-brain	O
-liposomes	O
-and	O
-BAR	O
-domain	O
-proteins	O
-that	O
-promote	O
-tubulation	O
-cluster	O
-on	O
-membranes	O
-at	O
-high	O
-densities	O
-.	O
-	O
-A	O
-substantial	O
-body	O
-of	O
-data	O
-has	O
-illuminated	O
-the	O
-complex	O
-regulation	O
-of	O
-WASP	O
-/	O
-N	O
--	O
-WASP	O
-proteins	O
-,	O
-and	O
-current	O
-evidence	O
-suggests	O
-that	O
-these	O
-allosteric	O
-activation	O
-mechanisms	O
-and	O
-oligomerization	O
-combine	O
-to	O
-regulate	O
-WASP	O
-activity	O
-,	O
-allowing	O
-the	O
-synchronization	O
-and	O
-integration	O
-of	O
-multiple	O
-potential	O
-activation	O
-signals	O
-(	O
-reviewed	O
-in	O
-Ref	O
-.).	O
-	O
-We	O
-envisage	O
-that	O
-TOCA1	O
-is	O
-first	O
-recruited	O
-to	O
-the	O
-appropriate	O
-membrane	O
-in	O
-response	O
-to	O
-PI	O
-(	O
-4	O
-,	O
-5	O
-)	O
-P2	O
-via	O
-its	O
-F	O
--	O
-BAR	O
-domain	O
-,	O
-where	O
-the	O
-local	O
-increase	O
-in	O
-concentration	O
-favors	O
-F	O
--	O
-BAR	O
--	O
-mediated	O
-dimerization	O
-of	O
-TOCA1	O
-.	O
-	O
-TOCA1	O
-can	O
-then	O
-recruit	O
-N	O
--	O
-WASP	O
-via	O
-an	O
-interaction	O
-between	O
-its	O
-SH3	O
-domain	O
-and	O
-the	O
-N	O
--	O
-WASP	O
-proline	O
--	O
-rich	O
-region	O
-.	O
-	O
-In	O
-a	O
-cellular	O
-context	O
-,	O
-full	O
--	O
-length	O
-TOCA1	O
-and	O
-N	O
--	O
-WASP	O
-are	O
-likely	O
-to	O
-have	O
-similar	O
-affinities	O
-for	O
-active	O
-Cdc42	O
-,	O
-but	O
-in	O
-the	O
-unfolded	O
-,	O
-active	O
-conformation	O
-,	O
-the	O
-affinity	O
-of	O
-N	O
--	O
-WASP	O
-for	O
-Cdc42	O
-dramatically	O
-increases	O
-.	O
-	O
-Our	O
-binding	O
-data	O
-suggest	O
-that	O
-TOCA1	O
-HR1	O
-binding	O
-is	O
-not	O
-allosterically	O
-regulated	O
-,	O
-and	O
-our	O
-NMR	O
-data	O
-,	O
-along	O
-with	O
-the	O
-high	O
-stability	O
-of	O
-TOCA1	O
-HR1	O
-,	O
-suggest	O
-that	O
-there	O
-is	O
-no	O
-widespread	O
-conformational	O
-change	O
-in	O
-the	O
-presence	O
-of	O
-Cdc42	O
-.	O
-	O
-As	O
-full	O
--	O
-length	O
-TOCA1	O
-and	O
-the	O
-isolated	O
-HR1	O
-domain	O
-bind	O
-Cdc42	O
-with	O
-similar	O
-affinities	O
-,	O
-the	O
-N	O
--	O
-WASP	O
--	O
-Cdc42	O
-interaction	O
-will	O
-be	O
-favored	O
-because	O
-the	O
-N	O
--	O
-WASP	O
-GBD	O
-can	O
-easily	O
-outcompete	O
-the	O
-TOCA1	O
-HR1	O
-for	O
-Cdc42	O
-.	O
-	O
-Potentially	O
-,	O
-the	O
-TOCA1	O
--	O
-Cdc42	O
-interaction	O
-functions	O
-to	O
-position	O
-N	O
--	O
-WASP	O
-and	O
-Cdc42	O
-such	O
-that	O
-they	O
-are	O
-poised	O
-to	O
-interact	O
-with	O
-high	O
-affinity	O
-.	O
-	O
-There	O
-is	O
-an	O
-advantage	O
-to	O
-such	O
-an	O
-effector	O
-handover	O
-,	O
-in	O
-that	O
-N	O
--	O
-WASP	O
-would	O
-only	O
-be	O
-robustly	O
-recruited	O
-when	O
-F	O
--	O
-BAR	O
-domains	O
-are	O
-already	O
-present	O
-.	O
-	O
-F	O
--	O
-BAR	O
-oligomerization	O
-is	O
-expected	O
-to	O
-occur	O
-following	O
-membrane	O
-binding	O
-,	O
-but	O
-a	O
-single	O
-monomer	O
-is	O
-shown	O
-for	O
-clarity	O
-.	O
-	O
-The	O
-HR1TOCA1	O
--	O
-Cdc42	O
-and	O
-SH3TOCA1	O
--	O
-N	O
--	O
-WASP	O
-interactions	O
-position	O
-Cdc42	O
-and	O
-N	O
--	O
-WASP	O
-for	O
-binding	O
-.	O
-	O
-Step	O
-4	O
-,	O
-the	O
-core	O
-CRIB	O
-binds	O
-with	O
-high	O
-affinity	O
-while	O
-the	O
-region	O
-C	O
--	O
-terminal	O
-to	O
-the	O
-CRIB	O
-displaces	O
-the	O
-TOCA1	O
-HR1	O
-domain	O
-and	O
-increases	O
-the	O
-affinity	O
-of	O
-the	O
-N	O
--	O
-WASP	O
--	O
-Cdc42	O
-interaction	O
-further	O
-.	O
-	O
-WH1	O
-,	O
-WASP	O
-homology	O
-1	O
-domain	O
-;	O
-PP	O
-,	O
-proline	O
--	O
-rich	O
-region	O
-;	O
-VCA	O
-,	O
-verprolin	O
-homology	O
-,	O
-cofilin	O
-homology	O
-,	O
-acidic	O
-region	O
-.	O
-	O
-We	O
-envisage	O
-a	O
-complex	O
-interplay	O
-of	O
-equilibria	O
-between	O
-free	O
-and	O
-bound	O
-,	O
-active	O
-and	O
-inactive	O
-Cdc42	O
-,	O
-TOCA	O
-family	O
-,	O
-and	O
-WASP	O
-family	O
-proteins	O
-,	O
-facilitating	O
-a	O
-tightly	O
-spatially	O
-and	O
-temporally	O
-regulated	O
-pathway	O
-requiring	O
-numerous	O
-simultaneous	O
-events	O
-in	O
-order	O
-to	O
-achieve	O
-appropriate	O
-and	O
-robust	O
-activation	O
-of	O
-the	O
-downstream	O
-pathway	O
-.	O
-	O
-Acetyl	O
--	O
-CoA	O
-carboxylases	O
-(	O
-ACCs	O
-)	O
-catalyse	O
-the	O
-committed	O
-step	O
-in	O
-fatty	O
--	O
-acid	O
-biosynthesis	O
-:	O
-the	O
-ATP	O
--	O
-dependent	O
-carboxylation	O
-of	O
-acetyl	O
--	O
-CoA	O
-to	O
-malonyl	O
--	O
-CoA	O
-.	O
-They	O
-are	O
-important	O
-regulatory	O
-hubs	O
-for	O
-metabolic	O
-control	O
-and	O
-relevant	O
-drug	O
-targets	O
-for	O
-the	O
-treatment	O
-of	O
-the	O
-metabolic	O
-syndrome	O
-and	O
-cancer	O
-.	O
-	O
-Combining	O
-the	O
-yeast	O
-CD	O
-structure	O
-with	O
-intermediate	O
-and	O
-low	O
--	O
-resolution	O
-data	O
-of	O
-larger	B-mutant
-fragments	I-mutant
-up	O
-to	O
-intact	O
-ACCs	O
-provides	O
-a	O
-comprehensive	O
-characterization	O
-of	O
-the	O
-dynamic	O
-fungal	O
-ACC	O
-architecture	O
-.	O
-	O
-In	O
-addition	O
-to	O
-the	O
-canonical	O
-ACC	O
-components	O
-,	O
-eukaryotic	O
-ACCs	O
-contain	O
-two	O
-non	O
--	O
-catalytic	O
-regions	O
-,	O
-the	O
-large	O
-central	O
-domain	O
-(	O
-CD	O
-)	O
-and	O
-the	O
-BC	O
-–	O
-CT	O
-interaction	O
-domain	O
-(	O
-BT	O
-).	O
-	O
-The	O
-function	O
-of	O
-this	O
-domain	O
-remains	O
-poorly	O
-characterized	O
-,	O
-although	O
-phosphorylation	O
-of	O
-several	O
-serine	O
-residues	O
-in	O
-the	O
-CD	O
-regulates	O
-ACC	O
-activity	O
-.	O
-	O
-Of	O
-these	O
-,	O
-only	O
-Ser1157	O
-is	O
-highly	O
-conserved	O
-in	O
-fungal	O
-ACC	O
-and	O
-aligns	O
-to	O
-Ser1216	O
-in	O
-human	O
-ACC1	O
-.	O
-	O
-Integrating	O
-these	O
-data	O
-with	O
-small	O
--	O
-angle	O
-X	O
--	O
-ray	O
-scattering	O
-(	O
-SAXS	O
-)	O
-and	O
-electron	O
-microscopy	O
-(	O
-EM	O
-)	O
-observations	O
-yield	O
-a	O
-comprehensive	O
-representation	O
-of	O
-the	O
-dynamic	O
-structure	O
-and	O
-regulation	O
-of	O
-fungal	O
-ACC	O
-.	O
-	O
-First	O
-,	O
-we	O
-focused	O
-on	O
-structure	O
-determination	O
-of	O
-the	O
-82	O
--	O
-kDa	O
-CD	O
-.	O
-	O
-Close	O
-structural	O
-homologues	O
-could	O
-not	O
-be	O
-found	O
-for	O
-the	O
-CDN	O
-or	O
-the	O
-CDC	O
-domains	O
-.	O
-	O
-To	O
-define	O
-the	O
-functional	O
-state	O
-of	O
-insect	O
--	O
-cell	O
--	O
-expressed	O
-ACC	O
-variants	O
-,	O
-we	O
-employed	O
-mass	O
-spectrometry	O
-(	O
-MS	O
-)	O
-for	O
-phosphorylation	O
-site	O
-detection	O
-.	O
-	O
-The	O
-SceCD	O
-structure	O
-thus	O
-authentically	O
-represents	O
-the	O
-state	O
-of	O
-SceACC	O
-,	O
-where	O
-the	O
-enzyme	O
-is	O
-inhibited	O
-by	O
-SNF1	O
--	O
-dependent	O
-phosphorylation	O
-.	O
-	O
-Each	O
-of	O
-the	O
-four	O
-CD	O
-domains	O
-in	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-individually	O
-resembles	O
-the	O
-corresponding	O
-SceCD	O
-domain	O
-;	O
-however	O
-,	O
-human	O
-and	O
-yeast	O
-CDs	O
-exhibit	O
-distinct	O
-overall	O
-structures	O
-.	O
-	O
-In	O
-agreement	O
-with	O
-their	O
-tight	O
-interaction	O
-in	O
-SceCD	O
-,	O
-the	O
-relative	O
-spatial	O
-arrangement	O
-of	O
-CDL	O
-and	O
-CDC1	O
-is	O
-preserved	O
-in	O
-HsaBT	B-mutant
--	I-mutant
-CD	I-mutant
-,	O
-but	O
-the	O
-human	O
-CDL	O
-/	O
-CDC1	O
-didomain	O
-is	O
-tilted	O
-by	O
-30	O
-°	O
-based	O
-on	O
-a	O
-superposition	O
-of	O
-human	O
-and	O
-yeast	O
-CDC2	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-It	O
-resembles	O
-the	O
-BT	O
-of	O
-propionyl	O
--	O
-CoA	O
-carboxylase	O
-;	O
-only	O
-the	O
-four	O
-C	O
--	O
-terminal	O
-strands	O
-of	O
-the	O
-β	O
--	O
-barrel	O
-are	O
-slightly	O
-tilted	O
-.	O
-	O
-The	O
-absence	O
-of	O
-the	O
-regulatory	O
-loop	O
-might	O
-be	O
-linked	O
-to	O
-the	O
-less	O
--	O
-restrained	O
-interface	O
-of	O
-CDL	O
-/	O
-CDC1	O
-and	O
-CDC2	O
-and	O
-altered	O
-relative	O
-orientations	O
-of	O
-these	O
-domains	O
-.	O
-	O
-To	O
-further	O
-obtain	O
-insights	O
-into	O
-the	O
-functional	O
-architecture	O
-of	O
-fungal	O
-ACC	O
-,	O
-we	O
-characterized	O
-larger	B-mutant
-multidomain	I-mutant
-fragments	I-mutant
-up	O
-to	O
-the	O
-intact	O
-enzymes	O
-.	O
-	O
-No	O
-crystals	O
-diffracting	O
-to	O
-sufficient	O
-resolution	O
-were	O
-obtained	O
-for	O
-larger	B-mutant
-BC	I-mutant
--	I-mutant
-containing	I-mutant
-fragments	I-mutant
-,	O
-or	O
-for	O
-full	O
--	O
-length	O
-Cth	O
-or	O
-SceACC	O
-.	O
-	O
-However	O
-,	O
-molecular	O
-replacement	O
-did	O
-not	O
-reveal	O
-a	O
-unique	O
-positioning	O
-of	O
-the	O
-BC	O
-domain	O
-.	O
-	O
-Indeed	O
-,	O
-the	O
-comparison	O
-of	O
-the	O
-positioning	O
-of	O
-eight	O
-instances	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-part	O
-of	O
-CD	O
-relative	O
-to	O
-CT	O
-in	O
-crystal	O
-structures	O
-determined	O
-here	O
-,	O
-reveals	O
-flexible	O
-interdomain	O
-linking	O
-(	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-Conformational	O
-variability	O
-in	O
-the	O
-CD	O
-thus	O
-contributes	O
-considerably	O
-to	O
-variations	O
-in	O
-the	O
-spacing	O
-between	O
-the	O
-BC	O
-and	O
-CT	O
-domains	O
-,	O
-and	O
-may	O
-extend	O
-to	O
-distance	O
-variations	O
-beyond	O
-the	O
-mobility	O
-range	O
-of	O
-the	O
-flexibly	O
-tethered	O
-BCCP	O
-.	O
-	O
-SAXS	O
-analysis	O
-of	O
-CthACC	O
-agrees	O
-with	O
-a	O
-dimeric	O
-state	O
-and	O
-an	O
-elongated	O
-shape	O
-with	O
-a	O
-maximum	O
-extent	O
-of	O
-350	O
-Å	O
-(	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-The	O
-flexibility	O
-in	O
-the	O
-CDC2	O
-/	O
-CT	O
-hinge	O
-appears	O
-substantially	O
-larger	O
-than	O
-the	O
-variations	O
-observed	O
-in	O
-the	O
-set	O
-of	O
-crystal	O
-structures	O
-.	O
-	O
-The	O
-phosphorylated	O
-regulatory	O
-loop	O
-binds	O
-to	O
-an	O
-allosteric	O
-site	O
-at	O
-the	O
-interface	O
-of	O
-two	O
-non	O
--	O
-catalytic	O
-domains	O
-and	O
-restricts	O
-conformational	O
-freedom	O
-at	O
-several	O
-hinges	O
-in	O
-the	O
-dynamic	O
-ACC	O
-.	O
-	O
-(	O
-b	O
-)	O
-Cartoon	O
-representation	O
-of	O
-the	O
-SceCD	O
-crystal	O
-structure	O
-.	O
-	O
-(	O
-c	O
-)	O
-Superposition	O
-of	O
-CDC1	O
-and	O
-CDC2	O
-reveals	O
-highly	O
-conserved	O
-folds	O
-.	O
-(	O
-d	O
-)	O
-The	O
-regulatory	O
-loop	O
-with	O
-the	O
-phosphorylated	O
-Ser1157	O
-is	O
-bound	O
-into	O
-a	O
-crevice	O
-between	O
-CDC1	O
-and	O
-CDC2	O
-,	O
-the	O
-conserved	O
-residues	O
-Arg1173	O
-and	O
-Arg1260	O
-coordinate	O
-the	O
-phosphoryl	O
--	O
-group	O
-.	O
-	O
-The	O
-range	O
-of	O
-hinge	O
-bending	O
-is	O
-indicated	O
-and	O
-the	O
-connection	O
-points	O
-between	O
-CDC2	O
-and	O
-CT	O
-(	O
-blue	O
-)	O
-as	O
-well	O
-as	O
-between	O
-CDC1	O
-and	O
-CDC2	O
-(	O
-green	O
-and	O
-grey	O
-)	O
-are	O
-marked	O
-as	O
-spheres	O
-.	O
-	O
-The	O
-connection	O
-points	O
-from	O
-CDC1	O
-to	O
-CDC2	O
-and	O
-to	O
-CDL	O
-are	O
-represented	O
-by	O
-green	O
-spheres	O
-.	O
-	O
-The	O
-domains	O
-are	O
-labelled	O
-and	O
-the	O
-distances	O
-between	O
-the	O
-N	O
-termini	O
-of	O
-CDN	O
-(	O
-spheres	O
-)	O
-in	O
-the	O
-compared	O
-structures	O
-are	O
-indicated	O
-.	O
-	O
-The	O
-two	O
-kinases	O
-exhibit	O
-nearly	O
-identical	O
-overall	O
-architecture	O
-,	O
-with	O
-both	O
-kinases	O
-possessing	O
-ATP	O
-hydrolysis	O
-activity	O
-in	O
-the	O
-absence	O
-of	O
-substrates	O
-.	O
-	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-display	O
-a	O
-sequence	O
-identity	O
-of	O
-44	O
-.	O
-9	O
-%,	O
-and	O
-belong	O
-to	O
-the	O
-ribulokinase	O
--	O
-like	O
-carbohydrate	O
-kinases	O
-,	O
-a	O
-sub	O
--	O
-family	O
-of	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-.	O
-	O
-However	O
-,	O
-the	O
-function	O
-of	O
-XK	O
--	O
-1	O
-(	O
-At2g21370	O
-)	O
-inside	O
-the	O
-chloroplast	O
-stroma	O
-has	O
-remained	O
-unknown	O
-.	O
-	O
-Among	O
-all	O
-these	O
-structural	O
-elements	O
-,	O
-α4	O
-/	O
-α5	O
-/	O
-α11	O
-/	O
-α18	O
-,	O
-β3	O
-/	O
-β2	O
-/	O
-β1	O
-/	O
-β6	O
-/	O
-β19	O
-/	O
-β20	O
-/	O
-β17	O
-and	O
-α21	O
-/	O
-α32	O
-form	O
-three	O
-patches	O
-,	O
-referred	O
-to	O
-as	O
-A1	O
-,	O
-B1	O
-and	O
-A2	O
-,	O
-exhibiting	O
-the	O
-core	O
-region	O
-.	O
-	O
-The	O
-structures	O
-most	O
-closely	O
-related	O
-to	O
-SePSK	O
-are	O
-xylulose	O
-kinase	O
-,	O
-glycerol	O
-kinase	O
-and	O
-ribulose	O
-kinase	O
-,	O
-implying	O
-that	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-might	O
-function	O
-similarly	O
-to	O
-these	O
-kinases	O
-.	O
-	O
-To	O
-further	O
-identify	O
-the	O
-actual	O
-substrate	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-,	O
-five	O
-different	O
-sugar	O
-molecules	O
-,	O
-including	O
-D	O
--	O
-ribulose	O
-,	O
-L	O
--	O
-ribulose	O
-,	O
-D	O
--	O
-xylulose	O
-,	O
-L	O
--	O
-xylulose	O
-and	O
-Glycerol	O
-,	O
-were	O
-used	O
-in	O
-enzymatic	O
-activity	O
-assays	O
-.	O
-	O
-While	O
-the	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-SePSK	O
-greatly	O
-increases	O
-upon	O
-addition	O
-of	O
-D	O
--	O
-ribulose	O
-(	O
-DR	O
-).	O
-	O
-(	O
-B	O
-)	O
-The	O
-ATP	O
-hydrolysis	O
-activity	O
-of	O
-SePSK	O
-with	O
-addition	O
-of	O
-five	O
-different	O
-substrates	O
-.	O
-	O
-To	O
-obtain	O
-more	O
-detailed	O
-information	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-in	O
-complex	O
-with	O
-ATP	O
-,	O
-we	O
-soaked	O
-the	O
-apo	O
--	O
-crystals	O
-in	O
-the	O
-reservoir	O
-adding	O
-cofactor	O
-ATP	O
-,	O
-and	O
-obtained	O
-the	O
-structures	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-bound	O
-with	O
-ATP	O
-at	O
-the	O
-resolution	O
-of	O
-2	O
-.	O
-3	O
-Å	O
-and	O
-1	O
-.	O
-8	O
-Å	O
-,	O
-respectively	O
-.	O
-	O
-Thus	O
-the	O
-two	O
-structures	O
-were	O
-named	O
-ADP	O
--	O
-SePSK	O
-and	O
-ADP	O
--	O
-AtXK	O
--	O
-1	O
-,	O
-respectively	O
-.	O
-	O
-Structure	O
-of	O
-SePSK	O
-in	O
-complex	O
-with	O
-AMP	O
--	O
-PNP	O
-.	O
-	O
-(	O
-A	O
-)	O
-The	O
-electron	O
-density	O
-of	O
-AMP	O
--	O
-PNP	O
-.	O
-	O
-The	O
-AMP	O
--	O
-PNP	O
-is	O
-depicted	O
-as	O
-sticks	O
-with	O
-its	O
-ǀFoǀ	O
--	O
-ǀFcǀ	O
-map	O
-contoured	O
-at	O
-3	O
-σ	O
-shown	O
-as	O
-cyan	O
-mesh	O
-.	O
-	O
-(	O
-B	O
-)	O
-The	O
-AMP	O
--	O
-PNP	O
-binding	O
-pocket	O
-.	O
-	O
-The	O
-AMP	O
--	O
-PNP	O
-and	O
-coordinated	O
-residues	O
-are	O
-shown	O
-as	O
-sticks	O
-.	O
-	O
-The	O
-potential	O
-substrate	O
-binding	O
-site	O
-in	O
-SePSK	O
-	O
-The	O
-RBL1	O
-and	O
-RBL2	O
-are	O
-depicted	O
-as	O
-sticks	O
-.	O
-(	O
-B	O
-)	O
-Interaction	O
-of	O
-two	O
-D	O
--	O
-ribulose	O
-molecules	O
-(	O
-RBL1	O
-and	O
-RBL2	O
-)	O
-with	O
-SePSK	O
-.	O
-	O
-The	O
-RBL	O
-molecules	O
-(	O
-carbon	O
-atoms	O
-colored	O
-yellow	O
-)	O
-and	O
-amino	O
-acid	O
-residues	O
-of	O
-SePSK	O
-(	O
-carbon	O
-atoms	O
-colored	O
-green	O
-)	O
-involved	O
-in	O
-RBL	O
-interaction	O
-are	O
-shown	O
-as	O
-sticks	O
-.	O
-	O
-The	O
-hydroxyl	O
-group	O
-of	O
-Ser12	O
-coordinates	O
-with	O
-O2	O
-of	O
-RBL2	O
-.	O
-	O
-Structural	O
-comparison	O
-of	O
-SePSK	O
-and	O
-AtXK	O
--	O
-1	O
-showed	O
-that	O
-while	O
-the	O
-RBL1	O
-binding	O
-pocket	O
-is	O
-conserved	O
-,	O
-the	O
-RBL2	O
-pocket	O
-is	O
-disrupted	O
-in	O
-AtXK	O
--	O
-1	O
-structure	O
-,	O
-despite	O
-the	O
-fact	O
-that	O
-the	O
-residues	O
-interacting	O
-with	O
-RBL2	O
-are	O
-highly	O
-conserved	O
-between	O
-the	O
-two	O
-proteins	O
-.	O
-	O
-In	O
-the	O
-RBL	O
--	O
-SePSK	O
-structure	O
-,	O
-a	O
-2	O
-.	O
-6	O
-Å	O
-hydrogen	O
-bond	O
-is	O
-present	O
-between	O
-RBL2	O
-and	O
-Ser12	O
-(	O
-Fig	O
-4B	O
-),	O
-while	O
-in	O
-the	O
-AtXK	O
--	O
-1	O
-structure	O
-this	O
-hydrogen	O
-bond	O
-with	O
-the	O
-corresponding	O
-residue	O
-(	O
-Ser22	O
-)	O
-is	O
-broken	O
-.	O
-	O
-This	O
-change	O
-might	O
-be	O
-the	O
-reason	O
-that	O
-AtXK	O
--	O
-1	O
-only	O
-shows	O
-limited	O
-increasing	O
-in	O
-its	O
-ATP	O
-hydrolysis	O
-ability	O
-upon	O
-adding	O
-D	O
--	O
-ribulose	O
-as	O
-a	O
-substrate	O
-after	O
-comparing	O
-with	O
-SePSK	O
-(	O
-Fig	O
-2C	O
-).	O
-	O
-The	O
-results	O
-showed	O
-that	O
-the	O
-affinity	O
-of	O
-D8A	B-mutant
--	O
-SePSK	O
-with	O
-D	O
--	O
-ribulose	O
-is	O
-weaker	O
-than	O
-that	O
-of	O
-WT	O
-with	O
-a	O
-reduction	O
-of	O
-approx	O
-.	O
-	O
-This	O
-distance	O
-between	O
-RBL2	O
-and	O
-AMP	O
--	O
-PNP	O
--	O
-γ	O
--	O
-phosphate	O
-is	O
-close	O
-enough	O
-to	O
-facilitate	O
-phosphate	O
-transferring	O
-.	O
-	O
-Together	O
-,	O
-our	O
-superposition	O
-results	O
-provided	O
-snapshots	O
-of	O
-the	O
-conformational	O
-changes	O
-at	O
-different	O
-catalytic	O
-stages	O
-of	O
-SePSK	O
-and	O
-potentially	O
-revealed	O
-the	O
-closed	O
-form	O
-of	O
-SePSK	O
-.	O
-	O
-In	O
-summary	O
-,	O
-our	O
-structural	O
-and	O
-enzymatic	O
-analyses	O
-provide	O
-evidence	O
-that	O
-SePSK	O
-shows	O
-D	O
--	O
-ribulose	O
-kinase	O
-activity	O
-,	O
-and	O
-exhibits	O
-the	O
-conserved	O
-features	O
-of	O
-FGGY	O
-family	O
-carbohydrate	O
-kinases	O
-.	O
-	O
-Three	O
-conserved	O
-residues	O
-in	O
-SePSK	O
-were	O
-identified	O
-to	O
-be	O
-essential	O
-for	O
-this	O
-function	O
-.	O
-	O
-We	O
-now	O
-present	O
-cryo	O
--	O
-electron	O
-microscopy	O
-3D	O
-reconstructions	O
-of	O
-the	O
-E	O
-.	O
-coli	O
-LdcI	O
-and	O
-LdcC	O
-,	O
-and	O
-an	O
-improved	O
-map	O
-of	O
-the	O
-LdcI	O
-bound	O
-to	O
-the	O
-LARA	O
-domain	O
-of	O
-RavA	O
-,	O
-at	O
-pH	O
-optimal	O
-for	O
-their	O
-enzymatic	O
-activity	O
-.	O
-	O
-They	O
-counteract	O
-acid	O
-stress	O
-experienced	O
-by	O
-the	O
-bacterium	O
-in	O
-the	O
-host	O
-digestive	O
-and	O
-urinary	O
-tract	O
-,	O
-and	O
-in	O
-particular	O
-in	O
-the	O
-extremely	O
-acidic	O
-stomach	O
-.	O
-	O
-Monomers	O
-tightly	O
-associate	O
-via	O
-their	O
-core	O
-domains	O
-into	O
-2	O
--	O
-fold	O
-symmetrical	O
-dimers	O
-with	O
-two	O
-complete	O
-active	O
-sites	O
-,	O
-and	O
-further	O
-build	O
-a	O
-toroidal	O
-D5	O
--	O
-symmetrical	O
-structure	O
-held	O
-by	O
-the	O
-wing	O
-and	O
-core	O
-domain	O
-interactions	O
-around	O
-the	O
-central	O
-pore	O
-,	O
-with	O
-the	O
-CTDs	O
-at	O
-the	O
-periphery	O
-.	O
-	O
-This	O
-allowed	O
-us	O
-to	O
-make	O
-a	O
-pseudoatomic	O
-model	O
-of	O
-the	O
-whole	O
-assembly	O
-,	O
-underpinned	O
-by	O
-a	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcI	O
--	O
-LARA	O
-complex	O
-(	O
-with	O
-LARA	O
-standing	O
-for	O
-LdcI	O
-associating	O
-domain	O
-of	O
-RavA	O
-),	O
-and	O
-to	O
-identify	O
-conformational	O
-rearrangements	O
-and	O
-specific	O
-elements	O
-essential	O
-for	O
-complex	O
-formation	O
-.	O
-	O
-The	O
-main	O
-determinants	O
-of	O
-the	O
-LdcI	O
--	O
-RavA	O
-cage	O
-assembly	O
-appeared	O
-to	O
-be	O
-the	O
-N	O
--	O
-terminal	O
-loop	O
-of	O
-the	O
-LARA	O
-domain	O
-of	O
-RavA	O
-and	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-LdcI	O
-.	O
-	O
-Finally	O
-,	O
-we	O
-performed	O
-multiple	O
-sequence	O
-alignment	O
-of	O
-22	O
-lysine	O
-decarboxylases	O
-from	O
-Enterobacteriaceae	O
-containing	O
-the	O
-ravA	O
--	O
-viaA	O
-operon	O
-in	O
-their	O
-genome	O
-.	O
-	O
-Significant	O
-differences	O
-between	O
-these	O
-pseudoatomic	O
-models	O
-can	O
-be	O
-interpreted	O
-as	O
-movements	O
-between	O
-specific	O
-biological	O
