diff --git "a/train.tsv" "b/train.tsv"
new file mode 100644--- /dev/null
+++ "b/train.tsv"
@@ -0,0 +1,144533 @@
+words	labels
+Molecular	O
+Dissection	O
+of	O
+Xyloglucan	O
+Recognition	O
+in	O
+a	O
+Prominent	O
+Human	O
+Gut	O
+Symbiont	O
+	O
+Polysaccharide	O
+utilization	O
+loci	O
+(	O
+PUL	O
+)	O
+within	O
+the	O
+genomes	O
+of	O
+resident	O
+human	O
+gut	O
+Bacteroidetes	O
+are	O
+central	O
+to	O
+the	O
+metabolism	O
+of	O
+the	O
+otherwise	O
+indigestible	O
+complex	O
+carbohydrates	O
+known	O
+as	O
+“	O
+dietary	O
+fiber	O
+.”	O
+However	O
+,	O
+functional	O
+characterization	O
+of	O
+PUL	O
+lags	O
+significantly	O
+behind	O
+sequencing	O
+efforts	O
+,	O
+which	O
+limits	O
+physiological	O
+understanding	O
+of	O
+the	O
+human	O
+-	O
+bacterial	O
+symbiosis	O
+.	O
+	O
+In	O
+particular	O
+,	O
+the	O
+molecular	O
+basis	O
+of	O
+complex	O
+polysaccharide	O
+recognition	O
+,	O
+an	O
+essential	O
+prerequisite	O
+to	O
+hydrolysis	O
+by	O
+cell	O
+surface	O
+glycosidases	O
+and	O
+subsequent	O
+metabolism	O
+,	O
+is	O
+generally	O
+poorly	O
+understood	O
+.	O
+	O
+Here	O
+,	O
+we	O
+present	O
+the	O
+biochemical	O
+,	O
+structural	O
+,	O
+and	O
+reverse	O
+genetic	O
+characterization	O
+of	O
+two	O
+unique	O
+cell	O
+surface	O
+glycan	O
+-	O
+binding	O
+proteins	O
+(	O
+SGBPs	O
+)	O
+encoded	O
+by	O
+a	O
+xyloglucan	O
+utilization	O
+locus	O
+(	O
+XyGUL	O
+)	O
+from	O
+Bacteroides	O
+ovatus	O
+,	O
+which	O
+are	O
+integral	O
+to	O
+growth	O
+on	O
+this	O
+key	O
+dietary	O
+vegetable	O
+polysaccharide	O
+.	O
+	O
+Biochemical	O
+analysis	O
+reveals	O
+that	O
+these	O
+outer	O
+membrane	O
+-	O
+anchored	O
+proteins	O
+are	O
+in	O
+fact	O
+exquisitely	O
+specific	O
+for	O
+the	O
+highly	O
+branched	O
+xyloglucan	O
+(	O
+XyG	O
+)	O
+polysaccharide	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+SGBP	O
+-	O
+A	O
+,	O
+a	O
+SusD	O
+homolog	O
+,	O
+with	O
+a	O
+bound	O
+XyG	O
+tetradecasaccharide	O
+reveals	O
+an	O
+extended	O
+carbohydrate	O
+-	O
+binding	O
+platform	O
+that	O
+primarily	O
+relies	O
+on	O
+recognition	O
+of	O
+the	O
+β	O
+-	O
+glucan	O
+backbone	O
+.	O
+	O
+The	O
+unique	O
+,	O
+tetra	O
+-	O
+modular	O
+structure	O
+of	O
+SGBP	O
+-	O
+B	O
+is	O
+comprised	O
+of	O
+tandem	O
+Ig	O
+-	O
+like	O
+folds	O
+,	O
+with	O
+XyG	O
+binding	O
+mediated	O
+at	O
+the	O
+distal	O
+C	O
+-	O
+terminal	O
+domain	O
+.	O
+	O
+Despite	O
+displaying	O
+similar	O
+affinities	O
+for	O
+XyG	O
+,	O
+reverse	O
+-	O
+genetic	O
+analysis	O
+reveals	O
+that	O
+SGBP	O
+-	O
+B	O
+is	O
+only	O
+required	O
+for	O
+the	O
+efficient	O
+capture	O
+of	O
+smaller	O
+oligosaccharides	O
+,	O
+whereas	O
+the	O
+presence	O
+of	O
+SGBP	O
+-	O
+A	O
+is	O
+more	O
+critical	O
+than	O
+its	O
+carbohydrate	O
+-	O
+binding	O
+ability	O
+for	O
+growth	O
+on	O
+XyG	O
+.	O
+Together	O
+,	O
+these	O
+data	O
+demonstrate	O
+that	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+play	O
+complementary	O
+,	O
+specialized	O
+roles	O
+in	O
+carbohydrate	O
+capture	O
+by	O
+B	O
+.	O
+ovatus	O
+and	O
+elaborate	O
+a	O
+model	O
+of	O
+how	O
+vegetable	O
+xyloglucans	O
+are	O
+accessed	O
+by	O
+the	O
+Bacteroidetes	O
+.	O
+	O
+Our	O
+combined	O
+analysis	O
+illuminates	O
+new	O
+fundamental	O
+aspects	O
+of	O
+complex	O
+polysaccharide	O
+recognition	O
+,	O
+cleavage	O
+,	O
+and	O
+import	O
+at	O
+the	O
+Bacteroidetes	O
+cell	O
+surface	O
+that	O
+may	O
+facilitate	O
+the	O
+development	O
+of	O
+prebiotics	O
+to	O
+target	O
+this	O
+phylum	O
+of	O
+gut	O
+bacteria	O
+.	O
+	O
+This	O
+microbial	O
+community	O
+is	O
+largely	O
+bacterial	O
+,	O
+with	O
+the	O
+Bacteroidetes	O
+,	O
+Firmicutes	O
+,	O
+and	O
+Actinobacteria	O
+comprising	O
+the	O
+dominant	O
+phyla	O
+.	O
+	O
+However	O
+,	O
+there	O
+is	O
+a	O
+paucity	O
+of	O
+data	O
+regarding	O
+how	O
+the	O
+vast	O
+array	O
+of	O
+complex	O
+carbohydrate	O
+structures	O
+are	O
+selectively	O
+recognized	O
+and	O
+imported	O
+by	O
+members	O
+of	O
+the	O
+microbiota	O
+,	O
+a	O
+critical	O
+process	O
+that	O
+enables	O
+these	O
+organisms	O
+to	O
+thrive	O
+in	O
+the	O
+competitive	O
+gut	O
+environment	O
+.	O
+	O
+The	O
+human	O
+gut	O
+bacteria	O
+Bacteroidetes	O
+share	O
+a	O
+profound	O
+capacity	O
+for	O
+dietary	O
+glycan	O
+degradation	O
+,	O
+with	O
+many	O
+species	O
+containing	O
+>	O
+250	O
+predicted	O
+carbohydrate	O
+-	O
+active	O
+enzymes	O
+(	O
+CAZymes	O
+),	O
+compared	O
+to	O
+50	O
+to	O
+100	O
+within	O
+many	O
+Firmicutes	O
+and	O
+only	O
+17	O
+in	O
+the	O
+human	O
+genome	O
+devoted	O
+toward	O
+carbohydrate	O
+utilization	O
+.	O
+	O
+A	O
+remarkable	O
+feature	O
+of	O
+the	O
+Bacteroidetes	O
+is	O
+the	O
+packaging	O
+of	O
+genes	O
+for	O
+carbohydrate	O
+catabolism	O
+into	O
+discrete	O
+polysaccharide	O
+utilization	O
+loci	O
+(	O
+PUL	O
+),	O
+which	O
+are	O
+transcriptionally	O
+regulated	O
+by	O
+specific	O
+substrate	O
+signatures	O
+.	O
+	O
+The	O
+Sus	O
+includes	O
+a	O
+lipid	O
+-	O
+anchored	O
+,	O
+outer	O
+membrane	O
+endo	O
+-	O
+amylase	O
+,	O
+SusG	O
+;	O
+a	O
+TonB	O
+-	O
+dependent	O
+transporter	O
+(	O
+TBDT	O
+),	O
+SusC	O
+,	O
+which	O
+imports	O
+oligosaccharides	O
+with	O
+the	O
+help	O
+of	O
+an	O
+associated	O
+starch	O
+-	O
+binding	O
+protein	O
+,	O
+SusD	O
+;	O
+two	O
+additional	O
+carbohydrate	O
+-	O
+binding	O
+lipoproteins	O
+,	O
+SusE	O
+and	O
+SusF	O
+;	O
+and	O
+two	O
+periplasmic	O
+exo	O
+-	O
+glucosidases	O
+,	O
+SusA	O
+and	O
+SusB	O
+,	O
+which	O
+generate	O
+glucose	O
+for	O
+transport	O
+into	O
+the	O
+cytoplasm	O
+.	O
+	O
+The	O
+importance	O
+of	O
+PUL	O
+as	O
+a	O
+successful	O
+evolutionary	O
+strategy	O
+is	O
+underscored	O
+by	O
+the	O
+observation	O
+that	O
+Bacteroidetes	O
+such	O
+as	O
+B	O
+.	O
+thetaiotaomicron	O
+and	O
+Bacteroides	O
+ovatus	O
+devote	O
+~	O
+18	O
+%	O
+of	O
+their	O
+genomes	O
+to	O
+these	O
+systems	O
+.	O
+	O
+Moving	O
+beyond	O
+seminal	O
+genomic	O
+and	O
+transcriptomic	O
+analyses	O
+,	O
+the	O
+current	O
+state	O
+-	O
+of	O
+-	O
+the	O
+-	O
+art	O
+PUL	O
+characterization	O
+involves	O
+combined	O
+reverse	O
+-	O
+genetic	O
+,	O
+biochemical	O
+,	O
+and	O
+structural	O
+studies	O
+to	O
+illuminate	O
+the	O
+molecular	O
+details	O
+of	O
+PUL	O
+function	O
+.	O
+	O
+Cleavage	O
+sites	O
+for	O
+BoXyGUL	O
+glycosidases	O
+(	O
+GHs	O
+)	O
+are	O
+indicated	O
+for	O
+solanaceous	O
+xyloglucan	O
+.	O
+(	O
+B	O
+)	O
+BtSus	O
+and	O
+BoXyGUL	O
+.	O
+(	O
+C	O
+)	O
+Localization	O
+of	O
+BoXyGUL	O
+-	O
+encoded	O
+proteins	O
+in	O
+cellular	O
+membranes	O
+and	O
+concerted	O
+modes	O
+of	O
+action	O
+in	O
+the	O
+degradation	O
+of	O
+xyloglucans	O
+to	O
+monosaccharides	O
+.	O
+	O
+XyG	O
+variants	O
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+constitute	O
+up	O
+to	O
+25	O
+%	O
+of	O
+the	O
+dry	O
+weight	O
+of	O
+common	O
+vegetables	O
+.	O
+	O
+Analogous	O
+to	O
+the	O
+Sus	O
+locus	O
+,	O
+the	O
+xyloglucan	O
+utilization	O
+locus	O
+(	O
+XyGUL	O
+)	O
+encodes	O
+a	O
+cohort	O
+of	O
+carbohydrate	O
+-	O
+binding	O
+,	O
+-	O
+hydrolyzing	O
+,	O
+and	O
+-	O
+importing	O
+proteins	O
+(	O
+Fig	O
+.	O
+1B	O
+and	O
+C	O
+).	O
+	O
+The	O
+number	O
+of	O
+glycoside	O
+hydrolases	O
+(	O
+GHs	O
+)	O
+encoded	O
+by	O
+the	O
+XyGUL	O
+is	O
+,	O
+however	O
+,	O
+more	O
+expansive	O
+than	O
+that	O
+by	O
+the	O
+Sus	O
+locus	O
+(	O
+Fig	O
+.	O
+1B	O
+),	O
+which	O
+reflects	O
+the	O
+greater	O
+complexity	O
+of	O
+glycosidic	O
+linkages	O
+found	O
+in	O
+XyG	O
+vis	O
+-	O
+à	O
+-	O
+vis	O
+starch	O
+.	O
+	O
+In	O
+the	O
+archetypal	O
+starch	O
+utilization	O
+system	O
+of	O
+B	O
+.	O
+thetaiotaomicron	O
+,	O
+starch	O
+binding	O
+to	O
+the	O
+cell	O
+surface	O
+is	O
+mediated	O
+at	O
+eight	O
+distinct	O
+starch	O
+-	O
+binding	O
+sites	O
+distributed	O
+among	O
+four	O
+surface	O
+glycan	O
+-	O
+binding	O
+proteins	O
+(	O
+SGBPs	O
+):	O
+two	O
+within	O
+the	O
+amylase	O
+SusG	O
+,	O
+one	O
+within	O
+SusD	O
+,	O
+two	O
+within	O
+SusE	O
+,	O
+and	O
+three	O
+within	O
+SusF	O
+.	O
+The	O
+functional	O
+redundancy	O
+of	O
+many	O
+of	O
+these	O
+sites	O
+is	O
+high	O
+:	O
+whereas	O
+SusD	O
+is	O
+essential	O
+for	O
+growth	O
+on	O
+starch	O
+,	O
+combined	O
+mutations	O
+of	O
+the	O
+SusE	O
+,	O
+SusF	O
+,	O
+and	O
+SusG	O
+binding	O
+sites	O
+are	O
+required	O
+to	O
+impair	O
+growth	O
+on	O
+the	O
+polysaccharide	O
+.	O
+	O
+Bacteroidetes	O
+PUL	O
+ubiquitously	O
+encode	O
+homologs	O
+of	O
+SusC	O
+and	O
+SusD	O
+,	O
+as	O
+well	O
+as	O
+proteins	O
+whose	O
+genes	O
+are	O
+immediately	O
+downstream	O
+of	O
+susD	O
+,	O
+akin	O
+to	O
+susE	O
+/	O
+F	O
+,	O
+and	O
+these	O
+are	O
+typically	O
+annotated	O
+as	O
+“	O
+putative	O
+lipoproteins	O
+”.	O
+	O
+The	O
+genes	O
+coding	O
+for	O
+these	O
+proteins	O
+,	O
+sometimes	O
+referred	O
+to	O
+as	O
+“	O
+susE	O
+/	O
+F	O
+positioned	O
+,”	O
+display	O
+products	O
+with	O
+a	O
+wide	O
+variation	O
+in	O
+amino	O
+acid	O
+sequence	O
+and	O
+which	O
+have	O
+little	O
+or	O
+no	O
+homology	O
+to	O
+other	O
+PUL	O
+-	O
+encoded	O
+proteins	O
+or	O
+known	O
+carbohydrate	O
+-	O
+binding	O
+proteins	O
+.	O
+	O
+We	O
+describe	O
+here	O
+the	O
+detailed	O
+functional	O
+and	O
+structural	O
+characterization	O
+of	O
+the	O
+noncatalytic	O
+SGBPs	O
+encoded	O
+by	O
+Bacova_02651	O
+and	O
+Bacova_02650	O
+of	O
+the	O
+XyGUL	O
+,	O
+here	O
+referred	O
+to	O
+as	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+,	O
+to	O
+elucidate	O
+their	O
+molecular	O
+roles	O
+in	O
+carbohydrate	O
+acquisition	O
+in	O
+vivo	O
+.	O
+	O
+Combined	O
+biochemical	O
+,	O
+structural	O
+,	O
+and	O
+reverse	O
+-	O
+genetic	O
+approaches	O
+clearly	O
+illuminate	O
+the	O
+distinct	O
+,	O
+yet	O
+complementary	O
+,	O
+functions	O
+that	O
+these	O
+two	O
+proteins	O
+play	O
+in	O
+XyG	O
+recognition	O
+as	O
+it	O
+impacts	O
+the	O
+physiology	O
+of	O
+B	O
+.	O
+ovatus	O
+.	O
+	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+are	O
+cell	O
+-	O
+surface	O
+-	O
+localized	O
+,	O
+xyloglucan	O
+-	O
+specific	O
+binding	O
+proteins	O
+.	O
+	O
+SGBP	O
+-	O
+A	O
+,	O
+encoded	O
+by	O
+the	O
+XyGUL	O
+locus	O
+tag	O
+Bacova_02651	O
+(	O
+Fig	O
+.	O
+1B	O
+),	O
+shares	O
+26	O
+%	O
+amino	O
+acid	O
+sequence	O
+identity	O
+(	O
+40	O
+%	O
+similarity	O
+)	O
+with	O
+its	O
+homolog	O
+,	O
+B	O
+.	O
+thetaiotaomicron	O
+SusD	O
+,	O
+and	O
+similar	O
+homology	O
+with	O
+the	O
+SusD	O
+-	O
+like	O
+proteins	O
+encoded	O
+within	O
+syntenic	O
+XyGUL	O
+identified	O
+in	O
+our	O
+earlier	O
+work	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+SGBP	O
+-	O
+B	O
+,	O
+encoded	O
+by	O
+locus	O
+tag	O
+Bacova_02650	O
+,	O
+displays	O
+little	O
+sequence	O
+similarity	O
+to	O
+the	O
+products	O
+of	O
+similarly	O
+positioned	O
+genes	O
+in	O
+syntenic	O
+XyGUL	O
+nor	O
+to	O
+any	O
+other	O
+gene	O
+product	O
+among	O
+the	O
+diversity	O
+of	O
+Bacteroidetes	O
+PUL	O
+.	O
+	O
+Whereas	O
+sequence	O
+similarity	O
+among	O
+SusC	O
+/	O
+SusD	O
+homolog	O
+pairs	O
+often	O
+serves	O
+as	O
+a	O
+hallmark	O
+for	O
+PUL	O
+identification	O
+,	O
+the	O
+sequence	O
+similarities	O
+of	O
+downstream	O
+genes	O
+encoding	O
+SGBPs	O
+are	O
+generally	O
+too	O
+low	O
+to	O
+allow	O
+reliable	O
+bioinformatic	O
+classification	O
+of	O
+their	O
+products	O
+into	O
+protein	O
+families	O
+,	O
+let	O
+alone	O
+prediction	O
+of	O
+function	O
+.	O
+	O
+Hence	O
+,	O
+there	O
+is	O
+a	O
+critical	O
+need	O
+for	O
+the	O
+elucidation	O
+of	O
+detailed	O
+structure	O
+-	O
+function	O
+relationships	O
+among	O
+PUL	O
+SGBPs	O
+,	O
+in	O
+light	O
+of	O
+the	O
+manifold	O
+glycan	O
+structures	O
+in	O
+nature	O
+.	O
+	O
+Immunofluorescence	O
+of	O
+formaldehyde	O
+-	O
+fixed	O
+,	O
+nonpermeabilized	O
+cells	O
+grown	O
+in	O
+minimal	O
+medium	O
+with	O
+XyG	O
+as	O
+the	O
+sole	O
+carbon	O
+source	O
+to	O
+induce	O
+XyGUL	O
+expression	O
+,	O
+reveals	O
+that	O
+both	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+are	O
+presented	O
+on	O
+the	O
+cell	O
+surface	O
+by	O
+N	O
+-	O
+terminal	O
+lipidation	O
+,	O
+as	O
+predicted	O
+by	O
+signal	O
+peptide	O
+analysis	O
+with	O
+SignalP	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+visualized	O
+by	O
+immunofluorescence	O
+.	O
+	O
+Formalin	O
+-	O
+fixed	O
+,	O
+nonpermeabilized	O
+B	O
+.	O
+ovatus	O
+cells	O
+were	O
+grown	O
+in	O
+minimal	O
+medium	O
+plus	O
+XyG	O
+,	O
+probed	O
+with	O
+custom	O
+rabbit	O
+antibodies	O
+to	O
+SGBP	O
+-	O
+A	O
+or	O
+SGBP	O
+-	O
+B	O
+,	O
+and	O
+then	O
+stained	O
+with	O
+Alexa	O
+Fluor	O
+488	O
+goat	O
+anti	O
+-	O
+rabbit	O
+IgG	O
+.	O
+(	O
+A	O
+)	O
+Overlay	O
+of	O
+bright	O
+-	O
+field	O
+and	O
+FITC	O
+images	O
+of	O
+B	O
+.	O
+ovatus	O
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+A	O
+.	O
+(	O
+B	O
+)	O
+Overlay	O
+of	O
+bright	O
+-	O
+field	O
+and	O
+FITC	O
+images	O
+of	O
+B	O
+.	O
+ovatus	O
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+.	O
+(	O
+C	O
+)	O
+Bright	O
+-	O
+field	O
+image	O
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+antibodies	O
+.	O
+	O
+(	O
+D	O
+)	O
+FITC	O
+images	O
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+antibodies	O
+.	O
+	O
+Cells	O
+lacking	O
+SGBP	O
+-	O
+A	O
+(	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+)	O
+do	O
+not	O
+grow	O
+on	O
+XyG	O
+and	O
+therefore	O
+could	O
+not	O
+be	O
+tested	O
+in	O
+parallel	O
+.	O
+	O
+Additional	O
+affinity	O
+PAGE	O
+analysis	O
+(	O
+Fig	O
+.	O
+3	O
+)	O
+demonstrates	O
+that	O
+SGBP	O
+-	O
+A	O
+also	O
+has	O
+moderate	O
+affinity	O
+for	O
+the	O
+artificial	O
+soluble	O
+cellulose	O
+derivative	O
+hydroxyethyl	O
+cellulose	O
+[	O
+HEC	O
+;	O
+a	O
+β	O
+(	O
+1	O
+→	O
+4	O
+)-	O
+glucan	O
+]	O
+and	O
+limited	O
+affinity	O
+for	O
+mixed	O
+-	O
+linkage	O
+β	O
+(	O
+1	O
+→	O
+3	O
+)/	O
+β	O
+(	O
+1	O
+→	O
+4	O
+)-	O
+glucan	O
+(	O
+MLG	O
+)	O
+and	O
+glucomannan	O
+(	O
+GM	O
+;	O
+mixed	O
+glucosyl	O
+and	O
+mannosyl	O
+backbone	O
+),	O
+which	O
+together	O
+indicate	O
+general	O
+binding	O
+to	O
+polysaccharide	O
+backbone	O
+residues	O
+and	O
+major	O
+contributions	O
+from	O
+side	O
+-	O
+chain	O
+recognition	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+SGBP	O
+-	O
+B	O
+bound	O
+to	O
+HEC	O
+more	O
+weakly	O
+than	O
+SGBP	O
+-	O
+A	O
+and	O
+did	O
+not	O
+bind	O
+to	O
+MLG	O
+or	O
+GM	O
+.	O
+	O
+Neither	O
+SGBP	O
+recognized	O
+galactomannan	O
+(	O
+GGM	O
+),	O
+starch	O
+,	O
+carboxymethylcellulose	O
+,	O
+or	O
+mucin	O
+(	O
+see	O
+Fig	O
+.	O
+S1	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+	O
+Notably	O
+,	O
+the	O
+absence	O
+of	O
+carbohydrate	O
+-	O
+binding	O
+modules	O
+in	O
+the	O
+GHs	O
+encoded	O
+by	O
+the	O
+XyGUL	O
+implies	O
+that	O
+noncatalytic	O
+recognition	O
+of	O
+xyloglucan	O
+is	O
+mediated	O
+entirely	O
+by	O
+SGBP	O
+-	O
+A	O
+and	O
+-	O
+B	O
+.	O
+	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+preferentially	O
+bind	O
+xyloglucan	O
+.	O
+	O
+Affinity	O
+electrophoresis	O
+(	O
+10	O
+%	O
+acrylamide	O
+)	O
+of	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+with	O
+BSA	O
+as	O
+a	O
+control	O
+protein	O
+.	O
+	O
+All	O
+samples	O
+were	O
+loaded	O
+on	O
+the	O
+same	O
+gel	O
+next	O
+to	O
+the	O
+BSA	O
+controls	O
+;	O
+thin	O
+black	O
+lines	O
+indicate	O
+where	O
+intervening	O
+lanes	O
+were	O
+removed	O
+from	O
+the	O
+final	O
+image	O
+for	O
+both	O
+space	O
+and	O
+clarity	O
+.	O
+	O
+The	O
+percentage	O
+of	O
+polysaccharide	O
+incorporated	O
+into	O
+each	O
+native	O
+gel	O
+is	O
+displayed	O
+.	O
+	O
+The	O
+vanguard	O
+endo	O
+-	O
+xyloglucanase	O
+of	O
+the	O
+XyGUL	O
+,	O
+BoGH5	O
+,	O
+preferentially	O
+cleaves	O
+the	O
+polysaccharide	O
+at	O
+unbranched	O
+glucosyl	O
+residues	O
+to	O
+generate	O
+xylogluco	O
+-	O
+oligosaccharides	O
+(	O
+XyGOs	O
+)	O
+comprising	O
+a	O
+Glc4	O
+backbone	O
+with	O
+variable	O
+side	O
+-	O
+chain	O
+galactosylation	O
+(	O
+XyGO1	O
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+;	O
+n	O
+=	O
+1	O
+)	O
+as	O
+the	O
+limit	O
+of	O
+digestion	O
+products	O
+in	O
+vitro	O
+;	O
+controlled	O
+digestion	O
+and	O
+fractionation	O
+by	O
+size	O
+exclusion	O
+chromatography	O
+allow	O
+the	O
+production	O
+of	O
+higher	O
+-	O
+order	O
+oligosaccharides	O
+(	O
+e	O
+.	O
+g	O
+.,	O
+XyGO2	O
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+;	O
+n	O
+=	O
+2	O
+).	O
+	O
+ITC	O
+demonstrates	O
+that	O
+SGBP	O
+-	O
+A	O
+binds	O
+to	O
+XyG	O
+polysaccharide	O
+and	O
+XyGO2	O
+(	O
+based	O
+on	O
+a	O
+Glc8	O
+backbone	O
+)	O
+with	O
+essentially	O
+equal	O
+affinities	O
+,	O
+while	O
+no	O
+binding	O
+of	O
+XyGO1	O
+(	O
+Glc4	O
+backbone	O
+)	O
+was	O
+detectable	O
+(	O
+Table	O
+1	O
+;	O
+see	O
+Fig	O
+.	O
+S2	O
+and	O
+S3	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+	O
+Together	O
+,	O
+these	O
+data	O
+clearly	O
+suggest	O
+that	O
+polysaccharide	O
+binding	O
+of	O
+both	O
+SGBPs	O
+is	O
+fulfilled	O
+by	O
+a	O
+dimer	O
+of	O
+the	O
+minimal	O
+repeat	O
+,	O
+corresponding	O
+to	O
+XyGO2	O
+(	O
+cf	O
+.	O
+	O
+The	O
+observation	O
+by	O
+affinity	O
+PAGE	O
+that	O
+these	O
+proteins	O
+specifically	O
+recognize	O
+XyG	O
+is	O
+further	O
+substantiated	O
+by	O
+their	O
+lack	O
+of	O
+binding	O
+for	O
+the	O
+undecorated	O
+oligosaccharide	O
+cellotetraose	O
+(	O
+Table	O
+1	O
+;	O
+see	O
+Fig	O
+.	O
+S3	O
+).	O
+	O
+Furthermore	O
+,	O
+SGBP	O
+-	O
+A	O
+binds	O
+cellohexaose	O
+with	O
+~	O
+770	O
+-	O
+fold	O
+weaker	O
+affinity	O
+than	O
+XyG	O
+,	O
+while	O
+SGBP	O
+-	O
+B	O
+displays	O
+no	O
+detectable	O
+binding	O
+to	O
+this	O
+linear	O
+hexasaccharide	O
+.	O
+	O
+To	O
+provide	O
+molecular	O
+-	O
+level	O
+insight	O
+into	O
+how	O
+the	O
+XyGUL	O
+SGBPs	O
+equip	O
+B	O
+.	O
+ovatus	O
+to	O
+specifically	O
+harvest	O
+XyG	O
+from	O
+the	O
+gut	O
+environment	O
+,	O
+we	O
+performed	O
+X	O
+-	O
+ray	O
+crystallography	O
+analysis	O
+of	O
+both	O
+SGBP	O
+-	O
+A	O
+and	O
+SGPB	O
+-	O
+B	O
+in	O
+oligosaccharide	O
+-	O
+complex	O
+forms	O
+.	O
+	O
+Summary	O
+of	O
+thermodynamic	O
+parameters	O
+for	O
+wild	O
+-	O
+type	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+obtained	O
+by	O
+isothermal	O
+titration	O
+calorimetry	O
+at	O
+25	O
+°	O
+Ca	O
+	O
+Carbohydrate	O
+Ka	O
+(	O
+M	O
+−	O
+1	O
+)	O
+ΔG	O
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+ΔH	O
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+TΔS	O
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+SGBP	O
+-	O
+A	O
+SGBP	O
+-	O
+B	O
+SGBP	O
+-	O
+A	O
+SGBP	O
+-	O
+B	O
+SGBP	O
+-	O
+A	O
+SGBP	O
+-	O
+B	O
+SGBP	O
+-	O
+A	O
+SGBP	O
+-	O
+B	O
+XyGb	O
+(	O
+4	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+)	O
+×	O
+105	O
+(	O
+5	O
+.	O
+7	O
+±	O
+0	O
+.	O
+2	O
+)	O
+×	O
+104	O
+−	O
+7	O
+.	O
+7	O
+−	O
+6	O
+.	O
+5	O
+−	O
+14	O
+±	O
+3	O
+−	O
+14	O
+±	O
+2	O
+−	O
+6	O
+.	O
+5	O
+−	O
+7	O
+.	O
+6	O
+XyGO2c	O
+3	O
+.	O
+0	O
+×	O
+105	O
+2	O
+.	O
+0	O
+×	O
+104	O
+−	O
+7	O
+.	O
+5	O
+−	O
+5	O
+.	O
+9	O
+−	O
+17	O
+.	O
+2	O
+−	O
+17	O
+.	O
+6	O
+−	O
+9	O
+.	O
+7	O
+−	O
+11	O
+.	O
+7	O
+XyGO1	O
+NBd	O
+(	O
+2	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+)	O
+×	O
+103	O
+NB	O
+−	O
+4	O
+.	O
+6	O
+NB	O
+−	O
+4	O
+.	O
+4	O
+±	O
+0	O
+.	O
+2	O
+NB	O
+0	O
+.	O
+2	O
+Cellohexaose	O
+568	O
+.	O
+0	O
+±	O
+291	O
+.	O
+0	O
+NB	O
+−	O
+3	O
+.	O
+8	O
+NB	O
+−	O
+16	O
+±	O
+8	O
+NB	O
+−	O
+12	O
+.	O
+7	O
+NB	O
+Cellotetraose	O
+NB	O
+NB	O
+NB	O
+NB	O
+NB	O
+NB	O
+NB	O
+NB	O
+	O
+SGBP	O
+-	O
+A	O
+is	O
+a	O
+SusD	O
+homolog	O
+with	O
+an	O
+extensive	O
+glycan	O
+-	O
+binding	O
+platform	O
+.	O
+	O
+Specifically	O
+,	O
+SGBP	O
+-	O
+A	O
+overlays	O
+B	O
+.	O
+thetaiotaomicron	O
+SusD	O
+(	O
+BtSusD	O
+)	O
+with	O
+a	O
+root	O
+mean	O
+square	O
+deviation	O
+(	O
+RMSD	O
+)	O
+value	O
+of	O
+2	O
+.	O
+2	O
+Å	O
+for	O
+363	O
+Cα	O
+pairs	O
+,	O
+which	O
+is	O
+notable	O
+given	O
+the	O
+26	O
+%	O
+amino	O
+acid	O
+identity	O
+(	O
+40	O
+%	O
+similarity	O
+)	O
+between	O
+these	O
+homologs	O
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+	O
+The	O
+SGBP	O
+-	O
+A	O
+:	O
+XyGO2	O
+complex	O
+superimposes	O
+closely	O
+with	O
+the	O
+apo	O
+structure	O
+(	O
+RMSD	O
+of	O
+0	O
+.	O
+6	O
+Å	O
+)	O
+and	O
+demonstrates	O
+that	O
+no	O
+major	O
+conformational	O
+change	O
+occurs	O
+upon	O
+substrate	O
+binding	O
+;	O
+small	O
+deviations	O
+in	O
+the	O
+orientation	O
+of	O
+several	O
+surface	O
+loops	O
+are	O
+likely	O
+the	O
+result	O
+of	O
+differential	O
+crystal	O
+packing	O
+.	O
+	O
+It	O
+is	O
+particularly	O
+notable	O
+that	O
+although	O
+the	O
+location	O
+of	O
+the	O
+ligand	O
+-	O
+binding	O
+site	O
+is	O
+conserved	O
+between	O
+SGBP	O
+-	O
+A	O
+and	O
+SusD	O
+,	O
+that	O
+of	O
+SGBP	O
+-	O
+A	O
+displays	O
+an	O
+~	O
+29	O
+-	O
+Å	O
+-	O
+long	O
+aromatic	O
+platform	O
+to	O
+accommodate	O
+the	O
+extended	O
+,	O
+linear	O
+XyG	O
+chain	O
+(	O
+see	O
+reference	O
+for	O
+a	O
+review	O
+of	O
+XyG	O
+secondary	O
+structure	O
+),	O
+versus	O
+the	O
+shorter	O
+,	O
+~	O
+18	O
+-	O
+Å	O
+-	O
+long	O
+,	O
+site	O
+within	O
+SusD	O
+that	O
+complements	O
+the	O
+helical	O
+conformation	O
+of	O
+amylose	O
+(	O
+Fig	O
+.	O
+4C	O
+and	O
+D	O
+).	O
+	O
+Molecular	O
+structure	O
+of	O
+SGBP	O
+-	O
+A	O
+(	O
+Bacova_02651	O
+).	O
+(	O
+A	O
+)	O
+Overlay	O
+of	O
+SGBP	O
+-	O
+A	O
+from	O
+the	O
+apo	O
+(	O
+rainbow	O
+)	O
+and	O
+XyGO2	O
+(	O
+gray	O
+)	O
+structures	O
+.	O
+	O
+An	O
+omit	O
+map	O
+(	O
+2σ	O
+)	O
+for	O
+XyGO2	O
+(	O
+orange	O
+and	O
+red	O
+sticks	O
+)	O
+is	O
+displayed	O
+.	O
+	O
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+omit	O
+map	O
+as	O
+in	O
+panel	O
+A	O
+,	O
+rotated	O
+90	O
+°	O
+clockwise	O
+.	O
+	O
+(	O
+C	O
+)	O
+Overlay	O
+of	O
+the	O
+Cα	O
+backbones	O
+of	O
+SGBP	O
+-	O
+A	O
+(	O
+black	O
+)	O
+with	O
+XyGO2	O
+(	O
+orange	O
+and	O
+red	O
+spheres	O
+)	O
+and	O
+BtSusD	O
+(	O
+blue	O
+)	O
+with	O
+maltoheptaose	O
+(	O
+pink	O
+and	O
+red	O
+spheres	O
+),	O
+highlighting	O
+the	O
+conservation	O
+of	O
+the	O
+glycan	O
+-	O
+binding	O
+site	O
+location	O
+.	O
+	O
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+of	O
+the	O
+SGBP	O
+-	O
+A	O
+(	O
+black	O
+and	O
+orange	O
+)	O
+and	O
+SusD	O
+(	O
+blue	O
+and	O
+pink	O
+)	O
+glycan	O
+-	O
+binding	O
+sites	O
+.	O
+	O
+The	O
+backbone	O
+glucose	O
+residues	O
+are	O
+numbered	O
+from	O
+the	O
+nonreducing	O
+end	O
+;	O
+xylose	O
+residues	O
+are	O
+labeled	O
+X1	O
+and	O
+X2	O
+.	O
+	O
+Indeed	O
+,	O
+the	O
+electron	O
+density	O
+for	O
+the	O
+ligand	O
+suggests	O
+some	O
+disorder	O
+,	O
+which	O
+may	O
+arise	O
+from	O
+multiple	O
+oligosaccharide	O
+orientations	O
+along	O
+the	O
+binding	O
+site	O
+.	O
+	O
+Three	O
+aromatic	O
+residues	O
+—	O
+W82	O
+,	O
+W283	O
+,	O
+W306	O
+—	O
+comprise	O
+the	O
+flat	O
+platform	O
+that	O
+stacks	O
+along	O
+the	O
+naturally	O
+twisted	O
+β	O
+-	O
+glucan	O
+backbone	O
+(	O
+Fig	O
+.	O
+4E	O
+).	O
+	O
+Contrasting	O
+with	O
+the	O
+clear	O
+importance	O
+of	O
+these	O
+hydrophobic	O
+interactions	O
+,	O
+there	O
+are	O
+remarkably	O
+few	O
+hydrogen	O
+-	O
+bonding	O
+interactions	O
+with	O
+the	O
+ligand	O
+,	O
+which	O
+are	O
+provided	O
+by	O
+R65	O
+,	O
+N83	O
+,	O
+and	O
+S308	O
+,	O
+which	O
+are	O
+proximal	O
+to	O
+Glc5	O
+and	O
+Glc3	O
+.	O
+	O
+Most	O
+surprising	O
+in	O
+light	O
+of	O
+the	O
+saccharide	O
+-	O
+binding	O
+data	O
+,	O
+however	O
+,	O
+was	O
+a	O
+lack	O
+of	O
+extensive	O
+recognition	O
+of	O
+the	O
+XyG	O
+side	O
+chains	O
+;	O
+only	O
+Y84	O
+appeared	O
+to	O
+provide	O
+a	O
+hydrophobic	O
+interface	O
+for	O
+a	O
+xylosyl	O
+residue	O
+(	O
+Xyl1	O
+).	O
+	O
+Summary	O
+of	O
+thermodynamic	O
+parameters	O
+for	O
+site	O
+-	O
+directed	O
+mutants	O
+of	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+obtained	O
+by	O
+ITC	O
+with	O
+XyG	O
+at	O
+25	O
+°	O
+Ca	O
+	O
+Weak	O
+binding	O
+represents	O
+a	O
+Ka	O
+of	O
+<	O
+500	O
+M	O
+−	O
+1	O
+.	O
+	O
+Ka	O
+fold	O
+change	O
+=	O
+Ka	O
+of	O
+wild	O
+-	O
+type	O
+protein	O
+/	O
+Ka	O
+of	O
+mutant	O
+protein	O
+for	O
+xyloglucan	O
+binding	O
+.	O
+	O
+SGBP	O
+-	O
+B	O
+has	O
+a	O
+multimodular	O
+structure	O
+with	O
+a	O
+single	O
+,	O
+C	O
+-	O
+terminal	O
+glycan	O
+-	O
+binding	O
+domain	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+full	O
+-	O
+length	O
+SGBP	O
+-	O
+B	O
+in	O
+complex	O
+with	O
+XyGO2	O
+(	O
+2	O
+.	O
+37	O
+Å	O
+,	O
+Rwork	O
+=	O
+19	O
+.	O
+9	O
+%,	O
+Rfree	O
+=	O
+23	O
+.	O
+9	O
+%,	O
+residues	O
+34	O
+to	O
+489	O
+)	O
+(	O
+Table	O
+2	O
+)	O
+revealed	O
+an	O
+extended	O
+structure	O
+composed	O
+of	O
+three	O
+tandem	O
+immunoglobulin	O
+(	O
+Ig	O
+)-	O
+like	O
+domains	O
+(	O
+domains	O
+A	O
+,	O
+B	O
+,	O
+and	O
+C	O
+)	O
+followed	O
+at	O
+the	O
+C	O
+terminus	O
+by	O
+a	O
+novel	O
+xyloglucan	O
+-	O
+binding	O
+domain	O
+(	O
+domain	O
+D	O
+)	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+	O
+These	O
+domains	O
+also	O
+display	O
+similarity	O
+to	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sandwich	O
+domains	O
+of	O
+many	O
+GH13	O
+enzymes	O
+,	O
+including	O
+the	O
+cyclodextrin	O
+glucanotransferase	O
+of	O
+Geobacillus	O
+stearothermophilus	O
+(	O
+Fig	O
+.	O
+5B	O
+).	O
+	O
+Such	O
+domains	O
+are	O
+not	O
+typically	O
+involved	O
+in	O
+carbohydrate	O
+binding	O
+.	O
+	O
+Indeed	O
+,	O
+visual	O
+inspection	O
+of	O
+the	O
+SGBP	O
+-	O
+B	O
+structure	O
+,	O
+as	O
+well	O
+as	O
+individual	O
+production	O
+of	O
+the	O
+A	O
+and	O
+B	O
+domains	O
+and	O
+affinity	O
+PAGE	O
+analysis	O
+(	O
+see	O
+Fig	O
+.	O
+S5	O
+in	O
+the	O
+supplemental	O
+material	O
+),	O
+indicates	O
+that	O
+these	O
+domains	O
+do	O
+not	O
+contribute	O
+to	O
+XyG	O
+capture	O
+.	O
+	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+production	O
+of	O
+the	O
+fused	B-mutant
+domains	I-mutant
+C	I-mutant
+and	I-mutant
+D	I-mutant
+in	O
+tandem	O
+(	O
+SGBP	O
+-	O
+B	O
+residues	O
+230	O
+to	O
+489	O
+)	O
+retains	O
+complete	O
+binding	O
+of	O
+xyloglucan	O
+in	O
+vitro	O
+,	O
+with	O
+the	O
+observed	O
+slight	O
+increase	O
+in	O
+affinity	O
+likely	O
+arising	O
+from	O
+a	O
+reduced	O
+potential	O
+for	O
+steric	O
+hindrance	O
+of	O
+the	O
+smaller	O
+protein	O
+construct	O
+during	O
+polysaccharide	O
+interactions	O
+(	O
+Table	O
+3	O
+).	O
+	O
+While	O
+neither	O
+the	O
+full	O
+-	O
+length	O
+protein	O
+nor	O
+domain	O
+D	O
+displays	O
+structural	O
+homology	O
+to	O
+known	O
+XyG	O
+-	O
+binding	O
+proteins	O
+,	O
+the	O
+topology	O
+of	O
+SGBP	O
+-	O
+B	O
+resembles	O
+the	O
+xylan	O
+-	O
+binding	O
+protein	O
+Bacova_04391	O
+(	O
+PDB	O
+3ORJ	O
+)	O
+encoded	O
+within	O
+a	O
+xylan	O
+-	O
+targeting	O
+PUL	O
+of	O
+B	O
+.	O
+ovatus	O
+(	O
+Fig	O
+.	O
+5C	O
+).	O
+	O
+The	O
+structure	O
+-	O
+based	O
+alignment	O
+of	O
+these	O
+proteins	O
+reveals	O
+17	O
+%	O
+sequence	O
+identity	O
+,	O
+with	O
+a	O
+core	O
+RMSD	O
+of	O
+3	O
+.	O
+6	O
+Å	O
+for	O
+253	O
+aligned	O
+residues	O
+.	O
+	O
+Multimodular	O
+structure	O
+of	O
+SGBP	O
+-	O
+B	O
+(	O
+Bacova_02650	O
+).	O
+(	O
+A	O
+)	O
+Full	O
+-	O
+length	O
+structure	O
+of	O
+SGBP	O
+-	O
+B	O
+,	O
+color	O
+coded	O
+by	O
+domain	O
+as	O
+indicated	O
+.	O
+	O
+An	O
+omit	O
+map	O
+(	O
+2σ	O
+)	O
+for	O
+XyGO2	O
+is	O
+displayed	O
+to	O
+highlight	O
+the	O
+location	O
+of	O
+the	O
+glycan	O
+-	O
+binding	O
+site	O
+.	O
+	O
+(	O
+B	O
+)	O
+Overlay	O
+of	O
+SGBP	O
+-	O
+B	O
+domains	O
+A	O
+,	O
+B	O
+,	O
+and	O
+C	O
+(	O
+colored	O
+as	O
+in	O
+panel	O
+A	O
+),	O
+with	O
+a	O
+C	O
+-	O
+terminal	O
+Ig	O
+-	O
+like	O
+domain	O
+of	O
+the	O
+G	O
+.	O
+stearothermophilus	O
+cyclodextrin	O
+glucanotransferase	O
+(	O
+PDB	O
+1CYG	O
+[	O
+residues	O
+375	O
+to	O
+493	O
+])	O
+in	O
+green	O
+.	O
+(	O
+C	O
+)	O
+Cα	O
+overlay	O
+of	O
+SGBP	O
+-	O
+B	O
+(	O
+gray	O
+)	O
+and	O
+Bacova_04391	O
+(	O
+PDB	O
+3ORJ	O
+)	O
+(	O
+pink	O
+).	O
+	O
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+omit	O
+map	O
+for	O
+the	O
+XyGO2	O
+ligand	O
+,	O
+contoured	O
+at	O
+2σ	O
+.	O
+(	O
+E	O
+)	O
+Stereo	O
+view	O
+of	O
+the	O
+xyloglucan	O
+-	O
+binding	O
+site	O
+of	O
+SGBP	O
+-	O
+B	O
+,	O
+displaying	O
+all	O
+residues	O
+within	O
+4	O
+Å	O
+of	O
+the	O
+ligand	O
+.	O
+	O
+The	O
+backbone	O
+glucose	O
+residues	O
+are	O
+numbered	O
+from	O
+the	O
+nonreducing	O
+end	O
+,	O
+xylose	O
+residues	O
+are	O
+shown	O
+as	O
+X1	O
+,	O
+X2	O
+,	O
+and	O
+X3	O
+,	O
+potential	O
+hydrogen	O
+-	O
+bonding	O
+interactions	O
+are	O
+shown	O
+as	O
+dashed	O
+lines	O
+,	O
+and	O
+the	O
+distance	O
+is	O
+shown	O
+in	O
+angstroms	O
+.	O
+	O
+Domains	O
+A	O
+,	O
+B	O
+,	O
+and	O
+C	O
+do	O
+not	O
+pack	O
+against	O
+each	O
+other	O
+.	O
+	O
+Moreover	O
+,	O
+the	O
+five	O
+-	O
+residue	O
+linkers	O
+between	O
+these	O
+first	O
+three	O
+domains	O
+all	O
+feature	O
+a	O
+proline	O
+as	O
+the	O
+middle	O
+residue	O
+,	O
+suggesting	O
+significant	O
+conformational	O
+rigidity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+	O
+Any	O
+mobility	O
+of	O
+SGBP	O
+-	O
+B	O
+on	O
+the	O
+surface	O
+of	O
+the	O
+cell	O
+(	O
+beyond	O
+lateral	O
+diffusion	O
+within	O
+the	O
+membrane	O
+)	O
+is	O
+likely	O
+imparted	O
+by	O
+the	O
+eight	O
+-	O
+residue	O
+linker	O
+that	O
+spans	O
+the	O
+predicted	O
+lipidated	O
+Cys	O
+(	O
+C28	O
+)	O
+and	O
+the	O
+first	O
+β	O
+-	O
+strand	O
+of	O
+domain	O
+A	O
+.	O
+Other	O
+outer	O
+membrane	O
+proteins	O
+from	O
+various	O
+Sus	O
+-	O
+like	O
+systems	O
+possess	O
+a	O
+similar	O
+10	O
+-	O
+to	O
+20	O
+-	O
+amino	O
+-	O
+acid	O
+flexible	O
+linker	O
+between	O
+the	O
+lipidated	O
+Cys	O
+that	O
+tethers	O
+the	O
+protein	O
+to	O
+the	O
+outside	O
+the	O
+cell	O
+and	O
+the	O
+first	O
+secondary	O
+structure	O
+element	O
+.	O
+	O
+Analogously	O
+,	O
+the	O
+outer	O
+membrane	O
+-	O
+anchored	O
+endo	O
+-	O
+xyloglucanase	O
+BoGH5	O
+of	O
+the	O
+XyGUL	O
+contains	O
+a	O
+100	O
+-	O
+amino	O
+-	O
+acid	O
+,	O
+all	O
+-	O
+β	O
+-	O
+strand	O
+,	O
+N	O
+-	O
+terminal	O
+module	O
+and	O
+flexible	O
+linker	O
+that	O
+imparts	O
+conformational	O
+flexibility	O
+and	O
+distances	O
+the	O
+catalytic	O
+module	O
+from	O
+the	O
+cell	O
+surface	O
+.	O
+	O
+XyG	O
+binds	O
+to	O
+domain	O
+D	O
+of	O
+SGBP	O
+-	O
+B	O
+at	O
+the	O
+concave	O
+interface	O
+of	O
+the	O
+top	O
+β	O
+-	O
+sheet	O
+,	O
+with	O
+binding	O
+mediated	O
+by	O
+loops	O
+connecting	O
+the	O
+β	O
+-	O
+strands	O
+.	O
+	O
+Six	O
+glucosyl	O
+residues	O
+,	O
+comprising	O
+the	O
+main	O
+chain	O
+,	O
+and	O
+three	O
+branching	O
+xylosyl	O
+residues	O
+of	O
+XyGO2	O
+can	O
+be	O
+modeled	O
+in	O
+the	O
+density	O
+(	O
+Fig	O
+.	O
+5D	O
+;	O
+cf	O
+.	O
+	O
+The	O
+aromatic	O
+platform	O
+created	O
+by	O
+W330	O
+,	O
+W364	O
+,	O
+and	O
+Y363	O
+spans	O
+four	O
+glucosyl	O
+residues	O
+,	O
+compared	O
+to	O
+the	O
+longer	O
+platform	O
+of	O
+SGBP	O
+-	O
+A	O
+,	O
+which	O
+supports	O
+six	O
+glucosyl	O
+residues	O
+(	O
+Fig	O
+.	O
+5E	O
+).	O
+	O
+The	O
+Y363A	B-mutant
+site	O
+-	O
+directed	O
+mutant	O
+of	O
+SGBP	O
+-	O
+B	O
+displays	O
+a	O
+20	O
+-	O
+fold	O
+decrease	O
+in	O
+the	O
+Ka	O
+for	O
+XyG	O
+,	O
+while	O
+the	O
+W364A	B-mutant
+mutant	O
+lacks	O
+XyG	O
+binding	O
+(	O
+Table	O
+3	O
+;	O
+see	O
+Fig	O
+.	O
+S6	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+	O
+There	O
+are	O
+no	O
+additional	O
+contacts	O
+between	O
+the	O
+protein	O
+and	O
+the	O
+β	O
+-	O
+glucan	O
+backbone	O
+and	O
+surprisingly	O
+few	O
+interactions	O
+with	O
+the	O
+side	O
+-	O
+chain	O
+xylosyl	O
+residues	O
+,	O
+despite	O
+that	O
+fact	O
+that	O
+ITC	O
+data	O
+demonstrate	O
+that	O
+SGBP	O
+-	O
+B	O
+does	O
+not	O
+measurably	O
+bind	O
+the	O
+cellohexaose	O
+(	O
+Table	O
+1	O
+).	O
+	O
+F414	O
+stacks	O
+with	O
+the	O
+xylosyl	O
+residue	O
+of	O
+Glc3	O
+,	O
+while	O
+Q407	O
+is	O
+positioned	O
+for	O
+hydrogen	O
+bonding	O
+with	O
+the	O
+O4	O
+of	O
+xylosyl	O
+residue	O
+Xyl1	O
+.	O
+	O
+Surprisingly	O
+,	O
+an	O
+F414A	B-mutant
+mutant	O
+of	O
+SGBP	O
+-	O
+B	O
+displays	O
+only	O
+a	O
+mild	O
+3	O
+-	O
+fold	O
+decrease	O
+in	O
+the	O
+Ka	O
+value	O
+for	O
+XyG	O
+,	O
+again	O
+suggesting	O
+that	O
+glycan	O
+recognition	O
+is	O
+primarily	O
+mediated	O
+via	O
+contact	O
+with	O
+the	O
+β	O
+-	O
+glucan	O
+backbone	O
+(	O
+Table	O
+3	O
+;	O
+see	O
+Fig	O
+.	O
+S6	O
+).	O
+	O
+Additional	O
+residues	O
+surrounding	O
+the	O
+binding	O
+site	O
+,	O
+including	O
+Y369	O
+and	O
+E412	O
+,	O
+may	O
+contribute	O
+to	O
+the	O
+recognition	O
+of	O
+more	O
+highly	O
+decorated	O
+XyG	O
+,	O
+but	O
+precisely	O
+how	O
+this	O
+is	O
+mediated	O
+is	O
+presently	O
+unclear	O
+.	O
+	O
+The	O
+CD	O
+domains	O
+of	O
+the	O
+truncated	O
+and	O
+full	O
+-	O
+length	O
+proteins	O
+superimpose	O
+with	O
+a	O
+0	O
+.	O
+4	O
+-	O
+Å	O
+RMSD	O
+of	O
+the	O
+Cα	O
+backbone	O
+,	O
+with	O
+no	O
+differences	O
+in	O
+the	O
+position	O
+of	O
+any	O
+of	O
+the	O
+glycan	O
+-	O
+binding	O
+residues	O
+(	O
+see	O
+Fig	O
+.	O
+S7A	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+	O
+While	O
+density	O
+is	O
+observed	O
+for	O
+XyGO2	O
+,	O
+the	O
+ligand	O
+could	O
+not	O
+be	O
+unambiguously	O
+modeled	O
+into	O
+this	O
+density	O
+to	O
+achieve	O
+a	O
+reasonable	O
+fit	O
+between	O
+the	O
+X	O
+-	O
+ray	O
+data	O
+and	O
+the	O
+known	O
+stereochemistry	O
+of	O
+the	O
+sugar	O
+(	O
+see	O
+Fig	O
+.	O
+S7B	O
+and	O
+C	O
+).	O
+	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+have	O
+distinct	O
+,	O
+coordinated	O
+functions	O
+in	O
+vivo	O
+.	O
+	O
+To	O
+disentangle	O
+the	O
+functions	O
+of	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+in	O
+XyG	O
+recognition	O
+and	O
+uptake	O
+,	O
+we	O
+created	O
+individual	O
+in	O
+-	O
+frame	O
+deletion	O
+and	O
+complementation	O
+mutant	O
+strains	O
+of	O
+B	O
+.	O
+ovatus	O
+.	O
+	O
+Growth	O
+on	O
+glucose	O
+displayed	O
+the	O
+shortest	O
+lag	O
+time	O
+for	O
+each	O
+strain	O
+,	O
+and	O
+so	O
+lag	O
+times	O
+were	O
+normalized	O
+for	O
+each	O
+carbohydrate	O
+by	O
+subtracting	O
+the	O
+lag	O
+time	O
+of	O
+that	O
+strain	O
+in	O
+glucose	O
+(	O
+Fig	O
+.	O
+6	O
+;	O
+see	O
+Fig	O
+.	O
+S8	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+	O
+A	O
+strain	O
+in	O
+which	O
+the	O
+entire	O
+XyGUL	O
+is	O
+deleted	O
+displays	O
+a	O
+lag	O
+of	O
+24	O
+.	O
+5	O
+h	O
+during	O
+growth	O
+on	O
+glucose	O
+compared	O
+to	O
+the	O
+isogenic	O
+parental	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+Δtdk	B-mutant
+strain	O
+,	O
+for	O
+which	O
+exponential	O
+growth	O
+lags	O
+for	O
+19	O
+.	O
+8	O
+h	O
+(	O
+see	O
+Fig	O
+.	O
+S8D	O
+).	O
+	O
+It	O
+is	O
+unknown	O
+whether	O
+this	O
+is	O
+because	O
+cultures	O
+were	O
+not	O
+normalized	O
+by	O
+the	O
+starting	O
+optical	O
+density	O
+(	O
+OD	O
+)	O
+or	O
+viable	O
+cells	O
+or	O
+reflects	O
+a	O
+minor	O
+defect	O
+for	O
+glucose	O
+utilization	O
+.	O
+	O
+The	O
+former	O
+seems	O
+more	O
+likely	O
+as	O
+the	O
+growth	O
+rates	O
+are	O
+nearly	O
+identical	O
+for	O
+these	O
+strains	O
+on	O
+glucose	O
+and	O
+xylose	O
+.	O
+	O
+The	O
+ΔXyGUL	B-mutant
+and	O
+WT	O
+Δtdk	B-mutant
+strains	O
+display	O
+normalized	O
+lag	O
+times	O
+on	O
+xylose	O
+within	O
+experimental	O
+error	O
+,	O
+and	O
+curiously	O
+some	O
+of	O
+the	O
+mutant	O
+and	O
+complemented	O
+strains	O
+display	O
+a	O
+nominally	O
+shorter	O
+lag	O
+time	O
+on	O
+xylose	O
+than	O
+the	O
+WT	O
+Δtdk	B-mutant
+strain	O
+.	O
+	O
+The	O
+reason	O
+for	O
+this	O
+observation	O
+on	O
+XyGO2	O
+is	O
+unclear	O
+,	O
+as	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+mutant	O
+does	O
+not	O
+have	O
+a	O
+significantly	O
+different	O
+growth	O
+rate	O
+from	O
+the	O
+WT	O
+on	O
+XyGO2	O
+.	O
+	O
+Growth	O
+of	O
+select	O
+XyGUL	O
+mutants	O
+on	O
+xyloglucan	O
+and	O
+oligosaccharides	O
+.	O
+	O
+B	O
+.	O
+ovatus	O
+mutants	O
+were	O
+created	O
+in	O
+a	O
+thymidine	B-mutant
+kinase	I-mutant
+deletion	I-mutant
+(	O
+Δtdk	B-mutant
+)	O
+mutant	O
+as	O
+described	O
+previously	O
+.	O
+	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+denotes	O
+the	O
+Bacova_02651	O
+(	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+)	O
+allele	O
+,	O
+and	O
+the	O
+GH9	O
+gene	O
+is	O
+Bacova_02649	O
+.	O
+	O
+Solid	O
+bars	O
+indicate	O
+conditions	O
+that	O
+are	O
+not	O
+statistically	O
+significant	O
+from	O
+the	O
+WT	O
+Δtdk	B-mutant
+cultures	O
+grown	O
+on	O
+the	O
+indicated	O
+carbohydrate	O
+,	O
+while	O
+open	O
+bars	O
+indicate	O
+a	O
+P	O
+value	O
+of	O
+<	O
+0	O
+.	O
+005	O
+compared	O
+to	O
+the	O
+WT	O
+Δtdk	B-mutant
+strain	O
+.	O
+	O
+Conditions	O
+denoted	O
+by	O
+the	O
+same	O
+letter	O
+(	O
+b	O
+,	O
+c	O
+,	O
+or	O
+d	O
+)	O
+are	O
+not	O
+statistically	O
+significant	O
+from	O
+each	O
+other	O
+but	O
+are	O
+significantly	O
+different	O
+from	O
+the	O
+condition	O
+labeled	O
+“	O
+a	O
+.”	O
+Complementation	O
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+and	O
+ΔSBGP	B-mutant
+-	I-mutant
+B	I-mutant
+was	O
+performed	O
+by	O
+allelic	O
+exchange	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+genes	O
+back	O
+into	O
+the	O
+genome	O
+for	O
+expression	O
+via	O
+the	O
+native	O
+promoter	O
+:	O
+these	O
+growth	O
+curves	O
+,	O
+quantified	O
+rates	O
+and	O
+lag	O
+times	O
+are	O
+displayed	O
+in	O
+Fig	O
+.	O
+S8	O
+in	O
+the	O
+supplemental	O
+material	O
+.	O
+	O
+Fig	O
+.	O
+1B	O
+)	O
+was	O
+completely	O
+incapable	O
+of	O
+growth	O
+on	O
+XyG	O
+,	O
+XyGO1	O
+,	O
+and	O
+XyGO2	O
+,	O
+indicating	O
+that	O
+SGBP	O
+-	O
+A	O
+is	O
+essential	O
+for	O
+XyG	O
+utilization	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+This	O
+result	O
+mirrors	O
+our	O
+previous	O
+data	O
+for	O
+the	O
+canonical	O
+Sus	O
+of	O
+B	O
+.	O
+thetaiotaomicron	O
+,	O
+which	O
+revealed	O
+that	O
+a	O
+homologous	O
+ΔsusD	B-mutant
+mutant	O
+is	O
+unable	O
+to	O
+grow	O
+on	O
+starch	O
+or	O
+malto	O
+-	O
+oligosaccharides	O
+,	O
+despite	O
+normal	O
+cell	O
+surface	O
+expression	O
+of	O
+all	O
+other	O
+PUL	O
+-	O
+encoded	O
+proteins	O
+.	O
+	O
+More	O
+recently	O
+,	O
+we	O
+demonstrated	O
+that	O
+this	O
+phenotype	O
+is	O
+due	O
+to	O
+the	O
+loss	O
+of	O
+the	O
+physical	O
+presence	O
+of	O
+SusD	O
+;	O
+complementation	O
+of	O
+ΔsusD	B-mutant
+with	O
+SusD	B-mutant
+*,	I-mutant
+a	O
+triple	O
+site	O
+-	O
+directed	O
+mutant	O
+(	O
+W96A	B-mutant
+W320A	B-mutant
+Y296A	B-mutant
+)	O
+that	O
+ablates	O
+glycan	O
+binding	O
+,	O
+restores	O
+B	O
+.	O
+thetaiotaomicron	O
+growth	O
+on	O
+malto	O
+-	O
+oligosaccharides	O
+and	O
+starch	O
+when	O
+sus	O
+transcription	O
+is	O
+induced	O
+by	O
+maltose	O
+addition	O
+.	O
+	O
+Similarly	O
+,	O
+the	O
+function	O
+of	O
+SGBP	O
+-	O
+A	O
+extends	O
+beyond	O
+glycan	O
+binding	O
+.	O
+	O
+Complementation	O
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+with	O
+the	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+(	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+)	O
+variant	O
+,	O
+which	O
+does	O
+not	O
+bind	O
+XyG	O
+,	O
+supports	O
+growth	O
+on	O
+XyG	O
+and	O
+XyGOs	O
+(	O
+Fig	O
+.	O
+6	O
+;	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*),	I-mutant
+with	O
+growth	O
+rates	O
+that	O
+are	O
+~	O
+70	O
+%	O
+that	O
+of	O
+the	O
+WT	O
+.	O
+	O
+In	O
+previous	O
+studies	O
+,	O
+we	O
+observed	O
+that	O
+carbohydrate	O
+binding	O
+by	O
+SusD	O
+enhanced	O
+the	O
+sensitivity	O
+of	O
+the	O
+cells	O
+to	O
+limiting	O
+concentrations	O
+of	O
+malto	O
+-	O
+oligosaccharides	O
+by	O
+several	O
+orders	O
+of	O
+magnitude	O
+,	O
+such	O
+that	O
+the	O
+addition	O
+of	O
+0	O
+.	O
+5	O
+g	O
+/	O
+liter	O
+maltose	O
+was	O
+required	O
+to	O
+restore	O
+growth	O
+of	O
+the	O
+ΔsusD	B-mutant
+::	O
+SusD	B-mutant
+*	I-mutant
+strain	O
+on	O
+starch	O
+,	O
+which	O
+nonetheless	O
+occurred	O
+following	O
+an	O
+extended	O
+lag	O
+phase	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+strain	O
+does	O
+not	O
+display	O
+an	O
+extended	O
+lag	O
+time	O
+on	O
+any	O
+of	O
+the	O
+xyloglucan	O
+substrates	O
+compared	O
+to	O
+the	O
+WT	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+However	O
+,	O
+the	O
+modest	O
+rate	O
+defect	O
+displayed	O
+by	O
+the	O
+SGBP	O
+-	O
+A	O
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+strain	O
+suggests	O
+that	O
+recognition	O
+of	O
+XyG	O
+and	O
+product	O
+import	O
+is	O
+somewhat	O
+less	O
+efficient	O
+in	O
+these	O
+cells	O
+.	O
+	O
+10	O
+-	O
+fold	O
+more	O
+weakly	O
+than	O
+XyGO2	O
+and	O
+XyG	O
+(	O
+Fig	O
+.	O
+6	O
+;	O
+Table	O
+1	O
+).	O
+	O
+As	O
+such	O
+,	O
+the	O
+data	O
+suggest	O
+that	O
+SGBP	O
+-	O
+A	O
+can	O
+compensate	O
+for	O
+the	O
+loss	O
+of	O
+function	O
+of	O
+SGBP	O
+-	O
+B	O
+on	O
+longer	O
+oligo	O
+-	O
+and	O
+polysaccharides	O
+,	O
+while	O
+SGBP	O
+-	O
+B	O
+may	O
+adapt	O
+the	O
+cell	O
+to	O
+recognize	O
+smaller	O
+oligosaccharides	O
+efficiently	O
+.	O
+	O
+Indeed	O
+,	O
+a	O
+double	O
+mutant	O
+,	O
+consisting	O
+of	O
+a	O
+crippled	O
+SGBP	O
+-	O
+A	O
+and	O
+a	O
+deletion	O
+of	O
+SGBP	O
+-	O
+B	O
+(	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*/	I-mutant
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+),	O
+exhibits	O
+an	O
+extended	O
+lag	O
+time	O
+on	O
+both	O
+XyG	O
+and	O
+XyGO2	O
+,	O
+as	O
+well	O
+as	O
+XyGO1	O
+.	O
+	O
+This	O
+additional	O
+role	O
+of	O
+SGBP	O
+-	O
+B	O
+is	O
+especially	O
+notable	O
+in	O
+the	O
+context	O
+of	O
+studies	O
+on	O
+BtSusE	O
+and	O
+BtSusF	O
+(	O
+positioned	O
+similarly	O
+in	O
+the	O
+archetypal	O
+Sus	O
+locus	O
+)	O
+(	O
+Fig	O
+.	O
+1B	O
+),	O
+for	O
+which	O
+growth	O
+defects	O
+on	O
+starch	O
+or	O
+malto	O
+-	O
+oligosaccharides	O
+have	O
+never	O
+been	O
+observed	O
+.	O
+	O
+However	O
+,	O
+combined	O
+deletion	O
+of	O
+the	O
+genes	O
+encoding	O
+GH9	O
+(	O
+encoded	O
+by	O
+Bacova_02649	O
+)	O
+and	O
+SGBP	O
+-	O
+B	O
+does	O
+not	O
+exacerbate	O
+the	O
+growth	O
+defect	O
+on	O
+XyGO1	O
+(	O
+Fig	O
+.	O
+6	O
+;	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+/	O
+ΔGH9	B-mutant
+).	O
+	O
+The	O
+necessity	O
+of	O
+SGBP	O
+-	O
+B	O
+is	O
+elevated	O
+in	O
+the	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+strain	O
+,	O
+as	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*/	I-mutant
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+mutant	O
+displays	O
+an	O
+extended	O
+lag	O
+during	O
+growth	O
+on	O
+XyG	O
+and	O
+xylogluco	O
+-	O
+oligosaccharides	O
+,	O
+while	O
+growth	O
+rate	O
+differences	O
+are	O
+more	O
+subtle	O
+.	O
+	O
+The	O
+precise	O
+reason	O
+for	O
+this	O
+lag	O
+is	O
+unclear	O
+,	O
+but	O
+recapitulating	O
+our	O
+findings	O
+on	O
+the	O
+role	O
+of	O
+SusD	O
+in	O
+malto	O
+-	O
+oligosaccharide	O
+sensing	O
+in	O
+B	O
+.	O
+thetaiotaomicron	O
+,	O
+this	O
+extended	O
+lag	O
+may	O
+be	O
+due	O
+to	O
+inefficient	O
+import	O
+and	O
+thus	O
+sensing	O
+of	O
+xyloglucan	O
+in	O
+the	O
+environment	O
+in	O
+the	O
+absence	O
+of	O
+glycan	O
+binding	O
+by	O
+essential	O
+SGBPs	O
+.	O
+	O
+Our	O
+previous	O
+work	O
+demonstrates	O
+that	O
+B	O
+.	O
+ovatus	O
+cells	O
+grown	O
+in	O
+minimal	O
+medium	O
+plus	O
+glucose	O
+express	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	O
+transcript	O
+.	O
+	O
+Thus	O
+,	O
+in	O
+our	O
+experiments	O
+,	O
+we	O
+presume	O
+that	O
+each	O
+strain	O
+,	O
+initially	O
+grown	O
+in	O
+glucose	O
+,	O
+expresses	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	O
+transcript	O
+and	O
+thus	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	O
+-	O
+encoded	O
+surface	O
+proteins	O
+,	O
+including	O
+the	O
+vanguard	O
+GH5	O
+.	O
+	O
+Presumably	O
+without	O
+glycan	O
+binding	O
+by	O
+the	O
+SGBPs	O
+,	O
+the	O
+GH5	O
+protein	O
+cannot	O
+efficiently	O
+process	O
+xyloglucan	O
+,	O
+and	O
+/	O
+or	O
+the	O
+lack	O
+of	O
+SGBP	O
+function	O
+prevents	O
+efficient	O
+capture	O
+and	O
+import	O
+of	O
+the	O
+processed	O
+oligosaccharides	O
+.	O
+	O
+In	O
+the	O
+BtSus	O
+,	O
+SusD	O
+and	O
+the	O
+TBDT	O
+SusC	O
+interact	O
+,	O
+and	O
+we	O
+speculate	O
+that	O
+this	O
+interaction	O
+is	O
+necessary	O
+for	O
+glycan	O
+uptake	O
+,	O
+as	O
+suggested	O
+by	O
+the	O
+fact	O
+that	O
+a	O
+ΔsusD	B-mutant
+mutant	O
+cannot	O
+grow	O
+on	O
+starch	O
+,	O
+but	O
+a	O
+ΔsusD	B-mutant
+::	O
+SusD	B-mutant
+*	I-mutant
+strain	O
+regains	O
+this	O
+ability	O
+if	O
+a	O
+transcriptional	O
+activator	O
+of	O
+the	O
+sus	O
+operon	O
+is	O
+supplied	O
+.	O
+	O
+However	O
+,	O
+unlike	O
+the	O
+Sus	O
+,	O
+in	O
+which	O
+elimination	O
+of	O
+SusE	O
+and	O
+SusF	O
+does	O
+not	O
+affect	O
+growth	O
+on	O
+starch	O
+,	O
+SGBP	O
+-	O
+B	O
+appears	O
+to	O
+have	O
+a	O
+dedicated	O
+role	O
+in	O
+growth	O
+on	O
+small	O
+xylogluco	O
+-	O
+oligosaccharides	O
+.	O
+	O
+The	O
+ability	O
+of	O
+gut	O
+-	O
+adapted	O
+microorganisms	O
+to	O
+thrive	O
+in	O
+the	O
+gastrointestinal	O
+tract	O
+is	O
+critically	O
+dependent	O
+upon	O
+their	O
+ability	O
+to	O
+efficiently	O
+recognize	O
+,	O
+cleave	O
+,	O
+and	O
+import	O
+glycans	O
+.	O
+	O
+The	O
+human	O
+gut	O
+,	O
+in	O
+particular	O
+,	O
+is	O
+a	O
+densely	O
+packed	O
+ecosystem	O
+with	O
+hundreds	O
+of	O
+species	O
+,	O
+in	O
+which	O
+there	O
+is	O
+potential	O
+for	O
+both	O
+competition	O
+and	O
+synergy	O
+in	O
+the	O
+utilization	O
+of	O
+different	O
+substrates	O
+.	O
+	O
+Recent	O
+work	O
+has	O
+elucidated	O
+that	O
+Bacteroidetes	O
+cross	O
+-	O
+feed	O
+during	O
+growth	O
+on	O
+many	O
+glycans	O
+;	O
+the	O
+glycoside	O
+hydrolases	O
+expressed	O
+by	O
+one	O
+species	O
+liberate	O
+oligosaccharides	O
+for	O
+consumption	O
+by	O
+other	O
+members	O
+of	O
+the	O
+community	O
+.	O
+	O
+Here	O
+,	O
+we	O
+demonstrate	O
+that	O
+the	O
+surface	O
+glycan	O
+binding	O
+proteins	O
+encoded	O
+within	O
+the	O
+BoXyGUL	O
+play	O
+unique	O
+and	O
+essential	O
+roles	O
+in	O
+the	O
+acquisition	O
+of	O
+the	O
+ubiquitous	O
+and	O
+abundant	O
+vegetable	O
+polysaccharide	O
+xyloglucan	O
+.	O
+	O
+Yet	O
+,	O
+a	O
+number	O
+of	O
+questions	O
+remain	O
+regarding	O
+the	O
+molecular	O
+interplay	O
+of	O
+SGBPs	O
+with	O
+their	O
+cotranscribed	O
+cohort	O
+of	O
+glycoside	O
+hydrolases	O
+and	O
+TonB	O
+-	O
+dependent	O
+transporters	O
+.	O
+	O
+A	O
+direct	O
+interaction	O
+between	O
+the	O
+BtSusC	O
+TBDT	O
+and	O
+the	O
+SusD	O
+SGBP	O
+has	O
+been	O
+previously	O
+demonstrated	O
+,	O
+as	O
+has	O
+an	O
+interaction	O
+between	O
+the	O
+homologous	O
+components	O
+encoded	O
+by	O
+an	O
+N	O
+-	O
+glycan	O
+-	O
+scavenging	O
+PUL	O
+of	O
+Capnocytophaga	O
+canimorsus	O
+.	O
+	O
+It	O
+is	O
+yet	O
+presently	O
+unclear	O
+whether	O
+this	O
+interaction	O
+is	O
+static	O
+or	O
+dynamic	O
+and	O
+to	O
+what	O
+extent	O
+the	O
+association	O
+of	O
+cognate	O
+TBDT	O
+/	O
+SGBPs	O
+is	O
+dependent	O
+upon	O
+the	O
+structure	O
+of	O
+the	O
+carbohydrate	O
+to	O
+be	O
+imported	O
+.	O
+	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+there	O
+is	O
+clear	O
+evidence	O
+for	O
+independent	O
+TBDTs	O
+in	O
+Bacteroidetes	O
+that	O
+do	O
+not	O
+require	O
+SGBP	O
+association	O
+for	O
+activity	O
+.	O
+	O
+For	O
+example	O
+,	O
+it	O
+was	O
+recently	O
+demonstrated	O
+that	O
+expression	O
+of	O
+nanO	O
+,	O
+which	O
+encodes	O
+a	O
+SusC	O
+-	O
+like	O
+TBDT	O
+as	O
+part	O
+of	O
+a	O
+sialic	O
+-	O
+acid	O
+-	O
+targeting	O
+PUL	O
+from	O
+B	O
+.	O
+fragilis	O
+,	O
+restored	O
+growth	O
+on	O
+this	O
+monosaccharide	O
+in	O
+a	O
+mutant	O
+strain	O
+of	O
+E	O
+.	O
+coli	O
+.	O
+	O
+In	O
+this	O
+instance	O
+,	O
+coexpression	O
+of	O
+the	O
+susD	O
+-	O
+like	O
+gene	O
+nanU	O
+was	O
+not	O
+required	O
+,	O
+nor	O
+did	O
+the	O
+expression	O
+of	O
+the	O
+nanU	O
+gene	O
+enhance	O
+growth	O
+kinetics	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+strict	O
+dependence	O
+on	O
+a	O
+SusD	O
+-	O
+like	O
+SGBP	O
+for	O
+glycan	O
+uptake	O
+in	O
+the	O
+Bacteroidetes	O
+may	O
+be	O
+variable	O
+and	O
+substrate	O
+dependent	O
+.	O
+	O
+Such	O
+is	O
+the	O
+case	O
+for	O
+XyGUL	O
+from	O
+related	O
+Bacteroides	O
+species	O
+,	O
+which	O
+may	O
+encode	O
+either	O
+one	O
+or	O
+two	O
+of	O
+these	O
+predicted	O
+SGBPs	O
+,	O
+and	O
+these	O
+proteins	O
+vary	O
+considerably	O
+in	O
+length	O
+.	O
+	O
+The	O
+extremely	O
+low	O
+similarity	O
+of	O
+these	O
+SGBPs	O
+is	O
+striking	O
+in	O
+light	O
+of	O
+the	O
+moderate	O
+sequence	O
+conservation	O
+observed	O
+among	O
+homologous	O
+GHs	O
+in	O
+syntenic	O
+PUL	O
+.	O
+	O
+This	O
+,	O
+together	O
+with	O
+the	O
+observation	O
+that	O
+these	O
+SGBPs	O
+,	O
+as	O
+exemplified	O
+by	O
+BtSusE	O
+and	O
+BtSusF	O
+and	O
+the	O
+XyGUL	O
+SGBP	O
+-	O
+B	O
+of	O
+the	O
+present	O
+study	O
+,	O
+are	O
+expendable	O
+for	O
+polysaccharide	O
+growth	O
+,	O
+implies	O
+a	O
+high	O
+degree	O
+of	O
+evolutionary	O
+flexibility	O
+to	O
+enhance	O
+glycan	O
+capture	O
+at	O
+the	O
+cell	O
+surface	O
+.	O
+	O
+However	O
+,	O
+the	O
+natural	O
+diversity	O
+of	O
+these	O
+proteins	O
+represents	O
+a	O
+rich	O
+source	O
+for	O
+the	O
+discovery	O
+of	O
+unique	O
+carbohydrate	O
+-	O
+binding	O
+motifs	O
+to	O
+both	O
+inform	O
+gut	O
+microbiology	O
+and	O
+generate	O
+new	O
+,	O
+specific	O
+carbohydrate	O
+analytical	O
+reagents	O
+.	O
+	O
+In	O
+conclusion	O
+,	O
+the	O
+present	O
+study	O
+further	O
+illuminates	O
+the	O
+essential	O
+role	O
+that	O
+surface	O
+-	O
+glycan	O
+binding	O
+proteins	O
+play	O
+in	O
+facilitating	O
+the	O
+catabolism	O
+of	O
+complex	O
+dietary	O
+carbohydrates	O
+by	O
+Bacteroidetes	O
+.	O
+	O
+The	O
+ability	O
+of	O
+our	O
+resident	O
+gut	O
+bacteria	O
+to	O
+recognize	O
+polysaccharides	O
+is	O
+the	O
+first	O
+committed	O
+step	O
+of	O
+glycan	O
+consumption	O
+by	O
+these	O
+organisms	O
+,	O
+a	O
+critical	O
+process	O
+that	O
+influences	O
+the	O
+community	O
+structure	O
+and	O
+thus	O
+the	O
+metabolic	O
+output	O
+(	O
+i	O
+.	O
+e	O
+.,	O
+short	O
+-	O
+chain	O
+fatty	O
+acid	O
+and	O
+metabolite	O
+profile	O
+)	O
+of	O
+these	O
+organisms	O
+.	O
+	O
+Inhibiting	O
+complex	O
+IL	O
+-	O
+17A	O
+and	O
+IL	O
+-	O
+17RA	O
+interactions	O
+with	O
+a	O
+linear	O
+peptide	O
+	O
+IL	O
+-	O
+17A	O
+is	O
+a	O
+pro	O
+-	O
+inflammatory	O
+cytokine	O
+that	O
+has	O
+been	O
+implicated	O
+in	O
+autoimmune	O
+and	O
+inflammatory	O
+diseases	O
+.	O
+	O
+HAP	O
+binds	O
+specifically	O
+to	O
+IL	O
+-	O
+17A	O
+and	O
+inhibits	O
+the	O
+interaction	O
+of	O
+the	O
+cytokine	O
+with	O
+its	O
+receptor	O
+,	O
+IL	O
+-	O
+17RA	O
+.	O
+	O
+Crystal	O
+structure	O
+studies	O
+revealed	O
+that	O
+two	O
+HAP	O
+molecules	O
+bind	O
+to	O
+one	O
+IL	O
+-	O
+17A	O
+dimer	O
+symmetrically	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+portions	O
+of	O
+HAP	O
+form	O
+a	O
+β	O
+-	O
+strand	O
+that	O
+inserts	O
+between	O
+two	O
+IL	O
+-	O
+17A	O
+monomers	O
+while	O
+the	O
+C	O
+-	O
+terminal	O
+section	O
+forms	O
+an	O
+α	O
+helix	O
+that	O
+directly	O
+blocks	O
+IL	O
+-	O
+17RA	O
+from	O
+binding	O
+to	O
+the	O
+same	O
+region	O
+of	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+IL	O
+-	O
+17A	O
+signals	O
+through	O
+a	O
+specific	O
+cell	O
+surface	O
+receptor	O
+complex	O
+which	O
+consists	O
+of	O
+IL	O
+-	O
+17RA	O
+and	O
+IL	O
+-	O
+17RC	O
+.	O
+	O
+IL	O
+-	O
+17A	O
+’	O
+s	O
+downstream	O
+signaling	O
+leads	O
+to	O
+increased	O
+production	O
+of	O
+inflammatory	O
+cytokines	O
+such	O
+as	O
+IL	O
+-	O
+6	O
+,	O
+IL	O
+-	O
+8	O
+,	O
+CCL	O
+-	O
+20	O
+and	O
+CXCL1	O
+by	O
+various	O
+mechanisms	O
+including	O
+stimulation	O
+of	O
+transcription	O
+and	O
+stabilization	O
+of	O
+mRNA	O
+.	O
+	O
+Although	O
+various	O
+cell	O
+types	O
+have	O
+been	O
+reported	O
+to	O
+express	O
+IL	O
+-	O
+17RA	O
+,	O
+the	O
+highest	O
+responses	O
+to	O
+IL	O
+-	O
+17A	O
+come	O
+from	O
+epithelial	O
+cells	O
+,	O
+endothelial	O
+cells	O
+,	O
+keratinocytes	O
+and	O
+fibroblasts	O
+.	O
+	O
+IL	O
+-	O
+17A	O
+and	O
+its	O
+signaling	O
+is	O
+important	O
+in	O
+host	O
+defense	O
+against	O
+certain	O
+fungal	O
+and	O
+bacterial	O
+infections	O
+as	O
+demonstrated	O
+by	O
+patients	O
+with	O
+autoantibodies	O
+against	O
+IL	O
+-	O
+17A	O
+and	O
+IL	O
+-	O
+17F	O
+,	O
+or	O
+with	O
+inborn	O
+errors	O
+of	O
+IL	O
+-	O
+17	O
+immunity	O
+.	O
+	O
+In	O
+addition	O
+to	O
+its	O
+physiological	O
+role	O
+,	O
+IL	O
+-	O
+17A	O
+is	O
+a	O
+key	O
+pathogenic	O
+factor	O
+in	O
+inflammatory	O
+and	O
+autoimmune	O
+diseases	O
+.	O
+	O
+In	O
+phase	O
+II	O
+and	O
+III	O
+clinical	O
+trials	O
+,	O
+neutralizing	O
+monoclonal	O
+antibodies	O
+against	O
+IL	O
+-	O
+17A	O
+(	O
+secukinumab	O
+and	O
+ixekizumab	O
+)	O
+or	O
+its	O
+receptor	O
+IL	O
+-	O
+17RA	O
+(	O
+brodalumab	O
+)	O
+are	O
+highly	O
+efficacious	O
+in	O
+treating	O
+moderate	O
+to	O
+severe	O
+plaque	O
+psoriasis	O
+and	O
+psoriatic	O
+arthritis	O
+.	O
+	O
+Secukinumab	O
+has	O
+been	O
+approved	O
+recently	O
+as	O
+a	O
+new	O
+psoriasis	O
+drug	O
+by	O
+the	O
+US	O
+Food	O
+and	O
+Drug	O
+Administration	O
+(	O
+Cosentyx	O
+™).	O
+	O
+In	O
+addition	O
+to	O
+psoriasis	O
+and	O
+psoriatic	O
+arthritis	O
+,	O
+IL	O
+-	O
+17A	O
+blockade	O
+has	O
+also	O
+shown	O
+preclinical	O
+and	O
+clinical	O
+efficacies	O
+in	O
+ankylosing	O
+spondylitis	O
+and	O
+rheumatoid	O
+arthritis	O
+.	O
+	O
+Among	O
+IL	O
+-	O
+17	O
+cytokines	O
+,	O
+IL	O
+-	O
+17A	O
+and	O
+IL	O
+-	O
+17F	O
+share	O
+the	O
+highest	O
+homology	O
+.	O
+	O
+Structures	O
+are	O
+known	O
+for	O
+apo	O
+IL	O
+-	O
+17F	O
+and	O
+its	O
+complex	O
+with	O
+IL	O
+-	O
+17RA	O
+,	O
+for	O
+apo	O
+IL	O
+-	O
+17A	O
+,	O
+its	O
+complex	O
+with	O
+an	O
+antibody	O
+Fab	O
+,	O
+and	O
+its	O
+complex	O
+with	O
+IL	O
+-	O
+17RA	O
+.	O
+	O
+Developing	O
+small	O
+molecules	O
+targeting	O
+protein	O
+-	O
+protein	O
+interactions	O
+is	O
+difficult	O
+with	O
+particular	O
+challenges	O
+associated	O
+with	O
+the	O
+large	O
+,	O
+shallow	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+interfaces	O
+.	O
+	O
+Our	O
+efforts	O
+resulted	O
+in	O
+discovery	O
+of	O
+a	O
+high	O
+affinity	O
+IL	O
+-	O
+17A	O
+peptide	O
+antagonist	O
+(	O
+HAP	O
+),	O
+which	O
+we	O
+attempted	O
+to	O
+increase	O
+the	O
+functional	O
+production	O
+and	O
+pharmacokinetics	O
+after	O
+fusing	O
+HAP	O
+to	O
+antibodies	O
+for	O
+evaluation	O
+as	O
+a	O
+bispecific	O
+therapeutic	O
+in	O
+animal	O
+studies	O
+.	O
+	O
+Unfortunately	O
+,	O
+this	O
+past	O
+work	O
+revealed	O
+stability	O
+issues	O
+of	O
+the	O
+uncapped	O
+HAP	O
+in	O
+cell	O
+culture	O
+Here	O
+,	O
+we	O
+provide	O
+the	O
+details	O
+of	O
+the	O
+discovery	O
+and	O
+optimization	O
+that	O
+led	O
+to	O
+HAP	O
+and	O
+report	O
+the	O
+complex	O
+structure	O
+of	O
+IL	O
+-	O
+17A	O
+with	O
+HAP	O
+,	O
+which	O
+provides	O
+structure	O
+based	O
+rationalization	O
+of	O
+peptide	O
+optimization	O
+and	O
+structure	O
+activity	O
+relationship	O
+(	O
+SAR	O
+).	O
+	O
+Single	O
+clones	O
+were	O
+isolated	O
+and	O
+sub	O
+-	O
+cultured	O
+in	O
+growth	O
+medium	O
+,	O
+and	O
+culture	O
+supernatants	O
+were	O
+used	O
+in	O
+an	O
+enzyme	O
+-	O
+linked	O
+immunosorbent	O
+assay	O
+(	O
+ELISA	O
+)	O
+to	O
+identify	O
+specific	O
+IL	O
+-	O
+17A	O
+-	O
+binding	O
+clones	O
+.	O
+	O
+Approximately	O
+10	O
+%	O
+of	O
+the	O
+clones	O
+that	O
+specifically	O
+bound	O
+to	O
+IL	O
+-	O
+17A	O
+also	O
+prevented	O
+the	O
+cytokine	O
+from	O
+binding	O
+to	O
+IL	O
+-	O
+17RA	O
+.	O
+	O
+A	O
+15	O
+-	O
+mer	O
+linear	O
+peptide	O
+1	O
+was	O
+shown	O
+to	O
+block	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+binding	O
+with	O
+an	O
+IC50	O
+of	O
+80	O
+nM	O
+in	O
+the	O
+competition	O
+ELISA	O
+assay	O
+(	O
+Table	O
+1	O
+).	O
+	O
+This	O
+peptide	O
+was	O
+then	O
+tested	O
+in	O
+a	O
+cell	O
+-	O
+based	O
+functional	O
+assay	O
+wherein	O
+production	O
+of	O
+GRO	O
+-	O
+α	O
+in	O
+BJ	O
+human	O
+fibroblast	O
+cells	O
+was	O
+measured	O
+as	O
+a	O
+function	O
+of	O
+IL	O
+-	O
+17A	O
+stimulation	O
+using	O
+1	O
+ng	O
+/	O
+ml	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Peptide	O
+1	O
+was	O
+found	O
+to	O
+be	O
+active	O
+in	O
+this	O
+functional	O
+assay	O
+with	O
+an	O
+IC50	O
+of	O
+370	O
+nM	O
+.	O
+	O
+Optimization	O
+of	O
+IL	O
+-	O
+17A	O
+peptide	O
+inhibitors	O
+	O
+A	O
+SAR	O
+campaign	O
+was	O
+undertaken	O
+to	O
+improve	O
+the	O
+potency	O
+of	O
+peptide	O
+1	O
+.	O
+	O
+When	O
+alanine	O
+was	O
+already	O
+present	O
+(	O
+positions	O
+7	O
+and	O
+15	O
+),	O
+substitution	O
+was	O
+made	O
+with	O
+lysine	O
+(	O
+Table	O
+1	O
+,	O
+peptides	O
+3	O
+–	O
+17	O
+).	O
+	O
+Positions	O
+1	O
+,	O
+2	O
+,	O
+4	O
+,	O
+5	O
+,	O
+7	O
+,	O
+14	O
+and	O
+15	O
+were	O
+shown	O
+to	O
+be	O
+amenable	O
+to	O
+substitution	O
+without	O
+significant	O
+loss	O
+(	O
+less	O
+than	O
+3	O
+-	O
+fold	O
+)	O
+of	O
+binding	O
+affinity	O
+as	O
+measured	O
+by	O
+the	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+competition	O
+ELISA	O
+.	O
+	O
+In	O
+order	O
+to	O
+rapidly	O
+evaluate	O
+the	O
+effects	O
+of	O
+substitution	O
+of	O
+natural	O
+amino	O
+acids	O
+at	O
+tolerant	O
+positions	O
+identified	O
+by	O
+the	O
+alanine	O
+scan	O
+,	O
+the	O
+lead	O
+sequence	O
+was	O
+subjected	O
+to	O
+site	O
+-	O
+specific	O
+saturation	O
+mutagenesis	O
+using	O
+MBP	O
+.	O
+	O
+Each	O
+of	O
+the	O
+seven	O
+positions	O
+identified	O
+by	O
+the	O
+alanine	O
+scan	O
+was	O
+individually	O
+modified	O
+while	O
+keeping	O
+the	O
+rest	O
+of	O
+the	O
+sequence	O
+constant	O
+.	O
+	O
+Peptides	O
+with	O
+beneficial	O
+point	O
+mutations	O
+at	O
+positions	O
+2	O
+,	O
+5	O
+,	O
+and	O
+14	O
+were	O
+synthesized	O
+and	O
+evaluated	O
+in	O
+the	O
+competition	O
+ELISA	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Two	O
+of	O
+the	O
+changes	O
+,	O
+V2H	B-mutant
+(	O
+18	O
+)	O
+or	O
+V2T	B-mutant
+(	O
+21	O
+)	O
+displayed	O
+improved	O
+binding	O
+in	O
+the	O
+competition	O
+ELISA	O
+.	O
+	O
+Introduction	O
+of	O
+a	O
+methionine	O
+(	O
+27	O
+)	O
+or	O
+a	O
+carboxamide	O
+(	O
+28	O
+and	O
+29	O
+)	O
+at	O
+position	O
+14	O
+was	O
+shown	O
+to	O
+improve	O
+the	O
+binding	O
+affinity	O
+of	O
+the	O
+lead	O
+peptide	O
+.	O
+	O
+In	O
+general	O
+,	O
+there	O
+was	O
+good	O
+agreement	O
+between	O
+the	O
+respective	O
+binding	O
+affinities	O
+of	O
+the	O
+synthesized	O
+peptides	O
+and	O
+their	O
+MBP	O
+fusion	O
+counterparts	O
+,	O
+except	O
+for	O
+substitution	O
+of	O
+valine	O
+at	O
+position	O
+2	O
+to	O
+a	O
+tryptophan	O
+(	O
+22	O
+),	O
+which	O
+resulted	O
+in	O
+a	O
+fivefold	O
+loss	O
+of	O
+affinity	O
+,	O
+for	O
+the	O
+free	O
+peptide	O
+when	O
+compared	O
+with	O
+the	O
+MBP	O
+fusion	O
+.	O
+	O
+Combining	O
+the	O
+key	O
+amino	O
+-	O
+acid	O
+residues	O
+identified	O
+by	O
+SAR	O
+into	O
+a	O
+single	O
+peptide	O
+sequence	O
+resulted	O
+in	O
+peptide	O
+30	O
+,	O
+named	O
+high	O
+affinity	O
+peptide	O
+(	O
+HAP	O
+),	O
+that	O
+was	O
+found	O
+to	O
+inhibit	O
+IL	O
+-	O
+17A	O
+signaling	O
+in	O
+a	O
+BJ	O
+human	O
+fibroblast	O
+cell	O
+assay	O
+with	O
+an	O
+IC50	O
+of	O
+17	O
+nM	O
+,	O
+a	O
+more	O
+than	O
+20	O
+-	O
+fold	O
+improvement	O
+over	O
+the	O
+phage	O
+peptide	O
+1	O
+(	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Figure	O
+S2	O
+).	O
+	O
+We	O
+also	O
+examined	O
+the	O
+effect	O
+of	O
+removing	O
+the	O
+acetyl	O
+group	O
+at	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+HAP	O
+(	O
+which	O
+is	O
+present	O
+in	O
+all	O
+the	O
+peptides	O
+made	O
+,	O
+see	O
+Supplementary	O
+Material	O
+).	O
+	O
+The	O
+un	O
+-	O
+capped	O
+peptide	O
+(	O
+31	O
+)	O
+had	O
+an	O
+IC50	O
+of	O
+420	O
+nM	O
+in	O
+the	O
+cell	O
+-	O
+based	O
+assay	O
+.	O
+	O
+The	O
+loss	O
+of	O
+cellular	O
+activity	O
+of	O
+31	O
+was	O
+most	O
+likely	O
+due	O
+to	O
+the	O
+degradation	O
+of	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+31	O
+,	O
+since	O
+peptide	O
+31	O
+was	O
+shown	O
+to	O
+be	O
+able	O
+to	O
+bind	O
+to	O
+IL	O
+-	O
+17A	O
+with	O
+similar	O
+affinity	O
+as	O
+HAP	O
+itself	O
+.	O
+	O
+Furthermore	O
+,	O
+our	O
+previous	O
+work	O
+had	O
+reported	O
+that	O
+in	O
+antibody	O
+fusions	O
+the	O
+uncapped	O
+peptide	O
+was	O
+degraded	O
+under	O
+cell	O
+assay	O
+conditions	O
+with	O
+removal	O
+of	O
+the	O
+first	O
+1	O
+-	O
+3	O
+residues	O
+to	O
+inactive	O
+products	O
+with	O
+the	O
+same	O
+N	O
+-	O
+terminal	O
+sequences	O
+as	O
+peptides	O
+32	O
+–	O
+34	O
+.	O
+	O
+C	O
+-	O
+terminal	O
+truncations	O
+showed	O
+a	O
+more	O
+gradual	O
+reduction	O
+in	O
+activity	O
+(	O
+35	O
+–	O
+37	O
+;	O
+Table	O
+2	O
+).	O
+	O
+After	O
+deletion	O
+of	O
+three	O
+amino	O
+acids	O
+from	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+(	O
+37	O
+),	O
+the	O
+peptide	O
+is	O
+no	O
+longer	O
+active	O
+.	O
+	O
+We	O
+reasoned	O
+that	O
+since	O
+the	O
+IL	O
+-	O
+17A	O
+protein	O
+is	O
+almost	O
+exclusively	O
+present	O
+in	O
+a	O
+dimeric	O
+form	O
+,	O
+dimerizing	O
+the	O
+IL	O
+-	O
+17A	O
+binding	O
+peptides	O
+could	O
+result	O
+in	O
+an	O
+improvement	O
+in	O
+binding	O
+affinity	O
+and	O
+inhibitory	O
+activity	O
+.	O
+	O
+Homodimers	O
+of	O
+HAP	O
+were	O
+made	O
+through	O
+attachment	O
+of	O
+polyethylene	O
+glycol	O
+(	O
+PEG	O
+)	O
+spacers	O
+of	O
+different	O
+lengths	O
+at	O
+amino	O
+acids	O
+4	O
+,	O
+7	O
+and	O
+14	O
+,	O
+as	O
+these	O
+positions	O
+were	O
+identified	O
+in	O
+the	O
+alanine	O
+scan	O
+analysis	O
+as	O
+not	O
+contributing	O
+significantly	O
+to	O
+the	O
+activity	O
+,	O
+and	O
+at	O
+each	O
+N	O
+-	O
+terminus	O
+(	O
+Supplementary	O
+Table	O
+S2	O
+).	O
+	O
+Due	O
+to	O
+the	O
+high	O
+reactivity	O
+of	O
+the	O
+pentafluoroester	O
+(	O
+PFP	O
+)	O
+group	O
+used	O
+as	O
+the	O
+activating	O
+group	O
+in	O
+the	O
+PEG	O
+,	O
+the	O
+histidine	O
+at	O
+position	O
+2	O
+and	O
+the	O
+lysine	O
+at	O
+position	O
+15	O
+were	O
+replaced	O
+with	O
+threonine	O
+and	O
+dimethyllysine	O
+respectively	O
+to	O
+prevent	O
+formation	O
+of	O
+side	O
+products	O
+,	O
+which	O
+resulted	O
+in	O
+peptide	O
+38	O
+that	O
+was	O
+comparable	O
+in	O
+activity	O
+with	O
+HAP	O
+.	O
+	O
+This	O
+exercise	O
+revealed	O
+that	O
+several	O
+dimeric	O
+peptides	O
+with	O
+the	O
+longer	O
+PEG21	O
+spacer	O
+were	O
+significantly	O
+more	O
+potent	O
+than	O
+the	O
+monomer	O
+peptide	O
+in	O
+the	O
+cell	O
+-	O
+based	O
+assay	O
+(	O
+Supplementary	O
+Table	O
+S2	O
+).	O
+	O
+The	O
+species	O
+cross	O
+-	O
+reactivity	O
+of	O
+the	O
+dimeric	O
+peptide	O
+45	O
+and	O
+HAP	O
+were	O
+assessed	O
+in	O
+a	O
+murine	O
+functional	O
+cell	O
+assay	O
+using	O
+15	O
+ng	O
+/	O
+ml	O
+murine	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Peptide	O
+45	O
+blocked	O
+the	O
+receptor	O
+binding	O
+of	O
+murine	O
+IL	O
+-	O
+17A	O
+although	O
+with	O
+potency	O
+two	O
+orders	O
+of	O
+magnitude	O
+weaker	O
+than	O
+that	O
+observed	O
+against	O
+human	O
+IL	O
+-	O
+17A	O
+(	O
+IC50	O
+=	O
+41	O
+nM	O
+vs	O
+IC50	O
+=	O
+0	O
+.	O
+1	O
+nM	O
+,	O
+respectively	O
+).	O
+	O
+The	O
+monomer	O
+HAP	O
+was	O
+much	O
+weaker	O
+(	O
+IC50	O
+>	O
+1	O
+μM	O
+)	O
+in	O
+inhibiting	O
+murine	O
+IL	O
+-	O
+17A	O
+signaling	O
+(	O
+Supplementary	O
+Figure	O
+S4	O
+).	O
+	O
+Although	O
+the	O
+dimeric	O
+peptide	O
+45	O
+is	O
+much	O
+more	O
+potent	O
+than	O
+HAP	O
+in	O
+the	O
+cell	O
+-	O
+based	O
+assay	O
+,	O
+in	O
+subsequent	O
+studies	O
+we	O
+decided	O
+to	O
+focus	O
+our	O
+efforts	O
+solely	O
+on	O
+characterizations	O
+of	O
+the	O
+monomeric	O
+peptide	O
+HAP	O
+in	O
+hopes	O
+to	O
+identify	O
+smaller	O
+peptide	O
+inhibitors	O
+containing	O
+the	O
+best	O
+minimal	O
+functional	O
+group	O
+.	O
+	O
+HAP	O
+binds	O
+to	O
+immobilized	O
+human	O
+IL	O
+-	O
+17A	O
+homodimer	O
+tightly	O
+(	O
+Table	O
+3	O
+).	O
+	O
+It	O
+has	O
+slightly	O
+weaker	O
+affinity	O
+for	O
+human	O
+IL	O
+-	O
+17A	O
+/	O
+F	O
+heterodimer	O
+and	O
+>	O
+10	O
+fold	O
+weaker	O
+affinity	O
+for	O
+mouse	O
+IL	O
+-	O
+17A	O
+(	O
+Table	O
+3	O
+).	O
+	O
+HAP	O
+does	O
+not	O
+show	O
+significant	O
+binding	O
+to	O
+immobilized	O
+human	O
+IL	O
+-	O
+17F	O
+homodimer	O
+or	O
+IL	O
+-	O
+17RA	O
+at	O
+concentrations	O
+up	O
+to	O
+100	O
+nM	O
+.	O
+	O
+Additionally	O
+,	O
+we	O
+investigated	O
+the	O
+antagonism	O
+of	O
+the	O
+human	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+interaction	O
+by	O
+HAP	O
+using	O
+orthogonal	O
+methods	O
+including	O
+SPR	O
+and	O
+Förster	O
+resonance	O
+energy	O
+transfer	O
+(	O
+FRET	O
+)	O
+competition	O
+assays	O
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+C	O
+).	O
+	O
+In	O
+both	O
+assays	O
+,	O
+incubation	O
+of	O
+IL	O
+-	O
+17A	O
+with	O
+HAP	O
+effectively	O
+blocks	O
+the	O
+binding	O
+of	O
+IL	O
+-	O
+17A	O
+to	O
+immobilized	O
+IL	O
+-	O
+17RA	O
+with	O
+similar	O
+sub	O
+-	O
+nM	O
+IC50	O
+(	O
+Table	O
+3	O
+).	O
+	O
+While	O
+either	O
+IL	O
+-	O
+17A	O
+or	O
+TNF	O
+-	O
+α	O
+alone	O
+can	O
+stimulate	O
+the	O
+release	O
+of	O
+multiple	O
+inflammatory	O
+cytokines	O
+,	O
+when	O
+acting	O
+together	O
+they	O
+can	O
+synergistically	O
+enhance	O
+each	O
+other	O
+’	O
+s	O
+effects	O
+(	O
+Supplementary	O
+Figure	O
+S5	O
+).	O
+	O
+These	O
+integrative	O
+responses	O
+to	O
+IL	O
+-	O
+17A	O
+and	O
+TNF	O
+-	O
+α	O
+in	O
+human	O
+keratinocytes	O
+have	O
+been	O
+reported	O
+to	O
+account	O
+for	O
+key	O
+inflammatory	O
+pathogenic	O
+circuits	O
+in	O
+psoriasis	O
+.	O
+	O
+Thus	O
+,	O
+we	O
+chose	O
+to	O
+study	O
+HAP	O
+’	O
+s	O
+efficacy	O
+in	O
+blocking	O
+the	O
+production	O
+of	O
+IL	O
+-	O
+8	O
+,	O
+IL	O
+-	O
+6	O
+and	O
+CCL	O
+-	O
+20	O
+by	O
+primary	O
+human	O
+keratinocytes	O
+stimulated	O
+by	O
+IL	O
+-	O
+17A	O
+in	O
+the	O
+presence	O
+of	O
+TNF	O
+-	O
+α	O
+,	O
+an	O
+assay	O
+which	O
+may	O
+be	O
+more	O
+disease	O
+-	O
+relevant	O
+.	O
+	O
+HAP	O
+inhibits	O
+the	O
+production	O
+of	O
+all	O
+three	O
+cytokines	O
+in	O
+a	O
+dose	O
+-	O
+dependent	O
+fashion	O
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+	O
+Significantly	O
+,	O
+the	O
+baseline	O
+levels	O
+of	O
+IL	O
+-	O
+8	O
+,	O
+IL	O
+-	O
+6	O
+and	O
+CCL	O
+-	O
+20	O
+stimulated	O
+by	O
+TNF	O
+-	O
+α	O
+alone	O
+are	O
+not	O
+inhibited	O
+by	O
+HAP	O
+,	O
+further	O
+indicating	O
+the	O
+selectivity	O
+of	O
+HAP	O
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+	O
+Such	O
+pharmacological	O
+selectivity	O
+may	O
+be	O
+important	O
+to	O
+suppress	O
+inflammatory	O
+pathogenic	O
+circuits	O
+in	O
+psoriasis	O
+,	O
+while	O
+sparing	O
+the	O
+anti	O
+-	O
+infectious	O
+immune	O
+responses	O
+produced	O
+by	O
+TNF	O
+-	O
+α	O
+.	O
+	O
+As	O
+a	O
+reference	O
+,	O
+a	O
+commercial	O
+anti	O
+-	O
+IL	O
+-	O
+17A	O
+antibody	O
+(	O
+R	O
+&	O
+D	O
+Systems	O
+)	O
+inhibits	O
+the	O
+production	O
+of	O
+IL	O
+-	O
+8	O
+with	O
+an	O
+IC50	O
+of	O
+13	O
+(±	O
+6	O
+)	O
+nM	O
+(	O
+N	O
+=	O
+3	O
+).	O
+	O
+Indeed	O
+,	O
+the	O
+IC50	O
+was	O
+14	O
+(±	O
+9	O
+)	O
+nM	O
+(	O
+N	O
+=	O
+12	O
+)	O
+for	O
+HAP	O
+inhibition	O
+of	O
+IL	O
+-	O
+8	O
+production	O
+when	O
+only	O
+5	O
+ng	O
+/	O
+ml	O
+IL	O
+-	O
+17A	O
+was	O
+used	O
+in	O
+this	O
+assay	O
+.	O
+	O
+Similar	O
+to	O
+keratinocytes	O
+assay	O
+results	O
+,	O
+while	O
+HAP	O
+inhibits	O
+IL	O
+-	O
+17A	O
+stimulated	O
+IL	O
+-	O
+6	O
+production	O
+by	O
+BJ	O
+human	O
+fibroblast	O
+potently	O
+(	O
+IC50	O
+of	O
+17	O
+nM	O
+),	O
+it	O
+does	O
+not	O
+inhibit	O
+TNF	O
+-	O
+α	O
+stimulated	O
+IL	O
+-	O
+6	O
+production	O
+at	O
+concentrations	O
+up	O
+to	O
+10	O
+μM	O
+(	O
+Supplementary	O
+Figure	O
+S2	O
+).	O
+	O
+Extensive	O
+crystallization	O
+trials	O
+,	O
+either	O
+by	O
+co	O
+-	O
+crystallization	O
+or	O
+by	O
+soaking	O
+HAP	O
+into	O
+preformed	O
+apo	O
+IL	O
+-	O
+17A	O
+crystals	O
+,	O
+failed	O
+to	O
+lead	O
+to	O
+an	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+complex	O
+crystals	O
+.	O
+	O
+We	O
+theorized	O
+that	O
+HAP	O
+binding	O
+induced	O
+large	O
+conformational	O
+changes	O
+in	O
+IL	O
+-	O
+17A	O
+that	O
+led	O
+to	O
+the	O
+difficulty	O
+of	O
+getting	O
+an	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+binary	O
+complex	O
+crystal	O
+.	O
+	O
+We	O
+hypothesized	O
+that	O
+HAP	O
+may	O
+target	O
+the	O
+N	O
+-	O
+terminal	O
+of	O
+IL	O
+-	O
+17A	O
+which	O
+is	O
+known	O
+to	O
+be	O
+more	O
+flexible	O
+than	O
+its	O
+C	O
+-	O
+terminal	O
+and	O
+conformational	O
+changes	O
+needed	O
+for	O
+HAP	O
+binding	O
+may	O
+be	O
+more	O
+likely	O
+there	O
+.	O
+	O
+We	O
+designed	O
+an	O
+antibody	O
+Fab	O
+known	O
+to	O
+target	O
+the	O
+C	O
+-	O
+terminal	O
+half	O
+of	O
+IL	O
+-	O
+17A	O
+based	O
+on	O
+a	O
+published	O
+IL	O
+-	O
+17A	O
+/	O
+Fab	O
+complex	O
+crystal	O
+structure	O
+,	O
+and	O
+produced	O
+it	O
+in	O
+HEK293	O
+cells	O
+.	O
+	O
+In	O
+an	O
+SPR	O
+assay	O
+HAP	O
+and	O
+this	O
+Fab	O
+were	O
+able	O
+to	O
+co	O
+-	O
+bind	O
+IL	O
+-	O
+17A	O
+without	O
+large	O
+changes	O
+in	O
+their	O
+binding	O
+affinities	O
+and	O
+kinetics	O
+,	O
+confirming	O
+our	O
+hypothesis	O
+(	O
+Supplementary	O
+Figure	O
+S6	O
+).	O
+	O
+These	O
+were	O
+,	O
+respectively	O
+,	O
+a	O
+presumably	O
+more	O
+homogeneous	O
+form	O
+of	O
+IL	O
+-	O
+17A	O
+that	O
+lacked	O
+the	O
+disordered	O
+N	O
+-	O
+terminal	O
+peptide	O
+and	O
+a	O
+full	O
+-	O
+length	O
+form	O
+of	O
+the	O
+cytokine	O
+with	O
+a	O
+full	O
+complement	O
+of	O
+disulfide	O
+bonds	O
+.	O
+	O
+Both	O
+complexes	O
+crystallized	O
+in	O
+the	O
+space	O
+group	O
+of	O
+P321	O
+,	O
+with	O
+half	O
+the	O
+complex	O
+(	O
+1	O
+Fab	O
+/	O
+1	O
+IL	O
+-	O
+17A	O
+monomer	O
+/	O
+1	O
+HAP	O
+)	O
+in	O
+the	O
+asymmetric	O
+unit	O
+.	O
+	O
+The	O
+intact	O
+complex	O
+can	O
+be	O
+generated	O
+by	O
+applying	O
+crystallographic	O
+2	O
+-	O
+fold	O
+symmetry	O
+.	O
+	O
+Electron	O
+densities	O
+for	O
+HAP	O
+residues	O
+Ile1	O
+-	O
+Asn14	O
+were	O
+readily	O
+interpretable	O
+with	O
+the	O
+exception	O
+of	O
+Lys15	O
+,	O
+which	O
+is	O
+disordered	O
+.	O
+	O
+When	O
+considering	O
+the	O
+protein	O
+,	O
+the	O
+complex	O
+structure	O
+containing	O
+the	O
+full	O
+length	O
+IL	O
+-	O
+17A	O
+is	O
+identical	O
+to	O
+that	O
+of	O
+the	O
+truncated	O
+IL	O
+-	O
+17A	O
+,	O
+with	O
+the	O
+exception	O
+of	O
+Cys106	O
+(	O
+Ser106	O
+in	O
+the	O
+truncated	O
+IL	O
+-	O
+17A	O
+),	O
+which	O
+is	O
+disordered	O
+.	O
+	O
+Cys106	O
+is	O
+covalently	O
+linked	O
+to	O
+Cys10	O
+that	O
+resides	O
+in	O
+the	O
+disordered	O
+N	O
+-	O
+terminal	O
+peptide	O
+in	O
+the	O
+full	O
+length	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Overall	O
+structure	O
+of	O
+Fab	O
+/	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+complex	O
+	O
+In	O
+a	O
+similar	O
+manner	O
+to	O
+the	O
+published	O
+structure	O
+of	O
+Fab	O
+/	O
+IL	O
+-	O
+17A	O
+complex	O
+,	O
+two	O
+Fab	O
+molecules	O
+bind	O
+symmetrically	O
+to	O
+the	O
+C	O
+-	O
+terminal	O
+of	O
+the	O
+cytokine	O
+dimer	O
+,	O
+interacting	O
+with	O
+epitopes	O
+from	O
+both	O
+monomers	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+	O
+Based	O
+on	O
+disclosed	O
+epitopes	O
+of	O
+Secukinumab	O
+and	O
+Ixekizumab	O
+,	O
+HAP	O
+binds	O
+to	O
+IL	O
+-	O
+17A	O
+at	O
+an	O
+area	O
+that	O
+is	O
+also	O
+different	O
+from	O
+those	O
+of	O
+those	O
+two	O
+antibodies	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+5	O
+residues	O
+of	O
+HAP	O
+,	O
+1IHVTI	O
+,	O
+form	O
+an	O
+amphipathic	O
+β	O
+-	O
+strand	O
+that	O
+inserts	O
+between	O
+β	O
+-	O
+strand	O
+4	O
+of	O
+one	O
+IL	O
+-	O
+17A	O
+monomer	O
+and	O
+β	O
+-	O
+strand	O
+0	O
+(	O
+the	O
+first	O
+ordered	O
+peptide	O
+of	O
+IL	O
+-	O
+17A	O
+)	O
+of	O
+the	O
+second	O
+monomer	O
+.	O
+	O
+This	O
+β	O
+-	O
+strand	O
+is	O
+parallel	O
+to	O
+both	O
+strands	O
+0	O
+and	O
+4	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+	O
+Strands	O
+0	O
+of	O
+two	O
+IL	O
+-	O
+17A	O
+monomer	O
+are	O
+antiparallel	O
+,	O
+as	O
+appeared	O
+in	O
+other	O
+IL	O
+-	O
+17A	O
+structures	O
+.	O
+	O
+As	O
+a	O
+comparison	O
+,	O
+an	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+complex	O
+structure	O
+(	O
+PDB	O
+code	O
+4HSA	O
+)	O
+is	O
+also	O
+shown	O
+with	O
+IL	O
+-	O
+17A	O
+in	O
+the	O
+same	O
+orientation	O
+(	O
+Fig	O
+.	O
+2C	O
+).	O
+	O
+IL	O
+-	O
+17RA	O
+binds	O
+IL	O
+-	O
+17A	O
+at	O
+three	O
+regions	O
+on	O
+the	O
+IL	O
+-	O
+17A	O
+homodimer	O
+.	O
+	O
+HAP	O
+binds	O
+IL	O
+-	O
+17A	O
+at	O
+region	O
+I	O
+.	O
+Region	O
+I	O
+is	O
+formed	O
+by	O
+residues	O
+at	O
+the	O
+ends	O
+of	O
+β	O
+strands	O
+0	O
+and	O
+4	O
+,	O
+and	O
+from	O
+loops	O
+1	O
+–	O
+2	O
+and	O
+3	O
+–	O
+4	O
+of	O
+IL	O
+-	O
+17A	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+The	O
+most	O
+significant	O
+interactions	O
+between	O
+the	O
+α	O
+helix	O
+of	O
+HAP	O
+and	O
+IL	O
+-	O
+17A	O
+involve	O
+Trp12	O
+of	O
+HAP	O
+,	O
+which	O
+binds	O
+in	O
+a	O
+hydrophobic	O
+pocket	O
+in	O
+IL	O
+-	O
+17A	O
+formed	O
+by	O
+the	O
+side	O
+chains	O
+of	O
+Phe110	O
+,	O
+Tyr62	O
+,	O
+Pro59	O
+and	O
+the	O
+hydrophobic	O
+portion	O
+of	O
+the	O
+Arg101	O
+side	O
+chain	O
+(	O
+Fig	O
+.	O
+3A	O
+).	O
+	O
+The	O
+Trp12	O
+side	O
+chain	O
+of	O
+HAP	O
+donates	O
+a	O
+hydrogen	O
+bond	O
+to	O
+the	O
+main	O
+chain	O
+oxygen	O
+of	O
+Pro69	O
+of	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+The	O
+positively	O
+charged	O
+Arg101	O
+side	O
+chain	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+engages	O
+in	O
+a	O
+charge	O
+-	O
+helix	O
+dipole	O
+interaction	O
+with	O
+the	O
+main	O
+chain	O
+oxygen	O
+of	O
+Trp12	O
+.	O
+	O
+Additionally	O
+,	O
+Leu9	O
+and	O
+Ile13	O
+of	O
+the	O
+HAP	O
+have	O
+hydrophobic	O
+interactions	O
+with	O
+IL	O
+-	O
+17A	O
+,	O
+and	O
+the	O
+Asp8	O
+side	O
+chain	O
+has	O
+hydrogen	O
+bond	O
+and	O
+ion	O
+pair	O
+interactions	O
+with	O
+Tyr62	O
+and	O
+Lys114	O
+of	O
+IL	O
+-	O
+17A	O
+,	O
+respectively	O
+.	O
+	O
+In	O
+region	O
+I	O
+,	O
+an	O
+IL	O
+-	O
+17RA	O
+peptide	O
+interacts	O
+with	O
+IL	O
+-	O
+17A	O
+in	O
+a	O
+very	O
+similar	O
+fashion	O
+to	O
+the	O
+α	O
+-	O
+helix	O
+of	O
+HAP	O
+.	O
+	O
+The	O
+IL	O
+-	O
+17RA	O
+peptide	O
+has	O
+sequences	O
+of	O
+27LDDSWI	O
+,	O
+and	O
+part	O
+of	O
+the	O
+peptide	O
+is	O
+also	O
+α	O
+-	O
+helical	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+	O
+Leu7	O
+,	O
+Trp31	O
+and	O
+Ile32	O
+of	O
+IL	O
+-	O
+17RA	O
+interact	O
+very	O
+similarly	O
+with	O
+the	O
+same	O
+residues	O
+of	O
+IL	O
+-	O
+17A	O
+as	O
+Leu9	O
+,	O
+Trp12	O
+and	O
+Ile13	O
+of	O
+HAP	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+	O
+In	O
+this	O
+sense	O
+,	O
+the	O
+α	O
+-	O
+helix	O
+of	O
+HAP	O
+with	O
+a	O
+sequence	O
+of	O
+9LWDWI	O
+is	O
+a	O
+good	O
+mimetic	O
+of	O
+the	O
+27LDDSWI	O
+peptide	O
+of	O
+IL	O
+-	O
+17RA	O
+.	O
+	O
+The	O
+β	O
+-	O
+strand	O
+of	O
+HAP	O
+has	O
+no	O
+equivalent	O
+in	O
+IL	O
+-	O
+17RA	O
+.	O
+	O
+The	O
+amphipathic	O
+β	O
+-	O
+strand	O
+of	O
+HAP	O
+orients	O
+the	O
+hydrophilic	O
+side	O
+chains	O
+of	O
+His2	O
+and	O
+Thr4	O
+outwards	O
+,	O
+and	O
+the	O
+hydrophobic	O
+side	O
+chains	O
+of	O
+Ile1	O
+,	O
+Val3	O
+and	O
+Ile5	O
+inward	O
+(	O
+Fig	O
+.	O
+3A	O
+).	O
+	O
+β	O
+-	O
+strand	O
+0	O
+in	O
+IL	O
+-	O
+17A	O
+is	O
+also	O
+amphipathic	O
+with	O
+the	O
+sequence	O
+of	O
+21TVMVNLNI	O
+.	O
+	O
+In	O
+all	O
+IL	O
+-	O
+17A	O
+structures	O
+obtained	O
+to	O
+date	O
+,	O
+β	O
+-	O
+strand	O
+0	O
+orients	O
+the	O
+hydrophilic	O
+side	O
+chains	O
+of	O
+Thr21	O
+,	O
+Asn25	O
+and	O
+Asn27	O
+outward	O
+,	O
+and	O
+the	O
+hydrophobic	O
+side	O
+chains	O
+of	O
+Val22	O
+,	O
+Val24	O
+,	O
+Leu26	O
+and	O
+Ile28	O
+inward	O
+.	O
+	O
+The	O
+binding	O
+pocket	O
+occupied	O
+by	O
+either	O
+Trp12	O
+of	O
+HAP	O
+or	O
+Trp31	O
+of	O
+IL	O
+-	O
+17RA	O
+is	O
+not	O
+formed	O
+in	O
+the	O
+apo	O
+IL	O
+-	O
+17A	O
+structure	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+	O
+Particularly	O
+for	O
+HAP	O
+,	O
+β	O
+-	O
+strands	O
+0	O
+have	O
+to	O
+shift	O
+out	O
+of	O
+the	O
+hydrophobic	O
+cleft	O
+formed	O
+by	O
+the	O
+main	O
+body	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+by	O
+as	O
+much	O
+as	O
+10	O
+Å	O
+between	O
+Cα	O
+atoms	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+	O
+Disruptions	O
+of	O
+the	O
+apo	O
+IL	O
+-	O
+17A	O
+structure	O
+by	O
+HAP	O
+binding	O
+are	O
+apparently	O
+compensated	O
+for	O
+by	O
+formation	O
+of	O
+the	O
+new	O
+interactions	O
+that	O
+involve	O
+almost	O
+the	O
+entire	O
+HAP	O
+molecule	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+	O
+The	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+complex	O
+structure	O
+obtained	O
+is	O
+very	O
+consistent	O
+with	O
+the	O
+observed	O
+SAR	O
+of	O
+our	O
+identified	O
+peptide	O
+inhibitors	O
+,	O
+explaining	O
+well	O
+how	O
+the	O
+evolution	O
+of	O
+the	O
+initial	O
+phage	O
+peptide	O
+1	O
+to	O
+HAP	O
+and	O
+45	O
+improved	O
+its	O
+potency	O
+(	O
+Supplementary	O
+Figure	O
+S7	O
+).	O
+	O
+The	O
+important	O
+interactions	O
+involving	O
+Trp12	O
+of	O
+HAP	O
+explain	O
+the	O
+>	O
+90	O
+times	O
+drop	O
+in	O
+potency	O
+of	O
+the	O
+W12A	B-mutant
+variant	O
+(	O
+6	O
+vs	O
+1	O
+,	O
+Table	O
+1	O
+).	O
+	O
+The	O
+amphipathic	O
+nature	O
+of	O
+the	O
+HAP	O
+β	O
+-	O
+strand	O
+explains	O
+the	O
+preference	O
+of	O
+the	O
+hydrophilic	O
+residues	O
+at	O
+the	O
+2	O
+and	O
+4	O
+positions	O
+of	O
+peptides	O
+(	O
+14	O
+,	O
+18	O
+,	O
+19	O
+,	O
+21	O
+and	O
+23	O
+vs	O
+1	O
+and	O
+22	O
+,	O
+Table	O
+1	O
+).	O
+	O
+All	O
+N	O
+-	O
+terminal	O
+residues	O
+of	O
+HAP	O
+are	O
+part	O
+of	O
+the	O
+β	O
+-	O
+sheet	O
+with	O
+β	O
+-	O
+stands	O
+0	O
+and	O
+4	O
+of	O
+IL	O
+-	O
+17A	O
+,	O
+which	O
+explains	O
+why	O
+removal	O
+of	O
+the	O
+first	O
+1	O
+–	O
+3	O
+residues	O
+completely	O
+abolishes	O
+the	O
+ability	O
+of	O
+HAP	O
+to	O
+block	O
+IL	O
+-	O
+17A	O
+cell	O
+signaling	O
+(	O
+31	O
+,	O
+32	O
+and	O
+33	O
+,	O
+Table	O
+2	O
+).	O
+	O
+Each	O
+peptide	O
+monomer	O
+in	O
+45	O
+may	O
+not	O
+necessarily	O
+be	O
+more	O
+potent	O
+than	O
+HAP	O
+,	O
+but	O
+two	O
+monomer	O
+peptides	O
+within	O
+the	O
+same	O
+molecule	O
+that	O
+can	O
+simultaneously	O
+bind	O
+to	O
+IL	O
+-	O
+17A	O
+can	O
+greatly	O
+improve	O
+its	O
+potency	O
+due	O
+to	O
+avidity	O
+effects	O
+.	O
+	O
+HAP	O
+targets	O
+region	O
+I	O
+of	O
+IL	O
+-	O
+17A	O
+,	O
+an	O
+area	O
+that	O
+has	O
+the	O
+least	O
+sequence	O
+conservation	O
+in	O
+IL	O
+-	O
+17	O
+cytokines	O
+.	O
+	O
+This	O
+lack	O
+of	O
+sequence	O
+conservation	O
+in	O
+the	O
+HAP	O
+binding	O
+site	O
+explains	O
+the	O
+observed	O
+specificity	O
+of	O
+HAP	O
+binding	O
+to	O
+human	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+This	O
+Phe	O
+-	O
+Phe	O
+motif	O
+is	O
+missing	O
+in	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Sequence	O
+alignments	O
+between	O
+human	O
+and	O
+mouse	O
+IL	O
+-	O
+17A	O
+indicated	O
+that	O
+among	O
+IL	O
+-	O
+17A	O
+residues	O
+that	O
+interacting	O
+with	O
+HAP	O
+,	O
+majority	O
+differences	O
+occur	O
+in	O
+strand	O
+0	O
+of	O
+IL	O
+-	O
+17A	O
+which	O
+interacts	O
+with	O
+the	O
+N	O
+-	O
+terminal	O
+β	O
+-	O
+strand	O
+of	O
+HAP	O
+.	O
+	O
+In	O
+human	O
+IL	O
+-	O
+17A	O
+the	O
+sequences	O
+are	O
+21TVMVNLNI	O
+,	O
+and	O
+in	O
+mouse	O
+they	O
+are	O
+21NVKVNLKV	O
+.	O
+	O
+Using	O
+a	O
+combination	O
+of	O
+phage	O
+display	O
+and	O
+SAR	O
+we	O
+have	O
+discovered	O
+novel	O
+peptides	O
+that	O
+are	O
+IL	O
+-	O
+17A	O
+antagonists	O
+.	O
+	O
+One	O
+of	O
+those	O
+peptides	O
+,	O
+HAP	O
+,	O
+also	O
+shows	O
+activity	O
+in	O
+inhibiting	O
+the	O
+production	O
+of	O
+multiple	O
+inflammatory	O
+cytokines	O
+by	O
+primary	O
+human	O
+keratinocytes	O
+stimulated	O
+by	O
+IL	O
+-	O
+17A	O
+and	O
+TNF	O
+-	O
+α	O
+,	O
+a	O
+disease	O
+relevant	O
+-	O
+model	O
+.	O
+	O
+With	O
+two	O
+HAP	O
+molecules	O
+covering	O
+both	O
+faces	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+dimer	O
+,	O
+HAP	O
+can	O
+block	O
+IL	O
+-	O
+17RA	O
+approaching	O
+from	O
+either	O
+face	O
+.	O
+	O
+To	O
+form	O
+the	O
+1	O
+:	O
+2	O
+complex	O
+observed	O
+in	O
+crystal	O
+structure	O
+,	O
+it	O
+is	O
+important	O
+that	O
+there	O
+is	O
+no	O
+strong	O
+negative	O
+cooperativity	O
+in	O
+the	O
+binding	O
+of	O
+two	O
+HAP	O
+molecules	O
+.	O
+	O
+In	O
+fact	O
+,	O
+in	O
+native	O
+electrospray	O
+ionization	O
+mass	O
+spectrometry	O
+analysis	O
+only	O
+1	O
+:	O
+2	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+complex	O
+was	O
+observed	O
+even	O
+when	O
+IL	O
+-	O
+17A	O
+was	O
+in	O
+excess	O
+(	O
+Supplementary	O
+Figure	O
+S8	O
+),	O
+indicating	O
+a	O
+positive	O
+binding	O
+cooperativity	O
+that	O
+favors	O
+inhibition	O
+of	O
+IL	O
+-	O
+17RA	O
+binding	O
+by	O
+HAP	O
+.	O
+	O
+Due	O
+to	O
+the	O
+discontinuous	O
+nature	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+binding	O
+interface	O
+,	O
+it	O
+is	O
+classified	O
+as	O
+having	O
+tertiary	O
+structural	O
+epitopes	O
+on	O
+both	O
+binding	O
+partners	O
+,	O
+and	O
+is	O
+therefore	O
+hard	O
+to	O
+target	O
+using	O
+small	O
+molecules	O
+.	O
+	O
+Our	O
+studies	O
+of	O
+HAP	O
+demonstrated	O
+an	O
+uncommon	O
+mode	O
+of	O
+action	O
+for	O
+a	O
+peptide	O
+in	O
+inhibiting	O
+such	O
+a	O
+difficult	O
+protein	O
+-	O
+protein	O
+interaction	O
+target	O
+,	O
+and	O
+suggest	O
+further	O
+possible	O
+improvements	O
+in	O
+its	O
+binding	O
+potency	O
+.	O
+	O
+Homo	O
+-	O
+dimerization	O
+of	O
+HAP	O
+(	O
+45	O
+)	O
+achieved	O
+sub	O
+-	O
+nanomolar	O
+potency	O
+against	O
+human	O
+IL	O
+-	O
+17A	O
+in	O
+cell	O
+assay	O
+.	O
+	O
+In	O
+the	O
+crystal	O
+structure	O
+,	O
+the	O
+distance	O
+between	O
+the	O
+carbonyl	O
+of	O
+Asn14	O
+of	O
+one	O
+HAP	O
+molecule	O
+and	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+the	O
+second	O
+is	O
+only	O
+15	O
+.	O
+7	O
+Å	O
+,	O
+suggesting	O
+the	O
+potential	O
+for	O
+more	O
+potent	O
+dimeric	O
+peptides	O
+to	O
+be	O
+designed	O
+by	O
+using	O
+linkers	O
+of	O
+different	O
+lengths	O
+at	O
+different	O
+positions	O
+.	O
+	O
+Another	O
+direction	O
+of	O
+improving	O
+HAP	O
+is	O
+by	O
+reducing	O
+its	O
+size	O
+.	O
+	O
+In	O
+summary	O
+,	O
+these	O
+peptide	O
+-	O
+based	O
+anti	O
+-	O
+IL	O
+-	O
+17A	O
+modalities	O
+could	O
+be	O
+further	O
+developed	O
+as	O
+alternative	O
+therapeutic	O
+options	O
+to	O
+the	O
+reported	O
+monoclonal	O
+antibodies	O
+.	O
+	O
+We	O
+are	O
+also	O
+very	O
+interested	O
+in	O
+finding	O
+non	O
+-	O
+peptidic	O
+small	O
+molecule	O
+IL	O
+-	O
+17A	O
+antagonists	O
+,	O
+and	O
+HAP	O
+can	O
+be	O
+used	O
+as	O
+an	O
+excellent	O
+tool	O
+peptide	O
+.	O
+	O
+The	O
+strategy	O
+utilized	O
+in	O
+generating	O
+the	O
+complex	O
+structures	O
+of	O
+HAP	O
+may	O
+also	O
+be	O
+useful	O
+for	O
+enabling	O
+structure	O
+based	O
+design	O
+of	O
+some	O
+known	O
+small	O
+molecule	O
+IL	O
+-	O
+17A	O
+antagonists	O
+.	O
+	O
+Binding	O
+of	O
+HAP	O
+to	O
+IL	O
+-	O
+17A	O
+and	O
+inhibition	O
+of	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+are	O
+measured	O
+by	O
+SPR	O
+,	O
+FRET	O
+and	O
+cell	O
+-	O
+based	O
+assays	O
+.	O
+	O
+(	O
+A	O
+)	O
+Typical	O
+SPR	O
+sensorgrams	O
+(	O
+black	O
+)	O
+of	O
+HAP	O
+at	O
+indicated	O
+concentrations	O
+binding	O
+to	O
+biotinylated	O
+human	O
+IL	O
+-	O
+17A	O
+immobilized	O
+on	O
+a	O
+streptavidin	O
+chip	O
+surface	O
+,	O
+fitted	O
+with	O
+single	O
+site	O
+binding	O
+model	O
+curves	O
+(	O
+red	O
+).	O
+	O
+Kinetic	O
+parameters	O
+(	O
+ka	O
+,	O
+kd	O
+)	O
+were	O
+obtained	O
+by	O
+a	O
+global	O
+fit	O
+using	O
+three	O
+concentrations	O
+in	O
+triplicate	O
+.	O
+	O
+Data	O
+are	O
+mean	O
+and	O
+error	O
+bars	O
+of	O
++/−	O
+standard	O
+deviation	O
+of	O
+three	O
+measurements	O
+.	O
+(	O
+C	O
+)	O
+Inhibition	O
+of	O
+IL	O
+-	O
+17A	O
+and	O
+IL	O
+-	O
+17RA	O
+binding	O
+by	O
+HAP	O
+measured	O
+by	O
+FRET	O
+assay	O
+.	O
+	O
+Data	O
+are	O
+mean	O
+and	O
+error	O
+bars	O
+of	O
++/−	O
+standard	O
+deviation	O
+from	O
+299	O
+experiments	O
+,	O
+each	O
+performed	O
+in	O
+duplicate	O
+.	O
+(	O
+D	O
+)	O
+Example	O
+of	O
+HAP	O
+selective	O
+inhibition	O
+of	O
+the	O
+production	O
+of	O
+IL	O
+-	O
+8	O
+(	O
+triangles	O
+),	O
+IL	O
+-	O
+6	O
+(	O
+squares	O
+)	O
+and	O
+CCL	O
+-	O
+20	O
+(	O
+circles	O
+)	O
+by	O
+primary	O
+human	O
+keratinocyte	O
+cells	O
+synergistically	O
+stimulated	O
+by	O
+100	O
+ng	O
+/	O
+ml	O
+IL	O
+-	O
+17A	O
+and	O
+10	O
+ng	O
+/	O
+ml	O
+TNF	O
+-	O
+α	O
+.	O
+	O
+HAP	O
+does	O
+not	O
+inhibit	O
+the	O
+baseline	O
+production	O
+of	O
+IL	O
+-	O
+6	O
+,	O
+IL	O
+-	O
+8	O
+and	O
+CCL	O
+-	O
+20	O
+stimulated	O
+by	O
+10	O
+ng	O
+/	O
+ml	O
+TNF	O
+-	O
+α	O
+alone	O
+(	O
+gray	O
+lines	O
+and	O
+symbols	O
+).	O
+	O
+Two	O
+HAP	O
+molecules	O
+are	O
+colored	O
+blue	O
+and	O
+red	O
+,	O
+and	O
+IL	O
+-	O
+17A	O
+monomers	O
+are	O
+colored	O
+ice	O
+blue	O
+and	O
+pink	O
+,	O
+respectively	O
+.	O
+	O
+(	O
+A	O
+)	O
+Overview	O
+of	O
+the	O
+distinct	O
+binding	O
+sites	O
+of	O
+Fab	O
+and	O
+HAP	O
+to	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+(	O
+B	O
+)	O
+Close	O
+-	O
+in	O
+view	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+structure	O
+.	O
+	O
+IL	O
+-	O
+17A	O
+β	O
+-	O
+strands	O
+are	O
+labelled	O
+.	O
+	O
+Each	O
+of	O
+the	O
+two	O
+bound	O
+HAP	O
+interacts	O
+with	O
+both	O
+monomers	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+dimer	O
+.	O
+	O
+Three	O
+distinct	O
+areas	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+interface	O
+are	O
+labeled	O
+.	O
+	O
+Mechanism	O
+of	O
+the	O
+inhibition	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+interaction	O
+by	O
+HAP	O
+.	O
+	O
+IL	O
+-	O
+17A	O
+dimer	O
+is	O
+in	O
+surface	O
+presentation	O
+(	O
+β	O
+-	O
+strands	O
+0	O
+shown	O
+as	O
+ribbons	O
+for	O
+clarity	O
+).	O
+	O
+HAP	O
+residues	O
+as	O
+well	O
+as	O
+key	O
+IL	O
+-	O
+17A	O
+residues	O
+are	O
+labeled	O
+.	O
+	O
+For	O
+clarity	O
+,	O
+a	O
+few	O
+HAP	O
+residues	O
+are	O
+also	O
+shown	O
+in	O
+stick	O
+model	O
+with	O
+carbon	O
+atoms	O
+colored	O
+green	O
+,	O
+oxygen	O
+in	O
+red	O
+and	O
+nitrogen	O
+in	O
+blue	O
+.	O
+	O
+(	O
+B	O
+)	O
+I	O
+-	O
+17RA	O
+(	O
+ribbon	O
+in	O
+gold	O
+)	O
+peptide	O
+Leu27	O
+-	O
+Ile32	O
+binds	O
+to	O
+the	O
+same	O
+area	O
+as	O
+the	O
+HAP	O
+α	O
+-	O
+helix	O
+.	O
+	O
+Trp31	O
+of	O
+IL	O
+-	O
+17RA	O
+binds	O
+to	O
+the	O
+same	O
+pocket	O
+in	O
+IL	O
+-	O
+17A	O
+as	O
+Trp12	O
+of	O
+HAP	O
+.	O
+(	O
+C	O
+)	O
+As	O
+illustrated	O
+by	O
+overlay	O
+a	O
+single	O
+HAP	O
+molecule	O
+and	O
+β	O
+-	O
+strands	O
+0	O
+(	O
+grey	O
+)	O
+of	O
+the	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+complex	O
+in	O
+the	O
+apo	O
+IL	O
+-	O
+17A	O
+structure	O
+,	O
+conformational	O
+changes	O
+in	O
+region	O
+I	O
+of	O
+IL	O
+-	O
+17A	O
+are	O
+needed	O
+for	O
+binding	O
+of	O
+both	O
+the	O
+β	O
+-	O
+stand	O
+and	O
+α	O
+-	O
+helix	O
+of	O
+the	O
+HAP	O
+.	O
+	O
+Molecular	O
+Basis	O
+of	O
+Ligand	O
+-	O
+Dependent	O
+Regulation	O
+of	O
+NadR	O
+,	O
+the	O
+Transcriptional	O
+Repressor	O
+of	O
+Meningococcal	O
+Virulence	O
+Factor	O
+NadA	O
+	O
+Neisseria	O
+adhesin	O
+A	O
+(	O
+NadA	O
+)	O
+is	O
+present	O
+on	O
+the	O
+meningococcal	O
+surface	O
+and	O
+contributes	O
+to	O
+adhesion	O
+to	O
+and	O
+invasion	O
+of	O
+human	O
+cells	O
+.	O
+	O
+NadA	O
+is	O
+also	O
+one	O
+of	O
+three	O
+recombinant	O
+antigens	O
+in	O
+the	O
+recently	O
+-	O
+approved	O
+Bexsero	O
+vaccine	O
+,	O
+which	O
+protects	O
+against	O
+serogroup	O
+B	O
+meningococcus	O
+.	O
+	O
+In	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+hydroxyphenylacetate	O
+(	O
+4	O
+-	O
+HPA	O
+),	O
+a	O
+catabolite	O
+present	O
+in	O
+human	O
+saliva	O
+both	O
+under	O
+physiological	O
+conditions	O
+and	O
+during	O
+bacterial	O
+infection	O
+,	O
+the	O
+binding	O
+of	O
+NadR	O
+to	O
+the	O
+nadA	O
+promoter	O
+is	O
+attenuated	O
+and	O
+nadA	O
+expression	O
+is	O
+induced	O
+.	O
+	O
+To	O
+gain	O
+insights	O
+into	O
+the	O
+regulation	O
+of	O
+NadR	O
+mediated	O
+by	O
+4	O
+-	O
+HPA	O
+,	O
+we	O
+combined	O
+structural	O
+,	O
+biochemical	O
+,	O
+and	O
+mutagenesis	O
+studies	O
+.	O
+	O
+In	O
+particular	O
+,	O
+two	O
+new	O
+crystal	O
+structures	O
+of	O
+ligand	O
+-	O
+free	O
+and	O
+ligand	O
+-	O
+bound	O
+NadR	O
+revealed	O
+(	O
+i	O
+)	O
+the	O
+molecular	O
+basis	O
+of	O
+‘	O
+conformational	O
+selection	O
+’	O
+by	O
+which	O
+a	O
+single	O
+molecule	O
+of	O
+4	O
+-	O
+HPA	O
+binds	O
+and	O
+stabilizes	O
+dimeric	O
+NadR	O
+in	O
+a	O
+conformation	O
+unsuitable	O
+for	O
+DNA	O
+-	O
+binding	O
+,	O
+(	O
+ii	O
+)	O
+molecular	O
+explanations	O
+for	O
+the	O
+binding	O
+specificities	O
+of	O
+different	O
+hydroxyphenylacetate	O
+ligands	O
+,	O
+including	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+which	O
+is	O
+produced	O
+during	O
+inflammation	O
+,	O
+(	O
+iii	O
+)	O
+the	O
+presence	O
+of	O
+a	O
+leucine	O
+residue	O
+essential	O
+for	O
+dimerization	O
+and	O
+conserved	O
+in	O
+many	O
+MarR	O
+family	O
+proteins	O
+,	O
+and	O
+(	O
+iv	O
+)	O
+four	O
+residues	O
+(	O
+His7	O
+,	O
+Ser9	O
+,	O
+Asn11	O
+and	O
+Phe25	O
+),	O
+which	O
+are	O
+involved	O
+in	O
+binding	O
+4	O
+-	O
+HPA	O
+,	O
+and	O
+were	O
+confirmed	O
+in	O
+vitro	O
+to	O
+have	O
+key	O
+roles	O
+in	O
+the	O
+regulatory	O
+mechanism	O
+in	O
+bacteria	O
+.	O
+	O
+Overall	O
+,	O
+this	O
+study	O
+deepens	O
+our	O
+molecular	O
+understanding	O
+of	O
+the	O
+sophisticated	O
+regulatory	O
+mechanisms	O
+of	O
+the	O
+expression	O
+of	O
+nadA	O
+and	O
+other	O
+genes	O
+governed	O
+by	O
+NadR	O
+,	O
+dependent	O
+on	O
+interactions	O
+with	O
+niche	O
+-	O
+specific	O
+signal	O
+molecules	O
+that	O
+may	O
+play	O
+important	O
+roles	O
+during	O
+meningococcal	O
+pathogenesis	O
+.	O
+	O
+The	O
+Bexsero	O
+vaccine	O
+protects	O
+against	O
+MenB	O
+and	O
+has	O
+recently	O
+been	O
+approved	O
+in	O
+>	O
+35	O
+countries	O
+worldwide	O
+.	O
+	O
+Neisseria	O
+adhesin	O
+A	O
+(	O
+NadA	O
+)	O
+present	O
+on	O
+the	O
+meningococcal	O
+surface	O
+can	O
+mediate	O
+binding	O
+to	O
+human	O
+cells	O
+and	O
+is	O
+one	O
+of	O
+the	O
+three	O
+MenB	O
+vaccine	O
+protein	O
+antigens	O
+.	O
+	O
+A	O
+deep	O
+understanding	O
+of	O
+nadA	O
+expression	O
+is	O
+therefore	O
+important	O
+,	O
+otherwise	O
+the	O
+contribution	O
+of	O
+NadA	O
+to	O
+vaccine	O
+-	O
+induced	O
+protection	O
+against	O
+meningococcal	O
+meningitis	O
+may	O
+be	O
+underestimated	O
+.	O
+	O
+These	O
+findings	O
+shed	O
+light	O
+on	O
+the	O
+regulation	O
+of	O
+NadR	O
+,	O
+a	O
+key	O
+MarR	O
+-	O
+family	O
+virulence	O
+factor	O
+of	O
+this	O
+important	O
+human	O
+pathogen	O
+.	O
+	O
+The	O
+‘	O
+Reverse	O
+Vaccinology	O
+’	O
+approach	O
+was	O
+pioneered	O
+to	O
+identify	O
+antigens	O
+for	O
+a	O
+protein	O
+-	O
+based	O
+vaccine	O
+against	O
+serogroup	O
+B	O
+Neisseria	O
+meningitidis	O
+(	O
+MenB	O
+),	O
+a	O
+human	O
+pathogen	O
+causing	O
+potentially	O
+-	O
+fatal	O
+sepsis	O
+and	O
+invasive	O
+meningococcal	O
+disease	O
+.	O
+	O
+Indeed	O
+,	O
+Reverse	O
+Vaccinology	O
+identified	O
+Neisseria	O
+adhesin	O
+A	O
+(	O
+NadA	O
+),	O
+a	O
+surface	O
+-	O
+exposed	O
+protein	O
+involved	O
+in	O
+epithelial	O
+cell	O
+invasion	O
+and	O
+found	O
+in	O
+~	O
+30	O
+%	O
+of	O
+clinical	O
+isolates	O
+.	O
+	O
+Recently	O
+,	O
+we	O
+reported	O
+the	O
+crystal	O
+structure	O
+of	O
+NadA	O
+,	O
+providing	O
+insights	O
+into	O
+its	O
+biological	O
+and	O
+immunological	O
+functions	O
+.	O
+	O
+Recombinant	O
+NadA	O
+elicits	O
+a	O
+strong	O
+bactericidal	O
+immune	O
+response	O
+and	O
+is	O
+therefore	O
+included	O
+in	O
+the	O
+Bexsero	O
+vaccine	O
+that	O
+protects	O
+against	O
+MenB	O
+and	O
+which	O
+was	O
+recently	O
+approved	O
+in	O
+over	O
+35	O
+countries	O
+worldwide	O
+.	O
+	O
+Previous	O
+studies	O
+revealed	O
+that	O
+nadA	O
+expression	O
+levels	O
+are	O
+mainly	O
+regulated	O
+by	O
+the	O
+Neisseria	O
+adhesin	O
+A	O
+Regulator	O
+(	O
+NadR	O
+).	O
+	O
+Studies	O
+of	O
+NadR	O
+also	O
+have	O
+broader	O
+implications	O
+,	O
+since	O
+a	O
+genome	O
+-	O
+wide	O
+analysis	O
+of	O
+MenB	O
+wild	O
+-	O
+type	O
+and	O
+nadR	O
+knock	O
+-	O
+out	O
+strains	O
+revealed	O
+that	O
+NadR	O
+influences	O
+the	O
+regulation	O
+of	O
+>	O
+30	O
+genes	O
+,	O
+including	O
+maf	O
+genes	O
+,	O
+from	O
+the	O
+multiple	O
+adhesin	O
+family	O
+.	O
+	O
+These	O
+genes	O
+encode	O
+a	O
+wide	O
+variety	O
+of	O
+proteins	O
+connected	O
+to	O
+many	O
+biological	O
+processes	O
+contributing	O
+to	O
+bacterial	O
+survival	O
+,	O
+adaptation	O
+in	O
+the	O
+host	O
+niche	O
+,	O
+colonization	O
+and	O
+invasion	O
+.	O
+	O
+NadR	O
+belongs	O
+to	O
+the	O
+MarR	O
+(	O
+Multiple	O
+Antibiotic	O
+Resistance	O
+Regulator	O
+)	O
+family	O
+,	O
+a	O
+group	O
+of	O
+ligand	O
+-	O
+responsive	O
+transcriptional	O
+regulators	O
+ubiquitous	O
+in	O
+bacteria	O
+and	O
+archaea	O
+.	O
+	O
+MarR	O
+family	O
+proteins	O
+can	O
+promote	O
+bacterial	O
+survival	O
+in	O
+the	O
+presence	O
+of	O
+antibiotics	O
+,	O
+toxic	O
+chemicals	O
+,	O
+organic	O
+solvents	O
+or	O
+reactive	O
+oxygen	O
+species	O
+and	O
+can	O
+regulate	O
+virulence	O
+factor	O
+expression	O
+.	O
+	O
+MarR	O
+homologues	O
+can	O
+act	O
+either	O
+as	O
+transcriptional	O
+repressors	O
+or	O
+as	O
+activators	O
+.	O
+	O
+Although	O
+>	O
+50	O
+MarR	O
+family	O
+structures	O
+are	O
+known	O
+,	O
+a	O
+molecular	O
+understanding	O
+of	O
+their	O
+ligand	O
+-	O
+dependent	O
+regulatory	O
+mechanisms	O
+is	O
+still	O
+limited	O
+,	O
+often	O
+hampered	O
+by	O
+lack	O
+of	O
+identification	O
+of	O
+their	O
+ligands	O
+and	O
+/	O
+or	O
+DNA	O
+targets	O
+.	O
+	O
+A	O
+potentially	O
+interesting	O
+exception	O
+comes	O
+from	O
+the	O
+ligand	O
+-	O
+free	O
+and	O
+salicylate	O
+-	O
+bound	O
+forms	O
+of	O
+the	O
+Methanobacterium	O
+thermoautotrophicum	O
+protein	O
+MTH313	O
+which	O
+revealed	O
+that	O
+two	O
+salicylate	O
+molecules	O
+bind	O
+to	O
+one	O
+MTH313	O
+dimer	O
+and	O
+induce	O
+large	O
+conformational	O
+changes	O
+,	O
+apparently	O
+sufficient	O
+to	O
+prevent	O
+DNA	O
+binding	O
+.	O
+	O
+However	O
+,	O
+it	O
+is	O
+unknown	O
+whether	O
+salicylate	O
+is	O
+a	O
+relevant	O
+in	O
+vivo	O
+ligand	O
+of	O
+either	O
+of	O
+these	O
+two	O
+proteins	O
+,	O
+which	O
+share	O
+~	O
+20	O
+%	O
+sequence	O
+identity	O
+with	O
+NadR	O
+,	O
+rendering	O
+unclear	O
+the	O
+interpretation	O
+of	O
+these	O
+findings	O
+in	O
+relation	O
+to	O
+the	O
+regulatory	O
+mechanisms	O
+of	O
+NadR	O
+or	O
+other	O
+MarR	O
+family	O
+proteins	O
+.	O
+	O
+NadR	O
+binds	O
+nadA	O
+on	O
+three	O
+different	O
+operators	O
+(	O
+OpI	O
+,	O
+OpII	O
+and	O
+OpIII	O
+).	O
+	O
+4	O
+-	O
+HPA	O
+is	O
+a	O
+small	O
+molecule	O
+derived	O
+from	O
+mammalian	O
+aromatic	O
+amino	O
+acid	O
+catabolism	O
+and	O
+is	O
+released	O
+in	O
+human	O
+saliva	O
+,	O
+where	O
+it	O
+has	O
+been	O
+detected	O
+at	O
+micromolar	O
+concentration	O
+.	O
+	O
+In	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+,	O
+NadR	O
+is	O
+unable	O
+to	O
+bind	O
+the	O
+nadA	O
+promoter	O
+and	O
+nadA	O
+gene	O
+expression	O
+is	O
+induced	O
+.	O
+	O
+In	O
+vivo	O
+,	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+in	O
+the	O
+host	O
+niche	O
+of	O
+N	O
+.	O
+meningitidis	O
+serves	O
+as	O
+an	O
+inducer	O
+of	O
+NadA	O
+production	O
+,	O
+thereby	O
+promoting	O
+bacterial	O
+adhesion	O
+to	O
+host	O
+cells	O
+.	O
+	O
+Further	O
+,	O
+we	O
+recently	O
+reported	O
+that	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+,	O
+produced	O
+during	O
+inflammation	O
+,	O
+is	O
+another	O
+inducer	O
+of	O
+nadA	O
+expression	O
+.	O
+	O
+Extending	O
+our	O
+previous	O
+studies	O
+based	O
+on	O
+hydrogen	O
+-	O
+deuterium	O
+exchange	O
+mass	O
+spectrometry	O
+(	O
+HDX	O
+-	O
+MS	O
+),	O
+here	O
+we	O
+sought	O
+to	O
+reveal	O
+the	O
+molecular	O
+mechanisms	O
+and	O
+effects	O
+of	O
+NadR	O
+/	O
+HPA	O
+interactions	O
+via	O
+X	O
+-	O
+ray	O
+crystallography	O
+,	O
+NMR	O
+spectroscopy	O
+and	O
+complementary	O
+biochemical	O
+and	O
+in	O
+vivo	O
+mutagenesis	O
+studies	O
+.	O
+	O
+Standard	O
+chromatographic	O
+techniques	O
+were	O
+used	O
+to	O
+obtain	O
+a	O
+highly	O
+purified	O
+sample	O
+of	O
+NadR	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+	O
+These	O
+data	O
+showed	O
+that	O
+NadR	O
+was	O
+dimeric	O
+in	O
+solution	O
+,	O
+since	O
+the	O
+theoretical	O
+molecular	O
+mass	O
+of	O
+the	O
+NadR	O
+dimer	O
+is	O
+33	O
+.	O
+73	O
+kDa	O
+;	O
+and	O
+,	O
+there	O
+was	O
+no	O
+change	O
+in	O
+oligomeric	O
+state	O
+on	O
+addition	O
+of	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+The	O
+thermal	O
+stability	O
+of	O
+NadR	O
+was	O
+examined	O
+using	O
+differential	O
+scanning	O
+calorimetry	O
+(	O
+DSC	O
+).	O
+	O
+The	O
+Tm	O
+of	O
+NadR	O
+was	O
+67	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+°	O
+C	O
+in	O
+the	O
+absence	O
+of	O
+ligand	O
+,	O
+and	O
+was	O
+unaffected	O
+by	O
+salicylate	O
+.	O
+	O
+However	O
+,	O
+an	O
+increased	O
+thermal	O
+stability	O
+was	O
+induced	O
+by	O
+4	O
+-	O
+HPA	O
+and	O
+,	O
+to	O
+a	O
+lesser	O
+extent	O
+,	O
+by	O
+3	O
+-	O
+HPA	O
+.	O
+	O
+Interestingly	O
+,	O
+NadR	O
+displayed	O
+the	O
+greatest	O
+Tm	O
+increase	O
+upon	O
+addition	O
+of	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+1B	O
+).	O
+	O
+Stability	O
+of	O
+NadR	O
+is	O
+increased	O
+by	O
+small	O
+molecule	O
+ligands	O
+.	O
+	O
+Melting	O
+-	O
+point	O
+(	O
+Tm	O
+)	O
+and	O
+its	O
+ligand	O
+-	O
+induced	O
+increase	O
+(	O
+ΔTm	O
+)	O
+derived	O
+from	O
+DSC	O
+thermostability	O
+experiments	O
+.	O
+	O
+Dissociation	O
+constants	O
+(	O
+KD	O
+)	O
+of	O
+the	O
+NadR	O
+/	O
+ligand	O
+interactions	O
+from	O
+SPR	O
+steady	O
+-	O
+state	O
+binding	O
+experiments	O
+.	O
+	O
+Ligand	O
+Tm	O
+(°	O
+C	O
+)	O
+ΔTm	O
+(°	O
+C	O
+)	O
+KD	O
+(	O
+mM	O
+)	O
+No	O
+ligand	O
+67	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+n	O
+.	O
+a	O
+.	O
+n	O
+.	O
+a	O
+.	O
+	O
+NadR	O
+displays	O
+distinct	O
+binding	O
+affinities	O
+for	O
+hydroxyphenylacetate	O
+ligands	O
+	O
+The	O
+SPR	O
+sensorgrams	O
+revealed	O
+very	O
+fast	O
+association	O
+and	O
+dissociation	O
+events	O
+,	O
+typical	O
+of	O
+small	O
+molecule	O
+ligands	O
+,	O
+thus	O
+prohibiting	O
+a	O
+detailed	O
+study	O
+of	O
+binding	O
+kinetics	O
+.	O
+	O
+3	O
+-	O
+HPA	O
+showed	O
+a	O
+weaker	O
+interaction	O
+,	O
+with	O
+a	O
+KD	O
+of	O
+2	O
+.	O
+7	O
+mM	O
+,	O
+while	O
+salicylate	O
+showed	O
+only	O
+a	O
+very	O
+weak	O
+response	O
+that	O
+did	O
+not	O
+reach	O
+saturation	O
+,	O
+indicating	O
+a	O
+non	O
+-	O
+specific	O
+interaction	O
+with	O
+NadR	O
+.	O
+A	O
+ranking	O
+of	O
+these	O
+KD	O
+values	O
+showed	O
+that	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+was	O
+the	O
+tightest	O
+binder	O
+,	O
+and	O
+thus	O
+matched	O
+the	O
+ranking	O
+of	O
+ligand	O
+-	O
+induced	O
+Tm	O
+increases	O
+observed	O
+in	O
+the	O
+DSC	O
+experiments	O
+.	O
+	O
+Although	O
+these	O
+KD	O
+values	O
+indicate	O
+rather	O
+weak	O
+interactions	O
+,	O
+they	O
+are	O
+similar	O
+to	O
+the	O
+values	O
+reported	O
+previously	O
+for	O
+the	O
+MarR	O
+/	O
+salicylate	O
+interaction	O
+(	O
+KD	O
+~	O
+1	O
+mM	O
+)	O
+and	O
+the	O
+MTH313	O
+/	O
+salicylate	O
+interaction	O
+(	O
+KD	O
+2	O
+–	O
+3	O
+mM	O
+),	O
+and	O
+approximately	O
+20	O
+-	O
+fold	O
+tighter	O
+than	O
+the	O
+ST1710	O
+/	O
+salicylate	O
+interaction	O
+(	O
+KD	O
+~	O
+20	O
+mM	O
+).	O
+	O
+Crystal	O
+structures	O
+of	O
+holo	O
+-	O
+NadR	O
+and	O
+apo	O
+-	O
+NadR	O
+	O
+First	O
+,	O
+we	O
+crystallized	O
+NadR	O
+(	O
+a	O
+selenomethionine	O
+-	O
+labelled	O
+derivative	O
+)	O
+in	O
+the	O
+presence	O
+of	O
+a	O
+200	O
+-	O
+fold	O
+molar	O
+excess	O
+of	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+The	O
+structure	O
+of	O
+the	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+complex	O
+was	O
+determined	O
+at	O
+2	O
+.	O
+3	O
+Å	O
+resolution	O
+using	O
+a	O
+combination	O
+of	O
+the	O
+single	O
+-	O
+wavelength	O
+anomalous	O
+dispersion	O
+(	O
+SAD	O
+)	O
+and	O
+molecular	O
+replacement	O
+(	O
+MR	O
+)	O
+methods	O
+,	O
+and	O
+was	O
+refined	O
+to	O
+R	O
+work	O
+/	O
+R	O
+free	O
+values	O
+of	O
+20	O
+.	O
+9	O
+/	O
+26	O
+.	O
+0	O
+%	O
+(	O
+Table	O
+2	O
+).	O
+	O
+Despite	O
+numerous	O
+attempts	O
+,	O
+we	O
+were	O
+unable	O
+to	O
+obtain	O
+high	O
+-	O
+quality	O
+crystals	O
+of	O
+NadR	O
+complexed	O
+with	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+,	O
+3	O
+,	O
+4	O
+-	O
+HPA	O
+,	O
+3	O
+-	O
+HPA	O
+or	O
+DNA	O
+targets	O
+.	O
+	O
+However	O
+,	O
+it	O
+was	O
+eventually	O
+possible	O
+to	O
+crystallize	O
+apo	O
+-	O
+NadR	O
+,	O
+and	O
+the	O
+structure	O
+was	O
+determined	O
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+by	O
+MR	O
+methods	O
+using	O
+the	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+complex	O
+as	O
+the	O
+search	O
+model	O
+.	O
+	O
+The	O
+apo	O
+-	O
+NadR	O
+structure	O
+was	O
+refined	O
+to	O
+R	O
+work	O
+/	O
+R	O
+free	O
+values	O
+of	O
+19	O
+.	O
+1	O
+/	O
+26	O
+.	O
+8	O
+%	O
+(	O
+Table	O
+2	O
+).	O
+	O
+The	O
+asymmetric	O
+unit	O
+of	O
+the	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+crystals	O
+(	O
+holo	O
+-	O
+NadR	O
+)	O
+contained	O
+one	O
+NadR	O
+homodimer	O
+,	O
+while	O
+the	O
+apo	O
+-	O
+NadR	O
+crystals	O
+contained	O
+two	O
+homodimers	O
+.	O
+	O
+Moreover	O
+,	O
+our	O
+SE	O
+-	O
+HPLC	O
+/	O
+MALLS	O
+analyses	O
+(	O
+see	O
+above	O
+)	O
+revealed	O
+that	O
+in	O
+solution	O
+NadR	O
+is	O
+dimeric	O
+,	O
+and	O
+previous	O
+studies	O
+using	O
+native	O
+mass	O
+spectrometry	O
+(	O
+MS	O
+)	O
+revealed	O
+dimers	O
+,	O
+not	O
+tetramers	O
+.	O
+	O
+The	O
+NadR	O
+homodimer	O
+bound	O
+to	O
+4	O
+-	O
+HPA	O
+has	O
+a	O
+dimerization	O
+interface	O
+mostly	O
+involving	O
+the	O
+top	O
+of	O
+its	O
+‘	O
+triangular	O
+’	O
+form	O
+,	O
+while	O
+the	O
+two	O
+DNA	O
+-	O
+binding	O
+domains	O
+are	O
+located	O
+at	O
+the	O
+base	O
+(	O
+Fig	O
+2A	O
+).	O
+	O
+High	O
+-	O
+quality	O
+electron	O
+density	O
+maps	O
+allowed	O
+clear	O
+identification	O
+of	O
+the	O
+bound	O
+ligand	O
+,	O
+4	O
+-	O
+HPA	O
+(	O
+Fig	O
+2B	O
+).	O
+	O
+The	O
+overall	O
+structure	O
+of	O
+NadR	O
+shows	O
+dimensions	O
+of	O
+~	O
+50	O
+×	O
+65	O
+×	O
+50	O
+Å	O
+and	O
+a	O
+large	O
+homodimer	O
+interface	O
+that	O
+buries	O
+a	O
+total	O
+surface	O
+area	O
+of	O
+~	O
+4800	O
+Å2	O
+.	O
+	O
+Each	O
+NadR	O
+monomer	O
+consists	O
+of	O
+six	O
+α	O
+-	O
+helices	O
+and	O
+two	O
+short	O
+β	O
+-	O
+strands	O
+,	O
+with	O
+helices	O
+α1	O
+,	O
+α5	O
+,	O
+and	O
+α6	O
+forming	O
+the	O
+dimer	O
+interface	O
+.	O
+	O
+Together	O
+,	O
+these	O
+structural	O
+elements	O
+constitute	O
+the	O
+winged	O
+helix	O
+-	O
+turn	O
+-	O
+helix	O
+(	O
+wHTH	O
+)	O
+DNA	O
+-	O
+binding	O
+domain	O
+and	O
+,	O
+together	O
+with	O
+the	O
+dimeric	O
+organization	O
+,	O
+are	O
+the	O
+hallmarks	O
+of	O
+MarR	O
+family	O
+structures	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+NadR	O
+in	O
+complex	O
+with	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+(	O
+A	O
+)	O
+The	O
+holo	O
+-	O
+NadR	O
+homodimer	O
+is	O
+depicted	O
+in	O
+green	O
+and	O
+blue	O
+for	O
+chains	O
+A	O
+and	O
+B	O
+respectively	O
+,	O
+while	O
+yellow	O
+sticks	O
+depict	O
+the	O
+4	O
+-	O
+HPA	O
+ligand	O
+(	O
+labelled	O
+).	O
+	O
+For	O
+simplicity	O
+,	O
+secondary	O
+structure	O
+elements	O
+are	O
+labelled	O
+for	O
+chain	O
+B	O
+only	O
+.	O
+	O
+Red	O
+dashes	O
+show	O
+hypothetical	O
+positions	O
+of	O
+chain	O
+B	O
+residues	O
+88	O
+–	O
+90	O
+that	O
+were	O
+not	O
+modeled	O
+due	O
+to	O
+lack	O
+of	O
+electron	O
+density	O
+.	O
+	O
+(	O
+B	O
+)	O
+A	O
+zoom	O
+into	O
+the	O
+pocket	O
+occupied	O
+by	O
+4	O
+-	O
+HPA	O
+shows	O
+that	O
+the	O
+ligand	O
+contacts	O
+both	O
+chains	O
+A	O
+and	O
+B	O
+;	O
+blue	O
+mesh	O
+shows	O
+electron	O
+density	O
+around	O
+4	O
+-	O
+HPA	O
+calculated	O
+from	O
+a	O
+composite	O
+omit	O
+map	O
+(	O
+omitting	O
+4	O
+-	O
+HPA	O
+),	O
+using	O
+phenix	O
+.	O
+	O
+A	O
+single	O
+conserved	O
+leucine	O
+residue	O
+(	O
+L130	O
+)	O
+is	O
+crucial	O
+for	O
+dimerization	O
+	O
+The	O
+NadR	O
+dimer	O
+interface	O
+is	O
+formed	O
+by	O
+at	O
+least	O
+32	O
+residues	O
+,	O
+which	O
+establish	O
+numerous	O
+inter	O
+-	O
+chain	O
+salt	O
+bridges	O
+or	O
+hydrogen	O
+bonds	O
+,	O
+and	O
+many	O
+hydrophobic	O
+packing	O
+interactions	O
+(	O
+Fig	O
+3A	O
+and	O
+3B	O
+).	O
+	O
+To	O
+determine	O
+which	O
+residues	O
+were	O
+most	O
+important	O
+for	O
+dimerization	O
+,	O
+we	O
+studied	O
+the	O
+interface	O
+in	O
+silico	O
+and	O
+identified	O
+several	O
+residues	O
+as	O
+potential	O
+mediators	O
+of	O
+key	O
+stabilizing	O
+interactions	O
+.	O
+	O
+Each	O
+mutant	O
+NadR	O
+protein	O
+was	O
+purified	O
+,	O
+and	O
+then	O
+its	O
+oligomeric	O
+state	O
+was	O
+examined	O
+by	O
+analytical	O
+SE	O
+-	O
+HPLC	O
+.	O
+	O
+Almost	O
+all	O
+the	O
+mutants	O
+showed	O
+the	O
+same	O
+elution	O
+profile	O
+as	O
+the	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+NadR	O
+protein	O
+.	O
+	O
+Only	O
+the	O
+L130K	B-mutant
+mutation	O
+induced	O
+a	O
+notable	O
+change	O
+in	O
+the	O
+oligomeric	O
+state	O
+of	O
+NadR	O
+(	O
+Fig	O
+3C	O
+).	O
+	O
+Further	O
+,	O
+in	O
+SE	O
+-	O
+MALLS	O
+analyses	O
+,	O
+the	O
+L130K	B-mutant
+mutant	O
+displayed	O
+two	O
+distinct	O
+species	O
+in	O
+solution	O
+,	O
+approximately	O
+80	O
+%	O
+being	O
+monomeric	O
+(	O
+a	O
+19	O
+kDa	O
+species	O
+),	O
+and	O
+only	O
+20	O
+%	O
+retaining	O
+the	O
+typical	O
+native	O
+dimeric	O
+state	O
+(	O
+a	O
+35	O
+kDa	O
+species	O
+)	O
+(	O
+Fig	O
+3D	O
+),	O
+demonstrating	O
+that	O
+Leu130	O
+is	O
+crucial	O
+for	O
+stable	O
+dimerization	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+most	O
+of	O
+the	O
+other	O
+residues	O
+identified	O
+in	O
+the	O
+NadR	O
+dimer	O
+interface	O
+were	O
+poorly	O
+conserved	O
+in	O
+the	O
+MarR	O
+family	O
+.	O
+	O
+Analysis	O
+of	O
+the	O
+NadR	O
+dimer	O
+interface	O
+.	O
+	O
+(	O
+A	O
+)	O
+Both	O
+orientations	O
+show	O
+chain	O
+A	O
+,	O
+green	O
+backbone	O
+ribbon	O
+,	O
+colored	O
+red	O
+to	O
+highlight	O
+all	O
+locations	O
+involved	O
+in	O
+dimerization	O
+;	O
+namely	O
+,	O
+inter	O
+-	O
+chain	O
+salt	O
+bridges	O
+or	O
+hydrogen	O
+bonds	O
+involving	O
+Q4	O
+,	O
+S5	O
+,	O
+K6	O
+,	O
+H7	O
+,	O
+S9	O
+,	O
+I10	O
+,	O
+N11	O
+,	O
+I15	O
+,	O
+Q16	O
+,	O
+R18	O
+,	O
+D36	O
+,	O
+R43	O
+,	O
+A46	O
+,	O
+Q59	O
+,	O
+C61	O
+,	O
+Y104	O
+,	O
+D112	O
+,	O
+R114	O
+,	O
+Y115	O
+,	O
+D116	O
+,	O
+E119	O
+,	O
+K126	O
+,	O
+E136	O
+,	O
+E141	O
+,	O
+N145	O
+,	O
+and	O
+the	O
+hydrophobic	O
+packing	O
+interactions	O
+involving	O
+I10	O
+,	O
+I12	O
+,	O
+L14	O
+,	O
+I15	O
+,	O
+R18	O
+,	O
+Y115	O
+,	O
+I118	O
+,	O
+L130	O
+,	O
+L133	O
+,	O
+L134	O
+and	O
+L137	O
+.	O
+	O
+Chain	O
+B	O
+,	O
+grey	O
+surface	O
+,	O
+is	O
+marked	O
+blue	O
+to	O
+highlight	O
+residues	O
+probed	O
+by	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+(	O
+E136	O
+only	O
+makes	O
+a	O
+salt	O
+bridge	O
+with	O
+K126	O
+,	O
+therefore	O
+it	O
+was	O
+sufficient	O
+to	O
+make	O
+the	O
+K126A	B-mutant
+mutation	O
+to	O
+assess	O
+the	O
+importance	O
+of	O
+this	O
+ionic	O
+interaction	O
+;	O
+the	O
+H7	O
+position	O
+is	O
+labelled	O
+for	O
+monomer	O
+A	O
+,	O
+since	O
+electron	O
+density	O
+was	O
+lacking	O
+for	O
+monomer	O
+B	O
+).	O
+(	O
+B	O
+)	O
+A	O
+zoom	O
+into	O
+the	O
+environment	O
+of	O
+helix	O
+α6	O
+to	O
+show	O
+how	O
+residue	O
+L130	O
+chain	O
+B	O
+(	O
+blue	O
+side	O
+chain	O
+)	O
+is	O
+a	O
+focus	O
+of	O
+hydrophobic	O
+packing	O
+interactions	O
+with	O
+L130	O
+,	O
+L133	O
+,	O
+L134	O
+and	O
+L137	O
+of	O
+chain	O
+A	O
+(	O
+red	O
+side	O
+chains	O
+).	O
+	O
+(	O
+C	O
+)	O
+SE	O
+-	O
+HPLC	O
+analyses	O
+of	O
+all	O
+mutant	O
+forms	O
+of	O
+NadR	O
+are	O
+compared	O
+with	O
+the	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+protein	O
+.	O
+	O
+The	O
+WT	O
+and	O
+most	O
+of	O
+the	O
+mutants	O
+show	O
+a	O
+single	O
+elution	O
+peak	O
+with	O
+an	O
+absorbance	O
+maximum	O
+at	O
+17	O
+.	O
+5	O
+min	O
+.	O
+	O
+The	O
+holo	O
+-	O
+NadR	O
+structure	O
+presents	O
+only	O
+one	O
+occupied	O
+ligand	O
+-	O
+binding	O
+pocket	O
+	O
+The	O
+tunnel	O
+was	O
+lined	O
+with	O
+rather	O
+hydrophobic	O
+amino	O
+acids	O
+,	O
+and	O
+did	O
+not	O
+contain	O
+water	O
+molecules	O
+.	O
+	O
+Unexpectedly	O
+,	O
+only	O
+one	O
+monomer	O
+of	O
+the	O
+holo	O
+-	O
+NadR	O
+homodimer	O
+contained	O
+4	O
+-	O
+HPA	O
+in	O
+the	O
+binding	O
+pocket	O
+,	O
+whereas	O
+the	O
+corresponding	O
+pocket	O
+of	O
+the	O
+other	O
+monomer	O
+was	O
+unoccupied	O
+by	O
+ligand	O
+,	O
+despite	O
+the	O
+large	O
+excess	O
+of	O
+4	O
+-	O
+HPA	O
+used	O
+in	O
+the	O
+crystallization	O
+conditions	O
+.	O
+	O
+Inspection	O
+of	O
+the	O
+protein	O
+-	O
+ligand	O
+interaction	O
+network	O
+revealed	O
+no	O
+bonds	O
+from	O
+NadR	O
+backbone	O
+groups	O
+to	O
+the	O
+ligand	O
+,	O
+but	O
+several	O
+key	O
+side	O
+chain	O
+mediated	O
+hydrogen	O
+(	O
+H	O
+)-	O
+bonds	O
+and	O
+ionic	O
+interactions	O
+,	O
+most	O
+notably	O
+between	O
+the	O
+carboxylate	O
+group	O
+of	O
+4	O
+-	O
+HPA	O
+and	O
+Ser9	O
+of	O
+chain	O
+A	O
+(	O
+SerA9	O
+),	O
+and	O
+chain	O
+B	O
+residues	O
+TrpB39	O
+,	O
+ArgB43	O
+and	O
+TyrB115	O
+(	O
+Fig	O
+4A	O
+).	O
+	O
+Atomic	O
+details	O
+of	O
+NadR	O
+/	O
+HPA	O
+interactions	O
+.	O
+	O
+A	O
+)	O
+A	O
+stereo	O
+-	O
+view	O
+zoom	O
+into	O
+the	O
+binding	O
+pocket	O
+showing	O
+side	O
+chain	O
+sticks	O
+for	O
+all	O
+interactions	O
+between	O
+NadR	O
+and	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+4	O
+-	O
+HPA	O
+is	O
+shown	O
+in	O
+yellow	O
+sticks	O
+,	O
+with	O
+oxygen	O
+atoms	O
+in	O
+red	O
+.	O
+	O
+A	O
+water	O
+molecule	O
+is	O
+shown	O
+by	O
+the	O
+red	O
+sphere	O
+.	O
+	O
+H	O
+-	O
+bonds	O
+up	O
+to	O
+3	O
+.	O
+6Å	O
+are	O
+shown	O
+as	O
+dashed	O
+lines	O
+.	O
+	O
+The	O
+entire	O
+set	O
+of	O
+residues	O
+making	O
+H	O
+-	O
+bonds	O
+or	O
+non	O
+-	O
+bonded	O
+contacts	O
+with	O
+4	O
+-	O
+HPA	O
+is	O
+as	O
+follows	O
+:	O
+SerA9	O
+,	O
+AsnA11	O
+,	O
+LeuB21	O
+,	O
+MetB22	O
+,	O
+PheB25	O
+,	O
+LeuB29	O
+,	O
+AspB36	O
+,	O
+TrpB39	O
+,	O
+ArgB43	O
+,	O
+ValB111	O
+and	O
+TyrB115	O
+(	O
+automated	O
+analysis	O
+performed	O
+using	O
+PDBsum	O
+and	O
+verified	O
+manually	O
+).	O
+	O
+Side	O
+chains	O
+mediating	O
+hydrophobic	O
+interactions	O
+are	O
+shown	O
+in	O
+orange	O
+.	O
+(	O
+B	O
+)	O
+A	O
+model	O
+was	O
+prepared	O
+to	O
+visualize	O
+putative	O
+interactions	O
+of	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+(	O
+pink	O
+)	O
+with	O
+NadR	O
+,	O
+revealing	O
+the	O
+potential	O
+for	O
+additional	O
+contacts	O
+(	O
+dashed	O
+lines	O
+)	O
+of	O
+the	O
+chloro	O
+moiety	O
+(	O
+green	O
+stick	O
+)	O
+with	O
+LeuB29	O
+and	O
+AspB36	O
+.	O
+	O
+Notably	O
+,	O
+the	O
+phenyl	O
+ring	O
+of	O
+PheB25	O
+was	O
+positioned	O
+parallel	O
+to	O
+the	O
+phenyl	O
+ring	O
+of	O
+4	O
+-	O
+HPA	O
+,	O
+potentially	O
+forming	O
+π	O
+-	O
+π	O
+parallel	O
+-	O
+displaced	O
+stacking	O
+interactions	O
+.	O
+	O
+Consequently	O
+,	O
+residues	O
+in	O
+the	O
+4	O
+-	O
+HPA	O
+binding	O
+pocket	O
+are	O
+mostly	O
+contributed	O
+by	O
+NadR	O
+chain	O
+B	O
+,	O
+and	O
+effectively	O
+created	O
+a	O
+polar	O
+‘	O
+floor	O
+’	O
+and	O
+a	O
+hydrophobic	O
+‘	O
+ceiling	O
+’,	O
+which	O
+house	O
+the	O
+ligand	O
+.	O
+	O
+Collectively	O
+,	O
+this	O
+mixed	O
+network	O
+of	O
+polar	O
+and	O
+hydrophobic	O
+interactions	O
+endows	O
+NadR	O
+with	O
+a	O
+strong	O
+recognition	O
+pattern	O
+for	O
+HPAs	O
+,	O
+with	O
+additional	O
+medium	O
+-	O
+range	O
+interactions	O
+potentially	O
+established	O
+with	O
+the	O
+hydroxyl	O
+group	O
+at	O
+the	O
+4	O
+-	O
+position	O
+.	O
+	O
+Structure	O
+-	O
+activity	O
+relationships	O
+:	O
+molecular	O
+basis	O
+of	O
+enhanced	O
+stabilization	O
+by	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+	O
+We	O
+modelled	O
+the	O
+binding	O
+of	O
+other	O
+HPAs	O
+by	O
+in	O
+silico	O
+superposition	O
+onto	O
+4	O
+-	O
+HPA	O
+in	O
+the	O
+holo	O
+-	O
+NadR	O
+structure	O
+,	O
+and	O
+thereby	O
+obtained	O
+molecular	O
+explanations	O
+for	O
+the	O
+binding	O
+specificities	O
+of	O
+diverse	O
+ligands	O
+.	O
+	O
+For	O
+example	O
+,	O
+similar	O
+to	O
+4	O
+-	O
+HPA	O
+,	O
+the	O
+binding	O
+of	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+could	O
+involve	O
+multiple	O
+bonds	O
+towards	O
+the	O
+carboxylate	O
+group	O
+of	O
+the	O
+ligand	O
+and	O
+some	O
+to	O
+the	O
+4	O
+-	O
+hydroxyl	O
+group	O
+.	O
+	O
+Additionally	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+LeuB29	O
+and	O
+AspB36	O
+would	O
+be	O
+only	O
+2	O
+.	O
+6	O
+–	O
+3	O
+.	O
+5	O
+Å	O
+from	O
+the	O
+chlorine	O
+atom	O
+,	O
+thus	O
+providing	O
+van	O
+der	O
+Waals	O
+’	O
+interactions	O
+or	O
+H	O
+-	O
+bonds	O
+to	O
+generate	O
+the	O
+additional	O
+binding	O
+affinity	O
+observed	O
+for	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+(	O
+Fig	O
+4B	O
+).	O
+	O
+Finally	O
+,	O
+salicylate	O
+is	O
+presumably	O
+unable	O
+to	O
+specifically	O
+bind	O
+NadR	O
+due	O
+to	O
+the	O
+2	O
+-	O
+hydroxyl	O
+substitution	O
+and	O
+the	O
+shorter	O
+aliphatic	O
+chain	O
+connecting	O
+its	O
+carboxylate	O
+group	O
+(	O
+Fig	O
+1A	O
+):	O
+the	O
+compound	O
+simply	O
+seems	O
+too	O
+small	O
+to	O
+simultaneously	O
+establish	O
+the	O
+network	O
+of	O
+beneficial	O
+bonds	O
+observed	O
+in	O
+the	O
+NadR	O
+/	O
+HPA	O
+interactions	O
+.	O
+	O
+Analysis	O
+of	O
+the	O
+pockets	O
+reveals	O
+the	O
+molecular	O
+basis	O
+for	O
+asymmetric	O
+binding	O
+and	O
+stoichiometry	O
+	O
+However	O
+,	O
+studies	O
+based	O
+on	O
+tryptophan	O
+fluorescence	O
+were	O
+confounded	O
+by	O
+the	O
+fluorescence	O
+of	O
+the	O
+HPA	O
+ligands	O
+,	O
+and	O
+isothermal	O
+titration	O
+calorimetry	O
+(	O
+ITC	O
+)	O
+was	O
+unfeasible	O
+due	O
+to	O
+the	O
+need	O
+for	O
+very	O
+high	O
+concentrations	O
+of	O
+NadR	O
+in	O
+the	O
+ITC	O
+chamber	O
+(	O
+due	O
+to	O
+the	O
+relatively	O
+low	O
+affinity	O
+),	O
+which	O
+exceeded	O
+the	O
+solubility	O
+limits	O
+of	O
+the	O
+protein	O
+.	O
+	O
+However	O
+,	O
+it	O
+was	O
+possible	O
+to	O
+calculate	O
+the	O
+binding	O
+stoichiometry	O
+of	O
+the	O
+NadR	O
+-	O
+HPA	O
+interactions	O
+using	O
+an	O
+SPR	O
+-	O
+based	O
+approach	O
+.	O
+	O
+This	O
+approach	O
+relies	O
+on	O
+the	O
+assumption	O
+that	O
+the	O
+captured	O
+protein	O
+(‘	O
+the	O
+ligand	O
+’,	O
+according	O
+to	O
+SPR	O
+conventions	O
+)	O
+is	O
+100	O
+%	O
+active	O
+and	O
+freely	O
+-	O
+accessible	O
+to	O
+potential	O
+interactors	O
+(‘	O
+the	O
+analytes	O
+’).	O
+	O
+Overall	O
+,	O
+the	O
+superposition	O
+revealed	O
+a	O
+high	O
+degree	O
+of	O
+structural	O
+similarity	O
+(	O
+Cα	O
+root	O
+mean	O
+square	O
+deviation	O
+(	O
+rmsd	O
+)	O
+of	O
+1	O
+.	O
+5Å	O
+),	O
+though	O
+on	O
+closer	O
+inspection	O
+a	O
+rotational	O
+difference	O
+of	O
+~	O
+9	O
+degrees	O
+along	O
+the	O
+long	O
+axis	O
+of	O
+helix	O
+α6	O
+was	O
+observed	O
+,	O
+suggesting	O
+that	O
+4	O
+-	O
+HPA	O
+induced	O
+a	O
+slight	O
+conformational	O
+change	O
+(	O
+Fig	O
+5A	O
+).	O
+	O
+Most	O
+notably	O
+,	O
+atomic	O
+clashes	O
+between	O
+the	O
+ligand	O
+and	O
+the	O
+side	O
+chains	O
+of	O
+MetA22	O
+,	O
+PheA25	O
+and	O
+ArgA43	O
+would	O
+occur	O
+if	O
+4	O
+-	O
+HPA	O
+were	O
+present	O
+in	O
+the	O
+monomer	O
+A	O
+pocket	O
+(	O
+Fig	O
+5B	O
+).	O
+	O
+Subsequently	O
+,	O
+analyses	O
+of	O
+the	O
+pockets	O
+in	O
+apo	O
+-	O
+NadR	O
+revealed	O
+that	O
+in	O
+the	O
+absence	O
+of	O
+ligand	O
+the	O
+long	O
+Arg43	O
+side	O
+chain	O
+was	O
+always	O
+in	O
+the	O
+open	O
+‘	O
+outward	O
+’	O
+position	O
+compatible	O
+with	O
+binding	O
+to	O
+the	O
+4	O
+-	O
+HPA	O
+carboxylate	O
+group	O
+.	O
+	O
+Structural	O
+differences	O
+of	O
+NadR	O
+in	O
+ligand	O
+-	O
+bound	O
+or	O
+free	O
+forms	O
+.	O
+	O
+(	O
+A	O
+)	O
+Aligned	O
+monomers	O
+of	O
+holo	O
+-	O
+NadR	O
+(	O
+chain	O
+A	O
+:	O
+green	O
+;	O
+chain	O
+B	O
+:	O
+blue	O
+),	O
+reveal	O
+major	O
+overall	O
+differences	O
+by	O
+the	O
+shift	O
+of	O
+helix	O
+α6	O
+.	O
+(	O
+B	O
+)	O
+Comparison	O
+of	O
+the	O
+two	O
+binding	O
+pockets	O
+in	O
+holo	O
+-	O
+NadR	O
+shows	O
+that	O
+in	O
+the	O
+ligand	O
+-	O
+free	O
+monomer	O
+A	O
+(	O
+green	O
+)	O
+residues	O
+Met22	O
+,	O
+Phe25	O
+and	O
+Arg43	O
+adopt	O
+‘	O
+inward	O
+’	O
+positions	O
+(	O
+highlighted	O
+by	O
+arrows	O
+)	O
+compared	O
+to	O
+the	O
+ligand	O
+-	O
+occupied	O
+pocket	O
+(	O
+blue	O
+residues	O
+);	O
+these	O
+‘	O
+inward	O
+’	O
+conformations	O
+appear	O
+unfavorable	O
+for	O
+binding	O
+of	O
+4	O
+-	O
+HPA	O
+due	O
+to	O
+clashes	O
+with	O
+the	O
+4	O
+-	O
+hydroxyl	O
+group	O
+,	O
+the	O
+phenyl	O
+ring	O
+and	O
+the	O
+carboxylate	O
+group	O
+,	O
+respectively	O
+.	O
+	O
+In	O
+these	O
+crystals	O
+,	O
+the	O
+ArgA43	O
+side	O
+chain	O
+showed	O
+two	O
+alternate	O
+conformations	O
+,	O
+modelled	O
+with	O
+50	O
+%	O
+occupancy	O
+in	O
+each	O
+state	O
+,	O
+as	O
+indicated	O
+by	O
+the	O
+two	O
+‘	O
+mirrored	O
+’	O
+arrows	O
+.	O
+	O
+Finally	O
+,	O
+we	O
+applied	O
+15N	O
+heteronuclear	O
+solution	O
+NMR	O
+spectroscopy	O
+to	O
+examine	O
+the	O
+interaction	O
+of	O
+4	O
+-	O
+HPA	O
+with	O
+apo	O
+NadR	O
+.	O
+We	O
+collected	O
+NMR	O
+spectra	O
+on	O
+NadR	O
+in	O
+the	O
+presence	O
+and	O
+absence	O
+of	O
+4	O
+-	O
+HPA	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+	O
+The	O
+1H	O
+-	O
+15N	O
+TROSY	O
+-	O
+HSQC	O
+spectrum	O
+of	O
+apo	O
+-	O
+NadR	O
+,	O
+acquired	O
+at	O
+25	O
+°	O
+C	O
+,	O
+displayed	O
+approximately	O
+140	O
+distinct	O
+peaks	O
+(	O
+Fig	O
+6A	O
+),	O
+most	O
+of	O
+which	O
+correspond	O
+to	O
+backbone	O
+amide	O
+N	O
+-	O
+H	O
+groups	O
+.	O
+	O
+Upon	O
+the	O
+addition	O
+of	O
+4	O
+-	O
+HPA	O
+,	O
+over	O
+45	O
+peaks	O
+showed	O
+chemical	O
+shift	O
+perturbations	O
+,	O
+i	O
+.	O
+e	O
+.	O
+changed	O
+position	O
+in	O
+the	O
+spectrum	O
+or	O
+disappeared	O
+,	O
+while	O
+the	O
+remaining	O
+peaks	O
+remained	O
+unchanged	O
+.	O
+	O
+This	O
+observation	O
+showed	O
+that	O
+4	O
+-	O
+HPA	O
+was	O
+able	O
+to	O
+bind	O
+NadR	O
+and	O
+induce	O
+notable	O
+changes	O
+in	O
+specific	O
+regions	O
+of	O
+the	O
+protein	O
+.	O
+	O
+NMR	O
+spectra	O
+of	O
+NadR	O
+in	O
+the	O
+presence	O
+and	O
+absence	O
+of	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+(	O
+A	O
+)	O
+Superposition	O
+of	O
+two	O
+1H	O
+-	O
+15N	O
+TROSY	O
+-	O
+HSQC	O
+spectra	O
+recorded	O
+at	O
+25	O
+°	O
+C	O
+on	O
+apo	O
+-	O
+NadR	O
+(	O
+cyan	O
+)	O
+and	O
+on	O
+NadR	O
+in	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+(	O
+red	O
+).	O
+	O
+The	O
+spectra	O
+acquired	O
+at	O
+10	O
+°	O
+C	O
+are	O
+excluded	O
+from	O
+panel	O
+A	O
+for	O
+simplicity	O
+.	O
+	O
+However	O
+,	O
+in	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+,	O
+the	O
+1H	O
+-	O
+15N	O
+TROSY	O
+-	O
+HSQC	O
+spectrum	O
+of	O
+NadR	O
+displayed	O
+approximately	O
+140	O
+peaks	O
+,	O
+as	O
+for	O
+apo	O
+-	O
+NadR	O
+,	O
+i	O
+.	O
+e	O
+.	O
+two	O
+distinct	O
+stable	O
+conformations	O
+(	O
+that	O
+might	O
+have	O
+potentially	O
+revealed	O
+the	O
+molecular	O
+asymmetry	O
+observed	O
+crystallographically	O
+)	O
+were	O
+not	O
+notable	O
+.	O
+	O
+These	O
+doubled	O
+peaks	O
+may	O
+therefore	O
+reveal	O
+that	O
+the	O
+cooler	O
+temperature	O
+partially	O
+trapped	O
+the	O
+existence	O
+in	O
+solution	O
+of	O
+two	O
+distinct	O
+states	O
+,	O
+in	O
+presence	O
+or	O
+absence	O
+of	O
+4	O
+-	O
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+,	O
+with	O
+minor	O
+conformational	O
+differences	O
+occurring	O
+at	O
+least	O
+in	O
+proximity	O
+to	O
+the	O
+binding	O
+pocket	O
+.	O
+	O
+Apo	O
+-	O
+NadR	O
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+reveal	O
+intrinsic	O
+conformational	O
+flexibility	O
+	O
+The	O
+apo	O
+-	O
+NadR	O
+crystal	O
+structure	O
+contained	O
+two	O
+homodimers	O
+in	O
+the	O
+asymmetric	O
+unit	O
+(	O
+chains	O
+A	O
++	O
+B	O
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+chains	O
+C	O
++	O
+D	O
+).	O
+	O
+Upon	O
+overall	O
+structural	O
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+,	O
+these	O
+dimers	O
+revealed	O
+a	O
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+minor	O
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+in	O
+the	O
+α6	O
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+(	O
+a	O
+major	O
+component	O
+of	O
+the	O
+dimer	O
+interface	O
+)	O
+and	O
+the	O
+helices	O
+α4	O
+-	O
+α5	O
+(	O
+the	O
+DNA	O
+binding	O
+region	O
+),	O
+and	O
+an	O
+rmsd	O
+of	O
+1	O
+.	O
+55Å	O
+(	O
+Fig	O
+7A	O
+).	O
+	O
+The	O
+slightly	O
+larger	O
+rmsd	O
+between	O
+the	O
+two	O
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+-	O
+homodimers	O
+,	O
+rather	O
+than	O
+between	O
+apo	O
+-	O
+and	O
+holo	O
+-	O
+homodimers	O
+,	O
+further	O
+indicate	O
+that	O
+apo	O
+-	O
+NadR	O
+possesses	O
+a	O
+notable	O
+degree	O
+of	O
+intrinsic	O
+conformational	O
+flexibility	O
+.	O
+	O
+Overall	O
+apo	O
+-	O
+and	O
+holo	O
+-	O
+NadR	O
+structures	O
+are	O
+similar	O
+.	O
+	O
+(	O
+A	O
+)	O
+Pairwise	O
+alignment	O
+of	O
+the	O
+two	O
+distinct	O
+apo	O
+-	O
+NadR	O
+homodimers	O
+(	O
+AB	O
+and	O
+CD	O
+)	O
+present	O
+in	O
+the	O
+apo	O
+-	O
+NadR	O
+crystals	O
+.	O
+(	O
+B	O
+)	O
+Alignment	O
+of	O
+the	O
+holo	O
+-	O
+NadR	O
+homodimer	O
+(	O
+green	O
+and	O
+blue	O
+chains	O
+)	O
+onto	O
+the	O
+apo	O
+-	O
+NadR	O
+homodimers	O
+.	O
+	O
+Here	O
+,	O
+larger	O
+differences	O
+are	O
+observed	O
+in	O
+the	O
+α6	O
+helices	O
+(	O
+top	O
+).	O
+	O
+4	O
+-	O
+HPA	O
+stabilizes	O
+concerted	O
+conformational	O
+changes	O
+in	O
+NadR	O
+that	O
+prevent	O
+DNA	O
+-	O
+binding	O
+	O
+To	O
+further	O
+investigate	O
+the	O
+conformational	O
+rearrangements	O
+of	O
+NadR	O
+,	O
+we	O
+performed	O
+local	O
+structural	O
+alignments	O
+using	O
+only	O
+a	O
+subset	O
+of	O
+residues	O
+in	O
+the	O
+DNA	O
+-	O
+binding	O
+helix	O
+(	O
+α4	O
+).	O
+	O
+By	O
+selecting	O
+and	O
+aligning	O
+residues	O
+Arg64	O
+-	O
+Ala77	O
+of	O
+one	O
+α4	O
+helix	O
+per	O
+dimer	O
+,	O
+superposition	O
+of	O
+the	O
+holo	O
+-	O
+homodimer	O
+onto	O
+the	O
+two	O
+apo	O
+-	O
+homodimers	O
+revealed	O
+differences	O
+in	O
+the	O
+monomer	O
+conformations	O
+of	O
+each	O
+structure	O
+.	O
+	O
+While	O
+one	O
+monomer	O
+from	O
+each	O
+structure	O
+was	O
+closely	O
+superimposable	O
+(	O
+Fig	O
+8A	O
+,	O
+left	O
+side	O
+),	O
+the	O
+second	O
+monomer	O
+displayed	O
+quite	O
+large	O
+differences	O
+(	O
+Fig	O
+8A	O
+,	O
+right	O
+side	O
+).	O
+	O
+Most	O
+notably	O
+,	O
+the	O
+position	O
+of	O
+the	O
+DNA	O
+-	O
+binding	O
+helix	O
+α4	O
+shifted	O
+by	O
+as	O
+much	O
+as	O
+6	O
+Å	O
+(	O
+Fig	O
+8B	O
+).	O
+	O
+Accordingly	O
+,	O
+helix	O
+α4	O
+was	O
+also	O
+found	O
+to	O
+be	O
+one	O
+of	O
+the	O
+most	O
+dynamic	O
+regions	O
+in	O
+previous	O
+HDX	O
+-	O
+MS	O
+analyses	O
+of	O
+apo	O
+-	O
+NadR	O
+in	O
+solution	O
+.	O
+	O
+The	O
+α4	O
+helices	O
+aligned	O
+closely	O
+,	O
+Cα	O
+rmsd	O
+0	O
+.	O
+2Å	O
+for	O
+14	O
+residues	O
+.	O
+	O
+(	O
+B	O
+)	O
+The	O
+relative	O
+positions	O
+of	O
+the	O
+α4	O
+helices	O
+of	O
+the	O
+4	O
+-	O
+HPA	O
+-	O
+bound	O
+holo	O
+homodimer	O
+chain	O
+B	O
+(	O
+blue	O
+),	O
+and	O
+of	O
+apo	O
+homodimers	O
+AB	O
+and	O
+CD	O
+(	O
+showing	O
+chains	O
+B	O
+and	O
+D	O
+)	O
+in	O
+pale	O
+blue	O
+.	O
+	O
+Dashes	O
+indicate	O
+the	O
+Ala77	O
+Cα	O
+atoms	O
+,	O
+in	O
+the	O
+most	O
+highly	O
+shifted	O
+region	O
+of	O
+the	O
+‘	O
+non	O
+-	O
+fixed	O
+’	O
+α4	O
+helix	O
+.	O
+	O
+(	O
+C	O
+)	O
+The	O
+double	O
+-	O
+stranded	O
+DNA	O
+molecule	O
+(	O
+grey	O
+cartoon	O
+)	O
+from	O
+the	O
+OhrR	O
+-	O
+ohrA	O
+complex	O
+is	O
+shown	O
+after	O
+superposition	O
+with	O
+NadR	O
+,	O
+to	O
+highlight	O
+the	O
+expected	O
+positions	O
+of	O
+the	O
+NadR	O
+α4	O
+helices	O
+in	O
+the	O
+DNA	O
+major	O
+grooves	O
+.	O
+	O
+For	O
+clarity	O
+,	O
+only	O
+the	O
+α4	O
+helices	O
+are	O
+shown	O
+in	O
+panels	O
+(	O
+B	O
+)	O
+and	O
+(	O
+C	O
+).	O
+(	O
+D	O
+)	O
+Upon	O
+comparison	O
+with	O
+the	O
+experimentally	O
+-	O
+determined	O
+OhrR	O
+:	O
+ohrA	O
+structure	O
+(	O
+grey	O
+),	O
+the	O
+α4	O
+helix	O
+of	O
+holo	O
+-	O
+NadR	O
+(	O
+blue	O
+)	O
+is	O
+shifted	O
+~	O
+8Å	O
+out	O
+of	O
+the	O
+major	O
+groove	O
+.	O
+	O
+In	O
+summary	O
+,	O
+compared	O
+to	O
+ligand	O
+-	O
+stabilized	O
+holo	O
+-	O
+NadR	O
+,	O
+apo	O
+-	O
+NadR	O
+displayed	O
+an	O
+intrinsic	O
+flexibility	O
+focused	O
+in	O
+the	O
+DNA	O
+-	O
+binding	O
+region	O
+.	O
+	O
+This	O
+was	O
+also	O
+evident	O
+in	O
+the	O
+greater	O
+disorder	O
+(	O
+i	O
+.	O
+e	O
+.	O
+less	O
+well	O
+-	O
+defined	O
+electron	O
+density	O
+)	O
+in	O
+the	O
+β1	O
+-	O
+β2	O
+loops	O
+of	O
+the	O
+apo	O
+dimers	O
+(	O
+density	O
+for	O
+16	O
+residues	O
+per	O
+dimer	O
+was	O
+missing	O
+)	O
+compared	O
+to	O
+the	O
+holo	O
+dimer	O
+(	O
+density	O
+for	O
+only	O
+3	O
+residues	O
+was	O
+missing	O
+).	O
+	O
+In	O
+holo	O
+-	O
+NadR	O
+,	O
+the	O
+distance	O
+separating	O
+the	O
+two	O
+DNA	O
+-	O
+binding	O
+α4	O
+helices	O
+was	O
+32	O
+Å	O
+,	O
+while	O
+in	O
+apo	O
+-	O
+NadR	O
+it	O
+was	O
+29	O
+Å	O
+for	O
+homodimer	O
+AB	O
+,	O
+and	O
+34	O
+Å	O
+for	O
+homodimer	O
+CD	O
+(	O
+Fig	O
+8C	O
+).	O
+	O
+Pairwise	O
+superpositions	O
+showed	O
+that	O
+the	O
+NadR	O
+apo	O
+-	O
+homodimer	O
+AB	O
+was	O
+the	O
+most	O
+similar	O
+to	O
+OhrR	O
+(	O
+rmsd	O
+2	O
+.	O
+6	O
+Å	O
+),	O
+while	O
+the	O
+holo	O
+-	O
+homodimer	O
+was	O
+the	O
+most	O
+divergent	O
+(	O
+rmsd	O
+3	O
+.	O
+3	O
+Å	O
+)	O
+(	O
+Fig	O
+8C	O
+).	O
+	O
+Assuming	O
+the	O
+same	O
+DNA	O
+-	O
+binding	O
+mechanism	O
+is	O
+used	O
+by	O
+OhrR	O
+and	O
+NadR	O
+,	O
+the	O
+apo	O
+-	O
+homodimer	O
+AB	O
+seems	O
+ideally	O
+pre	O
+-	O
+configured	O
+for	O
+DNA	O
+binding	O
+,	O
+while	O
+4	O
+-	O
+HPA	O
+appeared	O
+to	O
+stabilize	O
+holo	O
+-	O
+NadR	O
+in	O
+a	O
+conformation	O
+poorly	O
+suited	O
+for	O
+DNA	O
+binding	O
+.	O
+	O
+Specifically	O
+,	O
+in	O
+addition	O
+to	O
+the	O
+different	O
+inter	O
+-	O
+helical	O
+translational	O
+distances	O
+,	O
+the	O
+α4	O
+helices	O
+in	O
+the	O
+holo	O
+-	O
+NadR	O
+homodimer	O
+were	O
+also	O
+reoriented	O
+,	O
+resulting	O
+in	O
+movement	O
+of	O
+α4	O
+out	O
+of	O
+the	O
+major	O
+groove	O
+,	O
+by	O
+up	O
+to	O
+8Å	O
+,	O
+and	O
+presumably	O
+preventing	O
+efficient	O
+DNA	O
+binding	O
+in	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+(	O
+Fig	O
+8D	O
+).	O
+	O
+NadR	O
+residues	O
+His7	O
+,	O
+Ser9	O
+,	O
+Asn11	O
+and	O
+Phe25	O
+are	O
+essential	O
+for	O
+regulation	O
+of	O
+NadA	O
+expression	O
+in	O
+vivo	O
+	O
+While	O
+previous	O
+studies	O
+had	O
+correctly	O
+suggested	O
+the	O
+involvement	O
+of	O
+several	O
+NadR	O
+residues	O
+in	O
+ligand	O
+binding	O
+,	O
+the	O
+crystal	O
+structures	O
+presented	O
+here	O
+revealed	O
+additional	O
+residues	O
+with	O
+previously	O
+unknown	O
+roles	O
+in	O
+dimerization	O
+and	O
+/	O
+or	O
+binding	O
+to	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+To	O
+explore	O
+the	O
+functional	O
+involvement	O
+of	O
+these	O
+residues	O
+,	O
+we	O
+characterized	O
+the	O
+behavior	O
+of	O
+four	O
+new	O
+NadR	O
+mutants	O
+(	O
+H7A	B-mutant
+,	O
+S9A	B-mutant
+,	O
+N11A	B-mutant
+and	O
+F25A	B-mutant
+)	O
+in	O
+an	O
+in	O
+vivo	O
+assay	O
+using	O
+the	O
+previously	O
+described	O
+MC58	B-mutant
+-	I-mutant
+Δ1843	I-mutant
+nadR	O
+-	O
+null	O
+mutant	O
+strain	O
+,	O
+which	O
+was	O
+complemented	O
+either	O
+by	O
+wild	O
+-	O
+type	O
+nadR	O
+or	O
+by	O
+the	O
+nadR	O
+mutants	O
+.	O
+	O
+NadA	O
+protein	O
+abundance	O
+levels	O
+were	O
+assessed	O
+by	O
+Western	O
+blotting	O
+to	O
+evaluate	O
+the	O
+ability	O
+of	O
+the	O
+NadR	O
+mutants	O
+to	O
+repress	O
+the	O
+nadA	O
+promoter	O
+,	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+The	O
+nadR	O
+H7A	B-mutant
+,	O
+S9A	B-mutant
+and	O
+F25A	B-mutant
+complemented	O
+strains	O
+showed	O
+hyper	O
+-	O
+repression	O
+of	O
+nadA	O
+expression	O
+in	O
+vivo	O
+,	O
+i	O
+.	O
+e	O
+.	O
+these	O
+mutants	O
+repressed	O
+nadA	O
+more	O
+efficiently	O
+than	O
+the	O
+NadR	O
+WT	O
+protein	O
+,	O
+either	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+4	O
+-	O
+HPA	O
+,	O
+while	O
+complementation	O
+with	O
+wild	O
+-	O
+type	O
+nadR	O
+resulted	O
+in	O
+high	O
+production	O
+of	O
+NadA	O
+only	O
+in	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+(	O
+Fig	O
+9	O
+).	O
+	O
+Interestingly	O
+,	O
+and	O
+on	O
+the	O
+contrary	O
+,	O
+the	O
+nadR	O
+N11A	B-mutant
+complemented	O
+strain	O
+showed	O
+hypo	O
+-	O
+repression	O
+(	O
+i	O
+.	O
+e	O
+.	O
+exhibited	O
+high	O
+expression	O
+of	O
+nadA	O
+both	O
+in	O
+absence	O
+and	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+).	O
+	O
+Structure	O
+-	O
+based	O
+point	O
+mutations	O
+shed	O
+light	O
+on	O
+ligand	O
+-	O
+induced	O
+regulation	O
+of	O
+NadR	O
+.	O
+	O
+Complementation	O
+of	O
+ΔNadR	B-mutant
+with	O
+WT	O
+NadR	O
+enables	O
+induction	O
+of	O
+nadA	O
+expression	O
+by	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+A	O
+detailed	O
+understanding	O
+of	O
+the	O
+in	O
+vitro	O
+repression	O
+of	O
+nadA	O
+expression	O
+by	O
+the	O
+transcriptional	O
+regulator	O
+NadR	O
+is	O
+important	O
+,	O
+both	O
+because	O
+it	O
+is	O
+a	O
+relevant	O
+disease	O
+-	O
+related	O
+model	O
+of	O
+how	O
+small	O
+-	O
+molecule	O
+ligands	O
+can	O
+regulate	O
+MarR	O
+family	O
+proteins	O
+and	O
+thereby	O
+impact	O
+bacterial	O
+virulence	O
+,	O
+and	O
+because	O
+nadA	O
+expression	O
+levels	O
+are	O
+linked	O
+to	O
+the	O
+prediction	O
+of	O
+vaccine	O
+coverage	O
+.	O
+	O
+The	O
+repressive	O
+activity	O
+of	O
+NadR	O
+can	O
+be	O
+relieved	O
+by	O
+hydroxyphenylacetate	O
+(	O
+HPA	O
+)	O
+ligands	O
+,	O
+and	O
+HDX	O
+-	O
+MS	O
+studies	O
+previously	O
+indicated	O
+that	O
+4	O
+-	O
+HPA	O
+stabilizes	O
+dimeric	O
+NadR	O
+in	O
+a	O
+configuration	O
+incompatible	O
+with	O
+DNA	O
+binding	O
+.	O
+	O
+Despite	O
+these	O
+and	O
+other	O
+studies	O
+,	O
+the	O
+molecular	O
+mechanisms	O
+by	O
+which	O
+ligands	O
+regulate	O
+MarR	O
+family	O
+proteins	O
+are	O
+relatively	O
+poorly	O
+understood	O
+and	O
+likely	O
+differ	O
+depending	O
+on	O
+the	O
+specific	O
+ligand	O
+.	O
+	O
+Firstly	O
+,	O
+we	O
+confirmed	O
+that	O
+NadR	O
+is	O
+dimeric	O
+in	O
+solution	O
+and	O
+demonstrated	O
+that	O
+it	O
+retains	O
+its	O
+dimeric	O
+state	O
+in	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+,	O
+indicating	O
+that	O
+induction	O
+of	O
+a	O
+monomeric	O
+status	O
+is	O
+not	O
+the	O
+manner	O
+by	O
+which	O
+4	O
+-	O
+HPA	O
+regulates	O
+NadR	O
+.	O
+These	O
+observations	O
+were	O
+in	O
+agreement	O
+with	O
+(	O
+i	O
+)	O
+a	O
+previous	O
+study	O
+of	O
+NadR	O
+performed	O
+using	O
+SEC	O
+and	O
+mass	O
+spectrometry	O
+,	O
+and	O
+(	O
+ii	O
+)	O
+crystallographic	O
+studies	O
+showing	O
+that	O
+several	O
+MarR	O
+homologues	O
+are	O
+dimeric	O
+.	O
+	O
+We	O
+also	O
+used	O
+structure	O
+-	O
+guided	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+to	O
+identify	O
+an	O
+important	O
+conserved	O
+residue	O
+,	O
+Leu130	O
+,	O
+which	O
+stabilizes	O
+the	O
+NadR	O
+dimer	O
+interface	O
+,	O
+knowledge	O
+of	O
+which	O
+may	O
+also	O
+inform	O
+future	O
+studies	O
+to	O
+explore	O
+the	O
+regulatory	O
+mechanisms	O
+of	O
+other	O
+MarR	O
+family	O
+proteins	O
+.	O
+	O
+Secondly	O
+,	O
+we	O
+assessed	O
+the	O
+thermal	O
+stability	O
+and	O
+unfolding	O
+of	O
+NadR	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+ligands	O
+.	O
+	O
+All	O
+DSC	O
+profiles	O
+showed	O
+a	O
+single	O
+peak	O
+,	O
+suggesting	O
+that	O
+a	O
+single	O
+unfolding	O
+event	O
+simultaneously	O
+disrupted	O
+the	O
+dimer	O
+and	O
+the	O
+monomer	O
+.	O
+	O
+HPA	O
+ligands	O
+specifically	O
+increased	O
+the	O
+stability	O
+of	O
+NadR	O
+.	O
+The	O
+largest	O
+effects	O
+were	O
+induced	O
+by	O
+the	O
+naturally	O
+-	O
+occurring	O
+compounds	O
+4	O
+-	O
+HPA	O
+and	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+,	O
+which	O
+,	O
+in	O
+SPR	O
+assays	O
+,	O
+were	O
+found	O
+to	O
+bind	O
+NadR	O
+with	O
+KD	O
+values	O
+of	O
+1	O
+.	O
+5	O
+mM	O
+and	O
+1	O
+.	O
+1	O
+mM	O
+,	O
+respectively	O
+.	O
+	O
+Although	O
+these	O
+NadR	O
+/	O
+HPA	O
+interactions	O
+appeared	O
+rather	O
+weak	O
+,	O
+their	O
+distinct	O
+affinities	O
+and	O
+specificities	O
+matched	O
+their	O
+in	O
+vitro	O
+effects	O
+and	O
+their	O
+biological	O
+relevance	O
+appears	O
+similar	O
+to	O
+previous	O
+proposals	O
+that	O
+certain	O
+small	O
+molecules	O
+,	O
+including	O
+some	O
+antibiotics	O
+,	O
+in	O
+the	O
+millimolar	O
+concentration	O
+range	O
+may	O
+be	O
+broad	O
+inhibitors	O
+of	O
+MarR	O
+family	O
+proteins	O
+.	O
+	O
+Indeed	O
+,	O
+4	O
+-	O
+HPA	O
+is	O
+found	O
+in	O
+human	O
+saliva	O
+and	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+is	O
+produced	O
+during	O
+inflammatory	O
+processes	O
+,	O
+suggesting	O
+that	O
+these	O
+natural	O
+ligands	O
+are	O
+encountered	O
+by	O
+N	O
+.	O
+meningitidis	O
+in	O
+the	O
+mucosa	O
+of	O
+the	O
+oropharynx	O
+during	O
+infections	O
+.	O
+	O
+It	O
+is	O
+also	O
+possible	O
+that	O
+NadR	O
+responds	O
+to	O
+currently	O
+unidentified	O
+HPA	O
+analogues	O
+.	O
+	O
+Indeed	O
+,	O
+in	O
+the	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+complex	O
+there	O
+was	O
+a	O
+water	O
+molecule	O
+close	O
+to	O
+the	O
+carboxylate	O
+group	O
+and	O
+also	O
+a	O
+small	O
+unfilled	O
+tunnel	O
+~	O
+5Å	O
+long	O
+,	O
+both	O
+factors	O
+suggesting	O
+that	O
+alternative	O
+larger	O
+ligands	O
+could	O
+occupy	O
+the	O
+pocket	O
+.	O
+	O
+The	O
+ability	O
+to	O
+respond	O
+to	O
+various	O
+ligands	O
+might	O
+enable	O
+NadR	O
+in	O
+vivo	O
+to	O
+orchestrate	O
+multiple	O
+response	O
+mechanisms	O
+and	O
+modulate	O
+expression	O
+of	O
+genes	O
+other	O
+than	O
+nadA	O
+.	O
+Ultimately	O
+,	O
+confirmation	O
+of	O
+the	O
+relevance	O
+of	O
+each	O
+ligand	O
+will	O
+require	O
+a	O
+deeper	O
+understanding	O
+of	O
+the	O
+available	O
+concentration	O
+in	O
+vivo	O
+in	O
+the	O
+host	O
+niche	O
+during	O
+bacterial	O
+colonization	O
+and	O
+inflammation	O
+.	O
+	O
+Here	O
+,	O
+we	O
+determined	O
+the	O
+first	O
+crystal	O
+structures	O
+of	O
+apo	O
+-	O
+NadR	O
+and	O
+holo	O
+-	O
+NadR	O
+.	O
+These	O
+experimentally	O
+-	O
+determined	O
+structures	O
+enabled	O
+a	O
+new	O
+detailed	O
+characterization	O
+of	O
+the	O
+ligand	O
+-	O
+binding	O
+pocket	O
+.	O
+	O
+In	O
+holo	O
+-	O
+NadR	O
+,	O
+4	O
+-	O
+HPA	O
+interacted	O
+directly	O
+with	O
+at	O
+least	O
+11	O
+polar	O
+and	O
+hydrophobic	O
+residues	O
+.	O
+	O
+Several	O
+,	O
+but	O
+not	O
+all	O
+,	O
+of	O
+these	O
+interactions	O
+were	O
+predicted	O
+previously	O
+by	O
+homology	O
+modelling	O
+combined	O
+with	O
+ligand	O
+docking	O
+in	O
+silico	O
+.	O
+	O
+Subsequently	O
+,	O
+we	O
+established	O
+the	O
+functional	O
+importance	O
+of	O
+His7	O
+,	O
+Ser9	O
+,	O
+Asn11	O
+and	O
+Phe25	O
+in	O
+the	O
+in	O
+vitro	O
+response	O
+of	O
+meningococcus	O
+to	O
+4	O
+-	O
+HPA	O
+,	O
+via	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+.	O
+	O
+More	O
+unexpectedly	O
+,	O
+the	O
+crystal	O
+structure	O
+revealed	O
+that	O
+only	O
+one	O
+molecule	O
+of	O
+4	O
+-	O
+HPA	O
+was	O
+bound	O
+per	O
+NadR	O
+dimer	O
+.	O
+	O
+We	O
+also	O
+used	O
+heteronuclear	O
+NMR	O
+spectroscopy	O
+to	O
+detect	O
+substantial	O
+conformational	O
+changes	O
+of	O
+NadR	O
+occurring	O
+in	O
+solution	O
+upon	O
+addition	O
+of	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+Moreover	O
+,	O
+NMR	O
+spectra	O
+at	O
+10	O
+°	O
+C	O
+suggested	O
+the	O
+existence	O
+of	O
+two	O
+distinct	O
+conformations	O
+of	O
+NadR	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+ligand	O
+-	O
+binding	O
+pocket	O
+.	O
+	O
+More	O
+powerfully	O
+,	O
+our	O
+unique	O
+crystallographic	O
+observation	O
+of	O
+this	O
+‘	O
+occupied	O
+vs	O
+unoccupied	O
+site	O
+’	O
+asymmetry	O
+in	O
+the	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+interaction	O
+is	O
+,	O
+to	O
+our	O
+knowledge	O
+,	O
+the	O
+first	O
+example	O
+reported	O
+for	O
+a	O
+MarR	O
+family	O
+protein	O
+.	O
+	O
+Such	O
+a	O
+mechanism	O
+indicates	O
+negative	O
+cooperativity	O
+,	O
+which	O
+may	O
+enhance	O
+the	O
+ligand	O
+-	O
+responsiveness	O
+of	O
+NadR	O
+.	O
+	O
+Comparisons	O
+of	O
+the	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+complex	O
+with	O
+available	O
+MarR	O
+family	O
+/	O
+salicylate	O
+complexes	O
+revealed	O
+that	O
+4	O
+-	O
+HPA	O
+has	O
+a	O
+previously	O
+unobserved	O
+binding	O
+mode	O
+.	O
+	O
+Briefly	O
+,	O
+in	O
+the	O
+M	O
+.	O
+thermoautotrophicum	O
+MTH313	O
+dimer	O
+,	O
+one	O
+molecule	O
+of	O
+salicylate	O
+binds	O
+in	O
+the	O
+pocket	O
+of	O
+each	O
+monomer	O
+,	O
+though	O
+with	O
+two	O
+rather	O
+different	O
+positions	O
+and	O
+orientations	O
+,	O
+only	O
+one	O
+of	O
+which	O
+(	O
+site	O
+-	O
+1	O
+)	O
+is	O
+thought	O
+to	O
+be	O
+biologically	O
+relevant	O
+(	O
+Fig	O
+10A	O
+).	O
+	O
+In	O
+NadR	O
+,	O
+the	O
+single	O
+molecule	O
+of	O
+4	O
+-	O
+HPA	O
+binds	O
+in	O
+a	O
+position	O
+distinctly	O
+different	O
+from	O
+the	O
+salicylate	O
+binding	O
+site	O
+:	O
+translated	O
+by	O
+>	O
+10	O
+Å	O
+and	O
+with	O
+a	O
+180	O
+°	O
+inverted	O
+orientation	O
+(	O
+Fig	O
+10C	O
+).	O
+	O
+(	O
+A	O
+)	O
+A	O
+structural	O
+alignment	O
+of	O
+MTH313	O
+chains	O
+A	O
+and	O
+B	O
+shows	O
+that	O
+salicylate	O
+is	O
+bound	O
+in	O
+distinct	O
+locations	O
+in	O
+each	O
+monomer	O
+;	O
+site	O
+-	O
+1	O
+(	O
+thought	O
+to	O
+be	O
+the	O
+biologically	O
+relevant	O
+site	O
+)	O
+and	O
+site	O
+-	O
+2	O
+differ	O
+by	O
+~	O
+7Å	O
+(	O
+indicated	O
+by	O
+black	O
+dotted	O
+line	O
+)	O
+and	O
+also	O
+by	O
+ligand	O
+orientation	O
+.	O
+	O
+(	O
+C	O
+)	O
+Addition	O
+of	O
+holo	O
+-	O
+NadR	O
+(	O
+chain	O
+B	O
+,	O
+blue	O
+)	O
+to	O
+the	O
+alignment	O
+reveals	O
+that	O
+bound	O
+4	O
+-	O
+HPA	O
+differs	O
+in	O
+position	O
+by	O
+>	O
+10	O
+Å	O
+compared	O
+to	O
+salicylate	O
+,	O
+and	O
+adopts	O
+a	O
+novel	O
+orientation	O
+.	O
+	O
+Interestingly	O
+,	O
+a	O
+crystal	O
+structure	O
+was	O
+previously	O
+reported	O
+for	O
+a	O
+functionally	O
+-	O
+uncharacterized	O
+meningococcal	O
+homologue	O
+of	O
+NadR	O
+,	O
+termed	O
+NMB1585	O
+,	O
+which	O
+shares	O
+16	O
+%	O
+sequence	O
+identity	O
+with	O
+NadR	O
+.	O
+The	O
+two	O
+structures	O
+can	O
+be	O
+closely	O
+aligned	O
+(	O
+rmsd	O
+2	O
+.	O
+3	O
+Å	O
+),	O
+but	O
+NMB1585	O
+appears	O
+unsuited	O
+for	O
+binding	O
+HPAs	O
+,	O
+since	O
+its	O
+corresponding	O
+‘	O
+pocket	O
+’	O
+region	O
+is	O
+occupied	O
+by	O
+several	O
+bulky	O
+hydrophobic	O
+side	O
+chains	O
+.	O
+	O
+It	O
+can	O
+be	O
+speculated	O
+that	O
+MarR	O
+family	O
+members	O
+have	O
+evolved	O
+separately	O
+to	O
+engage	O
+distinct	O
+signaling	O
+molecules	O
+,	O
+thus	O
+enabling	O
+bacteria	O
+to	O
+use	O
+the	O
+overall	O
+conserved	O
+MarR	O
+scaffold	O
+to	O
+adapt	O
+and	O
+respond	O
+to	O
+diverse	O
+changing	O
+environmental	O
+stimuli	O
+experienced	O
+in	O
+their	O
+natural	O
+niches	O
+.	O
+	O
+The	O
+apo	O
+-	O
+NadR	O
+crystal	O
+structures	O
+revealed	O
+two	O
+dimers	O
+with	O
+slightly	O
+different	O
+conformations	O
+,	O
+most	O
+divergent	O
+in	O
+the	O
+DNA	O
+-	O
+binding	O
+domain	O
+.	O
+	O
+It	O
+is	O
+not	O
+unusual	O
+for	O
+a	O
+crystal	O
+structure	O
+to	O
+reveal	O
+multiple	O
+copies	O
+of	O
+the	O
+same	O
+protein	O
+in	O
+very	O
+slightly	O
+different	O
+conformations	O
+,	O
+which	O
+are	O
+likely	O
+representative	O
+of	O
+the	O
+lowest	O
+-	O
+energy	O
+conformations	O
+sampled	O
+by	O
+the	O
+dynamic	O
+ensemble	O
+of	O
+molecular	O
+states	O
+occurring	O
+in	O
+solution	O
+,	O
+and	O
+which	O
+likely	O
+have	O
+only	O
+small	O
+energetic	O
+differences	O
+,	O
+as	O
+described	O
+previously	O
+for	O
+MexR	O
+(	O
+a	O
+MarR	O
+protein	O
+)	O
+or	O
+more	O
+recently	O
+for	O
+the	O
+solute	O
+-	O
+binding	O
+protein	O
+FhuD2	O
+.	O
+	O
+Further	O
+,	O
+the	O
+holo	O
+-	O
+NadR	O
+structure	O
+was	O
+overall	O
+more	O
+different	O
+from	O
+the	O
+two	O
+apo	O
+-	O
+NadR	O
+structures	O
+(	O
+rmsd	O
+values	O
+~	O
+1	O
+.	O
+3Å	O
+),	O
+suggesting	O
+that	O
+the	O
+ligand	O
+selected	O
+and	O
+stabilized	O
+yet	O
+another	O
+conformation	O
+of	O
+NadR	O
+.	O
+These	O
+observations	O
+suggest	O
+that	O
+4	O
+-	O
+HPA	O
+,	O
+and	O
+potentially	O
+other	O
+similar	O
+ligands	O
+,	O
+can	O
+shift	O
+the	O
+molecular	O
+equilibrium	O
+,	O
+changing	O
+the	O
+energy	O
+barriers	O
+that	O
+separate	O
+active	O
+and	O
+inactive	O
+states	O
+,	O
+and	O
+stabilizing	O
+the	O
+specific	O
+conformation	O
+of	O
+NadR	O
+poorly	O
+suited	O
+to	O
+bind	O
+DNA	O
+.	O
+	O
+Comparisons	O
+of	O
+the	O
+apo	O
+-	O
+and	O
+holo	O
+-	O
+NadR	O
+structures	O
+revealed	O
+that	O
+the	O
+largest	O
+differences	O
+occurred	O
+in	O
+the	O
+DNA	O
+-	O
+binding	O
+helix	O
+α4	O
+.	O
+	O
+The	O
+shift	O
+of	O
+helix	O
+α4	O
+in	O
+holo	O
+-	O
+NadR	O
+was	O
+also	O
+accompanied	O
+by	O
+rearrangements	O
+at	O
+the	O
+dimer	O
+interface	O
+,	O
+involving	O
+helices	O
+α1	O
+,	O
+α5	O
+,	O
+and	O
+α6	O
+,	O
+and	O
+this	O
+holo	O
+-	O
+form	O
+appeared	O
+poorly	O
+suited	O
+for	O
+DNA	O
+-	O
+binding	O
+when	O
+compared	O
+with	O
+the	O
+known	O
+OhrR	O
+:	O
+ohrA	O
+complex	O
+.	O
+	O
+One	O
+of	O
+the	O
+two	O
+conformations	O
+of	O
+apo	O
+-	O
+NadR	O
+appeared	O
+ideally	O
+suited	O
+for	O
+DNA	O
+-	O
+binding	O
+.	O
+	O
+Overall	O
+,	O
+these	O
+analyses	O
+suggest	O
+that	O
+the	O
+apo	O
+-	O
+NadR	O
+dimer	O
+has	O
+a	O
+pre	O
+-	O
+existing	O
+equilibrium	O
+that	O
+samples	O
+a	O
+variety	O
+of	O
+conformations	O
+,	O
+some	O
+of	O
+which	O
+are	O
+compatible	O
+with	O
+DNA	O
+binding	O
+.	O
+	O
+Subsequently	O
+,	O
+upon	O
+ligand	O
+binding	O
+,	O
+holo	O
+-	O
+NadR	O
+adopts	O
+a	O
+structure	O
+less	O
+suited	O
+for	O
+DNA	O
+-	O
+binding	O
+and	O
+this	O
+conformation	O
+is	O
+selected	O
+and	O
+stabilized	O
+by	O
+a	O
+network	O
+of	O
+protein	O
+-	O
+ligand	O
+interactions	O
+and	O
+concomitant	O
+rearrangements	O
+at	O
+the	O
+NadR	O
+holo	O
+dimer	O
+interface	O
+.	O
+	O
+In	O
+an	O
+alternative	O
+and	O
+less	O
+extensive	O
+manner	O
+,	O
+the	O
+binding	O
+of	O
+two	O
+salicylate	O
+molecules	O
+to	O
+the	O
+M	O
+.	O
+thermoautotrophicum	O
+protein	O
+MTH313	O
+appeared	O
+to	O
+induce	O
+large	O
+changes	O
+in	O
+the	O
+wHTH	O
+domain	O
+,	O
+which	O
+was	O
+associated	O
+with	O
+reduced	O
+DNA	O
+-	O
+binding	O
+activity	O
+.	O
+	O
+Here	O
+we	O
+have	O
+presented	O
+two	O
+new	O
+crystal	O
+structures	O
+of	O
+the	O
+transcription	O
+factor	O
+,	O
+NadR	O
+,	O
+which	O
+regulates	O
+expression	O
+of	O
+the	O
+meningococcal	O
+surface	O
+protein	O
+,	O
+virulence	O
+factor	O
+and	O
+vaccine	O
+antigen	O
+NadA	O
+.	O
+Detailed	O
+structural	O
+analyses	O
+provided	O
+a	O
+molecular	O
+explanation	O
+for	O
+the	O
+ligand	O
+-	O
+responsive	O
+regulation	O
+by	O
+NadR	O
+on	O
+the	O
+majority	O
+of	O
+the	O
+promoters	O
+of	O
+meningococcal	O
+genes	O
+regulated	O
+by	O
+NadR	O
+,	O
+including	O
+nadA	O
+.	O
+Intriguingly	O
+,	O
+NadR	O
+exhibits	O
+a	O
+reversed	O
+regulatory	O
+mechanism	O
+on	O
+a	O
+second	O
+class	O
+of	O
+promoters	O
+,	O
+including	O
+mafA	O
+of	O
+the	O
+multiple	O
+adhesin	O
+family	O
+–	O
+i	O
+.	O
+e	O
+.	O
+NadR	O
+represses	O
+these	O
+genes	O
+in	O
+the	O
+presence	O
+but	O
+not	O
+absence	O
+of	O
+4	O
+-	O
+HPA	O
+.	O
+	O
+Ultimately	O
+,	O
+knowledge	O
+of	O
+the	O
+ligand	O
+-	O
+dependent	O
+activity	O
+of	O
+NadR	O
+will	O
+continue	O
+to	O
+deepen	O
+our	O
+understanding	O
+of	O
+nadA	O
+expression	O
+levels	O
+,	O
+which	O
+influence	O
+meningococcal	O
+pathogenesis	O
+.	O
+	O
+The	O
+structure	O
+of	O
+NMB1585	O
+,	O
+a	O
+MarR	O
+-	O
+family	O
+regulator	O
+from	O
+Neisseria	O
+meningitidis	O
+	O
+The	O
+nuclear	O
+hormone	O
+receptor	O
+RORγ	O
+regulates	O
+transcriptional	O
+genes	O
+involved	O
+in	O
+the	O
+production	O
+of	O
+the	O
+pro	O
+-	O
+inflammatory	O
+interleukin	O
+IL	O
+-	O
+17	O
+which	O
+has	O
+been	O
+linked	O
+to	O
+autoimmune	O
+diseases	O
+such	O
+as	O
+rheumatoid	O
+arthritis	O
+,	O
+multiple	O
+sclerosis	O
+and	O
+inflammatory	O
+bowel	O
+disease	O
+.	O
+	O
+This	O
+transcriptional	O
+activity	O
+of	O
+RORγ	O
+is	O
+modulated	O
+through	O
+a	O
+protein	O
+-	O
+protein	O
+interaction	O
+involving	O
+the	O
+activation	O
+function	O
+2	O
+(	O
+AF2	O
+)	O
+helix	O
+on	O
+the	O
+ligand	O
+binding	O
+domain	O
+of	O
+RORγ	O
+and	O
+a	O
+conserved	O
+LXXLL	O
+helix	O
+motif	O
+on	O
+coactivator	O
+proteins	O
+.	O
+	O
+We	O
+identified	O
+a	O
+novel	O
+series	O
+of	O
+synthetic	O
+benzoxazinone	O
+ligands	O
+having	O
+an	O
+agonist	O
+(	O
+BIO592	O
+)	O
+and	O
+inverse	O
+agonist	O
+(	O
+BIO399	O
+)	O
+mode	O
+of	O
+action	O
+in	O
+a	O
+FRET	O
+based	O
+assay	O
+.	O
+	O
+We	O
+show	O
+that	O
+the	O
+AF2	O
+helix	O
+of	O
+RORγ	O
+is	O
+proteolytically	O
+sensitive	O
+when	O
+inverse	O
+agonist	O
+BIO399	O
+binds	O
+.	O
+	O
+Using	O
+x	O
+-	O
+ray	O
+crystallography	O
+we	O
+show	O
+how	O
+small	O
+modifications	O
+on	O
+the	O
+benzoxazinone	O
+agonist	O
+BIO592	O
+trigger	O
+inverse	O
+agonism	O
+of	O
+RORγ	O
+.	O
+	O
+The	O
+proteolytic	O
+sensitivity	O
+of	O
+the	O
+AF2	O
+helix	O
+of	O
+RORγ	O
+demonstrates	O
+that	O
+it	O
+destabilizes	O
+upon	O
+BIO399	O
+inverse	O
+agonist	O
+binding	O
+perturbing	O
+the	O
+coactivator	O
+protein	O
+binding	O
+site	O
+.	O
+	O
+Even	O
+though	O
+a	O
+high	O
+degree	O
+of	O
+sequence	O
+similarity	O
+exists	O
+between	O
+the	O
+RORs	O
+,	O
+their	O
+functional	O
+roles	O
+in	O
+regulation	O
+for	O
+physiological	O
+processes	O
+involved	O
+in	O
+development	O
+and	O
+immunity	O
+are	O
+distinct	O
+.	O
+	O
+During	O
+development	O
+,	O
+RORγ	O
+regulates	O
+the	O
+transcriptional	O
+genes	O
+involved	O
+in	O
+the	O
+functioning	O
+of	O
+multiple	O
+pro	O
+-	O
+inflammatory	O
+lymphocyte	O
+lineages	O
+including	O
+T	O
+helper	O
+cells	O
+(	O
+TH17cells	O
+)	O
+which	O
+are	O
+necessary	O
+for	O
+IL	O
+-	O
+17	O
+production	O
+.	O
+	O
+IL	O
+-	O
+17	O
+is	O
+a	O
+pro	O
+-	O
+inflammatory	O
+interleukin	O
+linked	O
+to	O
+autoimmune	O
+diseases	O
+such	O
+as	O
+rheumatoid	O
+arthritis	O
+,	O
+multiple	O
+sclerosis	O
+and	O
+inflammatory	O
+bowel	O
+disease	O
+;	O
+making	O
+its	O
+transcriptional	O
+regulation	O
+through	O
+RORγ	O
+an	O
+attractive	O
+therapeutic	O
+target	O
+.	O
+	O
+RORγ	O
+consists	O
+of	O
+an	O
+N	O
+-	O
+terminal	O
+DNA	O
+binding	O
+domain	O
+(	O
+DBD	O
+)	O
+connected	O
+to	O
+a	O
+C	O
+-	O
+terminal	O
+ligand	O
+binding	O
+domain	O
+(	O
+LBD	O
+)	O
+via	O
+a	O
+flexible	O
+hinge	O
+region	O
+.	O
+	O
+The	O
+DBD	O
+is	O
+composed	O
+of	O
+two	O
+zinc	O
+fingers	O
+that	O
+allow	O
+it	O
+to	O
+interact	O
+with	O
+specifically	O
+encoded	O
+regions	O
+on	O
+the	O
+DNA	O
+called	O
+the	O
+nuclear	O
+receptor	O
+response	O
+elements	O
+.	O
+	O
+The	O
+LBD	O
+consists	O
+of	O
+a	O
+coactivator	O
+protein	O
+binding	O
+pocket	O
+and	O
+a	O
+hydrophobic	O
+ligand	O
+binding	O
+site	O
+(	O
+LBS	O
+)	O
+which	O
+are	O
+responsible	O
+for	O
+regulating	O
+transcription	O
+.	O
+	O
+The	O
+coactivator	O
+binding	O
+pocket	O
+of	O
+RORγ	O
+recognizes	O
+a	O
+conserved	O
+helix	O
+motif	O
+LXXLL	O
+(	O
+where	O
+X	O
+can	O
+be	O
+any	O
+amino	O
+acid	O
+)	O
+on	O
+transcriptional	O
+coactivator	O
+complexes	O
+and	O
+recruits	O
+it	O
+to	O
+activate	O
+transcription	O
+.	O
+	O
+In	O
+RORγ	O
+,	O
+the	O
+conformation	O
+of	O
+the	O
+AF2	O
+helix	O
+required	O
+to	O
+form	O
+the	O
+coactivator	O
+binding	O
+pocket	O
+is	O
+mediated	O
+by	O
+a	O
+salt	O
+bridge	O
+between	O
+His479	O
+and	O
+Tyr502	O
+in	O
+addition	O
+to	O
+π	O
+-	O
+π	O
+interactions	O
+between	O
+Tyr502	O
+and	O
+Phe506	O
+.	O
+	O
+The	O
+conformation	O
+of	O
+the	O
+AF2	O
+helix	O
+can	O
+be	O
+modulated	O
+through	O
+targeted	O
+ligands	O
+which	O
+bind	O
+the	O
+LBS	O
+and	O
+increase	O
+the	O
+binding	O
+of	O
+the	O
+coactivator	O
+protein	O
+(	O
+agonists	O
+)	O
+or	O
+disrupt	O
+binding	O
+(	O
+inverse	O
+agonists	O
+)	O
+thereby	O
+enhancing	O
+or	O
+inhibiting	O
+transcription	O
+.	O
+	O
+Since	O
+RORγ	O
+has	O
+been	O
+demonstrated	O
+to	O
+play	O
+an	O
+important	O
+role	O
+in	O
+pro	O
+-	O
+inflammatory	O
+gene	O
+expression	O
+patterns	O
+implicated	O
+in	O
+several	O
+major	O
+autoimmune	O
+diseases	O
+,	O
+our	O
+aim	O
+was	O
+to	O
+develop	O
+RORγ	O
+inverse	O
+agonists	O
+that	O
+would	O
+help	O
+down	O
+regulate	O
+pro	O
+-	O
+inflammatory	O
+gene	O
+transcription	O
+.	O
+	O
+Finally	O
+,	O
+comparing	O
+binding	O
+modes	O
+of	O
+our	O
+benzoxazinone	O
+RORγ	O
+crystal	O
+structures	O
+to	O
+other	O
+ROR	O
+structures	O
+,	O
+we	O
+hypothesize	O
+a	O
+new	O
+mode	O
+of	O
+action	O
+for	O
+achieving	O
+inverse	O
+agonism	O
+and	O
+selectivity	O
+.	O
+	O
+Interestingly	O
+,	O
+the	O
+structural	O
+difference	O
+between	O
+the	O
+agonist	O
+BIO592	O
+and	O
+inverse	O
+agonist	O
+BIO399	O
+was	O
+minor	O
+;	O
+with	O
+the	O
+2	O
+,	O
+3	O
+-	O
+dihydrobenzo	O
+[	O
+1	O
+,	O
+4	O
+]	O
+oxazepin	O
+-	O
+4	O
+-	O
+one	O
+ring	O
+system	O
+of	O
+BIO399	O
+being	O
+3	O
+atoms	O
+larger	O
+than	O
+the	O
+benzo	O
+[	O
+1	O
+,	O
+4	O
+]	O
+oxazine	O
+-	O
+3	O
+-	O
+one	O
+ring	O
+system	O
+of	O
+BIO592	O
+.	O
+	O
+In	O
+order	O
+to	O
+understand	O
+how	O
+small	O
+changes	O
+in	O
+the	O
+core	O
+ring	O
+system	O
+leads	O
+to	O
+inverse	O
+agonism	O
+,	O
+we	O
+wanted	O
+to	O
+structurally	O
+determine	O
+the	O
+binding	O
+mode	O
+of	O
+both	O
+BIO592	O
+and	O
+BIO399	O
+in	O
+the	O
+LBS	O
+of	O
+RORγ	O
+using	O
+x	O
+-	O
+ray	O
+crystallography	O
+.	O
+	O
+Structure	O
+of	O
+the	O
+RORγ518	O
+-	O
+BIO592	O
+-	O
+EBI96	O
+ternary	O
+complex	O
+is	O
+in	O
+a	O
+transcriptionally	O
+active	O
+conformation	O
+	O
+RORγ518	O
+bound	O
+to	O
+agonist	O
+BIO592	O
+was	O
+crystallized	O
+with	O
+a	O
+truncated	O
+form	O
+of	O
+the	O
+coactivator	O
+peptide	O
+EBI96	O
+to	O
+a	O
+resolution	O
+of	O
+2	O
+.	O
+6	O
+Å	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+The	O
+hydrogen	O
+bond	O
+between	O
+His479	O
+and	O
+Tyr502	O
+has	O
+been	O
+reported	O
+to	O
+be	O
+critical	O
+for	O
+RORγ	O
+agonist	O
+activity	O
+.	O
+	O
+Disrupting	O
+this	O
+interaction	O
+through	O
+mutagenesis	O
+reduced	O
+transcriptional	O
+activity	O
+of	O
+RORγ	O
+.	O
+	O
+This	O
+reduced	O
+transcriptional	O
+activity	O
+has	O
+been	O
+attributed	O
+to	O
+the	O
+inability	O
+of	O
+the	O
+AF2	O
+helix	O
+to	O
+complete	O
+the	O
+formation	O
+of	O
+the	O
+coactivator	O
+binding	O
+pocket	O
+necessary	O
+for	O
+coactivator	O
+proteins	O
+to	O
+bind	O
+.	O
+	O
+This	O
+interaction	O
+is	O
+further	O
+stabilized	O
+through	O
+a	O
+conserved	O
+charged	O
+clamp	O
+wherein	O
+the	O
+backbone	O
+amide	O
+of	O
+Tyr7	O
+and	O
+carbonyl	O
+of	O
+Leu11	O
+of	O
+EBI96	O
+form	O
+hydrogen	O
+bonds	O
+with	O
+Glu504	O
+(	O
+helix12	O
+)	O
+and	O
+Lys336	O
+(	O
+helix3	O
+)	O
+of	O
+RORγ	O
+.	O
+	O
+Formation	O
+of	O
+this	O
+charged	O
+clamp	O
+is	O
+essential	O
+for	O
+RORγ	O
+’	O
+s	O
+function	O
+for	O
+playing	O
+a	O
+role	O
+in	O
+transcriptional	O
+activation	O
+and	O
+this	O
+has	O
+been	O
+corroborated	O
+through	O
+mutagenic	O
+studies	O
+in	O
+this	O
+region	O
+.	O
+	O
+BIO592	O
+binds	O
+in	O
+a	O
+collapsed	O
+conformation	O
+stabilizing	O
+the	O
+agonist	O
+conformation	O
+of	O
+RORγ	O
+	O
+a	O
+Collapsed	O
+binding	O
+mode	O
+of	O
+agonist	O
+BIO592	O
+in	O
+the	O
+hydrophobic	O
+LBS	O
+of	O
+RORγ	O
+.	O
+	O
+The	O
+sulfonyl	O
+group	O
+faces	O
+the	O
+entrance	O
+of	O
+the	O
+pocket	O
+,	O
+while	O
+the	O
+CF3	O
+makes	O
+a	O
+hydrophobic	O
+contact	O
+with	O
+Ala327	O
+.	O
+	O
+RORγ	O
+AF2	O
+helix	O
+is	O
+sensitive	O
+to	O
+proteolysis	O
+in	O
+the	O
+presence	O
+of	O
+Inverse	O
+Agonist	O
+BIO399	O
+	O
+Next	O
+,	O
+we	O
+attempted	O
+co	O
+-	O
+crystallization	O
+with	O
+the	O
+inverse	O
+agonist	O
+BIO399	O
+.	O
+	O
+However	O
+,	O
+extensive	O
+crystallization	O
+efforts	O
+with	O
+BIO399	O
+and	O
+RORγ518	O
+or	O
+other	O
+AF2	O
+intact	O
+constructs	O
+did	O
+not	O
+produce	O
+crystals	O
+.	O
+	O
+We	O
+hypothesized	O
+that	O
+the	O
+RORγ518	O
+coactivator	O
+peptide	O
+interaction	O
+in	O
+the	O
+FRET	O
+assay	O
+was	O
+disrupted	O
+upon	O
+BIO399	O
+binding	O
+and	O
+that	O
+a	O
+conformational	O
+rearrangement	O
+of	O
+the	O
+AF2	O
+helix	O
+could	O
+have	O
+occurred	O
+,	O
+hindering	O
+crystallization	O
+.	O
+	O
+The	O
+unfolding	O
+of	O
+the	O
+AF2	O
+helix	O
+has	O
+been	O
+observed	O
+for	O
+other	O
+nuclear	O
+hormone	O
+receptors	O
+when	O
+bound	O
+to	O
+an	O
+inverse	O
+agonist	O
+or	O
+antagonist	O
+.	O
+	O
+We	O
+used	O
+partial	O
+proteolysis	O
+in	O
+combination	O
+with	O
+mass	O
+spectrometry	O
+to	O
+determine	O
+if	O
+BIO399	O
+was	O
+causing	O
+the	O
+AF2	O
+helix	O
+to	O
+unfold	O
+.	O
+	O
+Results	O
+of	O
+the	O
+Actinase	O
+E	O
+proteolysis	O
+experiments	O
+on	O
+RORγ518	O
+,	O
+the	O
+ternary	O
+complex	O
+of	O
+RORγ518	O
+with	O
+agonist	O
+BIO592	O
+and	O
+coactivator	O
+EBI96	O
+,	O
+or	O
+in	O
+the	O
+presence	O
+of	O
+inverse	O
+agonist	O
+BIO399	O
+supported	O
+our	O
+hypothesis	O
+.	O
+	O
+Analysis	O
+of	O
+the	O
+fragmentation	O
+pattern	O
+showed	O
+minimal	O
+proteolytic	O
+removal	O
+of	O
+the	O
+AF2	O
+helix	O
+by	O
+Actinase	O
+E	O
+on	O
+RORγ518	O
+alone	O
+(	O
+ending	O
+at	O
+504	O
+to	O
+506	O
+)	O
+and	O
+the	O
+ternary	O
+complex	O
+remained	O
+primarily	O
+intact	O
+(	O
+ending	O
+at	O
+515	O
+/	O
+518	O
+)	O
+(	O
+Additional	O
+file	O
+4	O
+).	O
+	O
+We	O
+attributed	O
+the	O
+inability	O
+to	O
+form	O
+crystals	O
+to	O
+the	O
+unfolding	O
+of	O
+the	O
+AF2	O
+helix	O
+induced	O
+by	O
+BIO399	O
+.	O
+	O
+AF2	O
