diff --git "a/annotation_IOB/all.tsv" "b/annotation_IOB/all.tsv"
new file mode 100644--- /dev/null
+++ "b/annotation_IOB/all.tsv"
@@ -0,0 +1,135766 @@
+Molecular	O
+Dissection	O
+of	O
+Xyloglucan	B-chemical
+Recognition	O
+in	O
+a	O
+Prominent	O
+Human	B-species
+Gut	O
+Symbiont	O
+
+Polysaccharide	B-gene
+utilization	I-gene
+loci	I-gene
+(	O
+PUL	B-gene
+)	O
+within	O
+the	O
+genomes	O
+of	O
+resident	O
+human	B-species
+gut	O
+Bacteroidetes	B-taxonomy_domain
+are	O
+central	O
+to	O
+the	O
+metabolism	O
+of	O
+the	O
+otherwise	O
+indigestible	O
+complex	O
+carbohydrates	B-chemical
+known	O
+as	O
+“	O
+dietary	O
+fiber	O
+.”	O
+However	O
+,	O
+functional	O
+characterization	O
+of	O
+PUL	B-gene
+lags	O
+significantly	O
+behind	O
+sequencing	O
+efforts	O
+,	O
+which	O
+limits	O
+physiological	O
+understanding	O
+of	O
+the	O
+human	B-species
+-	O
+bacterial	B-taxonomy_domain
+symbiosis	O
+.	O
+
+In	O
+particular	O
+,	O
+the	O
+molecular	O
+basis	O
+of	O
+complex	B-chemical
+polysaccharide	I-chemical
+recognition	O
+,	O
+an	O
+essential	O
+prerequisite	O
+to	O
+hydrolysis	O
+by	O
+cell	O
+surface	O
+glycosidases	B-protein_type
+and	O
+subsequent	O
+metabolism	O
+,	O
+is	O
+generally	O
+poorly	O
+understood	O
+.	O
+
+Here	O
+,	O
+we	O
+present	O
+the	O
+biochemical	B-experimental_method
+,	I-experimental_method
+structural	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+reverse	I-experimental_method
+genetic	I-experimental_method
+characterization	I-experimental_method
+of	O
+two	O
+unique	O
+cell	B-protein_type
+surface	I-protein_type
+glycan	I-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+(	O
+SGBPs	B-protein_type
+)	O
+encoded	O
+by	O
+a	O
+xyloglucan	B-gene
+utilization	I-gene
+locus	I-gene
+(	O
+XyGUL	B-gene
+)	O
+from	O
+Bacteroides	B-species
+ovatus	I-species
+,	O
+which	O
+are	O
+integral	O
+to	O
+growth	O
+on	O
+this	O
+key	O
+dietary	O
+vegetable	B-taxonomy_domain
+polysaccharide	B-chemical
+.	O
+
+Biochemical	B-experimental_method
+analysis	I-experimental_method
+reveals	O
+that	O
+these	O
+outer	B-protein_type
+membrane	I-protein_type
+-	I-protein_type
+anchored	I-protein_type
+proteins	I-protein_type
+are	O
+in	O
+fact	O
+exquisitely	O
+specific	O
+for	O
+the	O
+highly	O
+branched	O
+xyloglucan	B-chemical
+(	O
+XyG	B-chemical
+)	O
+polysaccharide	B-chemical
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+a	O
+SusD	B-protein
+homolog	O
+,	O
+with	O
+a	O
+bound	B-protein_state
+XyG	B-chemical
+tetradecasaccharide	B-chemical
+reveals	O
+an	O
+extended	O
+carbohydrate	B-site
+-	I-site
+binding	I-site
+platform	I-site
+that	O
+primarily	O
+relies	O
+on	O
+recognition	O
+of	O
+the	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+backbone	O
+.	O
+
+The	O
+unique	O
+,	O
+tetra	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+structure	B-evidence
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+is	O
+comprised	O
+of	O
+tandem	B-structure_element
+Ig	I-structure_element
+-	I-structure_element
+like	I-structure_element
+folds	I-structure_element
+,	O
+with	O
+XyG	B-chemical
+binding	O
+mediated	O
+at	O
+the	O
+distal	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+.	O
+
+Despite	O
+displaying	O
+similar	O
+affinities	B-evidence
+for	O
+XyG	B-chemical
+,	O
+reverse	B-experimental_method
+-	I-experimental_method
+genetic	I-experimental_method
+analysis	I-experimental_method
+reveals	O
+that	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+is	O
+only	O
+required	O
+for	O
+the	O
+efficient	O
+capture	O
+of	O
+smaller	O
+oligosaccharides	B-chemical
+,	O
+whereas	O
+the	O
+presence	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+is	O
+more	O
+critical	O
+than	O
+its	O
+carbohydrate	B-chemical
+-	O
+binding	O
+ability	O
+for	O
+growth	O
+on	O
+XyG	B-chemical
+.	O
+Together	O
+,	O
+these	O
+data	O
+demonstrate	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+play	O
+complementary	O
+,	O
+specialized	O
+roles	O
+in	O
+carbohydrate	B-chemical
+capture	O
+by	O
+B	B-species
+.	I-species
+ovatus	I-species
+and	O
+elaborate	O
+a	O
+model	O
+of	O
+how	O
+vegetable	B-taxonomy_domain
+xyloglucans	B-chemical
+are	O
+accessed	O
+by	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+.	O
+
+The	O
+Bacteroidetes	B-taxonomy_domain
+are	O
+dominant	O
+bacteria	B-taxonomy_domain
+in	O
+the	O
+human	B-species
+gut	O
+that	O
+are	O
+responsible	O
+for	O
+the	O
+digestion	O
+of	O
+the	O
+complex	B-chemical
+polysaccharides	I-chemical
+that	O
+constitute	O
+“	O
+dietary	O
+fiber	O
+.”	O
+Although	O
+this	O
+symbiotic	O
+relationship	O
+has	O
+been	O
+appreciated	O
+for	O
+decades	O
+,	O
+little	O
+is	O
+currently	O
+known	O
+about	O
+how	O
+Bacteroidetes	B-taxonomy_domain
+seek	O
+out	O
+and	O
+bind	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+polysaccharides	B-chemical
+as	O
+a	O
+necessary	O
+first	O
+step	O
+in	O
+their	O
+metabolism	O
+.	O
+
+Here	O
+,	O
+we	O
+provide	O
+the	O
+first	O
+biochemical	B-experimental_method
+,	I-experimental_method
+crystallographic	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+genetic	I-experimental_method
+insight	I-experimental_method
+into	O
+how	O
+two	O
+surface	B-protein_type
+glycan	I-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+from	O
+the	O
+complex	O
+Bacteroides	B-species
+ovatus	I-species
+xyloglucan	B-gene
+utilization	I-gene
+locus	I-gene
+(	O
+XyGUL	B-gene
+)	O
+enable	O
+recognition	O
+and	O
+uptake	O
+of	O
+this	O
+ubiquitous	O
+vegetable	B-taxonomy_domain
+polysaccharide	B-chemical
+.	O
+
+Our	O
+combined	O
+analysis	O
+illuminates	O
+new	O
+fundamental	O
+aspects	O
+of	O
+complex	B-chemical
+polysaccharide	I-chemical
+recognition	O
+,	O
+cleavage	O
+,	O
+and	O
+import	O
+at	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+cell	O
+surface	O
+that	O
+may	O
+facilitate	O
+the	O
+development	O
+of	O
+prebiotics	O
+to	O
+target	O
+this	O
+phylum	O
+of	O
+gut	O
+bacteria	B-taxonomy_domain
+.	O
+
+The	O
+human	B-species
+gut	O
+microbiota	B-taxonomy_domain
+influences	O
+the	O
+course	O
+of	O
+human	B-species
+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
+
+This	O
+microbial	B-taxonomy_domain
+community	O
+is	O
+largely	O
+bacterial	B-taxonomy_domain
+,	O
+with	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+,	O
+Firmicutes	B-taxonomy_domain
+,	O
+and	O
+Actinobacteria	B-taxonomy_domain
+comprising	O
+the	O
+dominant	O
+phyla	O
+.	O
+
+The	O
+ability	O
+to	O
+acquire	O
+energy	O
+from	O
+carbohydrates	B-chemical
+of	O
+dietary	O
+or	O
+host	O
+origin	O
+is	O
+central	O
+to	O
+the	O
+adaptation	O
+of	O
+human	B-species
+gut	O
+bacterial	B-taxonomy_domain
+species	O
+to	O
+their	O
+niche	O
+.	O
+
+More	O
+importantly	O
+,	O
+this	O
+makes	O
+diet	O
+a	O
+tractable	O
+way	O
+to	O
+manipulate	O
+the	O
+abundance	O
+and	O
+metabolic	O
+output	O
+of	O
+the	O
+microbiota	B-taxonomy_domain
+toward	O
+improved	O
+human	B-species
+health	O
+.	O
+
+However	O
+,	O
+there	O
+is	O
+a	O
+paucity	O
+of	O
+data	O
+regarding	O
+how	O
+the	O
+vast	O
+array	O
+of	O
+complex	B-chemical
+carbohydrate	I-chemical
+structures	O
+are	O
+selectively	O
+recognized	O
+and	O
+imported	O
+by	O
+members	O
+of	O
+the	O
+microbiota	B-taxonomy_domain
+,	O
+a	O
+critical	O
+process	O
+that	O
+enables	O
+these	O
+organisms	O
+to	O
+thrive	O
+in	O
+the	O
+competitive	O
+gut	O
+environment	O
+.	O
+
+The	O
+human	B-species
+gut	O
+bacteria	B-taxonomy_domain
+Bacteroidetes	B-taxonomy_domain
+share	O
+a	O
+profound	O
+capacity	O
+for	O
+dietary	O
+glycan	B-chemical
+degradation	O
+,	O
+with	O
+many	O
+species	O
+containing	O
+>	O
+250	O
+predicted	O
+carbohydrate	O
+-	O
+active	O
+enzymes	O
+(	O
+CAZymes	O
+),	O
+compared	O
+to	O
+50	O
+to	O
+100	O
+within	O
+many	O
+Firmicutes	B-taxonomy_domain
+and	O
+only	O
+17	O
+in	O
+the	O
+human	B-species
+genome	O
+devoted	O
+toward	O
+carbohydrate	O
+utilization	O
+.	O
+
+A	O
+remarkable	O
+feature	O
+of	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+is	O
+the	O
+packaging	O
+of	O
+genes	O
+for	O
+carbohydrate	O
+catabolism	O
+into	O
+discrete	O
+polysaccharide	B-gene
+utilization	I-gene
+loci	I-gene
+(	O
+PUL	B-gene
+),	O
+which	O
+are	O
+transcriptionally	O
+regulated	O
+by	O
+specific	O
+substrate	O
+signatures	O
+.	O
+
+The	O
+archetypal	O
+PUL	B-gene
+-	O
+encoded	O
+system	O
+is	O
+the	O
+starch	B-complex_assembly
+utilization	I-complex_assembly
+system	I-complex_assembly
+(	O
+Sus	B-complex_assembly
+)	O
+(	O
+Fig	O
+.	O
+1B	O
+)	O
+of	O
+Bacteroides	B-species
+thetaiotaomicron	I-species
+.	O
+
+The	O
+Sus	B-complex_assembly
+includes	O
+a	O
+lipid	B-protein_state
+-	I-protein_state
+anchored	I-protein_state
+,	O
+outer	O
+membrane	O
+endo	B-protein_type
+-	I-protein_type
+amylase	I-protein_type
+,	O
+SusG	B-protein
+;	O
+a	O
+TonB	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+transporter	I-protein_type
+(	O
+TBDT	B-protein_type
+),	O
+SusC	B-protein
+,	O
+which	O
+imports	O
+oligosaccharides	B-chemical
+with	O
+the	O
+help	O
+of	O
+an	O
+associated	O
+starch	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+,	O
+SusD	B-protein
+;	O
+two	O
+additional	O
+carbohydrate	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+lipoproteins	I-protein_type
+,	O
+SusE	B-protein
+and	O
+SusF	B-protein
+;	O
+and	O
+two	O
+periplasmic	O
+exo	B-protein_type
+-	I-protein_type
+glucosidases	I-protein_type
+,	O
+SusA	B-protein
+and	O
+SusB	B-protein
+,	O
+which	O
+generate	O
+glucose	B-chemical
+for	O
+transport	O
+into	O
+the	O
+cytoplasm	O
+.	O
+
+The	O
+importance	O
+of	O
+PUL	B-gene
+as	O
+a	O
+successful	O
+evolutionary	O
+strategy	O
+is	O
+underscored	O
+by	O
+the	O
+observation	O
+that	O
+Bacteroidetes	B-taxonomy_domain
+such	O
+as	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+and	O
+Bacteroides	B-species
+ovatus	I-species
+devote	O
+~	O
+18	O
+%	O
+of	O
+their	O
+genomes	O
+to	O
+these	O
+systems	O
+.	O
+
+Moving	O
+beyond	O
+seminal	O
+genomic	O
+and	O
+transcriptomic	O
+analyses	O
+,	O
+the	O
+current	O
+state	O
+-	O
+of	O
+-	O
+the	O
+-	O
+art	O
+PUL	B-gene
+characterization	O
+involves	O
+combined	O
+reverse	B-experimental_method
+-	I-experimental_method
+genetic	I-experimental_method
+,	I-experimental_method
+biochemical	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+studies	I-experimental_method
+to	O
+illuminate	O
+the	O
+molecular	O
+details	O
+of	O
+PUL	B-gene
+function	O
+.	O
+
+Xyloglucan	B-chemical
+and	O
+the	O
+Bacteroides	B-species
+ovatus	I-species
+xyloglucan	B-gene
+utilization	I-gene
+locus	I-gene
+(	O
+XyGUL	B-gene
+).	O
+(	O
+A	O
+)	O
+Representative	O
+structures	B-evidence
+of	O
+common	O
+xyloglucans	B-chemical
+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
+
+Cleavage	O
+sites	O
+for	O
+BoXyGUL	B-gene
+glycosidases	B-protein_type
+(	O
+GHs	B-protein_type
+)	O
+are	O
+indicated	O
+for	O
+solanaceous	B-taxonomy_domain
+xyloglucan	B-chemical
+.	O
+(	O
+B	O
+)	O
+BtSus	B-gene
+and	O
+BoXyGUL	B-gene
+.	O
+(	O
+C	O
+)	O
+Localization	O
+of	O
+BoXyGUL	B-gene
+-	O
+encoded	O
+proteins	O
+in	O
+cellular	O
+membranes	O
+and	O
+concerted	O
+modes	O
+of	O
+action	O
+in	O
+the	O
+degradation	O
+of	O
+xyloglucans	B-chemical
+to	O
+monosaccharides	O
+.	O
+
+The	O
+location	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+/	O
+B	B-protein
+is	O
+presented	O
+in	O
+this	O
+work	O
+;	O
+the	O
+location	O
+of	O
+GH5	B-protein
+has	O
+been	O
+empirically	O
+determined	O
+,	O
+and	O
+the	O
+enzymes	O
+have	O
+been	O
+placed	O
+based	O
+upon	O
+their	O
+predicted	O
+cellular	O
+location	O
+.	O
+
+We	O
+recently	O
+reported	O
+the	O
+detailed	O
+molecular	O
+characterization	O
+of	O
+a	O
+PUL	B-gene
+that	O
+confers	O
+the	O
+ability	O
+of	O
+the	O
+human	B-species
+gut	O
+commensal	O
+B	B-species
+.	I-species
+ovatus	I-species
+ATCC	I-species
+8483	I-species
+to	O
+grow	O
+on	O
+a	O
+prominent	O
+family	O
+of	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+glycans	B-chemical
+,	O
+the	O
+xyloglucans	B-chemical
+(	O
+XyG	B-chemical
+).	O
+
+XyG	B-chemical
+variants	O
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+constitute	O
+up	O
+to	O
+25	O
+%	O
+of	O
+the	O
+dry	O
+weight	O
+of	O
+common	O
+vegetables	B-taxonomy_domain
+.	O
+
+Analogous	O
+to	O
+the	O
+Sus	B-gene
+locus	I-gene
+,	O
+the	O
+xyloglucan	B-gene
+utilization	I-gene
+locus	I-gene
+(	O
+XyGUL	B-gene
+)	O
+encodes	O
+a	O
+cohort	O
+of	O
+carbohydrate	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+,	I-protein_type
+-	I-protein_type
+hydrolyzing	I-protein_type
+,	I-protein_type
+and	I-protein_type
+-	I-protein_type
+importing	I-protein_type
+proteins	I-protein_type
+(	O
+Fig	O
+.	O
+1B	O
+and	O
+C	O
+).	O
+
+The	O
+number	O
+of	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+(	O
+GHs	B-protein_type
+)	O
+encoded	O
+by	O
+the	O
+XyGUL	B-gene
+is	O
+,	O
+however	O
+,	O
+more	O
+expansive	O
+than	O
+that	O
+by	O
+the	O
+Sus	B-gene
+locus	I-gene
+(	O
+Fig	O
+.	O
+1B	O
+),	O
+which	O
+reflects	O
+the	O
+greater	O
+complexity	O
+of	O
+glycosidic	O
+linkages	O
+found	O
+in	O
+XyG	B-chemical
+vis	O
+-	O
+à	O
+-	O
+vis	O
+starch	B-chemical
+.	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	B-protein_type
+,	O
+a	O
+key	O
+outstanding	O
+question	O
+regarding	O
+this	O
+locus	O
+is	O
+how	O
+XyG	B-chemical
+recognition	O
+is	O
+mediated	O
+at	O
+the	O
+cell	O
+surface	O
+.	O
+
+In	O
+the	O
+archetypal	O
+starch	B-complex_assembly
+utilization	I-complex_assembly
+system	I-complex_assembly
+of	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+,	O
+starch	O
+binding	O
+to	O
+the	O
+cell	O
+surface	O
+is	O
+mediated	O
+at	O
+eight	O
+distinct	O
+starch	B-site
+-	I-site
+binding	I-site
+sites	I-site
+distributed	O
+among	O
+four	O
+surface	B-protein_type
+glycan	I-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+(	O
+SGBPs	B-protein_type
+):	O
+two	O
+within	O
+the	O
+amylase	B-protein_type
+SusG	B-protein
+,	O
+one	O
+within	O
+SusD	B-protein
+,	O
+two	O
+within	O
+SusE	B-protein
+,	O
+and	O
+three	O
+within	O
+SusF	B-protein
+.	O
+The	O
+functional	O
+redundancy	O
+of	O
+many	O
+of	O
+these	O
+sites	O
+is	O
+high	O
+:	O
+whereas	O
+SusD	B-protein
+is	O
+essential	O
+for	O
+growth	O
+on	O
+starch	B-chemical
+,	O
+combined	O
+mutations	O
+of	O
+the	O
+SusE	B-protein
+,	O
+SusF	B-protein
+,	O
+and	O
+SusG	B-protein
+binding	B-site
+sites	I-site
+are	O
+required	O
+to	O
+impair	O
+growth	O
+on	O
+the	O
+polysaccharide	B-chemical
+.	O
+
+Bacteroidetes	B-taxonomy_domain
+PUL	B-gene
+ubiquitously	O
+encode	O
+homologs	O
+of	O
+SusC	B-protein
+and	O
+SusD	B-protein
+,	O
+as	O
+well	O
+as	O
+proteins	O
+whose	O
+genes	O
+are	O
+immediately	O
+downstream	O
+of	O
+susD	B-gene
+,	O
+akin	O
+to	O
+susE	B-gene
+/	I-gene
+F	I-gene
+,	O
+and	O
+these	O
+are	O
+typically	O
+annotated	O
+as	O
+“	O
+putative	B-protein_state
+lipoproteins	B-protein_type
+”.	O
+
+The	O
+genes	O
+coding	O
+for	O
+these	O
+proteins	O
+,	O
+sometimes	O
+referred	O
+to	O
+as	O
+“	O
+susE	B-gene
+/	I-gene
+F	I-gene
+positioned	O
+,”	O
+display	O
+products	O
+with	O
+a	O
+wide	O
+variation	O
+in	O
+amino	O
+acid	O
+sequence	O
+and	O
+which	O
+have	O
+little	O
+or	O
+no	O
+homology	O
+to	O
+other	O
+PUL	B-gene
+-	O
+encoded	O
+proteins	O
+or	O
+known	O
+carbohydrate	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+.	O
+
+As	O
+the	O
+Sus	B-complex_assembly
+SGBPs	B-protein_type
+remain	O
+the	O
+only	O
+structurally	O
+characterized	O
+cohort	O
+to	O
+date	O
+,	O
+we	O
+therefore	O
+wondered	O
+whether	O
+such	O
+glycan	B-chemical
+binding	O
+and	O
+function	O
+are	O
+extended	O
+to	O
+other	O
+PUL	B-gene
+that	O
+target	O
+more	O
+complex	O
+and	O
+heterogeneous	O
+polysaccharides	B-chemical
+,	O
+such	O
+as	O
+XyG	B-chemical
+.	O
+
+We	O
+describe	O
+here	O
+the	O
+detailed	O
+functional	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+the	O
+noncatalytic	B-protein_state
+SGBPs	B-protein_type
+encoded	O
+by	O
+Bacova_02651	B-gene
+and	O
+Bacova_02650	B-gene
+of	O
+the	O
+XyGUL	B-gene
+,	O
+here	O
+referred	O
+to	O
+as	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+to	O
+elucidate	O
+their	O
+molecular	O
+roles	O
+in	O
+carbohydrate	O
+acquisition	O
+in	O
+vivo	O
+.	O
+
+Combined	O
+biochemical	B-experimental_method
+,	I-experimental_method
+structural	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+reverse	I-experimental_method
+-	I-experimental_method
+genetic	I-experimental_method
+approaches	I-experimental_method
+clearly	O
+illuminate	O
+the	O
+distinct	O
+,	O
+yet	O
+complementary	O
+,	O
+functions	O
+that	O
+these	O
+two	O
+proteins	O
+play	O
+in	O
+XyG	B-chemical
+recognition	O
+as	O
+it	O
+impacts	O
+the	O
+physiology	O
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+.	O
+
+These	O
+data	O
+extend	O
+our	O
+current	O
+understanding	O
+of	O
+the	O
+Sus	O
+-	O
+like	O
+glycan	B-chemical
+uptake	O
+paradigm	O
+within	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+and	O
+reveals	O
+how	O
+the	O
+complex	O
+dietary	O
+polysaccharide	B-chemical
+xyloglucan	B-chemical
+is	O
+recognized	O
+at	O
+the	O
+cell	O
+surface	O
+.	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+are	O
+cell	B-protein_type
+-	I-protein_type
+surface	I-protein_type
+-	I-protein_type
+localized	I-protein_type
+,	I-protein_type
+xyloglucan	I-protein_type
+-	I-protein_type
+specific	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+.	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+encoded	O
+by	O
+the	O
+XyGUL	B-gene
+locus	O
+tag	O
+Bacova_02651	B-gene
+(	O
+Fig	O
+.	O
+1B	O
+),	O
+shares	O
+26	O
+%	O
+amino	O
+acid	O
+sequence	O
+identity	O
+(	O
+40	O
+%	O
+similarity	O
+)	O
+with	O
+its	O
+homolog	O
+,	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+SusD	B-protein
+,	O
+and	O
+similar	O
+homology	O
+with	O
+the	O
+SusD	B-protein_type
+-	I-protein_type
+like	I-protein_type
+proteins	I-protein_type
+encoded	O
+within	O
+syntenic	O
+XyGUL	B-gene
+identified	O
+in	O
+our	O
+earlier	O
+work	O
+.	O
+
+In	O
+contrast	O
+,	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+encoded	O
+by	O
+locus	O
+tag	O
+Bacova_02650	B-gene
+,	O
+displays	O
+little	O
+sequence	O
+similarity	O
+to	O
+the	O
+products	O
+of	O
+similarly	O
+positioned	O
+genes	O
+in	O
+syntenic	O
+XyGUL	B-gene
+nor	O
+to	O
+any	O
+other	O
+gene	O
+product	O
+among	O
+the	O
+diversity	O
+of	O
+Bacteroidetes	B-taxonomy_domain
+PUL	B-gene
+.	O
+
+Whereas	O
+sequence	O
+similarity	O
+among	O
+SusC	B-protein
+/	O
+SusD	B-protein
+homolog	O
+pairs	O
+often	O
+serves	O
+as	O
+a	O
+hallmark	O
+for	O
+PUL	B-gene
+identification	O
+,	O
+the	O
+sequence	O
+similarities	O
+of	O
+downstream	O
+genes	O
+encoding	O
+SGBPs	B-protein_type
+are	O
+generally	O
+too	O
+low	O
+to	O
+allow	O
+reliable	O
+bioinformatic	O
+classification	O
+of	O
+their	O
+products	O
+into	O
+protein	O
+families	O
+,	O
+let	O
+alone	O
+prediction	O
+of	O
+function	O
+.	O
+
+Hence	O
+,	O
+there	O
+is	O
+a	O
+critical	O
+need	O
+for	O
+the	O
+elucidation	O
+of	O
+detailed	O
+structure	O
+-	O
+function	O
+relationships	O
+among	O
+PUL	B-gene
+SGBPs	B-protein_type
+,	O
+in	O
+light	O
+of	O
+the	O
+manifold	O
+glycan	B-chemical
+structures	O
+in	O
+nature	O
+.	O
+
+Immunofluorescence	B-experimental_method
+of	O
+formaldehyde	O
+-	O
+fixed	O
+,	O
+nonpermeabilized	O
+cells	O
+grown	O
+in	O
+minimal	O
+medium	O
+with	O
+XyG	B-chemical
+as	O
+the	O
+sole	O
+carbon	O
+source	O
+to	O
+induce	O
+XyGUL	B-gene
+expression	O
+,	O
+reveals	O
+that	O
+both	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+are	O
+presented	O
+on	O
+the	O
+cell	O
+surface	O
+by	O
+N	O
+-	O
+terminal	O
+lipidation	B-ptm
+,	O
+as	O
+predicted	O
+by	O
+signal	O
+peptide	O
+analysis	O
+with	O
+SignalP	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+
+Here	O
+,	O
+the	O
+SGBPs	B-protein_type
+very	O
+likely	O
+work	O
+in	O
+concert	O
+with	O
+the	O
+cell	B-protein_type
+-	I-protein_type
+surface	I-protein_type
+-	I-protein_type
+localized	I-protein_type
+endo	I-protein_type
+-	I-protein_type
+xyloglucanase	I-protein_type
+B	B-species
+.	I-species
+ovatus	I-species
+GH5	B-protein
+(	O
+BoGH5	B-protein
+)	O
+to	O
+recruit	O
+and	O
+cleave	O
+XyG	B-chemical
+for	O
+subsequent	O
+periplasmic	O
+import	O
+via	O
+the	O
+SusC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+TBDT	I-protein_type
+of	O
+the	O
+XyGUL	B-gene
+(	O
+Fig	O
+.	O
+1B	O
+and	O
+C	O
+).	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+visualized	O
+by	O
+immunofluorescence	B-experimental_method
+.	O
+
+Formalin	O
+-	O
+fixed	O
+,	O
+nonpermeabilized	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+were	O
+grown	O
+in	O
+minimal	O
+medium	O
+plus	O
+XyG	B-chemical
+,	O
+probed	O
+with	O
+custom	O
+rabbit	O
+antibodies	O
+to	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+or	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+and	O
+then	O
+stained	O
+with	O
+Alexa	O
+Fluor	O
+488	O
+goat	O
+anti	O
+-	O
+rabbit	O
+IgG	O
+.	O
+(	O
+A	O
+)	O
+Overlay	B-experimental_method
+of	O
+bright	B-evidence
+-	I-evidence
+field	I-evidence
+and	I-evidence
+FITC	I-evidence
+images	I-evidence
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+A	O
+.	O
+(	O
+B	O
+)	O
+Overlay	B-experimental_method
+of	O
+bright	B-evidence
+-	I-evidence
+field	I-evidence
+and	I-evidence
+FITC	I-evidence
+images	I-evidence
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+.	O
+(	O
+C	O
+)	O
+Bright	B-evidence
+-	I-evidence
+field	I-evidence
+image	I-evidence
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+antibodies	O
+.	O
+
+(	O
+D	O
+)	O
+FITC	B-evidence
+images	I-evidence
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+antibodies	O
+.	O
+
+Cells	O
+lacking	B-protein_state
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+)	O
+do	O
+not	O
+grow	O
+on	O
+XyG	B-chemical
+and	O
+therefore	O
+could	O
+not	O
+be	O
+tested	O
+in	O
+parallel	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	B-protein_type
+hydrolases	I-protein_type
+of	O
+the	O
+XyGUL	B-gene
+,	O
+we	O
+reported	O
+preliminary	O
+affinity	B-experimental_method
+PAGE	I-experimental_method
+and	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+ITC	B-experimental_method
+)	O
+data	O
+indicating	O
+that	O
+both	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+are	O
+competent	O
+xyloglucan	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+(	O
+affinity	B-evidence
+constant	I-evidence
+[	O
+Ka	B-evidence
+]	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
+
+Additional	O
+affinity	B-experimental_method
+PAGE	I-experimental_method
+analysis	O
+(	O
+Fig	O
+.	O
+3	O
+)	O
+demonstrates	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+also	O
+has	O
+moderate	O
+affinity	O
+for	O
+the	O
+artificial	O
+soluble	O
+cellulose	O
+derivative	O
+hydroxyethyl	B-chemical
+cellulose	I-chemical
+[	O
+HEC	B-chemical
+;	O
+a	O
+β	B-chemical
+(	I-chemical
+1	I-chemical
+→	I-chemical
+4	I-chemical
+)-	I-chemical
+glucan	I-chemical
+]	O
+and	O
+limited	O
+affinity	O
+for	O
+mixed	B-chemical
+-	I-chemical
+linkage	I-chemical
+β	I-chemical
+(	I-chemical
+1	I-chemical
+→	I-chemical
+3	I-chemical
+)/	I-chemical
+β	I-chemical
+(	I-chemical
+1	I-chemical
+→	I-chemical
+4	I-chemical
+)-	I-chemical
+glucan	I-chemical
+(	O
+MLG	B-chemical
+)	O
+and	O
+glucomannan	B-chemical
+(	O
+GM	B-chemical
+;	O
+mixed	O
+glucosyl	B-chemical
+and	O
+mannosyl	B-chemical
+backbone	O
+),	O
+which	O
+together	O
+indicate	O
+general	O
+binding	O
+to	O
+polysaccharide	B-chemical
+backbone	O
+residues	O
+and	O
+major	O
+contributions	O
+from	O
+side	O
+-	O
+chain	O
+recognition	O
+.	O
+
+In	O
+contrast	O
+,	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+bound	O
+to	O
+HEC	B-chemical
+more	O
+weakly	O
+than	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+did	O
+not	O
+bind	O
+to	O
+MLG	B-chemical
+or	O
+GM	B-chemical
+.	O
+
+Neither	O
+SGBP	B-protein_type
+recognized	O
+galactomannan	B-chemical
+(	O
+GGM	B-chemical
+),	O
+starch	B-chemical
+,	O
+carboxymethylcellulose	B-chemical
+,	O
+or	O
+mucin	B-chemical
+(	O
+see	O
+Fig	O
+.	O
+S1	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+Together	O
+,	O
+these	O
+results	O
+highlight	O
+the	O
+high	O
+specificities	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+for	O
+XyG	B-chemical
+,	O
+which	O
+is	O
+concordant	O
+with	O
+their	O
+association	O
+with	O
+XyG	B-protein_type
+-	I-protein_type
+specific	I-protein_type
+GHs	I-protein_type
+in	O
+the	O
+XyGUL	B-gene
+,	O
+as	O
+well	O
+as	O
+transcriptomic	O
+analysis	O
+indicating	O
+that	O
+B	B-species
+.	I-species
+ovatus	I-species
+has	O
+discrete	O
+PUL	B-gene
+for	O
+MLG	B-chemical
+,	O
+GM	B-chemical
+,	O
+and	O
+GGM	B-chemical
+(	O
+11	O
+).	O
+
+Notably	O
+,	O
+the	O
+absence	O
+of	O
+carbohydrate	B-site
+-	I-site
+binding	I-site
+modules	I-site
+in	O
+the	O
+GHs	B-protein_type
+encoded	O
+by	O
+the	O
+XyGUL	B-gene
+implies	O
+that	O
+noncatalytic	O
+recognition	O
+of	O
+xyloglucan	B-chemical
+is	O
+mediated	O
+entirely	O
+by	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+-	B-protein
+B	I-protein
+.	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+preferentially	O
+bind	O
+xyloglucan	B-chemical
+.	O
+
+Affinity	B-experimental_method
+electrophoresis	I-experimental_method
+(	O
+10	O
+%	O
+acrylamide	O
+)	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+with	O
+BSA	B-protein
+as	O
+a	O
+control	O
+protein	O
+.	O
+
+All	O
+samples	O
+were	O
+loaded	O
+on	O
+the	O
+same	O
+gel	O
+next	O
+to	O
+the	O
+BSA	B-protein
+controls	O
+;	O
+thin	O
+black	O
+lines	O
+indicate	O
+where	O
+intervening	O
+lanes	O
+were	O
+removed	O
+from	O
+the	O
+final	O
+image	O
+for	O
+both	O
+space	O
+and	O
+clarity	O
+.	O
+
+The	O
+percentage	O
+of	O
+polysaccharide	B-chemical
+incorporated	O
+into	O
+each	O
+native	O
+gel	O
+is	O
+displayed	O
+.	O
+
+The	O
+vanguard	O
+endo	B-protein_type
+-	I-protein_type
+xyloglucanase	I-protein_type
+of	O
+the	O
+XyGUL	B-gene
+,	O
+BoGH5	B-protein
+,	O
+preferentially	O
+cleaves	O
+the	O
+polysaccharide	B-chemical
+at	O
+unbranched	O
+glucosyl	B-chemical
+residues	O
+to	O
+generate	O
+xylogluco	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+(	O
+XyGOs	B-chemical
+)	O
+comprising	O
+a	O
+Glc4	B-structure_element
+backbone	I-structure_element
+with	O
+variable	B-structure_element
+side	I-structure_element
+-	I-structure_element
+chain	I-structure_element
+galactosylation	I-structure_element
+(	O
+XyGO1	B-chemical
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+;	O
+n	O
+=	O
+1	O
+)	O
+as	O
+the	O
+limit	O
+of	O
+digestion	O
+products	O
+in	O
+vitro	O
+;	O
+controlled	B-experimental_method
+digestion	I-experimental_method
+and	I-experimental_method
+fractionation	I-experimental_method
+by	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+allow	O
+the	O
+production	O
+of	O
+higher	O
+-	O
+order	O
+oligosaccharides	B-chemical
+(	O
+e	O
+.	O
+g	O
+.,	O
+XyGO2	B-chemical
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+;	O
+n	O
+=	O
+2	O
+).	O
+
+ITC	B-experimental_method
+demonstrates	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+binds	O
+to	O
+XyG	B-chemical
+polysaccharide	B-chemical
+and	O
+XyGO2	B-chemical
+(	O
+based	O
+on	O
+a	O
+Glc8	B-structure_element
+backbone	I-structure_element
+)	O
+with	O
+essentially	O
+equal	O
+affinities	B-evidence
+,	O
+while	O
+no	O
+binding	O
+of	O
+XyGO1	B-chemical
+(	O
+Glc4	B-structure_element
+backbone	I-structure_element
+)	O
+was	O
+detectable	O
+(	O
+Table	O
+1	O
+;	O
+see	O
+Fig	O
+.	O
+S2	O
+and	O
+S3	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+Similarly	O
+,	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+also	O
+bound	B-protein_state
+to	I-protein_state
+XyG	B-chemical
+and	O
+XyGO2	B-chemical
+with	O
+approximately	O
+equal	O
+affinities	B-evidence
+,	O
+although	O
+in	O
+both	O
+cases	O
+,	O
+Ka	B-evidence
+values	O
+were	O
+nearly	O
+10	O
+-	O
+fold	O
+lower	O
+than	O
+those	O
+for	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+.	O
+Also	O
+in	O
+contrast	O
+to	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+also	O
+bound	B-protein_state
+to	I-protein_state
+XyGO1	B-chemical
+,	O
+yet	O
+the	O
+affinity	B-evidence
+for	O
+this	O
+minimal	B-structure_element
+repeating	I-structure_element
+unit	I-structure_element
+was	O
+poor	O
+,	O
+with	O
+a	O
+Ka	B-evidence
+value	O
+of	O
+ca	O
+.	O
+1	O
+order	O
+of	O
+magnitude	O
+lower	O
+than	O
+for	O
+XyG	B-chemical
+and	O
+XyGO2	B-chemical
+.	O
+
+Together	O
+,	O
+these	O
+data	O
+clearly	O
+suggest	O
+that	O
+polysaccharide	B-chemical
+binding	O
+of	O
+both	O
+SGBPs	B-protein_type
+is	O
+fulfilled	O
+by	O
+a	O
+dimer	B-oligomeric_state
+of	O
+the	O
+minimal	B-structure_element
+repeat	I-structure_element
+,	O
+corresponding	O
+to	O
+XyGO2	B-chemical
+(	O
+cf	O
+.	O
+
+The	O
+observation	O
+by	O
+affinity	B-experimental_method
+PAGE	I-experimental_method
+that	O
+these	O
+proteins	O
+specifically	O
+recognize	O
+XyG	B-chemical
+is	O
+further	O
+substantiated	O
+by	O
+their	O
+lack	O
+of	O
+binding	O
+for	O
+the	O
+undecorated	O
+oligosaccharide	B-chemical
+cellotetraose	B-chemical
+(	O
+Table	O
+1	O
+;	O
+see	O
+Fig	O
+.	O
+S3	O
+).	O
+
+Furthermore	O
+,	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+binds	O
+cellohexaose	B-chemical
+with	O
+~	O
+770	O
+-	O
+fold	O
+weaker	O
+affinity	B-evidence
+than	O
+XyG	B-chemical
+,	O
+while	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+displays	O
+no	O
+detectable	O
+binding	O
+to	O
+this	O
+linear	O
+hexasaccharide	B-chemical
+.	O
+
+To	O
+provide	O
+molecular	O
+-	O
+level	O
+insight	O
+into	O
+how	O
+the	O
+XyGUL	B-gene
+SGBPs	B-protein_type
+equip	O
+B	B-species
+.	I-species
+ovatus	I-species
+to	O
+specifically	O
+harvest	O
+XyG	B-chemical
+from	O
+the	O
+gut	O
+environment	O
+,	O
+we	O
+performed	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+analysis	O
+of	O
+both	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGPB	B-protein
+-	I-protein
+B	I-protein
+in	O
+oligosaccharide	B-complex_assembly
+-	I-complex_assembly
+complex	I-complex_assembly
+forms	I-complex_assembly
+.	O
+
+Summary	O
+of	O
+thermodynamic	O
+parameters	O
+for	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+obtained	O
+by	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+at	O
+25	O
+°	O
+Ca	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+is	O
+a	O
+SusD	B-protein
+homolog	O
+with	O
+an	O
+extensive	O
+glycan	B-site
+-	I-site
+binding	I-site
+platform	I-site
+.	O
+
+As	O
+anticipated	O
+by	O
+sequence	O
+similarity	O
+,	O
+the	O
+high	O
+-	O
+resolution	O
+tertiary	O
+structure	B-evidence
+of	O
+apo	B-protein_state
+-	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+1	O
+.	O
+36	O
+Å	O
+,	O
+Rwork	B-evidence
+=	O
+14	O
+.	O
+7	O
+%,	O
+Rfree	B-evidence
+=	O
+17	O
+.	O
+4	O
+%,	O
+residues	O
+28	B-residue_range
+to	I-residue_range
+546	I-residue_range
+)	O
+(	O
+Table	O
+2	O
+)	O
+displays	O
+the	O
+canonical	O
+“	B-structure_element
+SusD	I-structure_element
+-	I-structure_element
+like	I-structure_element
+”	I-structure_element
+protein	I-structure_element
+fold	I-structure_element
+dominated	O
+by	O
+four	O
+tetratrico	B-structure_element
+-	I-structure_element
+peptide	I-structure_element
+repeat	I-structure_element
+(	O
+TPR	B-structure_element
+)	O
+motifs	O
+that	O
+cradle	O
+the	O
+rest	O
+of	O
+the	O
+structure	B-evidence
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+
+Specifically	O
+,	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+overlays	B-experimental_method
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+SusD	B-protein
+(	O
+BtSusD	B-protein
+)	O
+with	O
+a	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+(	O
+RMSD	B-evidence
+)	O
+value	O
+of	O
+2	O
+.	O
+2	O
+Å	O
+for	O
+363	O
+Cα	O
+pairs	O
+,	O
+which	O
+is	O
+notable	O
+given	O
+the	O
+26	O
+%	O
+amino	O
+acid	O
+identity	O
+(	O
+40	O
+%	O
+similarity	O
+)	O
+between	O
+these	O
+homologs	O
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+
+Cocrystallization	B-experimental_method
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+with	O
+XyGO2	B-chemical
+generated	O
+a	O
+substrate	B-complex_assembly
+complex	I-complex_assembly
+structure	B-evidence
+(	O
+2	O
+.	O
+3	O
+Å	O
+,	O
+Rwork	B-evidence
+=	O
+21	O
+.	O
+8	O
+%,	O
+Rfree	B-evidence
+=	O
+24	O
+.	O
+8	O
+%,	O
+residues	O
+36	B-residue_range
+to	I-residue_range
+546	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+4A	O
+and	O
+B	O
+;	O
+Table	O
+2	O
+)	O
+that	O
+revealed	O
+the	O
+distinct	O
+binding	B-site
+-	I-site
+site	I-site
+architecture	O
+of	O
+the	O
+XyG	B-protein_type
+binding	I-protein_type
+protein	I-protein_type
+.	O
+
+The	O
+SGBP	B-complex_assembly
+-	I-complex_assembly
+A	I-complex_assembly
+:	I-complex_assembly
+XyGO2	I-complex_assembly
+complex	O
+superimposes	B-experimental_method
+closely	O
+with	O
+the	O
+apo	B-protein_state
+structure	B-evidence
+(	O
+RMSD	B-evidence
+of	O
+0	O
+.	O
+6	O
+Å	O
+)	O
+and	O
+demonstrates	O
+that	O
+no	O
+major	O
+conformational	O
+change	O
+occurs	O
+upon	O
+substrate	O
+binding	O
+;	O
+small	O
+deviations	O
+in	O
+the	O
+orientation	O
+of	O
+several	O
+surface	O
+loops	O
+are	O
+likely	O
+the	O
+result	O
+of	O
+differential	O
+crystal	O
+packing	O
+.	O
+
+It	O
+is	O
+particularly	O
+notable	O
+that	O
+although	O
+the	O
+location	O
+of	O
+the	O
+ligand	B-site
+-	I-site
+binding	I-site
+site	I-site
+is	O
+conserved	B-protein_state
+between	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SusD	B-protein
+,	O
+that	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+displays	O
+an	O
+~	O
+29	O
+-	O
+Å	O
+-	O
+long	O
+aromatic	B-site
+platform	I-site
+to	O
+accommodate	O
+the	O
+extended	O
+,	O
+linear	O
+XyG	B-chemical
+chain	O
+(	O
+see	O
+reference	O
+for	O
+a	O
+review	O
+of	O
+XyG	B-chemical
+secondary	O
+structure	O
+),	O
+versus	O
+the	O
+shorter	O
+,	O
+~	O
+18	O
+-	O
+Å	O
+-	O
+long	O
+,	O
+site	B-site
+within	O
+SusD	B-protein
+that	O
+complements	O
+the	O
+helical	O
+conformation	O
+of	O
+amylose	B-chemical
+(	O
+Fig	O
+.	O
+4C	O
+and	O
+D	O
+).	O
+
+Molecular	O
+structure	B-evidence
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+Bacova_02651	B-gene
+).	O
+(	O
+A	O
+)	O
+Overlay	B-experimental_method
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+from	O
+the	O
+apo	B-protein_state
+(	O
+rainbow	O
+)	O
+and	O
+XyGO2	B-chemical
+(	O
+gray	O
+)	O
+structures	B-evidence
+.	O
+
+The	O
+apo	B-protein_state
+structure	B-evidence
+is	O
+color	O
+ramped	O
+from	O
+blue	O
+to	O
+red	O
+.	O
+
+An	O
+omit	B-evidence
+map	I-evidence
+(	O
+2σ	O
+)	O
+for	O
+XyGO2	B-chemical
+(	O
+orange	O
+and	O
+red	O
+sticks	O
+)	O
+is	O
+displayed	O
+.	O
+
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+omit	B-evidence
+map	I-evidence
+as	O
+in	O
+panel	O
+A	O
+,	O
+rotated	O
+90	O
+°	O
+clockwise	O
+.	O
+
+(	O
+C	O
+)	O
+Overlay	B-experimental_method
+of	O
+the	O
+Cα	O
+backbones	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+black	O
+)	O
+with	O
+XyGO2	B-chemical
+(	O
+orange	O
+and	O
+red	O
+spheres	O
+)	O
+and	O
+BtSusD	B-protein
+(	O
+blue	O
+)	O
+with	O
+maltoheptaose	B-chemical
+(	O
+pink	O
+and	O
+red	O
+spheres	O
+),	O
+highlighting	O
+the	O
+conservation	O
+of	O
+the	O
+glycan	B-site
+-	I-site
+binding	I-site
+site	I-site
+location	O
+.	O
+
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+of	O
+the	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+black	O
+and	O
+orange	O
+)	O
+and	O
+SusD	B-protein
+(	O
+blue	O
+and	O
+pink	O
+)	O
+glycan	B-site
+-	I-site
+binding	I-site
+sites	I-site
+.	O
+
+The	O
+approximate	O
+length	O
+of	O
+each	O
+glycan	B-site
+-	I-site
+binding	I-site
+site	I-site
+is	O
+displayed	O
+,	O
+colored	O
+to	O
+match	O
+the	O
+protein	B-evidence
+structures	I-evidence
+.	O
+(	O
+E	O
+)	O
+Stereo	O
+view	O
+of	O
+the	O
+xyloglucan	B-site
+-	I-site
+binding	I-site
+site	I-site
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+displaying	O
+all	O
+residues	O
+within	O
+4	O
+Å	O
+of	O
+the	O
+ligand	O
+.	O
+
+The	O
+backbone	O
+glucose	B-chemical
+residues	O
+are	O
+numbered	O
+from	O
+the	O
+nonreducing	O
+end	O
+;	O
+xylose	B-chemical
+residues	O
+are	O
+labeled	O
+X1	B-residue_name_number
+and	O
+X2	B-residue_name_number
+.	O
+
+Seven	O
+of	O
+the	O
+eight	O
+backbone	O
+glucosyl	B-chemical
+residues	O
+of	O
+XyGO2	B-chemical
+could	O
+be	O
+convincingly	O
+modeled	O
+in	O
+the	O
+ligand	B-evidence
+electron	I-evidence
+density	I-evidence
+,	O
+and	O
+only	O
+two	O
+α	B-chemical
+(	I-chemical
+1	I-chemical
+→	I-chemical
+6	I-chemical
+)-	I-chemical
+linked	I-chemical
+xylosyl	I-chemical
+residues	O
+were	O
+observed	O
+(	O
+Fig	O
+.	O
+4B	O
+;	O
+cf	O
+.	O
+
+Indeed	O
+,	O
+the	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+ligand	O
+suggests	O
+some	O
+disorder	O
+,	O
+which	O
+may	O
+arise	O
+from	O
+multiple	O
+oligosaccharide	B-chemical
+orientations	O
+along	O
+the	O
+binding	B-site
+site	I-site
+.	O
+
+Three	O
+aromatic	O
+residues	O
+—	O
+W82	B-residue_name_number
+,	O
+W283	B-residue_name_number
+,	O
+W306	B-residue_name_number
+—	O
+comprise	O
+the	O
+flat	B-site
+platform	I-site
+that	O
+stacks	O
+along	O
+the	O
+naturally	O
+twisted	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+backbone	O
+(	O
+Fig	O
+.	O
+4E	O
+).	O
+
+The	O
+functional	O
+importance	O
+of	O
+this	O
+platform	B-site
+is	O
+underscored	O
+by	O
+the	O
+observation	O
+that	O
+the	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+mutant	B-protein_state
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+designated	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*,	I-mutant
+is	O
+completely	B-protein_state
+devoid	I-protein_state
+of	I-protein_state
+XyG	I-protein_state
+affinity	I-protein_state
+(	O
+Table	O
+3	O
+;	O
+see	O
+Fig	O
+.	O
+S4	O
+in	O
+the	O
+supplemental	O
+material	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	B-protein_state
+displays	O
+a	O
+significant	O
+4	O
+.	O
+9	O
+-	O
+fold	O
+decrease	O
+in	O
+the	O
+Ka	B-evidence
+value	O
+for	O
+XyG	B-chemical
+,	O
+while	O
+the	O
+W306A	B-mutant
+substitution	B-experimental_method
+completely	O
+abolishes	B-protein_state
+XyG	I-protein_state
+binding	I-protein_state
+.	O
+
+Contrasting	O
+with	O
+the	O
+clear	O
+importance	O
+of	O
+these	O
+hydrophobic	O
+interactions	O
+,	O
+there	O
+are	O
+remarkably	O
+few	O
+hydrogen	O
+-	O
+bonding	O
+interactions	O
+with	O
+the	O
+ligand	B-chemical
+,	O
+which	O
+are	O
+provided	O
+by	O
+R65	B-residue_name_number
+,	O
+N83	B-residue_name_number
+,	O
+and	O
+S308	B-residue_name_number
+,	O
+which	O
+are	O
+proximal	O
+to	O
+Glc5	B-residue_name_number
+and	O
+Glc3	B-residue_name_number
+.	O
+
+Most	O
+surprising	O
+in	O
+light	O
+of	O
+the	O
+saccharide	B-evidence
+-	I-evidence
+binding	I-evidence
+data	I-evidence
+,	O
+however	O
+,	O
+was	O
+a	O
+lack	O
+of	O
+extensive	O
+recognition	O
+of	O
+the	O
+XyG	B-chemical
+side	O
+chains	O
+;	O
+only	O
+Y84	B-residue_name_number
+appeared	O
+to	O
+provide	O
+a	O
+hydrophobic	B-site
+interface	I-site
+for	O
+a	O
+xylosyl	B-chemical
+residue	O
+(	O
+Xyl1	B-residue_name_number
+).	O
+
+Summary	O
+of	O
+thermodynamic	O
+parameters	O
+for	O
+site	O
+-	O
+directed	O
+mutants	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+obtained	O
+by	O
+ITC	B-experimental_method
+with	O
+XyG	B-chemical
+at	O
+25	O
+°	O
+Ca	O
+
+Protein	O
+name	O
+Ka	B-evidence
+ΔG	O
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+ΔH	B-evidence
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+TΔS	B-evidence
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+Fold	O
+changeb	O
+M	O
+−	O
+1	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+)	O
+ND	O
+NB	O
+NB	O
+NB	O
+NB	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+W82A	B-mutant
+)	O
+c	O
+4	O
+.	O
+9	O
+9	O
+.	O
+1	O
+×	O
+104	O
+−	O
+6	O
+.	O
+8	O
+−	O
+6	O
+.	O
+3	O
+0	O
+.	O
+5	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+W306	B-residue_name_number
+)	O
+ND	O
+NB	O
+NB	O
+NB	O
+NB	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+230	B-residue_range
+–	I-residue_range
+489	I-residue_range
+)	O
+0	O
+.	O
+7	O
+(	O
+8	O
+.	O
+6	O
+±	O
+0	O
+.	O
+20	O
+)	O
+×	O
+104	O
+−	O
+6	O
+.	O
+7	O
+−	O
+14	O
+.	O
+9	O
+±	O
+0	O
+.	O
+1	O
+−	O
+8	O
+.	O
+2	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+Y363A	B-mutant
+)	O
+19	O
+.	O
+7	O
+(	O
+2	O
+.	O
+9	O
+±	O
+0	O
+.	O
+10	O
+)	O
+×	O
+103	O
+−	O
+4	O
+.	O
+7	O
+−	O
+18	O
+.	O
+1	O
+±	O
+0	O
+.	O
+1	O
+−	O
+13	O
+.	O
+3	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+W364A	B-mutant
+)	O
+ND	O
+Weak	O
+Weak	O
+Weak	O
+Weak	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+F414A	B-mutant
+)	O
+3	O
+.	O
+2	O
+(	O
+1	O
+.	O
+80	O
+±	O
+0	O
+.	O
+03	O
+)	O
+×	O
+104	O
+−	O
+5	O
+.	O
+8	O
+−	O
+11	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+−	O
+5	O
+.	O
+6	O
+
+Binding	O
+thermodynamics	O
+are	O
+based	O
+on	O
+the	O
+concentration	O
+of	O
+the	O
+binding	O
+unit	O
+,	O
+XyGO2	B-chemical
+.	O
+
+Weak	O
+binding	O
+represents	O
+a	O
+Ka	B-evidence
+of	O
+<	O
+500	O
+M	O
+−	O
+1	O
+.	O
+
+Ka	B-evidence
+fold	O
+change	O
+=	O
+Ka	B-evidence
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+protein	O
+/	O
+Ka	B-evidence
+of	O
+mutant	O
+protein	O
+for	O
+xyloglucan	B-chemical
+binding	O
+.	O
+
+SGBP	B-protein
+-	I-protein
+B	I-protein
+has	O
+a	O
+multimodular	O
+structure	O
+with	O
+a	O
+single	O
+,	O
+C	O
+-	O
+terminal	O
+glycan	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+SGBP	B-protein
+-	I-protein
+B	I-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+XyGO2	B-chemical
+(	O
+2	O
+.	O
+37	O
+Å	O
+,	O
+Rwork	B-evidence
+=	O
+19	O
+.	O
+9	O
+%,	O
+Rfree	B-evidence
+=	O
+23	O
+.	O
+9	O
+%,	O
+residues	O
+34	B-residue_range
+to	I-residue_range
+489	I-residue_range
+)	O
+(	O
+Table	O
+2	O
+)	O
+revealed	O
+an	O
+extended	O
+structure	B-evidence
+composed	O
+of	O
+three	O
+tandem	B-structure_element
+immunoglobulin	I-structure_element
+(	I-structure_element
+Ig	I-structure_element
+)-	I-structure_element
+like	I-structure_element
+domains	I-structure_element
+(	O
+domains	O
+A	B-structure_element
+,	O
+B	B-structure_element
+,	O
+and	O
+C	B-structure_element
+)	O
+followed	O
+at	O
+the	O
+C	O
+terminus	O
+by	O
+a	O
+novel	O
+xyloglucan	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+domain	O
+D	B-structure_element
+)	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+Domains	O
+A	B-structure_element
+,	O
+B	B-structure_element
+,	O
+and	O
+C	B-structure_element
+display	O
+similar	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+folds	I-structure_element
+;	O
+domains	O
+B	B-structure_element
+(	O
+residues	O
+134	B-residue_range
+to	I-residue_range
+230	I-residue_range
+)	O
+and	O
+C	B-structure_element
+(	O
+residues	O
+231	B-residue_range
+to	I-residue_range
+313	I-residue_range
+)	O
+can	O
+be	O
+superimposed	B-experimental_method
+onto	O
+domain	O
+A	B-structure_element
+(	O
+residues	O
+34	B-residue_range
+to	I-residue_range
+133	I-residue_range
+)	O
+with	O
+RMSDs	B-evidence
+of	O
+1	O
+.	O
+1	O
+and	O
+1	O
+.	O
+2	O
+Å	O
+,	O
+respectively	O
+,	O
+for	O
+47	O
+atom	O
+pairs	O
+(	O
+23	O
+%	O
+and	O
+16	O
+%	O
+sequence	O
+identity	O
+,	O
+respectively	O
+).	O
+
+These	B-structure_element
+domains	I-structure_element
+also	O
+display	O
+similarity	O
+to	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+domains	I-structure_element
+of	O
+many	O
+GH13	B-protein_type
+enzymes	I-protein_type
+,	O
+including	O
+the	O
+cyclodextrin	B-protein_type
+glucanotransferase	I-protein_type
+of	O
+Geobacillus	B-species
+stearothermophilus	I-species
+(	O
+Fig	O
+.	O
+5B	O
+).	O
+
+Such	B-structure_element
+domains	I-structure_element
+are	O
+not	O
+typically	O
+involved	O
+in	O
+carbohydrate	B-chemical
+binding	O
+.	O
+
+Indeed	O
+,	O
+visual	B-experimental_method
+inspection	I-experimental_method
+of	O
+the	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+structure	B-evidence
+,	O
+as	O
+well	O
+as	O
+individual	O
+production	O
+of	O
+the	O
+A	B-structure_element
+and	O
+B	B-structure_element
+domains	O
+and	O
+affinity	B-experimental_method
+PAGE	I-experimental_method
+analysis	O
+(	O
+see	O
+Fig	O
+.	O
+S5	O
+in	O
+the	O
+supplemental	O
+material	O
+),	O
+indicates	O
+that	O
+these	O
+domains	O
+do	O
+not	O
+contribute	O
+to	O
+XyG	B-chemical
+capture	O
+.	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+production	B-experimental_method
+of	O
+the	O
+fused	B-mutant
+domains	I-mutant
+C	I-mutant
+and	I-mutant
+D	I-mutant
+in	O
+tandem	O
+(	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+residues	O
+230	B-residue_range
+to	I-residue_range
+489	I-residue_range
+)	O
+retains	O
+complete	O
+binding	O
+of	O
+xyloglucan	B-chemical
+in	O
+vitro	O
+,	O
+with	O
+the	O
+observed	O
+slight	O
+increase	O
+in	O
+affinity	O
+likely	O
+arising	O
+from	O
+a	O
+reduced	O
+potential	O
+for	O
+steric	O
+hindrance	O
+of	O
+the	O
+smaller	O
+protein	O
+construct	O
+during	O
+polysaccharide	B-chemical
+interactions	O
+(	O
+Table	O
+3	O
+).	O
+
+While	O
+neither	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+nor	O
+domain	O
+D	B-structure_element
+displays	O
+structural	O
+homology	O
+to	O
+known	O
+XyG	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+,	O
+the	O
+topology	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+resembles	O
+the	O
+xylan	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+Bacova_04391	B-protein
+(	O
+PDB	O
+3ORJ	O
+)	O
+encoded	O
+within	O
+a	O
+xylan	B-chemical
+-	O
+targeting	O
+PUL	B-gene
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+(	O
+Fig	O
+.	O
+5C	O
+).	O
+
+The	O
+structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+alignment	I-experimental_method
+of	O
+these	O
+proteins	O
+reveals	O
+17	O
+%	O
+sequence	O
+identity	O
+,	O
+with	O
+a	O
+core	O
+RMSD	B-evidence
+of	O
+3	O
+.	O
+6	O
+Å	O
+for	O
+253	O
+aligned	O
+residues	O
+.	O
+
+While	O
+there	O
+is	O
+no	O
+substrate	O
+-	O
+complexed	O
+structure	O
+of	O
+Bacova_04391	B-protein
+available	O
+,	O
+the	O
+binding	B-site
+site	I-site
+is	O
+predicted	O
+to	O
+include	O
+W241	B-residue_name_number
+and	O
+Y404	B-residue_name_number
+,	O
+which	O
+are	O
+proximal	O
+to	O
+the	O
+XyGO	B-site
+binding	I-site
+site	I-site
+in	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+.	O
+However	O
+,	O
+the	O
+opposing	B-protein_state
+,	I-protein_state
+clamp	I-protein_state
+-	I-protein_state
+like	I-protein_state
+arrangement	I-protein_state
+of	O
+these	B-structure_element
+residues	I-structure_element
+in	O
+Bacova_04391	B-protein
+is	O
+clearly	O
+distinct	O
+from	O
+the	O
+planar	B-site
+surface	I-site
+arrangement	I-site
+of	O
+the	O
+residues	B-structure_element
+that	O
+interact	O
+with	O
+XyG	B-chemical
+in	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+described	O
+below	O
+).	O
+
+Multimodular	O
+structure	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+Bacova_02650	B-gene
+).	O
+(	O
+A	O
+)	O
+Full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+structure	B-evidence
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+color	O
+coded	O
+by	O
+domain	O
+as	O
+indicated	O
+.	O
+
+Prolines	B-residue_name
+between	O
+domains	O
+are	O
+indicated	O
+as	O
+spheres	O
+.	O
+
+An	O
+omit	B-evidence
+map	I-evidence
+(	O
+2σ	O
+)	O
+for	O
+XyGO2	B-chemical
+is	O
+displayed	O
+to	O
+highlight	O
+the	O
+location	O
+of	O
+the	O
+glycan	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+
+(	O
+B	O
+)	O
+Overlay	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+domains	O
+A	B-structure_element
+,	O
+B	B-structure_element
+,	O
+and	O
+C	B-structure_element
+(	O
+colored	O
+as	O
+in	O
+panel	O
+A	O
+),	O
+with	O
+a	O
+C	O
+-	O
+terminal	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domain	I-structure_element
+of	O
+the	O
+G	B-species
+.	I-species
+stearothermophilus	I-species
+cyclodextrin	B-protein_type
+glucanotransferase	I-protein_type
+(	O
+PDB	O
+1CYG	O
+[	O
+residues	O
+375	B-residue_range
+to	I-residue_range
+493	I-residue_range
+])	O
+in	O
+green	O
+.	O
+(	O
+C	O
+)	O
+Cα	O
+overlay	B-experimental_method
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+gray	O
+)	O
+and	O
+Bacova_04391	B-protein
+(	O
+PDB	O
+3ORJ	O
+)	O
+(	O
+pink	O
+).	O
+
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+omit	B-evidence
+map	I-evidence
+for	O
+the	O
+XyGO2	B-chemical
+ligand	O
+,	O
+contoured	O
+at	O
+2σ	O
+.	O
+(	O
+E	O
+)	O
+Stereo	O
+view	O
+of	O
+the	O
+xyloglucan	B-site
+-	I-site
+binding	I-site
+site	I-site
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+displaying	O
+all	O
+residues	O
+within	O
+4	O
+Å	O
+of	O
+the	O
+ligand	O
+.	O
+
+The	O
+backbone	O
+glucose	B-chemical
+residues	O
+are	O
+numbered	O
+from	O
+the	O
+nonreducing	O
+end	O
+,	O
+xylose	B-chemical
+residues	O
+are	O
+shown	O
+as	O
+X1	B-residue_name_number
+,	O
+X2	B-residue_name_number
+,	O
+and	O
+X3	B-residue_name_number
+,	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
+
+Inspection	O
+of	O
+the	O
+tertiary	O
+structure	B-evidence
+indicates	O
+that	O
+domains	O
+C	B-structure_element
+and	O
+D	B-structure_element
+are	O
+effectively	O
+inseparable	O
+,	O
+with	O
+a	O
+contact	O
+interface	O
+of	O
+396	O
+Å2	O
+.	O
+
+Domains	O
+A	B-structure_element
+,	O
+B	B-structure_element
+,	O
+and	O
+C	B-structure_element
+do	O
+not	O
+pack	O
+against	O
+each	O
+other	O
+.	O
+
+Moreover	O
+,	O
+the	O
+five	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+linkers	I-structure_element
+between	O
+these	O
+first	O
+three	O
+domains	O
+all	O
+feature	O
+a	O
+proline	B-residue_name
+as	O
+the	O
+middle	B-structure_element
+residue	I-structure_element
+,	O
+suggesting	O
+significant	O
+conformational	O
+rigidity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+Despite	O
+the	O
+lack	O
+of	O
+sequence	O
+and	O
+structural	O
+conservation	O
+,	O
+a	O
+similarly	O
+positioned	O
+proline	B-residue_name
+joins	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domains	I-structure_element
+of	O
+the	O
+xylan	O
+-	O
+binding	O
+Bacova_04391	B-protein
+and	O
+the	O
+starch	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+SusE	B-protein
+and	O
+SusF	B-protein
+.	O
+We	O
+speculate	O
+that	O
+this	O
+is	O
+a	O
+biologically	O
+important	O
+adaptation	O
+that	O
+serves	O
+to	O
+project	O
+the	O
+glycan	B-site
+binding	I-site
+site	I-site
+of	O
+these	O
+proteins	O
+far	O
+from	O
+the	O
+membrane	O
+surface	O
+.	O
+
+Any	O
+mobility	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+on	O
+the	O
+surface	O
+of	O
+the	O
+cell	O
+(	O
+beyond	O
+lateral	O
+diffusion	O
+within	O
+the	O
+membrane	O
+)	O
+is	O
+likely	O
+imparted	O
+by	O
+the	O
+eight	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+linker	I-structure_element
+that	O
+spans	O
+the	O
+predicted	O
+lipidated	B-protein_state
+Cys	B-residue_name
+(	O
+C28	B-residue_name_number
+)	O
+and	O
+the	O
+first	B-structure_element
+β	I-structure_element
+-	I-structure_element
+strand	I-structure_element
+of	O
+domain	O
+A	B-structure_element
+.	O
+Other	O
+outer	B-protein_type
+membrane	I-protein_type
+proteins	I-protein_type
+from	O
+various	O
+Sus	B-complex_assembly
+-	I-complex_assembly
+like	I-complex_assembly
+systems	I-complex_assembly
+possess	O
+a	O
+similar	O
+10	B-structure_element
+-	I-structure_element
+to	I-structure_element
+20	I-structure_element
+-	I-structure_element
+amino	I-structure_element
+-	I-structure_element
+acid	I-structure_element
+flexible	I-structure_element
+linker	I-structure_element
+between	O
+the	O
+lipidated	B-protein_state
+Cys	B-residue_name
+that	O
+tethers	O
+the	O
+protein	O
+to	O
+the	O
+outside	O
+the	O
+cell	O
+and	O
+the	O
+first	O
+secondary	O
+structure	O
+element	O
+.	O
+
+Analogously	O
+,	O
+the	O
+outer	B-protein_state
+membrane	I-protein_state
+-	I-protein_state
+anchored	I-protein_state
+endo	B-protein_type
+-	I-protein_type
+xyloglucanase	I-protein_type
+BoGH5	B-protein
+of	O
+the	O
+XyGUL	B-gene
+contains	O
+a	O
+100	B-structure_element
+-	I-structure_element
+amino	I-structure_element
+-	I-structure_element
+acid	I-structure_element
+,	I-structure_element
+all	I-structure_element
+-	I-structure_element
+β	I-structure_element
+-	I-structure_element
+strand	I-structure_element
+,	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+module	I-structure_element
+and	O
+flexible	B-structure_element
+linker	I-structure_element
+that	O
+imparts	O
+conformational	O
+flexibility	O
+and	O
+distances	O
+the	O
+catalytic	B-structure_element
+module	I-structure_element
+from	O
+the	O
+cell	O
+surface	O
+.	O
+
+XyG	B-chemical
+binds	B-protein_state
+to	I-protein_state
+domain	O
+D	B-structure_element
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+at	O
+the	O
+concave	B-site
+interface	I-site
+of	O
+the	O
+top	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+,	O
+with	O
+binding	O
+mediated	O
+by	O
+loops	B-structure_element
+connecting	O
+the	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+.	O
+
+Six	O
+glucosyl	B-chemical
+residues	O
+,	O
+comprising	O
+the	O
+main	O
+chain	O
+,	O
+and	O
+three	O
+branching	O
+xylosyl	B-chemical
+residues	O
+of	O
+XyGO2	B-chemical
+can	O
+be	O
+modeled	O
+in	O
+the	O
+density	B-evidence
+(	O
+Fig	O
+.	O
+5D	O
+;	O
+cf	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	B-chemical
+-	I-chemical
+and	I-chemical
+xylogluco	I-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+.	O
+
+The	O
+aromatic	B-site
+platform	I-site
+created	O
+by	O
+W330	B-residue_name_number
+,	O
+W364	B-residue_name_number
+,	O
+and	O
+Y363	B-residue_name_number
+spans	O
+four	O
+glucosyl	B-chemical
+residues	O
+,	O
+compared	O
+to	O
+the	O
+longer	B-protein_state
+platform	B-site
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+which	O
+supports	O
+six	O
+glucosyl	B-chemical
+residues	O
+(	O
+Fig	O
+.	O
+5E	O
+).	O
+
+The	O
+Y363A	B-mutant
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutant	I-experimental_method
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+displays	O
+a	O
+20	O
+-	O
+fold	O
+decrease	O
+in	O
+the	O
+Ka	B-evidence
+for	O
+XyG	B-chemical
+,	O
+while	O
+the	O
+W364A	B-mutant
+mutant	B-protein_state
+lacks	B-protein_state
+XyG	I-protein_state
+binding	I-protein_state
+(	O
+Table	O
+3	O
+;	O
+see	O
+Fig	O
+.	O
+S6	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+There	O
+are	O
+no	O
+additional	O
+contacts	O
+between	O
+the	O
+protein	O
+and	O
+the	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+backbone	O
+and	O
+surprisingly	O
+few	O
+interactions	O
+with	O
+the	O
+side	O
+-	O
+chain	O
+xylosyl	B-chemical
+residues	O
+,	O
+despite	O
+that	O
+fact	O
+that	O
+ITC	B-experimental_method
+data	O
+demonstrate	O
+that	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+does	O
+not	O
+measurably	O
+bind	O
+the	O
+cellohexaose	B-chemical
+(	O
+Table	O
+1	O
+).	O
+
+F414	B-residue_name_number
+stacks	O
+with	O
+the	O
+xylosyl	B-chemical
+residue	O
+of	O
+Glc3	B-residue_name_number
+,	O
+while	O
+Q407	B-residue_name_number
+is	O
+positioned	O
+for	O
+hydrogen	O
+bonding	O
+with	O
+the	O
+O4	O
+of	O
+xylosyl	B-chemical
+residue	O
+Xyl1	B-residue_name_number
+.	O
+
+Surprisingly	O
+,	O
+an	O
+F414A	B-mutant
+mutant	B-protein_state
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+displays	O
+only	O
+a	O
+mild	O
+3	O
+-	O
+fold	O
+decrease	O
+in	O
+the	O
+Ka	B-evidence
+value	O
+for	O
+XyG	B-chemical
+,	O
+again	O
+suggesting	O
+that	O
+glycan	B-chemical
+recognition	O
+is	O
+primarily	O
+mediated	O
+via	O
+contact	O
+with	O
+the	O
+β	O
+-	O
+glucan	O
+backbone	O
+(	O
+Table	O
+3	O
+;	O
+see	O
+Fig	O
+.	O
+S6	O
+).	O
+
+Additional	O
+residues	B-structure_element
+surrounding	O
+the	O
+binding	B-site
+site	I-site
+,	O
+including	O
+Y369	B-residue_name_number
+and	O
+E412	B-residue_name_number
+,	O
+may	O
+contribute	O
+to	O
+the	O
+recognition	O
+of	O
+more	O
+highly	O
+decorated	O
+XyG	B-chemical
+,	O
+but	O
+precisely	O
+how	O
+this	O
+is	O
+mediated	O
+is	O
+presently	O
+unclear	O
+.	O
+
+Hoping	O
+to	O
+achieve	O
+a	O
+higher	O
+-	O
+resolution	O
+view	O
+of	O
+the	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+–	O
+xyloglucan	B-chemical
+interaction	O
+,	O
+we	O
+solved	B-experimental_method
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+fused	B-mutant
+CD	I-mutant
+domains	I-mutant
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+XyGO2	B-chemical
+(	O
+1	O
+.	O
+57	O
+Å	O
+,	O
+Rwork	B-evidence
+=	O
+15	O
+.	O
+6	O
+%,	O
+Rfree	B-evidence
+=	O
+17	O
+.	O
+1	O
+%,	O
+residues	O
+230	B-residue_range
+to	I-residue_range
+489	I-residue_range
+)	O
+(	O
+Table	O
+2	O
+).	O
+
+The	O
+CD	B-structure_element
+domains	I-structure_element
+of	O
+the	O
+truncated	B-protein_state
+and	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+proteins	O
+superimpose	B-experimental_method
+with	O
+a	O
+0	O
+.	O
+4	O
+-	O
+Å	O
+RMSD	B-evidence
+of	O
+the	O
+Cα	O
+backbone	O
+,	O
+with	O
+no	O
+differences	O
+in	O
+the	O
+position	O
+of	O
+any	O
+of	O
+the	O
+glycan	B-site
+-	I-site
+binding	I-site
+residues	I-site
+(	O
+see	O
+Fig	O
+.	O
+S7A	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+While	O
+density	B-evidence
+is	O
+observed	O
+for	O
+XyGO2	B-chemical
+,	O
+the	O
+ligand	O
+could	O
+not	O
+be	O
+unambiguously	O
+modeled	O
+into	O
+this	O
+density	B-evidence
+to	O
+achieve	O
+a	O
+reasonable	O
+fit	O
+between	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+and	O
+the	O
+known	O
+stereochemistry	O
+of	O
+the	O
+sugar	O
+(	O
+see	O
+Fig	O
+.	O
+S7B	O
+and	O
+C	O
+).	O
+
+While	O
+this	O
+may	O
+occur	O
+for	O
+a	O
+number	O
+of	O
+reasons	O
+in	O
+crystal	B-evidence
+structures	I-evidence
+,	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	B-chemical
+averaged	O
+throughout	O
+the	O
+lattice	O
+.	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+have	O
+distinct	O
+,	O
+coordinated	O
+functions	O
+in	O
+vivo	O
+.	O
+
+The	O
+similarity	O
+of	O
+the	O
+glycan	B-chemical
+specificity	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+presents	O
+an	O
+intriguing	O
+conundrum	O
+regarding	O
+their	O
+individual	O
+roles	O
+in	O
+XyG	B-chemical
+utilization	O
+by	O
+B	B-species
+.	I-species
+ovatus	I-species
+.	O
+
+To	O
+disentangle	O
+the	O
+functions	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+in	O
+XyG	B-chemical
+recognition	O
+and	O
+uptake	O
+,	O
+we	O
+created	O
+individual	O
+in	B-experimental_method
+-	I-experimental_method
+frame	I-experimental_method
+deletion	I-experimental_method
+and	I-experimental_method
+complementation	I-experimental_method
+mutant	I-experimental_method
+strains	O
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+.	O
+
+In	O
+these	O
+growth	B-experimental_method
+experiments	I-experimental_method
+,	O
+overnight	O
+cultures	O
+of	O
+strains	O
+grown	O
+on	O
+minimal	O
+medium	O
+plus	O
+glucose	B-chemical
+were	O
+back	O
+-	O
+diluted	O
+1	O
+:	O
+100	O
+-	O
+fold	O
+into	O
+minimal	O
+medium	O
+containing	O
+5	O
+mg	O
+/	O
+ml	O
+of	O
+the	O
+reported	O
+carbohydrate	B-chemical
+.	O
+
+Growth	O
+on	O
+glucose	B-chemical
+displayed	O
+the	O
+shortest	O
+lag	B-evidence
+time	I-evidence
+for	O
+each	O
+strain	O
+,	O
+and	O
+so	O
+lag	B-evidence
+times	I-evidence
+were	O
+normalized	O
+for	O
+each	O
+carbohydrate	B-chemical
+by	O
+subtracting	O
+the	O
+lag	B-evidence
+time	I-evidence
+of	O
+that	O
+strain	O
+in	O
+glucose	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+;	O
+see	O
+Fig	O
+.	O
+S8	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+A	O
+strain	O
+in	O
+which	O
+the	O
+entire	O
+XyGUL	B-gene
+is	O
+deleted	B-experimental_method
+displays	O
+a	O
+lag	B-evidence
+of	O
+24	O
+.	O
+5	O
+h	O
+during	O
+growth	O
+on	O
+glucose	B-chemical
+compared	O
+to	O
+the	O
+isogenic	O
+parental	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+Δtdk	B-mutant
+strain	O
+,	O
+for	O
+which	O
+exponential	O
+growth	O
+lags	B-evidence
+for	O
+19	O
+.	O
+8	O
+h	O
+(	O
+see	O
+Fig	O
+.	O
+S8D	O
+).	O
+
+It	O
+is	O
+unknown	O
+whether	O
+this	O
+is	O
+because	O
+cultures	O
+were	O
+not	O
+normalized	O
+by	O
+the	O
+starting	O
+optical	O
+density	O
+(	O
+OD	O
+)	O
+or	O
+viable	O
+cells	O
+or	O
+reflects	O
+a	O
+minor	O
+defect	O
+for	O
+glucose	B-chemical
+utilization	O
+.	O
+
+The	O
+former	O
+seems	O
+more	O
+likely	O
+as	O
+the	O
+growth	O
+rates	O
+are	O
+nearly	O
+identical	O
+for	O
+these	O
+strains	O
+on	O
+glucose	B-chemical
+and	O
+xylose	B-chemical
+.	O
+
+The	O
+ΔXyGUL	B-mutant
+and	O
+WT	B-protein_state
+Δtdk	B-mutant
+strains	O
+display	O
+normalized	O
+lag	B-evidence
+times	I-evidence
+on	O
+xylose	B-chemical
+within	O
+experimental	O
+error	O
+,	O
+and	O
+curiously	O
+some	O
+of	O
+the	O
+mutant	O
+and	O
+complemented	O
+strains	O
+display	O
+a	O
+nominally	O
+shorter	O
+lag	B-evidence
+time	I-evidence
+on	O
+xylose	B-chemical
+than	O
+the	O
+WT	B-protein_state
+Δtdk	B-mutant
+strain	O
+.	O
+
+Complementation	B-experimental_method
+of	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+strain	O
+(	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+)	O
+restores	O
+growth	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+rates	O
+on	O
+xyloglucan	B-chemical
+and	O
+XyGO1	B-chemical
+,	O
+yet	O
+the	O
+calculated	O
+rate	O
+of	O
+the	O
+complemented	O
+strain	O
+is	O
+~	O
+72	O
+%	O
+that	O
+of	O
+the	O
+WT	B-protein_state
+Δtdk	B-mutant
+strain	O
+on	O
+XyGO2	B-chemical
+;	O
+similar	O
+results	O
+were	O
+obtained	O
+for	O
+the	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+complemented	O
+strain	O
+despite	O
+the	O
+fact	O
+that	O
+the	O
+growth	O
+curves	O
+do	O
+not	O
+appear	O
+much	O
+different	O
+(	O
+see	O
+Fig	O
+.	O
+S8C	O
+and	O
+F	O
+).	O
+
+The	O
+reason	O
+for	O
+this	O
+observation	O
+on	O
+XyGO2	B-chemical
+is	O
+unclear	O
+,	O
+as	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+mutant	B-protein_state
+does	O
+not	O
+have	O
+a	O
+significantly	O
+different	O
+growth	O
+rate	O
+from	O
+the	O
+WT	B-protein_state
+on	O
+XyGO2	B-chemical
+.	O
+
+Growth	O
+of	O
+select	O
+XyGUL	B-gene
+mutants	O
+on	O
+xyloglucan	B-chemical
+and	O
+oligosaccharides	B-chemical
+.	O
+
+B	B-species
+.	I-species
+ovatus	I-species
+mutants	O
+were	O
+created	O
+in	O
+a	O
+thymidine	B-mutant
+kinase	I-mutant
+deletion	I-mutant
+(	O
+Δtdk	B-mutant
+)	O
+mutant	O
+as	O
+described	O
+previously	O
+.	O
+
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+denotes	O
+the	O
+Bacova_02651	B-gene
+(	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+)	O
+allele	O
+,	O
+and	O
+the	O
+GH9	B-protein
+gene	O
+is	O
+Bacova_02649	B-gene
+.	O
+
+Growth	O
+was	O
+measured	O
+over	O
+time	O
+in	O
+minimal	O
+medium	O
+containing	O
+(	O
+A	O
+)	O
+XyG	B-chemical
+,	O
+(	O
+B	O
+)	O
+XyGO2	B-chemical
+,	O
+(	O
+C	O
+)	O
+XyGO1	B-chemical
+,	O
+(	O
+D	O
+)	O
+glucose	B-chemical
+,	O
+and	O
+(	O
+E	O
+)	O
+xylose	B-chemical
+.	O
+
+In	O
+panel	O
+F	O
+,	O
+the	O
+growth	O
+rate	O
+of	O
+each	O
+strain	O
+on	O
+the	O
+five	O
+carbon	O
+sources	O
+is	O
+displayed	O
+,	O
+and	O
+in	O
+panel	O
+G	O
+,	O
+the	O
+normalized	O
+lag	B-evidence
+time	I-evidence
+of	O
+each	O
+culture	O
+,	O
+relative	O
+to	O
+its	O
+growth	O
+on	O
+glucose	B-chemical
+,	O
+is	O
+displayed	O
+.	O
+
+Solid	O
+bars	O
+indicate	O
+conditions	O
+that	O
+are	O
+not	O
+statistically	O
+significant	O
+from	O
+the	O
+WT	B-protein_state
+Δtdk	B-mutant
+cultures	O
+grown	O
+on	O
+the	O
+indicated	O
+carbohydrate	B-chemical
+,	O
+while	O
+open	O
+bars	O
+indicate	O
+a	O
+P	O
+value	O
+of	O
+<	O
+0	O
+.	O
+005	O
+compared	O
+to	O
+the	O
+WT	B-protein_state
+Δtdk	B-mutant
+strain	O
+.	O
+
+Conditions	O
+denoted	O
+by	O
+the	O
+same	O
+letter	O
+(	O
+b	O
+,	O
+c	O
+,	O
+or	O
+d	O
+)	O
+are	O
+not	O
+statistically	O
+significant	O
+from	O
+each	O
+other	O
+but	O
+are	O
+significantly	O
+different	O
+from	O
+the	O
+condition	O
+labeled	O
+“	O
+a	O
+.”	O
+Complementation	O
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+and	O
+ΔSBGP	B-mutant
+-	I-mutant
+B	I-mutant
+was	O
+performed	O
+by	O
+allelic	O
+exchange	O
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+genes	O
+back	O
+into	O
+the	O
+genome	O
+for	O
+expression	O
+via	O
+the	O
+native	O
+promoter	O
+:	O
+these	O
+growth	O
+curves	O
+,	O
+quantified	O
+rates	O
+and	O
+lag	B-evidence
+times	I-evidence
+are	O
+displayed	O
+in	O
+Fig	O
+.	O
+S8	O
+in	O
+the	O
+supplemental	O
+material	O
+.	O
+
+The	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+(	O
+ΔBacova_02651	B-mutant
+)	O
+strain	O
+(	O
+cf	O
+.	O
+
+Fig	O
+.	O
+1B	O
+)	O
+was	O
+completely	O
+incapable	O
+of	O
+growth	O
+on	O
+XyG	B-chemical
+,	O
+XyGO1	B-chemical
+,	O
+and	O
+XyGO2	B-chemical
+,	O
+indicating	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+is	O
+essential	O
+for	O
+XyG	B-chemical
+utilization	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+This	O
+result	O
+mirrors	O
+our	O
+previous	O
+data	O
+for	O
+the	O
+canonical	O
+Sus	B-complex_assembly
+of	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+,	O
+which	O
+revealed	O
+that	O
+a	O
+homologous	O
+ΔsusD	B-mutant
+mutant	B-protein_state
+is	O
+unable	O
+to	O
+grow	O
+on	O
+starch	B-chemical
+or	O
+malto	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+,	O
+despite	O
+normal	O
+cell	O
+surface	O
+expression	O
+of	O
+all	O
+other	O
+PUL	B-gene
+-	O
+encoded	O
+proteins	O
+.	O
+
+More	O
+recently	O
+,	O
+we	O
+demonstrated	O
+that	O
+this	O
+phenotype	O
+is	O
+due	O
+to	O
+the	O
+loss	O
+of	O
+the	O
+physical	O
+presence	O
+of	O
+SusD	B-protein
+;	O
+complementation	B-experimental_method
+of	O
+ΔsusD	B-mutant
+with	O
+SusD	B-mutant
+*,	I-mutant
+a	O
+triple	B-protein_state
+site	I-protein_state
+-	I-protein_state
+directed	I-protein_state
+mutant	I-protein_state
+(	O
+W96A	B-mutant
+W320A	B-mutant
+Y296A	B-mutant
+)	O
+that	O
+ablates	B-protein_state
+glycan	I-protein_state
+binding	I-protein_state
+,	O
+restores	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+growth	O
+on	O
+malto	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+and	O
+starch	B-chemical
+when	O
+sus	B-gene
+transcription	O
+is	O
+induced	O
+by	O
+maltose	B-chemical
+addition	O
+.	O
+
+Similarly	O
+,	O
+the	O
+function	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+extends	O
+beyond	O
+glycan	B-chemical
+binding	O
+.	O
+
+Complementation	B-experimental_method
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+with	O
+the	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+(	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+)	O
+variant	O
+,	O
+which	O
+does	O
+not	B-protein_state
+bind	I-protein_state
+XyG	B-chemical
+,	O
+supports	O
+growth	O
+on	O
+XyG	B-chemical
+and	O
+XyGOs	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+;	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*),	I-mutant
+with	O
+growth	O
+rates	O
+that	O
+are	O
+~	O
+70	O
+%	O
+that	O
+of	O
+the	O
+WT	B-protein_state
+.	O
+
+In	O
+previous	O
+studies	O
+,	O
+we	O
+observed	O
+that	O
+carbohydrate	B-chemical
+binding	O
+by	O
+SusD	B-protein
+enhanced	O
+the	O
+sensitivity	O
+of	O
+the	O
+cells	O
+to	O
+limiting	O
+concentrations	O
+of	O
+malto	O
+-	O
+oligosaccharides	O
+by	O
+several	O
+orders	O
+of	O
+magnitude	O
+,	O
+such	O
+that	O
+the	O
+addition	O
+of	O
+0	O
+.	O
+5	O
+g	O
+/	O
+liter	O
+maltose	B-chemical
+was	O
+required	O
+to	O
+restore	O
+growth	O
+of	O
+the	O
+ΔsusD	B-mutant
+::	O
+SusD	B-mutant
+*	I-mutant
+strain	O
+on	O
+starch	B-chemical
+,	O
+which	O
+nonetheless	O
+occurred	O
+following	O
+an	O
+extended	O
+lag	B-evidence
+phase	I-evidence
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+strain	O
+does	O
+not	O
+display	O
+an	O
+extended	O
+lag	B-evidence
+time	I-evidence
+on	O
+any	O
+of	O
+the	O
+xyloglucan	B-chemical
+substrates	O
+compared	O
+to	O
+the	O
+WT	B-protein_state
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+specific	O
+glycan	B-chemical
+signal	O
+that	O
+upregulates	O
+BoXyGUL	B-gene
+is	O
+currently	O
+unknown	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	B-chemical
+binding	O
+by	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+enhances	O
+transcriptional	O
+activation	O
+of	O
+the	O
+XyGUL	B-gene
+.	O
+
+However	O
+,	O
+the	O
+modest	O
+rate	O
+defect	O
+displayed	O
+by	O
+the	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+strain	O
+suggests	O
+that	O
+recognition	O
+of	O
+XyG	B-chemical
+and	O
+product	O
+import	O
+is	O
+somewhat	O
+less	O
+efficient	O
+in	O
+these	O
+cells	O
+.	O
+
+Intriguingly	O
+,	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+strain	O
+(	O
+ΔBacova_02650	B-mutant
+)	O
+(	O
+cf	O
+.	O
+
+Fig	O
+.	O
+1B	O
+)	O
+exhibited	O
+a	O
+minor	O
+growth	O
+defect	O
+on	O
+both	O
+XyG	B-chemical
+and	O
+XyGO2	B-chemical
+,	O
+with	O
+rates	O
+84	O
+.	O
+6	O
+%	O
+and	O
+93	O
+.	O
+9	O
+%	O
+that	O
+of	O
+the	O
+WT	B-protein_state
+Δtdk	B-mutant
+strain	O
+.	O
+
+However	O
+,	O
+growth	O
+of	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+strain	O
+on	O
+XyGO1	B-chemical
+was	O
+54	O
+.	O
+2	O
+%	O
+the	O
+rate	O
+of	O
+the	O
+parental	O
+strain	O
+,	O
+despite	O
+the	O
+fact	O
+that	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+binds	O
+this	O
+substrate	O
+ca	O
+.	O
+
+10	O
+-	O
+fold	O
+more	O
+weakly	O
+than	O
+XyGO2	B-chemical
+and	O
+XyG	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+;	O
+Table	O
+1	O
+).	O
+
+As	O
+such	O
+,	O
+the	O
+data	O
+suggest	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+can	O
+compensate	O
+for	O
+the	O
+loss	O
+of	O
+function	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+on	O
+longer	O
+oligo	B-chemical
+-	I-chemical
+and	I-chemical
+polysaccharides	I-chemical
+,	O
+while	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+may	O
+adapt	O
+the	O
+cell	O
+to	O
+recognize	O
+smaller	O
+oligosaccharides	B-chemical
+efficiently	O
+.	O
+
+Indeed	O
+,	O
+a	O
+double	B-protein_state
+mutant	I-protein_state
+,	O
+consisting	O
+of	O
+a	O
+crippled	B-protein_state
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+a	O
+deletion	B-experimental_method
+of	I-experimental_method
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*/	I-mutant
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+),	O
+exhibits	O
+an	O
+extended	O
+lag	B-evidence
+time	I-evidence
+on	O
+both	O
+XyG	B-chemical
+and	O
+XyGO2	B-chemical
+,	O
+as	O
+well	O
+as	O
+XyGO1	B-chemical
+.	O
+
+Taken	O
+together	O
+,	O
+the	O
+data	O
+indicate	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+functionally	O
+complement	O
+each	O
+other	O
+in	O
+the	O
+capture	O
+of	O
+XyG	B-chemical
+polysaccharide	B-chemical
+,	O
+while	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+may	O
+allow	O
+B	B-species
+.	I-species
+ovatus	I-species
+to	O
+scavenge	O
+smaller	O
+XyGOs	B-chemical
+liberated	O
+by	O
+other	O
+gut	O
+commensals	O
+.	O
+
+This	O
+additional	O
+role	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+is	O
+especially	O
+notable	O
+in	O
+the	O
+context	O
+of	O
+studies	O
+on	O
+BtSusE	B-protein
+and	O
+BtSusF	B-protein
+(	O
+positioned	O
+similarly	O
+in	O
+the	O
+archetypal	O
+Sus	B-gene
+locus	I-gene
+)	O
+(	O
+Fig	O
+.	O
+1B	O
+),	O
+for	O
+which	O
+growth	O
+defects	O
+on	O
+starch	B-chemical
+or	O
+malto	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+have	O
+never	O
+been	O
+observed	O
+.	O
+
+Beyond	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+we	O
+speculated	O
+that	O
+the	O
+catalytically	B-protein_state
+feeble	I-protein_state
+endo	B-protein_type
+-	I-protein_type
+xyloglucanase	I-protein_type
+GH9	B-protein
+,	O
+which	O
+is	O
+expendable	O
+for	O
+growth	O
+in	O
+the	O
+presence	O
+of	O
+GH5	B-protein
+,	O
+might	O
+also	O
+play	O
+a	O
+role	O
+in	O
+glycan	B-chemical
+binding	O
+to	O
+the	O
+cell	O
+surface	O
+.	O
+
+However	O
+,	O
+combined	B-experimental_method
+deletion	I-experimental_method
+of	I-experimental_method
+the	I-experimental_method
+genes	I-experimental_method
+encoding	I-experimental_method
+GH9	B-protein
+(	O
+encoded	O
+by	O
+Bacova_02649	B-gene
+)	O
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+does	O
+not	O
+exacerbate	O
+the	O
+growth	O
+defect	O
+on	O
+XyGO1	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+;	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+/	O
+ΔGH9	B-mutant
+).	O
+
+The	O
+necessity	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+is	O
+elevated	O
+in	O
+the	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+strain	O
+,	O
+as	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*/	I-mutant
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+mutant	B-protein_state
+displays	O
+an	O
+extended	O
+lag	B-evidence
+during	O
+growth	O
+on	O
+XyG	B-chemical
+and	O
+xylogluco	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+,	O
+while	O
+growth	O
+rate	O
+differences	O
+are	O
+more	O
+subtle	O
+.	O
+
+The	O
+precise	O
+reason	O
+for	O
+this	O
+lag	B-evidence
+is	O
+unclear	O
+,	O
+but	O
+recapitulating	O
+our	O
+findings	O
+on	O
+the	O
+role	O
+of	O
+SusD	B-protein
+in	O
+malto	B-chemical
+-	I-chemical
+oligosaccharide	I-chemical
+sensing	O
+in	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+,	O
+this	O
+extended	O
+lag	B-evidence
+may	O
+be	O
+due	O
+to	O
+inefficient	O
+import	O
+and	O
+thus	O
+sensing	O
+of	O
+xyloglucan	B-chemical
+in	O
+the	O
+environment	O
+in	O
+the	O
+absence	O
+of	O
+glycan	B-chemical
+binding	O
+by	O
+essential	O
+SGBPs	B-protein_type
+.	O
+
+Our	O
+previous	O
+work	O
+demonstrates	O
+that	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+grown	O
+in	O
+minimal	O
+medium	O
+plus	O
+glucose	B-chemical
+express	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	B-gene
+transcript	O
+.	O
+
+Thus	O
+,	O
+in	O
+our	O
+experiments	O
+,	O
+we	O
+presume	O
+that	O
+each	O
+strain	O
+,	O
+initially	O
+grown	O
+in	O
+glucose	B-chemical
+,	O
+expresses	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	B-gene
+transcript	O
+and	O
+thus	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	B-gene
+-	O
+encoded	O
+surface	O
+proteins	O
+,	O
+including	O
+the	O
+vanguard	O
+GH5	B-protein
+.	O
+
+Presumably	O
+without	O
+glycan	B-chemical
+binding	O
+by	O
+the	O
+SGBPs	B-protein_type
+,	O
+the	O
+GH5	B-protein
+protein	O
+cannot	O
+efficiently	O
+process	O
+xyloglucan	B-chemical
+,	O
+and	O
+/	O
+or	O
+the	O
+lack	O
+of	O
+SGBP	B-protein_type
+function	O
+prevents	O
+efficient	O
+capture	O
+and	O
+import	O
+of	O
+the	O
+processed	O
+oligosaccharides	B-chemical
+.	O
+
+It	O
+may	O
+then	O
+be	O
+that	O
+only	O
+after	O
+a	O
+sufficient	O
+amount	O
+of	O
+glycan	B-chemical
+is	O
+processed	O
+and	O
+imported	O
+by	O
+the	O
+cell	O
+is	O
+XyGUL	B-gene
+upregulated	O
+and	O
+exponential	O
+growth	O
+on	O
+the	O
+glycan	B-chemical
+can	O
+begin	O
+.	O
+
+We	O
+hypothesize	O
+that	O
+during	O
+exponential	O
+growth	O
+the	O
+essential	O
+role	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+extends	O
+beyond	O
+glycan	B-chemical
+recognition	O
+,	O
+perhaps	O
+due	O
+to	O
+a	O
+critical	O
+interaction	O
+with	O
+the	O
+TBDT	B-protein_type
+.	O
+
+In	O
+the	O
+BtSus	B-gene
+,	O
+SusD	B-protein
+and	O
+the	O
+TBDT	B-protein_type
+SusC	B-protein
+interact	O
+,	O
+and	O
+we	O
+speculate	O
+that	O
+this	O
+interaction	O
+is	O
+necessary	O
+for	O
+glycan	B-chemical
+uptake	O
+,	O
+as	O
+suggested	O
+by	O
+the	O
+fact	O
+that	O
+a	O
+ΔsusD	B-mutant
+mutant	B-protein_state
+cannot	O
+grow	O
+on	O
+starch	B-chemical
+,	O
+but	O
+a	O
+ΔsusD	B-mutant
+::	O
+SusD	B-mutant
+*	I-mutant
+strain	O
+regains	O
+this	O
+ability	O
+if	O
+a	O
+transcriptional	B-protein_type
+activator	I-protein_type
+of	O
+the	O
+sus	B-gene
+operon	I-gene
+is	O
+supplied	O
+.	O
+
+Likewise	O
+,	O
+such	O
+cognate	O
+interactions	O
+between	O
+homologous	O
+protein	O
+pairs	O
+such	O
+as	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+its	O
+TBDT	B-protein_type
+may	O
+underlie	O
+our	O
+observation	O
+that	O
+a	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+mutant	B-protein_state
+cannot	O
+grow	O
+on	O
+xyloglucan	B-chemical
+.	O
+
+However	O
+,	O
+unlike	O
+the	O
+Sus	B-complex_assembly
+,	O
+in	O
+which	O
+elimination	B-experimental_method
+of	I-experimental_method
+SusE	B-protein
+and	O
+SusF	B-protein
+does	O
+not	O
+affect	O
+growth	O
+on	O
+starch	B-chemical
+,	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+appears	O
+to	O
+have	O
+a	O
+dedicated	O
+role	O
+in	O
+growth	O
+on	O
+small	O
+xylogluco	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+.	O
+
+The	O
+ability	O
+of	O
+gut	O
+-	O
+adapted	O
+microorganisms	B-taxonomy_domain
+to	O
+thrive	O
+in	O
+the	O
+gastrointestinal	O
+tract	O
+is	O
+critically	O
+dependent	O
+upon	O
+their	O
+ability	O
+to	O
+efficiently	O
+recognize	O
+,	O
+cleave	O
+,	O
+and	O
+import	O
+glycans	B-chemical
+.	O
+
+The	O
+human	B-species
+gut	O
+,	O
+in	O
+particular	O
+,	O
+is	O
+a	O
+densely	O
+packed	O
+ecosystem	O
+with	O
+hundreds	O
+of	O
+species	O
+,	O
+in	O
+which	O
+there	O
+is	O
+potential	O
+for	O
+both	O
+competition	O
+and	O
+synergy	O
+in	O
+the	O
+utilization	O
+of	O
+different	O
+substrates	O
+.	O
+
+Recent	O
+work	O
+has	O
+elucidated	O
+that	O
+Bacteroidetes	B-taxonomy_domain
+cross	O
+-	O
+feed	O
+during	O
+growth	O
+on	O
+many	O
+glycans	B-chemical
+;	O
+the	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+expressed	O
+by	O
+one	O
+species	O
+liberate	O
+oligosaccharides	B-chemical
+for	O
+consumption	O
+by	O
+other	O
+members	O
+of	O
+the	O
+community	O
+.	O
+
+Thus	O
+,	O
+understanding	O
+glycan	B-chemical
+capture	O
+at	O
+the	O
+cell	O
+surface	O
+is	O
+fundamental	O
+to	O
+explaining	O
+,	O
+and	O
+eventually	O
+predicting	O
+,	O
+how	O
+the	O
+carbohydrate	O
+content	O
+of	O
+the	O
+diet	O
+shapes	O
+the	O
+gut	O
+community	O
+structure	O
+as	O
+well	O
+as	O
+its	O
+causative	O
+health	O
+effects	O
+.	O
+
+Here	O
+,	O
+we	O
+demonstrate	O
+that	O
+the	O
+surface	B-protein_type
+glycan	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+encoded	O
+within	O
+the	O
+BoXyGUL	B-gene
+play	O
+unique	O
+and	O
+essential	O
+roles	O
+in	O
+the	O
+acquisition	O
+of	O
+the	O
+ubiquitous	O
+and	O
+abundant	O
+vegetable	B-taxonomy_domain
+polysaccharide	B-chemical
+xyloglucan	B-chemical
+.	O
+
+Yet	O
+,	O
+a	O
+number	O
+of	O
+questions	O
+remain	O
+regarding	O
+the	O
+molecular	O
+interplay	O
+of	O
+SGBPs	B-protein_type
+with	O
+their	O
+cotranscribed	O
+cohort	O
+of	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+and	O
+TonB	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+transporters	I-protein_type
+.	O
+
+A	O
+particularly	O
+understudied	O
+aspect	O
+of	O
+glycan	B-chemical
+utilization	O
+is	O
+the	O
+mechanism	O
+of	O
+import	O
+via	O
+TBDTs	B-protein_type
+(	O
+SusC	B-protein
+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	B-gene
+.	O
+
+PUL	B-gene
+-	O
+encoded	O
+TBDTs	B-protein_type
+in	O
+Bacteroidetes	B-taxonomy_domain
+are	O
+larger	O
+than	O
+the	O
+well	O
+-	O
+characterized	O
+iron	B-protein_type
+-	I-protein_type
+targeting	I-protein_type
+TBDTs	I-protein_type
+from	O
+many	O
+Proteobacteria	B-taxonomy_domain
+and	O
+are	O
+further	O
+distinguished	O
+as	O
+the	O
+only	O
+known	O
+glycan	B-protein_type
+-	I-protein_type
+importing	I-protein_type
+TBDTs	I-protein_type
+coexpressed	O
+with	O
+an	O
+SGBP	B-protein_type
+.	O
+
+A	O
+direct	O
+interaction	O
+between	O
+the	O
+BtSusC	B-protein
+TBDT	B-protein_type
+and	O
+the	O
+SusD	B-protein
+SGBP	B-protein_type
+has	O
+been	O
+previously	O
+demonstrated	O
+,	O
+as	O
+has	O
+an	O
+interaction	O
+between	O
+the	O
+homologous	O
+components	O
+encoded	O
+by	O
+an	O
+N	O
+-	O
+glycan	B-chemical
+-	O
+scavenging	O
+PUL	B-gene
+of	O
+Capnocytophaga	B-species
+canimorsus	I-species
+.	O
+
+Our	O
+observation	O
+here	O
+that	O
+the	O
+physical	O
+presence	O
+of	O
+the	O
+SusD	B-protein
+homolog	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+independent	O
+of	O
+XyG	B-chemical
+-	O
+binding	O
+ability	O
+,	O
+is	O
+both	O
+necessary	O
+and	O
+sufficient	O
+for	O
+XyG	B-chemical
+utilization	O
+further	O
+supports	O
+a	O
+model	O
+of	O
+glycan	B-chemical
+import	O
+whereby	O
+the	O
+SusC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+TBDTs	I-protein_type
+and	O
+the	O
+SusD	B-protein_type
+-	I-protein_type
+like	I-protein_type
+SGBPs	I-protein_type
+must	O
+be	O
+intimately	O
+associated	O
+to	O
+support	O
+glycan	B-chemical
+uptake	O
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+
+It	O
+is	O
+yet	O
+presently	O
+unclear	O
+whether	O
+this	O
+interaction	O
+is	O
+static	O
+or	O
+dynamic	O
+and	O
+to	O
+what	O
+extent	O
+the	O
+association	O
+of	O
+cognate	O
+TBDT	B-protein_type
+/	O
+SGBPs	B-protein_type
+is	O
+dependent	O
+upon	O
+the	O
+structure	O
+of	O
+the	O
+carbohydrate	B-chemical
+to	O
+be	O
+imported	O
+.	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+there	O
+is	O
+clear	O
+evidence	O
+for	O
+independent	O
+TBDTs	B-protein_type
+in	O
+Bacteroidetes	B-taxonomy_domain
+that	O
+do	O
+not	O
+require	O
+SGBP	B-protein_type
+association	O
+for	O
+activity	O
+.	O
+
+For	O
+example	O
+,	O
+it	O
+was	O
+recently	O
+demonstrated	O
+that	O
+expression	O
+of	O
+nanO	B-gene
+,	O
+which	O
+encodes	O
+a	O
+SusC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+TBDT	I-protein_type
+as	O
+part	O
+of	O
+a	O
+sialic	O
+-	O
+acid	O
+-	O
+targeting	O
+PUL	B-gene
+from	O
+B	B-species
+.	I-species
+fragilis	I-species
+,	O
+restored	O
+growth	O
+on	O
+this	O
+monosaccharide	B-chemical
+in	O
+a	O
+mutant	O
+strain	O
+of	O
+E	B-species
+.	I-species
+coli	I-species
+.	O
+
+In	O
+this	O
+instance	O
+,	O
+coexpression	O
+of	O
+the	O
+susD	B-gene
+-	O
+like	O
+gene	O
+nanU	B-gene
+was	O
+not	O
+required	O
+,	O
+nor	O
+did	O
+the	O
+expression	O
+of	O
+the	O
+nanU	B-gene
+gene	O
+enhance	O
+growth	O
+kinetics	O
+.	O
+
+Similarly	O
+,	O
+the	O
+deletion	O
+of	O
+BT1762	B-gene
+encoding	O
+a	O
+fructan	B-protein_type
+-	I-protein_type
+targeting	I-protein_type
+SusD	I-protein_type
+-	I-protein_type
+like	I-protein_type
+protein	I-protein_type
+in	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+did	O
+not	O
+result	O
+in	O
+a	O
+dramatic	O
+loss	O
+of	O
+growth	O
+on	O
+fructans	B-chemical
+.	O
+
+Thus	O
+,	O
+the	O
+strict	O
+dependence	O
+on	O
+a	O
+SusD	B-protein_type
+-	I-protein_type
+like	I-protein_type
+SGBP	I-protein_type
+for	O
+glycan	B-chemical
+uptake	O
+in	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+may	O
+be	O
+variable	O
+and	O
+substrate	O
+dependent	O
+.	O
+
+Furthermore	O
+,	O
+considering	O
+the	O
+broader	O
+distribution	O
+of	O
+TBDTs	B-protein_type
+in	O
+PUL	B-gene
+lacking	O
+SGBPs	B-protein_type
+(	O
+sometimes	O
+known	O
+as	O
+carbohydrate	B-gene
+utilization	I-gene
+containing	I-gene
+TBDT	I-gene
+[	I-gene
+CUT	I-gene
+]	I-gene
+loci	I-gene
+;	O
+see	O
+reference	O
+and	O
+reviewed	O
+in	O
+reference	O
+)	O
+across	O
+bacterial	B-taxonomy_domain
+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	B-taxonomy_domain
+.	O
+
+Equally	O
+intriguing	O
+is	O
+the	O
+observation	O
+that	O
+while	O
+SusD	B-protein_type
+-	I-protein_type
+like	I-protein_type
+proteins	I-protein_type
+such	O
+as	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+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	B-protein_type
+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	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+lipoproteins	I-protein_type
+with	O
+little	O
+or	O
+no	O
+sequence	O
+or	O
+structural	O
+conservation	O
+,	O
+even	O
+among	O
+syntenic	O
+PUL	B-gene
+that	O
+target	O
+the	O
+same	O
+polysaccharide	B-chemical
+.	O
+
+Such	O
+is	O
+the	O
+case	O
+for	O
+XyGUL	B-gene
+from	O
+related	O
+Bacteroides	B-taxonomy_domain
+species	O
+,	O
+which	O
+may	O
+encode	O
+either	O
+one	O
+or	O
+two	O
+of	O
+these	O
+predicted	O
+SGBPs	B-protein_type
+,	O
+and	O
+these	O
+proteins	O
+vary	O
+considerably	O
+in	O
+length	O
+.	O
+
+The	O
+extremely	O
+low	O
+similarity	O
+of	O
+these	O
+SGBPs	B-protein_type
+is	O
+striking	O
+in	O
+light	O
+of	O
+the	O
+moderate	O
+sequence	O
+conservation	O
+observed	O
+among	O
+homologous	O
+GHs	B-protein_type
+in	O
+syntenic	O
+PUL	B-gene
+.	O
+
+This	O
+,	O
+together	O
+with	O
+the	O
+observation	O
+that	O
