diff --git "a/dev.tsv" "b/dev.tsv"
new file mode 100644--- /dev/null
+++ "b/dev.tsv"
@@ -0,0 +1,30806 @@
+words	labels
+The	O
+human	O
+gut	O
+microbiota	O
+influences	O
+the	O
+course	O
+of	O
+human	O
+development	O
+and	O
+health	O
+,	O
+playing	O
+key	O
+roles	O
+in	O
+immune	O
+stimulation	O
+,	O
+intestinal	O
+cell	O
+proliferation	O
+,	O
+and	O
+metabolic	O
+balance	O
+.	O
+	O
+The	O
+ability	O
+to	O
+acquire	O
+energy	O
+from	O
+carbohydrates	O
+of	O
+dietary	O
+or	O
+host	O
+origin	O
+is	O
+central	O
+to	O
+the	O
+adaptation	O
+of	O
+human	O
+gut	O
+bacterial	O
+species	O
+to	O
+their	O
+niche	O
+.	O
+	O
+Xyloglucan	O
+and	O
+the	O
+Bacteroides	O
+ovatus	O
+xyloglucan	O
+utilization	O
+locus	O
+(	O
+XyGUL	O
+).	O
+(	O
+A	O
+)	O
+Representative	O
+structures	O
+of	O
+common	O
+xyloglucans	O
+using	O
+the	O
+Consortium	O
+for	O
+Functional	O
+Glycomics	O
+Symbol	O
+Nomenclature	O
+(	O
+http	O
+://	O
+www	O
+.	O
+functionalglycomics	O
+.	O
+org	O
+/	O
+static	O
+/	O
+consortium	O
+/	O
+Nomenclature	O
+.	O
+shtml	O
+).	O
+	O
+Whereas	O
+our	O
+previous	O
+study	O
+focused	O
+on	O
+the	O
+characterization	O
+of	O
+the	O
+linkage	O
+specificity	O
+of	O
+these	O
+GHs	O
+,	O
+a	O
+key	O
+outstanding	O
+question	O
+regarding	O
+this	O
+locus	O
+is	O
+how	O
+XyG	O
+recognition	O
+is	O
+mediated	O
+at	O
+the	O
+cell	O
+surface	O
+.	O
+	O
+Here	O
+,	O
+the	O
+SGBPs	O
+very	O
+likely	O
+work	O
+in	O
+concert	O
+with	O
+the	O
+cell	O
+-	O
+surface	O
+-	O
+localized	O
+endo	O
+-	O
+xyloglucanase	O
+B	O
+.	O
+ovatus	O
+GH5	O
+(	O
+BoGH5	O
+)	O
+to	O
+recruit	O
+and	O
+cleave	O
+XyG	O
+for	O
+subsequent	O
+periplasmic	O
+import	O
+via	O
+the	O
+SusC	O
+-	O
+like	O
+TBDT	O
+of	O
+the	O
+XyGUL	O
+(	O
+Fig	O
+.	O
+1B	O
+and	O
+C	O
+).	O
+	O
+In	O
+our	O
+initial	O
+study	O
+focused	O
+on	O
+the	O
+functional	O
+characterization	O
+of	O
+the	O
+glycoside	O
+hydrolases	O
+of	O
+the	O
+XyGUL	O
+,	O
+we	O
+reported	O
+preliminary	O
+affinity	O
+PAGE	O
+and	O
+isothermal	O
+titration	O
+calorimetry	O
+(	O
+ITC	O
+)	O
+data	O
+indicating	O
+that	O
+both	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+are	O
+competent	O
+xyloglucan	O
+-	O
+binding	O
+proteins	O
+(	O
+affinity	O
+constant	O
+[	O
+Ka	O
+]	O
+values	O
+of	O
+3	O
+.	O
+74	O
+×	O
+105	O
+M	O
+−	O
+1	O
+and	O
+4	O
+.	O
+98	O
+×	O
+104	O
+M	O
+−	O
+1	O
+,	O
+respectively	O
+[	O
+23	O
+]).	O
+	O
+Together	O
+,	O
+these	O
+results	O
+highlight	O
+the	O
+high	O
+specificities	O
+of	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+for	O
+XyG	O
+,	O
+which	O
+is	O
+concordant	O
+with	O
+their	O
+association	O
+with	O
+XyG	O
+-	O
+specific	O
+GHs	O
+in	O
+the	O
+XyGUL	O
+,	O
+as	O
+well	O
+as	O
+transcriptomic	O
+analysis	O
+indicating	O
+that	O
+B	O
+.	O
+ovatus	O
+has	O
+discrete	O
+PUL	O
+for	O
+MLG	O
+,	O
+GM	O
+,	O
+and	O
+GGM	O
+(	O
+11	O
+).	O
+	O
+The	O
+apo	O
+structure	O
+is	O
+color	O
+ramped	O
+from	O
+blue	O
+to	O
+red	O
+.	O
+	O
+The	O
+approximate	O
+length	O
+of	O
+each	O
+glycan	O
+-	O
+binding	O
+site	O
+is	O
+displayed	O
+,	O
+colored	O
+to	O
+match	O
+the	O
+protein	O
+structures	O
+.	O
+(	O
+E	O
+)	O
+Stereo	O
+view	O
+of	O
+the	O
+xyloglucan	O
+-	O
+binding	O
+site	O
+of	O
+SGBP	O
+-	O
+A	O
+,	O
+displaying	O
+all	O
+residues	O
+within	O
+4	O
+Å	O
+of	O
+the	O
+ligand	O
+.	O
+	O
+Potential	O
+hydrogen	O
+-	O
+bonding	O
+interactions	O
+are	O
+shown	O
+as	O
+dashed	O
+lines	O
+,	O
+and	O
+the	O
+distance	O
+is	O
+shown	O
+in	O
+angstroms	O
+.	O
+	O
+Dissection	O
+of	O
+the	O
+individual	O
+contribution	O
+of	O
+these	O
+residues	O
+reveals	O
+that	O
+the	O
+W82A	B-mutant
+mutant	O
+displays	O
+a	O
+significant	O
+4	O
+.	O
+9	O
+-	O
+fold	O
+decrease	O
+in	O
+the	O
+Ka	O
+value	O
+for	O
+XyG	O
+,	O
+while	O
+the	O
+W306A	B-mutant
+substitution	O
+completely	O
+abolishes	O
+XyG	O
+binding	O
+.	O
+	O
+Prolines	O
+between	O
+domains	O
+are	O
+indicated	O
+as	O
+spheres	O
+.	O
+	O
+The	O
+backbone	O
+is	O
+flat	O
+,	O
+with	O
+less	O
+of	O
+the	O
+“	O
+twisted	O
+-	O
+ribbon	O
+”	O
+geometry	O
+observed	O
+in	O
+some	O
+cello	O
+-	O
+and	O
+xylogluco	O
+-	O
+oligosaccharides	O
+.	O
+	O
+Hoping	O
+to	O
+achieve	O
+a	O
+higher	O
+-	O
+resolution	O
+view	O
+of	O
+the	O
+SGBP	O
+-	O
+B	O
+–	O
+xyloglucan	O
+interaction	O
+,	O
+we	O
+solved	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+fused	B-mutant
+CD	I-mutant
+domains	I-mutant
+in	O
+complex	O
+with	O
+XyGO2	O
+(	O
+1	O
+.	O
+57	O
+Å	O
+,	O
+Rwork	O
+=	O
+15	O
+.	O
+6	O
+%,	O
+Rfree	O
+=	O
+17	O
+.	O
+1	O
+%,	O
+residues	O
+230	O
+to	O
+489	O
+)	O
+(	O
+Table	O
+2	O
+).	O
+	O
+While	O
+this	O
+may	O
+occur	O
+for	O
+a	O
+number	O
+of	O
+reasons	O
+in	O
+crystal	O
+structures	O
+,	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+poor	O
+ligand	O
+density	O
+even	O
+at	O
+higher	O
+resolution	O
+is	O
+due	O
+to	O
+movement	O
+or	O
+multiple	O
+orientations	O
+of	O
+the	O
+sugar	O
+averaged	O
+throughout	O
+the	O
+lattice	O
+.	O
+	O
+The	O
+similarity	O
+of	O
+the	O
+glycan	O
+specificity	O
+of	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+presents	O
+an	O
+intriguing	O
+conundrum	O
+regarding	O
+their	O
+individual	O
+roles	O
+in	O
+XyG	O
+utilization	O
+by	O
+B	O
+.	O
+ovatus	O
+.	O
+	O
+The	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+(	O
+ΔBacova_02651	B-mutant
+)	O
+strain	O
+(	O
+cf	O
+.	O
+	O
+The	O
+specific	O
+glycan	O
+signal	O
+that	O
+upregulates	O
+BoXyGUL	O
+is	O
+currently	O
+unknown	O
+.	O
+	O
+From	O
+our	O
+present	O
+data	O
+,	O
+we	O
+cannot	O
+eliminate	O
+the	O
+possibility	O
+that	O
+the	O
+glycan	O
+binding	O
+by	O
+SGBP	O
+-	O
+A	O
+enhances	O
+transcriptional	O
+activation	O
+of	O
+the	O
+XyGUL	O
+.	O
+	O
+Beyond	O
+SGBP	O
+-	O
+A	O
+and	O
+SGBP	O
+-	O
+B	O
+,	O
+we	O
+speculated	O
+that	O
+the	O
+catalytically	O
+feeble	O
+endo	O
+-	O
+xyloglucanase	O
+GH9	O
+,	O
+which	O
+is	O
+expendable	O
+for	O
+growth	O
+in	O
+the	O
+presence	O
+of	O
+GH5	O
+,	O
+might	O
+also	O
+play	O
+a	O
+role	O
+in	O
+glycan	O
+binding	O
+to	O
+the	O
+cell	O
+surface	O
+.	O
+	O
+We	O
+hypothesize	O
+that	O
+during	O
+exponential	O
+growth	O
+the	O
+essential	O
+role	O
+of	O
+SGBP	O
+-	O
+A	O
+extends	O
+beyond	O
+glycan	O
+recognition	O
+,	O
+perhaps	O
+due	O
+to	O
+a	O
+critical	O
+interaction	O
+with	O
+the	O
+TBDT	O
+.	O
+	O
+A	O
+particularly	O
+understudied	O
+aspect	O
+of	O
+glycan	O
+utilization	O
+is	O
+the	O
+mechanism	O
+of	O
+import	O
+via	O
+TBDTs	O
+(	O
+SusC	O
+homologs	O
+)	O
+(	O
+Fig	O
+.	O
+1	O
+),	O
+which	O
+are	O
+ubiquitous	O
+and	O
+defining	O
+components	O
+of	O
+all	O
+PUL	O
+.	O
+	O
+Similarly	O
+,	O
+the	O
+deletion	O
+of	O
+BT1762	O
+encoding	O
+a	O
+fructan	O
+-	O
+targeting	O
+SusD	O
+-	O
+like	O
+protein	O
+in	O
+B	O
+.	O
+thetaiotaomicron	O
+did	O
+not	O
+result	O
+in	O
+a	O
+dramatic	O
+loss	O
+of	O
+growth	O
+on	O
+fructans	O
+.	O
+	O
+Furthermore	O
+,	O
+considering	O
+the	O
+broader	O
+distribution	O
+of	O
+TBDTs	O
+in	O
+PUL	O
+lacking	O
+SGBPs	O
+(	O
+sometimes	O
+known	O
+as	O
+carbohydrate	O
+utilization	O
+containing	O
+TBDT	O
+[	O
+CUT	O
+]	O
+loci	O
+;	O
+see	O
+reference	O
+and	O
+reviewed	O
+in	O
+reference	O
+)	O
+across	O
+bacterial	O
+phyla	O
+,	O
+it	O
+appears	O
+that	O
+the	O
+intimate	O
+biophysical	O
+association	O
+of	O
+these	O
+substrate	O
+-	O
+transport	O
+and	O
+-	O
+binding	O
+proteins	O
+is	O
+the	O
+result	O
+of	O
+specific	O
+evolution	O
+within	O
+the	O
+Bacteroidetes	O
+.	O
+	O
+Equally	O
+intriguing	O
+is	O
+the	O
+observation	O
+that	O
+while	O
+SusD	O
+-	O
+like	O
+proteins	O
+such	O
+as	O
+SGBP	O
+-	O
+A	O
+share	O
+moderate	O
+primary	O
+and	O
+high	O
+tertiary	O
+structural	O
+conservation	O
+,	O
+the	O
+genes	O
+for	O
+the	O
+SGBPs	O
+encoded	O
+immediately	O
+downstream	O
+(	O
+Fig	O
+.	O
+1B	O
+[	O
+sometimes	O
+referred	O
+to	O
+as	O
+“	O
+susE	O
+positioned	O
+”])	O
+encode	O
+glycan	O
+-	O
+binding	O
+lipoproteins	O
+with	O
+little	O
+or	O
+no	O
+sequence	O
+or	O
+structural	O
+conservation	O
+,	O
+even	O
+among	O
+syntenic	O
+PUL	O
+that	O
+target	O
+the	O
+same	O
+polysaccharide	O
+.	O
+	O
+Because	O
+the	O
+intestinal	O
+ecosystem	O
+is	O
+a	O
+dense	O
+consortium	O
+of	O
+bacteria	O
+that	O
+must	O
+compete	O
+for	O
+their	O
+nutrients	O
+,	O
+these	O
+multimodular	O
+SGBPs	O
+may	O
+reflect	O
+ongoing	O
+evolutionary	O
+experiments	O
+to	O
+enhance	O
+glycan	O
+uptake	O
+efficiency	O
+.	O
+	O
+Whether	O
+organisms	O
+that	O
+express	O
+longer	O
+SGBPs	O
+,	O
+extending	O
+further	O
+above	O
+the	O
+cell	O
+surface	O
+toward	O
+the	O
+extracellular	O
+environment	O
+,	O
+are	O
+better	O
+equipped	O
+to	O
+compete	O
+for	O
+available	O
+carbohydrates	O
+is	O
+presently	O
+unknown	O
+.	O
+	O
+Monoclonal	O
+antibodies	O
+inhibiting	O
+IL	O
+-	O
+17A	O
+signaling	O
+have	O
+demonstrated	O
+remarkable	O
+efficacy	O
+,	O
+but	O
+an	O
+oral	O
+therapy	O
+is	O
+still	O
+lacking	O
+.	O
+	O
+Tested	O
+in	O
+primary	O
+human	O
+cells	O
+,	O
+HAP	O
+blocked	O
+the	O
+production	O
+of	O
+multiple	O
+inflammatory	O
+cytokines	O
+.	O
+	O
+These	O
+polypeptides	O
+form	O
+covalent	O
+homodimers	O
+,	O
+and	O
+IL	O
+-	O
+17A	O
+and	O
+IL	O
+-	O
+17F	O
+also	O
+form	O
+an	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17F	O
+hetereodimer	O
+.	O
+	O
+In	O
+these	O
+structures	O
+,	O
+both	O
+IL	O
+-	O
+17A	O
+and	O
+IL	O
+-	O
+17F	O
+adopt	O
+a	O
+cysteine	O
+-	O
+knot	O
+fold	O
+with	O
+two	O
+intramolecular	O
+disulfides	O
+and	O
+two	O
+interchain	O
+disulfide	O
+bonds	O
+that	O
+covalently	O
+link	O
+two	O
+monomers	O
+.	O
+	O
+Identification	O
+of	O
+IL	O
+-	O
+17A	O
+peptide	O
+inhibitors	O
+	O
+Peptides	O
+specifically	O
+binding	O
+to	O
+human	O
+IL	O
+-	O
+17A	O
+were	O
+identified	O
+from	O
+phage	O
+panning	O
+using	O
+cyclic	O
+and	O
+linear	O
+peptide	O
+libraries	O
+(	O
+Supplementary	O
+Figure	O
+S1	O
+).	O
+	O
+The	O
+positive	O
+binding	O
+supernatants	O
+were	O
+tested	O
+for	O
+the	O
+ability	O
+to	O
+block	O
+biotinylated	O
+IL	O
+-	O
+17A	O
+signaling	O
+through	O
+IL	O
+-	O
+17RA	O
+in	O
+an	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+competition	O
+ELISA	O
+assay	O
+where	O
+unlabeled	O
+IL	O
+-	O
+17A	O
+was	O
+used	O
+as	O
+positive	O
+control	O
+to	O
+inhibit	O
+biotinylated	O
+IL	O
+-	O
+17A	O
+binding	O
+.	O
+	O
+An	O
+alanine	O
+scan	O
+of	O
+peptide	O
+2	O
+,	O
+an	O
+analogue	O
+of	O
+1	O
+with	O
+a	O
+lysine	O
+to	O
+arginine	O
+substitution	O
+at	O
+position	O
+14	O
+,	O
+was	O
+initiated	O
+.	O
+	O
+Modifications	O
+at	O
+positions	O
+2	O
+and	O
+14	O
+were	O
+shown	O
+to	O
+display	O
+improvement	O
+in	O
+binding	O
+affinity	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+	O
+In	O
+this	O
+work	O
+,	O
+32	O
+–	O
+34	O
+are	O
+capped	O
+by	O
+protective	O
+acetyl	O
+group	O
+and	O
+reflect	O
+the	O
+same	O
+inactivity	O
+as	O
+reported	O
+.	O
+	O
+Peptide	O
+45	O
+,	O
+dimerized	O
+via	O
+attachment	O
+of	O
+a	O
+PEG21	O
+spacer	O
+at	O
+position	O
+14	O
+(	O
+Supplementary	O
+Scheme	O
+S1	O
+and	O
+Figure	O
+S3	O
+),	O
+was	O
+the	O
+most	O
+potent	O
+with	O
+cellular	O
+IC50	O
+of	O
+0	O
+.	O
+1	O
+nM	O
+.	O
+This	O
+significant	O
+improvement	O
+in	O
+antagonism	O
+was	O
+not	O
+seen	O
+in	O
+the	O
+peptide	O
+monomer	O
+functionalized	O
+with	O
+a	O
+PEG21	O
+group	O
+at	O
+position	O
+14	O
+as	O
+peptide	O
+48	O
+had	O
+an	O
+IC50	O
+of	O
+21	O
+nM	O
+(	O
+Supplementary	O
+Scheme	O
+S2	O
+).	O
+	O
+To	O
+further	O
+characterize	O
+the	O
+interaction	O
+of	O
+HAP	O
+with	O
+IL	O
+-	O
+17A	O
+,	O
+we	O
+set	O
+out	O
+to	O
+determine	O
+its	O
+in	O
+vitro	O
+binding	O
+affinity	O
+,	O
+specificity	O
+and	O
+kinetic	O
+profile	O
+using	O
+Surface	O
+Plasmon	O
+Resonance	O
+(	O
+SPR	O
+)	O
+methods	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+	O
+HAP	O
+blocks	O
+IL	O
+-	O
+17A	O
+signaling	O
+in	O
+a	O
+human	O
+primary	O
+cell	O
+assay	O
+	O
+In	O
+patients	O
+,	O
+the	O
+concentration	O
+of	O
+IL	O
+-	O
+17A	O
+in	O
+psoriatic	O
+lesions	O
+is	O
+reported	O
+to	O
+be	O
+0	O
+.	O
+01	O
+ng	O
+/	O
+ml	O
+,	O
+well	O
+below	O
+the	O
+EC50	O
+(	O
+5	O
+–	O
+10ng	O
+/	O
+ml	O
+)	O
+of	O
+IL	O
+-	O
+17A	O
+induced	O
+IL	O
+-	O
+8	O
+production	O
+in	O
+vitro	O
+.	O
+	O
+It	O
+is	O
+known	O
+that	O
+an	O
+antibody	O
+antigen	O
+-	O
+binding	O
+fragment	O
+(	O
+Fab	O
+)	O
+can	O
+be	O
+used	O
+as	O
+crystallization	O
+chaperones	O
+in	O
+crystallizing	O
+difficult	O
+targets	O
+.	O
+	O
+Furthermore	O
+,	O
+since	O
+it	O
+binds	O
+to	O
+an	O
+area	O
+far	O
+away	O
+from	O
+that	O
+of	O
+HAP	O
+(	O
+see	O
+below	O
+),	O
+this	O
+Fab	O
+should	O
+have	O
+minimum	O
+effects	O
+on	O
+HAP	O
+binding	O
+conformation	O
+.	O
+	O
+Crystals	O
+of	O
+Fab	O
+/	O
+IL	O
+-	O
+17A	O
+/	O
+HAP	O
+ternary	O
+complex	O
+were	O
+obtained	O
+readily	O
+in	O
+crystallization	O
+screens	O
+.	O
+	O
+Crystallization	O
+of	O
+IL	O
+-	O
+17A	O
+and	O
+its	O
+binding	O
+partners	O
+was	O
+accomplished	O
+using	O
+two	O
+forms	O
+of	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Both	O
+structures	O
+were	O
+solved	O
+by	O
+molecular	O
+replacement	O
+.	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+8	O
+residues	O
+of	O
+the	O
+HAP	O
+that	O
+are	O
+ordered	O
+in	O
+the	O
+structure	O
+,	O
+7ADLWDWIN	O
+,	O
+form	O
+an	O
+amphipathic	O
+α	O
+-	O
+helix	O
+interacting	O
+with	O
+the	O
+second	O
+IL	O
+-	O
+17A	O
+monomer	O
+.	O
+	O
+Pro6	O
+of	O
+HAP	O
+makes	O
+a	O
+transition	O
+between	O
+the	O
+N	O
+-	O
+terminal	O
+β	O
+-	O
+strand	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+α	O
+-	O
+helix	O
+of	O
+HAP	O
+.	O
+	O
+Conformational	O
+changes	O
+in	O
+region	O
+I	O
+induced	O
+by	O
+HAP	O
+binding	O
+alone	O
+may	O
+allosterically	O
+affect	O
+IL	O
+-	O
+17RA	O
+binding	O
+,	O
+but	O
+more	O
+importantly	O
+,	O
+the	O
+α	O
+-	O
+helix	O
+of	O
+HAP	O
+directly	O
+competes	O
+with	O
+IL	O
+-	O
+17RA	O
+for	O
+binding	O
+to	O
+IL	O
+-	O
+17A	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+However	O
+,	O
+it	O
+mimics	O
+the	O
+β	O
+-	O
+strand	O
+0	O
+of	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Conformational	O
+changes	O
+of	O
+IL	O
+-	O
+17A	O
+are	O
+needed	O
+for	O
+both	O
+HAP	O
+and	O
+IL	O
+-	O
+17RA	O
+to	O
+bind	O
+to	O
+that	O
+region	O
+.	O
+	O
+During	O
+IL	O
+-	O
+17A	O
+signaling	O
+,	O
+IL	O
+-	O
+17A	O
+binds	O
+to	O
+one	O
+copy	O
+of	O
+IL	O
+-	O
+17RA	O
+and	O
+one	O
+copy	O
+of	O
+IL	O
+-	O
+17RC	O
+.	O
+	O
+HAP	O
+,	O
+with	O
+only	O
+15	O
+residues	O
+,	O
+can	O
+achieve	O
+almost	O
+the	O
+same	O
+binding	O
+affinity	O
+as	O
+the	O
+much	O
+larger	O
+IL	O
+-	O
+17RA	O
+molecule	O
+,	O
+indicating	O
+a	O
+more	O
+efficient	O
+way	O
+of	O
+binding	O
+to	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+As	O
+demonstrated	O
+by	O
+the	O
+crystal	O
+structure	O
+,	O
+binding	O
+of	O
+the	O
+α	O
+-	O
+helix	O
+of	O
+HAP	O
+should	O
+be	O
+sufficient	O
+for	O
+preventing	O
+IL	O
+-	O
+17RA	O
+binding	O
+to	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Theoretically	O
+,	O
+it	O
+is	O
+possible	O
+to	O
+design	O
+chemicals	O
+such	O
+as	O
+stapled	O
+α	O
+-	O
+helical	O
+peptides	O
+to	O
+block	O
+α	O
+-	O
+helix	O
+-	O
+mediated	O
+IL	O
+-	O
+17A	O
+/	O
+IL	O
+-	O
+17RA	O
+interactions	O
+.	O
+	O
+(	O
+A	O
+)	O
+HAP	O
+binds	O
+at	O
+region	O
+I	O
+of	O
+IL	O
+-	O
+17A	O
+.	O
+	O
+Polar	O
+interactions	O
+are	O
+shown	O
+in	O
+dashes	O
+.	O
+	O
+Notice	O
+that	O
+the	O
+Trp	O
+binding	O
+pocket	O
+for	O
+W12	O
+of	O
+HAP	O
+or	O
+W31	O
+of	O
+IL	O
+-	O
+17RA	O
+is	O
+missing	O
+in	O
+the	O
+apo	O
+structure	O
+.	O
+	O
+It	O
+is	O
+therefore	O
+important	O
+to	O
+understand	O
+the	O
+mechanisms	O
+which	O
+regulate	O
+nadA	O
+expression	O
+levels	O
+,	O
+which	O
+are	O
+predominantly	O
+controlled	O
+by	O
+the	O
+transcriptional	O
+regulator	O
+NadR	O
+(	O
+Neisseria	O
+adhesin	O
+A	O
+Regulator	O
+)	O
+both	O
+in	O
+vitro	O
+and	O
+in	O
+vivo	O
+.	O
+	O
+NadR	O
+binds	O
+the	O
+nadA	O
+promoter	O
+and	O
+represses	O
+gene	O
+transcription	O
+.	O
+	O
+Serogroup	O
+B	O
+meningococcus	O
+(	O
+MenB	O
+)	O
+causes	O
+fatal	O
+sepsis	O
+and	O
+invasive	O
+meningococcal	O
+disease	O
+,	O
+particularly	O
+in	O
+young	O
+children	O
+and	O
+adolescents	O
+,	O
+as	O
+highlighted	O
+by	O
+recent	O
+MenB	O
+outbreaks	O
+in	O
+universities	O
+of	O
+the	O
+United	O
+States	O
+and	O
+Canada	O
+.	O
+	O
+The	O
+amount	O
+of	O
+NadA	O
+exposed	O
+on	O
+the	O
+meningococcal	O
+surface	O
+also	O
+influences	O
+the	O
+antibody	O
+-	O
+mediated	O
+serum	O
+bactericidal	O
+response	O
+measured	O
+in	O
+vitro	O
+.	O
+	O
+Although	O
+additional	O
+factors	O
+influence	O
+nadA	O
+expression	O
+,	O
+we	O
+focused	O
+on	O
+its	O
+regulation	O
+by	O
+NadR	O
+,	O
+the	O
+major	O
+mediator	O
+of	O
+NadA	O
+phase	O
+variable	O
+expression	O
+.	O
+	O
+However	O
+,	O
+the	O
+homologous	O
+archeal	O
+Sulfolobus	O
+tokodaii	O
+protein	O
+ST1710	O
+presented	O
+essentially	O
+the	O
+same	O
+structure	O
+in	O
+ligand	O
+-	O
+free	O
+and	O
+salicylate	O
+-	O
+bound	O
+forms	O
+,	O
+apparently	O
+contrasting	O
+the	O
+mechanism	O
+proposed	O
+for	O
+MTH313	O
+.	O
+	O
+Despite	O
+these	O
+apparent	O
+differences	O
+,	O
+MTH313	O
+and	O
+ST1710	O
+bind	O
+salicylate	O
+in	O
+approximately	O
+the	O
+same	O
+site	O
+,	O
+between	O
+their	O
+dimerization	O
+and	O
+DNA	O
+-	O
+binding	O
+domains	O
+.	O
+	O
+We	O
+obtained	O
+detailed	O
+new	O
+insights	O
+into	O
+ligand	O
+specificity	O
+,	O
+how	O
+the	O
+ligand	O
+allosterically	O
+influences	O
+the	O
+DNA	O
+-	O
+binding	O
+ability	O
+of	O
+NadR	O
+,	O
+and	O
+the	O
+regulation	O
+of	O
+nadA	O
+expression	O
+,	O
+thus	O
+also	O
+providing	O
+a	O
+deeper	O
+structural	O
+understanding	O
+of	O
+the	O
+ligand	O
+-	O
+responsive	O
+MarR	O
+super	O
+-	O
+family	O
+.	O
+	O
+NadR	O
+is	O
+dimeric	O
+and	O
+is	O
+stabilized	O
+by	O
+specific	O
+hydroxyphenylacetate	O
+ligands	O
+	O
+Recombinant	O
+NadR	O
+was	O
+produced	O
+in	O
+E	O
+.	O
+coli	O
+using	O
+an	O
+expression	O
+construct	O
+prepared	O
+from	O
+N	O
+.	O
+meningitidis	O
+serogroup	O
+B	O
+strain	O
+MC58	O
+.	O
+	O
+Since	O
+ligand	O
+-	O
+binding	O
+often	O
+increases	O
+protein	O
+stability	O
+,	O
+we	O
+also	O
+investigated	O
+the	O
+effect	O
+of	O
+various	O
+HPAs	O
+(	O
+Fig	O
+1A	O
+)	O
+on	O
+the	O
+melting	O
+temperature	O
+(	O
+Tm	O
+)	O
+of	O
+NadR	O
+.	O
+As	O
+a	O
+control	O
+of	O
+specificity	O
+,	O
+we	O
+also	O
+tested	O
+salicylate	O
+,	O
+a	O
+known	O
+ligand	O
+of	O
+some	O
+MarR	O
+proteins	O
+previously	O
+reported	O
+to	O
+increase	O
+the	O
+Tm	O
+of	O
+ST1710	O
+and	O
+MTH313	O
+.	O
+	O
+3	O
+-	O
+HPA	O
+70	O
+.	O
+0	O
+±	O
+0	O
+.	O
+1	O
+2	O
+.	O
+7	O
+2	O
+.	O
+7	O
+±	O
+0	O
+.	O
+1	O
+4	O
+-	O
+HPA	O
+70	O
+.	O
+7	O
+±	O
+0	O
+.	O
+1	O
+3	O
+.	O
+3	O
+1	O
+.	O
+5	O
+±	O
+0	O
+.	O
+1	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+71	O
+.	O
+3	O
+±	O
+0	O
+.	O
+2	O
+3	O
+.	O
+9	O
+1	O
+.	O
+1	O
+±	O
+0	O
+.	O
+1	O
+	O
+To	O
+further	O
+investigate	O
+the	O
+binding	O
+of	O
+HPAs	O
+to	O
+NadR	O
+,	O
+we	O
+used	O
+surface	O
+plasmon	O
+resonance	O
+(	O
+SPR	O
+).	O
+	O
+Data	O
+collection	O
+and	O
+refinement	O
+statistics	O
+for	O
+NadR	O
+structures	O
+.	O
+	O
+In	O
+the	O
+apo	O
+-	O
+NadR	O
+crystals	O
+,	O
+the	O
+two	O
+homodimers	O
+were	O
+related	O
+by	O
+a	O
+rotation	O
+of	O
+~	O
+90	O
+°;	O
+the	O
+observed	O
+association	O
+of	O
+the	O
+two	O
+dimers	O
+was	O
+presumably	O
+merely	O
+an	O
+effect	O
+of	O
+crystal	O
+packing	O
+,	O
+since	O
+the	O
+interface	O
+between	O
+the	O
+two	O
+homodimers	O
+is	O
+small	O
+(<	O
+550	O
+Å2	O
+of	O
+buried	O
+surface	O
+area	O
+),	O
+and	O
+is	O
+not	O
+predicted	O
+to	O
+be	O
+physiologically	O
+relevant	O
+by	O
+the	O
+PISA	O
+software	O
+.	O
+	O
+Helices	O
+α3	O
+and	O
+α4	O
+form	O
+a	O
+helix	O
+-	O
+turn	O
+-	O
+helix	O
+motif	O
+,	O
+followed	O
+by	O
+the	O
+“	O
+wing	O
+motif	O
+”	O
+comprised	O
+of	O
+two	O
+short	O
+antiparallel	O
+β	O
+-	O
+strands	O
+(	O
+β1	O
+-	O
+β2	O
+)	O
+linked	O
+by	O
+a	O
+relatively	O
+long	O
+and	O
+flexible	O
+loop	O
+.	O
+	O
+Interestingly	O
+,	O
+in	O
+the	O
+α4	O
+-	O
+β2	O
+region	O
+,	O
+the	O
+stretch	O
+of	O
+residues	O
+from	O
+R64	O
+-	O
+R91	O
+presents	O
+seven	O
+positively	O
+-	O
+charged	O
+side	O
+chains	O
+,	O
+all	O
+available	O
+for	O
+potential	O
+interactions	O
+with	O
+DNA	O
+.	O
+	O
+Using	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+,	O
+a	O
+panel	O
+of	O
+eight	O
+mutant	O
+NadR	O
+proteins	O
+was	O
+prepared	O
+(	O
+including	O
+mutations	O
+H7A	B-mutant
+,	O
+S9A	B-mutant
+,	O
+N11A	B-mutant
+,	O
+D112A	B-mutant
+,	O
+R114A	B-mutant
+,	O
+Y115A	B-mutant
+,	O
+K126A	B-mutant
+,	O
+L130K	B-mutant
+and	O
+L133K	B-mutant
+),	O
+sufficient	O
+to	O
+explore	O
+the	O
+entire	O
+dimer	O
+interface	O
+.	O
+	O
+It	O
+is	O
+notable	O
+that	O
+L130	O
+is	O
+usually	O
+present	O
+as	O
+Leu	O
+,	O
+or	O
+an	O
+alternative	O
+bulky	O
+hydrophobic	O
+amino	O
+acid	O
+(	O
+e	O
+.	O
+g	O
+.	O
+Phe	O
+,	O
+Val	O
+),	O
+in	O
+many	O
+MarR	O
+family	O
+proteins	O
+,	O
+suggesting	O
+a	O
+conserved	O
+role	O
+in	O
+stabilizing	O
+the	O
+dimer	O
+interface	O
+.	O
+	O
+The	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+structure	O
+revealed	O
+the	O
+ligand	O
+-	O
+binding	O
+site	O
+nestled	O
+between	O
+the	O
+dimerization	O
+and	O
+DNA	O
+-	O
+binding	O
+domains	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+The	O
+binding	O
+pocket	O
+was	O
+almost	O
+entirely	O
+filled	O
+by	O
+4	O
+-	O
+HPA	O
+and	O
+one	O
+water	O
+molecule	O
+,	O
+although	O
+there	O
+also	O
+remained	O
+a	O
+small	O
+tunnel	O
+2	O
+-	O
+4Å	O
+in	O
+diameter	O
+and	O
+5	O
+-	O
+6Å	O
+long	O
+leading	O
+from	O
+the	O
+pocket	O
+(	O
+proximal	O
+to	O
+the	O
+4	O
+-	O
+hydroxyl	O
+position	O
+)	O
+to	O
+the	O
+protein	O
+surface	O
+.	O
+	O
+Green	O
+and	O
+blue	O
+ribbons	O
+depict	O
+NadR	O
+chains	O
+A	O
+and	O
+B	O
+,	O
+respectively	O
+.	O
+	O
+Residues	O
+AsnA11	O
+and	O
+ArgB18	O
+likely	O
+make	O
+indirect	O
+yet	O
+local	O
+contributions	O
+to	O
+ligand	O
+binding	O
+,	O
+mainly	O
+by	O
+stabilizing	O
+the	O
+position	O
+of	O
+AspB36	O
+.	O
+	O
+List	O
+of	O
+4	O
+-	O
+HPA	O
+atoms	O
+bound	O
+to	O
+NadR	O
+via	O
+ionic	O
+interactions	O
+and	O
+/	O
+or	O
+H	O
+-	O
+bonds	O
+.	O
+	O
+4	O
+-	O
+HPA	O
+atom	O
+NadR	O
+residue	O
+/	O
+atom	O
+Distance	O
+(	O
+Å	O
+)	O
+O2	O
+TrpB39	O
+/	O
+NE1	O
+2	O
+.	O
+83	O
+O2	O
+ArgB43	O
+/	O
+NH1	O
+2	O
+.	O
+76	O
+O1	O
+ArgB43	O
+/	O
+NH1	O
+3	O
+.	O
+84	O
+O1	O
+SerA9	O
+/	O
+OG	O
+2	O
+.	O
+75	O
+O1	O
+TyrB115	O
+/	O
+OH	O
+2	O
+.	O
+50	O
+O2	O
+Water	O
+(*	O
+Ser9	O
+/	O
+Asn11	O
+)	O
+2	O
+.	O
+88	O
+OH	O
+AspB36	O
+/	O
+OD1	O
+/	O
+OD2	O
+3	O
+.	O
+6	O
+/	O
+3	O
+.	O
+7	O
+	O
+*	O
+Bond	O
+distance	O
+between	O
+the	O
+ligand	O
+carboxylate	O
+group	O
+and	O
+the	O
+water	O
+molecule	O
+,	O
+which	O
+in	O
+turn	O
+makes	O
+H	O
+-	O
+bond	O
+to	O
+the	O
+SerA9	O
+and	O
+AsnA11	O
+side	O
+chains	O
+.	O
+	O
+In	O
+SPR	O
+,	O
+the	O
+signal	O
+measured	O
+is	O
+proportional	O
+to	O
+the	O
+total	O
+molecular	O
+mass	O
+proximal	O
+to	O
+the	O
+sensor	O
+surface	O
+;	O
+consequently	O
+,	O
+if	O
+the	O
+molecular	O
+weights	O
+of	O
+the	O
+interactors	O
+are	O
+known	O
+,	O
+then	O
+the	O
+stoichiometry	O
+of	O
+the	O
+resulting	O
+complex	O
+can	O
+be	O
+determined	O
+.	O
+	O
+The	O
+stoichiometry	O
+of	O
+the	O
+NadR	O
+-	O
+HPA	O
+interactions	O
+was	O
+determined	O
+using	O
+Eq	O
+1	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+),	O
+and	O
+revealed	O
+stoichiometries	O
+of	O
+1	O
+.	O
+13	O
+for	O
+4	O
+-	O
+HPA	O
+,	O
+1	O
+.	O
+02	O
+for	O
+3	O
+-	O
+HPA	O
+,	O
+and	O
+1	O
+.	O
+21	O
+for	O
+3Cl	O
+,	O
+4	O
+-	O
+HPA	O
+,	O
+strongly	O
+suggesting	O
+that	O
+one	O
+NadR	O
+dimer	O
+bound	O
+to	O
+1	O
+HPA	O
+analyte	O
+molecule	O
+.	O
+	O
+Indeed	O
+,	O
+we	O
+noted	O
+interesting	O
+differences	O
+in	O
+the	O
+side	O
+chains	O
+of	O
+Met22	O
+,	O
+Phe25	O
+and	O
+Arg43	O
+,	O
+which	O
+in	O
+monomer	O
+B	O
+are	O
+used	O
+to	O
+contact	O
+the	O
+ligand	O
+while	O
+in	O
+monomer	O
+A	O
+they	O
+partially	O
+occupied	O
+the	O
+pocket	O
+and	O
+collectively	O
+reduced	O
+its	O
+volume	O
+significantly	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+apo	O
+-	O
+form	O
+Met22	O
+and	O
+Phe25	O
+residues	O
+were	O
+still	O
+encroaching	O
+the	O
+spaces	O
+of	O
+the	O
+4	O
+-	O
+hydroxyl	O
+group	O
+and	O
+the	O
+phenyl	O
+ring	O
+of	O
+the	O
+ligand	O
+,	O
+respectively	O
+(	O
+Fig	O
+5C	O
+).	O
+	O
+The	O
+‘	O
+outward	O
+’	O
+position	O
+of	O
+Arg43	O
+generated	O
+an	O
+open	O
+apo	O
+-	O
+form	O
+pocket	O
+with	O
+volume	O
+approximately	O
+380Å3	O
+.	O
+	O
+Taken	O
+together	O
+,	O
+these	O
+observations	O
+suggest	O
+that	O
+Arg43	O
+is	O
+a	O
+major	O
+determinant	O
+of	O
+ligand	O
+binding	O
+,	O
+and	O
+that	O
+its	O
+‘	O
+inward	O
+’	O
+position	O
+inhibits	O
+the	O
+binding	O
+of	O
+4	O
+-	O
+HPA	O
+to	O
+the	O
+empty	O
+pocket	O
+of	O
+holo	O
+-	O
+NadR	O
+.	O
+	O
+The	O
+inner	O
+conformer	O
+is	O
+the	O
+one	O
+that	O
+would	O
+display	O
+major	O
+clashes	O
+if	O
+4	O
+-	O
+HPA	O
+were	O
+present	O
+.	O
+(	O
+C	O
+)	O
+Comparison	O
+of	O
+the	O
+empty	O
+pocket	O
+from	O
+holo	O
+-	O
+NadR	O
+(	O
+green	O
+residues	O
+)	O
+with	O
+the	O
+four	O
+empty	O
+pockets	O
+of	O
+apo	O
+-	O
+NadR	O
+(	O
+grey	O
+residues	O
+),	O
+shows	O
+that	O
+in	O
+the	O
+absence	O
+of	O
+4	O
+-	O
+HPA	O
+the	O
+Arg43	O
+side	O
+chain	O
+is	O
+always	O
+observed	O
+in	O
+the	O
+‘	O
+outward	O
+’	O
+conformation	O
+.	O
+	O
+The	O
+broad	O
+spectral	O
+dispersion	O
+and	O
+the	O
+number	O
+of	O
+peaks	O
+observed	O
+,	O
+which	O
+is	O
+close	O
+to	O
+the	O
+number	O
+of	O
+expected	O
+backbone	O
+amide	O
+N	O
+-	O
+H	O
+groups	O
+for	O
+this	O
+polypeptide	O
+,	O
+confirmed	O
+that	O
+apo	O
+-	O
+NadR	O
+is	O
+well	O
+-	O
+folded	O
+under	O
+these	O
+conditions	O
+and	O
+exhibits	O
+one	O
+conformation	O
+appreciable	O
+on	O
+the	O
+NMR	O
+timescale	O
+,	O
+i	O
+.	O
+e	O
+.	O
+in	O
+the	O
+NMR	O
+experiments	O
+at	O
+25	O
+°	O
+C	O
+,	O
+two	O
+or	O
+more	O
+distinct	O
+conformations	O
+of	O
+apo	O
+-	O
+NadR	O
+monomers	O
+were	O
+not	O
+readily	O
+apparent	O
+.	O
+	O
+(	O
+B	O
+,	O
+C	O
+)	O
+Overlay	O
+of	O
+selected	O
+regions	O
+of	O
+the	O
+1H	O
+-	O
+15N	O
+TROSY	O
+-	O
+HSQC	O
+spectra	O
+acquired	O
+at	O
+25	O
+°	O
+C	O
+of	O
+apo	O
+-	O
+NadR	O
+(	O
+cyan	O
+)	O
+and	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+(	O
+red	O
+)	O
+superimposed	O
+with	O
+the	O
+spectra	O
+acquired	O
+at	O
+10	O
+°	O
+C	O
+of	O
+apo	O
+-	O
+NadR	O
+(	O
+blue	O
+)	O
+and	O
+NadR	O
+/	O
+4	O
+-	O
+HPA	O
+(	O
+green	O
+).	O
+	O
+Considering	O
+the	O
+small	O
+size	O
+,	O
+fast	O
+diffusion	O
+and	O
+relatively	O
+low	O
+binding	O
+affinity	O
+of	O
+4	O
+-	O
+HPA	O
+,	O
+it	O
+would	O
+not	O
+be	O
+surprising	O
+if	O
+the	O
+ligand	O
+associates	O
+and	O
+dissociates	O
+rapidly	O
+on	O
+the	O
+NMR	O
+time	O
+scale	O
+,	O
+resulting	O
+in	O
+only	O
+one	O
+set	O
+of	O
+peaks	O
+whose	O
+chemical	O
+shifts	O
+represent	O
+the	O
+average	O
+environment	O
+of	O
+the	O
+bound	O
+and	O
+unbound	O
+states	O
+.	O
+	O
+Interestingly	O
+,	O
+by	O
+cooling	O
+the	O
+samples	O
+to	O
+10	O
+°	O
+C	O
+,	O
+we	O
+observed	O
+that	O
+a	O
+number	O
+of	O
+those	O
+peaks	O
+strongly	O
+affected	O
+by	O
+4	O
+-	O
+HPA	O
+(	O
+and	O
+therefore	O
+likely	O
+to	O
+be	O
+in	O
+the	O
+ligand	O
+-	O
+binding	O
+site	O
+)	O
+demonstrated	O
+evidence	O
+of	O
+peak	O
+splitting	O
+,	O
+i	O
+.	O
+e	O
+.	O
+a	O
+tendency	O
+to	O
+become	O
+two	O
+distinct	O
+peaks	O
+rather	O
+than	O
+one	O
+single	O
+peak	O
+(	O
+Fig	O
+6B	O
+and	O
+6C	O
+).	O
+	O
+Similarly	O
+,	O
+the	O
+entire	O
+holo	O
+-	O
+homodimer	O
+could	O
+be	O
+closely	O
+superposed	O
+onto	O
+each	O
+of	O
+the	O
+apo	O
+-	O
+homodimers	O
+,	O
+showing	O
+rmsd	O
+values	O
+of	O
+1	O
+.	O
+29Å	O
+and	O
+1	O
+.	O
+31Å	O
+,	O
+and	O
+with	O
+more	O
+notable	O
+differences	O
+in	O
+the	O
+α6	O
+helix	O
+positions	O
+(	O
+Fig	O
+7B	O
+).	O
+	O
+Structural	O
+comparisons	O
+of	O
+NadR	O
+and	O
+modelling	O
+of	O
+interactions	O
+with	O
+DNA	O
+.	O
+	O
+However	O
+,	O
+structural	O
+comparisons	O
+revealed	O
+that	O
+the	O
+shift	O
+of	O
+holo	O
+-	O
+NadR	O
+helix	O
+α4	O
+induced	O
+by	O
+the	O
+presence	O
+of	O
+4	O
+-	O
+HPA	O
+was	O
+also	O
+accompanied	O
+by	O
+several	O
+changes	O
+at	O
+the	O
+holo	O
+dimer	O
+interface	O
+,	O
+while	O
+such	O
+extensive	O
+structural	O
+differences	O
+were	O
+not	O
+observed	O
+in	O
+the	O
+apo	O
+dimer	O
+interfaces	O
+,	O
+particularly	O
+notable	O
+when	O
+comparing	O
+the	O
+α6	O
+helices	O
+(	O
+S3	O
+Fig	O
+).	O
+	O
+Interestingly	O
+,	O
+OhrR	O
+contacts	O
+ohrA	O
+across	O
+22	O
+base	O
+pairs	O
+(	O
+bp	O
+),	O
+and	O
+similarly	O
+the	O
+main	O
+NadR	O
+target	O
+sites	O
+identified	O
+in	O
+the	O
+nadA	O
+promoter	O
+(	O
+the	O
+operators	O
+Op	O
+I	O
+and	O
+Op	O
+II	O
+)	O
+both	O
+span	O
+22	O
+bp	O
+.	O
+	O
+When	O
+aligned	O
+with	O
+OhrR	O
+,	O
+the	O
+apo	O
+-	O
+homodimer	O
+CD	O
+presented	O
+yet	O
+another	O
+different	O
+intermediate	O
+conformation	O
+(	O
+rmsd	O
+2	O
+.	O
+9Å	O
+),	O
+apparently	O
+not	O
+ideally	O
+pre	O
+-	O
+configured	O
+for	O
+DNA	O
+binding	O
+,	O
+but	O
+which	O
+in	O
+solution	O
+can	O
+presumably	O
+readily	O
+adopt	O
+the	O
+AB	O
+conformation	O
+due	O
+to	O
+the	O
+intrinsic	O
+flexibility	O
+described	O
+above	O
+.	O
+	O
+Western	O
+blot	O
+analyses	O
+of	O
+wild	O
+-	O
+type	O
+(	O
+WT	O
+)	O
+strain	O
+(	O
+lanes	O
+1	O
+–	O
+2	O
+)	O
+or	O
+isogenic	O
+nadR	O
+knockout	O
+strains	O
+(	O
+ΔNadR	B-mutant
+)	O
+complemented	O
+to	O
+express	O
+the	O
+indicated	O
+NadR	O
+WT	O
+or	O
+mutant	O
+proteins	O
+(	O
+lanes	O
+3	O
+–	O
+12	O
+)	O
+or	O
+not	O
+complemented	O
+(	O
+lanes	O
+13	O
+–	O
+14	O
+),	O
+grown	O
+in	O
+the	O
+presence	O
+(	O
+even	O
+lanes	O
+)	O
+or	O
+absence	O
+(	O
+odd	O
+lanes	O
+)	O
+of	O
+5mM	O
+4	O
+-	O
+HPA	O
+,	O
+showing	O
+NadA	O
+and	O
+NadR	O
+expression	O
+.	O
+	O
+The	O
+H7A	B-mutant
+,	O
+S9A	B-mutant
+and	O
+F25A	B-mutant
+mutants	O
+efficiently	O
+repress	O
+nadA	O
+expression	O
+but	O
+are	O
+less	O
+ligand	O
+-	O
+responsive	O
+than	O
+WT	O
+NadR	O
+.	O
+The	O
+N11A	B-mutant
+mutant	O
+does	O
+not	O
+efficiently	O
+repress	O
+nadA	O
+expression	O
+either	O
+in	O
+presence	O
+or	O
+absence	O
+of	O
+4	O
+-	O
+HPA	O
+.	O
+(	O
+The	O
+protein	O
+abundance	O
+levels	O
+of	O
+the	O
+meningococcal	O
+factor	O
+H	O
+binding	O
+protein	O
+(	O
+fHbp	O
+)	O
+were	O
+used	O
+as	O
+a	O
+gel	O
+loading	O
+control	O
+).	O
+	O
+NadA	O
+is	O
+a	O
+surface	O
+-	O
+exposed	O
+meningococcal	O
+protein	O
+contributing	O
+to	O
+pathogenesis	O
+,	O
+and	O
+is	O
+one	O
+of	O
+three	O
+main	O
+antigens	O
+present	O
+in	O
+the	O
+vaccine	O
+Bexsero	O
+.	O
+	O
+We	O
+confirmed	O
+this	O
+stoichiometry	O
+in	O
+solution	O
+using	O
+SPR	O
+methods	O
+.	O
+	O
+Structural	O
+analyses	O
+suggested	O
+that	O
+‘	O
+inward	O
+’	O
+side	O
+chain	O
+positions	O
+of	O
+Met22	O
+,	O
+Phe25	O
+and	O
+especially	O
+Arg43	O
+precluded	O
+binding	O
+of	O
+a	O
+second	O
+ligand	O
+molecule	O
+.	O
+	O
+In	O
+the	O
+S	O
+.	O
+tokodaii	O
+protein	O
+ST1710	O
+,	O
+salicylate	O
+binds	O
+to	O
+the	O
+same	O
+position	O
+in	O
+each	O
+monomer	O
+of	O
+the	O
+dimer	O
+,	O
+in	O
+a	O
+site	O
+equivalent	O
+to	O
+the	O
+putative	O
+biologically	O
+relevant	O
+site	O
+of	O
+MTH313	O
+(	O
+Fig	O
+10B	O
+).	O
+	O
+Unlike	O
+other	O
+MarR	O
+family	O
+proteins	O
+which	O
+revealed	O
+multiple	O
+ligand	O
+binding	O
+interactions	O
+,	O
+we	O
+observed	O
+only	O
+1	O
+molecule	O
+of	O
+4	O
+-	O
+HPA	O
+bound	O
+to	O
+NadR	O
+,	O
+suggesting	O
+a	O
+more	O
+specific	O
+and	O
+less	O
+promiscuous	O
+interaction	O
+.	O
+	O
+NadR	O
+shows	O
+a	O
+ligand	O
+binding	O
+site	O
+distinct	O
+from	O
+other	O
+MarR	O
+homologues	O
+.	O
+	O
+Alternatively	O
+,	O
+it	O
+is	O
+possible	O
+that	O
+other	O
+MarR	O
+homologues	O
+(	O
+e	O
+.	O
+g	O
+.	O
+NMB1585	O
+)	O
+may	O
+have	O
+no	O
+extant	O
+functional	O
+binding	O
+pocket	O
+and	O
+thus	O
+may	O
+have	O
+lost	O
+the	O
+ability	O
+to	O
+respond	O
+to	O
+a	O
+ligand	O
+,	O
+acting	O
+instead	O
+as	O
+constitutive	O
+DNA	O
+-	O
+binding	O