+truncated	O
+RORγ	O
+BIO399	O
+complex	O
+is	O
+more	O
+amenable	O
+to	O
+crystallization	O
+	O
+a	O
+The	O
+binary	O
+structure	O
+of	O
+AF2	O
+-	O
+truncated	O
+RORγ	O
+and	O
+BIO399	O
+.	O
+	O
+b	O
+The	O
+superposition	O
+of	O
+inverse	O
+agonist	O
+BIO399	O
+(	O
+Cyan	O
+)	O
+and	O
+agonist	O
+BIO592	O
+(	O
+Green	O
+).	O
+	O
+c	O
+Movement	O
+of	O
+Met358	O
+and	O
+His479	O
+in	O
+the	O
+BIO399	O
+(	O
+Cyan	O
+)	O
+and	O
+BIO592	O
+(	O
+Green	O
+)	O
+structures	O
+	O
+The	O
+Actinase	O
+E	O
+treated	O
+RORγ518	O
+BIO399	O
+ternary	O
+complex	O
+(	O
+aeRORγ493	O
+/	O
+4	O
+)	O
+co	O
+-	O
+crystallized	O
+readily	O
+in	O
+several	O
+PEG	O
+based	O
+conditions	O
+.	O
+	O
+The	O
+structure	O
+of	O
+aeRORγ493	O
+/	O
+4	O
+BIO399	O
+complex	O
+was	O
+solved	O
+to	O
+2	O
+.	O
+3	O
+Å	O
+and	O
+adopted	O
+a	O
+similar	O
+core	O
+fold	O
+to	O
+the	O
+BIO592	O
+agonist	O
+crystal	O
+structure	O
+(	O
+Fig	O
+.	O
+5a	O
+,	O
+Additional	O
+file	O
+3	O
+).	O
+	O
+Inverse	O
+agonist	O
+BIO399	O
+uses	O
+Met358	O
+as	O
+a	O
+trigger	O
+for	O
+inverse	O
+agonism	O
+	O
+The	O
+majority	O
+of	O
+the	O
+side	O
+chains	O
+within	O
+4	O
+Å	O
+of	O
+BIO399	O
+and	O
+BIO592	O
+adopt	O
+similar	O
+rotomer	O
+conformations	O
+with	O
+the	O
+exceptions	O
+of	O
+Met358	O
+and	O
+His479	O
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+	O
+The	O
+difference	O
+density	O
+map	O
+showed	O
+clear	O
+positive	O
+density	O
+for	O
+Met358	O
+in	O
+an	O
+alternate	O
+rotomer	O
+conformation	O
+compared	O
+to	O
+the	O
+one	O
+observed	O
+in	O
+the	O
+molecular	O
+replacement	O
+model	O
+or	O
+the	O
+other	O
+agonist	O
+containing	O
+models	O
+(	O
+Additional	O
+file	O
+6	O
+).	O
+	O
+We	O
+tried	O
+to	O
+refine	O
+Met358	O
+in	O
+the	O
+same	O
+conformation	O
+as	O
+the	O
+molecular	O
+replacement	O
+model	O
+or	O
+the	O
+other	O
+agonist	O
+containing	O
+models	O
+,	O
+but	O
+the	O
+results	O
+clearly	O
+indicated	O
+that	O
+this	O
+was	O
+not	O
+possible	O
+,	O
+thus	O
+confirming	O
+the	O
+new	O
+rotamer	O
+conformation	O
+for	O
+the	O
+Met358	O
+sidechain	O
+in	O
+the	O
+inverse	O
+agonist	O
+bound	O
+structure	O
+.	O
+	O
+The	O
+change	O
+in	O
+rotomer	O
+conformation	O
+of	O
+Met358	O
+between	O
+the	O
+agonist	O
+and	O
+inverse	O
+agonist	O
+structures	O
+is	O
+attributed	O
+to	O
+the	O
+gem	O
+-	O
+dimethyl	O
+group	O
+on	O
+the	O
+larger	O
+7	O
+membered	O
+benzoxazinone	O
+ring	O
+system	O
+of	O
+BIO399	O
+.	O
+	O
+The	O
+comparison	O
+of	O
+the	O
+two	O
+structures	O
+shows	O
+that	O
+the	O
+agonist	O
+conformation	O
+observed	O
+in	O
+the	O
+BIO592	O
+structure	O
+would	O
+be	O
+perturbed	O
+by	O
+BIO399	O
+pushing	O
+Met358	O
+into	O
+Phe506	O
+of	O
+the	O
+AF2	O
+helix	O
+indicating	O
+that	O
+Met358	O
+is	O
+a	O
+trigger	O
+for	O
+inducing	O
+inverse	O
+agonism	O
+in	O
+RORγ	O
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+	O
+b	O
+Overlay	O
+of	O
+M358	O
+in	O
+RORγ	O
+structure	O
+BIO596	O
+(	O
+Green	O
+),	O
+BIO399	O
+(	O
+Cyan	O
+),	O
+Digoxin	O
+(	O
+Yellow	O
+),	O
+Compound	O
+2	O
+(	O
+Grey	O
+),	O
+Compound	O
+48	O
+(	O
+Salmon	O
+)	O
+and	O
+Compound	O
+4j	O
+(	O
+Orange	O
+)	O
+	O
+The	O
+co	O
+-	O
+crystal	O
+structure	O
+of	O
+RORγ	O
+with	O
+T0901317	O
+(	O
+PDB	O
+code	O
+:	O
+4NB6	O
+),	O
+an	O
+inverse	O
+agonist	O
+of	O
+RORγ	O
+(	O
+IC50	O
+of	O
+54nM	O
+in	O
+an	O
+SRC1	O
+displacement	O
+FRET	O
+assay	O
+and	O
+an	O
+IC50	O
+of	O
+59nM	O
+in	O
+our	O
+FRET	O
+assay	O
+(	O
+Additional	O
+file	O
+7	O
+))	O
+shows	O
+that	O
+it	O
+adopts	O
+a	O
+collapsed	O
+conformation	O
+similar	O
+to	O
+the	O
+structure	O
+of	O
+BIO399	O
+described	O
+here	O
+.	O
+	O
+The	O
+two	O
+compounds	O
+superimpose	O
+with	O
+an	O
+RMSD	O
+of	O
+0	O
+.	O
+81	O
+Å	O
+(	O
+Fig	O
+.	O
+6a	O
+).	O
+	O
+The	O
+CF3	O
+group	O
+on	O
+the	O
+hexafluoropropanol	O
+group	O
+of	O
+T0901317	O
+was	O
+reported	O
+to	O
+fit	O
+the	O
+electron	O
+density	O
+in	O
+two	O
+conformations	O
+one	O
+of	O
+which	O
+pushes	O
+Met358	O
+into	O
+the	O
+vicinity	O
+of	O
+Phe506	O
+in	O
+the	O
+RORγ	O
+BIO592	O
+agonist	O
+structure	O
+.	O
+	O
+Co	O
+-	O
+crystal	O
+structures	O
+of	O
+RORγ	O
+have	O
+been	O
+generated	O
+with	O
+several	O
+potent	O
+inverse	O
+agonists	O
+adopting	O
+a	O
+linear	O
+conformation	O
+distinct	O
+from	O
+the	O
+collapsed	O
+conformations	O
+seen	O
+for	O
+BIO399	O
+and	O
+T090131718	O
+.	O
+	O
+BIO399	O
+neither	O
+orients	O
+the	O
+sidechain	O
+of	O
+Trp317	O
+toward	O
+Tyr502	O
+nor	O
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+His479	O
+suggesting	O
+its	O
+mode	O
+of	O
+action	O
+is	O
+distinct	O
+from	O
+linear	O
+inverse	O
+agonists	O
+(	O
+Additional	O
+file	O
+8	O
+).	O
+	O
+In	O
+the	O
+linear	O
+inverse	O
+agonist	O
+crystal	O
+structures	O
+the	O
+side	O
+chain	O
+of	O
+Met358	O
+resides	O
+in	O
+a	O
+similar	O
+position	O
+as	O
+the	O
+rotomer	O
+observed	O
+in	O
+RORγ	O
+agonist	O
+structures	O
+with	O
+BIO592	O
+described	O
+here	O
+or	O
+as	O
+observed	O
+in	O
+the	O
+hydroxycholesterol	O
+derivatives	O
+and	O
+therefore	O
+would	O
+not	O
+trigger	O
+inverse	O
+agonism	O
+with	O
+these	O
+ligands	O
+(	O
+Fig	O
+.	O
+6b	O
+).	O
+	O
+BIO399	O
+shows	O
+selectivity	O
+for	O
+RORγ	O
+over	O
+RORα	O
+and	O
+RORβ	O
+in	O
+a	O
+GAL4	O
+Cellular	O
+Reporter	O
+Assay	O
+	O
+a	O
+Overlay	O
+of	O
+RORα	O
+(	O
+yellow	O
+),	O
+β	O
+(	O
+pink	O
+)	O
+and	O
+γ	O
+(	O
+cyan	O
+)	O
+showing	O
+side	O
+chain	O
+differences	O
+at	O
+Met358	O
+inverse	O
+agonism	O
+trigger	O
+position	O
+and	O
+(	O
+b	O
+)	O
+around	O
+the	O
+benzoxazinone	O
+ring	O
+system	O
+of	O
+BIO399	O
+	O
+In	O
+order	O
+to	O
+assess	O
+the	O
+in	O
+vivo	O
+selectivity	O
+profile	O
+of	O
+BIO399	O
+a	O
+cellular	O
+reporter	O
+assay	O
+was	O
+implemented	O
+where	O
+the	O
+ligand	O
+binding	O
+domains	O
+of	O
+ROR	O
+α	O
+,	O
+β	O
+and	O
+γ	O
+were	O
+fused	O
+to	O
+the	O
+DNA	O
+binding	O
+domain	O
+of	O
+the	O
+transcriptional	O
+factor	O
+GAL4	O
+.	O
+	O
+The	O
+ROR	O
+-	O
+GAL4	O
+fusion	O
+proteins	O
+were	O
+expressed	O
+in	O
+cells	O
+with	O
+the	O
+luciferase	O
+reporter	O
+gene	O
+under	O
+the	O
+control	O
+of	O
+a	O
+GAL4	O
+promoter	O
+.	O
+	O
+BIO399	O
+inhibited	O
+the	O
+luciferase	O
+activity	O
+when	O
+added	O
+to	O
+the	O
+cells	O
+expressing	O
+the	O
+RORγ	O
+-	O
+GAL4	O
+fusion	O
+with	O
+an	O
+in	O
+vivo	O
+IC50	O
+of	O
+42	O
+.	O
+5nM	O
+while	O
+showing	O
+>	O
+235	O
+and	O
+28	O
+fold	O
+selectivity	O
+over	O
+cells	O
+expressing	O
+GAL4	O
+fused	O
+to	O
+the	O
+LBD	O
+of	O
+ROR	O
+α	O
+or	O
+β	O
+,	O
+respectively	O
+(	O
+Table	O
+1	O
+).	O
+	O
+The	O
+LBS	O
+of	O
+RORs	O
+share	O
+a	O
+high	O
+degree	O
+of	O
+similarity	O
+.	O
+	O
+This	O
+selectivity	O
+profile	O
+for	O
+BIO399	O
+is	O
+attributed	O
+to	O
+the	O
+shorter	O
+leucine	O
+side	O
+chain	O
+in	O
+RORα	O
+and	O
+β	O
+which	O
+would	O
+not	O
+reach	O
+the	O
+phenylalanine	O
+on	O
+the	O
+AF2	O
+helix	O
+further	O
+underscoring	O
+the	O
+role	O
+of	O
+Met358	O
+as	O
+a	O
+trigger	O
+for	O
+RORγ	O
+specific	O
+inverse	O
+agonism	O
+(	O
+Fig	O
+.	O
+7a	O
+).	O
+	O
+We	O
+hypothesize	O
+that	O
+the	O
+two	O
+phenylalanine	O
+residues	O
+in	O
+the	O
+LBS	O
+of	O
+RORα	O
+occlude	O
+the	O
+dihydrobenzoxazepinone	O
+ring	O
+system	O
+of	O
+BIO399	O
+from	O
+binding	O
+it	O
+and	O
+responsible	O
+for	O
+the	O
+increase	O
+in	O
+selectivity	O
+for	O
+RORα	O
+over	O
+β	O
+.	O
+	O
+We	O
+have	O
+identified	O
+a	O
+novel	O
+series	O
+of	O
+synthetic	O
+benzoxazinone	O
+ligands	O
+which	O
+modulate	O
+the	O
+transcriptional	O
+activity	O
+of	O
+RORγ	O
+in	O
+a	O
+FRET	O
+based	O
+assay	O
+.	O
+	O
+Using	O
+partial	O
+proteolysis	O
+we	O
+show	O
+a	O
+conformational	O
+change	O
+which	O
+destabilizes	O
+the	O
+AF2	O
+helix	O
+of	O
+RORγ	O
+when	O
+the	O
+inverse	O
+agonist	O
+BIO399	O
+binds	O
+.	O
+	O
+The	O
+two	O
+RORγ	O
+co	O
+-	O
+crystal	O
+structures	O
+reported	O
+here	O
+show	O
+how	O
+a	O
+small	O
+change	O
+to	O
+the	O
+core	O
+ring	O
+system	O
+can	O
+modulate	O
+the	O
+mode	O
+of	O
+action	O
+from	O
+agonist	O
+(	O
+BIO592	O
+)	O
+to	O
+inverse	O
+agonism	O
+(	O
+BIO399	O
+).	O
+	O
+Finally	O
+,	O
+we	O
+are	O
+reporting	O
+a	O
+newly	O
+identified	O
+trigger	O
+for	O
+achieving	O
+RORγ	O
+specific	O
+inverse	O
+agonism	O
+in	O
+an	O
+in	O
+vivo	O
+setting	O
+through	O
+Met358	O
+which	O
+perturbs	O
+the	O
+agonist	O
+conformation	O
+of	O
+the	O
+AF2	O
+helix	O
+and	O
+prevents	O
+coactivator	O
+protein	O
+binding	O
+.	O
+	O
+Bacterial	O
+Microcompartments	O
+(	O
+BMCs	O
+)	O
+are	O
+proteinaceous	O
+organelles	O
+that	O
+encapsulate	O
+critical	O
+segments	O
+of	O
+autotrophic	O
+and	O
+heterotrophic	O
+metabolic	O
+pathways	O
+;	O
+they	O
+are	O
+functionally	O
+diverse	O
+and	O
+are	O
+found	O
+across	O
+23	O
+different	O
+phyla	O
+.	O
+	O
+The	O
+core	O
+enzyme	O
+phosphotransacylase	O
+(	O
+PTAC	O
+)	O
+recycles	O
+Coenzyme	O
+A	O
+and	O
+generates	O
+an	O
+acyl	O
+phosphate	O
+that	O
+can	O
+serve	O
+as	O
+an	O
+energy	O
+source	O
+.	O
+	O
+The	O
+PTAC	O
+predominantly	O
+associated	O
+with	O
+metabolosomes	O
+(	O
+PduL	O
+)	O
+has	O
+no	O
+sequence	O
+homology	O
+to	O
+the	O
+PTAC	O
+ubiquitous	O
+among	O
+fermentative	O
+bacteria	O
+(	O
+Pta	O
+).	O
+	O
+Here	O
+,	O
+we	O
+report	O
+two	O
+high	O
+-	O
+resolution	O
+PduL	O
+crystal	O
+structures	O
+with	O
+bound	O
+substrates	O
+.	O
+	O
+The	O
+PduL	O
+fold	O
+is	O
+unrelated	O
+to	O
+that	O
+of	O
+Pta	O
+;	O
+it	O
+contains	O
+a	O
+dimetal	O
+active	O
+site	O
+involved	O
+in	O
+a	O
+catalytic	O
+mechanism	O
+distinct	O
+from	O
+that	O
+of	O
+the	O
+housekeeping	O
+PTAC	O
+.	O
+	O
+The	O
+PduL	O
+structure	O
+,	O
+in	O
+the	O
+context	O
+of	O
+the	O
+catalytic	O
+core	O
+,	O
+completes	O
+our	O
+understanding	O
+of	O
+the	O
+structural	O
+basis	O
+of	O
+cofactor	O
+recycling	O
+in	O
+the	O
+metabolosome	O
+lumen	O
+.	O
+	O
+This	O
+study	O
+describes	O
+the	O
+structure	O
+of	O
+a	O
+novel	O
+phosphotransacylase	O
+enzyme	O
+that	O
+facilitates	O
+the	O
+recycling	O
+of	O
+the	O
+essential	O
+cofactor	O
+acetyl	O
+-	O
+CoA	O
+within	O
+a	O
+bacterial	O
+organelle	O
+and	O
+discusses	O
+the	O
+properties	O
+of	O
+the	O
+enzyme	O
+'	O
+s	O
+active	O
+site	O
+and	O
+how	O
+it	O
+is	O
+packaged	O
+into	O
+the	O
+organelle	O
+.	O
+	O
+The	O
+phosphotransacylase	O
+(	O
+Pta	O
+)	O
+enzyme	O
+catalyzes	O
+the	O
+conversion	O
+between	O
+acyl	O
+-	O
+CoA	O
+and	O
+acyl	O
+-	O
+phosphate	O
+.	O
+	O
+This	O
+reaction	O
+directly	O
+links	O
+an	O
+acyl	O
+-	O
+CoA	O
+with	O
+ATP	O
+generation	O
+via	O
+substrate	O
+-	O
+level	O
+phosphorylation	O
+,	O
+producing	O
+short	O
+-	O
+chain	O
+fatty	O
+acids	O
+(	O
+e	O
+.	O
+g	O
+.,	O
+acetate	O
+),	O
+and	O
+also	O
+provides	O
+a	O
+path	O
+for	O
+short	O
+-	O
+chain	O
+fatty	O
+acids	O
+to	O
+enter	O
+central	O
+metabolism	O
+.	O
+	O
+Due	O
+to	O
+this	O
+key	O
+function	O
+,	O
+Pta	O
+is	O
+conserved	O
+across	O
+the	O
+bacterial	O
+kingdom	O
+.	O
+	O
+Recently	O
+,	O
+a	O
+new	O
+type	O
+of	O
+phosphotransacylase	O
+was	O
+described	O
+that	O
+shares	O
+no	O
+evolutionary	O
+relation	O
+to	O
+Pta	O
+.	O
+	O
+Not	O
+only	O
+does	O
+PduL	O
+facilitate	O
+substrate	O
+level	O
+phosphorylation	O
+,	O
+but	O
+it	O
+also	O
+is	O
+critical	O
+for	O
+cofactor	O
+recycling	O
+within	O
+,	O
+and	O
+product	O
+efflux	O
+from	O
+,	O
+the	O
+organelle	O
+.	O
+	O
+We	O
+solved	O
+the	O
+structure	O
+of	O
+this	O
+convergent	O
+phosphotransacylase	O
+and	O
+show	O
+that	O
+it	O
+is	O
+completely	O
+structurally	O
+different	O
+from	O
+Pta	O
+,	O
+including	O
+its	O
+active	O
+site	O
+architecture	O
+.	O
+	O
+Bacterial	O
+Microcompartments	O
+(	O
+BMCs	O
+)	O
+are	O
+organelles	O
+that	O
+encapsulate	O
+enzymes	O
+for	O
+sequential	O
+biochemical	O
+reactions	O
+within	O
+a	O
+protein	O
+shell	O
+.	O
+	O
+The	O
+shell	O
+is	O
+typically	O
+composed	O
+of	O
+three	O
+types	O
+of	O
+protein	O
+subunits	O
+,	O
+which	O
+form	O
+either	O
+hexagonal	O
+(	O
+BMC	O
+-	O
+H	O
+and	O
+BMC	O
+-	O
+T	O
+)	O
+or	O
+pentagonal	O
+(	O
+BMC	O
+-	O
+P	O
+)	O
+tiles	O
+that	O
+assemble	O
+into	O
+a	O
+polyhedral	O
+shell	O
+.	O
+	O
+The	O
+vitamin	O
+B12	O
+-	O
+dependent	O
+propanediol	O
+-	O
+utilizing	O
+(	O
+PDU	O
+)	O
+BMC	O
+was	O
+one	O
+of	O
+the	O
+first	O
+functionally	O
+characterized	O
+catabolic	O
+BMCs	O
+;	O
+subsequently	O
+,	O
+other	O
+types	O
+have	O
+been	O
+implicated	O
+in	O
+the	O
+degradation	O
+of	O
+ethanolamine	O
+,	O
+choline	O
+,	O
+fucose	O
+,	O
+rhamnose	O
+,	O
+and	O
+ethanol	O
+,	O
+all	O
+of	O
+which	O
+produce	O
+different	O
+aldehyde	O
+intermediates	O
+(	O
+Table	O
+1	O
+).	O
+	O
+More	O
+recently	O
+,	O
+bioinformatic	O
+studies	O
+have	O
+demonstrated	O
+the	O
+widespread	O
+distribution	O
+of	O
+BMCs	O
+among	O
+diverse	O
+bacterial	O
+phyla	O
+and	O
+grouped	O
+them	O
+into	O
+23	O
+different	O
+functional	O
+types	O
+.	O
+	O
+The	O
+reactions	O
+carried	O
+out	O
+in	O
+the	O
+majority	O
+of	O
+catabolic	O
+BMCs	O
+(	O
+also	O
+known	O
+as	O
+metabolosomes	O
+)	O
+fit	O
+a	O
+generalized	O
+biochemical	O
+paradigm	O
+for	O
+the	O
+oxidation	O
+of	O
+aldehydes	O
+(	O
+Fig	O
+1	O
+).	O
+	O
+This	O
+involves	O
+a	O
+BMC	O
+-	O
+encapsulated	O
+signature	O
+enzyme	O
+that	O
+generates	O
+a	O
+toxic	O
+and	O
+/	O
+or	O
+volatile	O
+aldehyde	O
+that	O
+the	O
+BMC	O
+shell	O
+sequesters	O
+from	O
+the	O
+cytosol	O
+.	O
+	O
+These	O
+two	O
+cofactors	O
+are	O
+relatively	O
+large	O
+,	O
+and	O
+their	O
+diffusion	O
+across	O
+the	O
+protein	O
+shell	O
+is	O
+thought	O
+to	O
+be	O
+restricted	O
+,	O
+necessitating	O
+their	O
+regeneration	O
+within	O
+the	O
+BMC	O
+lumen	O
+.	O
+	O
+The	O
+final	O
+product	O
+of	O
+the	O
+BMC	O
+,	O
+an	O
+acyl	O
+-	O
+phosphate	O
+,	O
+can	O
+then	O
+be	O
+used	O
+to	O
+generate	O
+ATP	O
+via	O
+acyl	O
+kinase	O
+,	O
+or	O
+revert	O
+back	O
+to	O
+acyl	O
+-	O
+CoA	O
+by	O
+Pta	O
+for	O
+biosynthesis	O
+.	O
+	O
+Collectively	O
+,	O
+the	O
+aldehyde	O
+and	O
+alcohol	O
+dehydrogenases	O
+,	O
+as	O
+well	O
+as	O
+the	O
+PTAC	O
+,	O
+constitute	O
+the	O
+common	O
+metabolosome	O
+core	O
+.	O
+	O
+General	O
+biochemical	O
+model	O
+of	O
+aldehyde	O
+-	O
+degrading	O
+BMCs	O
+(	O
+metabolosomes	O
+)	O
+illustrating	O
+the	O
+common	O
+metabolosome	O
+core	O
+enzymes	O
+and	O
+reactions	O
+.	O
+	O
+Characterized	O
+and	O
+predicted	O
+catabolic	O
+BMC	O
+(	O
+metabolosome	O
+)	O
+types	O
+that	O
+represent	O
+the	O
+aldehyde	O
+-	O
+degrading	O
+paradigm	O
+(	O
+for	O
+definition	O
+of	O
+types	O
+see	O
+Kerfeld	O
+and	O
+Erbilgin	O
+).	O
+	O
+Name	O
+PTAC	O
+Type	O
+Sequestered	O
+Aldehyde	O
+PDU	O
+*	O
+PduL	O
+propionaldehyde	O
+EUT1	O
+PTA_PTB	O
+acetaldehyde	O
+EUT2	O
+PduL	O
+acetaldehyde	O
+ETU	O
+None	O
+acetaldehyde	O
+GRM1	O
+/	O
+CUT	O
+PduL	O
+acetaldehyde	O
+GRM2	O
+PduL	O
+acetaldehyde	O
+GRM3	O
+*,	O
+4	O
+PduL	O
+propionaldehyde	O
+GRM5	O
+/	O
+GRP	O
+PduL	O
+propionaldehyde	O
+PVM	O
+*	O
+PduL	O
+lactaldehyde	O
+RMM1	O
+,	O
+2	O
+None	O
+unknown	O
+SPU	O
+PduL	O
+unknown	O
+	O
+*	O
+PduL	O
+from	O
+these	O
+functional	O
+types	O
+of	O
+metabolosomes	O
+were	O
+purified	O
+in	O
+this	O
+study	O
+.	O
+	O
+The	O
+concerted	O
+functioning	O
+of	O
+a	O
+PTAC	O
+and	O
+an	O
+acetate	O
+kinase	O
+(	O
+Ack	O
+)	O
+is	O
+crucial	O
+for	O
+ATP	O
+generation	O
+in	O
+the	O
+fermentation	O
+of	O
+pyruvate	O
+to	O
+acetate	O
+(	O
+see	O
+Reactions	O
+1	O
+and	O
+2	O
+).	O
+	O
+Both	O
+enzymes	O
+are	O
+,	O
+however	O
+,	O
+not	O
+restricted	O
+to	O
+fermentative	O
+organisms	O
+.	O
+	O
+Reaction	O
+1	O
+:	O
+acetyl	O
+-	O
+S	O
+-	O
+CoA	O
++	O
+Pi	O
+←→	O
+acetyl	O
+phosphate	O
++	O
+CoA	O
+-	O
+SH	O
+(	O
+PTAC	O
+)	O
+	O
+Reaction	O
+2	O
+:	O
+acetyl	O
+phosphate	O
++	O
+ADP	O
+←→	O
+acetate	O
++	O
+ATP	O
+(	O
+Ack	O
+)	O
+	O
+Pta	O
+has	O
+been	O
+extensively	O
+characterized	O
+due	O
+to	O
+its	O
+key	O
+role	O
+in	O
+fermentation	O
+.	O
+	O
+More	O
+recently	O
+,	O
+a	O
+second	O
+type	O
+of	O
+PTAC	O
+without	O
+any	O
+sequence	O
+homology	O
+to	O
+Pta	O
+was	O
+identified	O
+.	O
+	O
+This	O
+protein	O
+,	O
+PduL	O
+(	O
+Pfam	O
+domain	O
+PF06130	O
+),	O
+was	O
+shown	O
+to	O
+catalyze	O
+the	O
+conversion	O
+of	O
+propionyl	O
+-	O
+CoA	O
+to	O
+propionyl	O
+-	O
+phosphate	O
+and	O
+is	O
+associated	O
+with	O
+a	O
+BMC	O
+involved	O
+in	O
+propanediol	O
+utilization	O
+,	O
+the	O
+PDU	O
+BMC	O
+.	O
+	O
+Both	O
+pduL	O
+and	O
+pta	O
+genes	O
+can	O
+be	O
+found	O
+in	O
+genetic	O
+loci	O
+of	O
+functionally	O
+distinct	O
+BMCs	O
+,	O
+although	O
+the	O
+PduL	O
+type	O
+is	O
+much	O
+more	O
+prevalent	O
+,	O
+being	O
+found	O
+in	O
+all	O
+but	O
+one	O
+type	O
+of	O
+metabolosome	O
+locus	O
+:	O
+EUT1	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Furthermore	O
+,	O
+in	O
+the	O
+Integrated	O
+Microbial	O
+Genomes	O
+Database	O
+,	O
+91	O
+%	O
+of	O
+genomes	O
+that	O
+encode	O
+PF06130	O
+also	O
+encode	O
+genes	O
+for	O
+shell	O
+proteins	O
+.	O
+	O
+As	O
+a	O
+member	O
+of	O
+the	O
+core	O
+biochemical	O
+machinery	O
+of	O
+functionally	O
+diverse	O
+aldehyde	O
+-	O
+oxidizing	O
+metabolosomes	O
+,	O
+PduL	O
+must	O
+have	O
+a	O
+certain	O
+level	O
+of	O
+substrate	O
+plasticity	O
+(	O
+see	O
+Table	O
+1	O
+)	O
+that	O
+is	O
+not	O
+required	O
+of	O
+Pta	O
+,	O
+which	O
+has	O
+generally	O
+been	O
+observed	O
+to	O
+prefer	O
+acetyl	O
+-	O
+CoA	O
+.	O
+PduL	O
+from	O
+the	O
+PDU	O
+BMC	O
+of	O
+Salmonella	O
+enterica	O
+favors	O
+propionyl	O
+-	O
+CoA	O
+over	O
+acetyl	O
+-	O
+CoA	O
+,	O
+and	O
+it	O
+is	O
+likely	O
+that	O
+PduL	O
+orthologs	O
+in	O
+functionally	O
+diverse	O
+BMCs	O
+would	O
+have	O
+substrate	O
+preferences	O
+for	O
+other	O
+CoA	O
+derivatives	O
+.	O
+	O
+EPs	O
+have	O
+also	O
+been	O
+observed	O
+to	O
+cause	O
+proteins	O
+to	O
+aggregate	O
+,	O
+and	O
+this	O
+has	O
+recently	O
+been	O
+suggested	O
+to	O
+be	O
+functionally	O
+relevant	O
+as	O
+an	O
+initial	O
+step	O
+in	O
+metabolosome	O
+assembly	O
+,	O
+in	O
+which	O
+a	O
+multifunctional	O
+protein	O
+core	O
+is	O
+formed	O
+,	O
+around	O
+which	O
+the	O
+shell	O
+assembles	O
+.	O
+	O
+Of	O
+the	O
+three	O
+common	O
+metabolosome	O
+core	O
+enzymes	O
+,	O
+crystal	O
+structures	O
+are	O
+available	O
+for	O
+both	O
+the	O
+alcohol	O
+and	O
+aldehyde	O
+dehydrogenases	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+structure	O
+of	O
+PduL	O
+,	O
+the	O
+PTAC	O
+found	O
+in	O
+the	O
+vast	O
+majority	O
+of	O
+catabolic	O
+BMCs	O
+,	O
+has	O
+not	O
+been	O
+determined	O
+.	O
+	O
+This	O
+is	O
+a	O
+major	O
+gap	O
+in	O
+our	O
+understanding	O
+of	O
+metabolosome	O
+-	O
+encapsulated	O
+biochemistry	O
+and	O
+cofactor	O
+recycling	O
+.	O
+	O
+Moreover	O
+,	O
+it	O
+will	O
+be	O
+useful	O
+for	O
+guiding	O
+efforts	O
+to	O
+engineer	O
+novel	O
+BMC	O
+cores	O
+for	O
+biotechnological	O
+applications	O
+.	O
+	O
+No	O
+available	O
+protein	O
+structures	O
+contain	O
+the	O
+PF06130	O
+domain	O
+,	O
+and	O
+homology	O
+searches	O
+using	O
+the	O
+primary	O
+structure	O
+of	O
+PduL	O
+do	O
+not	O
+return	O
+any	O
+significant	O
+results	O
+that	O
+would	O
+allow	O
+prediction	O
+of	O
+the	O
+structure	O
+.	O
+	O
+Moreover	O
+,	O
+the	O
+evident	O
+novelty	O
+of	O
+PduL	O
+makes	O
+its	O
+structure	O
+interesting	O
+in	O
+the	O
+context	O
+of	O
+convergent	O
+evolution	O
+of	O
+PTAC	O
+function	O
+;	O
+to	O
+-	O
+date	O
+,	O
+only	O
+the	O
+Pta	O
+active	O
+site	O
+and	O
+catalytic	O
+mechanism	O
+is	O
+known	O
+.	O
+	O
+We	O
+propose	O
+a	O
+catalytic	O
+mechanism	O
+analogous	O
+but	O
+yet	O
+distinct	O
+from	O
+the	O
+ubiquitous	O
+Pta	O
+enzyme	O
+,	O
+highlighting	O
+the	O
+functional	O
+convergence	O
+of	O
+two	O
+enzymes	O
+with	O
+completely	O
+different	O
+structures	O
+and	O
+metal	O
+requirements	O
+.	O
+	O
+We	O
+also	O
+investigate	O
+the	O
+quaternary	O
+structures	O
+of	O
+three	O
+different	O
+PduL	O
+homologs	O
+and	O
+situate	O
+our	O
+findings	O
+in	O
+the	O
+context	O
+of	O
+organelle	O
+biogenesis	O
+in	O
+functionally	O
+diverse	O
+BMCs	O
+.	O
+	O
+We	O
+cloned	O
+,	O
+expressed	O
+,	O
+and	O
+purified	O
+three	O
+different	O
+PduL	O
+homologs	O
+from	O
+functionally	O
+distinct	O
+BMCs	O
+(	O
+Table	O
+1	O
+):	O
+from	O
+the	O
+well	O
+-	O
+studied	O
+pdu	O
+locus	O
+in	O
+S	O
+.	O
+enterica	O
+Typhimurium	O
+LT2	O
+(	O
+sPduL	O
+),	O
+from	O
+the	O
+recently	O
+characterized	O
+pvm	O
+locus	O
+in	O
+Planctomyces	O
+limnophilus	O
+(	O
+pPduL	O
+),	O
+and	O
+from	O
+the	O
+grm3	O
+locus	O
+in	O
+Rhodopseudomonas	O
+palustris	O
+BisB18	O
+(	O
+rPduL	O
+).	O
+	O
+While	O
+purifying	O
+full	O
+-	O
+length	O
+sPduL	O
+,	O
+we	O
+observed	O
+a	O
+tendency	O
+to	O
+aggregation	O
+as	O
+described	O
+previously	O
+,	O
+with	O
+a	O
+large	O
+fraction	O
+of	O
+the	O
+expressed	O
+protein	O
+found	O
+in	O
+the	O
+insoluble	O
+fraction	O
+in	O
+a	O
+white	O
+,	O
+cake	O
+-	O
+like	O
+pellet	O
+.	O
+	O
+Similar	O
+differences	O
+in	O
+solubility	O
+were	O
+observed	O
+for	O
+pPduL	O
+and	O
+rPduL	O
+when	O
+comparing	O
+EP	O
+-	O
+truncated	O
+forms	O
+to	O
+the	O
+full	O
+-	O
+length	O
+protein	O
+,	O
+but	O
+none	O
+were	O
+quite	O
+as	O
+dramatic	O
+as	O
+for	O
+sPduL	O
+.	O
+We	O
+confirmed	O
+that	O
+all	O
+homologs	O
+were	O
+active	O
+(	O
+S1a	O
+and	O
+S1b	O
+Fig	O
+).	O
+	O
+Among	O
+these	O
+,	O
+we	O
+were	O
+only	O
+able	O
+to	O
+obtain	O
+diffraction	O
+-	O
+quality	O
+crystals	O
+of	O
+rPduL	O
+after	O
+removing	O
+the	O
+N	O
+-	O
+terminal	O
+putative	O
+EP	O
+(	O
+33	O
+amino	O
+acids	O
+,	O
+also	O
+see	O
+Fig	O
+2a	O
+)	O
+(	O
+rPduLΔEP	B-mutant
+).	O
+	O
+Truncated	O
+rPduLΔEP	B-mutant
+had	O
+comparable	O
+enzymatic	O
+activity	O
+to	O
+the	O
+full	O
+-	O
+length	O
+enzyme	O
+(	O
+S1a	O
+Fig	O
+).	O
+	O
+Structural	O
+overview	O
+of	O
+R	O
+.	O
+palustris	O
+PduL	O
+from	O
+the	O
+grm3	O
+locus	O
+.	O
+	O
+(	O
+a	O
+)	O
+Primary	O
+and	O
+secondary	O
+structure	O
+of	O
+rPduL	O
+(	O
+tubes	O
+represent	O
+α	O
+-	O
+helices	O
+,	O
+arrows	O
+β	O
+-	O
+sheets	O
+and	O
+dashed	O
+line	O
+residues	O
+disordered	O
+in	O
+the	O
+structure	O
+.	O
+	O
+The	O
+first	O
+33	O
+amino	O
+acids	O
+are	O
+present	O
+only	O
+in	O
+the	O
+wildtype	O
+construct	O
+and	O
+contains	O
+the	O
+predicted	O
+EP	O
+alpha	O
+helix	O
+,	O
+α0	O
+);	O
+the	O
+truncated	O
+rPduLΔEP	B-mutant
+that	O
+was	O
+crystallized	O
+begins	O
+with	O
+M	O
+-	O
+G	O
+-	O
+V	O
+.	O
+Coloring	O
+is	O
+according	O
+to	O
+structural	O
+domains	O
+(	O
+domain	O
+1	O
+D36	O
+-	O
+N46	O
+/	O
+Q155	O
+-	O
+C224	O
+,	O
+blue	O
+;	O
+loop	O
+insertion	O
+G61	O
+-	O
+E81	O
+,	O
+grey	O
+;	O
+domain	O
+2	O
+R47	O
+-	O
+F60	O
+/	O
+E82	O
+-	O
+A154	O
+,	O
+red	O
+).	O
+	O
+Metal	O
+coordination	O
+residues	O
+are	O
+highlighted	O
+in	O
+light	O
+blue	O
+and	O
+CoA	O
+contacting	O
+residues	O
+in	O
+magenta	O
+,	O
+residues	O
+contacting	O
+the	O
+CoA	O
+of	O
+the	O
+other	O
+chain	O
+are	O
+also	O
+outlined	O
+.	O
+	O
+(	O
+b	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+structure	O
+colored	O
+by	O
+domains	O
+and	O
+including	O
+secondary	O
+structure	O
+numbering	O
+.	O
+	O
+Coenzyme	O
+A	O
+is	O
+shown	O
+in	O
+magenta	O
+sticks	O
+and	O
+Zinc	O
+(	O
+grey	O
+)	O
+as	O
+spheres	O
+.	O
+	O
+We	O
+collected	O
+a	O
+native	O
+dataset	O
+from	O
+rPduLΔEP	B-mutant
+crystals	O
+diffracting	O
+to	O
+a	O
+resolution	O
+of	O
+1	O
+.	O
+54	O
+Å	O
+(	O
+Table	O
+2	O
+).	O
+	O
+Using	O
+a	O
+mercury	O
+-	O
+derivative	O
+crystal	O
+form	O
+diffracting	O
+to	O
+1	O
+.	O
+99	O
+Å	O
+(	O
+Table	O
+2	O
+),	O
+we	O
+obtained	O
+high	O
+quality	O
+electron	O
+density	O
+for	O
+model	O
+building	O
+and	O
+used	O
+the	O
+initial	O
+model	O
+to	O
+refine	O
+against	O
+the	O
+native	O
+data	O
+to	O
+Rwork	O
+/	O
+Rfree	O
+values	O
+of	O
+18	O
+.	O
+9	O
+/	O
+22	O
+.	O
+1	O
+%.	O
+	O
+There	O
+are	O
+two	O
+PduL	O
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+of	O
+the	O
+P212121	O
+unit	O
+cell	O
+.	O
+	O
+We	O
+were	O
+able	O
+to	O
+fit	O
+all	O
+of	O
+the	O
+primary	O
+structure	O
+of	O
+PduLΔEP	B-mutant
+into	O
+the	O
+electron	O
+density	O
+with	O
+the	O
+exception	O
+of	O
+three	O
+amino	O
+acids	O
+at	O
+the	O
+N	O
+-	O
+terminus	O
+and	O
+two	O
+amino	O
+acids	O
+at	O
+the	O
+C	O
+-	O
+terminus	O
+(	O
+Fig	O
+2a	O
+);	O
+the	O
+model	O
+is	O
+of	O
+excellent	O
+quality	O
+(	O
+Table	O
+2	O
+).	O
+	O
+Structurally	O
+,	O
+PduL	O
+consists	O
+of	O
+two	O
+domains	O
+(	O
+Fig	O
+2	O
+,	O
+blue	O
+/	O
+red	O
+),	O
+each	O
+a	O
+beta	O
+-	O
+barrel	O
+that	O
+is	O
+capped	O
+on	O
+both	O
+ends	O
+by	O
+short	O
+α	O
+-	O
+helices	O
+.	O
+	O
+β	O
+-	O
+Barrel	O
+2	O
+consists	O
+mainly	O
+of	O
+the	O
+central	O
+segment	O
+of	O
+primary	O
+structure	O
+(	O
+β2	O
+,	O
+β5	O
+–	O
+β9	O
+;	O
+residues	O
+47	O
+–	O
+60	O
+and	O
+82	O
+–	O
+154	O
+)	O
+(	O
+Fig	O
+2	O
+,	O
+red	O
+),	O
+but	O
+is	O
+interrupted	O
+by	O
+a	O
+short	O
+two	O
+-	O
+strand	O
+beta	O
+sheet	O
+(	O
+β3	O
+-	O
+β4	O
+,	O
+residues	O
+61	O
+–	O
+81	O
+).	O
+	O
+This	O
+β	O
+-	O
+sheet	O
+is	O
+involved	O
+in	O
+contacts	O
+between	O
+the	O
+two	O
+domains	O
+and	O
+forms	O
+a	O
+lid	O
+over	O
+the	O
+active	O
+site	O
+.	O
+	O
+Residues	O
+in	O
+this	O
+region	O
+(	O
+Gln42	O
+,	O
+Pro43	O
+,	O
+Gly44	O
+),	O
+covering	O
+the	O
+active	O
+site	O
+,	O
+are	O
+strongly	O
+conserved	O
+(	O
+Fig	O
+3	O
+).	O
+	O
+This	O
+structural	O
+arrangement	O
+is	O
+completely	O
+different	O
+from	O
+the	O
+functionally	O
+related	O
+Pta	O
+,	O
+which	O
+is	O
+composed	O
+of	O
+two	O
+domains	O
+,	O
+each	O
+consisting	O
+of	O
+a	O
+central	O
+flat	O
+beta	O
+sheet	O
+with	O
+alpha	O
+-	O
+helices	O
+on	O
+the	O
+top	O
+and	O
+bottom	O
+.	O
+	O
+Residues	O
+100	O
+%	O
+conserved	O
+across	O
+all	O
+PduL	O
+homologs	O
+in	O
+our	O
+dataset	O
+are	O
+noted	O
+with	O
+an	O
+asterisk	O
+,	O
+and	O
+residues	O
+conserved	O
+in	O
+over	O
+90	O
+%	O
+of	O
+sequences	O
+are	O
+noted	O
+with	O
+a	O
+colon	O
+.	O
+	O
+There	O
+are	O
+two	O
+PduL	O
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+forming	O
+a	O
+butterfly	O
+-	O
+shaped	O
+dimer	O
+(	O
+Fig	O
+4c	O
+).	O
+	O
+Consistent	O
+with	O
+this	O
+,	O
+results	O
+from	O
+size	O
+exclusion	O
+chromatography	O
+of	O
+rPduLΔEP	B-mutant
+suggest	O
+that	O
+it	O
+is	O
+a	O
+dimer	O
+in	O
+solution	O
+(	O
+Fig	O
+5e	O
+).	O
+	O
+The	O
+interface	O
+between	O
+the	O
+two	O
+chains	O
+buries	O
+882	O
+Å2	O
+per	O
+monomer	O
+and	O
+is	O
+mainly	O
+formed	O
+by	O
+α	O
+-	O
+helices	O
+2	O
+and	O
+4	O
+and	O
+parts	O
+of	O
+β	O
+-	O
+sheets	O
+12	O
+and	O
+14	O
+,	O
+as	O
+well	O
+as	O
+a	O
+π	O
+–	O
+π	O
+stacking	O
+of	O
+the	O
+adenine	O
+moiety	O
+of	O
+CoA	O
+with	O
+Phe116	O
+of	O
+the	O
+adjacent	O
+chain	O
+(	O
+Fig	O
+4c	O
+).	O
+	O
+The	O
+folds	O
+of	O
+the	O
+two	O
+chains	O
+in	O
+the	O
+asymmetric	O
+unit	O
+are	O
+very	O
+similar	O
+,	O
+superimposing	O
+with	O
+a	O
+rmsd	O
+of	O
+0	O
+.	O
+16	O
+Å	O
+over	O
+2	O
+,	O
+306	O
+aligned	O
+atom	O
+pairs	O
+.	O
+	O
+The	O
+peripheral	O
+helices	O
+and	O
+the	O
+short	O
+antiparallel	O
+β3	O
+–	O
+4	O
+sheet	O
+mediate	O
+most	O
+of	O
+the	O
+crystal	O
+contacts	O
+.	O
+	O
+Details	O
+of	O
+active	O
+site	O
+,	O
+dimeric	O
+assembly	O
+,	O
+and	O
+sequence	O
+conservation	O
+of	O
+PduL	O
+.	O
+	O
+(	O
+a	O
+,	O
+b	O
+)	O
+Proposed	O
+active	O
+site	O
+of	O
+PduL	O
+with	O
+relevant	O
+residues	O
+shown	O
+as	O
+sticks	O
+in	O
+atom	O
+coloring	O
+(	O
+nitrogen	O
+blue	O
+,	O
+oxygen	O
+red	O
+,	O
+sulfur	O
+yellow	O
+),	O
+zinc	O
+as	O
+grey	O
+colored	O
+spheres	O
+and	O
+coordinating	O
+ordered	O
+water	O
+molecules	O
+in	O
+red	O
+.	O
+	O
+(	O
+c	O
+)	O
+View	O
+of	O
+the	O
+dimer	O
+in	O
+the	O
+asymmetric	O
+unit	O
+from	O
+the	O
+side	O
+,	O
+domains	O
+1	O
+and	O
+2	O
+colored	O
+as	O
+in	O
+Fig	O
+2	O
+and	O
+the	O
+two	O
+chains	O
+differentiated	O
+by	O
+blue	O
+/	O
+red	O
+versus	O
+slate	O
+/	O
+firebrick	O
+.	O
+	O
+(	O
+d	O
+)	O
+Surface	O
+representation	O
+of	O
+the	O
+structure	O
+with	O
+indicated	O
+conservation	O
+(	O
+red	O
+:	O
+high	O
+,	O
+white	O
+:	O
+intermediate	O
+,	O
+yellow	O
+:	O
+low	O
+).	O
+	O
+All	O
+chromatograms	O
+are	O
+cropped	O
+to	O
+show	O
+only	O
+the	O
+linear	O
+range	O
+of	O
+separation	O
+based	O
+on	O
+standard	O
+runs	O
+,	O
+shown	O
+in	O
+black	O
+squares	O
+with	O
+a	O
+dashed	O
+linear	O
+trend	O
+line	O
+.	O
+	O
+Active	O
+Site	O
+Properties	O
+	O
+CoA	O
+and	O
+the	O
+metal	O
+ions	O
+bind	O
+between	O
+the	O
+two	O
+domains	O
+,	O
+presumably	O
+in	O
+the	O
+active	O
+site	O
+(	O
+Figs	O
+2b	O
+and	O
+4a	O
+).	O
+	O
+To	O
+identify	O
+the	O
+bound	O
+metals	O
+,	O
+we	O
+performed	O
+an	O
+X	O
+-	O
+ray	O
+fluorescence	O
+scan	O
+on	O
+the	O
+crystals	O
+at	O
+various	O
+wavelengths	O
+(	O
+corresponding	O
+to	O
+the	O
+K	O
+-	O
+edges	O
+of	O
+Mn	O
+,	O
+Fe	O
+,	O
+Co	O
+,	O
+Ni	O
+,	O
+Cu	O
+,	O
+and	O
+Zn	O
+).	O
+	O
+There	O
+was	O
+a	O
+large	O
+signal	O
+at	O
+the	O
+zinc	O
+edge	O
+,	O
+and	O
+we	O
+tested	O
+for	O
+the	O
+presence	O
+of	O
+zinc	O
+by	O
+collecting	O
+full	O
+data	O
+sets	O
+before	O
+and	O
+after	O
+the	O
+Zn	O
+K	O
+-	O
+edge	O
+(	O
+1	O
+.	O
+2861	O
+and	O
+1	O
+.	O
+2822	O
+Å	O
+,	O
+respectively	O
+).	O
+	O
+The	O
+large	O
+differences	O
+between	O
+the	O
+anomalous	O
+signals	O
+confirm	O
+the	O
+presence	O
+of	O
+zinc	O
+at	O
+both	O
+metal	O
+sites	O
+(	O
+S3	O
+Fig	O
+).	O
+	O
+The	O
+first	O
+zinc	O
+ion	O
+(	O
+Zn1	O
+)	O
+is	O
+in	O
+a	O
+tetrahedral	O
+coordination	O
+state	O
+with	O
+His48	O
+,	O
+His50	O
+,	O
+Glu109	O
+,	O
+and	O
+the	O
+CoA	O
+sulfur	O
+(	O
+Fig	O
+4a	O
+).	O
+	O
+The	O
+nitrogen	O
+atom	O
+coordinating	O
+the	O
+zinc	O
+is	O
+the	O
+Nε	O
+in	O
+each	O
+histidine	O
+residue	O
+,	O
+as	O
+is	O
+typical	O
+for	O
+this	O
+interaction	O
+.	O
+	O
+The	O
+phosphate	O
+-	O
+bound	O
+structure	O
+aligns	O
+well	O
+with	O
+the	O
+CoA	O
+-	O
+bound	O
+structure	O
+(	O
+0	O
+.	O
+43	O
+Å	O
+rmsd	O
+over	O
+2	O
+,	O
+361	O
+atoms	O
+for	O
+the	O
+monomer	O
+,	O
+0	O
+.	O
+83	O
+Å	O
+over	O
+5	O
+,	O
+259	O
+aligned	O
+atoms	O
+for	O
+the	O
+dimer	O
+).	O
+	O
+Conserved	O
+Arg103	O
+seems	O
+to	O
+be	O
+involved	O
+in	O
+maintaining	O
+the	O
+phosphate	O
+in	O
+that	O
+position	O
+.	O
+	O
+An	O
+additional	O
+phosphate	O
+molecule	O
+is	O
+bound	O
+at	O
+a	O
+crystal	O
+contact	O
+interface	O
+,	O
+perhaps	O
+accounting	O
+for	O
+the	O
+14	O
+Å	O
+shorter	O
+c	O
+-	O
+axis	O
+in	O
+the	O
+phosphate	O
+-	O
+bound	O
+crystal	O
+form	O
+(	O
+Table	O
+2	O
+).	O
+	O
+Interestingly	O
+,	O
+some	O
+of	O
+the	O
+residues	O
+important	O
+for	O
+dimerization	O
+of	O
+rPduL	O
+,	O
+particularly	O
+Phe116	O
+,	O
+are	O
+poorly	O
+conserved	O
+across	O
+PduL	O
+homologs	O
+associated	O
+with	O
+functionally	O
+diverse	O
+BMCs	O
+(	O
+Figs	O
+4c	O
+and	O
+3	O
+),	O
+suggesting	O
+that	O
+they	O
+may	O
+have	O
+alternative	O
+oligomeric	O
+states	O
+.	O
+	O
+We	O
+tested	O
+this	O
+hypothesis	O
+by	O
+performing	O
+size	O
+exclusion	O
+chromatography	O
+on	O
+both	O
+full	O
+-	O
+length	O
+and	O
+truncated	O
+variants	O
+(	O
+lacking	O
+the	O
+EP	O
+,	O
+ΔEP	B-mutant
+)	O
+of	O
+sPduL	O
+,	O
+rPduL	O
+,	O
+and	O
+pPduL	O
+.	O
+These	O
+three	O
+homologs	O
+are	O
+found	O
+in	O
+functionally	O
+distinct	O
+BMCs	O
+(	O
+Table	O
+1	O
+).	O
+	O
+It	O
+has	O
+been	O
+proposed	O
+that	O
+the	O
+catabolic	O
+BMCs	O
+may	O
+assemble	O
+in	O
+a	O
+core	O
+-	O
+first	O
+manner	O
+,	O
+with	O
+the	O
+luminal	O
+enzymes	O
+(	O
+signature	O
+enzyme	O
+,	O
+aldehyde	O
+,	O
+and	O
+alcohol	O
+dehydrogenases	O
+and	O
+the	O
+BMC	O
+PTAC	O
+)	O
+forming	O
+an	O
+initial	O
+bolus	O
+,	O
+or	O
+prometabolosome	O
+,	O
+around	O
+which	O
+a	O
+shell	O
+assembles	O
+.	O
+	O
+We	O
+found	O
+that	O
+not	O
+only	O
+did	O
+the	O
+different	O
+orthologs	O
+appear	O
+to	O
+assemble	O
+into	O
+different	O
+oligomeric	O
+states	O
+,	O
+but	O
+that	O
+quaternary	O
+structure	O
+was	O
+dependent	O
+on	O
+whether	O
+or	O
+not	O
+the	O
+EP	O
+was	O
+present	O
+.	O
+	O
+Full	O
+-	O
+length	O
+sPduL	O
+was	O
+unstable	O
+in	O
+solution	O
+—	O
+precipitating	O
+over	O
+time	O
+—	O
+and	O
+eluted	O
+throughout	O
+the	O
+entire	O
+volume	O
+of	O
+a	O
+size	O
+exclusion	O
+column	O
+,	O
+indicating	O
+it	O
+was	O
+nonspecifically	O
+aggregating	O
+.	O
+	O
+However	O
+,	O
+when	O
+the	O
+putative	O
+EP	O
+(	O
+residues	O
+1	O
+–	O
+27	O
+)	O
+was	O
+removed	O
+(	O
+sPduL	B-mutant
+ΔEP	I-mutant
+),	O
+the	O
+truncated	O
+protein	O
+was	O
+stable	O
+and	O
+eluted	O
+as	O
+a	O
+single	O
+peak	O
+(	O
+Fig	O
+5a	O
+)	O
+consistent	O
+with	O
+the	O
+size	O
+of	O
+a	O
+monomer	O
+(	O
+Fig	O
+5d	O
+,	O
+blue	O
+curve	O
+).	O
+	O
+In	O
+contrast	O
+,	O
+both	O
+full	O
+-	O
+length	O
+rPduL	O
+and	O
+pPduL	O
+appeared	O
+to	O
+exist	O
+in	O
+two	O
+distinct	O
+oligomeric	O
+states	O
+(	O
+Fig	O
+5b	O
+and	O
+5c	O
+respectively	O
+,	O
+orange	O
+curves	O
+),	O
+one	O
+form	O
+of	O
+the	O
+approximate	O
+size	O
+of	O
+a	O
+dimer	O
+and	O
+the	O
+second	O
+,	O
+a	O
+higher	O
+molecular	O
+weight	O
+oligomer	O
+(~	O
+150	O
+kDa	O
+).	O
+	O
+Upon	O
+deletion	O
+of	O
+the	O
+putative	O
+EP	O
+(	O
+residues	O
+1	O
+–	O
+47	O
+for	O
+rPduL	O
+,	O
+and	O
+1	O
+–	O
+20	O
+for	O
+pPduL	O
+),	O
+there	O
+was	O
+a	O
+distinct	O
+change	O
+in	O
+the	O
+elution	O
+profiles	O
+(	O
+Fig	O
+5b	O
+and	O
+5c	O
+respectively	O
+,	O
+blue	O
+curves	O
+).	O
+	O
+In	O
+contrast	O
+,	O
+rPduLΔEP	B-mutant
+eluted	O
+as	O
+one	O
+smaller	O
+oligomer	O
+,	O
+possibly	O
+a	O
+dimer	O
+.	O
+	O
+We	O
+also	O
+analyzed	O
+purified	O
+rPduL	O
+and	O
+rPduLΔEP	B-mutant
+by	O
+size	O
+exclusion	O
+chromatography	O
+coupled	O
+with	O
+multiangle	O
+light	O
+scattering	O
+(	O
+SEC	O
+-	O
+MALS	O
+)	O
+for	O
+a	O
+complementary	O
+approach	O
+to	O
+assessing	O
+oligomeric	O
+state	O
+.	O
+	O
+SEC	O
+-	O
+MALS	O
+analysis	O
+of	O
+rPdulΔEP	B-mutant
+is	O
+consistent	O
+with	O
+a	O
+dimer	O
+(	O
+as	O
+observed	O
+in	O
+the	O
+crystal	O
+structure	O
+)	O
+with	O
+a	O
+weighted	O
+average	O
+(	O
+Mw	O
+)	O
+and	O
+number	O
+average	O
+(	O
+Mn	O
+)	O
+of	O
+the	O
+molar	O
+mass	O
+of	O
+58	O
+.	O
+4	O
+kDa	O
++/−	O
+11	O
+.	O
+2	O
+%	O
+and	O
+58	O
+.	O
+8	O
+kDa	O
++/−	O
+10	O
+.	O
+9	O
+%,	O
+respectively	O
+(	O
+S4a	O
+Fig	O
+).	O
+	O
+This	O
+corresponds	O
+to	O
+an	O
+oligomeric	O
+state	O
+of	O
+six	O
+subunits	O
+(	O
+calculated	O
+molecular	O
+weight	O
+of	O
+144	O
+kDa	O
+).	O
+	O
+Collectively	O
+,	O
+these	O
+data	O
+strongly	O
+suggest	O
+that	O
+the	O
+N	O
+-	O
+terminal	O
+EP	O
+of	O
+PduL	O
+plays	O
+a	O
+role	O
+in	O
+defining	O
+the	O
+quaternary	O
+structure	O
+of	O
+the	O
+protein	O
+.	O
+	O
+The	O
+BMC	O
+shell	O
+not	O
+only	O
+sequesters	O
+specific	O
+enzymes	O
+but	O
+also	O
+their	O
+cofactors	O
+,	O
+thereby	O
+establishing	O
+a	O
+private	O
+cofactor	O
+pool	O
+dedicated	O
+to	O
+the	O
+encapsulated	O
+reactions	O
+.	O
+	O
+In	O
+catabolic	O
+BMCs	O
+,	O
+CoA	O
+and	O
+NAD	O
++	O
+must	O
+be	O
+continually	O
+recycled	O
+within	O
+the	O
+organelle	O
+(	O
+Fig	O
+1	O
+).	O
+	O
+Curiously	O
+,	O
+while	O
+the	O
+housekeeping	O
+Pta	O
+could	O
+provide	O
+this	O
+function	O
+,	O
+and	O
+indeed	O
+does	O
+so	O
+in	O
+the	O
+case	O
+of	O
+one	O
+type	O
+of	O
+ethanolamine	O
+-	O
+utilizing	O
+(	O
+EUT	O
+)	O
+BMC	O
+,	O
+the	O
+evolutionarily	O
+unrelated	O
+PduL	O
+fulfills	O
+this	O
+function	O
+for	O
+the	O
+majority	O
+of	O
+metabolosomes	O
+using	O
+a	O
+novel	O
+structure	O
+and	O
+active	O
+site	O
+for	O
+convergent	O
+evolution	O
+of	O
+function	O
+.	O
+	O
+The	O
+Tertiary	O
+Structure	O
+of	O
+PduL	O
+Is	O
+Formed	O
+by	O
+Discontinuous	O
+Segments	O
+of	O
+Primary	O
+Structure	O
+	O
+The	O
+structure	O
+of	O
+PduL	O
+consists	O
+of	O
+two	O
+β	O
+-	O
+barrel	O
+domains	O
+capped	O
+by	O
+short	O
+alpha	O
+helical	O
+segments	O
+(	O
+Fig	O
+2b	O
+).	O
+	O
+The	O
+two	O
+domains	O
+are	O
+structurally	O
+very	O
+similar	O
+(	O
+superimposing	O
+with	O
+a	O
+rmsd	O
+of	O
+1	O
+.	O
+34	O
+Å	O
+(	O
+over	O
+123	O
+out	O
+of	O
+320	O
+/	O
+348	O
+aligned	O
+backbone	O
+atoms	O
+,	O
+S5a	O
+Fig	O
+).	O
+	O
+However	O
+,	O
+the	O
+amino	O
+acid	O
+sequences	O
+of	O
+the	O
+two	O
+domains	O
+are	O
+only	O
+16	O
+%	O
+identical	O
+(	O
+mainly	O
+the	O
+RHxH	O
+motif	O
+,	O
+β2	O
+and	O
+β10	O
+),	O
+and	O
+34	O
+%	O
+similar	O
+.	O
+	O
+Our	O
+structure	O
+reveals	O
+that	O
+the	O
+two	O
+assigned	O
+PF06130	O
+domains	O
+(	O
+Fig	O
+3	O
+)	O
+do	O
+not	O
+form	O
+structurally	O
+discrete	O
+units	O
+;	O
+this	O
+reduces	O
+the	O
+apparent	O
+sequence	O
+conservation	O
+at	O
+the	O
+level	O
+of	O
+primary	O
+structure	O
+.	O
+	O
+One	O
+strand	O
+of	O
+the	O
+domain	O
+1	O
+beta	O
+barrel	O
+(	O
+shown	O
+in	O
+blue	O
+in	O
+Fig	O
+2	O
+)	O
+is	O
+contributed	O
+by	O
+the	O
+N	O
+-	O
+terminus	O
+,	O
+while	O
+the	O
+rest	O
+of	O
+the	O
+domain	O
+is	O
+formed	O
+by	O
+the	O
+residues	O
+from	O
+the	O
+C	O
+-	O
+terminal	O
+half	O
+of	O
+the	O
+protein	O
+.	O
+	O
+When	O
+aligned	O
+by	O
+structure	O
+,	O
+the	O
+β1	O
+strand	O
+of	O
+the	O
+first	O
+domain	O
+(	O
+Fig	O
+2a	O
+and	O
+2b	O
+,	O
+blue	O
+)	O
+corresponds	O
+to	O
+the	O
+final	O
+strand	O
+of	O
+the	O
+second	O
+domain	O
+(	O
+β9	O
+),	O
+effectively	O
+making	O
+the	O
+domains	O
+continuous	O
+if	O
+the	O
+first	O
+strand	O
+was	O
+transplanted	O
+to	O
+the	O
+C	O
+-	O
+terminus	O
+.	O
+	O
+The	O
+closest	O
+structural	O
+homolog	O
+of	O
+the	O
+PduL	O
+barrel	O
+domain	O
+is	O
+a	O
+subdomain	O
+of	O
+a	O
+multienzyme	O
+complex	O
+,	O
+the	O
+alpha	O
+subunit	O
+of	O
+ethylbenzene	O
+dehydrogenase	O
+(	O
+S5b	O
+Fig	O
+,	O
+rmsd	O
+of	O
+2	O
+.	O
+26	O
+Å	O
+over	O
+226	O
+aligned	O
+atoms	O
+consisting	O
+of	O
+one	O
+beta	O
+barrel	O
+and	O
+one	O
+capping	O
+helix	O
+).	O
+	O
+In	O
+contrast	O
+to	O
+PduL	O
+,	O
+there	O
+is	O
+only	O
+one	O
+barrel	O
+present	O
+in	O
+ethylbenzene	O
+dehydrogenase	O
+,	O
+and	O
+there	O
+is	O
+no	O
+comparable	O
+active	O
+site	O
+arrangement	O
+.	O
+	O
+The	O
+PduL	O
+signature	O
+primary	O
+structure	O
+,	O
+two	O
+PF06130	O
+domains	O
+,	O
+occurs	O
+in	O
+some	O
+multidomain	O
+proteins	O
+,	O
+most	O
+of	O
+them	O
+annotated	O
+as	O
+Acks	O
+,	O
+suggesting	O
+that	O
+PduL	O
+may	O
+also	O
+replace	O
+Pta	O
+in	O
+variants	O
+of	O
+the	O
+phosphotransacetylase	O
+-	O
+Ack	O
+pathway	O
+.	O
+	O
+These	O
+PduL	O
+homologs	O
+lack	O
+EPs	O
+,	O
+and	O
+their	O
+fusion	O
+to	O
+Ack	O
+may	O
+have	O
+evolved	O
+as	O
+a	O
+way	O
+to	O
+facilitate	O
+substrate	O
+channeling	O
+between	O
+the	O
+two	O
+enzymes	O
+.	O
+	O
+For	O
+BMC	O
+-	O
+encapsulated	O
+proteins	O
+to	O
+properly	O
+function	O
+together	O
+,	O
+they	O
+must	O
+be	O
+targeted	O
+to	O
+the	O
+lumen	O
+and	O
+assemble	O
+into	O
+an	O
+organization	O
+that	O
+facilitates	O
+substrate	O
+/	O
+product	O
+channeling	O
+among	O
+the	O
+different	O
+catalytic	O
+sites	O
+of	O
+the	O
+signature	O
+and	O
+core	O
+enzymes	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+extension	O
+on	O
+PduL	O
+homologs	O
+may	O
+serve	O
+both	O
+of	O
+these	O
+functions	O
+.	O
+	O
+The	O
+extension	O
+shares	O
+many	O
+features	O
+with	O
+previously	O
+characterized	O
+EPs	O
+:	O
+it	O
+is	O
+present	O
+only	O
+in	O
+homologs	O
+associated	O
+with	O
+BMC	O
+loci	O
+,	O
+and	O
+it	O
+is	O
+predicted	O
+to	O
+form	O
+an	O
+amphipathic	O
+α	O
+-	O
+helix	O
+.	O
+	O
+Moreover	O
+,	O
+its	O
+removal	O
+affects	O
+the	O
+oligomeric	O
+state	O
+of	O
+the	O
+protein	O
+.	O
+	O
+EP	O
+-	O
+mediated	O
+oligomerization	O
+has	O
+been	O
+observed	O
+for	O
+the	O
+signature	O
+and	O
+core	O
+BMC	O
+enzymes	O
+;	O
+for	O
+example	O
+,	O
+full	O
+-	O
+length	O
+propanediol	O
+dehydratase	O
+and	O
+ethanolamine	O
+ammonia	O
+-	O
+lyase	O
+(	O
+signature	O
+enzymes	O
+for	O
+PDU	O
+and	O
+EUT	O
+BMCs	O
+)	O
+subunits	O
+are	O
+also	O
+insoluble	O
+,	O
+but	O
+become	O
+soluble	O
+upon	O
+removal	O
+of	O
+the	O
+predicted	O
+EP	O
+.	O
+	O
+sPduL	O
+has	O
+also	O
+previously	O
+been	O
+reported	O
+to	O
+localize	O
+to	O
+inclusion	O
+bodies	O
+when	O
+overexpressed	O
+;	O
+we	O
+show	O
+here	O
+that	O
+this	O
+is	O
+dependent	O
+on	O
+the	O
+presence	O
+of	O
+the	O
+EP	O
+.	O
+	O
+This	O
+propensity	O
+of	O
+the	O
+EP	O
+to	O
+cause	O
+proteins	O
+to	O
+form	O
+complexes	O
+(	O
+Fig	O
+5	O
+)	O
+might	O
+not	O
+be	O
+a	O
+coincidence	O
+,	O
+but	O
+could	O
+be	O
+a	O
+necessary	O
+step	O
+in	O
+the	O
+assembly	O
+of	O
+BMCs	O
+.	O
+	O
+Structured	O
+aggregation	O
+of	O
+the	O
+core	O
+enzymes	O
+has	O
+been	O
+proposed	O
+to	O
+be	O
+the	O
+initial	O
+step	O
+in	O
+metabolosome	O
+assembly	O
+and	O
+is	O
+known	O
+to	O
+be	O
+the	O
+first	O
+step	O
+of	O
+β	O
+-	O
+carboxysome	O
+biogenesis	O
+,	O
+where	O
+the	O
+core	O
+enzyme	O
+Ribulose	O
+Bisphosphate	O
+Carboxylase	O
+/	O
+Oxygenase	O
+(	O
+RuBisCO	O
+)	O
+is	O
+aggregated	O
+by	O
+the	O
+CcmM	O
+protein	O
+.	O
+	O
+Likewise	O
+,	O
+CsoS2	O
+,	O
+a	O
+protein	O
+in	O
+the	O
+α	O
+-	O
+carboxysome	O
+core	O
+,	O
+also	O
+aggregates	O
+when	O
+purified	O
+and	O
+is	O
+proposed	O
+to	O
+facilitate	O
+the	O
+nucleation	O
+and	O
+encapsulation	O
+of	O
+RuBisCO	O
+molecules	O
+in	O
+the	O
+lumen	O
+of	O
+the	O
+organelle	O
+.	O
+	O
+This	O
+role	O
+for	O
+EPs	O
+in	O
+BMC	O
+assembly	O
+is	O
+in	O
+addition	O
+to	O
+their	O
+interaction	O
+with	O
+shell	O
+proteins	O
+.	O
+	O
+Our	O
+PduL	O
+crystals	O
+contained	O
+CoA	O
+that	O
+was	O
+captured	O
+from	O
+the	O
+Escherichia	O
+coli	O
+cytosol	O
+,	O
+indicating	O
+that	O
+the	O
+“	O
+ground	O
+state	O
+”	O
+of	O
+PduL	O
+is	O
+in	O
+the	O
+CoA	O
+-	O
+bound	O
+form	O
+;	O
+this	O
+could	O
+provide	O
+an	O
+elegantly	O
+simple	O
+means	O
+of	O
+guaranteeing	O
+a	O
+1	O
+:	O
+1	O
+ratio	O
+of	O
+CoA	O
+:	O
+PduL	O
+within	O
+the	O
+metabolosome	O
+lumen	O
+.	O
+	O
+Active	O
+Site	O
+Identification	O
+and	O
+Structural	O
+Insights	O
+into	O
+Catalysis	O
+	O
+The	O
+active	O
+site	O
+of	O
+PduL	O
+is	O
+formed	O
+at	O
+the	O
+interface	O
+of	O
+the	O
+two	O
+structural	O
+domains	O
+(	O
+Fig	O
+2b	O
+).	O
+	O
+As	O
+expected	O
+,	O
+the	O
+amino	O
+acid	O
+sequence	O
+conservation	O
+is	O
+highest	O
+in	O
+the	O
+region	O
+around	O
+the	O
+proposed	O
+active	O
+site	O
+(	O
+Fig	O
+4d	O
+);	O
+highly	O
+conserved	O
+residues	O
+are	O
+also	O
+involved	O
+in	O
+CoA	O
+binding	O
+(	O
+Figs	O
+2a	O
+and	O
+3	O
+,	O
+residues	O
+Ser45	O
+,	O
+Lys70	O
+,	O
+Arg97	O
+,	O
+Leu99	O
+,	O
+His204	O
+,	O
+Asn211	O
+).	O
+	O
+All	O
+of	O
+the	O
+metal	O
+-	O
+coordinating	O
+residues	O
+(	O
+Fig	O
+2a	O
+)	O
+are	O
+absolutely	O
+conserved	O
+,	O
+implicating	O
+them	O
+in	O
+catalysis	O
+or	O
+the	O
+correct	O
+spatial	O
+orientation	O
+of	O
+the	O
+substrates	O
+.	O
+	O
+Arg103	O
+,	O
+which	O
+contacts	O
+the	O
+phosphate	O
+(	O
+Fig	O
+4b	O
+),	O
+is	O
+present	O
+in	O
+all	O
+PduL	O
+homologs	O
+.	O
+	O
+The	O
+close	O
+resemblance	O
+between	O
+the	O
+structures	O
+binding	O
+CoA	O
+and	O
+phosphate	O
+likely	O
+indicates	O
+that	O
+no	O
+large	O
+changes	O
+in	O
+protein	O
+conformation	O
+are	O
+involved	O
+in	O
+catalysis	O
+,	O
+and	O
+that	O
+our	O
+crystal	O
+structures	O
+are	O
+representative	O
+of	O
+the	O
+active	O
+form	O
+.	O
+	O
+There	O
+is	O
+a	O
+pocket	O
+nearby	O
+the	O
+active	O
+site	O
+between	O
+the	O
+well	O
+-	O
+conserved	O
+residues	O
+Ser45	O
+and	O
+Ala154	O
+,	O
+which	O
+could	O
+accommodate	O
+the	O
+propionyl	O
+group	O
+(	O
+S6	O
+Fig	O
+).	O
+	O
+A	O
+homology	O
+model	O
+of	O
+sPduL	O
+indicates	O
+that	O
+the	O
+residues	O
+making	O
+up	O
+this	O
+pocket	O
+and	O
+the	O
+surrounding	O
+active	O
+site	O
+region	O
+are	O
+identical	O
+to	O
+that	O
+of	O
+rPduL	O
+,	O
+which	O
+is	O
+not	O
+surprising	O
+,	O
+because	O
+these	O
+two	O
+homologs	O
+presumably	O
+have	O
+the	O
+same	O
+propionyl	O
+-	O
+CoA	O
+substrate	O
+.	O
+	O
+The	O
+homology	O
+model	O
+of	O
+pPduL	O
+also	O
+has	O
+identical	O
+residues	O
+making	O
+up	O
+the	O
+pocket	O
+,	O
+but	O
+with	O
+a	O
+key	O
+difference	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+active	O
+site	O
+:	O
+Gln77	O
+of	O
+rPduL	O
+is	O
+replaced	O
+by	O
+a	O
+tyrosine	O
+(	O
+Tyr77	O
+)	O
+in	O
+pPduL	O
+.	O
+The	O
+physiological	O
+substrate	O
+of	O
+pPduL	O
+(	O
+Table	O
+1	O
+)	O
+is	O
+thought	O
+to	O
+be	O
+lactyl	O
+-	O
+CoA	O
+,	O
+which	O
+contains	O
+an	O
+additional	O
+hydroxyl	O
+group	O
+relative	O
+to	O
+propionyl	O
+-	O
+CoA	O
+.	O
+The	O
+presence	O
+of	O
+an	O
+aromatic	O
+residue	O
+at	O
+this	O
+position	O
+may	O
+underlie	O
+the	O
+substrate	O
+preference	O
+of	O
+the	O
+PduL	O
+enzyme	O
+from	O
+the	O
+pvm	O
+locus	O
+.	O
+	O
+The	O
+catalytic	O
+mechanism	O
+of	O
+Pta	O
+involves	O
+the	O
+abstraction	O
+of	O
+a	O
+thiol	O
+hydrogen	O
+by	O
+an	O
+aspartate	O
+residue	O
+,	O
+resulting	O
+in	O
+the	O
+nucleophilic	O
+attack	O
+of	O
+thiolate	O
+upon	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+acetyl	O
+-	O
+phosphate	O
+,	O
+oriented	O
+by	O
+an	O
+arginine	O
+and	O
+stabilized	O
+by	O
+a	O
+serine	O
+—	O
+there	O
+are	O
+no	O
+metals	O
+involved	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+in	O
+the	O
+rPduL	O
+structure	O
+,	O
+there	O
+are	O
+no	O
+conserved	O
+aspartate	O
+residues	O
+in	O
+or	O
+around	O
+the	O
+active	O
+site	O
+,	O
+and	O
+the	O
+only	O
+well	O
+-	O
+conserved	O
+glutamate	O
+residue	O
+in	O
+the	O
+active	O
+site	O
+is	O
+involved	O
+in	O
+coordinating	O
+one	O
+of	O
+the	O
+metal	O
+ions	O
+.	O
+	O
+These	O
+observations	O
+strongly	O
+suggest	O
+that	O
+an	O
+acidic	O
+residue	O
+is	O
+not	O
+directly	O
+involved	O
+in	O
+catalysis	O
+by	O
+PduL	O
+.	O
+Instead	O
+,	O
+the	O
+dimetal	O
+active	O
+site	O
+of	O
+PduL	O
+may	O
+create	O
+a	O
+nucleophile	O
+from	O
+one	O
+of	O
+the	O
+hydroxyl	O
+groups	O
+on	O
+free	O
+phosphate	O
+to	O
+attack	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+the	O
+thioester	O
+bond	O
+of	O
+an	O
+acyl	O
+-	O
+CoA	O
+.	O
+In	O
+the	O
+reverse	O
+direction	O
+,	O
+the	O
+metal	O
+ion	O
+(	O
+s	O
+)	O
+could	O
+stabilize	O
+the	O
+thiolate	O
+anion	O
+that	O
+would	O
+attack	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+an	O
+acyl	O
+-	O
+phosphate	O
+;	O
+a	O
+similar	O
+mechanism	O
+has	O
+been	O
+described	O
+for	O
+phosphatases	O
+where	O
+hydroxyl	O
+groups	O
+or	O
+hydroxide	O
+ions	O
+can	O
+act	O
+as	O
+a	O
+base	O
+when	O
+coordinated	O
+by	O
+a	O
+dimetal	O
+active	O
+site	O
+.	O
+	O
+Our	O
+structures	O
+provide	O
+the	O
+foundation	O
+for	O
+studies	O
+to	O
+elucidate	O
+the	O
+details	O
+of	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+PduL	O
+.	O
+Conserved	O
+residues	O
+in	O
+the	O
+active	O
+site	O
+that	O
+may	O
+contribute	O
+to	O
+substrate	O
+binding	O
+and	O
+/	O
+or	O
+transition	O
+state	O
+stabilization	O
+include	O
+Ser127	O
+,	O
+Arg103	O
+,	O
+Arg194	O
+,	O
+Gln107	O
+,	O
+Gln74	O
+,	O
+and	O
+Gln	O
+/	O
+Glu77	O
+.	O
+	O
+In	O
+the	O
+phosphate	O
+-	O
+bound	O
+crystal	O
+structure	O
+,	O
+Ser127	O
+and	O
+Arg103	O
+appear	O
+to	O
+position	O
+the	O
+phosphate	O
+(	O
+Fig	O
+4b	O
+).	O
+	O
+Alternatively	O
+,	O
+Arg103	O
+might	O
+act	O
+as	O
+a	O
+base	O
+to	O
+render	O
+the	O
+phosphate	O
+more	O
+nucleophilic	O
+.	O
+	O
+The	O
+functional	O
+groups	O
+of	O
+Gln74	O
+,	O
+Gln	O
+/	O
+Glu77	O
+,	O
+and	O
+Arg194	O
+are	O
+directed	O
+away	O
+from	O
+the	O
+active	O
+site	O
+in	O
+both	O
+CoA	O
+and	O
+phosphate	O
+-	O
+bound	O
+crystal	O
+structures	O
+and	O
+do	O
+not	O
+appear	O
+to	O
+be	O
+involved	O
+in	O
+hydrogen	O
+bonding	O
+with	O
+these	O
+substrates	O
+,	O
+although	O
+they	O
+could	O
+be	O
+important	O
+for	O
+positioning	O
+an	O
+acyl	O
+-	O
+phosphate	O
+.	O
+	O
+This	O
+hypothesis	O
+is	O
+strengthened	O
+by	O
+the	O
+fact	O
+that	O
+the	O
+CoA	O
+-	O
+bound	O
+crystals	O
+were	O
+obtained	O
+without	O
+added	O
+CoA	O
+,	O
+indicating	O
+that	O
+the	O
+protein	O
+bound	O
+CoA	O
+from	O
+the	O
+E	O
+.	O
+coli	O
+expression	O
+strain	O
+and	O
+retained	O
+it	O
+throughout	O
+purification	O
+and	O
+crystallization	O
+.	O
+	O
+Functional	O
+,	O
+but	O
+Not	O
+Structural	O
+,	O
+Convergence	O
+of	O
+PduL	O
+and	O
+Pta	O
+	O
+PduL	O
+and	O
+Pta	O
+are	O
+mechanistically	O
+and	O
+structurally	O
+distinct	O
+enzymes	O
+that	O
+catalyze	O
+the	O
+same	O
+reaction	O
+,	O
+a	O
+prime	O
+example	O
+of	O
+evolutionary	O
+convergence	O
+upon	O
+a	O
+function	O
+.	O
+	O
+There	O
+are	O
+several	O
+examples	O
+of	O
+such	O
+functional	O
+convergence	O
+of	O
+enzymes	O
+,	O
+although	O
+typically	O
+the	O
+enzymes	O
+have	O
+independently	O
+evolved	O
+similar	O
+,	O
+or	O
+even	O
+identical	O
+active	O
+sites	O
+;	O
+for	O
+example	O
+,	O
+the	O
+carbonic	O
+anhydrase	O
+family	O
+.	O
+	O
+However	O
+,	O
+apparently	O
+less	O
+frequent	O
+is	O
+functional	O
+convergence	O
+that	O
+is	O
+supported	O
+by	O
+distinctly	O
+different	O
+active	O
+sites	O
+and	O
+accordingly	O
+catalytic	O
+mechanism	O
+,	O
+as	O
+revealed	O
+by	O
+comparison	O
+of	O
+the	O
+structures	O
+of	O
+Pta	O
+and	O
+PduL	O
+.	O
+One	O
+well	O
+-	O
+studied	O
+example	O
+of	O
+this	O
+is	O
+the	O
+β	O
+-	O
+lactamase	O
+family	O
+of	O
+enzymes	O
+,	O
+in	O
+which	O
+the	O
+active	O
+site	O
+of	O
+Class	O
+A	O
+and	O
+Class	O
+C	O
+enzymes	O
+involve	O
+serine	O
+-	O
+based	O
+catalysis	O
+,	O
+but	O
+Class	O
+B	O
+enzymes	O
+are	O
+metalloproteins	O
+.	O
+	O
+This	O
+is	O
+not	O
+surprising	O
+,	O
+as	O
+β	O
+-	O
+lactamases	O
+are	O
+not	O
+so	O
+widespread	O
+among	O
+bacteria	O
+and	O
+therefore	O
+would	O
+be	O
+expected	O
+to	O
+have	O
+evolved	O
+independently	O
+several	O
+times	O
+as	O
+a	O
+defense	O
+mechanism	O
+against	O
+β	O
+-	O
+lactam	O
+antibiotics	O
+.	O
+	O
+However	O
+,	O
+nearly	O
+all	O
+bacteria	O
+encode	O
+Pta	O
+,	O
+and	O
+it	O
+is	O
+not	O
+immediately	O
+clear	O
+why	O
+the	O
+Pta	O
+/	O
+PduL	O
+functional	O
+convergence	O
+should	O
+have	O
+evolved	O
+:	O
+it	O
+would	O
+seem	O
+to	O
+be	O
+evolutionarily	O
+more	O
+resourceful	O
+for	O
+the	O
+Pta	O
+-	O
+encoding	O
+gene	O
+to	O
+be	O
+duplicated	O
+and	O
+repurposed	O
+for	O
+BMCs	O
+,	O
+as	O
+is	O
+apparently	O
+the	O
+case	O
+in	O
+one	O
+type	O
+of	O
+BMC	O
+—	O
+EUT1	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Further	O
+biochemical	O
+comparison	O
+between	O
+the	O
+two	O
+PTACs	O
+will	O
+likely	O
+yield	O
+exciting	O
+results	O
+that	O
+could	O
+answer	O
+this	O
+evolutionary	O
+question	O
+.	O
+	O
+BMCs	O
+are	O
+now	O
+known	O
+to	O
+be	O
+widespread	O
+among	O
+the	O
+bacteria	O
+and	O
+are	O
+involved	O
+in	O
+critical	O
+segments	O
+of	O
+both	O
+autotrophic	O
+and	O
+heterotrophic	O
+biochemical	O
+pathways	O
+that	O
+confer	O
+to	O
+the	O
+host	O
+organism	O
+a	O
+competitive	O
+(	O
+metabolic	O
+)	O
+advantage	O
+in	O
+select	O
+niches	O
+.	O
+	O
+As	O
+one	O
+of	O
+the	O
+three	O
+common	O
+metabolosome	O
+core	O
+enzymes	O
+,	O
+the	O
+structure	O
+of	O
+PduL	O
+provides	O
+a	O
+key	O
+missing	O
+piece	O
+to	O
+our	O
+structural	O
+picture	O
+of	O
+the	O
+shared	O
+core	O
+biochemistry	O
+(	O
+Fig	O
+1	O
+)	O
+of	O
+functionally	O
+diverse	O
+catabolic	O
+BMCs	O
+.	O
+	O
+We	O
+have	O
+observed	O
+the	O
+oligomeric	O
+state	O
+differences	O
+of	O
+PduL	O
+to	O
+correlate	O
+with	O
+the	O
+presence	O
+of	O
+an	O
+EP	O
+,	O
+providing	O
+new	O
+insight	O
+into	O
+the	O
+function	O
+of	O
+this	O
+sequence	O
+extension	O
+in	O
+BMC	O
+assembly	O
+.	O
+	O
+Moreover	O
+,	O
+our	O
+results	O
+suggest	O
+a	O
+means	O
+for	O
+Coenzyme	O
+A	O
+incorporation	O
+during	O
+metabolosome	O
+biogenesis	O
+.	O
+	O
+The	O
+fact	O
+that	O
+PduL	O
+is	O
+confined	O
+almost	O
+exclusively	O
+to	O
+metabolosomes	O
+can	O
+be	O
+used	O
+to	O
+develop	O
+an	O
+inhibitor	O
+that	O
+blocks	O
+only	O
+PduL	O
+and	O
+not	O
+Pta	O
+as	O
+a	O
+way	O
+to	O
+selectively	O
+disrupt	O
+BMC	O
+-	O
+based	O
+metabolism	O
+,	O
+while	O
+not	O
+affecting	O
+most	O
+commensal	O
+organisms	O
+that	O
+require	O
+PTAC	O
+activity	O
+.	O
+	O
+Biochemistry	O
+and	O
+Crystal	O
+Structure	O
+of	O
+Ectoine	O
+Synthase	O
+:	O
+A	O
+Metal	O
+-	O
+Containing	O
+Member	O
+of	O
+the	O
+Cupin	O
+Superfamily	O
+	O
+Ectoine	O
+is	O
+a	O
+compatible	O
+solute	O
+and	O
+chemical	O
+chaperone	O
+widely	O
+used	O
+by	O
+members	O
+of	O
+the	O
+Bacteria	O
+and	O
+a	O
+few	O
+Archaea	O
+to	O
+fend	O
+-	O
+off	O
+the	O
+detrimental	O
+effects	O
+of	O
+high	O
+external	O
+osmolarity	O
+on	O
+cellular	O
+physiology	O
+and	O
+growth	O
+.	O
+	O
+Ectoine	O
+synthase	O
+(	O
+EctC	O
+)	O
+catalyzes	O
+the	O
+last	O
+step	O
+in	O
+ectoine	O
+production	O
+and	O
+mediates	O
+the	O
+ring	O
+closure	O
+of	O
+the	O
+substrate	O
+N	O
+-	O
+gamma	O
+-	O
+acetyl	O
+-	O
+L	O
+-	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyric	O
+acid	O
+through	O
+a	O
+water	O
+elimination	O
+reaction	O
+.	O
+	O
+However	O
+,	O
+the	O
+crystal	O
+structure	O
+of	O
+ectoine	O
+synthase	O
+is	O
+not	O
+known	O
+and	O
+a	O
+clear	O
+understanding	O
+of	O
+how	O
+its	O
+fold	O
+contributes	O
+to	O
+enzyme	O
+activity	O
+is	O
+thus	O
+lacking	O
+.	O
+	O
+Using	O
+the	O
+ectoine	O
+synthase	O
+from	O
+the	O
+cold	O
+-	O
+adapted	O
+marine	O
+bacterium	O
+Sphingopyxis	O
+alaskensis	O
+(	O
+Sa	O
+),	O
+we	O
+report	O
+here	O
+both	O
+a	O
+detailed	O
+biochemical	O
+characterization	O
+of	O
+the	O
+EctC	O
+enzyme	O
+and	O
+the	O
+high	O
+-	O
+resolution	O
+crystal	O
+structure	O
+of	O
+its	O
+apo	O
+-	O
+form	O
+.	O
+	O
+Structural	O
+analysis	O
+classified	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+as	O
+a	O
+member	O
+of	O
+the	O
+cupin	O
+superfamily	O
+.	O
+	O
+The	O
+interface	O
+of	O
+the	O
+dimer	O
+assembly	O
+is	O
+shaped	O
+through	O
+backbone	O
+-	O
+contacts	O
+and	O
+weak	O
+hydrophobic	O
+interactions	O
+mediated	O
+by	O
+two	O
+beta	O
+-	O
+sheets	O
+within	O
+each	O
+monomer	O
+.	O
+	O
+We	O
+found	O
+that	O
+EctC	O
+not	O
+only	O
+effectively	O
+converts	O
+its	O
+natural	O
+substrate	O
+N	O
+-	O
+gamma	O
+-	O
+acetyl	O
+-	O
+L	O
+-	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyric	O
+acid	O
+into	O
+ectoine	O
+through	O
+a	O
+cyclocondensation	O
+reaction	O
+,	O
+but	O
+that	O
+it	O
+can	O
+also	O
+use	O
+the	O
+isomer	O
+N	O
+-	O
+alpha	O
+-	O
+acetyl	O
+-	O
+L	O
+-	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyric	O
+acid	O
+as	O
+its	O
+substrate	O
+,	O
+albeit	O
+with	O
+substantially	O
+reduced	O
+catalytic	O
+efficiency	O
+.	O
+	O
+An	O
+assessment	O
+of	O
+enzyme	O
+activity	O
+and	O
+iron	O
+content	O
+of	O
+these	O
+mutants	O
+give	O
+important	O
+clues	O
+for	O
+understanding	O
+the	O
+architecture	O
+of	O
+the	O
+active	O
+site	O
+positioned	O
+within	O
+the	O
+core	O
+of	O
+the	O
+EctC	O
+cupin	O
+barrel	O
+.	O
+	O
+Ectoine	O
+[(	O
+S	O
+)-	O
+2	O
+-	O
+methyl	O
+-	O
+1	O
+,	O
+4	O
+,	O
+5	O
+,	O
+6	O
+-	O
+tetrahydropyrimidine	O
+-	O
+4	O
+-	O
+carboxylic	O
+acid	O
+]	O
+and	O
+its	O
+derivative	O
+5	O
+-	O
+hydroxyectoine	O
+[(	O
+4S	O
+,	O
+5S	O
+)-	O
+5	O
+-	O
+hydroxy	O
+-	O
+2	O
+-	O
+methyl	O
+-	O
+1	O
+,	O
+4	O
+,	O
+5	O
+,	O
+6	O
+-	O
+tetrahydropyrimidine	O
+-	O
+4	O
+-	O
+carboxylic	O
+acid	O
+]	O
+are	O
+such	O
+compatible	O
+solutes	O
+.	O
+	O
+Both	O
+marine	O
+and	O
+terrestrial	O
+microorganisms	O
+produce	O
+them	O
+widely	O
+in	O
+response	O
+to	O
+osmotic	O
+or	O
+temperature	O
+stress	O
+.	O
+	O
+Synthesis	O
+of	O
+ectoine	O
+occurs	O
+from	O
+the	O
+intermediate	O
+metabolite	O
+L	O
+-	O
+aspartate	O
+-	O
+ß	O
+-	O
+semialdehyde	O
+and	O
+comprises	O
+the	O
+sequential	O
+activities	O
+of	O
+three	O
+enzymes	O
+:	O
+L	O
+-	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyrate	O
+transaminase	O
+(	O
+EctB	O
+;	O
+EC	O
+2	O
+.	O
+6	O
+.	O
+1	O
+.	O
+76	O
+),	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyrate	O
+acetyltransferase	O
+(	O
+EctA	O
+;	O
+EC	O
+2	O
+.	O
+3	O
+.	O
+1	O
+.	O
+178	O
+),	O
+and	O
+ectoine	O
+synthase	O
+(	O
+EctC	O
+;	O
+EC	O
+4	O
+.	O
+2	O
+.	O
+1	O
+.	O
+108	O
+)	O
+(	O
+Fig	O
+1	O
+).	O
+	O
+The	O
+ectoine	O
+derivative	O
+5	O
+-	O
+hydroxyectoine	O
+,	O
+a	O
+highly	O
+effective	O
+stress	O
+protectant	O
+in	O
+its	O
+own	O
+right	O
+,	O
+is	O
+synthesized	O
+by	O
+a	O
+substantial	O
+subgroup	O
+of	O
+the	O
+ectoine	O
+producers	O
+.	O
+	O
+The	O
+remarkable	O
+function	O
+preserving	O
+effects	O
+of	O
+ectoines	O
+for	O
+macromolecules	O
+and	O
+cells	O
+,	O
+frequently	O
+also	O
+addressed	O
+as	O
+chemical	O
+chaperones	O
+,	O
+led	O
+to	O
+a	O
+substantial	O
+interest	O
+in	O
+exploiting	O
+these	O
+compounds	O
+for	O
+biotechnological	O
+purposes	O
+and	O
+medical	O
+applications	O
+.	O
+	O
+Biosynthetic	O
+routes	O
+for	O
+ectoine	O
+and	O
+5	O
+-	O
+hydroxyectoine	O
+.	O
+	O
+Here	O
+we	O
+focus	O
+on	O
+ectoine	O
+synthase	O
+(	O
+EctC	O
+),	O
+the	O
+key	O
+enzyme	O
+of	O
+the	O
+ectoine	O
+biosynthetic	O
+route	O
+(	O
+Fig	O
+1	O
+).	O
+	O
+Biochemical	O
+characterizations	O
+of	O
+ectoine	O
+synthases	O
+from	O
+the	O
+extremophiles	O
+Halomonas	O
+elongata	O
+,	O
+Methylomicrobium	O
+alcaliphilum	O
+,	O
+and	O
+Acidiphilium	O
+cryptum	O
+,	O
+and	O
+from	O
+the	O
+nitrifying	O
+archaeon	O
+Nitrosopumilus	O
+maritimus	O
+have	O
+been	O
+carried	O
+out	O
+.	O
+	O
+Each	O
+of	O
+these	O
+enzymes	O
+catalyzes	O
+as	O
+their	O
+main	O
+activity	O
+the	O
+cyclization	O
+of	O
+N	O
+-	O
+γ	O
+-	O
+acetyl	O
+-	O
+L	O
+-	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyric	O
+acid	O
+(	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+),	O
+the	O
+reaction	O
+product	O
+of	O
+the	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyrate	O
+acetyltransferase	O
+(	O
+EctA	O
+),	O
+to	O
+ectoine	O
+with	O
+the	O
+concomitant	O
+release	O
+of	O
+a	O
+water	O
+molecule	O
+(	O
+Fig	O
+1	O
+).	O
+	O
+In	O
+side	O
+reactions	O
+,	O
+EctC	O
+can	O
+promote	O
+the	O
+formation	O
+of	O
+the	O
+synthetic	O
+compatible	O
+solute	O
+5	O
+-	O
+amino	O
+-	O
+3	O
+,	O
+4	O
+-	O
+dihydro	O
+-	O
+2H	O
+-	O
+pyrrole	O
+-	O
+2	O
+-	O
+carboxylate	O
+(	O
+ADPC	O
+)	O
+through	O
+the	O
+cyclic	O
+condensation	O
+of	O
+two	O
+glutamine	O
+molecules	O
+and	O
+it	O
+also	O
+possesses	O
+a	O
+minor	O
+hydrolytic	O
+activity	O
+for	O
+ectoine	O
+and	O
+synthetic	O
+ectoine	O
+derivatives	O
+with	O
+either	O
+reduced	O
+or	O
+expanded	O
+ring	O
+sizes	O
+.	O
+	O
+Although	O
+progress	O
+has	O
+been	O
+made	O
+with	O
+respect	O
+to	O
+the	O
+biochemical	O
+characterization	O
+of	O
+ectoine	O
+synthase	O
+,	O
+a	O
+clear	O
+understanding	O
+of	O
+how	O
+its	O
+structure	O
+contributes	O
+to	O
+its	O
+enzyme	O
+activity	O
+and	O
+reaction	O
+mechanism	O
+is	O
+still	O
+lacking	O
+.	O
+With	O
+this	O
+in	O
+mind	O
+,	O
+we	O
+have	O
+biochemically	O
+characterized	O
+the	O
+ectoine	O
+synthase	O
+from	O
+the	O
+cold	O
+-	O
+adapted	O
+marine	O
+bacterium	O
+Sphingopyxis	O
+alaskensis	O
+(	O
+Sa	O
+).	O
+	O
+We	O
+demonstrate	O
+here	O
+for	O
+the	O
+first	O
+time	O
+that	O
+the	O
+ectoine	O
+synthase	O
+is	O
+a	O
+metal	O
+-	O
+dependent	O
+enzyme	O
+,	O
+with	O
+iron	O
+as	O
+the	O
+most	O
+likely	O
+physiologically	O
+relevant	O
+co	O
+-	O
+factor	O
+.	O
+	O
+Overproduction	O
+,	O
+purification	O
+and	O
+oligomeric	O
+state	O
+of	O
+the	O
+ectoine	O
+synthase	O
+in	O
+solution	O
+	O
+We	O
+focused	O
+our	O
+biochemical	O
+and	O
+structural	O
+studies	O
+on	O
+the	O
+ectoine	O
+synthase	O
+from	O
+S	O
+.	O
+alaskensis	O
+[(	O
+Sa	O
+)	O
+EctC	O
+],	O
+a	O
+cold	O
+-	O
+adapted	O
+marine	O
+ultra	O
+-	O
+microbacterium	O
+,	O
+from	O
+which	O
+we	O
+recently	O
+also	O
+determined	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+ectoine	O
+hydroxylase	O
+(	O
+EctD	O
+)	O
+in	O
+complex	O
+with	O
+either	O
+its	O
+substrate	O
+or	O
+its	O
+reaction	O
+product	O
+.	O
+	O
+We	O
+expressed	O
+a	O
+codon	O
+-	O
+optimized	O
+version	O
+of	O
+the	O
+S	O
+.	O
+alaskensis	O
+ectC	O
+gene	O
+in	O
+E	O
+.	O
+coli	O
+to	O
+produce	O
+a	O
+recombinant	O
+protein	O
+with	O
+a	O
+carboxy	O
+-	O
+terminally	O
+attached	O
+Strep	O
+-	O
+tag	O
+II	O
+affinity	O
+peptide	O
+to	O
+allow	O
+purification	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+-	O
+Strep	O
+-	O
+Tag	O
+-	O
+II	O
+protein	O
+by	O
+affinity	O
+chromatography	O
+.	O
+	O
+Conventional	O
+size	O
+-	O
+exclusion	O
+chromatography	O
+(	O
+SEC	O
+)	O
+has	O
+already	O
+shown	O
+that	O
+(	O
+Sa	O
+)	O
+EctC	O
+preparations	O
+produced	O
+in	O
+this	O
+fashion	O
+are	O
+homogeneous	O
+and	O
+that	O
+the	O
+protein	O
+forms	O
+dimers	O
+in	O
+solution	O
+.	O
+	O
+High	O
+performance	O
+liquid	O
+chromatography	O
+coupled	O
+with	O
+multi	O
+-	O
+angle	O
+light	O
+-	O
+scattering	O
+detection	O
+(	O
+HPLC	O
+-	O
+MALS	O
+)	O
+experiments	O
+carried	O
+out	O
+here	O
+confirmed	O
+that	O
+the	O
+purified	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+was	O
+mono	O
+-	O
+disperse	O
+and	O
+possessed	O
+a	O
+molecular	O
+mass	O
+of	O
+33	O
+.	O
+0	O
+±	O
+2	O
+.	O
+3	O
+kDa	O
+(	O
+S2b	O
+Fig	O
+).	O
+	O
+This	O
+value	O
+corresponds	O
+very	O
+well	O
+with	O
+the	O
+theoretically	O
+calculated	O
+molecular	O
+mass	O
+of	O
+an	O
+(	O
+Sa	O
+)	O
+EctC	O
+dimer	O
+(	O
+molecular	O
+mass	O
+of	O
+the	O
+monomer	O
+,	O
+including	O
+the	O
+Strep	O
+-	O
+tag	O
+II	O
+affinity	O
+peptide	O
+:	O
+16	O
+.	O
+3	O
+kDa	O
+).	O
+	O
+Such	O
+a	O
+quaternary	O
+assembly	O
+as	O
+dimer	O
+has	O
+also	O
+been	O
+reported	O
+for	O
+the	O
+EctC	O
+proteins	O
+from	O
+H	O
+.	O
+elongata	O
+and	O
+N	O
+.	O
+maritimus	O
+.	O
+	O
+The	O
+EctA	O
+-	O
+produced	O
+substrate	O
+of	O
+the	O
+ectoine	O
+synthase	O
+,	O
+N	O
+-	O
+γ	O
+-	O
+acetyl	O
+-	O
+L	O
+-	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyric	O
+acid	O
+(	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+)	O
+(	O
+Fig	O
+1	O
+),	O
+is	O
+commercially	O
+not	O
+available	O
+.	O
+	O
+We	O
+used	O
+alkaline	O
+hydrolysis	O
+of	O
+ectoine	O
+and	O
+subsequent	O
+chromatography	O
+on	O
+silica	O
+gel	O
+columns	O
+to	O
+obtain	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+in	O
+chemically	O
+highly	O
+purified	O
+form	O
+(	O
+S1a	O
+Fig	O
+).	O
+	O
+This	O
+procedure	O
+also	O
+yielded	O
+the	O
+isomer	O
+of	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+,	O
+N	O
+-	O
+α	O
+-	O
+acetyl	O
+-	O
+L	O
+-	O
+2	O
+,	O
+4	O
+-	O
+diaminobutyric	O
+acid	O
+(	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+)	O
+(	O
+S1b	O
+Fig	O
+).	O
+	O
+Using	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+as	O
+the	O
+substrate	O
+,	O
+we	O
+initially	O
+evaluated	O
+a	O
+set	O
+of	O
+biochemical	O
+parameters	O
+of	O
+the	O
+recombinant	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+S	O
+.	O
+alaskensis	O
+,	O
+from	O
+which	O
+the	O
+studied	O
+ectoine	O
+synthase	O
+was	O
+originally	O
+derived	O
+,	O
+is	O
+a	O
+microorganism	O
+that	O
+is	O
+well	O
+-	O
+adapted	O
+to	O
+a	O
+life	O
+in	O
+permanently	O
+cold	O
+ocean	O
+waters	O
+.	O
+	O
+Consistent	O
+with	O
+the	O
+physicochemical	O
+attributes	O
+of	O
+this	O
+habitat	O
+,	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+was	O
+already	O
+enzymatically	O
+active	O
+at	O
+5	O
+°	O
+C	O
+,	O
+had	O
+a	O
+temperature	O
+optimum	O
+of	O
+15	O
+°	O
+C	O
+and	O
+was	O
+able	O
+to	O
+function	O
+over	O
+a	O
+broad	O
+range	O
+of	O
+temperatures	O
+(	O
+S3a	O
+Fig	O
+).	O
+	O
+It	O
+possessed	O
+an	O
+alkaline	O
+pH	O
+optimum	O
+of	O
+8	O
+.	O
+5	O
+(	O
+S3b	O
+Fig	O
+),	O
+a	O
+value	O
+similar	O
+to	O
+the	O
+ectoine	O
+synthases	O
+from	O
+the	O
+halo	O
+-	O
+tolerant	O
+H	O
+.	O
+elongata	O
+(	O
+pH	O
+optimum	O
+of	O
+8	O
+.	O
+5	O
+to	O
+9	O
+.	O
+0	O
+),	O
+the	O
+alkaliphile	O
+M	O
+.	O
+alcaliphilum	O
+(	O
+pH	O
+optimum	O
+of	O
+9	O
+.	O
+0	O
+),	O
+and	O
+the	O
+acidophile	O
+Acidiphilium	O
+cryptum	O
+(	O
+pH	O
+optimum	O
+of	O
+8	O
+.	O
+5	O
+to	O
+9	O
+.	O
+0	O
+),	O
+whereas	O
+the	O
+EctC	O
+protein	O
+from	O
+N	O
+.	O
+maritimus	O
+has	O
+a	O
+neutral	O
+pH	O
+optimum	O
+(	O
+pH	O
+7	O
+.	O
+0	O
+).	O
+	O
+The	O
+salinity	O
+of	O
+the	O
+assay	O
+buffer	O
+had	O
+a	O
+significant	O
+influence	O
+on	O
+the	O
+maximal	O
+enzyme	O
+activity	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+An	O
+increase	O
+in	O
+either	O
+the	O
+NaCl	O
+or	O
+the	O
+KCl	O
+concentration	O
+led	O
+to	O
+an	O
+approximately	O
+5	O
+-	O
+fold	O
+enhancement	O
+of	O
+the	O
+ectoine	O
+synthase	O
+activity	O
+.	O
+	O
+The	O
+maximum	O
+enzyme	O
+activity	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+occurred	O
+around	O
+250	O
+mM	O
+NaCl	O
+or	O
+KCl	O
+,	O
+respectively	O
+.	O
+	O
+Considerations	O
+based	O
+on	O
+bioinformatics	O
+suggests	O
+that	O
+EctC	O
+belongs	O
+to	O
+the	O
+cupin	O
+superfamily	O
+.	O
+	O
+Most	O
+of	O
+these	O
+proteins	O
+contain	O
+catalytically	O
+important	O
+transition	O
+state	O
+metals	O
+such	O
+as	O
+iron	O
+,	O
+copper	O
+,	O
+zinc	O
+,	O
+manganese	O
+,	O
+cobalt	O
+,	O
+or	O
+nickel	O
+.	O
+	O
+Cupins	O
+contain	O
+two	O
+conserved	O
+motifs	O
+:	O
+G	O
+(	O
+X	O
+)	O
+5HXH	O
+(	O
+X	O
+)	O
+3	O
+,	O
+4E	O
+(	O
+X	O
+)	O
+6G	O
+and	O
+G	O
+(	O
+X	O
+)	O
+5PXG	O
+(	O
+X	O
+)	O
+2H	O
+(	O
+X	O
+)	O
+3N	O
+(	O
+the	O
+letters	O
+in	O
+bold	O
+represent	O
+those	O
+residues	O
+that	O
+often	O
+coordinate	O
+the	O
+metal	O
+).	O
+	O
+Inspection	O
+of	O
+a	O
+previous	O
+alignment	O
+of	O
+the	O
+amino	O
+acid	O
+sequences	O
+of	O
+440	O
+EctC	O
+-	O
+type	O
+proteins	O
+revealed	O
+that	O
+the	O
+canonical	O
+metal	O
+-	O
+binding	O
+motif	O
+(	O
+s	O
+)	O
+of	O
+cupin	O
+-	O
+type	O
+proteins	O
+is	O
+not	O
+conserved	O
+among	O
+members	O
+of	O
+the	O
+extended	O
+ectoine	O
+synthase	O
+protein	O
+family	O
+.	O
+	O
+An	O
+abbreviated	O
+alignment	O
+of	O
+the	O
+amino	O
+acid	O
+sequence	O
+of	O
+EctC	O
+-	O
+type	O
+proteins	O
+is	O
+shown	O
+in	O
+Fig	O
+2	O
+.	O
+	O
+Abbreviated	O
+alignment	O
+of	O
+EctC	O
+-	O
+type	O
+proteins	O
+.	O
+	O
+Strictly	O
+conserved	O
+amino	O
+acid	O
+residues	O
+are	O
+shown	O
+in	O
+yellow	O
+.	O
+	O
+Dots	O
+shown	O
+above	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+sequence	O
+indicate	O
+residues	O
+likely	O
+to	O
+be	O
+involved	O
+in	O
+iron	O
+-	O
+binding	O
+(	O
+red	O
+),	O
+ligand	O
+-	O
+binding	O
+(	O
+green	O
+)	O
+and	O
+stabilization	O
+of	O
+the	O
+loop	O
+-	O
+architecture	O
+(	O
+blue	O
+).	O
+	O
+The	O
+conserved	O
+residue	O
+Tyr	O
+-	O
+52	O
+with	O
+so	O
+-	O
+far	O
+undefined	O
+functions	O
+is	O
+indicated	O
+by	O
+a	O
+green	O
+dot	O
+circled	O
+in	O
+red	O
+.	O
+	O
+Secondary	O
+structural	O
+elements	O
+(	O
+α	O
+-	O
+helices	O
+and	O
+β	O
+-	O
+sheets	O
+)	O
+found	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structure	O
+are	O
+projected	O
+onto	O
+the	O
+amino	O
+acid	O
+sequences	O
+of	O
+EctC	O
+-	O
+type	O
+proteins	O
+.	O
+	O
+For	O
+this	O
+analysis	O
+we	O
+used	O
+recombinant	O
+(	O
+Sa	O
+)	O
+EctC	O
+preparations	O
+from	O
+three	O
+independent	O
+protein	O
+overproduction	O
+and	O
+purification	O
+experiments	O
+.	O
+	O
+The	O
+ICP	O
+-	O
+MS	O
+analyses	O
+yielded	O
+an	O
+iron	O
+content	O
+of	O
+0	O
+.	O
+66	O
+±	O
+0	O
+.	O
+06	O
+mol	O
+iron	O
+per	O
+mol	O
+of	O
+protein	O
+and	O
+the	O
+used	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+preparations	O
+also	O
+contained	O
+a	O
+minor	O
+amount	O
+of	O
+zinc	O
+(	O
+0	O
+.	O
+08	O
+mol	O
+zinc	O
+per	O
+mol	O
+of	O
+protein	O
+).	O
+	O
+All	O
+other	O
+assayed	O
+metals	O
+(	O
+copper	O
+and	O
+nickel	O
+)	O
+were	O
+only	O
+present	O
+in	O
+trace	O
+amounts	O
+(	O
+0	O
+.	O
+01	O
+mol	O
+metal	O
+per	O
+mol	O
+of	O
+protein	O
+,	O
+respectively	O
+).	O
+	O
+The	O
+presence	O
+of	O
+iron	O
+in	O
+these	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+preparations	O
+was	O
+further	O
+confirmed	O
+by	O
+a	O
+colorimetric	O
+method	O
+that	O
+is	O
+based	O
+on	O
+an	O
+iron	O
+-	O
+complexing	O
+reagent	O
+;	O
+this	O
+procedure	O
+yielded	O
+an	O
+iron	O
+-	O
+content	O
+of	O
+0	O
+.	O
+84	O
+±	O
+0	O
+.	O
+05	O
+mol	O
+per	O
+mol	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+Hence	O
+,	O
+both	O
+ICP	O
+-	O
+MS	O
+and	O
+the	O
+colorimetric	O
+method	O
+clearly	O
+established	O
+that	O
+the	O
+recombinantly	O
+produced	O
+ectoine	O
+synthase	O
+from	O
+S	O
+.	O
+alaskensis	O
+is	O
+an	O
+iron	O
+-	O
+containing	O
+protein	O
+.	O
+	O
+The	O
+reason	O
+for	O
+this	O
+difference	O
+is	O
+not	O
+known	O
+,	O
+but	O
+indicates	O
+that	O
+the	O
+well	O
+established	O
+colorimetric	O
+assay	O
+probably	O
+overestimates	O
+the	O
+iron	O
+content	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+preparations	O
+to	O
+a	O
+certain	O
+degree	O
+.	O
+	O
+The	O
+iron	O
+detected	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+preparations	O
+could	O
+serve	O
+a	O
+structural	O
+role	O
+,	O
+or	O
+most	O
+likely	O
+,	O
+could	O
+be	O
+critical	O
+for	O
+enzyme	O
+catalysis	O
+as	O
+is	O
+the	O
+case	O
+for	O
+many	O
+members	O
+of	O
+the	O
+cupin	O
+superfamily	O
+.	O
+	O
+The	O
+addition	O
+of	O
+very	O
+low	O
+concentrations	O
+of	O
+EDTA	O
+(	O
+0	O
+.	O
+05	O
+mM	O
+)	O
+to	O
+the	O
+EctC	O
+enzyme	O
+already	O
+led	O
+to	O
+a	O
+noticeable	O
+inhibition	O
+of	O
+the	O
+ectoine	O
+synthase	O
+activity	O
+and	O
+the	O
+presence	O
+of	O
+1	O
+mM	O
+EDTA	O
+completely	O
+inhibited	O
+the	O
+enzyme	O
+(	O
+Fig	O
+3a	O
+).	O
+	O
+(	O
+a	O
+)	O
+Impact	O
+of	O
+the	O
+iron	O
+-	O
+chelator	O
+EDTA	O
+on	O
+the	O
+enzyme	O
+activity	O
+of	O
+the	O
+purified	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+Metal	O
+depletion	O
+and	O
+reconstitution	O
+experiments	O
+with	O
+(	O
+b	O
+)	O
+stoichiometric	O
+and	O
+(	O
+c	O
+)	O
+excess	O
+amounts	O
+of	O
+metals	O
+.	O
+	O
+The	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+was	O
+present	O
+at	O
+a	O
+concentration	O
+of	O
+10	O
+μM	O
+.	O
+The	O
+level	O
+of	O
+enzyme	O
+activity	O
+given	O
+in	O
+(	O
+b	O
+)	O
+is	O
+benchmarked	O
+relative	O
+to	O
+that	O
+of	O
+ectoine	O
+synthase	O
+enzyme	O
+assays	O
+in	O
+which	O
+1	O
+mM	O
+FeCl2	O
+was	O
+added	O
+.	O
+	O
+The	O
+addition	O
+of	O
+FeCl2	O
+to	O
+the	O
+enzyme	O
+assay	O
+restored	O
+enzyme	O
+activity	O
+to	O
+about	O
+38	O
+%,	O
+whereas	O
+the	O
+addition	O
+of	O
+ZnCl2	O
+or	O
+CoCl2	O
+rescued	O
+(	O
+Sa	O
+)	O
+EctC	O
+enzyme	O
+activity	O
+only	O
+to	O
+5	O
+%	O
+and	O
+3	O
+%,	O
+respectively	O
+.	O
+	O
+All	O
+other	O
+tested	O
+metals	O
+,	O
+including	O
+Fe3	O
++,	O
+were	O
+unable	O
+to	O
+restore	O
+activity	O
+(	O
+Fig	O
+3b	O
+).	O
+	O
+When	O
+the	O
+concentration	O
+of	O
+the	O
+various	O
+metals	O
+in	O
+the	O
+enzyme	O
+assay	O
+was	O
+increased	O
+100	O
+-	O
+fold	O
+,	O
+Fe2	O
++	O
+exhibited	O
+again	O
+the	O
+strongest	O
+stimulating	O
+effect	O
+on	O
+enzyme	O
+activity	O
+,	O
+and	O
+rescued	O
+enzyme	O
+activity	O
+to	O
+a	O
+degree	O
+similar	O
+to	O
+that	O
+exhibited	O
+by	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+preparations	O
+that	O
+had	O
+not	O
+been	O
+inactivated	O
+through	O
+EDTA	O
+treatment	O
+(	O
+Fig	O
+3c	O
+).	O
+	O
+However	O
+,	O
+a	O
+large	O
+molar	O
+excess	O
+of	O
+other	O
+transition	O
+-	O
+state	O
+metals	O
+(	O
+zinc	O
+,	O
+cobalt	O
+,	O
+nickel	O
+,	O
+copper	O
+,	O
+and	O
+manganese	O
+)	O
+typically	O
+found	O
+in	O
+members	O
+of	O
+the	O
+cupin	O
+superfamily	O
+allowed	O
+the	O
+partial	O
+rescue	O
+of	O
+ectoine	O
+synthase	O
+activity	O
+as	O
+well	O
+(	O
+Fig	O
+3c	O
+).	O
+	O
+This	O
+is	O
+in	O
+line	O
+with	O
+literature	O
+data	O
+showing	O
+that	O
+cupin	O
+-	O
+type	O
+enzymes	O
+are	O
+often	O
+promiscuous	O
+with	O
+respect	O
+to	O
+the	O
+use	O
+of	O
+the	O
+catalytically	O
+important	O
+metal	O
+.	O
+	O
+Kinetic	O
+parameters	O
+of	O
+EctC	O
+for	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+and	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+	O
+Based	O
+on	O
+the	O
+data	O
+presented	O
+in	O
+S3	O
+Fig	O
+,	O
+we	O
+formulated	O
+an	O
+optimized	O
+activity	O
+assay	O
+for	O
+the	O
+ectoine	O
+synthase	O
+of	O
+S	O
+.	O
+alaskensis	O
+and	O
+used	O
+it	O
+to	O
+determined	O
+the	O
+kinetic	O
+parameters	O
+for	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+enzyme	O
+for	O
+both	O
+its	O
+natural	O
+substrate	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+and	O
+the	O
+isomer	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+.	O
+	O
+Given	O
+the	O
+chemical	O
+relatedness	O
+of	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+to	O
+the	O
+natural	O
+substrate	O
+(	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+)	O
+of	O
+the	O
+ectoine	O
+synthase	O
+(	O
+S1a	O
+and	O
+S1b	O
+Fig	O
+),	O
+we	O
+wondered	O
+whether	O
+(	O
+Sa	O
+)	O
+EctC	O
+could	O
+also	O
+use	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+to	O
+produce	O
+ectoine	O
+.	O
+	O
+However	O
+,	O
+both	O
+the	O
+affinity	O
+(	O
+Km	O
+)	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+and	O
+its	O
+catalytic	O
+efficiency	O
+(	O
+kcat	O
+/	O
+Km	O
+)	O
+were	O
+strongly	O
+reduced	O
+in	O
+comparison	O
+with	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+.	O
+	O
+Both	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+and	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+are	O
+concomitantly	O
+formed	O
+during	O
+the	O
+enzymatic	O
+hydrolysis	O
+of	O
+the	O
+ectoine	O
+ring	O
+during	O
+catabolism	O
+.	O
+	O
+Our	O
+finding	O
+that	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+is	O
+a	O
+substrate	O
+for	O
+ectoine	O
+synthase	O
+has	O
+bearings	O
+for	O
+an	O
+understanding	O
+of	O
+the	O
+physiology	O
+of	O
+those	O
+microorganisms	O
+that	O
+can	O
+both	O
+synthesize	O
+and	O
+catabolize	O
+ectoine	O
+.	O
+	O
+However	O
+,	O
+these	O
+types	O
+of	O
+microorganisms	O
+should	O
+still	O
+be	O
+able	O
+to	O
+largely	O
+avoid	O
+a	O
+futile	O
+cycle	O
+since	O
+the	O
+affinity	O
+of	O
+ectoine	O
+synthase	O
+for	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+and	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+,	O
+and	O
+its	O
+catalytic	O
+efficiency	O
+for	O
+the	O
+two	O
+compounds	O
+,	O
+differs	O
+substantially	O
+(	O
+S4a	O
+and	O
+S4b	O
+Fig	O
+).	O
+	O
+Crystallization	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+	O
+Since	O
+no	O
+crystal	O
+structure	O
+of	O
+ectoine	O
+synthase	O
+has	O
+been	O
+reported	O
+,	O
+we	O
+set	O
+out	O
+to	O
+crystallize	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+Attempts	O
+to	O
+obtain	O
+crystals	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+in	O
+complex	O
+either	O
+with	O
+its	O
+substrate	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+or	O
+its	O
+reaction	O
+product	O
+ectoine	O
+were	O
+not	O
+successful	O
+.	O
+	O
+Attempts	O
+to	O
+solve	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+by	O
+molecular	O
+replacement	O
+has	O
+previously	O
+failed	O
+.	O
+	O
+However	O
+,	O
+we	O
+were	O
+able	O
+to	O
+obtain	O
+crystals	O
+of	O
+form	O
+B	O
+that	O
+were	O
+derivatized	O
+with	O
+mercury	O
+and	O
+these	O
+diffracted	O
+up	O
+to	O
+2	O
+.	O
+8	O
+Å	O
+(	O
+S1	O
+Table	O
+).	O
+	O
+This	O
+dataset	O
+was	O
+used	O
+to	O
+derive	O
+an	O
+initial	O
+structural	O
+model	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+,	O
+which	O
+in	O
+turn	O
+was	O
+employed	O
+as	O
+a	O
+template	O
+for	O
+molecular	O
+replacement	O
+to	O
+phase	O
+the	O
+native	O
+dataset	O
+(	O
+2	O
+.	O
+0	O
+Å	O
+)	O
+of	O
+crystal	O
+form	O
+B	O
+.	O
+After	O
+several	O
+rounds	O
+of	O
+manual	O
+model	O
+building	O
+and	O
+refinement	O
+,	O
+four	O
+monomers	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+were	O
+identified	O
+and	O
+the	O
+crystal	O
+structure	O
+was	O
+refined	O
+to	O
+a	O
+final	O
+Rcryst	O
+of	O
+21	O
+.	O
+1	O
+%	O
+and	O
+an	O
+Rfree	O
+of	O
+24	O
+.	O
+8	O
+%	O
+(	O
+S1	O
+Table	O
+).	O
+	O
+The	O
+two	O
+EctC	O
+structures	O
+that	O
+we	O
+determined	O
+revealed	O
+that	O
+the	O
+ectoine	O
+synthase	O
+belongs	O
+to	O
+the	O
+cupin	O
+superfamily	O
+with	O
+respect	O
+to	O
+its	O
+overall	O
+fold	O
+(	O
+Fig	O
+4a	O
+–	O
+4c	O
+).	O
+	O
+However	O
+,	O
+they	O
+represent	O
+two	O
+different	O
+states	O
+of	O
+the	O
+137	O
+amino	O
+acids	O
+comprising	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+First	O
+,	O
+the	O
+1	O
+.	O
+2	O
+Å	O
+structure	O
+reveals	O
+the	O
+spatial	O
+configuration	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+ranging	O
+from	O
+amino	O
+acid	O
+Met	O
+-	O
+1	O
+to	O
+Glu	O
+-	O
+115	O
+;	O
+hence	O
+,	O
+it	O
+lacks	O
+22	O
+amino	O
+acids	O
+at	O
+the	O
+carboxy	O
+-	O
+terminus	O
+of	O
+the	O
+authentic	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+In	O
+this	O
+structure	O
+no	O
+metal	O
+co	O
+-	O
+factor	O
+was	O
+identified	O
+.	O
+	O
+The	O
+second	O
+crystal	O
+structure	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+was	O
+solved	O
+at	O
+a	O
+resolution	O
+of	O
+2	O
+.	O
+0	O
+Å	O
+and	O
+contained	O
+four	O
+molecules	O
+of	O
+the	O
+protein	O
+in	O
+the	O
+asymmetric	O
+unit	O
+of	O
+which	O
+protomer	O
+A	O
+comprised	O
+amino	O
+acid	O
+Met	O
+-	O
+1	O
+to	O
+Gly	O
+-	O
+121	O
+and	O
+adopts	O
+a	O
+closed	O
+conformation	O
+.	O
+	O
+Hence	O
+,	O
+it	O
+still	O
+lacks	O
+16	O
+amino	O
+acid	O
+residues	O
+of	O
+the	O
+carboxy	O
+-	O
+terminus	O
+of	O
+the	O
+authentic	O
+137	O
+amino	O
+acids	O
+comprising	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+We	O
+therefore	O
+cannot	O
+exclude	O
+that	O
+this	O
+crystal	O
+structure	O
+does	O
+not	O
+represent	O
+the	O
+fully	O
+closed	O
+state	O
+of	O
+the	O
+ectoine	O
+synthase	O
+;	O
+consequently	O
+,	O
+we	O
+tentatively	O
+termed	O
+it	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+.	O
+	O
+Overall	O
+structure	O
+of	O
+the	O
+“	O
+open	O
+”	O
+and	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+crystal	O
+structures	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+.	O
+	O
+(	O
+a	O
+)	O
+The	O
+overall	O
+structure	O
+of	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+resolved	O
+at	O
+2	O
+.	O
+0	O
+Å	O
+is	O
+depicted	O
+in	O
+green	O
+in	O
+a	O
+cartoon	O
+(	O
+upper	O
+panel	O
+)	O
+and	O
+surface	O
+(	O
+lower	O
+panel	O
+)	O
+representation	O
+.	O
+	O
+(	O
+b	O
+)	O
+The	O
+overall	O
+structure	O
+of	O
+the	O
+“	O
+open	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+was	O
+resolved	O
+at	O
+1	O
+.	O
+2	O
+Å	O
+and	O
+is	O
+depicted	O
+in	O
+yellow	O
+in	O
+a	O
+cartoon	O
+(	O
+upper	O
+panel	O
+)	O
+and	O
+surface	O
+(	O
+lower	O
+panel	O
+)	O
+representation	O
+.	O
+	O
+The	O
+overall	O
+structure	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+is	O
+basically	O
+the	O
+same	O
+in	O
+both	O
+crystals	O
+except	O
+for	O
+the	O
+carboxy	O
+-	O
+terminus	O
+,	O
+which	O
+covers	O
+the	O
+entry	O
+of	O
+one	O
+side	O
+of	O
+the	O
+cupin	O
+barrel	O
+from	O
+the	O
+surroundings	O
+in	O
+monomer	O
+A	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+.	O
+	O
+This	O
+is	O
+reflected	O
+by	O
+the	O
+calculated	O
+root	O
+mean	O
+square	O
+deviation	O
+(	O
+RMSD	O
+)	O
+of	O
+the	O
+Cα	O
+atoms	O
+that	O
+was	O
+about	O
+0	O
+.	O
+56	O
+Å	O
+(	O
+over	O
+117	O
+residues	O
+)	O
+when	O
+the	O
+four	O
+“	O
+open	O
+”	O
+monomers	O
+were	O
+compared	O
+with	O
+each	O
+other	O
+.	O
+	O
+However	O
+,	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+monomer	O
+has	O
+a	O
+slightly	O
+higher	O
+RMSD	O
+of	O
+1	O
+.	O
+4	O
+Å	O
+(	O
+over	O
+117	O
+residues	O
+)	O
+when	O
+compared	O
+with	O
+the	O
+“	O
+open	O
+”	O
+2	O
+.	O
+0	O
+Å	O
+structure	O
+.	O
+	O
+Therefore	O
+,	O
+we	O
+describe	O
+in	O
+the	O
+following	O
+the	O
+overall	O
+structure	O
+for	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+form	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+and	O
+subsequently	O
+highlight	O
+the	O
+structural	O
+differences	O
+between	O
+the	O
+“	O
+open	O
+”	O
+and	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+forms	O
+in	O
+more	O
+detail	O
+.	O
+	O
+The	O
+β	O
+-	O
+strands	O
+form	O
+two	O
+anti	O
+-	O
+parallel	O
+β	O
+-	O
+sheets	O
+:	O
+β2	O
+β3	O
+,	O
+β4	O
+,	O
+β11	O
+,	O
+β6	O
+,	O
+and	O
+β9	O
+,	O
+and	O
+a	O
+smaller	O
+three	O
+-	O
+stranded	O
+β	O
+-	O
+sheet	O
+(	O
+β7	O
+,	O
+β8	O
+,	O
+and	O
+β10	O
+),	O
+respectively	O
+.	O
+	O
+These	O
+two	O
+β	O
+-	O
+sheets	O
+pack	O
+against	O
+each	O
+other	O
+,	O
+forming	O
+a	O
+cup	O
+-	O
+shaped	O
+β	O
+-	O
+sandwich	O
+with	O
+a	O
+topology	O
+characteristic	O
+for	O
+the	O
+cupin	O
+-	O
+fold	O
+.	O
+	O
+In	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+,	O
+a	O
+longer	O
+carboxy	O
+-	O
+terminal	O
+tail	O
+is	O
+visible	O
+in	O
+the	O
+electron	O
+density	O
+,	O
+folding	O
+into	O
+a	O
+small	O
+helix	O
+(	O
+α	O
+-	O
+II	O
+)	O
+that	O
+closes	O
+the	O
+active	O
+site	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+(	O
+Fig	O
+4a	O
+).	O
+	O
+Structural	O
+comparison	O
+analyses	O
+using	O
+the	O
+DALI	O
+server	O
+revealed	O
+that	O
+(	O
+Sa	O
+)	O
+EctC	O
+adopts	O
+a	O
+fold	O
+similar	O
+to	O
+other	O
+members	O
+of	O
+the	O
+cupin	O
+superfamily	O
+.	O
+	O
+The	O
+highest	O
+structural	O
+similarities	O
+are	O
+observed	O
+for	O
+the	O
+Cupin	O
+2	O
+conserved	O
+barrel	O
+domain	O
+protein	O
+(	O
+YP_751781	O
+.	O
+1	O
+)	O
+from	O
+Shewanella	O
+frigidimarina	O
+(	O
+PDB	O
+accession	O
+code	O
+:	O
+2PFW	O
+)	O
+with	O
+a	O
+Z	O
+-	O
+score	O
+of	O
+13	O
+.	O
+1	O
+and	O
+an	O
+RMSD	O
+of	O
+2	O
+.	O
+2	O
+Å	O
+over	O
+104	O
+Cα	O
+-	O
+atoms	O
+(	O
+structural	O
+data	O
+for	O
+this	O
+protein	O
+have	O
+been	O
+deposited	O
+in	O
+the	O
+PDB	O
+but	O
+no	O
+publication	O
+connected	O
+to	O
+this	O
+structure	O
+is	O
+currently	O
+available	O
+),	O
+a	O
+manganese	O
+-	O
+containing	O
+cupin	O
+(	O
+TM1459	O
+)	O
+from	O
+Thermotoga	O
+maritima	O
+(	O
+PDB	O
+accession	O
+code	O
+:	O
+1VJ2	O
+)	O
+with	O
+a	O
+Z	O
+-	O
+score	O
+of	O
+12	O
+.	O
+8	O
+and	O
+an	O
+RMSD	O
+of	O
+2	O
+.	O
+0	O
+Å	O
+over	O
+103	O
+Cα	O
+-	O
+atoms	O
+,	O
+the	O
+cyclase	O
+RemF	O
+from	O
+Streptomyces	O
+resistomycificus	O
+(	O
+PDB	O
+accession	O
+code	O
+:	O
+3HT1	O
+with	O
+a	O
+Z	O
+-	O
+score	O
+of	O
+11	O
+.	O
+9	O
+and	O
+an	O
+RMSD	O
+of	O
+1	O
+.	O
+9	O
+Å	O
+over	O
+102	O
+Cα	O
+-	O
+atoms	O
+),	O
+and	O
+an	O
+auxin	O
+-	O
+binding	O
+protein	O
+1	O
+from	O
+Zea	O
+mays	O
+(	O
+PDB	O
+accession	O
+code	O
+:	O
+1LR5	O
+)	O
+with	O
+an	O
+Z	O
+-	O
+score	O
+of	O
+11	O
+.	O
+8	O
+and	O
+an	O
+RMSD	O
+of	O
+2	O
+.	O
+8	O
+Å	O
+over	O
+104	O
+Cα	O
+-	O
+atoms	O
+).	O
+	O
+Next	O
+to	O
+RemF	O
+and	O
+the	O
+aldos	O
+-	O
+2	O
+-	O
+ulose	O
+dehydratase	O
+/	O
+isomerase	O
+,	O
+the	O
+ectoine	O
+synthase	O
+is	O
+only	O
+the	O
+third	O
+characterized	O
+dehydratase	O
+within	O
+the	O
+cupin	O
+superfamily	O
+.	O
+	O
+In	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+crystal	O
+structure	O
+,	O
+(	O
+Sa	O
+)	O
+EctC	O
+has	O
+crystallized	O
+as	O
+a	O
+dimer	O
+of	O
+dimers	O
+within	O
+the	O
+asymmetric	O
+unit	O
+.	O
+	O
+This	O
+dimer	O
+(	O
+Fig	O
+5a	O
+and	O
+5b	O
+)	O
+is	O
+composed	O
+of	O
+two	O
+monomers	O
+arranged	O
+in	O
+a	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+orientation	O
+and	O
+is	O
+stabilized	O
+via	O
+strong	O
+interactions	O
+mediated	O
+by	O
+two	O
+antiparallel	O
+β	O
+-	O
+strands	O
+,	O
+β	O
+-	O
+strand	O
+β1	O
+(	O
+sequence	O
+1MIVRN5	O
+)	O
+from	O
+monomer	O
+A	O
+and	O
+β	O
+-	O
+strand	O
+β8	O
+from	O
+monomer	O
+B	O
+(	O
+sequence	O
+82GVMYAL87	O
+)	O
+(	O
+Fig	O
+5c	O
+).	O
+	O
+The	O
+strong	O
+interactions	O
+between	O
+these	O
+β	O
+-	O
+strands	O
+rely	O
+primarily	O
+on	O
+backbone	O
+contacts	O
+.	O
+	O
+In	O
+addition	O
+to	O
+these	O
+interactions	O
+,	O
+some	O
+weaker	O
+hydrophobic	O
+interactions	O
+are	O
+also	O
+observed	O
+between	O
+the	O
+two	O
+monomers	O
+in	O
+some	O
+loops	O
+connecting	O
+the	O
+β	O
+-	O
+strands	O
+.	O
+	O
+Both	O
+values	O
+fall	O
+within	O
+the	O
+range	O
+for	O
+known	O
+functional	O
+dimers	O
+.	O
+	O
+Crystal	O
+structure	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+.	O
+	O
+(	O
+a	O
+)	O
+Top	O
+-	O
+view	O
+of	O
+the	O
+dimer	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+The	O
+position	O
+of	O
+the	O
+water	O
+molecule	O
+,	O
+described	O
+in	O
+detail	O
+in	O
+the	O
+text	O
+,	O
+is	O
+shown	O
+in	O
+one	O
+of	O
+the	O
+monomers	O
+as	O
+an	O
+orange	O
+sphere	O
+.	O
+(	O
+b	O
+)	O
+Side	O
+-	O
+view	O
+of	O
+a	O
+(	O
+Sa	O
+)	O
+EctC	O
+dimer	O
+allowing	O
+an	O
+assessment	O
+of	O
+the	O
+dimer	O
+interface	O
+formed	O
+by	O
+two	O
+β	O
+-	O
+strands	O
+of	O
+each	O
+monomer	O
+.	O
+	O
+(	O
+c	O
+)	O
+Close	O
+-	O
+up	O
+representation	O
+of	O
+the	O
+dimer	O
+interface	O
+mediated	O
+by	O
+beta	O
+-	O
+strand	O
+β1	O
+and	O
+β6	O
+.	O
+	O
+Indeed	O
+,	O
+a	O
+similar	O
+dimer	O
+configuration	O
+to	O
+the	O
+one	O
+described	O
+for	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+is	O
+observed	O
+with	O
+the	O
+same	O
+monomer	O
+-	O
+monomer	O
+interactions	O
+mediated	O
+by	O
+the	O
+two	O
+β	O
+-	O
+sheets	O
+.	O
+	O
+The	O
+crystallographic	O
+two	O
+-	O
+fold	O
+axis	O
+present	O
+within	O
+the	O
+crystal	O
+symmetry	O
+is	O
+located	O
+exactly	O
+in	O
+between	O
+the	O
+two	O
+monomers	O
+,	O
+resulting	O
+in	O
+a	O
+monomer	O
+within	O
+the	O
+asymmetric	O
+unit	O
+.	O
+	O
+Hence	O
+,	O
+the	O
+same	O
+dimer	O
+observed	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+can	O
+also	O
+be	O
+observed	O
+in	O
+the	O
+“	O
+open	O
+”	O
+structure	O
+.	O
+	O
+Interestingly	O
+,	O
+the	O
+proteins	O
+identified	O
+by	O
+the	O
+above	O
+-	O
+described	O
+DALI	O
+search	O
+not	O
+only	O
+have	O
+folds	O
+similar	O
+to	O
+EctC	O
+,	O
+but	O
+are	O
+also	O
+functional	O
+dimers	O
+that	O
+adopt	O
+similar	O
+monomer	O
+-	O
+monomer	O
+interactions	O
+within	O
+the	O
+dimer	O
+assembly	O
+as	O
+deduced	O
+from	O
+the	O
+inspection	O
+of	O
+the	O
+corresponding	O
+PDB	O
+files	O
+(	O
+2PFW	O
+,	O
+3HT1	O
+,	O
+1VJ2	O
+,	O
+1LR5	O
+).	O
+	O
+Structural	O
+rearrangements	O
+of	O
+the	O
+flexible	O
+(	O
+Sa	O
+)	O
+EctC	O
+carboxy	O
+-	O
+terminus	O
+	O
+The	O
+cupin	O
+core	O
+represents	O
+the	O
+structural	O
+framework	O
+of	O
+ectoine	O
+synthase	O
+(	O
+Figs	O
+4	O
+and	O
+5	O
+).	O
+	O
+The	O
+major	O
+difference	O
+in	O
+the	O
+two	O
+crystal	O
+structures	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+reported	O
+here	O
+is	O
+the	O
+orientation	O
+of	O
+the	O
+carboxy	O
+-	O
+terminus	O
+.	O
+	O
+Some	O
+amino	O
+acids	O
+located	O
+in	O
+the	O
+carboxy	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+137	O
+amino	O
+acids	O
+comprising	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+are	O
+highly	O
+conserved	O
+(	O
+Fig	O
+2	O
+)	O
+within	O
+the	O
+extended	O
+EctC	O
+protein	O
+family	O
+.	O
+	O
+At	O
+the	O
+end	O
+of	O
+β	O
+-	O
+strand	O
+β11	O
+,	O
+two	O
+consecutive	O
+conserved	O
+proline	O
+residues	O
+(	O
+Pro	O
+-	O
+109	O
+and	O
+Pro	O
+-	O
+110	O
+)	O
+are	O
+present	O
+that	O
+are	O
+responsible	O
+for	O
+a	O
+turn	O
+in	O
+the	O
+main	O
+chain	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+In	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+,	O
+the	O
+visible	O
+electron	O
+density	O
+of	O
+the	O
+carboxy	O
+-	O
+terminus	O
+is	O
+extended	O
+by	O
+7	O
+amino	O
+acid	O
+residues	O
+and	O
+ends	O
+at	O
+position	O
+Gly	O
+-	O
+121	O
+.	O
+	O
+Furthermore	O
+,	O
+this	O
+helix	O
+is	O
+stabilized	O
+via	O
+interactions	O
+with	O
+the	O
+loop	O
+region	O
+between	O
+β	O
+-	O
+strands	O
+β4	O
+and	O
+β6	O
+,	O
+thereby	O
+inducing	O
+a	O
+structural	O
+rearrangement	O
+.	O
+	O
+This	O
+induces	O
+the	O
+formation	O
+of	O
+β	O
+-	O
+strand	O
+β5	O
+,	O
+which	O
+is	O
+not	O
+present	O
+when	O
+the	O
+small	O
+C	O
+-	O
+terminal	O
+helix	O
+is	O
+absent	O
+as	O
+observed	O
+in	O
+the	O
+“	O
+open	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+.	O
+	O
+The	O
+position	O
+of	O
+this	O
+His	O
+residue	O
+is	O
+slightly	O
+shifted	O
+in	O
+both	O
+(	O
+Sa	O
+)	O
+EctC	O
+structures	O
+,	O
+likely	O
+the	O
+result	O
+of	O
+the	O
+formation	O
+of	O
+β	O
+-	O
+strand	O
+β5	O
+.	O
+	O
+The	O
+consecutive	O
+Pro	O
+-	O
+109	O
+and	O
+Pro	O
+-	O
+110	O
+residues	O
+found	O
+at	O
+the	O
+end	O
+of	O
+β	O
+-	O
+strand	O
+β11are	O
+highly	O
+conserved	O
+in	O
+EctC	O
+-	O
+type	O
+proteins	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+They	O
+are	O
+responsible	O
+for	O
+redirecting	O
+the	O
+main	O
+chain	O
+of	O
+the	O
+remaining	O
+carboxy	O
+-	O
+terminus	O
+(	O
+27	O
+amino	O
+acid	O
+residues	O
+)	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+to	O
+close	O
+the	O
+cupin	O
+fold	O
+.	O
+	O
+In	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+this	O
+results	O
+in	O
+a	O
+complete	O
+closure	O
+of	O
+the	O
+entry	O
+of	O
+the	O
+cupin	O
+barrel	O
+(	O
+Fig	O
+4a	O
+to	O
+4c	O
+).	O
+	O
+A	O
+search	O
+for	O
+partners	O
+interacting	O
+with	O
+Pro	O
+-	O
+109	O
+revealed	O
+that	O
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+interacts	O
+via	O
+its	O
+backbone	O
+oxygen	O
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+the	O
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+His	O
+-	O
+55	O
+as	O
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+in	O
+both	O
+the	O
+“	O
+open	O
+”	O
+and	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structures	O
+.	O
+	O
+The	O
+Pro	O
+-	O
+109	O
+/	O
+His	O
+-	O
+55	O
+interaction	O
+ensures	O
+the	O
+stable	O
+orientation	O
+of	O
+both	O
+proline	O
+residues	O
+at	O
+the	O
+end	O
+of	O
+β	O
+-	O
+strand	O
+β11	O
+.	O
+	O
+In	O
+addition	O
+to	O
+the	O
+interactions	O
+between	O
+Pro	O
+-	O
+109	O
+and	O
+His	O
+-	O
+55	O
+,	O
+the	O
+carboxy	O
+-	O
+terminal	O
+region	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+is	O
+held	O
+in	O
+position	O
+via	O
+an	O
+interaction	O
+of	O
+Glu	O
+-	O
+115	O
+with	O
+His	O
+-	O
+55	O
+,	O
+which	O
+stabilizes	O
+the	O
+conformation	O
+of	O
+the	O
+small	O
+helix	O
+in	O
+the	O
+carboxy	O
+-	O
+terminus	O
+further	O
+.	O
+	O
+Architecture	O
+of	O
+the	O
+presumed	O
+metal	O
+-	O
+binding	O
+site	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+and	O
+its	O
+flexible	O
+carboxy	O
+-	O
+terminus	O
+.	O
+	O
+(	O
+a	O
+)	O
+The	O
+described	O
+water	O
+molecule	O
+(	O
+depicted	O
+as	O
+orange	O
+sphere	O
+)	O
+is	O
+bound	O
+via	O
+interactions	O
+with	O
+the	O
+side	O
+chains	O
+of	O
+Glu	O
+-	O
+57	O
+,	O
+Tyr	O
+-	O
+85	O
+,	O
+and	O
+His	O
+-	O
+93	O
+.	O
+	O
+The	O
+position	O
+occupied	O
+by	O
+this	O
+water	O
+molecule	O
+represents	O
+probably	O
+the	O
+position	O
+of	O
+the	O
+Fe2	O
++	O
+cofactor	O
+in	O
+the	O
+active	O
+side	O
+of	O
+the	O
+ectoine	O
+synthase	O
+.	O
+	O
+His	O
+-	O
+55	O
+interacts	O
+with	O
+the	O
+double	O
+proline	O
+motif	O
+(	O
+Pro	O
+-	O
+109	O
+and	O
+Pro	O
+-	O
+110	O
+).	O
+	O
+It	O
+is	O
+further	O
+stabilized	O
+via	O
+an	O
+interaction	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+Glu	O
+-	O
+115	O
+which	O
+is	O
+localized	O
+in	O
+the	O
+flexible	O
+carboxy	O
+-	O
+terminus	O
+(	O
+colored	O
+in	O
+orange	O
+)	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+that	O
+is	O
+visible	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+.	O
+	O
+Since	O
+(	O
+Sa	O
+)	O
+EctC	O
+is	O
+a	O
+metal	O
+containing	O
+protein	O
+(	O
+Fig	O
+3	O
+),	O
+we	O
+tried	O
+to	O
+fit	O
+either	O
+Fe2	O
++,	O
+or	O
+Zn2	O
++	O
+ions	O
+into	O
+this	O
+density	O
+and	O
+also	O
+refined	O
+occupancy	O
+.	O
+	O
+Only	O
+the	O
+refinement	O
+of	O
+Fe2	O
++	O
+resulted	O
+in	O
+a	O
+visibly	O
+improved	O
+electron	O
+density	O
+,	O
+however	O
+with	O
+a	O
+low	O
+degree	O
+of	O
+occupancy	O
+.	O
+	O
+This	O
+possible	O
+iron	O
+molecule	O
+is	O
+bound	O
+via	O
+interactions	O
+with	O
+Glu	O
+-	O
+57	O
+,	O
+Tyr	O
+-	O
+85	O
+and	O
+His	O
+-	O
+93	O
+(	O
+Fig	O
+6a	O
+and	O
+6b	O
+).	O
+	O
+The	O
+distance	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+these	O
+residues	O
+and	O
+the	O
+(	O
+putative	O
+)	O
+iron	O
+co	O
+-	O
+factor	O
+is	O
+3	O
+.	O
+1	O
+Å	O
+for	O
+Glu	O
+-	O
+57	O
+,	O
+2	O
+.	O
+9	O
+Å	O
+for	O
+Tyr	O
+-	O
+85	O
+,	O
+and	O
+2	O
+.	O
+9	O
+Å	O
+for	O
+His	O
+-	O
+93	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+position	O
+of	O
+this	O
+water	O
+molecule	O
+is	O
+described	O
+in	O
+more	O
+detail	O
+below	O
+and	O
+is	O
+highlighted	O
+in	O
+Figs	O
+5a	O
+and	O
+5b	O
+and	O
+6a	O
+and	O
+6b	O
+as	O
+a	O
+sphere	O
+.	O
+	O
+Interestingly	O
+,	O
+all	O
+three	O
+amino	O
+acids	O
+coordinating	O
+this	O
+water	O
+molecule	O
+are	O
+strictly	O
+conserved	O
+within	O
+an	O
+alignment	O
+of	O
+440	O
+members	O
+of	O
+the	O
+EctC	O
+protein	O
+family	O
+(	O
+for	O
+an	O
+abbreviated	O
+alignment	O
+of	O
+EctC	O
+-	O
+type	O
+proteins	O
+see	O
+Fig	O
+2	O
+).	O
+	O
+In	O
+the	O
+“	O
+open	O
+”	O
+structure	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+,	O
+electron	O
+density	O
+is	O
+visible	O
+where	O
+the	O
+presumptive	O
+iron	O
+is	O
+positioned	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+.	O
+	O
+However	O
+,	O
+this	O
+electron	O
+density	O
+fits	O
+perfectly	O
+to	O
+a	O
+water	O
+molecule	O
+and	O
+not	O
+to	O
+an	O
+iron	O
+,	O
+and	O
+the	O
+water	O
+molecule	O
+was	O
+clearly	O
+visible	O
+after	O
+the	O
+refinement	O
+at	O
+this	O
+high	O
+resolution	O
+(	O
+1	O
+.	O
+2	O
+Å	O
+)	O
+of	O
+the	O
+“	O
+open	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+.	O
+	O
+In	O
+a	O
+superimposition	O
+of	O
+both	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structures	O
+,	O
+the	O
+spatial	O
+arrangements	O
+of	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+three	O
+amino	O
+acids	O
+(	O
+Glu	O
+-	O
+57	O
+,	O
+Tyr	O
+-	O
+85	O
+,	O
+and	O
+His	O
+-	O
+93	O
+)	O
+likely	O
+to	O
+contact	O
+the	O
+iron	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+match	O
+nicely	O
+with	O
+those	O
+of	O
+the	O
+corresponding	O
+residues	O
+of	O
+the	O
+“	O
+iron	O
+-	O
+free	O
+”	O
+“	O
+open	O
+”	O
+structure	O
+(	O
+Fig	O
+6b	O
+).	O
+	O
+In	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+,	O
+the	O
+hydroxyl	O
+-	O
+group	O
+of	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+Tyr	O
+-	O
+52	O
+points	O
+towards	O
+the	O
+iron	O
+(	O
+Fig	O
+6a	O
+and	O
+6b	O
+),	O
+but	O
+the	O
+corresponding	O
+distance	O
+(	O
+3	O
+.	O
+9	O
+Å	O
+)	O
+makes	O
+it	O
+highly	O
+unlikely	O
+that	O
+Tyr	O
+-	O
+52	O
+is	O
+directly	O
+involved	O
+in	O
+metal	O
+binding	O
+.	O
+	O
+Nevertheless	O
+,	O
+its	O
+substitution	O
+by	O
+an	O
+Ala	O
+residue	O
+causes	O
+a	O
+strong	O
+decrease	O
+in	O
+iron	O
+-	O
+content	O
+and	O
+enzyme	O
+activity	O
+of	O
+the	O
+mutant	O
+protein	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Since	O
+Tyr	O
+-	O
+52	O
+is	O
+strictly	O
+conserved	O
+in	O
+an	O
+alignment	O
+of	O
+440	O
+EctC	O
+-	O
+type	O
+proteins	O
+(	O
+Fig	O
+2	O
+),	O
+we	O
+speculate	O
+that	O
+it	O
+might	O
+be	O
+involved	O
+in	O
+contacting	O
+the	O
+substrate	O
+of	O
+the	O
+ectoine	O
+synthase	O
+and	O
+that	O
+the	O
+absence	O
+of	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+in	O
+our	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structures	O
+might	O
+endow	O
+the	O
+side	O
+chain	O
+of	O
+Tyr	O
+-	O
+52	O
+with	O
+extra	O
+spatial	O
+flexibility	O
+.	O
+	O
+To	O
+further	O
+analyze	O
+the	O
+putative	O
+iron	O
+binding	O
+site	O
+(	O
+Fig	O
+6a	O
+),	O
+we	O
+performed	O
+structure	O
+-	O
+guided	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+and	O
+assessed	O
+the	O
+resulting	O
+(	O
+Sa	O
+)	O
+EctC	O
+variants	O
+for	O
+their	O
+iron	O
+content	O
+and	O
+studied	O
+their	O
+enzyme	O
+activity	O
+.	O
+	O
+When	O
+those	O
+three	O
+residues	O
+(	O
+Glu	O
+-	O
+57	O
+,	O
+Tyr	O
+-	O
+85	O
+,	O
+His	O
+-	O
+93	O
+)	O
+that	O
+likely	O
+form	O
+the	O
+mono	O
+-	O
+nuclear	O
+iron	O
+center	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structure	O
+were	O
+individually	O
+replaced	O
+by	O
+an	O
+Ala	O
+residue	O
+,	O
+both	O
+the	O
+catalytic	O
+activity	O
+and	O
+the	O
+iron	O
+content	O
+of	O
+the	O
+mutant	O
+proteins	O
+was	O
+strongly	O
+reduced	O
+(	O
+Table	O
+1	O
+).	O
+	O
+For	O
+some	O
+of	O
+the	O
+presumptive	O
+iron	O
+-	O
+coordinating	O
+residues	O
+,	O
+additional	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+experiments	O
+were	O
+carried	O
+out	O
+.	O
+	O
+To	O
+verify	O
+the	O
+importance	O
+of	O
+the	O
+negative	O
+charge	O
+in	O
+the	O
+position	O
+of	O
+Glu	O
+-	O
+57	O
+,	O
+we	O
+created	O
+an	O
+Asp	O
+variant	O
+.	O
+	O
+This	O
+mutant	O
+protein	O
+rescued	O
+the	O
+enzyme	O
+activity	O
+and	O
+iron	O
+content	O
+of	O
+the	O
+Ala	O
+substitution	O
+substantially	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Collectively	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+the	O
+hydroxyl	O
+group	O
+of	O
+the	O
+Tyr	O
+-	O
+85	O
+side	O
+chain	O
+is	O
+needed	O
+for	O
+the	O
+binding	O
+of	O
+the	O
+iron	O
+(	O
+Fig	O
+6a	O
+).	O
+	O
+We	O
+also	O
+replaced	O
+the	O
+presumptive	O
+iron	O
+-	O
+binding	O
+residue	O
+His	O
+-	O
+93	O
+by	O
+an	O
+Asn	O
+residue	O
+,	O
+yielding	O
+a	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+variant	O
+that	O
+possessed	O
+an	O
+enzyme	O
+activity	O
+of	O
+23	O
+%	O
+and	O
+iron	O
+content	O
+of	O
+only	O
+14	O
+%	O
+relative	O
+to	O
+that	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+protein	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Collectively	O
+,	O
+the	O
+data	O
+addressing	O
+the	O
+functionality	O
+of	O
+the	O
+putative	O
+iron	O
+-	O
+coordinating	O
+residues	O
+(	O
+Glu	O
+-	O
+57	O
+,	O
+Tyr	O
+-	O
+85	O
+,	O
+His	O
+-	O
+93	O
+)	O
+buttress	O
+our	O
+notion	O
+that	O
+the	O
+Fe2	O
++	O
+present	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+is	O
+of	O
+catalytic	O
+importance	O
+.	O
+	O
+A	O
+chemically	O
+undefined	O
+ligand	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+provides	O
+clues	O
+for	O
+the	O
+binding	O
+of	O
+the	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+substrate	O
+	O
+Despite	O
+considerable	O
+efforts	O
+,	O
+either	O
+by	O
+trying	O
+co	O
+-	O
+crystallization	O
+or	O
+soaking	O
+experiments	O
+,	O
+we	O
+were	O
+not	O
+able	O
+to	O
+obtain	O
+a	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structures	O
+that	O
+contained	O
+either	O
+the	O
+substrate	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+,	O
+or	O
+ectoine	O
+,	O
+the	O
+reaction	O
+product	O
+of	O
+ectoine	O
+synthase	O
+(	O
+Fig	O
+1	O
+).	O
+	O
+However	O
+,	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+where	O
+the	O
+carboxy	O
+-	O
+terminal	O
+loop	O
+is	O
+largely	O
+resolved	O
+,	O
+a	O
+long	O
+stretched	O
+electron	O
+density	O
+feature	O
+was	O
+detected	O
+in	O
+the	O
+predicted	O
+active	O
+site	O
+of	O
+the	O
+enzyme	O
+;	O
+it	O
+remained	O
+visible	O
+after	O
+crystallographic	O
+refinement	O
+.	O
+	O
+We	O
+tried	O
+to	O
+fit	O
+all	O
+compounds	O
+used	O
+in	O
+the	O
+buffers	O
+during	O
+purification	O
+and	O
+crystallization	O
+into	O
+the	O
+observed	O
+electron	O
+density	O
+,	O
+but	O
+none	O
+matched	O
+.	O
+	O
+This	O
+observation	O
+indicates	O
+that	O
+the	O
+chemically	O
+undefined	O
+ligand	O
+was	O
+either	O
+trapped	O
+by	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+during	O
+its	O
+heterologous	O
+production	O
+in	O
+E	O
+.	O
+coli	O
+or	O
+during	O
+crystallization	O
+.	O
+	O
+Estimating	O
+from	O
+the	O
+dimensions	O
+of	O
+the	O
+electron	O
+density	O
+feature	O
+,	O
+we	O
+modeled	O
+the	O
+chemically	O
+undefined	O
+compound	O
+trapped	O
+by	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+as	O
+a	O
+hexane	O
+-	O
+1	O
+,	O
+6	O
+-	O
+diol	O
+molecule	O
+(	O
+PDB	O
+identifier	O
+:	O
+HEZ	O
+)	O
+to	O
+best	O
+fit	O
+the	O
+observed	O
+electron	O
+density	O
+.	O
+	O
+However	O
+,	O
+to	O
+the	O
+best	O
+of	O
+our	O
+knowledge	O
+,	O
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+-	O
+1	O
+,	O
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+-	O
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+is	O
+not	O
+part	O
+of	O
+the	O
+E	O
+.	O
+coli	O
+metabolome	O
+.	O
+	O
+We	O
+note	O
+that	O
+both	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
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+hexane	O
+-	O
+1	O
+,	O
+6	O
+-	O
+diol	O
+are	O
+both	O
+C6	O
+-	O
+compounds	O
+and	O
+display	O
+similar	O
+length	O
+(	O
+Fig	O
+7a	O
+).	O
+	O
+A	O
+chemically	O
+undefined	O
+ligand	O
+is	O
+captured	O
+in	O
+the	O
+active	O
+site	O
+of	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structure	O
+.	O
+	O
+(	O
+a	O
+)	O
+The	O
+observed	O
+electron	O
+density	O
+in	O
+the	O
+active	O
+site	O
+of	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+is	O
+modeled	O
+as	O
+a	O
+hexane	O
+-	O
+1	O
+,	O
+6	O
+-	O
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+and	O
+compared	O
+with	O
+the	O
+electron	O
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+the	O
+N	O
+-	O
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+-	O
+ADABA	O
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+the	O
+ectoine	O
+synthase	O
+to	O
+emphasize	O
+the	O
+similarity	O
+in	O
+size	O
+of	O
+these	O
+compounds	O
+.	O
+	O
+(	O
+b	O
+)	O
+The	O
+presumable	O
+binding	O
+site	O
+of	O
+the	O
+iron	O
+co	O
+-	O
+factor	O
+and	O
+of	O
+the	O
+modeled	O
+hexane	O
+-	O
+1	O
+,	O
+6	O
+-	O
+diol	O
+molecule	O
+is	O
+depicted	O
+.	O
+	O
+The	O
+amino	O
+acid	O
+side	O
+chains	O
+involved	O
+in	O
+iron	O
+-	O
+ligand	O
+binding	O
+are	O
+colored	O
+in	O
+blue	O
+and	O
+those	O
+involved	O
+in	O
+the	O
+binding	O
+of	O
+the	O
+chemically	O
+undefined	O
+ligand	O
+are	O
+colored	O
+in	O
+green	O
+using	O
+a	O
+ball	O
+and	O
+stick	O
+representation	O
+.	O
+	O
+The	O
+flexible	O
+carboxy	O
+-	O
+terminal	O
+loop	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+is	O
+highlighted	O
+in	O
+orange	O
+.	O
+	O
+We	O
+refined	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+with	O
+the	O
+trapped	O
+compound	O
+,	O
+and	O
+by	O
+doing	O
+so	O
+,	O
+the	O
+refinement	O
+parameters	O
+(	O
+especially	O
+R	O
+-	O
+and	O
+Rfree	O
+-	O
+factor	O
+)	O
+dropped	O
+by	O
+1	O
+.	O
+5	O
+%.	O
+	O
+Remarkably	O
+,	O
+all	O
+of	O
+these	O
+residues	O
+are	O
+highly	O
+conserved	O
+throughout	O
+the	O
+extended	O
+EctC	O
+protein	O
+family	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+Structure	O
+-	O
+guided	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+of	O
+the	O
+catalytic	O
+core	O
+of	O
+the	O
+ectoine	O
+synthase	O
+	O
+In	O
+a	O
+previous	O
+alignment	O
+of	O
+the	O
+amino	O
+acid	O
+sequences	O
+of	O
+440	O
+EctC	O
+-	O
+type	O
+proteins	O
+,	O
+13	O
+amino	O
+acids	O
+were	O
+identified	O
+as	O
+strictly	O
+conserved	O
+residues	O
+.	O
+	O
+Amino	O
+acid	O
+residues	O
+Gly	O
+-	O
+64	O
+,	O
+Pro	O
+-	O
+109	O
+,	O
+and	O
+Gly	O
+-	O
+113	O
+likely	O
+fulfill	O
+structural	O
+roles	O
+since	O
+they	O
+are	O
+positioned	O
+either	O
+at	O
+the	O
+end	O
+or	O
+at	O
+the	O
+beginning	O
+of	O
+β	O
+-	O
+strands	O
+and	O
+α	O
+-	O
+helices	O
+.	O
+	O
+We	O
+considered	O
+the	O
+remaining	O
+ten	O
+residues	O
+as	O
+important	O
+either	O
+for	O
+ligand	O
+binding	O
+,	O
+for	O
+catalysis	O
+,	O
+or	O
+for	O
+the	O
+structurally	O
+correct	O
+orientation	O
+of	O
+the	O
+flexible	O
+carboxy	O
+-	O
+terminus	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+In	O
+view	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+with	O
+the	O
+serendipitously	O
+trapped	O
+compound	O
+(	O
+Fig	O
+7b	O
+),	O
+we	O
+probed	O
+the	O
+functional	O
+importance	O
+of	O
+the	O
+seven	O
+residues	O
+that	O
+contact	O
+this	O
+ligand	O
+by	O
+structure	O
+-	O
+guided	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+(	O
+Table	O
+1	O
+).	O
+	O
+We	O
+benchmarked	O
+the	O
+activity	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+variants	O
+in	O
+a	O
+single	O
+time	O
+-	O
+point	O
+enzyme	O
+assay	O
+under	O
+conditions	O
+where	O
+10	O
+μM	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+converted	O
+almost	O
+completely	O
+the	O
+supplied	O
+10	O
+mM	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+substrate	O
+to	O
+9	O
+.	O
+33	O
+mM	O
+ectoine	O
+within	O
+a	O
+time	O
+frame	O
+of	O
+20	O
+min	O
+.	O
+	O
+In	O
+addition	O
+,	O
+we	O
+determined	O
+the	O
+iron	O
+content	O
+of	O
+each	O
+of	O
+the	O
+mutant	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+by	O
+a	O
+colorimetric	O
+assay	O
+(	O
+Table	O
+1	O
+).	O
+	O
+The	O
+side	O
+chains	O
+of	O
+the	O
+evolutionarily	O
+conserved	O
+Trp	O
+-	O
+21	O
+,	O
+Ser	O
+-	O
+23	O
+,	O
+Thr	O
+-	O
+40	O
+,	O
+Cys	O
+-	O
+105	O
+,	O
+and	O
+Phe	O
+-	O
+107	O
+residues	O
+(	O
+Fig	O
+2	O
+)	O
+make	O
+contacts	O
+with	O
+the	O
+chemically	O
+undefined	O
+ligand	O
+that	O
+we	O
+observed	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+(	O
+Fig	O
+7b	O
+).	O
+	O
+Thr	O
+-	O
+40	O
+is	O
+positioned	O
+on	O
+β	O
+-	O
+strand	O
+β5	O
+and	O
+its	O
+side	O
+chain	O
+protrudes	O
+into	O
+the	O
+lumen	O
+of	O
+the	O
+cupin	O
+barrel	O
+formed	O
+by	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+(	O
+Fig	O
+7b	O
+).	O
+	O
+We	O
+also	O
+replaced	O
+Phe	O
+-	O
+107	O
+with	O
+either	O
+an	O
+Tyr	O
+or	O
+an	O
+Trp	O
+residue	O
+:	O
+the	O
+Phe	B-mutant
+-	I-mutant
+107	I-mutant
+/	I-mutant
+Tyr	I-mutant
+substitution	O
+possessed	O
+near	O
+wild	O
+-	O
+type	O
+enzyme	O
+activity	O
+(	O
+about	O
+95	O
+%)	O
+and	O
+the	O
+full	O
+iron	O
+content	O
+,	O
+but	O
+the	O
+Phe	B-mutant
+-	I-mutant
+107	I-mutant
+/	I-mutant
+Trp	I-mutant
+substitution	O
+possessed	O
+only	O
+12	O
+%	O
+enzyme	O
+activity	O
+and	O
+72	O
+%	O
+iron	O
+content	O
+compared	O
+to	O
+the	O
+wild	O
+-	O
+type	O
+protein	O
+.	O
+	O
+The	O
+properties	O
+of	O
+these	O
+mutant	O
+proteins	O
+indicate	O
+that	O
+the	O
+aromatic	O
+side	O
+chain	O
+at	O
+position	O
+107	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+is	O
+of	O
+importance	O
+but	O
+that	O
+a	O
+substitution	O
+with	O
+a	O
+bulky	O
+aromatic	O
+side	O
+chain	O
+is	O
+strongly	O
+detrimental	O
+to	O
+enzyme	O
+activity	O
+and	O
+concomitantly	O
+moderately	O
+impairs	O
+iron	O
+binding	O
+.	O
+	O
+Replacement	O
+of	O
+the	O
+only	O
+Cys	O
+residue	O
+in	O
+(	O
+Sa	O
+)	O
+EctC	O
+(	O
+Cys	O
+-	O
+105	O
+;	O
+Fig	O
+2	O
+)	O
+by	O
+a	O
+Ser	O
+residue	O
+,	O
+a	O
+configuration	O
+that	O
+is	O
+naturally	O
+found	O
+in	O
+two	O
+EctC	O
+proteins	O
+among	O
+440	O
+inspected	O
+amino	O
+acid	O
+sequences	O
+,	O
+yielded	O
+a	O
+(	O
+Sa	O
+)	O
+EctC	O
+variant	O
+with	O
+84	O
+%	O
+wild	O
+-	O
+type	O
+activity	O
+and	O
+an	O
+iron	O
+content	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+protein	O
+.	O
+	O
+Since	O
+the	O
+side	O
+-	O
+chains	O
+of	O
+Cys	O
+residues	O
+are	O
+chemically	O
+reactive	O
+and	O
+often	O
+participate	O
+in	O
+enzyme	O
+catalysis	O
+,	O
+Cys	O
+-	O
+105	O
+(	O
+or	O
+Ser	O
+-	O
+105	O
+)	O
+might	O
+serve	O
+such	O
+a	O
+role	O
+for	O
+ectoine	O
+synthase	O
+.	O
+	O
+Based	O
+on	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structures	O
+that	O
+we	O
+present	O
+here	O
+,	O
+we	O
+can	O
+currently	O
+not	O
+firmly	O
+understand	O
+why	O
+the	O
+replacement	O
+of	O
+Tyr	O
+-	O
+52	O
+by	O
+Ala	O
+impairs	O
+enzyme	O
+function	O
+and	O
+iron	O
+content	O
+so	O
+drastically	O
+(	O
+Table	O
+1	O
+).	O
+	O
+This	O
+is	O
+different	O
+for	O
+the	O
+His	B-mutant
+-	I-mutant
+55	I-mutant
+/	I-mutant
+Ala	I-mutant
+substitution	O
+.	O
+	O
+The	O
+individual	O
+substitution	O
+of	O
+either	O
+Glu	O
+-	O
+115	O
+or	O
+His	O
+-	O
+55	O
+by	O
+an	O
+Ala	O
+residue	O
+is	O
+predicted	O
+to	O
+disrupt	O
+this	O
+interactive	O
+network	O
+and	O
+therefore	O
+should	O
+affect	O
+enzyme	O
+activity	O
+.	O
+	O
+Indeed	O
+,	O
+the	O
+Glu	B-mutant
+-	I-mutant
+115	I-mutant
+/	I-mutant
+Ala	I-mutant
+and	O
+the	O
+His	B-mutant
+-	I-mutant
+55	I-mutant
+/	I-mutant
+Ala	I-mutant
+substitutions	O
+possessed	O
+only	O
+21	O
+%	O
+and	O
+16	O
+%	O
+activity	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+protein	O
+,	O
+respectively	O
+(	O
+Table	O
+1	O
+).	O
+	O
+We	O
+also	O
+replaced	O
+Glu	O
+-	O
+115	O
+with	O
+a	O
+negatively	O
+charged	O
+residue	O
+(	O
+Asp	O
+);	O
+this	O
+(	O
+Sa	O
+)	O
+EctC	O
+variant	O
+possessed	O
+wild	O
+-	O
+type	O
+levels	O
+of	O
+iron	O
+and	O
+still	O
+exhibited	O
+77	O
+%	O
+of	O
+wild	O
+-	O
+type	O
+enzyme	O
+activity	O
+.	O
+	O
+Collectively	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+the	O
+correct	O
+positioning	O
+of	O
+the	O
+carboxy	O
+-	O
+terminus	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+is	O
+of	O
+structural	O
+and	O
+functional	O
+importance	O
+for	O
+the	O
+activity	O
+of	O
+the	O
+ectoine	O
+synthase	O
+.	O
+	O
+Residues	O
+Leu	O
+-	O
+87	O
+and	O
+Asp	O
+-	O
+91	O
+are	O
+highly	O
+conserved	O
+in	O
+the	O
+ectoine	O
+synthase	O
+protein	O
+family	O
+.	O
+	O
+The	O
+replacement	O
+of	O
+Leu	O
+-	O
+87	O
+by	O
+Ala	O
+led	O
+to	O
+a	O
+substantial	O
+drop	O
+in	O
+enzyme	O
+activity	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Conversely	O
+,	O
+the	O
+replacement	O
+of	O
+Asp	O
+-	O
+91	O
+by	O
+Ala	O
+and	O
+Glu	O
+,	O
+resulted	O
+in	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+variants	O
+with	O
+80	O
+%	O
+and	O
+98	O
+%	O
+enzyme	O
+activity	O
+,	O
+respectively	O
+(	O
+Table	O
+1	O
+).	O
+	O
+We	O
+currently	O
+cannot	O
+comment	O
+on	O
+possible	O
+functional	O
+role	O
+Asp	O
+-	O
+91	O
+.	O
+	O
+However	O
+,	O
+Leu	O
+-	O
+87	O
+is	O
+positioned	O
+at	O
+the	O
+end	O
+of	O
+one	O
+of	O
+the	O
+β	O
+-	O
+sheets	O
+that	O
+form	O
+the	O
+dimer	O
+interface	O
+(	O
+Fig	O
+5c	O
+)	O
+and	O
+it	O
+might	O
+therefore	O
+possess	O
+a	O
+structural	O
+role	O
+.	O
+	O
+It	O
+is	O
+also	O
+located	O
+near	O
+Tyr	O
+-	O
+85	O
+,	O
+one	O
+of	O
+the	O
+residues	O
+that	O
+probably	O
+coordinate	O
+the	O
+iron	O
+molecule	O
+with	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+active	O
+site	O
+(	O
+Fig	O
+6a	O
+)	O
+and	O
+therefore	O
+might	O
+exert	O
+indirect	O
+effects	O
+.	O
+	O
+We	O
+note	O
+that	O
+His	O
+-	O
+117	O
+is	O
+located	O
+close	O
+to	O
+the	O
+chemically	O
+undefined	O
+ligand	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+(	O
+Fig	O
+7b	O
+)	O
+and	O
+might	O
+thus	O
+play	O
+a	O
+role	O
+in	O
+contacting	O
+the	O
+natural	O
+substrate	O
+of	O
+the	O
+ectoine	O
+synthase	O
+.	O
+	O
+Both	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+variants	O
+exhibited	O
+wild	O
+-	O
+type	O
+level	O
+enzyme	O
+activities	O
+and	O
+possessed	O
+a	O
+iron	O
+content	O
+matching	O
+that	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+(	O
+Table	O
+1	O
+).	O
+	O
+This	O
+illustrates	O
+that	O
+not	O
+every	O
+amino	O
+acid	O
+substitution	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+leads	O
+to	O
+an	O
+indiscriminate	O
+impairment	O
+of	O
+enzyme	O
+function	O
+and	O
+iron	O
+content	O
+.	O
+	O
+The	O
+crystallographic	O
+data	O
+presented	O
+here	O
+firmly	O
+identify	O
+ectoine	O
+synthase	O
+(	O
+EctC	O
+),	O
+an	O
+enzyme	O
+critical	O
+for	O
+the	O
+production	O
+of	O
+the	O
+microbial	O
+cytoprotectant	O
+and	O
+chemical	O
+chaperone	O
+ectoine	O
+,	O
+as	O
+a	O
+new	O
+member	O
+of	O
+the	O
+cupin	O
+superfamily	O
+.	O
+	O
+The	O
+overall	O
+fold	O
+and	O
+bowl	O
+shape	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+(	O
+Figs	O
+4	O
+and	O
+5	O
+)	O
+with	O
+its	O
+11	O
+β	O
+-	O
+strands	O
+(	O
+β1	O
+-	O
+β11	O
+)	O
+and	O
+two	O
+α	O
+-	O
+helices	O
+(	O
+α	O
+-	O
+I	O
+and	O
+α	O
+-	O
+II	O
+)	O
+closely	O
+adheres	O
+to	O
+the	O
+design	O
+principles	O
+typically	O
+found	O
+in	O
+crystal	O
+structures	O
+of	O
+cupins	O
+.	O
+	O
+In	O
+addition	O
+to	O
+the	O
+ectoine	O
+synthase	O
+,	O
+the	O
+polyketide	O
+cyclase	O
+RemF	O
+is	O
+the	O
+only	O
+other	O
+currently	O
+known	O
+cupin	O
+-	O
+related	O
+enzyme	O
+that	O
+catalyze	O
+a	O
+cyclocondensation	O
+reaction	O
+although	O
+the	O
+substrates	O
+of	O
+EctC	O
+and	O
+RemF	O
+are	O
+rather	O
+different	O
+.	O
+	O
+The	O
+pro	O
+-	O
+and	O
+eukaryotic	O
+members	O
+of	O
+the	O
+cupin	O
+superfamily	O
+perform	O
+a	O
+variety	O
+of	O
+both	O
+enzymatic	O
+and	O
+non	O
+-	O
+enzymatic	O
+functions	O
+that	O
+are	O
+built	O
+upon	O
+a	O
+common	O
+structural	O
+scaffold	O
+.	O
+	O
+Most	O
+cupins	O
+contain	O
+transition	O
+state	O
+metals	O
+that	O
+can	O
+promote	O
+different	O
+types	O
+of	O
+chemical	O
+reactions	O
+.	O
+	O
+We	O
+report	O
+here	O
+for	O
+the	O
+first	O
+time	O
+that	O
+the	O
+ectoine	O
+synthase	O
+is	O
+a	O
+metal	O
+-	O
+dependent	O
+enzyme	O
+.	O
+	O
+ICP	O
+-	O
+MS	O
+,	O
+metal	O
+-	O
+depletion	O
+and	O
+reconstitution	O
+experiments	O
+(	O
+Fig	O
+3	O
+)	O
+consistently	O
+identify	O
+iron	O
+as	O
+the	O
+biologically	O
+most	O
+relevant	O
+metal	O
+for	O
+the	O
+EctC	O
+-	O
+catalyzed	O
+cyclocondensation	O
+reaction	O
+.	O
+	O
+However	O
+,	O
+as	O
+observed	O
+with	O
+other	O
+cupins	O
+,	O
+EctC	O
+is	O
+a	O
+somewhat	O
+promiscuous	O
+enzyme	O
+as	O
+far	O
+as	O
+the	O
+catalytically	O
+important	O
+metal	O
+is	O
+concerned	O
+when	O
+they	O
+are	O
+provided	O
+in	O
+large	O
+molar	O
+excess	O
+(	O
+Fig	O
+3c	O
+).	O
+	O
+Although	O
+some	O
+uncertainty	O
+remains	O
+with	O
+respect	O
+to	O
+the	O
+precise	O
+identity	O
+of	O
+amino	O
+acid	O
+residues	O
+that	O
+participate	O
+in	O
+metal	O
+binding	O
+by	O
+(	O
+Sa	O
+)	O
+EctC	O
+,	O
+our	O
+structure	O
+-	O
+guided	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+experiments	O
+targeting	O
+the	O
+presumptive	O
+iron	O
+-	O
+binding	O
+residues	O
+(	O
+Fig	O
+6a	O
+and	O
+6b	O
+)	O
+demonstrate	O
+that	O
+none	O
+of	O
+them	O
+can	O
+be	O
+spared	O
+(	O
+Table	O
+1	O
+).	O
+	O
+The	O
+three	O
+residues	O
+(	O
+Glu	O
+-	O
+57	O
+,	O
+Tyr	O
+-	O
+85	O
+,	O
+His	O
+-	O
+93	O
+)	O
+that	O
+we	O
+deem	O
+to	O
+form	O
+it	O
+(	O
+Figs	O
+6	O
+and	O
+7b	O
+)	O
+are	O
+strictly	O
+conserved	O
+in	O
+a	O
+large	O
+collection	O
+of	O
+EctC	O
+-	O
+type	O
+proteins	O
+originating	O
+from	O
+16	O
+bacterial	O
+and	O
+three	O
+archaeal	O
+phyla	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+We	O
+also	O
+show	O
+here	O
+for	O
+the	O
+first	O
+time	O
+that	O
+,	O
+in	O
+addition	O
+to	O
+its	O
+natural	O
+substrate	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+,	O
+EctC	O
+also	O
+converts	O
+the	O
+isomer	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+into	O
+ectoine	O
+,	O
+albeit	O
+with	O
+a	O
+73	O
+-	O
+fold	O
+reduced	O
+catalytic	O
+efficiency	O
+(	O
+S3a	O
+and	O
+S3b	O
+Fig	O
+).	O
+	O
+Our	O
+finding	O
+that	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+serves	O
+as	O
+a	O
+substrate	O
+for	O
+ectoine	O
+synthase	O
+has	O
+physiologically	O
+relevant	O
+ramifications	O
+for	O
+those	O
+microorganisms	O
+that	O
+can	O
+both	O
+synthesize	O
+and	O
+catabolize	O
+ectoine	O
+,	O
+since	O
+they	O
+need	O
+to	O
+prevent	O
+a	O
+futile	O
+cycle	O
+of	O
+synthesis	O
+and	O
+degradation	O
+when	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+is	O
+produced	O
+as	O
+an	O
+intermediate	O
+in	O
+the	O
+catabolic	O
+route	O
+.	O
+	O
+Although	O
+we	O
+cannot	O
+identify	O
+the	O
+true	O
+chemical	O
+nature	O
+of	O
+the	O
+C6	O
+compound	O
+that	O
+was	O
+trapped	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+nor	O
+its	O
+precise	O
+origin	O
+,	O
+we	O
+treated	O
+this	O
+compound	O
+as	O
+a	O
+proxy	O
+for	O
+the	O
+natural	O
+substrate	O
+of	O
+ectoine	O
+synthase	O
+,	O
+which	O
+is	O
+a	O
+C6	O
+compound	O
+as	O
+well	O
+(	O
+Fig	O
+7a	O
+).	O
+	O
+Indeed	O
+,	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+of	O
+those	O
+five	O
+residues	O
+that	O
+contact	O
+the	O
+unknown	O
+C6	O
+compound	O
+(	O
+Fig	O
+7b	O
+)	O
+yielded	O
+(	O
+Sa	O
+)	O
+EctC	O
+variants	O
+with	O
+strongly	O
+impaired	O
+enzyme	O
+function	O
+but	O
+near	O
+wild	O
+-	O
+type	O
+levels	O
+of	O
+iron	O
+(	O
+Table	O
+1	O
+).	O
+	O
+We	O
+therefore	O
+surmise	O
+that	O
+our	O
+crystallographic	O
+data	O
+and	O
+the	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+study	O
+reported	O
+here	O
+provide	O
+a	O
+structural	O
+and	O
+functional	O
+view	O
+into	O
+the	O
+architecture	O
+of	O
+the	O
+EctC	O
+active	O
+site	O
+(	O
+Fig	O
+7b	O
+).	O
+	O
+The	O
+ectoine	O
+synthase	O
+from	O
+the	O
+cold	O
+-	O
+adapted	O
+marine	O
+bacterium	O
+S	O
+.	O
+alaskensis	O
+can	O
+be	O
+considered	O
+as	O
+a	O
+psychrophilic	O
+enzyme	O
+(	O
+S3a	O
+Fig	O
+),	O
+types	O
+of	O
+proteins	O
+with	O
+a	O
+considerable	O
+structural	O
+flexibility	O
+.	O
+	O
+It	O
+is	O
+hoped	O
+that	O
+these	O
+can	O
+be	O
+further	O
+employed	O
+to	O
+obtain	O
+EctC	O
+crystal	O
+structures	O
+with	O
+either	O
+the	O
+substrate	O
+or	O
+the	O
+reaction	O
+product	O
+.	O
+	O
+Together	O
+with	O
+our	O
+finding	O
+that	O
+ectoine	O
+synthase	O
+is	O
+metal	O
+dependent	O
+,	O
+these	O
+crystal	O
+structures	O
+should	O
+allow	O
+a	O
+more	O
+detailed	O
+understanding	O
+of	O
+the	O
+chemistry	O
+underlying	O
+the	O
+EctC	O
+-	O
+catalyzed	O
+cyclocondensation	O
+reaction	O
+.	O
+	O
+Regnase	O
+-	O
+1	O
+is	O
+an	O
+RNase	O
+that	O
+directly	O
+cleaves	O
+mRNAs	O
+of	O
+inflammatory	O
+genes	O
+such	O
+as	O
+IL	O
+-	O
+6	O
+and	O
+IL	O
+-	O
+12p40	O
+,	O
+and	O
+negatively	O
+regulates	O
+cellular	O
+inflammatory	O
+responses	O
+.	O
+	O
+Here	O
+,	O
+we	O
+report	O
+the	O
+structures	O
+of	O
+four	O
+domains	O
+of	O
+Regnase	O
+-	O
+1	O
+from	O
+Mus	O
+musculus	O
+—	O
+the	O
+N	O
+-	O
+terminal	O
+domain	O
+(	O
+NTD	O
+),	O
+PilT	O
+N	O
+-	O
+terminus	O
+like	O
+(	O
+PIN	O
+)	O
+domain	O
+,	O
+zinc	O
+finger	O
+(	O
+ZF	O
+)	O
+domain	O
+and	O
+C	O
+-	O
+terminal	O
+domain	O
+(	O
+CTD	O
+).	O
+	O
+The	O
+PIN	O
+domain	O
+harbors	O
+the	O
+RNase	O
+catalytic	O
+center	O
+;	O
+however	O
+,	O
+it	O
+is	O
+insufficient	O
+for	O
+enzymatic	O
+activity	O
+.	O
+	O
+We	O
+found	O
+that	O
+the	O
+NTD	O
+associates	O
+with	O
+the	O
+PIN	O
+domain	O
+and	O
+significantly	O
+enhances	O
+its	O
+RNase	O
+activity	O
+.	O
+	O
+The	O
+PIN	O
+domain	O
+forms	O
+a	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+oligomer	O
+and	O
+the	O
+dimer	O
+interface	O
+overlaps	O
+with	O
+the	O
+NTD	O
+binding	O
+site	O
+.	O
+	O
+These	O
+results	O
+suggest	O
+that	O
+Regnase	O
+-	O
+1	O
+RNase	O
+activity	O
+is	O
+tightly	O
+controlled	O
+by	O
+both	O
+intramolecular	O
+(	O
+NTD	O
+-	O
+PIN	O
+)	O
+and	O
+intermolecular	O
+(	O
+PIN	O
+-	O
+PIN	O
+)	O
+interactions	O
+.	O
+	O
+The	O
+initial	O
+sensing	O
+of	O
+infection	O
+is	O
+mediated	O
+by	O
+a	O
+set	O
+of	O
+pattern	O
+-	O
+recognition	O
+receptors	O
+(	O
+PRRs	O
+)	O
+such	O
+Toll	O
+-	O
+like	O
+receptors	O
+(	O
+TLRs	O
+)	O
+and	O
+the	O
+intracellular	O
+signaling	O
+cascades	O
+triggered	O
+by	O
+TLRs	O
+evoke	O
+transcriptional	O
+expression	O
+of	O
+inflammatory	O
+mediators	O
+that	O
+coordinate	O
+the	O
+elimination	O
+of	O
+pathogens	O
+and	O
+infected	O
+cells	O
+.	O
+	O
+Regnase	O
+-	O
+1	O
+(	O
+also	O
+known	O
+as	O
+Zc3h12a	O
+and	O
+MCPIP1	O
+)	O
+is	O
+an	O
+RNase	O
+whose	O
+expression	O
+level	O
+is	O
+stimulated	O
+by	O
+lipopolysaccharides	O
+and	O
+prevents	O
+autoimmune	O
+diseases	O
+by	O
+directly	O
+controlling	O
+the	O
+stability	O
+of	O
+mRNAs	O
+of	O
+inflammatory	O
+genes	O
+such	O
+as	O
+interleukin	O
+(	O
+IL	O
+)-	O
+6	O
+,	O
+IL	O
+-	O
+1β	O
+,	O
+IL	O
+-	O
+2	O
+,	O
+and	O
+IL	O
+-	O
+12p40	O
+.	O
+	O
+Regnase	O
+-	O
+1	O
+accelerates	O
+target	O
+mRNA	O
+degradation	O
+via	O
+their	O
+3	O
+′-	O
+terminal	O
+untranslated	O
+region	O
+(	O
+3	O
+′	O
+UTR	O
+),	O
+and	O
+also	O
+degrades	O
+its	O
+own	O
+mRNA	O
+.	O
+	O
+Recently	O
+,	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+Regnase	O
+-	O
+1	O
+PIN	O
+domain	O
+derived	O
+from	O
+Homo	O
+sapiens	O
+was	O
+reported	O
+.	O
+	O
+The	O
+structure	O
+combined	O
+with	O
+functional	O
+analyses	O
+revealed	O
+that	O
+four	O
+catalytically	O
+important	O
+Asp	O
+residues	O
+form	O
+the	O
+catalytic	O
+center	O
+and	O
+stabilize	O
+Mg2	O
++	O
+binding	O
+that	O
+is	O
+crucial	O
+for	O
+RNase	O
+activity	O
+.	O
+	O
+Several	O
+CCCH	O
+-	O
+type	O
+ZF	O
+motifs	O
+in	O
+RNA	O
+-	O
+binding	O
+proteins	O
+have	O
+been	O
+reported	O
+to	O
+directly	O
+bind	O
+RNA	O
+.	O
+	O
+In	O
+addition	O
+,	O
+Regnase	O
+-	O
+1	O
+has	O
+been	O
+predicted	O
+to	O
+possess	O
+other	O
+domains	O
+in	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+regions	O
+.	O
+	O
+However	O
+,	O
+the	O
+structure	O
+and	O
+function	O
+of	O
+the	O
+ZF	O
+domain	O
+,	O
+N	O
+-	O
+terminal	O
+domain	O
+(	O
+NTD	O
+)	O
+and	O
+C	O
+-	O
+terminal	O
+domain	O
+(	O
+CTD	O
+)	O
+of	O
+Regnase	O
+-	O
+1	O
+have	O
+not	O
+been	O
+solved	O
+.	O
+	O
+Here	O
+,	O
+we	O
+performed	O
+structural	O
+and	O
+functional	O
+analyses	O
+of	O
+individual	O
+domains	O
+of	O
+Regnase	O
+-	O
+1	O
+derived	O
+from	O
+Mus	O
+musculus	O
+in	O
+order	O
+to	O
+understand	O
+the	O
+catalytic	O
+activity	O
+in	O
+vitro	O
+.	O
+	O
+Our	O
+data	O
+revealed	O
+that	O
+the	O
+catalytic	O
+activity	O
+of	O
+Regnase	O
+-	O
+1	O
+is	O
+regulated	O
+through	O
+both	O
+intra	O
+and	O
+intermolecular	O
+domain	O
+interactions	O
+in	O
+vitro	O
+.	O
+	O
+The	O
+NTD	O
+plays	O
+a	O
+crucial	O
+role	O
+in	O
+efficient	O
+cleavage	O
+of	O
+target	O
+mRNA	O
+,	O
+through	O
+intramolecular	O
+NTD	O
+-	O
+PIN	O
+interactions	O
+.	O
+	O
+Our	O
+findings	O
+suggest	O
+that	O
+Regnase	O
+-	O
+1	O
+cleaves	O
+its	O
+target	O
+mRNA	O
+by	O
+an	O
+NTD	O
+-	O
+activated	O
+functional	O
+PIN	O
+dimer	O
+,	O
+while	O
+the	O
+ZF	O
+increases	O
+RNA	O
+affinity	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+PIN	O
+dimer	O
+.	O
+	O
+We	O
+analyzed	O
+Rengase	O
+-	O
+1	O
+derived	O
+from	O
+Mus	O
+musculus	O
+and	O
+solved	O
+the	O
+structures	O
+of	O
+the	O
+four	O
+domains	O
+;	O
+NTD	O
+,	O
+PIN	O
+,	O
+ZF	O
+,	O
+and	O
+CTD	O
+individually	O
+by	O
+X	O
+-	O
+ray	O
+crystallography	O
+or	O
+NMR	O
+(	O
+Fig	O
+.	O
+1a	O
+–	O
+e	O
+).	O
+	O
+X	O
+-	O
+ray	O
+crystallography	O
+was	O
+attempted	O
+for	O
+the	O
+fragment	O
+containing	O
+both	O
+the	O
+PIN	O
+and	O
+ZF	O
+domains	O
+,	O
+however	O
+,	O
+electron	O
+density	O
+was	O
+observed	O
+only	O
+for	O
+the	O
+PIN	O
+domain	O
+(	O
+Fig	O
+.	O
+1c	O
+),	O
+consistent	O
+with	O
+a	O
+previous	O
+report	O
+on	O
+Regnase	O
+-	O
+1	O
+derived	O
+from	O
+Homo	O
+sapiens	O
+.	O
+	O
+This	O
+suggests	O
+that	O
+the	O
+PIN	O
+and	O
+ZF	O
+domains	O
+exist	O
+independently	O
+without	O
+interacting	O
+with	O
+each	O
+other	O
+.	O
+	O
+The	O
+NTD	O
+and	O
+CTD	O
+are	O
+both	O
+composed	O
+of	O
+three	O
+α	O
+helices	O
+,	O
+and	O
+structurally	O
+resemble	O
+ubiquitin	O
+conjugating	O
+enzyme	O
+E2	O
+K	O
+(	O
+PDB	O
+ID	O
+:	O
+3K9O	O
+)	O
+and	O
+ubiquitin	O
+associated	O
+protein	O
+1	O
+(	O
+PDB	O
+ID	O
+:	O
+4AE4	O
+),	O
+respectively	O
+,	O
+according	O
+to	O
+the	O
+Dali	O
+server	O
+.	O
+	O
+Contribution	O
+of	O
+each	O
+domain	O
+of	O
+Regnase	O
+-	O
+1	O
+to	O
+the	O
+mRNA	O
+binding	O
+activity	O
+	O
+First	O
+,	O
+we	O
+evaluated	O
+a	O
+role	O
+of	O
+the	O
+NTD	O
+and	O
+ZF	O
+domains	O
+for	O
+mRNA	O
+binding	O
+by	O
+an	O
+in	O
+vitro	O
+gel	O
+shift	O
+assay	O
+(	O
+Fig	O
+.	O
+1f	O
+).	O
+	O
+Fluorescently	O
+5	O
+′-	O
+labeled	O
+RNA	O
+corresponding	O
+to	O
+nucleotides	O
+82	O
+–	O
+106	O
+of	O
+the	O
+IL	O
+-	O
+6	O
+mRNA	O
+3	O
+′	O
+UTR	O
+and	O
+the	O
+catalytically	O
+inactive	O
+mutant	O
+(	O
+D226N	B-mutant
+and	O
+D244N	B-mutant
+)	O
+of	O
+Regnase	O
+-	O
+1	O
+—	O
+hereafter	O
+referred	O
+to	O
+as	O
+the	O
+DDNN	B-mutant
+mutant	O
+—	O
+were	O
+utilized	O
+.	O
+	O
+Upon	O
+addition	O
+of	O
+a	O
+larger	O
+amount	O
+of	O
+Regnase	O
+-	O
+1	O
+,	O
+the	O
+fluorescence	O
+of	O
+free	O
+RNA	O
+decreased	O
+,	O
+indicating	O
+that	O
+Regnase	O
+-	O
+1	O
+bound	O
+to	O
+the	O
+RNA	O
+.	O
+	O
+While	O
+the	O
+RNA	O
+binding	O
+ability	O
+was	O
+not	O
+significantly	O
+changed	O
+in	O
+the	O
+presence	O
+of	O
+NTD	O
+,	O
+it	O
+increased	O
+in	O
+the	O
+presence	O
+of	O
+the	O
+ZF	O
+domain	O
+(	O
+Fig	O
+.	O
+1f	O
+,	O
+g	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+Direct	O
+binding	O
+of	O
+the	O
+ZF	O
+domain	O
+and	O
+RNA	O
+were	O
+confirmed	O
+by	O
+NMR	O
+spectral	O
+changes	O
+.	O
+	O
+The	O
+fitting	O
+of	O
+the	O
+titration	O
+curve	O
+of	O
+Y314	O
+resulted	O
+in	O
+an	O
+apparent	O
+dissociation	O
+constant	O
+(	O
+Kd	O
+)	O
+of	O
+10	O
+±	O
+1	O
+.	O
+1	O
+μM	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+In	O
+order	O
+to	O
+characterize	O
+the	O
+role	O
+of	O
+each	O
+domain	O
+in	O
+the	O
+RNase	O
+activity	O
+of	O
+Regnase	O
+-	O
+1	O
+,	O
+we	O
+performed	O
+an	O
+in	O
+vitro	O
+cleavage	O
+assay	O
+using	O
+fluorescently	O
+5	O
+′-	O
+labeled	O
+RNA	O
+corresponding	O
+to	O
+nucleotides	O
+82	O
+–	O
+106	O
+of	O
+the	O
+IL	O
+-	O
+6	O
+mRNA	O
+3	O
+′	O
+UTR	O
+(	O
+Fig	O
+.	O
+1g	O
+).	O
+	O
+Regnase	O
+-	O
+1	O
+constructs	O
+consisting	O
+of	O
+NTD	B-mutant
+-	I-mutant
+PIN	I-mutant
+-	I-mutant
+ZF	I-mutant
+completely	O
+cleaved	O
+the	O
+target	O
+mRNA	O
+and	O
+generated	O
+the	O
+cleaved	O
+products	O
+.	O
+	O
+The	O
+apparent	O
+half	O
+-	O
+life	O
+(	O
+T1	O
+/	O
+2	O
+)	O
+of	O
+the	O
+RNase	O
+activity	O
+was	O
+about	O
+20	O
+minutes	O
+.	O
+	O
+Taken	O
+together	O
+with	O
+the	O
+results	O
+in	O
+the	O
+previous	O
+section	O
+,	O
+we	O
+conclude	O
+that	O
+the	O
+NTD	O
+is	O
+crucial	O
+for	O
+the	O
+RNase	O
+activity	O
+of	O
+Regnase	O
+-	O
+1	O
+in	O
+vitro	O
+,	O
+although	O
+it	O
+does	O
+not	O
+contribute	O
+to	O
+the	O
+direct	O
+mRNA	O
+binding	O
+.	O
+	O
+During	O
+purification	O
+by	O
+gel	O
+filtration	O
+,	O
+the	O
+PIN	O
+domain	O
+exhibited	O
+extremely	O
+asymmetric	O
+elution	O
+peaks	O
+in	O
+a	O
+concentration	O
+dependent	O
+manner	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+We	O
+found	O
+that	O
+the	O
+PIN	O
+domain	O
+formed	O
+a	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+oligomer	O
+that	O
+was	O
+commonly	O
+observed	O
+in	O
+all	O
+three	O
+crystal	O
+forms	O
+in	O
+spite	O
+of	O
+the	O
+different	O
+crystallization	O
+conditions	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+single	O
+mutations	O
+of	O
+side	O
+chains	O
+involved	O
+in	O
+the	O
+PIN	O
+–	O
+PIN	O
+oligomeric	O
+interaction	O
+resulted	O
+in	O
+monomer	O
+formation	O
+,	O
+judging	O
+from	O
+gel	O
+filtration	O
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+).	O
+	O
+Wild	O
+type	O
+and	O
+monomeric	O
+PIN	O
+mutants	O
+(	O
+P212A	B-mutant
+and	O
+D278R	B-mutant
+)	O
+were	O
+also	O
+analyzed	O
+by	O
+NMR	O
+.	O
+	O
+The	O
+spectra	O
+indicate	O
+that	O
+the	O
+dimer	O
+interface	O
+of	O
+the	O
+wild	O
+type	O
+PIN	O
+domain	O
+were	O
+significantly	O
+broadened	O
+compared	O
+to	O
+the	O
+monomeric	O
+mutants	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+These	O
+results	O
+indicate	O
+that	O
+the	O
+PIN	O
+domain	O
+forms	O
+a	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+oligomer	O
+in	O
+solution	O
+similar	O
+to	O
+the	O
+crystal	O
+structure	O
+.	O
+	O
+Interestingly	O
+,	O
+the	O
+monomeric	O
+PIN	O
+mutants	O
+P212A	B-mutant
+,	O
+R214A	B-mutant
+,	O
+and	O
+D278R	B-mutant
+had	O
+no	O
+significant	O
+RNase	O
+activity	O
+for	O
+IL	O
+-	O
+6	O
+mRNA	O
+in	O
+vitro	O
+(	O
+Fig	O
+.	O
+2c	O
+).	O
+	O
+The	O
+side	O
+chains	O
+of	O
+these	O
+residues	O
+point	O
+away	O
+from	O
+the	O
+catalytic	O
+center	O
+on	O
+the	O
+same	O
+molecule	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+While	O
+the	O
+NTD	O
+does	O
+not	O
+contribute	O
+to	O
+RNA	O
+binding	O
+(	O
+Fig	O
+.	O
+1f	O
+,	O
+g	O
+,	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+),	O
+it	O
+increases	O
+the	O
+RNase	O
+activity	O
+of	O
+Regnase	O
+-	O
+1	O
+(	O
+Fig	O
+.	O
+1h	O
+).	O
+	O
+In	O
+order	O
+to	O
+gain	O
+insight	O
+into	O
+the	O
+molecular	O
+mechanism	O
+of	O
+the	O
+NTD	O
+-	O
+mediated	O
+enhancement	O
+of	O
+Regnase	O
+-	O
+1	O
+RNase	O
+activity	O
+,	O
+we	O
+further	O
+investigated	O
+the	O
+domain	O
+-	O
+domain	O
+interaction	O
+between	O
+the	O
+NTD	O
+and	O
+the	O
+PIN	O
+domain	O
+using	O
+NMR	O
+.	O
+	O
+We	O
+used	O
+the	O
+catalytically	O
+inactive	O
+monomeric	O
+PIN	O
+mutant	O
+possessing	O
+both	O
+the	O
+DDNN	B-mutant
+and	O
+D278R	B-mutant
+mutations	O
+to	O
+avoid	O
+dimer	O
+formation	O
+of	O
+the	O
+PIN	O
+domain	O
+.	O
+	O
+The	O
+NMR	O
+signals	O
+from	O
+the	O
+PIN	O
+domain	O
+(	O
+residues	O
+V177	O
+,	O
+F210	O
+-	O
+T211	O
+,	O
+R214	O
+,	O
+F228	O
+-	O
+L232	O
+,	O
+and	O
+F234	O
+-	O
+S236	O
+)	O
+exhibited	O
+significant	O
+chemical	O
+shift	O
+changes	O
+upon	O
+addition	O
+of	O
+the	O
+NTD	O
+(	O
+Fig	O
+.	O
+3a	O
+).	O
+	O
+These	O
+results	O
+clearly	O
+indicate	O
+a	O
+direct	O
+interaction	O
+between	O
+the	O
+PIN	O
+domain	O
+and	O
+the	O
+NTD	O
+.	O
+	O
+Based	O
+on	O
+the	O
+titration	O
+curve	O
+for	O
+the	O
+chemical	O
+shift	O
+changes	O
+of	O
+L58	O
+,	O
+the	O
+apparent	O
+Kd	O
+between	O
+the	O
+isolated	O
+NTD	O
+and	O
+PIN	O
+was	O
+estimated	O
+to	O
+be	O
+110	O
+±	O
+5	O
+.	O
+8	O
+μM	O
+.	O
+Considering	O
+the	O
+fact	O
+that	O
+the	O
+NTD	O
+and	O
+PIN	O
+domains	O
+are	O
+attached	O
+by	O
+a	O
+linker	O
+,	O
+the	O
+actual	O
+binding	O
+affinity	O
+is	O
+expected	O
+much	O
+higher	O
+in	O
+the	O
+native	O
+protein	O
+.	O
+	O
+Mapping	O
+the	O
+residues	O
+with	O
+chemical	O
+shift	O
+changes	O
+reveals	O
+the	O
+putative	O
+PIN	O
+/	O
+NTD	O
+interface	O
+,	O
+which	O
+includes	O
+a	O
+helix	O
+that	O
+harbors	O
+catalytic	O
+residues	O
+D225	O
+and	O
+D226	O
+on	O
+the	O
+PIN	O
+domain	O
+(	O
+Fig	O
+.	O
+3a	O
+).	O
+	O
+Interestingly	O
+,	O
+the	O
+putative	O
+binding	O
+site	O
+for	O
+the	O
+NTD	O
+overlaps	O
+with	O
+the	O
+PIN	O
+-	O
+PIN	O
+dimer	O
+interface	O
+,	O
+implying	O
+that	O
+NTD	O
+binding	O
+can	O
+“	O
+terminate	O
+”	O
+PIN	O
+-	O
+PIN	O
+oligomerization	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+An	O
+in	O
+silico	O
+docking	O
+of	O
+the	O
+NTD	O
+and	O
+PIN	O
+domains	O
+using	O
+chemical	O
+shift	O
+restraints	O
+provided	O
+a	O
+model	O
+consistent	O
+with	O
+the	O
+NMR	O
+experiments	O
+(	O
+Fig	O
+.	O
+3c	O
+).	O
+	O
+To	O
+gain	O
+insight	O
+into	O
+the	O
+residues	O
+critical	O
+for	O
+Regnase	O
+-	O
+1	O
+RNase	O
+activity	O
+,	O
+each	O
+basic	O
+or	O
+aromatic	O
+residue	O
+located	O
+around	O
+the	O
+catalytic	O
+site	O
+of	O
+the	O
+PIN	O
+oligomer	O
+was	O
+mutated	O
+to	O
+alanine	O
+,	O
+and	O
+the	O
+oligomerization	O
+and	O
+RNase	O
+activity	O
+were	O
+investigated	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+From	O
+the	O
+gel	O
+filtration	O
+assays	O
+,	O
+all	O
+mutants	O
+except	O
+R214A	B-mutant
+formed	O
+dimers	O
+,	O
+suggesting	O
+that	O
+any	O
+lack	O
+of	O
+RNase	O
+activity	O
+in	O
+the	O
+mutants	O
+,	O
+except	O
+R214A	B-mutant
+,	O
+was	O
+directly	O
+due	O
+to	O
+mutational	O
+effects	O
+of	O
+the	O
+specific	O
+residues	O
+and	O
+not	O
+to	O
+abrogation	O
+of	O
+dimer	O
+formation	O
+.	O
+	O
+The	O
+W182A	B-mutant
+,	O
+R183A	B-mutant
+,	O
+and	O
+R214A	B-mutant
+mutants	O
+markedly	O
+lost	O
+cleavage	O
+activity	O
+for	O
+IL	O
+-	O
+6	O
+mRNA	O
+as	O
+well	O
+as	O
+for	O
+Regnase	O
+-	O
+1	O
+mRNA	O
+.	O
+	O
+The	O
+K184A	B-mutant
+,	O
+R215A	B-mutant
+,	O
+and	O
+R220A	B-mutant
+mutants	O
+moderately	O
+but	O
+significantly	O
+decreased	O
+the	O
+cleavage	O
+activity	O
+for	O
+both	O
+target	O
+mRNAs	O
+.	O
+	O
+The	O
+importance	O
+of	O
+K219	O
+and	O
+R247	O
+was	O
+slightly	O
+different	O
+for	O
+IL	O
+-	O
+6	O
+and	O
+Regnase	O
+-	O
+1	O
+mRNA	O
+;	O
+both	O
+K219	O
+and	O
+R247	O
+were	O
+more	O
+important	O
+in	O
+the	O
+cleavage	O
+of	O
+IL	O
+-	O
+6	O
+mRNA	O
+than	O
+for	O
+Regnase	O
+-	O
+1	O
+mRNA	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+R214	O
+is	O
+important	O
+for	O
+oligomerization	O
+of	O
+the	O
+PIN	O
+domain	O
+and	O
+the	O
+“	O
+secondary	O
+”	O
+chain	O
+’	O
+s	O
+residue	O
+R214	O
+is	O
+also	O
+positioned	O
+near	O
+the	O
+“	O
+primary	O
+”	O
+chain	O
+’	O
+s	O
+active	O
+site	O
+within	O
+the	O
+dimer	O
+interface	O
+.	O
+	O
+If	O
+this	O
+model	O
+is	O
+correct	O
+,	O
+then	O
+we	O
+reasoned	O
+that	O
+a	O
+catalytically	O
+inactive	O
+PIN	O
+and	O
+a	O
+PIN	O
+lacking	O
+the	O
+putative	O
+RNA	O
+-	O
+binding	O
+residues	O
+ought	O
+to	O
+be	O
+inactive	O
+in	O
+isolation	O
+but	O
+become	O
+active	O
+when	O
+mixed	O
+together	O
+.	O
+	O
+In	O
+order	O
+to	O
+test	O
+this	O
+hypothesis	O
+,	O
+we	O
+performed	O
+in	O
+vitro	O
+cleavage	O
+assays	O
+using	O
+combinations	O
+of	O
+Regnase	O
+-	O
+1	O
+mutants	O
+that	O
+had	O
+no	O
+or	O
+decreased	O
+RNase	O
+activities	O
+by	O
+themselves	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+These	O
+were	O
+paired	O
+with	O
+a	O
+DDNN	B-mutant
+mutant	O
+that	O
+had	O
+no	O
+RNase	O
+activity	O
+by	O
+itself	O
+.	O
+	O
+When	O
+any	O
+members	O
+of	O
+the	O
+two	O
+groups	O
+are	O
+mixed	O
+,	O
+two	O
+kinds	O
+of	O
+heterodimers	O
+can	O
+be	O
+formed	O
+:	O
+one	O
+is	O
+composed	O
+of	O
+a	O
+DDNN	B-mutant
+primary	O
+PIN	O
+and	O
+a	O
+basic	O
+residue	O
+mutant	O
+secondary	O
+PIN	O
+and	O
+is	O
+expected	O
+to	O
+exhibit	O
+no	O
+RNase	O
+activity	O
+;	O
+the	O
+other	O
+is	O
+composed	O
+of	O
+a	O
+basic	O
+residue	O
+mutant	O
+primary	O
+PIN	O
+and	O
+a	O
+DDNN	B-mutant
+secondary	O
+PIN	O
+and	O
+is	O
+predicted	O
+to	O
+rescue	O
+RNase	O
+activity	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+	O
+When	O
+we	O
+compared	O
+the	O
+fluorescence	O
+intensity	O
+of	O
+uncleaved	O
+IL	O
+-	O
+6	O
+mRNA	O
+,	O
+basic	O
+residue	O
+mutants	O
+W182A	B-mutant
+,	O
+K184A	B-mutant
+,	O
+R214A	B-mutant
+,	O
+and	O
+R220A	B-mutant
+were	O
+rescued	O
+upon	O
+addition	O
+of	O
+the	O
+DDNN	B-mutant
+mutant	O
+(	O
+Fig	O
+.	O
+5b	O
+).	O
+	O
+Consistently	O
+,	O
+when	O
+we	O
+compared	O
+the	O
+fluorescence	O
+intensity	O
+of	O
+the	O
+uncleaved	O
+Regnase	O
+-	O
+1	O
+mRNA	O
+,	O
+basic	O
+residue	O
+mutants	O
+K184A	B-mutant
+and	O
+R214A	B-mutant
+were	O
+rescued	O
+upon	O
+addition	O
+of	O
+the	O
+DDNN	B-mutant
+mutant	O
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+	O
+Rescue	O
+of	O
+K184A	B-mutant
+and	O
+R214A	B-mutant
+by	O
+the	O
+DDNN	B-mutant
+mutant	O
+was	O
+also	O
+confirmed	O
+by	O
+a	O
+significant	O
+increase	O
+in	O
+the	O
+cleaved	O
+products	O
+.	O
+	O
+This	O
+is	O
+particularly	O
+significant	O
+because	O
+the	O
+side	O
+chains	O
+of	O
+K184	O
+and	O
+R214	O
+in	O
+the	O
+primary	O
+PIN	O
+are	O
+oriented	O
+away	O
+from	O
+their	O
+own	O
+catalytic	O
+center	O
+,	O
+while	O
+those	O
+in	O
+the	O
+secondary	O
+PIN	O
+face	O
+toward	O
+the	O
+catalytic	O
+center	O
+of	O
+the	O
+primary	O
+PIN	O
+.	O
+	O
+R214	O
+is	O
+an	O
+important	O
+residue	O
+for	O
+dimer	O
+formation	O
+as	O
+shown	O
+in	O
+Fig	O
+.	O
+2	O
+,	O
+therefore	O
+,	O
+R214A	B-mutant
+in	O
+the	O
+secondary	O
+PIN	O
+cannot	O
+dimerize	O
+.	O
+	O
+Taken	O
+together	O
+,	O
+the	O
+rescue	O
+experiments	O
+above	O
+support	O
+the	O
+proposed	O
+model	O
+in	O
+which	O
+the	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+dimer	O
+is	O
+functional	O
+in	O
+vitro	O
+.	O
+	O
+Although	O
+the	O
+function	O
+of	O
+the	O
+CTD	O
+remains	O
+elusive	O
+,	O
+we	O
+revealed	O
+the	O
+functions	O
+of	O
+the	O
+NTD	O
+,	O
+PIN	O
+,	O
+and	O
+ZF	O
+domains	O
+.	O
+	O
+A	O
+Regnase	O
+-	O
+1	O
+construct	O
+consisting	O
+of	O
+PIN	O
+and	O
+ZF	O
+domains	O
+derived	O
+from	O
+Mus	O
+musculus	O
+was	O
+crystallized	O
+;	O
+however	O
+,	O
+the	O
+electron	O
+density	O
+of	O
+the	O
+ZF	O
+domain	O
+was	O
+low	O
+,	O
+indicating	O
+that	O
+the	O
+ZF	O
+domain	O
+is	O
+highly	O
+mobile	O
+in	O
+the	O
+absence	O
+of	O
+target	O
+mRNA	O
+or	O
+possibly	O
+other	O
+protein	O
+-	O
+protein	O
+interactions	O
+.	O
+	O
+Our	O
+NMR	O
+experiments	O
+confirmed	O
+direct	O
+binding	O
+of	O
+the	O
+ZF	O
+domain	O
+to	O
+IL	O
+-	O
+6	O
+mRNA	O
+with	O
+a	O
+Kd	O
+of	O
+10	O
+±	O
+1	O
+.	O
+1	O
+μM	O
+.	O
+Furthermore	O
+,	O
+an	O
+in	O
+vitro	O
+gel	O
+shift	O
+assay	O
+indicated	O
+that	O
+Regnase	O
+-	O
+1	O
+containing	O
+the	O
+ZF	O
+domain	O
+enhanced	O
+target	O
+mRNA	O
+-	O
+binding	O
+,	O
+but	O
+the	O
+protein	O
+-	O
+RNA	O
+complex	O
+remained	O
+in	O
+the	O
+bottom	O
+of	O
+the	O
+well	O
+without	O
+entering	O
+into	O
+the	O
+polyacrylamide	O
+gel	O
+.	O
+	O
+These	O
+results	O
+indicate	O
+that	O
+Regnase	O
+-	O
+1	O
+directly	O
+binds	O
+to	O
+RNA	O
+and	O
+precipitates	O
+under	O
+such	O
+experimental	O
+conditions	O
+.	O
+	O
+Due	O
+to	O
+this	O
+limitation	O
+,	O
+it	O
+is	O
+difficult	O
+to	O
+perform	O
+further	O
+structural	O
+analyses	O
+of	O
+mRNA	O
+-	O
+Regnase	O
+-	O
+1	O
+complexes	O
+by	O
+X	O
+-	O
+ray	O
+crystallography	O
+or	O
+NMR	O
+.	O
+	O
+Rao	O
+and	O
+co	O
+-	O
+workers	O
+previously	O
+argued	O
+that	O
+PIN	O
+dimerization	O
+is	O
+likely	O
+to	O
+be	O
+a	O
+crystallographic	O
+artifact	O
+with	O
+no	O
+physiological	O
+significance	O
+,	O
+since	O
+monomers	O
+were	O
+dominant	O
+in	O
+their	O
+analytical	O
+ultra	O
+-	O
+centrifugation	O
+experiments	O
+.	O
+	O
+This	O
+inconsistency	O
+might	O
+be	O
+due	O
+to	O
+difference	O
+in	O
+the	O
+analytical	O
+methods	O
+and	O
+/	O
+or	O
+protein	O
+concentrations	O
+used	O
+in	O
+each	O
+experiment	O
+,	O
+since	O
+the	O
+oligomer	O
+formation	O
+of	O
+PIN	O
+was	O
+dependent	O
+on	O
+the	O
+protein	O
+concentration	O
+in	O
+our	O
+study	O
+.	O
+	O
+Single	O
+mutations	O
+to	O
+residues	O
+involved	O
+in	O
+the	O
+putative	O
+oligomeric	O
+interaction	O
+of	O
+PIN	O
+monomerized	O
+as	O
+expected	O
+and	O
+these	O
+mutants	O
+lost	O
+their	O
+RNase	O
+activity	O
+as	O
+well	O
+.	O
+	O
+Based	O
+on	O
+these	O
+observations	O
+,	O
+we	O
+concluded	O
+that	O
+PIN	O
+-	O
+PIN	O
+dimer	O
+formation	O
+is	O
+critical	O
+for	O
+Regnase	O
+-	O
+1	O
+RNase	O
+activity	O
+in	O
+vitro	O
+.	O
+	O
+Within	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+PIN	O
+dimer	O
+,	O
+the	O
+Regnase	O
+-	O
+1	O
+specific	O
+basic	O
+regions	O
+in	O
+both	O
+the	O
+“	O
+primary	O
+”	O
+and	O
+“	O
+secondary	O
+”	O
+PINs	O
+are	O
+located	O
+around	O
+the	O
+catalytic	O
+site	O
+of	O
+the	O
+primary	O
+PIN	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+Moreover	O
+,	O
+our	O
+structure	O
+-	O
+based	O
+mutational	O
+analyses	O
+showed	O
+these	O
+two	O
+Regnase	O
+-	O
+1	O
+specific	O
+basic	O
+regions	O
+were	O
+essential	O
+for	O
+target	O
+mRNA	O
+cleavage	O
+in	O
+vitro	O
+.	O
+	O
+The	O
+cleavage	O
+assay	O
+also	O
+showed	O
+that	O
+the	O
+NTD	O
+is	O
+crucial	O
+for	O
+efficient	O
+mRNA	O
+cleavage	O
+.	O
+	O
+While	O
+further	O
+analyses	O
+are	O
+necessary	O
+to	O
+prove	O
+this	O
+point	O
+,	O
+our	O
+preliminary	O
+docking	O
+and	O
+molecular	O
+dynamics	O
+simulations	O
+indicate	O
+that	O
+NTD	O
+-	O
+binding	O
+rearranges	O
+the	O
+catalytic	O
+residues	O
+of	O
+the	O
+PIN	O
+domain	O
+toward	O
+an	O
+active	O
+conformation	O
+suitable	O
+for	O
+binding	O
+Mg2	O
++.	O
+	O
+In	O
+this	O
+context	O
+,	O
+it	O
+is	O
+interesting	O
+that	O
+,	O
+in	O
+response	O
+to	O
+TCR	O
+stimulation	O
+,	O
+Malt1	O
+cleaves	O
+Regnase	O
+-	O
+1	O
+at	O
+R111	O
+to	O
+control	O
+immune	O
+responses	O
+in	O
+vivo	O
+.	O
+	O
+This	O
+result	O
+is	O
+consistent	O
+with	O
+a	O
+model	O
+in	O
+which	O
+the	O
+NTD	O
+acts	O
+as	O
+an	O
+enhancer	O
+,	O
+and	O
+cleavage	O
+of	O
+the	O
+linker	O
+lowers	O
+enzymatic	O
+activity	O
+dramatically	O
+.	O
+	O
+We	O
+incorporated	O
+information	O
+from	O
+the	O
+cleavage	O
+site	O
+of	O
+IL	O
+-	O
+6	O
+mRNA	O
+in	O
+vitro	O
+is	O
+indicated	O
+by	O
+denaturing	O
+polyacrylamide	O
+gel	O
+electrophoresis	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+,	O
+b	O
+).	O
+	O
+The	O
+docking	O
+result	O
+revealed	O
+multiple	O
+RNA	O
+binding	O
+modes	O
+that	O
+satisfied	O
+the	O
+experimental	O
+results	O
+in	O
+vitro	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+,	O
+d	O
+),	O
+however	O
+,	O
+it	O
+should	O
+be	O
+noted	O
+that	O
+,	O
+in	O
+vivo	O
+,	O
+there	O
+would	O
+likely	O
+be	O
+many	O
+other	O
+RNA	O
+-	O
+binding	O
+proteins	O
+that	O
+would	O
+protect	O
+loop	O
+regions	O
+from	O
+cleavage	O
+by	O
+Regnase	O
+-	O
+1	O
+.	O
+	O
+In	O
+the	O
+absence	O
+of	O
+target	O
+mRNA	O
+,	O
+the	O
+PIN	O
+domain	O
+forms	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+oligomers	O
+at	O
+high	O
+concentration	O
+.	O
+	O
+A	O
+fully	O
+active	O
+catalytic	O
+center	O
+can	O
+be	O
+formed	O
+only	O
+when	O
+the	O
+NTD	O
+associates	O
+with	O
+the	O
+oligomer	O
+surface	O
+of	O
+the	O
+PIN	O
+domain	O
+,	O
+which	O
+terminates	O
+the	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+oligomer	O
+formation	O
+in	O
+one	O
+direction	O
+(	O
+primary	O
+PIN	O
+),	O
+and	O
+forms	O
+a	O
+functional	O
+dimer	O
+together	O
+with	O
+the	O
+neighboring	O
+PIN	O
+(	O
+secondary	O
+PIN	O
+).	O
+	O
+(	O
+a	O
+)	O
+Domain	O
+architecture	O
+of	O
+Regnase	O
+-	O
+1	O
+.	O
+(	O
+b	O
+)	O
+Solution	O
+structure	O
+of	O
+the	O
+NTD	O
+.	O
+(	O
+c	O
+)	O
+Crystal	O
+structure	O
+of	O
+the	O
+PIN	O
+domain	O
+.	O
+	O
+Catalytic	O
+Asp	O
+residues	O
+were	O
+shown	O
+in	O
+sticks	O
+.	O
+	O
+(	O
+d	O
+)	O
+Solution	O
+structure	O
+of	O
+the	O
+ZF	O
+domain	O
+.	O
+	O
+Three	O
+Cys	O
+residues	O
+and	O
+one	O
+His	O
+residue	O
+responsible	O
+for	O
+Zn2	O
++-	O
+binding	O
+were	O
+shown	O
+in	O
+sticks	O
+.	O
+	O
+(	O
+e	O
+)	O
+Solution	O
+structure	O
+of	O
+the	O
+CTD	O
+.	O
+	O
+All	O
+the	O
+structures	O
+were	O
+colored	O
+in	O
+rainbow	O
+from	O
+N	O
+-	O
+terminus	O
+(	O
+blue	O
+)	O
+to	O
+C	O
+-	O
+terminus	O
+(	O
+red	O
+).	O
+	O
+(	O
+f	O
+)	O
+In	O
+vitro	O
+gel	O
+shift	O
+binding	O
+assay	O
+between	O
+Regnase	O
+-	O
+1	O
+and	O
+IL	O
+-	O
+6	O
+mRNA	O
+.	O
+	O
+(	O
+g	O
+)	O
+Binding	O
+of	O
+Regnase	O
+-	O
+1	O
+and	O
+IL	O
+-	O
+6	O
+mRNA	O
+was	O
+plotted	O
+.	O
+	O
+(	O
+h	O
+)	O
+In	O
+vitro	O
+cleavage	O
+assay	O
+of	O
+Regnase	O
+-	O
+1	O
+to	O
+IL	O
+-	O
+6	O
+mRNA	O
+.	O
+	O
+Fluorescence	O
+intensity	O
+of	O
+the	O
+uncleaved	O
+IL	O
+-	O
+6	O
+mRNA	O
+was	O
+indicated	O
+as	O
+the	O
+percentage	O
+against	O
+that	O
+in	O
+the	O
+absence	O
+of	O
+Regnase	O
+-	O
+1	O
+.	O
+	O
+Head	O
+-	O
+to	O
+-	O
+tail	O
+oligomer	O
+formation	O
+of	O
+the	O
+PIN	O
+domain	O
+is	O
+crucial	O
+for	O
+the	O
+RNase	O
+activity	O
+of	O
+Regnase	O
+-	O
+1	O
+.	O
+	O
+(	O
+a	O
+)	O
+Gel	O
+filtration	O
+analyses	O
+of	O
+the	O
+PIN	O
+domain	O
+.	O
+	O
+Catalytic	O
+residues	O
+and	O
+mutated	O
+residues	O
+were	O
+shown	O
+in	O
+sticks	O
+.	O
+	O
+Residues	O
+important	O
+for	O
+the	O
+oligomeric	O
+interaction	O
+were	O
+colored	O
+red	O
+,	O
+while	O
+R215	O
+that	O
+was	O
+dispensable	O
+for	O
+the	O
+oligomeric	O
+interaction	O
+was	O
+colored	O
+blue	O
+.	O
+(	O
+c	O
+)	O
+RNase	O
+activity	O
+of	O
+monomeric	O
+mutants	O
+for	O
+IL	O
+-	O
+6	O
+mRNA	O
+was	O
+analyzed	O
+.	O
+	O
+Pro	O
+and	O
+the	O
+residues	O
+without	O
+analysis	O
+were	O
+colored	O
+black	O
+and	O
+gray	O
+,	O
+respectively	O
+.	O
+	O
+(	O
+b	O
+)	O
+NMR	O
+analyses	O
+of	O
+the	O
+PIN	O
+-	O
+binding	O
+to	O
+the	O
+NTD	O
+.	O
+	O
+The	O
+residues	O
+with	O
+significant	O
+chemical	O
+shift	O
+changes	O
+were	O
+labeled	O
+in	O
+the	O
+overlaid	O
+spectra	O
+(	O
+left	O
+)	O
+and	O
+colored	O
+red	O
+,	O
+yellow	O
+,	O
+or	O
+green	O
+on	O
+the	O
+surface	O
+and	O
+ribbon	O
+structure	O
+of	O
+the	O
+NTD	O
+.	O
+	O
+S62	O
+was	O
+colored	O
+gray	O
+and	O
+excluded	O
+from	O
+the	O
+analysis	O
+,	O
+due	O
+to	O
+low	O
+signal	O
+intensity	O
+.	O
+	O
+(	O
+c	O
+)	O
+Docking	O
+model	O
+of	O
+the	O
+NTD	O
+and	O
+the	O
+PIN	O
+domain	O
+.	O
+	O
+Critical	O
+residues	O
+in	O
+the	O
+PIN	O
+domain	O
+for	O
+the	O
+RNase	O
+activity	O
+of	O
+Regnase	O
+-	O
+1	O
+.	O
+	O
+(	O
+a	O
+)	O
+In	O
+vitro	O
+cleavage	O
+assay	O
+of	O
+basic	O
+residue	O
+mutants	O
+for	O
+IL	O
+-	O
+6	O
+mRNA	O
+.	O
+	O
+The	O
+fluorescence	O
+intensity	O
+of	O
+the	O
+uncleaved	O
+mRNA	O
+was	O
+quantified	O
+and	O
+the	O
+results	O
+were	O
+mapped	O
+on	O
+the	O
+PIN	O
+dimer	O
+structure	O
+.	O
+	O
+Heterodimer	O
+formation	O
+by	O
+combination	O
+of	O
+the	O
+Regnase	O
+-	O
+1	O
+basic	O
+residue	O
+mutants	O
+and	O
+the	O
+DDNN	B-mutant
+mutant	O
+restored	O
+the	O
+RNase	O
+activity	O
+.	O
+	O
+(	O
+a	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+concept	O
+of	O
+the	O
+experiment	O
+.	O
+(	O
+b	O
+)	O
+In	O
+vitro	O
+cleavage	O
+assay	O
+of	O
+Regnase	O
+-	O
+1	O
+for	O
+IL	O
+-	O
+6	O
+mRNA	O
+.	O
+	O
+The	O
+fluorescence	O
+intensity	O
+of	O
+the	O
+uncleaved	O
+mRNA	O
+was	O
+quantified	O
+and	O
+the	O
+results	O
+were	O
+mapped	O
+on	O
+the	O
+PIN	O
+dimer	O
+.	O
+	O
+The	O
+mutations	O
+whose	O
+RNase	O
+activities	O
+were	O
+restored	O
+in	O
+the	O
+presence	O
+of	O
+DDNN	B-mutant
+mutant	O
+were	O
+colored	O
+in	O
+red	O
+or	O
+yellow	O
+on	O
+the	O
+primary	O
+PIN	O
+.	O
+	O
+Schematic	O
+representation	O
+of	O
+regulation	O
+of	O
+the	O
+Regnase	O
+-	O
+1	O
+catalytic	O
+activity	O
+through	O
+the	O
+domain	O
+-	O
+domain	O
+interactions	O
+.	O
+	O
+Clan	O
+CD	O
+cysteine	O
+peptidases	O
+,	O
+a	O
+structurally	O
+related	O
+group	O
+of	O
+peptidases	O
+that	O
+include	O
+mammalian	O
+caspases	O
+,	O
+exhibit	O
+a	O
+wide	O
+range	O
+of	O
+important	O
+functions	O
+,	O
+along	O
+with	O
+a	O
+variety	O
+of	O
+specificities	O
+and	O
+activation	O
+mechanisms	O
+.	O
+	O
+However	O
+,	O
+for	O
+the	O
+clostripain	O
+family	O
+(	O
+denoted	O
+C11	O
+),	O
+little	O
+is	O
+currently	O
+known	O
+.	O
+	O
+Here	O
+,	O
+we	O
+describe	O
+the	O
+first	O
+crystal	O
+structure	O
+of	O
+a	O
+C11	O
+protein	O
+from	O
+the	O
+human	O
+gut	O
+bacterium	O
+,	O
+Parabacteroides	O
+merdae	O
+(	O
+PmC11	O
+),	O
+determined	O
+to	O
+1	O
+.	O
+7	O
+-	O
+Å	O
+resolution	O
+.	O
+	O
+PmC11	O
+is	O
+a	O
+monomeric	O
+cysteine	O
+peptidase	O
+that	O
+comprises	O
+an	O
+extended	O
+caspase	O
+-	O
+like	O
+α	O
+/	O
+β	O
+/	O
+α	O
+sandwich	O
+and	O
+an	O
+unusual	O
+C	O
+-	O
+terminal	O
+domain	O
+.	O
+	O
+It	O
+shares	O
+core	O
+structural	O
+elements	O
+with	O
+clan	O
+CD	O
+cysteine	O
+peptidases	O
+but	O
+otherwise	O
+structurally	O
+differs	O
+from	O
+the	O
+other	O
+families	O
+in	O
+the	O
+clan	O
+.	O
+	O
+These	O
+studies	O
+also	O
+revealed	O
+a	O
+well	O
+ordered	O
+break	O
+in	O
+the	O
+polypeptide	O
+chain	O
+at	O
+Lys147	O
+,	O
+resulting	O
+in	O
+a	O
+large	O
+conformational	O
+rearrangement	O
+close	O
+to	O
+the	O
+active	O
+site	O
+.	O
+	O
+Biochemical	O
+and	O
+kinetic	O
+analysis	O
+revealed	O
+Lys147	O
+to	O
+be	O
+an	O
+intramolecular	O
+processing	O
+site	O
+at	O
+which	O
+cleavage	O
+is	O
+required	O
+for	O
+full	O
+activation	O
+of	O
+the	O
+enzyme	O
+,	O
+suggesting	O
+an	O
+autoinhibitory	O
+mechanism	O
+for	O
+self	O
+-	O
+preservation	O
+.	O
+	O
+PmC11	O
+has	O
+an	O
+acidic	O
+binding	O
+pocket	O
+and	O
+a	O
+preference	O
+for	O
+basic	O
+substrates	O
+,	O
+and	O
+accepts	O
+substrates	O
+with	O
+Arg	O
+and	O
+Lys	O
+in	O
+P1	O
+and	O
+does	O
+not	O
+require	O
+Ca2	O
++	O
+for	O
+activity	O
+.	O
+	O
+Collectively	O
+,	O
+these	O
+data	O
+provide	O
+insights	O
+into	O
+the	O
+mechanism	O
+and	O
+activity	O
+of	O
+PmC11	O
+and	O
+a	O
+detailed	O
+framework	O
+for	O
+studies	O
+on	O
+C11	O
+peptidases	O
+from	O
+other	O
+phylogenetic	O
+kingdoms	O
+.	O
+	O
+Cysteine	O
+peptidases	O
+play	O
+crucial	O
+roles	O
+in	O
+the	O
+virulence	O
+of	O
+bacterial	O
+and	O
+other	O
+eukaryotic	O
+pathogens	O
+.	O
+	O
+Clan	O
+CD	O
+families	O
+are	O
+typically	O
+described	O
+using	O
+the	O
+name	O
+of	O
+their	O
+archetypal	O
+,	O
+or	O
+founding	O
+,	O
+member	O
+and	O
+also	O
+given	O
+an	O
+identification	O
+number	O
+preceded	O
+by	O
+a	O
+“	O
+C	O
+,”	O
+to	O
+denote	O
+cysteine	O
+peptidase	O
+.	O
+	O
+Although	O
+seven	O
+families	O
+(	O
+C14	O
+is	O
+additionally	O
+split	O
+into	O
+three	O
+subfamilies	O
+)	O
+have	O
+been	O
+described	O
+for	O
+this	O
+clan	O
+,	O
+crystal	O
+structures	O
+have	O
+only	O
+been	O
+determined	O
+from	O
+four	O
+:	O
+legumain	O
+(	O
+C13	O
+),	O
+caspase	O
+(	O
+C14a	O
+),	O
+paracaspase	O
+(	O
+C14b	O
+(	O
+P	O
+),	O
+metacaspase	O
+(	O
+C14b	O
+(	O
+M	O
+),	O
+gingipain	O
+(	O
+C25	O
+),	O
+and	O
+the	O
+cysteine	O
+peptidase	O
+domain	O
+(	O
+CPD	O
+)	O
+of	O
+various	O
+toxins	O
+(	O
+C80	O
+).	O
+	O
+No	O
+structural	O
+information	O
+is	O
+available	O
+for	O
+clostripain	O
+(	O
+C11	O
+),	O
+separase	O
+(	O
+C50	O
+),	O
+or	O
+PrtH	O
+-	O
+peptidase	O
+(	O
+C85	O
+).	O
+	O
+Clan	O
+CD	O
+enzymes	O
+have	O
+a	O
+highly	O
+conserved	O
+His	O
+/	O
+Cys	O
+catalytic	O
+dyad	O
+and	O
+exhibit	O
+strict	O
+specificity	O
+for	O
+the	O
+P1	O
+residue	O
+of	O
+their	O
+substrates	O
+.	O
+	O
+The	O
+archetypal	O
+and	O
+arguably	O
+most	O
+notable	O
+family	O
+in	O
+the	O
+clan	O
+is	O
+that	O
+of	O
+the	O
+mammalian	O
+caspases	O
+(	O
+C14a	O
+),	O
+although	O
+clan	O
+CD	O
+members	O
+are	O
+distributed	O
+throughout	O
+the	O
+entire	O
+phylogenetic	O
+kingdom	O
+and	O
+are	O
+often	O
+required	O
+in	O
+fundamental	O
+biological	O
+processes	O
+.	O
+	O
+Interestingly	O
+,	O
+little	O
+is	O
+known	O
+about	O
+the	O
+structure	O
+or	O
+function	O
+of	O
+the	O
+C11	O
+proteins	O
+,	O
+despite	O
+their	O
+widespread	O
+distribution	O
+and	O
+its	O
+archetypal	O
+member	O
+,	O
+clostripain	O
+from	O
+Clostridium	O
+histolyticum	O
+,	O
+first	O
+reported	O
+in	O
+the	O
+literature	O
+in	O
+1938	O
+.	O
+	O
+Clostripain	O
+has	O
+been	O
+described	O
+as	O
+an	O
+arginine	O
+-	O
+specific	O
+peptidase	O
+with	O
+a	O
+requirement	O
+for	O
+Ca2	O
++	O
+and	O
+loss	O
+of	O
+an	O
+internal	O
+nonapeptide	O
+for	O
+full	O
+activation	O
+;	O
+lack	O
+of	O
+structural	O
+information	O
+on	O
+the	O
+family	O
+appears	O
+to	O
+have	O
+prohibited	O
+further	O
+investigation	O
+.	O
+	O
+As	O
+part	O
+of	O
+an	O
+ongoing	O
+project	O
+to	O
+characterize	O
+commensal	O
+bacteria	O
+in	O
+the	O
+microbiome	O
+that	O
+inhabit	O
+the	O
+human	O
+gut	O
+,	O
+the	O
+structure	O
+of	O
+C11	O
+peptidase	O
+,	O
+PmC11	O
+,	O
+from	O
+Parabacteroides	O
+merdae	O
+was	O
+determined	O
+using	O
+the	O
+Joint	O
+Center	O
+for	O
+Structural	O
+Genomics	O
+(	O
+JCSG	O
+)	O
+4	O
+HTP	O
+structural	O
+biology	O
+pipeline	O
+.	O
+	O
+A	O
+single	O
+cleavage	O
+was	O
+observed	O
+in	O
+the	O
+polypeptide	O
+chain	O
+at	O
+Lys147	O
+(	O
+Fig	O
+.	O
+1	O
+,	O
+A	O
+and	O
+B	O
+),	O
+where	O
+both	O
+ends	O
+of	O
+the	O
+cleavage	O
+site	O
+are	O
+fully	O
+visible	O
+and	O
+well	O
+ordered	O
+in	O
+the	O
+electron	O
+density	O
+.	O
+	O
+Helix	O
+α3	O
+sits	O
+at	O
+the	O
+end	O
+of	O
+the	O
+loop	O
+following	O
+β5	O
+(	O
+L5	O
+),	O
+just	O
+preceding	O
+the	O
+Lys147	O
+cleavage	O
+site	O
+,	O
+with	O
+both	O
+L5	O
+and	O
+α3	O
+pointing	O
+away	O
+from	O
+the	O
+central	O
+β	O
+-	O
+sheet	O
+and	O
+toward	O
+the	O
+CTD	O
+,	O
+which	O
+starts	O
+with	O
+α8	O
+.	O
+	O
+Crystal	O
+structure	O
+of	O
+a	O
+C11	O
+peptidase	O
+from	O
+P	O
+.	O
+merdae	O
+.	O
+	O
+A	O
+,	O
+primary	O
+sequence	O
+alignment	O
+of	O
+PmC11	O
+(	O
+Uniprot	O
+ID	O
+A7A9N3	O
+)	O
+and	O
+clostripain	O
+(	O
+Uniprot	O
+ID	O
+P09870	O
+)	O
+from	O
+C	O
+.	O
+histolyticum	O
+with	O
+identical	O
+residues	O
+highlighted	O
+in	O
+gray	O
+shading	O
+.	O
+	O
+The	O
+secondary	O
+structure	O
+of	O
+PmC11	O
+from	O
+the	O
+crystal	O
+structure	O
+is	O
+mapped	O
+onto	O
+its	O
+sequence	O
+with	O
+the	O
+position	O
+of	O
+the	O
+PmC11	O
+catalytic	O
+dyad	O
+,	O
+autocatalytic	O
+cleavage	O
+site	O
+(	O
+Lys147	O
+),	O
+and	O
+S1	O
+binding	O
+pocket	O
+Asp	O
+(	O
+Asp177	O
+)	O
+highlighted	O
+by	O
+a	O
+red	O
+star	O
+,	O
+a	O
+red	O
+downturned	O
+triangle	O
+,	O
+and	O
+a	O
+red	O
+upturned	O
+triangle	O
+,	O
+respectively	O
+.	O
+	O
+Connecting	O
+loops	O
+are	O
+colored	O
+gray	O
+,	O
+the	O
+main	O
+β	O
+-	O
+sheet	O
+is	O
+in	O
+orange	O
+,	O
+with	O
+other	O
+strands	O
+in	O
+olive	O
+,	O
+α	O
+-	O
+helices	O
+are	O
+in	O
+blue	O
+,	O
+and	O
+the	O
+nonapeptide	O
+linker	O
+of	O
+clostripain	O
+that	O
+is	O
+excised	O
+upon	O
+autocleavage	O
+is	O
+underlined	O
+in	O
+red	O
+.	O
+	O
+Sequences	O
+around	O
+the	O
+catalytic	O
+site	O
+of	O
+clostripain	O
+and	O
+PmC11	O
+align	O
+well	O
+.	O
+	O
+B	O
+,	O
+topology	O
+diagram	O
+of	O
+PmC11	O
+colored	O
+as	O
+in	O
+A	O
+except	O
+that	O
+additional	O
+(	O
+non	O
+-	O
+core	O
+)	O
+β	O
+-	O
+strands	O
+are	O
+in	O
+yellow	O
+.	O
+	O
+Helices	O
+found	O
+on	O
+either	O
+side	O
+of	O
+the	O
+central	O
+β	O
+-	O
+sheet	O
+are	O
+shown	O
+above	O
+and	O
+below	O
+the	O
+sheet	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+position	O
+of	O
+the	O
+catalytic	O
+dyad	O
+(	O
+H	O
+,	O
+C	O
+)	O
+and	O
+the	O
+processing	O
+site	O
+(	O
+Lys147	O
+)	O
+are	O
+highlighted	O
+.	O
+	O
+Helices	O
+(	O
+1	O
+–	O
+14	O
+)	O
+and	O
+β	O
+-	O
+strands	O
+(	O
+1	O
+–	O
+9	O
+and	O
+A	O
+-	O
+F	O
+)	O
+are	O
+numbered	O
+from	O
+the	O
+N	O
+terminus	O
+.	O
+	O
+C	O
+,	O
+tertiary	O
+structure	O
+of	O
+PmC11	O
+.	O
+	O
+The	O
+N	O
+and	O
+C	O
+termini	O
+(	O
+N	O
+and	O
+C	O
+)	O