+these	O
+SGBPs	B-protein_type
+,	O
+as	O
+exemplified	O
+by	O
+BtSusE	B-protein
+and	O
+BtSusF	B-protein
+and	O
+the	O
+XyGUL	B-gene
+SGBP	B-protein
+-	I-protein
+B	I-protein
+of	O
+the	O
+present	O
+study	O
+,	O
+are	O
+expendable	O
+for	O
+polysaccharide	B-chemical
+growth	O
+,	O
+implies	O
+a	O
+high	O
+degree	O
+of	O
+evolutionary	O
+flexibility	O
+to	O
+enhance	O
+glycan	B-chemical
+capture	O
+at	O
+the	O
+cell	O
+surface	O
+.	O
+
+Because	O
+the	O
+intestinal	O
+ecosystem	O
+is	O
+a	O
+dense	O
+consortium	O
+of	O
+bacteria	B-taxonomy_domain
+that	O
+must	O
+compete	O
+for	O
+their	O
+nutrients	O
+,	O
+these	O
+multimodular	O
+SGBPs	B-protein_type
+may	O
+reflect	O
+ongoing	O
+evolutionary	O
+experiments	O
+to	O
+enhance	O
+glycan	B-chemical
+uptake	O
+efficiency	O
+.	O
+
+Whether	O
+organisms	O
+that	O
+express	O
+longer	O
+SGBPs	B-protein_type
+,	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	B-chemical
+is	O
+presently	O
+unknown	O
+.	O
+
+However	O
+,	O
+the	O
+natural	O
+diversity	O
+of	O
+these	O
+proteins	O
+represents	O
+a	O
+rich	O
+source	O
+for	O
+the	O
+discovery	O
+of	O
+unique	O
+carbohydrate	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+motifs	I-structure_element
+to	O
+both	O
+inform	O
+gut	O
+microbiology	O
+and	O
+generate	O
+new	O
+,	O
+specific	O
+carbohydrate	B-chemical
+analytical	O
+reagents	O
+.	O
+
+In	O
+conclusion	O
+,	O
+the	O
+present	O
+study	O
+further	O
+illuminates	O
+the	O
+essential	O
+role	O
+that	O
+surface	B-protein_type
+-	I-protein_type
+glycan	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+play	O
+in	O
+facilitating	O
+the	O
+catabolism	O
+of	O
+complex	O
+dietary	O
+carbohydrates	B-chemical
+by	O
+Bacteroidetes	B-taxonomy_domain
+.	O
+
+The	O
+ability	O
+of	O
+our	O
+resident	O
+gut	O
+bacteria	B-taxonomy_domain
+to	O
+recognize	O
+polysaccharides	B-chemical
+is	O
+the	O
+first	O
+committed	O
+step	O
+of	O
+glycan	B-chemical
+consumption	O
+by	O
+these	O
+organisms	O
+,	O
+a	O
+critical	O
+process	O
+that	O
+influences	O
+the	O
+community	O
+structure	O
+and	O
+thus	O
+the	O
+metabolic	O
+output	O
+(	O
+i	O
+.	O
+e	O
+.,	O
+short	O
+-	O
+chain	O
+fatty	O
+acid	O
+and	O
+metabolite	O
+profile	O
+)	O
+of	O
+these	O
+organisms	O
+.	O
+
+A	O
+molecular	O
+understanding	O
+of	O
+glycan	B-chemical
+uptake	O
+by	O
+human	B-species
+gut	O
+bacteria	B-taxonomy_domain
+is	O
+therefore	O
+central	O
+to	O
+the	O
+development	O
+of	O
+strategies	O
+to	O
+improve	O
+human	B-species
+health	O
+through	O
+manipulation	O
+of	O
+the	O
+microbiota	B-taxonomy_domain
+.	O
+
+Mucosal	O
+glycan	B-chemical
+foraging	O
+enhances	O
+fitness	O
+and	O
+transmission	O
+of	O
+a	O
+saccharolytic	O
+human	O
+gut	O
+bacterial	O
+symbiont	O
+
+Molecular	O
+Basis	O
+of	O
+Ligand	O
+-	O
+Dependent	O
+Regulation	O
+of	O
+NadR	B-protein
+,	O
+the	O
+Transcriptional	B-protein_type
+Repressor	I-protein_type
+of	O
+Meningococcal	B-taxonomy_domain
+Virulence	O
+Factor	O
+NadA	B-protein
+
+Neisseria	B-protein
+adhesin	I-protein
+A	I-protein
+(	O
+NadA	B-protein
+)	O
+is	O
+present	O
+on	O
+the	O
+meningococcal	B-taxonomy_domain
+surface	O
+and	O
+contributes	O
+to	O
+adhesion	O
+to	O
+and	O
+invasion	O
+of	O
+human	B-species
+cells	O
+.	O
+
+NadA	B-protein
+is	O
+also	O
+one	O
+of	O
+three	O
+recombinant	O
+antigens	O
+in	O
+the	O
+recently	O
+-	O
+approved	O
+Bexsero	O
+vaccine	O
+,	O
+which	O
+protects	O
+against	O
+serogroup	B-taxonomy_domain
+B	I-taxonomy_domain
+meningococcus	I-taxonomy_domain
+.	O
+
+The	O
+amount	O
+of	O
+NadA	B-protein
+on	O
+the	O
+bacterial	B-taxonomy_domain
+surface	O
+is	O
+of	O
+direct	O
+relevance	O
+in	O
+the	O
+constant	O
+battle	O
+of	O
+host	O
+-	O
+pathogen	O
+interactions	O
+:	O
+it	O
+influences	O
+the	O
+ability	O
+of	O
+the	O
+pathogen	O
+to	O
+engage	O
+human	B-species
+cell	O
+surface	O
+-	O
+exposed	O
+receptors	O
+and	O
+,	O
+conversely	O
+,	O
+the	O
+bacterial	B-taxonomy_domain
+susceptibility	O
+to	O
+the	O
+antibody	O
+-	O
+mediated	O
+immune	O
+response	O
+.	O
+
+It	O
+is	O
+therefore	O
+important	O
+to	O
+understand	O
+the	O
+mechanisms	O
+which	O
+regulate	O
+nadA	B-gene
+expression	O
+levels	O
+,	O
+which	O
+are	O
+predominantly	O
+controlled	O
+by	O
+the	O
+transcriptional	B-protein_type
+regulator	I-protein_type
+NadR	B-protein
+(	O
+Neisseria	B-protein
+adhesin	I-protein
+A	I-protein
+Regulator	I-protein
+)	O
+both	O
+in	O
+vitro	O
+and	O
+in	O
+vivo	O
+.	O
+
+NadR	B-protein
+binds	O
+the	O
+nadA	B-gene
+promoter	O
+and	O
+represses	O
+gene	O
+transcription	O
+.	O
+
+In	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+hydroxyphenylacetate	I-chemical
+(	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+),	O
+a	O
+catabolite	O
+present	O
+in	O
+human	B-species
+saliva	O
+both	O
+under	O
+physiological	O
+conditions	O
+and	O
+during	O
+bacterial	B-taxonomy_domain
+infection	O
+,	O
+the	O
+binding	O
+of	O
+NadR	B-protein
+to	O
+the	O
+nadA	B-gene
+promoter	O
+is	O
+attenuated	O
+and	O
+nadA	B-gene
+expression	O
+is	O
+induced	O
+.	O
+
+NadR	B-protein
+also	O
+mediates	O
+ligand	O
+-	O
+dependent	O
+regulation	O
+of	O
+many	O
+other	O
+meningococcal	B-taxonomy_domain
+genes	O
+,	O
+for	O
+example	O
+the	O
+highly	O
+-	O
+conserved	O
+multiple	O
+adhesin	O
+family	O
+(	O
+maf	O
+)	O
+genes	O
+,	O
+which	O
+encode	O
+proteins	O
+emerging	O
+with	O
+important	O
+roles	O
+in	O
+host	O
+-	O
+pathogen	O
+interactions	O
+,	O
+immune	O
+evasion	O
+and	O
+niche	O
+adaptation	O
+.	O
+
+To	O
+gain	O
+insights	O
+into	O
+the	O
+regulation	O
+of	O
+NadR	B-protein
+mediated	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+we	O
+combined	O
+structural	B-experimental_method
+,	I-experimental_method
+biochemical	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+mutagenesis	I-experimental_method
+studies	I-experimental_method
+.	O
+
+In	O
+particular	O
+,	O
+two	O
+new	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+ligand	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+NadR	B-protein
+revealed	O
+(	O
+i	O
+)	O
+the	O
+molecular	O
+basis	O
+of	O
+‘	O
+conformational	O
+selection	O
+’	O
+by	O
+which	O
+a	O
+single	O
+molecule	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+binds	O
+and	O
+stabilizes	O
+dimeric	B-oligomeric_state
+NadR	B-protein
+in	O
+a	O
+conformation	O
+unsuitable	O
+for	O
+DNA	O
+-	O
+binding	O
+,	O
+(	O
+ii	O
+)	O
+molecular	O
+explanations	O
+for	O
+the	O
+binding	O
+specificities	O
+of	O
+different	O
+hydroxyphenylacetate	B-chemical
+ligands	O
+,	O
+including	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+which	O
+is	O
+produced	O
+during	O
+inflammation	O
+,	O
+(	O
+iii	O
+)	O
+the	O
+presence	O
+of	O
+a	O
+leucine	B-residue_name
+residue	O
+essential	O
+for	O
+dimerization	O
+and	O
+conserved	B-protein_state
+in	O
+many	O
+MarR	B-protein_type
+family	O
+proteins	O
+,	O
+and	O
+(	O
+iv	O
+)	O
+four	O
+residues	O
+(	O
+His7	B-residue_name_number
+,	O
+Ser9	B-residue_name_number
+,	O
+Asn11	B-residue_name_number
+and	O
+Phe25	B-residue_name_number
+),	O
+which	O
+are	O
+involved	O
+in	O
+binding	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+and	O
+were	O
+confirmed	O
+in	O
+vitro	O
+to	O
+have	O
+key	O
+roles	O
+in	O
+the	O
+regulatory	O
+mechanism	O
+in	O
+bacteria	B-taxonomy_domain
+.	O
+
+Overall	O
+,	O
+this	O
+study	O
+deepens	O
+our	O
+molecular	O
+understanding	O
+of	O
+the	O
+sophisticated	O
+regulatory	O
+mechanisms	O
+of	O
+the	O
+expression	O
+of	O
+nadA	B-gene
+and	O
+other	O
+genes	O
+governed	O
+by	O
+NadR	B-protein
+,	O
+dependent	O
+on	O
+interactions	O
+with	O
+niche	O
+-	O
+specific	O
+signal	O
+molecules	O
+that	O
+may	O
+play	O
+important	O
+roles	O
+during	O
+meningococcal	B-taxonomy_domain
+pathogenesis	O
+.	O
+
+Serogroup	B-taxonomy_domain
+B	I-taxonomy_domain
+meningococcus	I-taxonomy_domain
+(	O
+MenB	B-species
+)	O
+causes	O
+fatal	O
+sepsis	O
+and	O
+invasive	O
+meningococcal	B-taxonomy_domain
+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	B-species
+outbreaks	O
+in	O
+universities	O
+of	O
+the	O
+United	O
+States	O
+and	O
+Canada	O
+.	O
+
+The	O
+Bexsero	O
+vaccine	O
+protects	O
+against	O
+MenB	B-species
+and	O
+has	O
+recently	O
+been	O
+approved	O
+in	O
+>	O
+35	O
+countries	O
+worldwide	O
+.	O
+
+Neisseria	B-protein
+adhesin	I-protein
+A	I-protein
+(	O
+NadA	B-protein
+)	O
+present	O
+on	O
+the	O
+meningococcal	B-taxonomy_domain
+surface	O
+can	O
+mediate	O
+binding	O
+to	O
+human	B-species
+cells	O
+and	O
+is	O
+one	O
+of	O
+the	O
+three	O
+MenB	B-species
+vaccine	O
+protein	O
+antigens	O
+.	O
+
+The	O
+amount	O
+of	O
+NadA	B-protein
+exposed	O
+on	O
+the	O
+meningococcal	B-taxonomy_domain
+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
+
+A	O
+deep	O
+understanding	O
+of	O
+nadA	B-gene
+expression	O
+is	O
+therefore	O
+important	O
+,	O
+otherwise	O
+the	O
+contribution	O
+of	O
+NadA	B-protein
+to	O
+vaccine	O
+-	O
+induced	O
+protection	O
+against	O
+meningococcal	B-taxonomy_domain
+meningitis	O
+may	O
+be	O
+underestimated	O
+.	O
+
+The	O
+abundance	O
+of	O
+surface	O
+-	O
+exposed	O
+NadA	B-protein
+is	O
+regulated	O
+by	O
+the	O
+ligand	B-protein_type
+-	I-protein_type
+responsive	I-protein_type
+transcriptional	I-protein_type
+repressor	I-protein_type
+NadR	B-protein
+.	O
+Here	O
+,	O
+we	O
+present	O
+functional	B-evidence
+,	I-evidence
+biochemical	I-evidence
+and	I-evidence
+high	I-evidence
+-	I-evidence
+resolution	I-evidence
+structural	I-evidence
+data	I-evidence
+on	O
+NadR	B-protein
+.	O
+Our	O
+studies	O
+provide	O
+detailed	O
+insights	O
+into	O
+how	O
+small	O
+molecule	O
+ligands	O
+,	O
+such	O
+as	O
+hydroxyphenylacetate	B-chemical
+derivatives	O
+,	O
+found	O
+in	O
+relevant	O
+host	O
+niches	O
+,	O
+modulate	O
+the	O
+structure	O
+and	O
+activity	O
+of	O
+NadR	B-protein
+,	O
+by	O
+‘	O
+conformational	O
+selection	O
+’	O
+of	O
+inactive	B-protein_state
+forms	O
+.	O
+
+These	O
+findings	O
+shed	O
+light	O
+on	O
+the	O
+regulation	O
+of	O
+NadR	B-protein
+,	O
+a	O
+key	O
+MarR	B-protein_type
+-	O
+family	O
+virulence	O
+factor	O
+of	O
+this	O
+important	O
+human	B-species
+pathogen	O
+.	O
+
+The	O
+‘	O
+Reverse	B-experimental_method
+Vaccinology	I-experimental_method
+’	O
+approach	O
+was	O
+pioneered	O
+to	O
+identify	O
+antigens	O
+for	O
+a	O
+protein	O
+-	O
+based	O
+vaccine	O
+against	O
+serogroup	B-species
+B	I-species
+Neisseria	I-species
+meningitidis	I-species
+(	O
+MenB	B-species
+),	O
+a	O
+human	B-species
+pathogen	O
+causing	O
+potentially	O
+-	O
+fatal	O
+sepsis	O
+and	O
+invasive	O
+meningococcal	B-taxonomy_domain
+disease	O
+.	O
+
+Indeed	O
+,	O
+Reverse	B-experimental_method
+Vaccinology	I-experimental_method
+identified	O
+Neisseria	B-protein
+adhesin	I-protein
+A	I-protein
+(	O
+NadA	B-protein
+),	O
+a	O
+surface	O
+-	O
+exposed	O
+protein	O
+involved	O
+in	O
+epithelial	O
+cell	O
+invasion	O
+and	O
+found	O
+in	O
+~	O
+30	O
+%	O
+of	O
+clinical	O
+isolates	O
+.	O
+
+Recently	O
+,	O
+we	O
+reported	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+NadA	B-protein
+,	O
+providing	O
+insights	O
+into	O
+its	O
+biological	O
+and	O
+immunological	O
+functions	O
+.	O
+
+Recombinant	O
+NadA	B-protein
+elicits	O
+a	O
+strong	O
+bactericidal	O
+immune	O
+response	O
+and	O
+is	O
+therefore	O
+included	O
+in	O
+the	O
+Bexsero	O
+vaccine	O
+that	O
+protects	O
+against	O
+MenB	B-species
+and	O
+which	O
+was	O
+recently	O
+approved	O
+in	O
+over	O
+35	O
+countries	O
+worldwide	O
+.	O
+
+Previous	O
+studies	O
+revealed	O
+that	O
+nadA	B-gene
+expression	O
+levels	O
+are	O
+mainly	O
+regulated	O
+by	O
+the	O
+Neisseria	B-protein
+adhesin	I-protein
+A	I-protein
+Regulator	I-protein
+(	O
+NadR	B-protein
+).	O
+
+Although	O
+additional	O
+factors	O
+influence	O
+nadA	B-gene
+expression	O
+,	O
+we	O
+focused	O
+on	O
+its	O
+regulation	O
+by	O
+NadR	B-protein
+,	O
+the	O
+major	O
+mediator	O
+of	O
+NadA	B-protein
+phase	O
+variable	O
+expression	O
+.	O
+
+Studies	O
+of	O
+NadR	B-protein
+also	O
+have	O
+broader	O
+implications	O
+,	O
+since	O
+a	O
+genome	O
+-	O
+wide	O
+analysis	O
+of	O
+MenB	B-species
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+nadR	B-gene
+knock	B-protein_state
+-	I-protein_state
+out	I-protein_state
+strains	O
+revealed	O
+that	O
+NadR	B-protein
+influences	O
+the	O
+regulation	O
+of	O
+>	O
+30	O
+genes	O
+,	O
+including	O
+maf	O
+genes	O
+,	O
+from	O
+the	O
+multiple	O
+adhesin	B-protein_type
+family	O
+.	O
+
+These	O
+genes	O
+encode	O
+a	O
+wide	O
+variety	O
+of	O
+proteins	O
+connected	O
+to	O
+many	O
+biological	O
+processes	O
+contributing	O
+to	O
+bacterial	B-taxonomy_domain
+survival	O
+,	O
+adaptation	O
+in	O
+the	O
+host	O
+niche	O
+,	O
+colonization	O
+and	O
+invasion	O
+.	O
+
+NadR	B-protein
+belongs	O
+to	O
+the	O
+MarR	B-protein_type
+(	O
+Multiple	B-protein_type
+Antibiotic	I-protein_type
+Resistance	I-protein_type
+Regulator	I-protein_type
+)	O
+family	O
+,	O
+a	O
+group	O
+of	O
+ligand	B-protein_type
+-	I-protein_type
+responsive	I-protein_type
+transcriptional	I-protein_type
+regulators	I-protein_type
+ubiquitous	O
+in	O
+bacteria	B-taxonomy_domain
+and	O
+archaea	B-taxonomy_domain
+.	O
+
+MarR	B-protein_type
+family	O
+proteins	O
+can	O
+promote	O
+bacterial	B-taxonomy_domain
+survival	O
+in	O
+the	O
+presence	O
+of	O
+antibiotics	O
+,	O
+toxic	O
+chemicals	O
+,	O
+organic	O
+solvents	O
+or	O
+reactive	O
+oxygen	O
+species	O
+and	O
+can	O
+regulate	O
+virulence	O
+factor	O
+expression	O
+.	O
+
+MarR	B-protein_type
+homologues	O
+can	O
+act	O
+either	O
+as	O
+transcriptional	O
+repressors	O
+or	O
+as	O
+activators	O
+.	O
+
+Although	O
+>	O
+50	O
+MarR	B-protein_type
+family	O
+structures	B-evidence
+are	O
+known	O
+,	O
+a	O
+molecular	O
+understanding	O
+of	O
+their	O
+ligand	O
+-	O
+dependent	O
+regulatory	O
+mechanisms	O
+is	O
+still	O
+limited	O
+,	O
+often	O
+hampered	O
+by	O
+lack	O
+of	O
+identification	O
+of	O
+their	O
+ligands	O
+and	O
+/	O
+or	O
+DNA	O
+targets	O
+.	O
+
+A	O
+potentially	O
+interesting	O
+exception	O
+comes	O
+from	O
+the	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+salicylate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+forms	O
+of	O
+the	O
+Methanobacterium	B-species
+thermoautotrophicum	I-species
+protein	O
+MTH313	B-protein
+which	O
+revealed	O
+that	O
+two	O
+salicylate	B-chemical
+molecules	O
+bind	O
+to	O
+one	O
+MTH313	B-protein
+dimer	B-oligomeric_state
+and	O
+induce	O
+large	O
+conformational	O
+changes	O
+,	O
+apparently	O
+sufficient	O
+to	O
+prevent	O
+DNA	O
+binding	O
+.	O
+
+However	O
+,	O
+the	O
+homologous	O
+archeal	B-taxonomy_domain
+Sulfolobus	B-species
+tokodaii	I-species
+protein	O
+ST1710	B-protein
+presented	O
+essentially	O
+the	O
+same	O
+structure	B-evidence
+in	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+salicylate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+forms	O
+,	O
+apparently	O
+contrasting	O
+the	O
+mechanism	O
+proposed	O
+for	O
+MTH313	B-protein
+.	O
+
+Despite	O
+these	O
+apparent	O
+differences	O
+,	O
+MTH313	B-protein
+and	O
+ST1710	B-protein
+bind	O
+salicylate	B-chemical
+in	O
+approximately	O
+the	O
+same	O
+site	O
+,	O
+between	O
+their	O
+dimerization	B-structure_element
+and	I-structure_element
+DNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domains	I-structure_element
+.	O
+
+However	O
+,	O
+it	O
+is	O
+unknown	O
+whether	O
+salicylate	B-chemical
+is	O
+a	O
+relevant	O
+in	O
+vivo	O
+ligand	O
+of	O
+either	O
+of	O
+these	O
+two	O
+proteins	O
+,	O
+which	O
+share	O
+~	O
+20	O
+%	O
+sequence	O
+identity	O
+with	O
+NadR	B-protein
+,	O
+rendering	O
+unclear	O
+the	O
+interpretation	O
+of	O
+these	O
+findings	O
+in	O
+relation	O
+to	O
+the	O
+regulatory	O
+mechanisms	O
+of	O
+NadR	B-protein
+or	O
+other	O
+MarR	B-protein_type
+family	O
+proteins	O
+.	O
+
+NadR	B-protein
+binds	O
+nadA	B-gene
+on	O
+three	O
+different	O
+operators	O
+(	O
+OpI	O
+,	O
+OpII	O
+and	O
+OpIII	O
+).	O
+
+The	O
+DNA	O
+-	O
+binding	O
+activity	O
+of	O
+NadR	B-protein
+is	O
+attenuated	O
+in	O
+vitro	O
+upon	O
+addition	O
+of	O
+various	O
+hydroxyphenylacetate	B-chemical
+(	O
+HPA	B-chemical
+)	O
+derivatives	O
+,	O
+including	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+is	O
+a	O
+small	O
+molecule	O
+derived	O
+from	O
+mammalian	B-taxonomy_domain
+aromatic	O
+amino	O
+acid	O
+catabolism	O
+and	O
+is	O
+released	O
+in	O
+human	B-species
+saliva	O
+,	O
+where	O
+it	O
+has	O
+been	O
+detected	O
+at	O
+micromolar	O
+concentration	O
+.	O
+
+In	O
+the	O
+presence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+NadR	B-protein
+is	O
+unable	O
+to	O
+bind	O
+the	O
+nadA	B-gene
+promoter	O
+and	O
+nadA	B-gene
+gene	O
+expression	O
+is	O
+induced	O
+.	O
+
+In	O
+vivo	O
+,	O
+the	O
+presence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+the	O
+host	O
+niche	O
+of	O
+N	B-species
+.	I-species
+meningitidis	I-species
+serves	O
+as	O
+an	O
+inducer	O
+of	O
+NadA	B-protein
+production	O
+,	O
+thereby	O
+promoting	O
+bacterial	B-taxonomy_domain
+adhesion	O
+to	O
+host	O
+cells	O
+.	O
+
+Further	O
+,	O
+we	O
+recently	O
+reported	O
+that	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+produced	O
+during	O
+inflammation	O
+,	O
+is	O
+another	O
+inducer	O
+of	O
+nadA	B-gene
+expression	O
+.	O
+
+Extending	O
+our	O
+previous	O
+studies	O
+based	O
+on	O
+hydrogen	B-experimental_method
+-	I-experimental_method
+deuterium	I-experimental_method
+exchange	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+HDX	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+),	O
+here	O
+we	O
+sought	O
+to	O
+reveal	O
+the	O
+molecular	O
+mechanisms	O
+and	O
+effects	O
+of	O
+NadR	B-protein
+/	O
+HPA	B-chemical
+interactions	O
+via	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+,	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+and	O
+complementary	O
+biochemical	B-experimental_method
+and	I-experimental_method
+in	I-experimental_method
+vivo	I-experimental_method
+mutagenesis	I-experimental_method
+studies	I-experimental_method
+.	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	B-protein
+,	O
+and	O
+the	O
+regulation	O
+of	O
+nadA	B-gene
+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	B-protein_type
+super	O
+-	O
+family	O
+.	O
+
+Moreover	O
+,	O
+these	O
+findings	O
+are	O
+important	O
+because	O
+the	O
+activity	O
+of	O
+NadR	B-protein
+impacts	O
+the	O
+potential	O
+coverage	O
+provided	O
+by	O
+anti	O
+-	O
+NadA	B-protein
+antibodies	O
+elicited	O
+by	O
+the	O
+Bexsero	O
+vaccine	O
+and	O
+influences	O
+host	O
+-	O
+bacteria	B-taxonomy_domain
+interactions	O
+that	O
+contribute	O
+to	O
+meningococcal	B-taxonomy_domain
+pathogenesis	O
+.	O
+
+NadR	B-protein
+is	O
+dimeric	B-oligomeric_state
+and	O
+is	O
+stabilized	O
+by	O
+specific	O
+hydroxyphenylacetate	B-chemical
+ligands	O
+
+Recombinant	O
+NadR	B-protein
+was	O
+produced	O
+in	O
+E	B-species
+.	I-species
+coli	I-species
+using	O
+an	O
+expression	B-experimental_method
+construct	I-experimental_method
+prepared	O
+from	O
+N	B-species
+.	I-species
+meningitidis	I-species
+serogroup	I-species
+B	I-species
+strain	I-species
+MC58	I-species
+.	O
+
+Standard	O
+chromatographic	O
+techniques	O
+were	O
+used	O
+to	O
+obtain	O
+a	O
+highly	O
+purified	O
+sample	O
+of	O
+NadR	B-protein
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+
+In	O
+analytical	B-experimental_method
+size	I-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+high	I-experimental_method
+-	I-experimental_method
+performance	I-experimental_method
+liquid	I-experimental_method
+chromatography	I-experimental_method
+(	O
+SE	B-experimental_method
+-	I-experimental_method
+HPLC	I-experimental_method
+)	O
+experiments	O
+coupled	O
+with	O
+multi	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+laser	I-experimental_method
+light	I-experimental_method
+scattering	I-experimental_method
+(	O
+MALLS	B-experimental_method
+),	O
+NadR	B-protein
+presented	O
+a	O
+single	O
+species	O
+with	O
+an	O
+absolute	O
+molecular	O
+mass	O
+of	O
+35	O
+kDa	O
+(	O
+S1	O
+Fig	O
+).	O
+
+These	O
+data	O
+showed	O
+that	O
+NadR	B-protein
+was	O
+dimeric	B-oligomeric_state
+in	O
+solution	O
+,	O
+since	O
+the	O
+theoretical	O
+molecular	O
+mass	O
+of	O
+the	O
+NadR	B-protein
+dimer	B-oligomeric_state
+is	O
+33	O
+.	O
+73	O
+kDa	O
+;	O
+and	O
+,	O
+there	O
+was	O
+no	O
+change	O
+in	O
+oligomeric	O
+state	O
+on	O
+addition	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+The	O
+thermal	O
+stability	O
+of	O
+NadR	B-protein
+was	O
+examined	O
+using	O
+differential	B-experimental_method
+scanning	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+DSC	B-experimental_method
+).	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	B-chemical
+(	O
+Fig	O
+1A	O
+)	O
+on	O
+the	O
+melting	B-evidence
+temperature	I-evidence
+(	O
+Tm	B-evidence
+)	O
+of	O
+NadR	B-protein
+.	O
+As	O
+a	O
+control	O
+of	O
+specificity	O
+,	O
+we	O
+also	O
+tested	O
+salicylate	B-chemical
+,	O
+a	O
+known	O
+ligand	O
+of	O
+some	O
+MarR	B-protein_type
+proteins	O
+previously	O
+reported	O
+to	O
+increase	O
+the	O
+Tm	B-evidence
+of	O
+ST1710	B-protein
+and	O
+MTH313	B-protein
+.	O
+
+The	O
+Tm	B-evidence
+of	O
+NadR	B-protein
+was	O
+67	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+°	O
+C	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ligand	I-protein_state
+,	O
+and	O
+was	O
+unaffected	O
+by	O
+salicylate	B-chemical
+.	O
+
+However	O
+,	O
+an	O
+increased	O
+thermal	O
+stability	O
+was	O
+induced	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+and	O
+,	O
+to	O
+a	O
+lesser	O
+extent	O
+,	O
+by	O
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+Interestingly	O
+,	O
+NadR	B-protein
+displayed	O
+the	O
+greatest	O
+Tm	B-evidence
+increase	O
+upon	O
+addition	O
+of	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+1B	O
+).	O
+
+Stability	O
+of	O
+NadR	B-protein
+is	O
+increased	O
+by	O
+small	O
+molecule	O
+ligands	O
+.	O
+
+(	O
+A	O
+)	O
+Molecular	O
+structures	O
+of	O
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+MW	O
+152	O
+.	O
+2	O
+),	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+MW	O
+152	O
+.	O
+2	O
+),	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+MW	O
+186	O
+.	O
+6	O
+)	O
+and	O
+salicylic	B-chemical
+acid	I-chemical
+(	O
+MW	O
+160	O
+.	O
+1	O
+).	O
+(	O
+B	O
+)	O
+DSC	B-experimental_method
+profiles	B-evidence
+,	O
+colored	O
+as	O
+follows	O
+:	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+violet	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+salicylate	I-complex_assembly
+(	O
+red	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+3	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+green	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+blue	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+3Cl	I-complex_assembly
+,	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+pink	O
+).	O
+
+All	O
+DSC	B-experimental_method
+profiles	B-evidence
+are	O
+representative	O
+of	O
+triplicate	O
+experiments	O
+.	O
+
+Melting	B-evidence
+-	I-evidence
+point	I-evidence
+(	O
+Tm	B-evidence
+)	O
+and	O
+its	O
+ligand	O
+-	O
+induced	O
+increase	O
+(	O
+ΔTm	B-evidence
+)	O
+derived	O
+from	O
+DSC	B-experimental_method
+thermostability	B-experimental_method
+experiments	I-experimental_method
+.	O
+
+Dissociation	B-evidence
+constants	I-evidence
+(	O
+KD	B-evidence
+)	O
+of	O
+the	O
+NadR	B-protein
+/	O
+ligand	O
+interactions	O
+from	O
+SPR	B-experimental_method
+steady	I-experimental_method
+-	I-experimental_method
+state	I-experimental_method
+binding	I-experimental_method
+experiments	I-experimental_method
+.	O
+
+Ligand	O
+Tm	B-evidence
+(°	O
+C	O
+)	O
+ΔTm	B-evidence
+(°	O
+C	O
+)	O
+KD	B-evidence
+(	O
+mM	O
+)	O
+No	O
+ligand	O
+67	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+n	O
+.	O
+a	O
+.	O
+n	O
+.	O
+a	O
+.	O
+
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+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	B-chemical
+-	I-chemical
+HPA	I-chemical
+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	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+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
+
+NadR	B-protein
+displays	O
+distinct	O
+binding	B-evidence
+affinities	I-evidence
+for	O
+hydroxyphenylacetate	B-chemical
+ligands	O
+
+To	O
+further	O
+investigate	O
+the	O
+binding	O
+of	O
+HPAs	B-chemical
+to	O
+NadR	B-protein
+,	O
+we	O
+used	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+(	O
+SPR	B-experimental_method
+).	O
+
+The	O
+SPR	B-experimental_method
+sensorgrams	B-evidence
+revealed	O
+very	O
+fast	O
+association	O
+and	O
+dissociation	O
+events	O
+,	O
+typical	O
+of	O
+small	O
+molecule	O
+ligands	O
+,	O
+thus	O
+prohibiting	O
+a	O
+detailed	O
+study	O
+of	O
+binding	O
+kinetics	O
+.	O
+
+However	O
+,	O
+steady	B-experimental_method
+-	I-experimental_method
+state	I-experimental_method
+SPR	I-experimental_method
+analyses	O
+of	O
+the	O
+NadR	B-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+interactions	O
+allowed	O
+determination	O
+of	O
+the	O
+equilibrium	B-evidence
+dissociation	I-evidence
+constants	I-evidence
+(	O
+KD	B-evidence
+)	O
+(	O
+Table	O
+1	O
+and	O
+S2	O
+Fig	O
+).	O
+
+The	O
+interactions	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+and	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+with	O
+NadR	B-protein
+exhibited	O
+KD	B-evidence
+values	O
+of	O
+1	O
+.	O
+5	O
+mM	O
+and	O
+1	O
+.	O
+1	O
+mM	O
+,	O
+respectively	O
+.	O
+
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+showed	O
+a	O
+weaker	O
+interaction	O
+,	O
+with	O
+a	O
+KD	B-evidence
+of	O
+2	O
+.	O
+7	O
+mM	O
+,	O
+while	O
+salicylate	B-chemical
+showed	O
+only	O
+a	O
+very	O
+weak	O
+response	O
+that	O
+did	O
+not	O
+reach	O
+saturation	O
+,	O
+indicating	O
+a	O
+non	O
+-	O
+specific	O
+interaction	O
+with	O
+NadR	B-protein
+.	O
+A	O
+ranking	O
+of	O
+these	O
+KD	B-evidence
+values	O
+showed	O
+that	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+was	O
+the	O
+tightest	O
+binder	O
+,	O
+and	O
+thus	O
+matched	O
+the	O
+ranking	O
+of	O
+ligand	O
+-	O
+induced	O
+Tm	B-evidence
+increases	O
+observed	O
+in	O
+the	O
+DSC	B-experimental_method
+experiments	O
+.	O
+
+Although	O
+these	O
+KD	B-evidence
+values	O
+indicate	O
+rather	O
+weak	O
+interactions	O
+,	O
+they	O
+are	O
+similar	O
+to	O
+the	O
+values	O
+reported	O
+previously	O
+for	O
+the	O
+MarR	B-protein_type
+/	O
+salicylate	B-chemical
+interaction	O
+(	O
+KD	O
+~	O
+1	O
+mM	O
+)	O
+and	O
+the	O
+MTH313	B-protein
+/	O
+salicylate	B-chemical
+interaction	O
+(	O
+KD	O
+2	O
+–	O
+3	O
+mM	O
+),	O
+and	O
+approximately	O
+20	O
+-	O
+fold	O
+tighter	O
+than	O
+the	O
+ST1710	B-protein
+/	O
+salicylate	B-chemical
+interaction	O
+(	O
+KD	O
+~	O
+20	O
+mM	O
+).	O
+
+Crystal	B-evidence
+structures	I-evidence
+of	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+and	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+
+To	O
+fully	O
+characterize	O
+the	O
+NadR	B-protein
+/	O
+HPA	B-chemical
+interactions	O
+,	O
+we	O
+sought	O
+to	O
+determine	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+NadR	B-protein
+in	O
+ligand	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+(	O
+holo	B-protein_state
+)	O
+and	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+(	O
+apo	B-protein_state
+)	O
+forms	O
+.	O
+
+First	O
+,	O
+we	O
+crystallized	B-experimental_method
+NadR	B-protein
+(	O
+a	O
+selenomethionine	B-experimental_method
+-	I-experimental_method
+labelled	I-experimental_method
+derivative	I-experimental_method
+)	O
+in	O
+the	O
+presence	O
+of	O
+a	O
+200	O
+-	O
+fold	O
+molar	O
+excess	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+The	O
+structure	B-evidence
+of	O
+the	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+complex	O
+was	O
+determined	O
+at	O
+2	O
+.	O
+3	O
+Å	O
+resolution	O
+using	O
+a	O
+combination	O
+of	O
+the	O
+single	B-experimental_method
+-	I-experimental_method
+wavelength	I-experimental_method
+anomalous	I-experimental_method
+dispersion	I-experimental_method
+(	O
+SAD	B-experimental_method
+)	O
+and	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+(	O
+MR	B-experimental_method
+)	O
+methods	O
+,	O
+and	O
+was	O
+refined	O
+to	O
+R	B-evidence
+work	I-evidence
+/	I-evidence
+R	I-evidence
+free	I-evidence
+values	O
+of	O
+20	O
+.	O
+9	O
+/	O
+26	O
+.	O
+0	O
+%	O
+(	O
+Table	O
+2	O
+).	O
+
+Despite	O
+numerous	O
+attempts	O
+,	O
+we	O
+were	O
+unable	O
+to	O
+obtain	O
+high	O
+-	O
+quality	O
+crystals	B-evidence
+of	O
+NadR	B-protein
+complexed	B-protein_state
+with	I-protein_state
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+or	O
+DNA	O
+targets	O
+.	O
+
+However	O
+,	O
+it	O
+was	O
+eventually	O
+possible	O
+to	O
+crystallize	B-experimental_method
+apo	B-protein_state
+-	O
+NadR	B-protein
+,	O
+and	O
+the	O
+structure	B-evidence
+was	O
+determined	O
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+by	O
+MR	B-experimental_method
+methods	O
+using	O
+the	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+complex	O
+as	O
+the	O
+search	O
+model	O
+.	O
+
+The	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+structure	B-evidence
+was	O
+refined	O
+to	O
+R	B-evidence
+work	I-evidence
+/	I-evidence
+R	I-evidence
+free	I-evidence
+values	O
+of	O
+19	O
+.	O
+1	O
+/	O
+26	O
+.	O
+8	O
+%	O
+(	O
+Table	O
+2	O
+).	O
+
+Data	O
+collection	O
+and	O
+refinement	O
+statistics	O
+for	O
+NadR	B-protein
+structures	B-evidence
+.	O
+
+The	O
+asymmetric	O
+unit	O
+of	O
+the	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+crystals	B-evidence
+(	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+)	O
+contained	O
+one	O
+NadR	B-protein
+homodimer	B-oligomeric_state
+,	O
+while	O
+the	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+crystals	B-evidence
+contained	O
+two	O
+homodimers	B-oligomeric_state
+.	O
+
+In	O
+the	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+crystals	B-evidence
+,	O
+the	O
+two	O
+homodimers	B-oligomeric_state
+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	B-oligomeric_state
+was	O
+presumably	O
+merely	O
+an	O
+effect	O
+of	O
+crystal	O
+packing	O
+,	O
+since	O
+the	O
+interface	B-site
+between	O
+the	O
+two	O
+homodimers	B-oligomeric_state
+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
+
+Moreover	O
+,	O
+our	O
+SE	B-experimental_method
+-	I-experimental_method
+HPLC	I-experimental_method
+/	I-experimental_method
+MALLS	I-experimental_method
+analyses	O
+(	O
+see	O
+above	O
+)	O
+revealed	O
+that	O
+in	O
+solution	O
+NadR	B-protein
+is	O
+dimeric	B-oligomeric_state
+,	O
+and	O
+previous	O
+studies	O
+using	O
+native	B-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+MS	B-experimental_method
+)	O
+revealed	O
+dimers	B-oligomeric_state
+,	O
+not	O
+tetramers	B-oligomeric_state
+.	O
+
+The	O
+NadR	B-protein
+homodimer	B-oligomeric_state
+bound	B-protein_state
+to	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+has	O
+a	O
+dimerization	B-site
+interface	I-site
+mostly	O
+involving	O
+the	O
+top	O
+of	O
+its	O
+‘	O
+triangular	B-protein_state
+’	O
+form	O
+,	O
+while	O
+the	O
+two	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domains	I-structure_element
+are	O
+located	O
+at	O
+the	O
+base	O
+(	O
+Fig	O
+2A	O
+).	O
+
+High	O
+-	O
+quality	O
+electron	B-evidence
+density	I-evidence
+maps	I-evidence
+allowed	O
+clear	O
+identification	O
+of	O
+the	O
+bound	B-protein_state
+ligand	O
+,	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+Fig	O
+2B	O
+).	O
+
+The	O
+overall	O
+structure	B-evidence
+of	O
+NadR	B-protein
+shows	O
+dimensions	O
+of	O
+~	O
+50	O
+×	O
+65	O
+×	O
+50	O
+Å	O
+and	O
+a	O
+large	O
+homodimer	B-site
+interface	I-site
+that	O
+buries	O
+a	O
+total	O
+surface	O
+area	O
+of	O
+~	O
+4800	O
+Å2	O
+.	O
+
+Each	O
+NadR	B-protein
+monomer	B-oligomeric_state
+consists	O
+of	O
+six	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+two	O
+short	B-structure_element
+β	I-structure_element
+-	I-structure_element
+strands	I-structure_element
+,	O
+with	O
+helices	B-structure_element
+α1	B-structure_element
+,	O
+α5	B-structure_element
+,	O
+and	O
+α6	B-structure_element
+forming	O
+the	O
+dimer	B-site
+interface	I-site
+.	O
+
+Helices	B-structure_element
+α3	B-structure_element
+and	O
+α4	B-structure_element
+form	O
+a	O
+helix	B-structure_element
+-	I-structure_element
+turn	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+motif	I-structure_element
+,	O
+followed	O
+by	O
+the	O
+“	O
+wing	B-structure_element
+motif	I-structure_element
+”	O
+comprised	O
+of	O
+two	O
+short	B-structure_element
+antiparallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+strands	I-structure_element
+(	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+)	O
+linked	O
+by	O
+a	O
+relatively	O
+long	O
+and	O
+flexible	O
+loop	B-structure_element
+.	O
+
+Interestingly	O
+,	O
+in	O
+the	O
+α4	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+region	I-structure_element
+,	O
+the	O
+stretch	O
+of	O
+residues	O
+from	O
+R64	B-residue_range
+-	I-residue_range
+R91	I-residue_range
+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	B-chemical
+.	O
+
+Together	O
+,	O
+these	O
+structural	O
+elements	O
+constitute	O
+the	O
+winged	B-structure_element
+helix	I-structure_element
+-	I-structure_element
+turn	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+(	O
+wHTH	B-structure_element
+)	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+and	O
+,	O
+together	O
+with	O
+the	O
+dimeric	B-oligomeric_state
+organization	O
+,	O
+are	O
+the	O
+hallmarks	O
+of	O
+MarR	B-protein_type
+family	O
+structures	B-evidence
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+NadR	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+(	O
+A	O
+)	O
+The	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+homodimer	B-oligomeric_state
+is	O
+depicted	O
+in	O
+green	O
+and	O
+blue	O
+for	O
+chains	B-structure_element
+A	I-structure_element
+and	I-structure_element
+B	I-structure_element
+respectively	O
+,	O
+while	O
+yellow	O
+sticks	O
+depict	O
+the	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+ligand	O
+(	O
+labelled	O
+).	O
+
+For	O
+simplicity	O
+,	O
+secondary	O
+structure	O
+elements	O
+are	O
+labelled	O
+for	O
+chain	B-structure_element
+B	I-structure_element
+only	O
+.	O
+
+Red	O
+dashes	O
+show	O
+hypothetical	O
+positions	O
+of	O
+chain	B-structure_element
+B	I-structure_element
+residues	O
+88	B-residue_range
+–	I-residue_range
+90	I-residue_range
+that	O
+were	O
+not	O
+modeled	O
+due	O
+to	O
+lack	O
+of	O
+electron	B-evidence
+density	I-evidence
+.	O
+
+(	O
+B	O
+)	O
+A	O
+zoom	O
+into	O
+the	O
+pocket	B-site
+occupied	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+shows	O
+that	O
+the	O
+ligand	O
+contacts	O
+both	O
+chains	B-structure_element
+A	I-structure_element
+and	I-structure_element
+B	I-structure_element
+;	O
+blue	O
+mesh	O
+shows	O
+electron	B-evidence
+density	I-evidence
+around	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+calculated	O
+from	O
+a	O
+composite	B-evidence
+omit	I-evidence
+map	I-evidence
+(	O
+omitting	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+),	O
+using	O
+phenix	B-experimental_method
+.	O
+
+The	O
+map	B-evidence
+is	O
+contoured	O
+at	O
+1σ	O
+and	O
+the	O
+figure	O
+was	O
+prepared	O
+with	O
+a	O
+density	B-evidence
+mesh	I-evidence
+carve	O
+factor	O
+of	O
+1	O
+.	O
+7	O
+,	O
+using	O
+Pymol	O
+(	O
+www	O
+.	O
+pymol	O
+.	O
+org	O
+).	O
+
+A	O
+single	O
+conserved	B-protein_state
+leucine	B-residue_name
+residue	O
+(	O
+L130	B-residue_name_number
+)	O
+is	O
+crucial	O
+for	O
+dimerization	O
+
+The	O
+NadR	B-protein
+dimer	B-site
+interface	I-site
+is	O
+formed	O
+by	O
+at	O
+least	O
+32	O
+residues	O
+,	O
+which	O
+establish	O
+numerous	O
+inter	O
+-	O
+chain	O
+salt	O
+bridges	O
+or	O
+hydrogen	O
+bonds	O
+,	O
+and	O
+many	O
+hydrophobic	O
+packing	O
+interactions	O
+(	O
+Fig	O
+3A	O
+and	O
+3B	O
+).	O
+
+To	O
+determine	O
+which	O
+residues	O
+were	O
+most	O
+important	O
+for	O
+dimerization	O
+,	O
+we	O
+studied	O
+the	O
+interface	B-site
+in	O
+silico	O
+and	O
+identified	O
+several	O
+residues	O
+as	O
+potential	O
+mediators	O
+of	O
+key	O
+stabilizing	O
+interactions	O
+.	O
+
+Using	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+,	O
+a	O
+panel	O
+of	O
+eight	O
+mutant	B-protein_state
+NadR	B-protein
+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	B-site
+interface	I-site
+.	O
+
+Each	O
+mutant	B-protein_state
+NadR	B-protein
+protein	O
+was	O
+purified	O
+,	O
+and	O
+then	O
+its	O
+oligomeric	O
+state	O
+was	O
+examined	O
+by	O
+analytical	B-experimental_method
+SE	I-experimental_method
+-	I-experimental_method
+HPLC	I-experimental_method
+.	O
+
+Almost	O
+all	O
+the	O
+mutants	O
+showed	O
+the	O
+same	O
+elution	O
+profile	O
+as	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+NadR	B-protein
+protein	O
+.	O
+
+Only	O
+the	O
+L130K	B-mutant
+mutation	O
+induced	O
+a	O
+notable	O
+change	O
+in	O
+the	O
+oligomeric	O
+state	O
+of	O
+NadR	B-protein
+(	O
+Fig	O
+3C	O
+).	O
+
+Further	O
+,	O
+in	O
+SE	B-experimental_method
+-	I-experimental_method
+MALLS	I-experimental_method
+analyses	O
+,	O
+the	O
+L130K	B-mutant
+mutant	B-protein_state
+displayed	O
+two	O
+distinct	O
+species	O
+in	O
+solution	O
+,	O
+approximately	O
+80	O
+%	O
+being	O
+monomeric	B-oligomeric_state
+(	O
+a	O
+19	O
+kDa	O
+species	O
+),	O
+and	O
+only	O
+20	O
+%	O
+retaining	O
+the	O
+typical	O
+native	O
+dimeric	B-oligomeric_state
+state	O
+(	O
+a	O
+35	O
+kDa	O
+species	O
+)	O
+(	O
+Fig	O
+3D	O
+),	O
+demonstrating	O
+that	O
+Leu130	B-residue_name_number
+is	O
+crucial	O
+for	O
+stable	O
+dimerization	O
+.	O
+
+It	O
+is	O
+notable	O
+that	O
+L130	B-residue_name_number
+is	O
+usually	O
+present	O
+as	O
+Leu	B-residue_name
+,	O
+or	O
+an	O
+alternative	O
+bulky	O
+hydrophobic	O
+amino	O
+acid	O
+(	O
+e	O
+.	O
+g	O
+.	O
+Phe	B-residue_name
+,	O
+Val	B-residue_name
+),	O
+in	O
+many	O
+MarR	B-protein_type
+family	O
+proteins	O
+,	O
+suggesting	O
+a	O
+conserved	B-protein_state
+role	O
+in	O
+stabilizing	O
+the	O
+dimer	B-site
+interface	I-site
+.	O
+
+In	O
+contrast	O
+,	O
+most	O
+of	O
+the	O
+other	O
+residues	O
+identified	O
+in	O
+the	O
+NadR	B-protein
+dimer	B-site
+interface	I-site
+were	O
+poorly	B-protein_state
+conserved	I-protein_state
+in	O
+the	O
+MarR	B-protein_type
+family	O
+.	O
+
+Analysis	O
+of	O
+the	O
+NadR	B-protein
+dimer	B-site
+interface	I-site
+.	O
+
+(	O
+A	O
+)	O
+Both	O
+orientations	O
+show	O
+chain	B-structure_element
+A	I-structure_element
+,	O
+green	O
+backbone	O
+ribbon	O
+,	O
+colored	O
+red	O
+to	O
+highlight	O
+all	O
+locations	O
+involved	O
+in	O
+dimerization	O
+;	O
+namely	O
+,	O
+inter	O
+-	O
+chain	O
+salt	O
+bridges	O
+or	O
+hydrogen	O
+bonds	O
+involving	O
+Q4	B-residue_name_number
+,	O
+S5	B-residue_name_number
+,	O
+K6	B-residue_name_number
+,	O
+H7	B-residue_name_number
+,	O
+S9	B-residue_name_number
+,	O
+I10	B-residue_name_number
+,	O
+N11	B-residue_name_number
+,	O
+I15	B-residue_name_number
+,	O
+Q16	B-residue_name_number
+,	O
+R18	B-residue_name_number
+,	O
+D36	B-residue_name_number
+,	O
+R43	B-residue_name_number
+,	O
+A46	B-residue_name_number
+,	O
+Q59	B-residue_name_number
+,	O
+C61	B-residue_name_number
+,	O
+Y104	B-residue_name_number
+,	O
+D112	B-residue_name_number
+,	O
+R114	B-residue_name_number
+,	O
+Y115	B-residue_name_number
+,	O
+D116	B-residue_name_number
+,	O
+E119	B-residue_name_number
+,	O
+K126	B-residue_name_number
+,	O
+E136	B-residue_name_number
+,	O
+E141	B-residue_name_number
+,	O
+N145	B-residue_name_number
+,	O
+and	O
+the	O
+hydrophobic	O
+packing	O
+interactions	O
+involving	O
+I10	B-residue_name_number
+,	O
+I12	B-residue_name_number
+,	O
+L14	B-residue_name_number
+,	O
+I15	B-residue_name_number
+,	O
+R18	B-residue_name_number
+,	O
+Y115	B-residue_name_number
+,	O
+I118	B-residue_name_number
+,	O
+L130	B-residue_name_number
+,	O
+L133	B-residue_name_number
+,	O
+L134	B-residue_name_number
+and	O
+L137	B-residue_name_number
+.	O
+
+Chain	B-structure_element
+B	I-structure_element
+,	O
+grey	O
+surface	O
+,	O
+is	O
+marked	O
+blue	O
+to	O
+highlight	O
+residues	O
+probed	O
+by	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+(	O
+E136	B-residue_name_number
+only	O
+makes	O
+a	O
+salt	O
+bridge	O
+with	O
+K126	B-residue_name_number
+,	O
+therefore	O
+it	O
+was	O
+sufficient	O
+to	O
+make	O
+the	O
+K126A	B-mutant
+mutation	O
+to	O
+assess	O
+the	O
+importance	O
+of	O
+this	O
+ionic	O
+interaction	O
+;	O
+the	O
+H7	B-residue_name_number
+position	O
+is	O
+labelled	O
+for	O
+monomer	B-oligomeric_state
+A	B-structure_element
+,	O
+since	O
+electron	B-evidence
+density	I-evidence
+was	O
+lacking	O
+for	O
+monomer	B-oligomeric_state
+B	B-structure_element
+).	O
+(	O
+B	O
+)	O
+A	O
+zoom	O
+into	O
+the	O
+environment	O
+of	O
+helix	B-structure_element
+α6	B-structure_element
+to	O
+show	O
+how	O
+residue	O
+L130	B-residue_name_number
+chain	B-structure_element
+B	I-structure_element
+(	O
+blue	O
+side	O
+chain	O
+)	O
+is	O
+a	O
+focus	O
+of	O
+hydrophobic	O
+packing	O
+interactions	O
+with	O
+L130	B-residue_name_number
+,	O
+L133	B-residue_name_number
+,	O
+L134	B-residue_name_number
+and	O
+L137	B-residue_name_number
+of	O
+chain	B-structure_element
+A	I-structure_element
+(	O
+red	O
+side	O
+chains	O
+).	O
+
+(	O
+C	O
+)	O
+SE	B-experimental_method
+-	I-experimental_method
+HPLC	I-experimental_method
+analyses	O
+of	O
+all	O
+mutant	B-protein_state
+forms	O
+of	O
+NadR	B-protein
+are	O
+compared	O
+with	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+protein	O
+.	O
+
+The	O
+WT	B-protein_state
+and	O
+most	O
+of	O
+the	O
+mutants	O
+show	O
+a	O
+single	O
+elution	O
+peak	O
+with	O
+an	O
+absorbance	O
+maximum	O
+at	O
+17	O
+.	O
+5	O
+min	O
+.	O
+
+Only	O
+the	O
+mutation	O
+L130K	B-mutant
+has	O
+a	O
+noteworthy	O
+effect	O
+on	O
+the	O
+oligomeric	O
+state	O
+,	O
+inducing	O
+a	O
+second	O
+peak	O
+with	O
+a	O
+longer	O
+retention	O
+time	O
+and	O
+a	O
+second	O
+peak	O
+maximum	O
+at	O
+18	O
+.	O
+6	O
+min	O
+.	O
+
+To	O
+a	O
+much	O
+lesser	O
+extent	O
+,	O
+the	O
+L133K	B-mutant
+mutation	O
+also	O
+appears	O
+to	O
+induce	O
+a	O
+‘	O
+shoulder	O
+’	O
+to	O
+the	O
+main	O
+peak	O
+,	O
+suggesting	O
+very	O
+weak	O
+ability	O
+to	O
+disrupt	O
+the	O
+dimer	B-oligomeric_state
+.	O
+(	O
+D	O
+)	O
+SE	B-experimental_method
+-	I-experimental_method
+HPLC	I-experimental_method
+/	I-experimental_method
+MALLS	I-experimental_method
+analyses	O
+of	O
+the	O
+L130K	B-mutant
+mutant	B-protein_state
+,	O
+shows	O
+20	O
+%	O
+dimer	B-oligomeric_state
+and	O
+80	O
+%	O
+monomer	B-oligomeric_state
+.	O
+
+The	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+structure	B-evidence
+presents	O
+only	O
+one	O
+occupied	O
+ligand	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+
+The	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+structure	B-evidence
+revealed	O
+the	O
+ligand	B-site
+-	I-site
+binding	I-site
+site	I-site
+nestled	O
+between	O
+the	O
+dimerization	B-structure_element
+and	I-structure_element
+DNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domains	I-structure_element
+(	O
+Fig	O
+2	O
+).	O
+
+The	O
+ligand	O
+showed	O
+a	O
+different	O
+position	O
+and	O
+orientation	O
+compared	O
+to	O
+salicylate	B-chemical
+complexed	B-protein_state
+with	I-protein_state
+MTH313	B-protein
+and	O
+ST1710	B-protein
+(	O
+see	O
+Discussion	O
+).	O
+
+The	O
+binding	B-site
+pocket	I-site
+was	O
+almost	O
+entirely	O
+filled	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+and	O
+one	O
+water	B-chemical
+molecule	O
+,	O
+although	O
+there	O
+also	O
+remained	O
+a	O
+small	O
+tunnel	B-site
+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	B-site
+(	O
+proximal	O
+to	O
+the	O
+4	O
+-	O
+hydroxyl	O
+position	O
+)	O
+to	O
+the	O
+protein	O
+surface	O
+.	O
+
+The	O
+tunnel	B-site
+was	O
+lined	O
+with	O
+rather	O
+hydrophobic	O
+amino	O
+acids	O
+,	O
+and	O
+did	O
+not	O
+contain	O
+water	B-chemical
+molecules	O
+.	O
+
+Unexpectedly	O
+,	O
+only	O
+one	O
+monomer	B-oligomeric_state
+of	O
+the	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+homodimer	B-oligomeric_state
+contained	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+the	O
+binding	B-site
+pocket	I-site
+,	O
+whereas	O
+the	O
+corresponding	O
+pocket	B-site
+of	O
+the	O
+other	O
+monomer	B-oligomeric_state
+was	O
+unoccupied	O
+by	O
+ligand	O
+,	O
+despite	O
+the	O
+large	O
+excess	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+used	O
+in	O
+the	O
+crystallization	O
+conditions	O
+.	O
+
+Inspection	O
+of	O
+the	O
+protein	B-site
+-	I-site
+ligand	I-site
+interaction	I-site
+network	I-site
+revealed	O
+no	O
+bonds	O
+from	O
+NadR	B-protein
+backbone	O
+groups	O
+to	O
+the	O
+ligand	O
+,	O
+but	O
+several	O
+key	O
+side	O
+chain	O
+mediated	O
+hydrogen	O
+(	O
+H	O
+)-	O
+bonds	O
+and	O
+ionic	O
+interactions	O
+,	O
+most	O
+notably	O
+between	O
+the	O
+carboxylate	O
+group	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+and	O
+Ser9	B-residue_name_number
+of	O
+chain	B-structure_element
+A	I-structure_element
+(	O
+SerA9	B-residue_name_number
+),	O
+and	O
+chain	B-structure_element
+B	I-structure_element
+residues	O
+TrpB39	B-residue_name_number
+,	O
+ArgB43	B-residue_name_number
+and	O
+TyrB115	B-residue_name_number
+(	O
+Fig	O
+4A	O
+).	O
+
+At	O
+the	O
+other	O
+‘	O
+end	O
+’	O
+of	O
+the	O
+ligand	O
+,	O
+the	O
+4	O
+-	O
+hydroxyl	O
+group	O
+was	O
+proximal	O
+to	O
+AspB36	B-residue_name_number
+,	O
+with	O
+which	O
+it	O
+may	O
+establish	O
+an	O
+H	O
+-	O
+bond	O
+(	O
+see	O
+bond	O
+distances	O
+in	O
+Table	O
+3	O
+).	O
+
+The	O
+water	B-chemical
+molecule	O
+observed	O
+in	O
+the	O
+pocket	O
+was	O
+bound	O
+by	O
+the	O
+carboxylate	O
+group	O
+and	O
+the	O
+side	O
+chains	O
+of	O
+SerA9	B-residue_name_number
+and	O
+AsnA11	B-residue_name_number
+.	O
+
+Atomic	O
+details	O
+of	O
+NadR	B-protein
+/	O
+HPA	B-chemical
+interactions	O
+.	O
+
+A	O
+)	O
+A	O
+stereo	O
+-	O
+view	O
+zoom	O
+into	O
+the	O
+binding	B-site
+pocket	I-site
+showing	O
+side	O
+chain	O
+sticks	O
+for	O
+all	O
+interactions	O
+between	O
+NadR	B-protein
+and	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+Green	O
+and	O
+blue	O
+ribbons	O
+depict	O
+NadR	B-protein
+chains	B-structure_element
+A	I-structure_element
+and	I-structure_element
+B	I-structure_element
+,	O
+respectively	O
+.	O
+
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+is	O
+shown	O
+in	O
+yellow	O
+sticks	O
+,	O
+with	O
+oxygen	O
+atoms	O
+in	O
+red	O
+.	O
+
+A	O
+water	B-chemical
+molecule	O
+is	O
+shown	O
+by	O
+the	O
+red	O
+sphere	O
+.	O
+
+The	O
+entire	O
+set	O
+of	O
+residues	O
+making	O
+H	O
+-	O
+bonds	O
+or	O
+non	O
+-	O
+bonded	O
+contacts	O
+with	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+is	O
+as	O
+follows	O
+:	O
+SerA9	B-residue_name_number
+,	O
+AsnA11	B-residue_name_number
+,	O
+LeuB21	B-residue_name_number
+,	O
+MetB22	B-residue_name_number
+,	O
+PheB25	B-residue_name_number
+,	O
+LeuB29	B-residue_name_number
+,	O
+AspB36	B-residue_name_number
+,	O
+TrpB39	B-residue_name_number
+,	O
+ArgB43	B-residue_name_number
+,	O
+ValB111	B-residue_name_number
+and	O
+TyrB115	B-residue_name_number
+(	O
+automated	O
+analysis	O
+performed	O
+using	O
+PDBsum	B-experimental_method
+and	O
+verified	O
+manually	O
+).	O
+
+Residues	O
+AsnA11	B-residue_name_number
+and	O
+ArgB18	B-residue_name_number
+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	B-residue_name_number
+.	O
+
+Side	O
+chains	O
+mediating	O
+hydrophobic	O
+interactions	O
+are	O
+shown	O
+in	O
+orange	O
+.	O
+(	O
+B	O
+)	O
+A	O
+model	O
+was	O
+prepared	O
+to	O
+visualize	O
+putative	O
+interactions	O
+of	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+pink	O
+)	O
+with	O
+NadR	B-protein
+,	O
+revealing	O
+the	O
+potential	O
+for	O
+additional	O
+contacts	O
+(	O
+dashed	O
+lines	O
+)	O
+of	O
+the	O
+chloro	O
+moiety	O
+(	O
+green	O
+stick	O
+)	O
+with	O
+LeuB29	B-residue_name_number
+and	O
+AspB36	B-residue_name_number
+.	O
+
+List	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+atoms	O
+bound	O
+to	O
+NadR	B-protein
+via	O
+ionic	O
+interactions	O
+and	O
+/	O
+or	O
+H	O
+-	O
+bonds	O
+.	O
+
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+atom	O
+NadR	B-protein
+residue	O
+/	O
+atom	O
+Distance	O
+(	O
+Å	O
+)	O
+O2	O
+TrpB39	B-residue_name_number
+/	O
+NE1	O
+2	O
+.	O
+83	O
+O2	O
+ArgB43	B-residue_name_number
+/	O
+NH1	O
+2	O
+.	O
+76	O
+O1	O
+ArgB43	B-residue_name_number
+/	O
+NH1	O
+3	O
+.	O
+84	O
+O1	O
+SerA9	B-residue_name_number
+/	O
+OG	O
+2	O
+.	O
+75	O
+O1	O
+TyrB115	B-residue_name_number
+/	O
+OH	O
+2	O
+.	O
+50	O
+O2	O
+Water	B-chemical
+(*	O
+Ser9	B-residue_name_number
+/	O
+Asn11	B-residue_name_number
+)	O
+2	O
+.	O
+88	O
+OH	O
+AspB36	B-residue_name_number
+/	O
+OD1	O
+/	O
+OD2	O
+3	O
+.	O
+6	O
+/	O
+3	O
+.	O
+7	O
+
+*	O
+Bond	O
+distance	O
+between	O
+the	O
+ligand	O
+carboxylate	O
+group	O
+and	O
+the	O
+water	B-chemical
+molecule	O
+,	O
+which	O
+in	O
+turn	O
+makes	O
+H	O
+-	O
+bond	O
+to	O
+the	O
+SerA9	B-residue_name_number
+and	O
+AsnA11	B-residue_name_number
+side	O
+chains	O
+.	O
+
+In	O
+addition	O
+to	O
+the	O
+H	O
+-	O
+bonds	O
+involving	O
+the	O
+carboxylate	O
+and	O
+hydroxyl	O
+groups	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+binding	O
+of	O
+the	O
+phenyl	O
+moiety	O
+appeared	O
+to	O
+be	O
+stabilized	O
+by	O
+several	O
+van	O
+der	O
+Waals	O
+’	O
+contacts	O
+,	O
+particularly	O
+those	O
+involving	O
+the	O
+hydrophobic	O
+side	O
+chain	O
+atoms	O
+of	O
+LeuB21	B-residue_name_number
+,	O
+MetB22	B-residue_name_number
+,	O
+PheB25	B-residue_name_number
+,	O
+LeuB29	B-residue_name_number
+and	O
+ValB111	B-residue_name_number
+(	O
+Fig	O
+4A	O
+).	O
+
+Notably	O
+,	O
+the	O
+phenyl	O
+ring	O
+of	O
+PheB25	B-residue_name_number
+was	O
+positioned	O
+parallel	O
+to	O
+the	O
+phenyl	O
+ring	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+potentially	O
+forming	O
+π	O
+-	O
+π	O
+parallel	O
+-	O
+displaced	O
+stacking	O
+interactions	O
+.	O
+
+Consequently	O
+,	O
+residues	O
+in	O
+the	O
+4	B-site
+-	I-site
+HPA	I-site
+binding	I-site
+pocket	I-site
+are	O
+mostly	O
+contributed	O
+by	O
+NadR	B-protein
+chain	B-structure_element
+B	I-structure_element
+,	O
+and	O
+effectively	O
+created	O
+a	O
+polar	O
+‘	O
+floor	O
+’	O
+and	O
+a	O
+hydrophobic	O
+‘	O
+ceiling	O
+’,	O
+which	O
+house	O
+the	O
+ligand	O
+.	O
+
+Collectively	O
+,	O
+this	O
+mixed	O
+network	O
+of	O
+polar	O
+and	O
+hydrophobic	O
+interactions	O
+endows	O
+NadR	B-protein
+with	O
+a	O
+strong	O
+recognition	O
+pattern	O
+for	O
+HPAs	B-chemical
+,	O
+with	O
+additional	O
+medium	O
+-	O
+range	O
+interactions	O
+potentially	O
+established	O
+with	O
+the	O
+hydroxyl	O
+group	O
+at	O
+the	O
+4	O
+-	O
+position	O
+.	O
+
+Structure	O
+-	O
+activity	O
+relationships	O
+:	O
+molecular	O
+basis	O
+of	O
+enhanced	O
+stabilization	O
+by	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+
+We	O
+modelled	B-experimental_method
+the	O
+binding	O
+of	O
+other	O
+HPAs	B-chemical
+by	O
+in	B-experimental_method
+silico	I-experimental_method
+superposition	I-experimental_method
+onto	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+the	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+structure	B-evidence
+,	O
+and	O
+thereby	O
+obtained	O
+molecular	O
+explanations	O
+for	O
+the	O
+binding	O
+specificities	O
+of	O
+diverse	O
+ligands	O
+.	O
+
+For	O
+example	O
+,	O
+similar	O
+to	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+the	O
+binding	O
+of	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+could	O
+involve	O
+multiple	O
+bonds	O
+towards	O
+the	O
+carboxylate	O
+group	O
+of	O
+the	O
+ligand	O
+and	O
+some	O
+to	O
+the	O
+4	O
+-	O
+hydroxyl	O
+group	O
+.	O
+
+Additionally	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+LeuB29	B-residue_name_number
+and	O
+AspB36	B-residue_name_number
+would	O
+be	O
+only	O
+2	O
+.	O
+6	O
+–	O
+3	O
+.	O
+5	O
+Å	O
+from	O
+the	O
+chlorine	O
+atom	O
+,	O
+thus	O
+providing	O
+van	O
+der	O
+Waals	O
+’	O
+interactions	O
+or	O
+H	O
+-	O
+bonds	O
+to	O
+generate	O
+the	O
+additional	O
+binding	B-evidence
+affinity	I-evidence
+observed	O
+for	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+Fig	O
+4B	O
+).	O
+
+The	O
+presence	O
+of	O
+a	O
+single	O
+hydroxyl	O
+group	O
+at	O
+position	O
+2	O
+,	O
+as	O
+in	O
+2	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+rather	O
+than	O
+at	O
+position	O
+4	O
+,	O
+would	O
+eliminate	O
+the	O
+possibility	O
+of	O
+favorable	O
+interactions	O
+with	O
+AspB36	B-residue_name_number
+,	O
+resulting	O
+in	O
+the	O
+lack	O
+of	O
+NadR	B-protein
+regulation	O
+by	O
+2	B-chemical
+-	I-chemical
+HPA	I-chemical
+described	O
+previously	O
+.	O
+
+Finally	O
+,	O
+salicylate	B-chemical
+is	O
+presumably	O
+unable	O
+to	O
+specifically	O
+bind	O
+NadR	B-protein
+due	O
+to	O
+the	O
+2	O
+-	O
+hydroxyl	O
+substitution	O
+and	O
+the	O
+shorter	O
+aliphatic	O
+chain	O
+connecting	O
+its	O
+carboxylate	O
+group	O
+(	O
+Fig	O
+1A	O
+):	O
+the	O
+compound	O
+simply	O
+seems	O
+too	O
+small	O
+to	O
+simultaneously	O
+establish	O
+the	O
+network	O
+of	O
+beneficial	O
+bonds	O
+observed	O
+in	O
+the	O
+NadR	B-protein
+/	O
+HPA	B-chemical
+interactions	O
+.	O
+
+Analysis	O
+of	O
+the	O
+pockets	B-site
+reveals	O
+the	O
+molecular	O
+basis	O
+for	O
+asymmetric	O
+binding	O
+and	O
+stoichiometry	O
+
+However	O
+,	O
+studies	O
+based	O
+on	O
+tryptophan	B-experimental_method
+fluorescence	I-experimental_method
+were	O
+confounded	O
+by	O
+the	O
+fluorescence	O
+of	O
+the	O
+HPA	B-chemical
+ligands	O
+,	O
+and	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+ITC	B-experimental_method
+)	O
+was	O
+unfeasible	O
+due	O
+to	O
+the	O
+need	O
+for	O
+very	O
+high	O
+concentrations	O
+of	O
+NadR	B-protein
+in	O
+the	O
+ITC	B-experimental_method
+chamber	O
+(	O
+due	O
+to	O
+the	O
+relatively	O
+low	O
+affinity	O
+),	O
+which	O
+exceeded	O
+the	O
+solubility	O
+limits	O
+of	O
+the	O
+protein	O
+.	O
+
+However	O
+,	O
+it	O
+was	O
+possible	O
+to	O
+calculate	O
+the	O
+binding	B-evidence
+stoichiometry	I-evidence
+of	O
+the	O
+NadR	B-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+interactions	O
+using	O
+an	O
+SPR	B-experimental_method
+-	O
+based	O
+approach	O
+.	O
+
+In	O
+SPR	B-experimental_method
+,	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
+
+This	O
+approach	O
+relies	O
+on	O
+the	O
+assumption	O
+that	O
+the	O
+captured	O
+protein	O
+(‘	O
+the	O
+ligand	O
+’,	O
+according	O
+to	O
+SPR	B-experimental_method
+conventions	O
+)	O
+is	O
+100	O
+%	O
+active	O
+and	O
+freely	O
+-	O
+accessible	O
+to	O
+potential	O
+interactors	O
+(‘	O
+the	O
+analytes	O
+’).	O
+
+Firstly	O
+,	O
+NadR	B-protein
+is	O
+expected	O
+to	O
+be	O
+covalently	O
+immobilized	O
+on	O
+the	O
+sensor	O
+chip	O
+as	O
+a	O
+dimer	B-oligomeric_state
+in	O
+random	O
+orientations	O
+,	O
+since	O
+it	O
+is	O
+a	O
+stable	B-protein_state
+dimer	B-oligomeric_state
+in	O
+solution	O
+and	O
+has	O
+sixteen	O
+lysines	B-residue_name
+well	O
+-	O
+distributed	O
+around	O
+its	O
+surface	O
+,	O
+all	O
+able	O
+to	O
+act	O
+as	O
+potential	O
+sites	O
+for	O
+amine	O
+coupling	O
+to	O
+the	O
+chip	O
+,	O
+and	O
+none	O
+of	O
+which	O
+are	O
+close	O
+to	O
+the	O
+ligand	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+Secondly	O
+,	O
+the	O
+HPA	B-chemical
+analytes	O
+are	O
+all	O
+very	O
+small	O
+(	O
+MW	O
+150	O
+–	O
+170	O
+,	O
+Fig	O
+1A	O
+)	O
+and	O
+therefore	O
+are	O
+expected	O
+to	O
+be	O
+able	O
+to	O
+diffuse	O
+readily	O
+into	O
+all	O
+potential	O
+binding	B-site
+sites	I-site
+,	O
+irrespective	O
+of	O
+the	O
+random	O
+orientations	O
+of	O
+the	O
+immobilized	O
+NadR	B-protein
+dimers	B-oligomeric_state
+on	O
+the	O
+chip	O
+.	O
+
+The	O
+stoichiometry	O
+of	O
+the	O
+NadR	B-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+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	B-evidence
+of	O
+1	O
+.	O
+13	O
+for	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+1	O
+.	O
+02	O
+for	O
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+and	O
+1	O
+.	O
+21	O
+for	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+strongly	O
+suggesting	O
+that	O
+one	O
+NadR	B-protein
+dimer	B-oligomeric_state
+bound	B-protein_state
+to	I-protein_state
+1	O
+HPA	B-chemical
+analyte	O
+molecule	O
+.	O
+
+The	O
+crystallographic	B-evidence
+data	I-evidence
+,	O
+supported	O
+by	O
+the	O
+SPR	B-experimental_method
+studies	O
+of	O
+binding	B-evidence
+stoichiometry	I-evidence
+,	O
+revealed	O
+the	O
+lack	O
+of	O
+a	O
+second	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+molecule	O
+in	O
+the	O
+homodimer	B-oligomeric_state
+,	O
+suggesting	O
+negative	O
+co	O
+-	O
+operativity	O
+,	O
+a	O
+phenomenon	O
+previously	O
+described	O
+for	O
+the	O
+MTH313	B-protein
+/	O
+salicylate	B-chemical
+interaction	O
+and	O
+for	O
+other	O
+MarR	B-protein_type
+family	O
+proteins	O
+.	O
+
+To	O
+explore	O
+the	O
+molecular	O
+basis	O
+of	O
+asymmetry	O
+in	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+,	O
+we	O
+superposed	B-experimental_method
+its	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+monomer	B-oligomeric_state
+(	O
+chain	B-structure_element
+A	I-structure_element
+)	O
+onto	O
+the	O
+ligand	B-protein_state
+-	I-protein_state
+occupied	I-protein_state
+monomer	B-oligomeric_state
+(	O
+chain	B-structure_element
+B	I-structure_element
+).	O
+
+Overall	O
+,	O
+the	O
+superposition	B-experimental_method
+revealed	O
+a	O
+high	O
+degree	O
+of	O
+structural	O
+similarity	O
+(	O
+Cα	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+(	O
+rmsd	B-evidence
+)	O
+of	O
+1	O
+.	O
+5Å	O
+),	O
+though	O
+on	O
+closer	O
+inspection	O
+a	O
+rotational	O
+difference	O
+of	O
+~	O
+9	O
+degrees	O
+along	O
+the	O
+long	O
+axis	O
+of	O
+helix	B-structure_element
+α6	B-structure_element
+was	O
+observed	O
+,	O
+suggesting	O
+that	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+induced	O
+a	O
+slight	O
+conformational	O
+change	O
+(	O
+Fig	O
+5A	O
+).	O
+
+However	O
+,	O
+since	O
+residues	O
+of	O
+helix	B-structure_element
+α6	B-structure_element
+were	O
+not	O
+directly	O
+involved	O
+in	O
+ligand	O
+binding	O
+,	O
+an	O
+explanation	O
+for	O
+the	O
+lack	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+monomer	B-oligomeric_state
+A	B-structure_element
+did	O
+not	O
+emerge	O
+by	O
+analyzing	O
+only	O
+these	O
+backbone	O
+atom	O
+positions	O
+,	O
+suggesting	O
+that	O
+a	O
+more	O
+complex	O
+series	O
+of	O
+allosteric	O
+events	O
+may	O
+occur	O
+.	O
+
+Indeed	O
+,	O
+we	O
+noted	O
+interesting	O
+differences	O
+in	O
+the	O
+side	O
+chains	O
+of	O
+Met22	B-residue_name_number
+,	O
+Phe25	B-residue_name_number
+and	O
+Arg43	B-residue_name_number
+,	O
+which	O
+in	O
+monomer	B-oligomeric_state
+B	B-structure_element
+are	O
+used	O
+to	O
+contact	O
+the	O
+ligand	O
+while	O
+in	O
+monomer	B-oligomeric_state
+A	B-structure_element
+they	O
+partially	O
+occupied	O
+the	O
+pocket	B-site
+and	O
+collectively	O
+reduced	O
+its	O
+volume	O
+significantly	O
+.	O
+
+Specifically	O
+,	O
+upon	O
+analysis	O
+with	O
+the	O
+CASTp	B-experimental_method
+software	O
+,	O
+the	O
+pocket	B-site
+in	O
+chain	B-structure_element
+B	I-structure_element
+containing	O
+the	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+exhibited	O
+a	O
+total	O
+volume	O
+of	O
+approximately	O
+370	O
+Å3	O
+,	O
+while	O
+the	O
+pocket	B-site
+in	O
+chain	B-structure_element
+A	I-structure_element
+was	O
+occupied	O
+by	O
+these	O
+three	O
+side	O
+chains	O
+that	O
+adopted	O
+‘	O
+inward	B-protein_state
+’	O
+positions	O
+and	O
+thereby	O
+divided	O
+the	O
+space	O
+into	O
+a	O
+few	O
+much	O
+smaller	O
+pockets	O
+,	O
+each	O
+with	O
+volume	O
+<	O
+50	O
+Å3	O
+,	O
+evidently	O
+rendering	O
+chain	B-structure_element
+A	I-structure_element
+unfavorable	O
+for	O
+ligand	O
+binding	O
+.	O
+
+Most	O
+notably	O
+,	O
+atomic	O
+clashes	O
+between	O
+the	O
+ligand	O
+and	O
+the	O
+side	O
+chains	O
+of	O
+MetA22	B-residue_name_number
+,	O
+PheA25	B-residue_name_number
+and	O
+ArgA43	B-residue_name_number
+would	O
+occur	O
+if	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+were	O
+present	O
+in	O
+the	O
+monomer	B-oligomeric_state
+A	B-structure_element
+pocket	B-site
+(	O
+Fig	O
+5B	O
+).	O
+
+Subsequently	O
+,	O
+analyses	O
+of	O
+the	O
+pockets	B-site
+in	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+revealed	O
+that	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ligand	I-protein_state
+the	O
+long	O
+Arg43	B-residue_name_number
+side	O
+chain	O
+was	O
+always	O
+in	O
+the	O
+open	O
+‘	O
+outward	B-protein_state
+’	O
+position	O
+compatible	O
+with	O
+binding	O
+to	O
+the	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+carboxylate	O
+group	O
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+apo	B-protein_state
+-	O
+form	O
+Met22	B-residue_name_number
+and	O
+Phe25	B-residue_name_number
+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
+
+The	O
+‘	O
+outward	B-protein_state
+’	O
+position	O
+of	O
+Arg43	B-residue_name_number
+generated	O
+an	O
+open	B-protein_state
+apo	B-protein_state
+-	O
+form	O
+pocket	B-site
+with	O
+volume	O
+approximately	O
+380Å3	O
+.	O
+
+Taken	O
+together	O
+,	O
+these	O
+observations	O
+suggest	O
+that	O
+Arg43	B-residue_name_number
+is	O
+a	O
+major	O
+determinant	O
+of	O
+ligand	O
+binding	O
+,	O
+and	O
+that	O
+its	O
+‘	O
+inward	B-protein_state
+’	O
+position	O
+inhibits	O
+the	O
+binding	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+to	O
+the	O
+empty	O
+pocket	B-site
+of	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+.	O
+
+Structural	O
+differences	O
+of	O
+NadR	B-protein
+in	O
+ligand	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+or	O
+free	B-protein_state
+forms	O
+.	O
+
+(	O
+A	O
+)	O
+Aligned	B-experimental_method
+monomers	B-oligomeric_state
+of	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+chain	B-structure_element
+A	I-structure_element
+:	O
+green	O
+;	O
+chain	B-structure_element
+B	I-structure_element
+:	O
+blue	O
+),	O
+reveal	O
+major	O
+overall	O
+differences	O
+by	O
+the	O
+shift	O
+of	O
+helix	B-structure_element
+α6	B-structure_element
+.	O
+(	O
+B	O
+)	O
+Comparison	B-experimental_method
+of	O
+the	O
+two	O
+binding	B-site
+pockets	I-site
+in	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+shows	O
+that	O
+in	O
+the	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+monomer	B-oligomeric_state
+A	B-structure_element
+(	O
+green	O
+)	O
+residues	O
+Met22	B-residue_name_number
+,	O
+Phe25	B-residue_name_number
+and	O
+Arg43	B-residue_name_number
+adopt	O
+‘	O
+inward	B-protein_state
+’	O
+positions	O
+(	O
+highlighted	O
+by	O
+arrows	O
+)	O
+compared	O
+to	O
+the	O
+ligand	B-protein_state
+-	I-protein_state
+occupied	I-protein_state
+pocket	B-site
+(	O
+blue	O
+residues	O
+);	O
+these	O
+‘	O
+inward	B-protein_state
+’	O
+conformations	O
+appear	O
+unfavorable	O
+for	O
+binding	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+due	O
+to	O
+clashes	O
+with	O
+the	O
+4	O
+-	O
+hydroxyl	O
+group	O
+,	O
+the	O
+phenyl	O
+ring	O
+and	O
+the	O
+carboxylate	O
+group	O
+,	O
+respectively	O
+.	O
+
+In	O
+these	O
+crystals	B-evidence
+,	O
+the	O
+ArgA43	B-residue_name_number
+side	O
+chain	O
+showed	O
+two	O
+alternate	O
+conformations	O
+,	O
+modelled	O
+with	O
+50	O
+%	O
+occupancy	O
+in	O
+each	O
+state	O
+,	O
+as	O
+indicated	O
+by	O
+the	O
+two	O
+‘	O
+mirrored	O
+’	O
+arrows	O
+.	O
+
+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	B-chemical
+-	I-chemical
+HPA	I-chemical
+were	O
+present	O
+.	O
+(	O
+C	O
+)	O
+Comparison	O
+of	O
+the	O
+empty	O
+pocket	B-site
+from	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+green	O
+residues	O
+)	O
+with	O
+the	O
+four	O
+empty	O
+pockets	B-site
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+grey	O
+residues	O
+),	O
+shows	O
+that	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+the	O
+Arg43	B-residue_name_number
+side	O
+chain	O
+is	O
+always	O
+observed	O
+in	O
+the	O
+‘	O
+outward	B-protein_state
+’	O
+conformation	O
+.	O
+
+Finally	O
+,	O
+we	O
+applied	O
+15N	B-experimental_method
+heteronuclear	I-experimental_method
+solution	I-experimental_method
+NMR	I-experimental_method
+spectroscopy	I-experimental_method
+to	O
+examine	O
+the	O
+interaction	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+with	O
+apo	B-protein_state
+NadR	B-protein
+.	O
+We	O
+collected	O
+NMR	B-experimental_method
+spectra	B-evidence
+on	O
+NadR	B-protein
+in	B-protein_state
+the	I-protein_state
+presence	I-protein_state
+and	O
+absence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+
+The	O
+1H	B-experimental_method
+-	I-experimental_method
+15N	I-experimental_method
+TROSY	I-experimental_method
+-	I-experimental_method
+HSQC	I-experimental_method
+spectrum	B-evidence
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+,	O
+acquired	O
+at	O
+25	O
+°	O
+C	O
+,	O
+displayed	O
+approximately	O
+140	O
+distinct	O
+peaks	O
+(	O
+Fig	O
+6A	O
+),	O
+most	O
+of	O
+which	O
+correspond	O
+to	O
+backbone	O
+amide	O
+N	O
+-	O
+H	O
+groups	O
+.	O
+
+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	B-protein_state
+-	O
+NadR	B-protein
+is	O
+well	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+under	O
+these	O
+conditions	O
+and	O
+exhibits	O
+one	O
+conformation	O
+appreciable	O
+on	O
+the	O
+NMR	B-experimental_method
+timescale	O
+,	O
+i	O
+.	O
+e	O
+.	O
+in	O
+the	O
+NMR	B-experimental_method
+experiments	O
+at	O
+25	O
+°	O
+C	O
+,	O
+two	O
+or	O
+more	O
+distinct	O
+conformations	O
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+monomers	B-oligomeric_state
+were	O
+not	O
+readily	O
+apparent	O
+.	O
+
+Upon	O
+the	O
+addition	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+over	O
+45	O
+peaks	O
+showed	O
+chemical	O
+shift	O
+perturbations	O
+,	O
+i	O
+.	O
+e	O
+.	O
+changed	O
+position	O
+in	O
+the	O
+spectrum	O
+or	O
+disappeared	O
+,	O
+while	O
+the	O
+remaining	O
+peaks	O
+remained	O
+unchanged	O
+.	O
+
+This	O
+observation	O
+showed	O
+that	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+was	O
+able	O
+to	O
+bind	O
+NadR	B-protein
+and	O
+induce	O
+notable	O
+changes	O
+in	O
+specific	O
+regions	O
+of	O
+the	O
+protein	O
+.	O
+
+NMR	B-experimental_method
+spectra	B-evidence
+of	O
+NadR	B-protein
+in	B-protein_state
+the	I-protein_state
+presence	I-protein_state
+and	O
+absence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+(	O
+A	O
+)	O
+Superposition	B-experimental_method
+of	O
+two	O
+1H	B-experimental_method
+-	I-experimental_method
+15N	I-experimental_method
+TROSY	I-experimental_method
+-	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+recorded	O
+at	O
+25	O
+°	O
+C	O
+on	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+cyan	O
+)	O
+and	O
+on	O
+NadR	B-protein
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+red	O
+).	O
+
+(	O
+B	O
+,	O
+C	O
+)	O
+Overlay	B-experimental_method
+of	O
+selected	O
+regions	O
+of	O
+the	O
+1H	B-experimental_method
+-	I-experimental_method
+15N	I-experimental_method
+TROSY	I-experimental_method
+-	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+acquired	O
+at	O
+25	O
+°	O
+C	O
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+cyan	O
+)	O
+and	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+red	O
+)	O
+superimposed	B-experimental_method
+with	O
+the	O
+spectra	B-evidence
+acquired	O
+at	O
+10	O
+°	O
+C	O
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+blue	O
+)	O
+and	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+green	O
+).	O
+
+The	O
+spectra	B-evidence
+acquired	O
+at	O
+10	O
+°	O
+C	O
+are	O
+excluded	O
+from	O
+panel	O
+A	O
+for	O
+simplicity	O
+.	O
+
+However	O
+,	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+the	O
+1H	B-experimental_method
+-	I-experimental_method
+15N	I-experimental_method
+TROSY	I-experimental_method
+-	I-experimental_method
+HSQC	I-experimental_method
+spectrum	B-evidence
+of	O
+NadR	B-protein
+displayed	O
+approximately	O
+140	O
+peaks	O
+,	O
+as	O
+for	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+,	O
+i	O
+.	O
+e	O
+.	O
+two	O
+distinct	O
+stable	O
+conformations	O
+(	O
+that	O
+might	O
+have	O
+potentially	O
+revealed	O
+the	O
+molecular	O
+asymmetry	O
+observed	O
+crystallographically	B-experimental_method
+)	O
+were	O
+not	O
+notable	O
+.	O
+
+Considering	O
+the	O
+small	O
+size	O
+,	O
+fast	O
+diffusion	O
+and	O
+relatively	O
+low	O
+binding	B-evidence
+affinity	I-evidence
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	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	B-experimental_method
+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	B-protein_state
+and	O
+unbound	B-protein_state
+states	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	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+and	O
+therefore	O
+likely	O
+to	O
+be	O
+in	O
+the	O
+ligand	B-site
+-	I-site
+binding	I-site
+site	I-site
+)	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
+
+These	O
+doubled	O
+peaks	O
+may	O
+therefore	O
+reveal	O
+that	O
+the	O
+cooler	O
+temperature	O
+partially	O
+trapped	O
+the	O
+existence	O
+in	O
+solution	O
+of	O
+two	O
+distinct	O
+states	O
+,	O
+in	O
+presence	B-protein_state
+or	O
+absence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+with	O
+minor	O
+conformational	O
+differences	O
+occurring	O
+at	O
+least	O
+in	O
+proximity	O
+to	O
+the	O
+binding	B-site
+pocket	I-site
+.	O
+
+Although	O
+more	O
+comprehensive	O
+NMR	B-experimental_method
+experiments	O
+and	O
+full	O
+chemical	O
+shift	O
+assignment	O
+of	O
+the	O
+spectra	B-evidence
+would	O
+be	O
+required	O
+to	O
+precisely	O
+define	O
+this	O
+multi	O
+-	O
+state	O
+behavior	O
+,	O
+the	O
+NMR	B-experimental_method
+data	O
+clearly	O
+demonstrate	O
+that	O
+NadR	B-protein
+exhibits	O
+conformational	O
+flexibility	O
+which	O
+is	O
+modulated	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+solution	O
+.	O
+
+Apo	B-protein_state
+-	O
+NadR	B-protein
+structures	B-evidence
+reveal	O
+intrinsic	O
+conformational	O
+flexibility	O
+
+The	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+crystal	B-evidence
+structure	I-evidence
+contained	O
+two	O
+homodimers	B-oligomeric_state
+in	O
+the	O
+asymmetric	O
+unit	O
+(	O
+chains	B-structure_element
+A	I-structure_element
++	I-structure_element
+B	I-structure_element
+and	O
+chains	B-structure_element
+C	I-structure_element
++	I-structure_element
+D	I-structure_element
+).	O
+
+Upon	O
+overall	O
+structural	B-experimental_method
+superposition	I-experimental_method
+,	O
+these	O
+dimers	B-oligomeric_state
+revealed	O
+a	O
+few	O
+minor	O
+differences	O
+in	O
+the	O
+α6	B-structure_element
+helix	I-structure_element
+(	O
+a	O
+major	O
+component	O
+of	O
+the	O
+dimer	B-site
+interface	I-site
+)	O
+and	O
+the	O
+helices	B-structure_element
+α4	B-structure_element
+-	I-structure_element
+α5	I-structure_element
+(	O
+the	O
+DNA	B-site
+binding	I-site
+region	I-site
+),	O
+and	O
+an	O
+rmsd	B-evidence
+of	O
+1	O
+.	O
+55Å	O
+(	O
+Fig	O
+7A	O
+).	O
+
+Similarly	O
+,	O
+the	O
+entire	O
+holo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+could	O
+be	O
+closely	B-experimental_method
+superposed	I-experimental_method
+onto	O
+each	O
+of	O
+the	O
+apo	B-protein_state
+-	O
+homodimers	B-oligomeric_state
+,	O
+showing	O
+rmsd	B-evidence
+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	B-structure_element
+helix	I-structure_element
+positions	O
+(	O
+Fig	O
+7B	O
+).	O
+
+The	O
+slightly	O
+larger	O
+rmsd	B-evidence
+between	O
+the	O
+two	O
+apo	B-protein_state
+-	O
+homodimers	B-oligomeric_state
+,	O
+rather	O
+than	O
+between	O
+apo	B-protein_state
+-	O
+and	O
+holo	B-protein_state
+-	O
+homodimers	B-oligomeric_state
+,	O
+further	O
+indicate	O
+that	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+possesses	O
+a	O
+notable	O
+degree	O
+of	O
+intrinsic	O
+conformational	O
+flexibility	O
+.	O
+
+Overall	O
+apo	B-protein_state
+-	O
+and	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+structures	B-evidence
+are	O
+similar	O
+.	O
+
+(	O
+A	O
+)	O
+Pairwise	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+two	O
+distinct	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+homodimers	B-oligomeric_state
+(	O
+AB	B-structure_element
+and	O
+CD	B-structure_element
+)	O
+present	O
+in	O
+the	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+crystals	B-evidence
+.	O
+(	O
+B	O
+)	O
+Alignment	B-experimental_method
+of	O
+the	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+homodimer	B-oligomeric_state
+(	O
+green	O
+and	O
+blue	O
+chains	O
+)	O
+onto	O
+the	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+homodimers	B-oligomeric_state
+.	O
+
+Here	O
+,	O
+larger	O
+differences	O
+are	O
+observed	O
+in	O
+the	O
+α6	B-structure_element
+helices	I-structure_element
+(	O
+top	O
+).	O
+
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+stabilizes	O
+concerted	O
+conformational	O
+changes	O
+in	O
+NadR	B-protein
+that	O
+prevent	O
+DNA	O
+-	O
+binding	O
+
+To	O
+further	O
+investigate	O
+the	O
+conformational	O
+rearrangements	O
+of	O
+NadR	B-protein
+,	O
+we	O
+performed	O
+local	B-experimental_method
+structural	I-experimental_method
+alignments	I-experimental_method
+using	O
+only	O
+a	O
+subset	O
+of	O
+residues	O
+in	O
+the	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+helix	I-structure_element
+(	O
+α4	B-structure_element
+).	O
+
+By	O
+selecting	B-experimental_method
+and	O
+aligning	B-experimental_method
+residues	O
+Arg64	B-residue_range
+-	I-residue_range
+Ala77	I-residue_range
+of	O
+one	O
+α4	B-structure_element
+helix	I-structure_element
+per	O
+dimer	B-oligomeric_state
+,	O
+superposition	B-experimental_method
+of	O
+the	O
+holo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+onto	O
+the	O
+two	O
+apo	B-protein_state
+-	O
+homodimers	B-oligomeric_state
+revealed	O
+differences	O
+in	O
+the	O
+monomer	B-oligomeric_state
+conformations	O
+of	O
+each	O
+structure	B-evidence
+.	O
+
+While	O
+one	O
+monomer	B-oligomeric_state
+from	O
+each	O
+structure	B-evidence
+was	O
+closely	O
+superimposable	O
+(	O
+Fig	O
+8A	O
+,	O
+left	O
+side	O
+),	O
+the	O
+second	O
+monomer	B-oligomeric_state
+displayed	O
+quite	O
+large	O
+differences	O
+(	O
+Fig	O
+8A	O
+,	O
+right	O
+side	O
+).	O
+
+Most	O
+notably	O
+,	O
+the	O
+position	O
+of	O
+the	O
+DNA	B-chemical
+-	O
+binding	O
+helix	B-structure_element
+α4	B-structure_element
+shifted	O
+by	O
+as	O
+much	O
+as	O
+6	O
+Å	O
+(	O
+Fig	O
+8B	O
+).	O
+
+Accordingly	O
+,	O
+helix	B-structure_element
+α4	B-structure_element
+was	O
+also	O
+found	O
+to	O
+be	O
+one	O
+of	O
+the	O
+most	O
+dynamic	O
+regions	O
+in	O
+previous	O
+HDX	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+analyses	O
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+in	O
+solution	O
+.	O
+
+Structural	B-experimental_method
+comparisons	I-experimental_method
+of	O
+NadR	B-protein
+and	O
+modelling	O
+of	O
+interactions	O
+with	O
+DNA	B-chemical
+.	O
+
+(	O
+A	O
+)	O
+The	O
+holo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+structure	B-evidence
+is	O
+shown	O
+as	O
+green	O
+and	O
+blue	O
+cartoons	O
+,	O
+for	O
+chain	B-structure_element
+A	I-structure_element
+and	I-structure_element
+B	I-structure_element
+,	O
+respectively	O
+,	O
+while	O
+the	O
+two	O
+homodimers	B-oligomeric_state
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+are	O
+both	O
+cyan	O
+and	O
+pale	O
+blue	O
+for	O
+chains	O
+A	B-structure_element
+/	I-structure_element
+C	I-structure_element
+and	O
+B	B-structure_element
+/	I-structure_element
+D	I-structure_element
+,	O
+respectively	O
+.	O
+
+The	O
+three	O
+homodimers	B-oligomeric_state
+(	O
+chains	O
+AB	B-structure_element
+holo	B-protein_state
+,	O
+AB	B-structure_element
+apo	B-protein_state
+,	O
+and	O
+CD	B-structure_element
+apo	B-protein_state
+)	O
+were	O
+overlaid	B-experimental_method
+by	O
+structural	B-experimental_method
+alignment	I-experimental_method
+exclusively	O
+of	O
+all	O
+heavy	O
+atoms	O
+in	O
+residues	O
+R64	B-residue_range
+-	I-residue_range
+A77	I-residue_range
+(	O
+shown	O
+in	O
+red	O
+,	O
+with	O
+side	O
+chain	O
+sticks	O
+)	O
+of	O
+chains	O
+A	B-structure_element
+holo	B-protein_state
+,	O
+A	B-structure_element
+apo	B-protein_state
+,	O
+and	O
+C	B-structure_element
+apo	B-protein_state
+,	O
+belonging	O
+to	O
+helix	B-structure_element
+α4	B-structure_element
+(	O
+left	O
+).	O
+
+The	O
+α4	B-structure_element
+helices	I-structure_element
+aligned	O
+closely	O
+,	O
+Cα	O
+rmsd	B-evidence
+0	O
+.	O
+2Å	O
+for	O
+14	O
+residues	O
+.	O
+
+(	O
+B	O
+)	O
+The	O
+relative	O
+positions	O
+of	O
+the	O
+α4	B-structure_element
+helices	I-structure_element
+of	O
+the	O
+4	B-protein_state
+-	I-protein_state
+HPA	I-protein_state
+-	I-protein_state
+bound	I-protein_state
+holo	B-protein_state
+homodimer	B-oligomeric_state
+chain	B-structure_element
+B	I-structure_element
+(	O
+blue	O
+),	O
+and	O
+of	O
+apo	B-protein_state
+homodimers	B-oligomeric_state
+AB	B-structure_element
+and	O
+CD	B-structure_element
+(	O
+showing	O
+chains	B-structure_element
+B	I-structure_element
+and	I-structure_element
+D	I-structure_element
+)	O
+in	O
+pale	O
+blue	O
+.	O
+
+Dashes	O
+indicate	O
+the	O
+Ala77	B-residue_name_number
+Cα	O
+atoms	O
+,	O
+in	O
+the	O
+most	O
+highly	O
+shifted	O
+region	O
+of	O
+the	O
+‘	O
+non	O
+-	O
+fixed	O
+’	O
+α4	B-structure_element
+helix	I-structure_element
+.	O
+
+(	O
+C	O
+)	O
+The	O
+double	O
+-	O
+stranded	O
+DNA	B-chemical
+molecule	O
+(	O
+grey	O
+cartoon	O
+)	O
+from	O
+the	O
+OhrR	B-complex_assembly
+-	I-complex_assembly
+ohrA	I-complex_assembly
+complex	O
+is	O
+shown	O
+after	O
+superposition	B-experimental_method
+with	O
+NadR	B-protein
+,	O
+to	O
+highlight	O
+the	O
+expected	O
+positions	O
+of	O
+the	O
+NadR	B-protein
+α4	B-structure_element
+helices	I-structure_element
+in	O
+the	O
+DNA	B-chemical
+major	O
+grooves	O
+.	O
+
+For	O
+clarity	O
+,	O
+only	O
+the	O
+α4	B-structure_element
+helices	I-structure_element
+are	O
+shown	O
+in	O
+panels	O
+(	O
+B	O
+)	O
+and	O
+(	O
+C	O
+).	O
+(	O
+D	O
+)	O
+Upon	O
+comparison	O
+with	O
+the	O
+experimentally	O
+-	O
+determined	O
+OhrR	B-complex_assembly
+:	I-complex_assembly
+ohrA	I-complex_assembly
+structure	B-evidence
+(	O
+grey	O
+),	O
+the	O
+α4	B-structure_element
+helix	I-structure_element
+of	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+blue	O
+)	O
+is	O
+shifted	O
+~	O
+8Å	O
+out	O
+of	O
+the	O
+major	O
+groove	O
+.	O
+
+However	O
+,	O
+structural	B-experimental_method
+comparisons	I-experimental_method
+revealed	O
+that	O
+the	O
+shift	O
+of	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+helix	B-structure_element
+α4	B-structure_element
+induced	O
+by	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+was	O
+also	O
+accompanied	O
+by	O
+several	O
+changes	O
+at	O
+the	O
+holo	B-protein_state
+dimer	B-site
+interface	I-site
+,	O
+while	O
+such	O
+extensive	O
+structural	O
+differences	O
+were	O
+not	O
+observed	O
+in	O
+the	O
+apo	B-protein_state
+dimer	B-site
+interfaces	I-site
+,	O
+particularly	O
+notable	O
+when	O
+comparing	O
+the	O
+α6	B-structure_element
+helices	I-structure_element
+(	O
+S3	O
+Fig	O
+).	O
+
+In	O
+summary	O
+,	O
+compared	O
+to	O
+ligand	B-protein_state
+-	I-protein_state
+stabilized	I-protein_state
+holo	B-protein_state
+-	O
+NadR	B-protein
+,	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+displayed	O
+an	O
+intrinsic	O
+flexibility	O
+focused	O
+in	O
+the	O
+DNA	B-site
+-	I-site
+binding	I-site
+region	I-site
+.	O
+
+This	O
+was	O
+also	O
+evident	O
+in	O
+the	O
+greater	O
+disorder	O
+(	O
+i	O
+.	O
+e	O
+.	O
+less	O
+well	O
+-	O
+defined	O
+electron	B-evidence
+density	I-evidence
+)	O
+in	O
+the	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+loops	I-structure_element
+of	O
+the	O
+apo	B-protein_state
+dimers	B-oligomeric_state
+(	O
+density	B-evidence
+for	O
+16	O
+residues	O
+per	O
+dimer	B-oligomeric_state
+was	O
+missing	O
+)	O
+compared	O
+to	O
+the	O
+holo	B-protein_state
+dimer	B-oligomeric_state
+(	O
+density	B-evidence
+for	O
+only	O
+3	O
+residues	O
+was	O
+missing	O
+).	O
+
+In	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+,	O
+the	O
+distance	O
+separating	O
+the	O
+two	O
+DNA	O
+-	O
+binding	O
+α4	B-structure_element
+helices	I-structure_element
+was	O
+32	O
+Å	O
+,	O
+while	O
+in	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+it	O
+was	O
+29	O
+Å	O
+for	O
+homodimer	B-oligomeric_state
+AB	B-structure_element
+,	O
+and	O
+34	O
+Å	O
+for	O
+homodimer	B-oligomeric_state
+CD	B-structure_element
+(	O
+Fig	O
+8C	O
+).	O
+
+Thus	O
+,	O
+the	O
+apo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+AB	B-structure_element
+presented	O
+the	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+helices	I-structure_element
+in	O
+a	O
+conformation	O
+similar	O
+to	O
+that	O
+observed	O
+in	O
+the	O
+protein	O
+:	O
+DNA	O
+complex	O
+of	O
+OhrR	B-complex_assembly
+:	I-complex_assembly
+ohrA	I-complex_assembly
+from	O
+Bacillus	B-species
+subtilis	I-species
+(	O
+Fig	O
+8C	O
+).	O
+
+Interestingly	O
+,	O
+OhrR	B-protein
+contacts	O
+ohrA	B-gene
+across	O
+22	O
+base	O
+pairs	O
+(	O
+bp	O
+),	O
+and	O
+similarly	O
+the	O
+main	O
+NadR	B-protein
+target	B-site
+sites	I-site
+identified	O
+in	O
+the	O
+nadA	B-gene
+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
+
+Pairwise	B-experimental_method
+superpositions	I-experimental_method
+showed	O
+that	O
+the	O
+NadR	B-protein
+apo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+AB	B-structure_element
+was	O
+the	O
+most	O
+similar	O
+to	O
+OhrR	B-protein
+(	O
+rmsd	B-evidence
+2	O
+.	O
+6	O
+Å	O
+),	O
+while	O
+the	O
+holo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+was	O
+the	O
+most	O
+divergent	O
+(	O
+rmsd	B-evidence
+3	O
+.	O
+3	O
+Å	O
+)	O
+(	O
+Fig	O
+8C	O
+).	O
+
+Assuming	O
+the	O
+same	O
+DNA	B-chemical
+-	O
+binding	O
+mechanism	O
+is	O
+used	O
+by	O
+OhrR	B-protein
+and	O
+NadR	B-protein
+,	O
+the	O
+apo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+AB	B-structure_element
+seems	O
+ideally	O
+pre	O
+-	O
+configured	O
+for	O
+DNA	B-chemical
+binding	O
+,	O
+while	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+appeared	O
+to	O
+stabilize	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+in	O
+a	O
+conformation	O
+poorly	O
+suited	O
+for	O
+DNA	B-chemical
+binding	O
+.	O
+
+Specifically	O
+,	O
+in	O
+addition	O
+to	O
+the	O
+different	O
+inter	B-evidence
+-	I-evidence
+helical	I-evidence
+translational	I-evidence
+distances	I-evidence
+,	O
+the	O
+α4	B-structure_element
+helices	I-structure_element
+in	O
+the	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+homodimer	B-oligomeric_state
+were	O
+also	O
+reoriented	O
+,	O
+resulting	O
+in	O
+movement	O
+of	O
+α4	B-structure_element
+out	O
+of	O
+the	O
+major	O
+groove	O
+,	O
+by	O
+up	O
+to	O
+8Å	O
+,	O
+and	O
+presumably	O
+preventing	O
+efficient	O
+DNA	B-chemical
+binding	O
+in	O
+the	O
+presence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+Fig	O
+8D	O
+).	O
+
+When	O
+aligned	B-experimental_method
+with	O
+OhrR	B-protein
+,	O
+the	O
+apo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+CD	B-structure_element
+presented	O
+yet	O
+another	O
+different	O
+intermediate	O
+conformation	O
+(	O
+rmsd	B-evidence
+2	O
+.	O
+9Å	O
+),	O
+apparently	O
+not	O
+ideally	O
+pre	O
+-	O
+configured	O
+for	O
+DNA	B-chemical
+binding	O
+,	O
+but	O
+which	O
+in	O
+solution	O
+can	O
+presumably	O
+readily	O
+adopt	O
+the	O
+AB	B-structure_element
+conformation	O
+due	O
+to	O
+the	O
+intrinsic	O
+flexibility	O
+described	O
+above	O
+.	O
+
+NadR	B-protein
+residues	O
+His7	B-residue_name_number
+,	O
+Ser9	B-residue_name_number
+,	O
+Asn11	B-residue_name_number
+and	O
+Phe25	B-residue_name_number
+are	O
+essential	O
+for	O
+regulation	O
+of	O
+NadA	B-protein
+expression	O
+in	O
+vivo	O
+
+While	O
+previous	O
+studies	O
+had	O
+correctly	O
+suggested	O
+the	O
+involvement	O
+of	O
+several	O
+NadR	B-protein
+residues	O
+in	O
+ligand	O
+binding	O
+,	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+presented	O
+here	O
+revealed	O
+additional	O
+residues	O
+with	O
+previously	O
+unknown	O
+roles	O
+in	O
+dimerization	O
+and	O
+/	O
+or	O
+binding	O