-states	O
-of	O
-the	O
-proteins	O
-as	O
-described	O
-below	O
-.	O
-	O
-Both	O
-visual	O
-inspection	O
-(	O
-Fig	O
-.	O
-2	O
-)	O
-and	O
-RMSD	O
-calculations	O
-(	O
-Table	O
-S2	O
-)	O
-show	O
-that	O
-globally	O
-the	O
-three	O
-structures	O
-at	O
-active	O
-pH	O
-(	O
-LdcIa	O
-,	O
-LdcI	O
--	O
-LARA	O
-and	O
-LdcC	O
-)	O
-are	O
-more	O
-similar	O
-to	O
-each	O
-other	O
-than	O
-to	O
-the	O
-structure	O
-determined	O
-at	O
-high	O
-pH	O
-conditions	O
-(	O
-LdcIi	O
-).	O
-	O
-The	O
-core	O
-domain	O
-is	O
-built	O
-by	O
-the	O
-PLP	O
--	O
-binding	O
-subdomain	O
-(	O
-PLP	O
--	O
-SD	O
-,	O
-residues	O
-184	O
-–	O
-417	O
-)	O
-flanked	O
-by	O
-two	O
-smaller	O
-subdomains	O
-rich	O
-in	O
-partly	O
-disordered	O
-loops	O
-–	O
-the	O
-linker	O
-region	O
-(	O
-residues	O
-130	O
-–	O
-183	O
-)	O
-and	O
-the	O
-subdomain	O
-4	O
-(	O
-residues	O
-418	O
-–	O
-563	O
-).	O
-	O
-In	O
-particular	O
-,	O
-transition	O
-from	O
-LdcIi	O
-to	O
-LdcI	O
--	O
-LARA	O
-involves	O
-~	O
-3	O
-.	O
-5	O
-Å	O
-and	O
-~	O
-4	O
-.	O
-5	O
-Å	O
-shifts	O
-away	O
-from	O
-the	O
-5	O
--	O
-fold	O
-axis	O
-in	O
-the	O
-active	O
-site	O
-α	O
--	O
-helices	O
-spanning	O
-residues	O
-218	O
-–	O
-232	O
-and	O
-246	O
-–	O
-254	O
-respectively	O
-(	O
-Fig	O
-.	O
-3C	O
-–	O
-E	O
-).	O
-	O
-At	O
-this	O
-resolution	O
-,	O
-the	O
-apo	O
--	O
-LdcIi	O
-and	O
-ppGpp	O
--	O
-LdcIi	O
-structures	O
-(	O
-both	O
-solved	O
-at	O
-pH	O
-8	O
-.	O
-5	O
-)	O
-appeared	O
-indistinguishable	O
-except	O
-for	O
-the	O
-presence	O
-of	O
-ppGpp	O
-(	O
-Fig	O
-.	O
-S11	O
-in	O
-ref	O
-.	O
-).	O
-	O
-Yet	O
-the	O
-superposition	O
-of	O
-the	O
-decamers	O
-lays	O
-bare	O
-a	O
-progressive	O
-movement	O
-of	O
-the	O
-CTD	O
-as	O
-a	O
-whole	O
-upon	O
-enzyme	O
-activation	O
-by	O
-pH	O
-and	O
-the	O
-binding	O
-of	O
-LARA	O
-.	O
-	O
-On	O
-the	O
-contrary	O
-,	O
-introduction	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-of	O
-LdcI	O
-into	O
-LdcC	O
-led	O
-to	O
-an	O
-assembly	O
-of	O
-the	O
-LdcCI	O
--	O
-RavA	O
-complex	O
-.	O
-	O
-(	O
-A	O
-,	O
-C	O
-,	O
-E	O
-)	O
-cryoEM	O
-map	O
-of	O
-the	O
-LdcC	O
-(	O
-A	O
-),	O
-LdcIa	O
-(	O
-C	O
-)	O
-and	O
-LdcI	O
--	O
-LARA	O
-(	O
-E	O
-)	O
-decamers	O
-with	O
-one	O
-protomer	O
-in	O
-light	O
-grey	O
-.	O
-	O
-Only	O
-one	O
-of	O
-the	O
-two	O
-rings	O
-of	O
-the	O
-double	O
-toroid	O
-is	O
-shown	O
-for	O
-clarity	O
-.	O
-	O
-Conformational	O
-rearrangements	O
-in	O
-the	O
-enzyme	O
-active	O
-site	O
-.	O
-	O
-(	O
-A	O
-)	O
-A	O
-slice	O
-through	O
-the	O
-pseudoatomic	O
-models	O
-of	O
-the	O
-LdcIa	O
-(	O
-purple	O
-)	O
-and	O
-LdcC	O
-(	O
-green	O
-)	O
-monomers	O
-extracted	O
-from	O
-the	O
-superimposed	O
-decamers	O
-(	O
-Fig	O
-.	O
-2	O
-).	O
-(	O
-B	O
-)	O
-The	O
-C	O
--	O
-terminal	O
-β	O
--	O
-sheet	O
-in	O
-LdcIa	O
-and	O
-LdcC	O
-enlarged	O
-from	O
-(	O
-A	O
-,	O
-C	O
-)	O
-Exchanged	O
-primary	O
-sequences	O
-(	O
-capital	O
-letters	O
-)	O
-and	O
-their	O
-immediate	O
-vicinity	O
-(	O
-lower	O
-case	O
-letters	O
-)	O
-colored	O
-as	O
-in	O
-(	O
-A	O
-,	O
-B	O
-),	O
-with	O
-the	O
-corresponding	O
-secondary	O
-structure	O
-elements	O
-and	O
-the	O
-amino	O
-acid	O
-numbering	O
-shown	O
-.	O
-	O
-(	O
-A	O
-)	O
-Maximum	O
-likelihood	O
-tree	O
-with	O
-the	O
-“	O
-LdcC	O
--	O
-like	O
-”	O
-and	O
-the	O
-“	O
-LdcI	O
--	O
-like	O
-”	O
-groups	O
-highlighted	O
-in	O
-green	O
-and	O
-pink	O
-,	O
-respectively	O
-.	O
-	O
-Structural	O
-basis	O
-for	O
-Mep2	O
-ammonium	O
-transceptor	O
-activation	O
-by	O
-phosphorylation	O
-	O
-Mep2	O
-proteins	O
-are	O
-fungal	O
-transceptors	O
-that	O
-play	O
-an	O
-important	O
-role	O
-as	O
-ammonium	O
-sensors	O
-in	O
-fungal	O
-development	O
-.	O
-	O
-While	O
-most	O
-studies	O
-have	O
-focused	O
-on	O
-the	O
-Saccharomyces	O
-cerevisiae	O
-transceptors	O
-for	O
-phosphate	O
-(	O
-Pho84	O
-),	O
-amino	O
-acids	O
-(	O
-Gap1	O
-)	O
-and	O
-ammonium	O
-(	O
-Mep2	O
-),	O
-transceptors	O
-are	O
-found	O
-in	O
-higher	O
-eukaryotes	O
-as	O
-well	O
-(	O
-for	O
-example	O
-,	O
-the	O
-mammalian	O
-SNAT2	O
-amino	O
--	O
-acid	O
-transporter	O
-and	O
-the	O
-GLUT2	O
-glucose	O
-transporter	O
-).	O
-	O
-Of	O
-these	O
-,	O
-only	O
-Mep2	O
-proteins	O
-function	O
-as	O
-ammonium	O
-receptors	O
-/	O
-sensors	O
-in	O
-fungal	O
-development	O
-.	O
-	O
-In	O
-bacteria	O
-,	O
-amt	O
-genes	O
-are	O
-present	O
-in	O
-an	O
-operon	O
-with	O
-glnK	O
-,	O
-encoding	O
-a	O
-PII	O
--	O
-like	O
-signal	O
-transduction	O
-class	O
-protein	O
-.	O
-	O
-Under	O
-conditions	O
-of	O
-nitrogen	O
-limitation	O
-,	O
-GlnK	O
-becomes	O
-uridylated	O
-,	O
-blocking	O
-its	O
-ability	O
-to	O
-bind	O
-and	O
-inhibit	O
-Amt	O
-proteins	O
-.	O
-	O
-(	O
-root	O
-mean	O
-square	O
-deviation	O
-)=	O
-0	O
-.	O
-7	O
-Å	O
-for	O
-434	O
-residues	O
-),	O
-with	O
-the	O
-main	O
-differences	O
-confined	O
-to	O
-the	O
-N	O
-terminus	O
-and	O
-the	O
-CTR	O
-(	O
-Fig	O
-.	O
-1	O
-).	O
-	O
-The	O
-N	O
-termini	O
-of	O
-the	O
-Mep2	O
-proteins	O
-are	O
-∼	O
-20	O
-–	O
-25	O
-residues	O
-longer	O
-compared	O
-with	O
-their	O
-bacterial	O
-counterparts	O
-(	O
-Figs	O
-1	O
-and	O
-2	O
-),	O
-substantially	O
-increasing	O
-the	O
-size	O
-of	O
-the	O
-extracellular	O
-domain	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-vestibule	O
-and	O
-the	O
-resulting	O
-inter	O
--	O
-monomer	O
-interactions	O
-likely	O
-increase	O
-the	O
-stability	O
-of	O
-the	O
-Mep2	O
-trimer	O
-,	O
-in	O
-support	O
-of	O
-data	O
-for	O
-plant	O
-AMT	O
-proteins	O
-.	O
-	O
-The	O
-head	O
-group	O
-of	O
-Arg54	O
-has	O
-moved	O
-∼	O
-11	O
-Å	O
-relative	O
-to	O
-that	O
-in	O
-Amt	O
--	O
-1	O
-,	O
-whereas	O
-the	O
-shift	O
-of	O
-the	O
-head	O
-group	O
-of	O
-the	O
-variable	O
-Lys55	O
-residue	O
-is	O
-almost	O
-20	O
-Å	O
-.	O
-The	O
-side	O
-chain	O
-of	O
-Lys56	O
-in	O
-the	O
-basic	O
-motif	O
-points	O
-in	O
-an	O
-opposite	O
-direction	O
-in	O
-the	O
-Mep2	O
-structures	O
-compared	O
-with	O
-that	O
-of	O
-,	O
-for	O
-example	O
-,	O
-Amt	O
--	O
-1	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-Significantly	O
-,	O
-this	O
-is	O
-also	O
-true	O
-for	O
-ScMep2	O
-,	O
-which	O
-was	O
-crystallized	O
-in	O
-the	O
-presence	O
-of	O
-0	O
-.	O
-2	O
-M	O
-ammonium	O
-ions	O
-(	O
-see	O
-Methods	O
-section	O
-).	O
-	O
-In	O
-Mep2	O
-,	O
-the	O
-CTR	O
-has	O
-moved	O
-away	O
-and	O
-makes	O
-relatively	O
-few	O
-contacts	O
-with	O
-the	O
-main	O
-body	O
-of	O
-the	O
-transporter	O
-,	O
-generating	O
-a	O
-more	O
-elongated	O
-protein	O
-(	O
-Figs	O
-1	O
-and	O
-4	O
-).	O
-	O
-These	O
-residues	O
-include	O
-those	O
-of	O
-the	O
-‘	O
-ExxGxD	O
-'	O
-motif	O
-,	O
-which	O
-when	O
-mutated	O
-generate	O
-inactive	O
-transporters	O
-.	O
-	O
-In	O
-Amt	O
--	O
-1	O
-and	O
-other	O
-bacterial	O
-ammonium	O
-transporters	O
-,	O
-these	O
-CTR	O
-residues	O
-interact	O
-with	O
-residues	O
-within	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-the	O
-protein	O
-.	O
-	O
-At	O
-the	O
-other	O
-end	O
-of	O
-ICL3	O
-,	O
-the	O
-backbone	O
-carbonyl	O
-groups	O
-of	O
-Gly172	O
-and	O
-Lys173	O
-are	O
-hydrogen	O
-bonded	O
-to	O
-the	O
-side	O
-chain	O
-of	O
-Arg370	O
-.	O
-	O
-This	O
-interaction	O
-in	O
-the	O
-centre	O
-of	O
-the	O
-protein	O
-may	O
-be	O
-particularly	O
-important	O
-to	O
-stabilize	O
-the	O
-open	O
-conformations	O
-of	O
-ammonium	O
-transporters	O
-.	O
-	O
-Where	O
-is	O
-the	O
-AI	O
-region	O
-and	O
-the	O
-Npr1	O
-phosphorylation	O
-site	O
-located	O
-?	O
-Our	O
-structures	O
-reveal	O
-that	O
-surprisingly	O
-,	O
-the	O
-AI	O
-region	O
-is	O
-folded	O
-back	O
-onto	O
-the	O
-CTR	O
-and	O
-is	O
-not	O
-located	O
-near	O
-the	O
-centre	O
-of	O
-the	O
-trimer	O
-as	O
-expected	O
-from	O
-the	O
-bacterial	O
-structures	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-The	O
-AI	O
-regions	O
-have	O
-very	O
-similar	O
-conformations	O
-in	O
-CaMep2	O
-and	O
-ScMep2	O
-,	O
-despite	O
-considerable	O
-differences	O
-in	O
-the	O
-rest	O
-of	O
-the	O
-CTR	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-This	O
-makes	O
-sense	O
-since	O
-the	O
-proteins	O
-were	O
-expressed	O
-in	O
-rich	O
-medium	O
-and	O
-confirms	O
-the	O
-recent	O
-suggestion	O
-by	O
-Boeckstaens	O
-et	O
-al	O
-.	O
-that	O
-the	O
-non	O
--	O
-phosphorylated	O
-form	O
-of	O
-Mep2	O
-corresponds	O
-to	O
-the	O
-inactive	O
-state	O
-.	O
-	O
-The	O
-peripheral	O
-location	O
-and	O
-disorder	O
-of	O
-the	O
-CTR	O
-beyond	O
-the	O
-kinase	O
-target	O
-site	O
-should	O
-facilitate	O
-the	O
-phosphorylation	O
-by	O
-Npr1	O
-.	O
-	O
-Mep2	O
-lacking	O
-the	O
-AI	O
-region	O
-is	O
-conformationally	O
-heterogeneous	O
-	O
-Density	O
-for	O
-ICL3	O
-and	O
-the	O
-CTR	O
-beyond	O
-residue	O
-Arg415	O
-is	O
-missing	O
-in	O
-the	O
-442Δ	B-mutant
-mutant	O
-,	O
-and	O
-the	O
-density	O
-for	O
-the	O
-other	O
-ICLs	O
-including	O
-ICL1	O
-is	O
-generally	O
-poor	O
-with	O
-visible	O
-parts	O
-of	O
-the	O
-structure	O
-having	O
-high	O
-B	O
--	O
-factors	O
-(	O
-Fig	O
-.	O
-7	O
-).	O
-	O
-Why	O
-then	O
-does	O
-this	O
-mutant	O
-appear	O
-to	O
-be	O
-constitutively	O
-active	O
-?	O
-We	O
-propose	O
-two	O
-possibilities	O
-.	O
-	O
-The	O
-latter	O
-model	O
-would	O
-fit	O
-well	O
-with	O
-the	O
-NH3	O
-/	O
-H	O
-+	O
-symport	O
-model	O
-in	O
-which	O
-the	O
-proton	O
-is	O
-relayed	O
-by	O
-the	O
-twin	O
--	O
-His	O
-motif	O
-.	O
-	O
-To	O
-test	O
-this	O
-hypothesis	O
-,	O
-we	O
-determined	O
-the	O
-structure	O
-of	O
-the	O
-phosphorylation	O
--	O
-mimicking	O
-R452D	B-mutant
-/	I-mutant
-S453D	I-mutant
-protein	O
-(	O
-hereafter	O
-termed	O
-‘	O
-DD	B-mutant
-mutant	I-mutant
-'),	O
-using	O
-data	O
-to	O
-a	O
-resolution	O
-of	O
-2	O
-.	O
-4	O
-Å	O
-.	O
-The	O
-additional	O
-mutation	O
-of	O
-the	O
-arginine	O
-preceding	O
-the	O
-phosphorylation	O
-site	O
-was	O
-introduced	O
-(	O
-i	O
-)	O
-to	O
-increase	O
-the	O
-negative	O
-charge	O
-density	O
-and	O
-make	O
-it	O
-more	O
-comparable	O
-to	O
-a	O
-phosphate	O
-at	O
-neutral	O
-pH	O
-,	O
-and	O
-(	O
-ii	O
-)	O
-to	O
-further	O
-destabilize	O
-the	O
-interactions	O
-of	O
-the	O
-AI	O
-region	O
-with	O
-the	O
-main	O
-body	O
-of	O
-the	O
-transporter	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-In	O
-addition	O
-,	O
-residues	O
-Glu420	O
--	O
-Leu423	O
-including	O
-Glu421	O
-of	O
-the	O
-ExxGxD	O
-motif	O
-are	O
-now	O
-disordered	O
-(	O
-Fig	O
-.	O
-8	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-The	O
-protein	O
-backbone	O
-has	O
-an	O
-average	O
-r	O
-.	O
-m	O
-.	O
-s	O
-.	O
-d	O
-.	O
-of	O
-only	O
-∼	O
-3	O
-Å	O
-during	O
-the	O
-200	O
--	O
-ns	O
-simulation	O
-,	O
-indicating	O
-that	O
-the	O
-protein	O
-is	O
-stable	O
-.	O
-	O
-There	O
-is	O
-flexibility	O
-in	O
-the	O
-side	O
-chains	O
-of	O
-the	O
-acidic	O
-residues	O
-so	O
-that	O
-they	O
-are	O
-able	O
-to	O
-form	O
-stable	O
-hydrogen	O
-bonds	O
-with	O
-Ser453	O
-.	O
-	O
-For	O
-example	O
-,	O
-the	O
-distance	O
-between	O
-the	O
-Asp453	O
-acidic	O
-oxygens	O
-and	O
-the	O
-Glu420	O
-acidic	O
-oxygens	O
-increases	O
-from	O
-∼	O
-7	O
-to	O
->	O
-22	O
-Å	O
-after	O
-200	O
-ns	O
-simulations	O
-,	O
-and	O
-thus	O
-these	O
-residues	O
-are	O
-not	O
-interacting	O
-.	O
-	O
-The	O
-distance	O
-between	O
-the	O
-phosphate	O
-of	O
-Sep453	O
-and	O
-the	O
-acidic	O
-oxygen	O
-atoms	O
-of	O
-Glu420	O
-is	O
-initially	O
-∼	O
-11	O
-Å	O
-,	O
-but	O
-increases	O
-to	O
->	O
-30	O
-Å	O
-after	O
-200	O
-ns	O
-.	O
-	O
-More	O
-specifically	O
-,	O
-the	O
-close	O
-interactions	O
-between	O
-the	O
-CTR	O
-and	O
-ICL1	O
-/	O
-ICL3	O
-present	O
-in	O
-open	O
-transporters	O
-are	O
-disrupted	O
-,	O
-causing	O
-ICL3	O
-to	O
-move	O
-outwards	O
-and	O
-block	O
-the	O
-channel	O
-(	O
-Figs	O
-4	O
-and	O
-9a	O
-).	O
-	O
-However	O
-,	O
-even	O
-the	O
-otherwise	O
-highly	O
-similar	O
-Mep2	O
-proteins	O
-of	O
-S	O
-.	O
-cerevisiae	O
-and	O
-C	O
-.	O
-albicans	O
-have	O
-different	O
-structures	O
-for	O
-their	O
-CTRs	O
-(	O
-Fig	O
-.	O
-1	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-In	O
-addition	O
-,	O
-the	O
-considerable	O
-differences	O
-between	O
-structurally	O
-resolved	O
-CTR	O
-domains	O
-means	O
-that	O
-the	O
-exact	O
-environment	O
-of	O
-T460	O
-in	O
-Amt	O
--	O
-1	O
-;	O
-1	O
-is	O
-also	O
-not	O
-known	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-(	O
-a	O
-)	O
-The	O
-triple	B-mutant
-mepΔ	I-mutant
-strain	O
-(	O
-black	O
-)	O
-and	O
-triple	O
-mepΔ	O
-npr1Δ	O
-strain	O
-(	O
-grey	O
-)	O
-containing	O
-plasmids	O
-expressing	O
-WT	O
-and	O
-variant	B-mutant
-ScMep2	I-mutant
-were	O
-grown	O
-on	O
-minimal	O
-medium	O
-containing	O
-1	O
-mM	O
-ammonium	O
-sulphate	O
-.	O
-	O
-The	O
-numbering	O
-is	O
-for	O
-CaMep2	O
-.	O
-	O
-Channel	O
-closures	O
-in	O
-Mep2	O
-.	O
-	O
-2Fo	O
-–	O
-Fc	O
-electron	O
-density	O
-(	O
-contoured	O
-at	O
-1	O
-.	O
-0	O
-σ	O
-)	O
-for	O
-residues	O
-Tyr49	O
-and	O
-His342	O
-is	O
-shown	O
-for	O
-the	O
-truncation	O
-mutant	O
-.	O
-	O
-The	O
-arrow	O
-indicates	O
-the	O
-phosphorylation	O
-site	O
-.	O
-	O
-Upon	O
-phosphorylation	O
-and	O
-mimicked	O
-by	O
-the	O
-CaMep2	O
-S453D	B-mutant
-and	O
-DD	B-mutant
-mutants	I-mutant
-(	O
-ii	O
-),	O
-the	O
-region	O
-around	O
-the	O
-ExxGxD	O
-motif	O
-undergoes	O
-a	O
-conformational	O
-change	O
-that	O
-results	O
-in	O
-the	O
-CTR	O
-interacting	O
-with	O
-the	O
-inward	O
--	O
-moving	O
-ICL3	O
-,	O
-opening	O
-the	O
-channel	O
-(	O
-full	O
-circle	O
-)	O
-(	O
-iii	O
-).	O
-	O
-Once	O
-a	O
-candidate	O
-antibody	O
-is	O
-identified	O
-,	O
-protein	O
-engineering	O
-is	O
-usually	O
-required	O
-to	O
-produce	O
-a	O
-molecule	O
-with	O
-the	O
-right	O
-biophysical	O
-and	O
-functional	O
-properties	O
-.	O
-	O
-The	O
-sequence	O
-diversity	O
-of	O
-the	O
-CDR	O
-regions	O
-presents	O
-a	O
-substantial	O
-challenge	O
-to	O
-antibody	O
-modeling	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-CDRs	O
-L1	O
-,	O
-L2	O
-,	O
-L3	O
-,	O
-H1	O
-and	O
-H2	O
-,	O
-no	O
-canonical	O
-structures	O
-have	O
-been	O
-observed	O
-for	O
-CDR	O
-H3	O
-,	O
-which	O
-is	O
-the	O
-most	O
-variable	O
-in	O
-length	O
-and	O
-amino	O
-acid	O
-sequence	O
-.	O
-	O
-Some	O
-clustering	O
-of	O
-conformations	O
-was	O
-observed	O
-for	O
-the	O
-shortest	O
-lengths	O
-;	O
-however	O
-,	O
-for	O
-the	O
-longer	O
-loops	O
-,	O
-only	O
-the	O
-portions	O
-nearest	O
-the	O
-framework	O
-(	O
-torso	O
-,	O
-stem	O
-or	O
-anchor	O
-region	O
-)	O
-were	O
-found	O
-to	O
-have	O
-defined	O
-conformations	O
-.	O
-	O
-Current	O
-antibody	O
-modeling	O
-approaches	O
-take	O
-advantage	O
-of	O
-the	O
-most	O
-recent	O
-advances	O
-in	O
-homology	O
-modeling	O
-,	O
-the	O
-evolving	O
-understanding	O
-of	O
-the	O
-CDR	O
-canonical	O
-structures	O
-,	O
-the	O
-emerging	O
-rules	O
-for	O
-CDR	O
-H3	O
-modeling	O
-and	O
-the	O
-growing	O
-body	O
-of	O
-antibody	O
-structural	O
-data	O
-available	O
-from	O
-the	O
-PDB	O
-.	O
-	O
-To	O
-support	O
-antibody	O
-engineering	O
-and	O
-therapeutic	O
-development	O
-efforts	O
-,	O
-a	O
-phage	O
-library	O
-was	O
-designed	O
-and	O
-constructed	O
-based	O
-on	O
-a	O
-limited	O
-number	O
-of	O
-scaffolds	O
-built	O
-with	O
-frequently	O
-used	O
-human	O
-germ	O
--	O
-line	O
-IGV	O
-and	O
-IGJ	O
-gene	O
-segments	O
-that	O
-encode	O
-antigen	O
-combining	O
-sites	O
-suitable	O
-for	O
-recognition	O
-of	O
-peptides	O
-and	O
-proteins	O
-.	O
-	O
-Variations	O
-occur	O
-in	O
-the	O
-pH	O
-(	O
-buffer	O
-)	O
-and	O
-the	O
-additives	O
-,	O
-and	O
-,	O
-in	O
-group	O
-3	O
-,	O
-PEG	O
-3350	O
-is	O
-the	O
-precipitant	O
-for	O
-one	O
-variants	O
-while	O
-ammonium	O
-sulfate	O
-is	O
-the	O
-precipitant	O
-for	O
-the	O
-other	O
-two	O
-.	O
-	O
-Apart	O
-from	O
-the	O
-C	O
--	O
-terminus	O
-,	O
-only	O
-a	O
-few	O
-surface	O
-residues	O
-in	O
-LC	O
-are	O
-disordered	O
-.	O
-	O
-The	O
-HCs	O
-feature	O
-the	O
-largest	O
-number	O
-of	O
-disordered	O
-residues	O
-,	O
-with	O
-the	O
-lower	O
-resolution	O
-structures	O
-having	O
-the	O
-most	O
-.	O
-	O
-CDR	O
-H1	O
-and	O
-CDR	O
-H2	O
-also	O
-show	O
-some	O
-degree	O
-of	O
-disorder	O
-,	O
-but	O
-to	O
-a	O
-lesser	O
-extent	O
-.	O
-	O
-Three	O
-of	O
-the	O
-HCs	O
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-,	O
-have	O
-the	O
-same	O
-canonical	O
-structure	O
-,	O
-H1	B-mutant
--	I-mutant
-13	I-mutant
--	I-mutant
-1	I-mutant
-,	O
-and	O
-the	O
-backbone	O
-conformations	O
-are	O
-tightly	O
-clustered	O
-for	O
-each	O
-set	O
-of	O
-Fab	O
-structures	O
-as	O
-reflected	O
-in	O
-the	O
-rmsd	O
-values	O
-(	O
-Fig	O
-.	O
-1B	O
--	O
-D	O
-).	O
-	O
-Each	O
-of	O
-the	O
-4	O
-HCs	O
-adopts	O
-only	O
-one	O
-canonical	O
-structure	O
-regardless	O
-of	O
-the	O
-pairing	O
-LC	O
-.	O
-	O
-Germlines	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-have	O
-the	O
-same	O
-canonical	O
-structure	O
-assignment	O
-H2	B-mutant
--	I-mutant
-10	I-mutant
--	I-mutant
-1	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-has	O
-H2	B-mutant
--	I-mutant
-10	I-mutant
--	I-mutant
-2	I-mutant
-,	O
-and	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-has	O
-H2	B-mutant
--	I-mutant
-9	I-mutant
--	I-mutant
-3	I-mutant
-.	O
-	O
-Germlines	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-are	O
-unique	O
-in	O
-the	O
-human	O
-repertoire	O
-in	O
-having	O
-an	O
-Ala	O
-at	O
-position	O
-71	O
-that	O
-leaves	O
-enough	O
-space	O
-for	O
-H	O
--	O
-Pro52a	O
-to	O
-pack	O
-deeper	O
-against	O
-CDR	O
-H4	O
-so	O
-that	O
-the	O
-following	O
-residues	O
-53	O
-and	O
-54	O
-point	O
-toward	O
-the	O
-putative	O
-antigen	O
-.	O
-	O
-However	O
-,	O
-there	O
-is	O
-a	O
-significant	O
-shift	O
-of	O
-the	O
-CDR	O
-as	O
-a	O
-rigid	O
-body	O
-when	O
-the	O
-2	O
-sets	O
-are	O
-superimposed	O
-.	O
-	O
-Germline	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-has	O
-Ala	O
-at	O
-position	O
-33	O
-whereas	O
-in	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-position	O
-33	O
-is	O
-occupied	O
-by	O
-a	O
-bulky	O
-Trp	O
-,	O
-which	O
-stacks	O
-against	O
-H	O
--	O
-Tyr52	O
-and	O
-drives	O
-CDR	O
-H2	O
-away	O
-from	O
-the	O
-center	O
-.	O
-	O
-For	O
-the	O
-remaining	O
-2	O
-,	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-has	O
-2	O
-different	O
-assignments	O
-,	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-1	I-mutant
-and	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-2	I-mutant
-,	O
-while	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-has	O
-a	O
-single	O
-assignment	O
-,	O
-L1	B-mutant
--	I-mutant
-17	I-mutant
--	I-mutant
-1	I-mutant
-.	O
-	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-is	O
-the	O
-most	O
-variable	O
-in	O
-CDR	O
-L1	O
-among	O
-the	O
-4	O
-germlines	O
-as	O
-indicated	O
-by	O
-an	O
-rmsd	O
-of	O
-0	O
-.	O
-54	O
-Å	O
-(	O
-Fig	O
-.	O
-3C	O
-).	O
-	O
-The	O
-third	O
-structure	O
-,	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-has	O
-CDR	O
-L1	O
-as	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-2	I-mutant