+of	O
+PmC11	O
+along	O
+with	O
+the	O
+central	O
+β	O
+-	O
+sheet	O
+(	O
+1	O
+–	O
+9	O
+),	O
+helix	O
+α5	O
+,	O
+and	O
+helices	O
+α8	O
+,	O
+α11	O
+,	O
+and	O
+α13	O
+from	O
+the	O
+C	O
+-	O
+terminal	O
+domain	O
+,	O
+are	O
+all	O
+labeled	O
+.	O
+	O
+Loops	O
+are	O
+colored	O
+gray	O
+,	O
+the	O
+main	O
+β	O
+-	O
+sheet	O
+is	O
+in	O
+orange	O
+,	O
+with	O
+other	O
+β	O
+-	O
+strands	O
+in	O
+yellow	O
+,	O
+and	O
+α	O
+-	O
+helices	O
+are	O
+in	O
+blue	O
+.	O
+	O
+Of	O
+the	O
+interacting	O
+secondary	O
+structure	O
+elements	O
+,	O
+α5	O
+is	O
+perhaps	O
+the	O
+most	O
+interesting	O
+.	O
+	O
+This	O
+helix	O
+makes	O
+a	O
+total	O
+of	O
+eight	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+CTD	O
+,	O
+including	O
+one	O
+salt	O
+bridge	O
+(	O
+Arg191	O
+-	O
+Asp255	O
+)	O
+and	O
+is	O
+surrounded	O
+by	O
+the	O
+CTD	O
+on	O
+one	O
+side	O
+and	O
+the	O
+main	O
+core	O
+of	O
+the	O
+enzyme	O
+on	O
+the	O
+other	O
+,	O
+acting	O
+like	O
+a	O
+linchpin	O
+holding	O
+both	O
+components	O
+together	O
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+	O
+PmC11	O
+is	O
+,	O
+as	O
+expected	O
+,	O
+most	O
+structurally	O
+similar	O
+to	O
+other	O
+members	O
+of	O
+clan	O
+CD	O
+with	O
+the	O
+top	O
+hits	O
+in	O
+a	O
+search	O
+of	O
+known	O
+structures	O
+being	O
+caspase	O
+-	O
+7	O
+,	O
+gingipain	O
+-	O
+K	O
+,	O
+and	O
+legumain	O
+(	O
+PBD	O
+codes	O
+4hq0	O
+,	O
+4tkx	O
+,	O
+and	O
+4aw9	O
+,	O
+respectively	O
+)	O
+(	O
+Table	O
+2	O
+).	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+domain	O
+is	O
+unique	O
+to	O
+PmC11	O
+within	O
+clan	O
+CD	O
+and	O
+structure	O
+comparisons	O
+for	O
+this	O
+domain	O
+alone	O
+does	O
+not	O
+produce	O
+any	O
+hits	O
+in	O
+the	O
+PDB	O
+(	O
+DaliLite	O
+,	O
+PDBeFold	O
+),	O
+suggesting	O
+a	O
+completely	O
+novel	O
+fold	O
+.	O
+	O
+As	O
+the	O
+archetypal	O
+and	O
+arguably	O
+most	O
+well	O
+studied	O
+member	O
+of	O
+clan	O
+CD	O
+,	O
+the	O
+caspases	O
+were	O
+used	O
+as	O
+the	O
+basis	O
+to	O
+investigate	O
+the	O
+structure	O
+/	O
+function	O
+relationships	O
+in	O
+PmC11	O
+,	O
+with	O
+caspase	O
+-	O
+7	O
+as	O
+the	O
+representative	O
+member	O
+.	O
+	O
+Six	O
+of	O
+the	O
+central	O
+β	O
+-	O
+strands	O
+in	O
+PmC11	O
+(	O
+β1	O
+–	O
+β2	O
+and	O
+β5	O
+–	O
+β8	O
+)	O
+share	O
+the	O
+same	O
+topology	O
+as	O
+the	O
+six	O
+-	O
+stranded	O
+β	O
+-	O
+sheet	O
+found	O
+in	O
+caspases	O
+,	O
+with	O
+strands	O
+β3	O
+,	O
+β4	O
+,	O
+and	O
+β9	O
+located	O
+on	O
+the	O
+outside	O
+of	O
+this	O
+core	O
+structure	O
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+box	O
+).	O
+	O
+Summary	O
+of	O
+PDBeFOLD	O
+superposition	O
+of	O
+structures	O
+found	O
+to	O
+be	O
+most	O
+similar	O
+to	O
+PmC11	O
+in	O
+the	O
+PBD	O
+based	O
+on	O
+DaliLite	O
+	O
+Biochemical	O
+and	O
+structural	O
+characterization	O
+of	O
+PmC11	O
+.	O
+	O
+A	O
+,	O
+ribbon	O
+representation	O
+of	O
+the	O
+overall	O
+structure	O
+of	O
+PmC11	O
+illustrating	O
+the	O
+catalytic	O
+site	O
+,	O
+cleavage	O
+site	O
+displacement	O
+,	O
+and	O
+potential	O
+S1	O
+binding	O
+site	O
+.	O
+	O
+The	O
+overall	O
+structure	O
+of	O
+PmC11	O
+is	O
+shown	O
+in	O
+gray	O
+,	O
+looking	O
+down	O
+into	O
+the	O
+catalytic	O
+site	O
+with	O
+the	O
+catalytic	O
+dyad	O
+in	O
+red	O
+.	O
+	O
+The	O
+two	O
+ends	O
+of	O
+the	O
+autolytic	O
+cleavage	O
+site	O
+(	O
+Lys147	O
+and	O
+Ala148	O
+,	O
+green	O
+)	O
+are	O
+displaced	O
+by	O
+19	O
+.	O
+5	O
+Å	O
+(	O
+thin	O
+black	O
+line	O
+)	O
+from	O
+one	O
+another	O
+and	O
+residues	O
+in	O
+the	O
+potential	O
+substrate	O
+binding	O
+pocket	O
+are	O
+highlighted	O
+in	O
+blue	O
+.	O
+	O
+PmC11	O
+migrates	O
+as	O
+a	O
+monomer	O
+with	O
+a	O
+molecular	O
+mass	O
+around	O
+41	O
+kDa	O
+calculated	O
+from	O
+protein	O
+standards	O
+of	O
+known	O
+molecular	O
+weights	O
+.	O
+	O
+Elution	O
+fractions	O
+across	O
+the	O
+major	O
+peak	O
+(	O
+1	O
+–	O
+6	O
+)	O
+were	O
+analyzed	O
+by	O
+SDS	O
+-	O
+PAGE	O
+on	O
+a	O
+4	O
+–	O
+12	O
+%	O
+gel	O
+in	O
+MES	O
+buffer	O
+.	O
+	O
+C	O
+,	O
+the	O
+active	O
+form	O
+of	O
+PmC11	O
+and	O
+two	O
+mutants	O
+,	O
+PmC11C179A	B-mutant
+(	O
+C	O
+)	O
+and	O
+PmC11K147A	B-mutant
+(	O
+K	O
+),	O
+were	O
+examined	O
+by	O
+SDS	O
+-	O
+PAGE	O
+(	O
+lane	O
+1	O
+)	O
+and	O
+Western	O
+blot	O
+analysis	O
+using	O
+an	O
+anti	O
+-	O
+His	O
+antibody	O
+(	O
+lane	O
+2	O
+)	O
+show	O
+that	O
+PmC11	O
+autoprocesses	O
+,	O
+whereas	O
+mutants	O
+,	O
+PmC11C179A	B-mutant
+and	O
+PmC11K147A	B-mutant
+,	O
+do	O
+not	O
+show	O
+autoprocessing	O
+in	O
+vitro	O
+.	O
+	O
+Km	O
+and	O
+Vmax	O
+of	O
+PmC11	O
+and	O
+K147A	B-mutant
+mutant	O
+were	O
+determined	O
+by	O
+monitoring	O
+change	O
+in	O
+the	O
+fluorescence	O
+corresponding	O
+to	O
+AMC	O
+release	O
+from	O
+Bz	O
+-	O
+R	O
+-	O
+AMC	O
+.	O
+	O
+E	O
+,	O
+intermolecular	O
+processing	O
+of	O
+PmC11C179A	B-mutant
+by	O
+PmC11	O
+.	O
+	O
+PmC11C179A	O
+(	O
+20	O
+μg	O
+)	O
+was	O
+incubated	O
+overnight	O
+at	O
+37	O
+°	O
+C	O
+with	O
+increasing	O
+amounts	O
+of	O
+processed	O
+PmC11	O
+and	O
+analyzed	O
+on	O
+a	O
+10	O
+%	O
+SDS	O
+-	O
+PAGE	O
+gel	O
+.	O
+	O
+A	O
+single	O
+lane	O
+of	O
+20	O
+μg	O
+of	O
+active	O
+PmC11	O
+(	O
+labeled	O
+20	O
+)	O
+is	O
+shown	O
+for	O
+comparison	O
+.	O
+	O
+The	O
+position	O
+of	O
+the	O
+catalytic	O
+dyad	O
+,	O
+one	O
+potential	O
+key	O
+substrate	O
+binding	O
+residue	O
+Asp177	O
+,	O
+and	O
+the	O
+ends	O
+of	O
+the	O
+cleavage	O
+site	O
+Lys147	O
+and	O
+Ala148	O
+are	O
+indicated	O
+.	O
+	O
+Five	O
+of	O
+the	O
+α	O
+-	O
+helices	O
+surrounding	O
+the	O
+β	O
+-	O
+sheet	O
+of	O
+PmC11	O
+(	O
+α1	O
+,	O
+α2	O
+,	O
+α4	O
+,	O
+α6	O
+,	O
+and	O
+α7	O
+)	O
+are	O
+found	O
+in	O
+similar	O
+positions	O
+to	O
+the	O
+five	O
+structurally	O
+conserved	O
+helices	O
+in	O
+caspases	O
+and	O
+other	O
+members	O
+of	O
+clan	O
+CD	O
+,	O
+apart	O
+from	O
+family	O
+C80	O
+.	O
+	O
+Other	O
+than	O
+its	O
+more	O
+extended	O
+β	O
+-	O
+sheet	O
+,	O
+PmC11	O
+differs	O
+most	O
+significantly	O
+from	O
+other	O
+clan	O
+CD	O
+members	O
+at	O
+its	O
+C	O
+terminus	O
+,	O
+where	O
+the	O
+CTD	O
+contains	O
+a	O
+further	O
+seven	O
+α	O
+-	O
+helices	O
+and	O
+four	O
+β	O
+-	O
+strands	O
+after	O
+β8	O
+.	O
+	O
+Autoprocessing	O
+of	O
+PmC11	O
+	O
+Purification	O
+of	O
+recombinant	O
+PmC11	O
+(	O
+molecular	O
+mass	O
+=	O
+42	O
+.	O
+6	O
+kDa	O
+)	O
+revealed	O
+partial	O
+processing	O
+into	O
+two	O
+cleavage	O
+products	O
+of	O
+26	O
+.	O
+4	O
+and	O
+16	O
+.	O
+2	O
+kDa	O
+,	O
+related	O
+to	O
+the	O
+observed	O
+cleavage	O
+at	O
+Lys147	O
+in	O
+the	O
+crystal	O
+structure	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+	O
+The	O
+single	O
+cleavage	O
+site	O
+of	O
+PmC11	O
+at	O
+Lys147	O
+is	O
+found	O
+immediately	O
+after	O
+α3	O
+,	O
+in	O
+loop	O
+L5	O
+within	O
+the	O
+central	O
+β	O
+-	O
+sheet	O
+(	O
+Figs	O
+.	O
+1	O
+,	O
+A	O
+and	O
+B	O
+,	O
+and	O
+2A	O
+).	O
+	O
+The	O
+two	O
+ends	O
+of	O
+the	O
+cleavage	O
+site	O
+are	O
+remarkably	O
+well	O
+ordered	O
+in	O
+the	O
+crystal	O
+structure	O
+and	O
+displaced	O
+from	O
+one	O
+another	O
+by	O
+19	O
+.	O
+5	O
+Å	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+	O
+Moreover	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+side	O
+of	O
+the	O
+cleavage	O
+site	O
+resides	O
+near	O
+the	O
+catalytic	O
+dyad	O
+with	O
+Ala148	O
+being	O
+4	O
+.	O
+5	O
+and	O
+5	O
+.	O
+7	O
+Å	O
+from	O
+His133	O
+and	O
+Cys179	O
+,	O
+respectively	O
+.	O
+	O
+Consequently	O
+,	O
+it	O
+appears	O
+feasible	O
+that	O
+the	O
+helix	O
+attached	O
+to	O
+Lys147	O
+(	O
+α3	O
+)	O
+could	O
+be	O
+responsible	O
+for	O
+steric	O
+autoinhibition	O
+of	O
+PmC11	O
+when	O
+Lys147	O
+is	O
+covalently	O
+bonded	O
+to	O
+Ala148	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+cleavage	O
+would	O
+be	O
+required	O
+for	O
+full	O
+activation	O
+of	O
+PmC11	O
+.	O
+	O
+To	O
+investigate	O
+this	O
+possibility	O
+,	O
+two	O
+mutant	O
+forms	O
+of	O
+the	O
+enzyme	O
+were	O
+created	O
+:	O
+PmC11C179A	B-mutant
+(	O
+a	O
+catalytically	O
+inactive	O
+mutant	O
+)	O
+and	O
+PmC11K147A	B-mutant
+(	O
+a	O
+cleavage	O
+-	O
+site	O
+mutant	O
+).	O
+	O
+Initial	O
+SDS	O
+-	O
+PAGE	O
+and	O
+Western	O
+blot	O
+analysis	O
+of	O
+both	O
+mutants	O
+revealed	O
+no	O
+discernible	O
+processing	O
+occurred	O
+as	O
+compared	O
+with	O
+active	O
+PmC11	O
+(	O
+Fig	O
+.	O
+2C	O
+).	O
+	O
+The	O
+PmC11K147A	B-mutant
+mutant	O
+enzyme	O
+had	O
+a	O
+markedly	O
+different	O
+reaction	O
+rate	O
+(	O
+Vmax	O
+)	O
+compared	O
+with	O
+WT	O
+,	O
+where	O
+the	O
+reaction	O
+velocity	O
+of	O
+PmC11	O
+was	O
+10	O
+times	O
+greater	O
+than	O
+that	O
+of	O
+PmC11K147A	B-mutant
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+	O
+Taken	O
+together	O
+,	O
+these	O
+data	O
+reveal	O
+that	O
+PmC11	O
+requires	O
+processing	O
+at	O
+Lys147	O
+for	O
+optimum	O
+activity	O
+.	O
+	O
+To	O
+investigate	O
+whether	O
+processing	O
+is	O
+a	O
+result	O
+of	O
+intra	O
+-	O
+or	O
+intermolecular	O
+cleavage	O
+,	O
+the	O
+PmC11C179A	B-mutant
+mutant	O
+was	O
+incubated	O
+with	O
+increasing	O
+concentrations	O
+of	O
+processed	O
+and	O
+activated	O
+PmC11	O
+.	O
+	O
+These	O
+studies	O
+revealed	O
+that	O
+there	O
+was	O
+no	O
+apparent	O
+cleavage	O
+of	O
+PmC11C179A	B-mutant
+by	O
+the	O
+active	O
+enzyme	O
+at	O
+low	O
+concentrations	O
+of	O
+PmC11	O
+and	O
+that	O
+only	O
+limited	O
+cleavage	O
+was	O
+observed	O
+when	O
+the	O
+ratio	O
+of	O
+active	O
+enzyme	O
+(	O
+PmC11	O
+:	O
+PmC11C179A	B-mutant
+)	O
+was	O
+increased	O
+to	O
+∼	O
+1	O
+:	O
+10	O
+and	O
+1	O
+:	O
+4	O
+,	O
+with	O
+complete	O
+cleavage	O
+observed	O
+at	O
+a	O
+ratio	O
+of	O
+1	O
+:	O
+1	O
+(	O
+Fig	O
+.	O
+2E	O
+).	O
+	O
+This	O
+suggests	O
+that	O
+cleavage	O
+of	O
+PmC11C179A	B-mutant
+was	O
+most	O
+likely	O
+an	O
+effect	O
+of	O
+the	O
+increasing	O
+concentration	O
+of	O
+PmC11	O
+and	O
+intermolecular	O
+cleavage	O
+.	O
+	O
+Collectively	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+the	O
+pro	O
+-	O
+form	O
+of	O
+PmC11	O
+is	O
+autoinhibited	O
+by	O
+a	O
+section	O
+of	O
+L5	O
+blocking	O
+access	O
+to	O
+the	O
+active	O
+site	O
+,	O
+prior	O
+to	O
+intramolecular	O
+cleavage	O
+at	O
+Lys147	O
+.	O
+	O
+This	O
+cleavage	O
+subsequently	O
+allows	O
+movement	O
+of	O
+the	O
+region	O
+containing	O
+Lys147	O
+and	O
+the	O
+active	O
+site	O
+to	O
+open	O
+up	O
+for	O
+substrate	O
+access	O
+.	O
+	O
+Substrate	O
+Specificity	O
+of	O
+PmC11	O
+	O
+The	O
+autocatalytic	O
+cleavage	O
+of	O
+PmC11	O
+at	O
+Lys147	O
+(	O
+sequence	O
+KLK	O
+∧	O
+A	O
+)	O
+demonstrates	O
+that	O
+the	O
+enzyme	O
+accepts	O
+substrates	O
+with	O
+Lys	O
+in	O
+the	O
+P1	O
+position	O
+.	O
+	O
+The	O
+catalytic	O
+dyad	O
+of	O
+PmC11	O
+sits	O
+near	O
+the	O
+bottom	O
+of	O
+an	O
+open	O
+pocket	O
+on	O
+the	O
+surface	O
+of	O
+the	O
+enzyme	O
+at	O
+a	O
+conserved	O
+location	O
+in	O
+the	O
+clan	O
+CD	O
+family	O
+.	O
+	O
+The	O
+PmC11	O
+structure	O
+reveals	O
+that	O
+the	O
+catalytic	O
+dyad	O
+forms	O
+part	O
+of	O
+a	O
+large	O
+acidic	O
+pocket	O
+(	O
+Fig	O
+.	O
+2G	O
+),	O
+consistent	O
+with	O
+a	O
+binding	O
+site	O
+for	O
+a	O
+basic	O
+substrate	O
+.	O
+	O
+This	O
+pocket	O
+is	O
+lined	O
+with	O
+the	O
+potential	O
+functional	O
+side	O
+chains	O
+of	O
+Asn50	O
+,	O
+Asp177	O
+,	O
+and	O
+Thr204	O
+with	O
+Gly134	O
+,	O
+Asp207	O
+,	O
+and	O
+Met205	O
+also	O
+contributing	O
+to	O
+the	O
+pocket	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+	O
+Interestingly	O
+,	O
+these	O
+residues	O
+are	O
+in	O
+regions	O
+that	O
+are	O
+structurally	O
+similar	O
+to	O
+those	O
+involved	O
+in	O
+the	O
+S1	O
+binding	O
+pockets	O
+of	O
+other	O
+clan	O
+CD	O
+members	O
+(	O
+shown	O
+in	O
+Ref	O
+.).	O
+	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+was	O
+also	O
+shown	O
+to	O
+bind	O
+to	O
+the	O
+enzyme	O
+:	O
+a	O
+size	O
+-	O
+shift	O
+was	O
+observed	O
+,	O
+by	O
+SDS	O
+-	O
+PAGE	O
+analysis	O
+,	O
+in	O
+the	O
+larger	O
+processed	O
+product	O
+of	O
+PmC11	O
+suggesting	O
+that	O
+the	O
+inhibitor	O
+bound	O
+to	O
+the	O
+active	O
+site	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+	O
+A	O
+structure	O
+overlay	O
+of	O
+PmC11	O
+with	O
+the	O
+MALT1	O
+-	O
+paracacaspase	O
+(	O
+MALT1	O
+-	O
+P	O
+),	O
+in	O
+complex	O
+with	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+,	O
+revealed	O
+that	O
+the	O
+PmC11	O
+dyad	O
+sits	O
+in	O
+a	O
+very	O
+similar	O
+position	O
+to	O
+that	O
+of	O
+active	O
+MALT1	O
+-	O
+P	O
+and	O
+that	O
+Asn50	O
+,	O
+Asp177	O
+,	O
+and	O
+Asp207	O
+superimpose	O
+well	O
+with	O
+the	O
+principal	O
+MALT1	O
+-	O
+P	O
+inhibitor	O
+binding	O
+residues	O
+(	O
+Asp365	O
+,	O
+Asp462	O
+,	O
+and	O
+Glu500	O
+,	O
+respectively	O
+(	O
+VRPR	O
+-	O
+FMK	O
+from	O
+MALT1	O
+-	O
+P	O
+with	O
+the	O
+corresponding	O
+PmC11	O
+residues	O
+from	O
+the	O
+structural	O
+overlay	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+1D	O
+),	O
+as	O
+described	O
+in	O
+Ref	O
+.).	O
+	O
+However	O
+,	O
+this	O
+loop	O
+has	O
+been	O
+shown	O
+to	O
+be	O
+important	O
+for	O
+substrate	O
+binding	O
+in	O
+clan	O
+CD	O
+and	O
+this	O
+residue	O
+could	O
+easily	O
+rotate	O
+and	O
+be	O
+involved	O
+in	O
+substrate	O
+binding	O
+in	O
+PmC11	O
+.	O
+	O
+Thus	O
+,	O
+Asn50	O
+,	O
+Asp177	O
+,	O
+and	O
+Asp207	O
+are	O
+most	O
+likely	O
+responsible	O
+for	O
+the	O
+substrate	O
+specificity	O
+of	O
+PmC11	O
+.	O
+	O
+PmC11	O
+binds	O
+and	O
+is	O
+inhibited	O
+by	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+and	O
+does	O
+not	O
+require	O
+Ca2	O
++	O
+for	O
+activity	O
+.	O
+	O
+Cleavage	O
+of	O
+Bz	O
+-	O
+R	O
+-	O
+AMC	O
+by	O
+PmC11	O
+was	O
+measured	O
+in	O
+a	O
+fluorometric	O
+activity	O
+assay	O
+with	O
+(+,	O
+purple	O
+)	O
+and	O
+without	O
+(−,	O
+red	O
+)	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+.	O
+	O
+PmC11	O
+was	O
+incubated	O
+with	O
+(+)	O
+or	O
+without	O
+(−)	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+and	O
+the	O
+samples	O
+analyzed	O
+on	O
+a	O
+10	O
+%	O
+SDS	O
+-	O
+PAGE	O
+gel	O
+.	O
+	O
+A	O
+size	O
+shift	O
+can	O
+be	O
+observed	O
+in	O
+the	O
+larger	O
+processed	O
+product	O
+of	O
+PmC11	O
+(	O
+26	O
+.	O
+1	O
+kDa	O
+).	O
+	O
+C	O
+,	O
+PmC11	O
+with	O
+the	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+from	O
+the	O
+MALT1	O
+-	O
+paracacaspase	O
+(	O
+MALT1	O
+-	O
+P	O
+)	O
+superimposed	O
+.	O
+	O
+A	O
+three	O
+-	O
+dimensional	O
+structural	O
+overlay	O
+of	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+from	O
+the	O
+MALT1	O
+-	O
+P	O
+complex	O
+onto	O
+PmC11	O
+.	O
+	O
+Residues	O
+surrounding	O
+the	O
+inhibitor	O
+are	O
+labeled	O
+and	O
+represent	O
+potentially	O
+important	O
+binding	O
+site	O
+residues	O
+,	O
+labeled	O
+in	O
+black	O
+and	O
+shown	O
+in	O
+an	O
+atomic	O
+representation	O
+.	O
+	O
+Furthermore	O
+,	O
+Cu2	O
++,	O
+Fe2	O
++,	O
+and	O
+Zn2	O
++	O
+appear	O
+to	O
+inhibit	O
+PmC11	O
+.	O
+	O
+Comparison	O
+with	O
+Clostripain	O
+	O
+Clostripain	O
+from	O
+C	O
+.	O
+histolyticum	O
+is	O
+the	O
+founding	O
+member	O
+of	O
+the	O
+C11	O
+family	O
+of	O
+peptidases	O
+and	O
+contains	O
+an	O
+additional	O
+149	O
+residues	O
+compared	O
+with	O
+PmC11	O
+.	O
+	O
+A	O
+multiple	O
+sequence	O
+alignment	O
+revealed	O
+that	O
+most	O
+of	O
+the	O
+secondary	O
+structural	O
+elements	O
+are	O
+conserved	O
+between	O
+the	O
+two	O
+enzymes	O
+,	O
+although	O
+they	O
+are	O
+only	O
+∼	O
+23	O
+%	O
+identical	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+	O
+Nevertheless	O
+,	O
+PmC11	O
+may	O
+be	O
+a	O
+good	O
+model	O
+for	O
+the	O
+core	O
+structure	O
+of	O
+clostripain	O
+.	O
+	O
+In	O
+addition	O
+,	O
+the	O
+predicted	O
+primary	O
+S1	O
+-	O
+binding	O
+residue	O
+in	O
+PmC11	O
+Asp177	O
+also	O
+overlays	O
+with	O
+the	O
+residue	O
+predicted	O
+to	O
+be	O
+the	O
+P1	O
+specificity	O
+determining	O
+residue	O
+in	O
+clostripain	O
+(	O
+Asp229	O
+,	O
+Fig	O
+.	O
+1A	O
+).	O
+	O
+Surprisingly	O
+,	O
+Ca2	O
++	O
+did	O
+not	O
+enhance	O
+PmC11	O
+activity	O
+and	O
+,	O
+furthermore	O
+,	O
+other	O
+divalent	O
+cations	O
+,	O
+Mg2	O
++,	O
+Mn2	O
++,	O
+Co2	O
++,	O
+Fe2	O
++,	O
+Zn2	O
++,	O
+and	O
+Cu2	O
++,	O
+were	O
+not	O
+necessary	O
+for	O
+PmC11	O
+activity	O
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+PmC11	O
+now	O
+provides	O
+three	O
+-	O
+dimensional	O
+information	O
+for	O
+a	O
+member	O
+of	O
+the	O
+clostripain	O
+C11	O
+family	O
+of	O
+cysteine	O
+peptidases	O
+.	O
+	O
+The	O
+structural	O
+similarity	O
+of	O
+PmC11	O
+with	O
+its	O
+nearest	O
+structural	O
+neighbors	O
+in	O
+the	O
+PDB	O
+is	O
+decidedly	O
+low	O
+,	O
+overlaying	O
+better	O
+with	O
+six	O
+-	O
+stranded	O
+caspase	O
+-	O
+7	O
+than	O
+any	O
+of	O
+the	O
+other	O
+larger	O
+members	O
+of	O
+the	O
+clan	O
+(	O
+Table	O
+2	O
+).	O
+	O
+The	O
+substrate	O
+specificity	O
+of	O
+PmC11	O
+is	O
+Arg	O
+/	O
+Lys	O
+and	O
+the	O
+crystal	O
+structure	O
+revealed	O
+an	O
+acidic	O
+pocket	O
+for	O
+specific	O
+binding	O
+of	O
+such	O
+basic	O
+substrates	O
+.	O
+	O
+PmC11	O
+differs	O
+from	O
+clostripain	O
+in	O
+that	O
+is	O
+does	O
+not	O
+appear	O
+to	O
+require	O
+divalent	O
+cations	O
+for	O
+activation	O
+.	O
+	O
+To	O
+date	O
+,	O
+the	O
+effector	O
+caspases	O
+are	O
+the	O
+only	O
+group	O
+of	O
+enzymes	O
+that	O
+require	O
+cleavage	O
+of	O
+a	O
+loop	O
+within	O
+the	O
+central	O
+β	O
+-	O
+sheet	O
+.	O
+	O
+All	O
+other	O
+clan	O
+CD	O
+members	O
+requiring	O
+cleavage	O
+for	O
+full	O
+activation	O
+do	O
+so	O
+at	O
+sites	O
+external	O
+to	O
+their	O
+central	O
+sheets	O
+.	O
+	O
+In	O
+addition	O
+,	O
+several	O
+members	O
+of	O
+clan	O
+CD	O
+exhibit	O
+self	O
+-	O
+inhibition	O
+,	O
+whereby	O
+regions	O
+of	O
+the	O
+enzyme	O
+block	O
+access	O
+to	O
+the	O
+active	O
+site	O
+.	O
+	O
+The	O
+structure	O
+of	O
+PmC11	O
+gives	O
+the	O
+first	O
+insight	O
+into	O
+this	O
+class	O
+of	O
+relatively	O
+unexplored	O
+family	O
+of	O
+proteins	O
+and	O
+should	O
+allow	O
+important	O
+catalytic	O
+and	O
+substrate	O
+binding	O
+residues	O
+to	O
+be	O
+identified	O
+in	O
+a	O
+variety	O
+of	O
+orthologues	O
+.	O
+	O
+Ribosome	O
+biogenesis	O
+factor	O
+Tsr3	O
+is	O
+the	O
+aminocarboxypropyl	O
+transferase	O
+responsible	O
+for	O
+18S	O
+rRNA	O
+hypermodification	O
+in	O
+yeast	O
+and	O
+humans	O
+	O
+Here	O
+we	O
+identify	O
+the	O
+cytoplasmic	O
+ribosome	O
+biogenesis	O
+protein	O
+Tsr3	O
+as	O
+the	O
+responsible	O
+enzyme	O
+in	O
+yeast	O
+and	O
+human	O
+cells	O
+.	O
+	O
+In	O
+functionally	O
+impaired	O
+Tsr3	O
+-	O
+mutants	O
+,	O
+a	O
+reduced	O
+level	O
+of	O
+acp	O
+modification	O
+directly	O
+correlates	O
+with	O
+increased	O
+20S	O
+pre	O
+-	O
+rRNA	O
+accumulation	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+archaeal	O
+Tsr3	O
+homologs	O
+revealed	O
+the	O
+same	O
+fold	O
+as	O
+in	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+-	O
+methyltransferases	O
+but	O
+a	O
+distinct	O
+SAM	O
+binding	O
+mode	O
+.	O
+	O
+This	O
+unique	O
+SAM	O
+binding	O
+mode	O
+explains	O
+why	O
+Tsr3	O
+transfers	O
+the	O
+acp	O
+and	O
+not	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	O
+to	O
+its	O
+substrate	O
+.	O
+	O
+Structurally	O
+,	O
+Tsr3	O
+therefore	O
+represents	O
+a	O
+novel	O
+class	O
+of	O
+acp	O
+transferase	O
+enzymes	O
+.	O
+	O
+Eukaryotic	O
+ribosome	O
+biogenesis	O
+is	O
+highly	O
+complex	O
+and	O
+requires	O
+a	O
+large	O
+number	O
+of	O
+non	O
+-	O
+ribosomal	O
+proteins	O
+and	O
+small	O
+non	O
+-	O
+coding	O
+RNAs	O
+in	O
+addition	O
+to	O
+ribosomal	O
+RNAs	O
+(	O
+rRNAs	O
+)	O
+and	O
+proteins	O
+.	O
+	O
+During	O
+eukaryotic	O
+ribosome	O
+biogenesis	O
+several	O
+dozens	O
+of	O
+rRNA	O
+nucleotides	O
+become	O
+chemically	O
+modified	O
+.	O
+	O
+The	O
+most	O
+abundant	O
+rRNA	O
+modifications	O
+are	O
+methylations	O
+at	O
+the	O
+2	O
+′-	O
+OH	O
+ribose	O
+moieties	O
+and	O
+isomerizations	O
+of	O
+uridine	O
+residues	O
+to	O
+pseudouridine	O
+,	O
+catalyzed	O
+by	O
+small	O
+nucleolar	O
+ribonucleoprotein	O
+particles	O
+(	O
+snoRNPs	O
+).	O
+	O
+Ribosomal	O
+RNA	O
+modifications	O
+have	O
+been	O
+suggested	O
+to	O
+optimize	O
+ribosome	O
+function	O
+,	O
+although	O
+in	O
+most	O
+cases	O
+this	O
+remains	O
+to	O
+be	O
+clearly	O
+established	O
+.	O
+	O
+Most	O
+modified	O
+rRNA	O
+nucleotides	O
+cluster	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+decoding	O
+or	O
+the	O
+peptidyl	O
+transferase	O
+center	O
+,	O
+suggesting	O
+an	O
+influence	O
+on	O
+ribosome	O
+functionality	O
+and	O
+stability	O
+.	O
+	O
+The	O
+chemically	O
+most	O
+complex	O
+modification	O
+is	O
+located	O
+in	O
+the	O
+loop	O
+capping	O
+helix	O
+31	O
+of	O
+18S	O
+rRNA	O
+(	O
+Supplementary	O
+Figure	O
+S1B	O
+).	O
+	O
+There	O
+a	O
+uridine	O
+(	O
+U1191	O
+in	O
+yeast	O
+)	O
+is	O
+modified	O
+to	O
+1	O
+-	O
+methyl	O
+-	O
+3	O
+-(	O
+3	O
+-	O
+amino	O
+-	O
+3	O
+-	O
+carboxypropyl	O
+)-	O
+pseudouridine	O
+(	O
+m1acp3Ψ	O
+,	O
+Figure	O
+1A	O
+).	O
+	O
+This	O
+base	O
+modification	O
+was	O
+first	O
+described	O
+in	O
+1968	O
+for	O
+hamster	O
+cells	O
+and	O
+is	O
+conserved	O
+in	O
+eukaryotes	O
+.	O
+	O
+This	O
+hypermodified	O
+nucleotide	O
+,	O
+which	O
+is	O
+located	O
+at	O
+the	O
+P	O
+-	O
+site	O
+tRNA	O
+,	O
+is	O
+synthesized	O
+in	O
+three	O
+steps	O
+beginning	O
+with	O
+the	O
+snR35	O
+H	O
+/	O
+ACA	O
+snoRNP	O
+guided	O
+conversion	O
+of	O
+uridine	O
+into	O
+pseudouridine	O
+.	O
+	O
+Methylation	O
+can	O
+only	O
+occur	O
+once	O
+pseudouridylation	O
+has	O
+taken	O
+place	O
+,	O
+as	O
+the	O
+latter	O
+reaction	O
+generates	O
+the	O
+substrate	O
+for	O
+the	O
+former	O
+.	O
+	O
+The	O
+final	O
+acp	O
+modification	O
+leading	O
+to	O
+N1	O
+-	O
+methyl	O
+-	O
+N3	O
+-	O
+aminocarboxypropyl	O
+-	O
+pseudouridine	O
+occurs	O
+late	O
+during	O
+40S	O
+biogenesis	O
+in	O
+the	O
+cytoplasm	O
+,	O
+while	O
+the	O
+two	O
+former	O
+reactions	O
+are	O
+taking	O
+place	O
+in	O
+the	O
+nucleolus	O
+and	O
+nucleus	O
+,	O
+and	O
+is	O
+independent	O
+from	O
+pseudouridylation	O
+or	O
+methylation	O
+.	O
+	O
+Both	O
+the	O
+methyl	O
+and	O
+the	O
+acp	O
+group	O
+are	O
+derived	O
+from	O
+S	O
+-	O
+adenosylmethionine	O
+(	O
+SAM	O
+),	O
+but	O
+the	O
+enzyme	O
+responsible	O
+for	O
+acp	O
+modification	O
+remained	O
+elusive	O
+for	O
+more	O
+than	O
+40	O
+years	O
+.	O
+	O
+Tsr3	O
+is	O
+necessary	O
+for	O
+acp	O
+modification	O
+of	O
+18S	O
+rRNA	O
+in	O
+yeast	O
+and	O
+human	O
+.	O
+(	O
+A	O
+)	O
+Hypermodified	O
+nucleotide	O
+m1acp3Ψ	O
+is	O
+synthesized	O
+in	O
+three	O
+steps	O
+:	O
+pseudouridylation	O
+catalyzed	O
+by	O
+snoRNP35	O
+,	O
+N1	O
+-	O
+methylation	O
+catalyzed	O
+by	O
+methyltransferase	O
+Nep1	O
+and	O
+N3	O
+-	O
+acp	O
+modification	O
+catalyzed	O
+by	O
+Tsr3	O
+.	O
+	O
+(	O
+B	O
+)	O
+RP	O
+-	O
+HPLC	O
+elution	O
+profile	O
+of	O
+yeast	O
+18S	O
+rRNA	O
+nucleosides	O
+.	O
+	O
+Wild	O
+type	O
+(	O
+WT	O
+)	O
+and	O
+plasmid	O
+encoded	O
+18S	O
+rRNA	O
+(	O
+U1191U	B-mutant
+)	O
+show	O
+the	O
+14C	O
+-	O
+acp	O
+signal	O
+,	O
+whereas	O
+the	O
+14C	O
+-	O
+acp	O
+signal	O
+is	O
+missing	O
+in	O
+the	O
+U1191A	B-mutant
+mutant	O
+plasmid	O
+encoded	O
+18S	O
+rRNA	O
+(	O
+U1191A	B-mutant
+)	O
+and	O
+Δtsr3	B-mutant
+mutants	O
+(	O
+Δtsr3	B-mutant
+).	O
+	O
+All	O
+samples	O
+were	O
+loaded	O
+on	O
+the	O
+gel	O
+with	O
+two	O
+different	O
+amounts	O
+of	O
+5	O
+and	O
+10	O
+μl	O
+.	O
+(	O
+D	O
+)	O
+Primer	O
+extension	O
+analysis	O
+of	O
+acp	O
+modification	O
+in	O
+yeast	O
+18S	O
+rRNA	O
+(	O
+right	O
+gel	O
+)	O
+including	O
+a	O
+sequencing	O
+ladder	O
+(	O
+left	O
+gel	O
+).	O
+	O
+The	O
+primer	O
+extension	O
+stop	O
+at	O
+nucleotide	O
+1191	O
+is	O
+missing	O
+exclusively	O
+in	O
+Δtsr3	B-mutant
+mutants	O
+and	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+recombinants	O
+.	O
+	O
+(	O
+E	O
+)	O
+Primer	O
+extension	O
+analysis	O
+of	O
+human	O
+18S	O
+rRNA	O
+after	O
+siRNA	O
+knockdown	O
+of	O
+HsNEP1	O
+/	O
+EMG1	O
+(	O
+541	O
+,	O
+542	O
+and	O
+543	O
+)	O
+and	O
+HsTSR3	O
+(	O
+544	O
+and	O
+545	O
+)	O
+(	O
+right	O
+gel	O
+),	O
+including	O
+a	O
+sequencing	O
+ladder	O
+(	O
+left	O
+gel	O
+).	O
+	O
+Only	O
+a	O
+few	O
+acp	O
+transferring	O
+enzymes	O
+have	O
+been	O
+characterized	O
+until	O
+now	O
+.	O
+	O
+During	O
+the	O
+biosynthesis	O
+of	O
+wybutosine	O
+,	O
+a	O
+tricyclic	O
+nucleoside	O
+present	O
+in	O
+eukaryotic	O
+and	O
+archaeal	O
+phenylalanine	O
+tRNA	O
+,	O
+Tyw2	O
+(	O
+Trm12	O
+in	O
+yeast	O
+)	O
+transfers	O
+an	O
+acp	O
+group	O
+from	O
+SAM	O
+to	O
+an	O
+acidic	O
+carbon	O
+atom	O
+.	O
+	O
+Archaeal	O
+Tyw2	O
+has	O
+a	O
+structure	O
+very	O
+similar	O
+to	O
+Rossmann	O
+-	O
+fold	O
+(	O
+class	O
+I	O
+)	O
+RNA	O
+-	O
+methyltransferases	O
+,	O
+but	O
+its	O
+distinctive	O
+SAM	O
+-	O
+binding	O
+mode	O
+enables	O
+the	O
+transfer	O
+of	O
+the	O
+acp	O
+group	O
+instead	O
+of	O
+the	O
+methyl	O
+group	O
+of	O
+the	O
+cofactor	O
+.	O
+	O
+Another	O
+acp	O
+modification	O
+has	O
+been	O
+described	O
+in	O
+the	O
+diphtamide	O
+biosynthesis	O
+pathway	O
+,	O
+where	O
+an	O
+acp	O
+group	O
+is	O
+transferred	O
+from	O
+SAM	O
+to	O
+the	O
+carbon	O
+atom	O
+of	O
+a	O
+histidine	O
+residue	O
+of	O
+eukaryotic	O
+translation	O
+elongation	O
+factor	O
+2	O
+by	O
+use	O
+of	O
+a	O
+radical	O
+mechanism	O
+.	O
+	O
+In	O
+a	O
+recent	O
+bioinformatic	O
+study	O
+,	O
+the	O
+uncharacterized	O
+yeast	O
+gene	O
+YOR006c	O
+was	O
+predicted	O
+to	O
+be	O
+involved	O
+in	O
+ribosome	O
+biogenesis	O
+.	O
+	O
+It	O
+is	O
+highly	O
+conserved	O
+among	O
+eukaryotes	O
+and	O
+archaea	O
+(	O
+Supplementary	O
+Figure	O
+S1A	O
+)	O
+and	O
+its	O
+deletion	O
+leads	O
+to	O
+an	O
+accumulation	O
+of	O
+the	O
+20S	O
+pre	O
+-	O
+rRNA	O
+precursor	O
+of	O
+18S	O
+rRNA	O
+,	O
+suggesting	O
+an	O
+influence	O
+on	O
+D	O
+-	O
+site	O
+cleavage	O
+during	O
+the	O
+maturation	O
+of	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+.	O
+	O
+On	O
+this	O
+basis	O
+,	O
+YOR006C	O
+was	O
+renamed	O
+‘	O
+Twenty	O
+S	O
+rRNA	O
+accumulation	O
+3	O
+′	O
+(	O
+TSR3	O
+).	O
+	O
+However	O
+,	O
+its	O
+function	O
+remained	O
+unclear	O
+although	O
+recently	O
+a	O
+putative	O
+nuclease	O
+function	O
+during	O
+18S	O
+rRNA	O
+maturation	O
+was	O
+predicted	O
+.	O
+	O
+Here	O
+,	O
+we	O
+identify	O
+Tsr3	O
+as	O
+the	O
+long	O
+-	O
+sought	O
+acp	O
+transferase	O
+that	O
+catalyzes	O
+the	O
+last	O
+step	O
+in	O
+the	O
+biosynthesis	O
+of	O
+the	O
+hypermodified	O
+nucleotide	O
+m1acp3Ψ	O
+in	O
+yeast	O
+and	O
+human	O
+cells	O
+.	O
+	O
+Furthermore	O
+using	O
+catalytically	O
+defective	O
+mutants	O
+of	O
+yeast	O
+Tsr3	O
+we	O
+demonstrated	O
+that	O
+the	O
+acp	O
+modification	O
+is	O
+required	O
+for	O
+18S	O
+rRNA	O
+maturation	O
+.	O
+	O
+Surprisingly	O
+,	O
+the	O
+crystal	O
+structures	O
+of	O
+archaeal	O
+homologs	O
+revealed	O
+that	O
+Tsr3	O
+is	O
+structurally	O
+similar	O
+to	O
+the	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+methyltransferases	O
+.	O
+	O
+Interestingly	O
+,	O
+the	O
+two	O
+structurally	O
+very	O
+different	O
+enzymes	O
+use	O
+similar	O
+strategies	O
+in	O
+binding	O
+the	O
+SAM	O
+-	O
+cofactor	O
+in	O
+order	O
+to	O
+ensure	O
+that	O
+in	O
+contrast	O
+to	O
+methyltransferases	O
+the	O
+acp	O
+and	O
+not	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	O
+is	O
+transferred	O
+to	O
+the	O
+substrate	O
+.	O
+	O
+Tsr3	O
+is	O
+the	O
+enzyme	O
+responsible	O
+for	O
+18S	O
+rRNA	O
+acp	O
+modification	O
+in	O
+yeast	O
+and	O
+humans	O
+	O
+The	O
+S	O
+.	O
+cerevisiae	O
+18S	O
+rRNA	O
+acp	O
+transferase	O
+was	O
+identified	O
+in	O
+a	O
+systematic	O
+genetic	O
+screen	O
+where	O
+numerous	O
+deletion	O
+mutants	O
+from	O
+the	O
+EUROSCARF	O
+strain	O
+collection	O
+(	O
+www	O
+.	O
+euroscarf	O
+.	O
+de	O
+)	O
+were	O
+analyzed	O
+by	O
+HPLC	O
+for	O
+alterations	O
+in	O
+18S	O
+rRNA	O
+base	O
+modifications	O
+.	O
+	O
+For	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+strain	O
+the	O
+HPLC	O
+elution	O
+profile	O
+of	O
+18S	O
+rRNA	O
+nucleosides	O
+(	O
+Figure	O
+1B	O
+)	O
+was	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+pseudouridine	O
+-	O
+N1	O
+methyltransferase	O
+mutant	O
+Δnep1	B-mutant
+,	O
+where	O
+a	O
+shoulder	O
+at	O
+∼	O
+7	O
+.	O
+4	O
+min	O
+elution	O
+time	O
+was	O
+missing	O
+in	O
+the	O
+elution	O
+profile	O
+.	O
+	O
+In	O
+order	O
+to	O
+directly	O
+analyze	O
+the	O
+presence	O
+of	O
+the	O
+acp	O
+modification	O
+of	O
+nucleotide	O
+1191	O
+we	O
+used	O
+an	O
+in	O
+vivo14C	O
+incorporation	O
+assay	O
+with	O
+1	O
+-	O
+14C	O
+-	O
+methionine	O
+.	O
+	O
+No	O
+radioactive	O
+labeling	O
+was	O
+detected	O
+in	O
+the	O
+18S	B-mutant
+U1191A	I-mutant
+mutant	O
+which	O
+served	O
+as	O
+a	O
+control	O
+for	O
+the	O
+specificity	O
+of	O
+the	O
+14C	O
+-	O
+aminocarboxypropyl	O
+incorporation	O
+.	O
+	O
+As	O
+previously	O
+shown	O
+,	O
+only	O
+the	O
+acp	O
+but	O
+none	O
+of	O
+the	O
+other	O
+modifications	O
+at	O
+U1191	O
+of	O
+yeast	O
+18S	O
+rRNA	O
+blocks	O
+reverse	O
+transcriptase	O
+activity	O
+.	O
+	O
+Therefore	O
+the	O
+presence	O
+of	O
+the	O
+acp	O
+modification	O
+can	O
+be	O
+directly	O
+assessed	O
+by	O
+primer	O
+extension	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+in	O
+a	O
+Δtsr3	B-mutant
+mutant	O
+no	O
+primer	O
+extension	O
+stop	O
+signal	O
+was	O
+present	O
+at	O
+this	O
+position	O
+.	O
+	O
+In	O
+a	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+double	O
+deletion	O
+strain	O
+the	O
+18S	O
+rRNA	O
+contains	O
+an	O
+unmodified	O
+U	O
+and	O
+the	O
+primer	O
+extension	O
+stop	O
+signal	O
+was	O
+missing	O
+(	O
+Figure	O
+1D	O
+).	O
+	O
+The	O
+Tsr3	O
+protein	O
+is	O
+highly	O
+conserved	O
+in	O
+yeast	O
+and	O
+humans	O
+(	O
+50	O
+%	O
+identity	O
+).	O
+	O
+After	O
+siRNA	O
+-	O
+mediated	O
+depletion	O
+of	O
+Tsr3	O
+in	O
+human	O
+colon	O
+carcinoma	O
+HCT116	O
+(+/+)	O
+cells	O
+the	O
+acp	O
+primer	O
+extension	O
+arrest	O
+was	O
+reduced	O
+in	O
+comparison	O
+to	O
+cells	O
+transfected	O
+with	O
+a	O
+non	O
+-	O
+targeting	O
+scramble	O
+siRNA	O
+control	O
+(	O
+Figure	O
+1E	O
+,	O
+compare	O
+lanes	O
+544	O
+and	O
+scramble	O
+).	O
+	O
+This	O
+suggests	O
+that	O
+low	O
+residual	O
+levels	O
+of	O
+HsTsr3	O
+are	O
+sufficient	O
+to	O
+modify	O
+the	O
+RNA	O
+.	O
+	O
+Thus	O
+,	O
+HsTsr3	O
+is	O
+also	O
+responsible	O
+for	O
+the	O
+acp	O
+modification	O
+of	O
+18S	O
+rRNA	O
+nucleotide	O
+Ψ1248	O
+in	O
+helix	O
+31	O
+.	O
+	O
+Similar	O
+to	O
+yeast	O
+,	O
+siRNA	O
+-	O
+mediated	O
+depletion	O
+of	O
+the	O
+Ψ1248	O
+N1	O
+-	O
+methyltransferase	O
+Nep1	O
+/	O
+Emg1	O
+had	O
+no	O
+influence	O
+on	O
+the	O
+primer	O
+extension	O
+arrest	O
+(	O
+Figure	O
+1E	O
+).	O
+	O
+Although	O
+the	O
+acp	O
+modification	O
+of	O
+18S	O
+rRNA	O
+is	O
+highly	O
+conserved	O
+in	O
+eukaryotes	O
+,	O
+yeast	O
+Δtsr3	B-mutant
+mutants	O
+showed	O
+only	O
+a	O
+minor	O
+growth	O
+defect	O
+.	O
+	O
+However	O
+,	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+was	O
+synthetic	O
+sick	O
+with	O
+a	O
+Δsnr35	B-mutant
+deletion	O
+preventing	O
+pseudouridylation	O
+and	O
+Nep1	O
+-	O
+catalyzed	O
+methylation	O
+of	O
+nucleotide	O
+1191	O
+(	O
+Figure	O
+2A	O
+).	O
+	O
+Interestingly	O
+,	O
+no	O
+increased	O
+growth	O
+defect	O
+could	O
+be	O
+observed	O
+for	O
+Δtsr3	B-mutant
+Δnep1	I-mutant
+recombinants	O
+containing	O
+the	O
+nep1	O
+suppressor	O
+mutation	O
+Δnop6	B-mutant
+as	O
+well	O
+as	O
+for	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+Δnep1	I-mutant
+recombinants	O
+with	O
+unmodified	O
+U1191	O
+(	O
+Supplementary	O
+Figure	O
+S2D	O
+and	O
+E	O
+).	O
+	O
+The	O
+Δtsr3	B-mutant
+deletion	O
+is	O
+synthetic	O
+sick	O
+with	O
+a	O
+Δsnr35	B-mutant
+deletion	O
+preventing	O
+U1191	O
+pseudouridylation	O
+.	O
+	O
+(	O
+B	O
+)	O
+In	O
+agar	O
+diffusion	O
+assays	O
+the	O
+yeast	O
+Δtsr3	B-mutant
+deletion	O
+mutant	O
+shows	O
+a	O
+hypersensitivity	O
+against	O
+paromomycin	O
+and	O
+hygromycin	O
+B	O
+which	O
+is	O
+further	O
+increased	O
+by	O
+recombination	O
+with	O
+Δsnr35	B-mutant
+.	O
+(	O
+C	O
+)	O
+Northern	O
+blot	O
+analysis	O
+with	O
+an	O
+ITS1	O
+hybridization	O
+probe	O
+after	O
+siRNA	O
+depletion	O
+of	O
+HsTSR3	O
+(	O
+siRNAs	O
+544	O
+and	O
+545	O
+)	O
+and	O
+a	O
+scrambled	O
+siRNA	O
+as	O
+control	O
+.	O
+	O
+The	O
+accumulation	O
+of	O
+18SE	O
+and	O
+47S	O
+and	O
+/	O
+or	O
+45S	O
+pre	O
+-	O
+RNAs	O
+is	O
+enforced	O
+upon	O
+HsTSR3	O
+depletion	O
+.	O
+	O
+Right	O
+gel	O
+:	O
+Ethidium	O
+bromide	O
+staining	O
+showing	O
+18S	O
+and	O
+28S	O
+rRNAs	O
+.	O
+	O
+(	O
+D	O
+)	O
+Cytoplasmic	O
+localization	O
+of	O
+yeast	O
+Tsr3	O
+shown	O
+by	O
+fluorescence	O
+microscopy	O
+of	O
+GFP	B-mutant
+-	I-mutant
+fused	I-mutant
+Tsr3	I-mutant
+.	O
+	O
+From	O
+left	O
+to	O
+right	O
+:	O
+differential	O
+interference	O
+contrast	O
+(	O
+DIC	O
+),	O
+green	O
+fluorescence	O
+of	O
+GFP	B-mutant
+-	I-mutant
+Tsr3	I-mutant
+,	O
+red	O
+fluorescence	O
+of	O
+Nop56	B-mutant
+-	I-mutant
+mRFP	I-mutant
+as	O
+nucleolar	O
+marker	O
+,	O
+and	O
+merge	O
+of	O
+GFP	B-mutant
+-	I-mutant
+Tsr3	I-mutant
+/	O
+Nop56	B-mutant
+-	I-mutant
+mRFP	I-mutant
+with	O
+DIC	O
+.	O
+(	O
+E	O
+)	O
+Elution	O
+profile	O
+(	O
+A254	O
+)	O
+after	O
+sucrose	O
+gradient	O
+separation	O
+of	O
+yeast	O
+ribosomal	O
+subunits	O
+and	O
+polysomes	O
+(	O
+upper	O
+part	O
+)	O
+and	O
+western	O
+blot	O
+analysis	O
+of	O
+3xHA	O
+tagged	O
+Tsr3	O
+(	O
+Tsr3	B-mutant
+-	I-mutant
+3xHA	I-mutant
+)	O
+after	O
+SDS	O
+-	O
+PAGE	O
+separation	O
+of	O
+polysome	O
+profile	O
+fractions	O
+taken	O
+every	O
+20	O
+s	O
+(	O
+lower	O
+part	O
+).	O
+	O
+The	O
+influence	O
+of	O
+the	O
+acp	O
+modification	O
+of	O
+nucleotide	O
+1191	O
+on	O
+ribosome	O
+function	O
+was	O
+analyzed	O
+by	O
+treating	O
+Δtsr3	B-mutant
+mutants	O
+with	O
+protein	O
+synthesis	O
+inhibitors	O
+.	O
+	O
+Similar	O
+to	O
+a	O
+temperature	O
+-	O
+sensitive	O
+nep1	O
+mutant	O
+,	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+caused	O
+hypersensitivity	O
+to	O
+paromomycin	O
+and	O
+,	O
+to	O
+a	O
+lesser	O
+extent	O
+,	O
+to	O
+hygromycin	O
+B	O
+(	O
+Figure	O
+2B	O
+),	O
+but	O
+not	O
+to	O
+G418	O
+or	O
+cycloheximide	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+	O
+In	O
+a	O
+yeast	O
+Δtsr3	B-mutant
+strain	O
+as	O
+well	O
+as	O
+in	O
+the	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+recombinant	O
+20S	O
+pre	O
+-	O
+rRNA	O
+accumulated	O
+significantly	O
+and	O
+the	O
+level	O
+of	O
+mature	O
+18S	O
+rRNA	O
+was	O
+reduced	O
+(	O
+Supplementary	O
+Figures	O
+S2C	O
+and	O
+S3D	O
+),	O
+as	O
+reported	O
+previously	O
+.	O
+	O
+A	O
+minor	O
+effect	O
+on	O
+20S	O
+rRNA	O
+accumulation	O
+was	O
+also	O
+observed	O
+for	O
+Δsnr35	B-mutant
+,	O
+but	O
+-	O
+probably	O
+due	O
+to	O
+different	O
+strain	O
+backgrounds	O
+–	O
+to	O
+a	O
+weaker	O
+extent	O
+than	O
+described	O
+earlier	O
+.	O
+	O
+In	O
+human	O
+cells	O
+,	O
+the	O
+depletion	O
+of	O
+HsTsr3	O
+in	O
+HCT116	O
+(+/+)	O
+cells	O
+caused	O
+an	O
+accumulation	O
+of	O
+the	O
+human	O
+20S	O
+pre	O
+-	O
+rRNA	O
+equivalent	O
+18S	O
+-	O
+E	O
+suggesting	O
+an	O
+evolutionary	O
+conserved	O
+role	O
+of	O
+Tsr3	O
+in	O
+the	O
+late	O
+steps	O
+of	O
+18S	O
+rRNA	O
+processing	O
+(	O
+Figure	O
+2C	O
+and	O
+Supplementary	O
+Figure	O
+S2B	O
+).	O
+	O
+In	O
+polysome	O
+profiles	O
+,	O
+a	O
+reduced	O
+level	O
+of	O
+80S	O
+ribosomes	O
+and	O
+a	O
+strong	O
+signal	O
+for	O
+free	O
+60S	O
+subunits	O
+was	O
+observed	O
+in	O
+line	O
+with	O
+the	O
+40S	O
+subunit	O
+deficiency	O
+(	O
+Supplementary	O
+Figure	O
+S2G	O
+).	O
+	O
+Cellular	O
+localization	O
+of	O
+Tsr3	O
+in	O
+S	O
+.	O
+cerevisiae	O
+	O
+Fluorescence	O
+microscopy	O
+of	O
+GFP	O
+-	O
+tagged	O
+Tsr3	O
+localized	O
+the	O
+fusion	O
+protein	O
+in	O
+the	O
+cytoplasm	O
+of	O
+yeast	O
+cells	O
+and	O
+no	O
+co	O
+-	O
+localization	O
+with	O
+the	O
+nucleolar	O
+marker	O
+protein	O
+Nop56	O
+could	O
+be	O
+observed	O
+(	O
+Figure	O
+2D	O
+).	O
+	O
+This	O
+agrees	O
+with	O
+previous	O
+biochemical	O
+data	O
+suggesting	O
+that	O
+the	O
+acp	O
+modification	O
+of	O
+18S	O
+rRNA	O
+occurs	O
+late	O
+during	O
+40S	O
+subunit	O
+biogenesis	O
+in	O
+the	O
+cytoplasm	O
+,	O
+and	O
+makes	O
+an	O
+additional	O
+nuclear	O
+localization	O
+as	O
+reported	O
+in	O
+a	O
+previous	O
+large	O
+-	O
+scale	O
+analysis	O
+unlikely	O
+.	O
+	O
+Searches	O
+for	O
+sequence	O
+homologs	O
+of	O
+S	O
+.	O
+cerevisiae	O
+Tsr3	O
+(	O
+ScTsr3	O
+)	O
+by	O
+us	O
+and	O
+others	O
+revealed	O
+that	O
+the	O
+genomes	O
+of	O
+many	O
+archaea	O
+contain	O
+genes	O
+encoding	O
+Tsr3	O
+-	O
+like	O
+proteins	O
+.	O
+	O
+To	O
+locate	O
+the	O
+domains	O
+most	O
+important	O
+for	O
+Tsr3	O
+activity	O
+,	O
+ScTsr3	O
+fragments	O
+of	O
+different	O
+lengths	O
+containing	O
+the	O
+highly	O
+conserved	O
+central	O
+part	O
+were	O
+expressed	O
+in	O
+a	O
+Δtsr3	B-mutant
+mutant	O
+(	O
+Figure	O
+3A	O
+)	O
+and	O
+analyzed	O
+by	O
+primer	O
+extension	O
+(	O
+Figure	O
+3B	O
+)	O
+and	O
+Northern	O
+blotting	O
+(	O
+Figure	O
+3C	O
+).	O
+	O
+TSR3	O
+fragments	O
+of	O
+different	O
+length	O
+were	O
+expressed	O
+under	O
+the	O
+native	O
+promotor	O
+from	O
+multicopy	O
+plasmids	O
+in	O
+a	O
+Δtsr3	B-mutant
+deletion	O
+strain	O
+.	O
+	O
+(	O
+B	O
+)	O
+Primer	O
+extension	O
+analysis	O
+of	O
+18S	O
+rRNA	O
+acp	O
+modification	O
+in	O
+yeast	O
+cells	O
+expressing	O
+the	O
+indicated	O
+TSR3	O
+fragments	O
+.	O
+	O
+N	O
+-	O
+terminal	O
+deletions	O
+of	O
+36	O
+or	O
+45	O
+amino	O
+acids	O
+and	O
+C	O
+-	O
+terminal	O
+deletions	O
+of	O
+43	O
+or	O
+76	O
+residues	O
+show	O
+a	O
+primer	O
+extension	O
+stop	O
+comparable	O
+to	O
+the	O
+wild	O
+type	O
+.	O
+	O
+Tsr3	O
+fragments	O
+37	O
+–	O
+223	O
+or	O
+46	O
+–	O
+223	O
+cause	O
+a	O
+nearly	O
+complete	O
+loss	O
+of	O
+the	O
+arrest	O
+signal	O
+.	O
+	O
+The	O
+box	O
+highlights	O
+the	O
+shortest	O
+Tsr3	O
+fragment	O
+(	O
+aa	O
+46	O
+–	O
+270	O
+)	O
+with	O
+wild	O
+type	O
+activity	O
+(	O
+strong	O
+primer	O
+extension	O
+block	O
+).	O
+(	O
+C	O
+)	O
+Northern	O
+blot	O
+analysis	O
+of	O
+20S	O
+pre	O
+-	O
+rRNA	O
+accumulation	O
+.	O
+	O
+A	O
+weak	O
+20S	O
+rRNA	O
+signal	O
+,	O
+indicating	O
+normal	O
+processing	O
+,	O
+is	O
+observed	O
+for	O
+Tsr3	O
+fragment	O
+46	O
+–	O
+270	O
+(	O
+highlighted	O
+in	O
+a	O
+box	O
+)	O
+showing	O
+its	O
+functionality	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+archaeal	O
+homologs	O
+correspond	O
+to	O
+the	O
+functional	O
+core	O
+of	O
+Tsr3	O
+.	O
+	O
+In	O
+order	O
+to	O
+define	O
+the	O
+structural	O
+basis	O
+for	O
+Tsr3	O
+function	O
+,	O
+homologs	O
+from	O
+thermophilic	O
+archaea	O
+were	O
+screened	O
+for	O
+crystallization	O
+.	O
+	O
+Well	O
+diffracting	O
+crystals	O
+were	O
+obtained	O
+for	O
+Tsr3	O
+homologs	O
+from	O
+the	O
+two	O
+crenarchaeal	O
+species	O
+Vulcanisaeta	O
+distributa	O
+(	O
+VdTsr3	O
+)	O
+and	O
+Sulfolobus	O
+solfataricus	O
+(	O
+SsTsr3	O
+)	O
+which	O
+share	O
+36	O
+%	O
+(	O
+VdTsr3	O
+)	O
+and	O
+38	O
+%	O
+(	O
+SsTsr3	O
+)	O
+identity	O
+with	O
+the	O
+ScTsr3	O
+core	O
+region	O
+(	O
+ScTsr3	O
+aa	O
+46	O
+–	O
+223	O
+).	O
+	O
+While	O
+for	O
+S	O
+.	O
+solfataricus	O
+the	O
+existence	O
+of	O
+a	O
+modified	O
+nucleotide	O
+of	O
+unknown	O
+chemical	O
+composition	O
+in	O
+the	O
+loop	O
+capping	O
+helix	O
+31	O
+of	O
+its	O
+16S	O
+rRNA	O
+has	O
+been	O
+demonstrated	O
+,	O
+no	O
+information	O
+regarding	O
+rRNA	O
+modifications	O
+is	O
+yet	O
+available	O
+for	O
+V	O
+.	O
+distributa	O
+.	O
+	O
+Crystals	O
+of	O
+VdTsr3	O
+diffracted	O
+to	O
+a	O
+resolution	O
+of	O
+1	O
+.	O
+6	O
+Å	O
+whereas	O
+crystals	O
+of	O
+SsTsr3	O
+diffracted	O
+to	O
+2	O
+.	O
+25	O
+Å	O
+.	O
+Serendipitously	O
+,	O
+VdTsr3	O
+was	O
+purified	O
+and	O
+crystallized	O
+in	O
+complex	O
+with	O
+endogenous	O
+(	O
+E	O
+.	O
+coli	O
+)	O
+SAM	O
+(	O
+Supplementary	O
+Figure	O
+S4	O
+)	O
+while	O
+SsTsr3	O
+crystals	O
+contained	O
+the	O
+protein	O
+in	O
+the	O
+apo	O
+state	O
+.	O
+	O
+The	O
+structure	O
+of	O
+VdTsr3	O
+was	O
+solved	O
+ab	O
+initio	O
+,	O
+by	O
+single	O
+-	O
+wavelength	O
+anomalous	O
+diffraction	O
+phasing	O
+(	O
+Se	O
+-	O
+SAD	O
+)	O
+with	O
+Se	O
+containing	O
+derivatives	O
+(	O
+selenomethionine	O
+and	O
+seleno	O
+-	O
+substituted	O
+SAM	O
+).	O
+	O
+The	O
+structure	O
+of	O
+SsTsr3	O
+was	O
+solved	O
+by	O
+molecular	O
+replacement	O
+using	O
+VdTsr3	O
+as	O
+a	O
+search	O
+model	O
+(	O
+see	O
+Supplementary	O
+Table	O
+S1	O
+for	O
+data	O
+collection	O
+and	O
+refinement	O
+statistics	O
+).	O
+	O
+The	O
+structure	O
+of	O
+VdTsr3	O
+can	O
+be	O
+divided	O
+into	O
+two	O
+domains	O
+(	O
+Figure	O
+4A	O
+).	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+domain	O
+(	O
+aa	O
+1	O
+–	O
+92	O
+)	O
+has	O
+a	O
+mixed	O
+α	O
+/	O
+β	O
+-	O
+structure	O
+centered	O
+around	O
+a	O
+five	O
+-	O
+stranded	O
+all	O
+-	O
+parallel	O
+β	O
+-	O
+sheet	O
+(	O
+Figure	O
+4B	O
+)	O
+with	O
+the	O
+strand	O
+order	O
+β5	O
+↑-	O
+β3	O
+↑-	O
+β4	O
+↑-	O
+β1	O
+↑-	O
+β2	O
+↑.	O
+The	O
+loops	O
+connecting	O
+β1	O
+and	O
+β2	O
+,	O
+β3	O
+and	O
+β4	O
+and	O
+β4	O
+and	O
+β5	O
+include	O
+α	O
+-	O
+helices	O
+α1	O
+,	O
+α2	O
+and	O
+α3	O
+,	O
+respectively	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+VdTsr3	O
+structure	O
+contains	O
+a	O
+deep	O
+trefoil	O
+knot	O
+.	O
+	O
+The	O
+structure	O
+of	O
+SsTsr3	O
+in	O
+the	O
+apo	O
+state	O
+is	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+VdTsr3	O
+(	O
+Figure	O
+4C	O
+)	O
+with	O
+an	O
+RMSD	O
+for	O
+equivalent	O
+Cα	O
+atoms	O
+of	O
+1	O
+.	O
+1	O
+Å	O
+.	O
+The	O
+only	O
+significant	O
+difference	O
+in	O
+the	O
+global	O
+structure	O
+of	O
+the	O
+two	O
+proteins	O
+is	O
+the	O
+presence	O
+of	O
+an	O
+extended	O
+α	O
+-	O
+helix	O
+α8	O
+and	O
+the	O
+absence	O
+of	O
+α	O
+-	O
+helix	O
+α9	O
+in	O
+SsTsr3	O
+.	O
+	O
+β	O
+-	O
+strands	O
+are	O
+colored	O
+in	O
+crimson	O
+whereas	O
+α	O
+-	O
+helices	O
+in	O
+the	O
+N	O
+-	O
+terminal	O
+domain	O
+are	O
+colored	O
+light	O
+blue	O
+and	O
+α	O
+-	O
+helices	O
+in	O
+the	O
+C	O
+-	O
+terminal	O
+domain	O
+are	O
+colored	O
+dark	O
+blue	O
+.	O
+	O
+The	O
+locations	O
+of	O
+the	O
+α	O
+-	O
+helix	O
+α8	O
+which	O
+is	O
+longer	O
+in	O
+SsTsr3	O
+and	O
+of	O
+α	O
+-	O
+helix	O
+α9	O
+which	O
+is	O
+only	O
+present	O
+in	O
+VdTsr3	O
+are	O
+indicated	O
+.	O
+(	O
+D	O
+)	O
+Secondary	O
+structure	O
+cartoon	O
+(	O
+left	O
+)	O
+of	O
+S	O
+.	O
+pombe	O
+Trm10	O
+(	O
+pdb4jwf	O
+)—	O
+the	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+methyltransferase	O
+structurally	O
+most	O
+similar	O
+to	O
+Tsr3	O
+and	O
+superposition	O
+of	O
+the	O
+VdTsr3	O
+and	O
+Trm10	O
+X	O
+-	O
+ray	O
+structures	O
+(	O
+right	O
+).	O
+(	O
+E	O
+)	O
+Analytical	O
+gel	O
+filtration	O
+profiles	O
+for	O
+VdTsr3	O
+(	O
+red	O
+)	O
+and	O
+SsTsr3	O
+(	O
+blue	O
+)	O
+show	O
+that	O
+both	O
+proteins	O
+are	O
+monomeric	O
+in	O
+solution	O
+.	O
+	O
+Vd	O
+,	O
+Vulcanisaeta	O
+distributa	O
+;	O
+Ss	O
+,	O
+Sulfolobus	O
+solfataricus	O
+.	O
+	O
+However	O
+,	O
+no	O
+structural	O
+similarity	O
+to	O
+an	O
+RLI	O
+-	O
+domain	O
+was	O
+detectable	O
+.	O
+	O
+This	O
+is	O
+in	O
+accordance	O
+with	O
+the	O
+functional	O
+analysis	O
+of	O
+alanine	O
+replacement	O
+mutations	O
+of	O
+cysteine	O
+residues	O
+in	O
+ScTsr3	O
+(	O
+Supplementary	O
+Figure	O
+S3	O
+).	O
+	O
+The	O
+β	O
+-	O
+strand	O
+topology	O
+and	O
+the	O
+deep	O
+C	O
+-	O
+terminal	O
+trefoil	O
+knot	O
+of	O
+archaeal	O
+Tsr3	O
+are	O
+the	O
+structural	O
+hallmarks	O
+of	O
+the	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+-	O
+methyltransferase	O
+fold	O
+.	O
+	O
+The	O
+closest	O
+structural	O
+homolog	O
+identified	O
+in	O
+a	O
+DALI	O
+search	O
+is	O
+the	O
+tRNA	O
+methyltransferase	O
+Trm10	O
+(	O
+DALI	O
+Z	O
+-	O
+score	O
+6	O
+.	O
+8	O
+)	O
+which	O
+methylates	O
+the	O
+N1	O
+nitrogen	O
+of	O
+G9	O
+/	O
+A9	O
+in	O
+many	O
+archaeal	O
+and	O
+eukaryotic	O
+tRNAs	O
+by	O
+using	O
+SAM	O
+as	O
+the	O
+methyl	O
+group	O
+donor	O
+.	O
+	O
+Interestingly	O
+,	O
+Nep1	O
+—	O
+the	O
+enzyme	O
+preceding	O
+Tsr3	O
+in	O
+the	O
+biosynthetic	O
+pathway	O
+for	O
+the	O
+synthesis	O
+of	O
+m1acp3Ψ	O
+—	O
+also	O
+belongs	O
+to	O
+the	O
+SPOUT	O
+-	O
+class	O
+of	O
+RNA	O
+methyltransferases	O
+.	O
+	O
+However	O
+,	O
+the	O
+structural	O
+similarities	O
+between	O
+Nep1	O
+and	O
+Tsr3	O
+(	O
+DALI	O
+Z	O
+-	O
+score	O
+4	O
+.	O
+4	O
+)	O
+are	O
+less	O
+pronounced	O
+than	O
+between	O
+Tsr3	O
+and	O
+Trm10	O
+.	O
+	O
+Most	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+-	O
+methyltransferases	O
+are	O
+homodimers	O
+.	O
+	O
+Gel	O
+filtration	O
+experiments	O
+with	O
+both	O
+VdTsr3	O
+and	O
+SsTsr3	O
+(	O
+Figure	O
+4E	O
+)	O
+showed	O
+that	O
+both	O
+proteins	O
+are	O
+monomeric	O
+in	O
+solution	O
+thereby	O
+extending	O
+the	O
+structural	O
+similarities	O
+to	O
+Trm10	O
+.	O
+	O
+So	O
+far	O
+,	O
+structural	O
+information	O
+is	O
+only	O
+available	O
+for	O
+one	O
+other	O
+enzyme	O
+that	O
+transfers	O
+the	O
+acp	O
+group	O
+from	O
+SAM	O
+to	O
+an	O
+RNA	O
+nucleotide	O
+.	O
+	O
+This	O
+enzyme	O
+,	O
+Tyw2	O
+,	O
+is	O
+part	O
+of	O
+the	O
+biosynthesis	O
+pathway	O
+of	O
+wybutosine	O
+nucleotides	O
+in	O
+tRNAs	O
+.	O
+	O
+Instead	O
+,	O
+Tyw2	O
+has	O
+a	O
+fold	O
+typical	O
+for	O
+the	O
+class	O
+-	O
+I	O
+-	O
+or	O
+Rossmann	O
+-	O
+fold	O
+class	O
+of	O
+methyltransferases	O
+(	O
+Supplementary	O
+Figure	O
+S5B	O
+).	O
+	O
+Cofactor	O
+binding	O
+of	O
+Tsr3	O
+	O
+The	O
+SAM	O
+-	O
+binding	O
+site	O
+of	O
+Tsr3	O
+is	O
+located	O
+in	O
+a	O
+deep	O
+crevice	O
+between	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+domains	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+trefoil	O
+knot	O
+as	O
+typical	O
+for	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+-	O
+methyltransferases	O
+(	O
+Figure	O
+4A	O
+).	O
+	O
+The	O
+adenine	O
+base	O
+of	O
+the	O
+cofactor	O
+is	O
+recognized	O
+by	O
+hydrogen	O
+bonds	O
+between	O
+its	O
+N1	O
+nitrogen	O
+and	O
+the	O
+backbone	O
+amide	O
+of	O
+L93	O
+directly	O
+preceding	O
+β5	O
+as	O
+well	O
+as	O
+between	O
+its	O
+N6	O
+-	O
+amino	O
+group	O
+and	O
+the	O
+backbone	O
+carbonyl	O
+group	O
+of	O
+Y108	O
+located	O
+in	O
+the	O
+loop	O
+connecting	O
+β5	O
+in	O
+the	O
+N	O
+-	O
+terminal	O
+and	O
+α4	O
+in	O
+the	O
+C	O
+-	O
+terminal	O
+domain	O
+(	O
+Figure	O
+5A	O
+).	O
+	O
+Furthermore	O
+,	O
+the	O
+adenine	O
+base	O
+of	O
+SAM	O
+is	O
+involved	O
+in	O
+hydrophobic	O
+packing	O
+interactions	O
+with	O
+the	O
+side	O
+chains	O
+of	O
+L45	O
+(	O
+β3	O
+),	O
+P47	O
+and	O
+W73	O
+(	O
+α3	O
+)	O
+in	O
+the	O
+N	O
+-	O
+terminal	O
+domain	O
+as	O
+well	O
+as	O
+with	O
+L93	O
+,	O
+L110	O
+(	O
+both	O
+in	O
+the	O
+loop	O
+connecting	O
+β5	O
+and	O
+α4	O
+)	O
+and	O
+A115	O
+(	O
+α5	O
+)	O
+in	O
+the	O
+C	O
+-	O
+terminal	O
+domain	O
+.	O
+	O
+The	O
+acp	O
+side	O
+chain	O
+of	O
+SAM	O
+is	O
+fixed	O
+in	O
+position	O
+by	O
+hydrogen	O
+bonding	O
+of	O
+its	O
+carboxylate	O
+group	O
+to	O
+the	O
+backbone	O
+amide	O
+and	O
+the	O
+side	O
+chain	O
+hydroxyl	O
+group	O
+of	O
+T19	O
+in	O
+α1	O
+as	O
+well	O
+as	O
+the	O
+backbone	O
+amide	O
+group	O
+of	O
+T112	O
+in	O
+α4	O
+(	O
+C	O
+-	O
+terminal	O
+domain	O
+).	O
+	O
+Most	O
+importantly	O
+,	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	O
+is	O
+buried	O
+in	O
+a	O
+hydrophobic	O
+pocket	O
+formed	O
+by	O
+the	O
+sidechains	O
+of	O
+W73	O
+and	O
+A76	O
+both	O
+located	O
+in	O
+α3	O
+(	O
+Figure	O
+5A	O
+and	O
+B	O
+).	O
+	O
+SAM	O
+-	O
+binding	O
+by	O
+Tsr3	O
+.	O
+	O
+Nitrogen	O
+atoms	O
+are	O
+dark	O
+blue	O
+,	O
+oxygen	O
+atoms	O
+red	O
+,	O
+sulfur	O
+atoms	O
+orange	O
+,	O
+carbon	O
+atoms	O
+of	O
+the	O
+protein	O
+light	O
+blue	O
+and	O
+carbon	O
+atoms	O
+of	O
+SAM	O
+yellow	O
+.	O
+	O
+(	O
+B	O
+)	O
+Solvent	O
+accessibility	O
+of	O
+the	O
+acp	O
+group	O
+of	O
+SAM	O
+bound	O
+to	O
+VdTsr3	O
+.	O
+	O
+The	O
+solvent	O
+accessible	O
+surface	O
+of	O
+the	O
+protein	O
+is	O
+shown	O
+in	O
+semitransparent	O
+gray	O
+whereas	O
+SAM	O
+is	O
+show	O
+in	O
+a	O
+stick	O
+representation	O
+.	O
+	O
+A	O
+red	O
+arrow	O
+indicates	O
+the	O
+reactive	O
+CH2	O
+-	O
+moiety	O
+of	O
+the	O
+acp	O
+group	O
+.	O
+(	O
+C	O
+)	O
+Solvent	O
+accessibility	O
+of	O
+the	O
+SAM	O
+methyl	O
+group	O
+for	O
+SAM	O
+bound	O
+to	O
+the	O
+RNA	O
+methyltransferase	O
+Trm10	O
+.	O
+	O
+Tryptophan	O
+fluorescence	O
+quenching	O
+curves	O
+upon	O
+addition	O
+of	O
+SAM	O
+(	O
+blue	O
+),	O
+5	O
+′-	O
+methyl	O
+-	O
+thioadenosine	O
+(	O
+red	O
+)	O
+and	O
+SAH	O
+(	O
+black	O
+).	O
+	O
+Radioactively	O
+labeled	O
+SAM	O
+is	O
+retained	O
+on	O
+a	O
+filter	O
+in	O
+the	O
+presence	O
+of	O
+SsTsr3	O
+.	O
+	O
+Addition	O
+of	O
+unlabeled	O
+SAM	O
+competes	O
+with	O
+the	O
+binding	O
+of	O
+labeled	O
+SAM	O
+.	O
+	O
+A	O
+W66A	B-mutant
+-	O
+mutant	O
+of	O
+SsTsr3	O
+(	O
+W73	O
+in	O
+VdTsr3	O
+)	O
+does	O
+not	O
+bind	O
+SAM	O
+.	O
+	O
+(	O
+F	O
+)	O
+Primer	O
+extension	O
+(	O
+upper	O
+left	O
+)	O
+shows	O
+a	O
+strongly	O
+reduced	O
+acp	O
+modification	O
+of	O
+yeast	O
+18S	O
+rRNA	O
+in	O
+Δtsr3	B-mutant
+cells	O
+expressing	O
+Tsr3	B-mutant
+-	I-mutant
+S62D	I-mutant
+,	O
+-	B-mutant
+E111A	I-mutant
+or	O
+–	B-mutant
+W114A	I-mutant
+.	O
+	O
+3xHA	O
+tagged	O
+Tsr3	O
+mutants	O
+are	O
+expressed	O
+comparable	O
+to	O
+the	O
+wild	O
+type	O
+as	O
+shown	O
+by	O
+western	O
+blot	O
+(	O
+lower	O
+left	O
+).	O
+	O
+Binding	O
+affinities	O
+for	O
+SAM	O
+and	O
+its	O
+analogs	O
+5	O
+′-	O
+methylthioadenosin	O
+and	O
+SAH	O
+to	O
+SsTsr3	O
+were	O
+measured	O
+using	O
+tryptophan	O
+fluorescence	O
+quenching	O
+.	O
+	O
+VdTsr3	O
+could	O
+not	O
+be	O
+used	O
+in	O
+these	O
+experiments	O
+since	O
+we	O
+could	O
+not	O
+purify	O
+it	O
+in	O
+a	O
+stable	O
+SAM	O
+-	O
+free	O
+form	O
+.	O
+	O
+S	O
+-	O
+adenosylhomocysteine	O
+which	O
+lacks	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	O
+binds	O
+with	O
+significantly	O
+lower	O
+affinity	O
+(	O
+KD	O
+=	O
+55	O
+.	O
+5	O
+μM	O
+)	O
+(	O
+Figure	O
+5D	O
+).	O
+	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+the	O
+loss	O
+of	O
+hydrogen	O
+bonds	O
+between	O
+the	O
+acp	O
+sidechain	O
+carboxylate	O
+group	O
+and	O
+the	O
+protein	O
+appears	O
+to	O
+be	O
+thermodynamically	O
+less	O
+important	O
+but	O
+these	O
+hydrogen	O
+bonds	O
+might	O
+play	O
+a	O
+crucial	O
+role	O
+for	O
+the	O
+proper	O
+orientation	O
+of	O
+the	O
+cofactor	O
+side	O
+chain	O
+in	O
+the	O
+substrate	O
+binding	O
+pocket	O
+.	O
+	O
+Accordingly	O
+,	O
+a	O
+W66A	B-mutant
+-	O
+mutation	O
+(	O
+W73	O
+in	O
+VdTsr3	O
+)	O
+of	O
+SsTsr3	O
+significantly	O
+diminished	O
+SAM	O
+-	O
+binding	O
+in	O
+a	O
+filter	O
+binding	O
+assay	O
+compared	O
+to	O
+the	O
+wild	O
+type	O
+(	O
+Figure	O
+5E	O
+).	O
+	O
+Nevertheless	O
+,	O
+a	O
+mutation	O
+of	O
+the	O
+equivalent	O
+position	O
+S62	O
+of	O
+ScTsr3	O
+to	O
+D	O
+,	O
+but	O
+not	O
+to	O
+A	O
+,	O
+resulted	O
+in	O
+reduced	O
+acp	O
+modification	O
+in	O
+vivo	O
+,	O
+as	O
+shown	O
+by	O
+primer	O
+extension	O
+analysis	O
+(	O
+Figure	O
+5F	O
+).	O
+	O
+The	O
+acp	O
+-	O
+transfer	O
+reaction	O
+catalyzed	O
+by	O
+Tsr3	O
+most	O
+likely	O
+requires	O
+the	O
+presence	O
+of	O
+a	O
+catalytic	O
+base	O
+in	O
+order	O
+to	O
+abstract	O
+a	O
+proton	O
+from	O
+the	O
+N3	O
+imino	O
+group	O
+of	O
+the	O
+modified	O
+pseudouridine	O
+.	O
+	O
+The	O
+side	O
+chain	O
+of	O
+D70	O
+(	O
+VdTsr3	O
+)	O
+located	O
+in	O
+β4	O
+is	O
+∼	O
+5	O
+Å	O
+away	O
+from	O
+the	O
+SAM	O
+sulfur	O
+atom	O
+.	O
+	O
+This	O
+residue	O
+is	O
+conserved	O
+as	O
+D	O
+or	O
+E	O
+both	O
+in	O
+archaeal	O
+and	O
+eukaryotic	O
+Tsr3	O
+homologs	O
+.	O
+	O
+However	O
+,	O
+the	O
+mutation	O
+of	O
+the	O
+corresponding	O
+residue	O
+of	O
+ScTsr3	O
+(	O
+E111A	B-mutant
+)	O
+leads	O
+to	O
+a	O
+significant	O
+decrease	O
+of	O
+the	O
+acp	O
+transferase	O
+activity	O
+in	O
+vivo	O
+(	O
+Figure	O
+5F	O
+).	O
+	O
+RNA	O
+-	O
+binding	O
+of	O
+Tsr3	O
+	O
+Furthermore	O
+,	O
+a	O
+negatively	O
+charged	O
+MES	O
+-	O
+ion	O
+is	O
+found	O
+in	O
+the	O
+crystal	O
+structure	O
+of	O
+VdTsr3	O
+complexed	O
+to	O
+the	O
+side	O
+chain	O
+of	O
+K22	O
+in	O
+helix	O
+α1	O
+.	O
+	O
+Helix	O
+α1	O
+contains	O
+two	O
+more	O
+positively	O
+charged	O
+amino	O
+acids	O
+K17	O
+and	O
+R25	O
+as	O
+does	O
+the	O
+loop	O
+preceding	O
+it	O
+(	O
+R9	O
+).	O
+	O
+A	O
+second	O
+cluster	O
+of	O
+positively	O
+charged	O
+residues	O
+is	O
+found	O
+in	O
+or	O
+near	O
+helix	O
+α3	O
+(	O
+K74	O
+,	O
+R75	O
+,	O
+K82	O
+,	O
+R85	O
+and	O
+K87	O
+).	O
+	O
+Some	O
+of	O
+these	O
+amino	O
+acids	O
+are	O
+conserved	O
+between	O
+archaeal	O
+and	O
+eukaryotic	O
+Tsr3	O
+(	O
+Supplementary	O
+Figure	O
+S1A	O
+).	O
+	O
+A	O
+triple	O
+mutation	O
+of	O
+the	O
+conserved	O
+positively	O
+charged	O
+residues	O
+R60	O
+,	O
+K65	O
+and	O
+R131	O
+to	O
+A	O
+in	O
+ScTsr3	O
+resulted	O
+in	O
+a	O
+protein	O
+with	O
+a	O
+significantly	O
+impaired	O
+acp	O
+transferase	O
+activity	O
+in	O
+vivo	O
+(	O
+Figure	O
+6D	O
+)	O
+in	O
+line	O
+with	O
+an	O
+important	O
+functional	O
+role	O
+for	O
+these	O
+positively	O
+charged	O
+residues	O
+.	O
+	O
+(	O
+A	O
+)	O
+Electrostatic	O
+charge	O
+distribution	O
+on	O
+the	O
+surface	O
+of	O
+VdTsr3	O
+.	O
+	O
+SAM	O
+is	O
+shown	O
+in	O
+a	O
+stick	O
+representation	O
+.	O
+	O
+Conserved	O
+basic	O
+amino	O
+acids	O
+are	O
+labeled	O
+.	O
+(	O
+B	O
+)	O
+Comparison	O
+of	O
+the	O
+secondary	O
+structures	O
+of	O
+helix	O
+31	O
+from	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+rRNAs	O
+in	O
+S	O
+.	O
+cerevisiae	O
+and	O
+S	O
+.	O
+solfataricus	O
+with	O
+the	O
+location	O
+of	O
+the	O
+hypermodified	O
+nucleotide	O
+indicated	O
+in	O
+red	O
+.	O
+	O
+For	O
+S	O
+.	O
+solfataricus	O
+the	O
+chemical	O
+identity	O
+of	O
+the	O
+hypermodified	O
+nucleotide	O
+is	O
+not	O
+known	O
+but	O
+the	O
+existence	O
+of	O
+NEP1	O
+and	O
+TSR3	O
+homologs	O
+suggest	O
+that	O
+it	O
+is	O
+indeed	O
+N1	O
+-	O
+methyl	O
+-	O
+N3	O
+-	O
+acp	O
+-	O
+pseudouridine	O
+.	O
+	O
+(	O
+C	O
+)	O
+Binding	O
+of	O
+SsTsr3	O
+to	O
+RNA	O
+.	O
+	O
+5	O
+′-	O
+fluoresceine	O
+labeled	O
+RNA	O
+oligonucleotides	O
+corresponding	O
+either	O
+to	O
+the	O
+native	O
+(	O
+20mer	O
+–	O
+see	O
+inset	O
+)	O
+or	O
+a	O
+stabilized	O
+(	O
+20mer_GC	O
+-	O
+inset	O
+)	O
+helix	O
+31	O
+of	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+rRNA	O
+from	O
+S	O
+.	O
+solfataricus	O
+were	O
+titrated	O
+with	O
+increasing	O
+amounts	O
+of	O
+SsTsr3	O
+and	O
+the	O
+changes	O
+in	O
+the	O
+fluoresceine	O
+fluorescence	O
+anisotropy	O
+were	O
+measured	O
+and	O
+fitted	O
+to	O
+a	O
+binding	O
+curve	O
+(	O
+20mer	O
+–	O
+red	O
+,	O
+20mer_GC	O
+–	O
+blue	O
+).	O
+	O
+Oligo	O
+-	O
+U9	O
+-	O
+RNA	O
+was	O
+used	O
+for	O
+comparison	O
+(	O
+black	O
+).	O
+	O
+The	O
+20mer_GC	O
+RNA	O
+was	O
+also	O
+titrated	O
+with	O
+SsTsr3	O
+in	O
+the	O
+presence	O
+of	O
+2	O
+mM	O
+SAM	O
+(	O
+purple	O
+).	O
+(	O
+D	O
+)	O
+Mutants	O
+of	O
+ScTsr3	O
+R60	O
+,	O
+K65	O
+or	O
+R131	O
+(	O
+equivalent	O
+to	O
+K17	O
+,	O
+K22	O
+and	O
+R91	O
+in	O
+VdTsr3	O
+)	O
+expressed	O
+in	O
+Δtsr3	B-mutant
+yeast	O
+cells	O
+show	O
+a	O
+primer	O
+extension	O
+stop	O
+comparable	O
+to	O
+the	O
+wild	O
+type	O
+.	O
+	O
+Combination	O
+of	O
+the	O
+three	O
+point	O
+mutations	O
+(	O
+R60A	B-mutant
+/	O
+K65A	B-mutant
+/	O
+R131A	B-mutant
+)	O
+leads	O
+to	O
+a	O
+strongly	O
+reduced	O
+acp	O
+modification	O
+of	O
+18S	O
+rRNA	O
+.	O
+	O
+SsTsr3	O
+in	O
+the	O
+apo	O
+state	O
+bound	O
+a	O
+20mer	O
+RNA	O
+corresponding	O
+to	O
+helix	O
+31	O
+of	O
+S	O
+.	O
+solfataricus	O
+16S	O
+rRNA	O
+(	O
+Figure	O
+6B	O
+)	O
+with	O
+a	O
+KD	O
+of	O
+1	O
+.	O
+9	O
+μM	O
+and	O
+to	O
+a	O
+version	O
+of	O
+this	O
+hairpin	O
+stabilized	O
+by	O
+additional	O
+GC	O
+base	O
+pairs	O
+(	O
+20mer	O
+-	O
+GC	O
+)	O
+with	O
+a	O
+KD	O
+of	O
+0	O
+.	O
+6	O
+μM	O
+(	O
+Figure	O
+6C	O
+).	O
+	O
+U1191	O
+is	O
+the	O
+only	O
+hypermodified	O
+base	O
+in	O
+the	O
+yeast	O
+18S	O
+rRNA	O
+and	O
+is	O
+strongly	O
+conserved	O
+in	O
+eukaryotes	O
+.	O
+	O
+The	O
+formation	O
+of	O
+1	O
+-	O
+methyl	O
+-	O
+3	O
+-(	O
+3	O
+-	O
+amino	O
+-	O
+3	O
+-	O
+carboxypropyl	O
+)-	O
+pseudouridine	O
+(	O
+m1acp3Ψ	O
+)	O
+is	O
+very	O
+complex	O
+requiring	O
+three	O
+successive	O
+modification	O
+reactions	O
+involving	O
+one	O
+H	O
+/	O
+ACA	O
+snoRNP	O
+(	O
+snR35	O
+)	O
+and	O
+two	O
+protein	O
+enzymes	O
+(	O
+Nep1	O
+/	O
+Emg1	O
+and	O
+Tsr3	O
+).	O
+	O
+The	O
+m1acp3Ψ	O
+base	O
+is	O
+located	O
+at	O
+the	O
+tip	O
+of	O
+helix	O
+31	O
+on	O
+the	O
+18S	O
+rRNA	O
+(	O
+Supplementary	O
+Figure	O
+S1B	O
+)	O
+which	O
+,	O
+together	O
+with	O
+helices	O
+18	O
+,	O
+24	O
+,	O
+34	O
+and	O
+44	O
+,	O
+contribute	O
+to	O
+building	O
+the	O
+decoding	O
+center	O
+of	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+.	O
+	O
+As	O
+shown	O
+here	O
+TSR3	O
+encodes	O
+the	O
+transferase	O
+catalyzing	O
+the	O
+acp	O
+modification	O
+as	O
+the	O
+last	O
+step	O
+in	O
+the	O
+biosynthesis	O
+of	O
+m1acp3Ψ	O
+in	O
+yeast	O
+and	O
+human	O
+cells	O
+.	O
+	O
+Unexpectedly	O
+,	O
+archaeal	O
+Tsr3	O
+has	O
+a	O
+structure	O
+similar	O
+to	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+methyltransferases	O
+,	O
+and	O
+it	O
+is	O
+the	O
+first	O
+example	O
+for	O
+an	O
+enzyme	O
+of	O
+this	O
+class	O
+transferring	O
+an	O
+acp	O
+group	O
+,	O
+due	O
+to	O
+a	O
+modified	O
+SAM	O
+-	O
+binding	O
+pocket	O
+that	O
+exposes	O
+the	O
+acp	O
+instead	O
+of	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	O
+to	O
+its	O
+RNA	O
+substrate	O
+.	O
+	O
+Similar	O
+to	O
+the	O
+structurally	O
+unrelated	O
+Rossmann	O
+-	O
+fold	O
+Tyw2	O
+acp	O
+transferase	O
+,	O
+the	O
+SAM	O
+methyl	O
+group	O
+of	O
+Tsr3	O
+is	O
+bound	O
+in	O
+an	O
+inaccessible	O
+hydrophobic	O
+pocket	O
+whereas	O
+the	O
+acp	O
+side	O
+chain	O
+becomes	O
+accessible	O
+for	O
+a	O
+nucleophilic	O
+attack	O
+by	O
+the	O
+N3	O
+of	O
+pseudouridine	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+in	O
+the	O
+structurally	O
+closely	O
+related	O
+RNA	O
+methyltransferase	O
+Trm10	O
+the	O
+methyl	O
+group	O
+of	O
+the	O
+cofactor	O
+SAM	O
+is	O
+accessible	O
+whereas	O
+its	O
+acp	O
+side	O
+chain	O
+is	O
+buried	O
+inside	O
+the	O
+protein	O
+.	O
+	O
+Thus	O
+,	O
+additional	O
+examples	O
+for	O
+acp	O
+transferase	O
+enzymes	O
+might	O
+be	O
+found	O
+with	O
+similarities	O
+to	O
+other	O
+structural	O
+classes	O
+of	O
+methyltransferases	O
+.	O
+	O
+This	O
+suggests	O
+that	O
+Tsr3	O
+is	O
+not	O
+stably	O
+incorporated	O
+into	O
+pre	O
+-	O
+ribosomal	O
+particles	O
+and	O
+that	O
+its	O
+binding	O
+to	O
+the	O
+nascent	O
+ribosomal	O
+subunit	O
+possibly	O
+requires	O
+additional	O
+interactions	O
+with	O
+other	O
+pre	O
+-	O
+ribosomal	O
+components	O
+.	O
+	O
+Consistently	O
+,	O
+in	O
+sucrose	O
+gradient	O
+analysis	O
+,	O
+Tsr3	O
+was	O
+found	O
+in	O
+low	O
+-	O
+molecular	O
+weight	O
+fractions	O
+rather	O
+than	O
+with	O
+pre	O
+-	O
+ribosome	O
+containing	O
+high	O
+-	O
+molecular	O
+weight	O
+fractions	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+several	O
+enzymes	O
+that	O
+catalyze	O
+base	O
+specific	O
+modifications	O
+in	O
+rRNAs	O
+Tsr3	O
+is	O
+not	O
+an	O
+essential	O
+protein	O
+.	O
+	O
+Typically	O
+,	O
+other	O
+small	O
+subunit	O
+rRNA	O
+methyltransferases	O
+as	O
+Dim1	O
+,	O
+Bud23	O
+and	O
+Nep1	O
+/	O
+Emg1	O
+carry	O
+dual	O
+functions	O
+,	O
+in	O
+ribosome	O
+biogenesis	O
+and	O
+rRNA	O
+modification	O
+,	O
+and	O
+it	O
+is	O
+their	O
+involvement	O
+in	O
+pre	O
+-	O
+RNA	O
+processing	O
+that	O
+is	O
+essential	O
+rather	O
+than	O
+their	O
+RNA	O
+-	O
+methylating	O
+activity	O
+(,	O
+discussed	O
+in	O
+7	O
+).	O
+	O
+In	O
+contrast	O
+,	O
+for	O
+several	O
+Tsr3	O
+mutants	O
+(	O
+SAM	O
+-	O
+binding	O
+and	O
+cysteine	O
+mutations	O
+)	O
+we	O
+found	O
+a	O
+systematic	O
+correlation	O
+between	O
+the	O
+loss	O
+of	O
+acp	O
+modification	O
+and	O
+the	O
+efficiency	O
+of	O
+18S	O
+rRNA	O
+maturation	O
+.	O
+	O
+Apart	O
+from	O
+most	O
+of	O
+the	O
+ribosomal	O
+proteins	O
+,	O
+cytoplasmic	O
+pre	O
+-	O
+40S	O
+particles	O
+contain	O
+20S	O
+rRNA	O
+and	O
+at	O
+least	O
+seven	O
+non	O
+-	O
+ribosomal	O
+proteins	O
+including	O
+the	O
+D	O
+-	O
+site	O
+endonuclease	O
+Nob1	O
+as	O
+well	O
+as	O
+Tsr1	O
+,	O
+a	O
+putative	O
+GTPase	O
+and	O
+Rio2	O
+which	O
+block	O
+the	O
+mRNA	O
+channel	O
+and	O
+the	O
+initiator	O
+tRNA	O
+binding	O
+site	O
+,	O
+respectively	O
+,	O
+thus	O
+preventing	O
+translation	O
+initiation	O
+.	O
+	O
+Finally	O
+,	O
+termination	O
+factor	O
+Rli1	O
+,	O
+an	O
+ATPase	O
+,	O
+promotes	O
+the	O
+dissociation	O
+of	O
+assembly	O
+factors	O
+and	O
+the	O
+80S	O
+-	O
+like	O
+complex	O
+dissociates	O
+and	O
+releases	O
+the	O
+mature	O
+40S	O
+subunit	O
+.	O
+	O
+Interestingly	O
+,	O
+differences	O
+in	O
+the	O
+level	O
+of	O
+acp	O
+modification	O
+were	O
+demonstrated	O
+for	O
+different	O
+steps	O
+of	O
+the	O
+cytoplasmic	O
+pre	O
+-	O
+40S	O
+subunit	O
+maturation	O
+after	O
+analyzing	O
+purified	O
+20S	O
+pre	O
+-	O
+rRNAs	O
+using	O
+different	O
+purification	O
+bait	O
+proteins	O
+.	O
+	O
+Early	O
+cytoplasmic	O
+pre	O
+-	O
+40S	O
+subunits	O
+still	O
+containing	O
+the	O
+ribosome	O
+assembly	O
+factors	O
+Tsr1	O
+,	O
+Ltv1	O
+,	O
+Enp1	O
+and	O
+Rio2	O
+were	O
+not	O
+or	O
+only	O
+partially	O
+acp	O
+modified	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+late	O
+pre	O
+-	O
+40S	O
+subunits	O
+containing	O
+Nob1	O
+and	O
+Rio1	O
+or	O
+already	O
+associated	O
+with	O
+60S	O
+subunits	O
+in	O
+80S	O
+-	O
+like	O
+particles	O
+showed	O
+acp	O
+modification	O
+levels	O
+comparable	O
+to	O
+mature	O
+40S	O
+subunits	O
+.	O
+	O
+These	O
+data	O
+and	O
+the	O
+finding	O
+that	O
+a	O
+missing	O
+acp	O
+modification	O
+hinders	O
+pre	O
+-	O
+20S	O
+rRNA	O
+processing	O
+,	O
+suggest	O
+that	O
+the	O
+acp	O
+modification	O
+together	O
+with	O
+the	O
+release	O
+of	O
+Rio2	O
+promotes	O
+the	O
+formation	O
+of	O
+the	O
+decoding	O
+site	O
+and	O
+thus	O
+D	O
+-	O
+site	O
+cleavage	O
+by	O
+Nob1	O
+.	O
+	O
+The	O
+interrelation	O
+between	O
+acp	O
+modification	O
+and	O
+Rio2	O
+release	O
+is	O
+also	O
+supported	O
+by	O
+CRAC	O
+analysis	O
+showing	O
+that	O
+Rio2	O
+binds	O
+to	O
+helix	O
+31	O
+next	O
+to	O
+the	O
+Ψ1191	O
+residue	O
+that	O
+receives	O
+the	O
+acp	O
+modification	O
+.	O
+	O
+Therefore	O
+,	O
+Rio2	O
+either	O
+blocks	O
+the	O
+access	O
+of	O
+Tsr3	O
+to	O
+helix	O
+31	O
+,	O
+and	O
+acp	O
+modification	O
+can	O
+only	O
+occur	O
+after	O
+Rio2	O
+is	O
+released	O
+,	O
+or	O
+the	O
+acp	O
+modification	O
+of	O
+m1Ψ1191	O
+and	O
+putative	O
+subsequent	O
+conformational	O
+changes	O
+of	O
+20S	O
+rRNA	O
+weaken	O
+the	O
+binding	O
+of	O
+Rio2	O
+to	O
+helix	O
+31	O
+and	O
+support	O
+its	O
+release	O
+from	O
+the	O
+pre	O
+-	O
+rRNA	O
+.	O
+	O
+In	O
+summary	O
+,	O
+by	O
+identifying	O
+Tsr3	O
+as	O
+the	O
+enzyme	O
+responsible	O
+for	O
+introducing	O
+the	O
+acp	O
+group	O
+to	O
+the	O
+hypermodified	O
+m1acp3Ψ	O
+nucleotide	O
+at	O
+position	O
+1191	O
+(	O
+yeast	O
+)/	O
+1248	O
+(	O
+humans	O
+)	O
+of	O
+18S	O
+rRNA	O
+we	O
+added	O
+one	O
+of	O
+the	O
+last	O
+remaining	O
+pieces	O
+to	O
+the	O
+puzzle	O
+of	O
+eukaryotic	O
+small	O
+ribosomal	O
+subunit	O
+rRNA	O
+modifications	O
+.	O
+	O
+Structural	O
+insights	O
+into	O
+the	O
+regulatory	O
+mechanism	O
+of	O
+the	O
+Pseudomonas	O
+aeruginosa	O
+YfiBNR	O
+system	O
+	O
+YfiBNR	O
+is	O
+a	O
+recently	O
+identified	O
+bis	O
+-(	O
+3	O
+’-	O
+5	O
+’)-	O
+cyclic	O
+dimeric	O
+GMP	O
+(	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+)	O
+signaling	O
+system	O
+in	O
+opportunistic	O
+pathogens	O
+.	O
+	O
+In	O
+response	O
+to	O
+cell	O
+stress	O
+,	O
+YfiB	O
+in	O
+the	O
+outer	O
+membrane	O
+can	O
+sequester	O
+the	O
+periplasmic	O
+protein	O
+YfiR	O
+,	O
+releasing	O
+its	O
+inhibition	O
+of	O
+YfiN	O
+on	O
+the	O
+inner	O
+membrane	O
+and	O
+thus	O
+provoking	O
+the	O
+diguanylate	O
+cyclase	O
+activity	O
+of	O
+YfiN	O
+to	O
+induce	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+production	O
+.	O
+	O
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	O
+structures	O
+of	O
+YfiB	O
+alone	O
+and	O
+of	O
+an	O
+active	O
+mutant	O
+YfiBL43P	B-mutant
+complexed	O
+with	O
+YfiR	O
+with	O
+2	O
+:	O
+2	O
+stoichiometry	O
+.	O
+	O
+Structural	O
+analyses	O
+revealed	O
+that	O
+in	O
+contrast	O
+to	O
+the	O
+compact	O
+conformation	O
+of	O
+the	O
+dimeric	O
+YfiB	O
+alone	O
+,	O
+YfiBL43P	B-mutant
+adopts	O
+a	O
+stretched	O
+conformation	O
+allowing	O
+activated	O
+YfiB	O
+to	O
+penetrate	O
+the	O
+peptidoglycan	O
+(	O
+PG	O
+)	O
+layer	O
+and	O
+access	O
+YfiR	O
+.	O
+YfiBL43P	B-mutant
+shows	O
+a	O
+more	O
+compact	O
+PG	O
+-	O
+binding	O
+pocket	O
+and	O
+much	O
+higher	O
+PG	O
+binding	O
+affinity	O
+than	O
+wild	O
+-	O
+type	O
+YfiB	O
+,	O
+suggesting	O
+a	O
+tight	O
+correlation	O
+between	O
+PG	O
+binding	O
+and	O
+YfiB	O
+activation	O
+.	O
+	O
+Based	O
+on	O
+the	O
+structural	O
+and	O
+biochemical	O
+data	O
+,	O
+we	O
+propose	O
+an	O
+updated	O
+regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	O
+system	O
+.	O
+	O
+Bis	O
+-(	O
+3	O
+’-	O
+5	O
+’)-	O
+cyclic	O
+dimeric	O
+GMP	O
+(	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+)	O
+is	O
+a	O
+ubiquitous	O
+second	O
+messenger	O
+that	O
+bacteria	O
+use	O
+to	O
+facilitate	O
+behavioral	O
+adaptations	O
+to	O
+their	O
+ever	O
+-	O
+changing	O
+environment	O
+.	O
+	O
+Intriguingly	O
+,	O
+studies	O
+in	O
+diverse	O
+species	O
+have	O
+revealed	O
+that	O
+a	O
+single	O
+bacterium	O
+can	O
+have	O
+dozens	O
+of	O
+DGCs	O
+and	O
+PDEs	O
+(	O
+Hickman	O
+et	O
+al	O
+.,;	O
+Kirillina	O
+et	O
+al	O
+.,;	O
+Kulasakara	O
+et	O
+al	O
+.,;	O
+Tamayo	O
+et	O
+al	O
+.,).	O
+	O
+In	O
+Pseudomonas	O
+aeruginosa	O
+in	O
+particular	O
+,	O
+42	O
+genes	O
+containing	O
+putative	O
+DGCs	O
+and	O
+/	O
+or	O
+PDEs	O
+were	O
+identified	O
+(	O
+Kulasakara	O
+et	O
+al	O
+.,).	O
+	O
+The	O
+functional	O
+role	O
+of	O
+a	O
+number	O
+of	O
+downstream	O
+effectors	O
+of	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+has	O
+been	O
+characterized	O
+as	O
+affecting	O
+exopolysaccharide	O
+(	O
+EPS	O
+)	O
+production	O
+,	O
+transcription	O
+,	O
+motility	O
+,	O
+and	O
+surface	O
+attachment	O
+(	O
+Caly	O
+et	O
+al	O
+.,;	O
+Camilli	O
+and	O
+Bassler	O
+,;	O
+Ha	O
+and	O
+O	O
+’	O
+Toole	O
+,;	O
+Pesavento	O
+and	O
+Hengge	O
+,).	O
+	O
+However	O
+,	O
+due	O
+to	O
+the	O
+intricacy	O
+of	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+signaling	O
+networks	O
+and	O
+the	O
+diversity	O
+of	O
+experimental	O
+cues	O
+,	O
+the	O
+detailed	O
+mechanisms	O
+by	O
+which	O
+these	O
+signaling	O
+pathways	O
+specifically	O
+sense	O
+and	O
+integrate	O
+different	O
+inputs	O
+remain	O
+largely	O
+elusive	O
+.	O
+	O
+Biofilm	O
+formation	O
+protects	O
+pathogenic	O
+bacteria	O
+from	O
+antibiotic	O
+treatment	O
+,	O
+and	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+-	O
+regulated	O
+biofilm	O
+formation	O
+has	O
+been	O
+extensively	O
+studied	O
+in	O
+P	O
+.	O
+aeruginosa	O
+(	O
+Evans	O
+,;	O
+Kirisits	O
+et	O
+al	O
+.,;	O
+Malone	O
+,;	O
+Reinhardt	O
+et	O
+al	O
+.,).	O
+	O
+In	O
+the	O
+lungs	O
+of	O
+cystic	O
+fibrosis	O
+(	O
+CF	O
+)	O
+patients	O
+,	O
+adherent	O
+biofilm	O
+formation	O
+and	O
+the	O
+appearance	O
+of	O
+small	O
+colony	O
+variant	O
+(	O
+SCV	O
+)	O
+morphologies	O
+of	O
+P	O
+.	O
+aeruginosa	O
+correlate	O
+with	O
+prolonged	O
+persistence	O
+of	O
+infection	O
+and	O
+poor	O
+lung	O
+function	O
+(	O
+Govan	O
+and	O
+Deretic	O
+,;	O
+Haussler	O
+et	O
+al	O
+.,;	O
+Haussler	O
+et	O
+al	O
+.,;	O
+Parsek	O
+and	O
+Singh	O
+,;	O
+Smith	O
+et	O
+al	O
+.,).	O
+	O
+Recently	O
+,	O
+Malone	O
+and	O
+coworkers	O
+identified	O
+the	O
+tripartite	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+signaling	O
+module	O
+system	O
+YfiBNR	O
+(	O
+also	O
+known	O
+as	O
+AwsXRO	O
+(	O
+Beaumont	O
+et	O
+al	O
+.,;	O
+Giddens	O
+et	O
+al	O
+.,)	O
+or	O
+Tbp	O
+(	O
+Ueda	O
+and	O
+Wood	O
+,))	O
+by	O
+genetic	O
+screening	O
+for	O
+mutants	O
+that	O
+displayed	O
+SCV	O
+phenotypes	O
+in	O
+P	O
+.	O
+aeruginosa	O
+PAO1	O
+(	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+YfiB	O
+is	O
+an	O
+OmpA	O
+/	O
+Pal	O
+-	O
+like	O
+outer	O
+-	O
+membrane	O
+lipoprotein	O
+(	O
+Parsons	O
+et	O
+al	O
+.,)	O
+that	O
+can	O
+activate	O
+YfiN	O
+by	O
+sequestering	O
+YfiR	O
+(	O
+Malone	O
+et	O
+al	O
+.,)	O
+in	O
+an	O
+unknown	O
+manner	O
+.	O
+	O
+It	O
+is	O
+also	O
+proposed	O
+that	O
+the	O
+sequestration	O
+of	O
+YfiR	O
+by	O
+YfiB	O
+can	O
+be	O
+induced	O
+by	O
+certain	O
+YfiB	O
+-	O
+mediated	O
+cell	O
+wall	O
+stress	O
+,	O
+and	O
+mutagenesis	O
+studies	O
+revealed	O
+a	O
+number	O
+of	O
+activation	O
+residues	O
+of	O
+YfiB	O
+that	O
+were	O
+located	O
+in	O
+close	O
+proximity	O
+to	O
+the	O
+predicted	O
+first	O
+helix	O
+of	O
+the	O
+periplasmic	O
+domain	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+In	O
+the	O
+present	O
+study	O
+,	O
+we	O
+solved	O
+the	O
+crystal	O
+structures	O
+of	O
+an	O
+N	O
+-	O
+terminal	O
+truncated	O
+form	O
+of	O
+YfiB	O
+(	O
+34	O
+–	O
+168	O
+)	O
+and	O
+YfiR	O
+in	O
+complex	O
+with	O
+an	O
+active	O
+mutant	O
+YfiBL43P	B-mutant
+.	O
+	O
+Most	O
+recently	O
+,	O
+Li	O
+and	O
+coworkers	O
+reported	O
+the	O
+crystal	O
+structures	O
+of	O
+YfiB	O
+(	O
+27	O
+–	O
+168	O
+)	O
+alone	O
+and	O
+YfiRC71S	B-mutant
+in	O
+complex	O
+with	O
+YfiB	O
+(	O
+59	O
+–	O
+168	O
+)	O
+(	O
+Li	O
+et	O
+al	O
+.,).	O
+	O
+Compared	O
+with	O
+the	O
+reported	O
+complex	O
+structure	O
+,	O
+YfiBL43P	B-mutant
+in	O
+our	O
+YfiB	O
+-	O
+YfiR	O
+complex	O
+structure	O
+has	O
+additional	O
+visible	O
+N	O
+-	O
+terminal	O
+residues	O
+44	O
+–	O
+58	O
+that	O
+are	O
+shown	O
+to	O
+play	O
+essential	O
+roles	O
+in	O
+YfiB	O
+activation	O
+and	O
+biofilm	O
+formation	O
+.	O
+	O
+Therefore	O
+,	O
+we	O
+are	O
+able	O
+to	O
+visualize	O
+the	O
+detailed	O
+allosteric	O
+arrangement	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+structure	O
+of	O
+YfiB	O
+and	O
+its	O
+important	O
+role	O
+in	O
+YfiB	O
+-	O
+YfiR	O
+interaction	O
+.	O
+	O
+In	O
+addition	O
+,	O
+we	O
+found	O
+that	O
+the	O
+YfiBL43P	B-mutant
+shows	O
+a	O
+much	O
+higher	O
+PG	O
+-	O
+binding	O
+affinity	O
+than	O
+wild	O
+-	O
+type	O
+YfiB	O
+,	O
+most	O
+likely	O
+due	O
+to	O
+its	O
+more	O
+compact	O
+PG	O
+-	O
+binding	O
+pocket	O
+.	O
+	O
+Moreover	O
+,	O
+we	O
+found	O
+that	O
+Vitamin	O
+B6	O
+(	O
+VB6	O
+)	O
+or	O
+L	O
+-	O
+Trp	O
+can	O
+bind	O
+YfiR	O
+with	O
+an	O
+affinity	O
+in	O
+the	O
+ten	O
+millimolar	O
+range	O
+.	O
+	O
+Together	O
+with	O
+functional	O
+data	O
+,	O
+these	O
+results	O
+provide	O
+new	O
+mechanistic	O
+insights	O
+into	O
+how	O
+activated	O
+YfiB	O
+sequesters	O
+YfiR	O
+and	O
+releases	O
+the	O
+suppression	O
+of	O
+YfiN	O
+.	O
+These	O
+findings	O
+may	O
+facilitate	O
+the	O
+development	O
+and	O
+optimization	O
+of	O
+anti	O
+-	O
+biofilm	O
+drugs	O
+for	O
+the	O
+treatment	O
+of	O
+chronic	O
+infections	O
+.	O
+	O
+Overall	O
+structure	O
+of	O
+YfiB	O
+	O
+We	O
+obtained	O
+two	O
+crystal	O
+forms	O
+of	O
+YfiB	O
+(	O
+residues	O
+34	O
+–	O
+168	O
+,	O
+lacking	O
+the	O
+signal	O
+peptide	O
+from	O
+residues	O
+1	O
+–	O
+26	O
+and	O
+periplasmic	O
+residues	O
+27	O
+–	O
+33	O
+),	O
+crystal	O
+forms	O
+I	O
+and	O
+II	O
+,	O
+belonging	O
+to	O
+space	O
+groups	O
+P21	O
+and	O
+P41	O
+,	O
+respectively	O
+.	O
+	O
+Overall	O
+structure	O
+of	O
+YfiB	O
+.	O
+(	O
+A	O
+)	O
+The	O
+overall	O
+structure	O
+of	O
+the	O
+YfiB	O
+monomer	O
+.	O
+(	O
+B	O
+)	O
+A	O
+topology	O
+diagram	O
+of	O
+the	O
+YfiB	O
+monomer	O
+.	O
+(	O
+C	O
+and	O
+D	O
+)	O
+The	O
+analytical	O
+ultracentrifugation	O
+experiment	O
+results	O
+for	O
+the	O
+wild	O
+-	O
+type	O
+YfiB	O
+and	O
+YfiBL43P	B-mutant
+	O
+Two	O
+dimeric	O
+types	O
+of	O
+YfiB	O
+dimer	O
+.	O
+(	O
+A	O
+–	O
+C	O
+)	O
+The	O
+“	O
+head	O
+to	O
+head	O
+”	O
+dimer	O
+.	O
+	O
+(	O
+A	O
+)	O
+and	O
+(	O
+E	O
+)	O
+indicate	O
+the	O
+front	O
+views	O
+of	O
+the	O
+two	O
+dimers	O
+,	O
+(	O
+B	O
+)	O
+and	O
+(	O
+F	O
+)	O
+indicate	O
+the	O
+top	O
+views	O
+of	O
+the	O
+two	O
+dimers	O
+,	O
+and	O
+(	O
+C	O
+)	O
+and	O
+(	O
+D	O
+)	O
+indicate	O
+the	O
+details	O
+of	O
+the	O
+two	O
+dimeric	O
+interfaces	O
+	O
+In	O
+addition	O
+,	O
+there	O
+is	O
+a	O
+short	O
+helix	O
+turn	O
+connecting	O
+the	O
+β4	O
+strand	O
+and	O
+α4	O
+helix	O
+(	O
+Fig	O
+.	O
+1A	O
+and	O
+1B	O
+).	O
+	O
+Each	O
+crystal	O
+form	O
+contains	O
+three	O
+different	O
+dimeric	O
+types	O
+of	O
+YfiB	O
+,	O
+two	O
+of	O
+which	O
+are	O
+present	O
+in	O
+both	O
+,	O
+suggesting	O
+that	O
+the	O
+rest	O
+of	O
+the	O
+dimeric	O
+types	O
+may	O
+result	O
+from	O
+crystal	O
+packing	O
+.	O
+	O
+Here	O
+,	O
+we	O
+refer	O
+to	O
+the	O
+two	O
+dimeric	O
+types	O
+as	O
+“	O
+head	O
+to	O
+head	O
+”	O
+and	O
+“	O
+back	O
+to	O
+back	O
+”	O
+according	O
+to	O
+the	O
+interacting	O
+mode	O
+(	O
+Fig	O
+.	O
+2A	O
+and	O
+2E	O
+),	O
+with	O
+the	O
+total	O
+buried	O
+surface	O
+areas	O
+being	O
+316	O
+.	O
+8	O
+Å2	O
+and	O
+554	O
+.	O
+3	O
+Å2	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+“	O
+head	O
+to	O
+head	O
+”	O
+dimer	O
+exhibits	O
+a	O
+clamp	O
+shape	O
+.	O
+	O
+The	O
+dimeric	O
+interaction	O
+is	O
+mainly	O
+hydrophilic	O
+,	O
+occurring	O
+among	O
+the	O
+main	O
+-	O
+chain	O
+and	O
+side	O
+-	O
+chain	O
+atoms	O
+of	O
+N68	O
+,	O
+L69	O
+,	O
+D70	O
+and	O
+R71	O
+on	O
+the	O
+α2	O
+-	O
+α3	O
+loops	O
+and	O
+R116	O
+and	O
+S120	O
+on	O
+the	O
+α4	O
+helices	O
+of	O
+both	O
+molecules	O
+,	O
+resulting	O
+in	O
+a	O
+complex	O
+hydrogen	O
+bond	O
+network	O
+(	O
+Fig	O
+.	O
+2D	O
+–	O
+F	O
+).	O
+	O
+The	O
+YfiB	O
+-	O
+YfiR	O
+interaction	O
+	O
+The	O
+YfiBL43P	B-mutant
+molecules	O
+are	O
+shown	O
+in	O
+cyan	O
+and	O
+yellow	O
+.	O
+	O
+The	O
+YfiR	O
+molecules	O
+are	O
+shown	O
+in	O
+green	O
+and	O
+magenta	O
+.	O
+	O
+To	O
+illustrate	O
+the	O
+differences	O
+between	O
+apo	O
+YfiB	O
+and	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+,	O
+the	O
+apo	O
+YfiB	O
+is	O
+shown	O
+in	O
+pink	O
+,	O
+except	O
+residues	O
+34	O
+–	O
+70	O
+are	O
+shown	O
+in	O
+red	O
+,	O
+whereas	O
+the	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+is	O
+shown	O
+in	O
+cyan	O
+,	O
+except	O
+residues	O
+44	O
+–	O
+70	O
+are	O
+shown	O
+in	O
+blue	O
+.	O
+(	O
+C	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+differences	O
+between	O
+apo	O
+YfiB	O
+and	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+.	O
+	O
+The	O
+key	O
+residues	O
+in	O
+apo	O
+YfiB	O
+are	O
+shown	O
+in	O
+red	O
+and	O
+those	O
+in	O
+YfiBL43P	B-mutant
+are	O
+shown	O
+in	O
+blue	O
+.	O
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+views	O
+showing	O
+interactions	O
+in	O
+regions	O
+I	O
+and	O
+II	O
+.	O
+	O
+YfiBL43P	B-mutant
+and	O
+YfiR	O
+are	O
+shown	O
+in	O
+cyan	O
+and	O
+green	O
+,	O
+respectively	O
+.	O
+(	O
+E	O
+and	O
+F	O
+)	O
+The	O
+conserved	O
+surface	O
+in	O
+YfiR	O
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	O
+.	O
+(	O
+G	O
+)	O
+The	O
+residues	O
+of	O
+YfiR	O
+responsible	O
+for	O
+interacting	O
+with	O
+YfiB	O
+are	O
+shown	O
+in	O
+green	O
+sticks	O
+,	O
+and	O
+the	O
+proposed	O
+YfiN	O
+-	O
+interacting	O
+residues	O
+are	O
+shown	O
+in	O
+yellow	O
+sticks	O
+.	O
+	O
+The	O
+red	O
+sticks	O
+,	O
+which	O
+represent	O
+the	O
+YfiB	O
+-	O
+interacting	O
+residues	O
+,	O
+are	O
+also	O
+responsible	O
+for	O
+the	O
+proposed	O
+interactions	O
+with	O
+YfiN	O
+	O
+To	O
+gain	O
+structural	O
+insights	O
+into	O
+the	O
+YfiB	O
+-	O
+YfiR	O
+interaction	O
+,	O
+we	O
+co	O
+-	O
+expressed	O
+YfiB	O
+(	O
+residues	O
+34	O
+–	O
+168	O
+)	O
+and	O
+YfiR	O
+(	O
+residues	O
+35	O
+–	O
+190	O
+,	O
+lacking	O
+the	O
+signal	O
+peptide	O
+),	O
+but	O
+failed	O
+to	O
+obtain	O
+the	O
+complex	O
+,	O
+in	O
+accordance	O
+with	O
+a	O
+previous	O
+report	O
+in	O
+which	O
+no	O
+stable	O
+complex	O
+of	O
+YfiB	O
+-	O
+YfiR	O
+was	O
+observed	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+It	O
+is	O
+likely	O
+that	O
+these	O
+residues	O
+may	O
+be	O
+involved	O
+in	O
+the	O
+conformational	O
+changes	O
+of	O
+YfiB	O
+that	O
+are	O
+related	O
+to	O
+YfiR	O
+sequestration	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+	O
+Therefore	O
+,	O
+we	O
+constructed	O
+two	O
+such	O
+single	O
+mutants	O
+of	O
+YfiB	O
+(	O
+YfiBL43P	B-mutant
+and	O
+YfiBF48S	B-mutant
+).	O
+	O
+As	O
+expected	O
+,	O
+both	O
+mutants	O
+form	O
+a	O
+stable	O
+complex	O
+with	O
+YfiR	O
+.	O
+Finally	O
+,	O
+we	O
+crystalized	O
+YfiR	O
+in	O
+complex	O
+with	O
+the	O
+YfiBL43P	B-mutant
+mutant	O
+and	O
+solved	O
+the	O
+structure	O
+at	O
+1	O
+.	O
+78	O
+Å	O
+resolution	O
+by	O
+molecular	O
+replacement	O
+using	O
+YfiR	O
+and	O
+YfiB	O
+as	O
+models	O
+.	O
+	O
+The	O
+YfiR	O
+dimer	O
+in	O
+the	O
+complex	O
+is	O
+identical	O
+to	O
+the	O
+non	O
+-	O
+oxidized	O
+YfiR	O
+dimer	O
+alone	O
+(	O
+Yang	O
+et	O
+al	O
+.,),	O
+with	O
+only	O
+Cys145	O
+-	O
+Cys152	O
+of	O
+the	O
+two	O
+disulfide	O
+bonds	O
+well	O
+formed	O
+,	O
+suggesting	O
+Cys71	O
+-	O
+Cys110	O
+disulfide	O
+bond	O
+formation	O
+is	O
+not	O
+essential	O
+for	O
+forming	O
+YfiB	O
+-	O
+YfiR	O
+complex	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+structural	O
+conformation	O
+of	O
+YfiBL43P	B-mutant
+,	O
+from	O
+the	O
+foremost	O
+N	O
+-	O
+terminus	O
+to	O
+residue	O
+D70	O
+,	O
+is	O
+significantly	O
+altered	O
+compared	O
+with	O
+that	O
+of	O
+the	O
+apo	O
+YfiB	O
+.	O
+The	O
+majority	O
+of	O
+the	O
+α1	O
+helix	O
+(	O
+residues	O
+34	O
+–	O
+43	O
+)	O
+is	O
+invisible	O
+on	O
+the	O
+electron	O
+density	O
+map	O
+,	O
+and	O
+the	O
+α2	O
+helix	O
+and	O
+β1	O
+and	O
+β2	O
+strands	O
+are	O
+rearranged	O
+to	O
+form	O
+a	O
+long	O
+loop	O
+containing	O
+two	O
+short	O
+α	O
+-	O
+helix	O
+turns	O
+(	O
+Fig	O
+.	O
+3B	O
+and	O
+3C	O
+),	O
+thus	O
+embracing	O
+the	O
+YfiR	O
+dimer	O
+.	O
+	O
+The	O
+observed	O
+changes	O
+in	O
+conformation	O
+of	O
+YfiB	O
+and	O
+the	O
+results	O
+of	O
+mutagenesis	O
+suggest	O
+a	O
+mechanism	O
+by	O
+which	O
+YfiB	O
+sequesters	O
+YfiR	O
+.	O
+	O
+The	O
+YfiB	O
+-	O
+YfiR	O
+interface	O
+can	O
+be	O
+divided	O
+into	O
+two	O
+regions	O
+(	O
+Fig	O
+.	O
+3A	O
+and	O
+3D	O
+).	O
+	O
+Region	O
+I	O
+is	O
+formed	O
+by	O
+numerous	O
+main	O
+-	O
+chain	O
+and	O
+side	O
+-	O
+chain	O
+hydrophilic	O
+interactions	O
+between	O
+residues	O
+E45	O
+,	O
+G47	O
+and	O
+E53	O
+from	O
+the	O
+N	O
+-	O
+terminal	O
+extended	O
+loop	O
+of	O
+YfiB	O
+and	O
+residues	O
+S57	O
+,	O
+R60	O
+,	O
+A89	O
+and	O
+H177	O
+from	O
+YfiR	O
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+i	O
+)).	O
+	O
+In	O
+region	O
+II	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+R96	O
+,	O
+E98	O
+and	O
+E157	O
+from	O
+YfiB	O
+interact	O
+with	O
+the	O
+side	O
+chains	O
+of	O
+E163	O
+,	O
+S146	O
+and	O
+R171	O
+from	O
+YfiR	O
+,	O
+respectively	O
+.	O
+	O
+Additionally	O
+,	O
+the	O
+main	O
+chains	O
+of	O
+I163	O
+and	O
+V165	O
+from	O
+YfiB	O
+form	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+main	O
+chains	O
+of	O
+L166	O
+and	O
+A164	O
+from	O
+YfiR	O
+,	O
+respectively	O
+,	O
+and	O
+the	O
+main	O
+chain	O
+of	O
+P166	O
+from	O
+YfiB	O
+interacts	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+R185	O
+from	O
+YfiR	O
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+II	O
+).	O
+	O
+Based	O
+on	O
+the	O
+observations	O
+that	O
+two	O
+separated	O
+YfiBL43P	B-mutant
+molecules	O
+form	O
+a	O
+2	O
+:	O
+2	O
+complex	O
+structure	O
+with	O
+YfiR	O
+dimer	O
+,	O
+we	O
+performed	O
+an	O
+analytical	O
+ultracentrifugation	O
+experiment	O
+to	O
+check	O
+the	O
+oligomeric	O
+states	O
+of	O
+wild	O
+-	O
+type	O
+YfiB	O
+and	O
+YfiBL43P	B-mutant
+.	O
+	O
+The	O
+results	O
+showed	O
+that	O
+wild	O
+-	O
+type	O
+YfiB	O
+exists	O
+in	O
+both	O
+monomeric	O
+and	O
+dimeric	O
+states	O
+in	O
+solution	O
+,	O
+while	O
+YfiBL43P	B-mutant
+primarily	O
+adopts	O
+the	O
+monomer	O
+state	O
+in	O
+solution	O
+(	O
+Fig	O
+.	O
+1C	O
+–	O
+D	O
+).	O
+	O
+For	O
+simplicity	O
+,	O
+we	O
+only	O
+discuss	O
+the	O
+“	O
+head	O
+to	O
+head	O
+”	O
+dimer	O
+in	O
+the	O
+following	O
+text	O
+.	O
+	O
+The	O
+PG	O
+-	O
+binding	O
+site	O
+in	O
+YfiB	O
+.	O
+(	O
+A	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+PG	O
+-	O
+binding	O
+sites	O
+of	O
+the	O
+H	O
+.	O
+influenzae	O
+Pal	O
+/	O
+PG	O
+-	O
+P	O
+complex	O
+and	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+complexed	O
+with	O
+sulfate	O
+ions	O
+.	O
+	O
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+showing	O
+the	O
+key	O
+residues	O
+of	O
+Pal	O
+interacting	O
+with	O
+the	O
+m	O
+-	O
+Dap5	O
+ε	O
+-	O
+carboxylate	O
+group	O
+of	O
+PG	O
+-	O
+P	O
+.	O
+Pal	O
+is	O
+shown	O
+in	O
+wheat	O
+and	O
+PG	O
+-	O
+P	O
+is	O
+in	O
+magenta	O
+.	O
+	O
+(	O
+C	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+showing	O
+the	O
+key	O
+residues	O
+of	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+interacting	O
+with	O
+a	O
+sulfate	O
+ion	O
+.	O
+	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+is	O
+shown	O
+in	O
+cyan	O
+;	O
+the	O
+sulfate	O
+ion	O
+,	O
+in	O
+green	O
+;	O
+and	O
+the	O
+water	O
+molecule	O
+,	O
+in	O
+yellow	O
+.	O
+(	O
+D	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+PG	O
+-	O
+binding	O
+sites	O
+of	O
+apo	O
+YfiB	O
+and	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+,	O
+the	O
+key	O
+residues	O
+are	O
+shown	O
+in	O
+stick	O
+.	O
+	O
+Apo	O
+YfiB	O
+is	O
+shown	O
+in	O
+yellow	O
+and	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+in	O
+cyan	O
+.	O
+(	O
+E	O
+and	O
+F	O
+)	O
+MST	O
+data	O
+and	O
+analysis	O
+for	O
+binding	O
+affinities	O
+of	O
+(	O
+E	O
+)	O
+YfiB	O
+wild	O
+-	O
+type	O
+and	O
+(	O
+F	O
+)	O
+YfiBL43P	B-mutant
+with	O
+PG	O
+.	O
+(	O
+G	O
+)	O
+The	O
+sequence	O
+alignment	O
+of	O
+P	O
+.	O
+aeruginosa	O
+and	O
+E	O
+.	O
+coli	O
+sources	O
+of	O
+YfiB	O
+,	O
+Pal	O
+and	O
+the	O
+periplasmic	O
+domain	O
+of	O
+OmpA	O
+	O
+PG	O
+-	O
+associated	O
+lipoprotein	O
+(	O
+Pal	O
+)	O
+is	O
+highly	O
+conserved	O
+in	O
+Gram	O
+-	O
+negative	O
+bacteria	O
+and	O
+anchors	O
+to	O
+the	O
+outer	O
+membrane	O
+through	O
+an	O
+N	O
+-	O
+terminal	O
+lipid	O
+attachment	O
+and	O
+to	O
+PG	O
+layer	O
+through	O
+its	O
+periplasmic	O
+domain	O
+,	O
+which	O
+is	O
+implicated	O
+in	O
+maintaining	O
+outer	O
+membrane	O
+integrity	O
+.	O
+	O
+Previous	O
+homology	O
+modeling	O
+studies	O
+suggested	O
+that	O
+YfiB	O
+contains	O
+a	O
+Pal	O
+-	O
+like	O
+PG	O
+-	O
+binding	O
+site	O
+(	O
+Parsons	O
+et	O
+al	O
+.,),	O
+and	O
+the	O
+mutation	O
+of	O
+two	O
+residues	O
+at	O
+this	O
+site	O
+,	O
+D102	O
+and	O
+G105	O
+,	O
+reduces	O
+the	O
+ability	O
+for	O
+biofilm	O
+formation	O
+and	O
+surface	O
+attachment	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+Structural	O
+superposition	O
+between	O
+YfiBL43P	B-mutant
+and	O
+Haemophilus	O
+influenzae	O
+Pal	O
+complexed	O
+with	O
+biosynthetic	O
+peptidoglycan	O
+precursor	O
+(	O
+PG	O
+-	O
+P	O
+),	O
+UDP	O
+-	O
+N	O
+-	O
+acetylmuramyl	O
+-	O
+L	O
+-	O
+Ala	O
+-	O
+α	O
+-	O
+D	O
+-	O
+Glu	O
+-	O
+m	O
+-	O
+Dap	O
+-	O
+D	O
+-	O
+Ala	O
+-	O
+D	O
+-	O
+Ala	O
+(	O
+m	O
+-	O
+Dap	O
+is	O
+meso	O
+-	O
+diaminopimelate	O
+)	O
+(	O
+PDB	O
+code	O
+:	O
+2aiz	O
+)	O
+(	O
+Parsons	O
+et	O
+al	O
+.,),	O
+revealed	O
+that	O
+the	O
+sulfate	O
+ion	O
+is	O
+located	O
+at	O
+the	O
+position	O
+of	O
+the	O
+m	O
+-	O
+Dap5	O
+ϵ	O
+-	O
+carboxylate	O
+group	O
+in	O
+the	O
+Pal	O
+/	O
+PG	O
+-	O
+P	O
+complex	O
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+	O
+Similarly	O
+,	O
+in	O
+the	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+structure	O
+,	O
+the	O
+sulfate	O
+ion	O
+interacts	O
+with	O
+the	O
+side	O
+-	O
+chain	O
+atoms	O
+of	O
+D102	O
+(	O
+corresponding	O
+to	O
+D71	O
+in	O
+Pal	O
+)	O
+and	O
+R117	O
+(	O
+corresponding	O
+to	O
+R86	O
+in	O
+Pal	O
+)	O
+and	O
+the	O
+main	O
+-	O
+chain	O
+amide	O
+of	O
+N68	O
+(	O
+corresponding	O
+to	O
+D37	O
+in	O
+Pal	O
+).	O
+	O
+In	O
+addition	O
+,	O
+sequence	O
+alignment	O
+of	O
+YfiB	O
+with	O
+Pal	O
+and	O
+the	O
+periplasmic	O
+domain	O
+of	O
+OmpA	O
+(	O
+proteins	O
+containing	O
+PG	O
+-	O
+binding	O
+site	O
+)	O
+showed	O
+that	O
+N68	O
+and	O
+D102	O
+are	O
+highly	O
+conserved	O
+(	O
+Fig	O
+.	O
+4G	O
+,	O
+blue	O
+stars	O
+),	O
+suggesting	O
+that	O
+these	O
+residues	O
+contribute	O
+to	O
+the	O
+PG	O
+-	O
+binding	O
+ability	O
+of	O
+YfiB	O
+.	O
+	O
+Interestingly	O
+,	O
+superposition	O
+of	O
+apo	O
+YfiB	O
+with	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+revealed	O
+that	O
+the	O
+PG	O
+-	O
+binding	O
+region	O
+is	O
+largely	O
+altered	O
+mainly	O
+due	O
+to	O
+different	O
+conformation	O
+of	O
+the	O
+N68	O
+containing	O
+loop	O
+.	O
+	O
+Compared	O
+to	O
+YfiBL43P	B-mutant
+,	O
+the	O
+N68	O
+-	O
+containing	O
+loop	O
+of	O
+the	O
+apo	O
+YfiB	O
+flips	O
+away	O
+about	O
+7	O
+Å	O
+,	O
+and	O
+D102	O
+and	O
+R117	O
+swing	O
+slightly	O
+outward	O
+;	O
+thus	O
+,	O
+the	O
+PG	O
+-	O
+binding	O
+pocket	O
+is	O
+enlarged	O
+with	O
+no	O
+sulfate	O
+ion	O
+or	O
+water	O
+bound	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+	O
+Therefore	O
+,	O
+we	O
+proposed	O
+that	O
+the	O
+PG	O
+-	O
+binding	O
+ability	O
+of	O
+inactive	O
+YfiB	O
+might	O
+be	O
+weaker	O
+than	O
+that	O
+of	O
+active	O
+YfiB	O
+.	O
+To	O
+validate	O
+this	O
+,	O
+we	O
+performed	O
+a	O
+microscale	O
+thermophoresis	O
+(	O
+MST	O
+)	O
+assay	O
+to	O
+measure	O
+the	O
+binding	O
+affinities	O
+of	O
+PG	O
+to	O
+wild	O
+-	O
+type	O
+YfiB	O
+and	O
+YfiBL43P	B-mutant
+,	O
+respectively	O
+.	O
+	O
+As	O
+the	O
+experiment	O
+is	O
+performed	O
+in	O
+the	O
+absence	O
+of	O
+YfiR	O
+,	O
+it	O
+suggests	O
+that	O
+an	O
+increase	O
+in	O
+the	O
+PG	O
+-	O
+binding	O
+affinity	O
+of	O
+YfiB	O
+is	O
+not	O
+a	O
+result	O
+of	O
+YfiB	O
+-	O
+YfiR	O
+interaction	O
+and	O
+is	O
+highly	O
+coupled	O
+to	O
+the	O
+activation	O
+of	O
+YfiB	O
+characterized	O
+by	O
+a	O
+stretched	O
+N	O
+-	O
+terminal	O
+conformation	O
+.	O
+	O
+The	O
+conserved	O
+surface	O
+in	O
+YfiR	O
+is	O
+functional	O
+for	O
+binding	O
+YfiB	O
+and	O
+YfiN	O
+	O
+Interestingly	O
+,	O
+the	O
+majority	O
+of	O
+this	O
+conserved	O
+surface	O
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	O
+(	O
+Fig	O
+.	O
+3E	O
+and	O
+3F	O
+).	O
+	O
+Malone	O
+JG	O
+et	O
+al	O
+.	O
+have	O
+reported	O
+that	O
+F151	O
+,	O
+E163	O
+,	O
+I169	O
+and	O
+Q187	O
+,	O
+located	O
+near	O
+the	O
+C	O
+-	O
+terminus	O
+of	O
+YfiR	O
+,	O
+comprise	O
+a	O
+putative	O
+YfiN	O
+binding	O
+site	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+Interestingly	O
+,	O
+these	O
+residues	O
+are	O
+part	O
+of	O
+the	O
+conserved	O
+surface	O
+of	O
+YfiR	O
+(	O
+Fig	O
+.	O
+3G	O
+).	O
+	O
+F151	O
+,	O
+E163	O
+and	O
+I169	O
+form	O
+a	O
+hydrophobic	O
+core	O
+while	O
+,	O
+Q187	O
+is	O
+located	O
+at	O
+the	O
+end	O
+of	O
+the	O
+α6	O
+helix	O
+.	O
+	O
+Collectively	O
+,	O
+a	O
+part	O
+of	O
+the	O
+YfiB	O
+-	O
+YfiR	O
+interface	O
+overlaps	O
+with	O
+the	O
+proposed	O
+YfiR	O
+-	O
+YfiN	O
+interface	O
+,	O
+suggesting	O
+alteration	O
+in	O
+the	O
+association	O
+-	O
+disassociation	O
+equilibrium	O
+of	O
+YfiR	O
+-	O
+YfiN	O
+and	O
+hence	O
+the	O
+ability	O
+of	O
+YfiB	O
+to	O
+sequester	O
+YfiR	O
+.	O
+	O
+YfiR	O
+binds	O
+small	O
+molecules	O
+	O
+Previous	O
+studies	O
+indicated	O
+that	O
+YfiR	O
+constitutes	O
+a	O
+YfiB	O
+-	O
+independent	O
+sensing	O
+device	O
+that	O
+can	O
+activate	O
+YfiN	O
+in	O
+response	O
+to	O
+the	O
+redox	O
+status	O
+of	O
+the	O
+periplasm	O
+,	O
+and	O
+we	O
+have	O
+reported	O
+YfiR	O
+structures	O
+in	O
+both	O
+the	O
+non	O
+-	O
+oxidized	O
+and	O
+the	O
+oxidized	O
+states	O
+earlier	O
+,	O
+revealing	O
+that	O
+the	O
+Cys145	O
+-	O
+Cys152	O
+disulfide	O
+bond	O
+plays	O
+an	O
+essential	O
+role	O
+in	O
+maintaining	O
+the	O
+correct	O
+folding	O
+of	O
+YfiR	O
+(	O
+Yang	O
+et	O
+al	O
+.,).	O
+	O
+However	O
+,	O
+whether	O
+YfiR	O
+is	O
+involved	O
+in	O
+other	O
+regulatory	O
+mechanisms	O
+is	O
+still	O
+an	O
+open	O
+question	O
+.	O
+	O
+Overall	O
+Structures	O
+of	O
+VB6	O
+-	O
+bound	O
+and	O
+Trp	O
+-	O
+bound	O
+YfiR	O
+.	O
+(	O
+A	O
+)	O
+Superposition	O
+of	O
+the	O
+overall	O
+structures	O
+of	O
+VB6	O
+-	O
+bound	O
+and	O
+Trp	O
+-	O
+bound	O
+YfiR	O
+.	O
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+views	O
+showing	O
+the	O
+key	O
+residues	O
+of	O
+YfiR	O
+that	O
+bind	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+.	O
+	O
+The	O
+electron	O
+densities	O
+of	O
+VB6	O
+and	O
+Trp	O
+are	O
+countered	O
+at	O
+3	O
+.	O
+0σ	O
+and	O
+2	O
+.	O
+3σ	O
+,	O
+respectively	O
+,	O
+in	O
+|	O
+Fo	O
+|-|	O
+Fc	O
+|	O
+maps	O
+.	O
+(	O
+C	O
+)	O
+Superposition	O
+of	O
+the	O
+hydrophobic	O
+pocket	O
+of	O
+YfiR	O
+with	O
+VB6	O
+,	O
+L	O
+-	O
+Trp	O
+and	O
+F57	O
+of	O
+YfiB	O
+	O
+For	O
+this	O
+purpose	O
+,	O
+we	O
+co	O
+-	O
+crystallized	O
+YfiR	O
+or	O
+soaked	O
+YfiR	O
+crystals	O
+with	O
+different	O
+small	O
+molecules	O
+,	O
+including	O
+L	O
+-	O
+Trp	O
+and	O
+B	O
+-	O
+group	O
+vitamins	O
+.	O
+	O
+Fortunately	O
+,	O
+we	O
+found	O
+obvious	O
+small	O
+-	O
+molecule	O
+density	O
+in	O
+the	O
+VB6	O
+-	O
+bound	O
+and	O
+Trp	O
+-	O
+bound	O
+YfiR	O
+crystal	O
+structures	O
+(	O
+Fig	O
+.	O
+5A	O
+and	O
+5B	O
+),	O
+and	O
+in	O
+both	O
+structures	O
+,	O
+the	O
+YfiR	O
+dimers	O
+resemble	O
+the	O
+oxidized	O
+YfiR	O
+structure	O
+in	O
+which	O
+both	O
+two	O
+disulfide	O
+bonds	O
+are	O
+well	O
+formed	O
+(	O
+Yang	O
+et	O
+al	O
+.,).	O
+	O
+Functional	O
+analysis	O
+of	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+.	O
+(	O
+A	O
+and	O
+B	O
+)	O
+The	O
+effect	O
+of	O
+increasing	O
+concentrations	O
+of	O
+VB6	O
+or	O
+L	O
+-	O
+Trp	O
+on	O
+YfiBL43P	B-mutant
+-	O
+induced	O
+attachment	O
+(	O
+bars	O
+).	O
+	O
+(	O
+C	O
+and	O
+D	O
+)	O
+BIAcore	O
+data	O
+and	O
+analysis	O
+for	O
+binding	O
+affinities	O
+of	O
+(	O
+C	O
+)	O
+VB6	O
+and	O
+(	O
+D	O
+)	O
+L	O
+-	O
+Trp	O
+with	O
+YfiR	O
+.	O
+(	O
+E	O
+–	O
+G	O
+)	O
+ITC	O
+data	O
+and	O
+analysis	O
+for	O
+titration	O
+of	O
+(	O
+E	O
+)	O
+YfiB	O
+wild	O
+-	O
+type	O
+,	O
+(	O
+F	O
+)	O
+YfiBL43P	O
+,	O
+and	O
+(	O
+G	O
+)	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+into	O
+YfiR	O
+	O
+Interestingly	O
+,	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+were	O
+found	O
+to	O
+occupy	O
+the	O
+same	O
+hydrophobic	O
+pocket	O
+,	O
+formed	O
+by	O
+L166	O
+/	O
+I169	O
+/	O
+V176	O
+/	O
+P178	O
+/	O
+L181	O
+of	O
+YfiR	O
+,	O
+which	O
+is	O
+also	O
+a	O
+binding	O
+pocket	O
+for	O
+F57	O
+of	O
+YfiB	O
+,	O
+as	O
+observed	O
+in	O
+the	O
+YfiB	O
+-	O
+YfiR	O
+complex	O
+(	O
+Fig	O
+.	O
+5C	O
+).	O
+	O
+The	O
+results	O
+showed	O
+Kd	O
+values	O
+of	O
+1	O
+.	O
+4	O
+×	O
+10	O
+−	O
+7	O
+mol	O
+/	O
+L	O
+and	O
+5	O
+.	O
+3	O
+×	O
+10	O
+−	O
+7	O
+mol	O
+/	O
+L	O
+for	O
+YfiBL43P	B-mutant
+and	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+,	O
+respectively	O
+,	O
+revealing	O
+that	O
+the	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+mutant	O
+caused	O
+a	O
+3	O
+.	O
+8	O
+-	O
+fold	O
+reduction	O
+in	O
+the	O
+binding	O
+affinity	O
+compared	O
+with	O
+the	O
+YfiBL43P	B-mutant
+mutant	O
+(	O
+Fig	O
+.	O
+6F	O
+and	O
+6G	O
+).	O
+	O
+In	O
+parallel	O
+,	O
+to	O
+better	O
+understand	O
+the	O
+putative	O
+functional	O
+role	O
+of	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+,	O
+yfiB	O
+was	O
+deleted	O
+in	O
+a	O
+PAO1	O
+wild	O
+-	O
+type	O
+strain	O
+,	O
+and	O
+a	O
+construct	O
+expressing	O
+the	O
+YfiBL43P	B-mutant
+mutant	O
+was	O
+transformed	O
+into	O
+the	O
+PAO1	O
+ΔyfiB	B-mutant
+strain	O
+to	O
+trigger	O
+YfiBL43P	B-mutant
+-	O
+induced	O
+biofilm	O
+formation	O
+.	O
+	O
+Growth	O
+and	O
+surface	O
+attachment	O
+assays	O
+were	O
+carried	O
+out	O
+for	O
+the	O
+yfiB	B-mutant
+-	I-mutant
+L43P	I-mutant
+strain	O
+in	O
+the	O
+presence	O
+of	O
+increasing	O
+concentrations	O
+of	O
+VB6	O
+or	O
+L	O
+-	O
+Trp	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+.	O
+6A	O
+and	O
+6B	O
+,	O
+the	O
+over	O
+-	O
+expression	O
+of	O
+YfiBL43P	B-mutant
+induced	O
+strong	O
+surface	O
+attachment	O
+and	O
+much	O
+slower	O
+growth	O
+of	O
+the	O
+yfiB	B-mutant
+-	I-mutant
+L43P	I-mutant
+strain	O
+,	O
+and	O
+as	O
+expected	O
+,	O
+a	O
+certain	O
+amount	O
+of	O
+VB6	O
+or	O
+L	O
+-	O
+Trp	O
+(	O
+4	O
+–	O
+6	O
+mmol	O
+/	O
+L	O
+for	O
+VB6	O
+and	O
+6	O
+–	O
+10	O
+mmol	O
+/	O
+L	O
+for	O
+L	O
+-	O
+Trp	O
+)	O
+could	O
+reduce	O
+the	O
+surface	O
+attachment	O
+.	O
+	O
+Interestingly	O
+,	O
+at	O
+a	O
+concentration	O
+higher	O
+than	O
+8	O
+mmol	O
+/	O
+L	O
+,	O
+VB6	O
+lost	O
+its	O
+ability	O
+to	O
+inhibit	O
+biofilm	O
+formation	O
+,	O
+implying	O
+that	O
+the	O
+VB6	O
+-	O
+involving	O
+regulatory	O
+mechanism	O
+is	O
+highly	O
+complicated	O
+and	O
+remains	O
+to	O
+be	O
+further	O
+investigated	O
+.	O
+	O
+Of	O
+note	O
+,	O
+both	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+have	O
+been	O
+reported	O
+to	O
+correlate	O
+with	O
+biofilm	O
+formation	O
+in	O
+certain	O
+Gram	O
+-	O
+negative	O
+bacteria	O
+(	O
+Grubman	O
+et	O
+al	O
+.,;	O
+Shimazaki	O
+et	O
+al	O
+.,).	O
+	O
+In	O
+Helicobacter	O
+pylori	O
+in	O
+particular	O
+,	O
+VB6	O
+biosynthetic	O
+enzymes	O
+act	O
+as	O
+novel	O
+virulence	O
+factors	O
+,	O
+and	O
+VB6	O
+is	O
+required	O
+for	O
+full	O
+motility	O
+and	O
+virulence	O
+(	O
+Grubman	O
+et	O
+al	O
+.,).	O
+	O
+In	O
+E	O
+.	O
+coli	O
+,	O
+mutants	O
+with	O
+decreased	O
+tryptophan	O
+synthesis	O
+show	O
+greater	O
+biofilm	O
+formation	O
+,	O
+and	O
+matured	O
+biofilm	O
+is	O
+degraded	O
+by	O
+L	O
+-	O
+tryptophan	O
+addition	O
+(	O
+Shimazaki	O
+et	O
+al	O
+.,).	O
+	O
+To	O
+answer	O
+the	O
+question	O
+whether	O
+competition	O
+of	O
+VB6	O
+or	O
+L	O
+-	O
+Trp	O
+for	O
+the	O
+YfiB	O
+F57	O
+-	O
+binding	O
+pocket	O
+of	O
+YfiR	O
+plays	O
+an	O
+essential	O
+role	O
+in	O
+inhibiting	O
+biofilm	O
+formation	O
+,	O
+we	O
+measured	O
+the	O
+binding	O
+affinities	O
+of	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+for	O
+YfiR	O
+via	O
+BIAcore	O
+experiments	O
+.	O
+	O
+The	O
+results	O
+showed	O
+relatively	O
+weak	O
+Kd	O
+values	O
+of	O
+35	O
+.	O
+2	O
+mmol	O
+/	O
+L	O
+and	O
+76	O
+.	O
+9	O
+mmol	O
+/	O
+L	O
+for	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+,	O
+respectively	O
+(	O
+Fig	O
+.	O
+6C	O
+and	O
+6D	O
+).	O
+	O
+Based	O
+on	O
+our	O
+results	O
+,	O
+we	O
+concluded	O
+that	O
+VB6	O
+or	O
+L	O
+-	O
+Trp	O
+can	O
+bind	O
+to	O
+YfiR	O
+,	O
+however	O
+,	O
+VB6	O
+or	O
+L	O
+-	O
+Trp	O
+alone	O
+may	O
+have	O
+little	O
+effects	O
+in	O
+interrupting	O
+the	O
+YfiB	O
+-	O
+YfiR	O
+interaction	O
+,	O
+the	O
+mechanism	O
+by	O
+which	O
+VB6	O
+or	O
+L	O
+-	O
+Trp	O
+inhibits	O
+biofilm	O
+formation	O
+remains	O
+unclear	O
+and	O
+requires	O
+further	O
+investigation	O
+.	O
+	O
+In	O
+addition	O
+to	O
+the	O
+preceding	O
+8	O
+aa	O
+loop	O
+(	O
+from	O
+the	O
+lipid	O
+acceptor	O
+Cys26	O
+to	O
+Gly34	O
+),	O
+the	O
+full	O
+length	O
+of	O
+the	O
+periplasmic	O
+portion	O
+of	O
+apo	O
+YfiB	O
+can	O
+reach	O
+approximately	O
+60	O
+Å	O
+.	O
+It	O
+was	O
+reported	O
+that	O
+the	O
+distance	O
+between	O
+the	O
+outer	O
+membrane	O
+and	O
+the	O
+cell	O
+wall	O
+is	O
+approximately	O
+50	O
+Å	O
+and	O
+that	O
+the	O
+thickness	O
+of	O
+the	O
+PG	O
+layer	O
+is	O
+approximately	O
+70	O
+Å	O
+(	O
+Matias	O
+et	O
+al	O
+.,).	O
+	O
+Thus	O
+,	O
+YfiB	O
+alone	O
+represents	O
+an	O
+inactive	O
+form	O
+that	O
+may	O
+only	O
+partially	O
+insert	O
+into	O
+the	O
+PG	O
+matrix	O
+.	O
+	O
+By	O
+contrast	O
+,	O
+YfiR	O
+-	O
+bound	O
+YfiBL43P	B-mutant
+(	O
+residues	O
+44	O
+–	O
+168	O
+)	O
+has	O
+a	O
+stretched	O
+conformation	O
+of	O
+approximately	O
+55	O
+Å	O
+in	O
+length	O
+.	O
+	O
+In	O
+addition	O
+to	O
+the	O
+17	O
+preceding	O
+intracellular	O
+residues	O
+(	O
+from	O
+the	O
+lipid	O
+acceptor	O
+Cys26	O
+to	O
+Leu43	O
+),	O
+the	O
+length	O
+of	O
+the	O
+intracellular	O
+portion	O
+of	O
+active	O
+YfiB	O
+may	O
+extend	O
+over	O
+100	O
+Å	O
+,	O
+assuming	O
+a	O
+fully	O
+stretched	O
+conformation	O
+.	O
+	O
+Regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	O
+tripartite	O
+system	O
+.	O
+	O
+The	O
+periplasmic	O
+domain	O
+of	O
+YfiB	O
+and	O
+the	O
+YfiB	O
+-	O
+YfiR	O
+complex	O
+are	O
+depicted	O
+according	O
+to	O
+the	O
+crystal	O
+structures	O
+.	O
+	O
+The	O
+lipid	O
+acceptor	O
+Cys26	O
+is	O
+indicated	O
+as	O
+blue	O
+ball	O
+.	O
+	O
+The	O
+loop	O
+connecting	O
+Cys26	O
+and	O
+Gly34	O
+of	O
+YfiB	O
+is	O
+modeled	O
+.	O
+	O
+The	O
+PAS	O
+domain	O
+of	O
+YfiN	O
+is	O
+shown	O
+as	O
+pink	O
+oval	O
+.	O
+	O
+These	O
+results	O
+,	O
+together	O
+with	O
+our	O
+observation	O
+that	O
+activated	O
+YfiB	O
+has	O
+a	O
+much	O
+higher	O
+cell	O
+wall	O
+binding	O
+affinity	O
+,	O
+and	O
+previous	O
+mutagenesis	O
+data	O
+showing	O
+that	O
+(	O
+1	O
+)	O
+both	O
+PG	O
+binding	O
+and	O
+membrane	O
+anchoring	O
+are	O
+required	O
+for	O
+YfiB	O
+activity	O
+and	O
+(	O
+2	O
+)	O
+activating	O
+mutations	O
+possessing	O
+an	O
+altered	O
+N	O
+-	O
+terminal	O
+loop	O
+length	O
+are	O
+dominant	O
+over	O
+the	O
+loss	O
+of	O
+PG	O
+binding	O
+(	O
+Malone	O
+et	O
+al	O
+.,),	O
+suggest	O
+an	O
+updated	O
+regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	O
+system	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+	O
+This	O
+allows	O
+the	O
+C	O
+-	O
+terminal	O
+portion	O
+of	O
+the	O
+membrane	O
+-	O
+anchored	O
+YfiB	O
+to	O
+reach	O
+,	O
+bind	O
+and	O
+penetrate	O
+the	O
+cell	O
+wall	O
+and	O
+sequester	O
+the	O
+YfiR	O
+dimer	O
+.	O
+	O
+The	O
+mechanism	O
+by	O
+which	O
+activated	O
+YfiB	O
+relieves	O
+the	O
+repression	O
+of	O
+YfiN	O
+may	O
+be	O
+applicable	O
+to	O
+the	O
+YfiBNR	O
+system	O
+in	O
+other	O
+bacteria	O
+and	O
+to	O
+analogous	O
+outside	O
+-	O
+in	O
+signaling	O
+for	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+production	O
+,	O
+which	O
+in	O
+turn	O
+may	O
+be	O
+relevant	O
+to	O
+the	O
+development	O
+of	O
+drugs	O
+that	O
+can	O
+circumvent	O
+complicated	O
+antibiotic	O
+resistance	O
+.	O
+	O
+X	O
+-	O
+ray	O
+Crystallographic	O
+Structures	O
+of	O
+a	O
+Trimer	O
+,	O
+Dodecamer	O
+,	O
+and	O
+Annular	O
+Pore	O
+Formed	O
+by	O
+an	O
+Aβ17	O
+–	O
+36	O
+β	O
+-	O
+Hairpin	O
+	O
+This	O
+paper	O
+presents	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structures	O
+of	O
+oligomers	O
+formed	O
+by	O
+a	O
+20	O
+-	O
+residue	O
+peptide	O
+segment	O
+derived	O
+from	O
+Aβ	O
+.	O
+	O
+The	O
+development	O
+of	O
+a	O
+peptide	O
+in	O
+which	O
+Aβ17	O
+–	O
+36	O
+is	O
+stabilized	O
+as	O
+a	O
+β	O
+-	O
+hairpin	O
+is	O
+described	O
+,	O
+and	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structures	O
+of	O
+oligomers	O
+it	O
+forms	O
+are	O
+reported	O
+.	O
+	O
+Two	O
+covalent	O
+constraints	O
+act	O
+in	O
+tandem	O
+to	O
+stabilize	O
+the	O
+Aβ17	O
+–	O
+36	O
+peptide	O
+in	O
+a	O
+hairpin	O
+conformation	O
+:	O
+a	O
+δ	O
+-	O
+linked	O
+ornithine	O
+turn	O
+connecting	O
+positions	O
+17	O
+and	O
+36	O
+to	O
+create	O
+a	O
+macrocycle	O
+and	O
+an	O
+intramolecular	O
+disulfide	O
+linkage	O
+between	O
+positions	O
+24	O
+and	O
+29	O
+.	O
+	O
+An	O
+N	O
+-	O
+methyl	O
+group	O
+at	O
+position	O
+33	O
+blocks	O
+uncontrolled	O
+aggregation	O
+.	O
+	O
+The	O
+peptide	O
+readily	O
+crystallizes	O
+as	O
+a	O
+folded	O
+β	O
+-	O
+hairpin	O
+,	O
+which	O
+assembles	O
+hierarchically	O
+in	O
+the	O
+crystal	O
+lattice	O
+.	O
+	O
+Three	O
+β	O
+-	O
+hairpin	O
+monomers	O
+assemble	O
+to	O
+form	O
+a	O
+triangular	O
+trimer	O
+,	O
+four	O
+trimers	O
+assemble	O
+in	O
+a	O
+tetrahedral	O
+arrangement	O
+to	O
+form	O
+a	O
+dodecamer	O
+,	O
+and	O
+five	O
+dodecamers	O
+pack	O
+together	O
+to	O
+form	O
+an	O
+annular	O
+pore	O
+.	O
+	O
+This	O
+hierarchical	O
+assembly	O
+provides	O
+a	O
+model	O
+,	O
+in	O
+which	O
+full	O
+-	O
+length	O
+Aβ	O
+transitions	O
+from	O
+an	O
+unfolded	O
+monomer	O
+to	O
+a	O
+folded	O
+β	O
+-	O
+hairpin	O
+,	O
+which	O
+assembles	O
+to	O
+form	O
+oligomers	O
+that	O
+further	O
+pack	O
+to	O
+form	O
+an	O
+annular	O
+pore	O
+.	O
+	O
+High	O
+-	O
+resolution	O
+structures	O
+of	O
+oligomers	O
+formed	O
+by	O
+the	O
+β	O
+-	O
+amyloid	O
+peptide	O
+Aβ	O
+are	O
+desperately	O
+needed	O
+to	O
+understand	O
+the	O
+molecular	O
+basis	O
+of	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+and	O
+ultimately	O
+develop	O
+preventions	O
+or	O
+treatments	O
+.	O
+	O
+In	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+,	O
+monomeric	O
+Aβ	O
+aggregates	O
+to	O
+form	O
+soluble	O
+low	O
+molecular	O
+weight	O
+oligomers	O
+,	O
+such	O
+as	O
+dimers	O
+,	O
+trimers	O
+,	O
+tetramers	O
+,	O
+hexamers	O
+,	O
+nonamers	O
+,	O
+and	O
+dodecamers	O
+,	O
+as	O
+well	O
+as	O
+high	O
+molecular	O
+weight	O
+aggregates	O
+,	O
+such	O
+as	O
+annular	O
+protofibrils	O
+.	O
+	O
+Mouse	O
+models	O
+for	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+have	O
+helped	O
+shape	O
+our	O
+current	O
+understanding	O
+about	O
+the	O
+Aβ	O
+oligomerization	O
+that	O
+precedes	O
+neurodegeneration	O
+.	O
+	O
+A	O
+56	O
+kDa	O
+soluble	O
+oligomer	O
+identified	O
+by	O
+SDS	O
+-	O
+PAGE	O
+was	O
+found	O
+to	O
+be	O
+especially	O
+important	O
+within	O
+this	O
+mixture	O
+.	O
+	O
+This	O
+oligomer	O
+was	O
+termed	O
+Aβ	O
+*	O
+56	O
+and	O
+appears	O
+to	O
+be	O
+a	O
+dodecamer	O
+of	O
+Aβ	O
+.	O
+	O
+Purified	O
+Aβ	O
+*	O
+56	O
+injected	O
+intercranially	O
+into	O
+healthy	O
+rats	O
+was	O
+found	O
+to	O
+impair	O
+memory	O
+,	O
+providing	O
+evidence	O
+that	O
+this	O
+Aβ	O
+oligomer	O
+may	O
+cause	O
+memory	O
+loss	O
+in	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+.	O
+	O
+Treatment	O
+of	O
+the	O
+mixture	O
+of	O
+low	O
+molecular	O
+weight	O
+oligomers	O
+with	O
+hexafluoroisopropanol	O
+resulted	O
+in	O
+the	O
+dissociation	O
+of	O
+the	O
+putative	O
+dodecamers	O
+,	O
+nonamers	O
+,	O
+and	O
+hexamers	O
+into	O
+trimers	O
+and	O
+monomers	O
+,	O
+suggesting	O
+that	O
+trimers	O
+may	O
+be	O
+the	O
+building	O
+block	O
+of	O
+the	O
+dodecamers	O
+,	O
+nonamers	O
+,	O
+and	O
+hexamers	O
+.	O
+	O
+Recently	O
+,	O
+Aβ	O
+trimers	O
+and	O
+Aβ	O
+*	O
+56	O
+were	O
+identified	O
+in	O
+the	O
+brains	O
+of	O
+cognitively	O
+normal	O
+humans	O
+and	O
+were	O
+found	O
+to	O
+increase	O
+with	O
+age	O
+.	O
+	O
+APFs	O
+were	O
+first	O
+discovered	O
+in	O
+vitro	O
+using	O
+chemically	O
+synthesized	O
+Aβ	O
+that	O
+aggregated	O
+into	O
+porelike	O
+structures	O
+that	O
+could	O
+be	O
+observed	O
+by	O
+atomic	O
+force	O
+microscopy	O
+(	O
+AFM	O
+)	O
+and	O
+transmission	O
+electron	O
+microscopy	O
+(	O
+TEM	O
+).	O
+	O
+The	O
+sizes	O
+of	O
+APFs	O
+prepared	O
+in	O
+vitro	O
+vary	O
+among	O
+different	O
+studies	O
+.	O
+	O
+Quist	O
+et	O
+al	O
+.	O
+observed	O
+APFs	O
+with	O
+an	O
+outer	O
+diameter	O
+of	O
+16	O
+nm	O
+embedded	O
+in	O
+a	O
+lipid	O
+bilayer	O
+.	O
+	O
+Although	O
+the	O
+APFs	O
+in	O
+these	O
+studies	O
+differ	O
+in	O
+size	O
+,	O
+they	O
+share	O
+a	O
+similar	O
+annular	O
+morphology	O
+and	O
+appear	O
+to	O
+be	O
+composed	O
+of	O
+smaller	O
+oligomers	O
+.	O
+	O
+APFs	O
+have	O
+also	O
+been	O
+observed	O
+in	O
+the	O
+brains	O
+of	O
+APP23	O
+transgenic	O
+mice	O
+by	O
+immunofluorescence	O
+with	O
+an	O
+anti	O
+-	O
+APF	O
+antibody	O
+and	O
+were	O
+found	O
+to	O
+accumulate	O
+in	O
+neuronal	O
+processes	O
+and	O
+synapses	O
+.	O
+	O
+In	O
+a	O
+subsequent	O
+study	O
+,	O
+APFs	O
+were	O
+isolated	O
+from	O
+the	O
+brains	O
+of	O
+Alzheimer	O
+’	O
+s	O
+patients	O
+by	O
+immunoprecipitation	O
+with	O
+an	O
+anti	O
+-	O
+APF	O
+antibody	O
+.	O
+	O
+Dimers	O
+of	O
+Aβ	O
+have	O
+also	O
+been	O
+isolated	O
+from	O
+the	O
+brains	O
+of	O
+Alzheimer	O
+’	O
+s	O
+patients	O
+.−	O
+Aβ	O
+dimers	O
+inhibit	O
+long	O
+-	O
+term	O
+potentiation	O
+in	O
+mice	O
+and	O
+promote	O
+hyperphosphorylation	O
+of	O
+the	O
+microtubule	O
+-	O
+associated	O
+protein	O
+tau	O
+,	O
+leading	O
+to	O
+neuritic	O
+damage	O
+.	O
+	O
+Aβ	O
+dimers	O
+have	O
+only	O
+been	O
+isolated	O
+from	O
+human	O
+or	O
+transgenic	O
+mouse	O
+brains	O
+that	O
+contain	O
+the	O
+pathognomonic	O
+fibrillar	O
+Aβ	O
+plaques	O
+associated	O
+with	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+endogenous	O
+rise	O
+of	O
+Aβ	O
+dimers	O
+in	O
+the	O
+brains	O
+of	O
+Tg2576	O
+and	O
+J20	O
+transgenic	O
+mice	O
+coincides	O
+with	O
+the	O
+deposition	O
+of	O
+Aβ	O
+plaques	O
+.	O
+	O
+The	O
+approach	O
+of	O
+isolating	O
+and	O
+characterizing	O
+Aβ	O
+oligomers	O
+has	O
+not	O
+provided	O
+any	O
+high	O
+-	O
+resolution	O
+structures	O
+of	O
+Aβ	O
+oligomers	O
+.	O
+	O
+Techniques	O
+such	O
+as	O
+SDS	O
+-	O
+PAGE	O
+,	O
+TEM	O
+,	O
+and	O
+AFM	O
+have	O
+only	O
+provided	O
+information	O
+about	O
+the	O
+molecular	O
+weights	O
+,	O
+sizes	O
+,	O
+morphologies	O
+,	O
+and	O
+stoichiometry	O
+of	O
+Aβ	O
+oligomers	O
+.	O
+	O
+High	O
+-	O
+resolution	O
+structural	O
+studies	O
+of	O
+Aβ	O
+have	O
+primarily	O
+focused	O
+on	O
+Aβ	O
+fibrils	O
+and	O
+Aβ	O
+monomers	O
+.	O
+	O
+Solid	O
+-	O
+state	O
+NMR	O
+spectroscopy	O
+studies	O
+of	O
+Aβ	O
+fibrils	O
+revealed	O
+that	O
+Aβ	O
+fibrils	O
+are	O
+generally	O
+composed	O
+of	O
+extended	O
+networks	O
+of	O
+in	O
+-	O
+register	O
+parallel	O
+β	O
+-	O
+sheets	O
+.��	O
+X	O
+-	O
+ray	O
+crystallographic	O
+studies	O
+using	O
+fragments	O
+of	O
+Aβ	O
+have	O
+provided	O
+additional	O
+information	O
+about	O
+how	O
+Aβ	O
+fibrils	O
+pack	O
+.	O
+	O
+Solution	O
+-	O
+phase	O
+NMR	O
+and	O
+solid	O
+-	O
+state	O
+NMR	O
+have	O
+been	O
+used	O
+to	O
+study	O
+the	O
+structures	O
+of	O
+the	O
+Aβ	O
+monomers	O
+within	O
+oligomeric	O
+assemblies	O
+.−	O
+A	O
+major	O
+finding	O
+from	O
+these	O
+studies	O
+is	O
+that	O
+oligomeric	O
+assemblies	O
+of	O
+Aβ	O
+are	O
+primarily	O
+composed	O
+of	O
+antiparallel	O
+β	O
+-	O
+sheets	O
+.	O
+	O
+The	O
+structure	O
+revealed	O
+that	O
+monomeric	O
+Aβ	O
+forms	O
+a	O
+β	O
+-	O
+hairpin	O
+when	O
+bound	O
+to	O
+the	O
+affibody	O
+.	O
+	O
+Locking	O
+Aβ	O
+into	O
+a	O
+β	O
+-	O
+hairpin	O
+structure	O
+resulted	O
+in	O
+the	O
+formation	O
+Aβ	O
+oligomers	O
+,	O
+which	O
+were	O
+observed	O
+by	O
+size	O
+exclusion	O
+chromatography	O
+(	O
+SEC	O
+)	O
+and	O
+SDS	O
+-	O
+PAGE	O
+.	O
+	O
+The	O
+oligomers	O
+with	O
+a	O
+molecular	O
+weight	O
+of	O
+∼	O
+100	O
+kDa	O
+that	O
+were	O
+isolated	O
+by	O
+SEC	O
+were	O
+toxic	O
+toward	O
+neuronally	O
+derived	O
+SH	O
+-	O
+SY5Y	O
+cells	O
+.	O
+	O
+This	O
+study	O
+provides	O
+evidence	O
+for	O
+the	O
+role	O
+of	O
+β	O
+-	O
+hairpin	O
+structure	O
+in	O
+Aβ	O
+oligomerization	O
+and	O
+neurotoxicity	O
+.	O
+	O
+Inspired	O
+by	O
+these	O
+β	O
+-	O
+hairpin	O
+structures	O
+,	O
+our	O
+laboratory	O
+developed	O
+a	O
+macrocyclic	O
+β	O
+-	O
+sheet	O
+peptide	O
+derived	O
+from	O
+Aβ17	O
+–	O
+36	O
+designed	O
+to	O
+mimic	O
+an	O
+Aβ	O
+β	O
+-	O
+hairpin	O
+and	O
+reported	O
+its	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+.	O
+	O
+This	O
+peptide	O
+(	O
+peptide	B-mutant
+1	I-mutant
+)	O
+consists	O
+of	O
+two	O
+β	O
+-	O
+strands	O
+comprising	O
+Aβ17	O
+–	O
+23	O
+and	O
+Aβ30	O
+–	O
+36	O
+covalently	O
+linked	O
+by	O
+two	O
+δ	O
+-	O
+linked	O
+ornithine	O
+(	O
+δOrn	O
+)	O
+β	O
+-	O
+turn	O
+mimics	O
+.	O
+	O
+The	O
+δOrn	O
+that	O
+connects	O
+residues	O
+D23	O
+and	O
+A30	O
+replaces	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+.	O
+	O
+The	O
+δOrn	O
+that	O
+connects	O
+residues	O
+L17	O
+and	O
+V36	O
+enforces	O
+β	O
+-	O
+hairpin	O
+structure	O
+.	O
+	O
+We	O
+incorporated	O
+an	O
+N	O
+-	O
+methyl	O
+group	O
+at	O
+position	O
+G33	O
+to	O
+prevent	O
+uncontrolled	O
+aggregation	O
+and	O
+precipitation	O
+of	O
+the	O
+peptide	O
+.	O
+	O
+To	O
+improve	O
+the	O
+solubility	O
+of	O
+the	O
+peptide	O
+we	O
+replaced	O
+M35	O
+with	O
+the	O
+hydrophilic	O
+isostere	O
+of	O
+methionine	O
+,	O
+ornithine	O
+(	O
+α	O
+-	O
+linked	O
+)	O
+(	O
+Figure	O
+1B	O
+).	O
+	O
+The	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+1	I-mutant
+reveals	O
+that	O
+it	O
+folds	O
+to	O
+form	O
+a	O
+β	O
+-	O
+hairpin	O
+that	O
+assembles	O
+to	O
+form	O
+trimers	O
+and	O
+that	O
+the	O
+trimers	O
+further	O
+assemble	O
+to	O
+form	O
+hexamers	O
+and	O
+dodecamers	O
+.	O
+	O
+(	O
+B	O
+)	O
+Chemical	O
+structure	O
+of	O
+peptide	B-mutant
+1	I-mutant
+illustrating	O
+Aβ17	O
+–	O
+23	O
+and	O
+Aβ30	O
+–	O
+36	O
+,	O
+M35Orn	O
+,	O
+the	O
+N	O
+-	O
+methyl	O
+group	O
+,	O
+and	O
+the	O
+δ	O
+-	O
+linked	O
+ornithine	O
+turns	O
+.	O
+(	O
+C	O
+)	O
+Chemical	O
+structure	O
+of	O
+peptide	B-mutant
+2	I-mutant
+illustrating	O
+Aβ17	O
+–	O
+36	O
+,	O
+the	O
+N	O
+-	O
+methyl	O
+group	O
+,	O
+the	O
+disulfide	O
+bond	O
+across	O
+positions	O
+24	O
+and	O
+29	O
+,	O
+and	O
+the	O
+δ	O
+-	O
+linked	O
+ornithine	O
+turn	O
+.	O
+	O
+Our	O
+design	O
+of	O
+peptide	B-mutant
+1	I-mutant
+omitted	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+.	O
+	O
+To	O
+visualize	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+,	O
+we	O
+performed	O
+replica	O
+-	O
+exchange	O
+molecular	O
+dynamics	O
+(	O
+REMD	O
+)	O
+simulations	O
+on	O
+Aβ17	O
+–	O
+36	O
+using	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+coordinates	O
+of	O
+Aβ17	O
+–	O
+23	O
+and	O
+Aβ30	O
+–	O
+36	O
+from	O
+peptide	B-mutant
+1	I-mutant
+.	O
+	O
+These	O
+studies	O
+provided	O
+a	O
+working	O
+model	O
+for	O
+a	O
+trimer	O
+of	O
+Aβ17	O
+–	O
+36	O
+β	O
+-	O
+hairpins	O
+and	O
+demonstrated	O
+that	O
+the	O
+trimer	O
+should	O
+be	O
+capable	O
+of	O
+accommodating	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+.	O
+	O
+In	O
+the	O
+current	O
+study	O
+we	O
+set	O
+out	O
+to	O
+restore	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+,	O
+reintroduce	O
+the	O
+methionine	O
+residue	O
+at	O
+position	O
+35	O
+,	O
+and	O
+determine	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structures	O
+of	O
+oligomers	O
+that	O
+form	O
+.	O
+	O
+We	O
+designed	O
+peptide	B-mutant
+2	I-mutant
+as	O
+a	O
+homologue	O
+of	O
+peptide	B-mutant
+1	I-mutant
+that	O
+embodies	O
+these	O
+ideas	O
+.	O
+	O
+Here	O
+,	O
+we	O
+describe	O
+the	O
+development	O
+of	O
+peptide	B-mutant
+2	I-mutant
+and	O
+report	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structures	O
+of	O
+the	O
+trimer	O
+,	O
+dodecamer	O
+,	O
+and	O
+annular	O
+pore	O
+observed	O
+within	O
+the	O
+crystal	O
+structure	O
+.	O
+	O
+Development	O
+of	O
+Peptide	B-mutant
+2	I-mutant
+	O
+We	O
+developed	O
+peptide	B-mutant
+2	I-mutant
+from	O
+peptide	B-mutant
+1	I-mutant
+by	O
+an	O
+iterative	O
+process	O
+,	O
+in	O
+which	O
+we	O
+first	O
+attempted	O
+to	O
+restore	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+without	O
+a	O
+disulfide	O
+linkage	O
+.	O
+	O
+We	O
+envisioned	O
+peptide	B-mutant
+3	I-mutant
+as	O
+a	O
+homologue	O
+of	O
+peptide	B-mutant
+1	I-mutant
+with	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+in	O
+place	O
+of	O
+the	O
+δOrn	O
+that	O
+connects	O
+D23	O
+and	O
+A30	O
+and	O
+p	O
+-	O
+iodophenylalanine	O
+(	O
+FI	O
+)	O
+in	O
+place	O
+of	O
+F19	O
+.	O
+	O
+We	O
+routinely	O
+use	O
+p	O
+-	O
+iodophenylalanine	O
+to	O
+determine	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+phases	O
+.	O
+	O
+We	O
+postulate	O
+that	O
+the	O
+loss	O
+of	O
+the	O
+δOrn	O
+constraint	O
+leads	O
+to	O
+conformational	O
+heterogeneity	O
+that	O
+prevents	O
+peptide	B-mutant
+3	I-mutant
+from	O
+crystallizing	O
+.	O
+	O
+We	O
+designed	O
+peptide	B-mutant
+4	I-mutant
+to	O
+embody	O
+this	O
+idea	O
+,	O
+mutating	O
+Val24	O
+and	O
+Gly29	O
+to	O
+cysteine	O
+and	O
+forming	O
+an	O
+interstrand	O
+disulfide	O
+linkage	O
+.	O
+	O
+Residues	O
+V24	O
+and	O
+G29	O
+form	O
+a	O
+non	O
+-	O
+hydrogen	O
+-	O
+bonded	O
+pair	O
+,	O
+which	O
+can	O
+readily	O
+accommodate	O
+disulfide	O
+linkages	O
+in	O
+antiparallel	O
+β	O
+-	O
+sheets	O
+.	O
+	O
+Disulfide	O
+bonds	O
+across	O
+non	O
+-	O
+hydrogen	O
+-	O
+bonded	O
+pairs	O
+stabilize	O
+β	O
+-	O
+hairpins	O
+,	O
+while	O
+disulfide	O
+bonds	O
+across	O
+hydrogen	O
+-	O
+bonded	O
+pairs	O
+do	O
+not	O
+.	O
+	O
+We	O
+were	O
+gratified	O
+to	O
+find	O
+that	O
+peptide	B-mutant
+4	I-mutant
+afforded	O
+crystals	O
+suitable	O
+for	O
+X	O
+-	O
+ray	O
+crystallography	O
+.	O
+	O
+As	O
+the	O
+next	O
+step	O
+in	O
+the	O
+iterative	O
+process	O
+,	O
+we	O
+determined	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+this	O
+peptide	O
+(	O
+PDB	O
+5HOW	O
+).	O
+	O
+After	O
+determining	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+4	I-mutant
+we	O
+reintroduced	O
+the	O
+native	O
+phenylalanine	O
+at	O
+position	O
+19	O
+and	O
+the	O
+methionine	O
+at	O
+position	O
+35	O
+to	O
+afford	O
+peptide	B-mutant
+2	I-mutant
+.	O
+	O
+We	O
+completed	O
+the	O
+iterative	O
+process	O
+—	O
+from	O
+1	O
+to	O
+3	O
+to	O
+4	O
+to	O
+2	O
+—	O
+by	O
+successfully	O
+determining	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+2	I-mutant
+(	O
+PDB	O
+5HOX	O
+and	O
+5HOY	O
+).	O
+	O
+The	O
+following	O
+sections	O
+describe	O
+the	O
+synthesis	O
+of	O
+peptides	B-mutant
+2	I-mutant
+–	I-mutant
+4	I-mutant
+and	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+2	I-mutant
+.	O
+	O
+Synthesis	O
+of	O
+Peptides	B-mutant
+2	I-mutant
+–	I-mutant
+4	I-mutant
+	O
+We	O
+synthesized	O
+peptides	B-mutant
+2	I-mutant
+–	I-mutant
+4	I-mutant
+by	O
+similar	O
+procedures	O
+to	O
+those	O
+we	O
+have	O
+developed	O
+for	O
+other	O
+macrocyclic	O
+peptides	O
+.	O
+	O
+We	O
+used	O
+acid	O
+-	O
+stable	O
+Acm	O
+-	O
+protected	O
+cysteine	O
+residues	O
+at	O
+positions	O
+24	O
+and	O
+29	O
+and	O
+removed	O
+the	O
+Acm	O
+groups	O
+by	O
+oxidation	O
+with	O
+I2	O
+in	O
+aqueous	O
+acetic	O
+acid	O
+to	O
+afford	O
+the	O
+disulfide	O
+linkage	O
+.	O
+	O
+Peptides	B-mutant
+2	I-mutant
+–	I-mutant
+4	I-mutant
+were	O
+purified	O
+by	O
+RP	O
+-	O
+HPLC	O
+.	O
+	O
+We	O
+screened	O
+crystallization	O
+conditions	O
+for	O
+peptide	B-mutant
+4	I-mutant
+in	O
+a	O
+96	O
+-	O
+well	O
+-	O
+plate	O
+format	O
+using	O
+three	O
+different	O
+Hampton	O
+Research	O
+crystallization	O
+kits	O
+(	O
+Crystal	O
+Screen	O
+,	O
+Index	O
+,	O
+and	O
+PEG	O
+/	O
+Ion	O
+)	O
+with	O
+three	O
+ratios	O
+of	O
+peptide	O
+and	O
+mother	O
+liquor	O
+per	O
+condition	O
+(	O
+864	O
+experiments	O
+).	O
+	O
+Peptide	B-mutant
+4	I-mutant
+afforded	O
+crystals	O
+in	O
+a	O
+single	O
+set	O
+of	O
+conditions	O
+containing	O
+HEPES	O
+buffer	O
+and	O
+Jeffamine	O
+M	O
+-	O
+600	O
+—	O
+the	O
+same	O
+crystallization	O
+conditions	O
+that	O
+afforded	O
+crystals	O
+of	O
+peptide	B-mutant
+1	I-mutant
+.	O
+	O
+Peptide	B-mutant
+2	I-mutant
+also	O
+afforded	O
+crystals	O
+in	O
+these	O
+conditions	O
+.	O
+	O
+We	O
+further	O
+optimized	O
+these	O
+conditions	O
+to	O
+rapidly	O
+(∼	O
+72	O
+h	O
+)	O
+yield	O
+crystals	O
+suitable	O
+for	O
+X	O
+-	O
+ray	O
+crystallography	O
+.	O
+	O
+The	O
+optimized	O
+conditions	O
+consist	O
+of	O
+0	O
+.	O
+1	O
+M	O
+HEPES	O
+at	O
+pH	O
+6	O
+.	O
+4	O
+with	O
+31	O
+%	O
+Jeffamine	O
+M	O
+-	O
+600	O
+for	O
+peptide	B-mutant
+4	I-mutant
+and	O
+0	O
+.	O
+1	O
+M	O
+HEPES	O
+pH	O
+7	O
+.	O
+1	O
+with	O
+29	O
+%	O
+Jeffamine	O
+M	O
+-	O
+600	O
+for	O
+peptide	B-mutant
+2	I-mutant
+.	O
+	O
+Crystal	O
+diffraction	O
+data	O
+for	O
+peptide	B-mutant
+2	I-mutant
+were	O
+also	O
+collected	O
+at	O
+the	O
+Advanced	O
+Light	O
+Source	O
+at	O
+Lawrence	O
+Berkeley	O
+National	O
+Laboratory	O
+with	O
+a	O
+synchrotron	O
+source	O
+at	O
+1	O
+.	O
+00	O
+Å	O
+wavelength	O
+to	O
+achieve	O
+higher	O
+resolution	O
+.	O
+	O
+Phases	O
+for	O
+peptide	B-mutant
+4	I-mutant
+were	O
+determined	O
+by	O
+single	O
+-	O
+wavelength	O
+anomalous	O
+diffraction	O
+(	O
+SAD	O
+)	O
+phasing	O
+by	O
+using	O
+the	O
+coordinates	O
+of	O
+the	O
+iodine	O
+anomalous	O
+signal	O
+from	O
+p	O
+-	O
+iodophenylalanine	O
+.	O
+	O
+Phases	O
+for	O
+peptide	B-mutant
+2	I-mutant
+were	O
+determined	O
+by	O
+isomorphous	O
+replacement	O
+of	O
+peptide	B-mutant
+4	I-mutant
+.	O
+	O
+The	O
+structures	O
+of	O
+peptides	B-mutant
+2	I-mutant
+and	I-mutant
+4	I-mutant
+were	O
+solved	O
+and	O
+refined	O
+in	O
+the	O
+P6122	O
+space	O
+group	O
+.	O
+	O
+The	O
+asymmetric	O
+unit	O
+of	O
+each	O
+peptide	O
+consists	O
+of	O
+six	O
+monomers	O
+,	O
+arranged	O
+as	O
+two	O
+trimers	O
+.	O
+	O
+Peptides	B-mutant
+2	I-mutant
+and	I-mutant
+4	I-mutant
+form	O
+morphologically	O
+identical	O
+structures	O
+and	O
+assemblies	O
+in	O
+the	O
+crystal	O
+lattice	O
+.	O
+	O
+The	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+2	I-mutant
+reveals	O
+that	O
+it	O
+folds	O
+to	O
+form	O
+a	O
+twisted	O
+β	O
+-	O
+hairpin	O
+comprising	O
+two	O
+β	O
+-	O
+strands	O
+connected	O
+by	O
+a	O
+loop	O
+(	O
+Figure	O
+2A	O
+).	O
+	O
+Eight	O
+residues	O
+make	O
+up	O
+each	O
+surface	O
+of	O
+the	O
+β	O
+-	O
+hairpin	O
+:	O
+L17	O
+,	O
+F19	O
+,	O
+A21	O
+,	O
+D23	O
+,	O
+A30	O
+,	O
+I32	O
+,	O
+L34	O
+,	O
+and	O
+V36	O
+make	O
+up	O
+one	O
+surface	O
+;	O
+V18	O
+,	O
+F20	O
+,	O
+E22	O
+,	O
+C24	O
+,	O
+C29	O
+,	O
+I31	O
+,	O
+G33	O
+,	O
+and	O
+M35	O
+make	O
+up	O
+the	O
+other	O
+surface	O
+.	O
+	O
+The	O
+β	O
+-	O
+strands	O
+of	O
+the	O
+monomers	O
+in	O
+the	O
+asymmetric	O
+unit	O
+are	O
+virtually	O
+identical	O
+,	O
+differing	O
+primarily	O
+in	O
+rotamers	O
+of	O
+F20	O
+,	O
+E22	O
+,	O
+C24	O
+,	O
+C29	O
+,	O
+I31	O
+,	O
+and	O
+M35	O
+(	O
+Figure	O
+S1	O
+).	O
+	O
+The	O
+disulfide	O
+linkages	O
+suffered	O
+radiation	O
+damage	O
+under	O
+synchrotron	O
+radiation	O
+.	O
+	O
+We	O
+refined	O
+three	O
+of	O
+the	O
+β	O
+-	O
+hairpins	O
+with	O
+intact	O
+disulfide	O
+linkages	O
+and	O
+three	O
+with	O
+thiols	O
+to	O
+represent	O
+cleaved	O
+disulfide	O
+linkages	O
+in	O
+the	O
+synchrotron	O
+data	O
+set	O
+(	O
+PDB	O
+5HOX	O
+).	O
+	O
+No	O
+evidence	O
+for	O
+cleavage	O
+of	O
+the	O
+disulfides	O
+was	O
+observed	O
+in	O
+the	O
+refinement	O
+of	O
+the	O
+data	O
+set	O
+collected	O
+on	O
+the	O
+X	O
+-	O
+ray	O
+diffractometer	O
+,	O
+and	O
+we	O
+refined	O
+all	O
+disulfide	O
+linkages	O
+as	O
+intact	O
+(	O
+PDB	O
+5HOY	O
+).	O
+	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+2	I-mutant
+(	O
+PDB	O
+5HOX	O
+,	O
+synchrotron	O
+data	O
+set	O
+).	O
+(	O
+A	O
+)	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+a	O
+representative	O
+β	O
+-	O
+hairpin	O
+monomer	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+.	O
+(	O
+B	O
+)	O
+Overlay	O
+of	O
+the	O
+six	O
+β	O
+-	O
+hairpin	O
+monomers	O
+in	O
+the	O
+asymmetric	O
+unit	O
+.	O
+	O
+The	O
+β	O
+-	O
+hairpins	O
+are	O
+shown	O
+as	O
+cartoons	O
+to	O
+illustrate	O
+the	O
+differences	O
+in	O
+the	O
+Aβ25	O
+–	O
+28	O
+loops	O
+.	O
+	O
+The	O
+Aβ25	O
+–	O
+28	O
+loops	O
+of	O
+the	O
+six	O
+monomers	O
+within	O
+the	O
+asymmetric	O
+unit	O
+vary	O
+substantially	O
+in	O
+backbone	O
+geometry	O
+and	O
+side	O
+chain	O
+rotamers	O
+(	O
+Figures	O
+2B	O
+and	O
+S1	O
+).	O
+	O
+The	O
+electron	O
+density	O
+for	O
+the	O
+loops	O
+is	O
+weak	O
+and	O
+diffuse	O
+compared	O
+to	O
+the	O
+electron	O
+density	O
+for	O
+the	O
+β	O
+-	O
+strands	O
+.	O
+	O
+Trimer	O
+	O
+Peptide	B-mutant
+2	I-mutant
+forms	O
+a	O
+trimer	O
+,	O
+much	O
+like	O
+that	O
+which	O
+we	O
+observed	O
+previously	O
+for	O
+peptide	B-mutant
+1	I-mutant
+,	O
+in	O
+which	O
+three	O
+β	O
+-	O
+hairpins	O
+assemble	O
+to	O
+form	O
+an	O
+equilateral	O
+triangle	O
+(	O
+Figure	O
+3A	O
+).	O
+	O
+The	O
+disulfide	O
+bonds	O
+between	O
+residues	O
+24	O
+and	O
+29	O
+are	O
+adjacent	O
+to	O
+the	O
+structural	O
+core	O
+of	O
+the	O
+trimer	O
+and	O
+do	O
+not	O
+make	O
+any	O
+substantial	O
+intermolecular	O
+contacts	O
+.	O
+	O
+Two	O
+crystallographically	O
+distinct	O
+trimers	O
+comprise	O
+the	O
+peptide	O
+portion	O
+of	O
+the	O
+asymmetric	O
+unit	O
+.	O
+	O
+The	O
+two	O
+trimers	O
+are	O
+almost	O
+identical	O
+in	O
+structure	O
+,	O
+differing	O
+slightly	O
+among	O
+side	O
+chain	O
+rotamers	O
+and	O
+loop	O
+conformations	O
+.	O
+	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+the	O
+trimer	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+.	O
+(	O
+A	O
+)	O
+Triangular	O
+trimer	O
+.	O
+	O
+The	O
+three	O
+water	O
+molecules	O
+in	O
+the	O
+center	O
+hole	O
+of	O
+the	O
+trimer	O
+are	O
+shown	O
+as	O
+spheres	O
+.	O
+(	O
+B	O
+)	O
+Detailed	O
+view	O
+of	O
+the	O
+intermolecular	O
+hydrogen	O
+bonds	O
+between	O
+the	O
+main	O
+chains	O
+of	O
+V18	O
+and	O
+E22	O
+and	O
+δOrn	O
+and	O
+C24	O
+,	O
+at	O
+the	O
+three	O
+corners	O
+of	O
+the	O
+triangular	O
+trimer	O
+.	O
+(	O
+C	O
+)	O
+The	O
+F19	O
+face	O
+of	O
+the	O
+trimer	O
+,	O
+with	O
+key	O
+side	O
+chains	O
+shown	O
+as	O
+spheres	O
+.	O
+(	O
+D	O
+)	O
+The	O
+F20	O
+face	O
+of	O
+the	O
+trimer	O
+,	O
+with	O
+key	O
+side	O
+chains	O
+as	O
+spheres	O
+.	O
+	O
+A	O
+network	O
+of	O
+18	O
+intermolecular	O
+hydrogen	O
+bonds	O
+helps	O
+stabilize	O
+the	O
+trimer	O
+.	O
+	O
+Three	O
+ordered	O
+water	O
+molecules	O
+fill	O
+the	O
+hole	O
+in	O
+the	O
+center	O
+of	O
+the	O
+trimer	O
+,	O
+hydrogen	O
+bonding	O
+to	O
+each	O
+other	O
+and	O
+to	O
+the	O
+main	O
+chain	O
+of	O
+F20	O
+(	O
+Figure	O
+3A	O
+).	O
+	O
+Hydrophobic	O
+contacts	O
+between	O
+residues	O
+at	O
+the	O
+three	O
+corners	O
+of	O
+the	O
+trimer	O
+,	O
+where	O
+the	O
+β	O
+-	O
+hairpins	O
+meet	O
+,	O
+further	O
+stabilize	O
+the	O
+trimer	O
+.	O
+	O
+At	O
+each	O
+corner	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+residues	O
+L17	O
+,	O
+F19	O
+,	O
+and	O
+V36	O
+of	O
+one	O
+β	O
+-	O
+hairpin	O
+pack	O
+against	O
+the	O
+side	O
+chains	O
+of	O
+residues	O
+A21	O
+,	O
+I32	O
+,	O
+L34	O
+,	O
+and	O
+also	O
+D23	O
+of	O
+the	O
+adjacent	O
+β	O
+-	O
+hairpin	O
+to	O
+create	O
+a	O
+hydrophobic	O
+cluster	O
+(	O
+Figure	O
+3C	O
+).	O
+The	O
+three	O
+hydrophobic	O
+clusters	O
+create	O
+a	O
+large	O
+hydrophobic	O
+surface	O
+on	O
+one	O
+face	O
+of	O
+the	O
+trimer	O
+.	O
+	O
+In	O
+subsequent	O
+discussion	O
+,	O
+we	O
+designate	O
+the	O
+former	O
+surface	O
+the	O
+“	O
+F19	O
+face	O
+”	O
+and	O
+the	O
+latter	O
+surface	O
+the	O
+“	O
+F20	O
+face	O
+”.	O
+	O
+Four	O
+trimers	O
+assemble	O
+to	O
+form	O
+a	O
+dodecamer	O
+.	O
+	O
+Each	O
+of	O
+the	O
+12	O
+β	O
+-	O
+hairpins	O
+constitutes	O
+an	O
+edge	O
+of	O
+the	O
+octahedron	O
+,	O
+and	O
+the	O
+triangular	O
+trimers	O
+occupy	O
+four	O
+of	O
+the	O
+eight	O
+faces	O
+of	O
+the	O
+octahedron	O
+.	O
+	O
+Figure	O
+4A	O
+illustrates	O
+the	O
+octahedral	O
+shape	O
+of	O
+the	O
+dodecamer	O
+.	O
+	O
+Figure	O
+4B	O
+illustrates	O
+the	O
+tetrahedral	O
+arrangement	O
+of	O
+the	O
+four	O
+trimers	O
+.	O
+	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+the	O
+dodecamer	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+.	O
+(	O
+A	O
+)	O
+View	O
+of	O
+the	O
+dodecamer	O
+that	O
+illustrates	O
+the	O
+octahedral	O
+shape	O
+.	O
+(	O
+B	O
+)	O
+View	O
+of	O
+the	O
+dodecamer	O
+that	O
+illustrates	O
+the	O
+tetrahedral	O
+arrangement	O
+of	O
+the	O
+four	O
+trimers	O
+that	O
+comprise	O
+the	O
+dodecamer	O
+.	O
+(	O
+C	O
+)	O
+View	O
+of	O
+two	O
+trimer	O
+subunits	O
+from	O
+inside	O
+the	O
+cavity	O
+of	O
+the	O
+dodecamer	O
+.	O
+	O
+The	O
+F19	O
+faces	O
+of	O
+the	O
+trimers	O
+line	O
+the	O
+interior	O
+of	O
+the	O
+dodecamer	O
+.	O
+	O
+At	O
+the	O
+six	O
+vertices	O
+,	O
+hydrophobic	O
+packing	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+L17	O
+,	O
+L34	O
+,	O
+and	O
+V36	O
+helps	O
+stabilize	O
+the	O
+dodecamer	O
+(	O
+Figures	O
+4C	O
+and	O
+D	O
+).	O
+	O
+Salt	O
+bridges	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+D23	O
+and	O
+δOrn	O
+at	O
+the	O
+vertices	O
+further	O
+stabilize	O
+the	O
+dodecamer	O
+.	O
+	O
+Each	O
+of	O
+the	O
+six	O
+vertices	O
+includes	O
+two	O
+Aβ25	O
+–	O
+28	O
+loops	O
+that	O
+extend	O
+past	O
+the	O
+core	O
+of	O
+the	O
+dodecamer	O
+without	O
+making	O
+any	O
+substantial	O
+intermolecular	O
+contacts	O
+.	O
+	O
+The	O
+exterior	O
+of	O
+the	O
+dodecamer	O
+displays	O
+four	O
+F20	O
+faces	O
+(	O
+Figure	O
+S3	O
+).	O
+	O
+Although	O
+the	O
+asymmetric	O
+unit	O
+comprises	O
+half	O
+a	O
+dodecamer	O
+,	O
+the	O
+crystal	O
+lattice	O
+may	O
+be	O
+thought	O
+of	O
+as	O
+being	O
+built	O
+of	O
+dodecamers	O
+.	O
+	O
+The	O
+shape	O
+and	O
+length	O
+of	O
+the	O
+electron	O
+density	O
+is	O
+consistent	O
+with	O
+the	O
+structure	O
+of	O
+Jeffamine	O
+M	O
+-	O
+600	O
+,	O
+which	O
+is	O
+an	O
+essential	O
+component	O
+of	O
+the	O
+crystallization	O
+conditions	O
+.	O
+	O
+The	O
+Jeffamine	O
+M	O
+-	O
+600	O
+appears	O
+to	O
+stabilize	O
+the	O
+dodecamer	O
+by	O
+occupying	O
+the	O
+central	O
+cavity	O
+and	O
+making	O
+hydrophobic	O
+contacts	O
+with	O
+residues	O
+lining	O
+the	O
+cavity	O
+(	O
+Figure	O
+S3	O
+).	O
+	O
+In	O
+a	O
+dodecamer	O
+formed	O
+by	O
+full	O
+-	O
+length	O
+Aβ	O
+,	O
+the	O
+hydrophobic	O
+C	O
+-	O
+terminal	O
+residues	O
+(	O
+Aβ37	O
+–	O
+40	O
+or	O
+Aβ37	O
+–	O
+42	O
+)	O
+might	O
+play	O
+a	O
+similar	O
+role	O
+in	O
+filling	O
+the	O
+dodecamer	O
+and	O
+thus	O
+create	O
+a	O
+packed	O
+hydrophobic	O
+core	O
+within	O
+the	O
+central	O
+cavity	O
+of	O
+the	O
+dodecamer	O
+.	O
+	O
+Five	O
+dodecamers	O
+assemble	O
+to	O
+form	O
+an	O
+annular	O
+porelike	O
+structure	O
+(	O
+Figure	O
+5A	O
+).	O
+	O
+Two	O
+morphologically	O
+distinct	O
+interactions	O
+between	O
+trimers	O
+occur	O
+at	O
+the	O
+interfaces	O
+of	O
+the	O
+five	O
+dodecamers	O
+:	O
+one	O
+in	O
+which	O
+the	O
+trimers	O
+are	O
+eclipsed	O
+(	O
+Figure	O
+5B	O
+),	O
+and	O
+one	O
+in	O
+which	O
+the	O
+trimers	O
+are	O
+staggered	O
+(	O
+Figure	O
+5C	O
+).	O
+	O
+Hydrophobic	O
+packing	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+F20	O
+,	O
+I31	O
+,	O
+and	O
+E22	O
+stabilizes	O
+these	O
+interfaces	O
+(	O
+Figure	O
+5D	O
+and	O
+E	O
+).	O
+	O
+The	O
+annular	O
+pore	O
+contains	O
+three	O
+eclipsed	O
+interfaces	O
+and	O
+two	O
+staggered	O
+interfaces	O
+.	O
+	O
+The	O
+staggered	O
+interfaces	O
+occur	O
+between	O
+dodecamers	O
+2	O
+and	O
+3	O
+and	O
+4	O
+and	O
+5	O
+.	O
+	O
+The	O
+annular	O
+pore	O
+is	O
+not	O
+completely	O
+flat	O
+,	O
+instead	O
+,	O
+adopting	O
+a	O
+slightly	O
+puckered	O
+shape	O
+,	O
+which	O
+accommodates	O
+the	O
+eclipsed	O
+and	O
+staggered	O
+interfaces	O
+.	O
+	O
+Ten	O
+Aβ25	O
+–	O
+28	O
+loops	O
+from	O
+the	O
+vertices	O
+of	O
+the	O
+five	O
+dodecamers	O
+line	O
+the	O
+hole	O
+in	O
+the	O
+center	O
+of	O
+the	O
+pore	O
+.	O
+	O
+The	O
+hydrophilic	O
+side	O
+chains	O
+of	O
+S26	O
+,	O
+N27	O
+,	O
+and	O
+K28	O
+decorate	O
+the	O
+hole	O
+.	O
+	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+the	O
+annular	O
+pore	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+.	O
+(	O
+A	O
+)	O
+Annular	O
+porelike	O
+structure	O
+illustrating	O
+the	O
+relationship	O
+of	O
+the	O
+five	O
+dodecamers	O
+that	O
+form	O
+the	O
+pore	O
+(	O
+top	O
+view	O
+).	O
+	O
+(	O
+B	O
+)	O
+Eclipsed	O
+interface	O
+between	O
+dodecamers	O
+1	O
+and	O
+2	O
+(	O
+side	O
+view	O
+).	O
+	O
+The	O
+same	O
+staggered	O
+interface	O
+also	O
+occurs	O
+between	O
+dodecamers	O
+4	O
+and	O
+5	O
+.	O
+(	O
+D	O
+)	O
+Eclipsed	O
+interface	O
+between	O
+dodecamers	O
+1	O
+and	O
+5	O
+(	O
+top	O
+view	O
+).	O
+	O
+The	O
+annular	O
+pore	O
+is	O
+comparable	O
+in	O
+size	O
+to	O
+other	O
+large	O
+protein	O
+assemblies	O
+.	O
+	O
+The	O
+diameter	O
+of	O
+the	O
+hole	O
+in	O
+the	O
+center	O
+of	O
+the	O
+pore	O
+is	O
+∼	O
+2	O
+nm	O
+.	O
+	O
+The	O
+thickness	O
+of	O
+the	O
+pore	O
+is	O
+∼	O
+5	O
+nm	O
+,	O
+which	O
+is	O
+comparable	O
+to	O
+that	O
+of	O
+a	O
+lipid	O
+bilayer	O
+membrane	O
+.	O
+	O
+Rather	O
+,	O
+the	O
+crystal	O
+lattice	O
+is	O
+composed	O
+of	O
+conjoined	O
+annular	O
+pores	O
+in	O
+which	O
+all	O
+four	O
+F20	O
+faces	O
+on	O
+the	O
+surface	O
+of	O
+each	O
+dodecamer	O
+contact	O
+F20	O
+faces	O
+on	O
+other	O
+dodecamers	O
+(	O
+Figure	O
+S4	O
+).	O
+	O
+The	O
+crystal	O
+lattice	O
+shows	O
+how	O
+the	O
+dodecamers	O
+can	O
+further	O
+assemble	O
+to	O
+form	O
+larger	O
+structures	O
+.	O
+	O
+Each	O
+dodecamer	O
+may	O
+be	O
+thought	O
+of	O
+as	O
+a	O
+tetravalent	O
+building	O
+block	O
+with	O
+the	O
+potential	O
+to	O
+assemble	O
+on	O
+all	O
+four	O
+faces	O
+to	O
+form	O
+higher	O
+-	O
+order	O
+supramolecular	O
+assemblies	O
+.	O
+	O
+The	O
+X	O
+-	O
+ray	O
+crystallographic	O
+study	O
+of	O
+peptide	B-mutant
+2	I-mutant
+described	O
+here	O
+provides	O
+high	O
+-	O
+resolution	O
+structures	O
+of	O
+oligomers	O
+formed	O
+by	O
+an	O
+Aβ17	O
+–	O
+36	O
+β	O
+-	O
+hairpin	O
+.	O
+	O
+Model	O
+for	O
+the	O
+hierarchical	O
+assembly	O
+of	O
+an	O
+Aβ	O
+β	O
+-	O
+hairpin	O
+into	O
+a	O
+trimer	O
+,	O
+dodecamer	O
+,	O
+and	O
+annular	O
+pore	O
+based	O
+on	O
+the	O
+crystallographic	O
+assembly	O
+of	O
+peptide	B-mutant
+2	I-mutant
+.	O
+	O
+Three	O
+β	O
+-	O
+hairpin	O
+monomers	O
+assemble	O
+to	O
+form	O
+a	O
+triangular	O
+trimer	O
+.	O
+	O
+The	O
+molecular	O
+weights	O
+shown	O
+correspond	O
+to	O
+an	O
+Aβ42	O
+monomer	O
+(∼	O
+4	O
+.	O
+5	O
+kDa	O
+),	O
+an	O
+Aβ42	O
+trimer	O
+(∼	O
+13	O
+.	O
+5	O
+kDa	O
+),	O
+an	O
+Aβ42	O
+dodecamer	O
+(∼	O
+54	O
+kDa	O
+),	O
+and	O
+an	O
+Aβ42	O
+annular	O
+pore	O
+composed	O
+of	O
+five	O
+dodecamers	O
+(∼	O
+270	O
+kDa	O
+).	O
+	O
+Two	O
+general	O
+types	O
+of	O
+endogenous	O
+Aβ	O
+oligomers	O
+have	O
+been	O
+observed	O
+:	O
+Aβ	O
+oligomers	O
+that	O
+occur	O
+on	O
+a	O
+pathway	O
+to	O
+fibrils	O
+,	O
+or	O
+“	O
+fibrillar	O
+oligomers	O
+”,	O
+and	O
+Aβ	O
+oligomers	O
+that	O
+evade	O
+a	O
+fibrillar	O
+fate	O
+,	O
+or	O
+“	O
+nonfibrillar	O
+oligomers	O
+”.−	O
+Fibrillar	O
+oligomers	O
+accumulate	O
+in	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+later	O
+than	O
+nonfibrillar	O
+oligomers	O
+and	O
+coincide	O
+with	O
+the	O
+deposition	O
+of	O
+plaques	O
+.	O
+	O
+Nonfibrillar	O
+oligomers	O
+accumulate	O
+early	O
+in	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+before	O
+plaque	O
+deposition	O
+.	O
+	O
+Fibrillar	O
+oligomers	O
+are	O
+recognized	O
+by	O
+the	O
+OC	O
+antibody	O
+but	O
+not	O
+the	O
+A11	O
+antibody	O
+,	O
+whereas	O
+nonfibrillar	O
+oligomers	O
+are	O
+recognized	O
+by	O
+the	O
+A11	O
+antibody	O
+but	O
+not	O
+the	O
+OC	O
+antibody	O
+.	O
+	O
+Larson	O
+and	O
+Lesné	O
+proposed	O
+a	O
+model	O
+for	O
+the	O
+endogenous	O
+production	O
+of	O
+nonfibrillar	O
+oligomers	O
+that	O
+explains	O
+these	O
+observations	O
+.	O
+	O
+In	O
+this	O
+model	O
+,	O
+folded	O
+Aβ	O
+monomer	O
+assembles	O
+into	O
+a	O
+trimer	O
+,	O
+the	O
+trimer	O
+further	O
+assembles	O
+into	O
+hexamers	O
+and	O
+dodecamers	O
+,	O
+and	O
+the	O
+dodecamers	O
+further	O
+assemble	O
+to	O
+form	O
+annular	O
+protofibrils	O
+.	O
+	O
+The	O
+crystallographically	O
+observed	O
+annular	O
+pore	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+is	O
+morphologically	O
+similar	O
+to	O
+the	O
+APFs	O
+formed	O
+by	O
+full	O
+-	O
+length	O
+Aβ	O
+.	O
+	O
+The	O
+annular	O
+pore	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+is	O
+comparable	O
+in	O
+size	O
+to	O
+the	O
+APFs	O
+prepared	O
+in	O
+vitro	O
+or	O
+isolated	O
+from	O
+Alzheimer	O
+’	O
+s	O
+brains	O
+(	O
+Figure	O
+7	O
+and	O
+Table	O
+1	O
+).	O
+	O
+The	O
+varying	O
+sizes	O
+of	O
+APFs	O
+formed	O
+by	O
+full	O
+-	O
+length	O
+Aβ	O
+might	O
+result	O
+from	O
+differences	O
+in	O
+the	O
+number	O
+of	O
+oligomer	O
+subunits	O
+comprising	O
+each	O
+APF	O
+.	O
+	O
+Although	O
+the	O
+annular	O
+pore	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+contains	O
+five	O
+dodecamer	O
+subunits	O
+,	O
+pores	O
+containing	O
+fewer	O
+or	O
+more	O
+subunits	O
+can	O
+easily	O
+be	O
+envisioned	O
+.	O
+	O
+The	O
+dodecamers	O
+that	O
+comprise	O
+the	O
+annular	O
+pore	O
+exhibit	O
+two	O
+modes	O
+of	O
+assembly	O
+—	O
+eclipsed	O
+interactions	O
+and	O
+staggered	O
+interactions	O
+between	O
+the	O
+F20	O
+faces	O
+of	O
+trimers	O
+within	O
+dodecamers	O
+.	O
+	O
+Surface	O
+views	O
+of	O
+the	O
+annular	O
+pore	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+.	O
+(	O
+A	O
+)	O
+Top	O
+view	O
+.	O
+	O
+annular	O
+pore	O
+source	O
+outer	O
+diameter	O
+inner	O
+diameter	O
+observation	O
+method	O
+peptide	O
+2	O
+∼	O
+11	O
+–	O
+12	O
+nm	O
+∼	O
+2	O
+nm	O
+X	O
+-	O
+ray	O
+crystallography	O
+synthetic	O
+Aβ	O
+7	O
+–	O
+10	O
+nm	O
+1	O
+.	O
+5	O
+–	O
+2	O
+nm	O
+TEM	O
+synthetic	O
+Aβ	O
+16	O
+nm	O
+not	O
+reported	O
+AFM	O
+synthetic	O
+Aβ	O
+8	O
+–	O
+25	O
+nm	O
+not	O
+reported	O
+TEM	O
+Alzheimer	O
+’	O
+s	O
+brain	O
+11	O
+–	O
+14	O
+nm	O
+2	O
+.	O
+5	O
+–	O
+4	O
+nm	O
+TEM	O
+	O
+Dot	O
+blot	O
+analysis	O
+shows	O
+that	O
+peptide	B-mutant
+2	I-mutant
+is	O
+reactive	O
+toward	O
+the	O
+A11	O
+antibody	O
+(	O
+Figure	O
+S5	O
+).	O
+	O
+This	O
+reactivity	O
+suggests	O
+that	O
+peptide	B-mutant
+2	I-mutant
+forms	O
+oligomers	O
+in	O
+solution	O
+that	O
+share	O
+structural	O
+similarities	O
+to	O
+the	O
+nonfibrillar	O
+oligomers	O
+formed	O
+by	O
+full	O
+-	O
+length	O
+Aβ	O
+.	O
+	O
+Further	O
+studies	O
+are	O
+needed	O
+to	O
+elucidate	O
+the	O
+species	O
+that	O
+peptide	B-mutant
+2	I-mutant
+forms	O
+in	O
+solution	O
+and	O
+to	O
+study	O
+their	O
+biological	O
+properties	O
+.	O
+	O
+The	O
+difficulty	O
+in	O
+studying	O
+the	O
+oligomers	O
+formed	O
+in	O
+solution	O
+may	O
+reflect	O
+the	O
+propensity	O
+of	O
+the	O
+dodecamer	O
+to	O
+assemble	O
+on	O
+all	O
+four	O
+F20	O
+faces	O
+.	O
+	O
+The	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+and	O
+A11	O
+reactivity	O
+of	O
+peptide	B-mutant
+2	I-mutant
+support	O
+the	O
+model	O
+proposed	O
+by	O
+Larsen	O
+and	O
+Lesné	O
+and	O
+suggest	O
+that	O
+β	O
+-	O
+hairpins	O
+constitute	O
+a	O
+fundamental	O
+building	O
+block	O
+for	O
+nonfibrillar	O
+oligomers	O
+.	O
+	O
+What	O
+makes	O
+β	O
+-	O
+hairpins	O
+special	O
+is	O
+that	O
+three	O
+β	O
+-	O
+hairpins	O
+can	O
+nestle	O
+together	O
+to	O
+form	O
+trimers	O
+,	O
+stabilized	O
+by	O
+a	O
+network	O
+of	O
+hydrogen	O
+bonds	O
+and	O
+hydrophobic	O
+interactions	O
+.	O
+	O
+The	O
+foldon	O
+domain	O
+of	O
+bacteriophage	O
+T4	O
+fibritin	O
+is	O
+composed	O
+of	O
+three	O
+β	O
+-	O
+hairpins	O
+that	O
+assemble	O
+into	O
+a	O
+triangular	O
+trimer	O
+similar	O
+to	O
+the	O
+triangular	O
+trimer	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+.	O
+	O
+Additionally	O
+,	O
+our	O
+research	O
+group	O
+has	O
+observed	O
+a	O
+similar	O
+assembly	O
+of	O
+a	O
+β	O
+-	O
+hairpin	O
+peptide	O
+derived	O
+from	O
+β2	O
+-	O
+microglobulin	O
+.	O
+	O
+Although	O
+we	O
+began	O
+these	O
+studies	O
+with	O
+a	O
+relatively	O
+simple	O
+hypothesis	O
+—	O
+that	O
+the	O
+trimers	O
+and	O
+dodecamers	O
+formed	O
+by	O
+peptide	B-mutant
+1	I-mutant
+could	O
+accommodate	O
+the	O
+Aβ24	O
+–	O
+29	O
+loop	O
+—	O
+an	O
+even	O
+more	O
+exciting	O
+finding	O
+has	O
+emerged	O
+—	O
+that	O
+the	O
+dodecamers	O
+can	O
+assemble	O
+to	O
+form	O
+annular	O
+pores	O
+.	O
+	O
+This	O
+finding	O
+could	O
+not	O
+have	O
+been	O
+anticipated	O
+from	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+1	I-mutant
+and	O
+reveals	O
+a	O
+new	O
+level	O
+of	O
+hierarchical	O
+assembly	O
+that	O
+recapitulates	O
+micrographic	O
+observations	O
+of	O
+annular	O
+protofibrils	O
+.	O
+	O
+The	O
+crystallographically	O
+observed	O
+dodecamer	O
+,	O
+in	O
+turn	O
+,	O
+recapitulates	O
+the	O
+observation	O
+of	O
+Aβ	O
+*	O
+56	O
+,	O
+which	O
+appears	O
+to	O
+be	O
+a	O
+dodecamer	O
+of	O
+Aβ	O
+.	O
+	O
+The	O
+crystallographically	O
+observed	O
+trimer	O
+recapitulates	O
+the	O
+Aβ	O
+trimers	O
+that	O
+are	O
+observed	O
+even	O
+before	O
+the	O
+onset	O
+of	O
+symptoms	O
+in	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+.	O
+	O
+Predictive	O
+features	O
+of	O
+ligand	O
+‐	O
+specific	O
+signaling	O
+through	O
+the	O
+estrogen	O
+receptor	O
+	O
+Some	O
+estrogen	O
+receptor	O
+‐	O
+α	O
+(	O
+ERα	O
+)‐	O
+targeted	O
+breast	O
+cancer	O
+therapies	O
+such	O
+as	O
+tamoxifen	O
+have	O
+tissue	O
+‐	O
+selective	O
+or	O
+cell	O
+‐	O
+specific	O
+activities	O
+,	O
+while	O
+others	O
+have	O
+similar	O
+activities	O
+in	O
+different	O
+cell	O
+types	O
+.	O
+	O
+To	O
+identify	O
+biophysical	O
+determinants	O
+of	O
+cell	O
+‐	O
+specific	O
+signaling	O
+and	O
+breast	O
+cancer	O
+cell	O
+proliferation	O
+,	O
+we	O
+synthesized	O
+241	O
+ERα	O
+ligands	O
+based	O
+on	O
+19	O
+chemical	O
+scaffolds	O
+,	O
+and	O
+compared	O
+ligand	O
+response	O
+using	O
+quantitative	O
+bioassays	O
+for	O
+canonical	O
+ERα	O
+activities	O
+and	O
+X	O
+‐	O
+ray	O
+crystallography	O
+.	O
+	O
+For	O
+some	O
+ligand	O
+series	O
+,	O
+a	O
+single	O
+inter	O
+‐	O
+atomic	O
+distance	O
+in	O
+the	O
+ligand	O
+‐	O
+binding	O
+domain	O
+predicted	O
+their	O
+proliferative	O
+effects	O
+.	O
+	O
+Thus	O
+,	O
+incorporating	O
+systems	O
+structural	O
+analyses	O
+with	O
+quantitative	O
+chemical	O
+biology	O
+reveals	O
+how	O
+ligands	O
+can	O
+achieve	O
+distinct	O
+allosteric	O
+signaling	O
+outcomes	O
+through	O
+ERα	O
+.	O
+	O
+Many	O
+drugs	O
+are	O
+small	O
+‐	O
+molecule	O
+ligands	O
+of	O
+allosteric	O
+signaling	O
+proteins	O
+,	O
+including	O
+G	O
+protein	O
+‐	O
+coupled	O
+receptors	O
+(	O
+GPCRs	O
+)	O
+and	O
+nuclear	O
+receptors	O
+such	O
+as	O
+ERα	O
+.	O
+	O
+Small	O
+‐	O
+molecule	O
+ligands	O
+control	O
+receptor	O
+activity	O
+by	O
+modulating	O
+recruitment	O
+of	O
+effector	O
+enzymes	O
+to	O
+distal	O
+regions	O
+of	O
+the	O
+receptor	O
+,	O
+relative	O
+to	O
+the	O
+ligand	O
+‐	O
+binding	O
+site	O
+.	O
+	O
+Allosteric	O
+control	O
+of	O
+ERα	O
+activity	O
+	O
+Chemical	O
+structures	O
+of	O
+some	O
+common	O
+ERα	O
+ligands	O
+.	O
+	O
+Schematic	O
+illustration	O
+of	O
+the	O
+canonical	O
+ERα	O
+signaling	O
+pathway	O
+.	O
+	O
+ERα	O
+contains	O
+structurally	O
+conserved	O
+globular	O
+domains	O
+of	O
+the	O
+nuclear	O
+receptor	O
+superfamily	O
+,	O
+including	O
+a	O
+DNA	O
+‐	O
+binding	O
+domain	O
+(	O
+DBD	O
+)	O
+that	O
+is	O
+connected	O
+by	O
+a	O
+flexible	O
+hinge	O
+region	O
+to	O
+the	O
+ligand	O
+‐	O
+binding	O
+domain	O
+(	O
+LBD	O
+),	O
+as	O
+well	O
+as	O
+unstructured	O
+AB	O
+and	O
+F	O
+domains	O
+at	O
+its	O
+amino	O
+and	O
+carboxyl	O
+termini	O
+,	O
+respectively	O
+(	O
+Fig	O
+1B	O
+).	O
+	O
+The	O
+LBD	O
+contains	O
+a	O
+ligand	O
+‐	O
+dependent	O
+coactivator	O
+‐	O
+binding	O
+site	O
+called	O
+activation	O
+function	O
+‐	O
+2	O
+(	O
+AF	O
+‐	O
+2	O
+).	O
+	O
+AF	O
+‐	O
+1	O
+and	O
+AF	O
+‐	O
+2	O
+bind	O
+distinct	O
+but	O
+overlapping	O
+sets	O
+of	O
+coregulators	O
+(	O
+Webb	O
+et	O
+al	O
+,	O
+1998	O
+;	O
+Endoh	O
+et	O
+al	O
+,	O
+1999	O
+;	O
+Delage	O
+‐	O
+Mourroux	O
+et	O
+al	O
+,	O
+2000	O
+;	O
+Yi	O
+et	O
+al	O
+,	O
+2015	O
+).	O
+	O
+AF	O
+‐	O
+2	O
+binds	O
+the	O
+signature	O
+LxxLL	O
+motif	O
+peptides	O
+of	O
+coactivators	O
+such	O
+as	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+(	O
+also	O
+known	O
+as	O
+SRC	O
+‐	O
+1	O
+/	O
+2	O
+/	O
+3	O
+).	O
+	O
+Yet	O
+,	O
+it	O
+is	O
+unknown	O
+how	O
+different	O
+ERα	O
+ligands	O
+control	O
+AF	O
+‐	O
+1	O
+through	O
+the	O
+LBD	O
+,	O
+and	O
+whether	O
+this	O
+inter	O
+‐	O
+domain	O
+communication	O
+is	O
+required	O
+for	O
+cell	O
+‐	O
+specific	O
+signaling	O
+or	O
+anti	O
+‐	O
+proliferative	O
+responses	O
+.	O
+	O
+In	O
+the	O
+canonical	O
+model	O
+of	O
+the	O
+ERα	O
+signaling	O
+pathway	O
+(	O
+Fig	O
+1C	O
+),	O
+E2	O
+‐	O
+bound	O
+ERα	O
+forms	O
+a	O
+homodimer	O
+that	O
+binds	O
+DNA	O
+at	O
+estrogen	O
+‐	O
+response	O
+elements	O
+(	O
+EREs	O
+),	O
+recruits	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+(	O
+Metivier	O
+et	O
+al	O
+,	O
+2003	O
+;	O
+Johnson	O
+&	O
+O	O
+'	O
+Malley	O
+,	O
+2012	O
+),	O
+and	O
+activates	O
+the	O
+GREB1	O
+gene	O
+,	O
+which	O
+is	O
+required	O
+for	O
+proliferation	O
+of	O
+ERα	O
+‐	O
+positive	O
+breast	O
+cancer	O
+cells	O
+(	O
+Ghosh	O
+et	O
+al	O
+,	O
+2000	O
+;	O
+Rae	O
+et	O
+al	O
+,	O
+2005	O
+;	O
+Deschenes	O
+et	O
+al	O
+,	O
+2007	O
+;	O
+Liu	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+	O
+Our	O
+long	O
+‐	O
+term	O
+goal	O
+is	O
+to	O
+be	O
+able	O
+to	O
+predict	O
+proliferative	O
+or	O
+anti	O
+‐	O
+proliferative	O
+activity	O
+of	O
+a	O
+ligand	O
+in	O
+different	O
+tissues	O
+from	O
+its	O
+crystal	O
+structure	O
+by	O
+identifying	O
+different	O
+structural	O
+perturbations	O
+that	O
+lead	O
+to	O
+specific	O
+signaling	O
+outcomes	O
+.	O
+	O
+We	O
+also	O
+determined	O
+the	O
+structures	O
+of	O
+76	O
+distinct	O
+ERα	O
+LBD	O
+complexes	O
+bound	O
+to	O
+different	O
+ligand	O
+types	O
+,	O
+which	O
+allowed	O
+us	O
+to	O
+understand	O
+how	O
+diverse	O
+ligand	O
+scaffolds	O
+distort	O
+the	O
+active	O
+conformation	O
+of	O
+the	O
+ERα	O
+LBD	O
+.	O
+	O
+Our	O
+findings	O
+here	O
+indicate	O
+that	O
+specific	O
+structural	O
+perturbations	O
+can	O
+be	O
+tied	O
+to	O
+ligand	O
+‐	O
+selective	O
+domain	O
+usage	O
+and	O
+signaling	O
+patterns	O
+,	O
+thus	O
+providing	O
+a	O
+framework	O
+for	O
+structure	O
+‐	O
+based	O
+design	O
+of	O
+improved	O
+breast	O
+cancer	O
+therapeutics	O
+,	O
+and	O
+understanding	O
+the	O
+different	O
+phenotypic	O
+effects	O
+of	O
+environmental	O
+estrogens	O
+.	O
+	O
+High	O
+‐	O
+throughput	O
+screens	O
+for	O
+ERα	O
+ligand	O
+profiling	O
+	O
+Summary	O
+of	O
+ligand	O
+screening	O
+assays	O
+used	O
+to	O
+measure	O
+ER	O
+‐	O
+mediated	O
+activities	O
+.	O
+	O
+ERE	O
+,	O
+estrogen	O
+‐	O
+response	O
+element	O
+;	O
+Luc	O
+,	O
+luciferase	O
+reporter	O
+gene	O
+;	O
+M2H	O
+,	O
+mammalian	O
+2	O
+‐	O
+hybrid	O
+;	O
+UAS	O
+,	O
+upstream	O
+‐	O
+activating	O
+sequence	O
+.	O
+	O
+Strength	O
+of	O
+AF	O
+‐	O
+1	O
+signaling	O
+does	O
+not	O
+determine	O
+cell	O
+‐	O
+specific	O
+signaling	O
+	O
+To	O
+compare	O
+ERα	O
+signaling	O
+induced	O
+by	O
+diverse	O
+ligand	O
+types	O
+,	O
+we	O
+synthesized	O
+and	O
+assayed	O
+a	O
+library	O
+of	O
+241	O
+ERα	O
+ligands	O
+containing	O
+19	O
+distinct	O
+molecular	O
+scaffolds	O
+.	O
+	O
+These	O
+include	O
+15	O
+indirect	O
+modulator	O
+series	O
+,	O
+which	O
+lack	O
+a	O
+SERM	O
+‐	O
+like	O
+side	O
+chain	O
+and	O
+modulate	O
+coactivator	O
+binding	O
+indirectly	O
+from	O
+the	O
+ligand	O
+‐	O
+binding	O
+pocket	O
+(	O
+Fig	O
+2A	O
+–	O
+E	O
+;	O
+Dataset	O
+EV1	O
+)	O
+(	O
+Zheng	O
+et	O
+al	O
+,	O
+2012	O
+)	O
+(	O
+Zhu	O
+et	O
+al	O
+,	O
+2012	O
+)	O
+(	O
+Muthyala	O
+et	O
+al	O
+,	O
+2003	O
+;	O
+Seo	O
+et	O
+al	O
+,	O
+2006	O
+)	O
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+)	O
+(	O
+Wang	O
+et	O
+al	O
+,	O
+2012	O
+)	O
+(	O
+Liao	O
+et	O
+al	O
+,	O
+2014	O
+)	O
+(	O
+Min	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+	O
+We	O
+also	O
+generated	O
+four	O
+direct	O
+modulator	O
+series	O
+with	O
+side	O
+chains	O
+designed	O
+to	O
+directly	O
+dislocate	O
+h12	O
+and	O
+thereby	O
+completely	O
+occlude	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+(	O
+Fig	O
+2C	O
+and	O
+E	O
+;	O
+Dataset	O
+EV1	O
+)	O
+(	O
+Kieser	O
+et	O
+al	O
+,	O
+2010	O
+).	O
+	O
+Ligand	O
+profiling	O
+using	O
+our	O
+quantitative	O
+bioassays	O
+revealed	O
+a	O
+wide	O
+range	O
+of	O
+ligand	O
+‐	O
+induced	O
+GREB1	O
+expression	O
+,	O
+reporter	O
+gene	O
+activities	O
+,	O
+ERα	O
+‐	O
+coactivator	O
+interactions	O
+,	O
+and	O
+proliferative	O
+effects	O
+on	O
+MCF	O
+‐	O
+7	O
+breast	O
+cancer	O
+cells	O
+(	O
+Figs	O
+EV1	O
+and	O
+EV2A	O
+–	O
+J	O
+).	O
+	O
+This	O
+wide	O
+variance	O
+enabled	O
+us	O
+to	O
+probe	O
+specific	O
+features	O
+of	O
+ERα	O
+signaling	O
+using	O
+ligand	O
+class	O
+analyses	O
+,	O
+and	O
+identify	O
+signaling	O
+patterns	O
+shared	O
+by	O
+specific	O
+ligand	O
+series	O
+or	O
+scaffolds	O
+.	O
+	O
+Classes	O
+of	O
+compounds	O
+in	O
+the	O
+ERα	O
+ligand	O
+library	O
+	O
+Structural	O
+details	O
+of	O
+the	O
+ERα	O
+LBD	O
+bound	O
+to	O
+the	O
+indicated	O
+ligands	O
+.	O
+	O
+Unlike	O
+E2	O
+(	O
+PDB	O
+1GWR	O
+),	O
+TAM	O
+is	O
+a	O
+direct	O
+modulator	O
+with	O
+a	O
+BSC	O
+that	O
+dislocates	O
+h12	O
+to	O
+block	O
+the	O
+NCOA2	O
+‐	O
+binding	O
+site	O
+(	O
+PDB	O
+3ERT	O
+).	O
+	O
+ERα	O
+ligands	O
+induced	O
+a	O
+range	O
+of	O
+agonist	O
+activity	O
+profiles	O
+	O
+To	O
+this	O
+end	O
+,	O
+we	O
+compared	O
+the	O
+average	O
+ligand	O
+‐	O
+induced	O
+GREB1	O
+mRNA	O
+levels	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+and	O
+3	O
+×	O
+ERE	O
+‐	O
+Luc	O
+reporter	O
+gene	O
+activity	O
+in	O
+Ishikawa	O
+endometrial	O
+cancer	O
+cells	O
+(	O
+E	O
+‐	O
+Luc	O
+)	O
+or	O
+in	O
+HepG2	O
+cells	O
+transfected	O
+with	O
+wild	O
+‐	O
+type	O
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+(	O
+L	O
+‐	O
+Luc	O
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+‐	O
+WT	O
+)	O
+(	O
+Figs	O
+3A	O
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+EV2A	O
+–	O
+C	O
+).	O
+	O
+Direct	O
+modulators	O
+showed	O
+significant	O
+differences	O
+in	O
+average	O
+activity	O
+between	O
+cell	O
+types	O
+except	O
+OBHS	O
+‐	O
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+analogs	O
+,	O
+which	O
+had	O
+similar	O
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+agonist	O
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+in	O
+the	O
+three	O
+cell	O
+types	O
+.	O
+	O
+While	O
+it	O
+was	O
+known	O
+that	O
+direct	O
+modulators	O
+such	O
+as	O
+tamoxifen	O
+drive	O
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+‐	O
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+signaling	O
+,	O
+these	O
+experiments	O
+reveal	O
+that	O
+indirect	O
+modulators	O
+also	O
+drive	O
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+,	O
+since	O
+eight	O
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+fourteen	O
+classes	O
+showed	O
+significant	O
+differences	O
+in	O
+average	O
+activity	O
+(	O
+Figs	O
+3A	O
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+EV2A	O
+–	O
+C	O
+).	O
+	O
+(	O
+A	O
+)	O
+Ligand	O
+‐	O
+specific	O
+ERα	O
+activities	O
+in	O
+HepG2	O
+,	O
+Ishikawa	O
+and	O
+MCF	O
+‐	O
+7	O
+cells	O
+.	O
+	O
+The	O
+ligand	O
+‐	O
+induced	O
+L	O
+‐	O
+Luc	O
+ERα	O
+‐	O
+WT	O
+and	O
+E	O
+‐	O
+Luc	O
+activities	O
+and	O
+GREB1	O
+mRNA	O
+levels	O
+are	O
+shown	O
+by	O
+scaffold	O
+(	O
+mean	O
++	O
+SD	O
+).	O
+	O
+Significant	O
+sensitivity	O
+to	O
+AB	O
+domain	O
+deletion	O
+was	O
+determined	O
+by	O
+Student	O
+'	O
+s	O
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+‐	O
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+(	O
+n	O
+=	O
+number	O
+of	O
+ligands	O
+per	O
+scaffold	O
+in	O
+Fig	O
+2	O
+).	O
+	O
+Correlation	O
+and	O
+regression	O
+analyses	O
+in	O
+a	O
+large	O
+test	O
+set	O
+.	O
+	O
+In	O
+cluster	O
+2	O
+,	O
+only	O
+one	O
+of	O
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+comparisons	O
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+a	O
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+positive	O
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+,	O
+while	O
+none	O
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+significant	O
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+cluster	O
+3	O
+.	O
++,	O
+statistically	O
+significant	O
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+AB	O
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+F	O
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+.	O
+	O
+To	O
+test	O
+this	O
+idea	O
+,	O
+we	O
+compared	O
+the	O
+average	O
+L	O
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+each	O
+scaffold	O
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+HepG2	O
+cells	O
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+wild	O
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+the	O
+AB	O
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+(	O
+Figs	O
+1B	O
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+).	O
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+similar	O
+L	O
+‐	O
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+‐	O
+WT	O
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+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activities	O
+,	O
+tamoxifen	O
+showed	O
+complete	O
+loss	O
+of	O
+activity	O
+without	O
+the	O
+AB	O
+domain	O
+(	O
+Fig	O
+EV1B	O
+).	O
+	O
+These	O
+“	O
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+direct	O
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+,	O
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+were	O
+not	O
+limited	O
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+scaffolds	O
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+cell	O
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+signaling	O
+(	O
+Fig	O
+3A	O
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+B	O
+).	O
+	O
+For	O
+each	O
+ligand	O
+class	O
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+scaffold	O
+,	O
+we	O
+calculated	O
+the	O
+Pearson	O
+'	O
+s	O
+correlation	O
+coefficient	O
+,	O
+r	O
+,	O
+for	O
+pairwise	O
+comparison	O
+of	O
+activity	O
+profiles	O
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+(	O
+GREB1	O
+),	O
+liver	O
+(	O
+L	O
+‐	O
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+),	O
+and	O
+endometrial	O
+cells	O
+(	O
+E	O
+‐	O
+Luc	O
+).	O
+	O
+We	O
+also	O
+calculated	O
+the	O
+coefficient	O
+of	O
+determination	O
+,	O
+r	O
+2	O
+,	O
+which	O
+describes	O
+the	O
+percentage	O
+of	O
+variance	O
+in	O
+a	O
+dependent	O
+variable	O
+such	O
+as	O
+proliferation	O
+that	O
+can	O
+be	O
+predicted	O
+by	O
+an	O
+independent	O
+variable	O
+such	O
+as	O
+GREB1	O
+expression	O
+.	O
+	O
+We	O
+present	O
+both	O
+calculations	O
+as	O
+r	O
+2	O
+to	O
+readily	O
+compare	O
+signaling	O
+specificities	O
+using	O
+a	O
+heat	O
+map	O
+on	O
+which	O
+the	O
+red	O
+–	O
+yellow	O
+palette	O
+indicates	O
+significant	O
+positive	O
+correlations	O
+(	O
+P	O
+≤	O
+0	O
+.	O
+05	O
+,	O
+F	O
+‐	O
+test	O
+for	O
+nonzero	O
+slope	O
+),	O
+while	O
+the	O
+blue	O
+palette	O
+denotes	O
+negative	O
+correlations	O
+(	O
+Fig	O
+3C	O
+–	O
+F	O
+).	O
+	O
+Correlation	O
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+OBHS	O
+‐	O
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+activity	O
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+cell	O
+types	O
+.	O
+	O
+Correlation	O
+analysis	O
+of	O
+L	O
+‐	O
+Luc	O
+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activity	O
+versus	O
+endogenous	O
+ERα	O
+activity	O
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+OBHS	O
+analogs	O
+.	O
+	O
+Correlation	O
+analysis	O
+of	O
+L	O
+‐	O
+Luc	O
+ERα	B-mutant
+‐	I-mutant
+ΔF	I-mutant
+activity	O
+versus	O
+endogenous	O
+ERα	O
+activities	O
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+OBHS	O
+analogs	O
+.	O
+	O
+Correlation	O
+analysis	O
+of	O
+MCF	O
+‐	O
+7	O
+cell	O
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+versus	O
+NCOA2	O
+/	O
+3	O
+recruitment	O
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+GREB1	O
+levels	O
+observed	O
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+response	O
+to	O
+(	O
+G	O
+)	O
+OBHS	O
+‐	O
+N	O
+and	O
+(	O
+H	O
+)	O
+OBHS	O
+‐	O
+BSC	O
+analogs	O
+.	O
+	O
+Scaffolds	O
+in	O
+cluster	O
+1	O
+exhibited	O
+strongly	O
+correlated	O
+GREB1	O
+levels	O
+,	O
+E	O
+‐	O
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+L	O
+‐	O
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+the	O
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+types	O
+(	O
+Fig	O
+3C	O
+lanes	O
+1	O
+–	O
+4	O
+),	O
+suggesting	O
+these	O
+ligands	O
+use	O
+similar	O
+ERα	O
+signaling	O
+pathways	O
+in	O
+the	O
+breast	O
+,	O
+endometrial	O
+,	O
+and	O
+liver	O
+cell	O
+types	O
+.	O
+	O
+This	O
+cluster	O
+includes	O
+WAY	O
+‐	O
+C	O
+,	O
+OBHS	O
+,	O
+OBHS	O
+‐	O
+N	O
+,	O
+and	O
+triaryl	O
+‐	O
+ethylene	O
+analogs	O
+,	O
+all	O
+of	O
+which	O
+are	O
+indirect	O
+modulators	O
+.	O
+	O
+For	O
+example	O
+,	O
+3	O
+,	O
+4	O
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+,	O
+furan	O
+,	O
+and	O
+S	O
+‐	O
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+‐	O
+2	O
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+positively	O
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+levels	O
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+E	O
+‐	O
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+not	O
+L	O
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+activity	O
+(	O
+Fig	O
+3C	O
+lanes	O
+5	O
+–	O
+7	O
+).	O
+	O
+In	O
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+,	O
+WAY	O
+dimer	O
+and	O
+WAY	O
+‐	O
+D	O
+analogs	O
+drove	O
+positively	O
+correlated	O
+GREB1	O
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+L	O
+‐	O
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+‐	O
+WT	O
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+E	O
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+(	O
+Fig	O
+3C	O
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+8	O
+and	O
+9	O
+).	O
+	O
+This	O
+cluster	O
+includes	O
+two	O
+direct	O
+modulator	O
+scaffolds	O
+(	O
+OBHS	O
+‐	O
+ASC	O
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+OBHS	O
+‐	O
+BSC	O
+),	O
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+five	O