+regulatory	O
+proteins	O
+.	O
+	O
+The	O
+noted	O
+flexibility	O
+may	O
+also	O
+explain	O
+how	O
+NadR	O
+can	O
+adapt	O
+to	O
+bind	O
+various	O
+DNA	O
+target	O
+sequences	O
+with	O
+slightly	O
+different	O
+structural	O
+features	O
+.	O
+	O
+Like	O
+other	O
+nuclear	O
+hormone	O
+receptors	O
+,	O
+RORγ	O
+’	O
+s	O
+helix12	O
+which	O
+makes	O
+up	O
+the	O
+C	O
+-	O
+termini	O
+of	O
+the	O
+LBD	O
+is	O
+an	O
+essential	O
+part	O
+of	O
+the	O
+coactivator	O
+binding	O
+pocket	O
+and	O
+is	O
+commonly	O
+referred	O
+to	O
+as	O
+the	O
+activation	O
+function	O
+helix	O
+2	O
+(	O
+AF2	O
+).	O
+	O
+FRET	O
+results	O
+for	O
+agonist	O
+BIO592	O
+(	O
+a	O
+)	O
+and	O
+Inverse	O
+Agonist	O
+BIO399	O
+(	O
+b	O
+)	O
+	O
+a	O
+The	O
+ternary	O
+structure	O
+of	O
+RORγ518	O
+BIO592	O
+and	O
+EBI96	O
+.	O
+	O
+b	O
+RORγ	O
+AF2	O
+helix	O
+in	O
+the	O
+agonist	O
+conformation	O
+.	O
+	O
+The	O
+structure	O
+of	O
+the	O
+ternary	O
+complex	O
+had	O
+features	O
+similar	O
+to	O
+other	O
+ROR	O
+agonist	O
+coactivator	O
+structures	O
+in	O
+a	O
+transcriptionally	O
+active	O
+canonical	O
+three	O
+layer	O
+helix	O
+fold	O
+with	O
+the	O
+AF2	O
+helix	O
+in	O
+the	O
+agonist	O
+conformation	O
+.	O
+	O
+The	O
+agonist	O
+conformation	O
+is	O
+stabilized	O
+by	O
+a	O
+hydrogen	O
+bond	O
+between	O
+His479	O
+and	O
+Tyr502	O
+,	O
+in	O
+addition	O
+to	O
+π	O
+-	O
+π	O
+interactions	O
+between	O
+His479	O
+,	O
+Tyr502	O
+and	O
+Phe506	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+Electron	O
+density	O
+for	O
+the	O
+coactivator	O
+peptide	O
+EBI96	O
+was	O
+observed	O
+for	O
+residues	O
+EFPYLLSLLG	O
+which	O
+formed	O
+a	O
+α	O
+-	O
+helix	O
+stabilized	O
+through	O
+hydrophobic	O
+interactions	O
+with	O
+the	O
+coactivator	O
+binding	O
+pocket	O
+on	O
+RORγ	O
+(	O
+Fig	O
+.	O
+2c	O
+).	O
+	O
+b	O
+Benzoxazinone	O
+ring	O
+system	O
+of	O
+agonist	O
+BIO592	O
+packing	O
+against	O
+His479	O
+of	O
+RORγ	O
+stabilizing	O
+agonist	O
+conformation	O
+of	O
+the	O
+AF2	O
+helix	O
+	O
+BIO592	O
+bound	O
+in	O
+a	O
+collapsed	O
+conformational	O
+state	O
+in	O
+the	O
+LBS	O
+of	O
+RORγ	O
+with	O
+the	O
+xylene	O
+ring	O
+positioned	O
+at	O
+the	O
+bottom	O
+of	O
+the	O
+pocket	O
+making	O
+hydrophobic	O
+interactions	O
+with	O
+Val376	O
+,	O
+Phe378	O
+,	O
+Phe388	O
+and	O
+Phe401	O
+,	O
+with	O
+the	O
+ethyl	O
+-	O
+benzoxazinone	O
+ring	O
+making	O
+several	O
+hydrophobic	O
+interactions	O
+with	O
+Trp317	O
+,	O
+Leu324	O
+,	O
+Met358	O
+,	O
+Leu391	O
+,	O
+Ile	O
+400	O
+and	O
+His479	O
+(	O
+Fig	O
+.	O
+3a	O
+,	O
+Additional	O
+file	O
+3	O
+).	O
+	O
+Hydrophobic	O
+interaction	O
+between	O
+the	O
+ethyl	O
+group	O
+of	O
+the	O
+benzoxazinone	O
+and	O
+His479	O
+reinforce	O
+the	O
+His479	O
+sidechain	O
+position	O
+for	O
+making	O
+the	O
+hydrogen	O
+bond	O
+with	O
+Tyr502	O
+thereby	O
+stabilizing	O
+the	O
+agonist	O
+conformation	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+	O
+However	O
+,	O
+in	O
+the	O
+presence	O
+of	O
+inverse	O
+agonist	O
+BIO399	O
+,	O
+the	O
+proteolytic	O
+pattern	O
+showed	O
+significantly	O
+less	O
+protection	O
+,	O
+albeit	O
+the	O
+products	O
+were	O
+more	O
+heterogeneous	O
+(	O
+majority	O
+ending	O
+at	O
+494	O
+/	O
+495	O
+),	O
+indicating	O
+the	O
+destabilization	O
+of	O
+the	O
+AF2	O
+helix	O
+compared	O
+to	O
+either	O
+the	O
+APO	O
+or	O
+ternary	O
+agonist	O
+complex	O
+(	O
+Fig	O
+.	O
+4	O
+,	O
+Additional	O
+file	O
+5	O
+).	O
+	O
+a	O
+Overlay	O
+of	O
+RORγ	O
+structures	O
+bound	O
+to	O
+BIO596	O
+(	O
+Green	O
+),	O
+BIO399	O
+(	O
+Cyan	O
+)	O
+and	O
+T0901317	O
+(	O
+Pink	O
+).	O
+	O
+We	O
+hypothesize	O
+that	O
+since	O
+the	O
+Met358	O
+sidechain	O
+conformation	O
+in	O
+the	O
+T0901317	O
+RORγ	O
+structure	O
+is	O
+not	O
+in	O
+the	O
+BIO399	O
+conformation	O
+,	O
+this	O
+difference	O
+could	O
+account	O
+for	O
+the	O
+10	O
+-	O
+fold	O
+reduction	O
+in	O
+the	O
+inverse	O
+agonism	O
+for	O
+T0901317	O
+compared	O
+to	O
+BIO399	O
+in	O
+the	O
+FRET	O
+assay	O
+.	O
+	O
+The	O
+inverse	O
+agonist	O
+activity	O
+for	O
+these	O
+compounds	O
+has	O
+been	O
+attributed	O
+to	O
+orientating	O
+Trp317	O
+to	O
+clash	O
+with	O
+Tyr502	O
+or	O
+a	O
+direct	O
+inverse	O
+agonist	O
+hydrogen	O
+bonding	O
+event	O
+with	O
+His479	O
+,	O
+both	O
+of	O
+which	O
+would	O
+perturb	O
+the	O
+agonist	O
+conformation	O
+of	O
+RORγ	O
+.	O
+	O
+GAL4	O
+cell	O
+assay	O
+selectivity	O
+profile	O
+for	O
+BIO399	O
+toward	O
+RORα	O
+and	O
+RORβ	O
+in	O
+GAL4	O
+	O
+Furthermore	O
+,	O
+RORα	O
+contains	O
+two	O
+phenylalanine	O
+residues	O
+in	O
+its	O
+LBS	O
+whereas	O
+RORβ	O
+and	O
+γ	O
+have	O
+a	O
+leucine	O
+in	O
+the	O
+same	O
+position	O
+(	O
+Fig	O
+.	O
+6b	O
+).	O
+	O
+In	O
+metabolism	O
+,	O
+molecules	O
+with	O
+“	O
+high	O
+-	O
+energy	O
+”	O
+bonds	O
+(	O
+e	O
+.	O
+g	O
+.,	O
+ATP	O
+and	O
+Acetyl	O
+~	O
+CoA	O
+)	O
+are	O
+critical	O
+for	O
+both	O
+catabolic	O
+and	O
+anabolic	O
+processes	O
+.	O
+	O
+The	O
+facets	O
+of	O
+the	O
+shell	O
+are	O
+composed	O
+primarily	O
+of	O
+hexamers	O
+that	O
+are	O
+typically	O
+perforated	O
+by	O
+pores	O
+lined	O
+with	O
+highly	O
+conserved	O
+,	O
+polar	O
+residues	O
+that	O
+presumably	O
+function	O
+as	O
+the	O
+conduits	O
+for	O
+metabolites	O
+into	O
+and	O
+out	O
+of	O
+the	O
+shell	O
+.	O
+	O
+Substrates	O
+and	O
+cofactors	O
+involving	O
+the	O
+PTAC	O
+reaction	O
+are	O
+shown	O
+in	O
+red	O
+;	O
+other	O
+substrates	O
+and	O
+enzymes	O
+are	O
+shown	O
+in	O
+black	O
+,	O
+and	O
+other	O
+cofactors	O
+are	O
+shown	O
+in	O
+gray	O
+.	O
+	O
+The	O
+activities	O
+of	O
+core	O
+enzymes	O
+are	O
+not	O
+confined	O
+to	O
+BMC	O
+-	O
+associated	O
+functions	O
+:	O
+aldehyde	O
+and	O
+alcohol	O
+dehydrogenases	O
+are	O
+utilized	O
+in	O
+diverse	O
+metabolic	O
+reactions	O
+,	O
+and	O
+PTAC	O
+catalyzes	O
+a	O
+key	O
+biochemical	O
+reaction	O
+in	O
+the	O
+process	O
+of	O
+obtaining	O
+energy	O
+during	O
+fermentation	O
+.	O
+	O
+This	O
+occurs	O
+,	O
+for	O
+example	O
+,	O
+during	O
+acetoclastic	O
+methanogenesis	O
+in	O
+the	O
+archaeal	O
+Methanosarcina	O
+species	O
+.	O
+	O
+Another	O
+distinctive	O
+feature	O
+of	O
+BMC	O
+-	O
+associated	O
+PduL	O
+homologs	O
+is	O
+an	O
+N	O
+-	O
+terminal	O
+encapsulation	O
+peptide	O
+(	O
+EP	O
+)	O
+that	O
+is	O
+thought	O
+to	O
+“	O
+target	O
+”	O
+proteins	O
+for	O
+encapsulation	O
+by	O
+the	O
+BMC	O
+shell	O
+.	O
+	O
+EPs	O
+are	O
+frequently	O
+found	O
+on	O
+BMC	O
+-	O
+associated	O
+proteins	O
+and	O
+have	O
+been	O
+shown	O
+to	O
+interact	O
+with	O
+shell	O
+proteins	O
+.	O
+	O
+Here	O
+we	O
+report	O
+high	O
+-	O
+resolution	O
+crystal	O
+structures	O
+of	O
+a	O
+PduL	O
+-	O
+type	O
+PTAC	O
+in	O
+both	O
+CoA	O
+-	O
+and	O
+phosphate	O
+-	O
+bound	O
+forms	O
+,	O
+completing	O
+our	O
+understanding	O
+of	O
+the	O
+structural	O
+basis	O
+of	O
+catalysis	O
+by	O
+the	O
+metabolosome	O
+common	O
+core	O
+enzymes	O
+.	O
+	O
+β	O
+-	O
+Barrel	O
+1	O
+consists	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+β	O
+strand	O
+and	O
+β	O
+strands	O
+from	O
+the	O
+C	O
+-	O
+terminal	O
+half	O
+of	O
+the	O
+polypeptide	O
+chain	O
+(	O
+β1	O
+,	O
+β10	O
+-	O
+β14	O
+;	O
+residues	O
+37	O
+–	O
+46	O
+and	O
+155	O
+–	O
+224	O
+).	O
+	O
+Primary	O
+structure	O
+conservation	O
+of	O
+the	O
+PduL	O
+protein	O
+family	O
+.	O
+	O
+Sequence	O
+logo	O
+calculated	O
+from	O
+the	O
+multiple	O
+sequence	O
+alignment	O
+of	O
+PduL	O
+homologs	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+),	O
+but	O
+not	O
+including	O
+putative	O
+EP	O
+sequences	O
+.	O
+	O
+The	O
+position	O
+numbers	O
+shown	O
+correspond	O
+to	O
+the	O
+residue	O
+numbering	O
+of	O
+rPduL	O
+;	O
+note	O
+that	O
+some	O
+positions	O
+in	O
+the	O
+logo	O
+represent	O
+gaps	O
+in	O
+the	O
+rPduL	O
+sequence	O
+.	O
+	O
+The	O
+asterisk	O
+and	O
+double	O
+arrow	O
+marks	O
+the	O
+location	O
+of	O
+the	O
+π	O
+–	O
+π	O
+interaction	O
+between	O
+F116	O
+and	O
+the	O
+CoA	O
+base	O
+of	O
+the	O
+other	O
+dimer	O
+chain	O
+.	O
+	O
+Size	O
+exclusion	O
+chromatography	O
+of	O
+PduL	O
+homologs	O
+.	O
+	O
+(	O
+a	O
+)–(	O
+c	O
+):	O
+Chromatograms	O
+of	O
+sPduL	O
+(	O
+a	O
+),	O
+rPduL	O
+(	O
+b	O
+),	O
+and	O
+pPduL	O
+(	O
+c	O
+)	O
+with	O
+(	O
+orange	O
+)	O
+or	O
+without	O
+(	O
+blue	O
+)	O
+the	O
+predicted	O
+EP	O
+,	O
+post	O
+-	O
+nickel	O
+affinity	O
+purification	O
+,	O
+applied	O
+over	O
+a	O
+preparative	O
+size	O
+exclusion	O
+column	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+	O
+The	O
+second	O
+(	O
+Zn2	O
+)	O
+is	O
+in	O
+octahedral	O
+coordination	O
+by	O
+three	O
+conserved	O
+histidine	O
+residues	O
+(	O
+His157	O
+,	O
+His159	O
+and	O
+His204	O
+)	O
+as	O
+well	O
+as	O
+three	O
+water	O
+molecules	O
+(	O
+Fig	O
+4a	O
+).	O
+	O
+When	O
+the	O
+crystals	O
+were	O
+soaked	O
+in	O
+a	O
+sodium	O
+phosphate	O
+solution	O
+for	O
+2	O
+d	O
+prior	O
+to	O
+data	O
+collection	O
+,	O
+the	O
+CoA	O
+dissociates	O
+,	O
+and	O
+density	O
+for	O
+a	O
+phosphate	O
+molecule	O
+is	O
+visible	O
+at	O
+the	O
+active	O
+site	O
+(	O
+Table	O
+2	O
+,	O
+Fig	O
+4b	O
+).	O
+	O
+Oligomeric	O
+States	O
+of	O
+PduL	O
+Orthologs	O
+Are	O
+Influenced	O
+by	O
+the	O
+EP	O
+	O
+Given	O
+the	O
+diversity	O
+of	O
+signature	O
+enzymes	O
+(	O
+Table	O
+1	O
+),	O
+it	O
+is	O
+plausible	O
+that	O
+PduL	O
+orthologs	O
+may	O
+adopt	O
+different	O
+oligomeric	O
+states	O
+that	O
+reflect	O
+the	O
+differences	O
+in	O
+the	O
+proteins	O
+being	O
+packaged	O
+with	O
+them	O
+in	O
+the	O
+organelle	O
+lumen	O
+.	O
+	O
+pPduLΔEP	B-mutant
+eluted	O
+as	O
+two	O
+smaller	O
+forms	O
+,	O
+possibly	O
+corresponding	O
+to	O
+a	O
+trimer	O
+and	O
+a	O
+monomer	O
+.	O
+	O
+Homologs	O
+of	O
+the	O
+predominant	O
+cofactor	O
+utilizer	O
+(	O
+aldehyde	O
+dehydrogenase	O
+)	O
+and	O
+NAD	O
++	O
+regenerator	O
+(	O
+alcohol	O
+dehydrogenase	O
+)	O
+have	O
+been	O
+structurally	O
+characterized	O
+,	O
+but	O
+until	O
+now	O
+structural	O
+information	O
+was	O
+lacking	O
+for	O
+PduL	O
+,	O
+which	O
+recycles	O
+CoA	O
+in	O
+the	O
+organelle	O
+lumen	O
+.	O
+	O
+Refined	O
+domain	O
+assignment	O
+based	O
+on	O
+our	O
+structure	O
+should	O
+be	O
+able	O
+to	O
+predict	O
+domains	O
+of	O
+PF06130	O
+homologs	O
+much	O
+more	O
+accurately	O
+.	O
+	O
+Implications	O
+for	O
+Metabolosome	O
+Core	O
+Assembly	O
+	O
+Free	O
+CoA	O
+and	O
+NAD	O
++/	O
+H	O
+could	O
+potentially	O
+be	O
+bound	O
+to	O
+the	O
+enzymes	O
+as	O
+the	O
+core	O
+assembles	O
+and	O
+is	O
+encapsulated	O
+.	O
+	O
+The	O
+free	O
+CoA	O
+-	O
+bound	O
+form	O
+is	O
+presumably	O
+poised	O
+for	O
+attack	O
+upon	O
+an	O
+acyl	O
+-	O
+phosphate	O
+,	O
+indicating	O
+that	O
+the	O
+enzyme	O
+initially	O
+binds	O
+CoA	O
+as	O
+opposed	O
+to	O
+acyl	O
+-	O
+phosphate	O
+.	O
+	O
+The	O
+phosphate	O
+-	O
+bound	O
+structure	O
+indicates	O
+that	O
+in	O
+the	O
+opposite	O
+reaction	O
+direction	O
+phosphate	O
+is	O
+bound	O
+first	O
+,	O
+and	O
+then	O
+an	O
+acyl	O
+-	O
+CoA	O
+enters	O
+.	O
+	O
+The	O
+two	O
+crystal	O
+structures	O
+that	O
+we	O
+report	O
+here	O
+for	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+(	O
+with	O
+resolutions	O
+of	O
+1	O
+.	O
+2	O
+Å	O
+and	O
+2	O
+.	O
+0	O
+Å	O
+,	O
+respectively	O
+),	O
+and	O
+data	O
+derived	O
+from	O
+extensive	O
+site	O
+-	O
+directed	O
+mutagenesis	O
+experiments	O
+targeting	O
+evolutionarily	O
+highly	O
+conserved	O
+residues	O
+within	O
+the	O
+extended	O
+EctC	O
+protein	O
+family	O
+,	O
+provide	O
+a	O
+first	O
+view	O
+into	O
+the	O
+architecture	O
+of	O
+the	O
+catalytic	O
+core	O
+of	O
+the	O
+ectoine	O
+synthase	O
+.	O
+	O
+The	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+was	O
+overproduced	O
+and	O
+isolated	O
+with	O
+good	O
+yields	O
+(	O
+30	O
+–	O
+40	O
+mg	O
+L	O
+-	O
+1	O
+of	O
+culture	O
+)	O
+and	O
+purity	O
+(	O
+S2a	O
+Fig	O
+).	O
+	O
+Biochemical	O
+properties	O
+of	O
+the	O
+ectoine	O
+synthase	O
+	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+has	O
+so	O
+far	O
+not	O
+been	O
+considered	O
+as	O
+a	O
+substrate	O
+for	O
+EctC	O
+,	O
+but	O
+microorganisms	O
+that	O
+use	O
+ectoine	O
+as	O
+a	O
+nutrient	O
+produce	O
+it	O
+as	O
+an	O
+intermediate	O
+during	O
+catabolism	O
+.	O
+	O
+The	O
+stimulation	O
+of	O
+EctC	O
+enzyme	O
+activity	O
+by	O
+salts	O
+has	O
+previously	O
+also	O
+been	O
+observed	O
+for	O
+other	O
+ectoine	O
+synthases	O
+.	O
+	O
+Since	O
+variations	O
+of	O
+the	O
+above	O
+-	O
+described	O
+metal	O
+-	O
+binding	O
+motif	O
+occur	O
+frequently	O
+,	O
+we	O
+experimentally	O
+investigated	O
+the	O
+presence	O
+and	O
+nature	O
+of	O
+the	O
+metal	O
+that	O
+might	O
+be	O
+contained	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+by	O
+inductive	O
+-	O
+coupled	O
+plasma	O
+mass	O
+spectrometry	O
+(	O
+ICP	O
+-	O
+MS	O
+).	O
+	O
+We	O
+note	O
+in	O
+this	O
+context	O
+,	O
+that	O
+the	O
+values	O
+obtained	O
+for	O
+the	O
+iron	O
+content	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+proteins	O
+varied	O
+by	O
+approximately	O
+10	O
+to	O
+20	O
+%	O
+between	O
+the	O
+two	O
+methods	O
+.	O
+	O
+Dependency	O
+of	O
+the	O
+ectoine	O
+synthase	O
+activity	O
+on	O
+metals	O
+.	O
+	O
+We	O
+then	O
+took	O
+such	O
+an	O
+inactivated	O
+enzyme	O
+preparation	O
+,	O
+removed	O
+the	O
+EDTA	O
+by	O
+dialysis	O
+,	O
+and	O
+added	O
+stoichiometric	O
+amounts	O
+(	O
+10	O
+μM	O
+)	O
+of	O
+various	O
+metals	O
+to	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+enzyme	O
+.	O
+	O
+Hence	O
+,	O
+N	O
+-	O
+α	O
+-	O
+ADABA	O
+is	O
+a	O
+newly	O
+recognized	O
+substrate	O
+for	O
+ectoine	O
+synthase	O
+.	O
+	O
+The	O
+Km	O
+dropped	O
+fife	O
+-	O
+fold	O
+from	O
+4	O
+.	O
+9	O
+±	O
+0	O
+.	O
+5	O
+mM	O
+to	O
+25	O
+.	O
+4	O
+±	O
+2	O
+.	O
+9	O
+mM	O
+,	O
+and	O
+the	O
+catalytic	O
+efficiency	O
+was	O
+reduced	O
+from	O
+1	O
+.	O
+47	O
+mM	O
+-	O
+1	O
+s	O
+-	O
+1	O
+to	O
+0	O
+.	O
+02	O
+mM	O
+-	O
+1	O
+s	O
+-	O
+1	O
+,	O
+a	O
+73	O
+-	O
+fold	O
+decrease	O
+.	O
+	O
+Finally	O
+,	O
+a	O
+monomer	O
+of	O
+this	O
+structure	O
+was	O
+used	O
+as	O
+a	O
+template	O
+for	O
+molecular	O
+replacement	O
+to	O
+phase	O
+the	O
+high	O
+-	O
+resolution	O
+(	O
+1	O
+.	O
+2	O
+Å	O
+)	O
+dataset	O
+of	O
+crystal	O
+form	O
+A	O
+,	O
+which	O
+was	O
+subsequently	O
+refined	O
+to	O
+a	O
+final	O
+Rcryst	O
+of	O
+12	O
+.	O
+4	O
+%	O
+and	O
+an	O
+Rfree	O
+of	O
+14	O
+.	O
+9	O
+%	O
+(	O
+S1	O
+Table	O
+).	O
+	O
+This	O
+structure	O
+adopts	O
+an	O
+open	O
+conformation	O
+with	O
+respect	O
+to	O
+the	O
+typical	O
+fold	O
+of	O
+cupin	O
+barrels	O
+and	O
+is	O
+therefore	O
+termed	O
+in	O
+the	O
+following	O
+the	O
+“	O
+open	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+(	O
+Fig	O
+4b	O
+).	O
+	O
+Interestingly	O
+,	O
+the	O
+three	O
+other	O
+monomers	O
+present	O
+in	O
+the	O
+asymmetric	O
+unit	O
+all	O
+range	O
+from	O
+Met	O
+-	O
+1	O
+to	O
+Glu	O
+-	O
+115	O
+and	O
+adopt	O
+a	O
+conformation	O
+similar	O
+to	O
+the	O
+“	O
+open	O
+”	O
+EctC	O
+structure	O
+.	O
+	O
+The	O
+structure	O
+of	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+consists	O
+of	O
+11	O
+β	O
+-	O
+strands	O
+(	O
+β1	O
+-	O
+β11	O
+)	O
+and	O
+two	O
+α	O
+-	O
+helices	O
+(	O
+α	O
+-	O
+I	O
+and	O
+α	O
+-	O
+II	O
+)	O
+(	O
+Fig	O
+4a	O
+).	O
+	O
+Our	O
+data	O
+classify	O
+EctC	O
+,	O
+in	O
+addition	O
+to	O
+the	O
+polyketide	O
+cyclase	O
+RemF	O
+,	O
+as	O
+the	O
+second	O
+known	O
+cupin	O
+-	O
+related	O
+enzyme	O
+that	O
+catalyze	O
+a	O
+cyclocondensation	O
+reaction	O
+.	O
+	O
+Analysis	O
+of	O
+the	O
+EctC	O
+dimer	O
+interface	O
+as	O
+observed	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structure	O
+	O
+It	O
+is	O
+worth	O
+mentioning	O
+that	O
+β	O
+-	O
+strand	O
+β5	O
+is	O
+located	O
+next	O
+to	O
+His	O
+-	O
+93	O
+,	O
+which	O
+in	O
+all	O
+likelihood	O
+involved	O
+in	O
+metal	O
+binding	O
+(	O
+see	O
+below	O
+).	O
+	O
+In	O
+the	O
+“	O
+open	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structure	O
+,	O
+both	O
+proline	O
+residues	O
+are	O
+visible	O
+in	O
+the	O
+electron	O
+density	O
+;	O
+however	O
+,	O
+almost	O
+directly	O
+after	O
+Pro	O
+-	O
+110	O
+,	O
+the	O
+electron	O
+density	O
+is	O
+drastically	O
+diminished	O
+caused	O
+by	O
+the	O
+flexibility	O
+of	O
+the	O
+carboxy	O
+-	O
+terminus	O
+.	O
+	O
+Since	O
+these	O
+proline	O
+residues	O
+are	O
+followed	O
+by	O
+the	O
+carboxy	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+,	O
+the	O
+interaction	O
+of	O
+His	O
+-	O
+55	O
+with	O
+Pro	O
+-	O
+109	O
+will	O
+likely	O
+play	O
+a	O
+substantial	O
+role	O
+in	O
+spatially	O
+orienting	O
+this	O
+very	O
+flexible	O
+part	O
+of	O
+the	O
+protein	O
+.	O
+	O
+The	O
+interaction	O
+between	O
+Glu	O
+-	O
+115	O
+and	O
+His	O
+-	O
+55	O
+is	O
+only	O
+visible	O
+in	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+where	O
+the	O
+partially	O
+extended	O
+carboxy	O
+-	O
+terminus	O
+is	O
+resolved	O
+in	O
+the	O
+electron	O
+density	O
+.	O
+	O
+(	O
+b	O
+)	O
+An	O
+overlay	O
+of	O
+the	O
+“	O
+open	O
+”	O
+(	O
+colored	O
+in	O
+light	O
+blue	O
+)	O
+and	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+colored	O
+in	O
+green	O
+)	O
+structure	O
+of	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+.	O
+	O
+The	O
+putative	O
+iron	O
+binding	O
+site	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+	O
+In	O
+the	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+structure	O
+of	O
+(	O
+Sa	O
+)	O
+EctC	O
+,	O
+each	O
+of	O
+the	O
+four	O
+monomers	O
+in	O
+the	O
+asymmetric	O
+unit	O
+contains	O
+a	O
+relative	O
+strong	O
+electron	O
+density	O
+positioned	O
+within	O
+the	O
+cupin	O
+barrel	O
+.	O
+	O
+Of	O
+note	O
+is	O
+the	O
+different	O
+spatial	O
+arrangement	O
+of	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+Tyr	O
+-	O
+52	O
+(	O
+located	O
+in	O
+a	O
+loop	O
+after	O
+the	O
+end	O
+of	O
+β	O
+-	O
+strand	O
+β5	O
+)	O
+in	O
+the	O
+“	O
+open	O
+”	O
+and	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+structures	O
+.	O
+	O
+It	O
+becomes	O
+apparent	O
+from	O
+an	O
+overlay	O
+of	O
+the	O
+“	O
+open	O
+”	O
+and	O
+“	O
+semi	O
+-	O
+closed	O
+”	O
+(	O
+Sa	O
+)	O
+EctC	O
+crystal	O
+structures	O
+that	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+Tyr	O
+-	O
+52	O
+rotates	O
+away	O
+from	O
+the	O
+position	O
+of	O
+the	O
+presumptive	O
+iron	O
+,	O
+whereas	O
+the	O
+side	O
+-	O
+chains	O
+of	O
+those	O
+residues	O
+that	O
+probably	O
+contacting	O
+the	O
+metal	O
+directly	O
+[	O
+Glu	O
+-	O
+57	O
+,	O
+Tyr	O
+-	O
+85	O
+,	O
+and	O
+His	O
+-	O
+93	O
+],	O
+remain	O
+in	O
+place	O
+(	O
+Fig	O
+6a	O
+and	O
+6b	O
+).	O
+	O
+We	O
+also	O
+replaced	O
+Tyr	O
+-	O
+85	O
+with	O
+either	O
+a	O
+Phe	O
+or	O
+a	O
+Trp	O
+residue	O
+and	O
+both	O
+mutant	O
+proteins	O
+largely	O
+lost	O
+their	O
+catalytic	O
+activity	O
+and	O
+iron	O
+content	O
+(	O
+Table	O
+1	O
+)	O
+despite	O
+the	O
+fact	O
+that	O
+these	O
+substitutions	O
+were	O
+conservative	O
+.	O
+	O
+Since	O
+we	O
+used	O
+PEG	O
+molecules	O
+in	O
+the	O
+crystallization	O
+conditions	O
+,	O
+the	O
+observed	O
+density	O
+might	O
+stem	O
+from	O
+an	O
+ordered	O
+part	O
+of	O
+a	O
+PEG	O
+molecule	O
+,	O
+or	O
+low	O
+molecular	O
+weight	O
+PEG	O
+species	O
+that	O
+might	O
+have	O
+been	O
+present	O
+in	O
+the	O
+PEG	O
+preparation	O
+used	O
+in	O
+our	O
+experiments	O
+.	O
+	O
+Despite	O
+these	O
+notable	O
+limitations	O
+,	O
+we	O
+considered	O
+the	O
+serendipitously	O
+trapped	O
+compound	O
+as	O
+a	O
+mock	O
+ligand	O
+that	O
+might	O
+provide	O
+useful	O
+insights	O
+into	O
+the	O
+spatial	O
+positioning	O
+of	O
+the	O
+true	O
+EctC	O
+substrate	O
+and	O
+those	O
+residues	O
+that	O
+coordinate	O
+it	O
+within	O
+the	O
+ectoine	O
+synthase	O
+active	O
+site	O
+.	O
+	O
+The	O
+electron	O
+density	O
+was	O
+calculated	O
+as	O
+an	O
+omit	O
+map	O
+and	O
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+.	O
+	O
+We	O
+also	O
+calculated	O
+an	O
+omit	O
+map	O
+and	O
+the	O
+electron	O
+density	O
+reappeared	O
+(	O
+Fig	O
+7b	O
+).	O
+	O
+These	O
+correspond	O
+to	O
+amino	O
+acids	O
+Thr	O
+-	O
+40	O
+,	O
+Tyr	O
+-	O
+52	O
+,	O
+His	O
+-	O
+55	O
+,	O
+Glu	O
+-	O
+57	O
+,	O
+Gly	O
+-	O
+64	O
+,	O
+Tyr	O
+-	O
+85	O
+-	O
+Leu	O
+-	O
+87	O
+,	O
+His	O
+-	O
+93	O
+,	O
+Phe	O
+-	O
+107	O
+,	O
+Pro	O
+-	O
+109	O
+,	O
+Gly	O
+-	O
+113	O
+,	O
+Glu	O
+-	O
+115	O
+,	O
+and	O
+His	O
+-	O
+117	O
+in	O
+the	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+(	O
+Fig	O
+2	O
+).	O
+	O
+Each	O
+of	O
+these	O
+mutant	O
+(	O
+Sa	O
+)	O
+EctC	O
+proteins	O
+was	O
+overproduced	O
+in	O
+E	O
+.	O
+coli	O
+and	O
+purified	O
+by	O
+affinity	O
+chromatography	O
+;	O
+they	O
+all	O
+yielded	O
+pure	O
+and	O
+stable	O
+protein	O
+preparations	O
+.	O
+	O
+We	O
+replaced	O
+each	O
+of	O
+these	O
+residues	O
+with	O
+an	O
+Ala	O
+residue	O
+and	O
+found	O
+that	O
+none	O
+of	O
+them	O
+had	O
+an	O
+influence	O
+on	O
+the	O
+iron	O
+content	O
+of	O
+the	O
+mutant	O
+proteins	O
+.	O
+	O
+Each	O
+of	O
+these	O
+residues	O
+is	O
+evolutionarily	O
+highly	O
+conserved	O
+.	O
+	O
+His	O
+-	O
+117	O
+is	O
+a	O
+strictly	O
+conserved	O
+residue	O
+and	O
+its	O
+substitution	O
+by	O
+an	O
+Ala	O
+residue	O
+results	O
+in	O
+a	O
+drop	O
+of	O
+enzyme	O
+activity	O
+(	O
+down	O
+to	O
+44	O
+%)	O
+and	O
+an	O
+iron	O
+content	O
+of	O
+83	O
+%	O
+(	O
+Table	O
+1	O
+).	O
+	O
+As	O
+an	O
+internal	O
+control	O
+for	O
+our	O
+mutagenesis	O
+experiments	O
+,	O
+we	O
+also	O
+substituted	O
+Thr	O
+-	O
+41	O
+and	O
+His	O
+-	O
+51	O
+,	O
+two	O
+residues	O
+that	O
+are	O
+not	O
+evolutionarily	O
+conserved	O
+in	O
+EctC	O
+-	O
+type	O
+proteins	O
+with	O
+Ala	O
+residues	O
+.	O
+	O
+Hence	O
+,	O
+the	O
+active	O
+site	O
+of	O
+ectoine	O
+synthase	O
+must	O
+possess	O
+a	O
+certain	O
+degree	O
+of	O
+structural	O
+plasticity	O
+,	O
+a	O
+notion	O
+that	O
+is	O
+supported	O
+by	O
+the	O
+report	O
+on	O
+the	O
+EctC	O
+-	O
+catalyzed	O
+formation	O
+of	O
+the	O
+synthetic	O
+compatible	O
+solute	O
+ADPC	O
+through	O
+the	O
+cyclic	O
+condensation	O
+of	O
+two	O
+glutamine	O
+molecules	O
+.	O
+	O
+We	O
+assumed	O
+that	O
+its	O
+location	O
+and	O
+mode	O
+of	O
+binding	O
+gives	O
+,	O
+in	O
+all	O
+likelihood	O
+,	O
+clues	O
+as	O
+to	O
+the	O
+position	O
+of	O
+the	O
+true	O
+substrate	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+within	O
+the	O
+EctC	O
+active	O
+site	O
+.	O
+	O
+This	O
+probably	O
+worked	O
+to	O
+the	O
+detriment	O
+of	O
+our	O
+efforts	O
+in	O
+solving	O
+crystal	O
+structures	O
+of	O
+the	O
+full	O
+-	O
+length	O
+(	O
+Sa	O
+)	O
+EctC	O
+protein	O
+in	O
+complex	O
+with	O
+either	O
+N	O
+-	O
+γ	O
+-	O
+ADABA	O
+or	O
+ectoine	O
+.	O
+	O
+Interestingly	O
+,	O
+mutations	O
+blocking	O
+PIN	O
+oligomerization	O
+had	O
+no	O
+RNase	O
+activity	O
+,	O
+indicating	O
+that	O
+both	O
+oligomerization	O
+and	O
+NTD	O
+binding	O
+are	O
+crucial	O
+for	O
+RNase	O
+activity	O
+in	O
+vitro	O
+.	O
+	O
+Regnase	O
+-	O
+1	O
+is	O
+a	O
+member	O
+of	O
+Regnase	O
+family	O
+and	O
+is	O
+composed	O
+of	O
+a	O
+PilT	O
+N	O
+-	O
+terminus	O
+like	O
+(	O
+PIN	O
+)	O
+domain	O
+followed	O
+by	O
+a	O
+CCCH	O
+-	O
+type	O
+zinc	O
+–	O
+finger	O
+(	O
+ZF	O
+)	O
+domain	O
+,	O
+which	O
+are	O
+conserved	O
+among	O
+Regnase	O
+family	O
+members	O
+.	O
+	O
+Moreover	O
+,	O
+Regnase	O
+-	O
+1	O
+functions	O
+as	O
+a	O
+dimer	O
+through	O
+intermolecular	O
+PIN	O
+-	O
+PIN	O
+interactions	O
+during	O
+cleavage	O
+of	O
+target	O
+mRNA	O
+.	O
+	O
+Although	O
+the	O
+PIN	O
+domain	O
+is	O
+responsible	O
+for	O
+the	O
+catalytic	O
+activity	O
+of	O
+Regnase	O
+-	O
+1	O
+,	O
+the	O
+roles	O
+of	O
+the	O
+other	O
+domains	O
+are	O
+largely	O
+unknown	O
+.	O
+	O
+These	O
+results	O
+indicate	O
+that	O
+not	O
+only	O
+the	O
+PIN	O
+but	O
+also	O
+the	O
+ZF	O
+domain	O
+contribute	O
+to	O
+RNA	O
+binding	O
+,	O
+while	O
+the	O
+NTD	O
+is	O
+not	O
+likely	O
+to	O
+be	O
+involved	O
+in	O
+direct	O
+interaction	O
+with	O
+RNA	O
+.	O
+	O
+Regnase	O
+-	O
+1	O
+lacking	O
+the	O
+ZF	O
+domain	O
+generated	O
+a	O
+smaller	O
+but	O
+appreciable	O
+amount	O
+of	O
+cleaved	O
+product	O
+(	O
+T1	O
+/	O
+2	O
+~	O
+70	O
+minutes	O
+),	O
+while	O
+those	O
+lacking	O
+the	O
+NTD	O
+did	O
+not	O
+generate	O
+cleaved	O
+products	O
+(	O
+T1	O
+/	O
+2	O
+>	O
+90	O
+minutes	O
+).	O
+	O
+Dimer	O
+formation	O
+of	O
+the	O
+PIN	O
+domains	O
+	O
+Domain	O
+-	O
+domain	O
+interaction	O
+between	O
+the	O
+NTD	O
+and	O
+the	O
+PIN	O
+domain	O
+	O
+Residues	O
+critical	O
+for	O
+Regnase	O
+-	O
+1	O
+RNase	O
+activity	O
+	O
+The	O
+other	O
+mutated	O
+residues	O
+—	O
+K152	O
+,	O
+R158	O
+,	O
+R188	O
+,	O
+R200	O
+,	O
+K204	O
+,	O
+K206	O
+,	O
+K257	O
+,	O
+and	O
+R258	O
+—	O
+were	O
+not	O
+critical	O
+for	O
+RNase	O
+activity	O
+.	O
+	O
+One	O
+group	O
+consisted	O
+of	O
+catalytically	O
+active	O
+PIN	O
+domains	O
+with	O
+mutation	O
+of	O
+basic	O
+residues	O
+found	O
+in	O
+the	O
+previous	O
+section	O
+to	O
+confer	O
+decreased	O
+RNase	O
+activity	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+According	O
+to	O
+the	O
+proposed	O
+model	O
+,	O
+an	O
+R214A	B-mutant
+PIN	O
+domain	O
+can	O
+only	O
+form	O
+a	O
+dimer	O
+when	O
+the	O
+DDNN	B-mutant
+PIN	O
+acts	O
+as	O
+the	O
+secondary	O
+PIN	O
+.	O
+	O
+The	O
+previously	O
+reported	O
+crystal	O
+structure	O
+of	O
+the	O
+Regnase	O
+-	O
+1	O
+PIN	O
+domain	O
+derived	O
+from	O
+Homo	O
+sapiens	O
+is	O
+nearly	O
+identical	O
+to	O
+the	O
+one	O
+derived	O
+from	O
+Mus	O
+musculus	O
+in	O
+this	O
+study	O
+,	O
+with	O
+a	O
+backbone	O
+RMSD	O
+of	O
+0	O
+.	O
+2	O
+Å	O
+.	O
+The	O
+amino	O
+acid	O
+sequences	O
+corresponding	O
+to	O
+PIN	O
+(	O
+residues	O
+134	O
+–	O
+295	O
+)	O
+are	O
+the	O
+two	O
+non	O
+-	O
+identical	O
+residues	O
+are	O
+substituted	O
+with	O
+similar	O
+amino	O
+acids	O
+.	O
+	O
+Since	O
+the	O
+NMR	O
+spectra	O
+of	O
+monomeric	O
+mutants	O
+overlaps	O
+with	O
+those	O
+of	O
+the	O
+oligomeric	O
+forms	O
+,	O
+it	O
+is	O
+unlikely	O
+that	O
+the	O
+tertiary	O
+structure	O
+of	O
+the	O
+monomeric	O
+mutants	O
+were	O
+affected	O
+by	O
+the	O
+mutations	O
+.	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4b	O
+,	O
+c	O
+).	O
+	O
+Moreover	O
+,	O
+we	O
+found	O
+that	O
+the	O
+NTD	O
+associates	O
+with	O
+the	O
+oligomeric	O
+surface	O
+of	O
+the	O
+primary	O
+PIN	O
+,	O
+docking	O
+to	O
+a	O
+helix	O
+that	O
+harbors	O
+its	O
+catalytic	O
+residues	O
+(	O
+Figs	O
+2b	O
+and	O
+3a	O
+).	O
+	O
+The	O
+affinity	O
+of	O
+the	O
+domain	O
+-	O
+domain	O
+interaction	O
+between	O
+two	O
+PIN	O
+domains	O
+(	O
+Kd	O
+=	O
+~	O
+10	O
+−	O
+4	O
+M	O
+)	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+NTD	O
+-	O
+PIN	O
+(	O
+Kd	O
+=	O
+110	O
+±	O
+5	O
+.	O
+8	O
+μM	O
+)	O
+interactions	O
+;	O
+however	O
+,	O
+the	O
+covalent	O
+connection	O
+corresponding	O
+to	O
+residues	O
+90	O
+–	O
+133	O
+between	O
+the	O
+NTD	O
+and	O
+the	O
+primary	O
+PIN	O
+will	O
+greatly	O
+enhance	O
+the	O
+intramolecular	O
+domain	O
+interaction	O
+in	O
+the	O
+case	O
+of	O
+full	O
+-	O
+length	O
+Regnase	O
+-	O
+1	O
+.	O
+	O
+Based	O
+on	O
+these	O
+structural	O
+and	O
+functional	O
+analyses	O
+of	O
+Regnase	O
+-	O
+1	O
+domain	O
+-	O
+domain	O
+interactions	O
+,	O
+we	O
+performed	O
+docking	O
+simulations	O
+of	O
+the	O
+NTD	O
+,	O
+PIN	O
+dimer	O
+,	O
+and	O
+IL	O
+-	O
+6	O
+mRNA	O
+.	O
+	O
+The	O
+overall	O
+model	O
+of	O
+regulation	O
+of	O
+Regnase	O
+-	O
+1	O
+RNase	O
+activity	O
+through	O
+domain	O
+-	O
+domain	O
+interactions	O
+in	O
+vitro	O
+is	O
+summarized	O
+in	O
+Fig	O
+.	O
+6	O
+.	O
+	O
+Structural	O
+and	O
+functional	O
+analyses	O
+of	O
+Regnase	O
+-	O
+1	O
+.	O
+	O
+Fluorescence	O
+intensity	O
+of	O
+the	O
+free	O
+IL	O
+-	O
+6	O
+in	O
+each	O
+sample	O
+was	O
+indicated	O
+as	O
+the	O
+percentage	O
+against	O
+that	O
+in	O
+the	O
+absence	O
+of	O
+Regnase	O
+-	O
+1	O
+.	O
+	O
+Domain	O
+-	O
+domain	O
+interaction	O
+between	O
+the	O
+NTD	O
+and	O
+the	O
+PIN	O
+domain	O
+.	O
+	O
+Residues	O
+in	O
+close	O
+proximity	O
+(<	O
+5	O
+Å	O
+)	O
+to	O
+each	O
+other	O
+in	O
+the	O
+docking	O
+structure	O
+were	O
+colored	O
+yellow	O
+.	O
+	O
+Mutated	O
+basic	O
+residues	O
+were	O
+shown	O
+in	O
+sticks	O
+and	O
+those	O
+with	O
+significantly	O
+reduced	O
+RNase	O
+activities	O
+were	O
+colored	O
+red	O
+or	O
+yellow	O
+.	O
+	O
+(	O
+c	O
+)	O
+In	O
+vitro	O
+cleavage	O
+assay	O
+of	O
+Regnase	O
+-	O
+1	O
+for	O
+Regnase	O
+-	O
+1	O
+mRNA	O
+.	O
+	O
+Crystal	O
+Structure	O
+and	O
+Activity	O
+Studies	O
+of	O
+the	O
+C11	O
+Cysteine	O
+Peptidase	O
+from	O
+Parabacteroides	O
+merdae	O
+in	O
+the	O
+Human	O
+Gut	O
+Microbiome	O
+*	O
+	O
+However	O
+,	O
+despite	O
+these	O
+similarities	O
+,	O
+clan	O
+CD	O
+forms	O
+a	O
+functionally	O
+diverse	O
+group	O
+of	O
+enzymes	O
+:	O
+the	O
+overall	O
+structural	O
+diversity	O
+between	O
+(	O
+and	O
+at	O
+times	O
+within	O
+)	O
+the	O
+various	O
+families	O
+provides	O
+these	O
+peptidases	O
+with	O
+a	O
+wide	O
+variety	O
+of	O
+substrate	O
+specificities	O
+and	O
+activation	O
+mechanisms	O
+.	O
+	O
+Structure	O
+of	O
+PmC11	O
+	O
+The	O
+central	O
+nine	O
+-	O
+stranded	O
+β	O
+-	O
+sheet	O
+(	O
+β1	O
+–	O
+β9	O
+)	O
+of	O
+PmC11	O
+consists	O
+of	O
+six	O
+parallel	O
+and	O
+three	O
+anti	O
+-	O
+parallel	O
+β	O
+-	O
+strands	O
+with	O
+4	O
+↑	O
+3	O
+↓	O
+2	O
+↑	O
+1	O
+↑	O
+5	O
+↑	O
+6	O
+↑	O
+7	O
+↓	O
+8	O
+↓	O
+9	O
+↑	O
+topology	O
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+and	O
+the	O
+overall	O
+structure	O
+includes	O
+14	O
+α	O
+-	O
+helices	O
+with	O
+six	O
+(	O
+α1	O
+–	O
+α2	O
+and	O
+α4	O
+–	O
+α7	O
+)	O
+closely	O
+surrounding	O
+the	O
+β	O
+-	O
+sheet	O
+in	O
+an	O
+approximately	O
+parallel	O
+orientation	O
+.	O
+	O
+Helices	O
+α1	O
+,	O
+α7	O
+,	O
+and	O
+α6	O
+are	O
+located	O
+on	O
+one	O
+side	O
+of	O
+the	O
+β	O
+-	O
+sheet	O
+with	O
+α2	O
+,	O
+α4	O
+,	O
+and	O
+α5	O
+on	O
+the	O
+opposite	O
+side	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+	O
+The	O
+core	O
+caspase	O
+-	O
+fold	O
+is	O
+highlighted	O
+in	O
+a	O
+box	O
+.	O
+	O
+The	O
+CTD	O
+of	O
+PmC11	O
+is	O
+composed	O
+of	O
+a	O
+tight	O
+helical	O
+bundle	O
+formed	O
+from	O
+helices	O
+α8	O
+–	O
+α14	O
+and	O
+includes	O
+strands	O
+βC	O
+and	O
+βF	O
+,	O
+and	O
+β	O
+-	O
+hairpin	O
+βD	O
+–	O
+βE	O
+.	O
+The	O
+CTD	O
+sits	O
+entirely	O
+on	O
+one	O
+side	O
+of	O
+the	O
+enzyme	O
+interacting	O
+only	O
+with	O
+α3	O
+,	O
+α5	O
+,	O
+β9	O
+,	O
+and	O
+the	O
+loops	O
+surrounding	O
+β8	O
+.	O
+	O
+D	O
+,	O
+cysteine	O
+peptidase	O
+activity	O
+of	O
+PmC11	O
+.	O
+	O
+Inactive	O
+PmC11C179A	B-mutant
+was	O
+not	O
+processed	O
+to	O
+a	O
+major	O
+extent	O
+by	O
+active	O
+PmC11	O
+until	O
+around	O
+a	O
+ratio	O
+of	O
+1	O
+:	O
+4	O
+(	O
+5	O
+μg	O
+of	O
+active	O
+PmC11	O
+).	O
+	O
+F	O
+,	O
+activity	O
+of	O
+PmC11	O
+against	O
+basic	O
+substrates	O
+.	O
+	O
+G	O
+,	O
+electrostatic	O
+surface	O
+potential	O
+of	O
+PmC11	O
+shown	O
+in	O
+a	O
+similar	O
+orientation	O
+,	O
+where	O
+blue	O
+and	O
+red	O
+denote	O
+positively	O
+and	O
+negatively	O
+charged	O
+surface	O
+potential	O
+,	O
+respectively	O
+,	O
+contoured	O
+at	O
+±	O
+5	O
+kT	O
+/	O
+e	O
+.	O
+	O
+Asp177	O
+is	O
+located	O
+near	O
+the	O
+catalytic	O
+cysteine	O
+and	O
+is	O
+conserved	O
+throughout	O
+the	O
+C11	O
+family	O
+,	O
+suggesting	O
+it	O
+is	O
+the	O
+primary	O
+S1	O
+binding	O
+site	O
+residue	O
+.	O
+	O
+Asp177	O
+is	O
+highly	O
+conserved	O
+throughout	O
+the	O
+clan	O
+CD	O
+C11	O
+peptidases	O
+and	O
+is	O
+thought	O
+to	O
+be	O
+primarily	O
+responsible	O
+for	O
+substrate	O
+specificity	O
+of	O
+the	O
+clan	O
+CD	O
+enzymes	O
+,	O
+as	O
+also	O
+illustrated	O
+from	O
+the	O
+proximity	O
+of	O
+these	O
+residues	O
+relative	O
+to	O
+the	O
+inhibitor	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+when	O
+PmC11	O
+is	O
+overlaid	O
+on	O
+the	O
+MALT1	O
+-	O
+P	O
+structure	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+	O
+A	O
+,	O
+PmC11	O
+activity	O
+is	O
+inhibited	O
+by	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+.	O
+	O
+B	O
+,	O
+gel	O
+-	O
+shift	O
+assay	O
+reveals	O
+that	O
+Z	O
+-	O
+VRPR	O
+-	O
+FMK	O
+binds	O
+to	O
+PmC11	O
+.	O
+	O
+The	O
+primary	O
+structural	O
+alignment	O
+also	O
+shows	O
+that	O
+the	O
+catalytic	O
+dyad	O
+in	O
+PmC11	O
+is	O
+structurally	O
+conserved	O
+in	O