-,	O
-which	O
-deviates	O
-from	O
-L1	B-mutant
--	I-mutant
-12	I-mutant
--	I-mutant
-1	I-mutant
-at	O
-residues	O
-29	O
--	O
-32	O
-,	O
-i	O
-.	O
-e	O
-.,	O
-at	O
-the	O
-site	O
-of	O
-insertion	O
-with	O
-respect	O
-to	O
-the	O
-11	O
--	O
-residue	O
-CDR	O
-.	O
-	O
-The	O
-fourth	O
-member	O
-of	O
-the	O
-set	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-was	O
-crystallized	O
-with	O
-2	O
-Fabs	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-As	O
-mentioned	O
-earlier	O
-,	O
-all	O
-16	O
-Fabs	O
-have	O
-the	O
-same	O
-CDR	O
-H3	O
-,	O
-for	O
-which	O
-the	O
-amino	O
-acid	O
-sequence	O
-is	O
-derived	O
-from	O
-the	O
-anti	O
--	O
-CCL2	O
-antibody	O
-CNTO	O
-888	O
-.	O
-	O
-The	O
-variations	O
-in	O
-CDR	O
-H3	O
-conformation	O
-are	O
-illustrated	O
-in	O
-Fig	O
-.	O
-6	O
-for	O
-the	O
-18	O
-Fab	O
-structures	O
-that	O
-have	O
-ordered	O
-backbone	O
-atoms	O
-.	O
-	O
-(	O
-B	O
-)	O
-The	O
-“	O
-extended	O
-”	O
-CDR	O
-H3	O
-of	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-with	O
-green	O
-carbon	O
-atoms	O
-and	O
-yellow	O
-dashed	O
-lines	O
-connecting	O
-the	O
-H	O
--	O
-bond	O
-pairs	O
-for	O
-Asp101	O
-OD1	O
-and	O
-OD2	O
-and	O
-Trp103	O
-NE1	O
-.	O
-	O
-The	O
-remaining	O
-8	O
-Fabs	O
-can	O
-be	O
-grouped	O
-into	O
-5	O
-different	O
-conformational	O
-classes	O
-.	O
-	O
-Position	O
-43	O
-may	O
-be	O
-alternatively	O
-occupied	O
-by	O
-Ser	O
-,	O
-Val	O
-or	O
-Pro	O
-(	O
-as	O
-in	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-),	O
-but	O
-the	O
-hydrophobic	O
-interaction	O
-with	O
-H	O
--	O
-Tyr91	O
-is	O
-preserved	O
-.	O
-	O
-In	O
-most	O
-of	O
-the	O
-structures	O
-,	O
-it	O
-has	O
-the	O
-χ2	O
-angle	O
-of	O
-∼	O
-80	O
-°,	O
-while	O
-the	O
-ring	O
-is	O
-flipped	O
-over	O
-(	O
-χ2	O
-=	O
-−	O
-100	O
-°)	O
-in	O
-H5	O
--	O
-51	O
-:	O
-L3	O
-:	O
-11	O
-and	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-.	O
-	O
-In	O
-fact	O
-,	O
-the	O
-parameter	O
-values	O
-for	O
-the	O
-set	O
-of	O
-16	O
-Fabs	O
-are	O
-in	O
-the	O
-middle	O
-of	O
-the	O
-distribution	O
-observed	O
-for	O
-351	O
-non	O
--	O
-redundant	O
-antibody	O
-structures	O
-determined	O
-at	O
-3	O
-.	O
-0	O
-Å	O
-resolution	O
-or	O
-better	O
-.	O
-	O
-An	O
-illustration	O
-of	O
-the	O
-difference	O
-in	O
-tilt	O
-angle	O
-for	O
-2	O
-pairs	O
-of	O
-variants	O
-by	O
-the	O
-superposition	O
-of	O
-the	O
-VH	O
-domains	O
-of	O
-(	O
-A	O
-)	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-on	O
-that	O
-of	O
-H5	O
--	O
-51	O
-:	O
-L1	O
--	O
-39	O
-(	O
-the	O
-VL	O
-domain	O
-is	O
-off	O
-by	O
-a	O
-rigid	O
--	O
-body	O
-roatation	O
-of	O
-10	O
-.	O
-5	O
-°)	O
-and	O
-(	O
-B	O
-)	O
-H1	O
--	O
-69	O
-:	O
-L4	O
--	O
-1	O
-on	O
-that	O
-of	O
-H5	O
--	O
-51	O
-:	O
-L1	O
--	O
-39	O
-(	O
-the	O
-VL	O
-domain	O
-is	O
-off	O
-by	O
-a	O
-rigid	O
--	O
-body	O
-roatation	O
-of	O
-1	O
-.	O
-6	O
-°).	O
-	O
-One	O
-of	O
-the	O
-2	O
-structures	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-,	O
-has	O
-its	O
-CDR	O
-H3	O
-in	O
-the	O
-‘	O
-extended	O
-’	O
-conformation	O
-;	O
-the	O
-other	O
-structure	O
-has	O
-it	O
-in	O
-the	O
-‘	O
-kinked	O
-’	O
-conformation	O
-.	O
-	O
-VH	O
-:	O
-VL	O
-buried	O
-surface	O
-area	O
-and	O
-complementarity	O
-	O
-Residues	O
-in	O
-CDR	O
-H3	O
-are	O
-missing	O
-:	O
-YGE	O
-in	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-11	O
-,	O
-GIY	O
-in	O
-H5	O
--	O
-51	O
-:	O
-L3	O
--	O
-20	O
-.	O
-	O
-This	O
-is	O
-the	O
-first	O
-report	O
-of	O
-a	O
-systematic	O
-structural	O
-investigation	O
-of	O
-a	O
-phage	O
-germline	O
-library	O
-.	O
-	O
-The	O
-16	O
-Fab	O
-structures	O
-offer	O
-a	O
-unique	O
-look	O
-at	O
-all	O
-pairings	O
-of	O
-4	O
-different	O
-HCs	O
-(	O
-H1	B-mutant
--	I-mutant
-69	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-23	I-mutant
-,	O
-H3	B-mutant
--	I-mutant
-53	I-mutant
-,	O
-and	O
-H5	B-mutant
--	I-mutant
-51	I-mutant
-)	O
-and	O
-4	O
-different	O
-LCs	O
-(	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-,	O
-L3	B-mutant
--	I-mutant
-11	I-mutant
-,	O
-L3	B-mutant
--	I-mutant
-20	I-mutant
-and	O
-L4	B-mutant
--	I-mutant
-1	I-mutant
-),	O
-all	O
-with	O
-the	O
-same	O
-CDR	O
-H3	O
-.	O
-	O
-Having	O
-all	O
-16	O
-VH	O
-:	O
-VL	O
-pairs	O
-with	O
-the	O
-same	O
-CDR	O
-H3	O
-provides	O
-some	O
-insights	O
-into	O
-why	O
-molecular	O
-modeling	O
-efforts	O
-of	O
-CDR	O
-H3	O
-have	O
-proven	O
-so	O
-difficult	O
-.	O
-	O
-Thus	O
-,	O
-it	O
-is	O
-likely	O
-that	O
-the	O
-CDR	O
-H3	O
-conformation	O
-is	O
-dependent	O
-upon	O
-2	O
-dominating	O
-factors	O
-:	O
-1	O
-)	O
-amino	O
-acid	O
-sequence	O
-;	O
-and	O
-2	O
-)	O
-VH	O
-and	O
-VL	O
-context	O
-.	O
-	O
-This	O
-subset	O
-also	O
-has	O
-2	O
-structures	O
-with	O
-2	O
-Fab	O
-copies	O
-in	O
-the	O
-asymmetric	O
-unit	O
-.	O
-	O
-The	O
-same	O
-variability	O
-is	O
-observed	O
-for	O
-the	O
-sets	O
-of	O
-variants	O
-composed	O
-of	O
-one	O
-LC	O
-paired	O
-with	O
-each	O
-of	O
-the	O
-4	O
-HCs	O
-.	O
-	O
-As	O
-noted	O
-in	O
-the	O
-Results	O
-section	O
-,	O
-the	O
-2	O
-variants	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-23	O
-:	O
-L3	O
--	O
-20	O
-,	O
-are	O
-outliers	O
-in	O
-terms	O
-of	O
-the	O
-tilt	O
-angle	O
-;	O
-at	O
-the	O
-same	O
-time	O
-,	O
-both	O
-have	O
-the	O
-smallest	O
-VH	O
-:	O
-VL	O
-interface	O
-.	O
-	O
-Other	O
-germlines	O
-have	O
-bulky	O
-residues	O
-,	O
-Tyr	O
-,	O
-Arg	O
-and	O
-Trp	O
-,	O
-at	O
-these	O
-positions	O
-,	O
-whereas	O
-L1	B-mutant
--	I-mutant
-39	I-mutant
-has	O
-Ser	O
-and	O
-Thr	O
-.	O
-	O
-A	O
-more	O
-compact	O
-CDR	O
-L3	O
-may	O
-be	O
-beneficial	O
-in	O
-this	O
-situation	O
-.	O
-	O
-Yet	O
-,	O
-for	O
-the	O
-2	O
-antibodies	O
-,	O
-the	O
-total	O
-gain	O
-in	O
-stability	O
-merits	O
-the	O
-domain	O
-repacking	O
-.	O
-	O
-Quite	O
-unexpectedly	O
-,	O
-2	O
-of	O
-the	O
-variants	O
-,	O
-H1	O
--	O
-69	O
-:	O
-L3	O
--	O
-20	O
-and	O
-H3	O
--	O
-53	O
-:	O
-L4	O
--	O
-1	O
-,	O
-have	O
-the	O
-‘	O
-extended	O
-’	O
-stem	O
-region	O
-differing	O
-from	O
-the	O
-other	O
-14	O
-that	O
-have	O
-a	O
-‘	O
-kinked	O
-’	O
-stem	O
-region	O
-.	O
-	O
-From	O
-this	O
-point	O
-of	O
-view	O
-,	O
-a	O
-novel	O
-approach	O
-to	O
-design	O
-combinatorial	O
-antibody	O
-libraries	O
-would	O
-be	O
-to	O
-cover	O
-the	O
-range	O
-of	O
-CDR	O
-conformations	O
-that	O
-may	O
-not	O
-necessarily	O
-coincide	O
-with	O
-the	O
-germline	O
-usage	O
-in	O
-the	O
-human	O
-repertoire	O
-.	O
-	O
-This	O
-study	O
-resulted	O
-in	O
-a	O
-series	O
-of	O
-snapshots	O
-depicting	O
-the	O
-various	O
-folding	O
-states	O
-of	O
-Im7	O
-while	O
-bound	O
-to	O
-Spy	O
-.	O
-	O
-Recent	O
-advances	O
-in	O
-X	O
--	O
-ray	O
-crystallography	O
-and	O
-NMR	O
-spectroscopy	O
-continue	O
-to	O
-improve	O
-our	O
-ability	O
-to	O
-analyze	O
-biomolecules	O
-that	O
-exist	O
-in	O
-multiple	O
-conformations	O
-.	O
-	O
-X	O
--	O
-ray	O
-crystallography	O
-has	O
-historically	O
-provided	O
-valuable	O
-information	O
-on	O
-small	O
--	O
-scale	O
-conformational	O
-changes	O
-,	O
-but	O
-observing	O
-large	O
--	O
-amplitude	O
-heterogeneous	O
-conformational	O
-changes	O
-often	O
-falls	O
-beyond	O
-the	O
-reach	O
-of	O
-current	O
-crystallographic	O
-techniques	O
-.	O
-	O
-However	O
-,	O
-modeling	O
-of	O
-the	O
-substrate	O
-in	O
-the	O
-complex	O
-proved	O
-to	O
-be	O
-a	O
-substantial	O
-challenge	O
-,	O
-as	O
-the	O
-electron	O
-density	O
-of	O
-the	O
-substrate	O
-was	O
-discontinuous	O
-and	O
-fragmented	O
-.	O
-	O
-To	O
-determine	O
-the	O
-structure	O
-of	O
-the	O
-substrate	O
-portion	O
-of	O
-these	O
-Spy	O
-:	O
-substrate	O
-complexes	O
-,	O
-we	O
-conceived	O
-of	O
-an	O
-approach	O
-that	O
-we	O
-term	O
-READ	O
-,	O
-for	O
-Residual	O
-Electron	O
-and	O
-Anomalous	O
-Density	O
-.	O
-	O
-Its	O
-strong	O
-anomalous	O
-scattering	O
-allowed	O
-us	O
-to	O
-track	O
-the	O
-positions	O
-of	O
-these	O
-individual	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-residues	O
-one	O
-at	O
-a	O
-time	O
-,	O
-potentially	O
-even	O
-if	O
-the	O
-residue	O
-was	O
-found	O
-in	O
-several	O
-locations	O
-in	O
-the	O
-same	O
-crystal	O
-.	O
-	O
-Together	O
-,	O
-these	O
-results	O
-indicated	O
-that	O
-the	O
-Im7	O
-substrate	O
-binds	O
-Spy	O
-in	O
-multiple	O
-conformations	O
-.	O
-	O
-To	O
-generate	O
-an	O
-accurate	O
-depiction	O
-of	O
-the	O
-chaperone	O
--	O
-substrate	O
-interactions	O
-,	O
-we	O
-devised	O
-a	O
-selection	O
-protocol	O
-based	O
-on	O
-a	O
-sample	O
--	O
-and	O
--	O
-select	O
-procedure	O
-employed	O
-in	O
-NMR	O
-spectroscopy	O
-.	O
-	O
-The	O
-coarse	O
--	O
-grained	O
-simulations	O
-are	O
-based	O
-on	O
-a	O
-single	O
--	O
-residue	O
-resolution	O
-model	O
-for	O
-protein	O
-folding	O
-and	O
-were	O
-extended	O
-here	O
-to	O
-describe	O
-Spy	O
--	O
-Im76	O
--	O
-45	O
-binding	O
-events	O
-(	O
-Online	O
-Methods	O
-).	O
-	O
-To	O
-accomplish	O
-this	O
-task	O
-,	O
-we	O
-generated	O
-a	O
-compressed	O
-version	O
-of	O
-the	O
-experimental	O
-2mFo	O
-−	O
-DFc	O
-electron	O
-density	O
-map	O
-for	O
-use	O
-in	O
-the	O
-selection	O
-.	O
-	O
-We	O
-constructed	O
-a	O
-contact	O
-map	O
-of	O
-the	O
-complex	O
-,	O
-which	O
-shows	O
-the	O
-frequency	O
-of	O
-interactions	O
-for	O
-chaperone	O
--	O
-substrate	O
-residue	O
-pairs	O
-(	O
-Fig	O
-.	O
-4	O
-).	O
-	O
-The	O
-Spy	O
--	O
-contacting	O
-residues	O
-comprise	O
-a	O
-mixture	O
-of	O
-charged	O
-,	O
-polar	O
-,	O
-and	O
-hydrophobic	O
-residues	O
-.	O
-	O
-Once	O
-the	O
-substrate	O
-begins	O
-to	O
-fold	O
-within	O
-this	O
-protected	O
-environment	O
-,	O
-it	O
-progressively	O
-buries	O
-its	O
-own	O
-hydrophobic	O
-residues	O
-,	O
-and	O
-its	O
-interactions	O
-with	O
-the	O
-chaperone	O
-shift	O
-towards	O
-becoming	O
-more	O
-electrostatic	O
-.	O
-	O
-Residues	O
-Asp32	O
-and	O
-Asp35	O
-are	O
-close	O
-to	O
-each	O
-other	O
-in	O
-the	O
-folded	O
-state	O
-of	O
-Im7	O
-.	O
-	O
-This	O
-proximity	O
-likely	O
-causes	O
-electrostatic	O
-repulsion	O
-that	O
-destabilizes	O
-Im7	O
-’	O
-s	O
-native	O
-state	O
-.	O
-	O
-In	O
-conjunction	O
-with	O
-our	O
-bound	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-ensemble	O
-,	O
-these	O
-mutants	O
-now	O
-allowed	O
-us	O
-to	O
-investigate	O
-structural	O
-features	O
-important	O
-to	O
-chaperone	O
-function	O
-.	O
-	O
-Despite	O
-extensive	O
-studies	O
-,	O
-exactly	O
-how	O
-complex	O
-chaperone	O
-machines	O
-help	O
-proteins	O
-fold	O
-remains	O
-controversial	O
-.	O
-	O
-Heterogeneous	O
-dynamic	O
-complexes	O
-or	O
-disordered	O
-regions	O
-of	O
-single	O
-proteins	O
-,	O
-once	O
-considered	O
-solely	O
-approachable	O
-by	O
-NMR	O
-spectroscopy	O
-,	O
-can	O
-now	O
-be	O
-visualized	O
-through	O
-X	O
--	O
-ray	O
-crystallography	O
-.	O
-	O
-Flowchart	O
-of	O
-the	O
-READ	O
-sample	O
--	O
-and	O
--	O
-select	O
-process	O
-.	O
-	O
-(	O
-a	O
-)	O
-Spy	O
-:	O
-Im76	O
--	O
-45	O
-contact	O
-map	O
-projected	O
-onto	O
-the	O
-bound	O
-Spy	O
-dimer	O
-(	O
-above	O
-)	O
-and	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-(	O
-below	O
-)	O
-structures	O
-.	O
-	O
-(	O
-a	O
-)	O
-Overlay	O
-of	O
-apo	O
-Spy	O
-(	O
-PDB	O
-ID	O
-:	O
-3O39	O
-,	O
-gray	O
-)	O
-and	O
-bound	O
-Spy	O
-(	O
-green	O
-).	O
-(	O
-b	O
-)	O
-Overlay	O
-of	O
-WT	O
-Spy	O
-bound	O
-to	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-(	O
-green	O
-),	O
-H96L	B-mutant
-Spy	O
-bound	O
-to	O
-Im7	O
-L18A	B-mutant
-L19	B-mutant
-AL13A	I-mutant
-(	O
-blue	O
-),	O
-H96L	B-mutant
-Spy	O
-bound	O
-to	O
-WT	O
-Im7	O
-(	O
-yellow	O
-),	O
-and	O
-WT	O
-Spy	O
-bound	O
-to	O
-casein	O
-(	O
-salmon	O
-).	O
-(	O
-c	O
-)	O
-Competition	O
-assay	O
-showing	O
-Im76	B-mutant
--	I-mutant
-45	I-mutant
-competes	O
-with	O
-Im7	O
-L18A	B-mutant
-L19A	B-mutant
-L37A	B-mutant
-H40W	B-mutant
-for	O
-the	O
-same	O
-binding	O
-site	O
-on	O
-Spy	O
-(	O
-further	O
-substrate	O
-competition	O
-assays	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-8	O
-).	O
-	O
-(	O
-b	O
-)	O
-F115	O
-and	O
-L32	O
-tether	O
-Spy	O
-’	O
-s	O
-linker	O
-region	O
-to	O
-its	O
-cradle	O
-,	O
-decreasing	O
-Spy	O
-activity	O
-by	O
-limiting	O
-linker	O
-region	O
-flexibility	O
-.	O
-	O
-Despite	O
-a	O
-long	O
-history	O
-of	O
-physiological	O
-and	O
-functional	O
-studies	O
-,	O
-the	O
-molecular	O
-mechanism	O
-of	O
-NCX	O
-has	O
-been	O
-elusive	O
-,	O
-owing	O
-to	O
-the	O
-lack	O
-of	O
-structural	O
-information	O
-.	O
-	O
-In	O
-this	O
-study	O
-,	O
-we	O
-set	O
-out	O
-to	O
-determine	O
-the	O
-structures	O
-of	O
-outward	O
--	O
-facing	O
-wild	O
--	O
-type	O
-NCX_Mj	O
-in	O
-complex	O
-with	O
-Na	O
-+,	O
-Ca2	O
-+	O
-and	O
-Sr2	O
-+,	O
-at	O
-various	O
-concentrations	O
-.	O
-	O
-Extracellular	O
-Na	O
-+	O
-binding	O
-	O
-To	O
-conclusively	O
-clarify	O
-this	O
-assignment	O
-,	O
-we	O
-first	O
-set	O
-out	O
-to	O
-examine	O
-the	O
-Na	O
-+	O
-occupancy	O
-of	O
-these	O
-sites	O
-without	O
-Ca2	O
-+.	O
-	O
-X	O
--	O
-ray	O
-diffraction	O
-of	O
-these	O
-soaked	O
-crystals	O
-revealed	O
-a	O
-Na	O
-+-	O
-dependent	O
-variation	O
-in	O
-the	O
-electron	O
--	O
-density	O
-distribution	O
-at	O
-sites	O
-Sext	O
-,	O
-SCa	O
-and	O
-Sint	O
-,	O
-indicating	O
-a	O
-Na	O
-+	O
-occupancy	O
-change	O
-(	O
-Fig	O
-.	O
-1c	O
-).	O
-	O
-Indeed	O
-,	O
-two	O
-observations	O
-indicate	O
-that	O
-a	O
-water	O
-molecule	O
-rather	O
-than	O
-a	O
-Na	O
-+	O
-ion	O
-occupies	O
-Smid	O
-,	O
-as	O
-was	O
-predicted	O
-in	O
-a	O
-recent	O
-simulation	O
-study	O
-.	O
-	O
-When	O
-Na	O
-+	O
-binds	O
-to	O
-Sext	O
-at	O
-high	O
-concentrations	O
-,	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-TM7	O
-is	O
-bent	O
-into	O
-two	O
-short	O
-helices	O
-,	O
-TM7a	O
-and	O
-TM7b	O
-(	O
-Fig	O
-.	O
-2a	O
-).	O
-	O
-TM7b	O
-occludes	O
-the	O
-four	O
-central	O
-binding	O
-sites	O
-from	O
-the	O
-external	O
-solution	O
-,	O
-with	O
-the	O
-backbone	O
-carbonyl	O
-of	O
-Ala206	O
-coordinating	O
-the	O
-Na	O
-+	O
-ion	O
-(	O
-Fig	O
-.	O
-2b	O
--	O
-d	O
-).	O
-	O
-Extracellular	O
-Ca2	O
-+	O
-and	O
-Sr2	O
-+	O
-binding	O
-and	O
-their	O
-competition	O
-with	O
-Na	O
-+	O
-	O
-Binding	O
-of	O
-Ca2	O
-+	O
-to	O
-both	O
-sites	O
-simultaneously	O
-is	O
-highly	O
-improbable	O
-due	O
-to	O
-their	O
-close	O
-proximity	O
-,	O
-and	O
-at	O
-least	O
-one	O
-water	O
-molecule	O
-can	O
-be	O
-discerned	O
-coordinating	O
-the	O
-ion	O
-(	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-Indeed	O
-,	O
-in	O
-most	O
-NCX	O
-proteins	O
-Asp240	O
-is	O
-substituted	O
-by	O
-Asn	O
-,	O
-which	O
-would	O
-likely	O
-weaken	O
-or	O
-abrogate	O
-Ca2	O
-+	O
-binding	O
-to	O
-Smid	O
-.	O
-	O
-Although	O
-the	O
-binding	O
-sites	O
-are	O
-thus	O
-fully	O
-accessible	O
-to	O
-the	O
-external	O
-solution	O
-(	O
-Fig	O
-.	O
-3e	O
-),	O
-the	O
-lack	O
-of	O
-electron	O
-density	O
-therein	O
-indicates	O
-no	O
-ions	O
-or	O
-ordered	O
-solvent	O
-molecules	O
-.	O
-	O
-Such	O
-interpretation	O
-would	O
-be	O
-consistent	O
-with	O
-the	O
-computer	O
-simulations	O
-reported	O
-below	O
-.	O
-	O
-That	O
-secondary	O
--	O
-active	O
-transporters	O
-are	O
-able	O
-to	O
-harness	O
-an	O
-electrochemical	O
-gradient	O
-of	O
-one	O
-substrate	O
-to	O
-power	O
-the	O
-uphill	O
-transport	O
-of	O
-another	O
-relies	O
-on	O
-a	O
-seemingly	O
-simple	O
-principle	O
-:	O
-they	O
-must	O
-not	O
-transition	O
-between	O
-outward	O
--	O
-and	O
-inward	O
--	O
-open	O
-conformations	O
-unless	O
-in	O
-two	O
-precise	O
-substrate	O
-occupancy	O
-states	O
-.	O
-	O
-As	O
-it	O
-happens	O
-,	O
-the	O
-results	O
-confirm	O
-that	O
-the	O
-structures	O
-now	O
-available	O
-are	O
-representing	O
-interconverting	O
-states	O
-of	O
-the	O
-functional	O
-cycle	O
-of	O
-NCX_Mj	O
-,	O
-while	O
-revealing	O
-how	O
-the	O
-alternating	O
--	O
-access	O
-mechanism	O
-is	O
-controlled	O
-by	O
-the	O
-ion	O
--	O
-occupancy	O
-state	O
-.	O
-	O
-This	O
-distortion	O
-occludes	O
-Sext	O
-from	O
-the	O
-exterior	O
-(	O
-Fig	O
-.	O
-4d	O
-,	O
-4h	O
--	O
-i	O
-)	O
-and	O
-appears	O
-to	O
-be	O
-induced	O
-by	O
-the	O
-Na	O
-+	O
-ion	O
-itself	O
-,	O
-which	O
-pulls	O
-the	O
-carbonyl	O
-group	O
-of	O
-A206	O
-into	O
-its	O
-coordination	O
-sphere	O
-(	O
-Fig	O
-.	O
-4g	O
-).	O
-	O
-When	O
-all	O
-Na	O
-+	O
-sites	O
-are	O
-occupied	O
-,	O
-the	O
-global	O
-free	O
--	O
-energy	O
-minimum	O
-corresponds	O
-to	O
-a	O
-conformation	O
-in	O
-which	O
-the	O
-ions	O
-are	O
-maximally	O
-coordinated	O
-by	O
-the	O
-protein	O
-(	O
-Fig	O
-.	O
-5a	O
-,	O
-5c	O
-);	O
-TM7ab	O
-is	O
-bent	O
-and	O
-packs	O
-closely	O
-with	O
-TM2	O
-and	O
-TM3	O
-,	O
-and	O
-so	O
-the	O
-binding	O
-sites	O
-are	O
-occluded	O
-from	O
-the	O
-solvent	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-The	O
-Na	O
-+	O
-ion	O
-at	O
-Sext	O
-remains	O
-fully	O
-coordinated	O
-,	O
-but	O
-an	O
-ordered	O
-water	O
-molecule	O
-now	O
-mediates	O
-its	O
-interaction	O
-with	O
-A206	O
-:	O
-O	O
-,	O
-relieving	O
-the	O
-strain	O
-on	O
-the	O
-F202	O
-:	O
-O	O
-–	O
-A206	O
-:	O
-N	O
-hydrogen	O
--	O
-bond	O
-(	O
-Fig	O
-.	O
-5c	O
-).	O
-	O
-Interestingly	O
-,	O
-this	O
-doubly	O
-occupied	O
-state	O
-can	O
-also	O
-access	O
-conformations	O
-in	O
-which	O
-the	O
-second	O
-aqueous	O
-channel	O
-mentioned	O
-above	O
-,	O
-i	O
-.	O
-e	O
-.	O
-leading	O
-to	O
-SCa	O
-between	O
-TM7	O
-and	O
-TM2	O
-and	O
-over	O
-the	O
-gating	O
-helices	O
-TM1	O
-and	O
-TM6	O
-,	O
-also	O
-becomes	O
-open	O
-(	O
-Fig	O
-.	O
-5b	O
--	O
-c	O
-).	O
-	O
-This	O
-processivity	O
-is	O
-logical	O
-since	O
-three	O
-Na	O
-+	O
-ions	O
-are	O
-involved	O
-,	O
-but	O
-also	O
-implies	O
-that	O
-in	O
-the	O
-Ca2	O
-+-	O
-bound	O
-state	O
-,	O
-which	O
-includes	O
-a	O
-single	O
-ion	O
-,	O
-the	O
-transporter	O
-ought	O
-to	O
-be	O
-able	O
-to	O
-access	O
-all	O
-three	O
-major	O
-conformations	O
-,	O
-i	O
-.	O
-e	O
-.	O
-the	O
-outward	O
--	O
-open	O
-state	O
-,	O
-in	O
-order	O
-to	O
-release	O
-(	O
-or	O
-re	O
--	O
-bind	O
-)	O
-Ca2	O
-+,	O
-but	O
-also	O
-the	O
-occluded	O
-conformation	O
-,	O
-and	O
-thus	O
-the	O
-semi	O
--	O
-open	O
-intermediate	O
-,	O
-in	O
-order	O
-to	O
-transition	O
-to	O
-the	O
-inward	O
--	O
-open	O
-state	O
-.	O
-	O
-By	O
-contrast	O
-,	O
-occupancy	O
-by	O
-H	O
-+,	O
-which	O
-as	O
-mentioned	O
-are	O
-not	O
-transported	O
-,	O
-might	O
-be	O
-compatible	O
-with	O
-a	O
-semi	O
--	O
-open	O
-state	O
-as	O
-well	O
-as	O
-with	O
-the	O
-fully	O
-open	O
-conformation	O
-,	O
-but	O
-should	O
-not	O
-be	O
-conducive	O
-to	O
-occlusion	O
-.	O
-	O
-This	O
-occluded	O
-conformation	O
-,	O
-which	O
-is	O
-a	O
-necessary	O
-intermediate	O
-between	O
-the	O
-outward	O
-and	O
-inward	O
--	O
-open	O
-states	O
-,	O
-and	O
-which	O
-entails	O
-the	O
-internal	O
-dehydration	O
-of	O
-the	O
-protein	O
-,	O
-is	O
-only	O
-attainable	O
-upon	O
-complete	O
-occupancy	O
-of	O
-the	O
-binding	O
-sites	O
-.	O
-	O
-The	O
-most	O
-apparent	O
-of	O
-these	O
-changes	O
-involves	O
-the	O
-N	O
--	O
-terminal	O
-half	O
-of	O