+indirect	O
+modulator	O
+scaffolds	O
+(	O
+A	O
+‐	O
+CD	O
+,	O
+cyclofenil	O
+,	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+,	O
+imine	O
+,	O
+and	O
+imidazopyridine	O
+).	O
+	O
+These	O
+results	O
+suggest	O
+that	O
+addition	O
+of	O
+an	O
+extended	O
+side	O
+chain	O
+to	O
+an	O
+ERα	O
+ligand	O
+scaffold	O
+is	O
+sufficient	O
+to	O
+induce	O
+cell	O
+‐	O
+specific	O
+signaling	O
+,	O
+where	O
+the	O
+relative	O
+activity	O
+profiles	O
+of	O
+the	O
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+ligands	O
+change	O
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+cell	O
+types	O
+.	O
+	O
+This	O
+is	O
+demonstrated	O
+by	O
+directly	O
+comparing	O
+the	O
+signaling	O
+specificities	O
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+(	O
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+,	O
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+)	O
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+(	O
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+modulator	O
+,	O
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+the	O
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+(	O
+Fig	O
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+,	O
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+OBHS	O
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+sufficient	O
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+abolish	O
+these	O
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+(	O
+Figs	O
+3C	O
+lanes	O
+1	O
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+,	O
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+).	O
+	O
+Modulation	O
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+signaling	O
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+AF	O
+‐	O
+1	O
+	O
+To	O
+evaluate	O
+the	O
+role	O
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+AF	O
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+,	O
+we	O
+compared	O
+activity	O
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+truncated	O
+ERα	O
+constructs	O
+in	O
+HepG2	O
+liver	O
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+endogenous	O
+ERα	O
+activity	O
+in	O
+the	O
+other	O
+cell	O
+types	O
+.	O
+	O
+The	O
+positive	O
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+the	O
+L	O
+‐	O
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+E	O
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+in	O
+cluster	O
+1	O
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+the	O
+AB	O
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+,	O
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+F	O
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+(	O
+Fig	O
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+	O
+This	O
+demonstrates	O
+that	O
+the	O
+signaling	O
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+underlying	O
+these	O
+positive	O
+correlations	O
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+an	O
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+(	O
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++	O
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+E2	O
+.	O
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+Despite	O
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+nearly	O
+complete	O
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+,	O
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+still	O
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+(	O
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+that	O
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+be	O
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+by	O
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+,	O
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+(	O
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+suggest	O
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++	O
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+assays	O
+led	O
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+,	O
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+,	O
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+other	O
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+(	O
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+,	O
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+,	O
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+(	O
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+,	O
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+(	O
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+different	O
+cell	O
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+numbers	O
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+samples	O
+,	O
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+showed	O
+r	O
+2	O
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+0	O
+.	O
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+,	O
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+high	O
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+(	O
+Fig	O
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+	O
+Kinetic	O
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+NCOA3	O
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+‐	O
+7	O
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+stimulated	O
+with	O
+E2	O
+or	O
+the	O
+indicated	O
+WAY	O
+‐	O
+C	O
+analog	O
+.	O
+	O
+NCOA3	O
+occupancy	O
+at	O
+GREB1	O
+was	O
+compared	O
+by	O
+ChIP	O
+assay	O
+45	O
+min	O
+after	O
+stimulation	O
+with	O
+vehicle	O
+,	O
+E2	O
+,	O
+or	O
+the	O
+WAY	O
+‐	O
+C	O
+analogs	O
+.	O
+	O
+In	O
+panel	O
+(	O
+B	O
+),	O
+the	O
+average	O
+recruitment	O
+of	O
+two	O
+biological	O
+replicates	O
+are	O
+shown	O
+as	O
+mean	O
++	O
+SEM	O
+,	O
+and	O
+the	O
+Z	O
+‐	O
+score	O
+is	O
+indicated	O
+.	O
+	O
+In	O
+panel	O
+(	O
+C	O
+),	O
+correlation	O
+analysis	O
+was	O
+performed	O
+for	O
+two	O
+biological	O
+replicates	O
+.	O
+	O
+Linear	O
+regression	O
+analyses	O
+comparing	O
+the	O
+ability	O
+of	O
+NCOA3	O
+recruitment	O
+,	O
+measured	O
+by	O
+ChIP	O
+or	O
+M2H	O
+,	O
+to	O
+predict	O
+other	O
+agonist	O
+activities	O
+of	O
+WAY	O
+‐	O
+C	O
+analogs	O
+.	O
+*	O
+Significant	O
+positive	O
+correlation	O
+(	O
+F	O
+‐	O
+test	O
+for	O
+nonzero	O
+slope	O
+,	O
+P	O
+‐	O
+value	O
+).	O
+	O
+The	O
+M2H	O
+assay	O
+for	O
+NCOA3	O
+recruitment	O
+broadly	O
+correlated	O
+with	O
+the	O
+other	O
+assays	O
+,	O
+and	O
+was	O
+predictive	O
+for	O
+GREB1	O
+expression	O
+and	O
+cell	O
+proliferation	O
+(	O
+Fig	O
+3E	O
+).	O
+	O
+Thus	O
+,	O
+the	O
+simplified	O
+coactivator	O
+‐	O
+binding	O
+assay	O
+showed	O
+much	O
+greater	O
+predictive	O
+power	O
+than	O
+the	O
+ChIP	O
+assay	O
+for	O
+ligand	O
+‐	O
+specific	O
+effects	O
+on	O
+GREB1	O
+expression	O
+and	O
+cell	O
+proliferation	O
+.	O
+	O
+ERβ	O
+activity	O
+is	O
+not	O
+an	O
+independent	O
+predictor	O
+of	O
+cell	O
+‐	O
+specific	O
+activity	O
+	O
+One	O
+difference	O
+between	O
+MCF	O
+‐	O
+7	O
+breast	O
+cancer	O
+cells	O
+and	O
+Ishikawa	O
+endometrial	O
+cancer	O
+cells	O
+is	O
+the	O
+contribution	O
+of	O
+ERβ	O
+to	O
+estrogenic	O
+response	O
+,	O
+as	O
+Ishikawa	O
+cells	O
+may	O
+express	O
+ERβ	O
+(	O
+Bhat	O
+&	O
+Pezzuto	O
+,	O
+2001	O
+).	O
+	O
+When	O
+overexpressed	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+,	O
+ERβ	O
+alters	O
+E2	O
+‐	O
+induced	O
+expression	O
+of	O
+only	O
+a	O
+subset	O
+of	O
+ERα	O
+‐	O
+target	O
+genes	O
+(	O
+Wu	O
+et	O
+al	O
+,	O
+2011	O
+),	O
+raising	O
+the	O
+possibility	O
+that	O
+ligand	O
+‐	O
+induced	O
+ERβ	O
+activity	O
+may	O
+contribute	O
+to	O
+E	O
+‐	O
+Luc	O
+activities	O
+,	O
+and	O
+thus	O
+underlie	O
+the	O
+lack	O
+of	O
+correlation	O
+between	O
+the	O
+E	O
+‐	O
+Luc	O
+and	O
+L	O
+‐	O
+Luc	O
+ERα	O
+‐	O
+WT	O
+activities	O
+or	O
+GREB1	O
+levels	O
+induced	O
+by	O
+cell	O
+‐	O
+specific	O
+modulators	O
+in	O
+cluster	O
+2	O
+and	O
+cluster	O
+3	O
+(	O
+Fig	O
+3C	O
+).	O
+	O
+To	O
+test	O
+this	O
+idea	O
+,	O
+we	O
+determined	O
+the	O
+L	O
+‐	O
+Luc	O
+ERβ	O
+activity	O
+profiles	O
+of	O
+the	O
+ligands	O
+(	O
+Fig	O
+EV1	O
+).	O
+	O
+For	O
+most	O
+scaffolds	O
+,	O
+L	O
+‐	O
+Luc	O
+ERβ	O
+and	O
+E	O
+‐	O
+Luc	O
+activities	O
+were	O
+not	O
+correlated	O
+,	O
+except	O
+for	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+and	O
+cyclofenil	O
+analogs	O
+,	O
+which	O
+showed	O
+moderate	O
+but	O
+significant	O
+correlations	O
+(	O
+Fig	O
+EV4A	O
+).	O
+	O
+Nevertheless	O
+,	O
+the	O
+E	O
+‐	O
+Luc	O
+activities	O
+of	O
+both	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+and	O
+cyclofenil	O
+analogs	O
+were	O
+better	O
+predicted	O
+by	O
+their	O
+L	O
+‐	O
+Luc	O
+ERα	O
+‐	O
+WT	O
+than	O
+L	O
+‐	O
+Luc	O
+ERβ	O
+activities	O
+(	O
+Fig	O
+EV4A	O
+and	O
+B	O
+).	O
+	O
+ERβ	O
+activity	O
+in	O
+HepG2	O
+cells	O
+rarely	O
+correlates	O
+with	O
+E	O
+‐	O
+Luc	O
+activity	O
+.	O
+	O
+Data	O
+information	O
+:	O
+The	O
+r	O
+2	O
+and	O
+P	O
+values	O
+for	O
+the	O
+indicated	O
+correlations	O
+are	O
+shown	O
+in	O
+both	O
+panels	O
+.	O
+*	O
+Significant	O
+positive	O
+correlation	O
+(	O
+F	O
+‐	O
+test	O
+for	O
+nonzero	O
+slope	O
+,	O
+P	O
+‐	O
+value	O
+)	O
+	O
+To	O
+overcome	O
+barriers	O
+to	O
+crystallization	O
+of	O
+ERα	O
+LBD	O
+complexes	O
+,	O
+we	O
+developed	O
+a	O
+conformation	O
+‐	O
+trapping	O
+X	O
+‐	O
+ray	O
+crystallography	O
+approach	O
+using	O
+the	O
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+mutation	O
+(	O
+Nettles	O
+et	O
+al	O
+,	O
+2008	O
+;	O
+Bruning	O
+et	O
+al	O
+,	O
+2010	O
+;	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+	O
+Eleven	O
+of	O
+these	O
+structures	O
+have	O
+been	O
+published	O
+,	O
+while	O
+65	O
+are	O
+new	O
+,	O
+including	O
+the	O
+DES	O
+‐	O
+bound	O
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+LBD	O
+.	O
+	O
+We	O
+present	O
+57	O
+of	O
+these	O
+new	O
+structures	O
+here	O
+(	O
+Dataset	O
+EV2	O
+),	O
+while	O
+the	O
+remaining	O
+eight	O
+new	O
+structures	O
+bound	O
+to	O
+OBHS	O
+‐	O
+N	O
+analogs	O
+will	O
+be	O
+published	O
+elsewhere	O
+(	O
+S	O
+.	O
+Srinivasan	O
+et	O
+al	O
+,	O
+in	O
+preparation	O
+).	O
+	O
+Examining	O
+many	O
+closely	O
+related	O
+structures	O
+allows	O
+us	O
+to	O
+visualize	O
+subtle	O
+structural	O
+differences	O
+,	O
+in	O
+effect	O
+using	O
+X	O
+‐	O
+ray	O
+crystallography	O
+as	O
+a	O
+systems	O
+biology	O
+tool	O
+.	O
+	O
+Based	O
+on	O
+our	O
+original	O
+OBHS	O
+structure	O
+,	O
+the	O
+OBHS	O
+,	O
+OBHS	O
+‐	O
+N	O
+,	O
+and	O
+triaryl	O
+‐	O
+ethylene	O
+compounds	O
+were	O
+modified	O
+with	O
+h11	O
+‐	O
+directed	O
+pendant	O
+groups	O
+(	O
+Zheng	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Zhu	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Liao	O
+et	O
+al	O
+,	O
+2014	O
+).	O
+	O
+Superposing	O
+the	O
+LBDs	O
+based	O
+on	O
+the	O
+class	O
+of	O
+bound	O
+ligands	O
+provides	O
+an	O
+ensemble	O
+view	O
+of	O
+the	O
+structural	O
+variance	O
+and	O
+clarifies	O
+what	O
+part	O
+of	O
+the	O
+ligand	O
+‐	O
+binding	O
+pocket	O
+is	O
+differentially	O
+perturbed	O
+or	O
+targeted	O
+.	O
+	O
+For	O
+the	O
+triaryl	O
+‐	O
+ethylene	O
+analogs	O
+,	O
+the	O
+displacement	O
+of	O
+h11	O
+was	O
+in	O
+a	O
+perpendicular	O
+direction	O
+,	O
+away	O
+from	O
+Ile424	O
+in	O
+h8	O
+and	O
+toward	O
+h12	O
+.	O
+	O
+Structure	O
+‐	O
+class	O
+analysis	O
+of	O
+triaryl	O
+‐	O
+ethylene	O
+analogs	O
+.	O
+	O
+Triaryl	O
+‐	O
+ethylene	O
+analogs	O
+bound	O
+to	O
+the	O
+superposed	O
+crystal	O
+structures	O
+of	O
+the	O
+ERα	O
+LBD	O
+are	O
+shown	O
+.	O
+	O
+Arrows	O
+indicate	O
+chemical	O
+variance	O
+in	O
+the	O
+orientation	O
+of	O
+the	O
+different	O
+h11	O
+‐	O
+directed	O
+ligand	O
+side	O
+groups	O
+(	O
+PDB	O
+5DK9	O
+,	O
+5DKB	O
+,	O
+5DKE	O
+,	O
+5DKG	O
+,	O
+5DKS	O
+,	O
+5DL4	O
+,	O
+5DLR	O
+,	O
+5DMC	O
+,	O
+5DMF	O
+and	O
+5DP0	O
+).	O
+	O
+Triaryl	O
+‐	O
+ethylene	O
+analogs	O
+induce	O
+variance	O
+of	O
+ERα	O
+conformations	O
+at	O
+the	O
+C	O
+‐	O
+terminal	O
+region	O
+of	O
+h11	O
+.	O
+	O
+Panel	O
+(	O
+B	O
+)	O
+shows	O
+the	O
+crystal	O
+structure	O
+of	O
+a	O
+triaryl	O
+‐	O
+ethylene	O
+analog	O
+‐	O
+bound	O
+ERα	O
+LBD	O
+(	O
+PDB	O
+5DLR	O
+).	O
+	O
+This	O
+region	O
+was	O
+expanded	O
+in	O
+panel	O
+(	O
+C	O
+),	O
+where	O
+the	O
+10	O
+triaryl	O
+‐	O
+ethylene	O
+analog	O
+‐	O
+bound	O
+ERα	O
+LBD	O
+structures	O
+(	O
+see	O
+Datasets	O
+EV1	O
+and	O
+EV2	O
+)	O
+were	O
+superposed	O
+to	O
+show	O
+variations	O
+in	O
+the	O
+h11	O
+C	O
+‐	O
+terminus	O
+(	O
+PDB	O
+5DK9	O
+,	O
+5DKB	O
+,	O
+5DKE	O
+,	O
+5DKG	O
+,	O
+5DKS	O
+,	O
+5DL4	O
+,	O
+5DLR	O
+,	O
+5DMC	O
+,	O
+5DMF	O
+,	O
+and	O
+5DP0	O
+).	O
+	O
+ERα	O
+LBDs	O
+in	O
+complex	O
+with	O
+diethylstilbestrol	O
+(	O
+DES	O
+)	O
+or	O
+a	O
+triaryl	O
+‐	O
+ethylene	O
+analog	O
+were	O
+superposed	O
+to	O
+show	O
+that	O
+the	O
+ligand	O
+‐	O
+induced	O
+difference	O
+in	O
+h11	O
+conformation	O
+is	O
+transmitted	O
+to	O
+the	O
+C	O
+‐	O
+terminus	O
+of	O
+h12	O
+(	O
+PDB	O
+4ZN7	O
+,	O
+5DMC	O
+).	O
+	O
+Inter	O
+‐	O
+atomic	O
+distances	O
+predict	O
+the	O
+proliferative	O
+effects	O
+of	O
+specific	O
+ligand	O
+series	O
+.	O
+	O
+Ile424	O
+–	O
+His524	O
+distance	O
+measured	O
+in	O
+the	O
+crystal	O
+structures	O
+correlates	O
+with	O
+the	O
+proliferative	O
+effect	O
+of	O
+triaryl	O
+‐	O
+ethylene	O
+analogs	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+Leu354	O
+–	O
+Leu525	O
+distance	O
+correlates	O
+with	O
+the	O
+proliferative	O
+effects	O
+of	O
+OBHS	O
+‐	O
+N	O
+analogs	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+.	O
+	O
+Structure	O
+‐	O
+class	O
+analysis	O
+of	O
+WAY	O
+‐	O
+C	O
+analogs	O
+.	O
+	O
+WAY	O
+‐	O
+C	O
+side	O
+groups	O
+subtly	O
+nudge	O
+h12	O
+Leu540	O
+.	O
+	O
+ERα	O
+LBD	O
+structures	O
+bound	O
+to	O
+4	O
+distinct	O
+WAY	O
+‐	O
+C	O
+analogs	O
+were	O
+superposed	O
+(	O
+PDB	O
+4	O
+IU7	O
+,	O
+4IV4	O
+,	O
+4IVW	O
+,	O
+4IW6	O
+)	O
+(	O
+see	O
+Datasets	O
+EV1	O
+and	O
+EV2	O
+).	O
+	O
+Structure	O
+‐	O
+class	O
+analysis	O
+of	O
+indirect	O
+modulators	O
+	O
+Structure	O
+‐	O
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+analysis	O
+of	O
+indirect	O
+modulators	O
+in	O
+cluster	O
+1	O
+.	O
+	O
+Crystal	O
+structures	O
+of	O
+the	O
+ERα	O
+LBD	O
+bound	O
+to	O
+OBHS	O
+and	O
+OBHS	O
+‐	O
+N	O
+analogs	O
+were	O
+superposed	O
+.	O
+	O
+Arrows	O
+indicate	O
+chemical	O
+variance	O
+in	O
+the	O
+orientation	O
+of	O
+the	O
+different	O
+h11	O
+‐	O
+directed	O
+ligand	O
+side	O
+groups	O
+.	O
+	O
+Panel	O
+(	O
+B	O
+)	O
+shows	O
+the	O
+ligand	O
+‐	O
+induced	O
+conformational	O
+variation	O
+at	O
+the	O
+C	O
+‐	O
+terminal	O
+region	O
+of	O
+h11	O
+(	O
+OBHS	O
+:	O
+PDB	O
+4ZN9	O
+,	O
+4ZNH	O
+,	O
+4ZNS	O
+,	O
+4ZNT	O
+,	O
+4ZNU	O
+,	O
+4ZNV	O
+,	O
+and	O
+4ZNW	O
+;	O
+OBHS	O
+‐	O
+N	O
+:	O
+PDB	O
+4ZUB	O
+,	O
+4ZUC	O
+,	O
+4ZWH	O
+,	O
+4ZWK	O
+,	O
+5BNU	O
+,	O
+5BP6	O
+,	O
+5BPR	O
+,	O
+and	O
+5BQ4	O
+).	O
+	O
+Structure	O
+‐	O
+class	O
+analysis	O
+of	O
+indirect	O
+modulators	O
+in	O
+clusters	O
+2	O
+and	O
+3	O
+.	O
+	O
+Crystal	O
+structures	O
+of	O
+the	O
+ERα	O
+LBD	O
+bound	O
+to	O
+ligands	O
+with	O
+cell	O
+‐	O
+specific	O
+activities	O
+were	O
+superposed	O
+.	O
+	O
+The	O
+bound	O
+ligands	O
+are	O
+shown	O
+,	O
+and	O
+arrows	O
+indicate	O
+considerable	O
+variation	O
+in	O
+the	O
+orientation	O
+of	O
+the	O
+different	O
+h3	O
+‐,	O
+h8	O
+‐,	O
+h11	O
+‐,	O
+or	O
+h12	O
+‐	O
+directed	O
+ligand	O
+side	O
+groups	O
+.	O
+	O
+As	O
+visualized	O
+in	O
+four	O
+LBD	O
+structures	O
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+),	O
+WAY	O
+‐	O
+C	O
+analogs	O
+were	O
+designed	O
+with	O
+small	O
+substitutions	O
+that	O
+slightly	O
+nudge	O
+h12	O
+Leu540	O
+,	O
+without	O
+exiting	O
+the	O
+ligand	O
+‐	O
+binding	O
+pocket	O
+(	O
+Fig	O
+5G	O
+and	O
+H	O
+).	O
+	O
+Therefore	O
+,	O
+changing	O
+h12	O
+dynamics	O
+maintains	O
+the	O
+canonical	O
+signaling	O
+pathway	O
+defined	O
+by	O
+E2	O
+(	O
+Fig	O
+1D	O
+)	O
+to	O
+support	O
+AF	O
+‐	O
+2	O
+‐	O
+driven	O
+signaling	O
+and	O
+recruit	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+for	O
+GREB1	O
+‐	O
+stimulated	O
+proliferation	O
+.	O
+	O
+Ligands	O
+with	O
+cell	O
+‐	O
+specific	O
+activity	O
+alter	O
+the	O
+shape	O
+of	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+	O
+These	O
+structures	O
+demonstrated	O
+that	O
+cell	O
+‐	O
+specific	O
+activity	O
+derived	O
+from	O
+altering	O
+the	O
+shape	O
+of	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+without	O
+an	O
+extended	O
+side	O
+chain	O
+.	O
+	O
+For	O
+instance	O
+,	O
+S	O
+‐	O
+OBHS	O
+‐	O
+2	O
+and	O
+S	O
+‐	O
+OBHS	O
+‐	O
+3	O
+analogs	O
+(	O
+Fig	O
+2	O
+)	O
+had	O
+similar	O
+ERα	O
+activity	O
+profiles	O
+in	O
+the	O
+different	O
+cell	O
+types	O
+(	O
+Fig	O
+EV2A	O
+–	O
+C	O
+),	O
+but	O
+the	O
+2	O
+‐	O
+versus	O
+3	O
+‐	O
+methyl	O
+substituted	O
+phenol	O
+rings	O
+altered	O
+the	O
+correlated	O
+signaling	O
+patterns	O
+in	O
+different	O
+cell	O
+types	O
+(	O
+Fig	O
+3B	O
+lanes	O
+7	O
+and	O
+12	O
+).	O
+	O
+This	O
+difference	O
+in	O
+ligand	O
+positioning	O
+altered	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+via	O
+a	O
+shift	O
+in	O
+the	O
+N	O
+‐	O
+terminus	O
+of	O
+h12	O
+,	O
+which	O
+directly	O
+contacts	O
+the	O
+coactivator	O
+.	O
+	O
+This	O
+effect	O
+is	O
+evident	O
+in	O
+a	O
+single	O
+structure	O
+due	O
+to	O
+its	O
+1	O
+Å	O
+magnitude	O
+(	O
+Fig	O
+6A	O
+and	O
+B	O
+).	O
+	O
+The	O
+shifts	O
+in	O
+h12	O
+residues	O
+Asp538	O
+and	O
+Leu539	O
+led	O
+to	O
+rotation	O
+of	O
+the	O
+coactivator	O
+peptide	O
+(	O
+Fig	O
+6C	O
+).	O
+	O
+Thus	O
+,	O
+cell	O
+‐	O
+specific	O
+activity	O
+can	O
+stem	O
+from	O
+perturbation	O
+of	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+without	O
+an	O
+extended	O
+side	O
+chain	O
+,	O
+which	O
+presumably	O
+alters	O
+the	O
+receptor	O
+–	O
+coregulator	O
+interaction	O
+profile	O
+.	O
+	O
+S	O
+‐	O
+OBHS	O
+‐	O
+2	O
+/	O
+3	O
+analogs	O
+subtly	O
+distort	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+.	O
+	O
+Panel	O
+(	O
+A	O
+)	O
+shows	O
+the	O
+crystal	O
+structure	O
+of	O
+an	O
+S	O
+‐	O
+OBHS	O
+‐	O
+3	O
+‐	O
+bound	O
+ERα	O
+LBD	O
+(	O
+PDB	O
+5DUH	O
+).	O
+	O
+The	O
+h3	O
+–	O
+h12	O
+interface	O
+(	O
+circled	O
+)	O
+at	O
+AF	O
+‐	O
+2	O
+(	O
+pink	O
+)	O
+was	O
+expanded	O
+in	O
+panels	O
+(	O
+B	O
+,	O
+C	O
+).	O
+	O
+The	O
+S	O
+‐	O
+OBHS	O
+‐	O
+2	O
+/	O
+3	O
+‐	O
+bound	O
+ERα	O
+LBDs	O
+were	O
+superposed	O
+to	O
+show	O
+shifts	O
+in	O
+h3	O
+(	O
+panel	O
+B	O
+)	O
+and	O
+the	O
+NCOA2	O
+peptide	O
+docked	O
+at	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+(	O
+panel	O
+C	O
+).	O
+	O
+Crystal	O
+structures	O
+show	O
+that	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+analogs	O
+shift	O
+h3	O
+and	O
+h11	O
+further	O
+apart	O
+compared	O
+to	O
+an	O
+A	O
+‐	O
+CD	O
+‐	O
+ring	O
+estrogen	O
+(	O
+PDB	O
+4PPS	O
+,	O
+5DRM	O
+,	O
+5DRJ	O
+).	O
+	O
+Average	O
+(	O
+mean	O
++	O
+SEM	O
+)	O
+α	O
+‐	O
+carbon	O
+distance	O
+measured	O
+from	O
+h3	O
+Thr347	O
+to	O
+h11	O
+Leu525	O
+of	O
+A	O
+‐	O
+CD	O
+‐,	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+‐,	O
+and	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+‐	O
+bound	O
+ERα	O
+LBDs	O
+.	O
+	O
+*	O
+Two	O
+‐	O
+tailed	O
+Student	O
+'	O
+s	O
+t	O
+‐	O
+test	O
+,	O
+P	O
+=	O
+0	O
+.	O
+002	O
+(	O
+PDB	O
+A	O
+‐	O
+CD	O
+:	O
+5DI7	O
+,	O
+5DID	O
+,	O
+5DIE	O
+,	O
+5DIG	O
+,	O
+and	O
+4PPS	O
+;	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+:	O
+4IWC	O
+,	O
+5DRM	O
+,	O
+and	O
+5DRJ	O
+;	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+:	O
+5DTV	O
+and	O
+5DU5	O
+).	O
+	O
+Crystal	O
+structures	O
+show	O
+that	O
+a	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+analog	O
+shifts	O
+h3	O
+(	O
+F	O
+)	O
+and	O
+the	O
+NCOA2	O
+(	O
+G	O
+)	O
+peptide	O
+compared	O
+to	O
+an	O
+A	O
+‐	O
+CD	O
+‐	O
+ring	O
+estrogen	O
+(	O
+PDB	O
+4PPS	O
+,	O
+5DTV	O
+).	O
+	O
+Hierarchical	O
+clustering	O
+of	O
+ligand	O
+‐	O
+specific	O
+binding	O
+of	O
+154	O
+interacting	O
+peptides	O
+to	O
+the	O
+ERα	O
+LBD	O
+was	O
+performed	O
+in	O
+triplicate	O
+by	O
+MARCoNI	O
+analysis	O
+.	O
+	O
+These	O
+compounds	O
+bind	O
+the	O
+LBD	O
+in	O
+an	O
+unusual	O
+fashion	O
+because	O
+they	O
+have	O
+a	O
+phenol	O
+‐	O
+to	O
+‐	O
+phenol	O
+length	O
+of	O
+~	O
+12	O
+Å	O
+,	O
+which	O
+is	O
+longer	O
+than	O
+steroids	O
+and	O
+other	O
+prototypical	O
+ERα	O
+agonists	O
+that	O
+are	O
+~	O
+10	O
+Å	O
+in	O
+length	O
+.	O
+	O
+One	O
+phenol	O
+pushed	O
+further	O
+toward	O
+h3	O
+(	O
+Fig	O
+6D	O
+),	O
+while	O
+the	O
+other	O
+phenol	O
+pushed	O
+toward	O
+the	O
+C	O
+‐	O
+terminus	O
+of	O
+h11	O
+to	O
+a	O
+greater	O
+extent	O
+than	O
+A	O
+‐	O
+CD	O
+‐	O
+ring	O
+estrogens	O
+(	O
+Nwachukwu	O
+et	O
+al	O
+,	O
+2014	O
+),	O
+which	O
+are	O
+close	O
+structural	O
+analogs	O
+of	O
+E2	O
+that	O
+lack	O
+a	O
+B	O
+‐	O
+ring	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+To	O
+quantify	O
+this	O
+difference	O
+,	O
+we	O
+compared	O
+the	O
+distance	O
+between	O
+α	O
+‐	O
+carbons	O
+at	O
+h3	O
+Thr347	O
+and	O
+h11	O
+Leu525	O
+in	O
+the	O
+set	O
+of	O
+structures	O
+containing	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+analogs	O
+(	O
+n	O
+=	O
+3	O
+)	O
+or	O
+A	O
+‐	O
+CD	O
+‐	O
+ring	O
+analogs	O
+(	O
+n	O
+=	O
+5	O
+)	O
+(	O
+Fig	O
+6E	O
+).	O
+	O
+We	O
+observed	O
+a	O
+difference	O
+of	O
+0	O
+.	O
+4	O
+Å	O
+that	O
+was	O
+significant	O
+(	O
+two	O
+‐	O
+tailed	O
+Student	O
+'	O
+s	O
+t	O
+‐	O
+test	O
+,	O
+P	O
+=	O
+0	O
+.	O
+002	O
+)	O
+due	O
+to	O
+the	O
+very	O
+tight	O
+clustering	O
+of	O
+the	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+‐	O
+induced	O
+LBD	O
+conformation	O
+.	O
+	O
+The	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+and	O
+3	O
+,	O
+4	O
+‐	O
+DTP	O
+scaffolds	O
+are	O
+isomeric	O
+,	O
+but	O
+with	O
+aryl	O
+groups	O
+at	O
+obtuse	O
+and	O
+acute	O
+angles	O
+,	O
+respectively	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+ERα	O
+in	O
+complex	O
+with	O
+a	O
+3	O
+,	O
+4	O
+‐	O
+DTP	O
+is	O
+unknown	O
+;	O
+however	O
+,	O
+we	O
+solved	O
+two	O
+crystal	O
+structures	O
+of	O
+ERα	O
+bound	O
+to	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+analogs	O
+and	O
+one	O
+structure	O
+containing	O
+a	O
+furan	O
+ligand	O
+—	O
+all	O
+of	O
+which	O
+have	O
+a	O
+3	O
+,	O
+4	O
+‐	O
+diaryl	O
+configuration	O
+(	O
+Fig	O
+2	O
+;	O
+Datasets	O
+EV1	O
+and	O
+EV2	O
+).	O
+	O
+In	O
+these	O
+structures	O
+,	O
+the	O
+A	O
+‐	O
+ring	O
+mimetic	O
+of	O
+the	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+scaffold	O
+bound	O
+h3	O
+Glu353	O
+as	O
+expected	O
+,	O
+but	O
+the	O
+other	O
+phenol	O
+wrapped	O
+around	O
+h3	O
+to	O
+form	O
+a	O
+hydrogen	O
+bond	O
+with	O
+Thr347	O
+,	O
+indicating	O
+a	O
+change	O
+in	O
+binding	O
+epitopes	O
+in	O
+the	O
+ERα	O
+ligand	O
+‐	O
+binding	O
+pocket	O
+(	O
+Fig	O
+6F	O
+).	O
+	O
+The	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+analogs	O
+also	O
+induced	O
+a	O
+shift	O
+in	O
+h3	O
+positioning	O
+,	O
+which	O
+translated	O
+again	O
+into	O
+a	O
+shift	O
+in	O
+the	O
+bound	O
+coactivator	O
+peptide	O
+(	O
+Fig	O
+6F	O
+).	O
+	O
+To	O
+test	O
+whether	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+shows	O
+changes	O
+in	O
+shape	O
+in	O
+solution	O
+,	O
+we	O
+used	O
+the	O
+microarray	O
+assay	O
+for	O
+real	O
+‐	O
+time	O
+coregulator	O
+–	O
+nuclear	O
+receptor	O
+interaction	O
+(	O
+MARCoNI	O
+)	O
+analysis	O
+(	O
+Aarts	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+	O
+Here	O
+,	O
+the	O
+ligand	O
+‐	O
+dependent	O
+interactions	O
+of	O
+the	O
+ERα	O
+LBD	O
+with	O
+over	O
+150	O
+distinct	O
+LxxLL	O
+motif	O
+peptides	O
+were	O
+assayed	O
+to	O
+define	O
+structural	O
+fingerprints	O
+for	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+,	O
+in	O
+a	O
+manner	O
+similar	O
+to	O
+the	O
+use	O
+of	O
+phage	O
+display	O
+peptides	O
+as	O
+structural	O
+probes	O
+(	O
+Connor	O
+et	O
+al	O
+,	O
+2001	O
+).	O
+	O
+Despite	O
+the	O
+similar	O
+average	O
+activities	O
+of	O
+these	O
+ligand	O
+classes	O
+(	O
+Fig	O
+3A	O
+and	O
+B	O
+),	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+and	O
+3	O
+,	O
+4	O
+‐	O
+DTP	O
+analogs	O
+displayed	O
+remarkably	O
+different	O
+peptide	O
+recruitment	O
+patterns	O
+(	O
+Fig	O
+6H	O
+),	O
+consistent	O
+with	O
+the	O
+structural	O
+analyses	O
+.	O
+	O
+However	O
+,	O
+there	O
+was	O
+a	O
+unique	O
+cluster	O
+of	O
+peptides	O
+that	O
+were	O
+recruited	O
+by	O
+E2	O
+but	O
+not	O
+the	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+analogs	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+3	O
+,	O
+4	O
+‐	O
+DTP	O
+analogs	O
+dismissed	O
+most	O
+of	O
+the	O
+peptides	O
+from	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+(	O
+Fig	O
+6H	O
+).	O
+	O
+Thus	O
+,	O
+the	O
+isomeric	O
+attachment	O
+of	O
+diaryl	O
+groups	O
+to	O
+the	O
+thiophene	O
+core	O
+changed	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
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+inside	O
+the	O
+ligand	O
+‐	O
+binding	O
+pocket	O
+,	O
+as	O
+predicted	O
+by	O
+the	O
+crystal	O
+structures	O
+.	O
+	O
+Together	O
+,	O
+these	O
+findings	O
+suggest	O
+that	O
+without	O
+an	O
+extended	O
+side	O
+chain	O
+,	O
+cell	O
+‐	O
+specific	O
+activity	O
+stems	O
+from	O
+different	O
+coregulator	O
+recruitment	O
+profiles	O
+,	O
+due	O
+to	O
+unique	O
+ligand	O
+‐	O
+induced	O
+conformations	O
+of	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+,	O
+in	O
+addition	O
+to	O
+differential	O
+usage	O
+of	O
+AF	O
+‐	O
+1	O
+.	O
+	O
+Indirect	O
+modulators	O
+in	O
+cluster	O
+1	O
+avoid	O
+this	O
+by	O
+perturbing	O
+the	O
+h11	O
+–	O
+h12	O
+interface	O
+,	O
+and	O
+modulating	O
+the	O
+dynamics	O
+of	O
+h12	O
+without	O
+changing	O
+the	O
+shape	O
+of	O
+AF	O
+‐	O
+2	O
+when	O
+stabilized	O
+.	O
+	O
+Our	O
+goal	O
+was	O
+to	O
+identify	O
+a	O
+minimal	O
+set	O
+of	O
+predictors	O
+that	O
+would	O
+link	O
+specific	O
+structural	O
+perturbations	O
+to	O
+ERα	O
+signaling	O
+pathways	O
+that	O
+control	O
+cell	O
+‐	O
+specific	O
+signaling	O
+and	O
+proliferation	O
+.	O
+	O
+We	O
+found	O
+a	O
+very	O
+strong	O
+set	O
+of	O
+predictors	O
+,	O
+where	O
+ligands	O
+in	O
+cluster	O
+1	O
+,	O
+defined	O
+by	O
+similar	O
+signaling	O
+across	O
+cell	O
+types	O
+,	O
+showed	O
+indirect	O
+modulation	O
+of	O
+h12	O
+dynamics	O
+via	O
+the	O
+h11	O
+–	O
+12	O
+interface	O
+or	O
+slight	O
+contact	O
+with	O
+h12	O
+.	O
+	O
+This	O
+perturbation	O
+determined	O
+proliferation	O
+that	O
+correlated	O
+strongly	O
+with	O
+AF	O
+‐	O
+2	O
+activity	O
+,	O
+recruitment	O
+of	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+family	O
+members	O
+,	O
+and	O
+induction	O
+of	O
+the	O
+GREB1	O
+gene	O
+,	O
+consistent	O
+with	O
+the	O
+canonical	O
+ERα	O
+signaling	O
+pathway	O
+(	O
+Fig	O
+1D	O
+).	O
+	O
+For	O
+ligands	O
+in	O
+cluster	O
+1	O
+,	O
+deletion	O
+of	O
+AF	O
+‐	O
+1	O
+reduced	O
+activity	O
+to	O
+varying	O
+degrees	O
+,	O
+but	O
+did	O
+not	O
+change	O
+the	O
+underlying	O
+signaling	O
+patterns	O
+established	O
+through	O
+AF	O
+‐	O
+2	O
+.	O
+	O
+Compared	O
+to	O
+cluster	O
+1	O
+,	O
+the	O
+structural	O
+rules	O
+are	O
+less	O
+clear	O
+in	O
+clusters	O
+2	O
+and	O
+3	O
+,	O
+but	O
+a	O
+number	O
+of	O
+indirect	O
+modulator	O
+classes	O
+perturbed	O
+the	O
+LBD	O
+conformation	O
+at	O
+the	O
+intersection	O
+of	O
+h3	O
+,	O
+the	O
+h12	O
+N	O
+‐	O
+terminus	O
+,	O
+and	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+.	O
+	O
+Ligands	O
+in	O
+these	O
+classes	O
+altered	O
+the	O
+shape	O
+of	O
+AF	O
+‐	O
+2	O
+to	O
+affect	O
+coregulator	O
+preferences	O
+.	O
+	O
+For	O
+direct	O
+and	O
+indirect	O
+modulators	O
+in	O
+cluster	O
+2	O
+or	O
+3	O
+,	O
+the	O
+canonical	O
+ERα	O
+signaling	O
+pathway	O
+involving	O
+recruitment	O
+of	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+and	O
+induction	O
+of	O
+GREB1	O
+did	O
+not	O
+generally	O
+predict	O
+their	O
+proliferative	O
+effects	O
+,	O
+indicating	O
+an	O
+alternate	O
+causal	O
+model	O
+(	O
+Fig	O
+1E	O
+).	O
+	O
+These	O
+principles	O
+outlined	O
+above	O
+provide	O
+a	O
+structural	O
+basis	O
+for	O
+how	O
+the	O
+ligand	O
+–	O
+receptor	O
+interface	O
+leads	O
+to	O
+different	O
+signaling	O
+specificities	O
+through	O
+AF	O
+‐	O
+1	O
+and	O
+AF	O
+‐	O
+2	O
+.	O
+	O
+It	O
+is	O
+noteworthy	O
+that	O
+regulation	O
+of	O
+h12	O
+dynamics	O
+indirectly	O
+through	O
+h11	O
+can	O
+virtually	O
+abolish	O
+AF	O
+‐	O
+2	O
+activity	O
+,	O
+and	O
+yet	O
+still	O
+drive	O
+robust	O
+transcriptional	O
+activity	O
+through	O
+AF	O
+‐	O
+1	O
+,	O
+as	O
+demonstrated	O
+with	O
+the	O
+OBHS	O
+series	O
+.	O
+	O
+This	O
+finding	O
+can	O
+be	O
+explained	O
+by	O
+the	O
+fact	O
+that	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+contain	O
+distinct	O
+binding	O
+sites	O
+for	O
+interaction	O
+with	O
+AF	O
+‐	O
+1	O
+and	O
+AF	O
+‐	O
+2	O
+(	O
+McInerney	O
+et	O
+al	O
+,	O
+1996	O
+;	O
+Webb	O
+et	O
+al	O
+,	O
+1998	O
+),	O
+which	O
+allows	O
+ligands	O
+to	O
+nucleate	O
+ERα	O
+–	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+interaction	O
+through	O
+AF	O
+‐	O
+2	O
+,	O
+and	O
+reinforce	O
+this	O
+interaction	O
+with	O
+additional	O
+binding	O
+to	O
+AF	O
+‐	O
+1	O
+.	O
+	O
+Completely	O
+blocking	O
+AF	O
+‐	O
+2	O
+with	O
+an	O
+extended	O
+side	O
+chain	O
+or	O
+altering	O
+the	O
+shape	O
+of	O
+AF	O
+‐	O
+2	O
+changes	O
+the	O
+preference	O
+away	O
+from	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+for	O
+determining	O
+GREB1	O
+levels	O
+and	O
+proliferation	O
+of	O
+breast	O
+cancer	O
+cells	O
+.	O
+	O
+AF	O
+‐	O
+2	O
+blockade	O
+also	O
+allows	O
+AF	O
+‐	O
+1	O
+to	O
+function	O
+independently	O
+,	O
+which	O
+is	O
+important	O
+since	O
+AF	O
+‐	O
+1	O
+drives	O
+tissue	O
+‐	O
+selective	O
+effects	O
+in	O
+vivo	O
+.	O
+	O
+This	O
+was	O
+demonstrated	O
+with	O
+AF	O
+‐	O
+1	O
+knockout	O
+mice	O
+that	O
+show	O
+E2	O
+‐	O
+dependent	O
+vascular	O
+protection	O
+,	O
+but	O
+not	O
+uterine	O
+proliferation	O
+,	O
+thus	O
+highlighting	O
+the	O
+role	O
+of	O
+AF	O
+‐	O
+1	O
+in	O
+tissue	O
+‐	O
+selective	O
+or	O
+cell	O
+‐	O
+specific	O
+signaling	O
+(	O
+Billon	O
+‐	O
+Gales	O
+et	O
+al	O
+,	O
+2009	O
+;	O
+Abot	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+	O
+Here	O
+,	O
+we	O
+examined	O
+many	O
+LBD	O
+structures	O
+and	O
+tested	O
+several	O
+variables	O
+that	O
+were	O
+not	O
+predictive	O
+,	O
+including	O
+ERβ	O
+activity	O
+,	O
+the	O
+strength	O
+of	O
+AF	O
+‐	O
+1	O
+signaling	O
+,	O
+and	O
+NCOA3	O
+occupancy	O
+at	O
+the	O
+GREB1	O
+gene	O
+.	O
+	O
+Similarly	O
+,	O
+we	O
+visualized	O
+structures	O
+to	O
+identify	O
+patterns	O
+.	O
+	O
+For	O
+example	O
+,	O
+phage	O
+display	O
+was	O
+used	O
+to	O
+identify	O
+the	O
+androgen	O
+receptor	O
+interactome	O
+,	O
+which	O
+was	O
+cloned	O
+into	O
+an	O
+M2H	O
+library	O
+and	O
+used	O
+to	O
+identify	O
+clusters	O
+of	O
+ligand	O
+‐	O
+selective	O
+interactions	O
+(	O
+Norris	O
+et	O
+al	O
+,	O
+2009	O
+).	O
+	O
+We	O
+have	O
+identified	O
+atomic	O
+vectors	O
+for	O
+the	O
+OBHS	O
+‐	O
+N	O
+and	O
+triaryl	O
+‐	O
+ethylene	O
+classes	O
+that	O
+predict	O
+ligand	O
+response	O
+(	O
+Fig	O
+5E	O
+and	O
+F	O
+).	O
+	O
+Indeed	O
+,	O
+the	O
+most	O
+anti	O
+‐	O
+proliferative	O
+compound	O
+in	O
+the	O
+OBHS	O
+‐	O
+N	O
+series	O
+had	O
+a	O
+fulvestrant	O
+‐	O
+like	O
+profile	O
+across	O
+a	O
+battery	O
+of	O
+assays	O
+(	O
+S	O
+.	O
+Srinivasan	O
+et	O
+al	O
+,	O
+in	O
+preparation	O
+).	O
+	O
+Secondly	O
+,	O
+our	O
+finding	O
+that	O
+WAY	O
+‐	O
+C	O
+compounds	O
+do	O
+not	O
+rely	O
+of	O
+AF	O
+‐	O
+1	O
+for	O
+signaling	O
+efficacy	O
+may	O
+derive	O
+from	O
+the	O
+slight	O
+contacts	O
+with	O
+h12	O
+observed	O
+in	O
+crystal	O
+structures	O
+(	O
+Figs	O
+3B	O
+and	O
+5H	O
+),	O
+unlike	O
+other	O
+compounds	O
+in	O
+cluster	O
+1	O
+that	O
+dislocate	O
+h11	O
+and	O
+rely	O
+on	O
+AF	O
+‐	O
+1	O
+for	O
+signaling	O
+efficacy	O
+(	O
+Figs	O
+3B	O
+and	O
+5C	O
+,	O
+and	O
+EV5B	O
+).	O
+	O
+Investigation	O
+of	O
+the	O
+Interaction	O
+between	O
+Cdc42	O
+and	O
+Its	O
+Effector	O
+TOCA1	O
+	O
+Transducer	O
+of	O
+Cdc42	O
+-	O
+dependent	O
+actin	O
+assembly	O
+protein	O
+1	O
+(	O
+TOCA1	O
+)	O
+is	O
+an	O
+effector	O
+of	O
+the	O
+Rho	O
+family	O
+small	O
+G	O
+protein	O
+Cdc42	O
+.	O
+	O
+It	O
+contains	O
+a	O
+membrane	O
+-	O
+deforming	O
+F	O
+-	O
+BAR	O
+domain	O
+as	O
+well	O
+as	O
+a	O
+Src	O
+homology	O
+3	O
+(	O
+SH3	O
+)	O
+domain	O
+and	O
+a	O
+G	O
+protein	O
+-	O
+binding	O
+homology	O
+region	O
+1	O
+(	O
+HR1	O
+)	O
+domain	O
+.	O
+	O
+TOCA1	O
+binding	O
+to	O
+Cdc42	O
+leads	O
+to	O
+actin	O
+rearrangements	O
+,	O
+which	O
+are	O
+thought	O
+to	O
+be	O
+involved	O
+in	O
+processes	O
+such	O
+as	O
+endocytosis	O
+,	O
+filopodia	O
+formation	O
+,	O
+and	O
+cell	O
+migration	O
+.	O
+	O
+We	O
+have	O
+also	O
+investigated	O
+the	O
+binding	O
+of	O
+the	O
+TOCA	O
+HR1	O
+domain	O
+to	O
+Cdc42	O
+and	O
+the	O
+potential	O
+ternary	O
+complex	O
+between	O
+Cdc42	O
+and	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+regions	O
+of	O
+TOCA1	O
+and	O
+a	O
+member	O
+of	O
+the	O
+Wiskott	O
+-	O
+Aldrich	O
+syndrome	O
+protein	O
+family	O
+,	O
+N	O
+-	O
+WASP	O
+.	O
+	O
+TOCA1	O
+binds	O
+Cdc42	O
+with	O
+micromolar	O
+affinity	O
+,	O
+in	O
+contrast	O
+to	O
+the	O
+nanomolar	O
+affinity	O
+of	O
+the	O
+N	O
+-	O
+WASP	O
+G	O
+protein	O
+-	O
+binding	O
+region	O
+for	O
+Cdc42	O
+.	O
+	O
+The	O
+Ras	O
+superfamily	O
+of	O
+small	O
+GTPases	O
+comprises	O
+over	O
+150	O
+members	O
+that	O
+regulate	O
+a	O
+multitude	O
+of	O
+cellular	O
+processes	O
+in	O
+eukaryotes	O
+.	O
+	O
+All	O
+members	O
+share	O
+a	O
+well	O
+defined	O
+core	O
+structure	O
+of	O
+∼	O
+20	O
+kDa	O
+known	O
+as	O
+the	O
+G	O
+domain	O
+,	O
+which	O
+is	O
+responsible	O
+for	O
+guanine	O
+nucleotide	O
+binding	O
+.	O
+	O
+The	O
+guanine	O
+nucleotide	O
+exchange	O
+factors	O
+mediate	O
+formation	O
+of	O
+the	O
+active	O
+state	O
+by	O
+promoting	O
+the	O
+dissociation	O
+of	O
+GDP	O
+,	O
+allowing	O
+GTP	O
+to	O
+bind	O
+.	O
+	O
+The	O
+GTPase	O
+-	O
+activating	O
+proteins	O
+stimulate	O
+the	O
+rate	O
+of	O
+intrinsic	O
+GTP	O
+hydrolysis	O
+,	O
+mediating	O
+the	O
+return	O
+to	O
+the	O
+inactive	O
+state	O
+(	O
+reviewed	O
+in	O
+Ref	O
+.).	O
+	O
+The	O
+overall	O
+conformation	O
+of	O
+small	O
+G	O
+proteins	O
+in	O
+the	O
+active	O
+and	O
+inactive	O
+states	O
+is	O
+similar	O
+,	O
+but	O
+they	O
+differ	O
+significantly	O
+in	O
+two	O
+main	O
+regions	O
+known	O
+as	O
+switch	O
+I	O
+and	O
+switch	O
+II	O
+.	O
+	O
+The	O
+structures	O
+of	O
+more	O
+than	O
+60	O
+small	O
+G	O
+protein	O
+·	O
+effector	O
+complexes	O
+have	O
+been	O
+solved	O
+,	O
+and	O
+,	O
+not	O
+surprisingly	O
+,	O
+the	O
+switch	O
+regions	O
+have	O
+been	O
+implicated	O
+in	O
+a	O
+large	O
+proportion	O
+of	O
+the	O
+G	O
+protein	O
+-	O
+effector	O
+interactions	O
+(	O
+reviewed	O
+in	O
+Ref	O
+.).	O
+	O
+However	O
+,	O
+because	O
+each	O
+of	O
+the	O
+150	O
+members	O
+of	O
+the	O
+superfamily	O
+interacts	O
+with	O
+multiple	O
+effectors	O
+,	O
+there	O
+are	O
+still	O
+a	O
+huge	O
+number	O
+of	O
+known	O
+G	O
+protein	O
+-	O
+effector	O
+interactions	O
+that	O
+have	O
+not	O
+yet	O
+been	O
+studied	O
+structurally	O
+.	O
+	O
+The	O
+Rho	O
+family	O
+comprises	O
+20	O
+members	O
+,	O
+of	O
+which	O
+three	O
+,	O
+RhoA	O
+,	O
+Rac1	O
+,	O
+and	O
+Cdc42	O
+,	O
+have	O
+been	O
+relatively	O
+well	O
+studied	O
+.	O
+	O
+N	O
+-	O
+WASP	O
+exists	O
+in	O
+an	O
+autoinhibited	O
+conformation	O
+,	O
+which	O
+is	O
+released	O
+upon	O
+PI	O
+(	O
+4	O
+,	O
+5	O
+)	O
+P2	O
+and	O
+Cdc42	O
+binding	O
+or	O
+by	O
+other	O
+factors	O
+,	O
+such	O
+as	O
+phosphorylation	O
+.	O
+	O
+The	O
+importance	O
+of	O
+TOCA1	O
+in	O
+actin	O
+polymerization	O
+has	O
+been	O
+demonstrated	O
+in	O
+a	O
+range	O
+of	O
+in	O
+vitro	O
+and	O
+in	O
+vivo	O
+studies	O
+,	O
+but	O
+the	O
+exact	O
+role	O
+of	O
+TOCA1	O
+in	O
+the	O
+many	O
+pathways	O
+involving	O
+actin	O
+assembly	O
+remains	O
+unclear	O
+.	O
+	O
+The	O
+most	O
+widely	O
+studied	O
+role	O
+of	O
+TOCA1	O
+is	O
+in	O
+membrane	O
+invagination	O
+and	O
+endocytosis	O
+,	O
+although	O
+it	O
+has	O
+also	O
+been	O
+implicated	O
+in	O
+filopodia	O
+formation	O
+,	O
+neurite	O
+elongation	O
+,	O
+transcriptional	O
+reprogramming	O
+via	O
+nuclear	O
+actin	O
+,	O
+and	O
+interaction	O
+with	O
+ZO	O
+-	O
+1	O
+at	O
+tight	O
+junctions	O
+.	O
+	O
+TOCA1	O
+comprises	O
+an	O
+N	O
+-	O
+terminal	O
+F	O
+-	O
+BAR	O
+domain	O
+,	O
+a	O
+central	O
+homology	O
+region	O
+1	O
+(	O
+HR1	O
+)	O
+domain	O
+,	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+SH3	O
+domain	O
+.	O
+	O
+The	O
+F	O
+-	O
+BAR	O
+domain	O
+is	O
+a	O
+known	O
+dimerization	O
+,	O
+membrane	O
+-	O
+binding	O
+,	O
+and	O
+membrane	O
+-	O
+deforming	O
+module	O
+found	O
+in	O
+a	O
+number	O
+of	O
+cell	O
+signaling	O
+proteins	O
+.	O
+	O
+Other	O
+HR1	O
+domains	O
+studied	O
+so	O
+far	O
+,	O
+including	O
+those	O
+from	O
+the	O
+PRK	O
+family	O
+,	O
+have	O
+been	O
+found	O
+to	O
+bind	O
+their	O
+cognate	O
+Rho	O
+family	O
+G	O
+protein	O
+-	O
+binding	O
+partner	O
+with	O
+high	O
+specificity	O
+and	O
+affinities	O
+in	O
+the	O
+nanomolar	O
+range	O
+.	O
+	O
+The	O
+structures	O
+of	O
+the	O
+PRK1	O
+HR1a	O
+domain	O
+in	O
+complex	O
+with	O
+RhoA	O
+and	O
+the	O
+HR1b	O
+domain	O
+in	O
+complex	O
+with	O
+Rac1	O
+show	O
+that	O
+the	O
+HR1	O
+domain	O
+comprises	O
+an	O
+anti	O
+-	O
+parallel	O
+coiled	O
+-	O
+coil	O
+that	O
+interacts	O
+with	O
+its	O
+G	O
+protein	O
+binding	O
+partner	O
+via	O
+both	O
+helices	O
+.	O
+	O
+The	O
+coiled	O
+-	O
+coil	O
+fold	O
+is	O
+shared	O
+by	O
+the	O
+HR1	O
+domain	O
+of	O
+the	O
+TOCA	O
+family	O
+protein	O
+,	O
+CIP4	O
+,	O
+and	O
+,	O
+based	O
+on	O
+sequence	O
+homology	O
+,	O
+by	O
+TOCA1	O
+itself	O
+.	O
+	O
+These	O
+HR1	O
+domains	O
+,	O
+however	O
+,	O
+show	O
+specificity	O
+for	O
+Cdc42	O
+,	O
+rather	O
+than	O
+RhoA	O
+or	O
+Rac1	O
+.	O
+	O
+How	O
+different	O
+HR1	O
+domain	O
+proteins	O
+distinguish	O
+their	O
+specific	O
+G	O
+protein	O
+partners	O
+remains	O
+only	O
+partially	O
+understood	O
+,	O
+and	O
+structural	O
+characterization	O
+of	O
+a	O
+novel	O
+G	O
+protein	O
+-	O
+HR1	O
+domain	O
+interaction	O
+would	O
+add	O
+to	O
+the	O
+growing	O
+body	O
+of	O
+information	O
+pertaining	O
+to	O
+these	O
+protein	O
+complexes	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+biological	O
+function	O
+of	O
+the	O
+interaction	O
+between	O
+TOCA1	O
+and	O
+Cdc42	O
+remains	O
+poorly	O
+understood	O
+,	O
+and	O
+so	O
+far	O
+there	O
+has	O
+been	O
+no	O
+biophysical	O
+or	O
+structural	O
+insight	O
+.	O
+	O
+There	O
+is	O
+some	O
+evidence	O
+for	O
+a	O
+ternary	O
+complex	O
+between	O
+Cdc42	O
+,	O
+N	O
+-	O
+WASP	O
+,	O
+and	O
+TOCA1	O
+,	O
+but	O
+there	O
+was	O
+no	O
+direct	O
+demonstration	O
+of	O
+simultaneous	O
+contacts	O
+between	O
+the	O
+two	O
+effectors	O
+and	O
+a	O
+single	O
+molecule	O
+of	O
+Cdc42	O
+.	O
+	O
+Nonetheless	O
+,	O
+the	O
+substantial	O
+difference	O
+between	O
+the	O
+structures	O
+of	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+regions	O
+of	O
+the	O
+two	O
+effectors	O
+is	O
+intriguing	O
+and	O
+implies	O
+that	O
+they	O
+bind	O
+to	O
+Cdc42	O
+quite	O
+differently	O
+,	O
+providing	O
+motivation	O
+for	O
+investigating	O
+the	O
+possibility	O
+that	O
+Cdc42	O
+can	O
+bind	O
+both	O
+effectors	O
+concurrently	O
+.	O
+	O
+WASP	O
+interacts	O
+with	O
+Cdc42	O
+via	O
+a	O
+conserved	O
+,	O
+unstructured	O
+binding	O
+motif	O
+known	O
+as	O
+the	O
+Cdc42	O
+-	O
+and	O
+Rac	O
+-	O
+interactive	O
+binding	O
+region	O
+(	O
+CRIB	O
+),	O
+which	O
+forms	O
+an	O
+intermolecular	O
+β	O
+-	O
+sheet	O
+,	O
+expanding	O
+the	O
+anti	O
+-	O
+parallel	O
+β2	O
+and	O
+β3	O
+strands	O
+of	O
+Cdc42	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+TOCA	O
+family	O
+proteins	O
+are	O
+thought	O
+to	O
+interact	O
+via	O
+the	O
+HR1	O
+domain	O
+,	O
+which	O
+may	O
+form	O
+a	O
+triple	O
+coiled	O
+-	O
+coil	O
+with	O
+switch	O
+II	O
+of	O
+Rac1	O
+,	O
+like	O
+the	O
+HR1b	O
+domain	O
+of	O
+PRK1	O
+.	O
+	O
+Here	O
+,	O
+we	O
+present	O
+the	O
+solution	O
+NMR	O
+structure	O
+of	O
+the	O
+HR1	O
+domain	O
+of	O
+TOCA1	O
+,	O
+providing	O
+the	O
+first	O
+structural	O
+data	O
+for	O
+this	O
+protein	O
+.	O
+	O
+We	O
+also	O
+present	O
+data	O
+pertaining	O
+to	O
+binding	O
+of	O
+the	O
+TOCA	O
+HR1	O
+domain	O
+to	O
+Cdc42	O
+,	O
+which	O
+is	O
+the	O
+first	O
+biophysical	O
+description	O
+of	O
+an	O
+HR1	O
+domain	O
+binding	O
+this	O
+particular	O
+Rho	O
+family	O
+small	O
+G	O
+protein	O
+.	O
+	O
+Finally	O
+,	O
+we	O
+investigate	O
+the	O
+potential	O
+ternary	O
+complex	O
+between	O
+Cdc42	O
+and	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+regions	O
+of	O
+TOCA1	O
+and	O
+N	O
+-	O
+WASP	O
+,	O
+contributing	O
+to	O
+our	O
+understanding	O
+of	O
+G	O
+protein	O
+-	O
+effector	O
+interactions	O
+as	O
+well	O
+as	O
+the	O
+roles	O
+of	O
+Cdc42	O
+,	O
+N	O
+-	O
+WASP	O
+,	O
+and	O
+TOCA1	O
+in	O
+the	O
+pathways	O
+that	O
+govern	O
+actin	O
+dynamics	O
+.	O
+	O
+TOCA1	O
+was	O
+identified	O
+in	O
+Xenopus	O
+extracts	O
+as	O
+a	O
+protein	O
+necessary	O
+for	O
+Cdc42	O
+-	O
+dependent	O
+actin	O
+assembly	O
+and	O
+was	O
+shown	O
+to	O
+bind	O
+to	O
+Cdc42	O
+·	O
+GTPγS	O
+but	O
+not	O
+to	O
+Cdc42	O
+·	O
+GDP	O
+or	O
+to	O
+Rac1	O
+and	O
+RhoA	O
+.	O
+Given	O
+its	O
+homology	O
+to	O
+other	O
+Rho	O
+family	O
+binding	O
+modules	O
+,	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+HR1	O
+domain	O
+of	O
+TOCA1	O
+is	O
+sufficient	O
+to	O
+bind	O
+Cdc42	O
+.	O
+	O
+The	O
+C	O
+.	O
+elegans	O
+TOCA1	O
+orthologues	O
+also	O
+bind	O
+to	O
+Cdc42	O
+via	O
+their	O
+consensus	O
+HR1	O
+domain	O
+.	O
+	O
+The	O
+HR1	O
+domains	O
+from	O
+the	O
+PRK	O
+family	O
+bind	O
+their	O
+G	O
+protein	O
+partners	O
+with	O
+a	O
+high	O
+affinity	O
+,	O
+exhibiting	O
+a	O
+range	O
+of	O
+submicromolar	O
+dissociation	O
+constants	O
+(	O
+Kd	O
+)	O
+as	O
+low	O
+as	O
+26	O
+nm	O
+.	O
+	O
+A	O
+Kd	O
+in	O
+the	O
+nanomolar	O
+range	O
+was	O
+therefore	O
+expected	O
+for	O
+the	O
+interaction	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+with	O
+Cdc42	O
+.	O
+	O
+We	O
+generated	O
+an	O
+X	O
+.	O
+tropicalis	O
+TOCA1	O
+HR1	O
+domain	O
+construct	O
+encompassing	O
+residues	O
+330	O
+–	O
+426	O
+.	O
+	O
+This	O
+region	O
+comprises	O
+the	O
+complete	O
+HR1	O
+domain	O
+based	O
+on	O
+secondary	O
+structure	O
+predictions	O
+and	O
+sequence	O
+alignments	O
+with	O
+another	O
+TOCA	O
+family	O
+member	O
+,	O
+CIP4	O
+,	O
+whose	O
+structure	O
+has	O
+been	O
+determined	O
+.	O
+	O
+The	O
+interaction	O
+between	O
+[	O
+3H	O
+]	O
+GTP	O
+·	O
+Cdc42	O
+and	O
+a	O
+C	O
+-	O
+terminally	O
+His	O
+-	O
+tagged	O
+TOCA1	O
+HR1	O
+domain	O
+construct	O
+was	O
+investigated	O
+using	O
+SPA	O
+.	O
+	O
+The	O
+binding	O
+isotherm	O
+for	O
+the	O
+interaction	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+1A	O
+,	O
+together	O
+with	O
+the	O
+Cdc42	O
+-	O
+PAK	O
+interaction	O
+as	O
+a	O
+positive	O
+control	O
+.	O
+	O
+The	O
+binding	O
+of	O
+TOCA1	O
+HR1	O
+to	O
+Cdc42	O
+was	O
+unexpectedly	O
+weak	O
+,	O
+with	O
+a	O
+Kd	O
+of	O
+>	O
+1	O
+μm	O
+.	O
+	O
+It	O
+was	O
+not	O
+possible	O
+to	O
+estimate	O
+the	O
+Kd	O
+more	O
+accurately	O
+using	O
+direct	O
+SPA	O
+experiments	O
+,	O
+because	O
+saturation	O
+could	O
+not	O
+be	O
+reached	O
+due	O
+to	O
+nonspecific	O
+signal	O
+at	O
+higher	O
+protein	O
+concentrations	O
+.	O
+	O
+The	O
+TOCA1	O
+HR1	O
+-	O
+Cdc42	O
+interaction	O
+is	O
+low	O
+affinity	O
+.	O
+	O
+The	O
+SPA	O
+signal	O
+was	O
+corrected	O
+by	O
+subtraction	O
+of	O
+control	O
+data	O
+with	O
+no	O
+GST	B-mutant
+-	I-mutant
+PAK	I-mutant
+or	O
+HR1	B-mutant
+-	I-mutant
+His6	I-mutant
+.	O
+	O
+The	O
+Kd	O
+values	O
+derived	O
+for	O
+the	O
+TOCA1	O
+HR1	O
+with	O
+Cdc42Δ7	B-mutant
+and	O
+full	O
+-	O
+length	O
+Cdc42	O
+were	O
+6	O
+.	O
+05	O
+±	O
+1	O
+.	O
+96	O
+and	O
+5	O
+.	O
+39	O
+±	O
+1	O
+.	O
+69	O
+μm	O
+,	O
+respectively	O
+.	O
+	O
+It	O
+was	O
+possible	O
+that	O
+the	O
+low	O
+affinity	O
+observed	O
+was	O
+due	O
+to	O
+negative	O
+effects	O
+of	O
+immobilization	O
+of	O
+the	O
+HR1	O
+domain	O
+,	O
+so	O
+other	O
+methods	O
+were	O
+employed	O
+,	O
+which	O
+utilized	O
+untagged	O
+proteins	O
+.	O
+	O
+Isothermal	O
+titration	O
+calorimetry	O
+was	O
+carried	O
+out	O
+,	O
+but	O
+no	O
+heat	O
+changes	O
+were	O
+observed	O
+at	O
+a	O
+range	O
+of	O
+concentrations	O
+and	O
+temperatures	O
+(	O
+data	O
+not	O
+shown	O
+),	O
+suggesting	O
+that	O
+the	O
+interaction	O
+is	O
+predominantly	O
+entropically	O
+driven	O
+.	O
+	O
+A	O
+complex	O
+of	O
+a	O
+GST	O
+fusion	O
+of	O
+the	O
+GBD	O
+of	O
+ACK	O
+,	O
+which	O
+binds	O
+with	O
+a	O
+high	O
+affinity	O
+to	O
+Cdc42	O
+,	O
+with	O
+radiolabeled	O
+[	O
+3H	O
+]	O
+GTP	O
+·	O
+Cdc42	O
+was	O
+preformed	O
+,	O
+and	O
+the	O
+effect	O
+of	O
+increasing	O
+concentrations	O
+of	O
+untagged	O
+TOCA1	O
+HR1	O
+domain	O
+was	O
+examined	O
+.	O
+	O
+Competition	O
+of	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+GBD	O
+bound	O
+to	O
+[	O
+3H	O
+]	O
+GTP	O
+·	O
+Cdc42	O
+by	O
+free	O
+ACK	O
+GBD	O
+was	O
+used	O
+as	O
+a	O
+control	O
+and	O
+to	O
+establish	O
+the	O
+value	O
+of	O
+background	O
+counts	O
+when	O
+Cdc42	O
+is	O
+fully	O
+displaced	O
+.	O
+	O
+The	O
+data	O
+were	O
+fitted	O
+to	O
+a	O
+binding	O
+isotherm	O
+describing	O
+competition	O
+.	O
+	O
+The	O
+Cdc42	O
+construct	O
+used	O
+in	O
+the	O
+binding	O
+assays	O
+has	O
+seven	O
+residues	O
+deleted	O
+from	O
+the	O
+C	O
+terminus	O
+to	O
+facilitate	O
+purification	O
+.	O
+	O
+As	O
+the	O
+observed	O
+affinity	O
+between	O
+TOCA1	O
+HR1	O
+and	O
+Cdc42	O
+was	O
+much	O
+lower	O
+than	O
+expected	O
+,	O
+we	O
+reasoned	O
+that	O
+the	O
+C	O
+terminus	O
+of	O
+Cdc42	O
+might	O
+be	O
+necessary	O
+for	O
+a	O
+high	O
+affinity	O
+interaction	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+Cdc42	O
+is	O
+not	O
+required	O
+for	O
+maximal	O
+binding	O
+of	O
+TOCA1	O
+HR1	O
+.	O
+	O
+Indeed	O
+,	O
+GST	O
+pull	O
+-	O
+downs	O
+performed	O
+with	O
+in	O
+vitro	O
+translated	O
+human	O
+TOCA1	O
+fragments	O
+had	O
+suggested	O
+that	O
+residues	O
+N	O
+-	O
+terminal	O
+to	O
+the	O
+HR1	O
+domain	O
+may	O
+be	O
+required	O
+to	O
+stabilize	O
+the	O
+HR1	O
+domain	O
+structure	O
+.	O
+	O
+TOCA1	O
+dimerizes	O
+via	O
+its	O
+F	O
+-	O
+BAR	O
+domain	O
+,	O
+which	O
+could	O
+also	O
+affect	O
+Cdc42	O
+binding	O
+,	O
+for	O
+example	O
+by	O
+presenting	O
+two	O
+HR1	O
+domains	O
+for	O
+Cdc42	O
+interactions	O
+.	O
+	O
+Various	O
+TOCA1	O
+fragments	O
+(	O
+Fig	O
+.	O
+2A	O
+)	O
+were	O
+therefore	O
+assessed	O
+for	O
+binding	O
+to	O
+full	O
+-	O
+length	O
+Cdc42	O
+by	O
+direct	O
+SPA	O
+.	O
+	O
+The	O
+isolated	O
+F	O
+-	O
+BAR	O
+domain	O
+showed	O
+no	O
+binding	O
+to	O
+full	O
+-	O
+length	O
+Cdc42	O
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+	O
+The	O
+HR1	B-mutant
+-	I-mutant
+SH3	I-mutant
+protein	O
+could	O
+not	O
+be	O
+purified	O
+to	O
+homogeneity	O
+as	O
+a	O
+fusion	O
+protein	O
+,	O
+so	O
+it	O
+was	O
+assayed	O
+in	O
+competition	O
+assays	O
+after	O
+cleavage	O
+of	O
+the	O
+His	O
+tag	O
+.	O
+	O
+Taken	O
+together	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+is	O
+sufficient	O
+for	O
+maximal	O
+binding	O
+and	O
+that	O
+this	O
+binding	O
+is	O
+of	O
+a	O
+relatively	O
+low	O
+affinity	O
+compared	O
+with	O
+many	O
+other	O
+Cdc42	O
+·	O
+effector	O
+complexes	O
+.	O
+	O
+The	O
+Cdc42	O
+-	O
+HR1	O
+interaction	O
+is	O
+of	O
+low	O
+affinity	O
+in	O
+the	O
+context	O
+of	O
+full	O
+-	O
+length	O
+protein	O
+and	O
+in	O
+TOCA1	O
+paralogues	O
+.	O
+	O
+A	O
+,	O
+diagram	O
+illustrating	O
+the	O
+TOCA1	O
+constructs	O
+assayed	O
+for	O
+Cdc42	O
+binding	O
+.	O
+	O
+The	O
+SPA	O
+signal	O
+was	O
+corrected	O
+by	O
+subtraction	O
+of	O
+control	O
+data	O
+with	O
+no	O
+fusion	O
+protein	O
+.	O
+	O
+C	O
+–	O
+E	O
+,	O
+representative	O
+examples	O
+of	O
+competition	O
+SPA	O
+experiments	O
+carried	O
+out	O
+with	O
+the	O
+indicated	O
+concentrations	O
+of	O
+the	O
+TOCA1	O
+HR1	B-mutant
+-	I-mutant
+SH3	I-mutant
+construct	O
+titrated	O
+into	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+and	O
+30	O
+nm	O
+Cdc42Δ7Q61L	O
+·[	O
+3H	O
+]	O
+GTP	O
+(	O
+C	O
+)	O
+or	O
+HR1CIP4	O
+(	O
+D	O
+)	O
+or	O
+HR1FBP17	O
+(	O
+E	O
+)	O
+titrated	O
+into	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+and	O
+30	O
+nm	O
+Cdc42FLQ61L	O
+·[	O
+3H	O
+]	O
+GTP	O
+.	O
+	O
+The	O
+low	O
+affinity	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+-	O
+Cdc42	O
+interaction	O
+raised	O
+the	O
+question	O
+of	O
+whether	O
+the	O
+other	O
+known	O
+Cdc42	O
+-	O
+binding	O
+TOCA	O
+family	O
+proteins	O
+,	O
+FBP17	O
+and	O
+CIP4	O
+,	O
+also	O
+bind	O
+weakly	O
+.	O
+	O
+The	O
+HR1	O
+domains	O
+from	O
+FBP17	O
+and	O
+CIP4	O
+were	O
+purified	O
+and	O
+assayed	O
+for	O
+Cdc42	O
+binding	O
+in	O
+competition	O
+SPAs	O
+,	O
+analogous	O
+to	O
+those	O
+carried	O
+out	O
+with	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+.	O
+	O
+The	O
+affinities	O
+of	O
+both	O
+the	O
+FBP17	O
+and	O
+CIP4	O
+HR1	O
+domains	O
+were	O
+also	O
+in	O
+the	O
+low	O
+micromolar	O
+range	O
+(	O
+10	O
+and	O
+5	O
+μm	O
+,	O
+respectively	O
+)	O
+(	O
+Fig	O
+.	O
+2	O
+,	O
+D	O
+and	O
+E	O
+),	O
+suggesting	O
+that	O
+low	O
+affinity	O
+interactions	O
+with	O
+Cdc42	O
+are	O
+a	O
+common	O
+feature	O
+within	O
+the	O
+TOCA	O
+family	O
+.	O
+	O
+Structure	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+Domain	O
+	O
+Because	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+was	O
+sufficient	O
+for	O
+maximal	O
+Cdc42	O
+-	O
+binding	O
+,	O
+we	O
+used	O
+this	O
+construct	O
+for	O
+structural	O
+studies	O
+.	O
+	O
+Initial	O
+experiments	O
+were	O
+performed	O
+with	O
+TOCA1	O
+residues	O
+324	O
+–	O
+426	O
+,	O
+but	O
+we	O
+observed	O
+that	O
+the	O
+N	O
+terminus	O
+was	O
+cleaved	O
+during	O
+purification	O
+to	O
+yield	O
+a	O
+new	O
+N	O
+terminus	O
+at	O
+residue	O
+330	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+	O
+We	O
+therefore	O
+engineered	O
+a	O
+construct	O
+comprising	O
+residues	O
+330	O
+–	O
+426	O
+to	O
+produce	O
+the	O
+minimal	O
+,	O
+stable	O
+HR1	O
+domain	O
+.	O
+	O
+2	O
+,	O
+778	O
+non	O
+-	O
+degenerate	O
+NOE	O
+restraints	O
+were	O
+used	O
+in	O
+initial	O
+structure	O
+calculations	O
+(	O
+1	O
+,	O
+791	O
+unambiguous	O
+and	O
+987	O
+ambiguous	O
+),	O
+derived	O
+from	O
+three	O
+-	O
+dimensional	O
+15N	O
+-	O
+separated	O
+NOESY	O
+and	O
+13C	O
+-	O
+separated	O
+NOESY	O
+experiments	O
+.	O
+	O
+100	O
+structures	O
+were	O
+calculated	O
+in	O
+the	O
+final	O
+iteration	O
+;	O
+the	O
+50	O
+lowest	O
+energy	O
+structures	O
+were	O
+water	O
+-	O
+refined	O
+;	O
+and	O
+of	O
+these	O
+,	O
+the	O
+35	O
+lowest	O
+energy	O
+structures	O
+were	O
+analyzed	O
+.	O
+	O
+Table	O
+1	O
+indicates	O
+that	O
+the	O
+HR1	O
+domain	O
+structure	O
+is	O
+well	O
+defined	O
+by	O
+the	O
+NMR	O
+data	O
+.	O
+	O
+a	O
+<	O
+SA	O
+>,	O
+the	O
+average	O
+root	O
+mean	O
+square	O
+deviations	O
+for	O
+the	O
+ensemble	O
+±	O
+S	O
+.	O
+D	O
+.	O
+	O
+b	O
+<	O
+SA	O
+>	O
+c	O
+,	O
+values	O
+for	O
+the	O
+structure	O
+that	O
+is	O
+closest	O
+to	O
+the	O
+mean	O
+.	O
+	O
+The	O
+structure	O
+closest	O
+to	O
+the	O
+mean	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+3A	O
+.	O
+	O
+The	O
+two	O
+α	O
+-	O
+helices	O
+of	O
+the	O
+HR1	O
+domain	O
+interact	O
+to	O
+form	O
+an	O
+anti	O
+-	O
+parallel	O
+coiled	O
+-	O
+coil	O
+with	O
+a	O
+slight	O
+left	O
+-	O
+handed	O
+twist	O
+,	O
+reminiscent	O
+of	O
+the	O
+HR1	O
+domains	O
+of	O
+CIP4	O
+(	O
+PDB	O
+code	O
+2KE4	O
+)	O
+and	O
+PRK1	O
+(	O
+PDB	O
+codes	O
+1CXZ	O
+and	O
+1URF	O
+).	O
+	O
+A	O
+,	O
+the	O
+backbone	O
+trace	O
+of	O
+the	O
+35	O
+lowest	O
+energy	O
+structures	O
+of	O
+the	O
+HR1	O
+domain	O
+overlaid	O
+with	O
+the	O
+structure	O
+closest	O
+to	O
+the	O
+mean	O
+is	O
+shown	O
+alongside	O
+a	O
+schematic	O
+representation	O
+of	O
+the	O
+structure	O
+closest	O
+to	O
+the	O
+mean	O
+.	O
+	O
+Flexible	O
+regions	O
+at	O
+the	O
+N	O
+and	O
+C	O
+termini	O
+(	O
+residues	O
+330	O
+–	O
+333	O
+and	O
+421	O
+–	O
+426	O
+)	O
+are	O
+omitted	O
+for	O
+clarity	O
+.	O
+	O
+The	O
+secondary	O
+structure	O
+was	O
+deduced	O
+using	O
+Stride	O
+,	O
+based	O
+on	O
+the	O
+Ramachandran	O
+angles	O
+,	O
+and	O
+is	O
+indicated	O
+as	O
+follows	O
+:	O
+gray	O
+,	O
+turn	O
+;	O
+yellow	O
+,	O
+α	O
+-	O
+helix	O
+;	O
+blue	O
+,	O
+310	O
+helix	O
+;	O
+white	O
+,	O
+coil	O
+.	O
+	O
+C	O
+,	O
+a	O
+close	O
+-	O
+up	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+of	O
+TOCA1	O
+HR1	O
+,	O
+indicating	O
+some	O
+of	O
+the	O
+NOEs	O
+defining	O
+its	O
+position	O
+with	O
+respect	O
+to	O
+the	O
+two	O
+α	O
+-	O
+helices	O
+.	O
+	O
+Dotted	O
+lines	O
+,	O
+NOE	O
+restraints	O
+.	O
+	O
+D	O
+,	O
+a	O
+close	O
+-	O
+up	O
+of	O
+the	O
+interhelix	O
+loop	O
+region	O
+showing	O
+some	O
+of	O
+the	O
+contacts	O
+between	O
+the	O
+loop	O
+and	O
+helix	O
+1	O
+.	O
+	O
+In	O
+the	O
+HR1a	O
+domain	O
+of	O
+PRK1	O
+,	O
+a	O
+region	O
+N	O
+-	O
+terminal	O
+to	O
+helix	O
+1	O
+forms	O
+a	O
+short	O
+α	O
+-	O
+helix	O
+,	O
+which	O
+packs	O
+against	O
+both	O
+helices	O
+of	O
+the	O
+HR1	O
+domain	O
+.	O
+	O
+This	O
+region	O
+of	O
+TOCA1	O
+HR1	O
+(	O
+residues	O
+334	O
+–	O
+340	O
+)	O
+is	O
+well	O
+defined	O
+in	O
+the	O
+family	O
+of	O
+structures	O
+(	O
+Fig	O
+.	O
+3A	O
+)	O
+but	O
+does	O
+not	O
+form	O
+an	O
+α	O
+-	O
+helix	O
+.	O
+	O
+It	O
+instead	O
+forms	O
+a	O
+series	O
+of	O
+turns	O
+,	O
+defined	O
+by	O
+NOE	O
+restraints	O
+observed	O
+between	O
+residues	O
+separated	O
+by	O
+one	O
+(	O
+residues	O
+332	O
+–	O
+334	O
+,	O
+333	O
+–	O
+335	O
+,	O
+etc	O
+.)	O
+or	O
+two	O
+(	O
+residues	O
+337	O
+–	O
+340	O
+)	O
+residues	O
+in	O
+the	O
+sequence	O
+and	O
+the	O
+φ	O
+and	O
+ψ	O
+angles	O
+,	O
+assessed	O
+using	O
+Stride	O
+.	O
+	O
+These	O
+turns	O
+cause	O
+the	O
+chain	O
+to	O
+reverse	O
+direction	O
+,	O
+allowing	O
+the	O
+N	O
+-	O
+terminal	O
+segment	O
+(	O
+residues	O
+334	O
+–	O
+340	O
+)	O
+to	O
+contact	O
+both	O
+helices	O
+of	O
+the	O
+HR1	O
+domain	O
+.	O
+	O
+Long	O
+range	O
+NOEs	O
+were	O
+observed	O
+linking	O
+Leu	O
+-	O
+334	O
+,	O
+Glu	O
+-	O
+335	O
+,	O
+and	O
+Asp	O
+-	O
+336	O
+with	O
+Trp	O
+-	O
+413	O
+of	O
+helix	O
+2	O
+,	O
+Leu	O
+-	O
+334	O
+with	O
+Lys	O
+-	O
+409	O
+of	O
+helix	O
+2	O
+,	O
+and	O
+Phe	O
+-	O
+337	O
+and	O
+Ser	O
+-	O
+338	O
+with	O
+Arg	O
+-	O
+345	O
+,	O
+Arg	O
+-	O
+348	O
+,	O
+and	O
+Leu	O
+-	O
+349	O
+of	O
+helix	O
+1	O
+.	O
+	O
+The	O
+two	O
+α	O
+-	O
+helices	O
+of	O
+TOCA1	O
+HR1	O
+are	O
+separated	O
+by	O
+a	O
+long	O
+loop	O
+of	O
+10	O
+residues	O
+(	O
+residues	O
+380	O
+–	O
+389	O
+)	O
+that	O
+contains	O
+two	O
+short	O
+310	O
+helices	O
+(	O
+residues	O
+381	O
+–	O
+383	O
+and	O
+386	O
+–	O
+389	O
+).	O
+	O
+Interestingly	O
+,	O
+side	O
+chains	O
+of	O
+residues	O
+within	O
+the	O
+loop	O
+region	O
+point	O
+back	O
+toward	O
+helix	O
+1	O
+;	O
+for	O
+example	O
+,	O
+there	O
+are	O
+numerous	O
+distinct	O
+NOEs	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+Asn	O
+-	O
+380	O
+and	O
+Met	O
+-	O
+383	O
+of	O
+the	O
+loop	O
+region	O
+and	O
+Tyr	O
+-	O
+377	O
+and	O
+Val	O
+-	O
+376	O
+of	O
+helix	O
+1	O
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+	O
+The	O
+backbone	O
+NH	O
+and	O
+CHα	O
+groups	O
+of	O
+Gly	O
+-	O
+384	O
+and	O
+Asp	O
+-	O
+385	O
+also	O
+show	O
+NOEs	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+Tyr	O
+-	O
+377	O
+.	O
+	O
+Mapping	O
+the	O
+TOCA1	O
+and	O
+Cdc42	O
+Binding	O
+Interfaces	O
+	O
+The	O
+HR1TOCA1	O
+-	O
+Cdc42	O
+interface	O
+was	O
+investigated	O
+using	O
+NMR	O
+spectroscopy	O
+.	O
+	O
+A	O
+comparison	O
+of	O
+the	O
+15N	O
+HSQC	O
+spectra	O
+of	O
+free	O
+HR1	O
+and	O
+HR1	O
+in	O
+the	O
+presence	O
+of	O
+excess	O
+Cdc42	O
+shows	O
+that	O
+although	O
+some	O
+peaks	O
+were	O
+shifted	O
+,	O
+several	O
+were	O
+much	O
+broader	O
+in	O
+the	O
+complex	O
+,	O
+and	O
+a	O
+considerable	O
+subset	O
+had	O
+disappeared	O
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+	O
+This	O
+behavior	O
+cannot	O
+be	O
+explained	O
+by	O
+the	O
+increase	O
+in	O
+molecular	O
+mass	O
+(	O
+from	O
+12	O
+to	O
+33	O
+kDa	O
+)	O
+when	O
+Cdc42	O
+binds	O
+and	O
+is	O
+more	O
+likely	O
+to	O
+be	O
+due	O
+to	O
+conformational	O
+exchange	O
+.	O
+	O
+Overall	O
+chemical	O
+shift	O
+perturbations	O
+(	O
+CSPs	O
+)	O
+were	O
+calculated	O
+for	O
+each	O
+residue	O
+,	O
+whereas	O
+those	O
+that	O
+had	O
+disappeared	O
+were	O
+assigned	O
+a	O
+shift	O
+change	O
+of	O
+0	O
+.	O
+2	O
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+	O
+A	O
+peak	O
+that	O
+disappeared	O
+or	O
+had	O
+a	O
+CSP	O
+above	O
+the	O
+mean	O
+CSP	O
+for	O
+the	O
+spectrum	O
+was	O
+considered	O
+to	O
+be	O
+significantly	O
+affected	O
+.	O
+	O
+Mapping	O
+the	O
+binding	O
+surface	O
+of	O
+Cdc42	O
+onto	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+.	O
+	O
+A	O
+,	O
+the	O
+15N	O
+HSQC	O
+of	O
+200	O
+μm	O
+TOCA1	O
+HR1	O
+domain	O
+is	O
+shown	O
+in	O
+the	O
+free	O
+form	O
+(	O
+black	O
+)	O
+and	O
+in	O
+the	O
+presence	O
+of	O
+a	O
+4	O
+-	O
+fold	O
+molar	O
+excess	O
+of	O
+Cdc42Δ7Q61L	O
+·	O
+GMPPNP	O
+(	O
+red	O
+).	O
+	O
+The	O
+mean	O
+CSP	O
+is	O
+marked	O
+with	O
+a	O
+red	O
+line	O
+.	O
+	O
+Those	O
+that	O
+were	O
+not	O
+traceable	O
+due	O
+to	O
+spectral	O
+overlap	O
+were	O
+assigned	O
+a	O
+CSP	O
+of	O
+zero	O
+and	O
+are	O
+marked	O
+with	O
+an	O
+asterisk	O
+below	O
+the	O
+bar	O
+.	O
+	O
+C	O
+,	O
+a	O
+schematic	O
+representation	O
+of	O
+the	O
+HR1	O
+domain	O
+.	O
+	O
+Residues	O
+with	O
+significantly	O
+affected	O
+backbone	O
+and	O
+side	O
+chain	O
+groups	O
+that	O
+are	O
+solvent	O
+-	O
+accessible	O
+are	O
+colored	O
+red	O
+.	O
+	O
+A	O
+close	O
+-	O
+up	O
+of	O
+the	O
+binding	O
+region	O
+is	O
+shown	O
+,	O
+with	O
+affected	O
+side	O
+chain	O
+heavy	O
+atoms	O
+shown	O
+as	O
+sticks	O
+.	O
+	O
+D	O
+,	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+region	O
+is	O
+marked	O
+in	O
+red	O
+onto	O
+structures	O
+of	O
+the	O
+HR1	O
+domains	O
+as	O
+indicated	O
+.	O
+	O
+15N	O
+HSQC	O
+shift	O
+mapping	O
+experiments	O
+report	O
+on	O
+changes	O
+to	O
+amide	O
+groups	O
+,	O
+which	O
+are	O
+mainly	O
+inaccessible	O
+because	O
+they	O
+are	O
+buried	O
+inside	O
+the	O
+helices	O
+and	O
+are	O
+involved	O
+in	O
+hydrogen	O
+bonds	O
+.	O
+	O
+Therefore	O
+,	O
+13C	O
+HSQC	O
+and	O
+methyl	O
+-	O
+selective	O
+SOFAST	O
+-	O
+HMQC	O
+experiments	O
+were	O
+also	O
+recorded	O
+on	O
+15N	O
+,	O
+13C	O
+-	O
+labeled	O
+TOCA1	O
+HR1	O
+to	O
+yield	O
+more	O
+information	O
+on	O
+side	O
+chain	O
+involvement	O
+.	O
+	O
+TOCA1	O
+residues	O
+whose	O
+signals	O
+were	O
+affected	O
+by	O
+Cdc42	O
+binding	O
+were	O
+mapped	O
+onto	O
+the	O
+structure	O
+of	O
+TOCA1	O
+HR1	O
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+	O
+The	O
+changes	O
+were	O
+localized	O
+to	O
+one	O
+end	O
+of	O
+the	O
+coiled	O
+-	O
+coil	O
+,	O
+and	O
+the	O
+binding	O
+site	O
+appeared	O
+to	O
+include	O
+residues	O
+from	O
+both	O
+α	O
+-	O
+helices	O
+and	O
+the	O
+loop	O
+region	O
+that	O
+joins	O
+them	O
+.	O
+	O
+The	O
+residues	O
+in	O
+the	O
+interhelical	O
+loop	O
+and	O
+helix	O
+1	O
+that	O
+contact	O
+each	O
+other	O
+(	O
+Fig	O
+.	O
+3D	O
+)	O
+show	O
+shift	O
+changes	O
+in	O
+their	O
+backbone	O
+NH	O
+and	O
+side	O
+chains	O
+in	O
+the	O
+presence	O
+of	O
+Cdc42	O
+.	O
+	O
+For	O
+example	O
+,	O
+the	O
+side	O
+chain	O
+of	O
+Asn	O
+-	O
+380	O
+and	O
+the	O
+backbones	O
+of	O
+Val	O
+-	O
+376	O
+and	O
+Tyr	O
+-	O
+377	O
+were	O
+significantly	O
+affected	O
+but	O
+are	O
+all	O
+buried	O
+in	O
+the	O
+free	O
+TOCA1	O
+HR1	O
+structure	O
+,	O
+indicating	O
+that	O
+local	O
+conformational	O
+changes	O
+in	O
+the	O
+loop	O
+may	O
+facilitate	O
+complex	O
+formation	O
+.	O
+	O
+The	O
+chemical	O
+shift	O
+mapping	O
+data	O
+indicate	O
+that	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+region	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+is	O
+broadly	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+CIP4	O
+and	O
+PRK1	O
+HR1	O
+domains	O
+(	O
+Figs	O
+.	O
+3B	O
+and	O
+4D	O
+).	O
+	O
+As	O
+was	O
+the	O
+case	O
+when	O
+labeled	O
+HR1	O
+was	O
+observed	O
+,	O
+several	O
+peaks	O
+were	O
+shifted	O
+in	O
+the	O
+complex	O
+,	O
+but	O
+many	O
+disappeared	O
+,	O
+indicating	O
+exchange	O
+on	O
+an	O
+unfavorable	O
+,	O
+millisecond	O
+time	O
+scale	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+	O
+Detailed	O
+side	O
+chain	O
+data	O
+could	O
+not	O
+be	O
+obtained	O
+for	O
+all	O
+residues	O
+due	O
+to	O
+spectral	O
+overlap	O
+,	O
+but	O
+constant	O
+time	O
+13C	O
+HSQC	O
+and	O
+methyl	O
+-	O
+selective	O
+SOFAST	O
+-	O
+HMQC	O
+experiments	O
+provided	O
+further	O
+information	O
+on	O
+certain	O
+well	O
+resolved	O
+side	O
+chains	O
+(	O
+marked	O
+with	O
+green	O
+asterisks	O
+in	O
+Fig	O
+.	O
+5B	O
+).	O
+	O
+Mapping	O
+the	O
+binding	O
+surface	O
+of	O
+the	O
+HR1	O
+domain	O
+onto	O
+Cdc42	O
+.	O
+	O
+B	O
+,	O
+CSPs	O
+are	O
+shown	O
+for	O
+backbone	O
+NH	O
+groups	O
+.	O
+	O
+Residues	O
+with	O
+disappeared	O
+peaks	O
+in	O
+13C	O
+HSQC	O
+experiments	O
+are	O
+marked	O
+on	O
+the	O
+chart	O
+with	O
+green	O
+asterisks	O
+.	O
+	O
+Residues	O
+with	O
+either	O
+side	O
+chain	O
+or	O
+backbone	O
+groups	O
+affected	O
+are	O
+colored	O
+blue	O
+if	O
+buried	O
+and	O
+yellow	O
+if	O
+solvent	O
+-	O
+accessible	O
+.	O
+	O
+The	O
+flexible	O
+switch	O
+regions	O
+are	O
+circled	O
+.	O
+	O
+As	O
+many	O
+of	O
+the	O
+peaks	O
+disappeared	O
+,	O
+the	O
+mean	O
+chemical	O
+shift	O
+change	O
+was	O
+relatively	O
+low	O
+,	O
+so	O
+a	O
+threshold	O
+of	O
+the	O
+mean	O
+plus	O
+one	O
+S	O
+.	O
+D	O
+.	O
+value	O
+was	O
+used	O
+to	O
+define	O
+a	O
+significant	O
+CSP	O
+.	O
+	O
+Parts	O
+of	O
+the	O
+switch	O
+regions	O
+(	O
+Fig	O
+.	O
+5	O
+,	O
+B	O
+and	O
+C	O
+)	O
+are	O
+invisible	O
+in	O
+NMR	O
+spectra	O
+recorded	O
+on	O
+free	O
+Cdc42	O
+due	O
+to	O
+conformational	O
+exchange	O
+.	O
+	O
+These	O
+switch	O
+regions	O
+become	O
+visible	O
+in	O
+Cdc42	O
+and	O
+other	O
+small	O
+G	O
+protein	O
+·	O
+effector	O
+complexes	O
+due	O
+to	O
+a	O
+decrease	O
+in	O
+conformational	O
+freedom	O
+upon	O
+complex	O
+formation	O
+.	O
+	O
+The	O
+switch	O
+regions	O
+of	O
+Cdc42	O
+did	O
+not	O
+,	O
+however	O
+,	O
+become	O
+visible	O
+in	O
+the	O
+presence	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+.	O
+	O
+Indeed	O
+,	O
+Ser	O
+-	O
+30	O
+of	O
+switch	O
+I	O
+and	O
+Arg	O
+-	O
+66	O
+,	O
+Arg	O
+-	O
+68	O
+,	O
+Leu	O
+-	O
+70	O
+,	O
+and	O
+Ser	O
+-	O
+71	O
+of	O
+switch	O
+II	O
+are	O
+visible	O
+in	O
+free	O
+Cdc42	O
+but	O
+disappear	O
+in	O
+the	O
+presence	O
+of	O
+the	O
+HR1	O
+domain	O
+.	O
+	O
+Nevertheless	O
+,	O
+mapping	O
+of	O
+the	O
+affected	O
+residues	O
+onto	O
+the	O
+NMR	O
+structure	O
+of	O
+free	O
+Cdc42Δ7Q61L	O
+·	O
+GMPPNP	O
+(	O
+Fig	O
+.	O
+5C	O
+)	O
+8	O
+shows	O
+that	O
+,	O
+although	O
+they	O
+are	O
+relatively	O
+widespread	O
+compared	O
+with	O
+changes	O
+in	O
+the	O
+HR1	O
+domain	O
+,	O
+in	O
+general	O
+,	O
+they	O
+are	O
+on	O
+the	O
+face	O
+of	O
+the	O
+protein	O
+that	O
+includes	O
+the	O
+switches	O
+.	O
+	O
+Although	O
+the	O
+binding	O
+interface	O
+may	O
+be	O
+overestimated	O
+,	O
+this	O
+suggests	O
+that	O
+the	O
+switch	O
+regions	O
+are	O
+involved	O
+in	O
+binding	O
+to	O
+TOCA1	O
+.	O
+	O
+The	O
+Cdc42	O
+·	O
+HR1TOCA1	O
+complex	O
+was	O
+not	O
+amenable	O
+to	O
+full	O
+structural	O
+analysis	O
+due	O
+to	O
+the	O
+weak	O
+interaction	O
+and	O
+the	O
+extensive	O
+exchange	O
+broadening	O
+seen	O
+in	O
+the	O
+NMR	O
+experiments	O
+.	O
+	O
+The	O
+orientation	O
+of	O
+the	O
+HR1	O
+domain	O
+with	O
+respect	O
+to	O
+Cdc42	O
+cannot	O
+be	O
+definitively	O
+concluded	O
+in	O
+the	O
+absence	O
+of	O
+unambiguous	O
+distance	O
+restraints	O
+;	O
+hence	O
+,	O
+HADDOCK	O
+produced	O
+a	O
+set	O
+of	O
+models	O
+in	O
+which	O
+the	O
+HR1	O
+domain	O
+contacts	O
+the	O
+same	O
+surface	O
+on	O
+Cdc42	O
+but	O
+is	O
+in	O
+various	O
+orientations	O
+with	O
+respect	O
+to	O
+Cdc42	O
+.	O
+	O
+The	O
+cluster	O
+with	O
+the	O
+lowest	O
+root	O
+mean	O
+square	O
+deviation	O
+from	O
+the	O
+lowest	O
+energy	O
+structure	O
+is	O
+assumed	O
+to	O
+be	O
+the	O
+best	O
+model	O
+.	O
+	O
+By	O
+these	O
+criteria	O
+,	O
+in	O
+the	O
+best	O
+model	O
+,	O
+the	O
+HR1	O
+domain	O
+is	O
+in	O
+a	O
+similar	O
+orientation	O
+to	O
+the	O
+HR1a	O
+domain	O
+of	O
+PRK1	O
+bound	O
+to	O
+RhoA	O
+and	O
+the	O
+HR1b	O
+domain	O
+bound	O
+to	O
+Rac1	O
+.	O
+	O
+A	O
+representative	O
+model	O
+from	O
+this	O
+cluster	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+6A	O
+alongside	O
+the	O
+Rac1	O
+-	O
+HR1b	O
+structure	O
+(	O
+PDB	O
+code	O
+2RMK	O
+)	O
+in	O
+Fig	O
+.	O
+6B	O
+.	O
+	O
+Model	O
+of	O
+Cdc42	O
+·	O
+HR1	O
+complex	O
+.	O
+	O
+A	O
+,	O
+a	O
+representative	O
+model	O
+of	O
+the	O
+Cdc42	O
+·	O
+HR1	O
+complex	O
+from	O
+the	O
+cluster	O
+closest	O
+to	O
+the	O
+lowest	O
+energy	O
+model	O
+produced	O
+using	O
+HADDOCK	O
+.	O
+	O
+Residues	O
+of	O
+Cdc42	O
+that	O
+are	O
+affected	O
+in	O
+the	O
+presence	O
+of	O
+the	O
+HR1	O
+domain	O
+but	O
+are	O
+not	O
+in	O
+close	O
+proximity	O
+to	O
+it	O
+are	O
+colored	O
+in	O
+red	O
+and	O
+labeled	O
+.	O
+	O
+B	O
+,	O
+structure	O
+of	O
+Rac1	O
+in	O
+complex	O
+with	O
+the	O
+HR1b	O
+domain	O
+of	O
+PRK1	O
+(	O
+PDB	O
+code	O
+2RMK	O
+).	O
+	O
+C	O
+,	O
+sequence	O
+alignment	O
+of	O
+RhoA	O
+,	O
+Cdc42	O
+and	O
+Rac1	O
+.	O
+	O
+Contact	O
+residues	O
+of	O
+RhoA	O
+and	O
+Rac1	O
+to	O
+PRK1	O
+HR1a	O
+and	O
+HR1b	O
+,	O
+respectively	O
+,	O
+are	O
+colored	O
+cyan	O
+.	O
+	O
+Residues	O
+of	O
+Cdc42	O
+that	O
+disappear	O
+or	O
+show	O
+chemical	O
+shift	O
+changes	O
+in	O
+the	O
+presence	O
+of	O
+TOCA1	O
+are	O
+colored	O
+cyan	O
+if	O
+also	O
+identified	O
+as	O
+contacts	O
+in	O
+RhoA	O
+and	O
+Rac1	O
+and	O
+yellow	O
+if	O
+they	O
+are	O
+not	O
+.	O
+	O
+A	O
+sequence	O
+alignment	O
+of	O
+RhoA	O
+,	O
+Cdc42	O
+,	O
+and	O
+Rac1	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+6C	O
+.	O
+	O
+The	O
+RhoA	O
+and	O
+Rac1	O
+contact	O
+residues	O
+in	O
+the	O
+switch	O
+regions	O
+are	O
+invisible	O
+in	O
+the	O
+spectra	O
+of	O
+Cdc42	O
+,	O
+but	O
+they	O
+are	O
+generally	O
+conserved	O
+between	O
+all	O
+three	O
+G	O
+proteins	O
+.	O
+	O
+Several	O
+Cdc42	O
+residues	O
+identified	O
+by	O
+chemical	O
+shift	O
+mapping	O
+are	O
+not	O
+in	O
+close	O
+contact	O
+in	O
+the	O
+Cdc42	O
+·	O
+TOCA1	O
+model	O
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+	O
+Other	O
+residues	O
+that	O
+are	O
+affected	O
+in	O
+the	O
+Cdc42	O
+·	O
+TOCA1	O
+complex	O
+but	O
+that	O
+do	O
+not	O
+correspond	O
+to	O
+contact	O
+residues	O
+of	O
+RhoA	O
+or	O
+Rac1	O
+(	O
+Fig	O
+.	O
+6C	O
+)	O
+include	O
+Gln	O
+-	O
+2Cdc42	O
+,	O
+Lys	O
+-	O
+16Cdc42	O
+,	O
+Thr	O
+-	O
+52Cdc42	O
+,	O
+and	O
+Arg	O
+-	O
+68Cdc42	O
+.	O
+	O
+From	O
+the	O
+known	O
+interactions	O
+and	O
+effects	O
+of	O
+the	O
+proteins	O
+in	O
+biological	O
+systems	O
+,	O
+it	O
+has	O
+been	O
+suggested	O
+that	O
+TOCA1	O
+and	O
+N	O
+-	O
+WASP	O
+could	O
+bind	O
+Cdc42	O
+simultaneously	O
+.	O
+	O
+Studies	O
+in	O
+CHO	O
+cells	O
+indicated	O
+that	O
+a	O
+Cdc42	O
+·	O
+N	O
+-	O
+WASP	O
+·	O
+TOCA1	O
+complex	O
+existed	O
+because	O
+FRET	O
+was	O
+observed	O
+between	O
+RFP	O
+-	O
+TOCA1	O
+and	O
+GFP	O
+-	O
+N	O
+-	O
+WASP	O
+,	O
+and	O
+the	O
+efficiency	O
+was	O
+decreased	O
+when	O
+an	O
+N	O
+-	O
+WASP	O
+mutant	O
+was	O
+used	O
+that	O
+no	O
+longer	O
+binds	O
+Cdc42	O
+.	O
+	O
+An	O
+overlay	O
+of	O
+the	O
+HADDOCK	O
+model	O
+of	O
+the	O
+Cdc42	O
+·	O
+HR1TOCA1	O
+complex	O
+and	O
+the	O
+structure	O
+of	O
+Cdc42	O
+in	O
+complex	O
+with	O
+the	O
+GBD	O
+of	O
+the	O
+N	O
+-	O
+WASP	O
+homologue	O
+,	O
+WASP	O
+(	O
+PDB	O
+code	O
+1CEE	O
+),	O
+shows	O
+that	O
+the	O
+HR1	O
+and	O
+GBD	O
+binding	O
+sites	O
+only	O
+partly	O
+overlap	O
+,	O
+and	O
+,	O
+therefore	O
+,	O
+a	O
+ternary	O
+complex	O
+remained	O
+possible	O
+(	O
+Fig	O
+.	O
+7A	O
+).	O
+	O
+Interestingly	O
+,	O
+the	O
+presence	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+would	O
+not	O
+prevent	O
+the	O
+core	O
+CRIB	O
+of	O
+WASP	O
+from	O
+binding	O
+to	O
+Cdc42	O
+,	O
+although	O
+the	O
+regions	O
+C	O
+-	O
+terminal	O
+to	O
+the	O
+CRIB	O
+that	O
+are	O
+required	O
+for	O
+high	O
+affinity	O
+binding	O
+of	O
+WASP	O
+would	O
+interfere	O
+sterically	O
+with	O
+the	O
+TOCA1	O
+HR1	O
+.	O
+	O
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+basic	O
+region	O
+in	O
+WASP	O
+including	O
+three	O
+lysines	O
+(	O
+residues	O
+230	O
+–	O
+232	O
+),	O
+N	O
+-	O
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+to	O
+the	O
+core	O
+CRIB	O
+,	O
+has	O
+been	O
+implicated	O
+in	O
+an	O
+electrostatic	O
+steering	O
+mechanism	O
+,	O
+and	O
+these	O
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+be	O
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+the	O
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+TOCA1	O
+HR1	O
+(	O
+Fig	O
+.	O
+7A	O
+).	O
+	O
+The	O
+N	O
+-	O
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+TOCA1	O
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+.	O
+	O
+The	O
+core	O
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+WASP	O
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+,	O
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+its	O
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+shown	O
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+orange	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+required	O
+for	O
+maximal	O
+affinity	O
+is	O
+shown	O
+in	O
+cyan	O
+.	O
+	O
+A	O
+semitransparent	O
+surface	O
+representation	O
+of	O
+Cdc42	O
+and	O
+WASP	O
+is	O
+shown	O
+overlaid	O
+with	O
+the	O
+schematic	O
+.	O
+	O
+C	O
+,	O
+Selected	O
+regions	O
+of	O
+the	O
+15N	O
+HSQC	O
+of	O
+145	O
+μm	O
+Cdc42Δ7Q61L	O
+·	O
+GMPPNP	O
+with	O
+the	O
+indicated	O
+ratios	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+,	O
+the	O
+N	O
+-	O
+WASP	O
+GBD	O
+,	O
+or	O
+both	O
+,	O
+showing	O
+that	O
+the	O
+TOCA	O
+HR1	O
+domain	O
+does	O
+not	O
+displace	O
+the	O
+N	O
+-	O
+WASP	O
+GBD	O
+.	O
+	O
+D	O
+,	O
+selected	O
+regions	O
+of	O
+the	O
+15N	O
+HSQC	O
+of	O
+600	O
+μm	O
+TOCA1	O
+HR1	O
+domain	O
+in	O
+complex	O
+with	O
+Cdc42	O
+in	O
+the	O
+absence	O
+and	O
+presence	O
+of	O
+the	O
+N	O
+-	O
+WASP	O
+GBD	O
+,	O
+showing	O
+displacement	O
+of	O
+Cdc42	O
+from	O
+the	O
+HR1	O
+domain	O
+by	O
+N	O
+-	O
+WASP	O
+.	O
+	O
+An	O
+N	O
+-	O
+WASP	O
+GBD	O
+construct	O
+was	O
+produced	O
+,	O
+and	O
+its	O
+affinity	O
+for	O
+Cdc42	O
+was	O
+measured	O
+by	O
+competition	O
+SPA	O
+(	O
+Fig	O
+.	O
+7B	O
+).	O
+	O
+The	O
+Kd	O
+that	O
+was	O
+determined	O
+(	O
+37	O
+nm	O
+)	O
+is	O
+consistent	O
+with	O
+the	O
+previously	O
+reported	O
+affinity	O
+.	O
+	O
+Unlabeled	O
+HR1TOCA1	O
+was	O
+then	O
+added	O
+to	O
+the	O
+Cdc42	O
+·	O
+N	O
+-	O
+WASP	O
+complex	O
+,	O
+and	O
+no	O
+changes	O
+were	O
+seen	O
+,	O
+suggesting	O
+that	O
+the	O
+N	O
+-	O
+WASP	O
+GBD	O
+was	O
+not	O
+displaced	O
+even	O
+in	O
+the	O
+presence	O
+of	O
+a	O
+5	O
+-	O
+fold	O
+excess	O
+of	O
+HR1TOCA1	O
+.	O
+	O
+These	O
+experiments	O
+were	O
+recorded	O
+at	O
+sufficiently	O
+high	O
+protein	O
+concentrations	O
+(	O
+145	O
+μm	O
+Cdc42	O
+,	O
+145	O
+μm	O
+N	O
+-	O
+WASP	O
+GBD	O
+,	O
+725	O
+μm	O
+TOCA1	O
+HR1	O
+domain	O
+)	O
+to	O
+be	O
+far	O
+in	O
+excess	O
+of	O
+the	O
+Kd	O
+values	O
+of	O
+the	O
+individual	O
+interactions	O
+(	O
+TOCA1	O
+Kd	O
+≈	O
+5	O
+μm	O
+,	O
+N	O
+-	O
+WASP	O
+Kd	O
+=	O
+37	O
+nm	O
+).	O
+	O
+Furthermore	O
+,	O
+15N	O
+-	O
+TOCA1	O
+HR1	O
+was	O
+monitored	O
+in	O
+the	O
+presence	O
+of	O
+unlabeled	O
+Cdc42Δ7Q61L	O
+·	O
+GMPPNP	O
+(	O
+1	O
+:	O
+1	O
+)	O
+before	O
+and	O
+after	O
+the	O
+addition	O
+of	O
+0	O
+.	O
+25	O
+and	O
+1	O
+.	O
+0	O
+eq	O
+of	O
+unlabeled	O
+N	O
+-	O
+WASP	O
+GBD	O
+.	O
+	O
+When	O
+in	O
+fast	O
+exchange	O
+,	O
+the	O
+NMR	O
+signal	O
+represents	O
+a	O
+population	O
+-	O
+weighted	O
+average	O
+between	O
+free	O
+and	O
+bound	O
+states	O
+,	O
+so	O
+the	O
+intermediate	O
+spectrum	O
+indicates	O
+that	O
+the	O
+population	O
+comprises	O
+a	O
+mixture	O
+of	O
+free	O
+and	O
+bound	O
+HR1	O
+domain	O
+.	O
+	O
+Again	O
+,	O
+the	O
+experiments	O
+were	O
+recorded	O
+on	O
+protein	O
+samples	O
+far	O
+in	O
+excess	O
+of	O
+the	O
+individual	O
+Kd	O
+values	O
+(	O
+600	O
+μm	O
+each	O
+protein	O
+).	O
+	O
+These	O
+data	O
+indicate	O
+that	O
+the	O
+HR1	O
+domain	O
+is	O
+displaced	O
+from	O
+Cdc42	O
+by	O
+N	O
+-	O
+WASP	O
+and	O
+that	O
+a	O
+ternary	O
+complex	O
+comprising	O
+TOCA1	O
+HR1	O
+,	O
+N	O
+-	O
+WASP	O
+GBD	O
+,	O
+and	O
+Cdc42	O
+is	O
+not	O
+formed	O
+.	O
+	O
+To	O
+extend	O
+these	O
+studies	O
+to	O
+a	O
+more	O
+complex	O
+system	O
+and	O
+to	O
+assess	O
+the	O
+ability	O
+of	O
+TOCA1	O
+HR1	O
+to	O
+compete	O
+with	O
+full	O
+-	O
+length	O
+N	O
+-	O
+WASP	O
+,	O
+pyrene	O
+actin	O
+assays	O
+were	O
+employed	O
+.	O
+	O
+These	O
+assays	O
+,	O
+described	O
+in	O
+detail	O
+elsewhere	O
+,	O
+were	O
+carried	O
+out	O
+using	O
+pyrene	O
+actin	O
+-	O
+supplemented	O
+Xenopus	O
+extracts	O
+into	O
+which	O
+exogenous	O
+TOCA1	O
+HR1	O
+domain	O
+or	O
+N	O
+-	O
+WASP	O
+GBD	O
+was	O
+added	O
+,	O
+to	O
+assess	O
+their	O
+effects	O
+on	O
+actin	O
+polymerization	O
+.	O
+	O
+Actin	O
+polymerization	O
+triggered	O
+by	O
+the	O
+addition	O
+of	O
+PI	O
+(	O
+4	O
+,	O
+5	O
+)	O
+P2	O
+-	O
+containing	O
+liposomes	O
+has	O
+previously	O
+been	O
+shown	O
+to	O
+depend	O
+on	O
+TOCA1	O
+and	O
+N	O
+-	O
+WASP	O
+.	O
+	O
+The	O
+addition	O
+of	O
+the	O
+isolated	O
+N	O
+-	O
+WASP	O
+GBD	O
+significantly	O
+inhibited	O
+the	O
+polymerization	O
+of	O
+actin	O
+at	O
+concentrations	O
+as	O
+low	O
+as	O
+100	O
+nm	O
+and	O
+completely	O
+abolished	O
+polymerization	O
+at	O
+higher	O
+concentrations	O
+(	O
+Fig	O
+.	O
+8	O
+).	O
+	O
+The	O
+addition	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+to	O
+100	O
+μm	O
+had	O
+no	O
+significant	O
+effect	O
+on	O
+the	O
+rate	O
+of	O
+actin	O
+polymerization	O
+or	O
+maximum	O
+fluorescence	O
+.	O
+	O
+Actin	O
+polymerization	O
+downstream	O
+of	O
+Cdc42	O
+·	O
+N	O
+-	O
+WASP	O
+·	O
+TOCA1	O
+is	O
+inhibited	O
+by	O
+excess	O
+N	O
+-	O
+WASP	O
+GBD	O
+but	O
+not	O
+by	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+.	O
+	O
+The	O
+Cdc42	O
+-	O
+TOCA1	O
+Interaction	O
+	O
+A	O
+single	O
+binding	O
+interface	O
+on	O
+both	O
+the	O
+HR1	O
+domain	O
+and	O
+Cdc42	O
+can	O
+be	O
+concluded	O
+from	O
+the	O
+data	O
+presented	O
+here	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+interfaces	O
+are	O
+comparable	O
+with	O
+those	O
+of	O
+other	O
+G	O
+protein	O
+-	O
+HR1	O
+interactions	O
+(	O
+Fig	O
+.	O
+4	O
+),	O
+and	O
+the	O
+lowest	O
+energy	O
+model	O
+produced	O
+in	O
+rigid	O
+body	O
+docking	O
+resembles	O
+previously	O
+studied	O
+G	O
+protein	O
+·	O
+HR1	O
+complexes	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+It	O
+seems	O
+,	O
+therefore	O
+,	O
+that	O
+the	O
+interaction	O
+,	O
+despite	O
+its	O
+relatively	O
+low	O
+affinity	O
+,	O
+is	O
+specific	O
+and	O
+sterically	O
+similar	O
+to	O
+other	O
+HR1	O
+domain	O
+-	O
+G	O
+protein	O
+interactions	O
+.	O
+	O
+A	O
+short	O
+region	O
+N	O
+-	O
+terminal	O
+to	O
+the	O
+coiled	O
+-	O
+coil	O
+exhibits	O
+a	O
+series	O
+of	O
+turns	O
+and	O
+contacts	O
+residues	O
+of	O
+both	O
+helices	O
+of	O
+the	O
+coiled	O
+-	O
+coil	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+The	O
+corresponding	O
+sequence	O
+in	O
+CIP4	O
+also	O
+includes	O
+a	O
+series	O
+of	O
+turns	O
+but	O
+is	O
+flexible	O
+,	O
+whereas	O
+in	O
+the	O
+HR1a	O
+domain	O
+of	O
+PRK1	O
+,	O
+the	O
+equivalent	O
+region	O
+adopts	O
+an	O
+α	O
+-	O
+helical	O
+structure	O
+that	O
+packs	O
+against	O
+the	O
+coiled	O
+-	O
+coil	O
+.	O
+	O
+The	O
+contacts	O
+between	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+and	O
+the	O
+coiled	O
+-	O
+coil	O
+are	O
+predominantly	O
+hydrophobic	O
+in	O
+both	O
+cases	O
+,	O
+but	O
+sequence	O
+-	O
+specific	O
+contacts	O
+do	O
+not	O
+appear	O
+to	O
+be	O
+conserved	O
+.	O
+	O
+This	O
+region	O
+is	O
+distant	O
+from	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+interface	O
+of	O
+the	O
+HR1	O
+domains	O
+,	O
+so	O
+the	O
+structural	O
+differences	O
+may	O
+relate	O
+to	O
+the	O
+structure	O
+and	O
+regulation	O
+of	O
+these	O
+domains	O
+rather	O
+than	O
+their	O
+G	O
+protein	O
+interactions	O
+.	O
+	O
+TOCA1	O
+and	O
+CIP4	O
+both	O
+bind	O
+weakly	O
+to	O
+Cdc42	O
+,	O
+whereas	O
+the	O
+HR1a	O
+domain	O
+of	O
+PRK1	O
+binds	O
+tightly	O
+to	O
+RhoA	O
+and	O
+Rac1	O
+,	O
+and	O
+the	O
+HR1b	O
+domain	O
+binds	O
+to	O
+Rac1	O
+.	O
+	O
+The	O
+structural	O
+features	O
+shared	O
+by	O
+TOCA1	O
+and	O
+CIP4	O
+may	O
+therefore	O
+be	O
+related	O
+to	O
+Cdc42	O
+binding	O
+specificity	O
+and	O
+the	O
+low	O
+affinities	O
+.	O
+	O
+In	O
+free	O
+TOCA1	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+interhelical	O
+region	O
+make	O
+extensive	O
+contacts	O
+with	O
+residues	O
+in	O
+helix	O
+1	O
+.	O
+	O
+The	O
+lowest	O
+energy	O
+model	O
+produced	O
+by	O
+HADDOCK	O
+using	O
+ambiguous	O
+interaction	O
+restraints	O
+from	O
+the	O
+titration	O
+data	O
+resembled	O
+the	O
+NMR	O
+structures	O
+of	O
+RhoA	O
+and	O
+Rac1	O
+in	O
+complex	O
+with	O
+their	O
+HR1	O
+domain	O
+partners	O
+.	O
+	O
+Phe	O
+-	O
+56Cdc42	O
+,	O
+which	O
+is	O
+a	O
+Trp	O
+in	O
+both	O
+Rac1	O
+and	O
+RhoA	O
+(	O
+Fig	O
+.	O
+6C	O
+),	O
+is	O
+thought	O
+to	O
+pack	O
+behind	O
+switch	O
+I	O
+when	O
+Cdc42	O
+interacts	O
+with	O
+ACK	O
+,	O
+maintaining	O
+the	O
+switch	O
+in	O
+a	O
+binding	O
+-	O
+competent	O
+orientation	O
+.	O
+	O
+This	O
+residue	O
+has	O
+also	O
+been	O
+identified	O
+as	O
+important	O
+for	O
+Cdc42	O
+-	O
+WASP	O
+binding	O
+.	O
+	O
+Phe	O
+-	O
+56Cdc42	O
+is	O
+therefore	O
+likely	O
+to	O
+be	O
+involved	O
+in	O
+the	O
+Cdc42	O
+-	O
+TOCA1	O
+interaction	O
+,	O
+probably	O
+by	O
+stabilizing	O
+the	O
+position	O
+of	O
+switch	O
+I	O
+.	O
+	O
+Gln	O
+-	O
+2Cdc42	O
+,	O
+which	O
+has	O
+also	O
+been	O
+identified	O
+as	O
+a	O
+contact	O
+residue	O
+in	O
+the	O
+Cdc42	O
+·	O
+ACK	O
+complex	O
+,	O
+contacts	O
+Val	O
+-	O
+376TOCA1	O
+and	O
+Asn	O
+-	O
+380TOCA1	O
+in	O
+the	O
+model	O
+and	O
+disrupts	O
+the	O
+contacts	O
+between	O
+the	O
+interhelical	O
+loop	O
+and	O
+the	O
+first	O
+helix	O
+of	O
+the	O
+TOCA1	O
+coiled	O
+-	O
+coil	O
+.	O
+	O
+Thr	O
+-	O
+52Cdc42	O
+,	O
+which	O
+has	O
+also	O
+been	O
+identified	O
+as	O
+making	O
+minor	O
+contacts	O
+with	O
+ACK	O
+,	O
+falls	O
+near	O
+the	O
+side	O
+chains	O
+of	O
+HR1TOCA1	O
+helix	O
+1	O
+,	O
+particularly	O
+Lys	O
+-	O
+372TOCA1	O
+,	O
+whereas	O
+the	O
+equivalent	O
+position	O
+in	O
+Rac1	O
+is	O
+Asn	O
+-	O
+52Rac1	O
+.	O
+	O
+N52T	B-mutant
+is	O
+one	O
+of	O
+a	O
+combination	O
+of	O
+seven	O
+residues	O
+found	O
+to	O
+confer	O
+ACK	O
+binding	O
+on	O
+Rac1	O
+and	O
+so	O
+may	O
+represent	O
+a	O
+specific	O
+Cdc42	O
+-	O
+effector	O
+contact	O
+residue	O
+.	O
+	O
+The	O
+position	O
+equivalent	O
+to	O
+Lys	O
+-	O
+372TOCA1	O
+in	O
+PRK1	O
+is	O
+Glu	O
+-	O
+58HR1a	O
+or	O
+Gln	O
+-	O
+151HR1b	O
+.	O
+	O
+Thr	O
+-	O
+52Cdc42	O
+-	O
+Lys	O
+-	O
+372TOCA1	O
+may	O
+therefore	O
+represent	O
+a	O
+specific	O
+Cdc42	O
+-	O
+HR1TOCA1	O
+contact	O
+.	O
+	O
+Arg	O
+-	O
+68Cdc42	O
+of	O
+switch	O
+II	O
+is	O
+positioned	O
+close	O
+to	O
+Glu	O
+-	O
+395TOCA1	O
+(	O
+Fig	O
+.	O
+6D	O
+),	O
+suggesting	O
+a	O
+direct	O
+electrostatic	O
+contact	O
+between	O
+switch	O
+II	O
+of	O
+Cdc42	O
+and	O
+helix	O
+2	O
+of	O
+the	O
+HR1	O
+domain	O
+.	O
+	O
+The	O
+importance	O
+of	O
+this	O
+residue	O
+in	O
+the	O
+Cdc42	O
+-	O
+TOCA1	O
+interaction	O
+remains	O
+unclear	O
+,	O
+although	O
+its	O
+mutation	O
+reduces	O
+binding	O
+to	O
+RhoGAP	O
+,	O
+suggesting	O
+that	O
+it	O
+can	O
+be	O
+involved	O
+in	O
+Cdc42	O
+interactions	O
+.	O
+	O
+The	O
+solution	O
+structure	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+presented	O
+here	O
+,	O
+along	O
+with	O
+the	O
+model	O
+of	O
+the	O
+HR1TOCA1	O
+·	O
+Cdc42	O
+complex	O
+is	O
+consistent	O
+with	O
+a	O
+conserved	O
+mode	O
+of	O
+binding	O
+across	O
+the	O
+known	O
+HR1	O
+domain	O
+-	O
+Rho	O
+family	O
+interactions	O
+,	O
+despite	O
+their	O
+differing	O
+affinities	O
+.	O
+	O
+We	O
+have	O
+previously	O
+postulated	O
+that	O
+the	O
+inherent	O
+flexibility	O
+of	O
+HR1	O
+domains	O
+contributes	O
+to	O
+their	O
+ability	O
+to	O
+bind	O
+to	O
+different	O
+Rho	O
+family	O
+G	O
+proteins	O
+,	O
+with	O
+Rho	O
+-	O
+binding	O
+HR1	O
+domains	O
+displaying	O
+increased	O
+flexibility	O
+,	O
+reflected	O
+in	O
+their	O
+lower	O
+melting	O
+temperatures	O
+(	O
+Tm	O
+)	O
+and	O
+Rac	O
+binders	O
+being	O
+more	O
+rigid	O
+.	O
+	O
+The	O
+Tm	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+is	O
+61	O
+.	O
+9	O
+°	O
+C	O
+(	O
+data	O
+not	O
+shown	O
+),	O
+which	O
+is	O
+the	O
+highest	O
+Tm	O
+that	O
+we	O
+have	O
+measured	O
+for	O
+an	O
+HR1	O
+domain	O
+thus	O
+far	O
+.	O
+	O
+An	O
+investigation	O
+into	O
+the	O
+local	O
+motions	O
+,	O
+particularly	O
+in	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+regions	O
+,	O
+may	O
+offer	O
+further	O
+insight	O
+into	O
+the	O
+differential	O
+specificities	O
+and	O
+affinities	O
+of	O
+G	O
+protein	O
+-	O
+HR1	O
+domain	O
+interactions	O
+.	O
+	O
+The	O
+low	O
+affinity	O
+of	O
+the	O
+Cdc42	O
+-	O
+HR1TOCA1	O
+interaction	O
+is	O
+consistent	O
+with	O
+a	O
+tightly	O
+spatially	O
+and	O
+temporally	O
+regulated	O
+pathway	O
+,	O
+requiring	O
+combinatorial	O
+signals	O
+leading	O
+to	O
+a	O
+series	O
+of	O
+coincident	O
+weak	O
+interactions	O
+that	O
+elicit	O
+full	O
+activation	O
+.	O
+	O
+The	O
+HR1	O
+domains	O
+from	O
+other	O
+TOCA	O
+family	O
+members	O
+,	O
+CIP4	O
+and	O
+FBP17	O
+,	O
+also	O
+bind	O
+at	O
+low	O
+micromolar	O
+affinities	O
+to	O
+Cdc42	O
+,	O
+so	O
+the	O
+low	O
+affinity	O
+interaction	O
+appears	O
+to	O
+be	O
+commonplace	O
+among	O
+this	O
+family	O
+of	O
+HR1	O
+domain	O
+proteins	O
+,	O
+in	O
+contrast	O
+to	O
+the	O
+PRK	O
+family	O
+.	O
+	O
+Evidence	O
+suggests	O
+that	O
+the	O
+TOCA	O
+family	O
+of	O
+proteins	O
+are	O
+recruited	O
+to	O
+the	O
+membrane	O
+via	O
+an	O
+interaction	O
+between	O
+their	O
+F	O
+-	O
+BAR	O
+domain	O
+and	O
+specific	O
+signaling	O
+lipids	O
+.	O
+	O
+For	O
+example	O
+,	O
+electrostatic	O
+interactions	O
+between	O
+the	O
+F	O
+-	O
+BAR	O
+domain	O
+and	O
+the	O
+membrane	O
+are	O
+required	O
+for	O
+TOCA1	O
+recruitment	O
+to	O
+membrane	O
+vesicles	O
+and	O
+tubules	O
+,	O
+and	O
+TOCA1	O
+-	O
+dependent	O
+actin	O
+polymerization	O
+is	O
+known	O
+to	O
+depend	O
+specifically	O
+on	O
+PI	O
+(	O
+4	O
+,	O
+5	O
+)	O
+P2	O
+.	O
+	O
+Once	O
+at	O
+the	O
+membrane	O
+,	O
+high	O
+local	O
+concentrations	O
+of	O
+TOCA1	O
+could	O
+exceed	O
+the	O
+Kd	O
+of	O
+F	O
+-	O
+BAR	O
+dimerization	O
+(	O
+likely	O
+to	O
+be	O
+comparable	O
+with	O
+that	O
+of	O
+the	O
+FCHo2	O
+F	O
+-	O
+BAR	O
+domain	O
+(	O
+2	O
+.	O
+5	O
+μm	O
+))	O
+and	O
+that	O
+of	O
+the	O
+Cdc42	O
+-	O
+HR1TOCA1	O
+interaction	O
+.	O
+	O
+Cdc42	O
+-	O
+HR1TOCA1	O
+binding	O
+would	O
+then	O
+be	O
+favorable	O
+,	O
+as	O
+long	O
+as	O
+coincident	O
+activation	O
+of	O
+Cdc42	O
+had	O
+occurred	O
+,	O
+leading	O
+to	O
+stabilization	O
+of	O
+TOCA1	O
+at	O
+the	O
+membrane	O
+and	O
+downstream	O
+activation	O
+of	O
+N	O
+-	O
+WASP	O
+.	O
+	O
+It	O
+has	O
+been	O
+postulated	O
+that	O
+WASP	O
+and	O
+N	O
+-	O
+WASP	O
+exist	O
+in	O
+equilibrium	O
+between	O
+folded	O
+(	O
+inactive	O
+)	O
+and	O
+unfolded	O
+(	O
+active	O
+)	O
+forms	O
+,	O
+and	O
+the	O
+affinity	O
+of	O
+Cdc42	O
+for	O
+the	O
+unfolded	O
+WASP	O
+proteins	O
+is	O
+significantly	O
+enhanced	O
+.	O
+	O
+The	O
+unfolded	O
+,	O
+high	O
+affinity	O
+state	O
+of	O
+WASP	O
+is	O
+represented	O
+by	O
+a	O
+short	O
+peptide	O
+,	O
+the	O
+GBD	O
+,	O
+which	O
+binds	O
+with	O
+a	O
+low	O
+nanomolar	O
+affinity	O
+to	O
+Cdc42	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+best	O
+estimate	O
+of	O
+the	O
+affinity	O
+of	O
+full	O
+-	O
+length	O
+WASP	O
+for	O
+Cdc42	O
+is	O
+low	O
+micromolar	O
+.	O
+	O
+In	O
+the	O
+inactive	O
+state	O
+of	O
+WASP	O
+,	O
+the	O
+actin	O
+-	O
+and	O
+Arp2	O
+/	O
+3	O
+-	O
+binding	O
+VCA	O
+domain	O
+contacts	O
+the	O
+GBD	O
+,	O
+competing	O
+for	O
+Cdc42	O
+binding	O
+.	O
+	O
+The	O
+high	O
+affinity	O
+of	O
+Cdc42	O
+for	O
+the	O
+unfolded	O
+,	O
+active	O
+form	O
+pushes	O
+the	O
+equilibrium	O
+in	O
+favor	O
+of	O
+(	O
+N	O
+-)	O
+WASP	O
+activation	O
+.	O
+	O
+Binding	O
+of	O
+PI	O
+(	O
+4	O
+,	O
+5	O
+)	O
+P2	O
+to	O
+the	O
+basic	O
+region	O
+just	O
+N	O
+-	O
+terminal	O
+to	O
+the	O
+GBD	O
+further	O
+favors	O
+the	O
+active	O
+conformation	O
+.	O
+	O
+Cdc42	O
+is	O
+activated	O
+in	O
+response	O
+to	O
+co	O
+-	O
+incident	O
+signals	O
+and	O
+can	O
+then	O
+bind	O
+to	O
+TOCA1	O
+,	O
+further	O
+stabilizing	O
+TOCA1	O
+at	O
+the	O
+membrane	O
+.	O
+	O
+The	O
+recruitment	O
+of	O
+N	O
+-	O
+WASP	O
+alone	O
+and	O
+of	O
+the	O
+N	O
+-	O
+WASP	O
+·	O
+WIP	O
+complex	O
+by	O
+TOCA1	O
+and	O
+FBP17	O
+has	O
+been	O
+demonstrated	O
+.	O
+	O
+It	O
+may	O
+therefore	O
+be	O
+envisaged	O
+that	O
+WIP	O
+and	O
+TOCA1	O
+exert	O
+opposing	O
+allosteric	O
+effects	O
+on	O
+N	O
+-	O
+WASP	O
+,	O
+with	O
+TOCA1	O
+favoring	O
+the	O
+unfolded	O
+,	O
+active	O
+conformation	O
+of	O
+N	O
+-	O
+WASP	O
+and	O
+increasing	O
+its	O
+affinity	O
+for	O
+Cdc42	O
+.	O
+	O
+TOCA1	O
+may	O
+also	O
+activate	O
+N	O
+-	O
+WASP	O
+by	O
+effective	O
+oligomerization	O
+because	O
+clustering	O
+of	O
+TOCA1	O
+at	O
+the	O
+membrane	O
+following	O
+coincident	O
+interactions	O
+with	O
+PI	O
+(	O
+4	O
+,	O
+5	O
+)	O
+P2	O
+and	O
+Cdc42	O
+would	O
+in	O
+turn	O
+lead	O
+to	O
+clustering	O
+of	O
+N	O
+-	O
+WASP	O
+,	O
+in	O
+addition	O
+to	O
+pushing	O
+the	O
+equilibrium	O
+toward	O
+the	O
+unfolded	O
+,	O
+active	O
+state	O
+.	O
+	O
+In	O
+such	O
+an	O
+array	O
+of	O
+molecules	O
+localized	O
+to	O
+a	O
+discrete	O
+region	O
+of	O
+the	O
+membrane	O
+,	O
+it	O
+is	O
+plausible	O
+that	O
+WASP	O
+could	O
+bind	O
+to	O
+a	O
+second	O
+Cdc42	O
+molecule	O
+rather	O
+than	O
+displacing	O
+TOCA1	O
+from	O
+its	O
+cognate	O
+Cdc42	O
+.	O
+	O
+Our	O
+NMR	O
+and	O
+affinity	O
+data	O
+,	O
+however	O
+,	O
+are	O
+consistent	O
+with	O
+displacement	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+by	O
+the	O
+N	O
+-	O
+WASP	O
+GBD	O
+.	O
+	O
+Furthermore	O
+,	O
+TOCA1	O
+is	O
+required	O
+for	O
+Cdc42	O
+-	O
+mediated	O
+activation	O
+of	O
+N	O
+-	O
+WASP	O
+·	O
+WIP	O
+,	O
+implying	O
+that	O
+it	O
+may	O
+not	O
+be	O
+possible	O
+for	O
+Cdc42	O
+to	O
+bind	O
+and	O
+activate	O
+N	O
+-	O
+WASP	O
+prior	O
+to	O
+TOCA1	O
+-	O
+Cdc42	O
+binding	O
+.	O
+	O
+In	O
+light	O
+of	O
+this	O
+,	O
+we	O
+favor	O
+an	O
+“	O
+effector	O
+handover	O
+”	O
+scheme	O
+whereby	O
+TOCA1	O
+interacts	O
+with	O
+Cdc42	O
+prior	O
+to	O
+N	O
+-	O
+WASP	O
+activation	O
+,	O
+after	O
+which	O
+N	O
+-	O
+WASP	O
+displaces	O
+TOCA1	O
+from	O
+its	O
+bound	O
+Cdc42	O
+in	O
+order	O
+to	O
+be	O
+fully	O
+activated	O
+rather	O
+than	O
+binding	O
+a	O
+second	O
+Cdc42	O
+molecule	O
+.	O
+	O
+The	O
+concomitant	O
+release	O
+of	O
+TOCA1	O
+from	O
+Cdc42	O
+while	O
+still	O
+bound	O
+to	O
+N	O
+-	O
+WASP	O
+presumably	O
+enhances	O
+the	O
+ability	O
+of	O
+TOCA1	O
+to	O
+further	O
+activate	O
+N	O
+-	O
+WASP	O
+·	O
+WIP	O
+-	O
+induced	O
+actin	O
+polymerization	O
+.	O
+	O
+Hence	O
+,	O
+actin	O
+polymerization	O
+cannot	O
+occur	O
+until	O
+F	O
+-	O
+BAR	O
+domains	O
+are	O
+poised	O
+for	O
+membrane	O
+distortion	O
+.	O
+	O
+Our	O
+model	O
+of	O
+the	O
+Cdc42	O
+·	O
+HR1TOCA1	O
+complex	O
+indicates	O
+a	O
+mechanism	O
+by	O
+which	O
+such	O
+a	O
+handover	O
+could	O
+take	O
+place	O
+(	O
+Fig	O
+.	O
+9	O
+)	O
+because	O
+it	O
+shows	O
+that	O
+the	O
+effector	O
+binding	O
+sites	O
+only	O
+partially	O
+overlap	O
+on	O
+Cdc42	O
+.	O
+	O
+The	O
+lysine	O
+residues	O
+thought	O
+to	O
+be	O
+involved	O
+in	O
+an	O
+electrostatic	O
+steering	O
+mechanism	O
+in	O
+WASP	O
+-	O
+Cdc42	O
+binding	O
+are	O
+conserved	O
+in	O
+N	O
+-	O
+WASP	O
+and	O
+would	O
+be	O
+able	O
+to	O
+interact	O
+with	O
+Cdc42	O
+even	O
+when	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+is	O
+already	O
+bound	O
+.	O
+	O
+It	O
+has	O
+been	O
+postulated	O
+that	O
+the	O
+initial	O
+interactions	O
+between	O
+this	O
+basic	O
+region	O
+and	O
+Cdc42	O
+could	O
+stabilize	O
+the	O
+active	O
+conformation	O
+of	O
+WASP	O
+,	O
+leading	O
+to	O
+high	O
+affinity	O
+binding	O
+between	O
+the	O
+core	O
+CRIB	O
+and	O
+Cdc42	O
+.	O
+	O
+The	O
+region	O
+C	O
+-	O
+terminal	O
+to	O
+the	O
+core	O
+CRIB	O
+,	O
+required	O
+for	O
+maximal	O
+affinity	O
+binding	O
+,	O
+would	O
+then	O
+fully	O
+displace	O
+the	O
+TOCA1	O
+HR1	O
+.	O
+	O
+A	O
+simplified	O
+model	O
+of	O
+the	O
+early	O
+stages	O
+of	O
+Cdc42	O
+·	O
+N	O
+-	O
+WASP	O
+·	O
+TOCA1	O
+-	O
+dependent	O
+actin	O
+polymerization	O
+.	O
+	O
+Step	O
+2	O
+,	O
+N	O
+-	O
+WASP	O
+exists	O
+in	O
+an	O
+inactive	O
+,	O
+folded	O
+conformation	O
+.	O
+	O
+The	O
+TOCA1	O
+SH3	O
+domain	O
+interacts	O
+with	O
+N	O
+-	O
+WASP	O
+,	O
+causing	O
+an	O
+activatory	O
+allosteric	O
+effect	O
+.	O
+	O
+Step	O
+3	O
+,	O
+electrostatic	O
+interactions	O
+between	O
+Cdc42	O
+and	O
+the	O
+basic	O
+region	O
+upstream	O
+of	O
+the	O
+CRIB	O
+initiate	O
+Cdc42	O
+·	O
+N	O
+-	O
+WASP	O
+binding	O
+.	O
+	O
+The	O
+VCA	O
+domain	O
+is	O
+released	O
+for	O
+downstream	O
+interactions	O
+,	O
+and	O
+actin	O
+polymerization	O
+proceeds	O
+.	O
+	O
+In	O
+conclusion	O
+,	O
+the	O
+data	O
+presented	O
+here	O
+show	O
+that	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+is	O
+sufficient	O
+for	O
+Cdc42	O
+binding	O
+in	O
+vitro	O
+and	O
+that	O
+the	O
+interaction	O
+is	O
+of	O
+micromolar	O
+affinity	O
+,	O
+lower	O
+than	O
+that	O
+of	O
+other	O
+G	O
+protein	O
+-	O
+HR1	O
+domain	O
+interactions	O
+.	O
+	O
+The	O
+analogous	O
+HR1	O
+domains	O
+from	O
+other	O
+TOCA1	O
+family	O
+members	O
+,	O
+FBP17	O
+and	O
+CIP4	O
+,	O
+also	O
+exhibit	O
+micromolar	O
+affinity	O
+for	O
+Cdc42	O
+.	O
+	O
+A	O
+role	O
+for	O
+the	O
+TOCA1	O
+-,	O
+FBP17	O
+-,	O
+and	O
+CIP4	O
+-	O
+Cdc42	O
+interactions	O
+in	O
+the	O
+recruitment	O
+of	O
+these	O
+proteins	O
+to	O
+the	O
+membrane	O
+therefore	O
+appears	O
+unlikely	O
+.	O
+	O
+Instead	O
+,	O
+our	O
+findings	O
+agree	O
+with	O
+earlier	O
+suggestions	O
+that	O
+the	O
+F	O
+-	O
+BAR	O
+domain	O
+is	O
+responsible	O
+for	O
+membrane	O
+recruitment	O
+.	O
+	O
+The	O
+role	O
+of	O
+the	O
+Cdc42	O
+-	O
+TOCA1	O
+interaction	O
+remains	O
+somewhat	O
+elusive	O
+,	O
+but	O
+it	O
+may	O
+serve	O
+to	O
+position	O
+activated	O
+Cdc42	O
+and	O
+N	O
+-	O
+WASP	O
+to	O
+allow	O
+full	O
+activation	O
+of	O
+N	O
+-	O
+WASP	O
+and	O
+as	O
+such	O
+serve	O
+to	O
+couple	O
+F	O
+-	O
+BAR	O
+-	O
+mediated	O
+membrane	O
+deformation	O
+with	O
+N	O
+-	O
+WASP	O
+activation	O
+.	O
+	O
+Our	O
+data	O
+are	O
+therefore	O
+easily	O
+reconciled	O
+with	O
+the	O
+dynamic	O
+instability	O
+models	O
+described	O
+in	O
+relation	O
+to	O
+the	O
+formation	O
+of	O
+endocytic	O
+vesicles	O
+and	O
+with	O
+the	O
+current	O
+data	O
+pertaining	O
+to	O
+the	O
+complex	O
+activation	O
+of	O
+WASP	O
+/	O
+N	O
+-	O
+WASP	O
+pathways	O
+by	O
+allosteric	O
+and	O
+oligomeric	O
+effects	O
+.	O
+	O
+We	O
+therefore	O
+postulate	O
+an	O
+effector	O
+handover	O
+mechanism	O
+based	O
+on	O
+current	O
+evidence	O
+surrounding	O
+WASP	O
+/	O
+N	O
+-	O
+WASP	O
+activation	O
+and	O
+our	O
+model	O
+of	O
+the	O
+Cdc42	O
+·	O
+HR1TOCA1	O
+complex	O
+.	O
+	O
+The	O
+displacement	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+from	O
+Cdc42	O
+by	O
+N	O
+-	O
+WASP	O
+may	O
+represent	O
+a	O
+unidirectional	O
+step	O
+in	O
+the	O
+pathway	O
+of	O
+Cdc42	O
+·	O
+N	O
+-	O
+WASP	O
+·	O
+TOCA1	O
+-	O
+dependent	O
+actin	O
+assembly	O
+.	O
+	O
+The	O
+dynamic	O
+organization	O
+of	O
+fungal	O
+acetyl	O
+-	O
+CoA	O
+carboxylase	O
+	O
+Eukaryotic	O
+ACCs	O
+are	O
+single	O
+-	O
+chain	O
+multienzymes	O
+characterized	O
+by	O
+a	O
+large	O
+,	O
+non	O
+-	O
+catalytic	O
+central	O
+domain	O
+(	O
+CD	O
+),	O
+whose	O
+role	O
+in	O
+ACC	O
+regulation	O
+remains	O
+poorly	O
+characterized	O
+.	O
+	O
+Here	O
+we	O
+report	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+yeast	O
+ACC	O
+CD	O
+,	O
+revealing	O
+a	O
+unique	O
+four	O
+-	O
+domain	O
+organization	O
+.	O
+	O
+A	O
+regulatory	O
+loop	O
+,	O
+which	O
+is	O
+phosphorylated	O
+at	O
+the	O
+key	O
+functional	O
+phosphorylation	O
+site	O
+of	O
+fungal	O
+ACC	O
+,	O
+wedges	O
+into	O
+a	O
+crevice	O
+between	O
+two	O
+domains	O
+of	O
+CD	O
+.	O
+	O
+Acetyl	O
+-	O
+CoA	O
+carboxylases	O
+are	O
+central	O
+regulatory	O
+hubs	O
+of	O
+fatty	O
+acid	O
+metabolism	O
+and	O
+are	O
+important	O
+targets	O
+for	O
+drug	O
+development	O
+in	O
+obesity	O
+and	O
+cancer	O
+.	O
+	O
+Here	O
+,	O
+the	O
+authors	O
+demonstrate	O
+that	O
+the	O
+regulation	O
+of	O
+these	O
+highly	O
+dynamic	O
+enzymes	O
+in	O
+fungi	O
+is	O
+governed	O
+by	O
+a	O
+mechanism	O
+based	O
+on	O
+phosphorylation	O
+-	O
+dependent	O
+conformational	O
+variability	O
+.	O
+	O
+Biotin	O
+-	O
+dependent	O
+acetyl	O
+-	O
+CoA	O
+carboxylases	O
+(	O
+ACCs	O
+)	O
+are	O
+essential	O
+enzymes	O
+that	O
+catalyse	O
+the	O
+ATP	O
+-	O
+dependent	O
+carboxylation	O
+of	O
+acetyl	O
+-	O
+CoA	O
+to	O
+malonyl	O
+-	O
+CoA	O
+.	O
+This	O
+reaction	O
+provides	O
+the	O
+committed	O
+activated	O
+substrate	O
+for	O
+the	O
+biosynthesis	O
+of	O
+fatty	O
+acids	O
+via	O
+fatty	O
+-	O
+acid	O
+synthase	O
+.	O
+	O
+By	O
+catalysing	O
+this	O
+rate	O
+-	O
+limiting	O
+step	O
+in	O
+fatty	O
+-	O
+acid	O
+biosynthesis	O
+,	O
+ACC	O
+plays	O
+a	O
+key	O
+role	O
+in	O
+anabolic	O
+metabolism	O
+.	O
+	O
+ACC	O
+inhibition	O
+and	O
+knock	O
+-	O
+out	O
+studies	O
+show	O
+the	O
+potential	O
+of	O
+targeting	O
+ACC	O
+for	O
+treatment	O
+of	O
+the	O
+metabolic	O
+syndrome	O
+.	O
+	O
+Furthermore	O
+,	O
+elevated	O
+ACC	O
+activity	O
+is	O
+observed	O
+in	O
+malignant	O
+tumours	O
+.	O
+	O
+A	O
+direct	O
+link	O
+between	O
+ACC	O
+and	O
+cancer	O
+is	O
+provided	O
+by	O
+cancer	O
+-	O
+associated	O
+mutations	B-mutant
+in	O
+the	O
+breast	O
+cancer	O
+susceptibility	O
+gene	O
+1	O
+(	O
+BRCA1	O
+),	O
+which	O
+relieve	O
+inhibitory	O
+interactions	O
+of	O
+BRCA1	O
+with	O
+ACC	O
+.	O
+	O
+Thus	O
+,	O
+ACC	O
+is	O
+a	O
+relevant	O
+drug	O
+target	O
+for	O
+type	O
+2	O
+diabetes	O
+and	O
+cancer	O
+.	O
+	O
+Microbial	O
+ACCs	O
+are	O
+also	O
+the	O
+principal	O
+target	O
+of	O
+antifungal	O
+and	O
+antibiotic	O
+compounds	O
+,	O
+such	O
+as	O
+Soraphen	O
+A	O
+.	O
+	O
+The	O
+principal	O
+functional	O
+protein	O
+components	O
+of	O
+ACCs	O
+have	O
+been	O
+described	O
+already	O
+in	O
+the	O
+late	O
+1960s	O
+for	O
+Escherichia	O
+coli	O
+(	O
+E	O
+.	O
+coli	O
+)	O
+ACC	O
+:	O
+Biotin	O
+carboxylase	O
+(	O
+BC	O
+)	O
+catalyses	O
+the	O
+ATP	O
+-	O
+dependent	O
+carboxylation	O
+of	O
+a	O
+biotin	O
+moiety	O
+,	O
+which	O
+is	O
+covalently	O
+linked	O
+to	O
+the	O
+biotin	O
+carboxyl	O
+carrier	O
+protein	O
+(	O
+BCCP	O
+).	O
+	O
+Carboxyltransferase	O
+(	O
+CT	O
+)	O
+transfers	O
+the	O
+activated	O
+carboxyl	O
+group	O
+from	O
+carboxybiotin	O
+to	O
+acetyl	O
+-	O
+CoA	O
+to	O
+yield	O
+malonyl	O
+-	O
+CoA	O
+.	O
+Prokaryotic	O
+ACCs	O
+are	O
+transient	O
+assemblies	O
+of	O
+individual	O
+BC	O
+,	O
+CT	O
+and	O
+BCCP	O
+subunits	O
+.	O
+	O
+Eukaryotic	O
+ACCs	O
+,	O
+instead	O
+,	O
+are	O
+multienzymes	O
+,	O
+which	O
+integrate	O
+all	O
+functional	O
+components	O
+into	O
+a	O
+single	O
+polypeptide	O
+chain	O
+of	O
+∼	O
+2	O
+,	O
+300	O
+amino	O
+acids	O
+.	O
+	O
+Human	O
+ACC	O
+occurs	O
+in	O
+two	O
+closely	O
+related	O
+isoforms	O
+,	O
+ACC1	O
+and	O
+2	O
+,	O
+located	O
+in	O
+the	O
+cytosol	O
+and	O
+at	O
+the	O
+outer	O
+mitochondrial	O
+membrane	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+CD	O
+comprises	O
+one	O
+-	O
+third	O
+of	O
+the	O
+protein	O
+and	O
+is	O
+a	O
+unique	O
+feature	O
+of	O
+eukaryotic	O
+ACCs	O
+without	O
+homologues	O
+in	O
+other	O
+proteins	O
+.	O
+	O
+The	O
+BT	O
+domain	O
+has	O
+been	O
+visualized	O
+in	O
+bacterial	O
+carboxylases	O
+,	O
+where	O
+it	O
+mediates	O
+contacts	O
+between	O
+α	O
+-	O
+and	O
+β	O
+-	O
+subunits	O
+.	O
+	O
+Structural	O
+studies	O
+on	O
+the	O
+functional	O
+architecture	O
+of	O
+intact	O
+ACCs	O
+have	O
+been	O
+hindered	O
+by	O
+their	O
+huge	O
+size	O
+and	O
+pronounced	O
+dynamics	O
+,	O
+as	O
+well	O
+as	O
+the	O
+transient	O
+assembly	O
+mode	O
+of	O
+bacterial	O
+ACCs	O
+.	O
+	O
+However	O
+,	O
+crystal	O
+structures	O
+of	O
+individual	O
+components	O
+or	O
+domains	O
+from	O
+prokaryotic	O
+and	O
+eukaryotic	O
+ACCs	O
+,	O
+respectively	O
+,	O
+have	O
+been	O
+solved	O
+.	O
+	O
+The	O
+structure	O
+determination	O
+of	O
+the	O
+holoenzymes	O
+of	O
+bacterial	O
+biotin	O
+-	O
+dependent	O
+carboxylases	O
+,	O
+which	O
+lack	O
+the	O
+characteristic	O
+CD	O
+,	O
+such	O
+as	O
+the	O
+pyruvate	O
+carboxylase	O
+(	O
+PC	O
+),	O
+propionyl	O
+-	O
+CoA	O
+carboxylase	O
+,	O
+3	O
+-	O
+methyl	O
+-	O
+crotonyl	O
+-	O
+CoA	O
+carboxylase	O
+and	O
+a	O
+long	O
+-	O
+chain	O
+acyl	O
+-	O
+CoA	O
+carboxylase	O
+revealed	O
+strikingly	O
+divergent	O
+architectures	O
+despite	O
+a	O
+general	O
+conservation	O
+of	O
+all	O
+functional	O
+components	O
+.	O
+	O
+In	O
+these	O
+structures	O
+,	O
+the	O
+BC	O
+and	O
+CT	O
+active	O
+sites	O
+are	O
+at	O
+distances	O
+between	O
+40	O
+and	O
+80	O
+Å	O
+,	O
+such	O
+that	O
+substrate	O
+transfer	O
+could	O
+be	O
+mediated	O
+solely	O
+by	O
+the	O
+mobility	O
+of	O
+the	O
+flexibly	O
+tethered	O
+BCCP	O
+.	O
+	O
+Human	O
+ACC1	O
+is	O
+regulated	O
+allosterically	O
+,	O
+via	O
+specific	O
+protein	O
+–	O
+protein	O
+interactions	O
+,	O
+and	O
+by	O
+reversible	O
+phosphorylation	O
+.	O
+	O
+Dynamic	O
+polymerization	O
+of	O
+human	O
+ACC1	O
+is	O
+linked	O
+to	O
+increased	O
+activity	O
+and	O
+is	O
+regulated	O
+allosterically	O
+by	O
+the	O
+activator	O
+citrate	O
+and	O
+the	O
+inhibitor	O
+palmitate	O
+,	O
+or	O
+by	O
+binding	O
+of	O
+the	O
+small	O
+protein	O
+MIG	O
+-	O
+12	O
+(	O
+ref	O
+.).	O
+	O
+Human	O
+ACC1	O
+is	O
+further	O
+regulated	O
+by	O
+specific	O
+phosphorylation	O
+-	O
+dependent	O
+binding	O
+of	O
+BRCA1	O
+to	O
+Ser1263	O
+in	O
+the	O
+CD	O
+.	O
+	O
+Furthermore	O
+,	O
+phosphorylation	O
+by	O
+AMP	O
+-	O
+activated	O
+protein	O
+kinase	O
+(	O
+AMPK	O
+)	O
+and	O
+cAMP	O
+-	O
+dependent	O
+protein	O
+kinase	O
+(	O
+PKA	O
+)	O
+leads	O
+to	O
+a	O
+decrease	O
+in	O
+ACC1	O
+activity	O
+.	O
+	O
+AMPK	O
+phosphorylates	O
+ACC1	O
+in	O
+vitro	O
+at	O
+Ser80	O
+,	O
+Ser1201	O
+and	O
+Ser1216	O
+and	O
+PKA	O
+at	O
+Ser78	O
+and	O
+Ser1201	O
+.	O
+	O
+However	O
+,	O
+regulatory	O
+effects	O
+on	O
+ACC1	O
+activity	O
+are	O
+mainly	O
+mediated	O
+by	O
+phosphorylation	O
+of	O
+Ser80	O
+and	O
+Ser1201	O
+(	O
+refs	O
+).	O
+	O
+Phosphorylated	O
+Ser80	O
+,	O
+which	O
+is	O
+highly	O
+conserved	O
+only	O
+in	O
+higher	O
+eukaryotes	O
+,	O
+presumably	O
+binds	O
+into	O
+the	O
+Soraphen	O
+A	O
+-	O
+binding	O
+pocket	O
+.	O
+	O
+The	O
+regulatory	O
+Ser1201	O
+shows	O
+only	O
+moderate	O
+conservation	O
+across	O
+higher	O
+eukaryotes	O
+,	O
+while	O
+the	O
+phosphorylated	O
+Ser1216	O
+is	O
+highly	O
+conserved	O
+across	O
+all	O
+eukaryotes	O
+.	O
+	O
+However	O
+,	O
+no	O
+effect	O
+of	O
+Ser1216	O
+phosphorylation	O
+on	O
+ACC	O
+activity	O
+has	O
+been	O
+reported	O
+in	O
+higher	O
+eukaryotes	O
+.	O
+	O
+For	O
+fungal	O
+ACC	O
+,	O
+neither	O
+spontaneous	O
+nor	O
+inducible	O
+polymerization	O
+has	O
+been	O
+detected	O
+despite	O
+considerable	O
+sequence	O
+conservation	O
+to	O
+human	O
+ACC1	O
+.	O
+	O
+The	O
+BRCA1	O
+-	O
+interacting	O
+phosphoserine	O
+position	O
+is	O
+not	O
+conserved	O
+in	O
+fungal	O
+ACC	O
+,	O
+and	O
+no	O
+other	O
+phospho	O
+-	O
+dependent	O
+protein	O
+–	O
+protein	O
+interactions	O
+of	O
+fungal	O
+ACC	O
+have	O
+been	O
+described	O
+.	O
+	O
+In	O
+yeast	O
+ACC	O
+,	O
+phosphorylation	O
+sites	O
+have	O
+been	O
+identified	O
+at	O
+Ser2	O
+,	O
+Ser735	O
+,	O
+Ser1148	O
+,	O
+Ser1157	O
+and	O
+Ser1162	O
+(	O
+ref	O
+.).	O
+	O
+Despite	O
+the	O
+outstanding	O
+relevance	O
+of	O
+ACC	O
+in	O
+primary	O
+metabolism	O
+and	O
+disease	O
+,	O
+the	O
+dynamic	O
+organization	O
+and	O
+regulation	O
+of	O
+the	O
+giant	O
+eukaryotic	O
+,	O
+and	O
+in	O
+particular	O
+fungal	O
+ACC	O
+,	O
+remain	O
+poorly	O
+characterized	O
+.	O
+	O
+Here	O
+we	O
+provide	O
+the	O
+structure	O
+of	O
+Saccharomyces	O
+cerevisiae	O
+(	O
+Sce	O
+)	O
+ACC	O
+CD	O
+,	O
+intermediate	O
+-	O
+and	O
+low	O
+-	O
+resolution	O
+structures	O
+of	O
+human	O
+(	O
+Hsa	O
+)	O
+ACC	O
+CD	O
+and	O
+larger	B-mutant
+fragments	I-mutant
+of	O
+fungal	O
+ACC	O
+from	O
+Chaetomium	O
+thermophilum	O
+(	O
+Cth	O
+;	O
+Fig	O
+.	O
+1a	O
+).	O
+	O
+The	O
+overall	O
+extent	O
+of	O
+the	O
+SceCD	O
+is	O
+70	O
+by	O
+75	O
+Å	O
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+,	O
+b	O
+),	O
+and	O
+the	O
+attachment	O
+points	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+26	O
+-	O
+residue	O
+linker	O
+to	O
+the	O
+BCCP	O
+domain	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+CT	O
+domain	O
+are	O
+separated	O
+by	O
+46	O
+Å	O
+(	O
+the	O
+N	O
+-	O
+and	O
+C	O
+termini	O
+are	O
+indicated	O
+with	O
+spheres	O
+in	O
+Fig	O
+.	O
+1b	O
+).	O
+	O
+CDN	O
+adopts	O
+a	O
+letter	O
+C	O
+shape	O
+,	O
+where	O
+one	O
+of	O
+the	O
+ends	O
+is	O
+a	O
+regular	O
+four	O
+-	O
+helix	O
+bundle	O
+(	O
+Nα3	O
+-	O
+6	O
+),	O
+the	O
+other	O
+end	O
+is	O
+a	O
+helical	O
+hairpin	O
+(	O
+Nα8	O
+,	O
+9	O
+)	O
+and	O
+the	O
+bridging	O
+region	O
+comprises	O
+six	O
+helices	O
+(	O
+Nα1	O
+,	O
+2	O
+,	O
+7	O
+,	O
+10	O
+–	O
+12	O
+).	O
+	O
+CDL	O
+is	O
+composed	O
+of	O
+a	O
+small	O
+,	O
+irregular	O
+four	O
+-	O
+helix	O
+bundle	O
+(	O
+Lα1	O
+–	O
+4	O
+)	O
+and	O
+tightly	O
+interacts	O
+with	O
+the	O
+open	O
+face	O
+of	O
+CDC1	O
+via	O
+an	O
+interface	O
+of	O
+1	O
+,	O
+300	O
+Å2	O
+involving	O
+helices	O
+Lα3	O
+and	O
+Lα4	O
+.	O
+	O
+CDC2	O
+is	O
+extended	O
+at	O
+its	O
+C	O
+terminus	O
+by	O
+an	O
+additional	O
+β	O
+-	O
+strand	O
+and	O
+an	O
+irregular	O
+β	O
+-	O
+hairpin	O
+.	O
+	O
+A	O
+regulatory	O
+loop	O
+mediates	O
+interdomain	O
+interactions	O
+	O
+In	O
+insect	O
+-	O
+cell	O
+-	O
+expressed	O
+full	O
+-	O
+length	O
+SceACC	O
+,	O
+the	O
+highly	O
+conserved	O
+Ser1157	O
+is	O
+the	O
+only	O
+fully	O
+occupied	O
+phosphorylation	O
+site	O
+with	O
+functional	O
+relevance	O
+in	O
+S	O
+.	O
+cerevisiae	O
+.	O
+	O
+Additional	O
+phosphorylation	O
+was	O
+detected	O
+for	O
+Ser2101	O
+and	O
+Tyr2179	O
+;	O
+however	O
+,	O
+these	O
+sites	O
+are	O
+neither	O
+conserved	O
+across	O
+fungal	O
+ACC	O
+nor	O
+natively	O
+phosphorylated	O
+in	O
+yeast	O
+.	O
+	O
+MS	O
+analysis	O
+of	O
+dissolved	O
+crystals	O
+confirmed	O
+the	O
+phosphorylated	O
+state	O
+of	O
+Ser1157	O
+also	O
+in	O
+SceCD	O
+crystals	O
+.	O
+	O
+In	O
+the	O
+SceCD	O
+crystal	O
+structure	O
+,	O
+the	O
+phosphorylated	O
+Ser1157	O
+resides	O
+in	O
+a	O
+regulatory	O
+36	O
+-	O
+amino	O
+-	O
+acid	O
+loop	O
+between	O
+strands	O
+β2	O
+and	O
+β3	O
+of	O
+CDC1	O
+(	O
+Fig	O
+.	O
+1b	O
+,	O
+d	O
+),	O
+which	O
+contains	O
+two	O
+additional	O
+less	O
+-	O
+conserved	O
+phosphorylation	O
+sites	O
+(	O
+Ser1148	O
+and	O
+Ser1162	O
+)	O
+confirmed	O
+in	O
+yeast	O
+,	O
+but	O
+not	O
+occupied	O
+here	O
+.	O
+	O
+This	O
+regulatory	O
+loop	O
+wedges	O
+between	O
+the	O
+CDC1	O
+and	O
+CDC2	O
+domains	O
+and	O
+provides	O
+the	O
+largest	O
+contribution	O
+to	O
+the	O
+interdomain	O
+interface	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+regulatory	O
+loop	O
+also	O
+directly	O
+contacts	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+CDC2	O
+leading	O
+into	O
+CT	O
+.	O
+	O
+Phosphoserine	O
+1157	O
+is	O
+tightly	O
+bound	O
+by	O
+two	O
+highly	O
+conserved	O
+arginines	O
+(	O
+Arg1173	O
+and	O
+Arg1260	O
+)	O
+of	O
+CDC1	O
+(	O
+Fig	O
+.	O
+1d	O
+).	O
+	O
+Already	O
+the	O
+binding	O
+of	O
+phosphorylated	O
+Ser1157	O
+apparently	O
+stabilizes	O
+the	O
+regulatory	O
+loop	O
+conformation	O
+;	O
+the	O
+accessory	O
+phosphorylation	O
+sites	O
+Ser1148	O
+and	O
+Ser1162	O
+in	O
+the	O
+same	O
+loop	O
+may	O
+further	O
+modulate	O
+the	O
+strength	O
+of	O
+interaction	O
+between	O
+the	O
+regulatory	O
+loop	O
+and	O
+the	O
+CDC1	O
+and	O
+CDC2	O
+domains	O
+.	O
+	O
+Phosphorylation	O
+of	O
+the	O
+regulatory	O
+loop	O
+thus	O
+determines	O
+interdomain	O
+interactions	O
+of	O
+CDC1	O
+and	O
+CDC2	O
+,	O
+suggesting	O
+that	O
+it	O
+may	O
+exert	O
+its	O
+regulatory	O
+function	O
+by	O
+modifying	O
+the	O
+overall	O
+structure	O
+and	O
+dynamics	O
+of	O
+the	O
+CD	O
+.	O
+	O
+The	O
+functional	O
+role	O
+of	O
+Ser1157	O
+was	O
+confirmed	O
+by	O
+an	O
+activity	O
+assay	O
+based	O
+on	O
+the	O
+incorporation	O
+of	O
+radioactive	O
+carbonate	O
+into	O
+acid	O
+non	O
+-	O
+volatile	O
+material	O
+.	O
+	O
+The	O
+values	O
+obtained	O
+for	O
+dephosphorylated	O
+SceACC	O
+are	O
+comparable	O
+to	O
+earlier	O
+measurements	O
+of	O
+non	O
+-	O
+phosphorylated	O
+yeast	O
+ACC	O
+expressed	O
+in	O
+E	O
+.	O
+coli	O
+.	O
+	O
+The	O
+variable	O
+CD	O
+is	O
+conserved	O
+between	O
+yeast	O
+and	O
+human	O
+	O
+To	O
+compare	O
+the	O
+organization	O
+of	O
+fungal	O
+and	O
+human	O
+ACC	O
+CD	O
+,	O
+we	O
+determined	O
+the	O
+structure	O
+of	O
+a	O
+human	O
+ACC1	B-mutant
+fragment	I-mutant
+that	O
+comprises	O
+the	O
+BT	O
+and	O
+CD	O
+domains	O
+(	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+),	O
+but	O
+lacks	O
+the	O
+mobile	O
+BCCP	O
+in	O
+between	O
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+	O
+An	O
+experimentally	O
+phased	O
+map	O
+was	O
+obtained	O
+at	O
+3	O
+.	O
+7	O
+Å	O
+resolution	O
+for	O
+a	O
+cadmium	O
+-	O
+derivatized	O
+crystal	O
+and	O
+was	O
+interpreted	O
+by	O
+a	O
+poly	O
+-	O
+alanine	O
+model	O
+(	O
+Fig	O
+.	O
+1e	O
+and	O
+Table	O
+1	O
+).	O
+	O
+With	O
+CDL	O
+/	O
+CDC1	O
+superposed	O
+,	O
+CDN	O
+in	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+is	O
+rotated	O
+by	O
+160	O
+°	O
+around	O
+a	O
+hinge	O
+at	O
+the	O
+connection	O
+of	O
+CDN	O
+/	O
+CDL	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+).	O
+	O
+This	O
+rotation	O
+displaces	O
+the	O
+N	O
+terminus	O
+of	O
+CDN	O
+in	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+by	O
+51	O
+Å	O
+compared	O
+with	O
+SceCD	O
+,	O
+resulting	O
+in	O
+a	O
+separation	O
+of	O
+the	O
+attachment	O
+points	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+linker	O
+to	O
+the	O
+BCCP	O
+domain	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+CT	O
+domain	O
+by	O
+67	O
+Å	O
+(	O
+the	O
+attachment	O
+points	O
+are	O
+indicated	O
+with	O
+spheres	O
+in	O
+Fig	O
+.	O
+1e	O
+).	O
+	O
+The	O
+BT	O
+domain	O
+of	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+consists	O
+of	O
+a	O
+helix	O
+that	O
+is	O
+surrounded	O
+at	O
+its	O
+N	O
+terminus	O
+by	O
+an	O
+antiparallel	O
+eight	O
+-	O
+stranded	O
+β	O
+-	O
+barrel	O
+.	O
+	O
+On	O
+the	O
+basis	O
+of	O
+MS	O
+analysis	O
+of	O
+insect	O
+-	O
+cell	O
+-	O
+expressed	O
+human	O
+full	O
+-	O
+length	O
+ACC	O
+,	O
+Ser80	O
+shows	O
+the	O
+highest	O
+degree	O
+of	O
+phosphorylation	O
+(	O
+90	O
+%).	O
+	O
+Ser29	O
+and	O
+Ser1263	O
+,	O
+implicated	O
+in	O
+insulin	O
+-	O
+dependent	O
+phosphorylation	O
+and	O
+BRCA1	O
+binding	O
+,	O
+respectively	O
+,	O
+are	O
+phosphorylated	O
+at	O
+intermediate	O
+levels	O
+(	O
+40	O
+%).	O
+	O
+The	O
+highly	O
+conserved	O
+Ser1216	O
+(	O
+corresponding	O
+to	O
+S	O
+.	O
+cerevisiae	O
+Ser1157	O
+),	O
+as	O
+well	O
+as	O
+Ser1201	O
+,	O
+both	O
+in	O
+the	O
+regulatory	O
+loop	O
+discussed	O
+above	O
+,	O
+are	O
+not	O
+phosphorylated	O
+.	O
+	O
+However	O
+,	O
+residual	O
+phosphorylation	O
+levels	O
+were	O
+detected	O
+for	O
+Ser1204	O
+(	O
+7	O
+%)	O
+and	O
+Ser1218	O
+(	O
+7	O
+%)	O
+in	O
+the	O
+same	O
+loop	O
+.	O
+	O
+MS	O
+analysis	O
+of	O
+the	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+crystallization	O
+sample	O
+reveals	O
+partial	O
+proteolytic	O
+digestion	O
+of	O
+the	O
+regulatory	O
+loop	O
+.	O
+	O
+Accordingly	O
+,	O
+most	O
+of	O
+this	O
+loop	O
+is	O
+not	O
+represented	O
+in	O
+the	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+crystal	O
+structure	O
+.	O
+	O
+Besides	O
+the	O
+regulatory	O
+loop	O
+,	O
+also	O
+the	O
+phosphopeptide	O
+target	O
+region	O
+for	O
+BRCA1	O
+interaction	O
+is	O
+not	O
+resolved	O
+presumably	O
+because	O
+of	O
+pronounced	O
+flexibility	O
+.	O
+	O
+At	O
+the	O
+level	O
+of	O
+isolated	O
+yeast	O
+and	O
+human	O
+CD	O
+,	O
+the	O
+structural	O
+analysis	O
+indicates	O
+the	O
+presence	O
+of	O
+at	O
+least	O
+two	O
+hinges	O
+,	O
+one	O
+with	O
+large	O
+-	O
+scale	O
+flexibility	O
+at	O
+the	O
+CDN	O
+/	O
+CDL	O
+connection	O
+,	O
+and	O
+one	O
+with	O
+tunable	O
+plasticity	O
+between	O
+CDL	O
+/	O
+CDC1	O
+and	O
+CDC2	O
+,	O
+plausibly	O
+affected	O
+by	O
+phosphorylation	O
+in	O
+the	O
+regulatory	O
+loop	O
+region	O
+.	O
+	O
+The	O
+integration	O
+of	O
+CD	O
+into	O
+the	O
+fungal	O
+ACC	O
+multienzyme	O
+	O
+Using	O
+molecular	O
+replacement	O
+based	O
+on	O
+fungal	O
+ACC	O
+CD	O
+and	O
+CT	O
+models	O
+,	O
+we	O
+obtained	O
+structures	O
+of	O
+a	O
+variant	B-mutant
+comprising	O
+CthCT	O
+and	O
+CDC1	O
+/	O
+CDC2	O
+in	O
+two	O
+crystal	O
+forms	O
+at	O
+resolutions	O
+of	O
+3	O
+.	O
+6	O
+and	O
+4	O
+.	O
+5	O
+Å	O
+(	O
+CthCD	B-mutant
+-	I-mutant
+CTCter1	I-mutant
+/	I-mutant
+2	I-mutant
+),	O
+respectively	O
+,	O
+as	O
+well	O
+as	O
+of	O
+a	O
+CthCT	O
+linked	O
+to	O
+the	O
+entire	O
+CD	O
+at	O
+7	O
+.	O
+2	O
+Å	O
+resolution	O
+(	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+;	O
+Figs	O
+1a	O
+and	O
+2	O
+,	O
+Table	O
+1	O
+).	O
+	O
+For	O
+CthΔBCCP	B-mutant
+,	O
+crystals	O
+diffracting	O
+to	O
+8	O
+.	O
+4	O
+Å	O
+resolution	O
+were	O
+obtained	O
+.	O
+	O
+Owing	O
+to	O
+the	O
+limited	O
+resolution	O
+the	O
+discussion	O
+of	O
+structures	O
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+CthΔBCCP	B-mutant
+is	O
+restricted	O
+to	O
+the	O
+analysis	O
+of	O
+domain	O
+localization	O
+.	O
+	O
+Still	O
+,	O
+these	O
+structures	O
+contribute	O
+considerably	O
+to	O
+the	O
+visualization	O
+of	O
+an	O
+intrinsically	O
+dynamic	O
+fungal	O
+ACC	O
+.	O
+	O
+In	O
+all	O
+these	O
+crystal	O
+structures	O
+,	O
+the	O
+CT	O
+domains	O
+build	O
+a	O
+canonical	O
+head	O
+-	O
+to	O
+-	O
+tail	O
+dimer	O
+,	O
+with	O
+active	O
+sites	O
+formed	O
+by	O
+contributions	O
+from	O
+both	O
+protomers	O
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+).	O
+	O
+The	O
+connection	O
+of	O
+CD	O
+and	O
+CT	O
+is	O
+provided	O
+by	O
+a	O
+10	O
+-	O
+residue	O
+peptide	O
+stretch	O
+,	O
+which	O
+links	O
+the	O
+N	O
+terminus	O
+of	O
+CT	O
+to	O
+the	O
+irregular	O
+β	O
+-	O
+hairpin	O
+/	O
+β	O
+-	O
+strand	O
+extension	O
+of	O
+CDC2	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+).	O
+	O
+CD	O
+/	O
+CT	O
+contacts	O
+are	O
+only	O
+formed	O
+in	O
+direct	O
+vicinity	O
+of	O
+the	O
+covalent	O
+linkage	O
+and	O
+involve	O
+the	O
+β	O
+-	O
+hairpin	O
+extension	O
+of	O
+CDC2	O
+as	O
+well	O
+as	O
+the	O
+loop	O
+between	O
+strands	O
+β2	O
+/	O
+β3	O
+of	O
+the	O
+CT	O
+N	O
+-	O
+lobe	O
+,	O
+which	O
+contains	O
+a	O
+conserved	O
+RxxGxN	O
+motif	O
+.	O
+	O
+The	O
+neighbouring	O
+loop	O
+on	O
+the	O
+CT	O
+side	O
+(	O
+between	O
+CT	O
+β1	O
+/	O
+β2	O
+)	O
+is	O
+displaced	O
+by	O
+2	O
+.	O
+5	O
+Å	O
+compared	O
+to	O
+isolated	O
+CT	O
+structures	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3c	O
+).	O
+	O
+On	O
+the	O
+basis	O
+of	O
+an	O
+interface	O
+area	O
+of	O
+∼	O
+600	O
+Å2	O
+and	O
+its	O
+edge	O
+-	O
+to	O
+-	O
+edge	O
+connection	O
+characteristics	O
+,	O
+the	O
+interface	O
+between	O
+CT	O
+and	O
+CD	O
+might	O
+be	O
+classified	O
+as	O
+conformationally	O
+variable	O
+.	O
+	O
+The	O
+CDC2	O
+/	O
+CT	O
+interface	O
+acts	O
+as	O
+a	O
+true	O
+hinge	O
+with	O
+observed	O
+rotation	O
+up	O
+to	O
+16	O
+°,	O
+which	O
+results	O
+in	O
+a	O
+translocation	O
+of	O
+the	O
+distal	O
+end	O
+of	O
+CDC2	O
+by	O
+8	O
+Å	O
+.	O
+	O
+The	O
+interface	O
+between	O
+CDC2	O
+and	O
+CDL	O
+/	O
+CDC1	O
+,	O
+which	O
+is	O
+mediated	O
+by	O
+the	O
+phosphorylated	O
+regulatory	O
+loop	O
+in	O
+the	O
+SceCD	O
+structure	O
+,	O
+is	O
+less	O
+variable	O
+than	O
+the	O
+CD	O
+–	O
+CT	O
+junction	O
+,	O
+and	O
+permits	O
+only	O
+limited	O
+rotation	O
+and	O
+tilting	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+	O
+Analysis	O
+of	O
+the	O
+impact	O
+of	O
+phosphorylation	O
+on	O
+the	O
+interface	O
+between	O
+CDC2	O
+and	O
+CDL	O
+/	O
+CDC1	O
+in	O
+CthACC	B-mutant
+variant	I-mutant
+structures	O
+is	O
+precluded	O
+by	O
+the	O
+limited	O
+crystallographic	O
+resolution	O
+.	O
+	O
+However	O
+,	O
+MS	O
+analysis	O
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+CthΔBCCP	B-mutant
+constructs	O
+revealed	O
+between	O
+60	O
+and	O
+70	O
+%	O
+phosphorylation	O
+of	O
+Ser1170	O
+(	O
+corresponding	O
+to	O
+SceACC	O
+Ser1157	O
+).	O
+	O
+The	O
+CDN	O
+domain	O
+positioning	O
+relative	O
+to	O
+CDL	O
+/	O
+CDC1	O
+is	O
+highly	O
+variable	O
+with	O
+three	O
+main	O
+orientations	O
+observed	O
+in	O
+the	O
+structures	O
+of	O
+SceCD	O
+and	O
+the	O
+larger	B-mutant
+CthACC	I-mutant
+fragments	I-mutant
+:	O
+CDN	O
+tilts	O
+,	O
+resulting	O
+in	O
+a	O
+displacement	O
+of	O
+its	O
+N	O
+terminus	O
+by	O
+23	O
+Å	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+observed	O
+in	O
+both	O
+protomers	O
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+one	O
+protomer	O
+of	O
+CthΔBCCP	B-mutant
+,	O
+denoted	O
+as	O
+CthCD	B-mutant
+-	I-mutant
+CT1	I-mutant
+/	I-mutant
+2	I-mutant
+and	O
+CthΔBCCP1	B-mutant
+,	O
+respectively	O
+).	O
+	O
+In	O
+addition	O
+,	O
+CDN	O
+can	O
+rotate	O
+around	O
+hinges	O
+in	O
+the	O
+connection	O
+between	O
+CDN	O
+/	O
+CDL	O
+by	O
+70	O
+°	O
+(	O
+Fig	O
+.	O
+4b	O
+,	O
+observed	O
+in	O
+the	O
+second	O
+protomer	O
+of	O
+CthΔBCCP	B-mutant
+,	O
+denoted	O
+as	O
+CthΔBCCP2	B-mutant
+)	O
+and	O
+160	O
+°	O
+(	O
+Fig	O
+.	O
+4c	O
+,	O
+observed	O
+in	O
+SceCD	O
+)	O
+leading	O
+to	O
+displacement	O
+of	O
+the	O
+anchor	O
+site	O
+for	O
+the	O
+BCCP	O
+linker	O
+by	O
+up	O
+to	O
+33	O
+and	O
+40	O
+Å	O
+,	O
+respectively	O
+.	O
+	O
+On	O
+the	O
+basis	O
+of	O
+the	O
+occurrence	O
+of	O
+related	O
+conformational	O
+changes	O
+between	O
+fungal	O
+and	O
+human	O
+ACC	B-mutant
+fragments	I-mutant
+,	O
+the	O
+observed	O
+set	O
+of	O
+conformations	O
+may	O
+well	O
+represent	O
+general	O
+states	O
+present	O
+in	O
+all	O
+eukaryotic	O
+ACCs	O
+.	O
+	O
+Large	O
+-	O
+scale	O
+conformational	O
+variability	O
+of	O
+fungal	O
+ACC	O
+	O
+To	O
+obtain	O
+a	O
+comprehensive	O
+view	O
+of	O
+fungal	O
+ACC	O
+dynamics	O
+in	O
+solution	O
+,	O
+we	O
+employed	O
+SAXS	O
+and	O
+EM	O
+.	O
+	O
+The	O
+smooth	O
+appearance	O
+of	O
+scattering	O
+curves	O
+and	O
+derived	O
+distance	O
+distributions	O
+might	O
+indicate	O
+substantial	O
+interdomain	O
+flexibility	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+–	O
+c	O
+).	O
+	O
+Direct	O
+observation	O
+of	O
+individual	O
+full	O
+-	O
+length	O
+CthACC	O
+particles	O
+,	O
+according	O
+to	O
+MS	O
+results	O
+predominantly	O
+in	O
+a	O
+phosphorylated	O
+low	O
+-	O
+activity	O
+state	O
+,	O
+in	O
+negative	O
+stain	O
+EM	O
+reveals	O
+a	O
+large	O
+set	O
+of	O
+conformations	O
+from	O
+rod	O
+-	O
+like	O
+extended	O
+to	O
+U	O
+-	O
+shaped	O
+particles	O
+.	O
+	O
+Class	O
+averages	O
+,	O
+obtained	O
+by	O
+maximum	O
+-	O
+likelihood	O
+-	O
+based	O
+two	O
+-	O
+dimensional	O
+(	O
+2D	O
+)	O
+classification	O
+,	O
+are	O
+focused	O
+on	O
+the	O
+dimeric	O
+CT	O
+domain	O
+and	O
+the	O
+full	O
+BC	B-mutant
+–	I-mutant
+BCCP	I-mutant
+–	I-mutant
+CD	I-mutant
+domain	O
+of	O
+only	O
+one	O
+protomer	O
+,	O
+due	O
+to	O
+the	O
+non	O
+-	O
+coordinated	O
+motions	O
+of	O
+the	O
+lateral	O
+BC	O
+/	O
+CD	O
+regions	O
+relative	O
+to	O
+the	O
+CT	O
+dimer	O
+.	O
+	O
+They	O
+identify	O
+the	O
+connections	O
+between	O
+CDN	O
+/	O
+CDL	O
+and	O
+between	O
+CDC2	O
+/	O
+CT	O
+as	O
+major	O
+contributors	O
+to	O
+conformational	O
+heterogeneity	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+	O
+The	O
+BC	O
+domain	O
+is	O
+not	O
+completely	O
+disordered	O
+,	O
+but	O
+laterally	O
+attached	O
+to	O
+BT	O
+/	O
+CDN	O
+in	O
+a	O
+generally	O
+conserved	O
+position	O
+,	O
+albeit	O
+with	O
+increased	O
+flexibility	O
+.	O
+	O
+Furthermore	O
+,	O
+based	O
+on	O
+an	O
+average	O
+length	O
+of	O
+the	O
+BCCP	O
+–	O
+CD	O
+linker	O
+in	O
+fungal	O
+ACC	O
+of	O
+26	O
+amino	O
+acids	O
+,	O
+mobility	O
+of	O
+the	O
+BCCP	O
+alone	O
+would	O
+not	O
+be	O
+sufficient	O
+to	O
+bridge	O
+the	O
+active	O
+sites	O
+of	O
+BC	O
+and	O
+CT	O
+.	O
+	O
+The	O
+most	O
+relevant	O
+candidate	O
+site	O
+for	O
+mediating	O
+such	O
+additional	O
+flexibility	O
+and	O
+permitting	O
+an	O
+extended	O
+set	O
+of	O
+conformations	O
+is	O
+the	O
+CDC1	O
+/	O
+CDC2	O
+interface	O
+,	O
+which	O
+is	O
+rigidified	O
+by	O
+the	O
+Ser1157	O
+-	O
+phosphorylated	O
+regulatory	O
+loop	O
+,	O
+as	O
+depicted	O
+in	O
+the	O
+SceCD	O
+crystal	O
+structure	O
+.	O
+	O
+Altogether	O
+,	O
+the	O
+architecture	O
+of	O
+fungal	O
+ACC	O
+is	O
+based	O
+on	O
+the	O
+central	O
+dimeric	O
+CT	O
+domain	O
+(	O
+Fig	O
+.	O
+4d	O
+).	O
+	O
+The	O
+CD	O
+has	O
+no	O
+direct	O
+role	O
+in	O
+substrate	O
+recognition	O
+or	O
+catalysis	O
+but	O
+contributes	O
+to	O
+the	O
+regulation	O
+of	O
+all	O
+eukaryotic	O
+ACCs	O
+.	O
+	O
+In	O
+higher	O
+eukaryotic	O
+ACCs	O
+,	O
+regulation	O
+via	O
+phosphorylation	O
+is	O
+achieved	O
+by	O
+combining	O
+the	O
+effects	O
+of	O
+phosphorylation	O
+at	O
+Ser80	O
+,	O
+Ser1201	O
+and	O
+Ser1263	O
+.	O
+	O
+In	O
+fungal	O
+ACC	O
+,	O
+however	O
+,	O
+Ser1157	O
+in	O
+the	O
+regulatory	O
+loop	O
+of	O
+the	O
+CD	O
+is	O
+the	O
+only	O
+phosphorylation	O
+site	O
+that	O
+has	O
+been	O
+demonstrated	O
+to	O
+be	O
+both	O
+phosphorylated	O
+in	O
+vivo	O
+and	O
+involved	O
+in	O
+the	O
+regulation	O
+of	O
+ACC	O
+activity	O
+.	O
+	O
+In	O
+its	O
+phosphorylated	O
+state	O
+,	O
+the	O
+regulatory	O
+loop	O
+containing	O
+Ser1157	O
+wedges	O
+between	O
+CDC1	O
+/	O
+CDC2	O
+and	O
+presumably	O
+limits	O
+the	O
+conformational	O
+freedom	O
+at	O
+this	O
+interdomain	O
+interface	O
+.	O
+	O
+However	O
+,	O
+flexibility	O
+at	O
+this	O
+hinge	O
+may	O
+be	O
+required	O
+for	O
+full	O
+ACC	O
+activity	O
+,	O
+as	O
+the	O
+distances	O
+between	O
+the	O
+BCCP	O
+anchor	O
+points	O
+and	O
+the	O
+active	O
+sites	O
+of	O
+BC	O
+and	O
+CT	O
+observed	O
+here	O
+are	O
+such	O
+large	O
+that	O
+mobility	O
+of	O
+the	O
+BCCP	O
+alone	O
+is	O
+not	O
+sufficient	O
+for	O
+substrate	O
+transfer	O
+.	O
+	O
+The	O
+current	O
+data	O
+thus	O
+suggest	O
+that	O
+regulation	O
+of	O
+fungal	O
+ACC	O
+is	O
+mediated	O
+by	O
+controlling	O
+the	O
+dynamics	O
+of	O
+the	O
+unique	O
+CD	O
+,	O
+rather	O
+than	O
+directly	O
+affecting	O
+catalytic	O
+turnover	O
+at	O
+the	O
+active	O
+sites	O
+of	O
+BC	O
+and	O
+CT	O
+.	O
+	O
+A	O
+comparison	O
+between	O
+fungal	O
+and	O
+human	O
+ACC	O
+will	O
+help	O
+to	O
+further	O
+discriminate	O
+mechanistic	O
+differences	O
+that	O
+contribute	O
+to	O
+the	O
+extended	O
+control	O
+and	O
+polymerization	O
+of	O
+human	O
+ACC	O
+.	O
+	O
+Most	O
+recently	O
+,	O
+a	O
+crystal	O
+structure	O
+of	O
+near	O
+full	O
+-	O
+length	O
+non	O
+-	O
+phosphorylated	O
+ACC	O
+from	O
+S	O
+.	O
+cerevisae	O
+(	O
+lacking	O
+only	O
+21	O
+N	O
+-	O
+terminal	O
+amino	O
+acids	O
+,	O
+here	O
+denoted	O
+as	O
+flACC	O
+)	O
+was	O
+published	O
+by	O
+Wei	O
+and	O
+Tong	O
+.	O
+	O
+In	O
+flACC	O
+,	O
+the	O
+ACC	O
+dimer	O
+obeys	O
+twofold	O
+symmetry	O
+and	O
+assembles	O
+in	O
+a	O
+triangular	O
+architecture	O
+with	O
+dimeric	O
+BC	O
+domains	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+).	O
+	O
+In	O
+their	O
+study	O
+,	O
+mutational	O
+data	O
+indicate	O
+a	O
+requirement	O
+for	O
+BC	O
+dimerization	O
+for	O
+catalytic	O
+activity	O
+.	O
+	O
+The	O
+transition	O
+from	O
+the	O
+elongated	O
+open	O
+shape	O
+,	O
+observed	O
+in	O
+our	O
+experiments	O
+,	O
+towards	O
+a	O
+compact	O
+triangular	O
+shape	O
+is	O
+based	O
+on	O
+an	O
+intricate	O
+interplay	O
+of	O
+several	O
+hinge	O
+-	O
+bending	O
+motions	O
+in	O
+the	O
+CD	O
+(	O
+Fig	O
+.	O
+4d	O
+).	O
+	O
+Comparison	O
+of	O
+flACC	O
+with	O
+our	O
+CthΔBCCP	B-mutant
+structure	O
+reveals	O
+the	O
+CDC2	O
+/	O
+CT	O
+hinge	O
+as	O
+a	O
+major	O
+contributor	O
+to	O
+conformational	O
+flexibility	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+	O
+In	O
+flACC	O
+,	O
+CDC2	O
+rotates	O
+∼	O
+120	O
+°	O
+with	O
+respect	O
+to	O
+the	O
+CT	O
+domain	O
+.	O
+	O
+On	O
+the	O
+basis	O
+of	O
+a	O
+superposition	O
+of	O
+CDC2	O
+,	O
+CDC1	O
+of	O
+the	O
+phosphorylated	O
+SceCD	O
+is	O
+rotated	O
+by	O
+30	O
+°	O
+relative	O
+to	O
+CDC1	O
+of	O
+the	O
+non	O
+-	O
+phosphorylated	O
+flACC	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5d	O
+),	O
+similar	O
+to	O
+what	O
+we	O
+have	O
+observed	O
+for	O
+the	O
+non	O
+-	O
+phosphorylated	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+).	O
+	O
+When	O
+inspecting	O
+all	O
+individual	O
+protomer	O
+and	O
+fragment	B-mutant
+structures	O
+in	O
+their	O
+study	O
+,	O
+Wei	O
+and	O
+Tong	O
+also	O
+identify	O
+the	O
+CDN	O
+/	O
+CDC1	O
+connection	O
+as	O
+a	O
+highly	O
+flexible	O
+hinge	O
+,	O
+in	O
+agreement	O
+with	O
+our	O
+observations	O
+.	O
+	O
+Only	O
+in	O
+three	O
+out	O
+of	O
+eight	O
+observed	O
+protomers	O
+a	O
+short	O
+peptide	O
+stretch	O
+(	O
+including	O
+Ser1157	O
+)	O
+was	O
+modelled	O
+.	O
+	O
+In	O
+those	O
+instances	O
+the	O
+Ser1157	O
+residue	O
+is	O
+located	O
+at	O
+a	O
+distance	O
+of	O
+14	O
+–	O
+20	O
+Å	O
+away	O
+from	O
+the	O
+location	O
+of	O
+the	O
+phosphorylated	O
+serine	O
+observed	O
+here	O
+,	O
+based	O
+on	O
+superposition	O
+of	O
+either	O
+CDC1	O
+or	O
+CDC2	O
+.	O
+	O
+Applying	O
+the	O
+conformation	O
+of	O
+the	O
+CDC1	O
+/	O
+CDC2	O
+hinge	O
+observed	O
+in	O
+SceCD	O
+on	O
+flACC	O
+leads	O
+to	O
+CDN	O
+sterically	O
+clashing	O
+with	O
+CDC2	O
+and	O
+BT	O
+/	O
+CDN	O
+clashing	O
+with	O
+CT	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+).	O
+	O
+In	O
+addition	O
+,	O
+EM	O
+micrographs	O
+of	O
+phosphorylated	O
+and	O
+dephosphorylated	O
+SceACC	O
+display	O
+for	O
+both	O
+samples	O
+mainly	O
+elongated	O
+and	O
+U	O
+-	O
+shaped	O
+conformations	O
+and	O
+reveal	O
+no	O
+apparent	O
+differences	O
+in	O
+particle	O
+shape	O
+distributions	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+	O
+This	O
+implicates	O
+that	O
+the	O
+triangular	O
+shape	O
+with	O
+dimeric	O
+BC	O
+domains	O
+has	O
+a	O
+low	O
+population	O
+also	O
+in	O
+the	O
+active	O
+form	O
+,	O
+even	O
+though	O
+a	O
+biasing	O
+influence	O
+of	O
+grid	O
+preparation	O
+cannot	O
+be	O
+excluded	O
+completely	O
+.	O
+	O
+Large	O
+-	O
+scale	O
+conformational	O
+variability	O
+has	O
+also	O
+been	O
+observed	O
+in	O
+most	O
+other	O
+carrier	O
+protein	O
+-	O
+based	O
+multienzymes	O
+,	O
+including	O
+polyketide	O
+and	O
+fatty	O
+-	O
+acid	O
+synthases	O
+(	O
+with	O
+the	O
+exception	O
+of	O
+fungal	O
+-	O
+type	O
+fatty	O
+-	O
+acid	O
+synthases	O
+),	O
+non	O
+-	O
+ribosomal	O
+peptide	O
+synthetases	O
+and	O
+the	O
+pyruvate	O
+dehydrogenase	O
+complexes	O
+,	O
+although	O
+based	O
+on	O
+completely	O
+different	O
+architectures	O
+.	O
+	O
+Together	O
+,	O
+this	O
+structural	O
+information	O
+suggests	O
+that	O
+variable	O
+carrier	O
+protein	O
+tethering	O
+is	O
+not	O
+sufficient	O
+for	O
+efficient	O
+substrate	O
+transfer	O
+and	O
+catalysis	O
+in	O
+any	O
+of	O
+these	O
+systems	O
+.	O
+	O
+The	O
+determination	O
+of	O
+a	O
+set	O
+of	O
+crystal	O
+structures	O
+of	O
+SceACC	O
+in	O
+two	O
+states	O
+,	O
+unphosphorylated	O
+and	O
+phosphorylated	O
+at	O
+the	O
+major	O
+regulatory	O
+site	O
+Ser1157	O
+,	O
+provides	O
+a	O
+unique	O
+depiction	O
+of	O
+multienzyme	O
+regulation	O
+by	O
+post	O
+-	O
+translational	O
+modification	O
+(	O
+Fig	O
+.	O
+4d	O
+).	O
+	O
+It	O
+disfavours	O
+the	O
+adoption	O
+of	O
+a	O
+rare	O
+,	O
+compact	O
+conformation	O
+,	O
+in	O
+which	O
+intramolecular	O
+dimerization	O
+of	O
+the	O
+BC	O
+domains	O
+results	O
+in	O
+catalytic	O
+turnover	O
+.	O
+	O
+The	O
+regulation	O
+of	O
+activity	O
+thus	O
+results	O
+from	O
+restrained	O
+large	O
+-	O
+scale	O
+conformational	O
+dynamics	O
+rather	O
+than	O
+a	O
+direct	O
+or	O
+indirect	O
+influence	O
+on	O
+active	O
+site	O
+structure	O
+.	O
+	O
+To	O
+our	O
+best	O
+knowledge	O
+,	O
+ACC	O
+is	O
+the	O
+first	O
+multienzyme	O
+for	O
+which	O
+such	O
+a	O
+phosphorylation	O
+-	O
+dependent	O
+mechanical	O
+control	O
+mechanism	O
+has	O
+been	O
+visualized	O
+.	O
+	O
+However	O
+,	O
+the	O
+example	O
+of	O
+ACC	O
+now	O
+demonstrates	O
+the	O
+possibility	O
+of	O
+regulating	O
+activity	O
+by	O
+controlled	O
+dynamics	O
+of	O
+non	O
+-	O
+enzymatic	O
+linker	O
+regions	O
+also	O
+in	O
+other	O
+families	O
+of	O
+carrier	O
+-	O
+dependent	O
+multienzymes	O
+.	O
+	O
+The	O
+phosphorylated	O
+central	O
+domain	O
+of	O
+yeast	O
+ACC	O
+.	O
+	O
+(	O
+a	O
+)	O
+Schematic	O
+overview	O
+of	O
+the	O
+domain	O
+organization	O
+of	O
+eukaryotic	O
+ACCs	O
+.	O
+	O
+Crystallized	O
+constructs	O
+are	O
+indicated	O
+.	O
+	O
+CDN	O
+is	O
+linked	O
+by	O
+a	O
+four	O
+-	O
+helix	O
+bundle	O
+(	O
+CDL	O
+)	O
+to	O
+two	O
+α	O
+–	O
+β	O
+-	O
+fold	O
+domains	O
+(	O
+CDC1	O
+and	O
+CDC2	O
+).	O
+	O
+The	O
+regulatory	O
+loop	O
+is	O
+shown	O
+as	O
+bold	O
+cartoon	O
+,	O
+and	O
+the	O
+phosphorylated	O
+Ser1157	O
+is	O
+marked	O
+by	O
+a	O
+red	O
+triangle	O
+.	O
+	O
+(	O
+e	O
+)	O
+Structural	O
+overview	O
+of	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+.	O
+	O
+The	O
+attachment	O
+points	O
+to	O
+the	O
+N	O
+-	O
+terminal	O
+BCCP	O
+domain	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+CT	O
+domain	O
+are	O
+indicated	O
+with	O
+spheres	O
+.	O
+	O
+Architecture	O
+of	O
+the	O
+CD	O
+–	O
+CT	O
+core	O
+of	O
+fungal	O
+ACC	O
+.	O
+	O
+One	O
+protomer	O
+is	O
+shown	O
+in	O
+colour	O
+and	O
+one	O
+in	O
+grey	O
+.	O
+	O
+Individual	O
+domains	O
+are	O
+labelled	O
+;	O
+the	O
+active	O
+site	O
+of	O
+CT	O
+and	O
+the	O
+position	O
+of	O
+the	O
+conserved	O
+regulatory	O
+phosphoserine	O
+site	O
+based	O
+on	O
+SceCD	O
+are	O
+indicated	O
+by	O
+an	O
+asterisk	O
+and	O
+a	O
+triangle	O
+,	O
+respectively	O
+.	O
+	O
+Variability	O
+of	O
+the	O
+connections	O
+of	O
+CDC2	O
+to	O
+CT	O
+and	O
+CDC1	O
+in	O
+fungal	O
+ACC	O
+.	O
+	O
+(	O
+a	O
+)	O
+Hinge	O
+properties	O
+of	O
+the	O
+CDC2	O
+–	O
+CT	O
+connection	O
+analysed	O
+by	O
+a	O
+CT	O
+-	O
+based	O
+superposition	O
+of	O
+eight	O
+instances	O
+of	O
+the	O
+CDC2	B-mutant
+-	I-mutant
+CT	I-mutant
+segment	I-mutant
+.	O
+	O
+For	O
+clarity	O
+,	O
+only	O
+one	O
+protomer	O
+of	O
+CthCD	B-mutant
+-	I-mutant
+CTCter1	I-mutant
+is	O
+shown	O
+in	O
+full	O
+colour	O
+as	O
+reference	O
+.	O
+	O
+For	O
+other	O
+instances	O
+,	O
+CDC2	O
+domains	O
+are	O
+shown	O
+in	O
+transparent	O
+tube	O
+representation	O
+with	O
+only	O
+one	O
+helix	O
+each	O
+highlighted	O
+.	O
+	O
+Representation	O
+as	O
+in	O
+a	O
+,	O
+but	O
+the	O
+CDC1	O
+and	O
+CDC2	O
+are	O
+superposed	O
+based	O
+on	O
+CDC2	O
+.	O
+	O
+One	O
+protomer	O
+of	O
+CthΔBCCP	B-mutant
+is	O
+shown	O
+in	O
+colour	O
+,	O
+the	O
+CDL	O
+domains	O
+are	O
+omitted	O
+for	O
+clarity	O
+and	O
+the	O
+position	O
+of	O
+the	O
+phosphorylated	O
+serine	O
+based	O
+on	O
+SceCD	O
+is	O
+indicated	O
+with	O
+a	O
+red	O
+triangle	O
+.	O
+	O
+(	O
+a	O
+–	O
+c	O
+)	O
+Large	O
+-	O
+scale	O
+conformational	O
+variability	O
+of	O
+the	O
+CDN	O
+domain	O
+relative	O
+to	O
+the	O
+CDL	O
+/	O
+CDC1	O
+domain	O
+.	O
+	O
+Domains	O
+other	O
+than	O
+CDN	O
+and	O
+CDL	O
+/	O
+CDC1	O
+are	O
+omitted	O
+for	O
+clarity	O
+.	O
+	O
+(	O
+d	O
+)	O
+Schematic	O
+model	O
+of	O
+fungal	O
+ACC	O
+showing	O
+the	O
+intrinsic	O
+,	O
+regulated	O
+flexibility	O
+of	O
+CD	O
+in	O
+the	O
+phosphorylated	O
+inhibited	O
+or	O
+the	O
+non	O
+-	O
+phosphorylated	O
+activated	O
+state	O
+.	O
+	O
+Flexibility	O
+of	O
+the	O
+CDC2	O
+/	O
+CT	O
+and	O
+CDN	O
+/	O
+CDL	O
+hinges	O
+is	O
+illustrated	O
+by	O
+arrows	O
+.	O
+	O
+The	O
+Ser1157	O
+phosphorylation	O
+site	O
+and	O
+the	O
+regulatory	O
+loop	O
+are	O
+schematically	O
+indicated	O
+in	O
+magenta	O
+.	O
+	O
+The	O
+genome	O
+of	O
+the	O
+Synechococcus	O
+elongatus	O
+strain	O
+PCC	O
+7942	O
+encodes	O
+a	O
+putative	O
+sugar	O
+kinase	O
+(	O
+SePSK	O
+),	O
+which	O
+shares	O
+44	O
+.	O
+9	O
+%	O
+sequence	O
+identity	O
+with	O
+the	O
+xylulose	O
+kinase	O
+-	O
+1	O
+(	O
+AtXK	O
+-	O
+1	O
+)	O
+from	O
+Arabidopsis	O
+thaliana	O
+.	O
+	O
+Sequence	O
+alignment	O
+suggests	O
+that	O
+both	O
+kinases	O
+belong	O
+to	O
+the	O
+ribulokinase	O
+-	O
+like	O
+carbohydrate	O
+kinases	O
+,	O
+a	O
+sub	O
+-	O
+family	O
+of	O
+FGGY	O
+family	O
+carbohydrate	O
+kinases	O
+.	O
+	O
+In	O
+addition	O
+,	O
+our	O
+enzymatic	O
+assays	O
+suggested	O
+that	O
+SePSK	O
+has	O
+the	O
+capability	O
+to	O
+phosphorylate	O
+D	O
+-	O
+ribulose	O
+.	O
+	O
+In	O
+order	O
+to	O
+understand	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	O
+,	O
+we	O
+solved	O
+the	O
+structure	O
+of	O
+SePSK	O
+in	O
+complex	O
+with	O
+D	O
+-	O
+ribulose	O
+and	O
+found	O
+two	O
+potential	O
+substrate	O
+binding	O
+pockets	O
+in	O
+SePSK	O
+.	O
+	O
+Using	O
+mutation	O
+and	O
+activity	O
+analysis	O
+,	O
+we	O
+further	O
+verified	O
+the	O
+key	O
+residues	O
+important	O
+for	O
+its	O
+catalytic	O
+activity	O
+.	O
+	O
+Moreover	O
+,	O
+our	O
+structural	O
+comparison	O
+with	O
+other	O
+family	O
+members	O
+suggests	O
+that	O
+there	O
+are	O
+major	O
+conformational	O
+changes	O
+in	O
+SePSK	O
+upon	O
+substrate	O
+binding	O
+,	O
+facilitating	O
+the	O
+catalytic	O
+process	O
+.	O
+	O
+Together	O
+,	O
+these	O
+results	O
+provide	O
+important	O
+information	O
+for	O
+a	O
+more	O
+detailed	O
+understanding	O
+of	O
+the	O
+cofactor	O
+and	O
+substrate	O
+binding	O
+mode	O
+as	O
+well	O
+as	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	O
+,	O
+and	O
+possible	O
+similarities	O
+with	O
+its	O
+plant	O
+homologue	O
+AtXK	O
+-	O
+1	O
+.	O
+	O
+Carbohydrates	O
+are	O
+essential	O
+cellular	O
+compounds	O
+involved	O
+in	O
+the	O
+metabolic	O
+processes	O
+present	O
+in	O
+all	O
+organisms	O
+.	O
+	O
+The	O
+FGGY	O
+family	O
+carbohydrate	O
+kinases	O
+contain	O
+different	O
+types	O
+of	O
+sugar	O
+kinases	O
+,	O
+all	O
+of	O
+which	O
+possess	O
+different	O
+catalytic	O
+substrates	O
+with	O
+preferences	O
+for	O
+short	O
+-	O
+chained	O
+sugar	O
+substrates	O
+,	O
+ranging	O
+from	O
+triose	O
+to	O
+heptose	O
+.	O
+	O
+These	O
+sugar	O
+substrates	O
+include	O
+L	O
+-	O
+ribulose	O
+,	O
+erythritol	O
+,	O
+L	O
+-	O
+fuculose	O
+,	O
+D	O
+-	O
+glycerol	O
+,	O
+D	O
+-	O
+gluconate	O
+,	O
+L	O
+-	O
+xylulose	O
+,	O
+D	O
+-	O
+ribulose	O
+,	O
+L	O
+-	O
+rhamnulose	O
+and	O
+D	O
+-	O
+xylulose	O
+.	O
+	O
+Structures	O
+reported	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+of	O
+the	O
+FGGY	O
+family	O
+carbohydrate	O
+kinases	O
+exhibit	O
+a	O
+similar	O
+overall	O
+architecture	O
+containing	O
+two	O
+protein	O
+domains	O
+,	O
+one	O
+of	O
+which	O
+is	O
+responsible	O
+for	O
+the	O
+binding	O
+of	O
+substrate	O
+,	O
+while	O
+the	O
+second	O
+is	O
+used	O
+for	O
+binding	O
+cofactor	O
+ATP	O
+.	O
+	O
+While	O
+the	O
+binding	O
+pockets	O
+for	O
+substrates	O
+are	O
+at	O
+the	O
+same	O
+position	O
+,	O
+each	O
+FGGY	O
+family	O
+carbohydrate	O
+kinases	O
+uses	O
+different	O
+substrate	O
+-	O
+binding	O
+residues	O
+,	O
+resulting	O
+in	O
+high	O
+substrate	O
+specificity	O
+.	O
+	O
+Synpcc7942_2462	O
+from	O
+the	O
+cyanobacteria	O
+Synechococcus	O
+elongatus	O
+PCC	O
+7942	O
+encodes	O
+a	O
+putative	O
+sugar	O
+kinase	O
+(	O
+SePSK	O
+),	O
+and	O
+this	O
+kinase	O
+contains	O
+426	O
+amino	O
+acids	O
+.	O
+	O
+The	O
+At2g21370	O
+gene	O
+product	O
+from	O
+Arabidopsis	O
+thaliana	O
+,	O
+xylulose	O
+kinase	O
+-	O
+1	O
+(	O
+AtXK	O
+-	O
+1	O
+),	O
+whose	O
+mature	O
+form	O
+contains	O
+436	O
+amino	O
+acids	O
+,	O
+is	O
+located	O
+in	O
+the	O
+chloroplast	O
+(	O
+ChloroP	O
+1	O
+.	O
+1	O
+Server	O
+).	O
+	O
+Members	O
+of	O
+this	O
+sub	O
+-	O
+family	O
+are	O
+responsible	O
+for	O
+the	O
+phosphorylation	O
+of	O
+sugars	O
+similar	O
+to	O
+L	O
+-	O
+ribulose	O
+and	O
+D	O
+-	O
+ribulose	O
+.	O
+	O
+The	O
+sequence	O
+and	O
+the	O
+substrate	O
+specificity	O
+of	O
+ribulokinase	O
+-	O
+like	O
+carbohydrate	O
+kinases	O
+are	O
+different	O
+,	O
+but	O
+they	O
+share	O
+the	O
+common	O
+folding	O
+feature	O
+with	O
+two	O
+domains	O
+.	O
+	O
+Domain	O
+I	O
+exhibits	O
+a	O
+ribonuclease	O
+H	O
+-	O
+like	O
+folding	O
+pattern	O
+,	O
+and	O
+is	O
+responsible	O
+for	O
+the	O
+substrate	O
+binding	O
+,	O
+while	O
+domain	O
+II	O
+possesses	O
+an	O
+actin	O
+-	O
+like	O
+ATPase	O
+domain	O
+that	O
+binds	O
+cofactor	O
+ATP	O
+.	O
+	O
+Two	O
+possible	O
+xylulose	O
+kinases	O
+(	O
+xylulose	O
+kinase	O
+-	O
+1	O
+:	O
+XK	O
+-	O
+1	O
+and	O
+xylulose	O
+kinase	O
+-	O
+2	O
+:	O
+XK	O
+-	O
+2	O
+)	O
+from	O
+Arabidopsis	O
+thaliana	O
+were	O
+previously	O
+proposed	O
+.	O
+	O
+It	O
+was	O
+shown	O
+that	O
+XK	O
+-	O
+2	O
+(	O
+At5g49650	O
+)	O
+located	O
+in	O
+the	O
+cytosol	O
+is	O
+indeed	O
+xylulose	O
+kinase	O
+.	O
+	O
+SePSK	O
+from	O
+Synechococcus	O
+elongatus	O
+strain	O
+PCC	O
+7942	O
+is	O
+the	O
+homolog	O
+of	O
+AtXK	O
+-	O
+1	O
+,	O
+though	O
+its	O
+physiological	O
+function	O
+and	O
+substrates	O
+remain	O
+unclear	O
+.	O
+	O
+In	O
+order	O
+to	O
+obtain	O
+functional	O
+and	O
+structural	O
+information	O
+about	O
+these	O
+two	O
+proteins	O
+,	O
+here	O
+we	O
+reported	O
+the	O
+crystal	O
+structures	O
+of	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+.	O
+	O
+Overall	O
+structures	O
+of	O
+apo	O
+-	O
+SePSK	O
+and	O
+apo	O
+-	O
+AtXK	O
+-	O
+1	O
+	O
+The	O
+attempt	O
+to	O
+solve	O
+the	O
+SePSK	O
+structure	O
+by	O
+molecular	O
+replacement	O
+method	O
+failed	O
+with	O
+ribulokinase	O
+from	O
+Bacillus	O
+halodurans	O
+(	O
+PDB	O
+code	O
+:	O
+3QDK	O
+,	O
+15	O
+.	O
+7	O
+%	O
+sequence	O
+identity	O
+)	O
+as	O
+an	O
+initial	O
+model	O
+.	O
+	O
+We	O
+therefore	O
+used	O
+single	O
+isomorphous	O
+replacement	O
+anomalous	O
+scattering	O
+method	O
+(	O
+SIRAS	O
+)	O
+for	O
+successful	O
+solution	O
+of	O
+the	O
+apo	O
+-	O
+SePSK	O
+structure	O
+at	O
+a	O
+resolution	O
+of	O
+2	O
+.	O
+3	O
+Å	O
+.	O
+Subsequently	O
+,	O
+the	O
+apo	O
+-	O
+SePSK	O
+structure	O
+was	O
+used	O
+as	O
+molecular	O
+replacement	O
+model	O
+to	O
+solve	O
+all	O
+other	O
+structures	O
+identified	O
+in	O
+this	O
+study	O
+.	O
+	O
+Our	O
+structural	O
+analysis	O
+showed	O
+that	O
+apo	O
+-	O
+SePSK	O
+consists	O
+of	O
+one	O
+SePSK	O
+protein	O
+molecule	O
+in	O
+an	O
+asymmetric	O
+unit	O
+.	O
+	O
+The	O
+amino	O
+-	O
+acid	O
+residues	O
+were	O
+traced	O
+from	O
+Val2	O
+to	O
+His419	O
+,	O
+except	O
+for	O
+the	O
+Met1	O
+residue	O
+and	O
+the	O
+seven	O
+residues	O
+at	O
+the	O
+C	O
+-	O
+termini	O
+.	O
+	O
+Domain	O
+I	O
+consists	O
+of	O
+non	O
+-	O
+contiguous	O
+portions	O
+of	O
+the	O
+polypeptide	O
+chains	O
+(	O
+aa	O
+.	O
+	O
+402	O
+–	O
+419	O
+),	O
+exhibiting	O
+11	O
+α	O
+-	O
+helices	O
+and	O
+11	O
+β	O
+-	O
+sheets	O
+.	O
+	O
+In	O
+addition	O
+,	O
+four	O
+β	O
+-	O
+sheets	O
+(	O
+β7	O
+,	O
+β10	O
+,	O
+β12	O
+and	O
+β16	O
+)	O
+and	O
+five	O
+α	O
+-	O
+helices	O
+(	O
+α8	O
+,	O
+α9	O
+,	O
+α13	O
+,	O
+α14	O
+and	O
+α15	O
+)	O
+flank	O
+the	O
+left	O
+side	O
+of	O
+the	O
+core	O
+region	O
+.	O
+	O
+Domain	O
+II	O
+is	O
+comprised	O
+of	O
+aa	O
+.	O
+	O
+229	O
+–	O
+401	O
+and	O
+classified	O
+into	O
+B2	O
+(	O
+β31	O
+/	O
+β29	O
+/	O
+β22	O
+/	O
+β23	O
+/	O
+β25	O
+/	O
+β24	O
+)	O
+and	O
+A3	O
+(	O
+α26	O
+/	O
+α27	O
+/	O
+α28	O
+/	O
+α30	O
+)	O
+(	O
+Fig	O
+1A	O
+and	O
+S1	O
+Fig	O
+).	O
+	O
+In	O
+the	O
+SePSK	O
+structure	O
+,	O
+B1	O
+and	O
+B2	O
+are	O
+sandwiched	O
+by	O
+A1	O
+,	O
+A2	O
+and	O
+A3	O
+,	O
+and	O
+the	O
+whole	O
+structure	O
+shows	O
+the	O
+A1	O
+/	O
+B1	O
+/	O
+A2	O
+/	O
+B2	O
+/	O
+A3	O
+(	O
+α	O
+/	O
+β	O
+/	O
+α	O
+/	O
+β	O
+/	O
+α	O
+)	O
+folding	O
+pattern	O
+,	O
+which	O
+is	O
+in	O
+common	O
+with	O
+other	O
+members	O
+of	O
+FGGY	O
+family	O
+carbohydrate	O
+kinases	O
+(	O
+S2	O
+Fig	O
+).	O
+	O
+The	O
+overall	O
+folding	O
+of	O
+SePSK	O
+resembles	O
+a	O
+clip	O
+,	O
+with	O
+A2	O
+of	O
+domain	O
+I	O
+acting	O
+as	O
+a	O
+hinge	O
+region	O
+.	O
+	O
+Overall	O
+structures	O
+of	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+.	O
+	O
+(	O
+A	O
+)	O
+Three	O
+-	O
+dimensional	O
+structure	O
+of	O
+apo	O
+-	O
+SePSK	O
+.	O
+	O
+The	O
+secondary	O
+structural	O
+elements	O
+are	O
+indicated	O
+(	O
+α	O
+-	O
+helix	O
+:	O
+cyan	O
+,	O
+β	O
+-	O
+sheet	O
+:	O
+yellow	O
+).	O
+	O
+(	O
+B	O
+)	O
+Three	O
+-	O
+dimensional	O
+structure	O
+of	O
+apo	O
+-	O
+AtXK	O
+-	O
+1	O
+.	O
+	O
+Apo	O
+-	O
+AtXK	O
+-	O
+1	O
+exhibits	O
+a	O
+folding	O
+pattern	O
+similar	O
+to	O
+that	O
+of	O
+SePSK	O
+in	O
+line	O
+with	O
+their	O
+high	O
+sequence	O
+identity	O
+(	O
+Fig	O
+1B	O
+and	O
+S1	O
+Fig	O
+).	O
+	O
+However	O
+,	O
+superposition	O
+of	O
+structures	O
+of	O
+AtXK	O
+-	O
+1	O
+and	O
+SePSK	O
+shows	O
+some	O
+differences	O
+,	O
+especially	O
+at	O
+the	O
+loop	O
+regions	O
+.	O
+	O
+A	O
+considerable	O
+difference	O
+is	O
+found	O
+in	O
+the	O
+loop3	O
+linking	O
+β3	O
+and	O
+α4	O
+,	O
+which	O
+is	O
+stretched	O
+out	O
+in	O
+the	O
+AtXK	O
+-	O
+1	O
+structure	O
+,	O
+while	O
+in	O
+the	O
+SePSK	O
+structure	O
+,	O
+it	O
+is	O
+bent	O
+back	O
+towards	O
+the	O
+inner	O
+part	O
+.	O
+	O
+The	O
+corresponding	O
+residues	O
+between	O
+these	O
+two	O
+structures	O
+(	O
+SePSK	O
+-	O
+Lys35	O
+and	O
+AtXK	O
+-	O
+1	O
+-	O
+Lys48	O
+)	O
+have	O
+a	O
+distance	O
+of	O
+15	O
+.	O
+4	O
+Å	O
+(	O
+S3	O
+Fig	O
+).	O
+	O
+Activity	O
+assays	O
+of	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+	O
+In	O
+order	O
+to	O
+understand	O
+the	O
+function	O
+of	O
+these	O
+two	O
+kinases	O
+,	O
+we	O
+performed	O
+structural	O
+comparison	O
+using	O
+Dali	O
+server	O
+.	O
+	O
+We	O
+first	O
+tested	O
+whether	O
+both	O
+enzymes	O
+possessed	O
+ATP	O
+hydrolysis	O
+activity	O
+in	O
+the	O
+absence	O
+of	O
+substrates	O
+.	O
+	O
+This	O
+finding	O
+is	O
+in	O
+agreement	O
+with	O
+a	O
+previous	O
+result	O
+showing	O
+that	O
+xylulose	O
+kinase	O
+(	O
+PDB	O
+code	O
+:	O
+2ITM	O
+)	O
+possessed	O
+ATP	O
+hydrolysis	O
+activity	O
+without	O
+adding	O
+substrate	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+2B	O
+,	O
+the	O
+ATP	O
+hydrolysis	O
+activity	O
+of	O
+SePSK	O
+greatly	O
+increased	O
+upon	O
+adding	O
+D	O
+-	O
+ribulose	O
+than	O
+adding	O
+other	O
+potential	O
+substrates	O
+,	O
+suggesting	O
+that	O
+it	O
+has	O
+D	O
+-	O
+ribulose	O
+kinase	O
+activity	O
+.	O
+	O
+In	O
+contrary	O
+,	O
+limited	O
+increasing	O
+of	O
+ATP	O
+hydrolysis	O
+activity	O
+was	O
+detected	O
+for	O
+AtXK	O
+-	O
+1	O
+upon	O
+addition	O
+of	O
+D	O
+-	O
+ribulose	O
+(	O
+Fig	O
+2C	O
+),	O
+despite	O
+its	O
+structural	O
+similarity	O
+with	O
+SePSK	O
+.	O
+	O
+The	O
+enzymatic	O
+activity	O
+assays	O
+of	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+.	O
+	O
+The	O
+substrates	O
+are	O
+DR	O
+(	O
+D	O
+-	O
+ribulose	O
+),	O
+LR	O
+(	O
+L	O
+-	O
+ribulose	O
+),	O
+DX	O
+(	O
+D	O
+-	O
+xylulose	O
+),	O
+LX	O
+(	O
+L	O
+-	O
+xylulose	O
+)	O
+and	O
+GLY	O
+(	O
+Glycerol	O
+).	O
+(	O
+C	O
+)	O
+The	O
+ATP	O
+hydrolysis	O
+activity	O
+of	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+with	O
+or	O
+without	O
+D	O
+-	O
+ribulose	O
+.	O
+(	O
+D	O
+)	O
+The	O
+ATP	O
+hydrolysis	O
+activity	O
+of	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+and	O
+single	O
+-	O
+site	O
+mutants	O
+of	O
+SePSK	O
+.	O
+	O
+Three	O
+single	O
+-	O
+site	O
+mutants	O
+of	O
+SePSK	O
+are	O
+D8A	B-mutant
+-	O
+SePSK	O
+,	O
+T11A	B-mutant
+-	O
+SePSK	O
+and	O
+D221A	B-mutant
+-	O
+SePSK	O
+.	O
+	O
+The	O
+ATP	O
+hydrolysis	O
+activity	O
+measured	O
+via	O
+luminescent	O
+ADP	O
+-	O
+Glo	O
+assay	O
+(	O
+Promega	O
+).	O
+	O
+To	O
+understand	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	O
+,	O
+we	O
+performed	O
+structural	O
+comparisons	O
+among	O
+xylulose	O
+kinase	O
+,	O
+glycerol	O
+kinase	O
+,	O
+ribulose	O
+kinase	O
+and	O
+SePSK	O
+.	O
+	O
+Our	O
+results	O
+suggested	O
+that	O
+three	O
+conserved	O
+residues	O
+(	O
+D8	O
+,	O
+T11	O
+and	O
+D221	O
+of	O
+SePSK	O
+)	O
+play	O
+an	O
+important	O
+role	O
+in	O
+SePSK	O
+function	O
+.	O
+	O
+Mutations	O
+of	O
+the	O
+corresponding	O
+residue	O
+in	O
+xylulose	O
+kinase	O
+and	O
+glycerol	O
+kinase	O
+from	O
+Escherichia	O
+coli	O
+greatly	O
+reduced	O
+their	O
+activity	O
+.	O
+	O
+To	O
+identify	O
+the	O
+function	O
+of	O
+these	O
+three	O
+residues	O
+of	O
+SePSK	O
+,	O
+we	O
+constructed	O
+D8A	B-mutant
+,	O
+T11A	B-mutant
+and	O
+D221A	B-mutant
+mutants	O
+.	O
+	O
+Using	O
+enzymatic	O
+activity	O
+assays	O
+,	O
+we	O
+found	O
+that	O
+all	O
+of	O
+these	O
+mutants	O
+exhibit	O
+much	O
+lower	O
+activity	O
+of	O
+ATP	O
+hydrolysis	O
+after	O
+adding	O
+D	O
+-	O
+ribulose	O
+than	O
+that	O
+of	O
+wild	O
+type	O
+,	O
+indicating	O
+the	O
+possibility	O
+that	O
+these	O
+three	O
+residues	O
+are	O
+involved	O
+in	O
+the	O
+catalytic	O
+process	O
+of	O
+phosphorylation	O
+D	O
+-	O
+ribulose	O
+and	O
+are	O
+vital	O
+for	O
+the	O
+function	O
+of	O
+SePSK	O
+(	O
+Fig	O
+2D	O
+).	O
+	O
+In	O
+both	O
+structures	O
+,	O
+a	O
+strong	O
+electron	O
+density	O
+was	O
+found	O
+in	O
+the	O
+conserved	O
+ATP	O
+binding	O
+pocket	O
+,	O
+but	O
+can	O
+only	O
+be	O
+fitted	O
+with	O
+an	O
+ADP	O
+molecule	O
+(	O
+S4	O
+Fig	O
+).	O
+	O
+The	O
+extremely	O
+weak	O
+electron	O
+densities	O
+of	O
+ATP	O
+γ	O
+-	O
+phosphate	O
+in	O
+both	O
+structures	O
+suggest	O
+that	O
+the	O
+γ	O
+-	O
+phosphate	O
+group	O
+of	O
+ATP	O
+is	O
+either	O
+flexible	O
+or	O
+hydrolyzed	O
+by	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+.	O
+	O
+To	O
+avoid	O
+hydrolysis	O
+of	O
+ATP	O
+,	O
+we	O
+soaked	O
+the	O
+crystals	O
+of	O
+apo	O
+-	O
+SePSK	O
+and	O
+apo	O
+-	O
+AtXK	O
+-	O
+1	O
+into	O
+the	O
+reservoir	O
+adding	O
+AMP	O
+-	O
+PNP	O
+.	O
+	O
+However	O
+,	O
+we	O
+found	O
+that	O
+the	O
+electron	O
+densities	O
+of	O
+γ	O
+-	O
+phosphate	O
+group	O
+of	O
+AMP	O
+-	O
+PNP	O
+(	O
+AMP	O
+-	O
+PNP	O
+γ	O
+-	O
+phosphate	O
+)	O
+are	O
+still	O
+weak	O
+in	O
+the	O
+AMP	O
+-	O
+PNP	O
+-	O
+SePSK	O
+and	O
+AMP	O
+-	O
+PNP	O
+-	O
+AtXK	O
+-	O
+1	O
+structures	O
+,	O
+suggesting	O
+high	O
+flexibility	O
+of	O
+ATP	O
+-	O
+γ	O
+-	O
+phosphate	O
+.	O
+	O
+The	O
+γ	O
+-	O
+phosphate	O
+group	O
+of	O
+ATP	O
+is	O
+transferred	O
+to	O
+the	O
+sugar	O
+substrate	O
+during	O
+the	O
+reaction	O
+process	O
+,	O
+so	O
+this	O
+flexibility	O
+might	O
+be	O
+important	O
+for	O
+the	O
+ability	O
+of	O
+these	O
+kinases	O
+.	O
+	O
+The	O
+overall	O
+structures	O
+as	O
+well	O
+as	O
+the	O
+coordination	O
+modes	O
+of	O
+ADP	O
+and	O
+AMP	O
+-	O
+PNP	O
+in	O
+the	O
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+-	O
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+-	O
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+-	O
+1	O
+,	O
+ADP	O
+-	O
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+-	O
+1	O
+,	O
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+-	O
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+AMP	O
+-	O
+PNP	O
+-	O
+SePSK	O
+structures	O
+are	O
+nearly	O
+identical	O
+(	O
+S5	O
+Fig	O
+),	O
+therefore	O
+the	O
+structure	O
+of	O
+AMP	O
+-	O
+PNP	O
+-	O
+SePSK	O
+is	O
+used	O
+here	O
+to	O
+describe	O
+the	O
+structural	O
+details	O
+and	O
+to	O
+compare	O
+with	O
+those	O
+of	O
+other	O
+family	O
+members	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+3A	O
+,	O
+one	O
+SePSK	O
+protein	O
+molecule	O
+is	O
+in	O
+an	O
+asymmetric	O
+unit	O
+with	O
+one	O
+AMP	O
+-	O
+PNP	O
+molecule	O
+.	O
+	O
+The	O
+AMP	O
+-	O
+PNP	O
+is	O
+bound	O
+at	O
+the	O
+domain	O
+II	O
+,	O
+where	O
+it	O
+fits	O
+well	O
+inside	O
+a	O
+positively	O
+charged	O
+groove	O
+.	O
+	O
+The	O
+AMP	O
+-	O
+PNP	O
+binding	O
+pocket	O
+consists	O
+of	O
+four	O
+α	O
+-	O
+helices	O
+(	O
+α26	O
+,	O
+α28	O
+,	O
+α27	O
+and	O
+α30	O
+)	O
+and	O
+forms	O
+a	O
+shape	O
+resembling	O
+a	O
+half	O
+-	O
+fist	O
+(	O
+Fig	O
+3A	O
+and	O
+3B	O
+).	O
+	O
+The	O
+head	O
+group	O
+of	O
+the	O
+AMP	O
+-	O
+PNP	O
+is	O
+embedded	O
+in	O
+a	O
+pocket	O
+surrounded	O
+by	O
+Trp383	O
+,	O
+Asn380	O
+,	O
+Gly376	O
+and	O
+Gly377	O
+.	O
+	O
+The	O
+purine	O
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+of	O
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+-	O
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+in	O
+parallel	O
+to	O
+the	O
+indole	O
+ring	O
+of	O
+Trp383	O
+.	O
+	O
+In	O
+addition	O
+,	O
+it	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+with	O
+the	O
+side	O
+chain	O
+amide	O
+of	O
+Asn380	O
+(	O
+Fig	O
+3B	O
+).	O
+	O
+Together	O
+,	O
+this	O
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+shows	O
+that	O
+the	O
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+-	O
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+-	O
+β	O
+-	O
+phosphate	O
+is	O
+sticking	O
+out	O
+of	O
+the	O
+ATP	O
+binding	O
+pocket	O
+,	O
+thus	O
+the	O
+γ	O
+-	O
+phosphate	O
+group	O
+is	O
+at	O
+the	O
+empty	O
+space	O
+between	O
+domain	O
+I	O
+and	O
+domain	O
+II	O
+and	O
+is	O
+unconstrained	O
+in	O
+its	O
+movement	O
+by	O
+the	O
+protein	O
+.	O
+	O
+The	O
+SePSK	O
+structure	O
+is	O
+shown	O
+in	O
+the	O
+electrostatic	O
+potential	O
+surface	O
+mode	O
+.	O
+	O
+The	O
+head	O
+of	O
+AMP	O
+-	O
+PNP	O
+is	O
+sandwiched	O
+by	O
+four	O
+residues	O
+(	O
+Leu293	O
+,	O
+Gly376	O
+,	O
+Gly377	O
+and	O
+Trp383	O
+).	O
+	O
+The	O
+results	O
+from	O
+our	O
+activity	O
+assays	O
+suggested	O
+that	O
+SePSK	O
+has	O
+D	O
+-	O
+ribulose	O
+kinase	O
+activity	O
+.	O
+	O
+As	O
+shown	O
+in	O
+S6	O
+Fig	O
+,	O
+two	O
+residual	O
+electron	O
+densities	O
+are	O
+visible	O
+in	O
+domain	O
+I	O
+,	O
+which	O
+can	O
+be	O
+interpreted	O
+as	O
+two	O
+D	O
+-	O
+ribulose	O
+molecules	O
+with	O
+reasonable	O
+fit	O
+.	O
+	O
+7	O
+.	O
+1	O
+Å	O
+(	O
+RBL1	O
+-	O
+C4	O
+and	O
+RBL2	O
+-	O
+C1	O
+).	O
+	O
+RBL1	O
+is	O
+located	O
+in	O
+the	O
+pocket	O
+consisting	O
+of	O
+α21	O
+and	O
+the	O
+loop	O
+between	O
+β6	O
+and	O
+β7	O
+.	O
+	O
+The	O
+O4	O
+and	O
+O5	O
+of	O
+RBL1	O
+are	O
+coordinated	O
+with	O
+the	O
+side	O
+chain	O
+carboxyl	O
+group	O
+of	O
+Asp221	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+O2	O
+of	O
+RBL1	O
+interacts	O
+with	O
+the	O
+main	O
+chain	O
+amide	O
+nitrogen	O
+of	O
+Ser72	O
+(	O
+Fig	O
+4B	O
+).	O
+	O
+This	O
+pocket	O
+is	O
+at	O
+a	O
+similar	O
+position	O
+of	O
+substrate	O
+binding	O
+site	O
+of	O
+other	O
+sugar	O
+kinase	O
+,	O
+such	O
+as	O
+L	O
+-	O
+ribulokinase	O
+(	O
+PDB	O
+code	O
+:	O
+3QDK	O
+)	O
+(	O
+S7	O
+Fig	O
+).	O
+	O
+However	O
+,	O
+structural	O
+comparison	O
+shows	O
+that	O
+the	O
+substrate	O
+ligating	O
+residues	O
+between	O
+the	O
+two	O
+structures	O
+are	O
+not	O
+strictly	O
+conserved	O
+.	O
+	O
+Based	O
+on	O
+the	O
+structures	O
+,	O
+the	O
+ligating	O
+residues	O
+of	O
+RBL1	O
+in	O
+RBL	O
+-	O
+SePSK	O
+structure	O
+are	O
+Ser72	O
+,	O
+Asp221	O
+and	O
+Ser222	O
+,	O
+and	O
+the	O
+interacting	O
+residues	O
+of	O
+L	O
+-	O
+ribulose	O
+with	O
+L	O
+-	O
+ribulokinase	O
+are	O
+Ala96	O
+,	O
+Lys208	O
+,	O
+Asp274	O
+and	O
+Glu329	O
+(	O
+S7	O
+Fig	O
+).	O
+	O
+Glu329	O
+in	O
+3QDK	O
+has	O
+no	O
+counterpart	O
+in	O
+RBL	O
+-	O
+SePSK	O
+structure	O
+.	O
+	O
+In	O
+addition	O
+,	O
+although	O
+Lys208	O
+of	O
+L	O
+-	O
+ribulokinase	O
+has	O
+the	O
+corresponding	O
+residue	O
+(	O
+Lys163	O
+)	O
+in	O
+RBL	O
+-	O
+SePSK	O
+structure	O
+,	O
+the	O
+hydrogen	O
+bond	O
+of	O
+Lys163	O
+is	O
+broken	O
+because	O
+of	O
+the	O
+conformational	O
+change	O
+of	O
+two	O
+α	O
+-	O
+helices	O
+(	O
+α9	O
+and	O
+α13	O
+)	O
+of	O
+SePSK	O
+.	O
+	O
+The	O
+binding	O
+of	O
+D	O
+-	O
+ribulose	O
+(	O
+RBL	O
+)	O
+with	O
+SePSK	O
+.	O
+	O
+(	O
+A	O
+)	O
+The	O
+electrostatic	O
+potential	O
+surface	O
+map	O
+of	O
+RBL	O
+-	O
+SePSK	O
+and	O
+a	O
+zoom	O
+-	O
+in	O
+view	O
+of	O
+RBL	O
+binding	O
+site	O
+.	O
+	O
+Single	O
+-	O
+cycle	O
+kinetic	O
+data	O
+are	O
+reflecting	O
+the	O
+interaction	O
+of	O
+SePSK	O
+and	O
+D8A	B-mutant
+-	O
+SePSK	O
+with	O
+D	O
+-	O
+ribulose	O
+.	O
+	O
+It	O
+shows	O
+two	O
+experimental	O
+sensorgrams	O
+after	O
+minus	O
+the	O
+empty	O
+sensorgrams	O
+.	O
+	O
+The	O
+original	O
+data	O
+is	O
+shown	O
+as	O
+black	O
+curve	O
+,	O
+and	O
+the	O
+fitted	O
+data	O
+is	O
+shown	O
+as	O
+different	O
+color	O
+(	O
+wild	O
+type	O
+SePSK	O
+:	O
+red	O
+curve	O
+,	O
+D8A	B-mutant
+-	O
+SePSK	O
+:	O
+green	O
+curve	O
+).	O
+	O
+Dissociation	O
+rate	O
+constant	O
+of	O
+wild	O
+type	O
+and	O
+D8A	B-mutant
+-	O
+SePSK	O
+are	O
+3	O
+ms	O
+-	O
+1	O
+and	O
+9	O
+ms	O
+-	O
+1	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+binding	O
+pocket	O
+of	O
+RBL2	O
+with	O
+relatively	O
+weak	O
+electron	O
+density	O
+is	O
+near	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+of	O
+SePSK	O
+and	O
+is	O
+negatively	O
+charged	O
+.	O
+	O
+The	O
+side	O
+chain	O
+of	O
+Asp8	O
+interacts	O
+strongly	O
+with	O
+O3	O
+and	O
+O4	O
+of	O
+RBL2	O
+.	O
+	O
+The	O
+backbone	O
+amide	O
+nitrogens	O
+of	O
+Gly13	O
+and	O
+Arg15	O
+also	O
+keep	O
+hydrogen	O
+bonds	O
+with	O
+RBL2	O
+(	O
+Fig	O
+4B	O
+).	O
+	O
+This	O
+break	O
+is	O
+probably	O
+induced	O
+by	O
+the	O
+conformational	O
+change	O
+of	O
+the	O
+two	O
+β	O
+-	O
+sheets	O
+(	O
+β1	O
+and	O
+β2	O
+),	O
+with	O
+the	O
+result	O
+that	O
+the	O
+linking	O
+loop	O
+(	O
+loop	O
+1	O
+)	O
+is	O
+located	O
+further	O
+away	O
+from	O
+the	O
+RBL2	O
+binding	O
+site	O
+.	O
+	O
+Our	O
+SePSK	O
+structure	O
+shows	O
+that	O
+the	O
+Asp8	O
+residue	O
+forms	O
+strong	O
+hydrogen	O
+bond	O
+with	O
+RBL2	O
+(	O
+Fig	O
+4B	O
+).	O
+	O
+In	O
+addition	O
+,	O
+our	O
+enzymatic	O
+assays	O
+indicated	O
+that	O
+Asp8	O
+is	O
+important	O
+for	O
+the	O
+activity	O
+of	O
+SePSK	O
+(	O
+Fig	O
+2D	O
+).	O
+	O
+To	O
+further	O
+verified	O
+this	O
+result	O
+,	O
+we	O
+measured	O
+the	O
+binding	O
+affinity	O
+for	O
+D	O
+-	O
+ribulose	O
+of	O
+both	O
+wild	O
+type	O
+(	O
+WT	O
+)	O
+and	O
+D8A	B-mutant
+mutant	O
+of	O
+SePSK	O
+using	O
+a	O
+surface	O
+plasmon	O
+resonance	O
+method	O
+.	O
+	O
+Dissociation	O
+rate	O
+constant	O
+(	O
+Kd	O
+)	O
+of	O
+wild	O
+type	O
+and	O
+D8A	B-mutant
+-	O
+SePSK	O
+are	O
+3	O
+ms	O
+-	O
+1	O
+and	O
+9	O
+ms	O
+-	O
+1	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+results	O
+implied	O
+that	O
+the	O
+second	O
+RBL	O
+binding	O
+site	O
+plays	O
+a	O
+role	O
+in	O
+the	O
+D	O
+-	O
+ribulose	O
+kinase	O
+function	O
+of	O
+SePSK	O
+.	O
+	O
+However	O
+,	O
+considering	O
+the	O
+high	O
+concentration	O
+of	O
+D	O
+-	O
+ribulose	O
+used	O
+for	O
+crystal	O
+soaking	O
+,	O
+as	O
+well	O
+as	O
+the	O
+relatively	O
+weak	O
+electron	O
+density	O
+of	O
+RBL2	O
+,	O
+it	O
+is	O
+also	O
+possible	O
+that	O
+the	O
+second	O
+binding	O
+site	O
+of	O
+D	O
+-	O
+ribulose	O
+in	O
+SePSK	O
+is	O
+an	O
+artifact	O
+.	O
+	O
+Simulated	O
+conformational	O
+change	O
+of	O
+SePSK	O
+during	O
+the	O
+catalytic	O
+process	O
+	O
+It	O
+was	O
+reported	O
+earlier	O
+that	O
+the	O
+crossing	O
+angle	O
+between	O
+the	O
+domain	O
+I	O
+and	O
+domain	O
+II	O
+in	O
+FGGY	O
+family	O
+carbohydrate	O
+kinases	O
+is	O
+different	O
+.	O
+	O
+In	O
+addition	O
+,	O
+this	O
+difference	O
+may	O
+be	O
+caused	O
+by	O
+the	O
+binding	O
+of	O
+substrates	O
+and	O
+/	O
+or	O
+ATP	O
+.	O
+	O
+As	O
+reported	O
+previously	O
+,	O
+members	O
+of	O
+the	O
+sugar	O
+kinase	O
+family	O
+undergo	O
+a	O
+conformational	O
+change	O
+to	O
+narrow	O
+the	O
+crossing	O
+angle	O
+between	O
+two	O
+domains	O
+and	O
+reduce	O
+the	O
+distance	O
+between	O
+substrate	O
+and	O
+ATP	O
+in	O
+order	O
+to	O
+facilitate	O
+the	O
+catalytic	O
+reaction	O
+of	O
+phosphorylation	O
+of	O
+sugar	O
+substrates	O
+.	O
+	O
+After	O
+comparing	O
+structures	O
+of	O
+apo	O
+-	O
+SePSK	O
+,	O
+RBL	O
+-	O
+SePSK	O
+and	O
+AMP	O
+-	O
+PNP	O
+-	O
+SePSK	O
+,	O
+we	O
+noticed	O
+that	O
+these	O
+structures	O
+presented	O
+here	O
+are	O
+similar	O
+.	O
+	O
+Superposing	O
+the	O
+structures	O
+of	O
+RBL	O
+-	O
+SePSK	O
+and	O
+AMP	O
+-	O
+PNP	O
+-	O
+SePSK	O
+,	O
+the	O
+results	O
+show	O
+that	O
+the	O
+nearest	O
+distance	O
+between	O
+AMP	O
+-	O
+PNP	O
+γ	O