+clostripain	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+	O
+Unlike	O
+PmC11	O
+,	O
+clostripain	O
+has	O
+two	O
+cleavage	O
+sites	O
+(	O
+Arg181	O
+and	O
+Arg190	O
+),	O
+which	O
+results	O
+in	O
+the	O
+removal	O
+of	O
+a	O
+nonapeptide	O
+,	O
+and	O
+is	O
+required	O
+for	O
+full	O
+activation	O
+of	O
+the	O
+enzyme	O
+(	O
+highlighted	O
+in	O
+Fig	O
+.	O
+1A	O
+).	O
+	O
+Interestingly	O
+,	O
+Arg190	O
+was	O
+found	O
+to	O
+align	O
+with	O
+Lys147	O
+in	O
+PmC11	O
+.	O
+	O
+As	O
+studies	O
+on	O
+clostripain	O
+revealed	O
+addition	O
+of	O
+Ca2	O
++	O
+ions	O
+are	O
+required	O
+for	O
+full	O
+activation	O
+,	O
+the	O
+Ca2	O
++	O
+dependence	O
+of	O
+PmC11	O
+was	O
+examined	O
+.	O
+	O
+In	O
+support	O
+of	O
+these	O
+findings	O
+,	O
+EGTA	O
+did	O
+not	O
+inhibit	O
+PmC11	O
+suggesting	O
+that	O
+,	O
+unlike	O
+clostripain	O
+,	O
+PmC11	O
+does	O
+not	O
+require	O
+Ca2	O
++	O
+or	O
+other	O
+divalent	O
+cations	O
+,	O
+for	O
+activity	O
+.	O
+	O
+The	O
+enzyme	O
+exhibits	O
+all	O
+of	O
+the	O
+key	O
+structural	O
+elements	O
+of	O
+clan	O
+CD	O
+members	O
+,	O
+but	O
+is	O
+unusual	O
+in	O
+that	O
+it	O
+has	O
+a	O
+nine	O
+-	O
+stranded	O
+central	O
+β	O
+-	O
+sheet	O
+with	O
+a	O
+novel	O
+C	O
+-	O
+terminal	O
+domain	O
+.	O
+	O
+In	O
+addition	O
+,	O
+the	O
+structure	O
+suggested	O
+a	O
+mechanism	O
+of	O
+self	O
+-	O
+inhibition	O
+in	O
+both	O
+PmC11	O
+and	O
+clostripain	O
+and	O
+an	O
+activation	O
+mechanism	O
+that	O
+requires	O
+autoprocessing	O
+.	O
+	O
+This	O
+is	O
+also	O
+the	O
+case	O
+in	O
+PmC11	O
+,	O
+although	O
+the	O
+cleavage	O
+loop	O
+is	O
+structurally	O
+different	O
+to	O
+that	O
+found	O
+in	O
+the	O
+caspases	O
+and	O
+follows	O
+the	O
+catalytic	O
+His	O
+(	O
+Fig	O
+.	O
+1A	O
+),	O
+as	O
+opposed	O
+to	O
+the	O
+Cys	O
+in	O
+the	O
+caspases	O
+.	O
+	O
+Like	O
+PmC11	O
+,	O
+these	O
+structures	O
+show	O
+preformed	O
+catalytic	O
+machinery	O
+and	O
+,	O
+for	O
+a	O
+substrate	O
+to	O
+gain	O
+access	O
+,	O
+movement	O
+and	O
+/	O
+or	O
+cleavage	O
+of	O
+the	O
+blocking	O
+region	O
+is	O
+required	O
+.	O
+	O
+Indeed	O
+,	O
+insights	O
+gained	O
+from	O
+an	O
+analysis	O
+of	O
+the	O
+PmC11	O
+structure	O
+revealed	O
+the	O
+identity	O
+of	O
+the	O
+Trypanosoma	O
+brucei	O
+PNT1	O
+protein	O
+as	O
+a	O
+C11	O
+cysteine	O
+peptidase	O
+with	O
+an	O
+essential	O
+role	O
+in	O
+organelle	O
+replication	O
+.	O
+	O
+In	O
+addition	O
+,	O
+18S	O
+and	O
+25S	O
+(	O
+yeast	O
+)/	O
+28S	O
+(	O
+humans	O
+)	O
+rRNAs	O
+contain	O
+several	O
+base	O
+modifications	O
+catalyzed	O
+by	O
+site	O
+-	O
+specific	O
+and	O
+snoRNA	O
+-	O
+independent	O
+enzymes	O
+.	O
+	O
+In	O
+a	O
+second	O
+step	O
+,	O
+the	O
+essential	O
+SPOUT	O
+-	O
+class	O
+methyltransferase	O
+Nep1	O
+/	O
+Emg1	O
+modifies	O
+the	O
+pseudouridine	O
+to	O
+N1	O
+-	O
+methylpseudouridine	O
+.	O
+	O
+Hypermodified	O
+m1acp3Ψ	O
+elutes	O
+at	O
+7	O
+.	O
+4	O
+min	O
+(	O
+wild	O
+type	O
+,	O
+left	O
+profile	O
+)	O
+and	O
+is	O
+missing	O
+in	O
+Δtsr3	B-mutant
+(	O
+middle	O
+profile	O
+)	O
+and	O
+Δnep1	B-mutant
+Δnop6	I-mutant
+mutants	O
+(	O
+right	O
+profile	O
+).	O
+	O
+Upper	O
+lanes	O
+show	O
+the	O
+ethidium	O
+bromide	O
+staining	O
+of	O
+the	O
+18S	O
+rRNAs	O
+for	O
+quantification	O
+.	O
+	O
+The	O
+efficiency	O
+of	O
+siRNA	O
+mediated	O
+HsTSR3	O
+repression	O
+correlates	O
+with	O
+the	O
+primer	O
+extension	O
+signals	O
+(	O
+see	O
+Supplementary	O
+Figure	O
+S2A	O
+).	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+only	O
+other	O
+structurally	O
+characterized	O
+acp	O
+transferase	O
+enzyme	O
+Tyw2	O
+belongs	O
+to	O
+the	O
+Rossmann	O
+-	O
+fold	O
+class	O
+of	O
+methyltransferase	O
+proteins	O
+.	O
+	O
+Indeed	O
+,	O
+in	O
+wild	O
+-	O
+type	O
+yeast	O
+a	O
+strong	O
+primer	O
+extension	O
+stop	O
+signal	O
+occurred	O
+at	O
+position	O
+1192	O
+.	O
+	O
+As	O
+expected	O
+,	O
+in	O
+a	O
+Δsnr35	B-mutant
+deletion	O
+preventing	O
+pseudouridylation	O
+and	O
+N1	O
+-	O
+methylation	O
+(	O
+resulting	O
+in	O
+acp3U	O
+)	O
+as	O
+well	O
+as	O
+in	O
+a	O
+Δnep1	B-mutant
+deletion	O
+strain	O
+where	O
+pseudouridine	O
+is	O
+not	O
+methylated	O
+(	O
+resulting	O
+in	O
+acp3Ψ	O
+)	O
+a	O
+primer	O
+extension	O
+stop	O
+signal	O
+of	O
+similar	O
+intensity	O
+as	O
+in	O
+the	O
+wild	O
+type	O
+was	O
+observed	O
+.	O
+	O
+The	O
+efficiency	O
+of	O
+siRNA	O
+-	O
+mediated	O
+depletion	O
+was	O
+established	O
+by	O
+RT	O
+-	O
+qPCR	O
+and	O
+found	O
+to	O
+be	O
+very	O
+high	O
+with	O
+siRNA	O
+544	O
+(	O
+Supplementary	O
+Figure	O
+S2A	O
+,	O
+remaining	O
+TSR3	O
+mRNA	O
+level	O
+of	O
+2	O
+%).	O
+	O
+Phenotypic	O
+characterization	O
+of	O
+Δtsr3	B-mutant
+mutants	O
+	O
+Phenotypic	O
+characterization	O
+of	O
+yeast	O
+TSR3	O
+deletion	O
+(	O
+Δtrs3	B-mutant
+)	O
+and	O
+human	O
+TSR3	O
+depletion	O
+(	O
+siRNAs	O
+544	O
+and	O
+545	O
+)	O
+and	O
+cellular	O
+localization	O
+of	O
+yeast	O
+Tsr3	O
+.	O
+(	O
+A	O
+)	O
+Growth	O
+of	O
+yeast	O
+wild	O
+type	O
+,	O
+Δtsr3	B-mutant
+,	O
+Δsnr35	B-mutant
+and	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+segregants	O
+after	O
+meiosis	O
+and	O
+tetrad	O
+dissection	O
+of	O
+Δtsr3	B-mutant
+/	O
+TSR3	O
+Δsnr35	B-mutant
+/	O
+SNR35	O
+heterozygous	O
+diploids	O
+.	O
+	O
+Surprisingly	O
+,	O
+early	O
+nucleolar	O
+processing	O
+reactions	O
+were	O
+also	O
+inhibited	O
+,	O
+and	O
+this	O
+was	O
+observed	O
+in	O
+both	O
+yeast	O
+Δtsr3	B-mutant
+cells	O
+(	O
+see	O
+accumulation	O
+of	O
+35S	O
+in	O
+Supplementary	O
+Figure	O
+S2C	O
+)	O
+and	O
+Tsr3	O
+depleted	O
+human	O
+cells	O
+(	O
+see	O
+47S	O
+/	O
+45S	O
+accumulation	O
+in	O
+Figure	O
+2C	O
+and	O
+Northern	O
+blot	O
+quantification	O
+in	O
+Supplementary	O
+Figure	O
+S2B	O
+).	O
+	O
+Consistent	O
+with	O
+its	O
+role	O
+in	O
+late	O
+18S	O
+rRNA	O
+processing	O
+,	O
+TSR3	O
+deletion	O
+leads	O
+to	O
+a	O
+ribosomal	O
+subunit	O
+imbalance	O
+with	O
+a	O
+reduced	O
+40S	O
+to	O
+60S	O
+ratio	O
+of	O
+0	O
+.	O
+81	O
+(	O
+σ	O
+=	O
+0	O
+.	O
+024	O
+)	O
+which	O
+was	O
+further	O
+increased	O
+in	O
+a	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+recombinant	O
+to	O
+0	O
+.	O
+73	O
+(	O
+σ	O
+=	O
+0	O
+.	O
+023	O
+)	O
+(	O
+Supplementary	O
+Figure	O
+S2F	O
+).	O
+	O
+After	O
+polysome	O
+gradient	O
+separation	O
+C	O
+-	O
+terminally	O
+epitope	O
+-	O
+labeled	O
+Tsr3	B-mutant
+-	I-mutant
+3xHA	I-mutant
+was	O
+exclusively	O
+detectable	O
+in	O
+the	O
+low	O
+-	O
+density	O
+fraction	O
+(	O
+Figure	O
+2E	O
+).	O
+	O
+Such	O
+distribution	O
+on	O
+a	O
+density	O
+gradient	O
+suggests	O
+that	O
+Tsr3	O
+only	O
+interacts	O
+transiently	O
+with	O
+pre	O
+-	O
+40S	O
+subunits	O
+,	O
+which	O
+presumably	O
+explains	O
+why	O
+it	O
+was	O
+not	O
+characterized	O
+in	O
+pre	O
+-	O
+ribosome	O
+affinity	O
+purifications	O
+.	O
+	O
+Structure	O
+of	O
+Tsr3	O
+	O
+However	O
+,	O
+these	O
+archaeal	O
+homologs	O
+are	O
+significantly	O
+smaller	O
+than	O
+ScTsr3	O
+(∼	O
+190	O
+aa	O
+in	O
+archaea	O
+vs	O
+.	O
+313	O
+aa	O
+in	O
+yeast	O
+)	O
+due	O
+to	O
+shortened	O
+N	O
+-	O
+and	O
+C	O
+-	O
+termini	O
+(	O
+Supplementary	O
+Figure	O
+S1A	O
+).	O
+	O
+N	O
+-	O
+terminal	O
+truncations	O
+of	O
+up	O
+to	O
+45	O
+aa	O
+and	O
+C	O
+-	O
+terminal	O
+truncations	O
+of	O
+up	O
+to	O
+76	O
+aa	O
+mediated	O
+acp	O
+modification	O
+as	O
+efficiently	O
+as	O
+the	O
+full	O
+-	O
+length	O
+protein	O
+and	O
+no	O
+significant	O
+increased	O
+levels	O
+of	O
+20S	O
+pre	O
+-	O
+RNA	O
+were	O
+detected	O
+.	O
+	O
+Even	O
+a	O
+Tsr3	O
+fragment	O
+with	O
+a	O
+90	O
+aa	O
+C	O
+-	O
+terminal	O
+truncation	O
+showed	O
+a	O
+residual	O
+primer	O
+extension	O
+stop	O
+,	O
+whereas	O
+N	O
+-	O
+terminal	O
+truncations	O
+exceeding	O
+46	O
+aa	O
+almost	O
+completely	O
+abolished	O
+the	O
+primer	O
+extension	O
+arrest	O
+(	O
+Figure	O
+3B	O
+).	O
+	O
+Strong	O
+20S	O
+rRNA	O
+accumulation	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+is	O
+observed	O
+for	O
+Tsr3	O
+fragments	O
+37	O
+–	O
+223	O
+or	O
+46	O
+–	O
+223	O
+.	O
+	O
+We	O
+focused	O
+on	O
+archaeal	O
+species	O
+containing	O
+a	O
+putative	O
+Nep1	O
+homolog	O
+suggesting	O
+that	O
+these	O
+species	O
+are	O
+in	O
+principle	O
+capable	O
+of	O
+synthesizing	O
+N1	O
+-	O
+methyl	O
+-	O
+N3	O
+-	O
+acp	O
+-	O
+pseudouridine	O
+.	O
+	O
+The	O
+C	O
+-	O
+terminal	O
+domain	O
+(	O
+aa	O
+93	O
+–	O
+184	O
+)	O
+has	O
+a	O
+globular	O
+all	O
+α	O
+-	O
+helical	O
+structure	O
+comprising	O
+α	O
+-	O
+helices	O
+α4	O
+to	O
+α9	O
+.	O
+	O
+Remarkably	O
+,	O
+the	O
+entire	O
+C	O
+-	O
+terminal	O
+domain	O
+(	O
+92	O
+aa	O
+)	O
+of	O
+the	O
+protein	O
+is	O
+threaded	O
+through	O
+the	O
+loop	O
+which	O
+connects	O
+β	O
+-	O
+strand	O
+β3	O
+and	O
+α	O
+-	O
+helix	O
+α2	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+domain	O
+.	O
+	O
+Tsr3	O
+has	O
+a	O
+fold	O
+similar	O
+to	O
+SPOUT	O
+-	O
+class	O
+RNA	O
+methyltransferases	O
+.	O
+(	O
+A	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+X	O
+-	O
+ray	O
+structure	O
+of	O
+VdTsr3	O
+in	O
+two	O
+orientations	O
+.	O
+	O
+A	O
+red	O
+arrow	O
+marks	O
+the	O
+location	O
+of	O
+the	O
+topological	O
+knot	O
+in	O
+the	O
+structure	O
+.	O
+(	O
+B	O
+)	O
+Secondary	O
+structure	O
+representation	O
+of	O
+the	O
+VdTsr3	O
+structure	O
+.	O
+	O
+Structure	O
+predictions	O
+suggested	O
+that	O
+Tsr3	O
+might	O
+contain	O
+a	O
+so	O
+-	O
+called	O
+RLI	O
+domain	O
+which	O
+contains	O
+a	O
+‘	O
+bacterial	O
+like	O
+’	O
+ferredoxin	O
+fold	O
+and	O
+binds	O
+two	O
+iron	O
+-	O
+sulfur	O
+clusters	O
+through	O
+eight	O
+conserved	O
+cysteine	O
+residues	O
+.	O
+	O
+A	O
+notable	O
+exception	O
+is	O
+Trm10	O
+.	O
+	O
+However	O
+,	O
+there	O
+are	O
+no	O
+structural	O
+similarities	O
+between	O
+Tsr3	O
+and	O
+Tyw2	O
+,	O
+which	O
+contains	O
+an	O
+all	O
+-	O
+parallel	O
+β	O
+-	O
+sheet	O
+of	O
+a	O
+different	O
+topology	O
+and	O
+no	O
+knot	O
+structure	O
+.	O
+	O
+The	O
+ribose	O
+2	O
+′	O
+and	O
+3	O
+′	O
+hydroxyl	O
+groups	O
+of	O
+SAM	O
+are	O
+hydrogen	O
+bonded	O
+to	O
+the	O
+backbone	O
+carbonyl	O
+group	O
+of	O
+I69	O
+.	O
+	O
+Consequently	O
+,	O
+the	O
+accessibility	O
+of	O
+this	O
+methyl	O
+group	O
+for	O
+a	O
+nucleophilic	O
+attack	O
+is	O
+strongly	O
+reduced	O
+in	O
+comparison	O
+with	O
+RNA	O
+-	O
+methyltransferases	O
+such	O
+as	O
+Trm10	O
+(	O
+Figure	O
+5B	O
+,	O
+C	O
+).	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+acp	O
+side	O
+chain	O
+of	O
+SAM	O
+is	O
+accessible	O
+for	O
+reactions	O
+in	O
+the	O
+Tsr3	O
+-	O
+bound	O
+state	O
+(	O
+Figure	O
+5B	O
+).	O
+	O
+Hydrogen	O
+bonds	O
+are	O
+indicated	O
+by	O
+dashed	O
+lines	O
+.	O
+	O
+(	O
+E	O
+)	O
+Binding	O
+of	O
+14C	O
+-	O
+labeled	O
+SAM	O
+to	O
+SsTsr3	O
+.	O
+	O
+This	O
+correlates	O
+with	O
+a	O
+20S	O
+pre	O
+-	O
+rRNA	O
+accumulation	O
+comparable	O
+to	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+(	O
+right	O
+:	O
+northern	O
+blot	O
+).	O
+	O
+This	O
+suggests	O
+that	O
+the	O
+hydrophobic	O
+interaction	O
+between	O
+SAM	O
+'	O
+s	O
+methyl	O
+group	O
+and	O
+the	O
+hydrophobic	O
+pocket	O
+of	O
+Tsr3	O
+is	O
+thermodynamically	O
+important	O
+for	O
+the	O
+interaction	O
+.	O
+	O
+Furthermore	O
+,	O
+a	O
+W	O
+to	O
+A	O
+mutation	O
+at	O
+the	O
+equivalent	O
+position	O
+W114	O
+in	O
+ScTsr3	O
+strongly	O
+reduced	O
+the	O
+in	O
+vivo	O
+acp	O
+transferase	O
+activity	O
+(	O
+Figure	O
+5F	O
+).	O
+	O
+The	O
+side	O
+chain	O
+hydroxyl	O
+group	O
+of	O
+T19	O
+seems	O
+of	O
+minor	O
+importance	O
+for	O
+SAM	O
+binding	O
+since	O
+mutations	O
+of	O
+T17	O
+(	O
+T19	O
+in	O
+VdTsr3	O
+)	O
+to	O
+either	O
+A	O
+or	O
+D	O
+did	O
+not	O
+significantly	O
+influence	O
+the	O
+SAM	O
+-	O
+binding	O
+affinity	O
+of	O
+SsTsr3	O
+(	O
+KD	O
+'	O
+s	O
+=	O
+3	O
+.	O
+9	O
+or	O
+11	O
+.	O
+2	O
+mM	O
+,	O
+respectively	O
+).	O
+	O
+In	O
+the	O
+C	O
+-	O
+terminal	O
+domain	O
+,	O
+the	O
+surface	O
+exposed	O
+α	O
+-	O
+helices	O
+α5	O
+and	O
+α7	O
+carry	O
+a	O
+significant	O
+amount	O
+of	O
+positively	O
+charged	O
+amino	O
+acids	O
+.	O
+	O
+RNA	O
+-	O
+binding	O
+of	O
+Tsr3	O
+.	O
+	O
+Also	O
+shown	O
+in	O
+stick	O
+representation	O
+is	O
+a	O
+negatively	O
+charged	O
+MES	O
+ion	O
+.	O
+	O
+The	O
+presence	O
+of	O
+saturating	O
+amounts	O
+of	O
+SAM	O
+(	O
+2	O
+mM	O
+)	O
+did	O
+not	O
+have	O
+a	O
+significant	O
+influence	O
+on	O
+the	O
+RNA	O
+-	O
+affinity	O
+of	O
+SsTsr3	O
+(	O
+KD	O
+of	O
+1	O
+.	O
+7	O
+μM	O
+for	O
+the	O
+20mer	O
+-	O
+GC	O
+-	O
+RNA	O
+)	O
+suggesting	O
+no	O
+cooperativity	O
+in	O
+substrate	O
+binding	O
+.	O
+	O
+This	O
+suggests	O
+that	O
+enzymes	O
+with	O
+a	O
+SAM	O
+-	O
+dependent	O
+acp	O
+transferase	O
+activity	O
+might	O
+have	O
+evolved	O
+from	O
+SAM	O
+-	O
+dependent	O
+methyltransferases	O
+by	O
+slight	O
+modifications	O
+of	O
+the	O
+SAM	O
+-	O
+binding	O
+pocket	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+Nep1	O
+,	O
+the	O
+enzyme	O
+preceding	O
+Tsr3	O
+in	O
+the	O
+m1acp3Ψ	O
+biosynthesis	O
+pathway	O
+,	O
+Tsr3	O
+binds	O
+rather	O
+weakly	O
+and	O
+with	O
+little	O
+specificity	O
+to	O
+its	O
+isolated	O
+substrate	O
+RNA	O
+.	O
+	O
+Recently	O
+,	O
+structural	O
+,	O
+functional	O
+,	O
+and	O
+CRAC	O
+(	O
+cross	O
+-	O
+linking	O
+and	O
+cDNA	O
+analysis	O
+)	O
+experiments	O
+of	O
+late	O
+assembly	O
+factors	O
+involved	O
+in	O
+cytoplasmic	O
+processing	O
+of	O
+40S	O
+subunits	O
+,	O
+along	O
+with	O
+cryo	O
+-	O
+EM	O
+studies	O
+of	O
+the	O
+late	O
+pre	O
+-	O
+40S	O
+subunits	O
+have	O
+provided	O
+important	O
+insights	O
+into	O
+late	O
+pre	O
+-	O
+40S	O
+processing	O
+.	O
+	O
+The	O
+cleavage	O
+step	O
+most	O
+likely	O
+acts	O
+as	O
+a	O
+quality	O
+control	O
+check	O
+that	O
+ensures	O
+the	O
+proper	O
+40S	O
+subunit	O
+assembly	O
+with	O
+only	O
+completely	O
+processed	O
+precursors	O
+.	O
+	O
+The	O
+YfiBNR	O
+system	O
+contains	O
+three	O
+protein	O
+members	O
+and	O
+modulates	O
+intracellular	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+levels	O
+in	O
+response	O
+to	O
+signals	O
+received	O
+in	O
+the	O
+periplasm	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+More	O
+recently	O
+,	O
+this	O
+system	O
+was	O
+also	O
+reported	O
+in	O
+other	O
+Gram	O
+-	O
+negative	O
+bacteria	O
+,	O
+such	O
+as	O
+Escherichia	O
+coli	O
+(	O
+Hufnagel	O
+et	O
+al	O
+.,;	O
+Raterman	O
+et	O
+al	O
+.,;	O
+Sanchez	O
+-	O
+Torres	O
+et	O
+al	O
+.,),	O
+Klebsiella	O
+pneumonia	O
+(	O
+Huertas	O
+et	O
+al	O
+.,)	O
+and	O
+Yersinia	O
+pestis	O
+(	O
+Ren	O
+et	O
+al	O
+.,).	O
+	O
+Whether	O
+YfiB	O
+directly	O
+recruits	O
+YfiR	O
+or	O
+recruits	O
+YfiR	O
+via	O
+a	O
+third	O
+partner	O
+is	O
+an	O
+open	O
+question	O
+.	O
+	O
+It	O
+has	O
+been	O
+reported	O
+that	O
+the	O
+activation	O
+of	O
+YfiN	O
+may	O
+be	O
+induced	O
+by	O
+redox	O
+-	O
+driven	O
+misfolding	O
+of	O
+YfiR	O
+(	O
+Giardina	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+In	O
+addition	O
+,	O
+quorum	O
+sensing	O
+-	O
+related	O
+dephosphorylation	O
+of	O
+the	O
+PAS	O
+domain	O
+of	O
+YfiN	O
+may	O
+also	O
+be	O
+involved	O
+in	O
+the	O
+regulation	O
+(	O
+Ueda	O
+and	O
+Wood	O
+,;	O
+Xu	O
+et	O
+al	O
+.,).	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+YfiB	O
+monomer	O
+consists	O
+of	O
+a	O
+five	O
+-	O
+stranded	O
+β	O
+-	O
+sheet	O
+(	O
+β1	O
+-	O
+2	O
+-	O
+5	O
+-	O
+3	O
+-	O
+4	O
+)	O
+flanked	O
+by	O
+five	O
+α	O
+-	O
+helices	O
+(	O
+α1	O
+–	O
+5	O
+)	O
+on	O
+one	O
+side	O
+.	O
+	O
+The	O
+residues	O
+proposed	O
+to	O
+contribute	O
+to	O
+YfiB	O
+activation	O
+are	O
+illustrated	O
+in	O
+sticks	O
+.	O
+	O
+It	O
+has	O
+been	O
+reported	O
+that	O
+single	O
+mutants	O
+of	O
+Q39	O
+,	O
+L43	O
+,	O
+F48	O
+and	O
+W55	O
+contribute	O
+to	O
+YfiB	O
+activation	O
+leading	O
+to	O
+the	O
+induction	O
+of	O
+the	O
+SCV	O
+phenotype	O
+in	O
+P	O
+.	O
+aeruginosa	O
+PAO1	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+	O
+These	O
+two	O
+regions	O
+contribute	O
+a	O
+robust	O
+hydrogen	O
+-	O
+bonding	O
+network	O
+to	O
+the	O
+YfiB	O
+-	O
+YfiR	O
+interface	O
+,	O
+resulting	O
+in	O
+a	O
+tightly	O
+bound	O
+complex	O
+.	O
+	O
+Therefore	O
+,	O
+it	O
+is	O
+possible	O
+that	O
+both	O
+dimeric	O
+types	O
+might	O
+exist	O
+in	O
+solution	O
+.	O
+	O
+In	O
+the	O
+Pal	O
+/	O
+PG	O
+-	O
+P	O
+complex	O
+structure	O
+,	O
+the	O
+m	O
+-	O
+Dap5	O
+ϵ	O
+-	O
+carboxylate	O
+group	O
+interacts	O
+with	O
+the	O
+side	O
+-	O
+chain	O
+atoms	O
+of	O
+D71	O
+and	O
+the	O
+main	O
+-	O
+chain	O
+amide	O
+of	O
+D37	O
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+	O
+Calculation	O
+using	O
+the	O
+ConSurf	O
+Server	O
+(	O
+http	O
+://	O
+consurf	O
+.	O
+tau	O
+.	O
+ac	O
+.	O
+il	O
+/),	O
+which	O
+estimates	O
+the	O
+evolutionary	O
+conservation	O
+of	O
+amino	O
+acid	O
+positions	O
+and	O
+visualizes	O
+information	O
+on	O
+the	O
+structure	O
+surface	O
+,	O
+revealed	O
+a	O
+conserved	O
+surface	O
+on	O
+YfiR	O
+that	O
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	O
+(	O
+Fig	O
+.	O
+3E	O
+and	O
+3F	O
+).	O
+	O
+E163	O
+and	O
+I169	O
+are	O
+YfiB	O
+-	O
+interacting	O
+residues	O
+of	O
+YfiR	O
+,	O
+in	O
+which	O
+E163	O
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+R96	O
+of	O
+YfiB	O
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+II	O
+)	O
+and	O
+I169	O
+is	O
+involved	O
+in	O
+forming	O
+the	O
+L166	O
+/	O
+I169	O
+/	O
+V176	O
+/	O
+P178	O
+/	O
+L181	O
+hydrophobic	O
+core	O
+for	O
+anchoring	O
+F57	O
+of	O
+YfiB	O
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+ii	O
+)).	O
+	O
+Intriguingly	O
+,	O
+a	O
+Dali	O
+search	O
+(	O
+Holm	O
+and	O
+Rosenstrom	O
+,)	O
+indicated	O
+that	O
+the	O
+closest	O
+homologs	O
+of	O
+YfiR	O
+shared	O
+the	O
+characteristic	O
+of	O
+being	O
+able	O
+to	O
+bind	O
+several	O
+structurally	O
+similar	O
+small	O
+molecules	O
+,	O
+such	O
+as	O
+L	O
+-	O
+Trp	O
+,	O
+L	O
+-	O
+Phe	O
+,	O
+B	O
+-	O
+group	O
+vitamins	O
+and	O
+their	O
+analogs	O
+,	O
+encouraging	O
+us	O
+to	O
+test	O
+whether	O
+YfiR	O
+can	O
+recognize	O
+these	O
+molecules	O
+.	O
+	O
+Structural	O
+analyses	O
+revealed	O
+that	O
+the	O
+VB6	O
+and	O
+L	O
+-	O
+Trp	O
+molecules	O
+are	O
+bound	O
+at	O
+the	O
+periphery	O
+of	O
+the	O
+YfiR	O
+dimer	O
+,	O
+but	O
+not	O
+at	O
+the	O
+dimer	O
+interface	O
+.	O
+	O
+To	O
+evaluate	O
+the	O
+importance	O
+of	O
+F57	O
+in	O
+YfiBL43P	O
+-	O
+YfiR	O
+interaction	O
+,	O
+the	O
+binding	O
+affinities	O
+of	O
+YfiBL43P	B-mutant
+and	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+for	O
+YfiR	O
+were	O
+measured	O
+by	O
+isothermal	O
+titration	O
+calorimetry	O
+(	O
+ITC	O
+).	O
+	O
+Provided	O
+that	O
+the	O
+diameter	O
+of	O
+the	O
+widest	O
+part	O
+of	O
+the	O
+YfiB	O
+dimer	O
+is	O
+approximately	O
+64	O
+Å	O
+,	O
+which	O
+is	O
+slightly	O
+smaller	O
+than	O
+the	O
+smallest	O
+diameter	O
+of	O
+the	O
+PG	O
+pore	O
+(	O
+70	O
+Å	O
+)	O
+(	O
+Meroueh	O
+et	O
+al	O
+.,),	O
+the	O
+YfiB	O
+dimer	O
+should	O
+be	O
+able	O
+to	O
+penetrate	O
+the	O
+PG	O
+layer	O
+.	O
+	O
+Once	O
+activated	O
+by	O
+certain	O
+cell	O
+stress	O
+,	O
+the	O
+dimeric	O
+YfiB	O
+transforms	O
+from	O
+a	O
+compact	O
+conformation	O
+to	O
+a	O
+stretched	O
+conformation	O
+,	O
+allowing	O
+the	O
+periplasmic	O
+domain	O
+of	O
+the	O
+membrane	O
+-	O
+anchored	O
+YfiB	O
+to	O
+penetrate	O
+the	O
+cell	O
+wall	O
+and	O
+sequester	O
+the	O
+YfiR	O
+dimer	O
+,	O
+thus	O
+relieving	O
+the	O
+repression	O
+of	O
+YfiN	O
+	O
+The	O
+YfiBNR	O
+system	O
+provides	O
+a	O
+good	O
+example	O
+of	O
+a	O
+delicate	O
+homeostatic	O
+system	O
+that	O
+integrates	O
+multiple	O
+signals	O
+to	O
+regulate	O
+the	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+level	O
+.	O
+	O
+These	O
+APFs	O
+had	O
+an	O
+outer	O
+diameter	O
+that	O
+ranged	O
+from	O
+11	O
+–	O
+14	O
+nm	O
+and	O
+an	O
+inner	O
+diameter	O
+that	O
+ranged	O
+from	O
+2	O
+.	O
+5	O
+–	O
+4	O
+nm	O
+.	O
+	O
+These	O
+observations	O
+suggest	O
+that	O
+the	O
+Aβ	O
+trimers	O
+,	O
+hexamers	O
+,	O
+dodecamers	O
+,	O
+and	O
+related	O
+assemblies	O
+may	O
+be	O
+associated	O
+with	O
+presymptomatic	O
+neurodegeneration	O
+,	O
+while	O
+Aβ	O
+dimers	O
+are	O
+more	O
+closely	O
+associated	O
+with	O
+fibril	O
+formation	O
+and	O
+plaque	O
+deposition	O
+during	O
+symptomatic	O
+Alzheimer	O
+’	O
+s	O
+disease	O
+.−	O
+	O
+Many	O
+of	O
+these	O
+studies	O
+have	O
+reported	O
+the	O
+monomer	O
+subunit	O
+as	O
+adopting	O
+a	O
+β	O
+-	O
+hairpin	O
+conformation	O
+,	O
+in	O
+which	O
+the	O
+hydrophobic	O
+central	O
+and	O
+C	O
+-	O
+terminal	O
+regions	O
+form	O
+an	O
+antiparallel	O
+β	O
+-	O
+sheet	O
+.	O
+	O
+In	O
+2008	O
+,	O
+Hoyer	O
+et	O
+al	O
+.	O
+reported	O
+the	O
+NMR	O
+structure	O
+of	O
+an	O
+Aβ	O
+monomer	O
+bound	O
+to	O
+an	O
+artificial	O
+binding	O
+protein	O
+called	O
+an	O
+affibody	O
+(	O
+PDB	O
+2OTK	O
+).	O
+	O
+This	O
+Aβ	O
+β	O
+-	O
+hairpin	O
+encompasses	O
+residues	O
+17	O
+–	O
+37	O
+and	O
+contains	O
+two	O
+β	O
+-	O
+strands	O
+comprising	O
+Aβ17	O
+–	O
+24	O
+and	O
+Aβ30	O
+–	O
+37	O
+connected	O
+by	O
+an	O
+Aβ25	O
+–	O
+29	O
+loop	O
+.	O
+	O
+In	O
+a	O
+related	O
+study	O
+,	O
+Sandberg	O
+et	O
+al	O
+.	O
+constrained	O
+Aβ	O
+in	O
+a	O
+β	O
+-	O
+hairpin	O
+conformation	O
+by	O
+mutating	O
+residues	O
+A21	O
+and	O
+A30	O
+to	O
+cysteine	O
+and	O
+forming	O
+an	O
+intramolecular	O
+disulfide	O
+bond	O
+.	O
+	O
+After	O
+determining	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+the	O
+p	O
+-	O
+iodophenylalanine	O
+variant	O
+we	O
+attempt	O
+to	O
+determine	O
+the	O
+structure	O
+of	O
+the	O
+native	O
+phenylalanine	O
+compound	O
+by	O
+isomorphous	O
+replacement	O
+.	O
+	O
+Upon	O
+synthesizing	O
+peptide	B-mutant
+3	I-mutant
+,	O
+we	O
+found	O
+that	O
+it	O
+formed	O
+an	O
+amorphous	O
+precipitate	O
+in	O
+most	O
+crystallization	O
+conditions	O
+screened	O
+and	O
+failed	O
+to	O
+afford	O
+crystals	O
+in	O
+any	O
+condition	O
+.	O
+	O
+Although	O
+the	O
+disulfide	O
+bond	O
+between	O
+positions	O
+24	O
+and	O
+29	O
+helps	O
+stabilize	O
+the	O
+β	O
+-	O
+hairpin	O
+,	O
+it	O
+does	O
+not	O
+alter	O
+the	O
+charge	O
+or	O
+substantially	O
+change	O
+the	O
+hydrophobicity	O
+of	O
+the	O
+Aβ17	O
+–	O
+36	O
+β	O
+-	O
+hairpin	O
+.	O
+	O
+Crystallization	O
+,	O
+X	O
+-	O
+ray	O
+Crystallographic	O
+Data	O
+Collection	O
+,	O
+Data	O
+Processing	O
+,	O
+and	O
+Structure	O
+Determination	O
+of	O
+Peptides	B-mutant
+2	I-mutant
+and	I-mutant
+4	I-mutant
+	O
+Data	O
+from	O
+peptides	B-mutant
+4	I-mutant
+and	I-mutant
+2	I-mutant
+suitable	O
+for	O
+refinement	O
+at	O
+2	O
+.	O
+30	O
+Å	O
+were	O
+obtained	O
+from	O
+the	O
+diffractometer	O
+;	O
+data	O
+from	O
+peptide	B-mutant
+2	I-mutant
+suitable	O
+for	O
+refinement	O
+at	O
+1	O
+.	O
+90	O
+Å	O
+were	O
+obtained	O
+from	O
+the	O
+synchrotron	O
+.	O
+	O
+Peptide	B-mutant
+2	I-mutant
+assembles	O
+into	O
+oligomers	O
+similar	O
+in	O
+morphology	O
+to	O
+those	O
+formed	O
+by	O
+peptide	B-mutant
+1	I-mutant
+.	O
+	O
+Hydrogen	O
+bonding	O
+and	O
+hydrophobic	O
+interactions	O
+between	O
+residues	O
+on	O
+the	O
+β	O
+-	O
+strands	O
+comprising	O
+Aβ17	O
+–	O
+23	O
+and	O
+Aβ30	O
+–	O
+36	O
+stabilize	O
+the	O
+core	O
+of	O
+the	O
+trimer	O
+.	O
+	O
+At	O
+the	O
+corners	O
+of	O
+the	O
+trimer	O
+,	O
+the	O
+pairs	O
+of	O
+β	O
+-	O
+hairpin	O
+monomers	O
+form	O
+four	O
+hydrogen	O
+bonds	O
+:	O
+two	O
+between	O
+the	O
+main	O
+chains	O
+of	O
+V18	O
+and	O
+E22	O
+and	O
+two	O
+between	O
+δOrn	O
+and	O
+the	O
+main	O
+chain	O
+of	O
+C24	O
+(	O
+Figure	O
+3B	O
+).	O
+	O
+The	O
+other	O
+face	O
+of	O
+the	O
+trimer	O
+displays	O
+a	O
+smaller	O
+hydrophobic	O
+surface	O
+,	O
+which	O
+includes	O
+the	O
+side	O
+chains	O
+of	O
+residues	O
+V18	O
+,	O
+F20	O
+,	O
+and	O
+I31	O
+of	O
+the	O
+three	O
+β	O
+-	O
+hairpins	O
+(	O
+Figure	O
+3D	O
+).	O
+	O
+Dodecamer	O
+	O
+The	O
+four	O
+trimers	O
+arrange	O
+in	O
+a	O
+tetrahedral	O
+fashion	O
+,	O
+creating	O
+a	O
+central	O
+cavity	O
+inside	O
+the	O
+dodecamer	O
+.	O
+Because	O
+each	O
+trimer	O
+is	O
+triangular	O
+,	O
+the	O
+resulting	O
+arrangement	O
+resembles	O
+an	O
+octahedron	O
+.	O
+	O
+Residues	O
+L17	O
+,	O
+L34	O
+,	O
+and	O
+V36	O
+are	O
+shown	O
+as	O
+spheres	O
+,	O
+illustrating	O
+the	O
+hydrophobic	O
+packing	O
+that	O
+occurs	O
+at	O
+the	O
+six	O
+vertices	O
+of	O
+the	O
+dodecamer	O
+.	O
+(	O
+D	O
+)	O
+Detailed	O
+view	O
+of	O
+one	O
+of	O
+the	O
+six	O
+vertices	O
+of	O
+the	O
+dodecamer	O
+.	O
+	O
+The	O
+electron	O
+density	O
+map	O
+for	O
+the	O
+X	O
+-	O
+ray	O
+crystallographic	O
+structure	O
+of	O
+peptide	B-mutant
+2	I-mutant
+has	O
+long	O
+tubes	O
+of	O
+electron	O
+density	O
+inside	O
+the	O
+central	O
+cavity	O
+of	O
+the	O
+dodecamer	O
+.	O
+	O
+Although	O
+Jeffamine	O
+M	O
+-	O
+600	O
+is	O
+a	O
+heterogeneous	O
+mixture	O
+with	O
+varying	O
+chain	O
+lengths	O
+and	O
+stereochemistry	O
+,	O
+we	O
+modeled	O
+a	O
+single	O
+stereoisomer	O
+with	O
+nine	O
+propylene	O
+glycol	O
+units	O
+(	O
+n	O
+=	O
+9	O
+)	O
+to	O
+fit	O
+the	O
+electron	O
+density	O
+.	O
+	O
+Annular	O
+Pore	O
+	O
+The	O
+same	O
+eclipsed	O
+interface	O
+also	O
+occurs	O
+between	O
+dodecamers	O
+1	O
+and	O
+5	O
+and	O
+3	O
+and	O
+4	O
+.	O
+(	O
+C	O
+)	O
+Staggered	O
+interface	O
+between	O
+dodecamers	O
+2	O
+and	O
+3	O
+(	O
+side	O
+view	O
+).	O
+	O
+It	O
+is	O
+important	O
+to	O
+note	O
+that	O
+the	O
+annular	O
+pore	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+is	O
+not	O
+a	O
+discrete	O
+unit	O
+in	O
+the	O
+crystal	O
+lattice	O
+.	O
+	O
+The	O
+dodecamers	O
+further	O
+assemble	O
+to	O
+form	O
+an	O
+annular	O
+pore	O
+(	O
+Figure	O
+6	O
+).	O
+	O
+Monomeric	O
+Aβ	O
+folds	O
+to	O
+form	O
+a	O
+β	O
+-	O
+hairpin	O
+in	O
+which	O
+the	O
+hydrophobic	O
+central	O
+and	O
+C	O
+-	O
+terminal	O
+regions	O
+form	O
+an	O
+antiparallel	O
+β	O
+-	O
+sheet	O
+.	O
+	O
+Four	O
+triangular	O
+trimers	O
+assemble	O
+to	O
+form	O
+a	O
+dodecamer	O
+.	O
+	O
+Five	O
+dodecamers	O
+assemble	O
+to	O
+form	O
+an	O
+annular	O
+pore	O
+.	O
+	O
+These	O
+criteria	O
+have	O
+been	O
+used	O
+to	O
+classify	O
+the	O
+Aβ	O
+oligomers	O
+that	O
+accumulate	O
+in	O
+vivo	O
+.	O
+	O
+Aβ	O
+dimers	O
+have	O
+been	O
+classified	O
+as	O
+fibrillar	O
+oligomers	O
+,	O
+whereas	O
+Aβ	O
+trimers	O
+,	O
+Aβ	O
+*	O
+56	O
+,	O
+and	O
+APFs	O
+have	O
+been	O
+classified	O
+as	O
+nonfibrillar	O
+oligomers	O
+.	O
+	O
+The	O
+hierarchical	O
+assembly	O
+of	O
+peptide	B-mutant
+2	I-mutant
+is	O
+consistent	O
+with	O
+this	O
+model	O
+;	O
+and	O
+the	O
+trimer	O
+,	O
+dodecamer	O
+,	O
+and	O
+annular	O
+pore	O
+formed	O
+by	O
+peptide	B-mutant
+2	I-mutant
+may	O
+share	O
+similarities	O
+to	O
+the	O
+trimers	O
+,	O
+Aβ	O
+*	O
+56	O
+,	O
+and	O
+APFs	O
+observed	O
+in	O
+vivo	O
+.	O
+	O
+Annular	O
+Pores	O
+Formed	O
+by	O
+Aβ	O
+and	O
+Peptide	B-mutant
+2	I-mutant
+	O
+This	O
+mode	O
+of	O
+assembly	O
+is	O
+not	O
+unique	O
+to	O
+Aβ	O
+.	O
+	O
+We	O
+believe	O
+this	O
+iterative	O
+,	O
+“	O
+bottom	O
+up	O
+”	O
+approach	O
+of	O
+identifying	O
+the	O
+minimal	O
+modification	O
+required	O
+to	O
+crystallize	O
+Aβ	O
+peptides	O
+will	O
+ultimately	O
+allow	O
+larger	O
+fragments	O
+of	O
+Aβ	O
+to	O
+be	O
+crystallized	O
+,	O
+thus	O
+providing	O
+greater	O
+insights	O
+into	O
+the	O
+structures	O
+of	O
+Aβ	O
+oligomers	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+N	O
+‐	O
+terminal	O
+coactivator	O
+‐	O
+binding	O
+site	O
+,	O
+activation	O
+function	O
+‐	O
+1	O
+(	O
+AF	O
+‐	O
+1	O
+),	O
+determined	O
+cell	O
+‐	O
+specific	O
+signaling	O
+induced	O
+by	O
+ligands	O
+that	O
+used	O
+alternate	O
+mechanisms	O
+to	O
+control	O
+cell	O
+proliferation	O
+.	O
+	O
+ERα	O
+domain	O
+organization	O
+lettered	O
+,	O
+A	O
+‐	O
+F	O
+.	O
+DBD	O
+,	O
+DNA	O
+‐	O
+binding	O
+domain	O
+;	O
+LBD	O
+,	O
+ligand	O
+‐	O
+binding	O
+domain	O
+;	O
+AF	O
+,	O
+activation	O
+function	O
+	O
+Branched	O
+causality	O
+model	O
+for	O
+ERα	O
+‐	O
+mediated	O
+cell	O
+proliferation	O
+.	O
+	O
+However	O
+,	O
+the	O
+agonist	O
+activity	O
+of	O
+SERMs	O
+derives	O
+from	O
+activation	O
+function	O
+‐	O
+1	O
+(	O
+AF	O
+‐	O
+1	O
+)—	O
+a	O
+coactivator	O
+recruitment	O
+site	O
+located	O
+in	O
+the	O
+AB	O
+domain	O
+(	O
+Berry	O
+et	O
+al	O
+,	O
+1990	O
+;	O
+Shang	O
+&	O
+Brown	O
+,	O
+2002	O
+;	O
+Abot	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+	O
+The	O
+simplest	O
+response	O
+model	O
+for	O
+ligand	O
+‐	O
+specific	O
+proliferative	O
+effects	O
+is	O
+a	O
+linear	O
+causality	O
+model	O
+,	O
+where	O
+the	O
+degree	O
+of	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+recruitment	O
+determines	O
+GREB1	O
+expression	O
+,	O
+which	O
+in	O
+turn	O
+drives	O
+ligand	O
+‐	O
+specific	O
+cell	O
+proliferation	O
+(	O
+Fig	O
+1D	O
+).	O
+	O
+OBHS	O
+is	O
+an	O
+indirect	O
+modulator	O
+that	O
+dislocates	O
+the	O
+h11	O
+C	O
+‐	O
+terminus	O
+to	O
+destabilize	O
+the	O
+h11	O
+–	O
+h12	O
+interface	O
+(	O
+PDB	O
+4ZN9	O
+).	O
+	O
+The	O
+ERα	O
+ligand	O
+library	O
+contains	O
+241	O
+ligands	O
+representing	O
+15	O
+indirect	O
+modulator	O
+scaffolds	O
+,	O
+plus	O
+4	O
+direct	O
+modulator	O
+scaffolds	O
+.	O
+	O
+Ligand	O
+‐	O
+specific	O
+signaling	O
+underlies	O
+ERα	O
+‐	O
+mediated	O
+cell	O
+proliferation	O
+	O
+(	O
+B	O
+)	O
+Ligand	O
+class	O
+analysis	O
+of	O
+the	O
+L	O
+‐	O
+Luc	O
+ERα	O
+‐	O
+WT	O
+and	O
+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activities	O
+in	O
+HepG2	O
+cells	O
+.	O
+	O
+Deletion	O
+of	O
+the	O
+AB	O
+domain	O
+significantly	O
+reduced	O
+the	O
+average	O
+L	O
+‐	O
+Luc	O
+activities	O
+of	O
+14	O
+scaffolds	O
+(	O
+Student	O
+'	O
+s	O
+t	O
+‐	O
+test	O
+,	O
+P	O
+≤	O
+0	O
+.	O
+05	O
+)	O
+(	O
+Fig	O
+3B	O
+).	O
+	O
+The	O
+value	O
+of	O
+r	O
+ranges	O
+from	O
+−	O
+1	O
+to	O
+1	O
+,	O
+and	O
+it	O
+defines	O
+the	O
+extent	O
+to	O
+which	O
+the	O
+data	O
+fit	O
+a	O
+straight	O
+line	O
+when	O
+compounds	O
+show	O
+similar	O
+agonist	O
+/	O
+antagonist	O
+activity	O
+profiles	O
+between	O
+cell	O
+types	O
+(	O
+Fig	O
+EV3A	O
+).	O
+	O
+This	O
+cluster	O
+includes	O
+two	O
+classes	O
+of	O
+direct	O
+modulators	O
+(	O
+cyclofenil	O
+‐	O
+ASC	O
+and	O
+WAY	O
+dimer	O
+),	O
+and	O
+six	O
+classes	O
+of	O
+indirect	O
+modulators	O
+(	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+,	O
+3	O
+,	O
+4	O
+‐	O
+DTP	O
+,	O
+S	O
+‐	O
+OBHS	O
+‐	O
+2	O
+and	O
+S	O
+‐	O
+OBHS	O
+‐	O
+3	O
+,	O
+furan	O
+,	O
+and	O
+WAY	O
+‐	O
+D	O
+).	O
+	O
+In	O
+contrast	O
+,	O
+AF	O
+‐	O
+1	O
+was	O
+a	O
+determinant	O
+of	O
+signaling	O
+specificity	O
+for	O
+scaffolds	O
+in	O
+cluster	O
+2	O
+.	O
+	O
+For	O
+ligands	O
+in	O
+cluster	O
+3	O
+,	O
+we	O
+could	O
+not	O
+eliminate	O
+a	O
+role	O
+for	O
+AF	O
+‐	O
+1	O
+in	O
+determining	O
+signaling	O
+specificity	O
+,	O
+since	O
+this	O
+cluster	O
+lacked	O
+positively	O
+correlated	O
+activity	O
+profiles	O
+(	O
+Fig	O
+3C	O
+),	O
+and	O
+deletion	O
+of	O
+the	O
+AB	O
+or	O
+F	O
+domain	O
+rarely	O
+induced	O
+such	O
+correlations	O
+(	O
+Fig	O
+3D	O
+),	O
+except	O
+for	O
+A	O
+‐	O
+CD	O
+and	O
+OBHS	O
+‐	O
+ASC	O
+analogs	O
+,	O
+where	O
+deletion	O
+of	O
+the	O
+AB	O
+domain	O
+or	O
+F	O
+domain	O
+led	O
+to	O
+positive	O
+correlations	O
+with	O
+E	O
+‐	O
+Luc	O
+activity	O
+and	O
+/	O
+or	O
+GREB1	O
+levels	O
+(	O
+Fig	O
+3D	O
+lanes	O
+13	O
+and	O
+18	O
+).	O
+	O
+To	O
+determine	O
+mechanisms	O
+for	O
+ligand	O
+‐	O
+dependent	O
+control	O
+of	O
+breast	O
+cancer	O
+cell	O
+proliferation	O
+,	O
+we	O
+performed	O
+linear	O
+regression	O
+analyses	O
+across	O
+the	O
+19	O
+scaffolds	O
+using	O
+MCF	O
+‐	O
+7	O
+cell	O
+proliferation	O
+as	O
+the	O
+dependent	O
+variable	O
+,	O
+and	O
+the	O
+other	O
+activities	O
+as	O
+independent	O
+variables	O
+(	O
+Fig	O
+3F	O
+).	O
+	O
+The	O
+lack	O
+of	O
+significant	O
+predictors	O
+for	O
+OBHS	O
+analogs	O
+(	O
+Fig	O
+3F	O
+lane	O
+1	O
+)	O
+reflects	O
+their	O
+small	O
+range	O
+of	O
+proliferative	O
+effects	O
+on	O
+MCF	O
+‐	O
+7	O
+cells	O
+(	O
+Fig	O
+EV2I	O
+).	O
+	O
+The	O
+significant	O
+correlations	O
+with	O
+GREB1	O
+expression	O
+and	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+recruitment	O
+observed	O
+in	O
+this	O
+cluster	O
+are	O
+consistent	O
+with	O
+the	O
+canonical	O
+signaling	O
+model	O
+(	O
+Fig	O
+1D	O
+),	O
+where	O
+NCOA1	O
+/	O
+2	O
+/	O
+3	O
+recruitment	O
+determines	O