+to	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+To	O
+explore	O
+the	O
+functional	O
+involvement	O
+of	O
+these	O
+residues	O
+,	O
+we	O
+characterized	O
+the	O
+behavior	O
+of	O
+four	O
+new	O
+NadR	B-protein
+mutants	O
+(	O
+H7A	B-mutant
+,	O
+S9A	B-mutant
+,	O
+N11A	B-mutant
+and	O
+F25A	B-mutant
+)	O
+in	O
+an	O
+in	O
+vivo	O
+assay	O
+using	O
+the	O
+previously	O
+described	O
+MC58	B-mutant
+-	I-mutant
+Δ1843	I-mutant
+nadR	B-gene
+-	O
+null	O
+mutant	B-protein_state
+strain	O
+,	O
+which	O
+was	O
+complemented	O
+either	O
+by	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+nadR	B-gene
+or	O
+by	O
+the	O
+nadR	B-gene
+mutants	B-protein_state
+.	O
+
+NadA	B-protein
+protein	O
+abundance	O
+levels	O
+were	O
+assessed	O
+by	O
+Western	B-experimental_method
+blotting	I-experimental_method
+to	O
+evaluate	O
+the	O
+ability	O
+of	O
+the	O
+NadR	B-protein
+mutants	B-protein_state
+to	O
+repress	O
+the	O
+nadA	B-gene
+promoter	O
+,	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+The	O
+nadR	B-gene
+H7A	B-mutant
+,	O
+S9A	B-mutant
+and	O
+F25A	B-mutant
+complemented	O
+strains	O
+showed	O
+hyper	O
+-	O
+repression	O
+of	O
+nadA	B-gene
+expression	O
+in	O
+vivo	O
+,	O
+i	O
+.	O
+e	O
+.	O
+these	O
+mutants	O
+repressed	O
+nadA	B-gene
+more	O
+efficiently	O
+than	O
+the	O
+NadR	B-protein
+WT	B-protein_state
+protein	O
+,	O
+either	O
+in	O
+the	O
+presence	O
+or	O
+absence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+while	O
+complementation	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+nadR	B-gene
+resulted	O
+in	O
+high	O
+production	O
+of	O
+NadA	B-protein
+only	O
+in	O
+the	O
+presence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+Fig	O
+9	O
+).	O
+
+Interestingly	O
+,	O
+and	O
+on	O
+the	O
+contrary	O
+,	O
+the	O
+nadR	B-gene
+N11A	B-mutant
+complemented	O
+strain	O
+showed	O
+hypo	O
+-	O
+repression	O
+(	O
+i	O
+.	O
+e	O
+.	O
+exhibited	O
+high	O
+expression	O
+of	O
+nadA	B-gene
+both	O
+in	O
+absence	O
+and	O
+presence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+).	O
+
+This	O
+mutagenesis	B-experimental_method
+data	O
+revealed	O
+that	O
+NadR	B-protein
+residues	O
+His7	B-residue_name_number
+,	O
+Ser9	B-residue_name_number
+,	O
+Asn11	B-residue_name_number
+and	O
+Phe25	B-residue_name_number
+play	O
+key	O
+roles	O
+in	O
+the	O
+ligand	O
+-	O
+mediated	O
+regulation	O
+of	O
+NadR	B-protein
+;	O
+they	O
+are	O
+each	O
+involved	O
+in	O
+the	O
+controlled	O
+de	O
+-	O
+repression	O
+of	O
+the	O
+nadA	B-gene
+promoter	O
+and	O
+synthesis	O
+of	O
+NadA	B-protein
+in	O
+response	O
+to	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+vivo	O
+.	O
+
+Structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+point	I-experimental_method
+mutations	I-experimental_method
+shed	O
+light	O
+on	O
+ligand	O
+-	O
+induced	O
+regulation	O
+of	O
+NadR	B-protein
+.	O
+
+Western	B-experimental_method
+blot	I-experimental_method
+analyses	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+strain	O
+(	O
+lanes	O
+1	O
+–	O
+2	O
+)	O
+or	O
+isogenic	O
+nadR	B-gene
+knockout	O
+strains	O
+(	O
+ΔNadR	B-mutant
+)	O
+complemented	O
+to	O
+express	O
+the	O
+indicated	O
+NadR	B-protein
+WT	B-protein_state
+or	O
+mutant	B-protein_state
+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	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+showing	O
+NadA	B-protein
+and	O
+NadR	B-protein
+expression	O
+.	O
+
+Complementation	O
+of	O
+ΔNadR	B-mutant
+with	O
+WT	B-protein_state
+NadR	B-protein
+enables	O
+induction	O
+of	O
+nadA	B-gene
+expression	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+The	O
+H7A	B-mutant
+,	O
+S9A	B-mutant
+and	O
+F25A	B-mutant
+mutants	O
+efficiently	O
+repress	O
+nadA	B-gene
+expression	O
+but	O
+are	O
+less	O
+ligand	O
+-	O
+responsive	O
+than	O
+WT	B-protein_state
+NadR	B-protein
+.	O
+The	O
+N11A	B-mutant
+mutant	B-protein_state
+does	O
+not	O
+efficiently	O
+repress	O
+nadA	B-gene
+expression	O
+either	O
+in	O
+presence	O
+or	O
+absence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+(	O
+The	O
+protein	O
+abundance	O
+levels	O
+of	O
+the	O
+meningococcal	B-taxonomy_domain
+factor	B-protein
+H	I-protein
+binding	I-protein
+protein	I-protein
+(	O
+fHbp	B-protein
+)	O
+were	O
+used	O
+as	O
+a	O
+gel	O
+loading	O
+control	O
+).	O
+
+NadA	B-protein
+is	O
+a	O
+surface	O
+-	O
+exposed	O
+meningococcal	B-taxonomy_domain
+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
+
+A	O
+detailed	O
+understanding	O
+of	O
+the	O
+in	O
+vitro	O
+repression	O
+of	O
+nadA	B-gene
+expression	O
+by	O
+the	O
+transcriptional	B-protein_type
+regulator	I-protein_type
+NadR	B-protein
+is	O
+important	O
+,	O
+both	O
+because	O
+it	O
+is	O
+a	O
+relevant	O
+disease	O
+-	O
+related	O
+model	O
+of	O
+how	O
+small	O
+-	O
+molecule	O
+ligands	O
+can	O
+regulate	O
+MarR	B-protein_type
+family	O
+proteins	O
+and	O
+thereby	O
+impact	O
+bacterial	B-taxonomy_domain
+virulence	O
+,	O
+and	O
+because	O
+nadA	B-gene
+expression	O
+levels	O
+are	O
+linked	O
+to	O
+the	O
+prediction	O
+of	O
+vaccine	O
+coverage	O
+.	O
+
+The	O
+repressive	O
+activity	O
+of	O
+NadR	B-protein
+can	O
+be	O
+relieved	O
+by	O
+hydroxyphenylacetate	B-chemical
+(	O
+HPA	B-chemical
+)	O
+ligands	O
+,	O
+and	O
+HDX	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+studies	O
+previously	O
+indicated	O
+that	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+stabilizes	O
+dimeric	B-oligomeric_state
+NadR	B-protein
+in	O
+a	O
+configuration	O
+incompatible	O
+with	O
+DNA	O
+binding	O
+.	O
+
+Despite	O
+these	O
+and	O
+other	O
+studies	O
+,	O
+the	O
+molecular	O
+mechanisms	O
+by	O
+which	O
+ligands	O
+regulate	O
+MarR	B-protein_type
+family	O
+proteins	O
+are	O
+relatively	O
+poorly	O
+understood	O
+and	O
+likely	O
+differ	O
+depending	O
+on	O
+the	O
+specific	O
+ligand	O
+.	O
+
+Given	O
+the	O
+importance	O
+of	O
+NadR	B-protein
+-	O
+mediated	O
+regulation	O
+of	O
+NadA	B-protein
+levels	O
+in	O
+the	O
+contexts	O
+of	O
+meningococcal	B-taxonomy_domain
+pathogenesis	O
+,	O
+we	O
+sought	O
+to	O
+characterize	O
+NadR	B-protein
+,	O
+and	O
+its	O
+interaction	O
+with	O
+ligands	O
+,	O
+at	O
+atomic	O
+resolution	O
+.	O
+
+Firstly	O
+,	O
+we	O
+confirmed	O
+that	O
+NadR	B-protein
+is	O
+dimeric	B-oligomeric_state
+in	O
+solution	O
+and	O
+demonstrated	O
+that	O
+it	O
+retains	O
+its	O
+dimeric	B-oligomeric_state
+state	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+indicating	O
+that	O
+induction	O
+of	O
+a	O
+monomeric	B-oligomeric_state
+status	O
+is	O
+not	O
+the	O
+manner	O
+by	O
+which	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+regulates	O
+NadR	B-protein
+.	O
+These	O
+observations	O
+were	O
+in	O
+agreement	O
+with	O
+(	O
+i	O
+)	O
+a	O
+previous	O
+study	O
+of	O
+NadR	B-protein
+performed	O
+using	O
+SEC	B-experimental_method
+and	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+,	O
+and	O
+(	O
+ii	O
+)	O
+crystallographic	B-experimental_method
+studies	I-experimental_method
+showing	O
+that	O
+several	O
+MarR	B-protein_type
+homologues	O
+are	O
+dimeric	B-oligomeric_state
+.	O
+
+We	O
+also	O
+used	O
+structure	B-experimental_method
+-	I-experimental_method
+guided	I-experimental_method
+site	I-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+to	O
+identify	O
+an	O
+important	O
+conserved	B-protein_state
+residue	O
+,	O
+Leu130	B-residue_name_number
+,	O
+which	O
+stabilizes	O
+the	O
+NadR	B-protein
+dimer	B-site
+interface	I-site
+,	O
+knowledge	O
+of	O
+which	O
+may	O
+also	O
+inform	O
+future	O
+studies	O
+to	O
+explore	O
+the	O
+regulatory	O
+mechanisms	O
+of	O
+other	O
+MarR	B-protein_type
+family	O
+proteins	O
+.	O
+
+Secondly	O
+,	O
+we	O
+assessed	B-experimental_method
+the	I-experimental_method
+thermal	I-experimental_method
+stability	I-experimental_method
+and	O
+unfolding	O
+of	O
+NadR	B-protein
+in	B-protein_state
+the	I-protein_state
+presence	I-protein_state
+or	O
+absence	B-protein_state
+of	I-protein_state
+ligands	O
+.	O
+
+All	O
+DSC	B-experimental_method
+profiles	B-evidence
+showed	O
+a	O
+single	O
+peak	O
+,	O
+suggesting	O
+that	O
+a	O
+single	O
+unfolding	O
+event	O
+simultaneously	O
+disrupted	O
+the	O
+dimer	B-oligomeric_state
+and	O
+the	O
+monomer	B-oligomeric_state
+.	O
+
+HPA	O
+ligands	O
+specifically	O
+increased	O
+the	O
+stability	O
+of	O
+NadR	B-protein
+.	O
+The	O
+largest	O
+effects	O
+were	O
+induced	O
+by	O
+the	O
+naturally	O
+-	O
+occurring	O
+compounds	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+and	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+which	O
+,	O
+in	O
+SPR	B-experimental_method
+assays	I-experimental_method
+,	O
+were	O
+found	O
+to	O
+bind	O
+NadR	B-protein
+with	O
+KD	B-evidence
+values	O
+of	O
+1	O
+.	O
+5	O
+mM	O
+and	O
+1	O
+.	O
+1	O
+mM	O
+,	O
+respectively	O
+.	O
+
+Although	O
+these	O
+NadR	B-protein
+/	O
+HPA	O
+interactions	O
+appeared	O
+rather	O
+weak	O
+,	O
+their	O
+distinct	O
+affinities	O
+and	O
+specificities	O
+matched	O
+their	O
+in	O
+vitro	O
+effects	O
+and	O
+their	O
+biological	O
+relevance	O
+appears	O
+similar	O
+to	O
+previous	O
+proposals	O
+that	O
+certain	O
+small	O
+molecules	O
+,	O
+including	O
+some	O
+antibiotics	O
+,	O
+in	O
+the	O
+millimolar	O
+concentration	O
+range	O
+may	O
+be	O
+broad	O
+inhibitors	O
+of	O
+MarR	B-protein_type
+family	O
+proteins	O
+.	O
+
+Indeed	O
+,	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+is	O
+found	O
+in	O
+human	B-species
+saliva	O
+and	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+is	O
+produced	O
+during	O
+inflammatory	O
+processes	O
+,	O
+suggesting	O
+that	O
+these	O
+natural	O
+ligands	O
+are	O
+encountered	O
+by	O
+N	B-species
+.	I-species
+meningitidis	I-species
+in	O
+the	O
+mucosa	O
+of	O
+the	O
+oropharynx	O
+during	O
+infections	O
+.	O
+
+It	O
+is	O
+also	O
+possible	O
+that	O
+NadR	B-protein
+responds	O
+to	O
+currently	O
+unidentified	O
+HPA	B-chemical
+analogues	O
+.	O
+
+Indeed	O
+,	O
+in	O
+the	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+complex	O
+there	O
+was	O
+a	O
+water	B-chemical
+molecule	O
+close	O
+to	O
+the	O
+carboxylate	O
+group	O
+and	O
+also	O
+a	O
+small	O
+unfilled	O
+tunnel	B-site
+~	O
+5Å	O
+long	O
+,	O
+both	O
+factors	O
+suggesting	O
+that	O
+alternative	O
+larger	O
+ligands	O
+could	O
+occupy	O
+the	O
+pocket	O
+.	O
+
+The	O
+ability	O
+to	O
+respond	O
+to	O
+various	O
+ligands	O
+might	O
+enable	O
+NadR	B-protein
+in	O
+vivo	O
+to	O
+orchestrate	O
+multiple	O
+response	O
+mechanisms	O
+and	O
+modulate	O
+expression	O
+of	O
+genes	O
+other	O
+than	O
+nadA	B-gene
+.	O
+Ultimately	O
+,	O
+confirmation	O
+of	O
+the	O
+relevance	O
+of	O
+each	O
+ligand	O
+will	O
+require	O
+a	O
+deeper	O
+understanding	O
+of	O
+the	O
+available	O
+concentration	O
+in	O
+vivo	O
+in	O
+the	O
+host	O
+niche	O
+during	O
+bacterial	B-taxonomy_domain
+colonization	O
+and	O
+inflammation	O
+.	O
+
+Here	O
+,	O
+we	O
+determined	O
+the	O
+first	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+and	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+.	O
+These	O
+experimentally	O
+-	O
+determined	O
+structures	B-evidence
+enabled	O
+a	O
+new	O
+detailed	O
+characterization	O
+of	O
+the	O
+ligand	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+In	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+,	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+interacted	O
+directly	O
+with	O
+at	O
+least	O
+11	O
+polar	O
+and	O
+hydrophobic	O
+residues	O
+.	O
+
+Several	O
+,	O
+but	O
+not	O
+all	O
+,	O
+of	O
+these	O
+interactions	O
+were	O
+predicted	O
+previously	O
+by	O
+homology	B-experimental_method
+modelling	I-experimental_method
+combined	O
+with	O
+ligand	B-experimental_method
+docking	I-experimental_method
+in	O
+silico	O
+.	O
+
+Subsequently	O
+,	O
+we	O
+established	O
+the	O
+functional	O
+importance	O
+of	O
+His7	B-residue_name_number
+,	O
+Ser9	B-residue_name_number
+,	O
+Asn11	B-residue_name_number
+and	O
+Phe25	B-residue_name_number
+in	O
+the	O
+in	O
+vitro	O
+response	O
+of	O
+meningococcus	B-taxonomy_domain
+to	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+via	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+.	O
+
+More	O
+unexpectedly	O
+,	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+revealed	O
+that	O
+only	O
+one	O
+molecule	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+was	O
+bound	B-protein_state
+per	O
+NadR	B-protein
+dimer	B-oligomeric_state
+.	O
+
+We	O
+confirmed	O
+this	O
+stoichiometry	O
+in	O
+solution	O
+using	O
+SPR	B-experimental_method
+methods	O
+.	O
+
+We	O
+also	O
+used	O
+heteronuclear	B-experimental_method
+NMR	I-experimental_method
+spectroscopy	I-experimental_method
+to	O
+detect	O
+substantial	O
+conformational	O
+changes	O
+of	O
+NadR	B-protein
+occurring	O
+in	O
+solution	O
+upon	O
+addition	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+Moreover	O
+,	O
+NMR	B-experimental_method
+spectra	B-evidence
+at	O
+10	O
+°	O
+C	O
+suggested	O
+the	O
+existence	O
+of	O
+two	O
+distinct	O
+conformations	O
+of	O
+NadR	B-protein
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+ligand	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+More	O
+powerfully	O
+,	O
+our	O
+unique	O
+crystallographic	B-evidence
+observation	I-evidence
+of	O
+this	O
+‘	O
+occupied	B-protein_state
+vs	O
+unoccupied	B-protein_state
+site	O
+’	O
+asymmetry	O
+in	O
+the	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+interaction	O
+is	O
+,	O
+to	O
+our	O
+knowledge	O
+,	O
+the	O
+first	O
+example	O
+reported	O
+for	O
+a	O
+MarR	B-protein_type
+family	O
+protein	O
+.	O
+
+Structural	B-experimental_method
+analyses	I-experimental_method
+suggested	O
+that	O
+‘	O
+inward	B-protein_state
+’	O
+side	O
+chain	O
+positions	O
+of	O
+Met22	B-residue_name_number
+,	O
+Phe25	B-residue_name_number
+and	O
+especially	O
+Arg43	B-residue_name_number
+precluded	O
+binding	O
+of	O
+a	O
+second	O
+ligand	O
+molecule	O
+.	O
+
+Such	O
+a	O
+mechanism	O
+indicates	O
+negative	O
+cooperativity	O
+,	O
+which	O
+may	O
+enhance	O
+the	O
+ligand	O
+-	O
+responsiveness	O
+of	O
+NadR	B-protein
+.	O
+
+Comparisons	O
+of	O
+the	O
+NadR	B-complex_assembly
+/	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+complex	O
+with	O
+available	O
+MarR	B-protein_type
+family	O
+/	O
+salicylate	B-chemical
+complexes	O
+revealed	O
+that	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+has	O
+a	O
+previously	O
+unobserved	O
+binding	O
+mode	O
+.	O
+
+Briefly	O
+,	O
+in	O
+the	O
+M	B-species
+.	I-species
+thermoautotrophicum	I-species
+MTH313	B-protein
+dimer	B-oligomeric_state
+,	O
+one	O
+molecule	O
+of	O
+salicylate	B-chemical
+binds	O
+in	O
+the	O
+pocket	B-site
+of	O
+each	O
+monomer	B-oligomeric_state
+,	O
+though	O
+with	O
+two	O
+rather	O
+different	O
+positions	O
+and	O
+orientations	O
+,	O
+only	O
+one	O
+of	O
+which	O
+(	O
+site	B-site
+-	I-site
+1	I-site
+)	O
+is	O
+thought	O
+to	O
+be	O
+biologically	O
+relevant	O
+(	O
+Fig	O
+10A	O
+).	O
+
+In	O
+the	O
+S	B-species
+.	I-species
+tokodaii	I-species
+protein	O
+ST1710	B-protein
+,	O
+salicylate	B-chemical
+binds	O
+to	O
+the	O
+same	O
+position	O
+in	O
+each	O
+monomer	B-oligomeric_state
+of	O
+the	O
+dimer	B-oligomeric_state
+,	O
+in	O
+a	O
+site	O
+equivalent	O
+to	O
+the	O
+putative	O
+biologically	O
+relevant	O
+site	O
+of	O
+MTH313	B-protein
+(	O
+Fig	O
+10B	O
+).	O
+
+Unlike	O
+other	O
+MarR	B-protein_type
+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	B-chemical
+-	I-chemical
+HPA	I-chemical
+bound	B-protein_state
+to	I-protein_state
+NadR	B-protein
+,	O
+suggesting	O
+a	O
+more	O
+specific	O
+and	O
+less	O
+promiscuous	O
+interaction	O
+.	O
+
+In	O
+NadR	B-protein
+,	O
+the	O
+single	O
+molecule	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+binds	O
+in	O
+a	O
+position	O
+distinctly	O
+different	O
+from	O
+the	O
+salicylate	B-site
+binding	I-site
+site	I-site
+:	O
+translated	O
+by	O
+>	O
+10	O
+Å	O
+and	O
+with	O
+a	O
+180	O
+°	O
+inverted	O
+orientation	O
+(	O
+Fig	O
+10C	O
+).	O
+
+NadR	B-protein
+shows	O
+a	O
+ligand	B-site
+binding	I-site
+site	I-site
+distinct	O
+from	O
+other	O
+MarR	B-protein_type
+homologues	O
+.	O
+
+(	O
+A	O
+)	O
+A	O
+structural	B-experimental_method
+alignment	I-experimental_method
+of	O
+MTH313	B-protein
+chains	B-structure_element
+A	I-structure_element
+and	I-structure_element
+B	I-structure_element
+shows	O
+that	O
+salicylate	B-chemical
+is	O
+bound	B-protein_state
+in	O
+distinct	O
+locations	O
+in	O
+each	O
+monomer	B-oligomeric_state
+;	O
+site	B-site
+-	I-site
+1	I-site
+(	O
+thought	O
+to	O
+be	O
+the	O
+biologically	O
+relevant	O
+site	O
+)	O
+and	O
+site	B-site
+-	I-site
+2	I-site
+differ	O
+by	O
+~	O
+7Å	O
+(	O
+indicated	O
+by	O
+black	O
+dotted	O
+line	O
+)	O
+and	O
+also	O
+by	O
+ligand	O
+orientation	O
+.	O
+
+(	O
+B	O
+)	O
+A	O
+structural	B-experimental_method
+alignment	I-experimental_method
+of	O
+MTH313	B-protein
+chain	B-structure_element
+A	I-structure_element
+and	O
+ST1710	B-protein
+(	O
+pink	O
+)	O
+(	O
+Cα	O
+rmsd	B-evidence
+2	O
+.	O
+3Å	O
+),	O
+shows	O
+that	O
+they	O
+bind	O
+salicylate	B-chemical
+in	O
+equivalent	O
+sites	O
+(	O
+differing	O
+by	O
+only	O
+~	O
+3Å	O
+)	O
+and	O
+with	O
+the	O
+same	O
+orientation	O
+.	O
+
+(	O
+C	O
+)	O
+Addition	O
+of	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+chain	B-structure_element
+B	I-structure_element
+,	O
+blue	O
+)	O
+to	O
+the	O
+alignment	B-experimental_method
+reveals	O
+that	O
+bound	B-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+differs	O
+in	O
+position	O
+by	O
+>	O
+10	O
+Å	O
+compared	O
+to	O
+salicylate	B-chemical
+,	O
+and	O
+adopts	O
+a	O
+novel	O
+orientation	O
+.	O
+
+Interestingly	O
+,	O
+a	O
+crystal	B-evidence
+structure	I-evidence
+was	O
+previously	O
+reported	O
+for	O
+a	O
+functionally	O
+-	O
+uncharacterized	O
+meningococcal	B-taxonomy_domain
+homologue	O
+of	O
+NadR	B-protein
+,	O
+termed	O
+NMB1585	B-protein
+,	O
+which	O
+shares	O
+16	O
+%	O
+sequence	O
+identity	O
+with	O
+NadR	B-protein
+.	O
+The	O
+two	O
+structures	B-evidence
+can	O
+be	O
+closely	O
+aligned	O
+(	O
+rmsd	B-evidence
+2	O
+.	O
+3	O
+Å	O
+),	O
+but	O
+NMB1585	B-protein
+appears	O
+unsuited	O
+for	O
+binding	O
+HPAs	B-chemical
+,	O
+since	O
+its	O
+corresponding	O
+‘	B-site
+pocket	I-site
+’	O
+region	O
+is	O
+occupied	O
+by	O
+several	O
+bulky	O
+hydrophobic	O
+side	O
+chains	O
+.	O
+
+It	O
+can	O
+be	O
+speculated	O
+that	O
+MarR	B-protein_type
+family	O
+members	O
+have	O
+evolved	O
+separately	O
+to	O
+engage	O
+distinct	O
+signaling	O
+molecules	O
+,	O
+thus	O
+enabling	O
+bacteria	B-taxonomy_domain
+to	O
+use	O
+the	O
+overall	O
+conserved	O
+MarR	B-protein_type
+scaffold	O
+to	O
+adapt	O
+and	O
+respond	O
+to	O
+diverse	O
+changing	O
+environmental	O
+stimuli	O
+experienced	O
+in	O
+their	O
+natural	O
+niches	O
+.	O
+
+Alternatively	O
+,	O
+it	O
+is	O
+possible	O
+that	O
+other	O
+MarR	B-protein_type
+homologues	O
+(	O
+e	O
+.	O
+g	O
+.	O
+NMB1585	B-protein
+)	O
+may	O
+have	O
+no	O
+extant	O
+functional	O
+binding	B-site
+pocket	I-site
+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	B-chemical
+-	O
+binding	O
+regulatory	O
+proteins	O
+.	O
+
+The	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+crystal	B-evidence
+structures	I-evidence
+revealed	O
+two	O
+dimers	B-oligomeric_state
+with	O
+slightly	O
+different	O
+conformations	O
+,	O
+most	O
+divergent	O
+in	O
+the	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+.	O
+
+It	O
+is	O
+not	O
+unusual	O
+for	O
+a	O
+crystal	B-evidence
+structure	I-evidence
+to	O
+reveal	O
+multiple	O
+copies	O
+of	O
+the	O
+same	O
+protein	O
+in	O
+very	O
+slightly	O
+different	O
+conformations	O
+,	O
+which	O
+are	O
+likely	O
+representative	O
+of	O
+the	O
+lowest	O
+-	O
+energy	O
+conformations	O
+sampled	O
+by	O
+the	O
+dynamic	O
+ensemble	O
+of	O
+molecular	O
+states	O
+occurring	O
+in	O
+solution	O
+,	O
+and	O
+which	O
+likely	O
+have	O
+only	O
+small	O
+energetic	O
+differences	O
+,	O
+as	O
+described	O
+previously	O
+for	O
+MexR	B-protein
+(	O
+a	O
+MarR	B-protein_type
+protein	O
+)	O
+or	O
+more	O
+recently	O
+for	O
+the	O
+solute	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+FhuD2	B-protein
+.	O
+
+Further	O
+,	O
+the	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+structure	B-evidence
+was	O
+overall	O
+more	O
+different	O
+from	O
+the	O
+two	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+structures	B-evidence
+(	O
+rmsd	B-evidence
+values	O
+~	O
+1	O
+.	O
+3Å	O
+),	O
+suggesting	O
+that	O
+the	O
+ligand	O
+selected	O
+and	O
+stabilized	O
+yet	O
+another	O
+conformation	O
+of	O
+NadR	B-protein
+.	O
+These	O
+observations	O
+suggest	O
+that	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+and	O
+potentially	O
+other	O
+similar	O
+ligands	O
+,	O
+can	O
+shift	O
+the	O
+molecular	O
+equilibrium	O
+,	O
+changing	O
+the	O
+energy	O
+barriers	O
+that	O
+separate	O
+active	B-protein_state
+and	O
+inactive	B-protein_state
+states	O
+,	O
+and	O
+stabilizing	O
+the	O
+specific	O
+conformation	O
+of	O
+NadR	B-protein
+poorly	O
+suited	O
+to	O
+bind	O
+DNA	B-chemical
+.	O
+
+Comparisons	O
+of	O
+the	O
+apo	B-protein_state
+-	O
+and	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+structures	B-evidence
+revealed	O
+that	O
+the	O
+largest	O
+differences	O
+occurred	O
+in	O
+the	O
+DNA	B-chemical
+-	O
+binding	O
+helix	B-structure_element
+α4	B-structure_element
+.	O
+
+The	O
+shift	O
+of	O
+helix	B-structure_element
+α4	B-structure_element
+in	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+was	O
+also	O
+accompanied	O
+by	O
+rearrangements	O
+at	O
+the	O
+dimer	B-site
+interface	I-site
+,	O
+involving	O
+helices	B-structure_element
+α1	B-structure_element
+,	O
+α5	B-structure_element
+,	O
+and	O
+α6	B-structure_element
+,	O
+and	O
+this	O
+holo	B-protein_state
+-	O
+form	O
+appeared	O
+poorly	O
+suited	O
+for	O
+DNA	B-chemical
+-	O
+binding	O
+when	O
+compared	O
+with	O
+the	O
+known	O
+OhrR	B-complex_assembly
+:	I-complex_assembly
+ohrA	I-complex_assembly
+complex	O
+.	O
+
+While	O
+some	O
+flexibility	O
+of	O
+helix	B-structure_element
+α4	B-structure_element
+was	O
+also	O
+observed	O
+in	O
+the	O
+two	O
+apo	B-protein_state
+-	O
+structures	B-evidence
+,	O
+concomitant	O
+changes	O
+in	O
+the	O
+dimer	B-site
+interfaces	I-site
+were	O
+not	O
+observed	O
+,	O
+possibly	O
+due	O
+to	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ligand	I-protein_state
+.	O
+
+One	O
+of	O
+the	O
+two	O
+conformations	O
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+appeared	O
+ideally	O
+suited	O
+for	O
+DNA	B-chemical
+-	O
+binding	O
+.	O
+
+Overall	O
+,	O
+these	O
+analyses	O
+suggest	O
+that	O
+the	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+dimer	B-oligomeric_state
+has	O
+a	O
+pre	O
+-	O
+existing	O
+equilibrium	O
+that	O
+samples	O
+a	O
+variety	O
+of	O
+conformations	O
+,	O
+some	O
+of	O
+which	O
+are	O
+compatible	O
+with	O
+DNA	B-chemical
+binding	O
+.	O
+
+The	O
+noted	O
+flexibility	O
+may	O
+also	O
+explain	O
+how	O
+NadR	B-protein
+can	O
+adapt	O
+to	O
+bind	O
+various	O
+DNA	B-chemical
+target	O
+sequences	O
+with	O
+slightly	O
+different	O
+structural	O
+features	O
+.	O
+
+Subsequently	O
+,	O
+upon	O
+ligand	O
+binding	O
+,	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+adopts	O
+a	O
+structure	O
+less	O
+suited	O
+for	O
+DNA	B-chemical
+-	O
+binding	O
+and	O
+this	O
+conformation	O
+is	O
+selected	O
+and	O
+stabilized	O
+by	O
+a	O
+network	O
+of	O
+protein	O
+-	O
+ligand	O
+interactions	O
+and	O
+concomitant	O
+rearrangements	O
+at	O
+the	O
+NadR	B-protein
+holo	B-protein_state
+dimer	B-site
+interface	I-site
+.	O
+
+In	O
+an	O
+alternative	O
+and	O
+less	O
+extensive	O
+manner	O
+,	O
+the	O
+binding	O
+of	O
+two	O
+salicylate	B-chemical
+molecules	O
+to	O
+the	O
+M	B-species
+.	I-species
+thermoautotrophicum	I-species
+protein	O
+MTH313	B-protein
+appeared	O
+to	O
+induce	O
+large	O
+changes	O
+in	O
+the	O
+wHTH	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+was	O
+associated	O
+with	O
+reduced	O
+DNA	O
+-	O
+binding	O
+activity	O
+.	O
+
+Here	O
+we	O
+have	O
+presented	O
+two	O
+new	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+transcription	B-protein_type
+factor	I-protein_type
+,	O
+NadR	B-protein
+,	O
+which	O
+regulates	O
+expression	O
+of	O
+the	O
+meningococcal	B-taxonomy_domain
+surface	O
+protein	O
+,	O
+virulence	O
+factor	O
+and	O
+vaccine	O
+antigen	O
+NadA	B-protein
+.	O
+Detailed	O
+structural	B-experimental_method
+analyses	I-experimental_method
+provided	O
+a	O
+molecular	O
+explanation	O
+for	O
+the	O
+ligand	O
+-	O
+responsive	O
+regulation	O
+by	O
+NadR	B-protein
+on	O
+the	O
+majority	O
+of	O
+the	O
+promoters	O
+of	O
+meningococcal	B-taxonomy_domain
+genes	O
+regulated	O
+by	O
+NadR	B-protein
+,	O
+including	O
+nadA	B-gene
+.	O
+Intriguingly	O
+,	O
+NadR	B-protein
+exhibits	O
+a	O
+reversed	O
+regulatory	O
+mechanism	O
+on	O
+a	O
+second	O
+class	O
+of	O
+promoters	O
+,	O
+including	O
+mafA	B-gene
+of	O
+the	O
+multiple	O
+adhesin	O
+family	O
+–	O
+i	O
+.	O
+e	O
+.	O
+NadR	B-protein
+represses	O
+these	O
+genes	O
+in	O
+the	O
+presence	O
+but	O
+not	O
+absence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+The	O
+latter	O
+may	O
+influence	O
+the	O
+surface	O
+abundance	O
+or	O
+secretion	O
+of	O
+maf	O
+proteins	O
+,	O
+an	O
+emerging	O
+class	O
+of	O
+highly	B-protein_state
+conserved	I-protein_state
+meningococcal	B-taxonomy_domain
+putative	O
+adhesins	O
+and	O
+toxins	O
+with	O
+many	O
+important	O
+roles	O
+.	O
+
+Further	O
+work	O
+is	O
+required	O
+to	O
+investigate	O
+how	O
+the	O
+two	O
+different	O
+promoter	O
+types	O
+influence	O
+the	O
+ligand	O
+-	O
+responsiveness	O
+of	O
+NadR	B-protein
+during	O
+bacterial	B-taxonomy_domain
+infection	O
+and	O
+may	O
+provide	O
+insights	O
+into	O
+the	O
+regulatory	O
+mechanisms	O
+occurring	O
+during	O
+these	O
+host	O
+-	O
+pathogen	O
+interactions	O
+.	O
+
+Ultimately	O
+,	O
+knowledge	O
+of	O
+the	O
+ligand	O
+-	O
+dependent	O
+activity	O
+of	O
+NadR	B-protein
+will	O
+continue	O
+to	O
+deepen	O
+our	O
+understanding	O
+of	O
+nadA	B-gene
+expression	O
+levels	O
+,	O
+which	O
+influence	O
+meningococcal	B-taxonomy_domain
+pathogenesis	O
+.	O
+
+Structure	O
+of	O
+an	O
+OhrR	O
+-	O
+ohrA	B-gene
+operator	O
+complex	O
+reveals	O
+the	O
+DNA	O
+binding	O
+mechanism	O
+of	O
+the	O
+MarR	O
+family	O
+
+The	O
+structure	O
+of	O
+NMB1585	B-protein
+,	O
+a	O
+MarR	O
+-	O
+family	O
+regulator	O
+from	O
+Neisseria	O
+meningitidis	O
+
+The	O
+Structural	O
+Basis	O
+of	O
+Coenzyme	B-chemical
+A	I-chemical
+Recycling	O
+in	O
+a	O
+Bacterial	B-taxonomy_domain
+Organelle	O
+
+Bacterial	B-taxonomy_domain
+Microcompartments	B-complex_assembly
+(	O
+BMCs	B-complex_assembly
+)	O
+are	O
+proteinaceous	O
+organelles	O
+that	O
+encapsulate	O
+critical	O
+segments	O
+of	O
+autotrophic	O
+and	O
+heterotrophic	O
+metabolic	O
+pathways	O
+;	O
+they	O
+are	O
+functionally	O
+diverse	O
+and	O
+are	O
+found	O
+across	O
+23	O
+different	O
+phyla	O
+.	O
+
+The	O
+majority	O
+of	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+(	O
+metabolosomes	B-complex_assembly
+)	O
+compartmentalize	O
+a	O
+common	O
+core	O
+of	O
+enzymes	O
+to	O
+metabolize	O
+compounds	O
+via	O
+a	O
+toxic	O
+and	O
+/	O
+or	O
+volatile	O
+aldehyde	B-chemical
+intermediate	O
+.	O
+
+The	O
+core	O
+enzyme	O
+phosphotransacylase	B-protein_type
+(	O
+PTAC	B-protein_type
+)	O
+recycles	O
+Coenzyme	B-chemical
+A	I-chemical
+and	O
+generates	O
+an	O
+acyl	B-chemical
+phosphate	I-chemical
+that	O
+can	O
+serve	O
+as	O
+an	O
+energy	O
+source	O
+.	O
+
+The	O
+PTAC	B-protein_type
+predominantly	O
+associated	O
+with	O
+metabolosomes	B-complex_assembly
+(	O
+PduL	B-protein_type
+)	O
+has	O
+no	O
+sequence	O
+homology	O
+to	O
+the	O
+PTAC	B-protein_type
+ubiquitous	O
+among	O
+fermentative	B-taxonomy_domain
+bacteria	I-taxonomy_domain
+(	O
+Pta	B-protein_type
+).	O
+
+Here	O
+,	O
+we	O
+report	O
+two	O
+high	O
+-	O
+resolution	O
+PduL	B-protein_type
+crystal	B-evidence
+structures	I-evidence
+with	B-protein_state
+bound	I-protein_state
+substrates	I-protein_state
+.	O
+
+The	O
+PduL	B-protein_type
+fold	B-structure_element
+is	O
+unrelated	O
+to	O
+that	B-structure_element
+of	O
+Pta	B-protein_type
+;	O
+it	O
+contains	O
+a	O
+dimetal	B-site
+active	I-site
+site	I-site
+involved	O
+in	O
+a	O
+catalytic	O
+mechanism	O
+distinct	O
+from	O
+that	O
+of	O
+the	O
+housekeeping	B-protein_state
+PTAC	B-protein_type
+.	O
+
+Accordingly	O
+,	O
+PduL	B-protein_type
+and	O
+Pta	B-protein_type
+exemplify	O
+functional	O
+,	O
+but	O
+not	O
+structural	O
+,	O
+convergent	O
+evolution	O
+.	O
+
+The	O
+PduL	B-protein_type
+structure	B-evidence
+,	O
+in	O
+the	O
+context	O
+of	O
+the	O
+catalytic	O
+core	O
+,	O
+completes	O
+our	O
+understanding	O
+of	O
+the	O
+structural	O
+basis	O
+of	O
+cofactor	O
+recycling	O
+in	O
+the	O
+metabolosome	B-complex_assembly
+lumen	O
+.	O
+
+This	O
+study	O
+describes	O
+the	O
+structure	B-evidence
+of	O
+a	O
+novel	O
+phosphotransacylase	B-protein_type
+enzyme	O
+that	O
+facilitates	O
+the	O
+recycling	O
+of	O
+the	O
+essential	O
+cofactor	O
+acetyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+within	O
+a	O
+bacterial	B-taxonomy_domain
+organelle	O
+and	O
+discusses	O
+the	O
+properties	O
+of	O
+the	O
+enzyme	O
+'	O
+s	O
+active	B-site
+site	I-site
+and	O
+how	O
+it	O
+is	O
+packaged	O
+into	O
+the	O
+organelle	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	B-chemical
+and	O
+Acetyl	B-chemical
+~	I-chemical
+CoA	I-chemical
+)	O
+are	O
+critical	O
+for	O
+both	O
+catabolic	O
+and	O
+anabolic	O
+processes	O
+.	O
+
+The	O
+phosphotransacylase	B-protein_type
+(	O
+Pta	B-protein_type
+)	O
+enzyme	O
+catalyzes	O
+the	O
+conversion	O
+between	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+and	O
+acyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+.	O
+
+This	O
+reaction	O
+directly	O
+links	O
+an	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+with	O
+ATP	B-chemical
+generation	O
+via	O
+substrate	O
+-	O
+level	O
+phosphorylation	O
+,	O
+producing	O
+short	B-chemical
+-	I-chemical
+chain	I-chemical
+fatty	I-chemical
+acids	I-chemical
+(	O
+e	O
+.	O
+g	O
+.,	O
+acetate	B-chemical
+),	O
+and	O
+also	O
+provides	O
+a	O
+path	O
+for	O
+short	B-chemical
+-	I-chemical
+chain	I-chemical
+fatty	I-chemical
+acids	I-chemical
+to	O
+enter	O
+central	O
+metabolism	O
+.	O
+
+Due	O
+to	O
+this	O
+key	O
+function	O
+,	O
+Pta	O
+is	O
+conserved	B-protein_state
+across	O
+the	O
+bacterial	B-taxonomy_domain
+kingdom	I-taxonomy_domain
+.	O
+
+Recently	O
+,	O
+a	O
+new	O
+type	O
+of	O
+phosphotransacylase	B-protein_type
+was	O
+described	O
+that	O
+shares	O
+no	O
+evolutionary	O
+relation	O
+to	O
+Pta	B-protein_type
+.	O
+
+This	O
+enzyme	O
+,	O
+PduL	B-protein_type
+,	O
+is	O
+exclusively	B-protein_state
+associated	O
+with	O
+organelles	O
+called	O
+bacterial	B-taxonomy_domain
+microcompartments	B-complex_assembly
+,	O
+which	O
+are	O
+used	O
+to	O
+catabolize	O
+various	O
+compounds	O
+.	O
+
+Not	O
+only	O
+does	O
+PduL	B-protein_type
+facilitate	O
+substrate	O
+level	O
+phosphorylation	O
+,	O
+but	O
+it	O
+also	O
+is	O
+critical	O
+for	O
+cofactor	O
+recycling	O
+within	O
+,	O
+and	O
+product	O
+efflux	O
+from	O
+,	O
+the	O
+organelle	O
+.	O
+
+We	O
+solved	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+this	O
+convergent	B-protein_state
+phosphotransacylase	B-protein_type
+and	O
+show	O
+that	O
+it	O
+is	O
+completely	O
+structurally	O
+different	O
+from	O
+Pta	B-protein_type
+,	O
+including	O
+its	O
+active	B-site
+site	I-site
+architecture	O
+.	O
+
+Bacterial	B-taxonomy_domain
+Microcompartments	B-complex_assembly
+(	O
+BMCs	B-complex_assembly
+)	O
+are	O
+organelles	O
+that	O
+encapsulate	O
+enzymes	O
+for	O
+sequential	O
+biochemical	O
+reactions	O
+within	O
+a	O
+protein	O
+shell	B-structure_element
+.	O
+
+The	O
+shell	B-structure_element
+is	O
+typically	O
+composed	O
+of	O
+three	O
+types	O
+of	O
+protein	O
+subunits	O
+,	O
+which	O
+form	O
+either	O
+hexagonal	B-protein_state
+(	O
+BMC	B-complex_assembly
+-	I-complex_assembly
+H	I-complex_assembly
+and	O
+BMC	B-complex_assembly
+-	I-complex_assembly
+T	I-complex_assembly
+)	O
+or	O
+pentagonal	B-protein_state
+(	O
+BMC	B-complex_assembly
+-	I-complex_assembly
+P	I-complex_assembly
+)	O
+tiles	O
+that	O
+assemble	O
+into	O
+a	O
+polyhedral	B-protein_state
+shell	B-structure_element
+.	O
+
+The	O
+facets	O
+of	O
+the	O
+shell	B-structure_element
+are	O
+composed	O
+primarily	O
+of	O
+hexamers	B-oligomeric_state
+that	O
+are	O
+typically	O
+perforated	O
+by	O
+pores	B-site
+lined	O
+with	O
+highly	B-protein_state
+conserved	I-protein_state
+,	O
+polar	B-protein_state
+residues	B-structure_element
+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	B-structure_element
+.	O
+
+The	O
+vitamin	B-complex_assembly
+B12	I-complex_assembly
+-	I-complex_assembly
+dependent	I-complex_assembly
+propanediol	I-complex_assembly
+-	I-complex_assembly
+utilizing	I-complex_assembly
+(	I-complex_assembly
+PDU	I-complex_assembly
+)	I-complex_assembly
+BMC	I-complex_assembly
+was	O
+one	O
+of	O
+the	O
+first	O
+functionally	O
+characterized	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+;	O
+subsequently	O
+,	O
+other	O
+types	O
+have	O
+been	O
+implicated	O
+in	O
+the	O
+degradation	O
+of	O
+ethanolamine	B-chemical
+,	O
+choline	B-chemical
+,	O
+fucose	B-chemical
+,	O
+rhamnose	B-chemical
+,	O
+and	O
+ethanol	B-chemical
+,	O
+all	O
+of	O
+which	O
+produce	O
+different	O
+aldehyde	B-chemical
+intermediates	O
+(	O
+Table	O
+1	O
+).	O
+
+More	O
+recently	O
+,	O
+bioinformatic	B-experimental_method
+studies	I-experimental_method
+have	O
+demonstrated	O
+the	O
+widespread	O
+distribution	O
+of	O
+BMCs	B-complex_assembly
+among	O
+diverse	O
+bacterial	B-taxonomy_domain
+phyla	I-taxonomy_domain
+and	O
+grouped	O
+them	O
+into	O
+23	O
+different	O
+functional	O
+types	O
+.	O
+
+The	O
+reactions	O
+carried	O
+out	O
+in	O
+the	O
+majority	O
+of	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+(	O
+also	O
+known	O
+as	O
+metabolosomes	B-complex_assembly
+)	O
+fit	O
+a	O
+generalized	O
+biochemical	O
+paradigm	O
+for	O
+the	O
+oxidation	O
+of	O
+aldehydes	B-chemical
+(	O
+Fig	O
+1	O
+).	O
+
+This	O
+involves	O
+a	O
+BMC	B-complex_assembly
+-	O
+encapsulated	O
+signature	O
+enzyme	O
+that	O
+generates	O
+a	O
+toxic	O
+and	O
+/	O
+or	O
+volatile	O
+aldehyde	B-chemical
+that	O
+the	O
+BMC	B-complex_assembly
+shell	B-structure_element
+sequesters	O
+from	O
+the	O
+cytosol	O
+.	O
+
+The	O
+aldehyde	B-chemical
+is	O
+subsequently	O
+converted	O
+into	O
+an	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+by	O
+aldehyde	B-protein_type
+dehydrogenase	I-protein_type
+,	O
+which	O
+uses	O
+NAD	B-chemical
++	I-chemical
+and	O
+CoA	B-chemical
+as	O
+cofactors	O
+.	O
+
+These	O
+two	O
+cofactors	O
+are	O
+relatively	O
+large	O
+,	O
+and	O
+their	O
+diffusion	O
+across	O
+the	O
+protein	B-structure_element
+shell	I-structure_element
+is	O
+thought	O
+to	O
+be	O
+restricted	O
+,	O
+necessitating	O
+their	O
+regeneration	O
+within	O
+the	O
+BMC	B-complex_assembly
+lumen	O
+.	O
+
+NAD	B-chemical
++	I-chemical
+is	O
+recycled	O
+via	O
+alcohol	B-protein_type
+dehydrogenase	I-protein_type
+,	O
+and	O
+CoA	B-chemical
+is	O
+recycled	O
+via	O
+phosphotransacetylase	B-protein_type
+(	O
+PTAC	B-protein_type
+)	O
+(	O
+Fig	O
+1	O
+).	O
+
+The	O
+final	O
+product	O
+of	O
+the	O
+BMC	B-complex_assembly
+,	O
+an	O
+acyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+,	O
+can	O
+then	O
+be	O
+used	O
+to	O
+generate	O
+ATP	B-chemical
+via	O
+acyl	B-protein_type
+kinase	I-protein_type
+,	O
+or	O
+revert	O
+back	O
+to	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+by	O
+Pta	B-protein_type
+for	O
+biosynthesis	O
+.	O
+
+Collectively	O
+,	O
+the	O
+aldehyde	B-protein_type
+and	I-protein_type
+alcohol	I-protein_type
+dehydrogenases	I-protein_type
+,	O
+as	O
+well	O
+as	O
+the	O
+PTAC	B-protein_type
+,	O
+constitute	O
+the	O
+common	O
+metabolosome	B-complex_assembly
+core	O
+.	O
+
+General	O
+biochemical	O
+model	O
+of	O
+aldehyde	B-protein_state
+-	I-protein_state
+degrading	I-protein_state
+BMCs	B-complex_assembly
+(	O
+metabolosomes	B-complex_assembly
+)	O
+illustrating	O
+the	O
+common	O
+metabolosome	B-complex_assembly
+core	O
+enzymes	O
+and	O
+reactions	O
+.	O
+
+Substrates	O
+and	O
+cofactors	O
+involving	O
+the	O
+PTAC	B-protein_type
+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
+
+Characterized	O
+and	O
+predicted	O
+catabolic	B-protein_state
+BMC	B-complex_assembly
+(	O
+metabolosome	B-complex_assembly
+)	O
+types	O
+that	O
+represent	O
+the	O
+aldehyde	B-chemical
+-	O
+degrading	O
+paradigm	O
+(	O
+for	O
+definition	O
+of	O
+types	O
+see	O
+Kerfeld	O
+and	O
+Erbilgin	O
+).	O
+
+Name	O
+PTAC	B-protein_type
+Type	O
+Sequestered	O
+Aldehyde	B-chemical
+PDU	B-complex_assembly
+*	O
+PduL	B-protein_type
+propionaldehyde	B-chemical
+EUT1	B-complex_assembly
+PTA_PTB	B-protein_type
+acetaldehyde	B-chemical
+EUT2	B-complex_assembly
+PduL	B-protein_type
+acetaldehyde	B-chemical
+ETU	B-complex_assembly
+None	O
+acetaldehyde	B-chemical
+GRM1	B-complex_assembly
+/	I-complex_assembly
+CUT	I-complex_assembly
+PduL	B-protein_type
+acetaldehyde	B-chemical
+GRM2	B-complex_assembly
+PduL	B-protein_type
+acetaldehyde	B-chemical
+GRM3	B-complex_assembly
+*,	I-complex_assembly
+4	I-complex_assembly
+PduL	B-protein_type
+propionaldehyde	B-chemical
+GRM5	B-complex_assembly
+/	I-complex_assembly
+GRP	I-complex_assembly
+PduL	B-protein_type
+propionaldehyde	B-chemical
+PVM	B-complex_assembly
+*	O
+PduL	B-protein_type
+lactaldehyde	B-chemical
+RMM1	B-complex_assembly
+,	I-complex_assembly
+2	I-complex_assembly
+None	O
+unknown	O
+SPU	B-complex_assembly
+PduL	B-protein_type
+unknown	O
+
+*	O
+PduL	B-protein_type
+from	O
+these	O
+functional	O
+types	O
+of	O
+metabolosomes	B-complex_assembly
+were	O
+purified	O
+in	O
+this	O
+study	O
+.	O
+
+The	O
+activities	O
+of	O
+core	O
+enzymes	O
+are	O
+not	O
+confined	O
+to	O
+BMC	B-complex_assembly
+-	O
+associated	O
+functions	O
+:	O
+aldehyde	B-protein_type
+and	I-protein_type
+alcohol	I-protein_type
+dehydrogenases	I-protein_type
+are	O
+utilized	O
+in	O
+diverse	O
+metabolic	O
+reactions	O
+,	O
+and	O
+PTAC	B-protein_type
+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
+
+The	O
+concerted	O
+functioning	O
+of	O
+a	O
+PTAC	B-protein_type
+and	O
+an	O
+acetate	B-protein_type
+kinase	I-protein_type
+(	O
+Ack	B-protein_type
+)	O
+is	O
+crucial	O
+for	O
+ATP	B-chemical
+generation	O
+in	O
+the	O
+fermentation	O
+of	O
+pyruvate	B-chemical
+to	O
+acetate	B-chemical
+(	O
+see	O
+Reactions	O
+1	O
+and	O
+2	O
+).	O
+
+Both	O
+enzymes	O
+are	O
+,	O
+however	O
+,	O
+not	O
+restricted	O
+to	O
+fermentative	B-taxonomy_domain
+organisms	I-taxonomy_domain
+.	O
+
+They	O
+can	O
+also	O
+work	O
+in	O
+the	O
+reverse	O
+direction	O
+to	O
+activate	O
+acetate	B-chemical
+to	O
+the	O
+CoA	B-chemical
+-	I-chemical
+thioester	I-chemical
+.	O
+
+This	O
+occurs	O
+,	O
+for	O
+example	O
+,	O
+during	O
+acetoclastic	O
+methanogenesis	O
+in	O
+the	O
+archaeal	B-taxonomy_domain
+Methanosarcina	B-taxonomy_domain
+species	I-taxonomy_domain
+.	O
+
+Reaction	O
+1	O
+:	O
+acetyl	B-chemical
+-	I-chemical
+S	I-chemical
+-	I-chemical
+CoA	I-chemical
++	O
+Pi	B-chemical
+←→	O
+acetyl	B-chemical
+phosphate	I-chemical
++	O
+CoA	B-chemical
+-	I-chemical
+SH	I-chemical
+(	O
+PTAC	B-protein_type
+)	O
+
+Reaction	O
+2	O
+:	O
+acetyl	B-chemical
+phosphate	I-chemical
++	O
+ADP	B-chemical
+←→	O
+acetate	B-chemical
++	O
+ATP	B-chemical
+(	O
+Ack	B-protein_type
+)	O
+
+The	O
+canonical	O
+PTAC	B-protein_type
+,	O
+Pta	B-protein_type
+,	O
+is	O
+an	O
+ancient	O
+enzyme	O
+found	O
+in	O
+some	O
+eukaryotes	B-taxonomy_domain
+and	O
+archaea	B-taxonomy_domain
+,	O
+and	O
+widespread	O
+among	O
+the	O
+bacteria	B-taxonomy_domain
+;	O
+90	O
+%	O
+of	O
+the	O
+bacterial	B-taxonomy_domain
+genomes	O
+in	O
+the	O
+Integrated	O
+Microbial	O
+Genomes	O
+database	O
+contain	O
+a	O
+gene	O
+encoding	O
+the	O
+PTA_PTB	B-protein_type
+phosphotransacylase	I-protein_type
+(	O
+Pfam	O
+domain	O
+PF01515	B-structure_element
+).	O
+
+Pta	B-protein_type
+has	O
+been	O
+extensively	O
+characterized	O
+due	O
+to	O
+its	O
+key	O
+role	O
+in	O
+fermentation	O
+.	O
+
+More	O
+recently	O
+,	O
+a	O
+second	O
+type	O
+of	O
+PTAC	B-protein_type
+without	O
+any	O
+sequence	O
+homology	O
+to	O
+Pta	B-protein_type
+was	O
+identified	O
+.	O
+
+This	O
+protein	O
+,	O
+PduL	B-protein_type
+(	O
+Pfam	O
+domain	O
+PF06130	B-structure_element
+),	O
+was	O
+shown	O
+to	O
+catalyze	O
+the	O
+conversion	O
+of	O
+propionyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+propionyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+and	O
+is	O
+associated	O
+with	O
+a	O
+BMC	B-complex_assembly
+involved	O
+in	O
+propanediol	O
+utilization	O
+,	O
+the	O
+PDU	B-complex_assembly
+BMC	I-complex_assembly
+.	O
+
+Both	O
+pduL	B-gene
+and	O
+pta	B-gene
+genes	O
+can	O
+be	O
+found	O
+in	O
+genetic	O
+loci	O
+of	O
+functionally	O
+distinct	O
+BMCs	B-complex_assembly
+,	O
+although	O
+the	O
+PduL	B-protein_type
+type	O
+is	O
+much	O
+more	O
+prevalent	O
+,	O
+being	O
+found	O
+in	O
+all	O
+but	O
+one	O
+type	O
+of	O
+metabolosome	B-gene
+locus	I-gene
+:	O
+EUT1	B-gene
+(	O
+Table	O
+1	O
+).	O
+
+Furthermore	O
+,	O
+in	O
+the	O
+Integrated	O
+Microbial	O
+Genomes	O
+Database	O
+,	O
+91	O
+%	O
+of	O
+genomes	O
+that	O
+encode	O
+PF06130	B-structure_element
+also	O
+encode	O
+genes	O
+for	O
+shell	O
+proteins	O
+.	O
+
+As	O
+a	O
+member	O
+of	O
+the	O
+core	O
+biochemical	O
+machinery	O
+of	O
+functionally	O
+diverse	O
+aldehyde	B-protein_state
+-	I-protein_state
+oxidizing	I-protein_state
+metabolosomes	B-complex_assembly
+,	O
+PduL	B-protein_type
+must	O
+have	O
+a	O
+certain	O
+level	O
+of	O
+substrate	O
+plasticity	O
+(	O
+see	O
+Table	O
+1	O
+)	O
+that	O
+is	O
+not	O
+required	O
+of	O
+Pta	B-protein_type
+,	O
+which	O
+has	O
+generally	O
+been	O
+observed	O
+to	O
+prefer	O
+acetyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+PduL	B-protein_type
+from	O
+the	O
+PDU	B-complex_assembly
+BMC	I-complex_assembly
+of	O
+Salmonella	B-species
+enterica	I-species
+favors	O
+propionyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+over	O
+acetyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+,	O
+and	O
+it	O
+is	O
+likely	O
+that	O
+PduL	B-protein_type
+orthologs	O
+in	O
+functionally	O
+diverse	O
+BMCs	B-complex_assembly
+would	O
+have	O
+substrate	O
+preferences	O
+for	O
+other	O
+CoA	B-chemical
+derivatives	O
+.	O
+
+Another	O
+distinctive	O
+feature	O
+of	O
+BMC	B-protein_state
+-	I-protein_state
+associated	I-protein_state
+PduL	B-protein_type
+homologs	O
+is	O
+an	O
+N	O
+-	O
+terminal	O
+encapsulation	B-structure_element
+peptide	I-structure_element
+(	O
+EP	B-structure_element
+)	O
+that	O
+is	O
+thought	O
+to	O
+“	O
+target	O
+”	O
+proteins	O
+for	O
+encapsulation	O
+by	O
+the	O
+BMC	B-complex_assembly
+shell	B-structure_element
+.	O
+
+EPs	B-structure_element
+are	O
+frequently	O
+found	O
+on	O
+BMC	B-protein_type
+-	I-protein_type
+associated	I-protein_type
+proteins	I-protein_type
+and	O
+have	O
+been	O
+shown	O
+to	O
+interact	O
+with	O
+shell	O
+proteins	O
+.	O
+
+EPs	B-structure_element
+have	O
+also	O
+been	O
+observed	O
+to	O
+cause	O
+proteins	O
+to	O
+aggregate	O
+,	O
+and	O
+this	O
+has	O
+recently	O
+been	O
+suggested	O
+to	O
+be	O
+functionally	O
+relevant	O
+as	O
+an	O
+initial	O
+step	O
+in	O
+metabolosome	B-complex_assembly
+assembly	O
+,	O
+in	O
+which	O
+a	O
+multifunctional	O
+protein	O
+core	O
+is	O
+formed	O
+,	O
+around	O
+which	O
+the	O
+shell	B-structure_element
+assembles	O
+.	O
+
+Of	O
+the	O
+three	O
+common	O
+metabolosome	B-complex_assembly
+core	O
+enzymes	O
+,	O
+crystal	B-evidence
+structures	I-evidence
+are	O
+available	O
+for	O
+both	O
+the	O
+alcohol	B-protein_type
+and	I-protein_type
+aldehyde	I-protein_type
+dehydrogenases	I-protein_type
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+structure	B-evidence
+of	O
+PduL	B-protein_type
+,	O
+the	O
+PTAC	B-protein_type
+found	O
+in	O
+the	O
+vast	O
+majority	O
+of	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+,	O
+has	O
+not	O
+been	O
+determined	O
+.	O
+
+This	O
+is	O
+a	O
+major	O
+gap	O
+in	O
+our	O
+understanding	O
+of	O
+metabolosome	B-complex_assembly
+-	O
+encapsulated	O
+biochemistry	O
+and	O
+cofactor	O
+recycling	O
+.	O
+
+Moreover	O
+,	O
+it	O
+will	O
+be	O
+useful	O
+for	O
+guiding	O
+efforts	O
+to	O
+engineer	O
+novel	O
+BMC	B-complex_assembly
+cores	O
+for	O
+biotechnological	O
+applications	O
+.	O
+
+The	O
+primary	O
+structure	O
+of	O
+PduL	B-protein_type
+homologs	O
+is	O
+subdivided	O
+into	O
+two	O
+PF06130	B-structure_element
+domains	O
+,	O
+each	O
+roughly	O
+80	B-residue_range
+residues	I-residue_range
+in	I-residue_range
+length	I-residue_range
+.	O
+
+No	O
+available	O
+protein	O
+structures	O
+contain	O
+the	O
+PF06130	B-structure_element
+domain	O
+,	O
+and	O
+homology	B-experimental_method
+searches	I-experimental_method
+using	O
+the	O
+primary	O
+structure	O
+of	O
+PduL	B-protein_type
+do	O
+not	O
+return	O
+any	O
+significant	O
+results	O
+that	O
+would	O
+allow	O
+prediction	O
+of	O
+the	O
+structure	B-evidence
+.	O
+
+Moreover	O
+,	O
+the	O
+evident	O
+novelty	O
+of	O
+PduL	B-protein_type
+makes	O
+its	O
+structure	B-evidence
+interesting	O
+in	O
+the	O
+context	O
+of	O
+convergent	O
+evolution	O
+of	O
+PTAC	B-protein_type
+function	O
+;	O
+to	O
+-	O
+date	O
+,	O
+only	O
+the	O
+Pta	B-protein_type
+active	B-site
+site	I-site
+and	O
+catalytic	O
+mechanism	O
+is	O
+known	O
+.	O
+
+Here	O
+we	O
+report	O
+high	O
+-	O
+resolution	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+a	O
+PduL	B-protein_type
+-	I-protein_type
+type	I-protein_type
+PTAC	I-protein_type
+in	O
+both	O
+CoA	B-protein_state
+-	I-protein_state
+and	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+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	B-complex_assembly
+common	O
+core	O
+enzymes	O
+.	O
+
+We	O
+propose	O
+a	O
+catalytic	O
+mechanism	O
+analogous	O
+but	O
+yet	O
+distinct	O
+from	O
+the	O
+ubiquitous	O
+Pta	B-protein_type
+enzyme	O
+,	O
+highlighting	O
+the	O
+functional	O
+convergence	O
+of	O
+two	O
+enzymes	O
+with	O
+completely	O
+different	O
+structures	O
+and	O
+metal	O
+requirements	O
+.	O
+
+We	O
+also	O
+investigate	O
+the	O
+quaternary	O
+structures	O
+of	O
+three	O
+different	O
+PduL	B-protein_type
+homologs	O
+and	O
+situate	O
+our	O
+findings	O
+in	O
+the	O
+context	O
+of	O
+organelle	O
+biogenesis	O
+in	O
+functionally	O
+diverse	O
+BMCs	B-complex_assembly
+.	O
+
+Structure	B-experimental_method
+Determination	I-experimental_method
+of	O
+PduL	B-protein_type
+
+We	O
+cloned	B-experimental_method
+,	I-experimental_method
+expressed	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+purified	I-experimental_method
+three	O
+different	O
+PduL	B-protein_type
+homologs	O
+from	O
+functionally	O
+distinct	O
+BMCs	B-complex_assembly
+(	O
+Table	O
+1	O
+):	O
+from	O
+the	O
+well	O
+-	O
+studied	O
+pdu	B-gene
+locus	I-gene
+in	O
+S	B-species
+.	I-species
+enterica	I-species
+Typhimurium	I-species
+LT2	I-species
+(	O
+sPduL	B-protein
+),	O
+from	O
+the	O
+recently	O
+characterized	O
+pvm	B-gene
+locus	I-gene
+in	O
+Planctomyces	B-species
+limnophilus	I-species
+(	O
+pPduL	B-protein
+),	O
+and	O
+from	O
+the	O
+grm3	B-gene
+locus	I-gene
+in	O
+Rhodopseudomonas	B-species
+palustris	I-species
+BisB18	I-species
+(	O
+rPduL	B-protein
+).	O
+
+While	O
+purifying	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+sPduL	B-protein
+,	O
+we	O
+observed	O
+a	O
+tendency	O
+to	O
+aggregation	O
+as	O
+described	O
+previously	O
+,	O
+with	O
+a	O
+large	O
+fraction	O
+of	O
+the	O
+expressed	O
+protein	O
+found	O
+in	O
+the	O
+insoluble	O
+fraction	O
+in	O
+a	O
+white	O
+,	O
+cake	O
+-	O
+like	O
+pellet	O
+.	O
+
+Remarkably	O
+,	O
+after	O
+removing	B-experimental_method
+the	O
+N	O
+-	O
+terminal	O
+putative	O
+EP	B-structure_element
+(	O
+27	B-residue_range
+amino	I-residue_range
+acids	I-residue_range
+),	O
+most	O
+of	O
+the	O
+sPduLΔEP	B-mutant
+protein	O
+was	O
+in	O
+the	O
+soluble	O
+fraction	O
+upon	O
+cell	O
+lysis	O
+.	O
+
+Similar	O
+differences	O
+in	O
+solubility	O
+were	O
+observed	O
+for	O
+pPduL	B-protein
+and	O
+rPduL	B-protein
+when	O
+comparing	O
+EP	B-protein_state
+-	I-protein_state
+truncated	I-protein_state
+forms	O
+to	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+,	O
+but	O
+none	O
+were	O
+quite	O
+as	O
+dramatic	O
+as	O
+for	O
+sPduL	B-protein
+.	O
+We	O
+confirmed	O
+that	O
+all	O
+homologs	O
+were	O
+active	B-protein_state
+(	O
+S1a	O
+and	O
+S1b	O
+Fig	O
+).	O
+
+Among	O
+these	O
+,	O
+we	O
+were	O
+only	O
+able	O
+to	O
+obtain	O
+diffraction	B-evidence
+-	I-evidence
+quality	I-evidence
+crystals	I-evidence
+of	O
+rPduL	B-protein
+after	O
+removing	B-experimental_method
+the	O
+N	O
+-	O
+terminal	O
+putative	O
+EP	B-structure_element
+(	O
+33	B-residue_range
+amino	I-residue_range
+acids	I-residue_range
+,	O
+also	O
+see	O
+Fig	O
+2a	O
+)	O
+(	O
+rPduLΔEP	B-mutant
+).	O
+
+Truncated	B-protein_state
+rPduLΔEP	B-mutant
+had	O
+comparable	O
+enzymatic	O
+activity	O
+to	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	O
+(	O
+S1a	O
+Fig	O
+).	O
+
+Structural	O
+overview	O
+of	O
+R	B-species
+.	I-species
+palustris	I-species
+PduL	B-protein_type
+from	O
+the	O
+grm3	B-gene
+locus	I-gene
+.	O
+
+(	O
+a	O
+)	O
+Primary	O
+and	O
+secondary	O
+structure	O
+of	O
+rPduL	B-protein
+(	O
+tubes	O
+represent	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+,	O
+arrows	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+and	O
+dashed	O
+line	O
+residues	O
+disordered	O
+in	O
+the	O
+structure	B-evidence
+.	O
+
+The	O
+first	B-residue_range
+33	I-residue_range
+amino	I-residue_range
+acids	I-residue_range
+are	O
+present	O
+only	O
+in	O
+the	O
+wildtype	O
+construct	O
+and	O
+contains	O
+the	O
+predicted	O
+EP	B-structure_element
+alpha	B-structure_element
+helix	I-structure_element
+,	O
+α0	B-structure_element
+);	O
+the	O
+truncated	B-protein_state
+rPduLΔEP	B-mutant
+that	O
+was	O
+crystallized	B-experimental_method
+begins	O
+with	O
+M	B-residue_name
+-	O
+G	B-residue_name
+-	O
+V	B-residue_name
+.	O
+Coloring	O
+is	O
+according	O
+to	O
+structural	O
+domains	O
+(	O
+domain	B-structure_element
+1	I-structure_element
+D36	B-residue_range
+-	I-residue_range
+N46	I-residue_range
+/	O
+Q155	B-residue_range
+-	I-residue_range
+C224	I-residue_range
+,	O
+blue	O
+;	O
+loop	B-structure_element
+insertion	I-structure_element
+G61	B-residue_range
+-	I-residue_range
+E81	I-residue_range
+,	O
+grey	O
+;	O
+domain	B-structure_element
+2	I-structure_element
+R47	B-residue_range
+-	I-residue_range
+F60	I-residue_range
+/	O
+E82	B-residue_range
+-	I-residue_range
+A154	I-residue_range
+,	O
+red	O
+).	O
+
+Metal	B-site
+coordination	I-site
+residues	I-site
+are	O
+highlighted	O
+in	O
+light	O
+blue	O
+and	O
+CoA	B-site
+contacting	I-site
+residues	I-site
+in	O
+magenta	O
+,	O
+residues	O
+contacting	O
+the	O
+CoA	B-chemical
+of	O
+the	O
+other	O
+chain	O
+are	O
+also	O
+outlined	O
+.	O
+
+(	O
+b	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+structure	B-evidence
+colored	O
+by	O
+domains	O
+and	O
+including	O
+secondary	O
+structure	B-evidence
+numbering	O
+.	O
+
+Coenzyme	B-chemical
+A	I-chemical
+is	O
+shown	O
+in	O
+magenta	O
+sticks	O
+and	O
+Zinc	B-chemical
+(	O
+grey	O
+)	O
+as	O
+spheres	O
+.	O
+
+We	O
+collected	B-experimental_method
+a	I-experimental_method
+native	I-experimental_method
+dataset	I-experimental_method
+from	O
+rPduLΔEP	B-mutant
+crystals	B-evidence
+diffracting	O
+to	O
+a	O
+resolution	O
+of	O
+1	O
+.	O
+54	O
+Å	O
+(	O
+Table	O
+2	O
+).	O
+
+Using	O
+a	O
+mercury	B-experimental_method
+-	I-experimental_method
+derivative	I-experimental_method
+crystal	I-experimental_method
+form	O
+diffracting	O
+to	O
+1	O
+.	O
+99	O
+Å	O
+(	O
+Table	O
+2	O
+),	O
+we	O
+obtained	O
+high	O
+quality	O
+electron	B-evidence
+density	I-evidence
+for	O
+model	O
+building	O
+and	O
+used	O
+the	O
+initial	O
+model	O
+to	O
+refine	O
+against	O
+the	O
+native	O
+data	O
+to	O
+Rwork	B-evidence
+/	O
+Rfree	B-evidence
+values	O
+of	O
+18	O
+.	O
+9	O
+/	O
+22	O
+.	O
+1	O
+%.	O
+
+There	O
+are	O
+two	O
+PduL	B-protein_type
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+of	O
+the	O
+P212121	O
+unit	O
+cell	O
+.	O
+
+We	O
+were	O
+able	O
+to	O
+fit	O
+all	O
+of	O
+the	O
+primary	O
+structure	O
+of	O
+PduLΔEP	B-mutant
+into	O
+the	O
+electron	B-evidence
+density	I-evidence
+with	O
+the	O
+exception	O
+of	O
+three	O
+amino	O
+acids	O
+at	O
+the	O
+N	O
+-	O
+terminus	O
+and	O
+two	O
+amino	O
+acids	O
+at	O
+the	O
+C	O
+-	O
+terminus	O
+(	O
+Fig	O
+2a	O
+);	O
+the	O
+model	O
+is	O
+of	O
+excellent	O
+quality	O
+(	O
+Table	O
+2	O
+).	O
+
+A	O
+CoA	B-chemical
+cofactor	O
+as	O
+well	O
+as	O
+two	O
+metal	O
+ions	O
+are	O
+clearly	O
+resolved	O
+in	O
+the	O
+density	B-evidence
+(	O
+for	O
+omit	B-evidence
+maps	I-evidence
+of	O
+CoA	B-chemical
+see	O
+S2	O
+Fig	O
+).	O
+
+Structurally	O
+,	O
+PduL	B-protein_type
+consists	O
+of	O
+two	O
+domains	B-structure_element
+(	O
+Fig	O
+2	O
+,	O
+blue	O
+/	O
+red	O
+),	O
+each	O
+a	O
+beta	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+that	O
+is	O
+capped	O
+on	O
+both	O
+ends	O
+by	O
+short	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+.	O
+
+β	B-structure_element
+-	I-structure_element
+Barrel	I-structure_element
+1	I-structure_element
+consists	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+β	B-structure_element
+strand	I-structure_element
+and	O
+β	B-structure_element
+strands	I-structure_element
+from	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+the	O
+polypeptide	O
+chain	O
+(	O
+β1	B-structure_element
+,	O
+β10	B-structure_element
+-	I-structure_element
+β14	I-structure_element
+;	O
+residues	O
+37	B-residue_range
+–	I-residue_range
+46	I-residue_range
+and	O
+155	B-residue_range
+–	I-residue_range
+224	I-residue_range
+).	O
+
+β	B-structure_element
+-	I-structure_element
+Barrel	I-structure_element
+2	I-structure_element
+consists	O
+mainly	O
+of	O
+the	O
+central	O
+segment	O
+of	O
+primary	O
+structure	O
+(	O
+β2	B-structure_element
+,	O
+β5	B-structure_element
+–	I-structure_element
+β9	I-structure_element
+;	O
+residues	O
+47	B-residue_range
+–	I-residue_range
+60	I-residue_range
+and	O
+82	B-residue_range
+–	I-residue_range
+154	I-residue_range
+)	O
+(	O
+Fig	O
+2	O
+,	O
+red	O
+),	O
+but	O
+is	O
+interrupted	O
+by	O
+a	O
+short	B-structure_element
+two	I-structure_element
+-	I-structure_element
+strand	I-structure_element
+beta	I-structure_element
+sheet	I-structure_element
+(	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+,	O
+residues	O
+61	B-residue_range
+–	I-residue_range
+81	I-residue_range
+).	O
+
+This	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+is	O
+involved	O
+in	O
+contacts	O
+between	O
+the	O
+two	O
+domains	O
+and	O
+forms	O
+a	O
+lid	O
+over	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+Residues	O
+in	O
+this	O
+region	O
+(	O
+Gln42	B-residue_name_number
+,	O
+Pro43	B-residue_name_number
+,	O
+Gly44	B-residue_name_number
+),	O
+covering	O
+the	O
+active	B-site
+site	I-site
+,	O
+are	O
+strongly	B-protein_state
+conserved	I-protein_state
+(	O
+Fig	O
+3	O
+).	O
+
+This	O
+structural	O
+arrangement	O
+is	O
+completely	O
+different	O
+from	O
+the	O
+functionally	O
+related	O
+Pta	B-protein_type
+,	O
+which	O
+is	O
+composed	O
+of	O
+two	O
+domains	B-structure_element
+,	O
+each	O
+consisting	O
+of	O
+a	O
+central	O
+flat	O
+beta	B-structure_element
+sheet	I-structure_element
+with	O
+alpha	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+on	O
+the	O
+top	O
+and	O
+bottom	O
+.	O
+
+Primary	O
+structure	O
+conservation	O
+of	O
+the	O
+PduL	B-protein_type
+protein	O
+family	O
+.	O
+
+Sequence	O
+logo	O
+calculated	O
+from	O
+the	O
+multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+PduL	B-protein_type
+homologs	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+),	O
+but	O
+not	B-protein_state
+including	I-protein_state
+putative	O
+EP	B-structure_element
+sequences	O
+.	O
+
+Residues	O
+100	O
+%	O
+conserved	O
+across	O
+all	O
+PduL	B-protein_type
+homologs	O
+in	O
+our	O
+dataset	O
+are	O
+noted	O
+with	O
+an	O
+asterisk	O
+,	O
+and	O
+residues	O
+conserved	O
+in	O
+over	O
+90	O
+%	O
+of	O
+sequences	O
+are	O
+noted	O
+with	O
+a	O
+colon	O
+.	O
+
+The	O
+sequences	O
+aligning	O
+to	O
+the	O
+PF06130	B-structure_element
+domain	O
+(	O
+determined	O
+by	O
+BLAST	O
+)	O
+are	O
+highlighted	O
+in	O
+red	O
+and	O
+blue	O
+.	O
+
+The	O
+position	O
+numbers	O
+shown	O
+correspond	O
+to	O
+the	O
+residue	O
+numbering	O
+of	O
+rPduL	B-protein
+;	O
+note	O
+that	O
+some	O
+positions	O
+in	O
+the	O
+logo	O
+represent	O
+gaps	O
+in	O
+the	O
+rPduL	B-protein
+sequence	O
+.	O
+
+There	O
+are	O
+two	O
+PduL	B-protein_type
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+forming	O
+a	O
+butterfly	B-protein_state
+-	I-protein_state
+shaped	I-protein_state
+dimer	B-oligomeric_state
+(	O
+Fig	O
+4c	O
+).	O
+
+Consistent	O
+with	O
+this	O
+,	O
+results	O
+from	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+of	O
+rPduLΔEP	B-mutant
+suggest	O
+that	O
+it	O
+is	O
+a	O
+dimer	B-oligomeric_state
+in	O
+solution	O
+(	O
+Fig	O
+5e	O
+).	O
+
+The	O
+interface	B-site
+between	O
+the	O
+two	O
+chains	O
+buries	O
+882	O
+Å2	O
+per	O
+monomer	B-oligomeric_state
+and	O
+is	O
+mainly	O
+formed	O
+by	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+2	I-structure_element
+and	I-structure_element
+4	I-structure_element
+and	O
+parts	O
+of	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+12	I-structure_element
+and	I-structure_element
+14	I-structure_element
+,	O
+as	O
+well	O
+as	O
+a	O
+π	O
+–	O
+π	O
+stacking	O
+of	O
+the	O
+adenine	B-chemical
+moiety	O
+of	O
+CoA	B-chemical
+with	O
+Phe116	B-residue_name_number
+of	O
+the	O
+adjacent	O
+chain	O
+(	O
+Fig	O
+4c	O
+).	O
+
+The	O
+folds	O
+of	O
+the	O
+two	O
+chains	O
+in	O
+the	O
+asymmetric	O
+unit	O
+are	O
+very	O
+similar	O
+,	O
+superimposing	B-experimental_method
+with	O
+a	O
+rmsd	B-evidence
+of	O
+0	O
+.	O
+16	O
+Å	O
+over	O
+2	O
+,	O
+306	O
+aligned	O
+atom	O
+pairs	O
+.	O
+
+The	O
+peripheral	O
+helices	B-structure_element
+and	O
+the	O
+short	B-structure_element
+antiparallel	I-structure_element
+β3	I-structure_element
+–	I-structure_element
+4	I-structure_element
+sheet	I-structure_element
+mediate	O
+most	O
+of	O
+the	O
+crystal	O
+contacts	O
+.	O
+
+Details	O
+of	O
+active	B-site
+site	I-site
+,	O
+dimeric	B-oligomeric_state
+assembly	O
+,	O
+and	O
+sequence	O
+conservation	O
+of	O
+PduL	B-protein_type
+.	O
+
+(	O
+a	O
+,	O
+b	O
+)	O
+Proposed	O
+active	B-site
+site	I-site
+of	O
+PduL	B-protein_type
+with	O
+relevant	O
+residues	O
+shown	O
+as	O
+sticks	O
+in	O
+atom	O
+coloring	O
+(	O
+nitrogen	B-chemical
+blue	O
+,	O
+oxygen	B-chemical
+red	O
+,	O
+sulfur	B-chemical
+yellow	O
+),	O
+zinc	B-chemical
+as	O
+grey	O
+colored	O
+spheres	O
+and	O
+coordinating	O
+ordered	O
+water	B-chemical
+molecules	O
+in	O
+red	O
+.	O
+
+Distances	O
+between	O
+atom	O
+centers	O
+are	O
+indicated	O
+in	O
+Å	O
+.	O
+(	O
+a	O
+)	O
+Coenzyme	B-chemical
+A	I-chemical
+containing	O
+,	O
+(	O
+b	O
+)	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+.	O
+
+(	O
+c	O
+)	O
+View	O
+of	O
+the	O
+dimer	B-oligomeric_state
+in	O
+the	O
+asymmetric	O
+unit	O
+from	O
+the	O
+side	O
+,	O
+domains	B-structure_element
+1	I-structure_element
+and	I-structure_element
+2	I-structure_element
+colored	O
+as	O
+in	O
+Fig	O
+2	O
+and	O
+the	O
+two	O
+chains	O
+differentiated	O
+by	O
+blue	O
+/	O
+red	O
+versus	O
+slate	O
+/	O
+firebrick	O
+.	O
+
+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	B-residue_name_number
+and	O
+the	O
+CoA	B-chemical
+base	O
+of	O
+the	O
+other	O
+dimer	B-oligomeric_state
+chain	O
+.	O
+
+(	O
+d	O
+)	O
+Surface	O
+representation	O
+of	O
+the	O
+structure	B-evidence
+with	O
+indicated	O
+conservation	O
+(	O
+red	O
+:	O
+high	O
+,	O
+white	O
+:	O
+intermediate	O
+,	O
+yellow	O
+:	O
+low	O
+).	O
+
+Size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+of	O
+PduL	B-protein_type
+homologs	O
+.	O
+
+(	O
+a	O
+)–(	O
+c	O
+):	O
+Chromatograms	B-evidence
+of	O
+sPduL	B-protein
+(	O
+a	O
+),	O
+rPduL	B-protein
+(	O
+b	O
+),	O
+and	O
+pPduL	B-protein
+(	O
+c	O
+)	O
+with	O
+(	O
+orange	O
+)	O
+or	O
+without	O
+(	O
+blue	O
+)	O
+the	O
+predicted	O
+EP	B-structure_element
+,	O
+post	O
+-	O
+nickel	B-experimental_method
+affinity	I-experimental_method
+purification	I-experimental_method
+,	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
+d	O
+)–(	O
+f	O
+):	O
+Chromatograms	B-evidence
+of	O
+sPduL	B-protein
+(	O
+d	O
+),	O
+rPduL	B-protein
+(	O
+e	O
+),	O
+and	O
+pPduL	B-protein
+(	O
+f	O
+)	O
+post	O
+-	O
+preparative	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+with	O
+different	O
+size	O
+fractions	O
+separated	O
+,	O
+applied	O
+over	O
+an	O
+analytical	O
+size	O
+exclusion	O
+column	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+
+All	O
+chromatograms	B-evidence
+are	O
+cropped	O
+to	O
+show	O
+only	O
+the	O
+linear	O
+range	O
+of	O
+separation	O
+based	O
+on	O
+standard	O
+runs	O
+,	O
+shown	O
+in	O
+black	O
+squares	O
+with	O
+a	O
+dashed	O
+linear	O
+trend	O
+line	O
+.	O
+
+Active	B-site
+Site	I-site
+Properties	O
+
+CoA	B-chemical
+and	O
+the	O
+metal	O
+ions	O
+bind	O
+between	O
+the	O
+two	O
+domains	O
+,	O
+presumably	O
+in	O
+the	O
+active	B-site
+site	I-site
+(	O
+Figs	O
+2b	O
+and	O
+4a	O
+).	O
+
+To	O
+identify	O
+the	O
+bound	O
+metals	O
+,	O
+we	O
+performed	O
+an	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+fluorescence	I-experimental_method
+scan	I-experimental_method
+on	O
+the	O
+crystals	B-evidence
+at	O
+various	O
+wavelengths	O
+(	O
+corresponding	O
+to	O
+the	O
+K	O
+-	O
+edges	O
+of	O
+Mn	B-chemical
+,	O
+Fe	B-chemical
+,	O
+Co	B-chemical
+,	O
+Ni	B-chemical
+,	O
+Cu	B-chemical
+,	O
+and	O
+Zn	B-chemical
+).	O
+
+There	O
+was	O
+a	O
+large	O
+signal	O
+at	O
+the	O
+zinc	O
+edge	O
+,	O
+and	O
+we	O
+tested	O
+for	O
+the	O
+presence	O
+of	O
+zinc	B-chemical
+by	O
+collecting	B-experimental_method
+full	I-experimental_method
+data	I-experimental_method
+sets	I-experimental_method
+before	I-experimental_method
+and	I-experimental_method
+after	I-experimental_method
+the	I-experimental_method
+Zn	I-experimental_method
+K	I-experimental_method
+-	I-experimental_method
+edge	I-experimental_method
+(	I-experimental_method
+1	I-experimental_method
+.	I-experimental_method
+2861	I-experimental_method
+and	I-experimental_method
+1	I-experimental_method
+.	I-experimental_method
+2822	I-experimental_method
+Å	I-experimental_method
+,	O
+respectively	O
+).	O
+
+The	O
+large	O
+differences	O
+between	O
+the	O
+anomalous	O
+signals	O
+confirm	O
+the	O
+presence	O
+of	O
+zinc	B-chemical
+at	O
+both	O
+metal	O
+sites	O
+(	O
+S3	O
+Fig	O
+).	O
+
+The	O
+first	O
+zinc	B-chemical
+ion	O
+(	O
+Zn1	B-chemical
+)	O
+is	O
+in	O
+a	O
+tetrahedral	O
+coordination	O
+state	O
+with	O
+His48	B-residue_name_number
+,	O
+His50	B-residue_name_number
+,	O
+Glu109	B-residue_name_number
+,	O
+and	O
+the	O
+CoA	B-chemical
+sulfur	B-chemical
+(	O
+Fig	O
+4a	O
+).	O
+
+The	O
+second	O
+(	O
+Zn2	B-chemical
+)	O
+is	O
+in	O
+octahedral	O
+coordination	O
+by	O
+three	O
+conserved	B-protein_state
+histidine	B-residue_name
+residues	O
+(	O
+His157	B-residue_name_number
+,	O
+His159	B-residue_name_number
+and	O
+His204	B-residue_name_number
+)	O
+as	O
+well	O
+as	O
+three	O
+water	B-chemical
+molecules	O
+(	O
+Fig	O
+4a	O
+).	O
+
+The	O
+nitrogen	O
+atom	O
+coordinating	O
+the	O
+zinc	B-chemical
+is	O
+the	O
+Nε	O
+in	O
+each	O
+histidine	B-residue_name
+residue	O
+,	O
+as	O
+is	O
+typical	O
+for	O
+this	O
+interaction	O
+.	O
+
+When	O
+the	O
+crystals	B-experimental_method
+were	I-experimental_method
+soaked	I-experimental_method
+in	O
+a	O
+sodium	B-chemical
+phosphate	I-chemical
+solution	O
+for	O
+2	O
+d	O
+prior	O
+to	O
+data	O
+collection	O
+,	O
+the	O
+CoA	B-chemical
+dissociates	O
+,	O
+and	O
+density	B-evidence
+for	O
+a	O
+phosphate	B-chemical
+molecule	O
+is	O
+visible	O
+at	O
+the	O
+active	B-site
+site	I-site
+(	O
+Table	O
+2	O
+,	O
+Fig	O
+4b	O
+).	O
+
+The	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+aligns	B-experimental_method
+well	O
+with	O
+the	O
+CoA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+(	O
+0	O
+.	O
+43	O
+Å	O
+rmsd	B-evidence
+over	O
+2	O
+,	O
+361	O
+atoms	O
+for	O
+the	O
+monomer	B-oligomeric_state
+,	O
+0	O
+.	O
+83	O
+Å	O
+over	O
+5	O
+,	O
+259	O
+aligned	O
+atoms	O
+for	O
+the	O
+dimer	B-oligomeric_state
+).	O
+
+The	O
+phosphate	B-chemical
+contacts	O
+both	O
+zinc	B-chemical
+atoms	O
+(	O
+Fig	O
+4b	O
+)	O
+and	O
+replaces	O
+the	O
+coordination	O
+by	O
+CoA	B-chemical
+at	O
+Zn1	B-chemical
+;	O
+the	O
+coordination	O
+for	O
+Zn2	B-chemical
+changes	O
+from	O
+octahedral	O
+with	O
+three	O
+bound	O
+waters	B-chemical
+to	O
+tetrahedral	O
+with	O
+a	O
+phosphate	B-chemical
+ion	O
+as	O
+one	O
+of	O
+the	O
+ligands	O
+(	O
+Fig	O
+4b	O
+).	O
+
+Conserved	B-protein_state
+Arg103	B-residue_name_number
+seems	O
+to	O
+be	O
+involved	O
+in	O
+maintaining	O
+the	O
+phosphate	B-chemical
+in	O
+that	O
+position	O
+.	O
+
+The	O
+two	O
+zinc	B-chemical
+atoms	O
+are	O
+slightly	O
+closer	O
+together	O
+in	O
+the	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+form	O
+(	O
+5	O
+.	O
+8	O
+Å	O
+vs	O
+6	O
+.	O
+3	O
+Å	O
+),	O
+possibly	O
+due	O
+to	O
+the	O
+bridging	O
+effect	O
+of	O
+the	O
+phosphate	B-chemical
+.	O
+
+An	O
+additional	O
+phosphate	B-chemical
+molecule	O
+is	O
+bound	O
+at	O
+a	O
+crystal	O
+contact	O
+interface	O
+,	O
+perhaps	O
+accounting	O
+for	O
+the	O
+14	O
+Å	O
+shorter	O
+c	O
+-	O
+axis	O
+in	O
+the	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystal	O
+form	O
+(	O
+Table	O
+2	O
+).	O
+
+Oligomeric	O
+States	O
+of	O
+PduL	B-protein_type
+Orthologs	O
+Are	O
+Influenced	O
+by	O
+the	O
+EP	B-structure_element
+
+Interestingly	O
+,	O
+some	O
+of	O
+the	O
+residues	O
+important	O
+for	O
+dimerization	O
+of	O
+rPduL	B-protein
+,	O
+particularly	O
+Phe116	B-residue_name_number
+,	O
+are	O
+poorly	B-protein_state
+conserved	I-protein_state
+across	O
+PduL	B-protein_type
+homologs	O
+associated	O
+with	O
+functionally	O
+diverse	O
+BMCs	B-complex_assembly
+(	O
+Figs	O
+4c	O
+and	O
+3	O
+),	O
+suggesting	O
+that	O
+they	O
+may	O
+have	O
+alternative	O
+oligomeric	O
+states	O
+.	O
+
+We	O
+tested	O
+this	O
+hypothesis	O
+by	O
+performing	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+on	O
+both	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+and	O
+truncated	O
+variants	O
+(	O
+lacking	B-protein_state
+the	O
+EP	B-structure_element
+,	O
+ΔEP	B-mutant
+)	O
+of	O
+sPduL	B-protein
+,	O
+rPduL	B-protein
+,	O
+and	O
+pPduL	B-protein
+.	O
+These	O
+three	O
+homologs	O
+are	O
+found	O
+in	O
+functionally	O
+distinct	O
+BMCs	B-complex_assembly
+(	O
+Table	O
+1	O
+).	O
+
+It	O
+has	O
+been	O
+proposed	O
+that	O
+the	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+may	O
+assemble	O
+in	O
+a	O
+core	O
+-	O
+first	O
+manner	O
+,	O
+with	O
+the	O
+luminal	O
+enzymes	O
+(	O
+signature	O
+enzyme	O
+,	O
+aldehyde	B-protein_type
+,	I-protein_type
+and	I-protein_type
+alcohol	I-protein_type
+dehydrogenases	I-protein_type
+and	O
+the	O
+BMC	B-complex_assembly
+PTAC	B-protein_type
+)	O
+forming	O
+an	O
+initial	O
+bolus	O
+,	O
+or	O
+prometabolosome	O
+,	O
+around	O
+which	O
+a	O
+shell	B-structure_element
+assembles	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	B-protein_type
+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
+
+We	O
+found	O
+that	O
+not	O
+only	O
+did	O
+the	O
+different	O
+orthologs	O
+appear	O
+to	O
+assemble	O
+into	O
+different	O
+oligomeric	O
+states	O
+,	O
+but	O
+that	O
+quaternary	O
+structure	O
+was	O
+dependent	O
+on	O
+whether	O
+or	O
+not	O
+the	O
+EP	B-structure_element
+was	O
+present	O
+.	O
+
+Full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+sPduL	B-protein
+was	O
+unstable	O
+in	O
+solution	O
+—	O
+precipitating	O
+over	O
+time	O
+—	O
+and	O
+eluted	O
+throughout	O
+the	O
+entire	O
+volume	O
+of	O
+a	O
+size	O
+exclusion	O
+column	O
+,	O
+indicating	O
+it	O
+was	O
+nonspecifically	O
+aggregating	O
+.	O
+
+However	O
+,	O
+when	O
+the	O
+putative	O
+EP	B-structure_element
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+27	I-residue_range
+)	O
+was	O
+removed	B-experimental_method
+(	O
+sPduL	B-mutant
+ΔEP	I-mutant
+),	O
+the	O
+truncated	B-protein_state
+protein	O
+was	O
+stable	O
+and	O
+eluted	O
+as	O
+a	O
+single	O
+peak	O
+(	O
+Fig	O
+5a	O
+)	O
+consistent	O
+with	O
+the	O
+size	O
+of	O
+a	O
+monomer	B-oligomeric_state
+(	O
+Fig	O
+5d	O
+,	O
+blue	O
+curve	O
+).	O
+
+In	O
+contrast	O
+,	O
+both	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+rPduL	B-protein
+and	O
+pPduL	B-protein
+appeared	O
+to	O
+exist	O
+in	O
+two	O
+distinct	O
+oligomeric	O
+states	O
+(	O
+Fig	O
+5b	O
+and	O
+5c	O
+respectively	O
+,	O
+orange	O
+curves	O
+),	O
+one	O
+form	O
+of	O
+the	O
+approximate	O
+size	O
+of	O
+a	O
+dimer	B-oligomeric_state
+and	O
+the	O
+second	O
+,	O
+a	O
+higher	O
+molecular	O
+weight	O
+oligomer	B-oligomeric_state
+(~	O
+150	O
+kDa	O
+).	O
+
+Upon	O
+deletion	B-experimental_method
+of	O
+the	O
+putative	O
+EP	B-structure_element
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+47	I-residue_range
+for	O
+rPduL	B-protein
+,	O
+and	O
+1	B-residue_range
+–	I-residue_range
+20	I-residue_range
+for	O
+pPduL	B-protein
+),	O
+there	O
+was	O
+a	O
+distinct	O
+change	O
+in	O
+the	O
+elution	O
+profiles	O
+(	O
+Fig	O
+5b	O
+and	O
+5c	O
+respectively	O
+,	O
+blue	O
+curves	O
+).	O
+
+pPduLΔEP	B-mutant
+eluted	O
+as	O
+two	O
+smaller	O
+forms	O
+,	O
+possibly	O
+corresponding	O
+to	O
+a	O
+trimer	B-oligomeric_state
+and	O
+a	O
+monomer	B-oligomeric_state
+.	O
+
+In	O
+contrast	O
+,	O
+rPduLΔEP	B-mutant
+eluted	O
+as	O
+one	O
+smaller	O
+oligomer	O
+,	O
+possibly	O
+a	O
+dimer	B-oligomeric_state
+.	O
+
+We	O
+also	O
+analyzed	O
+purified	O
+rPduL	B-protein
+and	O
+rPduLΔEP	B-mutant
+by	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+coupled	O
+with	O
+multiangle	B-experimental_method
+light	I-experimental_method
+scattering	I-experimental_method
+(	O
+SEC	B-experimental_method
+-	I-experimental_method
+MALS	I-experimental_method
+)	O
+for	O
+a	O
+complementary	O
+approach	O
+to	O
+assessing	O
+oligomeric	O
+state	O
+.	O
+
+SEC	B-experimental_method
+-	I-experimental_method
+MALS	I-experimental_method
+analysis	O
+of	O
+rPdulΔEP	B-mutant
+is	O
+consistent	O
+with	O
+a	O
+dimer	B-oligomeric_state
+(	O
+as	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+)	O
+with	O
+a	O
+weighted	B-evidence
+average	I-evidence
+(	I-evidence
+Mw	I-evidence
+)	I-evidence
+and	I-evidence
+number	I-evidence
+average	I-evidence
+(	I-evidence
+Mn	I-evidence
+)	I-evidence
+of	I-evidence
+the	I-evidence
+molar	I-evidence
+mass	I-evidence
+of	O
+58	O
+.	O
+4	O
+kDa	O
++/−	O
+11	O
+.	O
+2	O
+%	O
+and	O
+58	O
+.	O
+8	O
+kDa	O
++/−	O
+10	O
+.	O
+9	O
+%,	O
+respectively	O
+(	O
+S4a	O
+Fig	O
+).	O
+
+rPduL	B-protein
+full	B-protein_state
+length	I-protein_state
+runs	O
+as	O
+Mw	B-evidence
+=	O
+140	O
+.	O
+3	O
+kDa	O
++/−	O
+1	O
+.	O
+2	O
+%	O
+and	O
+Mn	B-evidence
+=	O
+140	O
+.	O
+5	O
+kDa	O
++/−	O
+1	O
+.	O
+2	O
+%.	O
+
+This	O
+corresponds	O
+to	O
+an	O
+oligomeric	O
+state	O
+of	O
+six	B-oligomeric_state
+subunits	I-oligomeric_state
+(	O
+calculated	O
+molecular	B-evidence
+weight	I-evidence
+of	O
+144	O
+kDa	O
+).	O
+
+Collectively	O
+,	O
+these	O
+data	O
+strongly	O
+suggest	O
+that	O
+the	O
+N	O
+-	O
+terminal	O
+EP	B-structure_element
+of	O
+PduL	B-protein_type
+plays	O
+a	O
+role	O
+in	O
+defining	O
+the	O
+quaternary	O
+structure	O
+of	O
+the	O
+protein	O
+.	O
+
+The	O
+BMC	B-complex_assembly
+shell	B-structure_element
+not	O
+only	O
+sequesters	O
+specific	O
+enzymes	O
+but	O
+also	O
+their	O
+cofactors	O
+,	O
+thereby	O
+establishing	O
+a	O
+private	O
+cofactor	O
+pool	O
+dedicated	O
+to	O
+the	O
+encapsulated	O
+reactions	O
+.	O
+
+In	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+,	O
+CoA	B-chemical
+and	O
+NAD	B-chemical
++	I-chemical
+must	O
+be	O
+continually	O
+recycled	O
+within	O
+the	O
+organelle	O
+(	O
+Fig	O
+1	O
+).	O
+
+Homologs	O
+of	O
+the	O
+predominant	O
+cofactor	O
+utilizer	O
+(	O
+aldehyde	B-protein_type
+dehydrogenase	I-protein_type
+)	O
+and	O
+NAD	B-chemical
++	I-chemical
+regenerator	O
+(	O
+alcohol	B-protein_type
+dehydrogenase	I-protein_type
+)	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	B-protein_type
+,	O
+which	O
+recycles	O
+CoA	B-chemical
+in	O
+the	O
+organelle	O
+lumen	O
+.	O
+
+Curiously	O
+,	O
+while	O
+the	O
+housekeeping	B-protein_state
+Pta	B-protein_type
+could	O
+provide	O
+this	O
+function	O
+,	O
+and	O
+indeed	O
+does	O
+so	O
+in	O
+the	O
+case	O
+of	O
+one	O
+type	O
+of	O
+ethanolamine	B-complex_assembly
+-	I-complex_assembly
+utilizing	I-complex_assembly
+(	I-complex_assembly
+EUT	I-complex_assembly
+)	I-complex_assembly
+BMC	I-complex_assembly
+,	O
+the	O
+evolutionarily	O
+unrelated	O
+PduL	B-protein_type
+fulfills	O
+this	O
+function	O
+for	O
+the	O
+majority	O
+of	O
+metabolosomes	B-complex_assembly
+using	O
+a	O
+novel	O
+structure	B-evidence
+and	O
+active	B-site
+site	I-site
+for	O
+convergent	O
+evolution	O
+of	O
+function	O
+.	O
+
+The	O
+Tertiary	O
+Structure	O
+of	O
+PduL	B-protein_type
+Is	O
+Formed	O
+by	O
+Discontinuous	O
+Segments	O
+of	O
+Primary	O
+Structure	O
+
+The	O
+structure	B-evidence
+of	O
+PduL	B-protein_type
+consists	O
+of	O
+two	B-structure_element
+β	I-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domains	I-structure_element
+capped	O
+by	O
+short	B-structure_element
+alpha	I-structure_element
+helical	I-structure_element
+segments	I-structure_element
+(	O
+Fig	O
+2b	O
+).	O
+
+The	O
+two	O
+domains	O
+are	O
+structurally	O
+very	O
+similar	O
+(	O
+superimposing	B-experimental_method
+with	O
+a	O
+rmsd	B-evidence
+of	O
+1	O
+.	O
+34	O
+Å	O
+(	O
+over	O
+123	O
+out	O
+of	O
+320	O
+/	O
+348	O
+aligned	O
+backbone	O
+atoms	O
+,	O
+S5a	O
+Fig	O
+).	O
+
+However	O
+,	O
+the	O
+amino	O
+acid	O
+sequences	O
+of	O
+the	O
+two	O
+domains	O
+are	O
+only	O
+16	O
+%	O
+identical	O
+(	O
+mainly	O
+the	O
+RHxH	B-structure_element
+motif	I-structure_element
+,	O
+β2	B-structure_element
+and	O
+β10	B-structure_element
+),	O
+and	O
+34	O
+%	O
+similar	O
+.	O
+
+Our	O
+structure	B-evidence
+reveals	O
+that	O
+the	O
+two	O
+assigned	O
+PF06130	B-structure_element
+domains	O
+(	O
+Fig	O
+3	O
+)	O
+do	O
+not	O
+form	O
+structurally	O
+discrete	O
+units	O
+;	O
+this	O
+reduces	O
+the	O
+apparent	O
+sequence	O
+conservation	O
+at	O
+the	O
+level	O
+of	O
+primary	O
+structure	O
+.	O
+
+One	O
+strand	B-structure_element
+of	O
+the	O
+domain	B-structure_element
+1	I-structure_element
+beta	B-structure_element
+barrel	I-structure_element
+(	O
+shown	O
+in	O
+blue	O
+in	O
+Fig	O
+2	O
+)	O
+is	O
+contributed	O
+by	O
+the	O
+N	O
+-	O
+terminus	O
+,	O
+while	O
+the	O
+rest	O
+of	O
+the	O
+domain	O
+is	O
+formed	O
+by	O
+the	O
+residues	O
+from	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+the	O
+protein	B-protein_type
+.	O
+
+When	O
+aligned	B-experimental_method
+by	O
+structure	B-evidence
+,	O
+the	O
+β1	B-structure_element
+strand	I-structure_element
+of	O
+the	O
+first	B-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+2a	O
+and	O
+2b	O
+,	O
+blue	O
+)	O
+corresponds	O
+to	O
+the	O
+final	B-structure_element
+strand	I-structure_element
+of	O
+the	O
+second	B-structure_element
+domain	I-structure_element
+(	O
+β9	B-structure_element
+),	O
+effectively	O
+making	O
+the	O
+domains	O
+continuous	O
+if	O
+the	O
+first	O
+strand	O
+was	O
+transplanted	O
+to	O
+the	O
+C	O
+-	O
+terminus	O
+.	O
+
+Refined	O
+domain	O
+assignment	O
+based	O
+on	O
+our	O
+structure	B-evidence
+should	O
+be	O
+able	O
+to	O
+predict	O
+domains	O
+of	O
+PF06130	B-structure_element
+homologs	O
+much	O
+more	O
+accurately	O
+.	O
+
+The	O
+closest	O
+structural	O
+homolog	O
+of	O
+the	O
+PduL	B-protein_type
+barrel	B-structure_element
+domain	I-structure_element
+is	O
+a	O
+subdomain	O
+of	O
+a	O
+multienzyme	O
+complex	O
+,	O
+the	O
+alpha	B-structure_element
+subunit	I-structure_element
+of	O
+ethylbenzene	B-protein_type
+dehydrogenase	I-protein_type
+(	O
+S5b	O
+Fig	O
+,	O
+rmsd	B-evidence
+of	O
+2	O
+.	O
+26	O
+Å	O
+over	O
+226	O
+aligned	O
+atoms	O
+consisting	O
+of	O
+one	O
+beta	B-structure_element
+barrel	I-structure_element
+and	O
+one	O
+capping	B-structure_element
+helix	I-structure_element
+).	O
+
+In	O
+contrast	O
+to	O
+PduL	B-protein_type
+,	O
+there	O
+is	O
+only	O
+one	O
+barrel	B-structure_element
+present	O
+in	O
+ethylbenzene	B-protein_type
+dehydrogenase	I-protein_type
+,	O
+and	O
+there	O
+is	O
+no	O
+comparable	O
+active	B-site
+site	I-site
+arrangement	O
+.	O
+
+The	O
+PduL	B-protein_type
+signature	O
+primary	O
+structure	O
+,	O
+two	O
+PF06130	B-structure_element
+domains	O
+,	O
+occurs	O
+in	O
+some	O
+multidomain	O
+proteins	O
+,	O
+most	O
+of	O
+them	O
+annotated	O
+as	O
+Acks	B-protein_type
+,	O
+suggesting	O
+that	O
+PduL	B-protein_type
+may	O
+also	O
+replace	O
+Pta	B-protein_type
+in	O
+variants	O
+of	O
+the	O
+phosphotransacetylase	B-protein_type
+-	O
+Ack	B-protein_type
+pathway	O
+.	O
+
+These	O
+PduL	B-protein_type
+homologs	O
+lack	B-protein_state
+EPs	B-structure_element
+,	O
+and	O
+their	B-protein_type
+fusion	O
+to	O
+Ack	B-protein_type
+may	O
+have	O
+evolved	O
+as	O
+a	O
+way	O
+to	O
+facilitate	O
+substrate	O
+channeling	O
+between	O
+the	O
+two	O
+enzymes	O
+.	O
+
+Implications	O
+for	O
+Metabolosome	B-complex_assembly
+Core	O
+Assembly	O
+
+For	O
+BMC	B-complex_assembly
+-	O
+encapsulated	O
+proteins	O
+to	O
+properly	O
+function	O
+together	O
+,	O
+they	O
+must	O
+be	O
+targeted	O
+to	O
+the	O
+lumen	O
+and	O
+assemble	O
+into	O
+an	O
+organization	O
+that	O
+facilitates	O
+substrate	O
+/	O
+product	O
+channeling	O
+among	O
+the	O
+different	O
+catalytic	B-site
+sites	I-site
+of	O
+the	O
+signature	O
+and	O
+core	O
+enzymes	O
+.	O
+
+The	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extension	I-structure_element
+on	O
+PduL	B-protein_type
+homologs	O
+may	O
+serve	O
+both	O
+of	O
+these	O
+functions	O
+.	O
+
+The	B-structure_element
+extension	I-structure_element
+shares	O
+many	O
+features	O
+with	O
+previously	O
+characterized	O
+EPs	B-structure_element
+:	O
+it	O
+is	O
+present	O
+only	O
+in	O
+homologs	O
+associated	O
+with	O
+BMC	B-gene
+loci	I-gene
+,	O
+and	O
+it	O
+is	O
+predicted	O
+to	O
+form	O
+an	O
+amphipathic	B-protein_state
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+.	O
+
+Moreover	O
+,	O
+its	O
+removal	B-experimental_method
+affects	O
+the	O
+oligomeric	O
+state	O
+of	O
+the	O
+protein	O
+.	O
+
+EP	B-structure_element
+-	O
+mediated	O
+oligomerization	O
+has	O
+been	O
+observed	O
+for	O
+the	O
+signature	O
+and	O
+core	O
+BMC	B-complex_assembly
+enzymes	O
+;	O
+for	O
+example	O
+,	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+propanediol	B-protein_type
+dehydratase	I-protein_type
+and	O
+ethanolamine	B-protein_type
+ammonia	I-protein_type
+-	I-protein_type
+lyase	I-protein_type
+(	O
+signature	O
+enzymes	O
+for	O
+PDU	B-complex_assembly
+and	O
+EUT	B-complex_assembly
+BMCs	I-complex_assembly
+)	O
+subunits	O
+are	O
+also	O
+insoluble	O
+,	O
+but	O
+become	O
+soluble	O
+upon	O
+removal	O
+of	O
+the	O
+predicted	O
+EP	B-structure_element
+.	O
+
+sPduL	B-protein
+has	O
+also	O
+previously	O
+been	O
+reported	O
+to	O
+localize	O
+to	O
+inclusion	O
+bodies	O
+when	O
+overexpressed	B-experimental_method
+;	O
+we	O
+show	O
+here	O
+that	O
+this	O
+is	O
+dependent	O
+on	O
+the	O
+presence	O
+of	O
+the	O
+EP	B-structure_element
+.	O
+
+This	O
+propensity	O
+of	O
+the	O
+EP	B-structure_element
+to	O
+cause	O
+proteins	O
+to	O
+form	O
+complexes	O
+(	O
+Fig	O
+5	O
+)	O
+might	O
+not	O
+be	O
+a	O
+coincidence	O
+,	O
+but	O
+could	O
+be	O
+a	O
+necessary	O
+step	O
+in	O
+the	O
+assembly	O
+of	O
+BMCs	B-complex_assembly
+.	O
+
+Structured	O
+aggregation	O
+of	O
+the	O
+core	O
+enzymes	O
+has	O
+been	O
+proposed	O
+to	O
+be	O
+the	O
+initial	O
+step	O
+in	O
+metabolosome	B-complex_assembly
+assembly	O
+and	O
+is	O
+known	O
+to	O
+be	O
+the	O
+first	O
+step	O
+of	O
+β	O
+-	O
+carboxysome	O
+biogenesis	O
+,	O
+where	O
+the	O
+core	O
+enzyme	O
+Ribulose	B-protein_type
+Bisphosphate	I-protein_type
+Carboxylase	I-protein_type
+/	I-protein_type
+Oxygenase	I-protein_type
+(	O
+RuBisCO	B-protein_type
+)	O
+is	O
+aggregated	O
+by	O
+the	O
+CcmM	B-protein_type
+protein	O
+.	O
+
+Likewise	O
+,	O
+CsoS2	B-protein_type
+,	O
+a	O
+protein	O
+in	O
+the	O
+α	B-complex_assembly
+-	I-complex_assembly
+carboxysome	I-complex_assembly
+core	O
+,	O
+also	O
+aggregates	O
+when	O
+purified	O
+and	O
+is	O
+proposed	O
+to	O
+facilitate	O
+the	O
+nucleation	O
+and	O
+encapsulation	O
+of	O
+RuBisCO	B-protein_type
+molecules	O
+in	O
+the	O
+lumen	O
+of	O
+the	O
+organelle	O
+.	O
+
+This	O
+role	O
+for	O
+EPs	B-structure_element
+in	O
+BMC	B-complex_assembly
+assembly	O
+is	O
+in	O
+addition	O
+to	O
+their	O
+interaction	O
+with	O
+shell	O
+proteins	O
+.	O
+
+Moreover	O
+,	O
+the	O
+PduL	B-protein_type
+crystal	B-evidence
+structures	I-evidence
+offer	O
+a	O
+clue	O
+as	O
+to	O
+how	O
+required	O
+cofactors	O
+enter	O
+the	O
+BMC	B-complex_assembly
+lumen	O
+during	O
+assembly	O
+.	O
+
+Free	O
+CoA	B-chemical
+and	O
+NAD	B-chemical
++/	I-chemical
+H	B-chemical
+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
+
+Our	O
+PduL	B-protein_type
+crystals	B-evidence
+contained	O
+CoA	B-chemical
+that	O
+was	O
+captured	O
+from	O
+the	O
+Escherichia	B-species
+coli	I-species
+cytosol	O
+,	O
+indicating	O
+that	O
+the	O
+“	O
+ground	O
+state	O
+”	O
+of	O
+PduL	B-protein_type
+is	O
+in	O
+the	O
+CoA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+form	O
+;	O
+this	O
+could	O
+provide	O
+an	O
+elegantly	O
+simple	O
+means	O
+of	O
+guaranteeing	O
+a	O
+1	O
+:	O
+1	O
+ratio	O
+of	O
+CoA	B-complex_assembly
+:	I-complex_assembly
+PduL	I-complex_assembly
+within	O
+the	O
+metabolosome	B-complex_assembly
+lumen	O
+.	O
+
+Active	B-site
+Site	I-site
+Identification	O
+and	O
+Structural	O
+Insights	O
+into	O
+Catalysis	O
+
+The	O
+active	B-site
+site	I-site
+of	O
+PduL	B-protein_type
+is	O
+formed	O
+at	O
+the	O
+interface	B-site
+of	O
+the	O
+two	O
+structural	O
+domains	B-structure_element
+(	O
+Fig	O
+2b	O
+).	O
+
+As	O
+expected	O
+,	O
+the	O
+amino	O
+acid	O
+sequence	O
+conservation	O
+is	O
+highest	O
+in	O
+the	O
+region	O
+around	O
+the	O
+proposed	O
+active	B-site
+site	I-site
+(	O
+Fig	O
+4d	O
+);	O
+highly	B-protein_state
+conserved	I-protein_state
+residues	O
+are	O
+also	O
+involved	O
+in	O
+CoA	B-chemical
+binding	O
+(	O
+Figs	O
+2a	O
+and	O
+3	O
+,	O
+residues	O
+Ser45	B-residue_name_number
+,	O
+Lys70	B-residue_name_number
+,	O
+Arg97	B-residue_name_number
+,	O
+Leu99	B-residue_name_number
+,	O
+His204	B-residue_name_number
+,	O
+Asn211	B-residue_name_number
+).	O
+
+All	O
+of	O
+the	O
+metal	B-site
+-	I-site
+coordinating	I-site
+residues	I-site
+(	O
+Fig	O
+2a	O
+)	O
+are	O
+absolutely	B-protein_state
+conserved	I-protein_state
+,	O
+implicating	O
+them	O
+in	O
+catalysis	O
+or	O
+the	O
+correct	O
+spatial	O
+orientation	O
+of	O
+the	O
+substrates	O
+.	O
+
+Arg103	B-residue_name_number
+,	O
+which	O
+contacts	O
+the	O
+phosphate	B-chemical
+(	O
+Fig	O
+4b	O
+),	O
+is	O
+present	O
+in	O
+all	O
+PduL	B-protein_type
+homologs	O
+.	O
+
+The	O
+close	O
+resemblance	O
+between	O
+the	O
+structures	O
+binding	O
+CoA	B-chemical
+and	O
+phosphate	B-chemical
+likely	O
+indicates	O
+that	O
+no	O
+large	O
+changes	O
+in	O
+protein	O
+conformation	O
+are	O
+involved	O
+in	O
+catalysis	O
+,	O
+and	O
+that	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+are	O
+representative	O
+of	O
+the	O
+active	B-protein_state
+form	O
+.	O
+
+The	O
+native	O
+substrate	O
+for	O
+the	O
+forward	O
+reaction	O
+of	O
+rPduL	B-protein
+and	O
+pPduL	B-protein
+,	O
+propionyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+,	O
+most	O
+likely	O
+binds	O
+to	O
+the	O
+enzyme	O
+in	O
+the	O
+same	O
+way	O
+at	O
+the	O
+observed	O
+nucleotide	B-chemical
+and	O
+pantothenic	B-chemical
+acid	I-chemical
+moiety	O
+,	O
+but	O
+the	O
+propionyl	O
+group	O
+in	O
+the	O
+CoA	B-chemical
+-	I-chemical
+thioester	I-chemical
+might	O
+point	O
+in	O
+a	O
+different	O
+direction	O
+.	O
+
+There	O
+is	O
+a	O
+pocket	B-site
+nearby	O
+the	O
+active	B-site
+site	I-site
+between	O
+the	O
+well	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+residues	O
+Ser45	B-residue_name_number
+and	O
+Ala154	B-residue_name_number
+,	O
+which	O
+could	O
+accommodate	O
+the	O
+propionyl	O
+group	O
+(	O
+S6	O
+Fig	O
+).	O
+
+A	O
+homology	B-experimental_method
+model	I-experimental_method
+of	O
+sPduL	B-protein
+indicates	O
+that	O
+the	O
+residues	O
+making	O
+up	O
+this	O
+pocket	B-site
+and	O
+the	O
+surrounding	O
+active	B-site
+site	I-site
+region	O
+are	O
+identical	O
+to	O
+that	O
+of	O
+rPduL	B-protein
+,	O
+which	O
+is	O
+not	O
+surprising	O
+,	O
+because	O
+these	O
+two	O
+homologs	O
+presumably	O
+have	O
+the	O
+same	O
+propionyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+substrate	O
+.	O
+
+The	O
+homology	B-experimental_method
+model	I-experimental_method
+of	O
+pPduL	B-protein
+also	O
+has	O
+identical	O
+residues	O
+making	O
+up	O
+the	O
+pocket	B-site
+,	O
+but	O
+with	O
+a	O
+key	O
+difference	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+active	B-site
+site	I-site
+:	O
+Gln77	B-residue_name_number
+of	O
+rPduL	B-protein
+is	O
+replaced	O
+by	O
+a	O
+tyrosine	B-residue_name
+(	O
+Tyr77	B-residue_name_number
+)	O
+in	O
+pPduL	B-protein
+.	O
+The	O
+physiological	O
+substrate	O
+of	O
+pPduL	B-protein
+(	O
+Table	O
+1	O
+)	O
+is	O
+thought	O
+to	O
+be	O
+lactyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+,	O
+which	O
+contains	O
+an	O
+additional	O
+hydroxyl	O
+group	O
+relative	O
+to	O
+propionyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+The	O
+presence	O
+of	O
+an	O
+aromatic	B-protein_state
+residue	B-structure_element
+at	O
+this	O
+position	O
+may	O
+underlie	O
+the	O
+substrate	O
+preference	O
+of	O
+the	O
+PduL	B-protein_type
+enzyme	O
+from	O
+the	O
+pvm	B-gene
+locus	I-gene
+.	O
+
+Indeed	O
+,	O
+in	O
+the	O
+majority	O
+of	O
+PduLs	B-protein_type
+encoded	O
+in	O
+pvm	B-gene
+loci	I-gene
+,	O
+Gln77	B-residue_name_number
+is	O
+substituted	O
+by	O
+either	O
+a	O
+Tyr	B-residue_name
+or	O
+Phe	B-residue_name
+,	O
+whereas	O
+it	O
+is	O
+typically	O
+a	O
+Gln	B-residue_name
+or	O
+Glu	B-residue_name
+in	O
+PduLs	B-protein_type
+in	O
+all	O
+other	O
+BMC	B-complex_assembly
+types	O
+that	O
+degrade	O
+acetyl	B-chemical
+-	I-chemical
+or	O
+propionyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+A	O
+comparison	B-experimental_method
+of	O
+the	O
+PduL	B-protein_type
+active	B-site
+site	I-site
+to	O
+that	O
+of	O
+the	O
+functionally	O
+identical	O
+Pta	B-protein_type
+suggests	O
+that	O
+the	O
+two	O
+enzymes	O
+have	O
+distinctly	O
+different	O
+mechanisms	O
+.	O
+
+The	O
+catalytic	O
+mechanism	O
+of	O
+Pta	B-protein_type
+involves	O
+the	O
+abstraction	O
+of	O
+a	O
+thiol	O
+hydrogen	O
+by	O
+an	O
+aspartate	B-residue_name
+residue	O
+,	O
+resulting	O
+in	O
+the	O
+nucleophilic	O
+attack	O
+of	O
+thiolate	O
+upon	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+acetyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+,	O
+oriented	O
+by	O
+an	O
+arginine	B-residue_name
+and	O
+stabilized	O
+by	O
+a	O
+serine	B-residue_name
+—	O
+there	O
+are	O
+no	O
+metals	O
+involved	O
+.	O
+
+In	O
+contrast	O
+,	O
+in	O
+the	O
+rPduL	B-protein
+structure	B-evidence
+,	O
+there	O
+are	O
+no	O
+conserved	O
+aspartate	B-residue_name
+residues	O
+in	O
+or	O
+around	O
+the	O
+active	B-site
+site	I-site
+,	O
+and	O
+the	O
+only	O
+well	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+glutamate	B-residue_name
+residue	O
+in	O
+the	O
+active	B-site
+site	I-site
+is	O
+involved	O
+in	O
+coordinating	O
+one	O
+of	O
+the	O
+metal	O
+ions	O
+.	O
+
+These	O
+observations	O
+strongly	O
+suggest	O
+that	O
+an	O
+acidic	B-protein_state
+residue	B-structure_element
+is	O
+not	O
+directly	O
+involved	O
+in	O
+catalysis	O
+by	O
+PduL	B-protein_type
+.	O
+Instead	O
+,	O
+the	O
+dimetal	B-site
+active	I-site
+site	I-site
+of	O
+PduL	B-protein_type
+may	O
+create	O
+a	O
+nucleophile	O
+from	O
+one	O
+of	O
+the	O
+hydroxyl	O
+groups	O
+on	O
+free	O
+phosphate	B-chemical
+to	O
+attack	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+the	O
+thioester	O
+bond	O
+of	O
+an	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+In	O
+the	O
+reverse	O
+direction	O
+,	O
+the	O
+metal	O
+ion	O
+(	O
+s	O
+)	O
+could	O
+stabilize	O
+the	O
+thiolate	O
+anion	O
+that	O
+would	O
+attack	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+an	O
+acyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+;	O
+a	O
+similar	O
+mechanism	O
+has	O
+been	O
+described	O
+for	O
+phosphatases	B-protein_type
+where	O
+hydroxyl	O
+groups	O
+or	O
+hydroxide	O
+ions	O
+can	O
+act	O
+as	O
+a	O
+base	O
+when	O
+coordinated	O
+by	O
+a	O
+dimetal	B-site
+active	I-site
+site	I-site
+.	O
+
+Our	O
+structures	B-evidence
+provide	O
+the	O
+foundation	O
+for	O
+studies	O
+to	O
+elucidate	O
+the	O
+details	O
+of	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+PduL	B-protein_type
+.	O
+Conserved	B-protein_state
+residues	O
+in	O
+the	O
+active	B-site
+site	I-site
+that	O
+may	O
+contribute	O
+to	O
+substrate	O
+binding	O
+and	O
+/	O
+or	O
+transition	O
+state	O
+stabilization	O
+include	O
+Ser127	B-residue_name_number
+,	O
+Arg103	B-residue_name_number
+,	O
+Arg194	B-residue_name_number
+,	O
+Gln107	B-residue_name_number
+,	O
+Gln74	B-residue_name_number
+,	O
+and	O
+Gln	B-residue_name_number
+/	O
+Glu77	B-residue_name_number
+.	O
+
+In	O
+the	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystal	B-evidence
+structure	I-evidence
+,	O
+Ser127	B-residue_name_number
+and	O
+Arg103	B-residue_name_number
+appear	O
+to	O
+position	O
+the	O
+phosphate	B-chemical
+(	O
+Fig	O
+4b	O
+).	O
+
+Alternatively	O
+,	O
+Arg103	B-residue_name_number
+might	O
+act	O
+as	O
+a	O
+base	O
+to	O
+render	O
+the	O
+phosphate	B-chemical
+more	O
+nucleophilic	O
+.	O
+
+The	O
+functional	O
+groups	O
+of	O
+Gln74	B-residue_name_number
+,	O
+Gln	B-residue_name_number
+/	O
+Glu77	B-residue_name_number
+,	O
+and	O
+Arg194	B-residue_name_number
+are	O
+directed	O
+away	O
+from	O
+the	O
+active	B-site
+site	I-site
+in	O
+both	O
+CoA	B-protein_state
+and	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystal	B-evidence
+structures	I-evidence
+and	O
+do	O
+not	O
+appear	O
+to	O
+be	O
+involved	O
+in	O
+hydrogen	O
+bonding	O
+with	O
+these	O
+substrates	O
+,	O
+although	O
+they	O
+could	O
+be	O
+important	O
+for	O
+positioning	O
+an	O
+acyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+.	O
+
+The	O
+free	O
+CoA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+form	O
+is	O
+presumably	O
+poised	O
+for	O
+attack	O
+upon	O
+an	O
+acyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+,	O
+indicating	O
+that	O
+the	O
+enzyme	O
+initially	O
+binds	O
+CoA	B-chemical
+as	O
+opposed	O
+to	O
+acyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+.	O
+
+This	O
+hypothesis	O
+is	O
+strengthened	O
+by	O
+the	O
+fact	O
+that	O
+the	O
+CoA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystals	B-evidence
+were	O
+obtained	O
+without	O
+added	O
+CoA	B-chemical
+,	O
+indicating	O
+that	O
+the	O
+protein	O
+bound	B-protein_state
+CoA	B-chemical
+from	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+expression	O
+strain	O
+and	O
+retained	O
+it	O
+throughout	O
+purification	O
+and	O
+crystallization	O
+.	O
+
+The	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+indicates	O
+that	O
+in	O
+the	O
+opposite	O
+reaction	O
+direction	O
+phosphate	B-chemical
+is	O
+bound	O
+first	O
+,	O
+and	O
+then	O
+an	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+enters	O
+.	O
+
+The	O
+two	O
+high	O
+-	O
+resolution	O
+crystal	B-evidence
+structures	I-evidence
+presented	O
+here	O
+will	O
+serve	O
+as	O
+the	O
+foundation	O
+for	O
+mechanistic	O
+studies	O
+on	O
+this	O
+noncanonical	O
+PTAC	B-protein_type
+enzyme	O
+to	O
+determine	O
+how	O
+the	O
+dimetal	B-site
+active	I-site
+site	I-site
+functions	O
+to	O
+catalyze	O
+both	O
+forward	O
+and	O
+reverse	O
+reactions	O
+.	O
+
+Functional	O
+,	O
+but	O
+Not	O
+Structural	O
+,	O
+Convergence	O
+of	O
+PduL	B-protein_type
+and	O
+Pta	B-protein_type
+
+PduL	B-protein_type
+and	O
+Pta	B-protein_type
+are	O
+mechanistically	O
+and	O
+structurally	O
+distinct	O
+enzymes	O
+that	O
+catalyze	O
+the	O
+same	O
+reaction	O
+,	O
+a	O
+prime	O
+example	O
+of	O
+evolutionary	O
+convergence	O
+upon	O
+a	O
+function	O
+.	O
+
+There	O
+are	O
+several	O
+examples	O
+of	O
+such	O
+functional	O
+convergence	O
+of	O
+enzymes	O
+,	O
+although	O
+typically	O
+the	O
+enzymes	O
+have	O
+independently	O
+evolved	O
+similar	O
+,	O
+or	O
+even	O
+identical	O
+active	B-site
+sites	I-site
+;	O
+for	O
+example	O
+,	O
+the	O
+carbonic	B-protein_type
+anhydrase	I-protein_type
+family	O
+.	O
+
+However	O
+,	O
+apparently	O
+less	O
+frequent	O
+is	O
+functional	O
+convergence	O
+that	O
+is	O
+supported	O
+by	O
+distinctly	O
+different	O
+active	B-site
+sites	I-site
+and	O
+accordingly	O
+catalytic	O
+mechanism	O
+,	O
+as	O
+revealed	O
+by	O
+comparison	O
+of	O
+the	O
+structures	O
+of	O
+Pta	B-protein_type
+and	O
+PduL	B-protein_type
+.	O
+One	O
+well	O
+-	O
+studied	O
+example	O
+of	O
+this	O
+is	O
+the	O
+β	B-protein_type
+-	I-protein_type
+lactamase	I-protein_type
+family	O
+of	O
+enzymes	O
+,	O
+in	O
+which	O
+the	O
+active	B-site
+site	I-site
+of	O
+Class	O
+A	O
+and	O
+Class	O
+C	O
+enzymes	O
+involve	O
+serine	O
+-	O
+based	O
+catalysis	O
+,	O
+but	O
+Class	O
+B	O
+enzymes	O
+are	O
+metalloproteins	B-protein_type
+.	O
+
+This	O
+is	O
+not	O
+surprising	O
+,	O
+as	O
+β	B-protein_type
+-	I-protein_type
+lactamases	I-protein_type
+are	O
+not	O
+so	O
+widespread	O
+among	O
+bacteria	B-taxonomy_domain
+and	O
+therefore	O
+would	O
+be	O
+expected	O
+to	O
+have	O
+evolved	O
+independently	O
+several	O
+times	O
+as	O
+a	O
+defense	O
+mechanism	O
+against	O
+β	O
+-	O
+lactam	O
+antibiotics	O
+.	O
+
+However	O
+,	O
+nearly	O
+all	O
+bacteria	B-taxonomy_domain
+encode	O
+Pta	B-protein_type
+,	O
+and	O
+it	O
+is	O
+not	O
+immediately	O
+clear	O
+why	O
+the	O
+Pta	B-protein_type
+/	O
+PduL	B-protein_type
+functional	O
+convergence	O
+should	O
+have	O
+evolved	O
+:	O
+it	O
+would	O
+seem	O
+to	O
+be	O
+evolutionarily	O
+more	O
+resourceful	O
+for	O
+the	O
+Pta	B-gene
+-	I-gene
+encoding	I-gene
+gene	I-gene
+to	O
+be	O
+duplicated	O
+and	O
+repurposed	O
+for	O
+BMCs	B-complex_assembly
+,	O
+as	O
+is	O
+apparently	O
+the	O
+case	O
+in	O
+one	O
+type	O
+of	O
+BMC	B-complex_assembly
+—	I-complex_assembly
+EUT1	I-complex_assembly
+(	O
+Table	O
+1	O
+).	O
+
+There	O
+could	O
+be	O
+some	O
+intrinsic	O
+biochemical	O
+difference	O
+between	O
+the	O
+two	O
+enzymes	O
+that	O
+renders	O
+PduL	B-protein_type
+a	O
+more	O
+attractive	O
+candidate	O
+for	O
+encapsulation	O
+in	O
+a	O
+BMC	B-complex_assembly
+—	O
+for	O
+example	O
+,	O
+PduL	B-protein_type
+might	O
+be	O
+more	O
+amenable	O
+to	O
+tight	O
+packaging	O
+,	O
+or	O
+is	O
+better	O
+suited	O
+for	O
+the	O
+chemical	O
+microenvironment	O
+formed	O
+within	O
+the	O
+lumen	O
+of	O
+the	O
+BMC	B-complex_assembly
+,	O
+which	O
+can	O
+be	O
+quite	O
+different	O
+from	O
+the	O
+cytosol	O
+.	O
+
+Further	O
+biochemical	O
+comparison	O
+between	O
+the	O
+two	O
+PTACs	B-protein_type
+will	O
+likely	O
+yield	O
+exciting	O
+results	O
+that	O
+could	O
+answer	O
+this	O
+evolutionary	O
+question	O
+.	O
+
+BMCs	B-complex_assembly
+are	O
+now	O
+known	O
+to	O
+be	O
+widespread	O
+among	O
+the	O
+bacteria	B-taxonomy_domain
+and	O
+are	O
+involved	O
+in	O
+critical	O
+segments	O
+of	O
+both	O
+autotrophic	O
+and	O
+heterotrophic	O
+biochemical	O
+pathways	O
+that	O
+confer	O
+to	O
+the	O
+host	O
+organism	O
+a	O
+competitive	O
+(	O
+metabolic	O
+)	O
+advantage	O
+in	O
+select	O
+niches	O
+.	O
+
+As	O
+one	O
+of	O
+the	O
+three	O
+common	O
+metabolosome	B-complex_assembly
+core	O
+enzymes	O
+,	O
+the	O
+structure	B-evidence
+of	O
+PduL	B-protein_type
+provides	O
+a	O
+key	O
+missing	O
+piece	O
+to	O
+our	O
+structural	O
+picture	O
+of	O
+the	O
+shared	O
+core	O
+biochemistry	O
+(	O
+Fig	O
+1	O
+)	O
+of	O
+functionally	O
+diverse	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+.	O
+
+We	O
+have	O
+observed	O
+the	O
+oligomeric	O
+state	O
+differences	O
+of	O
+PduL	B-protein_type
+to	O
+correlate	O
+with	O
+the	O
+presence	O
+of	O
+an	O
+EP	B-structure_element
+,	O
+providing	O
+new	O
+insight	O
+into	O
+the	O
+function	O
+of	O
+this	O
+sequence	O
+extension	O
+in	O
+BMC	B-complex_assembly
+assembly	O
+.	O
+
+Moreover	O
+,	O
+our	O
+results	O
+suggest	O
+a	O
+means	O
+for	O
+Coenzyme	B-chemical
+A	I-chemical
+incorporation	O
+during	O
+metabolosome	B-complex_assembly
+biogenesis	O
+.	O
+
+A	O
+detailed	O
+understanding	O
+of	O
+the	O
+underlying	O
+principles	O
+governing	O
+the	O
+assembly	O
+and	O
+internal	O
+structural	O
+organization	O
+of	O
+BMCs	B-complex_assembly
+is	O
+a	O
+requisite	O
+for	O
+synthetic	O
+biologists	O
+to	O
+design	O
+custom	O
+nanoreactors	O
+that	O
+use	O
+BMC	B-complex_assembly
+architectures	O
+as	O
+a	O
+template	O
+.	O
+
+Furthermore	O
+,	O
+given	O
+the	O
+growing	O
+number	O
+of	O
+metabolosomes	B-complex_assembly
+implicated	O
+in	O
+pathogenesis	O
+,	O
+the	O
+PduL	B-protein_type
+structure	B-evidence
+will	O
+be	O
+useful	O
+in	O
+the	O
+development	O
+of	O
+therapeutics	O
+.	O
+
+The	O
+fact	O
+that	O
+PduL	B-protein_type
+is	O
+confined	O
+almost	O
+exclusively	O
+to	O
+metabolosomes	B-complex_assembly
+can	O
+be	O
+used	O
+to	O
+develop	O
+an	O
+inhibitor	O
+that	O
+blocks	O
+only	O
+PduL	B-protein_type
+and	O
+not	O
+Pta	B-protein_type
+as	O
+a	O
+way	O
+to	O
+selectively	O
+disrupt	O
+BMC	B-complex_assembly
+-	O
+based	O
+metabolism	O
+,	O
+while	O
+not	O
+affecting	O
+most	O
+commensal	O
+organisms	O
+that	O
+require	O
+PTAC	B-protein_type
+activity	O
+.	O
+
+Structural	O
+basis	O
+for	O
+the	O
+regulation	O
+of	O
+enzymatic	O
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+by	O
+domain	O
+-	O
+domain	O
+interactions	O
+
+Regnase	B-protein
+-	I-protein
+1	I-protein
+is	O
+an	O
+RNase	B-protein_type
+that	O
+directly	O
+cleaves	O
+mRNAs	B-chemical
+of	O
+inflammatory	O
+genes	O
+such	O
+as	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+and	O
+IL	B-protein_type
+-	I-protein_type
+12p40	I-protein_type
+,	O
+and	O
+negatively	O
+regulates	O
+cellular	O
+inflammatory	O
+responses	O
+.	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+structures	B-evidence
+of	O
+four	O
+domains	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+from	O
+Mus	B-species
+musculus	I-species
+—	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+NTD	B-structure_element
+),	O
+PilT	B-structure_element
+N	I-structure_element
+-	I-structure_element
+terminus	I-structure_element
+like	I-structure_element
+(	O
+PIN	B-structure_element
+)	O
+domain	O
+,	O
+zinc	B-structure_element
+finger	I-structure_element
+(	O
+ZF	B-structure_element
+)	O
+domain	O
+and	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+CTD	B-structure_element
+).	O
+
+The	O
+PIN	B-structure_element
+domain	O
+harbors	O
+the	O
+RNase	B-protein_type
+catalytic	B-site
+center	I-site
+;	O
+however	O
+,	O
+it	O
+is	O
+insufficient	O
+for	O
+enzymatic	O
+activity	O
+.	O
+
+We	O
+found	O
+that	O
+the	O
+NTD	B-structure_element
+associates	O
+with	O
+the	O
+PIN	B-structure_element
+domain	O
+and	O
+significantly	O
+enhances	O
+its	O
+RNase	B-protein_type
+activity	O
+.	O
+
+The	O
+PIN	B-structure_element
+domain	O
+forms	O
+a	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+oligomer	B-oligomeric_state
+and	O
+the	O
+dimer	B-site
+interface	I-site
+overlaps	O
+with	O
+the	O
+NTD	B-site
+binding	I-site
+site	I-site
+.	O
+
+Interestingly	O
+,	O
+mutations	B-experimental_method
+blocking	O
+PIN	B-structure_element
+oligomerization	O
+had	O
+no	O
+RNase	B-protein_type
+activity	O
+,	O
+indicating	O
+that	O
+both	O
+oligomerization	O
+and	O
+NTD	B-structure_element
+binding	O
+are	O
+crucial	O
+for	O
+RNase	B-protein_type
+activity	O
+in	O
+vitro	O
+.	O
+
+These	O
+results	O
+suggest	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+activity	O
+is	O
+tightly	O
+controlled	O
+by	O
+both	O
+intramolecular	O
+(	O
+NTD	B-structure_element
+-	O
+PIN	B-structure_element
+)	O
+and	O
+intermolecular	O
+(	O
+PIN	B-structure_element
+-	O
+PIN	B-structure_element
+)	O
+interactions	O
+.	O
+
+The	O
+initial	O
+sensing	O
+of	O
+infection	O
+is	O
+mediated	O
+by	O
+a	O
+set	O
+of	O
+pattern	B-protein_type
+-	I-protein_type
+recognition	I-protein_type
+receptors	I-protein_type
+(	O
+PRRs	B-protein_type
+)	O
+such	O
+Toll	B-protein_type
+-	I-protein_type
+like	I-protein_type
+receptors	I-protein_type
+(	O
+TLRs	B-protein_type
+)	O
+and	O
+the	O
+intracellular	O
+signaling	O
+cascades	O
+triggered	O
+by	O
+TLRs	B-protein_type
+evoke	O
+transcriptional	O
+expression	O
+of	O
+inflammatory	O
+mediators	O
+that	O
+coordinate	O
+the	O
+elimination	O
+of	O
+pathogens	O
+and	O
+infected	O
+cells	O
+.	O
+
+Regnase	B-protein
+-	I-protein
+1	I-protein
+(	O
+also	O
+known	O
+as	O
+Zc3h12a	B-protein
+and	O
+MCPIP1	B-protein
+)	O
+is	O
+an	O
+RNase	B-protein_type
+whose	O
+expression	O
+level	O
+is	O
+stimulated	O
+by	O
+lipopolysaccharides	B-chemical
+and	O
+prevents	O
+autoimmune	O
+diseases	O
+by	O
+directly	O
+controlling	O
+the	O
+stability	O
+of	O
+mRNAs	B-chemical
+of	O
+inflammatory	O
+genes	O
+such	O
+as	O
+interleukin	O
+(	B-protein_type
+IL	I-protein_type
+)-	I-protein_type
+6	I-protein_type
+,	O
+IL	B-protein_type
+-	I-protein_type
+1β	I-protein_type
+,	O
+IL	B-protein_type
+-	I-protein_type
+2	I-protein_type
+,	O
+and	O
+IL	B-protein_type
+-	I-protein_type
+12p40	I-protein_type
+.	O
+
+Regnase	B-protein
+-	I-protein
+1	I-protein
+accelerates	O
+target	O
+mRNA	B-chemical
+degradation	O
+via	O
+their	O
+3	B-structure_element
+′-	I-structure_element
+terminal	I-structure_element
+untranslated	I-structure_element
+region	I-structure_element
+(	O
+3	B-structure_element
+′	I-structure_element
+UTR	I-structure_element
+),	O
+and	O
+also	O
+degrades	O
+its	O
+own	O
+mRNA	B-chemical
+.	O
+
+Regnase	B-protein
+-	I-protein
+1	I-protein
+is	O
+a	O
+member	O
+of	O
+Regnase	B-protein_type
+family	I-protein_type
+and	O
+is	O
+composed	O
+of	O
+a	O
+PilT	B-structure_element
+N	I-structure_element
+-	I-structure_element
+terminus	I-structure_element
+like	I-structure_element
+(	O
+PIN	B-structure_element
+)	O
+domain	O
+followed	O
+by	O
+a	O
+CCCH	B-structure_element
+-	I-structure_element
+type	I-structure_element
+zinc	I-structure_element
+–	I-structure_element
+finger	I-structure_element
+(	O
+ZF	B-structure_element
+)	O
+domain	O
+,	O
+which	O
+are	O
+conserved	B-protein_state
+among	O
+Regnase	B-protein_type
+family	I-protein_type
+members	I-protein_type
+.	O
+
+Recently	O
+,	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+PIN	B-structure_element
+domain	O
+derived	O
+from	O
+Homo	B-species
+sapiens	I-species
+was	O
+reported	O
+.	O
+
+The	O
+structure	B-evidence
+combined	O
+with	O
+functional	O
+analyses	O
+revealed	O
+that	O
+four	O
+catalytically	O
+important	O
+Asp	B-residue_name
+residues	O
+form	O
+the	O
+catalytic	B-site
+center	I-site
+and	O
+stabilize	O
+Mg2	B-chemical
++	I-chemical
+binding	O
+that	O
+is	O
+crucial	O
+for	O
+RNase	B-protein_type
+activity	O
+.	O
+
+Several	O
+CCCH	B-structure_element
+-	I-structure_element
+type	I-structure_element
+ZF	I-structure_element
+motifs	I-structure_element
+in	O
+RNA	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+have	O
+been	O
+reported	O
+to	O
+directly	O
+bind	O
+RNA	B-chemical
+.	O
+
+In	O
+addition	O
+,	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+has	O
+been	O
+predicted	O
+to	O
+possess	O
+other	O
+domains	O
+in	O
+the	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+regions	I-structure_element
+.	O
+
+However	O
+,	O
+the	O
+structure	B-evidence
+and	O
+function	O
+of	O
+the	O
+ZF	B-structure_element
+domain	O
+,	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+NTD	B-structure_element
+)	O
+and	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+CTD	B-structure_element
+)	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+have	O
+not	O
+been	O
+solved	O
+.	O
+
+Here	O
+,	O
+we	O
+performed	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+analyses	I-experimental_method
+of	O
+individual	O
+domains	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+derived	O
+from	O
+Mus	B-species
+musculus	I-species
+in	O
+order	O
+to	O
+understand	O
+the	O
+catalytic	O
+activity	O
+in	O
+vitro	O
+.	O
+
+Our	O
+data	O
+revealed	O
+that	O
+the	O
+catalytic	O
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+is	O
+regulated	O
+through	O
+both	O
+intra	O
+and	O
+intermolecular	O
+domain	O
+interactions	O
+in	O
+vitro	O
+.	O
+
+The	O
+NTD	B-structure_element
+plays	O
+a	O
+crucial	O
+role	O
+in	O
+efficient	O
+cleavage	O
+of	O
+target	O
+mRNA	B-chemical
+,	O
+through	O
+intramolecular	O
+NTD	B-structure_element
+-	O
+PIN	B-structure_element
+interactions	O
+.	O
+
+Moreover	O
+,	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+functions	O
+as	O
+a	O
+dimer	B-oligomeric_state
+through	O
+intermolecular	O
+PIN	B-structure_element
+-	O
+PIN	B-structure_element
+interactions	O
+during	O
+cleavage	O
+of	O
+target	O
+mRNA	B-chemical
+.	O
+
+Our	O
+findings	O
+suggest	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+cleaves	O
+its	O
+target	O
+mRNA	B-chemical
+by	O
+an	O
+NTD	B-protein_state
+-	I-protein_state
+activated	I-protein_state
+functional	B-protein_state
+PIN	B-structure_element
+dimer	B-oligomeric_state
+,	O
+while	O
+the	O
+ZF	B-structure_element
+increases	O
+RNA	B-chemical
+affinity	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+PIN	B-structure_element
+dimer	B-oligomeric_state
+.	O
+
+Domain	O
+structures	B-evidence
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+
+We	O
+analyzed	O
+Rengase	B-protein
+-	I-protein
+1	I-protein
+derived	O
+from	O
+Mus	B-species
+musculus	I-species
+and	O
+solved	B-experimental_method
+the	O
+structures	B-evidence
+of	O
+the	O
+four	O
+domains	O
+;	O
+NTD	B-structure_element
+,	O
+PIN	B-structure_element
+,	O
+ZF	B-structure_element
+,	O
+and	O
+CTD	B-structure_element
+individually	O
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+or	O
+NMR	B-experimental_method
+(	O
+Fig	O
+.	O
+1a	O
+–	O
+e	O
+).	O
+
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+was	O
+attempted	O
+for	O
+the	O
+fragment	O
+containing	O
+both	O
+the	O
+PIN	B-structure_element
+and	O
+ZF	B-structure_element
+domains	O
+,	O
+however	O
+,	O
+electron	B-evidence
+density	I-evidence
+was	O
+observed	O
+only	O
+for	O
+the	O
+PIN	B-structure_element
+domain	O
+(	O
+Fig	O
+.	O
+1c	O
+),	O
+consistent	O
+with	O
+a	O
+previous	O
+report	O
+on	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+derived	O
+from	O
+Homo	B-species
+sapiens	I-species
+.	O
+
+This	O
+suggests	O
+that	O
+the	O
+PIN	B-structure_element
+and	O
+ZF	B-structure_element
+domains	O
+exist	O
+independently	O
+without	O
+interacting	O
+with	O
+each	O
+other	O
+.	O
+
+The	O
+domain	O
+structures	B-evidence
+of	O
+NTD	B-structure_element
+,	O
+ZF	B-structure_element
+,	O
+and	O
+CTD	B-structure_element
+were	O
+determined	O
+by	O
+NMR	B-experimental_method
+(	O
+Fig	O
+.	O
+1b	O
+,	O
+d	O
+,	O
+e	O
+).	O
+
+The	O
+NTD	B-structure_element
+and	O
+CTD	B-structure_element
+are	O
+both	O
+composed	O
+of	O
+three	O
+α	B-structure_element
+helices	I-structure_element
+,	O
+and	O
+structurally	O
+resemble	O
+ubiquitin	B-protein
+conjugating	I-protein
+enzyme	I-protein
+E2	I-protein
+K	I-protein
+(	O
+PDB	O
+ID	O
+:	O
+3K9O	O
+)	O
+and	O
+ubiquitin	B-protein
+associated	I-protein
+protein	I-protein
+1	I-protein
+(	O
+PDB	O
+ID	O
+:	O
+4AE4	O
+),	O
+respectively	O
+,	O
+according	O
+to	O
+the	O
+Dali	B-experimental_method
+server	I-experimental_method
+.	O
+
+Contribution	O
+of	O
+each	O
+domain	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+to	O
+the	O
+mRNA	B-chemical
+binding	O
+activity	O
+
+Although	O
+the	O
+PIN	B-structure_element
+domain	O
+is	O
+responsible	O
+for	O
+the	O
+catalytic	O
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+,	O
+the	O
+roles	O
+of	O
+the	O
+other	O
+domains	O
+are	O
+largely	O
+unknown	O
+.	O
+
+First	O
+,	O
+we	O
+evaluated	O
+a	O
+role	O
+of	O
+the	O
+NTD	B-structure_element
+and	O
+ZF	B-structure_element
+domains	O
+for	O
+mRNA	B-chemical
+binding	O
+by	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+gel	I-experimental_method
+shift	I-experimental_method
+assay	I-experimental_method
+(	O
+Fig	O
+.	O
+1f	O
+).	O
+
+Fluorescently	B-protein_state
+5	I-protein_state
+′-	I-protein_state
+labeled	I-protein_state
+RNA	B-chemical
+corresponding	O
+to	O
+nucleotides	O
+82	O
+–	O
+106	O
+of	O
+the	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+3	B-structure_element
+′	I-structure_element
+UTR	I-structure_element
+and	O
+the	O
+catalytically	O
+inactive	B-protein_state
+mutant	B-protein_state
+(	O
+D226N	B-mutant
+and	O
+D244N	B-mutant
+)	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+—	O
+hereafter	O
+referred	O
+to	O
+as	O
+the	O
+DDNN	B-mutant
+mutant	B-protein_state
+—	O
+were	O
+utilized	O
+.	O
+
+Upon	O
+addition	O
+of	O
+a	O
+larger	O
+amount	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+,	O
+the	O
+fluorescence	B-evidence
+of	O
+free	B-protein_state
+RNA	B-chemical
+decreased	O
+,	O
+indicating	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+RNA	B-chemical
+.	O
+
+Based	O
+on	O
+the	O
+decrease	O
+in	O
+the	O
+free	O
+RNA	B-chemical
+fluorescence	O
+band	O
+,	O
+we	O
+evaluated	O
+the	O
+contribution	O
+of	O
+each	O
+domain	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+to	O
+RNA	B-chemical
+binding	O
+.	O
+
+While	O
+the	O
+RNA	B-chemical
+binding	O
+ability	O
+was	O
+not	O
+significantly	O
+changed	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+NTD	B-structure_element
+,	O
+it	O
+increased	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+ZF	B-structure_element
+domain	O
+(	O
+Fig	O
+.	O
+1f	O
+,	O
+g	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+
+Direct	O
+binding	O
+of	O
+the	O
+ZF	B-structure_element
+domain	O
+and	O
+RNA	B-chemical
+were	O
+confirmed	O
+by	O
+NMR	B-experimental_method
+spectral	B-evidence
+changes	I-evidence
+.	O
+
+The	O
+fitting	O
+of	O
+the	O
+titration	B-evidence
+curve	I-evidence
+of	O
+Y314	B-residue_name_number
+resulted	O
+in	O
+an	O
+apparent	O
+dissociation	B-evidence
+constant	I-evidence
+(	O
+Kd	B-evidence
+)	O
+of	O
+10	O
+±	O
+1	O
+.	O
+1	O
+μM	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+
+These	O
+results	O
+indicate	O
+that	O
+not	O
+only	O
+the	O
+PIN	B-structure_element
+but	O
+also	O
+the	O
+ZF	B-structure_element
+domain	O
+contribute	O
+to	O
+RNA	B-chemical
+binding	O
+,	O
+while	O
+the	O
+NTD	B-structure_element
+is	O
+not	O
+likely	O
+to	O
+be	O
+involved	O
+in	O
+direct	O
+interaction	O
+with	O
+RNA	B-chemical
+.	O
+
+Contribution	O
+of	O
+each	O
+domain	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+to	O
+RNase	B-protein_type
+activity	O
+
+In	O
+order	O
+to	O
+characterize	O
+the	O
+role	O
+of	O
+each	O
+domain	O
+in	O
+the	O
+RNase	B-protein_type
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+,	O
+we	O
+performed	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+cleavage	I-experimental_method
+assay	I-experimental_method
+using	O
+fluorescently	B-protein_state
+5	I-protein_state
+′-	I-protein_state
+labeled	I-protein_state
+RNA	B-chemical
+corresponding	O
+to	O
+nucleotides	O
+82	O
+–	O
+106	O
+of	O
+the	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+3	B-structure_element
+′	I-structure_element
+UTR	I-structure_element
+(	O
+Fig	O
+.	O
+1g	O
+).	O
+
+Regnase	B-protein
+-	I-protein
+1	I-protein
+constructs	O
+consisting	O
+of	O
+NTD	B-mutant
+-	I-mutant
+PIN	I-mutant
+-	I-mutant
+ZF	I-mutant
+completely	O
+cleaved	O
+the	O
+target	O
+mRNA	B-chemical
+and	O
+generated	O
+the	O
+cleaved	O
+products	O
+.	O
+
+The	O
+apparent	O
+half	O
+-	O
+life	O
+(	O
+T1	O
+/	O
+2	O
+)	O
+of	O
+the	O
+RNase	B-protein_type
+activity	O
+was	O
+about	O
+20	O
+minutes	O
+.	O
+
+Regnase	B-protein
+-	I-protein
+1	I-protein
+lacking	B-protein_state
+the	O
+ZF	B-structure_element
+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	B-protein_state
+the	O
+NTD	B-structure_element
+did	O
+not	O
+generate	O
+cleaved	O
+products	O
+(	O
+T1	O
+/	O
+2	O
+>	O
+90	O
+minutes	O
+).	O
+
+It	O
+should	O
+be	O
+noted	O
+that	O
+NTD	B-mutant
+-	I-mutant
+PIN	I-mutant
+(	I-mutant
+DDNN	I-mutant
+)-	I-mutant
+ZF	I-mutant
+,	O
+which	O
+possesses	O
+the	O
+NTD	B-structure_element
+but	O
+lacks	B-protein_state
+the	O
+catalytic	B-site
+residues	I-site
+in	O
+PIN	B-structure_element
+,	O
+completely	O
+lost	O
+all	O
+RNase	B-protein_type
+activity	O
+(	O
+Fig	O
+.	O
+1g	O
+,	O
+right	O
+panel	O
+),	O
+as	O
+expected	O
+,	O
+confirming	O
+that	O
+the	O
+RNase	B-protein_type
+catalytic	B-site
+center	I-site
+is	O
+located	O
+in	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+Taken	O
+together	O
+with	O
+the	O
+results	O
+in	O
+the	O
+previous	O
+section	O
+,	O
+we	O
+conclude	O
+that	O
+the	O
+NTD	B-structure_element
+is	O
+crucial	O
+for	O
+the	O
+RNase	B-protein_type
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+in	O
+vitro	O
+,	O
+although	O
+it	O
+does	O
+not	O
+contribute	O
+to	O
+the	O
+direct	O
+mRNA	B-chemical
+binding	O
+.	O
+
+Dimer	B-oligomeric_state
+formation	O
+of	O
+the	O
+PIN	B-structure_element
+domains	O
+
+During	O
+purification	B-experimental_method
+by	O
+gel	B-experimental_method
+filtration	I-experimental_method
+,	O
+the	O
+PIN	B-structure_element
+domain	O
+exhibited	O
+extremely	O
+asymmetric	O
+elution	O
+peaks	O
+in	O
+a	O
+concentration	O
+dependent	O
+manner	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+
+By	O
+comparison	B-experimental_method
+with	I-experimental_method
+the	I-experimental_method
+elution	I-experimental_method
+volume	I-experimental_method
+of	I-experimental_method
+standard	I-experimental_method
+marker	I-experimental_method
+proteins	I-experimental_method
+,	O
+the	O
+PIN	B-structure_element
+domain	O
+was	O
+assumed	O
+to	O
+be	O
+in	O
+equilibrium	O
+between	O
+a	O
+monomer	B-oligomeric_state
+and	O
+a	O
+dimer	B-oligomeric_state
+in	O
+solution	O
+at	O
+concentrations	O
+in	O
+the	O
+20	O
+–	O
+200	O
+μM	O
+range	O
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+has	O
+been	O
+determined	O
+in	O
+three	O
+distinct	O
+crystal	B-evidence
+forms	I-evidence
+with	O
+a	O
+space	O
+group	O
+of	O
+P3121	O
+(	O
+form	O
+I	O
+in	O
+this	O
+study	O
+and	O
+PDB	O
+ID	O
+3V33	O
+),	O
+P3221	O
+(	O
+form	O
+II	O
+in	O
+this	O
+study	O
+),	O
+and	O
+P41	O
+(	O
+PDB	O
+ID	O
+3V32	O
+and	O
+3V34	O
+),	O
+respectively	O
+.	O
+
+We	O
+found	O
+that	O
+the	O
+PIN	B-structure_element
+domain	O
+formed	O
+a	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+oligomer	B-oligomeric_state
+that	O
+was	O
+commonly	O
+observed	O
+in	O
+all	O
+three	O
+crystal	B-evidence
+forms	I-evidence
+in	O
+spite	O
+of	O
+the	O
+different	O
+crystallization	O
+conditions	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+
+Mutation	B-experimental_method
+of	O
+Arg215	B-residue_name_number
+,	O
+whose	O
+side	O
+chain	O
+faces	O
+to	O
+the	O
+opposite	O
+side	O
+of	O
+the	O
+oligomeric	B-site
+surface	I-site
+,	O
+to	O
+Glu	B-residue_name
+preserved	O
+the	O
+monomer	B-oligomeric_state
+/	O
+dimer	B-oligomeric_state
+equilibrium	O
+,	O
+similar	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+single	B-experimental_method
+mutations	I-experimental_method
+of	O
+side	O
+chains	O
+involved	O
+in	O
+the	O
+PIN	B-structure_element
+–	O
+PIN	B-structure_element
+oligomeric	O
+interaction	O
+resulted	O
+in	O
+monomer	B-oligomeric_state
+formation	O
+,	O
+judging	O
+from	O
+gel	B-experimental_method
+filtration	I-experimental_method
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+).	O
+
+Wild	B-protein_state
+type	I-protein_state
+and	O
+monomeric	B-oligomeric_state
+PIN	B-structure_element
+mutants	B-protein_state
+(	O
+P212A	B-mutant
+and	O
+D278R	B-mutant
+)	O
+were	O
+also	O
+analyzed	O
+by	O
+NMR	B-experimental_method
+.	O
+
+The	O
+spectra	B-evidence
+indicate	O
+that	O
+the	O
+dimer	B-site
+interface	I-site
+of	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+PIN	B-structure_element
+domain	O
+were	O
+significantly	O
+broadened	O
+compared	O
+to	O
+the	O
+monomeric	B-oligomeric_state
+mutants	B-protein_state
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+
+These	O
+results	O
+indicate	O
+that	O
+the	O
+PIN	B-structure_element
+domain	O
+forms	O
+a	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+oligomer	B-oligomeric_state
+in	O
+solution	O
+similar	O
+to	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+Interestingly	O
+,	O
+the	O
+monomeric	B-oligomeric_state
+PIN	B-structure_element
+mutants	B-protein_state
+P212A	B-mutant
+,	O
+R214A	B-mutant
+,	O
+and	O
+D278R	B-mutant
+had	O
+no	O
+significant	O
+RNase	B-protein_type
+activity	O
+for	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+in	O
+vitro	O
+(	O
+Fig	O
+.	O
+2c	O
+).	O
+
+The	O
+side	O
+chains	O
+of	O
+these	O
+residues	O
+point	O
+away	O
+from	O
+the	O
+catalytic	B-site
+center	I-site
+on	O
+the	O
+same	O
+molecule	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+
+Therefore	O
+,	O
+we	O
+concluded	O
+that	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+PIN	B-structure_element
+dimerization	O
+,	O
+together	O
+with	O
+the	O
+NTD	B-structure_element
+,	O
+are	O
+required	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+activity	O
+in	O
+vitro	O
+.	O
+
+Domain	O
+-	O
+domain	O
+interaction	O
+between	O
+the	O
+NTD	B-structure_element
+and	O
+the	O
+PIN	B-structure_element
+domain	O
+
+While	O
+the	O
+NTD	B-structure_element
+does	O
+not	O
+contribute	O
+to	O
+RNA	B-chemical
+binding	O
+(	O
+Fig	O
+.	O
+1f	O
+,	O
+g	O
+,	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+),	O
+it	O
+increases	O
+the	O
+RNase	B-protein_type
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+(	O
+Fig	O
+.	O
+1h	O
+).	O
+
+In	O
+order	O
+to	O
+gain	O
+insight	O
+into	O
+the	O
+molecular	O
+mechanism	O
+of	O
+the	O
+NTD	B-structure_element
+-	O
+mediated	O
+enhancement	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+activity	O
+,	O
+we	O
+further	O
+investigated	O
+the	O
+domain	O
+-	O
+domain	O
+interaction	O
+between	O
+the	O
+NTD	B-structure_element
+and	O
+the	O
+PIN	B-structure_element
+domain	O
+using	O
+NMR	B-experimental_method
+.	O
+
+We	O
+used	O
+the	O
+catalytically	B-protein_state
+inactive	I-protein_state
+monomeric	B-oligomeric_state
+PIN	B-structure_element
+mutant	B-protein_state
+possessing	O
+both	O
+the	O
+DDNN	B-mutant
+and	O
+D278R	B-mutant
+mutations	O
+to	O
+avoid	O
+dimer	B-oligomeric_state
+formation	O
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+The	O
+NMR	B-experimental_method
+signals	O
+from	O
+the	O
+PIN	B-structure_element
+domain	O
+(	O
+residues	O
+V177	B-residue_name_number
+,	O
+F210	B-residue_range
+-	I-residue_range
+T211	I-residue_range
+,	O
+R214	B-residue_name_number
+,	O
+F228	B-residue_range
+-	I-residue_range
+L232	I-residue_range
+,	O
+and	O
+F234	B-residue_range
+-	I-residue_range
+S236	I-residue_range
+)	O
+exhibited	O
+significant	O
+chemical	O
+shift	O
+changes	O
+upon	O
+addition	B-experimental_method
+of	I-experimental_method
+the	O
+NTD	B-structure_element
+(	O
+Fig	O
+.	O
+3a	O
+).	O
+
+Likewise	O
+,	O
+upon	O
+addition	B-experimental_method
+of	I-experimental_method
+the	O
+PIN	B-structure_element
+domain	O
+,	O
+NMR	B-experimental_method
+signals	O
+derived	O
+from	O
+R56	B-residue_name_number
+,	O
+L58	B-residue_range
+-	I-residue_range
+G59	I-residue_range
+,	O
+and	O
+V86	B-residue_range
+-	I-residue_range
+H88	I-residue_range
+in	O
+the	O
+NTD	B-structure_element
+exhibited	O
+large	O
+chemical	O
+shift	O
+changes	O
+and	O
+residues	O
+D53	B-residue_name_number
+,	O
+F55	B-residue_name_number
+,	O
+K57	B-residue_name_number
+,	O
+Y60	B-residue_range
+-	I-residue_range
+S61	I-residue_range
+,	O
+V68	B-residue_name_number
+,	O
+T80	B-residue_range
+-	I-residue_range
+G83	I-residue_range
+,	O
+L85	B-residue_name_number
+,	O
+and	O
+G89	B-residue_name_number
+of	O
+the	O
+NTD	B-structure_element
+as	O
+well	O
+as	O
+side	O
+chain	O
+amide	O
+signals	O
+of	O
+N79	B-residue_name_number
+exhibited	O
+small	O
+but	O
+appreciable	O
+chemical	O
+shift	O
+changes	O
+(	O
+Fig	O
+.	O
+3b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+
+These	O
+results	O
+clearly	O
+indicate	O
+a	O
+direct	O
+interaction	O
+between	O
+the	O
+PIN	B-structure_element
+domain	O
+and	O
+the	O
+NTD	B-structure_element
+.	O
+
+Based	O
+on	O
+the	O
+titration	B-evidence
+curve	I-evidence
+for	O
+the	O
+chemical	B-evidence
+shift	I-evidence
+changes	I-evidence
+of	O
+L58	B-residue_name_number
+,	O
+the	O
+apparent	O
+Kd	B-evidence
+between	O
+the	O
+isolated	O
+NTD	B-structure_element
+and	O
+PIN	B-structure_element
+was	O
+estimated	O
+to	O
+be	O
+110	O
+±	O
+5	O
+.	O
+8	O
+μM	O
+.	O
+Considering	O
+the	O
+fact	O
+that	O
+the	O
+NTD	B-structure_element
+and	O
+PIN	B-structure_element
+domains	O
+are	O
+attached	O
+by	O
+a	O
+linker	B-structure_element
+,	O
+the	O
+actual	O
+binding	B-evidence
+affinity	I-evidence
+is	O
+expected	O
+much	O
+higher	O
+in	O
+the	O
+native	B-protein_state
+protein	O
+.	O
+
+Mapping	O
+the	O
+residues	O
+with	O
+chemical	O
+shift	O
+changes	O
+reveals	O
+the	O
+putative	O
+PIN	B-site
+/	I-site
+NTD	I-site
+interface	I-site
+,	O
+which	O
+includes	O
+a	O
+helix	B-structure_element
+that	O
+harbors	O
+catalytic	O
+residues	O
+D225	B-residue_name_number
+and	O
+D226	B-residue_name_number
+on	O
+the	O
+PIN	B-structure_element
+domain	O
+(	O
+Fig	O
+.	O
+3a	O
+).	O
+
+Interestingly	O
+,	O
+the	O
+putative	O
+binding	B-site
+site	I-site
+for	O
+the	O
+NTD	B-structure_element
+overlaps	O
+with	O
+the	O
+PIN	B-site
+-	I-site
+PIN	I-site
+dimer	I-site
+interface	I-site
+,	O
+implying	O
+that	O
+NTD	B-structure_element
+binding	O
+can	O
+“	O
+terminate	O
+”	O
+PIN	B-structure_element
+-	O
+PIN	B-structure_element
+oligomerization	O
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+
+An	O
+in	B-experimental_method
+silico	I-experimental_method
+docking	I-experimental_method
+of	O
+the	O
+NTD	B-structure_element
+and	O
+PIN	B-structure_element
+domains	O
+using	O
+chemical	B-evidence
+shift	I-evidence
+restraints	I-evidence
+provided	O
+a	O
+model	O
+consistent	O
+with	O
+the	O
+NMR	B-experimental_method
+experiments	O
+(	O
+Fig	O
+.	O
+3c	O
+).	O
+
+Residues	O
+critical	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+activity	O
+
+To	O
+gain	O
+insight	O
+into	O
+the	O
+residues	O
+critical	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+activity	O
+,	O
+each	O
+basic	O
+or	O
+aromatic	O
+residue	O
+located	O
+around	O
+the	O
+catalytic	B-site
+site	I-site
+of	O
+the	O
+PIN	B-structure_element
+oligomer	B-oligomeric_state
+was	O
+mutated	B-experimental_method
+to	I-experimental_method
+alanine	B-residue_name
+,	O
+and	O
+the	O
+oligomerization	O
+and	O
+RNase	B-protein_type
+activity	O
+were	O
+investigated	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+From	O
+the	O
+gel	B-experimental_method
+filtration	I-experimental_method
+assays	I-experimental_method
+,	O
+all	O
+mutants	B-protein_state
+except	O
+R214A	B-mutant
+formed	O
+dimers	B-oligomeric_state
+,	O
+suggesting	O
+that	O
+any	O
+lack	O
+of	O
+RNase	B-protein_type
+activity	O
+in	O
+the	O
+mutants	B-protein_state
+,	O
+except	O
+R214A	B-mutant
+,	O
+was	O
+directly	O
+due	O
+to	O
+mutational	O
+effects	O
+of	O
+the	O
+specific	O
+residues	O
+and	O
+not	O
+to	O
+abrogation	O
+of	O
+dimer	B-oligomeric_state
+formation	O
+.	O
+
+The	O
+W182A	B-mutant
+,	O
+R183A	B-mutant
+,	O
+and	O
+R214A	B-mutant
+mutants	B-protein_state
+markedly	O
+lost	O
+cleavage	O
+activity	O
+for	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+as	O
+well	O
+as	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+mRNA	B-chemical
+.	O
+
+The	O
+K184A	B-mutant
+,	O
+R215A	B-mutant
+,	O
+and	O
+R220A	B-mutant
+mutants	B-protein_state
+moderately	O
+but	O
+significantly	O
+decreased	O
+the	O
+cleavage	O
+activity	O
+for	O
+both	O
+target	O
+mRNAs	B-chemical
+.	O
+
+The	O
+importance	O
+of	O
+K219	B-residue_name_number
+and	O
+R247	B-residue_name_number
+was	O
+slightly	O
+different	O
+for	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+and	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+mRNA	B-chemical
+;	O
+both	O
+K219	B-residue_name_number
+and	O
+R247	B-residue_name_number
+were	O
+more	O
+important	O
+in	O
+the	O
+cleavage	O
+of	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+than	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+mRNA	B-chemical
+.	O
+
+The	O
+other	O
+mutated	O
+residues	O
+—	O
+K152	B-residue_name_number
+,	O
+R158	B-residue_name_number
+,	O
+R188	B-residue_name_number
+,	O
+R200	B-residue_name_number
+,	O
+K204	B-residue_name_number
+,	O
+K206	B-residue_name_number
+,	O
+K257	B-residue_name_number
+,	O
+and	O
+R258	B-residue_name_number
+—	O
+were	O
+not	O
+critical	O
+for	O
+RNase	B-protein_type
+activity	O
+.	O
+
+The	O
+importance	O
+of	O
+residues	O
+W182	B-residue_name_number
+and	O
+R183	B-residue_name_number
+can	O
+readily	O
+be	O
+understood	O
+in	O
+terms	O
+of	O
+the	O
+monomeric	B-oligomeric_state
+PIN	B-structure_element
+structure	B-evidence
+as	O
+they	O
+are	O
+located	O
+near	O
+to	O
+the	O
+RNase	B-protein_type
+catalytic	B-site
+site	I-site
+;	O
+however	O
+,	O
+the	O
+importance	O
+of	O
+residue	O
+K184	B-residue_name_number
+,	O
+which	O
+points	O
+away	O
+from	O
+the	O
+active	B-site
+site	I-site
+is	O
+more	O
+easily	O
+rationalized	O
+in	O
+terms	O
+of	O
+the	O
+oligomeric	O
+structure	B-evidence
+,	O
+in	O
+which	O
+the	O
+“	O
+secondary	O
+”	O
+chain	O
+’	O
+s	O
+residue	O
+K184	B-residue_name_number
+is	O
+positioned	O
+near	O
+the	O
+“	O
+primary	B-protein_state
+”	I-protein_state
+chain	O
+’	O
+s	O
+catalytic	B-site
+site	I-site
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+In	O
+contrast	O
+,	O
+R214	B-residue_name_number
+is	O
+important	O
+for	O
+oligomerization	O
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+and	O
+the	O
+“	O
+secondary	O
+”	O
+chain	O
+’	O
+s	O
+residue	O
+R214	B-residue_name_number
+is	O
+also	O
+positioned	O
+near	O
+the	O
+“	O
+primary	B-protein_state
+”	O
+chain	O
+’	O
+s	O
+active	B-site
+site	I-site
+within	O
+the	O
+dimer	B-site
+interface	I-site
+.	O
+
+It	O
+should	O
+be	O
+noted	O
+that	O
+the	O
+putative	B-site
+-	I-site
+RNA	I-site
+binding	I-site
+residues	I-site
+K184	B-residue_name_number
+and	O
+R214	B-residue_name_number
+are	O
+unique	O
+to	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+among	O
+PIN	B-structure_element
+domains	O
+.	O
+
+Molecular	O
+mechanism	O
+of	O
+target	O
+mRNA	B-chemical
+cleavage	O
+by	O
+the	O
+PIN	B-structure_element
+dimer	B-oligomeric_state
+
+Our	O
+mutational	B-experimental_method
+experiments	I-experimental_method
+indicated	O
+that	O
+the	O
+observed	O
+dimer	B-oligomeric_state
+is	O
+functional	O
+and	O
+that	O
+the	O
+role	O
+of	O
+the	O
+secondary	B-protein_state
+PIN	B-structure_element
+domain	O
+is	O
+to	O
+position	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+-	O
+unique	O
+RNA	B-site
+binding	I-site
+residues	I-site
+near	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+primary	B-protein_state
+PIN	B-structure_element
+domain	O
+.	O
+
+If	O
+this	O
+model	O
+is	O
+correct	O
+,	O
+then	O
+we	O
+reasoned	O
+that	O
+a	O
+catalytically	B-protein_state
+inactive	I-protein_state
+PIN	B-structure_element
+and	O
+a	O
+PIN	B-structure_element
+lacking	B-protein_state
+the	O
+putative	O
+RNA	B-site
+-	I-site
+binding	I-site
+residues	I-site
+ought	O
+to	O
+be	O
+inactive	B-protein_state
+in	O
+isolation	O
+but	O
+become	O
+active	B-protein_state
+when	O
+mixed	O
+together	O
+.	O
+
+In	O
+order	O
+to	O
+test	O
+this	O
+hypothesis	O
+,	O
+we	O
+performed	O
+in	B-experimental_method
+vitro	I-experimental_method
+cleavage	I-experimental_method
+assays	I-experimental_method
+using	O
+combinations	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+mutants	B-protein_state
+that	O
+had	O
+no	O
+or	O
+decreased	O
+RNase	B-protein_type
+activities	O
+by	O
+themselves	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+One	O
+group	O
+consisted	O
+of	O
+catalytically	B-protein_state
+active	I-protein_state
+PIN	B-structure_element
+domains	O
+with	O
+mutation	B-experimental_method
+of	I-experimental_method
+basic	O
+residues	O
+found	O
+in	O
+the	O
+previous	O
+section	O
+to	O
+confer	O
+decreased	O
+RNase	B-protein_type
+activity	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+These	O
+were	O
+paired	O
+with	O
+a	O
+DDNN	B-mutant
+mutant	B-protein_state
+that	O
+had	O
+no	O
+RNase	B-protein_type
+activity	O
+by	O
+itself	O
+.	O
+
+When	O
+any	O
+members	O
+of	O
+the	O
+two	O
+groups	O
+are	O
+mixed	O
+,	O
+two	O
+kinds	O
+of	O
+heterodimers	B-oligomeric_state
+can	O
+be	O
+formed	O
+:	O
+one	O
+is	O
+composed	O
+of	O
+a	O
+DDNN	B-mutant
+primary	B-protein_state
+PIN	B-structure_element
+and	O
+a	O
+basic	O
+residue	O
+mutant	B-protein_state
+secondary	B-protein_state
+PIN	B-structure_element
+and	O
+is	O
+expected	O
+to	O
+exhibit	O
+no	O
+RNase	B-protein_type
+activity	O
+;	O
+the	O
+other	O
+is	O
+composed	O
+of	O
+a	O
+basic	O
+residue	O
+mutant	B-protein_state
+primary	B-protein_state
+PIN	B-structure_element
+and	O
+a	O
+DDNN	B-mutant
+secondary	B-protein_state
+PIN	B-structure_element
+and	O
+is	O
+predicted	O
+to	O
+rescue	O
+RNase	B-protein_type
+activity	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+
+When	O
+we	O
+compared	O
+the	O
+fluorescence	B-evidence
+intensity	I-evidence
+of	O
+uncleaved	B-protein_state
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+,	O
+basic	O
+residue	O
+mutants	B-protein_state
+W182A	B-mutant
+,	O
+K184A	B-mutant
+,	O
+R214A	B-mutant
+,	O
+and	O
+R220A	B-mutant
+were	O
+rescued	O
+upon	O
+addition	O
+of	O
+the	O
+DDNN	B-mutant
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+5b	O
+).	O
+
+Consistently	O
+,	O
+when	O
+we	O
+compared	O
+the	O
+fluorescence	B-evidence
+intensity	I-evidence
+of	O
+the	O
+uncleaved	B-protein_state
+Regnase	B-protein
+-	I-protein
+1	I-protein
+mRNA	B-chemical
+,	O
+basic	O
+residue	O
+mutants	B-protein_state
+K184A	B-mutant
+and	O
+R214A	B-mutant
+were	O
+rescued	O
+upon	O
+addition	O
+of	O
+the	O
+DDNN	B-mutant
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+
+Rescue	O
+of	O
+K184A	B-mutant
+and	O
+R214A	B-mutant
+by	O
+the	O
+DDNN	B-mutant
+mutant	B-protein_state
+was	O
+also	O
+confirmed	O
+by	O
+a	O
+significant	O
+increase	O
+in	O
+the	O
+cleaved	O
+products	O
+.	O
+
+This	O
+is	O
+particularly	O
+significant	O
+because	O
+the	O
+side	O
+chains	O
+of	O
+K184	B-residue_name_number
+and	O
+R214	B-residue_name_number
+in	O
+the	O
+primary	B-protein_state
+PIN	B-structure_element
+are	O
+oriented	O
+away	O
+from	O
+their	O
+own	O
+catalytic	B-site
+center	I-site
+,	O
+while	O
+those	O
+in	O
+the	O
+secondary	B-protein_state
+PIN	B-structure_element
+face	O
+toward	O
+the	O
+catalytic	B-site
+center	I-site
+of	O
+the	O
+primary	B-protein_state
+PIN	B-structure_element
+.	O
+
+R214	B-residue_name_number
+is	O
+an	O
+important	O
+residue	O
+for	O
+dimer	B-oligomeric_state
+formation	O
+as	O
+shown	O
+in	O
+Fig	O
+.	O
+2	O
+,	O
+therefore	O
+,	O
+R214A	B-mutant
+in	O
+the	O
+secondary	B-protein_state
+PIN	B-structure_element
+cannot	O
+dimerize	O
+.	O
+
+According	O
+to	O
+the	O
+proposed	O
+model	O
+,	O
+an	O
+R214A	B-mutant
+PIN	B-structure_element
+domain	O
+can	O
+only	O
+form	O
+a	O
+dimer	B-oligomeric_state
+when	O
+the	O
+DDNN	B-mutant
+PIN	B-structure_element
+acts	O
+as	O
+the	O
+secondary	B-protein_state
+PIN	B-structure_element
+.	O
+
+Taken	O
+together	O
+,	O
+the	O
+rescue	O
+experiments	O
+above	O
+support	O
+the	O
+proposed	O
+model	O
+in	O
+which	O
+the	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+dimer	B-oligomeric_state
+is	O
+functional	O
+in	O
+vitro	O
+.	O
+
+We	O
+determined	O
+the	O
+individual	O
+domain	O
+structures	B-evidence
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+by	O
+NMR	B-experimental_method
+and	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+.	O
+
+Although	O
+the	O
+function	O
+of	O
+the	O
+CTD	B-structure_element
+remains	O
+elusive	O
+,	O
+we	O
+revealed	O
+the	O
+functions	O
+of	O
+the	O
+NTD	B-structure_element
+,	O
+PIN	B-structure_element
+,	O
+and	O
+ZF	B-structure_element
+domains	O
+.	O
+
+A	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+construct	O
+consisting	O
+of	O
+PIN	B-structure_element
+and	O
+ZF	B-structure_element
+domains	O
+derived	O
+from	O
+Mus	B-species
+musculus	I-species
+was	O
+crystallized	B-experimental_method
+;	O
+however	O
+,	O
+the	O
+electron	B-evidence
+density	I-evidence
+of	O
+the	O
+ZF	B-structure_element
+domain	O
+was	O
+low	O
+,	O
+indicating	O
+that	O
+the	O
+ZF	B-structure_element
+domain	O
+is	O
+highly	B-protein_state
+mobile	I-protein_state
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+target	O
+mRNA	B-chemical
+or	O
+possibly	O
+other	O
+protein	O
+-	O
+protein	O
+interactions	O
+.	O
+
+Our	O
+NMR	B-experimental_method
+experiments	O
+confirmed	O
+direct	O
+binding	O
+of	O
+the	O
+ZF	B-structure_element
+domain	O
+to	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+with	O
+a	O
+Kd	B-evidence
+of	O
+10	O
+±	O
+1	O
+.	O
+1	O
+μM	O
+.	O
+Furthermore	O
+,	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+gel	I-experimental_method
+shift	I-experimental_method
+assay	I-experimental_method
+indicated	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+containing	O
+the	O
+ZF	B-structure_element
+domain	O
+enhanced	O
+target	O
+mRNA	B-chemical
+-	O
+binding	O
+,	O
+but	O
+the	O
+protein	O
+-	O
+RNA	B-chemical
+complex	O
+remained	O
+in	O
+the	O
+bottom	O
+of	O
+the	O
+well	O
+without	O
+entering	O
+into	O
+the	O
+polyacrylamide	O
+gel	O
+.	O
+
+These	O
+results	O
+indicate	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+directly	O
+binds	O
+to	O
+RNA	B-chemical
+and	O
+precipitates	O
+under	O
+such	O
+experimental	O
+conditions	O
+.	O
+
+Due	O
+to	O
+this	O
+limitation	O
+,	O
+it	O
+is	O
+difficult	O
+to	O
+perform	O
+further	O
+structural	B-experimental_method
+analyses	I-experimental_method
+of	O
+mRNA	B-complex_assembly
+-	I-complex_assembly
+Regnase	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+complexes	O
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+or	O
+NMR	B-experimental_method
+.	O
+
+The	O
+previously	O
+reported	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+PIN	B-structure_element
+domain	O
+derived	O
+from	O
+Homo	B-species
+sapiens	I-species
+is	O
+nearly	O
+identical	O
+to	O
+the	O
+one	O
+derived	O
+from	O
+Mus	B-species
+musculus	I-species
+in	O
+this	O
+study	O
+,	O
+with	O
+a	O
+backbone	O
+RMSD	B-evidence
+of	O
+0	O
+.	O
+2	O
+Å	O
+.	O
+The	O
+amino	O
+acid	O
+sequences	O
+corresponding	O
+to	O
+PIN	B-structure_element
+(	O
+residues	O
+134	B-residue_range
+–	I-residue_range
+295	I-residue_range
+)	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
+
+Both	O
+the	O
+mouse	B-taxonomy_domain
+and	O
+human	B-species
+PIN	B-structure_element
+domains	O
+form	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+oligomers	B-oligomeric_state
+in	O
+three	O
+distinct	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+
+Rao	O
+and	O
+co	O
+-	O
+workers	O
+previously	O
+argued	O
+that	O
+PIN	B-structure_element
+dimerization	O
+is	O
+likely	O
+to	O
+be	O
+a	O
+crystallographic	O
+artifact	O
+with	O
+no	O
+physiological	O
+significance	O
+,	O
+since	O
+monomers	B-oligomeric_state
+were	O
+dominant	O
+in	O
+their	O
+analytical	B-experimental_method
+ultra	I-experimental_method
+-	I-experimental_method
+centrifugation	I-experimental_method
+experiments	O
+.	O
+
+In	O
+contrast	O
+,	O
+our	O
+gel	B-experimental_method
+filtration	I-experimental_method
+data	O
+,	O
+mutational	B-experimental_method
+analyses	I-experimental_method
+,	O
+and	O
+NMR	B-experimental_method
+spectra	B-evidence
+all	O
+indicate	O
+that	O
+the	O
+PIN	B-structure_element
+domain	O
+forms	O
+a	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+dimer	B-oligomeric_state
+in	O
+solution	O
+in	O
+a	O
+manner	O
+similar	O
+to	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+This	O
+inconsistency	O
+might	O
+be	O
+due	O
+to	O
+difference	O
+in	O
+the	O
+analytical	O
+methods	O
+and	O
+/	O
+or	O
+protein	O
+concentrations	O
+used	O
+in	O
+each	O
+experiment	O
+,	O
+since	O
+the	O
+oligomer	B-oligomeric_state
+formation	O
+of	O
+PIN	B-structure_element
+was	O
+dependent	O
+on	O
+the	O
+protein	O
+concentration	O
+in	O
+our	O
+study	O
+.	O
+
+Single	B-experimental_method
+mutations	I-experimental_method
+to	O
+residues	O
+involved	O
+in	O
+the	O
+putative	O
+oligomeric	O
+interaction	O
+of	O
+PIN	B-structure_element
+monomerized	B-oligomeric_state
+as	O
+expected	O
+and	O
+these	O
+mutants	B-protein_state
+lost	O
+their	O
+RNase	B-protein_type
+activity	O
+as	O
+well	O
+.	O
+
+Since	O
+the	O
+NMR	B-experimental_method
+spectra	B-evidence
+of	O
+monomeric	B-oligomeric_state
+mutants	B-protein_state
+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	B-oligomeric_state
+mutants	B-protein_state
+were	O
+affected	O
+by	O
+the	O
+mutations	O
+.	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4b	O
+,	O
+c	O
+).	O
+
+Based	O
+on	O
+these	O
+observations	O
+,	O
+we	O
+concluded	O
+that	O
+PIN	B-structure_element
+-	O
+PIN	B-structure_element
+dimer	B-oligomeric_state
+formation	O
+is	O
+critical	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+activity	O
+in	O
+vitro	O
+.	O
+
+Within	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+PIN	B-structure_element
+dimer	B-oligomeric_state
+,	O
+the	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+specific	O
+basic	O
+regions	O
+in	O
+both	O
+the	O
+“	O
+primary	B-protein_state
+”	O
+and	O
+“	O
+secondary	B-protein_state
+”	O
+PINs	B-structure_element
+are	O
+located	O
+around	O
+the	O
+catalytic	B-site
+site	I-site
+of	O
+the	O
+primary	O
+PIN	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+Moreover	O
+,	O
+our	O
+structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+mutational	I-experimental_method
+analyses	I-experimental_method
+showed	O
+these	O
+two	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+specific	O
+basic	O
+regions	O
+were	O
+essential	O
+for	O
+target	O
+mRNA	B-chemical
+cleavage	O
+in	O
+vitro	O
+.	O
+
+The	O
+cleavage	B-experimental_method
+assay	I-experimental_method
+also	O
+showed	O
+that	O
+the	O
+NTD	B-structure_element
+is	O
+crucial	O
+for	O
+efficient	O
+mRNA	B-chemical
+cleavage	O
+.	O
+
+Moreover	O
+,	O
+we	O
+found	O
+that	O
+the	O
+NTD	B-structure_element
+associates	O
+with	O
+the	O
+oligomeric	B-site
+surface	I-site
+of	O
+the	O
+primary	B-protein_state
+PIN	B-structure_element
+,	O
+docking	O
+to	O
+a	O
+helix	B-structure_element
+that	O
+harbors	O
+its	O
+catalytic	B-site
+residues	I-site
+(	O
+Figs	O
+2b	O
+and	O
+3a	O
+).	O
+
+Taken	O
+together	O
+,	O
+this	O
+suggests	O
+that	O
+the	O
+NTD	B-structure_element
+and	O
+the	O
+PIN	B-structure_element
+domain	O
+compete	O
+for	O
+a	O
+common	B-site
+binding	I-site
+site	I-site
+.	O
+
+The	O
+affinity	B-evidence
+of	O
+the	O
+domain	O
+-	O
+domain	O
+interaction	O
+between	O
+two	O
+PIN	B-structure_element
+domains	O
+(	O
+Kd	B-evidence
+=	O
+~	O
+10	O
+−	O
+4	O
+M	O
+)	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+NTD	B-structure_element
+-	O
+PIN	B-structure_element
+(	O
+Kd	B-evidence
+=	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	B-residue_range
+–	I-residue_range
+133	I-residue_range
+between	O
+the	O
+NTD	B-structure_element
+and	O
+the	O
+primary	B-protein_state
+PIN	B-structure_element
+will	O
+greatly	O
+enhance	O
+the	O
+intramolecular	O
+domain	O
+interaction	O
+in	O
+the	O
+case	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+While	O
+further	O
+analyses	O
+are	O
+necessary	O
+to	O
+prove	O
+this	O
+point	O
+,	O
+our	O
+preliminary	O
+docking	B-experimental_method
+and	I-experimental_method
+molecular	I-experimental_method
+dynamics	I-experimental_method
+simulations	I-experimental_method
+indicate	O
+that	O
+NTD	B-structure_element
+-	O
+binding	O
+rearranges	O
+the	O
+catalytic	B-site
+residues	I-site
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+toward	O
+an	O
+active	B-protein_state
+conformation	O
+suitable	O
+for	O
+binding	O
+Mg2	B-chemical
++.	I-chemical
+
+In	O
+this	O
+context	O
+,	O
+it	O
+is	O
+interesting	O
+that	O
+,	O
+in	O
+response	O
+to	O
+TCR	O
+stimulation	O
+,	O
+Malt1	B-protein
+cleaves	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+at	O
+R111	B-residue_name_number
+to	O
+control	O
+immune	O
+responses	O
+in	O
+vivo	O
+.	O
+
+This	O
+result	O
+is	O
+consistent	O
+with	O
+a	O
+model	O
+in	O
+which	O
+the	O
+NTD	B-structure_element
+acts	O
+as	O
+an	O
+enhancer	O
+,	O
+and	O
+cleavage	O
+of	O
+the	O
+linker	B-structure_element
+lowers	O
+enzymatic	O
+activity	O
+dramatically	O
+.	O
+
+Based	O
+on	O
+these	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+analyses	I-experimental_method
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+domain	O
+-	O
+domain	O
+interactions	O
+,	O
+we	O
+performed	O
+docking	B-experimental_method
+simulations	I-experimental_method
+of	O
+the	O
+NTD	B-structure_element
+,	O
+PIN	B-structure_element
+dimer	B-oligomeric_state
+,	O
+and	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+.	O
+
+We	O
+incorporated	O
+information	O
+from	O
+the	O
+cleavage	B-site
+site	I-site
+of	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+in	O
+vitro	O
+is	O
+indicated	O
+by	O
+denaturing	O
+polyacrylamide	B-experimental_method
+gel	I-experimental_method
+electrophoresis	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+,	O
+b	O
+).	O
+
+The	O
+docking	B-experimental_method
+result	O
+revealed	O
+multiple	O
+RNA	B-chemical
+binding	O
+modes	O
+that	O
+satisfied	O
+the	O
+experimental	O
+results	O
+in	O
+vitro	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+,	O
+d	O
+),	O
+however	O
+,	O
+it	O
+should	O
+be	O
+noted	O
+that	O
+,	O
+in	O
+vivo	O
+,	O
+there	O
+would	O
+likely	O
+be	O
+many	O
+other	O
+RNA	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+that	O
+would	O
+protect	O
+loop	B-structure_element
+regions	O
+from	O
+cleavage	O
+by	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+The	O
+overall	O
+model	O
+of	O
+regulation	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+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
+
+In	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+target	O
+mRNA	B-chemical
+,	O
+the	O
+PIN	B-structure_element
+domain	O
+forms	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+oligomers	B-oligomeric_state
+at	O
+high	O
+concentration	O
+.	O
+
+A	O
+fully	B-protein_state
+active	I-protein_state
+catalytic	B-site
+center	I-site
+can	O
+be	O
+formed	O
+only	O
+when	O
+the	O
+NTD	B-structure_element
+associates	O
+with	O
+the	O
+oligomer	B-oligomeric_state
+surface	O
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+,	O
+which	O
+terminates	O
+the	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+oligomer	B-oligomeric_state
+formation	O
+in	O
+one	O
+direction	O
+(	O
+primary	B-protein_state
+PIN	B-structure_element
+),	O
+and	O
+forms	O
+a	O
+functional	B-protein_state
+dimer	B-oligomeric_state
+together	O
+with	O
+the	O
+neighboring	O
+PIN	B-structure_element
+(	O
+secondary	B-protein_state
+PIN	B-structure_element
+).	O
+
+While	O
+further	O
+investigations	O
+on	O
+the	O
+domain	O
+-	O
+domain	O
+interactions	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+in	O
+vivo	O
+are	O
+necessary	O
+,	O
+these	O
+intramolecular	O
+and	O
+intermolecular	O
+domain	O
+interactions	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+appear	O
+to	O
+structurally	O
+constrain	O
+Regnase	B-protein
+-	I-protein
+1activity	I-protein
+,	O
+which	O
+,	O
+in	O
+turn	O
+,	O
+enables	O
+tight	O
+regulation	O
+of	O
+immune	O
+responses	O
+.	O
+
+For	O
+the	O
+domain	O
+-	O
+domain	O
+interaction	O
+analyses	O
+between	O
+the	O
+NTD	O
+and	O
+the	O
+PIN	O
+domain	O
+,	O
+1H	O
+-	O
+15N	O
+HSQC	O
+spectra	O
+of	O
+uniformly	O
+15N	O
+-	O
+labeled	O
+proteins	O
+in	O
+the	O
+concentration	O
+of	O
+100	O
+μM	O
+were	O
+obtained	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+3	O
+or	O
+6	O
+molar	O
+equivalents	O
+of	O
+unlabeled	O
+proteins	O
+.	O
+
+Structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+analyses	I-experimental_method
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+(	O
+a	O
+)	O
+Domain	O
+architecture	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+(	O
+b	O
+)	O
+Solution	B-evidence
+structure	I-evidence
+of	O
+the	O
+NTD	B-structure_element
+.	O
+(	O
+c	O
+)	O
+Crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+Catalytic	B-protein_state
+Asp	B-residue_name
+residues	O
+were	O
+shown	O
+in	O
+sticks	O
+.	O
+
+(	O
+d	O
+)	O
+Solution	B-evidence
+structure	I-evidence
+of	O
+the	O
+ZF	B-structure_element
+domain	O
+.	O
+
+Three	O
+Cys	B-residue_name
+residues	O
+and	O
+one	O
+His	B-residue_name
+residue	O
+responsible	O
+for	O
+Zn2	O
++-	O
+binding	O
+were	O
+shown	O
+in	O
+sticks	O
+.	O
+
+(	O
+e	O
+)	O
+Solution	B-evidence
+structure	I-evidence
+of	O
+the	O
+CTD	B-structure_element
+.	O
+
+All	O
+the	O
+structures	B-evidence
+were	O
+colored	O
+in	O
+rainbow	O
+from	O
+N	O
+-	O
+terminus	O
+(	O
+blue	O
+)	O
+to	O
+C	O
+-	O
+terminus	O
+(	O
+red	O
+).	O
+
+(	O
+f	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+gel	I-experimental_method
+shift	I-experimental_method
+binding	I-experimental_method
+assay	I-experimental_method
+between	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+and	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+.	O
+
+Fluorescence	B-evidence
+intensity	I-evidence
+of	O
+the	O
+free	B-protein_state
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+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	B-protein_state
+of	I-protein_state
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+(	O
+g	O
+)	O
+Binding	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+and	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+was	O
+plotted	O
+.	O
+
+The	O
+percentage	O
+of	O
+the	O
+bound	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+was	O
+calculated	O
+based	O
+on	O
+the	O
+fluorescence	B-evidence
+intensities	I-evidence
+of	O
+the	O
+free	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+quantified	O
+in	O
+(	O
+f	O
+).	O
+
+(	O
+h	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+cleavage	I-experimental_method
+assay	I-experimental_method
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+to	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+.	O
+
+Fluorescence	B-evidence
+intensity	I-evidence
+of	O
+the	O
+uncleaved	B-protein_state
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+was	O
+indicated	O
+as	O
+the	O
+percentage	O
+against	O
+that	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+Head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+oligomer	B-oligomeric_state
+formation	O
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+is	O
+crucial	O
+for	O
+the	O
+RNase	B-protein_type
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+(	O
+a	O
+)	O
+Gel	B-experimental_method
+filtration	I-experimental_method
+analyses	I-experimental_method
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+(	O
+b	O
+)	O
+Dimer	B-oligomeric_state
+structure	B-evidence
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+Two	O
+PIN	B-structure_element
+molecules	O
+in	O
+the	O
+crystal	B-evidence
+were	O
+colored	O
+white	O
+and	O
+green	O
+,	O
+respectively	O
+.	O
+
+Catalytic	B-site
+residues	I-site
+and	O
+mutated	O
+residues	O
+were	O
+shown	O
+in	O
+sticks	O
+.	O
+
+Residues	O
+important	O
+for	O
+the	O
+oligomeric	O
+interaction	O
+were	O
+colored	O
+red	O
+,	O
+while	O
+R215	B-residue_name_number
+that	O
+was	O
+dispensable	O
+for	O
+the	O
+oligomeric	O
+interaction	O
+was	O
+colored	O
+blue	O
+.	O
+(	O
+c	O
+)	O
+RNase	B-protein_type
+activity	O
+of	O
+monomeric	B-oligomeric_state
+mutants	B-protein_state
+for	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+was	O
+analyzed	O
+.	O
+
+Domain	O
+-	O
+domain	O
+interaction	O
+between	O
+the	O
+NTD	B-structure_element
+and	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+(	O
+a	O
+)	O
+NMR	B-experimental_method
+analyses	I-experimental_method
+of	O
+the	O
+NTD	B-structure_element
+-	O
+binding	O
+to	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+The	O
+residues	O
+with	O
+significant	O
+chemical	O
+shift	O
+changes	O
+were	O
+labeled	O
+in	O
+the	O
+overlaid	B-experimental_method
+spectra	B-evidence
+(	O
+left	O
+)	O
+and	O
+colored	O
+red	O
+on	O
+the	O
+surface	O
+and	O
+ribbon	O
+structure	O
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+(	O
+right	O
+).	O
+
+Pro	B-residue_name
+and	O
+the	O
+residues	O
+without	O
+analysis	O
+were	O
+colored	O
+black	O
+and	O
+gray	O
+,	O
+respectively	O
+.	O
+
+(	O
+b	O
+)	O
+NMR	B-experimental_method
+analyses	I-experimental_method
+of	O
+the	O
+PIN	B-structure_element
+-	O
+binding	O
+to	O
+the	O
+NTD	B-structure_element
+.	O
+
+The	O
+residues	O
+with	O
+significant	B-evidence
+chemical	I-evidence
+shift	I-evidence
+changes	I-evidence
+were	O
+labeled	O
+in	O
+the	O
+overlaid	B-experimental_method
+spectra	B-evidence
+(	O
+left	O
+)	O
+and	O
+colored	O
+red	O
+,	O
+yellow	O
+,	O
+or	O
+green	O
+on	O
+the	O
+surface	O
+and	O
+ribbon	O
+structure	O
+of	O
+the	O
+NTD	B-structure_element
+.	O
+
+S62	B-residue_name_number
+was	O
+colored	O
+gray	O
+and	O
+excluded	O
+from	O
+the	O
+analysis	O
+,	O
+due	O
+to	O
+low	O
+signal	O
+intensity	O
+.	O
+
+(	O
+c	O
+)	O
+Docking	O
+model	O
+of	O
+the	O
+NTD	B-structure_element
+and	O
+the	O
+PIN	B-structure_element
+domain	O
+.	O
+
+The	O
+NTD	B-structure_element
+and	O
+the	O
+PIN	B-structure_element
+domain	O
+are	O
+shown	O
+in	O
+cyan	O
+and	O
+white	O
+,	O
+respectively	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	B-evidence
+structure	I-evidence
+were	O
+colored	O
+yellow	O
+.	O
+
+Catalytic	B-site
+residues	I-site
+of	O
+the	O
+PIN	B-structure_element
+domain	O
+are	O
+shown	O
+in	O
+sticks	O
+,	O
+and	O
+the	O
+residues	O
+that	O
+exhibited	O
+significant	B-evidence
+chemical	I-evidence
+shift	I-evidence
+changes	I-evidence
+in	O
+(	O
+a	O
+,	O
+b	O
+)	O
+were	O
+labeled	O
+.	O
+
+Critical	O
+residues	O
+in	O
+the	O
+PIN	B-structure_element
+domain	O
+for	O
+the	O
+RNase	B-protein_type
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+(	O
+a	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+cleavage	I-experimental_method
+assay	I-experimental_method
+of	O
+basic	O
+residue	O
+mutants	B-protein_state
+for	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+.	O
+
+(	O
+b	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+cleavage	I-experimental_method
+assay	I-experimental_method
+of	O
+basic	O
+residue	O
+mutants	B-protein_state
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+mRNA	B-chemical
+.	O
+
+The	O
+fluorescence	B-evidence
+intensity	I-evidence
+of	O
+the	O
+uncleaved	B-protein_state
+mRNA	B-chemical
+was	O
+quantified	O
+and	O
+the	O
+results	O
+were	O
+mapped	O
+on	O
+the	O
+PIN	B-structure_element
+dimer	B-oligomeric_state
+structure	B-evidence
+.	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	B-protein_type
+activities	O
+were	O
+colored	O
+red	O
+or	O
+yellow	O
+.	O
+
+Heterodimer	O
+formation	O
+by	O
+combination	O
+of	O
+the	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+basic	O
+residue	O
+mutants	B-protein_state
+and	O
+the	O
+DDNN	B-mutant
+mutant	B-protein_state
+restored	O
+the	O
+RNase	B-protein_type
+activity	O
+.	O
+
+(	O
+a	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+concept	O
+of	O
+the	O
+experiment	O
+.	O
+(	O
+b	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+cleavage	I-experimental_method
+assay	I-experimental_method
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+for	O
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+.	O
+
+(	O
+c	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+cleavage	I-experimental_method
+assay	I-experimental_method
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+mRNA	B-chemical
+.	O
+
+The	O
+fluorescence	B-evidence
+intensity	I-evidence
+of	O
+the	O
+uncleaved	B-protein_state
+mRNA	B-chemical
+was	O
+quantified	O
+and	O
+the	O
+results	O
+were	O
+mapped	O
+on	O
+the	O
+PIN	B-structure_element
+dimer	B-oligomeric_state
+.	O
+
+The	O
+mutations	O
+whose	O
+RNase	B-protein_type
+activities	O
+were	O
+not	O
+increased	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+DDNN	B-mutant
+mutant	B-protein_state
+were	O
+colored	O
+in	O
+blue	O
+on	O
+the	O
+primary	O
+PIN	B-structure_element
+.	O
+
+The	O
+mutations	O
+whose	O
+RNase	B-protein_type
+activities	O
+were	O
+restored	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+DDNN	B-mutant
+mutant	B-protein_state
+were	O
+colored	O
+in	O
+red	O
+or	O
+yellow	O
+on	O
+the	O
+primary	O
+PIN	B-structure_element
+.	O
+
+Schematic	O
+representation	O
+of	O
+regulation	O
+of	O
+the	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+catalytic	O
+activity	O
+through	O
+the	O
+domain	O
+-	O
+domain	O
+interactions	O
+.	O
+
+Ribosome	B-protein_type
+biogenesis	I-protein_type
+factor	I-protein_type
+Tsr3	B-protein
+is	O
+the	O
+aminocarboxypropyl	B-protein_type
+transferase	I-protein_type
+responsible	O
+for	O
+18S	B-chemical
+rRNA	I-chemical
+hypermodification	O
+in	O
+yeast	B-taxonomy_domain
+and	O
+humans	B-species
+
+The	O
+chemically	O
+most	O
+complex	O
+modification	O
+in	O
+eukaryotic	B-taxonomy_domain
+rRNA	B-chemical
+is	O
+the	O
+conserved	B-protein_state
+hypermodified	B-protein_state
+nucleotide	B-chemical
+N1	B-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+N3	I-chemical
+-	I-chemical
+aminocarboxypropyl	I-chemical
+-	I-chemical
+pseudouridine	I-chemical
+(	O
+m1acp3Ψ	B-chemical
+)	O
+located	O
+next	O
+to	O
+the	O
+P	B-site
+-	I-site
+site	I-site
+tRNA	B-chemical
+on	O
+the	O
+small	O
+subunit	O
+18S	B-chemical
+rRNA	I-chemical
+.	O
+
+While	O
+S	B-chemical
+-	I-chemical
+adenosylmethionine	I-chemical
+was	O
+identified	O
+as	O
+the	O
+source	O
+of	O
+the	O
+aminocarboxypropyl	B-chemical
+(	O
+acp	B-chemical
+)	O
+group	O
+more	O
+than	O
+40	O
+years	O
+ago	O
+the	O
+enzyme	O
+catalyzing	O
+the	O
+acp	B-chemical
+transfer	O
+remained	O
+elusive	O
+.	O
+
+Here	O
+we	O
+identify	O
+the	O
+cytoplasmic	O
+ribosome	O
+biogenesis	O
+protein	O
+Tsr3	B-protein
+as	O
+the	O
+responsible	O
+enzyme	O
+in	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+cells	O
+.	O
+
+In	O
+functionally	O
+impaired	O
+Tsr3	B-protein
+-	O
+mutants	B-protein_state
+,	O
+a	O
+reduced	O
+level	O
+of	O
+acp	B-chemical
+modification	O
+directly	O
+correlates	O
+with	O
+increased	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+accumulation	O
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+archaeal	B-taxonomy_domain
+Tsr3	B-protein
+homologs	O
+revealed	O
+the	O
+same	O
+fold	O
+as	O
+in	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+-	I-protein_type
+methyltransferases	I-protein_type
+but	O
+a	O
+distinct	O
+SAM	B-site
+binding	I-site
+mode	I-site
+.	O
+
+This	O
+unique	O
+SAM	B-site
+binding	I-site
+mode	I-site
+explains	O
+why	O
+Tsr3	B-protein
+transfers	O
+the	O
+acp	B-chemical
+and	O
+not	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	B-chemical
+to	O
+its	O
+substrate	O
+.	O
+
+Structurally	O
+,	O
+Tsr3	B-protein
+therefore	O
+represents	O
+a	O
+novel	O
+class	O
+of	O
+acp	B-protein_type
+transferase	I-protein_type
+enzymes	O
+.	O
+
+Eukaryotic	B-taxonomy_domain
+ribosome	O
+biogenesis	O
+is	O
+highly	O
+complex	O
+and	O
+requires	O
+a	O
+large	O
+number	O
+of	O
+non	O
+-	O
+ribosomal	O
+proteins	O
+and	O
+small	B-chemical
+non	I-chemical
+-	I-chemical
+coding	I-chemical
+RNAs	I-chemical
+in	O
+addition	O
+to	O
+ribosomal	B-chemical
+RNAs	I-chemical
+(	O
+rRNAs	B-chemical
+)	O
+and	O
+proteins	O
+.	O
+
+During	O
+eukaryotic	B-taxonomy_domain
+ribosome	O
+biogenesis	O
+several	O
+dozens	O
+of	O
+rRNA	B-chemical
+nucleotides	B-chemical
+become	O
+chemically	O
+modified	O
+.	O
+
+The	O
+most	O
+abundant	O
+rRNA	B-chemical
+modifications	O
+are	O
+methylations	B-ptm
+at	O
+the	O
+2	O
+′-	O
+OH	O
+ribose	B-chemical
+moieties	O
+and	O
+isomerizations	O
+of	O
+uridine	B-chemical
+residues	O
+to	O
+pseudouridine	B-chemical
+,	O
+catalyzed	O
+by	O
+small	B-complex_assembly
+nucleolar	I-complex_assembly
+ribonucleoprotein	I-complex_assembly
+particles	I-complex_assembly
+(	O
+snoRNPs	B-complex_assembly
+).	O
+
+In	O
+addition	O
+,	O
+18S	B-chemical
+and	O
+25S	B-chemical
+(	O
+yeast	B-taxonomy_domain
+)/	O
+28S	B-chemical
+(	O
+humans	B-species
+)	O
+rRNAs	B-chemical
+contain	O
+several	O
+base	O
+modifications	O
+catalyzed	O
+by	O
+site	O
+-	O
+specific	O
+and	O
+snoRNA	B-chemical
+-	O
+independent	O
+enzymes	O
+.	O
+
+In	O
+Saccharomyces	B-species
+cerevisiae	I-species
+18S	B-chemical
+rRNA	I-chemical
+contains	O
+four	O
+base	O
+methylations	B-ptm
+,	O
+two	O
+acetylations	B-ptm
+and	O
+a	O
+single	O
+3	B-chemical
+-	I-chemical
+amino	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+carboxypropyl	I-chemical
+(	O
+acp	B-chemical
+)	O
+modification	O
+,	O
+whereas	O
+six	O
+base	O
+methylations	B-ptm
+are	O
+present	O
+in	O
+the	O
+25S	B-chemical
+rRNA	I-chemical
+.	O
+
+While	O
+in	O
+humans	B-species
+the	O
+18S	B-chemical
+rRNA	I-chemical
+base	O
+modifications	O
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+,	O
+only	O
+three	O
+of	O
+the	O
+yeast	B-taxonomy_domain
+base	O
+modifications	O
+catalyzed	O
+by	O
+ScRrp8	B-protein
+/	O
+HsNML	B-protein
+,	O
+ScRcm1	B-protein
+/	O
+HsNSUN5	B-protein
+and	O
+ScNop2	B-protein
+/	O
+HsNSUN1	B-protein
+are	O
+preserved	O
+in	O
+the	O
+corresponding	O
+human	B-species
+28S	B-chemical
+rRNA	I-chemical
+.	O
+
+Ribosomal	B-chemical
+RNA	I-chemical
+modifications	O
+have	O
+been	O
+suggested	O
+to	O
+optimize	O
+ribosome	O
+function	O
+,	O
+although	O
+in	O
+most	O
+cases	O
+this	O
+remains	O
+to	O
+be	O
+clearly	O
+established	O
+.	O
+
+They	O
+might	O
+contribute	O
+to	O
+increased	O
+RNA	B-chemical
+stability	O
+by	O
+providing	O
+additional	O
+hydrogen	O
+bonds	O
+(	O
+pseudouridines	B-chemical
+),	O
+improved	O
+base	O
+stacking	O
+(	O
+pseudouridines	B-chemical
+and	O
+base	B-ptm
+methylations	I-ptm
+)	O
+or	O
+an	O
+increased	O
+resistance	O
+against	O
+hydrolysis	O
+(	O
+ribose	B-ptm
+methylations	I-ptm
+).	O
+
+Most	O
+modified	O
+rRNA	B-chemical
+nucleotides	B-chemical
+cluster	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+decoding	B-site
+or	O
+the	O
+peptidyl	B-site
+transferase	I-site
+center	I-site
+,	O
+suggesting	O
+an	O
+influence	O
+on	O
+ribosome	O
+functionality	O
+and	O
+stability	O
+.	O
+
+Defects	O
+of	O
+rRNA	B-chemical
+modification	O
+enzymes	O
+often	O
+lead	O
+to	O
+disturbed	O
+ribosome	O
+biogenesis	O
+or	O
+functionally	O
+impaired	O
+ribosomes	O
+,	O
+although	O
+the	O
+lack	O
+of	O
+individual	O
+rRNA	B-chemical
+modifications	O
+often	O
+has	O
+no	O
+or	O
+only	O
+a	O
+slight	O
+influence	O
+on	O
+the	O
+cell	O
+.	O
+
+The	O
+chemically	O
+most	O
+complex	O
+modification	O
+is	O
+located	O
+in	O
+the	O
+loop	B-structure_element
+capping	I-structure_element
+helix	I-structure_element
+31	I-structure_element
+of	O
+18S	B-chemical
+rRNA	I-chemical
+(	O
+Supplementary	O
+Figure	O
+S1B	O
+).	O
+
+There	O
+a	O
+uridine	B-residue_name
+(	O
+U1191	B-residue_name_number
+in	O
+yeast	B-taxonomy_domain
+)	O
+is	O
+modified	O
+to	O
+1	B-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+3	I-chemical
+-(	I-chemical
+3	I-chemical
+-	I-chemical
+amino	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+carboxypropyl	I-chemical
+)-	I-chemical
+pseudouridine	I-chemical
+(	O
+m1acp3Ψ	B-chemical
+,	O
+Figure	O
+1A	O
+).	O
+
+This	O
+base	O
+modification	O
+was	O
+first	O
+described	O
+in	O
+1968	O
+for	O
+hamster	B-taxonomy_domain
+cells	O
+and	O
+is	O
+conserved	B-protein_state
+in	I-protein_state
+eukaryotes	B-taxonomy_domain
+.	O
+
+This	O
+hypermodified	B-protein_state
+nucleotide	B-chemical
+,	O
+which	O
+is	O
+located	O
+at	O
+the	O
+P	B-site
+-	I-site
+site	I-site
+tRNA	B-chemical
+,	O
+is	O
+synthesized	O
+in	O
+three	O
+steps	O
+beginning	O
+with	O
+the	O
+snR35	B-chemical
+H	B-structure_element
+/	I-structure_element
+ACA	I-structure_element
+snoRNP	B-complex_assembly
+guided	O
+conversion	O
+of	O
+uridine	B-chemical
+into	O
+pseudouridine	B-chemical
+.	O
+
+In	O
+a	O
+second	O
+step	O
+,	O
+the	O
+essential	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+methyltransferase	I-protein_type
+Nep1	B-protein
+/	O
+Emg1	B-protein
+modifies	O
+the	O
+pseudouridine	B-chemical
+to	O
+N1	B-chemical
+-	I-chemical
+methylpseudouridine	I-chemical
+.	O
+
+Methylation	B-ptm
+can	O
+only	O
+occur	O
+once	O
+pseudouridylation	B-ptm
+has	O
+taken	O
+place	O
+,	O
+as	O
+the	O
+latter	O
+reaction	O
+generates	O
+the	O
+substrate	O
+for	O
+the	O
+former	O
+.	O
+
+The	O
+final	O
+acp	B-chemical
+modification	O
+leading	O
+to	O
+N1	B-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+N3	I-chemical
+-	I-chemical
+aminocarboxypropyl	I-chemical
+-	I-chemical
+pseudouridine	I-chemical
+occurs	O
+late	O
+during	O
+40S	B-complex_assembly
+biogenesis	O
+in	O
+the	O
+cytoplasm	O
+,	O
+while	O
+the	O
+two	O
+former	O
+reactions	O
+are	O
+taking	O
+place	O
+in	O
+the	O
+nucleolus	O
+and	O
+nucleus	O
+,	O
+and	O
+is	O
+independent	O
+from	O
+pseudouridylation	B-ptm
+or	O
+methylation	O
+.	O
+
+Both	O
+the	O
+methyl	O
+and	O
+the	O
+acp	O
+group	O
+are	O
+derived	O
+from	O
+S	B-chemical
+-	I-chemical
+adenosylmethionine	I-chemical
+(	O
+SAM	B-chemical
+),	O
+but	O
+the	O
+enzyme	O
+responsible	O
+for	O
+acp	B-chemical
+modification	O
+remained	O
+elusive	O
+for	O
+more	O
+than	O
+40	O
+years	O
+.	O
+
+Tsr3	B-protein
+is	O
+necessary	O
+for	O
+acp	B-chemical
+modification	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+in	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+.	O
+(	O
+A	O
+)	O
+Hypermodified	B-protein_state
+nucleotide	B-chemical
+m1acp3Ψ	B-chemical
+is	O
+synthesized	O
+in	O
+three	O
+steps	O
+:	O
+pseudouridylation	B-ptm
+catalyzed	O
+by	O
+snoRNP35	B-complex_assembly
+,	O
+N1	B-ptm
+-	I-ptm
+methylation	I-ptm
+catalyzed	O
+by	O
+methyltransferase	B-protein_type
+Nep1	B-protein
+and	O
+N3	O
+-	O
+acp	B-chemical
+modification	O
+catalyzed	O
+by	O
+Tsr3	B-protein
+.	O
+
+The	O
+asterisk	O
+indicates	O
+the	O
+C1	O
+-	O
+atom	O
+labeled	O
+in	O
+the	O
+14C	B-experimental_method
+-	I-experimental_method
+incorporation	I-experimental_method
+assay	I-experimental_method
+.	O
+
+(	O
+B	O
+)	O
+RP	B-experimental_method
+-	I-experimental_method
+HPLC	I-experimental_method
+elution	B-evidence
+profile	I-evidence
+of	O
+yeast	B-taxonomy_domain
+18S	B-chemical
+rRNA	I-chemical
+nucleosides	B-chemical
+.	O
+
+Hypermodified	B-protein_state
+m1acp3Ψ	B-chemical
+elutes	O
+at	O
+7	O
+.	O
+4	O
+min	O
+(	O
+wild	B-protein_state
+type	I-protein_state
+,	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
+C	O
+)	O
+14C	B-chemical
+-	I-chemical
+acp	I-chemical
+labeling	O
+of	O
+18S	B-chemical
+rRNAs	I-chemical
+.	O
+
+Wild	B-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+and	O
+plasmid	O
+encoded	O
+18S	B-chemical
+rRNA	I-chemical
+(	O
+U1191U	B-mutant
+)	O
+show	O
+the	O
+14C	B-chemical
+-	I-chemical
+acp	I-chemical
+signal	O
+,	O
+whereas	O
+the	O
+14C	B-chemical
+-	I-chemical
+acp	I-chemical
+signal	O
+is	O
+missing	O
+in	O
+the	O
+U1191A	B-mutant
+mutant	B-protein_state
+plasmid	O
+encoded	O
+18S	B-chemical
+rRNA	I-chemical
+(	O
+U1191A	B-mutant
+)	O
+and	O
+Δtsr3	B-mutant
+mutants	O
+(	O
+Δtsr3	B-mutant
+).	O
+
+Upper	O
+lanes	O
+show	O
+the	O
+ethidium	B-chemical
+bromide	I-chemical
+staining	O
+of	O
+the	O
+18S	B-chemical
+rRNAs	I-chemical
+for	O
+quantification	O
+.	O
+
+All	O
+samples	O
+were	O
+loaded	O
+on	O
+the	O
+gel	O
+with	O
+two	O
+different	O
+amounts	O
+of	O
+5	O
+and	O
+10	O
+μl	O
+.	O
+(	O
+D	O
+)	O
+Primer	B-experimental_method
+extension	I-experimental_method
+analysis	I-experimental_method
+of	O
+acp	B-chemical
+modification	O
+in	O
+yeast	B-taxonomy_domain
+18S	B-chemical
+rRNA	I-chemical
+(	O
+right	O
+gel	O
+)	O
+including	O
+a	O
+sequencing	O
+ladder	O
+(	O
+left	O
+gel	O
+).	O
+
+The	O
+primer	O
+extension	O
+stop	O
+at	O
+nucleotide	O
+1191	B-residue_number
+is	O
+missing	O
+exclusively	O
+in	O
+Δtsr3	B-mutant
+mutants	O
+and	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+recombinants	O
+.	O
+
+(	O
+E	O
+)	O
+Primer	B-experimental_method
+extension	I-experimental_method
+analysis	I-experimental_method
+of	O
+human	B-species
+18S	B-chemical
+rRNA	I-chemical
+after	O
+siRNA	B-experimental_method
+knockdown	I-experimental_method
+of	O
+HsNEP1	B-protein
+/	O
+EMG1	B-protein
+(	O
+541	O
+,	O
+542	O
+and	O
+543	O
+)	O
+and	O
+HsTSR3	B-protein
+(	O
+544	O
+and	O
+545	O
+)	O
+(	O
+right	O
+gel	O
+),	O
+including	O
+a	O
+sequencing	O
+ladder	O
+(	O
+left	O
+gel	O
+).	O
+
+The	O
+primer	O
+extension	O
+arrest	O
+is	O
+reduced	O
+in	O
+HTC116	O
+cells	O
+transfected	O
+with	O
+siRNAs	B-chemical
+544	O
+and	O
+545	O
+.	O
+
+The	O
+efficiency	O
+of	O
+siRNA	B-chemical
+mediated	O
+HsTSR3	B-protein
+repression	O
+correlates	O
+with	O
+the	O
+primer	B-evidence
+extension	I-evidence
+signals	I-evidence
+(	O
+see	O
+Supplementary	O
+Figure	O
+S2A	O
+).	O
+
+Only	O
+a	O
+few	O
+acp	B-chemical
+transferring	O
+enzymes	O
+have	O
+been	O
+characterized	O
+until	O
+now	O
+.	O
+
+During	O
+the	O
+biosynthesis	O
+of	O
+wybutosine	B-chemical
+,	O
+a	O
+tricyclic	O
+nucleoside	B-chemical
+present	O
+in	O
+eukaryotic	B-taxonomy_domain
+and	O
+archaeal	B-taxonomy_domain
+phenylalanine	B-chemical
+tRNA	B-chemical
+,	O
+Tyw2	B-protein
+(	O
+Trm12	B-protein
+in	O
+yeast	B-taxonomy_domain
+)	O
+transfers	O
+an	O
+acp	B-chemical
+group	O
+from	O
+SAM	B-chemical
+to	O
+an	O
+acidic	O
+carbon	O
+atom	O
+.	O
+
+Archaeal	B-taxonomy_domain
+Tyw2	B-protein
+has	O
+a	O
+structure	B-evidence
+very	O
+similar	O
+to	O
+Rossmann	B-protein_type
+-	I-protein_type
+fold	I-protein_type
+(	I-protein_type
+class	I-protein_type
+I	I-protein_type
+)	I-protein_type
+RNA	I-protein_type
+-	I-protein_type
+methyltransferases	I-protein_type
+,	O
+but	O
+its	O
+distinctive	O
+SAM	B-site
+-	I-site
+binding	I-site
+mode	I-site
+enables	O
+the	O
+transfer	O
+of	O
+the	O
+acp	B-chemical
+group	O
+instead	O
+of	O
+the	O
+methyl	O
+group	O
+of	O
+the	O
+cofactor	O
+.	O
+
+Another	O
+acp	B-chemical
+modification	O
+has	O
+been	O
+described	O
+in	O
+the	O
+diphtamide	B-chemical
+biosynthesis	O
+pathway	O
+,	O
+where	O
+an	O
+acp	B-chemical
+group	O
+is	O
+transferred	O
+from	O
+SAM	B-chemical
+to	O
+the	O
+carbon	O
+atom	O
+of	O
+a	O
+histidine	B-residue_name
+residue	O
+of	O
+eukaryotic	B-taxonomy_domain
+translation	B-protein_type
+elongation	I-protein_type
+factor	I-protein_type
+2	I-protein_type
+by	O
+use	O
+of	O
+a	O
+radical	O
+mechanism	O
+.	O
+
+In	O
+a	O
+recent	O
+bioinformatic	O
+study	O
+,	O
+the	O
+uncharacterized	O
+yeast	B-taxonomy_domain
+gene	O
+YOR006c	B-gene
+was	O
+predicted	O
+to	O
+be	O
+involved	O
+in	O
+ribosome	O
+biogenesis	O
+.	O
+
+It	O
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+among	O
+eukaryotes	B-taxonomy_domain
+and	O
+archaea	B-taxonomy_domain
+(	O