+-	O
+phosphate	O
+and	O
+RBL1	O
+/	O
+RBL2	O
+is	O
+7	O
+.	O
+5	O
+Å	O
+(	O
+RBL1	O
+-	O
+O5	O
+)/	O
+6	O
+.	O
+7	O
+Å	O
+(	O
+RBL2	O
+-	O
+O1	O
+)	O
+(	O
+S8	O
+Fig	O
+).	O
+	O
+This	O
+distance	O
+is	O
+too	O
+long	O
+to	O
+transfer	O
+the	O
+γ	O
+-	O
+phosphate	O
+group	O
+from	O
+ATP	O
+to	O
+the	O
+substrate	O
+.	O
+	O
+Since	O
+the	O
+two	O
+domains	O
+of	O
+SePSK	O
+are	O
+widely	O
+separated	O
+in	O
+this	O
+structure	O
+,	O
+we	O
+hypothesize	O
+that	O
+our	O
+structures	O
+of	O
+SePSK	O
+represent	O
+its	O
+open	O
+form	O
+,	O
+and	O
+that	O
+a	O
+conformational	O
+rearrangement	O
+must	O
+occur	O
+to	O
+switch	O
+to	O
+the	O
+closed	O
+state	O
+in	O
+order	O
+to	O
+facilitate	O
+the	O
+catalytic	O
+process	O
+of	O
+phosphorylation	O
+of	O
+sugar	O
+substrates	O
+.	O
+	O
+For	O
+studying	O
+such	O
+potential	O
+conformational	O
+change	O
+,	O
+a	O
+simulation	O
+on	O
+the	O
+Hingeprot	O
+Server	O
+was	O
+performed	O
+to	O
+predict	O
+the	O
+movement	O
+of	O
+different	O
+SePSK	O
+domains	O
+.	O
+	O
+The	O
+results	O
+showed	O
+that	O
+domain	O
+I	O
+and	O
+domain	O
+II	O
+are	O
+closer	O
+to	O
+each	O
+other	O
+with	O
+Ala228	O
+and	O
+Thr401	O
+in	O
+A2	O
+as	O
+Hinge	O
+-	O
+residues	O
+.	O
+	O
+Based	O
+on	O
+the	O
+above	O
+results	O
+,	O
+SePSK	O
+is	O
+divided	O
+into	O
+two	O
+rigid	O
+parts	O
+.	O
+	O
+The	O
+domain	O
+I	O
+of	O
+RBL	O
+-	O
+SePSK	O
+(	O
+aa	O
+.	O
+1	O
+–	O
+228	O
+,	O
+aa	O
+.	O
+402	O
+–	O
+421	O
+)	O
+and	O
+the	O
+domain	O
+II	O
+of	O
+AMP	O
+-	O
+PNP	O
+-	O
+SePSK	O
+(	O
+aa	O
+.	O
+229	O
+–	O
+401	O
+)	O
+were	O
+superposed	O
+with	O
+structures	O
+,	O
+including	O
+apo	O
+-	O
+AtXK	O
+-	O
+1	O
+,	O
+apo	O
+-	O
+SePSK	O
+,	O
+xylulose	O
+kinase	O
+from	O
+Lactobacillus	O
+acidophilus	O
+(	O
+PDB	O
+code	O
+:	O
+3LL3	O
+)	O
+and	O
+the	O
+S58W	B-mutant
+mutant	O
+form	O
+of	O
+glycerol	O
+kinase	O
+from	O
+Escherichia	O
+coli	O
+(	O
+PDB	O
+code	O
+:	O
+1GLJ	O
+).	O
+	O
+The	O
+results	O
+of	O
+superposition	O
+displayed	O
+different	O
+crossing	O
+angle	O
+between	O
+these	O
+two	O
+domains	O
+.	O
+	O
+After	O
+superposition	O
+,	O
+the	O
+distances	O
+of	O
+AMP	O
+-	O
+PNP	O
+γ	O
+-	O
+phosphate	O
+and	O
+the	O
+fifth	O
+hydroxyl	O
+group	O
+of	O
+RBL1	O
+are	O
+7	O
+.	O
+9	O
+Å	O
+(	O
+superposed	O
+with	O
+AtXK	O
+-	O
+1	O
+),	O
+7	O
+.	O
+4	O
+Å	O
+(	O
+superposed	O
+with	O
+SePSK	O
+),	O
+6	O
+.	O
+6	O
+Å	O
+(	O
+superposed	O
+with	O
+3LL3	O
+)	O
+and	O
+6	O
+.	O
+1	O
+Å	O
+(	O
+superposed	O
+with	O
+1GLJ	O
+).	O
+	O
+Meanwhile	O
+,	O
+the	O
+distances	O
+of	O
+AMP	O
+-	O
+PNP	O
+γ	O
+-	O
+phosphate	O
+and	O
+the	O
+first	O
+hydroxyl	O
+group	O
+of	O
+RBL2	O
+are	O
+7	O
+.	O
+2	O
+Å	O
+(	O
+superposed	O
+with	O
+AtXK	O
+-	O
+1	O
+),	O
+6	O
+.	O
+7	O
+Å	O
+(	O
+superposed	O
+with	O
+SePSK	O
+),	O
+3	O
+.	O
+7	O
+Å	O
+(	O
+superposed	O
+with	O
+3LL3	O
+),	O
+until	O
+AMP	O
+-	O
+PNP	O
+γ	O
+-	O
+phosphate	O
+fully	O
+contacts	O
+RBL2	O
+after	O
+superposition	O
+with	O
+1GLJ	O
+(	O
+Fig	O
+5	O
+).	O
+	O
+Simulated	O
+conformational	O
+change	O
+of	O
+SePSK	O
+during	O
+the	O
+catalytic	O
+process	O
+.	O
+	O
+The	O
+structures	O
+are	O
+shown	O
+as	O
+cartoon	O
+and	O
+the	O
+ligands	O
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+	O
+Domain	O
+I	O
+from	O
+D	O
+-	O
+ribulose	O
+-	O
+SePSK	O
+(	O
+green	O
+)	O
+and	O
+Domain	O
+II	O
+from	O
+AMP	O
+-	O
+PNP	O
+-	O
+SePSK	O
+(	O
+cyan	O
+)	O
+are	O
+superposed	O
+with	O
+apo	O
+-	O
+AtXK	O
+-	O
+1	O
+(	O
+1st	O
+),	O
+apo	O
+-	O
+SePSK	O
+(	O
+2nd	O
+),	O
+3LL3	O
+(	O
+3rd	O
+)	O
+and	O
+1GLJ	O
+(	O
+4th	O
+),	O
+respectively	O
+.	O
+	O
+The	O
+numbers	O
+near	O
+the	O
+black	O
+dashed	O
+lines	O
+show	O
+the	O
+distances	O
+(	O
+Å	O
+)	O
+between	O
+two	O
+nearest	O
+atoms	O
+of	O
+RBL	O
+and	O
+AMP	O
+-	O
+PNP	O
+.	O
+	O
+Our	O
+results	O
+provide	O
+the	O
+detailed	O
+information	O
+about	O
+the	O
+interaction	O
+of	O
+SePSK	O
+with	O
+ATP	O
+and	O
+substrates	O
+.	O
+	O
+Moreover	O
+,	O
+structural	O
+superposition	O
+results	O
+enable	O
+us	O
+to	O
+visualize	O
+the	O
+conformational	O
+change	O
+of	O
+SePSK	O
+during	O
+the	O
+catalytic	O
+process	O
+.	O
+	O
+In	O
+conclusion	O
+,	O
+our	O
+results	O
+provide	O
+important	O
+information	O
+for	O
+a	O
+more	O
+detailed	O
+understanding	O
+of	O
+the	O
+mechanisms	O
+of	O
+SePSK	O
+and	O
+other	O
+members	O
+of	O
+FGGY	O
+family	O
+carbohydrate	O
+kinases	O
+.	O
+	O
+Structural	O
+insights	O
+into	O
+the	O
+Escherichia	O
+coli	O
+lysine	O
+decarboxylases	O
+and	O
+molecular	O
+determinants	O
+of	O
+interaction	O
+with	O
+the	O
+AAA	O
++	O
+ATPase	O
+RavA	O
+	O
+The	O
+inducible	O
+lysine	O
+decarboxylase	O
+LdcI	O
+is	O
+an	O
+important	O
+enterobacterial	O
+acid	O
+stress	O
+response	O
+enzyme	O
+whereas	O
+LdcC	O
+is	O
+its	O
+close	O
+paralogue	O
+thought	O
+to	O
+play	O
+mainly	O
+a	O
+metabolic	O
+role	O
+.	O
+	O
+Previously	O
+,	O
+we	O
+proposed	O
+a	O
+pseudoatomic	O
+model	O
+of	O
+the	O
+LdcI	O
+-	O
+RavA	O
+cage	O
+based	O
+on	O
+its	O
+cryo	O
+-	O
+electron	O
+microscopy	O
+map	O
+and	O
+crystal	O
+structures	O
+of	O
+an	O
+inactive	O
+LdcI	O
+decamer	O
+and	O
+a	O
+RavA	O
+monomer	O
+.	O
+	O
+Comparison	O
+with	O
+each	O
+other	O
+and	O
+with	O
+available	O
+structures	O
+uncovers	O
+differences	O
+between	O
+LdcI	O
+and	O
+LdcC	O
+explaining	O
+why	O
+only	O
+the	O
+acid	O
+stress	O
+response	O
+enzyme	O
+is	O
+capable	O
+of	O
+binding	O
+RavA	O
+.	O
+We	O
+identify	O
+interdomain	O
+movements	O
+associated	O
+with	O
+the	O
+pH	O
+-	O
+dependent	O
+enzyme	O
+activation	O
+and	O
+with	O
+the	O
+RavA	O
+binding	O
+.	O
+	O
+Enterobacterial	O
+inducible	O
+decarboxylases	O
+of	O
+basic	O
+amino	O
+acids	O
+lysine	O
+,	O
+arginine	O
+and	O
+ornithine	O
+have	O
+a	O
+common	O
+evolutionary	O
+origin	O
+and	O
+belong	O
+to	O
+the	O
+α	O
+-	O
+family	O
+of	O
+pyridoxal	O
+-	O
+5	O
+′-	O
+phosphate	O
+(	O
+PLP	O
+)-	O
+dependent	O
+enzymes	O
+.	O
+	O
+Each	O
+decarboxylase	O
+is	O
+induced	O
+by	O
+an	O
+excess	O
+of	O
+the	O
+target	O
+amino	O
+acid	O
+and	O
+a	O
+specific	O
+range	O
+of	O
+extracellular	O
+pH	O
+,	O
+and	O
+works	O
+in	O
+conjunction	O
+with	O
+a	O
+cognate	O
+inner	O
+membrane	O
+antiporter	O
+.	O
+	O
+Decarboxylation	O
+of	O
+the	O
+amino	O
+acid	O
+into	O
+a	O
+polyamine	O
+is	O
+catalysed	O
+by	O
+a	O
+PLP	O
+cofactor	O
+in	O
+a	O
+multistep	O
+reaction	O
+that	O
+consumes	O
+a	O
+cytoplasmic	O
+proton	O
+and	O
+produces	O
+a	O
+CO2	O
+molecule	O
+passively	O
+diffusing	O
+out	O
+of	O
+the	O
+cell	O
+,	O
+while	O
+the	O
+polyamine	O
+is	O
+excreted	O
+by	O
+the	O
+antiporter	O
+in	O
+exchange	O
+for	O
+a	O
+new	O
+amino	O
+acid	O
+substrate	O
+.	O
+	O
+Consequently	O
+,	O
+these	O
+enzymes	O
+buffer	O
+both	O
+the	O
+bacterial	O
+cytoplasm	O
+and	O
+the	O
+local	O
+extracellular	O
+environment	O
+.	O
+	O
+Inducible	O
+enterobacterial	O
+amino	O
+acid	O
+decarboxylases	O
+have	O
+been	O
+intensively	O
+studied	O
+since	O
+the	O
+early	O
+1940	O
+because	O
+the	O
+ability	O
+of	O
+bacteria	O
+to	O
+withstand	O
+acid	O
+stress	O
+can	O
+be	O
+linked	O
+to	O
+their	O
+pathogenicity	O
+in	O
+humans	O
+.	O
+	O
+Furthermore	O
+,	O
+both	O
+LdcI	O
+and	O
+the	O
+biosynthetic	O
+lysine	O
+decarboxylase	O
+LdcC	O
+of	O
+uropathogenic	O
+Escherichia	O
+coli	O
+(	O
+UPEC	O
+)	O
+appear	O
+to	O
+play	O
+an	O
+important	O
+role	O
+in	O
+increased	O
+resistance	O
+of	O
+this	O
+pathogen	O
+to	O
+nitrosative	O
+stress	O
+produced	O
+by	O
+nitric	O
+oxide	O
+and	O
+other	O
+damaging	O
+reactive	O
+nitrogen	O
+intermediates	O
+accumulating	O
+during	O
+the	O
+course	O
+of	O
+urinary	O
+tract	O
+infections	O
+(	O
+UTI	O
+).	O
+	O
+This	O
+effect	O
+is	O
+attributed	O
+to	O
+cadaverine	O
+,	O
+the	O
+diamine	O
+produced	O
+by	O
+decarboxylation	O
+of	O
+lysine	O
+by	O
+LdcI	O
+and	O
+LdcC	O
+,	O
+that	O
+was	O
+shown	O
+to	O
+enhance	O
+UPEC	O
+colonisation	O
+of	O
+the	O
+bladder	O
+.	O
+	O
+In	O
+addition	O
+,	O
+the	O
+biosynthetic	O
+E	O
+.	O
+coli	O
+lysine	O
+decarboxylase	O
+LdcC	O
+,	O
+long	O
+thought	O
+to	O
+be	O
+constitutively	O
+expressed	O
+in	O
+low	O
+amounts	O
+,	O
+was	O
+demonstrated	O
+to	O
+be	O
+strongly	O
+upregulated	O
+by	O
+fluoroquinolones	O
+via	O
+their	O
+induction	O
+of	O
+RpoS	O
+.	O
+A	O
+direct	O
+correlation	O
+between	O
+the	O
+level	O
+of	O
+cadaverine	O
+and	O
+the	O
+resistance	O
+of	O
+E	O
+.	O
+coli	O
+to	O
+these	O
+antibiotics	O
+commonly	O
+used	O
+as	O
+a	O
+first	O
+-	O
+line	O
+treatment	O
+of	O
+UTI	O
+could	O
+be	O
+established	O
+.	O
+	O
+Both	O
+acid	O
+pH	O
+and	O
+cadaverine	O
+induce	O
+closure	O
+of	O
+outer	O
+membrane	O
+porins	O
+thereby	O
+contributing	O
+to	O
+bacterial	O
+protection	O
+from	O
+acid	O
+stress	O
+,	O
+but	O
+also	O
+from	O
+certain	O
+antibiotics	O
+,	O
+by	O
+reduction	O
+in	O
+membrane	O
+permeability	O
+.	O
+	O
+Each	O
+monomer	O
+is	O
+composed	O
+of	O
+three	O
+domains	O
+–	O
+an	O
+N	O
+-	O
+terminal	O
+wing	O
+domain	O
+(	O
+residues	O
+1	O
+–	O
+129	O
+),	O
+a	O
+PLP	O
+-	O
+binding	O
+core	O
+domain	O
+(	O
+residues	O
+130	O
+–	O
+563	O
+),	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+domain	O
+(	O
+CTD	O
+,	O
+residues	O
+564	O
+–	O
+715	O
+).	O
+	O
+Ten	O
+years	O
+ago	O
+we	O
+showed	O
+that	O
+the	O
+E	O
+.	O
+coli	O
+AAA	O
++	O
+ATPase	O
+RavA	O
+,	O
+involved	O
+in	O
+multiple	O
+stress	O
+response	O
+pathways	O
+,	O
+tightly	O
+interacted	O
+with	O
+LdcI	O
+but	O
+was	O
+not	O
+capable	O
+of	O
+binding	O
+to	O
+LdcC	O
+.	O
+We	O
+described	O
+how	O
+two	O
+double	O
+pentameric	O
+rings	O
+of	O
+the	O
+LdcI	O
+tightly	O
+associate	O
+with	O
+five	O
+hexameric	O
+rings	O
+of	O
+RavA	O
+to	O
+form	O
+a	O
+unique	O
+cage	O
+-	O
+like	O
+architecture	O
+that	O
+enables	O
+the	O
+bacterium	O
+to	O
+withstand	O
+acid	O
+stress	O
+even	O
+under	O
+conditions	O
+of	O
+nutrient	O
+deprivation	O
+eliciting	O
+stringent	O
+response	O
+.	O
+	O
+Furthermore	O
+,	O
+we	O
+recently	O
+solved	O
+the	O
+structure	O
+of	O
+the	O
+E	O
+.	O
+coli	O
+LdcI	O
+-	O
+RavA	O
+complex	O
+by	O
+cryo	O
+-	O
+electron	O
+microscopy	O
+(	O
+cryoEM	O
+)	O
+and	O
+combined	O
+it	O
+with	O
+the	O
+crystal	O
+structures	O
+of	O
+the	O
+individual	O
+proteins	O
+.	O
+	O
+In	O
+spite	O
+of	O
+this	O
+wealth	O
+of	O
+structural	O
+information	O
+,	O
+the	O
+fact	O
+that	O
+LdcC	O
+does	O
+not	O
+interact	O
+with	O
+RavA	O
+,	O
+although	O
+the	O
+two	O
+lysine	O
+decarboxylases	O
+are	O
+69	O
+%	O
+identical	O
+and	O
+84	O
+%	O
+similar	O
+,	O
+and	O
+the	O
+physiological	O
+significance	O
+of	O
+the	O
+absence	O
+of	O
+this	O
+interaction	O
+remained	O
+unexplored	O
+.	O
+	O
+To	O
+solve	O
+this	O
+discrepancy	O
+,	O
+in	O
+the	O
+present	O
+work	O
+we	O
+provided	O
+a	O
+three	O
+-	O
+dimensional	O
+(	O
+3D	O
+)	O
+cryoEM	O
+reconstruction	O
+of	O
+LdcC	O
+and	O
+compared	O
+it	O
+with	O
+the	O
+available	O
+LdcI	O
+and	O
+LdcI	O
+-	O
+RavA	O
+structures	O
+.	O
+	O
+Given	O
+that	O
+the	O
+LdcI	O
+crystal	O
+structures	O
+were	O
+obtained	O
+at	O
+high	O
+pH	O
+where	O
+the	O
+enzyme	O
+is	O
+inactive	O
+(	O
+LdcIi	O
+,	O
+pH	O
+8	O
+.	O
+5	O
+),	O
+whereas	O
+the	O
+cryoEM	O
+reconstructions	O
+of	O
+LdcI	O
+-	O
+RavA	O
+and	O
+LdcI	O
+-	O
+LARA	O
+were	O
+done	O
+at	O
+acidic	O
+pH	O
+optimal	O
+for	O
+the	O
+enzymatic	O
+activity	O
+,	O
+for	O
+a	O
+meaningful	O
+comparison	O
+,	O
+we	O
+also	O
+produced	O
+a	O
+3D	O
+reconstruction	O
+of	O
+the	O
+LdcI	O
+at	O
+active	O
+pH	O
+(	O
+LdcIa	O
+,	O
+pH	O
+6	O
+.	O
+2	O
+).	O
+	O
+Remarkably	O
+,	O
+this	O
+analysis	O
+revealed	O
+that	O
+several	O
+specific	O
+residues	O
+in	O
+the	O
+above	O
+-	O
+mentioned	O
+β	O
+-	O
+sheet	O
+,	O
+independently	O
+of	O
+the	O
+rest	O
+of	O
+the	O
+protein	O
+sequence	O
+,	O
+are	O
+sufficient	O
+to	O
+define	O
+if	O
+a	O
+particular	O
+lysine	O
+decarboxylase	O
+should	O
+be	O
+classified	O
+as	O
+an	O
+“	O
+LdcC	O
+-	O
+like	O
+”	O
+or	O
+an	O
+“	O
+LdcI	O
+-	O
+like	O
+”.	O
+	O
+This	O
+fascinating	O
+parallelism	O
+between	O
+the	O
+propensity	O
+for	O
+RavA	O
+binding	O
+and	O
+the	O
+genetic	O
+environment	O
+of	O
+an	O
+enterobacterial	O
+lysine	O
+decarboxylase	O
+,	O
+as	O
+well	O
+as	O
+the	O
+high	O
+degree	O
+of	O
+conservation	O
+of	O
+this	O
+small	O
+structural	O
+motif	O
+,	O
+emphasize	O
+the	O
+functional	O
+importance	O
+of	O
+the	O
+interaction	O
+between	O
+biodegradative	O
+enterobacterial	O
+lysine	O
+decarboxylases	O
+and	O
+the	O
+AAA	O
++	O
+ATPase	O
+RavA	O
+.	O
+	O
+CryoEM	O
+3D	O
+reconstructions	O
+of	O
+LdcC	O
+,	O
+LdcIa	O
+and	O
+LdcI	O
+-	O
+LARA	O
+	O
+In	O
+the	O
+frame	O
+of	O
+this	O
+work	O
+,	O
+we	O
+produced	O
+two	O
+novel	O
+subnanometer	O
+resolution	O
+cryoEM	O
+reconstructions	O
+of	O
+the	O
+E	O
+.	O
+coli	O
+lysine	O
+decarboxylases	O
+at	O
+pH	O
+optimal	O
+for	O
+their	O
+enzymatic	O
+activity	O
+–	O
+a	O
+5	O
+.	O
+5	O
+Å	O
+resolution	O
+cryoEM	O
+map	O
+of	O
+the	O
+LdcC	O
+(	O
+pH	O
+7	O
+.	O
+5	O
+)	O
+for	O
+which	O
+no	O
+3D	O
+structural	O
+information	O
+has	O
+been	O
+previously	O
+available	O
+(	O
+Figs	O
+1A	O
+,	O
+B	O
+and	O
+S1	O
+),	O
+and	O
+a	O
+6	O
+.	O
+1	O
+Å	O
+resolution	O
+cryoEM	O
+map	O
+of	O
+the	O
+LdcIa	O
+,	O
+(	O
+pH	O
+6	O
+.	O
+2	O
+)	O
+(	O
+Figs	O
+1C	O
+,	O
+D	O
+and	O
+S2	O
+).	O
+	O
+The	O
+wing	O
+domains	O
+as	O
+a	O
+stable	O
+anchor	O
+at	O
+the	O
+center	O
+of	O
+the	O
+double	O
+-	O
+ring	O
+	O
+As	O
+a	O
+first	O
+step	O
+of	O
+a	O
+comparative	O
+analysis	O
+,	O
+we	O
+superimposed	O
+the	O
+three	O
+cryoEM	O
+reconstructions	O
+(	O
+LdcIa	O
+,	O
+LdcI	O
+-	O
+LARA	O
+and	O
+LdcC	O
+)	O
+and	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+LdcIi	O
+decamer	O
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Movie	O
+S1	O
+).	O
+	O
+This	O
+superposition	O
+reveals	O
+that	O
+the	O
+densities	O
+lining	O
+the	O
+central	O
+hole	O
+of	O
+the	O
+toroid	O
+are	O
+roughly	O
+at	O
+the	O
+same	O
+location	O
+,	O
+while	O
+the	O
+rest	O
+of	O
+the	O
+structure	O
+exhibits	O
+noticeable	O
+changes	O
+.	O
+	O
+Specifically	O
+,	O
+at	O
+the	O
+center	O
+of	O
+the	O
+double	O
+-	O
+ring	O
+the	O
+wing	O
+domains	O
+of	O
+the	O
+subunits	O
+provide	O
+the	O
+conserved	O
+basis	O
+for	O
+the	O
+assembly	O
+with	O
+the	O
+lowest	O
+root	O
+mean	O
+square	O
+deviation	O
+(	O
+RMSD	O
+)	O
+(	O
+between	O
+1	O
+.	O
+4	O
+and	O
+2	O
+Å	O
+for	O
+the	O
+Cα	O
+atoms	O
+only	O
+),	O
+whereas	O
+the	O
+peripheral	O
+CTDs	O
+containing	O
+the	O
+RavA	O
+binding	O
+interface	O
+manifest	O
+the	O
+highest	O
+RMSD	O
+(	O
+up	O
+to	O
+4	O
+.	O
+2	O
+Å	O
+)	O
+(	O
+Table	O
+S2	O
+).	O
+	O
+In	O
+addition	O
+,	O
+the	O
+wing	O
+domains	O
+of	O
+all	O
+structures	O
+are	O
+very	O
+similar	O
+,	O
+with	O
+the	O
+RMSD	O
+after	O
+optimal	O
+rigid	O
+body	O
+alignment	O
+(	O
+RMSDmin	O
+)	O
+less	O
+than	O
+1	O
+.	O
+1	O
+Å	O
+.	O
+Thus	O
+,	O
+taking	O
+the	O
+limited	O
+resolution	O
+of	O
+the	O
+cryoEM	O
+maps	O
+into	O
+account	O
+,	O
+we	O
+consider	O
+that	O
+the	O
+wing	O
+domains	O
+of	O
+all	O
+the	O
+four	O
+structures	O
+are	O
+essentially	O
+identical	O
+and	O
+that	O
+in	O
+the	O
+present	O
+study	O
+the	O
+RMSD	O
+of	O
+less	O
+than	O
+2	O
+Å	O
+can	O
+serve	O
+as	O
+a	O
+baseline	O
+below	O
+which	O
+differences	O
+may	O
+be	O
+assumed	O
+as	O
+insignificant	O
+.	O
+	O
+This	O
+preservation	O
+of	O
+the	O
+central	O
+part	O
+of	O
+the	O
+double	O
+-	O
+ring	O
+assembly	O
+may	O
+help	O
+the	O
+enzymes	O
+to	O
+maintain	O
+their	O
+decameric	O
+state	O
+upon	O
+activation	O
+and	O
+incorporation	O
+into	O
+the	O
+LdcI	O
+-	O
+RavA	O
+cage	O
+.	O
+	O
+The	O
+core	O
+domain	O
+and	O
+the	O
+active	O
+site	O
+rearrangements	O
+upon	O
+pH	O
+-	O
+dependent	O
+enzyme	O
+activation	O
+and	O
+LARA	O
+binding	O
+	O
+The	O
+decameric	O
+enzyme	O
+is	O
+built	O
+of	O
+five	O
+dimers	O
+associating	O
+into	O
+a	O
+5	O
+-	O
+fold	O
+symmetrical	O
+double	O
+-	O
+ring	O
+(	O
+two	O
+monomers	O
+making	O
+a	O
+dimer	O
+are	O
+delineated	O
+in	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+As	O
+common	O
+for	O
+the	O
+α	O
+family	O
+of	O
+the	O
+PLP	O
+-	O
+dependent	O
+decarboxylases	O
+,	O
+dimerization	O
+is	O
+required	O
+for	O
+the	O
+enzymatic	O
+activity	O
+because	O
+the	O
+active	O
+site	O
+is	O
+buried	O
+in	O
+the	O
+dimer	O
+interface	O
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+	O
+This	O
+interface	O
+is	O
+formed	O
+essentially	O
+by	O
+the	O
+core	O
+domains	O
+with	O
+some	O
+contribution	O
+of	O
+the	O
+CTDs	O
+.	O
+	O
+Zooming	O
+in	O
+the	O
+variations	O
+in	O
+the	O
+PLP	O
+-	O
+SD	O
+shows	O
+that	O
+most	O
+of	O
+the	O
+structural	O
+changes	O
+concern	O
+displacements	O
+in	O
+the	O
+active	O
+site	O
+(	O
+Fig	O
+.	O
+3C	O
+–	O
+F	O
+).	O
+	O
+The	O
+most	O
+conspicuous	O
+differences	O
+between	O
+the	O
+PLP	O
+-	O
+SDs	O
+can	O
+be	O
+linked	O
+to	O
+the	O
+pH	O
+-	O
+dependent	O
+activation	O
+of	O
+the	O
+enzymes	O
+.	O
+	O
+Between	O
+these	O
+two	O
+extremes	O
+,	O
+the	O
+PLP	O
+-	O
+SDs	O
+of	O
+LdcIa	O
+and	O
+LdcC	O
+are	O
+similar	O
+both	O
+in	O
+the	O
+context	O
+of	O
+the	O
+decamer	O
+(	O
+Fig	O
+.	O
+3F	O
+)	O
+and	O
+in	O
+terms	O
+of	O
+RMSDmin	O
+=	O
+0	O
+.	O
+9	O
+Å	O
+,	O
+which	O
+probably	O
+reflects	O
+the	O
+fact	O
+that	O
+,	O
+at	O
+the	O
+optimal	O
+pH	O
+,	O
+these	O
+lysine	O
+decarboxylases	O
+have	O
+a	O
+similar	O
+enzymatic	O
+activity	O
+.	O
+	O
+In	O
+addition	O
+,	O
+our	O
+earlier	O
+biochemical	O
+observation	O
+that	O
+the	O
+enzymatic	O
+activity	O
+of	O
+LdcIa	O
+is	O
+unaffected	O
+by	O
+RavA	O
+binding	O
+is	O
+consistent	O
+with	O
+the	O
+relatively	O
+small	O
+changes	O
+undergone	O
+by	O
+the	O
+active	O
+site	O
+upon	O
+transition	O
+from	O
+LdcIa	O
+to	O
+LdcI	O
+-	O
+LARA	O
+.	O
+	O
+Worthy	O
+of	O
+note	O
+,	O
+our	O
+previous	O
+comparison	O
+of	O
+the	O
+crystal	O
+structure	O
+of	O
+LdcIi	O
+with	O
+that	O
+of	O
+the	O
+inducible	O
+arginine	O
+decarboxylase	O
+AdiA	O
+revealed	O
+high	O
+conservation	O
+of	O
+the	O
+PLP	O
+-	O
+coordinating	O
+residues	O
+and	O
+identified	O
+a	O
+patch	O
+of	O
+negatively	O
+charged	O
+residues	O
+lining	O
+the	O
+active	O
+site	O
+channel	O
+as	O
+a	O
+potential	O
+binding	O
+site	O
+for	O
+the	O
+target	O
+amino	O
+acid	O
+substrate	O
+(	O
+Figs	O
+S3	O
+and	O
+S4	O
+in	O
+ref	O
+.).	O
+	O
+Rearrangements	O
+of	O
+the	O
+ppGpp	O
+binding	O
+pocket	O
+upon	O
+pH	O
+-	O
+dependent	O
+enzyme	O
+activation	O
+and	O
+LARA	O
+binding	O
+	O
+An	O
+inhibitor	O
+of	O
+the	O
+LdcI	O
+and	O
+LdcC	O
+activity	O
+,	O
+the	O
+stringent	O
+response	O
+alarmone	O
+ppGpp	O
+,	O
+is	O
+known	O
+to	O
+bind	O
+at	O
+the	O
+interface	O
+between	O
+neighboring	O
+monomers	O
+within	O
+each	O
+ring	O
+(	O
+Fig	O
+.	O
+S4	O
+).	O
+	O
+The	O
+ppGpp	O
+binding	O
+pocket	O
+is	O
+made	O
+up	O
+by	O
+residues	O
+from	O
+all	O
+domains	O
+and	O
+is	O
+located	O
+approximately	O
+30	O
+Å	O
+away	O
+from	O
+the	O
+PLP	O
+moiety	O
+.	O
+	O
+Whereas	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+ppGpp	O
+-	O
+LdcIi	O
+was	O
+solved	O
+to	O
+2	O
+Å	O
+resolution	O
+,	O
+only	O
+a	O
+4	O
+.	O
+1	O
+Å	O
+resolution	O
+structure	O
+of	O
+the	O
+ppGpp	O
+-	O
+free	O
+LdcIi	O
+could	O
+be	O
+obtained	O
+.	O
+	O
+Thus	O
+,	O
+we	O
+speculated	O
+that	O
+inhibition	O
+of	O
+LdcI	O
+by	O
+ppGpp	O
+would	O
+be	O
+accompanied	O
+by	O
+a	O
+transduction	O
+of	O
+subtle	O
+structural	O
+changes	O
+at	O
+the	O
+level	O
+of	O
+individual	O
+amino	O
+acid	O
+side	O
+chains	O
+between	O
+the	O
+ppGpp	O
+binding	O
+pocket	O
+and	O
+the	O
+active	O
+site	O
+of	O
+the	O
+enzyme	O
+.	O
+	O
+All	O
+our	O
+current	O
+cryoEM	O
+reconstructions	O
+of	O
+the	O
+lysine	O
+decarboxylases	O
+were	O
+obtained	O
+in	O
+the	O
+absence	O
+of	O
+ppGpp	O
+in	O
+order	O
+to	O
+be	O
+closer	O
+to	O
+the	O
+active	O
+state	O
+of	O
+the	O
+enzymes	O
+under	O
+study	O
+.	O
+	O
+The	O
+fact	O
+that	O
+interaction	O
+with	O
+RavA	O
+reduces	O
+the	O
+ppGpp	O
+affinity	O
+for	O
+LdcI	O
+despite	O
+the	O
+long	O
+distance	O
+of	O
+~	O
+30	O
+Å	O
+between	O
+the	O
+LARA	O
+domain	O
+binding	O
+site	O
+and	O
+the	O
+closest	O
+ppGpp	O
+binding	O
+pocket	O
+(	O
+Fig	O
+.	O
+S5	O
+)	O
+seems	O
+to	O
+favor	O
+an	O
+allosteric	O
+regulation	O
+mechanism	O
+.	O
+	O
+Interestingly	O
+,	O
+although	O
+a	O
+number	O
+of	O
+ppGpp	O
+binding	O
+residues	O
+are	O
+strictly	O
+conserved	O
+between	O
+LdcI	O
+and	O
+AdiA	O
+that	O
+also	O
+forms	O
+decamers	O
+at	O
+low	O
+pH	O
+optimal	O
+for	O
+its	O
+arginine	O
+decarboxylase	O
+activity	O
+,	O
+no	O
+ppGpp	O
+regulation	O
+of	O
+AdiA	O
+could	O
+be	O
+demonstrated	O
+.	O
+	O
+Indeed	O
+,	O
+all	O
+CTDs	O
+have	O
+very	O
+similar	O
+structures	O
+(	O
+RMSDmin	O
+<	O
+1	O
+Å	O
+).	O
+	O
+The	O
+LdcIi	O
+monomer	O
+is	O
+the	O
+most	O
+compact	O
+,	O
+whereas	O
+LdcIa	O
+and	O
+especially	O
+LdcI	O
+-	O
+LARA	O
+gradually	O
+extend	O
+their	O
+CTDs	O
+towards	O
+the	O
+LARA	O
+domain	O
+of	O
+RavA	O
+(	O
+Figs	O
+2	O
+and	O
+4	O
+).	O
+	O
+These	O
+small	O
+but	O
+noticeable	O
+swinging	O
+and	O
+stretching	O
+(	O
+up	O
+to	O
+~	O
+4	O
+Å	O
+)	O
+may	O
+be	O
+related	O
+to	O
+the	O
+incorporation	O
+of	O
+the	O
+LdcI	O
+decamer	O
+into	O
+the	O
+LdcI	O
+-	O
+RavA	O
+cage	O
+.	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+of	O
+a	O
+lysine	O
+decarboxylase	O
+as	O
+a	O
+major	O
+determinant	O
+of	O
+the	O
+interaction	O
+with	O
+RavA	O
+	O
+In	O
+our	O
+previous	O
+contribution	O
+,	O
+based	O
+on	O
+the	O
+fit	O
+of	O
+the	O
+LdcIi	O
+and	O
+the	O
+LARA	O
+crystal	O
+structures	O
+into	O
+the	O
+LdcI	O
+-	O
+LARA	O
+cryoEM	O
+density	O
+,	O
+we	O
+predicted	O
+that	O
+the	O
+LdcI	O
+-	O
+RavA	O
+interaction	O
+should	O
+involve	O
+the	O
+C	O
+-	O
+terminal	O
+two	O
+-	O
+stranded	O
+β	O
+-	O
+sheet	O
+of	O
+the	O
+LdcI	O
+.	O
+Our	O
+present	O
+cryoEM	O
+maps	O
+and	O
+pseudoatomic	O
+models	O
+provide	O
+first	O
+structure	O
+-	O
+based	O
+insights	O
+into	O
+the	O
+differences	O
+between	O
+the	O
+inducible	O
+and	O
+the	O
+constitutive	O
+lysine	O
+decarboxylases	O
+.	O
+	O
+Therefore	O
+,	O
+we	O
+wanted	O
+to	O
+check	O
+the	O
+influence	O
+of	O
+the	O
+primary	O
+sequence	O
+of	O
+the	O
+two	O
+proteins	O
+in	O
+this	O
+region	O
+on	O
+their	O
+ability	O
+to	O
+interact	O
+with	O
+RavA	O
+.	O
+To	O
+this	O
+end	O
+,	O
+we	O
+swapped	O
+the	O
+relevant	O
+β	O
+-	O
+sheets	O
+of	O
+the	O
+two	O
+proteins	O
+and	O
+produced	O
+their	O
+chimeras	B-mutant
+,	O
+namely	O
+LdcIC	B-mutant
+(	O
+i	O
+.	O
+e	O
+.	O
+LdcI	O
+with	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+of	O
+LdcC	O
+)	O
+and	O
+LdcCI	B-mutant
+(	O
+i	O
+.	O
+e	O
+.	O
+LdcC	O
+with	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+of	O
+LdcI	O
+)	O
+(	O
+Fig	O
+.	O
+5A	O
+–	O
+C	O
+).	O
+	O
+Both	B-mutant
+constructs	I-mutant
+could	O
+be	O
+purified	O
+and	O
+could	O
+form	O
+decamers	O
+visually	O
+indistinguishable	O
+from	O
+the	O
+wild	O
+-	O
+type	O
+proteins	O
+.	O
+	O
+As	O
+expected	O
+,	O
+binding	O
+of	O
+LdcI	O
+to	O
+RavA	O
+was	O
+completely	O
+abolished	O
+by	O
+this	O
+procedure	O
+and	O
+no	O
+LdcIC	O
+-	O
+RavA	O
+complex	O
+could	O
+be	O
+detected	O
+.	O
+	O
+On	O
+the	O
+negative	O
+stain	O
+EM	O
+grid	O
+,	O
+the	O
+chimeric	O
+cages	O
+appeared	O
+less	O
+rigid	O
+than	O
+the	O
+native	O
+LdcI	O
+-	O
+RavA	O
+,	O
+which	O
+probably	O
+means	O
+that	O
+the	O
+environment	O
+of	O
+the	O
+β	O
+-	O
+sheet	O
+contributes	O
+to	O
+the	O
+efficiency	O
+of	O
+the	O
+interaction	O
+and	O
+the	O
+stability	O
+of	O
+the	O
+entire	O
+architecture	O
+(	O
+Fig	O
+.	O
+5D	O
+–	O
+F	O
+).	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+of	O
+a	O
+lysine	O
+decarboxylase	O
+is	O
+a	O
+highly	O
+conserved	O
+signature	O
+allowing	O
+to	O
+distinguish	O
+between	O
+LdcI	O
+and	O
+LdcC	O
+	O
+Alignment	O
+of	O
+the	O
+primary	O
+sequences	O
+of	O
+the	O
+E	O
+.	O
+coli	O
+LdcI	O
+and	O
+LdcC	O
+shows	O
+that	O
+some	O
+amino	O
+acid	O
+residues	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+are	O
+the	O
+same	O
+in	O
+the	O
+two	O
+proteins	O
+,	O
+whereas	O
+others	O
+are	O
+notably	O
+different	O
+in	O
+chemical	O
+nature	O
+.	O
+	O
+Importantly	O
+,	O
+most	O
+of	O
+the	O
+amino	O
+acid	O
+differences	O
+between	O
+the	O
+two	O
+enzymes	O
+are	O
+located	O
+in	O
+this	O
+very	O
+region	O
+.	O
+	O
+Thus	O
+,	O
+to	O
+advance	O
+beyond	O
+our	O
+experimental	O
+confirmation	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+as	O
+a	O
+major	O
+determinant	O
+of	O
+the	O
+capacity	O
+of	O
+a	O
+particular	O
+lysine	O
+decarboxylase	O
+to	O
+form	O
+a	O
+cage	O
+with	O
+RavA	O
+,	O
+we	O
+set	O
+out	O
+to	O
+investigate	O
+whether	O
+certain	O
+residues	O
+in	O
+this	O
+β	O
+-	O
+sheet	O
+are	O
+conserved	O
+in	O
+lysine	O
+decarboxylases	O
+of	O
+different	O
+enterobacteria	O
+that	O
+have	O
+the	O
+ravA	O
+-	O
+viaA	O
+operon	O
+in	O
+their	O
+genome	O
+.	O
+	O
+We	O
+inspected	O
+the	O
+genetic	O
+environment	O
+of	O
+lysine	O
+decarboxylases	O
+from	O
+22	O
+enterobacterial	O
+species	O
+referenced	O
+in	O
+the	O
+NCBI	O
+database	O
+,	O
+corrected	O
+the	O
+gene	O
+annotation	O
+where	O
+necessary	O
+(	O
+Tables	O
+S3	O
+and	O
+S4	O
+),	O
+and	O
+performed	O
+multiple	O
+sequence	O
+alignment	O
+coupled	O
+to	O
+a	O
+phylogenetic	O
+analysis	O
+(	O
+see	O
+Methods	O
+).	O
+	O
+First	O
+of	O
+all	O
+,	O
+consensus	O
+sequence	O
+for	O
+the	O
+entire	O
+lysine	O
+decarboxylase	O
+family	O
+was	O
+derived	O
+.	O
+	O
+Second	O
+,	O
+the	O
+phylogenetic	O
+analysis	O
+clearly	O
+split	O
+the	O
+lysine	O
+decarboxylases	O
+into	O
+two	O
+groups	O
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+	O
+All	O
+lysine	O
+decarboxylases	O
+predicted	O
+to	O
+be	O
+“	O
+LdcI	O
+-	O
+like	O
+”	O
+or	O
+biodegradable	O
+based	O
+on	O
+their	O
+genetic	O
+environment	O
+,	O
+as	O
+for	O
+example	O
+their	O
+organization	O
+in	O
+an	O
+operon	O
+with	O
+a	O
+gene	O
+encoding	O
+the	O
+CadB	O
+antiporter	O
+(	O
+see	O
+Methods	O
+),	O
+were	O
+found	O
+in	O
+one	O
+group	O
+,	O
+whereas	O
+all	O
+enzymes	O
+predicted	O
+as	O
+“	O
+LdcC	O
+-	O
+like	O
+”	O
+or	O
+biosynthetic	O
+partitioned	O
+into	O
+another	O
+group	O
+.	O
+	O
+Thus	O
+,	O
+consensus	O
+sequences	O
+could	O
+also	O
+be	O
+determined	O
+for	O
+each	O
+of	O
+the	O
+two	O
+groups	O
+(	O
+Figs	O
+6B	O
+,	O
+C	O
+and	O
+S7	O
+).	O
+	O
+Inspection	O
+of	O
+these	O
+consensus	O
+sequences	O
+revealed	O
+important	O
+differences	O
+between	O
+the	O
+groups	O
+regarding	O
+charge	O
+,	O
+size	O
+and	O
+hydrophobicity	O
+of	O
+several	O
+residues	O
+precisely	O
+at	O
+the	O
+level	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+that	O
+is	O
+responsible	O
+for	O
+the	O
+interaction	O
+with	O
+RavA	O
+(	O
+Fig	O
+.	O
+6B	O
+–	O
+D	O
+).	O
+	O
+For	O
+example	O
+,	O
+in	O
+our	O
+previous	O
+study	O
+,	O
+site	O
+-	O
+directed	O
+mutations	O
+identified	O
+Y697	O
+as	O
+critically	O
+required	O
+for	O
+the	O
+RavA	O
+binding	O
+.	O
+	O
+Our	O
+current	O
+analysis	O
+shows	O
+that	O
+Y697	O
+is	O
+strictly	O
+conserved	O
+in	O
+the	O
+“	O
+LdcI	O
+-	O
+like	O
+”	O
+group	O
+whereas	O
+the	O
+“	O
+LdcC	O
+-	O
+like	O
+”	O
+enzymes	O
+always	O
+have	O
+a	O
+lysine	O
+in	O
+this	O
+position	O
+;	O
+it	O
+also	O
+uncovers	O
+several	O
+other	O
+residues	O
+potentially	O
+essential	O
+for	O
+the	O
+interaction	O
+with	O
+RavA	O
+which	O
+can	O
+now	O
+be	O
+addressed	O
+by	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+.	O
+	O
+The	O
+third	O
+and	O
+most	O
+remarkable	O
+finding	O
+was	O
+that	O
+exactly	O
+the	O
+same	O
+separation	O
+into	O
+“	O
+LdcI	O
+-	O
+like	O
+”	O
+and	O
+“	O
+LdcC	O
+”-	O
+like	O
+groups	O
+can	O
+be	O
+obtained	O
+based	O
+on	O
+a	O
+comparison	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheets	O
+only	O
+,	O
+without	O
+taking	O
+the	O
+rest	O
+of	O
+the	O
+primary	O
+sequence	O
+into	O
+account	O
+.	O
+	O
+Therefore	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+emerges	O
+as	O
+being	O
+a	O
+highly	O
+conserved	O
+signature	O
+sequence	O
+,	O
+sufficient	O
+to	O
+unambiguously	O
+discriminate	O
+between	O
+the	O
+“	O
+LdcI	O
+-	O
+like	O
+”	O
+and	O
+“	O
+LdcC	O
+-	O
+like	O
+”	O
+enterobacterial	O
+lysine	O
+decarboxylases	O
+independently	O
+of	O
+any	O
+other	O
+information	O
+(	O
+Figs	O
+6	O
+and	O
+S7	O
+).	O
+	O
+Thus	O
+,	O
+enterobacteria	O
+identified	O
+here	O
+(	O
+Fig	O
+.	O
+6	O
+,	O
+Table	O
+S4	O
+)	O
+appear	O
+to	O
+exert	O
+evolutionary	O
+pressure	O
+on	O
+the	O
+biodegradative	O
+lysine	O
+decarboxylase	O
+towards	O
+the	O
+RavA	O
+binding	O
+.	O
+	O
+The	O
+conformational	O
+rearrangements	O
+of	O
+LdcI	O
+upon	O
+enzyme	O
+activation	O
+and	O
+RavA	O
+binding	O
+revealed	O
+in	O
+this	O
+work	O
+,	O
+and	O
+our	O
+amazing	O
+finding	O
+that	O
+the	O
+molecular	O
+determinant	O
+of	O
+the	O
+LdcI	O
+-	O
+RavA	O
+interaction	O
+is	O
+the	O
+one	O
+that	O
+straightforwardly	O
+determines	O
+if	O
+a	O
+particular	O
+enterobacterial	O
+lysine	O
+decarboxylase	O
+belongs	O
+to	O
+“	O
+LdcI	O
+-	O
+like	O
+”	O
+or	O
+“	O
+LdcC	O
+-	O
+like	O
+”	O
+proteins	O
+,	O
+should	O
+give	O
+a	O
+new	O
+impetus	O
+to	O
+functional	O
+studies	O
+of	O
+the	O
+unique	O
+LdcI	O
+-	O
+RavA	O
+cage	O
+.	O
+	O
+Together	O
+with	O
+the	O
+apo	O
+-	O
+LdcI	O
+and	O
+ppGpp	O
+-	O
+LdcIi	O
+crystal	O
+structures	O
+,	O
+our	O
+cryoEM	O
+reconstructions	O
+provide	O
+a	O
+structural	O
+framework	O
+for	O
+future	O
+studies	O
+of	O
+structure	O
+-	O
+function	O
+relationships	O
+of	O
+lysine	O
+decarboxylases	O
+from	O
+other	O
+enterobacteria	O
+and	O
+even	O
+of	O
+their	O
+homologues	O
+outside	O
+Enterobacteriaceae	O
+.	O
+For	O
+example	O
+,	O
+the	O
+lysine	O
+decarboxylase	O
+of	O
+Eikenella	O
+corrodens	O
+is	O
+thought	O
+to	O
+play	O
+a	O
+major	O
+role	O
+in	O
+the	O
+periodontal	O
+disease	O
+and	O
+its	O
+inhibitors	O
+were	O
+shown	O
+to	O
+retard	O
+gingivitis	O
+development	O
+.	O
+	O
+Finally	O
+,	O
+cadaverine	O
+being	O
+an	O
+important	O
+platform	O
+chemical	O
+for	O
+the	O
+production	O
+of	O
+industrial	O
+polymers	O
+such	O
+as	O
+nylon	O
+,	O
+structural	O
+information	O
+is	O
+valuable	O
+for	O
+optimisation	O
+of	O
+bacterial	O
+lysine	O
+decarboxylases	O
+used	O
+for	O
+its	O
+production	O
+in	O
+biotechnology	O
+.	O
+	O
+3D	O
+cryoEM	O
+reconstructions	O
+of	O
+LdcC	O
+,	O
+LdcI	O
+-	O
+LARA	O
+and	O
+LdcIa	O
+.	O
+	O
+In	O
+the	O
+rest	O
+of	O
+the	O
+protomers	O
+,	O
+the	O
+wing	O
+,	O
+core	O
+and	O
+C	O
+-	O
+terminal	O
+domains	O
+are	O
+colored	O
+from	O
+light	O
+to	O
+dark	O
+in	O
+shades	O
+of	O
+green	O
+for	O
+LdcC	O
+(	O
+A	O
+),	O
+pink	O
+for	O
+LdcIa	O
+(	O
+C	O
+)	O
+and	O
+blue	O
+for	O
+LdcI	O
+in	O
+LdcI	O
+-	O
+LARA	O
+(	O
+E	O
+).	O
+	O
+In	O
+(	O
+E	O
+),	O
+the	O
+LARA	O
+domain	O
+density	O
+is	O
+shown	O
+in	O
+dark	O
+grey	O
+.	O
+	O
+Two	O
+monomers	O
+making	O
+a	O
+dimer	O
+are	O
+delineated	O
+.	O
+	O
+Scale	O
+bar	O
+50	O
+Å	O
+.	O
+(	O
+B	O
+,	O
+D	O
+,	O
+F	O
+)	O
+One	O
+protomer	O
+from	O
+the	O
+cryoEM	O
+map	O
+of	O
+the	O
+LdcC	O
+(	O
+B	O
+),	O
+LdcIa	O
+(	O
+D	O
+)	O
+and	O
+LdcI	O
+-	O
+LARA	O
+(	O
+F	O
+)	O
+in	O
+light	O
+grey	O
+with	O
+the	O
+pseudoatomic	O
+model	O
+represented	O
+as	O
+cartoons	O
+and	O
+colored	O
+as	O
+the	O
+densities	O
+in	O
+(	O
+A	O
+,	O
+C	O
+,	O
+E	O
+).	O
+	O
+Superposition	O
+of	O
+the	O
+pseudoatomic	O
+models	O
+of	O
+LdcC	O
+,	O
+LdcI	O
+from	O
+LdcI	O
+-	O
+LARA	O
+and	O
+LdcIa	O
+colored	O
+as	O
+in	O
+Fig	O
+.	O
+1	O
+,	O
+and	O
+the	O
+crystal	O
+structure	O
+of	O
+LdcIi	O
+in	O
+shades	O
+of	O
+yellow	O
+.	O
+	O
+The	O
+dashed	O
+circle	O
+indicates	O
+the	O
+central	O
+region	O
+that	O
+remains	O
+virtually	O
+unchanged	O
+between	O
+all	O
+the	O
+structures	O
+,	O
+while	O
+the	O
+periphery	O
+undergoes	O
+visible	O
+movements	O
+.	O
+	O
+(	O
+A	O
+)	O
+LdcIi	O
+crystal	O
+structure	O
+,	O
+with	O
+one	O
+ring	O
+represented	O
+as	O
+a	O
+grey	O
+surface	O
+and	O
+the	O
+second	O
+as	O
+a	O
+cartoon	O
+.	O
+	O
+A	O
+monomer	O
+with	O
+its	O
+PLP	O
+cofactor	O
+is	O
+delineated	O
+.	O
+	O
+The	O
+PLP	O
+moieties	O
+of	O
+the	O
+cartoon	O
+ring	O
+are	O
+shown	O
+in	O
+red	O
+.	O
+	O
+(	O
+B	O
+)	O
+The	O
+LdcIi	O
+dimer	O
+extracted	O
+from	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+decamer	O
+.	O
+	O
+One	O
+monomer	O
+is	O
+colored	O
+in	O
+shades	O
+of	O
+yellow	O
+as	O
+in	O
+Figs	O
+1	O
+and	O
+2	O
+,	O
+while	O
+the	O
+monomer	O
+related	O
+by	O
+C2	O
+symmetry	O
+is	O
+grey	O
+.	O
+	O
+The	O
+PLP	O
+is	O
+red	O
+.	O
+	O
+Stretching	O
+of	O
+the	O
+LdcI	O
+monomer	O
+upon	O
+pH	O
+-	O
+dependent	O
+enzyme	O
+activation	O
+and	O
+LARA	O
+binding	O
+.	O
+	O
+(	O
+A	O
+–	O
+C	O
+)	O
+A	O
+slice	O
+through	O
+the	O
+pseudoatomic	O
+models	O
+of	O
+the	O
+LdcI	O
+monomers	O
+extracted	O
+from	O
+the	O
+superimposed	O
+decamers	O
+(	O
+Fig	O
+.	O
+2	O
+)	O
+The	O
+rectangle	O
+indicates	O
+the	O
+regions	O
+enlarged	O
+in	O
+(	O
+D	O
+–	O
+F	O
+).	O
+	O
+(	O
+A	O
+)	O
+compares	O
+LdcIi	O
+(	O
+yellow	O
+)	O
+and	O
+LdcIa	O
+(	O
+pink	O
+),	O
+(	O
+B	O
+)	O
+compares	O
+LdcIa	O
+(	O
+pink	O
+)	O
+and	O
+LdcI	O
+-	O
+LARA	O
+(	O
+blue	O
+),	O
+and	O
+(	O
+C	O
+)	O
+compares	O
+LdcIi	O
+(	O
+yellow	O
+),	O
+LdcIa	O
+(	O
+pink	O
+)	O
+and	O
+LdcI	O
+-	O
+LARA	O
+(	O
+blue	O
+)	O
+simultaneously	O
+in	O
+order	O
+to	O
+show	O
+the	O
+progressive	O
+stretching	O
+described	O
+in	O
+the	O
+text	O
+.	O
+	O
+The	O
+cryoEM	O
+density	O
+of	O
+the	O
+LARA	O
+domain	O
+is	O
+represented	O
+as	O
+a	O
+grey	O
+surface	O
+to	O
+show	O
+the	O
+position	O
+of	O
+the	O
+binding	O
+site	O
+and	O
+the	O
+direction	O
+of	O
+the	O
+movement	O
+.	O
+	O
+(	O
+D	O
+–	O
+F	O
+)	O
+Inserts	O
+zooming	O
+at	O
+the	O
+CTD	O
+part	O
+in	O
+proximity	O
+of	O
+the	O
+LARA	O
+binding	O
+site	O
+.	O
+	O
+Analysis	O
+of	O
+the	O
+LdcIC	B-mutant
+and	O
+LdcCI	B-mutant
+chimeras	B-mutant
+.	O
+	O
+Sequence	O
+analysis	O
+of	O
+enterobacterial	O
+lysine	O
+decarboxylases	O
+.	O
+	O
+(	O
+B	O
+)	O
+Analysis	O
+of	O
+consensus	O
+“	O
+LdcI	O
+-	O
+like	O
+”	O
+and	O
+“	O
+LdcC	O
+-	O
+like	O
+”	O
+sequences	O
+around	O
+the	O
+first	O
+and	O
+second	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+strands	O
+.	O
+	O
+(	O
+C	O
+)	O
+Signature	O
+sequences	O
+of	O
+LdcI	O
+and	O
+LdcC	O
+in	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+.	O
+	O
+Polarity	O
+differences	O
+are	O
+highlighted	O
+.	O
+(	O
+D	O
+)	O
+Position	O
+and	O
+nature	O
+of	O
+these	O
+differences	O
+at	O
+the	O
+surface	O
+of	O
+the	O
+respective	O
+cryoEM	O
+maps	O
+with	O
+the	O
+color	O
+code	O
+as	O
+in	O
+B	O
+.	O
+See	O
+also	O
+Fig	O
+.	O
+S7	O
+and	O
+Tables	O
+S3	O
+and	O
+S4	O
+.	O
+	O
+Mep2	O
+activity	O
+is	O
+tightly	O
+regulated	O
+by	O
+phosphorylation	O
+,	O
+but	O
+how	O
+this	O
+is	O
+achieved	O
+at	O
+the	O
+molecular	O
+level	O
+is	O
+not	O
+clear	O
+.	O
+	O
+Here	O
+we	O
+report	O
+X	O
+-	O
+ray	O
+crystal	O
+structures	O
+of	O
+the	O
+Mep2	O
+orthologues	O
+from	O
+Saccharomyces	O
+cerevisiae	O
+and	O
+Candida	O
+albicans	O
+and	O
+show	O
+that	O
+under	O
+nitrogen	O
+-	O
+sufficient	O
+conditions	O
+the	O
+transporters	O
+are	O
+not	O
+phosphorylated	O
+and	O
+present	O
+in	O
+closed	O
+,	O
+inactive	O
+conformations	O
+.	O
+	O
+The	O
+phosphorylation	O
+site	O
+in	O
+the	O
+CTR	O
+is	O
+solvent	O
+accessible	O
+and	O
+located	O
+in	O
+a	O
+negatively	O
+charged	O
+pocket	O
+∼	O
+30	O
+Å	O
+away	O
+from	O
+the	O
+channel	O
+exit	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+phosphorylation	O
+-	O
+mimicking	O
+Mep2	B-mutant
+variants	I-mutant
+from	O
+C	O
+.	O
+albicans	O
+show	O
+large	O
+conformational	O
+changes	O
+in	O
+a	O
+conserved	O
+and	O
+functionally	O
+important	O
+region	O
+of	O
+the	O
+CTR	O
+.	O
+	O
+The	O
+results	O
+allow	O
+us	O
+to	O
+propose	O
+a	O
+model	O
+for	O
+regulation	O
+of	O
+eukaryotic	O
+ammonium	O
+transport	O
+by	O
+phosphorylation	O
+.	O
+	O
+Mep2	O
+proteins	O
+are	O
+tightly	O
+regulated	O
+fungal	O
+ammonium	O
+transporters	O
+.	O
+	O
+Transceptors	O
+are	O
+membrane	O
+proteins	O
+that	O
+function	O
+not	O
+only	O
+as	O
+transporters	O
+but	O
+also	O
+as	O
+receptors	O
+/	O
+sensors	O
+during	O
+nutrient	O
+sensing	O
+to	O
+activate	O
+downstream	O
+signalling	O
+pathways	O
+.	O
+	O
+One	O
+of	O
+the	O
+most	O
+important	O
+unresolved	O
+questions	O
+in	O
+the	O
+field	O
+is	O
+how	O
+the	O
+transceptors	O
+couple	O
+to	O
+downstream	O
+signalling	O
+pathways	O
+.	O
+	O
+One	O
+hypothesis	O
+is	O
+that	O
+downstream	O
+signalling	O
+is	O
+dependent	O
+on	O
+a	O
+specific	O
+conformation	O
+of	O
+the	O
+transporter	O
+.	O
+	O
+Mep2	O
+(	O
+methylammonium	O
+(	O
+MA	O
+)	O
+permease	O
+)	O
+proteins	O
+are	O
+ammonium	O
+transceptors	O
+that	O
+are	O
+ubiquitous	O
+in	O
+fungi	O
+.	O
+	O
+They	O
+belong	O
+to	O
+the	O
+Amt	O
+/	O
+Mep	O
+/	O
+Rh	O
+family	O
+of	O
+transporters	O
+that	O
+are	O
+present	O
+in	O
+all	O
+kingdoms	O
+of	O
+life	O
+and	O
+they	O
+take	O
+up	O
+ammonium	O
+from	O
+the	O
+extracellular	O
+environment	O
+.	O
+	O
+Fungi	O
+typically	O
+have	O
+more	O
+than	O
+one	O
+Mep	O
+paralogue	O
+,	O
+for	O
+example	O
+,	O
+Mep1	O
+-	O
+3	O
+in	O
+S	O
+.	O
+cerevisiae	O
+.	O
+	O
+Under	O
+conditions	O
+of	O
+nitrogen	O
+limitation	O
+,	O
+Mep2	O
+initiates	O
+a	O
+signalling	O
+cascade	O
+that	O
+results	O
+in	O
+a	O
+switch	O
+from	O
+the	O
+yeast	O
+form	O
+to	O
+filamentous	O
+(	O
+pseudohyphal	O
+)	O
+growth	O
+that	O
+may	O
+be	O
+required	O
+for	O
+fungal	O
+pathogenicity	O
+.	O
+	O
+As	O
+is	O
+the	O
+case	O
+for	O
+other	O
+transceptors	O
+,	O
+it	O
+is	O
+not	O
+clear	O
+how	O
+Mep2	O
+interacts	O
+with	O
+downstream	O
+signalling	O
+partners	O
+,	O
+but	O
+the	O
+protein	O
+kinase	O
+A	O
+and	O
+mitogen	O
+-	O
+activated	O
+protein	O
+kinase	O
+pathways	O
+have	O
+been	O
+proposed	O
+as	O
+downstream	O
+effectors	O
+of	O
+Mep2	O
+(	O
+refs	O
+).	O
+	O
+Compared	O
+with	O
+Mep1	O
+and	O
+Mep3	O
+,	O
+Mep2	O
+is	O
+highly	O
+expressed	O
+and	O
+functions	O
+as	O
+a	O
+low	O
+-	O
+capacity	O
+,	O
+high	O
+-	O
+affinity	O
+transporter	O
+in	O
+the	O
+uptake	O
+of	O
+MA	O
+.	O
+	O
+In	O
+addition	O
+,	O
+Mep2	O
+is	O
+also	O
+important	O
+for	O
+uptake	O
+of	O
+ammonium	O
+produced	O
+by	O
+growth	O
+on	O
+other	O
+nitrogen	O
+sources	O
+.	O
+	O
+By	O
+contrast	O
+,	O
+several	O
+bacterial	O
+Amt	O
+orthologues	O
+have	O
+been	O
+characterized	O
+in	O
+detail	O
+via	O
+high	O
+-	O
+resolution	O
+crystal	O
+structures	O
+and	O
+a	O
+number	O
+of	O
+molecular	O
+dynamics	O
+(	O
+MD	O
+)	O
+studies	O
+.	O
+	O
+All	O
+the	O
+solved	O
+structures	O
+including	O
+that	O
+of	O
+RhCG	O
+are	O
+very	O
+similar	O
+,	O
+establishing	O
+the	O
+basic	O
+architecture	O
+of	O
+ammonium	O
+transporters	O
+.	O
+	O
+The	O
+proteins	O
+form	O
+stable	O
+trimers	O
+,	O
+with	O
+each	O
+monomer	O
+having	O
+11	O
+transmembrane	O
+(	O
+TM	O
+)	O
+helices	O
+and	O
+a	O
+central	O
+channel	O
+for	O
+the	O
+transport	O
+of	O
+ammonium	O
+.	O
+	O
+All	O
+structures	O
+show	O
+the	O
+transporters	O
+in	O
+open	O
+conformations	O
+.	O
+	O
+Where	O
+earlier	O
+studies	O
+favoured	O
+the	O
+transport	O
+of	O
+ammonia	O
+gas	O
+,	O
+recent	O
+data	O
+and	O
+theoretical	O
+considerations	O
+suggest	O
+that	O
+Amt	O
+/	O
+Mep	O
+proteins	O
+are	O
+instead	O
+active	O
+,	O
+electrogenic	O
+transporters	O
+of	O
+either	O
+NH4	O
++	O
+(	O
+uniport	O
+)	O
+or	O
+NH3	O
+/	O
+H	O
++	O
+(	O
+symport	O
+).	O
+	O
+A	O
+highly	O
+conserved	O
+pair	O
+of	O
+channel	O
+-	O
+lining	O
+histidine	O
+residues	O
+dubbed	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+may	O
+serve	O
+as	O
+a	O
+proton	O
+relay	O
+system	O
+while	O
+NH3	O
+moves	O
+through	O
+the	O
+channel	O
+during	O
+NH3	O
+/	O
+H	O
++	O
+symport	O
+.	O
+	O
+Ammonium	O
+transport	O
+is	O
+tightly	O
+regulated	O
+.	O
+	O
+In	O
+animals	O
+,	O
+this	O
+is	O
+due	O
+to	O
+toxicity	O
+of	O
+elevated	O
+intracellular	O
+ammonium	O
+levels	O
+,	O
+whereas	O
+for	O
+microorganisms	O
+ammonium	O
+is	O
+a	O
+preferred	O
+nitrogen	O
+source	O
+.	O
+	O
+By	O
+binding	O
+tightly	O
+to	O
+Amt	O
+proteins	O
+without	O
+inducing	O
+a	O
+conformational	O
+change	O
+in	O
+the	O
+transporter	O
+,	O
+GlnK	O
+sterically	O
+blocks	O
+ammonium	O
+conductance	O
+when	O
+nitrogen	O
+levels	O
+are	O
+sufficient	O
+.	O
+	O
+Importantly	O
+,	O
+eukaryotes	O
+do	O
+not	O
+have	O
+GlnK	O
+orthologues	O
+and	O
+have	O
+a	O
+different	O
+mechanism	O
+for	O
+regulation	O
+of	O
+ammonium	O
+transport	O
+activity	O
+.	O
+	O
+In	O
+plants	O
+,	O
+transporter	O
+phosphorylation	O
+and	O
+dephosphorylation	O
+are	O
+known	O
+to	O
+regulate	O
+activity	O
+.	O
+	O
+In	O
+S	O
+.	O
+cerevisiae	O
+,	O
+phosphorylation	O
+of	O
+Ser457	O
+within	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+(	O
+CTR	O
+)	O
+in	O
+the	O
+cytoplasm	O
+was	O
+recently	O
+proposed	O
+to	O
+cause	O
+Mep2	O
+opening	O
+,	O
+possibly	O
+via	O
+inducing	O
+a	O
+conformational	O
+change	O
+.	O
+	O
+The	O
+structures	O
+are	O
+similar	O
+to	O
+each	O
+other	O
+but	O
+show	O
+considerable	O
+differences	O
+to	O
+all	O
+other	O
+ammonium	O
+transporter	O
+structures	O
+.	O
+	O
+The	O
+most	O
+striking	O
+difference	O
+is	O
+the	O
+fact	O
+that	O
+the	O
+Mep2	O
+proteins	O
+have	O
+closed	O
+conformations	O
+.	O
+	O
+The	O
+putative	O
+phosphorylation	O
+site	O
+is	O
+solvent	O
+accessible	O
+and	O
+located	O
+in	O
+a	O
+negatively	O
+charged	O
+pocket	O
+∼	O
+30	O
+Å	O
+away	O
+from	O
+the	O
+channel	O
+exit	O
+.	O
+	O
+The	O
+channels	O
+of	O
+phosphorylation	O
+-	O
+mimicking	O
+mutants	O
+of	O
+C	O
+.	O
+albicans	O
+Mep2	O
+are	O
+still	O
+closed	O
+but	O
+show	O
+large	O
+conformational	O
+changes	O
+within	O
+a	O
+conserved	O
+part	O
+of	O
+the	O
+CTR	O
+.	O
+	O
+Together	O
+with	O
+a	O
+structure	O
+of	O
+a	O
+C	O
+-	O
+terminal	O
+Mep2	B-mutant
+variant	I-mutant
+lacking	O
+the	O
+segment	O
+containing	O
+the	O
+phosphorylation	O
+site	O
+,	O
+the	O
+results	O
+allow	O
+us	O
+to	O
+propose	O
+a	O
+structural	O
+model	O
+for	O
+phosphorylation	O
+-	O
+based	O
+regulation	O
+of	O
+eukaryotic	O
+ammonium	O
+transport	O
+.	O
+	O
+General	O
+architecture	O
+of	O
+Mep2	O
+ammonium	O
+transceptors	O
+	O
+The	O
+Mep2	O
+protein	O
+of	O
+S	O
+.	O
+cerevisiae	O
+(	O
+ScMep2	O
+)	O
+was	O
+overexpressed	O
+in	O
+S	O
+.	O
+cerevisiae	O
+in	O
+high	O
+yields	O
+,	O
+enabling	O
+structure	O
+determination	O
+by	O
+X	O
+-	O
+ray	O
+crystallography	O
+using	O
+data	O
+to	O
+3	O
+.	O
+2	O
+Å	O
+resolution	O
+by	O
+molecular	O
+replacement	O
+(	O
+MR	O
+)	O
+with	O
+the	O
+archaebacterial	O
+Amt	O
+-	O
+1	O
+structure	O
+(	O
+see	O
+Methods	O
+section	O
+).	O
+	O
+Given	O
+that	O
+the	O
+modest	O
+resolution	O
+of	O
+the	O
+structure	O
+and	O
+the	O
+limited	O
+detergent	O
+stability	O
+of	O
+ScMep2	O
+would	O
+likely	O
+complicate	O
+structure	O
+–	O
+function	O
+studies	O
+,	O
+several	O
+other	O
+fungal	O
+Mep2	O
+orthologues	O
+were	O
+subsequently	O
+overexpressed	O
+and	O
+screened	O
+for	O
+diffraction	O
+-	O
+quality	O
+crystals	O
+.	O
+	O
+Of	O
+these	O
+,	O
+Mep2	O
+from	O
+C	O
+.	O
+albicans	O
+(	O
+CaMep2	O
+)	O
+showed	O
+superior	O
+stability	O
+in	O
+relatively	O
+harsh	O
+detergents	O
+such	O
+as	O
+nonyl	O
+-	O
+glucoside	O
+,	O
+allowing	O
+structure	O
+determination	O
+in	O
+two	O
+different	O
+crystal	O
+forms	O
+to	O
+high	O
+resolution	O
+(	O
+up	O
+to	O
+1	O
+.	O
+5	O
+Å	O
+).	O
+	O
+Electron	O
+density	O
+is	O
+visible	O
+for	O
+the	O
+entire	O
+polypeptide	O
+chains	O
+,	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+43	O
+(	O
+ScMep2	O
+)	O
+and	O
+25	O
+residues	O
+(	O
+CaMep2	O
+),	O
+which	O
+are	O
+poorly	O
+conserved	O
+and	O
+presumably	O
+disordered	O
+.	O
+	O
+Both	O
+Mep2	O
+proteins	O
+show	O
+the	O
+archetypal	O
+trimeric	O
+assemblies	O
+in	O
+which	O
+each	O
+monomer	O
+consists	O
+of	O
+11	O
+TM	O
+helices	O
+surrounding	O
+a	O
+central	O
+pore	O
+.	O
+	O
+Important	O
+functional	O
+features	O
+such	O
+as	O
+the	O
+extracellular	O
+ammonium	O
+binding	O
+site	O
+,	O
+the	O
+Phe	O
+gate	O
+and	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+within	O
+the	O
+hydrophobic	O
+channel	O
+are	O
+all	O
+very	O
+similar	O
+to	O
+those	O
+present	O
+in	O
+the	O
+bacterial	O
+transporters	O
+and	O
+RhCG	O
+.	O
+	O
+In	O
+the	O
+remainder	O
+of	O
+the	O
+manuscript	O
+,	O
+we	O
+will	O
+specifically	O
+discuss	O
+CaMep2	O
+due	O
+to	O
+the	O
+superior	O
+resolution	O
+of	O
+the	O
+structure	O
+.	O
+	O
+While	O
+the	O
+overall	O
+architecture	O
+of	O
+Mep2	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+prokaryotic	O
+transporters	O
+(	O
+Cα	O
+r	O
+.	O
+m	O
+.	O
+s	O
+.	O
+d	O
+.	O
+with	O
+Amt	O
+-	O
+1	O
+=	O
+1	O
+.	O
+4	O
+Å	O
+for	O
+361	O
+residues	O
+),	O
+there	O
+are	O
+large	O
+differences	O
+within	O
+the	O
+N	O
+terminus	O
+,	O
+intracellular	O
+loops	O
+(	O
+ICLs	O
+)	O
+ICL1	O
+and	O
+ICL3	O
+,	O
+and	O
+the	O
+CTR	O
+.	O
+	O
+Moreover	O
+,	O
+the	O
+N	O
+terminus	O
+of	O
+one	O
+monomer	O
+interacts	O
+with	O
+the	O
+extended	O
+extracellular	O
+loop	O
+ECL5	O
+of	O
+a	O
+neighbouring	O
+monomer	O
+.	O
+	O
+However	O
+,	O
+given	O
+that	O
+an	O
+N	O
+-	O
+terminal	O
+deletion	O
+mutant	O
+(	O
+2	B-mutant
+-	I-mutant
+27Δ	I-mutant
+)	O
+grows	O
+as	O
+well	O
+as	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+Mep2	O
+on	O
+minimal	O
+ammonium	O
+medium	O
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+),	O
+the	O
+importance	O
+of	O
+the	O
+N	O
+terminus	O
+for	O
+Mep2	O
+activity	O
+is	O
+not	O
+clear	O
+.	O
+	O
+Mep2	O
+channels	O
+are	O
+closed	O
+by	O
+a	O
+two	O
+-	O
+tier	O
+channel	O
+block	O
+	O
+In	O
+the	O
+vicinity	O
+of	O
+the	O
+Mep2	O
+channel	O
+exit	O
+,	O
+the	O
+cytoplasmic	O
+end	O
+of	O
+TM2	O
+has	O
+unwound	O
+,	O
+generating	O
+a	O
+longer	O
+ICL1	O
+even	O
+though	O
+there	O
+are	O
+no	O
+insertions	O
+in	O
+this	O
+region	O
+compared	O
+to	O
+the	O
+bacterial	O
+proteins	O
+(	O
+Figs	O
+2	O
+and	O
+4	O
+).	O
+	O
+The	O
+largest	O
+backbone	O
+movements	O
+of	O
+equivalent	O
+residues	O
+within	O
+ICL1	O
+are	O
+∼	O
+10	O
+Å	O
+,	O
+markedly	O
+affecting	O
+the	O
+conserved	O
+basic	O
+RxK	O
+motif	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+In	O
+addition	O
+to	O
+changing	O
+the	O
+RxK	O
+motif	O
+,	O
+the	O
+movement	O
+of	O
+ICL1	O
+has	O
+another	O
+,	O
+crucial	O
+functional	O
+consequence	O
+.	O
+	O
+At	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+TM1	O
+,	O
+the	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+group	O
+of	O
+the	O
+relatively	O
+conserved	O
+Tyr49	O
+(	O
+Tyr53	O
+in	O
+ScMep2	O
+)	O
+makes	O
+a	O
+strong	O
+hydrogen	O
+bond	O
+with	O
+the	O
+ɛ2	O
+nitrogen	O
+atom	O
+of	O
+the	O
+absolutely	O
+conserved	O
+His342	O
+of	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+(	O
+His348	O
+in	O
+ScMep2	O
+),	O
+closing	O
+the	O
+channel	O
+(	O
+Figs	O
+4	O
+and	O
+5	O
+).	O
+	O
+In	O
+bacterial	O
+Amt	O
+proteins	O
+,	O
+this	O
+Tyr	O
+side	O
+chain	O
+is	O
+rotated	O
+∼	O
+4	O
+Å	O
+away	O
+as	O
+a	O
+result	O
+of	O
+the	O
+different	O
+conformation	O
+of	O
+TM1	O
+,	O
+leaving	O
+the	O
+channel	O
+open	O
+and	O
+the	O
+histidine	O
+available	O
+for	O
+its	O
+putative	O
+role	O
+in	O
+substrate	O
+transport	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+Finally	O
+,	O
+the	O
+important	O
+ICL3	O
+linking	O
+the	O
+pseudo	O
+-	O
+symmetrical	O
+halves	O
+(	O
+TM1	O
+-	O
+5	O
+and	O
+TM6	O
+-	O
+10	O
+)	O
+of	O
+the	O
+transporter	O
+is	O
+also	O
+shifted	O
+up	O
+to	O
+∼	O
+10	O
+Å	O
+and	O
+forms	O
+an	O
+additional	O
+barrier	O
+that	O
+closes	O
+the	O
+channel	O
+on	O
+the	O
+cytoplasmic	O
+side	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+This	O
+two	O
+-	O
+tier	O
+channel	O
+block	O
+likely	O
+ensures	O
+that	O
+very	O
+little	O
+ammonium	O
+transport	O
+will	O
+take	O
+place	O
+under	O
+nitrogen	O
+-	O
+sufficient	O
+conditions	O
+.	O
+	O
+The	O
+closed	O
+state	O
+of	O
+the	O
+channel	O
+might	O
+also	O
+explain	O
+why	O
+no	O
+density	O
+,	O
+which	O
+could	O
+correspond	O
+to	O
+ammonium	O
+(	O
+or	O
+water	O
+),	O
+is	O
+observed	O
+in	O
+the	O
+hydrophobic	O
+part	O
+of	O
+the	O
+Mep2	O
+channel	O
+close	O
+to	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+.	O
+	O
+The	O
+final	O
+region	O
+in	O
+Mep2	O
+that	O
+shows	O
+large	O
+differences	O
+compared	O
+with	O
+the	O
+bacterial	O
+transporters	O
+is	O
+the	O
+CTR	O
+.	O
+	O
+By	O
+contrast	O
+,	O
+in	O
+the	O
+structures	O
+of	O
+bacterial	O
+proteins	O
+,	O
+the	O
+CTR	O
+is	O
+docked	O
+tightly	O
+onto	O
+the	O
+N	O
+-	O
+terminal	O
+half	O
+of	O
+the	O
+transporters	O
+(	O
+corresponding	O
+to	O
+TM1	O
+-	O
+5	O
+),	O
+resulting	O
+in	O
+a	O
+more	O
+compact	O
+structure	O
+.	O
+	O
+This	O
+is	O
+illustrated	O
+by	O
+the	O
+positions	O
+of	O
+the	O
+five	O
+universally	O
+conserved	O
+residues	O
+within	O
+the	O
+CTR	O
+,	O
+that	O
+is	O
+,	O
+Arg415	O
+(	O
+370	O
+),	O
+Glu421	O
+(	O
+376	O
+),	O
+Gly424	O
+(	O
+379	O
+),	O
+Asp426	O
+(	O
+381	O
+)	O
+and	O
+Tyr	O
+435	O
+(	O
+390	O
+)	O
+in	O
+CaMep2	O
+(	O
+Amt	O
+-	O
+1	O
+)	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+On	O
+one	O
+side	O
+,	O
+the	O
+Tyr390	O
+hydroxyl	O
+in	O
+Amt	O
+-	O
+1	O
+is	O
+hydrogen	O
+bonded	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+the	O
+conserved	O
+His185	O
+at	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+loop	O
+ICL3	O
+.	O
+	O
+Similar	O
+interactions	O
+were	O
+also	O
+modelled	O
+in	O
+the	O
+active	O
+,	O
+non	O
+-	O
+phosphorylated	O
+plant	O
+AtAmt	O
+-	O
+1	O
+;	O
+1	O
+structure	O
+(	O
+for	O
+example	O
+,	O
+Y467	O
+-	O
+H239	O
+and	O
+D458	O
+-	O
+K71	O
+).	O
+	O
+The	O
+result	O
+of	O
+these	O
+interactions	O
+is	O
+that	O
+the	O
+CTR	O
+‘	O
+hugs	O
+'	O
+the	O
+N	O
+-	O
+terminal	O
+half	O
+of	O
+the	O
+transporters	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+Also	O
+noteworthy	O
+is	O
+Asp381	O
+,	O
+the	O
+side	O
+chain	O
+of	O
+which	O
+interacts	O
+strongly	O
+with	O
+the	O
+positive	O
+dipole	O
+on	O
+the	O
+N	O
+-	O
+terminal	O
+end	O
+of	O
+TM2	O
+.	O
+	O
+In	O
+the	O
+Mep2	O
+structures	O
+,	O
+none	O
+of	O
+the	O
+interactions	O
+mentioned	O
+above	O
+are	O
+present	O
+.	O
+	O
+Recently	O
+Boeckstaens	O
+et	O
+al	O
+.	O
+provided	O
+evidence	O
+that	O
+Ser457	O
+in	O
+ScMep2	O
+(	O
+corresponding	O
+to	O
+Ser453	O
+in	O
+CaMep2	O
+)	O
+is	O
+phosphorylated	O
+by	O
+the	O
+TORC1	O
+effector	O
+kinase	O
+Npr1	O
+under	O
+nitrogen	O
+-	O
+limiting	O
+conditions	O
+.	O
+	O
+Conversely	O
+,	O
+the	O
+phosphorylation	O
+-	O
+mimicking	O
+S457D	B-mutant
+variant	O
+is	O
+active	O
+both	O
+in	O
+the	O
+triple	B-mutant
+mepΔ	I-mutant
+background	O
+and	O
+in	O
+a	O
+triple	B-mutant
+mepΔ	I-mutant
+npr1Δ	I-mutant
+strain	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+Mutation	O
+of	O
+other	O
+potential	O
+phosphorylation	O
+sites	O
+in	O
+the	O
+CTR	O
+did	O
+not	O
+support	O
+growth	O
+in	O
+the	O
+npr1Δ	B-mutant
+background	O
+.	O
+	O
+Collectively	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+phosphorylation	O
+of	O
+Ser457	O
+opens	O
+the	O
+Mep2	O
+channel	O
+to	O
+allow	O
+ammonium	O
+uptake	O
+.	O
+	O
+Ser457	O
+is	O
+located	O
+in	O
+a	O
+part	O
+of	O
+the	O
+CTR	O
+that	O
+is	O
+conserved	O
+in	O
+a	O
+subgroup	O
+of	O
+Mep2	O
+proteins	O
+,	O
+but	O
+which	O
+is	O
+not	O
+present	O
+in	O
+bacterial	O
+proteins	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+This	O
+segment	O
+(	O
+residues	O
+450	O
+–	O
+457	O
+in	O
+ScMep2	O
+and	O
+446	O
+–	O
+453	O
+in	O
+CaMep2	O
+)	O
+was	O
+dubbed	O
+an	O
+autoinhibitory	O
+(	O
+AI	O
+)	O
+region	O
+based	O
+on	O
+the	O
+fact	O
+that	O
+its	O
+removal	O
+generates	O
+an	O
+active	O
+transporter	O
+in	O
+the	O
+absence	O
+of	O
+Npr1	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+The	O
+AI	O
+region	O
+packs	O
+against	O
+the	O
+cytoplasmic	O
+ends	O
+of	O
+TM2	O
+and	O
+TM4	O
+,	O
+physically	O
+linking	O
+the	O
+main	O
+body	O
+of	O
+the	O
+transporter	O
+with	O
+the	O
+CTR	O
+via	O
+main	O
+chain	O
+interactions	O
+and	O
+side	O
+-	O
+chain	O
+interactions	O
+of	O
+Val447	O
+,	O
+Asp449	O
+,	O
+Pro450	O
+and	O
+Arg452	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+Strikingly	O
+,	O
+the	O
+Npr1	O
+target	O
+serine	O
+residue	O
+is	O
+located	O
+at	O
+the	O
+periphery	O
+of	O
+the	O
+trimer	O
+,	O
+far	O
+away	O
+(∼	O
+30	O
+Å	O
+)	O
+from	O
+any	O
+channel	O
+exit	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+Despite	O
+its	O
+location	O
+at	O
+the	O
+periphery	O
+of	O
+the	O
+trimer	O
+,	O
+the	O
+electron	O
+density	O
+for	O
+the	O
+serine	O
+is	O
+well	O
+defined	O
+in	O
+both	O
+Mep2	O
+structures	O
+and	O
+corresponds	O
+to	O
+the	O
+non	O
+-	O
+phosphorylated	O
+state	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+For	O
+ScMep2	O
+,	O
+Ser457	O
+is	O
+the	O
+most	O
+C	O
+-	O
+terminal	O
+residue	O
+for	O
+which	O
+electron	O
+density	O
+is	O
+visible	O
+,	O
+indicating	O
+that	O
+the	O
+region	O
+beyond	O
+Ser457	O
+is	O
+disordered	O
+.	O
+	O
+In	O
+CaMep2	O
+,	O
+the	O
+visible	O
+part	O
+of	O
+the	O
+sequence	O
+extends	O
+for	O
+two	O
+residues	O
+beyond	O
+Ser453	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+The	O
+disordered	O
+part	O
+of	O
+the	O
+CTR	O
+is	O
+not	O
+conserved	O
+in	O
+ammonium	O
+transporters	O
+(	O
+Fig	O
+.	O
+2	O
+),	O
+suggesting	O
+that	O
+it	O
+is	O
+not	O
+important	O
+for	O
+transport	O
+.	O
+	O
+Interestingly	O
+,	O
+a	O
+ScMep2	O
+457Δ	B-mutant
+truncation	O
+mutant	O
+in	O
+which	O
+a	O
+His	O
+-	O
+tag	O
+directly	O
+follows	O
+Ser457	O
+is	O
+highly	O
+expressed	O
+but	O
+has	O
+low	O
+activity	O
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+),	O
+suggesting	O
+that	O
+the	O
+His	O
+-	O
+tag	O
+interferes	O
+with	O
+phosphorylation	O
+by	O
+Npr1	O
+.	O
+	O
+The	O
+same	O
+mutant	B-mutant
+lacking	O
+the	O
+His	O
+-	O
+tag	O
+has	O
+WT	O
+properties	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+),	O
+confirming	O
+that	O
+the	O
+region	O
+following	O
+the	O
+phosphorylation	O
+site	O
+is	O
+dispensable	O
+for	O
+function	O
+.	O
+	O
+Given	O
+that	O
+Ser457	O
+/	O
+453	O
+is	O
+far	O
+from	O
+any	O
+channel	O
+exit	O
+(	O
+Fig	O
+.	O
+6	O
+),	O
+the	O
+crucial	O
+question	O
+is	O
+how	O
+phosphorylation	O
+opens	O
+the	O
+Mep2	O
+channel	O
+to	O
+generate	O
+an	O
+active	O
+transporter	O
+.	O
+	O
+Boeckstaens	O
+et	O
+al	O
+.	O
+proposed	O
+that	O
+phosphorylation	O
+does	O
+not	O
+affect	O
+channel	O
+activity	O
+directly	O
+,	O
+but	O
+instead	O
+relieves	O
+inhibition	O
+by	O
+the	O
+AI	O
+region	O
+.	O
+	O
+The	O
+data	O
+behind	O
+this	O
+hypothesis	O
+is	O
+the	O
+observation	O
+that	O
+a	O
+ScMep2	O
+449	B-mutant
+-	I-mutant
+485Δ	I-mutant
+deletion	O
+mutant	O
+lacking	O
+the	O
+AI	O
+region	O
+is	O
+highly	O
+active	O
+in	O
+MA	O
+uptake	O
+both	O
+in	O
+the	O
+triple	B-mutant
+mepΔ	I-mutant
+and	O
+triple	B-mutant
+mepΔ	I-mutant
+npr1Δ	I-mutant
+backgrounds	O
+,	O
+implying	O
+that	O
+this	O
+Mep2	B-mutant
+variant	I-mutant
+has	O
+a	O
+constitutively	O
+open	O
+channel	O
+.	O
+	O
+This	O
+is	O
+not	O
+unexpected	O
+given	O
+the	O
+fact	O
+that	O
+the	O
+AI	O
+region	O
+bridges	O
+the	O
+CTR	O
+and	O
+the	O
+main	O
+body	O
+of	O
+Mep2	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+Interestingly	O
+,	O
+however	O
+,	O
+the	O
+Tyr49	O
+-	O
+His342	O
+hydrogen	O
+bond	O
+that	O
+closes	O
+the	O
+channel	O
+in	O
+the	O
+WT	O
+protein	O
+is	O
+still	O
+present	O
+(	O
+Fig	O
+.	O
+7	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+The	O
+second	O
+possibility	O
+is	O
+that	O
+the	O
+Tyr	O
+–	O
+His	O
+hydrogen	O
+bond	O
+has	O
+to	O
+be	O
+disrupted	O
+by	O
+the	O
+incoming	O
+substrate	O
+to	O
+open	O
+the	O
+channel	O
+.	O
+	O
+The	O
+importance	O
+of	O
+the	O
+Tyr	O
+–	O
+His	O
+hydrogen	O
+bond	O
+is	O
+underscored	O
+by	O
+the	O
+fact	O
+that	O
+its	O
+removal	O
+in	O
+the	O
+ScMep2	O
+Y53A	B-mutant
+mutant	O
+results	O
+in	O
+a	O
+constitutively	O
+active	O
+transporter	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+Phosphorylation	O
+causes	O
+a	O
+conformational	O
+change	O
+in	O
+the	O
+CTR	O
+	O
+Do	O
+the	O
+Mep2	O
+structures	O
+provide	O
+any	O
+clues	O
+regarding	O
+the	O
+potential	O
+effect	O
+of	O
+phosphorylation	O
+?	O
+	O
+The	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+of	O
+Ser457	O
+/	O
+453	O
+is	O
+located	O
+in	O
+a	O
+well	O
+-	O
+defined	O
+electronegative	O
+pocket	O
+that	O
+is	O
+solvent	O
+accessible	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+The	O
+closest	O
+atoms	O
+to	O
+the	O
+serine	O
+hydroxyl	O
+group	O
+are	O
+the	O
+backbone	O
+carbonyl	O
+atoms	O
+of	O
+Asp419	O
+,	O
+Glu420	O
+and	O
+Glu421	O
+,	O
+which	O
+are	O
+3	O
+–	O
+4	O
+Å	O
+away	O
+.	O
+	O
+We	O
+therefore	O
+predict	O
+that	O
+phosphorylation	O
+of	O
+Ser453	O
+will	O
+result	O
+in	O
+steric	O
+clashes	O
+as	O
+well	O
+as	O
+electrostatic	O
+repulsion	O
+,	O
+which	O
+in	O
+turn	O
+might	O
+cause	O
+substantial	O
+conformational	O
+changes	O
+within	O
+the	O
+CTR	O
+.	O
+	O
+Unexpectedly	O
+,	O
+the	O
+AI	O
+segment	O
+containing	O
+the	O
+mutated	O
+residues	O
+has	O
+only	O
+undergone	O
+a	O
+slight	O
+shift	O
+compared	O
+with	O
+the	O
+WT	O
+protein	O
+(	O
+Fig	O
+.	O
+8	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+By	O
+contrast	O
+,	O
+the	O
+conserved	O
+part	O
+of	O
+the	O
+CTR	O
+has	O
+undergone	O
+a	O
+large	O
+conformational	O
+change	O
+involving	O
+formation	O
+of	O
+a	O
+12	O
+-	O
+residue	O
+-	O
+long	O
+α	O
+-	O
+helix	O
+from	O
+Leu427	O
+to	O
+Asp438	O
+.	O
+	O
+This	O
+is	O
+the	O
+first	O
+time	O
+a	O
+large	O
+conformational	O
+change	O
+has	O
+been	O
+observed	O
+in	O
+an	O
+ammonium	O
+transporter	O
+as	O
+a	O
+result	O
+of	O
+a	O
+mutation	O
+,	O
+and	O
+confirms	O
+previous	O
+hypotheses	O
+that	O
+phosphorylation	O
+causes	O
+structural	O
+changes	O
+in	O
+the	O
+CTR	O
+.	O
+	O
+To	O
+exclude	O
+the	O
+possibility	O
+that	O
+the	O
+additional	O
+R452D	B-mutant
+mutation	O
+is	O
+responsible	O
+for	O
+the	O
+observed	O
+changes	O
+,	O
+we	O
+also	O
+determined	O
+the	O
+structure	O
+of	O
+the	O
+‘	O
+single	B-mutant
+D	I-mutant
+'	O
+S453D	B-mutant
+mutant	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+,	O
+the	O
+consequence	O
+of	O
+the	O
+single	B-mutant
+D	I-mutant
+mutation	O
+is	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+DD	B-mutant
+substitution	I-mutant
+,	O
+with	O
+conformational	O
+changes	O
+and	O
+increased	O
+dynamics	O
+confined	O
+to	O
+the	O
+conserved	O
+part	O
+of	O
+the	O
+CTR	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+To	O
+supplement	O
+the	O
+crystal	O
+structures	O
+,	O
+we	O
+also	O
+performed	O
+modelling	O
+and	O
+MD	O
+studies	O
+of	O
+WT	O
+CaMep2	O
+,	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+and	O
+phosphorylated	O
+protein	O
+(	O
+S453J	B-mutant
+).	O
+	O
+In	O
+the	O
+WT	O
+structure	O
+,	O
+the	O
+acidic	O
+residues	O
+Asp419	O
+,	O
+Glu420	O
+and	O
+Glu421	O
+are	O
+within	O
+hydrogen	O
+bonding	O
+distance	O
+of	O
+Ser453	O
+.	O
+	O
+After	O
+200	O
+ns	O
+of	O
+MD	O
+simulation	O
+,	O
+the	O
+interactions	O
+between	O
+these	O
+residues	O
+and	O
+Ser453	O
+remain	O
+intact	O
+.	O
+	O
+In	O
+particular	O
+,	O
+persistent	O
+hydrogen	O
+bonds	O
+are	O
+observed	O
+between	O
+the	O
+Ser453	O
+hydroxyl	O
+group	O
+and	O
+the	O
+acidic	O
+group	O
+of	O
+Glu420	O
+,	O
+and	O
+also	O
+between	O
+the	O
+amine	O
+group	O
+of	O
+Ser453	O
+and	O
+the	O
+backbone	O
+carbonyl	O
+of	O
+Glu420	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+The	O
+DD	B-mutant
+mutant	I-mutant
+is	O
+also	O
+stable	O
+during	O
+the	O
+simulations	O
+,	O
+but	O
+the	O
+average	O
+backbone	O
+r	O
+.	O
+m	O
+.	O
+s	O
+.	O
+d	O
+of	O
+∼	O
+3	O
+.	O
+6	O
+Å	O
+suggests	O
+slightly	O
+more	O
+conformational	O
+flexibility	O
+than	O
+WT	O
+.	O
+	O
+As	O
+the	O
+simulation	O
+proceeds	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+acidic	O
+residues	O
+move	O
+away	O
+from	O
+Asp452	O
+and	O
+Asp453	O
+,	O
+presumably	O
+to	O
+avoid	O
+electrostatic	O
+repulsion	O
+.	O
+	O
+The	O
+protein	O
+is	O
+structurally	O
+stable	O
+throughout	O
+the	O
+simulation	O
+with	O
+little	O
+deviation	O
+in	O
+the	O
+other	O
+parts	O
+of	O
+the	O
+protein	O
+.	O
+	O
+Finally	O
+,	O
+the	O
+S453J	B-mutant
+mutant	O
+is	O
+also	O
+stable	O
+throughout	O
+the	O
+200	O
+-	O
+ns	O
+simulation	O
+and	O
+has	O
+an	O
+average	O
+backbone	O
+deviation	O
+of	O
+∼	O
+3	O
+.	O
+8	O
+Å	O
+,	O
+which	O
+is	O
+similar	O
+to	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+.	O
+	O
+The	O
+short	O
+helix	O
+formed	O
+by	O
+residues	O
+Leu427	O
+to	O
+Asp438	O
+unravels	O
+during	O
+the	O
+simulations	O
+to	O
+a	O
+disordered	O
+state	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+MD	O
+simulations	O
+support	O
+the	O
+notion	O
+from	O
+the	O
+crystal	O
+structures	O
+that	O
+phosphorylation	O
+generates	O
+conformational	O
+changes	O
+in	O
+the	O
+conserved	O
+part	O
+of	O
+the	O
+CTR	O
+.	O
+	O
+However	O
+,	O
+the	O
+conformational	O
+changes	O
+for	O
+the	O
+phosphomimetic	B-mutant
+mutants	I-mutant
+in	O
+the	O
+crystals	O
+are	O
+confined	O
+to	O
+the	O
+CTR	O
+(	O
+Fig	O
+.	O
+8	O
+),	O
+and	O
+the	O
+channels	O
+are	O
+still	O
+closed	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+One	O
+possible	O
+explanation	O
+is	O
+that	O
+the	O
+mutants	B-mutant
+do	O
+not	O
+accurately	O
+mimic	O
+a	O
+phosphoserine	O
+,	O
+but	O
+the	O
+observation	O
+that	O
+the	O
+S453D	B-mutant
+and	O
+DD	B-mutant
+mutants	I-mutant
+are	O
+fully	O
+active	O
+in	O
+the	O
+absence	O
+of	O
+Npr1	O
+suggests	O
+that	O
+the	O
+mutations	O
+do	O
+mimic	O
+the	O
+effect	O
+of	O
+phosphorylation	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+The	O
+fact	O
+that	O
+the	O
+S453D	B-mutant
+structure	O
+was	O
+obtained	O
+in	O
+the	O
+presence	O
+of	O
+10	O
+mM	O
+ammonium	O
+ions	O
+suggests	O
+that	O
+the	O
+crystallization	O
+process	O
+favours	O
+closed	O
+states	O
+of	O
+the	O
+Mep2	O
+channels	O
+.	O
+	O
+Knowledge	O
+about	O
+ammonium	O
+transporter	O
+structure	O
+has	O
+been	O
+obtained	O
+from	O
+experimental	O
+and	O
+theoretical	O
+studies	O
+on	O
+bacterial	O
+family	O
+members	O
+.	O
+	O
+In	O
+addition	O
+,	O
+a	O
+number	O
+of	O
+biochemical	O
+and	O
+genetic	O
+studies	O
+are	O
+available	O
+for	O
+bacterial	O
+,	O
+fungal	O
+and	O
+plant	O
+proteins	O
+.	O
+	O
+These	O
+efforts	O
+have	O
+advanced	O
+our	O
+knowledge	O
+considerably	O
+but	O
+have	O
+not	O
+yet	O
+yielded	O
+atomic	O
+-	O
+level	O
+answers	O
+to	O
+several	O
+important	O
+mechanistic	O
+questions	O
+,	O
+including	O
+how	O
+ammonium	O
+transport	O
+is	O
+regulated	O
+in	O
+eukaryotes	O
+and	O
+the	O
+mechanism	O
+of	O
+ammonium	O
+signalling	O
+.	O
+	O
+In	O
+Arabidopsis	O
+thaliana	O
+Amt	O
+-	O
+1	O
+;	O
+1	O
+,	O
+phosphorylation	O
+of	O
+the	O
+CTR	O
+residue	O
+T460	O
+under	O
+conditions	O
+of	O
+high	O
+ammonium	O
+inhibits	O
+transport	O
+activity	O
+,	O
+that	O
+is	O
+,	O
+the	O
+default	O
+(	O
+non	O
+-	O
+phosphorylated	O
+)	O
+state	O
+of	O
+the	O
+plant	O
+transporter	O
+is	O
+open	O
+.	O
+	O
+Interestingly	O
+,	O
+phosphomimetic	B-mutant
+mutations	I-mutant
+introduced	O
+into	O
+one	O
+monomer	O
+inactivate	O
+the	O
+entire	O
+trimer	O
+,	O
+indicating	O
+that	O
+(	O
+i	O
+)	O
+heterotrimerization	O
+occurs	O
+and	O
+(	O
+ii	O
+)	O
+the	O
+CTR	O
+mediates	O
+allosteric	O
+regulation	O
+of	O
+ammonium	O
+transport	O
+activity	O
+via	O
+phosphorylation	O
+.	O
+	O
+Owing	O
+to	O
+the	O
+lack	O
+of	O
+structural	O
+information	O
+for	O
+plant	O
+AMTs	O
+,	O
+the	O
+details	O
+of	O
+channel	O
+closure	O
+and	O
+inter	O
+-	O
+monomer	O
+crosstalk	O
+are	O
+not	O
+yet	O
+clear	O
+.	O
+	O
+Contrasting	O
+with	O
+the	O
+plant	O
+transporters	O
+,	O
+the	O
+inactive	O
+states	O
+of	O
+Mep2	O
+proteins	O
+under	O
+conditions	O
+of	O
+high	O
+ammonium	O
+are	O
+non	O
+-	O
+phosphorylated	O
+,	O
+with	O
+channels	O
+that	O
+are	O
+closed	O
+on	O
+the	O
+cytoplasmic	O
+side	O
+.	O
+	O
+In	O
+fungi	O
+,	O
+preventing	O
+ammonium	O
+entry	O
+via	O
+channel	O
+closure	O
+in	O
+ammonium	O
+transporters	O
+would	O
+be	O
+one	O
+way	O
+to	O
+alleviate	O
+ammonium	O
+toxicity	O
+,	O
+in	O
+addition	O
+to	O
+ammonium	O
+excretion	O
+via	O
+Ato	O
+transporters	O
+and	O
+amino	O
+-	O
+acid	O
+secretion	O
+.	O
+	O
+In	O
+addition	O
+,	O
+ICL1	O
+has	O
+shifted	O
+inwards	O
+to	O
+contribute	O
+to	O
+the	O
+channel	O
+closure	O
+by	O
+engaging	O
+His2	O
+from	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+via	O
+hydrogen	O
+bonding	O
+with	O
+a	O
+highly	O
+conserved	O
+tyrosine	O
+hydroxyl	O
+group	O
+.	O
+	O
+Upon	O
+phosphorylation	O
+by	O
+the	O
+Npr1	O
+kinase	O
+in	O
+response	O
+to	O
+nitrogen	O
+limitation	O
+,	O
+the	O
+region	O
+around	O
+the	O
+conserved	O
+ExxGxD	O
+motif	O
+undergoes	O
+a	O
+conformational	O
+change	O
+that	O
+opens	O
+the	O
+channel	O
+(	O
+Fig	O
+.	O
+9	O
+).	O
+	O
+Importantly	O
+,	O
+the	O
+structural	O
+similarities	O
+in	O
+the	O
+TM	O
+parts	O
+of	O
+Mep2	O
+and	O
+AfAmt	O
+-	O
+1	O
+(	O
+Fig	O
+.	O
+5a	O
+)	O
+suggest	O
+that	O
+channel	O
+opening	O
+/	O
+closure	O
+does	O
+not	O
+require	O
+substantial	O
+changes	O
+in	O
+the	O
+residues	O
+lining	O
+the	O
+channel	O
+.	O
+	O
+How	O
+exactly	O
+the	O
+channel	O
+opens	O
+and	O
+whether	O
+opening	O
+is	O
+intra	O
+-	O
+monomeric	O
+are	O
+still	O
+open	O
+questions	O
+;	O
+it	O
+is	O
+possible	O
+that	O
+the	O
+change	O
+in	O
+the	O
+CTR	O
+may	O
+disrupt	O
+its	O
+interactions	O
+with	O
+ICL3	O
+of	O
+the	O
+neighbouring	O
+monomer	O
+(	O
+Fig	O
+.	O
+9b	O
+),	O
+which	O
+could	O
+result	O
+in	O
+opening	O
+of	O
+the	O
+neighbouring	O
+channel	O
+via	O
+inward	O
+movement	O
+of	O
+its	O
+ICL3	O
+.	O
+	O
+Whether	O
+or	O
+not	O
+Mep2	O
+channel	O
+opening	O
+requires	O
+,	O
+in	O
+addition	O
+to	O
+phosphorylation	O
+,	O
+disruption	O
+of	O
+the	O
+Tyr	O
+–	O
+His2	O
+interaction	O
+by	O
+the	O
+ammonium	O
+substrate	O
+is	O
+not	O
+yet	O
+clear	O
+.	O
+	O
+Is	O
+our	O
+model	O
+for	O
+opening	O
+and	O
+closing	O
+of	O
+Mep2	O
+channels	O
+valid	O
+for	O
+other	O
+eukaryotic	O
+ammonium	O
+transporters	O
+?	O
+Our	O
+structural	O
+data	O
+support	O
+previous	O
+studies	O
+and	O
+clarify	O
+the	O
+central	O
+role	O
+of	O
+the	O
+CTR	O
+and	O
+cytoplasmic	O
+loops	O
+in	O
+the	O
+transition	O
+between	O
+closed	O
+and	O
+open	O
+states	O
+.	O
+	O
+In	O
+addition	O
+,	O
+the	O
+AI	O
+region	O
+of	O
+the	O
+CTR	O
+containing	O
+the	O
+Npr1	O
+kinase	O
+site	O
+is	O
+conserved	O
+in	O
+only	O
+a	O
+subset	O
+of	O
+fungal	O
+transporters	O
+,	O
+suggesting	O
+that	O
+the	O
+details	O
+of	O
+the	O
+structural	O
+changes	O
+underpinning	O
+regulation	O
+vary	O
+.	O
+	O
+Nevertheless	O
+,	O
+given	O
+the	O
+central	O
+role	O
+of	O
+absolutely	O
+conserved	O
+residues	O
+within	O
+the	O
+ICL1	O
+-	O
+ICL3	O
+-	O
+CTR	O
+interaction	O
+network	O
+(	O
+Fig	O
+.	O
+4	O
+),	O
+we	O
+propose	O
+that	O
+the	O
+structural	O
+basics	O
+of	O
+fungal	O
+ammonium	O
+transporter	O
+activation	O
+are	O
+conserved	O
+.	O
+	O
+The	O
+fact	O
+that	O
+Mep2	O
+orthologues	O
+of	O
+distantly	O
+related	O
+fungi	O
+are	O
+fully	O
+functional	O
+in	O
+ammonium	O
+transport	O
+and	O
+signalling	O
+in	O
+S	O
+.	O
+cerevisiae	O
+supports	O
+this	O
+notion	O
+.	O
+	O
+With	O
+regards	O
+to	O
+plant	O
+AMTs	O
+,	O
+it	O
+has	O
+been	O
+proposed	O
+that	O
+phosphorylation	O
+at	O
+T460	O
+generates	O
+conformational	O
+changes	O
+that	O
+would	O
+close	O
+the	O
+neighbouring	O
+pore	O
+via	O
+the	O
+C	O
+terminus	O
+.	O
+	O
+This	O
+assumption	O
+was	O
+based	O
+partly	O
+on	O
+a	O
+homology	O
+model	O
+for	O
+Amt	O
+-	O
+1	O
+;	O
+1	O
+based	O
+on	O
+the	O
+(	O
+open	O
+)	O
+archaebacterial	O
+AfAmt	O
+-	O
+1	O
+structure	O
+,	O
+which	O
+suggested	O
+that	O
+the	O
+C	O
+terminus	O
+of	O
+Amt	O
+-	O
+1	O
+;	O
+1	O
+would	O
+extend	O
+further	O
+to	O
+the	O
+neighbouring	O
+monomer	O
+.	O
+	O
+Our	O
+Mep2	O
+structures	O
+show	O
+that	O
+this	O
+assumption	O
+may	O
+not	O
+be	O
+correct	O
+(	O
+Fig	O
+.	O
+4	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+Based	O
+on	O
+the	O
+available	O
+structural	O
+information	O
+,	O
+we	O
+consider	O
+it	O
+more	O
+likely	O
+that	O
+phosphorylation	O
+-	O
+mediated	O
+pore	O
+closure	O
+in	O
+Amt	O
+-	O
+1	O
+;	O
+1	O
+is	O
+intra	O
+-	O
+monomeric	O
+,	O
+via	O
+disruption	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+CTR	O
+and	O
+ICL1	O
+/	O
+ICL3	O
+(	O
+for	O
+example	O
+,	O
+Y467	O
+-	O
+H239	O
+and	O
+D458	O
+-	O
+K71	O
+).	O
+	O
+There	O
+is	O
+generally	O
+no	O
+equivalent	O
+for	O
+CaMep2	O
+Tyr49	O
+in	O
+plant	O
+AMTs	O
+,	O
+indicating	O
+that	O
+a	O
+Tyr	O
+–	O
+His2	O
+hydrogen	O
+bond	O
+as	O
+observed	O
+in	O
+Mep2	O
+may	O
+not	O
+contribute	O
+to	O
+the	O
+closed	O
+state	O
+in	O
+plant	O
+transporters	O
+.	O
+	O
+We	O
+propose	O
+that	O
+intra	O
+-	O
+monomeric	O
+CTR	O
+-	O
+ICL1	O
+/	O
+ICL3	O
+interactions	O
+lie	O
+at	O
+the	O
+basis	O
+of	O
+regulation	O
+of	O
+both	O
+fungal	O
+and	O
+plant	O
+ammonium	O
+transporters	O
+;	O
+close	O
+interactions	O
+generate	O
+open	O
+channels	O
+,	O
+whereas	O
+the	O
+lack	O
+of	O
+‘	O
+intra	O
+-'	O
+interactions	O
+leads	O
+to	O
+inactive	O
+states	O
+.	O
+	O
+The	O
+need	O
+to	O
+regulate	O
+in	O
+opposite	O
+ways	O
+may	O
+be	O
+the	O
+reason	O
+why	O
+the	O
+phosphorylation	O
+sites	O
+are	O
+in	O
+different	O
+parts	O
+of	O
+the	O
+CTR	O
+,	O
+that	O
+is	O
+,	O
+centrally	O
+located	O
+close	O
+to	O
+the	O
+ExxGxD	O
+motif	O
+in	O
+AMTs	O
+and	O
+peripherally	O
+in	O
+Mep2	O
+.	O
+	O
+In	O
+this	O
+way	O
+,	O
+phosphorylation	O
+can	O
+either	O
+lead	O
+to	O
+channel	O
+closing	O
+(	O
+in	O
+the	O
+case	O
+of	O
+AMTs	O
+)	O
+or	O
+channel	O
+opening	O
+in	O
+the	O
+case	O
+of	O
+Mep2	O
+.	O
+	O
+Our	O
+model	O
+also	O
+provides	O
+an	O
+explanation	O
+for	O
+the	O
+observation	O
+that	O
+certain	B-mutant
+mutations	I-mutant
+within	O
+the	O
+CTR	O
+completely	O
+abolish	O
+transport	O
+activity	O
+.	O
+	O
+An	O
+example	O
+of	O
+an	O
+inactivating	O
+residue	O
+is	O
+the	O
+glycine	O
+of	O
+the	O
+ExxGxD	O
+motif	O
+of	O
+the	O
+CTR	O
+.	O
+	O
+Mutation	O
+of	O
+this	O
+residue	O
+(	O
+G393	O
+in	O
+EcAmtB	O
+;	O
+G456	O
+in	O
+AtAmt	O
+-	O
+1	O
+;	O
+1	O
+)	O
+inactivates	O
+transporters	O
+as	O
+diverse	O
+as	O
+Escherichia	O
+coli	O
+AmtB	O
+and	O
+A	O
+.	O
+thaliana	O
+Amt	O
+-	O
+1	O
+;	O
+1	O
+(	O
+refs	O
+).	O
+	O
+Such	O
+mutations	O
+likely	O
+cause	O
+structural	O
+changes	O
+in	O
+the	O
+CTR	O
+that	O
+prevent	O
+close	O
+contacts	O
+between	O
+the	O
+CTR	O
+and	O
+ICL1	O
+/	O
+ICL3	O
+,	O
+thereby	O
+stabilizing	O
+a	O
+closed	O
+state	O
+that	O
+may	O
+be	O
+similar	O
+to	O
+that	O
+observed	O
+in	O
+Mep2	O
+.	O
+	O
+Regulation	O
+and	O
+modulation	O
+of	O
+membrane	O
+transport	O
+by	O
+phosphorylation	O
+is	O
+known	O
+to	O
+occur	O
+in	O
+,	O
+for	O
+example	O
+,	O
+aquaporins	O
+and	O
+urea	O
+transporters	O
+,	O
+and	O
+is	O
+likely	O
+to	O
+be	O
+a	O
+common	O
+theme	O
+for	O
+eukaryotic	O
+channels	O
+and	O
+transporters	O
+.	O
+	O
+Recently	O
+,	O
+phosphorylation	O
+was	O
+also	O
+shown	O
+to	O
+modulate	O
+substrate	O
+affinity	O
+in	O
+nitrate	O
+transporters	O
+.	O
+	O
+With	O
+respect	O
+to	O
+ammonium	O
+transport	O
+,	O
+phosphorylation	O
+has	O
+thus	O
+far	O
+only	O
+been	O
+shown	O
+for	O
+A	O
+.	O
+thaliana	O
+AMTs	O
+and	O
+for	O
+S	O
+.	O
+cerevisiae	O
+Mep2	O
+(	O
+refs	O
+).	O
+	O
+However	O
+,	O
+the	O
+absence	O
+of	O
+GlnK	O
+proteins	O
+in	O
+eukaryotes	O
+suggests	O
+that	O
+phosphorylation	O
+-	O
+based	O
+regulation	O
+of	O
+ammonium	O
+transport	O
+may	O
+be	O
+widespread	O
+.	O
+	O
+With	O
+respect	O
+to	O
+Mep2	O
+-	O
+mediated	O
+signalling	O
+to	O
+induce	O
+pseudohyphal	O
+growth	O
+,	O
+two	O
+models	O
+have	O
+been	O
+put	O
+forward	O
+as	O
+to	O
+how	O
+this	O
+occurs	O
+and	O
+why	O
+it	O
+is	O
+specific	O
+to	O
+Mep2	O
+proteins	O
+.	O
+	O
+In	O
+one	O
+model	O
+,	O
+signalling	O
+is	O
+proposed	O
+to	O
+depend	O
+on	O
+the	O
+nature	O
+of	O
+the	O
+transported	O
+substrate	O
+,	O
+which	O
+might	O
+be	O
+different	O
+in	O
+certain	O
+subfamilies	O
+of	O
+ammonium	O
+transporters	O
+(	O
+for	O
+example	O
+,	O
+Mep1	O
+/	O
+Mep3	O
+versus	O
+Mep2	O
+).	O
+	O
+In	O
+the	O
+other	O
+model	O
+,	O
+signalling	O
+is	O
+thought	O
+to	O
+require	O
+a	O
+distinct	O
+conformation	O
+of	O
+the	O
+Mep2	O
+transporter	O
+occurring	O
+during	O
+the	O
+transport	O
+cycle	O
+.	O
+	O
+While	O
+the	O
+current	O
+study	O
+does	O
+not	O
+specifically	O
+address	O
+the	O
+mechanism	O
+of	O
+signalling	O
+underlying	O
+pseudohyphal	O
+growth	O
+,	O
+our	O
+structures	O
+do	O
+show	O
+that	O
+Mep2	O
+proteins	O
+can	O
+assume	O
+different	O
+conformations	O
+.	O
+	O
+It	O
+is	O
+clear	O
+that	O
+ammonium	O
+transport	O
+across	O
+biomembranes	O
+remains	O
+a	O
+fascinating	O
+and	O
+challenging	O
+field	O
+in	O
+large	O
+part	O
+due	O
+to	O
+the	O
+unique	O
+properties	O
+of	O
+the	O
+substrate	O
+.	O
+	O
+Our	O
+Mep2	O
+structural	O
+work	O
+now	O
+provides	O
+a	O
+foundation	O
+for	O
+future	O
+studies	O
+to	O
+uncover	O
+the	O
+details	O
+of	O
+the	O
+structural	O
+changes	O
+that	O
+occur	O
+during	O
+eukaryotic	O
+ammonium	O
+transport	O
+and	O
+signaling	O
+,	O
+and	O
+to	O
+assess	O
+the	O
+possibility	O
+to	O
+utilize	O
+small	O
+molecules	O
+to	O
+shut	O
+down	O
+ammonium	O
+sensing	O
+and	O
+downstream	O
+signalling	O
+pathways	O
+in	O
+pathogenic	O
+fungi	O
+.	O
+	O
+X	O
+-	O
+ray	O
+crystal	O
+structures	O
+of	O
+Mep2	O
+transceptors	O
+.	O
+	O
+The	O
+region	O
+showing	O
+ICL1	O
+(	O
+blue	O
+),	O
+ICL3	O
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	O
+(	O
+red	O
+)	O
+is	O
+boxed	O
+for	O
+comparison	O
+.	O
+	O
+(	O
+b	O
+)	O
+CaMep2	O
+trimer	O
+viewed	O
+from	O
+the	O
+intracellular	O
+side	O
+(	O
+right	O
+).	O
+	O
+The	O
+CTR	O
+is	O
+boxed	O
+.	O
+	O
+(	O
+c	O
+)	O
+Overlay	O
+of	O
+ScMep2	O
+(	O
+grey	O
+)	O
+and	O
+CaMep2	O
+(	O
+rainbow	O
+),	O
+illustrating	O
+the	O
+differences	O
+in	O
+the	O
+CTRs	O
+.	O
+	O
+Sequence	O
+conservation	O
+in	O
+ammonium	O
+transporters	O
+.	O
+	O
+ClustalW	O
+alignment	O
+of	O
+CaMep2	O
+,	O
+ScMep2	O
+,	O
+A	O
+.	O
+fulgidus	O
+Amt	O
+-	O
+1	O
+,	O
+E	O
+.	O
+coli	O
+AmtB	O
+and	O
+A	O
+.	O
+thaliana	O
+Amt	O
+-	O
+1	O
+;	O
+1	O
+.	O
+	O
+The	O
+conserved	O
+RxK	O
+motif	O
+in	O
+ICL1	O
+is	O
+boxed	O
+in	O
+blue	O
+,	O
+the	O
+ER	O
+motif	O
+in	O
+ICL2	O
+in	O
+cyan	O
+,	O
+the	O
+conserved	O
+ExxGxD	O
+motif	O
+of	O
+the	O
+CTR	O
+in	O
+red	O
+and	O
+the	O
+AI	O
+region	O
+in	O
+yellow	O
+.	O
+	O
+Coloured	O
+residues	O
+are	O
+functionally	O
+important	O
+and	O
+correspond	O
+to	O
+those	O
+of	O
+the	O
+Phe	O
+gate	O
+(	O
+blue	O
+),	O
+the	O
+binding	O
+site	O
+Trp	O
+residue	O
+(	O
+magenta	O
+)	O
+and	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+(	O
+red	O
+).	O
+	O
+The	O
+Npr1	O
+kinase	O
+site	O
+in	O
+the	O
+AI	O
+region	O
+is	O
+highlighted	O
+pink	O
+.	O
+	O
+The	O
+grey	O
+sequences	O
+at	O
+the	O
+C	O
+termini	O
+of	O
+CaMep2	O
+and	O
+ScMep2	O
+are	O
+not	O
+visible	O
+in	O
+the	O
+structures	O
+and	O
+are	O
+likely	O
+disordered	O
+.	O
+	O
+The	O
+quantified	O
+cell	O
+density	O
+reflects	O
+logarithmic	O
+growth	O
+after	O
+24	O
+h	O
+.	O
+Error	O
+bars	O
+are	O
+the	O
+s	O
+.	O
+d	O
+.	O
+for	O
+three	O
+replicates	O
+of	O
+each	O
+strain	O
+(	O
+b	O
+)	O
+The	O
+strains	O
+used	O
+in	O
+a	O
+were	O
+also	O
+serially	O
+diluted	O
+and	O
+spotted	O
+onto	O
+minimal	O
+agar	O
+plates	O
+containing	O
+glutamate	O
+(	O
+0	O
+.	O
+1	O
+%)	O
+or	O
+ammonium	O
+sulphate	O
+(	O
+1	O
+mM	O
+),	O
+and	O
+grown	O
+for	O
+3	O
+days	O
+at	O
+30	O
+°	O
+C	O
+.	O
+	O
+Structural	O
+differences	O
+between	O
+Mep2	O
+and	O
+bacterial	O
+ammonium	O
+transporters	O
+.	O
+	O
+(	O
+a	O
+)	O
+ICL1	O
+in	O
+AfAmt	O
+-	O
+1	O
+(	O
+light	O
+blue	O
+)	O
+and	O
+CaMep2	O
+(	O
+dark	O
+blue	O
+),	O
+showing	O
+unwinding	O
+and	O
+inward	O
+movement	O
+in	O
+the	O
+fungal	O
+protein	O
+.	O
+(	O
+b	O
+)	O
+Stereo	O
+diagram	O
+viewed	O
+from	O
+the	O
+cytosol	O
+of	O
+ICL1	O
+,	O
+ICL3	O
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	O
+(	O
+red	O
+)	O
+in	O
+AfAmt	O
+-	O
+1	O
+(	O
+light	O
+colours	O
+)	O
+and	O
+CaMep2	O
+(	O
+dark	O
+colours	O
+).	O
+	O
+The	O
+side	O
+chains	O
+of	O
+residues	O
+in	O
+the	O
+RxK	O
+motif	O
+as	O
+well	O
+as	O
+those	O
+of	O
+Tyr49	O
+and	O
+His342	O
+are	O
+labelled	O
+.	O
+	O
+(	O
+c	O
+)	O
+Conserved	O
+residues	O
+in	O
+ICL1	O
+-	O
+3	O
+and	O
+the	O
+CTR	O
+.	O
+	O
+Views	O
+from	O
+the	O
+cytosol	O
+for	O
+CaMep2	O
+(	O
+left	O
+)	O
+and	O
+AfAmt	O
+-	O
+1	O
+,	O
+highlighting	O
+the	O
+large	O
+differences	O
+in	O
+conformation	O
+of	O
+the	O
+conserved	O
+residues	O
+in	O
+ICL1	O
+(	O
+RxK	O
+motif	O
+;	O
+blue	O
+),	O
+ICL2	O
+(	O
+ER	O
+motif	O
+;	O
+cyan	O
+),	O
+ICL3	O
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	O
+(	O
+red	O
+).	O
+	O
+The	O
+labelled	O
+residues	O
+are	O
+analogous	O
+within	O
+both	O
+structures	O
+.	O
+	O
+In	O
+b	O
+and	O
+c	O
+,	O
+the	O
+centre	O
+of	O
+the	O
+trimer	O
+is	O
+at	O
+top	O
+.	O
+	O
+(	O
+a	O
+)	O
+Stereo	O
+superposition	O
+of	O
+AfAmt	O
+-	O
+1	O
+and	O
+CaMep2	O
+showing	O
+the	O
+residues	O
+of	O
+the	O
+Phe	O
+gate	O
+,	O
+His2	O
+of	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+and	O
+the	O
+tyrosine	O
+residue	O
+Y49	O
+in	O
+TM1	O
+that	O
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+His2	O
+in	O
+CaMep2	O
+.	O
+(	O
+b	O
+)	O
+Surface	O
+views	O
+from	O
+the	O
+side	O
+in	O
+rainbow	O
+colouring	O
+,	O
+showing	O
+the	O
+two	O
+-	O
+tier	O
+channel	O
+block	O
+(	O
+indicated	O
+by	O
+the	O
+arrows	O
+)	O
+in	O
+CaMep2	O
+.	O
+	O
+The	O
+Npr1	O
+kinase	O
+target	O
+Ser453	O
+is	O
+dephosphorylated	O
+and	O
+located	O
+in	O
+an	O
+electronegative	O
+pocket	O
+.	O
+	O
+(	O
+a	O
+)	O
+Stereoviews	O
+of	O
+CaMep2	O
+showing	O
+2Fo	O
+–	O
+Fc	O
+electron	O
+density	O
+(	O
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+)	O
+for	O
+CTR	O
+residues	O
+Asp419	O
+-	O
+Met422	O
+and	O
+for	O
+Tyr446	O
+-	O
+Thr455	O
+of	O
+the	O
+AI	O
+region	O
+.	O
+	O
+The	O
+phosphorylation	O
+target	O
+residue	O
+Ser453	O
+is	O
+labelled	O
+in	O
+bold	O
+.	O
+	O
+(	O
+c	O
+)	O
+Cytoplasmic	O
+view	O
+of	O
+the	O
+Mep2	O
+trimer	O
+indicating	O
+the	O
+large	O
+distance	O
+between	O
+Ser453	O
+and	O
+the	O
+channel	O
+exits	O
+(	O
+circles	O
+;	O
+Ile52	O
+lining	O
+the	O
+channel	O
+exit	O
+is	O
+shown	O
+).	O
+	O
+Effect	O
+of	O
+removal	O
+of	O
+the	O
+AI	O
+region	O
+on	O
+Mep2	O
+structure	O
+.	O
+	O
+(	O
+a	O
+)	O
+Side	O
+views	O
+for	O
+WT	O
+CaMep2	O
+(	O
+left	O
+)	O
+and	O
+the	O
+truncation	O
+mutant	O
+442Δ	B-mutant
+(	O
+right	O
+).	O
+	O
+The	O
+latter	O
+is	O
+shown	O
+as	O
+a	O
+putty	O
+model	O
+according	O
+to	O
+B	O
+-	O
+factors	O
+to	O
+illustrate	O
+the	O
+disorder	O
+in	O
+the	O
+protein	O
+on	O
+the	O
+cytoplasmic	O
+side	O
+.	O
+	O
+Phosphorylation	O
+causes	O
+conformational	O
+changes	O
+in	O
+the	O
+CTR	O
+.	O
+	O
+(	O
+a	O
+)	O
+Cytoplasmic	O
+view	O
+of	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+trimer	O
+,	O
+with	O
+WT	O
+CaMep2	O
+superposed	O
+in	O
+grey	O
+for	O
+one	O
+of	O
+the	O
+monomers	O
+.	O
+	O
+The	O
+AI	O
+region	O
+is	O
+coloured	O
+magenta	O
+.	O
+	O
+(	O
+b	O
+)	O
+Monomer	O
+side	O
+-	O
+view	O
+superposition	O
+of	O
+WT	O
+CaMep2	O
+and	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+,	O
+showing	O
+the	O
+conformational	O
+change	O
+and	O
+disorder	O
+around	O
+the	O
+ExxGxD	O
+motif	O
+.	O
+	O
+Side	O
+chains	O
+for	O
+residues	O
+452	O
+and	O
+453	O
+are	O
+shown	O
+as	O
+stick	O
+models	O
+.	O
+	O
+(	O
+a	O
+)	O
+In	O
+the	O
+closed	O
+,	O
+non	O
+-	O
+phosphorylated	O
+state	O
+(	O
+i	O
+),	O
+the	O
+CTR	O
+(	O
+magenta	O
+)	O
+and	O
+ICL3	O
+(	O
+green	O
+)	O
+are	O
+far	O
+apart	O
+with	O
+the	O
+latter	O
+blocking	O
+the	O
+intracellular	O
+channel	O
+exit	O
+(	O
+indicated	O
+with	O
+a	O
+hatched	O
+circle	O
+).	O
+	O
+The	O
+open	O
+-	O
+channel	O
+Mep2	O
+structure	O
+is	O
+represented	O
+by	O
+archaebacterial	O
+Amt	O
+-	O
+1	O
+and	O
+shown	O
+in	O
+lighter	O
+colours	O
+consistent	O
+with	O
+Fig	O
+.	O
+4	O
+.	O
+	O
+As	O
+discussed	O
+in	O
+the	O
+text	O
+,	O
+similar	O
+structural	O
+arrangements	O
+may	O
+occur	O
+in	O
+plant	O
+AMTs	O
+.	O
+	O
+In	O
+this	O
+case	O
+however	O
+,	O
+the	O
+open	O
+channel	O
+corresponds	O
+to	O
+the	O
+non	O
+-	O
+phosphorylated	O
+state	O
+;	O
+phosphorylation	O
+breaks	O
+the	O
+CTR	O
+–	O
+ICL3	O
+interactions	O
+leading	O
+to	O
+channel	O
+closure	O
+.	O
+(	O
+b	O
+)	O
+Model	O
+based	O
+on	O
+AMT	O
+transporter	O
+analogy	O
+showing	O
+how	O
+phosphorylation	O
+of	O
+a	O
+Mep2	O
+monomer	O
+might	O
+allosterically	O
+open	O
+channels	O
+in	O
+the	O
+entire	O
+trimer	O
+via	O
+disruption	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+CTR	O
+and	O
+ICL3	O
+of	O
+a	O
+neighbouring	O
+monomer	O
+(	O
+arrow	O
+).	O
+	O
+Structural	O
+diversity	O
+in	O
+a	O
+human	O
+antibody	O
+germline	O
+library	O
+	O
+All	O
+four	O
+heavy	O
+chains	O
+of	O
+the	O
+antigen	O
+-	O
+binding	O
+fragments	O
+(	O
+Fabs	O
+)	O
+have	O
+the	O
+same	O
+complementarity	O
+-	O
+determining	O
+region	O
+(	O
+CDR	O
+)	O
+H3	O
+that	O
+was	O
+reported	O
+in	O
+an	O
+earlier	O
+Fab	O
+structure	O
+.	O
+	O
+The	O
+structure	O
+analyses	O
+include	O
+comparisons	O
+of	O
+the	O
+overall	O
+structures	O
+,	O
+canonical	O
+structures	O
+of	O
+the	O
+CDRs	O
+and	O
+the	O
+VH	O
+:	O
+VL	O
+packing	O
+interactions	O
+.	O
+	O
+The	O
+CDR	O
+conformations	O
+for	O
+the	O
+most	O
+part	O
+are	O
+tightly	O
+clustered	O
+,	O
+especially	O
+for	O
+the	O
+ones	O
+with	O
+shorter	O
+lengths	O
+.	O
+	O
+The	O
+longer	O
+CDRs	O
+with	O
+tandem	O
+glycines	O
+or	O
+serines	O
+have	O
+more	O
+conformational	O
+diversity	O
+than	O
+the	O
+others	O
+.	O
+	O
+One	O
+conclusion	O
+is	O
+that	O
+the	O
+CDR	O
+H3	O
+conformations	O
+are	O
+influenced	O
+by	O
+both	O
+their	O
+amino	O
+acid	O
+sequence	O
+and	O
+their	O
+structural	O
+environment	O
+determined	O
+by	O
+the	O
+heavy	O
+and	O
+light	O
+chain	O
+pairing	O
+.	O
+	O
+The	O
+stem	O
+regions	O
+of	O
+14	O
+of	O
+the	O
+variant	O
+pairs	O
+are	O
+in	O
+the	O
+‘	O
+kinked	O
+’	O
+conformation	O
+,	O
+and	O
+only	O
+2	O
+are	O
+in	O
+the	O
+extended	O
+conformation	O
+.	O
+	O
+The	O
+packing	O
+of	O
+the	O
+VH	O
+and	O
+VL	O
+domains	O
+is	O
+consistent	O
+with	O
+our	O
+knowledge	O
+of	O
+antibody	O
+structure	O
+,	O
+and	O
+the	O
+tilt	O
+angles	O
+between	O
+these	O
+domains	O
+cover	O
+a	O
+range	O
+of	O
+11	O
+degrees	O
+.	O
+	O
+Two	O
+of	O
+16	O
+structures	O
+showed	O
+particularly	O
+large	O
+variations	O
+in	O
+the	O
+tilt	O
+angles	O
+when	O
+compared	O
+with	O
+the	O
+other	O
+pairings	O
+.	O
+	O
+At	O
+present	O
+,	O
+therapeutic	O
+antibodies	O
+are	O
+the	O
+largest	O
+class	O
+of	O
+biotherapeutic	O
+proteins	O
+that	O
+are	O
+in	O
+clinical	O
+trials	O
+.	O
+	O
+The	O
+use	O
+of	O
+monoclonal	O
+antibodies	O
+as	O
+therapeutics	O
+began	O
+in	O
+the	O
+early	O
+1980s	O
+,	O
+and	O
+their	O
+composition	O
+has	O
+transitioned	O
+from	O
+murine	O
+antibodies	O
+to	O
+generally	O
+less	O
+immunogenic	O
+humanized	O
+and	O
+human	O
+antibodies	O
+.	O
+	O
+The	O
+technologies	O
+currently	O
+used	O
+to	O
+obtain	O
+human	O
+antibodies	O
+include	O
+transgenic	O
+mice	O
+containing	O
+human	O
+antibody	O
+repertoires	O
+,	O
+cloning	O
+directly	O
+from	O
+human	O
+B	O
+cells	O
+,	O
+and	O
+in	O
+vitro	O
+selection	O
+from	O
+antibody	O
+libraries	O
+using	O
+various	O
+display	O
+technologies	O
+.	O
+	O
+All	O
+engineering	O
+efforts	O
+are	O
+guided	O
+by	O
+our	O
+understanding	O
+of	O
+the	O
+atomic	O
+structures	O
+of	O
+antibodies	O
+.	O
+	O
+In	O
+such	O
+efforts	O
+,	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+specific	O
+antibody	O
+may	O
+not	O
+be	O
+available	O
+,	O
+but	O
+modeling	O
+can	O
+be	O
+used	O
+to	O
+guide	O
+the	O
+engineering	O
+efforts	O
+.	O
+	O
+Today	O
+'	O
+s	O
+antibody	O
+modeling	O
+approaches	O
+,	O
+which	O
+normally	O
+focus	O
+on	O
+the	O
+variable	O
+region	O
+,	O
+are	O
+being	O
+developed	O
+by	O
+the	O
+application	O
+of	O
+structural	O
+principles	O
+and	O
+insights	O
+that	O
+are	O
+evolving	O
+as	O
+our	O
+knowledge	O
+of	O
+antibody	O
+structures	O
+continues	O
+to	O
+expand	O
+.	O
+	O
+Our	O
+current	O
+structural	O
+knowledge	O
+of	O
+antibodies	O
+is	O
+based	O
+on	O
+a	O
+multitude	O
+of	O
+studies	O
+that	O
+used	O
+many	O
+techniques	O
+to	O
+gain	O
+insight	O
+into	O
+the	O
+functional	O
+and	O
+structural	O
+properties	O
+of	O
+this	O
+class	O
+of	O
+macromolecule	O
+.	O
+	O
+Five	O
+different	O
+antibody	O
+isotypes	O
+occur	O
+,	O
+IgG	O
+,	O
+IgD	O
+,	O
+IgE	O
+,	O
+IgA	O
+and	O
+IgM	O
+,	O
+and	O
+each	O
+isotype	O
+has	O
+a	O
+unique	O
+role	O
+in	O
+the	O
+adaptive	O
+immune	O
+system	O
+.	O
+	O
+IgG	O
+,	O
+IgD	O
+and	O
+IgE	O
+isotypes	O
+are	O
+composed	O
+of	O
+2	O
+heavy	O
+chains	O
+(	O
+HCs	O
+)	O
+and	O
+2	O
+light	O
+chains	O
+(	O
+LCs	O
+)	O
+linked	O
+through	O
+disulfide	O
+bonds	O
+,	O
+while	O
+IgA	O
+and	O
+IgM	O
+are	O
+double	O
+and	O
+quintuple	O
+versions	O
+of	O
+antibodies	O
+,	O
+respectively	O
+.	O
+	O
+These	O
+multimeric	O
+forms	O
+are	O
+linked	O
+with	O
+an	O
+additional	O
+J	O
+chain	O
+.	O
+	O
+The	O
+LCs	O
+that	O
+associate	O
+with	O
+the	O
+HCs	O
+are	O
+divided	O
+into	O
+2	O
+functionally	O
+indistinguishable	O
+classes	O
+,	O
+κ	O
+and	O
+λ	O
+.	O
+	O
+Both	O
+κ	O
+and	O
+λ	O
+polypeptide	O
+chains	O
+are	O
+composed	O
+of	O
+a	O
+single	O
+V	O
+domain	O
+and	O
+a	O
+single	O
+C	O
+domain	O
+.	O
+	O
+The	O
+heavy	O
+and	O
+light	O
+chains	O
+are	O
+composed	O
+of	O
+structural	O
+domains	O
+that	O
+have	O
+∼	O
+110	O
+amino	O
+acid	O
+residues	O
+.	O
+	O
+All	O
+immunoglobulin	O
+chains	O
+have	O
+an	O
+N	O
+-	O
+terminal	O
+V	O
+domain	O
+followed	O
+by	O
+1	O
+to	O
+4	O
+C	O
+domains	O
+,	O
+depending	O
+upon	O
+the	O
+chain	O
+type	O
+.	O
+	O
+This	O
+site	O
+,	O
+which	O
+interacts	O
+with	O
+the	O
+antigen	O
+(	O
+or	O
+target	O
+),	O
+is	O
+the	O
+focus	O
+of	O
+current	O
+antibody	O
+modeling	O
+efforts	O
+.	O
+	O
+This	O
+interaction	O
+site	O
+is	O
+composed	O
+of	O
+6	O
+complementarity	O
+-	O
+determining	O
+regions	O
+(	O
+CDRs	O
+)	O
+that	O
+were	O
+identified	O
+in	O
+early	O
+antibody	O
+amino	O
+acid	O
+sequence	O
+analyses	O
+to	O
+be	O
+hypervariable	O
+in	O
+nature	O
+,	O
+and	O
+thus	O
+are	O
+responsible	O
+for	O
+the	O
+sequence	O
+and	O
+structural	O
+diversity	O
+of	O
+our	O
+antibody	O
+repertoire	O
+.	O
+	O
+However	O
+,	O
+an	O
+initial	O
+structural	O
+analysis	O
+of	O
+the	O
+combining	O
+sites	O
+of	O
+the	O
+small	O
+set	O
+of	O
+structures	O
+of	O
+immunoglobulin	O
+fragments	O
+available	O
+in	O
+the	O
+1980s	O
+found	O
+that	O
+5	O
+of	O
+the	O
+6	O
+hypervariable	O
+loops	O
+or	O
+CDRs	O
+had	O
+canonical	O
+structures	O
+(	O
+a	O
+limited	O
+set	O
+of	O
+main	O
+-	O
+chain	O
+conformations	O
+).	O
+	O
+A	O
+CDR	O
+canonical	O
+structure	O
+is	O
+defined	O
+by	O
+its	O
+length	O
+and	O
+conserved	O
+residues	O
+located	O
+in	O
+the	O
+hypervariable	O
+loop	O
+and	O
+framework	O
+residues	O
+(	O
+V	O
+-	O
+region	O
+residues	O
+that	O
+are	O
+not	O
+part	O
+of	O
+the	O
+CDRs	O
+).	O
+	O
+Furthermore	O
+,	O
+studies	O
+of	O
+antibody	O
+sequences	O
+revealed	O
+that	O
+the	O
+total	O
+number	O
+of	O
+canonical	O
+structures	O
+are	O
+limited	O
+for	O
+each	O
+CDR	O
+,	O
+indicating	O
+possibly	O
+that	O
+antigen	O
+recognition	O
+may	O
+be	O
+affected	O
+by	O
+structural	O
+restrictions	O
+at	O
+the	O
+antigen	O
+-	O
+binding	O
+site	O
+.	O
+	O
+Additional	O
+efforts	O
+have	O
+led	O
+to	O
+our	O
+current	O
+understanding	O
+that	O
+the	O
+LC	O
+CDRs	O
+L1	O
+,	O
+L2	O
+,	O
+and	O
+L3	O
+have	O
+preferred	O
+sets	O
+of	O
+canonical	O
+structures	O
+based	O
+on	O
+length	O
+and	O
+amino	O
+acid	O
+sequence	O
+composition	O
+.	O
+	O
+This	O
+was	O
+also	O
+found	O
+to	O
+be	O
+the	O
+case	O
+for	O
+the	O
+H1	O
+and	O
+H2	O
+CDRs	O
+.	O
+	O
+Classification	O
+schemes	O
+for	O
+the	O
+canonical	O
+structures	O
+of	O
+these	O
+5	O
+CDRs	O
+have	O
+emerged	O
+and	O
+evolved	O
+as	O
+the	O
+number	O
+of	O
+depositions	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+of	O
+Fab	O
+fragments	O
+of	O
+antibodies	O
+grow	O
+.	O
+	O
+Recently	O
+,	O
+a	O
+comprehensive	O
+CDR	O
+classification	O
+scheme	O
+was	O
+reported	O
+identifying	O
+72	O
+clusters	O
+of	O
+conformations	O
+observed	O
+in	O
+antibody	O
+structures	O
+.	O
+	O
+The	O
+knowledge	O
+and	O
+predictability	O
+of	O
+these	O
+CDR	O
+canonical	O
+structures	O
+have	O
+greatly	O
+advanced	O
+antibody	O
+modeling	O
+efforts	O
+.	O
+	O
+The	O
+cataloging	O
+and	O
+development	O
+of	O
+the	O
+rules	O
+for	O
+predicting	O
+the	O
+conformation	O
+of	O
+the	O
+anchor	O
+region	O
+of	O
+CDR	O
+H3	O
+continue	O
+to	O
+be	O
+refined	O
+,	O
+producing	O
+new	O
+insight	O
+into	O
+the	O
+CDR	O
+H3	O
+conformations	O
+and	O
+new	O
+tools	O
+for	O
+antibody	O
+engineering	O
+.	O
+	O
+Recent	O
+antibody	O
+modeling	O
+assessments	O
+show	O
+continued	O
+improvement	O
+in	O
+the	O
+quality	O
+of	O
+the	O
+models	O
+being	O
+generated	O
+by	O
+a	O
+variety	O
+of	O
+modeling	O
+methods	O
+.	O
+	O
+Although	O
+antibody	O
+modeling	O
+is	O
+improving	O
+,	O
+the	O
+latest	O
+assessment	O
+revealed	O
+a	O
+number	O
+of	O
+challenges	O
+that	O
+need	O
+to	O
+be	O
+overcome	O
+to	O
+provide	O
+accurate	O
+3	O
+-	O
+dimensional	O
+models	O
+of	O
+antibody	O
+V	O
+regions	O
+,	O
+including	O
+accuracies	O
+in	O
+the	O
+modeling	O
+of	O
+CDR	O
+H3	O
+.	O
+	O
+The	O
+need	O
+for	O
+improvement	O
+in	O
+this	O
+area	O
+was	O
+also	O
+highlighted	O
+in	O
+a	O
+recent	O
+study	O
+reporting	O
+an	O
+approach	O
+and	O
+results	O
+that	O
+may	O
+influence	O
+future	O
+antibody	O
+modeling	O
+efforts	O
+.	O
+	O
+One	O
+important	O
+finding	O
+of	O
+the	O
+antibody	O
+modeling	O
+assessments	O
+was	O
+that	O
+errors	O
+in	O
+the	O
+structural	O
+templates	O
+that	O
+are	O
+used	O
+as	O
+the	O
+basis	O
+for	O
+homology	O
+models	O
+can	O
+propagate	O
+into	O
+the	O
+final	O
+models	O
+,	O
+producing	O
+inaccuracies	O
+that	O
+may	O
+negatively	O
+influence	O
+the	O
+predictive	O
+nature	O
+of	O
+the	O
+V	O
+region	O
+model	O
+.	O
+	O
+This	O
+Fab	O
+library	O
+is	O
+composed	O
+of	O
+3	O
+HC	O
+germlines	O
+,	O
+IGHV1	B-mutant
+-	I-mutant
+69	I-mutant
+(	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+),	O
+IGHV3	B-mutant
+-	I-mutant
+23	I-mutant
+(	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+)	O
+and	O
+IGHV5	B-mutant
+-	I-mutant
+51	I-mutant
+(	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+),	O
+and	O
+4	O
+LC	O
+germlines	O
+(	O
+all	O
+κ	O
+),	O
+IGKV1	B-mutant
+-	I-mutant
+39	I-mutant
+(	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+),	O
+IGKV3	B-mutant
+-	I-mutant
+11	I-mutant
+(	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+),	O
+IGKV3	B-mutant
+-	I-mutant
+20	I-mutant
+(	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+)	O
+and	O
+IGKV4	B-mutant
+-	I-mutant
+1	I-mutant
+(	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+).	O
+	O
+Selection	O
+of	O
+these	O
+genes	O
+was	O
+based	O
+on	O
+the	O
+high	O
+frequency	O
+of	O
+their	O
+use	O
+and	O
+their	O
+cognate	O
+canonical	O
+structures	O
+that	O
+were	O
+found	O
+binding	O
+to	O
+peptides	O
+and	O
+proteins	O
+,	O
+as	O
+well	O
+as	O
+their	O
+ability	O
+to	O
+be	O
+expressed	O
+in	O
+bacteria	O
+and	O
+displayed	O
+on	O
+filamentous	O
+phage	O
+.	O
+	O
+The	O
+implementation	O
+of	O
+the	O
+library	O
+involves	O
+the	O
+diversification	O
+of	O
+the	O
+human	O
+germline	O
+genes	O
+to	O
+mimic	O
+that	O
+found	O
+in	O
+natural	O
+human	O
+libraries	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+determinations	O
+and	O
+structural	O
+analyses	O
+of	O
+all	O
+germline	O
+Fabs	O
+in	O
+the	O
+library	O
+described	O
+above	O
+along	O
+with	O
+the	O
+structures	O
+of	O
+a	O
+fourth	O
+HC	O
+germline	O
+,	O
+IGHV3	B-mutant
+-	I-mutant
+53	I-mutant
+(	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+),	O
+paired	O
+with	O
+the	O
+4	O
+LCs	O
+of	O
+the	O
+library	O
+have	O
+been	O
+carried	O
+out	O
+to	O
+support	O
+antibody	O
+therapeutic	O
+development	O
+.	O
+	O
+All	O
+16	O
+HCs	O
+of	O
+the	O
+Fabs	O
+have	O
+the	O
+same	O
+CDR	O
+H3	O
+that	O
+was	O
+reported	O
+in	O
+an	O
+earlier	O
+Fab	O
+structure	O
+.	O
+	O
+This	O
+is	O
+the	O
+first	O
+systematic	O
+study	O
+of	O
+the	O
+same	O
+VH	O
+and	O
+VL	O
+structures	O
+in	O
+the	O
+context	O
+of	O
+different	O
+pairings	O
+.	O
+	O
+The	O
+structure	O
+analyses	O
+include	O
+comparisons	O
+of	O
+the	O
+overall	O
+structures	O
+,	O
+canonical	O
+structures	O
+of	O
+the	O
+L1	O
+,	O
+L2	O
+,	O
+L3	O
+,	O
+H1	O
+and	O
+H2	O
+CDRs	O
+,	O
+the	O
+structures	O
+of	O
+all	O
+CDR	O
+H3s	O
+,	O
+and	O
+the	O
+VH	O
+:	O
+VL	O
+packing	O
+interactions	O
+.	O
+	O
+The	O
+structures	O
+and	O
+their	O
+analyses	O
+provide	O
+a	O
+foundation	O
+for	O
+future	O
+antibody	O
+engineering	O
+and	O
+structure	O
+determination	O
+efforts	O
+.	O
+	O
+Crystal	O
+structures	O
+	O
+Crystal	O
+data	O
+,	O
+X	O
+-	O
+ray	O
+data	O
+,	O
+and	O
+refinement	O
+statistics	O
+.	O
+	O
+(	O
+Continued	O
+)	O
+Crystal	O
+data	O
+,	O
+X	O
+-	O
+ray	O
+data	O
+,	O
+and	O
+refinement	O
+statistics	O
+.	O
+	O
+The	O
+crystal	O
+structures	O
+of	O
+a	O
+germline	O
+library	O
+composed	O
+of	O
+16	O
+Fabs	O
+generated	O
+by	O
+combining	O
+4	O
+HCs	O
+(	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+)	O
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+4	O
+LCs	O
+(	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+)	O
+have	O
+been	O
+determined	O
+.	O
+	O
+The	O
+Fab	O
+heavy	O
+and	O
+light	O
+chain	O
+sequences	O
+for	O
+the	O
+variants	O
+numbered	O
+according	O
+to	O
+Chothia	O
+are	O
+shown	O
+in	O
+Fig	O
+.	O
+S1	O
+.	O
+	O
+The	O
+four	O
+different	O
+HCs	O
+all	O
+have	O
+the	O
+same	O
+CDR	O
+H3	O
+sequence	O
+,	O
+ARYDGIYGELDF	O
+.	O
+	O
+These	O
+include	O
+(	O
+1	O
+)	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+11	O
+and	O
+H3	O
+-	O
+23	O
+:	O
+L4	O
+-	O
+1	O
+in	O
+P212121	O
+,	O
+(	O
+2	O
+)	O
+H3	O
+-	O
+53	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+H3	O
+-	O
+53	O
+:	O
+L3	O
+-	O
+11	O
+and	O
+H3	O
+-	O
+53	O
+:	O
+L3	O
+-	O
+20	O
+in	O
+P6522	O
+,	O
+and	O
+(	O
+3	O
+)	O
+H5	O
+-	O
+51	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+11	O
+and	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+20	O
+in	O
+P212121	O
+.	O
+	O
+The	O
+similarity	O
+in	O
+the	O
+crystal	O
+forms	O
+is	O
+attributed	O
+in	O
+part	O
+to	O
+cross	O
+-	O
+seeding	O
+using	O
+the	O
+microseed	O
+matrix	O
+screening	O
+for	O
+groups	O
+2	O
+and	O
+3	O
+.	O
+	O
+The	O
+number	O
+of	O
+Fab	O
+molecules	O
+in	O
+the	O
+crystallographic	O
+asymmetric	O
+unit	O
+varies	O
+from	O
+1	O
+(	O
+for	O
+12	O
+Fabs	O
+)	O
+to	O
+2	O
+(	O
+for	O
+4	O
+Fabs	O
+).	O
+	O
+Invariably	O
+,	O
+the	O
+HCs	O
+have	O
+more	O
+disorder	O
+than	O
+the	O
+LCs	O
+.	O
+	O
+For	O
+the	O
+LC	O
+,	O
+the	O
+disorder	O
+is	O
+observed	O
+at	O
+2	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+residues	O
+with	O
+few	O
+exceptions	O
+.	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+residues	O
+including	O
+the	O
+6xHis	O
+tags	O
+are	O
+disordered	O
+in	O
+all	O
+16	O
+structures	O
+.	O
+	O
+In	O
+addition	O
+to	O
+these	O
+,	O
+2	O
+primary	O
+disordered	O
+stretches	O
+of	O
+residues	O
+are	O
+observed	O
+in	O
+a	O
+number	O
+of	O
+structures	O
+(	O
+Table	O
+S1	O
+).	O
+	O
+One	O
+involves	O
+the	O
+loop	O
+connecting	O
+the	O
+first	O
+2	O
+β	O
+-	O
+strands	O
+of	O
+the	O
+constant	O
+domain	O
+(	O
+in	O
+all	O
+Fabs	O
+except	O
+H3	O
+-	O
+23	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+11	O
+and	O
+H3	O
+-	O
+53	O
+:	O
+L1	O
+-	O
+39	O
+).	O
+	O
+The	O
+other	O
+is	O
+located	O
+in	O
+CDR	O
+H3	O
+(	O
+in	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+11	O
+,	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+in	O
+one	O
+of	O
+2	O
+copies	O
+of	O
+H3	O
+-	O
+23	O
+:	O
+L4	O
+-	O
+1	O
+).	O
+	O
+CDR	O
+canonical	O
+structures	O
+	O
+Several	O
+CDR	O
+definitions	O
+have	O
+evolved	O
+over	O
+decades	O
+of	O
+antibody	O
+research	O
+.	O
+	O
+Depending	O
+on	O
+the	O
+focus	O
+of	O
+the	O
+study	O
+,	O
+the	O
+CDR	O
+boundaries	O
+differ	O
+slightly	O
+between	O
+various	O
+definitions	O
+.	O
+	O
+In	O
+this	O
+work	O
+,	O
+we	O
+use	O
+the	O
+CDR	O
+definition	O
+of	O
+North	O
+et	O
+al	O
+.,	O
+which	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+Martin	O
+with	O
+the	O
+following	O
+exceptions	O
+:	O
+1	O
+)	O
+CDRs	O
+H1	O
+and	O
+H3	O
+begin	O
+immediately	O
+after	O
+the	O
+Cys	O
+;	O
+and	O
+2	O
+)	O
+CDR	O
+L2	O
+includes	O
+an	O
+additional	O
+residue	O
+at	O
+the	O
+N	O
+-	O
+terminal	O
+side	O
+,	O
+typically	O
+Tyr	O
+.	O
+	O
+CDR	O
+H1	O
+	O
+The	O
+superposition	O
+of	O
+CDR	O
+H1	O
+backbones	O
+for	O
+all	O
+HC	O
+:	O
+LC	O
+pairs	O
+with	O
+heavy	O
+chains	O
+:	O
+(	O
+A	O
+)	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+(	O
+B	O
+)	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+(	O
+C	O
+)	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+(	O
+D	O
+)	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+.	O
+	O
+Assignments	O
+for	O
+2	O
+copies	O
+of	O
+the	O
+Fab	O
+in	O
+the	O
+asymmetric	O
+unit	O
+are	O
+given	O
+for	O
+5	O
+structures	O
+.	O
+	O
+No	O
+assignment	O
+(	O
+NA	O
+)	O
+for	O
+CDRs	O
+with	O
+missing	O
+residues	O
+.	O
+	O
+The	O
+four	O
+HCs	O
+feature	O
+CDR	O
+H1	O
+of	O
+the	O
+same	O
+length	O
+,	O
+and	O
+their	O
+sequences	O
+are	O
+highly	O
+similar	O
+(	O
+Table	O
+2	O
+).	O
+	O
+The	O
+CDR	O
+H1	O
+backbone	O
+conformations	O
+for	O
+all	O
+variants	O
+for	O
+each	O
+of	O
+the	O
+HCs	O
+are	O
+shown	O
+in	O
+Fig	O
+.	O
+1	O
+.	O
+	O
+Some	O
+deviation	O
+is	O
+observed	O
+for	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+,	O
+mostly	O
+due	O
+to	O
+H3	O
+-	O
+53	O
+:	O
+L4	O
+-	O
+1	O
+,	O
+which	O
+exhibits	O
+a	O
+significant	O
+degree	O
+of	O
+disorder	O
+in	O
+CDR	O
+H1	O
+.	O
+	O
+The	O
+electron	O
+density	O
+for	O
+the	O
+backbone	O
+is	O
+weak	O
+and	O
+discontinuous	O
+,	O
+and	O
+completely	O
+missing	O
+for	O
+several	O
+side	O
+chains	O
+.	O
+	O
+The	O
+CDR	O
+H1	O
+structures	O
+with	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+shown	O
+in	O
+Fig	O
+.	O
+1A	O
+are	O
+quite	O
+variable	O
+,	O
+both	O
+for	O
+the	O
+structures	O
+with	O
+different	O
+LCs	O
+and	O
+for	O
+the	O
+copies	O
+of	O
+the	O
+same	O
+Fab	O
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+11	O
+and	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+.	O
+	O
+In	O
+total	O
+,	O
+6	O
+independent	O
+Fab	O
+structures	O
+produce	O
+5	O
+different	O
+canonical	O
+structures	O
+,	O
+namely	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+3	I-mutant
+,	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+4	I-mutant
+,	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+6	I-mutant
+and	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+10	I-mutant
+.	O
+	O
+A	O
+major	O
+difference	O
+of	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+from	O
+the	O
+other	O
+germlines	O
+in	O
+the	O
+experimental	O
+data	O
+set	O
+is	O
+the	O
+presence	O
+of	O
+Gly	O
+instead	O
+of	O
+Phe	O
+or	O
+Tyr	O
+at	O
+position	O
+27	O
+(	O
+residue	O
+5	O
+of	O
+13	O
+in	O
+CDR	O
+H1	O
+).	O
+	O
+Glycine	O
+introduces	O
+the	O
+possibility	O
+of	O
+a	O
+higher	O
+degree	O
+of	O
+conformational	O
+flexibility	O
+that	O
+undoubtedly	O
+translates	O
+to	O
+the	O
+differences	O
+observed	O
+,	O
+and	O
+contributes	O
+to	O
+the	O
+elevated	O
+thermal	O
+parameters	O
+for	O
+the	O
+atoms	O
+in	O
+the	O
+amino	O
+acid	O
+residues	O
+in	O
+this	O
+region	O
+.	O
+	O
+The	O
+superposition	O
+of	O
+CDR	O
+H2	O
+backbones	O
+for	O
+all	O
+HC	O
+:	O
+LC	O
+pairs	O
+with	O
+heavy	O
+chains	O
+:	O
+(	O
+A	O
+)	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+(	O
+B	O
+)	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+(	O
+C	O
+)	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+(	O
+D	O
+)	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+.	O
+	O
+The	O
+canonical	O
+structures	O
+of	O
+CDR	O
+H2	O
+have	O
+fairly	O
+consistent	O
+conformations	O
+(	O
+Table	O
+2	O
+,	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+The	O
+conformations	O
+for	O
+all	O
+of	O
+these	O
+CDR	O
+H2s	O
+are	O
+tightly	O
+clustered	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+In	O
+one	O
+case	O
+,	O
+in	O
+the	O
+second	O
+Fab	O
+of	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+CDR	O
+H2	O
+is	O
+partially	O
+disordered	O
+(	O
+Δ55	B-mutant
+-	I-mutant
+60	I-mutant
+).	O
+	O
+Although	O
+three	O
+of	O
+the	O
+germlines	O
+have	O
+CDR	O
+H2	O
+of	O
+the	O
+same	O
+length	O
+,	O
+10	O
+residues	O
+,	O
+they	O
+adopt	O
+2	O
+distinctively	O
+different	O
+conformations	O
+depending	O
+mostly	O
+on	O
+the	O
+residue	O
+at	O
+position	O
+71	O
+from	O
+the	O
+so	O
+-	O
+called	O
+CDR	O
+H4	O
+.	O
+	O
+Arg71	O
+in	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+fills	O
+the	O
+space	O
+between	O
+CDRs	O
+H2	O
+and	O
+H4	O
+,	O
+and	O
+defines	O
+the	O
+conformation	O
+of	O
+the	O
+tip	O
+of	O
+CDR	O
+H2	O
+so	O
+that	O
+residue	O
+54	O
+points	O
+away	O
+from	O
+the	O
+antigen	O
+binding	O
+site	O
+.	O
+	O
+Conformations	O
+of	O
+CDR	O
+H2	O
+in	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+,	O
+both	O
+of	O
+which	O
+have	O
+canonical	O
+structure	O
+H2	B-mutant
+-	I-mutant
+10	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+show	O
+little	O
+deviation	O
+within	O
+each	O
+set	O
+of	O
+4	O
+structures	O
+.	O
+	O
+CDR	O
+L1	O
+	O
+The	O
+superposition	O
+of	O
+CDR	O
+L1	O
+backbones	O
+for	O
+all	O
+HC	O
+:	O
+LC	O
+pairs	O
+with	O
+light	O
+chains	O
+:	O
+(	O
+A	O
+)	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+(	O
+B	O
+)	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+(	O
+C	O
+)	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+(	O
+D	O
+)	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+.	O
+	O
+Of	O
+these	O
+LCs	O
+,	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+and	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+have	O
+the	O
+same	O
+canonical	O
+structure	O
+,	O
+L1	B-mutant
+-	I-mutant
+11	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+and	O
+superimpose	O
+very	O
+well	O
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+has	O
+the	O
+longest	O
+CDR	O
+L1	O
+,	O
+composed	O
+of	O
+17	O
+amino	O
+acid	O
+residues	O
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+	O
+Despite	O
+this	O
+,	O
+the	O
+conformations	O
+are	O
+tightly	O
+clustered	O
+(	O
+rmsd	O
+is	O
+0	O
+.	O
+20	O
+Å	O
+).	O
+	O
+The	O
+backbone	O
+conformations	O
+of	O
+the	O
+stem	O
+regions	O
+superimpose	O
+well	O
+.	O
+	O
+Some	O
+changes	O
+in	O
+conformation	O
+occur	O
+between	O
+residues	O
+30a	O
+and	O
+30f	O
+(	O
+residues	O
+8	O
+and	O
+13	O
+of	O
+17	O
+in	O
+CDR	O
+L1	O
+).	O
+	O
+This	O
+is	O
+the	O
+tip	O
+of	O
+the	O
+loop	O
+region	O
+,	O
+which	O
+appears	O
+to	O
+have	O
+similar	O
+conformations	O
+that	O
+fan	O
+out	O
+the	O
+structures	O
+because	O
+of	O
+the	O
+slight	O
+differences	O
+in	O
+torsion	O
+angles	O
+in	O
+the	O
+backbone	O
+near	O
+Tyr30a	O
+and	O
+Lys30f	O
+.	O
+	O
+The	O
+conformation	O
+of	O
+CDR	O
+L1	O
+in	O
+these	O
+2	O
+Fabs	O
+is	O
+slightly	O
+different	O
+,	O
+and	O
+both	O
+conformations	O
+fall	O
+somewhere	O
+between	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+1	I-mutant
+and	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+2	I-mutant
+.	O
+	O
+This	O
+reflects	O
+the	O
+lack	O
+of	O
+accuracy	O
+in	O
+the	O
+structure	O
+due	O
+to	O
+low	O
+resolution	O
+of	O
+the	O
+X	O
+-	O
+ray	O
+data	O
+(	O
+3	O
+.	O
+3	O
+Å	O
+).	O
+	O
+CDR	O
+L2	O
+	O
+The	O
+superposition	O
+of	O
+CDR	O
+L2	O
+backbones	O
+for	O
+all	O
+HC	O
+:	O
+LC	O
+pairs	O
+with	O
+light	O
+chains	O
+:	O
+(	O
+A	O
+)	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+(	O
+B	O
+)	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+(	O
+C	O
+)	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+(	O
+D	O
+)	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+.	O
+	O
+All	O
+four	O
+LCs	O
+have	O
+CDR	O
+L2	O
+of	O
+the	O
+same	O
+length	O
+and	O
+canonical	O
+structure	O
+,	O
+L2	B-mutant
+-	I-mutant
+8	I-mutant
+-	I-mutant
+1	I-mutant
+(	O
+Table	O
+2	O
+).	O
+	O
+The	O
+CDR	O
+L2	O
+conformations	O
+for	O
+each	O
+of	O
+the	O
+LCs	O
+paired	O
+with	O
+the	O
+4	O
+HCs	O
+are	O
+clustered	O
+more	O
+tightly	O
+than	O
+any	O
+of	O
+the	O
+other	O
+CDRs	O
+(	O
+rmsd	O
+values	O
+are	O
+in	O
+the	O
+range	O
+0	O
+.	O
+09	O
+-	O
+0	O
+.	O
+16	O
+Å	O
+),	O
+and	O
+all	O
+4	O
+sets	O
+have	O
+virtually	O
+the	O
+same	O
+conformation	O
+despite	O
+the	O
+sequence	O
+diversity	O
+of	O
+the	O
+loop	O
+.	O
+	O
+CDR	O
+L3	O
+	O
+The	O
+superposition	O
+of	O
+CDR	O
+L3	O
+backbones	O
+for	O
+all	O
+HC	O
+:	O
+LC	O
+pairs	O
+with	O
+light	O
+chains	O
+:	O
+(	O
+A	O
+)	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+(	O
+B	O
+)	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+(	O
+C	O
+)	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+(	O
+D	O
+)	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+.	O
+	O
+The	O
+conformations	O
+of	O
+CDR	O
+L3	O
+for	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+and	O
+particularly	O
+for	O
+L320	O
+,	O
+are	O
+not	O
+as	O
+tightly	O
+clustered	O
+as	O
+those	O
+of	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+(	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+The	O
+slight	O
+conformational	O
+variability	O
+occurs	O
+in	O
+the	O
+region	O
+of	O
+amino	O
+acid	O
+residues	O
+90	O
+-	O
+92	O
+,	O
+which	O
+is	O
+in	O
+contact	O
+with	O
+CDR	O
+H3	O
+.	O
+	O
+The	O
+loop	O
+and	O
+the	O
+2	O
+β	O
+-	O
+strands	O
+of	O
+the	O
+CDR	O
+H3	O
+in	O
+this	O
+‘	O
+parent	O
+’	O
+structure	O
+are	O
+stabilized	O
+by	O
+H	O
+-	O
+bonds	O
+between	O
+the	O
+carbonyl	O
+oxygen	O
+and	O
+peptide	O
+nitrogen	O
+atoms	O
+in	O
+the	O
+2	O
+strands	O
+.	O
+	O
+An	O
+interesting	O
+feature	O
+of	O
+these	O
+CDR	O
+H3	O
+structures	O
+is	O
+the	O
+presence	O
+of	O
+a	O
+water	O
+molecule	O
+that	O
+interacts	O
+with	O
+the	O
+peptide	O
+nitrogens	O
+and	O
+carbonyl	O
+oxygens	O
+near	O
+the	O
+bridging	O
+loop	O
+connecting	O
+the	O
+2	O
+β	O
+-	O
+strands	O
+.	O
+	O
+This	O
+water	O
+is	O
+present	O
+in	O
+both	O
+the	O
+bound	O
+(	O
+4DN4	O
+)	O
+and	O
+unbound	O
+(	O
+4DN3	O
+)	O
+forms	O
+of	O
+CNTO	O
+888	O
+.	O
+	O
+The	O
+stem	O
+region	O
+of	O
+CDR	O
+H3	O
+in	O
+the	O
+parental	O
+Fab	O
+is	O
+in	O
+a	O
+‘	O
+kinked	O
+’	O
+conformation	O
+,	O
+in	O
+which	O
+the	O
+indole	O
+nitrogen	O
+of	O
+Trp103	O
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+the	O
+carbonyl	O
+oxygen	O
+of	O
+Leu100b	O
+.	O
+	O
+The	O
+carboxyl	O
+group	O
+of	O
+Asp101	O
+forms	O
+a	O
+salt	O
+bridge	O
+with	O
+Arg94	O
+.	O
+	O
+Ribbon	O
+representations	O
+of	O
+(	O
+A	O
+)	O
+the	O
+superposition	O
+of	O
+all	O
+CDR	O
+H3s	O
+of	O
+the	O
+structures	O
+with	O
+complete	O
+backbone	O
+traces	O
+.	O
+(	O
+B	O
+)	O
+The	O
+CDR	O
+H3s	O
+rotated	O
+90	O
+°	O
+about	O
+the	O
+y	O
+axis	O
+of	O
+the	O
+page	O
+.	O
+	O
+The	O
+structure	O
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+each	O
+CDR	O
+H3	O
+is	O
+represented	O
+with	O
+a	O
+different	O
+color	O
+.	O
+	O
+Three	O
+of	O
+the	O
+21	O
+Fab	O
+structures	O
+(	O
+including	O
+multiple	O
+copies	O
+in	O
+the	O
+asymmetric	O
+unit	O
+),	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+11	O
+,	O
+H551	O
+:	O
+L3	O
+-	O
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+-	O
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+:	O
+L4	O
+-	O
+1	O
+(	O
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+the	O
+2	O
+Fabs	O
+),	O
+have	O
+missing	O
+(	O
+disordered	O
+)	O
+residues	O
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+the	O
+apex	O
+of	O
+the	O
+CDR	O
+loop	O
+.	O
+	O
+A	O
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+of	O
+representatives	O
+of	O
+the	O
+“	O
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+”	O
+and	O
+“	O
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+”	O
+structures	O
+.	O
+	O
+(	O
+A	O
+)	O
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+“	O
+kinked	O
+”	O
+CDR	O
+H3	O
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+H1	O
+-	O
+69	O
+:	O
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+-	O
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+purple	O
+carbon	O
+atoms	O
+and	O
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+lines	O
+connecting	O
+the	O
+H	O
+-	O
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+for	O
+Leu100b	O
+O	O
+and	O
+Trp103	O
+NE1	O
+,	O
+Arg94	O
+NE	O
+and	O
+Asp101	O
+OD1	O
+,	O
+and	O
+Arg94	O
+NH2	O
+and	O
+Asp101	O
+OD2	O
+.	O
+	O
+In	O
+10	O
+of	O
+the	O
+18	O
+Fab	O
+structures	O
+,	O
+H1	O
+-	O
+69	O
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+-	O
+39	O
+,	O
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+-	O
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+-	O
+69	O
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+-	O
+1	O
+,	O
+H3	O
+-	O
+23	O
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+-	O
+11	O
+(	O
+2	O
+Fabs	O
+),	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+H3	O
+-	O
+53	O
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+-	O
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+,	O
+H3	O
+-	O
+53	O
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+-	O
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+and	O
+H5	O
+-	O
+51	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+the	O
+CDRs	O
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+similar	O
+conformations	O
+to	O
+that	O
+found	O
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+4DN3	O
+.	O
+	O
+The	O
+bases	O
+of	O
+these	O
+structures	O
+have	O
+the	O
+‘	O
+kinked	O
+’	O
+conformation	O
+with	O
+the	O
+H	O
+-	O
+bond	O
+between	O
+Trp103	O
+and	O
+Leu100b	O
+.	O
+	O
+The	O
+largest	O
+backbone	O
+conformational	O
+deviation	O
+for	O
+the	O
+set	O
+is	O
+at	O
+Tyr99	O
+,	O
+where	O
+the	O
+C	O
+=	O
+O	O
+is	O
+rotated	O
+by	O
+90	O
+°	O
+relative	O
+to	O
+that	O
+observed	O
+in	O
+4DN3	O
+.	O
+	O
+Also	O
+,	O
+it	O
+is	O
+worth	O
+noting	O
+that	O
+only	O
+one	O
+of	O
+these	O
+structures	O
+,	O
+H1	O
+-	O
+69	O
+:	O
+L4	O
+-	O
+1	O
+,	O
+has	O
+the	O
+conserved	O
+water	O
+molecule	O
+in	O
+CDR	O
+H3	O
+observed	O
+in	O
+the	O
+4DN3	O
+and	O
+4DN4	O
+structures	O
+.	O
+	O
+The	O
+CDR	O
+H3	O
+for	O
+this	O
+structure	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+S3	O
+.	O
+	O
+The	O
+stem	O
+regions	O
+in	O
+these	O
+3	O
+cases	O
+are	O
+in	O
+the	O
+‘	O
+kinked	O
+’	O
+conformation	O
+consistent	O
+with	O
+that	O
+observed	O
+for	O
+4DN3	O
+.	O
+	O
+The	O
+five	O
+remaining	O
+Fabs	O
+,	O
+H5	O
+-	O
+51	O
+:	O
+L4	O
+-	O
+1	O
+(	O
+2	O
+copies	O
+),	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+(	O
+2	O
+copies	O
+)	O
+and	O
+H3	O
+-	O
+53	O
+:	O
+L4	O
+-	O
+1	O
+,	O
+have	O
+3	O
+different	O
+CDR	O
+H3	O
+conformations	O
+(	O
+Fig	O
+.	O
+S4	O
+).	O
+	O
+The	O
+stem	O
+regions	O
+of	O
+CDR	O
+H3	O
+for	O
+the	O
+H5	O
+-	O
+51	O
+:	O
+L4	O
+-	O
+1	O
+Fabs	O
+are	O
+in	O
+the	O
+‘	O
+kinked	O
+’	O
+conformation	O
+while	O
+,	O
+surprisingly	O
+,	O
+those	O
+of	O
+the	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
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+H3	O
+-	O
+53	O
+:	O
+L4	O
+-	O
+1	O
+are	O
+in	O
+the	O
+‘	O
+extended	O
+’	O
+conformation	O
+(	O
+Fig	O
+.	O
+7B	O
+).	O
+	O
+The	O
+two	O
+domains	O
+pack	O
+together	O
+such	O
+that	O
+the	O
+5	O
+-	O
+stranded	O
+β	O
+-	O
+sheets	O
+,	O
+which	O
+have	O
+hydrophobic	O
+surfaces	O
+,	O
+interact	O
+with	O
+each	O
+other	O
+bringing	O
+the	O
+CDRs	O
+from	O
+both	O
+the	O
+VH	O
+and	O
+VL	O
+domains	O
+into	O
+close	O
+proximity	O
+.	O
+	O
+The	O
+domain	O
+packing	O
+of	O
+the	O
+variants	O
+was	O
+assessed	O
+by	O
+computing	O
+the	O
+domain	O
+interface	O
+interactions	O
+,	O
+the	O
+VH	O
+:	O
+VL	O
+tilt	O
+angles	O
+,	O
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+buried	O
+surface	O
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+.	O
+	O
+VH	O
+:	O
+VL	O
+interface	O
+amino	O
+acid	O
+residue	O
+interactions	O
+	O
+The	O
+conserved	O
+VH	O
+:	O
+VL	O
+interactions	O
+as	O
+viewed	O
+along	O
+the	O
+VH	O
+/	O
+VL	O
+axis	O
+.	O
+	O
+The	O
+VH	O
+residues	O
+are	O
+in	O
+blue	O
+,	O
+the	O
+VL	O
+residues	O
+are	O
+in	O
+orange	O
+.	O
+	O
+The	O
+VH	O
+:	O
+VL	O
+interface	O
+is	O
+pseudosymmetric	O
+,	O
+and	O
+involves	O
+2	O
+stretches	O
+of	O
+the	O
+polypeptide	O
+chain	O
+from	O
+each	O
+domain	O
+,	O
+namely	O
+CDR3	O
+and	O
+the	O
+framework	O
+region	O
+between	O
+CDRs	O
+1	O
+and	O
+2	O
+.	O
+	O
+These	O
+stretches	O
+form	O
+antiparallel	O
+β	O
+-	O
+hairpins	O
+within	O
+the	O
+internal	O
+5	O
+-	O
+stranded	O
+β	O
+-	O
+sheet	O
+.	O
+	O
+There	O
+are	O
+a	O
+few	O
+principal	O
+inter	O
+-	O
+domain	O
+interactions	O
+that	O
+are	O
+conserved	O
+not	O
+only	O
+in	O
+the	O
+experimental	O
+set	O
+of	O
+16	O
+Fabs	O
+,	O
+but	O
+in	O
+all	O
+human	O
+antibodies	O
+.	O
+	O
+These	O
+core	O
+interactions	O
+provide	O
+enough	O
+stability	O
+to	O
+the	O
+VH	O
+:	O
+VL	O
+dimer	O
+so	O
+that	O
+additional	O
+VH	O
+-	O
+VL	O
+contacts	O
+can	O
+tolerate	O
+amino	O
+acid	O
+sequence	O
+variations	O
+in	O
+CDRs	O
+H3	O
+and	O
+L3	O
+that	O
+form	O
+part	O
+of	O
+the	O
+VH	O
+:	O
+VL	O
+interface	O
+.	O
+	O
+In	O
+total	O
+,	O
+about	O
+20	O
+residues	O
+are	O
+involved	O
+in	O
+the	O
+VH	O
+:	O
+VL	O
+interactions	O
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+each	O
+side	O
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+Fig	O
+.	O
+S5	O
+).	O
+	O
+Half	O
+of	O
+them	O
+are	O
+in	O
+the	O
+framework	O
+regions	O
+and	O
+those	O
+residues	O
+(	O
+except	O
+residue	O
+61	O
+in	O
+HC	O
+,	O
+which	O
+is	O
+actually	O
+in	O
+CDR2	O
+in	O
+Kabat	O
+'	O
+s	O
+definition	O
+)	O
+are	O
+conserved	O
+in	O
+the	O
+set	O
+of	O
+16	O
+Fabs	O
+.	O
+	O
+One	O
+notable	O
+exception	O
+is	O
+H	O
+-	O
+Trp47	O
+,	O
+which	O
+exhibits	O
+2	O
+conformations	O
+of	O
+the	O
+indole	O
+ring	O
+.	O
+	O
+Interestingly	O
+,	O
+these	O
+are	O
+the	O
+only	O
+2	O
+structures	O
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+residues	O
+missing	O
+in	O
+CDR	O
+H3	O
+because	O
+of	O
+disorder	O
+,	O
+although	O
+both	O
+structures	O
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+determined	O
+at	O
+high	O
+resolution	O
+and	O
+the	O
+rest	O
+of	O
+the	O
+structure	O
+is	O
+well	O
+defined	O
+.	O
+	O
+Apparently	O
+,	O
+residues	O
+flanking	O
+CDR	O
+H3	O
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+the	O
+2	O
+VH	O
+:	O
+VL	O
+pairings	O
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+inconsistent	O
+with	O
+any	O
+stable	O
+conformation	O
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+CDR	O
+H3	O
+,	O
+which	O
+translates	O
+into	O
+a	O
+less	O
+restricted	O
+conformational	O
+space	O
+for	O
+some	O
+of	O
+them	O
+,	O
+including	O
+H	O
+-	O
+Trp47	O
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+	O
+VH	O
+:	O
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+angles	O
+	O
+The	O
+relative	O
+orientation	O
+of	O
+VH	O
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+VL	O
+has	O
+been	O
+measured	O
+in	O
+a	O
+number	O
+of	O
+different	O
+ways	O
+.	O
+	O
+The	O
+four	O
+LCs	O
+all	O
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+classified	O
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+Type	O
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+have	O
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+position	O
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+,	O
+and	O
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+results	O
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+orientation	O
+parameter	O
+are	O
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+range	O
+of	O
+values	O
+of	O
+this	O
+type	O
+reported	O
+by	O
+Dunbar	O
+and	O
+co	O
+-	O
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+	O
+The	O
+only	O
+exception	O
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+,	O
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+toward	O
+smaller	O
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+the	O
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+value	O
+of	O
+70	O
+.	O
+8	O
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+to	O
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+at	O
+72	O
+°	O
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+the	O
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+This	O
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+the	O
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+The	O
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+approach	O
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+computing	O
+the	O
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+The	O
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+independent	O
+Fabs	O
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+-	O
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+,	O
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+-	O
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+-	O
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+,	O
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+,	O
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+-	O
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+)	O
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+	O
+This	O
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+its	O
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+.	O
+	O
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+,	O
+this	O
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+experimental	O
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+that	O
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+the	O
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+value	O
+by	O
+2	O
+.	O
+5	O
+standard	O
+deviations	O
+(	O
+Table	O
+S2	O
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+Differences	O
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+	O
+The	O
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+for	O
+all	O
+pairs	O
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+V	O
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+in	O
+Table	O
+3	O
+.	O
+	O
+The	O
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+differences	O
+in	O
+the	O
+tilt	O
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+between	O
+the	O
+Fabs	O
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+isomorphous	O
+crystal	O
+forms	O
+.	O
+	O
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+areas	O
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+.	O
+	O
+The	O
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+of	O
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+PISA	O
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+surface	O
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+calculation	O
+are	O
+shown	O
+in	O
+Table	O
+4	O
+.	O
+	O
+The	O
+interface	O
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+average	O
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+.	O
+	O
+Six	O
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+have	O
+CDR	O
+H3	O
+side	O
+chains	O
+or	O
+complete	O
+residues	O
+missing	O
+,	O
+and	O
+therefore	O
+their	O
+interfaces	O
+are	O
+much	O
+smaller	O
+than	O
+in	O
+the	O
+other	O
+10	O
+structures	O
+with	O
+complete	O
+CDRs	O
+(	O
+the	O
+results	O
+are	O
+provided	O
+for	O
+all	O
+Fabs	O
+for	O
+completeness	O
+).	O
+	O
+Among	O
+the	O
+complete	O
+structures	O
+,	O
+the	O
+interface	O
+areas	O
+range	O
+from	O
+684	O
+to	O
+836	O
+Å2	O
+.	O
+	O
+Interestingly	O
+,	O
+the	O
+2	O
+structures	O
+that	O
+have	O
+the	O
+largest	O
+tilt	O
+angle	O
+differences	O
+with	O
+the	O
+other	O
+variants	O
+,	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+have	O
+the	O
+smallest	O
+VH	O
+:	O
+VL	O
+interfaces	O
+,	O
+684	O
+and	O
+725	O
+Å2	O
+,	O
+respectively	O
+.	O
+	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+20	O
+is	O
+also	O
+unique	O
+in	O
+that	O
+it	O
+has	O
+the	O
+lowest	O
+value	O
+(	O
+0	O
+.	O
+676	O
+)	O
+of	O
+surface	O
+complementarity	O
+.	O
+	O
+Melting	O
+temperatures	O
+for	O
+the	O
+16	O
+Fabs	O
+.	O
+	O
+Colors	O
+:	O
+blue	O
+(	O
+Tm	O
+<	O
+70	O
+°	O
+C	O
+),	O
+green	O
+(	O
+70	O
+°	O
+C	O
+<	O
+Tm	O
+<	O
+73	O
+°	O
+C	O
+),	O
+yellow	O
+(	O
+73	O
+°	O
+C	O
+<	O
+Tm	O
+<	O
+78	O
+°	O
+C	O
+),	O
+orange	O
+(	O
+Tm	O
+>	O
+78	O
+°	O
+C	O
+).	O
+	O
+Melting	O
+temperatures	O
+(	O
+Tm	O
+)	O
+were	O
+measured	O
+for	O
+all	O
+Fabs	O
+using	O
+differential	O
+scanning	O
+calorimetry	O
+(	O
+Table	O
+5	O
+).	O
+	O
+It	O
+appears	O
+that	O
+for	O
+each	O
+given	O
+LC	O
+,	O
+the	O
+Fabs	O
+with	O
+germlines	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+are	O
+substantially	O
+more	O
+stable	O
+than	O
+those	O
+with	O
+germlines	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+.	O
+	O
+In	O
+addition	O
+,	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+provides	O
+a	O
+much	O
+higher	O
+degree	O
+of	O
+stabilization	O
+than	O
+the	O
+other	O
+3	O
+LC	O
+germlines	O
+when	O
+combined	O
+with	O
+any	O
+of	O
+the	O
+HCs	O
+.	O
+	O
+As	O
+a	O
+result	O
+,	O
+the	O
+Tm	O
+for	O
+pairs	O
+H1	O
+-	O
+69	O
+:	O
+L1	O
+-	O
+39	O
+and	O
+H3	O
+-	O
+23	O
+:	O
+L1	O
+-	O
+39	O
+is	O
+12	O
+-	O
+13	O
+°	O
+higher	O
+than	O
+for	O
+pairs	O
+H3	O
+-	O
+53	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+H3	O
+-	O
+53	O
+:	O
+L4	O
+-	O
+1	O
+,	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+H5	O
+-	O
+51	O
+:	O
+L4	O
+-	O
+1	O
+.	O
+	O
+These	O
+findings	O
+correlate	O
+well	O
+with	O
+the	O
+degree	O
+of	O
+conformational	O
+disorder	O
+observed	O
+in	O
+the	O
+crystal	O
+structures	O
+.	O
+	O
+No	O
+electron	O
+density	O
+is	O
+observed	O
+for	O
+a	O
+number	O
+of	O
+side	O
+chains	O
+in	O
+CDRs	O
+H3	O
+and	O
+L3	O
+in	O
+all	O
+Fabs	O
+with	O
+germline	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+,	O
+which	O
+indicates	O
+loose	O
+packing	O
+of	O
+the	O
+variable	O
+domains	O
+.	O
+	O
+All	O
+those	O
+molecules	O
+are	O
+relatively	O
+unstable	O
+,	O
+as	O
+is	O
+reflected	O
+in	O
+their	O
+low	O
+Tms	O
+.	O
+	O
+The	O
+structural	O
+data	O
+set	O
+taken	O
+as	O
+a	O
+whole	O
+provides	O
+insight	O
+into	O
+how	O
+the	O
+backbone	O
+conformations	O
+of	O
+the	O
+CDRs	O
+of	O
+a	O
+specific	O
+heavy	O
+or	O
+light	O
+chain	O
+vary	O
+when	O
+it	O
+is	O
+paired	O
+with	O
+4	O
+different	O
+light	O
+or	O
+heavy	O
+chains	O
+,	O
+respectively	O
+.	O
+	O
+A	O
+large	O
+variability	O
+in	O
+the	O
+CDR	O
+conformations	O
+for	O
+the	O
+sets	O
+of	O
+HCs	O
+and	O
+LCs	O
+is	O
+observed	O
+.	O
+	O
+In	O
+some	O
+cases	O
+the	O
+CDR	O
+conformations	O
+for	O
+all	O
+members	O
+of	O
+a	O
+set	O
+are	O
+virtually	O
+identical	O
+,	O
+for	O
+others	O
+subtle	O
+changes	O
+occur	O
+in	O
+a	O
+few	O
+members	O
+of	O
+a	O
+set	O
+,	O
+and	O
+in	O
+some	O
+cases	O
+larger	O
+deviations	O
+are	O
+observed	O
+within	O
+a	O
+set	O
+.	O
+	O
+The	O
+five	O
+variants	O
+that	O
+crystallized	O
+with	O
+2	O
+copies	O
+of	O
+the	O
+Fab	O
+in	O
+the	O
+asymmetric	O
+unit	O
+serve	O
+somewhat	O
+as	O
+controls	O
+for	O
+the	O
+influence	O
+of	O
+crystal	O
+packing	O
+on	O
+the	O
+conformations	O
+of	O
+the	O
+CDRs	O
+.	O
+	O
+In	O
+four	O
+of	O
+the	O
+5	O
+structures	O
+the	O
+CDR	O
+conformations	O
+are	O
+consistent	O
+.	O
+	O
+In	O
+only	O
+one	O
+case	O
+,	O
+that	O
+of	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+(	O
+the	O
+lowest	O
+resolution	O
+structure	O
+),	O
+do	O
+we	O
+see	O
+differences	O
+in	O
+the	O
+conformations	O
+of	O
+the	O
+2	O
+copies	O
+of	O
+CDRs	O
+H1	O
+and	O
+L1	O
+.	O
+	O
+This	O
+variability	O
+is	O
+likely	O
+a	O
+result	O
+of	O
+2	O
+factors	O
+,	O
+crystal	O
+packing	O
+interactions	O
+and	O
+internal	O
+instability	O
+of	O
+the	O
+variable	O
+domain	O
+.	O
+	O
+For	O
+the	O
+CDRs	O
+with	O
+canonical	O
+structures	O
+,	O
+the	O
+largest	O
+changes	O
+in	O
+conformation	O
+occur	O
+for	O
+CDR	O
+H1	O
+of	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+.	O
+	O
+The	O
+other	O
+2	O
+HCs	O
+,	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+,	O
+have	O
+canonical	O
+structures	O
+that	O
+are	O
+remarkably	O
+well	O
+conserved	O
+(	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+Of	O
+the	O
+4	O
+HCs	O
+,	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+has	O
+the	O
+greatest	O
+number	O
+of	O
+canonical	O
+structure	O
+assignments	O
+(	O
+Table	O
+2	O
+).	O
+	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+is	O
+unique	O
+in	O
+having	O
+a	O
+pair	O
+of	O
+glycine	O
+residues	O
+at	O
+positions	O
+26	O
+and	O
+27	O
+,	O
+which	O
+provide	O
+more	O
+conformational	O
+freedom	O
+in	O
+CDR	O
+H1	O
+.	O
+	O
+Besides	O
+IGHV1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+only	O
+the	O
+germlines	O
+of	O
+the	O
+VH4	O
+family	O
+possess	O
+double	O
+glycines	O
+in	O
+CDR	O
+H1	O
+,	O
+and	O
+it	O
+will	O
+be	O
+interesting	O
+to	O
+see	O
+if	O
+they	O
+are	O
+also	O
+conformationally	O
+unstable	O
+.	O
+	O
+As	O
+mentioned	O
+in	O
+the	O
+Results	O
+section	O
+,	O
+this	O
+data	O
+set	O
+is	O
+composed	O
+of	O
+21	O
+Fabs	O
+,	O
+since	O
+5	O
+of	O
+the	O
+16	O
+variants	O
+have	O
+2	O
+Fab	O
+copies	O
+in	O
+the	O
+asymmetric	O
+unit	O
+.	O
+	O
+For	O
+the	O
+18	O
+Fabs	O
+with	O
+complete	O
+backbone	O
+atoms	O
+for	O
+CDR	O
+H3	O
+,	O
+10	O
+have	O
+conformations	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+parent	O
+,	O
+while	O
+the	O
+others	O
+have	O
+significantly	O
+different	O
+conformations	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+More	O
+than	O
+half	O
+of	O
+the	O
+variants	O
+retain	O
+the	O
+conformation	O
+of	O
+the	O
+parent	O
+despite	O
+having	O
+differences	O
+in	O
+the	O
+VH	O
+:	O
+VL	O
+pairing	O
+.	O
+	O
+This	O
+subset	O
+includes	O
+2	O
+structures	O
+with	O
+2	O
+copies	O
+of	O
+the	O
+Fab	O
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+all	O
+of	O
+which	O
+are	O
+nearly	O
+identical	O
+in	O
+conformation	O
+.	O
+	O
+The	O
+remaining	O
+8	O
+structures	O
+exhibit	O
+“	O
+non	O
+-	O
+parental	O
+”	O
+conformations	O
+,	O
+indicating	O
+that	O
+the	O
+VH	O
+and	O
+VL	O
+context	O
+can	O
+also	O
+be	O
+a	O
+dominating	O
+factor	O
+influencing	O
+CDR	O
+H3	O
+.	O
+	O
+Interestingly	O
+,	O
+as	O
+described	O
+earlier	O
+,	O
+these	O
+2	O
+pairs	O
+differ	O
+in	O
+the	O
+stem	O
+regions	O
+with	O
+the	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+pair	O
+in	O
+the	O
+‘	O
+extended	O
+’	O
+conformation	O
+and	O
+H5	O
+-	O
+51	O
+:	O
+L4	O
+-	O
+1	O
+pair	O
+in	O
+the	O
+‘	O
+kinked	O
+’	O
+conformation	O
+.	O
+	O
+The	O
+CDR	O
+H3	O
+conformational	O
+analysis	O
+shows	O
+that	O
+,	O
+for	O
+each	O
+set	O
+of	O
+variants	O
+of	O
+one	O
+HC	O
+paired	O
+with	O
+the	O
+4	O
+different	O
+LCs	O
+,	O
+both	O
+“	O
+parental	O
+”	O
+and	O
+“	O
+non	O
+-	O
+parental	O
+”	O
+conformations	O
+are	O
+observed	O
+.	O
+	O
+Thus	O
+,	O
+no	O
+patterns	O
+of	O
+conformational	O
+preference	O
+for	O
+a	O
+particular	O
+HC	O
+or	O
+LC	O
+emerge	O
+to	O
+shed	O
+any	O
+direct	O
+light	O
+on	O
+what	O
+drives	O
+the	O
+conformational	O
+differences	O
+.	O
+	O
+This	O
+finding	O
+supports	O
+the	O
+hypothesis	O
+of	O
+Weitzner	O
+et	O
+al	O
+.	O
+that	O
+the	O
+H3	O
+conformation	O
+is	O
+controlled	O
+both	O
+by	O
+its	O
+sequence	O
+and	O
+its	O
+environment	O
+.	O
+	O
+In	O
+looking	O
+at	O
+a	O
+possible	O
+correlation	O
+between	O
+the	O
+tilt	O
+angle	O
+and	O
+the	O
+conformation	O
+of	O
+CDR	O
+H3	O
+,	O
+no	O
+clear	O
+trends	O
+are	O
+observed	O
+.	O
+	O
+Two	O
+variants	O
+,	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+have	O
+the	O
+largest	O
+differences	O
+in	O
+the	O
+tilt	O
+angles	O
+compared	O
+to	O
+other	O
+variants	O
+as	O
+seen	O
+in	O
+Table	O
+3	O
+.	O
+	O
+The	O
+absolute	O
+VH	O
+:	O
+VL	O
+orientation	O
+parameters	O
+for	O
+the	O
+2	O
+Fabs	O
+(	O
+Table	O
+S2	O
+)	O
+show	O
+significant	O
+deviation	O
+in	O
+HL	O
+,	O
+LC1	O
+and	O
+HC2	O
+values	O
+(	O
+2	O
+-	O
+3	O
+standard	O
+deviations	O
+from	O
+the	O
+mean	O
+).	O
+	O
+One	O
+of	O
+the	O
+variants	O
+,	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+has	O
+the	O
+CDR	O
+H3	O
+conformation	O
+similar	O
+to	O
+the	O
+parent	O
+,	O
+but	O
+the	O
+other	O
+,	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+is	O
+different	O
+.	O
+	O
+These	O
+smaller	O
+interfaces	O
+may	O
+perhaps	O
+translate	O
+to	O
+a	O
+significant	O
+deviation	O
+in	O
+how	O
+VH	O
+is	O
+oriented	O
+relative	O
+to	O
+VL	O
+than	O
+the	O
+other	O
+variants	O
+.	O
+	O
+These	O
+deviations	O
+from	O
+the	O
+other	O
+variants	O
+can	O
+also	O
+be	O
+seen	O
+to	O
+some	O
+extent	O
+in	O
+VH	O
+:	O
+VL	O
+orientation	O
+parameters	O
+in	O
+Table	O
+S2	O
+,	O
+as	O
+well	O
+as	O
+in	O
+the	O
+smaller	O
+number	O
+of	O
+residues	O
+involved	O
+in	O
+the	O
+VH	O
+:	O
+VL	O
+interfaces	O
+of	O
+these	O
+2	O
+variants	O
+(	O
+Fig	O
+.	O
+S5	O
+).	O
+	O
+These	O
+differences	O
+undoubtedly	O
+influence	O
+the	O
+conformation	O
+of	O
+the	O
+CDRs	O
+,	O
+in	O
+particular	O
+CDR	O
+H1	O
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+and	O
+CDR	O
+L1	O
+(	O
+Fig	O
+.	O
+3C	O
+),	O
+especially	O
+with	O
+the	O
+tandem	O
+glycines	O
+and	O
+multiple	O
+serines	O
+present	O
+,	O
+respectively	O
+.	O
+	O
+As	O
+indicated	O
+by	O
+the	O
+melting	O
+temperatures	O
+,	O
+germlines	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+for	O
+HC	O
+and	O
+germline	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+for	O
+LC	O
+produce	O
+more	O
+stable	O
+Fabs	O
+compared	O
+to	O
+the	O
+other	O
+germlines	O
+in	O
+the	O
+experimental	O
+set	O
+.	O
+	O
+One	O
+possible	O
+explanation	O
+of	O
+the	O
+clear	O
+preference	O
+of	O
+LC	O
+germline	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+is	O
+that	O
+CDR	O
+L3	O
+has	O
+smaller	O
+residues	O
+at	O
+positions	O
+91	O
+and	O
+94	O
+,	O
+allowing	O
+for	O
+more	O
+room	O
+to	O
+accommodate	O
+CDR	O
+H3	O
+.	O
+	O
+Various	O
+combinations	O
+of	O
+germline	O
+sequences	O
+for	O
+VL	O
+and	O
+VH	O
+impose	O
+certain	O
+constraints	O
+on	O
+CDR	O
+H3	O
+,	O
+which	O
+has	O
+to	O
+adapt	O
+to	O
+the	O
+environment	O
+.	O
+	O
+At	O
+the	O
+other	O
+end	O
+of	O
+the	O
+stability	O
+range	O
+is	O
+LC	O
+germline	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+,	O
+which	O
+yields	O
+antibodies	O
+with	O
+the	O
+lowest	O
+Tms	O
+.	O
+	O
+While	O
+pairings	O
+with	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+may	O
+be	O
+safely	O
+called	O
+a	O
+mismatch	O
+,	O
+those	O
+with	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+have	O
+Tms	O
+about	O
+5	O
+-	O
+6	O
+°	O
+higher	O
+.	O
+	O
+It	O
+is	O
+possible	O
+that	O
+by	O
+adopting	O
+extreme	O
+tilt	O
+angles	O
+the	O
+structure	O
+modulates	O
+CDR	O
+H3	O
+and	O
+its	O
+environment	O
+,	O
+which	O
+apparently	O
+cannot	O
+be	O
+achieved	O
+solely	O
+by	O
+conformational	O
+rearrangement	O
+of	O
+the	O
+CDR	O
+.	O
+	O
+Overall	O
+,	O
+the	O
+stability	O
+of	O
+the	O
+Fab	O
+,	O
+as	O
+measured	O
+by	O
+Tm	O
+,	O
+is	O
+a	O
+result	O
+of	O
+the	O
+mutual	O
+adjustment	O
+of	O
+the	O
+HC	O
+and	O
+LC	O
+variable	O
+domains	O
+and	O
+adjustment	O
+of	O
+CDR	O
+H3	O
+to	O
+the	O
+VH	O
+:	O
+VL	O
+cleft	O
+.	O
+	O
+The	O
+final	O
+conformation	O
+represents	O
+an	O
+energetic	O
+minimum	O
+;	O
+however	O
+,	O
+in	O
+most	O
+cases	O
+it	O
+is	O
+very	O
+shallow	O
+,	O
+so	O
+that	O
+a	O
+single	O
+mutation	O
+can	O
+cause	O
+a	O
+dramatic	O
+rearrangement	O
+of	O
+the	O
+structure	O
+.	O
+	O
+In	O
+summary	O
+,	O
+the	O
+analysis	O
+of	O
+this	O
+structural	O
+library	O
+of	O
+germline	O
+variants	O
+composed	O
+of	O
+all	O
+pairs	O
+of	O
+4	O
+HCs	O
+and	O
+4LCs	O
+,	O
+all	O
+with	O
+the	O
+same	O
+CDR	O
+H3	O
+,	O
+offers	O
+some	O
+unique	O
+insights	O
+into	O
+antibody	O
+structure	O
+and	O
+how	O
+pairing	O
+and	O
+sequence	O
+may	O
+influence	O
+,	O
+or	O
+not	O
+,	O
+the	O
+canonical	O
+structures	O
+of	O
+the	O
+L1	O
+,	O
+L2	O
+,	O
+L3	O
+,	O
+H1	O
+and	O
+H2	O
+CDRs	O
+.	O
+	O
+These	O
+data	O
+reveal	O
+the	O
+difficulty	O
+of	O
+modeling	O
+CDR	O
+H3	O
+accurately	O
+,	O
+as	O
+shown	O
+again	O
+in	O
+Antibody	O
+Modeling	O
+Assessment	O
+II	O
+.	O
+	O
+Furthermore	O
+,	O
+antibody	O
+CDRs	O
+,	O
+H3	O
+in	O
+particular	O
+,	O
+may	O
+go	O
+through	O
+conformational	O
+changes	O
+upon	O
+binding	O
+their	O
+targets	O
+,	O
+making	O
+structural	O
+prediction	O
+for	O
+docking	O
+purposes	O
+an	O
+even	O
+more	O
+difficult	O
+task	O
+.	O
+	O
+For	O
+those	O
+applications	O
+where	O
+accurate	O
+CDR	O
+structures	O
+are	O
+essential	O
+,	O
+such	O
+as	O
+docking	O
+,	O
+the	O
+results	O
+in	O
+this	O
+work	O
+demonstrate	O
+the	O
+importance	O
+of	O
+experimental	O
+structures	O
+.	O
+	O
+With	O
+the	O
+recent	O
+advances	O
+in	O
+expression	O
+and	O
+crystallization	O
+methods	O
+,	O
+Fab	O
+structures	O
+can	O
+be	O
+obtained	O
+rapidly	O
+.	O
+	O
+The	O
+set	O
+of	O
+16	O
+germline	O
+Fab	O
+structures	O
+offers	O
+a	O
+unique	O
+dataset	O
+to	O
+facilitate	O
+software	O
+development	O
+for	O
+antibody	O
+modeling	O
+.	O
+	O
+The	O
+results	O
+essentially	O
+support	O
+the	O
+underlying	O
+idea	O
+of	O
+canonical	O
+structures	O
+,	O
+indicating	O
+that	O
+most	O
+CDRs	O
+with	O
+germline	O
+sequences	O
+tend	O
+to	O
+adopt	O
+predefined	O
+conformations	O
+.	O
+	O
+This	O
+would	O
+insure	O
+more	O
+structural	O
+diversity	O
+,	O
+leading	O
+to	O
+a	O
+more	O
+diverse	O
+panel	O
+of	O
+antibodies	O
+that	O
+would	O
+bind	O
+to	O
+a	O
+broad	O
+spectrum	O
+of	O
+targets	O
+.	O
+	O
+Challenges	O
+in	O
+determining	O
+the	O
+structures	O
+of	O
+heterogeneous	O
+and	O
+dynamic	O
+protein	O
+complexes	O
+have	O
+greatly	O
+hampered	O
+past	O
+efforts	O
+to	O
+obtain	O
+a	O
+mechanistic	O
+understanding	O
+of	O
+many	O
+important	O
+biological	O
+processes	O
+.	O
+	O
+One	O
+such	O
+process	O
+is	O
+chaperone	O
+-	O
+assisted	O
+protein	O
+folding	O
+,	O
+where	O
+obtaining	O
+structural	O
+ensembles	O
+of	O
+chaperone	O
+:	O
+substrate	O
+complexes	O
+would	O
+ultimately	O
+reveal	O
+how	O
+chaperones	O
+help	O
+proteins	O
+fold	O
+into	O
+their	O
+native	O
+state	O
+.	O
+	O
+To	O
+address	O
+this	O
+problem	O
+,	O
+we	O
+devised	O
+a	O
+novel	O
+structural	O
+biology	O
+approach	O
+based	O
+on	O
+X	O
+-	O
+ray	O
+crystallography	O
+,	O
+termed	O
+Residual	O
+Electron	O
+and	O
+Anomalous	O
+Density	O
+(	O
+READ	O
+).	O
+	O
+READ	O
+enabled	O
+us	O
+to	O
+visualize	O
+even	O
+sparsely	O
+populated	O
+conformations	O
+of	O
+the	O
+substrate	O
+protein	O
+immunity	O
+protein	O
+7	O
+(	O
+Im7	O
+)	O
+in	O
+complex	O
+with	O
+the	O
+E	O
+.	O
+coli	O
+chaperone	O
+Spy	O
+.	O
+	O
+The	O
+ensemble	O
+shows	O
+that	O
+Spy	O
+-	O
+associated	O
+Im7	O
+samples	O
+conformations	O
+ranging	O
+from	O
+unfolded	O
+to	O
+partially	O
+folded	O
+and	O
+native	O
+-	O
+like	O
+states	O
+,	O
+and	O
+reveals	O
+how	O
+a	O
+substrate	O
+can	O
+explore	O
+its	O
+folding	O
+landscape	O
+while	O
+bound	O
+to	O
+a	O
+chaperone	O
+.	O
+	O
+High	O
+-	O
+resolution	O
+structural	O
+models	O
+of	O
+protein	O
+-	O
+protein	O
+interactions	O
+are	O
+critical	O
+for	O
+obtaining	O
+mechanistic	O
+insights	O
+into	O
+biological	O
+processes	O
+.	O
+	O
+However	O
+,	O
+many	O
+protein	O
+-	O
+protein	O
+interactions	O
+are	O
+highly	O
+dynamic	O
+,	O
+making	O
+it	O
+difficult	O
+to	O
+obtain	O
+high	O
+-	O
+resolution	O
+data	O
+.	O
+	O
+Particularly	O
+challenging	O
+are	O
+interactions	O
+of	O
+intrinsically	O
+or	O
+conditionally	O
+disordered	O
+sections	O
+of	O
+proteins	O
+with	O
+their	O
+partner	O
+proteins	O
+.	O
+	O
+It	O
+is	O
+clear	O
+that	O
+molecular	O
+chaperones	O
+aid	O
+in	O
+protein	O
+folding	O
+.	O
+	O
+Structural	O
+characterization	O
+of	O
+chaperone	O
+-	O
+assisted	O
+protein	O
+folding	O
+likely	O
+would	O
+help	O
+bring	O
+clarity	O
+to	O
+this	O
+question	O
+.	O
+	O
+Structural	O
+models	O
+of	O
+chaperone	O
+-	O
+substrate	O
+complexes	O
+have	O
+recently	O
+begun	O
+to	O
+provide	O
+information	O
+as	O
+to	O
+how	O
+a	O
+chaperone	O
+can	O
+recognize	O
+its	O
+substrate	O
+.	O
+	O
+However	O
+,	O
+the	O
+impact	O
+that	O
+chaperones	O
+have	O
+on	O
+their	O
+substrates	O
+,	O
+and	O
+how	O
+these	O
+interactions	O
+affect	O
+the	O
+folding	O
+process	O
+remain	O
+largely	O
+unknown	O
+.	O
+	O
+For	O
+most	O
+chaperones	O
+,	O
+it	O
+is	O
+still	O
+unclear	O
+whether	O
+the	O
+chaperone	O
+actively	O
+participates	O
+in	O
+and	O
+affects	O
+the	O
+folding	O
+of	O
+the	O
+substrate	O
+proteins	O
+,	O
+or	O
+merely	O
+provides	O
+a	O
+suitable	O
+microenvironment	O
+enabling	O
+the	O
+substrate	O
+to	O
+fold	O
+on	O
+its	O
+own	O
+.	O
+	O
+This	O
+is	O
+a	O
+truly	O
+fundamental	O
+question	O
+in	O
+the	O
+chaperone	O
+field	O
+,	O
+and	O
+one	O
+that	O
+has	O
+eluded	O
+the	O
+community	O
+largely	O
+because	O
+of	O
+the	O
+highly	O
+dynamic	O
+nature	O
+of	O
+the	O
+chaperone	O
+-	O
+substrate	O
+complexes	O
+.	O
+	O
+To	O
+address	O
+this	O
+question	O
+,	O
+we	O
+investigated	O
+the	O
+ATP	O
+-	O
+independent	O
+Escherichia	O
+coli	O
+periplasmic	O
+chaperone	O
+Spy	O
+.	O
+	O
+Spy	O
+prevents	O
+protein	O
+aggregation	O
+and	O
+aids	O
+in	O
+protein	O
+folding	O
+under	O
+various	O
+stress	O
+conditions	O
+,	O
+including	O
+treatment	O
+with	O
+tannin	O
+and	O
+butanol	O
+.	O
+	O
+We	O
+originally	O
+discovered	O
+Spy	O
+by	O
+its	O
+ability	O
+to	O
+stabilize	O
+the	O
+protein	O
+-	O
+folding	O
+model	O
+Im7	O
+in	O
+vivo	O
+and	O
+recently	O
+demonstrated	O
+that	O
+Im7	O
+folds	O
+while	O
+associated	O
+with	O
+Spy	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+Spy	O
+revealed	O
+that	O
+it	O
+forms	O
+a	O
+thin	O
+α	O
+-	O
+helical	O
+homodimeric	O
+cradle	O
+.	O
+	O
+Crosslinking	O
+and	O
+genetic	O
+experiments	O
+suggested	O
+that	O
+Spy	O
+interacts	O
+with	O
+substrates	O
+somewhere	O
+on	O
+its	O
+concave	O
+side	O
+.	O
+	O
+By	O
+using	O
+a	O
+novel	O
+X	O
+-	O
+ray	O
+crystallography	O
+-	O
+based	O
+approach	O
+to	O
+model	O
+disorder	O
+in	O
+crystal	O
+structures	O
+,	O
+we	O
+have	O
+now	O
+determined	O
+the	O
+high	O
+-	O
+resolution	O
+ensemble	O
+of	O
+the	O
+dynamic	O
+Spy	O
+:	O
+Im7	O
+complex	O
+.	O
+	O
+This	O
+work	O
+provides	O
+a	O
+detailed	O
+view	O
+of	O
+chaperone	O
+-	O
+mediated	O
+protein	O
+folding	O
+and	O
+shows	O
+how	O
+substrates	O
+like	O
+Im7	O
+find	O
+their	O
+native	O
+fold	O
+while	O
+bound	O
+to	O
+their	O
+chaperones	O
+.	O
+	O
+Crystallizing	O
+the	O
+Spy	O
+:	O
+Im7	O
+complex	O
+	O
+We	O
+reasoned	O
+that	O
+to	O
+obtain	O
+crystals	O
+of	O
+complexes	O
+between	O
+Spy	O
+(	O
+domain	O
+boundaries	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+)	O
+and	O
+its	O
+substrate	O
+proteins	O
+,	O
+our	O
+best	O
+approach	O
+was	O
+to	O
+identify	O
+crystallization	O
+conditions	O
+that	O
+yielded	O
+Spy	O
+crystals	O
+in	O
+the	O
+presence	O
+of	O
+protein	O
+substrates	O
+but	O
+not	O
+in	O
+their	O
+absence	O
+.	O
+	O
+We	O
+therefore	O
+screened	O
+crystallization	O
+conditions	O
+for	O
+Spy	O
+with	O
+four	O
+different	O
+substrate	O
+proteins	O
+:	O
+a	O
+fragment	O
+of	O
+the	O
+largely	O
+unfolded	O
+bovine	O
+α	O
+-	O
+casein	O
+protein	O
+,	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+E	O
+.	O
+coli	O
+Im7	O
+,	O
+an	O
+unfolded	O
+variant	O
+of	O
+Im7	O
+(	O
+L18A	B-mutant
+L19A	B-mutant
+L37A	B-mutant
+),	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+half	O
+of	O
+Im7	O
+(	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+),	O
+which	O
+encompasses	O
+the	O
+entire	O
+Spy	O
+-	O
+binding	O
+portion	O
+of	O
+Im7	O
+.	O
+	O
+We	O
+found	O
+conditions	O
+in	O
+which	O
+all	O
+four	O
+substrates	O
+co	O
+-	O
+crystallized	O
+with	O
+Spy	O
+,	O
+but	O
+in	O
+which	O
+Spy	O
+alone	O
+did	O
+not	O
+yield	O
+crystals	O
+.	O
+	O
+Subsequent	O
+crystal	O
+washing	O
+and	O
+dissolution	O
+experiments	O
+confirmed	O
+the	O
+presence	O
+of	O
+the	O
+substrates	O
+in	O
+the	O
+co	O
+-	O
+crystals	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+The	O
+crystals	O
+diffracted	O
+to	O
+~	O
+1	O
+.	O
+8	O
+Å	O
+resolution	O
+.	O
+	O
+We	O
+used	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+selenomethionine	O
+crystals	O
+for	O
+phasing	O
+with	O
+single	O
+-	O
+wavelength	O
+anomalous	O
+diffraction	O
+(	O
+SAD	O
+)	O
+experiments	O
+,	O
+and	O
+used	O
+this	O
+solution	O
+to	O
+build	O
+the	O
+well	O
+-	O
+ordered	O
+Spy	O
+portions	O
+of	O
+all	O
+four	O
+complexes	O
+.	O
+	O
+Even	O
+the	O
+minimal	O
+binding	O
+portion	O
+of	O
+Im7	O
+(	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+)	O
+showed	O
+highly	O
+dispersed	O
+electron	O
+density	O
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+	O
+We	O
+hypothesized	O
+that	O
+the	O
+fragmented	O
+density	O
+was	O
+due	O
+to	O
+multiple	O
+,	O
+partially	O
+occupied	O
+conformations	O
+of	O
+the	O
+substrate	O
+bound	O
+within	O
+the	O
+crystal	O
+.	O
+	O
+Such	O
+residual	O
+density	O
+is	O
+typically	O
+not	O
+considered	O
+usable	O
+by	O
+traditional	O
+X	O
+-	O
+ray	O
+crystallography	O
+methods	O
+.	O
+	O
+Thus	O
+,	O
+we	O
+developed	O
+a	O
+new	O
+approach	O
+to	O
+interpret	O
+the	O
+chaperone	O
+-	O
+bound	O
+substrate	O
+in	O
+multiple	O
+conformations	O
+.	O
+	O
+READ	O
+:	O
+a	O
+strategy	O
+to	O
+visualize	O
+heterogeneous	O
+and	O
+dynamic	O
+biomolecules	O
+	O
+We	O
+split	O
+this	O
+approach	O
+into	O
+five	O
+steps	O
+:	O
+(	O
+1	O
+)	O
+By	O
+using	O
+a	O
+well	O
+-	O
+diffracting	O
+Spy	O
+:	O
+substrate	O
+co	O
+-	O
+crystal	O
+,	O
+we	O
+first	O
+determined	O
+the	O
+structure	O
+of	O
+the	O
+folded	O
+domain	O
+of	O
+Spy	O
+and	O
+obtained	O
+high	O
+quality	O
+residual	O
+electron	O
+density	O
+within	O
+the	O
+dynamic	O
+regions	O
+of	O
+the	O
+substrate	O
+.	O
+	O
+(	O
+2	O
+)	O
+We	O
+then	O
+labeled	O
+individual	O
+residues	O
+in	O
+the	O
+flexible	O
+regions	O
+of	O
+the	O
+substrate	O
+with	O
+the	O
+strong	O
+anomalous	O
+scatterer	O
+iodine	O
+,	O
+which	O
+serves	O
+to	O
+locate	O
+these	O
+residues	O
+in	O
+three	O
+-	O
+dimensional	O
+space	O
+using	O
+their	O
+anomalous	O
+density	O
+.	O
+	O
+(	O
+3	O
+)	O
+We	O
+performed	O
+molecular	O
+dynamics	O
+(	O
+MD	O
+)	O
+simulations	O
+to	O
+generate	O
+a	O
+pool	O
+of	O
+energetically	O
+reasonable	O
+conformations	O
+of	O
+the	O
+dynamic	O
+complex	O
+and	O
+(	O
+4	O
+)	O
+applied	O
+a	O
+sample	O
+-	O
+and	O
+-	O
+select	O
+algorithm	O
+to	O
+determine	O
+the	O
+minimal	O
+set	O
+of	O
+substrate	O
+conformations	O
+that	O
+fit	O
+both	O
+the	O
+residual	O
+and	O
+anomalous	O
+density	O
+.	O
+	O
+The	O
+electron	O
+density	O
+then	O
+allowed	O
+us	O
+to	O
+connect	O
+the	O
+labeled	O
+residues	O
+of	O
+the	O
+substrate	O
+by	O
+confining	O
+the	O
+protein	O
+chain	O
+within	O
+regions	O
+of	O
+detectable	O
+density	O
+.	O
+	O
+In	O
+this	O
+way	O
+,	O
+the	O
+two	O
+forms	O
+of	O
+data	O
+together	O
+were	O
+able	O
+to	O
+describe	O
+multiple	O
+conformations	O
+of	O
+the	O
+substrate	O
+within	O
+the	O
+crystal	O
+.	O
+	O
+However	O
+,	O
+we	O
+believe	O
+that	O
+READ	O
+will	O
+prove	O
+generally	O
+applicable	O
+to	O
+visualizing	O
+heterogeneous	O
+and	O
+dynamic	O
+complexes	O
+that	O
+have	O
+previously	O
+escaped	O
+detailed	O
+structural	O
+analysis	O
+.	O
+	O
+Collecting	O
+READ	O
+data	O
+for	O
+the	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+complex	O
+	O
+To	O
+apply	O
+the	O
+READ	O
+technique	O
+to	O
+the	O
+folding	O
+mechanism	O
+employed	O
+by	O
+the	O
+chaperone	O
+Spy	O
+,	O
+we	O
+selected	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+for	O
+further	O
+investigation	O
+because	O
+NMR	O
+data	O
+suggested	O
+that	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+could	O
+recapitulate	O
+unfolded	O
+,	O
+partially	O
+folded	O
+,	O
+and	O
+native	O
+-	O
+like	O
+states	O
+of	O
+Im7	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+Moreover	O
+,	O
+binding	O
+experiments	O
+indicated	O
+that	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+comprises	O
+the	O
+entire	O
+Spy	O
+-	O
+binding	O
+region	O
+.	O
+	O
+To	O
+introduce	O
+the	O
+anomalous	O
+scatterer	O
+iodine	O
+,	O
+we	O
+replaced	O
+eight	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+residues	O
+with	O
+the	O
+non	O
+-	O
+canonical	O
+amino	O
+acid	O
+4	O
+-	O
+iodophenylalanine	O
+(	O
+pI	O
+-	O
+Phe	O
+).	O
+	O
+Consistent	O
+with	O
+our	O
+electron	O
+density	O
+map	O
+,	O
+we	O
+found	O
+that	O
+the	O
+majority	O
+of	O
+anomalous	O
+signals	O
+emerged	O
+in	O
+the	O
+cradle	O
+of	O
+Spy	O
+,	O
+implying	O
+that	O
+this	O
+is	O
+the	O
+likely	O
+Im7	O
+substrate	O
+binding	O
+site	O
+.	O
+	O
+Consistent	O
+with	O
+the	O
+fragmented	O
+density	O
+,	O
+however	O
+,	O
+we	O
+observed	O
+multiple	O
+iodine	O
+positions	O
+for	O
+seven	O
+of	O
+the	O
+eight	O
+substituted	O
+residues	O
+.	O
+	O
+READ	O
+sample	O
+-	O
+and	O
+-	O
+select	O
+procedure	O
+	O
+During	O
+each	O
+round	O
+of	O
+the	O
+selection	O
+,	O
+a	O
+genetic	O
+algorithm	O
+alters	O
+the	O
+ensemble	O
+and	O
+its	O
+agreement	O
+to	O
+the	O
+experimental	O
+data	O
+is	O
+re	O
+-	O
+evaluated	O
+.	O
+	O
+If	O
+successful	O
+,	O
+the	O
+selection	O
+identifies	O
+the	O
+smallest	O
+group	O
+of	O
+specific	O
+conformations	O
+that	O
+best	O
+fits	O
+the	O
+residual	O
+electron	O
+density	O
+and	O
+anomalous	O
+signals	O
+.	O
+	O
+This	O
+strategy	O
+allows	O
+a	O
+wide	O
+range	O
+of	O
+substrate	O
+conformations	O
+to	O
+interact	O
+with	O
+the	O
+chaperone	O
+.	O
+	O
+From	O
+the	O
+MD	O
+simulations	O
+,	O
+we	O
+extracted	O
+~	O
+10	O
+,	O
+000	O
+diverse	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+complexes	O
+to	O
+be	O
+used	O
+by	O
+the	O
+ensuing	O
+selection	O
+.	O
+	O
+Each	O
+complex	O
+within	O
+this	O
+pool	O
+comprises	O
+one	O
+Spy	O
+dimer	O
+bound	O
+to	O
+a	O
+single	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+substrate	O
+.	O
+	O
+This	O
+pool	O
+was	O
+then	O
+used	O
+by	O
+the	O
+selection	O
+algorithm	O
+to	O
+identify	O
+the	O
+minimal	O
+ensemble	O
+that	O
+best	O
+satisfies	O
+both	O
+the	O
+residual	O
+electron	O
+and	O
+anomalous	O
+crystallographic	O
+data	O
+.	O
+	O
+Simultaneously	O
+,	O
+it	O
+uses	O
+the	O
+residual	O
+electron	O
+density	O
+to	O
+help	O
+choose	O
+ensembles	O
+.	O
+	O
+This	O
+process	O
+provided	O
+us	O
+with	O
+a	O
+target	O
+map	O
+that	O
+the	O
+ensuing	O
+selection	O
+tried	O
+to	O
+recapitulate	O
+.	O
+	O
+To	O
+reduce	O
+the	O
+extent	O
+of	O
+3D	O
+space	O
+to	O
+be	O
+explored	O
+,	O
+this	O
+compressed	O
+map	O
+was	O
+created	O
+by	O
+only	O
+using	O
+density	O
+from	O
+regions	O
+of	O
+space	O
+significantly	O
+sampled	O
+by	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+in	O
+the	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+MD	O
+simulations	O
+.	O
+	O
+For	O
+each	O
+of	O
+the	O
+~	O
+10	O
+,	O
+000	O
+complexes	O
+in	O
+the	O
+coarse	O
+-	O
+grained	O
+MD	O
+pool	O
+,	O
+the	O
+electron	O
+density	O
+at	O
+the	O
+Cα	O
+positions	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+was	O
+extracted	O
+and	O
+used	O
+to	O
+construct	O
+an	O
+electron	O
+density	O
+map	O
+(	O
+Online	O
+Methods	O
+).	O
+	O
+These	O
+individual	O
+electron	O
+density	O
+maps	O
+from	O
+the	O
+separate	O
+conformers	O
+could	O
+then	O
+be	O
+combined	O
+(	O
+Fig	O
+.	O
+2	O
+)	O
+and	O
+compared	O
+to	O
+the	O
+averaged	O
+experimental	O
+electron	O
+density	O
+map	O
+as	O
+part	O
+of	O
+the	O
+selection	O
+algorithm	O
+.	O
+	O
+This	O
+approach	O
+allowed	O
+us	O
+to	O
+simultaneously	O
+use	O
+both	O
+the	O
+iodine	O
+anomalous	O
+signals	O
+and	O
+the	O
+residual	O
+electron	O
+density	O
+in	O
+the	O
+selection	O
+procedure	O
+.	O
+	O
+The	O
+selection	O
+resulted	O
+in	O
+small	O
+ensembles	O
+from	O
+the	O
+MD	O
+pool	O
+that	O
+best	O
+fit	O
+the	O
+READ	O
+data	O
+(	O
+Fig	O
+.	O
+1c	O
+,	O
+d	O
+).	O
+	O
+Before	O
+analyzing	O
+the	O
+details	O
+of	O
+the	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+complex	O
+,	O
+we	O
+first	O
+engaged	O
+in	O
+a	O
+series	O
+of	O
+validation	O
+tests	O
+to	O
+verify	O
+the	O
+ensemble	O
+and	O
+selection	O
+procedure	O
+(	O
+Supplementary	O
+Note	O
+1	O
+,	O
+Figures	O
+1c	O
+,	O
+d	O
+,	O
+Supplemental	O
+Figures	O
+5	O
+-	O
+7	O
+).	O
+	O
+Of	O
+note	O
+,	O
+the	O
+final	O
+six	O
+-	O
+membered	O
+ensemble	O
+was	O
+the	O
+largest	O
+ensemble	O
+that	O
+could	O
+simultaneously	O
+decrease	O
+the	O
+RFree	O
+and	O
+pass	O
+the	O
+10	O
+-	O
+fold	O
+cross	O
+-	O
+validation	O
+test	O
+.	O
+	O
+This	O
+ensemble	O
+depicts	O
+the	O
+conformations	O
+that	O
+the	O
+substrate	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+adopts	O
+while	O
+bound	O
+to	O
+the	O
+chaperone	O
+Spy	O
+(	O
+Fig	O
+.	O
+3	O
+Supplementary	O
+Movie	O
+1	O
+,	O
+and	O
+Table	O
+1	O
+).	O
+	O
+Our	O
+results	O
+showed	O
+that	O
+by	O
+using	O
+this	O
+novel	O
+READ	O
+approach	O
+,	O
+we	O
+were	O
+able	O
+to	O
+obtain	O
+structural	O
+information	O
+about	O
+the	O
+dynamic	O
+interaction	O
+of	O
+a	O
+chaperone	O
+with	O
+its	O
+substrate	O
+protein	O
+.	O
+	O
+We	O
+were	O
+particularly	O
+interested	O
+in	O
+finding	O
+answers	O
+to	O
+one	O
+of	O
+the	O
+most	O
+fundamental	O
+questions	O
+in	O
+chaperone	O
+biology	O
+—	O
+how	O
+does	O
+chaperone	O
+binding	O
+affect	O
+substrate	O
+structure	O
+and	O
+vice	O
+versa	O
+.	O
+	O
+By	O
+analyzing	O
+the	O
+individual	O
+structures	O
+of	O
+the	O
+six	O
+-	O
+member	O
+ensemble	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+bound	O
+to	O
+Spy	O
+,	O
+we	O
+observed	O
+that	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+takes	O
+on	O
+several	O
+different	O
+conformations	O
+while	O
+bound	O
+.	O
+	O
+We	O
+found	O
+these	O
+conformations	O
+to	O
+be	O
+highly	O
+heterogeneous	O
+and	O
+to	O
+include	O
+unfolded	O
+,	O
+partially	O
+folded	O
+,	O
+and	O
+native	O
+-	O
+like	O
+states	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+We	O
+found	O
+that	O
+the	O
+primary	O
+interaction	O
+sites	O
+on	O
+Spy	O
+reside	O
+at	O
+the	O
+N	O
+and	O
+C	O
+termini	O
+(	O
+Arg122	O
+,	O
+Thr124	O
+,	O
+and	O
+Phe29	O
+)	O
+as	O
+well	O
+as	O
+on	O
+the	O
+concave	O
+face	O
+of	O
+the	O
+chaperone	O
+(	O
+Arg61	O
+,	O
+Arg43	O
+,	O
+Lys47	O
+,	O
+His96	O
+,	O
+and	O
+Met46	O
+).	O
+	O
+Surprisingly	O
+,	O
+we	O
+noted	O
+that	O
+in	O
+the	O
+ensemble	O
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+interacts	O
+with	O
+only	O
+38	O
+%	O
+of	O
+the	O
+hydrophobic	O
+residues	O
+in	O
+the	O
+Spy	O
+cradle	O
+,	O
+but	O
+interacts	O
+with	O
+61	O
+%	O
+of	O
+the	O
+hydrophilic	O
+residues	O
+in	O
+the	O
+cradle	O
+.	O
+	O
+With	O
+respect	O
+to	O
+the	O
+substrate	O
+,	O
+we	O
+observed	O
+that	O
+nearly	O
+every	O
+residue	O
+in	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+is	O
+in	O
+contact	O
+with	O
+Spy	O
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+However	O
+,	O
+we	O
+did	O
+notice	O
+that	O
+despite	O
+this	O
+uniformity	O
+,	O
+regions	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+preferentially	O
+interact	O
+with	O
+different	O
+regions	O
+in	O
+Spy	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+	O
+For	O
+example	O
+,	O
+the	O
+N	O
+-	O
+terminal	O
+half	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+binds	O
+more	O
+consistently	O
+in	O
+the	O
+Spy	O
+cradle	O
+,	O
+whereas	O
+the	O
+C	O
+-	O
+terminal	O
+half	O
+predominantly	O
+binds	O
+to	O
+the	O
+outer	O
+edges	O
+of	O
+Spy	O
+’	O
+s	O
+concave	O
+surface	O
+.	O
+	O
+Not	O
+unexpectedly	O
+,	O
+we	O
+found	O
+that	O
+as	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+progresses	O
+from	O
+the	O
+unfolded	O
+to	O
+the	O
+native	O
+state	O
+,	O
+its	O
+interactions	O
+with	O
+Spy	O
+shift	O
+accordingly	O
+.	O
+	O
+Whereas	O
+the	O
+least	O
+-	O
+folded	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+pose	O
+in	O
+the	O
+ensemble	O
+forms	O
+the	O
+most	O
+hydrophobic	O
+contacts	O
+with	O
+Spy	O
+(	O
+Fig	O
+.	O
+3	O
+),	O
+the	O
+two	O
+most	O
+-	O
+folded	O
+conformations	O
+form	O
+the	O
+fewest	O
+hydrophobic	O
+contacts	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+Although	O
+we	O
+do	O