+GREB1	O
+expression	O
+,	O
+which	O
+then	O
+drives	O
+proliferation	O
+.	O
+	O
+3	O
+,	O
+4	O
+‐	O
+DTP	O
+,	O
+cyclofenil	O
+,	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+,	O
+and	O
+imidazopyridine	O
+analogs	O
+had	O
+NCOA1	O
+/	O
+3	O
+recruitment	O
+profiles	O
+that	O
+predicted	O
+their	O
+proliferative	O
+effects	O
+,	O
+without	O
+determining	O
+GREB1	O
+levels	O
+(	O
+Fig	O
+3E	O
+and	O
+F	O
+,	O
+lanes	O
+5	O
+and	O
+14	O
+–	O
+16	O
+).	O
+	O
+Therefore	O
+,	O
+we	O
+first	O
+performed	O
+a	O
+time	O
+‐	O
+course	O
+study	O
+,	O
+and	O
+found	O
+that	O
+E2	O
+and	O
+the	O
+WAY	O
+‐	O
+C	O
+analog	O
+,	O
+AAPII	O
+‐	O
+151	O
+‐	O
+4	O
+,	O
+induced	O
+recruitment	O
+of	O
+NCOA3	O
+to	O
+the	O
+GREB1	O
+promoter	O
+in	O
+a	O
+temporal	O
+cycle	O
+that	O
+peaked	O
+after	O
+45	O
+min	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+(	O
+Fig	O
+4A	O
+).	O
+	O
+However	O
+,	O
+the	O
+ChIP	O
+assays	O
+for	O
+WAY	O
+‐	O
+C	O
+‐	O
+induced	O
+recruitment	O
+of	O
+NCOA3	O
+to	O
+the	O
+GREB1	O
+promoter	O
+did	O
+not	O
+correlate	O
+with	O
+any	O
+of	O
+the	O
+other	O
+WAY	O
+‐	O
+C	O
+activity	O
+profiles	O
+(	O
+Fig	O
+4D	O
+),	O
+although	O
+the	O
+positive	O
+correlation	O
+between	O
+ChIP	O
+assays	O
+and	O
+NCOA3	O
+recruitment	O
+via	O
+M2H	O
+assay	O
+showed	O
+a	O
+trend	O
+toward	O
+significance	O
+with	O
+r	O
+2	O
+=	O
+0	O
+.	O
+36	O
+and	O
+P	O
+=	O
+0	O
+.	O
+09	O
+(	O
+F	O
+‐	O
+test	O
+for	O
+nonzero	O
+slope	O
+).	O
+	O
+ERβ	O
+activity	O
+is	O
+not	O
+an	O
+independent	O
+predictor	O
+of	O
+E	O
+‐	O
+Luc	O
+activity	O
+	O
+To	O
+further	O
+validate	O
+this	O
+approach	O
+,	O
+we	O
+solved	O
+the	O
+structure	O
+of	O
+the	O
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+LBD	O
+in	O
+complex	O
+with	O
+diethylstilbestrol	O
+(	O
+DES	O
+),	O
+which	O
+bound	O
+identically	O
+in	O
+the	O
+wild	O
+‐	O
+type	O
+and	O
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+LBDs	O
+,	O
+demonstrating	O
+again	O
+that	O
+this	O
+surface	O
+mutation	O
+stabilizes	O
+h12	O
+dynamics	O
+to	O
+facilitate	O
+crystallization	O
+without	O
+changing	O
+ligand	O
+binding	O
+(	O
+Appendix	O
+Fig	O
+S1A	O
+and	O
+B	O
+)	O
+(	O
+Nettles	O
+et	O
+al	O
+,	O
+2008	O
+;	O
+Bruning	O
+et	O
+al	O
+,	O
+2010	O
+;	O
+Delfosse	O
+et	O
+al	O
+,	O
+2012	O
+).	O
+	O
+Using	O
+this	O
+approach	O
+,	O
+we	O
+solved	O
+76	O
+ERα	O
+LBD	O
+structures	O
+in	O
+the	O
+active	O
+conformation	O
+and	O
+bound	O
+to	O
+ligands	O
+studied	O
+here	O
+(	O
+Appendix	O
+Fig	O
+S1C	O
+).	O
+	O
+The	O
+indirect	O
+modulator	O
+scaffolds	O
+in	O
+cluster	O
+1	O
+did	O
+not	O
+show	O
+cell	O
+‐	O
+specific	O
+signaling	O
+(	O
+Fig	O
+3C	O
+),	O
+but	O
+shared	O
+common	O
+structural	O
+perturbations	O
+that	O
+we	O
+designed	O
+to	O
+modulate	O
+h12	O
+dynamics	O
+.	O
+	O
+The	O
+24	O
+structures	O
+containing	O
+OBHS	O
+,	O
+OBHS	O
+‐	O
+N	O
+,	O
+or	O
+triaryl	O
+‐	O
+ethylene	O
+analogs	O
+showed	O
+structural	O
+diversity	O
+in	O
+the	O
+same	O
+part	O
+of	O
+the	O
+scaffolds	O
+(	O
+Figs	O
+5A	O
+and	O
+EV5A	O
+),	O
+and	O
+the	O
+same	O
+region	O
+of	O
+the	O
+LBD	O
+—	O
+the	O
+C	O
+‐	O
+terminal	O
+end	O
+of	O
+h11	O
+(	O
+Figs	O
+5B	O
+and	O
+C	O
+,	O
+and	O
+EV5B	O
+),	O
+which	O
+in	O
+turn	O
+nudges	O
+h12	O
+(	O
+Fig	O
+5C	O
+and	O
+D	O
+).	O
+	O
+We	O
+observed	O
+that	O
+the	O
+OBHS	O
+‐	O
+N	O
+analogs	O
+displaced	O
+h11	O
+along	O
+a	O
+vector	O
+away	O
+from	O
+Leu354	O
+in	O
+a	O
+region	O
+of	O
+h3	O
+that	O
+is	O
+unaffected	O
+by	O
+the	O
+ligands	O
+,	O
+and	O
+toward	O
+the	O
+dimer	O
+interface	O
+.	O
+	O
+Remarkably	O
+,	O
+these	O
+individual	O
+inter	O
+‐	O
+atomic	O
+distances	O
+showed	O
+a	O
+ligand	O
+class	O
+‐	O
+specific	O
+ability	O
+to	O
+significantly	O
+predict	O
+proliferative	O
+effects	O
+(	O
+Fig	O
+5E	O
+and	O
+F	O
+),	O
+demonstrating	O
+the	O
+feasibility	O
+of	O
+developing	O
+a	O
+minimal	O
+set	O
+of	O
+activity	O
+predictors	O
+from	O
+crystal	O
+structures	O
+.	O
+	O
+The	O
+h11	O
+–	O
+h12	O
+interface	O
+(	O
+circled	O
+)	O
+includes	O
+the	O
+C	O
+‐	O
+terminal	O
+part	O
+of	O
+h11	O
+.	O
+	O
+Direct	O
+modulators	O
+like	O
+tamoxifen	O
+drive	O
+AF	O
+‐	O
+1	O
+‐	O
+dependent	O
+cell	O
+‐	O
+specific	O
+activity	O
+by	O
+completely	O
+occluding	O
+AF	O
+‐	O
+2	O
+,	O
+but	O
+it	O
+is	O
+not	O
+known	O
+how	O
+indirect	O
+modulators	O
+produce	O
+cell	O
+‐	O
+specific	O
+ERα	O
+activity	O
+.	O
+	O
+The	O
+2F	O
+o	O
+‐	O
+F	O
+c	O
+electron	O
+density	O
+map	O
+and	O
+F	O
+o	O
+‐	O
+F	O
+c	O
+difference	O
+map	O
+of	O
+a	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+‐	O
+bound	O
+structure	O
+(	O
+PDB	O
+5DRJ	O
+)	O
+were	O
+contoured	O
+at	O
+1	O
+.	O
+0	O
+sigma	O
+and	O
+±	O
+3	O
+.	O
+0	O
+sigma	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+analogs	O
+showed	O
+perturbation	O
+of	O
+h11	O
+,	O
+as	O
+well	O
+as	O
+h3	O
+,	O
+which	O
+forms	O
+part	O
+of	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+.	O
+	O
+The	O
+shifts	O
+in	O
+h3	O
+suggest	O
+these	O
+compounds	O
+are	O
+positioned	O
+to	O
+alter	O
+coregulator	O
+preferences	O
+.	O
+	O
+Therefore	O
+,	O
+these	O
+indirect	O
+modulators	O
+,	O
+including	O
+S	O
+‐	O
+OBHS	O
+‐	O
+2	O
+,	O
+S	O
+‐	O
+OBHS	O
+‐	O
+3	O
+,	O
+2	O
+,	O
+5	O
+‐	O
+DTP	O
+,	O
+and	O
+3	O
+,	O
+4	O
+‐	O
+DTPD	O
+analogs	O
+—	O
+all	O
+of	O
+which	O
+show	O
+cell	O
+‐	O
+specific	O
+activity	O
+profiles	O
+—	O
+induced	O
+shifts	O
+in	O
+h3	O
+and	O
+h12	O
+that	O
+were	O
+transmitted	O
+to	O
+the	O
+coactivator	O
+peptide	O
+via	O
+an	O
+altered	O
+AF	O
+‐	O
+2	O
+surface	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+an	O
+extended	O
+side	O
+chain	O
+designed	O
+to	O
+directly	O
+reposition	O
+h12	O
+and	O
+completely	O
+disrupt	O
+the	O
+AF	O
+‐	O
+2	O
+surface	O
+results	O
+in	O
+cell	O
+‐	O
+specific	O
+signaling	O
+.	O
+	O
+If	O
+we	O
+calculated	O
+inter	O
+‐	O
+atomic	O
+distance	O
+matrices	O
+containing	O
+4	O
+,	O
+000	O
+atoms	O
+per	O
+structure	O
+×	O
+76	O
+ligand	O
+–	O
+receptor	O
+complexes	O
+,	O
+we	O
+would	O
+have	O
+3	O
+×	O
+105	O
+predictions	O
+.	O
+	O
+We	O
+have	O
+found	O
+that	O
+the	O
+TOCA1	O
+HR1	O
+,	O
+like	O
+the	O
+closely	O
+related	O
+CIP4	O
+HR1	O
+,	O
+has	O
+interesting	O
+structural	O
+features	O
+that	O
+are	O
+not	O
+observed	O
+in	O
+other	O
+HR1	O
+domains	O
+.	O
+	O
+NMR	O
+experiments	O
+show	O
+that	O
+the	O
+Cdc42	O
+-	O
+binding	O
+domain	O
+from	O
+N	O
+-	O
+WASP	O
+is	O
+able	O
+to	O
+displace	O
+TOCA1	O
+HR1	O
+from	O
+Cdc42	O
+,	O
+whereas	O
+the	O
+N	O
+-	O
+WASP	O
+domain	O
+but	O
+not	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+inhibits	O
+actin	O
+polymerization	O
+.	O
+	O
+This	O
+suggests	O
+that	O
+TOCA1	O
+binding	O
+to	O
+Cdc42	O
+is	O
+an	O
+early	O
+step	O
+in	O
+the	O
+Cdc42	O
+-	O
+dependent	O
+pathways	O
+that	O
+govern	O
+actin	O
+dynamics	O
+,	O
+and	O
+the	O
+differential	O
+binding	O
+affinities	O
+of	O
+the	O
+effectors	O
+facilitate	O
+a	O
+handover	O
+from	O
+TOCA1	O
+to	O
+N	O
+-	O
+WASP	O
+,	O
+which	O
+can	O
+then	O
+drive	O
+recruitment	O
+of	O
+the	O
+actin	O
+-	O
+modifying	O
+machinery	O
+.	O
+	O
+These	O
+molecular	O
+switches	O
+cycle	O
+between	O
+active	O
+,	O
+GTP	O
+-	O
+bound	O
+,	O
+and	O
+inactive	O
+,	O
+GDP	O
+-	O
+bound	O
+,	O
+states	O
+with	O
+the	O
+help	O
+of	O
+auxiliary	O
+proteins	O
+.	O
+	O
+In	O
+the	O
+active	O
+state	O
+,	O
+G	O
+proteins	O
+bind	O
+to	O
+an	O
+array	O
+of	O
+downstream	O
+effectors	O
+,	O
+through	O
+which	O
+they	O
+exert	O
+their	O
+extensive	O
+roles	O
+within	O
+the	O
+cell	O
+.	O
+	O
+RhoA	O
+acts	O
+to	O
+rearrange	O
+existing	O
+actin	O
+structures	O
+to	O
+form	O
+stress	O
+fibers	O
+,	O
+whereas	O
+Rac1	O
+and	O
+Cdc42	O
+promote	O
+de	O
+novo	O
+actin	O
+polymerization	O
+to	O
+form	O
+lamellipodia	O
+and	O
+filopodia	O
+,	O
+respectively	O
+.	O
+	O
+Following	O
+their	O
+release	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+regions	O
+of	O
+N	O
+-	O
+WASP	O
+are	O
+free	O
+to	O
+interact	O
+with	O
+G	O
+-	O
+actin	O
+and	O
+a	O
+known	O
+nucleator	O
+of	O
+actin	O
+assembly	O
+,	O
+the	O
+Arp2	O
+/	O
+3	O
+complex	O
+.	O
+	O
+The	O
+TOCA1	O
+SH3	O
+domain	O
+has	O
+many	O
+known	O
+binding	O
+partners	O
+,	O
+including	O
+N	O
+-	O
+WASP	O
+and	O
+dynamin	O
+.	O
+	O
+The	O
+HR1	O
+domain	O
+has	O
+been	O
+directly	O
+implicated	O
+in	O
+the	O
+interaction	O
+between	O
+TOCA1	O
+and	O
+Cdc42	O
+,	O
+representing	O
+the	O
+first	O
+Cdc42	O
+-	O
+HR1	O
+domain	O
+interaction	O
+to	O
+be	O
+identified	O
+.	O
+	O
+Both	O
+of	O
+the	O
+G	O
+protein	O
+switch	O
+regions	O
+are	O
+involved	O
+in	O
+the	O
+interaction	O
+.	O
+	O
+The	O
+interactions	O
+of	O
+TOCA1	O
+and	O
+N	O
+-	O
+WASP	O
+with	O
+Cdc42	O
+as	O
+well	O
+as	O
+with	O
+each	O
+other	O
+have	O
+raised	O
+questions	O
+as	O
+to	O
+whether	O
+the	O
+two	O
+Cdc42	O
+effectors	O
+can	O
+interact	O
+with	O
+a	O
+single	O
+molecule	O
+of	O
+Cdc42	O
+simultaneously	O
+.	O
+	O
+Cdc42	O
+-	O
+TOCA1	O
+Binding	O
+	O
+A	O
+,	O
+curves	O
+derived	O
+from	O
+direct	O
+binding	O
+assays	O
+in	O
+which	O
+the	O
+indicated	O
+concentrations	O
+of	O
+Cdc42Δ7Q61L	O
+·[	O
+3H	O
+]	O
+GTP	O
+were	O
+incubated	O
+with	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+PAK	I-mutant
+or	O
+HR1	B-mutant
+-	I-mutant
+His6	I-mutant
+in	O
+SPAs	O
+.	O
+	O
+The	O
+Kd	O
+values	O
+derived	O
+for	O
+the	O
+ACK	O
+GBD	O
+with	O
+Cdc42Δ7	B-mutant
+and	O
+full	O
+-	O
+length	O
+Cdc42	O
+were	O
+0	O
+.	O
+032	O
+±	O
+0	O
+.	O
+01	O
+and	O
+0	O
+.	O
+011	O
+±	O
+0	O
+.	O
+01	O
+μm	O
+,	O
+respectively	O
+.	O
+	O
+Other	O
+G	O
+protein	O
+-	O
+HR1	O
+domain	O
+interactions	O
+have	O
+also	O
+failed	O
+to	O
+show	O
+heat	O
+changes	O
+in	O
+our	O
+hands	O
+.	O
+7	O
+Infrared	O
+interferometry	O
+with	O
+immobilized	O
+Cdc42	O
+was	O
+also	O
+attempted	O
+but	O
+was	O
+unsuccessful	O
+for	O
+both	O
+TOCA1	O
+HR1	O
+and	O
+for	O
+the	O
+positive	O
+control	O
+,	O
+ACK	O
+.	O
+	O
+The	O
+affinity	O
+was	O
+therefore	O
+determined	O
+using	O
+competition	O
+SPAs	O
+.	O
+	O
+Free	O
+ACK	O
+competed	O
+with	O
+itself	O
+with	O
+an	O
+affinity	O
+of	O
+32	O
+nm	O
+,	O
+similar	O
+to	O
+the	O
+value	O
+obtained	O
+by	O
+direct	O
+binding	O
+of	O
+23	O
+nm	O
+.	O
+	O
+The	O
+TOCA1	O
+HR1	O
+domain	O
+also	O
+fully	O
+competed	O
+with	O
+the	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+but	O
+bound	O
+with	O
+an	O
+affinity	O
+of	O
+6	O
+μm	O
+(	O
+Fig	O
+.	O
+1	O
+,	O
+B	O
+and	O
+C	O
+),	O
+in	O
+agreement	O
+with	O
+the	O
+low	O
+affinity	O
+observed	O
+in	O
+the	O
+direct	O
+binding	O
+experiments	O
+.	O
+	O
+These	O
+residues	O
+are	O
+not	O
+generally	O
+required	O
+for	O
+G	O
+protein	O
+-	O
+effector	O
+interactions	O
+,	O
+including	O
+the	O
+interaction	O
+between	O
+RhoA	O
+and	O
+the	O
+PRK1	O
+HR1a	O
+domain	O
+.	O
+	O
+In	O
+contrast	O
+,	O
+the	O
+C	O
+terminus	O
+of	O
+Rac1	O
+contains	O
+a	O
+polybasic	O
+sequence	O
+,	O
+which	O
+is	O
+crucial	O
+for	O
+Rac1	O
+binding	O
+to	O
+the	O
+HR1b	O
+domain	O
+from	O
+PRK1	O
+.	O
+	O
+This	O
+construct	O
+competed	O
+with	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+GBD	O
+to	O
+give	O
+a	O
+similar	O
+affinity	O
+to	O
+the	O
+HR1	O
+domain	O
+alone	O
+(	O
+Kd	O
+=	O
+4	O
+.	O
+6	O
+±	O
+4	O
+μm	O
+;	O
+Fig	O
+.	O
+2C	O
+).	O
+	O
+Domain	O
+boundaries	O
+are	O
+derived	O
+from	O
+secondary	O
+structure	O
+predictions	O
+;	O
+B	O
+,	O
+binding	O
+curves	O
+derived	O
+from	O
+direct	O
+binding	O
+assays	O
+,	O
+in	O
+which	O
+the	O
+indicated	O
+concentrations	O
+of	O
+Cdc42Δ7Q61L	O
+·[	O
+3H	O
+]	O
+GTP	O
+were	O
+incubated	O
+with	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+or	O
+His	O
+-	O
+tagged	O
+TOCA1	O
+constructs	O
+,	O
+as	O
+indicated	O
+,	O
+in	O
+SPAs	O
+.	O
+	O
+The	O
+data	O
+were	O
+fitted	O
+to	O
+a	O
+binding	O
+isotherm	O
+to	O
+give	O
+an	O
+apparent	O
+Kd	O
+and	O
+are	O
+expressed	O
+as	O
+a	O
+percentage	O
+of	O
+the	O
+maximum	O
+signal	O
+.	O
+	O
+The	O
+structure	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+.	O
+	O
+B	O
+,	O
+a	O
+sequence	O
+alignment	O
+of	O
+the	O
+HR1	O
+domains	O
+from	O
+TOCA1	O
+,	O
+CIP4	O
+,	O
+and	O
+PRK1	O
+.	O
+	O
+Residues	O
+with	O
+significantly	O
+affected	O
+backbone	O
+or	O
+side	O
+chain	O
+chemical	O
+shifts	O
+when	O
+Cdc42	O
+bound	O
+and	O
+that	O
+are	O
+buried	O
+are	O
+colored	O
+dark	O
+blue	O
+,	O
+whereas	O
+those	O
+that	O
+are	O
+solvent	O
+-	O
+accessible	O
+are	O
+colored	O
+yellow	O
+.	O
+	O
+Side	O
+chains	O
+whose	O
+CH	O
+groups	O
+disappeared	O
+in	O
+the	O
+presence	O
+of	O
+Cdc42	O
+are	O
+marked	O
+on	O
+the	O
+graph	O
+in	O
+Fig	O
+.	O
+4B	O
+with	O
+green	O
+asterisks	O
+.	O
+	O
+The	O
+overall	O
+CSP	O
+was	O
+calculated	O
+for	O
+each	O
+residue	O
+.	O
+	O
+The	O
+red	O
+line	O
+indicates	O
+the	O
+mean	O
+CSP	O
+,	O
+plus	O
+one	O
+S	O
+.	O
+D	O
+.	O
+Residues	O
+that	O
+disappeared	O
+in	O
+the	O
+presence	O
+of	O
+Cdc42	O
+were	O
+assigned	O
+a	O
+CSP	O
+of	O
+0	O
+.	O
+1	O
+and	O
+are	O
+indicated	O
+with	O
+open	O
+bars	O
+.	O
+	O
+This	O
+suggests	O
+that	O
+the	O
+switch	O
+regions	O
+are	O
+not	O
+rigidified	O
+in	O
+the	O
+HR1	O
+complex	O
+and	O
+are	O
+still	O
+in	O
+conformational	O
+exchange	O
+.	O
+	O
+Modeling	O
+the	O
+Cdc42	O
+·	O
+TOCA1	O
+HR1	O
+Complex	O
+	O
+Cdc42	O
+is	O
+shown	O
+in	O
+cyan	O
+,	O
+and	O
+TOCA1	O
+is	O
+shown	O
+in	O
+purple	O
+.	O
+	O
+Some	O
+of	O
+these	O
+can	O
+be	O
+rationalized	O
+;	O
+for	O
+example	O
+,	O
+Thr	O
+-	O
+24Cdc42	O
+,	O
+Leu	O
+-	O
+160Cdc42	O
+,	O
+and	O
+Lys	O
+-	O
+163Cdc42	O
+all	O
+pack	O
+behind	O
+switch	O
+I	O
+and	O
+are	O
+likely	O
+to	O
+be	O
+affected	O
+by	O
+conformational	O
+changes	O
+within	O
+the	O
+switch	O
+,	O
+while	O
+Glu	O
+-	O
+95Cdc42	O
+and	O
+Lys	O
+-	O
+96Cdc42	O
+are	O
+in	O
+the	O
+helix	O
+behind	O
+switch	O
+II	O
+.	O
+	O
+Competition	O
+between	O
+N	O
+-	O
+WASP	O
+and	O
+TOCA1	O
+	O
+A	O
+,	O
+the	O
+model	O
+of	O
+the	O
+Cdc42	O
+·	O
+TOCA1	O
+HR1	O
+domain	O
+complex	O
+overlaid	O
+with	O
+the	O
+Cdc42	O
+-	O
+WASP	O
+structure	O
+.	O
+	O
+B	O
+,	O
+competition	O
+SPA	O
+experiments	O
+carried	O
+out	O
+with	O
+indicated	O
+concentrations	O
+of	O
+the	O
+N	O
+-	O
+WASP	O
+GBD	O
+construct	O
+titrated	O
+into	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+or	O
+GST	B-mutant
+-	I-mutant
+WASP	I-mutant
+GBD	O
+and	O
+30	O
+nm	O
+Cdc42Δ7Q61L	O
+·[	O
+3H	O
+]	O
+GTP	O
+.	O
+	O
+Unlabeled	O
+N	O
+-	O
+WASP	O
+GBD	O
+was	O
+titrated	O
+into	O
+15N	O
+-	O
+Cdc42Δ7Q61L	O
+·	O
+GMPPNP	O
+,	O
+and	O
+the	O
+backbone	O
+NH	O
+groups	O
+were	O
+monitored	O
+using	O
+HSQCs	O
+(	O
+Fig	O
+.	O
+7C	O
+).	O
+	O
+Actin	O
+polymerization	O
+in	O
+all	O
+cases	O
+was	O
+initiated	O
+by	O
+the	O
+addition	O
+of	O
+PI	O
+(	O
+4	O
+,	O
+5	O
+)	O
+P2	O
+-	O
+containing	O
+liposomes	O
+.	O
+	O
+Endogenous	O
+N	O
+-	O
+WASP	O
+is	O
+present	O
+at	O
+∼	O
+100	O
+nm	O
+in	O
+Xenopus	O
+extracts	O
+,	O
+whereas	O
+TOCA1	O
+is	O
+present	O
+at	O
+a	O
+10	O
+-	O
+fold	O
+lower	O
+concentration	O
+than	O
+N	O
+-	O
+WASP	O
+.	O
+	O
+This	O
+is	O
+consistent	O
+with	O
+endogenous	O
+N	O
+-	O
+WASP	O
+,	O
+activated	O
+by	O
+other	O
+components	O
+of	O
+the	O
+assay	O
+,	O
+outcompeting	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+for	O
+Cdc42	O
+binding	O
+.	O
+	O
+Fluorescence	O
+curves	O
+show	O
+actin	O
+polymerization	O
+in	O
+the	O
+presence	O
+of	O
+increasing	O
+concentrations	O
+of	O
+N	O
+-	O
+WASP	O
+GBD	O
+or	O
+TOCA1	O
+HR1	O
+domain	O
+as	O
+indicated	O
+.	O
+	O
+This	O
+is	O
+over	O
+100	O
+times	O
+lower	O
+than	O
+that	O
+of	O
+the	O
+N	O
+-	O
+WASP	O
+GBD	O
+(	O
+Kd	O
+=	O
+37	O
+nm	O
+)	O
+and	O
+considerably	O
+lower	O
+than	O
+other	O
+known	O
+G	O
+protein	O
+-	O
+HR1	O
+domain	O
+interactions	O
+.	O
+	O
+The	O
+TOCA1	O
+HR1	O
+domain	O
+is	O
+a	O
+left	O
+-	O
+handed	O
+coiled	O
+-	O
+coil	O
+comparable	O
+with	O
+other	O
+known	O
+HR1	O
+domains	O
+.	O
+	O
+The	O
+interhelical	O
+loops	O
+of	O
+TOCA1	O
+and	O
+CIP4	O
+differ	O
+from	O
+the	O
+same	O
+region	O
+in	O
+the	O
+HR1	O
+domains	O
+of	O
+PRK1	O
+in	O
+that	O
+they	O
+are	O
+longer	O
+and	O
+contain	O
+two	O
+short	O
+stretches	O
+of	O
+310	O
+-	O
+helix	O
+.	O
+	O
+This	O
+region	O
+lies	O
+within	O
+the	O
+G	O
+protein	O
+-	O
+binding	O
+surface	O
+of	O
+all	O
+of	O
+the	O
+HR1	O
+domains	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+	O
+Many	O
+of	O
+these	O
+residues	O
+are	O
+significantly	O
+affected	O
+in	O
+the	O
+presence	O
+of	O
+Cdc42	O
+,	O
+so	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+conformation	O
+of	O
+this	O
+loop	O
+is	O
+altered	O
+in	O
+the	O
+Cdc42	O
+complex	O
+.	O
+	O
+These	O
+observations	O
+therefore	O
+provide	O
+a	O
+molecular	O
+mechanism	O
+whereby	O
+mutation	O
+of	O
+Met383	O
+-	O
+Gly384	O
+-	O
+Asp385	O
+to	O
+Ile383	O
+-	O
+Ser384	O
+-	O
+Thr385	O
+abolishes	O
+TOCA1	O
+binding	O
+to	O
+Cdc42	O
+.	O
+	O
+For	O
+example	O
+,	O
+Phe	O
+-	O
+56Cdc42	O
+,	O
+which	O
+is	O
+not	O
+visible	O
+in	O
+free	O
+Cdc42	O
+or	O
+Cdc42	O
+·	O
+HR1TOCA1	O
+,	O
+is	O
+close	O
+to	O
+the	O
+TOCA1	O
+HR1	O
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+	O
+Some	O
+residues	O
+that	O
+are	O
+affected	O
+in	O
+the	O
+Cdc42	O
+·	O
+HR1TOCA1	O
+complex	O
+but	O
+do	O
+not	O
+correspond	O
+to	O
+contact	O
+residues	O
+of	O
+RhoA	O
+or	O
+Rac1	O
+(	O
+Fig	O
+.	O
+6C	O
+)	O
+may	O
+contact	O
+HR1TOCA1	O
+directly	O
+(	O
+Fig	O
+.	O
+6D	O
+).	O
+	O
+The	O
+weak	O
+binding	O
+prevented	O
+detailed	O
+structural	O
+and	O
+thermodynamic	O
+studies	O
+of	O
+the	O
+complex	O
+.	O
+	O
+Nonetheless	O
+,	O
+structural	O
+studies	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+,	O
+combined	O
+with	O
+chemical	O
+shift	O
+mapping	O
+,	O
+have	O
+highlighted	O
+some	O
+potentially	O
+interesting	O
+differences	O
+between	O
+Cdc42	O
+-	O
+HR1TOCA1	O
+and	O
+RhoA	O
+/	O
+Rac1	O
+-	O
+HR1PRK1	O
+binding	O
+.	O
+	O
+As	O
+such	O
+,	O
+the	O
+ability	O
+of	O
+the	O
+TOCA1	O
+HR1	O
+domain	O
+to	O
+bind	O
+to	O
+Cdc42	O
+(	O
+a	O
+close	O
+relative	O
+of	O
+Rac1	O
+rather	O
+than	O
+RhoA	O
+)	O
+fits	O
+this	O
+trend	O
+.	O
+	O
+The	O
+low	O
+affinity	O
+of	O
+the	O
+HR1TOCA1	O
+-	O
+Cdc42	O
+interaction	O
+in	O
+the	O
+context	O
+of	O
+the	O
+physiological	O
+concentration	O
+of	O
+TOCA1	O
+in	O
+Xenopus	O
+extracts	O
+(∼	O
+10	O
+nm	O
+)	O
+suggests	O
+that	O
+binding	O
+between	O
+TOCA1	O
+and	O
+Cdc42	O
+is	O
+likely	O
+to	O
+occur	O
+in	O
+vivo	O
+only	O
+when	O
+TOCA1	O
+is	O
+at	O
+high	O
+local	O
+concentrations	O
+and	O
+membrane	O
+-	O
+localized	O
+and	O
+therefore	O
+in	O
+close	O
+proximity	O
+to	O
+activated	O
+Cdc42	O
+.	O
+	O
+WIP	O
+inhibits	O
+the	O
+activation	O
+of	O
+N	O
+-	O
+WASP	O
+by	O
+Cdc42	O
+,	O
+an	O
+effect	O
+that	O
+is	O
+reversed	O
+by	O
+TOCA1	O
+.	O
+	O
+A	O
+combination	O
+of	O
+allosteric	O
+activation	O
+by	O
+PI	O
+(	O
+4	O
+,	O
+5	O
+)	O
+P2	O
+,	O
+activated	O
+Cdc42	O
+and	O
+TOCA1	O
+,	O
+and	O
+oligomeric	O
+activation	O
+implemented	O
+by	O
+TOCA1	O
+would	O
+lead	O
+to	O
+full	O
+activation	O
+of	O
+N	O
+-	O
+WASP	O
+and	O
+downstream	O
+actin	O
+polymerization	O
+.	O
+	O
+The	O
+commonly	O
+used	O
+MGD	B-mutant
+→	I-mutant
+IST	I-mutant
+(	O
+Cdc42	O
+-	O
+binding	O
+deficient	O
+)	O
+mutant	O
+of	O
+TOCA1	O
+has	O
+a	O
+reduced	O
+ability	O
+to	O
+activate	O
+the	O
+N	O
+-	O
+WASP	O
+·	O
+WIP	O
+complex	O
+,	O
+further	O
+indicating	O
+the	O
+importance	O
+of	O
+the	O
+Cdc42	O
+-	O
+HR1TOCA1	O
+interaction	O
+prior	O
+to	O
+downstream	O
+activation	O
+of	O
+N	O
+-	O
+WASP	O
+.	O
+	O
+Step	O
+1	O
+,	O
+TOCA1	O
+is	O
+recruited	O
+to	O
+the	O
+membrane	O
+via	O
+its	O
+F	O
+-	O
+BAR	O
+domain	O
+and	O
+/	O
+or	O
+Cdc42	O
+interactions	O
+.	O
+	O
+It	O
+is	O
+clear	O
+from	O
+the	O
+data	O
+presented	O
+here	O
+that	O
+TOCA1	O
+and	O
+N	O
+-	O
+WASP	O
+do	O
+not	O
+bind	O
+Cdc42	O
+simultaneously	O
+and	O
+that	O
+N	O
+-	O
+WASP	O
+is	O
+likely	O
+to	O
+outcompete	O
+TOCA1	O
+for	O
+Cdc42	O
+binding	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+related	O
+carboxylases	O
+,	O
+large	O
+-	O
+scale	O
+conformational	O
+changes	O
+are	O
+required	O
+for	O
+substrate	O
+turnover	O
+,	O
+and	O
+are	O
+mediated	O
+by	O
+the	O
+CD	O
+under	O
+phosphorylation	O
+control	O
+.	O
+	O
+BRCA1	O
+binds	O
+only	O
+to	O
+the	O
+phosphorylated	O
+form	O
+of	O
+ACC1	O
+and	O
+prevents	O
+ACC	O
+activation	O
+by	O
+phosphatase	O
+-	O
+mediated	O
+dephosphorylation	O
+.	O
+	O
+Its	O
+phosphorylation	O
+by	O
+the	O
+AMPK	O
+homologue	O
+SNF1	O
+results	O
+in	O
+strongly	O
+reduced	O
+ACC	O
+activity	O
+.	O
+	O
+The	O
+organization	O
+of	O
+the	O
+yeast	O
+ACC	O
+CD	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+the	O
+CD	O
+of	O
+SceACC	O
+(	O
+SceCD	O
+)	O
+was	O
+determined	O
+at	O
+3	O
+.	O
+0	O
+Å	O
+resolution	O
+by	O
+experimental	O
+phasing	O
+and	O
+refined	O
+to	O
+Rwork	O
+/	O
+Rfree	O
+=	O
+0	O
+.	O
+20	O
+/	O
+0	O
+.	O
+24	O
+(	O
+Table	O
+1	O
+).	O
+	O
+SceCD	O
+comprises	O
+four	O
+distinct	O
+domains	O
+,	O
+an	O
+N	O
+-	O
+terminal	O
+α	O
+-	O
+helical	O
+domain	O
+(	O
+CDN	O
+),	O
+and	O
+a	O
+central	O
+four	O
+-	O
+helix	O
+bundle	O
+linker	O
+domain	O
+(	O
+CDL	O
+),	O
+followed	O
+by	O
+two	O
+α	O
+–	O
+β	O
+-	O
+fold	O
+C	O
+-	O
+terminal	O
+domains	O
+(	O
+CDC1	O
+/	O
+CDC2	O
+).	O
+	O
+CDL	O
+does	O
+not	O
+interact	O
+with	O
+CDN	O
+apart	O
+from	O
+the	O
+covalent	O
+linkage	O
+and	O
+forms	O
+only	O
+a	O
+small	O
+contact	O
+to	O
+CDC2	O
+via	O
+a	O
+loop	O
+between	O
+Lα2	O
+/	O
+α3	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+end	O
+of	O
+Lα1	O
+,	O
+with	O
+an	O
+interface	O
+area	O
+of	O
+400	O
+Å2	O
+.	O
+	O
+CDC1	O
+/	O
+CDC2	O
+share	O
+a	O
+common	O
+fold	O
+;	O
+they	O
+are	O
+composed	O
+of	O
+six	O
+-	O
+stranded	O
+β	O
+-	O
+sheets	O
+flanked	O
+on	O
+one	O
+side	O
+by	O
+two	O
+long	O
+,	O
+bent	O
+helices	O
+inserted	O
+between	O
+strands	O
+β3	O
+/	O
+β4	O
+and	O
+β4	O
+/	O
+β5	O
+.	O
+	O
+On	O
+the	O
+basis	O
+of	O
+a	O
+root	O
+mean	O
+square	O
+deviation	O
+of	O
+main	O
+chain	O
+atom	O
+positions	O
+of	O
+2	O
+.	O
+2	O
+Å	O
+,	O
+CDC1	O
+/	O
+CDC2	O
+are	O
+structurally	O
+more	O
+closely	O
+related	O
+to	O
+each	O
+other	O
+than	O
+to	O
+any	O
+other	O
+protein	O
+(	O
+Fig	O
+.	O
+1c	O
+);	O
+they	O
+may	O
+thus	O
+have	O
+evolved	O
+by	O
+duplication	O
+.	O
+	O
+Phosphorylated	O
+SceACC	O
+shows	O
+only	O
+residual	O
+activity	O
+(	O
+kcat	O
+=	O
+0	O
+.	O
+4	O
+±	O
+0	O
+.	O
+2	O
+s	O
+−	O
+1	O
+,	O
+s	O
+.	O
+d	O
+.	O
+based	O
+on	O
+five	O
+replicate	O
+measurements	O
+),	O
+which	O
+increases	O
+16	O
+-	O
+fold	O
+(	O
+kcat	O
+=	O
+6	O
+.	O
+5	O
+±	O
+0	O
+.	O
+3	O
+s	O
+−	O
+1	O
+)	O
+after	O
+dephosphorylation	O
+with	O
+λ	O
+protein	O
+phosphatase	O
+.	O
+	O
+As	O
+a	O
+result	O
+,	O
+the	O
+N	O
+terminus	O
+of	O
+CDL	O
+at	O
+helix	O
+Lα1	O
+,	O
+which	O
+connects	O
+to	O
+CDN	O
+,	O
+is	O
+shifted	O
+by	O
+12	O
+Å	O
+.	O
+Remarkably	O
+,	O
+CDN	O
+of	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+adopts	O
+a	O
+completely	O
+different	O
+orientation	O
+compared	O
+with	O
+SceCD	O
+.	O
+	O
+To	O
+improve	O
+crystallizability	O
+,	O
+we	O
+generated	O
+ΔBCCP	B-mutant
+variants	I-mutant
+of	O
+full	O
+-	O
+length	O
+ACC	O
+,	O
+which	O
+,	O
+based	O
+on	O
+SAXS	O
+analysis	O
+,	O
+preserve	O
+properties	O
+of	O
+intact	O
+ACC	O
+(	O
+Supplementary	O
+Table	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+–	O
+c	O
+).	O
+	O
+The	O
+connecting	O
+region	O
+is	O
+remarkably	O
+similar	O
+in	O
+isolated	O
+CD	O
+and	O
+CthCD	B-mutant
+-	I-mutant
+CTCter	I-mutant
+structures	O
+,	O
+indicating	O
+inherent	O
+conformational	O
+stability	O
+.	O
+	O
+Surprisingly	O
+,	O
+in	O
+both	O
+the	O
+linear	O
+and	O
+U	O
+-	O
+shaped	O
+conformations	O
+,	O
+the	O
+approximate	O
+distances	O
+between	O
+the	O
+BC	O
+and	O
+CT	O
+active	O
+sites	O
+would	O
+remain	O
+larger	O
+than	O
+110	O
+Å	O
+.	O
+These	O
+observed	O
+distances	O
+are	O
+considerably	O
+larger	O
+than	O
+in	O
+static	O
+structures	O
+of	O
+any	O
+other	O
+related	O
+biotin	O
+-	O
+dependent	O
+carboxylase	O
+.	O
+	O
+The	O
+CD	O
+consists	O
+of	O
+four	O
+distinct	O
+subdomains	O
+and	O
+acts	O
+as	O
+a	O
+tether	O
+from	O
+the	O
+CT	O
+to	O
+the	O
+mobile	O
+BCCP	O
+and	O
+an	O
+oriented	O
+BC	O
+domain	O
+.	O
+	O
+A	O
+second	O
+hinge	O
+can	O
+be	O
+identified	O
+between	O
+CDC1	O
+/	O
+CDC2	O
+.	O
+	O
+The	O
+only	O
+bona	O
+fide	O
+regulatory	O
+phophorylation	O
+site	O
+of	O
+fungal	O
+ACC	O
+in	O
+the	O
+regulatory	O
+loop	O
+is	O
+directly	O
+participating	O
+in	O
+CDC1	O
+/	O
+CDC2	O
+domain	O
+interactions	O
+and	O
+thus	O
+stabilizes	O
+the	O
+hinge	O
+conformation	O
+.	O
+	O
+In	O
+flACC	O
+,	O
+the	O
+regulatory	O
+loop	O
+is	O
+mostly	O
+disordered	O
+,	O
+illustrating	O
+the	O
+increased	O
+flexibility	O
+due	O
+to	O
+the	O
+absence	O
+of	O
+the	O
+phosphoryl	O
+group	O
+.	O
+	O
+Thus	O
+,	O
+in	O
+accordance	O
+with	O
+the	O
+results	O
+presented	O
+here	O
+,	O
+phosphorylation	O
+of	O
+Ser1157	O
+in	O
+SceACC	O
+most	O
+likely	O
+limits	O
+flexibility	O
+in	O
+the	O
+CDC1	O
+/	O
+CDC2	O
+hinge	O
+such	O
+that	O
+activation	O
+through	O
+BC	O
+dimerization	O
+is	O
+not	O
+possible	O
+(	O
+Fig	O
+.	O
+4d	O
+),	O
+which	O
+however	O
+does	O
+not	O
+exclude	O
+intermolecular	O
+dimerization	O
+.	O
+	O
+Cartoon	O
+representation	O
+of	O
+crystal	O
+structures	O
+of	O
+multidomain	B-mutant
+constructs	I-mutant
+of	O
+CthACC	O
+.	O
+	O
+(	O
+b	O
+)	O
+The	O
+interdomain	O
+interface	O
+of	O
+CDC1	O
+and	O
+CDC2	O
+exhibits	O
+only	O
+limited	O
+plasticity	O
+.	O
+	O
+The	O
+conformational	O
+dynamics	O
+of	O
+fungal	O
+ACC	O
+.	O
+	O
+CthCD	B-mutant
+-	I-mutant
+CT1	I-mutant
+(	O
+in	O
+colour	O
+)	O
+serves	O
+as	O
+reference	O
+,	O
+the	O
+compared	O
+structures	O
+(	O
+as	O
+indicated	O
+,	O
+numbers	O
+after	O
+construct	O
+name	O
+differentiate	O
+between	O
+individual	O
+protomers	O
+)	O
+are	O
+shown	O
+in	O
+grey	O
+.	O
+	O
+Crystal	O
+Structures	O
+of	O
+Putative	O
+Sugar	O
+Kinases	O
+from	O
+Synechococcus	O
+Elongatus	O
+PCC	O
+7942	O
+and	O
+Arabidopsis	O
+Thaliana	O
+	O
+Here	O
+we	O
+solved	O
+the	O
+structures	O
+of	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+in	O
+both	O
+their	O
+apo	O
+forms	O
+and	O
+in	O
+complex	O
+with	O
+nucleotide	O
+substrates	O
+.	O
+	O
+Phosphorylation	O
+is	O
+one	O
+of	O
+the	O
+various	O
+pivotal	O
+modifications	O
+of	O
+carbohydrates	O
+,	O
+and	O
+is	O
+catalyzed	O
+by	O
+specific	O
+sugar	O
+kinases	O
+.	O
+	O
+These	O
+kinases	O
+exhibit	O
+considerable	O
+differences	O
+in	O
+their	O
+folding	O
+pattern	O
+and	O
+substrate	O
+specificity	O
+.	O
+	O
+Based	O
+on	O
+sequence	O
+analysis	O
+,	O
+they	O
+can	O
+be	O
+divided	O
+into	O
+four	O
+families	O
+,	O
+namely	O
+HSP	O
+70_NBD	O
+family	O
+,	O
+FGGY	O
+family	O
+,	O
+Mer_B	O
+like	O
+family	O
+and	O
+Parm_like	O
+family	O
+.	O
+	O
+Our	O
+findings	O
+provide	O
+new	O
+details	O
+of	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	O
+and	O
+lay	O
+the	O
+foundation	O
+for	O
+future	O
+studies	O
+into	O
+its	O
+homologs	O
+in	O
+eukaryotes	O
+.	O
+	O
+Apo	O
+-	O
+SePSK	O
+contains	O
+two	O
+domains	O
+referred	O
+to	O
+further	O
+on	O
+as	O
+domain	O
+I	O
+and	O
+domain	O
+II	O
+(	O
+Fig	O
+1A	O
+).	O
+	O
+2	O
+–	O
+228	O
+and	O
+aa	O
+.	O
+	O
+The	O
+secondary	O
+structural	O
+elements	O
+are	O
+indicated	O
+(	O
+α	O
+-	O
+helix	O
+:	O
+green	O
+,	O
+β	O
+-	O
+sheet	O
+:	O
+wheat	O
+).	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+2A	O
+,	O
+both	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+exhibited	O
+ATP	O
+hydrolysis	O
+activity	O
+.	O
+	O
+(	O
+A	O
+)	O
+The	O
+ATP	O
+hydrolysis	O
+activity	O
+of	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+.	O
+	O
+Both	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+showed	O
+ATP	O
+hydrolysis	O
+activity	O
+in	O
+the	O
+absence	O
+of	O
+substrate	O
+.	O
+	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+possess	O
+a	O
+similar	O
+ATP	O
+binding	O
+site	O
+	O
+This	O
+result	O
+was	O
+consistent	O
+with	O
+our	O
+enzymatic	O
+activity	O
+assays	O
+where	O
+SePSK	O
+and	O
+AtXK	O
+-	O
+1	O
+showed	O
+ATP	O
+hydrolysis	O
+activity	O
+without	O
+adding	O
+any	O
+substrates	O
+(	O
+Fig	O
+2A	O
+and	O
+2C	O
+).	O
+	O
+The	O
+tail	O
+of	O
+AMP	O
+-	O
+PNP	O
+points	O
+to	O
+the	O
+hinge	O
+region	O
+of	O
+SePSK	O
+,	O
+and	O
+its	O
+α	O
+-	O
+phosphate	O
+and	O
+β	O
+-	O
+phosphate	O
+groups	O
+are	O
+stabilized	O
+by	O
+Gly376	O
+and	O
+Ser243	O
+,	O
+respectively	O
+.	O
+	O
+The	O
+four	O
+α	O
+-	O
+helices	O
+(	O
+α26	O
+,	O
+α28	O
+,	O
+α27	O
+and	O
+α30	O
+)	O
+are	O
+labeled	O
+in	O
+red	O
+.	O
+	O
+To	O
+better	O
+understand	O
+the	O
+interaction	O
+pattern	O
+between	O
+SePSK	O
+and	O
+D	O
+-	O
+ribulose	O
+,	O
+the	O
+apo	O
+-	O
+SePSK	O
+crystals	O
+were	O
+soaked	O
+into	O
+the	O
+reservoir	O
+with	O
+10	O
+mM	O
+D	O
+-	O
+ribulose	O
+(	O
+RBL	O
+)	O
+and	O
+the	O
+RBL	O
+-	O
+SePSK	O
+structure	O
+was	O
+solved	O
+.	O
+	O
+As	O
+shown	O
+in	O
+Fig	O
+4A	O
+,	O
+the	O
+nearest	O
+distance	O
+between	O
+the	O
+carbon	O
+skeleton	O
+of	O
+two	O
+D	O
+-	O
+ribulose	O
+molecules	O
+are	O
+approx	O
+.	O
+	O
+The	O
+hydrogen	O
+bonds	O
+are	O
+indicated	O
+by	O
+the	O
+black	O
+dashed	O
+lines	O
+and	O
+the	O
+numbers	O
+near	O
+the	O
+dashed	O
+lines	O
+are	O
+the	O
+distances	O
+(	O
+Å	O
+).	O
+(	O
+C	O
+)	O
+The	O
+binding	O
+affinity	O
+assays	O
+of	O
+SePSK	O
+with	O
+D	O
+-	O
+ribulose	O
+.	O
+	O
+A	O
+unique	O
+macromolecular	O
+cage	O
+formed	O
+by	O
+two	O
+decamers	O
+of	O
+the	O
+Escherichia	O
+coli	O
+LdcI	O
+and	O
+five	O
+hexamers	O
+of	O
+the	O
+AAA	O
++	O
+ATPase	O
+RavA	O
+was	O
+shown	O
+to	O
+counteract	O
+acid	O
+stress	O
+under	O
+starvation	O
+.	O
+	O
+Multiple	O
+sequence	O
+alignment	O
+coupled	O
+to	O
+a	O
+phylogenetic	O
+analysis	O
+reveals	O
+that	O
+certain	O
+enterobacteria	O
+exert	O
+evolutionary	O
+pressure	O
+on	O
+the	O
+lysine	O
+decarboxylase	O
+towards	O
+the	O
+cage	O
+-	O
+like	O
+assembly	O
+with	O
+RavA	O
+,	O
+implying	O
+that	O
+this	O
+complex	O
+may	O
+have	O
+an	O
+important	O
+function	O
+under	O
+particular	O
+stress	O
+conditions	O
+.	O
+	O
+These	O
+amino	O
+acid	O
+decarboxylases	O
+are	O
+therefore	O
+called	O
+acid	O
+stress	O
+inducible	O
+or	O
+biodegradative	O
+to	O
+distinguish	O
+them	O
+from	O
+their	O
+biosynthetic	O
+lysine	O
+and	O
+ornithine	O
+decarboxylase	O
+paralogs	O
+catalysing	O
+the	O
+same	O
+reaction	O
+but	O
+responsible	O
+for	O
+the	O
+polyamine	O
+production	O
+at	O
+neutral	O
+pH	O
+.	O
+	O
+In	O
+particular	O
+,	O
+the	O
+inducible	O
+lysine	O
+decarboxylase	O
+LdcI	O
+(	O
+or	O
+CadA	O
+)	O
+attracts	O
+attention	O
+due	O
+to	O
+its	O
+broad	O
+pH	O
+range	O
+of	O
+activity	O
+and	O
+its	O
+capacity	O
+to	O
+promote	O
+survival	O
+and	O
+growth	O
+of	O
+pathogenic	O
+enterobacteria	O
+such	O
+as	O
+Salmonella	O
+enterica	O
+serovar	O
+Typhimurium	O
+,	O
+Vibrio	O
+cholerae	O
+and	O
+Vibrio	O
+vulnificus	O
+under	O
+acidic	O
+conditions	O
+.	O
+	O
+The	O
+crystal	O
+structure	O
+of	O
+the	O
+E	O
+.	O
+coli	O
+LdcI	O
+as	O
+well	O
+as	O
+its	O
+low	O
+resolution	O
+characterisation	O
+by	O
+electron	O
+microscopy	O
+(	O
+EM	O
+)	O
+showed	O
+that	O
+it	O
+is	O
+a	O
+decamer	O
+made	O
+of	O
+two	O
+pentameric	O
+rings	O
+.	O
+	O
+This	O
+comparison	O
+pinpointed	O
+differences	O
+between	O
+the	O
+biodegradative	O
+and	O
+the	O
+biosynthetic	O
+lysine	O
+decarboxylases	O
+and	O
+brought	O
+to	O
+light	O
+interdomain	O
+movements	O
+associated	O
+to	O
+pH	O
+-	O
+dependent	O
+enzyme	O
+activation	O
+and	O
+RavA	O
+binding	O
+,	O
+notably	O
+at	O
+the	O
+predicted	O
+RavA	O
+binding	O
+site	O
+at	O
+the	O
+level	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheet	O
+of	O
+LdcI	O
+.	O
+Consequently	O
+,	O
+we	O
+tested	O
+the	O
+capacity	O
+of	O
+cage	O
+formation	O
+by	O
+LdcI	B-mutant
+-	I-mutant
+LdcC	I-mutant
+chimeras	I-mutant
+where	O
+we	O
+interchanged	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+sheets	O
+in	O
+question	O
+.	O
+	O
+In	O
+addition	O
+,	O
+we	O
+improved	O
+our	O
+earlier	O
+cryoEM	O