+Supplementary	O
+Figure	O
+S1A	O
+)	O
+and	O
+its	O
+deletion	O
+leads	O
+to	O
+an	O
+accumulation	O
+of	O
+the	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+precursor	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+,	O
+suggesting	O
+an	O
+influence	O
+on	O
+D	B-site
+-	I-site
+site	I-site
+cleavage	O
+during	O
+the	O
+maturation	O
+of	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+.	O
+
+On	O
+this	O
+basis	O
+,	O
+YOR006C	B-gene
+was	O
+renamed	O
+‘	O
+Twenty	B-protein
+S	I-protein
+rRNA	I-protein
+accumulation	I-protein
+3	I-protein
+′	O
+(	O
+TSR3	B-protein
+).	O
+
+However	O
+,	O
+its	O
+function	O
+remained	O
+unclear	O
+although	O
+recently	O
+a	O
+putative	O
+nuclease	O
+function	O
+during	O
+18S	B-chemical
+rRNA	I-chemical
+maturation	O
+was	O
+predicted	O
+.	O
+
+Here	O
+,	O
+we	O
+identify	O
+Tsr3	B-protein
+as	O
+the	O
+long	O
+-	O
+sought	O
+acp	B-protein_type
+transferase	I-protein_type
+that	O
+catalyzes	O
+the	O
+last	O
+step	O
+in	O
+the	O
+biosynthesis	O
+of	O
+the	O
+hypermodified	B-protein_state
+nucleotide	B-chemical
+m1acp3Ψ	B-chemical
+in	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+cells	O
+.	O
+
+Furthermore	O
+using	O
+catalytically	B-protein_state
+defective	I-protein_state
+mutants	O
+of	O
+yeast	B-taxonomy_domain
+Tsr3	B-protein
+we	O
+demonstrated	O
+that	O
+the	O
+acp	B-chemical
+modification	O
+is	O
+required	O
+for	O
+18S	B-chemical
+rRNA	I-chemical
+maturation	O
+.	O
+
+Surprisingly	O
+,	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+archaeal	B-taxonomy_domain
+homologs	O
+revealed	O
+that	O
+Tsr3	B-protein
+is	O
+structurally	O
+similar	O
+to	O
+the	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+methyltransferases	I-protein_type
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+only	O
+other	O
+structurally	O
+characterized	O
+acp	B-protein_type
+transferase	I-protein_type
+enzyme	O
+Tyw2	B-protein
+belongs	O
+to	O
+the	O
+Rossmann	B-protein_type
+-	I-protein_type
+fold	I-protein_type
+class	I-protein_type
+of	I-protein_type
+methyltransferase	I-protein_type
+proteins	I-protein_type
+.	O
+
+Interestingly	O
+,	O
+the	O
+two	O
+structurally	O
+very	O
+different	O
+enzymes	O
+use	O
+similar	O
+strategies	O
+in	O
+binding	O
+the	O
+SAM	B-chemical
+-	O
+cofactor	O
+in	O
+order	O
+to	O
+ensure	O
+that	O
+in	O
+contrast	O
+to	O
+methyltransferases	B-protein_type
+the	O
+acp	B-chemical
+and	O
+not	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	B-chemical
+is	O
+transferred	O
+to	O
+the	O
+substrate	O
+.	O
+
+Tsr3	B-protein
+is	O
+the	O
+enzyme	O
+responsible	O
+for	O
+18S	B-chemical
+rRNA	I-chemical
+acp	B-chemical
+modification	O
+in	O
+yeast	B-taxonomy_domain
+and	O
+humans	B-species
+
+The	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+18S	B-protein_type
+rRNA	I-protein_type
+acp	I-protein_type
+transferase	I-protein_type
+was	O
+identified	O
+in	O
+a	O
+systematic	O
+genetic	O
+screen	O
+where	O
+numerous	O
+deletion	O
+mutants	O
+from	O
+the	O
+EUROSCARF	O
+strain	O
+collection	O
+(	O
+www	O
+.	O
+euroscarf	O
+.	O
+de	O
+)	O
+were	O
+analyzed	O
+by	O
+HPLC	B-experimental_method
+for	O
+alterations	O
+in	O
+18S	B-chemical
+rRNA	I-chemical
+base	O
+modifications	O
+.	O
+
+For	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+strain	O
+the	O
+HPLC	B-evidence
+elution	I-evidence
+profile	I-evidence
+of	O
+18S	B-chemical
+rRNA	I-chemical
+nucleosides	B-chemical
+(	O
+Figure	O
+1B	O
+)	O
+was	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+pseudouridine	B-protein_type
+-	I-protein_type
+N1	I-protein_type
+methyltransferase	I-protein_type
+mutant	B-protein_state
+Δnep1	B-mutant
+,	O
+where	O
+a	O
+shoulder	O
+at	O
+∼	O
+7	O
+.	O
+4	O
+min	O
+elution	O
+time	O
+was	O
+missing	O
+in	O
+the	O
+elution	O
+profile	O
+.	O
+
+As	O
+previously	O
+reported	O
+this	O
+shoulder	O
+was	O
+identified	O
+by	O
+ESI	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+as	O
+corresponding	O
+to	O
+m1acp3Ψ	B-chemical
+.	O
+
+In	O
+order	O
+to	O
+directly	O
+analyze	O
+the	O
+presence	O
+of	O
+the	O
+acp	B-chemical
+modification	O
+of	O
+nucleotide	B-chemical
+1191	B-residue_number
+we	O
+used	O
+an	O
+in	B-experimental_method
+vivo14C	I-experimental_method
+incorporation	I-experimental_method
+assay	I-experimental_method
+with	O
+1	B-chemical
+-	I-chemical
+14C	I-chemical
+-	I-chemical
+methionine	I-chemical
+.	O
+
+Whereas	O
+the	O
+acp	B-chemical
+labeling	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+was	O
+clearly	O
+present	O
+in	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+strain	O
+no	O
+radioactive	O
+labeling	O
+could	O
+be	O
+observed	O
+in	O
+a	O
+Δtsr3	B-mutant
+strain	O
+(	O
+Figure	O
+1C	O
+).	O
+
+No	O
+radioactive	O
+labeling	O
+was	O
+detected	O
+in	O
+the	O
+18S	B-mutant
+U1191A	I-mutant
+mutant	B-protein_state
+which	O
+served	O
+as	O
+a	O
+control	O
+for	O
+the	O
+specificity	O
+of	O
+the	O
+14C	B-chemical
+-	I-chemical
+aminocarboxypropyl	I-chemical
+incorporation	O
+.	O
+
+As	O
+previously	O
+shown	O
+,	O
+only	O
+the	O
+acp	B-chemical
+but	O
+none	O
+of	O
+the	O
+other	O
+modifications	O
+at	O
+U1191	B-residue_name_number
+of	O
+yeast	B-taxonomy_domain
+18S	B-chemical
+rRNA	I-chemical
+blocks	O
+reverse	O
+transcriptase	O
+activity	O
+.	O
+
+Therefore	O
+the	O
+presence	O
+of	O
+the	O
+acp	B-chemical
+modification	O
+can	O
+be	O
+directly	O
+assessed	O
+by	O
+primer	B-experimental_method
+extension	I-experimental_method
+.	O
+
+Indeed	O
+,	O
+in	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+yeast	B-taxonomy_domain
+a	O
+strong	O
+primer	B-evidence
+extension	I-evidence
+stop	I-evidence
+signal	I-evidence
+occurred	O
+at	O
+position	O
+1192	B-residue_number
+.	O
+
+In	O
+contrast	O
+,	O
+in	O
+a	O
+Δtsr3	B-mutant
+mutant	B-protein_state
+no	O
+primer	O
+extension	O
+stop	O
+signal	O
+was	O
+present	O
+at	O
+this	O
+position	O
+.	O
+
+As	O
+expected	O
+,	O
+in	O
+a	O
+Δsnr35	B-mutant
+deletion	B-experimental_method
+preventing	O
+pseudouridylation	B-ptm
+and	O
+N1	B-ptm
+-	I-ptm
+methylation	I-ptm
+(	O
+resulting	O
+in	O
+acp3U	B-chemical
+)	O
+as	O
+well	O
+as	O
+in	O
+a	O
+Δnep1	B-mutant
+deletion	O
+strain	O
+where	O
+pseudouridine	B-chemical
+is	O
+not	B-protein_state
+methylated	I-protein_state
+(	O
+resulting	O
+in	O
+acp3Ψ	B-chemical
+)	O
+a	O
+primer	B-evidence
+extension	I-evidence
+stop	I-evidence
+signal	I-evidence
+of	O
+similar	O
+intensity	O
+as	O
+in	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+was	O
+observed	O
+.	O
+
+In	O
+a	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+double	O
+deletion	O
+strain	O
+the	O
+18S	B-chemical
+rRNA	I-chemical
+contains	O
+an	O
+unmodified	B-protein_state
+U	B-chemical
+and	O
+the	O
+primer	O
+extension	O
+stop	O
+signal	O
+was	O
+missing	O
+(	O
+Figure	O
+1D	O
+).	O
+
+The	O
+Tsr3	B-protein
+protein	O
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+yeast	B-taxonomy_domain
+and	O
+humans	B-species
+(	O
+50	O
+%	O
+identity	O
+).	O
+
+Human	B-species
+18S	B-chemical
+rRNA	I-chemical
+has	O
+also	O
+been	O
+shown	O
+to	O
+contain	O
+m1acp3Ψ	B-ptm
+in	O
+the	O
+18S	B-chemical
+rRNA	I-chemical
+at	O
+position	O
+1248	B-residue_number
+.	O
+
+After	O
+siRNA	B-experimental_method
+-	I-experimental_method
+mediated	I-experimental_method
+depletion	I-experimental_method
+of	O
+Tsr3	B-protein
+in	O
+human	B-species
+colon	O
+carcinoma	O
+HCT116	O
+(+/+)	O
+cells	O
+the	O
+acp	B-evidence
+primer	I-evidence
+extension	I-evidence
+arrest	I-evidence
+was	O
+reduced	O
+in	O
+comparison	O
+to	O
+cells	O
+transfected	O
+with	O
+a	O
+non	O
+-	O
+targeting	O
+scramble	O
+siRNA	B-chemical
+control	O
+(	O
+Figure	O
+1E	O
+,	O
+compare	O
+lanes	O
+544	O
+and	O
+scramble	O
+).	O
+
+The	O
+efficiency	O
+of	O
+siRNA	B-chemical
+-	O
+mediated	O
+depletion	O
+was	O
+established	O
+by	O
+RT	B-experimental_method
+-	I-experimental_method
+qPCR	I-experimental_method
+and	O
+found	O
+to	O
+be	O
+very	O
+high	O
+with	O
+siRNA	B-chemical
+544	O
+(	O
+Supplementary	O
+Figure	O
+S2A	O
+,	O
+remaining	O
+TSR3	B-protein
+mRNA	O
+level	O
+of	O
+2	O
+%).	O
+
+By	O
+comparison	O
+,	O
+treating	O
+cells	O
+with	O
+siRNA	B-chemical
+545	O
+,	O
+which	O
+only	O
+reduced	O
+the	O
+TSR3	B-protein
+mRNA	O
+to	O
+20	O
+%,	O
+did	O
+not	O
+markedly	O
+reduced	O
+the	O
+acp	B-chemical
+signal	O
+.	O
+
+This	O
+suggests	O
+that	O
+low	O
+residual	O
+levels	O
+of	O
+HsTsr3	B-protein
+are	O
+sufficient	O
+to	O
+modify	O
+the	O
+RNA	B-chemical
+.	O
+
+Thus	O
+,	O
+HsTsr3	B-protein
+is	O
+also	O
+responsible	O
+for	O
+the	O
+acp	B-chemical
+modification	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+nucleotide	B-chemical
+Ψ1248	B-ptm
+in	O
+helix	B-structure_element
+31	I-structure_element
+.	O
+
+Similar	O
+to	O
+yeast	B-taxonomy_domain
+,	O
+siRNA	B-experimental_method
+-	I-experimental_method
+mediated	I-experimental_method
+depletion	I-experimental_method
+of	O
+the	O
+Ψ1248	B-protein_type
+N1	I-protein_type
+-	I-protein_type
+methyltransferase	I-protein_type
+Nep1	B-protein
+/	O
+Emg1	B-protein
+had	O
+no	O
+influence	O
+on	O
+the	O
+primer	B-evidence
+extension	I-evidence
+arrest	I-evidence
+(	O
+Figure	O
+1E	O
+).	O
+
+Phenotypic	O
+characterization	O
+of	O
+Δtsr3	B-mutant
+mutants	O
+
+Although	O
+the	O
+acp	B-chemical
+modification	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+eukaryotes	B-taxonomy_domain
+,	O
+yeast	B-taxonomy_domain
+Δtsr3	B-mutant
+mutants	O
+showed	O
+only	O
+a	O
+minor	O
+growth	O
+defect	O
+.	O
+
+However	O
+,	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+was	O
+synthetic	O
+sick	O
+with	O
+a	O
+Δsnr35	B-mutant
+deletion	O
+preventing	O
+pseudouridylation	B-ptm
+and	O
+Nep1	B-protein
+-	O
+catalyzed	O
+methylation	O
+of	O
+nucleotide	O
+1191	B-residue_number
+(	O
+Figure	O
+2A	O
+).	O
+
+Interestingly	O
+,	O
+no	O
+increased	O
+growth	O
+defect	O
+could	O
+be	O
+observed	O
+for	O
+Δtsr3	B-mutant
+Δnep1	I-mutant
+recombinants	O
+containing	O
+the	O
+nep1	B-gene
+suppressor	O
+mutation	O
+Δnop6	B-mutant
+as	O
+well	O
+as	O
+for	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+Δnep1	I-mutant
+recombinants	O
+with	O
+unmodified	B-protein_state
+U1191	B-residue_name_number
+(	O
+Supplementary	O
+Figure	O
+S2D	O
+and	O
+E	O
+).	O
+
+Phenotypic	O
+characterization	O
+of	O
+yeast	B-taxonomy_domain
+TSR3	B-protein
+deletion	O
+(	O
+Δtrs3	B-mutant
+)	O
+and	O
+human	B-species
+TSR3	B-protein
+depletion	O
+(	O
+siRNAs	B-chemical
+544	O
+and	O
+545	O
+)	O
+and	O
+cellular	O
+localization	O
+of	O
+yeast	B-taxonomy_domain
+Tsr3	B-protein
+.	O
+(	O
+A	O
+)	O
+Growth	O
+of	O
+yeast	B-taxonomy_domain
+wild	B-protein_state
+type	I-protein_state
+,	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	B-protein
+Δsnr35	B-mutant
+/	O
+SNR35	B-protein
+heterozygous	O
+diploids	O
+.	O
+
+The	O
+Δtsr3	B-mutant
+deletion	O
+is	O
+synthetic	O
+sick	O
+with	O
+a	O
+Δsnr35	B-mutant
+deletion	O
+preventing	O
+U1191	B-residue_name_number
+pseudouridylation	O
+.	O
+
+(	O
+B	O
+)	O
+In	O
+agar	B-experimental_method
+diffusion	I-experimental_method
+assays	I-experimental_method
+the	O
+yeast	B-taxonomy_domain
+Δtsr3	B-mutant
+deletion	B-protein_state
+mutant	I-protein_state
+shows	O
+a	O
+hypersensitivity	O
+against	O
+paromomycin	B-chemical
+and	O
+hygromycin	B-chemical
+B	I-chemical
+which	O
+is	O
+further	O
+increased	O
+by	O
+recombination	O
+with	O
+Δsnr35	B-mutant
+.	O
+(	O
+C	O
+)	O
+Northern	B-experimental_method
+blot	I-experimental_method
+analysis	I-experimental_method
+with	O
+an	O
+ITS1	O
+hybridization	O
+probe	O
+after	O
+siRNA	B-experimental_method
+depletion	I-experimental_method
+of	O
+HsTSR3	B-protein
+(	O
+siRNAs	B-chemical
+544	O
+and	O
+545	O
+)	O
+and	O
+a	O
+scrambled	O
+siRNA	B-chemical
+as	O
+control	O
+.	O
+
+The	O
+accumulation	O
+of	O
+18SE	B-chemical
+and	O
+47S	B-chemical
+and	O
+/	O
+or	O
+45S	B-chemical
+pre	I-chemical
+-	I-chemical
+RNAs	I-chemical
+is	O
+enforced	O
+upon	O
+HsTSR3	B-protein
+depletion	O
+.	O
+
+Right	O
+gel	O
+:	O
+Ethidium	O
+bromide	O
+staining	O
+showing	O
+18S	B-chemical
+and	O
+28S	B-chemical
+rRNAs	I-chemical
+.	O
+
+(	O
+D	O
+)	O
+Cytoplasmic	O
+localization	O
+of	O
+yeast	B-taxonomy_domain
+Tsr3	B-protein
+shown	O
+by	O
+fluorescence	B-experimental_method
+microscopy	I-experimental_method
+of	O
+GFP	B-mutant
+-	I-mutant
+fused	I-mutant
+Tsr3	I-mutant
+.	O
+
+From	O
+left	O
+to	O
+right	O
+:	O
+differential	B-experimental_method
+interference	I-experimental_method
+contrast	I-experimental_method
+(	O
+DIC	B-experimental_method
+),	O
+green	O
+fluorescence	O
+of	O
+GFP	B-mutant
+-	I-mutant
+Tsr3	I-mutant
+,	O
+red	O
+fluorescence	O
+of	O
+Nop56	B-mutant
+-	I-mutant
+mRFP	I-mutant
+as	O
+nucleolar	O
+marker	O
+,	O
+and	O
+merge	O
+of	O
+GFP	B-mutant
+-	I-mutant
+Tsr3	I-mutant
+/	O
+Nop56	B-mutant
+-	I-mutant
+mRFP	I-mutant
+with	O
+DIC	B-experimental_method
+.	O
+(	O
+E	O
+)	O
+Elution	B-evidence
+profile	I-evidence
+(	O
+A254	O
+)	O
+after	O
+sucrose	B-experimental_method
+gradient	I-experimental_method
+separation	I-experimental_method
+of	O
+yeast	B-taxonomy_domain
+ribosomal	B-complex_assembly
+subunits	I-complex_assembly
+and	O
+polysomes	B-complex_assembly
+(	O
+upper	O
+part	O
+)	O
+and	O
+western	B-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+3xHA	B-chemical
+tagged	O
+Tsr3	B-protein
+(	O
+Tsr3	B-mutant
+-	I-mutant
+3xHA	I-mutant
+)	O
+after	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+separation	O
+of	O
+polysome	O
+profile	O
+fractions	O
+taken	O
+every	O
+20	O
+s	O
+(	O
+lower	O
+part	O
+).	O
+
+The	O
+TSR3	B-protein
+gene	O
+was	O
+genetically	O
+modified	O
+at	O
+its	O
+native	O
+locus	O
+,	O
+resulting	O
+in	O
+a	O
+C	O
+-	O
+terminal	O
+fusion	B-protein_state
+of	O
+Tsr3	B-protein
+with	O
+a	O
+3xHA	B-chemical
+epitope	O
+expressed	O
+by	O
+the	O
+native	O
+promotor	O
+in	O
+yeast	B-taxonomy_domain
+strain	O
+CEN	O
+.	O
+BM258	O
+-	O
+5B	O
+.	O
+
+The	O
+influence	O
+of	O
+the	O
+acp	B-chemical
+modification	O
+of	O
+nucleotide	B-chemical
+1191	B-residue_number
+on	O
+ribosome	O
+function	O
+was	O
+analyzed	O
+by	O
+treating	O
+Δtsr3	B-mutant
+mutants	O
+with	O
+protein	O
+synthesis	O
+inhibitors	O
+.	O
+
+Similar	O
+to	O
+a	O
+temperature	O
+-	O
+sensitive	O
+nep1	B-gene
+mutant	B-protein_state
+,	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+caused	O
+hypersensitivity	O
+to	O
+paromomycin	B-chemical
+and	O
+,	O
+to	O
+a	O
+lesser	O
+extent	O
+,	O
+to	O
+hygromycin	B-chemical
+B	I-chemical
+(	O
+Figure	O
+2B	O
+),	O
+but	O
+not	O
+to	O
+G418	B-chemical
+or	O
+cycloheximide	B-chemical
+(	O
+data	O
+not	O
+shown	O
+).	O
+
+In	O
+accordance	O
+with	O
+the	O
+synthetic	O
+sick	O
+growth	O
+phenotype	O
+the	O
+paromomycin	B-chemical
+and	O
+hygromycin	B-chemical
+B	I-chemical
+hypersensitivity	O
+further	O
+increased	O
+in	O
+a	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+recombination	O
+strain	O
+(	O
+Figure	O
+2B	O
+).	O
+
+In	O
+a	O
+yeast	B-taxonomy_domain
+Δtsr3	B-mutant
+strain	O
+as	O
+well	O
+as	O
+in	O
+the	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+recombinant	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+accumulated	O
+significantly	O
+and	O
+the	O
+level	O
+of	O
+mature	O
+18S	B-chemical
+rRNA	I-chemical
+was	O
+reduced	O
+(	O
+Supplementary	O
+Figures	O
+S2C	O
+and	O
+S3D	O
+),	O
+as	O
+reported	O
+previously	O
+.	O
+
+A	O
+minor	O
+effect	O
+on	O
+20S	B-chemical
+rRNA	I-chemical
+accumulation	O
+was	O
+also	O
+observed	O
+for	O
+Δsnr35	B-mutant
+,	O
+but	O
+-	O
+probably	O
+due	O
+to	O
+different	O
+strain	O
+backgrounds	O
+–	O
+to	O
+a	O
+weaker	O
+extent	O
+than	O
+described	O
+earlier	O
+.	O
+
+In	O
+human	B-species
+cells	O
+,	O
+the	O
+depletion	B-experimental_method
+of	I-experimental_method
+HsTsr3	B-protein
+in	O
+HCT116	O
+(+/+)	O
+cells	O
+caused	O
+an	O
+accumulation	O
+of	O
+the	O
+human	B-species
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+equivalent	O
+18S	B-chemical
+-	I-chemical
+E	I-chemical
+suggesting	O
+an	O
+evolutionary	O
+conserved	O
+role	O
+of	O
+Tsr3	B-protein
+in	O
+the	O
+late	O
+steps	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+processing	O
+(	O
+Figure	O
+2C	O
+and	O
+Supplementary	O
+Figure	O
+S2B	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	B-taxonomy_domain
+Δtsr3	B-mutant
+cells	O
+(	O
+see	O
+accumulation	O
+of	O
+35S	B-complex_assembly
+in	O
+Supplementary	O
+Figure	O
+S2C	O
+)	O
+and	O
+Tsr3	B-protein
+depleted	O
+human	B-species
+cells	O
+(	O
+see	O
+47S	B-complex_assembly
+/	O
+45S	B-complex_assembly
+accumulation	O
+in	O
+Figure	O
+2C	O
+and	O
+Northern	B-experimental_method
+blot	I-experimental_method
+quantification	O
+in	O
+Supplementary	O
+Figure	O
+S2B	O
+).	O
+
+Consistent	O
+with	O
+its	O
+role	O
+in	O
+late	O
+18S	B-chemical
+rRNA	I-chemical
+processing	O
+,	O
+TSR3	B-protein
+deletion	O
+leads	O
+to	O
+a	O
+ribosomal	O
+subunit	O
+imbalance	O
+with	O
+a	O
+reduced	O
+40S	B-complex_assembly
+to	O
+60S	B-complex_assembly
+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
+
+In	O
+polysome	B-evidence
+profiles	I-evidence
+,	O
+a	O
+reduced	O
+level	O
+of	O
+80S	B-complex_assembly
+ribosomes	I-complex_assembly
+and	O
+a	O
+strong	O
+signal	O
+for	O
+free	O
+60S	B-complex_assembly
+subunits	O
+was	O
+observed	O
+in	O
+line	O
+with	O
+the	O
+40S	B-complex_assembly
+subunit	O
+deficiency	O
+(	O
+Supplementary	O
+Figure	O
+S2G	O
+).	O
+
+Cellular	O
+localization	O
+of	O
+Tsr3	B-protein
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+
+Fluorescence	B-experimental_method
+microscopy	I-experimental_method
+of	O
+GFP	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+Tsr3	B-protein
+localized	O
+the	O
+fusion	O
+protein	O
+in	O
+the	O
+cytoplasm	O
+of	O
+yeast	B-taxonomy_domain
+cells	O
+and	O
+no	O
+co	O
+-	O
+localization	O
+with	O
+the	O
+nucleolar	O
+marker	O
+protein	O
+Nop56	B-protein
+could	O
+be	O
+observed	O
+(	O
+Figure	O
+2D	O
+).	O
+
+This	O
+agrees	O
+with	O
+previous	O
+biochemical	O
+data	O
+suggesting	O
+that	O
+the	O
+acp	B-chemical
+modification	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+occurs	O
+late	O
+during	O
+40S	B-complex_assembly
+subunit	O
+biogenesis	O
+in	O
+the	O
+cytoplasm	O
+,	O
+and	O
+makes	O
+an	O
+additional	O
+nuclear	O
+localization	O
+as	O
+reported	O
+in	O
+a	O
+previous	O
+large	O
+-	O
+scale	O
+analysis	O
+unlikely	O
+.	O
+
+After	O
+polysome	B-experimental_method
+gradient	I-experimental_method
+separation	I-experimental_method
+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
+
+Such	O
+distribution	B-evidence
+on	I-evidence
+a	I-evidence
+density	I-evidence
+gradient	I-evidence
+suggests	O
+that	O
+Tsr3	B-protein
+only	O
+interacts	O
+transiently	O
+with	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+subunits	I-complex_assembly
+,	O
+which	O
+presumably	O
+explains	O
+why	O
+it	O
+was	O
+not	O
+characterized	O
+in	O
+pre	B-experimental_method
+-	I-experimental_method
+ribosome	I-experimental_method
+affinity	I-experimental_method
+purifications	I-experimental_method
+.	O
+
+Structure	B-evidence
+of	O
+Tsr3	B-protein
+
+Searches	O
+for	O
+sequence	O
+homologs	O
+of	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+Tsr3	B-protein
+(	O
+ScTsr3	B-protein
+)	O
+by	O
+us	O
+and	O
+others	O
+revealed	O
+that	O
+the	O
+genomes	O
+of	O
+many	O
+archaea	B-taxonomy_domain
+contain	O
+genes	O
+encoding	O
+Tsr3	B-protein_type
+-	I-protein_type
+like	I-protein_type
+proteins	I-protein_type
+.	O
+
+However	O
+,	O
+these	O
+archaeal	B-taxonomy_domain
+homologs	O
+are	O
+significantly	O
+smaller	O
+than	O
+ScTsr3	B-protein
+(∼	O
+190	O
+aa	O
+in	O
+archaea	B-taxonomy_domain
+vs	O
+.	O
+313	O
+aa	O
+in	O
+yeast	B-taxonomy_domain
+)	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
+
+To	O
+locate	O
+the	O
+domains	O
+most	O
+important	O
+for	O
+Tsr3	B-protein
+activity	O
+,	O
+ScTsr3	B-protein
+fragments	O
+of	O
+different	O
+lengths	O
+containing	O
+the	O
+highly	B-protein_state
+conserved	I-protein_state
+central	O
+part	O
+were	O
+expressed	B-experimental_method
+in	O
+a	O
+Δtsr3	B-mutant
+mutant	B-protein_state
+(	O
+Figure	O
+3A	O
+)	O
+and	O
+analyzed	O
+by	O
+primer	B-experimental_method
+extension	I-experimental_method
+(	O
+Figure	O
+3B	O
+)	O
+and	O
+Northern	B-experimental_method
+blotting	I-experimental_method
+(	O
+Figure	O
+3C	O
+).	O
+
+N	O
+-	O
+terminal	O
+truncations	B-experimental_method
+of	O
+up	O
+to	O
+45	B-residue_range
+aa	I-residue_range
+and	O
+C	O
+-	O
+terminal	O
+truncations	B-experimental_method
+of	O
+up	O
+to	O
+76	B-residue_range
+aa	I-residue_range
+mediated	O
+acp	B-chemical
+modification	O
+as	O
+efficiently	O
+as	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+and	O
+no	O
+significant	O
+increased	O
+levels	O
+of	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+RNA	I-chemical
+were	O
+detected	O
+.	O
+
+Even	O
+a	O
+Tsr3	B-protein
+fragment	O
+with	O
+a	O
+90	B-residue_range
+aa	I-residue_range
+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	B-residue_range
+aa	I-residue_range
+almost	O
+completely	O
+abolished	O
+the	O
+primer	O
+extension	O
+arrest	O
+(	O
+Figure	O
+3B	O
+).	O
+
+Domain	O
+characterization	O
+of	O
+yeast	B-taxonomy_domain
+Tsr3	B-protein
+and	O
+correlation	O
+of	O
+acp	B-chemical
+modification	O
+with	O
+late	O
+18S	B-chemical
+rRNA	I-chemical
+processing	O
+steps	O
+.	O
+(	O
+A	O
+)	O
+Scheme	O
+of	O
+the	O
+TSR3	B-protein
+gene	O
+with	O
+truncation	O
+positions	O
+in	O
+the	O
+open	O
+reading	O
+frame	O
+.	O
+
+TSR3	B-protein
+fragments	O
+of	O
+different	O
+length	O
+were	O
+expressed	O
+under	O
+the	O
+native	O
+promotor	O
+from	O
+multicopy	O
+plasmids	O
+in	O
+a	O
+Δtsr3	B-mutant
+deletion	O
+strain	O
+.	O
+
+(	O
+B	O
+)	O
+Primer	B-experimental_method
+extension	I-experimental_method
+analysis	I-experimental_method
+of	O
+18S	B-chemical
+rRNA	I-chemical
+acp	B-chemical
+modification	O
+in	O
+yeast	B-taxonomy_domain
+cells	O
+expressing	O
+the	O
+indicated	O
+TSR3	B-protein
+fragments	O
+.	O
+
+N	O
+-	O
+terminal	O
+deletions	B-experimental_method
+of	O
+36	B-residue_range
+or	O
+45	B-residue_range
+amino	O
+acids	O
+and	O
+C	O
+-	O
+terminal	O
+deletions	B-experimental_method
+of	O
+43	B-residue_range
+or	O
+76	B-residue_range
+residues	O
+show	O
+a	O
+primer	B-evidence
+extension	I-evidence
+stop	I-evidence
+comparable	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
+Tsr3	B-protein
+fragments	O
+37	B-residue_range
+–	I-residue_range
+223	I-residue_range
+or	O
+46	B-residue_range
+–	I-residue_range
+223	I-residue_range
+cause	O
+a	O
+nearly	O
+complete	O
+loss	O
+of	O
+the	O
+arrest	O
+signal	O
+.	O
+
+The	O
+box	O
+highlights	O
+the	O
+shortest	O
+Tsr3	B-protein
+fragment	O
+(	O
+aa	O
+46	B-residue_range
+–	I-residue_range
+270	I-residue_range
+)	O
+with	O
+wild	B-protein_state
+type	I-protein_state
+activity	O
+(	O
+strong	O
+primer	B-evidence
+extension	I-evidence
+block	I-evidence
+).	O
+(	O
+C	O
+)	O
+Northern	B-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+accumulation	O
+.	O
+
+A	O
+weak	O
+20S	B-chemical
+rRNA	I-chemical
+signal	O
+,	O
+indicating	O
+normal	O
+processing	O
+,	O
+is	O
+observed	O
+for	O
+Tsr3	B-protein
+fragment	O
+46	B-residue_range
+–	I-residue_range
+270	I-residue_range
+(	O
+highlighted	O
+in	O
+a	O
+box	O
+)	O
+showing	O
+its	O
+functionality	O
+.	O
+
+Strong	O
+20S	O
+rRNA	O
+accumulation	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+Δtsr3	B-mutant
+deletion	B-experimental_method
+is	O
+observed	O
+for	O
+Tsr3	B-protein
+fragments	O
+37	B-residue_range
+–	I-residue_range
+223	I-residue_range
+or	O
+46	B-residue_range
+–	I-residue_range
+223	I-residue_range
+.	O
+
+Thus	O
+,	O
+the	O
+archaeal	B-taxonomy_domain
+homologs	O
+correspond	O
+to	O
+the	O
+functional	O
+core	O
+of	O
+Tsr3	B-protein
+.	O
+
+In	O
+order	O
+to	O
+define	O
+the	O
+structural	O
+basis	O
+for	O
+Tsr3	B-protein
+function	O
+,	O
+homologs	O
+from	O
+thermophilic	B-taxonomy_domain
+archaea	I-taxonomy_domain
+were	O
+screened	O
+for	O
+crystallization	B-experimental_method
+.	O
+
+We	O
+focused	O
+on	O
+archaeal	B-taxonomy_domain
+species	O
+containing	O
+a	O
+putative	O
+Nep1	B-protein
+homolog	O
+suggesting	O
+that	O
+these	O
+species	O
+are	O
+in	O
+principle	O
+capable	O
+of	O
+synthesizing	O
+N1	B-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+N3	I-chemical
+-	I-chemical
+acp	I-chemical
+-	I-chemical
+pseudouridine	I-chemical
+.	O
+
+Well	O
+diffracting	O
+crystals	B-evidence
+were	O
+obtained	O
+for	O
+Tsr3	B-protein
+homologs	O
+from	O
+the	O
+two	O
+crenarchaeal	B-taxonomy_domain
+species	O
+Vulcanisaeta	B-species
+distributa	I-species
+(	O
+VdTsr3	B-protein
+)	O
+and	O
+Sulfolobus	B-species
+solfataricus	I-species
+(	O
+SsTsr3	B-protein
+)	O
+which	O
+share	O
+36	O
+%	O
+(	O
+VdTsr3	B-protein
+)	O
+and	O
+38	O
+%	O
+(	O
+SsTsr3	B-protein
+)	O
+identity	O
+with	O
+the	O
+ScTsr3	B-protein
+core	B-structure_element
+region	I-structure_element
+(	O
+ScTsr3	B-protein
+aa	O
+46	B-residue_range
+–	I-residue_range
+223	I-residue_range
+).	O
+
+While	O
+for	O
+S	B-species
+.	I-species
+solfataricus	I-species
+the	O
+existence	O
+of	O
+a	O
+modified	O
+nucleotide	B-chemical
+of	O
+unknown	O
+chemical	O
+composition	O
+in	O
+the	O
+loop	B-structure_element
+capping	I-structure_element
+helix	I-structure_element
+31	I-structure_element
+of	O
+its	O
+16S	B-chemical
+rRNA	I-chemical
+has	O
+been	O
+demonstrated	O
+,	O
+no	O
+information	O
+regarding	O
+rRNA	O
+modifications	O
+is	O
+yet	O
+available	O
+for	O
+V	B-species
+.	I-species
+distributa	I-species
+.	O
+
+Crystals	B-evidence
+of	O
+VdTsr3	B-protein
+diffracted	O
+to	O
+a	O
+resolution	O
+of	O
+1	O
+.	O
+6	O
+Å	O
+whereas	O
+crystals	B-evidence
+of	O
+SsTsr3	B-protein
+diffracted	O
+to	O
+2	O
+.	O
+25	O
+Å	O
+.	O
+Serendipitously	O
+,	O
+VdTsr3	B-protein
+was	O
+purified	O
+and	O
+crystallized	B-experimental_method
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+endogenous	B-protein_state
+(	O
+E	B-species
+.	I-species
+coli	I-species
+)	O
+SAM	B-chemical
+(	O
+Supplementary	O
+Figure	O
+S4	O
+)	O
+while	O
+SsTsr3	B-protein
+crystals	B-evidence
+contained	O
+the	O
+protein	O
+in	O
+the	O
+apo	B-protein_state
+state	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+VdTsr3	B-protein
+was	O
+solved	O
+ab	O
+initio	O
+,	O
+by	O
+single	B-experimental_method
+-	I-experimental_method
+wavelength	I-experimental_method
+anomalous	I-experimental_method
+diffraction	I-experimental_method
+phasing	I-experimental_method
+(	O
+Se	B-experimental_method
+-	I-experimental_method
+SAD	I-experimental_method
+)	O
+with	O
+Se	B-chemical
+containing	O
+derivatives	O
+(	O
+selenomethionine	B-chemical
+and	O
+seleno	B-chemical
+-	I-chemical
+substituted	I-chemical
+SAM	I-chemical
+).	O
+
+The	O
+structure	B-evidence
+of	O
+SsTsr3	B-protein
+was	O
+solved	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+using	O
+VdTsr3	B-protein
+as	O
+a	O
+search	O
+model	O
+(	O
+see	O
+Supplementary	O
+Table	O
+S1	O
+for	O
+data	O
+collection	O
+and	O
+refinement	O
+statistics	O
+).	O
+
+The	O
+structure	B-evidence
+of	O
+VdTsr3	B-protein
+can	O
+be	O
+divided	O
+into	O
+two	O
+domains	O
+(	O
+Figure	O
+4A	O
+).	O
+
+The	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+aa	O
+1	B-residue_range
+–	I-residue_range
+92	I-residue_range
+)	O
+has	O
+a	O
+mixed	O
+α	B-structure_element
+/	I-structure_element
+β	I-structure_element
+-	I-structure_element
+structure	I-structure_element
+centered	O
+around	O
+a	O
+five	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+all	I-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+Figure	O
+4B	O
+)	O
+with	O
+the	O
+strand	O
+order	O
+β5	B-structure_element
+↑-	I-structure_element
+β3	B-structure_element
+↑-	I-structure_element
+β4	B-structure_element
+↑-	I-structure_element
+β1	B-structure_element
+↑-	I-structure_element
+β2	B-structure_element
+↑.	I-structure_element
+The	O
+loops	B-structure_element
+connecting	O
+β1	B-structure_element
+and	O
+β2	B-structure_element
+,	O
+β3	B-structure_element
+and	O
+β4	B-structure_element
+and	O
+β4	B-structure_element
+and	O
+β5	B-structure_element
+include	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+α1	B-structure_element
+,	O
+α2	B-structure_element
+and	O
+α3	B-structure_element
+,	O
+respectively	O
+.	O
+
+The	O
+loop	B-structure_element
+connecting	O
+β2	B-structure_element
+and	O
+β3	B-structure_element
+contains	O
+a	O
+single	O
+turn	O
+of	O
+a	O
+310	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+.	O
+Helices	B-structure_element
+α1	B-structure_element
+and	O
+α2	B-structure_element
+are	O
+located	O
+on	O
+one	O
+side	O
+of	O
+the	O
+five	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+while	O
+α3	B-structure_element
+packs	O
+against	O
+the	O
+opposite	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+surface	O
+.	O
+
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+aa	O
+93	B-residue_range
+–	I-residue_range
+184	I-residue_range
+)	O
+has	O
+a	O
+globular	B-structure_element
+all	I-structure_element
+α	I-structure_element
+-	I-structure_element
+helical	I-structure_element
+structure	I-structure_element
+comprising	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+α4	B-structure_element
+to	I-structure_element
+α9	I-structure_element
+.	O
+
+Remarkably	O
+,	O
+the	O
+entire	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+92	B-residue_range
+aa	I-residue_range
+)	O
+of	O
+the	O
+protein	O
+is	O
+threaded	O
+through	O
+the	O
+loop	B-structure_element
+which	O
+connects	O
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+β3	B-structure_element
+and	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+α2	B-structure_element
+of	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+.	O
+
+Thus	O
+,	O
+the	O
+VdTsr3	B-protein
+structure	B-evidence
+contains	O
+a	O
+deep	B-structure_element
+trefoil	I-structure_element
+knot	I-structure_element
+.	O
+
+The	O
+structure	B-evidence
+of	O
+SsTsr3	B-protein
+in	O
+the	O
+apo	B-protein_state
+state	O
+is	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+VdTsr3	B-protein
+(	O
+Figure	O
+4C	O
+)	O
+with	O
+an	O
+RMSD	B-evidence
+for	O
+equivalent	O
+Cα	O
+atoms	O
+of	O
+1	O
+.	O
+1	O
+Å	O
+.	O
+The	O
+only	O
+significant	O
+difference	O
+in	O
+the	O
+global	O
+structure	B-evidence
+of	O
+the	O
+two	O
+proteins	O
+is	O
+the	O
+presence	O
+of	O
+an	O
+extended	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+α8	B-structure_element
+and	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+α9	B-structure_element
+in	O
+SsTsr3	B-protein
+.	O
+
+Tsr3	B-protein
+has	O
+a	O
+fold	O
+similar	O
+to	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+methyltransferases	I-protein_type
+.	O
+(	O
+A	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structure	I-evidence
+of	O
+VdTsr3	B-protein
+in	O
+two	O
+orientations	O
+.	O
+
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+are	O
+colored	O
+in	O
+crimson	O
+whereas	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+in	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+are	O
+colored	O
+light	O
+blue	O
+and	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+are	O
+colored	O
+dark	O
+blue	O
+.	O
+
+The	O
+bound	O
+S	B-chemical
+-	I-chemical
+adenosylmethionine	I-chemical
+is	O
+shown	O
+in	O
+a	O
+stick	O
+representation	O
+and	O
+colored	O
+by	O
+atom	O
+type	O
+.	O
+
+A	O
+red	O
+arrow	O
+marks	O
+the	O
+location	O
+of	O
+the	O
+topological	B-structure_element
+knot	I-structure_element
+in	O
+the	O
+structure	B-evidence
+.	O
+(	O
+B	O
+)	O
+Secondary	O
+structure	O
+representation	O
+of	O
+the	O
+VdTsr3	B-protein
+structure	B-evidence
+.	O
+
+The	O
+color	O
+coding	O
+is	O
+the	O
+same	O
+as	O
+in	O
+(	O
+A	O
+).	O
+(	O
+C	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structures	I-evidence
+of	O
+VdTsr3	B-protein
+in	O
+the	O
+SAM	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+state	O
+(	O
+red	O
+)	O
+and	O
+SsTsr3	B-protein
+(	O
+blue	O
+)	O
+in	O
+the	O
+apo	B-protein_state
+state	O
+.	O
+
+The	O
+locations	O
+of	O
+the	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+α8	B-structure_element
+which	O
+is	O
+longer	O
+in	O
+SsTsr3	B-protein
+and	O
+of	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+α9	B-structure_element
+which	O
+is	O
+only	O
+present	O
+in	O
+VdTsr3	B-protein
+are	O
+indicated	O
+.	O
+(	O
+D	O
+)	O
+Secondary	O
+structure	O
+cartoon	O
+(	O
+left	O
+)	O
+of	O
+S	B-species
+.	I-species
+pombe	I-species
+Trm10	B-protein
+(	O
+pdb4jwf	O
+)—	O
+the	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+methyltransferase	I-protein_type
+structurally	O
+most	O
+similar	O
+to	O
+Tsr3	B-protein
+and	O
+superposition	B-experimental_method
+of	O
+the	O
+VdTsr3	B-protein
+and	O
+Trm10	B-protein
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structures	I-evidence
+(	O
+right	O
+).	O
+(	O
+E	O
+)	O
+Analytical	B-experimental_method
+gel	I-experimental_method
+filtration	I-experimental_method
+profiles	B-evidence
+for	O
+VdTsr3	B-protein
+(	O
+red	O
+)	O
+and	O
+SsTsr3	B-protein
+(	O
+blue	O
+)	O
+show	O
+that	O
+both	O
+proteins	O
+are	O
+monomeric	B-oligomeric_state
+in	O
+solution	O
+.	O
+
+Vd	B-species
+,	O
+Vulcanisaeta	B-species
+distributa	I-species
+;	O
+Ss	B-species
+,	O
+Sulfolobus	B-species
+solfataricus	I-species
+.	O
+
+Structure	B-experimental_method
+predictions	I-experimental_method
+suggested	O
+that	O
+Tsr3	B-protein
+might	O
+contain	O
+a	O
+so	O
+-	O
+called	O
+RLI	B-structure_element
+domain	I-structure_element
+which	O
+contains	O
+a	O
+‘	O
+bacterial	B-structure_element
+like	I-structure_element
+’	I-structure_element
+ferredoxin	I-structure_element
+fold	I-structure_element
+and	O
+binds	O
+two	O
+iron	O
+-	O
+sulfur	O
+clusters	O
+through	O
+eight	O
+conserved	B-protein_state
+cysteine	B-residue_name
+residues	O
+.	O
+
+However	O
+,	O
+no	O
+structural	O
+similarity	O
+to	O
+an	O
+RLI	B-structure_element
+-	I-structure_element
+domain	I-structure_element
+was	O
+detectable	O
+.	O
+
+This	O
+is	O
+in	O
+accordance	O
+with	O
+the	O
+functional	O
+analysis	O
+of	O
+alanine	B-experimental_method
+replacement	I-experimental_method
+mutations	I-experimental_method
+of	O
+cysteine	B-residue_name
+residues	O
+in	O
+ScTsr3	B-protein
+(	O
+Supplementary	O
+Figure	O
+S3	O
+).	O
+
+The	O
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+topology	I-structure_element
+and	O
+the	O
+deep	O
+C	O
+-	O
+terminal	O
+trefoil	B-structure_element
+knot	I-structure_element
+of	O
+archaeal	B-taxonomy_domain
+Tsr3	B-protein
+are	O
+the	O
+structural	O
+hallmarks	O
+of	O
+the	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+-	I-protein_type
+methyltransferase	I-protein_type
+fold	O
+.	O
+
+The	O
+closest	O
+structural	O
+homolog	O
+identified	O
+in	O
+a	O
+DALI	B-experimental_method
+search	I-experimental_method
+is	O
+the	O
+tRNA	B-protein_type
+methyltransferase	I-protein_type
+Trm10	B-protein
+(	O
+DALI	B-evidence
+Z	I-evidence
+-	I-evidence
+score	I-evidence
+6	O
+.	O
+8	O
+)	O
+which	O
+methylates	O
+the	O
+N1	O
+nitrogen	O
+of	O
+G9	B-residue_name_number
+/	O
+A9	B-residue_name_number
+in	O
+many	O
+archaeal	B-taxonomy_domain
+and	O
+eukaryotic	B-taxonomy_domain
+tRNAs	B-chemical
+by	O
+using	O
+SAM	B-chemical
+as	O
+the	O
+methyl	O
+group	O
+donor	O
+.	O
+
+In	O
+comparison	O
+to	O
+Tsr3	B-protein
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+element	I-structure_element
+of	O
+Trm10	B-protein
+is	O
+extended	O
+by	O
+one	O
+additional	O
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+pairing	O
+to	O
+β2	B-structure_element
+.	O
+
+Furthermore	O
+,	O
+the	O
+trefoil	B-structure_element
+knot	I-structure_element
+of	O
+Trm10	B-protein
+is	O
+not	O
+as	O
+deep	O
+as	O
+that	O
+of	O
+Tsr3	B-protein
+(	O
+Figure	O
+4D	O
+).	O
+
+Interestingly	O
+,	O
+Nep1	B-protein
+—	O
+the	O
+enzyme	O
+preceding	O
+Tsr3	B-protein
+in	O
+the	O
+biosynthetic	O
+pathway	O
+for	O
+the	O
+synthesis	O
+of	O
+m1acp3Ψ	B-chemical
+—	O
+also	O
+belongs	O
+to	O
+the	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+of	I-protein_type
+RNA	I-protein_type
+methyltransferases	I-protein_type
+.	O
+
+However	O
+,	O
+the	O
+structural	O
+similarities	O
+between	O
+Nep1	B-protein
+and	O
+Tsr3	B-protein
+(	O
+DALI	B-evidence
+Z	I-evidence
+-	I-evidence
+score	I-evidence
+4	O
+.	O
+4	O
+)	O
+are	O
+less	O
+pronounced	O
+than	O
+between	O
+Tsr3	B-protein
+and	O
+Trm10	B-protein
+.	O
+
+Most	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+-	I-protein_type
+methyltransferases	I-protein_type
+are	O
+homodimers	B-oligomeric_state
+.	O
+
+A	O
+notable	O
+exception	O
+is	O
+Trm10	B-protein
+.	O
+
+Gel	B-experimental_method
+filtration	I-experimental_method
+experiments	O
+with	O
+both	O
+VdTsr3	B-protein
+and	O
+SsTsr3	B-protein
+(	O
+Figure	O
+4E	O
+)	O
+showed	O
+that	O
+both	O
+proteins	O
+are	O
+monomeric	B-oligomeric_state
+in	O
+solution	O
+thereby	O
+extending	O
+the	O
+structural	O
+similarities	O
+to	O
+Trm10	B-protein
+.	O
+
+So	O
+far	O
+,	O
+structural	O
+information	O
+is	O
+only	O
+available	O
+for	O
+one	O
+other	O
+enzyme	O
+that	O
+transfers	O
+the	O
+acp	B-chemical
+group	O
+from	O
+SAM	B-chemical
+to	O
+an	O
+RNA	B-chemical
+nucleotide	B-chemical
+.	O
+
+This	O
+enzyme	O
+,	O
+Tyw2	B-protein
+,	O
+is	O
+part	O
+of	O
+the	O
+biosynthesis	O
+pathway	O
+of	O
+wybutosine	B-chemical
+nucleotides	I-chemical
+in	O
+tRNAs	B-chemical
+.	O
+
+However	O
+,	O
+there	O
+are	O
+no	O
+structural	O
+similarities	O
+between	O
+Tsr3	B-protein
+and	O
+Tyw2	B-protein
+,	O
+which	O
+contains	O
+an	O
+all	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+a	O
+different	O
+topology	O
+and	O
+no	O
+knot	B-structure_element
+structure	I-structure_element
+.	O
+
+Instead	O
+,	O
+Tyw2	B-protein
+has	O
+a	O
+fold	O
+typical	O
+for	O
+the	O
+class	B-protein_type
+-	I-protein_type
+I	I-protein_type
+-	I-protein_type
+or	I-protein_type
+Rossmann	I-protein_type
+-	I-protein_type
+fold	I-protein_type
+class	I-protein_type
+of	I-protein_type
+methyltransferases	I-protein_type
+(	O
+Supplementary	O
+Figure	O
+S5B	O
+).	O
+
+Cofactor	O
+binding	O
+of	O
+Tsr3	B-protein
+
+The	O
+SAM	B-site
+-	I-site
+binding	I-site
+site	I-site
+of	O
+Tsr3	B-protein
+is	O
+located	O
+in	O
+a	O
+deep	O
+crevice	O
+between	O
+the	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domains	I-structure_element
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+trefoil	B-structure_element
+knot	I-structure_element
+as	O
+typical	O
+for	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+-	I-protein_type
+methyltransferases	I-protein_type
+(	O
+Figure	O
+4A	O
+).	O
+
+The	O
+adenine	B-chemical
+base	O
+of	O
+the	O
+cofactor	O
+is	O
+recognized	O
+by	O
+hydrogen	O
+bonds	O
+between	O
+its	O
+N1	O
+nitrogen	O
+and	O
+the	O
+backbone	O
+amide	O
+of	O
+L93	B-residue_name_number
+directly	O
+preceding	O
+β5	B-structure_element
+as	O
+well	O
+as	O
+between	O
+its	O
+N6	O
+-	O
+amino	O
+group	O
+and	O
+the	O
+backbone	O
+carbonyl	O
+group	O
+of	O
+Y108	B-residue_name_number
+located	O
+in	O
+the	O
+loop	B-structure_element
+connecting	O
+β5	B-structure_element
+in	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+and	O
+α4	B-structure_element
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+Figure	O
+5A	O
+).	O
+
+Furthermore	O
+,	O
+the	O
+adenine	B-chemical
+base	O
+of	O
+SAM	B-chemical
+is	O
+involved	O
+in	O
+hydrophobic	O
+packing	O
+interactions	O
+with	O
+the	O
+side	O
+chains	O
+of	O
+L45	B-residue_name_number
+(	O
+β3	B-structure_element
+),	O
+P47	B-residue_name_number
+and	O
+W73	B-residue_name_number
+(	O
+α3	B-structure_element
+)	O
+in	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+as	O
+well	O
+as	O
+with	O
+L93	B-residue_name_number
+,	O
+L110	B-residue_name_number
+(	O
+both	O
+in	O
+the	O
+loop	B-structure_element
+connecting	O
+β5	B-structure_element
+and	O
+α4	B-structure_element
+)	O
+and	O
+A115	B-residue_name_number
+(	O
+α5	B-structure_element
+)	O
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+ribose	B-chemical
+2	O
+′	O
+and	O
+3	O
+′	O
+hydroxyl	O
+groups	O
+of	O
+SAM	B-chemical
+are	O
+hydrogen	O
+bonded	O
+to	O
+the	O
+backbone	O
+carbonyl	O
+group	O
+of	O
+I69	B-residue_name_number
+.	O
+
+The	O
+acp	B-chemical
+side	O
+chain	O
+of	O
+SAM	B-chemical
+is	O
+fixed	O
+in	O
+position	O
+by	O
+hydrogen	O
+bonding	O
+of	O
+its	O
+carboxylate	O
+group	O
+to	O
+the	O
+backbone	O
+amide	O
+and	O
+the	O
+side	O
+chain	O
+hydroxyl	O
+group	O
+of	O
+T19	B-residue_name_number
+in	O
+α1	B-structure_element
+as	O
+well	O
+as	O
+the	O
+backbone	O
+amide	O
+group	O
+of	O
+T112	B-residue_name_number
+in	O
+α4	B-structure_element
+(	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+).	O
+
+Most	O
+importantly	O
+,	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	B-chemical
+is	O
+buried	O
+in	O
+a	O
+hydrophobic	B-site
+pocket	I-site
+formed	O
+by	O
+the	O
+sidechains	O
+of	O
+W73	B-residue_name_number
+and	O
+A76	B-residue_name_number
+both	O
+located	O
+in	O
+α3	B-structure_element
+(	O
+Figure	O
+5A	O
+and	O
+B	O
+).	O
+
+W73	B-residue_name_number
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+all	O
+known	O
+Tsr3	B-protein_type
+proteins	I-protein_type
+,	O
+whereas	O
+A76	B-residue_name_number
+can	O
+be	O
+replaced	O
+by	O
+other	O
+hydrophobic	O
+amino	B-chemical
+acids	I-chemical
+.	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	B-protein_type
+-	I-protein_type
+methyltransferases	I-protein_type
+such	O
+as	O
+Trm10	B-protein
+(	O
+Figure	O
+5B	O
+,	O
+C	O
+).	O
+
+In	O
+contrast	O
+,	O
+the	O
+acp	B-chemical
+side	O
+chain	O
+of	O
+SAM	B-chemical
+is	O
+accessible	O
+for	O
+reactions	O
+in	O
+the	O
+Tsr3	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+state	O
+(	O
+Figure	O
+5B	O
+).	O
+
+SAM	B-chemical
+-	O
+binding	O
+by	O
+Tsr3	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+SAM	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+of	O
+VdTsr3	B-protein
+.	O
+
+Nitrogen	O
+atoms	O
+are	O
+dark	O
+blue	O
+,	O
+oxygen	O
+atoms	O
+red	O
+,	O
+sulfur	B-chemical
+atoms	O
+orange	O
+,	O
+carbon	O
+atoms	O
+of	O
+the	O
+protein	O
+light	O
+blue	O
+and	O
+carbon	O
+atoms	O
+of	O
+SAM	B-chemical
+yellow	O
+.	O
+
+(	O
+B	O
+)	O
+Solvent	O
+accessibility	O
+of	O
+the	O
+acp	B-chemical
+group	O
+of	O
+SAM	B-chemical
+bound	B-protein_state
+to	I-protein_state
+VdTsr3	B-protein
+.	O
+
+The	O
+solvent	O
+accessible	O
+surface	O
+of	O
+the	O
+protein	O
+is	O
+shown	O
+in	O
+semitransparent	O
+gray	O
+whereas	O
+SAM	B-chemical
+is	O
+show	O
+in	O
+a	O
+stick	O
+representation	O
+.	O
+
+A	O
+red	O
+arrow	O
+indicates	O
+the	O
+reactive	O
+CH2	O
+-	O
+moiety	O
+of	O
+the	O
+acp	B-chemical
+group	O
+.	O
+(	O
+C	O
+)	O
+Solvent	O
+accessibility	O
+of	O
+the	O
+SAM	B-chemical
+methyl	O
+group	O
+for	O
+SAM	B-chemical
+bound	B-protein_state
+to	I-protein_state
+the	O
+RNA	B-protein_type
+methyltransferase	I-protein_type
+Trm10	B-protein
+.	O
+
+Bound	B-protein_state
+SAM	B-chemical
+was	O
+modelled	O
+based	O
+on	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structure	I-evidence
+of	O
+the	O
+Trm10	B-complex_assembly
+/	I-complex_assembly
+SAH	I-complex_assembly
+-	O
+complex	O
+(	O
+pdb4jwf	O
+).	O
+
+A	O
+red	O
+arrow	O
+indicates	O
+the	O
+SAM	B-chemical
+methyl	O
+group	O
+.	O
+(	O
+D	O
+)	O
+Binding	O
+of	O
+SAM	B-chemical
+analogs	O
+to	O
+SsTsr3	B-protein
+.	O
+
+Tryptophan	B-evidence
+fluorescence	I-evidence
+quenching	I-evidence
+curves	I-evidence
+upon	O
+addition	O
+of	O
+SAM	B-chemical
+(	O
+blue	O
+),	O
+5	B-chemical
+′-	I-chemical
+methyl	I-chemical
+-	I-chemical
+thioadenosine	I-chemical
+(	O
+red	O
+)	O
+and	O
+SAH	B-chemical
+(	O
+black	O
+).	O
+
+(	O
+E	O
+)	O
+Binding	O
+of	O
+14C	B-chemical
+-	I-chemical
+labeled	I-chemical
+SAM	I-chemical
+to	O
+SsTsr3	B-protein
+.	O
+
+Radioactively	O
+labeled	O
+SAM	B-chemical
+is	O
+retained	O
+on	O
+a	O
+filter	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+SsTsr3	B-protein
+.	O
+
+Addition	O
+of	O
+unlabeled	O
+SAM	B-chemical
+competes	O
+with	O
+the	O
+binding	O
+of	O
+labeled	O
+SAM	B-chemical
+.	O
+
+A	O
+W66A	B-mutant
+-	O
+mutant	B-protein_state
+of	O
+SsTsr3	B-protein
+(	O
+W73	B-residue_name_number
+in	O
+VdTsr3	B-protein
+)	O
+does	O
+not	O
+bind	O
+SAM	B-chemical
+.	O
+
+(	O
+F	O
+)	O
+Primer	B-experimental_method
+extension	I-experimental_method
+(	O
+upper	O
+left	O
+)	O
+shows	O
+a	O
+strongly	O
+reduced	O
+acp	B-chemical
+modification	O
+of	O
+yeast	B-taxonomy_domain
+18S	B-chemical
+rRNA	I-chemical
+in	O
+Δtsr3	B-mutant
+cells	O
+expressing	O
+Tsr3	B-mutant
+-	I-mutant
+S62D	I-mutant
+,	O
+-	B-mutant
+E111A	I-mutant
+or	O
+–	B-mutant
+W114A	I-mutant
+.	O
+
+This	O
+correlates	O
+with	O
+a	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+accumulation	O
+comparable	O
+to	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+(	O
+right	O
+:	O
+northern	B-experimental_method
+blot	I-experimental_method
+).	O
+
+3xHA	B-protein_state
+tagged	I-protein_state
+Tsr3	B-protein
+mutants	B-protein_state
+are	O
+expressed	O
+comparable	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+as	O
+shown	O
+by	O
+western	B-experimental_method
+blot	I-experimental_method
+(	O
+lower	O
+left	O
+).	O
+
+Binding	B-evidence
+affinities	I-evidence
+for	O
+SAM	B-chemical
+and	O
+its	O
+analogs	O
+5	B-chemical
+′-	I-chemical
+methylthioadenosin	I-chemical
+and	O
+SAH	B-chemical
+to	O
+SsTsr3	B-protein
+were	O
+measured	O
+using	O
+tryptophan	B-experimental_method
+fluorescence	I-experimental_method
+quenching	I-experimental_method
+.	O
+
+VdTsr3	B-protein
+could	O
+not	O
+be	O
+used	O
+in	O
+these	O
+experiments	O
+since	O
+we	O
+could	O
+not	O
+purify	O
+it	O
+in	O
+a	O
+stable	B-protein_state
+SAM	B-protein_state
+-	I-protein_state
+free	I-protein_state
+form	O
+.	O
+
+SsTsr3	B-protein
+bound	B-protein_state
+SAM	B-chemical
+with	O
+a	O
+KD	B-evidence
+of	O
+6	O
+.	O
+5	O
+μM	O
+,	O
+which	O
+is	O
+similar	O
+to	O
+SAM	B-evidence
+-	I-evidence
+KD	I-evidence
+'	I-evidence
+s	I-evidence
+reported	O
+for	O
+several	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+methyltransferases	I-protein_type
+.	O
+
+5	B-chemical
+′-	I-chemical
+methylthioadenosin	I-chemical
+—	O
+the	O
+reaction	O
+product	O
+after	O
+the	O
+acp	B-chemical
+-	O
+transfer	O
+—	O
+binds	O
+only	O
+∼	O
+2	O
+.	O
+5	O
+-	O
+fold	O
+weaker	O
+(	O
+KD	O
+=	O
+16	O
+.	O
+7	O
+μM	O
+)	O
+compared	O
+to	O
+SAM	B-chemical
+.	O
+
+S	B-chemical
+-	I-chemical
+adenosylhomocysteine	I-chemical
+which	O
+lacks	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	B-chemical
+binds	O
+with	O
+significantly	O
+lower	O
+affinity	B-evidence
+(	O
+KD	B-evidence
+=	O
+55	O
+.	O
+5	O
+μM	O
+)	O
+(	O
+Figure	O
+5D	O
+).	O
+
+This	O
+suggests	O
+that	O
+the	O
+hydrophobic	O
+interaction	O
+between	O
+SAM	B-chemical
+'	O
+s	O
+methyl	O
+group	O
+and	O
+the	O
+hydrophobic	B-site
+pocket	I-site
+of	O
+Tsr3	B-protein
+is	O
+thermodynamically	O
+important	O
+for	O
+the	O
+interaction	O
+.	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+the	O
+loss	O
+of	O
+hydrogen	O
+bonds	O
+between	O
+the	O
+acp	B-chemical
+sidechain	O
+carboxylate	O
+group	O
+and	O
+the	O
+protein	O
+appears	O
+to	O
+be	O
+thermodynamically	O
+less	O
+important	O
+but	O
+these	O
+hydrogen	O
+bonds	O
+might	O
+play	O
+a	O
+crucial	O
+role	O
+for	O
+the	O
+proper	O
+orientation	O
+of	O
+the	O
+cofactor	O
+side	O
+chain	O
+in	O
+the	O
+substrate	B-site
+binding	I-site
+pocket	I-site
+.	O
+
+Accordingly	O
+,	O
+a	O
+W66A	B-mutant
+-	O
+mutation	B-experimental_method
+(	O
+W73	B-residue_name_number
+in	O
+VdTsr3	B-protein
+)	O
+of	O
+SsTsr3	B-protein
+significantly	O
+diminished	O
+SAM	B-evidence
+-	I-evidence
+binding	I-evidence
+in	O
+a	O
+filter	B-experimental_method
+binding	I-experimental_method
+assay	I-experimental_method
+compared	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+Figure	O
+5E	O
+).	O
+
+Furthermore	O
+,	O
+a	O
+W	B-experimental_method
+to	I-experimental_method
+A	I-experimental_method
+mutation	I-experimental_method
+at	O
+the	O
+equivalent	O
+position	O
+W114	B-residue_name_number
+in	O
+ScTsr3	B-protein
+strongly	O
+reduced	O
+the	O
+in	O
+vivo	O
+acp	B-protein_type
+transferase	I-protein_type
+activity	O
+(	O
+Figure	O
+5F	O
+).	O
+
+The	O
+side	O
+chain	O
+hydroxyl	O
+group	O
+of	O
+T19	B-residue_name_number
+seems	O
+of	O
+minor	O
+importance	O
+for	O
+SAM	B-chemical
+binding	O
+since	O
+mutations	B-experimental_method
+of	O
+T17	B-residue_name_number
+(	O
+T19	B-residue_name_number
+in	O
+VdTsr3	B-protein
+)	O
+to	O
+either	O
+A	B-residue_name
+or	O
+D	B-residue_name
+did	O
+not	O
+significantly	O
+influence	O
+the	O
+SAM	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+SsTsr3	B-protein
+(	O
+KD	B-evidence
+'	O
+s	O
+=	O
+3	O
+.	O
+9	O
+or	O
+11	O
+.	O
+2	O
+mM	O
+,	O
+respectively	O
+).	O
+
+Nevertheless	O
+,	O
+a	O
+mutation	B-experimental_method
+of	O
+the	O
+equivalent	O
+position	O
+S62	B-residue_name_number
+of	O
+ScTsr3	B-protein
+to	O
+D	B-residue_name
+,	O
+but	O
+not	O
+to	O
+A	B-residue_name
+,	O
+resulted	O
+in	O
+reduced	O
+acp	B-chemical
+modification	O
+in	O
+vivo	O
+,	O
+as	O
+shown	O
+by	O
+primer	B-experimental_method
+extension	I-experimental_method
+analysis	I-experimental_method
+(	O
+Figure	O
+5F	O
+).	O
+
+The	O
+acp	B-chemical
+-	O
+transfer	O
+reaction	O
+catalyzed	O
+by	O
+Tsr3	B-protein
+most	O
+likely	O
+requires	O
+the	O
+presence	O
+of	O
+a	O
+catalytic	O
+base	O
+in	O
+order	O
+to	O
+abstract	O
+a	O
+proton	O
+from	O
+the	O
+N3	O
+imino	O
+group	O
+of	O
+the	O
+modified	O
+pseudouridine	B-chemical
+.	O
+
+The	O
+side	O
+chain	O
+of	O
+D70	B-residue_name_number
+(	O
+VdTsr3	B-protein
+)	O
+located	O
+in	O
+β4	B-structure_element
+is	O
+∼	O
+5	O
+Å	O
+away	O
+from	O
+the	O
+SAM	B-chemical
+sulfur	O
+atom	O
+.	O
+
+This	O
+residue	O
+is	O
+conserved	B-protein_state
+as	I-protein_state
+D	B-residue_name
+or	O
+E	B-residue_name
+both	O
+in	O
+archaeal	B-taxonomy_domain
+and	O
+eukaryotic	B-taxonomy_domain
+Tsr3	B-protein
+homologs	O
+.	O
+
+Mutations	B-experimental_method
+of	O
+the	O
+corresponding	O
+residue	O
+in	O
+SsTsr3	B-protein
+to	O
+A	B-residue_name
+(	O
+D63	B-residue_name_number
+)	O
+does	O
+not	O
+significantly	O
+alter	O
+the	O
+SAM	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+the	O
+protein	O
+(	O
+KD	B-evidence
+=	O
+11	O
+.	O
+0	O
+μM	O
+).	O
+
+However	O
+,	O
+the	O
+mutation	B-experimental_method
+of	O
+the	O
+corresponding	O
+residue	O
+of	O
+ScTsr3	B-protein
+(	O
+E111A	B-mutant
+)	O
+leads	O
+to	O
+a	O
+significant	O
+decrease	O
+of	O
+the	O
+acp	B-protein_type
+transferase	I-protein_type
+activity	O
+in	O
+vivo	O
+(	O
+Figure	O
+5F	O
+).	O
+
+RNA	O
+-	O
+binding	O
+of	O
+Tsr3	B-protein
+
+Analysis	B-experimental_method
+of	I-experimental_method
+the	I-experimental_method
+electrostatic	I-experimental_method
+surface	I-experimental_method
+properties	I-experimental_method
+of	O
+VdTsr3	B-protein
+clearly	O
+identified	O
+positively	B-site
+charged	I-site
+surface	I-site
+patches	I-site
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+SAM	B-site
+-	I-site
+binding	I-site
+site	I-site
+suggesting	O
+a	O
+putative	O
+RNA	B-site
+-	I-site
+binding	I-site
+site	I-site
+(	O
+Figure	O
+6A	O
+).	O
+
+Furthermore	O
+,	O
+a	O
+negatively	O
+charged	O
+MES	B-chemical
+-	O
+ion	O
+is	O
+found	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+VdTsr3	B-protein
+complexed	B-protein_state
+to	I-protein_state
+the	O
+side	O
+chain	O
+of	O
+K22	B-residue_name_number
+in	O
+helix	B-structure_element
+α1	B-structure_element
+.	O
+
+Its	O
+negatively	O
+charged	O
+sulfate	B-chemical
+group	O
+might	O
+mimic	O
+an	O
+RNA	B-chemical
+backbone	O
+phosphate	O
+.	O
+
+Helix	B-structure_element
+α1	B-structure_element
+contains	O
+two	O
+more	O
+positively	O
+charged	O
+amino	O
+acids	O
+K17	B-residue_name_number
+and	O
+R25	B-residue_name_number
+as	O
+does	O
+the	O
+loop	B-structure_element
+preceding	O
+it	O
+(	O
+R9	B-residue_name_number
+).	O
+
+A	O
+second	O
+cluster	O
+of	O
+positively	O
+charged	O
+residues	O
+is	O
+found	O
+in	O
+or	O
+near	O
+helix	B-structure_element
+α3	B-structure_element
+(	O
+K74	B-residue_name_number
+,	O
+R75	B-residue_name_number
+,	O
+K82	B-residue_name_number
+,	O
+R85	B-residue_name_number
+and	O
+K87	B-residue_name_number
+).	O
+
+Some	O
+of	O
+these	O
+amino	O
+acids	O
+are	O
+conserved	B-protein_state
+between	O
+archaeal	B-taxonomy_domain
+and	O
+eukaryotic	B-taxonomy_domain
+Tsr3	B-protein
+(	O
+Supplementary	O
+Figure	O
+S1A	O
+).	O
+
+In	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+,	O
+the	O
+surface	O
+exposed	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+α5	B-structure_element
+and	O
+α7	B-structure_element
+carry	O
+a	O
+significant	O
+amount	O
+of	O
+positively	O
+charged	O
+amino	O
+acids	O
+.	O
+
+A	O
+triple	B-experimental_method
+mutation	I-experimental_method
+of	O
+the	O
+conserved	B-protein_state
+positively	O
+charged	O
+residues	O
+R60	B-residue_name_number
+,	O
+K65	B-residue_name_number
+and	O
+R131	B-residue_name_number
+to	O
+A	B-residue_name
+in	O
+ScTsr3	B-protein
+resulted	O
+in	O
+a	O
+protein	O
+with	O
+a	O
+significantly	O
+impaired	O
+acp	B-protein_type
+transferase	I-protein_type
+activity	O
+in	O
+vivo	O
+(	O
+Figure	O
+6D	O
+)	O
+in	O
+line	O
+with	O
+an	O
+important	O
+functional	O
+role	O
+for	O
+these	O
+positively	O
+charged	O
+residues	O
+.	O
+
+RNA	O
+-	O
+binding	O
+of	O
+Tsr3	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Electrostatic	O
+charge	O
+distribution	O
+on	O
+the	O
+surface	O
+of	O
+VdTsr3	B-protein
+.	O
+
+SAM	B-chemical
+is	O
+shown	O
+in	O
+a	O
+stick	O
+representation	O
+.	O
+
+Also	O
+shown	O
+in	O
+stick	O
+representation	O
+is	O
+a	O
+negatively	O
+charged	O
+MES	B-chemical
+ion	O
+.	O
+
+Conserved	B-protein_state
+basic	O
+amino	B-chemical
+acids	I-chemical
+are	O
+labeled	O
+.	O
+(	O
+B	O
+)	O
+Comparison	O
+of	O
+the	O
+secondary	O
+structures	O
+of	O
+helix	B-structure_element
+31	I-structure_element
+from	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+rRNAs	B-chemical
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+and	O
+S	B-species
+.	I-species
+solfataricus	I-species
+with	O
+the	O
+location	O
+of	O
+the	O
+hypermodified	B-protein_state
+nucleotide	B-chemical
+indicated	O
+in	O
+red	O
+.	O
+
+For	O
+S	B-species
+.	I-species
+solfataricus	I-species
+the	O
+chemical	O
+identity	O
+of	O
+the	O
+hypermodified	B-protein_state
+nucleotide	B-chemical
+is	O
+not	O
+known	O
+but	O
+the	O
+existence	O
+of	O
+NEP1	B-protein
+and	O
+TSR3	B-protein
+homologs	O
+suggest	O
+that	O
+it	O
+is	O
+indeed	O
+N1	B-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+N3	I-chemical
+-	I-chemical
+acp	I-chemical
+-	I-chemical
+pseudouridine	I-chemical
+.	O
+
+(	O
+C	O
+)	O
+Binding	O
+of	O
+SsTsr3	B-protein
+to	O
+RNA	B-chemical
+.	O
+
+5	O
+′-	O
+fluoresceine	B-chemical
+labeled	O
+RNA	B-chemical
+oligonucleotides	O
+corresponding	O
+either	O
+to	O
+the	O
+native	B-protein_state
+(	O
+20mer	B-oligomeric_state
+–	O
+see	O
+inset	O
+)	O
+or	O
+a	O
+stabilized	B-protein_state
+(	O
+20mer_GC	B-oligomeric_state
+-	O
+inset	O
+)	O
+helix	B-structure_element
+31	I-structure_element
+of	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+rRNA	B-chemical
+from	O
+S	B-species
+.	I-species
+solfataricus	I-species
+were	O
+titrated	B-experimental_method
+with	I-experimental_method
+increasing	I-experimental_method
+amounts	I-experimental_method
+of	O
+SsTsr3	B-protein
+and	O
+the	O
+changes	O
+in	O
+the	O
+fluoresceine	B-chemical
+fluorescence	B-evidence
+anisotropy	I-evidence
+were	O
+measured	O
+and	O
+fitted	O
+to	O
+a	O
+binding	B-evidence
+curve	I-evidence
+(	O
+20mer	B-oligomeric_state
+–	O
+red	O
+,	O
+20mer_GC	B-oligomeric_state
+–	O
+blue	O
+).	O
+
+Oligo	B-chemical
+-	I-chemical
+U9	I-chemical
+-	I-chemical
+RNA	I-chemical
+was	O
+used	O
+for	O
+comparison	O
+(	O
+black	O
+).	O
+
+The	O
+20mer_GC	B-oligomeric_state
+RNA	B-chemical
+was	O
+also	O
+titrated	B-experimental_method
+with	O
+SsTsr3	B-protein
+in	O
+the	O
+presence	O
+of	O
+2	O
+mM	O
+SAM	B-chemical
+(	O
+purple	O
+).	O
+(	O
+D	O
+)	O
+Mutants	B-protein_state
+of	O
+ScTsr3	B-protein
+R60	B-residue_name_number
+,	O
+K65	B-residue_name_number
+or	O
+R131	B-residue_name_number
+(	O
+equivalent	O
+to	O
+K17	B-residue_name_number
+,	O
+K22	B-residue_name_number
+and	O
+R91	B-residue_name_number
+in	O
+VdTsr3	B-protein
+)	O
+expressed	B-experimental_method
+in	O
+Δtsr3	B-mutant
+yeast	B-taxonomy_domain
+cells	O
+show	O
+a	O
+primer	B-evidence
+extension	I-evidence
+stop	I-evidence
+comparable	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
+Combination	B-experimental_method
+of	I-experimental_method
+the	I-experimental_method
+three	I-experimental_method
+point	I-experimental_method
+mutations	I-experimental_method
+(	O
+R60A	B-mutant
+/	O
+K65A	B-mutant
+/	O
+R131A	B-mutant
+)	O
+leads	O
+to	O
+a	O
+strongly	O
+reduced	O
+acp	B-chemical
+modification	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+.	O
+
+In	O
+order	O
+to	O
+explore	O
+the	O
+RNA	O
+-	O
+ligand	O
+specificity	O
+of	O
+Tsr3	B-protein
+we	O
+titrated	B-experimental_method
+SsTsr3	B-protein
+prepared	O
+in	O
+RNase	B-protein_state
+-	I-protein_state
+free	I-protein_state
+form	O
+with	O
+5	O
+′-	O
+fluoresceine	B-chemical
+-	O
+labeled	O
+RNA	B-chemical
+and	O
+determined	O
+the	O
+affinity	B-evidence
+by	O
+fluorescence	B-experimental_method
+anisotropy	I-experimental_method
+measurements	I-experimental_method
+.	O
+
+SsTsr3	B-protein
+in	O
+the	O
+apo	B-protein_state
+state	O
+bound	B-protein_state
+a	O
+20mer	B-oligomeric_state
+RNA	B-chemical
+corresponding	O
+to	O
+helix	B-structure_element
+31	I-structure_element
+of	O
+S	B-species
+.	I-species
+solfataricus	I-species
+16S	B-chemical
+rRNA	I-chemical
+(	O
+Figure	O
+6B	O
+)	O
+with	O
+a	O
+KD	B-evidence
+of	O
+1	O
+.	O
+9	O
+μM	O
+and	O
+to	O
+a	O
+version	O
+of	O
+this	O
+hairpin	B-structure_element
+stabilized	O
+by	O
+additional	O
+GC	O
+base	O
+pairs	O
+(	O
+20mer	B-oligomeric_state
+-	I-oligomeric_state
+GC	I-oligomeric_state
+)	O
+with	O
+a	O
+KD	B-evidence
+of	O
+0	O
+.	O
+6	O
+μM	O
+(	O
+Figure	O
+6C	O
+).	O
+
+A	O
+single	O
+stranded	O
+oligoU	B-chemical
+-	I-chemical
+RNA	I-chemical
+bound	B-protein_state
+with	O
+a	O
+10	O
+-	O
+fold	O
+-	O
+reduced	O
+affinity	B-evidence
+(	O
+6	O
+.	O
+0	O
+μM	O
+).	O
+
+The	O
+presence	O
+of	O
+saturating	O
+amounts	O
+of	O
+SAM	B-chemical
+(	O
+2	O
+mM	O
+)	O
+did	O
+not	O
+have	O
+a	O
+significant	O
+influence	O
+on	O
+the	O
+RNA	B-evidence
+-	I-evidence
+affinity	I-evidence
+of	O
+SsTsr3	B-protein
+(	O
+KD	B-evidence
+of	O
+1	O
+.	O
+7	O
+μM	O
+for	O
+the	O
+20mer	B-oligomeric_state
+-	I-oligomeric_state
+GC	I-oligomeric_state
+-	O
+RNA	B-chemical
+)	O
+suggesting	O
+no	O
+cooperativity	O
+in	O
+substrate	O
+binding	O
+.	O
+
+U1191	B-residue_name_number
+is	O
+the	O
+only	O
+hypermodified	B-protein_state
+base	O
+in	O
+the	O
+yeast	B-taxonomy_domain
+18S	B-chemical
+rRNA	I-chemical
+and	O
+is	O
+strongly	B-protein_state
+conserved	I-protein_state
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+The	O
+formation	O
+of	O
+1	B-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+3	I-chemical
+-(	I-chemical
+3	I-chemical
+-	I-chemical
+amino	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+carboxypropyl	I-chemical
+)-	I-chemical
+pseudouridine	I-chemical
+(	O
+m1acp3Ψ	B-chemical
+)	O
+is	O
+very	O
+complex	O
+requiring	O
+three	O
+successive	O
+modification	O
+reactions	O
+involving	O
+one	O
+H	B-structure_element
+/	I-structure_element
+ACA	I-structure_element
+snoRNP	B-complex_assembly
+(	O
+snR35	B-protein
+)	O
+and	O
+two	O
+protein	O
+enzymes	O
+(	O
+Nep1	B-protein
+/	O
+Emg1	B-protein
+and	O
+Tsr3	B-protein
+).	O
+
+This	O
+makes	O
+it	O
+unique	O
+in	O
+eukaryotic	B-taxonomy_domain
+rRNA	B-chemical
+modification	O
+.	O
+
+The	O
+m1acp3Ψ	B-chemical
+base	O
+is	O
+located	O
+at	O
+the	O
+tip	O
+of	O
+helix	B-structure_element
+31	I-structure_element
+on	O
+the	O
+18S	B-chemical
+rRNA	I-chemical
+(	O
+Supplementary	O
+Figure	O
+S1B	O
+)	O
+which	O
+,	O
+together	O
+with	O
+helices	B-structure_element
+18	I-structure_element
+,	I-structure_element
+24	I-structure_element
+,	I-structure_element
+34	I-structure_element
+and	I-structure_element
+44	I-structure_element
+,	O
+contribute	O
+to	O
+building	O
+the	O
+decoding	O
+center	O
+of	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+.	O
+
+A	O
+similar	O
+modification	O
+(	O
+acp3U	B-chemical
+)	O
+was	O
+identified	O
+in	O
+Haloferax	B-species
+volcanii	I-species
+and	O
+corresponding	O
+modified	O
+nucleotides	B-chemical
+were	O
+also	O
+shown	O
+to	O
+occur	O
+in	O
+other	O
+archaea	B-taxonomy_domain
+.	O
+
+As	O
+shown	O
+here	O
+TSR3	B-protein
+encodes	O
+the	O
+transferase	O
+catalyzing	O
+the	O
+acp	B-chemical
+modification	O
+as	O
+the	O
+last	O
+step	O
+in	O
+the	O
+biosynthesis	O
+of	O
+m1acp3Ψ	B-chemical
+in	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+cells	O
+.	O
+
+Unexpectedly	O
+,	O
+archaeal	B-taxonomy_domain
+Tsr3	B-protein
+has	O
+a	O
+structure	B-evidence
+similar	O
+to	O
+SPOUT	B-protein_type
+-	I-protein_type
+class	I-protein_type
+RNA	I-protein_type
+methyltransferases	I-protein_type
+,	O
+and	O
+it	O
+is	O
+the	O
+first	O
+example	O
+for	O
+an	O
+enzyme	O
+of	O
+this	O
+class	O
+transferring	O
+an	O
+acp	B-chemical
+group	O
+,	O
+due	O
+to	O
+a	O
+modified	O
+SAM	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+that	O
+exposes	O
+the	O
+acp	B-chemical
+instead	O
+of	O
+the	O
+methyl	O
+group	O
+of	O
+SAM	B-chemical
+to	O
+its	O
+RNA	B-chemical
+substrate	O
+.	O
+
+Similar	O
+to	O
+the	O
+structurally	O
+unrelated	O
+Rossmann	B-protein_type
+-	I-protein_type
+fold	I-protein_type
+Tyw2	I-protein_type
+acp	I-protein_type
+transferase	I-protein_type
+,	O
+the	O
+SAM	B-chemical
+methyl	O
+group	O
+of	O
+Tsr3	B-protein
+is	O
+bound	O
+in	O
+an	O
+inaccessible	O
+hydrophobic	B-site
+pocket	I-site
+whereas	O
+the	O
+acp	B-chemical
+side	O
+chain	O
+becomes	O
+accessible	O
+for	O
+a	O
+nucleophilic	O
+attack	O
+by	O
+the	O
+N3	O
+of	O
+pseudouridine	B-chemical
+.	O
+
+In	O
+contrast	O
+,	O
+in	O
+the	O
+structurally	O
+closely	O
+related	O
+RNA	B-protein_type
+methyltransferase	I-protein_type
+Trm10	B-protein
+the	O
+methyl	O
+group	O
+of	O
+the	O
+cofactor	O
+SAM	B-chemical
+is	O
+accessible	O
+whereas	O
+its	O
+acp	B-chemical
+side	O
+chain	O
+is	O
+buried	O
+inside	O
+the	O
+protein	O
+.	O
+
+This	O
+suggests	O
+that	O
+enzymes	O
+with	O
+a	O
+SAM	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+acp	I-protein_type
+transferase	I-protein_type
+activity	O
+might	O
+have	O
+evolved	O
+from	O
+SAM	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+methyltransferases	I-protein_type
+by	O
+slight	O
+modifications	O
+of	O
+the	O
+SAM	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+Thus	O
+,	O
+additional	O
+examples	O
+for	O
+acp	B-protein_type
+transferase	I-protein_type
+enzymes	O
+might	O
+be	O
+found	O
+with	O
+similarities	O
+to	O
+other	O
+structural	O
+classes	O
+of	O
+methyltransferases	B-protein_type
+.	O
+
+In	O
+contrast	O
+to	O
+Nep1	B-protein
+,	O
+the	O
+enzyme	O
+preceding	O
+Tsr3	B-protein
+in	O
+the	O
+m1acp3Ψ	B-chemical
+biosynthesis	O
+pathway	O
+,	O
+Tsr3	B-protein
+binds	O
+rather	O
+weakly	O
+and	O
+with	O
+little	O
+specificity	O
+to	O
+its	O
+isolated	O
+substrate	O
+RNA	B-chemical
+.	O
+
+This	O
+suggests	O
+that	O
+Tsr3	B-protein
+is	O
+not	O
+stably	O
+incorporated	O
+into	O
+pre	B-complex_assembly
+-	I-complex_assembly
+ribosomal	I-complex_assembly
+particles	I-complex_assembly
+and	O
+that	O
+its	O
+binding	O
+to	O
+the	O
+nascent	O
+ribosomal	B-complex_assembly
+subunit	I-complex_assembly
+possibly	O
+requires	O
+additional	O
+interactions	O
+with	O
+other	O
+pre	O
+-	O
+ribosomal	O
+components	O
+.	O
+
+Consistently	O
+,	O
+in	O
+sucrose	B-experimental_method
+gradient	I-experimental_method
+analysis	I-experimental_method
+,	O
+Tsr3	B-protein
+was	O
+found	O
+in	O
+low	O
+-	O
+molecular	O
+weight	O
+fractions	O
+rather	O
+than	O
+with	O
+pre	B-complex_assembly
+-	I-complex_assembly
+ribosome	I-complex_assembly
+containing	O
+high	O
+-	O
+molecular	O
+weight	O
+fractions	O
+.	O
+
+In	O
+contrast	O
+to	O
+several	O
+enzymes	O
+that	O
+catalyze	O
+base	O
+specific	O
+modifications	O
+in	O
+rRNAs	B-chemical
+Tsr3	B-protein
+is	O
+not	O
+an	O
+essential	O
+protein	O
+.	O
+
+Typically	O
+,	O
+other	O
+small	B-protein_type
+subunit	I-protein_type
+rRNA	I-protein_type
+methyltransferases	I-protein_type
+as	O
+Dim1	B-protein
+,	O
+Bud23	B-protein
+and	O
+Nep1	B-protein
+/	O
+Emg1	B-protein
+carry	O
+dual	O
+functions	O
+,	O
+in	O
+ribosome	O
+biogenesis	O
+and	O
+rRNA	B-chemical
+modification	O
+,	O
+and	O
+it	O
+is	O
+their	O
+involvement	O
+in	O
+pre	B-chemical
+-	I-chemical
+RNA	I-chemical
+processing	O
+that	O
+is	O
+essential	O
+rather	O
+than	O
+their	O
+RNA	O
+-	O
+methylating	O
+activity	O
+(,	O
+discussed	O
+in	O
+7	O
+).	O
+
+In	O
+contrast	O
+,	O
+for	O
+several	O
+Tsr3	B-protein
+mutants	O
+(	O
+SAM	B-protein_state
+-	I-protein_state
+binding	I-protein_state
+and	O
+cysteine	B-protein_state
+mutations	I-protein_state
+)	O
+we	O
+found	O
+a	O
+systematic	O
+correlation	O
+between	O
+the	O
+loss	O
+of	O
+acp	B-chemical
+modification	O
+and	O
+the	O
+efficiency	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+maturation	O
+.	O
+
+This	O
+demonstrates	O
+that	O
+,	O
+unlike	O
+the	O
+other	O
+small	O
+subunit	O
+rRNA	B-chemical
+base	O
+modifications	O
+,	O
+the	O
+acp	B-chemical
+modification	O
+is	O
+required	O
+for	O
+efficient	O
+pre	B-chemical
+-	I-chemical
+rRNA	I-chemical
+processing	O
+.	O
+
+Recently	O
+,	O
+structural	B-experimental_method
+,	I-experimental_method
+functional	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+CRAC	I-experimental_method
+(	I-experimental_method
+cross	I-experimental_method
+-	I-experimental_method
+linking	I-experimental_method
+and	I-experimental_method
+cDNA	I-experimental_method
+analysis	I-experimental_method
+)	I-experimental_method
+experiments	I-experimental_method
+of	O
+late	O
+assembly	O
+factors	O
+involved	O
+in	O
+cytoplasmic	O
+processing	O
+of	O
+40S	B-complex_assembly
+subunits	I-complex_assembly
+,	O
+along	O
+with	O
+cryo	B-experimental_method
+-	I-experimental_method
+EM	I-experimental_method
+studies	O
+of	O
+the	O
+late	B-protein_state
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+subunits	I-complex_assembly
+have	O
+provided	O
+important	O
+insights	O
+into	O
+late	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+processing	O
+.	O
+
+Apart	O
+from	O
+most	O
+of	O
+the	O
+ribosomal	O
+proteins	O
+,	O
+cytoplasmic	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+particles	I-complex_assembly
+contain	O
+20S	B-chemical
+rRNA	I-chemical
+and	O
+at	O
+least	O
+seven	O
+non	B-protein_type
+-	I-protein_type
+ribosomal	I-protein_type
+proteins	I-protein_type
+including	O
+the	O
+D	B-protein_type
+-	I-protein_type
+site	I-protein_type
+endonuclease	I-protein_type
+Nob1	B-protein
+as	O
+well	O
+as	O
+Tsr1	B-protein
+,	O
+a	O
+putative	O
+GTPase	B-protein_type
+and	O
+Rio2	B-protein
+which	O
+block	O
+the	O
+mRNA	B-site
+channel	I-site
+and	O
+the	O
+initiator	B-site
+tRNA	I-site
+binding	I-site
+site	I-site
+,	O
+respectively	O
+,	O
+thus	O
+preventing	O
+translation	O
+initiation	O
+.	O
+
+After	O
+structural	O
+changes	O
+,	O
+possibly	O
+driven	O
+by	O
+GTP	B-chemical
+hydrolysis	O
+,	O
+which	O
+go	O
+together	O
+with	O
+the	O
+formation	O
+of	O
+the	O
+decoding	B-site
+site	I-site
+,	O
+the	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+becomes	O
+accessible	O
+for	O
+Nob1	B-protein
+cleavage	O
+at	O
+site	B-site
+D	I-site
+.	O
+This	O
+also	O
+involves	O
+joining	O
+of	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+and	O
+60S	B-complex_assembly
+subunits	I-complex_assembly
+to	O
+80S	B-complex_assembly
+-	I-complex_assembly
+like	I-complex_assembly
+particles	I-complex_assembly
+in	O
+a	O
+translation	O
+-	O
+like	O
+cycle	O
+promoted	O
+by	O
+eIF5B	B-protein
+.	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	B-complex_assembly
+subunit	I-complex_assembly
+assembly	O
+with	O
+only	O
+completely	O
+processed	O
+precursors	O
+.	O
+
+Finally	O
+,	O
+termination	B-protein_type
+factor	I-protein_type
+Rli1	B-protein
+,	O
+an	O
+ATPase	B-protein_type
+,	O
+promotes	O
+the	O
+dissociation	O
+of	O
+assembly	O
+factors	O
+and	O
+the	O
+80S	B-complex_assembly
+-	I-complex_assembly
+like	I-complex_assembly
+complex	I-complex_assembly
+dissociates	O
+and	O
+releases	O
+the	O
+mature	B-protein_state
+40S	B-complex_assembly
+subunit	I-complex_assembly
+.	O
+
+Interestingly	O
+,	O
+differences	O
+in	O
+the	O
+level	O
+of	O
+acp	B-chemical
+modification	O
+were	O
+demonstrated	O
+for	O
+different	O
+steps	O
+of	O
+the	O
+cytoplasmic	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+subunit	I-complex_assembly
+maturation	O
+after	O
+analyzing	O
+purified	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNAs	I-chemical
+using	O
+different	O
+purification	O
+bait	O
+proteins	O
+.	O
+
+Early	O
+cytoplasmic	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+subunits	I-complex_assembly
+still	O
+containing	O
+the	O
+ribosome	B-protein_type
+assembly	I-protein_type
+factors	I-protein_type
+Tsr1	B-protein
+,	O
+Ltv1	B-protein
+,	O
+Enp1	B-protein
+and	O
+Rio2	B-protein
+were	O
+not	O
+or	O
+only	O
+partially	O
+acp	B-protein_state
+modified	I-protein_state
+.	O
+
+In	O
+contrast	O
+,	O
+late	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+subunits	I-complex_assembly
+containing	O
+Nob1	B-protein
+and	O
+Rio1	B-protein
+or	O
+already	O
+associated	O
+with	O
+60S	B-complex_assembly
+subunits	I-complex_assembly
+in	O
+80S	B-complex_assembly
+-	I-complex_assembly
+like	I-complex_assembly
+particles	I-complex_assembly
+showed	O
+acp	B-chemical
+modification	O
+levels	O
+comparable	O
+to	O
+mature	B-protein_state
+40S	B-complex_assembly
+subunits	I-complex_assembly
+.	O
+
+Thus	O
+,	O
+the	O
+acp	B-chemical
+transfer	O
+to	O
+m1Ψ1191	B-residue_name_number
+occurs	O
+during	O
+the	O
+step	O
+at	O
+which	O
+Rio2	B-protein
+leaves	O
+the	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+particle	I-complex_assembly
+.	O
+
+These	O
+data	O
+and	O
+the	O
+finding	O
+that	O
+a	O
+missing	O
+acp	B-chemical
+modification	O
+hinders	O
+pre	B-chemical
+-	I-chemical
+20S	I-chemical
+rRNA	I-chemical
+processing	O
+,	O
+suggest	O
+that	O
+the	O
+acp	B-chemical
+modification	O
+together	O
+with	O
+the	O
+release	O
+of	O
+Rio2	B-protein
+promotes	O
+the	O
+formation	O
+of	O
+the	O
+decoding	B-site
+site	I-site
+and	O
+thus	O
+D	B-site
+-	I-site
+site	I-site
+cleavage	O
+by	O
+Nob1	B-protein
+.	O
+
+The	O
+interrelation	O
+between	O
+acp	B-chemical
+modification	O
+and	O
+Rio2	B-protein
+release	O
+is	O
+also	O
+supported	O
+by	O
+CRAC	B-experimental_method
+analysis	I-experimental_method
+showing	O
+that	O
+Rio2	B-protein
+binds	O
+to	O
+helix	B-structure_element
+31	I-structure_element
+next	O
+to	O
+the	O
+Ψ1191	B-residue_name_number
+residue	O
+that	O
+receives	O
+the	O
+acp	B-chemical
+modification	O
+.	O
+
+Therefore	O
+,	O
+Rio2	B-protein
+either	O
+blocks	O
+the	O
+access	O
+of	O
+Tsr3	B-protein
+to	O
+helix	B-structure_element
+31	I-structure_element
+,	O
+and	O
+acp	B-chemical
+modification	O
+can	O
+only	O
+occur	O
+after	O
+Rio2	B-protein
+is	O
+released	O
+,	O
+or	O
+the	O
+acp	B-chemical
+modification	O
+of	O
+m1Ψ1191	B-residue_name_number
+and	O
+putative	O
+subsequent	O
+conformational	O
+changes	O
+of	O
+20S	B-chemical
+rRNA	I-chemical
+weaken	O
+the	O
+binding	O
+of	O
+Rio2	B-protein
+to	O
+helix	B-structure_element
+31	I-structure_element
+and	O
+support	O
+its	O
+release	O
+from	O
+the	O
+pre	B-chemical
+-	I-chemical
+rRNA	I-chemical
+.	O
+
+In	O
+summary	O
+,	O
+by	O
+identifying	O
+Tsr3	B-protein
+as	O
+the	O
+enzyme	O
+responsible	O
+for	O
+introducing	O
+the	O
+acp	B-chemical
+group	O
+to	O
+the	O
+hypermodified	B-protein_state
+m1acp3Ψ	B-chemical
+nucleotide	B-chemical
+at	O
+position	O
+1191	B-residue_number
+(	O
+yeast	B-taxonomy_domain
+)/	O
+1248	B-residue_number
+(	O
+humans	B-species
+)	O
+of	O
+18S	B-chemical
+rRNA	I-chemical
+we	O
+added	O
+one	O
+of	O
+the	O
+last	O
+remaining	O
+pieces	O
+to	O
+the	O
+puzzle	O
+of	O
+eukaryotic	B-taxonomy_domain
+small	B-chemical
+ribosomal	I-chemical
+subunit	I-chemical
+rRNA	I-chemical
+modifications	O
+.	O
+
+The	O
+current	O
+data	O
+together	O
+with	O
+the	O
+finding	O
+that	O
+acp	B-chemical
+modification	O
+takes	O
+place	O
+at	O
+the	O
+very	O
+last	O
+step	O
+in	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+subunit	I-complex_assembly
+maturation	O
+indicate	O
+that	O
+the	O
+acp	B-chemical
+modification	O
+probably	O
+supports	O
+the	O
+formation	O
+of	O
+the	O
+decoding	B-site
+site	I-site
+and	O
+efficient	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+D	B-site
+-	I-site
+site	I-site
+cleavage	O
+.	O
+
+Furthermore	O
+,	O
+our	O
+structural	B-evidence
+data	I-evidence
+unravelled	O
+how	O
+the	O
+regioselectivity	O
+of	O
+SAM	B-chemical
+-	O
+dependent	O
+group	O
+transfer	O
+reactions	O
+can	O
+be	O
+tuned	O
+by	O
+distinct	O
+small	O
+evolutionary	O
+adaptions	O
+of	O
+the	O
+ligand	B-site
+binding	I-site
+pocket	I-site
+of	O
+SAM	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+enzymes	I-protein_type
+.	O
+
+Biosynthesis	O
+of	O
+a	O
+hypermodified	B-protein_state
+nucleotide	O
+in	O
+Saccharomyces	O
+carlsbergensis	O
+17S	O
+and	O
+HeLa	O
+-	O
+cell	O
+18S	O
+ribosomal	O
+ribonucleic	O
+acid	O
+
+Presence	O
+of	O
+a	O
+hypermodified	B-protein_state
+nucleotide	O
+in	O
+HeLa	O
+cell	O
+18	O
+S	O
+and	O
+Saccharomyces	O
+carlsbergensis	O
+17	O
+S	O
+ribosomal	O
+RNAs	O
+
+Mutations	O
+in	O
+the	O
+nucleolar	O
+proteins	O
+Tma23	O
+and	O
+Nop6	O
+suppress	O
+the	O
+malfunction	O
+of	O
+the	O
+Nep1	B-protein_type
+protein	O
+
+The	O
+yeast	O
+ribosome	O
+synthesis	O
+factor	O
+Emg1	B-protein_type
+is	O
+a	O
+novel	O
+member	O
+of	O
+the	O
+superfamily	O
+of	O
+alpha	O
+/	O
+beta	O
+knot	O
+fold	O
+methyltransferases	O
+
+Crystal	B-evidence
+Structure	I-evidence
+and	O
+Activity	B-experimental_method
+Studies	I-experimental_method
+of	O
+the	O
+C11	B-protein_type
+Cysteine	B-protein_type
+Peptidase	I-protein_type
+from	O
+Parabacteroides	B-species
+merdae	I-species
+in	O
+the	O
+Human	B-species
+Gut	O
+Microbiome	O
+*	O
+
+Clan	B-protein_type
+CD	I-protein_type
+cysteine	I-protein_type
+peptidases	I-protein_type
+,	O
+a	O
+structurally	O
+related	O
+group	O
+of	O
+peptidases	B-protein_type
+that	O
+include	O
+mammalian	B-taxonomy_domain
+caspases	B-protein_type
+,	O
+exhibit	O
+a	O
+wide	O
+range	O
+of	O
+important	O
+functions	O
+,	O
+along	O
+with	O
+a	O
+variety	O
+of	O
+specificities	O
+and	O
+activation	O
+mechanisms	O
+.	O
+
+However	O
+,	O
+for	O
+the	O
+clostripain	B-protein_type
+family	I-protein_type
+(	O
+denoted	O
+C11	B-protein_type
+),	O
+little	O
+is	O
+currently	O
+known	O
+.	O
+
+Here	O
+,	O
+we	O
+describe	O
+the	O
+first	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+C11	B-protein_type
+protein	O
+from	O
+the	O
+human	B-species
+gut	O
+bacterium	B-taxonomy_domain
+,	O
+Parabacteroides	B-species
+merdae	I-species
+(	O
+PmC11	B-protein
+),	O
+determined	O
+to	O
+1	O
+.	O
+7	O
+-	O
+Å	O
+resolution	O
+.	O
+
+PmC11	B-protein
+is	O
+a	O
+monomeric	B-oligomeric_state
+cysteine	B-protein_type
+peptidase	I-protein_type
+that	O
+comprises	O
+an	O
+extended	B-structure_element
+caspase	I-structure_element
+-	I-structure_element
+like	I-structure_element
+α	I-structure_element
+/	I-structure_element
+β	I-structure_element
+/	I-structure_element
+α	I-structure_element
+sandwich	I-structure_element
+and	O
+an	O
+unusual	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+.	O
+
+It	O
+shares	O
+core	O
+structural	O
+elements	O
+with	O
+clan	B-protein_type
+CD	I-protein_type
+cysteine	I-protein_type
+peptidases	I-protein_type
+but	O
+otherwise	O
+structurally	O
+differs	O
+from	O
+the	O
+other	O
+families	O
+in	O
+the	O
+clan	O
+.	O
+
+These	O
+studies	O
+also	O
+revealed	O
+a	O
+well	O
+ordered	O
+break	O
+in	O
+the	O
+polypeptide	O
+chain	O
+at	O
+Lys147	B-residue_name_number
+,	O
+resulting	O
+in	O
+a	O
+large	O
+conformational	O
+rearrangement	O
+close	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+Biochemical	B-experimental_method
+and	I-experimental_method
+kinetic	I-experimental_method
+analysis	I-experimental_method
+revealed	O
+Lys147	B-residue_name_number
+to	O
+be	O
+an	O
+intramolecular	B-site
+processing	I-site
+site	I-site
+at	O
+which	O
+cleavage	B-ptm
+is	O
+required	O
+for	O
+full	B-protein_state
+activation	I-protein_state
+of	O
+the	O
+enzyme	B-protein
+,	O
+suggesting	O
+an	O
+autoinhibitory	O
+mechanism	O
+for	O
+self	O
+-	O
+preservation	O
+.	O
+
+PmC11	B-protein
+has	O
+an	O
+acidic	B-site
+binding	I-site
+pocket	I-site
+and	O
+a	O
+preference	O
+for	O
+basic	O
+substrates	O
+,	O
+and	O
+accepts	O
+substrates	O
+with	O
+Arg	B-residue_name
+and	O
+Lys	B-residue_name
+in	O
+P1	B-residue_number
+and	O
+does	O
+not	O
+require	O
+Ca2	B-chemical
++	I-chemical
+for	O
+activity	O
+.	O
+
+Collectively	O
+,	O
+these	O
+data	O
+provide	O
+insights	O
+into	O
+the	O
+mechanism	O
+and	O
+activity	O
+of	O
+PmC11	B-protein
+and	O
+a	O
+detailed	O
+framework	O
+for	O
+studies	O
+on	O
+C11	B-protein_type
+peptidases	I-protein_type
+from	O
+other	O
+phylogenetic	O
+kingdoms	O
+.	O
+
+Cysteine	B-protein_type
+peptidases	I-protein_type
+play	O
+crucial	O
+roles	O
+in	O
+the	O
+virulence	O
+of	O
+bacterial	B-taxonomy_domain
+and	O
+other	O
+eukaryotic	B-taxonomy_domain
+pathogens	O
+.	O
+
+In	O
+the	O
+MEROPS	O
+peptidase	O
+database	O
+,	O
+clan	B-protein_type
+CD	I-protein_type
+contains	O
+groups	O
+(	O
+or	O
+families	O
+)	O
+of	O
+cysteine	B-protein_type
+peptidases	I-protein_type
+that	O
+share	O
+some	O
+highly	B-protein_state
+conserved	I-protein_state
+structural	O
+elements	O
+.	O
+
+Clan	B-protein_type
+CD	I-protein_type
+families	I-protein_type
+are	O
+typically	O
+described	O
+using	O
+the	O
+name	O
+of	O
+their	O
+archetypal	O
+,	O
+or	O
+founding	O
+,	O
+member	O
+and	O
+also	O
+given	O
+an	O
+identification	O
+number	O
+preceded	O
+by	O
+a	O
+“	O
+C	O
+,”	O
+to	O
+denote	O
+cysteine	B-protein_type
+peptidase	I-protein_type
+.	O
+
+Although	O
+seven	O
+families	O
+(	O
+C14	O
+is	O
+additionally	O
+split	O
+into	O
+three	O
+subfamilies	O
+)	O
+have	O
+been	O
+described	O
+for	O
+this	O
+clan	O
+,	O
+crystal	B-evidence
+structures	I-evidence
+have	O
+only	O
+been	O
+determined	O
+from	O
+four	O
+:	O
+legumain	B-protein
+(	O
+C13	B-protein_type
+),	O
+caspase	B-protein
+(	O
+C14a	B-protein_type
+),	O
+paracaspase	B-protein
+(	O
+C14b	B-protein_type
+(	I-protein_type
+P	I-protein_type
+),	O
+metacaspase	B-protein
+(	O
+C14b	B-protein_type
+(	I-protein_type
+M	I-protein_type
+),	O
+gingipain	B-protein
+(	O
+C25	B-protein_type
+),	O
+and	O
+the	O
+cysteine	B-structure_element
+peptidase	I-structure_element
+domain	I-structure_element
+(	O
+CPD	B-structure_element
+)	O
+of	O
+various	O
+toxins	O
+(	O
+C80	B-protein_type
+).	O
+
+No	O
+structural	O
+information	O
+is	O
+available	O
+for	O
+clostripain	B-protein
+(	O
+C11	B-protein_type
+),	O
+separase	B-protein
+(	O
+C50	B-protein_type
+),	O
+or	O
+PrtH	B-protein
+-	I-protein
+peptidase	I-protein
+(	O
+C85	B-protein_type
+).	O
+
+Clan	B-protein_type
+CD	I-protein_type
+enzymes	I-protein_type
+have	O
+a	O
+highly	B-protein_state
+conserved	I-protein_state
+His	B-site
+/	I-site
+Cys	I-site
+catalytic	I-site
+dyad	I-site
+and	O
+exhibit	O
+strict	O
+specificity	O
+for	O
+the	O
+P1	B-residue_number
+residue	O
+of	O
+their	O
+substrates	O
+.	O
+
+However	O
+,	O
+despite	O
+these	O
+similarities	O
+,	O
+clan	B-protein_type
+CD	I-protein_type
+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	B-protein_type
+with	O
+a	O
+wide	O
+variety	O
+of	O
+substrate	O
+specificities	O
+and	O
+activation	O
+mechanisms	O
+.	O
+
+The	O
+archetypal	O
+and	O
+arguably	O
+most	O
+notable	O
+family	O
+in	O
+the	O
+clan	O
+is	O
+that	O
+of	O
+the	O
+mammalian	B-taxonomy_domain
+caspases	B-protein_type
+(	O
+C14a	B-protein_type
+),	O
+although	O
+clan	B-protein_type
+CD	I-protein_type
+members	O
+are	O
+distributed	O
+throughout	O
+the	O
+entire	O
+phylogenetic	O
+kingdom	O
+and	O
+are	O
+often	O
+required	O
+in	O
+fundamental	O
+biological	O
+processes	O
+.	O
+
+Interestingly	O
+,	O
+little	O
+is	O
+known	O
+about	O
+the	O
+structure	O
+or	O
+function	O
+of	O
+the	O
+C11	B-protein_type
+proteins	O
+,	O
+despite	O
+their	O
+widespread	O
+distribution	O
+and	O
+its	O
+archetypal	O
+member	O
+,	O
+clostripain	B-protein
+from	O
+Clostridium	B-species
+histolyticum	I-species
+,	O
+first	O
+reported	O
+in	O
+the	O
+literature	O
+in	O
+1938	O
+.	O
+
+Clostripain	B-protein
+has	O
+been	O
+described	O
+as	O
+an	O
+arginine	B-protein_type
+-	I-protein_type
+specific	I-protein_type
+peptidase	I-protein_type
+with	O
+a	O
+requirement	O
+for	O
+Ca2	B-chemical
++	I-chemical
+and	O
+loss	O
+of	O
+an	O
+internal	B-structure_element
+nonapeptide	I-structure_element
+for	O
+full	B-protein_state
+activation	I-protein_state
+;	O
+lack	O
+of	O
+structural	O
+information	O
+on	O
+the	O
+family	O
+appears	O
+to	O
+have	O
+prohibited	O
+further	O
+investigation	O
+.	O
+
+As	O
+part	O
+of	O
+an	O
+ongoing	O
+project	O
+to	O
+characterize	O
+commensal	O
+bacteria	B-taxonomy_domain
+in	O
+the	O
+microbiome	O
+that	O
+inhabit	O
+the	O
+human	B-species
+gut	O
+,	O
+the	O
+structure	B-evidence
+of	O
+C11	B-protein_type
+peptidase	I-protein_type
+,	O
+PmC11	B-protein
+,	O
+from	O
+Parabacteroides	B-species
+merdae	I-species
+was	O
+determined	O
+using	O
+the	O
+Joint	O
+Center	O
+for	O
+Structural	O
+Genomics	O
+(	O
+JCSG	O
+)	O
+4	O
+HTP	O
+structural	O
+biology	O
+pipeline	O
+.	O
+
+The	O
+structure	B-experimental_method
+was	I-experimental_method
+analyzed	I-experimental_method
+,	O
+and	O
+the	O
+enzyme	O
+was	O
+biochemically	B-experimental_method
+characterized	I-experimental_method
+to	O
+provide	O
+the	O
+first	O
+structure	O
+/	O
+function	O
+correlation	O
+for	O
+a	O
+C11	B-protein_type
+peptidase	I-protein_type
+.	O
+
+Structure	B-evidence
+of	O
+PmC11	B-protein
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+catalytically	B-protein_state
+active	I-protein_state
+form	O
+of	O
+PmC11	B-protein
+revealed	O
+an	O
+extended	B-structure_element
+caspase	I-structure_element
+-	I-structure_element
+like	I-structure_element
+α	I-structure_element
+/	I-structure_element
+β	I-structure_element
+/	I-structure_element
+α	I-structure_element
+sandwich	I-structure_element
+architecture	O
+comprised	O
+of	O
+a	O
+central	O
+nine	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+,	O
+with	O
+an	O
+unusual	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+CTD	B-structure_element
+),	O
+starting	O
+at	O
+Lys250	B-residue_name_number
+.	O
+
+A	O
+single	B-ptm
+cleavage	I-ptm
+was	O
+observed	O
+in	O
+the	O
+polypeptide	O
+chain	O
+at	O
+Lys147	B-residue_name_number
+(	O
+Fig	O
+.	O
+1	O
+,	O
+A	O
+and	O
+B	O
+),	O
+where	O
+both	O
+ends	O
+of	O
+the	O
+cleavage	B-site
+site	I-site
+are	O
+fully	O
+visible	O
+and	O
+well	O
+ordered	O
+in	O
+the	O
+electron	B-evidence
+density	I-evidence
+.	O
+
+The	O
+central	O
+nine	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+β1	B-structure_element
+–	I-structure_element
+β9	I-structure_element
+)	O
+of	O
+PmC11	B-protein
+consists	O
+of	O
+six	O
+parallel	B-structure_element
+and	O
+three	O
+anti	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+strands	I-structure_element
+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	B-evidence
+includes	O
+14	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+with	O
+six	O
+(	O
+α1	B-structure_element
+–	I-structure_element
+α2	I-structure_element
+and	O
+α4	B-structure_element
+–	I-structure_element
+α7	I-structure_element
+)	O
+closely	O
+surrounding	O