+not	O
+yet	O
+have	O
+time	O
+resolution	O
+data	O
+of	O
+these	O
+various	O
+snapshots	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+,	O
+this	O
+ensemble	O
+illustrates	O
+how	O
+a	O
+substrate	O
+samples	O
+its	O
+folding	O
+landscape	O
+while	O
+bound	O
+to	O
+a	O
+chaperone	O
+.	O
+	O
+Spy	O
+changes	O
+conformation	O
+upon	O
+substrate	O
+binding	O
+	O
+Comparing	O
+the	O
+structure	O
+of	O
+Spy	O
+in	O
+its	O
+substrate	O
+-	O
+bound	O
+and	O
+apo	O
+states	O
+revealed	O
+that	O
+the	O
+Spy	O
+dimer	O
+also	O
+undergoes	O
+significant	O
+conformational	O
+changes	O
+upon	O
+substrate	O
+binding	O
+(	O
+Fig	O
+.	O
+5a	O
+and	O
+Supplementary	O
+Movie	O
+2	O
+).	O
+	O
+Upon	O
+substrate	O
+binding	O
+,	O
+the	O
+Spy	O
+dimer	O
+twists	O
+9	O
+°	O
+about	O
+its	O
+center	O
+relative	O
+to	O
+its	O
+apo	O
+form	O
+.	O
+	O
+This	O
+twist	O
+yields	O
+asymmetry	O
+and	O
+results	O
+in	O
+substantially	O
+different	O
+interaction	O
+patterns	O
+in	O
+the	O
+two	O
+Spy	O
+monomers	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+	O
+This	O
+increased	O
+disorder	O
+might	O
+explain	O
+how	O
+Spy	O
+is	O
+able	O
+to	O
+recognize	O
+and	O
+bind	O
+different	O
+substrates	O
+and	O
+/	O
+or	O
+differing	O
+conformations	O
+of	O
+the	O
+same	O
+substrate	O
+.	O
+	O
+The	O
+RMSD	O
+between	O
+the	O
+well	O
+-	O
+folded	O
+sections	O
+of	O
+Spy	O
+in	O
+the	O
+four	O
+chaperone	O
+-	O
+substrate	O
+complexes	O
+was	O
+very	O
+small	O
+,	O
+less	O
+than	O
+0	O
+.	O
+3	O
+Å	O
+.	O
+Combined	O
+with	O
+competition	O
+experiments	O
+showing	O
+that	O
+the	O
+substrates	O
+compete	O
+in	O
+solution	O
+for	O
+Spy	O
+binding	O
+(	O
+Fig	O
+.	O
+5c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+8	O
+),	O
+we	O
+conclude	O
+that	O
+all	O
+the	O
+tested	O
+substrates	O
+share	O
+the	O
+same	O
+overall	O
+Spy	O
+binding	O
+site	O
+.	O
+	O
+To	O
+shed	O
+light	O
+on	O
+how	O
+chaperones	O
+interact	O
+with	O
+their	O
+substrates	O
+,	O
+we	O
+developed	O
+a	O
+novel	O
+structural	O
+biology	O
+method	O
+(	O
+READ	O
+)	O
+and	O
+applied	O
+it	O
+to	O
+determine	O
+a	O
+conformational	O
+ensemble	O
+of	O
+the	O
+chaperone	O
+Spy	O
+bound	O
+to	O
+substrate	O
+.	O
+	O
+As	O
+a	O
+substrate	O
+,	O
+we	O
+used	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+,	O
+the	O
+chaperone	O
+-	O
+interacting	O
+portion	O
+of	O
+the	O
+protein	O
+-	O
+folding	O
+model	O
+protein	O
+Im7	O
+.	O
+	O
+In	O
+the	O
+chaperone	O
+-	O
+bound	O
+ensemble	O
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+samples	O
+unfolded	O
+,	O
+partially	O
+folded	O
+,	O
+and	O
+native	O
+-	O
+like	O
+states	O
+.	O
+	O
+The	O
+ensemble	O
+provides	O
+an	O
+unprecedented	O
+description	O
+of	O
+the	O
+conformations	O
+that	O
+a	O
+substrate	O
+assumes	O
+while	O
+exploring	O
+its	O
+chaperone	O
+-	O
+associated	O
+folding	O
+landscape	O
+.	O
+	O
+We	O
+recently	O
+showed	O
+that	O
+Im7	O
+can	O
+fold	O
+while	O
+remaining	O
+continuously	O
+bound	O
+to	O
+Spy	O
+.	O
+	O
+The	O
+structures	O
+of	O
+our	O
+ensemble	O
+agree	O
+well	O
+with	O
+lower	O
+-	O
+resolution	O
+crosslinking	O
+data	O
+,	O
+which	O
+indicate	O
+that	O
+chaperone	O
+-	O
+substrate	O
+interactions	O
+primarily	O
+occur	O
+on	O
+the	O
+concave	O
+surface	O
+of	O
+Spy	O
+.	O
+	O
+The	O
+ensemble	O
+suggests	O
+a	O
+model	O
+in	O
+which	O
+Spy	O
+provides	O
+an	O
+amphipathic	O
+surface	O
+that	O
+allows	O
+substrate	O
+proteins	O
+to	O
+assume	O
+different	O
+conformations	O
+while	O
+bound	O
+to	O
+the	O
+chaperone	O
+.	O
+	O
+This	O
+model	O
+is	O
+consistent	O
+with	O
+previous	O
+studies	O
+postulating	O
+that	O
+the	O
+flexible	O
+binding	O
+of	O
+chaperones	O
+allows	O
+for	O
+substrate	O
+protein	O
+folding	O
+.	O
+	O
+The	O
+amphipathic	O
+concave	O
+surface	O
+of	O
+Spy	O
+likely	O
+facilitates	O
+this	O
+flexible	O
+binding	O
+and	O
+may	O
+be	O
+a	O
+crucial	O
+feature	O
+for	O
+Spy	O
+and	O
+potentially	O
+other	O
+chaperones	O
+,	O
+allowing	O
+them	O
+to	O
+bind	O
+multiple	O
+conformations	O
+of	O
+many	O
+different	O
+substrates	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+Spy	O
+’	O
+s	O
+binding	O
+hotspots	O
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+displays	O
+substantially	O
+less	O
+specificity	O
+in	O
+its	O
+binding	O
+sites	O
+.	O
+	O
+This	O
+trend	O
+suggests	O
+that	O
+complex	O
+formation	O
+between	O
+an	O
+ATP	O
+-	O
+independent	O
+chaperone	O
+and	O
+its	O
+unfolded	O
+substrate	O
+may	O
+initially	O
+involve	O
+hydrophobic	O
+interactions	O
+,	O
+effectively	O
+shielding	O
+the	O
+exposed	O
+aggregation	O
+-	O
+sensitive	O
+hydrophobic	O
+regions	O
+in	O
+the	O
+substrate	O
+.	O
+	O
+Notably	O
+,	O
+the	O
+most	O
+frequent	O
+contacts	O
+between	O
+Spy	O
+and	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+are	O
+charge	O
+-	O
+charge	O
+interactions	O
+.	O
+	O
+The	O
+negatively	O
+charged	O
+Im7	O
+residues	O
+Glu21	O
+,	O
+Asp32	O
+,	O
+and	O
+Asp35	O
+reside	O
+on	O
+the	O
+surface	O
+of	O
+Im7	O
+and	O
+form	O
+interactions	O
+with	O
+Spy	O
+’	O
+s	O
+positively	O
+charged	O
+cradle	O
+in	O
+both	O
+the	O
+unfolded	O
+and	O
+native	O
+-	O
+like	O
+states	O
+.	O
+	O
+Interaction	O
+with	O
+Spy	O
+’	O
+s	O
+positively	O
+-	O
+charged	O
+residues	O
+likely	O
+relieves	O
+the	O
+charge	O
+repulsion	O
+between	O
+Asp32	O
+and	O
+Asp35	O
+,	O
+promoting	O
+their	O
+compaction	O
+into	O
+a	O
+helical	O
+conformation	O
+.	O
+	O
+As	O
+inter	O
+-	O
+molecular	O
+hydrophobic	O
+interactions	O
+between	O
+Spy	O
+and	O
+the	O
+substrate	O
+become	O
+progressively	O
+replaced	O
+by	O
+intra	O
+-	O
+molecular	O
+interactions	O
+within	O
+the	O
+substrate	O
+,	O
+the	O
+affinity	O
+between	O
+chaperone	O
+and	O
+substrates	O
+could	O
+decrease	O
+,	O
+eventually	O
+leading	O
+to	O
+release	O
+of	O
+the	O
+folded	O
+client	O
+protein	O
+.	O
+	O
+Recently	O
+,	O
+we	O
+employed	O
+a	O
+genetic	O
+selection	O
+system	O
+to	O
+improve	O
+the	O
+chaperone	O
+activity	O
+of	O
+Spy	O
+.	O
+	O
+This	O
+selection	O
+resulted	O
+in	O
+“	O
+Super	O
+Spy	O
+”	O
+variants	O
+that	O
+were	O
+more	O
+effective	O
+at	O
+both	O
+preventing	O
+aggregation	O
+and	O
+promoting	O
+protein	O
+folding	O
+.	O
+	O
+Our	O
+ensemble	O
+revealed	O
+that	O
+two	O
+of	O
+the	O
+Super	O
+Spy	O
+mutations	O
+(	O
+H96L	B-mutant
+and	O
+Q100L	B-mutant
+)	O
+form	O
+part	O
+of	O
+the	O
+chaperone	O
+contact	O
+surface	O
+that	O
+binds	O
+to	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+By	O
+sampling	O
+multiple	O
+conformations	O
+,	O
+this	O
+linker	O
+region	O
+may	O
+allow	O
+diverse	O
+substrate	O
+conformations	O
+to	O
+be	O
+accommodated	O
+.	O
+	O
+Other	O
+Super	O
+Spy	O
+mutations	O
+(	O
+F115I	B-mutant
+and	O
+F115L	B-mutant
+)	O
+caused	O
+increased	O
+flexibility	O
+but	O
+not	O
+tighter	O
+substrate	O
+binding	O
+.	O
+	O
+This	O
+residue	O
+does	O
+not	O
+directly	O
+contact	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+in	O
+our	O
+READ	O
+-	O
+derived	O
+ensemble	O
+.	O
+	O
+This	O
+interaction	O
+presumably	O
+reduces	O
+the	O
+mobility	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+helix	O
+.	O
+	O
+The	O
+F115I	B-mutant
+/	O
+L	B-mutant
+substitutions	O
+would	O
+replace	O
+these	O
+hydrogen	O
+bonds	O
+with	O
+hydrophobic	O
+interactions	O
+that	O
+have	O
+little	O
+angular	O
+dependence	O
+.	O
+	O
+As	O
+a	O
+result	O
+,	O
+the	O
+C	O
+-	O
+terminus	O
+,	O
+and	O
+possibly	O
+also	O
+the	O
+flexible	O
+linker	O
+,	O
+is	O
+likely	O
+to	O
+become	O
+more	O
+flexible	O
+and	O
+thus	O
+more	O
+accommodating	O
+of	O
+different	O
+conformations	O
+of	O
+substrates	O
+.	O
+	O
+Our	O
+study	O
+indicates	O
+that	O
+the	O
+chaperone	O
+Spy	O
+employs	O
+a	O
+simple	O
+surface	O
+binding	O
+approach	O
+that	O
+allows	O
+the	O
+substrate	O
+to	O
+explore	O
+various	O
+conformations	O
+and	O
+form	O
+transiently	O
+favorable	O
+interactions	O
+while	O
+being	O
+protected	O
+from	O
+aggregation	O
+.	O
+	O
+We	O
+speculate	O
+that	O
+many	O
+other	O
+chaperones	O
+could	O
+utilize	O
+a	O
+similar	O
+strategy	O
+.	O
+	O
+ATP	O
+and	O
+co	O
+-	O
+chaperone	O
+dependencies	O
+may	O
+have	O
+emerged	O
+later	O
+through	O
+evolution	O
+to	O
+better	O
+modulate	O
+and	O
+control	O
+chaperone	O
+action	O
+.	O
+	O
+In	O
+addition	O
+to	O
+insights	O
+into	O
+chaperone	O
+function	O
+,	O
+this	O
+work	O
+presents	O
+a	O
+new	O
+method	O
+for	O
+determining	O
+heterogeneous	O
+structural	O
+ensembles	O
+via	O
+a	O
+hybrid	O
+methodology	O
+of	O
+X	O
+-	O
+ray	O
+crystallography	O
+and	O
+computational	O
+modeling	O
+.	O
+	O
+Crystallographic	O
+data	O
+and	O
+ensemble	O
+selection	O
+.	O
+(	O
+a	O
+)	O
+2mFo	O
+−	O
+DFc	O
+omit	O
+map	O
+of	O
+residual	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+and	O
+flexible	O
+linker	O
+electron	O
+density	O
+contoured	O
+at	O
+0	O
+.	O
+5	O
+σ	O
+.	O
+	O
+This	O
+is	O
+the	O
+residual	O
+density	O
+that	O
+is	O
+used	O
+in	O
+the	O
+READ	O
+selection	O
+.	O
+	O
+(	O
+b	O
+)	O
+Composites	O
+of	O
+iodine	O
+positions	O
+detected	O
+from	O
+anomalous	O
+signals	O
+using	O
+pI	O
+-	O
+Phe	O
+substitutions	O
+,	O
+colored	O
+and	O
+numbered	O
+by	O
+sequence	O
+.	O
+	O
+Multiple	O
+iodine	O
+positions	O
+were	O
+detected	O
+for	O
+most	O
+residues	O
+.	O
+	O
+Agreement	O
+to	O
+the	O
+residual	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+electron	O
+density	O
+(	O
+c	O
+)	O
+and	O
+anomalous	O
+iodine	O
+signals	O
+(	O
+d	O
+)	O
+for	O
+ensembles	O
+of	O
+varying	O
+size	O
+generated	O
+by	O
+randomly	O
+choosing	O
+from	O
+the	O
+MD	O
+pool	O
+(	O
+blue	O
+)	O
+and	O
+from	O
+the	O
+selection	O
+procedure	O
+(	O
+black	O
+).	O
+	O
+The	O
+cost	O
+function	O
+,	O
+χ2	O
+,	O
+decreases	O
+as	O
+the	O
+agreement	O
+to	O
+the	O
+experimental	O
+data	O
+increases	O
+and	O
+is	O
+defined	O
+in	O
+the	O
+Online	O
+Methods	O
+.	O
+	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+ensemble	O
+,	O
+arranged	O
+by	O
+RMSD	O
+to	O
+native	O
+state	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+.	O
+Although	O
+the	O
+six	O
+-	O
+membered	O
+ensemble	O
+from	O
+the	O
+READ	O
+selection	O
+should	O
+be	O
+considered	O
+only	O
+as	O
+an	O
+ensemble	O
+,	O
+for	O
+clarity	O
+,	O
+the	O
+individual	O
+conformers	O
+are	O
+shown	O
+separately	O
+here	O
+.	O
+	O
+Atoms	O
+that	O
+were	O
+either	O
+not	O
+directly	O
+selected	O
+in	O
+the	O
+READ	O
+procedure	O
+,	O
+or	O
+whose	O
+position	O
+could	O
+not	O
+be	O
+justified	O
+based	O
+on	O
+agreement	O
+with	O
+the	O
+residual	O
+electron	O
+density	O
+were	O
+removed	O
+,	O
+leading	O
+to	O
+non	O
+-	O
+contiguous	O
+sections	O
+.	O
+	O
+Dashed	O
+lines	O
+connect	O
+non	O
+-	O
+contiguous	O
+segments	O
+of	O
+the	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+substrate	O
+.	O
+	O
+Residues	O
+of	O
+the	O
+Spy	O
+flexible	O
+linker	O
+region	O
+that	O
+fit	O
+the	O
+residual	O
+electron	O
+density	O
+are	O
+shown	O
+as	O
+larger	O
+gray	O
+spheres	O
+.	O
+	O
+Shown	O
+below	O
+each	O
+ensemble	O
+member	O
+is	O
+the	O
+RMSD	O
+of	O
+each	O
+conformer	O
+to	O
+the	O
+native	O
+state	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+,	O
+as	O
+well	O
+as	O
+the	O
+percentage	O
+of	O
+contacts	O
+between	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+and	O
+Spy	O
+that	O
+are	O
+hydrophobic	O
+.	O
+	O
+Contact	O
+maps	O
+of	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+complex	O
+.	O
+	O
+For	O
+clarity	O
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+is	O
+represented	O
+with	O
+a	O
+single	O
+conformation	O
+.	O
+	O
+As	O
+the	O
+residues	O
+involved	O
+in	O
+contacts	O
+are	O
+more	O
+evenly	O
+distributed	O
+in	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+compared	O
+to	O
+Spy	O
+,	O
+its	O
+contact	O
+map	O
+was	O
+amplified	O
+.	O
+(	O
+b	O
+)	O
+Detailed	O
+contact	O
+maps	O
+of	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+.	O
+	O
+Contacts	O
+to	O
+the	O
+two	O
+Spy	O
+monomers	O
+are	O
+depicted	O
+separately	O
+.	O
+	O
+Note	O
+that	O
+the	O
+flexible	O
+linker	O
+region	O
+of	O
+Spy	O
+(	O
+residues	O
+47	O
+–	O
+57	O
+)	O
+is	O
+not	O
+represented	O
+in	O
+the	O
+2D	O
+contact	O
+maps	O
+.	O
+	O
+Spy	O
+conformation	O
+changes	O
+upon	O
+substrate	O
+binding	O
+.	O
+	O
+Flexibility	O
+of	O
+Spy	O
+linker	O
+region	O
+and	O
+effect	O
+of	O
+Super	O
+Spy	O
+mutants	O
+.	O
+(	O
+a	O
+)	O
+The	O
+Spy	O
+linker	O
+region	O
+adopts	O
+one	O
+dominant	O
+conformation	O
+in	O
+its	O
+apo	O
+state	O
+(	O
+PDB	O
+ID	O
+3039	O
+,	O
+gray	O
+),	O
+but	O
+expands	O
+and	O
+adopts	O
+multiple	O
+conformations	O
+in	O
+bound	O
+states	O
+(	O
+green	O
+).	O
+	O
+The	O
+Super	O
+Spy	O
+mutants	O
+F115L	B-mutant
+,	O
+F115I	B-mutant
+,	O
+and	O
+L32P	B-mutant
+are	O
+proposed	O
+to	O
+gain	O
+activity	O
+by	O
+increasing	O
+the	O
+flexibility	O
+or	O
+size	O
+of	O
+this	O
+linker	O
+region	O
+.	O
+	O
+L32	O
+,	O
+F115	O
+,	O
+and	O
+Y104	O
+are	O
+rendered	O
+in	O
+purple	O
+to	O
+illustrate	O
+residues	O
+that	O
+are	O
+most	O
+affected	O
+by	O
+Super	O
+Spy	O
+mutations	O
+;	O
+CH	O
+⋯	O
+π	O
+hydrogen	O
+bonds	O
+are	O
+depicted	O
+by	O
+orange	O
+dashes	O
+.	O
+	O
+Mechanism	O
+of	O
+extracellular	O
+ion	O
+exchange	O
+and	O
+binding	O
+-	O
+site	O
+occlusion	O
+in	O
+the	O
+sodium	O
+-	O
+calcium	O
+exchanger	O
+	O
+Na	O
++/	O
+Ca2	O
++	O
+exchangers	O
+utilize	O
+the	O
+Na	O
++	O
+electrochemical	O
+gradient	O
+across	O
+the	O
+plasma	O
+membrane	O
+to	O
+extrude	O
+intracellular	O
+Ca2	O
++,	O
+and	O
+play	O
+a	O
+central	O
+role	O
+in	O
+Ca2	O
++	O
+homeostasis	O
+.	O
+	O
+Here	O
+,	O
+we	O
+elucidate	O
+their	O
+mechanisms	O
+of	O
+extracellular	O
+ion	O
+recognition	O
+and	O
+exchange	O
+through	O
+a	O
+structural	O
+analysis	O
+of	O
+the	O
+exchanger	O
+from	O
+Methanococcus	O
+jannaschii	O
+(	O
+NCX_Mj	O
+)	O
+bound	O
+to	O
+Na	O
++,	O
+Ca2	O
++	O
+or	O
+Sr2	O
++	O
+in	O
+various	O
+occupancies	O
+and	O
+in	O
+an	O
+apo	O
+state	O
+.	O
+	O
+This	O
+analysis	O
+defines	O
+the	O
+binding	O
+mode	O
+and	O
+relative	O
+affinity	O
+of	O
+these	O
+ions	O
+,	O
+establishes	O
+the	O
+structural	O
+basis	O
+for	O
+the	O
+anticipated	O
+3Na	O
++:	O
+1Ca2	O
++	O
+exchange	O
+stoichiometry	O
+,	O
+and	O
+reveals	O
+the	O
+conformational	O
+changes	O
+at	O
+the	O
+onset	O
+of	O
+the	O
+alternating	O
+-	O
+access	O
+transport	O
+mechanism	O
+.	O
+	O
+An	O
+independent	O
+analysis	O
+of	O
+the	O
+dynamics	O
+and	O
+conformational	O
+free	O
+-	O
+energy	O
+landscape	O
+of	O
+NCX_Mj	O
+in	O
+different	O
+ion	O
+-	O
+occupancy	O
+states	O
+,	O
+based	O
+on	O
+enhanced	O
+-	O
+sampling	O
+molecular	O
+-	O
+dynamics	O
+simulations	O
+,	O
+demonstrates	O
+that	O
+the	O
+crystal	O
+structures	O
+reflect	O
+mechanistically	O
+relevant	O
+,	O
+interconverting	O
+conformations	O
+.	O
+	O
+These	O
+calculations	O
+also	O
+reveal	O
+the	O
+mechanism	O
+by	O
+which	O
+the	O
+outward	O
+-	O
+to	O
+-	O
+inward	O
+transition	O
+is	O
+controlled	O
+by	O
+the	O
+ion	O
+-	O
+occupancy	O
+state	O
+,	O
+thereby	O
+explaining	O
+the	O
+emergence	O
+of	O
+strictly	O
+-	O
+coupled	O
+Na	O
++/	O
+Ca2	O
++	O
+antiport	O
+.	O
+	O
+Na	O
++/	O
+Ca2	O
++	O
+exchangers	O
+(	O
+NCX	O
+)	O
+play	O
+physiologically	O
+essential	O
+roles	O
+in	O
+Ca2	O
++	O
+signaling	O
+and	O
+homeostasis	O
+.	O
+	O
+NCX	O
+catalyzes	O
+the	O
+uphill	O
+extrusion	O
+of	O
+intracellular	O
+Ca2	O
++	O
+across	O
+the	O
+cell	O
+membrane	O
+,	O
+by	O
+coupling	O
+this	O
+process	O
+to	O
+the	O
+downhill	O
+permeation	O
+of	O
+Na	O
++	O
+into	O
+the	O
+cell	O
+,	O
+with	O
+a	O
+3	O
+Na	O
++	O
+to	O
+1	O
+Ca2	O
++	O
+stoichiometry	O
+.	O
+	O
+The	O
+basic	O
+functional	O
+unit	O
+for	O
+ion	O
+transport	O
+in	O
+NCX	O
+consists	O
+of	O
+ten	O
+membrane	O
+-	O
+spanning	O
+segments	O
+,	O
+comprising	O
+two	O
+homologous	O
+halves	O
+.	O
+	O
+Each	O
+of	O
+these	O
+halves	O
+contains	O
+a	O
+highly	O
+conserved	O
+region	O
+,	O
+referred	O
+to	O
+as	O
+α	O
+-	O
+repeat	O
+,	O
+known	O
+to	O
+be	O
+important	O
+for	O
+ion	O
+binding	O
+and	O
+translocation	O
+;	O
+in	O
+eukaryotic	O
+NCX	O
+,	O
+the	O
+two	O
+halves	O
+are	O
+connected	O
+by	O
+a	O
+large	O
+intracellular	O
+regulatory	O
+domain	O
+,	O
+which	O
+is	O
+absent	O
+in	O
+microbial	O
+NCX	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+Our	O
+recent	O
+atomic	O
+-	O
+resolution	O
+structure	O
+of	O
+NCX_Mj	O
+from	O
+Methanococcus	O
+jannaschii	O
+provided	O
+the	O
+first	O
+view	O
+of	O
+the	O
+basic	O
+functional	O
+unit	O
+of	O
+an	O
+NCX	O
+protein	O
+.	O
+	O
+This	O
+structure	O
+shows	O
+the	O
+exchanger	O
+in	O
+an	O
+outward	O
+-	O
+facing	O
+conformation	O
+and	O
+reveals	O
+four	O
+putative	O
+ion	O
+-	O
+binding	O
+sites	O
+,	O
+denominated	O
+internal	O
+(	O
+Sint	O
+),	O
+external	O
+(	O
+Sext	O
+),	O
+Ca2	O
++-	O
+binding	O
+(	O
+SCa	O
+)	O
+and	O
+middle	O
+(	O
+Smid	O
+),	O
+clustered	O
+in	O
+the	O
+center	O
+of	O
+the	O
+protein	O
+and	O
+occluded	O
+from	O
+the	O
+solvent	O
+(	O
+Fig	O
+.	O
+1a	O
+-	O
+b	O
+).	O
+	O
+These	O
+structures	O
+reveal	O
+the	O
+mode	O
+of	O
+recognition	O
+of	O
+these	O
+ions	O
+,	O
+their	O
+relative	O
+affinities	O
+,	O
+and	O
+the	O
+mechanism	O
+of	O
+extracellular	O
+ion	O
+exchange	O
+,	O
+for	O
+a	O
+well	O
+-	O
+defined	O
+,	O
+functional	O
+conformation	O
+in	O
+a	O
+membrane	O
+-	O
+like	O
+environment	O
+.	O
+	O
+An	O
+independent	O
+analysis	O
+based	O
+on	O
+molecular	O
+-	O
+dynamics	O
+simulations	O
+demonstrates	O
+that	O
+the	O
+structures	O
+capture	O
+mechanistically	O
+relevant	O
+states	O
+.	O
+	O
+These	O
+calculations	O
+also	O
+reveal	O
+how	O
+the	O
+ion	O
+occupancy	O
+state	O
+of	O
+the	O
+outward	O
+-	O
+facing	O
+exchanger	O
+determines	O
+the	O
+feasibility	O
+of	O
+the	O
+transition	O
+to	O
+the	O
+inward	O
+-	O
+facing	O
+conformation	O
+,	O
+thereby	O
+addressing	O
+a	O
+key	O
+outstanding	O
+question	O
+in	O
+secondary	O
+-	O
+active	O
+transport	O
+,	O
+namely	O
+how	O
+the	O
+transported	O
+substrates	O
+control	O
+the	O
+alternating	O
+-	O
+access	O
+mechanism	O
+.	O
+	O
+Crystals	O
+were	O
+grown	O
+in	O
+150	O
+mM	O
+NaCl	O
+using	O
+the	O
+lipidic	O
+cubic	O
+phase	O
+(	O
+LCP	O
+)	O
+technique	O
+.	O
+	O
+The	O
+crystallization	O
+solutions	O
+around	O
+the	O
+LCP	O
+droplets	O
+were	O
+then	O
+slowly	O
+replaced	O
+by	O
+solutions	O
+containing	O
+different	O
+concentrations	O
+of	O
+NaCl	O
+and	O
+EGTA	O
+(	O
+Methods	O
+).	O
+	O
+Occupancy	O
+refinement	O
+indicated	O
+two	O
+Na	O
++	O
+ions	O
+bind	O
+to	O
+Sint	O
+and	O
+SCa	O
+at	O
+low	O
+Na	O
++	O
+concentrations	O
+(	O
+Fig	O
+.	O
+1c	O
+),	O
+with	O
+a	O
+slight	O
+preference	O
+for	O
+Sint	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Binding	O
+of	O
+a	O
+third	O
+Na	O
++	O
+to	O
+Sext	O
+occurs	O
+at	O
+higher	O
+concentrations	O
+,	O
+as	O
+no	O
+density	O
+was	O
+observed	O
+there	O
+at	O
+10	O
+mM	O
+Na	O
++	O
+or	O
+lower	O
+(	O
+Fig	O
+.	O
+1c	O
+);	O
+Sext	O
+is	O
+however	O
+partially	O
+occupied	O
+at	O
+20	O
+mM	O
+Na	O
++,	O
+and	O
+fully	O
+occupied	O
+at	O
+150	O
+mM	O
+(	O
+Fig	O
+.	O
+1c	O
+).	O
+	O
+The	O
+Na	O
++	O
+occupation	O
+at	O
+SCa	O
+,	O
+compounded	O
+with	O
+the	O
+expected	O
+3Na	O
++:	O
+1Ca2	O
++	O
+stoichiometry	O
+,	O
+implies	O
+our	O
+previous	O
+assignment	O
+of	O
+the	O
+Smid	O
+site	O
+must	O
+be	O
+re	O
+-	O
+evaluated	O
+.	O
+	O
+Second	O
+,	O
+the	O
+protein	O
+coordination	O
+geometry	O
+at	O
+Smid	O
+is	O
+clearly	O
+suboptimal	O
+for	O
+Na	O
++	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+).	O
+	O
+The	O
+water	O
+molecule	O
+at	O
+Smid	O
+forms	O
+hydrogen	O
+-	O
+bonds	O
+with	O
+the	O
+highly	O
+conserved	O
+Glu54	O
+and	O
+Glu213	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+),	O
+stabilizing	O
+their	O
+orientation	O
+to	O
+properly	O
+coordinate	O
+multiple	O
+Na	O
++	O
+ions	O
+at	O
+Sext	O
+,	O
+SCa	O
+and	O
+Sint	O
+.	O
+	O
+It	O
+can	O
+be	O
+inferred	O
+from	O
+this	O
+assignment	O
+that	O
+Glu54	O
+and	O
+Glu213	O
+are	O
+ionized	O
+,	O
+while	O
+Asp240	O
+,	O
+which	O
+flanks	O
+Smid	O
+(	O
+and	O
+is	O
+replaced	O
+by	O
+Asn	O
+in	O
+eukaryotic	O
+NCX	O
+)	O
+would	O
+be	O
+protonated	O
+,	O
+as	O
+indicated	O
+by	O
+the	O
+abovementioned	O
+simulation	O
+study	O
+.	O
+	O
+Na	O
++-	O
+dependent	O
+conformational	O
+change	O
+	O
+The	O
+NCX_Mj	O
+structures	O
+in	O
+various	O
+Na	O
++	O
+concentrations	O
+also	O
+reveal	O
+that	O
+Na	O
++	O
+binding	O
+to	O
+Sext	O
+is	O
+coupled	O
+to	O
+a	O
+subtle	O
+but	O
+important	O
+conformational	O
+change	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+However	O
+,	O
+when	O
+Sext	O
+becomes	O
+empty	O
+at	O
+low	O
+Na	O
++	O
+concentrations	O
+,	O
+TM7a	O
+and	O
+TM7b	O
+become	O
+a	O
+continuous	O
+straight	O
+helix	O
+(	O
+Fig	O
+.	O
+2a	O
+),	O
+and	O
+the	O
+carbonyl	O
+group	O
+of	O
+Ala206	O
+retracts	O
+away	O
+(	O
+Fig	O
+.	O
+2b	O
+-	O
+d	O
+).	O
+	O
+TM7a	O
+also	O
+forms	O
+hydrophobic	O
+contacts	O
+with	O
+the	O
+C	O
+-	O
+terminal	O
+half	O
+of	O
+TM6	O
+.	O
+	O
+These	O
+contacts	O
+are	O
+absent	O
+in	O
+the	O
+structure	O
+with	O
+Na	O
++	O
+at	O
+Sext	O
+,	O
+in	O
+which	O
+there	O
+is	O
+an	O
+open	O
+gap	O
+between	O
+the	O
+two	O
+helices	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+This	O
+difference	O
+is	O
+noteworthy	O
+because	O
+TM6	O
+and	O
+TM1	O
+are	O
+believed	O
+to	O
+undergo	O
+a	O
+sliding	O
+motion	O
+,	O
+relative	O
+to	O
+the	O
+rest	O
+of	O
+the	O
+protein	O
+,	O
+when	O
+the	O
+transporter	O
+switches	O
+to	O
+the	O
+inward	O
+-	O
+facing	O
+conformation	O
+.	O
+	O
+The	O
+straightening	O
+of	O
+TM7ab	O
+also	O
+opens	O
+up	O
+a	O
+passageway	O
+from	O
+the	O
+external	O
+solution	O
+to	O
+Sext	O
+and	O
+Smid	O
+,	O
+while	O
+SCa	O
+and	O
+Sint	O
+remain	O
+occluded	O
+(	O
+Fig	O
+.	O
+2d	O
+).	O
+	O
+Thus	O
+,	O
+the	O
+structures	O
+at	O
+high	O
+and	O
+low	O
+Na	O
++	O
+concentrations	O
+represent	O
+the	O
+outward	O
+-	O
+facing	O
+occluded	O
+and	O
+partially	O
+open	O
+states	O
+,	O
+respectively	O
+.	O
+	O
+This	O
+conformational	O
+change	O
+is	O
+dependent	O
+on	O
+the	O
+Na	O
++	O
+occupancy	O
+of	O
+Sext	O
+and	O
+occurs	O
+when	O
+Na	O
++	O
+already	O
+occupies	O
+Sint	O
+and	O
+SCa	O
+.	O
+	O
+Our	O
+crystallographic	O
+titration	O
+experiment	O
+indicates	O
+that	O
+the	O
+K1	O
+/	O
+2	O
+of	O
+this	O
+Na	O
++-	O
+driven	O
+conformational	O
+transition	O
+is	O
+~	O
+20	O
+mM	O
+.	O
+At	O
+this	O
+concentration	O
+,	O
+Sext	O
+is	O
+partially	O
+occupied	O
+and	O
+the	O
+NCX_Mj	O
+crystal	O
+is	O
+a	O
+mixture	O
+of	O
+both	O
+the	O
+occluded	O
+and	O
+partially	O
+open	O
+conformations	O
+.	O
+	O
+The	O
+finding	O
+that	O
+the	O
+Na	O
++	O
+occupancy	O
+change	O
+from	O
+2	O
+to	O
+3	O
+ions	O
+coincides	O
+with	O
+a	O
+conformational	O
+change	O
+of	O
+the	O
+transporter	O
+also	O
+provides	O
+a	O
+rationale	O
+to	O
+the	O
+Hill	O
+coefficient	O
+of	O
+the	O
+Na	O
++-	O
+dependent	O
+activation	O
+process	O
+in	O
+eukaryotic	O
+NCX	O
+.	O
+	O
+To	O
+determine	O
+how	O
+Ca2	O
++	O
+binds	O
+to	O
+NCX_Mj	O
+and	O
+competes	O
+with	O
+Na	O
++,	O
+we	O
+first	O
+titrated	O
+the	O
+crystals	O
+with	O
+Sr2	O
++	O
+(	O
+Methods	O
+).	O
+	O
+Sr2	O
++	O
+is	O
+transported	O
+by	O
+NCX	O
+similarly	O
+to	O
+Ca2	O
++	O
+,	O
+and	O
+is	O
+distinguishable	O
+from	O
+Na	O
++	O
+by	O
+its	O
+greater	O
+electron	O
+-	O
+density	O
+intensity	O
+.	O
+	O
+Protein	O
+crystals	O
+soaked	O
+with	O
+10	O
+mM	O
+Sr2	O
++	O
+and	O
+2	O
+.	O
+5	O
+mM	O
+Na	O
++	O
+revealed	O
+a	O
+strong	O
+electron	O
+-	O
+density	O
+peak	O
+at	O
+site	O
+SCa	O
+,	O
+indicating	O
+binding	O
+of	O
+a	O
+single	O
+Sr2	O
++	O
+ion	O
+(	O
+Fig	O
+.	O
+3a	O
+).	O
+	O
+The	O
+Sr2	O
++-	O
+loaded	O
+NCX_Mj	O
+structure	O
+adopts	O
+the	O
+partially	O
+open	O
+conformation	O
+observed	O
+at	O
+low	O
+Na	O
++	O
+concentrations	O
+.	O
+	O
+Binding	O
+of	O
+Sr2	O
++,	O
+however	O
+,	O
+excludes	O
+Na	O
++	O
+entirely	O
+.	O
+	O
+Crystal	O
+titrations	O
+with	O
+decreasing	O
+Sr2	O
++	O
+or	O
+increasing	O
+Na	O
++	O
+demonstrated	O
+that	O
+Sr2	O
++	O
+binds	O
+to	O
+the	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+with	O
+low	O
+affinity	O
+,	O
+and	O
+that	O
+it	O
+can	O
+be	O
+out	O
+-	O
+competed	O
+by	O
+Na	O
++	O
+even	O
+at	O
+low	O
+concentrations	O
+(	O
+Supplementary	O
+Note	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+-	O
+b	O
+).	O
+	O
+Thus	O
+,	O
+in	O
+100	O
+mM	O
+Na	O
++	O
+and	O
+10	O
+mM	O
+Sr2	O
++,	O
+Na	O
++	O
+completely	O
+replaced	O
+Sr2	O
++	O
+(	O
+Fig	O
+.	O
+3a	O
+)	O
+and	O
+reverted	O
+NCX_Mj	O
+to	O
+the	O
+Na	O
++-	O
+loaded	O
+,	O
+fully	O
+occluded	O
+state	O
+.	O
+	O
+Similar	O
+titration	O
+experiments	O
+showed	O
+that	O
+Ca2	O
++	O
+and	O
+Sr2	O
++	O
+binding	O
+to	O
+NCX_Mj	O
+are	O
+not	O
+exactly	O
+alike	O
+The	O
+electron	O
+density	O
+distribution	O
+from	O
+crystals	O
+soaked	O
+in	O
+high	O
+Ca2	O
++	O
+and	O
+low	O
+Na	O
++,	O
+indicates	O
+that	O
+Ca2	O
++	O
+can	O
+bind	O
+to	O
+Smid	O
+as	O
+well	O
+as	O
+SCa	O
+,	O
+with	O
+a	O
+preference	O
+for	O
+SCa	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+	O
+The	O
+partial	O
+Ca2	O
++	O
+occupancy	O
+at	O
+Smid	O
+is	O
+likely	O
+caused	O
+by	O
+Asp240	O
+,	O
+which	O
+flanks	O
+this	O
+site	O
+and	O
+can	O
+in	O
+principle	O
+coordinate	O
+Ca2	O
++.	O
+	O
+Previous	O
+functional	O
+and	O
+computational	O
+studies	O
+,	O
+however	O
+,	O
+indicate	O
+Asp240	O
+becomes	O
+protonated	O
+during	O
+transport	O
+.	O
+	O
+SCa	O
+is	O
+therefore	O
+the	O
+functional	O
+Ca2	O
++	O
+site	O
+.	O
+	O
+Specifically	O
+,	O
+our	O
+crystallographic	O
+titration	O
+assay	O
+indicates	O
+Ca2	O
++	O
+binds	O
+with	O
+sub	O
+-	O
+millimolar	O
+affinity	O
+,	O
+in	O
+good	O
+agreement	O
+with	O
+the	O
+external	O
+apparent	O
+Ca2	O
++	O
+affinities	O
+deduced	O
+functionally	O
+for	O
+cardiac	O
+NCX	O
+(	O
+Km	O
+~	O
+0	O
+.	O
+32	O
+mM	O
+)	O
+and	O
+NCX_Mj	O
+(	O
+Km	O
+~	O
+0	O
+.	O
+175	O
+mM	O
+).	O
+	O
+Taken	O
+together	O
+,	O
+these	O
+crystal	O
+titration	O
+experiments	O
+demonstrate	O
+that	O
+the	O
+four	O
+binding	O
+sites	O
+in	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+exhibit	O
+different	O
+specificity	O
+:	O
+Sint	O
+and	O
+Sext	O
+are	O
+Na	O
++	O
+specific	O
+whereas	O
+SCa	O
+,	O
+previously	O
+hypothesized	O
+to	O
+be	O
+Ca2	O
++	O
+specific	O
+,	O
+can	O
+also	O
+bind	O
+Na	O
++,	O
+confirming	O
+our	O
+earlier	O
+simulation	O
+study	O
+,	O
+as	O
+well	O
+as	O
+Sr2	O
++;	O
+Smid	O
+can	O
+also	O
+transiently	O
+accommodate	O
+Ca2	O
++	O
+but	O
+during	O
+transport	O
+Smid	O
+is	O
+most	O
+likely	O
+occupied	O
+by	O
+water	O
+.	O
+	O
+The	O
+ion	O
+-	O
+binding	O
+sites	O
+in	O
+NCX_Mj	O
+can	O
+therefore	O
+accommodate	O
+up	O
+to	O
+three	O
+Na	O
++	O
+ions	O
+or	O
+a	O
+single	O
+divalent	O
+ion	O
+,	O
+and	O
+occupancy	O
+by	O
+Na	O
++	O
+and	O
+Ca2	O
++	O
+(	O
+or	O
+Sr2	O
++)	O
+are	O
+mutually	O
+exclusive	O
+,	O
+as	O
+was	O
+deduced	O
+for	O
+eukaryotic	O
+exchangers	O
+.	O
+	O
+A	O
+structure	O
+of	O
+NCX_Mj	O
+without	O
+Na	O
++	O
+or	O
+Ca2	O
++	O
+bound	O
+	O
+An	O
+apo	O
+state	O
+of	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+is	O
+likely	O
+to	O
+exist	O
+transiently	O
+in	O
+physiological	O
+conditions	O
+,	O
+despite	O
+the	O
+high	O
+amounts	O
+of	O
+extracellular	O
+Na	O
++	O
+(~	O
+150	O
+mM	O
+)	O
+and	O
+Ca2	O
++	O
+(~	O
+2	O
+mM	O
+).	O
+	O
+This	O
+structure	O
+is	O
+similar	O
+to	O
+the	O
+partially	O
+open	O
+structure	O
+with	O
+two	O
+Na	O
++	O
+or	O
+either	O
+one	O
+Ca2	O
++	O
+or	O
+one	O
+Sr2	O
++	O
+ion	O
+,	O
+with	O
+two	O
+noticeable	O
+differences	O
+.	O
+	O
+First	O
+,	O
+TM7ab	O
+along	O
+with	O
+the	O
+extracellular	O
+half	O
+of	O
+the	O
+TM6	O
+and	O
+TM1	O
+swing	O
+further	O
+away	O
+from	O
+the	O
+protein	O
+core	O
+(	O
+Fig	O
+.	O
+3c	O
+),	O
+resulting	O
+in	O
+a	O
+slightly	O
+wider	O
+passageway	O
+into	O
+the	O
+binding	O
+sites	O
+.	O
+	O
+Second	O
+,	O
+Glu54	O
+and	O
+Glu213	O
+side	O
+chains	O
+rotate	O
+away	O
+from	O
+the	O
+binding	O
+sites	O
+and	O
+appear	O
+to	O
+form	O
+hydrogen	O
+-	O
+bonds	O
+with	O
+residues	O
+involved	O
+in	O
+ion	O
+coordination	O
+in	O
+the	O
+fully	O
+Na	O
++-	O
+loaded	O
+structure	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+	O
+This	O
+apo	O
+structure	O
+might	O
+therefore	O
+represent	O
+the	O
+unloaded	O
+,	O
+open	O
+state	O
+of	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+.	O
+	O
+Alternatively	O
+,	O
+this	O
+structure	O
+might	O
+capture	O
+a	O
+fully	O
+protonated	O
+state	O
+of	O
+the	O
+transporter	O
+,	O
+to	O
+which	O
+Na	O
++	O
+and	O
+Ca2	O
++	O
+cannot	O
+bind	O
+.	O
+	O
+Indeed	O
+,	O
+transport	O
+assays	O
+of	O
+NCX_Mj	O
+have	O
+shown	O
+that	O
+even	O
+in	O
+the	O
+presence	O
+of	O
+Na	O
++	O
+or	O
+Ca2	O
++,	O
+low	O
+pH	O
+inactivates	O
+the	O
+transport	O
+cycle	O
+.	O
+	O
+Ion	O
+occupancy	O
+determines	O
+the	O
+free	O
+-	O
+energy	O
+landscape	O
+of	O
+NCX_Mj	O
+	O
+NCX	O
+must	O
+be	O
+loaded	O
+either	O
+with	O
+3	O
+Na	O
++	O
+or	O
+1	O
+Ca2	O
++,	O
+and	O
+therefore	O
+functions	O
+as	O
+an	O
+antiporter	O
+;	O
+symporters	O
+,	O
+by	O
+contrast	O
+,	O
+undergo	O
+the	O
+alternating	O
+-	O
+access	O
+transition	O
+only	O
+when	O
+all	O
+substrates	O
+and	O
+coupling	O
+ions	O
+are	O
+concurrently	O
+bound	O
+,	O
+or	O
+in	O
+the	O
+apo	O
+state	O
+.	O
+	O
+A	O
+series	O
+of	O
+exploratory	O
+MD	O
+simulations	O
+was	O
+initially	O
+carried	O
+out	O
+to	O
+examine	O
+what	O
+features	O
+of	O
+the	O
+NCX_Mj	O
+structure	O
+might	O
+depend	O
+on	O
+the	O
+ion	O
+-	O
+binding	O
+sites	O
+occupancy	O
+.	O
+	O
+Specifically	O
+,	O
+we	O
+first	O
+simulated	O
+the	O
+outward	O
+-	O
+occluded	O
+form	O
+,	O
+in	O
+the	O
+ion	O
+configuration	O
+we	O
+previously	O
+predicted	O
+,	O
+now	O
+confirmed	O
+by	O
+the	O
+high	O
+-	O
+Na	O
++	O
+crystal	O
+structure	O
+described	O
+above	O
+(	O
+Fig	O
+.	O
+1b	O
+).	O
+	O
+That	O
+is	O
+,	O
+Na	O
++	O
+ions	O
+occupy	O
+Sext	O
+,	O
+SCa	O
+,	O
+and	O
+Sint	O
+,	O
+while	O
+D240	O
+is	O
+protonated	O
+and	O
+a	O
+water	O
+molecule	O
+occupies	O
+Smid	O
+.	O
+	O
+The	O
+Na	O
++	O
+ion	O
+at	O
+Sext	O
+was	O
+then	O
+relocated	O
+from	O
+the	O
+site	O
+to	O
+the	O
+bulk	O
+solution	O
+(	O
+Methods	O
+),	O
+and	O
+this	O
+system	O
+was	O
+then	O
+allowed	O
+to	O
+evolve	O
+freely	O
+in	O
+time	O
+.	O
+	O
+The	O
+Na	O
++	O
+ions	O
+at	O
+SCa	O
+and	O
+Sint	O
+were	O
+displaced	O
+subsequently	O
+,	O
+and	O
+an	O
+analogous	O
+simulation	O
+was	O
+then	O
+carried	O
+out	O
+.	O
+	O
+The	O
+most	O
+notable	O
+change	O
+upon	O
+displacement	O
+of	O
+Na	O
++	O
+from	O
+Sext	O
+was	O
+the	O
+straightening	O
+of	O
+TM7ab	O
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+When	O
+3	O
+Na	O
++	O
+ions	O
+are	O
+bound	O
+,	O
+TM7ab	O
+primarily	O
+folds	O
+as	O
+two	O
+distinct	O
+,	O
+non	O
+-	O
+collinear	O
+α	O
+-	O
+helical	O
+fragments	O
+,	O
+owing	O
+to	O
+the	O
+loss	O
+of	O
+the	O
+backbone	O
+carbonyl	O
+-	O
+amide	O
+hydrogen	O
+-	O
+bonds	O
+between	O
+F202	O
+and	O
+A206	O
+,	O
+and	O
+T203	O
+and	O
+F207	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+	O
+With	O
+Sext	O
+empty	O
+,	O
+however	O
+,	O
+TM7ab	O
+forms	O
+a	O
+canonical	O
+α	O
+-	O
+helix	O
+(	O
+Fig	O
+.	O
+4a	O
+-	O
+b	O
+,	O
+4g	O
+),	O
+thereby	O
+creating	O
+an	O
+opening	O
+between	O
+TM3	O
+and	O
+TM7	O
+,	O
+which	O
+in	O
+turn	O
+allows	O
+water	O
+molecules	O
+from	O
+the	O
+external	O
+solution	O
+to	O
+reach	O
+into	O
+Sext	O
+(	O
+Fig	O
+.	O
+4e	O
+,	O
+4h	O
+-	O
+i	O
+),	O
+i	O
+.	O
+e	O
+.	O
+the	O
+transporter	O
+is	O
+no	O
+longer	O
+occluded	O
+.	O
+	O
+Displacement	O
+of	O
+Na	O
++	O
+from	O
+SCa	O
+and	O
+Sint	O
+induces	O
+further	O
+changes	O
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+	O
+Together	O
+,	O
+these	O
+changes	O
+open	O
+a	O
+second	O
+aqueous	O
+channel	O
+leading	O
+directly	O
+into	O
+SCa	O
+and	O
+Sint	O
+(	O
+Fig	O
+.	O
+4f	O
+,	O
+Fig	O
+.	O
+4h	O
+-	O
+i	O
+).	O
+	O
+The	O
+transporter	O
+thus	O
+becomes	O
+fully	O
+outward	O
+-	O
+open	O
+.	O
+	O
+As	O
+above	O
+,	O
+we	O
+initially	O
+examined	O
+three	O
+occupancy	O
+states	O
+,	O
+namely	O
+with	O
+Na	O
++	O
+in	O
+Sext	O
+,	O
+SCa	O
+and	O
+Sint	O
+,	O
+with	O
+Na	O
++	O
+only	O
+at	O
+SCa	O
+and	O
+Sint	O
+,	O
+and	O
+without	O
+Na	O
++.	O
+	O
+These	O
+calculations	O
+demonstrate	O
+that	O
+the	O
+Na	O
++	O
+occupancy	O
+state	O
+of	O
+the	O
+transporter	O
+has	O
+a	O
+profound	O
+effect	O
+on	O
+its	O
+conformational	O
+free	O
+-	O
+energy	O
+landscape	O
+.	O
+	O
+At	O
+a	O
+small	O
+energetic	O
+cost	O
+,	O
+however	O
+,	O
+the	O
+transporter	O
+can	O
+adopt	O
+a	O
+metastable	O
+‘	O
+half	O
+-	O
+open	O
+’	O
+conformation	O
+in	O
+which	O
+TM7ab	O
+is	O
+completely	O
+straight	O
+and	O
+Sext	O
+is	O
+open	O
+to	O
+the	O
+exterior	O
+(	O
+Fig	O
+.	O
+5a	O
+,	O
+5b	O
+).	O
+	O
+This	O
+semi	O
+-	O
+open	O
+conformation	O
+is	O
+nearly	O
+identical	O
+to	O
+that	O
+found	O
+to	O
+be	O
+the	O
+most	O
+probable	O
+when	O
+Na	O
++	O
+occupies	O
+only	O
+SCa	O
+and	O
+Sint	O
+(	O
+2	O
+×	O
+Na	O
++,	O
+Fig	O
+.	O
+5a	O
+),	O
+demonstrating	O
+that	O
+binding	O
+(	O
+or	O
+release	O
+)	O
+of	O
+Na	O
++	O
+to	O
+Sext	O
+occurs	O
+in	O
+this	O
+metastable	O
+conformation	O
+.	O
+	O
+Crucially	O
+,	O
+though	O
+,	O
+the	O
+free	O
+-	O
+energy	O
+landscape	O
+for	O
+this	O
+partially	O
+occupied	O
+state	O
+demonstrates	O
+that	O
+the	O
+occluded	O
+conformation	O
+is	O
+no	O
+longer	O
+energetically	O
+feasible	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+	O
+Displacement	O
+of	O
+the	O
+two	O
+remaining	O
+Na	O
++	O
+ions	O
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+SCa	O
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+Sint	O
+further	O
+reshapes	O
+the	O
+free	O
+-	O
+energy	O
+landscape	O
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+the	O
+transporter	O
+(	O
+No	O
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+,	O
+Fig	O
+.	O
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+),	O
+which	O
+now	O
+can	O
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+a	O
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+featuring	O
+the	O
+two	O
+aqueous	O
+channels	O
+(	O
+Fig	O
+.	O
+5b	O
+-	O
+c	O
+).	O
+	O
+The	O
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+to	O
+the	O
+occluded	O
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+in	O
+this	O
+apo	O
+state	O
+is	O
+again	O
+energetically	O
+unfeasible	O
+.	O
+	O
+From	O
+a	O
+mechanistic	O
+standpoint	O
+,	O
+it	O
+is	O
+satisfying	O
+to	O
+observe	O
+how	O
+the	O
+open	O
+and	O
+semi	O
+-	O
+open	O
+states	O
+are	O
+each	O
+compatible	O
+with	O
+two	O
+different	O
+Na	O
++	O
+occupancies	O
+,	O
+explaining	O
+how	O
+sequential	O
+Na	O
++	O
+binding	O
+to	O
+energetically	O
+accessible	O
+conformations	O
+(	O
+prior	O
+to	O
+those	O
+binding	O
+events	O
+)	O
+progressively	O
+reshape	O
+the	O
+free	O
+-	O
+energy	O
+landscape	O
+of	O
+the	O
+transporter	O
+;	O
+by	O
+contrast	O
+,	O
+the	O
+occluded	O
+conformation	O
+is	O
+forbidden	O
+unless	O
+the	O
+Na	O
++	O
+occupancy	O
+is	O
+complete	O
+.	O
+	O
+To	O
+assess	O
+this	O
+hypothesis	O
+,	O
+we	O
+carried	O
+out	O
+enhanced	O
+-	O
+sampling	O
+simulations	O
+for	O
+the	O
+Ca2	O
++	O
+and	O
+H	O
++-	O
+bound	O
+states	O
+of	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+analogous	O
+to	O
+those	O
+described	O
+above	O
+for	O
+Na	O
++	O
+(	O
+see	O
+Supplementary	O
+Note	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+-	O
+4	O
+for	O
+details	O
+on	O
+how	O
+the	O
+structures	O
+of	O
+the	O
+Ca2	O
++-	O
+bound	O
+state	O
+was	O
+predicted	O
+).	O
+	O
+The	O
+calculated	O
+free	O
+-	O
+energy	O
+landscape	O
+for	O
+Ca2	O
++-	O
+bound	O
+NCX_Mj	O
+confirms	O
+the	O
+hypothesis	O
+outlined	O
+above	O
+(	O
+1	O
+×	O
+Ca2	O
++,	O
+Fig	O
+.	O
+6a	O
+):	O
+consistent	O
+with	O
+the	O
+fact	O
+that	O
+NCX_Mj	O
+transports	O
+a	O
+single	O
+Ca2	O
++,	O
+the	O
+occluded	O
+,	O
+dehydrated	O
+conformation	O
+is	O
+one	O
+of	O
+the	O
+major	O
+energetic	O
+minima	O
+,	O
+but	O
+clearly	O
+the	O
+exchanger	O
+can	O
+also	O
+adopt	O
+the	O
+semi	O
+-	O
+open	O
+and	O
+open	O
+states	O
+that	O
+would	O
+be	O
+required	O
+for	O
+Ca2	O
++	O
+release	O
+and	O
+Na	O
++	O
+entry	O
+,	O
+via	O
+either	O
+of	O
+the	O
+aqueous	O
+access	O
+channels	O
+that	O
+lead	O
+to	O
+Sext	O
+and	O
+SCa	O
+(	O
+Fig	O
+.	O
+6b	O
+-	O
+c	O
+).	O
+	O
+By	O
+contrast	O
+,	O
+protonation	O
+of	O
+Glu54	O
+and	O
+Glu213	O
+makes	O
+the	O
+occluded	O
+conformation	O
+energetically	O
+unfeasible	O
+,	O
+consistent	O
+with	O
+the	O
+fact	O
+that	O
+NCX_Mj	O
+does	O
+not	O
+transport	O
+protons	O
+;	O
+in	O
+this	O
+H	O
++-	O
+bound	O
+state	O
+,	O
+though	O
+,	O
+the	O
+exchanger	O
+can	O
+adopt	O
+the	O
+semi	O
+-	O
+open	O
+conformation	O
+captured	O
+in	O
+the	O
+low	O
+pH	O
+,	O
+apo	O
+crystal	O
+structure	O
+(	O
+2	O
+×	O
+H	O
++,	O
+Fig	O
+.	O
+6a	O
+-	O
+c	O
+).	O
+	O
+Taken	O
+together	O
+,	O
+this	O
+systematic	O
+computational	O
+analysis	O
+of	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+clearly	O
+demonstrates	O
+that	O
+the	O
+alternating	O
+-	O
+access	O
+and	O
+ion	O
+-	O
+recognition	O
+mechanisms	O
+in	O
+this	O
+Na	O
++/	O
+Ca2	O
++	O
+exchanger	O
+are	O
+coupled	O
+through	O
+the	O
+influence	O
+that	O
+the	O
+bound	O
+ions	O
+have	O
+on	O
+the	O
+free	O
+-	O
+energy	O
+landscape	O
+of	O
+the	O
+protein	O
+,	O
+which	O
+in	O
+turn	O
+determines	O
+whether	O
+or	O
+not	O
+the	O
+occluded	O
+conformation	O
+is	O
+energetically	O
+feasible	O
+.	O
+	O
+The	O
+alternating	O
+-	O
+access	O
+hypothesis	O
+implicitly	O
+dictates	O
+that	O
+the	O
+switch	O
+between	O
+outward	O
+-	O
+and	O
+inward	O
+-	O
+open	O
+conformations	O
+of	O
+a	O
+given	O
+secondary	O
+-	O
+active	O
+transporter	O
+must	O
+not	O
+occur	O
+unless	O
+the	O
+appropriate	O
+type	O
+and	O
+number	O
+of	O
+substrates	O
+are	O
+recognized	O
+.	O
+	O
+Yet	O
+,	O
+when	O
+multiple	O
+species	O
+are	O
+to	O
+be	O
+co	O
+-	O
+translocated	O
+,	O
+by	O
+either	O
+an	O
+antiporter	O
+or	O
+a	O
+symporter	O
+,	O
+partial	O
+occupancies	O
+must	O
+not	O
+be	O
+conducive	O
+to	O
+the	O
+alternating	O
+-	O
+access	O
+switch	O
+.	O
+	O
+Here	O
+,	O
+we	O
+have	O
+provided	O
+novel	O
+insights	O
+into	O
+this	O
+intriguing	O
+mechanism	O
+of	O
+conformational	O
+control	O
+through	O
+structural	O
+studies	O
+and	O
+quantitative	O
+molecular	O
+simulations	O
+of	O
+a	O
+Na	O
++/	O
+Ca2	O
++	O
+exchanger	O
+.	O
+	O
+Specifically	O
+,	O
+our	O
+studies	O
+of	O
+NCX_Mj	O
+reveal	O
+the	O
+mechanism	O
+of	O
+forward	O
+ion	O
+exchange	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+	O
+Here	O
+,	O
+we	O
+demonstrate	O
+that	O
+conformational	O
+changes	O
+in	O
+the	O
+extracellular	O
+region	O
+of	O
+the	O
+TM2	O
+-	O
+TM3	O
+and	O
+TM7	O
+-	O
+TM8	O
+bundle	O
+precede	O
+and	O
+are	O
+necessary	O
+for	O
+the	O
+transition	O
+,	O
+and	O
+are	O
+associated	O
+with	O
+ion	O
+recognition	O
+and	O
+/	O
+or	O
+release	O
+.	O
+	O
+Interestingly	O
+,	O
+the	O
+bending	O
+of	O
+TM7	O
+associated	O
+with	O
+the	O
+occlusion	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+sites	O
+also	O
+unlocks	O
+its	O
+interaction	O
+with	O
+TM6	O
+,	O
+and	O
+thus	O
+enables	O
+TM6	O
+and	O
+TM1	O
+to	O
+freely	O
+slide	O
+to	O
+the	O
+inward	O
+-	O
+facing	O
+conformation	O
+.	O
+	O
+The	O
+crystal	O
+structures	O
+of	O
+NCX_Mj	O
+reported	O
+here	O
+,	O
+with	O
+either	O
+Na	O
++,	O
+Ca2	O
++,	O
+Sr2	O
++	O
+or	O
+H	O
++	O
+bound	O
+,	O
+capture	O
+the	O
+exchanger	O
+in	O
+different	O
+conformational	O
+states	O
+.	O
+	O
+These	O
+states	O
+can	O
+only	O
+represent	O
+a	O
+subset	O
+among	O
+all	O
+possible	O
+,	O
+but	O
+they	O
+ought	O
+to	O
+reflect	O
+inherent	O
+preferences	O
+of	O
+the	O
+transporter	O
+,	O
+modulated	O
+by	O
+the	O
+experimental	O
+conditions	O
+.	O
+	O
+For	O
+example	O
+,	O
+in	O
+the	O
+crystal	O
+of	O
+NCX_Mj	O
+in	O
+LCP	O
+,	O
+the	O
+extracellular	O
+half	O
+of	O
+the	O
+gating	O
+helices	O
+(	O
+TM6	O
+and	O
+TM1	O
+)	O
+form	O
+a	O
+lattice	O
+contact	O
+,	O
+which	O
+might	O
+ultimately	O
+restrict	O
+the	O
+degree	O
+of	O
+opening	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+sites	O
+in	O
+some	O
+cases	O
+(	O
+e	O
+.	O
+g	O
+.	O
+in	O
+the	O
+apo	O
+,	O
+low	O
+pH	O
+structure	O
+).	O
+	O
+Nonetheless	O
+,	O
+the	O
+calculated	O
+free	O
+-	O
+energy	O
+landscapes	O
+,	O
+derived	O
+without	O
+knowledge	O
+of	O
+the	O
+experimental	O
+data	O
+,	O
+reassuringly	O
+confirm	O
+that	O
+the	O
+crystallized	O
+structures	O
+correspond	O
+to	O
+mechanistically	O
+relevant	O
+,	O
+interconverting	O
+states	O
+.	O
+	O
+The	O
+simulations	O
+also	O
+demonstrate	O
+how	O
+this	O
+landscape	O
+is	O
+drastically	O
+re	O
+-	O
+shaped	O
+upon	O
+each	O
+ion	O
+-	O
+binding	O
+event	O
+.	O
+	O
+We	O
+posit	O
+that	O
+a	O
+similar	O
+principle	O
+might	O
+govern	O
+the	O
+alternating	O
+-	O
+access	O
+mechanism	O
+in	O
+other	O
+transporters	O
+;	O
+that	O
+is	O
+,	O
+we	O
+anticipate	O
+that	O
+for	O
+both	O
+symporters	O
+and	O
+antiporters	O
+,	O
+it	O
+is	O
+the	O
+feasibility	O
+of	O
+the	O
+occluded	O
+state	O
+,	O
+encoded	O
+in	O
+the	O
+protein	O
+conformational	O
+free	O
+-	O
+energy	O
+landscape	O
+and	O
+its	O
+dependence	O
+on	O
+substrate	O
+binding	O
+,	O
+that	O
+ultimately	O
+explains	O
+their	O
+specific	O
+coupling	O
+mechanisms	O
+.	O
+	O
+In	O
+multiple	O
+ways	O
+,	O
+our	O
+findings	O
+provide	O
+an	O
+explanation	O
+for	O
+,	O
+existing	O
+functional	O
+,	O
+biochemical	O
+and	O
+biophysical	O
+data	O
+for	O
+both	O
+NCX_Mj	O
+and	O
+its	O
+eukaryotic	O
+homologues	O
+.	O
+	O
+The	O
+crystallographic	O
+data	O
+also	O
+provides	O
+the	O
+long	O
+-	O
+sought	O
+structural	O
+basis	O
+for	O
+the	O
+‘	O
+two	O
+-	O
+site	O
+’	O
+model	O
+proposed	O
+to	O
+describe	O
+competitive	O
+cation	O
+binding	O
+in	O
+eukaryotic	O
+NCX	O
+,	O
+underscoring	O
+the	O
+relevance	O
+of	O
+these	O
+studies	O
+of	O
+NCX_Mj	O
+as	O
+a	O
+prototypical	O
+Na	O
++/	O
+Ca2	O
++	O
+exchanger	O
+.	O
+	O
+Interestingly	O
+,	O
+binding	O
+of	O
+Ca2	O
++	O
+to	O
+Smid	O
+appears	O
+to	O
+be	O
+also	O
+possible	O
+,	O
+but	O
+available	O
+evidence	O
+indicates	O
+that	O
+this	O
+event	O
+transiently	O
+blocks	O
+the	O
+exchange	O
+cycle	O
+.	O
+	O
+Indeed	O
+,	O
+structures	O
+of	O
+NCX_Mj	O
+bound	O
+to	O
+Cd2	O
++	O
+or	O
+Mn2	O
++,	O
+both	O
+of	O
+which	O
+inhibit	O
+transport	O
+,	O
+show	O
+these	O
+ions	O
+at	O
+Smid	O
+;	O
+by	O
+contrast	O
+,	O
+Sr2	O
++	O
+binds	O
+only	O
+to	O
+SCa	O
+,	O
+and	O
+accordingly	O
+,	O
+is	O
+transported	O
+by	O
+NCX	O
+similarly	O
+to	O
+calcium	O
+.	O
+	O
+In	O
+addition	O
+,	O
+the	O
+increased	O
+compactness	O
+of	O
+the	O
+protein	O
+tertiary	O
+structure	O
+in	O
+the	O
+occluded	O
+state	O
+would	O
+also	O
+slow	O
+down	O
+the	O
+dynamics	O
+of	O
+the	O
+secondary	O
+-	O
+structure	O
+elements	O
+,	O
+and	O
+thus	O
+further	O
+reduce	O
+the	O
+HDX	O
+rate	O
+.	O
+	O
+As	O
+the	O
+calculated	O
+free	O
+-	O
+energy	O
+landscapes	O
+show	O
+,	O
+Na	O
++	O
+and	O
+Ca2	O
++	O
+induce	O
+the	O
+occlusion	O
+of	O
+the	O
+transporter	O
+in	O
+a	O
+comparable	O
+manner	O
+,	O
+and	O
+yet	O
+the	O
+ion	O
+-	O
+bound	O
+states	O
+retain	O
+the	O
+ability	O
+to	O
+explore	O
+conformations	O
+that	O
+are	O
+partially	O
+or	O
+fully	O
+open	O
+to	O
+the	O
+extracellular	O
+solution	O
+,	O
+precisely	O
+so	O
+as	O
+to	O
+be	O
+able	O
+to	O
+unload	O
+and	O
+re	O
+-	O
+load	O
+the	O
+substrates	O
+.	O
+	O
+Na	O
++	O
+binding	O
+to	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+.	O
+	O
+(	O
+a	O
+)	O
+Overall	O
+structure	O
+of	O
+native	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+from	O
+crystals	O
+grown	O
+in	O
+150	O
+mM	O
+Na	O
++.	O
+	O
+Colored	O
+spheres	O
+represent	O
+the	O
+bound	O
+Na	O
++	O
+(	O
+green	O
+)	O
+and	O
+water	O
+(	O
+red	O
+).	O
+	O
+(	O
+b	O
+)	O
+Structural	O
+details	O
+and	O
+definition	O
+of	O
+the	O
+four	O
+central	O
+binding	O
+sites	O
+.	O
+	O
+The	O
+electron	O
+density	O
+(	O
+grey	O
+mesh	O
+,	O
+1	O
+.	O
+9	O
+Å	O
+Fo	O
+-	O
+Fc	O
+ion	O
+omit	O
+map	O
+contoured	O
+at	O
+4σ	O
+)	O
+at	O
+Smid	O
+was	O
+modeled	O
+as	O
+water	O
+(	O
+red	O
+sphere	O
+)	O
+and	O
+those	O
+at	O
+Sext	O
+,	O
+SCa	O
+and	O
+Sint	O
+as	O
+Na	O
++	O
+ions	O
+(	O
+green	O
+spheres	O
+).	O
+	O
+Further	O
+details	O
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+.	O
+(	O
+c	O
+)	O
+Concentration	O
+-	O
+dependent	O
+change	O
+in	O
+Na	O
++	O
+occupancy	O
+(	O
+see	O
+also	O
+Table	O
+1	O
+).	O
+	O
+All	O
+Fo	O
+–	O
+Fc	O
+ion	O
+-	O
+omit	O
+maps	O
+are	O
+calculated	O
+to	O
+2	O
+.	O
+4	O
+Å	O
+and	O
+contoured	O
+at	O
+3σ	O
+for	O
+comparison	O
+.	O
+	O
+The	O
+displacement	O
+of	O
+A206	O
+reflects	O
+the	O
+[	O
+Na	O
++]-	O
+dependent	O
+conformational	O
+change	O
+from	O
+the	O
+partially	O
+open	O
+to	O
+the	O
+occluded	O
+state	O
+(	O
+observed	O
+at	O
+low	O
+and	O
+high	O
+Na	O
++	O
+concentrations	O
+,	O
+respectively	O
+).	O
+	O
+At	O
+20	O
+mM	O
+Na	O
++,	O
+both	O
+conformations	O
+co	O
+-	O
+exist	O
+.	O
+	O
+Na	O
++-	O
+occupancy	O
+dependent	O
+conformational	O
+change	O
+in	O
+NCX_Mj	O
+.	O
+	O
+(	O
+a	O
+)	O
+Superimposition	O
+of	O
+the	O
+NCX_Mj	O
+crystal	O
+structures	O
+obtained	O
+in	O
+high	O
+(	O
+100	O
+mM	O
+,	O
+cyan	O
+cylinders	O
+)	O
+and	O
+low	O
+(	O
+10	O
+mM	O
+,	O
+brown	O
+cylinders	O
+)	O
+Na	O
++	O
+concentrations	O
+.	O
+	O
+(	O
+b	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+interface	O
+between	O
+TM6	O
+and	O
+TM7ab	O
+in	O
+the	O
+NCX_Mj	O
+structures	O
+obtained	O
+at	O
+high	O
+and	O
+low	O
+Na	O
++	O
+concentrations	O
+(	O
+top	O
+and	O
+lower	O
+panels	O
+,	O
+respectively	O
+).	O
+	O
+Residues	O
+forming	O
+van	O
+-	O
+der	O
+-	O
+Waals	O
+contacts	O
+in	O
+the	O
+structure	O
+at	O
+low	O
+Na	O
++	O
+concentration	O
+are	O
+shown	O
+in	O
+detail	O
+.	O
+	O
+T50	O
+and	O
+T209	O
+(	O
+labeled	O
+in	O
+red	O
+)	O
+coordinate	O
+Sr2	O
++	O
+through	O
+their	O
+backbone	O
+carbonyl	O
+-	O
+oxygen	O
+atoms	O
+.	O
+	O
+High	O
+Na	O
++	O
+concentration	O
+(	O
+100	O
+mM	O
+)	O
+completely	O
+displaces	O
+Sr2	O
++	O
+and	O
+reverts	O
+NCX_Mj	O
+to	O
+the	O
+occluded	O
+state	O
+(	O
+right	O
+panel	O
+).	O
+	O
+The	O
+contour	O
+level	O
+of	O
+the	O
+Fo	O
+–	O
+Fc	O
+omit	O
+map	O
+of	O
+the	O
+structure	O
+at	O
+high	O
+Na	O
++	O
+concentration	O
+was	O
+lowered	O
+(	O
+to	O
+4σ	O
+)	O
+so	O
+as	O
+to	O
+visualize	O
+the	O
+density	O
+from	O
+Na	O
++	O
+ions	O
+and	O
+H2O	O
+.	O
+	O
+The	O
+side	O
+chains	O
+of	O
+E54	O
+and	O
+E213	O
+from	O
+the	O
+low	O
+Na	O
++	O
+structure	O
+are	O
+also	O
+shown	O
+(	O
+light	O
+brown	O
+)	O
+for	O
+comparison	O
+.	O
+	O
+White	O
+spheres	O
+indicate	O
+the	O
+location	O
+Sint	O
+,	O
+Smid	O
+SCa	O
+.	O
+(	O
+e	O
+)	O
+Extracellular	O
+solvent	O
+accessibility	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+sites	O
+in	O
+apo	O
+NCX_Mj	O
+.	O
+	O
+Spontaneous	O
+changes	O
+in	O
+the	O
+structure	O
+of	O
+outward	O
+-	O
+occluded	O
+,	O
+fully	O
+Na	O
++-	O
+occupied	O
+NCX_Mj	O
+(	O
+PDB	O
+code	O
+3V5U	O
+)	O
+upon	O
+sequential	O
+displacement	O
+of	O
+Na	O
++.	O
+	O
+(	O
+c	O
+)	O
+Representative	O
+simulation	O
+snapshots	O
+(	O
+same	O
+as	O
+above	O
+)	O
+with	O
+Na	O
++	O
+bound	O
+at	O
+SCa	O
+and	O
+Sint	O
+(	O
+marine	O
+cartoons	O
+,	O
+yellow	O
+spheres	O
+)	O
+and	O
+without	O
+any	O
+Na	O
++	O
+bound	O
+(	O
+grey	O
+cartoons	O
+).	O
+	O
+(	O
+d	O
+)	O
+Close	O
+-	O
+up	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+region	O
+in	O
+the	O
+fully	O
+Na	O
++-	O
+occupied	O
+state	O
+.	O
+	O
+(	O
+f	O
+)	O
+Close	O
+-	O
+up	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+region	O
+in	O
+the	O
+Na	O
++-	O
+free	O
+state	O
+.	O
+(	O
+g	O
+-	O
+i	O
+)	O
+Analytical	O
+descriptors	O
+of	O
+the	O
+changes	O
+just	O
+described	O
+,	O
+calculated	O
+from	O
+the	O
+simulations	O
+of	O
+each	O
+Na	O
++-	O
+occupancy	O
+state	O
+depicted	O
+in	O
+panels	O
+(	O
+a	O
+-	O
+f	O
+).	O
+	O
+These	O
+descriptors	O
+were	O
+employed	O
+as	O
+collective	O
+variables	O
+in	O
+the	O
+Bias	O
+-	O
+Exchange	O
+Metadynamics	O
+simulations	O
+(	O
+Methods	O
+).	O
+	O
+Thermodynamic	O
+basis	O
+for	O
+the	O
+proposed	O
+mechanism	O
+of	O
+substrate	O
+control	O
+of	O
+the	O
+alternating	O
+-	O
+access	O
+transition	O
+of	O
+NCX	O
+.	O
+(	O
+a	O
+)	O
+Calculated	O
+conformational	O
+free	O
+-	O
+energy	O
+landscapes	O
+for	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+,	O
+for	O
+two	O
+different	O
+Na	O
++-	O
+occupancy	O
+states	O
+,	O
+and	O
+for	O
+a	O
+state	O
+with	O
+no	O
+ions	O
+bound	O
+.	O
+	O
+Black	O
+circles	O
+map	O
+the	O
+X	O
+-	O
+ray	O
+structures	O
+of	O
+NCX_Mj	O
+obtained	O
+at	O
+high	O
+and	O
+low	O
+Na	O
++	O
+concentration	O
+,	O
+as	O
+well	O
+as	O
+that	O
+at	O
+low	O
+pH	O
+,	O
+reported	O
+in	O
+this	O
+study	O
+.	O
+	O
+The	O
+two	O
+inverted	O
+-	O
+topology	O
+repeats	O
+in	O
+the	O
+transporter	O
+structure	O
+(	O
+transparent	O
+cartoons	O
+)	O
+are	O
+colored	O
+differently	O
+(	O
+TM1	O
+-	O
+5	O
+,	O
+orange	O
+;	O
+TM6	O
+-	O
+10	O
+,	O
+marine	O
+).	O
+	O
+(	O
+c	O
+)	O
+Close	O
+-	O
+up	O
+views	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+region	O
+in	O
+the	O
+same	O
+conformational	O
+free	O
+-	O
+energy	O
+minima	O
+.	O
+	O
+Key	O
+residues	O
+involved	O
+in	O
+Na	O
++	O
+and	O
+water	O
+coordination	O
+(	O
+W	O
+)	O
+are	O
+highlighted	O
+(	O
+sticks	O
+,	O
+black	O
+lines	O
+).	O
+	O
+The	O
+water	O
+-	O
+density	O
+maps	O
+in	O
+(	O
+b	O
+)	O
+is	O
+shown	O
+here	O
+as	O
+a	O
+grey	O
+mesh	O
+.	O
+	O
+Note	O
+D240	O
+is	O
+protonated	O
+,	O
+while	O
+E54	O
+and	O
+E213	O
+are	O
+ionized	O
+.	O
+	O
+Thermodynamic	O
+basis	O
+for	O
+the	O
+proposed	O
+mechanism	O
+of	O
+substrate	O
+control	O
+of	O
+the	O
+alternating	O
+-	O
+access	O
+transition	O
+of	O
+NCX	O
+.	O
+(	O
+a	O
+)	O
+Calculated	O
+free	O
+-	O
+energy	O
+landscapes	O
+for	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+,	O
+for	O
+the	O
+Ca2	O
++	O
+and	O
+the	O
+fully	O
+protonated	O
+state	O
+.	O
+	O
+The	O
+free	O
+energy	O
+is	O
+plotted	O
+as	O
+in	O
+Fig	O
+.	O
+5	O
+.	O
+	O
+For	O
+Ca2	O
++,	O
+a	O
+map	O
+is	O
+shown	O
+in	O
+which	O
+a	O
+correction	O
+for	O
+the	O
+charge	O
+-	O
+transfer	O
+between	O
+the	O
+ion	O
+and	O
+the	O
+protein	O
+is	O
+introduced	O
+,	O
+alongside	O
+the	O
+uncorrected	O
+map	O
+(	O
+see	O
+Supplementary	O
+Notes	O
+3	O
+-	O
+4	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+-	O
+6	O
+).	O
+	O
+The	O
+uncorrected	O
+map	O
+overstabilizes	O
+the	O
+open	O
+state	O
+relative	O
+to	O
+the	O
+semi	O
+-	O
+open	O
+and	O
+occluded	O
+because	O
+it	O
+also	O
+overestimates	O
+the	O
+cost	O
+of	O
+dehydration	O
+of	O
+the	O
+ion	O
+,	O
+once	O
+it	O
+is	O
+bound	O
+to	O
+the	O
+protein	O
+(	O
+this	O
+effect	O
+is	O
+negligible	O
+for	O
+Na	O
++).	O
+	O
+The	O
+Ca2	O
++	O
+ion	O
+is	O
+shown	O
+as	O
+a	O
+red	O
+sphere	O
+;	O
+the	O
+protein	O
+is	O
+shown	O
+as	O
+in	O
+Fig	O
+.	O
+5	O
+.	O
+(	O
+c	O
+)	O
+Close	O
+-	O
+up	O
+views	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+region	O
+in	O
+the	O
+same	O
+conformational	O
+free	O
+-	O
+energy	O
+minima	O
+.	O
+	O
+Key	O
+residues	O
+involved	O
+in	O
+Ca2	O
++	O
+and	O
+water	O
+coordination	O
+(	O
+W	O
+)	O
+are	O
+highlighted	O
+(	O
+sticks	O
+,	O
+black	O
+lines	O
+).	O
+	O
+In	O
+the	O
+occluded	O
+state	O
+with	O
+Ca2	O
++	O
+bound	O
+,	O
+helix	O
+TM7ab	O
+bends	O
+in	O
+the	O
+same	O
+way	O
+as	O
+in	O
+the	O
+fully	O
+occupied	O
+Na	O
++	O
+state	O
+,	O
+as	O
+the	O
+carbonyl	O
+of	O
+Ala206	O
+forms	O
+a	O
+hydrogen	O
+-	O
+bonding	O
+interaction	O
+with	O
+Ser210	O
+.	O
+	O
+Structural	O
+mechanism	O
+of	O
+extracellular	O
+forward	O
+ion	O
+exchange	O
+in	O
+NCX	O
+.	O
+	O
+The	O
+carbonyl	O
+groups	O
+of	O
+Ala47	O
+(	O
+on	O
+TM2b	O
+)	O
+and	O
+Ala206	O
+(	O
+on	O
+TM7b	O
+),	O
+and	O
+the	O
+side	O
+chains	O
+of	O
+Glu54	O
+(	O
+on	O
+TM2c	O
+)	O
+and	O
+Glu213	O
+(	O
+on	O
+TM7c	O
+)	O
+are	O
+highlighted	O
+;	O
+these	O
+are	O
+four	O
+of	O
+the	O
+key	O
+residues	O
+for	O
+ion	O
+chelation	O
+and	O
+conformational	O
+changes	O
+.	O
+	O
+The	O
+green	O
+open	O
+cylinders	O
+represent	O
+the	O
+gating	O
+helices	O
+TM1	O
+and	O
+TM6	O
+.	O
+	O
+Asterisks	O
+mark	O
+the	O
+states	O
+whose	O
+crystal	O
+structures	O
+have	O
+been	O
+determined	O
+in	O
+this	O
+study	O
+.	O
+	O
+These	O
+states	O
+and	O
+their	O
+connectivity	O
+can	O
+also	O
+be	O
+deduced	O
+from	O
+the	O
+calculated	O
+free	O
+-	O
+energy	O
+landscapes	O
+,	O
+which	O
+also	O
+reveal	O
+a	O
+Ca2	O
++-	O
+loaded	O
+outward	O
+-	O
+facing	O
+occluded	O
+state	O
+,	O
+and	O
+an	O
+unloaded	O
+,	O
+fully	O
+open	O
+state	O
+.	O
+	O
+How	O
+the	O
+essential	O
+pre	O
+-	O
+mRNA	O
+splicing	O
+factor	O
+U2AF65	O
+recognizes	O
+the	O
+polypyrimidine	O
+(	O
+Py	O
+)	O
+signals	O
+of	O
+the	O
+major	O
+class	O
+of	O
+3	O
+′	O
+splice	O
+sites	O
+in	O
+human	O
+gene	O
+transcripts	O
+remains	O
+incompletely	O
+understood	O
+.	O
+	O
+We	O
+determined	O
+four	O
+structures	O
+of	O
+an	O
+extended	O
+U2AF65	O
+–	O
+RNA	O
+-	O
+binding	O
+domain	O
+bound	O
+to	O
+Py	O
+-	O
+tract	O
+oligonucleotides	O
+at	O
+resolutions	O
+between	O
+2	O
+.	O
+0	O
+and	O
+1	O
+.	O
+5	O
+Å	O
+.	O
+These	O
+structures	O
+together	O
+with	O
+RNA	O
+binding	O
+and	O
+splicing	O
+assays	O
+reveal	O
+unforeseen	O
+roles	O
+for	O
+U2AF65	O
+inter	O
+-	O
+domain	O
+residues	O
+in	O
+recognizing	O
+a	O
+contiguous	O
+,	O
+nine	O
+-	O
+nucleotide	O
+Py	O
+tract	O
+.	O
+	O
+The	O
+U2AF65	O
+linker	O
+residues	O
+between	O
+the	O
+dual	O
+RNA	O
+recognition	O
+motifs	O
+(	O
+RRMs	O
+)	O
+recognize	O
+the	O
+central	O
+nucleotide	O
+,	O
+whereas	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+RRM	O
+extensions	O
+recognize	O
+the	O
+3	O
+′	O
+terminus	O
+and	O
+third	O
+nucleotide	O
+.	O
+	O
+Single	O
+-	O
+molecule	O
+FRET	O
+experiments	O
+suggest	O
+that	O
+conformational	O
+selection	O
+and	O
+induced	O
+fit	O
+of	O
+the	O
+U2AF65	O
+RRMs	O
+are	O
+complementary	O
+mechanisms	O
+for	O
+Py	O
+-	O
+tract	O
+association	O
+.	O
+	O
+Altogether	O
+,	O
+these	O
+results	O
+advance	O
+the	O
+mechanistic	O
+understanding	O
+of	O
+molecular	O
+recognition	O
+for	O
+a	O
+major	O
+class	O
+of	O
+splice	O
+site	O
+signals	O
+.	O
+	O
+The	O
+pre	O
+-	O
+mRNA	O
+splicing	O
+factor	O
+U2AF65	O
+recognizes	O
+3	O
+′	O
+splice	O
+sites	O
+in	O
+human	O
+gene	O
+transcripts	O
+,	O
+but	O
+the	O
+details	O
+are	O
+not	O
+fully	O
+understood	O
+.	O
+	O
+The	O
+differential	O
+skipping	O
+or	O
+inclusion	O
+of	O
+alternatively	O
+spliced	O
+pre	O
+-	O
+mRNA	O
+regions	O
+is	O
+a	O
+major	O
+source	O
+of	O
+diversity	O
+for	O
+nearly	O
+all	O
+human	O
+gene	O
+transcripts	O
+.	O
+	O
+At	O
+the	O
+3	O
+′	O
+splice	O
+site	O
+of	O
+the	O
+major	O
+intron	O
+class	O
+,	O
+these	O
+include	O
+a	O
+polypyrimidine	O
+(	O
+Py	O
+)	O
+tract	O
+comprising	O
+primarily	O
+Us	O
+or	O
+Cs	O
+,	O
+which	O
+is	O
+preceded	O
+by	O
+a	O
+branch	O
+point	O
+sequence	O
+(	O
+BPS	O
+)	O
+that	O
+ultimately	O
+serves	O
+as	O
+the	O
+nucleophile	O
+in	O
+the	O
+splicing	O
+reaction	O
+and	O
+an	O
+AG	O
+-	O
+dinucleotide	O
+at	O
+the	O
+3	O
+′	O
+splice	O
+site	O
+junction	O
+.	O
+	O
+Disease	O
+-	O
+causing	O
+mutations	O
+often	O
+compromise	O
+pre	O
+-	O
+mRNA	O
+splicing	O
+(	O
+reviewed	O
+in	O
+refs	O
+),	O
+yet	O
+a	O
+priori	O
+predictions	O
+of	O
+splice	O
+sites	O
+and	O
+the	O
+consequences	O
+of	O
+their	O
+mutations	O
+are	O
+challenged	O
+by	O
+the	O
+brevity	O
+and	O
+degeneracy	O
+of	O
+known	O
+splice	O
+site	O
+sequences	O
+.	O
+	O
+High	O
+-	O
+resolution	O
+structures	O
+of	O
+intact	O
+splicing	O
+factor	O
+–	O
+RNA	O
+complexes	O
+would	O
+offer	O
+key	O
+insights	O
+regarding	O
+the	O
+juxtaposition	O
+of	O
+the	O
+distinct	O
+splice	O
+site	O
+consensus	O
+sequences	O
+and	O
+their	O
+relationship	O
+to	O
+disease	O
+-	O
+causing	O
+point	O
+mutations	O
+.	O
+	O
+The	O
+early	O
+-	O
+stage	O
+pre	O
+-	O
+mRNA	O
+splicing	O
+factor	O
+U2AF65	O
+is	O
+essential	O
+for	O
+viability	O
+in	O
+vertebrates	O
+and	O
+other	O
+model	O
+organisms	O
+(	O
+for	O
+example	O
+,	O
+ref	O
+.).	O
+	O
+A	O
+tightly	O
+controlled	O
+assembly	O
+among	O
+U2AF65	O
+,	O
+the	O
+pre	O
+-	O
+mRNA	O
+,	O
+and	O
+partner	O
+proteins	O
+sequentially	O
+identifies	O
+the	O
+3	O
+′	O
+splice	O
+site	O
+and	O
+promotes	O
+association	O
+of	O
+the	O
+spliceosome	O
+,	O
+which	O
+ultimately	O
+accomplishes	O
+the	O
+task	O
+of	O
+splicing	O
+.	O
+	O
+Subsequently	O
+U2AF65	O
+recruits	O
+the	O
+U2	O
+small	O
+nuclear	O
+ribonucleoprotein	O
+particle	O
+(	O
+snRNP	O
+)	O
+and	O
+ultimately	O
+dissociates	O
+from	O
+the	O
+active	O
+spliceosome	O
+.	O
+	O
+Biochemical	O
+characterizations	O
+of	O
+U2AF65	O
+demonstrated	O
+that	O
+tandem	O
+RNA	O
+recognition	O
+motifs	O
+(	O
+RRM1	O
+and	O
+RRM2	O
+)	O
+recognize	O
+the	O
+Py	O
+tract	O
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+	O
+Milestone	O
+crystal	O
+structures	O
+of	O
+the	O
+core	O
+U2AF65	O
+RRM1	O
+and	O
+RRM2	O
+connected	O
+by	O
+a	O
+shortened	O
+inter	O
+-	O
+RRM	O
+linker	O
+(	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+)	O
+detailed	O
+a	O
+subset	O
+of	O
+nucleotide	O
+interactions	O
+with	O
+the	O
+individual	O
+U2AF65	O
+RRMs	O
+.	O
+	O
+A	O
+subsequent	O
+NMR	O
+structure	O
+characterized	O
+the	O
+side	O
+-	O
+by	O
+-	O
+side	O
+arrangement	O
+of	O
+the	O
+minimal	O
+U2AF65	O
+RRM1	O
+and	O
+RRM2	O
+connected	O
+by	O
+a	O
+linker	O
+of	O
+natural	O
+length	O
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+),	O
+yet	O
+depended	O
+on	O
+the	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+crystal	O
+structures	O
+for	O
+RNA	O
+interactions	O
+and	O
+an	O
+ab	O
+initio	O
+model	O
+for	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+conformation	O
+.	O
+	O
+Here	O
+,	O
+we	O
+use	O
+X	O
+-	O
+ray	O
+crystallography	O
+and	O
+biochemical	O
+studies	O
+to	O
+reveal	O
+new	O
+roles	O
+in	O
+Py	O
+-	O
+tract	O
+recognition	O
+for	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+and	O
+key	O
+residues	O
+surrounding	O
+the	O
+core	O
+U2AF65	O
+RRMs	O
+.	O
+	O
+Cognate	O
+U2AF65	O
+–	O
+Py	O
+-	O
+tract	O
+recognition	O
+requires	O
+RRM	O
+extensions	O
+	O
+The	O
+RNA	O
+affinity	O
+of	O
+the	O
+minimal	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+domain	O
+comprising	O
+the	O
+core	O
+RRM1	O
+–	O
+RRM2	O
+folds	O
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+,	O
+residues	O
+148	O
+–	O
+336	O
+)	O
+is	O
+relatively	O
+weak	O
+compared	O
+with	O
+full	O
+-	O
+length	O
+U2AF65	O
+(	O
+Fig	O
+.	O
+1a	O
+,	O
+b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+Historically	O
+,	O
+this	O
+difference	O
+was	O
+attributed	O
+to	O
+the	O
+U2AF65	O
+arginine	O
+–	O
+serine	O
+rich	O
+domain	O
+,	O
+which	O
+contacts	O
+pre	O
+-	O
+mRNA	O
+–	O
+U2	O
+snRNA	O
+duplexes	O
+outside	O
+of	O
+the	O
+Py	O
+tract	O
+.	O
+	O
+We	O
+noticed	O
+that	O
+the	O
+RNA	O
+-	O
+binding	O
+affinity	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+domain	O
+was	O
+greatly	O
+enhanced	O
+by	O
+the	O
+addition	O
+of	O
+seven	O
+and	O
+six	O
+residues	O
+at	O
+the	O
+respective	O
+N	O
+and	O
+C	O
+termini	O
+of	O
+the	O
+minimal	O
+RRM1	O
+and	O
+RRM2	O
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+residues	O
+141	O
+–	O
+342	O
+;	O
+Fig	O
+.	O
+1a	O
+).	O
+	O
+In	O
+a	O
+fluorescence	O
+anisotropy	O
+assay	O
+for	O
+binding	O
+a	O
+representative	O
+Py	O
+tract	O
+derived	O
+from	O
+the	O
+well	O
+-	O
+characterized	O
+splice	O
+site	O
+of	O
+the	O
+adenovirus	O
+major	O
+late	O
+promoter	O
+(	O
+AdML	O
+),	O
+the	O
+RNA	O
+affinity	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+increased	O
+by	O
+100	O
+-	O
+fold	O
+relative	O
+to	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+to	O
+comparable	O
+levels	O
+as	O
+full	O
+-	O
+length	O
+U2AF65	O
+(	O
+Fig	O
+.	O
+1b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+–	O
+d	O
+).	O
+	O
+U2AF65	O
+-	O
+bound	O
+Py	O
+tract	O
+comprises	O
+nine	O
+contiguous	O
+nucleotides	O
+	O
+To	O
+investigate	O
+the	O
+structural	O
+basis	O
+for	O
+cognate	O
+U2AF65	O
+recognition	O
+of	O
+a	O
+contiguous	O
+Py	O
+tract	O
+,	O
+we	O
+determined	O
+four	O
+crystal	O
+structures	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+bound	O
+to	O
+Py	O
+-	O
+tract	O
+oligonucleotides	O
+(	O
+Fig	O
+.	O
+2a	O
+;	O
+Table	O
+1	O
+).	O
+	O
+The	O
+protein	O
+and	O
+oligonucleotide	O
+conformations	O
+are	O
+nearly	O
+identical	O
+among	O
+the	O
+four	O
+new	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+The	O
+extensions	O
+at	O
+the	O
+N	O
+terminus	O
+of	O
+RRM1	O
+and	O
+C	O
+terminus	O
+of	O
+RRM2	O
+adopt	O
+well	O
+-	O
+ordered	O
+α	O
+-	O
+helices	O
+.	O
+	O
+The	O
+discovery	O
+of	O
+nine	O
+U2AF65	O
+-	O
+binding	O
+sites	O
+for	O
+contiguous	O
+Py	O
+-	O
+tract	O
+nucleotides	O
+was	O
+unexpected	O
+.	O
+	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+characterize	O
+ribose	O
+(	O
+r	O
+)	O
+nucleotides	O
+at	O
+all	O
+of	O
+the	O
+binding	O
+sites	O
+except	O
+the	O
+seventh	O
+and	O
+eighth	O
+deoxy	O
+-(	O
+d	O
+)	O
+U	O
+,	O
+which	O
+are	O
+likely	O
+to	O
+lack	O
+2	O
+′-	O
+hydroxyl	O
+contacts	O
+based	O
+on	O
+the	O
+RNA	O
+-	O
+bound	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structure	O
+.	O
+	O
+In	O
+striking	O
+departures	O
+from	O
+prior	O
+partial	O
+views	O
+,	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+reveal	O
+three	O
+unanticipated	O
+nucleotide	O
+-	O
+binding	O
+sites	O
+at	O
+the	O
+centre	O
+of	O
+the	O
+Py	O
+tract	O
+,	O
+as	O
+well	O
+as	O
+numerous	O
+new	O
+interactions	O
+that	O
+underlie	O
+cognate	O
+recognition	O
+of	O
+the	O
+Py	O
+tract	O
+(	O
+Fig	O
+.	O
+3a	O
+–	O
+h	O
+).	O
+	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+interacts	O
+with	O
+the	O
+Py	O
+tract	O
+	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM2	O
+,	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+and	O
+RRM1	O
+concomitantly	O
+recognize	O
+the	O
+three	O
+central	O
+nucleotides	O
+of	O
+the	O
+Py	O
+tract	O
+,	O
+which	O
+are	O
+likely	O
+to	O
+coordinate	O
+the	O
+conformational	O
+arrangement	O
+of	O
+these	O
+disparate	O
+portions	O
+of	O
+the	O
+protein	O
+.	O
+	O
+Residues	O
+in	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+comprise	O
+a	O
+centrally	O
+located	O
+binding	O
+site	O
+for	O
+the	O
+fifth	O
+nucleotide	O
+on	O
+the	O
+RRM2	O
+surface	O
+and	O
+abutting	O
+the	O
+RRM1	O
+/	O
+RRM2	O
+interface	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+	O
+The	O
+backbone	O
+amide	O
+of	O
+the	O
+linker	O
+V254	O
+and	O
+the	O
+carbonyl	O
+of	O
+T252	O
+engage	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+rU5	O
+-	O
+O4	O
+and	O
+-	O
+N3H	O
+atoms	O
+.	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+also	O
+participates	O
+in	O
+the	O
+preceding	O
+rU4	O
+-	O
+binding	O
+site	O
+,	O
+where	O
+the	O
+V254	O
+backbone	O
+carbonyl	O
+and	O
+D256	O
+carboxylate	O
+position	O
+the	O
+K260	O
+side	O
+chain	O
+to	O
+hydrogen	O
+bond	O
+with	O
+the	O
+rU4	O
+-	O
+O4	O
+(	O
+Fig	O
+.	O
+3c	O
+).	O
+	O
+At	O
+the	O
+opposite	O
+side	O
+of	O
+the	O
+central	O
+fifth	O
+nucleotide	O
+,	O
+the	O
+sixth	O
+rU6	O
+nucleotide	O
+is	O
+located	O
+at	O
+the	O
+inter	O
+-	O
+RRM1	O
+/	O
+RRM2	O
+interface	O
+(	O
+Fig	O
+.	O
+3e	O
+;	O
+Supplementary	O
+Movie	O
+1	O
+).	O
+	O
+The	O
+rU6	O
+base	O
+edge	O
+is	O
+relatively	O
+solvent	O
+exposed	O
+;	O
+accordingly	O
+,	O
+the	O
+rU6	O
+hydrogen	O
+bonds	O
+with	O
+U2AF65	O
+are	O
+water	O
+mediated	O
+apart	O
+from	O
+a	O
+single	O
+direct	O
+interaction	O
+by	O
+the	O
+RRM1	O
+-	O
+N196	O
+side	O
+chain	O
+.	O
+	O
+Mutagenesis	O
+of	O
+either	O
+V254	O
+in	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+to	O
+proline	O
+or	O
+RRM1	O
+–	O
+R227	O
+to	O
+alanine	O
+,	O
+which	O
+remove	O
+the	O
+hydrogen	O
+bond	O
+with	O
+the	O
+fifth	O
+uracil	O
+-	O
+O4	O
+or	O
+-	O
+O2	O
+,	O
+reduced	O
+the	O
+affinities	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+for	O
+the	O
+representative	O
+AdML	O
+Py	O
+tract	O
+by	O
+four	O
+-	O
+or	O
+five	O
+-	O
+fold	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+energetic	O
+penalties	O
+due	O
+to	O
+these	O
+mutations	O
+(	O
+ΔΔG	O
+0	O
+.	O
+8	O
+–	O
+0	O
+.	O
+9	O
+kcal	O
+mol	O
+−	O
+1	O
+)	O
+are	O
+consistent	O
+with	O
+the	O
+loss	O
+of	O
+each	O
+hydrogen	O
+bond	O
+with	O
+the	O
+rU5	O
+base	O
+and	O
+support	O
+the	O
+relevance	O
+of	O
+the	O
+central	O
+nucleotide	O
+interactions	O
+observed	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+.	O
+	O
+Rather	O
+than	O
+interacting	O
+with	O
+a	O
+new	O
+5	O
+′-	O
+terminal	O
+nucleotide	O
+as	O
+we	O
+had	O
+hypothesized	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+α	O
+-	O
+helix	O
+of	O
+RRM2	O
+instead	O
+folds	O
+across	O
+one	O
+surface	O
+of	O
+rU3	O
+in	O
+the	O
+third	O
+binding	O
+site	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+	O
+There	O
+,	O
+a	O
+salt	O
+bridge	O
+between	O
+the	O
+K340	O
+side	O
+chain	O
+and	O
+nucleotide	O
+phosphate	O
+,	O
+as	O
+well	O
+as	O
+G338	O
+-	O
+base	O
+stacking	O
+and	O
+a	O
+hydrogen	O
+bond	O
+between	O
+the	O
+backbone	O
+amide	O
+of	O
+G338	O
+and	O
+the	O
+rU3	O
+-	O
+O4	O
+,	O
+secure	O
+the	O
+RRM2	O
+extension	O
+.	O
+	O
+At	O
+the	O
+N	O
+terminus	O
+,	O
+the	O
+α	O
+-	O
+helical	O
+extension	O
+of	O
+U2AF65	O
+RRM1	O
+positions	O
+the	O
+Q147	O
+side	O
+chain	O
+to	O
+bridge	O
+the	O
+eighth	O
+and	O
+ninth	O
+nucleotides	O
+at	O
+the	O
+3	O
+′	O
+terminus	O
+of	O
+the	O
+Py	O
+tract	O
+(	O
+Fig	O
+.	O
+3f	O
+–	O
+h	O
+).	O
+	O
+The	O
+Q147	O
+residue	O
+participates	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+-	O
+N3H	O
+of	O
+the	O
+eighth	O
+uracil	O
+and	O
+-	O
+O2	O
+of	O
+the	O
+ninth	O
+pyrimidine	O
+.	O
+	O
+Versatile	O
+primary	O
+sequence	O
+of	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+reveal	O
+that	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+mediates	O
+an	O
+extensive	O
+interface	O
+with	O
+the	O
+second	O
+α	O
+-	O
+helix	O
+of	O
+RRM1	O
+,	O
+the	O
+β2	O
+/	O
+β3	O
+strands	O
+of	O
+RRM2	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+α	O
+-	O
+helical	O
+extension	O
+of	O
+RRM1	O
+.	O
+	O
+Altogether	O
+,	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+residues	O
+(	O
+R228	O
+–	O
+K260	O
+)	O
+bury	O
+2	O
+,	O
+800	O
+Å2	O
+of	O
+surface	O
+area	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+holo	O
+-	O
+protein	O
+,	O
+suggestive	O
+of	O
+a	O
+cognate	O
+interface	O
+compared	O
+with	O
+1	O
+,	O
+900	O
+Å2	O
+for	O
+a	O
+typical	O
+protein	O
+–	O
+protein	O
+complex	O
+.	O
+	O
+The	O
+path	O
+of	O
+the	O
+linker	O
+initiates	O
+at	O
+P229	O
+following	O
+the	O
+core	O
+RRM1	O
+β	O
+-	O
+strand	O
+,	O
+in	O
+a	O
+kink	O
+that	O
+is	O
+positioned	O
+by	O
+intra	O
+-	O
+molecular	O
+stacking	O
+among	O
+the	O
+consecutive	O
+R228	O
+,	O
+Y232	O
+and	O
+P234	O
+side	O
+chains	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+lower	O
+right	O
+).	O
+	O
+A	O
+second	O
+kink	O
+at	O
+P236	O
+,	O
+coupled	O
+with	O
+respective	O
+packing	O
+of	O
+the	O
+L235	O
+and	O
+M238	O
+side	O
+chains	O
+on	O
+the	O
+N	O
+-	O
+terminal	O
+α	O
+-	O
+helical	O
+RRM1	O
+extension	O
+and	O
+the	O
+core	O
+RRM1	O
+α2	O
+-	O
+helix	O
+,	O
+reverses	O
+the	O
+direction	O
+of	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+towards	O
+the	O
+RRM1	O
+/	O
+RRM2	O
+interface	O
+and	O
+away	O
+from	O
+the	O
+RNA	O
+-	O
+binding	O
+site	O
+.	O
+	O
+In	O
+the	O
+neighbouring	O
+apical	O
+region	O
+of	O
+the	O
+linker	O
+,	O
+the	O
+V244	O
+and	O
+V246	O
+side	O
+chains	O
+pack	O
+in	O
+a	O
+hydrophobic	O
+pocket	O
+between	O
+two	O
+α	O
+-	O
+helices	O
+of	O
+the	O
+core	O
+RRM1	O
+.	O
+	O
+The	O
+adjacent	O
+V249	O
+and	O
+V250	O
+are	O
+notable	O
+for	O
+their	O
+respective	O
+interactions	O
+that	O
+connect	O
+RRM1	O
+and	O
+RRM2	O
+at	O
+this	O
+distal	O
+interface	O
+from	O
+the	O
+RNA	O
+-	O
+binding	O
+site	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+top	O
+).	O
+	O
+A	O
+third	O
+kink	O
+stacks	O
+P247	O
+and	O
+G248	O
+with	O
+Y245	O
+and	O
+re	O
+-	O
+orients	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+linker	O
+towards	O
+the	O
+RRM2	O
+and	O
+bound	O
+RNA	O
+.	O
+	O
+Few	O
+direct	O
+contacts	O
+are	O
+made	O
+between	O
+the	O
+remaining	O
+residues	O
+of	O
+the	O
+linker	O
+and	O
+the	O
+U2AF65	O
+RRM2	O
+;	O
+instead	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+conformation	O
+of	O
+the	O
+linker	O
+appears	O
+primarily	O
+RNA	O
+mediated	O
+(	O
+Fig	O
+.	O
+3c	O
+,	O
+d	O
+).	O
+	O
+We	O
+investigated	O
+whether	O
+the	O
+observed	O
+contacts	O
+between	O
+the	O
+RRMs	O
+and	O
+linker	O
+were	O
+critical	O
+for	O
+RNA	O
+binding	O
+by	O
+structure	O
+-	O
+guided	O
+mutagenesis	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+	O
+We	O
+titrated	O
+these	O
+mutant	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+proteins	O
+into	O
+fluorescein	O
+-	O
+labelled	O
+AdML	O
+Py	O
+-	O
+tract	O
+RNA	O
+and	O
+fit	O
+the	O
+fluorescence	O
+anisotropy	O
+changes	O
+to	O
+obtain	O
+the	O
+apparent	O
+equilibrium	O
+affinities	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+–	O
+h	O
+).	O
+	O
+First	O
+,	O
+we	O
+replaced	O
+V249	O
+and	O
+V250	O
+at	O
+the	O
+RRM1	O
+/	O
+RRM2	O
+interface	O
+and	O
+V254	O
+at	O
+the	O
+bound	O
+RNA	O
+site	O
+with	O
+glycine	O
+(	O
+3Gly	B-mutant
+).	O
+	O
+A	O
+more	O
+conservative	O
+substitution	O
+of	O
+these	O
+five	O
+residues	O
+(	O
+251	O
+–	O
+255	O
+)	O
+with	O
+an	O
+unrelated	O
+sequence	O
+capable	O
+of	O
+backbone	O
+-	O
+mediated	O
+hydrogen	O
+bonds	O
+(	O
+STVVP	B-mutant
+>	I-mutant
+NLALA	I-mutant
+)	O
+confirmed	O
+the	O
+subtle	O
+impact	O
+of	O
+this	O
+versatile	O
+inter	O
+-	O
+RRM	O
+sequence	O
+on	O
+affinity	O
+for	O
+the	O
+AdML	O
+Py	O
+tract	O
+.	O
+	O
+Finally	O
+,	O
+to	O
+ensure	O
+that	O
+these	O
+selective	O
+mutations	O
+were	O
+sufficient	O
+to	O
+disrupt	O
+the	O
+linker	O
+/	O
+RRM	O
+contacts	O
+,	O
+we	O
+substituted	O
+glycine	O
+for	O
+the	O
+majority	O
+of	O
+buried	O
+hydrophobic	O
+residues	O
+in	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+(	O
+including	O
+M144	O
+,	O
+L235	O
+,	O
+M238	O
+,	O
+V244	O
+,	O
+V246	O
+,	O
+V249	O
+,	O
+V250	O
+,	O
+S251	O
+,	O
+T252	O
+,	O
+V253	O
+,	O
+V254	O
+,	O
+P255	O
+;	O
+called	O
+12Gly	B-mutant
+).	O
+	O
+Despite	O
+12	O
+concurrent	O
+mutations	O
+,	O
+the	O
+AdML	O
+RNA	O
+affinity	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+12Gly	I-mutant
+variant	O
+was	O
+reduced	O
+by	O
+only	O
+three	O
+-	O
+fold	O
+relative	O
+to	O
+the	O
+unmodified	O
+protein	O
+(	O
+Fig	O
+.	O
+4b	O
+),	O
+which	O
+is	O
+less	O
+than	O
+the	O
+penalty	O
+of	O
+the	O
+V254P	B-mutant
+mutation	O
+that	O
+disrupts	O
+the	O
+rU5	O
+hydrogen	O
+bond	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+i	O
+).	O
+	O
+To	O
+test	O
+the	O
+interplay	O
+of	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+with	O
+its	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+RRM	O
+extensions	O
+,	O
+we	O
+constructed	O
+an	O
+internal	O
+linker	O
+deletion	O
+of	O
+20	O
+-	O
+residues	O
+within	O
+the	O
+extended	O
+RNA	O
+-	O
+binding	O
+domain	O
+(	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+).	O
+	O
+We	O
+found	O
+that	O
+the	O
+affinity	O
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+for	O
+the	O
+AdML	O
+RNA	O
+was	O
+significantly	O
+reduced	O
+relative	O
+to	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+(	O
+four	O
+-	O
+fold	O
+,	O
+Figs	O
+1b	O
+and	O
+4b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4i	O
+).	O
+	O
+Yet	O
+,	O
+it	O
+is	O
+well	O
+known	O
+that	O
+the	O
+linker	O
+deletion	O
+in	O
+the	O
+context	O
+of	O
+the	O
+minimal	O
+RRM1	O
+–	O
+RRM2	O
+boundaries	O
+has	O
+no	O
+detectable	O
+effect	O
+on	O
+the	O
+RNA	O
+affinities	O
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+compared	O
+with	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+(	O
+refs	O
+;	O
+Figs	O
+1b	O
+and	O
+4b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4j	O
+).	O
+	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+suggest	O
+that	O
+an	O
+extended	O
+conformation	O
+of	O
+the	O
+truncated	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+inter	O
+-	O
+RRM	O
+linker	O
+would	O
+suffice	O
+to	O
+connect	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM1	O
+C	O
+terminus	O
+to	O
+the	O
+N	O
+terminus	O
+of	O
+RRM2	O
+(	O
+24	O
+Å	O
+distance	O
+between	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+R227	O
+-	O
+Cα	O
+–	O
+H259	O
+-	O
+Cα	O
+atoms	O
+),	O
+which	O
+agrees	O
+with	O
+the	O
+greater	O
+RNA	O
+affinities	O
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+dual	O
+RRMs	O
+compared	O
+with	O
+the	O
+individual	O
+U2AF65	O
+RRMs	O
+.	O
+	O
+However	O
+,	O
+stretching	O
+of	O
+the	O
+truncated	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+linker	O
+to	O
+connect	O
+the	O
+RRM	O
+termini	O
+is	O
+expected	O
+to	O
+disrupt	O
+its	O
+nucleotide	O
+interactions	O
+.	O
+	O
+Likewise	O
+,	O
+deletion	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	O
+extension	O
+in	O
+the	O
+shortened	O
+constructs	O
+would	O
+remove	O
+packing	O
+interactions	O
+that	O
+position	O
+the	O
+linker	O
+in	O
+a	O
+kinked	O
+turn	O
+following	O
+P229	O
+(	O
+Fig	O
+.	O
+4a	O
+),	O
+consistent	O
+with	O
+the	O
+lower	O
+RNA	O
+affinities	O
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+compared	O
+with	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+.	O
+	O
+Notably	O
+,	O
+the	O
+Q147A	B-mutant
+/	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+mutation	O
+reduced	O
+the	O
+RNA	O
+affinity	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+3Mut	I-mutant
+protein	O
+by	O
+30	O
+-	O
+fold	O
+more	O
+than	O
+would	O
+be	O
+expected	O
+based	O
+on	O
+simple	O
+addition	O
+of	O
+the	O
+ΔΔG	O
+'	O
+s	O
+for	O
+the	O
+single	O
+mutations	O
+.	O
+	O
+This	O
+difference	O
+indicates	O
+that	O
+the	O
+linearly	O
+distant	O
+regions	O
+of	O
+the	O
+U2AF65	O
+primary	O
+sequence	O
+,	O
+including	O
+Q147	O
+in	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	O
+extension	O
+and	O
+R227	O
+/	O
+V254	O
+in	O
+the	O
+N	O
+-/	O
+C	O
+-	O
+terminal	O
+linker	O
+regions	O
+at	O
+the	O
+fifth	O
+nucleotide	O
+site	O
+,	O
+cooperatively	O
+recognize	O
+the	O
+Py	O
+tract	O
+.	O
+	O
+Altogether	O
+,	O
+we	O
+conclude	O
+that	O
+the	O
+conformation	O
+of	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+is	O
+key	O
+for	O
+recognizing	O
+RNA	O
+and	O
+is	O
+positioned	O
+by	O
+the	O
+RRM	O
+extension	O
+but	O
+otherwise	O
+relatively	O
+independent	O
+of	O
+the	O
+side	O
+chain	O
+composition	O
+.	O
+	O
+The	O
+non	O
+-	O
+additive	O
+effects	O
+of	O
+the	O
+Q147A	B-mutant
+/	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+triple	O
+mutation	O
+,	O
+coupled	O
+with	O
+the	O
+context	O
+-	O
+dependent	O
+penalties	O
+of	O
+an	O
+internal	O
+U2AF65	O
+linker	O
+deletion	O
+,	O
+highlights	O
+the	O
+importance	O
+of	O
+the	O
+structural	O
+interplay	O
+among	O
+the	O
+U2AF65	O
+linker	O
+and	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+extensions	O
+flanking	O
+the	O
+core	O
+RRMs	O
+.	O
+	O
+As	O
+a	O
+representative	O
+splicing	O
+substrate	O
+,	O
+we	O
+utilized	O
+a	O
+well	O
+-	O
+characterized	O
+minigene	O
+splicing	O
+reporter	O
+(	O
+called	O
+pyPY	O
+)	O
+comprising	O
+a	O
+weak	O
+(	O
+that	O
+is	O
+,	O
+degenerate	O
+,	O
+py	O
+)	O
+and	O
+strong	O
+(	O
+that	O
+is	O
+,	O
+U	O
+-	O
+rich	O
+,	O
+PY	O
+)	O
+polypyrimidine	O
+tracts	O
+preceding	O
+two	O
+alternative	O
+splice	O
+sites	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+	O
+When	O
+co	O
+-	O
+transfected	O
+with	O
+an	O
+expression	O
+plasmid	O
+for	O
+wild	O
+-	O
+type	O
+U2AF65	O
+,	O
+use	O
+of	O
+the	O
+py	O
+splice	O
+site	O
+significantly	O
+increases	O
+(	O
+by	O
+more	O
+than	O
+five	O
+-	O
+fold	O
+)	O
+and	O
+as	O
+documented	O
+converts	O
+a	O
+fraction	O
+of	O
+the	O
+unspliced	O
+to	O
+spliced	O
+transcript	O
+.	O
+	O
+We	O
+introduced	O
+the	O
+triple	O
+mutation	O
+(	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+/	O
+Q147A	B-mutant
+)	O
+that	O
+significantly	O
+reduced	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+association	O
+with	O
+the	O
+Py	O
+tract	O
+(	O
+Fig	O
+.	O
+4b	O
+)	O
+in	O
+the	O
+context	O
+of	O
+full	O
+-	O
+length	O
+U2AF65	O
+(	O
+U2AF65	B-mutant
+-	I-mutant
+3Mut	I-mutant
+).	O
+	O
+Co	O
+-	O
+transfection	O
+of	O
+the	O
+U2AF65	B-mutant
+-	I-mutant
+3Mut	I-mutant
+with	O
+the	O
+pyPY	O
+splicing	O
+substrate	O
+significantly	O
+reduced	O
+splicing	O
+of	O
+the	O
+weak	O
+‘	O
+py	O
+'	O
+splice	O
+site	O
+relative	O
+to	O
+wild	O
+-	O
+type	O
+U2AF65	O
+(	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+	O
+We	O
+conclude	O
+that	O
+the	O
+Py	O
+-	O
+tract	O
+interactions	O
+with	O
+these	O
+residues	O
+of	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+and	O
+RRM	O
+extensions	O
+are	O
+important	O
+for	O
+splicing	O
+as	O
+well	O
+as	O
+for	O
+binding	O
+a	O
+representative	O
+of	O
+the	O
+major	O
+U2	O
+-	O
+class	O
+of	O
+splice	O
+sites	O
+.	O
+	O
+Sparse	O
+inter	O
+-	O
+RRM	O
+contacts	O
+underlie	O
+apo	O
+-	O
+U2AF65	O
+dynamics	O
+	O
+The	O
+direct	O
+interface	O
+between	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM1	O
+and	O
+RRM2	O
+is	O
+minor	O
+,	O
+burying	O
+265	O
+Å2	O
+of	O
+solvent	O
+accessible	O
+surface	O
+area	O
+compared	O
+with	O
+570	O
+Å2	O
+on	O
+average	O
+for	O
+a	O
+crystal	O
+packing	O
+interface	O
+.	O
+	O
+A	O
+handful	O
+of	O
+inter	O
+-	O
+RRM	O
+hydrogen	O
+bonds	O
+are	O
+apparent	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+RRM1	O
+-	O
+N155	O
+and	O
+RRM2	O
+-	O
+K292	O
+,	O
+RRM1	O
+-	O
+N155	O
+and	O
+RRM2	O
+-	O
+D272	O
+as	O
+well	O
+as	O
+the	O
+backbone	O
+atoms	O
+of	O
+RRM1	O
+-	O
+G221	O
+and	O
+RRM2	O
+-	O
+D273	O
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+	O
+This	O
+minor	O
+U2AF65	O
+RRM1	O
+/	O
+RRM2	O
+interface	O
+,	O
+coupled	O
+with	O
+the	O
+versatile	O
+sequence	O
+of	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+,	O
+highlighted	O
+the	O
+potential	O
+role	O
+for	O
+inter	O
+-	O
+RRM	O
+conformational	O
+dynamics	O
+in	O
+U2AF65	O
+-	O
+splice	O
+site	O
+recognition	O
+.	O
+	O
+Paramagnetic	O
+resonance	O
+enhancement	O
+(	O
+PRE	O
+)	O
+measurements	O
+previously	O
+had	O
+suggested	O
+a	O
+predominant	O
+back	O
+-	O
+to	O
+-	O
+back	O
+,	O
+or	O
+‘	O
+closed	O
+'	O
+conformation	O
+of	O
+the	O
+apo	O
+-	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+RRM1	O
+and	O
+RRM2	O
+in	O
+equilibrium	O
+with	O
+a	O
+minor	O
+‘	O
+open	O
+'	O
+conformation	O
+resembling	O
+the	O
+RNA	O
+-	O
+bound	O
+inter	O
+-	O
+RRM	O
+arrangement	O
+.	O
+	O
+Yet	O
+,	O
+small	O
+-	O
+angle	O
+X	O
+-	O
+ray	O
+scattering	O
+(	O
+SAXS	O
+)	O
+data	O
+indicated	O
+that	O
+both	O
+the	O
+minimal	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+longer	O
+constructs	O
+comprise	O
+a	O
+highly	O
+diverse	O
+continuum	O
+of	O
+conformations	O
+in	O
+the	O
+absence	O
+of	O
+RNA	O
+that	O
+includes	O
+the	O
+‘	O
+closed	O
+'	O
+and	O
+‘	O
+open	O
+'	O
+conformations	O
+.	O
+	O
+The	O
+inter	O
+-	O
+RRM	O
+dynamics	O
+of	O
+U2AF65	O
+were	O
+followed	O
+using	O
+FRET	O
+between	O
+fluorophores	O
+attached	O
+to	O
+RRM1	O
+and	O
+RRM2	O
+(	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+,	O
+Methods	O
+).	O
+	O
+The	O
+FRET	O
+efficiencies	O
+of	O
+either	O
+of	O
+these	O
+structurally	O
+characterized	O
+conformations	O
+also	O
+are	O
+expected	O
+to	O
+be	O
+significantly	O
+greater	O
+than	O
+elongated	O
+U2AF65	O
+conformations	O
+that	O
+lack	O
+inter	O
+-	O
+RRM	O
+contacts	O
+.	O
+	O
+We	O
+first	O
+characterized	O
+the	O
+conformational	O
+dynamics	O
+spectrum	O
+of	O
+U2AF65	O
+in	O
+the	O
+absence	O
+of	O
+RNA	O
+(	O
+Fig	O
+.	O
+6c	O
+,	O
+d	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+,	O
+b	O
+).	O
+	O
+Virtually	O
+no	O
+fluorescent	O
+molecules	O
+were	O
+detected	O
+in	O
+the	O
+absence	O
+of	O
+biotin	O
+-	O
+NTA	O
+/	O
+Ni	O
++	O
+2	O
+,	O
+which	O
+demonstrates	O
+the	O
+absence	O
+of	O
+detectable	O
+non	O
+-	O
+specific	O
+binding	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+to	O
+the	O
+slide	O
+.	O
+	O
+The	O
+FRET	O
+distribution	O
+histogram	O
+built	O
+from	O
+more	O
+than	O
+a	O
+thousand	O
+traces	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+in	O
+the	O
+absence	O
+of	O
+ligand	O
+showed	O
+an	O
+extremely	O
+broad	O
+distribution	O
+centred	O
+at	O
+a	O
+FRET	O
+efficiency	O
+of	O
+∼	O
+0	O
+.	O
+4	O
+(	O
+Fig	O
+.	O
+6d	O
+).	O
+	O
+Approximately	O
+40	O
+%	O
+of	O
+the	O
+smFRET	O
+traces	O
+showed	O
+apparent	O
+transitions	O
+between	O
+multiple	O
+FRET	O
+values	O
+(	O
+for	O
+example	O
+,	O
+Fig	O
+.	O
+6c	O
+).	O
+	O
+Despite	O
+the	O
+large	O
+width	O
+of	O
+the	O
+FRET	O
+-	O
+distribution	O
+histogram	O
+,	O
+the	O
+majority	O
+(	O
+80	O
+%)	O
+of	O
+traces	O
+that	O
+showed	O
+fluctuations	O
+sampled	O
+only	O
+two	O
+distinct	O
+FRET	O
+states	O
+(	O
+for	O
+example	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+	O
+Approximately	O
+70	O
+%	O
+of	O
+observed	O
+fluctuations	O
+were	O
+interchanges	O
+between	O
+the	O
+∼	O
+0	O
+.	O
+65	O
+and	O
+∼	O
+0	O
+.	O
+45	O
+FRET	O
+values	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7b	O
+).	O
+	O
+We	O
+cannot	O
+exclude	O
+a	O
+possibility	O
+that	O
+tethering	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+to	O
+the	O
+microscope	O
+slide	O
+introduces	O
+structural	O
+heterogeneity	O
+into	O
+the	O
+protein	O
+and	O
+,	O
+thus	O
+,	O
+contributes	O
+to	O
+the	O
+breadth	O
+of	O
+the	O
+FRET	O
+distribution	O
+histogram	O
+.	O
+	O
+We	O
+conclude	O
+that	O
+weak	O
+contacts	O
+between	O
+the	O
+U2AF65	O
+RRM1	O
+and	O
+RRM2	O
+permit	O
+dissociation	O
+of	O
+these	O
+RRMs	O
+in	O
+the	O
+absence	O
+of	O
+RNA	O
+.	O
+	O
+U2AF65	O
+conformational	O
+selection	O
+and	O
+induced	O
+fit	O
+by	O
+bound	O
+RNA	O
+	O
+Addition	O
+of	O
+the	O
+AdML	O
+RNA	O
+to	O
+tethered	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+selectively	O
+increases	O
+a	O
+fraction	O
+of	O
+molecules	O
+showing	O
+an	O
+∼	O
+0	O
+.	O
+45	O
+apparent	O
+FRET	O
+efficiency	O
+,	O
+suggesting	O
+that	O
+RNA	O
+binding	O
+stabilizes	O
+a	O
+single	O
+conformation	O
+,	O
+which	O
+corresponds	O
+to	O
+the	O
+0	O
+.	O
+45	O
+FRET	O
+state	O
+(	O
+Fig	O
+.	O
+6e	O
+,	O
+f	O
+).	O
+	O
+We	O
+tethered	O
+the	O
+AdML	O
+RNA	O
+to	O
+the	O
+slide	O
+via	O
+a	O
+biotinylated	O
+oligonucleotide	O
+DNA	O
+handle	O
+and	O
+added	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+in	O
+the	O
+absence	O
+of	O
+biotin	O
+-	O
+NTA	O
+resin	O
+(	O
+Fig	O
+.	O
+6g	O
+,	O
+h	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+	O
+A	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+was	O
+again	O
+predominant	O
+,	O
+indicating	O
+a	O
+similar	O
+RNA	O
+-	O
+bound	O
+conformation	O
+and	O
+structural	O
+dynamics	O
+for	O
+the	O
+untethered	O
+and	O
+tethered	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+).	O
+	O
+We	O
+introduced	O
+an	O
+rArA	O
+purine	O
+dinucleotide	O
+within	O
+a	O
+variant	O
+of	O
+the	O
+AdML	O
+Py	O
+tract	O
+(	O
+detailed	O
+in	O
+Methods	O
+).	O
+	O
+Insertion	O
+of	O
+adenine	O
+nucleotides	O
+decreased	O
+binding	O
+affinity	O
+of	O
+U2AF65	O
+to	O
+RNA	O
+by	O
+approximately	O
+five	O
+-	O
+fold	O
+.	O
+	O
+Nevertheless	O
+,	O
+in	O
+the	O
+presence	O
+of	O
+saturating	O
+concentrations	O
+of	O
+rArA	O
+-	O
+interrupted	O
+RNA	O
+slide	O
+-	O
+tethered	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+showed	O
+a	O
+prevalent	O
+∼	O
+0	O
+.	O
+45	O
+apparent	O
+FRET	O
+value	O
+(	O
+Fig	O
+.	O
+6i	O
+,	O
+j	O
+),	O
+which	O
+was	O
+also	O
+predominant	O
+in	O
+the	O
+presence	O
+of	O
+continuous	O
+Py	O
+tract	O
+.	O
+	O
+It	O
+should	O
+be	O
+noted	O
+that	O
+inferring	O
+distances	O
+from	O
+FRET	O
+values	O
+is	O
+prone	O
+to	O
+significant	O
+error	O
+because	O
+of	O
+uncertainties	O
+in	O
+the	O
+determination	O
+of	O
+fluorophore	O
+orientation	O
+factor	O
+κ2	O
+and	O
+Förster	O
+radius	O
+R0	O
+,	O
+the	O
+parameters	O
+used	O
+in	O
+distance	O
+calculations	O
+.	O
+	O
+The	O
+remaining	O
+∼	O
+30	O
+%	O
+of	O
+traces	O
+for	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+bound	O
+to	O
+the	O
+slide	O
+-	O
+tethered	O
+RNA	O
+showed	O
+fluctuations	O
+between	O
+distinct	O
+FRET	O
+values	O
+.	O
+	O
+The	O
+majority	O
+of	O
+traces	O
+that	O
+show	O
+fluctuations	O
+began	O
+at	O
+high	O
+(	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+)	O
+FRET	O
+value	O
+and	O
+transitioned	O
+to	O
+a	O
+∼	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+	O
+Although	O
+a	O
+compact	O
+conformation	O
+(	O
+or	O
+multiple	O
+conformations	O
+)	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+7	O
+–	O
+0	O
+.	O
+8	O
+FRET	O
+values	O
+can	O
+bind	O
+RNA	O
+,	O
+on	O
+RNA	O
+binding	O
+,	O
+these	O
+compact	O
+conformations	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+transition	O
+into	O
+a	O
+more	O
+stable	O
+structural	O
+state	O
+that	O
+corresponds	O
+to	O
+∼	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+and	O
+is	O
+likely	O
+similar	O
+to	O
+the	O
+side	O
+-	O
+by	O
+-	O
+side	O
+inter	O
+-	O
+RRM	O
+-	O
+arrangement	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+crystal	O
+structures	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+sequence	O
+of	O
+structural	O
+rearrangements	O
+of	O
+U2AF65	O
+observed	O
+in	O
+smFRET	O
+traces	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+)	O
+suggests	O
+that	O
+a	O
+‘	O
+conformational	O
+selection	O
+'	O
+mechanism	O
+of	O
+Py	O
+-	O
+tract	O
+recognition	O
+(	O
+that	O
+is	O
+,	O
+RNA	O
+ligand	O
+stabilization	O
+of	O
+a	O
+pre	O
+-	O
+configured	O
+U2AF65	O
+conformation	O
+)	O
+is	O
+complemented	O
+by	O
+‘	O
+induced	O
+fit	O
+'	O
+(	O
+that	O
+is	O
+,	O
+RNA	O
+-	O
+induced	O
+rearrangement	O
+of	O
+the	O
+U2AF65	O
+RRMs	O
+to	O
+achieve	O
+the	O
+final	O
+‘	O
+side	O
+-	O
+by	O
+-	O
+side	O
+'	O
+conformation	O
+),	O
+as	O
+discussed	O
+below	O
+.	O
+	O
+Several	O
+observations	O
+indicate	O
+that	O
+the	O
+numerous	O
+intramolecular	O
+contacts	O
+,	O
+here	O
+revealed	O
+among	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+and	O
+RRM1	O
+,	O
+RRM2	O
+,	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	O
+extension	O
+,	O
+synergistically	O
+coordinate	O
+U2AF65	O
+–	O
+Py	O
+-	O
+tract	O
+recognition	O
+.	O
+	O
+Likewise	O
+,	O
+deletion	O
+of	O
+20	O
+inter	O
+-	O
+RRM	O
+linker	O
+residues	O
+significantly	O
+reduces	O
+U2AF65	O
+–	O
+RNA	O
+binding	O
+only	O
+when	O
+introduced	O
+in	O
+the	O
+context	O
+of	O
+the	O
+longer	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+construct	O
+comprising	O
+the	O
+RRM	O
+extensions	O
+,	O
+which	O
+in	O
+turn	O
+position	O
+the	O
+linker	O
+for	O
+RNA	O
+interactions	O
+.	O
+	O
+Notably	O
+,	O
+a	O
+triple	O
+mutation	O
+of	O
+three	O
+residues	O
+(	O
+V254P	B-mutant
+,	O
+Q147A	B-mutant
+and	O
+R227A	B-mutant
+)	O
+in	O
+the	O
+respective	O
+inter	O
+-	O
+RRM	O
+linker	O
+,	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+extensions	O
+non	O
+-	O
+additively	O
+reduce	O
+RNA	O
+binding	O
+by	O
+150	O
+-	O
+fold	O
+.	O
+	O
+Altogether	O
+,	O
+these	O
+data	O
+indicate	O
+that	O
+interactions	O
+among	O
+the	O
+U2AF65	O
+RRM1	O
+/	O
+RRM2	O
+,	O
+inter	O
+-	O
+RRM	O
+linker	O
+,	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+extensions	O
+are	O
+mutually	O
+inter	O
+-	O
+dependent	O
+for	O
+cognate	O
+Py	O
+-	O
+tract	O
+recognition	O
+.	O
+	O
+The	O
+implications	O
+of	O
+this	O
+finding	O
+for	O
+U2AF65	O
+conservation	O
+and	O
+Py	O
+-	O
+tract	O
+recognition	O
+are	O
+detailed	O
+in	O
+the	O
+Supplementary	O
+Discussion	O
+.	O
+	O
+Recently	O
+,	O
+high	O
+-	O
+throughput	O
+sequencing	O
+studies	O
+have	O
+shown	O
+that	O
+somatic	O
+mutations	O
+in	O
+pre	O
+-	O
+mRNA	O
+splicing	O
+factors	O
+occur	O
+in	O
+the	O
+majority	O
+of	O
+patients	O
+with	O
+myelodysplastic	O
+syndrome	O
+(	O
+MDS	O
+).	O
+	O
+MDS	O
+-	O
+relevant	O
+mutations	O
+are	O
+common	O
+in	O
+the	O
+small	O
+U2AF	O
+subunit	O
+(	O
+U2AF35	O
+,	O
+or	O
+U2AF1	O
+),	O
+yet	O
+such	O
+mutations	O
+are	O
+rare	O
+in	O
+the	O
+large	O
+U2AF65	O
+subunit	O
+(	O
+also	O
+called	O
+U2AF2	O
+)—	O
+possibly	O
+due	O
+to	O
+the	O
+selective	O
+versus	O
+nearly	O
+universal	O
+requirements	O
+of	O
+these	O
+factors	O
+for	O
+splicing	O
+.	O
+	O
+A	O
+confirmed	O
+somatic	O
+mutation	O
+of	O
+U2AF65	O
+in	O
+patients	O
+with	O
+MDS	O
+,	O
+L187V	B-mutant
+,	O
+is	O
+located	O
+on	O
+a	O
+solvent	O
+-	O
+exposed	O
+surface	O
+of	O
+RRM1	O
+that	O
+is	O
+distinct	O
+from	O
+the	O
+RNA	O
+interface	O
+(	O
+Fig	O
+.	O
+7a	O
+).	O
+	O
+This	O
+L187	O
+surface	O
+is	O
+oriented	O
+towards	O
+the	O
+N	O
+terminus	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+construct	O
+,	O
+where	O
+it	O
+is	O
+expected	O
+to	O
+abut	O
+the	O
+U2AF35	O
+-	O
+binding	O
+site	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	O
+-	O
+length	O
+U2AF	O
+heterodimer	O
+.	O
+	O
+Likewise	O
+,	O
+an	O
+unconfirmed	O
+M144I	B-mutant
+mutation	O
+reported	O
+by	O
+the	O
+same	O
+group	O
+corresponds	O
+to	O
+the	O
+N	O
+-	O
+terminal	O
+residue	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+which	O
+is	O
+separated	O
+by	O
+only	O
+∼	O
+20	O
+residues	O
+from	O
+the	O
+U2AF35	O
+-	O
+binding	O
+site	O
+.	O
+	O
+Our	O
+smFRET	O
+results	O
+agree	O
+with	O
+prior	O
+NMR	O
+/	O
+PRE	O
+evidence	O
+for	O
+multi	O
+-	O
+domain	O
+conformational	O
+selection	O
+as	O
+one	O
+mechanistic	O
+basis	O
+for	O
+U2AF65	O
+–	O
+RNA	O
+association	O
+(	O
+Fig	O
+.	O
+7b	O
+).	O
+	O
+An	O
+∼	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+is	O
+likely	O
+to	O
+correspond	O
+to	O
+the	O
+U2AF65	O
+conformation	O
+visualized	O
+in	O
+our	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+crystal	O
+structures	O
+,	O
+in	O
+which	O
+the	O
+RRM1	O
+and	O
+RRM2	O
+bind	O
+side	O
+-	O
+by	O
+-	O
+side	O
+to	O
+the	O
+Py	O
+-	O
+tract	O
+oligonucleotide	O
+.	O
+	O
+The	O
+lesser	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+and	O
+0	O
+.	O
+2	O
+–	O
+0	O
+.	O
+3	O
+FRET	O
+values	O
+in	O
+the	O
+untethered	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+experiment	O
+could	O
+correspond	O
+to	O
+respective	O
+variants	O
+of	O
+the	O
+‘	O
+closed	O
+',	O
+back	O
+-	O
+to	O
+-	O
+back	O
+U2AF65	O
+conformations	O
+characterized	O
+by	O
+NMR	O
+/	O
+PRE	O
+data	O
+,	O
+or	O
+to	O
+extended	O
+U2AF65	O
+conformations	O
+,	O
+in	O
+which	O
+the	O
+intramolecular	O
+RRM1	O
+/	O
+RRM2	O
+interactions	O
+have	O
+dissociated	O
+the	O
+protein	O
+is	O
+bound	O
+to	O
+RNA	O
+via	O
+single	O
+RRMs	O
+.	O
+	O
+Notably	O
+,	O
+our	O
+smFRET	O
+results	O
+reveal	O
+that	O
+U2AF65	O
+–	O
+Py	O
+-	O
+tract	O
+recognition	O
+can	O
+be	O
+characterized	O
+by	O
+an	O
+‘	O
+extended	O
+conformational	O
+selection	O
+'	O
+model	O
+(	O
+Fig	O
+.	O
+7b	O
+).	O
+	O
+Here	O
+,	O
+the	O
+majority	O
+of	O
+changes	O
+in	O
+smFRET	O
+traces	O
+for	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+bound	O
+to	O
+slide	O
+-	O
+tethered	O
+RNA	O
+began	O
+at	O
+high	O
+(	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+)	O
+FRET	O
+value	O
+and	O
+transition	O
+to	O
+the	O
+predominant	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+	O
+As	O
+such	O
+,	O
+the	O
+smFRET	O
+approach	O
+reconciles	O
+prior	O
+inconsistencies	O
+between	O
+two	O
+major	O
+conformations	O
+that	O
+were	O
+detected	O
+by	O
+NMR	O
+/	O
+PRE	O
+experiments	O
+and	O
+a	O
+broad	O
+ensemble	O
+of	O
+diverse	O
+inter	O
+-	O
+RRM	O
+arrangements	O
+that	O
+fit	O
+the	O
+SAXS	O
+data	O
+for	O
+the	O
+apo	O
+-	O
+protein	O
+.	O
+	O
+Similar	O
+interdisciplinary	O
+structural	O
+approaches	O
+are	O
+likely	O
+to	O
+illuminate	O
+whether	O
+similar	O
+mechanistic	O
+bases	O
+for	O
+RNA	O
+binding	O
+are	O
+widespread	O
+among	O
+other	O
+members	O
+of	O
+the	O
+vast	O
+multi	O
+-	O
+RRM	O
+family	O
+.	O
+	O
+Based	O
+on	O
+(	O
+i	O
+)	O
+similar	O
+RNA	O
+affinities	O
+of	O
+U2AF65	O
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+(	O
+ii	O
+)	O
+indistinguishable	O
+conformations	O
+among	O
+four	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+in	O
+two	O
+different	O
+crystal	O
+packing	O
+arrangements	O
+and	O
+(	O
+iii	O
+)	O
+penalties	O
+of	O
+structure	O
+-	O
+guided	O
+mutations	O
+in	O
+RNA	O
+binding	O
+and	O
+splicing	O
+assays	O
+,	O
+we	O
+suggest	O
+that	O
+the	O
+extended	O
+inter	O
+-	O
+RRM	O
+regions	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+underlie	O
+cognate	O
+Py	O
+-	O
+tract	O
+recognition	O
+by	O
+the	O
+full	O
+-	O
+length	O
+U2AF65	O
+protein	O
+.	O
+	O
+Further	O
+research	O
+will	O
+be	O
+needed	O
+to	O
+understand	O
+the	O
+roles	O
+of	O
+SF1	O
+and	O
+U2AF35	O
+subunits	O
+in	O
+the	O
+conformational	O
+equilibria	O
+underlying	O
+U2AF65	O
+association	O
+with	O
+Py	O
+tracts	O
+.	O
+	O
+Moreover	O
+,	O
+structural	O
+differences	O
+among	O
+U2AF65	O
+homologues	O
+and	O
+paralogues	O
+may	O
+regulate	O
+splice	O
+site	O
+selection	O
+.	O
+	O
+Ultimately	O
+,	O
+these	O
+guidelines	O
+will	O
+assist	O
+the	O
+identification	O
+of	O
+3	O
+′	O
+splice	O
+sites	O
+and	O
+the	O
+relationship	O
+of	O
+disease	O
+-	O
+causing	O
+mutations	O
+to	O
+penalties	O
+for	O
+U2AF65	O
+association	O
+.	O
+	O
+(	O
+a	O
+)	O
+Domain	O
+organization	O
+of	O
+full	O
+-	O
+length	O
+(	O
+fl	O
+)	O
+U2AF65	O
+and	O
+constructs	O
+used	O
+for	O
+RNA	O
+binding	O
+and	O
+structural	O
+experiments	O
+.	O
+	O
+An	O
+internal	O
+deletion	O
+(	O
+d	B-mutant
+,	O
+Δ	B-mutant
+)	O
+of	O
+residues	O
+238	O
+–	O
+257	O
+removes	O
+a	O
+portion	O
+of	O
+the	O
+inter	O
+-	O
+RRM	O
+linker	O
+from	O
+the	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+constructs	O
+.	O
+	O
+The	O
+flU2AF65	O
+protein	O
+includes	O
+a	O
+heterodimerization	O
+domain	O
+of	O
+the	O
+U2AF35	O
+subunit	O
+to	O
+promote	O
+solubility	O
+and	O
+folding	O
+.	O
+	O
+The	O
+KD	O
+'	O
+s	O
+for	O
+binding	O
+5	O
+′-	O
+CCUUUUCCCCCCC	O
+-	O
+3	O
+′	O
+are	O
+:	O
+flU2AF65	O
+,	O
+41	O
+±	O
+2	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+31	O
+±	O
+3	O
+nM	O
+.	O
+The	O
+KD	O
+'	O
+s	O
+for	O
+binding	O
+5	O
+′-	O
+CCCCCCCUUUUCC	O
+-	O
+3	O
+′	O
+are	O
+:	O
+flU2AF65	O
+,	O
+414	O
+±	O
+12	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+417	O
+±	O
+10	O
+nM	O
+.	O
+Bar	O
+graphs	O
+are	O
+hatched	O
+to	O
+match	O
+the	O
+constructs	O
+shown	O
+in	O
+a	O
+.	O
+The	O
+average	O
+apparent	O
+equilibrium	O
+affinity	O
+(	O
+KA	O
+)	O
+and	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+for	O
+three	O
+independent	O
+titrations	O
+are	O
+plotted	O
+.	O
+	O
+RRM	O
+,	O
+RNA	O
+recognition	O
+motif	O
+;	O
+RS	O
+,	O
+arginine	O
+-	O
+serine	O
+rich	O
+;	O
+UHM	O
+,	O
+U2AF	O
+homology	O
+motif	O
+;	O
+ULM	O
+,	O
+U2AF	O
+ligand	O
+motif	O
+.	O
+	O
+(	O
+a	O
+)	O
+Alignment	O
+of	O
+oligonucleotide	O
+sequences	O
+that	O
+were	O
+co	O
+-	O
+crystallized	O
+in	O
+the	O
+indicated	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+.	O
+	O
+The	O
+regions	O
+of	O
+RRM1	O
+,	O
+RRM2	O
+and	O
+linker	O
+contacts	O
+are	O
+indicated	O
+above	O
+by	O
+respective	O
+black	O
+and	O
+blue	O
+arrows	O
+from	O
+N	O
+-	O
+to	O
+C	O
+-	O
+terminus	O
+.	O
+	O
+The	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+nucleotide	O
+-	O
+binding	O
+sites	O
+are	O
+given	O
+in	O
+parentheses	O
+(	O
+site	O
+4	O
+'	O
+interacts	O
+with	O
+dU2AF65	B-mutant
+RRM1	O
+and	O
+RRM2	O
+by	O
+crystallographic	O
+symmetry	O
+).	O
+	O
+Crystallographic	O
+statistics	O
+are	O
+given	O
+in	O
+Table	O
+1	O
+and	O
+the	O
+overall	O
+conformations	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+/	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	O
+are	O
+compared	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+.	O
+	O
+Representative	O
+views	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+interactions	O
+with	O
+each	O
+new	O
+nucleotide	O
+of	O
+the	O
+bound	O
+Py	O
+tract	O
+.	O
+	O
+New	O
+residues	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+are	O
+coloured	O
+a	O
+darker	O
+shade	O
+of	O
+blue	O
+,	O
+apart	O
+from	O
+residues	O
+that	O
+were	O
+tested	O
+by	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+,	O
+which	O
+are	O
+coloured	O
+yellow	O
+.	O
+	O
+The	O
+nucleotide	O
+-	O
+binding	O
+sites	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structure	O
+are	O
+compared	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+–	O
+h	O
+.	O
+The	O
+first	O
+and	O
+seventh	O
+U2AF651	O
+,	O
+2L	O
+-	O
+binding	O
+sites	O
+are	O
+unchanged	O
+from	O
+the	O
+prior	O
+dU2AF651	O
+,	O
+2	O
+–	O
+RNA	O
+structure	O
+and	O
+are	O
+portrayed	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+,	O
+f	O
+.	O
+The	O
+four	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+are	O
+similar	O
+with	O
+the	O
+exception	O
+of	O
+pH	O
+-	O
+dependent	O
+variations	O
+at	O
+the	O
+ninth	O
+site	O
+that	O
+are	O
+detailed	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+3i	O
+,	O
+j	O
+.	O
+The	O
+representative	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	O
+shown	O
+has	O
+the	O
+highest	O
+resolution	O
+and	O
+/	O
+or	O
+ribose	O
+nucleotide	O
+at	O
+the	O
+given	O
+site	O
+:	O
+(	O
+a	O
+)	O
+rU2	O
+of	O
+structure	O
+iv	O
+;	O
+(	O
+b	O
+)	O
+rU3	O
+of	O
+structure	O
+iii	O
+;	O
+(	O
+c	O
+)	O
+rU4	O
+of	O
+structure	O
+i	O
+;	O
+(	O
+d	O
+)	O
+rU5	O
+of	O
+structure	O
+iii	O
+;	O
+(	O
+e	O
+)	O
+rU6	O
+of	O
+structure	O
+ii	O
+;	O
+(	O
+f	O
+)	O
+dU8	O
+of	O
+structure	O
+iii	O
+;	O
+(	O
+g	O
+)	O
+dU9	O
+of	O
+structure	O
+iii	O
+;	O
+(	O
+h	O
+)	O
+rC9	O
+of	O
+structure	O
+iv	O
+.	O
+	O
+(	O
+i	O
+)	O
+Bar	O
+graph	O
+of	O
+apparent	O
+equilibrium	O
+affinities	O
+(	O
+KA	O
+)	O
+of	O
+the	O
+wild	O
+type	O
+(	O
+blue	O
+)	O
+and	O
+the	O
+indicated	O
+mutant	O
+(	O
+yellow	O
+)	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+proteins	O
+binding	O
+the	O
+AdML	O
+Py	O
+tract	O
+(	O
+5	O
+′-	O
+CCCUUUUUUUUCC	O
+-	O
+3	O
+′).	O
+	O
+The	O
+apparent	O
+equilibrium	O
+dissociation	O
+constants	O
+(	O
+KD	O
+)	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+mutant	O
+proteins	O
+are	O
+:	O
+wild	O
+type	O
+(	O
+WT	O
+),	O
+35	O
+±	O
+6	O
+nM	O
+;	O
+R227A	B-mutant
+,	O
+166	O
+±	O
+2	O
+nM	O
+;	O
+V254P	B-mutant
+,	O
+137	O
+±	O
+10	O
+nM	O
+;	O
+Q147A	B-mutant
+,	O
+171	O
+±	O
+21	O
+nM	O
+.	O
+The	O
+average	O
+KA	O
+and	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+for	O
+three	O
+independent	O
+titrations	O
+are	O
+plotted	O
+.	O
+	O
+The	O
+average	O
+fitted	O
+fluorescence	O
+anisotropy	O
+RNA	O
+-	O
+binding	O
+curves	O
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+–	O
+c	O
+.	O
+	O
+(	O
+a	O
+)	O
+Contacts	O
+of	O
+the	O
+U2AF65	O
+inter	O
+-	O
+RRM	O
+linker	O
+with	O
+the	O
+RRMs	O
+.	O
+	O
+A	O
+semi	O
+-	O
+transparent	O
+space	O
+-	O
+filling	O
+surface	O
+is	O
+shown	O
+for	O
+the	O
+RRM1	O
+(	O
+green	O
+)	O
+and	O
+RRM2	O
+(	O
+light	O
+blue	O
+).	O
+	O
+Residues	O
+V249	O
+,	O
+V250	O
+,	O
+V254	O
+(	O
+yellow	O
+)	O
+are	O
+mutated	O
+to	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+V254G	B-mutant
+in	O
+the	O
+3Gly	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+S251	O
+,	O
+T252	O
+,	O
+V253	O
+,	O
+P255	O
+(	O
+red	O
+)	O
+along	O
+with	O
+V254	O
+are	O
+mutated	O
+to	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+5Gly	B-mutant
+mutant	I-mutant
+or	O
+to	O
+S251N	B-mutant
+/	O
+T252L	B-mutant
+/	O
+V253A	B-mutant
+/	O
+V254L	B-mutant
+/	O
+P255A	B-mutant
+in	O
+the	O
+NLALA	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+M144	O
+,	O
+L235	O
+,	O
+M238	O
+,	O
+V244	O
+,	O
+V246	O
+(	O
+orange	O
+)	O
+along	O
+with	O
+V249	O
+,	O
+V250	O
+,	O
+S251	O
+,	O
+T252	O
+,	O
+V253	O
+,	O
+V254	O
+,	O
+P255	O
+are	O
+mutated	O
+to	O
+M144G	B-mutant
+/	O
+L235G	B-mutant
+/	O
+M238G	B-mutant
+/	O
+V244G	B-mutant
+/	O
+V246G	B-mutant
+/	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+12Gly	B-mutant
+mutant	I-mutant
+.	O
+	O
+Other	O
+linker	O
+residues	O
+are	O
+coloured	O
+either	O
+dark	O
+blue	O
+for	O
+new	O
+residues	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	O
+or	O
+light	O
+blue	O
+for	O
+the	O
+remaining	O
+inter	O
+-	O
+RRM	O
+residues	O
+.	O
+	O
+The	O
+central	O
+panel	O
+shows	O
+an	O
+overall	O
+view	O
+with	O
+stick	O
+diagrams	O
+for	O
+mutated	O
+residues	O
+;	O
+boxed	O
+regions	O
+are	O
+expanded	O
+to	O
+show	O
+the	O
+C	O
+-	O
+terminal	O
+(	O
+bottom	O
+left	O
+)	O
+and	O
+central	O
+linker	O
+regions	O
+(	O
+top	O
+)	O
+at	O
+the	O
+inter	O
+-	O
+RRM	O
+interfaces	O
+,	O
+and	O
+N	O
+-	O
+terminal	O
+linker	O
+region	O
+contacts	O
+with	O
+RRM1	O
+(	O
+bottom	O
+right	O
+).	O
+	O
+The	O
+fitted	O
+fluorescence	O
+anisotropy	O
+RNA	O
+-	O
+binding	O
+curves	O
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+–	O
+j	O
+.	O
+(	O
+c	O
+)	O
+Close	O
+view	O
+of	O
+the	O
+U2AF65	O
+RRM1	O
+/	O
+RRM2	O
+interface	O
+following	O
+a	O
+two	O
+-	O
+fold	O
+rotation	O
+about	O
+the	O
+x	O
+-	O
+axis	O
+relative	O
+to	O
+a	O
+.	O
+	O
+U2AF65	O
+inter	O
+-	O
+domain	O
+residues	O
+are	O
+important	O
+for	O
+splicing	O
+a	O
+representative	O
+pre	O
+-	O
+mRNA	O
+substrate	O
+in	O
+human	O
+cells	O
+.	O
+	O
+(	O
+a	O
+)	O
+Schematic	O
+diagram	O
+of	O
+the	O
+pyPY	O
+reporter	O
+minigene	O
+construct	O
+comprising	O
+two	O
+alternative	O
+splice	O
+sites	O
+preceded	O
+by	O
+either	O
+the	O
+weak	O
+IgM	O
+Py	O
+tract	O
+(	O
+py	O
+)	O
+or	O
+the	O
+strong	O
+AdML	O
+Py	O
+tract	O
+(	O
+PY	O
+)	O
+(	O
+sequences	O
+inset	O
+).	O
+	O
+(	O
+b	O
+)	O
+Representative	O
+RT	O
+-	O
+PCR	O
+of	O
+pyPY	O
+transcripts	O
+from	O
+HEK293T	O
+cells	O
+co	O
+-	O
+transfected	O
+with	O
+constructs	O
+encoding	O
+the	O
+pyPY	O
+minigene	O
+and	O
+either	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+U2AF65	O
+or	O
+a	O
+triple	O
+U2AF65	O
+mutant	O
+(	O
+3Mut	B-mutant
+)	O
+of	O
+Q147A	B-mutant
+,	O
+R227A	B-mutant
+and	O
+V254P	B-mutant
+residues	O
+.	O
+(	O
+c	O
+)	O
+A	O
+bar	O
+graph	O
+of	O
+the	O
+average	O
+percentage	O
+of	O
+the	O
+py	O
+-	O
+spliced	O
+mRNA	O
+relative	O
+to	O
+total	O
+detected	O
+pyPY	O
+transcripts	O
+(	O
+spliced	O
+and	O
+unspliced	O
+)	O
+for	O
+the	O
+corresponding	O
+gel	O
+lanes	O
+(	O
+black	O
+,	O
+no	O
+U2AF65	O
+added	O
+;	O
+white	O
+,	O
+WT	O
+U2AF65	O
+;	O
+grey	O
+,	O
+3Mut	B-mutant
+U2AF65	O
+).	O
+	O
+Protein	O
+overexpression	O
+and	O
+qRT	O
+-	O
+PCR	O
+results	O
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+.	O
+	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+proteins	O
+were	O
+doubly	O
+labelled	O
+at	O
+A181C	B-mutant
+/	O
+Q324C	B-mutant
+such	O
+that	O
+a	O
+mixture	O
+of	O
+Cy3	O
+/	O
+Cy5	O
+fluorophores	O
+are	O
+expected	O
+to	O
+be	O
+present	O
+at	O
+each	O
+site	O
+.	O
+	O
+(	O
+c	O
+–	O
+f	O
+,	O
+i	O
+,	O
+j	O
+)	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+protein	O
+was	O
+immobilized	O
+on	O
+the	O
+microscope	O
+slide	O
+via	O
+biotin	O
+-	O
+NTA	O
+/	O
+Ni	O
++	O
+2	O
+(	O
+orange	O
+line	O
+)	O
+on	O
+a	O
+neutravidin	O
+(	O
+black	O
+X	O
+)-	O
+biotin	O
+-	O
+PEG	O
+(	O
+orange	O
+triangle	O
+)-	O
+treated	O
+surface	O
+and	O
+imaged	O
+either	O
+in	O
+the	O
+absence	O
+of	O
+ligands	O
+(	O
+c	O
+,	O
+d	O
+),	O
+in	O
+the	O
+presence	O
+of	O
+5	O
+μM	O
+AdML	O
+Py	O
+-	O
+tract	O
+RNA	O
+(	O
+5	O
+′-	O
+CCUUUUUUUUCC	O
+-	O
+3	O
+′)	O
+(	O
+e	O
+,	O
+f	O
+),	O
+or	O
+in	O
+the	O
+presence	O
+of	O
+10	O
+μM	O
+adenosine	O
+-	O
+interrupted	O
+variant	O
+RNA	O
+(	O
+5	O
+′-	O
+CUUUUUAAUUUCCA	O
+-	O
+3	O
+′)	O
+(	O
+i	O
+,	O
+j	O
+).	O
+	O
+The	O
+untethered	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+protein	O
+(	O
+1	O
+nM	O
+)	O
+was	O
+added	O
+to	O
+AdML	O
+RNA	O
+–	O
+polyethylene	O
+-	O
+glycol	O
+-	O
+linker	O
+–	O
+DNA	O
+oligonucleotide	O
+(	O
+10	O
+nM	O
+),	O
+which	O
+was	O
+immobilized	O
+on	O
+the	O
+microscope	O
+slide	O
+by	O
+annealing	O
+with	O
+a	O
+complementary	O
+biotinyl	O
+-	O
+DNA	O
+oligonucleotide	O
+(	O
+black	O
+vertical	O
+line	O
+).	O
+	O
+Typical	O
+single	O
+-	O
+molecule	O
+FRET	O
+traces	O
+(	O
+c	O
+,	O
+e	O
+,	O
+g	O
+,	O
+i	O
+)	O
+show	O
+fluorescence	O
+intensities	O
+from	O
+Cy3	O
+(	O
+green	O
+)	O
+and	O
+Cy5	O
+(	O
+red	O
+)	O
+and	O
+the	O
+calculated	O
+apparent	O
+FRET	O
+efficiency	O
+(	O
+blue	O
+).	O
+	O
+N	O
+is	O
+the	O
+number	O
+of	O
+single	O
+-	O
+molecule	O
+traces	O
+compiled	O
+for	O
+each	O
+histogram	O
+.	O
+	O
+Schematic	O
+models	O
+of	O
+U2AF65	O
+recognizing	O
+the	O
+Py	O
+tract	O
+.	O
+	O
+(	O
+a	O
+)	O
+Diagram	O
+of	O
+the	O
+U2AF65	O
+,	O
+SF1	O
+and	O
+U2AF35	O
+splicing	O
+factors	O
+bound	O
+to	O
+the	O
+consensus	O
+elements	O
+of	O
+the	O
+3	O
+′	O
+splice	O
+site	O
+.	O
+	O
+MDS	O
+-	O
+relevant	O
+mutated	O
+residues	O
+of	O
+U2AF65	O
+are	O
+shown	O
+as	O
+yellow	O
+spheres	O
+(	O
+L187	O
+and	O
+M144	O
+).	O
+	O
+Alternatively	O
+,	O
+a	O
+conformation	O
+of	O
+U2AF65	O
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+can	O
+directly	O
+bind	O
+to	O
+RNA	O
+;	O
+RNA	O
+binding	O
+stabilizes	O
+the	O
+‘	O
+open	O
+',	O
+side	O
+-	O
+by	O
+-	O
+side	O
+conformation	O
+and	O
+thus	O
+shifts	O
+the	O
+U2AF65	O
+population	O
+towards	O
+the	O
+∼	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+.	O
+	O
+RRM1	O
+,	O
+green	O
+;	O
+RRM2	O
+,	O
+pale	O
+blue	O
+;	O
+RRM	O
+extensions	O
+/	O
+linker	O
+,	O
+blue	O
+.	O
+	O
+Systematic	O
+analysis	O
+of	O
+radiation	O
+damage	O
+within	O
+a	O
+protein	O
+–	O
+RNA	O
+complex	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+MGy	O
+)	O
+reveals	O
+significant	O
+differential	O
+susceptibility	O
+of	O
+RNA	O
+and	O
+protein	O
+.	O
+	O
+A	O
+new	O
+method	O
+of	O
+difference	O
+electron	O
+-	O
+density	O
+quantification	O
+is	O
+presented	O
+.	O
+	O
+Radiation	O
+damage	O
+during	O
+macromolecular	O
+X	O
+-	O
+ray	O
+crystallographic	O
+data	O
+collection	O
+is	O
+still	O
+the	O
+main	O
+impediment	O
+for	O
+many	O
+macromolecular	O
+structure	O
+determinations	O
+.	O
+	O
+Although	O
+this	O
+has	O
+been	O
+well	O
+characterized	O
+within	O
+protein	O
+crystals	O
+,	O
+far	O
+less	O
+is	O
+known	O
+about	O
+specific	O
+damage	O
+effects	O
+within	O
+the	O
+larger	O
+class	O
+of	O
+nucleoprotein	O
+complexes	O
+.	O
+	O
+Here	O
+,	O
+a	O
+methodology	O
+has	O
+been	O
+developed	O
+whereby	O
+per	O
+-	O
+atom	O
+density	O
+changes	O
+could	O
+be	O
+quantified	O
+with	O
+increasing	O
+dose	O
+over	O
+a	O
+wide	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+.	O
+0	O
+MGy	O
+)	O
+range	O
+and	O
+at	O
+higher	O
+resolution	O
+(	O
+1	O
+.	O
+98	O
+Å	O
+)	O
+than	O
+the	O
+previous	O
+systematic	O
+specific	O
+damage	O
+study	O
+on	O
+a	O
+protein	O
+–	O
+DNA	O
+complex	O
+.	O
+	O
+Specific	O
+damage	O
+manifestations	O
+were	O
+determined	O
+within	O
+the	O
+large	O
+trp	O
+RNA	O
+-	O
+binding	O
+attenuation	O
+protein	O
+(	O
+TRAP	O
+)	O
+bound	O
+to	O
+a	O
+single	O
+-	O
+stranded	O
+RNA	O
+that	O
+forms	O
+a	O
+belt	O
+around	O
+the	O
+protein	O
+.	O
+	O
+The	O
+11	O
+-	O
+fold	O
+symmetry	O
+within	O
+each	O
+TRAP	O
+ring	O
+permitted	O
+statistically	O
+significant	O
+analysis	O
+of	O
+the	O
+Glu	O
+and	O
+Asp	O
+damage	O
+patterns	O
+,	O
+with	O
+RNA	O
+binding	O
+unexpectedly	O
+being	O
+observed	O
+to	O
+protect	O
+these	O
+otherwise	O
+highly	O
+sensitive	O
+residues	O
+within	O
+the	O
+11	O
+RNA	O
+-	O
+binding	O
+pockets	O
+distributed	O
+around	O
+the	O
+outside	O
+of	O
+the	O
+protein	O
+molecule	O
+.	O
+	O
+Additionally	O
+,	O
+the	O
+method	O
+enabled	O
+a	O
+quantification	O
+of	O
+the	O
+reduction	O
+in	O
+radiation	O
+-	O
+induced	O
+Lys	O
+and	O
+Phe	O
+disordering	O
+upon	O
+RNA	O
+binding	O
+directly	O
+from	O
+the	O
+electron	O
+density	O
+.	O
+	O
+Significant	O
+progress	O
+has	O
+been	O
+made	O
+in	O
+recent	O
+years	O
+in	O
+understanding	O
+the	O
+inevitable	O
+manifestations	O
+of	O
+X	O
+-	O
+ray	O
+-	O
+induced	O
+RD	O
+within	O
+protein	O
+crystals	O
+,	O
+and	O
+there	O
+is	O
+now	O
+a	O
+body	O
+of	O
+literature	O
+on	O
+possible	O
+strategies	O
+to	O
+mitigate	O
+the	O
+effects	O
+of	O
+RD	O
+(	O
+e	O
+.	O
+g	O
+.	O
+Zeldin	O
+,	O
+Brockhauser	O
+et	O
+al	O
+.,	O
+2013	O
+;	O
+Bourenkov	O
+&	O
+Popov	O
+,	O
+2010	O
+).	O
+	O
+However	O
+,	O
+there	O
+is	O
+still	O
+no	O
+general	O
+consensus	O
+within	O
+the	O
+field	O
+on	O
+how	O
+to	O
+minimize	O
+RD	O
+during	O
+MX	O
+data	O
+collection	O
+,	O
+and	O
+debates	O
+on	O
+the	O
+dependence	O
+of	O
+RD	O
+progression	O
+on	O
+incident	O
+X	O
+-	O
+ray	O
+energy	O
+(	O
+Shimizu	O
+et	O
+al	O
+.,	O
+2007	O
+;	O
+Liebschner	O
+et	O
+al	O
+.,	O
+2015	O
+)	O
+and	O
+the	O
+efficacy	O
+of	O
+radical	O
+scavengers	O
+(	O
+Allan	O
+et	O
+al	O
+.,	O
+2013	O
+)	O
+have	O
+yet	O
+to	O
+be	O
+resolved	O
+.	O
+	O
+Global	O
+radiation	O
+damage	O
+is	O
+observed	O
+within	O
+reciprocal	O
+space	O
+as	O
+the	O
+overall	O
+decay	O
+of	O
+the	O
+summed	O
+intensity	O
+of	O
+reflections	O
+detected	O
+within	O
+the	O
+diffraction	O
+pattern	O
+as	O
+dose	O
+increases	O
+(	O
+Garman	O
+,	O
+2010	O
+;	O
+Murray	O
+&	O
+Garman	O
+,	O
+2002	O
+).	O
+	O
+At	O
+100	O
+K	O
+,	O
+an	O
+experimental	O
+dose	O
+limit	O
+of	O
+30	O
+MGy	O
+has	O
+been	O
+recommended	O
+as	O
+an	O
+upper	O
+limit	O
+beyond	O
+which	O
+the	O
+biological	O
+information	O
+derived	O
+from	O
+any	O
+macromolecular	O
+crystal	O
+may	O
+be	O
+compromised	O
+(	O
+Owen	O
+et	O
+al	O
+.,	O
+2006	O
+).	O
+	O
+Indeed	O
+,	O
+the	O
+C	O
+—	O
+Se	O
+bond	O
+in	O
+selenomethionine	O
+,	O
+the	O
+stability	O
+of	O
+which	O
+is	O
+key	O
+for	O
+the	O
+success	O
+of	O
+experimental	O
+phasing	O
+methods	O
+,	O
+can	O
+be	O
+cleaved	O
+at	O
+a	O
+dose	O
+as	O
+low	O
+as	O
+2	O
+MGy	O
+for	O
+a	O
+crystal	O
+maintained	O
+at	O
+100	O
+K	O
+(	O
+Holton	O
+,	O
+2007	O
+).	O
+	O
+Active	O
+-	O
+site	O
+residues	O
+appear	O
+to	O
+be	O
+particularly	O
+susceptible	O
+,	O
+particularly	O
+for	O
+photosensitive	O
+proteins	O
+and	O
+in	O
+instances	O
+where	O
+chemical	O
+strain	O
+is	O
+an	O
+intrinsic	O
+feature	O
+of	O
+the	O
+reaction	O
+mechanism	O
+.	O
+	O
+For	O
+instance	O
+,	O
+structure	O
+determination	O
+of	O
+the	O
+purple	O
+membrane	O
+protein	O
+bacterio	O
+­	O
+rhodopsin	O
+required	O
+careful	O
+corrections	O
+for	O
+radiation	O
+-	O
+induced	O
+structural	O
+changes	O
+before	O
+the	O
+correct	O
+photosensitive	O
+intermediate	O
+states	O
+could	O
+be	O
+isolated	O
+(	O
+Matsui	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+	O
+The	O
+significant	O
+chemical	O
+strain	O
+required	O
+for	O
+catalysis	O
+within	O
+the	O
+active	O
+site	O
+of	O
+phosphoserine	O
+aminotransferase	O
+has	O
+been	O
+observed	O
+to	O
+diminish	O
+during	O
+X	O
+-	O
+ray	O
+exposure	O
+(	O
+Dubnovitsky	O
+et	O
+al	O
+.,	O
+2005	O
+).	O
+	O
+Since	O
+the	O
+majority	O
+of	O
+SRD	O
+studies	O
+to	O
+date	O
+have	O
+focused	O
+on	O
+proteins	O
+,	O
+much	O
+less	O
+is	O
+known	O
+about	O
+the	O
+effects	O
+of	O
+X	O
+-	O
+ray	O
+irradiation	O
+on	O
+the	O
+wider	O
+class	O
+of	O
+crystalline	O
+nucleoprotein	O
+complexes	O
+or	O
+how	O
+to	O
+correct	O
+for	O
+such	O
+radiation	O
+-	O
+induced	O
+structural	O
+changes	O
+.	O
+	O
+It	O
+is	O
+essential	O
+to	O
+understand	O
+how	O
+these	O
+increasingly	O
+complex	O
+macromolecular	O
+structures	O
+are	O
+affected	O
+by	O
+the	O
+radiation	O
+used	O
+to	O
+solve	O
+them	O
+.	O
+	O
+Nucleoproteins	O
+also	O
+represent	O
+one	O
+of	O
+the	O
+main	O
+targets	O
+of	O
+radiotherapy	O
+,	O
+and	O
+an	O
+insight	O
+into	O
+the	O
+damage	O
+mechanisms	O
+induced	O
+by	O
+X	O
+-	O
+ray	O
+irradiation	O
+could	O
+inform	O
+innovative	O
+treatments	O
+.	O
+	O
+Investigations	O
+on	O
+sub	O
+-	O
+ionization	O
+-	O
+level	O
+LEEs	O
+(	O
+0	O
+–	O
+15	O
+eV	O
+)	O
+interacting	O
+with	O
+both	O
+dried	O
+and	O
+aqueous	O
+oligonucleotides	O
+(	O
+Alizadeh	O
+&	O
+Sanche	O
+,	O
+2014	O
+;	O
+Simons	O
+,	O
+2006	O
+)	O
+concluded	O
+that	O
+resonant	O
+electron	O
+attachment	O
+to	O
+DNA	O
+bases	O
+and	O
+the	O
+sugar	O
+-	O
+phosphate	O
+backbone	O
+could	O
+lead	O
+to	O
+the	O
+preferential	O
+cleavage	O
+of	O
+strong	O
+(∼	O
+4	O
+eV	O
+,	O
+385	O
+kJ	O
+mol	O
+−	O
+1	O
+)	O
+sugar	O
+-	O
+phosphate	O
+C	O
+—	O
+O	O
+covalent	O
+bonds	O
+within	O
+the	O
+DNA	O
+backbone	O
+and	O
+then	O
+base	O
+-	O
+sugar	O
+N1	O
+—	O
+C	O
+bonds	O
+,	O
+eventually	O
+leading	O
+to	O
+single	O
+-	O
+strand	O
+breakages	O
+(	O
+SSBs	O
+;	O
+Ptasińska	O
+&	O
+Sanche	O
+,	O
+2007	O
+).	O
+	O
+Electrons	O
+have	O
+been	O
+shown	O
+to	O
+be	O
+mobile	O
+at	O
+77	O
+K	O
+by	O
+electron	O
+spin	O
+resonance	O
+spectroscopy	O
+studies	O
+(	O
+Symons	O
+,	O
+1997	O
+;	O
+Jones	O
+et	O
+al	O
+.,	O
+1987	O
+),	O
+with	O
+rapid	O
+electron	O
+quantum	O
+tunnelling	O
+and	O
+positive	O
+hole	O
+migration	O
+along	O
+the	O
+protein	O
+backbone	O
+and	O
+through	O
+stacked	O
+DNA	O
+bases	O
+indicated	O
+as	O
+a	O
+dominant	O
+mechanism	O
+by	O
+which	O
+oxidative	O
+and	O
+reductive	O
+damage	O
+localizes	O
+at	O
+distances	O
+from	O
+initial	O
+ionization	O
+sites	O
+at	O
+100	O
+K	O
+(	O
+O	O
+’	O
+Neill	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+	O
+Only	O
+at	O
+doses	O
+above	O
+20	O
+MGy	O
+were	O
+precursors	O
+of	O
+phosphodiester	O
+-	O
+bond	O
+cleavage	O
+observed	O
+within	O
+AT	O
+-	O
+rich	O
+regions	O
+of	O
+the	O
+35	O
+-	O
+mer	O
+DNA	O
+.	O
+	O
+For	O
+crystalline	O
+complexes	O
+such	O
+as	O
+C	O
+.	O
+Esp1396I	O
+,	O
+whether	O
+the	O
+protein	O
+is	O
+intrinsically	O
+more	O
+susceptible	O
+to	O
+X	O
+-	O
+ray	O
+-	O
+induced	O
+damage	O
+or	O
+whether	O
+the	O
+protein	O
+scavenges	O
+electrons	O
+to	O
+protect	O
+the	O
+DNA	O
+remains	O
+unclear	O
+in	O
+the	O
+absence	O
+of	O
+a	O
+non	O
+-	O
+nucleic	O
+acid	O
+-	O
+bound	O
+protein	O
+control	O
+obtained	O
+under	O
+exactly	O
+the	O
+same	O
+crystallization	O
+and	O
+data	O
+-	O
+collection	O
+conditions	O
+.	O
+	O
+To	O
+monitor	O
+the	O
+effects	O
+of	O
+nucleic	O
+acid	O
+binding	O
+on	O
+protein	O
+damage	O
+susceptibility	O
+,	O
+a	O
+crystal	O
+containing	O
+two	O
+protein	O
+molecules	O
+per	O
+asymmetric	O
+unit	O
+,	O
+only	O
+one	O
+of	O
+which	O
+was	O
+bound	O
+to	O
+RNA	O
+,	O
+is	O
+reported	O
+here	O
+(	O
+Fig	O
+.	O
+1	O
+▸).	O
+	O
+TRAP	O
+consists	O
+of	O
+11	O
+identical	O
+subunits	O
+assembled	O
+into	O
+a	O
+ring	O
+with	O
+11	O
+-	O
+fold	O
+rotational	O
+symmetry	O
+.	O
+	O
+In	O
+this	O
+structure	O
+,	O
+the	O
+bases	O
+of	O
+the	O
+G1	O
+-	O
+A2	O
+-	O
+G3	O
+nucleotides	O
+form	O
+direct	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+protein	O
+,	O
+unlike	O
+the	O
+U4	O
+-	O
+U5	O
+nucleotides	O
+,	O
+which	O
+appear	O
+to	O
+be	O
+more	O
+flexible	O
+.	O
+	O
+Ten	O
+successive	O
+1	O
+.	O
+98	O
+Å	O
+resolution	O
+MX	O
+data	O
+sets	O
+were	O
+collected	O
+from	O
+the	O
+same	O
+TRAP	O
+–	O
+RNA	O
+crystal	O
+to	O
+analyse	O
+X	O
+-	O
+ray	O
+-	O
+induced	O
+structural	O
+changes	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+d	O
+1	O
+=	O
+1	O
+.	O
+3	O
+MGy	O
+to	O
+d	O
+10	O
+=	O
+25	O
+.	O
+0	O
+MGy	O
+).	O
+	O
+To	O
+avoid	O
+the	O
+previous	O
+necessity	O
+for	O
+visual	O
+inspection	O
+of	O
+electron	O
+-	O
+density	O
+maps	O
+to	O
+detect	O
+SRD	O
+sites	O
+,	O
+a	O
+computational	O
+approach	O
+was	O
+designed	O
+to	O
+quantify	O
+the	O
+electron	O
+-	O
+density	O
+change	O
+for	O
+each	O
+refined	O
+atom	O
+with	O
+increasing	O
+dose	O
+,	O
+thus	O
+providing	O
+a	O
+rapid	O
+systematic	O
+method	O
+for	O
+SRD	O
+study	O
+on	O
+such	O
+large	O
+multimeric	O
+complexes	O
+.	O
+	O
+By	O
+employing	O
+the	O
+high	O
+11	O
+-	O
+fold	O
+structural	O
+symmetry	O
+within	O
+each	O
+TRAP	O
+macromolecule	O
+,	O
+this	O
+approach	O
+permitted	O
+a	O
+thorough	O
+statistical	O
+quantification	O
+of	O
+the	O
+RD	O
+effects	O
+of	O
+RNA	O
+binding	O
+to	O
+TRAP	O
+.	O
+	O
+Per	O
+-	O
+atom	O
+quantification	O
+of	O
+electron	O
+density	O
+	O
+To	O
+quantify	O
+the	O
+exact	O
+effects	O
+of	O
+nucleic	O
+acid	O
+binding	O
+to	O
+a	O
+protein	O
+on	O
+SRD	O
+susceptibility	O
+,	O
+a	O
+high	O
+-	O
+throughput	O
+and	O
+automated	O
+pipeline	O
+was	O
+created	O
+to	O
+systematically	O
+calculate	O
+the	O
+electron	O
+-	O
+density	O
+change	O
+for	O
+every	O
+refined	O
+atom	O
+within	O
+the	O
+TRAP	O
+–	O
+RNA	O
+structure	O
+as	O
+a	O
+function	O
+of	O
+dose	O
+.	O
+	O
+This	O
+provides	O
+an	O
+atom	O
+-	O
+specific	O
+quantification	O
+of	O
+density	O
+–	O
+dose	O
+dynamics	O
+,	O
+which	O
+was	O
+previously	O
+lacking	O
+within	O
+the	O
+field	O
+.	O
+	O
+However	O
+,	O
+these	O
+σ	O
+levels	O
+depend	O
+on	O
+the	O
+standard	O
+deviation	O
+values	O
+of	O
+the	O
+map	O
+,	O
+which	O
+can	O
+deviate	O
+between	O
+data	O
+sets	O
+,	O
+and	O
+are	O
+thus	O
+unsuitable	O
+for	O
+quantitative	O
+comparison	O
+of	O
+density	O
+between	O
+different	O
+dose	O
+data	O
+sets	O
+.	O
+	O
+Instead	O
+,	O
+we	O
+use	O
+here	O
+a	O
+maximum	O
+density	O
+-	O
+loss	O
+metric	O
+(	O
+D	O
+loss	O
+),	O
+which	O
+is	O
+the	O
+per	O
+-	O
+atom	O
+equivalent	O
+of	O
+the	O
+magnitude	O
+of	O
+these	O
+negative	O
+Fourier	O
+difference	O
+map	O
+peaks	O
+in	O
+units	O
+of	O
+e	O
+Å	O
+−	O
+3	O
+.	O
+	O
+Large	O
+positive	O
+D	O
+loss	O
+values	O
+indicate	O
+radiation	O
+-	O
+induced	O
+atomic	O
+disordering	O
+reproducibly	O
+throughout	O
+the	O
+unit	O
+cells	O
+with	O
+respect	O
+to	O
+the	O
+initial	O
+low	O
+-	O
+dose	O
+data	O
+set	O
+.	O
+	O
+For	O
+each	O
+TRAP	O
+–	O
+RNA	O
+data	O
+set	O
+,	O
+the	O
+D	O
+loss	O
+metric	O
+successfully	O
+identified	O
+the	O
+recognized	O
+forms	O
+of	O
+protein	O
+SRD	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+a	O
+),	O
+with	O
+clear	O
+Glu	O
+and	O
+Asp	O
+side	O
+-	O
+chain	O
+decarboxylation	O
+even	O
+in	O
+the	O
+first	O
+difference	O
+map	O
+calculated	O
+(	O
+3	O
+.	O
+9	O
+MGy	O
+;	O
+Fig	O
+.	O
+3	O
+▸	O
+a	O
+).	O
+	O
+The	O
+main	O
+sequence	O
+of	O
+TRAP	O
+does	O
+not	O
+contain	O
+any	O
+Trp	O
+and	O
+Cys	O
+residues	O
+(	O
+and	O
+thus	O
+contains	O
+no	O
+disulfide	O
+bonds	O
+).	O
+	O
+The	O
+substrate	O
+Trp	O
+amino	O
+-	O
+acid	O
+ligands	O
+also	O
+exhibited	O
+disordering	O
+of	O
+the	O
+free	O
+terminal	O
+carboxyl	O
+groups	O
+at	O
+higher	O
+doses	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+a	O
+);	O
+however	O
+,	O
+no	O
+clear	O
+Fourier	O
+difference	O
+peaks	O
+could	O
+be	O
+observed	O
+visually	O
+.	O
+	O
+Even	O
+for	O
+radiation	O
+-	O
+insensitive	O
+residues	O
+(	O
+e	O
+.	O
+g	O
+.	O
+Gly	O
+)	O
+the	O
+average	O
+D	O
+loss	O
+increases	O
+with	O
+dose	O
+:	O
+this	O
+is	O
+the	O
+effect	O
+of	O
+global	O
+radiation	O
+damage	O
+,	O
+since	O
+as	O
+dose	O
+increases	O
+the	O
+electron	O
+density	O
+associated	O
+with	O
+each	O
+refined	O
+atom	O
+becomes	O
+weaker	O
+as	O
+the	O
+atomic	O
+occupancy	O
+decreases	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+b	O
+).	O
+	O
+Only	O
+Glu	O
+and	O
+Asp	O
+residues	O
+exhibit	O
+a	O
+rate	O
+of	O
+D	O
+loss	O
+increase	O
+that	O
+consistently	O
+exceeds	O
+the	O
+average	O
+decay	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+b	O
+,	O
+dashed	O
+line	O
+)	O
+at	O
+each	O
+dose	O
+.	O
+	O
+The	O
+rate	O
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+D	O
+loss	O
+(	O
+attributed	O
+to	O
+side	O
+-	O
+chain	O
+decarboxylation	O
+)	O
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+consistently	O
+larger	O
+for	O
+Glu	O
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+Asp	O
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+the	O
+large	O
+dose	O
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+(	O
+Fig	O
+.	O
+2	O
+▸	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+S3	O
+);	O
+this	O
+observation	O
+is	O
+consistent	O
+with	O
+our	O
+calculations	O
+on	O
+model	O
+systems	O
+(	O
+see	O
+above	O
+)	O
+that	O
+suggest	O
+that	O
+,	O
+without	O
+considering	O
+differential	O
+hydrogen	O
+-	O
+bonding	O
+environments	O
+,	O
+CO2	O
+loss	O
+is	O
+more	O
+exothermic	O
+by	O
+around	O
+8	O
+kJ	O
+mol	O
+−	O
+1	O
+from	O
+oxidized	O
+Glu	O
+residues	O
+than	O
+from	O
+their	O
+Asp	O
+counterparts	O
+.	O
+	O
+RNA	O
+is	O
+less	O
+susceptible	O
+to	O
+electron	O
+-	O
+density	O
+loss	O
+than	O
+protein	O
+within	O
+the	O
+TRAP	O
+–	O
+RNA	O
+complex	O
+	O
+Only	O
+at	O
+the	O
+highest	O
+doses	O
+investigated	O
+(>	O
+20	O
+MGy	O
+)	O
+was	O
+density	O
+loss	O
+observed	O
+at	O
+the	O
+RNA	O
+phosphate	O
+and	O
+C	O
+—	O
+O	O
+bonds	O
+of	O
+the	O
+phosphodiester	O
+backbone	O
+.	O
+	O
+However	O
+,	O
+the	O
+median	O
+D	O
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+was	O
+lower	O
+by	O
+a	O
+factor	O
+of	O
+>	O
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+Glu	O
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+-	O
+chain	O
+groups	O
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+25	O
+.	O
+0	O
+MGy	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+S4	O
+),	O
+and	O
+furthermore	O
+could	O
+not	O
+be	O
+numerically	O
+distinguished	O
+from	O
+Gly	O
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+atoms	O
+within	O
+TRAP	O
+,	O
+which	O
+are	O
+not	O
+radiation	O
+-	O
+sensitive	O
+at	O
+the	O
+doses	O
+tested	O
+here	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+S3	O
+).	O
+	O
+RNA	O
+binding	O
+protects	O
+radiation	O
+-	O
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+residues	O
+	O
+For	O
+the	O
+large	O
+number	O
+of	O
+acidic	O
+residues	O
+per	O
+TRAP	O
+ring	O
+(	O
+four	O
+Asp	O
+and	O
+six	O
+Glu	O
+residues	O
+per	O
+protein	O
+monomer	O
+),	O
+a	O
+strong	O
+dependence	O
+of	O
+decarboxylation	O
+susceptibility	O
+on	O
+local	O
+environment	O
+was	O
+observed	O
+(	O
+Fig	O
+.	O
+4	O
+▸).	O
+	O
+For	O
+each	O
+Glu	O
+Cδ	O
+or	O
+Asp	O
+Cγ	O
+atom	O
+,	O
+D	O
+loss	O
+provided	O
+a	O
+direct	O
+measure	O
+of	O
+the	O
+rate	O
+of	O
+side	O
+-	O
+chain	O
+carboxyl	O
+-	O
+group	O
+disordering	O
+and	O
+subsequent	O
+decarboxylation	O
+.	O
+	O
+For	O
+acidic	O
+residues	O
+with	O
+no	O
+differing	O
+interactions	O
+between	O
+nonbound	O
+and	O
+bound	O
+TRAP	O
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+a	O
+),	O
+similar	O
+damage	O
+was	O
+apparent	O
+between	O
+the	O
+two	O
+rings	O
+within	O
+the	O
+asymmetric	O
+unit	O
+,	O
+as	O
+expected	O
+.	O
+	O
+However	O
+,	O
+TRAP	O
+residues	O
+directly	O
+on	O
+the	O
+RNA	O
+-	O
+binding	O
+interfaces	O
+exhibited	O
+greater	O
+damage	O
+accumulation	O
+in	O
+nonbound	O
+TRAP	O
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+b	O
+),	O
+and	O
+for	O
+residues	O
+at	O
+the	O
+ring	O
+–	O
+ring	O
+interfaces	O
+(	O
+where	O
+crystal	O
+contacts	O
+were	O
+detected	O
+)	O
+bound	O
+TRAP	O
+exhibited	O
+enhanced	O
+SRD	O
+accumulation	O
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+c	O
+).	O
+	O
+Hotelling	O
+’	O
+s	O
+T	O
+-	O
+squared	O
+test	O
+(	O
+the	O
+multivariate	O
+counterpart	O
+of	O
+Student	O
+’	O
+s	O
+t	O
+-	O
+test	O
+)	O
+was	O
+used	O
+to	O
+reject	O
+the	O
+null	O
+hypothesis	O
+that	O
+the	O
+means	O
+of	O
+the	O
+D	O
+loss	O
+metric	O
+were	O
+equal	O
+for	O
+the	O
+bound	O
+and	O
+nonbound	O
+groups	O
+in	O
+Fig	O
+.	O
+5	O
+▸.	O
+	O
+A	O
+significant	O
+reduction	O
+in	O
+D	O
+loss	O
+is	O
+seen	O
+for	O
+Glu36	O
+in	O
+RNA	O
+-	O
+bound	O
+compared	O
+with	O
+nonbound	O
+TRAP	O
+,	O
+indicative	O
+of	O
+a	O
+lower	O
+rate	O
+of	O
+side	O
+-	O
+chain	O
+decarboxylation	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+a	O
+;	O
+p	O
+=	O
+6	O
+.	O
+06	O
+×	O
+10	O
+−	O
+5	O
+).	O
+	O
+In	O
+our	O
+analysis	O
+,	O
+Asp39	O
+in	O
+the	O
+TRAP	O
+–(	O
+GAGUU	O
+)	O
+10GAG	O
+structure	O
+appears	O
+to	O
+exhibit	O
+two	O
+distinct	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+G1	O
+base	O
+within	O
+each	O
+of	O
+the	O
+11	O
+TRAP	O
+–	O
+RNA	O
+interfaces	O
+,	O
+as	O
+does	O
+Glu36	O
+to	O
+G3	O
+;	O
+however	O
+,	O
+the	O
+reduction	O
+in	O
+density	O
+disordering	O
+upon	O
+RNA	O
+binding	O
+is	O
+far	O
+less	O
+significant	O
+for	O
+Asp39	O
+than	O
+for	O
+Glu36	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+b	O
+,	O
+p	O
+=	O
+0	O
+.	O
+0925	O
+).	O
+	O
+RNA	O
+binding	O
+reduces	O
+radiation	O
+-	O
+induced	O
+disorder	O
+on	O
+the	O
+atomic	O
+scale	O
+	O
+One	O
+oxygen	O
+(	O
+O	O
+∊	O
+1	O
+)	O
+of	O
+Glu42	O
+appears	O
+to	O
+form	O
+a	O
+hydrogen	O
+bond	O
+to	O
+a	O
+nearby	O
+water	O
+within	O
+each	O
+TRAP	O
+RNA	O
+-	O
+binding	O
+pocket	O
+,	O
+with	O
+the	O
+other	O
+(	O
+O	O
+∊	O
+2	O
+)	O
+being	O
+involved	O
+in	O
+a	O
+salt	O
+-	O
+bridge	O
+interaction	O
+with	O
+Arg58	O
+(	O
+Hopcroft	O
+et	O
+al	O
+.,	O
+2002	O
+;	O
+Antson	O
+et	O
+al	O
+.,	O
+1999	O
+).	O
+	O
+A	O
+significant	O
+difference	O
+was	O
+observed	O
+between	O
+the	O
+D	O
+loss	O
+dynamics	O
+for	O
+the	O
+nonbound	O
+/	O
+bound	O
+Glu42	O
+O	O
+∊	O
+1	O
+atoms	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+c	O
+;	O
+p	O
+=	O
+0	O
+.	O
+007	O
+)	O
+but	O
+not	O
+for	O
+the	O
+Glu42	O
+O	O
+∊	O
+2	O
+atoms	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+d	O
+;	O
+p	O
+=	O
+0	O
+.	O
+239	O
+),	O
+indicating	O
+that	O
+the	O
+stabilizing	O
+strength	O
+of	O
+this	O
+salt	O
+-	O
+bridge	O
+interaction	O
+was	O
+conserved	O
+upon	O
+RNA	O
+binding	O
+and	O
+that	O
+the	O
+water	O
+-	O
+mediated	O
+hydrogen	O
+bond	O
+had	O
+a	O
+greater	O
+relative	O
+susceptibility	O
+to	O
+atomic	O
+disordering	O
+in	O
+the	O
+absence	O
+of	O
+RNA	O
+.	O
+	O
+The	O
+density	O
+-	O
+change	O
+dynamics	O
+were	O
+statistically	O
+indistinguishable	O
+between	O
+bound	O
+and	O
+nonbound	O
+TRAP	O
+for	O
+each	O
+Glu42	O
+carboxyl	O
+group	O
+Cδ	O
+atom	O
+(	O
+p	O
+=	O
+0	O
+.	O
+435	O
+),	O
+indicating	O
+that	O
+upon	O
+RNA	O
+binding	O
+the	O
+conserved	O
+salt	O
+-	O
+bridge	O
+interaction	O
+ultimately	O
+dictated	O
+the	O
+overall	O
+Glu42	O
+decarboxylation	O
+rate	O
+.	O
+	O
+The	O
+RNA	O
+-	O
+stabilizing	O
+effect	O
+was	O
+not	O
+restricted	O
+to	O
+radiation	O
+-	O
+sensitive	O
+acidic	O
+residues	O
+.	O
+	O
+The	O
+side	O
+chain	O
+of	O
+Phe32	O
+stacks	O
+against	O
+the	O
+G3	O
+base	O
+within	O
+the	O
+11	O
+TRAP	O
+RNA	O
+-	O
+binding	O
+interfaces	O
+(	O
+Antson	O
+et	O
+al	O
+.,	O
+1999	O
+).	O
+	O
+The	O
+D	O
+loss	O
+for	O
+Lys37	O
+side	O
+-	O
+chain	O
+atoms	O
+was	O
+also	O
+reduced	O
+when	O
+stacked	O
+against	O
+the	O
+G1	O
+base	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+f	O
+;	O
+p	O
+=	O
+0	O
+.	O
+0243	O
+for	O
+Lys37	O
+C	O
+∊	O
+atoms	O
+).	O
+	O
+Here	O
+,	O
+MX	O
+radiation	O
+-	O
+induced	O
+specific	O
+structural	O
+changes	O
+within	O
+the	O
+large	O
+TRAP	O
+–	O
+RNA	O
+assembly	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+.	O
+0	O
+MGy	O
+)	O
+have	O
+been	O
+analysed	O
+using	O
+a	O
+high	O
+-	O
+throughput	O
+quantitative	O
+approach	O
+,	O
+providing	O
+a	O
+measure	O
+of	O
+the	O
+electron	O
+-	O
+density	O
+distribution	O
+for	O
+each	O
+refined	O
+atom	O
+with	O
+increasing	O
+dose	O
+,	O
+D	O
+loss	O
+.	O
+	O
+Compared	O
+with	O
+previous	O
+studies	O
+,	O
+the	O
+results	O
+provide	O
+a	O
+further	O
+step	O
+in	O
+the	O
+detailed	O
+characterization	O
+of	O
+SRD	O
+effects	O
+in	O
+MX	O
+.	O
+	O
+Here	O
+,	O
+it	O
+provided	O
+the	O
+precision	O
+required	O
+to	O
+quantify	O
+the	O
+role	O
+of	O
+RNA	O
+in	O
+the	O
+damage	O
+susceptibilities	O
+of	O
+equivalent	O
+atoms	O
+between	O
+RNA	O
+-	O
+bound	O
+and	O
+nonbound	O
+TRAP	O
+,	O
+but	O
+it	O
+is	O
+applicable	O
+to	O
+any	O
+MX	O
+SRD	O
+study	O
+.	O
+	O
+RNA	O
+backbone	O
+disordering	O
+thus	O
+appears	O
+to	O
+be	O
+the	O
+main	O
+radiation	O
+-	O
+induced	O
+effect	O
+in	O
+RNA	O
+,	O
+with	O
+the	O
+protein	O
+–	O
+base	O
+interactions	O
+maintained	O
+even	O
+at	O
+high	O
+doses	O
+(>	O
+20	O
+MGy	O
+).	O
+	O
+The	O
+U4	O
+phosphate	O
+exhibited	O
+marginally	O
+larger	O
+D	O
+loss	O
+values	O
+above	O
+20	O
+MGy	O
+than	O
+G1	O
+,	O
+A2	O
+and	O
+G3	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+S4	O
+).	O
+	O
+Since	O
+U4	O
+is	O
+the	O
+only	O
+refined	O
+nucleotide	O
+not	O
+to	O
+exhibit	O
+significant	O
+base	O
+–	O
+protein	O
+interactions	O
+around	O
+TRAP	O
+(	O
+with	O
+a	O
+water	O
+-	O
+mediated	O
+hydrogen	O
+bond	O
+detected	O
+in	O
+only	O
+three	O
+of	O
+the	O
+11	O
+subunits	O
+and	O
+a	O
+single	O
+Arg58	O
+hydrogen	O
+bond	O
+suggested	O
+in	O
+a	O
+further	O
+four	O
+subunits	O
+),	O
+this	O
+increased	O
+U4	O
+D	O
+loss	O
+can	O
+be	O
+explained	O
+owing	O
+to	O
+its	O
+greater	O
+flexibility	O
+.	O
+	O
+At	O
+25	O
+.	O
+0	O
+MGy	O
+,	O
+the	O
+magnitude	O
+of	O
+the	O
+RNA	O
+backbone	O
+D	O
+loss	O
+was	O
+of	O
+the	O
+same	O
+order	O
+as	O
+for	O
+the	O
+radiation	O
+-	O
+insensitive	O
+Gly	O
+Cα	O
+atoms	O
+and	O
+on	O
+average	O
+less	O
+than	O
+half	O
+that	O
+of	O
+the	O
+acidic	O
+residues	O
+of	O
+the	O
+protein	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+S3	O
+).	O
+	O
+Consequently	O
+,	O
+no	O
+clear	O
+single	O
+-	O
+strand	O
+breaks	O
+could	O
+be	O
+located	O
+,	O
+and	O
+since	O
+RNA	O
+-	O
+binding	O
+within	O
+the	O
+current	O
+TRAP	O
+–(	O
+GAGUU	O
+)	O
+10GAG	O
+complex	O
+is	O
+mediated	O
+predominantly	O
+through	O
+base	O
+–	O
+protein	O
+interactions	O
+,	O
+the	O
+biological	O
+integrity	O
+of	O
+the	O
+RNA	O
+complex	O
+was	O
+dictated	O
+by	O
+the	O
+rate	O
+at	O
+which	O
+protein	O
+decarboxylation	O
+occurred	O
+.	O
+	O
+RNA	O
+interacting	O
+with	O
+TRAP	O
+was	O
+shown	O
+to	O
+offer	O
+significant	O
+protection	O
+against	O
+radiation	O
+-	O
+induced	O
+structural	O
+changes	O
+.	O
+	O
+However	O
+,	O
+compared	O
+with	O
+Asp39	O
+,	O
+Glu36	O
+is	O
+strikingly	O
+less	O
+decarboxylated	O
+when	O
+bound	O
+to	O
+RNA	O
+(	O
+Fig	O
+.	O
+4	O
+▸).	O
+	O
+For	O
+Glu36	O
+and	O
+Asp39	O
+,	O
+no	O
+direct	O
+quantitative	O
+correlation	O
+could	O
+be	O
+established	O
+between	O
+hydrogen	O
+-	O
+bond	O
+length	O
+and	O
+D	O
+loss	O
+(	O
+linear	O
+R	O
+2	O
+of	O
+<	O
+0	O
+.	O
+23	O
+for	O
+all	O
+doses	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+S5	O
+).	O
+	O
+Thus	O
+,	O
+another	O
+factor	O
+must	O
+be	O
+responsible	O
+for	O
+this	O
+clear	O
+reduction	O
+in	O
+Glu36	O
+CO2	O
+decarboxyl	O
+­	O
+ation	O
+in	O
+RNA	O
+-	O
+bound	O
+TRAP	O
+.	O
+	O
+The	O
+Glu36	O
+carboxyl	O
+side	O
+chain	O
+also	O
+potentially	O
+forms	O
+hydrogen	O
+bonds	O
+to	O
+His34	O
+and	O
+Lys56	O
+,	O
+but	O
+since	O
+these	O
+interactions	O
+are	O
+conserved	O
+irrespective	O
+of	O
+G3	O
+nucleotide	O
+binding	O
+,	O
+this	O
+cannot	O
+directly	O
+account	O
+for	O
+the	O
+stabilization	O
+effect	O
+on	O
+Glu36	O
+in	O
+RNA	O
+-	O
+bound	O
+TRAP	O
+.	O
+	O
+When	O
+bound	O
+to	O
+RNA	O
+,	O
+the	O
+average	O
+solvent	O
+-	O
+accessible	O
+area	O
+of	O
+the	O
+Glu36	O
+side	O
+-	O
+chain	O
+O	O
+atoms	O
+is	O
+reduced	O
+from	O
+∼	O
+15	O
+to	O
+0	O
+Å2	O
+.	O
+	O
+We	O
+propose	O
+that	O
+with	O
+no	O
+solvent	O
+accessibility	O
+Glu36	O
+decarboxylation	O
+is	O
+inhibited	O
+,	O
+since	O
+the	O
+CO2	O
+-	O
+formation	O
+rate	O
+K	O
+2	O
+is	O
+greatly	O
+reduced	O
+,	O
+and	O
+suggest	O
+that	O
+steric	O
+hindrance	O
+prevents	O
+each	O
+radicalized	O
+Glu36	O
+CO2	O
+group	O
+from	O
+achieving	O
+the	O
+planar	O
+conformation	O
+required	O
+for	O
+complete	O
+dissociation	O
+from	O
+TRAP	O
+.	O
+	O
+The	O
+electron	O
+-	O
+recombination	O
+rate	O
+K	O
+−	O
+1	O
+remains	O
+high	O
+,	O
+however	O
+,	O
+owing	O
+to	O
+rapid	O
+electron	O
+migration	O
+through	O
+the	O
+protein	O
+–	O
+RNA	O
+complex	O
+to	O
+refill	O
+the	O
+Glu36	O
+positive	O
+hole	O
+(	O
+the	O
+precursor	O
+for	O
+Glu	O
+decarboxylation	O
+).	O
+	O
+Upon	O
+RNA	O
+binding	O
+,	O
+the	O
+Asp39	O
+side	O
+-	O
+chain	O
+carboxyl	O
+group	O
+solvent	O
+-	O
+accessible	O
+area	O
+changes	O
+from	O
+∼	O
+75	O
+to	O
+35	O
+Å2	O
+,	O
+still	O
+allowing	O
+a	O
+high	O
+CO2	O
+-	O
+formation	O
+rate	O
+K	O
+2	O
+.	O
+	O
+By	O
+comparing	O
+equivalent	O
+acidic	O
+residues	O
+with	O
+and	O
+without	O
+RNA	O
+,	O
+we	O
+have	O
+now	O
+deconvoluted	O
+the	O
+role	O
+of	O
+solvent	O
+accessibility	O
+from	O
+other	O
+local	O
+protein	O
+environment	O
+factors	O
+,	O
+and	O
+thus	O
+propose	O
+a	O
+suitable	O
+mechanism	O
+by	O
+which	O
+exceptionally	O
+low	O
+solvent	O
+accessibility	O
+can	O
+reduce	O
+the	O
+rate	O
+of	O
+decarboxylation	O
+.	O
+	O
+Apart	O
+from	O
+these	O
+RNA	O
+-	O
+binding	O
+interfaces	O
+,	O
+RNA	O
+binding	O
+was	O
+seen	O
+to	O
+enhance	O
+decarboxylation	O
+for	O
+residues	O
+Glu50	O
+,	O
+Glu71	O
+and	O
+Glu73	O
+,	O
+all	O
+of	O
+which	O
+are	O
+involved	O
+in	O
+crystal	O
+contacts	O
+between	O
+TRAP	O
+rings	O
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+c	O
+).	O
+	O
+However	O
+,	O
+for	O
+each	O
+of	O
+these	O
+residues	O
+the	O
+exact	O
+crystal	O
+contacts	O
+are	O
+not	O
+preserved	O
+between	O
+bound	O
+and	O
+nonbound	O
+TRAP	O
+or	O
+even	O
+between	O
+monomers	O
+within	O
+one	O
+TRAP	O
+ring	O
+.	O
+	O
+For	O
+example	O
+,	O
+in	O
+bound	O
+TRAP	O
+,	O
+Glu73	O
+hydrogen	O
+-	O
+bonds	O
+to	O
+a	O
+nearby	O
+lysine	O
+on	O
+each	O
+of	O
+the	O
+11	O
+subunits	O
+,	O
+whereas	O
+in	O
+nonbound	O
+TRAP	O
+no	O
+such	O
+interaction	O
+exists	O
+and	O
+Glu73	O
+interacts	O
+with	O
+a	O
+variable	O
+number	O
+of	O
+refined	O
+waters	O
+in	O
+each	O
+subunit	O
+.	O
+	O
+Radiation	O
+-	O
+induced	O
+side	O
+-	O
+chain	O
+conformational	O
+changes	O
+have	O
+been	O
+poorly	O
+characterized	O
+in	O
+previous	O
+SRD	O
+investigations	O
+owing	O
+to	O
+their	O
+strong	O
+dependence	O
+on	O
+packing	O
+density	O
+and	O
+geometric	O
+strain	O
+.	O
+	O
+Such	O
+structural	O
+changes	O
+are	O
+known	O
+to	O
+have	O
+significant	O
+roles	O
+within	O
+enzymatic	O
+pathways	O
+,	O
+and	O
+experimenters	O
+must	O
+be	O
+aware	O
+of	O
+these	O
+possible	O
+confounding	O
+factors	O
+when	O
+assigning	O
+true	O
+functional	O
+mechanisms	O
+using	O
+MX	O
+.	O
+	O
+Our	O
+results	O
+show	O
+that	O
+RNA	O
+binding	O
+to	O
+TRAP	O
+physically	O
+stabilizes	O
+non	O
+-	O
+acidic	O
+residues	O
+within	O
+the	O
+TRAP	O
+macromolecule	O
+,	O
+most	O
+notably	O
+Lys37	O
+and	O
+Phe32	O
+,	O
+which	O
+stack	O
+against	O
+the	O
+G1	O
+and	O
+G3	O
+bases	O
+,	O
+respectively	O
+.	O
+	O
+It	O
+has	O
+been	O
+suggested	O
+(	O
+Burmeister	O
+,	O
+2000	O
+)	O
+that	O
+Tyr	O
+residues	O
+can	O
+lose	O
+their	O
+aromatic	O
+–	O
+OH	O
+group	O
+owing	O
+to	O
+radiation	O
+-	O
+induced	O
+effects	O
+;	O
+however	O
+,	O
+no	O
+energetically	O
+favourable	O
+pathway	O
+for	O
+–	O
+OH	O
+cleavage	O
+exists	O
+and	O
+this	O
+has	O
+not	O
+been	O
+detected	O
+in	O
+aqueous	O
+radiation	O
+-	O
+chemistry	O
+studies	O
+.	O
+	O
+In	O
+TRAP	O
+,	O
+D	O
+loss	O
+increased	O
+at	O
+a	O
+similar	O
+rate	O
+for	O
+both	O
+the	O
+Tyr	O
+O	O
+atoms	O
+and	O
+aromatic	O
+ring	O
+atoms	O
+,	O
+suggesting	O
+that	O
+full	O
+ring	O
+conformational	O
+disordering	O
+is	O
+more	O
+likely	O
+.	O
+	O
+Indeed	O
+,	O
+no	O
+convincing	O
+reproducible	O
+Fourier	O
+difference	O
+peaks	O
+above	O
+the	O
+background	O
+map	O
+noise	O
+were	O
+observed	O
+around	O
+any	O
+Tyr	O
+terminal	O
+–	O
+OH	O
+groups	O
+.	O
+	O
+The	O
+RNA	O
+-	O
+stabilization	O
+effects	O
+on	O
+protein	O
+are	O
+observed	O
+at	O
+short	O
+ranges	O
+and	O
+are	O
+restricted	O
+to	O
+within	O
+the	O
+RNA	O
+-	O
+binding	O
+interfaces	O
+around	O
+the	O
+TRAP	O
+ring	O
+.	O
+	O
+An	O
+increase	O
+in	O
+the	O
+dose	O
+at	O
+which	O
+functionally	O
+important	O
+residues	O
+remain	O
+intact	O
+has	O
+biological	O
+ramifications	O
+for	O
+understanding	O
+the	O
+mechanisms	O
+at	O
+which	O
+ionizing	O
+radiation	O
+damage	O
+is	O
+mitigated	O
+within	O
+naturally	O
+forming	O
+DNA	O
+–	O
+protein	O
+and	O
+RNA	O
+–	O
+protein	O
+complexes	O
+.	O
+	O
+In	O
+these	O
+studies	O
+,	O
+the	O
+main	O
+damaging	O
+species	O
+is	O
+predicted	O
+to	O
+be	O
+the	O
+oxidizing	O
+hydroxyl	O
+radical	O
+produced	O
+through	O
+solvent	O
+irradiation	O
+,	O
+which	O
+is	O
+known	O
+to	O
+add	O
+to	O
+double	O
+covalent	O
+bonds	O
+within	O
+both	O
+DNA	O
+and	O
+RNA	O
+bases	O
+to	O
+induce	O
+strand	O
+breaks	O
+and	O
+base	O
+modification	O
+(	O
+Spotheim	O
+-	O
+Maurizot	O
+&	O
+Davídková	O
+,	O
+2011	O
+;	O
+Chance	O
+et	O
+al	O
+.,	O
+1997	O
+).	O
+	O
+It	O
+was	O
+suggested	O
+that	O
+physical	O
+screening	O
+of	O
+DNA	O
+by	O
+protein	O
+shielded	O
+the	O
+DNA	O
+–	O
+protein	O
+interaction	O
+sites	O
+from	O
+radical	O
+damage	O
+,	O
+yielding	O
+an	O
+extended	O
+life	O
+-	O
+dose	O
+for	O
+the	O
+nucleoprotein	O
+complex	O
+compared	O
+with	O
+separate	O
+protein	O
+and	O
+DNA	O
+constituents	O
+at	O
+RT	O
+.	O
+	O
+The	O
+results	O
+presented	O
+here	O
+suggest	O
+that	O
+biologically	O
+relevant	O
+nucleoprotein	O
+complexes	O
+also	O
+exhibit	O
+prolonged	O
+life	O
+-	O
+doses	O
+under	O
+the	O
+effect	O
+of	O
+LEE	O
+-	O
+induced	O
+structural	O
+changes	O
+,	O
+involving	O
+direct	O
+physical	O
+protection	O
+of	O
+key	O
+RNA	O
+-	O
+binding	O
+residues	O
+.	O
+	O
+Such	O
+reduced	O
+radiation	O
+-	O
+sensitivity	O
+in	O
+this	O
+case	O
+ensures	O
+that	O
+the	O
+interacting	O
+protein	O
+remains	O
+bound	O
+long	O
+enough	O
+to	O
+the	O
+RNA	O
+to	O
+complete	O
+its	O
+function	O
+,	O
+even	O
+whilst	O
+exposed	O
+to	O
+ionizing	O
+radiation	O
+.	O
+	O
+Within	O
+the	O
+nonbound	O
+TRAP	O
+macromolecule	O
+,	O
+the	O
+acidic	O
+residues	O
+within	O
+the	O
+unoccupied	O
+RNA	O
+-	O
+binding	O
+interfaces	O
+(	O
+Asp39	O
+,	O
+Glu36	O
+,	O
+Glu42	O
+)	O
+are	O
+notably	O
+amongst	O
+the	O
+most	O
+susceptible	O
+residues	O
+within	O
+the	O
+asymmetric	O
+unit	O
+(	O
+Fig	O
+.	O
+4	O
+▸).	O
+	O
+When	O
+exposed	O
+to	O
+X	O
+-	O
+rays	O
+,	O
+these	O
+residues	O
+will	O
+be	O
+preferentially	O
+damaged	O
+by	O
+X	O
+-	O
+rays	O
+and	O
+subsequently	O
+reduce	O
+the	O
+affinity	O
+with	O
+which	O
+TRAP	O
+binds	O
+to	O
+RNA	O
+.	O
+	O
+Within	O
+the	O
+cellular	O
+environment	O
+,	O
+this	O
+mechanism	O
+could	O
+reduce	O
+the	O
+risk	O
+that	O
+radiation	O
+-	O
+damaged	O
+proteins	O
+might	O
+bind	O
+to	O
+RNA	O
+,	O
+thus	O
+avoiding	O
+the	O
+detrimental	O
+introduction	O
+of	O
+incorrect	O
+DNA	O
+-	O
+repair	O
+,	O
+transcriptional	O
+and	O
+base	O
+-	O
+modification	O
+pathways	O
+.	O
+	O
+The	O
+TRAP	O
+–(	O
+GAGUU	O
+)	O
+10GAG	O
+complex	O
+asymmetric	O
+unit	O
+(	O
+PDB	O
+entry	O
+1gtf	O
+;	O
+Hopcroft	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+	O
+Bound	O
+tryptophan	O
+ligands	O
+are	O
+represented	O
+as	O
+coloured	O
+spheres	O
+.	O
+	O
+(	O
+a	O
+)	O
+Electron	O
+-	O
+density	O
+loss	O
+sites	O
+as	O
+indicated	O
+by	O
+D	O
+loss	O
+in	O
+the	O
+TRAP	O
+–	O
+RNA	O
+complex	O
+crystal	O
+by	O
+residue	O
+/	O
+nucleotide	O
+type	O
+for	O
+five	O
+doses	O
+[	O
+sites	O
+determined	O
+above	O
+the	O
+4	O
+×	O
+average	O
+D	O
+loss	O
+threshold	O
+,	O
+calculated	O
+over	O
+the	O
+TRAP	O
+–	O
+RNA	O
+structure	O
+for	O
+the	O
+first	O
+difference	O
+map	O
+:	O
+F	O
+obs	O
+(	O
+d	O
+2	O
+)	O
+−	O
+F	O
+obs	O
+(	O
+d	O
+1	O
+)].	O
+	O
+Only	O
+a	O
+subset	O
+of	O
+key	O
+TRAP	O
+residue	O
+types	O
+are	O
+included	O
+.	O
+	O
+The	O
+average	O
+D	O
+loss	O
+(	O
+calculated	O
+over	O
+the	O
+whole	O
+TRAP	O
+asymmetric	O
+unit	O
+)	O
+is	O
+shown	O
+at	O
+each	O
+dose	O
+(	O
+dashed	O
+line	O
+).	O
+	O
+In	O
+(	O
+a	O
+)	O
+clear	O
+difference	O
+density	O
+is	O
+observed	O
+around	O
+the	O
+Glu42	O
+carboxyl	O
+side	O
+chain	O
+in	O
+chain	O
+H	O
+,	O
+within	O
+the	O
+lowest	O
+dose	O
+difference	O
+map	O
+at	O
+d	O
+2	O
+=	O
+3	O
+.	O
+9	O
+MGy	O
+.	O
+	O
+Radiation	O
+-	O
+induced	O
+protein	O
+disordering	O
+is	O
+evident	O
+across	O
+the	O
+large	O
+dose	O
+range	O
+(	O
+b	O
+,	O
+c	O
+);	O
+in	O
+comparison	O
+,	O
+no	O
+clear	O
+deterioration	O
+of	O
+the	O
+RNA	O
+density	O
+was	O
+observed	O
+.	O
+	O
+D	O
+loss	O
+calculated	O
+for	O
+all	O
+side	O
+-	O
+chain	O
+carboxyl	O
+group	O
+Glu	O
+Cδ	O
+and	O
+Asp	O
+Cγ	O
+atoms	O
+within	O
+the	O
+TRAP	O
+–	O
+RNA	O
+complex	O
+for	O
+a	O
+dose	O
+of	O
+19	O
+.	O
+3	O
+MGy	O
+(	O
+d	O
+8	O
+).	O
+	O
+D	O
+loss	O
+against	O
+dose	O
+for	O
+(	O
+a	O
+)	O
+Glu36	O
+Cδ	O
+,	O
+(	O
+b	O
+)	O
+Asp39	O
+Cγ	O
+,	O
+(	O
+c	O
+)	O
+Glu42	O
+O	O
+∊	O
+1	O
+,	O
+(	O
+d	O
+)	O
+Glu42	O
+O	O
+∊	O
+2	O
+,	O
+(	O
+e	O
+)	O
+Phe32	O
+Cζ	O
+and	O
+(	O
+f	O
+)	O
+Lys37	O
+C	O
+∊	O
+atoms	O
+.	O
+	O
+95	O
+%	O
+CI	O
+are	O
+included	O
+for	O
+each	O
+set	O
+of	O
+11	O
+equivalent	O
+atoms	O
+grouped	O
+as	O
+bound	O
+/	O
+nonbound	O
+.	O
+	O
+RNA	O
+-	O
+binding	O
+interface	O
+interactions	O
+are	O
+shown	O
+for	O
+TRAP	O
+chain	O
+N	O
+,	O
+with	O
+the	O
+F	O
+obs	O
+(	O
+d	O
+7	O
+)	O
+−	O
+F	O
+obs	O
+(	O
+d	O
+1	O
+)	O
+Fourier	O
+difference	O
+map	O
+(	O
+dose	O
+16	O
+.	O
+7	O
+MGy	O
+)	O
+overlaid	O
+and	O
+contoured	O
+at	O
+a	O
+±	O
+4σ	O
+level	O
+.	O
+	O
+A	O
+conserved	O
+motif	O
+in	O
+JNK	O
+/	O
+p38	O
+-	O
+specific	O
+MAPK	O
+phosphatases	O
+as	O
+a	O
+determinant	O
+for	O
+JNK1	O
+recognition	O
+and	O
+inactivation	O
+	O
+Mitogen	O
+-	O
+activated	O
+protein	O
+kinases	O
+(	O
+MAPKs	O
+),	O
+important	O
+in	O
+a	O
+large	O
+array	O
+of	O
+signalling	O
+pathways	O
+,	O
+are	O
+tightly	O
+controlled	O
+by	O
+a	O
+cascade	O
+of	O
+protein	O
+kinases	O
+and	O
+by	O
+MAPK	O
+phosphatases	O
+(	O
+MKPs	O
+).	O
+	O
+MAPK	O
+signalling	O
+efficiency	O
+and	O
+specificity	O
+is	O
+modulated	O
+by	O
+protein	O
+–	O
+protein	O
+interactions	O
+between	O
+individual	O
+MAPKs	O
+and	O
+the	O
+docking	O
+motifs	O
+in	O
+cognate	O
+binding	O
+partners	O
+.	O
+	O
+Two	O
+types	O
+of	O
+docking	O
+interactions	O
+have	O
+been	O
+identified	O
+:	O
+D	O
+-	O
+motif	O
+-	O
+mediated	O
+interaction	O
+and	O
+FXF	O
+-	O
+docking	O
+interaction	O
+.	O
+	O
+Here	O
+we	O
+report	O
+the	O
+crystal	O
+structure	O
+of	O
+JNK1	O
+bound	O
+to	O
+the	O
+catalytic	O
+domain	O
+of	O
+MKP7	O
+at	O
+2	O
+.	O
+4	O
+-	O
+Å	O
+resolution	O
+,	O
+providing	O
+high	O
+-	O
+resolution	O
+structural	O
+insight	O
+into	O
+the	O
+FXF	O
+-	O
+docking	O
+interaction	O
+.	O
+	O
+The	O
+285FNFL288	O
+segment	O
+in	O
+MKP7	O
+directly	O
+binds	O
+to	O
+a	O
+hydrophobic	O
+site	O
+on	O
+JNK1	O
+that	O
+is	O
+near	O
+the	O
+MAPK	O
+insertion	O
+and	O
+helix	O
+αG	O
+.	O
+Biochemical	O
+studies	O
+further	O
+reveal	O
+that	O
+this	O
+highly	O
+conserved	O
+structural	O
+motif	O
+is	O
+present	O
+in	O
+all	O
+members	O
+of	O
+the	O
+MKP	O
+family	O
+,	O
+and	O
+the	O
+interaction	O
+mode	O
+is	O
+universal	O
+and	O
+critical	O
+for	O
+the	O
+MKP	O
+-	O
+MAPK	O
+recognition	O
+and	O
+biological	O
+function	O
+.	O
+	O
+The	O
+important	O
+MAPK	O
+family	O
+of	O
+signalling	O
+proteins	O
+is	O
+controlled	O
+by	O
+MAPK	O
+phosphatases	O
+(	O
+MKPs	O
+).	O
+	O
+Here	O
+,	O
+the	O
+authors	O
+report	O
+the	O
+structure	O
+of	O
+MKP7	O
+bound	O
+to	O
+JNK1	O
+and	O
+characterise	O
+the	O
+conserved	O
+MKP	O
+-	O
+MAPK	O
+interaction	O
+.	O
+	O
+The	O
+mitogen	O
+-	O
+activated	O
+protein	O
+kinases	O
+(	O
+MAPKs	O
+)	O
+are	O
+central	O
+components	O
+of	O
+the	O
+signal	O
+-	O
+transduction	O
+pathways	O
+,	O
+which	O
+mediate	O
+the	O
+cellular	O
+response	O
+to	O
+a	O
+variety	O
+of	O
+extracellular	O
+stimuli	O
+,	O
+ranging	O
+from	O
+growth	O
+factors	O
+to	O
+environmental	O
+stresses	O
+.	O
+	O
+The	O
+MAPK	O
+signalling	O
+pathways	O
+are	O
+evolutionally	O
+highly	O
+conserved	O
+.	O
+	O
+The	O
+basic	O
+assembly	O
+of	O
+MAPK	O
+pathways	O
+is	O
+a	O
+three	O
+-	O
+tier	O
+kinase	O
+module	O
+that	O
+establishes	O
+a	O
+sequential	O
+activation	O
+cascade	O
+:	O
+a	O
+MAPK	O
+kinase	O
+kinase	O
+activates	O
+a	O
+MAPK	O
+kinase	O
+,	O
+which	O
+in	O
+turn	O
+activates	O
+a	O
+MAPK	O
+.	O
+	O
+The	O
+MAPKs	O
+are	O
+activated	O
+by	O
+MAPK	O
+kinases	O
+that	O
+phosphorylate	O
+the	O
+MAPKs	O
+at	O
+conserved	O
+threonine	O
+and	O
+tyrosine	O
+residues	O
+within	O
+their	O
+activation	O
+loop	O
+.	O
+	O
+After	O
+activation	O
+,	O
+each	O
+MAPK	O
+phosphorylates	O
+a	O
+distinct	O
+set	O
+of	O
+protein	O
+substrates	O
+,	O
+which	O
+act	O
+as	O
+the	O
+critical	O
+effectors	O
+that	O
+enable	O
+cells	O
+to	O
+mount	O
+the	O
+appropriate	O
+responses	O
+to	O
+varied	O
+stimuli	O
+.	O
+	O
+MAPKs	O
+lie	O
+at	O
+the	O
+bottom	O
+of	O
+conserved	O
+three	O
+-	O
+component	O
+phosphorylation	O
+cascades	O
+and	O
+utilize	O
+docking	O
+interactions	O
+to	O
+link	O
+module	O
+components	O
+and	O
+bind	O
+substrates	O
+.	O
+	O
+Two	O
+types	O
+of	O
+docking	O
+motifs	O
+have	O
+been	O
+identified	O
+in	O
+MAPK	O
+substrates	O
+and	O
+cognate	O
+proteins	O
+:	O
+kinase	O
+-	O
+interacting	O
+motif	O
+(	O
+D	O
+-	O
+motif	O
+)	O
+and	O
+FXF	O
+-	O
+motif	O
+(	O
+also	O
+called	O
+DEF	O
+motif	O
+,	O
+docking	O
+site	O
+for	O
+ERK	O
+FXF	O
+).	O
+	O
+The	O
+best	O
+-	O
+studied	O
+docking	O
+interactions	O
+are	O
+those	O
+between	O
+MAP	O
+kinases	O
+and	O
+‘	O
+D	O
+-	O
+motifs	O
+',	O
+which	O
+consists	O
+of	O
+two	O
+or	O
+more	O
+basic	O
+residues	O
+followed	O
+by	O
+a	O
+short	O
+linker	O
+and	O
+a	O
+cluster	O
+of	O
+hydrophobic	O
+residues	O
+.	O
+	O
+The	O
+D	O
+-	O
+motif	O
+-	O
+docking	O
+site	O
+(	O
+D	O
+-	O
+site	O
+)	O
+in	O
+MAPKs	O
+is	O
+situated	O
+in	O
+a	O
+noncatalytic	O
+region	O
+opposite	O
+of	O
+the	O
+kinase	O
+catalytic	O
+pocket	O
+and	O
+is	O
+comprised	O
+of	O
+a	O
+highly	O
+acidic	O
+patch	O
+and	O
+a	O
+hydrophobic	O
+groove	O
+.	O
+	O
+D	O
+-	O
+motifs	O
+are	O
+found	O
+in	O
+many	O
+MAPK	O
+-	O
+interacting	O
+proteins	O
+,	O
+including	O
+substrates	O
+,	O
+activating	O
+kinases	O
+and	O
+inactivating	O
+phosphatases	O
+,	O
+as	O
+well	O
+as	O
+scaffolding	O
+proteins	O
+.	O
+	O
+A	O
+second	O
+docking	O
+motif	O
+for	O
+MAPKs	O
+consists	O
+of	O
+two	O
+Phe	O
+residues	O
+separated	O
+by	O
+one	O
+residue	O
+(	O
+FXF	O
+-	O
+motif	O
+).	O
+	O
+This	O
+motif	O
+has	O
+been	O
+observed	O
+in	O
+several	O
+MAPK	O
+substrates	O
+.	O
+	O
+The	O
+FXF	O
+-	O
+motif	O
+-	O
+binding	O
+site	O
+of	O
+ERK2	O
+has	O
+been	O
+mapped	O
+to	O
+a	O
+hydrophobic	O
+pocket	O
+formed	O
+between	O
+the	O
+P	O
++	O
+1	O
+site	O
+,	O
+αG	O
+helix	O
+and	O
+the	O
+MAPK	O
+insert	O
+.	O
+	O
+However	O
+,	O
+the	O
+generality	O
+and	O
+mechanism	O
+of	O
+the	O
+FXF	O
+-	O
+mediated	O
+interaction	O
+is	O
+unclear	O
+.	O
+	O
+The	O
+physiological	O
+outcome	O
+of	O
+MAPK	O
+signalling	O
+depends	O
+on	O
+both	O
+the	O
+magnitude	O
+and	O
+the	O
+duration	O
+of	O
+kinase	O
+activation	O
+.	O
+	O
+Downregulation	O
+of	O
+MAPK	O
+activity	O
+can	O
+be	O
+achieved	O
+through	O
+direct	O
+dephosphorylation	O
+of	O
+the	O
+phospho	O
+-	O
+threonine	O
+and	O
+/	O
+or	O
+tyrosine	O
+residues	O
+by	O
+various	O
+serine	O
+/	O
+threonine	O
+phosphatases	O
+,	O
+tyrosine	O
+phosphatases	O
+and	O
+dual	O
+-	O
+specificity	O
+phosphatases	O
+(	O
+DUSPs	O
+)	O
+termed	O
+MKPs	O
+.	O
+	O
+Dysregulated	O
+expression	O
+of	O
+MKPs	O
+has	O
+been	O
+associated	O
+with	O
+pathogenesis	O
+of	O
+various	O
+diseases	O
+,	O
+and	O
+understanding	O
+their	O
+precise	O
+recognition	O
+mechanism	O
+presents	O
+an	O
+important	O
+challenge	O
+and	O
+opportunity	O
+for	O
+drug	O
+development	O
+.	O
+	O
+Here	O
+,	O
+we	O
+present	O
+the	O
+crystal	O
+structure	O
+of	O
+JNK1	O
+in	O
+complex	O
+with	O
+the	O
+catalytic	O
+domain	O
+of	O
+MKP7	O
+.	O
+	O
+In	O
+the	O
+JNK1	O
+–	O
+MKP7	O
+complex	O
+,	O
+a	O
+hydrophobic	O
+motif	O
+(	O
+285FNFL288	O
+)	O
+that	O
+initiates	O
+the	O
+helix	O
+α5	O
+in	O
+the	O
+MKP7	O
+catalytic	O
+domain	O
+directly	O
+binds	O
+to	O
+the	O
+FXF	O
+-	O
+motif	O
+-	O
+binding	O
+site	O
+on	O
+JNK1	O
+,	O
+providing	O
+the	O
+structural	O
+insight	O
+into	O
+the	O
+classic	O
+FXF	O
+-	O
+type	O
+docking	O
+interaction	O
+.	O
+	O
+Thus	O
+,	O
+our	O
+study	O
+reveals	O
+a	O
+hitherto	O
+unrecognized	O
+interaction	O
+mode	O
+for	O
+encoding	O
+complex	O
+target	O
+specificity	O
+among	O
+MAPK	O
+isoforms	O
+.	O
+	O
+Interaction	O
+of	O
+JNK1	O
+with	O
+the	O
+MKP7	O
+catalytic	O
+domain	O
+	O
+DUSPs	O
+belong	O
+to	O
+the	O
+protein	O
+-	O
+tyrosine	O
+phosphatases	O
+(	O
+PTPase	O
+)	O
+superfamily	O
+,	O
+which	O
+is	O
+defined	O
+by	O
+the	O
+PTPase	O
+-	O
+signature	O
+motif	O
+CXXGXXR	O
+.	O
+	O
+MKPs	O
+represent	O
+a	O
+distinct	O
+subfamily	O
+within	O
+a	O
+larger	O
+group	O
+of	O
+DUSPs	O
+.	O
+	O
+All	O
+MKPs	O
+contain	O
+a	O
+highly	O
+conserved	O
+C	O
+-	O
+terminal	O
+catalytic	O
+domain	O
+(	O
+CD	O
+)	O
+and	O
+an	O
+N	O
+-	O
+terminal	O
+kinase	O
+-	O
+binding	O
+domain	O
+(	O
+KBD	O
+).	O
+	O
+The	O
+KBD	O
+is	O
+homologous	O
+to	O
+the	O
+rhodanese	O
+family	O
+and	O
+contains	O
+an	O
+intervening	O
+cluster	O
+of	O
+basic	O
+amino	O
+acids	O
+,	O
+which	O
+has	O
+been	O
+suggested	O
+to	O
+be	O
+important	O
+for	O
+interacting	O
+with	O
+the	O
+target	O
+MAPKs	O
+.	O
+	O
+Biochemical	O
+and	O
+structural	O
+studies	O
+have	O
+revealed	O
+that	O
+the	O
+KBD	O
+of	O
+MKPs	O
+is	O
+critical	O
+for	O
+MKP3	O
+docking	O
+to	O
+ERK2	O
+,	O
+and	O
+MKP5	O
+binding	O
+to	O
+p38α	O
+,	O
+although	O
+their	O
+binding	O
+mechanisms	O
+are	O
+completely	O
+different	O
+.	O
+	O
+However	O
+,	O
+it	O
+is	O
+unknown	O
+if	O
+other	O
+MAPKs	O
+can	O
+interact	O
+with	O
+the	O
+KBD	O
+of	O
+their	O
+cognate	O
+phosphatases	O
+in	O
+the	O
+same	O
+manner	O
+as	O
+observed	O
+for	O
+recognition	O
+of	O
+ERK2	O
+and	O
+p38α	O
+by	O
+their	O
+MKPs	O
+,	O
+or	O
+whether	O
+they	O
+recognize	O
+distinct	O
+docking	O
+motifs	O
+of	O
+MKPs	O
+.	O
+	O
+MKP7	O
+,	O
+the	O
+biggest	O
+molecule	O
+in	O
+the	O
+MKP	O
+family	O
+,	O
+selectively	O
+inactivates	O
+JNK	O
+and	O
+p38	O
+following	O
+stress	O
+activation	O
+.	O
+	O
+To	O
+quantitatively	O
+assess	O
+the	O
+contribution	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+domain	O
+to	O
+the	O
+MKP7	O
+-	O
+catalysed	O
+JNK1	O
+dephosphorylation	O
+,	O
+we	O
+first	O
+measured	O
+the	O
+kinetic	O
+parameters	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+truncation	O
+of	O
+MKP7	O
+(	O
+MKP7ΔC304	B-mutant
+,	O
+residues	O
+5	O
+–	O
+303	O
+)	O
+and	O
+MKP7	O
+-	O
+CD	O
+(	O
+residues	O
+156	O
+–	O
+301	O
+)	O
+towards	O
+phosphorylated	O
+JNK1	O
+(	O
+pJNK1	O
+).	O
+	O
+The	O
+kcat	O
+and	O
+Km	O
+of	O
+the	O
+MKP7	O
+-	O
+CD	O
+(	O
+0	O
+.	O
+028	O
+s	O
+−	O
+1	O
+and	O
+0	O
+.	O
+26	O
+μM	O
+)	O
+so	O
+determined	O
+were	O
+nearly	O
+identical	O
+to	O
+those	O
+of	O
+MKP7ΔC304	B-mutant
+(	O
+0	O
+.	O
+029	O
+s	O
+−	O
+1	O
+and	O
+0	O
+.	O
+27	O
+μM	O
+),	O
+indicating	O
+that	O
+the	O
+MKP7	O
+-	O
+KBD	O
+has	O
+no	O
+effect	O
+on	O
+enzyme	O
+catalysis	O
+.	O
+	O
+We	O
+next	O
+examined	O
+the	O
+interaction	O
+of	O
+JNK1	O
+with	O
+the	O
+CD	O
+and	O
+KBD	O
+of	O
+MKP7	O
+by	O
+gel	O
+filtration	O
+analysis	O
+.	O
+	O
+When	O
+3	O
+molar	O
+equivalents	O
+of	O
+CD	O
+were	O
+mixed	O
+with	O
+1	O
+molar	O
+equivalent	O
+of	O
+JNK1	O
+,	O
+a	O
+significant	O
+amount	O
+of	O
+CD	O
+co	O
+-	O
+migrated	O
+with	O
+JNK1	O
+to	O
+earlier	O
+fractions	O
+,	O
+and	O
+the	O
+excess	O
+amount	O
+of	O
+CD	O
+was	O
+eluted	O
+from	O
+the	O
+size	O
+exclusion	O
+column	O
+as	O
+a	O
+monomer	O
+,	O
+indicating	O
+stable	O
+complex	O
+formation	O
+(	O
+Fig	O
+.	O
+2c	O
+).	O
+	O
+In	O
+contrast	O
+,	O
+no	O
+KBD	O
+–	O
+JNK1	O
+complex	O
+was	O
+detected	O
+when	O
+3	O
+molar	O
+equivalents	O
+of	O
+KBD	O
+were	O
+mixed	O
+with	O
+1	O
+molar	O
+equivalent	O
+of	O
+JNK1	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+.	O
+2d	O
+,	O
+the	O
+CD	O
+of	O
+MKP7	O
+can	O
+be	O
+pulled	O
+down	O
+by	O
+JNK1	O
+,	O
+while	O
+the	O
+KBD	O
+failed	O
+to	O
+bind	O
+to	O
+the	O
+counterpart	O
+protein	O
+.	O
+	O
+Taken	O
+together	O
+,	O
+our	O
+data	O
+indicate	O
+that	O
+the	O
+CD	O
+of	O
+MKP7	O
+,	O
+but	O
+not	O
+the	O
+KBD	O
+domain	O
+,	O
+is	O
+responsible	O
+for	O
+JNK	O
+substrate	O
+-	O
+binding	O
+and	O
+enzymatic	O
+specificity	O
+.	O
+	O
+Crystal	O
+structure	O
+of	O
+JNK1	O
+in	O
+complex	O
+with	O
+the	O
+MKP7	O
+-	O
+CD	O
+	O
+To	O
+understand	O
+the	O
+molecular	O
+basis	O
+of	O
+JNK1	O
+recognition	O
+by	O
+MKP7	O
+,	O
+we	O
+determined	O
+the	O
+crystal	O
+structure	O
+of	O
+unphosphorylated	O
+JNK1	O
+in	O
+complex	O
+with	O
+the	O
+MKP7	O
+-	O
+CD	O
+(	O
+Fig	O
+.	O
+3a	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+and	O
+Table	O
+1	O
+).	O
+	O
+In	O
+the	O
+complex	O
+,	O
+JNK1	O
+has	O
+its	O
+characteristic	O