-TM7	O
-(	O
-TM7ab	O
-);	O
-together	O
-with	O
-more	O
-subtle	O
-displacements	O
-in	O
-TM2	O
-and	O
-TM3	O
-,	O
-this	O
-change	O
-in	O
-TM7ab	O
-correlates	O
-with	O
-the	O
-opening	O
-and	O
-closing	O
-of	O
-two	O
-distinct	O
-aqueous	O
-channels	O
-leading	O
-into	O
-the	O
-ion	O
--	O
-binding	O
-sites	O
-from	O
-the	O
-extracellular	O
-solution	O
-.	O
-	O
-The	O
-striking	O
-quantitative	O
-agreement	O
-between	O
-the	O
-ion	O
--	O
-binding	O
-affinities	O
-inferred	O
-from	O
-our	O
-crystallographic	O
-titrations	O
-and	O
-the	O
-Km	O
-and	O
-K1	O
-/	O
-2	O
-values	O
-previously	O
-deduced	O
-from	O
-functional	O
-assays	O
-has	O
-been	O
-discussed	O
-above	O
-.	O
-	O
-Specifically	O
-,	O
-our	O
-crystal	O
-titrations	O
-suggest	O
-that	O
-,	O
-during	O
-forward	O
-Na	O
-+/	O
-Ca2	O
-+	O
-exchange	O
-,	O
-sites	O
-Sint	O
-and	O
-SCa	O
-,	O
-which	O
-Ca2	O
-+	O
-and	O
-Na	O
-+	O
-compete	O
-for	O
-,	O
-can	O
-be	O
-grouped	O
-into	O
-one	O
-;	O
-Na	O
-+	O
-binding	O
-to	O
-these	O
-sites	O
-does	O
-not	O
-require	O
-high	O
-Na	O
-+	O
-concentrations	O
-,	O
-and	O
-two	O
-Na	O
-+	O
-ions	O
-along	O
-with	O
-a	O
-water	O
-molecule	O
-(	O
-at	O
-Smid	O
-)	O
-are	O
-sufficient	O
-to	O
-displace	O
-Ca2	O
-+,	O
-explaining	O
-the	O
-Hill	O
-coefficient	O
-of	O
-~	O
-2	O
-for	O
-Na	O
-+-	O
-dependent	O
-inhibition	O
-of	O
-Ca2	O
-+	O
-fluxes	O
-.	O
-	O
-No	O
-significant	O
-changes	O
-were	O
-observed	O
-in	O
-the	O
-side	O
--	O
-chains	O
-involved	O
-in	O
-ion	O
-or	O
-water	O
-coordination	O
-at	O
-the	O
-SCa	O
-,	O
-Sint	O
-and	O
-Smid	O
-sites	O
-.	O
-	O
-The	O
-vacant	O
-Sext	O
-site	O
-in	O
-the	O
-structure	O
-at	O
-low	O
-Na	O
-+	O
-concentration	O
-is	O
-indicated	O
-with	O
-a	O
-white	O
-sphere	O
-.	O
-	O
-(	O
-d	O
-)	O
-Extracellular	O
-solvent	O
-accessibility	O
-of	O
-the	O
-ion	O
-binding	O
-sites	O
-in	O
-the	O
-structures	O
-at	O
-high	O
-and	O
-low	O
-[	O
-Na	O
-+].	O
-	O
-Putative	O
-solvent	O
-channels	O
-are	O
-represented	O
-as	O
-light	O
--	O
-purple	O
-surfaces	O
-.	O
-	O
-Residues	O
-involved	O
-in	O
-Sr2	O
-+	O
-coordination	O
-are	O
-labeled	O
-.	O
-	O
-(	O
-b	O
-)	O
-Ca2	O
-+	O
-(	O
-tanned	O
-spheres	O
-)	O
-binds	O
-either	O
-to	O
-SCa	O
-or	O
-Smid	O
-in	O
-crystals	O
-titrated	O
-with	O
-10	O
-mM	O
-Ca2	O
-+	O
-and	O
-2	O
-.	O
-5	O
-mM	O
-Na	O
-+	O
-(	O
-see	O
-also	O
-Supplementary	O
-Fig	O
-.	O
-2	O
-).	O
-	O
-Approximate	O
-distances	O
-between	O
-TM2	O
-,	O
-TM3	O
-and	O
-TM7	O
-are	O
-indicated	O
-in	O
-Å	O
-.	O
-(	O
-e	O
-)	O
-Close	O
--	O
-up	O
-of	O
-the	O
-ion	O
--	O
-binding	O
-region	O
-in	O
-the	O
-partially	O
-Na	O
-+-	O
-occupied	O
-state	O
-.	O
-	O
-The	O
-water	O
--	O
-density	O
-maps	O
-in	O
-(	O
-b	O
-)	O
-are	O
-shown	O
-here	O
-as	O
-a	O
-grey	O
-mesh	O
-.	O
-	O
-An	O
-extended	O
-U2AF65	O
-–	O
-RNA	O
--	O
-binding	O
-domain	O
-recognizes	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-signal	O
-	O
-Initially	O
-U2AF65	O
-recognizes	O
-the	O
-Py	O
--	O
-tract	O
-splice	O
-site	O
-signal	O
-.	O
-	O
-As	O
-such	O
-,	O
-the	O
-molecular	O
-mechanisms	O
-for	O
-Py	O
--	O
-tract	O
-recognition	O
-by	O
-the	O
-intact	O
-U2AF65	O
-–	O
-RNA	O
--	O
-binding	O
-domain	O
-remained	O
-unknown	O
-.	O
-	O
-We	O
-use	O
-single	O
--	O
-molecule	O
-Förster	O
-resonance	O
-energy	O
-transfer	O
-(	O
-smFRET	O
-)	O
-to	O
-characterize	O
-the	O
-conformational	O
-dynamics	O
-of	O
-this	O
-extended	O
-U2AF65	O
-–	O
-RNA	O
--	O
-binding	O
-domain	O
-during	O
-Py	O
--	O
-tract	O
-recognition	O
-.	O
-	O
-The	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-RRM1	O
-and	O
-RRM2	O
-associate	O
-with	O
-the	O
-Py	O
-tract	O
-in	O
-a	O
-parallel	O
-,	O
-side	O
--	O
-by	O
--	O
-side	O
-arrangement	O
-(	O
-shown	O
-for	O
-representative	O
-structure	O
-iv	O
-in	O
-Fig	O
-.	O
-2b	O
-,	O
-c	O
-;	O
-Supplementary	O
-Movie	O
-1	O
-).	O
-	O
-An	O
-extended	O
-conformation	O
-of	O
-the	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-linker	O
-traverses	O
-across	O
-the	O
-α	O
--	O
-helical	O
-surface	O
-of	O
-RRM1	O
-and	O
-the	O
-central	O
-β	O
--	O
-strands	O
-of	O
-RRM2	O
-and	O
-is	O
-well	O
-defined	O
-in	O
-the	O
-electron	O
-density	O
-(	O
-Fig	O
-.	O
-2b	O
-).	O
-	O
-Both	O
-RRM1	O
-/	O
-RRM2	O
-extensions	O
-and	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-directly	O
-recognize	O
-the	O
-bound	O
-oligonucleotide	O
-.	O
-	O
-Based	O
-on	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structures	O
-,	O
-we	O
-originally	O
-hypothesized	O
-that	O
-the	O
-U2AF65	O
-RRMs	O
-would	O
-bind	O
-the	O
-minimal	O
-seven	O
-nucleotides	O
-observed	O
-in	O
-these	O
-structures	O
-.	O
-	O
-Surprisingly	O
-,	O
-the	O
-RRM2	O
-extension	O
-/	O
-inter	O
--	O
-RRM	O
-linker	O
-contribute	O
-new	O
-central	O
-nucleotide	O
--	O
-binding	O
-sites	O
-near	O
-the	O
-RRM1	O
-/	O
-RRM2	O
-junction	O
-and	O
-the	O
-RRM1	O
-extension	O
-recognizes	O
-the	O
-3	O
-′-	O
-terminal	O
-nucleotide	O
-(	O
-Fig	O
-.	O
-2c	O
-;	O
-Supplementary	O
-Movie	O
-1	O
-).	O
-	O
-Qualitatively	O
-,	O
-a	O
-subset	O
-of	O
-the	O
-U2AF651	O
-,	O
-2L	O
--	O
-nucleotide	O
--	O
-binding	O
-sites	O
-(	O
-sites	O
-1	O
-–	O
-3	O
-and	O
-7	O
-–	O
-9	O
-)	O
-share	O
-similar	O
-locations	O
-to	O
-those	O
-of	O
-the	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structures	O
-(	O
-Supplementary	O
-Figs	O
-2c	O
-,	O
-d	O
-and	O
-3	O
-).	O
-	O
-Otherwise	O
-,	O
-the	O
-rU4	O
-nucleotide	O
-packs	O
-against	O
-F304	O
-in	O
-the	O
-signature	O
-ribonucleoprotein	O
-consensus	O
-motif	O
-(	O
-RNP	O
-)-	O
-2	O
-of	O
-RRM2	O
-.	O
-	O
-This	O
-nucleotide	O
-twists	O
-to	O
-face	O
-away	O
-from	O
-the	O
-U2AF65	O
-linker	O
-and	O
-instead	O
-inserts	O
-the	O
-rU6	O
--	O
-uracil	O
-into	O
-a	O
-sandwich	O
-between	O
-the	O
-β2	O
-/	O
-β3	O
-loops	O
-of	O
-RRM1	O
-and	O
-RRM2	O
-.	O
-	O
-The	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-extensions	O
-of	O
-the	O
-U2AF65	O
-RRM1	O
-and	O
-RRM2	O
-directly	O
-contact	O
-the	O
-bound	O
-Py	O
-tract	O
-.	O
-	O
-Consequently	O
-,	O
-the	O
-U2AF651	O
-,	O
-2L	O
--	O
-bound	O
-rU2	O
--	O
-O4	O
-and	O
--	O
-N3H	O
-form	O
-dual	O
-hydrogen	O
-bonds	O
-with	O
-the	O
-K329	O
-backbone	O
-atoms	O
-(	O
-Fig	O
-.	O
-3a	O
-),	O
-rather	O
-than	O
-a	O
-single	O
-hydrogen	O
-bond	O
-with	O
-the	O
-K329	O
-side	O
-chain	O
-as	O
-in	O
-the	O
-prior	O
-dU2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-structure	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3b	O
-).	O
-	O
-The	O
-adjacent	O
-R146	O
-guanidinium	O
-group	O
-donates	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-3	O
-′-	O
-terminal	O
-ribose	O
--	O
-O2	O
-′	O
-and	O
-O3	O
-′	O
-atoms	O
-,	O
-where	O
-it	O
-could	O
-form	O
-a	O
-salt	O
-bridge	O
-with	O
-a	O
-phospho	O
--	O
-diester	O
-group	O
-in	O
-the	O
-context	O
-of	O
-a	O
-longer	O
-pre	O
--	O
-mRNA	O
-.	O
-	O
-We	O
-compare	O
-U2AF65	O
-interactions	O
-with	O
-uracil	O
-relative	O
-to	O
-cytosine	O
-pyrimidines	O
-at	O
-the	O
-ninth	O
-binding	O
-site	O
-in	O
-Fig	O
-.	O
-3g	O
-,	O
-h	O
-and	O
-the	O
-Supplementary	O
-Discussion	O
-.	O
-	O
-At	O
-the	O
-RNA	O
-surface	O
-,	O
-the	O
-key	O
-V254	O
-that	O
-recognizes	O
-the	O
-fifth	O
-uracil	O
-is	O
-secured	O
-via	O
-hydrophobic	O
-contacts	O
-between	O
-its	O
-side	O
-chain	O
-and	O
-the	O
-β	O
--	O
-sheet	O
-surface	O
-of	O
-RRM2	O
-,	O
-chiefly	O
-the	O
-consensus	O
-RNP1	O
--	O
-F304	O
-residue	O
-that	O
-stacks	O
-with	O
-the	O
-fourth	O
-uracil	O
-(	O
-Fig	O
-.	O
-4a	O
-,	O
-lower	O
-left	O
-).	O
-	O
-However	O
-,	O
-the	O
-resulting	O
-decrease	O
-in	O
-the	O
-AdML	O
-RNA	O
-affinity	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
--	I-mutant
-3Gly	I-mutant
-mutant	O
-relative	O
-to	O
-wild	O
--	O
-type	O
-protein	O
-was	O
-not	O
-significant	O
-(	O
-Fig	O
-.	O
-4b	O
-).	O
-	O
-In	O
-parallel	O
-,	O
-we	O
-replaced	O
-five	O
-linker	O
-residues	O
-(	O
-S251	O
-,	O
-T252	O
-,	O
-V253	O
-,	O
-V254	O
-and	O
-P255	O
-)	O
-at	O
-the	O
-fifth	O
-nucleotide	O
--	O
-binding	O
-site	O
-with	O
-glycines	O
-(	O
-5Gly	B-mutant
-)	O
-and	O
-also	O
-found	O
-that	O
-the	O
-RNA	O
-affinity	O
-of	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
--	I-mutant
-5Gly	I-mutant
-mutant	O
-likewise	O
-decreased	O
-only	O
-slightly	O
-relative	O
-to	O
-wild	O
--	O
-type	O
-protein	O
-.	O
-	O
-Importance	O
-of	O
-U2AF65	O
-–	O
-RNA	O
-contacts	O
-for	O
-pre	O
--	O
-mRNA	O
-splicing	O
-	O
-To	O
-complement	O
-the	O
-static	O
-portraits	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structure	O
-that	O
-we	O
-had	O
-determined	O
-by	O
-X	O
--	O
-ray	O
-crystallography	O
-,	O
-we	O
-used	O
-smFRET	O
-to	O
-characterize	O
-the	O
-probability	O
-distribution	O
-functions	O
-and	O
-time	O
-dependence	O
-of	O
-U2AF65	O
-inter	O
--	O
-RRM	O
-conformational	O
-dynamics	O
-in	O
-solution	O
-.	O
-	O
-Double	O
--	O
-cysteine	O
-variant	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-was	O
-modified	O
-with	O
-equimolar	O
-amount	O
-of	O
-Cy3	O
-and	O
-Cy5	O
-.	O
-	O
-Only	O
-traces	O
-that	O
-showed	O
-single	O
-photobleaching	O
-events	O
-for	O
-both	O
-donor	O
-and	O
-acceptor	O
-dyes	O
-and	O
-anti	O
--	O
-correlated	O
-changes	O
-in	O
-acceptor	O
-and	O
-donor	O
-fluorescence	O
-were	O
-included	O
-in	O
-smFRET	O
-data	O
-analysis	O
-.	O
-	O
-The	O
-double	O
--	O
-labelled	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-protein	O
-was	O
-tethered	O
-to	O
-a	O
-slide	O
-via	O
-biotin	O
--	O
-NTA	O
-/	O
-Ni	O
-+	O
-2	O
-resin	O
-.	O
-	O
-However	O
-,	O
-the	O
-presence	O
-of	O
-repetitive	O
-fluctuations	O
-between	O
-particular	O
-FRET	O
-values	O
-supports	O
-the	O
-hypothesis	O
-that	O
-RNA	O
--	O
-free	O
-U2AF65	O
-samples	O
-several	O
-distinct	O
-conformations	O
-.	O
-	O
-This	O
-result	O
-is	O
-consistent	O
-with	O
-the	O
-broad	O
-ensembles	O
-of	O
-extended	O
-solution	O
-conformations	O
-that	O
-best	O
-fit	O
-the	O
-SAXS	O
-data	O
-collected	O
-for	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-as	O
-well	O
-as	O
-for	O
-a	O
-longer	O
-construct	O
-(	O
-residues	O
-136	O
-–	O
-347	O
-).	O
-	O
-We	O
-next	O
-used	O
-smFRET	O
-to	O
-probe	O
-the	O
-conformational	O
-selection	O
-of	O
-distinct	O
-inter	O
--	O
-RRM	O
-arrangements	O
-following	O
-association	O
-of	O
-U2AF65	O
-with	O
-the	O
-AdML	O
-Py	O
--	O
-tract	O
-prototype	O
-.	O
-	O
-To	O
-assess	O
-the	O
-possible	O
-contributions	O
-of	O
-RNA	O
--	O
-free	O
-conformations	O
-of	O
-U2AF65	O
-and	O
-/	O
-or	O
-structural	O
-heterogeneity	O
-introduced	O
-by	O
-tethering	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-to	O
-the	O
-slide	O
-to	O
-the	O
-observed	O
-distribution	O
-of	O
-FRET	O
-values	O
-,	O
-we	O
-reversed	O
-the	O
-immobilization	O
-scheme	O
-.	O
-	O
-Therefore	O
-,	O
-RRM1	O
--	O
-to	O
--	O
-RRM2	O
-distance	O
-remains	O
-similar	O
-regardless	O
-of	O
-whether	O
-U2AF65	O
-is	O
-bound	O
-to	O
-interrupted	O
-or	O
-continuous	O
-Py	O
-tract	O
-.	O
-	O
-The	O
-inter	O
--	O
-fluorophore	O
-distances	O
-derived	O
-from	O
-the	O
-observed	O
-0	O
-.	O
-45	O
-FRET	O
-state	O
-agree	O
-with	O
-the	O
-distances	O
-between	O
-the	O
-α	O
--	O
-carbon	O
-atoms	O
-of	O
-the	O
-respective	O
-residues	O
-in	O
-the	O
-crystal	O
-structures	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-bound	O
-to	O
-Py	O
--	O
-tract	O
-oligonucleotides	O
-.	O
-	O
-Hidden	O
-Markov	O
-modelling	O
-analysis	O
-of	O
-smFRET	O
-traces	O
-suggests	O
-that	O
-RNA	O
--	O
-bound	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-can	O
-sample	O
-at	O
-least	O
-two	O
-other	O
-conformations	O
-corresponding	O
-to	O
-∼	O
-0	O
-.	O
-7	O
-–	O
-0	O
-.	O
-8	O
-and	O
-∼	O
-0	O
-.	O
-3	O
-FRET	O
-values	O
-in	O
-addition	O
-to	O
-the	O
-predominant	O
-conformation	O
-corresponding	O
-to	O
-the	O
-0	O
-.	O
-45	O
-FRET	O
-state	O
-.	O
-	O
-Truncation	O
-of	O
-U2AF65	O
-to	O
-the	O
-core	O
-RRM1	O
-–	O
-RRM2	O
-region	O
-reduces	O
-its	O
-RNA	O
-affinity	O
-by	O
-100	O
--	O
-fold	O
-.	O
-	O
-As	O
-such	O
-,	O
-we	O
-suggest	O
-that	O
-the	O
-MDS	O
--	O
-relevant	O
-U2AF65	O
-mutations	O
-contribute	O
-to	O
-MDS	O
-progression	O
-indirectly	O
-,	O
-by	O
-destabilizing	O
-a	O
-relevant	O
-conformation	O
-of	O
-the	O
-conjoined	O
-U2AF35	O
-subunit	O
-rather	O
-than	O
-affecting	O
-U2AF65	O
-functions	O
-in	O
-RNA	O
-binding	O
-or	O
-spliceosome	O
-recruitment	O
-per	O
-se	O
-.	O
-	O
-An	O
-increased	O
-prevalence	O
-of	O
-the	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-following	O
-U2AF65	O
-–	O
-RNA	O
-binding	O
-,	O
-coupled	O
-with	O
-the	O
-apparent	O
-absence	O
-of	O
-transitions	O
-in	O
-many	O
-∼	O
-0	O
-.	O
-45	O
--	O
-value	O
-single	O
-molecule	O
-traces	O
-(	O
-for	O
-example	O
-,	O
-Fig	O
-.	O
-6e	O
-),	O
-suggests	O
-a	O
-population	O
-shift	O
-in	O
-which	O
-RNA	O
-binds	O
-to	O
-(	O
-and	O
-draws	O
-the	O
-equilibrium	O
-towards	O
-)	O
-a	O
-pre	O
--	O
-configured	O
-inter	O
--	O
-RRM	O
-proximity	O
-that	O
-most	O
-often	O
-corresponds	O
-to	O
-the	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-.	O
-	O
-Examples	O
-of	O
-‘	O
-extended	O
-conformational	O
-selection	O
-'	O
-during	O
-ligand	O
-binding	O
-have	O
-been	O
-characterized	O
-for	O
-a	O
-growing	O
-number	O
-of	O
-macromolecules	O
-(	O
-for	O
-example	O
-,	O
-adenylate	O
-kinase	O
-,	O
-LAO	O
--	O
-binding	O
-protein	O
-,	O
-poly	O
--	O
-ubiquitin	O
-,	O
-maltose	O
--	O
-binding	O
-protein	O
-and	O
-the	O
-preQ1	O
-riboswitch	O
-,	O
-among	O
-others	O
-).	O
-	O
-These	O
-transitions	O
-could	O
-correspond	O
-to	O
-rearrangement	O
-from	O
-the	O
-‘	O
-closed	O
-'	O
-NMR	O
-/	O
-PRE	O
--	O
-based	O
-U2AF65	O
-conformation	O
-in	O
-which	O
-the	O
-RNA	O
--	O
-binding	O
-surface	O
-of	O
-only	O
-a	O
-single	O
-RRM	O
-is	O
-exposed	O
-and	O
-available	O
-for	O
-RNA	O
-binding	O
-,	O
-to	O
-the	O
-structural	O
-state	O
-seen	O
-in	O
-the	O
-side	O
--	O
-by	O
--	O
-side	O
-,	O
-RNA	O
--	O
-bound	O
-crystal	O
-structure	O
-.	O
-	O
-The	O
-finding	O
-that	O
-U2AF65	O
-recognizes	O
-a	O
-nine	O
-base	O
-pair	O
-Py	O
-tract	O
-contributes	O
-to	O
-an	O
-elusive	O
-‘	O
-code	O
-'	O
-for	O
-predicting	O
-splicing	O
-patterns	O
-from	O
-primary	O
-sequences	O
-in	O
-the	O
-post	O
--	O
-genomic	O
-era	O
-(	O
-reviewed	O
-in	O
-ref	O
-.).	O
-	O
-(	O
-b	O
-)	O
-Comparison	O
-of	O
-the	O
-apparent	O
-equilibrium	O
-affinities	O
-of	O
-various	O
-U2AF65	O
-constructs	O
-for	O
-binding	O
-the	O
-prototypical	O
-AdML	O
-Py	O
-tract	O
-(	O
-5	O
-′-	O
-CCCUUUUUUUUCC	O
--	O
-3	O
-′).	O
-	O
-The	O
-apparent	O
-equilibrium	O
-dissociation	O
-constants	O
-(	O
-KD	O
-)	O
-for	O
-binding	O
-the	O
-AdML	O
-13mer	O
-are	O
-as	O
-follows	O
-:	O
-flU2AF65	O
-,	O
-30	O
-±	O
-3	O
-nM	O
-;	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-,	O
-35	O
-±	O
-6	O
-nM	O
-;	O
-U2AF651	B-mutant
-,	I-mutant
-2	I-mutant
-,	O
-3	O
-,	O
-600	O
-±	O
-300	O
-nM	O
-.	O
-(	O
-c	O
-)	O
-Comparison	O
-of	O
-the	O
-RNA	O
-sequence	O
-specificities	O
-of	O
-flU2AF65	O
-and	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-constructs	O
-binding	O
-C	O
--	O
-rich	O
-Py	O
-tracts	O
-with	O
-4U	O
-'	O
-s	O
-embedded	O
-in	O
-either	O
-the	O
-5	O
-′-	O
-(	O
-light	O
-grey	O
-fill	O
-)	O
-or	O
-3	O
-′-	O
-(	O
-dark	O
-grey	O
-fill	O
-)	O
-regions	O
-.	O
-	O
-The	O
-purified	O
-protein	O
-and	O
-average	O
-fitted	O
-fluorescence	O
-anisotropy	O
-RNA	O
--	O
-binding	O
-curves	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-1	O
-.	O
-	O
-(	O
-b	O
-)	O
-Stereo	O
-views	O
-of	O
-a	O
-‘	O
-kicked	O
-'	O
-2	O
-|	O
-Fo	O
-|−|	O
-Fc	O
-|	O
-electron	O
-density	O
-map	O
-contoured	O
-at	O
-1σ	O
-for	O
-the	O
-inter	O
--	O
-RRM	O
-linker	O
-,	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-residues	O
-(	O
-blue	O
-)	O
-or	O
-bound	O
-oligonucleotide	O
-of	O
-a	O
-representative	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-structure	O
-(	O
-structure	O
-iv	O
-,	O
-bound	O
-to	O
-5	O
-′-(	O
-P	O
-)	O
-rUrUrUdUrUrU	O
-(	O
-BrdU	O
-)	O
-dUrC	O
-)	O
-(	O
-magenta	O
-).	O
-	O
-BrdU	O
-,	O
-5	O
--	O
-bromo	O
--	O
-deoxy	O
--	O
-uridine	O
-;	O
-d	O
-,	O
-deoxy	O
--	O
-ribose	O
-;	O
-P	O
--,	O
-5	O
-′-	O
-phosphorylation	O
-;	O
-r	O
-,	O
-ribose	O
-.	O
-	O
-The	O
-U2AF65	O
-linker	O
-/	O
-RRM	O
-and	O
-inter	O
--	O
-RRM	O
-interactions	O
-.	O
-	O
-RNA	O
-binding	O
-stabilizes	O
-the	O
-side	O
--	O
-by	O
--	O
-side	O
-conformation	O
-of	O
-U2AF65	O
-RRMs	O
-.	O
-	O
-Additional	O
-traces	O
-for	O
-untethered	O
-,	O
-RNA	O
--	O
-bound	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-are	O
-shown	O
-in	O
-Supplementary	O
-Fig	O
-.	O
-7c	O
-,	O
-d	O
-.	O
-Histograms	O
-(	O
-d	O
-,	O
-f	O
-,	O
-h	O
-,	O
-j	O
-)	O
-show	O
-the	O
-distribution	O
-of	O
-FRET	O
-values	O
-in	O
-RNA	O
--	O
-free	O
-,	O
-slide	O
--	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-d	O
-);	O
-AdML	O
-RNA	O
--	O
-bound	O
-,	O
-slide	O
--	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-f	O
-);	O
-AdML	O
-RNA	O
--	O
-bound	O
-,	O
-untethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-h	O
-)	O
-and	O
-adenosine	O
--	O
-interrupted	O
-RNA	O
--	O
-bound	O
-,	O
-slide	O
--	O
-tethered	O
-U2AF651	B-mutant
-,	I-mutant
-2LFRET	I-mutant
-(	O
-Cy3	O
-/	O
-Cy5	O
-)	O
-(	O
-j	O
-).	O
-	O
-A	O
-surface	O
-representation	O
-of	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-is	O
-shown	O
-bound	O
-to	O
-nine	O
-nucleotides	O
-(	O
-nt	O
-);	O
-the	O
-relative	O
-distances	O
-and	O
-juxtaposition	O
-of	O
-the	O
-branch	O
-point	O
-sequence	O
-(	O
-BPS	O
-)	O
-and	O
-consensus	O
-AG	O
-dinucleotide	O
-at	O
-the	O
-3	O
-′	O
-splice	O
-site	O
-are	O
-unknown	O
-.	O
-	O
-(	O
-b	O
-)	O
-Following	O
-binding	O
-to	O
-the	O
-Py	O
--	O
-tract	O
-RNA	O
-,	O
-a	O
-conformation	O
-corresponding	O
-to	O
-high	O
-FRET	O
-and	O
-consistent	O
-with	O
-the	O
-‘	O
-closed	O
-',	O
-back	O
--	O
-to	O
--	O
-back	O
-apo	O
--	O
-U2AF65	O
-model	O
-resulting	O
-from	O
-PRE	O
-/	O
-NMR	O
-characterization	O
-(	O
-PDB	O
-ID	O
-2YH0	O
-)	O
-often	O
-transitions	O
-to	O
-a	O
-conformation	O
-corresponding	O
-to	O
-∼	O
-0	O
-.	O
-45	O
-FRET	O
-value	O
-,	O
-which	O
-is	O
-consistent	O
-with	O
-‘	O
-open	O
-',	O
-side	O
--	O
-by	O
--	O
-side	O
-RRMs	O
-such	O
-as	O
-the	O
-U2AF651	B-mutant
-,	I-mutant
-2L	I-mutant
-crystal	O
-structures	O
-.	O
-	O
-Over	O
-a	O
-large	O
-dose	O
-range	O
-,	O
-the	O
-RNA	O
-was	O
-found	O
-to	O
-be	O
-far	O
-less	O
-susceptible	O
-to	O
-radiation	O
--	O
-induced	O
-chemical	O
-changes	O
-than	O
-the	O
-protein	O
-.	O
-	O
-Dose	O
-is	O
-defined	O
-as	O
-the	O
-absorbed	O
-energy	O
-per	O
-unit	O
-mass	O
-of	O
-crystal	O
-in	O
-grays	O
-(	O
-Gy	O
-;	O
-1	O
-Gy	O
-=	O
-1	O
-J	O
-kg	O
-−	O
-1	O
-),	O
-and	O
-is	O
-the	O
-metric	O
-against	O
-which	O
-damage	O
-progression	O
-should	O
-be	O
-monitored	O
-during	O
-MX	O
-data	O
-collection	O
-,	O
-as	O
-opposed	O
-to	O