+the	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+in	O
+an	O
+approximately	O
+parallel	O
+orientation	O
+.	O
+
+Helices	B-structure_element
+α1	B-structure_element
+,	O
+α7	B-structure_element
+,	O
+and	O
+α6	B-structure_element
+are	O
+located	O
+on	O
+one	O
+side	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+with	O
+α2	B-structure_element
+,	O
+α4	B-structure_element
+,	O
+and	O
+α5	B-structure_element
+on	O
+the	O
+opposite	O
+side	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+
+Helix	B-structure_element
+α3	B-structure_element
+sits	O
+at	O
+the	O
+end	O
+of	O
+the	O
+loop	B-structure_element
+following	O
+β5	B-structure_element
+(	O
+L5	B-structure_element
+),	O
+just	O
+preceding	O
+the	O
+Lys147	B-residue_name_number
+cleavage	B-site
+site	I-site
+,	O
+with	O
+both	O
+L5	B-structure_element
+and	O
+α3	B-structure_element
+pointing	O
+away	O
+from	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+and	O
+toward	O
+the	O
+CTD	B-structure_element
+,	O
+which	O
+starts	O
+with	O
+α8	B-structure_element
+.	O
+
+The	O
+structure	B-evidence
+also	O
+includes	O
+two	O
+short	O
+β	B-structure_element
+-	I-structure_element
+hairpins	I-structure_element
+(	O
+βA	B-structure_element
+–	I-structure_element
+βB	I-structure_element
+and	O
+βD	B-structure_element
+–	I-structure_element
+βE	I-structure_element
+)	O
+and	O
+a	O
+small	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+βC	B-structure_element
+–	I-structure_element
+βF	I-structure_element
+),	O
+which	O
+is	O
+formed	O
+from	O
+two	O
+distinct	O
+regions	O
+of	O
+the	O
+sequence	O
+(	O
+βC	B-structure_element
+precedes	O
+α11	B-structure_element
+,	O
+α12	B-structure_element
+and	O
+β9	B-structure_element
+,	O
+whereas	O
+βF	B-structure_element
+follows	O
+the	O
+βD	B-structure_element
+-	I-structure_element
+βE	I-structure_element
+hairpin	B-structure_element
+)	O
+in	O
+the	O
+middle	O
+of	O
+the	O
+CTD	B-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+).	O
+
+Crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+C11	B-protein_type
+peptidase	I-protein_type
+from	O
+P	B-species
+.	I-species
+merdae	I-species
+.	O
+
+A	O
+,	O
+primary	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+PmC11	B-protein
+(	O
+Uniprot	O
+ID	O
+A7A9N3	O
+)	O
+and	O
+clostripain	B-protein
+(	O
+Uniprot	O
+ID	O
+P09870	O
+)	O
+from	O
+C	B-species
+.	I-species
+histolyticum	I-species
+with	O
+identical	O
+residues	O
+highlighted	O
+in	O
+gray	O
+shading	O
+.	O
+
+The	O
+secondary	O
+structure	O
+of	O
+PmC11	B-protein
+from	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+is	O
+mapped	O
+onto	O
+its	O
+sequence	O
+with	O
+the	O
+position	O
+of	O
+the	O
+PmC11	B-protein
+catalytic	B-site
+dyad	I-site
+,	O
+autocatalytic	B-site
+cleavage	I-site
+site	I-site
+(	O
+Lys147	B-residue_name_number
+),	O
+and	O
+S1	B-site
+binding	I-site
+pocket	I-site
+Asp	B-residue_name
+(	O
+Asp177	B-residue_name_number
+)	O
+highlighted	O
+by	O
+a	O
+red	O
+star	O
+,	O
+a	O
+red	O
+downturned	O
+triangle	O
+,	O
+and	O
+a	O
+red	O
+upturned	O
+triangle	O
+,	O
+respectively	O
+.	O
+
+Connecting	O
+loops	B-structure_element
+are	O
+colored	O
+gray	O
+,	O
+the	O
+main	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+is	O
+in	O
+orange	O
+,	O
+with	O
+other	O
+strands	O
+in	O
+olive	O
+,	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+are	O
+in	O
+blue	O
+,	O
+and	O
+the	O
+nonapeptide	B-structure_element
+linker	I-structure_element
+of	O
+clostripain	B-protein
+that	O
+is	O
+excised	O
+upon	O
+autocleavage	B-ptm
+is	O
+underlined	O
+in	O
+red	O
+.	O
+
+Sequences	O
+around	O
+the	O
+catalytic	B-site
+site	I-site
+of	O
+clostripain	B-protein
+and	O
+PmC11	B-protein
+align	O
+well	O
+.	O
+
+B	O
+,	O
+topology	O
+diagram	O
+of	O
+PmC11	B-protein
+colored	O
+as	O
+in	O
+A	O
+except	O
+that	O
+additional	O
+(	O
+non	O
+-	O
+core	O
+)	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+are	O
+in	O
+yellow	O
+.	O
+
+Helices	O
+found	O
+on	O
+either	O
+side	O
+of	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+are	O
+shown	O
+above	O
+and	O
+below	O
+the	O
+sheet	B-structure_element
+,	O
+respectively	O
+.	O
+
+The	O
+position	O
+of	O
+the	O
+catalytic	B-site
+dyad	I-site
+(	O
+H	B-residue_name
+,	O
+C	B-residue_name
+)	O
+and	O
+the	O
+processing	B-site
+site	I-site
+(	O
+Lys147	B-residue_name_number
+)	O
+are	O
+highlighted	O
+.	O
+
+Helices	O
+(	O
+1	O
+–	O
+14	O
+)	O
+and	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+(	O
+1	O
+–	O
+9	O
+and	O
+A	O
+-	O
+F	O
+)	O
+are	O
+numbered	O
+from	O
+the	O
+N	O
+terminus	O
+.	O
+
+The	O
+core	B-structure_element
+caspase	I-structure_element
+-	I-structure_element
+fold	I-structure_element
+is	O
+highlighted	O
+in	O
+a	O
+box	O
+.	O
+
+C	O
+,	O
+tertiary	O
+structure	O
+of	O
+PmC11	B-protein
+.	O
+
+The	O
+N	O
+and	O
+C	O
+termini	O
+(	O
+N	O
+and	O
+C	O
+)	O
+of	O
+PmC11	B-protein
+along	O
+with	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+1	O
+–	O
+9	O
+),	O
+helix	B-structure_element
+α5	B-structure_element
+,	O
+and	O
+helices	B-structure_element
+α8	B-structure_element
+,	O
+α11	B-structure_element
+,	O
+and	O
+α13	B-structure_element
+from	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+,	O
+are	O
+all	O
+labeled	O
+.	O
+
+Loops	O
+are	O
+colored	O
+gray	O
+,	O
+the	O
+main	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+is	O
+in	O
+orange	O
+,	O
+with	O
+other	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+in	O
+yellow	O
+,	O
+and	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+are	O
+in	O
+blue	O
+.	O
+
+The	O
+CTD	B-structure_element
+of	O
+PmC11	B-protein
+is	O
+composed	O
+of	O
+a	O
+tight	B-structure_element
+helical	I-structure_element
+bundle	I-structure_element
+formed	O
+from	O
+helices	B-structure_element
+α8	B-structure_element
+–	I-structure_element
+α14	I-structure_element
+and	O
+includes	O
+strands	B-structure_element
+βC	B-structure_element
+and	O
+βF	B-structure_element
+,	O
+and	O
+β	B-structure_element
+-	I-structure_element
+hairpin	I-structure_element
+βD	B-structure_element
+–	I-structure_element
+βE	I-structure_element
+.	O
+The	O
+CTD	B-structure_element
+sits	O
+entirely	O
+on	O
+one	O
+side	O
+of	O
+the	O
+enzyme	O
+interacting	O
+only	O
+with	O
+α3	B-structure_element
+,	O
+α5	B-structure_element
+,	O
+β9	B-structure_element
+,	O
+and	O
+the	O
+loops	B-structure_element
+surrounding	O
+β8	B-structure_element
+.	O
+
+Of	O
+the	O
+interacting	O
+secondary	O
+structure	O
+elements	O
+,	O
+α5	B-structure_element
+is	O
+perhaps	O
+the	O
+most	O
+interesting	O
+.	O
+
+This	B-structure_element
+helix	I-structure_element
+makes	O
+a	O
+total	O
+of	O
+eight	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+CTD	B-structure_element
+,	O
+including	O
+one	O
+salt	O
+bridge	O
+(	O
+Arg191	B-residue_name_number
+-	O
+Asp255	B-residue_name_number
+)	O
+and	O
+is	O
+surrounded	O
+by	O
+the	O
+CTD	B-structure_element
+on	O
+one	O
+side	O
+and	O
+the	O
+main	B-structure_element
+core	I-structure_element
+of	O
+the	O
+enzyme	O
+on	O
+the	O
+other	O
+,	O
+acting	O
+like	O
+a	O
+linchpin	O
+holding	O
+both	O
+components	O
+together	O
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+
+PmC11	B-protein
+is	O
+,	O
+as	O
+expected	O
+,	O
+most	O
+structurally	O
+similar	O
+to	O
+other	O
+members	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+with	O
+the	O
+top	O
+hits	O
+in	O
+a	O
+search	O
+of	O
+known	O
+structures	B-evidence
+being	O
+caspase	B-protein
+-	I-protein
+7	I-protein
+,	O
+gingipain	B-protein
+-	I-protein
+K	I-protein
+,	O
+and	O
+legumain	B-protein
+(	O
+PBD	O
+codes	O
+4hq0	O
+,	O
+4tkx	O
+,	O
+and	O
+4aw9	O
+,	O
+respectively	O
+)	O
+(	O
+Table	O
+2	O
+).	O
+
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+is	O
+unique	O
+to	O
+PmC11	B-protein
+within	O
+clan	B-protein_type
+CD	I-protein_type
+and	O
+structure	B-experimental_method
+comparisons	I-experimental_method
+for	O
+this	B-structure_element
+domain	I-structure_element
+alone	I-structure_element
+does	O
+not	O
+produce	O
+any	O
+hits	O
+in	O
+the	O
+PDB	O
+(	O
+DaliLite	B-experimental_method
+,	O
+PDBeFold	B-experimental_method
+),	O
+suggesting	O
+a	O
+completely	O
+novel	O
+fold	O
+.	O
+
+As	O
+the	O
+archetypal	O
+and	O
+arguably	O
+most	O
+well	O
+studied	O
+member	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+,	O
+the	O
+caspases	B-protein_type
+were	O
+used	O
+as	O
+the	O
+basis	O
+to	O
+investigate	O
+the	O
+structure	O
+/	O
+function	O
+relationships	O
+in	O
+PmC11	B-protein
+,	O
+with	O
+caspase	B-protein
+-	I-protein
+7	I-protein
+as	O
+the	O
+representative	O
+member	O
+.	O
+
+Six	O
+of	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+in	O
+PmC11	B-protein
+(	O
+β1	B-structure_element
+–	I-structure_element
+β2	I-structure_element
+and	O
+β5	B-structure_element
+–	I-structure_element
+β8	I-structure_element
+)	O
+share	O
+the	O
+same	O
+topology	O
+as	O
+the	O
+six	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+found	O
+in	O
+caspases	B-protein_type
+,	O
+with	O
+strands	B-structure_element
+β3	B-structure_element
+,	O
+β4	B-structure_element
+,	O
+and	O
+β9	B-structure_element
+located	O
+on	O
+the	O
+outside	O
+of	O
+this	O
+core	B-structure_element
+structure	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+box	O
+).	O
+
+His133	B-residue_name_number
+and	O
+Cys179	B-residue_name_number
+were	O
+found	O
+at	O
+locations	O
+structurally	O
+homologous	O
+to	O
+the	O
+caspase	B-protein_type
+catalytic	B-site
+dyad	I-site
+,	O
+and	O
+other	O
+clan	B-protein_type
+CD	I-protein_type
+structures	B-evidence
+,	O
+at	O
+the	O
+C	O
+termini	O
+of	O
+strands	B-structure_element
+β5	B-structure_element
+and	O
+β6	B-structure_element
+,	O
+respectively	O
+(	O
+Figs	O
+.	O
+1	O
+,	O
+A	O
+and	O
+B	O
+,	O
+and	O
+2A	O
+).	O
+
+A	O
+multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+C11	B-protein_type
+proteins	O
+revealed	O
+that	O
+these	O
+residues	O
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+(	O
+data	O
+not	O
+shown	O
+).	O
+
+Summary	O
+of	O
+PDBeFOLD	B-experimental_method
+superposition	I-experimental_method
+of	O
+structures	O
+found	O
+to	O
+be	O
+most	O
+similar	O
+to	O
+PmC11	B-protein
+in	O
+the	O
+PBD	O
+based	O
+on	O
+DaliLite	B-experimental_method
+
+Biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+PmC11	B-protein
+.	O
+
+A	O
+,	O
+ribbon	O
+representation	O
+of	O
+the	O
+overall	O
+structure	O
+of	O
+PmC11	B-protein
+illustrating	O
+the	O
+catalytic	B-site
+site	I-site
+,	O
+cleavage	O
+site	O
+displacement	O
+,	O
+and	O
+potential	O
+S1	B-site
+binding	I-site
+site	I-site
+.	O
+
+The	O
+overall	O
+structure	B-evidence
+of	O
+PmC11	B-protein
+is	O
+shown	O
+in	O
+gray	O
+,	O
+looking	O
+down	O
+into	O
+the	O
+catalytic	B-site
+site	I-site
+with	O
+the	O
+catalytic	B-site
+dyad	I-site
+in	O
+red	O
+.	O
+
+The	O
+two	O
+ends	O
+of	O
+the	O
+autolytic	B-site
+cleavage	I-site
+site	I-site
+(	O
+Lys147	B-residue_name_number
+and	O
+Ala148	B-residue_name_number
+,	O
+green	O
+)	O
+are	O
+displaced	O
+by	O
+19	O
+.	O
+5	O
+Å	O
+(	O
+thin	O
+black	O
+line	O
+)	O
+from	O
+one	O
+another	O
+and	O
+residues	O
+in	O
+the	O
+potential	O
+substrate	B-site
+binding	I-site
+pocket	I-site
+are	O
+highlighted	O
+in	O
+blue	O
+.	O
+
+B	O
+,	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+of	O
+PmC11	B-protein
+.	O
+
+PmC11	O
+migrates	O
+as	O
+a	O
+monomer	B-oligomeric_state
+with	O
+a	O
+molecular	O
+mass	O
+around	O
+41	O
+kDa	O
+calculated	O
+from	O
+protein	O
+standards	O
+of	O
+known	O
+molecular	O
+weights	O
+.	O
+
+Elution	O
+fractions	O
+across	O
+the	O
+major	O
+peak	O
+(	O
+1	O
+–	O
+6	O
+)	O
+were	O
+analyzed	O
+by	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+on	O
+a	O
+4	O
+–	O
+12	O
+%	O
+gel	O
+in	O
+MES	O
+buffer	O
+.	O
+
+C	O
+,	O
+the	O
+active	B-protein_state
+form	O
+of	O
+PmC11	B-protein
+and	O
+two	O
+mutants	O
+,	O
+PmC11C179A	B-mutant
+(	O
+C	O
+)	O
+and	O
+PmC11K147A	B-mutant
+(	O
+K	O
+),	O
+were	O
+examined	O
+by	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+(	O
+lane	O
+1	O
+)	O
+and	O
+Western	B-experimental_method
+blot	I-experimental_method
+analysis	O
+using	O
+an	O
+anti	O
+-	O
+His	O
+antibody	O
+(	O
+lane	O
+2	O
+)	O
+show	O
+that	O
+PmC11	B-protein
+autoprocesses	B-ptm
+,	O
+whereas	O
+mutants	O
+,	O
+PmC11C179A	B-mutant
+and	O
+PmC11K147A	B-mutant
+,	O
+do	O
+not	O
+show	O
+autoprocessing	B-ptm
+in	O
+vitro	O
+.	O
+
+D	O
+,	O
+cysteine	O
+peptidase	O
+activity	O
+of	O
+PmC11	B-protein
+.	O
+
+Km	O
+and	O
+Vmax	B-evidence
+of	O
+PmC11	B-protein
+and	O
+K147A	B-mutant
+mutant	O
+were	O
+determined	O
+by	O
+monitoring	O
+change	O
+in	O
+the	O
+fluorescence	O
+corresponding	O
+to	O
+AMC	O
+release	O
+from	O
+Bz	B-chemical
+-	I-chemical
+R	I-chemical
+-	I-chemical
+AMC	I-chemical
+.	O
+
+E	O
+,	O
+intermolecular	B-ptm
+processing	I-ptm
+of	O
+PmC11C179A	B-mutant
+by	O
+PmC11	B-protein
+.	O
+
+PmC11C179A	O
+(	O
+20	O
+μg	O
+)	O
+was	O
+incubated	O
+overnight	O
+at	O
+37	O
+°	O
+C	O
+with	O
+increasing	O
+amounts	O
+of	O
+processed	O
+PmC11	B-protein
+and	O
+analyzed	O
+on	O
+a	O
+10	O
+%	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+gel	O
+.	O
+
+Inactive	O
+PmC11C179A	B-mutant
+was	O
+not	O
+processed	O
+to	O
+a	O
+major	O
+extent	O
+by	O
+active	B-protein_state
+PmC11	B-protein
+until	O
+around	O
+a	O
+ratio	O
+of	O
+1	O
+:	O
+4	O
+(	O
+5	O
+μg	O
+of	O
+active	B-protein_state
+PmC11	B-protein
+).	O
+
+A	O
+single	O
+lane	O
+of	O
+20	O
+μg	O
+of	O
+active	B-protein_state
+PmC11	B-protein
+(	O
+labeled	O
+20	O
+)	O
+is	O
+shown	O
+for	O
+comparison	O
+.	O
+
+F	O
+,	O
+activity	B-evidence
+of	O
+PmC11	B-protein
+against	O
+basic	O
+substrates	O
+.	O
+
+G	O
+,	O
+electrostatic	O
+surface	O
+potential	O
+of	O
+PmC11	B-protein
+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
+
+The	O
+position	O
+of	O
+the	O
+catalytic	B-site
+dyad	I-site
+,	O
+one	O
+potential	O
+key	B-site
+substrate	I-site
+binding	I-site
+residue	I-site
+Asp177	B-residue_name_number
+,	O
+and	O
+the	O
+ends	O
+of	O
+the	O
+cleavage	B-site
+site	I-site
+Lys147	B-residue_name_number
+and	O
+Ala148	B-residue_name_number
+are	O
+indicated	O
+.	O
+
+Five	O
+of	O
+the	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+surrounding	O
+the	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+PmC11	B-protein
+(	O
+α1	B-structure_element
+,	O
+α2	B-structure_element
+,	O
+α4	B-structure_element
+,	O
+α6	B-structure_element
+,	O
+and	O
+α7	B-structure_element
+)	O
+are	O
+found	O
+in	O
+similar	O
+positions	O
+to	O
+the	O
+five	O
+structurally	B-protein_state
+conserved	I-protein_state
+helices	B-structure_element
+in	O
+caspases	B-protein_type
+and	O
+other	O
+members	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+,	O
+apart	O
+from	O
+family	O
+C80	B-protein_type
+.	O
+
+Other	O
+than	O
+its	O
+more	O
+extended	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+,	O
+PmC11	B-protein
+differs	O
+most	O
+significantly	O
+from	O
+other	O
+clan	B-protein_type
+CD	I-protein_type
+members	O
+at	O
+its	O
+C	O
+terminus	O
+,	O
+where	O
+the	O
+CTD	B-structure_element
+contains	O
+a	O
+further	O
+seven	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+four	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+after	O
+β8	B-structure_element
+.	O
+
+Autoprocessing	B-ptm
+of	O
+PmC11	B-protein
+
+Purification	B-experimental_method
+of	O
+recombinant	O
+PmC11	B-protein
+(	O
+molecular	O
+mass	O
+=	O
+42	O
+.	O
+6	O
+kDa	O
+)	O
+revealed	O
+partial	O
+processing	O
+into	O
+two	O
+cleavage	O
+products	O
+of	O
+26	O
+.	O
+4	O
+and	O
+16	O
+.	O
+2	O
+kDa	O
+,	O
+related	O
+to	O
+the	O
+observed	O
+cleavage	B-ptm
+at	O
+Lys147	B-residue_name_number
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+Incubation	B-experimental_method
+of	O
+PmC11	B-protein
+at	O
+37	O
+°	O
+C	O
+for	O
+16	O
+h	O
+,	O
+resulted	O
+in	O
+a	O
+fully	B-protein_state
+processed	I-protein_state
+enzyme	O
+that	O
+remained	O
+as	O
+an	O
+intact	B-protein_state
+monomer	B-oligomeric_state
+when	O
+applied	O
+to	O
+a	O
+size	O
+-	O
+exclusion	O
+column	O
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+
+The	O
+single	O
+cleavage	B-site
+site	I-site
+of	O
+PmC11	B-protein
+at	O
+Lys147	B-residue_name_number
+is	O
+found	O
+immediately	O
+after	O
+α3	B-structure_element
+,	O
+in	O
+loop	B-structure_element
+L5	B-structure_element
+within	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+Figs	O
+.	O
+1	O
+,	O
+A	O
+and	O
+B	O
+,	O
+and	O
+2A	O
+).	O
+
+The	O
+two	O
+ends	O
+of	O
+the	O
+cleavage	B-site
+site	I-site
+are	O
+remarkably	O
+well	O
+ordered	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+and	O
+displaced	O
+from	O
+one	O
+another	O
+by	O
+19	O
+.	O
+5	O
+Å	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+Moreover	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+side	O
+of	O
+the	O
+cleavage	B-site
+site	I-site
+resides	O
+near	O
+the	O
+catalytic	B-site
+dyad	I-site
+with	O
+Ala148	B-residue_name_number
+being	O
+4	O
+.	O
+5	O
+and	O
+5	O
+.	O
+7	O
+Å	O
+from	O
+His133	B-residue_name_number
+and	O
+Cys179	B-residue_name_number
+,	O
+respectively	O
+.	O
+
+Consequently	O
+,	O
+it	O
+appears	O
+feasible	O
+that	O
+the	O
+helix	B-structure_element
+attached	O
+to	O
+Lys147	B-residue_name_number
+(	O
+α3	B-structure_element
+)	O
+could	O
+be	O
+responsible	O
+for	O
+steric	O
+autoinhibition	O
+of	O
+PmC11	B-protein
+when	O
+Lys147	B-residue_name_number
+is	O
+covalently	O
+bonded	O
+to	O
+Ala148	B-residue_name_number
+.	O
+
+Thus	O
+,	O
+the	O
+cleavage	B-ptm
+would	O
+be	O
+required	O
+for	O
+full	B-protein_state
+activation	I-protein_state
+of	O
+PmC11	B-protein
+.	O
+
+To	O
+investigate	O
+this	O
+possibility	O
+,	O
+two	O
+mutant	O
+forms	O
+of	O
+the	O
+enzyme	O
+were	O
+created	O
+:	O
+PmC11C179A	B-mutant
+(	O
+a	O
+catalytically	B-protein_state
+inactive	I-protein_state
+mutant	I-protein_state
+)	O
+and	O
+PmC11K147A	B-mutant
+(	O
+a	O
+cleavage	B-protein_state
+-	I-protein_state
+site	I-protein_state
+mutant	I-protein_state
+).	O
+
+Initial	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+and	O
+Western	B-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+both	O
+mutants	O
+revealed	O
+no	O
+discernible	O
+processing	O
+occurred	O
+as	O
+compared	O
+with	O
+active	B-protein_state
+PmC11	B-protein
+(	O
+Fig	O
+.	O
+2C	O
+).	O
+
+The	O
+PmC11K147A	B-mutant
+mutant	B-protein_state
+enzyme	O
+had	O
+a	O
+markedly	O
+different	O
+reaction	B-evidence
+rate	I-evidence
+(	O
+Vmax	B-evidence
+)	O
+compared	O
+with	O
+WT	B-protein_state
+,	O
+where	O
+the	O
+reaction	B-evidence
+velocity	I-evidence
+of	O
+PmC11	B-protein
+was	O
+10	O
+times	O
+greater	O
+than	O
+that	O
+of	O
+PmC11K147A	B-mutant
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+
+Taken	O
+together	O
+,	O
+these	O
+data	O
+reveal	O
+that	O
+PmC11	B-protein
+requires	O
+processing	O
+at	O
+Lys147	B-residue_name_number
+for	O
+optimum	O
+activity	O
+.	O
+
+To	O
+investigate	O
+whether	O
+processing	O
+is	O
+a	O
+result	O
+of	O
+intra	O
+-	O
+or	O
+intermolecular	O
+cleavage	O
+,	O
+the	O
+PmC11C179A	B-mutant
+mutant	B-protein_state
+was	O
+incubated	B-experimental_method
+with	I-experimental_method
+increasing	I-experimental_method
+concentrations	I-experimental_method
+of	O
+processed	B-protein_state
+and	O
+activated	B-protein_state
+PmC11	B-protein
+.	O
+
+These	O
+studies	O
+revealed	O
+that	O
+there	O
+was	O
+no	O
+apparent	O
+cleavage	O
+of	O
+PmC11C179A	B-mutant
+by	O
+the	O
+active	B-protein_state
+enzyme	O
+at	B-experimental_method
+low	I-experimental_method
+concentrations	I-experimental_method
+of	O
+PmC11	B-protein
+and	O
+that	O
+only	O
+limited	O
+cleavage	O
+was	O
+observed	O
+when	O
+the	O
+ratio	O
+of	O
+active	B-protein_state
+enzyme	O
+(	O
+PmC11	B-protein
+:	O
+PmC11C179A	B-mutant
+)	O
+was	O
+increased	B-experimental_method
+to	I-experimental_method
+∼	I-experimental_method
+1	I-experimental_method
+:	I-experimental_method
+10	I-experimental_method
+and	I-experimental_method
+1	I-experimental_method
+:	I-experimental_method
+4	I-experimental_method
+,	O
+with	O
+complete	O
+cleavage	O
+observed	O
+at	O
+a	O
+ratio	B-experimental_method
+of	I-experimental_method
+1	I-experimental_method
+:	I-experimental_method
+1	I-experimental_method
+(	O
+Fig	O
+.	O
+2E	O
+).	O
+
+This	O
+suggests	O
+that	O
+cleavage	B-ptm
+of	O
+PmC11C179A	B-mutant
+was	O
+most	O
+likely	O
+an	O
+effect	O
+of	O
+the	O
+increasing	O
+concentration	O
+of	O
+PmC11	B-protein
+and	O
+intermolecular	O
+cleavage	O
+.	O
+
+Collectively	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+the	O
+pro	B-protein_state
+-	I-protein_state
+form	I-protein_state
+of	O
+PmC11	B-protein
+is	O
+autoinhibited	B-protein_state
+by	O
+a	O
+section	O
+of	O
+L5	B-structure_element
+blocking	O
+access	O
+to	O
+the	O
+active	B-site
+site	I-site
+,	O
+prior	O
+to	O
+intramolecular	B-ptm
+cleavage	I-ptm
+at	O
+Lys147	B-residue_name_number
+.	O
+
+This	O
+cleavage	B-ptm
+subsequently	O
+allows	O
+movement	O
+of	O
+the	O
+region	O
+containing	O
+Lys147	B-residue_name_number
+and	O
+the	O
+active	B-site
+site	I-site
+to	O
+open	B-protein_state
+up	O
+for	O
+substrate	O
+access	O
+.	O
+
+Substrate	O
+Specificity	O
+of	O
+PmC11	B-protein
+
+The	O
+autocatalytic	B-ptm
+cleavage	I-ptm
+of	O
+PmC11	B-protein
+at	O
+Lys147	B-residue_name_number
+(	O
+sequence	O
+KLK	O
+∧	O
+A	O
+)	O
+demonstrates	O
+that	O
+the	O
+enzyme	O
+accepts	O
+substrates	O
+with	O
+Lys	B-residue_name
+in	O
+the	O
+P1	B-residue_number
+position	O
+.	O
+
+As	O
+expected	O
+,	O
+PmC11	B-protein
+showed	O
+no	O
+activity	O
+against	O
+substrates	O
+with	O
+Pro	B-residue_name
+or	O
+Asp	B-residue_name
+in	O
+P1	B-residue_number
+but	O
+was	O
+active	B-protein_state
+toward	O
+substrates	O
+with	O
+a	O
+basic	O
+residue	O
+in	O
+P1	B-residue_number
+such	O
+as	O
+Bz	B-chemical
+-	I-chemical
+R	I-chemical
+-	I-chemical
+AMC	I-chemical
+,	O
+Z	B-chemical
+-	I-chemical
+GGR	I-chemical
+-	I-chemical
+AMC	I-chemical
+,	O
+and	O
+BOC	B-chemical
+-	I-chemical
+VLK	I-chemical
+-	I-chemical
+AMC	I-chemical
+.	O
+
+The	O
+rate	O
+of	O
+cleavage	O
+was	O
+∼	O
+3	O
+-	O
+fold	O
+greater	O
+toward	O
+the	O
+single	O
+Arg	B-residue_name
+substrate	O
+Bz	B-chemical
+-	I-chemical
+R	I-chemical
+-	I-chemical
+AMC	I-chemical
+than	O
+for	O
+the	O
+other	O
+two	O
+(	O
+Fig	O
+.	O
+2F	O
+)	O
+and	O
+,	O
+unexpectedly	O
+,	O
+PmC11	B-protein
+showed	O
+no	O
+activity	O
+toward	O
+BOC	B-chemical
+-	I-chemical
+K	I-chemical
+-	I-chemical
+AMC	I-chemical
+.	O
+
+These	O
+results	O
+confirm	O
+that	O
+PmC11	B-protein
+accepts	O
+substrates	O
+containing	O
+Arg	B-residue_name
+or	O
+Lys	B-residue_name
+in	O
+P1	B-residue_number
+with	O
+a	O
+possible	O
+preference	O
+for	O
+Arg	B-residue_name
+.	O
+
+The	O
+catalytic	B-site
+dyad	I-site
+of	O
+PmC11	B-protein
+sits	O
+near	O
+the	O
+bottom	O
+of	O
+an	O
+open	B-protein_state
+pocket	B-site
+on	O
+the	O
+surface	O
+of	O
+the	O
+enzyme	O
+at	O
+a	O
+conserved	B-protein_state
+location	I-protein_state
+in	O
+the	O
+clan	O
+CD	B-protein_type
+family	I-protein_type
+.	O
+
+The	O
+PmC11	B-protein
+structure	B-evidence
+reveals	O
+that	O
+the	O
+catalytic	B-site
+dyad	I-site
+forms	O
+part	O
+of	O
+a	O
+large	O
+acidic	B-site
+pocket	I-site
+(	O
+Fig	O
+.	O
+2G	O
+),	O
+consistent	O
+with	O
+a	O
+binding	B-site
+site	I-site
+for	O
+a	O
+basic	O
+substrate	O
+.	O
+
+This	O
+pocket	B-site
+is	O
+lined	O
+with	O
+the	O
+potential	O
+functional	O
+side	O
+chains	O
+of	O
+Asn50	B-residue_name_number
+,	O
+Asp177	B-residue_name_number
+,	O
+and	O
+Thr204	B-residue_name_number
+with	O
+Gly134	B-residue_name_number
+,	O
+Asp207	B-residue_name_number
+,	O
+and	O
+Met205	B-residue_name_number
+also	O
+contributing	O
+to	O
+the	O
+pocket	B-site
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+Interestingly	O
+,	O
+these	O
+residues	O
+are	O
+in	O
+regions	O
+that	O
+are	O
+structurally	B-protein_state
+similar	I-protein_state
+to	O
+those	O
+involved	O
+in	O
+the	O
+S1	B-site
+binding	I-site
+pockets	I-site
+of	O
+other	O
+clan	B-protein_type
+CD	I-protein_type
+members	I-protein_type
+(	O
+shown	O
+in	O
+Ref	O
+.).	O
+
+Because	O
+PmC11	B-protein
+recognizes	O
+basic	O
+substrates	O
+,	O
+the	O
+tetrapeptide	O
+inhibitor	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+was	O
+tested	O
+as	O
+an	O
+enzyme	O
+inhibitor	O
+and	O
+was	O
+found	O
+to	O
+inhibit	B-protein_state
+both	O
+the	O
+autoprocessing	B-ptm
+and	O
+activity	O
+of	O
+PmC11	B-protein
+(	O
+Fig	O
+.	O
+3A	O
+).	O
+
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+was	O
+also	O
+shown	O
+to	O
+bind	O
+to	O
+the	O
+enzyme	O
+:	O
+a	O
+size	B-evidence
+-	I-evidence
+shift	I-evidence
+was	O
+observed	O
+,	O
+by	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+analysis	O
+,	O
+in	O
+the	O
+larger	O
+processed	O
+product	O
+of	O
+PmC11	B-protein
+suggesting	O
+that	O
+the	O
+inhibitor	B-protein_state
+bound	I-protein_state
+to	O
+the	O
+active	B-site
+site	I-site
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+
+A	O
+structure	B-experimental_method
+overlay	I-experimental_method
+of	O
+PmC11	B-protein
+with	O
+the	O
+MALT1	B-protein
+-	I-protein
+paracacaspase	I-protein
+(	O
+MALT1	B-protein
+-	I-protein
+P	I-protein
+),	O
+in	O
+complex	B-protein_state
+with	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+,	O
+revealed	O
+that	O
+the	O
+PmC11	B-protein
+dyad	B-site
+sits	O
+in	O
+a	O
+very	O
+similar	O
+position	O
+to	O
+that	O
+of	O
+active	B-protein_state
+MALT1	B-protein
+-	I-protein
+P	I-protein
+and	O
+that	O
+Asn50	B-residue_name_number
+,	O
+Asp177	B-residue_name_number
+,	O
+and	O
+Asp207	B-residue_name_number
+superimpose	O
+well	O
+with	O
+the	O
+principal	O
+MALT1	B-protein
+-	I-protein
+P	I-protein
+inhibitor	B-site
+binding	I-site
+residues	I-site
+(	O
+Asp365	B-residue_name_number
+,	O
+Asp462	B-residue_name_number
+,	O
+and	O
+Glu500	B-residue_name_number
+,	O
+respectively	O
+(	O
+VRPR	B-chemical
+-	I-chemical
+FMK	I-chemical
+from	O
+MALT1	B-protein
+-	I-protein
+P	I-protein
+with	O
+the	O
+corresponding	O
+PmC11	B-protein
+residues	O
+from	O
+the	O
+structural	B-experimental_method
+overlay	I-experimental_method
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+1D	O
+),	O
+as	O
+described	O
+in	O
+Ref	O
+.).	O
+
+Asp177	B-residue_name_number
+is	O
+located	O
+near	O
+the	O
+catalytic	B-protein_state
+cysteine	B-residue_name
+and	O
+is	O
+conserved	B-protein_state
+throughout	I-protein_state
+the	O
+C11	B-protein_type
+family	I-protein_type
+,	O
+suggesting	O
+it	O
+is	O
+the	O
+primary	O
+S1	B-site
+binding	I-site
+site	I-site
+residue	I-site
+.	O
+
+In	O
+the	O
+structure	B-evidence
+of	O
+PmC11	B-protein
+,	O
+Asp207	B-residue_name_number
+resides	O
+on	O
+a	O
+flexible	O
+loop	B-structure_element
+pointing	O
+away	O
+from	O
+the	O
+S1	B-site
+binding	I-site
+pocket	I-site
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+However	O
+,	O
+this	O
+loop	B-structure_element
+has	O
+been	O
+shown	O
+to	O
+be	O
+important	O
+for	O
+substrate	O
+binding	O
+in	O
+clan	B-protein_type
+CD	I-protein_type
+and	O
+this	O
+residue	O
+could	O
+easily	O
+rotate	O
+and	O
+be	O
+involved	O
+in	O
+substrate	O
+binding	O
+in	O
+PmC11	B-protein
+.	O
+
+Thus	O
+,	O
+Asn50	B-residue_name_number
+,	O
+Asp177	B-residue_name_number
+,	O
+and	O
+Asp207	B-residue_name_number
+are	O
+most	O
+likely	O
+responsible	O
+for	O
+the	O
+substrate	O
+specificity	O
+of	O
+PmC11	B-protein
+.	O
+
+Asp177	B-residue_name_number
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+throughout	O
+the	O
+clan	B-protein_type
+CD	I-protein_type
+C11	I-protein_type
+peptidases	I-protein_type
+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	B-protein_type
+CD	I-protein_type
+enzymes	I-protein_type
+,	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	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+when	O
+PmC11	B-protein
+is	O
+overlaid	B-experimental_method
+on	O
+the	O
+MALT1	B-protein
+-	I-protein
+P	I-protein
+structure	B-evidence
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+PmC11	B-protein
+binds	O
+and	O
+is	O
+inhibited	O
+by	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+and	O
+does	O
+not	O
+require	O
+Ca2	B-chemical
++	I-chemical
+for	O
+activity	O
+.	O
+
+A	O
+,	O
+PmC11	O
+activity	O
+is	O
+inhibited	O
+by	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+.	O
+
+Cleavage	O
+of	O
+Bz	B-chemical
+-	I-chemical
+R	I-chemical
+-	I-chemical
+AMC	I-chemical
+by	O
+PmC11	B-protein
+was	O
+measured	O
+in	O
+a	O
+fluorometric	B-experimental_method
+activity	I-experimental_method
+assay	I-experimental_method
+with	O
+(+,	O
+purple	O
+)	O
+and	O
+without	O
+(−,	O
+red	O
+)	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+.	O
+
+B	O
+,	O
+gel	B-experimental_method
+-	I-experimental_method
+shift	I-experimental_method
+assay	I-experimental_method
+reveals	O
+that	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+binds	O
+to	O
+PmC11	B-protein
+.	O
+
+PmC11	O
+was	O
+incubated	B-experimental_method
+with	O
+(+)	O
+or	O
+without	O
+(−)	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+and	O
+the	O
+samples	O
+analyzed	O
+on	O
+a	O
+10	O
+%	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+gel	O
+.	O
+
+A	O
+size	B-evidence
+shift	I-evidence
+can	O
+be	O
+observed	O
+in	O
+the	O
+larger	O
+processed	O
+product	O
+of	O
+PmC11	B-protein
+(	O
+26	O
+.	O
+1	O
+kDa	O
+).	O
+
+C	O
+,	O
+PmC11	B-protein
+with	O
+the	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+from	O
+the	O
+MALT1	B-protein
+-	I-protein
+paracacaspase	I-protein
+(	O
+MALT1	B-protein
+-	I-protein
+P	I-protein
+)	O
+superimposed	B-experimental_method
+.	O
+
+A	O
+three	B-experimental_method
+-	I-experimental_method
+dimensional	I-experimental_method
+structural	I-experimental_method
+overlay	I-experimental_method
+of	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+from	O
+the	O
+MALT1	B-protein
+-	I-protein
+P	I-protein
+complex	O
+onto	O
+PmC11	B-protein
+.	O
+
+The	O
+position	O
+and	O
+orientation	O
+of	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+was	O
+taken	O
+from	O
+superposition	B-experimental_method
+of	O
+the	O
+PmC11	B-protein
+and	O
+MALTI_P	B-protein
+structures	B-evidence
+and	O
+indicates	O
+the	O
+presumed	O
+active	B-site
+site	I-site
+of	O
+PmC11	B-protein
+.	O
+
+Residues	O
+surrounding	O
+the	O
+inhibitor	O
+are	O
+labeled	O
+and	O
+represent	O
+potentially	O
+important	O
+binding	B-site
+site	I-site
+residues	I-site
+,	O
+labeled	O
+in	O
+black	O
+and	O
+shown	O
+in	O
+an	O
+atomic	O
+representation	O
+.	O
+
+C	O
+,	O
+divalent	O
+cations	O
+do	O
+not	O
+increase	O
+the	O
+activity	O
+of	O
+PmC11	B-protein
+.	O
+
+The	O
+cleavage	O
+of	O
+Bz	B-chemical
+-	I-chemical
+R	I-chemical
+-	I-chemical
+AMC	I-chemical
+by	O
+PmC11	B-protein
+was	O
+measured	O
+in	O
+the	O
+presence	O
+of	O
+the	O
+cations	O
+Ca2	B-chemical
++,	I-chemical
+Mn2	B-chemical
++,	I-chemical
+Zn2	B-chemical
++,	I-chemical
+Co2	B-chemical
++,	I-chemical
+Cu2	B-chemical
++,	I-chemical
+Mg2	B-chemical
++,	I-chemical
+and	O
+Fe3	B-chemical
++	I-chemical
+with	O
+EGTA	B-chemical
+as	O
+a	O
+negative	O
+control	O
+,	O
+and	O
+relative	B-experimental_method
+fluorescence	I-experimental_method
+measured	I-experimental_method
+against	I-experimental_method
+time	I-experimental_method
+(	O
+min	O
+).	O
+
+The	O
+addition	B-experimental_method
+of	I-experimental_method
+cations	I-experimental_method
+produced	O
+no	O
+improvement	O
+in	O
+activity	O
+of	O
+PmC11	B-protein
+when	O
+compared	O
+in	O
+the	O
+presence	O
+of	O
+EGTA	B-chemical
+,	O
+suggesting	O
+that	O
+PmC11	B-protein
+does	O
+not	O
+require	O
+metal	O
+ions	O
+for	O
+proteolytic	O
+activity	O
+.	O
+
+Furthermore	O
+,	O
+Cu2	B-chemical
++,	I-chemical
+Fe2	B-chemical
++,	I-chemical
+and	O
+Zn2	B-chemical
++	I-chemical
+appear	O
+to	O
+inhibit	B-protein_state
+PmC11	B-protein
+.	O
+
+Comparison	O
+with	O
+Clostripain	B-protein
+
+Clostripain	B-protein
+from	O
+C	B-species
+.	I-species
+histolyticum	I-species
+is	O
+the	O
+founding	O
+member	O
+of	O
+the	O
+C11	B-protein_type
+family	I-protein_type
+of	O
+peptidases	B-protein_type
+and	O
+contains	O
+an	O
+additional	O
+149	B-residue_range
+residues	I-residue_range
+compared	O
+with	O
+PmC11	B-protein
+.	O
+
+A	O
+multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+revealed	O
+that	O
+most	O
+of	O
+the	O
+secondary	B-structure_element
+structural	I-structure_element
+elements	I-structure_element
+are	O
+conserved	B-protein_state
+between	O
+the	O
+two	O
+enzymes	O
+,	O
+although	O
+they	O
+are	O
+only	O
+∼	O
+23	O
+%	O
+identical	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+
+Nevertheless	O
+,	O
+PmC11	B-protein
+may	O
+be	O
+a	O
+good	O
+model	O
+for	O
+the	O
+core	O
+structure	O
+of	O
+clostripain	B-protein
+.	O
+
+The	O
+primary	B-experimental_method
+structural	I-experimental_method
+alignment	I-experimental_method
+also	O
+shows	O
+that	O
+the	O
+catalytic	B-site
+dyad	I-site
+in	O
+PmC11	B-protein
+is	O
+structurally	B-protein_state
+conserved	I-protein_state
+in	O
+clostripain	B-protein
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+
+Unlike	O
+PmC11	B-protein
+,	O
+clostripain	B-protein
+has	O
+two	O
+cleavage	B-site
+sites	I-site
+(	O
+Arg181	B-residue_name_number
+and	O
+Arg190	B-residue_name_number
+),	O
+which	O
+results	O
+in	O
+the	O
+removal	O
+of	O
+a	O
+nonapeptide	B-structure_element
+,	O
+and	O
+is	O
+required	O
+for	O
+full	B-protein_state
+activation	I-protein_state
+of	O
+the	O
+enzyme	O
+(	O
+highlighted	O
+in	O
+Fig	O
+.	O
+1A	O
+).	O
+
+Interestingly	O
+,	O
+Arg190	B-residue_name_number
+was	O
+found	O
+to	O
+align	O
+with	O
+Lys147	B-residue_name_number
+in	O
+PmC11	B-protein
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+predicted	O
+primary	O
+S1	B-site
+-	I-site
+binding	I-site
+residue	I-site
+in	O
+PmC11	B-protein
+Asp177	B-residue_name_number
+also	O
+overlays	B-experimental_method
+with	O
+the	O
+residue	O
+predicted	O
+to	O
+be	O
+the	O
+P1	B-site
+specificity	I-site
+determining	I-site
+residue	I-site
+in	O
+clostripain	B-protein
+(	O
+Asp229	B-residue_name_number
+,	O
+Fig	O
+.	O
+1A	O
+).	O
+
+As	O
+studies	O
+on	O
+clostripain	B-protein
+revealed	O
+addition	O
+of	O
+Ca2	B-chemical
++	I-chemical
+ions	O
+are	O
+required	O
+for	O
+full	B-protein_state
+activation	I-protein_state
+,	O
+the	O
+Ca2	B-chemical
++	I-chemical
+dependence	O
+of	O
+PmC11	B-protein
+was	O
+examined	O
+.	O
+
+Surprisingly	O
+,	O
+Ca2	B-chemical
++	I-chemical
+did	O
+not	O
+enhance	O
+PmC11	B-protein
+activity	O
+and	O
+,	O
+furthermore	O
+,	O
+other	O
+divalent	O
+cations	O
+,	O
+Mg2	B-chemical
++,	I-chemical
+Mn2	B-chemical
++,	I-chemical
+Co2	B-chemical
++,	I-chemical
+Fe2	B-chemical
++,	I-chemical
+Zn2	B-chemical
++,	I-chemical
+and	O
+Cu2	B-chemical
++,	I-chemical
+were	O
+not	O
+necessary	O
+for	O
+PmC11	B-protein
+activity	O
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+
+In	O
+support	O
+of	O
+these	O
+findings	O
+,	O
+EGTA	B-chemical
+did	O
+not	O
+inhibit	O
+PmC11	B-protein
+suggesting	O
+that	O
+,	O
+unlike	O
+clostripain	B-protein
+,	O
+PmC11	B-protein
+does	O
+not	O
+require	O
+Ca2	B-chemical
++	I-chemical
+or	O
+other	O
+divalent	O
+cations	O
+,	O
+for	O
+activity	O
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+PmC11	B-protein
+now	O
+provides	O
+three	O
+-	O
+dimensional	O
+information	O
+for	O
+a	O
+member	O
+of	O
+the	O
+clostripain	B-protein
+C11	B-protein_type
+family	I-protein_type
+of	O
+cysteine	B-protein_type
+peptidases	I-protein_type
+.	O
+
+The	O
+enzyme	O
+exhibits	O
+all	O
+of	O
+the	O
+key	O
+structural	O
+elements	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+members	I-protein_type
+,	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	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+with	O
+a	O
+novel	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+structural	O
+similarity	O
+of	O
+PmC11	B-protein
+with	O
+its	O
+nearest	O
+structural	O
+neighbors	O
+in	O
+the	O
+PDB	O
+is	O
+decidedly	O
+low	O
+,	O
+overlaying	O
+better	O
+with	O
+six	O
+-	O
+stranded	O
+caspase	B-protein
+-	I-protein
+7	I-protein
+than	O
+any	O
+of	O
+the	O
+other	O
+larger	O
+members	O
+of	O
+the	O
+clan	O
+(	O
+Table	O
+2	O
+).	O
+
+The	O
+substrate	O
+specificity	O
+of	O
+PmC11	B-protein
+is	O
+Arg	B-residue_name
+/	O
+Lys	B-residue_name
+and	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+revealed	O
+an	O
+acidic	B-site
+pocket	I-site
+for	O
+specific	O
+binding	O
+of	O
+such	O
+basic	O
+substrates	O
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+structure	B-evidence
+suggested	O
+a	O
+mechanism	O
+of	O
+self	O
+-	O
+inhibition	O
+in	O
+both	O
+PmC11	B-protein
+and	O
+clostripain	B-protein
+and	O
+an	O
+activation	O
+mechanism	O
+that	O
+requires	O
+autoprocessing	B-ptm
+.	O
+
+PmC11	B-protein
+differs	O
+from	O
+clostripain	B-protein
+in	O
+that	O
+is	O
+does	O
+not	O
+appear	O
+to	O
+require	O
+divalent	O
+cations	O
+for	O
+activation	O
+.	O
+
+Several	O
+other	O
+members	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+require	O
+processing	B-ptm
+for	O
+full	B-protein_state
+activation	I-protein_state
+including	O
+legumain	B-protein
+,	O
+gingipain	B-protein
+-	I-protein
+R	I-protein
+,	O
+MARTX	B-protein
+-	I-protein
+CPD	I-protein
+,	O
+and	O
+the	O
+effector	B-protein_type
+caspases	I-protein_type
+,	O
+e	O
+.	O
+g	O
+.	O
+caspase	B-protein
+-	I-protein
+7	I-protein
+.	O
+
+To	O
+date	O
+,	O
+the	O
+effector	B-protein_type
+caspases	I-protein_type
+are	O
+the	O
+only	O
+group	O
+of	O
+enzymes	O
+that	O
+require	O
+cleavage	B-ptm
+of	O
+a	O
+loop	B-structure_element
+within	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+.	O
+
+This	O
+is	O
+also	O
+the	O
+case	O
+in	O
+PmC11	B-protein
+,	O
+although	O
+the	O
+cleavage	B-ptm
+loop	B-structure_element
+is	O
+structurally	O
+different	O
+to	O
+that	O
+found	O
+in	O
+the	O
+caspases	B-protein_type
+and	O
+follows	O
+the	O
+catalytic	B-protein_state
+His	B-residue_name
+(	O
+Fig	O
+.	O
+1A	O
+),	O
+as	O
+opposed	O
+to	O
+the	O
+Cys	B-residue_name
+in	O
+the	O
+caspases	B-protein_type
+.	O
+
+All	O
+other	O
+clan	B-protein_type
+CD	I-protein_type
+members	I-protein_type
+requiring	O
+cleavage	B-ptm
+for	O
+full	B-protein_state
+activation	I-protein_state
+do	O
+so	O
+at	O
+sites	B-site
+external	O
+to	O
+their	O
+central	O
+sheets	B-structure_element
+.	O
+
+The	O
+caspases	B-protein_type
+and	O
+gingipain	B-protein
+-	I-protein
+R	I-protein
+both	O
+undergo	O
+intermolecular	B-ptm
+(	I-ptm
+trans	I-ptm
+)	I-ptm
+cleavage	I-ptm
+and	O
+legumain	B-protein
+and	O
+MARTX	B-protein
+-	I-protein
+CPD	I-protein
+are	O
+reported	O
+to	O
+perform	O
+intramolecular	B-ptm
+(	I-ptm
+cis	I-ptm
+)	I-ptm
+cleavage	I-ptm
+.	O
+
+In	O
+addition	O
+,	O
+several	O
+members	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+exhibit	O
+self	O
+-	O
+inhibition	O
+,	O
+whereby	O
+regions	B-structure_element
+of	O
+the	O
+enzyme	O
+block	O
+access	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+Like	O
+PmC11	B-protein
+,	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	B-ptm
+of	O
+the	O
+blocking	B-structure_element
+region	I-structure_element
+is	O
+required	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+PmC11	B-protein
+gives	O
+the	O
+first	O
+insight	O
+into	O
+this	O
+class	O
+of	O
+relatively	O
+unexplored	O
+family	O
+of	O
+proteins	O
+and	O
+should	O
+allow	O
+important	O
+catalytic	O
+and	O
+substrate	O
+binding	O
+residues	O
+to	O
+be	O
+identified	O
+in	O
+a	O
+variety	O
+of	O
+orthologues	O
+.	O
+
+Indeed	O
+,	O
+insights	O
+gained	O
+from	O
+an	O
+analysis	O
+of	O
+the	O
+PmC11	B-protein
+structure	B-evidence
+revealed	O
+the	O
+identity	O
+of	O
+the	O
+Trypanosoma	B-species
+brucei	I-species
+PNT1	B-protein
+protein	O
+as	O
+a	O
+C11	B-protein_type
+cysteine	I-protein_type
+peptidase	I-protein_type
+with	O
+an	O
+essential	O
+role	O
+in	O
+organelle	O
+replication	O
+.	O
+
+The	O
+PmC11	B-protein
+structure	B-evidence
+should	O
+provide	O
+a	O
+good	O
+basis	O
+for	O
+structural	B-experimental_method
+modeling	I-experimental_method
+and	O
+,	O
+given	O
+the	O
+importance	O
+of	O
+other	O
+clan	B-protein_type
+CD	I-protein_type
+enzymes	I-protein_type
+,	O
+this	O
+work	O
+should	O
+also	O
+advance	O
+the	O
+exploration	O
+of	O
+these	O
+peptidases	B-protein_type
+and	O
+potentially	O
+identify	O
+new	O
+biologically	O
+important	O
+substrates	O
+.	O
+
+Structural	O
+insights	O
+into	O
+the	O
+regulatory	O
+mechanism	O
+of	O
+the	O
+Pseudomonas	B-species
+aeruginosa	I-species
+YfiBNR	B-complex_assembly
+system	O
+
+YfiBNR	B-complex_assembly
+is	O
+a	O
+recently	O
+identified	O
+bis	B-chemical
+-(	I-chemical
+3	I-chemical
+’-	I-chemical
+5	I-chemical
+’)-	I-chemical
+cyclic	I-chemical
+dimeric	I-chemical
+GMP	I-chemical
+(	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+)	O
+signaling	O
+system	O
+in	O
+opportunistic	O
+pathogens	O
+.	O
+
+In	O
+response	O
+to	O
+cell	O
+stress	O
+,	O
+YfiB	B-protein
+in	O
+the	O
+outer	O
+membrane	O
+can	O
+sequester	O
+the	O
+periplasmic	O
+protein	O
+YfiR	B-protein
+,	O
+releasing	O
+its	O
+inhibition	O
+of	O
+YfiN	B-protein
+on	O
+the	O
+inner	O
+membrane	O
+and	O
+thus	O
+provoking	O
+the	O
+diguanylate	O
+cyclase	O
+activity	O
+of	O
+YfiN	B-protein
+to	O
+induce	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+production	O
+.	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+YfiB	B-protein
+alone	B-protein_state
+and	O
+of	O
+an	O
+active	B-protein_state
+mutant	B-protein_state
+YfiBL43P	B-mutant
+complexed	B-protein_state
+with	I-protein_state
+YfiR	B-protein
+with	O
+2	O
+:	O
+2	O
+stoichiometry	O
+.	O
+
+Structural	B-experimental_method
+analyses	I-experimental_method
+revealed	O
+that	O
+in	O
+contrast	O
+to	O
+the	O
+compact	B-protein_state
+conformation	I-protein_state
+of	O
+the	O
+dimeric	B-oligomeric_state
+YfiB	B-protein
+alone	B-protein_state
+,	O
+YfiBL43P	B-mutant
+adopts	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+allowing	O
+activated	B-protein_state
+YfiB	B-protein
+to	O
+penetrate	O
+the	O
+peptidoglycan	B-chemical
+(	O
+PG	B-chemical
+)	O
+layer	O
+and	O
+access	O
+YfiR	B-protein
+.	O
+YfiBL43P	B-mutant
+shows	O
+a	O
+more	O
+compact	O
+PG	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+and	O
+much	O
+higher	O
+PG	B-evidence
+binding	I-evidence
+affinity	I-evidence
+than	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+,	O
+suggesting	O
+a	O
+tight	O
+correlation	O
+between	O
+PG	O
+binding	O
+and	O
+YfiB	B-protein
+activation	O
+.	O
+
+In	O
+addition	O
+,	O
+our	O
+crystallographic	B-experimental_method
+analyses	I-experimental_method
+revealed	O
+that	O
+YfiR	B-protein
+binds	O
+Vitamin	B-chemical
+B6	I-chemical
+(	O
+VB6	B-chemical
+)	O
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+at	O
+a	O
+YfiB	B-site
+-	I-site
+binding	I-site
+site	I-site
+and	O
+that	O
+both	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+are	O
+able	O
+to	O
+reduce	O
+YfiBL43P	B-mutant
+-	O
+induced	O
+biofilm	O
+formation	O
+.	O
+
+Based	O
+on	O
+the	O
+structural	B-evidence
+and	I-evidence
+biochemical	I-evidence
+data	I-evidence
+,	O
+we	O
+propose	O
+an	O
+updated	O
+regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	B-complex_assembly
+system	O
+.	O
+
+Bis	B-chemical
+-(	I-chemical
+3	I-chemical
+’-	I-chemical
+5	I-chemical
+’)-	I-chemical
+cyclic	I-chemical
+dimeric	I-chemical
+GMP	I-chemical
+(	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+)	O
+is	O
+a	O
+ubiquitous	O
+second	O
+messenger	O
+that	O
+bacteria	B-taxonomy_domain
+use	O
+to	O
+facilitate	O
+behavioral	O
+adaptations	O
+to	O
+their	O
+ever	O
+-	O
+changing	O
+environment	O
+.	O
+
+An	O
+increase	O
+in	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+promotes	O
+biofilm	O
+formation	O
+,	O
+and	O
+a	O
+decrease	O
+results	O
+in	O
+biofilm	O
+degradation	O
+(	O
+Boehm	O
+et	O
+al	O
+.,;	O
+Duerig	O
+et	O
+al	O
+.,;	O
+Hickman	O
+et	O
+al	O
+.,;	O
+Jenal	O
+,;	O
+Romling	O
+et	O
+al	O
+.,).	O
+
+The	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+level	O
+is	O
+regulated	O
+by	O
+two	O
+reciprocal	O
+enzyme	O
+systems	O
+,	O
+namely	O
+,	O
+diguanylate	B-protein_type
+cyclases	I-protein_type
+(	O
+DGCs	B-protein_type
+)	O
+that	O
+synthesize	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+and	O
+phosphodiesterases	B-protein_type
+(	O
+PDEs	B-protein_type
+)	O
+that	O
+hydrolyze	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+(	O
+Kulasakara	O
+et	O
+al	O
+.,;	O
+Ross	O
+et	O
+al	O
+.,;	O
+Ross	O
+et	O
+al	O
+.,).	O
+Many	O
+of	O
+these	O
+enzymes	O
+are	O
+multiple	O
+-	O
+domain	O
+proteins	O
+containing	O
+a	O
+variable	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+that	O
+commonly	O
+acts	O
+as	O
+a	O
+signal	O
+sensor	O
+or	O
+transduction	O
+module	O
+,	O
+followed	O
+by	O
+the	O
+relatively	B-protein_state
+conserved	I-protein_state
+GGDEF	B-structure_element
+motif	I-structure_element
+in	O
+DGCs	B-protein_type
+or	O
+EAL	B-structure_element
+/	I-structure_element
+HD	I-structure_element
+-	I-structure_element
+GYP	I-structure_element
+domains	I-structure_element
+in	O
+PDEs	B-protein_type
+(	O
+Hengge	O
+,;	O
+Navarro	O
+et	O
+al	O
+.,;	O
+Schirmer	O
+and	O
+Jenal	O
+,).	O
+
+Intriguingly	O
+,	O
+studies	O
+in	O
+diverse	O
+species	O
+have	O
+revealed	O
+that	O
+a	O
+single	O
+bacterium	B-taxonomy_domain
+can	O
+have	O
+dozens	O
+of	O
+DGCs	B-protein_type
+and	O
+PDEs	B-protein_type
+(	O
+Hickman	O
+et	O
+al	O
+.,;	O
+Kirillina	O
+et	O
+al	O
+.,;	O
+Kulasakara	O
+et	O
+al	O
+.,;	O
+Tamayo	O
+et	O
+al	O
+.,).	O
+
+In	O
+Pseudomonas	B-species
+aeruginosa	I-species
+in	O
+particular	O
+,	O
+42	O
+genes	O
+containing	O
+putative	O
+DGCs	B-protein_type
+and	O
+/	O
+or	O
+PDEs	B-protein_type
+were	O
+identified	O
+(	O
+Kulasakara	O
+et	O
+al	O
+.,).	O
+
+The	O
+functional	O
+role	O
+of	O
+a	O
+number	O
+of	O
+downstream	O
+effectors	O
+of	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+has	O
+been	O
+characterized	O
+as	O
+affecting	O
+exopolysaccharide	B-chemical
+(	O
+EPS	B-chemical
+)	O
+production	O
+,	O
+transcription	O
+,	O
+motility	O
+,	O
+and	O
+surface	O
+attachment	O
+(	O
+Caly	O
+et	O
+al	O
+.,;	O
+Camilli	O
+and	O
+Bassler	O
+,;	O
+Ha	O
+and	O
+O	O
+’	O
+Toole	O
+,;	O
+Pesavento	O
+and	O
+Hengge	O
+,).	O
+
+However	O
+,	O
+due	O
+to	O
+the	O
+intricacy	O
+of	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+signaling	O
+networks	O
+and	O
+the	O
+diversity	O
+of	O
+experimental	O
+cues	O
+,	O
+the	O
+detailed	O
+mechanisms	O
+by	O
+which	O
+these	O
+signaling	O
+pathways	O
+specifically	O
+sense	O
+and	O
+integrate	O
+different	O
+inputs	O
+remain	O
+largely	O
+elusive	O
+.	O
+
+Biofilm	O
+formation	O
+protects	O
+pathogenic	O
+bacteria	B-taxonomy_domain
+from	O
+antibiotic	O
+treatment	O
+,	O
+and	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+-	O
+regulated	O
+biofilm	O
+formation	O
+has	O
+been	O
+extensively	O
+studied	O
+in	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+(	O
+Evans	O
+,;	O
+Kirisits	O
+et	O
+al	O
+.,;	O
+Malone	O
+,;	O
+Reinhardt	O
+et	O
+al	O
+.,).	O
+
+In	O
+the	O
+lungs	O
+of	O
+cystic	O
+fibrosis	O
+(	O
+CF	O
+)	O
+patients	O
+,	O
+adherent	O
+biofilm	O
+formation	O
+and	O
+the	O
+appearance	O
+of	O
+small	O
+colony	O
+variant	O
+(	O
+SCV	O
+)	O
+morphologies	O
+of	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+correlate	O
+with	O
+prolonged	O
+persistence	O
+of	O
+infection	O
+and	O
+poor	O
+lung	O
+function	O
+(	O
+Govan	O
+and	O
+Deretic	O
+,;	O
+Haussler	O
+et	O
+al	O
+.,;	O
+Haussler	O
+et	O
+al	O
+.,;	O
+Parsek	O
+and	O
+Singh	O
+,;	O
+Smith	O
+et	O
+al	O
+.,).	O
+
+Recently	O
+,	O
+Malone	O
+and	O
+coworkers	O
+identified	O
+the	O
+tripartite	B-protein_state
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+signaling	O
+module	O
+system	O
+YfiBNR	B-complex_assembly
+(	O
+also	O
+known	O
+as	O
+AwsXRO	B-complex_assembly
+(	O
+Beaumont	O
+et	O
+al	O
+.,;	O
+Giddens	O
+et	O
+al	O
+.,)	O
+or	O
+Tbp	B-complex_assembly
+(	O
+Ueda	O
+and	O
+Wood	O
+,))	O
+by	O
+genetic	B-experimental_method
+screening	I-experimental_method
+for	O
+mutants	O
+that	O
+displayed	O
+SCV	O
+phenotypes	O
+in	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+PAO1	I-species
+(	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+The	O
+YfiBNR	B-complex_assembly
+system	O
+contains	O
+three	O
+protein	O
+members	O
+and	O
+modulates	O
+intracellular	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+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
+
+More	O
+recently	O
+,	O
+this	O
+system	O
+was	O
+also	O
+reported	O
+in	O
+other	O
+Gram	B-taxonomy_domain
+-	I-taxonomy_domain
+negative	I-taxonomy_domain
+bacteria	I-taxonomy_domain
+,	O
+such	O
+as	O
+Escherichia	B-species
+coli	I-species
+(	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	B-species
+pneumonia	I-species
+(	O
+Huertas	O
+et	O
+al	O
+.,)	O
+and	O
+Yersinia	B-species
+pestis	I-species
+(	O
+Ren	O
+et	O
+al	O
+.,).	O
+
+YfiN	B-protein
+is	O
+an	O
+integral	O
+inner	O
+-	O
+membrane	O
+protein	O
+with	O
+two	O
+potential	O
+transmembrane	B-structure_element
+helices	I-structure_element
+,	O
+a	O
+periplasmic	O
+Per	B-structure_element
+-	I-structure_element
+Arnt	I-structure_element
+-	I-structure_element
+Sim	I-structure_element
+(	O
+PAS	B-structure_element
+)	O
+domain	O
+,	O
+and	O
+cytosolic	O
+domains	O
+containing	O
+a	O
+HAMP	B-structure_element
+domain	I-structure_element
+(	O
+mediate	O
+input	O
+-	O
+output	O
+signaling	O
+in	O
+histidine	B-protein_type
+kinases	I-protein_type
+,	O
+adenylyl	B-protein_type
+cyclases	I-protein_type
+,	O
+methyl	B-protein_type
+-	I-protein_type
+accepting	I-protein_type
+chemotaxis	I-protein_type
+proteins	I-protein_type
+,	O
+and	O
+phosphatases	B-protein_type
+)	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+GGDEF	B-structure_element
+domain	I-structure_element
+indicating	O
+a	O
+DGC	B-protein_type
+’	O
+s	O
+function	O
+(	O
+Giardina	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+YfiN	B-protein
+is	O
+repressed	B-protein_state
+by	I-protein_state
+specific	O
+interaction	O
+between	O
+its	O
+periplasmic	O
+PAS	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+periplasmic	O
+protein	O
+YfiR	B-protein
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+YfiB	B-protein
+is	O
+an	O
+OmpA	B-protein_type
+/	I-protein_type
+Pal	I-protein_type
+-	I-protein_type
+like	I-protein_type
+outer	O
+-	O
+membrane	O
+lipoprotein	B-protein_type
+(	O
+Parsons	O
+et	O
+al	O
+.,)	O
+that	O
+can	O
+activate	O
+YfiN	B-protein
+by	O
+sequestering	O
+YfiR	B-protein
+(	O
+Malone	O
+et	O
+al	O
+.,)	O
+in	O
+an	O
+unknown	O
+manner	O
+.	O
+
+Whether	O
+YfiB	B-protein
+directly	O
+recruits	O
+YfiR	B-protein
+or	O
+recruits	O
+YfiR	B-protein
+via	O
+a	O
+third	O
+partner	O
+is	O
+an	O
+open	O
+question	O
+.	O
+
+After	O
+the	O
+sequestration	O
+of	O
+YfiR	B-protein
+by	O
+YfiB	B-protein
+,	O
+the	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+produced	O
+by	O
+activated	B-protein_state
+YfiN	B-protein
+increases	O
+the	O
+biosynthesis	O
+of	O
+the	O
+Pel	B-chemical
+and	O
+Psl	B-chemical
+EPSs	B-chemical
+,	O
+resulting	O
+in	O
+the	O
+appearance	O
+of	O
+the	O
+SCV	O
+phenotype	O
+,	O
+which	O
+indicates	O
+enhanced	O
+biofilm	O
+formation	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+It	O
+has	O
+been	O
+reported	O
+that	O
+the	O
+activation	O
+of	O
+YfiN	B-protein
+may	O
+be	O
+induced	O
+by	O
+redox	O
+-	O
+driven	O
+misfolding	O
+of	O
+YfiR	B-protein
+(	O
+Giardina	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+It	O
+is	O
+also	O
+proposed	O
+that	O
+the	O
+sequestration	O
+of	O
+YfiR	B-protein
+by	O
+YfiB	B-protein
+can	O
+be	O
+induced	O
+by	O
+certain	O
+YfiB	B-protein
+-	O
+mediated	O
+cell	O
+wall	O
+stress	O
+,	O
+and	O
+mutagenesis	B-experimental_method
+studies	I-experimental_method
+revealed	O
+a	O
+number	O
+of	O
+activation	B-structure_element
+residues	I-structure_element
+of	O
+YfiB	B-protein
+that	O
+were	O
+located	O
+in	O
+close	O
+proximity	O
+to	O
+the	O
+predicted	B-protein_state
+first	B-structure_element
+helix	I-structure_element
+of	O
+the	O
+periplasmic	B-structure_element
+domain	I-structure_element
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+In	O
+addition	O
+,	O
+quorum	O
+sensing	O
+-	O
+related	O
+dephosphorylation	O
+of	O
+the	O
+PAS	B-structure_element
+domain	I-structure_element
+of	O
+YfiN	B-protein
+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
+
+Recently	O
+,	O
+we	O
+solved	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+YfiR	B-protein
+in	O
+both	O
+the	O
+non	B-protein_state
+-	I-protein_state
+oxidized	I-protein_state
+and	O
+the	O
+oxidized	B-protein_state
+states	O
+,	O
+revealing	O
+breakage	O
+/	O
+formation	O
+of	O
+one	O
+disulfide	B-ptm
+bond	I-ptm
+(	O
+Cys71	B-residue_name_number
+-	O
+Cys110	B-residue_name_number
+)	O
+and	O
+local	O
+conformational	O
+change	O
+around	O
+the	O
+other	O
+one	O
+(	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+),	O
+indicating	O
+that	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+plays	O
+an	O
+important	O
+role	O
+in	O
+maintaining	O
+the	O
+correct	O
+folding	O
+of	O
+YfiR	B-protein
+(	O
+Yang	O
+et	O
+al	O
+.,).	O
+
+In	O
+the	O
+present	O
+study	O
+,	O
+we	O
+solved	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+an	O
+N	O
+-	O
+terminal	O
+truncated	B-protein_state
+form	O
+of	O
+YfiB	B-protein
+(	O
+34	B-residue_range
+–	I-residue_range
+168	I-residue_range
+)	O
+and	O
+YfiR	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+an	O
+active	B-protein_state
+mutant	B-protein_state
+YfiBL43P	B-mutant
+.	O
+
+Most	O
+recently	O
+,	O
+Li	O
+and	O
+coworkers	O
+reported	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+YfiB	B-protein
+(	O
+27	B-residue_range
+–	I-residue_range
+168	I-residue_range
+)	O
+alone	B-protein_state
+and	O
+YfiRC71S	B-mutant
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+YfiB	B-protein
+(	O
+59	B-residue_range
+–	I-residue_range
+168	I-residue_range
+)	O
+(	O
+Li	O
+et	O
+al	O
+.,).	O
+
+Compared	O
+with	O
+the	O
+reported	O
+complex	O
+structure	O
+,	O
+YfiBL43P	B-mutant
+in	O
+our	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+structure	B-evidence
+has	O
+additional	O
+visible	O
+N	O
+-	O
+terminal	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+58	I-residue_range
+that	O
+are	O
+shown	O
+to	O
+play	O
+essential	O
+roles	O
+in	O
+YfiB	B-protein
+activation	O
+and	O
+biofilm	O
+formation	O
+.	O
+
+Therefore	O
+,	O
+we	O
+are	O
+able	O
+to	O
+visualize	O
+the	O
+detailed	O
+allosteric	O
+arrangement	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+structure	O
+of	O
+YfiB	B-protein
+and	O
+its	O
+important	O
+role	O
+in	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+.	O
+
+In	O
+addition	O
+,	O
+we	O
+found	O
+that	O
+the	O
+YfiBL43P	B-mutant
+shows	O
+a	O
+much	O
+higher	O
+PG	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+than	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+,	O
+most	O
+likely	O
+due	O
+to	O
+its	O
+more	O
+compact	O
+PG	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+Moreover	O
+,	O
+we	O
+found	O
+that	O
+Vitamin	B-chemical
+B6	I-chemical
+(	O
+VB6	B-chemical
+)	O
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+can	O
+bind	O
+YfiR	B-protein
+with	O
+an	O
+affinity	B-evidence
+in	O
+the	O
+ten	O
+millimolar	O
+range	O
+.	O
+
+Together	O
+with	O
+functional	O
+data	O
+,	O
+these	O
+results	O
+provide	O
+new	O
+mechanistic	O
+insights	O
+into	O
+how	O
+activated	B-protein_state
+YfiB	B-protein
+sequesters	O
+YfiR	B-protein
+and	O
+releases	O
+the	O
+suppression	O
+of	O
+YfiN	B-protein
+.	O
+These	O
+findings	O
+may	O
+facilitate	O
+the	O
+development	O
+and	O
+optimization	O
+of	O
+anti	O
+-	O
+biofilm	O
+drugs	O
+for	O
+the	O
+treatment	O
+of	O
+chronic	O
+infections	O
+.	O
+
+Overall	O
+structure	B-evidence
+of	O
+YfiB	B-protein
+
+We	O
+obtained	O
+two	O
+crystal	B-evidence
+forms	I-evidence
+of	O
+YfiB	B-protein
+(	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+168	I-residue_range
+,	O
+lacking	B-protein_state
+the	O
+signal	B-structure_element
+peptide	I-structure_element
+from	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+26	I-residue_range
+and	O
+periplasmic	O
+residues	O
+27	B-residue_range
+–	I-residue_range
+33	I-residue_range
+),	O
+crystal	O
+forms	O
+I	O
+and	O
+II	O
+,	O
+belonging	O
+to	O
+space	O
+groups	O
+P21	O
+and	O
+P41	O
+,	O
+respectively	O
+.	O
+
+Overall	O
+structure	B-evidence
+of	O
+YfiB	B-protein
+.	O
+(	O
+A	O
+)	O
+The	O
+overall	O
+structure	B-evidence
+of	O
+the	O
+YfiB	B-protein
+monomer	B-oligomeric_state
+.	O
+(	O
+B	O
+)	O
+A	O
+topology	O
+diagram	O
+of	O
+the	O
+YfiB	B-protein
+monomer	B-oligomeric_state
+.	O
+(	O
+C	O
+and	O
+D	O
+)	O
+The	O
+analytical	B-experimental_method
+ultracentrifugation	I-experimental_method
+experiment	O
+results	O
+for	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+and	O
+YfiBL43P	B-mutant
+
+Two	O
+dimeric	B-oligomeric_state
+types	O
+of	O
+YfiB	B-protein
+dimer	B-oligomeric_state
+.	O
+(	O
+A	O
+–	O
+C	O
+)	O
+The	O
+“	O
+head	B-protein_state
+to	I-protein_state
+head	I-protein_state
+”	O
+dimer	B-oligomeric_state
+.	O
+
+The	O
+“	O
+back	B-protein_state
+to	I-protein_state
+back	I-protein_state
+”	O
+dimer	B-oligomeric_state
+.	O
+
+(	O
+A	O
+)	O
+and	O
+(	O
+E	O
+)	O
+indicate	O
+the	O
+front	O
+views	O
+of	O
+the	O
+two	O
+dimers	B-oligomeric_state
+,	O
+(	O
+B	O
+)	O
+and	O
+(	O
+F	O
+)	O
+indicate	O
+the	O
+top	O
+views	O
+of	O
+the	O
+two	O
+dimers	B-oligomeric_state
+,	O
+and	O
+(	O
+C	O
+)	O
+and	O
+(	O
+D	O
+)	O
+indicate	O
+the	O
+details	O
+of	O
+the	O
+two	O
+dimeric	B-site
+interfaces	I-site
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+YfiB	B-protein
+monomer	B-oligomeric_state
+consists	O
+of	O
+a	O
+five	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+β1	B-structure_element
+-	I-structure_element
+2	I-structure_element
+-	I-structure_element
+5	I-structure_element
+-	I-structure_element
+3	I-structure_element
+-	I-structure_element
+4	I-structure_element
+)	O
+flanked	O
+by	O
+five	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+α1	B-structure_element
+–	I-structure_element
+5	I-structure_element
+)	O
+on	O
+one	O
+side	O
+.	O
+
+In	O
+addition	O
+,	O
+there	O
+is	O
+a	O
+short	O
+helix	B-structure_element
+turn	I-structure_element
+connecting	O
+the	O
+β4	B-structure_element
+strand	I-structure_element
+and	O
+α4	B-structure_element
+helix	I-structure_element
+(	O
+Fig	O
+.	O
+1A	O
+and	O
+1B	O
+).	O
+
+Each	O
+crystal	O
+form	O
+contains	O
+three	O
+different	O
+dimeric	B-oligomeric_state
+types	O
+of	O
+YfiB	B-protein
+,	O
+two	O
+of	O
+which	O
+are	O
+present	O
+in	O
+both	O
+,	O
+suggesting	O
+that	O
+the	O
+rest	O
+of	O
+the	O
+dimeric	B-oligomeric_state
+types	O
+may	O
+result	O
+from	O
+crystal	O
+packing	O
+.	O
+
+Here	O
+,	O
+we	O
+refer	O
+to	O
+the	O
+two	O
+dimeric	B-oligomeric_state
+types	O
+as	O
+“	O
+head	B-protein_state
+to	I-protein_state
+head	I-protein_state
+”	O
+and	O
+“	O
+back	B-protein_state
+to	I-protein_state
+back	I-protein_state
+”	O
+according	O
+to	O
+the	O
+interacting	O
+mode	O
+(	O
+Fig	O
+.	O
+2A	O
+and	O
+2E	O
+),	O
+with	O
+the	O
+total	O
+buried	O
+surface	O
+areas	O
+being	O
+316	O
+.	O
+8	O
+Å2	O
+and	O
+554	O
+.	O
+3	O
+Å2	O
+,	O
+respectively	O
+.	O
+
+The	O
+“	O
+head	B-protein_state
+to	I-protein_state
+head	I-protein_state
+”	O
+dimer	B-oligomeric_state
+exhibits	O
+a	O
+clamp	B-protein_state
+shape	I-protein_state
+.	O
+
+The	O
+dimerization	O
+occurs	O
+mainly	O
+via	O
+hydrophobic	O
+interactions	O
+formed	O
+by	O
+A37	B-residue_name_number
+and	O
+I40	B-residue_name_number
+on	O
+the	O
+α1	B-structure_element
+helices	I-structure_element
+,	O
+L50	B-residue_name_number
+on	O
+the	O
+β1	B-structure_element
+strands	I-structure_element
+,	O
+and	O
+W55	B-residue_name_number
+on	O
+the	O
+β2	B-structure_element
+strands	I-structure_element
+of	O
+both	O
+molecules	O
+,	O
+making	O
+a	O
+hydrophobic	B-site
+interacting	I-site
+core	I-site
+(	O
+Fig	O
+.	O
+2A	O
+–	O
+C	O
+).	O
+
+The	O
+“	O
+back	B-protein_state
+to	I-protein_state
+back	I-protein_state
+”	O
+dimer	B-oligomeric_state
+presents	O
+a	O
+Y	B-protein_state
+shape	I-protein_state
+.	O
+
+The	O
+dimeric	O
+interaction	O
+is	O
+mainly	O
+hydrophilic	O
+,	O
+occurring	O
+among	O
+the	O
+main	O
+-	O
+chain	O
+and	O
+side	O
+-	O
+chain	O
+atoms	O
+of	O
+N68	B-residue_name_number
+,	O
+L69	B-residue_name_number
+,	O
+D70	B-residue_name_number
+and	O
+R71	B-residue_name_number
+on	O
+the	O
+α2	B-structure_element
+-	I-structure_element
+α3	I-structure_element
+loops	I-structure_element
+and	O
+R116	B-residue_name_number
+and	O
+S120	B-residue_name_number
+on	O
+the	O
+α4	B-structure_element
+helices	I-structure_element
+of	O
+both	O
+molecules	O
+,	O
+resulting	O
+in	O
+a	O
+complex	O
+hydrogen	B-site
+bond	I-site
+network	I-site
+(	O
+Fig	O
+.	O
+2D	O
+–	O
+F	O
+).	O
+
+The	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+
+Overall	O
+structure	B-evidence
+of	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+and	O
+the	O
+conserved	B-site
+surface	I-site
+in	O
+YfiR	B-protein
+.	O
+(	O
+A	O
+)	O
+The	O
+overall	O
+structure	B-evidence
+of	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+.	O
+
+The	O
+YfiBL43P	B-mutant
+molecules	O
+are	O
+shown	O
+in	O
+cyan	O
+and	O
+yellow	O
+.	O
+
+The	O
+YfiR	B-protein
+molecules	O
+are	O
+shown	O
+in	O
+green	O
+and	O
+magenta	O
+.	O
+
+Two	O
+interacting	O
+regions	O
+are	O
+highlighted	O
+by	O
+red	O
+rectangles	O
+.	O
+(	O
+B	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+apo	B-protein_state
+YfiB	B-protein
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+.	O
+
+To	O
+illustrate	O
+the	O
+differences	O
+between	O
+apo	B-protein_state
+YfiB	B-protein
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+,	O
+the	O
+apo	B-protein_state
+YfiB	B-protein
+is	O
+shown	O
+in	O
+pink	O
+,	O
+except	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+70	I-residue_range
+are	O
+shown	O
+in	O
+red	O
+,	O
+whereas	O
+the	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+is	O
+shown	O
+in	O
+cyan	O
+,	O
+except	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+70	I-residue_range
+are	O
+shown	O
+in	O
+blue	O
+.	O
+(	O
+C	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+differences	O
+between	O
+apo	B-protein_state
+YfiB	B-protein
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+.	O
+
+The	O
+residues	O
+proposed	O
+to	O
+contribute	O
+to	O
+YfiB	B-protein
+activation	O
+are	O
+illustrated	O
+in	O
+sticks	O
+.	O
+
+The	O
+key	O
+residues	O
+in	O
+apo	B-protein_state
+YfiB	B-protein
+are	O
+shown	O
+in	O
+red	O
+and	O
+those	O
+in	O
+YfiBL43P	B-mutant
+are	O
+shown	O
+in	O
+blue	O
+.	O
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+views	O
+showing	O
+interactions	O
+in	O
+regions	B-structure_element
+I	I-structure_element
+and	I-structure_element
+II	I-structure_element
+.	O
+
+YfiBL43P	B-mutant
+and	O
+YfiR	B-protein
+are	O
+shown	O
+in	O
+cyan	O
+and	O
+green	O
+,	O
+respectively	O
+.	O
+(	O
+E	O
+and	O
+F	O
+)	O
+The	O
+conserved	B-site
+surface	I-site
+in	O
+YfiR	B-protein
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	B-protein
+.	O
+(	O
+G	O
+)	O
+The	O
+residues	B-structure_element
+of	O
+YfiR	B-protein
+responsible	O
+for	O
+interacting	O
+with	O
+YfiB	B-protein
+are	O
+shown	O
+in	O
+green	O
+sticks	O
+,	O
+and	O
+the	O
+proposed	O
+YfiN	B-site
+-	I-site
+interacting	I-site
+residues	I-site
+are	O
+shown	O
+in	O
+yellow	O
+sticks	O
+.	O
+
+The	O
+red	O
+sticks	O
+,	O
+which	O
+represent	O
+the	O
+YfiB	B-site
+-	I-site
+interacting	I-site
+residues	I-site
+,	O
+are	O
+also	O
+responsible	O
+for	O
+the	O
+proposed	O
+interactions	O
+with	O
+YfiN	B-protein
+
+To	O
+gain	O
+structural	O
+insights	O
+into	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+,	O
+we	O
+co	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+YfiB	B-protein
+(	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+168	I-residue_range
+)	O
+and	O
+YfiR	B-protein
+(	O
+residues	O
+35	B-residue_range
+–	I-residue_range
+190	I-residue_range
+,	O
+lacking	B-protein_state
+the	O
+signal	B-structure_element
+peptide	I-structure_element
+),	O
+but	O
+failed	O
+to	O
+obtain	O
+the	O
+complex	O
+,	O
+in	O
+accordance	O
+with	O
+a	O
+previous	O
+report	O
+in	O
+which	O
+no	B-protein_state
+stable	I-protein_state
+complex	O
+of	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+was	O
+observed	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+It	O
+has	O
+been	O
+reported	O
+that	O
+single	B-experimental_method
+mutants	I-experimental_method
+of	I-experimental_method
+Q39	B-residue_name_number
+,	O
+L43	B-residue_name_number
+,	O
+F48	B-residue_name_number
+and	O
+W55	B-residue_name_number
+contribute	O
+to	O
+YfiB	B-protein
+activation	O
+leading	O
+to	O
+the	O
+induction	O
+of	O
+the	O
+SCV	O
+phenotype	O
+in	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+PAO1	I-species
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+It	O
+is	O
+likely	O
+that	O
+these	O
+residues	O
+may	O
+be	O
+involved	O
+in	O
+the	O
+conformational	O
+changes	O
+of	O
+YfiB	B-protein
+that	O
+are	O
+related	O
+to	O
+YfiR	B-protein
+sequestration	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+Therefore	O
+,	O
+we	O
+constructed	B-experimental_method
+two	I-experimental_method
+such	I-experimental_method
+single	I-experimental_method
+mutants	I-experimental_method
+of	O
+YfiB	B-protein
+(	O
+YfiBL43P	B-mutant
+and	O
+YfiBF48S	B-mutant
+).	O
+
+As	O
+expected	O
+,	O
+both	O
+mutants	O
+form	O
+a	O
+stable	B-protein_state
+complex	B-protein_state
+with	I-protein_state
+YfiR	B-protein
+.	O
+Finally	O
+,	O
+we	O
+crystalized	B-experimental_method
+YfiR	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+YfiBL43P	B-mutant
+mutant	B-protein_state
+and	O
+solved	O
+the	O
+structure	B-evidence
+at	O
+1	O
+.	O
+78	O
+Å	O
+resolution	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+using	O
+YfiR	B-protein
+and	O
+YfiB	B-protein
+as	O
+models	O
+.	O
+
+The	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+is	O
+a	O
+2	O
+:	O
+2	O
+heterotetramer	B-oligomeric_state
+(	O
+Fig	O
+.	O
+3A	O
+)	O
+in	O
+which	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+is	O
+clamped	O
+by	O
+two	O
+separated	O
+YfiBL43P	B-mutant
+molecules	O
+with	O
+a	O
+total	O
+buried	O
+surface	O
+area	O
+of	O
+3161	O
+.	O
+2	O
+Å2	O
+.	O
+
+The	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+in	O
+the	O
+complex	O
+is	O
+identical	O
+to	O
+the	O
+non	B-protein_state
+-	I-protein_state
+oxidized	I-protein_state
+YfiR	B-protein
+dimer	B-oligomeric_state
+alone	B-protein_state
+(	O
+Yang	O
+et	O
+al	O
+.,),	O
+with	O
+only	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+of	O
+the	O
+two	O
+disulfide	B-ptm
+bonds	I-ptm
+well	O
+formed	O
+,	O
+suggesting	O
+Cys71	B-residue_name_number
+-	O
+Cys110	B-residue_name_number
+disulfide	B-ptm
+bond	I-ptm
+formation	O
+is	O
+not	O
+essential	O
+for	O
+forming	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+.	O
+
+The	O
+N	O
+-	O
+terminal	O
+structural	O
+conformation	O
+of	O
+YfiBL43P	B-mutant
+,	O
+from	O
+the	O
+foremost	O
+N	O
+-	O
+terminus	O
+to	O
+residue	O
+D70	B-residue_name_number
+,	O
+is	O
+significantly	O
+altered	O
+compared	O
+with	O
+that	O
+of	O
+the	O
+apo	B-protein_state
+YfiB	B-protein
+.	O
+The	O
+majority	O
+of	O
+the	O
+α1	B-structure_element
+helix	I-structure_element
+(	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+43	I-residue_range
+)	O
+is	O
+invisible	O
+on	O
+the	O
+electron	B-evidence
+density	I-evidence
+map	I-evidence
+,	O
+and	O
+the	O
+α2	B-structure_element
+helix	I-structure_element
+and	O
+β1	B-structure_element
+and	O
+β2	B-structure_element
+strands	I-structure_element
+are	O
+rearranged	O
+to	O
+form	O
+a	O
+long	O
+loop	B-structure_element
+containing	O
+two	O
+short	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+turns	I-structure_element
+(	O
+Fig	O
+.	O
+3B	O
+and	O
+3C	O
+),	O
+thus	O
+embracing	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+.	O
+
+The	O
+observed	O
+changes	O
+in	O
+conformation	O
+of	O
+YfiB	B-protein
+and	O
+the	O
+results	O
+of	O
+mutagenesis	B-experimental_method
+suggest	O
+a	O
+mechanism	O
+by	O
+which	O
+YfiB	B-protein
+sequesters	O
+YfiR	B-protein
+.	O
+
+The	O
+YfiB	B-site
+-	I-site
+YfiR	I-site
+interface	I-site
+can	O
+be	O
+divided	O
+into	O
+two	O
+regions	O
+(	O
+Fig	O
+.	O
+3A	O
+and	O
+3D	O
+).	O
+
+Region	B-structure_element
+I	I-structure_element
+is	O
+formed	O
+by	O
+numerous	O
+main	O
+-	O
+chain	O
+and	O
+side	O
+-	O
+chain	O
+hydrophilic	O
+interactions	O
+between	O
+residues	O
+E45	B-residue_name_number
+,	O
+G47	B-residue_name_number
+and	O
+E53	B-residue_name_number
+from	O
+the	O
+N	O
+-	O
+terminal	O
+extended	O
+loop	B-structure_element
+of	O
+YfiB	B-protein
+and	O
+residues	O
+S57	B-residue_name_number
+,	O
+R60	B-residue_name_number
+,	O
+A89	B-residue_name_number
+and	O
+H177	B-residue_name_number
+from	O
+YfiR	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+i	O
+)).	O
+
+Additionally	O
+,	O
+three	O
+hydrophobic	B-site
+anchoring	I-site
+sites	I-site
+exist	O
+in	O
+region	B-structure_element
+I	I-structure_element
+.	O
+The	O
+residues	O
+F48	B-residue_name_number
+and	O
+W55	B-residue_name_number
+of	O
+YfiB	B-protein
+are	O
+inserted	O
+into	O
+the	O
+hydrophobic	B-site
+cores	I-site
+mainly	O
+formed	O
+by	O
+the	O
+main	O
+chain	O
+and	O
+side	O
+chain	O
+carbon	O
+atoms	O
+of	O
+residues	O
+S57	B-residue_name_number
+/	O
+Q88	B-residue_name_number
+/	O
+A89	B-residue_name_number
+/	O
+N90	B-residue_name_number
+and	O
+R60	B-residue_name_number
+/	O
+R175	B-residue_name_number
+/	O
+H177	B-residue_name_number
+of	O
+YfiR	B-protein
+,	O
+respectively	O
+;	O
+and	O
+F57	B-residue_name_number
+of	O
+YfiB	B-protein
+is	O
+inserted	O
+into	O
+the	O
+hydrophobic	B-site
+pocket	I-site
+formed	O
+by	O
+L166	B-residue_name_number
+/	O
+I169	B-residue_name_number
+/	O
+V176	B-residue_name_number
+/	O
+P178	B-residue_name_number
+/	O
+L181	B-residue_name_number
+of	O
+YfiR	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+ii	O
+)).	O
+
+In	O
+region	B-structure_element
+II	I-structure_element
+,	O
+the	O
+side	O
+chains	O
+of	O
+R96	B-residue_name_number
+,	O
+E98	B-residue_name_number
+and	O
+E157	B-residue_name_number
+from	O
+YfiB	B-protein
+interact	O
+with	O
+the	O
+side	O
+chains	O
+of	O
+E163	B-residue_name_number
+,	O
+S146	B-residue_name_number
+and	O
+R171	B-residue_name_number
+from	O
+YfiR	B-protein
+,	O
+respectively	O
+.	O
+
+Additionally	O
+,	O
+the	O
+main	O
+chains	O
+of	O
+I163	B-residue_name_number
+and	O
+V165	B-residue_name_number
+from	O
+YfiB	B-protein
+form	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+main	O
+chains	O
+of	O
+L166	B-residue_name_number
+and	O
+A164	B-residue_name_number
+from	O
+YfiR	B-protein
+,	O
+respectively	O
+,	O
+and	O
+the	O
+main	O
+chain	O
+of	O
+P166	B-residue_name_number
+from	O
+YfiB	B-protein
+interacts	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+R185	B-residue_name_number
+from	O
+YfiR	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+II	O
+).	O
+
+These	O
+two	O
+regions	O
+contribute	O
+a	O
+robust	O
+hydrogen	B-site
+-	I-site
+bonding	I-site
+network	I-site
+to	O
+the	O
+YfiB	B-site
+-	I-site
+YfiR	I-site
+interface	I-site
+,	O
+resulting	O
+in	O
+a	O
+tightly	O
+bound	O
+complex	O
+.	O
+
+Based	O
+on	O
+the	O
+observations	O
+that	O
+two	O
+separated	O
+YfiBL43P	B-mutant
+molecules	O
+form	O
+a	O
+2	O
+:	O
+2	O
+complex	O
+structure	B-evidence
+with	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+,	O
+we	O
+performed	O
+an	O
+analytical	B-experimental_method
+ultracentrifugation	I-experimental_method
+experiment	O
+to	O
+check	O
+the	O
+oligomeric	O
+states	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+and	O
+YfiBL43P	B-mutant
+.	O
+
+The	O
+results	O
+showed	O
+that	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+exists	O
+in	O
+both	O
+monomeric	B-oligomeric_state
+and	O
+dimeric	B-oligomeric_state
+states	O
+in	O
+solution	O
+,	O
+while	O
+YfiBL43P	B-mutant
+primarily	O
+adopts	O
+the	O
+monomer	B-oligomeric_state
+state	O
+in	O
+solution	O
+(	O
+Fig	O
+.	O
+1C	O
+–	O
+D	O
+).	O
+
+This	O
+suggests	O
+that	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+YfiB	B-protein
+plays	O
+an	O
+important	O
+role	O
+in	O
+forming	O
+the	O
+dimeric	B-oligomeric_state
+YfiB	B-protein
+in	O
+solution	O
+and	O
+that	O
+the	O
+conformational	O
+change	O
+of	O
+residue	O
+L43	B-residue_name_number
+is	O
+associated	O
+with	O
+the	O
+stretch	O
+of	O
+the	O
+N	O
+-	O
+terminus	O
+and	O
+opening	O
+of	O
+the	O
+dimer	B-oligomeric_state
+.	O
+
+Therefore	O
+,	O
+it	O
+is	O
+possible	O
+that	O
+both	O
+dimeric	B-oligomeric_state
+types	O
+might	O
+exist	O
+in	O
+solution	O
+.	O
+
+For	O
+simplicity	O
+,	O
+we	O
+only	O
+discuss	O
+the	O
+“	O
+head	B-protein_state
+to	I-protein_state
+head	I-protein_state
+”	O
+dimer	B-oligomeric_state
+in	O
+the	O
+following	O
+text	O
+.	O
+
+The	O
+PG	B-site
+-	I-site
+binding	I-site
+site	I-site
+of	O
+YfiB	B-protein
+
+The	O
+PG	B-site
+-	I-site
+binding	I-site
+site	I-site
+in	O
+YfiB	B-protein
+.	O
+(	O
+A	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+PG	B-site
+-	I-site
+binding	I-site
+sites	I-site
+of	O
+the	O
+H	B-species
+.	I-species
+influenzae	I-species
+Pal	B-complex_assembly
+/	I-complex_assembly
+PG	I-complex_assembly
+-	I-complex_assembly
+P	I-complex_assembly
+complex	O
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+complexed	B-protein_state
+with	I-protein_state
+sulfate	B-chemical
+ions	O
+.	O
+
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+showing	O
+the	O
+key	O
+residues	O
+of	O
+Pal	B-protein_type
+interacting	O
+with	O
+the	O
+m	B-chemical
+-	I-chemical
+Dap5	I-chemical
+ε	I-chemical
+-	I-chemical
+carboxylate	I-chemical
+group	O
+of	O
+PG	B-chemical
+-	I-chemical
+P	I-chemical
+.	O
+Pal	B-protein_type
+is	O
+shown	O
+in	O
+wheat	O
+and	O
+PG	B-chemical
+-	I-chemical
+P	I-chemical
+is	O
+in	O
+magenta	O
+.	O
+
+(	O
+C	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+showing	O
+the	O
+key	O
+residues	O
+of	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+interacting	O
+with	O
+a	O
+sulfate	B-chemical
+ion	O
+.	O
+
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+is	O
+shown	O
+in	O
+cyan	O
+;	O
+the	O
+sulfate	B-chemical
+ion	O
+,	O
+in	O
+green	O
+;	O
+and	O
+the	O
+water	B-chemical
+molecule	O
+,	O
+in	O
+yellow	O
+.	O
+(	O
+D	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+PG	B-site
+-	I-site
+binding	I-site
+sites	I-site
+of	O
+apo	B-protein_state
+YfiB	B-protein
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+,	O
+the	O
+key	O
+residues	O
+are	O
+shown	O
+in	O
+stick	O
+.	O
+
+Apo	B-protein_state
+YfiB	B-protein
+is	O
+shown	O
+in	O
+yellow	O
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+in	O
+cyan	O
+.	O
+(	O
+E	O
+and	O
+F	O
+)	O
+MST	B-experimental_method
+data	O
+and	O
+analysis	O
+for	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+(	O
+E	O
+)	O
+YfiB	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+(	O
+F	O
+)	O
+YfiBL43P	B-mutant
+with	O
+PG	B-chemical
+.	O
+(	O
+G	O
+)	O
+The	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+and	O
+E	B-species
+.	I-species
+coli	I-species
+sources	O
+of	O
+YfiB	B-protein
+,	O
+Pal	B-protein_type
+and	O
+the	O
+periplasmic	B-structure_element
+domain	I-structure_element
+of	O
+OmpA	B-protein_type
+
+PG	B-protein_type
+-	I-protein_type
+associated	I-protein_type
+lipoprotein	I-protein_type
+(	O
+Pal	B-protein_type
+)	O
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+Gram	B-taxonomy_domain
+-	I-taxonomy_domain
+negative	I-taxonomy_domain
+bacteria	I-taxonomy_domain
+and	O
+anchors	O
+to	O
+the	O
+outer	O
+membrane	O
+through	O
+an	O
+N	O
+-	O
+terminal	O
+lipid	O
+attachment	O
+and	O
+to	O
+PG	O
+layer	O
+through	O
+its	O
+periplasmic	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+is	O
+implicated	O
+in	O
+maintaining	O
+outer	O
+membrane	O
+integrity	O
+.	O
+
+Previous	O
+homology	B-experimental_method
+modeling	I-experimental_method
+studies	O
+suggested	O
+that	O
+YfiB	B-protein
+contains	O
+a	O
+Pal	B-site
+-	I-site
+like	I-site
+PG	I-site
+-	I-site
+binding	I-site
+site	I-site
+(	O
+Parsons	O
+et	O
+al	O
+.,),	O
+and	O
+the	O
+mutation	B-experimental_method
+of	I-experimental_method
+two	I-experimental_method
+residues	I-experimental_method
+at	O
+this	O
+site	O
+,	O
+D102	B-residue_name_number
+and	O
+G105	B-residue_name_number
+,	O
+reduces	O
+the	O
+ability	O
+for	O
+biofilm	O
+formation	O
+and	O
+surface	O
+attachment	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+In	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+,	O
+one	O
+sulfate	B-chemical
+ion	O
+is	O
+found	O
+at	O
+the	O
+bottom	O
+of	O
+each	O
+YfiBL43P	B-mutant
+molecule	O
+(	O
+Fig	O
+.	O
+3A	O
+)	O
+and	O
+forms	O
+a	O
+strong	O
+hydrogen	O
+bond	O
+with	O
+D102	B-residue_name_number
+of	O
+YfiBL43P	B-mutant
+(	O
+Fig	O
+.	O
+4A	O
+and	O
+4C	O
+).	O
+
+Structural	B-experimental_method
+superposition	I-experimental_method
+between	O
+YfiBL43P	B-mutant
+and	O
+Haemophilus	B-species
+influenzae	I-species
+Pal	B-protein_type
+complexed	B-protein_state
+with	I-protein_state
+biosynthetic	O
+peptidoglycan	B-chemical
+precursor	I-chemical
+(	O
+PG	B-chemical
+-	I-chemical
+P	I-chemical
+),	O
+UDP	B-chemical
+-	I-chemical
+N	I-chemical
+-	I-chemical
+acetylmuramyl	I-chemical
+-	I-chemical
+L	I-chemical
+-	I-chemical
+Ala	I-chemical
+-	I-chemical
+α	I-chemical
+-	I-chemical
+D	I-chemical
+-	I-chemical
+Glu	I-chemical
+-	I-chemical
+m	I-chemical
+-	I-chemical
+Dap	I-chemical
+-	I-chemical
+D	I-chemical
+-	I-chemical
+Ala	I-chemical
+-	I-chemical
+D	I-chemical
+-	I-chemical
+Ala	I-chemical
+(	O
+m	B-chemical
+-	I-chemical
+Dap	I-chemical
+is	O
+meso	B-chemical
+-	I-chemical
+diaminopimelate	I-chemical
+)	O
+(	O
+PDB	O
+code	O
+:	O
+2aiz	O
+)	O
+(	O
+Parsons	O
+et	O
+al	O
+.,),	O
+revealed	O
+that	O
+the	O
+sulfate	B-chemical
+ion	O
+is	O
+located	O
+at	O
+the	O
+position	O
+of	O
+the	O
+m	B-chemical
+-	I-chemical
+Dap5	I-chemical
+ϵ	I-chemical
+-	I-chemical
+carboxylate	I-chemical
+group	O
+in	O
+the	O
+Pal	B-complex_assembly
+/	I-complex_assembly
+PG	I-complex_assembly
+-	I-complex_assembly
+P	I-complex_assembly
+complex	O
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+
+In	O
+the	O
+Pal	B-complex_assembly
+/	I-complex_assembly
+PG	I-complex_assembly
+-	I-complex_assembly
+P	I-complex_assembly
+complex	O
+structure	B-evidence
+,	O
+the	O
+m	B-chemical
+-	I-chemical
+Dap5	I-chemical
+ϵ	I-chemical
+-	I-chemical
+carboxylate	I-chemical
+group	O
+interacts	O
+with	O
+the	O
+side	O
+-	O
+chain	O
+atoms	O
+of	O
+D71	B-residue_name_number
+and	O
+the	O
+main	O
+-	O
+chain	O
+amide	O
+of	O
+D37	B-residue_name_number
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+
+Similarly	O
+,	O
+in	O
+the	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+structure	B-evidence
+,	O
+the	O
+sulfate	B-chemical
+ion	O
+interacts	O
+with	O
+the	O
+side	O
+-	O
+chain	O
+atoms	O
+of	O
+D102	B-residue_name_number
+(	O
+corresponding	O
+to	O
+D71	B-residue_name_number
+in	O
+Pal	B-protein_type
+)	O
+and	O
+R117	B-residue_name_number
+(	O
+corresponding	O
+to	O
+R86	B-residue_name_number
+in	O
+Pal	B-protein_type
+)	O
+and	O
+the	O
+main	O
+-	O
+chain	O
+amide	O
+of	O
+N68	B-residue_name_number
+(	O
+corresponding	O
+to	O
+D37	B-residue_name_number
+in	O
+Pal	B-protein_type
+).	O
+
+Moreover	O
+,	O
+a	O
+water	B-chemical
+molecule	O
+was	O
+found	O
+to	O
+bridge	O
+the	O
+sulfate	B-chemical
+ion	O
+and	O
+the	O
+side	O
+chains	O
+of	O
+N67	B-residue_name_number
+and	O
+D102	B-residue_name_number
+,	O
+strengthening	O
+the	O
+hydrogen	B-site
+bond	I-site
+network	I-site
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+
+In	O
+addition	O
+,	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+YfiB	B-protein
+with	O
+Pal	B-protein_type
+and	O
+the	O
+periplasmic	B-structure_element
+domain	I-structure_element
+of	O
+OmpA	B-protein_type
+(	O
+proteins	O
+containing	O
+PG	B-site
+-	I-site
+binding	I-site
+site	I-site
+)	O
+showed	O
+that	O
+N68	B-residue_name_number
+and	O
+D102	B-residue_name_number
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+(	O
+Fig	O
+.	O
+4G	O
+,	O
+blue	O
+stars	O
+),	O
+suggesting	O
+that	O
+these	O
+residues	O
+contribute	O
+to	O
+the	O
+PG	O
+-	O
+binding	O
+ability	O
+of	O
+YfiB	B-protein
+.	O
+
+Interestingly	O
+,	O
+superposition	B-experimental_method
+of	O
+apo	B-protein_state
+YfiB	B-protein
+with	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+revealed	O
+that	O
+the	O
+PG	B-site
+-	I-site
+binding	I-site
+region	I-site
+is	O
+largely	O
+altered	O
+mainly	O
+due	O
+to	O
+different	B-protein_state
+conformation	I-protein_state
+of	O
+the	O
+N68	B-residue_name_number
+containing	O
+loop	B-structure_element
+.	O
+
+Compared	O
+to	O
+YfiBL43P	B-mutant
+,	O
+the	O
+N68	B-residue_name_number
+-	O
+containing	O
+loop	B-structure_element
+of	O
+the	O
+apo	B-protein_state
+YfiB	B-protein
+flips	O
+away	O
+about	O
+7	O
+Å	O
+,	O
+and	O
+D102	B-residue_name_number
+and	O
+R117	B-residue_name_number
+swing	O
+slightly	O
+outward	O
+;	O
+thus	O
+,	O
+the	O
+PG	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+is	O
+enlarged	O
+with	O
+no	O
+sulfate	B-chemical
+ion	O
+or	O
+water	B-chemical
+bound	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+
+Therefore	O
+,	O
+we	O
+proposed	O
+that	O
+the	O
+PG	B-chemical
+-	O
+binding	O
+ability	O
+of	O
+inactive	B-protein_state
+YfiB	B-protein
+might	O
+be	O
+weaker	O
+than	O
+that	O
+of	O
+active	B-protein_state
+YfiB	B-protein
+.	O
+To	O
+validate	O
+this	O
+,	O
+we	O
+performed	O
+a	O
+microscale	B-experimental_method
+thermophoresis	I-experimental_method
+(	O
+MST	B-experimental_method
+)	O
+assay	O
+to	O
+measure	O
+the	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+PG	B-chemical
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+and	O
+YfiBL43P	B-mutant
+,	O
+respectively	O
+.	O
+
+The	O
+results	O
+indicated	O
+that	O
+the	O
+PG	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+YfiBL43P	B-mutant
+is	O
+65	O
+.	O
+5	O
+μmol	O
+/	O
+L	O
+,	O
+which	O
+is	O
+about	O
+16	O
+-	O
+fold	O
+stronger	O
+than	O
+that	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+(	O
+Kd	O
+=	O
+1	O
+.	O
+1	O
+mmol	O
+/	O
+L	O
+)	O
+(	O
+Fig	O
+.	O
+4E	O
+–	O
+F	O
+).	O
+
+As	O
+the	O
+experiment	O
+is	O
+performed	O
+in	B-protein_state
+the	I-protein_state
+absence	I-protein_state
+of	I-protein_state
+YfiR	B-protein
+,	O
+it	O
+suggests	O
+that	O
+an	O
+increase	O
+in	O
+the	O
+PG	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+YfiB	B-protein
+is	O
+not	O
+a	O
+result	O
+of	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+and	O
+is	O
+highly	O
+coupled	O
+to	O
+the	O
+activation	O
+of	O
+YfiB	B-protein
+characterized	O
+by	O
+a	O
+stretched	B-protein_state
+N	I-protein_state
+-	I-protein_state
+terminal	I-protein_state
+conformation	I-protein_state
+.	O
+
+The	O
+conserved	B-site
+surface	I-site
+in	O
+YfiR	B-protein
+is	O
+functional	O
+for	O
+binding	O
+YfiB	B-protein
+and	O
+YfiN	B-protein
+
+Calculation	O
+using	O
+the	O
+ConSurf	B-experimental_method
+Server	I-experimental_method
+(	O
+http	O
+://	O
+consurf	O
+.	O
+tau	O
+.	O
+ac	O
+.	O
+il	O
+/),	O
+which	O
+estimates	O
+the	O
+evolutionary	B-evidence
+conservation	I-evidence
+of	O
+amino	O
+acid	O
+positions	O
+and	O
+visualizes	O
+information	O
+on	O
+the	O
+structure	B-site
+surface	I-site
+,	O
+revealed	O
+a	O
+conserved	B-site
+surface	I-site
+on	O
+YfiR	B-protein
+that	O
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	B-protein
+(	O
+Fig	O
+.	O
+3E	O
+and	O
+3F	O
+).	O
+
+Interestingly	O
+,	O
+the	O
+majority	O
+of	O
+this	O
+conserved	B-site
+surface	I-site
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	B-protein