+bilobal	O
+structure	O
+comprising	O
+an	O
+N	O
+-	O
+terminal	O
+lobe	O
+rich	O
+in	O
+β	O
+-	O
+sheet	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+lobe	O
+that	O
+is	O
+mostly	O
+α	O
+-	O
+helical	O
+.	O
+	O
+One	O
+side	O
+of	O
+the	O
+β	O
+-	O
+sheet	O
+is	O
+covered	O
+with	O
+two	O
+α	O
+-	O
+helices	O
+and	O
+the	O
+other	O
+is	O
+covered	O
+with	O
+four	O
+α	O
+-	O
+helices	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+	O
+MKP7	O
+-	O
+CD	O
+is	O
+positioned	O
+onto	O
+the	O
+JNK1	O
+molecule	O
+so	O
+that	O
+the	O
+active	O
+site	O
+of	O
+the	O
+phosphatase	O
+faces	O
+towards	O
+the	O
+activation	O
+segment	O
+.	O
+	O
+In	O
+an	O
+alignment	O
+of	O
+the	O
+structure	O
+of	O
+MKP7	O
+-	O
+CD	O
+with	O
+that	O
+of	O
+VHR	O
+,	O
+an	O
+atypical	O
+‘	O
+MKP	O
+'	O
+consisting	O
+of	O
+only	O
+a	O
+catalytic	O
+domain	O
+,	O
+119	O
+of	O
+147	O
+MKP7	O
+-	O
+CD	O
+residues	O
+could	O
+be	O
+superimposed	O
+with	O
+a	O
+r	O
+.	O
+m	O
+.	O
+s	O
+.	O
+d	O
+.	O
+(	O
+root	O
+mean	O
+squared	O
+deviation	O
+)	O
+of	O
+1	O
+.	O
+05	O
+Å	O
+(	O
+Fig	O
+.	O
+3c	O
+).	O
+	O
+The	O
+most	O
+striking	O
+difference	O
+is	O
+that	O
+helix	O
+α0	O
+and	O
+loop	O
+α0	O
+–	O
+β1	O
+of	O
+VHR	O
+are	O
+absent	O
+in	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+Another	O
+region	O
+that	O
+cannot	O
+be	O
+aligned	O
+with	O
+VHR	O
+is	O
+found	O
+in	O
+loop	O
+β3	O
+–	O
+β4	O
+.	O
+	O
+This	O
+loop	O
+is	O
+shortened	O
+by	O
+nine	O
+residues	O
+in	O
+MKP7	O
+-	O
+CD	O
+compared	O
+with	O
+that	O
+in	O
+VHR	O
+.	O
+	O
+Asp213	O
+in	O
+MKP7	O
+also	O
+adopts	O
+a	O
+position	O
+similar	O
+to	O
+that	O
+of	O
+Asp92	O
+in	O
+VHR	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+),	O
+indicating	O
+that	O
+Asp213	O
+is	O
+likely	O
+to	O
+function	O
+as	O
+the	O
+general	O
+acid	O
+in	O
+MKP7	O
+.	O
+	O
+We	O
+also	O
+observed	O
+the	O
+binding	O
+of	O
+a	O
+chloride	O
+ion	O
+in	O
+the	O
+active	O
+site	O
+of	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+It	O
+is	O
+located	O
+3	O
+.	O
+36	O
+Å	O
+from	O
+the	O
+Cys244	O
+side	O
+chain	O
+and	O
+makes	O
+electrostatic	O
+interactions	O
+with	O
+the	O
+dipole	O
+moment	O
+of	O
+helix	O
+α3	O
+and	O
+with	O
+several	O
+main	O
+-	O
+chain	O
+amide	O
+groups	O
+.	O
+	O
+Thus	O
+this	O
+chloride	O
+ion	O
+is	O
+a	O
+mimic	O
+for	O
+the	O
+phosphate	O
+group	O
+of	O
+the	O
+substrate	O
+,	O
+as	O
+revealed	O
+by	O
+a	O
+comparison	O
+with	O
+the	O
+structure	O
+of	O
+PTP1B	O
+in	O
+complex	O
+with	O
+phosphotyrosine	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+).	O
+	O
+Although	O
+the	O
+catalytically	O
+important	O
+residues	O
+in	O
+MKP7	O
+-	O
+CD	O
+are	O
+well	O
+aligned	O
+with	O
+those	O
+in	O
+VHR	O
+,	O
+the	O
+residues	O
+in	O
+the	O
+P	O
+-	O
+loop	O
+of	O
+MKP7	O
+are	O
+smaller	O
+and	O
+have	O
+a	O
+more	O
+hydrophobic	O
+character	O
+than	O
+those	O
+of	O
+VHR	O
+(	O
+Cys124	O
+-	O
+Arg125	O
+-	O
+Glu126	O
+-	O
+Gly127	O
+-	O
+Tyr128	O
+-	O
+Gly129	O
+-	O
+Arg130	O
+;	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+).	O
+	O
+The	O
+difference	O
+in	O
+the	O
+polarity	O
+/	O
+hydrophobicity	O
+of	O
+the	O
+surface	O
+may	O
+also	O
+point	O
+to	O
+the	O
+origin	O
+of	O
+the	O
+differences	O
+in	O
+the	O
+substrate	O
+-	O
+recognition	O
+mechanism	O
+for	O
+these	O
+two	O
+phosphatases	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1e	O
+,	O
+f	O
+).	O
+	O
+As	O
+a	O
+result	O
+,	O
+the	O
+buried	O
+solvent	O
+-	O
+accessible	O
+surface	O
+area	O
+is	O
+∼	O
+1	O
+,	O
+315	O
+Å	O
+.	O
+In	O
+the	O
+C	O
+-	O
+terminal	O
+domain	O
+,	O
+JNK1	O
+has	O
+an	O
+insertion	O
+after	O
+the	O
+helix	O
+αG	O
+.	O
+This	O
+insertion	O
+consists	O
+of	O
+two	O
+helices	O
+(	O
+α1L14	O
+and	O
+α2L14	O
+)	O
+that	O
+are	O
+common	O
+to	O
+all	O
+members	O
+of	O
+the	O
+MAPK	O
+family	O
+.	O
+	O
+The	O
+interactive	O
+surface	O
+in	O
+JNK1	O
+,	O
+formed	O
+by	O
+the	O
+helices	O
+αG	O
+and	O
+α2L14	O
+,	O
+displays	O
+a	O
+hydrophobic	O
+region	O
+,	O
+centred	O
+at	O
+Trp234	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+	O
+The	O
+MKP7	O
+-	O
+docking	O
+region	O
+includes	O
+two	O
+helices	O
+,	O
+α4	O
+and	O
+α5	O
+,	O
+and	O
+the	O
+general	O
+acid	O
+loop	O
+.	O
+	O
+The	O
+aromatic	O
+ring	O
+of	O
+Phe285	O
+on	O
+MKP7	O
+α5	O
+-	O
+helix	O
+is	O
+nestled	O
+in	O
+a	O
+hydrophobic	O
+pocket	O
+on	O
+JNK1	O
+,	O
+formed	O
+by	O
+side	O
+chains	O
+of	O
+Ile197	O
+,	O
+Leu198	O
+,	O
+Ile231	O
+,	O
+Trp234	O
+,	O
+Val256	O
+,	O
+Tyr259	O
+,	O
+Val260	O
+and	O
+the	O
+aliphatic	O
+portion	O
+of	O
+His230	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+f	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1g	O
+).	O
+	O
+In	O
+addition	O
+,	O
+there	O
+are	O
+hydrogen	O
+bonds	O
+between	O
+Ser282	O
+and	O
+Asn286	O
+of	O
+MKP7	O
+and	O
+His230	O
+and	O
+Thr255	O
+of	O
+JNK1	O
+,	O
+and	O
+the	O
+main	O
+chain	O
+of	O
+Phe215	O
+in	O
+the	O
+general	O
+acid	O
+loop	O
+of	O
+MKP7	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+the	O
+side	O
+chain	O
+of	O
+Gln253	O
+in	O
+JNK1	O
+.	O
+	O
+The	O
+second	O
+interactive	O
+area	O
+involves	O
+the	O
+α4	O
+helix	O
+of	O
+MKP7	O
+and	O
+charged	O
+/	O
+polar	O
+residues	O
+of	O
+JNK1	O
+(	O
+Fig	O
+.	O
+3e	O
+).	O
+	O
+Mutational	O
+analysis	O
+of	O
+the	O
+JNK1	O
+–	O
+MKP7	O
+docking	O
+interface	O
+	O
+To	O
+assess	O
+the	O
+importance	O
+of	O
+the	O
+aforementioned	O
+interactions	O
+,	O
+we	O
+generated	O
+a	O
+series	O
+of	O
+point	O
+mutations	O
+on	O
+the	O
+MKP7	O
+-	O
+CD	O
+and	O
+examined	O
+their	O
+effect	O
+on	O
+the	O
+MKP7	O
+-	O
+catalysed	O
+JNK1	O
+dephosphorylation	O
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+When	O
+the	O
+hydrophobic	O
+residues	O
+Phe285	O
+and	O
+Phe287	O
+on	O
+the	O
+α5	O
+of	O
+MKP7	O
+-	O
+CD	O
+were	O
+replaced	O
+by	O
+Asp	O
+or	O
+Ala	O
+,	O
+their	O
+phosphatase	O
+activities	O
+for	O
+JNK1	O
+dephosphorylation	O
+decreased	O
+∼	O
+10	O
+-	O
+fold	O
+.	O
+	O
+In	O
+comparison	O
+,	O
+replacement	O
+of	O
+the	O
+other	O
+residues	O
+(	O
+Phe215	O
+,	O
+Asp268	O
+,	O
+Lys275	O
+,	O
+Ser282	O
+,	O
+Asn286	O
+and	O
+Leu292	O
+)	O
+with	O
+an	O
+Ala	O
+or	O
+Asp	O
+individually	O
+led	O
+to	O
+a	O
+modest	O
+decrease	O
+in	O
+catalytic	O
+efficiencies	O
+,	O
+suggesting	O
+that	O
+this	O
+position	O
+may	O
+only	O
+affect	O
+some	O
+selectivity	O
+of	O
+MKP	O
+.	O
+	O
+Interestingly	O
+,	O
+mutation	O
+of	O
+Phe287	O
+results	O
+in	O
+a	O
+considerable	O
+loss	O
+of	O
+activity	O
+against	O
+pJNK1	O
+without	O
+altering	O
+the	O
+affinity	O
+of	O
+MKP7	O
+-	O
+CD	O
+for	O
+JNK1	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+We	O
+also	O
+generated	O
+a	O
+series	O
+of	O
+point	O
+mutations	O
+in	O
+the	O
+JNK1	O
+and	O
+assessed	O
+the	O
+effect	O
+on	O
+JNK1	O
+binding	O
+using	O
+the	O
+GST	O
+pull	O
+-	O
+down	O
+assay	O
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+	O
+Substitution	O
+at	O
+Asp229	O
+,	O
+Trp234	O
+,	O
+Thr255	O
+,	O
+Val256	O
+,	O
+Tyr259	O
+and	O
+Val260	O
+significantly	O
+reduced	O
+the	O
+binding	O
+affinity	O
+of	O
+MKP7	O
+-	O
+CD	O
+for	O
+JNK	O
+.	O
+	O
+Taken	O
+together	O
+,	O
+these	O
+results	O
+are	O
+consistent	O
+with	O
+the	O
+present	O
+crystallographic	O
+model	O
+,	O
+which	O
+reveal	O
+the	O
+hydrophobic	O
+contacts	O
+between	O
+the	O
+MKP7	O
+catalytic	O
+domain	O
+and	O
+JNK1	O
+have	O
+a	O
+predominant	O
+role	O
+in	O
+the	O
+enzyme	O
+–	O
+substrate	O
+interaction	O
+,	O
+and	O
+hydrophobic	O
+residue	O
+Phe285	O
+in	O
+the	O
+MKP7	O
+-	O
+CD	O
+is	O
+a	O
+key	O
+residue	O
+for	O
+its	O
+high	O
+-	O
+affinity	O
+binding	O
+to	O
+JNK1	O
+.	O
+	O
+This	O
+allosteric	O
+activation	O
+of	O
+MKP3	O
+has	O
+been	O
+well	O
+-	O
+documented	O
+in	O
+vitro	O
+using	O
+pNPP	O
+,	O
+a	O
+small	O
+-	O
+molecule	O
+phosphotyrosine	O
+analogue	O
+of	O
+its	O
+normal	O
+substrate	O
+.	O
+	O
+We	O
+then	O
+assayed	O
+pNPPase	O
+activities	O
+of	O
+MKP7ΔC304	B-mutant
+and	O
+MKP7	O
+-	O
+CD	O
+in	O
+the	O
+presence	O
+of	O
+JNK1	O
+.	O
+	O
+We	O
+therefore	O
+examined	O
+the	O
+effects	O
+of	O
+the	O
+MKP7	O
+-	O
+CD	O
+mutants	O
+on	O
+their	O
+pNPPase	O
+activities	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+.	O
+4f	O
+,	O
+all	O
+the	O
+mutants	O
+,	O
+except	O
+F287D	B-mutant
+/	I-mutant
+A	I-mutant
+,	O
+showed	O
+little	O
+or	O
+no	O
+activity	O
+change	O
+compared	O
+with	O
+the	O
+wild	O
+-	O
+type	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+In	O
+the	O
+JNK1	O
+/	O
+MKP7	O
+-	O
+CD	O
+complex	O
+structure	O
+,	O
+Phe287	O
+of	O
+MKP7	O
+does	O
+not	O
+make	O
+contacts	O
+with	O
+JNK1	O
+substrate	O
+.	O
+	O
+It	O
+penetrates	O
+into	O
+a	O
+pocket	O
+formed	O
+by	O
+residues	O
+from	O
+the	O
+P	O
+-	O
+loop	O
+and	O
+general	O
+acid	O
+loop	O
+and	O
+forms	O
+hydrophobic	O
+contacts	O
+with	O
+the	O
+aliphatic	O
+portions	O
+of	O
+side	O
+chains	O
+of	O
+Arg250	O
+,	O
+Glu217	O
+and	O
+Ile219	O
+,	O
+suggesting	O
+that	O
+Phe287	O
+in	O
+MKP7	O
+would	O
+play	O
+a	O
+similar	O
+role	O
+to	O
+that	O
+of	O
+its	O
+structural	O
+counterpart	O
+in	O
+the	O
+PTPs	O
+(	O
+Gln266	O
+in	O
+PTP1B	O
+)	O
+and	O
+VHR	O
+(	O
+Phe166	O
+in	O
+VHR	O
+)	O
+in	O
+the	O
+precise	O
+alignment	O
+of	O
+active	O
+-	O
+site	O
+residues	O
+in	O
+MKP7	O
+with	O
+respect	O
+to	O
+the	O
+substrate	O
+for	O
+efficient	O
+catalysis	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+).	O
+	O
+Kinase	O
+-	O
+associated	O
+phosphatase	O
+(	O
+KAP	O
+),	O
+a	O
+member	O
+of	O
+the	O
+DUSP	O
+family	O
+,	O
+plays	O
+a	O
+crucial	O
+role	O
+in	O
+cell	O
+cycle	O
+regulation	O
+by	O
+dephosphorylating	O
+the	O
+pThr160	O
+residue	O
+of	O
+CDK2	O
+(	O
+cyclin	O
+-	O
+dependent	O
+kinase	O
+2	O
+).	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+the	O
+CDK2	O
+/	O
+KAP	O
+complex	O
+has	O
+been	O
+determined	O
+at	O
+3	O
+.	O
+0	O
+Å	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+	O
+The	O
+interface	O
+between	O
+these	O
+two	O
+proteins	O
+consists	O
+of	O
+three	O
+discontinuous	O
+contact	O
+regions	O
+.	O
+	O
+There	O
+is	O
+a	O
+hydrogen	O
+bond	O
+between	O
+the	O
+main	O
+-	O
+chain	O
+nitrogen	O
+of	O
+Ile183	O
+(	O
+KAP	O
+)	O
+and	O
+side	O
+chain	O
+oxygen	O
+of	O
+Glu208	O
+(	O
+CDK2	O
+),	O
+and	O
+salt	O
+bridges	O
+between	O
+Lys184	O
+of	O
+KAP	O
+and	O
+Asp235	O
+of	O
+CDK2	O
+.	O
+	O
+The	O
+substitution	O
+of	O
+the	O
+two	O
+hydrophobic	O
+residues	O
+with	O
+charged	O
+/	O
+polar	O
+residues	O
+(	O
+F285I	B-mutant
+/	O
+N286K	B-mutant
+)	O
+seriously	O
+disrupts	O
+the	O
+hydrophobic	O
+interaction	O
+required	O
+for	O
+MKP7	O
+binding	O
+on	O
+JNK1	O
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+F	O
+-	O
+site	O
+interaction	O
+is	O
+crucial	O
+for	O
+JNK1	O
+inactivation	O
+in	O
+vivo	O
+	O
+JNK	O
+is	O
+activated	O
+following	O
+cellular	O
+exposure	O
+to	O
+a	O
+number	O
+of	O
+acute	O
+stimuli	O
+such	O
+as	O
+anisomycin	O
+,	O
+H2O2	O
+,	O
+ultraviolet	O
+light	O
+,	O
+sorbitol	O
+,	O
+DNA	O
+-	O
+damaging	O
+agents	O
+and	O
+several	O
+strong	O
+apoptosis	O
+inducers	O
+(	O
+etoposide	O
+,	O
+cisplatin	O
+and	O
+taxol	O
+).	O
+	O
+To	O
+assess	O
+the	O
+effects	O
+of	O
+MKP7	O
+and	O
+its	O
+mutants	O
+on	O
+the	O
+activation	O
+of	O
+endogenous	O
+JNK	O
+in	O
+vivo	O
+,	O
+HEK293T	O
+cells	O
+were	O
+transfected	O
+with	O
+blank	O
+vector	O
+or	O
+with	O
+HA	O
+-	O
+tagged	O
+constructs	O
+for	O
+full	O
+-	O
+length	O
+MKP7	O
+,	O
+MKP7ΔC304	B-mutant
+and	O
+MKP7	O
+-	O
+CD	O
+or	O
+MKP7	O
+mutants	O
+,	O
+and	O
+stimulated	O
+with	O
+ultraviolet	O
+or	O
+etoposide	O
+treatment	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+.	O
+6a	O
+–	O
+c	O
+,	O
+immunobloting	O
+showed	O
+similar	O
+expression	O
+levels	O
+for	O
+the	O
+different	O
+MKP7	O
+constructs	O
+in	O
+all	O
+the	O
+cells	O
+.	O
+	O
+Overexpressed	O
+full	O
+-	O
+length	O
+MKP7	O
+,	O
+MKP7ΔC304	B-mutant
+and	O
+MKP7	O
+-	O
+CD	O
+significantly	O
+reduced	O
+the	O
+endogenous	O
+level	O
+of	O
+phosphorylated	O
+JNK	O
+compared	O
+with	O
+vector	O
+-	O
+transfected	O
+cells	O
+.	O
+	O
+We	O
+next	O
+tested	O
+in	O
+vivo	O
+interactions	O
+between	O
+JNK1	O
+mutants	O
+and	O
+full	O
+-	O
+length	O
+MKP7	O
+by	O
+coimmunoprecipitation	O
+experiments	O
+under	O
+unstimulated	O
+conditions	O
+.	O
+	O
+When	O
+co	O
+-	O
+expressed	O
+in	O
+HEK293T	O
+cells	O
+,	O
+wild	O
+-	O
+type	O
+(	O
+HA	O
+)-	O
+JNK1	O
+was	O
+readily	O
+precipitated	O
+with	O
+(	O
+Myc	O
+)-	O
+MKP7	O
+(	O
+Fig	O
+.	O
+6d	O
+),	O
+indicating	O
+that	O
+MKP7	O
+binds	O
+dephosphorylated	O
+JNK1	O
+protein	O
+in	O
+vivo	O
+.	O
+	O
+Activation	O
+of	O
+the	O
+JNK	O
+signalling	O
+pathway	O
+is	O
+frequently	O
+associated	O
+with	O
+apoptotic	O
+cell	O
+death	O
+,	O
+and	O
+inhibition	O
+of	O
+JNK	O
+can	O
+prevent	O
+apoptotic	O
+death	O
+of	O
+multiple	O
+cells	O
+.	O
+	O
+To	O
+examine	O
+whether	O
+the	O
+inhibition	O
+of	O
+JNK	O
+activity	O
+by	O
+MKP7	O
+would	O
+provide	O
+protections	O
+against	O
+the	O
+apoptosis	O
+,	O
+we	O
+analysed	O
+the	O
+rate	O
+of	O
+apoptosis	O
+in	O
+ultraviolet	O
+-	O
+irradiated	O
+cells	O
+transfected	O
+with	O
+MKP7	O
+(	O
+wild	O
+type	O
+or	O
+mutants	O
+)	O
+by	O
+flow	O
+cytometry	O
+.	O
+	O
+The	O
+results	O
+showed	O
+similar	O
+apoptotic	O
+rates	O
+between	O
+cells	O
+transfected	O
+with	O
+blank	O
+vector	O
+or	O
+with	O
+MKP7	O
+(	O
+wild	O
+type	O
+or	O
+mutants	O
+)	O
+under	O
+unstimulated	O
+conditions	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+),	O
+while	O
+ultraviolet	O
+-	O
+irradiation	O
+significantly	O
+increased	O
+apoptotic	O
+rate	O
+in	O
+cells	O
+transfected	O
+with	O
+blank	O
+vector	O
+(	O
+Fig	O
+.	O
+6e	O
+).	O
+	O
+Expressions	O
+of	O
+wild	O
+-	O
+type	O
+MKP7	O
+,	O
+MKP7ΔC304	B-mutant
+and	O
+MKP7	O
+-	O
+CD	O
+significantly	O
+decreased	O
+the	O
+proportion	O
+of	O
+apoptotic	O
+cells	O
+after	O
+ultraviolet	O
+treatment	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+cells	O
+transfected	O
+with	O
+the	O
+MKP7	O
+FXF	O
+-	O
+motif	O
+mutants	O
+(	O
+F285D	B-mutant
+,	O
+F287D	B-mutant
+and	O
+L288D	B-mutant
+)	O
+showed	O
+little	O
+protective	O
+effect	O
+after	O
+ultraviolet	O
+treatment	O
+and	O
+similar	O
+levels	O
+of	O
+apoptosis	O
+rates	O
+were	O
+detected	O
+to	O
+cells	O
+transfected	O
+with	O
+control	O
+vectors	O
+(	O
+Fig	O
+.	O
+6e	O
+,	O
+f	O
+).	O
+	O
+Taken	O
+together	O
+,	O
+our	O
+results	O
+suggested	O
+that	O
+FXF	O
+-	O
+motif	O
+-	O
+mediated	O
+,	O
+rather	O
+than	O
+KBD	O
+-	O
+mediated	O
+,	O
+interaction	O
+is	O
+essential	O
+for	O
+MKP7	O
+to	O
+block	O
+ultraviolet	O
+-	O
+induced	O
+apoptosis	O
+.	O
+	O
+A	O
+similar	O
+docking	O
+mechanism	O
+for	O
+JNK1	O
+recognition	O
+by	O
+MKP5	O
+	O
+MKP5	O
+is	O
+unique	O
+among	O
+the	O
+MKPs	O
+in	O
+possessing	O
+an	O
+additional	O
+domain	O
+of	O
+unknown	O
+function	O
+at	O
+the	O
+N	O
+-	O
+terminus	O
+(	O
+Fig	O
+.	O
+7a	O
+).	O
+	O
+Deletion	O
+of	O
+the	O
+KBD	O
+in	O
+MKP5	O
+leads	O
+to	O
+a	O
+280	O
+-	O
+fold	O
+increase	O
+in	O
+Km	O
+for	O
+p38α	O
+substrate	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+human	O
+MKP5	O
+-	O
+CD	O
+has	O
+been	O
+determined	O
+.	O
+	O
+To	O
+address	O
+this	O
+issue	O
+,	O
+we	O
+first	O
+examined	O
+the	O
+docking	O
+ability	O
+of	O
+JNK1	O
+to	O
+the	O
+KBD	O
+and	O
+CD	O
+of	O
+MKP5	O
+using	O
+gel	O
+filtration	O
+analysis	O
+and	O
+pull	O
+-	O
+down	O
+assays	O
+.	O
+	O
+It	O
+can	O
+be	O
+seen	O
+from	O
+gel	O
+filtration	O
+experiments	O
+that	O
+JNK1	O
+can	O
+forms	O
+a	O
+stable	O
+heterodimer	O
+with	O
+MKP5	O
+-	O
+CD	O
+in	O
+solution	O
+,	O
+but	O
+no	O
+detectable	O
+interaction	O
+was	O
+found	O
+with	O
+the	O
+KBD	O
+domain	O
+(	O
+Fig	O
+.	O
+7d	O
+).	O
+	O
+The	O
+catalytic	O
+domain	O
+of	O
+MKP5	O
+,	O
+but	O
+not	O
+its	O
+KBD	O
+,	O
+was	O
+able	O
+to	O
+pull	O
+-	O
+down	O
+a	O
+detectable	O
+amount	O
+of	O
+JNK1	O
+(	O
+Fig	O
+.	O
+7e	O
+),	O
+implicating	O
+a	O
+different	O
+substrate	O
+-	O
+recognition	O
+mechanisms	O
+for	O
+p38	O
+and	O
+JNK	O
+MAPKs	O
+.	O
+	O
+To	O
+further	O
+test	O
+our	O
+hypothesis	O
+,	O
+we	O
+generated	O
+forms	O
+of	O
+MKP5	O
+-	O
+CD	O
+bearing	O
+mutations	O
+corresponding	O
+to	O
+the	O
+changes	O
+we	O
+made	O
+on	O
+MKP7	O
+-	O
+CD	O
+on	O
+the	O
+basis	O
+of	O
+sequence	O
+and	O
+structural	O
+alignment	O
+and	O
+examined	O
+their	O
+effects	O
+on	O
+the	O
+phosphatase	O
+activity	O
+.	O
+	O
+Taken	O
+together	O
+,	O
+our	O
+results	O
+suggest	O
+that	O
+MKP5	O
+binds	O
+JNK1	O
+in	O
+a	O
+docking	O
+mode	O
+similar	O
+to	O
+that	O
+in	O
+the	O
+JNK1	O
+–	O
+MKP7	O
+complex	O
+,	O
+and	O
+the	O
+detailed	O
+interaction	O
+model	O
+can	O
+be	O
+generated	O
+using	O
+molecular	O
+dynamics	O
+simulation	O
+based	O
+on	O
+the	O
+structure	O
+of	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+complex	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4b	O
+,	O
+c	O
+).	O
+	O
+In	O
+this	O
+model	O
+,	O
+the	O
+MKP5	O
+-	O
+CD	O
+adopts	O
+a	O
+conformation	O
+nearly	O
+identical	O
+to	O
+that	O
+in	O
+its	O
+unbound	O
+form	O
+,	O
+suggesting	O
+that	O
+the	O
+conformation	O
+of	O
+the	O
+catalytic	O
+domain	O
+undergoes	O
+little	O
+change	O
+,	O
+if	O
+any	O
+at	O
+all	O
+,	O
+upon	O
+JNK1	O
+binding	O
+.	O
+	O
+In	O
+particular	O
+,	O
+Leu449	O
+of	O
+MKP5	O
+,	O
+which	O
+is	O
+equivalent	O
+to	O
+the	O
+key	O
+residue	O
+Phe285	O
+of	O
+MKP7	O
+,	O
+buried	O
+deeply	O
+within	O
+the	O
+hydrophobic	O
+pocket	O
+of	O
+JNK1	O
+in	O
+the	O
+same	O
+way	O
+as	O
+Phe285	O
+in	O
+the	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+complex	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+).	O
+	O
+Despite	O
+the	O
+strong	O
+similarities	O
+between	O
+JNK1	O
+–	O
+MKP5	O
+-	O
+CD	O
+and	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+,	O
+however	O
+,	O
+there	O
+are	O
+differences	O
+.	O
+	O
+The	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+interaction	O
+is	O
+better	O
+and	O
+more	O
+extensive	O
+.	O
+	O
+Asp268	O
+of	O
+MKP7	O
+-	O
+CD	O
+forms	O
+salt	O
+bridge	O
+with	O
+JNK1	O
+Arg263	O
+,	O
+whereas	O
+the	O
+corresponding	O
+residue	O
+Thr432	O
+in	O
+MKP5	O
+-	O
+CD	O
+may	O
+not	O
+interact	O
+with	O
+JNK1	O
+.	O
+	O
+Crystal	O
+structures	O
+of	O
+ERK2	O
+bound	O
+with	O
+the	O
+D	O
+-	O
+motif	O
+sequences	O
+derived	O
+from	O
+MKP3	O
+and	O
+HePTP	O
+have	O
+been	O
+reported	O
+.	O
+	O
+These	O
+structures	O
+revealed	O
+that	O
+linear	O
+docking	O
+motifs	O
+in	O
+interacting	O
+proteins	O
+bind	O
+to	O
+a	O
+common	O
+docking	O
+site	O
+on	O
+MAPKs	O
+outside	O
+the	O
+kinase	O
+active	O
+site	O
+.	O
+	O
+The	O
+particular	O
+amino	O
+acids	O
+and	O
+their	O
+spacing	O
+within	O
+D	O
+-	O
+motif	O
+sequences	O
+and	O
+amino	O
+acid	O
+composition	O
+of	O
+the	O
+docking	O
+sites	O
+on	O
+MAPKs	O
+appear	O
+to	O
+determine	O
+the	O
+specificity	O
+of	O
+D	O
+-	O
+motifs	O
+for	O
+individual	O
+MAPKs	O
+.	O
+	O
+Recently	O
+,	O
+the	O
+crystal	O
+structure	O
+of	O
+a	O
+complex	O
+between	O
+the	O
+KBD	O
+of	O
+MKP5	O
+and	O
+p38α	O
+has	O
+been	O
+obtained	O
+.	O
+	O
+This	O
+complex	O
+has	O
+revealed	O
+a	O
+distinct	O
+interaction	O
+mode	O
+for	O
+MKP5	O
+.	O
+	O
+The	O
+KBD	O
+of	O
+MKP5	O
+binds	O
+to	O
+p38α	O
+in	O
+the	O
+opposite	O
+polypeptide	O
+direction	O
+compared	O
+with	O
+how	O
+the	O
+D	O
+-	O
+motif	O
+of	O
+MKP3	O
+binds	O
+to	O
+ERK2	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+the	O
+canonical	O
+D	O
+-	O
+motif	O
+-	O
+binding	O
+mode	O
+,	O
+separate	O
+helices	O
+,	O
+α2	O
+and	O
+α3	O
+′,	O
+in	O
+the	O
+KBD	O
+of	O
+MKP5	O
+engage	O
+the	O
+p38α	O
+-	O
+docking	O
+site	O
+.	O
+	O
+Further	O
+structural	O
+and	O
+biochemical	O
+studies	O
+indicate	O
+that	O
+KBD	O
+of	O
+MKP7	O
+may	O
+interact	O
+with	O
+p38α	O
+in	O
+a	O
+similar	O
+manner	O
+to	O
+that	O
+of	O
+MKP5	O
+.	O
+	O
+Taken	O
+together	O
+,	O
+these	O
+results	O
+suggest	O
+that	O
+MKP7	O
+utilizes	O
+a	O
+bipartite	O
+recognition	O
+mechanism	O
+to	O
+achieve	O
+the	O
+efficiency	O
+and	O
+fidelity	O
+of	O
+p38α	O
+signalling	O
+.	O
+	O
+In	O
+addition	O
+to	O
+the	O
+canonical	O
+D	O
+-	O
+site	O
+,	O
+the	O
+MAPK	O
+ERK2	O
+contains	O
+a	O
+second	O
+binding	O
+site	O
+utilized	O
+by	O
+transcription	O
+factor	O
+substrates	O
+and	O
+phosphatases	O
+,	O
+the	O
+FXF	O
+-	O
+motif	O
+-	O
+binding	O
+site	O
+(	O
+also	O
+called	O
+F	O
+-	O
+site	O
+),	O
+that	O
+is	O
+exposed	O
+in	O
+active	O
+ERK2	O
+and	O
+the	O
+D	O
+-	O
+motif	O
+peptide	O
+-	O
+induced	O
+conformation	O
+of	O
+MAPKs	O
+.	O
+	O
+MKP3	O
+is	O
+highly	O
+specific	O
+in	O
+dephosphorylating	O
+and	O
+inactivating	O
+ERK2	O
+,	O
+and	O
+the	O
+phosphatase	O
+activity	O
+of	O
+the	O
+MKP3	O
+-	O
+catalysed	O
+pNPP	O
+reaction	O
+can	O
+be	O
+markedly	O
+increased	O
+in	O
+the	O
+presence	O
+of	O
+ERK2	O
+(	O
+refs	O
+).	O
+	O
+Sequence	O
+alignment	O
+of	O
+all	O
+MKPs	O
+reveals	O
+a	O
+high	O
+degree	O
+of	O
+conservation	O
+of	O
+residues	O
+surrounding	O
+the	O
+interacting	O
+region	O
+observed	O
+in	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+complex	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+A	O
+comprehensive	O
+examination	O
+of	O
+the	O
+molecular	O
+basis	O
+of	O
+the	O
+specific	O
+ERK2	O
+recognition	O
+by	O
+MKP3	O
+is	O
+underway	O
+.	O
+	O
+The	O
+FXF	O
+-	O
+motif	O
+-	O
+mediated	O
+interaction	O
+is	O
+critical	O
+for	O
+both	O
+pERK2	O
+inactivation	O
+and	O
+ERK2	O
+-	O
+induced	O
+MKP3	O
+activation	O
+(	O
+manuscript	O
+in	O
+preparation	O
+).	O
+	O
+In	O
+summary	O
+,	O
+we	O
+have	O
+resolved	O
+the	O
+structure	O
+of	O
+JNK1	O
+in	O
+complex	O
+with	O
+the	O
+catalytic	O
+domain	O
+of	O
+MKP7	O
+.	O
+	O
+Results	O
+from	O
+biochemical	O
+characterization	O
+of	O
+the	O
+Phe285	O
+and	O
+Phe287	O
+MKP7	O
+mutants	O
+combined	O
+with	O
+structural	O
+information	O
+support	O
+the	O
+conclusion	O
+that	O
+the	O
+two	O
+Phe	O
+residues	O
+serve	O
+different	O
+roles	O
+in	O
+the	O
+catalytic	O
+reaction	O
+.	O
+	O
+This	O
+newly	O
+identified	O
+FXF	O
+-	O
+type	O
+motif	O
+is	O
+present	O
+in	O
+all	O
+MKPs	O
+,	O
+except	O
+that	O
+the	O
+residue	O
+at	O
+the	O
+first	O
+position	O
+in	O
+MKP5	O
+is	O
+an	O
+equivalent	O
+hydrophobic	O
+leucine	O
+residue	O
+(	O
+see	O
+also	O
+Fig	O
+.	O
+7f	O
+,	O
+g	O
+),	O
+suggesting	O
+that	O
+these	O
+two	O
+Phe	O
+residues	O
+would	O
+play	O
+a	O
+similar	O
+role	O
+in	O
+facilitating	O
+substrate	O
+recognition	O
+and	O
+catalysis	O
+,	O
+respectively	O
+.	O
+	O
+An	O
+important	O
+feature	O
+of	O
+MKP	O
+–	O
+JNK1	O
+interactions	O
+is	O
+that	O
+MKP7	O
+or	O
+MKP5	O
+only	O
+interact	O
+with	O
+the	O
+F	O
+-	O
+site	O
+of	O
+JNK1	O
+.	O
+	O
+One	O
+possible	O
+explanation	O
+is	O
+that	O
+JNK1	O
+needs	O
+to	O
+use	O
+the	O
+D	O
+-	O
+site	O
+to	O
+interact	O
+with	O
+JIP	O
+-	O
+1	O
+,	O
+a	O
+scaffold	O
+protein	O
+for	O
+JNK	O
+signalling	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+JNK	O
+-	O
+binding	O
+domain	O
+of	O
+JIP	O
+-	O
+1	O
+interacts	O
+with	O
+the	O
+D	O
+-	O
+site	O
+on	O
+JNK	O
+and	O
+this	O
+interaction	O
+is	O
+required	O
+for	O
+JIP	O
+-	O
+1	O
+-	O
+mediated	O
+enhancement	O
+of	O
+JNK	O
+activation	O
+.	O
+	O
+In	O
+addition	O
+,	O
+JIP	O
+-	O
+1	O
+can	O
+also	O
+associate	O
+with	O
+MKP7	O
+via	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+MKP7	O
+(	O
+ref	O
+.).	O
+	O
+Thus	O
+,	O
+our	O
+biochemical	O
+and	O
+structural	O
+data	O
+allow	O
+us	O
+to	O
+present	O
+a	O
+model	O
+for	O
+the	O
+JNK1	O
+–	O
+JIP	O
+-	O
+1	O
+–	O
+MKP7	O
+ternary	O
+complex	O
+and	O
+provide	O
+an	O
+important	O
+insight	O
+into	O
+the	O
+assembly	O
+and	O
+function	O
+of	O
+JNK	O
+signalling	O
+modules	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+The	O
+cDNAs	O
+of	O
+human	O
+MKP7	O
+and	O
+MKP5	O
+were	O
+kindly	O
+provided	O
+by	O
+Dr	O
+Mathijs	O
+Baens	O
+(	O
+University	O
+of	O
+Leuven	O
+)	O
+and	O
+Dr	O
+Eisuke	O
+Nishida	O
+(	O
+Kyoto	O
+University	O
+),	O
+respectively	O
+.	O
+	O
+The	O
+cDNAs	O
+of	O
+human	O
+ASK1	O
+,	O
+MKK4	O
+,	O
+MKK7	O
+and	O
+JNK1	O
+were	O
+kindly	O
+provided	O
+by	O
+Dr	O
+Zhenguo	O
+Wu	O
+(	O
+Hong	O
+Kong	O
+University	O
+of	O
+Science	O
+and	O
+Technology	O
+).	O
+	O
+The	O
+human	O
+full	O
+-	O
+length	O
+JNK1	O
+,	O
+MKK4	O
+,	O
+MKK7	O
+and	O
+the	O
+kinase	O
+domain	O
+of	O
+ASK1	O
+(	O
+659	O
+–	O
+951	O
+)	O
+were	O
+cloned	O
+into	O
+pGEX4T	O
+-	O
+2	O
+,	O
+pET15b	O
+and	O
+/	O
+or	O
+pET21b	O
+vectors	O
+to	O
+produce	O
+a	O
+GST	O
+-	O
+or	O
+His	O
+-	O
+tagged	O
+protein	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+unphosphorylated	O
+JNK1	O
+in	O
+complex	O
+with	O
+the	O
+catalytic	O
+domain	O
+of	O
+MKP7	O
+was	O
+refined	O
+to	O
+2	O
+.	O
+4	O
+Å	O
+resolution	O
+.	O
+	O
+Domain	O
+structures	O
+of	O
+ten	O
+human	O
+MKPs	O
+and	O
+the	O
+atypical	O
+VHR	O
+.	O
+	O
+On	O
+the	O
+basis	O
+of	O
+sequence	O
+similarity	O
+,	O
+protein	O
+structure	O
+,	O
+substrate	O
+specificity	O
+and	O
+subcellular	O
+localization	O
+,	O
+the	O
+ten	O
+members	O
+of	O
+MKP	O
+family	O
+can	O
+be	O
+divided	O
+into	O
+three	O
+groups	O
+.	O
+	O
+The	O
+first	O
+subfamily	O
+comprises	O
+MKP1	O
+,	O
+MKP2	O
+,	O
+PAC1	O
+and	O
+hVH3	O
+,	O
+which	O
+are	O
+inducible	O
+nuclear	O
+phosphatases	O
+and	O
+can	O
+dephosphorylate	O
+ERK	O
+(	O
+and	O
+JNK	O
+,	O
+p38	O
+)	O
+MAPKs	O
+.	O
+	O
+The	O
+second	O
+subfamily	O
+contains	O
+MKP3	O
+,	O
+MKP4	O
+and	O
+MKPX	O
+,	O
+which	O
+are	O
+cytoplasmic	O
+ERK	O
+-	O
+specific	O
+MKPs	O
+.	O
+	O
+The	O
+third	O
+subfamily	O
+comprises	O
+MKP5	O
+,	O
+MKP7	O
+and	O
+hVH5	O
+,	O
+which	O
+were	O
+located	O
+in	O
+both	O
+nucleus	O
+and	O
+cytoplasm	O
+,	O
+and	O
+selectively	O
+inactivate	O
+JNK	O
+and	O
+p38	O
+.	O
+	O
+All	O
+MKPs	O
+contain	O
+both	O
+the	O
+CD	O
+and	O
+KBD	O
+domains	O
+,	O
+whereas	O
+VHR	O
+,	O
+an	O
+atypical	O
+MKP	O
+,	O
+only	O
+contains	O
+a	O
+highly	O
+conserved	O
+catalytic	O
+domain	O
+.	O
+	O
+In	O
+addition	O
+to	O
+the	O
+CD	O
+and	O
+KBD	O
+,	O
+MKP7	O
+contains	O
+a	O
+unique	O
+long	O
+C	O
+-	O
+terminal	O
+region	O
+that	O
+contains	O
+NES	O
+,	O
+NLS	O
+and	O
+PEST	O
+motifs	O
+,	O
+which	O
+has	O
+no	O
+effect	O
+on	O
+the	O
+binding	O
+ability	O
+and	O
+phosphatase	O
+activity	O
+of	O
+MKP7	O
+toward	O
+MAPKs	O
+.	O
+	O
+NES	O
+,	O
+nuclear	O
+export	O
+signal	O
+;	O
+NLS	O
+,	O
+nuclear	O
+localization	O
+signal	O
+;	O
+PEST	O
+,	O
+C	O
+-	O
+terminal	O
+sequence	O
+rich	O
+in	O
+prolines	O
+,	O
+glutamates	O
+,	O
+serines	O
+and	O
+threonines	O
+.	O
+	O
+MKP7	O
+-	O
+CD	O
+is	O
+crucial	O
+for	O
+JNK1	O
+binding	O
+and	O
+enzyme	O
+catalysis	O
+.	O
+	O
+The	O
+KBD	O
+and	O
+CD	O
+of	O
+MKP7	O
+are	O
+shown	O
+in	O
+green	O
+and	O
+blue	O
+,	O
+and	O
+the	O
+N	O
+-	O
+lobe	O
+and	O
+C	O
+-	O
+lobe	O
+of	O
+JNK1	O
+are	O
+coloured	O
+in	O
+lemon	O
+and	O
+yellow	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+error	O
+bars	O
+represent	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+(	O
+c	O
+)	O
+Gel	O
+filtration	O
+analysis	O
+for	O
+interaction	O
+of	O
+JNK1	O
+with	O
+MKP7	O
+-	O
+CD	O
+and	O
+MKP7	O
+-	O
+KBD	O
+.	O
+	O
+(	O
+d	O
+)	O
+GST	O
+-	O
+mediated	O
+pull	O
+-	O
+down	O
+assay	O
+for	O
+interaction	O
+of	O
+JNK1	O
+with	O
+MKP7	O
+-	O
+CD	O
+and	O
+MKP7	O
+-	O
+KBD	O
+.	O
+	O
+The	O
+protein	O
+amounts	O
+of	O
+MKP7	O
+used	O
+are	O
+shown	O
+at	O
+the	O
+bottom	O
+.	O
+	O
+Structure	O
+of	O
+JNK1	O
+in	O
+complex	O
+with	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+(	O
+a	O
+)	O
+Ribbon	O
+diagram	O
+of	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+complex	O
+in	O
+two	O
+views	O
+related	O
+by	O
+a	O
+45	O
+°	O
+rotation	O
+around	O
+a	O
+vertical	O
+axis	O
+.	O
+(	O
+b	O
+)	O
+Structure	O
+of	O
+MKP7	O
+-	O
+CD	O
+with	O
+its	O
+active	O
+site	O
+highlight	O
+in	O
+cyan	O
+.	O
+	O
+The	O
+JNK1	O
+is	O
+shown	O
+in	O
+surface	O
+representation	O
+coloured	O
+according	O
+to	O
+electrostatic	O
+potential	O
+(	O
+positive	O
+,	O
+blue	O
+;	O
+negative	O
+,	O
+red	O
+).	O
+	O
+(	O
+e	O
+)	O
+Interaction	O
+networks	O
+mainly	O
+involving	O
+helices	O
+α4	O
+and	O
+α5	O
+from	O
+MKP7	O
+-	O
+CD	O
+,	O
+and	O
+αG	O
+and	O
+α2L14	O
+of	O
+JNK1	O
+.	O
+	O
+(	O
+f	O
+)	O
+The	O
+2Fo	O
+−	O
+Fc	O
+omit	O
+map	O
+(	O
+contoured	O
+at	O
+1	O
+.	O
+5σ	O
+)	O
+clearly	O
+shows	O
+electron	O
+density	O
+for	O
+the	O
+285FNFL288	O
+segment	O
+of	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+(	O
+a	O
+)	O
+Effects	O
+of	O
+mutations	O
+in	O
+MKP7	O
+-	O
+CD	O
+on	O
+the	O
+JNK1	O
+dephosphorylation	O
+(	O
+mean	O
+±	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.,	O
+n	O
+=	O
+3	O
+).	O
+	O
+Residues	O
+involved	O
+in	O
+hydrophobic	O
+and	O
+hydrophilic	O
+contacts	O
+are	O
+coloured	O
+in	O
+red	O
+and	O
+blue	O
+,	O
+respectively	O
+.	O
+(	O
+b	O
+)	O
+Gel	O
+filtration	O
+analysis	O
+for	O
+interaction	O
+of	O
+JNK1	O
+with	O
+MKP7	O
+-	O
+CD	O
+mutant	O
+F285D	B-mutant
+.	O
+	O
+Mutant	O
+F285D	B-mutant
+and	O
+JNK1	O
+were	O
+eluted	O
+as	O
+monomers	O
+,	O
+with	O
+the	O
+molecular	O
+masses	O
+of	O
+∼	O
+17	O
+and	O
+44	O
+kDa	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+top	O
+panel	O
+shows	O
+the	O
+relative	O
+affinities	O
+of	O
+MKP7	O
+-	O
+CD	O
+to	O
+JNK1	O
+mutants	O
+,	O
+with	O
+the	O
+affinity	O
+of	O
+wild	O
+-	O
+type	O
+JNK1	O
+defined	O
+as	O
+100	O
+%,	O
+the	O
+middle	O
+panel	O
+is	O
+the	O
+electrophoretic	O
+pattern	O
+of	O
+MKP7	O
+-	O
+CD	O
+and	O
+JNK1	O
+mutants	O
+after	O
+GST	O
+pull	O
+-	O
+down	O
+assays	O
+.	O
+	O
+The	O
+protein	O
+amounts	O
+of	O
+MKP7	O
+-	O
+CD	O
+used	O
+are	O
+shown	O
+at	O
+the	O
+bottom	O
+.	O
+(	O
+d	O
+)	O
+Circular	O
+dichroism	O
+spectra	O
+for	O
+MKP7	O
+-	O
+CD	O
+wild	O
+type	O
+and	O
+mutants	O
+.	O
+	O
+(	O
+f	O
+)	O
+Effects	O
+of	O
+mutations	O
+in	O
+MKP7	O
+-	O
+CD	O
+on	O
+the	O
+pNPP	O
+hydrolysis	O
+reaction	O
+(	O
+mean	O
+±	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.,	O
+n	O
+=	O
+3	O
+).	O
+	O
+Comparison	O
+of	O
+CDK2	O
+-	O
+KAP	O
+and	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+(	O
+a	O
+)	O
+Superposition	O
+of	O
+the	O
+complex	O
+structures	O
+of	O
+CDK2	O
+-	O
+KAP	O
+(	O
+PDB	O
+1FQ1	O
+)	O
+and	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+The	O
+interactions	O
+between	O
+these	O
+two	O
+proteins	O
+consist	O
+of	O
+three	O
+discontinuous	O
+contact	O
+regions	O
+,	O
+centred	O
+at	O
+the	O
+multiple	O
+hydrogen	O
+bonds	O
+between	O
+the	O
+pThr160	O
+of	O
+CDK2	O
+and	O
+the	O
+active	O
+site	O
+of	O
+KAP	O
+(	O
+region	O
+I	O
+).	O
+	O
+(	O
+b	O
+)	O
+Interactions	O
+networks	O
+at	O
+the	O
+auxiliary	O
+region	O
+II	O
+mainly	O
+involving	O
+helix	O
+α7	O
+from	O
+KAP	O
+and	O
+the	O
+αG	O
+helix	O
+and	O
+following	O
+L14	O
+loop	O
+of	O
+CDK2	O
+.	O
+	O
+Residues	O
+of	O
+KAP	O
+and	O
+CDK2	O
+are	O
+highlighted	O
+as	O
+green	O
+and	O
+red	O
+sticks	O
+,	O
+respectively	O
+.	O
+	O
+(	O
+d	O
+)	O
+Sequence	O
+alignment	O
+of	O
+the	O
+F	O
+-	O
+site	O
+regions	O
+on	O
+MAPKs	O
+.	O
+	O
+Residues	O
+of	O
+JNK1	O
+involved	O
+in	O
+recognition	O
+of	O
+MKP7	O
+are	O
+indicated	O
+by	O
+orange	O
+asterisks	O
+,	O
+and	O
+those	O
+forming	O
+the	O
+F	O
+-	O
+site	O
+are	O
+highlighted	O
+in	O
+yellow	O
+.	O
+	O
+FXF	O
+-	O
+motif	O
+is	O
+critical	O
+for	O
+controlling	O
+the	O
+phosphorylation	O
+of	O
+JNK	O
+and	O
+ultraviolet	O
+-	O
+induced	O
+apoptosis	O
+.	O
+	O
+(	O
+a	O
+–	O
+c	O
+)	O
+FXF	O
+-	O
+motif	O
+is	O
+essential	O
+for	O
+the	O
+dephosphorylation	O
+of	O
+JNK	O
+by	O
+MKP7	O
+.	O
+	O
+HEK293T	O
+cells	O
+were	O
+infected	O
+with	O
+lentiviruses	O
+expressing	O
+MKP7	O
+and	O
+its	O
+mutants	O
+(	O
+1	O
+.	O
+0	O
+μg	O
+).	O
+	O
+After	O
+36	O
+h	O
+infection	O
+,	O
+cells	O
+were	O
+untreated	O
+in	O
+a	O
+,	O
+stimulated	O
+with	O
+30	O
+μM	O
+etoposide	O
+for	O
+3	O
+h	O
+in	O
+b	O
+or	O
+irradiated	O
+with	O
+25	O
+J	O
+m	O
+−	O
+2	O
+ultraviolet	O
+light	O
+at	O
+30	O
+min	O
+before	O
+lysis	O
+in	O
+c	O
+.	O
+Whole	O
+-	O
+cell	O
+extracts	O
+were	O
+then	O
+immunoblotted	O
+with	O
+antibody	O
+indicated	O
+.	O
+	O
+HEK293T	O
+cells	O
+were	O
+co	O
+-	O
+transfected	O
+with	O
+MKP7	O
+full	O
+-	O
+length	O
+(	O
+1	O
+.	O
+0	O
+μg	O
+)	O
+and	O
+JNK1	O
+(	O
+wild	O
+type	O
+or	O
+mutants	O
+as	O
+indicated	O
+,	O
+1	O
+.	O
+0	O
+μg	O
+).	O
+	O
+Whole	O
+-	O
+cell	O
+extracts	O
+were	O
+then	O
+immunoprecipitated	O
+with	O
+antibody	O
+against	O
+Myc	O
+for	O
+MKP7	O
+;	O
+immunobloting	O
+was	O
+carried	O
+out	O
+with	O
+antibodies	O
+indicated	O
+.	O
+	O
+IP	O
+,	O
+immunoprecipitation	O
+;	O
+TCL	O
+,	O
+total	O
+cell	O
+lysate	O
+.	O
+	O
+HeLa	O
+cells	O
+were	O
+infected	O
+with	O
+lentiviruses	O
+expressing	O
+MKP7	O
+full	O
+-	O
+length	O
+and	O
+its	O
+mutants	O
+.	O
+	O
+Cells	O
+were	O
+then	O
+subjected	O
+to	O
+flow	O
+cytometry	O
+analysis	O
+.	O
+	O
+Apoptotic	O
+cells	O
+were	O
+determined	O
+by	O
+Annexin	O
+-	O
+V	O
+-	O
+APC	O
+/	O
+PI	O
+staining	O
+.	O
+	O
+Staining	O
+with	O
+both	O
+Annexin	O
+-	O
+V	O
+and	O
+PI	O
+indicate	O
+apoptosis	O
+(	O
+upper	O
+right	O
+quadrant	O
+).	O
+	O
+MKP5	O
+-	O
+CD	O
+is	O
+crucial	O
+for	O
+JNK1	O
+binding	O
+and	O
+enzyme	O
+catalysis	O
+.	O
+	O
+(	O
+a	O
+)	O
+Domain	O
+organization	O
+of	O
+human	O
+MKP5	O
+.	O
+	O
+The	O
+KBD	O
+and	O
+CD	O
+of	O
+MKP5	O
+are	O
+shown	O
+in	O
+brown	O
+and	O
+grey	O
+,	O
+respectively	O
+.	O
+(	O
+b	O
+)	O
+Plots	O
+of	O
+initial	O
+velocity	O
+of	O
+the	O
+MKP5	O
+-	O
+catalysed	O
+reaction	O
+versus	O
+phospho	O
+-	O
+JNK1	O
+concentration	O
+.	O
+	O
+The	O
+corresponding	O
+residues	O
+on	O
+MKP5	O
+are	O
+depicted	O
+as	O
+orange	O
+sticks	O
+,	O
+and	O
+MKP5	O
+residues	O
+numbers	O
+are	O
+in	O
+parentheses	O
+.	O
+	O
+(	O
+h	O
+)	O
+Pull	O
+-	O
+down	O
+assays	O
+of	O
+MKP5	O
+-	O
+CD	O
+by	O
+GST	O
+-	O
+tagged	O
+JNK1	O
+mutants	O
+.	O
+	O
+Plants	O
+constantly	O
+renew	O
+during	O
+their	O
+life	O
+cycle	O
+and	O
+thus	O
+require	O
+to	O
+shed	O
+senescent	O
+and	O
+damaged	O
+organs	O
+.	O
+	O
+Floral	O
+abscission	O
+is	O
+controlled	O
+by	O
+the	O
+leucine	O
+-	O
+rich	O
+repeat	O
+receptor	O
+kinase	O
+(	O
+LRR	O
+-	O
+RK	O
+)	O
+HAESA	O
+and	O
+the	O
+peptide	O
+hormone	O
+IDA	O
+.	O
+	O
+Here	O
+we	O
+show	O
+that	O
+IDA	O
+is	O
+sensed	O
+directly	O
+by	O
+the	O
+HAESA	O
+ectodomain	O
+.	O
+	O
+Crystal	O
+structures	O
+of	O
+HAESA	O
+in	O
+complex	O
+with	O
+IDA	O
+reveal	O
+a	O
+hormone	O
+binding	O
+pocket	O
+that	O
+accommodates	O
+an	O
+active	O
+dodecamer	O
+peptide	O
+.	O
+	O
+A	O
+central	O
+hydroxyproline	O
+residue	O
+anchors	O
+IDA	O
+to	O
+the	O
+receptor	O
+.	O
+	O
+This	O
+sequence	O
+pattern	O
+is	O
+conserved	O
+among	O
+diverse	O
+plant	O
+peptides	O
+,	O
+suggesting	O
+that	O
+plant	O
+peptide	O
+hormone	O
+receptors	O
+may	O
+share	O
+a	O
+common	O
+ligand	O
+binding	O
+mode	O
+and	O
+activation	O
+mechanism	O
+.	O
+	O
+However	O
+,	O
+the	O
+molecular	O
+details	O
+of	O
+how	O
+IDA	O
+triggers	O
+organ	O
+shedding	O
+are	O
+not	O
+clear	O
+.	O
+	O
+The	O
+shedding	O
+of	O
+floral	O
+organs	O
+(	O
+or	O
+leaves	O
+)	O
+can	O
+be	O
+easily	O
+studied	O
+in	O
+a	O
+model	O
+plant	O
+called	O
+Arabidopsis	O
+.	O
+	O
+Santiago	O
+et	O
+al	O
+.	O
+used	O
+protein	O
+biochemistry	O
+,	O
+structural	O
+biology	O
+and	O
+genetics	O
+to	O
+uncover	O
+how	O
+the	O
+IDA	O
+hormone	O
+activates	O
+HAESA	O
+.	O
+	O
+The	O
+experiments	O
+show	O
+that	O
+IDA	O
+binds	O
+directly	O
+to	O
+a	O
+canyon	O
+shaped	O
+pocket	O
+in	O
+HAESA	O
+that	O
+extends	O
+out	O
+from	O
+the	O
+surface	O
+of	O
+the	O
+cell	O
+.	O
+	O
+IDA	O
+binding	O
+to	O
+HAESA	O
+allows	O
+another	O
+receptor	O
+protein	O
+called	O
+SERK1	O
+to	O
+bind	O
+to	O
+HAESA	O
+,	O
+which	O
+results	O
+in	O
+the	O
+release	O
+of	O
+signals	O
+inside	O
+the	O
+cell	O
+that	O
+trigger	O
+the	O
+shedding	O
+of	O
+organs	O
+.	O
+	O
+The	O
+next	O
+step	O
+following	O
+on	O
+from	O
+this	O
+work	O
+is	O
+to	O
+understand	O
+what	O
+signals	O
+are	O
+produced	O
+when	O
+IDA	O
+activates	O
+HAESA	O
+.	O
+	O
+Another	O
+challenge	O
+will	O
+be	O
+to	O
+find	O
+out	O
+where	O
+IDA	O
+is	O
+produced	O
+in	O
+the	O
+plant	O
+and	O
+what	O
+causes	O
+it	O
+to	O
+accumulate	O
+in	O
+specific	O
+places	O
+in	O
+preparation	O
+for	O
+organ	O
+shedding	O
+.	O
+	O
+(	O
+A	O
+)	O
+SDS	O
+PAGE	O
+analysis	O
+of	O
+the	O
+purified	O
+Arabidopsis	O
+thaliana	O
+HAESA	O
+ectodomain	O
+(	O
+residues	O
+20	O
+–	O
+620	O
+)	O
+obtained	O
+by	O
+secreted	O
+expression	O
+in	O
+insect	O
+cells	O
+.	O
+	O
+The	O
+calculated	O
+molecular	O
+mass	O
+is	O
+65	O
+.	O
+7	O
+kDa	O
+,	O
+the	O
+actual	O
+molecular	O
+mass	O
+obtained	O
+by	O
+mass	O
+spectrometry	O
+is	O
+74	O
+,	O
+896	O
+Da	O
+,	O
+accounting	O
+for	O
+the	O
+N	O
+-	O
+glycans	O
+.	O
+(	O
+B	O
+)	O
+Ribbon	O
+diagrams	O
+showing	O
+front	O
+(	O
+left	O
+panel	O
+)	O
+and	O
+side	O
+views	O
+(	O
+right	O
+panel	O
+)	O
+of	O
+the	O
+isolated	O
+HAESA	O
+LRR	O
+domain	O
+.	O
+	O
+The	O
+N	O
+-	O
+(	O
+residues	O
+20	O
+–	O
+88	O
+)	O
+and	O
+C	O
+-	O
+terminal	O
+(	O
+residues	O
+593	O
+–	O
+615	O
+)	O
+capping	O
+domains	O
+are	O
+shown	O
+in	O
+yellow	O
+,	O
+the	O
+central	O
+21	O
+LRR	O
+motifs	O
+are	O
+in	O
+blue	O
+and	O
+disulphide	O
+bonds	O
+are	O
+highlighted	O
+in	O
+green	O
+(	O
+in	O
+bonds	O
+representation	O
+).	O
+(	O
+C	O
+)	O
+Structure	O
+based	O
+sequence	O
+alignment	O
+of	O
+the	O
+21	O
+leucine	O
+-	O
+rich	O
+repeats	O
+in	O
+HAESA	O
+with	O
+the	O
+plant	O
+LRR	O
+consensus	O
+sequence	O
+shown	O
+for	O
+comparison	O
+.	O
+	O
+Conserved	O
+hydrophobic	O
+residues	O
+are	O
+shaded	O
+in	O
+gray	O
+,	O
+N	O
+-	O
+glycosylation	O
+sites	O
+visible	O
+in	O
+our	O
+structures	O
+are	O
+highlighted	O
+in	O
+blue	O
+,	O
+cysteine	O
+residues	O
+involved	O
+in	O
+disulphide	O
+bridge	O
+formation	O
+in	O
+green	O
+.	O
+(	O
+D	O
+)	O
+Asn	O
+-	O
+linked	O
+glycans	O
+mask	O
+the	O
+N	O
+-	O
+terminal	O
+portion	O
+of	O
+the	O
+HAESA	O
+ectodomain	O
+.	O
+	O
+Oligomannose	O
+core	O
+structures	O
+(	O
+containing	O
+two	O
+N	O
+-	O
+actylglucosamines	O
+and	O
+three	O
+terminal	O
+mannose	O
+units	O
+)	O
+as	O
+found	O
+in	O
+Trichoplusia	O
+ni	O
+cells	O
+and	O
+in	O
+plants	O
+were	O
+modeled	O
+onto	O
+the	O
+seven	O
+glycosylation	O
+sites	O
+observed	O
+in	O
+our	O
+HAESA	O
+structures	O
+,	O
+to	O
+visualize	O
+the	O
+surface	O
+areas	O
+potentially	O
+not	O
+masked	O
+by	O
+carbohydrate	O
+.	O
+	O
+Hydrophobic	O
+contacts	O
+and	O
+a	O
+hydrogen	O
+-	O
+bond	O
+network	O
+mediate	O
+the	O
+interaction	O
+between	O
+HAESA	O
+and	O
+the	O
+peptide	O
+hormone	O
+IDA	O
+.	O
+	O
+(	O
+A	O
+)	O
+Details	O
+of	O
+the	O
+IDA	O
+binding	O
+pocket	O
+.	O
+	O
+HAESA	O
+is	O
+shown	O
+in	O
+blue	O
+(	O
+ribbon	O
+diagram	O
+),	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	O
+-	O
+His	O
+-	O
+Asn	O
+motif	O
+(	O
+left	O
+panel	O
+),	O
+the	O
+central	O
+Hyp	O
+anchor	O
+(	O
+center	O
+)	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+Pro	O
+-	O
+rich	O
+motif	O
+in	O
+IDA	O
+(	O
+right	O
+panel	O
+)	O
+are	O
+shown	O
+in	O
+yellow	O
+(	O
+in	O
+bonds	O
+representation	O
+).	O
+	O
+HAESA	O
+interface	O
+residues	O
+are	O
+shown	O
+as	O
+sticks	O
+,	O
+selected	O
+hydrogen	O
+bond	O
+interactions	O
+are	O
+denoted	O
+as	O
+dotted	O
+lines	O
+(	O
+in	O
+magenta	O
+).	O
+(	O
+B	O
+)	O
+View	O
+of	O
+the	O
+complete	O
+IDA	O
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+yellow	O
+)	O
+binding	O
+pocket	O
+in	O
+HAESA	O
+(	O
+surface	O
+view	O
+,	O
+in	O
+blue	O
+).	O
+	O
+Orientation	O
+as	O
+in	O
+(	O
+A	O
+).	O
+(	O
+C	O
+)	O
+Structure	O
+based	O
+sequence	O
+alignment	O
+of	O
+leucine	O
+-	O
+rich	O
+repeats	O
+in	O
+HAESA	O
+with	O
+the	O
+plant	O
+LRR	O
+consensus	O
+sequence	O
+shown	O
+for	O
+comparison	O
+.	O
+	O
+The	O
+IDA	O
+binding	O
+pocket	O
+covers	O
+LRRs	O
+2	O
+–	O
+14	O
+and	O
+all	O
+residues	O
+originate	O
+from	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+HAESA	O
+superhelix	O
+.	O
+	O
+The	O
+IDA	O
+-	O
+HAESA	O
+and	O
+SERK1	O
+-	O
+HAESA	O
+complex	O
+interfaces	O
+are	O
+conserved	O
+among	O
+HAESA	O
+and	O
+HAESA	O
+-	O
+like	O
+proteins	O
+from	O
+different	O
+plant	O
+species	O
+.	O
+	O
+Structure	O
+-	O
+based	O
+sequence	O
+alignment	O
+of	O
+the	O
+HAESA	O
+family	O
+members	O
+:	O
+Arabidopsis	O
+thaliana	O
+HAESA	O
+(	O
+Uniprot	O
+(	O
+http	O
+://	O
+www	O
+.	O
+uniprot	O
+.	O
+org	O
+)	O
+ID	O
+P47735	O
+),	O
+Arabidopsis	O
+thaliana	O
+HSL2	O
+(	O
+Uniprot	O
+ID	O
+C0LGX3	O
+),	O
+Capsella	O
+rubella	O
+HAESA	O
+(	O
+Uniprot	O
+ID	O
+R0F2U6	O
+),	O
+Citrus	O
+clementina	O
+HSL2	O
+(	O
+Uniprot	O
+ID	O
+V4U227	O
+),	O
+Vitis	O
+vinifera	O
+HAESA	O
+(	O
+Uniprot	O
+ID	O
+F6HM39	O
+).	O
+	O
+HAESA	O
+residues	O
+interacting	O
+with	O
+the	O
+IDA	O
+peptide	O
+and	O
+/	O
+or	O
+the	O
+SERK1	O
+co	O
+-	O
+receptor	O
+kinase	O
+ectodomain	O
+are	O
+highlighted	O
+in	O
+blue	O
+and	O
+orange	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+peptide	O
+hormone	O
+IDA	O
+binds	O
+to	O
+the	O
+HAESA	O
+LRR	O
+ectodomain	O
+.	O
+	O
+(	O
+A	O
+)	O
+Multiple	O
+sequence	O
+alignment	O
+of	O
+selected	O
+IDA	O
+family	O
+members	O
+.	O
+	O
+The	O
+conserved	O
+PIP	O
+motif	O
+is	O
+highlighted	O
+in	O
+yellow	O
+,	O
+the	O
+central	O
+Hyp	O
+in	O
+blue	O
+.	O
+	O
+The	O
+PKGV	O
+motif	O
+present	O
+in	O
+our	O
+N	O
+-	O
+terminally	O
+extended	O
+IDA	O
+peptide	O
+is	O
+highlighted	O
+in	O
+red	O
+.	O
+(	O
+B	O
+)	O
+Isothermal	O
+titration	O
+calorimetry	O
+of	O
+the	O
+HAESA	O
+ectodomain	O
+vs	O
+.	O
+IDA	O
+and	O
+including	O
+the	O
+synthetic	O
+peptide	O
+sequence	O
+.	O
+	O
+(	O
+C	O
+)	O
+Structure	O
+of	O
+the	O
+HAESA	O
+–	O
+IDA	O
+complex	O
+with	O
+HAESA	O
+shown	O
+in	O
+blue	O
+(	O
+ribbon	O
+diagram	O
+).	O
+	O
+The	O
+peptide	O
+binding	O
+pocket	O
+covers	O
+HAESA	O
+LRRs	O
+2	O
+–	O
+14	O
+.	O
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+entire	O
+IDA	O
+(	O
+in	O
+yellow	O
+)	O
+peptide	O
+binding	O
+site	O
+in	O
+HAESA	O
+(	O
+in	O
+blue	O
+).	O
+	O
+Details	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+central	O
+Hyp	O
+anchor	O
+in	O
+IDA	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	O
+-	O
+His	O
+-	O
+Asn	O
+motif	O
+with	O
+HAESA	O
+are	O
+highlighted	O
+in	O
+(	O
+E	O
+)	O
+and	O
+(	O
+F	O
+),	O
+respectively	O
+.	O
+	O
+During	O
+their	O
+growth	O
+,	O
+development	O
+and	O
+reproduction	O
+plants	O
+use	O
+cell	O
+separation	O
+processes	O
+to	O
+detach	O
+no	O
+-	O
+longer	O
+required	O
+,	O
+damaged	O
+or	O
+senescent	O
+organs	O
+.	O
+	O
+The	O
+LRR	O
+-	O
+RKs	O
+HAESA	O
+(	O
+greek	O
+:	O
+to	O
+adhere	O
+to	O
+)	O
+and	O
+HAESA	O
+-	O
+LIKE	O
+2	O
+(	O
+HSL2	O
+)	O
+redundantly	O
+control	O
+floral	O
+abscission	O
+.	O
+	O
+Loss	O
+-	O
+of	O
+-	O
+function	O
+of	O
+the	O
+secreted	O
+small	O
+protein	O
+INFLORESCENCE	O
+DEFICIENT	O
+IN	O
+ABSCISSION	O
+(	O
+IDA	O
+)	O
+causes	O
+floral	O
+organs	O
+to	O
+remain	O
+attached	O
+while	O
+its	O
+over	O
+-	O
+expression	O
+leads	O
+to	O
+premature	O
+shedding	O
+.	O
+	O
+Full	O
+-	O
+length	O
+IDA	O
+is	O
+proteolytically	O
+processed	O
+and	O
+a	O
+conserved	O
+stretch	O
+of	O
+20	O
+amino	O
+-	O
+acids	O
+(	O
+termed	O
+EPIP	O
+)	O
+can	O
+rescue	O
+the	O
+IDA	O
+loss	O
+-	O
+of	O
+-	O
+function	O
+phenotype	O
+(	O
+Figure	O
+1A	O
+).	O
+	O
+It	O
+has	O
+been	O
+demonstrated	O
+that	O
+a	O
+dodecamer	O
+peptide	O
+within	O
+EPIP	O
+is	O
+able	O
+to	O
+activate	O
+HAESA	O
+and	O
+HSL2	O
+in	O
+transient	O
+assays	O
+in	O
+tobacco	O
+cells	O
+.	O
+	O
+IDA	O
+directly	O
+binds	O
+to	O
+the	O
+LRR	O
+domain	O
+of	O
+HAESA	O
+	O
+Active	O
+IDA	O
+-	O
+family	O
+peptide	O
+hormones	O
+are	O
+hydroxyprolinated	O
+dodecamers	O
+.	O
+	O
+Note	O
+that	O
+Pro58IDA	O
+and	O
+Leu67IDA	O
+are	O
+the	O
+first	O
+residues	O
+defined	O
+by	O
+electron	O
+density	O
+when	O
+bound	O
+to	O
+the	O
+HAESA	O
+ectodomain	O
+.	O
+(	O
+D	O
+)	O
+Table	O
+summaries	O
+for	O
+equilibrium	O
+dissociation	O
+constants	O
+(	O
+Kd	O
+),	O
+binding	O
+enthalpies	O
+(	O
+ΔH	O
+),	O
+binding	O
+entropies	O
+(	O
+ΔS	O
+)	O
+and	O
+stoichoimetries	O
+(	O
+N	O
+)	O
+for	O
+different	O
+IDA	O
+peptides	O
+binding	O
+to	O
+the	O
+HAESA	O
+ectodomain	O
+(	O
+±	O
+fitting	O
+errors	O
+;	O
+n	O
+.	O
+d	O
+.	O
+	O
+no	O
+detectable	O
+binding	O
+).	O
+(	O
+E	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+active	O
+IDA	O
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+gray	O
+)	O
+and	O
+IDL1	O
+peptide	O
+(	O
+in	O
+yellow	O
+)	O
+hormones	O
+bound	O
+to	O
+the	O
+HAESA	O
+ectodomain	O
+.	O
+	O
+Root	O
+mean	O
+square	O
+deviation	O
+(	O
+r	O
+.	O
+m	O
+.	O
+s	O
+.	O
+d	O
+.)	O
+is	O
+1	O
+.	O
+0	O
+Å	O
+comparing	O
+100	O
+corresponding	O
+atoms	O
+.	O
+	O
+The	O
+receptor	O
+kinase	O
+SERK1	O
+acts	O
+as	O
+a	O
+HAESA	O
+co	O
+-	O
+receptor	O
+and	O
+promotes	O
+high	O
+-	O
+affinity	O
+IDA	O
+sensing	O
+.	O
+	O
+(	O
+A	O
+)	O
+Petal	O
+break	O
+-	O
+strength	O
+assays	O
+measure	O
+the	O
+force	O
+(	O
+expressed	O
+in	O
+gram	O
+equivalents	O
+)	O
+required	O
+to	O
+remove	O
+the	O
+petals	O
+from	O
+the	O
+flower	O
+of	O
+serk	O
+mutant	O
+plants	O
+compared	O
+to	O
+haesa	O
+/	O
+hsl2	O
+mutant	O
+and	O
+Col	O
+-	O
+0	O
+wild	O
+-	O
+type	O
+flowers	O
+.	O
+	O
+Petal	O
+break	O
+-	O
+strength	O
+was	O
+found	O
+significantly	O
+increased	O
+in	O
+almost	O
+all	O
+positions	O
+(	O
+indicated	O
+with	O
+a	O
+*)	O
+for	O
+haesa	O
+/	O
+hsl2	O
+and	O
+serk1	O
+-	O
+1	O
+mutant	O
+plants	O
+with	O
+respect	O
+to	O
+the	O
+Col	O
+-	O
+0	O
+control	O
+.	O
+	O
+The	O
+HAESA	O
+LRR	O
+domain	O
+elutes	O
+as	O
+a	O
+monomer	O
+(	O
+black	O
+dotted	O
+line	O
+),	O
+as	O
+does	O
+the	O
+isolated	O
+SERK1	O
+ectodomain	O
+(	O
+blue	O
+dotted	O
+line	O
+).	O
+	O
+A	O
+HAESA	O
+–	O
+IDA	O
+–	O
+SERK1	O
+complex	O
+elutes	O
+as	O
+an	O
+apparent	O
+heterodimer	O
+(	O
+red	O
+line	O
+),	O
+while	O
+a	O
+mixture	O
+of	O
+HAESA	O
+and	O
+SERK1	O
+yields	O
+two	O
+isolated	O
+peaks	O
+that	O
+correspond	O
+to	O
+monomeric	O
+HAESA	O
+and	O
+SERK1	O
+,	O
+respectively	O
+(	O
+black	O
+line	O
+).	O
+	O
+no	O
+detectable	O
+binding	O
+)	O
+(	O
+D	O
+)	O
+Analytical	O
+size	O
+-	O
+exclusion	O
+chromatography	O
+in	O
+the	O
+presence	O
+of	O
+the	O
+IDA	O
+Hyp64	O
+→	O
+Pro	O
+mutant	O
+peptide	O
+reveals	O
+no	O
+complex	O
+formation	O
+between	O
+HAESA	O
+and	O
+SERK1	O
+ectodomains	O
+.	O
+	O
+(	O
+E	O
+)	O
+In	O
+vitro	O
+kinase	O
+assays	O
+of	O
+the	O
+HAESA	O
+and	O
+SERK1	O
+kinase	O
+domains	O
+.	O
+	O
+Wild	O
+-	O
+type	O
+HAESA	O
+and	O
+SERK1	O
+kinase	O
+domains	O
+(	O
+KDs	O
+)	O
+exhibit	O
+auto	O
+-	O
+phosphorylation	O
+activities	O
+(	O
+lanes	O
+1	O
++	O
+3	O
+).	O
+	O
+Transphosphorylation	O
+activity	O
+from	O
+the	O
+active	O
+kinase	O
+to	O
+the	O
+mutated	O
+form	O
+can	O
+be	O
+observed	O
+in	O
+both	O
+directions	O
+(	O
+lanes	O
+5	O
++	O
+6	O
+).	O
+	O
+A	O
+Hyp	O
+-	O
+modified	O
+dodecamer	O
+comprising	O
+the	O
+highly	O
+conserved	O
+PIP	O
+motif	O
+in	O
+IDA	O
+(	O
+Figure	O
+1A	O
+)	O
+interacts	O
+with	O
+HAESA	O
+with	O
+1	O
+:	O
+1	O
+stoichiometry	O
+(	O
+N	O
+)	O
+and	O
+with	O
+a	O
+dissociation	O
+constant	O
+(	O
+Kd	O
+)	O
+of	O
+~	O
+20	O
+μM	O
+(	O
+Figure	O
+1B	O
+).	O
+	O
+The	O
+central	O
+Hyp64IDA	O
+is	O
+buried	O
+in	O
+a	O
+specific	O
+pocket	O
+formed	O
+by	O
+HAESA	O
+LRRs	O
+8	O
+–	O
+10	O
+,	O
+with	O
+its	O
+hydroxyl	O
+group	O
+establishing	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+strictly	O
+conserved	O
+Glu266HAESA	O
+and	O
+with	O
+a	O
+water	O
+molecule	O
+,	O
+which	O
+in	O
+turn	O
+is	O
+coordinated	O
+by	O
+the	O
+main	O
+chain	O
+oxygens	O
+of	O
+Phe289HAESA	O
+and	O
+Ser311HAESA	O
+(	O
+Figure	O
+1E	O
+;	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+).	O
+	O
+The	O
+restricted	O
+size	O
+of	O
+the	O
+Hyp	O
+pocket	O
+suggests	O
+that	O
+IDA	O
+does	O
+not	O
+require	O
+arabinosylation	O
+of	O
+Hyp64IDA	O
+for	O
+activity	O
+in	O
+vivo	O
+,	O
+a	O
+modification	O
+that	O
+has	O
+been	O
+reported	O
+for	O
+Hyp	O
+residues	O
+in	O
+plant	O
+CLE	O
+peptide	O
+hormones	O
+.	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+Arg	O
+-	O
+His	O
+-	O
+Asn	O
+motif	O
+in	O
+IDA	O
+maps	O
+to	O
+a	O
+cavity	O
+formed	O
+by	O
+HAESA	O
+LRRs	O
+11	O
+–	O
+14	O
+(	O
+Figure	O
+1D	O
+,	O
+F	O
+).	O
+	O
+The	O
+COO	O
+-	O
+group	O
+of	O
+Asn69IDA	O
+is	O
+in	O
+direct	O
+contact	O
+with	O
+Arg407HAESA	O
+and	O
+Arg409HAESA	O
+and	O
+HAESA	O
+cannot	O
+bind	O
+a	O
+C	O
+-	O
+terminally	O
+extended	O
+IDA	B-mutant
+-	I-mutant
+SFVN	I-mutant
+peptide	O
+(	O
+Figures	O
+1D	O
+,	O
+F	O
+,	O
+2D	O
+).	O
+	O
+This	O
+suggests	O
+that	O
+the	O
+conserved	O
+Asn69IDA	O
+may	O
+constitute	O
+the	O
+very	O
+C	O
+-	O
+terminus	O
+of	O
+the	O
+mature	O
+IDA	O
+peptide	O
+in	O
+planta	O
+and	O
+that	O
+active	O
+IDA	O
+is	O
+generated	O
+by	O
+proteolytic	O
+processing	O
+from	O
+a	O
+longer	O
+pre	O
+-	O
+protein	O
+.	O
+	O
+Mutation	O
+of	O
+Arg417HSL2	O
+(	O
+which	O
+corresponds	O
+to	O
+Arg409HAESA	O
+)	O
+causes	O
+a	O
+loss	O
+-	O
+of	O
+-	O
+function	O
+phenotype	O
+in	O
+HSL2	O
+,	O
+which	O
+indicates	O
+that	O
+the	O
+peptide	O
+binding	O
+pockets	O
+in	O
+different	O
+HAESA	O
+receptors	O
+have	O
+common	O
+structural	O
+and	O
+sequence	O
+features	O
+.	O
+	O
+Indeed	O
+,	O
+we	O
+find	O
+many	O
+of	O
+the	O
+residues	O
+contributing	O
+to	O
+the	O
+formation	O
+of	O
+the	O
+IDA	O
+binding	O
+surface	O
+in	O
+HAESA	O
+to	O
+be	O
+conserved	O
+in	O
+HSL2	O
+and	O
+in	O
+other	O
+HAESA	O
+-	O
+type	O
+receptors	O
+in	O
+different	O
+plant	O
+species	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+).	O
+	O
+A	O
+N	O
+-	O
+terminal	O
+Pro	O
+-	O
+rich	O
+motif	O
+in	O
+IDA	O
+makes	O
+contacts	O
+with	O
+LRRs	O
+2	O
+–	O
+6	O
+of	O
+the	O
+receptor	O
+(	O
+Figure	O
+1D	O
+,	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2A	O
+–	O
+C	O
+).	O
+	O
+HAESA	O
+specifically	O
+senses	O
+IDA	O
+-	O
+family	O
+dodecamer	O
+peptides	O
+	O
+We	O
+next	O
+investigated	O
+whether	O
+HAESA	O
+binds	O
+N	O
+-	O
+terminally	O
+extended	O
+versions	O
+of	O
+IDA	O
+.	O
+	O
+We	O
+obtained	O
+a	O
+structure	O
+of	O
+HAESA	O
+in	O
+complex	O
+with	O
+a	O
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+peptide	O
+at	O
+1	O
+.	O
+94	O
+Å	O
+resolution	O
+(	O
+Table	O
+2	O
+).	O
+	O
+In	O
+this	O
+structure	O
+,	O
+no	O
+additional	O
+electron	O
+density	O
+accounts	O
+for	O
+the	O
+PKGV	O
+motif	O
+at	O
+the	O
+IDA	O
+N	O
+-	O
+terminus	O
+(	O
+Figure	O
+2A	O
+,	O
+B	O
+).	O
+	O
+IDL1	O
+,	O
+which	O
+can	O
+rescue	O
+IDA	O
+loss	O
+-	O
+of	O
+-	O
+function	O
+mutants	O
+when	O
+introduced	O
+in	O
+abscission	O
+zone	O
+cells	O
+,	O
+can	O
+also	O
+be	O
+sensed	O
+by	O
+HAESA	O
+,	O
+albeit	O
+with	O
+lower	O
+affinity	O
+(	O
+Figure	O
+2D	O
+).	O
+	O
+A	O
+2	O
+.	O
+56	O
+Å	O
+co	O
+-	O
+crystal	O
+structure	O
+with	O
+IDL1	O
+reveals	O
+that	O
+different	O
+IDA	O
+family	O
+members	O
+use	O
+a	O
+common	O
+binding	O
+mode	O
+to	O
+interact	O
+with	O
+HAESA	O
+-	O
+type	O
+receptors	O
+(	O
+Figure	O
+2A	O
+–	O
+C	O
+,	O
+E	O
+,	O
+Table	O
+2	O
+).	O
+	O
+We	O
+do	O
+not	O
+detect	O
+interaction	O
+between	O
+HAESA	O
+and	O
+a	O
+synthetic	O
+peptide	O
+missing	O
+the	O
+C	O
+-	O
+terminal	O
+Asn69IDA	O
+(	O
+ΔN69	B-mutant
+),	O
+highlighting	O
+the	O
+importance	O
+of	O
+the	O
+polar	O
+interactions	O
+between	O
+the	O
+IDA	O
+carboxy	O
+-	O
+terminus	O
+and	O
+Arg407HAESA	O
+/	O
+Arg409HAESA	O
+(	O
+Figures	O
+1F	O
+,	O
+2D	O
+).	O
+	O
+The	O
+co	O
+-	O
+receptor	O
+kinase	O
+SERK1	O
+allows	O
+for	O
+high	O
+-	O
+affinity	O
+IDA	O
+sensing	O
+	O
+It	O
+has	O
+been	O
+recently	O
+reported	O
+that	O
+SOMATIC	O
+EMBRYOGENESIS	O
+RECEPTOR	O
+KINASES	O
+(	O
+SERKs	O
+)	O
+are	O
+positive	O
+regulators	O
+of	O
+floral	O
+abscission	O
+and	O
+can	O
+interact	O
+with	O
+HAESA	O
+and	O
+HSL2	O
+in	O
+an	O
+IDA	O
+-	O
+dependent	O
+manner	O
+.	O
+	O
+As	O
+all	O
+five	O
+SERK	O
+family	O
+members	O
+appear	O
+to	O
+be	O
+expressed	O
+in	O
+the	O
+Arabidopsis	O
+abscission	O
+zone	O
+,	O
+we	O
+quantified	O
+their	O
+relative	O
+contribution	O
+to	O
+floral	O
+abscission	O
+in	O
+Arabidopsis	O
+using	O
+a	O
+petal	O
+break	O
+-	O
+strength	O
+assay	O
+.	O
+	O
+We	O
+found	O
+that	O
+the	O
+force	O
+required	O
+to	O
+remove	O
+the	O
+petals	O
+of	O
+serk1	O
+-	O
+1	O
+mutants	O
+is	O
+significantly	O
+higher	O
+than	O
+that	O
+needed	O
+for	O
+wild	O
+-	O
+type	O
+plants	O
+,	O
+as	O
+previously	O
+observed	O
+for	O
+haesa	O
+/	O
+hsl2	O
+mutants	O
+,	O
+and	O
+that	O
+floral	O
+abscission	O
+is	O
+delayed	O
+in	O
+serk1	O
+-	O
+1	O
+(	O
+Figure	O
+3A	O
+).	O
+	O
+Possibly	O
+because	O
+SERKs	O
+have	O
+additional	O
+roles	O
+in	O
+plant	O
+development	O
+such	O
+as	O
+in	O
+pollen	O
+formation	O
+and	O
+brassinosteroid	O
+signaling	O
+,	O
+we	O
+found	O
+that	O
+higher	O
+-	O
+order	O
+SERK	O
+mutants	O
+exhibit	O
+pleiotropic	O
+phenotypes	O
+in	O
+the	O
+flower	O
+,	O
+rendering	O
+their	O
+analysis	O
+and	O
+comparison	O
+by	O
+quantitative	O
+petal	O
+break	O
+-	O
+strength	O
+assays	O
+difficult	O
+.	O
+	O
+We	O
+next	O
+quantified	O
+the	O
+contribution	O
+of	O
+SERK1	O
+to	O
+IDA	O
+recognition	O
+by	O
+HAESA	O
+.	O
+	O
+We	O
+next	O
+titrated	O
+SERK1	O
+into	O
+a	O
+solution	O
+containing	O
+only	O
+the	O
+HAESA	O
+ectodomain	O
+.	O
+	O
+In	O
+this	O
+case	O
+,	O
+there	O
+was	O
+no	O
+detectable	O
+interaction	O
+between	O
+receptor	O
+and	O
+co	O
+-	O
+receptor	O
+,	O
+while	O
+in	O
+the	O
+presence	O
+of	O
+IDA	O
+,	O
+SERK1	O
+strongly	O
+binds	O
+HAESA	O
+with	O
+a	O
+dissociation	O
+constant	O
+in	O
+the	O
+mid	O
+-	O
+nanomolar	O
+range	O
+(	O
+Figure	O
+3C	O
+).	O
+	O
+This	O
+suggests	O
+that	O
+IDA	O
+itself	O
+promotes	O
+receptor	O
+–	O
+co	O
+-	O
+receptor	O
+association	O
+,	O
+as	O
+previously	O
+described	O
+for	O
+the	O
+steroid	O
+hormone	O
+brassinolide	O
+and	O
+for	O
+other	O
+LRR	O
+-	O
+RK	O
+complexes	O
+.	O
+	O
+Consistently	O
+,	O
+the	O
+HAESA	O
+kinase	O
+domain	O
+can	O
+transphosphorylate	O
+SERK1	O
+and	O
+vice	O
+versa	O
+in	O
+in	O
+vitro	O
+transphosphorylation	O
+assays	O
+(	O
+Figure	O
+3E	O
+).	O
+	O
+SERK1	O
+senses	O
+a	O
+conserved	O
+motif	O
+in	O
+IDA	O
+family	O
+peptides	O
+	O
+(	O
+A	O
+)	O
+Overview	O
+of	O
+the	O
+ternary	O
+complex	O
+with	O
+HAESA	O
+in	O
+blue	O
+(	O
+surface	O
+representation	O
+),	O
+IDA	O
+in	O
+yellow	O
+(	O
+bonds	O
+representation	O
+)	O
+and	O
+SERK1	O
+in	O
+orange	O
+(	O
+surface	O
+view	O
+).	O
+(	O
+B	O
+)	O
+The	O
+HAESA	O
+ectodomain	O
+undergoes	O
+a	O
+conformational	O
+change	O
+upon	O
+SERK1	O
+co	O
+-	O
+receptor	O
+binding	O
+.	O
+	O
+Shown	O
+are	O
+Cα	O
+traces	O
+of	O
+a	O
+structural	O
+superposition	O
+of	O
+the	O
+unbound	O
+(	O
+yellow	O
+)	O
+and	O
+SERK1	O
+-	O
+bound	O
+(	O
+blue	O
+)	O
+HAESA	O
+ectodomains	O
+(	O
+r	O
+.	O
+m	O
+.	O
+s	O
+.	O
+d	O
+.	O
+is	O
+1	O
+.	O
+5	O
+Å	O
+between	O
+572	O
+corresponding	O
+Cα	O
+atoms	O
+).	O
+	O
+SERK1	O
+(	O
+in	O
+orange	O
+)	O
+and	O
+IDA	O
+(	O
+in	O
+red	O
+)	O
+are	O
+shown	O
+alongside	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+capping	O
+domain	O
+of	O
+SERK1	O
+(	O
+in	O
+orange	O
+)	O
+directly	O
+contacts	O
+the	O
+C	O
+-	O
+terminal	O
+part	O
+of	O
+IDA	O
+(	O
+in	O
+yellow	O
+,	O
+in	O
+bonds	O
+representation	O
+)	O
+and	O
+the	O
+receptor	O
+HAESA	O
+(	O
+in	O
+blue	O
+).	O
+	O
+Polar	O
+contacts	O
+of	O
+SERK1	O
+with	O
+IDA	O
+are	O
+shown	O
+in	O
+magenta	O
+,	O
+with	O
+the	O
+HAESA	O
+LRR	O
+domain	O
+in	O
+gray	O
+.	O
+(	O
+D	O
+)	O
+Details	O
+of	O
+the	O
+zipper	O
+-	O
+like	O
+SERK1	O
+-	O
+HAESA	O
+interface	O
+.	O
+	O
+Ribbon	O
+diagrams	O
+of	O
+HAESA	O
+(	O
+in	O
+blue	O
+)	O
+and	O
+SERK1	O
+(	O
+in	O
+orange	O
+)	O
+are	O
+shown	O
+with	O
+selected	O
+interface	O
+residues	O
+(	O
+in	O
+bonds	O
+representation	O
+).	O
+	O
+To	O
+understand	O
+in	O
+molecular	O
+terms	O
+how	O
+SERK1	O
+contributes	O
+to	O
+high	O
+-	O
+affinity	O
+IDA	O
+recognition	O
+,	O
+we	O
+solved	O
+a	O
+2	O
+.	O
+43	O
+Å	O
+crystal	O
+structure	O
+of	O
+the	O
+ternary	O
+HAESA	O
+–	O
+IDA	O
+–	O
+SERK1	O
+complex	O
+(	O
+Figure	O
+4A	O
+,	O
+Table	O
+2	O
+).	O
+	O
+HAESA	O
+LRRs	O
+16	O
+–	O
+21	O
+and	O
+its	O
+C	O
+-	O
+terminal	O
+capping	O
+domain	O
+undergo	O
+a	O
+conformational	O
+change	O
+upon	O
+SERK1	O
+binding	O
+(	O
+Figure	O
+4B	O
+).	O
+	O
+SERK1	O
+loop	O
+residues	O
+establish	O
+multiple	O
+hydrophobic	O
+and	O
+polar	O
+contacts	O
+with	O
+Lys66IDA	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	O
+-	O
+His	O
+-	O
+Asn	O
+motif	O
+in	O
+IDA	O
+(	O
+Figure	O
+4C	O
+).	O
+	O
+SERK1	O
+LRRs	O
+1	O
+–	O
+5	O
+and	O
+its	O
+C	O
+-	O
+terminal	O
+capping	O
+domain	O
+form	O
+an	O
+additional	O
+zipper	O
+-	O
+like	O
+interface	O
+with	O
+residues	O
+originating	O
+from	O
+HAESA	O
+LRRs	O
+15	O
+–	O
+21	O
+and	O
+from	O
+the	O
+HAESA	O
+C	O
+-	O
+terminal	O
+cap	O
+(	O
+Figure	O
+4D	O
+).	O
+	O
+SERK1	O
+binds	O
+HAESA	O
+using	O
+these	O
+two	O
+distinct	O
+interaction	O
+surfaces	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+),	O
+with	O
+the	O
+N	O
+-	O
+cap	O
+of	O
+the	O
+SERK1	O
+LRR	O
+domain	O
+partially	O
+covering	O
+the	O
+IDA	O
+peptide	O
+binding	O
+cleft	O
+.	O
+	O
+The	O
+IDA	O
+C	O
+-	O
+terminal	O
+motif	O
+is	O
+required	O
+for	O
+HAESA	O
+-	O
+SERK1	O
+complex	O
+formation	O
+and	O
+for	O
+IDA	O
+bioactivity	O
+.	O
+	O
+Purified	O
+HAESA	O
+and	O
+SERK1	O
+are	O
+~	O
+75	O
+and	O
+~	O
+28	O
+kDa	O
+,	O
+respectively	O
+.	O
+	O
+Left	O
+panel	O
+:	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+;	O
+center	O
+:	O
+IDA	B-mutant
+ΔN69	I-mutant
+,	O
+right	O
+panel	O
+:	O
+SDS	O
+-	O
+PAGE	O
+of	O
+peak	O
+fractions	O
+.	O
+	O
+Note	O
+that	O
+the	O
+HAESA	O
+and	O
+SERK1	O
+input	O
+lanes	O
+have	O
+already	O
+been	O
+shown	O
+in	O
+Figure	O
+3D	O
+.	O
+(	O
+B	O
+)	O
+Isothermal	O
+titration	O
+thermographs	O
+of	O
+wild	O
+-	O
+type	O
+and	O
+mutant	O
+IDA	O
+peptides	O
+titrated	O
+into	O
+a	O
+HAESA	O
+-	O
+SERK1	O
+mixture	O
+in	O
+the	O
+cell	O
+.	O
+	O
+Table	O
+summaries	O
+for	O
+calorimetric	O
+binding	O
+constants	O
+and	O
+stoichoimetries	O
+for	O
+different	O
+IDA	O
+peptides	O
+binding	O
+to	O
+the	O
+HAESA	O
+–	O
+SERK1	O
+ectodomain	O
+mixture	O
+(	O
+±	O
+fitting	O
+errors	O
+;	O
+n	O
+.	O
+d	O
+.	O
+	O
+(	O
+C	O
+)	O
+Quantitative	O
+petal	O
+break	O
+-	O
+strength	O
+assay	O
+for	O
+Col	O
+-	O
+0	O
+wild	O
+-	O
+type	O
+flowers	O
+and	O
+35S	O
+::	O
+IDA	O
+wild	O
+-	O
+type	O
+and	O
+35S	O
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+mutant	O
+flowers	O
+.	O
+	O
+35S	O
+::	O
+IDA	O
+plants	O
+showed	O
+significantly	O
+increased	O
+abscission	O
+compared	O
+to	O
+Col	O
+-	O
+0	O
+controls	O
+in	O
+inflorescence	O
+positions	O
+2	O
+and	O
+3	O
+(	O
+a	O
+).	O
+	O
+Up	O
+to	O
+inflorescence	O
+position	O
+4	O
+,	O
+petal	O
+break	O
+in	O
+35S	O
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+mutant	O
+plants	O
+was	O
+significantly	O
+increased	O
+compared	O
+to	O
+both	O
+Col	O
+-	O
+0	O
+control	O
+plants	O
+(	O
+b	O
+)	O
+and	O
+35S	O
+::	O
+IDA	O
+plants	O
+(	O
+c	O
+)	O
+(	O
+D	O
+)	O
+Normalized	O
+expression	O
+levels	O
+(	O
+relative	O
+expression	O
+±	O
+standard	O
+error	O
+;	O
+ida	O
+:	O
+-	O
+0	O
+.	O
+02	O
+±	O
+0	O
+.	O
+001	O
+;	O
+Col	O
+-	O
+0	O
+:	O
+1	O
+±	O
+0	O
+.	O
+11	O
+;	O
+35S	O
+::	O
+IDA	O
+124	O
+±	O
+0	O
+.	O
+75	O
+;	O
+35S	O
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+:	O
+159	O
+±	O
+0	O
+.	O
+58	O
+)	O
+of	O
+IDA	O
+wild	O
+-	O
+type	O
+and	O
+mutant	O
+transcripts	O
+in	O
+the	O
+35S	O
+promoter	O
+over	O
+-	O
+expression	O
+lines	O
+analyzed	O
+in	O
+(	O
+C	O
+).	O
+(	O
+E	O
+)	O
+Magnified	O
+view	O
+of	O
+representative	O
+abscission	O
+zones	O
+from	O
+35S	O
+::	O
+IDA	O
+,	O
+Col	O
+-	O
+0	O
+wild	O
+-	O
+type	O
+and	O
+35S	O
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+double	O
+-	O
+mutant	O
+T3	O
+transgenic	O
+lines	O
+.	O
+	O
+The	O
+four	O
+C	O
+-	O
+terminal	O
+residues	O
+in	O
+IDA	O
+(	O
+Lys66IDA	O
+-	O
+Asn69IDA	O
+)	O
+are	O
+conserved	O
+among	O
+IDA	O
+family	O
+members	O
+and	O
+are	O
+in	O
+direct	O
+contact	O
+with	O
+SERK1	O
+(	O
+Figures	O
+1A	O
+,	O
+4C	O
+).	O
+	O
+We	O
+thus	O
+assessed	O
+their	O
+contribution	O
+to	O
+HAESA	O
+–	O
+SERK1	O
+complex	O
+formation	O
+.	O
+	O
+Deletion	O
+of	O
+the	O
+buried	O
+Asn69IDA	O
+completely	O
+inhibits	O
+receptor	O
+–	O
+co	O
+-	O
+receptor	O
+complex	O
+formation	O
+and	O
+HSL2	O
+activation	O
+(	O
+Figure	O
+5A	O
+,	O
+B	O
+).	O
+	O
+A	O
+synthetic	O
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+mutant	O
+peptide	O
+(	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R66A	I-mutant
+)	O
+showed	O
+a	O
+10	O
+fold	O
+reduced	O
+binding	O
+affinity	O
+when	O
+titrated	O
+in	O
+a	O
+HAESA	O
+/	O
+SERK1	O
+protein	O
+solution	O
+(	O
+Figures	O
+5A	O
+,	O
+B	O
+,	O
+2D	O
+).	O
+	O
+Comparison	O
+of	O
+35S	O
+::	O
+IDA	O
+wild	O
+-	O
+type	O
+and	O
+mutant	O
+plants	O
+further	O
+indicates	O
+that	O
+mutation	O
+of	O
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+may	O
+cause	O
+a	O
+weak	O
+dominant	O
+negative	O
+effect	O
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+	O
+In	O
+agreement	O
+with	O
+our	O
+structures	O
+and	O
+biochemical	O
+assays	O
+,	O
+this	O
+experiment	O
+suggests	O
+a	O
+role	O
+of	O
+the	O
+conserved	O
+IDA	O
+C	O
+-	O
+terminus	O
+in	O
+the	O
+control	O
+of	O
+floral	O
+abscission	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+animal	O
+LRR	O
+receptors	O
+,	O
+plant	O
+LRR	O
+-	O
+RKs	O
+harbor	O
+spiral	O
+-	O
+shaped	O
+ectodomains	O
+and	O
+thus	O
+they	O
+require	O
+shape	O
+-	O
+complementary	O
+co	O
+-	O
+receptor	O
+proteins	O
+for	O
+receptor	O
+activation	O
+.	O
+	O
+SERK1	O
+has	O
+been	O
+previously	O
+reported	O
+as	O
+a	O
+positive	O
+regulator	O
+in	O
+plant	O
+embryogenesis	O
+,	O
+male	O
+sporogenesis	O
+,	O
+brassinosteroid	O
+signaling	O
+and	O
+in	O
+phytosulfokine	O
+perception	O
+.	O
+	O
+Recent	O
+findings	O
+by	O
+and	O
+our	O
+mechanistic	O
+studies	O
+now	O
+also	O
+support	O
+a	O
+positive	O
+role	O
+for	O
+SERK1	O
+in	O
+floral	O
+abscission	O
+.	O
+	O
+As	O
+serk1	O
+-	O
+1	O
+mutant	O
+plants	O
+show	O
+intermediate	O
+abscission	O
+phenotypes	O
+when	O
+compared	O
+to	O
+haesa	O
+/	O
+hsl2	O
+mutants	O
+,	O
+SERK1	O
+likely	O
+acts	O
+redundantly	O
+with	O
+other	O
+SERKs	O
+in	O
+the	O
+abscission	O
+zone	O
+(	O
+Figure	O
+3A	O
+).	O
+	O
+It	O
+has	O
+been	O
+previously	O
+suggested	O
+that	O
+SERK1	O
+can	O
+inhibit	O
+cell	O
+separation	O
+.	O
+	O
+However	O
+our	O
+results	O
+show	O
+that	O
+SERK1	O
+also	O
+can	O
+activate	O
+this	O
+process	O
+upon	O
+IDA	O
+sensing	O
+,	O
+indicating	O
+that	O
+SERKs	O
+may	O
+fulfill	O
+several	O
+different	O
+functions	O
+in	O
+the	O
+course	O
+of	O
+the	O
+abscission	O
+process	O
+.	O
+	O
+While	O
+the	O
+sequence	O
+of	O
+the	O
+mature	O
+IDA	O
+peptide	O
+has	O
+not	O
+been	O
+experimentally	O
+determined	O
+in	O
+planta	O
+,	O
+our	O
+HAESA	O
+-	O
+IDA	O
+complex	O
+structures	O
+and	O
+calorimetry	O
+assays	O
+suggest	O
+that	O
+active	O
+IDLs	O
+are	O
+hydroxyprolinated	O
+dodecamers	O
+.	O
+	O
+Our	O
+comparative	O
+structural	O
+and	O
+biochemical	O
+analysis	O
+further	O
+suggests	O
+that	O
+IDLs	O
+share	O
+a	O
+common	O
+receptor	O
+binding	O
+mode	O
+,	O
+but	O
+may	O
+preferably	O
+bind	O
+to	O
+HAESA	O
+,	O
+HSL1	O
+or	O
+HSL2	O
+in	O
+different	O
+plant	O
+tissues	O
+and	O
+organs	O
+.	O
+	O
+The	O
+fact	O
+that	O
+SERK1	O
+specifically	O
+interacts	O
+with	O
+the	O
+very	O
+C	O
+-	O
+terminus	O
+of	O
+IDLs	O
+may	O
+allow	O
+for	O
+the	O
+rational	O
+design	O
+of	O
+peptide	O
+hormone	O
+antagonists	O
+,	O
+as	O
+previously	O
+demonstrated	O
+for	O
+the	O
+brassinosteroid	O
+pathway	O
+.	O
+	O
+Importantly	O
+,	O
+our	O
+calorimetry	O
+assays	O
+reveal	O
+that	O
+the	O
+SERK1	O
+ectodomain	O
+binds	O
+HAESA	O
+with	O
+nanomolar	O
+affinity	O
+,	O
+but	O
+only	O
+in	O
+the	O
+presence	O
+of	O
+IDA	O
+(	O
+Figure	O
+3C	O
+).	O
+	O
+This	O
+ligand	O
+-	O
+induced	O
+formation	O
+of	O
+a	O
+receptor	O
+–	O
+co	O
+-	O
+receptor	O
+complex	O
+may	O
+allow	O
+the	O
+HAESA	O
+and	O
+SERK1	O
+kinase	O
+domains	O
+to	O
+efficiently	O
+trans	O
+-	O
+phosphorylate	O
+and	O
+activate	O
+each	O
+other	O
+in	O
+the	O
+cytoplasm	O
+.	O
+	O
+SERK1	O
+uses	O
+partially	O
+overlapping	O
+surface	O
+areas	O
+to	O
+activate	O
+different	O
+plant	O
+signaling	O
+receptors	O
+.	O
+	O
+(	O
+A	O
+)	O
+Structural	O
+comparison	O
+of	O
+plant	O
+steroid	O
+and	O
+peptide	O
+hormone	O
+membrane	O
+signaling	O
+complexes	O
+.	O
+	O
+Left	O
+panel	O
+:	O
+Ribbon	O
+diagram	O
+of	O
+HAESA	O
+(	O
+in	O
+blue	O
+),	O
+SERK1	O
+(	O
+in	O
+orange	O
+)	O
+and	O
+IDA	O
+(	O
+in	O
+bonds	O
+and	O
+surface	O
+represention	O
+).	O
+	O
+Right	O
+panel	O
+:	O
+Ribbon	O
+diagram	O
+of	O
+the	O
+plant	O
+steroid	O
+receptor	O
+BRI1	O
+(	O
+in	O
+blue	O
+)	O
+bound	O
+to	O
+brassinolide	O
+(	O
+in	O
+gray	O
+,	O
+in	O
+bonds	O
+representation	O
+)	O
+and	O
+to	O
+SERK1	O
+,	O
+shown	O
+in	O
+the	O
+same	O
+orientation	O
+(	O
+PDB	O
+-	O
+ID	O
+.	O
+4lsx	O
+).	O
+	O
+A	O
+ribbon	O
+diagram	O
+of	O
+SERK1	O
+in	O
+the	O
+same	O
+orientation	O
+is	O
+shown	O
+alongside	O
+.	O
+	O
+Residues	O
+interacting	O
+with	O
+the	O
+HAESA	O
+or	O
+BRI1	O
+LRR	O
+domains	O
+are	O
+shown	O
+in	O
+orange	O
+or	O
+magenta	O
+,	O
+respectively	O
+.	O
+	O
+Comparison	O
+of	O
+our	O
+HAESA	O
+–	O
+IDA	O
+–	O
+SERK1	O
+structure	O
+with	O
+the	O
+brassinosteroid	O
+receptor	O
+signaling	O
+complex	O
+,	O
+where	O
+SERK1	O
+also	O
+acts	O
+as	O
+co	O
+-	O
+receptor	O
+,	O
+reveals	O
+an	O
+overall	O
+conserved	O
+mode	O
+of	O
+SERK1	O
+binding	O
+,	O
+while	O
+the	O
+ligand	O
+binding	O
+pockets	O
+map	O
+to	O
+very	O
+different	O
+areas	O
+in	O
+the	O
+corresponding	O
+receptors	O
+(	O
+LRRs	O
+2	O
+–	O
+14	O
+;	O
+HAESA	O
+;	O
+LRRs	O
+21	O
+–	O
+25	O
+,	O
+BRI1	O
+)	O
+and	O
+may	O
+involve	O
+an	O
+island	O
+domain	O
+(	O
+BRI1	O
+)	O
+or	O
+not	O
+(	O
+HAESA	O
+)	O
+(	O
+Figure	O
+6A	O
+).	O
+	O
+Several	O
+residues	O
+in	O
+the	O
+SERK1	O
+N	O
+-	O
+terminal	O
+capping	O
+domain	O
+(	O
+Thr59SERK1	O
+,	O
+Phe61SERK1	O
+)	O
+and	O
+the	O
+LRR	O
+inner	O
+surface	O
+(	O
+Asp75SERK1	O
+,	O
+Tyr101SERK1	O
+,	O
+SER121SERK1	O
+,	O
+Phe145SERK1	O
+)	O
+contribute	O
+to	O
+the	O
+formation	O
+of	O
+both	O
+complexes	O
+(	O
+Figures	O
+4C	O
+,	O
+D	O
+,	O
+6B	O
+).	O
+	O
+These	O
+residues	O
+are	O
+not	O
+involved	O
+in	O
+the	O
+sensing	O
+of	O
+the	O
+steroid	O
+hormone	O
+brassinolide	O
+.	O
+	O
+In	O
+both	O
+cases	O
+however	O
+,	O
+the	O
+co	O
+-	O
+receptor	O
+completes	O
+the	O
+hormone	O
+binding	O
+pocket	O
+.	O
+	O
+This	O
+fact	O
+together	O
+with	O
+the	O
+largely	O
+overlapping	O
+SERK1	O
+binding	O
+surfaces	O
+in	O
+HAESA	O
+and	O
+BRI1	O
+allows	O
+us	O
+to	O
+speculate	O
+that	O
+SERK1	O
+may	O
+promote	O
+high	O
+-	O
+affinity	O
+peptide	O
+hormone	O
+and	O
+brassinosteroid	O
+sensing	O
+by	O
+simply	O
+slowing	O
+down	O
+dissociation	O
+of	O
+the	O
+ligand	O
+from	O
+its	O
+cognate	O
+receptor	O
+.	O
+	O
+The	O
+conserved	O
+(	O
+Arg	O
+)-	O
+His	O
+-	O
+Asn	O
+motif	O
+is	O
+highlighted	O
+in	O
+red	O
+,	O
+the	O
+central	O
+Hyp	O
+residue	O
+in	O
+IDLs	O
+and	O
+CLEs	O
+is	O
+marked	O
+in	O
+blue	O
+.	O
+	O
+Our	O
+experiments	O
+reveal	O
+that	O
+SERK1	O
+recognizes	O
+a	O
+C	O
+-	O
+terminal	O
+Arg	O
+-	O
+His	O
+-	O
+Asn	O
+motif	O
+in	O
+IDA	O
+.	O
+	O
+Among	O
+these	O
+are	O
+the	O
+CLE	O
+peptides	O
+regulating	O
+stem	O
+cell	O
+maintenance	O
+in	O
+the	O
+shoot	O
+and	O
+the	O
+root	O
+.	O
+	O
+Diverse	O
+plant	O
+peptide	O
+hormones	O
+may	O
+thus	O
+also	O
+bind	O
+their	O
+LRR	O
+-	O
+RK	O
+receptors	O
+in	O
+an	O
+extended	O
+conformation	O
+along	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+LRR	O
+domain	O
+and	O
+may	O
+also	O
+use	O
+small	O
+,	O
+shape	O
+-	O
+complementary	O
+co	O
+-	O
+receptors	O
+for	O
+high	O
+-	O
+affinity	O
+ligand	O
+binding	O
+and	O
+receptor	O
+activation	O
+.	O
+	O
+Ensemble	O
+cryo	O
+-	O
+EM	O
+uncovers	O
+inchworm	O
+-	O
+like	O
+translocation	O
+of	O
+a	O
+viral	O
+IRES	O
+through	O
+the	O
+ribosome	O
+	O
+Internal	O
+ribosome	O
+entry	O
+sites	O
+(	O
+IRESs	O
+)	O
+mediate	O
+cap	O
+-	O
+independent	O
+translation	O
+of	O
+viral	O
+mRNAs	O
+.	O
+	O
+The	O
+IRES	O
+rearranges	O
+from	O
+extended	O
+to	O
+bent	O
+to	O
+extended	O
+conformations	O
+.	O
+	O
+This	O
+inchworm	O
+-	O
+like	O
+movement	O
+is	O
+coupled	O
+with	O
+ribosomal	O
+inter	O
+-	O
+subunit	O
+rotation	O
+and	O
+40S	O
+head	O
+swivel	O
+.	O
+	O
+eEF2	O
+,	O
+attached	O
+to	O
+the	O
+60S	O
+subunit	O
+,	O
+slides	O
+along	O
+the	O
+rotating	O
+40S	O
+subunit	O
+to	O
+enter	O
+the	O
+A	O
+site	O
+.	O
+	O
+Its	O
+diphthamide	O
+-	O
+bearing	O
+tip	O
+at	O
+domain	O
+IV	O
+separates	O
+the	O
+tRNA	O
+-	O
+mRNA	O
+-	O
+like	O
+pseudoknot	O
+I	O
+(	O
+PKI	O
+)	O
+of	O
+the	O
+IRES	O
+from	O
+the	O
+decoding	O
+center	O
+.	O
+	O
+This	O
+unlocks	O
+40S	O
+domains	O
+,	O
+facilitating	O
+head	O
+swivel	O
+and	O
+biasing	O
+IRES	O
+translocation	O
+via	O
+hitherto	O
+-	O
+elusive	O
+intermediates	O
+with	O
+PKI	O
+captured	O
+between	O
+the	O
+A	O
+and	O
+P	O
+sites	O
+.	O
+	O
+Virus	O
+propagation	O
+relies	O
+on	O
+the	O
+host	O
+translational	O
+apparatus	O
+.	O
+	O
+To	O
+efficiently	O
+compete	O
+with	O
+host	O
+mRNAs	O
+and	O
+engage	O
+in	O
+translation	O
+under	O
+stress	O
+,	O
+some	O
+viral	O
+mRNAs	O
+undergo	O
+cap	O
+-	O
+independent	O
+translation	O
+.	O
+	O
+An	O
+IRES	O
+is	O
+located	O
+at	O
+the	O
+5	O
+’	O
+untranslated	O
+region	O
+of	O
+the	O
+viral	O
+mRNA	O
+,	O
+preceding	O
+an	O
+open	O
+reading	O
+frame	O
+(	O
+ORF	O
+).	O
+	O
+Subsequent	O
+binding	O
+of	O
+an	O
+elongator	O
+aminoacyl	O
+-	O
+tRNA	O
+to	O
+the	O
+ribosomal	O
+A	O
+site	O
+transitions	O
+the	O
+initiation	O
+complex	O
+into	O
+the	O
+elongation	O
+cycle	O
+of	O
+translation	O
+.	O
+	O
+Upon	O
+peptide	O
+bond	O
+formation	O
+,	O
+the	O
+two	O
+tRNAs	O
+and	O
+their	O
+respective	O
+mRNA	O
+codons	O
+translocate	O
+from	O
+the	O
+A	O
+and	O
+P	O
+to	O
+P	O
+and	O
+E	O
+(	O
+exit	O
+)	O
+sites	O
+,	O
+freeing	O
+the	O
+A	O
+site	O
+for	O
+the	O
+next	O
+elongator	O
+tRNA	O
+.	O
+	O
+The	O
+IGR	O
+IRES	O
+mRNAs	O
+do	O
+not	O
+contain	O
+an	O
+AUG	O
+start	O
+codon	O
+.	O
+	O
+The	O
+IGR	O
+-	O
+IRES	O
+-	O
+driven	O
+initiation	O
+does	O
+not	O
+involve	O
+initiator	O
+tRNAMet	O
+and	O
+initiation	O
+factors	O
+.	O
+	O
+As	O
+such	O
+,	O
+this	O
+group	O
+of	O
+IRESs	O
+represents	O
+the	O
+most	O
+streamlined	O
+mechanism	O
+of	O
+eukaryotic	O
+translation	O
+initiation	O
+.	O
+	O
+Early	O
+electron	O
+cryo	O
+-	O
+microscopy	O
+(	O
+cryo	O
+-	O
+EM	O
+)	O
+studies	O
+have	O
+found	O
+that	O
+the	O
+CrPV	O
+IRES	O
+packs	O
+in	O
+the	O
+ribosome	O
+intersubunit	O
+space	O
+.	O
+	O
+Recent	O
+cryo	O
+-	O
+EM	O
+structures	O
+of	O
+ribosome	O
+-	O
+bound	O
+TSV	O
+IRES	O
+and	O
+CrPV	O
+IRES	O
+revealed	O
+that	O
+IGR	O
+IRESs	O
+position	O
+the	O
+ORF	O
+by	O
+mimicking	O
+a	O
+translating	O
+ribosome	O
+bound	O
+with	O
+tRNA	O
+and	O
+mRNA	O
+.	O
+	O
+The	O
+~	O
+200	O
+-	O
+nt	O
+IRES	O
+RNAs	O
+span	O
+from	O
+the	O
+A	O
+site	O
+beyond	O
+the	O
+E	O
+site	O
+.	O
+	O
+A	O
+conserved	O
+tRNA	O
+-	O
+mRNA	O
+–	O
+like	O
+structural	O
+element	O
+of	O
+pseudoknot	O
+I	O
+(	O
+PKI	O
+)	O
+interacts	O
+with	O
+the	O
+decoding	O
+center	O
+in	O
+the	O
+A	O
+site	O
+of	O
+the	O
+40S	O
+subunit	O
+.	O
+	O
+The	O
+downstream	O
+initiation	O
+codon	O
+—	O
+coding	O
+for	O
+alanine	O
+—	O
+is	O
+placed	O
+in	O
+the	O
+mRNA	O
+tunnel	O
+,	O
+preceding	O
+the	O
+decoding	O
+center	O
+.	O
+	O
+PKI	O
+of	O
+IGR	O
+IRESs	O
+therefore	O
+mimics	O
+an	O
+A	O
+-	O
+site	O
+elongator	O
+tRNA	O
+interacting	O
+with	O
+an	O
+mRNA	O
+sense	O
+codon	O
+,	O
+but	O
+not	O
+a	O
+P	O
+-	O
+site	O
+initiator	O
+tRNAMet	O
+and	O
+the	O
+AUG	O
+start	O
+codon	O
+.	O
+	O
+A	O
+cryo	O
+-	O
+EM	O
+structure	O
+of	O
+the	O
+ribosome	O
+bound	O
+with	O
+a	O
+CrPV	O
+IRES	O
+and	O
+release	O
+factor	O
+eRF1	O
+occupying	O
+the	O
+A	O
+site	O
+provided	O
+insight	O
+into	O
+the	O
+post	O
+-	O
+translocation	O
+state	O
+.	O
+	O
+In	O
+this	O
+structure	O
+,	O
+PKI	O
+is	O
+positioned	O
+in	O
+the	O
+P	O
+site	O
+and	O
+the	O
+first	O
+mRNA	O
+codon	O
+is	O
+located	O
+in	O
+the	O
+A	O
+site	O
+.	O
+	O
+How	O
+the	O
+large	O
+IRES	O
+RNA	O
+translocates	O
+within	O
+the	O
+ribosome	O
+,	O
+allowing	O
+PKI	O
+translocation	O
+from	O
+the	O
+A	O
+to	O
+P	O
+site	O
+is	O
+not	O
+known	O
+.	O
+	O
+The	O
+structural	O
+similarity	O
+of	O
+PKI	O
+and	O
+the	O
+tRNA	O
+anticodon	O
+stem	O
+loop	O
+(	O
+ASL	O
+)	O
+bound	O
+to	O
+a	O
+codon	O
+suggests	O
+that	O
+their	O
+mechanisms	O
+of	O
+translocation	O
+are	O
+similar	O
+to	O
+some	O
+extent	O
+.	O
+	O
+Translocation	O
+of	O
+the	O
+IRES	O
+or	O
+tRNA	O
+-	O
+mRNA	O
+requires	O
+eukaryotic	O
+elongation	O
+factor	O
+2	O
+(	O
+eEF2	O
+),	O
+a	O
+structural	O
+and	O
+functional	O
+homolog	O
+of	O
+the	O
+well	O
+-	O
+studied	O
+bacterial	O
+EF	O
+-	O
+G	O
+.	O
+Pre	O
+-	O
+translocation	O
+tRNA	O
+-	O
+bound	O
+ribosomes	O
+contain	O
+a	O
+peptidyl	O
+-	O
+and	O
+deacyl	O
+-	O
+tRNA	O
+,	O
+both	O
+base	O
+-	O
+paired	O
+to	O
+mRNA	O
+codons	O
+in	O
+the	O
+A	O
+and	O
+P	O
+sites	O
+(	O
+termed	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+).	O
+	O
+Intersubunit	O
+rotation	O
+occurs	O
+spontaneously	O
+upon	O
+peptidyl	O
+transfer	O
+,	O
+and	O
+is	O
+coupled	O
+with	O
+formation	O
+of	O
+hybrid	O
+tRNA	O
+states	O
+.	O
+	O
+In	O
+the	O
+rotated	O
+pre	O
+-	O
+translocation	O
+ribosome	O
+,	O
+the	O
+peptidyl	O
+-	O
+tRNA	O
+binds	O
+the	O
+A	O
+site	O
+of	O
+the	O
+small	O
+subunit	O
+with	O
+its	O
+ASL	O
+and	O
+the	O
+P	O
+site	O
+of	O
+the	O
+large	O
+subunit	O
+with	O
+the	O
+CCA	O
+3	O
+’	O
+end	O
+(	O
+A	O
+/	O
+P	O
+hybrid	O
+state	O
+).	O
+	O
+The	O
+ribosome	O
+can	O
+undergo	O
+spontaneous	O
+,	O
+thermally	O
+-	O
+driven	O
+forward	O
+-	O
+reverse	O
+rotation	O
+that	O
+shifts	O
+the	O
+two	O
+tRNAs	O
+between	O
+the	O
+hybrid	O
+and	O
+'	O
+classical	O
+'	O
+states	O
+while	O
+the	O
+anticodon	O
+stem	O
+loops	O
+remain	O
+non	O
+-	O
+translocated	O
+.	O
+	O
+EF	O
+-	O
+G	O
+is	O
+thought	O
+to	O
+'	O
+unlock	O
+'	O
+the	O
+pre	O
+-	O
+translocation	O
+ribosome	O
+,	O
+allowing	O
+movement	O
+of	O
+the	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+,	O
+however	O
+the	O
+structural	O
+details	O
+of	O
+this	O
+unlocking	O
+are	O
+not	O
+known	O
+.	O
+	O
+The	O
+second	O
+large	O
+-	O
+scale	O
+rearrangement	O
+involves	O
+rotation	O
+,	O
+or	O
+swiveling	O
+,	O
+of	O
+the	O
+head	O
+of	O
+the	O
+small	O
+subunit	O
+relative	O
+to	O
+the	O
+body	O
+.	O
+	O
+The	O
+head	O
+can	O
+rotate	O
+by	O
+up	O
+to	O
+~	O
+20	O
+°	O
+around	O
+the	O
+axis	O
+nearly	O
+orthogonal	O
+to	O
+that	O
+of	O
+intersubunit	O
+rotation	O
+,	O
+in	O
+the	O
+absence	O
+of	O
+tRNA	O
+or	O
+in	O
+the	O
+presence	O
+of	O
+a	O
+single	O
+P	O
+/	O
+E	O
+tRNA	O
+and	O
+eEF2	O
+or	O
+EF	O
+-	O
+G	O
+.	O
+Förster	O
+resonance	O
+energy	O
+transfer	O
+(	O
+FRET	O
+)	O
+data	O
+suggest	O
+that	O
+head	O
+swivel	O
+of	O
+the	O
+rotated	O
+small	O
+subunit	O
+facilitates	O
+EF	O
+-	O
+G	O
+-	O
+mediated	O
+movement	O
+of	O
+2tRNA	O
+•	O
+mRNA	O
+.	O
+	O
+The	O
+structural	O
+role	O
+of	O
+head	O
+swivel	O
+is	O
+not	O
+fully	O
+understood	O
+.	O
+	O
+Whether	O
+and	O
+how	O
+the	O
+head	O
+swivel	O
+mediates	O
+tRNA	O
+transition	O
+from	O
+the	O
+A	O
+to	O
+P	O
+site	O
+remains	O
+unknown	O
+.	O
+	O
+Comparison	O
+of	O
+70S	O
+•	O
+2tRNA	O
+•	O
+mRNA	O
+and	O
+80S	O
+•	O
+IRES	O
+translocation	O
+complexes	O
+.	O
+	O
+Nucleotides	O
+C1054	O
+,	O
+G966	O
+and	O
+G693	O
+of	O
+16S	O
+rRNA	O
+are	O
+shown	O
+in	O
+black	O
+to	O
+denote	O
+the	O
+A	O
+,	O
+P	O
+and	O
+E	O
+sites	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+extents	O
+of	O
+the	O
+30S	O
+subunit	O
+rotation	O
+and	O
+head	O
+swivel	O
+relative	O
+to	O
+their	O
+positions	O
+in	O
+the	O
+post	O
+-	O
+translocation	O
+structure	O
+are	O
+shown	O
+with	O
+arrows	O
+.	O
+	O
+References	O
+and	O
+PDB	O
+codes	O
+of	O
+the	O
+structures	O
+are	O
+shown	O
+.	O
+	O
+(	O
+b	O
+)	O
+Structures	O
+of	O
+the	O
+80S	O
+•	O
+IRES	O
+complexes	O
+in	O
+the	O
+absence	O
+and	O
+presence	O
+of	O
+eEF2	O
+(	O
+this	O
+work	O
+).	O
+	O
+The	O
+large	O
+ribosomal	O
+subunit	O
+is	O
+shown	O
+in	O
+cyan	O
+;	O
+the	O
+small	O
+subunit	O
+in	O
+light	O
+yellow	O
+(	O
+head	O
+)	O
+and	O
+wheat	O
+-	O
+yellow	O
+(	O
+body	O
+);	O
+the	O
+TSV	O
+IRES	O
+in	O
+red	O
+,	O
+eEF2	O
+in	O
+green	O
+.	O
+	O
+Unresolved	O
+regions	O
+of	O
+the	O
+IRES	O
+in	O
+densities	O
+for	O
+Structures	O
+III	O
+and	O
+V	O
+are	O
+shown	O
+in	O
+gray	O
+.	O
+	O
+The	O
+extents	O
+of	O
+the	O
+40S	O
+subunit	O
+rotation	O
+and	O
+head	O
+swivel	O
+relative	O
+to	O
+their	O
+positions	O
+in	O
+the	O
+post	O
+-	O
+translocation	O
+structure	O
+are	O
+shown	O
+with	O
+arrows	O
+.	O
+	O
+3D	O
+classification	O
+using	O
+a	O
+3D	O
+mask	O
+around	O
+the	O
+40S	O
+head	O
+,	O
+TSV	O
+IRES	O
+and	O
+eEF2	O
+,	O
+of	O
+the	O
+4x	O
+binned	O
+stack	O
+was	O
+used	O
+to	O
+identify	O
+particles	O
+containing	O
+both	O
+the	O
+IRES	O
+and	O
+eEF2	O
+.	O
+	O
+Cryo	O
+-	O
+EM	O
+density	O
+of	O
+Structures	O
+I	O
+-	O
+V	O
+.	O
+	O
+In	O
+panels	O
+(	O
+a	O
+-	O
+e	O
+),	O
+the	O
+maps	O
+are	O
+segmented	O
+and	O
+colored	O
+as	O
+in	O
+Figure	O
+1	O
+.	O
+	O
+The	O
+maps	O
+in	O
+all	O
+panels	O
+were	O
+B	O
+-	O
+softened	O
+by	O
+applying	O
+a	O
+B	O
+-	O
+factor	O
+of	O
+30	O
+Å2	O
+.	O
+	O
+(	O
+a	O
+-	O
+e	O
+)	O
+Cryo	O
+-	O
+EM	O
+map	O
+of	O
+Structures	O
+I	O
+,	O
+II	O
+,	O
+III	O
+,	O
+IV	O
+and	O
+V	O
+.	O
+(	O
+f	O
+-	O
+j	O
+)	O
+Local	O
+resolution	O
+of	O
+unfiltered	O
+and	O
+unmasked	O
+cryo	O
+-	O
+EM	O
+reconstructions	O
+,	O
+assessed	O
+using	O
+Blocres	O
+from	O
+the	O
+BSoft	O
+package	O
+,	O
+for	O
+Structures	O
+I	O
+,	O
+II	O
+,	O
+III	O
+,	O
+IV	O
+and	O
+V	O
+.	O
+(	O
+k	O
+-	O
+o	O
+)	O
+Cryo	O
+-	O
+EM	O
+density	O
+for	O
+the	O
+TSV	O
+IRES	O
+(	O
+red	O
+model	O
+)	O
+and	O
+eEF2	O
+(	O
+green	O
+model	O
+)	O
+in	O
+Structures	O
+I	O
+,	O
+II	O
+,	O
+III	O
+,	O
+IV	O
+and	O
+V	O
+.	O
+(	O
+p	O
+)	O
+Fourier	O
+shell	O
+correlation	O
+(	O
+FSC	O
+)	O
+curves	O
+for	O
+Structures	O
+I	O
+-	O
+V	O
+.	O
+The	O
+horizontal	O
+axis	O
+is	O
+labeled	O
+with	O
+spatial	O
+frequency	O
+Å	O
+-	O
+1	O
+and	O
+with	O
+Å	O
+.	O
+The	O
+resolutions	O
+stated	O
+in	O
+the	O
+text	O
+correspond	O
+to	O
+an	O
+FSC	O
+threshold	O
+value	O
+of	O
+0	O
+.	O
+143	O
+,	O
+shown	O
+as	O
+a	O
+dotted	O
+line	O
+,	O
+for	O
+the	O
+FREALIGN	O
+-	O
+derived	O
+FSC	O
+('	O
+Part_FSC	O
+').	O
+	O
+Nucleotides	O
+C1274	O
+,	O
+U1191	O
+of	O
+the	O
+40S	O
+head	O
+and	O
+G904	O
+of	O
+the	O
+platform	O
+(	O
+C1054	O
+,	O
+G966	O
+and	O
+G693	O
+in	O
+E	O
+.	O
+coli	O
+16S	O
+rRNA	O
+)	O
+are	O
+shown	O
+in	O
+black	O
+to	O
+denote	O
+the	O
+A	O
+,	O
+P	O
+and	O
+E	O
+sites	O
+,	O
+respectively	O
+.	O
+	O
+(	O
+b	O
+)	O
+Schematic	O
+representation	O
+of	O
+the	O
+structures	O
+shown	O
+in	O
+panel	O
+a	O
+,	O
+denoting	O
+the	O
+conformations	O
+of	O
+the	O
+small	O
+subunit	O
+relative	O
+to	O
+the	O
+large	O
+subunit	O
+.	O
+	O
+A	O
+,	O
+P	O
+and	O
+E	O
+sites	O
+are	O
+shown	O
+as	O
+rectangles	O
+.	O
+	O
+We	O
+used	O
+cryo	O
+-	O
+EM	O
+to	O
+visualize	O
+80S	O
+•	O
+TSV	O
+IRES	O
+complexes	O
+formed	O
+in	O
+the	O
+presence	O
+of	O
+eEF2	O
+•	O
+GTP	O
+and	O
+the	O
+translation	O
+inhibitor	O
+sordarin	O
+,	O
+which	O
+stabilizes	O
+eEF2	O
+on	O
+the	O
+ribosome	O
+.	O
+	O
+Although	O
+the	O
+mechanism	O
+of	O
+sordarin	O
+action	O
+is	O
+not	O
+fully	O
+understood	O
+,	O
+the	O
+inhibitor	O
+does	O
+not	O
+affect	O
+the	O
+conformation	O
+of	O
+eEF2	O
+•	O
+GDPNP	O
+on	O
+the	O
+ribosome	O
+,	O
+rendering	O
+it	O
+an	O
+excellent	O
+tool	O
+in	O
+translocation	O
+studies	O
+.	O
+	O
+This	O
+ensemble	O
+of	O
+structures	O
+allowed	O
+us	O
+to	O
+reconstruct	O
+a	O
+sequence	O
+of	O
+steps	O
+in	O
+IRES	O
+translocation	O
+induced	O
+by	O
+eEF2	O
+.	O
+	O
+We	O
+used	O
+single	O
+-	O
+particle	O
+cryo	O
+-	O
+EM	O
+and	O
+maximum	O
+-	O
+likelihood	O
+image	O
+classification	O
+in	O
+FREALIGN	O
+to	O
+obtain	O
+three	O
+-	O
+dimensional	O
+density	O
+maps	O
+from	O
+a	O
+single	O
+specimen	O
+.	O
+	O
+The	O
+translocation	O
+complex	O
+was	O
+formed	O
+using	O
+S	O
+.	O
+cerevisiae	O
+80S	O
+ribosomes	O
+,	O
+Taura	O
+syndrome	O
+virus	O
+IRES	O
+,	O
+and	O
+S	O
+.	O
+cerevisiae	O
+eEF2	O
+in	O
+the	O
+presence	O
+of	O
+GTP	O
+and	O
+the	O
+eEF2	O
+-	O
+binding	O
+translation	O
+inhibitor	O
+sordarin	O
+.	O
+	O
+Unsupervised	O
+cryo	O
+-	O
+EM	O
+data	O
+classification	O
+was	O
+combined	O
+with	O
+the	O
+use	O
+of	O
+three	O
+-	O
+dimensional	O
+and	O
+two	O
+-	O
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+around	O
+the	O
+ribosomal	O
+A	O
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+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+	O
+Large	O
+-	O
+scale	O
+rearrangements	O
+in	O
+Structures	O
+I	O
+through	O
+V	O
+,	O
+coupled	O
+with	O
+the	O
+movement	O
+of	O
+PKI	O
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+A	O
+to	O
+P	O
+site	O
+and	O
+eEF2	O
+entry	O
+into	O
+the	O
+A	O
+site	O
+.	O
+	O
+(	O
+a	O
+)	O
+Rotational	O
+states	O
+of	O
+the	O
+40S	O
+subunit	O
+in	O
+the	O
+80S	O
+•	O
+IRES	O
+structure	O
+(	O
+INIT	O
+;	O
+PDB	O
+3J6Y	O
+)	O
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+in	O
+80S	O
+•	O
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+•	O
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+Structures	O
+I	O
+,	O
+II	O
+,	O
+III	O
+,	O
+IV	O
+and	O
+V	O
+(	O
+this	O
+work	O
+).	O
+	O
+For	O
+each	O
+structure	O
+,	O
+the	O
+triangle	O
+outlines	O
+the	O
+contours	O
+of	O
+the	O
+40S	O
+body	O
+;	O
+the	O
+lower	O
+angle	O
+illustrates	O
+the	O
+extent	O
+of	O
+intersubunit	O
+(	O
+body	O
+)	O
+rotation	O
+.	O
+	O
+The	O
+sizes	O
+of	O
+the	O
+arrows	O
+correspond	O
+to	O
+the	O
+extent	O
+of	O
+the	O
+head	O
+swivel	O
+(	O
+yellow	O
+)	O
+and	O
+subunit	O
+rotation	O
+(	O
+black	O
+).	O
+	O
+(	O
+b	O
+)	O
+Solvent	O
+view	O
+(	O
+opposite	O
+from	O
+that	O
+shown	O
+in	O
+(	O
+a	O
+))	O
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+structure	O
+(	O
+INIT	O
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+)	O
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+I	O
+,	O
+II	O
+,	O
+III	O
+,	O
+IV	O
+and	O
+V	O
+(	O
+this	O
+work	O
+).	O
+	O
+The	O
+structures	O
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+colored	O
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+in	O
+Figure	O
+1	O
+.	O
+	O
+(	O
+a	O
+)	O
+Comparison	O
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+-	O
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+Structures	O
+I	O
+through	O
+V	O
+,	O
+sampling	O
+a	O
+~	O
+10	O
+°	O
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+Structure	O
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+(	O
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+rotated	O
+)	O
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+Structure	O
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+(	O
+non	O
+-	O
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+	O
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+Structures	O
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+-	O
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+alignments	O
+of	O
+the	O
+25S	O
+ribosomal	O
+RNAs	O
+.	O
+	O
+(	O
+b	O
+)	O
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+graph	O
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+the	O
+angles	O
+characterizing	O
+the	O
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+Structures	O
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+.	O
+Measurements	O
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+two	O
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+•	O
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+(	O
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+)	O
+structures	O
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+included	O
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+comparison	O
+.	O
+	O
+(	O
+d	O
+)	O
+Comparison	O
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+conformations	O
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+L1	O
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+stalks	O
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+Structures	O
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+80S	O
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+and	O
+tRNA	O
+-	O
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+.	O
+	O
+Superpositions	O
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+performed	O
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+alignments	O
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+25S	O
+ribosomal	O
+RNAs	O
+.	O
+	O
+(	O
+e	O
+)	O
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+positions	O
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+PKI	O
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+residues	O
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+head	O
+(	O
+U1191	O
+)	O
+and	O
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+(	O
+C1637	O
+)	O
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+.	O
+(	O
+f	O
+and	O
+g	O
+)	O
+Close	O
+-	O
+up	O
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+rearrangements	O
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+the	O
+A	O
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+P	O
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+(	O
+INIT	O
+:	O
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+ID	O
+3J6Y	O
+)	O
+to	O
+the	O
+post	O
+-	O
+translocation	O
+Structure	O
+V	O
+.	O
+The	O
+fragment	O
+shown	O
+within	O
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+rectangle	O
+in	O
+panel	O
+f	O
+is	O
+magnified	O
+in	O
+panel	O
+g	O
+.	O
+Nucleotides	O
+of	O
+the	O
+40S	O
+body	O
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+shown	O
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+orange	O
+,	O
+40S	O
+head	O
+in	O
+yellow	O
+.	O
+	O
+Our	O
+structures	O
+represent	O
+hitherto	O
+uncharacterized	O
+translocation	O
+complexes	O
+of	O
+the	O
+TSV	O
+IRES	O
+captured	O
+within	O
+globally	O
+distinct	O
+80S	O
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+(	O
+Figures	O
+1b	O
+and	O
+2	O
+).	O
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+I	O
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+,	O
+according	O
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+-	O
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+-	O
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+(	O
+Figure	O
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+—	O
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+1	O
+).	O
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+Specifically	O
+,	O
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+,	O
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+Structure	O
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+(	O
+Figure	O
+4	O
+;	O
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+3	O
+—	O
+figure	O
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+1	O
+).	O
+	O
+Thus	O
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+I	O
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+the	O
+A	O
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+P	O
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+(	O
+Figure	O
+2	O
+—	O
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+data	O
+1	O
+),	O
+suggesting	O
+that	O
+these	O
+structures	O
+describe	O
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+of	O
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+.	O
+	O
+Structure	O
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+corresponds	O
+to	O
+the	O
+post	O
+-	O
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+state	O
+.	O
+	O
+Changes	O
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+ribosome	O
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+eEF2	O
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+through	O
+the	O
+ribosome	O
+	O
+Using	O
+the	O
+post	O
+-	O
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+S	O
+.	O
+cerevisiae	O
+80S	O
+ribosome	O
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+with	O
+the	O
+P	O
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+E	O
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+reference	O
+(	O
+80S	O
+•	O
+2tRNA	O
+•	O
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+),	O
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+which	O
+both	O
+the	O
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+and	O
+the	O
+head	O
+-	O
+body	O
+swivel	O
+are	O
+0	O
+°,	O
+we	O
+found	O
+that	O
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+ribosome	O
+adopts	O
+four	O
+globally	O
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+conformations	O
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+Structures	O
+I	O
+through	O
+V	O
+(	O
+Figure	O
+1b	O
+;	O
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+also	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1	O
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+Figure	O
+2	O
+—	O
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+1	O
+).	O
+	O
+Structure	O
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+comprises	O
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+most	O
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+(~	O
+10	O
+°),	O
+characteristic	O
+of	O
+pre	O
+-	O
+translocation	O
+hybrid	O
+-	O
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+	O
+From	O
+Structure	O
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+,	O
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+small	O
+subunit	O
+undergoes	O
+backward	O
+(	O
+reverse	O
+)	O
+rotation	O
+(	O
+Figure	O
+2b	O
+;	O
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+also	O
+Figure	O
+1	O
+—	O
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+supplement	O
+2	O
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+2	O
+—	O
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+1	O
+).	O
+	O
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+-	O
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+0	O
+.	O
+5	O
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+-	O
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+-	O
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+	O
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+,	O
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+~	O
+9	O
+°	O
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+covers	O
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+nearly	O
+complete	O
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+relative	O
+subunit	O
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+,	O
+similar	O
+to	O
+what	O
+was	O
+reported	O
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+-	O
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+,	O
+bacterial	O
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+	O
+The	O
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+that	O
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+(	O
+Figures	O
+2c	O
+and	O
+d	O
+;	O
+see	O
+also	O
+Figure	O
+2	O
+—	O
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+data	O
+1	O
+).	O
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+,	O
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+(~	O
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+-	O
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+to	O
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+-	O
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+.	O
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+-	O
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+(	O
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+toward	O
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+and	O
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+backs	O
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+(	O
+Figure	O
+2	O
+—	O
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+supplement	O
+1	O
+).	O
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+The	O
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+-	O
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+(	O
+12	O
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+-	O
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+(	O
+14	O
+°).	O
+	O
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+observed	O
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+-	O
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+,	O
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+which	O
+PKI	O
+transitions	O
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+the	O
+A	O
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+,	O
+while	O
+eEF2	O
+occupies	O
+the	O
+A	O
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+.	O
+	O
+By	O
+comparison	O
+,	O
+the	O
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+-	O
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+(	O
+4	O
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+,	O
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+absence	O
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+,	O
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+-	O
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+(~	O
+10	O
+°)	O
+(	O
+Figure	O
+2c	O
+).	O
+	O
+These	O
+observations	O
+suggest	O
+that	O
+eEF2	O
+is	O
+necessary	O
+for	O
+inducing	O
+or	O
+stabilizing	O
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+.	O
+	O
+(	O
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+)	O
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+,	O
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+-	O
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+)	O
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+-	O
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+(	O
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+)	O
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+,	O
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+(	O
+not	O
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+)	O
+(	O
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+)	O
+Positions	O
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+(	O
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+)	O
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+(	O
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+,	O
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+(	O
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+Superpositions	O
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+(	O
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+–	O
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+proteins	O
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+,	O
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+(	O
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+)	O
+Intra	O
+-	O
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+For	O
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+structure	O
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+light	O
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+.	O
+	O
+Superpositions	O
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+18S	O
+rRNA	O
+.	O
+	O
+(	O
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+)	O
+Positions	O
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+those	O
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+-	O
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+-	O
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+.	O
+(	O
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+)	O
+Positions	O
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+proteins	O
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+(	O
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+)	O
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+(	O
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+5	O
+′	O
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+(	O
+left	O
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+Positions	O
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+L1stalk	O
+,	O
+tRNA	O
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+IRES	O
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+to	O
+proteins	O
+uS7	O
+and	O
+eS25	O
+,	O
+in	O
+80S	O
+•	O
+tRNA	O
+structures	O
+and	O
+80S	O
+•	O
+IRES	O
+structures	O
+I	O
+and	O
+V	O
+(	O
+this	O
+work	O
+).	O
+	O
+The	O
+view	O
+shows	O
+the	O
+vicinity	O
+of	O
+the	O
+ribosomal	O
+E	O
+site	O
+.	O
+	O
+Interactions	O
+of	O
+the	O
+stem	O
+loops	O
+4	O
+and	O
+5	O
+of	O
+the	O
+TSV	O
+with	O
+proteins	O
+uS7	O
+and	O
+eS25	O
+.	O
+	O
+2668	O
+–	O
+2687	O
+)	O
+and	O
+protein	O
+uL5	O
+(	O
+collectively	O
+labeled	O
+as	O
+central	O
+protuberance	O
+,	O
+CP	O
+,	O
+in	O
+the	O
+upper	O
+-	O
+row	O
+first	O
+figure	O
+,	O
+and	O
+individually	O
+labeled	O
+in	O
+the	O
+lower	O
+-	O
+row	O
+first	O
+figure	O
+).	O
+	O
+Structures	O
+of	O
+80S	O
+•	O
+IRES	O
+complexes	O
+in	O
+the	O
+absence	O
+of	O
+eEF2	O
+(	O
+INIT	O
+;	O
+PDB	O
+3J6Y	O
+,)	O
+and	O
+in	O
+the	O
+presence	O
+of	O
+eEF2	O
+(	O
+this	O
+work	O
+)	O
+are	O
+shown	O
+in	O
+the	O
+upper	O
+row	O
+and	O
+labeled	O
+.	O
+	O
+Structures	O
+of	O
+the	O
+80S	O
+complexes	O
+with	O
+tRNAs	O
+are	O
+shown	O
+in	O
+the	O
+lower	O
+row	O
+in	O
+a	O
+view	O
+similar	O
+to	O
+that	O
+for	O
+the	O
+80S	O
+•	O
+IRES	O
+complex	O
+.	O
+	O
+(	O
+a	O
+)	O
+Secondary	O
+structure	O
+of	O
+the	O
+TSV	O
+IRES	O
+.	O
+	O
+The	O
+TSV	O
+IRES	O
+comprises	O
+two	O
+domains	O
+:	O
+the	O
+5	O
+'	O
+domain	O
+(	O
+blue	O
+)	O
+and	O
+the	O
+PKI	O
+domain	O
+(	O
+red	O
+).	O
+	O
+The	O
+open	O
+reading	O
+frame	O
+(	O
+gray	O
+)	O
+is	O
+immediately	O
+following	O
+pseudoknot	O
+I	O
+(	O
+PKI	O
+).	O
+	O
+(	O
+b	O
+)	O
+Three	O
+-	O
+dimensional	O
+structure	O
+of	O
+the	O
+TSV	O
+IRES	O
+(	O
+Structure	O
+II	O
+).	O
+	O
+(	O
+c	O
+)	O
+Positions	O
+of	O
+the	O
+IRES	O
+and	O
+eEF2	O
+on	O
+the	O
+small	O
+subunit	O
+in	O
+Structures	O
+I	O
+to	O
+V	O
+.	O
+The	O
+initiation	O
+-	O
+state	O
+IRES	O
+is	O
+shown	O
+in	O
+gray	O
+.	O
+	O
+The	O
+insert	O
+shows	O
+density	O
+for	O
+interaction	O
+of	O
+diphthamide	O
+699	O
+(	O
+eEF2	O
+;	O
+green	O
+)	O
+with	O
+the	O
+codon	O
+-	O
+anticodon	O
+-	O
+like	O
+helix	O
+(	O
+PKI	O
+;	O
+red	O
+)	O
+in	O
+Structure	O
+V	O
+.	O
+(	O
+d	O
+and	O
+e	O
+)	O
+Density	O
+of	O
+the	O
+P	O
+site	O
+in	O
+Structure	O
+V	O
+shows	O
+that	O
+interactions	O
+of	O
+PKI	O
+with	O
+the	O
+18S	O
+rRNA	O
+nucleotides	O
+(	O
+c	O
+)	O
+are	O
+nearly	O
+identical	O
+to	O
+those	O
+in	O
+the	O
+P	O
+site	O
+of	O
+the	O
+2tRNA	O
+•	O
+mRNA	O
+-	O
+bound	O
+70S	O
+ribosome	O
+(	O
+d	O
+).	O
+	O
+In	O
+each	O
+structure	O
+,	O
+the	O
+TSV	O
+IRES	O
+adopts	O
+a	O
+distinct	O
+conformation	O
+in	O
+the	O
+intersubunit	O
+space	O
+of	O
+the	O
+ribosome	O
+(	O
+Figures	O
+3	O
+and	O
+4	O
+).	O
+	O
+The	O
+IRES	O
+(	O
+nt	O
+6758	O
+–	O
+6952	O
+)	O
+consists	O
+of	O
+two	O
+globular	O
+parts	O
+(	O
+Figure	O
+3a	O
+):	O
+the	O
+5	O
+’-	O
+region	O
+(	O
+domains	O
+I	O
+and	O
+II	O
+,	O
+nt	O
+6758	O
+–	O
+6888	O
+)	O
+and	O
+the	O
+PKI	O
+domain	O
+(	O
+domain	O
+III	O
+,	O
+nt	O
+6889	O
+–	O
+6952	O
+).	O
+	O
+We	O
+collectively	O
+term	O
+domains	O
+I	O
+and	O
+II	O
+the	O
+5	O
+’	O
+domain	O
+.	O
+	O
+The	O
+PKI	O
+domain	O
+comprises	O
+PKI	O
+and	O
+stem	O
+loop	O
+3	O
+(	O
+SL3	O
+),	O
+which	O
+stacks	O
+on	O
+top	O
+of	O
+the	O
+stem	O
+of	O
+PKI	O
+.	O
+	O
+The	O
+6953GCU	O
+triplet	O
+immediately	O
+following	O
+the	O
+PKI	O
+domain	O
+is	O
+the	O
+first	O
+codon	O
+of	O
+the	O
+open	O
+reading	O
+frame	O
+.	O
+	O
+In	O
+the	O
+eEF2	O
+-	O
+free	O
+80S	O
+•	O
+IRES	O
+initiation	O
+complex	O
+(	O
+INIT	O
+),	O
+the	O
+bulk	O
+of	O
+the	O
+5	O
+’-	O
+domain	O
+(	O
+nt	O
+.	O
+	O
+6758	O
+–	O
+6888	O
+)	O
+binds	O
+near	O
+the	O
+E	O
+site	O
+,	O
+contacting	O
+the	O
+ribosome	O
+mostly	O
+by	O
+means	O
+of	O
+three	O
+protruding	O
+structural	O
+elements	O
+:	O
+the	O
+L1	O
+.	O
+1	O
+region	O
+and	O
+stem	O
+loops	O
+4	O
+and	O
+5	O
+(	O
+SL4	O
+and	O
+SL5	O
+).	O
+	O
+In	O
+Structures	O
+I	O
+to	O
+IV	O
+,	O
+these	O
+contacts	O
+remain	O
+as	O
+in	O
+the	O
+initiation	O
+complex	O
+(	O
+Figure	O
+1a	O
+).	O
+	O
+Specifically	O
+,	O
+the	O
+L1	O
+.	O
+1	O
+region	O
+interacts	O
+with	O
+the	O
+L1	O
+stalk	O
+of	O
+the	O
+large	O
+subunit	O
+,	O
+while	O
+SL4	O
+and	O
+SL5	O
+bind	O
+at	O
+the	O
+side	O
+of	O
+the	O
+40S	O
+head	O
+and	O
+interact	O
+with	O
+proteins	O
+uS7	O
+,	O
+uS11	O
+and	O
+eS25	O
+(	O
+Figure	O
+3	O
+��	O
+figure	O
+supplement	O
+2	O
+and	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+3	O
+;	O
+ribosomal	O
+proteins	O
+are	O
+termed	O
+according	O
+to	O
+).	O
+	O
+In	O
+Structures	O
+I	O
+-	O
+IV	O
+,	O
+the	O
+minor	O
+groove	O
+of	O
+SL4	O
+(	O
+at	O
+nt	O
+6840	O
+–	O
+6846	O
+)	O
+binds	O
+next	O
+to	O
+an	O
+α	O
+-	O
+helix	O
+of	O
+uS7	O
+,	O
+which	O
+is	O
+rich	O
+in	O
+positively	O
+charged	O
+residues	O
+(	O
+K212	O
+,	O
+K213	O
+,	O
+R219	O
+and	O
+K222	O
+).	O
+	O
+The	O
+tip	O
+of	O
+SL4	O
+binds	O
+in	O
+the	O
+vicinity	O
+of	O
+R157	O
+in	O
+the	O
+β	O
+-	O
+hairpin	O
+of	O
+uS7	O
+and	O
+of	O
+Y58	O
+in	O
+uS11	O
+.	O
+	O
+In	O
+Structure	O
+V	O
+,	O
+however	O
+,	O
+the	O
+density	O
+for	O
+SL5	O
+is	O
+missing	O
+suggesting	O
+that	O
+SL5	O
+is	O
+mobile	O
+,	O
+while	O
+weak	O
+SL4	O
+density	O
+suggests	O
+that	O
+SL4	O
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+shifted	O
+along	O
+the	O
+surface	O
+of	O
+uS7	O
+,	O
+~	O
+20	O
+Å	O
+away	O
+from	O
+its	O
+initial	O
+position	O
+(	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+2c	O
+).	O
+	O
+Inchworm	O
+-	O
+like	O
+translocation	O
+of	O
+the	O
+TSV	O
+IRES	O
+.	O
+	O
+Conformations	O
+and	O
+positions	O
+of	O
+the	O
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+initiation	O
+state	O
+and	O
+in	O
+Structures	O
+I	O
+-	O
+V	O
+are	O
+shown	O
+relative	O
+to	O
+those	O
+of	O
+the	O
+A	O
+-,	O
+P	O
+-	O
+and	O
+E	O
+-	O
+site	O
+tRNAs	O
+.	O
+	O
+Distances	O
+between	O
+nucleotides	O
+6848	O
+and	O
+6913	O
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+SL4	O
+and	O
+PKI	O
+,	O
+respectively	O
+,	O
+are	O
+shown	O
+(	O
+see	O
+also	O
+Figure	O
+2	O
+—	O
+source	O
+data	O
+1	O
+).	O
+	O
+The	O
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+of	O
+the	O
+IRES	O
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+considerably	O
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+the	O
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+Structures	O
+I	O
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+,	O
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+an	O
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+to	O
+compact	O
+to	O
+extended	O
+conformation	O
+(	O
+Figure	O
+4	O
+;	O
+see	O
+also	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+2a	O
+).	O
+	O
+Because	O
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+Structures	O
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+toward	O
+the	O
+P	O
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+,	O
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+the	O
+5	O
+’	O
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+unchanged	O
+near	O
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+,	O
+the	O
+distance	O
+between	O
+the	O
+domains	O
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+(	O
+Figure	O
+4	O
+).	O
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+In	O
+the	O
+80S	O
+•	O
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+initiation	O
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+,	O
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+A	O
+-	O
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+-	O
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+separated	O
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+SL4	O
+by	O
+almost	O
+50	O
+Å	O
+(	O
+Figure	O
+4	O
+).	O
+	O
+In	O
+Structures	O
+I	O
+and	O
+II	O
+,	O
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+retracted	O
+from	O
+the	O
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+Structures	O
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+,	O
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+domain	O
+approaches	O
+to	O
+within	O
+~	O
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+Å	O
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+SL4	O
+.	O
+	O
+Because	O
+the	O
+5	O
+’-	O
+domain	O
+in	O
+the	O
+following	O
+structure	O
+(	O
+V	O
+)	O
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+~	O
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+the	O
+40S	O
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+,	O
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+returns	O
+to	O
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+(~	O
+45	O
+Å	O
+)	O
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+is	O
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+that	O
+in	O
+the	O
+80S	O
+•	O
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+complex	O
+.	O
+	O
+2668	O
+–	O
+2687	O
+)	O
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+(	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+6	O
+).	O
+	O
+This	O
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+SL3	O
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+•	O
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+,	O
+in	O
+which	O
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+closely	O
+mimic	O
+the	O
+ASL	O
+and	O
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+of	O
+the	O
+A	O
+-	O
+site	O
+tRNA	O
+,	O
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+.	O
+	O
+In	O
+the	O
+highly	O
+bent	O
+Structures	O
+III	O
+and	O
+IV	O
+,	O
+the	O
+hinge	O
+region	O
+interacts	O
+with	O
+the	O
+universally	O
+conserved	O
+uL5	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+tail	O
+of	O
+eL42	O
+(	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+7	O
+).	O
+	O
+However	O
+,	O
+in	O
+the	O
+extended	O
+conformations	O
+,	O
+these	O
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+subunit	O
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+by	O
+more	O
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+10	O
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+,	O
+suggesting	O
+that	O
+an	O
+interaction	O
+between	O
+them	O
+stabilizes	O
+the	O
+bent	O
+conformations	O
+but	O
+not	O
+the	O
+extended	O
+ones	O
+.	O
+	O
+This	O
+loop	O
+is	O
+poorly	O
+resolved	O
+in	O
+Structures	O
+I	O
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+IV	O
+,	O
+suggesting	O
+conformational	O
+flexibility	O
+in	O
+agreement	O
+with	O
+structural	O
+studies	O
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+isolated	O
+PKI	O
+and	O
+biochemical	O
+studies	O
+of	O
+unbound	O
+IRESs	O
+.	O
+	O
+In	O
+Structure	O
+V	O
+,	O
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+3	O
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+bound	O
+in	O
+the	O
+40S	O
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+and	O
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+backbone	O
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+3	O
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+codon	O
+-	O
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+of	O
+PKI	O
+(	O
+at	O
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+.	O
+	O
+6945	O
+–	O
+6946	O
+)	O
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+with	O
+R148	O
+and	O
+R157	O
+in	O
+β	O
+-	O
+hairpin	O
+of	O
+uS7	O
+.	O
+	O
+This	O
+interpretation	O
+is	O
+consistent	O
+with	O
+the	O
+recent	O
+observation	O
+that	O
+alterations	O
+in	O
+loop	O
+3	O
+of	O
+the	O
+CrPV	O
+IRES	O
+result	O
+in	O
+decreased	O
+efficiency	O
+of	O
+translocation	O
+.	O
+	O
+eEF2	O
+structures	O
+	O
+Elements	O
+of	O
+the	O
+80S	O
+ribosome	O
+that	O
+contact	O
+eEF2	O
+in	O
+Structures	O
+I	O
+through	O
+V	O
+.	O
+	O
+The	O
+switch	O
+loop	O
+I	O
+in	O
+Structure	O
+I	O
+is	O
+shown	O
+in	O
+blue	O
+.	O
+	O
+The	O
+putative	O
+position	O
+of	O
+the	O
+switch	O
+loop	O
+I	O
+,	O
+unresolved	O
+in	O
+the	O
+density	O
+of	O
+Structure	O
+II	O
+,	O
+is	O
+shown	O
+with	O
+a	O
+dashed	O
+line	O
+.	O
+	O
+Colors	O
+for	O
+the	O
+ribosome	O
+and	O
+eEF2	O
+are	O
+as	O
+in	O
+Figure	O
+1	O
+.	O
+	O
+Conformations	O
+and	O
+interactions	O
+of	O
+eEF2	O
+.	O
+	O
+(	O
+a	O
+)	O
+Conformations	O
+of	O
+eEF2	O
+in	O
+Structures	O
+I	O
+-	O
+V	O
+and	O
+domain	O
+organization	O
+of	O
+eEF2	O
+are	O
+shown	O
+.	O
+	O
+Roman	O
+numerals	O
+denote	O
+eEF2	O
+domains	O
+.	O
+	O
+Superposition	O
+was	O
+obtained	O
+by	O
+structural	O
+alignment	O
+of	O
+domains	O
+I	O
+and	O
+II	O
+.	O
+	O
+(	O
+b	O
+)	O
+Elements	O
+of	O
+the	O
+80S	O
+ribosome	O
+in	O
+Structures	O
+I	O
+and	O
+V	O
+that	O
+contact	O
+eEF2	O
+.	O
+	O
+eEF2	O
+is	O
+shown	O
+in	O
+green	O
+,	O
+IRES	O
+RNA	O
+in	O
+red	O
+,	O
+40S	O
+subunit	O
+elements	O
+in	O
+orange	O
+,	O
+60S	O
+in	O
+cyan	O
+/	O
+teal	O
+.	O
+	O
+(	O
+d	O
+)	O
+Interactions	O
+of	O
+the	O
+GTPase	O
+domains	O
+with	O
+the	O
+40S	O
+and	O
+60S	O
+subunits	O
+in	O
+Structure	O
+I	O
+(	O
+colored	O
+in	O
+green	O
+/	O
+blue	O
+,	O
+eEF2	O
+;	O
+orange	O
+,	O
+40S	O
+;	O
+cyan	O
+/	O
+teal	O
+,	O
+60S	O
+)	O
+and	O
+in	O
+Structure	O
+II	O
+(	O
+gray	O
+).	O
+	O
+Switch	O
+loop	O
+I	O
+(	O
+SWI	O
+)	O
+in	O
+Structure	O
+I	O
+is	O
+in	O
+blue	O
+;	O
+dashed	O
+line	O
+shows	O
+the	O
+putative	O
+location	O
+of	O
+unresolved	O
+switch	O
+loop	O
+I	O
+in	O
+Structure	O
+II	O
+.	O
+	O
+(	O
+h	O
+)	O
+Cryo	O
+-	O
+EM	O
+density	O
+showing	O
+the	O
+sordarin	O
+-	O
+binding	O
+pocket	O
+of	O
+eEF2	O
+(	O
+Structure	O
+II	O
+).	O
+	O
+Sordarin	O
+is	O
+shown	O
+in	O
+pink	O
+with	O
+oxygen	O
+atoms	O
+in	O
+red	O
+.	O
+	O
+Elongation	O
+factor	O
+eEF2	O
+in	O
+all	O
+five	O
+structures	O
+is	O
+bound	O
+with	O
+GDP	O
+and	O
+sordarin	O
+(	O
+Figure	O
+5	O
+).	O
+	O
+The	O
+elongation	O
+factor	O
+consists	O
+of	O
+three	O
+dynamic	O
+superdomains	O
+:	O
+an	O
+N	O
+-	O
+terminal	O
+globular	O
+superdomain	O
+formed	O
+by	O
+the	O
+G	O
+(	O
+GTPase	O
+)	O
+domain	O
+(	O
+domain	O
+I	O
+)	O
+and	O
+domain	O
+II	O
+;	O
+a	O
+linker	O
+domain	O
+III	O
+;	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+superdomain	O
+comprising	O
+domains	O
+IV	O
+and	O
+V	O
+(	O
+Figure	O
+5a	O
+).	O
+	O
+Domain	O
+IV	O
+extends	O
+from	O
+the	O
+main	O
+body	O
+and	O
+is	O
+critical	O
+for	O
+translocation	O
+catalyzed	O
+by	O
+eEF2	O
+or	O
+EF	O
+-	O
+G	O
+.	O
+ADP	O
+-	O
+ribosylation	O
+of	O
+eEF2	O
+at	O
+the	O
+tip	O
+of	O
+domain	O
+IV	O
+or	O
+deletion	O
+of	O
+domain	O
+IV	O
+from	O
+EF	O
+-	O
+G	O
+abrogate	O
+translocation	O
+.	O
+	O
+In	O
+post	O
+-	O
+translocation	O
+-	O
+like	O
+80S	O
+•	O
+tRNA	O
+•	O
+eEF2	O
+complexes	O
+,	O
+domain	O
+IV	O
+binds	O
+in	O
+the	O
+40S	O
+A	O
+site	O
+,	O
+suggesting	O
+direct	O
+involvement	O
+of	O
+domain	O
+IV	O
+in	O
+translocation	O
+of	O
+tRNA	O
+from	O
+the	O
+A	O
+to	O
+P	O
+site	O
+.	O
+	O
+GDP	O
+in	O
+our	O
+structures	O
+is	O
+bound	O
+in	O
+the	O
+GTPase	O
+center	O
+(	O
+Figures	O
+5d	O
+,	O
+e	O
+and	O
+f	O
+)	O
+and	O
+sordarin	O
+is	O
+sandwiched	O
+between	O
+the	O
+β	O
+-	O
+platforms	O
+of	O
+domains	O
+III	O
+and	O
+V	O
+(	O
+Figures	O
+5g	O
+and	O
+h	O
+),	O
+as	O
+in	O
+the	O
+structure	O
+of	O
+free	O
+eEF2	O
+•	O
+sordarin	O
+complex	O
+.	O
+	O
+The	O
+global	O
+conformations	O
+of	O
+eEF2	O
+(	O
+Figure	O
+5a	O
+)	O
+are	O
+similar	O
+in	O
+these	O
+structures	O
+(	O
+all	O
+-	O
+atom	O
+RMSD	O
+≤	O
+2	O
+Å	O
+),	O
+but	O
+the	O
+positions	O
+of	O
+eEF2	O
+relative	O
+to	O
+the	O
+40S	O
+subunit	O
+differ	O
+substantially	O
+as	O
+a	O
+result	O
+of	O
+40S	O
+subunit	O
+rotation	O
+(	O
+Figure	O
+2	O
+—	O
+source	O
+data	O
+1	O
+).	O
+	O
+From	O
+Structure	O
+I	O
+to	O
+V	O
+,	O
+eEF2	O
+is	O
+rigidly	O
+attached	O
+to	O
+the	O
+GTPase	O
+-	O
+associated	O
+center	O
+of	O
+the	O
+60S	O
+subunit	O
+.	O
+	O
+The	O
+GTPase	O
+-	O
+associated	O
+center	O
+comprises	O
+the	O
+P	O
+stalk	O
+(	O
+L11	O
+and	O
+L7	O
+/	O
+L12	O
+stalk	O
+in	O
+bacteria	O
+)	O
+and	O
+the	O
+sarcin	O
+-	O
+ricin	O
+loop	O
+(	O
+SRL	O
+,	O
+nt	O
+3012	O
+–	O
+3042	O
+).	O
+	O
+The	O
+tips	O
+of	O
+25S	O
+rRNA	O
+helices	O
+43	O
+and	O
+44	O
+of	O
+the	O
+P	O
+stalk	O
+(	O
+nucleotides	O
+G1242	O
+and	O
+A1270	O
+,	O
+respectively	O
+)	O
+stack	O
+on	O
+V754	O
+and	O
+Y744	O
+of	O
+domain	O
+V	O
+.	O
+An	O
+αββ	O
+motif	O
+of	O
+the	O
+eukaryote	O
+-	O
+specific	O
+protein	O
+P0	O
+(	O
+aa	O
+126	O
+–	O
+154	O
+)	O
+packs	O
+in	O
+the	O
+crevice	O
+between	O
+the	O
+long	O
+α	O
+-	O
+helix	O
+D	O
+(	O
+aa	O
+172	O
+–	O
+188	O
+)	O
+of	O
+the	O
+GTPase	O
+domain	O
+and	O
+the	O
+β	O
+-	O
+sheet	O
+region	O
+(	O
+aa	O
+246	O
+–	O
+263	O
+)	O
+of	O
+the	O
+GTPase	O
+domain	O
+insert	O
+(	O
+or	O
+G	O
+’	O
+insert	O
+)	O
+of	O
+eEF2	O
+(	O
+secondary	O
+-	O
+structure	O
+nomenclatures	O
+for	O
+eEF2	O
+and	O
+EF	O
+-	O
+G	O
+are	O
+the	O
+same	O
+).	O
+	O
+Although	O
+the	O
+P	O
+/	O
+L11	O
+stalk	O
+is	O
+known	O
+to	O
+be	O
+dynamic	O
+,	O
+its	O
+position	O
+remains	O
+unchanged	O
+from	O
+Structure	O
+I	O
+to	O
+V	O
+:	O
+all	O
+-	O
+atom	O
+root	O
+-	O
+mean	O
+-	O
+square	O
+differences	O
+for	O
+the	O
+25S	O
+rRNA	O
+of	O
+the	O
+P	O
+stalk	O
+(	O
+nt	O
+1223	O
+–	O
+1286	O
+)	O
+are	O
+within	O
+2	O
+.	O
+5	O
+Å	O
+.	O
+However	O
+,	O
+with	O
+respect	O
+to	O
+its	O
+position	O
+in	O
+the	O
+80S	O
+•	O
+IRES	O
+complex	O
+in	O
+the	O
+absence	O
+of	O
+eEF2	O
+and	O
+in	O
+the	O
+80S	O
+•	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+,	O
+the	O
+P	O
+stalk	O
+is	O
+shifted	O
+by	O
+~	O
+13	O
+Å	O
+toward	O
+the	O
+A	O
+site	O
+(	O
+Figure	O
+2d	O
+).	O
+	O
+While	O
+the	O
+overall	O
+mode	O
+of	O
+this	O
+interaction	O
+is	O
+similar	O
+to	O
+that	O
+seen	O
+in	O
+70S	O
+•	O
+EF	O
+-	O
+G	O
+crystal	O
+structures	O
+,	O
+there	O
+is	O
+an	O
+important	O
+local	O
+difference	O
+between	O
+Structure	O
+I	O
+and	O
+Structures	O
+II	O
+-	O
+V	O
+in	O
+switch	O
+loop	O
+I	O
+,	O
+as	O
+discussed	O
+below	O
+.	O
+	O
+Structures	O
+I	O
+through	O
+V	O
+are	O
+shown	O
+.	O
+	O
+Electrostatic	O
+surface	O
+of	O
+eEF2	O
+is	O
+shown	O
+;	O
+negatively	O
+and	O
+positively	O
+charged	O
+regions	O
+are	O
+shown	O
+in	O
+red	O
+and	O
+blue	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+view	O
+was	O
+obtained	O
+by	O
+structural	O
+alignment	O
+of	O
+the	O
+18S	O
+rRNAs	O
+.	O
+	O
+The	O
+view	O
+was	O
+obtained	O
+by	O
+superpositions	O
+of	O
+the	O
+body	O
+domains	O
+of	O
+18S	O
+rRNAs	O
+.	O
+	O
+(	O
+c	O
+)	O
+Rearrangements	O
+,	O
+from	O
+Structure	O
+I	O
+through	O
+V	O
+,	O
+of	O
+a	O
+positively	O
+charged	O
+cluster	O
+of	O
+eEF2	O
+(	O
+K613	O
+,	O
+R617	O
+and	O
+R631	O
+)	O
+positioned	O
+over	O
+the	O
+phosphate	O
+backbone	O
+of	O
+18S	O
+helices	O
+33	O
+and	O
+34	O
+,	O
+suggesting	O
+a	O
+role	O
+of	O
+electrostatic	O
+interactions	O
+in	O
+eEF2	O
+diffusion	O
+over	O
+the	O
+40S	O
+surface	O
+.	O
+	O
+(	O
+d	O
+)	O
+Shift	O
+of	O
+the	O
+tip	O
+of	O
+domain	O
+III	O
+of	O
+eEF2	O
+,	O
+interacting	O
+with	O
+uS12	O
+upon	O
+reverse	O
+subunit	O
+rotation	O
+from	O
+Structure	O
+I	O
+to	O
+Structure	O
+V	O
+.	O
+Structure	O
+I	O
+colored	O
+as	O
+in	O
+Figure	O
+1	O
+,	O
+except	O
+uS12	O
+,	O
+which	O
+is	O
+in	O
+purple	O
+;	O
+Structure	O
+V	O
+is	O
+in	O
+gray	O
+.	O
+	O
+There	O
+are	O
+two	O
+modest	O
+but	O
+noticeable	O
+domain	O
+rearrangements	O
+between	O
+Structures	O
+I	O
+and	O
+V	O
+.	O
+Unlike	O
+in	O
+free	O
+eEF2	O
+,	O
+which	O
+can	O
+sample	O
+large	O
+movements	O
+of	O
+at	O
+least	O
+50	O
+Å	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+superdomain	O
+relative	O
+to	O
+the	O
+N	O
+-	O
+terminal	O
+superdomain	O
+(	O
+Figure	O
+5c	O
+),	O
+eEF2	O
+undergoes	O
+moderate	O
+repositioning	O
+of	O
+domain	O
+IV	O
+(~	O
+3	O
+Å	O
+;	O
+Figure	O
+5a	O
+)	O
+and	O
+domain	O
+III	O
+(~	O
+5	O
+Å	O
+;	O
+Figure	O
+6d	O
+).	O
+	O
+This	O
+limited	O
+flexibility	O
+of	O
+the	O
+ribosome	O
+-	O
+bound	O
+eEF2	O
+is	O
+likely	O
+the	O
+result	O
+of	O
+simultaneous	O
+fixation	O
+of	O
+eEF2	O
+superdomains	O
+,	O
+via	O
+domains	O
+I	O
+and	O
+V	O
+,	O
+by	O
+the	O
+GTPase	O
+-	O
+associated	O
+center	O
+of	O
+the	O
+large	O
+subunit	O
+.	O
+	O
+Domain	O
+IV	O
+of	O
+eEF2	O
+binds	O
+at	O
+the	O
+40S	O
+A	O
+site	O
+in	O
+Structures	O
+I	O
+to	O
+V	O
+but	O
+the	O
+mode	O
+of	O
+interaction	O
+differs	O
+in	O
+each	O
+complex	O
+(	O
+Figure	O
+6	O
+).	O
+	O
+eEF2	O
+settles	O
+into	O
+the	O
+A	O
+site	O
+from	O
+Structure	O
+I	O
+to	O
+V	O
+,	O
+as	O
+the	O
+tip	O
+of	O
+domain	O
+IV	O
+shifts	O
+by	O
+~	O
+10	O
+Å	O
+relative	O
+to	O
+the	O
+body	O
+and	O
+by	O
+~	O
+20	O
+Å	O
+relative	O
+to	O
+the	O
+swiveling	O
+head	O
+.	O
+	O
+Modest	O
+intra	O
+-	O
+eEF2	O
+shifts	O
+of	O
+domain	O
+IV	O
+between	O
+Structures	O
+I	O
+to	O
+V	O
+outline	O
+a	O
+stochastic	O
+trajectory	O
+(	O
+Figure	O
+5a	O
+),	O
+consistent	O
+with	O
+local	O
+adjustments	O
+of	O
+the	O
+domain	O
+in	O
+the	O
+A	O
+site	O
+.	O
+	O
+At	O
+the	O
+central	O
+region	O
+of	O
+eEF2	O
+,	O
+domains	O
+II	O
+and	O
+III	O
+contact	O
+the	O
+40S	O
+body	O
+(	O
+mainly	O
+at	O
+nucleotides	O
+48	O
+–	O
+52	O
+and	O
+429	O
+–	O
+432	O
+of	O
+18S	O
+rRNA	O
+helix	O
+5	O
+and	O
+uS12	O
+).	O
+	O
+Comparison	O
+of	O
+eEF2	O
+conformations	O
+reveals	O
+that	O
+in	O
+Structure	O
+V	O
+,	O
+domain	O
+III	O
+is	O
+displaced	O
+as	O
+a	O
+result	O
+of	O
+interaction	O
+with	O
+uS12	O
+,	O
+as	O
+discussed	O
+below	O
+.	O
+	O
+In	O
+summary	O
+,	O
+between	O
+Structures	O
+I	O
+and	O
+V	O
+,	O
+a	O
+step	O
+-	O
+wise	O
+translocation	O
+of	O
+PKI	O
+by	O
+~	O
+15	O
+Å	O
+from	O
+the	O
+A	O
+to	O
+P	O
+site	O
+-	O
+within	O
+the	O
+40S	O
+subunit	O
+–	O
+occurs	O
+simultaneously	O
+with	O
+the	O
+~	O
+11	O
+Å	O
+side	O
+-	O
+way	O
+entry	O
+of	O
+domain	O
+IV	O
+into	O
+the	O
+A	O
+site	O
+coupled	O
+with	O
+~	O
+3	O
+to	O
+5	O
+Å	O
+inter	O
+-	O
+domain	O
+rearrangements	O
+in	O
+eEF2	O
+.	O
+	O
+These	O
+shifts	O
+occur	O
+during	O
+the	O
+reverse	O
+rotation	O
+of	O
+the	O
+40S	O
+body	O
+coupled	O
+with	O
+the	O
+forward	O
+-	O
+then	O
+-	O
+reverse	O
+head	O
+swivel	O
+.	O
+	O
+To	O
+elucidate	O
+the	O
+detailed	O
+structural	O
+mechanism	O
+of	O
+IRES	O
+translocation	O
+and	O
+the	O
+roles	O
+of	O
+eEF2	O
+and	O
+ribosome	O
+rearrangements	O
+,	O
+we	O
+describe	O
+in	O
+the	O
+following	O
+sections	O
+the	O
+interactions	O
+of	O
+PKI	O
+and	O
+eEF2	O
+with	O
+the	O
+ribosomal	O
+A	O
+and	O
+P	O
+sites	O
+in	O
+Structures	O
+I	O
+through	O
+V	O
+(	O
+Figure	O
+2g	O
+;	O
+see	O
+also	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+	O
+Structure	O
+I	O
+represents	O
+a	O
+pre	O
+-	O
+translocation	O
+IRES	O
+and	O
+initial	O
+entry	O
+of	O
+eEF2	O
+in	O
+a	O
+GTP	O
+-	O
+like	O
+state	O
+	O
+In	O
+the	O
+fully	O
+rotated	O
+Structure	O
+I	O
+,	O
+PKI	O
+is	O
+shifted	O
+toward	O
+the	O
+P	O
+site	O
+by	O
+~	O
+3	O
+Å	O
+relative	O
+to	O
+its	O
+position	O
+in	O
+the	O
+initiation	O
+complex	O
+but	O
+maintains	O
+interactions	O
+with	O
+the	O
+partially	O
+swiveled	O
+head	O
+.	O
+	O
+The	O
+C1274	O
+:	O
+G6953	O
+base	O
+pair	O
+provides	O
+a	O
+stacking	O
+platform	O
+for	O
+the	O
+codon	O
+-	O
+anticodon	O
+–	O
+like	O
+helix	O
+of	O
+PKI	O
+.	O
+	O
+We	O
+therefore	O
+define	O
+C1274	O
+as	O
+the	O
+foundation	O
+of	O
+the	O
+'	O
+head	O
+A	O
+site	O
+'.	O
+	O
+Accordingly	O
+,	O
+we	O
+use	O
+U1191	O
+(	O
+G966	O
+in	O
+E	O
+.	O
+coli	O
+)	O
+and	O
+C1637	O
+(	O
+C1400	O
+in	O
+E	O
+.	O
+coli	O
+)	O
+as	O
+the	O
+reference	O
+points	O
+of	O
+the	O
+'	O
+head	O
+P	O
+site	O
+'	O
+and	O
+'	O
+body	O
+P	O
+site	O
+'	O
+(	O
+Figure	O
+2g	O
+),	O
+respectively	O
+,	O
+because	O
+these	O
+nucleotides	O
+form	O
+a	O
+stacking	O
+foundation	O
+for	O
+the	O
+fully	O
+translocated	O
+mRNA	O
+-	O
+tRNA	O
+helix	O
+in	O
+tRNA	O
+-	O
+bound	O
+structures	O
+and	O
+in	O
+our	O
+post	O
+-	O
+translocation	O
+Structure	O
+V	O
+discussed	O
+below	O
+.	O
+	O
+Interactions	O
+of	O
+the	O
+residues	O
+at	O
+the	O
+eEF2	O
+tip	O
+with	O
+the	O
+decoding	O
+center	O
+of	O
+the	O
+IRES	O
+-	O
+bound	O
+ribosome	O
+.	O
+	O
+Key	O
+elements	O
+of	O
+the	O
+decoding	O
+center	O
+of	O
+the	O
+'	O
+locked	O
+'	O
+initiation	O
+structure	O
+,	O
+'	O
+unlocked	O
+'	O
+Structure	O
+I	O
+,	O
+and	O
+post	O
+-	O
+translocation	O
+Structure	O
+V	O
+(	O
+this	O
+work	O
+)	O
+are	O
+shown	O
+.	O
+	O
+The	O
+histidine	O
+-	O
+diphthamide	O
+tip	O
+of	O
+eEF2	O
+is	O
+shown	O
+in	O
+green	O
+.	O
+	O
+Nucleotides	O
+of	O
+the	O
+18S	O
+rRNA	O
+body	O
+are	O
+in	O
+orange	O
+and	O
+head	O
+in	O
+yellow	O
+;	O
+25S	O
+rRNA	O
+nucleotide	O
+A2256	O
+is	O
+blue	O
+.	O
+	O
+A	O
+and	O
+P	O
+sites	O
+are	O
+schematically	O
+demarcated	O
+by	O
+dotted	O
+lines	O
+.	O
+	O
+The	O
+interaction	O
+of	O
+PKI	O
+with	O
+the	O
+40S	O
+body	O
+is	O
+substantially	O
+rearranged	O
+relative	O
+to	O
+that	O
+in	O
+the	O
+initiation	O
+state	O
+.	O
+	O
+In	O
+Structure	O
+I	O
+,	O
+PKI	O
+does	O
+not	O
+contact	O
+these	O
+nucleotides	O
+(	O
+Figures	O
+2g	O
+and	O
+7	O
+).	O
+	O
+The	O
+position	O
+of	O
+eEF2	O
+on	O
+the	O
+40S	O
+subunit	O
+of	O
+Structure	O
+I	O
+is	O
+markedly	O
+distinct	O
+from	O
+those	O
+in	O
+Structures	O
+II	O
+to	O
+V	O
+.	O
+The	O
+translocase	O
+interacts	O
+with	O
+the	O
+40S	O
+body	O
+but	O
+does	O
+not	O
+contact	O
+the	O
+head	O
+(	O
+Figures	O
+5b	O
+and	O
+6a	O
+;	O
+Figure	O
+5	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+	O
+The	O
+tip	O
+of	O
+domain	O
+IV	O
+is	O
+wedged	O
+between	O
+PKI	O
+and	O
+decoding	O
+-	O
+center	O
+nucleotides	O
+A1755	O
+and	O
+A1756	O
+,	O
+which	O
+are	O
+bulged	O
+out	O
+of	O
+h44	O
+.	O
+	O
+This	O
+tip	O
+contains	O
+the	O
+histidine	O
+-	O
+diphthamide	O
+triad	O
+(	O
+H583	O
+,	O
+H694	O
+and	O
+Diph699	O
+),	O
+which	O
+interacts	O
+with	O
+the	O
+codon	O
+-	O
+anticodon	O
+-	O
+like	O
+helix	O
+of	O
+PKI	O
+and	O
+A1756	O
+(	O
+Figure	O
+7	O
+).	O
+	O
+Diphthamide	O
+is	O
+a	O
+unique	O
+posttranslational	O
+modification	O
+conserved	O
+in	O
+archaeal	O
+and	O
+eukaryotic	O
+EF2	O
+(	O
+at	O
+residue	O
+699	O
+in	O
+S	O
+.	O
+cerevisiae	O
+)	O
+and	O
+involves	O
+addition	O
+of	O
+a	O
+~	O
+7	O
+-	O
+Å	O
+long	O
+3	O
+-	O
+carboxyamido	O
+-	O
+3	O
+-(	O
+trimethylamino	O
+)-	O
+propyl	O
+moiety	O
+to	O
+the	O
+histidine	O
+imidazole	O
+ring	O
+at	O
+CE1	O
+.	O
+	O
+The	O
+opposite	O
+surface	O
+of	O
+the	O
+tail	O
+is	O
+oriented	O
+toward	O
+the	O
+minor	O
+-	O
+groove	O
+side	O
+of	O
+the	O
+second	O
+base	O
+pair	O
+of	O
+the	O
+codon	O
+-	O
+anticodon	O
+helix	O
+(	O
+G6906	O
+:	O
+C6951	O
+).	O
+	O
+The	O
+splitting	O
+of	O
+the	O
+interaction	O
+of	O
+A1755	O
+-	O
+A1756	O
+and	O
+PKI	O
+is	O
+achieved	O
+by	O
+providing	O
+the	O
+histidine	O
+-	O
+diphthamine	O
+tip	O
+as	O
+a	O
+binding	O
+partner	O
+for	O
+both	O
+A1756	O
+and	O
+the	O
+minor	O
+groove	O
+of	O
+the	O
+codon	O
+-	O
+anticodon	O
+helix	O
+(	O
+Figure	O
+7	O
+).	O
+	O
+Unlike	O
+in	O
+Structures	O
+II	O
+to	O
+V	O
+,	O
+the	O
+conformation	O
+of	O
+the	O
+eEF2	O
+GTPase	O
+center	O
+in	O
+Structure	O
+I	O
+resembles	O
+that	O
+of	O
+a	O
+GTP	O
+-	O
+bound	O
+translocase	O
+(	O
+Figure	O
+5e	O
+).	O
+	O
+Switch	O
+loop	O
+II	O
+(	O
+aa	O
+105	O
+–	O
+110	O
+),	O
+which	O
+carries	O
+the	O
+catalytic	O
+H108	O
+(	O
+H92	O
+in	O
+E	O
+.	O
+coli	O
+EF	O
+-	O
+G	O
+;	O
+is	O
+well	O
+resolved	O
+in	O
+all	O
+five	O
+structures	O
+.	O
+	O
+The	O
+N	O
+-	O
+terminal	O
+part	O
+of	O
+the	O
+loop	O
+(	O
+aa	O
+50	O
+–	O
+60	O
+)	O
+is	O
+sandwiched	O
+between	O
+the	O
+tip	O
+of	O
+helix	O
+14	O
+(	O
+415CAAA418	O
+)	O
+of	O
+the	O
+18S	O
+rRNA	O
+of	O
+the	O
+40S	O
+subunit	O
+and	O
+helix	O
+A	O
+(	O
+aa	O
+32	O
+–	O
+42	O
+)	O
+of	O
+eEF2	O
+(	O
+Figure	O
+5d	O
+).	O
+	O
+Bulged	O
+A416	O
+interacts	O
+with	O
+the	O
+switch	O
+loop	O
+in	O
+the	O
+vicinity	O
+of	O
+D53	O
+.	O
+	O
+In	O
+Structure	O
+II	O
+,	O
+relative	O
+to	O
+Structure	O
+I	O
+,	O
+PKI	O
+is	O
+further	O
+shifted	O
+along	O
+the	O
+40S	O
+body	O
+,	O
+traversing	O
+~	O
+4	O
+Å	O
+toward	O
+the	O
+P	O
+site	O
+(	O
+Figures	O
+2e	O
+,	O
+f	O
+,	O
+and	O
+g	O
+),	O
+while	O
+stacking	O
+on	O
+C1274	O
+at	O
+the	O
+head	O
+A	O
+site	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+intermediate	O
+position	O
+of	O
+PKI	O
+is	O
+possible	O
+due	O
+to	O
+a	O
+large	O
+swivel	O
+of	O
+the	O
+head	O
+relative	O
+to	O
+the	O
+body	O
+,	O
+which	O
+brings	O
+the	O
+head	O
+A	O
+site	O
+close	O
+to	O
+the	O
+body	O
+P	O
+site	O
+.	O
+	O
+The	O
+head	O
+interface	O
+of	O
+domain	O
+IV	O
+interacts	O
+with	O
+the	O
+40S	O
+head	O
+(	O
+Figure	O
+6a	O
+).	O
+	O
+Here	O
+,	O
+a	O
+positively	O
+charged	O
+surface	O
+of	O
+eEF2	O
+,	O
+formed	O
+by	O
+K613	O
+,	O
+R617	O
+and	O
+R631	O
+contacts	O
+the	O
+phosphate	O
+backbone	O
+of	O
+helix	O
+33	O
+(	O
+Figures	O
+6c	O
+;	O
+see	O
+also	O
+Figure	O
+6	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+	O
+Consistent	O
+with	O
+the	O
+similar	O
+head	O
+swivels	O
+in	O
+Structure	O
+III	O
+and	O
+Structure	O
+II	O
+,	O
+relative	O
+positions	O
+of	O
+the	O
+40S	O
+head	O
+A	O
+site	O
+and	O
+body	O
+P	O
+site	O
+remain	O
+as	O
+in	O
+Structure	O
+II	O
+.	O
+	O
+The	O
+map	O
+allows	O
+placement	O
+of	O
+PKI	O
+at	O
+the	O
+body	O
+P	O
+site	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+).	O
+	O
+Thus	O
+,	O
+in	O
+Structure	O
+III	O
+,	O
+PKI	O
+has	O
+translocated	O
+along	O
+the	O
+40S	O
+body	O
+,	O
+but	O
+the	O
+head	O
+remains	O
+fully	O
+swiveled	O
+so	O
+that	O
+PKI	O
+is	O
+between	O
+the	O
+head	O
+A	O
+and	O
+P	O
+sites	O
+.	O
+	O
+Lower	O
+resolution	O
+of	O
+the	O
+map	O
+in	O
+this	O
+region	O
+suggests	O
+that	O
+PKI	O
+is	O
+somewhat	O
+destabilized	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+body	O
+P	O
+site	O
+in	O
+the	O
+absence	O
+of	O
+stacking	O
+with	O
+the	O
+foundations	O
+of	O
+the	O
+head	O
+A	O
+site	O
+(	O
+C1274	O
+)	O
+or	O
+P	O
+site	O
+(	O
+U1191	O
+).	O
+	O
+The	O
+position	O
+of	O
+eEF2	O
+is	O
+similar	O
+to	O
+that	O
+in	O
+Structure	O
+II	O
+.	O
+	O
+Structure	O
+IV	O
+represents	O
+a	O
+highly	O
+bent	O
+IRES	O
+with	O
+PKI	O
+partially	O
+accommodated	O
+in	O
+the	O
+P	O
+site	O
+	O
+Unwinding	O
+of	O
+the	O
+head	O
+moves	O
+the	O
+head	O
+P	O
+-	O
+site	O
+residue	O
+U1191	O
+and	O
+body	O
+P	O
+-	O
+site	O
+residue	O
+C1637	O
+closer	O
+together	O
+,	O
+resulting	O
+in	O
+a	O
+partially	O
+restored	O
+40S	O
+P	O
+site	O
+.	O
+	O
+Whereas	O
+C1637	O
+forms	O
+a	O
+stacking	O
+platform	O
+for	O
+the	O
+last	O
+base	O
+pair	O
+of	O
+PKI	O
+,	O
+U1191	O
+does	O
+not	O
+yet	O
+stack	O
+on	O
+PKI	O
+because	O
+the	O
+head	O
+remains	O
+partially	O
+swiveled	O
+.	O
+	O
+This	O
+renders	O
+PKI	O
+partially	O
+accommodated	O
+in	O
+the	O
+P	O
+site	O
+(	O
+Figure	O
+2g	O
+).	O
+	O
+This	O
+results	O
+in	O
+rearrangements	O
+of	O
+eEF2	O
+interactions	O
+with	O
+the	O
+head	O
+,	O
+allowing	O
+eEF2	O
+to	O
+advance	O
+further	O
+into	O
+the	O
+A	O
+site	O
+.	O
+	O
+To	O
+this	O
+end	O
+,	O
+the	O
+head	O
+-	O
+interacting	O
+interface	O
+of	O
+domain	O
+IV	O
+slides	O
+along	O
+the	O
+surface	O
+of	O
+the	O
+head	O
+by	O
+5	O
+Å	O
+.	O
+Helix	O
+A	O
+of	O
+domain	O
+IV	O
+is	O
+positioned	O
+next	O
+to	O
+the	O
+backbone	O
+of	O
+h34	O
+,	O
+with	O
+positively	O
+charged	O
+residues	O
+K613	O
+,	O
+R617	O
+and	O
+R631	O
+rearranged	O
+from	O
+the	O
+backbone	O
+of	O
+h33	O
+(	O
+Figure	O
+6c	O
+;	O
+see	O
+also	O
+Figure	O
+6	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+	O
+Structure	O
+V	O
+represents	O
+an	O
+extended	O
+IRES	O
+with	O
+PKI	O
+fully	O
+accommodated	O
+in	O
+the	O
+P	O
+site	O
+and	O
+domain	O
+IV	O
+of	O
+eEF2	O
+in	O
+the	O
+A	O
+site	O
+	O
+In	O
+the	O
+nearly	O
+non	O
+-	O
+rotated	O
+and	O
+non	O
+-	O
+swiveled	O
+ribosome	O
+conformation	O
+in	O
+Structure	O
+V	O
+closely	O
+resembling	O
+that	O
+of	O
+the	O
+post	O
+-	O
+translocation	O
+80S	O
+•	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+,	O
+PKI	O
+is	O
+fully	O
+accommodated	O
+in	O
+the	O
+P	O
+site	O
+.	O
+	O
+The	O
+codon	O
+-	O
+anticodon	O
+–	O
+like	O
+helix	O
+is	O
+stacked	O
+on	O
+P	O
+-	O
+site	O
+residues	O
+U1191	O
+and	O
+C1637	O
+(	O
+Figure	O
+3d	O
+),	O
+analogous	O
+to	O
+stacking	O
+of	O
+the	O
+tRNA	O
+-	O
+mRNA	O
+helix	O
+(	O
+Figure	O
+3e	O
+).	O
+	O
+A	O
+notable	O
+conformational	O
+change	O
+in	O
+eEF2	O
+from	O
+that	O
+in	O
+the	O
+preceding	O
+Structures	O
+is	O
+visible	O
+in	O
+the	O
+position	O
+of	O
+domain	O
+III	O
+,	O
+which	O
+contacts	O
+uS12	O
+(	O
+Figure	O
+6d	O
+).	O
+	O
+In	O
+this	O
+position	O
+,	O
+uS12	O
+forms	O
+extensive	O
+interactions	O
+with	O
+eEF2	O
+domains	O
+II	O
+and	O
+III	O
+.	O
+	O
+Specifically	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+tail	O
+of	O
+uS12	O
+packs	O
+against	O
+the	O
+β	O
+-	O
+barrel	O
+of	O
+domain	O
+II	O
+,	O
+while	O
+the	O
+β	O
+-	O
+barrel	O
+of	O
+uS12	O
+packs	O
+against	O
+helix	O
+A	O
+of	O
+domain	O
+III	O
+.	O
+	O
+Domain	O
+IV	O
+of	O
+eEF2	O
+is	O
+fully	O
+accommodated	O
+in	O
+the	O
+A	O
+site	O
+.	O
+	O
+The	O
+first	O
+codon	O
+of	O
+the	O
+open	O
+reading	O
+frame	O
+is	O
+also	O
+positioned	O
+in	O
+the	O
+A	O
+site	O
+,	O
+with	O
+bases	O
+exposed	O
+toward	O
+eEF2	O
+(	O
+Figure	O
+7	O
+),	O
+resembling	O
+the	O
+conformations	O
+of	O
+the	O
+A	O
+-	O
+site	O
+codons	O
+in	O
+EF	O
+-	O
+G	O
+-	O
+bound	O
+70S	O
+complexes	O
+.	O
+	O
+As	O
+in	O
+the	O
+preceding	O
+Structures	O
+,	O
+the	O
+histidine	O
+-	O
+diphthamide	O
+tip	O
+is	O
+bound	O
+in	O
+the	O
+minor	O
+groove	O
+of	O
+the	O
+P	O
+-	O
+site	O
+codon	O
+-	O
+anticodon	O
+helix	O
+.	O
+	O
+Diph699	O
+slightly	O
+rearranges	O
+,	O
+relative	O
+to	O
+that	O
+in	O
+Structure	O
+I	O
+(	O
+Figure	O
+7	O
+),	O
+and	O
+interacts	O
+with	O
+four	O
+out	O
+of	O
+six	O
+codon	O
+-	O
+anticodon	O
+nucleotides	O
+.	O
+	O
+The	O
+amide	O
+at	O
+the	O
+diphthamide	O
+end	O
+interacts	O
+with	O
+N2	O
+of	O
+G6906	O
+and	O
+O2	O
+and	O
+O2	O
+’	O
+of	O
+C6951	O
+(	O
+corresponding	O
+to	O
+nt	O
+2	O
+of	O
+the	O
+codon	O
+).	O
+	O
+The	O
+trimethylamino	O
+-	O
+group	O
+is	O
+positioned	O
+over	O
+the	O
+ribose	O
+of	O
+C6952	O
+(	O
+codon	O
+nt	O
+3	O
+).	O
+	O
+IRES	O
+translocation	O
+mechanism	O
+	O
+Animation	O
+showing	O
+the	O
+transition	O
+from	O
+the	O
+initiation	O
+80S	O
+•	O
+TSV	O
+IRES	O
+structures	O
+(	O
+Koh	O
+et	O
+al	O
+.,	O
+2014	O
+)	O
+to	O
+eEF2	O
+-	O
+bound	O
+Structures	O
+I	O
+through	O
+V	O
+(	O
+this	O
+work	O
+).	O
+	O
+In	O
+scenes	O
+1	O
+,	O
+2	O
+and	O
+3	O
+,	O
+nucleotides	O
+C1274	O
+,	O
+U1191	O
+of	O
+the	O
+40S	O
+head	O
+and	O
+G904	O
+of	O
+the	O
+40S	O
+platform	O
+are	O
+shown	O
+in	O
+black	O
+to	O
+denote	O
+the	O
+A	O
+,	O
+P	O
+and	O
+E	O
+sites	O
+,	O
+respectively	O
+.	O
+	O
+In	O
+scene	O
+4	O
+,	O
+C1274	O
+and	O
+U1191	O
+are	O
+labeled	O
+and	O
+shown	O
+in	O
+yellow	O
+;	O
+G577	O
+,	O
+A1755	O
+and	O
+A1756	O
+of	O
+the	O
+40S	O
+body	O
+A	O
+site	O
+and	O
+C1637	O
+of	O
+the	O
+body	O
+P	O
+site	O
+are	O
+labeled	O
+and	O
+shown	O
+in	O
+orange	O
+.	O
+	O
+In	O
+this	O
+work	O
+we	O
+have	O
+captured	O
+the	O
+structures	O
+of	O
+the	O
+TSV	O
+IRES	O
+,	O
+whose	O
+PKI	O
+samples	O
+positions	O
+between	O
+the	O
+A	O
+and	O
+P	O
+sites	O
+(	O
+Structures	O
+I	O
+–	O
+IV	O
+),	O
+as	O
+well	O
+as	O
+in	O
+the	O
+P	O
+site	O
+(	O
+Structure	O
+V	O
+).	O
+	O
+We	O
+propose	O
+that	O
+together	O
+with	O
+the	O
+previously	O
+reported	O
+initiation	O
+state	O
+,	O
+these	O
+structures	O
+represent	O
+the	O
+trajectory	O
+of	O
+eEF2	O
+-	O
+induced	O
+IRES	O
+translocation	O
+(	O
+shown	O
+as	O
+an	O
+animation	O
+in	O
+http	O
+://	O
+labs	O
+.	O
+umassmed	O
+.	O
+edu	O
+/	O
+korostelevlab	O
+/	O
+msc	O
+/	O
+iresmovie	O
+.	O
+gif	O
+and	O
+Video	O
+1	O
+).	O
+	O
+Furthermore	O
+,	O
+they	O
+provide	O
+insight	O
+into	O
+the	O
+mechanism	O
+of	O
+eEF2	O
+•	O
+GTP	O
+association	O
+with	O
+the	O
+pre	O
+-	O
+translocation	O
+ribosome	O
+and	O
+eEF2	O
+•	O
+GDP	O
+dissociation	O
+from	O
+the	O
+post	O
+-	O
+translocation	O
+ribosome	O
+,	O
+also	O
+delineating	O
+the	O
+mechanism	O
+of	O
+translation	O
+inhibition	O
+by	O
+the	O
+antifungal	O
+drug	O
+sordarin	O
+.	O
+	O
+In	O
+summary	O
+,	O
+the	O
+reported	O
+ensemble	O
+of	O
+structures	O
+substantially	O
+enhances	O
+our	O
+understanding	O
+of	O
+the	O
+translocation	O
+mechanism	O
+,	O
+including	O
+that	O
+of	O
+tRNAs	O
+as	O
+discussed	O
+below	O
+.	O
+	O
+Translocation	O
+of	O
+the	O
+TSV	O
+IRES	O
+on	O
+the	O
+40S	O
+subunit	O
+globally	O
+resembles	O
+a	O
+step	O
+of	O
+an	O
+inchworm	O
+(	O
+Figure	O
+4	O
+;	O
+see	O
+also	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+	O
+At	O
+the	O
+start	O
+(	O
+initiation	O
+state	O
+),	O
+the	O
+IRES	O
+adopts	O
+an	O
+extended	O
+conformation	O
+(	O
+extended	O
+inchworm	O
+).	O
+	O
+PKI	O
+,	O
+representing	O
+the	O
+hind	O
+end	O
+,	O
+is	O
+bound	O
+in	O
+the	O
+A	O
+site	O
+.	O
+	O
+This	O
+shortens	O
+the	O
+distance	O
+between	O
+PKI	O
+and	O
+SL4	O
+by	O
+up	O
+to	O
+20	O
+Å	O
+relative	O
+to	O
+the	O
+initiating	O
+IRES	O
+structure	O
+,	O
+resulting	O
+in	O
+a	O
+bent	O
+IRES	O
+conformation	O
+(	O
+bent	O
+inchworm	O
+).	O
+	O
+Finally	O
+(	O
+Structures	O
+IV	O
+to	O
+V	O
+),	O
+as	O
+the	O
+hind	O
+end	O
+is	O
+accommodated	O
+in	O
+the	O
+P	O
+site	O
+,	O
+the	O
+front	O
+'	O
+legs	O
+'	O
+advance	O
+by	O
+departing	O
+from	O
+their	O
+initial	O
+binding	O
+sites	O
+.	O
+	O
+This	O
+converts	O
+the	O
+IRES	O
+into	O
+an	O
+extended	O
+conformation	O
+,	O
+rendering	O
+the	O
+inchworm	O
+prepared	O
+for	O
+the	O
+next	O
+translocation	O
+step	O
+.	O
+	O
+In	O
+the	O
+post	O
+-	O
+translocation	O
+CrPV	O
+IRES	O
+structure	O
+,	O
+the	O
+5	O
+’-	O
+domain	O
+similarly	O
+protrudes	O
+between	O
+the	O
+subunits	O
+and	O
+interacts	O
+with	O
+the	O
+L1	O
+stalk	O
+,	O
+as	O
+in	O
+the	O
+initiation	O
+state	O
+for	O
+this	O
+IRES	O
+.	O
+	O
+This	O
+underlines	O
+structural	O
+similarity	O
+for	O
+the	O
+TSV	O
+and	O
+CrPV	O
+IRES	O
+translocation	O
+mechanisms	O
+.	O
+	O
+One	O
+of	O
+the	O
+mechanistic	O
+scenarios	O
+(	O
+discussed	O
+in	O
+)	O
+involves	O
+binding	O
+of	O
+the	O
+first	O
+aminoacyl	O
+-	O
+tRNA	O
+to	O
+the	O
+post	O
+-	O
+translocated	O
+IRES	O
+mRNA	O
+frame	O
+shifted	O
+by	O
+one	O
+nucleotide	O
+(	O
+predominantly	O
+a	O
++	O
+1	O
+frame	O
+shift	O
+).	O
+	O
+It	O
+is	O
+likely	O
+that	O
+alternative	O
+frame	O
+setting	O
+occurs	O
+following	O
+eEF2	O
+release	O
+and	O
+that	O
+this	O
+depends	O
+on	O
+transient	O
+displacement	O
+of	O
+the	O
+start	O
+codon	O
+in	O
+the	O
+decoding	O
+center	O
+,	O
+allowing	O
+binding	O
+of	O
+the	O
+corresponding	O
+amino	O
+acyl	O
+-	O
+tRNA	O
+to	O
+an	O
+off	O
+-	O
+frame	O
+codon	O
+.	O
+	O
+Further	O
+structural	O
+studies	O
+involving	O
+80S	O
+•	O
+IRES	O
+•	O
+tRNA	O
+complexes	O
+are	O
+necessary	O
+to	O
+understand	O
+the	O
+mechanisms	O
+underlying	O
+alternative	O
+reading	O
+frame	O
+selection	O
+.	O
+	O
+The	O
+presence	O
+of	O
+several	O
+translocation	O
+complexes	O
+in	O
+a	O
+single	O
+sample	O
+suggests	O
+that	O
+the	O
+structures	O
+represent	O
+equilibrium	O
+states	O
+of	O
+forward	O
+and	O
+reverse	O
+translocation	O
+of	O
+the	O
+IRES	O
+,	O
+which	O
+interconvert	O
+among	O
+each	O
+other	O
+.	O
+	O
+Specifically	O
+,	O
+biochemical	O
+toe	O
+-	O
+printing	O
+studies	O
+in	O
+the	O
+presence	O
+of	O
+eEF2	O
+•	O
+GTP	O
+identified	O
+IRES	O
+in	O
+a	O
+non	O
+-	O
+translocated	O
+position	O
+unless	O
+eEF1a	O
+•	O
+aa	O
+-	O
+tRNA	O
+is	O
+also	O
+present	O
+.	O
+	O
+These	O
+findings	O
+indicate	O
+that	O
+IRES	O
+translocation	O
+by	O
+eEF2	O
+is	O
+futile	O
+:	O
+the	O
+IRES	O
+returns	O
+to	O
+the	O
+A	O
+site	O
+upon	O
+releasing	O
+eEF2	O
+•	O
+GDP	O
+unless	O
+an	O
+amino	O
+-	O
+acyl	O
+tRNA	O
+enters	O
+the	O
+A	O
+site	O
+and	O
+blocks	O
+IRES	O
+back	O
+-	O
+translocation	O
+.	O
+	O
+This	O
+contrasts	O
+with	O
+the	O
+post	O
+-	O
+translocated	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+,	O
+in	O
+which	O
+the	O
+classical	O
+P	O
+and	O
+E	O
+-	O
+site	O
+tRNAs	O
+are	O
+stabilized	O
+in	O
+the	O
+non	O
+-	O
+rotated	O
+ribosome	O
+after	O
+translocase	O
+release	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+meta	O
+-	O
+stability	O
+of	O
+the	O
+post	O
+-	O
+translocation	O
+IRES	O
+is	O
+likely	O
+due	O
+to	O
+the	O
+absence	O
+of	O
+stabilizing	O
+structural	O
+features	O
+present	O
+in	O
+the	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+.	O
+	O
+Furthermore	O
+,	O
+interactions	O
+of	O
+SL4	O
+and	O
+SL5	O
+with	O
+the	O
+40S	O
+subunit	O
+likely	O
+contribute	O
+to	O
+stabilization	O
+of	O
+pre	O
+-	O
+translocation	O
+structures	O
+.	O
+	O
+Our	O
+structures	O
+delineate	O
+the	O
+mechanistic	O
+functions	O
+for	O
+intersubunit	O
+rotation	O
+and	O
+head	O
+swivel	O
+in	O
+translocation	O
+.	O
+	O
+Various	O
+degrees	O
+of	O
+intersubunit	O
+rotation	O
+have	O
+been	O
+observed	O
+in	O
+cryo	O
+-	O
+EM	O
+studies	O
+of	O
+the	O
+80S	O
+•	O
+IRES	O
+initiation	O
+complexes	O
+.	O
+	O
+This	O
+suggests	O
+that	O
+the	O
+subunits	O
+are	O
+capable	O
+of	O
+spontaneous	O
+rotation	O
+,	O
+as	O
+is	O
+the	O
+case	O
+for	O
+tRNA	O
+-	O
+bound	O
+pre	O
+-	O
+translocation	O
+complexes	O
+.	O
+	O
+The	O
+pre	O
+-	O
+translocation	O
+Structure	O
+I	O
+with	O
+eEF2	O
+least	O
+advanced	O
+into	O
+the	O
+A	O
+site	O
+adopts	O
+a	O
+fully	O
+rotated	O
+conformation	O
+.	O
+	O
+Reverse	O
+intersubunit	O
+rotation	O
+from	O
+Structure	O
+I	O
+to	O
+V	O
+shifts	O
+the	O
+translocation	O
+tunnel	O
+(	O
+the	O
+tunnel	O
+between	O
+the	O
+A	O
+,	O
+P	O
+and	O
+E	O
+sites	O
+)	O
+toward	O
+eEF2	O
+,	O
+which	O
+is	O
+rigidly	O
+attached	O
+to	O
+the	O
+60S	O
+subunit	O
+.	O
+	O
+This	O
+allows	O
+eEF2	O
+to	O
+move	O
+into	O
+the	O
+A	O
+site	O
+.	O
+	O
+As	O
+such	O
+,	O
+reverse	O
+intersubunit	O
+rotation	O
+facilitates	O
+full	O
+docking	O
+of	O
+eEF2	O
+in	O
+the	O
+A	O
+site	O
+.	O
+	O
+The	O
+head	O
+swivel	O
+allows	O
+gradual	O
+translocation	O
+of	O
+PKI	O
+to	O
+the	O
+P	O
+site	O
+,	O
+first	O
+with	O
+respect	O
+to	O
+the	O
+body	O
+and	O
+then	O
+to	O
+the	O
+head	O
+.	O
+	O
+The	O
+fully	O
+swiveled	O
+conformations	O
+of	O
+Structures	O
+II	O
+and	O
+III	O
+represent	O
+the	O
+mid	O
+-	O
+point	O
+of	O
+translocation	O
+,	O
+in	O
+which	O
+PKI	O
+relocates	O
+between	O
+the	O
+head	O
+A	O
+site	O
+and	O
+body	O
+P	O
+site	O
+.	O
+	O
+We	O
+note	O
+that	O
+such	O
+mid	O
+-	O
+states	O
+have	O
+not	O
+been	O
+observed	O
+for	O
+2tRNA	O
+•	O
+mRNA	O
+,	O
+but	O
+their	O
+formation	O
+can	O
+explain	O
+the	O
+formation	O
+of	O
+subsequent	O
+pe	O
+/	O
+E	O
+hybrid	O
+and	O
+ap	O
+/	O
+P	O
+chimeric	O
+structures	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+	O
+Reverse	O
+swivel	O
+from	O
+Structure	O
+III	O
+to	O
+V	O
+brings	O
+the	O
+head	O
+to	O
+the	O
+non	O
+-	O
+swiveled	O
+position	O
+,	O
+restoring	O
+the	O
+A	O
+and	O
+P	O
+sites	O
+on	O
+the	O
+small	O
+subunit	O
+.	O
+	O
+The	O
+functions	O
+of	O
+eEF2	O
+in	O
+translocation	O
+	O
+The	O
+structures	O
+,	O
+therefore	O
+,	O
+offer	O
+a	O
+unique	O
+opportunity	O
+to	O
+address	O
+the	O
+role	O
+of	O
+the	O
+elongation	O
+factors	O
+during	O
+translocation	O
+.	O
+	O
+As	O
+discussed	O
+above	O
+,	O
+the	O
+first	O
+role	O
+is	O
+to	O
+directly	O
+shift	O
+PKI	O
+out	O
+of	O
+the	O
+A	O
+site	O
+upon	O
+spontaneous	O
+reverse	O
+intersubunit	O
+rotation	O
+.	O
+	O
+The	O
+bulky	O
+ADP	O
+-	O
+ribosyl	O
+moiety	O
+at	O
+this	O
+position	O
+would	O
+disrupt	O
+the	O
+interaction	O
+,	O
+rendering	O
+eEF2	O
+unable	O
+to	O
+bind	O
+to	O
+the	O
+A	O
+site	O
+and	O
+/	O
+or	O
+stalled	O
+on	O
+ribosomes	O
+in	O
+a	O
+non	O
+-	O
+productive	O
+conformation	O
+.	O
+	O
+As	O
+eEF2	O
+shifts	O
+PKI	O
+toward	O
+the	O
+P	O
+site	O
+in	O
+the	O
+course	O
+of	O
+reverse	O
+intersubunit	O
+rotation	O
+,	O
+the	O
+60S	O
+-	O
+attached	O
+translocase	O
+migrates	O
+along	O
+the	O
+surface	O
+of	O
+the	O
+40S	O
+subunit	O
+,	O
+guided	O
+by	O
+electrostatic	O
+interactions	O
+.	O
+	O
+Positively	O
+-	O
+charged	O
+patches	O
+of	O
+domains	O
+II	O
+and	O
+III	O
+(	O
+R391	O
+,	O
+K394	O
+,	O
+R433	O
+,	O
+R510	O
+)	O
+and	O
+IV	O
+(	O
+K613	O
+,	O
+R617	O
+,	O
+R609	O
+,	O
+R631	O
+,	O
+K651	O
+)	O
+slide	O
+over	O
+rRNA	O
+of	O
+the	O
+40S	O
+body	O
+(	O
+h5	O
+)	O
+and	O
+head	O
+(	O
+h18	O
+and	O
+h33	O
+/	O
+h34	O
+),	O
+respectively	O
+.	O
+	O
+The	O
+Structures	O
+reveal	O
+hopping	O
+of	O
+the	O
+positive	O
+clusters	O
+over	O
+rRNA	O
+helices	O
+.	O
+	O
+For	O
+example	O
+,	O
+between	O
+Structures	O
+II	O
+and	O
+V	O
+,	O
+the	O
+K613	O
+/	O
+R617	O
+/	O
+R631	O
+cluster	O
+of	O
+domain	O
+IV	O
+hops	O
+by	O
+~	O
+19	O
+Å	O
+(	O
+for	O
+Cα	O
+of	O
+R617	O
+)	O
+from	O
+the	O
+phosphate	O
+backbone	O
+of	O
+h33	O
+(	O
+at	O
+nt	O
+1261	O
+–	O
+1264	O
+)	O
+to	O
+that	O
+of	O
+the	O
+neighboring	O
+h34	O
+(	O
+at	O
+nt	O
+1442	O
+–	O
+1445	O
+).	O
+	O
+Thus	O
+,	O
+sliding	O
+of	O
+eEF2	O
+involves	O
+reorganization	O
+of	O
+electrostatic	O
+,	O
+perhaps	O
+isoenergetic	O
+interactions	O
+,	O
+echoing	O
+those	O
+implied	O
+in	O
+extraordinarily	O
+fast	O
+ribosome	O
+inactivation	O
+rates	O
+by	O
+the	O
+small	O
+-	O
+protein	O
+ribotoxins	O
+and	O
+in	O
+fast	O
+protein	O
+association	O
+and	O
+diffusion	O
+along	O
+DNA	O
+.	O
+	O
+Comparison	O
+of	O
+our	O
+structures	O
+with	O
+the	O
+80S	O
+•	O
+IRES	O
+initiation	O
+structure	O
+reveals	O
+the	O
+structural	O
+basis	O
+for	O
+the	O
+second	O
+key	O
+function	O
+of	O
+the	O
+translocase	O
+:	O
+'	O
+unlocking	O
+'	O
+of	O
+intrasubunit	O
+rearrangements	O
+that	O
+are	O
+required	O
+for	O
+step	O
+-	O
+wise	O
+translocation	O
+of	O
+PKI	O
+on	O
+the	O
+small	O
+subunit	O
+.	O
+	O
+Whereas	O
+intersubunit	O
+rotation	O
+of	O
+the	O
+pre	O
+-	O
+translocation	O
+complex	O
+occurs	O
+spontaneously	O
+,	O
+the	O
+head	O
+swivel	O
+is	O
+induced	O
+by	O
+the	O
+eEF2	O
+/	O
+EF	O
+-	O
+G	O
+translocase	O
+,	O
+consistent	O
+with	O
+requirement	O
+of	O
+eEF2	O
+for	O
+unlocking	O
+.	O
+	O
+Structural	O
+studies	O
+revealed	O
+large	O
+head	O
+swivels	O
+in	O
+various	O
+70S	O
+•	O
+tRNA	O
+•	O
+EF	O
+-	O
+G	O
+and	O
+80S	O
+•	O
+tRNA	O
+•	O
+eEF2	O
+complexes	O
+,	O
+but	O
+not	O
+in	O
+'	O
+locked	O
+'	O
+complexes	O
+with	O
+the	O
+A	O
+site	O
+occupied	O
+by	O
+the	O
+tRNA	O
+in	O
+the	O
+absence	O
+of	O
+the	O
+translocase	O
+.	O
+	O
+Our	O
+structures	O
+suggest	O
+that	O
+eEF2	O
+induces	O
+head	O
+swivel	O
+by	O
+'	O
+unlocking	O
+'	O
+the	O
+head	O
+-	O
+body	O
+interactions	O
+(	O
+Figure	O
+7	O
+).	O
+	O
+Binding	O
+of	O
+the	O
+ASL	O
+to	O
+the	O
+A	O
+site	O
+is	O
+known	O
+from	O
+structural	O
+studies	O
+of	O
+bacterial	O
+ribosomes	O
+to	O
+result	O
+in	O
+'	O
+domain	O
+closure	O
+'	O
+of	O
+the	O
+small	O
+subunit	O
+,	O
+i	O
+.	O
+e	O
+.	O
+closer	O
+association	O
+of	O
+the	O
+head	O
+,	O
+shoulder	O
+and	O
+body	O
+domains	O
+.	O
+	O
+The	O
+domain	O
+closure	O
+'	O
+locks	O
+'	O
+cognate	O
+tRNA	O
+in	O
+the	O
+A	O
+site	O
+via	O
+stacking	O
+on	O
+the	O
+head	O
+A	O
+site	O
+(	O
+C1274	O
+in	O
+S	O
+.	O
+cerevisiae	O
+or	O
+C1054	O
+in	O
+E	O
+.	O
+coli	O
+)	O
+and	O
+interactions	O
+with	O
+the	O
+body	O
+A	O
+-	O
+site	O
+nucleotides	O
+A1755	O
+and	O
+A1756	O
+(	O
+A1492	O
+and	O
+A1493	O
+in	O
+E	O
+.	O
+coli	O
+).	O
+	O
+This	O
+'	O
+locked	O
+'	O
+state	O
+is	O
+identical	O
+to	O
+that	O
+observed	O
+for	O
+PKI	O
+in	O
+the	O
+80S	O
+•	O
+IRES	O
+initiation	O
+structures	O
+in	O
+the	O
+absence	O
+of	O
+eEF2	O
+.	O
+	O
+Structure	O
+I	O
+demonstrates	O
+that	O
+at	O
+an	O
+early	O
+pre	O
+-	O
+translocation	O
+step	O
+,	O
+the	O
+histidine	O
+-	O
+diphthamide	O
+tip	O
+of	O
+eEF2	O
+is	O
+wedged	O
+between	O
+A1755	O
+and	O
+A1756	O
+and	O
+PKI	O
+.	O
+	O
+Destabilization	O
+of	O
+the	O
+head	O
+-	O
+bound	O
+PKI	O
+at	O
+the	O
+body	O
+A	O
+site	O
+thus	O
+allows	O
+mobility	O
+of	O
+the	O
+head	O
+relative	O
+to	O
+the	O
+body	O
+.	O
+	O
+The	O
+histidine	O
+-	O
+diphthamide	O
+-	O
+induced	O
+disengagement	O
+of	O
+PKI	O
+from	O
+A1755	O
+and	O
+A1756	O
+therefore	O
+provides	O
+the	O
+structural	O
+definition	O
+for	O
+the	O
+'	O
+unlocking	O
+'	O
+mode	O
+of	O
+eEF2	O
+action	O
+.	O
+	O
+In	O
+summary	O
+,	O
+our	O
+structures	O
+are	O
+consistent	O
+with	O
+a	O
+model	O
+of	O
+eEF2	O
+-	O
+induced	O
+translocation	O
+in	O
+which	O
+both	O
+PKI	O
+and	O
+eEF2	O
+passively	O
+migrate	O
+into	O
+the	O
+P	O
+and	O
+A	O
+site	O
+,	O
+respectively	O
+,	O
+during	O
+spontaneous	O
+40S	O
+body	O
+rotation	O
+and	O
+head	O
+swivel	O
+,	O
+the	O
+latter	O
+being	O
+allowed	O
+by	O
+'	O
+unlocking	O
+'	O
+of	O
+the	O
+A	O
+site	O
+by	O
+eEF2	O
+.	O
+	O
+Observation	O
+of	O
+different	O
+PKI	O
+conformations	O
+sampling	O
+a	O
+range	O
+of	O
+positions	O
+between	O
+the	O
+A	O
+and	O
+P	O
+sites	O
+in	O
+the	O
+presence	O
+of	O
+eEF2	O
+•	O
+GDP	O
+implies	O
+that	O
+thermal	O
+fluctuations	O
+of	O
+the	O
+40S	O
+head	O
+domain	O
+are	O
+sufficient	O
+for	O
+translocation	O
+along	O
+the	O
+energetically	O
+flat	O
+trajectory	O
+.	O
+	O
+Insights	O
+into	O
+eEF2	O
+association	O
+with	O
+and	O
+dissociation	O
+from	O
+the	O
+ribosome	O
+	O
+The	O
+conformational	O
+rearrangements	O
+in	O
+eEF2	O
+from	O
+Structure	O
+I	O
+through	O
+Structure	O
+V	O
+provide	O
+insights	O
+into	O
+the	O
+mechanisms	O
+of	O
+eEF2	O
+association	O
+with	O
+the	O
+pre	O
+-	O
+translocation	O
+ribosome	O
+and	O
+dissociation	O
+from	O
+the	O
+post	O
+-	O
+translocation	O
+ribosome	O
+.	O
+	O
+In	O
+all	O
+five	O
+structures	O
+,	O
+the	O
+GTPase	O
+domain	O
+is	O
+attached	O
+to	O
+the	O
+P	O
+stalk	O
+and	O
+the	O
+sarcin	O
+-	O
+ricin	O
+loop	O
+.	O
+	O
+Here	O
+,	O
+switch	O
+loop	O
+I	O
+interacts	O
+with	O
+helix	O
+14	O
+(	O
+415CAAA418	O
+)	O
+of	O
+the	O
+18S	O
+rRNA	O
+.	O
+	O
+This	O
+stabilization	O
+renders	O
+the	O
+GTPase	O
+center	O
+to	O
+adopt	O
+a	O
+GTP	O
+-	O
+bound	O
+conformation	O
+,	O
+similar	O
+to	O
+those	O
+observed	O
+in	O
+other	O
+translational	O
+GTPases	O
+in	O
+the	O
+presence	O
+of	O
+GTP	O
+analogs	O
+and	O
+in	O
+the	O
+80S	O
+•	O
+eEF2	O
+complex	O
+bound	O
+with	O
+a	O
+transition	O
+-	O
+state	O
+mimic	O
+GDP	O
+•	O
+AlF4	O
+–.	O
+The	O
+switch	O
+loop	O
+contacts	O
+the	O
+base	O
+of	O
+A416	O
+(	O
+invariable	O
+A344	O
+in	O
+E	O
+.	O
+coli	O
+and	O
+A463	O
+in	O
+H	O
+.	O
+sapiens	O
+).	O
+	O
+This	O
+structural	O
+basis	O
+rationalizes	O
+the	O
+observation	O
+of	O
+transient	O
+stabilization	O
+of	O
+the	O
+rotated	O
+70S	O
+ribosome	O
+upon	O
+EF	O
+-	O
+G	O
+•	O
+GTP	O
+binding	O
+and	O
+prior	O
+to	O
+translocation	O
+.	O
+	O
+The	O
+least	O
+rotated	O
+conformation	O
+of	O
+the	O
+post	O
+-	O
+translocation	O
+Structure	O
+V	O
+suggests	O
+conformational	O
+changes	O
+that	O
+may	O
+trigger	O
+eEF2	O
+release	O
+from	O
+the	O
+ribosome	O
+at	O
+the	O
+end	O
+of	O
+translocation	O
+.	O
+	O
+The	O
+most	O
+pronounced	O
+inter	O
+-	O
+domain	O
+rearrangement	O
+in	O
+eEF2	O
+involves	O
+movement	O
+of	O
+domain	O
+III	O
+.	O
+	O
+In	O
+the	O
+rotated	O
+or	O
+mid	O
+-	O
+rotated	O
+Structures	O
+I	O
+through	O
+III	O
+,	O
+this	O
+domain	O
+remains	O
+rigidly	O
+associated	O
+with	O
+domain	O
+V	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+superdomain	O
+and	O
+does	O
+not	O
+undergo	O
+noticeable	O
+rearrangements	O
+.	O
+	O
+In	O
+Structure	O
+V	O
+,	O
+however	O
+,	O
+the	O
+tip	O
+of	O
+helix	O
+A	O
+of	O
+domain	O
+III	O
+is	O
+displaced	O
+toward	O
+domain	O
+I	O
+by	O
+~	O
+5	O
+Å	O
+relative	O
+to	O
+that	O
+in	O
+mid	O
+-	O
+rotated	O
+or	O
+fully	O
+rotated	O
+structures	O
+.	O
+	O
+Sordarin	O
+stabilizes	O
+GDP	O
+-	O
+bound	O
+eEF2	O
+on	O
+the	O
+ribosome	O
+	O
+Based	O
+on	O
+biochemical	O
+experiments	O
+,	O
+two	O
+alternative	O
+mechanisms	O
+of	O
+action	O
+were	O
+proposed	O
+:	O
+sordarin	O
+either	O
+prevents	O
+eEF2	O
+departure	O
+by	O
+inhibiting	O
+GTP	O
+hydrolysis	O
+or	O
+acts	O
+after	O
+GTP	O
+hydrolysis	O
+.	O
+	O
+Our	O
+structures	O
+therefore	O
+indicate	O
+that	O
+sordarin	O
+stalls	O
+eEF2	O
+on	O
+the	O
+ribosome	O
+in	O
+the	O
+GDP	O
+-	O
+bound	O
+form	O
+,	O
+i	O
+.	O
+e	O
+.	O
+following	O
+GTP	O
+hydrolysis	O
+and	O
+phosphate	O
+release	O
+.	O
+	O
+The	O
+mechanism	O
+of	O
+stalling	O
+is	O
+suggested	O
+by	O
+comparison	O
+of	O
+pre	O
+-	O
+translocation	O
+and	O
+post	O
+-	O
+translocation	O
+structures	O
+in	O
+our	O
+ensemble	O
+.	O
+	O
+In	O
+the	O
+nearly	O
+non	O
+-	O
+rotated	O
+post	O
+-	O
+translocation	O
+Structure	O
+V	O
+,	O
+the	O
+tip	O
+of	O
+domain	O
+III	O
+is	O
+shifted	O
+,	O
+however	O
+the	O
+interface	O
+between	O
+domains	O
+III	O
+and	O
+V	O
+remains	O
+unchanged	O
+,	O
+suggesting	O
+strong	O
+stabilization	O
+of	O
+this	O
+interface	O
+by	O
+sordarin	O
+.	O
+	O
+We	O
+note	O
+that	O
+Structure	O
+V	O
+is	O
+slightly	O
+more	O
+rotated	O
+than	O
+the	O
+80S	O
+•	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+in	O
+the	O
+absence	O
+of	O
+eEF2	O
+•	O
+sordarin	O
+,	O
+implying	O
+that	O
+sordarin	O
+interferes	O
+with	O
+the	O
+final	O
+stages	O
+of	O
+reverse	O
+rotation	O
+of	O
+the	O
+post	O
+-	O
+translocation	O
+ribosome	O
+.	O
+	O
+We	O
+propose	O
+that	O
+sordarin	O
+acts	O
+to	O
+prevent	O
+full	O
+reverse	O
+rotation	O
+and	O
+release	O
+of	O
+eEF2	O
+•	O
+GDP	O
+by	O
+stabilizing	O
+the	O
+interdomain	O
+interface	O
+and	O
+thus	O
+blocking	O
+uS12	O
+-	O
+induced	O
+disengagement	O
+of	O
+domain	O
+III	O
+from	O
+domain	O
+V	O
+.	O
+	O
+Because	O
+translocation	O
+of	O
+tRNA	O
+must	O
+involve	O
+large	O
+-	O
+scale	O
+dynamics	O
+,	O
+this	O
+step	O
+has	O
+long	O
+been	O
+regarded	O
+as	O
+the	O
+most	O
+puzzling	O
+step	O
+of	O
+translation	O
+.	O
+	O
+The	O
+structural	O
+understanding	O
+of	O
+ribosome	O
+and	O
+tRNA	O
+dynamics	O
+has	O
+been	O
+greatly	O
+aided	O
+by	O
+a	O
+wealth	O
+of	O
+X	O
+-	O
+ray	O
+and	O
+cryo	O
+-	O
+EM	O
+structures	O
+(	O
+reviewed	O
+in	O
+).	O
+	O
+However	O
+,	O
+visualization	O
+of	O
+the	O
+eEF2	O
+/	O
+EF	O
+-	O
+G	O
+-	O
+induced	O
+translocation	O
+is	O
+confined	O
+to	O
+very	O
+early	O
+pre	O
+-	O
+EF	O
+-	O
+G	O
+-	O
+entry	O
+states	O
+and	O
+late	O
+(	O
+almost	O
+translocated	O
+or	O
+fully	O
+translocated	O
+)	O
+states	O
+,	O
+leaving	O
+most	O
+of	O
+the	O
+path	O
+from	O
+the	O
+A	O
+to	O
+the	O
+P	O
+site	O
+uncharacterized	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+	O
+Our	O
+study	O
+provides	O
+new	O
+insights	O
+into	O
+the	O
+structural	O
+understanding	O
+of	O
+tRNA	O
+translocation	O
+.	O
+	O
+This	O
+is	O
+evident	O
+from	O
+the	O
+fact	O
+that	O
+ribosome	O
+rearrangements	O
+in	O
+translocation	O
+are	O
+inherent	O
+to	O
+the	O
+ribosome	O
+and	O
+likely	O
+occur	O
+in	O
+similar	O
+ways	O
+in	O
+both	O
+cases	O
+.	O
+	O
+For	O
+example	O
+,	O
+fluorescence	O
+and	O
+biochemical	O
+studies	O
+revealed	O
+that	O
+the	O
+early	O
+pre	O
+-	O
+translocation	O
+EF	O
+-	O
+G	O
+-	O
+bound	O
+ribosomes	O
+are	O
+fully	O
+rotated	O
+and	O
+translocation	O
+of	O
+the	O
+tRNA	O
+-	O
+mRNA	O
+complex	O
+occurs	O
+during	O
+reverse	O
+rotation	O
+of	O
+the	O
+small	O
+subunit	O
+,	O
+coupled	O
+with	O
+head	O
+swivel	O
+.	O
+	O
+The	O
+sequence	O
+of	O
+ribosome	O
+rearrangements	O
+during	O
+IRES	O
+translocation	O
+also	O
+agrees	O
+with	O
+that	O
+inferred	O
+from	O
+70S	O
+•	O
+EF	O
+-	O
+G	O
+structures	O
+,	O
+including	O
+those	O
+in	O
+which	O
+the	O
+A	O
+-	O
+to	O
+-	O
+P	O
+-	O
+site	O
+translocating	O
+tRNA	O
+was	O
+not	O
+present	O
+.	O
+	O
+Specifically	O
+,	O
+an	O
+earlier	O
+translocation	O
+intermediate	O
+ribosome	O
+(	O
+TIpre	O
+)	O
+was	O
+proposed	O
+to	O
+adopt	O
+a	O
+rotated	O
+(	O
+7	O
+–	O
+9	O
+°)	O
+body	O
+and	O
+a	O
+partly	O
+rotated	O
+head	O
+(	O
+5	O
+–	O
+7	O
+.	O
+5	O
+°),	O
+in	O
+agreement	O
+with	O
+the	O
+conformation	O
+of	O
+our	O
+Structure	O
+I	O
+.	O
+The	O
+most	O
+swiveled	O
+head	O
+(	O
+18	O
+–	O
+21	O
+°)	O
+was	O
+observed	O
+in	O
+a	O
+mid	O
+-	O
+rotated	O
+ribosome	O
+(	O
+3	O
+–	O
+5	O
+°)	O
+of	O
+a	O
+later	O
+translocation	O
+intermediate	O
+TIpost	O
+,	O
+similar	O
+to	O
+the	O
+conformation	O
+of	O
+our	O
+Structure	O
+III	O
+.	O
+	O
+Overall	O
+,	O
+these	O