+map	O
+of	O
+the	O
+LdcI	O
+-	O
+LARA	O
+complex	O
+from	O
+7	O
+.	O
+5	O
+Å	O
+to	O
+6	O
+.	O
+2	O
+Å	O
+resolution	O
+(	O
+Figs	O
+1E	O
+,	O
+F	O
+and	O
+S3	O
+).	O
+	O
+Based	O
+on	O
+these	O
+reconstructions	O
+,	O
+reliable	O
+pseudoatomic	O
+models	O
+of	O
+the	O
+three	O
+assemblies	O
+were	O
+obtained	O
+by	O
+flexible	O
+fitting	O
+of	O
+either	O
+the	O
+crystal	O
+structure	O
+of	O
+LdcIi	O
+or	O
+a	O
+derived	O
+structural	O
+homology	O
+model	O
+of	O
+LdcC	O
+(	O
+Table	O
+S1	O
+).	O
+	O
+The	O
+resolution	O
+of	O
+the	O
+cryoEM	O
+maps	O
+does	O
+not	O
+allow	O
+modeling	O
+the	O
+position	O
+of	O
+the	O
+PLP	O
+moiety	O
+and	O
+calls	O
+for	O
+caution	O
+in	O
+detailed	O
+mechanistic	O
+interpretations	O
+in	O
+terms	O
+of	O
+individual	O
+amino	O
+acids	O
+.	O
+	O
+While	O
+differences	O
+in	O
+the	O
+ppGpp	O
+binding	O
+site	O
+could	O
+indeed	O
+be	O
+visualized	O
+(	O
+Fig	O
+.	O
+S4	O
+),	O
+the	O
+level	O
+of	O
+resolution	O
+warns	O
+against	O
+speculations	O
+about	O
+their	O
+significance	O
+.	O
+	O
+Swinging	O
+and	O
+stretching	O
+of	O
+the	O
+CTDs	O
+upon	O
+pH	O
+-	O
+dependent	O
+LdcI	O
+activation	O
+and	O
+LARA	O
+binding	O
+	O
+Inspection	O
+of	O
+the	O
+superimposed	O
+decameric	O
+structures	O
+(	O
+Figs	O
+2	O
+and	O
+S6	O
+)	O
+suggests	O
+a	O
+depiction	O
+of	O
+the	O
+wing	O
+domains	O
+as	O
+an	O
+anchor	O
+around	O
+which	O
+the	O
+peripheral	O
+CTDs	O
+swing	O
+.	O
+	O
+This	O
+swinging	O
+movement	O
+seems	O
+to	O
+be	O
+mediated	O
+by	O
+the	O
+core	O
+domains	O
+and	O
+is	O
+accompanied	O
+by	O
+a	O
+stretching	O
+of	O
+the	O
+whole	O
+LdcI	O
+subunits	O
+attracted	O
+by	O
+the	O
+RavA	O
+magnets	O
+.	O
+	O
+Our	O
+structures	O
+show	O
+that	O
+this	O
+motif	O
+is	O
+not	O
+involved	O
+in	O
+the	O
+enzymatic	O
+activity	O
+or	O
+the	O
+oligomeric	O
+state	O
+of	O
+the	O
+proteins	O
+.	O
+	O
+One	O
+of	O
+the	O
+elucidated	O
+roles	O
+of	O
+the	O
+LdcI	O
+-	O
+RavA	O
+cage	O
+is	O
+to	O
+maintain	O
+LdcI	O
+activity	O
+under	O
+conditions	O
+of	O
+enterobacterial	O
+starvation	O
+by	O
+preventing	O
+LdcI	O
+inhibition	O
+by	O
+the	O
+stringent	O
+response	O
+alarmone	O
+ppGpp	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+recently	O
+documented	O
+interaction	O
+of	O
+both	O
+LdcI	O
+and	O
+RavA	O
+with	O
+specific	O
+subunits	O
+of	O
+the	O
+respiratory	O
+complex	O
+I	O
+,	O
+together	O
+with	O
+the	O
+unanticipated	O
+link	O
+between	O
+RavA	O
+and	O
+maturation	O
+of	O
+numerous	O
+iron	O
+-	O
+sulfur	O
+proteins	O
+,	O
+tend	O
+to	O
+suggest	O
+an	O
+additional	O
+intriguing	O
+function	O
+for	O
+this	O
+3	O
+.	O
+5	O
+MDa	O
+assembly	O
+.	O
+	O
+Besides	O
+,	O
+the	O
+structures	O
+and	O
+the	O
+pseudoatomic	O
+models	O
+of	O
+the	O
+active	O
+ppGpp	O
+-	O
+free	O
+states	O
+of	O
+both	O
+the	O
+biodegradative	O
+and	O
+the	O
+biosynthetic	O
+E	O
+.	O
+coli	O
+lysine	O
+decarboxylases	O
+offer	O
+an	O
+additional	O
+tool	O
+for	O
+analysis	O
+of	O
+their	O
+role	O
+in	O
+UPEC	O
+infectivity	O
+.	O
+	O
+The	O
+active	O
+site	O
+is	O
+boxed	O
+.	O
+	O
+(	O
+D	O
+,	O
+E	O
+)	O
+A	O
+gallery	O
+of	O
+negative	O
+stain	O
+EM	O
+images	O
+of	O
+(	O
+D	O
+)	O
+the	O
+wild	O
+type	O
+LdcI	O
+-	O
+RavA	O
+cage	O
+and	O
+(	O
+E	O
+)	O
+the	O
+LdcCI	B-mutant
+-	I-mutant
+RavA	I-mutant
+cage	I-mutant
+-	I-mutant
+like	I-mutant
+particles	I-mutant
+.	O
+(	O
+F	O
+)	O
+Some	O
+representative	O
+class	O
+averages	O
+of	O
+the	O
+LdcCI	B-mutant
+-	I-mutant
+RavA	I-mutant
+cage	I-mutant
+-	I-mutant
+like	I-mutant
+particles	I-mutant
+.	O
+	O
+Numbering	O
+as	O
+in	O
+E	O
+.	O
+coli	O
+.	O
+	O
+Relative	O
+to	O
+the	O
+open	O
+bacterial	O
+ammonium	O
+transporters	O
+,	O
+non	O
+-	O
+phosphorylated	O
+Mep2	O
+exhibits	O
+shifts	O
+in	O
+cytoplasmic	O
+loops	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+(	O
+CTR	O
+)	O
+to	O
+occlude	O
+the	O
+cytoplasmic	O
+exit	O
+of	O
+the	O
+channel	O
+and	O
+to	O
+interact	O
+with	O
+His2	O
+of	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+.	O
+	O
+Here	O
+,	O
+the	O
+authors	O
+report	O
+the	O
+crystal	O
+structures	O
+of	O
+closed	O
+states	O
+of	O
+Mep2	O
+proteins	O
+and	O
+propose	O
+a	O
+model	O
+for	O
+their	O
+regulation	O
+by	O
+comparing	O
+them	O
+with	O
+the	O
+open	O
+ammonium	O
+transporters	O
+of	O
+bacteria	O
+.	O
+	O
+A	O
+common	O
+feature	O
+of	O
+transceptors	O
+is	O
+that	O
+they	O
+are	O
+induced	O
+when	O
+cells	O
+are	O
+starved	O
+for	O
+their	O
+substrate	O
+.	O
+	O
+With	O
+the	O
+exception	O
+of	O
+the	O
+human	O
+RhCG	O
+structure	O
+,	O
+no	O
+structural	O
+information	O
+is	O
+available	O
+for	O
+eukaryotic	O
+ammonium	O
+transporters	O
+.	O
+	O
+To	O
+elucidate	O
+the	O
+mechanism	O
+of	O
+Mep2	O
+transport	O
+regulation	O
+,	O
+we	O
+present	O
+here	O
+X	O
+-	O
+ray	O
+crystal	O
+structures	O
+of	O
+the	O
+Mep2	O
+transceptors	O
+from	O
+S	O
+.	O
+cerevisiae	O
+and	O
+C	O
+.	O
+albicans	O
+.	O
+	O
+Despite	O
+different	O
+crystal	O
+packing	O
+(	O
+Supplementary	O
+Table	O
+1	O
+),	O
+the	O
+two	O
+CaMep2	O
+structures	O
+are	O
+identical	O
+to	O
+each	O
+other	O
+and	O
+very	O
+similar	O
+to	O
+ScMep2	O
+(	O
+Cα	O
+r	O
+.	O
+m	O
+.	O
+s	O
+.	O
+d	O
+.	O
+	O
+Unless	O
+specifically	O
+stated	O
+,	O
+the	O
+drawn	O
+conclusions	O
+also	O
+apply	O
+to	O
+ScMep2	O
+.	O
+	O
+Together	O
+with	O
+additional	O
+,	O
+smaller	O
+differences	O
+in	O
+other	O
+extracellular	O
+loops	O
+,	O
+these	O
+changes	O
+generate	O
+a	O
+distinct	O
+vestibule	O
+leading	O
+to	O
+the	O
+ammonium	O
+binding	O
+site	O
+that	O
+is	O
+much	O
+more	O
+pronounced	O
+than	O
+in	O
+the	O
+bacterial	O
+proteins	O
+.	O
+	O
+The	O
+largest	O
+differences	O
+between	O
+the	O
+Mep2	O
+structures	O
+and	O
+the	O
+other	O
+known	O
+ammonium	O
+transporter	O
+structures	O
+are	O
+located	O
+on	O
+the	O
+intracellular	O
+side	O
+of	O
+the	O
+membrane	O
+.	O
+	O
+ICL1	O
+has	O
+also	O
+moved	O
+inwards	O
+relative	O
+to	O
+its	O
+position	O
+in	O
+the	O
+bacterial	O
+Amts	O
+.	O
+	O
+Compared	O
+with	O
+ICL1	O
+,	O
+the	O
+backbone	O
+conformational	O
+changes	O
+observed	O
+for	O
+the	O
+neighbouring	O
+ICL2	O
+are	O
+smaller	O
+,	O
+but	O
+large	O
+shifts	O
+are	O
+nevertheless	O
+observed	O
+for	O
+the	O
+conserved	O
+residues	O
+Glu140	O
+and	O
+Arg141	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+Phosphorylation	O
+target	O
+site	O
+is	O
+at	O
+the	O
+periphery	O
+of	O
+Mep2	O
+	O
+In	O
+the	O
+absence	O
+of	O
+Npr1	O
+,	O
+plasmid	O
+-	O
+encoded	O
+WT	O
+Mep2	O
+in	O
+a	O
+S	O
+.	O
+cerevisiae	O
+mep1	B-mutant
+-	I-mutant
+3Δ	I-mutant
+strain	O
+(	O
+triple	B-mutant
+mepΔ	I-mutant
+)	O
+does	O
+not	O
+allow	O
+growth	O
+on	O
+low	O
+concentrations	O
+of	O
+ammonium	O
+,	O
+suggesting	O
+that	O
+the	O
+transporter	O
+is	O
+inactive	O
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+We	O
+obtained	O
+a	O
+similar	O
+result	O
+for	O
+ammonium	O
+uptake	O
+by	O
+the	O
+446Δ	B-mutant
+mutant	O
+(	O
+Fig	O
+.	O
+3	O
+),	O
+supporting	O
+the	O
+data	O
+from	O
+Marini	O
+et	O
+al	O
+.	O
+We	O
+then	O
+constructed	O
+and	O
+purified	O
+the	O
+analogous	O
+CaMep2	O
+442Δ	B-mutant
+truncation	O
+mutant	O
+and	O
+determined	O
+the	O
+crystal	O
+structure	O
+using	O
+data	O
+to	O
+3	O
+.	O
+4	O
+Å	O
+resolution	O
+.	O
+	O
+The	O
+structure	O
+shows	O
+that	O
+removal	O
+of	O
+the	O
+AI	O
+region	O
+markedly	O
+increases	O
+the	O
+dynamics	O
+of	O
+the	O
+cytoplasmic	O
+parts	O
+of	O
+the	O
+transporter	O
+.	O
+	O
+The	O
+first	O
+one	O
+is	O
+that	O
+the	O
+open	O
+state	O
+is	O
+disfavoured	O
+by	O
+crystallization	O
+because	O
+of	O
+lower	O
+stability	O
+or	O
+due	O
+to	O
+crystal	O
+packing	O
+constraints	O
+.	O
+	O
+The	O
+ammonium	O
+uptake	O
+activity	O
+of	O
+the	O
+S	O
+.	O
+cerevisiae	O
+version	O
+of	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+is	O
+the	O
+same	O
+as	O
+that	O
+of	O
+WT	O
+Mep2	O
+and	O
+the	O
+S453D	B-mutant
+mutant	O
+,	O
+indicating	O
+that	O
+the	O
+mutations	O
+do	O
+not	O
+affect	O
+transporter	O
+functionality	O
+in	O
+the	O
+triple	B-mutant
+mepΔ	I-mutant
+background	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+	O
+The	O
+movement	O
+of	O
+the	O
+acidic	O
+residues	O
+away	O
+from	O
+Arg452	O
+and	O
+Sep453	O
+is	O
+more	O
+pronounced	O
+in	O
+this	O
+simulation	O
+in	O
+comparison	O
+with	O
+the	O
+movement	O
+away	O
+from	O
+Asp452	O
+and	O
+Asp453	O
+in	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+.	O
+	O
+The	O
+reason	O
+why	O
+similar	O
+transporters	O
+such	O
+as	O
+A	O
+.	O
+thaliana	O
+Amt	O
+-	O
+1	O
+;	O
+1	O
+and	O
+Mep2	O
+are	O
+regulated	O
+in	O
+opposite	O
+ways	O
+by	O
+phosphorylation	O
+(	O
+inactivation	O
+in	O
+plants	O
+and	O
+activation	O
+in	O
+fungi	O
+)	O
+is	O
+not	O
+known	O
+.	O
+	O
+By	O
+determining	O
+the	O
+first	O
+structures	O
+of	O
+closed	O
+ammonium	O
+transporters	O
+and	O
+comparing	O
+those	O
+structures	O
+with	O
+the	O
+permanently	O
+open	O
+bacterial	O
+proteins	O
+,	O
+we	O
+demonstrate	O
+that	O
+Mep2	O
+channel	O
+closure	O
+is	O
+likely	O
+due	O
+to	O
+movements	O
+of	O
+the	O
+CTR	O
+and	O
+ICL1	O
+and	O
+ICL3	O
+.	O
+	O
+Owing	O
+to	O
+the	O
+crosstalk	O
+between	O
+monomers	O
+,	O
+a	O
+single	O
+phosphorylation	O
+event	O
+might	O
+lead	O
+to	O
+opening	O
+of	O
+the	O
+entire	O
+trimer	O
+,	O
+although	O
+this	O
+has	O
+not	O
+yet	O
+been	O
+tested	O
+(	O
+Fig	O
+.	O
+9b	O
+).	O
+	O
+It	O
+should	O
+also	O
+be	O
+noted	O
+that	O
+the	O
+tyrosine	O
+residue	O
+interacting	O
+with	O
+His2	O
+is	O
+highly	O
+conserved	O
+in	O
+fungal	O
+Mep2	O
+orthologues	O
+,	O
+suggesting	O
+that	O
+the	O
+Tyr	O
+–	O
+His2	O
+hydrogen	O
+bond	O
+might	O
+be	O
+a	O
+general	O
+way	O
+to	O
+close	O
+Mep2	O
+proteins	O
+.	O
+	O
+For	O
+example	O
+,	O
+NH3	O
+uniport	O
+or	O
+symport	O
+of	O
+NH3	O
+/	O
+H	O
++	O
+might	O
+result	O
+in	O
+changes	O
+in	O
+local	O
+pH	O
+,	O
+but	O
+NH4	O
++	O
+uniport	O
+might	O
+not	O
+,	O
+and	O
+this	O
+difference	O
+might	O
+determine	O
+signalling	O
+.	O
+	O
+(	O
+a	O
+)	O
+Monomer	O
+cartoon	O
+models	O
+viewed	O
+from	O
+the	O
+side	O
+for	O
+(	O
+left	O
+)	O
+A	O
+.	O
+fulgidus	O
+Amt	O
+-	O
+1	O
+(	O
+PDB	O
+ID	O
+2B2H	O
+),	O
+S	O
+.	O
+cerevisiae	O
+Mep2	O
+(	O
+middle	O
+)	O
+and	O
+C	O
+.	O
+albicans	O
+Mep2	O
+(	O
+right	O
+).	O
+	O
+One	O
+monomer	O
+is	O
+coloured	O
+as	O
+in	O
+a	O
+and	O
+one	O
+monomer	O
+is	O
+coloured	O
+by	O
+B	O
+-	O
+factor	O
+(	O
+blue	O
+,	O
+low	O
+;	O
+red	O
+;	O
+high	O
+).	O
+	O
+The	O
+secondary	O
+structure	O
+elements	O
+observed	O
+for	O
+CaMep2	O
+are	O
+indicated	O
+,	O
+with	O
+the	O
+numbers	O
+corresponding	O
+to	O
+the	O
+centre	O
+of	O
+the	O
+TM	O
+segment	O
+.	O
+	O
+Growth	O
+of	O
+ScMep2	B-mutant
+variants	I-mutant
+on	O
+low	O
+ammonium	O
+medium	O
+.	O
+	O
+(	O
+b	O
+)	O
+Overlay	O
+of	O
+the	O
+CTRs	O
+of	O
+ScMep2	O
+(	O
+grey	O
+)	O
+and	O
+CaMep2	O
+(	O
+green	O
+),	O
+showing	O
+the	O
+similar	O
+electronegative	O
+environment	O
+surrounding	O
+the	O
+phosphorylation	O
+site	O
+(	O
+P	O
+).	O
+	O
+The	O
+AI	O
+regions	O
+are	O
+coloured	O
+magenta	O
+.	O
+	O
+Missing	O
+regions	O
+are	O
+labelled	O
+.	O
+(	O
+b	O
+)	O
+Stereo	O
+superpositions	O
+of	O
+WT	O
+CaMep2	O
+and	O
+the	O
+truncation	O
+mutant	O
+.	O
+	O
+Schematic	O
+model	O
+for	O
+phosphorylation	O
+-	O
+based	O
+regulation	O
+of	O
+Mep2	O
+ammonium	O
+transporters	O
+.	O
+	O
+To	O
+support	O
+antibody	O
+therapeutic	O
+development	O
+,	O
+the	O
+crystal	O
+structures	O
+of	O
+a	O
+set	O
+of	O
+16	O
+germline	O
+variants	O
+composed	O
+of	O
+4	O
+different	O
+kappa	O
+light	O
+chains	O
+paired	O
+with	O
+4	O
+different	O
+heavy	O
+chains	O
+have	O
+been	O
+determined	O
+.	O
+	O
+CDR	O
+H3	O
+,	O
+despite	O
+having	O
+the	O
+same	O
+amino	O
+acid	O
+sequence	O
+,	O
+exhibits	O
+the	O
+largest	O
+conformational	O
+diversity	O
+.	O
+	O
+About	O
+half	O
+of	O
+the	O
+structures	O
+have	O
+CDR	O
+H3	O
+conformations	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+parent	O
+;	O
+the	O
+others	O
+diverge	O
+significantly	O
+.	O
+	O
+The	O
+structures	O
+and	O
+their	O
+analyses	O
+provide	O
+a	O
+rich	O
+foundation	O
+for	O
+future	O
+antibody	O
+modeling	O
+and	O
+engineering	O
+efforts	O
+.	O
+	O
+Isotypes	O
+IgG	O
+,	O
+IgD	O
+and	O
+IgA	O
+each	O
+have	O
+4	O
+domains	O
+,	O
+one	O
+variable	O
+(	O
+V	O
+)	O
+and	O
+3	O
+constant	O
+(	O
+C	O
+)	O
+domains	O
+,	O
+while	O
+IgE	O
+and	O
+IgM	O
+each	O
+have	O
+the	O
+same	O
+4	O
+domains	O
+along	O
+with	O
+an	O
+additional	O
+C	O
+domain	O
+.	O
+	O
+These	O
+domains	O
+have	O
+a	O
+common	O
+folding	O
+pattern	O
+often	O
+referred	O
+to	O
+as	O
+the	O
+“	O
+immunoglobulin	O
+fold	O
+,”	O
+formed	O
+by	O
+the	O
+packing	O
+together	O
+of	O
+2	O
+anti	O
+-	O
+parallel	O
+β	O
+-	O
+sheets	O
+.	O
+	O
+In	O
+antibodies	O
+,	O
+the	O
+heavy	O
+and	O
+light	O
+chain	O
+V	O
+domains	O
+pack	O
+together	O
+forming	O
+the	O
+antigen	O
+combining	O
+site	O
+.	O
+	O
+Later	O
+studies	O
+found	O
+that	O
+the	O
+CDR	O
+loop	O
+length	O
+is	O
+the	O
+primary	O
+determining	O
+factor	O
+of	O
+antigen	O
+-	O
+binding	O
+site	O
+topography	O
+because	O
+it	O
+is	O
+the	O
+primary	O
+factor	O
+for	O
+determining	O
+a	O
+canonical	O
+structure	O
+.	O
+	O
+In	O
+the	O
+torso	O
+region	O
+,	O
+2	O
+primary	O
+groups	O
+could	O
+be	O
+identified	O
+,	O
+which	O
+led	O
+to	O
+sequence	O
+-	O
+based	O
+rules	O
+that	O
+can	O
+predict	O
+with	O
+some	O
+degree	O
+of	O
+reliability	O
+the	O
+conformation	O
+of	O
+the	O
+stem	O
+region	O
+.	O
+	O
+The	O
+“	O
+kinked	O
+”	O
+or	O
+“	O
+bulged	O
+”	O
+conformation	O
+is	O
+the	O
+most	O
+prevalent	O
+,	O
+but	O
+an	O
+“	O
+extended	O
+”	O
+or	O
+“	O
+non	O
+-	O
+bulged	O
+”	O
+conformation	O
+is	O
+also	O
+,	O
+but	O
+less	O
+frequently	O
+,	O
+observed	O
+.	O
+	O
+Crystallization	O
+of	O
+the	O
+16	O
+Fabs	O
+was	O
+previously	O
+reported	O
+.	O
+	O
+Three	O
+sets	O
+of	O
+the	O
+crystals	O
+were	O
+isomorphous	O
+with	O
+nearly	O
+identical	O
+unit	O
+cells	O
+(	O
+Table	O
+1	O
+).	O
+	O
+The	O
+crystal	O
+structures	O
+of	O
+the	O
+16	O
+Fabs	O
+have	O
+been	O
+determined	O
+at	O
+resolutions	O
+ranging	O
+from	O
+3	O
+.	O
+3	O
+Å	O
+to	O
+1	O
+.	O
+65	O
+Å	O
+(	O
+Table	O
+1	O
+).	O
+	O
+Overall	O
+the	O
+structures	O
+are	O
+fairly	O
+complete	O
+,	O
+and	O
+,	O
+as	O
+can	O
+be	O
+expected	O
+,	O
+the	O
+models	O
+for	O
+the	O
+higher	O
+resolution	O
+structures	O
+are	O
+more	O
+complete	O
+than	O
+those	O
+for	O
+the	O
+lower	O
+resolution	O
+structures	O
+(	O
+Table	O
+S1	O
+).	O
+	O
+CDRs	O
+are	O
+defined	O
+using	O
+the	O
+Dunbrack	O
+convention	O
+[	O
+12	O
+].	O
+	O
+CDR	O
+H2	O
+	O
+Most	O
+likely	O
+this	O
+is	O
+the	O
+result	O
+of	O
+interaction	O
+of	O
+CDR	O
+H2	O
+with	O
+CDR	O
+H1	O
+,	O
+namely	O
+with	O
+the	O
+residue	O
+at	O
+position	O
+33	O
+(	O
+residue	O
+11	O
+of	O
+13	O
+in	O
+CDR	O
+H1	O
+).	O
+	O
+The	O
+four	O
+LC	O
+CDRs	O
+L1	O
+feature	O
+3	O
+different	O
+lengths	O
+(	O
+11	O
+,	O
+12	O
+and	O
+17	O
+residues	O
+)	O
+having	O
+a	O
+total	O
+of	O
+4	O
+different	O
+canonical	O
+structure	O
+assignments	O
+.	O
+	O
+Two	O
+structures	O
+,	O
+H3	O
+-	O
+53	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+20	O
+are	O
+assigned	O
+to	O
+canonical	O
+structure	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+1	I-mutant
+with	O
+virtually	O
+identical	O
+backbone	O
+conformations	O
+.	O
+	O
+As	O
+with	O
+CDR	O
+L2	O
+,	O
+all	O
+4	O
+LCs	O
+have	O
+CDR	O
+L3	O
+of	O
+the	O
+same	O
+length	O
+and	O
+canonical	O
+structure	O
+,	O
+L3	B-mutant
+-	I-mutant
+9	I-mutant
+-	I-mutant
+cis7	I-mutant
+-	I-mutant
+1	I-mutant
+(	O
+Table	O
+2	O
+).	O
+	O
+CDR	O
+H3	O
+conformational	O
+diversity	O
+	O
+Despite	O
+having	O
+the	O
+same	O
+amino	O
+acid	O
+sequence	O
+in	O
+all	O
+variants	O
+,	O
+CDR	O
+H3	O
+has	O
+the	O
+highest	O
+degree	O
+of	O
+structural	O
+diversity	O
+and	O
+disorder	O
+of	O
+all	O
+of	O
+the	O
+CDRs	O
+in	O
+the	O
+experimental	O
+set	O
+.	O
+	O
+Another	O
+four	O
+of	O
+the	O
+Fabs	O
+,	O
+H3	O
+-	O
+23	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+H3	O
+-	O
+53	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+H3	O
+-	O
+53	O
+:	O
+L3	O
+-	O
+11	O
+and	O
+H3	O
+-	O
+53	O
+:	O
+L4	O
+-	O
+1	O
+have	O
+missing	O
+side	O
+-	O
+chain	O
+atoms	O
+.	O
+	O
+A	O
+representative	O
+CDR	O
+H3	O
+structure	O
+for	O
+H1	O
+-	O
+69	O
+:	O
+L1	O
+-	O
+39	O
+illustrating	O
+this	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+7A	O
+.	O
+	O
+In	O
+fact	O
+,	O
+it	O
+is	O
+the	O
+only	O
+Fab	O
+in	O
+the	O
+set	O
+that	O
+has	O
+a	O
+water	O
+molecule	O
+present	O
+at	O
+this	O
+site	O
+.	O
+	O
+Three	O
+of	O
+the	O
+Fabs	O
+,	O
+H3	O
+-	O
+23	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+H3	O
+-	O
+23	O
+:	O
+L4	O
+-	O
+1	O
+and	O
+H3	O
+-	O
+53	O
+:	O
+L1	O
+-	O
+39	O
+,	O
+have	O
+distinctive	O
+conformations	O
+.	O
+	O
+VH	O
+:	O
+VL	O
+domain	O
+packing	O
+	O
+The	O
+VH	O
+and	O
+VL	O
+domains	O
+have	O
+a	O
+β	O
+-	O
+sandwich	O
+structure	O
+(	O
+also	O
+often	O
+referred	O
+as	O
+a	O
+Greek	O
+key	O
+motif	O
+)	O
+and	O
+each	O
+is	O
+composed	O
+of	O
+a	O
+4	O
+-	O
+stranded	O
+and	O
+a	O
+5	O
+-	O
+stranded	O
+antiparallel	O
+β	O
+-	O
+sheets	O
+.	O
+	O
+They	O
+include	O
+:	O
+1	O
+)	O
+a	O
+bidentate	O
+hydrogen	O
+bond	O
+between	O
+L	O
+-	O
+Gln38	O
+and	O
+H	O
+-	O
+Gln39	O
+;	O
+2	O
+)	O
+H	O
+-	O
+Leu45	O
+in	O
+a	O
+hydrophobic	O
+pocket	O
+between	O
+L	O
+-	O
+Phe98	O
+,	O
+L	O
+-	O
+Tyr87	O
+and	O
+L	O
+-	O
+Pro44	O
+;	O
+3	O
+)	O
+L	O
+-	O
+Pro44	O
+stacked	O
+against	O
+H	O
+-	O
+Trp103	O
+;	O
+and	O
+4	O
+)	O
+L	O
+-	O
+Ala43	O
+opposite	O
+the	O
+face	O
+of	O
+H	O
+-	O
+Tyr91	O
+(	O
+Fig	O
+.	O
+8	O
+).	O
+	O
+With	O
+the	O
+exception	O
+of	O
+L	O
+-	O
+Ala43	O
+,	O
+all	O
+other	O
+residues	O
+are	O
+conserved	O
+in	O
+human	O
+germlines	O
+.	O
+	O
+The	O
+first	O
+approach	O
+uses	O
+ABangles	O
+,	O
+the	O
+results	O
+of	O
+which	O
+are	O
+shown	O
+in	O
+Table	O
+S2	O
+.	O
+	O
+For	O
+structures	O
+with	O
+2	O
+copies	O
+of	O
+the	O
+Fab	O
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+only	O
+one	O
+structure	O
+was	O
+used	O
+.	O
+	O
+The	O
+largest	O
+deviations	O
+in	O
+the	O
+tilt	O
+angle	O
+,	O
+up	O
+to	O
+11	O
+.	O
+0	O
+°,	O
+are	O
+found	O
+for	O
+2	O
+structures	O
+,	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+that	O
+stand	O
+out	O
+from	O
+the	O
+other	O
+Fabs	O
+.	O
+	O
+Two	O
+examples	O
+illustrating	O
+large	O
+(	O
+10	O
+.	O
+5	O
+°)	O
+and	O
+small	O
+(	O
+1	O
+.	O
+6	O
+°)	O
+differences	O
+in	O
+the	O
+tilt	O
+angles	O
+are	O
+shown	O
+in	O
+Fig	O
+.	O
+9	O
+.	O
+	O
+Some	O
+side	O
+chain	O
+atoms	O
+in	O
+CDR	O
+H3	O
+are	O
+missing	O
+.	O
+	O
+Parts	O
+of	O
+CDR	O
+H3	O
+main	O
+chain	O
+are	O
+completely	O
+disordered	O
+,	O
+and	O
+were	O
+not	O
+modeled	O
+in	O
+Fabs	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+H5	O
+-	O
+51	O
+:	O
+L3	O
+-	O
+11	O
+that	O
+have	O
+the	O
+lowest	O
+Tms	O
+in	O
+the	O
+set	O
+.	O
+	O
+Pairing	O
+of	O
+different	O
+germlines	O
+yields	O
+antibodies	O
+with	O
+various	O
+degrees	O
+of	O
+stability	O
+.	O
+	O
+Curiously	O
+,	O
+the	O
+2	O
+Fabs	O
+,	O
+H1	O
+-	O
+69	O
+:	O
+L3	O
+-	O
+20	O
+and	O
+H3	O
+-	O
+23	O
+:	O
+L3	O
+-	O
+20	O
+,	O
+deviate	O
+markedly	O
+in	O
+their	O
+tilt	O
+angles	O
+from	O
+the	O
+rest	O
+of	O
+the	O
+panel	O
+.	O
+	O
+Note	O
+that	O
+most	O
+of	O
+the	O
+VH	O
+:	O
+VL	O
+interface	O
+residues	O
+are	O
+invariant	O
+;	O
+therefore	O
+,	O
+significant	O
+change	O
+of	O
+the	O
+tilt	O
+angle	O
+must	O
+come	O
+with	O
+a	O
+penalty	O
+in	O
+free	O
+energy	O
+.	O
+	O
+Comparison	O
+of	O
+the	O
+CDR	O
+H3s	O
+reveals	O
+a	O
+large	O
+set	O
+of	O
+variants	O
+with	O
+conformations	O
+similar	O
+to	O
+the	O
+parent	O
+,	O
+while	O
+a	O
+second	O
+set	O
+has	O
+significant	O
+conformational	O
+variability	O
+,	O
+indicating	O
+that	O
+both	O
+the	O
+sequence	O
+and	O
+the	O
+structural	O
+context	O
+define	O
+the	O
+CDR	O
+H3	O
+conformation	O
+.	O
+	O
+Fortunately	O
+,	O
+for	O
+most	O
+applications	O
+of	O
+antibody	O
+modeling	O
+,	O
+such	O
+as	O
+engineering	O
+affinity	O
+and	O
+biophysical	O
+properties	O
+,	O
+an	O
+accurate	O
+CDR	O
+H3	O
+structure	O
+is	O
+not	O
+always	O
+necessary	O
+.	O
+	O
+Visualizing	O
+chaperone	O
+-	O
+assisted	O
+protein	O
+folding	O
+	O
+NMR	O
+can	O
+theoretically	O
+be	O
+used	O
+to	O
+determine	O
+heterogeneous	O
+ensembles	O
+,	O
+but	O
+in	O
+practice	O
+,	O
+this	O
+proves	O
+to	O
+be	O
+very	O
+challenging	O
+.	O
+	O
+Importantly	O
+,	O
+even	O
+though	O
+we	O
+only	O
+labeled	O
+a	O
+subset	O
+of	O
+the	O
+residues	O
+in	O
+the	O
+flexible	O
+regions	O
+of	O
+the	O
+substrate	O
+with	O
+iodine	O
+,	O
+the	O
+residual	O
+electron	O
+density	O
+can	O
+provide	O
+spatial	O
+information	O
+on	O
+many	O
+of	O
+the	O
+other	O
+flexible	O
+residues	O
+.	O
+	O
+As	O
+described	O
+in	O
+detail	O
+below	O
+,	O
+we	O
+developed	O
+the	O
+READ	O
+method	O
+to	O
+uncover	O
+the	O
+ensemble	O
+of	O
+conformations	O
+that	O
+the	O
+Spy	O
+-	O
+binding	O
+domain	O
+of	O
+Im7	O
+(	O
+i	O
+.	O
+e	O
+.,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+)	O
+adopts	O
+while	O
+bound	O
+to	O
+Spy	O
+.	O
+	O
+We	O
+then	O
+co	O
+-	O
+crystallized	O
+Spy	O
+and	O
+the	O
+eight	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+peptides	O
+,	O
+each	O
+of	O
+which	O
+harbored	O
+an	O
+individual	O
+pI	O
+-	O
+Phe	O
+substitution	O
+at	O
+one	O
+distinct	O
+position	O
+,	O
+and	O
+collected	O
+anomalous	O
+data	O
+for	O
+all	O
+eight	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+complexes	O
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+Supplementary	O
+Table	O
+1	O
+Supplementary	O
+Dataset	O
+1	O
+,	O
+and	O
+Supplementary	O
+Table	O
+2	O
+).	O
+	O
+To	O
+determine	O
+the	O
+structural	O
+ensemble	O
+that	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+adopts	O
+while	O
+bound	O
+to	O
+Spy	O
+,	O
+we	O
+combined	O
+the	O
+residual	O
+electron	O
+density	O
+and	O
+the	O
+anomalous	O
+signals	O
+from	O
+our	O
+pI	O
+-	O
+Phe	O
+substituted	O
+Spy	O
+:	O
+Im76	O
+-	O
+45	O
+complexes	O
+.	O
+	O
+The	O
+READ	O
+sample	O
+-	O
+and	O
+-	O
+select	O
+algorithm	O
+is	O
+diagrammed	O
+in	O
+Fig	O
+.	O
+2	O
+.	O
+	O
+Prior	O
+to	O
+performing	O
+the	O
+selection	O
+,	O
+we	O
+generated	O
+a	O
+large	O
+and	O
+diverse	O
+pool	O
+of	O
+chaperone	O
+-	O
+substrate	O
+complexes	O
+using	O
+coarse	O
+-	O
+grained	O
+MD	O
+simulations	O
+in	O
+a	O
+pseudo	O
+-	O
+crystal	O
+environment	O
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+	O
+The	O
+initial	O
+conditions	O
+of	O
+the	O
+binding	O
+simulations	O
+are	O
+not	O
+biased	O
+toward	O
+a	O
+particular	O
+conformation	O
+of	O
+the	O
+substrate	O
+or	O
+any	O
+specific	O
+chaperone	O
+-	O
+substrate	O
+interaction	O
+(	O
+Online	O
+Methods	O
+).	O
+	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+binds	O
+and	O
+unbinds	O
+to	O
+Spy	O
+throughout	O
+the	O
+simulations	O
+.	O
+	O
+The	O
+anomalous	O
+scattering	O
+portion	O
+of	O
+the	O
+selection	O
+uses	O
+our	O
+basic	O
+knowledge	O
+of	O
+pI	O
+-	O
+Phe	O
+geometry	O
+:	O
+the	O
+iodine	O
+is	O
+separated	O
+from	O
+its	O
+respective	O
+Cα	O
+atom	O
+in	O
+each	O
+coarse	O
+-	O
+grained	O
+conformer	O
+by	O
+6	O
+.	O
+5	O
+Å	O
+.	O
+The	O
+selection	O
+then	O
+picks	O
+ensembles	O
+that	O
+best	O
+reproduce	O
+the	O
+collection	O
+of	O
+iodine	O
+anomalous	O
+signals	O
+.	O
+	O
+To	O
+make	O
+the	O
+electron	O
+density	O
+selection	O
+practical	O
+,	O
+we	O
+needed	O
+to	O
+develop	O
+a	O
+method	O
+to	O
+rapidly	O
+evaluate	O
+the	O
+agreement	O
+between	O
+the	O
+selected	O
+sub	O
+-	O
+ensembles	O
+and	O
+the	O
+experimental	O
+electron	O
+density	O
+on	O
+-	O
+the	O
+-	O
+fly	O
+during	O
+the	O
+selection	O
+procedure	O
+.	O
+	O
+Folding	O
+and	O
+interactions	O
+of	O
+Im7	O
+while	O
+bound	O
+to	O
+Spy	O
+	O
+The	O
+ensemble	O
+primarily	O
+encompasses	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+laying	O
+diagonally	O
+within	O
+the	O
+Spy	O
+cradle	O
+in	O
+several	O
+different	O
+orientations	O
+,	O
+but	O
+some	O
+conformations	O
+traverse	O
+as	O
+far	O
+as	O
+the	O
+tips	O
+or	O
+even	O
+extend	O
+over	O
+the	O
+side	O
+of	O
+the	O
+cradle	O
+(	O
+Figs	O
+.	O
+3	O
+,	O
+4a	O
+).	O
+	O
+This	O
+mixture	O
+suggests	O
+the	O
+importance	O
+of	O
+both	O
+electrostatic	O
+and	O
+hydrophobic	O
+components	O
+in	O
+binding	O
+the	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+ensemble	O
+.	O
+	O
+This	O
+shift	O
+in	O
+contacts	O
+is	O
+likely	O
+due	O
+to	O
+hydrophobic	O
+residues	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+preferentially	O
+forming	O
+intra	O
+-	O
+molecular	O
+contacts	O
+upon	O
+folding	O
+(	O
+i	O
+.	O
+e	O
+.,	O
+hydrophobic	O
+collapse	O
+),	O
+effectively	O
+removing	O
+themselves	O
+from	O
+the	O
+interaction	O
+sites	O
+.	O
+	O
+The	O
+diversity	O
+of	O
+conformations	O
+and	O
+binding	O
+sites	O
+observed	O
+here	O
+emphasizes	O
+the	O
+dynamic	O
+and	O
+heterogeneous	O
+nature	O
+of	O
+the	O
+chaperone	O
+-	O
+substrate	O
+ensemble	O
+.	O
+	O
+It	O
+is	O
+possible	O
+that	O
+this	O
+twist	O
+serves	O
+to	O
+increase	O
+heterogeneity	O
+in	O
+Spy	O
+by	O
+providing	O
+more	O
+binding	O
+poses	O
+.	O
+	O
+Additionally	O
+,	O
+we	O
+observed	O
+that	O
+the	O
+linker	O
+region	O
+(	O
+residues	O
+47	O
+–	O
+57	O
+)	O
+of	O
+Spy	O
+,	O
+which	O
+participates	O
+in	O
+substrate	O
+interaction	O
+,	O
+becomes	O
+mostly	O
+disordered	O
+upon	O
+binding	O
+the	O
+substrate	O
+.	O
+	O
+Importantly	O
+,	O
+we	O
+observed	O
+the	O
+same	O
+structural	O
+changes	O
+in	O
+Spy	O
+regardless	O
+of	O
+which	O
+of	O
+the	O
+four	O
+substrates	O
+was	O
+bound	O
+(	O
+Fig	O
+.	O
+5b	O
+,	O
+Table	O
+1	O
+).	O
+	O
+This	O
+substrate	O
+-	O
+chaperone	O
+ensemble	O
+helps	O
+accomplish	O
+the	O
+longstanding	O
+goal	O
+of	O
+obtaining	O
+a	O
+detailed	O
+view	O
+of	O
+how	O
+a	O
+chaperone	O
+aids	O
+protein	O
+folding	O
+.	O
+	O
+The	O
+high	O
+-	O
+resolution	O
+ensemble	O
+obtained	O
+here	O
+now	O
+provides	O
+insight	O
+into	O
+exactly	O
+how	O
+this	O
+occurs	O
+.	O
+	O
+Nearly	O
+all	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+residues	O
+come	O
+in	O
+contact	O
+with	O
+Spy	O
+.	O
+	O
+Unfolded	O
+substrate	O
+conformers	O
+interact	O
+with	O
+Spy	O
+through	O
+both	O
+hydrophobic	O
+and	O
+hydrophilic	O
+interactions	O
+,	O
+whereas	O
+the	O
+binding	O
+of	O
+native	O
+-	O
+like	O
+states	O
+is	O
+mainly	O
+hydrophilic	O
+.	O
+	O
+Previous	O
+analysis	O
+revealed	O
+that	O
+the	O
+Super	O
+Spy	O
+variants	O
+either	O
+bound	O
+Im7	O
+tighter	O
+than	O
+WT	O
+Spy	O
+,	O
+increased	O
+chaperone	O
+flexibility	O
+as	O
+measured	O
+via	O
+H	O
+/	O
+D	O
+exchange	O
+,	O
+or	O
+both	O
+.	O
+	O
+Moreover	O
+,	O
+our	O
+co	O
+-	O
+structure	O
+suggests	O
+that	O
+the	O
+L32P	B-mutant
+substitution	O
+,	O
+which	O
+increases	O
+Spy	O
+’	O
+s	O
+flexibility	O
+,	O
+could	O
+operate	O
+by	O
+unhinging	O
+the	O
+N	O
+-	O
+terminal	O
+helix	O
+and	O
+effectively	O
+expanding	O
+the	O
+size	O
+of	O
+the	O
+disordered	O
+linker	O
+.	O
+	O
+This	O
+possibility	O
+is	O
+supported	O
+by	O
+the	O
+Spy	O
+:	O
+substrate	O
+structures	O
+,	O
+in	O
+which	O
+the	O
+linker	O
+region	O
+becomes	O
+more	O
+flexible	O
+compared	O
+to	O
+the	O
+apo	O
+state	O
+(	O
+Fig	O
+.	O
+6a	O
+).	O
+	O
+Instead	O
+,	O
+when	O
+Spy	O
+is	O
+bound	O
+to	O
+substrate	O
+,	O
+F115	O
+engages	O
+in	O
+close	O
+CH	O
+⋯	O
+π	O
+hydrogen	O
+bonds	O
+with	O
+Tyr104	O
+(	O
+Fig	O
+.	O
+6b	O
+).	O
+	O
+Overall	O
+,	O
+comparison	O
+of	O
+our	O
+ensemble	O
+to	O
+the	O
+Super	O
+Spy	O
+variants	O
+provides	O
+specific	O
+examples	O
+to	O
+corroborate	O
+the	O
+importance	O
+of	O
+conformational	O
+flexibility	O
+in	O
+chaperone	O
+-	O
+substrate	O
+interactions	O
+.	O
+	O
+Spy	O
+is	O
+depicted	O
+as	O
+a	O
+gray	O
+surface	O
+and	O
+the	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+conformer	O
+is	O
+shown	O
+as	O
+orange	O
+balls	O
+.	O
+	O
+The	O
+frequency	O
+plotted	O
+is	O
+calculated	O
+as	O
+the	O
+average	O
+contact	O
+frequency	O
+from	O
+Spy	O
+to	O
+every	O
+residue	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+and	O
+vice	O
+-	O
+versa	O
+.	O
+	O
+The	O
+mechanism	O
+of	O
+NCX	O
+proteins	O
+is	O
+therefore	O
+highly	O
+likely	O
+to	O
+be	O
+consistent	O
+with	O
+the	O
+alternating	O
+-	O
+access	O
+model	O
+of	O
+secondary	O
+-	O
+active	O
+transport	O
+.	O
+	O
+With	O
+similar	O
+ion	O
+exchange	O
+properties	O
+to	O
+those	O
+of	O
+its	O
+eukaryotic	O
+counterparts	O
+,	O
+NCX_Mj	O
+provides	O
+a	O
+compelling	O
+model	O
+system	O
+to	O
+investigate	O
+the	O
+structural	O
+basis	O
+for	O
+the	O
+specificity	O
+,	O
+stoichiometry	O
+and	O
+mechanism	O
+of	O
+the	O
+ion	O
+-	O
+exchange	O
+reaction	O
+catalyzed	O
+by	O
+NCX	O
+.	O
+	O
+The	O
+assignment	O
+of	O
+the	O
+four	O
+central	O
+binding	O
+sites	O
+identified	O
+in	O
+the	O
+previously	O
+reported	O
+NCX_Mj	O
+structure	O
+was	O
+hampered	O
+by	O
+the	O
+presence	O
+of	O
+both	O
+Na	O
++	O
+and	O
+Ca2	O
++	O
+in	O
+the	O
+protein	O
+crystals	O
+.	O
+	O
+First	O
+,	O
+the	O
+electron	O
+density	O
+at	O
+Smid	O
+does	O
+not	O
+depend	O
+significantly	O
+on	O
+the	O
+Na	O
++	O
+concentration	O
+.	O
+	O
+This	O
+structurally	O
+-	O
+derived	O
+Na	O
++	O
+affinity	O
+agrees	O
+well	O
+with	O
+the	O
+external	O
+Na	O
++	O
+concentration	O
+required	O
+for	O
+NCX	O
+activation	O
+in	O
+eukaryotes	O
+.	O
+	O
+Similarly	O
+to	O
+Sr2	O
++,	O
+Ca2	O
++	O
+binds	O
+with	O
+low	O
+affinity	O
+to	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+and	O
+can	O
+be	O
+readily	O
+displaced	O
+by	O
+Na	O
++	O
+(	O
+Supplementary	O
+Note	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+).	O
+	O
+This	O
+finding	O
+is	O
+consistent	O
+with	O
+physiological	O
+and	O
+biochemical	O
+data	O
+for	O
+both	O
+eukaryotic	O
+NCX	O
+and	O
+NCX_Mj	O
+indicating	O
+that	O
+the	O
+apparent	O
+Ca2	O
++	O
+affinity	O
+is	O
+much	O
+lower	O
+on	O
+the	O
+extracellular	O
+than	O
+the	O
+cytoplasmic	O
+side	O
+.	O
+	O
+We	O
+were	O
+able	O
+to	O
+determine	O
+an	O
+apo	O
+-	O
+state	O
+structure	O
+of	O
+NCX_Mj	O
+,	O
+by	O
+crystallizing	O
+the	O
+protein	O
+at	O
+lower	O
+pH	O
+and	O
+in	O
+the	O
+absence	O
+of	O
+Na	O
++	O
+(	O
+Methods	O
+).	O
+	O
+To	O
+examine	O
+this	O
+central	O
+question	O
+,	O
+we	O
+sought	O
+to	O
+characterize	O
+the	O
+conformational	O
+free	O
+-	O
+energy	O
+landscape	O
+of	O
+NCX_Mj	O
+and	O
+to	O
+examine	O
+its	O
+dependence	O
+on	O
+the	O
+ion	O
+-	O
+occupancy	O
+state	O
+,	O
+using	O
+molecular	O
+dynamics	O
+(	O
+MD	O
+)	O
+simulations	O
+.	O
+	O
+This	O
+computational	O
+analysis	O
+was	O
+based	O
+solely	O
+on	O
+the	O
+published	O
+structure	O
+of	O
+NCX_Mj	O
+,	O
+independently	O
+of	O
+the	O
+crystallographic	O
+studies	O
+described	O
+above	O
+.	O
+	O
+These	O
+initial	O
+simulations	O
+revealed	O
+noticeable	O
+changes	O
+in	O
+the	O
+transporter	O
+,	O
+consistent	O
+with	O
+those	O
+observed	O
+in	O
+the	O
+new	O
+crystal	O
+structures	O
+.	O
+	O
+The	O
+most	O
+noticeable	O
+is	O
+an	O
+increased	O
+separation	O
+between	O
+TM7	O
+and	O
+TM2	O
+(	O
+Fig	O
+.	O
+4f	O
+),	O
+previously	O
+brought	O
+together	O
+by	O
+concurrent	O
+backbone	O
+interactions	O
+with	O
+the	O
+Na	O
++	O
+ion	O
+at	O
+SCa	O
+(	O
+Fig	O
+.	O
+4d	O
+-	O
+e	O
+).	O
+	O
+TM1	O
+and	O
+TM6	O
+also	O
+slide	O
+further	O
+towards	O
+the	O
+membrane	O
+center	O
+,	O
+relative	O
+to	O
+the	O
+outward	O
+-	O
+occluded	O
+state	O
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+	O
+To	O
+more	O
+rigorously	O
+characterize	O
+the	O
+influence	O
+of	O
+the	O
+ion	O
+-	O
+occupancy	O
+state	O
+on	O
+the	O
+conformational	O
+dynamics	O
+of	O
+the	O
+exchanger	O
+,	O
+we	O
+carried	O
+out	O
+a	O
+series	O
+of	O
+enhanced	O
+-	O
+sampling	O
+MD	O
+calculations	O
+designed	O
+to	O
+reversibly	O
+simulate	O
+the	O
+transition	O
+between	O
+the	O
+outward	O
+-	O
+occluded	O
+and	O
+fully	O
+outward	O
+-	O
+open	O
+states	O
+,	O
+and	O
+thus	O
+quantify	O
+the	O
+free	O
+-	O
+energy	O
+landscape	O
+encompassing	O
+these	O
+states	O
+(	O
+Methods	O
+).	O
+	O