+(	O
+Fig	O
+.	O
+3E	O
+and	O
+3F	O
+).	O
+
+Malone	O
+JG	O
+et	O
+al	O
+.	O
+have	O
+reported	O
+that	O
+F151	B-residue_name_number
+,	O
+E163	B-residue_name_number
+,	O
+I169	B-residue_name_number
+and	O
+Q187	B-residue_name_number
+,	O
+located	O
+near	O
+the	O
+C	O
+-	O
+terminus	O
+of	O
+YfiR	B-protein
+,	O
+comprise	O
+a	O
+putative	O
+YfiN	B-site
+binding	I-site
+site	I-site
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+Interestingly	O
+,	O
+these	O
+residues	O
+are	O
+part	O
+of	O
+the	O
+conserved	B-site
+surface	I-site
+of	O
+YfiR	B-protein
+(	O
+Fig	O
+.	O
+3G	O
+).	O
+
+F151	B-residue_name_number
+,	O
+E163	B-residue_name_number
+and	O
+I169	B-residue_name_number
+form	O
+a	O
+hydrophobic	B-site
+core	I-site
+while	O
+,	O
+Q187	B-residue_name_number
+is	O
+located	O
+at	O
+the	O
+end	O
+of	O
+the	O
+α6	B-structure_element
+helix	I-structure_element
+.	O
+
+E163	B-residue_name_number
+and	O
+I169	B-residue_name_number
+are	O
+YfiB	B-site
+-	I-site
+interacting	I-site
+residues	I-site
+of	O
+YfiR	B-protein
+,	O
+in	O
+which	O
+E163	B-residue_name_number
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+R96	B-residue_name_number
+of	O
+YfiB	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+II	O
+)	O
+and	O
+I169	B-residue_name_number
+is	O
+involved	O
+in	O
+forming	O
+the	O
+L166	B-residue_name_number
+/	O
+I169	B-residue_name_number
+/	O
+V176	B-residue_name_number
+/	O
+P178	B-residue_name_number
+/	O
+L181	B-residue_name_number
+hydrophobic	B-site
+core	I-site
+for	O
+anchoring	O
+F57	B-residue_name_number
+of	O
+YfiB	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+ii	O
+)).	O
+
+Collectively	O
+,	O
+a	O
+part	O
+of	O
+the	O
+YfiB	B-site
+-	I-site
+YfiR	I-site
+interface	I-site
+overlaps	O
+with	O
+the	O
+proposed	O
+YfiR	B-site
+-	I-site
+YfiN	I-site
+interface	I-site
+,	O
+suggesting	O
+alteration	O
+in	O
+the	O
+association	O
+-	O
+disassociation	O
+equilibrium	O
+of	O
+YfiR	B-protein
+-	O
+YfiN	B-protein
+and	O
+hence	O
+the	O
+ability	O
+of	O
+YfiB	B-protein
+to	O
+sequester	O
+YfiR	B-protein
+.	O
+
+YfiR	B-protein
+binds	O
+small	O
+molecules	O
+
+Previous	O
+studies	O
+indicated	O
+that	O
+YfiR	B-protein
+constitutes	O
+a	O
+YfiB	B-protein
+-	O
+independent	O
+sensing	O
+device	O
+that	O
+can	O
+activate	O
+YfiN	B-protein
+in	O
+response	O
+to	O
+the	O
+redox	O
+status	O
+of	O
+the	O
+periplasm	O
+,	O
+and	O
+we	O
+have	O
+reported	O
+YfiR	B-protein
+structures	B-evidence
+in	O
+both	O
+the	O
+non	B-protein_state
+-	I-protein_state
+oxidized	I-protein_state
+and	O
+the	O
+oxidized	B-protein_state
+states	O
+earlier	O
+,	O
+revealing	O
+that	O
+the	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+disulfide	B-ptm
+bond	I-ptm
+plays	O
+an	O
+essential	O
+role	O
+in	O
+maintaining	O
+the	O
+correct	O
+folding	O
+of	O
+YfiR	B-protein
+(	O
+Yang	O
+et	O
+al	O
+.,).	O
+
+However	O
+,	O
+whether	O
+YfiR	B-protein
+is	O
+involved	O
+in	O
+other	O
+regulatory	O
+mechanisms	O
+is	O
+still	O
+an	O
+open	O
+question	O
+.	O
+
+Overall	O
+Structures	B-evidence
+of	O
+VB6	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+and	O
+Trp	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiR	B-protein
+.	O
+(	O
+A	O
+)	O
+Superposition	B-experimental_method
+of	O
+the	O
+overall	O
+structures	B-evidence
+of	O
+VB6	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+and	O
+Trp	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiR	B-protein
+.	O
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+views	O
+showing	O
+the	O
+key	O
+residues	O
+of	O
+YfiR	B-protein
+that	O
+bind	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+.	O
+
+The	O
+electron	B-evidence
+densities	I-evidence
+of	O
+VB6	B-chemical
+and	O
+Trp	B-chemical
+are	O
+countered	O
+at	O
+3	O
+.	O
+0σ	O
+and	O
+2	O
+.	O
+3σ	O
+,	O
+respectively	O
+,	O
+in	O
+|	B-evidence
+Fo	I-evidence
+|-|	I-evidence
+Fc	I-evidence
+|	I-evidence
+maps	I-evidence
+.	O
+(	O
+C	O
+)	O
+Superposition	B-experimental_method
+of	O
+the	O
+hydrophobic	B-site
+pocket	I-site
+of	O
+YfiR	B-protein
+with	O
+VB6	B-chemical
+,	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+and	O
+F57	B-residue_name_number
+of	O
+YfiB	B-protein
+
+Intriguingly	O
+,	O
+a	O
+Dali	B-experimental_method
+search	I-experimental_method
+(	O
+Holm	O
+and	O
+Rosenstrom	O
+,)	O
+indicated	O
+that	O
+the	O
+closest	O
+homologs	O
+of	O
+YfiR	B-protein
+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	B-chemical
+-	I-chemical
+Trp	I-chemical
+,	O
+L	B-chemical
+-	I-chemical
+Phe	I-chemical
+,	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	B-protein
+can	O
+recognize	O
+these	O
+molecules	O
+.	O
+
+For	O
+this	O
+purpose	O
+,	O
+we	O
+co	B-experimental_method
+-	I-experimental_method
+crystallized	I-experimental_method
+YfiR	B-protein
+or	O
+soaked	B-experimental_method
+YfiR	B-protein
+crystals	B-evidence
+with	O
+different	O
+small	O
+molecules	O
+,	O
+including	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+and	O
+B	O
+-	O
+group	O
+vitamins	O
+.	O
+
+Fortunately	O
+,	O
+we	O
+found	O
+obvious	O
+small	B-evidence
+-	I-evidence
+molecule	I-evidence
+density	I-evidence
+in	O
+the	O
+VB6	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+and	O
+Trp	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiR	B-protein
+crystal	B-evidence
+structures	I-evidence
+(	O
+Fig	O
+.	O
+5A	O
+and	O
+5B	O
+),	O
+and	O
+in	O
+both	O
+structures	B-evidence
+,	O
+the	O
+YfiR	B-protein
+dimers	B-oligomeric_state
+resemble	O
+the	O
+oxidized	B-protein_state
+YfiR	B-protein
+structure	B-evidence
+in	O
+which	O
+both	O
+two	O
+disulfide	B-ptm
+bonds	I-ptm
+are	O
+well	O
+formed	O
+(	O
+Yang	O
+et	O
+al	O
+.,).	O
+
+Functional	O
+analysis	O
+of	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+.	O
+(	O
+A	O
+and	O
+B	O
+)	O
+The	O
+effect	B-experimental_method
+of	I-experimental_method
+increasing	I-experimental_method
+concentrations	I-experimental_method
+of	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+on	O
+YfiBL43P	B-mutant
+-	O
+induced	O
+attachment	O
+(	O
+bars	O
+).	O
+
+The	O
+relative	B-evidence
+optical	I-evidence
+density	I-evidence
+is	O
+represented	O
+as	O
+curves	O
+.	O
+
+Wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+is	O
+used	O
+as	O
+negative	O
+control	O
+.	O
+
+(	O
+C	O
+and	O
+D	O
+)	O
+BIAcore	B-experimental_method
+data	O
+and	O
+analysis	O
+for	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+(	O
+C	O
+)	O
+VB6	B-chemical
+and	O
+(	O
+D	O
+)	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+with	O
+YfiR	B-protein
+.	O
+(	O
+E	O
+–	O
+G	O
+)	O
+ITC	B-experimental_method
+data	O
+and	O
+analysis	O
+for	O
+titration	B-experimental_method
+of	O
+(	O
+E	O
+)	O
+YfiB	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+,	O
+(	O
+F	O
+)	O
+YfiBL43P	O
+,	O
+and	O
+(	O
+G	O
+)	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+into	O
+YfiR	B-protein
+
+Structural	B-experimental_method
+analyses	I-experimental_method
+revealed	O
+that	O
+the	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+molecules	O
+are	O
+bound	B-protein_state
+at	I-protein_state
+the	O
+periphery	O
+of	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+,	O
+but	O
+not	O
+at	O
+the	O
+dimer	B-site
+interface	I-site
+.	O
+
+Interestingly	O
+,	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+were	O
+found	O
+to	O
+occupy	O
+the	O
+same	O
+hydrophobic	B-site
+pocket	I-site
+,	O
+formed	O
+by	O
+L166	B-residue_name_number
+/	O
+I169	B-residue_name_number
+/	O
+V176	B-residue_name_number
+/	O
+P178	B-residue_name_number
+/	O
+L181	B-residue_name_number
+of	O
+YfiR	B-protein
+,	O
+which	O
+is	O
+also	O
+a	O
+binding	B-site
+pocket	I-site
+for	O
+F57	B-residue_name_number
+of	O
+YfiB	B-protein
+,	O
+as	O
+observed	O
+in	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+(	O
+Fig	O
+.	O
+5C	O
+).	O
+
+To	O
+evaluate	O
+the	O
+importance	O
+of	O
+F57	B-residue_name_number
+in	O
+YfiBL43P	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+,	O
+the	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+YfiBL43P	B-mutant
+and	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+for	O
+YfiR	B-protein
+were	O
+measured	O
+by	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+ITC	B-experimental_method
+).	O
+
+The	O
+results	O
+showed	O
+Kd	B-evidence
+values	O
+of	O
+1	O
+.	O
+4	O
+×	O
+10	O
+−	O
+7	O
+mol	O
+/	O
+L	O
+and	O
+5	O
+.	O
+3	O
+×	O
+10	O
+−	O
+7	O
+mol	O
+/	O
+L	O
+for	O
+YfiBL43P	B-mutant
+and	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+,	O
+respectively	O
+,	O
+revealing	O
+that	O
+the	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+mutant	B-protein_state
+caused	O
+a	O
+3	O
+.	O
+8	O
+-	O
+fold	O
+reduction	O
+in	O
+the	O
+binding	B-evidence
+affinity	I-evidence
+compared	O
+with	O
+the	O
+YfiBL43P	B-mutant
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+6F	O
+and	O
+6G	O
+).	O
+
+In	O
+parallel	O
+,	O
+to	O
+better	O
+understand	O
+the	O
+putative	O
+functional	O
+role	O
+of	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+,	O
+yfiB	B-gene
+was	O
+deleted	B-experimental_method
+in	O
+a	O
+PAO1	B-species
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+strain	O
+,	O
+and	O
+a	O
+construct	B-experimental_method
+expressing	I-experimental_method
+the	O
+YfiBL43P	B-mutant
+mutant	B-protein_state
+was	O
+transformed	B-experimental_method
+into	I-experimental_method
+the	O
+PAO1	B-species
+ΔyfiB	B-mutant
+strain	O
+to	O
+trigger	O
+YfiBL43P	B-mutant
+-	O
+induced	O
+biofilm	O
+formation	O
+.	O
+
+Growth	B-experimental_method
+and	I-experimental_method
+surface	I-experimental_method
+attachment	I-experimental_method
+assays	I-experimental_method
+were	O
+carried	O
+out	O
+for	O
+the	O
+yfiB	B-mutant
+-	I-mutant
+L43P	I-mutant
+strain	O
+in	O
+the	O
+presence	O
+of	O
+increasing	B-experimental_method
+concentrations	I-experimental_method
+of	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+.	O
+
+As	O
+shown	O
+in	O
+Fig	O
+.	O
+6A	O
+and	O
+6B	O
+,	O
+the	O
+over	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+YfiBL43P	B-mutant
+induced	O
+strong	O
+surface	O
+attachment	O
+and	O
+much	O
+slower	O
+growth	O
+of	O
+the	O
+yfiB	B-mutant
+-	I-mutant
+L43P	I-mutant
+strain	O
+,	O
+and	O
+as	O
+expected	O
+,	O
+a	O
+certain	O
+amount	O
+of	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+(	O
+4	O
+–	O
+6	O
+mmol	O
+/	O
+L	O
+for	O
+VB6	B-chemical
+and	O
+6	O
+–	O
+10	O
+mmol	O
+/	O
+L	O
+for	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+)	O
+could	O
+reduce	O
+the	O
+surface	O
+attachment	O
+.	O
+
+Interestingly	O
+,	O
+at	O
+a	O
+concentration	O
+higher	O
+than	O
+8	O
+mmol	O
+/	O
+L	O
+,	O
+VB6	B-chemical
+lost	O
+its	O
+ability	O
+to	O
+inhibit	O
+biofilm	O
+formation	O
+,	O
+implying	O
+that	O
+the	O
+VB6	B-chemical
+-	O
+involving	O
+regulatory	O
+mechanism	O
+is	O
+highly	O
+complicated	O
+and	O
+remains	O
+to	O
+be	O
+further	O
+investigated	O
+.	O
+
+Of	O
+note	O
+,	O
+both	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+have	O
+been	O
+reported	O
+to	O
+correlate	O
+with	O
+biofilm	O
+formation	O
+in	O
+certain	O
+Gram	B-taxonomy_domain
+-	I-taxonomy_domain
+negative	I-taxonomy_domain
+bacteria	I-taxonomy_domain
+(	O
+Grubman	O
+et	O
+al	O
+.,;	O
+Shimazaki	O
+et	O
+al	O
+.,).	O
+
+In	O
+Helicobacter	B-species
+pylori	I-species
+in	O
+particular	O
+,	O
+VB6	B-chemical
+biosynthetic	O
+enzymes	O
+act	O
+as	O
+novel	O
+virulence	O
+factors	O
+,	O
+and	O
+VB6	B-chemical
+is	O
+required	O
+for	O
+full	O
+motility	O
+and	O
+virulence	O
+(	O
+Grubman	O
+et	O
+al	O
+.,).	O
+
+In	O
+E	B-species
+.	I-species
+coli	I-species
+,	O
+mutants	O
+with	O
+decreased	O
+tryptophan	B-chemical
+synthesis	O
+show	O
+greater	O
+biofilm	O
+formation	O
+,	O
+and	O
+matured	O
+biofilm	O
+is	O
+degraded	O
+by	O
+L	B-chemical
+-	I-chemical
+tryptophan	I-chemical
+addition	O
+(	O
+Shimazaki	O
+et	O
+al	O
+.,).	O
+
+To	O
+answer	O
+the	O
+question	O
+whether	O
+competition	O
+of	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+for	O
+the	O
+YfiB	B-protein
+F57	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+of	O
+YfiR	B-protein
+plays	O
+an	O
+essential	O
+role	O
+in	O
+inhibiting	O
+biofilm	O
+formation	O
+,	O
+we	O
+measured	O
+the	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+for	O
+YfiR	B-protein
+via	O
+BIAcore	B-experimental_method
+experiments	O
+.	O
+
+The	O
+results	O
+showed	O
+relatively	O
+weak	O
+Kd	B-evidence
+values	O
+of	O
+35	O
+.	O
+2	O
+mmol	O
+/	O
+L	O
+and	O
+76	O
+.	O
+9	O
+mmol	O
+/	O
+L	O
+for	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+,	O
+respectively	O
+(	O
+Fig	O
+.	O
+6C	O
+and	O
+6D	O
+).	O
+
+Based	O
+on	O
+our	O
+results	O
+,	O
+we	O
+concluded	O
+that	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+can	O
+bind	O
+to	O
+YfiR	B-protein
+,	O
+however	O
+,	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+alone	B-protein_state
+may	O
+have	O
+little	O
+effects	O
+in	O
+interrupting	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+,	O
+the	O
+mechanism	O
+by	O
+which	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+inhibits	O
+biofilm	O
+formation	O
+remains	O
+unclear	O
+and	O
+requires	O
+further	O
+investigation	O
+.	O
+
+Previous	O
+studies	O
+suggested	O
+that	O
+in	O
+response	O
+to	O
+cell	O
+stress	O
+,	O
+YfiB	B-protein
+in	O
+the	O
+outer	O
+membrane	O
+sequesters	O
+the	O
+periplasmic	O
+protein	O
+YfiR	B-protein
+,	O
+releasing	O
+its	O
+inhibition	O
+of	O
+YfiN	B-protein
+on	O
+the	O
+inner	O
+membrane	O
+and	O
+thus	O
+inducing	O
+the	O
+diguanylate	O
+cyclase	O
+activity	O
+of	O
+YfiN	B-protein
+to	O
+allow	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+production	O
+(	O
+Giardina	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+YfiB	B-protein
+alone	B-protein_state
+and	O
+an	O
+active	B-protein_state
+mutant	B-protein_state
+YfiBL43P	B-mutant
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+YfiR	B-protein
+,	O
+indicating	O
+that	O
+YfiR	B-protein
+forms	O
+a	O
+2	O
+:	O
+2	O
+complex	B-protein_state
+with	I-protein_state
+YfiB	B-protein
+via	O
+a	O
+region	O
+composed	O
+of	O
+conserved	O
+residues	O
+.	O
+
+Our	O
+structural	B-experimental_method
+data	I-experimental_method
+analysis	I-experimental_method
+shows	O
+that	O
+the	O
+activated	B-protein_state
+YfiB	B-protein
+has	O
+an	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+portion	I-structure_element
+that	O
+is	O
+largely	O
+altered	O
+,	O
+adopting	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+compared	O
+with	O
+the	O
+compact	B-protein_state
+conformation	I-protein_state
+of	O
+the	O
+apo	B-protein_state
+YfiB	B-protein
+.	O
+The	O
+apo	B-protein_state
+YfiB	B-protein
+structure	B-evidence
+constructed	O
+beginning	O
+at	O
+residue	O
+34	B-residue_number
+has	O
+a	O
+compact	B-protein_state
+conformation	I-protein_state
+of	O
+approximately	O
+45	O
+Å	O
+in	O
+length	O
+.	O
+
+In	O
+addition	O
+to	O
+the	O
+preceding	B-residue_range
+8	I-residue_range
+aa	I-residue_range
+loop	B-structure_element
+(	O
+from	O
+the	O
+lipid	O
+acceptor	O
+Cys26	B-residue_range
+to	I-residue_range
+Gly34	I-residue_range
+),	O
+the	O
+full	B-protein_state
+length	I-protein_state
+of	O
+the	O
+periplasmic	O
+portion	O
+of	O
+apo	B-protein_state
+YfiB	B-protein
+can	O
+reach	O
+approximately	O
+60	O
+Å	O
+.	O
+It	O
+was	O
+reported	O
+that	O
+the	O
+distance	O
+between	O
+the	O
+outer	O
+membrane	O
+and	O
+the	O
+cell	O
+wall	O
+is	O
+approximately	O
+50	O
+Å	O
+and	O
+that	O
+the	O
+thickness	O
+of	O
+the	O
+PG	O
+layer	O
+is	O
+approximately	O
+70	O
+Å	O
+(	O
+Matias	O
+et	O
+al	O
+.,).	O
+
+Thus	O
+,	O
+YfiB	B-protein
+alone	B-protein_state
+represents	O
+an	O
+inactive	B-protein_state
+form	O
+that	O
+may	O
+only	O
+partially	O
+insert	O
+into	O
+the	O
+PG	O
+matrix	O
+.	O
+
+By	O
+contrast	O
+,	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+(	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+168	I-residue_range
+)	O
+has	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+of	O
+approximately	O
+55	O
+Å	O
+in	O
+length	O
+.	O
+
+In	O
+addition	O
+to	O
+the	O
+17	B-residue_range
+preceding	I-residue_range
+intracellular	I-residue_range
+residues	I-residue_range
+(	O
+from	O
+the	O
+lipid	O
+acceptor	O
+Cys26	B-residue_range
+to	I-residue_range
+Leu43	I-residue_range
+),	O
+the	O
+length	O
+of	O
+the	O
+intracellular	O
+portion	O
+of	O
+active	B-protein_state
+YfiB	B-protein
+may	O
+extend	O
+over	O
+100	O
+Å	O
+,	O
+assuming	O
+a	O
+fully	B-protein_state
+stretched	I-protein_state
+conformation	I-protein_state
+.	O
+
+Provided	O
+that	O
+the	O
+diameter	O
+of	O
+the	O
+widest	O
+part	O
+of	O
+the	O
+YfiB	B-protein
+dimer	B-oligomeric_state
+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	B-protein
+dimer	B-oligomeric_state
+should	O
+be	O
+able	O
+to	O
+penetrate	O
+the	O
+PG	O
+layer	O
+.	O
+
+Regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	B-complex_assembly
+tripartite	B-protein_state
+system	O
+.	O
+
+The	O
+periplasmic	B-structure_element
+domain	I-structure_element
+of	O
+YfiB	B-protein
+and	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+are	O
+depicted	O
+according	O
+to	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+The	O
+lipid	O
+acceptor	O
+Cys26	B-residue_name_number
+is	O
+indicated	O
+as	O
+blue	O
+ball	O
+.	O
+
+The	O
+loop	B-structure_element
+connecting	O
+Cys26	B-residue_name_number
+and	O
+Gly34	B-residue_name_number
+of	O
+YfiB	B-protein
+is	O
+modeled	O
+.	O
+
+The	O
+PAS	B-structure_element
+domain	I-structure_element
+of	O
+YfiN	B-protein
+is	O
+shown	O
+as	O
+pink	O
+oval	O
+.	O
+
+Once	O
+activated	B-protein_state
+by	O
+certain	O
+cell	O
+stress	O
+,	O
+the	O
+dimeric	B-oligomeric_state
+YfiB	B-protein
+transforms	O
+from	O
+a	O
+compact	B-protein_state
+conformation	I-protein_state
+to	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+,	O
+allowing	O
+the	O
+periplasmic	B-structure_element
+domain	I-structure_element
+of	O
+the	O
+membrane	B-protein_state
+-	I-protein_state
+anchored	I-protein_state
+YfiB	B-protein
+to	O
+penetrate	O
+the	O
+cell	O
+wall	O
+and	O
+sequester	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+,	O
+thus	O
+relieving	O
+the	O
+repression	O
+of	O
+YfiN	B-protein
+
+These	O
+results	O
+,	O
+together	O
+with	O
+our	O
+observation	O
+that	O
+activated	B-protein_state
+YfiB	B-protein
+has	O
+a	O
+much	O
+higher	O
+cell	B-evidence
+wall	I-evidence
+binding	I-evidence
+affinity	I-evidence
+,	O
+and	O
+previous	O
+mutagenesis	O
+data	O
+showing	O
+that	O
+(	O
+1	O
+)	O
+both	O
+PG	B-chemical
+binding	O
+and	O
+membrane	O
+anchoring	O
+are	O
+required	O
+for	O
+YfiB	B-protein
+activity	O
+and	O
+(	O
+2	O
+)	O
+activating	O
+mutations	O
+possessing	O
+an	O
+altered	O
+N	O
+-	O
+terminal	O
+loop	B-structure_element
+length	O
+are	O
+dominant	O
+over	O
+the	O
+loss	O
+of	O
+PG	B-chemical
+binding	O
+(	O
+Malone	O
+et	O
+al	O
+.,),	O
+suggest	O
+an	O
+updated	O
+regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	B-complex_assembly
+system	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+
+In	O
+this	O
+model	O
+,	O
+in	O
+response	O
+to	O
+a	O
+particular	O
+cell	O
+stress	O
+that	O
+is	O
+yet	O
+to	O
+be	O
+identified	O
+,	O
+the	O
+dimeric	B-oligomeric_state
+YfiB	B-protein
+is	O
+activated	B-protein_state
+from	O
+a	O
+compact	B-protein_state
+,	O
+inactive	B-protein_state
+conformation	B-protein_state
+to	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+,	O
+which	O
+possesses	O
+increased	O
+PG	B-chemical
+binding	O
+affinity	O
+.	O
+
+This	O
+allows	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+portion	I-structure_element
+of	O
+the	O
+membrane	B-protein_state
+-	I-protein_state
+anchored	I-protein_state
+YfiB	B-protein
+to	O
+reach	O
+,	O
+bind	O
+and	O
+penetrate	O
+the	O
+cell	O
+wall	O
+and	O
+sequester	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+.	O
+
+The	O
+YfiBNR	B-complex_assembly
+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	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+level	O
+.	O
+
+Homologs	O
+of	O
+the	O
+YfiBNR	B-complex_assembly
+system	O
+are	O
+functionally	B-protein_state
+conserved	I-protein_state
+in	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+(	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,),	O
+E	B-species
+.	I-species
+coli	I-species
+(	O
+Hufnagel	O
+et	O
+al	O
+.,;	O
+Raterman	O
+et	O
+al	O
+.,;	O
+Sanchez	O
+-	O
+Torres	O
+et	O
+al	O
+.,),	O
+K	B-species
+.	I-species
+pneumonia	I-species
+(	O
+Huertas	O
+et	O
+al	O
+.,)	O
+and	O
+Y	B-species
+.	I-species
+pestis	I-species
+(	O
+Ren	O
+et	O
+al	O
+.,),	O
+where	O
+they	O
+affect	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+production	O
+and	O
+biofilm	O
+formation	O
+.	O
+
+The	O
+mechanism	O
+by	O
+which	O
+activated	B-protein_state
+YfiB	B-protein
+relieves	O
+the	O
+repression	O
+of	O
+YfiN	B-protein
+may	O
+be	O
+applicable	O
+to	O
+the	O
+YfiBNR	B-complex_assembly
+system	O
+in	O
+other	O
+bacteria	B-taxonomy_domain
+and	O
+to	O
+analogous	O
+outside	O
+-	O
+in	O
+signaling	O
+for	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+production	O
+,	O
+which	O
+in	O
+turn	O
+may	O
+be	O
+relevant	O
+to	O
+the	O
+development	O
+of	O
+drugs	O
+that	O
+can	O
+circumvent	O
+complicated	O
+antibiotic	O
+resistance	O
+.	O
+
+Predictive	O
+features	O
+of	O
+ligand	O
+‐	O
+specific	O
+signaling	O
+through	O
+the	O
+estrogen	B-protein_type
+receptor	I-protein_type
+
+Some	O
+estrogen	B-protein
+receptor	I-protein
+‐	I-protein
+α	I-protein
+(	O
+ERα	B-protein
+)‐	O
+targeted	O
+breast	O
+cancer	O
+therapies	O
+such	O
+as	O
+tamoxifen	B-chemical
+have	O
+tissue	O
+‐	O
+selective	O
+or	O
+cell	O
+‐	O
+specific	O
+activities	O
+,	O
+while	O
+others	O
+have	O
+similar	O
+activities	O
+in	O
+different	O
+cell	O
+types	O
+.	O
+
+To	O
+identify	O
+biophysical	O
+determinants	O
+of	O
+cell	O
+‐	O
+specific	O
+signaling	O
+and	O
+breast	O
+cancer	O
+cell	O
+proliferation	O
+,	O
+we	O
+synthesized	B-experimental_method
+241	O
+ERα	B-protein
+ligands	O
+based	O
+on	O
+19	O
+chemical	O
+scaffolds	O
+,	O
+and	O
+compared	O
+ligand	O
+response	O
+using	O
+quantitative	B-experimental_method
+bioassays	I-experimental_method
+for	O
+canonical	O
+ERα	B-protein
+activities	O
+and	O
+X	B-experimental_method
+‐	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+.	O
+
+Ligands	O
+that	O
+regulate	O
+the	O
+dynamics	O
+and	O
+stability	O
+of	O
+the	O
+coactivator	B-site
+‐	I-site
+binding	I-site
+site	I-site
+in	O
+the	O
+C	O
+‐	O
+terminal	O
+ligand	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+,	O
+called	O
+activation	B-structure_element
+function	I-structure_element
+‐	I-structure_element
+2	I-structure_element
+(	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+),	O
+showed	O
+similar	O
+activity	O
+profiles	O
+in	O
+different	O
+cell	O
+types	O
+.	O
+
+Such	O
+ligands	O
+induced	O
+breast	O
+cancer	O
+cell	O
+proliferation	O
+in	O
+a	O
+manner	O
+that	O
+was	O
+predicted	O
+by	O
+the	O
+canonical	O
+recruitment	O
+of	O
+the	O
+coactivators	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+and	O
+induction	O
+of	O
+the	O
+GREB1	B-protein
+proliferative	O
+gene	O
+.	O
+
+For	O
+some	O
+ligand	O
+series	O
+,	O
+a	O
+single	O
+inter	B-evidence
+‐	I-evidence
+atomic	I-evidence
+distance	I-evidence
+in	O
+the	O
+ligand	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+predicted	O
+their	O
+proliferative	O
+effects	O
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+N	O
+‐	O
+terminal	O
+coactivator	B-site
+‐	I-site
+binding	I-site
+site	I-site
+,	O
+activation	B-structure_element
+function	I-structure_element
+‐	I-structure_element
+1	I-structure_element
+(	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+),	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
+
+Thus	O
+,	O
+incorporating	O
+systems	B-experimental_method
+structural	I-experimental_method
+analyses	I-experimental_method
+with	O
+quantitative	B-experimental_method
+chemical	I-experimental_method
+biology	I-experimental_method
+reveals	O
+how	O
+ligands	O
+can	O
+achieve	O
+distinct	O
+allosteric	O
+signaling	O
+outcomes	O
+through	O
+ERα	B-protein
+.	O
+
+Many	O
+drugs	O
+are	O
+small	O
+‐	O
+molecule	O
+ligands	O
+of	O
+allosteric	O
+signaling	O
+proteins	O
+,	O
+including	O
+G	B-protein_type
+protein	I-protein_type
+‐	I-protein_type
+coupled	I-protein_type
+receptors	I-protein_type
+(	O
+GPCRs	B-protein_type
+)	O
+and	O
+nuclear	B-protein_type
+receptors	I-protein_type
+such	O
+as	O
+ERα	B-protein
+.	O
+
+Small	O
+‐	O
+molecule	O
+ligands	O
+control	O
+receptor	O
+activity	O
+by	O
+modulating	O
+recruitment	O
+of	O
+effector	O
+enzymes	O
+to	O
+distal	O
+regions	O
+of	O
+the	O
+receptor	O
+,	O
+relative	O
+to	O
+the	O
+ligand	B-site
+‐	I-site
+binding	I-site
+site	I-site
+.	O
+
+For	O
+example	O
+,	O
+selective	O
+estrogen	B-protein_type
+receptor	I-protein_type
+modulators	I-protein_type
+(	O
+SERMs	B-protein_type
+)	O
+such	O
+as	O
+tamoxifen	B-chemical
+(	O
+Nolvadex	B-chemical
+®;	I-chemical
+AstraZeneca	O
+)	O
+or	O
+raloxifene	B-chemical
+(	O
+Evista	B-chemical
+®;	I-chemical
+Eli	O
+Lilly	O
+)	O
+(	O
+Fig	O
+1A	O
+)	O
+block	O
+the	O
+ERα	B-protein
+‐	O
+mediated	O
+proliferative	O
+effects	O
+of	O
+the	O
+native	O
+estrogen	B-chemical
+,	O
+17β	B-chemical
+‐	I-chemical
+estradiol	I-chemical
+(	O
+E2	B-chemical
+),	O
+on	O
+breast	O
+cancer	O
+cells	O
+,	O
+but	O
+promote	O
+beneficial	O
+estrogenic	O
+effects	O
+on	O
+bone	O
+mineral	O
+density	O
+and	O
+adverse	O
+estrogenic	O
+effects	O
+such	O
+as	O
+uterine	O
+proliferation	O
+,	O
+fatty	O
+liver	O
+,	O
+or	O
+stroke	O
+(	O
+Frolik	O
+et	O
+al	O
+,	O
+1996	O
+;	O
+Fisher	O
+et	O
+al	O
+,	O
+1998	O
+;	O
+McDonnell	O
+et	O
+al	O
+,	O
+2002	O
+;	O
+Jordan	O
+,	O
+2003	O
+).	O
+
+Allosteric	O
+control	O
+of	O
+ERα	B-protein
+activity	O
+
+Chemical	O
+structures	O
+of	O
+some	O
+common	O
+ERα	B-protein
+ligands	O
+.	O
+
+E2	B-chemical
+‐	O
+rings	O
+are	O
+numbered	O
+A	O
+‐	O
+D	O
+.	O
+The	O
+E	O
+‐	O
+ring	O
+is	O
+the	O
+common	O
+site	O
+of	O
+attachment	O
+for	O
+BSC	O
+found	O
+in	O
+many	O
+SERMS	B-protein_type
+.	O
+
+ERα	B-protein
+domain	O
+organization	O
+lettered	O
+,	O
+A	O
+‐	O
+F	O
+.	O
+DBD	B-structure_element
+,	O
+DNA	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+;	O
+LBD	B-structure_element
+,	O
+ligand	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+;	O
+AF	B-structure_element
+,	O
+activation	B-structure_element
+function	I-structure_element
+
+Schematic	O
+illustration	O
+of	O
+the	O
+canonical	O
+ERα	B-protein
+signaling	O
+pathway	O
+.	O
+
+Linear	O
+causality	O
+model	O
+for	O
+ERα	B-protein
+‐	O
+mediated	O
+cell	O
+proliferation	O
+.	O
+
+Branched	O
+causality	O
+model	O
+for	O
+ERα	B-protein
+‐	O
+mediated	O
+cell	O
+proliferation	O
+.	O
+
+ERα	B-protein
+contains	O
+structurally	B-protein_state
+conserved	I-protein_state
+globular	B-structure_element
+domains	I-structure_element
+of	O
+the	O
+nuclear	B-protein_type
+receptor	I-protein_type
+superfamily	I-protein_type
+,	O
+including	O
+a	O
+DNA	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+DBD	B-structure_element
+)	O
+that	O
+is	O
+connected	O
+by	O
+a	O
+flexible	B-protein_state
+hinge	B-structure_element
+region	I-structure_element
+to	O
+the	O
+ligand	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+LBD	B-structure_element
+),	O
+as	O
+well	O
+as	O
+unstructured	B-protein_state
+AB	B-structure_element
+and	O
+F	B-structure_element
+domains	O
+at	O
+its	O
+amino	O
+and	O
+carboxyl	O
+termini	O
+,	O
+respectively	O
+(	O
+Fig	O
+1B	O
+).	O
+
+The	O
+LBD	B-structure_element
+contains	O
+a	O
+ligand	O
+‐	O
+dependent	O
+coactivator	B-site
+‐	I-site
+binding	I-site
+site	I-site
+called	O
+activation	B-structure_element
+function	I-structure_element
+‐	I-structure_element
+2	I-structure_element
+(	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+).	O
+
+However	O
+,	O
+the	O
+agonist	O
+activity	O
+of	O
+SERMs	B-protein_type
+derives	O
+from	O
+activation	B-structure_element
+function	I-structure_element
+‐	I-structure_element
+1	I-structure_element
+(	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+)—	O
+a	O
+coactivator	B-site
+recruitment	I-site
+site	I-site
+located	O
+in	O
+the	O
+AB	B-structure_element
+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
+
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+and	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+bind	O
+distinct	O
+but	O
+overlapping	O
+sets	O
+of	O
+coregulators	O
+(	O
+Webb	O
+et	O
+al	O
+,	O
+1998	O
+;	O
+Endoh	O
+et	O
+al	O
+,	O
+1999	O
+;	O
+Delage	O
+‐	O
+Mourroux	O
+et	O
+al	O
+,	O
+2000	O
+;	O
+Yi	O
+et	O
+al	O
+,	O
+2015	O
+).	O
+
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+binds	O
+the	O
+signature	O
+LxxLL	B-structure_element
+motif	I-structure_element
+peptides	O
+of	O
+coactivators	O
+such	O
+as	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+(	O
+also	O
+known	O
+as	O
+SRC	B-protein
+‐	I-protein
+1	I-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+).	O
+
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+binds	O
+a	O
+separate	O
+surface	O
+on	O
+these	O
+coactivators	O
+(	O
+Webb	O
+et	O
+al	O
+,	O
+1998	O
+;	O
+Yi	O
+et	O
+al	O
+,	O
+2015	O
+).	O
+
+Yet	O
+,	O
+it	O
+is	O
+unknown	O
+how	O
+different	O
+ERα	B-protein
+ligands	O
+control	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+through	O
+the	O
+LBD	B-structure_element
+,	O
+and	O
+whether	O
+this	O
+inter	O
+‐	O
+domain	O
+communication	O
+is	O
+required	O
+for	O
+cell	O
+‐	O
+specific	O
+signaling	O
+or	O
+anti	O
+‐	O
+proliferative	O
+responses	O
+.	O
+
+In	O
+the	O
+canonical	O
+model	O
+of	O
+the	O
+ERα	B-protein
+signaling	O
+pathway	O
+(	O
+Fig	O
+1C	O
+),	O
+E2	B-protein_state
+‐	I-protein_state
+bound	I-protein_state
+ERα	B-protein
+forms	O
+a	O
+homodimer	B-oligomeric_state
+that	O
+binds	O
+DNA	O
+at	O
+estrogen	B-site
+‐	I-site
+response	I-site
+elements	I-site
+(	O
+EREs	B-site
+),	O
+recruits	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+(	O
+Metivier	O
+et	O
+al	O
+,	O
+2003	O
+;	O
+Johnson	O
+&	O
+O	O
+'	O
+Malley	O
+,	O
+2012	O
+),	O
+and	O
+activates	O
+the	O
+GREB1	B-protein
+gene	O
+,	O
+which	O
+is	O
+required	O
+for	O
+proliferation	O
+of	O
+ERα	B-protein
+‐	O
+positive	O
+breast	O
+cancer	O
+cells	O
+(	O
+Ghosh	O
+et	O
+al	O
+,	O
+2000	O
+;	O
+Rae	O
+et	O
+al	O
+,	O
+2005	O
+;	O
+Deschenes	O
+et	O
+al	O
+,	O
+2007	O
+;	O
+Liu	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+
+However	O
+,	O
+ERα	B-protein
+‐	O
+mediated	O
+proliferative	O
+responses	O
+vary	O
+in	O
+a	O
+ligand	O
+‐	O
+dependent	O
+manner	O
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+);	O
+thus	O
+,	O
+it	O
+is	O
+not	O
+known	O
+whether	O
+this	O
+canonical	O
+model	O
+is	O
+widely	O
+applicable	O
+across	O
+diverse	O
+ERα	B-protein
+ligands	O
+.	O
+
+Our	O
+long	O
+‐	O
+term	O
+goal	O
+is	O
+to	O
+be	O
+able	O
+to	O
+predict	O
+proliferative	O
+or	O
+anti	O
+‐	O
+proliferative	O
+activity	O
+of	O
+a	O
+ligand	O
+in	O
+different	O
+tissues	O
+from	O
+its	O
+crystal	B-evidence
+structure	I-evidence
+by	O
+identifying	O
+different	O
+structural	O
+perturbations	O
+that	O
+lead	O
+to	O
+specific	O
+signaling	O
+outcomes	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	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+determines	O
+GREB1	B-protein
+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
+
+In	O
+this	O
+signaling	O
+model	O
+,	O
+multiple	O
+coregulator	O
+binding	O
+events	O
+and	O
+target	O
+genes	O
+(	O
+Won	O
+Jeong	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Nwachukwu	O
+et	O
+al	O
+,	O
+2014	O
+),	O
+LBD	B-structure_element
+conformation	O
+,	O
+nucleocytoplasmic	O
+shuttling	O
+,	O
+the	O
+occupancy	O
+and	O
+dynamics	O
+of	O
+DNA	O
+binding	O
+,	O
+and	O
+other	O
+biophysical	O
+features	O
+could	O
+contribute	O
+independently	O
+to	O
+cell	O
+proliferation	O
+(	O
+Lickwar	O
+et	O
+al	O
+,	O
+2012	O
+).	O
+
+To	O
+test	O
+these	O
+signaling	O
+models	O
+,	O
+we	O
+profiled	O
+a	O
+diverse	O
+library	O
+of	O
+ERα	B-protein
+ligands	O
+using	O
+systems	O
+biology	O
+approaches	O
+to	O
+X	B-experimental_method
+‐	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+and	O
+chemical	B-experimental_method
+biology	I-experimental_method
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+),	O
+including	O
+a	O
+series	O
+of	O
+quantitative	O
+bioassays	O
+for	O
+ERα	B-protein
+function	O
+that	O
+were	O
+statistically	O
+robust	O
+and	O
+reproducible	O
+,	O
+based	O
+on	O
+the	O
+Z	B-evidence
+’‐	I-evidence
+statistic	I-evidence
+(	O
+Fig	O
+EV1A	O
+and	O
+B	O
+;	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+
+We	O
+also	O
+determined	B-experimental_method
+the	O
+structures	B-evidence
+of	O
+76	O
+distinct	O
+ERα	B-protein
+LBD	B-structure_element
+complexes	O
+bound	B-protein_state
+to	I-protein_state
+different	O
+ligand	O
+types	O
+,	O
+which	O
+allowed	O
+us	O
+to	O
+understand	O
+how	O
+diverse	O
+ligand	O
+scaffolds	O
+distort	O
+the	O
+active	B-protein_state
+conformation	O
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+.	O
+
+Our	O
+findings	O
+here	O
+indicate	O
+that	O
+specific	O
+structural	O
+perturbations	O
+can	O
+be	O
+tied	O
+to	O
+ligand	O
+‐	O
+selective	O
+domain	O
+usage	O
+and	O
+signaling	O
+patterns	O
+,	O
+thus	O
+providing	O
+a	O
+framework	O
+for	O
+structure	O
+‐	O
+based	O
+design	O
+of	O
+improved	O
+breast	O
+cancer	O
+therapeutics	O
+,	O
+and	O
+understanding	O
+the	O
+different	O
+phenotypic	O
+effects	O
+of	O
+environmental	O
+estrogens	B-chemical
+.	O
+
+High	O
+‐	O
+throughput	O
+screens	O
+for	O
+ERα	B-protein
+ligand	O
+profiling	O
+
+Summary	O
+of	O
+ligand	B-experimental_method
+screening	I-experimental_method
+assays	I-experimental_method
+used	O
+to	O
+measure	O
+ER	O
+‐	O
+mediated	O
+activities	O
+.	O
+
+ERE	B-structure_element
+,	O
+estrogen	B-structure_element
+‐	I-structure_element
+response	I-structure_element
+element	I-structure_element
+;	O
+Luc	B-experimental_method
+,	O
+luciferase	B-experimental_method
+reporter	I-experimental_method
+gene	I-experimental_method
+;	O
+M2H	B-experimental_method
+,	O
+mammalian	B-experimental_method
+2	I-experimental_method
+‐	I-experimental_method
+hybrid	I-experimental_method
+;	O
+UAS	B-structure_element
+,	O
+upstream	B-structure_element
+‐	I-structure_element
+activating	I-structure_element
+sequence	I-structure_element
+.	O
+
+Strength	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+signaling	O
+does	O
+not	O
+determine	O
+cell	O
+‐	O
+specific	O
+signaling	O
+
+To	O
+compare	O
+ERα	B-protein
+signaling	O
+induced	O
+by	O
+diverse	O
+ligand	O
+types	O
+,	O
+we	O
+synthesized	B-experimental_method
+and	I-experimental_method
+assayed	I-experimental_method
+a	O
+library	O
+of	O
+241	O
+ERα	B-protein
+ligands	O
+containing	O
+19	O
+distinct	O
+molecular	O
+scaffolds	O
+.	O
+
+These	O
+include	O
+15	O
+indirect	O
+modulator	O
+series	O
+,	O
+which	O
+lack	B-protein_state
+a	O
+SERM	B-protein_type
+‐	I-protein_type
+like	I-protein_type
+side	O
+chain	O
+and	O
+modulate	O
+coactivator	O
+binding	O
+indirectly	O
+from	O
+the	O
+ligand	B-site
+‐	I-site
+binding	I-site
+pocket	I-site
+(	O
+Fig	O
+2A	O
+–	O
+E	O
+;	O
+Dataset	O
+EV1	O
+)	O
+(	O
+Zheng	O
+et	O
+al	O
+,	O
+2012	O
+)	O
+(	O
+Zhu	O
+et	O
+al	O
+,	O
+2012	O
+)	O
+(	O
+Muthyala	O
+et	O
+al	O
+,	O
+2003	O
+;	O
+Seo	O
+et	O
+al	O
+,	O
+2006	O
+)	O
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+)	O
+(	O
+Wang	O
+et	O
+al	O
+,	O
+2012	O
+)	O
+(	O
+Liao	O
+et	O
+al	O
+,	O
+2014	O
+)	O
+(	O
+Min	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+
+We	O
+also	O
+generated	O
+four	O
+direct	O
+modulator	O
+series	O
+with	O
+side	O
+chains	O
+designed	O
+to	O
+directly	O
+dislocate	O
+h12	B-structure_element
+and	O
+thereby	O
+completely	O
+occlude	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+(	O
+Fig	O
+2C	O
+and	O
+E	O
+;	O
+Dataset	O
+EV1	O
+)	O
+(	O
+Kieser	O
+et	O
+al	O
+,	O
+2010	O
+).	O
+
+Ligand	B-experimental_method
+profiling	I-experimental_method
+using	O
+our	O
+quantitative	B-experimental_method
+bioassays	I-experimental_method
+revealed	O
+a	O
+wide	O
+range	O
+of	O
+ligand	O
+‐	O
+induced	O
+GREB1	B-protein
+expression	O
+,	O
+reporter	O
+gene	O
+activities	O
+,	O
+ERα	B-protein
+‐	O
+coactivator	O
+interactions	O
+,	O
+and	O
+proliferative	O
+effects	O
+on	O
+MCF	O
+‐	O
+7	O
+breast	O
+cancer	O
+cells	O
+(	O
+Figs	O
+EV1	O
+and	O
+EV2A	O
+–	O
+J	O
+).	O
+
+This	O
+wide	O
+variance	O
+enabled	O
+us	O
+to	O
+probe	O
+specific	O
+features	O
+of	O
+ERα	B-protein
+signaling	O
+using	O
+ligand	B-experimental_method
+class	I-experimental_method
+analyses	I-experimental_method
+,	O
+and	O
+identify	O
+signaling	O
+patterns	O
+shared	O
+by	O
+specific	O
+ligand	O
+series	O
+or	O
+scaffolds	O
+.	O
+
+Classes	O
+of	O
+compounds	O
+in	O
+the	O
+ERα	B-protein
+ligand	O
+library	O
+
+Structure	B-evidence
+of	O
+the	O
+E2	B-protein_state
+‐	I-protein_state
+bound	I-protein_state
+ERα	B-protein
+LBD	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+an	O
+NCOA2	B-protein
+peptide	O
+of	O
+(	O
+PDB	O
+1GWR	O
+).	O
+
+Structural	O
+details	O
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+the	O
+indicated	O
+ligands	O
+.	O
+
+Unlike	O
+E2	B-chemical
+(	O
+PDB	O
+1GWR	O
+),	O
+TAM	B-chemical
+is	O
+a	O
+direct	O
+modulator	O
+with	O
+a	O
+BSC	O
+that	O
+dislocates	O
+h12	B-structure_element
+to	O
+block	O
+the	O
+NCOA2	B-site
+‐	I-site
+binding	I-site
+site	I-site
+(	O
+PDB	O
+3ERT	O
+).	O
+
+OBHS	B-chemical
+is	O
+an	O
+indirect	O
+modulator	O
+that	O
+dislocates	O
+the	O
+h11	B-structure_element
+C	O
+‐	O
+terminus	O
+to	O
+destabilize	O
+the	O
+h11	B-site
+–	I-site
+h12	I-site
+interface	I-site
+(	O
+PDB	O
+4ZN9	O
+).	O
+
+The	O
+ERα	B-protein
+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
+
+ERα	B-protein
+ligands	O
+induced	O
+a	O
+range	O
+of	O
+agonist	O
+activity	O
+profiles	O
+
+To	O
+this	O
+end	O
+,	O
+we	O
+compared	O
+the	O
+average	O
+ligand	O
+‐	O
+induced	O
+GREB1	B-protein
+mRNA	O
+levels	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+and	O
+3	B-experimental_method
+×	I-experimental_method
+ERE	I-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+reporter	O
+gene	O
+activity	O
+in	O
+Ishikawa	O
+endometrial	O
+cancer	O
+cells	O
+(	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+)	O
+or	O
+in	O
+HepG2	O
+cells	O
+transfected	O
+with	O
+wild	B-protein_state
+‐	I-protein_state
+type	I-protein_state
+ERα	B-protein
+(	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+)	O
+(	O
+Figs	O
+3A	O
+and	O
+EV2A	O
+–	O
+C	O
+).	O
+
+Direct	O
+modulators	O
+showed	O
+significant	O
+differences	O
+in	O
+average	O
+activity	O
+between	O
+cell	O
+types	O
+except	O
+OBHS	B-chemical
+‐	I-chemical
+ASC	I-chemical
+analogs	O
+,	O
+which	O
+had	O
+similar	O
+low	O
+agonist	O
+activities	O
+in	O
+the	O
+three	O
+cell	O
+types	O
+.	O
+
+While	O
+it	O
+was	O
+known	O
+that	O
+direct	O
+modulators	O
+such	O
+as	O
+tamoxifen	B-chemical
+drive	O
+cell	O
+‐	O
+specific	O
+signaling	O
+,	O
+these	O
+experiments	O
+reveal	O
+that	O
+indirect	O
+modulators	O
+also	O
+drive	O
+cell	O
+‐	O
+specific	O
+signaling	O
+,	O
+since	O
+eight	O
+of	O
+fourteen	O
+classes	O
+showed	O
+significant	O
+differences	O
+in	O
+average	O
+activity	O
+(	O
+Figs	O
+3A	O
+and	O
+EV2A	O
+–	O
+C	O
+).	O
+
+Ligand	O
+‐	O
+specific	O
+signaling	O
+underlies	O
+ERα	B-protein
+‐	O
+mediated	O
+cell	O
+proliferation	O
+
+(	O
+A	O
+)	O
+Ligand	O
+‐	O
+specific	O
+ERα	B-protein
+activities	O
+in	O
+HepG2	O
+,	O
+Ishikawa	O
+and	O
+MCF	O
+‐	O
+7	O
+cells	O
+.	O
+
+The	O
+ligand	O
+‐	O
+induced	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+and	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activities	O
+and	O
+GREB1	B-protein
+mRNA	O
+levels	O
+are	O
+shown	O
+by	O
+scaffold	O
+(	O
+mean	O
++	O
+SD	O
+).	O
+
+(	O
+B	O
+)	O
+Ligand	O
+class	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+and	O
+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activities	O
+in	O
+HepG2	O
+cells	O
+.	O
+
+Significant	O
+sensitivity	O
+to	O
+AB	B-structure_element
+domain	O
+deletion	O
+was	O
+determined	O
+by	O
+Student	B-experimental_method
+'	I-experimental_method
+s	I-experimental_method
+t	I-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+(	O
+n	O
+=	O
+number	O
+of	O
+ligands	O
+per	O
+scaffold	O
+in	O
+Fig	O
+2	O
+).	O
+
+Correlation	B-experimental_method
+and	I-experimental_method
+regression	I-experimental_method
+analyses	I-experimental_method
+in	O
+a	O
+large	O
+test	O
+set	O
+.	O
+
+In	O
+cluster	O
+1	O
+,	O
+the	O
+first	O
+three	O
+comparisons	O
+(	O
+rows	O
+)	O
+showed	O
+significant	O
+positive	O
+correlations	O
+(	O
+F	B-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+for	O
+nonzero	O
+slope	O
+,	O
+P	B-evidence
+≤	O
+0	O
+.	O
+05	O
+).	O
+
+In	O
+cluster	O
+2	O
+,	O
+only	O
+one	O
+of	O
+these	O
+comparisons	O
+revealed	O
+a	O
+significant	O
+positive	O
+correlation	O
+,	O
+while	O
+none	O
+was	O
+significant	O
+in	O
+cluster	O
+3	O
+.	O
++,	O
+statistically	O
+significant	O
+correlations	O
+gained	O
+by	O
+deletion	B-experimental_method
+of	O
+the	O
+AB	B-structure_element
+or	O
+F	B-structure_element
+domains	O
+.	O
+
+−,	O
+significant	O
+correlations	O
+lost	O
+upon	O
+deletion	O
+of	O
+AB	B-structure_element
+or	O
+F	B-structure_element
+domains	O
+.	O
+
+Tamoxifen	B-chemical
+depends	O
+on	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+its	O
+cell	O
+‐	O
+specific	O
+activity	O
+(	O
+Sakamoto	O
+et	O
+al	O
+,	O
+2002	O
+);	O
+therefore	O
+,	O
+we	O
+asked	O
+whether	O
+cell	O
+‐	O
+specific	O
+signaling	O
+observed	O
+here	O
+is	O
+due	O
+to	O
+a	O
+similar	O
+dependence	O
+on	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+activity	O
+(	O
+Fig	O
+EV1	O
+).	O
+
+To	O
+test	O
+this	O
+idea	O
+,	O
+we	O
+compared	O
+the	O
+average	B-evidence
+L	I-evidence
+‐	I-evidence
+Luc	I-evidence
+activities	I-evidence
+of	O
+each	O
+scaffold	O
+in	O
+HepG2	O
+cells	O
+co	B-experimental_method
+‐	I-experimental_method
+transfected	I-experimental_method
+with	O
+wild	B-protein_state
+‐	I-protein_state
+type	I-protein_state
+ERα	B-protein
+or	O
+with	O
+ERα	B-protein
+lacking	B-protein_state
+the	I-protein_state
+AB	B-structure_element
+domain	O
+(	O
+Figs	O
+1B	O
+and	O
+EV1	O
+).	O
+
+While	O
+E2	B-chemical
+showed	O
+similar	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+and	O
+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activities	O
+,	O
+tamoxifen	B-chemical
+showed	O
+complete	O
+loss	O
+of	O
+activity	O
+without	B-protein_state
+the	O
+AB	B-structure_element
+domain	O
+(	O
+Fig	O
+EV1B	O
+).	O
+
+Deletion	B-experimental_method
+of	I-experimental_method
+the	O
+AB	B-structure_element
+domain	O
+significantly	O
+reduced	O
+the	O
+average	B-evidence
+L	I-evidence
+‐	I-evidence
+Luc	I-evidence
+activities	I-evidence
+of	O
+14	O
+scaffolds	O
+(	O
+Student	B-experimental_method
+'	I-experimental_method
+s	I-experimental_method
+t	I-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+,	O
+P	B-evidence
+≤	O
+0	O
+.	O
+05	O
+)	O
+(	O
+Fig	O
+3B	O
+).	O
+
+These	O
+“	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+‐	O
+sensitive	O
+”	O
+activities	O
+were	O
+exhibited	O
+by	O
+both	O
+direct	O
+and	O
+indirect	O
+modulators	O
+,	O
+and	O
+were	O
+not	O
+limited	O
+to	O
+scaffolds	O
+that	O
+showed	O
+cell	O
+‐	O
+specific	O
+signaling	O
+(	O
+Fig	O
+3A	O
+and	O
+B	O
+).	O
+
+Thus	O
+,	O
+the	O
+strength	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+signaling	O
+does	O
+not	O
+determine	O
+cell	O
+‐	O
+specific	O
+signaling	O
+.	O
+
+Identifying	O
+cell	O
+‐	O
+specific	O
+signaling	O
+clusters	O
+in	O
+ERα	B-protein
+ligand	O
+classes	O
+
+For	O
+each	O
+ligand	O
+class	O
+or	O
+scaffold	O
+,	O
+we	O
+calculated	O
+the	O
+Pearson	B-evidence
+'	I-evidence
+s	I-evidence
+correlation	I-evidence
+coefficient	I-evidence
+,	O
+r	B-evidence
+,	O
+for	O
+pairwise	O
+comparison	O
+of	O
+activity	O
+profiles	O
+in	O
+breast	O
+(	O
+GREB1	B-protein
+),	O
+liver	O
+(	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+),	O
+and	O
+endometrial	O
+cells	O
+(	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+).	O
+
+The	O
+value	O
+of	O
+r	B-evidence
+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
+
+We	O
+also	O
+calculated	O
+the	O
+coefficient	B-evidence
+of	I-evidence
+determination	I-evidence
+,	O
+r	B-evidence
+2	I-evidence
+,	O
+which	O
+describes	O
+the	O
+percentage	O
+of	O
+variance	O
+in	O
+a	O
+dependent	O
+variable	O
+such	O
+as	O
+proliferation	O
+that	O
+can	O
+be	O
+predicted	O
+by	O
+an	O
+independent	O
+variable	O
+such	O
+as	O
+GREB1	B-protein
+expression	O
+.	O
+
+We	O
+present	O
+both	O
+calculations	O
+as	O
+r	B-evidence
+2	I-evidence
+to	O
+readily	O
+compare	O
+signaling	O
+specificities	O
+using	O
+a	O
+heat	O
+map	O
+on	O
+which	O
+the	O
+red	O
+–	O
+yellow	O
+palette	O
+indicates	O
+significant	O
+positive	O
+correlations	O
+(	O
+P	B-evidence
+≤	O
+0	O
+.	O
+05	O
+,	O
+F	B-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+for	O
+nonzero	O
+slope	O
+),	O
+while	O
+the	O
+blue	O
+palette	O
+denotes	O
+negative	O
+correlations	O
+(	O
+Fig	O
+3C	O
+–	O
+F	O
+).	O
+
+The	O
+side	O
+chain	O
+of	O
+OBHS	B-chemical
+‐	I-chemical
+BSC	I-chemical
+analogs	O
+induces	O
+cell	O
+‐	O
+specific	O
+signaling	O
+
+Correlation	O
+analysis	O
+of	O
+OBHS	B-chemical
+versus	O
+OBHS	B-chemical
+‐	I-chemical
+BSC	I-chemical
+activity	O
+across	O
+cell	O
+types	O
+.	O
+
+Correlation	O
+analysis	O
+of	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activity	O
+versus	O
+endogenous	O
+ERα	B-protein
+activity	O
+of	O
+OBHS	B-chemical
+analogs	O
+.	O
+
+In	O
+panel	O
+(	O
+D	O
+),	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+activity	O
+from	O
+panel	O
+(	O
+B	O
+)	O
+is	O
+shown	O
+for	O
+comparison	O
+.	O
+
+Correlation	O
+analysis	O
+of	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-mutant
+‐	I-mutant
+ΔF	I-mutant
+activity	O
+versus	O
+endogenous	O
+ERα	B-protein
+activities	O
+of	O
+OBHS	B-chemical
+analogs	O
+.	O
+
+Correlation	O
+analysis	O
+of	O
+MCF	O
+‐	O
+7	O
+cell	O
+proliferation	O
+versus	O
+NCOA2	B-protein
+/	I-protein
+3	I-protein
+recruitment	O
+or	O
+GREB1	B-protein
+levels	O
+observed	O
+in	O
+response	O
+to	O
+(	O
+G	O
+)	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+and	O
+(	O
+H	O
+)	O
+OBHS	B-chemical
+‐	I-chemical
+BSC	I-chemical
+analogs	O
+.	O
+
+Scaffolds	O
+in	O
+cluster	O
+1	O
+exhibited	O
+strongly	O
+correlated	O
+GREB1	B-protein
+levels	O
+,	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+and	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+profiles	O
+across	O
+the	O
+three	O
+cell	O
+types	O
+(	O
+Fig	O
+3C	O
+lanes	O
+1	O
+–	O
+4	O
+),	O
+suggesting	O
+these	O
+ligands	O
+use	O
+similar	O
+ERα	B-protein
+signaling	O
+pathways	O
+in	O
+the	O
+breast	O
+,	O
+endometrial	O
+,	O
+and	O
+liver	O
+cell	O
+types	O
+.	O
+
+This	O
+cluster	O
+includes	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+,	O
+OBHS	B-chemical
+,	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+,	O
+and	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+,	O
+all	O
+of	O
+which	O
+are	O
+indirect	O
+modulators	O
+.	O
+
+This	O
+cluster	O
+includes	O
+two	O
+classes	O
+of	O
+direct	O
+modulators	O
+(	O
+cyclofenil	B-chemical
+‐	I-chemical
+ASC	I-chemical
+and	O
+WAY	B-chemical
+dimer	I-chemical
+),	O
+and	O
+six	O
+classes	O
+of	O
+indirect	O
+modulators	O
+(	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+2	I-chemical
+and	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+,	O
+furan	B-chemical
+,	O
+and	O
+WAY	B-chemical
+‐	I-chemical
+D	I-chemical
+).	O
+
+For	O
+example	O
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+furan	B-chemical
+,	O
+and	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+2	I-chemical
+drove	O
+positively	O
+correlated	O
+GREB1	B-protein
+levels	O
+and	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+but	O
+not	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+activity	O
+(	O
+Fig	O
+3C	O
+lanes	O
+5	O
+–	O
+7	O
+).	O
+
+In	O
+contrast	O
+,	O
+WAY	B-chemical
+dimer	I-chemical
+and	O
+WAY	B-chemical
+‐	I-chemical
+D	I-chemical
+analogs	O
+drove	O
+positively	O
+correlated	O
+GREB1	B-protein
+levels	O
+and	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+but	O
+not	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+(	O
+Fig	O
+3C	O
+lanes	O
+8	O
+and	O
+9	O
+).	O
+
+This	O
+cluster	O
+includes	O
+two	O
+direct	O
+modulator	O
+scaffolds	O
+(	O
+OBHS	B-chemical
+‐	I-chemical
+ASC	I-chemical
+and	O
+OBHS	B-chemical
+‐	I-chemical
+BSC	I-chemical
+),	O
+and	O
+five	O
+indirect	O
+modulator	O
+scaffolds	O
+(	O
+A	B-chemical
+‐	I-chemical
+CD	I-chemical
+,	O
+cyclofenil	B-chemical
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+,	O
+imine	B-chemical
+,	O
+and	O
+imidazopyridine	B-chemical
+).	O
+
+These	O
+results	O
+suggest	O
+that	O
+addition	O
+of	O
+an	O
+extended	O
+side	O
+chain	O
+to	O
+an	O
+ERα	B-protein
+ligand	O
+scaffold	O
+is	O
+sufficient	O
+to	O
+induce	O
+cell	O
+‐	O
+specific	O
+signaling	O
+,	O
+where	O
+the	O
+relative	O
+activity	O
+profiles	O
+of	O
+the	O
+individual	O
+ligands	O
+change	O
+between	O
+cell	O
+types	O
+.	O
+
+This	O
+is	O
+demonstrated	O
+by	O
+directly	O
+comparing	O
+the	O
+signaling	O
+specificities	O
+of	O
+matched	O
+OBHS	B-chemical
+(	O
+indirect	O
+modulator	O
+,	O
+cluster	O
+1	O
+)	O
+and	O
+OBHS	B-chemical
+‐	I-chemical
+BSC	I-chemical
+analogs	O
+(	O
+direct	O
+modulator	O
+,	O
+cluster	O
+3	O
+),	O
+which	O
+differ	O
+only	O
+in	O
+the	O
+basic	O
+side	O
+chain	O
+(	O
+Fig	O
+2E	O
+).	O
+
+The	O
+activities	O
+of	O
+OBHS	B-chemical
+analogs	O
+were	O
+positively	O
+correlated	O
+across	O
+the	O
+three	O
+cell	O
+types	O
+,	O
+but	O
+the	O
+side	O
+chain	O
+of	O
+OBHS	B-chemical
+‐	I-chemical
+BSC	I-chemical
+analogs	O
+was	O
+sufficient	O
+to	O
+abolish	O
+these	O
+correlations	O
+(	O
+Figs	O
+3C	O
+lanes	O
+1	O
+and	O
+19	O
+,	O
+and	O
+EV3A	O
+–	O
+C	O
+).	O
+
+Thus	O
+,	O
+examining	O
+the	O
+correlated	O
+patterns	O
+of	O
+ERα	B-protein
+activity	O
+within	O
+each	O
+scaffold	O
+demonstrates	O
+that	O
+an	O
+extended	O
+side	O
+chain	O
+is	O
+not	O
+required	O
+for	O
+cell	O
+‐	O
+specific	O
+signaling	O
+.	O
+
+Modulation	O
+of	O
+signaling	O
+specificity	O
+by	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+
+To	O
+evaluate	O
+the	O
+role	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+and	O
+the	O
+F	B-structure_element
+domain	O
+in	O
+ERα	B-protein
+signaling	O
+specificity	O
+,	O
+we	O
+compared	O
+activity	O
+of	O
+truncated	O
+ERα	B-protein
+constructs	O
+in	O
+HepG2	O
+liver	O
+cells	O
+with	O
+endogenous	O
+ERα	B-protein
+activity	O
+in	O
+the	O
+other	O
+cell	O
+types	O
+.	O
+
+The	O
+positive	O
+correlation	O
+between	O
+the	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+and	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activities	O
+or	O
+GREB1	B-protein
+levels	O
+induced	O
+by	O
+scaffolds	O
+in	O
+cluster	O
+1	O
+was	O
+generally	O
+retained	O
+without	O
+the	O
+AB	B-structure_element
+domain	O
+,	O
+or	O
+the	O
+F	B-structure_element
+domain	O
+(	O
+Fig	O
+3D	O
+lanes	O
+1	O
+–	O
+4	O
+).	O
+
+This	O
+demonstrates	O
+that	O
+the	O
+signaling	O
+specificities	O
+underlying	O
+these	O
+positive	O
+correlations	O
+are	O
+not	O
+modified	O
+by	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+.	O
+
+OBHS	B-chemical
+analogs	O
+showed	O
+an	O
+average	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activity	O
+of	O
+3	O
+.	O
+2	O
+%	O
+±	O
+3	O
+(	O
+mean	O
++	O
+SEM	O
+)	O
+relative	O
+to	O
+E2	B-chemical
+.	O
+
+Despite	O
+this	O
+nearly	O
+complete	O
+lack	O
+of	O
+activity	O
+,	O
+the	O
+pattern	O
+of	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-mutant
+‐	I-mutant
+ΔAB	I-mutant
+activity	O
+was	O
+still	O
+highly	O
+correlated	O
+with	O
+the	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+and	O
+GREB1	B-protein
+expression	O
+(	O
+Fig	O
+EV3D	O
+and	O
+E	O
+),	O
+demonstrating	O
+that	O
+very	O
+small	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+activities	O
+can	O
+be	O
+amplified	O
+by	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+to	O
+produce	O
+robust	O
+signals	O
+.	O
+
+Similarly	O
+,	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+F	B-structure_element
+domain	O
+did	O
+not	O
+abolish	O
+correlations	O
+between	O
+the	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+and	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+or	O
+GREB1	B-protein
+levels	O
+induced	O
+by	O
+OBHS	B-chemical
+analogs	O
+(	O
+Fig	O
+EV3F	O
+).	O
+
+These	O
+similar	O
+patterns	O
+of	O
+ligand	O
+activity	O
+in	O
+the	O
+wild	B-protein_state
+‐	I-protein_state
+type	I-protein_state
+and	O
+deletion	O
+mutants	B-protein_state
+suggest	O
+that	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+and	O
+the	O
+F	B-structure_element
+domain	O
+purely	O
+amplify	O
+the	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+activities	O
+of	O
+ligands	O
+in	O
+cluster	O
+1	O
+.	O
+
+In	O
+contrast	O
+,	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+was	O
+a	O
+determinant	O
+of	O
+signaling	O
+specificity	O
+for	O
+scaffolds	O
+in	O
+cluster	O
+2	O
+.	O
+
+Deletion	B-experimental_method
+of	I-experimental_method
+the	O
+AB	B-structure_element
+or	O
+F	B-structure_element
+domain	O
+altered	O
+correlations	O
+for	O
+six	O
+of	O
+the	O
+eight	O
+scaffolds	O
+in	O
+this	O
+cluster	O
+(	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+,	O
+WAY	B-chemical
+‐	I-chemical
+D	I-chemical
+,	O
+WAY	B-chemical
+dimer	I-chemical
+,	O
+and	O
+cyclofenil	B-chemical
+‐	I-chemical
+ASC	I-chemical
+)	O
+(	O
+Fig	O
+3D	O
+lanes	O
+5	O
+–	O
+12	O
+).	O
+
+Comparing	O
+Fig	O
+3C	O
+and	O
+D	O
+,	O
+the	O
++	O
+and	O
+−	O
+signs	O
+indicate	O
+where	O
+the	O
+deletion	B-experimental_method
+mutant	I-experimental_method
+assays	I-experimental_method
+led	O
+to	O
+a	O
+gain	O
+or	O
+loss	O
+of	O
+statically	O
+significant	O
+correlation	O
+,	O
+respectively	O
+.	O
+
+Thus	O
+,	O
+in	O
+cluster	O
+2	O
+,	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+substantially	O
+modulated	O
+the	O
+specificity	O
+of	O
+ligands	O
+with	O
+cell	O
+‐	O
+specific	O
+activity	O
+(	O
+Fig	O
+3D	O
+lanes	O
+5	O
+–	O
+12	O
+).	O
+
+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	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+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	B-experimental_method
+of	I-experimental_method
+the	O
+AB	B-structure_element
+or	O
+F	B-structure_element
+domain	O
+rarely	O
+induced	O
+such	O
+correlations	O
+(	O
+Fig	O
+3D	O
+),	O
+except	O
+for	O
+A	B-chemical
+‐	I-chemical
+CD	I-chemical
+and	O
+OBHS	B-chemical
+‐	I-chemical
+ASC	I-chemical
+analogs	O
+,	O
+where	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+AB	B-structure_element
+domain	O
+or	O
+F	B-structure_element
+domain	O
+led	O
+to	O
+positive	O
+correlations	O
+with	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+and	O
+/	O
+or	O
+GREB1	B-protein
+levels	O
+(	O
+Fig	O
+3D	O
+lanes	O
+13	O
+and	O
+18	O
+).	O
+
+Thus	O
+,	O
+ligands	O
+in	O
+cluster	O
+2	O
+rely	O
+on	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+both	O
+activity	O
+(	O
+Fig	O
+3B	O
+)	O
+and	O
+signaling	O
+specificity	O
+(	O
+Fig	O
+3D	O
+).	O
+
+Ligand	O
+‐	O
+specific	O
+control	O
+of	O
+GREB1	B-protein
+expression	O
+
+To	O
+determine	O
+whether	O
+ligand	O
+classes	O
+control	O
+expression	O
+of	O
+native	O
+ERα	B-protein
+target	O
+genes	O
+through	O
+the	O
+canonical	O
+linear	O
+signaling	O
+pathway	O
+,	O
+we	O
+performed	O
+pairwise	B-experimental_method
+linear	I-experimental_method
+regression	I-experimental_method
+analyses	I-experimental_method
+using	O
+ERα	B-complex_assembly
+–	I-complex_assembly
+NCOA1	I-complex_assembly
+/	I-complex_assembly
+2	I-complex_assembly
+/	I-complex_assembly
+3	I-complex_assembly
+interactions	O
+in	O
+M2H	B-experimental_method
+assay	I-experimental_method
+as	O
+independent	O
+predictors	O
+of	O
+GREB1	B-protein
+expression	O
+(	O
+the	O
+dependent	O
+variable	O
+)	O
+(	O
+Figs	O
+EV1	O
+and	O
+EV2A	O
+,	O
+F	O
+–	O
+H	O
+).	O
+
+In	O
+cluster	O
+1	O
+,	O
+the	O
+recruitment	O
+of	O
+NCOA1	B-protein
+and	O
+NCOA2	B-protein
+was	O
+highest	O
+for	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+,	O
+followed	O
+by	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+,	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+,	O
+and	O
+OBHS	B-chemical
+series	O
+,	O
+while	O
+for	O
+NCOA3	B-protein
+,	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+compounds	O
+induced	O
+the	O
+most	O
+recruitment	O
+and	O
+OBHS	B-chemical
+ligands	O
+were	O
+inverse	O
+agonists	O
+(	O
+Fig	O
+EV2F	O
+–	O
+H	O
+).	O
+
+The	O
+average	O
+induction	O
+of	O
+GREB1	B-protein
+by	O
+cluster	O
+1	O
+ligands	O
+showed	O
+greater	O
+variance	O
+,	O
+with	O
+a	O
+range	O
+between	O
+~	O
+25	O
+and	O
+~	O
+75	O
+%	O
+for	O
+OBHS	B-chemical
+and	O
+a	O
+range	O
+from	O
+full	O
+agonist	O
+to	O
+inverse	O
+agonist	O
+for	O
+the	O
+others	O
+in	O
+cluster	O
+1	O
+(	O
+Fig	O
+EV2A	O
+).	O
+
+GREB1	B-protein
+levels	O
+induced	O
+by	O
+OBHS	B-chemical
+analogs	O
+were	O
+determined	O
+by	O
+recruitment	O
+of	O
+NCOA1	B-protein
+but	O
+not	O
+NCOA2	B-protein
+/	I-protein
+3	I-protein
+(	O
+Fig	O
+3E	O
+lane	O
+1	O
+),	O
+suggesting	O
+that	O
+there	O
+may	O
+be	O
+alternate	O
+or	O
+preferential	O
+use	O
+of	O
+these	O
+coactivators	O
+by	O
+different	O
+classes	O
+.	O
+
+However	O
+,	O
+in	O
+cluster	O
+1	O
+,	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+generally	O
+predicted	O
+GREB1	B-protein
+levels	O
+(	O
+Fig	O
+3E	O
+lanes	O
+1	O
+–	O
+4	O
+),	O
+consistent	O
+with	O
+the	O
+canonical	O
+signaling	O
+model	O
+(	O
+Fig	O
+1D	O
+).	O
+
+For	O
+clusters	O
+2	O
+and	O
+3	O
+,	O
+GREB1	B-protein
+activity	O
+was	O
+generally	O
+not	O
+predicted	O
+by	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+.	O
+
+Direct	O
+modulators	O
+showed	O
+low	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+(	O
+Fig	O
+EV2F	O
+–	O
+H	O
+),	O
+but	O
+only	O
+OBHS	B-chemical
+‐	I-chemical
+ASC	I-chemical
+analogs	O
+had	O
+NCOA2	B-protein
+recruitment	O
+profiles	O
+that	O
+predicted	O
+a	O
+full	O
+range	O
+of	O
+effects	O
+on	O
+GREB1	B-protein
+levels	O
+(	O
+Figs	O
+3E	O
+lanes	O
+9	O
+,	O
+11	O
+,	O
+18	O
+–	O
+19	O
+,	O
+and	O
+EV2A	O
+).	O
+
+The	O
+indirect	O
+modulators	O
+in	O
+clusters	O
+2	O
+and	O
+3	O
+stimulated	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+and	O
+GREB1	B-protein
+expression	O
+with	O
+substantial	O
+variance	O
+(	O
+Figs	O
+3A	O
+and	O
+EV2F	O
+–	O
+H	O
+).	O
+
+However	O
+,	O
+ligand	O
+‐	O
+induced	O
+GREB1	B-protein
+levels	O
+were	O
+generally	O
+not	O
+determined	O
+by	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+(	O
+Fig	O
+3E	O
+lanes	O
+5	O
+–	O
+19	O
+),	O
+consistent	O
+with	O
+an	O
+alternate	O
+causality	O
+model	O
+(	O
+Fig	O
+1E	O
+).	O
+
+Out	O
+of	O
+11	O
+indirect	O
+modulator	O
+series	O
+in	O
+cluster	O
+2	O
+or	O
+3	O
+,	O
+only	O
+the	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+class	O
+had	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+profiles	O
+that	O
+predicted	O
+GREB1	B-protein
+levels	O
+(	O
+Fig	O
+3E	O
+lane	O
+12	O
+).	O
+
+These	O
+results	O
+suggest	O
+that	O
+compounds	O
+that	O
+show	O
+cell	O
+‐	O
+specific	O
+signaling	O
+do	O
+not	O
+activate	O
+GREB1	B-protein
+,	O
+or	O
+use	O
+coactivators	O
+other	O
+than	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+to	O
+control	O
+GREB1	B-protein
+expression	O
+(	O
+Fig	O
+1E	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	B-experimental_method
+regression	I-experimental_method
+analyses	I-experimental_method
+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
+
+In	O
+cluster	O
+1	O
+,	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+and	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activities	O
+,	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+,	O
+and	O
+GREB1	B-protein
+levels	O
+generally	O
+predicted	O
+the	O
+proliferative	O
+response	O
+(	O
+Fig	O
+3F	O
+lanes	O
+2	O
+–	O
+4	O
+).	O
+
+With	O
+the	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+compounds	O
+,	O
+NCOA3	B-protein
+and	O
+GREB1	B-protein
+showed	O
+near	O
+perfect	O
+prediction	O
+of	O
+proliferation	O
+(	O
+Fig	O
+EV3G	O
+),	O
+with	O
+unexplained	O
+variance	O
+similar	O
+to	O
+the	O
+noise	O
+in	O
+the	O
+assays	O
+.	O
+
+The	O
+lack	O
+of	O
+significant	O
+predictors	O
+for	O
+OBHS	B-chemical
+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
+
+The	O
+significant	O
+correlations	O
+with	O
+GREB1	B-protein
+expression	O
+and	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+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	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+determines	O
+GREB1	B-protein
+expression	O
+,	O
+which	O
+then	O
+drives	O
+proliferation	O
+.	O
+
+Despite	O
+this	O
+phenotypic	O
+variance	O
+,	O
+proliferation	O
+was	O
+not	O
+generally	O
+predicted	O
+by	O
+correlated	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+and	O
+GREB1	B-protein
+induction	O
+(	O
+Figs	O
+3F	O
+lanes	O
+5	O
+–	O
+19	O
+,	O
+and	O
+EV3H	O
+).	O
+
+Out	O
+of	O
+15	O
+ligand	O
+series	O
+in	O
+these	O
+clusters	O
+,	O
+only	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+induced	O
+a	O
+proliferative	O
+response	O
+that	O
+was	O
+predicted	O
+by	O
+GREB1	B-protein
+levels	O
+,	O
+which	O
+were	O
+not	O
+determined	O
+by	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+(	O
+Fig	O
+3E	O
+and	O
+F	O
+lane	O
+10	O
+).	O
+
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+cyclofenil	B-chemical
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+,	O
+and	O
+imidazopyridine	B-chemical
+analogs	O
+had	O
+NCOA1	B-protein
+/	I-protein
+3	I-protein
+recruitment	O
+profiles	O
+that	O
+predicted	O
+their	O
+proliferative	O
+effects	O
+,	O
+without	O
+determining	O
+GREB1	B-protein
+levels	O
+(	O
+Fig	O
+3E	O
+and	O
+F	O
+,	O
+lanes	O
+5	O
+and	O
+14	O
+–	O
+16	O
+).	O
+
+Similarly	O
+,	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+,	O
+cyclofenil	B-chemical
+‐	I-chemical
+ASC	I-chemical
+,	O
+and	O
+OBHS	B-chemical
+‐	I-chemical
+ASC	I-chemical
+had	O
+positively	O
+correlated	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+and	O
+GREB1	B-protein
+levels	O
+,	O
+but	O
+none	O
+of	O
+these	O
+activities	O
+determined	O
+their	O
+proliferative	O
+effects	O
+(	O
+Fig	O
+3E	O
+and	O
+F	O
+lanes	O
+11	O
+–	O
+12	O
+and	O
+18	O
+).	O
+
+For	O
+ligands	O
+that	O
+show	O
+cell	O
+‐	O
+specific	O
+signaling	O
+,	O
+ERα	B-protein
+‐	O
+mediated	O
+recruitment	O
+of	O
+other	O
+coregulators	O
+and	O
+activation	O
+of	O
+other	O
+target	O
+genes	O
+likely	O
+determine	O
+their	O
+proliferative	O
+effects	O
+on	O
+MCF	O
+‐	O
+7	O
+cells	O
+.	O
+
+NCOA3	B-protein
+occupancy	O
+at	O
+GREB1	B-protein
+did	O
+not	O
+predict	O
+the	O
+proliferative	O
+response	O
+
+We	O
+also	O
+questioned	O
+whether	O
+promoter	O
+occupancy	O
+by	O
+coactivators	O
+is	O
+statistically	O
+robust	O
+and	O
+reproducible	O
+for	O
+ligand	O
+class	O
+analysis	O
+using	O
+a	O
+chromatin	B-experimental_method
+immunoprecipitation	I-experimental_method
+(	I-experimental_method
+ChIP	I-experimental_method
+)‐	I-experimental_method
+based	I-experimental_method
+quantitative	I-experimental_method
+assay	I-experimental_method
+,	I-experimental_method
+and	O
+whether	O
+it	O
+has	O
+a	O
+better	O
+predictive	O
+power	O
+than	O
+the	O
+M2H	B-experimental_method
+assay	I-experimental_method
+.	O
+
+ERα	B-protein
+and	O
+NCOA3	B-protein
+cycle	O
+on	O
+and	O
+off	O
+the	O
+GREB1	B-protein
+promoter	O
+(	O
+Nwachukwu	O
+et	O
+al	O
+,	O
+2014	O
+).	O
+
+Therefore	O
+,	O
+we	O
+first	O
+performed	O
+a	O
+time	B-experimental_method
+‐	I-experimental_method
+course	I-experimental_method
+study	I-experimental_method
+,	O
+and	O
+found	O
+that	O
+E2	B-chemical
+and	O
+the	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analog	O
+,	O
+AAPII	B-chemical
+‐	I-chemical
+151	I-chemical
+‐	I-chemical
+4	I-chemical
+,	O
+induced	O
+recruitment	O
+of	O
+NCOA3	B-protein
+to	O
+the	O
+GREB1	B-protein
+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
+
+At	O
+this	O
+time	O
+point	O
+,	O
+other	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analogs	O
+also	O
+induced	O
+recruitment	O
+of	O
+NCOA3	B-protein
+at	O
+this	O
+site	O
+to	O
+varying	O
+degrees	O
+(	O
+Fig	O
+4B	O
+).	O
+
+The	O
+Z	B-evidence
+’	I-evidence
+for	O
+this	O
+assay	O
+was	O
+0	O
+.	O
+6	O
+,	O
+showing	O
+statistical	O
+robustness	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+).	O
+
+We	O
+prepared	O
+biological	O
+replicates	O
+with	O
+different	O
+cell	O
+passage	O
+numbers	O
+and	O
+separately	O
+prepared	O
+samples	O
+,	O
+which	O
+showed	O
+r	B-evidence
+2	I-evidence
+of	O
+0	O
+.	O
+81	O
+,	O
+demonstrating	O
+high	O
+reproducibility	O
+(	O
+Fig	O
+4C	O
+).	O
+
+NCOA3	B-protein
+occupancy	O
+at	O
+GREB1	B-protein
+is	O
+statistically	O
+robust	O
+but	O
+does	O
+not	O
+predict	O
+transcriptional	O
+activity	O
+
+Kinetic	B-experimental_method
+ChIP	I-experimental_method
+assay	I-experimental_method
+examining	O
+recruitment	O
+of	O
+NCOA3	B-protein
+to	O
+the	O
+GREB1	B-protein
+gene	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+stimulated	O
+with	O
+E2	B-chemical
+or	O
+the	O
+indicated	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analog	O
+.	O
+
+NCOA3	B-protein
+occupancy	O
+at	O
+GREB1	B-protein
+was	O
+compared	O
+by	O
+ChIP	B-experimental_method
+assay	I-experimental_method
+45	O
+min	O
+after	O
+stimulation	O
+with	O
+vehicle	O
+,	O
+E2	B-chemical
+,	O
+or	O
+the	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analogs	O
+.	O
+
+In	O
+panel	O
+(	O
+B	O
+),	O
+the	O
+average	O
+recruitment	O
+of	O
+two	O
+biological	O
+replicates	O
+are	O
+shown	O
+as	O
+mean	O
++	O
+SEM	O
+,	O
+and	O
+the	O
+Z	B-evidence
+‐	I-evidence
+score	I-evidence
+is	O
+indicated	O
+.	O
+
+In	O
+panel	O
+(	O
+C	O
+),	O
+correlation	B-experimental_method
+analysis	I-experimental_method
+was	O
+performed	O
+for	O
+two	O
+biological	O
+replicates	O
+.	O
+
+Linear	B-experimental_method
+regression	I-experimental_method
+analyses	I-experimental_method
+comparing	O
+the	O
+ability	O
+of	O
+NCOA3	B-protein
+recruitment	O
+,	O
+measured	O
+by	O
+ChIP	B-experimental_method
+or	O
+M2H	B-experimental_method
+,	O
+to	O
+predict	O
+other	O
+agonist	O
+activities	O
+of	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analogs	O
+.	O
+*	O
+Significant	O
+positive	O
+correlation	O
+(	O
+F	B-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+for	O
+nonzero	O
+slope	O
+,	O
+P	B-evidence
+‐	I-evidence
+value	I-evidence
+).	O
+
+The	O
+M2H	B-experimental_method
+assay	I-experimental_method
+for	O
+NCOA3	B-protein
+recruitment	O
+broadly	O
+correlated	O
+with	O
+the	O
+other	O
+assays	O
+,	O
+and	O
+was	O
+predictive	O
+for	O
+GREB1	B-protein
+expression	O
+and	O
+cell	O
+proliferation	O
+(	O
+Fig	O
+3E	O
+).	O
+
+However	O
+,	O
+the	O
+ChIP	B-experimental_method
+assays	I-experimental_method
+for	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+‐	O
+induced	O
+recruitment	O
+of	O
+NCOA3	B-protein
+to	O
+the	O
+GREB1	B-protein
+promoter	O
+did	O
+not	O
+correlate	O
+with	O
+any	O
+of	O
+the	O
+other	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+activity	O
+profiles	O
+(	O
+Fig	O
+4D	O
+),	O
+although	O
+the	O
+positive	O
+correlation	O
+between	O
+ChIP	B-experimental_method
+assays	I-experimental_method
+and	O
+NCOA3	B-protein
+recruitment	O
+via	O
+M2H	B-experimental_method
+assay	I-experimental_method
+showed	O
+a	O
+trend	O
+toward	O
+significance	O
+with	O
+r	B-evidence
+2	I-evidence
+=	O
+0	O
+.	O
+36	O
+and	O
+P	B-evidence
+=	O
+0	O
+.	O
+09	O
+(	O
+F	B-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+for	O
+nonzero	O
+slope	O
+).	O
+
+Thus	O
+,	O
+the	O
+simplified	O
+coactivator	B-experimental_method
+‐	I-experimental_method
+binding	I-experimental_method
+assay	I-experimental_method
+showed	O
+much	O
+greater	O
+predictive	O
+power	O
+than	O
+the	O
+ChIP	B-experimental_method
+assay	I-experimental_method
+for	O
+ligand	O
+‐	O
+specific	O
+effects	O
+on	O
+GREB1	B-protein
+expression	O
+and	O
+cell	O
+proliferation	O
+.	O
+
+ERβ	B-protein
+activity	O
+is	O
+not	O
+an	O
+independent	O
+predictor	O
+of	O
+cell	O
+‐	O
+specific	O
+activity	O
+
+One	O
+difference	O
+between	O
+MCF	O
+‐	O
+7	O
+breast	O
+cancer	O
+cells	O
+and	O
+Ishikawa	O
+endometrial	O
+cancer	O
+cells	O
+is	O
+the	O
+contribution	O
+of	O
+ERβ	B-protein
+to	O
+estrogenic	O
+response	O
+,	O
+as	O
+Ishikawa	O
+cells	O
+may	O
+express	O
+ERβ	B-protein
+(	O
+Bhat	O
+&	O
+Pezzuto	O
+,	O
+2001	O
+).	O
+
+When	O
+overexpressed	B-experimental_method
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+,	O
+ERβ	B-protein
+alters	O
+E2	B-chemical
+‐	O
+induced	O
+expression	O
+of	O
+only	O
+a	O
+subset	O
+of	O
+ERα	B-protein
+‐	O
+target	O
+genes	O
+(	O
+Wu	O
+et	O
+al	O
+,	O
+2011	O
+),	O
+raising	O
+the	O
+possibility	O
+that	O
+ligand	O
+‐	O
+induced	O
+ERβ	B-protein
+activity	O
+may	O
+contribute	O
+to	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activities	O
+,	O
+and	O
+thus	O
+underlie	O
+the	O
+lack	O
+of	O
+correlation	O
+between	O
+the	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+and	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+activities	O
+or	O
+GREB1	B-protein
+levels	O
+induced	O
+by	O
+cell	O
+‐	O
+specific	O
+modulators	O
+in	O
+cluster	O
+2	O
+and	O
+cluster	O
+3	O
+(	O
+Fig	O
+3C	O
+).	O
+
+To	O
+test	O
+this	O
+idea	O
+,	O
+we	O
+determined	O
+the	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERβ	O
+activity	O
+profiles	O
+of	O
+the	O
+ligands	O
+(	O
+Fig	O
+EV1	O
+).	O
+
+All	O
+direct	O
+modulator	O
+and	O
+two	O
+indirect	O
+modulator	O
+scaffolds	O
+(	O
+OBHS	B-chemical
+and	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+)	O
+lacked	O
+ERβ	O
+agonist	O
+activity	O
+.	O
+
+For	O
+most	O
+scaffolds	O
+,	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERβ	O
+and	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activities	O
+were	O
+not	O
+correlated	O
+,	O
+except	O
+for	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+cyclofenil	B-chemical
+analogs	O
+,	O
+which	O
+showed	O
+moderate	O
+but	O
+significant	O
+correlations	O
+(	O
+Fig	O
+EV4A	O
+).	O
+
+Nevertheless	O
+,	O
+the	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activities	O
+of	O
+both	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+cyclofenil	B-chemical
+analogs	O
+were	O
+better	O
+predicted	O
+by	O
+their	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+than	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERβ	B-protein
+activities	O
+(	O
+Fig	O
+EV4A	O
+and	O
+B	O
+).	O
+
+ERβ	B-protein
+activity	O
+is	O
+not	O
+an	O
+independent	O
+predictor	O
+of	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+
+ERβ	B-protein
+activity	O
+in	O
+HepG2	O
+cells	O
+rarely	O
+correlates	O
+with	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+.	O
+
+ERα	B-protein
+activity	O
+of	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+cyclofenil	B-chemical
+analogs	O
+correlates	O
+with	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+.	O
+
+Data	O
+information	O
+:	O
+The	O
+r	O
+2	O
+and	O
+P	B-evidence
+values	I-evidence
+for	O
+the	O
+indicated	O
+correlations	O
+are	O
+shown	O
+in	O
+both	O
+panels	O
+.	O
+*	O
+Significant	O
+positive	O
+correlation	O
+(	O
+F	B-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+for	O
+nonzero	O
+slope	O
+,	O
+P	B-evidence
+‐	I-evidence
+value	I-evidence
+)	O
+
+To	O
+overcome	O
+barriers	O
+to	O
+crystallization	B-experimental_method
+of	O
+ERα	B-protein
+LBD	B-structure_element
+complexes	O
+,	O
+we	O
+developed	O
+a	O
+conformation	B-experimental_method
+‐	I-experimental_method
+trapping	I-experimental_method
+X	I-experimental_method
+‐	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+approach	O
+using	O
+the	O
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+mutation	O
+(	O
+Nettles	O
+et	O
+al	O
+,	O
+2008	O
+;	O
+Bruning	O
+et	O
+al	O
+,	O
+2010	O
+;	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+
+To	O
+further	O
+validate	O
+this	O
+approach	O
+,	O
+we	O
+solved	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+the	O
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+LBD	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+diethylstilbestrol	B-chemical
+(	O
+DES	B-chemical
+),	O
+which	O
+bound	O
+identically	O
+in	O
+the	O
+wild	B-protein_state
+‐	I-protein_state
+type	I-protein_state
+and	O
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+LBDs	B-structure_element
+,	O
+demonstrating	O
+again	O
+that	O
+this	O
+surface	O
+mutation	O
+stabilizes	O
+h12	B-structure_element
+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
+
+Using	O
+this	O
+approach	O
+,	O
+we	O
+solved	B-experimental_method
+76	O
+ERα	B-protein
+LBD	B-structure_element
+structures	B-evidence
+in	O
+the	O
+active	B-protein_state
+conformation	I-protein_state
+and	O
+bound	B-protein_state
+to	I-protein_state
+ligands	I-protein_state
+studied	O
+here	O
+(	O
+Appendix	O
+Fig	O
+S1C	O
+).	O
+
+Eleven	O
+of	O
+these	O
+structures	B-evidence
+have	O
+been	O
+published	O
+,	O
+while	O
+65	O
+are	O
+new	O
+,	O
+including	O
+the	O
+DES	B-protein_state
+‐	I-protein_state
+bound	I-protein_state
+ERα	B-mutant
+‐	I-mutant
+Y537S	I-mutant
+LBD	B-structure_element
+.	O
+
+We	O
+present	O
+57	O
+of	O
+these	O
+new	O
+structures	B-evidence
+here	O
+(	O
+Dataset	O
+EV2	O
+),	O
+while	O
+the	O
+remaining	O
+eight	O
+new	O
+structures	B-evidence
+bound	B-protein_state
+to	I-protein_state
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+analogs	O
+will	O
+be	O
+published	O
+elsewhere	O
+(	O
+S	O
+.	O
+Srinivasan	O
+et	O
+al	O
+,	O
+in	O
+preparation	O
+).	O
+
+Examining	O
+many	O
+closely	O
+related	O
+structures	B-evidence
+allows	O
+us	O
+to	O
+visualize	O
+subtle	O
+structural	O
+differences	O
+,	O
+in	O
+effect	O
+using	O
+X	B-experimental_method
+‐	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+as	O
+a	O
+systems	O
+biology	O
+tool	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	B-structure_element
+dynamics	O
+.	O
+
+Based	O
+on	O
+our	O
+original	O
+OBHS	B-chemical
+structure	B-evidence
+,	O
+the	O
+OBHS	B-chemical
+,	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+,	O
+and	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+compounds	O
+were	O
+modified	O
+with	O
+h11	B-structure_element
+‐	O
+directed	O
+pendant	O
+groups	O
+(	O
+Zheng	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Zhu	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Liao	O
+et	O
+al	O
+,	O
+2014	O
+).	O
+
+Superposing	B-experimental_method
+the	O
+LBDs	B-structure_element
+based	O
+on	O
+the	O
+class	O
+of	O
+bound	O
+ligands	O
+provides	O
+an	O
+ensemble	O
+view	O
+of	O
+the	O
+structural	O
+variance	O
+and	O
+clarifies	O
+what	O
+part	O
+of	O
+the	O
+ligand	B-site
+‐	I-site
+binding	I-site
+pocket	I-site
+is	O
+differentially	O
+perturbed	O
+or	O
+targeted	O
+.	O
+
+The	O
+24	O
+structures	B-evidence
+containing	O
+OBHS	B-chemical
+,	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+,	O
+or	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+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	B-structure_element
+—	O
+the	O
+C	O
+‐	O
+terminal	O
+end	O
+of	O
+h11	B-structure_element
+(	O
+Figs	O
+5B	O
+and	O
+C	O
+,	O
+and	O
+EV5B	O
+),	O
+which	O
+in	O
+turn	O
+nudges	O
+h12	B-structure_element
+(	O
+Fig	O
+5C	O
+and	O
+D	O
+).	O
+
+We	O
+observed	O
+that	O
+the	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+analogs	O
+displaced	O
+h11	B-structure_element
+along	O
+a	O
+vector	O
+away	O
+from	O
+Leu354	B-residue_name_number
+in	O
+a	O
+region	O
+of	O
+h3	B-structure_element
+that	O
+is	O
+unaffected	O
+by	O
+the	O
+ligands	O
+,	O
+and	O
+toward	O
+the	O
+dimer	B-site
+interface	I-site
+.	O
+
+For	O
+the	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+,	O
+the	O
+displacement	O
+of	O
+h11	B-structure_element
+was	O
+in	O
+a	O
+perpendicular	O
+direction	O
+,	O
+away	O
+from	O
+Ile424	B-residue_name_number
+in	O
+h8	B-structure_element
+and	O
+toward	O
+h12	B-structure_element
+.	O
+
+Remarkably	O
+,	O
+these	O
+individual	O
+inter	B-evidence
+‐	I-evidence
+atomic	I-evidence
+distances	I-evidence
+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	B-evidence
+structures	I-evidence
+.	O
+
+Structure	B-experimental_method
+‐	I-experimental_method
+class	I-experimental_method
+analysis	I-experimental_method
+of	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+.	O
+
+Triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+superposed	B-experimental_method
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+are	O
+shown	O
+.	O
+
+Arrows	O
+indicate	O
+chemical	O
+variance	O
+in	O
+the	O
+orientation	O
+of	O
+the	O
+different	O
+h11	B-structure_element
+‐	O
+directed	O
+ligand	O
+side	O
+groups	O
+(	O
+PDB	O
+5DK9	O
+,	O
+5DKB	O
+,	O
+5DKE	O
+,	O
+5DKG	O
+,	O
+5DKS	O
+,	O
+5DL4	O
+,	O
+5DLR	O
+,	O
+5DMC	O
+,	O
+5DMF	O
+and	O
+5DP0	O
+).	O
+
+Triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+induce	O
+variance	O
+of	O
+ERα	B-protein
+conformations	O
+at	O
+the	O
+C	O
+‐	O
+terminal	O
+region	O
+of	O
+h11	B-structure_element
+.	O
+
+Panel	O
+(	O
+B	O
+)	O
+shows	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analog	O
+‐	O
+bound	O
+ERα	B-protein
+LBD	B-structure_element
+(	O
+PDB	O
+5DLR	O
+).	O
+
+The	O
+h11	B-site
+–	I-site
+h12	I-site
+interface	I-site
+(	O
+circled	O
+)	O
+includes	O
+the	O
+C	O
+‐	O
+terminal	O
+part	O
+of	O
+h11	B-structure_element
+.	O
+
+This	O
+region	O
+was	O
+expanded	O
+in	O
+panel	O
+(	O
+C	O
+),	O
+where	O
+the	O
+10	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analog	O
+‐	O
+bound	O
+ERα	B-protein
+LBD	B-structure_element
+structures	B-evidence
+(	O
+see	O
+Datasets	O
+EV1	O
+and	O
+EV2	O
+)	O
+were	O
+superposed	B-experimental_method
+to	O
+show	O
+variations	O
+in	O
+the	O
+h11	B-structure_element
+C	O
+‐	O
+terminus	O
+(	O
+PDB	O
+5DK9	O
+,	O
+5DKB	O
+,	O
+5DKE	O
+,	O
+5DKG	O
+,	O
+5DKS	O
+,	O
+5DL4	O
+,	O
+5DLR	O
+,	O
+5DMC	O
+,	O
+5DMF	O
+,	O
+and	O
+5DP0	O
+).	O
+
+ERα	B-protein
+LBDs	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+diethylstilbestrol	B-chemical
+(	O
+DES	B-chemical
+)	O
+or	O
+a	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analog	O
+were	O
+superposed	B-experimental_method
+to	O
+show	O
+that	O
+the	O
+ligand	O
+‐	O
+induced	O
+difference	O
+in	O
+h11	B-structure_element
+conformation	O
+is	O
+transmitted	O
+to	O
+the	O
+C	O
+‐	O
+terminus	O
+of	O
+h12	B-structure_element
+(	O
+PDB	O
+4ZN7	O
+,	O
+5DMC	O
+).	O
+
+Inter	B-evidence
+‐	I-evidence
+atomic	I-evidence
+distances	I-evidence
+predict	O
+the	O
+proliferative	O
+effects	O
+of	O
+specific	O
+ligand	O
+series	O
+.	O
+
+Ile424	B-residue_name_number
+–	O
+His524	B-residue_name_number
+distance	B-evidence
+measured	O
+in	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+correlates	O
+with	O
+the	O
+proliferative	O
+effect	O
+of	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+Leu354	B-residue_name_number
+–	O
+Leu525	B-residue_name_number
+distance	B-evidence
+correlates	O
+with	O
+the	O
+proliferative	O
+effects	O
+of	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+analogs	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+.	O
+
+Structure	B-experimental_method
+‐	I-experimental_method
+class	I-experimental_method
+analysis	I-experimental_method
+of	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analogs	O
+.	O
+
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+side	O
+groups	O
+subtly	O
+nudge	O
+h12	B-structure_element
+Leu540	B-residue_name_number
+.	O
+
+ERα	B-protein
+LBD	B-structure_element
+structures	B-evidence
+bound	B-protein_state
+to	I-protein_state
+4	O
+distinct	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analogs	O
+were	O
+superposed	B-experimental_method
+(	O
+PDB	O
+4	O
+IU7	O
+,	O
+4IV4	O
+,	O
+4IVW	O
+,	O
+4IW6	O
+)	O
+(	O
+see	O
+Datasets	O
+EV1	O
+and	O
+EV2	O
+).	O
+
+Structure	B-experimental_method
+‐	I-experimental_method
+class	I-experimental_method
+analysis	I-experimental_method
+of	O
+indirect	O
+modulators	O
+
+Structure	B-experimental_method
+‐	I-experimental_method
+class	I-experimental_method
+analysis	I-experimental_method
+of	O
+indirect	O
+modulators	O
+in	O
+cluster	O
+1	O
+.	O
+
+Crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+OBHS	B-chemical
+and	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+analogs	O
+were	O
+superposed	B-experimental_method
+.	O
+
+Arrows	O
+indicate	O
+chemical	O
+variance	O
+in	O
+the	O
+orientation	O
+of	O
+the	O
+different	O
+h11	B-structure_element
+‐	O
+directed	O
+ligand	O
+side	O
+groups	O
+.	O
+
+Panel	O
+(	O
+B	O
+)	O
+shows	O
+the	O
+ligand	O
+‐	O
+induced	O
+conformational	O
+variation	O
+at	O
+the	O
+C	O
+‐	O
+terminal	O
+region	O
+of	O
+h11	B-structure_element
+(	O
+OBHS	B-chemical
+:	O
+PDB	O
+4ZN9	O
+,	O
+4ZNH	O
+,	O
+4ZNS	O
+,	O
+4ZNT	O
+,	O
+4ZNU	O
+,	O
+4ZNV	O
+,	O
+and	O
+4ZNW	O
+;	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+:	O
+PDB	O
+4ZUB	O
+,	O
+4ZUC	O
+,	O
+4ZWH	O
+,	O
+4ZWK	O
+,	O
+5BNU	O
+,	O
+5BP6	O
+,	O
+5BPR	O
+,	O
+and	O
+5BQ4	O
+).	O
+
+Structure	B-experimental_method
+‐	I-experimental_method
+class	I-experimental_method
+analysis	I-experimental_method
+of	O
+indirect	O
+modulators	O
+in	O
+clusters	O
+2	O
+and	O
+3	O
+.	O
+
+Crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+ligands	O
+with	O
+cell	O
+‐	O
+specific	O
+activities	O
+were	O
+superposed	B-experimental_method
+.	O
+
+The	O
+bound	O
+ligands	O
+are	O
+shown	O
+,	O
+and	O
+arrows	O
+indicate	O
+considerable	O
+variation	O
+in	O
+the	O
+orientation	O
+of	O
+the	O
+different	O
+h3	B-structure_element
+‐,	O
+h8	B-structure_element
+‐,	O
+h11	B-structure_element
+‐,	O
+or	O
+h12	B-structure_element
+‐	O
+directed	O
+ligand	O
+side	O
+groups	O
+.	O
+
+As	O
+visualized	O
+in	O
+four	O
+LBD	B-structure_element
+structures	B-evidence
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+),	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analogs	O
+were	O
+designed	O
+with	O
+small	O
+substitutions	O
+that	O
+slightly	O
+nudge	O
+h12	B-structure_element
+Leu540	B-residue_name_number
+,	O
+without	O
+exiting	O
+the	O
+ligand	B-site
+‐	I-site
+binding	I-site
+pocket	I-site
+(	O
+Fig	O
+5G	O
+and	O
+H	O
+).	O
+
+Therefore	O
+,	O
+changing	O
+h12	B-structure_element
+dynamics	O
+maintains	O
+the	O
+canonical	O
+signaling	O
+pathway	O
+defined	O
+by	O
+E2	B-chemical
+(	O
+Fig	O
+1D	O
+)	O
+to	O
+support	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+‐	O
+driven	O
+signaling	O
+and	O
+recruit	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+for	O
+GREB1	B-protein
+‐	O
+stimulated	O
+proliferation	O
+.	O
+
+Ligands	O
+with	O
+cell	O
+‐	O
+specific	O
+activity	O
+alter	O
+the	O
+shape	O
+of	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+
+Direct	O
+modulators	O
+like	O
+tamoxifen	B-chemical
+drive	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+‐	O
+dependent	O
+cell	O
+‐	O
+specific	O
+activity	O
+by	O
+completely	O
+occluding	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+,	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	B-protein
+activity	O
+.	O
+
+Therefore	O
+,	O
+we	O
+examined	O
+another	O
+50	O
+LBD	B-structure_element
+structures	B-evidence
+containing	O
+ligands	O
+in	O
+clusters	O
+2	O
+and	O
+3	O
+.	O
+
+These	O
+structures	B-evidence
+demonstrated	O
+that	O
+cell	O
+‐	O
+specific	O
+activity	O
+derived	O
+from	O
+altering	O
+the	O
+shape	O
+of	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+without	O
+an	O
+extended	O
+side	O
+chain	O
+.	O
+
+Ligands	O
+in	O
+cluster	O
+2	O
+and	O
+cluster	O
+3	O
+showed	O
+conformational	O