+correlations	O
+suggest	O
+that	O
+the	O
+intermediate	O
+locations	O
+of	O
+the	O
+elusive	O
+A	O
+-	O
+to	O
+-	O
+P	O
+-	O
+site	O
+translocating	O
+tRNA	O
+are	O
+similar	O
+to	O
+those	O
+of	O
+PKI	O
+in	O
+our	O
+structures	O
+.	O
+	O
+Second	O
+,	O
+the	O
+structures	O
+clarify	O
+the	O
+structural	O
+basis	O
+of	O
+the	O
+often	O
+-	O
+used	O
+but	O
+structurally	O
+undefined	O
+terms	O
+'	O
+locking	O
+'	O
+and	O
+'	O
+unlocking	O
+'	O
+with	O
+respect	O
+to	O
+the	O
+pre	O
+-	O
+translocation	O
+complex	O
+(	O
+Figure	O
+6f	O
+).	O
+	O
+These	O
+interactions	O
+are	O
+maintained	O
+for	O
+the	O
+classical	O
+-	O
+and	O
+hybrid	O
+-	O
+state	O
+tRNAs	O
+in	O
+the	O
+spontaneously	O
+sampled	O
+non	O
+-	O
+rotated	O
+and	O
+rotated	O
+ribosomes	O
+,	O
+respectively	O
+.	O
+	O
+Unlocking	O
+involves	O
+separation	O
+of	O
+the	O
+codon	O
+-	O
+anticodon	O
+helix	O
+from	O
+the	O
+decoding	O
+center	O
+residues	O
+by	O
+the	O
+protruding	O
+tip	O
+of	O
+eEF2	O
+/	O
+EF	O
+-	O
+G	O
+(	O
+Figure	O
+7	O
+),	O
+occurring	O
+in	O
+the	O
+fully	O
+rotated	O
+ribosome	O
+at	O
+an	O
+early	O
+pre	O
+-	O
+translocation	O
+step	O
+.	O
+	O
+Third	O
+,	O
+our	O
+findings	O
+uncover	O
+a	O
+new	O
+role	O
+of	O
+the	O
+head	O
+swivel	O
+.	O
+	O
+Previous	O
+studies	O
+showed	O
+that	O
+this	O
+movement	O
+widens	O
+the	O
+constriction	O
+('	O
+gate	O
+')	O
+between	O
+the	O
+P	O
+and	O
+E	O
+sites	O
+,	O
+thus	O
+allowing	O
+the	O
+P	O
+-	O
+tRNA	O
+passage	O
+to	O
+the	O
+E	O
+site	O
+.	O
+	O
+In	O
+addition	O
+to	O
+the	O
+'	O
+gate	O
+-	O
+opening	O
+'	O
+role	O
+,	O
+we	O
+now	O
+show	O
+that	O
+the	O
+head	O
+swivel	O
+brings	O
+the	O
+head	O
+A	O
+site	O
+to	O
+the	O
+body	O
+P	O
+site	O
+,	O
+allowing	O
+a	O
+step	O
+-	O
+wise	O
+conveying	O
+of	O
+the	O
+codon	O
+-	O
+anticodon	O
+helix	O
+between	O
+the	O
+A	O
+and	O
+P	O
+sites	O
+.	O
+	O
+Our	O
+findings	O
+implicate	O
+,	O
+however	O
+,	O
+that	O
+the	O
+energy	O
+landscape	O
+is	O
+not	O
+completely	O
+flat	O
+and	O
+contains	O
+local	O
+minima	O
+for	O
+transient	O
+positions	O
+of	O
+the	O
+codon	O
+-	O
+anticodon	O
+helix	O
+between	O
+the	O
+A	O
+and	O
+P	O
+sites	O
+.	O
+	O
+The	O
+shift	O
+of	O
+the	O
+PKI	O
+with	O
+respect	O
+to	O
+the	O
+body	O
+occurs	O
+during	O
+forward	O
+head	O
+swivel	O
+in	O
+two	O
+major	O
+sub	O
+-	O
+steps	O
+of	O
+~	O
+4	O
+Å	O
+each	O
+(	O
+initiation	O
+complex	O
+to	O
+I	O
+,	O
+and	O
+I	O
+to	O
+II	O
+),	O
+after	O
+which	O
+PKI	O
+undergoes	O
+small	O
+shifts	O
+to	O
+settle	O
+in	O
+the	O
+body	O
+P	O
+site	O
+in	O
+Structures	O
+III	O
+,	O
+IV	O
+and	O
+V	O
+(	O
+Figure	O
+2	O
+—	O
+source	O
+data	O
+1	O
+).	O
+	O
+Movement	O
+of	O
+PKI	O
+relative	O
+to	O
+the	O
+head	O
+occurs	O
+during	O
+the	O
+subsequent	O
+reverse	O
+swivel	O
+in	O
+three	O
+3	O
+–	O
+7	O
+Å	O
+sub	O
+-	O
+steps	O
+(	O
+II	O
+to	O
+III	O
+to	O
+IV	O
+to	O
+V	O
+).	O
+	O
+Translation	O
+of	O
+viral	O
+mRNA	O
+	O
+Our	O
+work	O
+sheds	O
+light	O
+on	O
+the	O
+dynamic	O
+mechanism	O
+of	O
+cap	O
+-	O
+independent	O
+translation	O
+by	O
+IGR	O
+IRESs	O
+,	O
+tightly	O
+coupled	O
+with	O
+the	O
+universally	O
+conserved	O
+dynamic	O
+properties	O
+of	O
+the	O
+ribosome	O
+.	O
+	O
+Like	O
+in	O
+the	O
+2tRNA	O
+•	O
+mRNA	O
+translocating	O
+complex	O
+in	O
+which	O
+the	O
+two	O
+tRNAs	O
+move	O
+independently	O
+of	O
+each	O
+other	O
+,	O
+the	O
+PKI	O
+domain	O
+moves	O
+relative	O
+to	O
+the	O
+5	O
+´-	O
+domain	O
+,	O
+causing	O
+the	O
+IRES	O
+to	O
+undergo	O
+an	O
+inchworm	O
+-	O
+walk	O
+translocation	O
+.	O
+	O
+A	O
+large	O
+structural	O
+difference	O
+between	O
+the	O
+IRES	O
+and	O
+the	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+exists	O
+,	O
+however	O
+,	O
+in	O
+that	O
+the	O
+IRES	O
+lacks	O
+three	O
+out	O
+of	O
+six	O
+tRNA	O
+-	O
+like	O
+domains	O
+involved	O
+in	O
+tRNA	O
+translocation	O
+.	O
+	O
+Although	O
+structurally	O
+handicapped	O
+,	O
+the	O
+TSV	O
+IRES	O
+manages	O
+to	O
+translocate	O
+by	O
+employing	O
+ribosome	O
+dynamics	O
+that	O
+are	O
+remarkably	O
+similar	O
+to	O
+that	O
+in	O
+2tRNA	O
+•	O
+mRNA	O
+translocation	O
+.	O
+	O
+This	O
+property	O
+is	O
+rendered	O
+by	O
+the	O
+relative	O
+mobility	O
+of	O
+the	O
+three	O
+major	O
+building	O
+blocks	O
+,	O
+the	O
+60S	O
+subunit	O
+and	O
+the	O
+40S	O
+head	O
+and	O
+body	O
+,	O
+assisted	O
+by	O
+ligand	O
+-	O
+interacting	O
+extensions	O
+including	O
+the	O
+L1	O
+stalk	O
+and	O
+the	O
+P	O
+stalk	O
+.	O
+	O
+Viral	O
+mRNAs	O
+have	O
+evolved	O
+to	O
+adopt	O
+an	O
+atypical	O
+structure	O
+to	O
+employ	O
+the	O
+inherent	O
+ribosome	O
+dynamics	O
+,	O
+to	O
+be	O
+able	O
+to	O
+hijack	O
+the	O
+host	O
+translational	O
+machinery	O
+in	O
+a	O
+simple	O
+fashion	O
+.	O
+	O
+Ensemble	O
+cryo	O
+-	O
+EM	O
+	O
+High	O
+-	O
+resolution	O
+crystal	O
+structures	O
+,	O
+on	O
+the	O
+other	O
+hand	O
+,	O
+can	O
+provide	O
+static	O
+images	O
+of	O
+an	O
+assembly	O
+,	O
+and	O
+the	O
+structural	O
+dynamics	O
+can	O
+only	O
+be	O
+inferred	O
+by	O
+comparing	O
+structures	O
+that	O
+are	O
+usually	O
+obtained	O
+in	O
+different	O
+experiments	O
+and	O
+under	O
+different	O
+,	O
+often	O
+non	O
+-	O
+native	O
+,	O
+conditions	O
+.	O
+	O
+Cryo	O
+-	O
+EM	O
+offers	O
+the	O
+possibility	O
+of	O
+obtaining	O
+integrated	O
+information	O
+of	O
+both	O
+structure	O
+and	O
+dynamics	O
+as	O
+demonstrated	O
+in	O
+lower	O
+-	O
+resolution	O
+studies	O
+of	O
+bacterial	O
+ribosome	O
+complexes	O
+.	O
+	O
+This	O
+is	O
+presumably	O
+one	O
+of	O
+the	O
+reasons	O
+why	O
+most	O
+recent	O
+studies	O
+of	O
+ribosome	O
+complexes	O
+have	O
+focused	O
+on	O
+a	O
+single	O
+high	O
+-	O
+resolution	O
+structure	O
+despite	O
+the	O
+non	O
+-	O
+uniform	O
+local	O
+resolution	O
+of	O
+the	O
+maps	O
+that	O
+likely	O
+reflects	O
+structural	O
+heterogeneity	O
+.	O
+	O
+The	O
+computational	O
+efficiency	O
+of	O
+FREALIGN	O
+has	O
+allowed	O
+us	O
+to	O
+classify	O
+a	O
+relatively	O
+large	O
+dataset	O
+(	O
+1	O
+.	O
+1	O
+million	O
+particles	O
+)	O
+into	O
+15	O
+classes	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2	O
+)	O
+and	O
+obtain	O
+eight	O
+near	O
+-	O
+atomic	O
+-	O
+resolution	O
+structures	O
+from	O
+it	O
+.	O
+	O
+Therefore	O
+,	O
+cryo	O
+-	O
+EM	O
+has	O
+the	O
+potential	O
+to	O
+become	O
+a	O
+standard	O
+tool	O
+for	O
+uncovering	O
+detailed	O
+dynamic	O
+pathways	O
+of	O
+complex	O
+macromolecular	O
+machines	O
+.	O
+	O
+A	O
+unified	O
+mechanism	O
+for	O
+proteolysis	O
+and	O
+autocatalytic	O
+activation	O
+in	O
+the	O
+20S	O
+proteasome	O
+	O
+Biogenesis	O
+of	O
+the	O
+20S	O
+proteasome	O
+is	O
+tightly	O
+regulated	O
+.	O
+	O
+However	O
+,	O
+the	O
+trigger	O
+for	O
+the	O
+self	O
+-	O
+activation	O
+and	O
+the	O
+reason	O
+for	O
+the	O
+strict	O
+conservation	O
+of	O
+threonine	O
+as	O
+the	O
+active	O
+site	O
+nucleophile	O
+remain	O
+enigmatic	O
+.	O
+	O
+Here	O
+we	O
+use	O
+mutagenesis	O
+,	O
+X	O
+-	O
+ray	O
+crystallography	O
+and	O
+biochemical	O
+assays	O
+to	O
+suggest	O
+that	O
+Lys33	O
+initiates	O
+nucleophilic	O
+attack	O
+of	O
+the	O
+propeptide	O
+by	O
+deprotonating	O
+the	O
+Thr1	O
+hydroxyl	O
+group	O
+and	O
+that	O
+both	O
+residues	O
+together	O
+with	O
+Asp17	O
+are	O
+part	O
+of	O
+a	O
+catalytic	O
+triad	O
+.	O
+	O
+Substitution	O
+of	O
+Thr1	O
+by	O
+Cys	O
+disrupts	O
+the	O
+interaction	O
+with	O
+Lys33	O
+and	O
+inactivates	O
+the	O
+proteasome	O
+.	O
+	O
+Although	O
+a	O
+Thr1Ser	B-mutant
+mutant	O
+is	O
+active	O
+,	O
+it	O
+is	O
+less	O
+efficient	O
+compared	O
+with	O
+wild	O
+type	O
+because	O
+of	O
+the	O
+unfavourable	O
+orientation	O
+of	O
+Ser1	O
+towards	O
+incoming	O
+substrates	O
+.	O
+	O
+The	O
+proteasome	O
+,	O
+an	O
+essential	O
+molecular	O
+machine	O
+,	O
+is	O
+a	O
+threonine	O
+protease	O
+,	O
+but	O
+the	O
+evolution	O
+and	O
+the	O
+components	O
+of	O
+its	O
+proteolytic	O
+centre	O
+are	O
+unclear	O
+.	O
+	O
+Here	O
+,	O
+the	O
+authors	O
+use	O
+structural	O
+biology	O
+and	O
+biochemistry	O
+to	O
+investigate	O
+the	O
+role	O
+of	O
+proteasome	O
+active	O
+site	O
+residues	O
+on	O
+maturation	O
+and	O
+activity	O
+.	O
+	O
+The	O
+20S	O
+proteasome	O
+core	O
+particle	O
+(	O
+CP	O
+)	O
+is	O
+the	O
+key	O
+non	O
+-	O
+lysosomal	O
+protease	O
+of	O
+eukaryotic	O
+cells	O
+.	O
+	O
+Its	O
+seven	O
+different	O
+α	O
+and	O
+seven	O
+different	O
+β	O
+subunits	O
+assemble	O
+into	O
+four	O
+heptameric	O
+rings	O
+that	O
+are	O
+stacked	O
+on	O
+each	O
+other	O
+to	O
+form	O
+a	O
+hollow	O
+cylinder	O
+.	O
+	O
+While	O
+the	O
+inactive	O
+α	O
+subunits	O
+build	O
+the	O
+two	O
+outer	O
+rings	O
+,	O
+the	O
+β	O
+subunits	O
+form	O
+the	O
+inner	O
+rings	O
+.	O
+	O
+Only	O
+three	O
+out	O
+of	O
+the	O
+seven	O
+different	O
+β	O
+subunits	O
+,	O
+namely	O
+β1	O
+,	O
+β2	O
+and	O
+β5	O
+,	O
+bear	O
+N	O
+-	O
+terminal	O
+proteolytic	O
+active	O
+centres	O
+,	O
+and	O
+before	O
+CP	O
+maturation	O
+these	O
+are	O
+protected	O
+by	O
+propeptides	O
+.	O
+	O
+In	O
+the	O
+last	O
+stage	O
+of	O
+CP	O
+biogenesis	O
+,	O
+the	O
+prosegments	O
+are	O
+autocatalytically	O
+removed	O
+through	O
+nucleophilic	O
+attack	O
+by	O
+the	O
+active	O
+site	O
+residue	O
+Thr1	O
+on	O
+the	O
+preceding	O
+peptide	O
+bond	O
+involving	O
+Gly	O
+(-	O
+1	O
+).	O
+	O
+Release	O
+of	O
+the	O
+propeptides	O
+creates	O
+a	O
+functionally	O
+active	O
+CP	O
+that	O
+cleaves	O
+proteins	O
+into	O
+short	O
+peptides	O
+.	O
+	O
+Although	O
+the	O
+chemical	O
+nature	O
+of	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+and	O
+hence	O
+substrate	O
+preferences	O
+are	O
+unique	O
+to	O
+each	O
+of	O
+the	O
+distinct	O
+active	O
+β	O
+subunits	O
+,	O
+all	O
+active	O
+sites	O
+employ	O
+an	O
+identical	O
+reaction	O
+mechanism	O
+to	O
+hydrolyse	O
+peptide	O
+bonds	O
+.	O
+	O
+Nucleophilic	O
+attack	O
+of	O
+Thr1Oγ	O
+on	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+the	O
+scissile	O
+peptide	O
+bond	O
+creates	O
+a	O
+first	O
+cleavage	O
+product	O
+and	O
+a	O
+covalent	O
+acyl	O
+-	O
+enzyme	O
+intermediate	O
+.	O
+	O
+Hydrolysis	O
+of	O
+this	O
+complex	O
+by	O
+the	O
+addition	O
+of	O
+a	O
+nucleophilic	O
+water	O
+molecule	O
+regenerates	O
+the	O
+enzyme	O
+and	O
+releases	O
+the	O
+second	O
+peptide	O
+fragment	O
+.	O
+	O
+The	O
+proteasome	O
+belongs	O
+to	O
+the	O
+family	O
+of	O
+N	O
+-	O
+terminal	O
+nucleophilic	O
+(	O
+Ntn	O
+)	O
+hydrolases	O
+,	O
+and	O
+the	O
+free	O
+N	O
+-	O
+terminal	O
+amine	O
+group	O
+of	O
+Thr1	O
+was	O
+proposed	O
+to	O
+deprotonate	O
+the	O
+Thr1	O
+hydroxyl	O
+group	O
+to	O
+generate	O
+a	O
+nucleophilic	O
+Thr1Oγ	O
+for	O
+peptide	O
+-	O
+bond	O
+cleavage	O
+.	O
+	O
+An	O
+alternative	O
+candidate	O
+for	O
+deprotonating	O
+the	O
+Thr1	O
+hydroxyl	O
+group	O
+is	O
+the	O
+side	O
+chain	O
+of	O
+Lys33	O
+as	O
+it	O
+is	O
+within	O
+hydrogen	O
+-	O
+bonding	O
+distance	O
+to	O
+Thr1OH	O
+(	O
+2	O
+.	O
+7	O
+Å	O
+).	O
+	O
+In	O
+principle	O
+it	O
+could	O
+function	O
+as	O
+the	O
+general	O
+base	O
+during	O
+both	O
+autocatalytic	O
+removal	O
+of	O
+the	O
+propeptide	O
+and	O
+protein	O
+substrate	O
+cleavage	O
+.	O
+	O
+Here	O
+we	O
+provide	O
+experimental	O
+evidences	O
+for	O
+this	O
+distinct	O
+view	O
+of	O
+the	O
+proteasome	O
+active	O
+-	O
+site	O
+mechanism	O
+.	O
+	O
+Furthermore	O
+,	O
+we	O
+determine	O
+the	O
+advantages	O
+of	O
+Thr	O
+over	O
+Cys	O
+or	O
+Ser	O
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+using	O
+X	O
+-	O
+ray	O
+crystallography	O
+together	O
+with	O
+activity	O
+and	O
+inhibition	O
+assays	O
+.	O
+	O
+Inactivation	O
+of	O
+the	O
+active	O
+site	O
+Thr1	O
+by	O
+mutation	O
+to	O
+Ala	O
+has	O
+been	O
+used	O
+to	O
+study	O
+substrate	O
+specificity	O
+and	O
+the	O
+hierarchy	O
+of	O
+the	O
+proteasome	O
+active	O
+sites	O
+.	O
+	O
+Yeast	O
+strains	O
+carrying	O
+the	O
+single	O
+mutations	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+or	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+or	O
+both	O
+,	O
+are	O
+viable	O
+,	O
+even	O
+though	O
+one	O
+or	O
+two	O
+of	O
+the	O
+three	O
+distinct	O
+catalytic	O
+β	O
+subunits	O
+are	O
+disabled	O
+and	O
+carry	O
+remnants	O
+of	O
+their	O
+N	O
+-	O
+terminal	O
+propeptides	O
+(	O
+Table	O
+1	O
+).	O
+	O
+By	O
+contrast	O
+,	O
+the	O
+T1A	B-mutant
+mutation	O
+in	O
+subunit	O
+β5	O
+has	O
+been	O
+reported	O
+to	O
+be	O
+lethal	O
+or	O
+nearly	O
+so	O
+.	O
+	O
+Viability	O
+is	O
+restored	O
+if	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+subunit	O
+has	O
+its	O
+propeptide	O
+(	O
+pp	O
+)	O
+deleted	O
+but	O
+expressed	O
+separately	O
+in	O
+trans	O
+(	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+pp	O
+trans	O
+),	O
+although	O
+substantial	O
+phenotypic	O
+impairment	O
+remains	O
+(	O
+Table	O
+1	O
+).	O
+	O
+The	O
+extremely	O
+weak	O
+growth	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	O
+pp	O
+cis	O
+described	O
+by	O
+Chen	O
+and	O
+Hochstrasser	O
+compared	O
+with	O
+the	O
+inviability	O
+reported	O
+by	O
+Heinemeyer	O
+et	O
+al	O
+.	O
+prompted	O
+us	O
+to	O
+analyse	O
+this	O
+discrepancy	O
+.	O
+	O
+We	O
+also	O
+identified	O
+an	O
+additional	O
+point	O
+mutation	O
+K81R	B-mutant
+in	O
+subunit	O
+β5	O
+that	O
+was	O
+present	O
+in	O
+the	O
+allele	O
+used	O
+in	O
+ref	O
+..	O
+This	O
+single	O
+amino	O
+-	O
+acid	O
+exchange	O
+is	O
+located	O
+at	O
+the	O
+interface	O
+of	O
+the	O
+subunits	O
+α4	O
+,	O
+β4	O
+and	O
+β5	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+)	O
+and	O
+might	O
+weakly	O
+promote	O
+CP	O
+assembly	O
+by	O
+enhancing	O
+inter	O
+-	O
+subunit	O
+contacts	O
+.	O
+	O
+The	O
+slightly	O
+better	O
+growth	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	O
+allowed	O
+us	O
+to	O
+solve	O
+the	O
+crystal	O
+structure	O
+of	O
+a	O
+yeast	O
+proteasome	O
+(	O
+yCP	O
+)	O
+with	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutation	O
+,	O
+which	O
+is	O
+discussed	O
+in	O
+the	O
+following	O
+section	O
+(	O
+for	O
+details	O
+see	O
+Supplementary	O
+Note	O
+1	O
+).	O
+	O
+Propeptide	O
+conformation	O
+and	O
+triggering	O
+of	O
+autolysis	O
+	O
+In	O
+the	O
+final	O
+steps	O
+of	O
+proteasome	O
+biogenesis	O
+,	O
+the	O
+propeptides	O
+are	O
+autocatalytically	O
+cleaved	O
+from	O
+the	O
+mature	O
+β	O
+-	O
+subunit	O
+domains	O
+.	O
+	O
+Furthermore	O
+,	O
+it	O
+was	O
+observed	O
+that	O
+the	O
+prosegment	O
+forms	O
+an	O
+antiparallel	O
+β	O
+-	O
+sheet	O
+in	O
+the	O
+active	O
+site	O
+,	O
+and	O
+that	O
+Gly	O
+(-	O
+1	O
+)	O
+adopts	O
+a	O
+γ	O
+-	O
+turn	O
+conformation	O
+,	O
+which	O
+by	O
+definition	O
+is	O
+characterized	O
+by	O
+a	O
+hydrogen	O
+bond	O
+between	O
+Leu	O
+(-	O
+2	O
+)	O
+O	O
+and	O
+Thr1NH	O
+(	O
+ref	O
+.).	O
+	O
+In	O
+subunit	O
+β1	O
+,	O
+we	O
+found	O
+that	O
+Gly	O
+(-	O
+1	O
+)	O
+indeed	O
+forms	O
+a	O
+sharp	O
+turn	O
+,	O
+which	O
+relaxes	O
+on	O
+prosegment	O
+cleavage	O
+(	O
+Fig	O
+.	O
+1a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+However	O
+,	O
+the	O
+γ	O
+-	O
+turn	O
+conformation	O
+and	O
+the	O
+associated	O
+hydrogen	O
+bond	O
+initially	O
+proposed	O
+is	O
+for	O
+geometric	O
+and	O
+chemical	O
+reasons	O
+inappropriate	O
+and	O
+would	O
+not	O
+perfectly	O
+position	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+Gly	O
+(-	O
+1	O
+)	O
+for	O
+nucleophilic	O
+attack	O
+by	O
+Thr1	O
+.	O
+	O
+Regarding	O
+the	O
+β2	O
+propeptide	O
+,	O
+Thr	O
+(-	O
+2	O
+)	O
+occupies	O
+the	O
+S1	O
+pocket	O
+but	O
+is	O
+less	O
+deeply	O
+anchored	O
+compared	O
+with	O
+Leu	O
+(-	O
+2	O
+)	O
+in	O
+β1	O
+,	O
+which	O
+might	O
+be	O
+due	O
+to	O
+the	O
+rather	O
+large	O
+β2	O
+-	O
+S1	O
+pocket	O
+created	O
+by	O
+Gly45	O
+.	O
+	O
+Next	O
+,	O
+we	O
+examined	O
+the	O
+position	O
+of	O
+the	O
+β5	O
+propeptide	O
+in	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	O
+.	O
+	O
+Surprisingly	O
+,	O
+Gly	O
+(-	O
+1	O
+)	O
+is	O
+completely	O
+extended	O
+and	O
+forces	O
+the	O
+histidine	O
+side	O
+chain	O
+at	O
+position	O
+(-	O
+2	O
+)	O
+to	O
+occupy	O
+the	O
+S2	O
+instead	O
+of	O
+the	O
+S1	O
+pocket	O
+,	O
+thereby	O
+disrupting	O
+the	O
+antiparallel	O
+β	O
+-	O
+sheet	O
+.	O
+	O
+Nonetheless	O
+,	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+Gly	O
+(-	O
+1	O
+)	O
+would	O
+be	O
+ideally	O
+placed	O
+for	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	O
+(	O
+Fig	O
+.	O
+1c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+,	O
+d	O
+).	O
+	O
+As	O
+the	O
+K81R	B-mutant
+mutation	O
+is	O
+located	O
+far	O
+from	O
+the	O
+active	O
+site	O
+(	O
+Thr1Cα	O
+–	O
+Arg81Cα	O
+:	O
+24	O
+Å	O
+),	O
+any	O
+influence	O
+on	O
+propeptide	O
+conformation	O
+can	O
+be	O
+excluded	O
+.	O
+	O
+Processing	O
+of	O
+β	O
+-	O
+subunit	O
+precursors	O
+requires	O
+deprotonation	O
+of	O
+Thr1OH	O
+;	O
+however	O
+,	O
+the	O
+general	O
+base	O
+initiating	O
+autolysis	O
+is	O
+unknown	O
+.	O
+	O
+Remarkably	O
+,	O
+eukaryotic	O
+proteasomal	O
+β5	O
+subunits	O
+bear	O
+a	O
+His	O
+residue	O
+in	O
+position	O
+(-	O
+2	O
+)	O
+of	O
+the	O
+propeptide	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+).	O
+	O
+Gly	O
+(-	O
+1	O
+)	O
+and	O
+Phe	O
+/	O
+Lys	O
+(-	O
+2	O
+)	O
+were	O
+visualized	O
+at	O
+low	O
+occupancy	O
+,	O
+while	O
+Ala	O
+/	O
+Asn	O
+(-	O
+2	O
+)	O
+could	O
+not	O
+be	O
+assigned	O
+.	O
+	O
+This	O
+observation	O
+indicates	O
+a	O
+mixture	O
+of	O
+processed	O
+and	O
+unprocessed	O
+β5	O
+subunits	O
+and	O
+partially	O
+impaired	O
+autolysis	O
+,	O
+thereby	O
+excluding	O
+any	O
+essential	O
+role	O
+of	O
+residue	O
+(-	O
+2	O
+)	O
+as	O
+the	O
+general	O
+base	O
+.	O
+	O
+Leu	O
+(-	O
+2	O
+)	O
+is	O
+encoded	O
+in	O
+the	O
+yeast	O
+β1	O
+subunit	O
+precursor	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+);	O
+Thr	O
+(-	O
+2	O
+)	O
+is	O
+generally	O
+part	O
+of	O
+β2	O
+-	O
+propeptides	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+);	O
+and	O
+Ala	O
+(-	O
+2	O
+)	O
+was	O
+expected	O
+to	O
+fit	O
+the	O
+β5	O
+-	O
+S1	O
+pocket	O
+without	O
+inducing	O
+conformational	O
+changes	O
+of	O
+Met45	O
+,	O
+allowing	O
+it	O
+to	O
+accommodate	O
+‘	O
+β1	O
+-	O
+like	O
+'	O
+propeptide	O
+positioning	O
+.	O
+	O
+As	O
+expected	O
+from	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutants	O
+,	O
+the	O
+yeasts	O
+show	O
+severe	O
+growth	O
+phenotypes	O
+,	O
+with	O
+minor	O
+variations	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+and	O
+Table	O
+1	O
+).	O
+	O
+We	O
+determined	O
+crystal	O
+structures	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+A	I-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutants	O
+(	O
+Supplementary	O
+Table	O
+1	O
+).	O
+	O
+For	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+A	I-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+variant	O
+,	O
+only	O
+the	O
+residues	O
+Gly	O
+(-	O
+1	O
+)	O
+and	O
+Ala	O
+(-	O
+2	O
+)	O
+could	O
+be	O
+visualized	O
+,	O
+indicating	O
+that	O
+Ala	O
+(-	O
+2	O
+)	O
+leads	O
+to	O
+insufficient	O
+stabilization	O
+of	O
+the	O
+propeptide	O
+in	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+).	O
+	O
+Nevertheless	O
+,	O
+both	O
+Leu	O
+(-	O
+2	O
+)	O
+and	O
+Thr	O
+(-	O
+2	O
+)	O
+were	O
+found	O
+to	O
+occupy	O
+the	O
+S1	O
+specificity	O
+pocket	O
+formed	O
+by	O
+Met45	O
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4f	O
+–	O
+h	O
+).	O
+	O
+Since	O
+Gly	O
+(-	O
+1	O
+)	O
+adopts	O
+the	O
+same	O
+position	O
+in	O
+both	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+and	O
+mutant	O
+β5	O
+propeptides	O
+,	O
+and	O
+since	O
+in	O
+all	O
+cases	O
+its	O
+carbonyl	O
+carbon	O
+is	O
+perfectly	O
+placed	O
+for	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	O
+(	O
+Fig	O
+.	O
+2b	O
+),	O
+we	O
+propose	O
+that	O
+neither	O
+binding	O
+of	O
+residue	O
+(-	O
+2	O
+)	O
+to	O
+the	O
+S1	O
+pocket	O
+nor	O
+formation	O
+of	O
+the	O
+antiparallel	O
+β	O
+-	O
+sheet	O
+is	O
+essential	O
+for	O
+autolysis	O
+of	O
+the	O
+propeptide	O
+.	O
+	O
+Next	O
+,	O
+we	O
+determined	O
+the	O
+crystal	O
+structure	O
+of	O
+a	O
+chimeric	O
+yCP	O
+having	O
+the	O
+yeast	O
+β1	O
+-	O
+propeptide	O
+replaced	O
+by	O
+its	O
+β5	O
+counterpart	O
+.	O
+	O
+Although	O
+we	O
+observed	O
+fragments	O
+of	O
+2FO	O
+–	O
+FC	O
+electron	O
+density	O
+in	O
+the	O
+β1	O
+active	O
+site	O
+,	O
+the	O
+data	O
+were	O
+not	O
+interpretable	O
+.	O
+	O
+As	O
+proven	O
+by	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+crystal	O
+structures	O
+,	O
+Thr	O
+(-	O
+2	O
+)	O
+hydrogen	O
+bonds	O
+to	O
+Gly	O
+(-	O
+1	O
+)	O
+O	O
+.	O
+Although	O
+this	O
+interaction	O
+was	O
+not	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	O
+(	O
+Fig	O
+.	O
+2c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4c	O
+,	O
+i	O
+),	O
+exchange	O
+of	O
+Thr	O
+(-	O
+2	O
+)	O
+by	O
+Val	O
+in	O
+β2	O
+,	O
+a	O
+conservative	O
+mutation	O
+regarding	O
+size	O
+but	O
+drastic	O
+with	O
+respect	O
+to	O
+polarity	O
+,	O
+was	O
+found	O
+to	O
+inhibit	O
+maturation	O
+of	O
+this	O
+subunit	O
+(	O
+Fig	O
+.	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4e	O
+,	O
+j	O
+).	O
+	O
+In	O
+particular	O
+,	O
+Val	O
+(-	O
+2	O
+)	O
+is	O
+displaced	O
+from	O
+the	O
+S1	O
+site	O
+and	O
+Gly	O
+(-	O
+1	O
+)	O
+is	O
+severely	O
+shifted	O
+(	O
+movement	O
+of	O
+the	O
+carbonyl	O
+oxygen	O
+atom	O
+of	O
+3	O
+.	O
+8	O
+Å	O
+),	O
+thereby	O
+preventing	O
+nucleophilic	O
+attack	O
+of	O
+Thr1	O
+(	O
+Fig	O
+.	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4j	O
+,	O
+k	O
+).	O
+	O
+These	O
+results	O
+further	O
+confirm	O
+that	O
+correct	O
+positioning	O
+of	O
+the	O
+active	O
+-	O
+site	O
+residues	O
+and	O
+Gly	O
+(-	O
+1	O
+)	O
+is	O
+decisive	O
+for	O
+the	O
+maturation	O
+of	O
+the	O
+proteasome	O
+.	O
+	O
+The	O
+active	O
+site	O
+of	O
+the	O
+proteasome	O
+	O
+Proton	O
+shuttling	O
+from	O
+the	O
+proteasomal	O
+active	O
+site	O
+Thr1OH	O
+to	O
+Thr1NH2	O
+via	O
+a	O
+nucleophilic	O
+water	O
+molecule	O
+was	O
+suggested	O
+to	O
+initiate	O
+peptide	O
+-	O
+bond	O
+hydrolysis	O
+.	O
+	O
+A	O
+proposed	O
+catalytic	O
+tetrad	O
+model	O
+involving	O
+Thr1OH	O
+,	O
+Thr1NH2	O
+,	O
+Lys33NH2	O
+and	O
+Asp17Oδ	O
+,	O
+as	O
+well	O
+as	O
+a	O
+nucleophilic	O
+water	O
+molecule	O
+as	O
+the	O
+proton	O
+shuttle	O
+appeared	O
+to	O
+accommodate	O
+all	O
+possible	O
+views	O
+of	O
+the	O
+proteasomal	O
+active	O
+site	O
+.	O
+	O
+Twenty	O
+years	O
+later	O
+,	O
+with	O
+a	O
+plethora	O
+of	O
+yCP	O
+X	O
+-	O
+ray	O
+structures	O
+in	O
+hand	O
+,	O
+we	O
+decided	O
+to	O
+re	O
+-	O
+analyse	O
+the	O
+active	O
+site	O
+of	O
+the	O
+proteasome	O
+and	O
+to	O
+resolve	O
+the	O
+uncertainty	O
+regarding	O
+the	O
+nature	O
+of	O
+the	O
+general	O
+base	O
+.	O
+	O
+Mutation	O
+of	O
+β5	O
+-	O
+Lys33	O
+to	O
+Ala	O
+causes	O
+a	O
+strongly	O
+deleterious	O
+phenotype	O
+,	O
+and	O
+previous	O
+structural	O
+and	O
+biochemical	O
+analyses	O
+confirmed	O
+that	O
+this	O
+is	O
+caused	O
+by	O
+failure	O
+of	O
+propeptide	O
+cleavage	O
+,	O
+and	O
+consequently	O
+,	O
+lack	O
+of	O
+ChT	O
+-	O
+L	O
+activity	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+and	O
+Table	O
+1	O
+;	O
+for	O
+details	O
+see	O
+Supplementary	O
+Note	O
+1	O
+).	O
+	O
+This	O
+discrepancy	O
+in	O
+growth	O
+was	O
+traced	O
+to	O
+an	O
+additional	O
+point	O
+mutation	O
+L	B-mutant
+(-	I-mutant
+49	I-mutant
+)	I-mutant
+S	I-mutant
+in	O
+the	O
+β5	O
+-	O
+propeptide	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+mutant	O
+(	O
+see	O
+also	O
+Supplementary	O
+Note	O
+1	O
+).	O
+	O
+This	O
+structural	O
+alteration	O
+destroys	O
+active	O
+-	O
+site	O
+integrity	O
+and	O
+abolishes	O
+catalytic	O
+activity	O
+of	O
+the	O
+β5	O
+active	O
+site	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+).	O
+	O
+Additional	O
+proof	O
+for	O
+the	O
+key	O
+function	O
+of	O
+Lys33	O
+was	O
+obtained	O
+from	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+mutant	O
+,	O
+with	O
+the	O
+propeptide	O
+expressed	O
+separately	O
+from	O
+the	O
+main	O
+subunit	O
+(	O
+pp	O
+trans	O
+).	O
+	O
+The	O
+Thr1	O
+N	O
+terminus	O
+of	O
+this	O
+mutant	O
+is	O
+not	O
+blocked	O
+by	O
+the	O
+propeptide	O
+,	O
+yet	O
+its	O
+catalytic	O
+activity	O
+is	O
+reduced	O
+by	O
+∼	O
+83	O
+%	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6b	O
+).	O
+	O
+Consistent	O
+with	O
+this	O
+,	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	O
+trans	O
+mutant	O
+in	O
+complex	O
+with	O
+carfilzomib	O
+only	O
+showed	O
+partial	O
+occupancy	O
+of	O
+the	O
+ligand	O
+at	O
+the	O
+β5	O
+active	O
+sites	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+and	O
+Supplementary	O
+Table	O
+1	O
+).	O
+	O
+Since	O
+no	O
+acetylation	O
+of	O
+the	O
+Thr1	O
+N	O
+terminus	O
+was	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	O
+trans	O
+apo	O
+crystal	O
+structure	O
+,	O
+the	O
+reduced	O
+reactivity	O
+towards	O
+substrates	O
+and	O
+inhibitors	O
+indicates	O
+that	O
+Lys33NH2	O
+,	O
+rather	O
+than	O
+Thr1NH2	O
+,	O
+deprotonates	O
+and	O
+activates	O
+Thr1OH	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	O
+trans	O
+mutant	O
+without	O
+inhibitor	O
+revealed	O
+that	O
+Thr1Oγ	O
+strongly	O
+coordinates	O
+a	O
+well	O
+-	O
+defined	O
+water	O
+molecule	O
+(∼	O
+2	O
+Å	O
+;	O
+Fig	O
+.	O
+3c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5c	O
+,	O
+d	O
+).	O
+	O
+Remarkably	O
+,	O
+the	O
+solvent	O
+molecule	O
+occupies	O
+the	O
+position	O
+normally	O
+taken	O
+by	O
+Lys33NH2	O
+in	O
+the	O
+WT	O
+proteasome	O
+structure	O
+(	O
+Fig	O
+.	O
+3c	O
+),	O
+further	O
+corroborating	O
+the	O
+essential	O
+role	O
+of	O
+Lys33	O
+as	O
+the	O
+general	O
+base	O
+for	O
+autolysis	O
+and	O
+proteolysis	O
+.	O
+	O
+Conservative	O
+substitution	O
+of	O
+Lys33	O
+by	O
+Arg	O
+delays	O
+autolysis	O
+of	O
+the	O
+β5	O
+precursor	O
+and	O
+impairs	O
+yeast	O
+growth	O
+(	O
+for	O
+details	O
+see	O
+Supplementary	O
+Note	O
+1	O
+).	O
+	O
+While	O
+Thr1	O
+occupies	O
+the	O
+same	O
+position	O
+as	O
+in	O
+WT	O
+yCPs	O
+,	O
+Arg33	O
+is	O
+unable	O
+to	O
+hydrogen	O
+bond	O
+to	O
+Asp17	O
+,	O
+thereby	O
+inactivating	O
+the	O
+β5	O
+active	O
+site	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5e	O
+).	O
+	O
+The	O
+conservative	O
+mutation	O
+of	O
+Asp17	O
+to	O
+Asn	O
+in	O
+subunit	O
+β5	O
+of	O
+the	O
+yCP	O
+also	O
+provokes	O
+a	O
+severe	O
+growth	O
+defect	O
+(	O
+Supplementary	O
+Note	O
+1	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+and	O
+Table	O
+1	O
+).	O
+	O
+Notably	O
+,	O
+only	O
+with	O
+the	O
+additional	O
+point	O
+mutation	O
+L	B-mutant
+(-	I-mutant
+49	I-mutant
+)	I-mutant
+S	I-mutant
+present	O
+in	O
+the	O
+β5	O
+propeptide	O
+could	O
+we	O
+purify	O
+a	O
+small	O
+amount	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+mutant	O
+yCP	O
+.	O
+	O
+As	O
+determined	O
+by	O
+crystallographic	O
+analysis	O
+,	O
+this	O
+mutant	O
+β5	O
+subunit	O
+was	O
+partially	O
+processed	O
+(	O
+Table	O
+1	O
+)	O
+but	O
+displayed	O
+impaired	O
+reactivity	O
+towards	O
+the	O
+proteasome	O
+inhibitor	O
+carfilzomib	O
+compared	O
+with	O
+the	O
+subunits	O
+β1	O
+and	O
+β2	O
+,	O
+and	O
+with	O
+WT	O
+β5	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+	O
+Even	O
+though	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+pp	O
+trans	O
+yCP	O
+crystal	O
+structure	O
+appeared	O
+identical	O
+to	O
+the	O
+WT	O
+yCP	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7b	O
+),	O
+the	O
+co	O
+-	O
+crystal	O
+structure	O
+with	O
+the	O
+α	O
+′,	O
+β	O
+′	O
+epoxyketone	O
+inhibitor	O
+carfilzomib	O
+visualized	O
+only	O
+partial	O
+occupancy	O
+of	O
+the	O
+ligand	O
+in	O
+the	O
+β5	O
+active	O
+site	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+	O
+Autolysis	O
+and	O
+residual	O
+catalytic	O
+activity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+mutants	O
+may	O
+originate	O
+from	O
+the	O
+carbonyl	O
+group	O
+of	O
+Asn17	O
+,	O
+which	O
+albeit	O
+to	O
+a	O
+lower	O
+degree	O
+still	O
+can	O
+polarize	O
+Lys33	O
+for	O
+the	O
+activation	O
+of	O
+Thr1	O
+.	O
+	O
+In	O
+agreement	O
+,	O
+an	O
+E17A	B-mutant
+mutant	O
+in	O
+the	O
+proteasomal	O
+β	O
+-	O
+subunit	O
+of	O
+the	O
+archaeon	O
+Thermoplasma	O
+acidophilum	O
+prevents	O
+autolysis	O
+and	O
+catalysis	O
+.	O
+	O
+On	O
+the	O
+basis	O
+of	O
+these	O
+results	O
+,	O
+we	O
+propose	O
+that	O
+CPs	O
+from	O
+all	O
+domains	O
+of	O
+life	O
+use	O
+a	O
+catalytic	O
+triad	O
+consisting	O
+of	O
+Thr1	O
+,	O
+Lys33	O
+and	O
+Asp	O
+/	O
+Glu17	O
+for	O
+both	O
+autocatalytic	O
+precursor	O
+processing	O
+and	O
+proteolysis	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+	O
+This	O
+model	O
+is	O
+also	O
+consistent	O
+with	O
+the	O
+fact	O
+that	O
+no	O
+defined	O
+water	O
+molecule	O
+is	O
+observed	O
+in	O
+the	O
+mature	O
+WT	O
+proteasomal	O
+active	O
+site	O
+that	O
+could	O
+shuttle	O
+the	O
+proton	O
+from	O
+Thr1Oγ	O
+to	O
+Thr1NH2	O
+.	O
+	O
+To	O
+explore	O
+this	O
+active	O
+-	O
+site	O
+model	O
+further	O
+,	O
+we	O
+exchanged	O
+the	O
+conserved	O
+Asp166	O
+residue	O
+for	O
+Asn	O
+in	O
+the	O
+yeast	O
+β5	O
+subunit	O
+.	O
+	O
+X	O
+-	O
+ray	O
+data	O
+on	O
+the	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+mutant	O
+indicate	O
+that	O
+the	O
+β5	O
+propeptide	O
+is	O
+hydrolysed	O
+,	O
+but	O
+due	O
+to	O
+reorientation	O
+of	O
+Ser129OH	O
+,	O
+the	O
+interaction	O
+with	O
+Asn166Oδ	O
+is	O
+disrupted	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8a	O
+).	O
+	O
+Soaking	O
+the	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+crystals	O
+with	O
+carfilzomib	O
+and	O
+MG132	O
+resulted	O
+in	O
+covalent	O
+modification	O
+of	O
+Thr1	O
+at	O
+high	O
+occupancy	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+	O
+In	O
+the	O
+MG132	O
+-	O
+bound	O
+state	O
+,	O
+Thr1N	O
+is	O
+unmodified	O
+,	O
+and	O
+we	O
+again	O
+observe	O
+that	O
+Ser129	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+a	O
+water	O
+molecule	O
+instead	O
+of	O
+Asn166	O
+.	O
+	O
+Substitution	O
+of	O
+the	O
+active	O
+-	O
+site	O
+Thr1	O
+by	O
+Cys	O
+	O
+Mutation	O
+of	O
+Thr1	O
+to	O
+Cys	O
+inactivates	O
+the	O
+20S	O
+proteasome	O
+from	O
+the	O
+archaeon	O
+T	O
+.	O
+acidophilum	O
+.	O
+	O
+In	O
+yeast	O
+,	O
+this	O
+mutation	O
+causes	O
+a	O
+strong	O
+growth	O
+defect	O
+(	O
+Fig	O
+.	O
+4a	O
+and	O
+Table	O
+1	O
+),	O
+although	O
+the	O
+propeptide	O
+is	O
+hydrolysed	O
+,	O
+as	O
+shown	O
+here	O
+by	O
+its	O
+X	O
+-	O
+ray	O
+structure	O
+.	O
+	O
+In	O
+one	O
+of	O
+the	O
+two	O
+β5	O
+subunits	O
+,	O
+however	O
+,	O
+we	O
+found	O
+the	O
+cleaved	O
+propeptide	O
+still	O
+bound	O
+in	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+	O
+His	O
+(-	O
+2	O
+)	O
+occupies	O
+the	O
+S2	O
+pocket	O
+like	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	O
+,	O
+but	O
+in	O
+contrast	O
+to	O
+the	O
+latter	O
+,	O
+the	O
+propeptide	O
+in	O
+the	O
+T1C	B-mutant
+mutant	O
+adopts	O
+an	O
+antiparallel	O
+β	O
+-	O
+sheet	O
+conformation	O
+as	O
+known	O
+from	O
+inhibitors	O
+like	O
+MG132	O
+(	O
+Fig	O
+.	O
+4c	O
+–	O
+e	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9b	O
+).	O
+	O
+On	O
+the	O
+basis	O
+of	O
+the	O
+phenotype	O
+of	O
+the	O
+T1C	B-mutant
+mutant	O
+and	O
+the	O
+propeptide	O
+remnant	O
+identified	O
+in	O
+its	O
+active	O
+site	O
+,	O
+we	O
+suppose	O
+that	O
+autolysis	O
+is	O
+retarded	O
+and	O
+may	O
+not	O
+have	O
+been	O
+completed	O
+before	O
+crystallization	O
+.	O
+	O
+Despite	O
+propeptide	O
+hydrolysis	O
+,	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+active	O
+site	O
+is	O
+catalytically	O
+inactive	O
+(	O
+Fig	O
+.	O
+4b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9a	O
+).	O
+	O
+Moreover	O
+,	O
+the	O
+structural	O
+data	O
+reveal	O
+that	O
+the	O
+thiol	O
+group	O
+of	O
+Cys1	O
+is	O
+rotated	O
+by	O
+74	O
+°	O
+with	O
+respect	O
+to	O
+the	O
+hydroxyl	O
+side	O
+chain	O
+of	O
+Thr1	O
+(	O
+Fig	O
+.	O
+4f	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9b	O
+).	O
+	O
+Consequently	O
+,	O
+the	O
+hydrogen	O
+bond	O
+bridging	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+and	O
+Lys33	O
+in	O
+WT	O
+CPs	O
+is	O
+broken	O
+with	O
+Cys1	O
+.	O
+	O
+Notably	O
+,	O
+the	O
+2FO	O
+–	O
+FC	O
+electron	O
+-	O
+density	O
+map	O
+of	O
+the	O
+T1C	B-mutant
+mutant	O
+also	O
+indicates	O
+that	O
+Lys33NH2	O
+is	O
+disordered	O
+.	O
+	O
+The	O
+benefit	O
+of	O
+Thr	O
+over	O
+Ser	O
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+	O
+All	O
+proteasomes	O
+strictly	O
+employ	O
+threonine	O
+as	O
+the	O
+active	O
+-	O
+site	O
+residue	O
+instead	O
+of	O
+serine	O
+.	O
+	O
+To	O
+investigate	O
+the	O
+reason	O
+for	O
+this	O
+singularity	O
+,	O
+we	O
+analysed	O
+a	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	O
+,	O
+which	O
+is	O
+viable	O
+but	O
+suffers	O
+from	O
+growth	O
+defects	O
+(	O
+Fig	O
+.	O
+4a	O
+and	O
+Table	O
+1	O
+).	O
+	O
+By	O
+contrast	O
+,	O
+turnover	O
+of	O
+the	O
+substrate	O
+Z	O
+-	O
+GGL	O
+-	O
+pNA	O
+,	O
+used	O
+to	O
+monitor	O
+ChT	O
+-	O
+L	O
+activity	O
+in	O
+situ	O
+but	O
+in	O
+a	O
+less	O
+quantitative	O
+fashion	O
+,	O
+is	O
+not	O
+detectably	O
+impaired	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+9a	O
+).	O
+	O
+Crystal	O
+structure	O
+analysis	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	O
+confirmed	O
+precursor	O
+processing	O
+(	O
+Fig	O
+.	O
+4g	O
+),	O
+and	O
+ligand	O
+-	O
+complex	O
+structures	O
+with	O
+bortezomib	O
+and	O
+carfilzomib	O
+unambiguously	O
+corroborated	O
+the	O
+reactivity	O
+of	O
+Ser1	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+However	O
+,	O
+the	O
+apo	O
+crystal	O
+structure	O
+revealed	O
+that	O
+Ser1Oγ	O
+is	O
+turned	O
+away	O
+from	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+(	O
+Fig	O
+.	O
+4g	O
+).	O
+	O
+Because	O
+both	O
+conformations	O
+of	O
+Ser1Oγ	O
+are	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Lys33NH2	O
+(	O
+Fig	O
+.	O
+4h	O
+),	O
+the	O
+relay	O
+system	O
+is	O
+capable	O
+of	O
+hydrolysing	O
+peptide	O
+substrates	O
+,	O
+albeit	O
+at	O
+lower	O
+rates	O
+compared	O
+with	O
+Thr1	O
+.	O
+	O
+The	O
+active	O
+-	O
+site	O
+residue	O
+Thr1	O
+is	O
+fixed	O
+in	O
+its	O
+position	O
+,	O
+as	O
+its	O
+methyl	O
+group	O
+is	O
+engaged	O
+in	O
+hydrophobic	O
+interactions	O
+with	O
+Thr3	O
+and	O
+Ala46	O
+(	O
+Fig	O
+.	O
+4h	O
+).	O
+	O
+Consequently	O
+,	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Thr1	O
+requires	O
+no	O
+reorientation	O
+before	O
+substrate	O
+cleavage	O
+and	O
+is	O
+thus	O
+more	O
+catalytically	O
+efficient	O
+than	O
+Ser1	O
+.	O
+	O
+In	O
+vitro	O
+,	O
+the	O
+mutant	O
+proteasome	O
+is	O
+less	O
+susceptible	O
+to	O
+proteasome	O
+inhibition	O
+by	O
+bortezomib	O
+(	O
+3	O
+.	O
+7	O
+-	O
+fold	O
+)	O
+and	O
+carfilzomib	O
+(	O
+1	O
+.	O
+8	O
+-	O
+fold	O
+;	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+Nevertheless	O
+,	O
+inhibitor	O
+complex	O
+structures	O
+indicate	O
+identical	O
+binding	O
+modes	O
+compared	O
+with	O
+the	O
+WT	O
+yCP	O
+structures	O
+,	O
+with	O
+the	O
+same	O
+inhibitors	O
+.	O
+	O
+Notably	O
+,	O
+the	O
+affinity	O
+of	O
+the	O
+tetrapeptide	O
+carfilzomib	O
+is	O
+less	O
+impaired	O
+,	O
+as	O
+it	O
+is	O
+better	O
+stabilized	O
+in	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+than	O
+the	O
+dipeptide	O
+bortezomib	O
+,	O
+which	O
+lacks	O
+a	O
+defined	O
+P3	O
+site	O
+and	O
+has	O
+only	O
+a	O
+few	O
+interactions	O
+with	O
+the	O
+surrounding	O
+protein	O
+.	O
+	O
+Considered	O
+together	O
+,	O
+these	O
+results	O
+provide	O
+a	O
+plausible	O
+explanation	O
+for	O
+the	O
+invariance	O
+of	O
+threonine	O
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+in	O
+proteasomes	O
+in	O
+all	O
+three	O
+domains	O
+of	O
+life	O
+,	O
+as	O
+well	O
+as	O
+in	O
+proteasome	O
+-	O
+like	O
+proteases	O
+such	O
+as	O
+HslV	O
+(	O
+ref	O
+.).	O
+	O
+The	O
+β	O
+-	O
+subunit	O
+propeptides	O
+,	O
+particularly	O
+that	O
+of	O
+β5	O
+,	O
+are	O
+key	O
+factors	O
+that	O
+help	O
+drive	O
+proper	O
+assembly	O
+of	O
+the	O
+CP	O
+complex	O
+.	O
+	O
+By	O
+contrast	O
+,	O
+the	O
+prosegments	O
+of	O
+β	O
+subunits	O
+are	O
+dispensable	O
+for	O
+archaeal	O
+proteasome	O
+assembly	O
+,	O
+at	O
+least	O
+when	O
+heterologously	O
+expressed	O
+in	O
+Escherichia	O
+coli	O
+.	O
+	O
+In	O
+eukaryotes	O
+,	O
+deletion	O
+of	O
+or	O
+failure	O
+to	O
+cleave	O
+the	O
+β1	O
+and	O
+β2	O
+propeptides	O
+is	O
+well	O
+tolerated	O
+.	O
+	O
+These	O
+observations	O
+highlight	O
+the	O
+unique	O
+function	O
+and	O
+importance	O
+of	O
+the	O
+β5	O
+propeptide	O
+as	O
+well	O
+as	O
+the	O
+β5	O
+active	O
+site	O
+for	O
+maturation	O
+and	O
+function	O
+of	O
+the	O
+eukaryotic	O
+CP	O
+.	O
+	O
+Here	O
+we	O
+have	O
+described	O
+the	O
+atomic	O
+structures	O
+of	O
+various	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutants	O
+,	O
+which	O
+allowed	O
+for	O
+the	O
+first	O
+time	O
+visualization	O
+of	O
+the	O
+residual	O
+β5	O
+propeptide	O
+.	O
+	O
+From	O
+these	O
+data	O
+we	O
+conclude	O
+that	O
+only	O
+the	O
+positioning	O
+of	O
+Gly	O
+(-	O
+1	O
+)	O
+and	O
+Thr1	O
+as	O
+well	O
+as	O
+the	O
+integrity	O
+of	O
+the	O
+proteasomal	O
+active	O
+site	O
+are	O
+required	O
+for	O
+autolysis	O
+.	O
+	O
+In	O
+this	O
+regard	O
+,	O
+inappropriate	O
+N	O
+-	O
+acetylation	O
+of	O
+the	O
+Thr1	O
+N	O
+terminus	O
+cannot	O
+be	O
+removed	O
+by	O
+Thr1Oγ	O
+due	O
+to	O
+the	O
+rotational	O
+freedom	O
+and	O
+flexibility	O
+of	O
+the	O
+acetyl	O
+group	O
+.	O
+	O
+The	O
+propeptide	O
+needs	O
+some	O
+anchoring	O
+in	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+to	O
+properly	O
+position	O
+Gly	O
+(-	O
+1	O
+),	O
+but	O
+this	O
+seems	O
+to	O
+be	O
+independent	O
+of	O
+the	O
+orientation	O
+of	O
+residue	O
+(-	O
+2	O
+).	O
+	O
+Autolytic	O
+activation	O
+of	O
+the	O
+CP	O
+constitutes	O
+one	O
+of	O
+the	O
+final	O
+steps	O
+of	O
+proteasome	O
+biogenesis	O
+,	O
+but	O
+the	O
+trigger	O
+for	O
+propeptide	O
+cleavage	O
+had	O
+remained	O
+enigmatic	O
+.	O
+	O
+Thus	O
+,	O
+specific	O
+protein	O
+surroundings	O
+can	O
+significantly	O
+alter	O
+the	O
+chemical	O
+properties	O
+of	O
+amino	O
+acids	O
+such	O
+as	O
+Lys	O
+to	O
+function	O
+as	O
+an	O
+acid	O
+–	O
+base	O
+catalyst	O
+.	O
+	O
+Consistent	O
+with	O
+this	O
+model	O
+,	O
+the	O
+positively	O
+charged	O
+Thr1	O
+N	O
+terminus	O
+is	O
+engaged	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+inhibitory	O
+compounds	O
+like	O
+fellutamide	O
+B	O
+(	O
+ref	O
+.),	O
+α	O
+-	O
+ketoamides	O
+,	O
+homobelactosin	O
+C	O
+(	O
+ref	O
+.)	O
+and	O
+salinosporamide	O
+A	O
+(	O
+ref	O
+.).	O
+	O
+Furthermore	O
+,	O
+opening	O
+of	O
+the	O
+β	O
+-	O
+lactone	O
+compound	O
+omuralide	O
+by	O
+Thr1	O
+creates	O
+a	O
+C3	O
+-	O
+hydroxyl	O
+group	O
+,	O
+whose	O
+proton	O
+originates	O
+from	O
+Thr1NH3	O
++.	O
+	O
+The	O
+resulting	O
+uncharged	O
+Thr1NH2	O
+is	O
+hydrogen	O
+-	O
+bridged	O
+to	O
+the	O
+C3	O
+-	O
+OH	O
+group	O
+.	O
+	O
+In	O
+agreement	O
+,	O
+acetylation	O
+of	O
+the	O
+Thr1	O
+N	O
+terminus	O
+irreversibly	O
+blocks	O
+hydrolytic	O
+activity	O
+,	O
+and	O
+binding	O
+of	O
+substrates	O
+is	O
+prevented	O
+for	O
+steric	O
+reasons	O
+.	O
+	O
+By	O
+acting	O
+as	O
+a	O
+proton	O
+donor	O
+during	O
+catalysis	O
+,	O
+the	O
+Thr1	O
+N	O
+terminus	O
+may	O
+also	O
+favour	O
+cleavage	O
+of	O
+substrate	O
+peptide	O
+bonds	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+	O
+During	O
+autolysis	O
+the	O
+Thr1	O
+N	O
+terminus	O
+is	O
+engaged	O
+in	O
+a	O
+hydroxyoxazolidine	O
+ring	O
+intermediate	O
+(	O
+Fig	O
+.	O
+3d	O
+),	O
+which	O
+is	O
+unstable	O
+and	O
+short	O
+-	O
+lived	O
+.	O
+	O
+The	O
+mutation	O
+D166N	B-mutant
+lowers	O
+the	O
+pKa	O
+of	O
+Thr1N	O
+,	O
+which	O
+is	O
+thus	O
+more	O
+likely	O
+to	O
+exist	O
+in	O
+the	O
+uncharged	O
+deprotonated	O
+state	O
+(	O
+Thr1NH2	O
+).	O
+	O
+Hence	O
+,	O
+the	O
+proteasome	O
+can	O
+be	O
+viewed	O
+as	O
+having	O
+a	O
+second	O
+triad	O
+that	O
+is	O
+essential	O
+for	O
+efficient	O
+proteolysis	O
+.	O
+	O
+However	O
+,	O
+owing	O
+to	O
+Cys	O
+being	O
+a	O
+strong	O
+nucleophile	O
+,	O
+the	O
+propeptide	O
+can	O
+still	O
+be	O
+cleaved	O
+off	O
+over	O
+time	O
+.	O
+	O
+While	O
+only	O
+one	O
+single	O
+turnover	O
+is	O
+necessary	O
+for	O
+autolysis	O
+,	O
+continuous	O
+enzymatic	O
+activity	O
+is	O
+required	O
+for	O
+significant	O
+and	O
+detectable	O
+substrate	O
+hydrolysis	O
+.	O
+	O
+Notably	O
+,	O
+in	O
+the	O
+Ntn	O
+hydrolase	O
+penicillin	O
+acylase	O
+,	O
+substitution	O
+of	O
+the	O
+catalytic	O
+N	O
+-	O
+terminal	O
+Ser	O
+residue	O
+by	O
+Cys	O
+also	O
+inactivates	O
+the	O
+enzyme	O
+but	O
+still	O
+enables	O
+precursor	O
+processing	O
+.	O
+	O
+To	O
+investigate	O
+why	O
+the	O
+CP	O
+specifically	O
+employs	O
+threonine	O
+as	O
+its	O
+active	O
+-	O
+site	O
+residue	O
+,	O
+we	O
+used	O
+a	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	O
+of	O
+the	O
+yCP	O
+and	O
+characterized	O
+it	O
+biochemically	O
+and	O
+structurally	O
+.	O
+	O
+Activity	O
+assays	O
+with	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	O
+revealed	O
+reduced	O
+turnover	O
+of	O
+Suc	O
+-	O
+LLVY	O
+-	O
+AMC	O
+.	O
+	O
+We	O
+also	O
+observed	O
+slightly	O
+lower	O
+affinity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	O
+yCP	O
+for	O
+the	O
+Food	O
+and	O
+Drug	O
+Administration	O
+-	O
+approved	O
+proteasome	O
+inhibitors	O
+bortezomib	O
+and	O
+carfilzomib	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+Thr1	O
+,	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Ser1	O
+occupies	O
+the	O
+position	O
+of	O
+the	O
+Thr1	O
+methyl	O
+side	O
+chain	O
+in	O
+the	O
+WT	O
+enzyme	O
+,	O
+which	O
+requires	O
+its	O
+reorientation	O
+relative	O
+to	O
+the	O
+substrate	O
+to	O
+allow	O
+cleavage	O
+(	O
+Fig	O
+.	O
+4g	O
+,	O
+h	O
+).	O
+	O
+Similarly	O
+,	O
+although	O
+the	O
+serine	O
+mutant	O
+is	O
+active	O
+,	O
+threonine	O
+is	O
+more	O
+efficient	O
+in	O
+the	O
+context	O
+of	O
+the	O
+proteasome	O
+active	O
+site	O
+.	O
+	O
+The	O
+greater	O
+suitability	O
+of	O
+threonine	O
+for	O
+the	O
+proteasome	O
+active	O
+site	O
+,	O
+which	O
+has	O
+been	O
+noted	O
+in	O
+biochemical	O
+as	O
+well	O
+as	O
+in	O
+kinetic	O
+studies	O
+,	O
+constitutes	O
+a	O
+likely	O
+reason	O
+for	O
+the	O
+conservation	O
+of	O
+the	O
+Thr1	O
+residue	O
+in	O
+all	O
+proteasomes	O
+from	O
+bacteria	O
+to	O
+eukaryotes	O
+.	O
+	O
+Conformation	O
+of	O
+proteasomal	O
+propeptides	O
+.	O
+	O
+(	O
+a	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	O
+and	O
+the	O
+matured	O
+WT	O
+β1	O
+active	O
+-	O
+site	O
+Thr1	O
+.	O
+	O
+Only	O
+the	O
+residues	O
+(-	O
+5	O
+)	O
+to	O
+(-	O
+1	O
+)	O
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	O
+are	O
+displayed	O
+.	O
+	O
+(	O
+b	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	O
+and	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	O
+highlights	O
+subtle	O
+differences	O
+in	O
+their	O
+conformations	O
+,	O
+but	O
+illustrates	O
+that	O
+Ala1	O
+and	O
+Gly	O
+(-	O
+1	O
+)	O
+match	O
+well	O
+.	O
+	O
+Thr	O
+(-	O
+2	O
+)	O
+OH	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Gly	O
+(-	O
+1	O
+)	O
+O	O
+(∼	O
+2	O
+.	O
+8	O
+Å	O
+;	O
+black	O
+dashed	O
+line	O
+).	O
+	O
+(	O
+c	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+propeptide	O
+remnants	O
+depict	O
+their	O
+differences	O
+in	O
+conformation	O
+.	O
+	O
+Nonetheless	O
+,	O
+in	O
+all	O
+mutants	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+Gly	O
+(-	O
+1	O
+)	O
+is	O
+ideally	O
+placed	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	O
+.	O
+	O
+The	O
+hydrogen	O
+bond	O
+between	O
+Thr	O
+(-	O
+2	O
+)	O
+OH	O
+and	O
+Gly	O
+(-	O
+1	O
+)	O
+O	O
+(∼	O
+2	O
+.	O
+8	O
+Å	O
+)	O
+is	O
+indicated	O
+by	O
+a	O
+black	O
+dashed	O
+line	O
+.	O
+	O
+Mutations	O
+of	O
+residue	O
+(-	O
+2	O
+)	O
+and	O
+their	O
+influence	O
+on	O
+propeptide	O
+conformation	O
+and	O
+autolysis	O
+.	O
+	O
+(	O
+a	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	O
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	O
+propeptide	O
+.	O
+	O
+The	O
+(-	O
+2	O
+)	O
+residues	O
+of	O
+both	O
+prosegments	O
+point	O
+into	O
+the	O
+S1	O
+pocket	O
+.	O
+	O
+(	O
+c	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	O
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	O
+propeptide	O
+.	O
+	O
+Notably	O
+,	O
+Val	O
+(-	O
+2	O
+)	O
+of	O
+the	O
+latter	O
+does	O
+not	O
+occupy	O
+the	O
+S1	O
+pocket	O
+,	O
+thereby	O
+changing	O
+the	O
+orientation	O
+of	O
+Gly	O
+(-	O
+1	O
+)	O
+and	O
+preventing	O
+nucleophilic	O
+attack	O
+of	O
+Thr1Oγ	O
+on	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+Gly	O
+(-	O
+1	O
+).	O
+	O
+Architecture	O
+and	O
+proposed	O
+reaction	O
+mechanism	O
+of	O
+the	O
+proteasomal	O
+active	O
+site	O
+.	O
+	O
+(	O
+a	O
+)	O
+Hydrogen	O
+-	O
+bonding	O
+network	O
+at	O
+the	O
+mature	O
+WT	O
+β5	O
+proteasomal	O
+active	O
+site	O
+(	O
+dotted	O
+lines	O
+).	O
+	O
+Thr1OH	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Lys33NH2	O
+(	O
+2	O
+.	O
+7	O
+Å	O
+),	O
+which	O
+in	O
+turn	O
+interacts	O
+with	O
+Asp17Oδ	O
+.	O
+	O
+(	O
+c	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+WT	O
+β5	O
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	O
+trans	O
+mutant	O
+active	O
+site	O
+.	O
+	O
+Similarly	O
+to	O
+Lys33	O
+,	O
+the	O
+water	O
+molecule	O
+hydrogen	O
+bonds	O
+to	O
+Arg19O	O
+,	O
+Asp17Oδ	O
+and	O
+Thr1OH	O
+.	O
+	O
+(	O
+d	O
+)	O
+Proposed	O
+chemical	O
+reaction	O
+mechanism	O
+for	O
+autocatalytic	O
+precursor	O
+processing	O
+and	O
+proteolysis	O
+in	O
+the	O
+proteasome	O
+.	O
+	O
+The	O
+active	O
+-	O
+site	O
+Thr1	O
+is	O
+depicted	O
+in	O
+blue	O
+,	O
+the	O
+propeptide	O
+segment	O
+and	O
+the	O
+peptide	O
+substrate	O
+are	O
+coloured	O
+in	O
+green	O
+,	O
+whereas	O
+the	O
+scissile	O
+peptide	O
+bond	O
+is	O
+highlighted	O
+in	O
+red	O
+.	O
+	O
+Autolysis	O
+(	O
+left	O
+set	O
+of	O
+structures	O
+)	O
+is	O
+initiated	O
+by	O
+deprotonation	O
+of	O
+Thr1OH	O
+via	O
+Lys33NH2	O
+and	O
+the	O
+formation	O
+of	O
+a	O
+tetrahedral	O
+transition	O
+state	O
+.	O
+	O
+Collapse	O
+of	O
+the	O
+transition	O
+state	O
+frees	O
+the	O
+Thr1	O
+N	O
+terminus	O
+(	O
+by	O
+completing	O
+an	O
+N	O
+-	O
+to	O
+-	O
+O	O
+acyl	O
+shift	O
+of	O
+the	O
+propeptide	O
+),	O
+which	O
+is	O
+subsequently	O
+protonated	O
+by	O
+Asp166OH	O
+via	O
+Ser129OH	O
+.	O
+	O
+The	O
+resulting	O
+deprotonated	O
+Thr1NH2	O
+finally	O
+activates	O
+a	O
+water	O
+molecule	O
+for	O
+hydrolysis	O
+of	O
+the	O
+acyl	O
+-	O
+enzyme	O
+.	O
+	O
+(	O
+b	O
+)	O
+Purified	O
+WT	O
+and	O
+mutant	O
+proteasomes	O
+were	O
+tested	O
+for	O
+their	O
+chymotrypsin	O
+-	O
+like	O
+activity	O
+(	O
+β5	O
+)	O
+using	O
+the	O
+substrate	O
+Suc	O
+-	O
+LLVY	O
+-	O
+AMC	O
+.	O
+	O
+The	O
+prosegment	O
+is	O
+cleaved	O
+but	O
+still	O
+bound	O
+in	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+.	O
+	O
+Notably	O
+,	O
+His	O
+(-	O
+2	O
+)	O
+does	O
+not	O
+occupy	O
+the	O
+S1	O
+pocket	O
+formed	O
+by	O
+Met45	O
+,	O
+similar	O
+to	O
+what	O
+was	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	O
+.	O
+	O
+(	O
+d	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+mutant	O
+subunits	O
+onto	O
+the	O
+WT	O
+β5	O
+subunit	O
+.	O
+(	O
+e	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+propeptide	O
+onto	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+active	O
+site	O
+(	O
+blue	O
+)	O
+and	O
+the	O
+WT	O
+β5	O
+active	O
+site	O
+in	O
+complex	O
+with	O
+the	O
+proteasome	O
+inhibitor	O
+MG132	O
+(	O
+ref	O
+.).	O
+	O
+The	O
+inhibitor	O
+as	O
+well	O
+as	O
+the	O
+propeptides	O
+adopt	O
+similar	O
+conformations	O
+in	O
+the	O
+substrate	O
+-	O
+binding	O
+channel	O
+.	O
+	O
+(	O
+f	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+WT	O
+β5	O
+and	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+mutant	O
+active	O
+sites	O
+illustrates	O
+the	O
+different	O
+orientations	O
+of	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Thr1	O
+and	O
+the	O
+thiol	O
+side	O
+chain	O
+of	O
+Cys1	O
+.	O
+	O
+(	O
+g	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+WT	O
+β5	O
+and	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	O
+active	O
+sites	O
+reveals	O
+different	O
+orientations	O
+of	O
+the	O
+hydroxyl	O
+groups	O
+of	O
+Thr1	O
+and	O
+Ser1	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+2FO	O
+–	O
+FC	O
+electron	O
+-	O
+density	O
+map	O
+for	O
+Ser1	O
+(	O
+blue	O
+mesh	O
+contoured	O
+at	O
+1σ	O
+)	O
+is	O
+illustrated	O
+.	O
+	O
+(	O
+h	O
+)	O
+The	O
+methyl	O
+group	O
+of	O
+Thr1	O
+is	O
+anchored	O
+by	O
+hydrophobic	O
+interactions	O
+with	O
+Ala46Cβ	O
+and	O
+Thr3Cγ	O
+.	O
+	O
+Inhibition	O
+of	O
+WT	O
+and	O
+mutant	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+proteasomes	O
+by	O
+bortezomib	O
+and	O
+carfilzomib	O
+.	O
+	O
+Purified	O
+yeast	O
+proteasomes	O
+were	O
+tested	O
+for	O
+the	O
+susceptibility	O
+of	O
+their	O
+ChT	O
+-	O
+L	O
+(	O
+β5	O
+)	O
+activity	O
+to	O
+inhibition	O
+by	O
+bortezomib	O
+and	O
+carfilzomib	O
+using	O
+the	O
+substrate	O
+Suc	O
+-	O
+LLVY	O
+-	O
+AMC	O
+.	O
+	O
+IC50	O
+values	O
+were	O
+determined	O
+in	O
+triplicate	O
+;	O
+s	O
+.	O
+d	O
+.'	O
+s	O
+are	O
+indicated	O
+by	O
+error	O
+bars	O
+.	O
+	O
+Note	O
+that	O
+IC50	O
+values	O
+depend	O
+on	O
+time	O
+and	O
+enzyme	O
+concentration	O
+.	O
+	O
+Proteasomes	O
+(	O
+final	O
+concentration	O
+:	O
+66	O
+nM	O
+)	O
+were	O
+incubated	O
+with	O
+inhibitor	O
+for	O
+45	O
+min	O
+before	O
+substrate	O
+addition	O
+(	O
+final	O
+concentration	O
+:	O
+200	O
+μM	O
+).	O
+	O
+Structures	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	O
+in	O
+complex	O
+with	O
+both	O
+ligands	O
+(	O
+green	O
+)	O
+prove	O
+the	O
+reactivity	O
+of	O
+Ser1	O
+(	O
+right	O
+panel	O
+).	O
+	O
+The	O
+WT	O
+proteasome	O
+:	O
+inhibitor	O
+complex	O
+structures	O
+(	O
+inhibitor	O
+in	O
+grey	O
+;	O
+Thr1	O
+in	O
+black	O
+)	O
+are	O
+superimposed	O
+and	O
+demonstrate	O
+that	O
+mutation	O
+of	O
+Thr1	O
+to	O
+Ser	O
+does	O
+not	O
+affect	O
+the	O
+binding	O
+mode	O
+of	O
+bortezomib	O
+or	O
+carfilzomib	O
+.	O
+	O
+The	O
+discovery	O
+of	O
+new	O
+histone	O
+modifications	O
+is	O
+unfolding	O
+at	O
+startling	O
+rates	O
+,	O
+however	O
+,	O
+the	O
+identification	O
+of	O
+effectors	O
+capable	O
+of	O
+interpreting	O
+these	O
+modifications	O
+has	O
+lagged	O
+behind	O
+.	O
+	O
+Here	O
+we	O
+report	O
+the	O
+YEATS	O
+domain	O
+as	O
+an	O
+effective	O
+reader	O
+of	O
+histone	O
+lysine	O
+crotonylation	O
+–	O
+an	O
+epigenetic	O
+signature	O
+associated	O
+with	O
+active	O
+transcription	O
+.	O
+	O
+We	O
+show	O
+that	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+engages	O
+crotonyllysine	O
+via	O
+a	O
+unique	O
+π	O
+-	O
+π	O
+-	O
+π	O
+-	O
+stacking	O
+mechanism	O
+and	O
+that	O
+other	O
+YEATS	O
+domains	O
+have	O
+crotonyllysine	O
+binding	O
+activity	O
+.	O
+	O
+Crotonylation	O
+of	O
+lysine	O
+residues	O
+(	O
+crotonyllysine	O
+,	O
+Kcr	O
+)	O
+has	O
+emerged	O
+as	O
+one	O
+of	O
+the	O
+fundamental	O
+histone	O
+post	O
+-	O
+translational	O
+modifications	O
+(	O
+PTMs	O
+)	O
+found	O
+in	O
+mammalian	O
+chromatin	O
+.	O
+	O
+The	O
+crotonyllysine	O
+mark	O
+on	O
+histone	O
+H3K18	O
+is	O
+produced	O
+by	O
+p300	O
+,	O
+a	O
+histone	O
+acetyltransferase	O
+also	O
+responsible	O
+for	O
+acetylation	O
+of	O
+histones	O
+.	O
+	O
+While	O
+a	O
+number	O
+of	O
+acetyllysine	O
+readers	O
+have	O
+been	O
+identified	O
+and	O
+characterized	O
+,	O
+a	O
+specific	O
+reader	O
+of	O
+the	O
+crotonyllysine	O
+mark	O
+remains	O
+unknown	O
+(	O
+reviewed	O
+in	O
+).	O
+	O
+The	O
+family	O
+of	O
+acetyllysine	O
+readers	O
+has	O
+been	O
+expanded	O
+with	O
+the	O
+discovery	O
+that	O
+the	O
+YEATS	O
+(	O
+Yaf9	O
+,	O
+ENL	O
+,	O
+AF9	O
+,	O
+Taf14	O
+,	O
+Sas5	O
+)	O
+domains	O
+of	O
+human	O
+AF9	O
+and	O
+yeast	O
+Taf14	O
+are	O
+capable	O
+of	O
+recognizing	O
+the	O
+histone	O
+mark	O
+H3K9ac	O
+.	O
+	O
+The	O
+acetyllysine	O
+binding	O
+function	O
+of	O
+the	O
+AF9	O
+YEATS	O
+domain	O
+is	O
+essential	O
+for	O
+the	O
+recruitment	O
+of	O
+the	O
+histone	O
+methyltransferase	O
+DOT1L	O
+to	O
+H3K9ac	O
+-	O
+containing	O
+chromatin	O
+and	O
+for	O
+DOT1L	O
+-	O
+mediated	O
+H3K79	O
+methylation	O
+and	O
+transcription	O
+.	O
+	O
+Similarly	O
+,	O
+activation	O
+of	O
+a	O
+subset	O
+of	O
+genes	O
+and	O
+DNA	O
+damage	O
+repair	O
+in	O
+yeast	O
+require	O
+the	O
+acetyllysine	O
+binding	O
+activity	O
+of	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+.	O
+	O
+Consistent	O
+with	O
+its	O
+role	O
+in	O
+gene	O
+regulation	O
+,	O
+Taf14	O
+was	O
+identified	O
+as	O
+a	O
+core	O
+component	O
+of	O
+the	O
+transcription	O
+factor	O
+complexes	O
+TFIID	O
+and	O
+TFIIF	O
+.	O
+	O
+We	O
+found	O
+that	O
+H3K9cr	O
+is	O
+present	O
+in	O
+yeast	O
+and	O
+is	O
+dynamically	O
+regulated	O
+.	O
+	O
+To	O
+elucidate	O
+the	O
+molecular	O
+basis	O
+for	O
+recognition	O
+of	O
+the	O
+H3K9cr	O
+mark	O
+,	O
+we	O
+obtained	O
+a	O
+crystal	O
+structure	O
+of	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+in	O
+complex	O
+with	O
+H3K9cr5	O
+-	O
+13	O
+(	O
+residues	O
+5	O
+–	O
+13	O
+of	O
+H3	O
+)	O
+peptide	O
+(	O
+Fig	O
+.	O
+1	O
+,	O
+Supplementary	O
+Results	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+and	O
+Supplementary	O
+Table	O
+1	O
+).	O
+	O
+The	O
+Taf14	O
+YEATS	O
+domain	O
+adopts	O
+an	O
+immunoglobin	O
+-	O
+like	O
+β	O
+sandwich	O
+fold	O
+containing	O
+eight	O
+anti	O
+-	O
+parallel	O
+β	O
+strands	O
+linked	O
+by	O
+short	O
+loops	O
+that	O
+form	O
+a	O
+binding	O
+site	O
+for	O
+H3K9cr	O
+(	O
+Fig	O
+.	O
+1b	O
+).	O
+	O
+The	O
+H3K9cr	O
+peptide	O
+lays	O
+in	O
+an	O
+extended	O
+conformation	O
+in	O
+an	O
+orientation	O
+orthogonal	O
+to	O
+the	O
+β	O
+strands	O
+and	O
+is	O
+stabilized	O
+through	O
+an	O
+extensive	O
+network	O
+of	O
+direct	O
+and	O
+water	O
+-	O
+mediated	O
+hydrogen	O
+bonds	O
+and	O
+a	O
+salt	O
+bridge	O
+(	O
+Fig	O
+.	O
+1c	O
+).	O
+	O
+The	O
+fully	O
+extended	O
+side	O
+chain	O
+of	O
+K9cr	O
+transverses	O
+the	O
+narrow	O
+tunnel	O
+,	O
+crossing	O
+the	O
+β	O
+sandwich	O
+at	O
+right	O
+angle	O
+in	O
+a	O
+corkscrew	O
+-	O
+like	O
+manner	O
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Figure	O
+1b	O
+).	O
+	O
+The	O
+planar	O
+crotonyl	O
+group	O
+is	O
+inserted	O
+between	O
+Trp81	O
+and	O
+Phe62	O
+of	O
+the	O
+protein	O
+,	O
+the	O
+aromatic	O
+rings	O
+of	O
+which	O
+are	O
+positioned	O
+strictly	O
+parallel	O
+to	O
+each	O
+other	O
+and	O
+at	O
+equal	O
+distance	O
+from	O
+the	O
+crotonyl	O
+group	O
+,	O
+yielding	O
+a	O
+novel	O
+aromatic	O
+-	O
+amide	O
+/	O
+aliphatic	O
+-	O
+aromatic	O
+π	O
+-	O
+π	O
+-	O
+π	O
+-	O
+stacking	O
+system	O
+that	O
+,	O
+to	O
+our	O
+knowledge	O
+,	O
+has	O
+not	O
+been	O
+reported	O
+previously	O
+for	O
+any	O
+protein	O
+-	O
+protein	O
+interaction	O
+(	O
+Fig	O
+.	O
+1d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+).	O
+	O
+The	O
+side	O
+chain	O
+of	O
+Trp81	O
+appears	O
+to	O
+adopt	O
+two	O
+conformations	O
+,	O
+one	O
+of	O
+which	O
+provides	O
+maximum	O
+π	O
+-	O
+stacking	O
+with	O
+the	O
+alkene	O
+functional	O
+group	O
+while	O
+the	O
+other	O
+rotamer	O
+affords	O
+maximum	O
+π	O
+-	O
+stacking	O
+with	O
+the	O
+amide	O
+π	O
+electrons	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+).	O
+	O
+The	O
+dual	O
+conformation	O
+of	O
+Trp81	O
+is	O
+likely	O
+due	O
+to	O
+the	O
+conjugated	O
+nature	O
+of	O
+the	O
+C	O
+=	O
+C	O
+and	O
+C	O
+=	O
+O	O
+π	O
+-	O
+orbitals	O
+within	O
+the	O
+crotonyl	O
+functional	O
+group	O
+.	O
+	O
+In	O
+addition	O
+to	O
+π	O
+-	O
+π	O
+-	O
+π	O
+stacking	O
+,	O
+the	O
+crotonyl	O
+group	O
+is	O
+stabilized	O
+by	O
+a	O
+set	O
+of	O
+hydrogen	O
+bonds	O
+and	O
+electrostatic	O
+interactions	O
+.	O
+	O
+This	O
+provides	O
+the	O
+capability	O
+for	O
+the	O
+alkene	O
+moiety	O
+to	O
+form	O
+electrostatic	O
+contacts	O
+,	O
+as	O
+Cα	O
+and	O
+Cβ	O
+lay	O
+within	O
+electrostatic	O
+interaction	O
+distances	O
+of	O
+the	O
+carbonyl	O
+oxygen	O
+of	O
+Gln79	O
+and	O
+of	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Thr61	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+hydroxyl	O
+group	O
+of	O
+Thr61	O
+also	O
+participates	O
+in	O
+a	O
+hydrogen	O
+bond	O
+with	O
+the	O
+amide	O
+nitrogen	O
+of	O
+the	O
+K9cr	O
+side	O
+chain	O
+(	O
+Fig	O
+.	O
+1d	O
+).	O
+	O
+The	O
+fixed	O
+position	O
+of	O
+the	O
+Thr61	O
+hydroxyl	O
+group	O
+,	O
+which	O
+facilitates	O
+interactions	O
+with	O
+both	O
+the	O
+amide	O
+and	O
+Cα	O
+of	O
+K9cr	O
+,	O
+is	O
+achieved	O
+through	O
+a	O
+hydrogen	O
+bond	O
+with	O
+imidazole	O
+ring	O
+of	O
+His59	O
+.	O
+	O
+Extra	O
+stabilization	O
+of	O
+K9cr	O
+is	O
+attained	O
+by	O
+a	O
+hydrogen	O
+bond	O
+formed	O
+between	O
+its	O
+carbonyl	O
+oxygen	O
+and	O
+the	O
+backbone	O
+nitrogen	O
+of	O
+Trp81	O
+,	O
+as	O
+well	O
+as	O
+a	O
+water	O
+-	O
+mediated	O
+hydrogen	O
+bond	O
+with	O
+the	O
+backbone	O
+carbonyl	O
+group	O
+of	O
+Gly82	O
+(	O
+Fig	O
+1d	O
+).	O
+	O
+Binding	O
+of	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+to	O
+H3K9cr	O
+is	O
+robust	O
+.	O
+	O
+This	O
+value	O
+is	O
+in	O
+the	O
+range	O
+of	O
+binding	O
+affinities	O
+exhibited	O
+by	O
+the	O
+majority	O
+of	O
+histone	O
+readers	O
+,	O
+thus	O
+attesting	O
+to	O
+the	O
+physiological	O
+relevance	O
+of	O
+the	O
+H3K9cr	O
+recognition	O
+by	O
+Taf14	O
+.	O
+	O
+Both	O
+H3K9cr	O
+and	O
+H3K9ac	O
+were	O
+detected	O
+in	O
+yeast	O
+histones	O
+;	O
+to	O
+our	O
+knowledge	O
+,	O
+this	O
+is	O
+the	O
+first	O
+report	O
+of	O
+H3K9cr	O
+occurring	O
+in	O
+yeast	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Figure	O
+2a	O
+,	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3e	O
+,	O
+H3K9cr	O
+levels	O
+were	O
+abolished	O
+or	O
+reduced	O
+considerably	O
+in	O
+the	O
+HAT	O
+deletion	O
+strains	O
+,	O
+whereas	O
+they	O
+were	O
+dramatically	O
+increased	O
+in	O
+the	O
+HDAC	O
+deletion	O
+strains	O
+.	O
+	O
+We	O
+have	O
+previously	O
+shown	O
+that	O
+among	O
+acetylated	O
+histone	O
+marks	O
+,	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+prefers	O
+acetylated	O
+H3K9	O
+(	O
+also	O
+see	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+),	O
+however	O
+it	O
+binds	O
+to	O
+H3K9cr	O
+tighter	O
+.	O
+	O
+Binding	O
+of	O
+H3K9cr	O
+induced	O
+resonance	O
+changes	O
+in	O
+slow	O
+exchange	O
+regime	O
+on	O
+the	O
+NMR	O
+time	O
+scale	O
+,	O
+indicative	O
+of	O
+strong	O
+interaction	O
+.	O
+	O
+Furthermore	O
+,	O
+crosspeaks	O
+of	O
+Gly80	O
+and	O
+Trp81	O
+of	O
+the	O
+YEATS	O
+domain	O
+were	O
+uniquely	O
+perturbed	O
+by	O
+H3K9cr	O
+and	O
+H3K9ac	O
+,	O
+indicating	O
+a	O
+different	O
+chemical	O
+environment	O
+in	O
+the	O
+respective	O
+crotonyllysine	O
+and	O
+acetyllysine	O
+binding	O
+pockets	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+These	O
+differences	O
+support	O
+our	O
+model	O
+that	O
+Trp81	O
+adopts	O
+two	O
+conformations	O
+upon	O
+complex	O
+formation	O
+with	O
+the	O
+H3K9cr	O
+mark	O
+as	O
+compared	O
+to	O
+H3K9ac	O
+(	O
+Supplementary	O
+Figs	O
+.	O
+1c	O
+,	O
+d	O
+and	O
+4c	O
+).	O
+	O
+One	O
+of	O
+the	O
+conformations	O
+,	O
+characterized	O
+by	O
+the	O
+π	O
+stacking	O
+involving	O
+two	O
+aromatic	O
+residues	O
+and	O
+the	O
+alkene	O
+group	O
+,	O
+is	O
+observed	O
+only	O
+in	O
+the	O
+YEATS	O
+-	O
+H3K9cr	O
+complex	O
+.	O
+	O
+To	O
+establish	O
+whether	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+is	O
+able	O
+to	O
+recognize	O
+other	O
+recently	O
+identified	O
+acyllysine	O
+marks	O
+,	O
+we	O
+performed	O
+solution	O
+pull	O
+-	O
+down	O
+assays	O
+using	O
+H3	O
+peptides	O
+acetylated	O
+,	O
+propionylated	O
+,	O
+butyrylated	O
+,	O
+and	O
+crotonylated	O
+at	O
+lysine	O
+9	O
+(	O
+residues	O
+1	O
+–	O
+20	O
+of	O
+H3	O
+).	O
+	O
+As	O
+shown	O
+in	O
+Figure	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+,	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+binds	O
+more	O
+strongly	O
+to	O
+H3K9cr1	O
+-	O
+20	O
+,	O
+as	O
+compared	O
+to	O
+other	O
+acylated	O
+histone	O
+peptides	O
+.	O
+	O
+Addition	O
+of	O
+H3K9ac1	O
+-	O
+20	O
+,	O
+H3K9pr1	O
+-	O
+20	O
+,	O
+and	O
+H3K9bu1	O
+-	O
+20	O
+peptides	O
+caused	O
+chemical	O
+shift	O
+perturbations	O
+in	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+in	O
+intermediate	O
+exchange	O
+regime	O
+,	O
+implying	O
+that	O
+these	O
+interactions	O
+are	O
+weaker	O
+compared	O
+to	O
+the	O
+interaction	O
+with	O
+the	O
+H3K9cr1	O
+-	O
+20	O
+peptide	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+).	O
+	O
+We	O
+concluded	O
+that	O
+H3K9cr	O
+is	O
+the	O
+preferred	O
+target	O
+of	O
+this	O
+domain	O
+.	O
+	O
+From	O
+comparative	O
+structural	O
+analysis	O
+of	O
+the	O
+YEATS	O
+complexes	O
+,	O
+Gly80	O
+emerged	O
+as	O
+candidate	O
+residue	O
+potentially	O
+responsible	O
+for	O
+the	O
+preference	O
+for	O
+crotonyllysine	O
+.	O
+	O
+In	O
+attempt	O
+to	O
+generate	O
+a	O
+mutant	O
+capable	O
+of	O
+accommodating	O
+a	O
+short	O
+acetyl	O
+moiety	O
+but	O
+discriminating	O
+against	O
+a	O
+longer	O
+,	O
+planar	O
+crotonyl	O
+moiety	O
+,	O
+we	O
+mutated	O
+Gly80	O
+to	O
+more	O
+bulky	O
+residues	O
+,	O
+however	O
+all	O
+mutants	O
+of	O
+Gly80	O
+lost	O
+their	O
+binding	O
+activities	O
+towards	O
+either	O
+acylated	O
+peptide	O
+,	O
+suggesting	O
+that	O
+Gly80	O
+is	O
+absolutely	O
+required	O
+for	O
+the	O
+interaction	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+mutation	O
+of	O
+Val24	O
+,	O
+a	O
+residue	O
+located	O
+on	O
+another	O
+side	O
+of	O
+Trp81	O
+,	O
+had	O
+no	O
+effect	O
+on	O
+binding	O
+(	O
+Fig	O
+.	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+,	O
+c	O
+).	O
+	O
+We	O
+found	O
+that	O
+all	O
+YEATS	O
+domains	O
+tested	O
+are	O
+capable	O
+of	O
+binding	O
+to	O
+crotonyllysine	O
+peptides	O
+,	O
+though	O
+they	O
+display	O
+variable	O
+preferences	O
+for	O
+the	O
+acyl	O
+moieties	O
+.	O
+	O
+While	O
+YEATS2	O
+and	O
+ENL	O
+showed	O
+selectivity	O
+for	O
+the	O
+crotonylated	O
+peptides	O
+,	O
+GAS41	O
+and	O
+AF9	O
+bound	O
+acylated	O
+peptides	O
+almost	O
+equally	O
+well	O
+.	O
+	O
+Unlike	O
+the	O
+YEATS	O
+domain	O
+,	O
+a	O
+known	O
+acetyllysine	O
+reader	O
+,	O
+bromodomain	O
+,	O
+does	O
+not	O
+recognize	O
+crotonyllysine	O
+.	O
+	O
+We	O
+assayed	O
+a	O
+large	O
+set	O
+of	O
+BDs	O
+in	O
+pull	O
+-	O
+down	O
+experiments	O
+and	O
+found	O
+that	O
+this	O
+module	O
+is	O
+highly	O
+specific	O
+for	O
+acetyllysine	O
+and	O
+propionyllysine	O
+containing	O
+peptides	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+	O
+However	O
+,	O
+bromodomains	O
+did	O
+not	O
+interact	O
+(	O
+or	O
+associated	O
+very	O
+weakly	O
+)	O
+with	O
+longer	O
+acyl	O
+modifications	O
+,	O
+including	O
+crotonyllysine	O
+,	O
+as	O
+in	O
+the	O
+case	O
+of	O
+BDs	O
+of	O
+TAF1	O
+and	O
+BRD2	O
+,	O
+supporting	O
+recent	O
+reports	O
+.	O
+	O
+In	O
+conclusion	O
+,	O
+we	O
+have	O
+identified	O
+the	O
+YEATS	O
+domain	O
+of	O
+Taf14	O
+as	O
+the	O
+first	O
+reader	O
+of	O
+histone	O
+crotonylation	O
+.	O
+	O
+As	O
+we	O
+previously	O
+showed	O
+the	O
+importance	O
+of	O
+acyllysine	O
+binding	O
+by	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+for	O
+the	O
+DNA	O
+damage	O
+response	O
+and	O
+gene	O
+transcription	O
+,	O
+it	O
+will	O
+be	O
+essential	O
+in	O
+the	O
+future	O
+to	O
+define	O
+the	O
+physiological	O
+role	O
+of	O
+crotonyllysine	O
+recognition	O
+and	O
+to	O
+differentiate	O
+the	O
+activities	O
+of	O
+Taf14	O
+that	O
+are	O
+due	O
+to	O
+binding	O
+to	O
+crotonyllysine	O
+and	O
+acetyllysine	O
+modifications	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+functional	O
+significance	O
+of	O
+crotonyllysine	O
+recognition	O
+by	O
+other	O
+YEATS	O
+proteins	O
+will	O
+be	O
+of	O
+great	O
+importance	O
+to	O
+elucidate	O
+and	O
+compare	O
+.	O
+	O
+The	O
+structural	O
+mechanism	O
+for	O
+the	O
+recognition	O
+of	O
+H3K9cr	O
+	O
+(	O
+a	O
+)	O
+Chemical	O
+structure	O
+of	O
+crotonyllysine	O
+.	O
+(	O
+b	O
+)	O
+The	O
+crystal	O
+structure	O
+of	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+(	O
+wheat	O
+)	O
+in	O
+complex	O
+with	O
+the	O
+H3K9cr5	O
+-	O
+13	O
+peptide	O
+(	O
+green	O
+).	O
+(	O
+c	O
+)	O
+H3K9cr	O
+is	O
+stabilized	O
+via	O
+an	O
+extensive	O
+network	O
+of	O
+intermolecular	O
+electrostatic	O
+and	O
+polar	O
+interactions	O
+with	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+.	O
+	O
+(	O
+d	O
+)	O
+The	O
+π	O
+-	O
+π	O
+-	O
+π	O
+stacking	O
+mechanism	O
+involving	O
+the	O
+alkene	O
+moiety	O
+of	O
+crotonyllysine	O
+.	O
+	O
+(	O
+a	O
+,	O
+b	O
+)	O
+Western	O
+blot	O
+analysis	O
+comparing	O
+the	O
+levels	O
+of	O
+H3K9cr	O
+and	O
+H3K9ac	O
+in	O
+wild	O
+type	O
+(	O
+WT	O
+),	O
+HAT	O
+deletion	O
+,	O
+or	O
+HDAC	O
+deletion	O
+yeast	O
+strains	O
+.	O
+	O
+(	O
+c	O
+)	O
+Superimposed	O
+1H	O
+,	O
+15N	O
+HSQC	O
+spectra	O
+of	O
+Taf14	O
+YEATS	O
+recorded	O
+as	O
+H3K9cr5	O
+-	O
+13	O
+and	O
+H3K9ac5	O
+-	O
+13	O
+peptides	O
+were	O
+titrated	O
+in	O
+.	O
+	O
+Spectra	O
+are	O
+color	O
+coded	O
+according	O
+to	O
+the	O
+protein	O
+:	O
+peptide	O
+molar	O
+ratio	O
+.	O
+	O
+(	O
+d	O
+)	O
+Western	O
+blot	O
+analyses	O
+of	O
+peptide	O
+pull	O
+-	O
+down	O
+assays	O
+using	O
+wild	O
+-	O
+type	O
+and	O
+mutated	O
+Taf14	O
+YEATS	O
+domains	O
+and	O
+indicated	O
+peptides	O
+.	O
+	O
+In	O
+this	O
+data	O
+article	O
+,	O
+we	O
+report	O
+the	O
+solution	O
+NMR	O
+-	O
+derived	O
+structure	O
+of	O
+the	O
+Tom1	O
+GAT	O
+domain	O
+.	O
+	O
+The	O
+estimated	O
+protein	O
+structure	O
+exhibits	O
+a	O
+bundle	O
+of	O
+three	O
+helical	O
+elements	O
+.	O
+	O
+We	O
+compare	O
+the	O
+Tom1	O
+GAT	O
+structure	O
+with	O
+those	O
+structures	O
+corresponding	O
+to	O
+the	O
+Tollip	O
+TBD	O
+-	O
+and	O
+ubiquitin	O
+-	O
+bound	O
+states	O
+.	O
+	O
+NMR	O
+data	O
+was	O
+recorded	O
+using	O
+a	O
+Bruker	O
+800	O
+MHz	O
+Data	O
+format	O
+PDB	O
+format	O
+text	O
+file	O
+.	O
+	O
+Analyzed	O
+by	O
+CS	O
+-	O
+Rosetta	O
+,	O
+Protein	O
+Structure	O
+Validation	O
+Server	O
+(	O
+PSVS	O
+),	O
+NMRPipe	O
+,	O
+NMRDraw	O
+,	O
+and	O
+PyMol	O
+Experimental	O
+factors	O
+Recombinant	O
+human	O
+Tom1	O
+GAT	O
+domain	O
+was	O
+purified	O
+to	O
+homogeneity	O
+before	O
+use	O
+Experimental	O
+features	O
+Solution	O
+structure	O
+of	O
+Tom1	O
+GAT	O
+was	O
+determined	O
+from	O
+NMR	O
+chemical	O
+shift	O
+data	O
+Data	O
+source	O
+location	O
+Virginia	O
+and	O
+Colorado	O
+,	O
+United	O
+States	O
+.	O
+	O
+Tom1	O
+GAT	O
+structural	O
+data	O
+is	O
+publicly	O
+available	O
+in	O
+the	O
+RCSB	O
+Protein	O
+Data	O
+Bank	O
+(	O
+http	O
+://	O
+www	O
+.	O
+rscb	O
+.	O
+org	O
+/)	O
+under	O
+the	O
+accession	O
+number	O
+PDB	O
+:	O
+2n9d	O
+	O
+Tom1	O
+GAT	O
+can	O
+adopt	O
+distinct	O
+conformations	O
+upon	O
+ligand	O
+binding	O
+.	O
+	O
+Unlike	O
+ubiquitin	O
+binding	O
+,	O
+data	O
+suggest	O
+that	O
+conformational	O
+changes	O
+of	O
+the	O
+Tom1	O
+GAT	O
+α	O
+-	O
+helices	O
+1	O
+and	O
+2	O
+occur	O
+upon	O
+Tollip	O
+TBD	O
+binding	O
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+	O
+Representative	O
+far	O
+-	O
+UV	O
+CD	O
+spectrum	O
+of	O
+the	O
+His	O
+-	O
+Tom1	O
+GAT	O
+domain	O
+.	O
+	O
+(	O
+A	O
+)	O
+Stereo	O
+view	O
+displaying	O
+the	O
+best	O
+-	O
+fit	O
+backbone	O
+superposition	O
+of	O
+the	O
+refined	O
+structures	O
+for	O
+the	O
+Tom1	O
+GAT	O
+domain	O
+.	O
+	O
+(	O
+A	O
+)	O
+Two	O
+views	O
+of	O
+the	O
+superimposed	O
+structures	O
+of	O
+the	O
+Tom1	O
+GAT	O
+domain	O
+in	O
+the	O
+free	O
+state	O
+(	O
+gray	O
+)	O
+with	O
+that	O
+in	O
+the	O
+Tollip	O
+TBD	O
+-	O
+bound	O
+state	O
+(	O
+red	O
+).	O
+(	O
+B	O
+)	O
+Two	O
+views	O
+of	O
+the	O
+superimposed	O
+structures	O
+of	O
+the	O
+Tom1	O
+GAT	O
+domain	O
+(	O
+gray	O
+)	O
+with	O
+that	O
+in	O
+the	O
+Ub	O
+-	O
+bound	O
+state	O
+(	O
+green	O
+).	O
+	O
+NMR	O
+and	O
+refinement	O
+statistics	O
+for	O
+the	O
+Tom1	O
+GAT	O
+domain	O
+.	O
+	O
+Haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+/	O
+Sigma	O
+-	O
+2	O
+receptor	O
+facilitates	O
+cancer	O
+proliferation	O
+and	O
+chemoresistance	O
+	O
+Progesterone	O
+-	O
+receptor	O
+membrane	O
+component	O
+1	O
+(	O
+PGRMC1	O
+/	O
+Sigma	O
+-	O
+2	O
+receptor	O
+)	O
+is	O
+a	O
+haem	O
+-	O
+containing	O
+protein	O
+that	O
+interacts	O
+with	O
+epidermal	O
+growth	O
+factor	O
+receptor	O
+(	O
+EGFR	O
+)	O
+and	O
+cytochromes	O
+P450	O
+to	O
+regulate	O
+cancer	O
+proliferation	O
+and	O
+chemoresistance	O
+;	O
+its	O
+structural	O
+basis	O
+remains	O
+unknown	O
+.	O
+	O
+Here	O
+crystallographic	O
+analyses	O
+of	O
+the	O
+PGRMC1	O
+cytosolic	O
+domain	O
+at	O
+1	O
+.	O
+95	O
+Å	O
+resolution	O
+reveal	O
+that	O
+it	O
+forms	O
+a	O
+stable	O
+dimer	O
+through	O
+stacking	O
+interactions	O
+of	O
+two	O
+protruding	O
+haem	O
+molecules	O
+.	O
+	O
+The	O
+haem	O
+iron	O
+is	O
+five	O
+-	O
+coordinated	O
+by	O
+Tyr113	O
+,	O
+and	O
+the	O
+open	O
+surface	O
+of	O
+the	O
+haem	O
+mediates	O
+dimerization	O
+.	O
+	O
+Carbon	O
+monoxide	O
+(	O
+CO	O
+)	O
+interferes	O
+with	O
+PGRMC1	O
+dimerization	O
+by	O
+binding	O
+to	O
+the	O
+sixth	O
+coordination	O
+site	O
+of	O
+the	O
+haem	O
+.	O
+	O
+Haem	O
+-	O
+mediated	O
+PGRMC1	O
+dimerization	O
+is	O
+required	O
+for	O
+interactions	O
+with	O
+EGFR	O
+and	O
+cytochromes	O
+P450	O
+,	O
+cancer	O
+proliferation	O
+and	O
+chemoresistance	O
+against	O
+anti	O
+-	O
+cancer	O
+drugs	O
+;	O
+these	O
+events	O
+are	O
+attenuated	O
+by	O
+either	O
+CO	O
+or	O
+haem	O
+deprivation	O
+in	O
+cancer	O
+cells	O
+.	O
+	O
+This	O
+study	O
+demonstrates	O
+protein	O
+dimerization	O
+via	O
+haem	O
+–	O
+haem	O
+stacking	O
+,	O
+which	O
+has	O
+not	O
+been	O
+seen	O
+in	O
+eukaryotes	O
+,	O
+and	O
+provides	O
+insights	O
+into	O
+its	O
+functional	O
+significance	O
+in	O
+cancer	O
+.	O
+	O
+PGRMC1	O
+binds	O
+to	O
+EGFR	O
+and	O
+cytochromes	O
+P450	O
+,	O
+and	O
+is	O
+known	O
+to	O
+be	O
+involved	O
+in	O
+cancer	O
+proliferation	O
+and	O
+in	O
+drug	O
+resistance	O
+.	O
+	O
+Here	O
+,	O
+the	O
+authors	O
+determine	O
+the	O
+structure	O
+of	O
+the	O
+cytosolic	O
+domain	O
+of	O
+PGRMC1	O
+,	O
+which	O
+forms	O
+a	O
+dimer	O
+via	O
+haem	O
+–	O
+haem	O
+stacking	O
+,	O
+and	O
+propose	O
+how	O
+this	O
+interaction	O
+could	O
+be	O
+involved	O
+in	O
+its	O
+function	O
+.	O
+	O
+Increased	O
+dietary	O
+intake	O
+of	O
+haem	O
+is	O
+a	O
+risk	O
+factor	O
+for	O
+several	O
+types	O
+of	O
+cancer	O
+.	O
+	O
+Previous	O
+studies	O
+showed	O
+that	O
+deprivation	O
+of	O
+iron	O
+or	O
+haem	O
+suppresses	O
+tumourigenesis	O
+.	O
+	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+carbon	O
+monoxide	O
+(	O
+CO	O
+),	O
+the	O
+gaseous	O
+mediator	O
+generated	O
+by	O
+oxidative	O
+degradation	O
+of	O
+haem	O
+via	O
+haem	O
+oxygenase	O
+(	O
+HO	O
+),	O
+inhibits	O
+tumour	O
+growth	O
+.	O
+	O
+To	O
+gain	O
+insight	O
+into	O
+the	O
+underlying	O
+mechanisms	O
+,	O
+we	O
+took	O
+chemical	O
+biological	O
+approaches	O
+using	O
+affinity	O
+nanobeads	O
+carrying	O
+haem	O
+and	O
+identified	O
+progesterone	O
+-	O
+receptor	O
+membrane	O
+component	O
+1	O
+(	O
+PGRMC1	O
+)	O
+as	O
+a	O
+haem	O
+-	O
+binding	O
+protein	O
+from	O
+mouse	O
+liver	O
+extracts	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+PGRMC1	O
+is	O
+anchored	O
+to	O
+the	O
+cell	O
+membrane	O
+through	O
+the	O
+N	O
+-	O
+terminal	O
+transmembrane	O
+helix	O
+and	O
+interacts	O
+with	O
+epidermal	O
+growth	O
+factor	O
+receptor	O
+(	O
+EGFR	O
+)	O
+and	O
+cytochromes	O
+P450	O
+(	O
+ref	O
+).	O
+	O
+While	O
+PGRMC1	O
+is	O
+implicated	O
+in	O
+cell	O
+proliferation	O
+and	O
+cholesterol	O
+biosynthesis	O
+,	O
+the	O
+structural	O
+basis	O
+on	O
+which	O
+PGRMC1	O
+exerts	O
+its	O
+function	O
+remains	O
+largely	O
+unknown	O
+.	O
+	O
+Here	O
+we	O
+show	O
+that	O
+PGRMC1	O
+exhibits	O
+a	O
+unique	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+.	O
+	O
+The	O
+dimer	O
+binds	O
+to	O
+EGFR	O
+and	O
+cytochromes	O
+P450	O
+to	O
+enhance	O
+tumour	O
+cell	O
+proliferation	O
+and	O
+chemoresistance	O
+.	O
+	O
+The	O
+dimer	O
+is	O
+dissociated	O
+to	O
+monomers	O
+by	O
+physiological	O
+levels	O
+of	O
+CO	O
+,	O
+suggesting	O
+that	O
+PGRMC1	O
+serves	O
+as	O
+a	O
+CO	O
+-	O
+sensitive	O
+molecular	O
+switch	O
+regulating	O
+cancer	O
+cell	O
+proliferation	O
+.	O
+	O
+X	O
+-	O
+ray	O
+crystal	O
+structure	O
+of	O
+PGRMC1	O
+	O
+We	O
+solved	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+haem	O
+-	O
+bound	O
+PGRMC1	O
+cytosolic	O
+domain	O
+(	O
+a	O
+.	O
+a	O
+.	O
+72	O
+–	O
+195	O
+)	O
+at	O
+1	O
+.	O
+95	O
+Å	O
+resolution	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+In	O
+the	O
+presence	O
+of	O
+haem	O
+,	O
+PGRMC1	O
+forms	O
+a	O
+dimeric	O
+structure	O
+largely	O
+through	O
+hydrophobic	O
+interactions	O
+between	O
+the	O
+haem	O
+moieties	O
+of	O
+two	O
+monomers	O
+(	O
+Fig	O
+.	O
+1a	O
+,	O
+Table	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+;	O
+a	O
+stereo	O
+-	O
+structural	O
+image	O
+is	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+4	O
+).	O
+	O
+While	O
+the	O
+overall	O
+fold	O
+of	O
+PGRMC1	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+canonical	O
+cytochrome	O
+b5	O
+,	O
+their	O
+haem	O
+irons	O
+are	O
+coordinated	O
+differently	O
+.	O
+	O
+In	O
+cytochrome	O
+b5	O
+,	O
+the	O
+haem	O
+iron	O
+is	O
+six	O
+-	O
+coordinated	O
+by	O
+two	O
+axial	O
+histidine	O
+residues	O
+.	O
+	O
+A	O
+homologous	O
+helix	O
+that	O
+holds	O
+haem	O
+in	O
+cytochrome	O
+b5	O
+is	O
+longer	O
+,	O
+shifts	O
+away	O
+from	O
+haem	O
+,	O
+and	O
+does	O
+not	O
+form	O
+a	O
+coordinate	O
+bond	O
+in	O
+PGRMC1	O
+(	O
+Fig	O
+.	O
+1c	O
+).	O
+	O
+Contrary	O
+to	O
+our	O
+finding	O
+,	O
+Kaluka	O
+et	O
+al	O
+.	O
+recently	O
+reported	O
+that	O
+Tyr164	O
+of	O
+PGRMC1	O
+is	O
+the	O
+axial	O
+ligand	O
+of	O
+haem	O
+because	O
+mutation	O
+of	O
+this	O
+residue	O
+impairs	O
+haem	O
+binding	O
+.	O
+	O
+Our	O
+structural	O
+data	O
+revealed	O
+that	O
+Tyr164	O
+and	O
+a	O
+few	O
+other	O
+residues	O
+such	O
+as	O
+Tyr107	O
+and	O
+Lys163	O
+are	O
+in	O
+fact	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+haem	O
+propionates	O
+.	O
+	O
+This	O
+is	O
+consistent	O
+with	O
+observations	O
+by	O
+Min	O
+et	O
+al	O
+.	O
+that	O
+Tyr	O
+107	O
+and	O
+Tyr113	O
+of	O
+PGRMC1	O
+are	O
+involved	O
+in	O
+binding	O
+with	O
+haem	O
+.	O
+	O
+These	O
+amino	O
+acid	O
+residues	O
+are	O
+conserved	O
+among	O
+MAPR	O
+family	O
+members	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+),	O
+suggesting	O
+that	O
+these	O
+proteins	O
+share	O
+the	O
+ability	O
+to	O
+exhibit	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+.	O
+	O
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+exhibits	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+in	O
+solution	O
+	O
+In	O
+the	O
+PGRMC1	O
+crystal	O
+,	O
+two	O
+different	O
+types	O
+of	O
+crystal	O
+contacts	O
+(	O
+chain	O
+A	O
+–	O
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+″	O
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+A	O
+–	O
+B	O
+)	O
+were	O
+observed	O
+in	O
+addition	O
+to	O
+the	O
+haem	O
+-	O
+mediated	O
+dimer	O
+(	O
+chain	O
+A	O
+–	O
+A	O
+′)	O
+(	O
+Supplementary	O
+Figs	O
+3	O
+and	O
+6a	O
+).	O
+	O
+To	O
+confirm	O
+that	O
+haem	O
+-	O
+assisted	O
+dimerization	O
+of	O
+PGRMC1	O
+occurs	O
+in	O
+solution	O
+,	O
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+the	O
+structure	O
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+apo	O
+-	O
+and	O
+haem	O
+-	O
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+by	O
+two	O
+-	O
+dimensional	O
+nuclear	O
+magnetic	O
+resonance	O
+(	O
+NMR	O
+)	O
+using	O
+heteronuclear	O
+single	O
+-	O
+quantum	O
+coherence	O
+and	O
+transverse	O
+relaxation	O
+-	O
+optimized	O
+spectroscopy	O
+(	O
+Supplementary	O
+Figs	O
+6b	O
+and	O
+7	O
+).	O
+	O
+NMR	O
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+from	O
+some	O
+amino	O
+acid	O
+residues	O
+of	O
+PGRMC1	O
+disappeared	O
+due	O
+to	O
+the	O
+paramagnetic	O
+relaxation	O
+effect	O
+of	O
+haem	O
+(	O
+Supplementary	O
+Figs	O
+6b	O
+);	O
+these	O
+residues	O
+were	O
+located	O
+in	O
+the	O
+haem	O
+-	O
+binding	O
+region	O
+.	O
+	O
+When	O
+chemical	O
+shifts	O
+of	O
+apo	O
+-	O
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+haem	O
+-	O
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+forms	O
+of	O
+PGMRC1	O
+were	O
+compared	O
+,	O
+some	O
+amino	O
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+close	O
+to	O
+those	O
+which	O
+disappeared	O
+because	O
+of	O
+the	O
+paramagnetic	O
+relaxation	O
+effect	O
+of	O
+haem	O
+exhibit	O
+notable	O
+chemical	O
+shifts	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+;	O
+dark	O
+yellow	O
+).	O
+	O
+We	O
+also	O
+attempted	O
+to	O
+predict	O
+the	O
+secondary	O
+structure	O
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+PGRMC1	O
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+data	O
+by	O
+calculating	O
+with	O
+TALOS	O
++	O
+program	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8	O
+);	O
+the	O
+prediction	O
+suggested	O
+that	O
+the	O
+overall	O
+secondary	O
+structure	O
+is	O
+comparable	O
+between	O
+apo	O
+-	O
+and	O
+haem	O
+-	O
+bound	O
+forms	O
+of	O
+PGRMC1	O
+in	O
+solution	O
+.	O
+	O
+We	O
+analysed	O
+the	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+the	O
+PGRMC1	O
+cytosolic	O
+domain	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+)	O
+in	O
+solution	O
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Table	O
+2	O
+).	O
+	O
+Mass	O
+spectrometry	O
+(	O
+MS	O
+)	O
+analyses	O
+under	O
+non	O
+-	O
+denaturing	O
+condition	O
+demonstrated	O
+that	O
+the	O
+apo	O
+-	O
+monomer	O
+PGRMC1	O
+resulted	O
+in	O
+dimerization	O
+by	O
+binding	O
+with	O
+haem	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+This	O
+observation	O
+led	O
+us	O
+to	O
+examine	O
+whether	O
+or	O
+not	O
+the	O
+disulfide	O
+bond	O
+contributes	O
+to	O
+PGRMC1	O
+dimerization	O
+.	O
+	O
+MS	O
+analyses	O
+under	O
+non	O
+-	O
+denaturing	O
+conditions	O
+clearly	O
+showed	O
+that	O
+the	O
+Cys129Ser	B-mutant
+(	O
+C129S	B-mutant
+)	O
+mutant	O
+is	O
+dimerized	O
+in	O
+the	O
+presence	O
+of	O
+haem	O
+,	O
+indicating	O
+that	O
+the	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+of	O
+PGRMC1	O
+occurs	O
+independently	O
+of	O
+the	O
+disulfide	O
+bond	O
+formation	O
+via	O
+Cys129	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+Supporting	O
+this	O
+,	O
+MS	O
+analyses	O
+under	O
+denaturing	O
+conditions	O
+showed	O
+that	O
+haem	O
+-	O
+mediated	O
+PGRMC1	O
+dimer	O
+is	O
+completely	O
+dissociated	O
+into	O
+monomer	O
+,	O
+indicating	O
+that	O
+dimerization	O
+of	O
+this	O
+kind	O
+is	O
+not	O
+mediated	O
+by	O
+any	O
+covalent	O
+bond	O
+such	O
+as	O
+disulfide	O
+bond	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+9	O
+).	O
+	O
+We	O
+also	O
+analysed	O
+the	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+by	O
+diffusion	O
+-	O
+ordered	O
+NMR	O
+spectroscopy	O
+(	O
+DOSY	O
+)	O
+analyses	O
+(	O
+Table	O
+2	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+10	O
+).	O
+	O
+The	O
+results	O
+suggested	O
+that	O
+the	O
+hydrodynamic	O
+radius	O
+of	O
+haem	O
+-	O
+bound	O
+PGRMC1	O
+is	O
+larger	O
+than	O
+that	O
+of	O
+apo	O
+-	O
+PGRMC1	O
+.	O
+	O
+To	O
+further	O
+evaluate	O
+changes	O
+in	O
+molecular	O
+weights	O
+in	O
+dimerization	O
+of	O
+PGRMC1	O
+,	O
+sedimentation	O
+velocity	O
+analytical	O
+ultracentrifugation	O
+(	O
+SV	O
+-	O
+AUC	O
+)	O
+analysis	O
+was	O
+carried	O
+out	O
+.	O
+	O
+Whereas	O
+the	O
+wild	O
+-	O
+type	O
+(	O
+wt	O
+)	O
+apo	O
+-	O
+PGRMC1	O
+appeared	O
+at	O
+a	O
+1	O
+.	O
+9	O
+S	O
+peak	O
+as	O
+monomer	O
+,	O
+the	O
+haem	O
+-	O
+binding	O
+PGRMC1	O
+was	O
+converted	O
+into	O
+dimer	O
+at	O
+a	O
+3	O
+.	O
+1	O
+S	O
+peak	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+Similarly	O
+,	O
+the	O
+C129S	B-mutant
+mutant	O
+of	O
+PGRMC1	O
+converted	O
+from	O
+monomer	O
+to	O
+dimer	O
+by	O
+binding	O
+to	O
+haem	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+SV	O
+-	O
+AUC	O
+analyses	O
+also	O
+allowed	O
+us	O
+to	O
+examine	O
+the	O
+stability	O
+of	O
+haem	O
+/	O
+PGRMC1	O
+dimer	O
+.	O
+	O
+To	O
+this	O
+end	O
+,	O
+we	O
+used	O
+different	O
+concentrations	O
+(	O
+3	O
+.	O
+5	O
+–	O
+147	O
+μmol	O
+l	O
+−	O
+1	O
+)	O
+of	O
+haem	O
+-	O
+bound	O
+PGRMC1	O
+protein	O
+(	O
+a	O
+.	O
+a	O
+.	O
+72	O
+–	O
+195	O
+),	O
+which	O
+were	O
+identical	O
+to	O
+that	O
+used	O
+in	O
+the	O
+crystallographic	O
+analysis	O
+.	O
+	O
+The	O
+sedimentation	O
+coefficients	O
+calculated	O
+on	O
+the	O
+basis	O
+of	O
+the	O
+crystal	O
+structure	O
+were	O
+1	O
+.	O
+71	O
+S	O
+for	O
+monomer	O
+and	O
+2	O
+.	O
+56	O
+S	O
+for	O
+dimer	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+11	O
+,	O
+upper	O
+panel	O
+).	O
+	O
+The	O
+results	O
+showed	O
+that	O
+the	O
+PGRMC1	O
+dimer	O
+is	O
+not	O
+dissociated	O
+into	O
+monomer	O
+at	O
+all	O
+concentrations	O
+examined	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+11	O
+,	O
+lower	O
+panel	O
+),	O
+suggesting	O
+that	O
+the	O
+Kd	O
+value	O
+of	O
+haem	O
+-	O
+mediated	O
+dimer	O
+of	O
+PGRMC1	O
+is	O
+under	O
+3	O
+.	O
+5	O
+μmol	O
+l	O
+−	O
+1	O
+.	O
+	O
+We	O
+also	O
+showed	O
+by	O
+haem	O
+titration	O
+experiments	O
+that	O
+haem	O
+binding	O
+to	O
+PGRMC1	O
+was	O
+of	O
+low	O
+affinity	O
+with	O
+a	O
+Kd	O
+value	O
+of	O
+50	O
+nmol	O
+l	O
+−	O
+1	O
+;	O
+this	O
+is	O
+comparable	O
+with	O
+that	O
+of	O
+iron	O
+regulatory	O
+protein	O
+2	O
+,	O
+which	O
+is	O
+known	O
+to	O
+be	O
+regulated	O
+by	O
+intracellular	O
+levels	O
+of	O
+haem	O
+(	O
+Fig	O
+.	O
+2c	O
+and	O
+Supplementary	O
+Table	O
+1	O
+).	O
+	O
+These	O
+results	O
+raised	O
+the	O
+possibility	O
+that	O
+the	O
+function	O
+of	O
+PGRMC1	O
+is	O
+regulated	O
+by	O
+intracellular	O
+haem	O
+concentrations	O
+.	O
+	O
+CO	O
+inhibits	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+	O
+Crystallographic	O
+analyses	O
+revealed	O
+that	O
+Tyr113	O
+of	O
+PGRMC1	O
+is	O
+an	O
+axial	O
+ligand	O
+for	O
+haem	O
+and	O
+contributes	O
+to	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+	O
+Analysis	O
+of	O
+UV	O
+-	O
+visible	O
+spectra	O
+revealed	O
+that	O
+the	O
+heme	O
+of	O
+PGRMC1	O
+is	O
+reducible	O
+from	O
+ferric	O
+to	O
+ferrous	O
+state	O
+,	O
+thus	O
+allowing	O
+CO	O
+binding	O
+(	O
+Fig	O
+.	O
+3a	O
+).	O
+	O
+Analysis	O
+of	O
+the	O
+ferric	O
+form	O
+of	O
+PGRMC1	O
+using	O
+resonance	O
+Raman	O
+spectroscopy	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+13	O
+)	O
+showed	O
+that	O
+the	O
+relative	O
+intensity	O
+of	O
+oxidation	O
+and	O
+spin	O
+state	O
+marker	O
+bands	O
+(	O
+ν4	O
+and	O
+ν3	O
+)	O
+is	O
+close	O
+to	O
+1	O
+.	O
+0	O
+,	O
+which	O
+is	O
+consistent	O
+with	O
+it	O
+being	O
+a	O
+haem	O
+protein	O
+with	O
+a	O
+proximal	O
+Tyr	O
+coordination	O
+.	O
+	O
+A	O
+specific	O
+Raman	O
+shift	O
+peaking	O
+at	O
+vFe	O
+–	O
+CO	O
+=	O
+500	O
+cm	O
+−	O
+1	O
+demonstrated	O
+that	O
+the	O
+CO	O
+-	O
+bound	O
+haem	O
+of	O
+PGRMC1	O
+is	O
+six	O
+-	O
+coordinated	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+13	O
+).	O
+	O
+Since	O
+PGRMC1	O
+dimerization	O
+involves	O
+the	O
+open	O
+surface	O
+of	O
+haem	O
+on	O
+the	O
+opposite	O
+side	O
+of	O
+the	O
+axial	O
+Tyr113	O
+,	O
+no	O
+space	O
+for	O
+CO	O
+binding	O
+is	O
+available	O
+in	O
+the	O
+dimeric	O
+structure	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+	O
+This	O
+prompted	O
+us	O
+to	O
+ask	O
+if	O
+CO	O
+binding	O
+to	O
+haem	O
+causes	O
+dissociation	O
+of	O
+the	O
+PGRMC1	O
+dimer	O
+.	O
+	O
+Analysis	O
+by	O
+gel	O
+filtration	O
+chromatography	O
+revealed	O
+that	O
+the	O
+relative	O
+molecular	O
+sizes	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+and	O
+the	O
+C129S	B-mutant
+mutant	O
+of	O
+PGRMC1	O
+are	O
+increased	O
+by	O
+adding	O
+haem	O
+to	O
+apo	O
+-	O
+PGRMC1	O
+regardless	O
+of	O
+the	O
+oxidation	O
+state	O
+of	O
+the	O
+iron	O
+(	O
+Fig	O
+.	O
+3c	O
+),	O
+which	O
+is	O
+in	O
+agreement	O
+with	O
+the	O
+results	O
+in	O
+Table	O
+1	O
+.	O
+	O
+CO	O
+application	O
+to	O
+ferrous	O
+PGRMC1	O
+abolished	O
+the	O
+haem	O
+-	O
+dependent	O
+increase	O
+in	O
+its	O
+molecular	O
+size	O
+.	O
+	O
+Under	O
+this	O
+reducing	O
+condition	O
+in	O
+the	O
+presence	O
+of	O
+dithionite	O
+,	O
+analyses	O
+of	O
+UV	O
+-	O
+visible	O
+spectra	O
+indicated	O
+that	O
+CO	O
+-	O
+binding	O
+with	O
+haem	O
+-	O
+PGRMC1	O
+is	O
+stable	O
+,	O
+showing	O
+only	O
+20	O
+%	O
+reduction	O
+of	O
+the	O
+absorbance	O
+at	O
+412	O
+nm	O
+within	O
+2	O
+h	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+14	O
+).	O
+	O
+Furthermore	O
+,	O
+the	O
+Tyr113Phe	B-mutant
+(	O
+Y113F	B-mutant
+)	O
+mutant	O
+of	O
+PGRMC1	O
+was	O
+not	O
+responsive	O
+to	O
+haem	O
+.	O
+	O
+The	O
+peak	O
+corresponding	O
+to	O
+the	O
+haem	O
+/	O
+PGRMC1	O
+dimer	O
+was	O
+detected	O
+under	O
+reducing	O
+conditions	O
+in	O
+the	O
+presence	O
+of	O
+dithionite	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+15	O
+,	O
+middle	O
+panel	O
+).	O
+	O
+Under	O
+these	O
+circumstances	O
+,	O
+CO	O
+application	O
+induced	O
+dissociation	O
+of	O
+the	O
+haem	O
+-	O
+mediated	O
+dimers	O
+of	O
+PGRMC1	O
+to	O
+generate	O
+a	O
+peak	O
+of	O
+monomers	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+15	O
+,	O
+lower	O
+panel	O
+).	O
+	O
+These	O
+observations	O
+raised	O
+the	O
+transition	O
+model	O
+for	O
+structural	O
+regulation	O
+of	O
+PGRMC1	O
+in	O
+response	O
+to	O
+haem	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+	O
+As	O
+mentioned	O
+above	O
+,	O
+apo	O
+-	O
+PGRMC1	O
+exists	O
+as	O
+monomer	O
+.	O
+	O
+CO	O
+induces	O
+the	O
+dissociation	O
+of	O
+the	O
+haem	O
+-	O
+mediated	O
+dimer	O
+of	O
+PGRMC1	O
+by	O
+interfering	O
+with	O
+the	O
+haem	O
+-	O
+stacking	O
+interface	O
+via	O
+formation	O
+of	O
+the	O
+six	O
+-	O
+coordinated	O
+CO	O
+-	O
+haem	O
+-	O
+PGRMC1	O
+complex	O
+.	O
+	O
+Such	O
+a	O
+dynamic	O
+structural	O
+regulation	O
+led	O
+us	O
+to	O
+further	O
+examine	O
+the	O
+regulation	O
+of	O
+PGRMC1	O
+functions	O
+in	O
+cancer	O
+cells	O
+.	O
+	O
+PGRMC1	O
+dimerization	O
+is	O
+required	O
+for	O
+binding	O
+to	O
+EGFR	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+.	O
+4a	O
+,	O
+the	O
+cytosolic	O
+domain	O
+of	O
+wild	O
+-	O
+type	O
+PGRMC1	O
+,	O
+but	O
+not	O
+the	O
+Y113F	B-mutant
+mutant	O
+,	O
+interacted	O
+with	O
+purified	O
+EGFR	O
+in	O
+a	O
+haem	O
+-	O
+dependent	O
+manner	O
+.	O
+	O
+This	O
+interaction	O
+was	O
+disrupted	O
+by	O
+the	O
+ruthenium	O
+-	O
+based	O
+CO	O
+-	O
+releasing	O
+molecule	O
+,	O
+CORM3	O
+,	O
+but	O
+not	O
+by	O
+RuCl3	O
+as	O
+a	O
+control	O
+reagent	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+	O
+We	O
+further	O
+analysed	O
+the	O
+intracellular	O
+interaction	O
+between	O
+PGRMC1	O
+and	O
+EGFR	O
+.	O
+	O
+FLAG	O
+-	O
+tagged	O
+PGRMC1	O
+ectopically	O
+expressed	O
+in	O
+human	O
+colon	O
+cancer	O
+HCT116	O
+cells	O
+was	O
+immunoprecipitated	O
+with	O
+anti	O
+-	O
+FLAG	O
+antibody	O
+,	O
+and	O
+co	O
+-	O
+immunoprecipitated	O
+EGFR	O
+and	O
+endogenous	O
+PGRMC1	O
+binding	O
+to	O
+FLAG	O
+-	O
+PGRMC1	O
+were	O
+detected	O
+by	O
+Western	O
+blotting	O
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+	O
+The	O
+C129S	B-mutant
+mutant	O
+of	O
+PGRMC1	O
+also	O
+interacted	O
+with	O
+endogenous	O
+PGRMC1	O
+and	O
+EGFR	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+16	O
+).	O
+	O
+Whereas	O
+FLAG	O
+-	O
+tagged	O
+wild	O
+-	O
+type	O
+PGRMC1	O
+interacted	O
+with	O
+endogenous	O
+PGRMC1	O
+and	O
+EGFR	O
+,	O
+the	O
+Y113F	B-mutant
+mutant	O
+did	O
+not	O
+.	O
+	O
+As	O
+expected	O
+,	O
+SA	O
+significantly	O
+reduced	O
+PGRMC1	O
+dimerization	O
+and	O
+its	O
+interaction	O
+with	O
+EGFR	O
+(	O
+Fig	O
+.	O
+4e	O
+),	O
+indicating	O
+that	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+of	O
+PGMRC1	O
+is	O
+critical	O
+for	O
+its	O
+binding	O
+to	O
+EGFR	O
+.	O
+	O
+PGRMC1	O
+dimer	O
+facilitates	O
+EGFR	O
+-	O
+mediated	O
+cancer	O
+growth	O
+	O
+Next	O
+,	O
+we	O
+investigated	O
+the	O
+functional	O
+significance	O
+of	O
+PGRMC1	O
+dimerization	O
+in	O
+EGFR	O
+signaling	O
+.	O
+	O
+EGF	O
+-	O
+induced	O
+phosphorylations	O
+of	O
+EGFR	O
+and	O
+its	O
+downstream	O
+targets	O
+AKT	O
+and	O
+ERK	O
+were	O
+decreased	O
+by	O
+PGRMC1	O
+knockdown	O
+(	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+)	O
+(	O
+Fig	O
+.	O
+4f	O
+).	O
+	O
+Similarly	O
+,	O
+EGFR	O
+signaling	O
+was	O
+suppressed	O
+by	O
+treatment	O
+of	O
+HCT116	O
+cells	O
+with	O
+SA	O
+(	O
+Fig	O
+.	O
+4g	O
+)	O
+or	O
+CORM3	O
+(	O
+Fig	O
+.	O
+4h	O
+).	O
+	O
+These	O
+results	O
+suggested	O
+that	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+of	O
+PGRMC1	O
+is	O
+critical	O
+for	O
+EGFR	O
+signaling	O
+.	O
+	O
+To	O
+further	O
+investigate	O
+the	O
+role	O
+of	O
+the	O
+dimerized	O
+form	O
+of	O
+PGRMC1	O
+in	O
+cancer	O
+proliferation	O
+,	O
+we	O
+performed	O
+PGRMC1	O
+knockdown	O
+-	O
+rescue	O
+experiments	O
+using	O
+FLAG	O
+-	O
+tagged	O
+wild	O
+-	O
+type	O
+and	O
+Y113F	B-mutant
+PGRMC1	O
+expression	O
+vectors	O
+,	O
+in	O
+which	O
+silent	O
+mutations	O
+were	O
+introduced	O
+into	O
+the	O
+nucleotide	O
+sequence	O
+targeted	O
+by	O
+shRNA	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+	O
+Chemosensitivity	O
+enhancement	O
+by	O
+two	O
+different	O
+shRNAs	O
+to	O
+PGRMC1	O
+was	O
+seen	O
+also	O
+in	O
+HCT116	O
+cells	O
+and	O
+human	O
+hepatoma	O
+HuH7	O
+cells	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+17	O
+).	O
+	O
+Ten	O
+days	O
+after	O
+intra	O
+-	O
+splenic	O
+implantation	O
+of	O
+HCT116	O
+cells	O
+that	O
+were	O
+genetically	O
+tagged	O
+with	O
+a	O
+fluorescent	O
+protein	O
+Venus	O
+,	O
+the	O
+group	O
+implanted	O
+with	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+cells	O
+showed	O
+a	O
+significant	O
+decrease	O
+of	O
+liver	O
+metastasis	O
+in	O
+comparison	O
+with	O
+the	O
+control	O
+group	O
+(	O
+Fig	O
+.	O
+5d	O
+).	O
+	O
+Interaction	O
+of	O
+PGRMC1	O
+dimer	O
+with	O
+cytochromes	O
+P450	O
+	O
+Since	O
+PGRMC1	O
+has	O
+been	O
+shown	O
+to	O
+interact	O
+with	O
+cytochromes	O
+P450	O
+(	O
+ref	O
+),	O
+we	O
+investigated	O
+whether	O
+the	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+of	O
+PGRMC1	O
+is	O
+necessary	O
+for	O
+their	O
+interactions	O
+.	O
+	O
+Moreover	O
+,	O
+the	O
+interaction	O
+of	O
+PGRMC1	O
+with	O
+CYP1A2	O
+was	O
+blocked	O
+by	O
+CORM3	O
+under	O
+reducing	O
+conditions	O
+(	O
+Fig	O
+.	O
+6c	O
+),	O
+indicating	O
+that	O
+PGRMC1	O
+dimerization	O
+is	O
+necessary	O
+for	O
+its	O
+interaction	O
+with	O
+cytochromes	O
+P450	O
+.	O
+	O
+Doxorubicin	O
+is	O
+an	O
+anti	O
+-	O
+cancer	O
+reagent	O
+that	O
+is	O
+metabolized	O
+into	O
+inactive	O
+doxorubicinol	O
+by	O
+CYP2D6	O
+and	O
+CYP3A4	O
+(	O
+Fig	O
+.	O
+6d	O
+).	O
+	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+significantly	O
+suppressed	O
+the	O
+conversion	O
+of	O
+doxorubicin	O
+to	O
+doxorubicinol	O
+(	O
+Fig	O
+.	O
+6d	O
+)	O
+and	O
+increased	O
+sensitivity	O
+to	O
+doxorubicin	O
+(	O
+Fig	O
+.	O
+6e	O
+).	O
+	O
+This	O
+effect	O
+was	O
+reversed	O
+by	O
+co	O
+-	O
+expression	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+PGRMC1	O
+but	O
+not	O
+of	O
+the	O
+Y113F	B-mutant
+mutant	O
+,	O
+suggesting	O
+that	O
+PGRMC1	O
+enhances	O
+doxorubicin	O
+resistance	O
+of	O
+cancer	O
+cells	O
+by	O
+facilitating	O
+its	O
+degradation	O
+via	O
+cytochromes	O
+P450	O
+.	O
+	O
+To	O
+gain	O
+further	O
+insight	O
+into	O
+the	O
+interaction	O
+between	O
+PGRMC1	O
+and	O
+cytochromes	O
+P450	O
+,	O
+surface	O
+plasmon	O
+resonance	O
+analyses	O
+were	O
+conducted	O
+using	O
+recombinant	O
+CYP51	O
+and	O
+PGRMC1	O
+.	O
+	O
+This	O
+was	O
+based	O
+on	O
+a	O
+previous	O
+study	O
+showing	O
+that	O
+PGRMC1	O
+binds	O
+to	O
+CYP51	O
+and	O
+enhances	O
+cholesterol	O
+biosynthesis	O
+by	O
+CYP51	O
+(	O
+refs	O
+).	O
+	O
+CYP51	O
+interacted	O
+with	O
+PGRMC1	O
+in	O
+a	O
+concentration	O
+-	O
+dependent	O
+manner	O
+in	O
+the	O
+presence	O
+of	O
+haem	O
+,	O
+but	O
+not	O
+in	O
+its	O
+absence	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+19	O
+),	O
+suggesting	O
+the	O
+requirement	O
+for	O
+the	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+.	O
+	O
+This	O
+is	O
+the	O
+report	O
+showing	O
+crystallographic	O
+evidence	O
+that	O
+indicates	O
+roles	O
+of	O
+the	O
+direct	O
+haem	O
+–	O
+haem	O
+stacking	O
+in	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+in	O
+eukaryotes	O
+,	O
+although	O
+a	O
+few	O
+examples	O
+are	O
+known	O
+in	O
+bacteria	O
+.	O
+	O
+Recently	O
+,	O
+Peluso	O
+et	O
+al	O
+.	O
+reported	O
+that	O
+PGRMC1	O
+binds	O
+to	O
+PGRMC2	O
+,	O
+suggesting	O
+that	O
+MAPR	O
+family	O
+members	O
+may	O
+also	O
+undergo	O
+haem	O
+-	O
+mediated	O
+heterodimerization	O
+.	O
+	O
+While	O
+the	O
+effects	O
+of	O
+PGRMC1	O
+on	O
+cholesterol	O
+synthesis	O
+mediated	O
+by	O
+CYP51	O
+have	O
+been	O
+well	O
+documented	O
+in	O
+yeast	O
+and	O
+human	O
+cells	O
+,	O
+it	O
+has	O
+not	O
+been	O
+clear	O
+whether	O
+drug	O
+-	O
+metabolizing	O
+CYP	O
+activities	O
+are	O
+regulated	O
+by	O
+PGRMC1	O
+.	O
+	O
+Szczesna	O
+-	O
+Skorupa	O
+and	O
+Kemper	O
+reported	O
+that	O
+PGRMC1	O
+exhibited	O
+an	O
+inhibitory	O
+effect	O
+on	O
+CYP3A4	O
+drug	O
+metabolizing	O
+activity	O
+by	O
+competitively	O
+binding	O
+with	O
+cytochrome	O
+P450	O
+reductase	O
+(	O
+CPR	O
+)	O
+in	O
+HEK293	O
+or	O
+HepG2	O
+cells	O
+.	O
+	O
+Several	O
+other	O
+groups	O
+showed	O
+that	O
+PGRMC1	O
+enhanced	O
+chemoresistance	O
+in	O
+several	O
+cancer	O
+cells	O
+such	O
+as	O
+uterine	O
+sarcoma	O
+,	O
+breast	O
+cancer	O
+,	O
+endometrial	O
+tumour	O
+and	O
+ovarian	O
+cancer	O
+;	O
+however	O
+,	O
+no	O
+evidence	O
+of	O
+PGRMC1	O
+-	O
+dependent	O
+regulation	O
+of	O
+CYP	O
+activity	O
+was	O
+provided	O
+.	O
+	O
+Our	O
+results	O
+showed	O
+that	O
+PGRMC1	O
+contributes	O
+to	O
+enhancement	O
+of	O
+the	O
+doxorubicin	O
+metabolism	O
+,	O
+which	O
+is	O
+mediated	O
+by	O
+CYP2D6	O
+or	O
+CYP3A4	O
+in	O
+human	O
+colon	O
+cancer	O
+HCT116	O
+cells	O
+(	O
+Fig	O
+.	O
+6d	O
+).	O
+	O
+While	O
+the	O
+effects	O
+of	O
+structural	O
+diversity	O
+of	O
+CYP	O
+family	O
+proteins	O
+and	O
+interactions	O
+with	O
+different	O
+xenobiotic	O
+substrates	O
+should	O
+further	O
+be	O
+examined	O
+,	O
+the	O
+current	O
+results	O
+suggest	O
+that	O
+the	O
+interaction	O
+of	O
+drug	O
+-	O
+metabolizing	O
+CYPs	O
+with	O
+the	O
+haem	O
+-	O
+mediated	O
+dimer	O
+of	O
+PGRMC1	O
+plays	O
+a	O
+crucial	O
+role	O
+in	O
+regulating	O
+their	O
+activities	O
+.	O
+	O
+We	O
+showed	O
+that	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+of	O
+PGRMC1	O
+enhances	O
+proliferation	O
+and	O
+chemoresistance	O
+of	O
+cancer	O
+cells	O
+through	O
+binding	O
+to	O
+and	O
+regulating	O
+EGFR	O
+and	O
+cytochromes	O
+P450	O
+(	O
+illustrated	O
+in	O
+Fig	O
+.	O
+7	O
+).	O
+	O
+Since	O
+the	O
+haem	O
+-	O
+binding	O
+affinity	O
+of	O
+PGRMC1	O
+is	O
+lower	O
+than	O
+those	O
+of	O
+constitutive	O
+haem	O
+-	O
+binding	O
+proteins	O
+such	O
+as	O
+myoglobin	O
+,	O
+PGMRC1	O
+is	O
+probably	O
+interconverted	O
+between	O
+apo	O
+-	O
+monomer	O
+and	O
+haem	O
+-	O
+bound	O
+dimer	O
+forms	O
+in	O
+response	O
+to	O
+changes	O
+in	O
+the	O
+intracellular	O
+haem	O
+concentration	O
+.	O
+	O
+Considering	O
+microenvironments	O
+in	O
+and	O
+around	O
+malignant	O
+tumours	O
+,	O
+the	O
+haem	O
+concentration	O
+in	O
+cancer	O
+cells	O
+is	O
+likely	O
+to	O
+be	O
+elevated	O
+through	O
+multiple	O
+mechanisms	O
+,	O
+such	O
+as	O
+(	O
+i	O
+)	O
+an	O
+increased	O
+intake	O
+of	O
+haem	O
+,	O
+(	O
+ii	O
+)	O
+mutation	O
+of	O
+enzymes	O
+in	O
+TCA	O
+cycle	O
+(	O
+for	O
+example	O
+,	O
+fumarate	O
+hydratase	O
+)	O
+that	O
+increases	O
+the	O
+level	O
+of	O
+succinyl	O
+CoA	O
+,	O
+a	O
+substrate	O
+for	O
+haem	O
+biosynthesis	O
+and	O
+(	O
+iii	O
+)	O
+metastasis	O
+to	O
+haem	O
+-	O
+rich	O
+organs	O
+such	O
+as	O
+liver	O
+,	O
+brain	O
+and	O
+bone	O
+marrow	O
+.	O
+	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+excessive	O
+haem	O
+induces	O
+HO	O
+-	O
+1	O
+,	O
+the	O
+enzyme	O
+that	O
+oxidatively	O
+degrades	O
+haem	O
+and	O
+generates	O
+CO	O
+.	O
+	O
+Thus	O
+,	O
+HO	O
+-	O
+1	O
+induction	O
+in	O
+cancer	O
+cells	O
+may	O
+inhibit	O
+the	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+of	O
+PGRMC1	O
+through	O
+the	O
+production	O
+of	O
+CO	O
+and	O
+thereby	O
+suppress	O
+tumour	O
+progression	O
+.	O
+	O
+This	O
+idea	O
+is	O
+consistent	O
+with	O
+the	O
+observation	O
+that	O
+HO	O
+-	O
+1	O
+induction	O
+or	O
+CO	O
+inhibits	O
+tumour	O
+growth	O
+.	O
+	O
+Furthermore	O
+,	O
+Sigma	O
+-	O
+2	O
+ligand	O
+-	O
+binding	O
+is	O
+decreased	O
+in	O
+transgenic	O
+amyloid	O
+beta	O
+deposition	O
+model	O
+APP	O
+/	O
+PS1	O
+female	O
+mice	O
+.	O
+	O
+These	O
+results	O
+suggest	O
+a	O
+possible	O
+involvement	O
+of	O
+PGRMC1	O
+in	O
+Alzheimer	O
+'	O
+s	O
+disease	O
+.	O
+	O
+The	O
+roles	O
+of	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+in	O
+the	O
+functional	O
+regulation	O
+of	O
+its	O
+target	O
+proteins	O
+deserve	O
+further	O
+studies	O
+to	O
+find	O
+evidence	O
+that	O
+therapeutic	O
+interventions	O
+to	O
+interfere	O
+with	O
+the	O
+function	O
+of	O
+the	O
+dimer	O
+may	O
+control	O
+varied	O
+disease	O
+conditions	O
+.	O
+	O
+Two	O
+PGRMC1	O
+subunits	O
+(	O
+blue	O
+and	O
+green	O
+ribbons	O
+)	O
+dimerize	O
+via	O
+stacking	O
+of	O
+the	O
+haem	O
+molecules	O
+.	O
+	O
+(	O
+b	O
+)	O
+Haem	O
+coordination	O
+of	O
+PGRMC1	O
+with	O
+Tyr113	O
+.	O
+	O
+Comparison	O
+of	O
+PGRMC1	O
+(	O
+blue	O
+)	O
+and	O
+cytochrome	O
+b5	O
+(	O
+yellow	O
+,	O
+ID	O
+:	O
+3NER	O
+).	O
+(	O
+c	O
+)	O
+PGRMC1	O
+has	O
+a	O
+longer	O
+helix	O
+(	O
+a	O
+.	O
+a	O
+.	O
+147	O
+–	O
+163	O
+),	O
+which	O
+is	O
+shifted	O
+away	O
+from	O
+the	O
+haem	O
+(	O
+arrow	O
+).	O
+	O
+PGRCM1	O
+is	O
+dimerized	O
+by	O
+binding	O
+with	O
+haem	O
+.	O
+	O
+(	O
+a	O
+)	O
+Mass	O
+spectrometric	O
+analyses	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+(	O
+wt	O
+)	O
+PGRMC1	O
+or	O
+the	O
+C129S	B-mutant
+mutant	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+haem	O
+under	O
+non	O
+-	O
+denaturing	O
+condition	O
+.	O
+	O
+Both	O
+proteins	O
+had	O
+identical	O
+lengths	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+).	O
+	O
+SV	O
+-	O
+AUC	O
+experiments	O
+were	O
+performed	O
+with	O
+1	O
+.	O
+5	O
+mg	O
+ml	O
+−	O
+1	O
+of	O
+PGRMC1	O
+proteins	O
+.	O
+	O
+The	O
+major	O
+peak	O
+with	O
+sedimentation	O
+coefficient	O
+S20	O
+,	O
+w	O
+of	O
+1	O
+.	O
+9	O
+∼	O
+2	O
+.	O
+0	O
+S	O
+(	O
+monomer	O
+)	O
+or	O
+3	O
+.	O
+1	O
+S	O
+(	O
+dimer	O
+)	O
+was	O
+detected	O
+.	O
+	O
+(	O
+c	O
+)	O
+Difference	O
+absorption	O
+spectra	O
+of	O
+PGRMC1	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+)	O
+titrated	O
+with	O
+haem	O
+(	O
+left	O
+panel	O
+).	O
+	O
+The	O
+titration	O
+curve	O
+of	O
+haem	O
+to	O
+PGRMC1	O
+(	O
+right	O
+panel	O
+).	O
+	O
+The	O
+absorbance	O
+difference	O
+at	O
+400	O
+nm	O
+is	O
+plotted	O
+against	O
+the	O
+haem	O
+concentration	O
+.	O
+	O
+Carbon	O
+monoxide	O
+inhibits	O
+haem	O
+-	O
+dependent	O
+PGRMC1	O
+dimerization	O
+.	O
+	O
+(	O
+a	O
+)	O
+UV	O
+-	O
+visible	O
+absorption	O
+spectra	O
+of	O
+PGRMC1	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+).	O
+	O
+Measurements	O
+were	O
+performed	O
+in	O
+the	O
+presence	O
+of	O
+the	O
+oxidized	O
+form	O
+of	O
+haem	O
+(	O
+ferric	O
+),	O
+the	O
+reduced	O
+form	O
+of	O
+haem	O
+(	O
+ferrous	O
+)	O
+and	O
+the	O
+reduced	O
+form	O
+of	O
+haem	O
+plus	O
+CO	O
+gas	O
+(	O
+ferrous	O
++	O
+CO	O
+).	O
+	O
+(	O
+b	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+haem	O
+stacking	O
+.	O
+	O
+(	O
+c	O
+)	O
+Gel	O
+-	O
+filtration	O
+chromatography	O
+analyses	O
+of	O
+PGRMC1	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+)	O
+wild	O
+-	O
+type	O
+(	O
+wt	O
+)	O
+and	O
+the	O
+Y113F	B-mutant
+or	O
+C129S	B-mutant
+mutant	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+haem	O
+,	O
+dithionite	O
+and	O
+/	O
+or	O
+CO	O
+.	O
+(	O
+d	O
+)	O
+Transition	O
+model	O
+for	O
+structural	O
+regulation	O
+of	O
+PGRMC1	O
+in	O
+response	O
+to	O
+haem	O
+and	O
+CO	O
+.	O
+	O
+Haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+is	O
+necessary	O
+for	O
+tumour	O
+proliferation	O
+mediated	O
+by	O
+EGFR	O
+signalling	O
+.	O
+	O
+Input	O
+and	O
+bound	O
+proteins	O
+were	O
+detected	O
+by	O
+Western	O
+blotting	O
+.	O
+	O
+(	O
+b	O
+)	O
+In	O
+vitro	O
+binding	O
+assay	O
+was	O
+performed	O
+as	O
+in	O
+(	O
+a	O
+)	O
+using	O
+haem	O
+-	O
+bound	O
+FLAG	O
+-	O
+PGRMC1	O
+wt	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+)	O
+and	O
+purified	O
+EGFR	O
+with	O
+or	O
+without	O
+treatment	O
+of	O
+RuCl3	O
+and	O
+CORM3	O
+.	O
+	O
+(	O
+c	O
+)	O
+FLAG	O
+-	O
+PGRMC1	O
+wt	O
+or	O
+Y113F	B-mutant
+(	O
+full	O
+length	O
+)	O
+was	O
+over	O
+-	O
+expressed	O
+in	O
+HCT116	O
+cells	O
+and	O
+immunoprecipitated	O
+with	O
+anti	O
+-	O
+FLAG	O
+antibody	O
+-	O
+conjugated	O
+beads	O
+.	O
+	O
+Co	O
+-	O
+immunoprecipitated	O
+proteins	O
+(	O
+FLAG	O
+-	O
+PGRMC1	O
+,	O
+endogenous	O
+PGRMC1	O
+and	O
+EGFR	O
+)	O
+were	O
+detected	O
+with	O
+Western	O
+blotting	O
+by	O
+using	O
+anti	O
+-	O
+PGRMC1	O
+or	O
+anti	O
+-	O
+EGFR	O
+antibody	O
+.	O
+	O
+(	O
+d	O
+)	O
+HCT116	O
+cells	O
+were	O
+treated	O
+with	O
+or	O
+without	O
+250	O
+μmol	O
+l	O
+−	O
+1	O
+of	O
+succinylacetone	O
+(	O
+SA	O
+)	O
+for	O
+48	O
+h	O
+.	O
+The	O
+intracellular	O
+haem	O
+was	O
+extracted	O
+and	O
+quantified	O
+by	O
+reverse	O
+-	O
+phase	O
+HPLC	O
+.	O
+	O
+of	O
+four	O
+separate	O
+experiments	O
+.	O
+**	O
+P	O
+<	O
+0	O
+.	O
+01	O
+using	O
+unpaired	O
+Student	O
+'	O
+s	O
+t	O
+-	O
+test	O
+.	O
+(	O
+e	O
+)	O
+Co	O
+-	O
+immunoprecipitation	O
+assay	O
+was	O
+performed	O
+as	O
+in	O
+(	O
+c	O
+)	O
+with	O
+or	O
+without	O
+SA	O
+treatment	O
+in	O
+HCT116	O
+cells	O
+.	O
+	O
+Haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+accelerates	O
+tumour	O
+growth	O
+through	O
+the	O
+EGFR	O
+signaling	O
+pathway	O
+.	O
+	O
+(	O
+a	O
+)	O
+Nucleotide	O
+sequences	O
+of	O
+PGRMC1	O
+targeted	O
+by	O
+shRNA	O
+and	O
+of	O
+the	O
+shRNA	O
+-	O
+resistant	O
+full	O
+length	O
+PGRMC1	O
+expression	O
+vector	O
+.	O
+	O
+of	O
+four	O
+separate	O
+experiments	O
+.	O
+*	O
+P	O
+<	O
+0	O
+.	O
+01	O
+using	O
+ANOVA	O
+with	O
+Fischer	O
+'	O
+s	O
+LSD	O
+test	O
+.	O
+	O
+(	O
+c	O
+)	O
+Spheroid	O
+formation	O
+in	O
+control	O
+and	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+HCT116	O
+cells	O
+.	O
+	O
+Scale	O
+bar	O
+:	O
+0	O
+.	O
+1	O
+mm	O
+.	O
+(	O
+d	O
+)	O
+Tumour	O
+-	O
+bearing	O
+livers	O
+of	O
+NOG	O
+mice	O
+at	O
+10	O
+days	O
+after	O
+intrasplenic	O
+injection	O
+of	O
+HCT116	O
+(	O
+control	O
+)	O
+or	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+cells	O
+.	O
+	O
+of	O
+10	O
+separate	O
+experiments	O
+.	O
+*	O
+P	O
+<	O
+0	O
+.	O
+05	O
+using	O
+unpaired	O
+Student	O
+'	O
+s	O
+t	O
+-	O
+test	O
+.	O
+	O
+(	O
+d	O
+)	O
+Schematic	O
+illustration	O
+of	O
+doxorubicin	O
+metabolism	O
+is	O
+shown	O
+on	O
+the	O
+left	O
+.	O
+	O
+Doxorubicin	O
+was	O
+incubated	O
+with	O
+HCT116	O
+cells	O
+expressing	O
+control	O
+shRNA	O
+or	O
+shPGRMC1	O
+(	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+),	O
+and	O
+the	O
+doxorubicinol	O
+/	O
+doxorubicin	O
+ratios	O
+in	O
+cell	O
+pellets	O
+were	O
+determined	O
+using	O
+LC	O
+-	O
+MS	O
+.	O
+	O
+of	O
+four	O
+separate	O
+experiments	O
+.	O
+**	O
+P	O
+<	O
+0	O
+.	O
+01	O
+versus	O
+control	O
+using	O
+unpaired	O
+Student	O
+'	O
+s	O
+t	O
+-	O
+test	O
+.	O
+(	O
+e	O
+)	O
+Indicated	O
+amounts	O
+of	O
+doxorubicin	O
+were	O
+added	O
+to	O
+HCT116	O
+(	O
+control	O
+)	O
+cells	O
+,	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+cells	O
+,	O
+or	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+cells	O
+expressing	O
+shRNA	O
+-	O
+resistant	O
+full	O
+-	O
+length	O
+PGRMC1	O
+wt	O
+or	O
+Y113F	B-mutant
+,	O
+and	O
+cell	O
+viability	O
+was	O
+examined	O
+by	O
+MTT	O
+assay	O
+.	O
+	O
+Apo	O
+-	O
+PGRMC1	O
+exists	O
+as	O
+an	O
+inactive	O
+monomer	O
+.	O
+	O
+On	O
+binding	O
+to	O
+haem	O
+,	O
+PGRMC1	O
+forms	O
+a	O
+dimer	O
+through	O
+stacking	O
+interactions	O
+between	O
+the	O
+haem	O
+moieties	O
+,	O
+which	O
+enables	O
+PGRMC1	O
+to	O
+interact	O
+with	O
+EGFR	O
+and	O
+cytochromes	O
+P450	O
+,	O
+leading	O
+to	O
+an	O
+enhanced	O
+proliferation	O
+and	O
+chemoresistance	O
+of	O
+cancer	O
+cells	O
+.	O
+	O
+CO	O
+interferes	O
+with	O
+the	O
+stacking	O
+interactions	O
+of	O
+the	O
+haems	O
+and	O
+thereby	O
+inhibits	O
+PGRMC1	O
+functions	O
+.	O
+	O
+PGRMC1	O
+proteins	O
+exhibit	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+in	O
+solution	O
+.	O
+	O
+Apo	O
+form	O
+Haem	O
+-	O
+bound	O
+form	O
+Mass	O
+(	O
+Da	O
+)	O
+Mass	O
+(	O
+Da	O
+)	O
+aPGRMC1	O
+wt	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+)	O
+ESI	O
+-	O
+MS	O
+—	O
+17	O
+,	O
+844	O
+.	O
+14	O
+—	O
+36	O
+,	O
+920	O
+.	O
+19	O
+Theoretical	O
+17	O
+,	O
+843	O
+.	O
+65	O
+36	O
+,	O
+918	O
+.	O
+06	O
+Hydrodynamic	O
+radius	O
+10	O
+−	O
+9	O
+(	O
+m	O
+)	O
+MW	O
+(	O
+kDa	O
+)	O
+Hydrodynamic	O
+radius	O
+10	O
+−	O
+9	O
+(	O
+m	O
+)	O
+MW	O
+(	O
+kDa	O
+)	O
+DOSY	O
+2	O
+.	O
+04	O
+–	O
+2	O
+.	O
+15	O
+20	O
+2	O
+.	O
+94	O
+–	O
+3	O
+.	O
+02	O
+42	O
+S20	O
+,	O
+w	O
+(	O
+S	O
+)	O
+MW	O
+(	O
+kDa	O
+)	O
+S20	O
+,	O
+w	O
+(	O
+S	O
+)	O
+MW	O
+(	O
+kDa	O
+)	O
+SV	O
+-	O
+AUC	O
+1	O
+.	O
+9	O
+17	O
+.	O
+6	O
+3	O
+.	O
+1	O
+35	O
+.	O
+5	O
+bPGRMC1	O
+C129S	B-mutant
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+)	O
+ESI	O
+-	O
+MS	O
+—	O
+17	O
+,	O
+827	O
+.	O
+91	O
+—	O
+36	O
+,	O
+887	O
+.	O
+07	O
+Theoretical	O
+17	O
+,	O
+827	O
+.	O
+59	O
+36	O
+,	O
+885	O
+.	O
+6	O
+S20	O
+,	O
+w	O
+(	O
+S	O
+)	O
+MW	O
+(	O
+kDa	O
+)	O
+S20	O
+,	O
+w	O
+(	O
+S	O
+)	O
+MW	O
+(	O
+kDa	O
+)	O
+SV	O
+-	O
+AUC	O
+2	O
+.	O
+0	O
+18	O
+.	O
+1	O
+3	O
+.	O
+1	O
+35	O
+.	O
+8	O
+	O
+The	O
+protein	O
+sizes	O
+of	O
+the	O
+wt	O
+and	O
+C129S	B-mutant
+PGRMC1	O
+cytosolic	O
+domains	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+)	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+haem	O
+were	O
+estimated	O
+by	O
+ESI	O
+-	O
+MS	O
+,	O
+DOSY	O
+and	O
+SV	O
+-	O
+AUC	O
+.	O
+	O
+However	O
+,	O
+the	O
+mechanisms	O
+that	O
+allow	O
+the	O
+clonal	O
+T	O
+cell	O
+antigen	O
+receptor	O
+(	O
+TCR	O
+)	O
+to	O
+functionally	O
+engage	O
+multiple	O
+peptide	O
+–	O
+major	O
+histocompatibility	O
+complexes	O
+(	O
+pMHC	O
+)	O
+are	O
+unclear	O
+.	O
+	O
+Here	O
+,	O
+we	O
+studied	O
+multiligand	O
+discrimination	O
+by	O
+a	O
+human	O
+,	O
+preproinsulin	O
+reactive	O
+,	O
+MHC	O
+class	O
+-	O
+I	O
+–	O
+restricted	O
+CD8	O
++	O
+T	O
+cell	O
+clone	O
+(	O
+1E6	O
+)	O
+that	O
+can	O
+recognize	O
+over	O
+1	O
+million	O
+different	O
+peptides	O
+.	O
+	O
+Evaluation	O
+of	O
+these	O
+structures	O
+demonstrated	O
+that	O
+binding	O
+was	O
+stabilized	O
+through	O
+a	O
+conserved	O
+lock	O
+-	O
+and	O
+-	O
+key	O
+–	O
+like	O
+minimal	O
+binding	O
+footprint	O
+that	O
+enables	O
+1E6	O
+TCR	O
+to	O
+tolerate	O
+vast	O
+numbers	O
+of	O
+substitutions	O
+outside	O
+of	O
+this	O
+so	O
+-	O
+called	O
+hotspot	O
+.	O
+	O
+T	O
+cells	O
+perform	O
+an	O
+essential	O
+role	O
+in	O
+adaptive	O
+immunity	O
+by	O
+interrogating	O
+the	O
+host	O
+proteome	O
+for	O
+anomalies	O
+,	O
+classically	O
+by	O
+recognizing	O
+peptides	O
+bound	O
+in	O
+major	O
+histocompatibility	O
+(	O
+MHC	O
+)	O
+molecules	O
+at	O
+the	O
+cell	O
+surface	O
+.	O
+	O
+Recent	O
+data	O
+supports	O
+the	O
+notion	O
+that	O
+,	O
+to	O
+perform	O
+this	O
+role	O
+,	O
+the	O
+highly	O
+variable	O
+αβ	O
+T	O
+cell	O
+antigen	O
+receptor	O
+(	O
+TCR	O
+)	O
+must	O
+be	O
+able	O
+to	O
+recognize	O
+thousands	O
+,	O
+if	O
+not	O
+millions	O
+,	O
+of	O
+different	O
+peptide	O
+ligands	O
+.	O
+	O
+Several	O
+mechanisms	O
+,	O
+by	O
+which	O
+TCRs	O
+could	O
+bind	O
+to	O
+a	O
+large	O
+number	O
+of	O
+different	O
+peptide	O
+-	O
+MHC	O
+(	O
+pMHC	O
+),	O
+have	O
+been	O
+proposed	O
+.	O
+	O
+Structures	O
+of	O
+unligated	O
+and	O
+ligated	O
+TCRs	O
+have	O
+shown	O
+that	O
+the	O
+TCR	O
+complementarity	O
+determining	O
+region	O
+(	O
+CDR	O
+)	O
+loops	O
+can	O
+be	O
+flexible	O
+,	O
+perhaps	O
+enabling	O
+peptide	O
+binding	O
+using	O
+different	O
+loop	O
+conformations	O
+.	O
+	O
+Other	O
+studies	O
+,	O
+mainly	O
+in	O
+the	O
+murine	O
+system	O
+,	O
+have	O
+demonstrated	O
+that	O
+the	O
+same	O
+TCR	O
+can	O
+interact	O
+with	O
+different	O
+pMHCs	O
+using	O
+a	O
+common	O
+or	O
+divergent	O
+modality	O
+.	O
+	O
+Recent	O
+studies	O
+in	O
+model	O
+murine	O
+systems	O
+demonstrate	O
+that	O
+TCR	O
+cross	O
+-	O
+reactivity	O
+can	O
+be	O
+governed	O
+by	O
+recognition	O
+of	O
+a	O
+conserved	O
+region	O
+in	O
+the	O
+peptide	O
+that	O
+allows	O
+tolerance	O
+of	O
+peptide	O
+sequence	O
+variation	O
+outside	O
+of	O
+this	O
+hotspot	O
+.	O
+	O
+CD8	O
++	O
+T	O
+cells	O
+that	O
+recognize	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+–	O
+ALWGPDPAAA	O
+have	O
+been	O
+shown	O
+to	O
+populate	O
+insulitic	O
+lesions	O
+in	O
+patients	O
+with	O
+type	O
+1	O
+diabetes	O
+(	O
+T1D	O
+).	O
+	O
+We	O
+demonstrated	O
+that	O
+the	O
+TCR	O
+from	O
+the	O
+1E6	O
+T	O
+cell	O
+clone	O
+bound	O
+to	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+–	O
+ALWGPDPAAA	O
+using	O
+a	O
+limited	O
+footprint	O
+and	O
+very	O
+weak	O
+binding	O
+affinity	O
+.	O
+	O
+Here	O
+,	O
+we	O
+solved	O
+the	O
+structure	O
+of	O
+the	O
+1E6	O
+TCR	O
+with	O
+7	O
+altered	O
+peptide	O
+ligands	O
+(	O
+APLs	O
+)	O
+determined	O
+by	O
+our	O
+previously	O
+published	O
+combinatorial	O
+peptide	O
+library	O
+(	O
+CPL	O
+)	O
+screening	O
+,	O
+2	O
+of	O
+which	O
+mapped	O
+within	O
+human	O
+pathogens	O
+.	O
+	O
+We	O
+also	O
+solved	O
+the	O
+structure	O
+of	O
+each	O
+unligated	O
+APL	O
+to	O
+investigate	O
+whether	O
+structural	O
+changes	O
+occurred	O
+before	O
+or	O
+after	O
+binding	O
+—	O
+which	O
+,	O
+combined	O
+with	O
+an	O
+in	O
+-	O
+depth	O
+cellular	O
+and	O
+biophysical	O
+analysis	O
+of	O
+the	O
+1E6	O
+interaction	O
+with	O
+each	O
+APL	O
+,	O
+demonstrated	O
+the	O
+molecular	O
+mechanism	O
+mediating	O
+the	O
+high	O
+level	O
+of	O
+cross	O
+-	O
+reactivity	O
+exhibited	O
+by	O
+this	O
+preproinsulin	O
+-	O
+reactive	O
+human	O
+CD8	O
++	O
+T	O
+cell	O
+clone	O
+.	O
+	O
+The	O
+1E6	O
+T	O
+cell	O
+clone	O
+recognizes	O
+APLs	O
+across	O
+a	O
+large	O
+dynamic	O
+range	O
+.	O
+	O
+We	O
+have	O
+previously	O
+demonstrated	O
+that	O
+the	O
+1E6	O
+T	O
+cell	O
+clone	O
+can	O
+recognize	O
+over	O
+1	O
+million	O
+different	O
+peptides	O
+with	O
+a	O
+potency	O
+comparable	O
+with	O
+,	O
+or	O
+better	O
+than	O
+,	O
+the	O
+cognate	O
+preproinsulin	O
+peptide	O
+ALWGPDPAAA	O
+.	O
+	O
+From	O
+this	O
+large	O
+functional	O
+scan	O
+,	O
+we	O
+selected	O
+7	O
+different	O
+APLs	O
+that	O
+activated	O
+the	O
+1E6	O
+T	O
+cell	O
+clone	O
+across	O
+a	O
+wide	O
+(	O
+4	O
+-	O
+log	O
+)	O
+functional	O
+range	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Competitive	O
+functional	O
+testing	O
+revealed	O
+that	O
+the	O
+preproinsulin	O
+-	O
+derived	O
+sequence	O
+ALWGPDPAAA	O
+was	O
+one	O
+of	O
+the	O
+least	O
+potent	O
+targets	O
+for	O
+1E6	O
+,	O
+with	O
+only	O
+the	O
+MVWGPDPLYV	O
+and	O
+YLGGPDFPTI	O
+demonstrating	O
+a	O
+similar	O
+low	O
+-	O
+activity	O
+profile	O
+in	O
+MIP	O
+-	O
+1β	O
+secretion	O
+and	O
+target	O
+killing	O
+assays	O
+(	O
+Figure	O
+1	O
+,	O
+A	O
+and	O
+B	O
+).	O
+	O
+At	O
+the	O
+other	O
+end	O
+of	O
+the	O
+spectrum	O
+,	O
+the	O
+RQFGPDFPTI	O
+peptide	O
+stimulated	O
+MIP	O
+-	O
+1β	O
+release	O
+and	O
+killing	O
+by	O
+1E6	O
+at	O
+an	O
+exogenous	O
+peptide	O
+concentration	O
+2	O
+–	O
+3	O
+logs	O
+lower	O
+compared	O
+with	O
+ALWGPDPAAA	O
+.	O
+	O
+The	O
+pattern	O
+of	O
+peptide	O
+potency	O
+was	O
+closely	O
+mirrored	O
+by	O
+pMHC	O
+tetramer	O
+staining	O
+experiments	O
+(	O
+Figure	O
+1C	O
+and	O
+plots	O
+shown	O
+in	O
+Supplemental	O
+Figure	O
+1	O
+;	O
+supplemental	O
+material	O
+available	O
+online	O
+with	O
+this	O
+article	O
+;	O
+doi	O
+:	O
+10	O
+.	O
+1172	O
+/	O
+JCI85679DS1	O
+).	O
+	O
+Here	O
+,	O
+the	O
+A2	O
+-	O
+RQFGPDFPTI	O
+tetramer	O
+stained	O
+1E6	O
+with	O
+the	O
+greatest	O
+MFI	O
+,	O
+gradually	O
+decreasing	O
+to	O
+the	O
+weakest	O
+tetramers	O
+:	O
+A2	O
+-	O
+MVWGPDPLYV	O
+and	O
+-	O
+YLGGPDFPTI	O
+.	O
+	O
+To	O
+parallel	O
+the	O
+functional	O
+analysis	O
+,	O
+we	O
+also	O
+performed	O
+thermal	O
+melt	O
+(	O
+Tm	O
+)	O
+experiments	O
+using	O
+synchrotron	O
+radiation	O
+circular	O
+dichroism	O
+(	O
+SRCD	O
+)	O
+to	O
+investigate	O
+the	O
+stability	O
+of	O
+each	O
+APL	O
+(	O
+Figure	O
+1D	O
+).	O
+	O
+This	O
+pattern	O
+of	O
+stability	O
+did	O
+not	O
+correlate	O
+with	O
+the	O
+T	O
+cell	O
+activation	O
+or	O
+tetramer	O
+staining	O
+experiments	O
+,	O
+indicating	O
+that	O
+peptide	O
+binding	O
+to	O
+the	O
+MHC	O
+do	O
+not	O
+explain	O
+ligand	O
+potency	O
+.	O
+	O
+The	O
+1E6	O
+TCR	O
+can	O
+bind	O
+peptides	O
+with	O
+strong	O
+antipathogen	O
+-	O
+like	O
+affinities	O
+.	O
+	O
+We	O
+,	O
+and	O
+others	O
+,	O
+have	O
+previously	O
+demonstrated	O
+that	O
+antipathogenic	O
+TCRs	O
+tend	O
+to	O
+bind	O
+with	O
+stronger	O
+affinity	O
+compared	O
+with	O
+self	O
+-	O
+reactive	O
+TCRs	O
+,	O
+likely	O
+a	O
+consequence	O
+of	O
+the	O
+deletion	O
+of	O
+T	O
+cells	O
+with	O
+high	O
+-	O
+affinity	O
+self	O
+-	O
+reactive	O
+TCR	O
+during	O
+thymic	O
+selection	O
+.	O
+	O
+In	O
+accordance	O
+with	O
+this	O
+trend	O
+,	O
+the	O
+1E6	O
+TCR	O
+bound	O
+the	O
+natural	O
+preproinsulin	O
+peptide	O
+,	O
+ALWGPDPAAA	O
+,	O
+with	O
+the	O
+weakest	O
+affinity	O
+currently	O
+published	O
+for	O
+a	O
+human	O
+CD8	O
++	O
+T	O
+cell	O
+–	O
+derived	O
+TCR	O
+with	O
+a	O
+biologically	O
+relevant	O
+ligand	O
+(	O
+KD	O
+>	O
+200	O
+μM	O
+;	O
+KD	O
+,	O
+equilibrium	O
+binding	O
+constant	O
+).	O
+	O
+Surface	O
+plasmon	O
+resonance	O
+(	O
+SPR	O
+)	O
+analysis	O
+of	O
+the	O
+1E6	O
+TCR	O
+–	O
+pMHC	O
+interaction	O
+for	O
+all	O
+7	O
+APLs	O
+(	O
+Figure	O
+2	O
+,	O
+A	O
+–	O
+H	O
+)	O
+demonstrated	O
+that	O
+stronger	O
+binding	O
+affinity	O
+(	O
+represented	O
+as	O
+ΔG	O
+°,	O
+kcal	O
+/	O
+mol	O
+)	O
+correlated	O
+well	O
+with	O
+the	O
+EC50	O
+values	O
+(	O
+peptide	O
+concentration	O
+required	O
+to	O
+reach	O
+half	O
+-	O
+maximal	O
+1E6	O
+T	O
+cell	O
+killing	O
+)	O
+for	O
+each	O
+ligand	O
+,	O
+demonstrated	O
+by	O
+a	O
+Pearson	O
+’	O
+s	O
+correlation	O
+analysis	O
+value	O
+of	O
+0	O
+.	O
+8	O
+(	O
+P	O
+=	O
+0	O
+.	O
+01	O
+)	O
+(	O
+Figure	O
+2I	O
+).	O
+	O
+It	O
+should	O
+be	O
+noted	O
+that	O
+this	O
+correlation	O
+,	O
+although	O
+consistent	O
+with	O
+the	O
+T	O
+cell	O
+killing	O
+experiments	O
+,	O
+uses	O
+only	O
+approximate	O
+affinities	O
+calculated	O
+for	O
+the	O
+2	O
+weakest	O
+ligands	O
+.	O
+	O
+Third	O
+,	O
+the	O
+1E6	O
+TCR	O
+bound	O
+to	O
+A2	O
+-	O
+RQFGPDWIVA	O
+peptide	O
+,	O
+within	O
+the	O
+C	O
+.	O
+asparagiforme	O
+proteome	O
+,	O
+with	O
+approximately	O
+4	O
+-	O
+fold	O
+stronger	O
+affinity	O
+than	O
+A2	O
+-	O
+ALWGPDPAAA	O
+,	O
+demonstrating	O
+the	O
+potential	O
+for	O
+a	O
+pathogen	O
+-	O
+derived	O
+antigen	O
+to	O
+initiate	O
+a	O
+response	O
+to	O
+the	O
+self	O
+-	O
+derived	O
+sequence	O
+.	O
+	O
+Finally	O
+,	O
+these	O
+data	O
+demonstrate	O
+the	O
+largest	O
+range	O
+of	O
+binding	O
+affinities	O
+reported	O
+for	O
+a	O
+natural	O
+,	O
+endogenous	O
+human	O
+TCR	O
+of	O
+more	O
+than	O
+3	O
+logs	O
+of	O
+magnitude	O
+(	O
+A2	O
+-	O
+MVWGPDPLYV	O
+vs	O
+.	O
+A2	O
+-	O
+RQFGPDFPTI	O
+).	O
+	O
+In	O
+agreement	O
+with	O
+SPR	O
+experiments	O
+,	O
+the	O
+range	O
+of	O
+2D	O
+affinities	O
+we	O
+detected	O
+differed	O
+by	O
+around	O
+3	O
+logs	O
+,	O
+with	O
+the	O
+A2	O
+-	O
+MVWGPDPLYV	O
+generating	O
+the	O
+weakest	O
+2D	O
+affinity	O
+(	O
+2	O
+.	O
+6	O
+×	O
+10	O
+–	O
+5	O
+AcKa	O
+μm4	O
+)	O
+and	O
+A2	O
+-	O
+RQFGPDFPTI	O
+the	O
+strongest	O
+(	O
+4	O
+.	O
+5	O
+×	O
+10	O
+–	O
+2	O
+AcKa	O
+μm4	O
+)	O
+(	O
+Figure	O
+2J	O
+).	O
+	O
+Of	O
+note	O
+,	O
+these	O
+data	O
+demonstrate	O
+a	O
+close	O
+agreement	O
+between	O
+the	O
+3D	O
+affinity	O
+values	O
+generated	O
+using	O
+SPR	O
+and	O
+2D	O
+affinity	O
+values	O
+generated	O
+using	O
+adhesion	O
+frequency	O
+assays	O
+.	O
+	O
+The	O
+1E6	O
+TCR	O
+uses	O
+a	O
+consensus	O
+binding	O
+mode	O
+to	O
+engage	O
+multiple	O
+APLs	O
+.	O
+	O
+Our	O
+previous	O
+structure	O
+of	O
+the	O
+1E6	O
+-	O
+A2	O
+-	O
+ALWGPDPAAA	O
+complex	O
+demonstrated	O
+a	O
+limited	O
+binding	O
+footprint	O
+between	O
+the	O
+TCR	O
+and	O
+pMHC	O
+.	O
+	O
+In	O
+order	O
+to	O
+examine	O
+the	O
+mechanism	O
+by	O
+which	O
+the	O
+1E6	O
+TCR	O
+engaged	O
+a	O
+wide	O
+range	O
+of	O
+peptides	O
+with	O
+divergent	O
+binding	O
+affinities	O
+,	O
+we	O
+solved	O
+the	O
+structure	O
+of	O
+the	O
+1E6	O
+TCR	O
+in	O
+complex	O
+with	O
+all	O
+7	O
+APLs	O
+used	O
+in	O
+Figure	O
+2	O
+.	O
+	O
+All	O
+structures	O
+were	O
+solved	O
+in	O
+space	O
+group	O
+P1	O
+to	O
+2	O
+–	O
+3	O
+Å	O
+resolution	O
+with	O
+crystallographic	O
+Rwork	O
+/	O
+Rfree	O
+ratios	O
+within	O
+accepted	O
+limits	O
+as	O
+shown	O
+in	O
+the	O
+theoretically	O
+expected	O
+distribution	O
+(	O
+ref	O
+.	O
+and	O
+Supplemental	O
+Table	O
+1	O
+).	O
+	O
+The	O
+1E6	O
+TCR	O
+used	O
+a	O
+very	O
+similar	O
+overall	O
+binding	O
+modality	O
+to	O
+engage	O
+all	O
+of	O
+the	O
+APLs	O
+,	O
+with	O
+root	O
+mean	O
+square	O
+deviation	O
+ranging	O
+between	O
+0	O
+.	O
+81	O
+and	O
+1	O
+.	O
+12	O
+Å2	O
+(	O
+compared	O
+with	O
+1E6	O
+-	O
+A2	O
+-	O
+ALWGPDPAAA	O
+).	O
+	O
+The	O
+relatively	O
+broad	O
+range	O
+of	O
+buried	O
+surface	O
+areas	O
+(	O
+1	O
+,	O
+670	O
+–	O
+1	O
+,	O
+920	O
+Å2	O
+)	O
+did	O
+not	O
+correlate	O
+well	O
+with	O
+TCR	O
+binding	O
+affinity	O
+(	O
+Pearson	O
+’	O
+s	O
+correlation	O
+=	O
+0	O
+.	O
+45	O
+,	O
+P	O
+=	O
+0	O
+.	O
+2	O
+).	O
+	O
+The	O
+surface	O
+complementarity	O
+values	O
+(	O
+0	O
+.	O
+52	O
+–	O
+0	O
+.	O
+7	O
+)	O
+correlated	O
+slightly	O
+with	O
+affinity	O
+(	O
+Pearson	O
+’	O
+s	O
+correlation	O
+=	O
+0	O
+.	O
+7	O
+,	O
+P	O
+=	O
+0	O
+.	O
+05	O
+)	O
+but	O
+could	O
+not	O
+explain	O
+all	O
+differences	O
+in	O
+binding	O
+(	O
+Figure	O
+3A	O
+and	O
+Table	O
+2	O
+).	O
+	O
+The	O
+TCR	O
+CDR	O
+loops	O
+were	O
+in	O
+a	O
+very	O
+similar	O
+position	O
+in	O
+all	O
+complexes	O
+,	O
+apart	O
+from	O
+some	O
+slight	O
+deviations	O
+in	O
+the	O
+TCR	O
+β	O
+-	O
+chain	O
+(	O
+Figure	O
+3B	O
+);	O
+the	O
+peptides	O
+were	O
+all	O
+presented	O
+in	O
+a	O
+similar	O
+conformation	O
+(	O
+Figure	O
+3C	O
+);	O
+and	O
+there	O
+was	O
+minimal	O
+variation	O
+in	O
+crossing	O
+angles	O
+of	O
+the	O
+TCR	O
+(	O
+42	O
+.	O
+3	O
+°–	O
+45	O
+.	O
+6	O
+°)	O
+(	O
+Figure	O
+3D	O
+).	O
+	O
+However	O
+,	O
+subtle	O
+differences	O
+in	O
+the	O
+respective	O
+interfaces	O
+were	O
+apparent	O
+(	O
+discussed	O
+below	O
+)	O
+and	O
+resulted	O
+in	O
+altered	O
+binding	O
+affinities	O
+of	O
+the	O
+respective	O
+complexes	O
+.	O
+	O
+Interactions	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+different	O
+APLs	O
+are	O
+focused	O
+around	O
+a	O
+conserved	O
+GPD	O
+peptide	O
+motif	O
+.	O
+	O
+We	O
+next	O
+performed	O
+an	O
+in	O
+-	O
+depth	O
+atomic	O
+analysis	O
+of	O
+the	O
+contacts	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+each	O
+APL	O
+to	O
+determine	O
+the	O
+structural	O
+basis	O
+for	O
+the	O
+altered	O
+T	O
+cell	O
+peptide	O
+sensitivities	O
+and	O
+TCR	O
+binding	O
+affinities	O
+(	O
+Table	O
+2	O
+).	O
+	O
+Concomitant	O
+with	O
+our	O
+global	O
+analysis	O
+of	O
+1E6	O
+TCR	O
+binding	O
+to	O
+the	O
+APLs	O
+,	O
+we	O
+observed	O
+a	O
+common	O
+interaction	O
+element	O
+,	O
+consistent	O
+with	O
+our	O
+previous	O
+findings	O
+,	O
+that	O
+utilized	O
+TCR	O
+residues	O
+Tyr97α	O
+and	O
+Trp97β	O
+,	O
+forming	O
+an	O
+aromatic	O
+cap	O
+over	O
+a	O
+central	O
+GPD	O
+motif	O
+that	O
+was	O
+present	O
+in	O
+all	O
+of	O
+the	O
+APLs	O
+(	O
+Figure	O
+4	O
+).	O
+	O
+Interactions	O
+between	O
+these	O
+2	O
+TCR	O
+and	O
+3	O
+peptide	O
+residues	O
+accounted	O
+for	O
+41	O
+%–	O
+50	O
+%	O
+of	O
+the	O
+total	O
+contacts	O
+across	O
+all	O
+complexes	O
+(	O
+Table	O
+2	O
+),	O
+demonstrating	O
+the	O
+conserved	O
+peptide	O
+centric	O
+binding	O
+mode	O
+utilized	O
+by	O
+the	O
+1E6	O
+TCR	O
+.	O
+	O
+This	O
+fixed	O
+anchoring	O
+between	O
+the	O
+2	O
+molecules	O
+was	O
+important	O
+for	O
+stabilization	O
+of	O
+the	O
+TCR	O
+-	O
+pMHC	O
+complex	O
+,	O
+as	O
+—	O
+although	O
+other	O
+peptides	O
+without	O
+the	O
+‘	O
+GDP	O
+’	O
+motif	O
+were	O
+tested	O
+and	O
+shown	O
+to	O
+activate	O
+the	O
+1E6	O
+T	O
+cell	O
+clone	O
+—	O
+we	O
+were	O
+unable	O
+to	O
+measure	O
+robust	O
+affinities	O
+using	O
+SPR	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+	O
+These	O
+data	O
+support	O
+the	O
+requirement	O
+for	O
+a	O
+conserved	O
+interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+the	O
+GPD	O
+motif	O
+,	O
+as	O
+we	O
+observed	O
+in	O
+our	O
+previously	O
+published	O
+1E6	O
+-	O
+A2	O
+-	O
+ALWGPDPAAA	O
+structure	O
+.	O
+	O
+For	O
+example	O
+,	O
+the	O
+1E6	O
+TCR	O
+made	O
+only	O
+47	O
+peptide	O
+contacts	O
+with	O
+A2	O
+-	O
+MVWGPDPLYV	O
+(	O
+KD	O
+=	O
+~	O
+600	O
+μM	O
+)	O
+compared	O
+with	O
+63	O
+and	O
+57	O
+contacts	O
+with	O
+A2	O
+-	O
+YQFGPDFPIA	O
+(	O
+KD	O
+=	O
+7	O
+.	O
+4	O
+μM	O
+)	O
+and	O
+A2	O
+-	O
+RQFGPDFPTI	O
+(	O
+KD	O
+=	O
+0	O
+.	O
+5	O
+μM	O
+),	O
+respectively	O
+.	O
+	O
+For	O
+example	O
+,	O
+the	O
+1E6	O
+TCR	O
+made	O
+64	O
+peptide	O
+contacts	O
+with	O
+A2	O
+-	O
+YLGGPDFPTI	O
+(	O
+KD	O
+=	O
+~	O
+400	O
+μM	O
+)	O
+compared	O
+with	O
+43	O
+contacts	O
+with	O
+A2	O
+-	O
+RQWGPDPAAV	O
+(	O
+KD	O
+=	O
+7	O
+.	O
+8	O
+μM	O
+).	O
+	O
+The	O
+most	O
+important	O
+peptide	O
+modification	O
+in	O
+terms	O
+of	O
+generating	O
+new	O
+contacts	O
+was	O
+peptide	O
+position	O
+1	O
+.	O
+	O
+The	O
+stronger	O
+ligands	O
+all	O
+encoded	O
+larger	O
+side	O
+chains	O
+(	O
+Arg	O
+or	O
+Tyr	O
+)	O
+at	O
+peptide	O
+position	O
+1	O
+(	O
+Figure	O
+5	O
+,	O
+E	O
+–	O
+H	O
+),	O
+enabling	O
+interactions	O
+with	O
+1E6	O
+that	O
+were	O
+not	O
+present	O
+in	O
+the	O
+weaker	O
+APLs	O
+that	O
+lacked	O
+large	O
+side	O
+chains	O
+in	O
+this	O
+position	O
+(	O
+Figure	O
+5	O
+,	O
+A	O
+,	O
+C	O
+,	O
+and	O
+D	O
+).	O
+	O
+We	O
+have	O
+previously	O
+shown	O
+that	O
+the	O
+1E6	O
+TCR	O
+uses	O
+a	O
+rigid	O
+lock	O
+-	O
+and	O
+-	O
+key	O
+mechanism	O
+during	O
+binding	O
+to	O
+A2	O
+-	O
+ALWGPDPAAA	O
+.	O
+	O
+In	O
+order	O
+to	O
+determine	O
+whether	O
+any	O
+of	O
+the	O
+APLs	O
+required	O
+an	O
+induced	O
+fit	O
+mechanism	O
+during	O
+binding	O
+that	O
+could	O
+explain	O
+the	O
+difference	O
+in	O
+free	O
+binding	O
+energy	O
+(	O
+ΔG	O
+)	O
+between	O
+each	O
+complex	O
+(	O
+Table	O
+2	O
+),	O
+we	O
+solved	O
+the	O
+unligated	O
+structures	O
+of	O
+all	O
+7	O
+APLs	O
+(	O
+the	O
+A2	O
+-	O
+ALWGPDPAAA	O
+structure	O
+has	O
+been	O
+previously	O
+published	O
+and	O
+was	O
+used	O
+in	O
+this	O
+comparison	O
+,	O
+ref	O
+.)	O
+(	O
+Figure	O
+6	O
+and	O
+Supplemental	O
+Table	O
+2	O
+).	O
+	O
+This	O
+movement	O
+could	O
+result	O
+in	O
+an	O
+entropic	O
+penalty	O
+contributing	O
+to	O
+the	O
+weak	O
+TCR	O
+binding	O
+affinity	O
+we	O
+observed	O
+for	O
+this	O
+ligand	O
+.	O
+	O
+Additional	O
+small	O
+movements	O
+in	O
+the	O
+Cα	O
+backbone	O
+of	O
+the	O
+peptide	O
+around	O
+peptide	O
+residue	O
+Asp6	O
+were	O
+apparent	O
+in	O
+the	O
+A2	O
+-	O
+YLGGPDFPTI	O
+(	O
+KD	O
+=	O
+~	O
+400	O
+μM	O
+),	O
+A2	O
+-	O
+ALWGPDPAAA	O
+(	O
+KD	O
+=	O
+~	O
+208	O
+μM	O
+),	O
+and	O
+A2	O
+-	O
+RQFGPDWIVA	O
+(	O
+KD	O
+=	O
+44	O
+.	O
+4	O
+μM	O
+)	O
+structures	O
+(	O
+Figure	O
+6	O
+,	O
+B	O
+,	O
+C	O
+,	O
+and	O
+E	O
+).	O
+	O
+The	O
+unligated	O
+structures	O
+of	O
+A2	O
+-	O
+AQWGPDAAA	O
+,	O
+A2	O
+-	O
+RQWGPDPAAV	O
+,	O
+A2	O
+-	O
+YQFGPDFPIA	O
+,	O
+and	O
+A2	O
+-	O
+RQFGPDFPTI	O
+were	O
+virtually	O
+identical	O
+when	O
+in	O
+complex	O
+with	O
+1E6	O
+(	O
+Figure	O
+6	O
+,	O
+D	O
+and	O
+F	O
+–	O
+H	O
+).	O
+	O
+Apart	O
+from	O
+the	O
+case	O
+of	O
+A2	O
+-	O
+AQWGPDAAA	O
+(	O
+KD	O
+=	O
+61	O
+.	O
+9	O
+μM	O
+),	O
+these	O
+observations	O
+support	O
+the	O
+conclusion	O
+that	O
+the	O
+higher	O
+-	O
+affinity	O
+ligands	O
+required	O
+less	O
+conformational	O
+melding	O
+during	O
+binding	O
+,	O
+which	O
+could	O
+be	O
+energetically	O
+beneficial	O
+(	O
+lower	O
+entopic	O
+cost	O
+)	O
+during	O
+ligation	O
+with	O
+the	O
+1E6	O
+TCR	O
+.	O
+	O
+Peptide	O
+modifications	O
+alter	O
+the	O
+interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+the	O
+MHC	O
+surface	O
+.	O
+	O
+In	O
+addition	O
+to	O
+changes	O
+between	O
+the	O
+TCR	O
+and	O
+peptide	O
+component	O
+,	O
+we	O
+also	O
+observed	O
+that	O
+different	O
+APLs	O
+had	O
+different	O
+knock	O
+-	O
+on	O
+effects	O
+between	O
+the	O
+TCR	O
+and	O
+MHC	O
+.	O
+	O
+MHC	O
+residue	O
+Arg65	O
+that	O
+forms	O
+part	O
+of	O
+the	O
+MHC	O
+restriction	O
+triad	O
+(	O
+Arg65	O
+,	O
+Ala69	O
+,	O
+and	O
+Gln155	O
+)	O
+played	O
+a	O
+central	O
+role	O
+in	O
+TCR	O
+-	O
+MHC	O
+contacts	O
+,	O
+with	O
+Gln155	O
+playing	O
+a	O
+less	O
+important	O
+role	O
+and	O
+Ala69	O
+playing	O
+no	O
+role	O
+in	O
+binding	O
+at	O
+the	O
+interface	O
+(	O
+Figure	O
+7	O
+).	O
+	O
+Generally	O
+,	O
+the	O
+weaker	O
+-	O
+affinity	O
+APLs	O
+made	O
+fewer	O
+contacts	O
+with	O
+the	O
+MHC	O
+surface	O
+(	O
+27	O
+–	O
+29	O
+interactions	O
+)	O
+compared	O
+with	O
+the	O
+stronger	O
+-	O
+affinity	O
+APLs	O
+(	O
+29	O
+–	O
+35	O
+contacts	O
+),	O
+consistent	O
+with	O
+a	O
+better	O
+Pearson	O
+’	O
+s	O
+correlation	O
+value	O
+(	O
+0	O
+.	O
+55	O
+)	O
+compared	O
+with	O
+TCR	O
+-	O
+peptide	O
+interactions	O
+versus	O
+affinity	O
+(	O
+0	O
+.	O
+045	O
+).	O
+	O
+For	O
+instance	O
+,	O
+contacts	O
+were	O
+made	O
+between	O
+TCR	O
+residue	O
+Val53β	O
+and	O
+MHC	O
+residue	O
+Gln72	O
+in	O
+all	O
+APLs	O
+except	O
+for	O
+in	O
+the	O
+weakest	O
+affinity	O
+ligand	O
+pair	O
+,	O
+1E6	O
+-	O
+A2	O
+-	O
+MVWGPDPLYV	O
+,	O
+in	O
+which	O
+a	O
+subtle	O
+change	O
+in	O
+TCR	O
+conformation	O
+—	O
+probably	O
+mediated	O
+by	O
+different	O
+peptide	O
+contacts	O
+—	O
+abrogated	O
+this	O
+interaction	O
+(	O
+Figure	O
+7A	O
+).	O
+	O
+An	O
+energetic	O
+switch	O
+from	O
+unfavorable	O
+to	O
+favorable	O
+entropy	O
+(	O
+order	O
+-	O
+to	O
+-	O
+disorder	O
+)	O
+correlates	O
+with	O
+antigen	O
+potency	O
+.	O
+	O
+Our	O
+analysis	O
+of	O
+the	O
+contact	O
+network	O
+provided	O
+some	O
+clues	O
+that	O
+could	O
+explain	O
+the	O
+different	O
+antigen	O
+potencies	O
+and	O
+binding	O
+affinities	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+the	O
+different	O
+APLs	O
+.	O
+	O
+For	O
+example	O
+,	O
+the	O
+1E6	O
+TCR	O
+bound	O
+to	O
+A2	O
+-	O
+RQWGPDPAAV	O
+with	O
+the	O
+third	O
+strongest	O
+affinity	O
+(	O
+KD	O
+=	O
+7	O
+.	O
+8	O
+μM	O
+)	O
+but	O
+made	O
+fewer	O
+contacts	O
+than	O
+with	O
+A2	O
+-	O
+ALWGPDPAAA	O
+(	O
+KD	O
+=	O
+~	O
+208	O
+μM	O
+)	O
+(	O
+Table	O
+2	O
+).	O
+	O
+The	O
+weak	O
+binding	O
+affinity	O
+between	O
+1E6	O
+and	O
+A2	O
+-	O
+MVWGPDPLYV	O
+and	O
+A2	O
+-	O
+YLGGPDFPTI	O
+generated	O
+thermodynamic	O
+data	O
+that	O
+were	O
+not	O
+robust	O
+enough	O
+to	O
+gain	O
+insight	O
+into	O
+the	O
+enthalpic	O
+(	O
+ΔH	O
+°)	O
+and	O
+entropic	O
+(	O
+TΔS	O
+°)	O
+changes	O
+that	O
+contributed	O
+to	O
+the	O
+different	O
+binding	O
+affinities	O
+/	O
+potencies	O
+for	O
+each	O
+APL	O
+.	O
+	O
+For	O
+instance	O
+,	O
+the	O
+A2	O
+-	O
+ALWGPDPAAA	O
+,	O
+A2	O
+-	O
+AQWGPDAAA	O
+,	O
+and	O
+A2	O
+-	O
+RQFGPDWIVA	O
+(	O
+KD	O
+=	O
+~	O
+208	O
+μM	O
+,	O
+KD	O
+=	O
+61	O
+.	O
+9	O
+μM	O
+,	O
+and	O
+KD	O
+=	O
+44	O
+.	O
+4	O
+μM	O
+,	O
+respectively	O
+)	O
+were	O
+all	O
+entropically	O
+unfavorable	O
+(	O
+TΔS	O
+°	O
+=	O
+–	O
+2	O
+.	O
+9	O
+to	O
+–	O
+5	O
+.	O
+6	O
+kcal	O
+/	O
+mol	O
+),	O
+indicating	O
+a	O
+net	O
+change	O
+from	O
+disorder	O
+to	O
+order	O
+.	O
+	O
+Conversely	O
+,	O
+the	O
+stronger	O
+-	O
+affinity	O
+ligands	O
+A2	O
+-	O
+RQWGPDPAAV	O
+(	O
+KD	O
+=	O
+7	O
+.	O
+8	O
+μM	O
+),	O
+A2	O
+-	O
+YQFGPDFPIA	O
+(	O
+KD	O
+=	O
+7	O
+.	O
+4	O
+μM	O
+),	O
+and	O
+A2	O
+-	O
+RQFGPDFPTI	O
+(	O
+KD	O
+=	O
+0	O
+.	O
+5	O
+μM	O
+)	O
+exhibited	O
+favorable	O
+entropy	O
+(	O
+TΔS	O
+°	O
+=	O
+2	O
+.	O
+2	O
+to	O
+14	O
+.	O
+9	O
+kcal	O
+/	O
+mol	O
+),	O
+indicating	O
+an	O
+order	O
+-	O
+to	O
+-	O
+disorder	O
+change	O
+during	O
+binding	O
+,	O
+possibly	O
+through	O
+the	O
+expulsion	O
+of	O
+ordered	O
+water	O
+molecules	O
+.	O
+	O
+The	O
+potential	O
+requirement	O
+for	O
+a	O
+larger	O
+degree	O
+of	O
+induced	O
+fit	O
+during	O
+binding	O
+to	O
+these	O
+weaker	O
+-	O
+affinity	O
+ligands	O
+is	O
+consistent	O
+with	O
+the	O
+larger	O
+entropic	O
+penalties	O
+observed	O
+for	O
+these	O
+interactions	O
+.	O
+	O
+Potential	O
+epitopes	O
+for	O
+1E6	O
+TCR	O
+occur	O
+commonly	O
+in	O
+the	O
+viral	O
+proteome	O
+.	O
+	O
+Three	O
+hundred	O
+forty	O
+-	O
+two	O
+of	O
+these	O
+decamers	O
+conformed	O
+to	O
+the	O
+motif	O
+xxxGPDxxxx	O
+.	O
+	O
+Of	O
+these	O
+,	O
+53	O
+peptides	O
+contained	O
+the	O
+motif	O
+xOxGPDxxxO	O
+,	O
+where	O
+O	O
+is	O
+one	O
+of	O
+the	O
+hydrophobic	O
+amino	O
+acid	O
+residues	O
+A	O
+,	O
+V	O
+,	O
+I	O
+,	O
+L	O
+,	O
+M	O
+,	O
+Y	O
+,	O
+F	O
+,	O
+and	O
+W	O
+that	O
+might	O
+allow	O
+binding	O
+to	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+(	O
+Supplemental	O
+Table	O
+4	O
+).	O
+	O
+Thus	O
+,	O
+there	O
+are	O
+many	O
+pathogen	O
+-	O
+encoded	O
+peptides	O
+that	O
+could	O
+act	O
+as	O
+agonists	O
+for	O
+the	O
+1E6	O
+T	O
+cell	O
+beyond	O
+the	O
+MVWGPDPLYV	O
+and	O
+RQFGPDWIVA	O
+sequences	O
+studied	O
+here	O
+.	O
+	O
+Extension	O
+of	O
+these	O
+analyses	O
+to	O
+include	O
+the	O
+larger	O
+genomes	O
+of	O
+bacterial	O
+pathogens	O
+would	O
+be	O
+expected	O
+to	O
+considerably	O
+increase	O
+these	O
+numbers	O
+.	O
+	O
+The	O
+binding	O
+affinity	O
+of	O
+the	O
+1E6	O
+TCR	O
+interaction	O
+with	O
+A2	O
+-	O
+RQFGPDWIVA	O
+is	O
+considerably	O
+higher	O
+than	O
+with	O
+the	O
+disease	O
+-	O
+implicated	O
+A2	O
+-	O
+ALWGPDPAAA	O
+sequence	O
+(	O
+KD	O
+=	O
+44	O
+.	O
+4	O
+μM	O
+and	O
+KD	O
+>	O
+200	O
+μM	O
+,	O
+respectively	O
+),	O
+highlighting	O
+how	O
+a	O
+pathogen	O
+-	O
+derived	O
+sequence	O
+might	O
+be	O
+capable	O
+of	O
+priming	O
+a	O
+1E6	O
+-	O
+like	O
+T	O
+cell	O
+.	O
+	O
+T	O
+cell	O
+antigen	O
+discrimination	O
+is	O
+governed	O
+by	O
+an	O
+interaction	O
+between	O
+the	O
+clonally	O
+expressed	O
+TCR	O
+and	O
+pMHC	O
+,	O
+mediated	O
+by	O
+the	O
+chemical	O
+characteristics	O
+of	O
+the	O
+interacting	O
+molecules	O
+.	O
+	O
+It	O
+has	O
+recently	O
+become	O
+clear	O
+that	O
+TCR	O
+cross	O
+-	O
+reactivity	O
+with	O
+large	O
+numbers	O
+of	O
+different	O
+pMHC	O
+ligands	O
+is	O
+essential	O
+to	O
+plug	O
+holes	O
+in	O
+T	O
+cell	O
+immune	O
+coverage	O
+that	O
+pathogens	O
+could	O
+exploit	O
+.	O
+	O
+Flexibility	O
+at	O
+the	O
+interface	O
+between	O
+the	O
+TCR	O
+and	O
+pMHC	O
+,	O
+demonstrated	O
+in	O
+various	O
+studies	O
+,	O
+has	O
+been	O
+suggested	O
+as	O
+a	O
+mechanism	O
+mediating	O
+T	O
+cell	O
+cross	O
+-	O
+reactivity	O
+with	O
+multiple	O
+distinct	O
+epitopes	O
+.	O
+	O
+Focused	O
+binding	O
+around	O
+a	O
+minimal	O
+peptide	O
+motif	O
+has	O
+also	O
+been	O
+implicated	O
+as	O
+an	O
+alternative	O
+mechanism	O
+enabling	O
+TCR	O
+cross	O
+-	O
+reactivity	O
+.	O
+	O
+Notably	O
+among	O
+these	O
+studies	O
+,	O
+Garcia	O
+and	O
+colleagues	O
+recently	O
+used	O
+the	O
+alloreactive	O
+murine	O
+TCR	O
+-	O
+MHC	O
+pair	O
+of	O
+the	O
+42F3	O
+TCR	O
+and	O
+H2	O
+-	O
+Ld	O
+to	O
+demonstrate	O
+recognition	O
+of	O
+a	O
+large	O
+number	O
+of	O
+different	O
+peptides	O
+via	O
+conserved	O
+hotspot	O
+contacts	O
+with	O
+prominent	O
+up	O
+-	O
+facing	O
+peptide	O
+residues	O
+.	O
+	O
+First	O
+,	O
+we	O
+currently	O
+know	O
+nothing	O
+about	O
+how	O
+human	O
+MHCI	O
+–	O
+restricted	O
+TCRs	O
+mediate	O
+cross	O
+-	O
+reactivity	O
+in	O
+the	O
+context	O
+of	O
+a	O
+clinically	O
+relevant	O
+model	O
+of	O
+autoimmunity	O
+,	O
+thought	O
+to	O
+be	O
+a	O
+major	O
+pathway	O
+of	O
+disease	O
+initiation	O
+in	O
+several	O
+autoimmune	O
+diseases	O
+.	O
+	O
+Second	O
+,	O
+molecular	O
+studies	O
+have	O
+not	O
+yet	O
+revealed	O
+a	O
+broad	O
+set	O
+of	O
+rules	O
+that	O
+determine	O
+TCR	O
+cross	O
+-	O
+reactivity	O
+because	O
+,	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+allo	O
+–	O
+TCR	O
+-	O
+MHC	O
+pair	O
+of	O
+the	O
+42F3	O
+TCR	O
+and	O
+H2	O
+-	O
+Ld	O
+that	O
+did	O
+not	O
+encounter	O
+each	O
+other	O
+during	O
+T	O
+cell	O
+development	O
+,	O
+studies	O
+have	O
+been	O
+limited	O
+to	O
+structures	O
+of	O
+a	O
+TCR	O
+with	O
+only	O
+2	O
+or	O
+3	O
+different	O
+ligands	O
+.	O
+	O
+Here	O
+,	O
+we	O
+investigated	O
+a	O
+highly	O
+cross	O
+-	O
+reactive	O
+MHCI	O
+-	O
+restricted	O
+TCR	O
+isolated	O
+from	O
+a	O
+patient	O
+with	O
+T1D	O
+that	O
+recognizes	O
+an	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+–	O
+restricted	O
+preproinsulin	O
+signal	O
+peptide	O
+(	O
+ALWGPDPAAA15	O
+–	O
+24	O
+).	O
+	O
+Human	O
+CD8	O
++	O
+T	O
+cell	O
+clones	O
+expressing	O
+TCRs	O
+with	O
+this	O
+specificity	O
+mediate	O
+the	O
+destruction	O
+of	O
+β	O
+cells	O
+,	O
+have	O
+been	O
+found	O
+in	O
+islets	O
+early	O
+in	O
+infection	O
+,	O
+and	O
+are	O
+proposed	O
+to	O
+be	O
+a	O
+major	O
+driver	O
+of	O
+disease	O
+.	O
+	O
+We	O
+solved	O
+the	O
+structure	O
+of	O
+the	O
+1E6	O
+TCR	O
+with	O
+7	O
+APLs	O
+to	O
+enable	O
+a	O
+comprehensive	O
+analysis	O
+of	O
+the	O
+molecular	O
+basis	O
+of	O
+TCR	O
+degeneracy	O
+.	O
+	O
+Overall	O
+,	O
+the	O
+difference	O
+in	O
+antigen	O
+potency	O
+correlated	O
+well	O
+with	O
+the	O
+binding	O
+energy	O
+(	O
+ΔG	O
+°	O
+kcal	O
+/	O
+mol	O
+)	O
+of	O
+the	O
+1E6	O
+TCR	O
+for	O
+the	O
+different	O
+epitopes	O
+,	O
+which	O
+ranged	O
+from	O
+values	O
+of	O
+ΔG	O
+°	O
+=	O
+~–	O
+4	O
+.	O
+4	O
+to	O
+–	O
+8	O
+.	O
+6	O
+kcal	O
+/	O
+mol	O
+(	O
+calculated	O
+from	O
+3D	O
+affinity	O
+data	O
+)	O
+or	O
+2D	O
+affinity	O
+values	O
+of	O
+AcKa	O
+=	O
+2	O
+.	O
+5	O
+×	O
+10	O
+–	O
+5	O
+to	O
+4	O
+.	O
+4	O
+×	O
+10	O
+–	O
+2	O
+μm4	O
+.	O
+	O
+The	O
+weaker	O
+end	O
+of	O
+this	O
+spectrum	O
+extends	O
+our	O
+understanding	O
+of	O
+the	O
+limits	O
+in	O
+which	O
+T	O
+cells	O
+can	O
+functionally	O
+operate	O
+in	O
+terms	O
+of	O
+TCR	O
+3D	O
+binding	O
+affinity	O
+and	O
+is	O
+in	O
+line	O
+with	O
+the	O
+types	O
+of	O
+very	O
+low	O
+affinity	O
+,	O
+yet	O
+fully	O
+functional	O
+self	O
+-	O
+reactive	O
+CD8	O
++	O
+T	O
+cells	O
+we	O
+have	O
+observed	O
+in	O
+tumor	O
+-	O
+infiltrating	O
+lymphocytes	O
+.	O
+	O
+Previous	O
+studies	O
+of	O
+autoreactive	O
+TCRs	O
+have	O
+shown	O
+that	O
+their	O
+binding	O
+mode	O
+is	O
+generally	O
+atypical	O
+,	O
+either	O
+due	O
+to	O
+an	O
+unusual	O
+binding	O
+manner	O
+,	O
+weak	O
+TCR	O
+binding	O
+affinity	O
+,	O
+an	O
+unstable	O
+pMHC	O
+,	O
+or	O
+a	O
+combination	O
+of	O
+these	O
+factors	O
+.	O
+	O
+Our	O
+data	O
+demonstrate	O
+the	O
+potential	O
+for	O
+an	O
+autoreactive	O
+TCR	O
+to	O
+bind	O
+with	O
+a	O
+conventional	O
+binding	O
+mode	O
+to	O
+a	O
+stable	O
+pMHC	O
+with	O
+antipathogen	O
+-	O
+like	O
+affinity	O
+(	O
+KD	O
+=	O
+0	O
+.	O
+5	O
+μM	O
+)	O
+depending	O
+on	O
+the	O
+peptide	O
+sequence	O
+.	O
+	O
+Our	O
+structural	O
+analysis	O
+revealed	O
+that	O
+the	O
+1E6	O
+TCR	O
+bound	O
+with	O
+a	O
+conserved	O
+conformation	O
+across	O
+all	O
+APLs	O
+investigated	O
+.	O
+	O
+This	O
+binding	O
+orientation	O
+was	O
+mediated	O
+through	O
+a	O
+focused	O
+interaction	O
+with	O
+TCR	O
+residues	O
+Tyr97α	O
+and	O
+Trp97β	O
+that	O
+formed	O
+an	O
+aromatic	O
+cap	O
+over	O
+a	O
+central	O
+‘	O
+GDP	O
+’	O
+motif	O
+that	O
+was	O
+common	O
+to	O
+all	O
+APLs	O
+.	O
+	O
+This	O
+hotspot	O
+binding	O
+,	O
+defined	O
+as	O
+a	O
+localized	O
+cluster	O
+of	O
+interactions	O
+that	O
+dominate	O
+binding	O
+energy	O
+during	O
+protein	O
+-	O
+protein	O
+interactions	O
+,	O
+has	O
+been	O
+previously	O
+shown	O
+to	O
+contribute	O
+to	O
+TCR	O
+recognition	O
+of	O
+MHC	O
+as	O
+a	O
+mechanism	O
+that	O
+tunes	O
+T	O
+cell	O
+cross	O
+-	O
+reactivity	O
+by	O
+providing	O
+fixed	O
+anchor	O
+points	O
+that	O
+enable	O
+TCRs	O
+to	O
+tolerate	O
+a	O
+variable	O
+peptide	O
+cargo	O
+.	O
+	O
+Alternatively	O
+,	O
+interactions	O
+between	O
+the	O
+TCR	O
+and	O
+peptide	O
+have	O
+been	O
+shown	O
+to	O
+dominate	O
+the	O
+energetic	O
+landscape	O
+during	O
+ligand	O
+engagement	O
+,	O
+ensuring	O
+that	O
+T	O
+cells	O
+retain	O
+peptide	O
+specificity	O
+.	O
+	O
+The	O
+binding	O
+mechanism	O
+utilized	O
+by	O
+the	O
+1E6	O
+TCR	O
+during	O
+pMHC	O
+recognition	O
+is	O
+consistent	O
+with	O
+both	O
+of	O
+these	O
+models	O
+.	O
+	O
+Ligand	O
+engagement	O
+is	O
+dominated	O
+by	O
+peptide	O
+interactions	O
+,	O
+but	O
+hotspot	O
+-	O
+like	O
+interactions	O
+with	O
+the	O
+central	O
+GPD	O
+motif	O
+enable	O
+the	O
+1E6	O
+TCR	O
+to	O
+tolerate	O
+peptide	O
+residues	O
+that	O
+vary	O
+outside	O
+of	O
+this	O
+region	O
+,	O
+explaining	O
+how	O
+T	O
+cells	O
+expressing	O
+this	O
+TCR	O
+may	O
+cross	O
+-	O
+react	O
+with	O
+a	O
+large	O
+number	O
+of	O
+different	O
+peptides	O
+.	O
+	O
+In	O
+both	O
+of	O
+these	O
+examples	O
+,	O
+self	O
+-	O
+recognition	O
+is	O
+mediated	O
+by	O
+TCR	O
+residues	O
+with	O
+aromatic	O
+side	O
+chains	O
+.	O
+	O
+Combined	O
+with	O
+evidence	O
+demonstrating	O
+that	O
+aromatic	O
+side	O
+chains	O
+are	O
+conserved	O
+in	O
+the	O
+CDR2	O
+loops	O
+of	O
+TCRs	O
+from	O
+many	O
+species	O
+,	O
+we	O
+speculate	O
+that	O
+these	O
+aromatic	O
+residues	O
+could	O
+impart	O
+a	O
+level	O
+of	O
+“	O
+stickiness	O
+”	O
+to	O
+TCRs	O
+,	O
+which	O
+might	O
+be	O
+enriched	O
+in	O
+an	O
+autoimmune	O
+setting	O
+when	O
+the	O
+TCR	O
+often	O
+binds	O
+in	O
+a	O
+nonoptimal	O
+fashion	O
+.	O
+	O
+For	O
+example	O
+,	O
+all	O
+of	O
+the	O
+stronger	O
+ligands	O
+encoded	O
+larger	O
+side	O
+chains	O
+(	O
+Arg	O
+or	O
+Tyr	O
+)	O
+at	O
+peptide	O
+position	O
+1	O
+that	O
+enabled	O
+new	O
+interactions	O
+with	O
+1E6	O
+not	O
+present	O
+with	O
+the	O
+Ala	O
+at	O
+this	O
+position	O
+in	O
+the	O
+natural	O
+preproinsulin	O
+peptide	O
+.	O
+	O
+We	O
+have	O
+recently	O
+demonstrated	O
+how	O
+a	O
+suboptimal	O
+position	O
+2	O
+anchor	O
+in	O
+a	O
+melanoma	O
+-	O
+derived	O
+antigen	O
+can	O
+improve	O
+TCR	O
+binding	O
+through	O
+a	O
+similar	O
+mechanism	O
+.	O
+	O
+Early	O
+thermodynamic	O
+analysis	O
+of	O
+TCR	O
+-	O
+pMHC	O
+interactions	O
+suggested	O
+a	O
+common	O
+energetic	O
+signature	O
+,	O
+driven	O
+by	O
+favorable	O
+enthalpy	O
+(	O
+generally	O
+mediated	O
+through	O
+an	O
+increase	O
+in	O
+electrostatic	O
+interactions	O
+)	O
+and	O
+unfavorable	O
+entropy	O
+(	O
+changes	O
+from	O
+disorder	O
+to	O
+order	O
+).	O
+	O
+However	O
+,	O
+more	O
+recent	O
+data	O
+have	O
+shown	O
+that	O
+TCRs	O
+can	O
+utilize	O
+a	O
+range	O
+of	O
+energetic	O
+strategies	O
+during	O
+pMHC	O
+binding	O
+,	O
+currently	O
+with	O
+no	O
+obvious	O
+pattern	O
+in	O
+terms	O
+of	O
+TCR	O
+affinity	O
+,	O
+binding	O
+mechanism	O
+,	O
+or	O
+specificity	O
+(	O
+pathogen	O
+,	O
+cancer	O
+,	O
+or	O
+self	O
+-	O
+ligands	O
+).	O
+	O
+The	O
+weaker	O
+APL	O
+ligands	O
+were	O
+characterized	O
+by	O
+favorable	O
+enthalpy	O
+and	O
+unfavorable	O
+entropy	O
+,	O
+whereas	O
+the	O
+stronger	O
+ligands	O
+progressively	O
+shifted	O
+to	O
+favorable	O
+entropy	O
+.	O
+	O
+Thus	O
+,	O
+the	O
+enhanced	O
+antigen	O
+potency	O
+was	O
+probably	O
+mediated	O
+through	O
+a	O
+shift	O
+from	O
+an	O
+induced	O
+fit	O
+to	O
+a	O
+lock	O
+-	O
+and	O
+-	O
+key	O
+interaction	O
+between	O
+the	O
+stronger	O
+ligands	O
+(	O
+less	O
+requirement	O
+for	O
+energetically	O
+unfavorable	O
+disorder	O
+-	O
+to	O
+-	O
+order	O
+changes	O
+),	O
+resulting	O
+in	O
+a	O
+more	O
+energetically	O
+favorable	O
+ΔG	O
+value	O
+.	O
+	O
+Importantly	O
+,	O
+the	O
+preproinsulin	O
+-	O
+derived	O
+epitope	O
+was	O
+one	O
+of	O
+the	O
+least	O
+potent	O
+peptides	O
+,	O
+demonstrating	O
+that	O
+the	O
+1E6	O
+T	O
+cell	O
+clone	O
+had	O
+the	O
+ability	O
+to	O
+respond	O
+to	O
+different	O
+peptide	O
+sequences	O
+with	O
+far	O
+greater	O
+potency	O
+.	O
+	O
+The	O
+RQFGPDWIVA	O
+peptide	O
+,	O
+which	O
+was	O
+substantially	O
+more	O
+potent	O
+than	O
+the	O
+preproinsulin	O
+peptide	O
+,	O
+is	O
+within	O
+the	O
+proteome	O
+of	O
+a	O
+common	O
+human	O
+pathogen	O
+(	O
+C	O
+.	O
+asparagiforme	O
+),	O
+demonstrating	O
+the	O
+potential	O
+for	O
+an	O
+encounter	O
+between	O
+a	O
+naive	O
+1E6	O
+-	O
+like	O
+T	O
+cell	O
+and	O
+a	O
+foreign	O
+peptide	O
+with	O
+a	O
+more	O
+potent	O
+ligand	O
+that	O
+might	O
+then	O
+break	O
+self	O
+-	O
+tolerance	O
+.	O
+	O
+Further	O
+experiments	O
+will	O
+be	O
+required	O
+to	O
+determine	O
+whether	O
+any	O
+naturally	O
+presented	O
+,	O
+human	O
+pathogen	O
+–	O
+derived	O
+peptides	O
+act	O
+as	O
+active	O
+ligands	O
+for	O
+1E6	O
+,	O
+but	O
+our	O
+work	O
+presented	O
+here	O
+demonstrates	O
+that	O
+it	O
+is	O
+at	O
+least	O
+feasible	O
+for	O
+an	O
+autoimmune	O
+TCR	O
+to	O
+bind	O
+to	O
+a	O
+different	O
+peptide	O
+sequence	O
+that	O
+could	O
+be	O
+present	O
+in	O
+a	O
+pathogen	O
+proteome	O
+with	O
+substantially	O
+higher	O
+affinity	O
+and	O
+potency	O
+than	O
+the	O
+interaction	O
+it	O
+might	O
+use	O
+to	O
+attack	O
+self	O
+-	O
+tissue	O
+.	O
+	O
+In	O
+summary	O
+,	O
+this	O
+investigation	O
+into	O
+the	O
+molecular	O
+basis	O
+of	O
+T	O
+cell	O
+cross	O
+-	O
+reactivity	O
+using	O
+a	O
+clinically	O
+relevant	O
+cytotoxic	O
+CD8	O
++	O
+T	O
+cell	O
+clone	O
+that	O
+kills	O
+human	O
+pancreatic	O
+β	O
+cells	O
+provides	O
+answers	O
+to	O
+a	O
+number	O
+of	O
+previously	O
+outstanding	O
+questions	O
+.	O
+	O
+First	O
+,	O
+our	O
+data	O
+shows	O
+that	O
+a	O
+single	O
+TCR	O
+has	O
+the	O
+potential	O
+to	O
+functionally	O
+(	O
+assessed	O
+through	O
+T	O
+cell	O
+activation	O
+)	O
+bind	O
+to	O
+different	O
+ligands	O
+with	O
+affinities	O
+ranging	O
+across	O
+3	O
+orders	O
+of	O
+magnitude	O
+.	O
+	O
+Second	O
+,	O
+this	O
+is	O
+the	O
+first	O
+example	O
+in	O
+which	O
+ligands	O
+have	O
+been	O
+identified	O
+and	O
+characterized	O
+for	O
+a	O
+human	O
+autoreactive	O
+TCR	O
+that	O
+are	O
+substantially	O
+more	O
+potent	O
+than	O
+the	O
+natural	O
+self	O
+-	O
+ligand	O
+,	O
+demonstrating	O
+the	O
+potential	O
+for	O
+a	O
+pathogenic	O
+ligand	O
+to	O
+break	O
+self	O
+-	O
+tolerance	O
+and	O
+prime	O
+self	O
+-	O
+reactive	O
+T	O
+cells	O
+.	O
+	O
+Third	O
+,	O
+this	O
+first	O
+structural	O
+analysis	O
+of	O
+a	O
+cross	O
+-	O
+reactive	O
+human	O
+MHCI	O
+–	O
+restricted	O
+autoimmune	O
+TCR	O
+showed	O
+that	O
+degeneracy	O
+was	O
+mediated	O
+through	O
+TCR	O
+-	O
+pMHC	O
+anchoring	O
+by	O
+a	O
+conserved	O
+minimal	O
+binding	O
+peptide	O
+motif	O
+.	O
+	O
+Finally	O
+,	O
+TCR	O
+ligand	O
+discrimination	O
+was	O
+characterized	O
+by	O
+an	O
+energetic	O
+shift	O
+from	O
+an	O
+enthalpically	O
+to	O
+entropically	O
+driven	O
+interaction	O
+.	O
+	O
+Our	O
+demonstration	O
+of	O
+the	O
+molecular	O
+mechanism	O
+governing	O
+cross	O
+-	O
+reactivity	O
+by	O
+this	O
+preproinsulin	O
+reactive	O
+human	O
+CD8	O
++	O
+T	O
+cell	O
+clone	O
+supports	O
+the	O
+notion	O
+first	O
+put	O
+forward	O
+by	O
+Wucherpfennig	O
+and	O
+Strominger	O
+that	O
+molecular	O
+mimicry	O
+could	O
+mediate	O
+autoimmunity	O
+and	O
+has	O
+far	O
+-	O
+reaching	O
+implications	O
+for	O
+the	O
+complex	O
+nature	O
+of	O
+T	O
+cell	O
+antigen	O
+discrimination	O
+.	O
+	O
+The	O
+1E6	O
+T	O
+cell	O
+clone	O
+reacts	O
+with	O
+a	O
+broad	O
+sensitivity	O
+range	O
+to	O
+APLs	O
+.	O
+	O
+(	O
+A	O
+and	O
+B	O
+)	O
+The	O
+1E6	O
+T	O
+cell	O
+clone	O
+was	O
+tested	O
+in	O
+a	O
+peptide	O
+dilution	O
+assay	O
+,	O
+in	O
+triplicate	O
+,	O
+with	O
+MVWGPDPLYV	O
+(	O
+gray	O
+),	O
+YLGGPDFPTI	O
+(	O
+red	O
+),	O
+ALWGPDPAAA	O
+(	O
+blue	O
+),	O
+AQWGPDPAAA	O
+(	O
+green	O
+),	O
+RQFGPDWIVA	O
+(	O
+dark	O
+blue	O
+),	O
+RQWGPDPAAV	O
+(	O
+purple	O
+),	O
+YQFGPDFPTA	O
+(	O
+yellow	O
+),	O
+and	O
+RQFGPDFPTI	O
+(	O
+cyan	O
+)	O
+peptides	O
+presented	O
+by	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+–	O
+expressing	O
+C1R	O
+cells	O
+for	O
+release	O
+of	O
+MIP	O
+-	O
+1β	O
+(	O
+A	O
+)	O
+and	O
+killing	O
+(	O
+B	O
+).	O
+	O
+Tm	O
+values	O
+were	O
+calculated	O
+using	O
+a	O
+Boltzmann	O
+fit	O
+to	O
+each	O
+set	O
+of	O
+data	O
+.	O
+	O
+3D	O
+and	O
+2D	O
+binding	O
+analysis	O
+of	O
+the	O
+1E6	O
+TCR	O
+with	O
+A2	O
+-	O
+ALW	O
+and	O
+the	O
+APLs	O
+.	O
+	O
+(	O
+A	O
+–	O
+H	O
+)	O
+Binding	O
+affinity	O
+of	O
+the	O
+1E6	O
+TCR	O
+interaction	O
+at	O
+25	O
+°	O
+C	O
+using	O
+SPR	O
+.	O
+	O
+Eight	O
+serial	O
+dilutions	O
+of	O
+the	O
+1E6	O
+TCR	O
+were	O
+measured	O
+(	O
+shown	O
+in	O
+the	O
+inset	O
+);	O
+representative	O
+data	O
+from	O
+3	O
+independent	O
+experiments	O
+are	O
+plotted	O
+.	O
+	O
+The	O
+equilibrium	O
+binding	O
+constant	O
+(	O
+KD	O
+)	O
+values	O
+were	O
+calculated	O
+using	O
+a	O
+nonlinear	O
+curve	O
+fit	O
+(	O
+y	O
+=	O
+[	O
+P1x	O
+]/[	O
+P2	O
++	O
+X	O
+]).	O
+	O
+In	O
+order	O
+to	O
+calculate	O
+each	O
+response	O
+,	O
+the	O
+1E6	O
+TCR	O
+was	O
+also	O
+injected	O
+over	O
+a	O
+control	O
+sample	O
+(	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+–	O
+ILAKFLHWL	O
+)	O
+that	O
+was	O
+deducted	O
+from	O
+the	O
+experimental	O
+data	O
+.	O
+	O
+(	O
+A	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+MVWGPDPLYV	O
+(	O
+approximate	O
+value	O
+);	O
+(	O
+B	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+YLGGPDFPTI	O
+(	O
+approximate	O
+value	O
+);	O
+(	O
+C	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+ALWGPDPAAA	O
+;	O
+(	O
+D	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+AQWGPDPAAA	O
+;	O
+(	O
+E	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+RQFGPDWIVA	O
+;	O
+(	O
+F	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+RQWGPDPAAV	O
+;	O
+(	O
+G	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+YQFGPDFPTA	O
+;	O
+and	O
+(	O
+H	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+RQFGPDFPTI	O
+.	O
+(	O
+I	O
+)	O
+ΔG	O
+values	O
+,	O
+calculated	O
+from	O
+SPR	O
+experiments	O
+,	O
+plotted	O
+against	O
+1	O
+/	O
+EC50	O
+(	O
+the	O
+reciprocal	O
+peptide	O
+concentration	O
+required	O
+to	O
+reach	O
+half	O
+-	O
+maximal	O
+1E6	O
+T	O
+cell	O
+killing	O
+)	O
+showing	O
+Pearson	O
+’	O
+s	O
+coefficient	O
+analysis	O
+(	O
+r	O
+)	O
+and	O
+P	O
+value	O
+(	O
+including	O
+approximate	O
+values	O
+from	O
+A	O
+and	O
+B	O
+).	O
+	O
+(	O
+J	O
+)	O
+Effective	O
+2D	O
+affinity	O
+(	O
+AcKa	O
+)	O
+calculated	O
+using	O
+adhesion	O
+frequency	O
+assays	O
+,	O
+using	O
+at	O
+least	O
+5	O
+cell	O
+pairs	O
+,	O
+and	O
+calculated	O
+as	O
+an	O
+average	O
+of	O
+100	O
+cell	O
+cell	O
+contacts	O
+.	O
+	O
+The	O
+1E6	O
+TCR	O
+and	O
+each	O
+peptide	O
+are	O
+colored	O
+according	O
+to	O
+the	O
+APL	O
+used	O
+in	O
+the	O
+complex	O
+as	O
+in	O
+Figure	O
+1	O
+.	O
+(	O
+B	O
+)	O
+Position	O
+of	O
+the	O
+1E6	O
+TCR	O
+CDR	O
+loops	O
+(	O
+multicolored	O
+lines	O
+)	O
+in	O
+each	O
+complex	O
+.	O
+	O
+The	O
+ALWGPDPAAA	O
+peptide	O
+(	O
+green	O
+sticks	O
+)	O
+is	O
+shown	O
+in	O
+the	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+binding	O
+groove	O
+(	O
+gray	O
+surface	O
+).	O
+(	O
+C	O
+)	O
+The	O
+Cα	O
+backbone	O
+conformation	O
+of	O
+each	O
+APL	O
+(	O
+multicolored	O
+illustration	O
+)	O
+in	O
+the	O
+context	O
+of	O
+the	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+α1	O
+helices	O
+(	O
+gray	O
+illustration	O
+).	O
+(	O
+D	O
+)	O
+Crossing	O
+angle	O
+of	O
+the	O
+1E6	O
+TCR	O
+(	O
+multicolored	O
+lines	O
+)	O
+calculated	O
+using	O
+previously	O
+published	O
+parameters	O
+in	O
+the	O
+context	O
+of	O
+the	O
+ALWGPDPAAA	O
+peptide	O
+(	O
+green	O
+sticks	O
+)	O
+bound	O
+in	O
+the	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+binding	O
+groove	O
+(	O
+gray	O
+surface	O
+).	O
+	O
+A	O
+conserved	O
+interaction	O
+with	O
+a	O
+GPD	O
+motif	O
+underpins	O
+the	O
+1E6	O
+TCR	O
+interaction	O
+with	O
+the	O
+APLs	O
+.	O
+	O
+The	O
+rest	O
+of	O
+the	O
+peptide	O
+,	O
+and	O
+the	O
+MHCα1	O
+helix	O
+,	O
+are	O
+shown	O
+as	O
+a	O
+gray	O
+illustration	O
+.	O
+	O
+The	O
+1E6	O
+TCR	O
+makes	O
+distinct	O
+peptide	O
+contacts	O
+with	O
+peripheral	O
+APL	O
+residues	O
+.	O
+	O
+Boxes	O
+show	O
+total	O
+contacts	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+each	O
+peptide	O
+ligand	O
+.	O
+	O
+(	O
+D	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+green	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+AQWGPDPAAA	O
+(	O
+green	O
+illustration	O
+and	O
+sticks	O
+).	O
+(	O
+E	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+dark	O
+blue	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+RQFGPDWIVA	O
+(	O
+dark	O
+blue	O
+illustration	O
+and	O
+sticks	O
+).	O
+(	O
+F	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+purple	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+RQWGPDPAAV	O
+(	O
+purple	O
+illustration	O
+and	O
+sticks	O
+).	O
+(	O
+G	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+yellow	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+YQFGPDFPTA	O
+(	O
+yellow	O
+illustration	O
+and	O
+sticks	O
+).	O
+(	O
+H	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+cyan	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+RQFGPDFPTI	O
+(	O
+cyan	O
+illustration	O
+and	O
+sticks	O
+).	O
+	O
+Comparison	O
+of	O
+ligated	O
+and	O
+unligated	O
+APLs	O
+.	O
+	O
+Superposition	O
+of	O
+each	O
+APL	O
+in	O
+unligated	O
+form	O
+and	O
+ligated	O
+to	O
+the	O
+1E6	O
+TCR	O
+.	O
+	O
+All	O
+unligated	O
+pMHCs	O
+are	O
+shown	O
+as	O
+light	O
+green	O
+illustrations	O
+.	O
+	O
+Peptide	O
+sequences	O
+are	O
+shown	O
+underneath	O
+each	O
+structure	O
+aligned	O
+with	O
+the	O
+peptide	O
+structure	O
+.	O
+	O
+A	O
+large	O
+conformational	O
+shift	O
+was	O
+observed	O
+for	O
+Tyr8	O
+in	O
+the	O
+ligated	O
+versus	O
+unligated	O
+states	O
+(	O
+black	O
+circle	O
+).	O
+(	O
+B	O
+)	O
+A2	O
+-	O
+YLGGPDFPTI	O
+(	O
+red	O
+sticks	O
+).	O
+(	O
+C	O
+)	O
+A2	O
+-	O
+ALWGPDPAAA	O
+(	O
+blue	O
+sticks	O
+)	O
+reproduced	O
+from	O
+previous	O
+published	O
+data	O
+.	O
+(	O
+D	O
+)	O
+A2	O
+-	O
+AQWGPDPAAA	O
+(	O
+green	O
+sticks	O
+).	O
+(	O
+E	O
+)	O
+A2	O
+-	O
+RQFGPDWIVA	O
+(	O
+dark	O
+blue	O
+sticks	O
+).	O
+(	O
+F	O
+)	O
+A2	O
+-	O
+RQWGPDPAAV	O
+(	O
+purple	O
+sticks	O
+).	O
+(	O
+G	O
+)	O
+A2	O
+-	O
+YQFGPDFPTA	O
+(	O
+yellow	O
+sticks	O
+).	O
+(	O
+H	O
+)	O
+A2	O
+-	O
+RQFGPDFPTI	O
+(	O
+cyan	O
+sticks	O
+).	O
+	O
+Interactions	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+the	O
+MHC	O
+α1	O
+helix	O
+residues	O
+Arg65	O
+,	O
+Lys66	O
+,	O
+and	O
+Gln72	O
+.	O
+	O
+Hydrogen	O
+bonds	O
+are	O
+shown	O
+as	O
+red	O
+dotted	O
+lines	O
+;	O
+vdW	O
+contacts	O
+are	O
+shown	O
+as	O
+black	O
+dotted	O
+lines	O
+.	O
+	O
+MHCα1	O
+helix	O
+are	O
+shown	O
+in	O
+gray	O
+illustrations	O
+.	O
+	O
+Thermodynamic	O
+analysis	O
+of	O
+the	O
+1E6	O
+TCR	O
+with	O
+A2	O
+-	O
+ALWGPDPAAA	O
+and	O
+the	O
+APLs	O
+.	O
+	O
+Eight	O
+serial	O
+dilutions	O
+of	O
+the	O
+1E6	O
+TCR	O
+were	O
+injected	O
+,	O
+in	O
+duplicate	O
+,	O
+over	O
+each	O
+immobilized	O
+APL	O
+and	O
+A2	O
+-	O
+ALW	O
+at	O
+5	O
+°	O
+C	O
+,	O
+13	O
+°	O
+C	O
+,	O
+18	O
+°	O
+C	O
+,	O
+25	O
+°	O
+C	O
+,	O
+30	O
+°	O
+C	O
+,	O
+and	O
+37	O
+°	O
+C	O
+.	O
+	O
+The	O
+equilibrium	O
+binding	O
+constant	O
+(	O
+KD	O
+)	O
+values	O
+were	O
+calculated	O
+using	O
+a	O
+nonlinear	O
+curve	O
+fit	O
+(	O
+y	O
+=	O
+[	O
+P1x	O
+]/[	O
+P2	O
++	O
+X	O
+]),	O
+and	O
+thermodynamic	O
+parameters	O
+were	O
+calculated	O
+according	O
+to	O
+the	O
+Gibbs	O
+-	O
+Helmholtz	O
+equation	O
+(	O
+ΔG	O
+°	O
+=	O
+ΔH	O
+−	O
+TΔS	O
+°).	O
+	O
+The	O
+binding	O
+free	O
+energies	O
+,	O
+ΔG	O
+°	O
+(	O
+ΔG	O
+°	O
+=	O
+RTlnKD	O
+),	O
+were	O
+plotted	O
+against	O
+temperature	O
+(	O
+K	O
+)	O
+using	O
+nonlinear	O
+regression	O
+to	O
+fit	O
+the	O
+3	O
+-	O
+parameters	O
+van	O
+’	O
+t	O
+Hoff	O
+equation	O
+(	O
+RT	O
+ln	O
+KD	O
+=	O
+ΔH	O
+°	O
+–	O
+TΔS	O
+°	O
++	O
+ΔCp	O
+°[	O
+T	O
+-	O
+T0	O
+]	O
+–	O
+TΔCp	O
+°	O
+ln	O
+[	O
+T	O
+/	O
+T0	O
+]	O
+with	O
+T0	O
+=	O
+298	O
+K	O
+).	O
+	O
+(	O
+A	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+ALWGPDPAAA	O
+;	O
+(	O
+B	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+AQWGPDPAAA	O
+;	O
+(	O
+C	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+RQFGPDWIVA	O
+;	O
+(	O
+D	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+RQWGPDPAAV	O
+,	O
+(	O
+E	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+YQFGPDFPTA	O
+;	O
+and	O
+(	O
+F	O
+)	O
+1E6	O
+-	O
+A2	O
+-	O
+RQFGPDFPTI	O
+.	O
+	O
+1E6	O
+TCR	O
+-	O
+pMHC	O
+contacts	O
+,	O
+affinity	O
+measurements	O
+and	O
+thermodynamics	O
+	O