+It	O
+is	O
+however	O
+also	O
+non	O
+-	O
+trivial	O
+:	O
+antiporters	O
+,	O
+for	O
+example	O
+,	O
+do	O
+not	O
+undergo	O
+the	O
+alternating	O
+-	O
+access	O
+transition	O
+without	O
+a	O
+cargo	O
+,	O
+but	O
+this	O
+is	O
+precisely	O
+how	O
+membrane	O
+symporters	O
+reset	O
+their	O
+transport	O
+cycles	O
+.	O
+	O
+Similarly	O
+puzzling	O
+is	O
+that	O
+a	O
+given	O
+antiporter	O
+will	O
+undergo	O
+this	O
+transition	O
+upon	O
+recognition	O
+of	O
+substrates	O
+of	O
+different	O
+charge	O
+,	O
+size	O
+and	O
+number	O
+.	O
+	O
+The	O
+internal	O
+symmetry	O
+of	O
+outward	O
+-	O
+facing	O
+NCX_Mj	O
+and	O
+the	O
+inward	O
+-	O
+facing	O
+crystal	O
+structures	O
+of	O
+several	O
+Ca2	O
++/	O
+H	O
++	O
+exchangers	O
+indicate	O
+that	O
+the	O
+alternating	O
+-	O
+access	O
+mechanism	O
+of	O
+NCX	O
+proteins	O
+entails	O
+a	O
+sliding	O
+motion	O
+of	O
+TM1	O
+and	O
+TM6	O
+relative	O
+to	O
+the	O
+rest	O
+of	O
+the	O
+transporter	O
+.	O
+	O
+Indeed	O
+,	O
+we	O
+show	O
+that	O
+it	O
+is	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+the	O
+occluded	O
+state	O
+in	O
+this	O
+landscape	O
+that	O
+explains	O
+the	O
+antiport	O
+function	O
+of	O
+NCX_Mj	O
+and	O
+its	O
+3Na	O
++:	O
+1Ca2	O
++	O
+stoichiometry	O
+.	O
+	O
+Consistent	O
+with	O
+that	O
+finding	O
+,	O
+mutations	O
+that	O
+have	O
+been	O
+shown	O
+to	O
+inactivate	O
+or	O
+diminish	O
+the	O
+transport	O
+activity	O
+of	O
+NCX_Mj	O
+and	O
+cardiac	O
+NCX	O
+perfectly	O
+map	O
+to	O
+the	O
+first	O
+ion	O
+-	O
+coordination	O
+shell	O
+in	O
+our	O
+NCX_Mj	O
+structures	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4c	O
+-	O
+d	O
+).	O
+	O
+The	O
+Sext	O
+site	O
+,	O
+by	O
+contrast	O
+,	O
+might	O
+be	O
+thought	O
+as	O
+an	O
+activation	O
+site	O
+for	O
+inward	O
+Na	O
++	O
+translocation	O
+,	O
+since	O
+this	O
+is	O
+where	O
+the	O
+third	O
+Na	O
++	O
+ion	O
+binds	O
+at	O
+high	O
+Na	O
++	O
+concentration	O
+,	O
+enabling	O
+the	O
+transition	O
+to	O
+the	O
+occluded	O
+state	O
+.	O
+	O
+Lastly	O
+,	O
+our	O
+theory	O
+that	O
+occlusion	O
+of	O
+NCX_Mj	O
+is	O
+selectively	O
+induced	O
+upon	O
+Ca2	O
++	O
+or	O
+Na	O
++	O
+recognition	O
+is	O
+consonant	O
+with	O
+a	O
+recent	O
+analysis	O
+of	O
+the	O
+rate	O
+of	O
+hydrogen	O
+-	O
+deuterium	O
+exchange	O
+(	O
+HDX	O
+)	O
+in	O
+NCX_Mj	O
+,	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+these	O
+ions	O
+,	O
+in	O
+conditions	O
+that	O
+favor	O
+outward	O
+-	O
+facing	O
+conformations	O
+.	O
+	O
+Specifically	O
+,	O
+saturating	O
+amounts	O
+of	O
+Ca2	O
++	O
+or	O
+Na	O
++	O
+resulted	O
+in	O
+a	O
+noticeable	O
+slowdown	O
+in	O
+the	O
+HDX	O
+rate	O
+for	O
+extracellular	O
+portions	O
+of	O
+the	O
+α	O
+-	O
+repeat	O
+helices	O
+.	O
+	O
+We	O
+interpret	O
+these	O
+observations	O
+as	O
+reflecting	O
+that	O
+the	O
+solvent	O
+accessibility	O
+of	O
+the	O
+protein	O
+interior	O
+is	O
+diminished	O
+upon	O
+ion	O
+recognition	O
+,	O
+consistent	O
+with	O
+our	O
+finding	O
+that	O
+opening	O
+and	O
+closing	O
+of	O
+extracellular	O
+aqueous	O
+pathways	O
+to	O
+the	O
+ion	O
+-	O
+binding	O
+sites	O
+depend	O
+on	O
+ion	O
+occupancy	O
+state	O
+.	O
+	O
+Our	O
+data	O
+would	O
+also	O
+explain	O
+the	O
+observation	O
+that	O
+the	O
+reduction	O
+in	O
+the	O
+HDX	O
+rate	O
+is	O
+comparable	O
+for	O
+Na	O
++	O
+and	O
+Ca2	O
++,	O
+as	O
+well	O
+as	O
+the	O
+finding	O
+that	O
+the	O
+degree	O
+of	O
+deuterium	O
+incorporation	O
+remains	O
+non	O
+-	O
+negligible	O
+even	O
+under	O
+saturating	O
+ion	O
+concentrations	O
+.	O
+	O
+(	O
+c	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+Na	O
++-	O
+binding	O
+sites	O
+.	O
+	O
+Residues	O
+surrounding	O
+this	O
+site	O
+are	O
+also	O
+indicated	O
+;	O
+note	O
+A206	O
+(	O
+labeled	O
+in	O
+red	O
+)	O
+coordinates	O
+Na	O
++	O
+at	O
+Sext	O
+via	O
+its	O
+backbone	O
+carbonyl	O
+oxygen	O
+.	O
+	O
+Divalent	O
+cation	O
+binding	O
+and	O
+apo	O
+structure	O
+of	O
+NCX_Mj	O
+.	O
+(	O
+a	O
+)	O
+A	O
+single	O
+Sr2	O
++	O
+(	O
+dark	O
+blue	O
+sphere	O
+)	O
+binds	O
+at	O
+SCa	O
+in	O
+crystals	O
+titrated	O
+with	O
+10	O
+mM	O
+Sr2	O
++	O
+and	O
+2	O
+.	O
+5	O
+mM	O
+Na	O
++	O
+(	O
+see	O
+also	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+	O
+There	O
+are	O
+no	O
+significant	O
+changes	O
+in	O
+the	O
+side	O
+-	O
+chains	O
+involved	O
+in	O
+ion	O
+coordination	O
+,	O
+relative	O
+to	O
+the	O
+Na	O
++-	O
+bound	O
+state	O
+.	O
+	O
+The	O
+relative	O
+occupancies	O
+are	O
+55	O
+%	O
+and	O
+45	O
+%,	O
+respectively	O
+.	O
+(	O
+c	O
+)	O
+Superimposition	O
+of	O
+NCX_Mj	O
+structures	O
+obtained	O
+at	O
+low	O
+Na	O
++	O
+concentration	O
+(	O
+10	O
+mM	O
+)	O
+and	O
+pH	O
+6	O
+.	O
+5	O
+(	O
+brown	O
+)	O
+and	O
+in	O
+the	O
+absence	O
+of	O
+Na	O
++	O
+and	O
+pH	O
+4	O
+(	O
+light	O
+green	O
+),	O
+referred	O
+to	O
+as	O
+apo	O
+state	O
+.	O
+(	O
+d	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+sites	O
+in	O
+the	O
+apo	O
+(	O
+or	O
+high	O
+H	O
++)	O
+state	O
+.	O
+	O
+(	O
+a	O
+)	O
+Representative	O
+simulation	O
+snapshots	O
+of	O
+NCX_Mj	O
+(	O
+Methods	O
+)	O
+with	O
+Na	O
++	O
+bound	O
+at	O
+Sext	O
+,	O
+SCa	O
+and	O
+Sint	O
+(	O
+orange	O
+cartoons	O
+,	O
+green	O
+spheres	O
+)	O
+and	O
+with	O
+Na	O
++	O
+bound	O
+only	O
+at	O
+SCa	O
+and	O
+Sint	O
+(	O
+marine	O
+cartoons	O
+,	O
+yellow	O
+spheres	O
+)	O
+(	O
+b	O
+)	O
+Close	O
+-	O
+up	O
+of	O
+the	O
+backbone	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+half	O
+of	O
+TM7	O
+(	O
+TM7ab	O
+),	O
+in	O
+the	O
+same	O
+Na	O
++	O
+occupancy	O
+states	O
+depicted	O
+in	O
+(	O
+a	O
+).	O
+	O
+(	O
+g	O
+)	O
+Probability	O
+distributions	O
+of	O
+an	O
+analytical	O
+descriptor	O
+of	O
+the	O
+backbone	O
+hydrogen	O
+-	O
+bonding	O
+pattern	O
+in	O
+TM7ab	O
+(	O
+Eq	O
+.	O
+2	O
+).	O
+(	O
+h	O
+)	O
+Mean	O
+value	O
+(	O
+with	O
+standard	O
+deviation	O
+)	O
+of	O
+a	O
+quantitative	O
+descriptor	O
+of	O
+the	O
+solvent	O
+accessibility	O
+of	O
+the	O
+Sext	O
+site	O
+(	O
+Eq	O
+.	O
+1	O
+).	O
+(	O
+i	O
+)	O
+Mean	O
+value	O
+(	O
+with	O
+standard	O
+deviation	O
+)	O
+of	O
+a	O
+quantitative	O
+descriptor	O
+of	O
+the	O
+solvent	O
+accessibility	O
+of	O
+the	O
+SCa	O
+site	O
+(	O
+Eq	O
+.	O
+1	O
+).	O
+	O
+The	O
+free	O
+energy	O
+is	O
+plotted	O
+as	O
+a	O
+function	O
+of	O
+two	O
+coordinates	O
+,	O
+each	O
+describing	O
+the	O
+degree	O
+of	O
+opening	O
+of	O
+the	O
+aqueous	O
+channels	O
+leading	O
+to	O
+the	O
+Sext	O
+and	O
+SCa	O
+sites	O
+,	O
+respectively	O
+(	O
+see	O
+Methods	O
+).	O
+	O
+(	O
+b	O
+)	O
+Density	O
+isosurfaces	O
+for	O
+water	O
+molecules	O
+within	O
+12	O
+Å	O
+of	O
+the	O
+ion	O
+-	O
+binding	O
+region	O
+(	O
+grey	O
+volumes	O
+),	O
+for	O
+each	O
+of	O
+the	O
+major	O
+conformational	O
+free	O
+-	O
+energy	O
+minima	O
+in	O
+each	O
+ion	O
+-	O
+occupancy	O
+state	O
+.	O
+	O
+Na	O
++	O
+ions	O
+are	O
+shown	O
+as	O
+green	O
+spheres	O
+.	O
+	O
+Black	O
+circles	O
+map	O
+the	O
+crystal	O
+structures	O
+obtained	O
+at	O
+high	O
+Ca2	O
++	O
+concentration	O
+and	O
+at	O
+low	O
+pH	O
+(	O
+or	O
+high	O
+H	O
++)	O
+reported	O
+in	O
+this	O
+study	O
+.	O
+	O
+(	O
+b	O
+)	O
+Water	O
+-	O
+density	O
+isosurfaces	O
+analogous	O
+to	O
+those	O
+in	O
+Fig	O
+.	O
+5	O
+are	O
+shown	O
+for	O
+each	O
+of	O
+the	O
+major	O
+conformational	O
+free	O
+-	O
+energy	O
+minima	O
+in	O
+the	O
+free	O
+-	O
+energy	O
+maps	O
+.	O
+	O
+Here	O
+,	O
+the	O
+authors	O
+report	O
+U2AF65	O
+structures	O
+and	O
+single	O
+molecule	O
+FRET	O
+that	O
+reveal	O
+mechanistic	O
+insights	O
+into	O
+splice	O
+site	O
+recognition	O
+.	O
+	O
+The	O
+splice	O
+sites	O
+are	O
+marked	O
+by	O
+relatively	O
+short	O
+consensus	O
+sequences	O
+and	O
+are	O
+regulated	O
+by	O
+additional	O
+pre	O
+-	O
+mRNA	O
+motifs	O
+(	O
+reviewed	O
+in	O
+ref	O
+.).	O
+	O
+In	O
+turn	O
+,	O
+the	O
+ternary	O
+complex	O
+of	O
+U2AF65	O
+with	O
+SF1	O
+and	O
+U2AF35	O
+identifies	O
+the	O
+surrounding	O
+BPS	O
+and	O
+3	O
+′	O
+splice	O
+site	O
+junctions	O
+.	O
+	O
+Likewise	O
+,	O
+both	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+full	O
+-	O
+length	O
+U2AF65	O
+showed	O
+similar	O
+sequence	O
+specificity	O
+for	O
+U	O
+-	O
+rich	O
+stretches	O
+in	O
+the	O
+5	O
+′-	O
+region	O
+of	O
+the	O
+Py	O
+tract	O
+and	O
+promiscuity	O
+for	O
+C	O
+-	O
+rich	O
+regions	O
+in	O
+the	O
+3	O
+′-	O
+region	O
+(	O
+Fig	O
+.	O
+1c	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+1e	O
+–	O
+h	O
+).	O
+	O
+By	O
+sequential	O
+boot	O
+strapping	O
+(	O
+Methods	O
+),	O
+we	O
+optimized	O
+the	O
+oligonucleotide	O
+length	O
+,	O
+the	O
+position	O
+of	O
+a	O
+Br	O
+-	O
+dU	O
+,	O
+and	O
+the	O
+identity	O
+of	O
+the	O
+terminal	O
+nucleotide	O
+(	O
+rU	O
+,	O
+dU	O
+and	O
+rC	O
+)	O
+to	O
+achieve	O
+full	O
+views	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+bound	O
+to	O
+contiguous	O
+Py	O
+tracts	O
+at	O
+up	O
+to	O
+1	O
+.	O
+5	O
+Å	O
+resolution	O
+.	O
+	O
+We	O
+compare	O
+the	O
+global	O
+conformation	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+with	O
+the	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+crystal	O
+structure	O
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+NMR	O
+structure	O
+in	O
+the	O
+Supplementary	O
+Discussion	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+.	O
+	O
+Yet	O
+,	O
+only	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+interactions	O
+at	O
+sites	O
+1	O
+and	O
+7	O
+are	O
+nearly	O
+identical	O
+to	O
+those	O
+of	O
+the	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+,	O
+f	O
+).	O
+	O
+In	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+strand	O
+of	O
+RRM1	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+K225	O
+and	O
+R227	O
+donate	O
+additional	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+rU5	O
+-	O
+O2	O
+lone	O
+pair	O
+electrons	O
+.	O
+	O
+We	O
+tested	O
+the	O
+contribution	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+interactions	O
+with	O
+the	O
+new	O
+central	O
+nucleotide	O
+to	O
+Py	O
+-	O
+tract	O
+affinity	O
+(	O
+Fig	O
+.	O
+3i	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+	O
+U2AF65	O
+RRM	O
+extensions	O
+interact	O
+with	O
+the	O
+Py	O
+tract	O
+	O
+Indirectly	O
+,	O
+the	O
+additional	O
+contacts	O
+with	O
+the	O
+third	O
+nucleotide	O
+shift	O
+the	O
+rU2	O
+nucleotide	O
+in	O
+the	O
+second	O
+binding	O
+site	O
+closer	O
+to	O
+the	O
+C	O
+-	O
+terminal	O
+β	O
+-	O
+strand	O
+of	O
+RRM2	O
+.	O
+	O
+Consistent	O
+with	O
+loss	O
+of	O
+a	O
+hydrogen	O
+bond	O
+with	O
+the	O
+ninth	O
+pyrimidine	O
+-	O
+O2	O
+(	O
+ΔΔG	O
+1	O
+.	O
+0	O
+kcal	O
+mol	O
+−	O
+1	O
+),	O
+mutation	O
+of	O
+the	O
+Q147	O
+to	O
+an	O
+alanine	O
+reduced	O
+U2AF651	O
+,	O
+2L	O
+affinity	O
+for	O
+the	O
+AdML	O
+Py	O
+tract	O
+by	O
+five	O
+-	O
+fold	O
+(	O
+Fig	O
+.	O
+3i	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4c	O
+).	O
+	O
+We	O
+introduced	O
+glycine	O
+substitutions	O
+to	O
+maximally	O
+reduce	O
+the	O
+buried	O
+surface	O
+area	O
+without	O
+directly	O
+interfering	O
+with	O
+its	O
+hydrogen	O
+bonds	O
+between	O
+backbone	O
+atoms	O
+and	O
+the	O
+base	O
+.	O
+	O
+To	O
+further	O
+test	O
+cooperation	O
+among	O
+the	O
+U2AF65	O
+RRM	O
+extensions	O
+and	O
+inter	O
+-	O
+RRM	O
+linker	O
+for	O
+RNA	O
+recognition	O
+,	O
+we	O
+tested	O
+the	O
+impact	O
+of	O
+a	O
+triple	O
+Q147A	B-mutant
+/	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+mutation	O
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+3Mut	I-mutant
+)	O
+for	O
+RNA	O
+binding	O
+(	O
+Fig	O
+.	O
+4b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+).	O
+	O
+We	O
+proceeded	O
+to	O
+test	O
+the	O
+importance	O
+of	O
+new	O
+U2AF65	O
+–	O
+Py	O
+-	O
+tract	O
+interactions	O
+for	O
+splicing	O
+of	O
+a	O
+model	O
+pre	O
+-	O
+mRNA	O
+substrate	O
+in	O
+a	O
+human	O
+cell	O
+line	O
+(	O
+Fig	O
+.	O
+5	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+When	O
+transfected	O
+into	O
+HEK293T	O
+cells	O
+containing	O
+only	O
+endogenous	O
+U2AF65	O
+,	O
+the	O
+PY	O
+splice	O
+site	O
+is	O
+used	O
+and	O
+the	O
+remaining	O
+transcript	O
+remains	O
+unspliced	O
+.	O
+	O
+The	O
+strong	O
+PY	O
+splice	O
+site	O
+is	O
+insensitive	O
+to	O
+added	O
+U2AF65	O
+,	O
+suggesting	O
+that	O
+endogenous	O
+U2AF65	O
+levels	O
+are	O
+sufficient	O
+to	O
+saturate	O
+this	O
+site	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+).	O
+	O
+The	O
+positions	O
+of	O
+single	O
+cysteine	O
+mutations	O
+for	O
+fluorophore	O
+attachment	O
+(	O
+A181C	B-mutant
+in	O
+RRM1	O
+and	O
+Q324C	B-mutant
+in	O
+RRM2	O
+)	O
+were	O
+chosen	O
+based	O
+on	O
+inspection	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	O
+and	O
+the	O
+‘	O
+closed	O
+'	O
+model	O
+of	O
+apo	O
+-	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+.	O
+	O
+Criteria	O
+included	O
+(	O
+i	O
+)	O
+residue	O
+locations	O
+that	O
+are	O
+distant	O
+from	O
+and	O
+hence	O
+not	O
+expected	O
+to	O
+interfere	O
+with	O
+the	O
+RRM	O
+/	O
+RNA	O
+or	O
+inter	O
+-	O
+RRM	O
+interfaces	O
+,	O
+(	O
+ii	O
+)	O
+inter	O
+-	O
+dye	O
+distances	O
+(	O
+50	O
+Å	O
+for	O
+U2AF651	O
+,	O
+2L	O
+–	O
+Py	O
+tract	O
+and	O
+30	O
+Å	O
+for	O
+the	O
+closed	O
+apo	O
+-	O
+model	O
+)	O
+that	O
+are	O
+expected	O
+to	O
+be	O
+near	O
+the	O
+Förster	O
+radius	O
+(	O
+Ro	O
+)	O
+for	O
+the	O
+Cy3	O
+/	O
+Cy5	O
+pair	O
+(	O
+56	O
+Å	O
+),	O
+where	O
+changes	O
+in	O
+the	O
+efficiency	O
+of	O
+energy	O
+transfer	O
+are	O
+most	O
+sensitive	O
+to	O
+distance	O
+,	O
+and	O
+(	O
+iii	O
+)	O
+FRET	O
+efficiencies	O
+that	O
+are	O
+calculated	O
+to	O
+be	O
+significantly	O
+greater	O
+for	O
+the	O
+‘	O
+closed	O
+'	O
+apo	O
+-	O
+model	O
+as	O
+opposed	O
+to	O
+the	O
+‘	O
+open	O
+'	O
+RNA	O
+-	O
+bound	O
+structures	O
+(	O
+by	O
+∼	O
+30	O
+%).	O
+	O
+We	O
+examined	O
+the	O
+effect	O
+on	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+conformations	O
+of	O
+purine	O
+interruptions	O
+that	O
+often	O
+occur	O
+in	O
+relatively	O
+degenerate	O
+human	O
+Py	O
+tracts	O
+.	O
+	O
+Nevertheless	O
+,	O
+the	O
+predominant	O
+0	O
+.	O
+45	O
+FRET	O
+state	O
+in	O
+the	O
+presence	O
+of	O
+RNA	O
+agrees	O
+with	O
+the	O
+Py	O
+-	O
+tract	O
+-	O
+bound	O
+crystal	O
+structure	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+.	O
+	O
+Importantly	O
+,	O
+the	O
+majority	O
+of	O
+traces	O
+(∼	O
+70	O
+%)	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	O
+/	O
+Cy5	O
+)	O
+bound	O
+to	O
+the	O
+slide	O
+-	O
+tethered	O
+RNA	O
+lacked	O
+FRET	O
+fluctuations	O
+and	O
+predominately	O
+exhibited	O
+a	O
+∼	O
+0	O
+.	O
+45	O
+FRET	O
+value	O
+(	O
+for	O
+example	O
+,	O
+Fig	O
+.	O
+6g	O
+).	O
+	O
+The	O
+U2AF65	O
+structures	O
+and	O
+analyses	O
+presented	O
+here	O
+represent	O
+a	O
+successful	O
+step	O
+towards	O
+defining	O
+a	O
+molecular	O
+map	O
+of	O
+the	O
+3	O
+′	O
+splice	O
+site	O
+.	O
+	O
+The	O
+intact	O
+U2AF65	O
+RRM1	O
+/	O
+RRM2	O
+-	O
+containing	O
+domain	O
+and	O
+flanking	O
+residues	O
+are	O
+required	O
+for	O
+binding	O
+contiguous	O
+Py	O
+tracts	O
+.	O
+	O
+Structures	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+recognizing	O
+a	O
+contiguous	O
+Py	O
+tract	O
+.	O
+	O
+For	O
+clarity	O
+,	O
+we	O
+consistently	O
+number	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+nucleotide	O
+-	O
+binding	O
+sites	O
+from	O
+one	O
+to	O
+nine	O
+,	O
+although	O
+in	O
+some	O
+cases	O
+the	O
+co	O
+-	O
+crystallized	O
+oligonucleotide	O
+comprises	O
+eight	O
+nucleotides	O
+and	O
+as	O
+such	O
+leaves	O
+the	O
+first	O
+binding	O
+site	O
+empty	O
+.	O
+	O
+(	O
+b	O
+)	O
+Bar	O
+graph	O
+of	O
+apparent	O
+equilibrium	O
+affinities	O
+(	O
+KA	O
+)	O
+for	O
+the	O
+AdML	O
+Py	O
+tract	O
+(	O
+5	O
+′-	O
+CCCUUUUUUUUCC	O
+-	O
+3	O
+′)	O
+of	O
+the	O
+wild	O
+-	O
+type	O
+(	O
+blue	O
+)	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+protein	O
+compared	O
+with	O
+mutations	O
+of	O
+the	O
+residues	O
+shown	O
+in	O
+a	O
+:	O
+3Gly	B-mutant
+(	O
+yellow	O
+),	O
+5Gly	B-mutant
+(	O
+red	O
+),	O
+NLALA	B-mutant
+(	O
+hatched	O
+red	O
+),	O
+12Gly	B-mutant
+(	O
+orange	O
+)	O
+and	O
+the	O
+linker	O
+deletions	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+in	O
+the	O
+minimal	O
+RRM1	O
+–	O
+RRM2	O
+region	O
+(	O
+residues	O
+148	O
+–	O
+237	O
+,	O
+258	O
+–	O
+336	O
+)	O
+or	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+(	O
+residues	O
+141	O
+–	O
+237	O
+,	O
+258	O
+–	O
+342	O
+).	O
+	O
+The	O
+apparent	O
+equilibrium	O
+dissociation	O
+constants	O
+(	O
+KD	O
+)	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+mutant	O
+proteins	O
+are	O
+:	O
+wild	O
+type	O
+(	O
+WT	O
+),	O
+35	O
+±	O
+6	O
+nM	O
+;	O
+3Gly	B-mutant
+,	O
+47	O
+±	O
+4	O
+nM	O
+;	O
+5Gly	B-mutant
+,	O
+61	O
+±	O
+3	O
+nM	O
+;	O
+12Gly	B-mutant
+,	O
+88	O
+±	O
+21	O
+nM	O
+;	O
+NLALA	B-mutant
+,	O
+45	O
+±	O
+3	O
+nM	O
+;	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+123	O
+±	O
+5	O
+nM	O
+;	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+,	O
+5000	O
+±	O
+100	O
+nM	O
+;	O
+3Mut	B-mutant
+,	O
+5630	O
+±	O
+70	O
+nM	O
+.	O
+The	O
+average	O
+KA	O
+and	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+for	O
+three	O
+independent	O
+titrations	O
+are	O
+plotted	O
+.	O
+	O
+(	O
+a	O
+,	O
+b	O
+)	O
+Views	O
+of	O
+FRET	O
+pairs	O
+chosen	O
+to	O
+follow	O
+the	O
+relative	O
+movement	O
+of	O
+RRM1	O
+and	O
+RRM2	O
+on	O
+the	O
+crystal	O
+structure	O
+of	O
+‘	O
+side	O
+-	O
+by	O
+-	O
+side	O
+'	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRMs	O
+bound	O
+to	O
+a	O
+Py	O
+-	O
+tract	O
+oligonucleotide	O
+(	O
+a	O
+,	O
+representative	O
+structure	O
+iv	O
+)	O
+or	O
+‘	O
+closed	O
+'	O
+NMR	O
+/	O
+PRE	O
+-	O
+based	O
+model	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+(	O
+b	O
+,	O
+PDB	O
+ID	O
+2YH0	O
+)	O
+in	O
+identical	O
+orientations	O
+of	O
+RRM2	O
+.	O
+	O
+RNA	O
+protects	O
+a	O
+nucleoprotein	O
+complex	O
+against	O
+radiation	O
+damage	O
+	O
+The	O
+availability	O
+of	O
+two	O
+TRAP	O
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+of	O
+which	O
+only	O
+one	O
+contained	O
+bound	O
+RNA	O
+,	O
+allowed	O
+a	O
+controlled	O
+investigation	O
+into	O
+the	O
+exact	O
+role	O
+of	O
+RNA	O
+binding	O
+in	O
+protein	O
+specific	O
+damage	O
+susceptibility	O
+.	O
+	O
+With	O
+the	O
+wide	O
+use	O
+of	O
+high	O
+-	O
+flux	O
+third	O
+-	O
+generation	O
+synchrotron	O
+sources	O
+,	O
+radiation	O
+damage	O
+(	O
+RD	O
+)	O
+has	O
+once	O
+again	O
+become	O
+a	O
+dominant	O
+reason	O
+for	O
+the	O
+failure	O
+of	O
+structure	O
+determination	O
+using	O
+macromolecular	O
+crystallography	O
+(	O
+MX	O
+)	O
+in	O
+experiments	O
+conducted	O
+both	O
+at	O
+room	O
+temperature	O
+and	O
+under	O
+cryocooled	O
+conditions	O
+(	O
+100	O
+K	O
+).	O
+	O
+Specific	O
+radiation	O
+damage	O
+(	O
+SRD	O
+)	O
+is	O
+observed	O
+in	O
+the	O
+real	O
+-	O
+space	O
+electron	O
+density	O
+,	O
+and	O
+has	O
+been	O
+detected	O
+at	O
+much	O
+lower	O
+doses	O
+than	O
+any	O
+observable	O
+decay	O
+in	O
+the	O
+intensity	O
+of	O
+reflections	O
+.	O
+	O
+SRD	O
+has	O
+been	O
+well	O
+characterized	O
+in	O
+a	O
+large	O
+range	O
+of	O
+proteins	O
+,	O
+and	O
+is	O
+seen	O
+to	O
+follow	O
+a	O
+reproducible	O
+order	O
+:	O
+metallo	O
+-	O
+centre	O
+reduction	O
+,	O
+disulfide	O
+-	O
+bond	O
+cleavage	O
+,	O
+acidic	O
+residue	O
+decarboxylation	O
+and	O
+methionine	O
+methylthio	O
+cleavage	O
+(	O
+Ravelli	O
+&	O
+McSweeney	O
+,	O
+2000	O
+;	O
+Burmeister	O
+,	O
+2000	O
+;	O
+Weik	O
+et	O
+al	O
+.,	O
+2000	O
+;	O
+Yano	O
+et	O
+al	O
+.,	O
+2005	O
+).	O
+	O
+Understanding	O
+RD	O
+to	O
+such	O
+complexes	O
+is	O
+crucial	O
+,	O
+since	O
+DNA	O
+is	O
+rarely	O
+naked	O
+within	O
+a	O
+cell	O
+,	O
+instead	O
+dynamically	O
+interacting	O
+with	O
+proteins	O
+,	O
+facilitating	O
+replication	O
+,	O
+transcription	O
+,	O
+modification	O
+and	O
+DNA	O
+repair	O
+.	O
+	O
+As	O
+of	O
+early	O
+2016	O
+,	O
+>	O
+5400	O
+nucleoprotein	O
+complex	O
+structures	O
+have	O
+been	O
+deposited	O
+within	O
+the	O
+PDB	O
+,	O
+with	O
+91	O
+%	O
+solved	O
+by	O
+MX	O
+.	O
+	O
+Using	O
+newly	O
+developed	O
+methodology	O
+,	O
+we	O
+present	O
+a	O
+controlled	O
+SRD	O
+investigation	O
+at	O
+1	O
+.	O
+98	O
+Å	O
+resolution	O
+using	O
+a	O
+large	O
+(∼	O
+91	O
+kDa	O
+)	O
+crystalline	O
+protein	O
+–	O
+RNA	O
+complex	O
+:	O
+trp	O
+RNA	O
+-	O
+binding	O
+attenuation	O
+protein	O
+(	O
+TRAP	O
+)	O
+bound	O
+to	O
+a	O
+53	O
+bp	O
+RNA	O
+sequence	O
+(	O
+GAGUU	O
+)	O
+10GAG	O
+(	O
+PDB	O
+entry	O
+1gtf	O
+;	O
+Hopcroft	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+	O
+It	O
+binds	O
+with	O
+high	O
+affinity	O
+(	O
+K	O
+d	O
+≃	O
+1	O
+.	O
+0	O
+nM	O
+)	O
+to	O
+RNA	O
+segments	O
+containing	O
+11	O
+GAG	O
+/	O
+UAG	O
+triplets	O
+separated	O
+by	O
+two	O
+or	O
+three	O
+spacer	O
+nucleotides	O
+(	O
+Elliott	O
+et	O
+al	O
+.,	O
+2001	O
+)	O
+to	O
+regulate	O
+the	O
+transcription	O
+of	O
+tryptophan	O
+biosynthetic	O
+genes	O
+in	O
+Bacillus	O
+subtilis	O
+(	O
+Antson	O
+et	O
+al	O
+.,	O
+1999	O
+).	O
+	O
+Previous	O
+studies	O
+have	O
+characterized	O
+SRD	O
+sites	O
+by	O
+reporting	O
+magnitudes	O
+of	O
+F	O
+obs	O
+(	O
+d	O
+n	O
+)	O
+−	O
+F	O
+obs	O
+(	O
+d	O
+1	O
+)	O
+Fourier	O
+difference	O
+map	O
+peaks	O
+in	O
+terms	O
+of	O
+the	O
+sigma	O
+(	O
+σ	O
+)	O
+contour	O
+level	O
+(	O
+the	O
+number	O
+of	O
+standard	O
+deviations	O
+from	O
+the	O
+mean	O
+map	O
+electron	O
+-	O
+density	O
+value	O
+)	O
+at	O
+which	O
+peaks	O
+become	O
+visible	O
+.	O
+	O
+Visual	O
+inspection	O
+of	O
+Fourier	O
+difference	O
+maps	O
+illustrated	O
+the	O
+clear	O
+lack	O
+of	O
+RNA	O
+electron	O
+-	O
+density	O
+degradation	O
+with	O
+increasing	O
+dose	O
+compared	O
+with	O
+the	O
+obvious	O
+protein	O
+damage	O
+manifestations	O
+(	O
+Figs	O
+.	O
+3	O
+▸	O
+b	O
+and	O
+3	O
+▸	O
+c	O
+).	O
+	O
+For	O
+each	O
+TRAP	O
+ring	O
+subunit	O
+,	O
+the	O
+Glu36	O
+side	O
+-	O
+chain	O
+carboxyl	O
+group	O
+accepts	O
+a	O
+pair	O
+of	O
+hydrogen	O
+bonds	O
+from	O
+the	O
+two	O
+N	O
+atoms	O
+of	O
+the	O
+G3	O
+RNA	O
+base	O
+.	O
+	O
+With	O
+increasing	O
+dose	O
+,	O
+the	O
+D	O
+loss	O
+associated	O
+with	O
+the	O
+Phe32	O
+side	O
+chain	O
+was	O
+significantly	O
+reduced	O
+upon	O
+RNA	O
+binding	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+e	O
+;	O
+Phe32	O
+Cζ	O
+;	O
+p	O
+=	O
+0	O
+.	O
+0014	O
+),	O
+an	O
+indication	O
+that	O
+radiation	O
+-	O
+induced	O
+conformation	O
+disordering	O
+of	O
+Phe32	O
+had	O
+been	O
+reduced	O
+.	O
+	O
+The	O
+RNA	O
+was	O
+found	O
+to	O
+be	O
+substantially	O
+more	O
+radiation	O
+-	O
+resistant	O
+than	O
+the	O
+protein	O
+,	O
+even	O
+at	O
+the	O
+highest	O
+doses	O
+investigated	O
+(∼	O
+25	O
+.	O
+0	O
+MGy	O
+),	O
+which	O
+is	O
+in	O
+strong	O
+concurrence	O
+with	O
+our	O
+previous	O
+SRD	O
+investigation	O
+of	O
+the	O
+C	O
+.	O
+Esp1396I	O
+protein	O
+–	O
+DNA	O
+complex	O
+(	O
+Bury	O
+et	O
+al	O
+.,	O
+2015	O
+).	O
+	O
+Consistent	O
+with	O
+that	O
+study	O
+,	O
+at	O
+high	O
+doses	O
+of	O
+above	O
+∼	O
+20	O
+MGy	O
+,	O
+F	O
+obs	O
+(	O
+d	O
+n	O
+)	O
+−	O
+F	O
+obs	O
+(	O
+d	O
+1	O
+)	O
+map	O
+density	O
+was	O
+detected	O
+around	O
+P	O
+,	O
+O3	O
+′	O
+and	O
+O5	O
+′	O
+atoms	O
+of	O
+the	O
+RNA	O
+backbone	O
+,	O
+with	O
+no	O
+significant	O
+difference	O
+density	O
+localized	O
+to	O
+RNA	O
+ribose	O
+and	O
+basic	O
+subunits	O
+.	O
+	O
+This	O
+is	O
+in	O
+good	O
+agreement	O
+with	O
+previous	O
+mutagenesis	O
+and	O
+nucleoside	O
+analogue	O
+studies	O
+(	O
+Elliott	O
+et	O
+al	O
+.,	O
+2001	O
+),	O
+which	O
+indicated	O
+that	O
+the	O
+G1	O
+nucleotide	O
+does	O
+not	O
+bind	O
+to	O
+TRAP	O
+as	O
+strongly	O
+as	O
+do	O
+A2	O
+and	O
+G3	O
+,	O
+and	O
+plays	O
+little	O
+role	O
+in	O
+the	O
+high	O
+RNA	O
+-	O
+binding	O
+affinity	O
+of	O
+TRAP	O
+(	O
+K	O
+d	O
+≃	O
+1	O
+.	O
+1	O
+±	O
+0	O
+.	O
+4	O
+nM	O
+).	O
+	O
+The	O
+prevalence	O
+of	O
+radical	O
+attack	O
+from	O
+solvent	O
+channels	O
+surrounding	O
+the	O
+protein	O
+in	O
+the	O
+crystal	O
+is	O
+a	O
+questionable	O
+cause	O
+,	O
+considering	O
+previous	O
+observations	O
+indicating	O
+that	O
+the	O
+strongly	O
+oxidizing	O
+hydroxyl	O
+radical	O
+is	O
+immobile	O
+at	O
+100	O
+K	O
+(	O
+Allan	O
+et	O
+al	O
+.,	O
+2013	O
+;	O
+Owen	O
+et	O
+al	O
+.,	O
+2012	O
+).	O
+	O
+For	O
+example	O
+,	O
+Asp17	O
+is	O
+located	O
+∼	O
+6	O
+.	O
+8	O
+Å	O
+from	O
+the	O
+G1	O
+base	O
+,	O
+outside	O
+the	O
+RNA	O
+-	O
+binding	O
+interfaces	O
+,	O
+and	O
+has	O
+indistinguishable	O
+Cγ	O
+atom	O
+D	O
+loss	O
+dose	O
+-	O
+dynamics	O
+between	O
+RNA	O
+-	O
+bound	O
+and	O
+nonbound	O
+TRAP	O
+(	O
+p	O
+>	O
+0	O
+.	O
+9	O
+).	O
+	O
+This	O
+structure	O
+reveals	O
+the	O
+molecular	O
+mechanism	O
+underlying	O
+the	O
+docking	O
+interaction	O
+between	O
+MKP7	O
+and	O
+JNK1	O
+.	O
+	O
+On	O
+the	O
+basis	O
+of	O
+sequence	O
+similarity	O
+,	O
+substrate	O
+specificity	O
+and	O
+predominant	O
+subcellular	O
+localization	O
+,	O
+the	O
+MKP	O
+family	O
+can	O
+be	O
+further	O
+divided	O
+into	O
+three	O
+groups	O
+(	O
+Fig	O
+.	O
+1	O
+).	O
+	O
+In	O
+addition	O
+to	O
+the	O
+CD	O
+and	O
+KBD	O
+,	O
+MKP7	O
+has	O
+a	O
+long	O
+C	O
+-	O
+terminal	O
+region	O
+that	O
+contains	O
+both	O
+nuclear	O
+localization	O
+and	O
+export	O
+sequences	O
+by	O
+which	O
+MKP7	O
+shuttles	O
+between	O
+the	O
+nucleus	O
+and	O
+the	O
+cytoplasm	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+	O
+Thus	O
+,	O
+the	O
+kinetic	O
+data	O
+were	O
+analysed	O
+using	O
+the	O
+general	O
+initial	O
+velocity	O
+equation	O
+,	O
+taking	O
+substrate	O
+depletion	O
+into	O
+account	O
+:	O
+	O
+The	O
+overall	O
+folding	O
+of	O
+MKP7	O
+-	O
+CD	O
+is	O
+typical	O
+of	O
+DUSPs	O
+,	O
+with	O
+a	O
+central	O
+twisted	O
+five	O
+-	O
+stranded	O
+β	O
+-	O
+sheet	O
+surrounded	O
+by	O
+six	O
+α	O
+-	O
+helices	O
+.	O
+	O
+Since	O
+helix	O
+α0	O
+and	O
+the	O
+following	O
+loop	O
+α0	O
+–	O
+β1	O
+are	O
+known	O
+for	O
+a	O
+substrate	O
+-	O
+recognition	O
+motif	O
+of	O
+VHR	O
+and	O
+other	O
+phosphatases	O
+,	O
+the	O
+absence	O
+of	O
+these	O
+moieties	O
+implicates	O
+a	O
+different	O
+substrate	O
+-	O
+binding	O
+mode	O
+of	O
+MKP7	O
+.	O
+	O
+The	O
+MKP7	O
+-	O
+CD	O
+structure	O
+near	O
+the	O
+active	O
+site	O
+exhibits	O
+a	O
+typical	O
+active	O
+conformation	O
+as	O
+found	O
+in	O
+VHR	O
+and	O
+other	O
+PTPs	O
+.	O
+	O
+The	O
+catalytic	O
+residue	O
+,	O
+Cys244	O
+,	O
+is	O
+located	O
+just	O
+after	O
+strand	O
+β5	O
+and	O
+optimally	O
+positioned	O
+for	O
+nucleophilic	O
+attack	O
+.	O
+	O
+The	O
+carboxylate	O
+of	O
+Asp268	O
+in	O
+MKP7	O
+forms	O
+a	O
+salt	O
+bridge	O
+with	O
+side	O
+chain	O
+of	O
+Arg263	O
+in	O
+JNK1	O
+,	O
+and	O
+Lys275	O
+of	O
+MKP7	O
+forms	O
+a	O
+hydrogen	O
+bond	O
+and	O
+a	O
+salt	O
+bridge	O
+with	O
+Thr228	O
+and	O
+Asp229	O
+of	O
+JNK1	O
+,	O
+respectively	O
+.	O
+	O
+Gel	O
+filtration	O
+analysis	O
+further	O
+confirmed	O
+the	O
+key	O
+role	O
+of	O
+Phe285	O
+in	O
+the	O
+MKP7	O
+–	O
+JNK1	O
+interaction	O
+:	O
+no	O
+F285D	O
+–	O
+JNK1	O
+complex	O
+was	O
+detected	O
+when	O
+3	O
+molar	O
+equivalents	O
+of	O
+MKP7	O
+-	O
+CD	O
+(	O
+F285D	B-mutant
+)	O
+were	O
+mixed	O
+with	O
+1	O
+molar	O
+equivalent	O
+of	O
+JNK1	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+	O
+To	O
+determine	O
+whether	O
+the	O
+deficiencies	O
+in	O
+their	O
+abilities	O
+to	O
+bind	O
+partner	O
+proteins	O
+or	O
+carry	O
+out	O
+catalytic	O
+function	O
+are	O
+owing	O
+to	O
+misfolding	O
+of	O
+the	O
+purified	O
+mutant	O
+proteins	O
+,	O
+we	O
+also	O
+examined	O
+the	O
+folding	O
+properties	O
+of	O
+the	O
+JNK1	O
+and	O
+MKP7	O
+mutants	O
+with	O
+circular	O
+dichroism	O
+.	O
+	O
+The	O
+spectra	O
+of	O
+these	O
+mutants	O
+are	O
+similar	O
+to	O
+the	O
+wild	O
+-	O
+type	O
+proteins	O
+,	O
+indicating	O
+that	O
+these	O
+mutants	O
+fold	O
+as	O
+well	O
+as	O
+the	O
+wild	O
+-	O
+type	O
+proteins	O
+(	O
+Fig	O
+.	O
+4d	O
+,	O
+e	O
+).	O
+	O
+It	O
+has	O
+previously	O
+been	O
+reported	O
+that	O
+several	O
+cytosolic	O
+and	O
+inducible	O
+nuclear	O
+MKPs	O
+undergo	O
+catalytic	O
+activation	O
+upon	O
+interaction	O
+with	O
+the	O
+MAPK	O
+substrates	O
+.	O
+	O
+Incubation	O
+of	O
+MKP7	O
+with	O
+JNK1	O
+did	O
+not	O
+markedly	O
+stimulate	O
+the	O
+phosphatase	O
+activity	O
+,	O
+which	O
+is	O
+consistent	O
+with	O
+previous	O
+results	O
+that	O
+MKP7	O
+solely	O
+possesses	O
+the	O
+intrinsic	O
+activity	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+In	O
+addition	O
+,	O
+His230	O
+and	O
+Val256	O
+in	O
+JNK1	O
+are	O
+replaced	O
+by	O
+the	O
+negatively	O
+charged	O
+residues	O
+Glu208	O
+and	O
+Asp235	O
+in	O
+CDK2	O
+(	O
+Fig	O
+.	O
+5d	O
+),	O
+and	O
+the	O
+charge	O
+distribution	O
+on	O
+the	O
+CDK2	O
+interactive	O
+surface	O
+is	O
+quite	O
+different	O
+from	O
+that	O
+of	O
+JNK	O
+.	O
+	O
+These	O
+data	O
+indicated	O
+that	O
+a	O
+unique	O
+hydrophobic	O
+pocket	O
+formed	O
+between	O
+the	O
+MAPK	O
+insert	O
+and	O
+αG	O
+helix	O
+plays	O
+a	O
+major	O
+role	O
+in	O
+the	O
+substrate	O
+recognition	O
+by	O
+MKPs	O
+.	O
+	O
+The	O
+KBD	O
+of	O
+MKP5	O
+interacts	O
+with	O
+the	O
+D	O
+-	O
+site	O
+of	O
+p38α	O
+to	O
+mediate	O
+the	O
+enzyme	O
+–	O
+substrate	O
+interaction	O
+.	O
+	O
+The	O
+substrate	O
+specificity	O
+constant	O
+kcat	O
+/	O
+Km	O
+value	O
+for	O
+MKP5	O
+-	O
+CD	O
+was	O
+calculated	O
+as	O
+1	O
+.	O
+0	O
+×	O
+105	O
+M	O
+−	O
+1	O
+s	O
+−	O
+1	O
+,	O
+which	O
+is	O
+very	O
+close	O
+to	O
+that	O
+of	O
+MKP7	O
+-	O
+CD	O
+(	O
+1	O
+.	O
+07	O
+×	O
+105	O
+M	O
+−	O
+1	O
+s	O
+−	O
+1	O
+).	O
+	O
+Comparisons	O
+between	O
+catalytic	O
+domains	O
+structures	O
+of	O
+MKP5	O
+and	O
+MKP7	O
+reveal	O
+that	O
+the	O
+overall	O
+folds	O
+of	O
+the	O
+two	O
+proteins	O
+are	O
+highly	O
+similar	O
+,	O
+with	O
+only	O
+a	O
+few	O
+regions	O
+exhibiting	O
+small	O
+deviations	O
+(	O
+r	O
+.	O
+m	O
+.	O
+s	O
+.	O
+d	O
+.	O
+of	O
+0	O
+.	O
+79	O
+Å	O
+;	O
+Fig	O
+.	O
+7c	O
+).	O
+	O
+Given	O
+the	O
+distinct	O
+interaction	O
+mode	O
+revealed	O
+by	O
+the	O
+crystal	O
+structure	O
+of	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+,	O
+one	O
+obvious	O
+question	O
+is	O
+whether	O
+this	O
+is	O
+a	O
+general	O
+mechanism	O
+used	O
+by	O
+all	O
+members	O
+of	O
+the	O
+JNK	O
+-	O
+specific	O
+MKPs	O
+.	O
+	O
+Pull	O
+-	O
+down	O
+assays	O
+also	O
+confirmed	O
+the	O
+protein	O
+–	O
+protein	O
+interactions	O
+observed	O
+above	O
+.	O
+	O
+In	O
+addition	O
+,	O
+there	O
+were	O
+no	O
+significant	O
+differences	O
+in	O
+the	O
+CD	O
+spectra	O
+between	O
+wild	O
+-	O
+type	O
+and	O
+mutant	O
+proteins	O
+,	O
+indicating	O
+that	O
+the	O
+overall	O
+structures	O
+of	O
+these	O
+mutants	O
+did	O
+not	O
+change	O
+significantly	O
+from	O
+that	O
+of	O
+wild	O
+-	O
+type	O
+MKP5	O
+protein	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+In	O
+addition	O
+,	O
+the	O
+key	O
+interacting	O
+residues	O
+of	O
+MKP7	O
+-	O
+CD	O
+,	O
+Phe215	O
+,	O
+Leu267	O
+and	O
+Leu288	O
+,	O
+are	O
+replaced	O
+by	O
+less	O
+hydrophobic	O
+residues	O
+,	O
+Asn379	O
+,	O
+Met431	O
+and	O
+Met452	O
+in	O
+MKP5	O
+-	O
+CD	O
+(	O
+Fig	O
+.	O
+5c	O
+),	O
+respectively	O
+,	O
+which	O
+may	O
+result	O
+in	O
+weaker	O
+hydrophobic	O
+interactions	O
+between	O
+MKP5	O
+-	O
+CD	O
+and	O
+JNK1	O
+.	O
+	O
+The	O
+propagation	O
+of	O
+MAPK	O
+signals	O
+is	O
+attenuated	O
+through	O
+the	O
+actions	O
+of	O
+the	O
+MKPs	O
+.	O
+	O
+Most	O
+studies	O
+have	O
+focused	O
+on	O
+the	O
+dephosphorylation	O
+of	O
+MAPKs	O
+by	O
+phosphatases	O
+containing	O
+the	O
+‘	O
+kinase	O
+-	O
+interaction	O
+motif	O
+'	O
+(	O
+D	O
+-	O
+motif	O
+),	O
+including	O
+a	O
+group	O
+of	O
+DUSPs	O
+(	O
+MKPs	O
+)	O
+and	O
+a	O
+distinct	O
+subfamily	O
+of	O
+tyrosine	O
+phosphatases	O
+(	O
+HePTP	O
+,	O
+STEP	O
+and	O
+PTP	O
+-	O
+SL	O
+).	O
+	O
+In	O
+contrast	O
+to	O
+MKP5	O
+,	O
+removal	O
+of	O
+the	O
+KBD	O
+domain	O
+from	O
+MKP7	O
+does	O
+not	O
+drastically	O
+affect	O
+enzyme	O
+catalysis	O
+,	O
+and	O
+the	O
+kinetic	O
+parameters	O
+of	O
+MKP7	O
+-	O
+CD	O
+for	O
+p38α	O
+substrate	O
+are	O
+very	O
+similar	O
+to	O
+those	O
+for	O
+JNK1	O
+substrate	O
+.	O
+	O
+The	O
+MKP7	O
+-	O
+KBD	O
+docks	O
+to	O
+the	O
+D	O
+-	O
+site	O
+located	O
+on	O
+the	O
+back	O
+side	O
+of	O
+the	O
+p38α	O
+catalytic	O
+pocket	O
+for	O
+high	O
+-	O
+affinity	O