-time	O
-.	O
-	O
-There	O
-are	O
-a	O
-number	O
-of	O
-cases	O
-where	O
-SRD	O
-manifestations	O
-have	O
-compromised	O
-the	O
-biological	O
-information	O
-extracted	O
-from	O
-MX	O
--	O
-determined	O
-structures	O
-at	O
-much	O
-lower	O
-doses	O
-than	O
-the	O
-recommended	O
-30	O
-MGy	O
-limit	O
-,	O
-leading	O
-to	O
-false	O
-structural	O
-interpretations	O
-of	O
-protein	O
-mechanisms	O
-.	O
-	O
-The	O
-investigation	O
-of	O
-naturally	O
-forming	O
-nucleoprotein	O
-complexes	O
-circumvents	O
-the	O
-inherent	O
-challenges	O
-in	O
-making	O
-controlled	O
-comparisons	O
-of	O
-damage	O
-mechanisms	O
-between	O
-protein	O
-and	O
-nucleic	O
-acids	O
-crystallized	O
-separately	O
-.	O
-Recently	O
-,	O
-for	O
-a	O
-well	O
-characterized	O
-bacterial	O
-protein	O
-–	O
-DNA	O
-complex	O
-(	O
-C	O
-.	O
-Esp1396I	O
-;	O
-PDB	O
-entry	O
-3clc	O
-;	O
-resolution	O
-2	O
-.	O
-8	O
-Å	O
-;	O
-McGeehan	O
-et	O
-al	O
-.,	O
-2008	O
-)	O
-it	O
-was	O
-concluded	O
-that	O
-over	O
-a	O
-wide	O
-dose	O
-range	O
-(	O
-2	O
-.	O
-1	O
-–	O
-44	O
-.	O
-6	O
-MGy	O
-)	O
-the	O
-protein	O
-was	O
-far	O
-more	O
-susceptible	O
-to	O
-SRD	O
-than	O
-the	O
-DNA	O
-within	O
-the	O
-crystal	O
-(	O
-Bury	O
-et	O
-al	O
-.,	O
-2015	O
-).	O
-	O
-Three	O
-acidic	O
-residues	O
-(	O
-Glu36	O
-,	O
-Asp39	O
-and	O
-Glu42	O
-)	O
-are	O
-involved	O
-in	O
-RNA	O
-interactions	O
-within	O
-each	O
-of	O
-the	O
-11	O
-TRAP	O
-ring	O
-subunits	O
-,	O
-and	O
-Fig	O
-.	O
-5	O
-▸	O
-shows	O
-their	O
-density	O
-changes	O
-with	O
-increasing	O
-dose	O
-.	O
-	O
-Salt	O
--	O
-bridge	O
-interactions	O
-have	O
-previously	O
-been	O
-suggested	O
-to	O
-reduce	O
-the	O
-glutamate	O
-decarboxylation	O
-rate	O
-within	O
-the	O
-large	O
-(∼	O
-62	O
-.	O
-4	O
-kDa	O
-)	O
-myrosinase	O
-protein	O
-structure	O
-(	O
-Burmeister	O
-,	O
-2000	O
-).	O
-	O
-The	O
-extended	O
-aliphatic	O
-Lys37	O
-side	O
-chain	O
-stacks	O
-against	O
-the	O
-nearby	O
-G1	O
-base	O
-,	O
-making	O
-a	O
-series	O
-of	O
-nonpolar	O
-contacts	O
-within	O
-each	O
-RNA	O
--	O
-binding	O
-interface	O
-.	O
-	O
-Representative	O
-Phe32	O
-and	O
-Lys37	O
-atoms	O
-were	O
-selected	O
-to	O
-illustrate	O
-these	O
-trends	O
-.	O
-	O
-Our	O
-method	O
-­	O
-ology	O
-,	O
-which	O
-eliminated	O
-tedious	O
-and	O
-error	O
--	O
-prone	O
-visual	O
-inspection	O
-,	O
-permitted	O
-the	O
-determination	O
-on	O
-a	O
-per	O
--	O
-atom	O
-basis	O
-of	O
-the	O
-most	O
-damaged	O
-sites	O
-,	O
-as	O
-characterized	O
-by	O
-F	O
-obs	O
-(	O
-d	O
-n	O
-)	O
-−	O
-F	O
-obs	O
-(	O
-d	O
-1	O
-)	O
-Fourier	O
-difference	O
-map	O
-peaks	O
-between	O
-successive	O
-data	O
-sets	O
-collected	O
-from	O
-the	O
-same	O
-crystal	O
-.	O
-	O
-Both	O
-Glu36	O
-and	O
-Asp39	O
-bind	O
-directly	O
-to	O
-RNA	O
-,	O
-each	O
-through	O
-two	O
-hydrogen	O
-bonds	O
-to	O
-guanine	O
-bases	O
-(	O
-G3	O
-and	O
-G1	O
-,	O
-respectively	O
-).	O
-	O
-Observations	O
-of	O
-lower	O
-protein	O
-radiation	O
--	O
-sensitivity	O
-in	O
-DNA	O
--	O
-bound	O
-forms	O
-have	O
-been	O
-recorded	O
-in	O
-solution	O
-at	O
-RT	O
-at	O
-much	O
-lower	O
-doses	O
-(∼	O
-1	O
-kGy	O
-)	O
-than	O
-those	O
-used	O
-for	O
-typical	O
-MX	O
-experiments	O
-[	O
-e	O
-.	O
-g	O
-.	O
-an	O
-oestrogen	O
-response	O
-element	O
-–	O
-receptor	O
-complex	O
-(	O
-Stísová	O
-et	O
-al	O
-.,	O
-2006	O
-)	O
-and	O
-a	O
-DNA	O
-glycosylase	O
-and	O
-its	O
-abasic	O
-DNA	O
-target	O
-site	O
-(	O
-Gillard	O
-et	O
-al	O
-.,	O
-2004	O
-)].	O
-	O
-However	O
-,	O
-in	O
-the	O
-current	O
-MX	O
-study	O
-at	O
-100	O
-K	O
-,	O
-the	O
-main	O
-damaging	O
-species	O
-are	O
-believed	O
-to	O
-be	O
-migrating	O
-LEEs	O
-and	O
-holes	O
-produced	O
-directly	O
-within	O
-the	O
-protein	O
-–	O
-RNA	O
-components	O
-or	O
-in	O
-closely	O
-associated	O
-solvent	O
-.	O
-	O
-RNA	O
-is	O
-shown	O
-is	O
-yellow	O
-.	O
-	O
-(	O
-b	O
-)	O
-Average	O
-D	O
-loss	O
-for	O
-each	O
-residue	O
-/	O
-nucleotide	O
-type	O
-with	O
-respect	O
-to	O
-the	O
-DWD	O
-(	O
-diffraction	O
--	O
-weighted	O
-dose	O
-;	O
-Zeldin	O
-,	O
-Brock	O
-­	O
-hauser	O
-et	O
-al	O
-.,	O
-2013	O
-).	O
-	O
-Residues	O
-have	O
-been	O
-grouped	O
-by	O
-amino	O
--	O
-acid	O
-number	O
-,	O
-and	O
-split	O
-into	O
-bound	O
-and	O
-nonbound	O
-groupings	O
-,	O
-with	O
-each	O
-bar	O
-representing	O
-the	O
-mean	O
-calculated	O
-over	O
-11	O
-equivalent	O
-atoms	O
-around	O
-a	O
-TRAP	O
-ring	O
-.	O
-	O
-The	O
-three	O
-best	O
--	O
-characterized	O
-MAPK	O
-signalling	O
-pathways	O
-are	O
-mediated	O
-by	O
-the	O
-kinases	O
-extracellular	O
-signal	O
--	O
-regulated	O
-kinase	O
-(	O
-ERK	O
-),	O
-c	O
--	O
-Jun	O
-N	O
--	O
-terminal	O
-kinase	O
-(	O
-JNK	O
-)	O
-and	O
-p38	O
-.	O
-	O
-The	O
-ERK	O
-pathway	O
-is	O
-activated	O
-by	O
-various	O
-mitogens	O
-and	O
-phorbol	O
-esters	O
-,	O
-whereas	O
-the	O
-JNK	O
-and	O
-p38	O
-pathways	O
-are	O
-stimulated	O
-mainly	O
-by	O
-environmental	O
-stress	O
-and	O
-inflammatory	O
-cytokines	O
-.	O
-	O
-MKPs	O
-constitute	O
-a	O
-group	O
-of	O
-DUSPs	O
-that	O
-are	O
-characterized	O
-by	O
-their	O
-ability	O
-to	O
-dephosphorylate	O
-both	O
-phosphotyrosine	O
-and	O
-phosphoserine	O
-/	O
-phospho	O
--	O
-threonine	O
-residues	O
-within	O
-a	O
-substrate	O
-.	O
-	O
-Biochemical	O
-and	O
-modelling	O
-studies	O
-further	O
-demonstrate	O
-that	O
-the	O
-molecular	O
-interactions	O
-mediate	O
-this	O
-key	O
-element	O
-for	O
-substrate	O
-recognition	O
-are	O
-highly	O
-conserved	O
-among	O
-all	O
-MKP	O
--	O
-family	O
-members	O
-.	O
-	O
-In	O
-mammalian	O
-cells	O
-,	O
-the	O
-MKP	O
-subfamily	O
-includes	O
-10	O
-distinct	O
-catalytically	O
-active	O
-MKPs	O
-.	O
-	O
-Figure	O
-2b	O
-shows	O
-the	O
-variation	O
-of	O
-initial	O
-rates	O
-of	O
-the	O
-MKP7ΔC304	B-mutant
-and	O
-MKP7	O
--	O
-CD	O
--	O
-catalysed	O
-reaction	O
-with	O
-the	O
-concentration	O
-of	O
-phospho	O
--	O
-JNK1	O
-.	O
-Because	O
-the	O
-concentrations	O
-of	O
-MKP7	O
-and	O
-pJNK1	O
-were	O
-comparable	O
-in	O
-the	O
-reaction	O
-,	O
-the	O
-assumption	O
-that	O
-the	O
-free	O
--	O
-substrate	O
-concentration	O
-is	O
-equal	O
-to	O
-the	O
-total	O
-substrate	O
-concentration	O
-is	O
-not	O
-valid	O
-.	O
-	O
-To	O
-further	O
-confirm	O
-the	O
-JNK1	O
-–	O
-MKP7	O
--	O
-CD	O
-interaction	O
-,	O
-we	O
-performed	O
-a	O
-pull	O
--	O
-down	O
-assay	O
-using	O
-the	O
-purified	O
-proteins	O
-.	O
-	O
-The	O
-catalytic	O
-domain	O
-of	O
-MKP7	O
-interacts	O
-with	O
-JNK1	O
-through	O
-a	O
-contiguous	O
-surface	O
-area	O
-that	O
-is	O
-remote	O
-from	O
-the	O
-active	O
-site	O
-.	O
-	O
-The	O
-active	O
-site	O
-of	O
-MKP7	O
-consists	O
-of	O
-the	O
-phosphate	O
--	O
-binding	O
-loop	O
-(	O
-P	O
--	O
-loop	O
-,	O
-Cys244	O
--	O
-Leu245	O
--	O
-Ala246	O
--	O
-Gly247	O
--	O
-Ile248	O
--	O
-Ser249	O
--	O
-Arg250	O
-),	O
-and	O
-Asp213	O
-in	O
-the	O
-general	O
-acid	O
-loop	O
-(	O
-Fig	O
-.	O
-3b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-1b	O
-).	O
-	O
-The	O
-side	O
-chain	O
-of	O
-strictly	O
-conserved	O
-Arg250	O
-is	O
-oriented	O
-towards	O
-the	O
-negatively	O
-charged	O
-chloride	O
-,	O
-similar	O
-to	O
-the	O
-canonical	O
-phosphate	O
--	O
-coordinating	O
-conformation	O
-.	O
-	O
-In	O
-the	O
-complex	O
-,	O
-MKP7	O
--	O
-CD	O
-and	O
-JNK1	O
-form	O
-extensive	O
-protein	O
-–	O
-protein	O
-interactions	O
-involving	O
-the	O
-C	O
--	O
-terminal	O
-helices	O
-of	O
-MKP7	O
--	O
-CD	O
-and	O
-C	O
--	O
-lobe	O
-of	O
-JNK1	O
-(	O
-Fig	O
-.	O
-3d	O
-,	O
-e	O
-).	O
-	O
-Mutation	O
-of	O
-Leu288	O
-markedly	O
-reduced	O
-its	O
-solubility	O
-when	O
-expressed	O
-in	O
-Escherichia	O
-coli	O
-,	O
-resulting	O
-in	O
-the	O
-insoluble	O
-aggregation	O
-of	O
-the	O
-mutant	O
-protein	O
-.	O
-	O
-The	O
-small	O
-pNPP	O
-molecule	O
-binds	O
-directly	O
-at	O
-the	O
-enzyme	O
-active	O
-site	O
-and	O
-can	O
-be	O
-used	O
-to	O
-probe	O
-the	O
-reaction	O
-mechanism	O
-of	O
-protein	O
-phosphatases	O
-.	O
-	O
-Biochemical	O
-results	O
-suggested	O
-that	O
-the	O
-affinity	O
-and	O
-specificity	O
-between	O
-KAP	O
-and	O
-CDK2	O
-results	O
-from	O
-the	O
-recognition	O
-site	O
-comprising	O
-CDK2	O
-residues	O
-from	O
-the	O
-αG	O
-helix	O
-and	O
-L14	O
-loop	O
-and	O
-the	O
-N	O
--	O
-terminal	O
-helical	O
-region	O
-of	O
-KAP	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-Structural	O
-analysis	O
-and	O
-sequence	O
-alignment	O
-reveal	O
-that	O
-one	O
-of	O
-the	O
-few	O
-differences	O
-between	O
-MKP7	O
--	O
-CD	O
-and	O
-KAP	O
-in	O
-the	O
-substrate	O
--	O
-binding	O
-region	O
-is	O
-the	O
-presence	O
-of	O
-the	O
-motif	O
-FNFL	O
-in	O
-MKP7	O
--	O
-CD	O
-,	O
-which	O
-corresponds	O
-to	O
-IKQY	O
-in	O
-KAP	O
-(	O
-Fig	O
-.	O
-5c	O
-).	O
-	O
-Parallel	O
-experiments	O
-showed	O
-clearly	O
-that	O
-the	O
-D	O
--	O
-motif	O
-mutants	O
-(	O
-R56A	B-mutant
-/	O
-R57A	B-mutant
-and	O
-V63A	B-mutant
-/	O
-I65A	B-mutant
-)	O
-dephosphorylated	O
-JNK	O
-as	O
-did	O
-the	O
-wild	O
-type	O
-under	O
-the	O
-same	O
-conditions	O
-,	O
-further	O
-confirming	O
-that	O
-the	O
-MKP7	O
--	O
-KBD	O
-is	O
-not	O
-required	O
-for	O
-the	O
-JNK	O
-inactivation	O
-in	O
-vivo	O
-.	O
-	O
-Consistent	O
-with	O
-the	O
-in	O
-vitro	O
-data	O
-,	O
-the	O
-level	O
-of	O
-phosphorylated	O
-JNK	O
-was	O
-not	O
-or	O
-little	O
-altered	O
-in	O
-MKP7	O
-FXF	O
--	O
-motif	O
-mutants	O
-(	O
-F285D	B-mutant
-,	O
-F287D	B-mutant
-and	O
-L288D	B-mutant
-)-	O
-transfected	O
-cells	O
-,	O
-and	O
-the	O
-MKP7	O
-D268A	B-mutant
-and	O
-N286A	B-mutant
-mutants	O
-retained	O
-the	O
-ability	O
-to	O
-reduce	O
-the	O
-phosphorylation	O
-levels	O
-of	O
-JNK	O
-.	O
-	O
-In	O
-agreement	O
-with	O
-the	O
-in	O
-vitro	O
-pull	O
--	O
-down	O
-results	O
-,	O
-the	O
-mutants	O
-D229A	B-mutant
-,	O
-W234D	B-mutant
-and	O
-Y259D	B-mutant
-were	O
-not	O
-co	O
--	O
-precipitated	O
-with	O
-MKP7	O
-,	O
-and	O
-the	O
-I231D	B-mutant
-mutant	O
-had	O
-only	O
-little	O
-effect	O
-on	O
-the	O
-JNK1	O
-–	O
-MKP7	O
-interaction	O
-(	O
-Fig	O
-.	O
-6d	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-).	O
-	O
-Moreover	O
-,	O
-treatment	O
-of	O
-cells	O
-expressing	O
-MKP7	O
--	O
-KBD	O
-mutants	O
-(	O
-R56A	B-mutant
-/	O
-R57A	B-mutant
-and	O
-V63A	B-mutant
-/	O
-I65A	B-mutant
-)	O
-decreased	O
-the	O
-apoptosis	O
-rates	O
-to	O
-a	O
-similar	O
-extent	O
-as	O
-MKP7	O
-wild	O
-type	O
-did	O
-.	O
-	O
-MKP5	O
-belongs	O
-to	O
-the	O
-same	O
-subfamily	O
-as	O
-MKP7	O
-.	O
-	O
-In	O
-contrast	O
-to	O
-p38α	O
-substrate	O
-,	O
-deletion	O
-of	O
-the	O
-MKP5	O
--	O
-KBD	O
-had	O
-little	O
-effects	O
-on	O
-the	O
-kinetic	O
-parameters	O
-for	O
-the	O
-JNK1	O
-dephosphorylation	O
-,	O
-indicating	O
-that	O
-the	O
-KBD	O
-of	O
-MKP5	O
-is	O
-not	O
-required	O
-for	O
-the	O
-JNK1	O
-dephosphorylation	O
-(	O
-Fig	O
-.	O
-7b	O
-).	O
-	O
-As	O
-shown	O
-in	O
-Fig	O
-.	O
-7f	O
-,	O
-the	O
-T432A	B-mutant
-and	O
-L449F	B-mutant
-MKP5	O
-mutant	O
-showed	O
-little	O
-or	O
-no	O
-difference	O
-in	O
-phosphatase	O
-activity	O
-,	O
-whereas	O
-the	O
-other	O
-mutants	O
-showed	O
-reduced	O
-specific	O
-activities	O
-of	O
-MKP5	O
-.	O
-	O
-As	O
-in	O
-the	O
-case	O
-of	O
-MKP7	O
-,	O
-all	O
-the	O
-mutants	O
-,	O
-except	O
-F451D	B-mutant
-/	I-mutant
-A	I-mutant
-,	O
-showed	O
-no	O
-pNPPase	O
-activity	O
-changes	O
-compared	O
-with	O
-the	O
-wild	O
--	O
-type	O
-MKP5	O
--	O
-CD	O
-(	O
-Fig	O
-.	O
-7g	O
-),	O
-and	O
-the	O
-point	O
-mutations	O
-in	O
-JNK1	O
-also	O
-reduced	O
-the	O
-binding	O
-affinity	O
-of	O
-MKP5	O
--	O
-CD	O
-for	O
-JNK1	O
-(	O
-Fig	O
-.	O
-7h	O
-).	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-the	O
-experimental	O
-observation	O
-showing	O
-that	O
-JNK1	O
-binds	O
-to	O
-MKP7	O
--	O
-CD	O
-much	O
-more	O
-tightly	O
-than	O
-MKP5	O
--	O
-CD	O
-(	O
-Km	O
-value	O
-of	O
-MKP5	O
--	O
-CD	O
-for	O
-pJNK1	O
-substrate	O
-is	O
-∼	O
-20	O
--	O
-fold	O
-higher	O
-than	O
-that	O
-of	O
-MKP7	O
--	O
-CD	O
-).	O
-	O
-The	O
-MAPKs	O
-p38	O
-,	O
-ERK	O
-and	O
-JNK	O
-,	O
-are	O
-central	O
-to	O
-evolutionarily	O
-conserved	O
-signalling	O
-pathways	O
-that	O
-are	O
-present	O
-in	O
-all	O
-eukaryotic	O
-cells	O
-.	O
-	O
-Each	O
-MAPK	O
-cascade	O
-is	O
-activated	O
-in	O
-response	O
-to	O
-a	O
-diverse	O
-array	O
-of	O
-extracellular	O
-signals	O
-and	O
-culminates	O
-in	O
-the	O
-dual	O
--	O
-phosphorylation	O
-of	O
-a	O
-threonine	O
-and	O
-a	O
-tyrosine	O
-residue	O
-in	O
-the	O
-MAPK	O
--	O
-activation	O
-loop	O
-.	O
-	O
-This	O
-structure	O
-reveals	O
-an	O
-FXF	O
--	O
-docking	O
-interaction	O
-mode	O
-between	O
-MAPK	O
-and	O
-MKP	O
-.	O
-	O
-When	O
-MKP7	O
-is	O
-bound	O
-to	O
-JIP	O
--	O
-1	O
-,	O
-it	O
-reduces	O
-JNK	O
-activation	O
-,	O
-leading	O
-to	O
-reduced	O
-phosphorylation	O
-of	O
-the	O
-JNK	O
-target	O
-c	O
--	O
-Jun	O
-.	O
-	O
-The	O
-colour	O
-scheme	O
-is	O
-the	O
-same	O
-in	O
-the	O
-following	O
-figures	O
-unless	O
-indicated	O
-otherwise	O
-.	O
-(	O
-b	O
-)	O
-Plots	O
-of	O
-initial	O
-velocity	O
-of	O
-the	O
-MKP7	O
--	O
-catalysed	O
-reaction	O
-versus	O
-phospho	O
--	O
-JNK1	O
-concentration	O
-.	O
-	O
-The	O
-top	O
-panel	O
-shows	O
-the	O
-relative	O
-affinities	O
-of	O
-MKP7	O
--	O
-CD	O
-and	O
-MKP7	O
--	O
-KBD	O
-to	O
-JNK1	O
-,	O
-with	O
-the	O
-affinity	O
-of	O
-MKP7	O
--	O
-CD	O
-defined	O
-as	O
-100	O
-%;	O
-the	O
-middle	O
-panel	O
-is	O
-the	O
-electrophoretic	O
-pattern	O
-of	O
-MKP7	O
-and	O
-JNK1	O
-after	O
-GST	O
-pull	O
--	O
-down	O
-assays	O
-.	O
-	O
-Blue	O
-dashed	O
-lines	O
-represent	O
-polar	O
-interactions	O
-.	O
-	O
-The	O
-CDK2	O
-is	O
-shown	O
-in	O
-surface	O
-representation	O
-coloured	O
-according	O
-to	O
-the	O
-electrostatic	O
-potential	O
-(	O
-positive	O
-,	O
-blue	O
-;	O
-negative	O
-,	O
-red	O
-).	O
-	O
-Residues	O
-of	O
-MKP7	O
--	O
-CD	O
-involved	O
-in	O
-JNK1	O
-recognition	O
-are	O
-indicated	O
-by	O
-cyan	O
-asterisks	O
-,	O
-and	O
-the	O
-conserved	O
-FXF	O
--	O
-motif	O
-is	O
-highlighted	O
-in	O
-cyan	O
-.	O
-	O
-The	O
-secondary	O
-structure	O
-assignments	O
-of	O
-MKP7	O
--	O
-CD	O
-and	O
-KAP	O
-are	O
-shown	O
-above	O
-and	O
-below	O
-each	O
-sequence	O
-.	O
-	O
-Shown	O
-is	O
-a	O
-typical	O
-immunoblot	O
-for	O
-phosphorylated	O
-JNK	O
-from	O
-three	O
-independent	O
-experiments	O
-.	O
-	O
-The	O
-results	O
-using	O
-Annexin	O
--	O
-V	O
-stain	O
-for	O
-membrane	O
-phosphatidylserine	O
-eversion	O
-,	O
-combined	O
-with	O
-propidium	O
-iodide	O
-(	O
-PI	O
-)	O
-uptake	O
-to	O
-evaluate	O
-cells	O
-whose	O
-membranes	O
-had	O
-been	O
-compromised	O
-.	O
-	O
-The	O
-solid	O
-lines	O
-are	O
-best	O
--	O
-fitting	O
-results	O
-according	O
-to	O
-the	O
-Michaelis	O
-–	O
-Menten	O
-equation	O
-with	O
-Km	O
-and	O
-kcat	O
-values	O
-indicated	O
-.	O
-	O
-(	O
-d	O
-)	O
-Gel	O
-filtration	O
-analysis	O
-for	O
-interaction	O
-of	O
-JNK1	O
-with	O
-MKP5	O
--	O
-CD	O
-and	O
-MKP5	O
--	O
-KBD	O
-.	O
-(	O
-e	O
-)	O
-GST	O
--	O
-mediated	O
-pull	O
--	O
-down	O
-assays	O
-for	O
-interaction	O
-of	O
-JNK1	O
-with	O
-MKP5	O
--	O
-CD	O
-and	O
-MKP5	O
--	O
-KBD	O
-.	O
-	O
-The	O
-panels	O
-are	O
-arranged	O
-the	O
-same	O
-as	O
-in	O
-Fig	O
-.	O
-2d	O
-.	O
-(	O
-f	O
-)	O
-Effects	O
-of	O
-mutations	O
-in	O
-MKP5	O
--	O
-CD	O
-on	O
-the	O
-JNK1	O
-dephosphorylation	O
-(	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.,	O
-n	O
-=	O
-3	O
-).	O
-	O
-(	O
-g	O
-)	O
-Effects	O
-of	O
-mutations	O
-in	O
-MKP5	O
--	O
-CD	O
-on	O
-the	O
-pNPP	O
-hydrolysis	O
-reaction	O
-(	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-m	O
-.,	O
-n	O
-=	O
-3	O
-).	O
-	O
-The	O
-HAESA	O
-ectodomain	O
-folds	O
-into	O
-a	O
-superhelical	O
-assembly	O
-of	O
-21	O
-leucine	O
--	O
-rich	O
-repeats	O
-.	O
-	O
-The	O
-HAESA	O
-ectodomain	O
-is	O
-shown	O
-in	O
-blue	O
-(	O
-in	O
-surface	O
-representation	O
-),	O
-the	O
-glycan	O
-structures	O
-are	O
-shown	O
-in	O
-yellow	O
-.	O
-	O
-Residues	O
-mediating	O
-hydrophobic	O
-interactions	O
-with	O
-the	O
-IDA	O
-peptide	O
-are	O
-highlighted	O
-in	O
-blue	O
-,	O
-residues	O
-contributing	O
-to	O
-hydrogen	O
-bond	O
-interactions	O
-and	O
-/	O
-or	O
-salt	O
-bridges	O
-are	O
-shown	O
-in	O
-red	O
-.	O
-	O
-The	O
-alignment	O
-includes	O
-a	O
-secondary	O
-structure	O
-assignment	O
-calculated	O
-with	O
-the	O
-program	O
-DSSP	O
-and	O
-colored	O
-according	O
-to	O
-Figure	O
-1	O
-,	O
-with	O
-the	O
-N	O
--	O
-and	O
-C	O
--	O
-terminal	O
-caps	O
-and	O
-the	O
-21	O
-LRR	O
-motifs	O
-indicated	O
-in	O
-orange	O
-and	O
-blue	O
-,	O
-respectively	O
-.	O
-	O
-Void	O
-(	O
-V0	O
-)	O
-volume	O
-and	O
-total	O
-volume	O
-(	O
-Vt	O
-)	O
-are	O
-shown	O
-,	O
-together	O
-with	O
-elution	O
-volumes	O
-for	O
-molecular	O
-mass	O
-standards	O
-(	O
-A	O
-,	O
-Thyroglobulin	O
-,	O
-669	O
-,	O
-000	O
-Da	O
-;	O
-B	O
-,	O
-Ferritin	O
-,	O
-440	O
-,	O
-00	O
-Da	O
-,	O
-C	O
-,	O
-Aldolase	O
-,	O
-158	O
-,	O
-000	O
-Da	O
-;	O
-D	O
-,	O
-Conalbumin	O
-,	O
-75	O
-,	O
-000	O
-Da	O
-;	O
-E	O
-,	O
-Ovalbumin	O
-,	O
-44	O
-,	O
-000	O
-Da	O
-;	O
-F	O
-,	O
-Carbonic	O
-anhydrase	O
-,	O
-29	O
-,	O
-000	O
-Da	O
-).	O
-	O
-Mutant	O
-(	O
-m	O
-)	O
-versions	O
-,	O
-which	O
-carry	O
-point	O
-mutations	O
-in	O
-their	O
-active	O
-sites	O
-(	O
-Asp837HAESA	B-mutant
-→	I-mutant
-Asn	I-mutant
-,	O
-Asp447SERK1	B-mutant
-→	I-mutant
-Asn	I-mutant
-)	O
-possess	O
-no	O
-autophosphorylation	O
-activity	O
-(	O
-lanes	O
-2	O
-+	O
-4	O
-).	O
-	O
-We	O
-next	O
-determined	O
-crystal	O
-structures	O
-of	O
-the	O
-apo	O
-HAESA	O
-ectodomain	O
-and	O
-of	O
-a	O
-HAESA	O
--	O
-IDA	O
-complex	O
-,	O
-at	O
-1	O
-.	O
-74	O
-and	O
-1	O
-.	O
-86	O
-Å	O
-resolution	O
-,	O
-respectively	O
-(	O
-Figure	O
-1C	O
-;	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1B	O
-–	O
-D	O
-;	O
-Tables	O
-1	O
-,	O
-2	O
-).	O
-	O
-We	O
-next	O
-tested	O
-if	O
-HAESA	O
-binds	O
-other	O
-IDA	O
-peptide	O
-family	O
-members	O
-.	O
-	O
-Notably	O
-,	O
-HAESA	O
-can	O
-discriminate	O
-between	O
-IDLs	O
-and	O
-functionally	O
-unrelated	O
-dodecamer	O
-peptides	O
-with	O
-Hyp	O
-modifications	O
-,	O
-such	O
-as	O
-CLV3	O
-(	O
-Figures	O
-2D	O
-,	O
-7	O
-).	O
-	O
-Our	O
-experiments	O
-suggest	O
-that	O
-among	O
-the	O
-SERK	O
-family	O
-members	O
-,	O
-SERK1	O
-is	O
-a	O
-positive	O
-regulator	O
-of	O
-floral	O
-abscission	O
-.	O
-	O
-We	O
-thus	O
-focused	O
-on	O
-analyzing	O
-the	O
-contribution	O
-of	O
-SERK1	O
-to	O
-HAESA	O
-ligand	O
-sensing	O
-and	O
-receptor	O
-activation	O
-.	O
-	O
-Our	O
-calorimetry	O
-experiments	O
-now	O
-reveal	O
-that	O
-SERKs	O
-may	O
-render	O
-HAESA	O
-,	O
-and	O
-potentially	O
-other	O
-receptor	O
-kinases	O
-,	O
-competent	O
-for	O
-high	O
--	O
-affinity	O
-sensing	O
-of	O
-their	O
-cognate	O
-ligands	O
-.	O
-	O
-Together	O
-,	O
-our	O
-genetic	O
-and	O
-biochemical	O
-experiments	O
-implicate	O
-SERK1	O
-as	O
-a	O
-HAESA	O
-co	O
--	O
-receptor	O
-in	O
-the	O
-Arabidopsis	O
-abscission	O
-zone	O
-.	O
-	O
-The	O
-conformational	O
-change	O
-in	O
-the	O
-C	O
--	O
-terminal	O
-LRRs	O
-and	O
-capping	O
