diff --git "a/test.tsv" "b/test.tsv"
new file mode 100644--- /dev/null
+++ "b/test.tsv"
@@ -0,0 +1,31923 @@
+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
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+)	O
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+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
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+,	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
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+A	O
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+(	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