+association	O
+,	O
+whereas	O
+the	O
+interaction	O
+of	O
+the	O
+MKP7	O
+-	O
+CD	O
+with	O
+another	O
+p38α	O
+structural	O
+region	O
+,	O
+which	O
+is	O
+close	O
+to	O
+the	O
+activation	O
+loop	O
+,	O
+may	O
+not	O
+only	O
+stabilize	O
+binding	O
+but	O
+also	O
+provide	O
+contacts	O
+crucial	O
+for	O
+organizing	O
+the	O
+MKP7	O
+active	O
+site	O
+with	O
+respect	O
+to	O
+the	O
+phosphoreceptor	O
+in	O
+the	O
+p38α	O
+substrate	O
+for	O
+efficient	O
+dephosphorylation	O
+.	O
+	O
+This	O
+hydrophobic	O
+site	O
+was	O
+first	O
+identified	O
+by	O
+changes	O
+in	O
+deuterium	O
+exchange	O
+profiles	O
+,	O
+and	O
+is	O
+near	O
+the	O
+MAPK	O
+insertion	O
+and	O
+helix	O
+αG	O
+.	O
+Interestingly	O
+,	O
+many	O
+of	O
+the	O
+equivalent	O
+residues	O
+in	O
+JNK1	O
+,	O
+important	O
+for	O
+MKP7	O
+-	O
+CD	O
+recognition	O
+,	O
+are	O
+also	O
+used	O
+for	O
+substrate	O
+binding	O
+by	O
+ERK2	O
+(	O
+ref	O
+.),	O
+indicating	O
+that	O
+this	O
+site	O
+is	O
+overlapped	O
+with	O
+the	O
+DEF	O
+-	O
+site	O
+previously	O
+identified	O
+in	O
+ERK2	O
+(	O
+Fig	O
+.	O
+5d	O
+).	O
+	O
+Therefore	O
+,	O
+it	O
+is	O
+tempting	O
+to	O
+speculate	O
+that	O
+the	O
+catalytic	O
+domain	O
+of	O
+MKP3	O
+may	O
+bind	O
+to	O
+ERK2	O
+in	O
+a	O
+manner	O
+analogous	O
+to	O
+the	O
+way	O
+by	O
+which	O
+MKP7	O
+-	O
+CD	O
+binds	O
+to	O
+JNK1	O
+.	O
+	O
+The	O
+ongoing	O
+work	O
+demonstrates	O
+that	O
+although	O
+the	O
+overall	O
+interaction	O
+modes	O
+are	O
+similar	O
+between	O
+the	O
+JNK1	O
+–	O
+MKP7	O
+-	O
+CD	O
+and	O
+ERK2	O
+–	O
+MKP3	O
+-	O
+CD	O
+complexes	O
+,	O
+the	O
+ERK2	O
+–	O
+MKP3	O
+-	O
+CD	O
+interaction	O
+is	O
+less	O
+extensive	O
+and	O
+helix	O
+α4	O
+from	O
+MKP3	O
+-	O
+CD	O
+does	O
+not	O
+interact	O
+directly	O
+with	O
+ERK2	O
+.	O
+	O
+Phe285	O
+is	O
+essential	O
+for	O
+JNK1	O
+substrate	O
+binding	O
+,	O
+whereas	O
+Phe287	O
+plays	O
+a	O
+role	O
+for	O
+the	O
+precise	O
+alignment	O
+of	O
+active	O
+-	O
+site	O
+residues	O
+,	O
+which	O
+are	O
+important	O
+for	O
+transition	O
+-	O
+state	O
+stabilization	O
+.	O
+	O
+(	O
+a	O
+)	O
+Domain	O
+organization	O
+of	O
+human	O
+MKP7	O
+and	O
+JNK1	O
+.	O
+	O
+The	O
+2Fo	O
+−	O
+Fc	O
+omit	O
+map	O
+(	O
+contoured	O
+at	O
+1	O
+.	O
+5σ	O
+)	O
+for	O
+the	O
+P	O
+-	O
+loop	O
+of	O
+MKP7	O
+-	O
+CD	O
+is	O
+shown	O
+at	O
+inset	O
+of	O
+b	O
+.	O
+(	O
+c	O
+)	O
+Structure	O
+of	O
+VHR	O
+with	O
+its	O
+active	O
+site	O
+highlighted	O
+in	O
+marine	O
+blue	O
+.	O
+(	O
+d	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+JNK1	O
+–	O
+MKP7	O
+interface	O
+showing	O
+interacting	O
+amino	O
+acids	O
+of	O
+JNK1	O
+(	O
+orange	O
+)	O
+and	O
+MKP7	O
+-	O
+CD	O
+(	O
+cyan	O
+).	O
+	O
+MKP7	O
+-	O
+CD	O
+is	O
+shown	O
+in	O
+surface	O
+representation	O
+coloured	O
+according	O
+to	O
+electrostatic	O
+potential	O
+(	O
+positive	O
+,	O
+blue	O
+;	O
+negative	O
+,	O
+red	O
+).	O
+	O
+Mutational	O
+analysis	O
+on	O
+interactions	O
+between	O
+MKP7	O
+-	O
+CD	O
+and	O
+JNK1	O
+.	O
+	O
+However	O
+,	O
+in	O
+contrast	O
+to	O
+the	O
+wild	O
+-	O
+type	O
+MKP7	O
+-	O
+CD	O
+,	O
+mutant	O
+F285D	B-mutant
+did	O
+not	O
+co	O
+-	O
+migrate	O
+with	O
+JNK1	O
+.	O
+	O
+(	O
+c	O
+)	O
+Pull	O
+-	O
+down	O
+assays	O
+of	O
+MKP7	O
+-	O
+CD	O
+by	O
+GST	O
+-	O
+tagged	O
+JNK1	O
+mutants	O
+.	O
+	O
+Measurements	O
+were	O
+averaged	O
+for	O
+three	O
+scans	O
+.	O
+(	O
+e	O
+)	O
+Circular	O
+dichroism	O
+spectra	O
+for	O
+JNK1	O
+wild	O
+type	O
+and	O
+mutants	O
+.	O
+	O
+The	O
+N	O
+-	O
+lobe	O
+and	O
+C	O
+-	O
+lobe	O
+of	O
+CDK2	O
+are	O
+coloured	O
+in	O
+grey	O
+and	O
+pink	O
+,	O
+respectively	O
+,	O
+and	O
+KAP	O
+is	O
+coloured	O
+in	O
+green	O
+.	O
+	O
+Interestingly	O
+,	O
+the	O
+recognition	O
+of	O
+CDK2	O
+by	O
+KAP	O
+is	O
+augmented	O
+by	O
+a	O
+similar	O
+interface	O
+as	O
+that	O
+observed	O
+in	O
+the	O
+complex	O
+of	O
+JNK1	O
+and	O
+MKP7	O
+-	O
+CD	O
+(	O
+region	O
+II	O
+).	O
+	O
+One	O
+remarkable	O
+difference	O
+between	O
+these	O
+two	O
+kinase	O
+-	O
+phosphatase	O
+complexes	O
+is	O
+that	O
+helix	O
+α6	O
+of	O
+KAP	O
+(	O
+corresponding	O
+to	O
+helix	O
+α4	O
+of	O
+MKP7	O
+-	O
+CD	O
+)	O
+plays	O
+little	O
+,	O
+if	O
+any	O
+,	O
+role	O
+in	O
+the	O
+formation	O
+of	O
+a	O
+stable	O
+heterodimer	O
+of	O
+CDK2	O
+and	O
+KAP	O
+.	O
+(	O
+c	O
+)	O
+Sequence	O
+alignment	O
+of	O
+the	O
+JNK	O
+-	O
+interacting	O
+regions	O
+on	O
+MKPs	O
+.	O
+	O
+(	O
+d	O
+)	O
+F	O
+-	O
+site	O
+is	O
+required	O
+for	O
+JNK1	O
+to	O
+interact	O
+with	O
+MKP7	O
+.	O
+	O
+(	O
+e	O
+)	O
+Effect	O
+of	O
+MKP7	O
+(	O
+wild	O
+type	O
+or	O
+mutants	O
+)	O
+expression	O
+on	O
+ultraviolet	O
+-	O
+induced	O
+apoptosis	O
+.	O
+	O
+(	O
+f	O
+)	O
+Statistical	O
+analysis	O
+of	O
+apoptotic	O
+cells	O
+(	O
+mean	O
+±	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.,	O
+n	O
+=	O
+3	O
+),	O
+*	O
+P	O
+<	O
+0	O
+.	O
+05	O
+,	O
+***	O
+P	O
+<	O
+0	O
+.	O
+001	O
+(	O
+ANOVA	O
+followed	O
+by	O
+Tukey	O
+'	O
+s	O
+test	O
+).	O
+	O
+The	O
+error	O
+bars	O
+represent	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+(	O
+c	O
+)	O
+Structural	O
+comparison	O
+of	O
+the	O
+JNK	O
+-	O
+interacting	O
+residues	O
+on	O
+MKP5	O
+-	O
+CD	O
+(	O
+PDB	O
+1ZZW	O
+)	O
+and	O
+MKP7	O
+-	O
+CD	O
+.	O
+	O
+Mechanistic	O
+insight	O
+into	O
+a	O
+peptide	O
+hormone	O
+signaling	O
+complex	O
+mediating	O
+floral	O
+organ	O
+abscission	O
+	O
+It	O
+is	O
+unknown	O
+how	O
+expression	O
+of	O
+IDA	O
+in	O
+the	O
+abscission	O
+zone	O
+leads	O
+to	O
+HAESA	O
+activation	O
+.	O
+	O
+The	O
+HAESA	O
+co	O
+-	O
+receptor	O
+SERK1	O
+,	O
+a	O
+positive	O
+regulator	O
+of	O
+the	O
+floral	O
+abscission	O
+pathway	O
+,	O
+allows	O
+for	O
+high	O
+-	O
+affinity	O
+sensing	O
+of	O
+the	O
+peptide	O
+hormone	O
+by	O
+binding	O
+to	O
+an	O
+Arg	O
+-	O
+His	O
+-	O
+Asn	O
+motif	O
+in	O
+IDA	O
+.	O
+	O
+Plants	O
+can	O
+shed	O
+their	O
+leaves	O
+,	O
+flowers	O
+or	O
+other	O
+organs	O
+when	O
+they	O
+no	O
+longer	O
+need	O
+them	O
+.	O
+But	O
+how	O
+does	O
+a	O
+leaf	O
+or	O
+a	O
+flower	O
+know	O
+when	O
+to	O
+let	O
+go	O
+?	O
+A	O
+receptor	O
+protein	O
+called	O
+HAESA	O
+is	O
+found	O
+on	O
+the	O
+surface	O
+of	O
+the	O
+cells	O
+that	O
+surround	O
+a	O
+future	O
+break	O
+point	O
+on	O
+the	O
+plant	O
+.	O
+When	O
+its	O
+time	O
+to	O
+shed	O
+an	O
+organ	O
+,	O
+a	O
+hormone	O
+called	O
+IDA	O
+instructs	O
+HAESA	O
+to	O
+trigger	O
+the	O
+shedding	O
+process	O
+.	O
+	O
+Cysteine	O
+residues	O
+engaged	O
+in	O
+disulphide	O
+bonds	O
+are	O
+depicted	O
+in	O
+green	O
+.	O
+	O
+IDA	O
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+surface	O
+view	O
+included	O
+)	O
+is	O
+depicted	O
+in	O
+yellow	O
+.	O
+	O
+Hydrogren	O
+bonds	O
+are	O
+depicted	O
+as	O
+dotted	O
+lines	O
+(	O
+in	O
+magenta	O
+),	O
+a	O
+water	O
+molecule	O
+is	O
+shown	O
+as	O
+a	O
+red	O
+sphere	O
+.	O
+	O
+Abscission	O
+of	O
+floral	O
+organs	O
+in	O
+Arabidopsis	O
+is	O
+a	O
+model	O
+system	O
+to	O
+study	O
+these	O
+cell	O
+separation	O
+processes	O
+in	O
+molecular	O
+detail	O
+.	O
+	O
+This	O
+sequence	O
+motif	O
+is	O
+highly	O
+conserved	O
+among	O
+IDA	O
+family	O
+members	O
+(	O
+IDA	O
+-	O
+LIKE	O
+PROTEINS	O
+,	O
+IDLs	O
+)	O
+and	O
+contains	O
+a	O
+central	O
+Pro	O
+residue	O
+,	O
+presumed	O
+to	O
+be	O
+post	O
+-	O
+translationally	O
+modified	O
+to	O
+hydroxyproline	O
+(	O
+Hyp	O
+;	O
+Figure	O
+1A	O
+).	O
+	O
+The	O
+available	O
+genetic	O
+and	O
+biochemical	O
+evidence	O
+suggests	O
+that	O
+IDA	O
+and	O
+HAESA	O
+together	O
+control	O
+floral	O
+abscission	O
+,	O
+but	O
+it	O
+is	O
+poorly	O
+understood	O
+if	O
+IDA	O
+is	O
+directly	O
+sensed	O
+by	O
+the	O
+receptor	O
+kinase	O
+HAESA	O
+and	O
+how	O
+IDA	O
+binding	O
+at	O
+the	O
+cell	O
+surface	O
+would	O
+activate	O
+the	O
+receptor	O
+.	O
+	O
+Close	O
+-	O
+up	O
+views	O
+of	O
+(	O
+A	O
+)	O
+IDA	O
+,	O
+(	O
+B	O
+)	O
+the	O
+N	O
+-	O
+terminally	O
+extended	O
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+and	O
+(	O
+C	O
+)	O
+IDL1	O
+bound	O
+to	O
+the	O
+HAESA	O
+hormone	O
+binding	O
+pocket	O
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+yellow	O
+)	O
+and	O
+including	O
+simulated	O
+annealing	O
+2Fo	O
+–	O
+Fc	O
+omit	O
+electron	O
+density	O
+maps	O
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+.	O
+	O
+(	O
+B	O
+)	O
+Analytical	O
+size	O
+-	O
+exclusion	O
+chromatography	O
+.	O
+	O
+A	O
+SDS	O
+PAGE	O
+of	O
+the	O
+peak	O
+fractions	O
+is	O
+shown	O
+alongside	O
+.	O
+	O
+Purified	O
+HAESA	O
+and	O
+SERK1	O
+are	O
+~	O
+75	O
+and	O
+~	O
+28	O
+kDa	O
+,	O
+respectively	O
+.	O
+(	O
+C	O
+)	O
+Isothermal	O
+titration	O
+calorimetry	O
+of	O
+wild	O
+-	O
+type	O
+and	O
+Hyp64	O
+→	O
+Pro	O
+IDA	O
+versus	O
+the	O
+HAESA	O
+and	O
+SERK1	O
+ectodomains	O
+.	O
+	O
+The	O
+titration	O
+of	O
+IDA	O
+wild	O
+-	O
+type	O
+versus	O
+the	O
+isolated	O
+HAESA	O
+ectodomain	O
+from	O
+Figure	O
+1B	O
+is	O
+shown	O
+for	O
+comparison	O
+(	O
+red	O
+line	O
+;	O
+n	O
+.	O
+d	O
+.	O
+	O
+We	O
+purified	O
+the	O
+HAESA	O
+ectodomain	O
+(	O
+residues	O
+20	O
+–	O
+620	O
+)	O
+from	O
+baculovirus	O
+-	O
+infected	O
+insect	O
+cells	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1A	O
+,	O
+see	O
+Materials	O
+and	O
+methods	O
+)	O
+and	O
+quantified	O
+the	O
+interaction	O
+of	O
+the	O
+~	O
+75	O
+kDa	O
+glycoprotein	O
+with	O
+synthetic	O
+IDA	O
+peptides	O
+using	O
+isothermal	O
+titration	O
+calorimetry	O
+(	O
+ITC	O
+).	O
+	O
+IDA	O
+binds	O
+in	O
+a	O
+completely	O
+extended	O
+conformation	O
+along	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+HAESA	O
+ectodomain	O
+,	O
+covering	O
+LRRs	O
+2	O
+–	O
+14	O
+(	O
+Figure	O
+1C	O
+,	O
+D	O
+,	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+	O
+Other	O
+hydrophobic	O
+and	O
+polar	O
+interactions	O
+are	O
+mediated	O
+by	O
+Ser62IDA	O
+,	O
+Ser65IDA	O
+and	O
+by	O
+backbone	O
+atoms	O
+along	O
+the	O
+IDA	O
+peptide	O
+(	O
+Figure	O
+1D	O
+,	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2A	O
+–	O
+C	O
+).	O
+	O
+Consistently	O
+,	O
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+and	O
+IDA	O
+have	O
+similar	O
+binding	O
+affinities	O
+in	O
+our	O
+ITC	O
+assays	O
+,	O
+further	O
+indicating	O
+that	O
+HAESA	O
+senses	O
+a	O
+dodecamer	O
+peptide	O
+comprising	O
+residues	O
+58	O
+-	O
+69IDA	O
+(	O
+Figure	O
+2D	O
+).	O
+	O
+Replacing	O
+Hyp64IDA	O
+,	O
+which	O
+is	O
+common	O
+to	O
+all	O
+IDLs	O
+,	O
+with	O
+proline	O
+impairs	O
+the	O
+interaction	O
+with	O
+the	O
+receptor	O
+,	O
+as	O
+does	O
+the	O
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+double	O
+-	O
+mutant	O
+discussed	O
+below	O
+(	O
+Figure	O
+1A	O
+,	O
+2D	O
+).	O
+	O
+Our	O
+binding	O
+assays	O
+reveal	O
+that	O
+IDA	O
+family	O
+peptides	O
+are	O
+sensed	O
+by	O
+the	O
+isolated	O
+HAESA	O
+ectodomain	O
+with	O
+relatively	O
+weak	O
+binding	O
+affinities	O
+(	O
+Figures	O
+1B	O
+,	O
+2A	O
+–	O
+D	O
+).	O
+	O
+The	O
+serk2	O
+-	O
+2	O
+,	O
+serk3	O
+-	O
+1	O
+,	O
+serk4	O
+-	O
+1	O
+and	O
+serk5	O
+-	O
+1	O
+mutant	O
+lines	O
+showed	O
+a	O
+petal	O
+break	O
+-	O
+strength	O
+profile	O
+not	O
+significantly	O
+different	O
+from	O
+wild	O
+-	O
+type	O
+plants	O
+.	O
+	O
+In	O
+vitro	O
+,	O
+the	O
+LRR	O
+ectodomain	O
+of	O
+SERK1	O
+(	O
+residues	O
+24	O
+–	O
+213	O
+)	O
+forms	O
+stable	O
+,	O
+IDA	O
+-	O
+dependent	O
+heterodimeric	O
+complexes	O
+with	O
+HAESA	O
+in	O
+size	O
+exclusion	O
+chromatography	O
+experiments	O
+(	O
+Figure	O
+3B	O
+).	O
+	O
+We	O
+found	O
+that	O
+HAESA	O
+senses	O
+IDA	O
+with	O
+a	O
+~	O
+60	O
+fold	O
+higher	O
+binding	O
+affinity	O
+in	O
+the	O
+presence	O
+of	O
+SERK1	O
+,	O
+suggesting	O
+that	O
+SERK1	O
+is	O
+involved	O
+in	O
+the	O
+specific	O
+recognition	O
+of	O
+the	O
+peptide	O
+hormone	O
+(	O
+Figure	O
+3C	O
+).	O
+	O
+Importantly	O
+,	O
+hydroxyprolination	O
+of	O
+IDA	O
+is	O
+critical	O
+for	O
+HAESA	O
+-	O
+IDA	O
+-	O
+SERK1	O
+complex	O
+formation	O
+(	O
+Figure	O
+3C	O
+,	O
+D	O
+).	O
+	O
+Upon	O
+IDA	O
+binding	O
+at	O
+the	O
+cell	O
+surface	O
+,	O
+the	O
+kinase	O
+domains	O
+of	O
+HAESA	O
+and	O
+SERK1	O
+,	O
+which	O
+have	O
+been	O
+shown	O
+to	O
+be	O
+active	O
+protein	O
+kinases	O
+,	O
+may	O
+interact	O
+in	O
+the	O
+cytoplasm	O
+to	O
+activate	O
+each	O
+other	O
+.	O
+	O
+Crystal	O
+structure	O
+of	O
+a	O
+HAESA	O
+–	O
+IDA	O
+–	O
+SERK1	O
+signaling	O
+complex	O
+.	O
+	O
+Polar	O
+interactions	O
+are	O
+highlighted	O
+as	O
+dotted	O
+lines	O
+(	O
+in	O
+magenta	O
+).	O
+	O
+(	O
+A	O
+)	O
+Size	O
+exclusion	O
+chromatography	O
+experiments	O
+similar	O
+to	O
+Figure	O
+3B	O
+,	O
+D	O
+reveal	O
+that	O
+IDA	O
+mutant	O
+peptides	O
+targeting	O
+the	O
+C	O
+-	O
+terminal	O
+motif	O
+do	O
+not	O
+form	O
+biochemically	O
+stable	O
+HAESA	O
+-	O
+IDA	O
+-	O
+SERK1	O
+complexes	O
+.	O
+	O
+We	O
+over	O
+-	O
+expressed	O
+full	O
+-	O
+length	O
+wild	O
+-	O
+type	O
+IDA	O
+or	O
+this	O
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+double	O
+-	O
+mutant	O
+to	O
+similar	O
+levels	O
+in	O
+Col	O
+-	O
+0	O
+Arabidopsis	O
+plants	O
+(	O
+Figure	O
+5D	O
+).	O
+	O
+We	O
+found	O
+that	O
+over	O
+-	O
+expression	O
+of	O
+wild	O
+-	O
+type	O
+IDA	O
+leads	O
+to	O
+early	O
+floral	O
+abscission	O
+and	O
+an	O
+enlargement	O
+of	O
+the	O
+abscission	O
+zone	O
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+	O
+It	O
+will	O
+be	O
+thus	O
+interesting	O
+to	O
+see	O
+if	O
+proteolytic	O
+processing	O
+of	O
+full	O
+-	O
+length	O
+IDA	O
+in	O
+vivo	O
+is	O
+regulated	O
+in	O
+a	O
+cell	O
+-	O
+type	O
+or	O
+tissue	O
+-	O
+specific	O
+manner	O
+.	O
+	O
+This	O
+observation	O
+is	O
+consistent	O
+with	O
+our	O
+complex	O
+structure	O
+in	O
+which	O
+receptor	O
+and	O
+co	O
+-	O
+receptor	O
+together	O
+form	O
+the	O
+IDA	O
+binding	O
+pocket	O
+.	O
+	O
+In	O
+addition	O
+,	O
+residues	O
+53	O
+-	O
+55SERK1	O
+from	O
+the	O
+SERK1	O
+N	O
+-	O
+terminal	O
+cap	O
+mediate	O
+specific	O
+interactions	O
+with	O
+the	O
+IDA	O
+peptide	O
+(	O
+Figures	O
+4C	O
+,	O
+6B	O
+).	O
+	O
+Different	O
+plant	O
+peptide	O
+hormone	O
+families	O
+contain	O
+a	O
+C	O
+-	O
+terminal	O
+(	O
+Arg	O
+)-	O
+His	O
+-	O
+Asn	O
+motif	O
+,	O
+which	O
+in	O
+IDA	O
+represents	O
+the	O
+co	O
+-	O
+receptor	O
+recognition	O
+site	O
+.	O
+	O
+Importantly	O
+,	O
+this	O
+motif	O
+can	O
+also	O
+be	O
+found	O
+in	O
+other	O
+peptide	O
+hormone	O
+families	O
+(	O
+Figure	O
+7	O
+).	O
+	O
+Using	O
+electron	O
+cryo	O
+-	O
+microscopy	O
+of	O
+a	O
+single	O
+specimen	O
+,	O
+we	O
+present	O
+five	O
+ribosome	O
+structures	O
+formed	O
+with	O
+the	O
+Taura	O
+syndrome	O
+virus	O
+IRES	O
+and	O
+translocase	O
+eEF2	O
+•	O
+GTP	O
+bound	O
+with	O
+sordarin	O
+.	O
+	O
+The	O
+structures	O
+suggest	O
+missing	O
+links	O
+in	O
+our	O
+understanding	O
+of	O
+tRNA	O
+translocation	O
+.	O
+	O
+To	O
+this	O
+end	O
+,	O
+internal	O
+ribosome	O
+entry	O
+site	O
+(	O
+IRES	O
+)	O
+RNAs	O
+are	O
+employed	O
+(	O
+reviewed	O
+in	O
+.	O
+	O
+An	O
+unusual	O
+strategy	O
+of	O
+initiation	O
+is	O
+used	O
+by	O
+intergenic	O
+-	O
+region	O
+(	O
+IGR	O
+)	O
+IRESs	O
+found	O
+in	O
+Dicistroviridae	O
+arthropod	O
+-	O
+infecting	O
+viruses	O
+.	O
+	O
+These	O
+include	O
+shrimp	O
+-	O
+infecting	O
+Taura	O
+syndrome	O
+virus	O
+(	O
+TSV	O
+),	O
+and	O
+insect	O
+viruses	O
+Plautia	O
+stali	O
+intestine	O
+virus	O
+(	O
+PSIV	O
+)	O
+and	O
+Cricket	O
+paralysis	O
+virus	O
+(	O
+CrPV	O
+).	O
+	O
+A	O
+recent	O
+demonstration	O
+of	O
+bacterial	O
+translation	O
+initiation	O
+by	O
+an	O
+IGR	O
+IRES	O
+indicates	O
+that	O
+the	O
+IRESs	O
+take	O
+advantage	O
+of	O
+conserved	O
+structural	O
+and	O
+dynamic	O
+properties	O
+of	O
+the	O
+ribosome	O
+.	O
+	O
+For	O
+a	O
+cognate	O
+aminoacyl	O
+-	O
+tRNA	O
+to	O
+bind	O
+the	O
+first	O
+viral	O
+mRNA	O
+codon	O
+,	O
+PKI	O
+has	O
+to	O
+be	O
+translocated	O
+from	O
+the	O
+A	O
+site	O
+,	O
+so	O
+that	O
+the	O
+first	O
+codon	O
+can	O
+be	O
+presented	O
+in	O
+the	O
+A	O
+site	O
+.	O
+	O
+First	O
+,	O
+studies	O
+of	O
+bacterial	O
+ribosomes	O
+showed	O
+that	O
+a	O
+~	O
+10	O
+°	O
+rotation	O
+of	O
+the	O
+small	O
+subunit	O
+relative	O
+to	O
+the	O
+large	O
+subunit	O
+,	O
+known	O
+as	O
+intersubunit	O
+rotation	O
+,	O
+or	O
+ratcheting	O
+,	O
+is	O
+required	O
+for	O
+translocation	O
+.	O
+	O
+Schematic	O
+of	O
+cryo	O
+-	O
+EM	O
+refinement	O
+and	O
+classification	O
+procedures	O
+.	O
+	O
+All	O
+particles	O
+were	O
+initially	O
+aligned	O
+to	O
+a	O
+single	O
+model	O
+.	O
+	O
+All	O
+measurements	O
+are	O
+relative	O
+to	O
+the	O
+non	O
+-	O
+rotated	O
+80S	O
+•	O
+2tRNA	O
+•	O
+mRNA	O
+structure	O
+.	O
+	O
+This	O
+approach	O
+revealed	O
+five	O
+80S	O
+•	O
+IRES	O
+•	O
+eEF2	O
+•	O
+GDP	O
+structures	O
+at	O
+average	O
+resolutions	O
+of	O
+3	O
+.	O
+5	O
+to	O
+4	O
+.	O
+2	O
+Å	O
+,	O
+sufficient	O
+to	O
+locate	O
+IRES	O
+domains	O
+and	O
+to	O
+resolve	O
+individual	O
+residues	O
+in	O
+the	O
+core	O
+regions	O
+of	O
+the	O
+ribosome	O
+and	O
+eEF2	O
+(	O
+Figures	O
+3c	O
+,	O
+d	O
+,	O
+and	O
+5f	O
+,	O
+h	O
+;	O
+see	O
+also	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2	O
+and	O
+Figure	O
+5	O
+—	O
+figure	O
+supplement	O
+2	O
+),	O
+including	O
+the	O
+post	O
+-	O
+translational	O
+modification	O
+diphthamide	O
+699	O
+(	O
+Figure	O
+3c	O
+).	O
+	O
+The	O
+views	O
+were	O
+obtained	O
+by	O
+structural	O
+alignment	O
+of	O
+the	O
+25S	O
+rRNAs	O
+;	O
+the	O
+sarcin	O
+-	O
+ricin	O
+loop	O
+(	O
+SRL	O
+)	O
+of	O
+25S	O
+rRNA	O
+is	O
+shown	O
+in	O
+gray	O
+for	O
+reference	O
+.	O
+	O
+(	O
+c	O
+)	O
+Comparison	O
+of	O
+the	O
+40S	O
+conformations	O
+in	O
+Structures	O
+I	O
+through	O
+V	O
+shows	O
+distinct	O
+positions	O
+of	O
+the	O
+head	O
+relative	O
+to	O
+the	O
+body	O
+of	O
+the	O
+40S	O
+subunit	O
+(	O
+head	O
+swivel	O
+).	O
+	O
+Conformation	O
+of	O
+the	O
+non	O
+-	O
+swiveled	O
+40S	O
+subunit	O
+in	O
+the	O
+S	O
+.	O
+cerevisiae	O
+80S	O
+ribosome	O
+bound	O
+with	O
+two	O
+tRNAs	O
+is	O
+shown	O
+for	O
+reference	O
+(	O
+blue	O
+).	O
+	O
+The	O
+central	O
+protuberance	O
+(	O
+CP	O
+)	O
+is	O
+labeled	O
+.	O
+	O
+Structures	O
+II	O
+and	O
+III	O
+are	O
+in	O
+mid	O
+-	O
+rotation	O
+conformations	O
+(~	O
+5	O
+°).	O
+	O
+IRES	O
+rearrangements	O
+	O
+Position	O
+and	O
+interactions	O
+of	O
+loop	O
+3	O
+(	O
+variable	O
+loop	O
+region	O
+)	O
+of	O
+the	O
+PKI	O
+domain	O
+in	O
+Structure	O
+V	O
+(	O
+this	O
+work	O
+)	O
+resembles	O
+those	O
+of	O
+the	O
+anticodon	O
+stem	O
+loop	O
+of	O
+the	O
+E	O
+-	O
+site	O
+tRNA	O
+(	O
+blue	O
+)	O
+in	O
+the	O
+80S	O
+•	O
+2tRNA	O
+•	O
+mRNA	O
+complex	O
+.	O
+	O
+Structures	O
+of	O
+translocation	O
+complexes	O
+of	O
+the	O
+bacterial	O
+70S	O
+ribosome	O
+bound	O
+with	O
+two	O
+tRNAs	O
+and	O
+yeast	O
+80S	O
+complexes	O
+with	O
+tRNAs	O
+are	O
+shown	O
+in	O
+the	O
+upper	O
+row	O
+and	O
+labeled	O
+.	O
+	O
+Positions	O
+of	O
+the	O
+IRES	O
+relative	O
+to	O
+eEF2	O
+and	O
+elements	O
+of	O
+the	O
+ribosome	O
+in	O
+Structures	O
+I	O
+through	O
+V	O
+.	O
+	O
+The	O
+minor	O
+groove	O
+of	O
+SL5	O
+(	O
+at	O
+nt	O
+6862	O
+–	O
+6868	O
+)	O
+contacts	O
+the	O
+positively	O
+charged	O
+region	O
+of	O
+eS25	O
+(	O
+R49	O
+,	O
+R58	O
+and	O
+R68	O
+)	O
+(	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+4	O
+).	O
+	O
+The	O
+view	O
+was	O
+obtained	O
+by	O
+structural	O
+alignment	O
+of	O
+the	O
+body	O
+domains	O
+of	O
+18S	O
+rRNAs	O
+of	O
+the	O
+corresponding	O
+80S	O
+structures	O
+.	O
+	O
+Rearrangements	O
+of	O
+the	O
+IRES	O
+involve	O
+restructuring	O
+of	O
+several	O
+interactions	O
+with	O
+the	O
+ribosome	O
+.	O
+	O
+In	O
+Structure	O
+I	O
+,	O
+SL3	O
+of	O
+the	O
+PKI	O
+domain	O
+is	O
+positioned	O
+between	O
+the	O
+A	O
+-	O
+site	O
+finger	O
+(	O
+nt	O
+1008	O
+–	O
+1043	O
+of	O
+25S	O
+rRNA	O
+)	O
+and	O
+the	O
+P	O
+site	O
+of	O
+the	O
+60S	O
+subunit	O
+,	O
+comprising	O
+helix	O
+84	O
+of	O
+25S	O
+rRNA	O
+(	O
+nt	O
+.	O
+	O
+The	O
+second	O
+set	O
+of	O
+major	O
+structural	O
+changes	O
+involves	O
+interaction	O
+of	O
+the	O
+P	O
+site	O
+region	O
+of	O
+the	O
+large	O
+subunit	O
+with	O
+the	O
+hinge	O
+point	O
+of	O
+the	O
+IRES	O
+bending	O
+between	O
+the	O
+5	O
+´	O
+domain	O
+and	O
+the	O
+PKI	O
+domain	O
+(	O
+nt	O
+.	O
+6886	O
+–	O
+6890	O
+).	O
+	O
+The	O
+view	O
+and	O
+colors	O
+are	O
+as	O
+in	O
+Figure	O
+5b	O
+:	O
+eEF2	O
+is	O
+shown	O
+in	O
+green	O
+,	O
+IRES	O
+RNA	O
+in	O
+red	O
+,	O
+40S	O
+subunit	O
+elements	O
+in	O
+orange	O
+,	O
+60S	O
+in	O
+cyan	O
+/	O
+teal	O
+.	O
+	O
+Cryo	O
+-	O
+EM	O
+density	O
+of	O
+the	O
+GTPase	O
+region	O
+in	O
+Structures	O
+I	O
+and	O
+II	O
+.	O
+	O
+Repositioning	O
+(	O
+sliding	O
+)	O
+of	O
+the	O
+positively	O
+-	O
+charged	O
+cluster	O
+of	O
+domain	O
+IV	O
+of	O
+eEF2	O
+over	O
+the	O
+phosphate	O
+backbone	O
+(	O
+red	O
+)	O
+of	O
+the	O
+18S	O
+helices	O
+33	O
+and	O
+34	O
+.	O
+	O
+Interactions	O
+of	O
+eEF2	O
+with	O
+the	O
+40S	O
+subunit	O
+.	O
+	O
+From	O
+Structure	O
+I	O
+to	O
+V	O
+,	O
+these	O
+central	O
+domains	O
+migrate	O
+by	O
+~	O
+10	O
+Å	O
+along	O
+the	O
+40S	O
+surface	O
+(	O
+Figure	O
+6c	O
+).	O
+	O
+At	O
+the	O
+head	O
+,	O
+C1274	O
+of	O
+the	O
+18S	O
+rRNA	O
+(	O
+C1054	O
+in	O
+E	O
+.	O
+coli	O
+)	O
+base	O
+pairs	O
+with	O
+the	O
+first	O
+nucleotide	O
+of	O
+the	O
+ORF	O
+immediately	O
+downstream	O
+of	O
+PKI	O
+.	O
+	O
+The	O
+codon	O
+-	O
+anticodon	O
+-	O
+like	O
+helix	O
+of	O
+PKI	O
+is	O
+shown	O
+in	O
+red	O
+,	O
+the	O
+downstream	O
+first	O
+codon	O
+of	O
+the	O
+ORF	O
+in	O
+magenta	O
+.	O
+	O
+Histidines	O
+583	O
+and	O
+694	O
+interact	O
+with	O
+the	O
+phosphate	O
+backbone	O
+of	O
+the	O
+anticodon	O
+-	O
+like	O
+strand	O
+(	O
+at	O
+G6907	O
+and	O
+C6908	O
+).	O
+	O
+Thus	O
+,	O
+in	O
+comparison	O
+with	O
+the	O
+initiation	O
+state	O
+,	O
+the	O
+histidine	O
+-	O
+diphthamide	O
+tip	O
+of	O
+eEF2	O
+replaces	O
+the	O
+codon	O
+-	O
+anticodon	O
+–	O
+like	O
+helix	O
+of	O
+PKI	O
+.	O
+	O
+The	O
+histidine	O
+resides	O
+next	O
+to	O
+the	O
+backbone	O
+of	O
+G3028	O
+of	O
+the	O
+sarcin	O
+-	O
+ricin	O
+loop	O
+and	O
+near	O
+the	O
+diphosphate	O
+of	O
+GDP	O
+(	O
+Figure	O
+5e	O
+).	O
+	O
+By	O
+contrast	O
+,	O
+switch	O
+loop	O
+I	O
+(	O
+aa	O
+50	O
+–	O
+70	O
+in	O
+S	O
+.	O
+cerevisiae	O
+eEF2	O
+)	O
+is	O
+resolved	O
+only	O
+in	O
+Structure	O
+I	O
+(	O
+Figure	O
+5	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+	O
+As	O
+such	O
+,	O
+the	O
+conformations	O
+of	O
+SWI	O
+and	O
+the	O
+GTPase	O
+center	O
+in	O
+general	O
+are	O
+similar	O
+to	O
+those	O
+observed	O
+in	O
+ribosome	O
+-	O
+bound	O
+EF	O
+-	O
+Tu	O
+and	O
+EF	O
+-	O
+G	O
+in	O
+the	O
+presence	O
+of	O
+GTP	O
+analogs	O
+.	O
+	O
+Structure	O
+II	O
+reveals	O
+PKI	O
+between	O
+the	O
+body	O
+A	O
+and	O
+P	O
+sites	O
+and	O
+eEF2	O
+partially	O
+advanced	O
+into	O
+the	O
+A	O
+site	O
+	O
+Domain	O
+IV	O
+of	O
+eEF2	O
+is	O
+further	O
+entrenched	O
+in	O
+the	O
+A	O
+site	O
+by	O
+~	O
+3	O
+Å	O
+relative	O
+to	O
+the	O
+body	O
+and	O
+~	O
+8	O
+Å	O
+relative	O
+to	O
+the	O
+head	O
+,	O
+preserving	O
+its	O
+interactions	O
+with	O
+PKI	O
+.	O
+	O
+In	O
+Structure	O
+IV	O
+,	O
+the	O
+40S	O
+subunit	O
+is	O
+almost	O
+non	O
+-	O
+rotated	O
+relative	O
+to	O
+the	O
+60S	O
+subunit	O
+,	O
+and	O
+the	O
+40S	O
+head	O
+is	O
+mid	O
+-	O
+swiveled	O
+.	O
+	O
+In	O
+Structure	O
+V	O
+,	O
+protein	O
+uS12	O
+is	O
+shifted	O
+along	O
+with	O
+the	O
+40S	O
+body	O
+as	O
+a	O
+result	O
+of	O
+intersubunit	O
+rotation	O
+.	O
+	O
+This	O
+shifts	O
+the	O
+tip	O
+of	O
+helix	O
+A	O
+of	O
+domain	O
+III	O
+(	O
+at	O
+aa	O
+500	O
+)	O
+by	O
+~	O
+5	O
+Å	O
+(	O
+relative	O
+to	O
+that	O
+in	O
+Structure	O
+I	O
+)	O
+toward	O
+domain	O
+I	O
+.	O
+Although	O
+domain	O
+III	O
+remains	O
+in	O
+contact	O
+with	O
+domain	O
+V	O
+,	O
+the	O
+shift	O
+occurs	O
+in	O
+the	O
+direction	O
+that	O
+could	O
+eventually	O
+disconnect	O
+the	O
+β	O
+-	O
+platforms	O
+of	O
+these	O
+domains	O
+.	O
+	O
+The	O
+imidazole	O
+moiety	O
+stacks	O
+on	O
+G6907	O
+(	O
+corresponding	O
+to	O
+nt	O
+36	O
+in	O
+the	O
+tRNA	O
+anticodon	O
+)	O
+and	O
+hydrogen	O
+bonds	O
+with	O
+O2	O
+’	O
+of	O
+G6906	O
+(	O
+nt	O
+35	O
+of	O
+tRNA	O
+).	O
+	O
+The	O
+front	O
+'	O
+legs	O
+'	O
+(	O
+SL4	O
+and	O
+SL5	O
+)	O
+of	O
+the	O
+5	O
+’-	O
+domain	O
+(	O
+front	O
+end	O
+)	O
+are	O
+attached	O
+to	O
+the	O
+40S	O
+head	O
+proteins	O
+uS7	O
+,	O
+uS11	O
+and	O
+eS25	O
+(	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+	O
+Notably	O
+,	O
+at	O
+all	O
+steps	O
+,	O
+the	O
+head	O
+of	O
+the	O
+IRES	O
+inchworm	O
+(	O
+L1	O
+.	O
+1	O
+region	O
+)	O
+is	O
+supported	O
+by	O
+the	O
+mobile	O
+L1	O
+stalk	O
+.	O
+	O
+Partitioned	O
+roles	O
+of	O
+40S	O
+subunit	O
+rearrangements	O
+	O
+Specifically	O
+,	O
+intersubunit	O
+rotation	O
+allows	O
+eEF2	O
+entry	O
+into	O
+the	O
+A	O
+site	O
+,	O
+while	O
+the	O
+head	O
+swivel	O
+mediates	O
+PKI	O
+translocation	O
+.	O
+	O
+Because	O
+the	O
+histidine	O
+-	O
+diphthamide	O
+tip	O
+of	O
+eEF2	O
+(	O
+H583	O
+,	O
+H694	O
+and	O
+Diph699	O
+)	O
+attaches	O
+to	O
+the	O
+codon	O
+-	O
+anticodon	O
+-	O
+like	O
+helix	O
+of	O
+PKI	O
+,	O
+eEF2	O
+appears	O
+to	O
+directly	O
+force	O
+PKI	O
+out	O
+of	O
+the	O
+A	O
+site	O
+.	O
+	O
+To	O
+our	O
+knowledge	O
+,	O
+our	O
+work	O
+provides	O
+the	O
+first	O
+high	O
+-	O
+resolution	O
+view	O
+of	O
+the	O
+dynamics	O
+of	O
+a	O
+ribosomal	O
+translocase	O
+that	O
+is	O
+inferred	O
+from	O
+an	O
+ensemble	O
+of	O
+structures	O
+sampled	O
+under	O
+uniform	O
+conditions	O
+.	O
+	O
+While	O
+the	O
+ribosome	O
+itself	O
+has	O
+the	O
+capacity	O
+to	O
+translocate	O
+in	O
+the	O
+absence	O
+of	O
+the	O
+translocase	O
+,	O
+spontaneous	O
+translocation	O
+is	O
+slow	O
+.	O
+	O
+The	O
+80S	O
+•	O
+IRES	O
+•	O
+eEF2	O
+structures	O
+reported	O
+here	O
+suggest	O
+two	O
+main	O
+roles	O
+for	O
+eEF2	O
+in	O
+translocation	O
+.	O
+	O
+In	O
+our	O
+structures	O
+,	O
+the	O
+tip	O
+of	O
+domain	O
+IV	O
+docks	O
+next	O
+to	O
+PKI	O
+,	O
+with	O
+diphthamide	O
+699	O
+fit	O
+into	O
+the	O
+minor	O
+groove	O
+of	O
+the	O
+codon	O
+-	O
+anticodon	O
+-	O
+like	O
+helix	O
+of	O
+PKI	O
+(	O
+Figure	O
+7	O
+).	O
+	O
+This	O
+arrangement	O
+rationalizes	O
+inactivation	O
+of	O
+eEF2	O
+by	O
+diphtheria	O
+toxin	O
+,	O
+which	O
+catalyzes	O
+ADP	O
+-	O
+ribosylation	O
+of	O
+the	O
+diphthamide	O
+(	O
+reviewed	O
+in	O
+).	O
+	O
+The	O
+enzyme	O
+ADP	O
+-	O
+ribosylates	O
+the	O
+NE2	O
+atom	O
+of	O
+the	O
+imidazole	O
+ring	O
+,	O
+which	O
+in	O
+our	O
+structures	O
+interacts	O
+with	O
+the	O
+first	O
+two	O
+residues	O
+of	O
+the	O
+anticodon	O
+-	O
+like	O
+strand	O
+of	O
+PKI	O
+.	O
+	O
+However	O
+,	O
+the	O
+structural	O
+and	O
+mechanistic	O
+definitions	O
+of	O
+the	O
+locked	O
+and	O
+unlocked	O
+states	O
+have	O
+remained	O
+unclear	O
+,	O
+ranging	O
+from	O
+the	O
+globally	O
+distinct	O
+ribosome	O
+conformations	O
+to	O
+unknown	O
+local	O
+rearrangements	O
+,	O
+e	O
+.	O
+g	O
+.	O
+those	O
+in	O
+the	O
+decoding	O
+center	O
+.	O
+	O
+FRET	O
+data	O
+indicate	O
+that	O
+translocation	O
+of	O
+2tRNA	O
+•	O
+mRNA	O
+on	O
+the	O
+70S	O
+ribosome	O
+requires	O
+a	O
+forward	O
+-	O
+and	O
+-	O
+reverse	O
+head	O
+swivel	O
+,	O
+which	O
+may	O
+be	O
+related	O
+to	O
+the	O
+unlocking	O
+phenomenon	O
+.	O
+	O
+Mutations	O
+of	O
+residues	O
+flanking	O
+A344	O
+in	O
+E	O
+.	O
+coli	O
+16S	O
+rRNA	O
+modestly	O
+inhibit	O
+translation	O
+but	O
+do	O
+not	O
+specifically	O
+affect	O
+EF	O
+-	O
+G	O
+-	O
+mediated	O
+translocation	O
+.	O
+	O
+However	O
+,	O
+the	O
+effect	O
+of	O
+A344	O
+mutation	O
+on	O
+translation	O
+was	O
+not	O
+addressed	O
+in	O
+that	O
+study	O
+,	O
+leaving	O
+the	O
+question	O
+open	O
+whether	O
+this	O
+residue	O
+is	O
+critical	O
+for	O
+eEF2	O
+/	O
+EF	O
+-	O
+G	O
+function	O
+.	O
+	O
+The	O
+interaction	O
+between	O
+h14	O
+and	O
+switch	O
+loop	O
+I	O
+is	O
+not	O
+resolved	O
+in	O
+Structures	O
+II	O
+to	O
+V	O
+,	O
+in	O
+all	O
+of	O
+which	O
+the	O
+small	O
+subunit	O
+is	O
+partially	O
+rotated	O
+or	O
+non	O
+-	O
+rotated	O
+,	O
+so	O
+that	O
+helix	O
+14	O
+is	O
+placed	O
+at	O
+least	O
+6	O
+Å	O
+farther	O
+from	O
+eEF2	O
+(	O
+Figure	O
+5d	O
+).	O
+	O
+We	O
+conclude	O
+that	O
+unlike	O
+other	O
+conformations	O
+of	O
+the	O
+ribosome	O
+,	O
+the	O
+fully	O
+rotated	O
+40S	O
+subunit	O
+of	O
+the	O
+pre	O
+-	O
+translocation	O
+ribosome	O
+provides	O
+an	O
+interaction	O
+surface	O
+,	O
+complementing	O
+the	O
+P	O
+stalk	O
+and	O
+SRL	O
+,	O
+for	O
+binding	O
+of	O
+the	O
+GTP	O
+-	O
+bound	O
+translocase	O
+.	O
+	O
+This	O
+displacement	O
+is	O
+caused	O
+by	O
+the	O
+8	O
+Å	O
+movement	O
+of	O
+the	O
+40S	O
+body	O
+protein	O
+uS12	O
+upon	O
+reverse	O
+intersubunit	O
+rotation	O
+from	O
+Structure	O
+I	O
+to	O
+V	O
+(	O
+Figure	O
+6d	O
+).	O
+	O
+As	O
+we	O
+discuss	O
+below	O
+,	O
+Structure	O
+V	O
+captures	O
+a	O
+'	O
+pre	O
+-	O
+unstacking	O
+'	O
+state	O
+due	O
+to	O
+stabilization	O
+of	O
+the	O
+interface	O
+between	O
+domains	O
+III	O
+and	O
+V	O
+by	O
+sordarin	O
+.	O
+	O
+Intersubunit	O
+rearrangements	O
+and	O
+tRNA	O
+hybrid	O
+states	O
+have	O
+been	O
+proposed	O
+to	O
+play	O
+key	O
+roles	O
+half	O
+a	O
+century	O
+ago	O
+.	O
+	O
+Despite	O
+an	O
+impressive	O
+body	O
+of	O
+biochemical	O
+,	O
+fluorescence	O
+and	O
+structural	O
+data	O
+accumulated	O
+since	O
+then	O
+,	O
+translocation	O
+remains	O
+the	O
+least	O
+understood	O
+step	O
+of	O
+elongation	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+step	O
+-	O
+wise	O
+coupling	O
+of	O
+ribosome	O
+dynamics	O
+with	O
+IRES	O
+translocation	O
+is	O
+overall	O
+consistent	O
+with	O
+that	O
+observed	O
+for	O
+2tRNA	O
+•	O
+mRNA	O
+translocation	O
+in	O
+solution	O
+.	O
+	O
+We	O
+deem	O
+the	O
+pre	O
+-	O
+translocation	O
+complex	O
+locked	O
+,	O
+because	O
+the	O
+A	O
+-	O
+site	O
+bound	O
+ASL	O
+-	O
+mRNA	O
+is	O
+stabilized	O
+by	O
+interactions	O
+with	O
+the	O
+decoding	O
+center	O
+.	O
+	O
+This	O
+unlatches	O
+the	O
+head	O
+,	O
+allowing	O
+creation	O
+of	O
+hitherto	O
+elusive	O
+intermediate	O
+tRNA	O
+positions	O
+during	O
+spontaneous	O
+reverse	O
+body	O
+rotation	O
+.	O
+	O
+Finally	O
+,	O
+the	O
+similar	O
+populations	O
+of	O
+particles	O
+(	O
+within	O
+a	O
+2X	O
+range	O
+)	O
+in	O
+our	O
+80S	O
+•	O
+IRES	O
+•	O
+eEF2	O
+reconstructions	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2	O
+)	O
+suggest	O
+that	O
+the	O
+intermediate	O
+translocation	O
+states	O
+sample	O
+several	O
+energetically	O
+similar	O
+and	O
+interconverting	O
+conformations	O
+.	O
+	O
+The	O
+cryo	O
+-	O
+EM	O
+structures	O
+demonstrate	O
+that	O
+the	O
+TSV	O
+IRES	O
+structurally	O
+and	O
+dynamically	O
+represents	O
+a	O
+chimera	O
+of	O
+the	O
+2tRNA	O
+•	O
+mRNA	O
+translocating	O
+complex	O
+(	O
+A	O
+/	O
+P	O
+-	O
+tRNA	O
+•	O
+P	O
+/	O
+E	O
+-	O
+tRNA	O
+•	O
+mRNA	O
+).	O
+	O
+Intergenic	O
+IRESs	O
+,	O
+in	O
+turn	O
+,	O
+represent	O
+a	O
+striking	O
+example	O
+of	O
+convergent	O
+molecular	O
+evolution	O
+.	O
+	O
+This	O
+work	O
+provides	O
+insights	O
+into	O
+the	O
+basic	O
+mechanism	O
+of	O
+proteolysis	O
+and	O
+propeptide	O
+autolysis	O
+,	O
+as	O
+well	O
+as	O
+the	O
+evolutionary	O
+pressures	O
+that	O
+drove	O
+the	O
+proteasome	O
+to	O
+become	O
+a	O
+threonine	O
+protease	O
+.	O
+	O
+Data	O
+from	O
+biochemical	O
+and	O
+structural	O
+analyses	O
+of	O
+proteasome	O
+variants	O
+with	O
+mutations	O
+in	O
+the	O
+β5	O
+propeptide	O
+and	O
+the	O
+active	O
+site	O
+strongly	O
+support	O
+the	O
+model	O
+and	O
+deliver	O
+novel	O
+insights	O
+into	O
+the	O
+structural	O
+constraints	O
+required	O
+for	O
+the	O
+autocatalytic	O
+activation	O
+of	O
+the	O
+proteasome	O
+.	O
+	O
+Proteasome	O
+-	O
+mediated	O
+degradation	O
+of	O
+cell	O
+-	O
+cycle	O
+regulators	O
+and	O
+potentially	O
+toxic	O
+misfolded	O
+proteins	O
+is	O
+required	O
+for	O
+the	O
+viability	O
+of	O
+eukaryotic	O
+cells	O
+.	O
+	O
+These	O
+results	O
+indicate	O
+that	O
+the	O
+β1	O
+and	O
+β2	O
+proteolytic	O
+activities	O
+are	O
+not	O
+essential	O
+for	O
+cell	O
+survival	O
+.	O
+	O
+Our	O
+present	O
+crystallographic	O
+analysis	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+pp	O
+trans	O
+mutant	O
+demonstrates	O
+that	O
+the	O
+mutation	O
+per	O
+se	O
+does	O
+not	O
+structurally	O
+alter	O
+the	O
+catalytic	O
+active	O