-domain	O
-is	O
-indicated	O
-by	O
-an	O
-arrow	O
-.	O
-(	O
-C	O
-)	O
-SERK1	O
-forms	O
-an	O
-integral	O
-part	O
-of	O
-the	O
-receptor	O
-'	O
-s	O
-peptide	O
-binding	O
-pocket	O
-.	O
-	O
-The	O
-SERK1	O
-ectodomain	O
-interacts	O
-with	O
-the	O
-IDA	O
-peptide	O
-binding	O
-site	O
-using	O
-a	O
-loop	O
-region	O
-(	O
-residues	O
-51	O
--	O
-59SERK1	O
-)	O
-from	O
-its	O
-N	O
--	O
-terminal	O
-cap	O
-(	O
-Figure	O
-4A	O
-,	O
-C	O
-).	O
-	O
-Deletion	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-Asn69IDA	O
-completely	O
-inhibits	O
-complex	O
-formation	O
-.	O
-	O
-15	O
-out	O
-of	O
-15	O
-35S	O
-::	O
-IDA	O
-plants	O
-,	O
-0	O
-out	O
-of	O
-15	O
-Col	O
--	O
-0	O
-plants	O
-and	O
-0	O
-out	O
-of	O
-15	O
-35S	O
-::	O
-IDA	B-mutant
-K66A	I-mutant
-/	I-mutant
-R67A	I-mutant
-double	O
--	O
-mutant	O
-plants	O
-,	O
-showed	O
-an	O
-enlarged	O
-abscission	O
-zone	O
-,	O
-respectively	O
-(	O
-3	O
-independent	O
-lines	O
-were	O
-analyzed	O
-).	O
-	O
-In	O
-contrast	O
-,	O
-over	O
--	O
-expression	O
-of	O
-the	O
-IDA	B-mutant
-Lys66IDA	I-mutant
-/	I-mutant
-Arg67IDA	I-mutant
-→	I-mutant
-Ala	I-mutant
-double	O
-mutant	O
-significantly	O
-delays	O
-floral	O
-abscission	O
-when	O
-compared	O
-to	O
-wild	O
--	O
-type	O
-control	O
-plants	O
-,	O
-suggesting	O
-that	O
-the	O
-mutant	O
-IDA	O
-peptide	O
-has	O
-reduced	O
-activity	O
-in	O
-planta	O
-(	O
-Figure	O
-5C	O
-–	O
-E	O
-).	O
-	O
-For	O
-a	O
-rapidly	O
-growing	O
-number	O
-of	O
-plant	O
-signaling	O
-pathways	O
-,	O
-SERK	O
-proteins	O
-act	O
-as	O
-these	O
-essential	O
-co	O
--	O
-receptors	O
-(;	O
-).	O
-	O
-The	O
-central	O
-Hyp	O
-residue	O
-in	O
-IDA	O
-is	O
-found	O
-buried	O
-in	O
-the	O
-HAESA	O
-peptide	O
-binding	O
-surface	O
-and	O
-thus	O
-this	O
-post	O
--	O
-translational	O
-modification	O
-may	O
-regulate	O
-IDA	O
-bioactivity	O
-.	O
-	O
-In	O
-our	O
-quantitative	O
-biochemical	O
-assays	O
-,	O
-the	O
-presence	O
-of	O
-SERK1	O
-dramatically	O
-increases	O
-the	O
-HAESA	O
-binding	O
-specificity	O
-and	O
-affinity	O
-for	O
-IDA	O
-.	O
-	O
-It	O
-is	O
-of	O
-note	O
-that	O
-our	O
-reported	O
-binding	O
-affinities	O
-for	O
-IDA	O
-and	O
-SERK1	O
-have	O
-been	O
-measured	O
-using	O
-synthetic	O
-peptides	O
-and	O
-the	O
-isolated	O
-HAESA	O
-and	O
-SERK1	O
-ectodomains	O
-,	O
-and	O
-thus	O
-might	O
-differ	O
-in	O
-the	O
-context	O
-of	O
-the	O
-full	O
--	O
-length	O
-,	O
-membrane	O
--	O
-embedded	O
-signaling	O
-complex	O
-.	O
-	O
-(	O
-B	O
-)	O
-View	O
-of	O
-the	O
-inner	O
-surface	O
-of	O
-the	O
-SERK1	O
-LRR	O
-domain	O
-(	O
-PDB	O
--	O
-ID	O
-4lsc	O
-,	O
-surface	O
-representation	O
-,	O
-in	O
-gray	O
-).	O
-	O
-Structure	O
--	O
-guided	O
-multiple	O
-sequence	O
-alignment	O
-of	O
-IDA	O
-and	O
-IDA	O
--	O
-like	O
-peptides	O
-with	O
-other	O
-plant	O
-peptide	O
-hormone	O
-families	O
-,	O
-including	O
-CLAVATA3	O
-–	O
-EMBRYO	O
-SURROUNDING	O
-REGION	O
--	O
-RELATED	O
-(	O
-CLV3	O
-/	O
-CLE	O
-),	O
-ROOT	O
-GROWTH	O
-FACTOR	O
-–	O
-GOLVEN	O
-(	O
-RGF	O
-/	O
-GLV	O
-),	O
-PRECURSOR	O
-GENE	O
-PROPEP1	O
-(	O
-PEP1	O
-)	O
-from	O
-Arabidopsis	O
-thaliana	O
-.	O
-	O
-It	O
-is	O
-interesting	O
-to	O
-note	O
-,	O
-that	O
-CLEs	O
-in	O
-their	O
-mature	O
-form	O
-are	O
-also	O
-hydroxyprolinated	O
-dodecamers	O
-,	O
-which	O
-bind	O
-to	O
-a	O
-surface	O
-area	O
-in	O
-the	O
-BARELY	O
-ANY	O
-MERISTEM	O
-1	O
-receptor	O
-that	O
-would	O
-correspond	O
-to	O
-part	O
-of	O
-the	O
-IDA	O
-binding	O
-cleft	O
-in	O
-HAESA	O
-.	O
-	O
-The	O
-structures	O
-suggest	O
-a	O
-trajectory	O
-of	O
-IRES	O
-translocation	O
-,	O
-required	O
-for	O
-translation	O
-initiation	O
-,	O
-and	O
-provide	O
-an	O
-unprecedented	O
-view	O
-of	O
-eEF2	O
-dynamics	O
-.	O
-	O
-To	O
-initiate	O
-translation	O
-,	O
-a	O
-structured	O
-IRES	O
-RNA	O
-interacts	O
-with	O
-the	O
-40S	O
-subunit	O
-or	O
-the	O
-80S	O
-ribosome	O
-,	O
-resulting	O
-in	O
-precise	O
-positioning	O
-of	O
-the	O
-downstream	O
-start	O
-codon	O
-in	O
-the	O
-small	O
-40S	O
-subunit	O
-.	O
-	O
-The	O
-canonical	O
-scenario	O
-of	O
-cap	O
--	O
-dependent	O
-and	O
-IRES	O
--	O
-dependent	O
-initiation	O
-involves	O
-positioning	O
-of	O
-the	O
-AUG	O
-start	O
-codon	O
-and	O
-the	O
-initiator	O
-tRNAMet	O
-in	O
-the	O
-ribosomal	O
-peptidyl	O
--	O
-tRNA	O
-(	O
-P	O
-)	O
-site	O
-,	O
-facilitated	O
-by	O
-interaction	O
-with	O
-initiation	O
-factors	O
-.	O
-	O
-The	O
-codon	O
--	O
-anticodon	O
--	O
-like	O
-helix	O
-of	O
-PKI	O
-is	O
-stabilized	O
-by	O
-interactions	O
-with	O
-the	O
-universally	O
-conserved	O
-decoding	O
--	O
-center	O
-nucleotides	O
-G577	O
-,	O
-A1755	O
-and	O
-A1756	O
-(	O
-G530	O
-,	O
-A1492	O
-and	O
-A1493	O
-in	O
-E	O
-.	O
-coli	O
-16S	O
-ribosomal	O
-RNA	O
-,	O
-or	O
-rRNA	O
-).	O
-	O
-How	O
-this	O
-non	O
--	O
-canonical	O
-initiation	O
-complex	O
-transitions	O
-to	O
-the	O
-elongation	O
-step	O
-is	O
-not	O
-fully	O
-understood	O
-.	O
-	O
-Translocation	O
-of	O
-2tRNA	O
-•	O
-mRNA	O
-involves	O
-two	O
-major	O
-large	O
--	O
-scale	O
-ribosome	O
-rearrangements	O
-(	O
-Figure	O
-1	O
-—	O
-figure	O
-supplement	O
-1	O
-)	O
-(	O
-reviewed	O
-in	O
-).	O
-	O
-Concurrently	O
-,	O
-the	O
-deacyl	O
--	O
-tRNA	O
-interacts	O
-with	O
-the	O
-P	O
-site	O
-of	O
-the	O
-small	O
-subunit	O
-and	O
-the	O
-E	O
-site	O
-of	O
-the	O
-large	O
-subunit	O
-(	O
-P	O
-/	O
-E	O
-hybrid	O
-state	O
-).	O
-	O
-Binding	O
-of	O
-EF	O
--	O
-G	O
-next	O
-to	O
-the	O
-A	O
-site	O
-and	O
-reverse	O
-rotation	O
-of	O
-the	O
-small	O
-subunit	O
-results	O
-in	O
-translocation	O
-of	O
-both	O
-ASLs	O
-on	O
-the	O
-small	O
-subunit	O
-.	O
-	O
-Structures	O
-of	O
-the	O
-70S	O
-•	O
-EF	O
--	O
-G	O
-complex	O
-bound	O
-with	O
-two	O
-nearly	O
-translocated	O
-tRNAs	O
-,	O
-exhibit	O
-a	O
-large	O
-18	O
-°	O
-to	O
-21	O
-°	O
-head	O
-swivel	O
-in	O
-a	O
-mid	O
--	O
-rotated	O
-subunit	O
-,	O
-whereas	O
-no	O
-head	O
-swivel	O
-is	O
-observed	O
-in	O
-the	O
-fully	O
-rotated	O
-pre	O
--	O
-translocation	O
-or	O
-in	O
-the	O
-non	O
--	O
-rotated	O
-post	O
--	O
-translocation	O
-70S	O
-•	O
-2tRNA	O
-•	O
-EF	O
--	O
-G	O
-structures	O
-.	O
-	O
-The	O
-head	O
-swivel	O
-was	O
-proposed	O
-to	O
-facilitate	O
-transition	O
-of	O
-the	O
-tRNA	O
-from	O
-the	O
-P	O
-to	O
-E	O
-site	O
-by	O
-widening	O
-a	O
-constriction	O
-between	O
-these	O
-sites	O
-on	O
-the	O
-30S	O
-subunit	O
-.	O
-	O
-This	O
-widening	O
-allows	O
-the	O
-ASL	O
-to	O
-sample	O
-positions	O
-between	O
-the	O
-P	O
-and	O
-E	O
-sites	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structures	O
-of	O
-bacterial	O
-70S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-translocation	O
-complexes	O
-,	O
-ordered	O
-according	O
-to	O
-the	O
-position	O
-of	O
-the	O
-translocating	O
-A	O
-->	O
-P	O
-tRNA	O
-(	O
-orange	O
-).	O
-	O
-The	O
-large	O
-ribosomal	O
-subunit	O
-is	O
-shown	O
-in	O
-cyan	O
-;	O
-the	O
-small	O
-subunit	O
-in	O
-light	O
-yellow	O
-(	O
-head	O
-)	O
-and	O
-wheat	O
--	O
-yellow	O
-(	O
-body	O
-),	O
-elongation	O
-factor	O
-G	O
-(	O
-EF	O
--	O
-G	O
-)	O
-is	O
-shown	O
-in	O
-green	O
-.	O
-	O
-Nucleotides	O
-C1274	O
-,	O
-U1191	O
-of	O
-the	O
-40S	O
-head	O
-and	O
-G904	O
-of	O
-the	O
-platform	O
-(	O
-corresponding	O
-to	O
-C1054	O
-,	O
-G966	O
-and	O
-G693	O
-in	O
-E	O
-.	O
-coli	O
-16S	O
-rRNA	O
-)	O
-are	O
-shown	O
-in	O
-black	O
-to	O
-denote	O
-the	O
-A	O
-,	O
-P	O
-and	O
-E	O
-sites	O
-,	O
-respectively	O
-.	O
-	O
-Subsequent	O
-3D	O
-classification	O
-using	O
-a	O
-2D	O
-mask	O
-comprising	O
-PKI	O
-and	O
-domain	O
-IV	O
-of	O
-eEF2	O
-yielded	O
-5	O
-'	O
-purified	O
-'	O
-classes	O
-representing	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-Sub	O
--	O
-classification	O
-of	O
-each	O
-class	O
-did	O
-not	O
-yield	O
-additional	O
-classes	O
-,	O
-but	O
-helped	O
-improve	O
-density	O
-in	O
-the	O
-PKI	O
-region	O
-of	O
-class	O
-III	O
-(	O
-estimated	O
-resolution	O
-and	O
-percentage	O
-of	O
-particles	O
-in	O
-the	O
-sub	O
--	O
-classified	O
-reconstruction	O
-are	O
-shown	O
-in	O
-parentheses	O
-).	O
-	O
-Cryo	O
--	O
-EM	O
-structures	O
-of	O
-the	O
-80S	O
-•	O
-TSV	O
-IRES	O
-bound	O
-with	O
-eEF2	O
-•	O
-GDP	O
-•	O
-sordarin	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-In	O
-all	O
-panels	O
-,	O
-the	O
-large	O
-ribosomal	O
-subunit	O
-is	O
-shown	O
-in	O
-cyan	O
-;	O
-the	O
-small	O
-subunit	O
-in	O
-light	O
-yellow	O
-(	O
-head	O
-)	O
-and	O
-wheat	O
--	O
-yellow	O
-(	O
-body	O
-);	O
-the	O
-TSV	O
-IRES	O
-in	O
-red	O
-,	O
-eEF2	O
-in	O
-green	O
-.	O
-	O
-We	O
-sought	O
-to	O
-address	O
-the	O
-following	O
-questions	O
-by	O
-structural	O
-visualization	O
-of	O
-80S	O
-•	O
-IRES	O
-•	O
-eEF2	O
-translocation	O
-complexes	O
-:	O
-(	O
-1	O
-)	O
-How	O
-does	O
-a	O
-large	O
-IRES	O
-RNA	O
-move	O
-through	O
-the	O
-restricted	O
-intersubunit	O
-space	O
-,	O
-bringing	O
-PKI	O
-from	O
-the	O
-A	O
-to	O
-P	O
-site	O
-of	O
-the	O
-small	O
-subunit	O
-?	O
-(	O
-2	O
-)	O
-How	O
-does	O
-eEF2	O
-mediate	O
-IRES	O
-translocation	O
-?	O
-(	O
-3	O
-)	O
-Does	O
-IRES	O
-translocation	O
-involve	O
-large	O
-rearrangements	O
-in	O
-the	O
-ribosome	O
-,	O
-similar	O
-to	O
-tRNA	O
-translocation	O
-?	O
-(	O
-4	O
-)	O
-What	O
-,	O
-if	O
-any	O
-,	O
-is	O
-the	O
-mechanistic	O
-role	O
-of	O
-40S	O
-head	O
-rotation	O
-in	O
-IRES	O
-translocation	O
-?	O
-	O
-Maximum	O
--	O
-likelihood	O
-classification	O
-using	O
-FREALIGN	O
-identified	O
-five	O
-IRES	O
--	O
-eEF2	O
--	O
-bound	O
-ribosome	O
-structures	O
-within	O
-a	O
-single	O
-sample	O
-(	O
-Figures	O
-1	O
-and	O
-2	O
-).	O
-	O
-The	O
-structures	O
-differ	O
-in	O
-the	O
-positions	O
-and	O
-conformations	O
-of	O
-ribosomal	O
-subunits	O
-(	O
-Figures	O
-1b	O
-and	O
-2	O
-),	O
-IRES	O
-RNA	O
-(	O
-Figures	O
-3	O
-and	O
-4	O
-)	O
-and	O
-eEF2	O
-(	O
-Figures	O
-5	O
-and	O
-6	O
-).	O
-	O
-18S	O
-ribosomal	O
-RNA	O
-is	O
-shown	O
-and	O
-ribosomal	O
-proteins	O
-are	O
-omitted	O
-for	O
-clarity	O
-.	O
-	O
-The	O
-superpositions	O
-of	O
-structures	O
-were	O
-performed	O
-by	O
-structural	O
-alignments	O
-of	O
-the	O
-18S	O
-ribosomal	O
-RNAs	O
-excluding	O
-the	O
-head	O
-region	O
-(	O
-nt	O
-1150	O
-–	O
-1620	O
-).	O
-	O
-Structure	O
-IV	O
-adopts	O
-a	O
-slightly	O
-rotated	O
-conformation	O
-(~	O
-1	O
-°).	O
-	O
-40S	O
-head	O
-swivel	O
-	O
-Comparison	O
-of	O
-the	O
-TSV	O
-IRES	O
-and	O
-eEF2	O
-positions	O
-in	O
-Structures	O
-I	O
-through	O
-V	O
-.	O
-	O
-In	O
-all	O
-panels	O
-,	O
-superpositions	O
-were	O
-obtained	O
-by	O
-structural	O
-alignments	O
-of	O
-the	O
-18S	O
-rRNAs	O
-.	O
-	O
-Ribosomal	O
-proteins	O
-of	O
-the	O
-initiation	O
-state	O
-are	O
-shown	O
-in	O
-gray	O
-for	O
-comparison	O
-.	O
-	O
-Loop	O
-1	O
-.	O
-1	O
-and	O
-stem	O
-loops	O
-4	O
-and	O
-5	O
-of	O
-the	O
-IRES	O
-are	O
-labeled	O
-.	O
-	O
-Positions	O
-of	O
-tRNAs	O
-and	O
-the	O
-TSV	O
-IRES	O
-relative	O
-to	O
-the	O
-A	O
--	O
-site	O
-finger	O
-(	O
-blue	O
-,	O
-nt	O
-1008	O
-–	O
-1043	O
-of	O
-25S	O
-rRNA	O
-)	O
-and	O
-the	O
-P	O
-site	O
-of	O
-the	O
-large	O
-subunit	O
-,	O
-comprising	O
-helix	O
-84	O
-of	O
-25S	O
-rRNA	O
-(	O
-nt	O
-.	O
-	O
-Structures	O
-of	O
-80S	O
-•	O
-IRES	O
-complexes	O
-in	O
-the	O
-absence	O
-of	O
-eEF2	O
-(	O
-INIT	O
-;	O
-PDB	O
-3J6Y	O
-,)	O
-and	O
-in	O
-the	O
-presence	O
-of	O
-eEF2	O
-(	O
-this	O
-work	O
-)	O
-are	O
-shown	O
-in	O
-the	O
-lower	O
-row	O
-and	O
-labeled	O
-.	O
-	O
-Interactions	O
-of	O
-the	O
-TSV	O
-IRES	O
-with	O
-uL5	O
-and	O
-eL42	O
-.	O
-	O
-Pseudoknots	O
-and	O
-stem	O
-loops	O
-are	O
-labeled	O
-and	O
-colored	O
-as	O
-in	O
-(	O
-a	O
-).	O
-	O
-The	O
-L1	O
-.	O
-1	O
-region	O
-remains	O
-in	O
-contact	O
-with	O
-the	O
-L1	O
-stalk	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-3	O
-).	O
-	O
-As	O
-such	O
-,	O
-the	O
-transition	O
-from	O
-the	O
-initiation	O
-state	O
-to	O
-Structure	O
-I	O
-involves	O
-repositioning	O
-of	O
-SL3	O
-around	O
-the	O
-A	O
--	O
-site	O
-finger	O
-,	O
-resembling	O
-the	O
-transition	O
-between	O
-the	O
-pre	O
--	O
-translocation	O
-A	O
-/	O
-P	O
-and	O
-A	O
-/	O
-P	O
-*	O
-tRNA	O
-.	O
-	O
-Another	O
-local	O
-rearrangement	O
-concerns	O
-loop	O
-3	O
-,	O
-also	O
-known	O
-as	O
-the	O
-variable	O
-loop	O
-region	O
-,	O
-which	O
-connects	O
-the	O
-ASL	O
--	O
-and	O
-mRNA	O
--	O
-like	O
-parts	O
-of	O
-PKI	O
-.	O
-	O
-The	O
-interaction	O
-of	O
-loop	O
-3	O
-backbone	O
-with	O
-uS7	O
-resembles	O
-that	O
-of	O
-the	O
-anticodon	O
--	O
-stem	O
-loop	O
-of	O
-E	O
--	O
-site	O
-tRNA	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-80S	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-structure	O
-(	O
-Figure	O
-3	O
-—	O
-figure	O
-supplement	O
-5	O
-).	O
-	O
-Ordering	O
-of	O
-loop	O
-3	O
-suggests	O
-that	O
-this	O
-flexible	O
-region	O
-contributes	O
-to	O
-the	O
-stabilization	O
-of	O
-the	O
-PKI	O
-domain	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-state	O
-.	O
-	O
-(	O
-c	O
-)	O
-Comparison	O
-of	O
-conformations	O
-of	O
-eEF2	O
-•	O
-sordarin	O
-in	O
-Structure	O
-I	O
-(	O
-light	O
-green	O
-)	O
-with	O
-those	O
-of	O
-free	O
-apo	O
--	O
-eEF2	O
-(	O
-magenta	O
-)	O
-and	O
-eEF2	O
-•	O
-sordarin	O
-(	O
-teal	O
-).	O
-	O
-Superposition	O
-was	O
-obtained	O
-by	O
-structural	O
-alignment	O
-of	O
-the	O
-25S	O
-rRNAs	O
-.	O
-	O
-(	O
-e	O
-)	O
-Comparison	O
-of	O
-the	O
-GTP	O
--	O
-like	O
-conformation	O
-of	O
-eEF2	O
-•	O
-GDP	O
-in	O
-Structure	O
-I	O
-(	O
-light	O
-green	O
-)	O
-with	O
-those	O
-of	O
-70S	O
--	O
-bound	O
-elongation	O
-factors	O
-EF	O
--	O
-Tu	O
-•	O
-GDPCP	O
-(	O
-teal	O
-)	O
-and	O
-EF	O
--	O
-G	O
-•	O
-GDP	O
-•	O
-fusidic	O
-acid	O
-(	O
-magenta	O
-;	O
-fusidic	O
-acid	O
-not	O
-shown	O
-).	O
-(	O
-f	O
-)	O
-Cryo	O
--	O
-EM	O
-density	O
-showing	O
-guanosine	O
-diphosphate	O
-bound	O
-in	O
-the	O
-GTPase	O
-center	O
-(	O
-green	O
-)	O
-next	O
-to	O
-the	O
-sarcin	O
--	O
-ricin	O
-loop	O
-of	O
-25S	O
-rRNA	O
-(	O
-cyan	O
-)	O
-of	O
-Structure	O
-II	O
-.	O
-(	O
-g	O
-)	O
-Comparison	O
-of	O
-the	O
-sordarin	O
--	O
-binding	O
-sites	O
-in	O
-the	O
-ribosome	O
--	O
-bound	O
-(	O
-light	O
-green	O
-;	O
-Structure	O
-II	O
-)	O
-and	O
-isolated	O
-eEF2	O
-(	O
-teal	O
-).	O
-	O
-The	O
-sarcin	O
--	O
-ricin	O
-loop	O
-interacts	O
-with	O
-the	O
-GTP	O
--	O
-binding	O
-site	O
-of	O
-eEF2	O
-(	O
-Figures	O
-5d	O
-and	O
-f	O
-).	O
-	O
-(	O
-a	O
-)	O
-eEF2	O
-(	O
-green	O
-)	O
-interacts	O
-only	O
-with	O
-the	O
-body	O
-in	O
-Structure	O
-I	O
-(	O
-eEF2	O
-domains	O
-are	O
-labeled	O
-with	O
-roman	O
-numerals	O
-in	O
-white	O
-),	O
-and	O
-with	O
-both	O
-the	O
-head	O
-and	O
-body	O
-in	O
-Structures	O
-II	O
-through	O
-V	O
-.	O
-Colors	O
-are	O
-as	O
-in	O
-Figure	O
-1	O
-,	O
-except	O
-for	O
-the	O
-40S	O
-structural	O
-elements	O
-that	O
-contact	O
-eEF2	O
-,	O
-which	O
-are	O
-labeled	O
-and	O
-shown	O
-in	O
-purple	O
-.	O
-(	O
-b	O
-)	O
-Entry	O
-of	O
-eEF2	O
-into	O
-the	O
-40S	O
-A	O
-site	O
-,	O
-from	O
-Structure	O
-I	O
-through	O
-V	O
-.	O
-Distances	O
-to	O
-the	O
-A	O
--	O
-site	O
-accommodated	O
-eEF2	O
-(	O
-Structure	O
-V	O
-)	O
-are	O
-shown	O
-.	O
-	O
-Because	O
-eEF2	O
-is	O
-rigidly	O
-attached	O
-to	O
-the	O
-60S	O
-subunit	O
-and	O
-does	O
-not	O
-undergo	O
-large	O
-inter	O
--	O
-subunit	O
-rearrangements	O
-,	O
-gradual	O
-entry	O
-of	O
-domain	O
-IV	O
-into	O
-the	O
-A	O
-site	O
-between	O
-Structures	O
-I	O
-and	O
-V	O
-is	O
-due	O
-to	O
-40S	O
-subunit	O
-rotation	O
-and	O
-head	O
-swivel	O
-.	O
-	O
-In	O
-the	O
-latter	O
-,	O
-PKI	O
-is	O
-stabilized	O
-by	O
-interactions	O
-with	O
-the	O
-universally	O
-conserved	O
-decoding	O
--	O
-center	O
-nucleotides	O
-G577	O
-,	O
-A1755	O
-and	O
-A1756	O
-('	O
-body	O
-A	O
-site	O
-'),	O
-as	O
-in	O
-the	O
-A	O
--	O
-site	O
-tRNA	O
-bound	O
-complexes	O
-.	O
-	O
-Domain	O
-IV	O
-is	O
-partially	O
-engaged	O
-with	O
-the	O
-body	O
-A	O
-site	O
-.	O
-	O
-The	O
-trimethylamino	O
-end	O
-of	O
-Diph699	O
-packs	O
-over	O
-A1756	O
-(	O
-Figure	O
-7	O
-).	O
-	O
-In	O
-translational	O
-GTPases	O
-,	O
-switch	O
-loops	O
-I	O
-and	O
-II	O
-are	O
-involved	O
-in	O
-the	O
-GTPase	O
-activity	O
-(	O
-reviewed	O
-in	O
-).	O
-	O
-Next	O
-to	O
-GDP	O
-,	O
-the	O
-C	O
--	O
-terminal	O
-part	O
-of	O
-the	O
-switch	O
-loop	O
-(	O
-aa	O
-61	O
-–	O
-67	O
-)	O
-adopts	O
-a	O
-helical	O
-fold	O
-.	O
-	O
-The	O
-decoding	O
-center	O
-residues	O
-A1755	O
-and	O
-A1756	O
-are	O
-rearranged	O
-to	O
-pack	O
-inside	O
-helix	O
-44	O
-,	O
-making	O
-room	O
-for	O
-eEF2	O
-.	O
-	O
-This	O
-conformation	O
-of	O
-decoding	O
-center	O
-residues	O
-is	O
-also	O
-observed	O
-in	O
-the	O
-absence	O
-of	O
-A	O
--	O
-site	O
-ligands	O
-.	O
-	O
-Structure	O
-III	O
-represents	O
-a	O
-highly	O
-bent	O
-IRES	O
-with	O
-PKI	O
-captured	O
-between	O
-the	O
-head	O
-A	O
-and	O
-P	O
-sites	O
-	O
-Among	O
-the	O
-five	O
-structures	O
-,	O
-the	O
-PKI	O
-domain	O
-is	O
-least	O
-ordered	O
-in	O
-Structure	O
-III	O
-and	O
-lacks	O
-density	O
-for	O
-SL3	O
-.	O
-	O
-Unwinding	O
-of	O
-the	O
-40S	O
-head	O
-also	O
-positions	O
-the	O
-head	O
-A	O
-site	O
-closer	O
-to	O
-the	O
-body	O
-A	O
-site	O
-.	O
-	O
-Four	O
-views	O
-(	O
-scenes	O
-)	O
-are	O
-shown	O
-:	O
-(	O
-1	O
-)	O
-A	O
-view	O
-down	O
-the	O
-intersubunit	O
-space	O
-,	O
-with	O
-the	O
-head	O
-of	O
-the	O
-40S	O
-subunit	O
-oriented	O
-toward	O
-a	O
-viewer	O
-,	O
-as	O
-in	O
-Figure	O
-1a	O
-;	O
-(	O
-2	O
-)	O
-A	O
-view	O
-at	O
-the	O
-solvent	O
-side	O
-of	O
-the	O
-40S	O
-subunit	O
-,	O
-with	O
-the	O
-40S	O
-head	O
-shown	O
-at	O
-the	O
-top	O
-,	O
-as	O
-in	O
-Figure	O
-2	O
-—	O
-figure	O
-supplement	O
-1	O
-;	O
-(	O
-3	O
-)	O
-A	O
-view	O
-down	O
-at	O
-the	O
-subunit	O
-interface	O
-of	O
-the	O
-40S	O
-subunit	O
-;	O
-(	O
-4	O
-)	O
-A	O
-close	O
--	O
-up	O
-view	O
-of	O
-the	O
-decoding	O
-center	O
-(	O
-A	O
-site	O
-)	O
-and	O
-the	O
-P	O
-site	O
-,	O
-as	O
-in	O
-Figure	O
-2g	O
-.	O
-Each	O
-scene	O
-is	O
-shown	O
-twice	O
-.	O
-	O
-Our	O
-structures	O
-reveal	O
-previously	O
-unseen	O
-intermediate	O
-states	O
-of	O
-eEF2	O
-or	O
-EF	O
--	O
-G	O
-engagement	O
-with	O
-the	O
-A	O
-site	O
-,	O
-providing	O
-the	O
-structural	O
-basis	O
-for	O
-the	O
-mechanism	O
-of	O
-translocase	O
-action	O
-.	O
-	O
-In	O
-the	O
-first	O
-sub	O
--	O
-step	O
-(	O
-Structures	O
-I	O
-to	O
-IV	O
-),	O
-the	O
-hind	O
-end	O
-advances	O
-from	O
-the	O
-A	O
-to	O
-the	O
-P	O
-site	O
-and	O
-approaches	O
-the	O
-front	O
-end	O
-,	O
-which	O
-remains	O
-attached	O
-to	O
-the	O
-40S	O
-surface	O
-.	O
-	O
-Upon	O
-translocation	O
-,	O
-the	O
-GCU	O
-start	O
-codon	O
-is	O
-positioned	O
-in	O
-the	O
-A	O
-site	O
-(	O
-Structure	O
-V	O
-),	O
-ready	O
-for	O
-interaction	O
-with	O
-Ala	O
--	O
-tRNAAla	O
-upon	O
-eEF2	O
-departure	O
-.	O
-	O
-Recent	O
-studies	O
-have	O
-shown	O
-that	O
-in	O
-some	O
-cases	O
-a	O
-fraction	O
-of	O
-IGR	O
-IRES	O
--	O
-driven	O
-translation	O
-results	O
-from	O
-an	O
-alternative	O
-reading	O
-frame	O
-,	O
-which	O
-is	O
-shifted	O
-by	O
-one	O
-nucleotide	O
-relative	O
-to	O
-the	O
-normal	O
-ORF	O
-.	O
-	O
-In	O
-our	O
-structures	O
-,	O
-the	O
-IRES	O
-presents	O
-to	O
-the	O
-decoding	O
-center	O
-a	O
-pre	O
--	O
-translocated	O
-or	O
-fully	O
-translocated	O
-ORF	O
-,	O
-rather	O
-than	O
-a	O
-+	O
-1	O
-(	O
-more	O
-translocated	O
-)	O
-ORF	O
-,	O
-suggesting	O
-that	O
-eEF2	O
-does	O
-not	O
-induce	O
-a	O
-highly	O
-populated	O
-fraction	O
-of	O
-+	O
-1	O
-shifted	O
-IRES	O
-mRNAs	O
-.	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-the	O
-observations	O
-that	O
-the	O
-intergenic	O
-IRESs	O
-are	O
-prone	O
-to	O
-reverse	O
-translocation	O
-.	O
-	O
-In	O
-the	O
-initiation	O
-state	O
-,	O
-the	O
-IRES	O
-resembles	O
-a	O
-pre	O
--	O
-translocation	O
-2tRNA	O
-•	O
-mRNA	O
-complex	O
-reduced	O
-to	O
-the	O
-A	O
-/	O
-P	O
--	O
-tRNA	O
-anticodon	O
--	O
-stem	O
-loop	O
-and	O
-elbow	O
-in	O
-the	O
-A	O
-site	O
-and	O
-the	O
-P	O
-/	O
-E	O
--	O
-tRNA	O
-elbow	O
-contacting	O
-the	O
-L1	O
-stalk	O
-.	O
-	O
-Because	O
-the	O
-anticodon	O
--	O
-stem	O
-loop	O
-of	O
-the	O
-A	O
--	O
-tRNA	O
-is	O
-sufficient	O
-for	O
-translocation	O
-completion	O
-,	O
-we	O
-ascribe	O
-the	O
-meta	O
--	O
-stability	O
-of	O
-the	O
-post	O
--	O
-translocation	O
-IRES	O
-to	O
-the	O
-absence	O
-of	O
-the	O
-P	O
-/	O
-E	O
--	O
-tRNA	O
-elements	O
-,	O
-either	O
-the	O
-ASL	O
-or	O
-the	O
-acceptor	O
-arm	O
-,	O
-or	O
-both	O
-.	O
-	O
-Translocases	O
-are	O
-efficient	O
-enzymes	O
-.	O
-	O
-EF	O
--	O
-G	O
-enhances	O
-the	O
-translocation	O
-rate	O
-by	O
-several	O
-orders	O
-of	O
-magnitude	O
-,	O
-aided	O
-by	O
-an	O
-additional	O
-2	O
--	O
-to	O
-50	O
--	O
-fold	O
-boost	O
-from	O
-GTP	O
-hydrolysis	O
-.	O
-	O
-Due	O
-to	O
-the	O
-lack	O
-of	O
-structures	O
-of	O
-translocation	O
-intermediates	O
-,	O
-the	O
-mechanistic	O
-role	O
-of	O
-eEF2	O
-/	O
-EF	O
--	O
-G	O
-is	O
-not	O
-fully	O
-understood	O
-.	O
-	O
-The	O
-unlocking	O
-model	O
-of	O
-the	O
-ribosome	O
-•	O
-2tRNA	O
-•	O
-mRNA	O
-pre	O
--	O
-translocation	O
-complex	O
-has	O
-been	O
-proposed	O
-decades	O
-ago	O
-and	O
-functional	O
-requirement	O
-of	O
-the	O
-translocase	O
-in	O
-this	O
-process	O
-has	O
-been	O
-implicated	O
-.	O
-	O
-This	O
-destabilization	O
-allows	O
-PKI	O
-to	O
-detach	O
-from	O
-the	O
-body	O
-A	O
-site	O
-upon	O
-spontaneous	O
-reverse	O
-40S	O
-body	O
-rotation	O
-,	O
-while	O
-maintaining	O
-interactions	O
-with	O
-the	O
-head	O
-A	O
-site	O
-.	O
-	O
-In	O
-the	O
-fully	O
--	O
-rotated	O
-pre	O
--	O
-translocation	O
--	O
-like	O
-Structure	O
-I	O
-,	O
-an	O
-additional	O
-interaction	O
-exists	O
-.	O
-	O
-We	O
-propose	O
-that	O
-the	O
-shift	O
-of	O
-domain	O
-III	O
-by	O
-uS12	O
-initiates	O
-intra	O
--	O
-domain	O
-rearrangements	O
-in	O
-eEF2	O
-,	O
-which	O
-unstack	O
-the	O
-β	O
--	O
-platform	O
-of	O
-domain	O
-III	O
-from	O
-that	O
-of	O
-domain	O
-V	O
-.	O
-This	O
-would	O
-result	O
-in	O
-a	O
-conformation	O
-characteristic	O
-of	O
-free	O
-eEF2	O
-and	O
-EF	O
--	O
-G	O
-in	O
-which	O
-the	O
-β	O
--	O
-platforms	O
-are	O
-nearly	O
-perpendicular	O
-.	O
-	O
-Sordarin	O
-is	O
-a	O
-potent	O
-antifungal	O
-antibiotic	O
-that	O
-inhibits	O
-translation	O
-.	O
-	O
-Although	O
-our	O
-complex	O
-was	O
-assembled	O
-using	O
-eEF2	O
-•	O
-GTP	O
-,	O
-density	O
-maps	O
-clearly	O
-show	O
-GDP	O
-and	O
-Mg2	O
-+	O
-in	O
-each	O
-structure	O
-(	O
-Figure	O
-5g	O
-).	O
-	O
-In	O
-all	O
-five	O
-structures	O
-,	O
-sordarin	O
-is	O
-bound	O
-between	O
-domains	O
-III	O
-and	O
-V	O
-of	O
-eEF2	O
-,	O
-stabilized	O
-by	O
-hydrophobic	O
-interactions	O
-identical	O
-to	O
-those	O
-in	O
-the	O
-isolated	O
-eEF2	O
-•	O
-sordarin	O
-complex	O
-(	O
-Figures	O
-5g	O
-and	O
-h	O
-).	O
-	O
-Implications	O
-for	O
-tRNA	O
-and	O
-mRNA	O
-translocation	O
-during	O
-translation	O
-	O
-First	O
-,	O
-we	O
-propose	O
-that	O
-tRNA	O
-and	O
-IRES	O
-translocations	O
-occur	O
-via	O
-the	O
-same	O
-general	O
-trajectory	O
-.	O
-	O
-This	O
-is	O
-consistent	O
-with	O
-the	O
-idea	O
-of	O
-a	O
-rather	O
-flat	O
-energy	O
-landscape	O
-of	O
-translocation	O
-,	O
-suggested	O
-by	O
-recent	O
-work	O
-that	O
-measured	O
-mechanical	O
-work	O
-produced	O
-by	O
-the	O
-ribosome	O
-during	O
-translocation	O
-.	O
-	O
-We	O
-note	O
-that	O
-four	O
-of	O
-our	O
-near	O
--	O
-atomic	O
-resolution	O
-maps	O
-comprised	O
-~	O
-30	O
-,	O
-000	O
-particles	O
-each	O
-,	O
-the	O
-minimum	O
-number	O
-required	O
-for	O
-a	O
-near	O
--	O
-atomic	O
--	O
-resolution	O
-reconstruction	O
-of	O
-the	O
-ribosome	O
-.	O
-	O
-This	O
-difference	O
-likely	O
-accounts	O
-for	O
-the	O
-inefficient	O
-translocation	O
-of	O
-the	O
-IRES	O
-,	O
-which	O
-is	O
-difficult	O
-to	O
-stabilize	O
-in	O
-the	O
-post	O
--	O
-translocation	O
-state	O
-and	O
-therefore	O
-is	O
-prone	O
-to	O
-reverse	O
-translocation	O
-.	O
-	O
-The	O
-uniformity	O
-of	O
-ribosome	O
-dynamics	O
-underscores	O
-the	O
-idea	O
-that	O
-translocation	O
-is	O
-an	O
-inherent	O
-and	O
-structurally	O
--	O
-optimized	O
-property	O
-of	O
-the	O
-ribosome	O
-,	O
-supported	O
-also	O
-by	O
-translocation	O
-activity	O
-in	O
-the	O
-absence	O
-of	O
-the	O
-elongation	O
-factor	O
-.	O
-	O
-Our	O
-current	O
-understanding	O
-of	O
-macromolecular	O
-machines	O
-,	O
-such	O
-as	O
-the	O
-ribosome	O
-,	O
-is	O
-often	O
-limited	O
-by	O
-a	O
-gap	O
-between	O
-biophysical	O
-/	O
-biochemical	O
-studies	O
-and	O
-structural	O
-studies	O
-.	O
-	O
-For	O
-example	O
-,	O
-Förster	O
-resonance	O
-energy	O
-transfer	O
-can	O
-provide	O
-insight	O
-into	O
-the	O
-macromolecular	O
-dynamics	O
-of	O
-an	O
-assembly	O
-at	O
-the	O
-single	O
--	O
-molecule	O
-level	O
-but	O
-is	O
-limited	O
-to	O
-specifically	O
-labeled	O
-locations	O
-within	O
-the	O
-assembly	O
-.	O
-	O
-The	O
-classification	O
-,	O
-which	O
-followed	O
-an	O
-initial	O
-alignment	O
-of	O
-all	O
-particles	O
-to	O
-a	O
-single	O
-reference	O
-,	O
-required	O
-about	O
-130	O
-,	O
-000	O
-CPU	O
-hours	O
-or	O
-about	O
-five	O
-to	O
-six	O
-full	O
-days	O
-on	O
-a	O
-1000	O
--	O
-CPU	O
-cluster	O
-.	O
-	O
-The	O
-N	O
--	O
-terminal	O
-propeptides	O
-protecting	O
-the	O
-active	O
--	O
-site	O
-threonines	O
-are	O
-autocatalytically	O
-released	O
-only	O
-on	O
-completion	O
-of	O
-assembly	O
-.	O
-	O
-This	O
-mechanism	O
-,	O
-however	O
-,	O
-cannot	O
-explain	O
-autocatalytic	O
-precursor	O
-processing	O
-because	O
-in	O
-the	O
-immature	O
-active	O
-sites	O
-,	O
-Thr1N	O
-is	O
-part	O
-of	O
-the	O
-peptide	O
-bond	O
-with	O
-Gly	O
-(-	O
-1	O
-),	O
-the	O
-bond	O
-that	O
-needs	O
-to	O
-be	O
-hydrolysed	O
-.	O
-	O
-Inactivation	O
-of	O
-proteasome	O
-subunits	O
-by	O
-T1A	B-mutant
-mutations	O
-	O
-Sequencing	O
-of	O
-the	O
-plasmids	O
-,	O
-testing	O
-them	O
-in	O
-both	O
-published	O
-yeast	O
-strain	O
-backgrounds	O
-and	O
-site	O
--	O
-directed	O
-mutagenesis	O
-revealed	O
-that	O
-the	O
-β5	B-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-pp	O
-cis	O
-is	O
-viable	O
-,	O
-but	O
-suffers	O
-from	O
-a	O
-marked	O
-growth	O
-defect	O
-that	O
-requires	O
-extended	O
-incubation	O
-of	O
-4	O
-–	O
-5	O
-days	O
-for	O
-initial	O
-colony	O
-formation	O
-(	O
-Table	O
-1	O
-and	O
-Supplementary	O
-Methods	O
-).	O
-	O
-For	O
-subunit	O
-β1	O
-,	O
-this	O
-process	O
-was	O
-previously	O
-inferred	O
-to	O
-require	O
-that	O
-the	O
-propeptide	O
-residue	O
-at	O
-position	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-subunit	O
-precursor	O
-occupies	O
-the	O
-S1	O
-specificity	O
-pocket	O
-of	O
-the	O
-substrate	O
--	O
-binding	O
-channel	O
-formed	O
-by	O
-amino	O
-acid	O
-45	O
-(	O
-for	O
-details	O
-see	O
-Supplementary	O
-Note	O
-2	O
-).	O
-	O
-Here	O
-we	O
-again	O
-analysed	O
-the	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
-mutant	O
-crystallographically	O
-but	O
-in	O
-addition	O
-determined	O
-the	O
-structures	O
-of	O
-the	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-single	O
-and	O
-β1	B-mutant
--	I-mutant
-T1A	I-mutant
--	I-mutant
-β2	I-mutant
--	I-mutant
-T1A	I-mutant
-double	O
-mutants	O
-(	O
-Protein	O
-Data	O
-Bank	O
-(	O
-PDB	O
-)	O
-entry	O
-codes	O
-are	O
-provided	O
-in	O
-Supplementary	O
-Table	O
-1	O
-).	O
-	O
-Instead	O
-,	O
-the	O
-plasticity	O
-of	O
-the	O
-β5	O
-S1	O
-pocket	O
-caused	O
-by	O
-the	O
-rotational	O
-flexibility	O
-of	O
-Met45	O
-might	O
-prevent	O
-stable	O
-accommodation	O
-of	O
-His	O
-(-	O
-2	O
-)	O
-in	O
-the	O
-S1	O
-site	O
-and	O
-thus	O
-also	O
-promote	O
-its	O
-immediate	O
-release	O
-after	O
-autolysis	O
-.	O
-	O
-Structural	O
-analyses	O
-revealed	O
-that	O
-the	O
-propeptides	O
-of	O
-all	O
-mutant	O
-yCPs	O
-shared	O
-residual	O
-2FO	O
-–	O
-FC	O
-electron	O
-densities	O
-.	O
-	O
-By	O
-contrast	O
-,	O
-the	O
-prosegments	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-L	I-mutant
--	I-mutant
-T1A	I-mutant
-and	O
-the	O
-β5	B-mutant
--	I-mutant
-H	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-T	I-mutant
--	I-mutant
-T1A	I-mutant
-mutants	O
-were	O
-significantly	O
-better	O
-resolved	O
-in	O
-the	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-maps	O
-yet	O
-not	O
-at	O
-full	O
-occupancy	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-4b	O
-,	O
-c	O
-and	O
-Supplementary	O
-Table	O
-1	O
-),	O
-suggesting	O
-that	O
-the	O
-natural	O
-propeptide	O
-bearing	O
-His	O
-(-	O
-2	O
-)	O
-is	O
-most	O
-favourable	O
-.	O
-	O
-This	O
-result	O
-proves	O
-that	O
-the	O
-naturally	O
-occurring	O
-His	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-β5	O
-propeptide	O
-does	O
-not	O
-stably	O
-fit	O
-into	O
-the	O
-S1	O
-site	O
-.	O
-	O
-Bearing	O
-in	O
-mind	O
-that	O
-in	O
-contrast	O
-to	O
-Thr	O
-(-	O
-2	O
-)	O
-in	O
-β2	O
-,	O
-Leu	O
-(-	O
-2	O
-)	O
-in	O
-subunit	O
-β1	O
-is	O
-not	O
-conserved	O
-among	O
-species	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-3a	O
-),	O
-we	O
-created	O
-a	O
-β2	B-mutant
--	I-mutant
-T	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-V	I-mutant
-proteasome	O
-mutant	O
-.	O
-	O
-However	O
-,	O
-in	O
-the	O
-immature	O
-particle	O
-Thr1NH2	O
-is	O
-blocked	O
-by	O
-the	O
-propeptide	O
-and	O
-cannot	O
-activate	O
-Thr1Oγ	O
-.	O
-	O
-Instead	O
-,	O
-Lys33NH2	O
-,	O
-which	O
-is	O
-in	O
-hydrogen	O
--	O
-bonding	O
-distance	O
-to	O
-Thr1Oγ	O
-(	O
-2	O
-.	O
-7	O
-Å	O
-)	O
-in	O
-all	O
-catalytically	O
-active	O
-β	O
-subunits	O
-(	O
-Fig	O
-.	O
-3a	O
-,	O
-b	O
-),	O
-was	O
-proposed	O
-to	O
-serve	O
-as	O
-the	O
-proton	O
-acceptor	O
-.	O
-	O
-This	O
-water	O
-hydrogen	O
-bonds	O
-also	O
-to	O
-Arg19O	O
-(∼	O
-3	O
-.	O
-0	O
-Å	O
-)	O
-and	O
-Asp17Oδ	O
-(∼	O
-3	O
-.	O
-0	O
-Å	O
-),	O
-and	O
-thereby	O
-presumably	O
-enables	O
-residual	O
-activity	O
-of	O
-the	O
-mutant	O
-.	O
-	O
-The	O
-ChT	O
--	O
-L	O
-activity	O
-of	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-pp	O
-in	O
-trans	O
-CP	O
-towards	O
-the	O
-canonical	O
-β5	O
-model	O
-substrates	O
-N	O
--	O
-succinyl	O
--	O
-Leu	O
--	O
-Leu	O
--	O
-Val	O
--	O
-Tyr	O
--	O
-7	O
--	O
-amino	O
--	O
-4	O
--	O
-methylcoumarin	O
-(	O
-Suc	O
--	O
-LLVY	O
--	O
-AMC	O
-)	O
-and	O
-carboxybenzyl	O
--	O
-Gly	O
--	O
-Gly	O
--	O
-Leu	O
--	O
-para	O
--	O
-nitroanilide	O
-(	O
-Z	O
--	O
-GGL	O
--	O
-pNA	O
-)	O
-was	O
-severely	O
-reduced	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6b	O
-),	O
-confirming	O
-that	O
-Asp17	O
-is	O
-of	O
-fundamental	O
-importance	O
-for	O
-the	O
-catalytic	O
-activity	O
-of	O
-the	O
-mature	O
-proteasome	O
-.	O
-	O
-Strikingly	O
-,	O
-although	O
-the	O
-X	O
--	O
-ray	O
-data	O
-on	O
-the	O
-β5	B-mutant
--	I-mutant
-D17N	I-mutant
-mutant	O
-with	O
-the	O
-propeptide	O
-expressed	O
-in	O
-cis	O
-and	O
-in	O
-trans	O
-looked	O
-similar	O
-,	O
-there	O
-was	O
-a	O
-pronounced	O
-difference	O
-in	O
-their	O
-growth	O
-phenotypes	O
-observed	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-7b	O
-).	O
-	O
-The	O
-β5	B-mutant
--	I-mutant
-D166N	I-mutant
-pp	O
-cis	O
-yeast	O
-mutant	O
-is	O
-significantly	O
-impaired	O
-in	O
-growth	O
-and	O
-its	O
-ChT	O
--	O
-L	O
-activity	O
-is	O
-drastically	O
-reduced	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-,	O
-b	O
-and	O
-Table	O
-1	O
-).	O
-	O
-The	O
-hydrogen	O
-bonds	O
-involving	O
-Ser169OH	O
-are	O
-intact	O
-and	O
-may	O
-account	O
-for	O
-residual	O
-substrate	O
-turnover	O
-.	O
-	O
-Together	O
-,	O
-these	O
-observations	O
-suggest	O
-that	O
-efficient	O
-peptide	O
--	O
-bond	O
-hydrolysis	O
-requires	O
-that	O
-Lys33NH2	O
-hydrogen	O
-bonds	O
-to	O
-the	O
-active	O
-site	O
-nucleophile	O
-.	O
-	O
-Activity	O
-assays	O
-with	O
-the	O
-β5	O
--	O
-specific	O
-substrate	O
-Suc	O
--	O
-LLVY	O
--	O
-AMC	O
-demonstrated	O
-that	O
-the	O
-ChT	O
--	O
-L	O
-activity	O
-of	O
-the	O
-T1S	B-mutant
-mutant	O
-is	O
-reduced	O
-by	O
-40	O
-–	O
-45	O
-%	O
-compared	O
-with	O
-WT	O
-proteasomes	O
-depending	O
-on	O
-the	O
-incubation	O
-temperature	O
-(	O
-Fig	O
-.	O
-4b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-9c	O
-).	O
-	O
-Compared	O
-with	O
-Thr1Oγ	O
-in	O
-WT	O
-CP	O
-structures	O
-,	O
-Ser1Oγ	O
-is	O
-rotated	O
-by	O
-60	O
-°.	O
-	O
-In	O
-addition	O
-,	O
-they	O
-prevent	O
-irreversible	O
-inactivation	O
-of	O
-the	O
-Thr1	O
-N	O
-terminus	O
-by	O
-N	O
--	O
-acetylation	O
-.	O
-	O
-However	O
-,	O
-removal	O
-of	O
-the	O
-β5	O
-prosegment	O
-or	O
-any	O
-interference	O
-with	O
-its	O
-cleavage	O
-causes	O
-severe	O
-phenotypic	O
-defects	O
-.	O
-	O
-On	O
-the	O
-basis	O
-of	O
-the	O
-numerous	O
-CP	O
-:	O
-ligand	O
-complexes	O
-solved	O
-during	O
-the	O
-past	O
-18	O
-years	O
-and	O
-in	O
-the	O
-current	O
-study	O
-,	O
-we	O
-provide	O
-a	O
-revised	O
-interpretation	O
-of	O
-proteasome	O
-active	O
--	O
-site	O
-architecture	O
-.	O
-	O
-We	O
-propose	O
-a	O
-catalytic	O
-triad	O
-for	O
-the	O
-active	O
-site	O
-of	O
-the	O
-CP	O
-consisting	O
-of	O
-residues	O
-Thr1	O
-,	O
-Lys33	O
-and	O
-Asp	O
-/	O
-Glu17	O
-,	O
-which	O
-are	O
-conserved	O
-among	O
-all	O
-proteolytically	O
-active	O
-eukaryotic	O
-,	O
-bacterial	O
-and	O
-archaeal	O
-proteasome	O
-subunits	O
-.	O
-	O
-Cleavage	O
-of	O
-the	O
-scissile	O
-peptide	O
-bond	O
-requires	O
-protonation	O
-of	O
-the	O
-emerging	O
-free	O
-amine	O
-,	O
-and	O
-in	O
-the	O
-proteasome	O
-,	O
-the	O
-Thr1	O
-amine	O
-group	O
-is	O
-likely	O
-to	O
-assume	O
-this	O
-function	O
-.	O
-	O
-Analogously	O
-,	O
-Thr1NH3	O
-+	O
-might	O
-promote	O
-the	O
-bivalent	O
-reaction	O
-mode	O
-of	O
-epoxyketone	O
-inhibitors	O
-by	O
-protonating	O
-the	O
-epoxide	O
-moiety	O
-to	O
-create	O
-a	O
-positively	O
-charged	O
-trivalent	O
-oxygen	O
-atom	O
-that	O
-is	O
-subsequently	O
-nucleophilically	O
-attacked	O
-by	O
-Thr1NH2	O
-.	O
-	O
-The	O
-residues	O
-Ser129	O
-and	O
-Asp166	O
-are	O
-expected	O
-to	O
-increase	O
-the	O
-pKa	O
-value	O
-of	O
-Thr1N	O
-,	O
-thereby	O
-favouring	O
-its	O
-charged	O
-state	O
-.	O
-	O
-Consistent	O
-with	O
-playing	O
-an	O
-essential	O
-role	O
-in	O
-proton	O
-shuttling	O
-,	O
-the	O
-mutation	O
-D166A	B-mutant
-prevents	O
-autolysis	O
-of	O
-the	O
-archaeal	O
-CP	O
-and	O
-the	O
-exchange	O
-D166N	B-mutant
-impairs	O
-catalytic	O
-activity	O
-of	O
-the	O
-yeast	O
-CP	O
-about	O
-60	O
-%.	O
-	O
-While	O
-Lys33NH2	O
-and	O
-Asp17Oδ	O
-are	O
-required	O
-to	O
-deprotonate	O
-the	O
-Thr1	O
-hydroxyl	O
-side	O
-chain	O
-,	O
-Ser129OH	O
-and	O
-Asp166OH	O
-serve	O
-to	O
-protonate	O
-the	O
-N	O
--	O
-terminal	O
-amine	O
-group	O
-of	O
-Thr1	O
-.	O
-	O
-Structural	O
-analyses	O
-support	O
-these	O
-findings	O
-with	O
-the	O
-T1S	B-mutant
-mutant	O
-and	O
-provide	O
-an	O
-explanation	O
-for	O
-the	O
-strict	O
-use	O
-of	O
-Thr	O
-residues	O
-in	O
-proteasomes	O
-.	O
-	O
-Notably	O
-,	O
-proteolytically	O
-active	O
-proteasome	O
-subunits	O
-from	O
-archaea	O
-,	O
-yeast	O
-and	O
-mammals	O
-,	O
-including	O
-constitutive	O
-,	O
-immuno	O
--	O
-and	O
-thymoproteasome	O
-subunits	O
-,	O
-either	O
-encode	O
-Thr	O
-or	O
-Ile	O
-at	O
-position	O
-3	O
-,	O
-indicating	O
-the	O
-importance	O
-of	O
-the	O
-Cγ	O
-for	O
-fixing	O
-the	O
-position	O
-of	O
-the	O
-nucleophilic	O
-Thr1	O
-.	O
-	O
-The	O
-major	O
-determinant	O
-of	O
-the	O
-S1	O
-specificity	O
-pocket	O
-,	O
-residue	O
-45	O
-,	O
-is	O
-depicted	O
-.	O
-	O
-Note	O
-the	O
-tight	O
-conformation	O
-of	O
-Gly	O
-(-	O
-1	O
-)	O
-and	O
-Ala1	O
-before	O
-propeptide	O
-removal	O
-(	O
-G	O
-(-	O
-1	O
-)	O
-turn	O
-;	O
-cyan	O
-double	O
-arrow	O
-)	O
-compared	O
-with	O
-the	O
-relaxed	O
-,	O
-processed	O
-WT	O
-active	O
--	O
-site	O
-Thr1	O
-(	O
-red	O
-double	O
-arrow	O
-).	O
-	O
-The	O
-black	O
-arrow	O
-indicates	O
-the	O
-attack	O
-of	O
-Thr1Oγ	O
-onto	O
-the	O
-carbonyl	O
-carbon	O
-atom	O
-of	O
-Gly	O
-(-	O
-1	O
-).	O
-	O
-While	O
-residue	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-β1	O
-and	O
-β2	O
-prosegments	O
-fit	O
-the	O
-S1	O
-pocket	O
-,	O
-His	O
-(-	O
-2	O
-)	O
-of	O
-the	O
-β5	O
-propeptide	O
-occupies	O
-the	O
-S2	O
-pocket	O
-.	O
-	O
-The	O
-(-	O
-2	O
-)	O
-residues	O
-of	O
-both	O
-prosegments	O
-point	O
-into	O
-the	O
-S1	O
-pocket	O
-,	O
-but	O
-only	O
-Thr	O
-(-	O
-2	O
-)	O
-OH	O
-of	O
-β2	O
-forms	O
-a	O
-hydrogen	O
-bridge	O
-to	O
-Gly	O
-(-	O
-1	O
-)	O
-O	O
-(	O
-black	O
-dashed	O
-line	O
-).	O
-	O
-(	O
-d	O
-)	O
-Structural	O
-superposition	O
-of	O
-the	O
-matured	O
-β2	O
-active	O
-site	O
-,	O
-the	O
-WT	O
-β2	B-mutant
--	I-mutant
-T1A	I-mutant
-propeptide	O
-and	O
-the	O
-β2	B-mutant
--	I-mutant
-T	I-mutant
-(-	I-mutant
-2	I-mutant
-)	I-mutant
-V	I-mutant
-mutant	O
-propeptide	O
-.	O
-	O
-The	O
-Thr1	O
-N	O
-terminus	O
-is	O
-engaged	O
-in	O
-hydrogen	O
-bonds	O
-with	O
-Ser129Oγ	O
-,	O
-the	O
-carbonyl	O
-oxygen	O
-of	O
-residue	O
-168	O
-,	O
-Ser169Oγ	O
-and	O
-Asp166Oδ	O
-.	O
-(	O
-b	O
-)	O
-The	O
-orientations	O
-of	O
-the	O
-active	O
--	O
-site	O
-residues	O
-involved	O
-in	O
-hydrogen	O
-bonding	O
-are	O
-strictly	O
-conserved	O
-in	O
-each	O
-proteolytic	O
-centre	O
-,	O
-as	O
-shown	O
-by	O
-superposition	O
-of	O
-the	O
-β	O
-subunits	O
-.	O
-	O
-In	O
-the	O
-latter	O
-,	O
-a	O
-water	O
-molecule	O
-(	O
-red	O
-sphere	O
-)	O
-is	O
-found	O
-at	O
-the	O
-position	O
-where	O
-in	O
-the	O
-WT	O
-structure	O
-the	O
-side	O
-chain	O
-amine	O
-group	O
-of	O
-Lys33	O
-is	O
-located	O
-.	O
-	O
-Note	O
-,	O
-the	O
-strong	O
-interaction	O
-with	O
-the	O
-water	O
-molecule	O
-causes	O
-a	O
-minor	O
-shift	O
-of	O
-Thr1	O
-,	O
-while	O
-all	O
-other	O
-active	O
--	O
-site	O
-residues	O
-remain	O
-in	O
-place	O
-.	O
-	O
-The	O
-charged	O
-Thr1	O
-N	O
-terminus	O
-may	O
-engage	O
-in	O
-the	O
-orientation	O
-of	O
-the	O
-amide	O
-moiety	O
-and	O
-donate	O
-a	O
-proton	O
-to	O
-the	O
-emerging	O
-N	O
-terminus	O
-of	O
-the	O
-C	O
--	O
-terminal	O
-cleavage	O
-product	O
-.	O
-	O
-The	O
-2FO	O
-–	O
-FC	O
-electron	O
--	O
-density	O
-maps	O
-(	O
-blue	O
-mesh	O
-)	O
-for	O
-Ser1	O
-(	O
-brown	O
-)	O
-and	O
-the	O