+site	O
+and	O
+that	O
+the	O
+trans	O
+-	O
+expressed	O
+β5	O
+propeptide	O
+is	O
+not	O
+bound	O
+in	O
+the	O
+β5	O
+substrate	O
+-	O
+binding	O
+channel	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+).	O
+	O
+Thr	O
+(-	O
+2	O
+)	O
+positions	O
+Gly	O
+(-	O
+1	O
+)	O
+O	O
+via	O
+hydrogen	O
+bonding	O
+(∼	O
+2	O
+.	O
+8	O
+Å	O
+)	O
+in	O
+a	O
+perfect	O
+trajectory	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	O
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+	O
+As	O
+histidine	O
+commonly	O
+functions	O
+as	O
+a	O
+proton	O
+shuttle	O
+in	O
+the	O
+catalytic	O
+triads	O
+of	O
+serine	O
+proteases	O
+,	O
+we	O
+investigated	O
+the	O
+role	O
+of	O
+His	O
+(-	O
+2	O
+)	O
+in	O
+processing	O
+of	O
+the	O
+β5	O
+propeptide	O
+by	O
+exchanging	O
+it	O
+for	O
+Asn	O
+,	O
+Lys	O
+,	O
+Phe	O
+and	O
+Ala	O
+.	O
+All	O
+yeast	O
+mutants	O
+were	O
+viable	O
+at	O
+30	O
+°	O
+C	O
+,	O
+but	O
+suffered	O
+from	O
+growth	O
+defects	O
+at	O
+37	O
+°	O
+C	O
+with	O
+the	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+N	I-mutant
+and	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+F	I-mutant
+mutants	O
+being	O
+most	O
+affected	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+and	O
+Table	O
+1	O
+).	O
+	O
+In	O
+agreement	O
+,	O
+the	O
+chymotrypsin	O
+-	O
+like	O
+(	O
+ChT	O
+-	O
+L	O
+)	O
+activity	O
+of	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+N	I-mutant
+and	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+F	I-mutant
+mutant	O
+yCPs	O
+was	O
+impaired	O
+in	O
+situ	O
+and	O
+in	O
+vitro	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3c	O
+).	O
+	O
+Next	O
+,	O
+we	O
+examined	O
+the	O
+effect	O
+of	O
+residue	O
+(-	O
+2	O
+)	O
+on	O
+the	O
+orientation	O
+of	O
+the	O
+propeptide	O
+by	O
+creating	O
+mutants	O
+that	O
+combine	O
+the	O
+T1A	B-mutant
+(	O
+K81R	B-mutant
+)	O
+mutation	O
+(	O
+s	O
+)	O
+with	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+,	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+or	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+A	I-mutant
+substitutions	O
+.	O
+	O
+Notably	O
+,	O
+the	O
+2FO	O
+–	O
+FC	O
+electron	O
+-	O
+density	O
+map	O
+displays	O
+a	O
+different	O
+orientation	O
+for	O
+the	O
+β2	O
+propeptide	O
+than	O
+has	O
+been	O
+observed	O
+for	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+proteasome	O
+.	O
+	O
+The	O
+phenotype	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+mutant	O
+was	O
+however	O
+less	O
+pronounced	O
+than	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+yeast	O
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+Structural	O
+comparison	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+L	I-mutant
+(-	I-mutant
+49	I-mutant
+)	I-mutant
+S	I-mutant
+-	I-mutant
+K33A	I-mutant
+and	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+active	O
+sites	O
+revealed	O
+that	O
+mutation	O
+of	O
+Lys33	O
+to	O
+Ala	O
+creates	O
+a	O
+cavity	O
+that	O
+is	O
+filled	O
+with	O
+Thr1	O
+and	O
+the	O
+remnant	O
+propeptide	O
+.	O
+	O
+In	O
+contrast	O
+to	O
+the	O
+cis	O
+-	O
+construct	O
+,	O
+expression	O
+of	O
+the	O
+β5	O
+propeptide	O
+in	O
+trans	O
+allowed	O
+straightforward	O
+isolation	O
+and	O
+crystallization	O
+of	O
+the	O
+D17N	B-mutant
+mutant	O
+proteasome	O
+.	O
+	O
+This	O
+observation	O
+is	O
+consistent	O
+with	O
+a	O
+strongly	O
+reduced	O
+reactivity	O
+of	O
+β5	O
+-	O
+Thr1	O
+and	O
+the	O
+crystal	O
+structure	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+pp	O
+cis	O
+mutant	O
+in	O
+complex	O
+with	O
+carfilzomib	O
+.	O
+	O
+Asp166Oδ	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Thr1NH2	O
+via	O
+Ser129OH	O
+and	O
+Ser169OH	O
+,	O
+and	O
+therefore	O
+was	O
+proposed	O
+to	O
+be	O
+involved	O
+in	O
+catalysis	O
+.	O
+	O
+Instead	O
+,	O
+a	O
+water	O
+molecule	O
+is	O
+bound	O
+to	O
+Ser129OH	O
+and	O
+Thr1NH2	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8b	O
+),	O
+which	O
+may	O
+enable	O
+precursor	O
+processing	O
+.	O
+	O
+In	O
+the	O
+carfilzomib	O
+complex	O
+structure	O
+,	O
+Thr1Oγ	O
+and	O
+Thr1N	O
+incorporate	O
+into	O
+a	O
+morpholine	O
+ring	O
+structure	O
+and	O
+Ser129	O
+adopts	O
+its	O
+WT	O
+-	O
+like	O
+orientation	O
+.	O
+	O
+Whereas	O
+Asn	O
+can	O
+to	O
+some	O
+degree	O
+replace	O
+Asp166	O
+due	O
+to	O
+its	O
+carbonyl	O
+group	O
+in	O
+the	O
+side	O
+chain	O
+,	O
+Ala	O
+at	O
+this	O
+position	O
+was	O
+found	O
+to	O
+prevent	O
+both	O
+autolysis	O
+and	O
+catalysis	O
+.	O
+	O
+These	O
+results	O
+suggest	O
+that	O
+Asp166	O
+and	O
+Ser129	O
+function	O
+as	O
+a	O
+proton	O
+shuttle	O
+and	O
+affect	O
+the	O
+protonation	O
+state	O
+of	O
+Thr1N	O
+during	O
+autolysis	O
+and	O
+catalysis	O
+.	O
+	O
+Owing	O
+to	O
+the	O
+unequal	O
+positions	O
+of	O
+the	O
+two	O
+β5	O
+subunits	O
+within	O
+the	O
+CP	O
+in	O
+the	O
+crystal	O
+lattice	O
+,	O
+maturation	O
+and	O
+propeptide	O
+displacement	O
+may	O
+occur	O
+at	O
+different	O
+timescales	O
+in	O
+the	O
+two	O
+subunits	O
+.	O
+	O
+In	O
+agreement	O
+,	O
+soaking	O
+crystals	O
+with	O
+the	O
+CP	O
+inhibitors	O
+bortezomib	O
+or	O
+carfilzomib	O
+modifies	O
+only	O
+the	O
+β1	O
+and	O
+β2	O
+active	O
+sites	O
+,	O
+while	O
+leaving	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+proteolytic	O
+centres	O
+unmodified	O
+even	O
+though	O
+they	O
+are	O
+only	O
+partially	O
+occupied	O
+by	O
+the	O
+cleaved	O
+propeptide	O
+remnant	O
+.	O
+	O
+In	O
+agreement	O
+,	O
+at	O
+an	O
+elevated	O
+growing	O
+temperature	O
+of	O
+37	O
+°	O
+C	O
+the	O
+T1S	B-mutant
+mutant	O
+is	O
+unable	O
+to	O
+grow	O
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+	O
+Hence	O
+,	O
+the	O
+mean	O
+residence	O
+time	O
+of	O
+carfilzomib	O
+at	O
+the	O
+active	O
+site	O
+is	O
+prolonged	O
+and	O
+the	O
+probability	O
+to	O
+covalently	O
+react	O
+with	O
+Ser1	O
+is	O
+increased	O
+.	O
+	O
+The	O
+20S	O
+proteasome	O
+CP	O
+is	O
+the	O
+major	O
+non	O
+-	O
+lysosomal	O
+protease	O
+in	O
+eukaryotic	O
+cells	O
+,	O
+and	O
+its	O
+assembly	O
+is	O
+highly	O
+organized	O
+.	O
+	O
+Depending	O
+on	O
+the	O
+(-	O
+2	O
+)	O
+residue	O
+we	O
+observed	O
+various	O
+propeptide	O
+conformations	O
+,	O
+but	O
+Gly	O
+(-	O
+1	O
+)	O
+is	O
+in	O
+all	O
+structures	O
+perfectly	O
+located	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	O
+,	O
+although	O
+it	O
+does	O
+not	O
+adopt	O
+the	O
+tight	O
+turn	O
+observed	O
+for	O
+the	O
+prosegment	O
+of	O
+subunit	O
+β1	O
+.	O
+	O
+Lys33NH2	O
+is	O
+expected	O
+to	O
+act	O
+as	O
+the	O
+proton	O
+acceptor	O
+during	O
+autocatalytic	O
+removal	O
+of	O
+the	O
+propeptides	O
+,	O
+as	O
+well	O
+as	O
+during	O
+substrate	O
+proteolysis	O
+,	O
+while	O
+Asp17Oδ	O
+orients	O
+Lys33NH2	O
+and	O
+makes	O
+it	O
+more	O
+prone	O
+to	O
+protonation	O
+by	O
+raising	O
+its	O
+pKa	O
+(	O
+hydrogen	O
+bond	O
+distance	O
+:	O
+Lys33NH3	O
++–	O
+Asp17Oδ	O
+:	O
+2	O
+.	O
+9	O
+Å	O
+).	O
+	O
+Analogously	O
+to	O
+the	O
+proteasome	O
+,	O
+a	O
+Thr	O
+–	O
+Lys	O
+–	O
+Asp	O
+triad	O
+is	O
+also	O
+found	O
+in	O
+L	O
+-	O
+asparaginase	O
+.	O
+	O
+In	O
+this	O
+new	O
+view	O
+of	O
+the	O
+proteasomal	O
+active	O
+site	O
+,	O
+the	O
+positively	O
+charged	O
+Thr1NH3	O
++-	O
+terminus	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+amide	O
+nitrogen	O
+of	O
+incoming	O
+peptide	O
+substrates	O
+and	O
+stabilizes	O
+as	O
+well	O
+as	O
+activates	O
+them	O
+for	O
+the	O
+endoproteolytic	O
+cleavage	O
+by	O
+Thr1Oγ	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+	O
+Breakdown	O
+of	O
+this	O
+tetrahedral	O
+transition	O
+state	O
+releases	O
+the	O
+Thr1	O
+N	O
+terminus	O
+that	O
+is	O
+protonated	O
+by	O
+aspartic	O
+acid	O
+166	O
+via	O
+Ser129OH	O
+to	O
+yield	O
+Thr1NH3	O
++.	O
+	O
+This	O
+interpretation	O
+agrees	O
+with	O
+the	O
+strongly	O
+reduced	O
+catalytic	O
+activity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+mutant	O
+on	O
+the	O
+one	O
+hand	O
+,	O
+and	O
+the	O
+ability	O
+to	O
+react	O
+readily	O
+with	O
+carfilzomib	O
+on	O
+the	O
+other	O
+.	O
+	O
+In	O
+accord	O
+with	O
+the	O
+proposed	O
+Thr1	O
+–	O
+Lys33	O
+–	O
+Asp17	O
+catalytic	O
+triad	O
+,	O
+crystallographic	O
+data	O
+on	O
+the	O
+proteolytically	O
+inactive	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+mutant	O
+demonstrate	O
+that	O
+the	O
+interaction	O
+of	O
+Lys33NH2	O
+and	O
+Cys1	O
+is	O
+broken	O
+.	O
+	O
+Thr1	O
+is	O
+well	O
+anchored	O
+in	O
+the	O
+active	O
+site	O
+by	O
+hydrophobic	O
+interactions	O
+of	O
+its	O
+Cγ	O
+methyl	O
+group	O
+with	O
+Ala46	O
+(	O
+Cβ	O
+),	O
+Lys33	O
+(	O
+carbon	O
+side	O
+chain	O
+)	O
+and	O
+Thr3	O
+(	O
+Cγ	O
+).	O
+	O
+Notably	O
+,	O
+in	O
+the	O
+threonine	O
+aspartase	O
+Taspase1	O
+,	O
+mutation	O
+of	O
+the	O
+active	O
+-	O
+site	O
+Thr234	O
+to	O
+Ser	O
+also	O
+places	O
+the	O
+side	O
+chain	O
+in	O
+the	O
+position	O
+of	O
+the	O
+methyl	O
+group	O
+of	O
+Thr234	O
+in	O
+the	O
+WT	O
+,	O
+thereby	O
+reducing	O
+catalytic	O
+activity	O
+.	O
+	O
+(	O
+b	O
+)	O
+Structural	O
+superposition	O
+of	O
+the	O
+β5	O
+propeptides	O
+in	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+β5	B-mutant
+-(	I-mutant
+H	I-mutant
+-	I-mutant
+2	I-mutant
+)	I-mutant
+A	I-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+and	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	O
+proteasomes	O
+.	O
+	O
+While	O
+the	O
+residues	O
+(-	O
+2	O
+)	O
+to	O
+(-	O
+4	O
+)	O
+vary	O
+in	O
+their	O
+conformation	O
+,	O
+Gly	O
+(-	O
+1	O
+)	O
+and	O
+Ala1	O
+are	O
+located	O
+in	O
+all	O
+structures	O
+at	O
+the	O
+same	O
+positions	O
+.	O
+	O
+The	O
+strictly	O
+conserved	O
+oxyanion	O
+hole	O
+Gly47NH	O
+stabilizing	O
+the	O
+negatively	O
+charged	O
+intermediate	O
+is	O
+illustrated	O
+as	O
+a	O
+semicircle	O
+.	O
+	O
+Next	O
+,	O
+Thr1NH2	O
+polarizes	O
+a	O
+water	O
+molecule	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+of	O
+the	O
+acyl	O
+-	O
+enzyme	O
+intermediate	O
+.	O
+	O
+On	O
+hydrolysis	O
+of	O
+the	O
+latter	O
+,	O
+the	O
+active	O
+-	O
+site	O
+Thr1	O
+is	O
+ready	O
+for	O
+catalysis	O
+(	O
+right	O
+set	O
+of	O
+structures	O
+).	O
+	O
+The	O
+proteasome	O
+favours	O
+threonine	O
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+.	O
+	O
+(	O
+a	O
+)	O
+Growth	O
+tests	O
+by	O
+serial	O
+dilution	O
+of	O
+WT	O
+and	O
+pre2	O
+(	O
+β5	O
+)	O
+mutant	O
+yeast	O
+cultures	O
+reveal	O
+growth	O
+defects	O
+of	O
+the	O
+active	O
+-	O
+site	O
+mutants	O
+under	O
+the	O
+indicated	O
+conditions	O
+after	O
+2	O
+days	O
+(	O
+2	O
+d	O
+)	O
+of	O
+incubation	O
+.	O
+	O
+(	O
+c	O
+)	O
+Illustration	O
+of	O
+the	O
+2FO	O
+–	O
+FC	O
+electron	O
+-	O
+density	O
+map	O
+(	O
+blue	O
+mesh	O
+contoured	O
+at	O
+1σ	O
+)	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+propeptide	O
+fragment	O
+.	O
+	O
+Ser1	O
+lacks	O
+this	O
+stabilization	O
+and	O
+is	O
+therefore	O
+rotated	O
+by	O
+60	O
+°.	O
+	O
+Inhibition	O
+assays	O
+(	O
+left	O
+panel	O
+).	O
+	O
+p300	O
+-	O
+catalyzed	O
+histone	O
+crotonylation	O
+,	O
+which	O
+is	O
+likely	O
+metabolically	O
+regulated	O
+,	O
+stimulates	O
+transcription	O
+to	O
+a	O
+greater	O
+degree	O
+than	O
+p300	O
+-	O
+catalyzed	O
+acetylation	O
+.	O
+	O
+The	O
+discovery	O
+of	O
+individual	O
+biological	O
+roles	O
+for	O
+the	O
+crotonyllysine	O
+and	O
+acetyllysine	O
+marks	O
+suggests	O
+that	O
+these	O
+PTMs	O
+can	O
+be	O
+read	O
+by	O
+distinct	O
+readers	O
+.	O
+	O
+However	O
+,	O
+Taf14	O
+is	O
+also	O
+found	O
+in	O
+a	O
+number	O
+of	O
+chromatin	O
+-	O
+remodeling	O
+complexes	O
+(	O
+i	O
+.	O
+e	O
+.,	O
+INO80	O
+,	O
+SWI	O
+/	O
+SNF	O
+and	O
+RSC	O
+)	O
+and	O
+the	O
+histone	O
+acetyltransferase	O
+complex	O
+NuA3	O
+,	O
+indicating	O
+a	O
+multifaceted	O
+role	O
+of	O
+Taf14	O
+in	O
+transcriptional	O
+regulation	O
+and	O
+chromatin	O
+biology	O
+.	O
+	O
+In	O
+this	O
+study	O
+,	O
+we	O
+identified	O
+the	O
+Taf14	O
+YEATS	O
+domain	O
+as	O
+a	O
+reader	O
+of	O
+crotonyllysine	O
+that	O
+binds	O
+to	O
+histone	O
+H3	O
+crotonylated	O
+at	O
+lysine	O
+9	O
+(	O
+H3K9cr	O
+)	O
+via	O
+a	O
+distinctive	O
+binding	O
+mechanism	O
+.	O
+	O
+This	O
+distinctive	O
+mechanism	O
+was	O
+corroborated	O
+through	O
+mapping	O
+the	O
+Taf14	O
+YEATS	O
+-	O
+H3K9cr	O
+binding	O
+interface	O
+in	O
+solution	O
+using	O
+NMR	O
+chemical	O
+shift	O
+perturbation	O
+analysis	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+).	O
+	O
+To	O
+determine	O
+whether	O
+H3K9cr	O
+is	O
+present	O
+in	O
+yeast	O
+,	O
+we	O
+generated	O
+whole	O
+cell	O
+extracts	O
+from	O
+logarithmically	O
+growing	O
+yeast	O
+cells	O
+and	O
+subjected	O
+them	O
+to	O
+Western	O
+blot	O
+analysis	O
+using	O
+antibodies	O
+directed	O
+towards	O
+H3K9cr	O
+,	O
+H3K9ac	O
+and	O
+H3	O
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Table	O
+2	O
+).	O
+	O
+We	O
+next	O
+asked	O
+if	O
+H3K9cr	O
+is	O
+regulated	O
+by	O
+the	O
+actions	O
+of	O
+histone	O
+acetyltransferases	O
+(	O
+HATs	O
+)	O
+and	O
+histone	O
+deacetylases	O
+(	O
+HDACs	O
+).	O
+	O
+Furthermore	O
+,	O
+fluctuations	O
+in	O
+the	O
+H3K9cr	O
+levels	O
+were	O
+more	O
+substantial	O
+than	O
+fluctuations	O
+in	O
+the	O
+corresponding	O
+H3K9ac	O
+levels	O
+.	O
+	O
+Together	O
+,	O
+these	O
+results	O
+reveal	O
+that	O
+H3K9cr	O
+is	O
+a	O
+dynamic	O
+mark	O
+of	O
+chromatin	O
+in	O
+yeast	O
+and	O
+suggest	O
+an	O
+important	O
+role	O
+for	O
+this	O
+modification	O
+in	O
+transcription	O
+as	O
+it	O
+is	O
+regulated	O
+by	O
+HATs	O
+and	O
+HDACs	O
+.	O
+	O
+The	O
+selectivity	O
+of	O
+Taf14	O
+towards	O
+crotonyllysine	O
+was	O
+substantiated	O
+by	O
+1H	O
+,	O
+15N	O
+HSQC	O
+experiments	O
+,	O
+in	O
+which	O
+either	O
+H3K9cr5	O
+-	O
+13	O
+or	O
+H3K9ac5	O
+-	O
+13	O
+peptide	O
+was	O
+titrated	O
+into	O
+the	O
+15N	O
+-	O
+labeled	O
+Taf14	O
+YEATS	O
+domain	O
+(	O
+Fig	O
+.	O
+2c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+	O
+To	O
+determine	O
+if	O
+the	O
+binding	O
+to	O
+crotonyllysine	O
+is	O
+conserved	O
+,	O
+we	O
+tested	O
+human	O
+YEATS	O
+domains	O
+by	O
+pull	O
+-	O
+down	O
+experiments	O
+using	O
+singly	O
+and	O
+multiply	O
+acetylated	O
+,	O
+propionylated	O
+,	O
+butyrylated	O
+,	O
+and	O
+crotonylated	O
+histone	O
+peptides	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+	O
+These	O
+results	O
+demonstrate	O
+that	O
+the	O
+YEATS	O
+domain	O
+is	O
+currently	O
+the	O
+sole	O
+reader	O
+of	O
+crotonyllysine	O
+.	O
+	O
+The	O
+unique	O
+and	O
+previously	O
+unobserved	O
+aromatic	O
+-	O
+amide	O
+/	O
+aliphatic	O
+-	O
+aromatic	O
+π	O
+-	O
+π	O
+-	O
+π	O
+-	O
+stacking	O
+mechanism	O
+facilitates	O
+the	O
+specific	O
+recognition	O
+of	O
+the	O
+crotonyl	O
+moiety	O
+.	O
+	O
+We	O
+further	O
+demonstrate	O
+that	O
+H3K9cr	O
+exists	O
+in	O
+yeast	O
+and	O
+is	O
+dynamically	O
+regulated	O
+by	O
+HATs	O
+and	O
+HDACs	O
+.	O
+	O
+Total	O
+H3	O
+was	O
+used	O
+as	O
+a	O
+loading	O
+control	O
+.	O
+	O
+Structure	O
+of	O
+the	O
+GAT	O
+domain	O
+of	O
+the	O
+endosomal	O
+adapter	O
+protein	O
+Tom1	O
+	O
+The	O
+Tom1	O
+GAT	O
+domain	O
+solution	O
+structure	O
+will	O
+provide	O
+additional	O
+tools	O
+for	O
+modulating	O
+its	O
+biological	O
+function	O
+.	O
+	O
+A	O
+conformational	O
+response	O
+of	O
+the	O
+Tom1	O
+GAT	O
+domain	O
+upon	O
+Tollip	O
+TBD	O
+binding	O
+can	O
+serve	O
+as	O
+an	O
+example	O
+to	O
+explain	O
+mutually	O
+exclusive	O
+ligand	O
+binding	O
+events	O
+.	O
+	O
+The	O
+Tom1	O
+GAT	O
+structural	O
+restraints	O
+yielded	O
+ten	O
+helical	O
+structures	O
+(	O
+Fig	O
+.	O
+2A	O
+,	O
+B	O
+)	O
+with	O
+a	O
+root	O
+mean	O
+square	O
+deviation	O
+(	O
+RMSD	O
+)	O
+of	O
+0	O
+.	O
+9	O
+Å	O
+for	O
+backbone	O
+and	O
+1	O
+.	O
+3	O
+Å	O
+for	O
+all	O
+heavy	O
+atoms	O
+(	O
+Table	O
+1	O
+)	O
+and	O
+estimated	O
+the	O
+presence	O
+of	O
+three	O
+helices	O
+spanning	O
+residues	O
+Q216	O
+-	O
+E240	O
+(	O
+α	O
+-	O
+helix	O
+1	O
+),	O
+P248	O
+-	O
+Q274	O
+(	O
+α	O
+-	O
+helix	O
+2	O
+),	O
+and	O
+E278	O
+-	O
+T306	O
+(	O
+α	O
+-	O
+helix	O
+3	O
+).	O
+	O
+NMR	O
+structural	O
+statistics	O
+for	O
+lowest	O
+energy	O
+conformers	O
+of	O
+Tom1	O
+GAT	O
+using	O
+PSVS	O
+.	O
+	O
+Much	O
+attention	O
+has	O
+been	O
+paid	O
+to	O
+the	O
+roles	O
+of	O
+haem	O
+-	O
+iron	O
+in	O
+cancer	O
+development	O
+.	O
+	O
+Thus	O
+,	O
+a	O
+tenuous	O
+balance	O
+between	O
+free	O
+haem	O
+and	O
+CO	O
+plays	O
+key	O
+roles	O
+in	O
+cancer	O
+development	O
+and	O
+chemoresistance	O
+,	O
+although	O
+the	O
+underlying	O
+mechanisms	O
+are	O
+not	O
+fully	O
+understood	O
+.	O
+	O
+Consequently	O
+,	O
+the	O
+five	O
+-	O
+coordinated	O
+haem	O
+of	O
+PGRMC1	O
+has	O
+an	O
+open	O
+surface	O
+that	O
+allows	O
+its	O
+dimerization	O
+through	O
+hydrophobic	O
+haem	O
+–	O
+haem	O
+stacking	O
+.	O
+	O
+Furthermore	O
+,	O
+free	O
+energy	O
+of	O
+dissociation	O
+predicted	O
+by	O
+PISA	O
+suggested	O
+that	O
+the	O
+haem	O
+-	O
+mediated	O
+dimer	O
+is	O
+stable	O
+in	O
+solution	O
+while	O
+the	O
+other	O
+potential	O
+interactions	O
+are	O
+not	O
+.	O
+	O
+A	O
+value	O
+of	O
+this	O
+kind	O
+implies	O
+that	O
+the	O
+PGRMC1	O
+dimer	O
+is	O
+more	O
+stable	O
+than	O
+other	O
+dimers	O
+of	O
+extracellular	O
+domain	O
+of	O
+membrane	O
+proteins	O
+such	O
+as	O
+Toll	O
+like	O
+receptor	O
+9	O
+(	O
+dimerization	O
+Kd	O
+of	O
+20	O
+μmol	O
+l	O
+−	O
+1	O
+)	O
+(	O
+ref	O
+.)	O
+and	O
+plexin	O
+A2	O
+receptor	O
+(	O
+dimerization	O
+Kd	O
+higher	O
+than	O
+300	O
+μmol	O
+l	O
+−	O
+1	O
+)	O
+(	O
+ref	O
+.).	O
+	O
+These	O
+results	O
+suggest	O
+that	O
+CO	O
+favours	O
+the	O
+six	O
+-	O
+coordinate	O
+form	O
+of	O
+haem	O
+and	O
+interferes	O
+with	O
+the	O
+haem	O
+-	O
+mediated	O
+dimerization	O
+of	O
+PGRMC1	O
+.	O
+	O
+Because	O
+PGRMC1	O
+is	O
+known	O
+to	O
+interact	O
+with	O
+EGFR	O
+and	O
+to	O
+accelerate	O
+tumour	O
+progression	O
+,	O
+we	O
+examined	O
+the	O
+effect	O
+of	O
+haem	O
+-	O
+dependent	O
+dimerization	O
+of	O
+PGRMC1	O
+on	O
+its	O
+interaction	O
+with	O
+EGFR	O
+by	O
+using	O
+purified	O
+proteins	O
+.	O
+	O
+We	O
+also	O
+examined	O
+the	O
+effect	O
+of	O
+succinylacetone	O
+(	O
+SA	O
+),	O
+an	O
+inhibitor	O
+of	O
+haem	O
+biosynthesis	O
+(	O
+Fig	O
+.	O
+4d	O
+).	O
+	O
+Thus	O
+,	O
+PGRMC1	O
+dimerization	O
+is	O
+important	O
+for	O
+cancer	O
+cell	O
+proliferation	O
+and	O
+chemoresistance	O
+.	O
+	O
+We	O
+examined	O
+the	O
+role	O
+of	O
+PGRMC1	O
+in	O
+metastatic	O
+progression	O
+by	O
+xenograft	O
+transplantation	O
+assays	O
+using	O
+super	O
+-	O
+immunodeficient	O
+NOD	O
+/	O
+scid	O
+/	O
+γnull	O
+(	O
+NOG	O
+)	O
+mice	O
+.	O
+	O
+Recombinant	O
+CYP1A2	O
+and	O
+CYP3A4	O
+including	O
+a	O
+microsomal	O
+formulation	O
+containing	O
+cytochrome	O
+b5	O
+and	O
+cytochrome	O
+P450	O
+reductase	O
+,	O
+drug	O
+-	O
+metabolizing	O
+cytochromes	O
+P450	O
+,	O
+interacted	O
+with	O
+wild	O
+-	O
+type	O
+PGRMC1	O
+,	O
+but	O
+not	O
+with	O
+the	O
+Y113F	B-mutant
+mutant	O
+,	O
+in	O
+a	O
+haem	O
+-	O
+dependent	O
+manner	O
+(	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+).	O
+	O
+Enhanced	O
+doxorubicin	O
+sensitivity	O
+was	O
+modestly	O
+but	O
+significantly	O
+induced	O
+by	O
+PGRMC1	B-mutant
+-	I-mutant
+KD	I-mutant
+.	O
+	O
+Recently	O
+,	O
+Lucas	O
+et	O
+al	O
+.	O
+reported	O
+that	O
+translationally	O
+-	O
+controlled	O
+tumour	O
+protein	O
+was	O
+dimerized	O
+by	O
+binding	O
+with	O
+haem	O
+,	O
+but	O
+its	O
+structural	O
+basis	O
+remains	O
+unclear	O
+.	O
+	O
+Sequence	O
+alignments	O
+show	O
+that	O
+haem	O
+-	O
+binding	O
+residues	O
+(	O
+Tyr113	O
+,	O
+Tyr107	O
+,	O
+Lys163	O
+and	O
+Tyr164	O
+)	O
+in	O
+PGRMC1	O
+are	O
+conserved	O
+among	O
+MAPR	O
+proteins	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+	O
+Moreover	O
+,	O
+exposure	O
+of	O
+cancer	O
+cells	O
+to	O
+stimuli	O
+such	O
+as	O
+hypoxia	O
+,	O
+radiation	O
+and	O
+chemotherapy	O
+causes	O
+cell	O
+damages	O
+and	O
+leads	O
+to	O
+protein	O
+degradation	O
+,	O
+resulting	O
+in	O
+increased	O
+levels	O
+of	O
+TCA	O
+cycle	O
+intermediates	O
+and	O
+in	O
+an	O
+enhanced	O
+haem	O
+biosynthesis	O
+.	O
+	O
+(	O
+a	O
+)	O
+FLAG	O
+-	O
+PGRMC1	O
+wild	O
+-	O
+type	O
+(	O
+wt	O
+)	O
+and	O
+Y113F	B-mutant
+mutant	O
+proteins	O
+(	O
+a	O
+.	O
+a	O
+.	O
+44	O
+–	O
+195	O
+),	O
+in	O
+either	O
+apo	O
+-	O
+or	O
+haem	O
+-	O
+bound	O
+form	O
+,	O
+were	O
+incubated	O
+with	O
+purified	O
+EGFR	O
+and	O
+co	O
+-	O
+immunoprecipitated	O
+with	O
+anti	O
+-	O
+FLAG	O
+antibody	O
+-	O
+conjugated	O
+beads	O
+.	O
+	O
+(	O
+g	O
+,	O
+h	O
+)	O
+HCT116	O
+cells	O
+were	O
+treated	O
+with	O
+or	O
+without	O
+EGF	O
+,	O
+SA	O
+,	O
+RuCl3	O
+and	O
+CORM3	O
+as	O
+indicated	O
+,	O
+and	O
+components	O
+of	O
+the	O
+EGFR	O
+signaling	O
+pathway	O
+were	O
+detected	O
+by	O
+Western	O
+blotting	O
+.	O
+	O
+(	O
+c	O
+)	O
+Binding	O
+assay	O
+was	O
+performed	O
+as	O
+in	O
+(	O
+a	O
+)	O
+using	O
+haem	O
+-	O
+bound	O
+FLAG	O
+-	O
+PGRMC1	O
+wt	O
+and	O
+CYP1A2	O
+with	O
+or	O
+without	O
+RuCl3	O
+and	O
+CORM3	O
+.	O
+	O
+We	O
+generated	O
+high	O
+-	O
+resolution	O
+structures	O
+of	O
+the	O
+1E6	O
+TCR	O
+bound	O
+to	O
+7	O
+altered	O
+peptide	O
+ligands	O
+,	O
+including	O
+a	O
+pathogen	O
+-	O
+derived	O
+peptide	O
+that	O
+was	O
+an	O
+order	O
+of	O
+magnitude	O
+more	O
+potent	O
+than	O
+the	O
+natural	O
+self	O
+-	O
+peptide	O
+.	O
+	O
+Highly	O
+potent	O
+antigens	O
+of	O
+the	O
+1E6	O
+TCR	O
+engaged	O
+with	O
+a	O
+strong	O
+antipathogen	O
+-	O
+like	O
+binding	O
+affinity	O
+;	O
+this	O
+engagement	O
+was	O
+governed	O
+though	O
+an	O
+energetic	O
+switch	O
+from	O
+an	O
+enthalpically	O
+to	O
+entropically	O
+driven	O
+interaction	O
+compared	O
+with	O
+the	O
+natural	O
+autoimmune	O
+ligand	O
+.	O
+	O
+This	O
+ability	O
+is	O
+required	O
+to	O
+enable	O
+the	O
+estimated	O
+25	O
+million	O
+distinct	O
+TCRs	O
+expressed	O
+in	O
+humans	O
+to	O
+provide	O
+effective	O
+immune	O
+coverage	O
+against	O
+all	O
+possible	O
+foreign	O
+peptide	O
+antigens	O
+.	O
+	O
+The	O
+RQFGPDWIVA	O
+sequence	O
+(	O
+present	O
+in	O
+C	O
+.	O
+asparagiforme	O
+)	O
+activated	O
+the	O
+1E6	O
+T	O
+cell	O
+with	O
+around	O
+1	O
+log	O
+–	O
+greater	O
+potency	O
+compared	O
+with	O
+ALWGPDPAAA	O
+.	O
+	O
+The	O
+range	O
+of	O
+Tm	O
+was	O
+between	O
+49	O
+.	O
+4	O
+°	O
+C	O
+(	O
+RQFGPDWIVA	O
+)	O
+and	O
+60	O
+.	O
+3	O
+°	O
+C	O
+(	O
+YQFGPDFPIA	O
+),	O
+with	O
+an	O
+average	O
+approximately	O
+55	O
+°	O
+C	O
+,	O
+similar	O
+to	O
+our	O
+previous	O
+findings	O
+.	O
+	O
+First	O
+,	O
+the	O
+1E6	O
+T	O
+cell	O
+could	O
+still	O
+functionally	O
+respond	O
+to	O
+peptide	O
+when	O
+the	O
+TCR	O
+binding	O
+affinity	O
+was	O
+extremely	O
+weak	O
+,	O
+e	O
+.	O
+g	O
+.,	O
+the	O
+1E6	O
+TCR	O
+binding	O
+affinity	O
+for	O
+the	O
+A2	O
+-	O
+MVWGPDPLYV	O
+peptide	O
+was	O
+KD	O
+=	O
+~	O
+600	O
+μM	O
+.	O
+Second	O
+,	O
+the	O
+1E6	O
+TCR	O
+bound	O
+to	O
+A2	O
+-	O
+RQFGPDFPTI	O
+with	O
+KD	O
+=	O
+0	O
+.	O
+5	O
+μM	O
+,	O
+equivalent	O
+to	O
+the	O
+binding	O
+affinity	O
+of	O
+the	O
+very	O
+strongest	O
+antipathogen	O
+TCRs	O
+.	O
+	O
+To	O
+confirm	O
+the	O
+affinity	O
+spread	O
+detected	O
+by	O
+SPR	O
+,	O
+and	O
+to	O
+evaluate	O
+whether	O
+experiments	O
+performed	O
+using	O
+soluble	O
+molecules	O
+were	O
+biologically	O
+relevant	O
+to	O
+events	O
+at	O
+the	O
+T	O
+cell	O
+surface	O
+,	O
+we	O
+determined	O
+the	O
+effective	O
+2D	O
+affinity	O
+of	O
+each	O
+APL	O
+using	O
+an	O
+adhesion	O
+frequency	O
+assay	O
+in	O
+which	O
+a	O
+human	O
+rbc	O
+coated	O
+in	O
+pMHC	O
+acted	O
+as	O
+an	O
+adhesion	O
+sensor	O
+.	O
+	O
+As	O
+with	O
+the	O
+3D	O
+affinity	O
+measurements	O
+,	O
+the	O
+2D	O
+affinity	O
+measurements	O
+correlated	O
+well	O
+with	O
+the	O
+EC50	O
+values	O
+for	O
+each	O
+ligand	O
+(	O
+Figure	O
+2K	O
+)	O
+demonstrating	O
+a	O
+strong	O
+correlation	O
+(	O
+Pearson	O
+’	O
+s	O
+correlation	O
+=	O
+0	O
+.	O
+8	O
+,	O
+P	O
+=	O
+0	O
+.	O
+01	O
+)	O
+between	O
+T	O
+cell	O
+antigen	O
+sensitivity	O
+and	O
+TCR	O
+binding	O
+affinity	O
+.	O
+	O
+Overall	O
+,	O
+the	O
+1E6	O
+TCR	O
+used	O
+a	O
+canonical	O
+binding	O
+mode	O
+to	O
+engage	O
+each	O
+APL	O
+with	O
+the	O
+TCR	O
+α	O
+-	O
+chain	O
+positioned	O
+over	O
+the	O
+MHC	O
+class	O
+I	O
+(	O
+MHCI	O
+)	O
+α2	O
+-	O
+helix	O
+and	O
+the	O
+TCR	O
+β	O
+-	O
+chain	O
+over	O
+the	O
+MHCI	O
+α	O
+-	O
+1	O
+helix	O
+,	O
+straddling	O
+the	O
+peptide	O
+cargo	O
+.	O
+	O
+Focused	O
+hotspot	O
+binding	O
+around	O
+a	O
+conserved	O
+GPD	O
+motif	O
+enables	O
+the	O
+1E6	O
+TCR	O
+to	O
+tolerate	O
+peptide	O
+degeneracy	O
+.	O
+	O
+Although	O
+the	O
+number	O
+of	O
+peptide	O
+contacts	O
+was	O
+a	O
+good	O
+predictor	O
+of	O
+TCR	O
+binding	O
+affinity	O
+for	O
+some	O
+of	O
+the	O
+APLs	O
+,	O
+for	O
+others	O
+,	O
+the	O
+correlation	O
+was	O
+poor	O
+(	O
+Pearson	O
+’	O
+s	O
+correlation	O
+=	O
+0	O
+.	O
+045	O
+,	O
+P	O
+=	O
+0	O
+.	O
+92	O
+),	O
+possibly	O
+because	O
+of	O
+different	O
+resolutions	O
+for	O
+each	O
+complex	O
+structure	O
+.	O
+	O
+The	O
+unligated	O
+A2	O
+-	O
+MVWGPDPLYV	O
+(	O
+KD	O
+=	O
+~	O
+600	O
+μM	O
+)	O
+structure	O
+revealed	O
+that	O
+the	O
+side	O
+chain	O
+Tyr9	O
+swung	O
+around	O
+8	O
+Å	O
+in	O
+the	O
+complex	O
+structure	O
+,	O
+subsequently	O
+making	O
+contacts	O
+with	O
+TCR	O
+residues	O
+Asp30β	O
+and	O
+Asn51β	O
+(	O
+Figure	O
+6A	O
+and	O
+Figure	O
+5A	O
+,	O
+respectively	O
+).	O
+	O
+The	O
+overall	O
+free	O
+binding	O
+energies	O
+(	O
+ΔG	O
+°)	O
+were	O
+between	O
+–	O
+4	O
+.	O
+4	O
+and	O
+–	O
+8	O
+.	O
+6	O
+kcal	O
+/	O
+mol	O
+,	O
+reflecting	O
+the	O
+wide	O
+range	O
+of	O
+TCR	O
+binding	O
+affinities	O
+we	O
+observed	O
+for	O
+the	O
+different	O
+APLs	O
+.	O
+	O
+The	O
+enthalpic	O
+contribution	O
+in	O
+each	O
+complex	O
+did	O
+not	O
+follow	O
+a	O
+clear	O
+trend	O
+with	O
+affinity	O
+,	O
+with	O
+all	O
+but	O
+the	O
+1E6	O
+-	O
+A2	O
+-	O
+RQFGPDFPTI	O
+interaction	O
+(	O
+ΔH	O
+°	O
+=	O
+6	O
+.	O
+3	O
+kcal	O
+/	O
+mol	O
+)	O
+generating	O
+an	O
+energetically	O
+favorable	O
+enthalpy	O
+value	O
+(	O
+ΔH	O
+°	O
+=	O
+–	O
+3	O
+.	O
+7	O
+to	O
+–	O
+11	O
+.	O
+4	O
+kcal	O
+/	O
+mol	O
+);	O
+this	O
+indicated	O
+a	O
+net	O
+gain	O
+in	O
+electrostatic	O
+interactions	O
+during	O
+complex	O
+formation	O
+.	O
+	O
+Furthermore	O
+,	O
+the	O
+structures	O
+of	O
+the	O
+unligated	O
+pMHCs	O
+demonstrated	O
+that	O
+,	O
+for	O
+these	O
+stronger	O
+-	O
+affinity	O
+ligands	O
+,	O
+there	O
+was	O
+less	O
+conformational	O
+difference	O
+between	O
+the	O
+TCR	O
+ligated	O
+pMHCs	O
+compared	O
+with	O
+the	O
+weaker	O
+-	O
+affinity	O
+ligands	O
+(	O
+Figure	O
+6	O
+).	O
+	O
+Sethi	O
+and	O
+colleagues	O
+recently	O
+demonstrated	O
+that	O
+the	O
+MHCII	O
+-	O
+restricted	O
+Hy	O
+.	O
+1B11	O
+TCR	O
+,	O
+which	O
+was	O
+isolated	O
+from	O
+a	O
+patient	O
+with	O
+multiple	O
+sclerosis	O
+,	O
+could	O
+anchor	O
+into	O
+a	O
+deep	O
+pocket	O
+formed	O
+from	O
+peptide	O
+residues	O
+2	O
+,	O
+3	O
+,	O
+and	O
+5	O
+(	O
+from	O
+MBP85	O
+–	O
+99	O
+bound	O
+to	O
+HLA	O
+-	O
+DQ1	O
+).	O
+	O
+Although	O
+the	O
+1E6	O
+T	O
+cell	O
+was	O
+able	O
+to	O
+activate	O
+weakly	O
+with	O
+peptides	O
+that	O
+lacked	O
+this	O
+motif	O
+,	O
+we	O
+were	O
+unable	O
+to	O
+robustly	O
+measure	O
+binding	O
+affinities	O
+or	O
+generate	O
+complex	O
+structures	O
+with	O
+these	O
+ligands	O
+,	O
+highlighting	O
+the	O
+central	O
+role	O
+of	O
+this	O
+interaction	O
+during	O
+1E6	O
+T	O
+cell	O
+antigen	O
+recognition	O
+.	O
+	O
+These	O
+findings	O
+are	O
+also	O
+analogous	O
+to	O
+the	O
+observed	O
+binding	O
+mode	O
+of	O
+the	O
+Hy	O
+.	O
+1B11	O
+TCR	O
+,	O
+in	O
+which	O
+one	O
+aromatic	O
+residue	O
+of	O
+the	O
+TCR	O
+CDR3α	O
+loop	O
+anchored	O
+into	O
+a	O
+pocket	O
+created	O
+by	O
+a	O
+conserved	O
+peptide	O
+motif	O
+.	O
+	O
+Despite	O
+some	O
+weak	O
+statistical	O
+correlation	O
+between	O
+the	O
+surface	O
+complementarity	O
+(	O
+SC	O
+)	O
+and	O
+affinity	O
+,	O
+closer	O
+inspection	O
+of	O
+the	O
+interface	O
+revealed	O
+no	O
+obvious	O
+structural	O
+signature	O
+that	O
+could	O
+definitively	O
+explain	O
+the	O
+differences	O
+in	O
+antigen	O
+potency	O
+and	O
+TCR	O
+binding	O
+strength	O
+between	O
+the	O
+different	O
+ligands	O
+.	O
+	O
+However	O
+,	O
+similar	O
+to	O
+our	O
+findings	O
+in	O
+other	O
+systems	O
+,	O
+modifications	O
+to	O
+residues	O
+outside	O
+of	O
+the	O
+canonical	O
+central	O
+peptide	O
+bulge	O
+were	O
+important	O
+for	O
+generating	O
+new	O
+interactions	O
+.	O
+	O
+These	O
+data	O
+also	O
+explain	O
+our	O
+previous	O
+findings	O
+that	O
+alteration	O
+of	O
+the	O
+anchor	O
+residue	O
+at	O
+peptide	O
+position	O
+2	O
+(	O
+Leu	B-mutant
+-	I-mutant
+Gln	I-mutant
+)	O
+has	O
+a	O
+direct	O
+effect	O
+on	O
+1E6	O
+TCR	O
+binding	O
+affinity	O
+because	O
+our	O
+structural	O
+analysis	O
+demonstrated	O
+that	O
+1E6	O
+made	O
+3	O
+additional	O
+bonds	O
+with	O
+A2	O
+-	O
+AQWGPDPAAA	O
+compared	O
+with	O
+A2	O
+-	O
+ALWGPDPAAA	O
+,	O
+consistent	O
+with	O
+the	O
+>	O
+3	O
+-	O
+fold	O
+stronger	O
+binding	O
+affinity	O
+.	O
+	O
+Although	O
+no	O
+energetic	O
+signature	O
+appears	O
+to	O
+exist	O
+for	O
+different	O
+TCRs	O
+,	O
+we	O
+used	O
+thermodynamic	O
+analysis	O
+here	O
+to	O
+explore	O
+whether	O
+changes	O
+in	O
+energetics	O
+could	O
+help	O
+explain	O
+ligand	O
+discrimination	O
+by	O
+a	O
+single	O
+TCR	O
+.	O
+	O
+This	O
+analysis	O
+demonstrated	O
+a	O
+strong	O
+relationship	O
+(	O
+according	O
+to	O
+the	O
+Pearson	O
+’	O
+s	O
+correlation	O
+analysis	O
+)	O
+between	O
+the	O
+energetic	O
+signature	O
+used	O
+by	O
+the	O
+1E6	O
+TCR	O
+and	O
+the	O
+sensitivity	O
+of	O
+the	O
+1E6	O
+T	O
+cell	O
+clone	O
+to	O
+different	O
+APLs	O
+.	O
+	O
+(	O
+C	O
+)	O
+The	O
+1E6	O
+T	O
+cell	O
+clone	O
+was	O
+stained	O
+,	O
+in	O
+duplicate	O
+,	O
+with	O
+tetramers	O
+composed	O
+of	O
+each	O
+APL	O
+(	O
+colored	O
+as	O
+above	O
+)	O
+presented	O
+by	O
+HLA	O
+-	O
+A	O
+*	O
+0201	O
+.	O
+(	O
+D	O
+)	O
+The	O
+stability	O
+of	O
+each	O
+APL	O
+(	O
+colored	O
+as	O
+above	O
+)	O
+was	O
+tested	O
+,	O
+in	O
+duplicate	O
+,	O
+using	O
+CD	O
+by	O
+recording	O
+the	O
+peak	O
+at	O
+218	O
+nm	O
+absorbance	O
+from	O
+5	O
+°	O
+C	O
+–	O
+90	O
+°	O
+C	O
+.	O
+	O
+The	O
+1E6	O
+TCR	O
+uses	O
+a	O
+conserved	O
+binding	O
+mode	O
+to	O
+engage	O
+A2	O
+-	O
+ALWGPDPAAA	O
+and	O
+the	O
+APLs	O
+.	O
+	O
+Interaction	O
+between	O
+1E6	O
+TCR	O
+(	O
+gray	O
+illustration	O
+)	O
+residues	O
+Tyr97α	O
+and	O
+Tyr97β	O
+(	O
+the	O
+position	O
+of	O
+these	O
+side	O
+chains	O
+in	O
+the	O
+TCR	O
+in	O
+complex	O
+with	O
+all	O
+7	O
+APLs	O
+,	O
+and	O
+the	O
+previously	O
+reported	O
+A2	O
+-	O
+ALWGPDPAAA	O
+epitope	O
+,	O
+is	O
+shown	O
+in	O
+multicolored	O
+sticks	O
+;	O
+ref	O
+.)	O
+and	O
+the	O
+GPD	O
+peptide	O
+motif	O
+(	O
+the	O
+position	O
+of	O
+these	O
+side	O
+chains	O
+in	O
+all	O
+7	O
+APLs	O
+and	O
+A2	O
+-	O
+ALWGPDPAAA	O
+in	O
+complex	O
+with	O
+the	O
+1E6	O
+TCR	O
+is	O
+shown	O
+in	O
+multicolored	O
+sticks	O
+).	O
+	O
+Interactions	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+peptide	O
+residues	O
+outside	O
+of	O
+the	O
+conserved	O
+GPD	O
+motif	O
+.	O
+	O
+Hydrogen	O
+bonds	O
+are	O
+shown	O
+as	O
+red	O
+dotted	O
+lines	O
+;	O
+van	O
+der	O
+Waals	O
+(	O
+vdW	O
+)	O
+contacts	O
+are	O
+shown	O
+as	O
+black	O
+dotted	O
+lines	O
+.	O
+	O
+(	O
+A	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+black	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+MVWGPDPLYV	O
+(	O
+black	O
+illustration	O
+and	O
+sticks	O
+).	O
+(	O
+B	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+red	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+YLGGPDFPTI	O
+(	O
+red	O
+illustration	O
+and	O
+sticks	O
+).	O
+(	O
+C	O
+)	O
+Interaction	O
+between	O
+the	O
+1E6	O
+TCR	O
+(	O
+blue	O
+illustration	O
+and	O
+sticks	O
+)	O
+and	O
+A2	O
+-	O
+ALWGPDPAAA	O
+(	O
+blue	O
+illustration	O
+and	O
+sticks	O
+)	O
+reproduced	O
+from	O
+previous	O
+published	O
+data	O
+.	O
+	O
+The	O
+1E6	O
+TCR	O
+makes	O
+distinct	O
+peptide	O
+contacts	O
+with	O
+the	O
+MHC	O
+surface	O
+depending	O
+on	O
+the	O
+peptide	O
+cargo	O
+.	O
+	O
+Boxes	O
+show	O
+total	O
+contacts	O
+between	O
+the	O
+1E6	O
+TCR	O
+and	O
+these	O
+key	O
+residues	O
+(	O
+green	O
+boxes	O
+are	O
+MHC	O
+residues	O
+;	O
+white	O
+boxes	O
+are	O
+TCR	O
+residues	O
+).	O
+	O