-covalently	O
-bound	O
-ligands	O
-(	O
-green	O
-;	O
-only	O
-the	O
-P1	O
-site	O
-(	O
-Leu1	O
-)	O
-is	O
-shown	O
-)	O
-are	O
-contoured	O
-at	O
-1σ	O
-.	O
-	O
-The	O
-Taf14	O
-YEATS	O
-domain	O
-is	O
-a	O
-reader	O
-of	O
-histone	O
-crotonylation	O
-	O
-Owing	O
-to	O
-some	O
-differences	O
-in	O
-their	O
-genomic	O
-distribution	O
-,	O
-the	O
-crotonyllysine	O
-and	O
-acetyllysine	O
-(	O
-Kac	O
-)	O
-modifications	O
-have	O
-been	O
-linked	O
-to	O
-distinct	O
-functional	O
-outcomes	O
-.	O
-	O
-A	O
-recent	O
-survey	O
-of	O
-bromodomains	O
-(	O
-BDs	O
-)	O
-demonstrates	O
-that	O
-only	O
-one	O
-BD	O
-associates	O
-very	O
-weakly	O
-with	O
-a	O
-crotonylated	O
-peptide	O
-,	O
-however	O
-it	O
-binds	O
-more	O
-tightly	O
-to	O
-acetylated	O
-peptides	O
-,	O
-inferring	O
-that	O
-bromodomains	O
-do	O
-not	O
-possess	O
-physiologically	O
-relevant	O
-crotonyllysine	O
-binding	O
-activity	O
-.	O
-	O
-The	O
-most	O
-striking	O
-feature	O
-of	O
-the	O
-crotonyllysine	O
-recognition	O
-mechanism	O
-is	O
-the	O
-unique	O
-coordination	O
-of	O
-crotonylated	O
-lysine	O
-residue	O
-.	O
-	O
-The	O
-π	O
-bond	O
-conjugation	O
-of	O
-the	O
-crotonyl	O
-group	O
-gives	O
-rise	O
-to	O
-a	O
-dipole	O
-moment	O
-of	O
-the	O
-alkene	O
-moiety	O
-,	O
-resulting	O
-in	O
-a	O
-partial	O
-positive	O
-charge	O
-on	O
-the	O
-β	O
--	O
-carbon	O
-(	O
-Cβ	O
-)	O
-and	O
-a	O
-partial	O
-negative	O
-charge	O
-on	O
-the	O
-α	O
--	O
-carbon	O
-(	O
-Cα	O
-).	O
-	O
-The	O
-dissociation	O
-constant	O
-(	O
-Kd	O
-)	O
-for	O
-the	O
-Taf14	O
-YEATS	O
--	O
-H3K9cr5	O
--	O
-13	O
-complex	O
-was	O
-found	O
-to	O
-be	O
-9	O
-.	O
-5	O
-μM	O
-,	O
-as	O
-measured	O
-by	O
-fluorescence	O
-spectroscopy	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-2c	O
-).	O
-	O
-Towards	O
-this	O
-end	O
-,	O
-we	O
-probed	O
-extracts	O
-derived	O
-from	O
-yeast	O
-cells	O
-in	O
-which	O
-major	O
-yeast	O
-HATs	O
-(	O
-HAT1	O
-,	O
-Gcn5	O
-,	O
-and	O
-Rtt109	O
-)	O
-or	O
-HDACs	O
-(	O
-Rpd3	O
-,	O
-Hos1	O
-,	O
-and	O
-Hos2	O
-)	O
-were	O
-deleted	O
-.	O
-	O
-In	O
-contrast	O
-,	O
-binding	O
-of	O
-H3K9ac	O
-resulted	O
-in	O
-an	O
-intermediate	O
-exchange	O
-,	O
-which	O
-is	O
-characteristic	O
-of	O
-a	O
-weaker	O
-association	O
-.	O
-	O
-The	O
-preference	O
-for	O
-H3K9cr	O
-over	O
-H3K9ac	O
-,	O
-H3K9pr	O
-and	O
-H3K9bu	O
-was	O
-supported	O
-by	O
-1H	O
-,	O
-15N	O
-HSQC	O
-titration	O
-experiments	O
-.	O
-	O
-H3K9cr	O
-is	O
-a	O
-selective	O
-target	O
-of	O
-the	O
-Taf14	O
-YEATS	O
-domain	O
-	O
-Cellular	O
-homeostasis	O
-requires	O
-correct	O
-delivery	O
-of	O
-cell	O
--	O
-surface	O
-receptor	O
-proteins	O
-(	O
-cargo	O
-)	O
-to	O
-their	O
-target	O
-subcellular	O
-compartments	O
-.	O
-	O
-The	O
-adapter	O
-proteins	O
-Tom1	O
-and	O
-Tollip	O
-are	O
-involved	O
-in	O
-sorting	O
-of	O
-ubiquitinated	O
-cargo	O
-in	O
-endosomal	O
-compartments	O
-.	O
-	O
-Recruitment	O
-of	O
-Tom1	O
-to	O
-the	O
-endosomal	O
-compartments	O
-is	O
-mediated	O
-by	O
-its	O
-GAT	O
-domain	O
-’	O
-s	O
-association	O
-to	O
-Tollip	O
-’	O
-s	O
-Tom1	O
--	O
-binding	O
-domain	O
-(	O
-TBD	O
-).	O
-	O
-Subject	O
-area	O
-Biology	O
-More	O
-specific	O
-subject	O
-area	O
-Structural	O
-biology	O
-Type	O
-of	O
-data	O
-Table	O
-,	O
-text	O
-file	O
-,	O
-graph	O
-,	O
-figures	O
-How	O
-data	O
-was	O
-acquired	O
-Circular	O
-dichroism	O
-and	O
-NMR	O
-.	O
-	O
-Analysis	O
-of	O
-the	O
-far	O
--	O
-UV	O
-circular	O
-dichroism	O
-(	O
-CD	O
-)	O
-spectrum	O
-of	O
-the	O
-Tom	O
-1	O
-GAT	O
-domain	O
-(	O
-Fig	O
-.	O
-1	O
-)	O
-predicts	O
-58	O
-.	O
-7	O
-%	O
-α	O
--	O
-helix	O
-,	O
-3	O
-%	O
-β	O
--	O
-strand	O
-,	O
-15	O
-.	O
-5	O
-%	O
-turn	O
-,	O
-and	O
-22	O
-.	O
-8	O
-%	O
-disordered	O
-regions	O
-.	O
-	O
-Helices	O
-are	O
-shown	O
-in	O
-orange	O
-,	O
-whereas	O
-loops	O
-are	O
-colored	O
-in	O
-green	O
-.	O
-(	O
-B	O
-)	O
-Ribbon	O
-illustration	O
-of	O
-the	O
-Tom1	O
-GAT	O
-domain	O
-.	O
-	O
-deviations	O
-were	O
-obtained	O
-by	O
-superimposing	O
-residues	O
-215	O
-–	O
-309	O
-of	O
-Tom1	O
-GAT	O
-among	O
-10	O
-lowest	O
-energy	O
-refined	O
-structures	O
-.	O
-	O
-PGRMC1	O
-is	O
-a	O
-member	O
-of	O
-the	O
-membrane	O
--	O
-associated	O
-progesterone	O
-receptor	O
-(	O
-MAPR	O
-)	O
-family	O
-with	O
-a	O
-cytochrome	O
-b5	O
--	O
-like	O
-haem	O
--	O
-binding	O
-region	O
-,	O
-and	O
-is	O
-known	O
-to	O
-be	O
-highly	O
-expressed	O
-in	O
-various	O
-types	O
-of	O
-cancers	O
-.	O
-	O
-These	O
-histidines	O
-are	O
-missing	O
-in	O
-PGRMC1	O
-,	O
-and	O
-the	O
-haem	O
-iron	O
-is	O
-five	O
--	O
-coordinated	O
-by	O
-Tyr113	O
-(	O
-Y113	O
-)	O
-alone	O
-(	O
-Fig	O
-.	O
-1b	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-3	O
-).	O
-	O
-However	O
-,	O
-at	O
-the	O
-interfaces	O
-of	O
-the	O
-other	O
-possible	O
-dimeric	O
-structures	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-6a	O
-,	O
-chain	O
-A	O
-–	O
-A	O
-″;	O
-cyan	O
-and	O
-chain	O
-A	O
-–	O
-B	O
-;	O
-violet	O
-),	O
-no	O
-significant	O
-difference	O
-was	O
-observed	O
-.	O
-	O
-It	O
-should	O
-be	O
-noted	O
-that	O
-a	O
-disulfide	O
-bond	O
-between	O
-two	O
-Cys129	O
-residues	O
-is	O
-observed	O
-in	O
-the	O
-crystal	O
-of	O
-PGRMC1	O
-(	O
-Fig	O
-.	O
-1a	O
-),	O
-while	O
-Cys129	O
-is	O
-not	O
-conserved	O
-among	O
-the	O
-MAPR	O
-family	O
-proteins	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-5a	O
-).	O
-	O
-The	O
-current	O
-analytical	O
-data	O
-confirmed	O
-that	O
-apo	O
--	O
-PGRMC1	O
-monomer	O
-converts	O
-into	O
-dimer	O
-by	O
-binding	O
-to	O
-haem	O
-in	O
-solution	O
-(	O
-Table	O
-2	O
-).	O
-	O
-Furthermore	O
-,	O
-the	O
-UV	O
--	O
-visible	O
-spectrum	O
-of	O
-the	O
-wild	O
-type	O
-PGRMC1	O
-was	O
-the	O
-same	O
-as	O
-that	O
-of	O
-the	O
-C129S	B-mutant
-mutant	O
-of	O
-PGRMC1	O
-,	O
-and	O
-the	O
-R	O
-/	O
-Z	O
-ratio	O
-determined	O
-by	O
-the	O
-intensities	O
-between	O
-the	O
-Soret	O
-band	O
-(	O
-394	O
-nm	O
-)	O
-peak	O
-and	O
-the	O
-274	O
--	O
-nm	O
-peak	O
-showed	O
-that	O
-these	O
-proteins	O
-were	O
-fully	O
-loaded	O
-with	O
-haem	O
-(	O
-Supplementary	O
-Fig	O
-.	O
-12	O
-).	O
-	O
-To	O
-examine	O
-the	O
-inhibitory	O
-effects	O
-of	O
-CO	O
-on	O
-haem	O
--	O
-mediated	O
-PGRMC1	O
-dimerization	O
-,	O
-SV	O
--	O
-AUC	O
-analysis	O
-was	O
-carried	O
-out	O
-.	O
-	O
-By	O
-binding	O
-with	O
-haem	O
-(	O
-binding	O
-Kd	O
-=	O
-50	O
-nmol	O
-l	O
-−	O
-1	O
-),	O
-PGRMC1	O
-forms	O
-a	O
-stable	O
-dimer	O
-(	O
-dimerization	O
-Kd	O
-<<	O
-3	O
-.	O
-5	O
-μmol	O
-l	O
-−	O
-1	O
-)	O
-through	O
-stacking	O
-of	O
-the	O
-two	O
-open	O
-surfaces	O
-of	O
-the	O
-five	O
--	O
-coordinated	O
-haem	O
-molecules	O
-in	O
-each	O
-monomer	O
-.	O
-	O
-While	O
-proliferation	O
-of	O
-HCT116	O
-cells	O
-was	O
-not	O
-affected	O
-by	O
-knocking	O
-down	O
-PGRMC1	O
-,	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-were	O
-more	O
-sensitive	O
-to	O
-the	O
-EGFR	O
-inhibitor	O
-erlotinib	O
-than	O
-control	O
-HCT116	O
-cells	O
-,	O
-and	O
-the	O
-knockdown	O
-effect	O
-was	O
-reversed	O
-by	O
-co	O
--	O
-expression	O
-of	O
-shRNA	O
--	O
-resistant	O
-wild	O
--	O
-type	O
-PGRMC1	O
-but	O
-not	O
-of	O
-the	O
-Y113F	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-5b	O
-).	O
-	O
-Furthermore	O
-,	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-inhibited	O
-spheroid	O
-formation	O
-of	O
-HCT116	O
-cells	O
-in	O
-culture	O
-,	O
-and	O
-this	O
-inhibition	O
-was	O
-reversed	O
-by	O
-co	O
--	O
-expression	O
-of	O
-wild	O
--	O
-type	O
-PGRMC1	O
-but	O
-not	O
-of	O
-the	O
-Y113F	B-mutant
-mutant	O
-(	O
-Fig	O
-.	O
-5c	O
-and	O
-Supplementary	O
-Fig	O
-.	O
-18	O
-).	O
-	O
-The	O
-Kd	O
-value	O
-of	O
-PGRMC1	O
-binding	O
-to	O
-CYP51	O
-was	O
-in	O
-a	O
-micromolar	O
-range	O
-and	O
-comparable	O
-with	O
-those	O
-of	O
-other	O
-haem	O
-proteins	O
-,	O
-such	O
-as	O
-cytochrome	O
-P450	O
-reductase	O
-and	O
-neuroglobin	O
-/	O
-Gαi1	O
-(	O
-ref	O
-.),	O
-suggesting	O
-that	O
-haem	O
--	O
-dependent	O
-PGRMC1	O
-interaction	O
-with	O
-CYP51	O
-is	O
-biologically	O
-relevant	O
-.	O
-	O
-In	O
-this	O
-study	O
-,	O
-we	O
-showed	O
-that	O
-PGRMC1	O
-dimerizes	O
-by	O
-stacking	O
-interactions	O
-of	O
-haem	O
-molecules	O
-from	O
-each	O
-monomer	O
-.	O
-	O
-In	O
-the	O
-current	O
-study	O
-,	O
-the	O
-Y113	O
-residue	O
-plays	O
-a	O
-crucial	O
-role	O
-for	O
-the	O
-haem	O
--	O
-dependent	O
-dimerization	O
-of	O
-PGRMC1	O
-and	O
-resultant	O
-regulation	O
-of	O
-cancer	O
-proliferation	O
-and	O
-chemoresistance	O
-(	O
-Figs	O
-5c	O
-and	O
-6e	O
-).	O
-	O
-Since	O
-the	O
-Y113	O
-residue	O
-is	O
-involved	O
-in	O
-the	O
-putative	O
-consensus	O
-motif	O
-of	O
-phosphorylation	O
-by	O
-tyrosine	O
-kinases	O
-such	O
-as	O
-Abl	O
-and	O
-Lck	O
-,	O
-we	O
-investigated	O
-whether	O
-phosphorylated	O
-Y113	O
-is	O
-present	O
-in	O
-HCT116	O
-cells	O
-by	O
-ESI	O
--	O
-MS	O
-analysis	O
-.	O
-	O
-We	O
-showed	O
-that	O
-the	O
-haem	O
--	O
-mediated	O
-dimer	O
-of	O
-PGRMC1	O
-enables	O
-interaction	O
-with	O
-different	O
-subclasses	O
-of	O
-cytochromes	O
-P450	O
-(	O
-CYP	O
-)	O
-(	O
-Fig	O
-.	O
-6	O
-).	O
-	O
-On	O
-the	O
-other	O
-hand	O
-,	O
-Oda	O
-et	O
-al	O
-.	O
-reported	O
-that	O
-PGRMC1	O
-had	O
-no	O
-effect	O
-to	O
-CYP2E1	O
-and	O
-CYP3A4	O
-activities	O
-in	O
-HepG2	O
-cell	O
-.	O
-	O
-Besides	O
-the	O
-regulatory	O
-roles	O
-of	O
-PGRMC1	O
-/	O
-Sigma	O
--	O
-2	O
-receptor	O
-in	O
-proliferation	O
-and	O
-chemoresistance	O
-in	O
-cancer	O
-cells	O
-(	O
-ref	O
-.),	O
-recent	O
-reports	O
-show	O
-that	O
-PGRMC1	O
-is	O
-able	O
-to	O
-bind	O
-to	O
-amyloid	O
-beta	O
-oligomer	O
-to	O
-enhance	O
-its	O
-neurotoxicity	O
-.	O
-	O
-Alzheimer	O
-'	O
-s	O
-therapeutics	O
-targeting	O
-amyloid	O
-beta	O
-1	O
--	O
-42	O
-oligomers	O
-II	O
-:	O
-Sigma	O
--	O
-2	O
-/	O
-PGRMC1	O
-receptors	O
-mediate	O
-Abeta	O
-42	O
-oligomer	O
-binding	O
-and	O
-synaptotoxicity	O
-	O
-X	O
--	O
-ray	O
-crystal	O
-structure	O
-of	O
-PGRMC1	O
-.	O
-	O
-(	O
-a	O
-)	O
-Structure	O
-of	O
-the	O
-PGRMC1	O
-dimer	O
-formed	O
-through	O
-stacked	O
-haems	O
-.	O
-	O
-(	O
-b	O
-)	O
-SV	O
--	O
-AUC	O
-analyses	O
-of	O
-the	O
-wt	O
--	O
-PGRMC1	O
-and	O
-the	O
-C129S	B-mutant
-mutant	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-)	O
-in	O
-the	O
-presence	O
-or	O
-absence	O
-of	O
-haem	O
-.	O
-	O
-(	O
-f	O
-)	O
-HCT116	O
-cells	O
-expressing	O
-control	O
-shRNA	O
-or	O
-those	O
-knocking	O
-down	O
-PGRMC1	O
-(	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-)	O
-were	O
-treated	O
-with	O
-EGF	O
-or	O
-left	O
-untreated	O
-,	O
-and	O
-components	O
-of	O
-the	O
-EGFR	O
-signaling	O
-pathway	O
-were	O
-detected	O
-by	O
-Western	O
-blotting	O
-.	O
-	O
-Stable	O
-PGRMC1	B-mutant
--	I-mutant
-knockdown	I-mutant
-(	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-)	O
-HCT116	O
-cells	O
-were	O
-transiently	O
-transfected	O
-with	O
-the	O
-shRNA	O
--	O
-resistant	O
-expression	O
-vector	O
-of	O
-wild	O
--	O
-type	O
-PGRMC1	O
-(	O
-wt	O
-)	O
-or	O
-the	O
-Y113F	B-mutant
-mutant	O
-(	O
-Y113F	B-mutant
-).	O
-	O
-(	O
-b	O
-)	O
-Erlotinib	O
-was	O
-added	O
-to	O
-HCT116	O
-(	O
-control	O
-)	O
-cells	O
-,	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-or	O
-PGRMC1	B-mutant
--	I-mutant
-KD	I-mutant
-cells	O
-expressing	O
-shRNA	O
--	O
-resistant	O
-PGRMC1	O
-wt	O
-or	O
-Y113F	B-mutant
-,	O
-and	O
-cell	O
-viability	O
-was	O
-examined	O
-by	O
-MTT	O
-assay	O
-.	O
-	O
-The	O
-graph	O
-represents	O
-mean	O
-±	O
-s	O
-.	O
-e	O
-.	O
-of	O
-each	O
-spheroid	O
-size	O
-.	O
-*	O
-P	O
-<	O
-0	O
-.	O
-01	O
-using	O
-ANOVA	O
-with	O
-Fischer	O
-'	O
-s	O
-LSD	O
-test	O
-.	O
-	O
-Haem	O
--	O
-dependent	O
-PGRMC1	O
-dimerization	O
-enhances	O
-tumour	O
-chemoresistance	O
-through	O
-interaction	O
-with	O
-cytochromes	O
-P450	O
-.	O
-	O
-(	O
-a	O
-,	O
-b	O
-)	O
-FLAG	O
--	O
-PGRMC1	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-)	O
-and	O
-Y113F	B-mutant
-mutant	O
-proteins	O
-(	O
-a	O
-.	O
-a	O
-.	O
-44	O
-–	O
-195	O
-),	O
-in	O
-either	O
-apo	O
-or	O
-haem	O
--	O
-bound	O
-form	O
-,	O
-were	O
-incubated	O
-with	O
-CYP1A2	O
-(	O
-a	O
-)	O
-or	O
-CYP3A4	O
-(	O
-b	O
-)	O
-and	O
-immunoprecipitated	O
-with	O
-anti	O
--	O
-FLAG	O
-antibody	O
--	O
-conjugated	O
-beads	O
-.	O
-	O
-Schematic	O
-diagram	O
-for	O
-the	O
-regulation	O
-of	O
-PGRMC1	O
-functions	O
-.	O
-	O
-Differences	O
-in	O
-molecular	O
-weights	O
-of	O
-the	O
-wild	O
--	O
-type	O
-(	O
-wt	O
-;	O
-a	O
-)	O
-and	O
-the	O
-C129S	B-mutant
-mutant	O
-(	O
-b	O
-)	O
-PGRMC1	O
-proteins	O
-in	O
-the	O
-absence	O
-(	O
-apo	O
-form	O
-)	O
-or	O
-the	O
-presence	O
-of	O
-haem	O
-(	O
-haem	O
--	O
-bound	O
-form	O
-).	O
-	O
-Hotspot	O
-autoimmune	O
-T	O
-cell	O
-receptor	O
-binding	O
-underlies	O
-pathogen	O
-and	O
-insulin	O
-peptide	O
-cross	O
--	O
-reactivity	O
-	O
-Both	O
-MHC	O
-and	O
-peptide	O
-have	O
-also	O
-been	O
-shown	O
-to	O
-undergo	O
-structural	O
-changes	O
-upon	O
-TCR	O
-binding	O
-,	O
-mediating	O
-an	O
-induced	O
-fit	O
-between	O
-the	O
-TCR	O
-and	O
-pMHC	O
-.	O
-	O
-We	O
-recently	O
-reported	O
-that	O
-the	O
-1E6	O
-human	O
-CD8	O
-+	O
-T	O
-cell	O
-clone	O
-—	O
-which	O
-mediates	O
-the	O
-destruction	O
-of	O
-β	O
-cells	O
-through	O
-the	O
-recognition	O
-of	O
-a	O
-major	O
-,	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-–	O
-restricted	O
-,	O
-preproinsulin	O
-signal	O
-peptide	O
-(	O
-ALWGPDPAAA15	O
-–	O
-24	O
-)	O
-—	O
-can	O
-recognize	O
-upwards	O
-of	O
-1	O
-million	O
-different	O
-peptides	O
-.	O
-	O
-This	O
-first	O
-experimental	O
-evidence	O
-of	O
-a	O
-high	O
-level	O
-of	O
-CD8	O
-+	O
-T	O
-cell	O
-cross	O
--	O
-reactivity	O
-in	O
-a	O
-human	O
-autoimmune	O
-disease	O
-system	O
-hinted	O
-toward	O
-molecular	O
-mimicry	O
-by	O
-a	O
-more	O
-potent	O
-pathogenic	O
-peptide	O
-as	O
-a	O
-potential	O
-mechanism	O
-leading	O
-to	O
-β	O
-cell	O
-destruction	O
-.	O
-	O
-These	O
-APLs	O
-differed	O
-from	O
-the	O
-natural	O
-preproinsulin	O
-peptide	O
-by	O
-up	O
-to	O
-7	O
-of	O
-10	O
-residues	O
-.	O
-	O
-Two	O
-of	O
-these	O
-peptides	O
-,	O
-MVWGPDPLYV	O
-and	O
-RQFGPDWIVA	O
-(	O
-bold	O
-text	O
-signifies	O
-amino	O
-acids	O
-that	O
-are	O
-different	O
-from	O
-the	O
-index	O
-preproinsulin	O
-–	O
-derived	O
-sequence	O
-),	O
-are	O
-contained	O
-within	O
-the	O
-proteomes	O
-of	O
-the	O
-human	O
-pathogens	O
-Bacteroides	O
-fragilis	O
-/	O
-thetaiotaomicron	O
-and	O
-Clostridium	O
-asparagiforme	O
-,	O
-respectively	O
-.	O
-	O
-The	O
-low	O
-number	O
-of	O
-contacts	O
-between	O
-the	O
-2	O
-molecules	O
-most	O
-likely	O
-contributed	O
-to	O
-the	O
-weak	O
-binding	O
-affinity	O
-of	O
-the	O
-interaction	O
-.	O
-	O
-Although	O
-the	O
-1E6	O
-TCR	O
-formed	O
-a	O
-similar	O
-overall	O
-interaction	O
-with	O
-each	O
-APL	O
-,	O
-the	O
-stabilization	O
-between	O
-the	O
-TCR	O
-and	O
-the	O
-GPD	O
-motif	O
-enabled	O
-fine	O
-differences	O
-in	O
-the	O
-contact	O
-network	O
-with	O
-both	O
-the	O
-peptide	O
-and	O
-MHC	O
-surface	O
-that	O
-allowed	O
-discrimination	O
-between	O
-each	O
-ligand	O
-(	O
-Figure	O
-5	O
-).	O
-	O
-These	O
-data	O
-demonstrated	O
-that	O
-the	O
-unligated	O
-structure	O
-of	O
-the	O
-1E6	O
-TCR	O
-was	O
-virtually	O
-identical	O
-to	O
-its	O
-ligated	O
-counterparts	O
-.	O
-	O
-Thus	O
-,	O
-we	O
-performed	O
-an	O
-in	O
--	O
-depth	O
-thermodynamic	O
-analysis	O
-of	O
-6	O
-of	O
-the	O
-ligands	O
-under	O
-investigation	O
-(	O
-Figure	O
-8	O
-and	O
-Supplemental	O
-Table	O
-3	O
-).	O
-	O
-However	O
-,	O
-there	O
-was	O
-a	O
-clear	O
-switch	O
-in	O
-entropy	O
-between	O
-the	O
-weaker	O
--	O
-affinity	O
-and	O
-stronger	O
--	O
-affinity	O
-ligands	O
-,	O
-indicated	O
-by	O
-a	O
-strong	O
-Pearson	O
-’	O
-s	O
-correlation	O
-value	O
-between	O
-entropy	O
-and	O
-affinity	O
-(	O
-Pearson	O
-’	O
-s	O
-correlation	O
-value	O
-0	O
-.	O
-93	O
-,	O
-P	O
-=	O
-0	O
-.	O
-007	O
-).	O
-	O
-We	O
-searched	O
-a	O
-database	O
-of	O
-over	O
-1	O
-,	O
-924	O
-,	O
-572	O
-unique	O
-decamer	O
-peptides	O
-from	O
-the	O
-proteome	O
-of	O
-viral	O
-pathogens	O
-that	O
-are	O
-known	O
-,	O
-or	O
-strongly	O
-suspected	O
-,	O
-to	O
-infect	O
-humans	O
-.	O
-	O
-This	O
-notion	O
-is	O
-attractive	O
-because	O
-the	O
-CDR	O
-loops	O
-,	O
-which	O
-form	O
-the	O
-TCR	O
-antigen	O
--	O
-binding	O
-site	O
-,	O
-are	O
-usually	O
-the	O
-most	O
-flexible	O
-part	O
-of	O
-the	O
-TCR	O
-and	O
-have	O
-the	O
-ability	O
-to	O
-mold	O
-around	O
-differently	O
-shaped	O
-ligands	O
-.	O
-	O
-This	O
-motif	O
-was	O
-conserved	O
-in	O
-at	O
-least	O
-2	O
-potential	O
-foreign	O
-peptides	O
-,	O
-originating	O
-from	O
-Herpes	O
-simplex	O
-virus	O
-and	O
-Pseudomonas	O
-aeruginosa	O
-,	O
-enabling	O
-TCR	O
-recognition	O
-of	O
-foreign	O
-epitopes	O
-.	O
-	O
-We	O
-have	O
-previously	O
-demonstrated	O
-the	O
-importance	O
-of	O
-the	O
-GPD	O
-motif	O
-using	O
-a	O
-peptide	O
-library	O
-scan	O
-,	O
-as	O
-well	O
-as	O
-a	O
-CPL	O
-scan	O
-approach	O
-.	O
-	O
-These	O
-results	O
-challenge	O
-the	O
-notion	O
-that	O
-the	O
-most	O
-potent	O
-peptide	O
-antigens	O
-exhibit	O
-the	O
-greatest	O
-pMHC	O
-stability	O
-and	O
-have	O
-implications	O
-for	O
-the	O
-design	O
-of	O
-anchor	O
-residue	O
-–	O
-modified	O
-heteroclitic	O
-peptides	O
-for	O
-vaccination	O
-.	O
-	O
-These	O
-parameters	O
-aligned	O
-well	O
-with	O
-structural	O
-data	O
-,	O
-demonstrating	O
-that	O
-TCRs	O
-engaged	O
-pMHC	O
-using	O
-an	O
-induced	O
-fit	O
-binding	O
-mode	O
-.	O
-	O
-These	O
-differences	O
-were	O
-consistent	O
-with	O
-a	O
-greater	O
-degree	O
-of	O
-movement	O
-between	O
-the	O
-unligated	O
-and	O
-ligated	O
-pMHCs	O
-for	O
-the	O
-weaker	O
-ligands	O
-,	O
-suggesting	O
-a	O
-greater	O
-requirement	O
-for	O
-disorder	O
--	O
-to	O
--	O
-order	O
-changes	O
-during	O
-TCR	O
-binding	O
-.	O
-	O
-Indeed	O
-,	O
-we	O
-found	O
-over	O
-50	O
-decamer	O
-peptides	O
-from	O
-the	O
-proteome	O
-of	O
-likely	O
-,	O
-or	O
-known	O
-,	O
-human	O
-viral	O
-pathogens	O
-alone	O
-that	O
-contained	O
-both	O
-the	O
-conserved	O
-central	O
-GPD	O
-motif	O
-and	O
-anchor	O
-residues	O
-at	O
-positions	O
-2	O
-and	O
-10	O
-that	O
-would	O
-enable	O
-binding	O
-to	O
-HLA	O
--	O
-A	O
-*	O
-02	O
-:	O
-01	O
-.	O
-	O
-(	O
-K	O
-)	O
-Effective	O
-2D	O
-affinity	O
-plotted	O
-against	O
-1	O
-/	O
-EC50	O
-showing	O
-Pearson	O
-’	O
-s	O
-coefficient	O
-analysis	O
-(	O
-r	O
-)	O
-and	O
-P	O
-value	O
-.	O
-	O
-(	O
-A	O
-)	O
-Superposition	O
-of	O
-the	O
-1E6	O
-TCR	O
-(	O
-multicolored	O
-illustration	O
-)	O
-in	O
-complex	O
-with	O
-all	O
-7	O
-APLs	O
-(	O
-multicolored	O
-sticks	O
-)	O
-and	O
-the	O
-A2	O
--	O
-ALWGPDPAAA	O
-ligand	O
-using	O
-the	O
-HLA	O
--	O
-A	O
-*	O
-0201	O
-(	O
-gray	O
-illustration	O
-)	O
-molecule	O
-to	O
-align	O
-all	O
-of	O
-the	O
-structures	O
-.	O
-	O
-The	O
-MHCα1	O
-helix	O
-is	O
-shown	O
-in	O
-gray	O
-illustrations	O
-.	O
-	O
-(	O
-A	O
-)	O
-A2	O
--	O
-MVWGPDPLYV	O
-(	O
-black	O
-sticks	O
-).	O
-	O