+heterogeneity	O
+in	O
+parts	O
+of	O
+the	O
+scaffold	O
+that	O
+were	O
+directed	O
+toward	O
+multiple	O
+regions	O
+of	O
+the	O
+receptor	O
+including	O
+h3	B-structure_element
+,	O
+h8	B-structure_element
+,	O
+h11	B-structure_element
+,	O
+h12	B-structure_element
+,	O
+and	O
+/	O
+or	O
+the	O
+β	B-structure_element
+‐	I-structure_element
+sheets	I-structure_element
+(	O
+Fig	O
+EV5C	O
+–	O
+G	O
+).	O
+
+For	O
+instance	O
+,	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+2	I-chemical
+and	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+analogs	O
+(	O
+Fig	O
+2	O
+)	O
+had	O
+similar	O
+ERα	B-protein
+activity	O
+profiles	O
+in	O
+the	O
+different	O
+cell	O
+types	O
+(	O
+Fig	O
+EV2A	O
+–	O
+C	O
+),	O
+but	O
+the	O
+2	O
+‐	O
+versus	O
+3	O
+‐	O
+methyl	O
+substituted	O
+phenol	O
+rings	O
+altered	O
+the	O
+correlated	O
+signaling	O
+patterns	O
+in	O
+different	O
+cell	O
+types	O
+(	O
+Fig	O
+3B	O
+lanes	O
+7	O
+and	O
+12	O
+).	O
+
+This	O
+difference	O
+in	O
+ligand	O
+positioning	O
+altered	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+via	O
+a	O
+shift	O
+in	O
+the	O
+N	O
+‐	O
+terminus	O
+of	O
+h12	B-structure_element
+,	O
+which	O
+directly	O
+contacts	O
+the	O
+coactivator	O
+.	O
+
+This	O
+effect	O
+is	O
+evident	O
+in	O
+a	O
+single	O
+structure	B-evidence
+due	O
+to	O
+its	O
+1	O
+Å	O
+magnitude	O
+(	O
+Fig	O
+6A	O
+and	O
+B	O
+).	O
+
+The	O
+shifts	O
+in	O
+h12	B-structure_element
+residues	O
+Asp538	B-residue_name_number
+and	O
+Leu539	B-residue_name_number
+led	O
+to	O
+rotation	O
+of	O
+the	O
+coactivator	O
+peptide	O
+(	O
+Fig	O
+6C	O
+).	O
+
+Thus	O
+,	O
+cell	O
+‐	O
+specific	O
+activity	O
+can	O
+stem	O
+from	O
+perturbation	O
+of	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+without	O
+an	O
+extended	O
+side	O
+chain	O
+,	O
+which	O
+presumably	O
+alters	O
+the	O
+receptor	O
+–	O
+coregulator	O
+interaction	O
+profile	O
+.	O
+
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+2	I-chemical
+/	I-chemical
+3	I-chemical
+analogs	O
+subtly	O
+distort	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+.	O
+
+Panel	O
+(	O
+A	O
+)	O
+shows	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+an	O
+S	B-protein_state
+‐	I-protein_state
+OBHS	I-protein_state
+‐	I-protein_state
+3	I-protein_state
+‐	I-protein_state
+bound	I-protein_state
+ERα	B-protein
+LBD	B-structure_element
+(	O
+PDB	O
+5DUH	O
+).	O
+
+The	O
+h3	B-site
+–	I-site
+h12	I-site
+interface	I-site
+(	O
+circled	O
+)	O
+at	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+(	O
+pink	O
+)	O
+was	O
+expanded	O
+in	O
+panels	O
+(	O
+B	O
+,	O
+C	O
+).	O
+
+The	O
+S	B-protein_state
+‐	I-protein_state
+OBHS	I-protein_state
+‐	I-protein_state
+2	I-protein_state
+/	I-protein_state
+3	I-protein_state
+‐	I-protein_state
+bound	I-protein_state
+ERα	B-protein
+LBDs	B-structure_element
+were	O
+superposed	B-experimental_method
+to	O
+show	O
+shifts	O
+in	O
+h3	B-structure_element
+(	O
+panel	O
+B	O
+)	O
+and	O
+the	O
+NCOA2	B-protein
+peptide	O
+docked	O
+at	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+(	O
+panel	O
+C	O
+).	O
+
+Crystal	B-evidence
+structures	I-evidence
+show	O
+that	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+shift	O
+h3	B-structure_element
+and	O
+h11	B-structure_element
+further	O
+apart	O
+compared	O
+to	O
+an	O
+A	O
+‐	O
+CD	O
+‐	O
+ring	O
+estrogen	B-chemical
+(	O
+PDB	O
+4PPS	O
+,	O
+5DRM	O
+,	O
+5DRJ	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	B-protein_state
+,	I-protein_state
+5	I-protein_state
+‐	I-protein_state
+DTP	I-protein_state
+‐	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+(	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
+
+Average	O
+(	O
+mean	O
++	O
+SEM	O
+)	O
+α	B-evidence
+‐	I-evidence
+carbon	I-evidence
+distance	I-evidence
+measured	O
+from	O
+h3	B-structure_element
+Thr347	B-residue_name_number
+to	O
+h11	B-structure_element
+Leu525	B-residue_name_number
+of	O
+A	B-protein_state
+‐	I-protein_state
+CD	I-protein_state
+‐,	I-protein_state
+2	I-protein_state
+,	I-protein_state
+5	I-protein_state
+‐	I-protein_state
+DTP	I-protein_state
+‐,	I-protein_state
+and	I-protein_state
+3	I-protein_state
+,	I-protein_state
+4	I-protein_state
+‐	I-protein_state
+DTPD	I-protein_state
+‐	I-protein_state
+bound	I-protein_state
+ERα	B-protein
+LBDs	B-structure_element
+.	O
+
+*	O
+Two	O
+‐	O
+tailed	O
+Student	B-experimental_method
+'	I-experimental_method
+s	I-experimental_method
+t	I-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+,	O
+P	B-evidence
+=	O
+0	O
+.	O
+002	O
+(	O
+PDB	O
+A	B-chemical
+‐	I-chemical
+CD	I-chemical
+:	O
+5DI7	O
+,	O
+5DID	O
+,	O
+5DIE	O
+,	O
+5DIG	O
+,	O
+and	O
+4PPS	O
+;	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+:	O
+4IWC	O
+,	O
+5DRM	O
+,	O
+and	O
+5DRJ	O
+;	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+:	O
+5DTV	O
+and	O
+5DU5	O
+).	O
+
+Crystal	B-evidence
+structures	I-evidence
+show	O
+that	O
+a	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+analog	O
+shifts	O
+h3	B-structure_element
+(	O
+F	B-structure_element
+)	O
+and	O
+the	O
+NCOA2	B-protein
+(	O
+G	O
+)	O
+peptide	O
+compared	O
+to	O
+an	O
+A	B-chemical
+‐	I-chemical
+CD	I-chemical
+‐	O
+ring	O
+estrogen	B-chemical
+(	O
+PDB	O
+4PPS	O
+,	O
+5DTV	O
+).	O
+
+Hierarchical	B-experimental_method
+clustering	I-experimental_method
+of	O
+ligand	O
+‐	O
+specific	O
+binding	O
+of	O
+154	O
+interacting	O
+peptides	O
+to	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+was	O
+performed	O
+in	O
+triplicate	O
+by	O
+MARCoNI	B-experimental_method
+analysis	I-experimental_method
+.	O
+
+The	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+showed	O
+perturbation	O
+of	O
+h11	B-structure_element
+,	O
+as	O
+well	O
+as	O
+h3	B-structure_element
+,	O
+which	O
+forms	O
+part	O
+of	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+.	O
+
+These	O
+compounds	O
+bind	O
+the	O
+LBD	B-structure_element
+in	O
+an	O
+unusual	O
+fashion	O
+because	O
+they	O
+have	O
+a	O
+phenol	O
+‐	O
+to	O
+‐	O
+phenol	O
+length	O
+of	O
+~	O
+12	O
+Å	O
+,	O
+which	O
+is	O
+longer	O
+than	O
+steroids	O
+and	O
+other	O
+prototypical	O
+ERα	B-protein
+agonists	O
+that	O
+are	O
+~	O
+10	O
+Å	O
+in	O
+length	O
+.	O
+
+One	O
+phenol	O
+pushed	O
+further	O
+toward	O
+h3	B-structure_element
+(	O
+Fig	O
+6D	O
+),	O
+while	O
+the	O
+other	O
+phenol	O
+pushed	O
+toward	O
+the	O
+C	O
+‐	O
+terminus	O
+of	O
+h11	B-structure_element
+to	O
+a	O
+greater	O
+extent	O
+than	O
+A	B-chemical
+‐	I-chemical
+CD	I-chemical
+‐	O
+ring	O
+estrogens	B-chemical
+(	O
+Nwachukwu	O
+et	O
+al	O
+,	O
+2014	O
+),	O
+which	O
+are	O
+close	O
+structural	O
+analogs	O
+of	O
+E2	B-chemical
+that	O
+lack	O
+a	O
+B	O
+‐	O
+ring	O
+(	O
+Fig	O
+2	O
+).	O
+
+To	O
+quantify	O
+this	O
+difference	O
+,	O
+we	O
+compared	O
+the	O
+distance	B-evidence
+between	O
+α	O
+‐	O
+carbons	O
+at	O
+h3	B-structure_element
+Thr347	B-residue_name_number
+and	O
+h11	B-structure_element
+Leu525	B-residue_name_number
+in	O
+the	O
+set	O
+of	O
+structures	B-evidence
+containing	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+(	O
+n	O
+=	O
+3	O
+)	O
+or	O
+A	B-chemical
+‐	I-chemical
+CD	I-chemical
+‐	O
+ring	O
+analogs	O
+(	O
+n	O
+=	O
+5	O
+)	O
+(	O
+Fig	O
+6E	O
+).	O
+
+We	O
+observed	O
+a	O
+difference	O
+of	O
+0	O
+.	O
+4	O
+Å	O
+that	O
+was	O
+significant	O
+(	O
+two	O
+‐	O
+tailed	O
+Student	B-experimental_method
+'	I-experimental_method
+s	I-experimental_method
+t	I-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+,	O
+P	B-evidence
+=	O
+0	O
+.	O
+002	O
+)	O
+due	O
+to	O
+the	O
+very	O
+tight	O
+clustering	O
+of	O
+the	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+‐	O
+induced	O
+LBD	B-structure_element
+conformation	O
+.	O
+
+The	O
+shifts	O
+in	O
+h3	B-structure_element
+suggest	O
+these	O
+compounds	O
+are	O
+positioned	O
+to	O
+alter	O
+coregulator	O
+preferences	O
+.	O
+
+The	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+scaffolds	O
+are	O
+isomeric	O
+,	O
+but	O
+with	O
+aryl	O
+groups	O
+at	O
+obtuse	O
+and	O
+acute	O
+angles	O
+,	O
+respectively	O
+(	O
+Fig	O
+2	O
+).	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+ERα	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+is	O
+unknown	O
+;	O
+however	O
+,	O
+we	O
+solved	B-experimental_method
+two	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+ERα	B-protein
+bound	B-protein_state
+to	I-protein_state
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+analogs	O
+and	O
+one	O
+structure	B-evidence
+containing	O
+a	O
+furan	B-chemical
+ligand	O
+—	O
+all	O
+of	O
+which	O
+have	O
+a	O
+3	O
+,	O
+4	O
+‐	O
+diaryl	O
+configuration	O
+(	O
+Fig	O
+2	O
+;	O
+Datasets	O
+EV1	O
+and	O
+EV2	O
+).	O
+
+In	O
+these	O
+structures	B-evidence
+,	O
+the	O
+A	O
+‐	O
+ring	O
+mimetic	O
+of	O
+the	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+scaffold	O
+bound	O
+h3	B-structure_element
+Glu353	B-residue_name_number
+as	O
+expected	O
+,	O
+but	O
+the	O
+other	O
+phenol	O
+wrapped	O
+around	O
+h3	B-structure_element
+to	O
+form	O
+a	O
+hydrogen	O
+bond	O
+with	O
+Thr347	B-residue_name_number
+,	O
+indicating	O
+a	O
+change	O
+in	O
+binding	O
+epitopes	O
+in	O
+the	O
+ERα	B-protein
+ligand	B-site
+‐	I-site
+binding	I-site
+pocket	I-site
+(	O
+Fig	O
+6F	O
+).	O
+
+The	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+analogs	O
+also	O
+induced	O
+a	O
+shift	O
+in	O
+h3	B-structure_element
+positioning	O
+,	O
+which	O
+translated	O
+again	O
+into	O
+a	O
+shift	O
+in	O
+the	O
+bound	O
+coactivator	O
+peptide	O
+(	O
+Fig	O
+6F	O
+).	O
+
+Therefore	O
+,	O
+these	O
+indirect	O
+modulators	O
+,	O
+including	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+2	I-chemical
+,	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+,	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+and	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+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	B-structure_element
+and	O
+h12	B-structure_element
+that	O
+were	O
+transmitted	O
+to	O
+the	O
+coactivator	O
+peptide	O
+via	O
+an	O
+altered	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+.	O
+
+To	O
+test	O
+whether	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+shows	O
+changes	O
+in	O
+shape	O
+in	O
+solution	O
+,	O
+we	O
+used	O
+the	O
+microarray	B-experimental_method
+assay	I-experimental_method
+for	I-experimental_method
+real	I-experimental_method
+‐	I-experimental_method
+time	I-experimental_method
+coregulator	I-experimental_method
+–	I-experimental_method
+nuclear	I-experimental_method
+receptor	I-experimental_method
+interaction	I-experimental_method
+(	O
+MARCoNI	B-experimental_method
+)	O
+analysis	O
+(	O
+Aarts	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+
+Here	O
+,	O
+the	O
+ligand	O
+‐	O
+dependent	O
+interactions	O
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+with	O
+over	O
+150	O
+distinct	O
+LxxLL	B-structure_element
+motif	I-structure_element
+peptides	O
+were	O
+assayed	O
+to	O
+define	O
+structural	O
+fingerprints	O
+for	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+,	O
+in	O
+a	O
+manner	O
+similar	O
+to	O
+the	O
+use	O
+of	O
+phage	B-experimental_method
+display	I-experimental_method
+peptides	I-experimental_method
+as	O
+structural	O
+probes	O
+(	O
+Connor	O
+et	O
+al	O
+,	O
+2001	O
+).	O
+
+Despite	O
+the	O
+similar	O
+average	O
+activities	O
+of	O
+these	O
+ligand	O
+classes	O
+(	O
+Fig	O
+3A	O
+and	O
+B	O
+),	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+displayed	O
+remarkably	O
+different	O
+peptide	O
+recruitment	O
+patterns	O
+(	O
+Fig	O
+6H	O
+),	O
+consistent	O
+with	O
+the	O
+structural	B-experimental_method
+analyses	I-experimental_method
+.	O
+
+Hierarchical	B-experimental_method
+clustering	I-experimental_method
+revealed	O
+that	O
+many	O
+of	O
+the	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+recapitulated	O
+most	O
+of	O
+the	O
+peptide	O
+recruitment	O
+and	O
+dismissal	O
+patterns	O
+observed	O
+with	O
+E2	B-chemical
+(	O
+Fig	O
+6H	O
+).	O
+
+However	O
+,	O
+there	O
+was	O
+a	O
+unique	O
+cluster	O
+of	O
+peptides	O
+that	O
+were	O
+recruited	O
+by	O
+E2	B-chemical
+but	O
+not	O
+the	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+.	O
+
+In	O
+contrast	O
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+dismissed	O
+most	O
+of	O
+the	O
+peptides	O
+from	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+(	O
+Fig	O
+6H	O
+).	O
+
+Thus	O
+,	O
+the	O
+isomeric	O
+attachment	O
+of	O
+diaryl	O
+groups	O
+to	O
+the	O
+thiophene	B-chemical
+core	O
+changed	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+from	O
+inside	O
+the	O
+ligand	B-site
+‐	I-site
+binding	I-site
+pocket	I-site
+,	O
+as	O
+predicted	O
+by	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Together	O
+,	O
+these	O
+findings	O
+suggest	O
+that	O
+without	O
+an	O
+extended	O
+side	O
+chain	O
+,	O
+cell	O
+‐	O
+specific	O
+activity	O
+stems	O
+from	O
+different	O
+coregulator	O
+recruitment	O
+profiles	O
+,	O
+due	O
+to	O
+unique	O
+ligand	O
+‐	O
+induced	O
+conformations	O
+of	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+,	O
+in	O
+addition	O
+to	O
+differential	O
+usage	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+.	O
+
+Indirect	O
+modulators	O
+in	O
+cluster	O
+1	O
+avoid	O
+this	O
+by	O
+perturbing	O
+the	O
+h11	B-site
+–	I-site
+h12	I-site
+interface	I-site
+,	O
+and	O
+modulating	O
+the	O
+dynamics	O
+of	O
+h12	B-structure_element
+without	O
+changing	O
+the	O
+shape	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+when	O
+stabilized	O
+.	O
+
+Our	O
+goal	O
+was	O
+to	O
+identify	O
+a	O
+minimal	O
+set	O
+of	O
+predictors	O
+that	O
+would	O
+link	O
+specific	O
+structural	O
+perturbations	O
+to	O
+ERα	B-protein
+signaling	O
+pathways	O
+that	O
+control	O
+cell	O
+‐	O
+specific	O
+signaling	O
+and	O
+proliferation	O
+.	O
+
+We	O
+found	O
+a	O
+very	O
+strong	O
+set	O
+of	O
+predictors	O
+,	O
+where	O
+ligands	O
+in	O
+cluster	O
+1	O
+,	O
+defined	O
+by	O
+similar	O
+signaling	O
+across	O
+cell	O
+types	O
+,	O
+showed	O
+indirect	O
+modulation	O
+of	O
+h12	B-structure_element
+dynamics	O
+via	O
+the	O
+h11	B-site
+–	I-site
+12	I-site
+interface	I-site
+or	O
+slight	O
+contact	O
+with	O
+h12	B-structure_element
+.	O
+
+This	O
+perturbation	O
+determined	O
+proliferation	O
+that	O
+correlated	O
+strongly	O
+with	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+activity	O
+,	O
+recruitment	O
+of	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+family	O
+members	O
+,	O
+and	O
+induction	O
+of	O
+the	O
+GREB1	B-protein
+gene	O
+,	O
+consistent	O
+with	O
+the	O
+canonical	O
+ERα	B-protein
+signaling	O
+pathway	O
+(	O
+Fig	O
+1D	O
+).	O
+
+For	O
+ligands	O
+in	O
+cluster	O
+1	O
+,	O
+deletion	B-experimental_method
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+reduced	O
+activity	O
+to	O
+varying	O
+degrees	O
+,	O
+but	O
+did	O
+not	O
+change	O
+the	O
+underlying	O
+signaling	O
+patterns	O
+established	O
+through	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+.	O
+
+In	O
+contrast	O
+,	O
+an	O
+extended	O
+side	O
+chain	O
+designed	O
+to	O
+directly	O
+reposition	O
+h12	B-structure_element
+and	O
+completely	O
+disrupt	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+results	O
+in	O
+cell	O
+‐	O
+specific	O
+signaling	O
+.	O
+
+Compared	O
+to	O
+cluster	O
+1	O
+,	O
+the	O
+structural	O
+rules	O
+are	O
+less	O
+clear	O
+in	O
+clusters	O
+2	O
+and	O
+3	O
+,	O
+but	O
+a	O
+number	O
+of	O
+indirect	O
+modulator	O
+classes	O
+perturbed	O
+the	O
+LBD	B-structure_element
+conformation	O
+at	O
+the	O
+intersection	O
+of	O
+h3	B-structure_element
+,	O
+the	O
+h12	B-structure_element
+N	O
+‐	O
+terminus	O
+,	O
+and	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+.	O
+
+Ligands	O
+in	O
+these	O
+classes	O
+altered	O
+the	O
+shape	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+to	O
+affect	O
+coregulator	O
+preferences	O
+.	O
+
+For	O
+direct	O
+and	O
+indirect	O
+modulators	O
+in	O
+cluster	O
+2	O
+or	O
+3	O
+,	O
+the	O
+canonical	O
+ERα	B-protein
+signaling	O
+pathway	O
+involving	O
+recruitment	O
+of	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+and	O
+induction	O
+of	O
+GREB1	B-protein
+did	O
+not	O
+generally	O
+predict	O
+their	O
+proliferative	O
+effects	O
+,	O
+indicating	O
+an	O
+alternate	O
+causal	O
+model	O
+(	O
+Fig	O
+1E	O
+).	O
+
+These	O
+principles	O
+outlined	O
+above	O
+provide	O
+a	O
+structural	O
+basis	O
+for	O
+how	O
+the	O
+ligand	B-site
+–	I-site
+receptor	I-site
+interface	I-site
+leads	O
+to	O
+different	O
+signaling	O
+specificities	O
+through	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+and	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+.	O
+
+It	O
+is	O
+noteworthy	O
+that	O
+regulation	O
+of	O
+h12	B-structure_element
+dynamics	O
+indirectly	O
+through	O
+h11	B-structure_element
+can	O
+virtually	O
+abolish	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+activity	O
+,	O
+and	O
+yet	O
+still	O
+drive	O
+robust	O
+transcriptional	O
+activity	O
+through	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+,	O
+as	O
+demonstrated	O
+with	O
+the	O
+OBHS	B-chemical
+series	O
+.	O
+
+This	O
+finding	O
+can	O
+be	O
+explained	O
+by	O
+the	O
+fact	O
+that	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+contain	O
+distinct	O
+binding	B-site
+sites	I-site
+for	O
+interaction	O
+with	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+and	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+(	O
+McInerney	O
+et	O
+al	O
+,	O
+1996	O
+;	O
+Webb	O
+et	O
+al	O
+,	O
+1998	O
+),	O
+which	O
+allows	O
+ligands	O
+to	O
+nucleate	O
+ERα	B-complex_assembly
+–	I-complex_assembly
+NCOA1	I-complex_assembly
+/	I-complex_assembly
+2	I-complex_assembly
+/	I-complex_assembly
+3	I-complex_assembly
+interaction	O
+through	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+,	O
+and	O
+reinforce	O
+this	O
+interaction	O
+with	O
+additional	O
+binding	O
+to	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+.	O
+
+Completely	O
+blocking	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+with	O
+an	O
+extended	O
+side	O
+chain	O
+or	O
+altering	O
+the	O
+shape	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+changes	O
+the	O
+preference	O
+away	O
+from	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+for	O
+determining	O
+GREB1	B-protein
+levels	O
+and	O
+proliferation	O
+of	O
+breast	O
+cancer	O
+cells	O
+.	O
+
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+blockade	O
+also	O
+allows	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+to	O
+function	O
+independently	O
+,	O
+which	O
+is	O
+important	O
+since	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+drives	O
+tissue	O
+‐	O
+selective	O
+effects	O
+in	O
+vivo	O
+.	O
+
+This	O
+was	O
+demonstrated	O
+with	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+knockout	O
+mice	O
+that	O
+show	O
+E2	B-chemical
+‐	O
+dependent	O
+vascular	O
+protection	O
+,	O
+but	O
+not	O
+uterine	O
+proliferation	O
+,	O
+thus	O
+highlighting	O
+the	O
+role	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+in	O
+tissue	O
+‐	O
+selective	O
+or	O
+cell	O
+‐	O
+specific	O
+signaling	O
+(	O
+Billon	O
+‐	O
+Gales	O
+et	O
+al	O
+,	O
+2009	O
+;	O
+Abot	O
+et	O
+al	O
+,	O
+2013	O
+).	O
+
+Here	O
+,	O
+we	O
+examined	O
+many	O
+LBD	B-structure_element
+structures	B-evidence
+and	O
+tested	O
+several	O
+variables	O
+that	O
+were	O
+not	O
+predictive	O
+,	O
+including	O
+ERβ	B-protein
+activity	O
+,	O
+the	O
+strength	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+signaling	O
+,	O
+and	O
+NCOA3	B-protein
+occupancy	O
+at	O
+the	O
+GREB1	B-protein
+gene	O
+.	O
+
+Similarly	O
+,	O
+we	O
+visualized	O
+structures	B-evidence
+to	O
+identify	O
+patterns	O
+.	O
+
+For	O
+example	O
+,	O
+phage	B-experimental_method
+display	I-experimental_method
+was	O
+used	O
+to	O
+identify	O
+the	O
+androgen	O
+receptor	O
+interactome	O
+,	O
+which	O
+was	O
+cloned	O
+into	O
+an	O
+M2H	B-experimental_method
+library	O
+and	O
+used	O
+to	O
+identify	O
+clusters	O
+of	O
+ligand	O
+‐	O
+selective	O
+interactions	O
+(	O
+Norris	O
+et	O
+al	O
+,	O
+2009	O
+).	O
+
+Also	O
+,	O
+we	O
+have	O
+used	O
+siRNA	B-experimental_method
+screening	I-experimental_method
+to	O
+identify	O
+a	O
+number	O
+of	O
+coregulators	O
+required	O
+for	O
+ERα	B-protein
+‐	O
+mediated	O
+repression	O
+of	O
+the	O
+IL	O
+‐	O
+6	O
+gene	O
+(	O
+Nwachukwu	O
+et	O
+al	O
+,	O
+2014	O
+).	O
+
+If	O
+we	O
+calculated	O
+inter	B-evidence
+‐	I-evidence
+atomic	I-evidence
+distance	I-evidence
+matrices	I-evidence
+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
+
+We	O
+have	O
+identified	O
+atomic	B-evidence
+vectors	I-evidence
+for	O
+the	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+and	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+classes	O
+that	O
+predict	O
+ligand	O
+response	O
+(	O
+Fig	O
+5E	O
+and	O
+F	O
+).	O
+
+Indeed	O
+,	O
+the	O
+most	O
+anti	O
+‐	O
+proliferative	O
+compound	O
+in	O
+the	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+series	O
+had	O
+a	O
+fulvestrant	O
+‐	O
+like	O
+profile	O
+across	O
+a	O
+battery	O
+of	O
+assays	O
+(	O
+S	O
+.	O
+Srinivasan	O
+et	O
+al	O
+,	O
+in	O
+preparation	O
+).	O
+
+Secondly	O
+,	O
+our	O
+finding	O
+that	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+compounds	O
+do	O
+not	O
+rely	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+signaling	O
+efficacy	O
+may	O
+derive	O
+from	O
+the	O
+slight	O
+contacts	O
+with	O
+h12	B-structure_element
+observed	O
+in	O
+crystal	B-evidence
+structures	I-evidence
+(	O
+Figs	O
+3B	O
+and	O
+5H	O
+),	O
+unlike	O
+other	O
+compounds	O
+in	O
+cluster	O
+1	O
+that	O
+dislocate	O
+h11	B-structure_element
+and	O
+rely	O
+on	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+signaling	O
+efficacy	O
+(	O
+Figs	O
+3B	O
+and	O
+5C	O
+,	O
+and	O
+EV5B	O
+).	O
+
+Some	O
+of	O
+these	O
+ligands	O
+altered	O
+the	O
+shape	O
+of	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+by	O
+perturbing	O
+the	O
+h3	B-site
+–	I-site
+h12	I-site
+interface	I-site
+,	O
+thus	O
+providing	O
+a	O
+route	O
+to	O
+new	O
+SERM	O
+‐	O
+like	O
+activity	O
+profiles	O
+by	O
+combining	O
+indirect	O
+and	O
+direct	O
+modulation	O
+of	O
+receptor	O
+structure	O
+.	O
+
+Incorporation	O
+of	O
+statistical	O
+approaches	O
+to	O
+understand	O
+relationships	O
+between	O
+structure	O
+and	O
+signaling	O
+variables	O
+moves	O
+us	O
+toward	O
+predictive	O
+models	O
+for	O
+complex	O
+ERα	B-protein
+‐	O
+mediated	O
+responses	O
+such	O
+as	O
+in	O
+vivo	O
+uterine	O
+proliferation	O
+or	O
+tumor	O
+growth	O
+,	O
+and	O
+more	O
+generally	O
+toward	O
+structure	O
+‐	O
+based	O
+design	O
+for	O
+other	O
+allosteric	O
+drug	O
+targets	O
+including	O
+GPCRs	B-protein_type
+and	O
+other	O
+nuclear	B-protein_type
+receptors	I-protein_type
+.	O
+
+Investigation	O
+of	O
+the	O
+Interaction	O
+between	O
+Cdc42	B-protein
+and	O
+Its	O
+Effector	O
+TOCA1	B-protein
+
+Transducer	B-protein
+of	I-protein
+Cdc42	I-protein
+-	I-protein
+dependent	I-protein
+actin	I-protein
+assembly	I-protein
+protein	I-protein
+1	I-protein
+(	O
+TOCA1	B-protein
+)	O
+is	O
+an	O
+effector	O
+of	O
+the	O
+Rho	B-protein_type
+family	I-protein_type
+small	I-protein_type
+G	I-protein_type
+protein	I-protein_type
+Cdc42	B-protein
+.	O
+
+It	O
+contains	O
+a	O
+membrane	O
+-	O
+deforming	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+as	O
+well	O
+as	O
+a	O
+Src	B-structure_element
+homology	I-structure_element
+3	I-structure_element
+(	O
+SH3	B-structure_element
+)	O
+domain	O
+and	O
+a	O
+G	B-structure_element
+protein	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+homology	I-structure_element
+region	I-structure_element
+1	I-structure_element
+(	O
+HR1	B-structure_element
+)	O
+domain	O
+.	O
+
+TOCA1	B-protein
+binding	O
+to	O
+Cdc42	B-protein
+leads	O
+to	O
+actin	O
+rearrangements	O
+,	O
+which	O
+are	O
+thought	O
+to	O
+be	O
+involved	O
+in	O
+processes	O
+such	O
+as	O
+endocytosis	O
+,	O
+filopodia	O
+formation	O
+,	O
+and	O
+cell	O
+migration	O
+.	O
+
+We	O
+have	O
+solved	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+of	O
+TOCA1	B-protein
+,	O
+providing	O
+the	O
+first	O
+structural	B-evidence
+data	I-evidence
+for	O
+this	O
+protein	O
+.	O
+
+We	O
+have	O
+found	O
+that	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+,	O
+like	O
+the	O
+closely	O
+related	O
+CIP4	B-protein
+HR1	B-structure_element
+,	O
+has	O
+interesting	O
+structural	O
+features	O
+that	O
+are	O
+not	O
+observed	O
+in	O
+other	O
+HR1	B-structure_element
+domains	O
+.	O
+
+We	O
+have	O
+also	O
+investigated	O
+the	O
+binding	O
+of	O
+the	O
+TOCA	B-protein
+HR1	B-structure_element
+domain	O
+to	O
+Cdc42	B-protein
+and	O
+the	O
+potential	O
+ternary	O
+complex	O
+between	O
+Cdc42	B-protein
+and	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+regions	I-site
+of	O
+TOCA1	B-protein
+and	O
+a	O
+member	O
+of	O
+the	O
+Wiskott	B-protein_type
+-	I-protein_type
+Aldrich	I-protein_type
+syndrome	I-protein_type
+protein	I-protein_type
+family	I-protein_type
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+TOCA1	B-protein
+binds	O
+Cdc42	B-protein
+with	O
+micromolar	O
+affinity	O
+,	O
+in	O
+contrast	O
+to	O
+the	O
+nanomolar	O
+affinity	O
+of	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+region	I-site
+for	O
+Cdc42	B-protein
+.	O
+
+NMR	B-experimental_method
+experiments	O
+show	O
+that	O
+the	O
+Cdc42	B-site
+-	I-site
+binding	I-site
+domain	I-site
+from	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+is	O
+able	O
+to	O
+displace	O
+TOCA1	B-protein
+HR1	B-structure_element
+from	O
+Cdc42	B-protein
+,	O
+whereas	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+domain	O
+but	O
+not	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+inhibits	O
+actin	O
+polymerization	O
+.	O
+
+This	O
+suggests	O
+that	O
+TOCA1	B-protein
+binding	O
+to	O
+Cdc42	B-protein
+is	O
+an	O
+early	O
+step	O
+in	O
+the	O
+Cdc42	B-protein
+-	O
+dependent	O
+pathways	O
+that	O
+govern	O
+actin	O
+dynamics	O
+,	O
+and	O
+the	O
+differential	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+the	O
+effectors	O
+facilitate	O
+a	O
+handover	O
+from	O
+TOCA1	B-protein
+to	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+which	O
+can	O
+then	O
+drive	O
+recruitment	O
+of	O
+the	O
+actin	O
+-	O
+modifying	O
+machinery	O
+.	O
+
+The	O
+Ras	B-protein_type
+superfamily	I-protein_type
+of	O
+small	B-protein_type
+GTPases	I-protein_type
+comprises	O
+over	O
+150	O
+members	O
+that	O
+regulate	O
+a	O
+multitude	O
+of	O
+cellular	O
+processes	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+The	O
+superfamily	O
+can	O
+be	O
+divided	O
+into	O
+five	O
+families	O
+based	O
+on	O
+structural	O
+and	O
+functional	O
+similarities	O
+:	O
+Ras	B-protein_type
+,	O
+Rho	B-protein_type
+,	O
+Rab	B-protein_type
+,	O
+Arf	B-protein_type
+,	O
+and	O
+Ran	B-protein_type
+.	O
+
+All	O
+members	O
+share	O
+a	O
+well	O
+defined	O
+core	O
+structure	O
+of	O
+∼	O
+20	O
+kDa	O
+known	O
+as	O
+the	O
+G	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+is	O
+responsible	O
+for	O
+guanine	B-chemical
+nucleotide	I-chemical
+binding	O
+.	O
+
+These	O
+molecular	O
+switches	O
+cycle	O
+between	O
+active	B-protein_state
+,	O
+GTP	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+,	O
+and	O
+inactive	B-protein_state
+,	O
+GDP	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+,	O
+states	O
+with	O
+the	O
+help	O
+of	O
+auxiliary	O
+proteins	O
+.	O
+
+The	O
+guanine	B-protein_type
+nucleotide	I-protein_type
+exchange	I-protein_type
+factors	I-protein_type
+mediate	O
+formation	O
+of	O
+the	O
+active	B-protein_state
+state	O
+by	O
+promoting	O
+the	O
+dissociation	O
+of	O
+GDP	B-chemical
+,	O
+allowing	O
+GTP	B-chemical
+to	O
+bind	O
+.	O
+
+The	O
+GTPase	B-protein_type
+-	I-protein_type
+activating	I-protein_type
+proteins	I-protein_type
+stimulate	O
+the	O
+rate	O
+of	O
+intrinsic	O
+GTP	B-chemical
+hydrolysis	O
+,	O
+mediating	O
+the	O
+return	O
+to	O
+the	O
+inactive	B-protein_state
+state	O
+(	O
+reviewed	O
+in	O
+Ref	O
+.).	O
+
+The	O
+overall	O
+conformation	O
+of	O
+small	B-protein_type
+G	I-protein_type
+proteins	I-protein_type
+in	O
+the	O
+active	B-protein_state
+and	O
+inactive	B-protein_state
+states	O
+is	O
+similar	O
+,	O
+but	O
+they	O
+differ	O
+significantly	O
+in	O
+two	O
+main	O
+regions	O
+known	O
+as	O
+switch	B-site
+I	I-site
+and	O
+switch	B-site
+II	I-site
+.	O
+
+These	O
+regions	O
+are	O
+responsible	O
+for	O
+“	O
+sensing	O
+”	O
+the	O
+nucleotide	O
+state	O
+,	O
+with	O
+the	O
+GTP	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+state	O
+showing	O
+greater	O
+rigidity	O
+and	O
+the	O
+GDP	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+state	O
+adopting	O
+a	O
+more	O
+relaxed	O
+conformation	O
+(	O
+reviewed	O
+in	O
+Ref	O
+.).	O
+
+In	O
+the	O
+active	B-protein_state
+state	O
+,	O
+G	B-protein_type
+proteins	I-protein_type
+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
+
+The	O
+structures	B-evidence
+of	O
+more	O
+than	O
+60	O
+small	O
+G	B-protein_type
+protein	I-protein_type
+·	O
+effector	O
+complexes	O
+have	O
+been	O
+solved	B-experimental_method
+,	O
+and	O
+,	O
+not	O
+surprisingly	O
+,	O
+the	O
+switch	B-site
+regions	I-site
+have	O
+been	O
+implicated	O
+in	O
+a	O
+large	O
+proportion	O
+of	O
+the	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+effector	O
+interactions	O
+(	O
+reviewed	O
+in	O
+Ref	O
+.).	O
+
+However	O
+,	O
+because	O
+each	O
+of	O
+the	O
+150	O
+members	O
+of	O
+the	O
+superfamily	O
+interacts	O
+with	O
+multiple	O
+effectors	O
+,	O
+there	O
+are	O
+still	O
+a	O
+huge	O
+number	O
+of	O
+known	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+effector	O
+interactions	O
+that	O
+have	O
+not	O
+yet	O
+been	O
+studied	O
+structurally	O
+.	O
+
+The	O
+Rho	B-protein_type
+family	I-protein_type
+comprises	O
+20	O
+members	O
+,	O
+of	O
+which	O
+three	O
+,	O
+RhoA	B-protein
+,	O
+Rac1	B-protein
+,	O
+and	O
+Cdc42	B-protein
+,	O
+have	O
+been	O
+relatively	O
+well	O
+studied	O
+.	O
+
+RhoA	B-protein
+acts	O
+to	O
+rearrange	O
+existing	O
+actin	O
+structures	O
+to	O
+form	O
+stress	O
+fibers	O
+,	O
+whereas	O
+Rac1	B-protein
+and	O
+Cdc42	B-protein
+promote	O
+de	O
+novo	O
+actin	O
+polymerization	O
+to	O
+form	O
+lamellipodia	O
+and	O
+filopodia	O
+,	O
+respectively	O
+.	O
+
+A	O
+number	O
+of	O
+RhoA	B-protein
+and	O
+Rac1	B-protein
+effector	O
+proteins	O
+,	O
+including	O
+the	O
+formins	O
+and	O
+members	O
+of	O
+the	O
+protein	B-protein_type
+kinase	I-protein_type
+C	I-protein_type
+-	I-protein_type
+related	I-protein_type
+kinase	I-protein_type
+(	O
+PRK	B-protein_type
+)	O
+6	B-protein_type
+family	O
+,	O
+along	O
+with	O
+Cdc42	B-protein
+effectors	O
+,	O
+including	O
+the	O
+Wiskott	B-protein_type
+-	I-protein_type
+Aldrich	I-protein_type
+syndrome	I-protein_type
+(	O
+WASP	B-protein_type
+)	O
+family	O
+and	O
+the	O
+transducer	O
+of	O
+Cdc42	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+actin	I-protein_type
+assembly	I-protein_type
+(	O
+TOCA	B-protein_type
+)	O
+family	O
+,	O
+have	O
+also	O
+been	O
+linked	O
+to	O
+the	O
+pathways	O
+that	O
+govern	O
+cytoskeletal	O
+dynamics	O
+.	O
+
+Cdc42	B-protein
+effectors	O
+,	O
+TOCA1	B-protein
+and	O
+the	O
+ubiquitously	O
+expressed	O
+member	O
+of	O
+the	O
+WASP	B-protein_type
+family	I-protein_type
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+have	O
+been	O
+implicated	O
+in	O
+the	O
+regulation	O
+of	O
+actin	O
+polymerization	O
+downstream	O
+of	O
+Cdc42	B-protein
+and	O
+phosphatidylinositol	B-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+-	I-chemical
+bisphosphate	I-chemical
+(	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+).	O
+
+N	B-protein
+-	I-protein
+WASP	I-protein
+exists	O
+in	O
+an	O
+autoinhibited	B-protein_state
+conformation	I-protein_state
+,	O
+which	O
+is	O
+released	O
+upon	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+and	O
+Cdc42	B-protein
+binding	O
+or	O
+by	O
+other	O
+factors	O
+,	O
+such	O
+as	O
+phosphorylation	O
+.	O
+
+Following	O
+their	O
+release	O
+,	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+regions	I-structure_element
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+are	O
+free	O
+to	O
+interact	O
+with	O
+G	B-protein_type
+-	I-protein_type
+actin	I-protein_type
+and	O
+a	O
+known	O
+nucleator	O
+of	O
+actin	O
+assembly	O
+,	O
+the	O
+Arp2	B-complex_assembly
+/	I-complex_assembly
+3	I-complex_assembly
+complex	O
+.	O
+
+The	O
+importance	O
+of	O
+TOCA1	B-protein
+in	O
+actin	O
+polymerization	O
+has	O
+been	O
+demonstrated	O
+in	O
+a	O
+range	O
+of	O
+in	O
+vitro	O
+and	O
+in	O
+vivo	O
+studies	O
+,	O
+but	O
+the	O
+exact	O
+role	O
+of	O
+TOCA1	B-protein
+in	O
+the	O
+many	O
+pathways	O
+involving	O
+actin	O
+assembly	O
+remains	O
+unclear	O
+.	O
+
+The	O
+most	O
+widely	O
+studied	O
+role	O
+of	O
+TOCA1	B-protein
+is	O
+in	O
+membrane	O
+invagination	O
+and	O
+endocytosis	O
+,	O
+although	O
+it	O
+has	O
+also	O
+been	O
+implicated	O
+in	O
+filopodia	O
+formation	O
+,	O
+neurite	O
+elongation	O
+,	O
+transcriptional	O
+reprogramming	O
+via	O
+nuclear	O
+actin	O
+,	O
+and	O
+interaction	O
+with	O
+ZO	B-protein
+-	I-protein
+1	I-protein
+at	O
+tight	O
+junctions	O
+.	O
+
+TOCA1	B-protein
+comprises	O
+an	O
+N	O
+-	O
+terminal	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+,	O
+a	O
+central	B-structure_element
+homology	I-structure_element
+region	I-structure_element
+1	I-structure_element
+(	O
+HR1	B-structure_element
+)	O
+domain	O
+,	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+SH3	B-structure_element
+domain	O
+.	O
+
+The	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+is	O
+a	O
+known	O
+dimerization	O
+,	O
+membrane	O
+-	O
+binding	O
+,	O
+and	O
+membrane	O
+-	O
+deforming	O
+module	O
+found	O
+in	O
+a	O
+number	O
+of	O
+cell	O
+signaling	O
+proteins	O
+.	O
+
+The	O
+TOCA1	B-protein
+SH3	B-structure_element
+domain	O
+has	O
+many	O
+known	O
+binding	O
+partners	O
+,	O
+including	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+dynamin	B-protein
+.	O
+
+The	O
+HR1	B-structure_element
+domain	O
+has	O
+been	O
+directly	O
+implicated	O
+in	O
+the	O
+interaction	O
+between	O
+TOCA1	B-protein
+and	O
+Cdc42	B-protein
+,	O
+representing	O
+the	O
+first	O
+Cdc42	B-protein
+-	O
+HR1	B-structure_element
+domain	O
+interaction	O
+to	O
+be	O
+identified	O
+.	O
+
+Other	O
+HR1	B-structure_element
+domains	O
+studied	O
+so	O
+far	O
+,	O
+including	O
+those	O
+from	O
+the	O
+PRK	B-protein_type
+family	I-protein_type
+,	O
+have	O
+been	O
+found	O
+to	O
+bind	O
+their	O
+cognate	O
+Rho	O
+family	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+binding	O
+partner	O
+with	O
+high	O
+specificity	O
+and	O
+affinities	B-evidence
+in	O
+the	O
+nanomolar	O
+range	O
+.	O
+
+The	O
+structures	B-evidence
+of	O
+the	O
+PRK1	B-protein
+HR1a	B-structure_element
+domain	O
+in	O
+complex	B-protein_state
+with	I-protein_state
+RhoA	B-protein
+and	O
+the	O
+HR1b	B-structure_element
+domain	O
+in	O
+complex	B-protein_state
+with	I-protein_state
+Rac1	B-protein
+show	O
+that	O
+the	O
+HR1	B-structure_element
+domain	O
+comprises	O
+an	O
+anti	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+coiled	I-structure_element
+-	I-structure_element
+coil	I-structure_element
+that	O
+interacts	O
+with	O
+its	O
+G	B-protein_type
+protein	I-protein_type
+binding	O
+partner	O
+via	O
+both	O
+helices	B-structure_element
+.	O
+
+Both	O
+of	O
+the	O
+G	B-site
+protein	I-site
+switch	I-site
+regions	I-site
+are	O
+involved	O
+in	O
+the	O
+interaction	O
+.	O
+
+The	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+fold	I-structure_element
+is	O
+shared	O
+by	O
+the	O
+HR1	B-structure_element
+domain	O
+of	O
+the	O
+TOCA	B-protein_type
+family	I-protein_type
+protein	I-protein_type
+,	O
+CIP4	B-protein
+,	O
+and	O
+,	O
+based	O
+on	O
+sequence	O
+homology	O
+,	O
+by	O
+TOCA1	B-protein
+itself	O
+.	O
+
+These	O
+HR1	B-structure_element
+domains	O
+,	O
+however	O
+,	O
+show	O
+specificity	O
+for	O
+Cdc42	B-protein
+,	O
+rather	O
+than	O
+RhoA	B-protein
+or	O
+Rac1	B-protein
+.	O
+
+How	O
+different	O
+HR1	B-structure_element
+domain	O
+proteins	O
+distinguish	O
+their	O
+specific	O
+G	B-protein_type
+protein	I-protein_type
+partners	O
+remains	O
+only	O
+partially	O
+understood	O
+,	O
+and	O
+structural	O
+characterization	O
+of	O
+a	O
+novel	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+HR1	B-structure_element
+domain	O
+interaction	O
+would	O
+add	O
+to	O
+the	O
+growing	O
+body	O
+of	O
+information	O
+pertaining	O
+to	O
+these	O
+protein	O
+complexes	O
+.	O
+
+Furthermore	O
+,	O
+the	O
+biological	O
+function	O
+of	O
+the	O
+interaction	O
+between	O
+TOCA1	B-protein
+and	O
+Cdc42	B-protein
+remains	O
+poorly	O
+understood	O
+,	O
+and	O
+so	O
+far	O
+there	O
+has	O
+been	O
+no	O
+biophysical	O
+or	O
+structural	O
+insight	O
+.	O
+
+The	O
+interactions	O
+of	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+with	O
+Cdc42	B-protein
+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	B-protein
+effectors	O
+can	O
+interact	O
+with	O
+a	O
+single	O
+molecule	O
+of	O
+Cdc42	B-protein
+simultaneously	O
+.	O
+
+There	O
+is	O
+some	O
+evidence	O
+for	O
+a	O
+ternary	O
+complex	O
+between	O
+Cdc42	B-protein
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+and	O
+TOCA1	B-protein
+,	O
+but	O
+there	O
+was	O
+no	O
+direct	O
+demonstration	O
+of	O
+simultaneous	O
+contacts	O
+between	O
+the	O
+two	O
+effectors	O
+and	O
+a	O
+single	O
+molecule	O
+of	O
+Cdc42	B-protein
+.	O
+
+Nonetheless	O
+,	O
+the	O
+substantial	O
+difference	O
+between	O
+the	O
+structures	B-evidence
+of	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+regions	I-site
+of	O
+the	O
+two	O
+effectors	O
+is	O
+intriguing	O
+and	O
+implies	O
+that	O
+they	O
+bind	O
+to	O
+Cdc42	B-protein
+quite	O
+differently	O
+,	O
+providing	O
+motivation	O
+for	O
+investigating	O
+the	O
+possibility	O
+that	O
+Cdc42	B-protein
+can	O
+bind	O
+both	O
+effectors	O
+concurrently	O
+.	O
+
+WASP	B-protein_type
+interacts	O
+with	O
+Cdc42	B-protein
+via	O
+a	O
+conserved	B-protein_state
+,	O
+unstructured	B-structure_element
+binding	I-structure_element
+motif	I-structure_element
+known	O
+as	O
+the	O
+Cdc42	B-structure_element
+-	I-structure_element
+and	I-structure_element
+Rac	I-structure_element
+-	I-structure_element
+interactive	I-structure_element
+binding	I-structure_element
+region	I-structure_element
+(	O
+CRIB	B-structure_element
+),	O
+which	O
+forms	O
+an	O
+intermolecular	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+,	O
+expanding	O
+the	O
+anti	O
+-	O
+parallel	O
+β2	B-structure_element
+and	I-structure_element
+β3	I-structure_element
+strands	I-structure_element
+of	O
+Cdc42	B-protein
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+TOCA	B-protein_type
+family	I-protein_type
+proteins	I-protein_type
+are	O
+thought	O
+to	O
+interact	O
+via	O
+the	O
+HR1	B-structure_element
+domain	O
+,	O
+which	O
+may	O
+form	O
+a	O
+triple	B-structure_element
+coiled	I-structure_element
+-	I-structure_element
+coil	I-structure_element
+with	O
+switch	B-site
+II	I-site
+of	O
+Rac1	B-protein
+,	O
+like	O
+the	O
+HR1b	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+.	O
+
+Here	O
+,	O
+we	O
+present	O
+the	O
+solution	B-experimental_method
+NMR	I-experimental_method
+structure	B-evidence
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+of	O
+TOCA1	B-protein
+,	O
+providing	O
+the	O
+first	O
+structural	B-evidence
+data	I-evidence
+for	O
+this	O
+protein	O
+.	O
+
+We	O
+also	O
+present	O
+data	O
+pertaining	O
+to	O
+binding	O
+of	O
+the	O
+TOCA	B-protein_type
+HR1	B-structure_element
+domain	O
+to	O
+Cdc42	B-protein
+,	O
+which	O
+is	O
+the	O
+first	O
+biophysical	O
+description	O
+of	O
+an	O
+HR1	B-structure_element
+domain	O
+binding	O
+this	O
+particular	O
+Rho	B-protein_type
+family	I-protein_type
+small	I-protein_type
+G	I-protein_type
+protein	I-protein_type
+.	O
+
+Finally	O
+,	O
+we	O
+investigate	O
+the	O
+potential	O
+ternary	O
+complex	O
+between	O
+Cdc42	B-protein
+and	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+regions	I-site
+of	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+contributing	O
+to	O
+our	O
+understanding	O
+of	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+effector	O
+interactions	O
+as	O
+well	O
+as	O
+the	O
+roles	O
+of	O
+Cdc42	B-protein
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+and	O
+TOCA1	B-protein
+in	O
+the	O
+pathways	O
+that	O
+govern	O
+actin	O
+dynamics	O
+.	O
+
+Cdc42	B-protein
+-	O
+TOCA1	B-protein
+Binding	O
+
+TOCA1	B-protein
+was	O
+identified	O
+in	O
+Xenopus	B-taxonomy_domain
+extracts	O
+as	O
+a	O
+protein	O
+necessary	O
+for	O
+Cdc42	B-protein
+-	O
+dependent	O
+actin	O
+assembly	O
+and	O
+was	O
+shown	O
+to	O
+bind	O
+to	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+GTPγS	I-complex_assembly
+but	O
+not	O
+to	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+GDP	I-complex_assembly
+or	O
+to	O
+Rac1	B-protein
+and	O
+RhoA	B-protein
+.	O
+Given	O
+its	O
+homology	O
+to	O
+other	O
+Rho	B-site
+family	I-site
+binding	I-site
+modules	I-site
+,	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+HR1	B-structure_element
+domain	O
+of	O
+TOCA1	B-protein
+is	O
+sufficient	O
+to	O
+bind	O
+Cdc42	B-protein
+.	O
+
+The	O
+C	B-species
+.	I-species
+elegans	I-species
+TOCA1	B-protein
+orthologues	O
+also	O
+bind	O
+to	O
+Cdc42	B-protein
+via	O
+their	O
+consensus	O
+HR1	B-structure_element
+domain	O
+.	O
+
+The	O
+HR1	B-structure_element
+domains	O
+from	O
+the	O
+PRK	B-protein_type
+family	I-protein_type
+bind	O
+their	O
+G	B-protein_type
+protein	I-protein_type
+partners	O
+with	O
+a	O
+high	O
+affinity	O
+,	O
+exhibiting	O
+a	O
+range	O
+of	O
+submicromolar	O
+dissociation	B-evidence
+constants	I-evidence
+(	O
+Kd	B-evidence
+)	O
+as	O
+low	O
+as	O
+26	O
+nm	O
+.	O
+
+A	O
+Kd	B-evidence
+in	O
+the	O
+nanomolar	O
+range	O
+was	O
+therefore	O
+expected	O
+for	O
+the	O
+interaction	O
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+with	O
+Cdc42	B-protein
+.	O
+
+We	O
+generated	O
+an	O
+X	B-species
+.	I-species
+tropicalis	I-species
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+construct	O
+encompassing	O
+residues	O
+330	B-residue_range
+–	I-residue_range
+426	I-residue_range
+.	O
+
+This	O
+region	O
+comprises	O
+the	O
+complete	O
+HR1	B-structure_element
+domain	O
+based	O
+on	O
+secondary	O
+structure	O
+predictions	O
+and	O
+sequence	B-experimental_method
+alignments	I-experimental_method
+with	O
+another	O
+TOCA	B-protein_type
+family	I-protein_type
+member	O
+,	O
+CIP4	B-protein
+,	O
+whose	O
+structure	B-evidence
+has	O
+been	O
+determined	O
+.	O
+
+The	O
+interaction	O
+between	O
+[	B-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+·	I-complex_assembly
+Cdc42	I-complex_assembly
+and	O
+a	O
+C	O
+-	O
+terminally	O
+His	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+construct	O
+was	O
+investigated	O
+using	O
+SPA	B-experimental_method
+.	O
+
+The	O
+binding	B-evidence
+isotherm	I-evidence
+for	O
+the	O
+interaction	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+1A	O
+,	O
+together	O
+with	O
+the	O
+Cdc42	B-protein
+-	O
+PAK	B-protein
+interaction	O
+as	O
+a	O
+positive	O
+control	O
+.	O
+
+The	O
+binding	O
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+to	O
+Cdc42	B-protein
+was	O
+unexpectedly	O
+weak	O
+,	O
+with	O
+a	O
+Kd	B-evidence
+of	O
+>	O
+1	O
+μm	O
+.	O
+
+It	O
+was	O
+not	O
+possible	O
+to	O
+estimate	O
+the	O
+Kd	B-evidence
+more	O
+accurately	O
+using	O
+direct	O
+SPA	B-experimental_method
+experiments	O
+,	O
+because	O
+saturation	O
+could	O
+not	O
+be	O
+reached	O
+due	O
+to	O
+nonspecific	O
+signal	O
+at	O
+higher	O
+protein	O
+concentrations	O
+.	O
+
+The	O
+TOCA1	B-protein
+HR1	B-structure_element
+-	O
+Cdc42	B-protein
+interaction	O
+is	O
+low	O
+affinity	O
+.	O
+
+A	O
+,	O
+curves	O
+derived	O
+from	O
+direct	B-experimental_method
+binding	I-experimental_method
+assays	I-experimental_method
+in	O
+which	O
+the	O
+indicated	O
+concentrations	O
+of	O
+Cdc42Δ7Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+were	O
+incubated	B-experimental_method
+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	B-experimental_method
+.	O
+
+The	O
+SPA	B-experimental_method
+signal	O
+was	O
+corrected	O
+by	O
+subtraction	O
+of	O
+control	O
+data	O
+with	O
+no	O
+GST	B-mutant
+-	I-mutant
+PAK	I-mutant
+or	O
+HR1	B-mutant
+-	I-mutant
+His6	I-mutant
+.	O
+
+The	O
+data	O
+were	O
+fitted	O
+to	O
+a	O
+binding	B-evidence
+isotherm	I-evidence
+to	O
+give	O
+an	O
+apparent	O
+Kd	B-evidence
+and	O
+are	O
+expressed	O
+as	O
+a	O
+percentage	O
+of	O
+the	O
+maximum	O
+signal	O
+;	O
+B	O
+and	O
+C	O
+,	O
+competition	B-experimental_method
+SPA	I-experimental_method
+experiments	O
+were	O
+carried	O
+out	O
+with	O
+the	O
+indicated	O
+concentrations	O
+of	O
+ACK	B-protein
+GBD	B-structure_element
+(	O
+B	O
+)	O
+or	O
+HR1	B-structure_element
+domain	O
+(	O
+C	O
+)	O
+titrated	B-experimental_method
+into	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+and	O
+either	O
+30	O
+nm	O
+Cdc42Δ7Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+or	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+.	O
+
+The	O
+Kd	B-evidence
+values	O
+derived	O
+for	O
+the	O
+ACK	B-protein
+GBD	B-structure_element
+with	O
+Cdc42Δ7	B-mutant
+and	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42	B-protein
+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
+
+The	O
+Kd	B-evidence
+values	O
+derived	O
+for	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+with	O
+Cdc42Δ7	B-mutant
+and	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42	B-protein
+were	O
+6	O
+.	O
+05	O
+±	O
+1	O
+.	O
+96	O
+and	O
+5	O
+.	O
+39	O
+±	O
+1	O
+.	O
+69	O
+μm	O
+,	O
+respectively	O
+.	O
+
+It	O
+was	O
+possible	O
+that	O
+the	O
+low	O
+affinity	O
+observed	O
+was	O
+due	O
+to	O
+negative	O
+effects	O
+of	O
+immobilization	O
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+,	O
+so	O
+other	O
+methods	O
+were	O
+employed	O
+,	O
+which	O
+utilized	O
+untagged	B-protein_state
+proteins	O
+.	O
+
+Isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+was	O
+carried	O
+out	O
+,	O
+but	O
+no	O
+heat	O
+changes	O
+were	O
+observed	O
+at	O
+a	O
+range	O
+of	O
+concentrations	O
+and	O
+temperatures	O
+(	O
+data	O
+not	O
+shown	O
+),	O
+suggesting	O
+that	O
+the	O
+interaction	O
+is	O
+predominantly	O
+entropically	O
+driven	O
+.	O
+
+Other	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+HR1	B-structure_element
+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	B-experimental_method
+interferometry	I-experimental_method
+with	O
+immobilized	B-protein_state
+Cdc42	B-protein
+was	O
+also	O
+attempted	O
+but	O
+was	O
+unsuccessful	O
+for	O
+both	O
+TOCA1	B-protein
+HR1	B-structure_element
+and	O
+for	O
+the	O
+positive	O
+control	O
+,	O
+ACK	B-protein
+.	O
+
+The	O
+affinity	B-evidence
+was	O
+therefore	O
+determined	O
+using	O
+competition	B-experimental_method
+SPAs	I-experimental_method
+.	O
+
+A	O
+complex	O
+of	O
+a	O
+GST	B-experimental_method
+fusion	I-experimental_method
+of	O
+the	O
+GBD	B-structure_element
+of	O
+ACK	B-protein
+,	O
+which	O
+binds	O
+with	O
+a	O
+high	O
+affinity	O
+to	O
+Cdc42	B-protein
+,	O
+with	O
+radiolabeled	O
+[	B-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+·	I-complex_assembly
+Cdc42	I-complex_assembly
+was	O
+preformed	O
+,	O
+and	O
+the	O
+effect	O
+of	O
+increasing	B-experimental_method
+concentrations	I-experimental_method
+of	O
+untagged	B-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+was	O
+examined	O
+.	O
+
+Competition	O
+of	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+GBD	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+[	B-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+·	I-complex_assembly
+Cdc42	I-complex_assembly
+by	O
+free	B-protein_state
+ACK	B-protein
+GBD	B-structure_element
+was	O
+used	O
+as	O
+a	O
+control	O
+and	O
+to	O
+establish	O
+the	O
+value	O
+of	O
+background	O
+counts	O
+when	O
+Cdc42	B-protein
+is	O
+fully	O
+displaced	O
+.	O
+
+The	O
+data	O
+were	O
+fitted	O
+to	O
+a	O
+binding	B-evidence
+isotherm	I-evidence
+describing	O
+competition	O
+.	O
+
+Free	B-protein_state
+ACK	B-protein
+competed	O
+with	O
+itself	O
+with	O
+an	O
+affinity	B-evidence
+of	O
+32	O
+nm	O
+,	O
+similar	O
+to	O
+the	O
+value	O
+obtained	O
+by	O
+direct	B-experimental_method
+binding	I-experimental_method
+of	O
+23	O
+nm	O
+.	O
+
+The	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+also	O
+fully	O
+competed	O
+with	O
+the	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+but	O
+bound	B-protein_state
+with	O
+an	O
+affinity	B-evidence
+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	B-evidence
+observed	O
+in	O
+the	O
+direct	B-experimental_method
+binding	I-experimental_method
+experiments	I-experimental_method
+.	O
+
+The	O
+Cdc42	B-protein
+construct	O
+used	O
+in	O
+the	O
+binding	B-experimental_method
+assays	I-experimental_method
+has	O
+seven	B-residue_range
+residues	I-residue_range
+deleted	B-experimental_method
+from	O
+the	O
+C	O
+terminus	O
+to	O
+facilitate	O
+purification	O
+.	O
+
+These	O
+residues	O
+are	O
+not	O
+generally	O
+required	O
+for	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+effector	O
+interactions	O
+,	O
+including	O
+the	O
+interaction	O
+between	O
+RhoA	B-protein
+and	O
+the	O
+PRK1	B-protein
+HR1a	B-structure_element
+domain	O
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+C	O
+terminus	O
+of	O
+Rac1	B-protein
+contains	O
+a	O
+polybasic	O
+sequence	O
+,	O
+which	O
+is	O
+crucial	O
+for	O
+Rac1	B-protein
+binding	O
+to	O
+the	O
+HR1b	B-structure_element
+domain	O
+from	O
+PRK1	B-protein
+.	O
+
+As	O
+the	O
+observed	O
+affinity	B-evidence
+between	O
+TOCA1	B-protein
+HR1	B-structure_element
+and	O
+Cdc42	B-protein
+was	O
+much	O
+lower	O
+than	O
+expected	O
+,	O
+we	O
+reasoned	O
+that	O
+the	O
+C	O
+terminus	O
+of	O
+Cdc42	B-protein
+might	O
+be	O
+necessary	O
+for	O
+a	O
+high	O
+affinity	B-evidence
+interaction	O
+.	O
+
+The	O
+binding	B-experimental_method
+experiments	I-experimental_method
+were	O
+repeated	O
+with	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+[	B-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+·	I-complex_assembly
+Cdc42	I-complex_assembly
+,	O
+but	O
+the	O
+affinity	B-evidence
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+for	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42	B-protein
+was	O
+similar	O
+to	O
+its	O
+affinity	B-evidence
+for	O
+truncated	B-protein_state
+Cdc42	B-protein
+(	O
+Kd	B-evidence
+≈	O
+5	O
+μm	O
+;	O
+Fig	O
+.	O
+1C	O
+).	O
+
+Thus	O
+,	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+Cdc42	B-protein
+is	O
+not	O
+required	O
+for	O
+maximal	O
+binding	O
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+.	O
+
+Another	O
+possible	O
+explanation	O
+for	O
+the	O
+low	O
+affinities	B-evidence
+observed	O
+was	O
+that	O
+the	O
+HR1	B-structure_element
+domain	O
+alone	B-protein_state
+is	O
+not	O
+sufficient	O
+for	O
+maximal	O
+binding	O
+of	O
+the	O
+TOCA	B-protein_type
+proteins	I-protein_type
+to	O
+Cdc42	B-protein
+and	O
+that	O
+the	O
+other	O
+domains	O
+are	O
+required	O
+.	O
+
+Indeed	O
+,	O
+GST	B-experimental_method
+pull	I-experimental_method
+-	I-experimental_method
+downs	I-experimental_method
+performed	O
+with	O
+in	O
+vitro	O
+translated	O
+human	B-species
+TOCA1	B-protein
+fragments	O
+had	O
+suggested	O
+that	O
+residues	O
+N	O
+-	O
+terminal	O
+to	O
+the	O
+HR1	B-structure_element
+domain	O
+may	O
+be	O
+required	O
+to	O
+stabilize	O
+the	O
+HR1	B-structure_element
+domain	O
+structure	O
+.	O
+
+Furthermore	O
+,	O
+both	O
+BAR	B-structure_element
+and	O
+SH3	B-structure_element
+domains	O
+have	O
+been	O
+implicated	O
+in	O
+interactions	O
+with	O
+small	O
+G	B-protein_type
+proteins	I-protein_type
+(	O
+e	O
+.	O
+g	O
+.	O
+the	O
+BAR	B-structure_element
+domain	O
+of	O
+Arfaptin2	B-protein
+binds	O
+to	O
+Rac1	B-protein
+and	O
+Arl1	B-protein
+),	O
+while	O
+an	O
+SH3	B-structure_element
+domain	O
+mediates	O
+the	O
+interaction	O
+between	O
+Rac1	B-protein
+and	O
+the	O
+guanine	B-protein
+nucleotide	I-protein
+exchange	I-protein
+factor	I-protein
+,	O
+β	B-protein
+-	I-protein
+PIX	I-protein
+.	O
+
+TOCA1	B-protein
+dimerizes	B-oligomeric_state
+via	O
+its	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+,	O
+which	O
+could	O
+also	O
+affect	O
+Cdc42	B-protein
+binding	O
+,	O
+for	O
+example	O
+by	O
+presenting	O
+two	O
+HR1	B-structure_element
+domains	O
+for	O
+Cdc42	B-protein
+interactions	O
+.	O
+
+Various	O
+TOCA1	B-protein
+fragments	O
+(	O
+Fig	O
+.	O
+2A	O
+)	O
+were	O
+therefore	O
+assessed	O
+for	O
+binding	O
+to	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42	B-protein
+by	O
+direct	O
+SPA	B-experimental_method
+.	O
+
+The	O
+isolated	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+showed	O
+no	O
+binding	O
+to	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42	B-protein
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+
+Full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+TOCA1	B-protein
+and	O
+ΔSH3	B-mutant
+TOCA1	B-protein
+bound	B-protein_state
+with	O
+micromolar	O
+affinity	O
+(	O
+Fig	O
+.	O
+2B	O
+),	O
+in	O
+a	O
+similar	O
+manner	O
+to	O
+the	O
+isolated	O
+HR1	B-structure_element
+domain	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+
+The	O
+HR1	B-mutant
+-	I-mutant
+SH3	I-mutant
+protein	O
+could	O
+not	O
+be	O
+purified	O
+to	O
+homogeneity	O
+as	O
+a	O
+fusion	O
+protein	O
+,	O
+so	O
+it	O
+was	O
+assayed	O
+in	O
+competition	B-experimental_method
+assays	I-experimental_method
+after	O
+cleavage	O
+of	O
+the	O
+His	O
+tag	O
+.	O
+
+This	O
+construct	O
+competed	O
+with	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+GBD	B-structure_element
+to	O
+give	O
+a	O
+similar	O
+affinity	O
+to	O
+the	O
+HR1	B-structure_element
+domain	O
+alone	B-protein_state
+(	O
+Kd	B-evidence
+=	O
+4	O
+.	O
+6	O
+±	O
+4	O
+μm	O
+;	O
+Fig	O
+.	O
+2C	O
+).	O
+
+Taken	O
+together	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+is	O
+sufficient	O
+for	O
+maximal	O
+binding	O
+and	O
+that	O
+this	O
+binding	O
+is	O
+of	O
+a	O
+relatively	O
+low	O
+affinity	O
+compared	O
+with	O
+many	O
+other	O
+Cdc42	B-protein
+·	O
+effector	O
+complexes	O
+.	O
+
+The	O
+Cdc42	B-complex_assembly
+-	I-complex_assembly
+HR1	I-complex_assembly
+interaction	O
+is	O
+of	O
+low	O
+affinity	O
+in	O
+the	O
+context	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+and	O
+in	O
+TOCA1	B-protein
+paralogues	O
+.	O
+
+A	O
+,	O
+diagram	O
+illustrating	O
+the	O
+TOCA1	B-protein
+constructs	O
+assayed	O
+for	O
+Cdc42	B-protein
+binding	O
+.	O
+
+Domain	O
+boundaries	O
+are	O
+derived	O
+from	O
+secondary	O
+structure	O
+predictions	O
+;	O
+B	O
+,	O
+binding	B-evidence
+curves	I-evidence
+derived	O
+from	O
+direct	B-experimental_method
+binding	I-experimental_method
+assays	I-experimental_method
+,	O
+in	O
+which	O
+the	O
+indicated	O
+concentrations	O
+of	O
+Cdc42Δ7Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+were	O
+incubated	B-experimental_method
+with	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+or	O
+His	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+TOCA1	B-protein
+constructs	O
+,	O
+as	O
+indicated	O
+,	O
+in	O
+SPAs	B-experimental_method
+.	O
+
+The	O
+SPA	B-experimental_method
+signal	O
+was	O
+corrected	O
+by	O
+subtraction	O
+of	O
+control	O
+data	O
+with	O
+no	O
+fusion	O
+protein	O
+.	O
+
+The	O
+data	O
+were	O
+fitted	O
+to	O
+a	O
+binding	B-evidence
+isotherm	I-evidence
+to	O
+give	O
+an	O
+apparent	O
+Kd	B-evidence
+and	O
+are	O
+expressed	O
+as	O
+a	O
+percentage	O
+of	O
+the	O
+maximum	O
+signal	O
+.	O
+
+C	O
+–	O
+E	O
+,	O
+representative	O
+examples	O
+of	O
+competition	B-experimental_method
+SPA	I-experimental_method
+experiments	O
+carried	O
+out	O
+with	O
+the	O
+indicated	O
+concentrations	O
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-mutant
+-	I-mutant
+SH3	I-mutant
+construct	O
+titrated	B-experimental_method
+into	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+and	O
+30	O
+nm	O
+Cdc42Δ7Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+(	O
+C	O
+)	O
+or	O
+HR1CIP4	B-structure_element
+(	O
+D	O
+)	O
+or	O
+HR1FBP17	B-structure_element
+(	O
+E	O
+)	O
+titrated	B-experimental_method
+into	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+and	O
+30	O
+nm	O
+Cdc42FLQ61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+.	O
+
+The	O
+low	O
+affinity	O
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+-	O
+Cdc42	B-protein
+interaction	O
+raised	O
+the	O
+question	O
+of	O
+whether	O
+the	O
+other	O
+known	O
+Cdc42	B-protein
+-	O
+binding	O
+TOCA	B-protein_type
+family	I-protein_type
+proteins	I-protein_type
+,	O
+FBP17	B-protein
+and	O
+CIP4	B-protein
+,	O
+also	O
+bind	O
+weakly	O
+.	O
+
+The	O
+HR1	B-structure_element
+domains	O
+from	O
+FBP17	B-protein
+and	O
+CIP4	B-protein
+were	O
+purified	B-experimental_method
+and	O
+assayed	O
+for	O
+Cdc42	B-protein
+binding	O
+in	O
+competition	B-experimental_method
+SPAs	I-experimental_method
+,	O
+analogous	O
+to	O
+those	O
+carried	O
+out	O
+with	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+.	O
+
+The	O
+affinities	B-evidence
+of	O
+both	O
+the	O
+FBP17	B-protein
+and	O
+CIP4	B-protein
+HR1	B-structure_element
+domains	O
+were	O
+also	O
+in	O
+the	O
+low	O
+micromolar	O
+range	O
+(	O
+10	O
+and	O
+5	O
+μm	O
+,	O
+respectively	O
+)	O
+(	O
+Fig	O
+.	O
+2	O
+,	O
+D	O
+and	O
+E	O
+),	O
+suggesting	O
+that	O
+low	O
+affinity	O
+interactions	O
+with	O
+Cdc42	B-protein
+are	O
+a	O
+common	O
+feature	O
+within	O
+the	O
+TOCA	B-protein_type
+family	I-protein_type
+.	O
+
+Structure	B-evidence
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+Domain	O
+
+Because	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+was	O
+sufficient	O
+for	O
+maximal	O
+Cdc42	B-protein
+-	O
+binding	O
+,	O
+we	O
+used	O
+this	O
+construct	O
+for	O
+structural	O
+studies	O
+.	O
+
+Initial	O
+experiments	O
+were	O
+performed	O
+with	O
+TOCA1	B-protein
+residues	O
+324	B-residue_range
+–	I-residue_range
+426	I-residue_range
+,	O
+but	O
+we	O
+observed	O
+that	O
+the	O
+N	O
+terminus	O
+was	O
+cleaved	O
+during	O
+purification	O
+to	O
+yield	O
+a	O
+new	O
+N	O
+terminus	O
+at	O
+residue	O
+330	B-residue_number
+(	O
+data	O
+not	O
+shown	O
+).	O
+
+We	O
+therefore	O
+engineered	O
+a	O
+construct	O
+comprising	O
+residues	O
+330	B-residue_range
+–	I-residue_range
+426	I-residue_range
+to	O
+produce	O
+the	O
+minimal	B-protein_state
+,	O
+stable	B-protein_state
+HR1	B-structure_element
+domain	O
+.	O
+
+2	O
+,	O
+778	O
+non	O
+-	O
+degenerate	O
+NOE	B-evidence
+restraints	I-evidence
+were	O
+used	O
+in	O
+initial	O
+structure	B-experimental_method
+calculations	I-experimental_method
+(	O
+1	O
+,	O
+791	O
+unambiguous	O
+and	O
+987	O
+ambiguous	O
+),	O
+derived	O
+from	O
+three	O
+-	O
+dimensional	O
+15N	B-experimental_method
+-	I-experimental_method
+separated	I-experimental_method
+NOESY	I-experimental_method
+and	O
+13C	B-experimental_method
+-	I-experimental_method
+separated	I-experimental_method
+NOESY	I-experimental_method
+experiments	O
+.	O
+
+There	O
+were	O
+1	O
+,	O
+845	O
+unambiguous	O
+NOEs	B-evidence
+and	O
+757	O
+ambiguous	O
+NOEs	B-evidence
+after	O
+eight	O
+iterations	O
+.	O
+
+100	O
+structures	B-evidence
+were	O
+calculated	B-experimental_method
+in	O
+the	O
+final	O
+iteration	O
+;	O
+the	O
+50	O
+lowest	O
+energy	O
+structures	B-evidence
+were	O
+water	O
+-	O
+refined	O
+;	O
+and	O
+of	O
+these	O
+,	O
+the	O
+35	O
+lowest	O
+energy	O
+structures	B-evidence
+were	O
+analyzed	O
+.	O
+
+Table	O
+1	O
+indicates	O
+that	O
+the	O
+HR1	B-structure_element
+domain	O
+structure	B-evidence
+is	O
+well	O
+defined	O
+by	O
+the	O
+NMR	B-experimental_method
+data	O
+.	O
+
+a	O
+<	O
+SA	O
+>,	O
+the	O
+average	B-evidence
+root	I-evidence
+mean	I-evidence
+square	I-evidence
+deviations	I-evidence
+for	O
+the	O
+ensemble	O
+±	O
+S	O
+.	O
+D	O
+.	O
+
+b	O
+<	O
+SA	O
+>	O
+c	O
+,	O
+values	O
+for	O
+the	O
+structure	B-evidence
+that	O
+is	O
+closest	O
+to	O
+the	O
+mean	O
+.	O
+
+The	O
+structure	B-evidence
+closest	O
+to	O
+the	O
+mean	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+3A	O
+.	O
+
+The	O
+two	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+interact	O
+to	O
+form	O
+an	O
+anti	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+coiled	I-structure_element
+-	I-structure_element
+coil	I-structure_element
+with	O
+a	O
+slight	O
+left	O
+-	O
+handed	O
+twist	O
+,	O
+reminiscent	O
+of	O
+the	O
+HR1	B-structure_element
+domains	O
+of	O
+CIP4	B-protein
+(	O
+PDB	O
+code	O
+2KE4	O
+)	O
+and	O
+PRK1	B-protein
+(	O
+PDB	O
+codes	O
+1CXZ	O
+and	O
+1URF	O
+).	O
+
+A	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+illustrating	O
+the	O
+secondary	O
+structure	O
+elements	O
+of	O
+the	O
+TOCA1	B-protein
+and	O
+CIP4	B-protein
+HR1	B-structure_element
+domains	O
+and	O
+the	O
+HR1a	B-structure_element
+and	O
+HR1b	B-structure_element
+domains	O
+from	O
+PRK1	B-protein
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+3B	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+.	O
+
+A	O
+,	O
+the	O
+backbone	O
+trace	B-evidence
+of	O
+the	O
+35	O
+lowest	O
+energy	O
+structures	B-evidence
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+overlaid	O
+with	O
+the	O
+structure	B-evidence
+closest	O
+to	O
+the	O
+mean	O
+is	O
+shown	O
+alongside	O
+a	O
+schematic	O
+representation	O
+of	O
+the	O
+structure	B-evidence
+closest	O
+to	O
+the	O
+mean	O
+.	O
+
+Flexible	O
+regions	O
+at	O
+the	O
+N	O
+and	O
+C	O
+termini	O
+(	O
+residues	O
+330	B-residue_range
+–	I-residue_range
+333	I-residue_range
+and	O
+421	B-residue_range
+–	I-residue_range
+426	I-residue_range
+)	O
+are	O
+omitted	O
+for	O
+clarity	O
+.	O
+
+B	O
+,	O
+a	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+HR1	B-structure_element
+domains	O
+from	O
+TOCA1	B-protein
+,	O
+CIP4	B-protein
+,	O
+and	O
+PRK1	B-protein
+.	O
+
+The	O
+secondary	O
+structure	O
+was	O
+deduced	O
+using	O
+Stride	B-experimental_method
+,	O
+based	O
+on	O
+the	O
+Ramachandran	B-evidence
+angles	I-evidence
+,	O
+and	O
+is	O
+indicated	O
+as	O
+follows	O
+:	O
+gray	O
+,	O
+turn	O
+;	O
+yellow	O
+,	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+;	O
+blue	O
+,	O
+310	B-structure_element
+helix	I-structure_element
+;	O
+white	O
+,	O
+coil	O
+.	O
+
+C	O
+,	O
+a	O
+close	O
+-	O
+up	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+,	O
+indicating	O
+some	O
+of	O
+the	O
+NOEs	B-evidence
+defining	O
+its	O
+position	O
+with	O
+respect	O
+to	O
+the	O
+two	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+.	O
+
+Dotted	O
+lines	O
+,	O
+NOE	B-evidence
+restraints	I-evidence
+.	O
+
+D	O
+,	O
+a	O
+close	O
+-	O
+up	O
+of	O
+the	O
+interhelix	B-structure_element
+loop	I-structure_element
+region	O
+showing	O
+some	O
+of	O
+the	O
+contacts	O
+between	O
+the	O
+loop	B-structure_element
+and	O
+helix	B-structure_element
+1	I-structure_element
+.	O
+
+In	O
+the	O
+HR1a	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+,	O
+a	O
+region	O
+N	O
+-	O
+terminal	O
+to	O
+helix	B-structure_element
+1	I-structure_element
+forms	O
+a	O
+short	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+,	O
+which	O
+packs	O
+against	O
+both	O
+helices	O
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+.	O
+
+This	O
+region	O
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+(	O
+residues	O
+334	B-residue_range
+–	I-residue_range
+340	I-residue_range
+)	O
+is	O
+well	O
+defined	O
+in	O
+the	O
+family	O
+of	O
+structures	B-evidence
+(	O
+Fig	O
+.	O
+3A	O
+)	O
+but	O
+does	O
+not	O
+form	O
+an	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+.	O
+
+It	O
+instead	O
+forms	O
+a	O
+series	O
+of	O
+turns	O
+,	O
+defined	O
+by	O
+NOE	B-evidence
+restraints	I-evidence
+observed	O
+between	O
+residues	O
+separated	O
+by	O
+one	O
+(	O
+residues	O
+332	B-residue_range
+–	I-residue_range
+334	I-residue_range
+,	O
+333	B-residue_range
+–	I-residue_range
+335	I-residue_range
+,	O
+etc	O
+.)	O
+or	O
+two	O
+(	O
+residues	O
+337	B-residue_range
+–	I-residue_range
+340	I-residue_range
+)	O
+residues	O
+in	O
+the	O
+sequence	O
+and	O
+the	O
+φ	B-evidence
+and	I-evidence
+ψ	I-evidence
+angles	I-evidence
+,	O
+assessed	O
+using	O
+Stride	B-experimental_method
+.	O
+
+These	O
+turns	O
+cause	O
+the	O
+chain	O
+to	O
+reverse	O
+direction	O
+,	O
+allowing	O
+the	O
+N	O
+-	O
+terminal	O
+segment	O
+(	O
+residues	O
+334	B-residue_range
+–	I-residue_range
+340	I-residue_range
+)	O
+to	O
+contact	O
+both	O
+helices	O
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+.	O
+
+Long	O
+range	O
+NOEs	B-evidence
+were	O
+observed	O
+linking	O
+Leu	B-residue_name_number
+-	I-residue_name_number
+334	I-residue_name_number
+,	O
+Glu	B-residue_name_number
+-	I-residue_name_number
+335	I-residue_name_number
+,	O
+and	O
+Asp	B-residue_name_number
+-	I-residue_name_number
+336	I-residue_name_number
+with	O
+Trp	B-residue_name_number
+-	I-residue_name_number
+413	I-residue_name_number
+of	O
+helix	B-structure_element
+2	I-structure_element
+,	O
+Leu	B-residue_name_number
+-	I-residue_name_number
+334	I-residue_name_number
+with	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+409	I-residue_name_number
+of	O
+helix	B-structure_element
+2	I-structure_element
+,	O
+and	O
+Phe	B-residue_name_number
+-	I-residue_name_number
+337	I-residue_name_number
+and	O
+Ser	B-residue_name_number
+-	I-residue_name_number
+338	I-residue_name_number
+with	O
+Arg	B-residue_name_number
+-	I-residue_name_number
+345	I-residue_name_number
+,	O
+Arg	B-residue_name_number
+-	I-residue_name_number
+348	I-residue_name_number
+,	O
+and	O
+Leu	B-residue_name_number
+-	I-residue_name_number
+349	I-residue_name_number
+of	O
+helix	B-structure_element
+1	I-structure_element
+.	O
+
+The	O
+two	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+are	O
+separated	O
+by	O
+a	O
+long	O
+loop	B-structure_element
+of	O
+10	O
+residues	O
+(	O
+residues	O
+380	B-residue_range
+–	I-residue_range
+389	I-residue_range
+)	O
+that	O
+contains	O
+two	O
+short	B-structure_element
+310	I-structure_element
+helices	I-structure_element
+(	O
+residues	O
+381	B-residue_range
+–	I-residue_range
+383	I-residue_range
+and	O
+386	B-residue_range
+–	I-residue_range
+389	I-residue_range
+).	O
+
+Interestingly	O
+,	O
+side	O
+chains	O
+of	O
+residues	O
+within	O
+the	O
+loop	B-structure_element
+region	I-structure_element
+point	O
+back	O
+toward	O
+helix	B-structure_element
+1	I-structure_element
+;	O
+for	O
+example	O
+,	O
+there	O
+are	O
+numerous	O
+distinct	O
+NOEs	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+Asn	B-residue_name_number
+-	I-residue_name_number
+380	I-residue_name_number
+and	O
+Met	B-residue_name_number
+-	I-residue_name_number
+383	I-residue_name_number
+of	O
+the	O
+loop	B-structure_element
+region	I-structure_element
+and	O
+Tyr	B-residue_name_number
+-	I-residue_name_number
+377	I-residue_name_number
+and	O
+Val	B-residue_name_number
+-	I-residue_name_number
+376	I-residue_name_number
+of	O
+helix	B-structure_element
+1	I-structure_element
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+
+The	O
+backbone	O
+NH	O
+and	O
+CHα	O
+groups	O
+of	O
+Gly	B-residue_name_number
+-	I-residue_name_number
+384	I-residue_name_number
+and	O
+Asp	B-residue_name_number
+-	I-residue_name_number
+385	I-residue_name_number
+also	O
+show	O
+NOEs	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+Tyr	B-residue_name_number
+-	I-residue_name_number
+377	I-residue_name_number
+.	O
+
+Mapping	O
+the	O
+TOCA1	B-protein
+and	O
+Cdc42	B-site
+Binding	I-site
+Interfaces	I-site
+
+The	O
+HR1TOCA1	B-site
+-	I-site
+Cdc42	I-site
+interface	I-site
+was	O
+investigated	O
+using	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+.	O
+
+A	O
+series	O
+of	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+experiments	O
+was	O
+recorded	O
+on	O
+15N	B-chemical
+-	O
+labeled	B-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+increasing	B-experimental_method
+concentrations	I-experimental_method
+of	O
+unlabeled	B-protein_state
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+to	O
+map	O
+the	O
+Cdc42	B-site
+-	I-site
+binding	I-site
+surface	I-site
+.	O
+
+A	O
+comparison	O
+of	O
+the	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+of	O
+free	B-protein_state
+HR1	B-structure_element
+and	O
+HR1	B-structure_element
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+excess	O
+Cdc42	B-protein
+shows	O
+that	O
+although	O
+some	O
+peaks	O
+were	O
+shifted	O
+,	O
+several	O
+were	O
+much	O
+broader	O
+in	O
+the	O
+complex	O
+,	O
+and	O
+a	O
+considerable	O
+subset	O
+had	O
+disappeared	O
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+
+This	O
+behavior	O
+cannot	O
+be	O
+explained	O
+by	O
+the	O
+increase	O
+in	O
+molecular	O
+mass	O
+(	O
+from	O
+12	O
+to	O
+33	O
+kDa	O
+)	O
+when	O
+Cdc42	B-protein
+binds	O
+and	O
+is	O
+more	O
+likely	O
+to	O
+be	O
+due	O
+to	O
+conformational	O
+exchange	O
+.	O
+
+Overall	O
+chemical	B-experimental_method
+shift	I-experimental_method
+perturbations	I-experimental_method
+(	O
+CSPs	B-experimental_method
+)	O
+were	O
+calculated	O
+for	O
+each	O
+residue	O
+,	O
+whereas	O
+those	O
+that	O
+had	O
+disappeared	O
+were	O
+assigned	O
+a	O
+shift	O
+change	O
+of	O
+0	O
+.	O
+2	O
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+
+A	O
+peak	O
+that	O
+disappeared	O
+or	O
+had	O
+a	O
+CSP	B-experimental_method
+above	O
+the	O
+mean	O
+CSP	B-experimental_method
+for	O
+the	O
+spectrum	O
+was	O
+considered	O
+to	O
+be	O
+significantly	O
+affected	O
+.	O
+
+Mapping	O
+the	O
+binding	B-site
+surface	I-site
+of	O
+Cdc42	B-protein
+onto	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+.	O
+
+A	O
+,	O
+the	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+of	O
+200	O
+μm	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+is	O
+shown	O
+in	O
+the	O
+free	B-protein_state
+form	I-protein_state
+(	O
+black	O
+)	O
+and	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+a	O
+4	O
+-	O
+fold	O
+molar	O
+excess	O
+of	O
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+(	O
+red	O
+).	O
+
+B	O
+,	O
+CSPs	B-experimental_method
+were	O
+calculated	O
+as	O
+described	O
+under	O
+“	O
+Experimental	O
+Procedures	O
+”	O
+and	O
+are	O
+shown	O
+for	O
+backbone	O
+and	O
+side	O
+chain	O
+NH	O
+groups	O
+.	O
+
+The	O
+mean	O
+CSP	B-experimental_method
+is	O
+marked	O
+with	O
+a	O
+red	O
+line	O
+.	O
+
+Residues	O
+that	O
+disappeared	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Cdc42	B-protein
+were	O
+assigned	O
+a	O
+CSP	B-experimental_method
+of	O
+0	O
+.	O
+2	O
+but	O
+were	O
+excluded	O
+when	O
+calculating	O
+the	O
+mean	O
+CSP	B-experimental_method
+and	O
+are	O
+indicated	O
+with	O
+open	O
+bars	O
+.	O
+
+Those	O
+that	O
+were	O
+not	O
+traceable	O
+due	O
+to	O
+spectral	O
+overlap	O
+were	O
+assigned	O
+a	O
+CSP	B-experimental_method
+of	O
+zero	O
+and	O
+are	O
+marked	O
+with	O
+an	O
+asterisk	O
+below	O
+the	O
+bar	O
+.	O
+
+Residues	O
+with	O
+affected	O
+side	O
+chain	O
+CSPs	B-experimental_method
+derived	O
+from	O
+13C	B-experimental_method
+HSQCs	I-experimental_method
+are	O
+marked	O
+with	O
+green	O
+asterisks	O
+above	O
+the	O
+bars	O
+.	O
+
+C	O
+,	O
+a	O
+schematic	O
+representation	O
+of	O
+the	O
+HR1	B-structure_element
+domain	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	B-protein_state
+bound	I-protein_state
+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	B-protein_state
+-	I-protein_state
+accessible	I-protein_state
+are	O
+colored	O
+yellow	O
+.	O
+
+Residues	O
+with	O
+significantly	O
+affected	O
+backbone	O
+and	O
+side	O
+chain	O
+groups	O
+that	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+accessible	I-protein_state
+are	O
+colored	O
+red	O
+.	O
+
+A	O
+close	O
+-	O
+up	O
+of	O
+the	O
+binding	B-site
+region	I-site
+is	O
+shown	O
+,	O
+with	O
+affected	O
+side	O
+chain	O
+heavy	O
+atoms	O
+shown	O
+as	O
+sticks	O
+.	O
+
+D	O
+,	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+region	I-site
+is	O
+marked	O
+in	O
+red	O
+onto	O
+structures	B-evidence
+of	O
+the	O
+HR1	B-structure_element
+domains	O
+as	O
+indicated	O
+.	O
+
+15N	B-experimental_method
+HSQC	I-experimental_method
+shift	I-experimental_method
+mapping	I-experimental_method
+experiments	O
+report	O
+on	O
+changes	O
+to	O
+amide	O
+groups	O
+,	O
+which	O
+are	O
+mainly	O
+inaccessible	O
+because	O
+they	O
+are	O
+buried	O
+inside	O
+the	O
+helices	B-structure_element
+and	O
+are	O
+involved	O
+in	O
+hydrogen	O
+bonds	O
+.	O
+
+Therefore	O
+,	O
+13C	B-experimental_method
+HSQC	I-experimental_method
+and	O
+methyl	B-experimental_method
+-	I-experimental_method
+selective	I-experimental_method
+SOFAST	I-experimental_method
+-	I-experimental_method
+HMQC	I-experimental_method
+experiments	O
+were	O
+also	O
+recorded	O
+on	O
+15N	B-chemical
+,	O
+13C	B-chemical
+-	O
+labeled	B-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+to	O
+yield	O
+more	O
+information	O
+on	O
+side	O
+chain	O
+involvement	O
+.	O
+
+Side	O
+chains	O
+whose	O
+CH	O
+groups	O
+disappeared	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Cdc42	B-protein
+are	O
+marked	O
+on	O
+the	O
+graph	O
+in	O
+Fig	O
+.	O
+4B	O
+with	O
+green	O
+asterisks	O
+.	O
+
+TOCA1	B-protein
+residues	O
+whose	O
+signals	O
+were	O
+affected	O
+by	O
+Cdc42	B-protein
+binding	O
+were	O
+mapped	O
+onto	O
+the	O
+structure	B-evidence
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+
+The	O
+changes	O
+were	O
+localized	O
+to	O
+one	O
+end	O
+of	O
+the	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+,	O
+and	O
+the	O
+binding	B-site
+site	I-site
+appeared	O
+to	O
+include	O
+residues	O
+from	O
+both	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+the	O
+loop	B-structure_element
+region	I-structure_element
+that	O
+joins	O
+them	O
+.	O
+
+The	O
+residues	O
+in	O
+the	O
+interhelical	B-structure_element
+loop	I-structure_element
+and	O
+helix	B-structure_element
+1	I-structure_element
+that	O
+contact	O
+each	O
+other	O
+(	O
+Fig	O
+.	O
+3D	O
+)	O
+show	O
+shift	O
+changes	O
+in	O
+their	O
+backbone	O
+NH	O
+and	O
+side	O
+chains	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Cdc42	B-protein
+.	O
+
+For	O
+example	O
+,	O
+the	O
+side	O
+chain	O
+of	O
+Asn	B-residue_name_number
+-	I-residue_name_number
+380	I-residue_name_number
+and	O
+the	O
+backbones	O
+of	O
+Val	B-residue_name_number
+-	I-residue_name_number
+376	I-residue_name_number
+and	O
+Tyr	B-residue_name_number
+-	I-residue_name_number
+377	I-residue_name_number
+were	O
+significantly	O
+affected	O
+but	O
+are	O
+all	O
+buried	O
+in	O
+the	O
+free	B-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+structure	B-evidence
+,	O
+indicating	O
+that	O
+local	O
+conformational	O
+changes	O
+in	O
+the	O
+loop	B-structure_element
+may	O
+facilitate	O
+complex	O
+formation	O
+.	O
+
+The	O
+chemical	B-experimental_method
+shift	I-experimental_method
+mapping	I-experimental_method
+data	O
+indicate	O
+that	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+region	I-site
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+is	O
+broadly	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+CIP4	B-protein
+and	O
+PRK1	B-protein
+HR1	B-structure_element
+domains	O
+(	O
+Figs	O
+.	O
+3B	O
+and	O
+4D	O
+).	O
+
+The	O
+corresponding	O
+15N	B-experimental_method
+and	O
+13C	B-experimental_method
+NMR	I-experimental_method
+experiments	O
+were	O
+also	O
+recorded	O
+on	O
+15N	B-chemical
+-	O
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+or	O
+15N	B-chemical
+/	O
+13C	B-chemical
+-	O
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+unlabeled	B-protein_state
+HR1	B-structure_element
+domain	O
+.	O
+
+The	O
+overall	O
+CSP	B-experimental_method
+was	O
+calculated	O
+for	O
+each	O
+residue	O
+.	O
+
+As	O
+was	O
+the	O
+case	O
+when	O
+labeled	B-protein_state
+HR1	B-structure_element
+was	O
+observed	O
+,	O
+several	O
+peaks	O
+were	O
+shifted	O
+in	O
+the	O
+complex	O
+,	O
+but	O
+many	O
+disappeared	O
+,	O
+indicating	O
+exchange	O
+on	O
+an	O
+unfavorable	O
+,	O
+millisecond	O
+time	O
+scale	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+Detailed	O
+side	O
+chain	O
+data	O
+could	O
+not	O
+be	O
+obtained	O
+for	O
+all	O
+residues	O
+due	O
+to	O
+spectral	O
+overlap	O
+,	O
+but	O
+constant	B-experimental_method
+time	I-experimental_method
+13C	I-experimental_method
+HSQC	I-experimental_method
+and	O
+methyl	B-experimental_method
+-	I-experimental_method
+selective	I-experimental_method
+SOFAST	I-experimental_method
+-	I-experimental_method
+HMQC	I-experimental_method
+experiments	O
+provided	O
+further	O
+information	O
+on	O
+certain	O
+well	O
+resolved	O
+side	O
+chains	O
+(	O
+marked	O
+with	O
+green	O
+asterisks	O
+in	O
+Fig	O
+.	O
+5B	O
+).	O
+
+Mapping	O
+the	O
+binding	B-site
+surface	I-site
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+onto	O
+Cdc42	B-protein
+.	O
+
+A	O
+,	O
+the	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+of	O
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+is	O
+shown	O
+in	O
+its	O
+free	B-protein_state
+form	I-protein_state
+(	O
+black	O
+)	O
+and	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+excess	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+(	O
+1	O
+:	O
+2	O
+.	O
+2	O
+,	O
+red	O
+).	O
+
+B	O
+,	O
+CSPs	B-experimental_method
+are	O
+shown	O
+for	O
+backbone	O
+NH	O
+groups	O
+.	O
+
+The	O
+red	O
+line	O
+indicates	O
+the	O
+mean	O
+CSP	B-experimental_method
+,	O
+plus	O
+one	O
+S	O
+.	O
+D	O
+.	O
+Residues	O
+that	O
+disappeared	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Cdc42	B-protein
+were	O
+assigned	O
+a	O
+CSP	B-experimental_method
+of	O
+0	O
+.	O
+1	O
+and	O
+are	O
+indicated	O
+with	O
+open	O
+bars	O
+.	O
+
+Residues	O
+with	O
+disappeared	O
+peaks	O
+in	O
+13C	B-experimental_method
+HSQC	I-experimental_method
+experiments	O
+are	O
+marked	O
+on	O
+the	O
+chart	O
+with	O
+green	O
+asterisks	O
+.	O
+
+C	O
+,	O
+the	O
+residues	O
+with	O
+significantly	O
+affected	O
+backbone	O
+and	O
+side	O
+chain	O
+groups	O
+are	O
+highlighted	O
+on	O
+an	O
+NMR	B-experimental_method
+structure	B-evidence
+of	O
+free	B-protein_state
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+;	O
+those	O
+that	O
+are	O
+buried	O
+are	O
+colored	O
+dark	O
+blue	O
+,	O
+whereas	O
+those	O
+that	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+accessible	I-protein_state
+are	O
+colored	O
+red	O
+.	O
+
+Residues	O
+with	O
+either	O
+side	O
+chain	O
+or	O
+backbone	O
+groups	O
+affected	O
+are	O
+colored	O
+blue	O
+if	O
+buried	O
+and	O
+yellow	O
+if	O
+solvent	B-protein_state
+-	I-protein_state
+accessible	I-protein_state
+.	O
+
+Residues	O
+without	O
+information	O
+from	O
+shift	B-experimental_method
+mapping	I-experimental_method
+are	O
+colored	O
+gray	O
+.	O
+
+The	O
+flexible	B-protein_state
+switch	B-site
+regions	I-site
+are	O
+circled	O
+.	O
+
+As	O
+many	O
+of	O
+the	O
+peaks	O
+disappeared	O
+,	O
+the	O
+mean	B-evidence
+chemical	I-evidence
+shift	I-evidence
+change	I-evidence
+was	O
+relatively	O
+low	O
+,	O
+so	O
+a	O
+threshold	O
+of	O
+the	O
+mean	O
+plus	O
+one	O
+S	O
+.	O
+D	O
+.	O
+value	O
+was	O
+used	O
+to	O
+define	O
+a	O
+significant	O
+CSP	B-experimental_method
+.	O
+
+Parts	O
+of	O
+the	O
+switch	B-site
+regions	I-site
+(	O
+Fig	O
+.	O
+5	O
+,	O
+B	O
+and	O
+C	O
+)	O
+are	O
+invisible	O
+in	O
+NMR	B-experimental_method
+spectra	B-evidence
+recorded	O
+on	O
+free	B-protein_state
+Cdc42	B-protein
+due	O
+to	O
+conformational	O
+exchange	O
+.	O
+
+These	O
+switch	B-site
+regions	I-site
+become	O
+visible	O
+in	O
+Cdc42	B-protein
+and	O
+other	O
+small	O
+G	B-protein_type
+protein	I-protein_type
+·	O
+effector	O
+complexes	O
+due	O
+to	O
+a	O
+decrease	O
+in	O
+conformational	O
+freedom	O
+upon	O
+complex	O
+formation	O
+.	O
+
+The	O
+switch	B-site
+regions	I-site
+of	O
+Cdc42	B-protein
+did	O
+not	O
+,	O
+however	O
+,	O
+become	O
+visible	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+.	O
+
+Indeed	O
+,	O
+Ser	B-residue_name_number
+-	I-residue_name_number
+30	I-residue_name_number
+of	O
+switch	B-site
+I	I-site
+and	O
+Arg	B-residue_name_number
+-	I-residue_name_number
+66	I-residue_name_number
+,	O
+Arg	B-residue_name_number
+-	I-residue_name_number
+68	I-residue_name_number
+,	O
+Leu	B-residue_name_number
+-	I-residue_name_number
+70	I-residue_name_number
+,	O
+and	O
+Ser	B-residue_name_number
+-	I-residue_name_number
+71	I-residue_name_number
+of	O
+switch	B-site
+II	I-site
+are	O
+visible	O
+in	O
+free	B-protein_state
+Cdc42	B-protein
+but	O
+disappear	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+HR1	B-structure_element
+domain	O
+.	O
+
+This	O
+suggests	O
+that	O
+the	O
+switch	B-site
+regions	I-site
+are	O
+not	O
+rigidified	O
+in	O
+the	O
+HR1	B-structure_element
+complex	O
+and	O
+are	O
+still	O
+in	O
+conformational	O
+exchange	O
+.	O
+
+Nevertheless	O
+,	O
+mapping	O
+of	O
+the	O
+affected	O
+residues	O
+onto	O
+the	O
+NMR	B-experimental_method
+structure	B-evidence
+of	O
+free	B-protein_state
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+(	O
+Fig	O
+.	O
+5C	O
+)	O
+8	O
+shows	O
+that	O
+,	O
+although	O
+they	O
+are	O
+relatively	O
+widespread	O
+compared	O
+with	O
+changes	O
+in	O
+the	O
+HR1	B-structure_element
+domain	O
+,	O
+in	O
+general	O
+,	O
+they	O
+are	O
+on	O
+the	O
+face	O
+of	O
+the	O
+protein	O
+that	O
+includes	O
+the	O
+switches	B-site
+.	O
+
+Although	O
+the	O
+binding	B-site
+interface	I-site
+may	O
+be	O
+overestimated	O
+,	O
+this	O
+suggests	O
+that	O
+the	O
+switch	B-site
+regions	I-site
+are	O
+involved	O
+in	O
+binding	O
+to	O
+TOCA1	B-protein
+.	O
+
+Modeling	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+HR1	I-complex_assembly
+Complex	O
+
+The	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1TOCA1	I-complex_assembly
+complex	O
+was	O
+not	O
+amenable	O
+to	O
+full	O
+structural	O
+analysis	O
+due	O
+to	O
+the	O
+weak	O
+interaction	O
+and	O
+the	O
+extensive	O
+exchange	O
+broadening	O
+seen	O
+in	O
+the	O
+NMR	B-experimental_method
+experiments	O
+.	O
+
+HADDOCK	B-experimental_method
+was	O
+therefore	O
+used	O
+to	O
+perform	O
+rigid	O
+body	B-experimental_method
+docking	I-experimental_method
+based	O
+on	O
+the	O
+structures	B-evidence
+of	O
+free	B-protein_state
+HR1	B-structure_element
+domain	O
+and	O
+Cdc42	B-protein
+and	O
+ambiguous	O
+interaction	O
+restraints	O
+derived	O
+from	O
+the	O
+titration	B-experimental_method
+experiments	I-experimental_method
+described	O
+above	O
+.	O
+
+The	O
+orientation	O
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+with	O
+respect	O
+to	O
+Cdc42	B-protein
+cannot	O
+be	O
+definitively	O
+concluded	O
+in	O
+the	O
+absence	O
+of	O
+unambiguous	O
+distance	O
+restraints	O
+;	O
+hence	O
+,	O
+HADDOCK	B-experimental_method
+produced	O
+a	O
+set	O
+of	O
+models	O
+in	O
+which	O
+the	O
+HR1	B-structure_element
+domain	O
+contacts	O
+the	O
+same	O
+surface	O
+on	O
+Cdc42	B-protein
+but	O
+is	O
+in	O
+various	O
+orientations	O
+with	O
+respect	O
+to	O
+Cdc42	B-protein
+.	O
+
+The	O
+cluster	O
+with	O
+the	O
+lowest	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+from	O
+the	O
+lowest	O
+energy	O
+structure	B-evidence
+is	O
+assumed	O
+to	O
+be	O
+the	O
+best	O
+model	O
+.	O
+
+By	O
+these	O
+criteria	O
+,	O
+in	O
+the	O
+best	O
+model	O
+,	O
+the	O
+HR1	B-structure_element
+domain	O
+is	O
+in	O
+a	O
+similar	O
+orientation	O
+to	O
+the	O
+HR1a	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+bound	B-protein_state
+to	I-protein_state
+RhoA	B-protein
+and	O
+the	O
+HR1b	B-structure_element
+domain	O
+bound	B-protein_state
+to	I-protein_state
+Rac1	B-protein
+.	O
+
+A	O
+representative	O
+model	O
+from	O
+this	O
+cluster	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+6A	O
+alongside	O
+the	O
+Rac1	B-complex_assembly
+-	I-complex_assembly
+HR1b	I-complex_assembly
+structure	B-evidence
+(	O
+PDB	O
+code	O
+2RMK	O
+)	O
+in	O
+Fig	O
+.	O
+6B	O
+.	O
+
+Model	O
+of	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1	I-complex_assembly
+complex	O
+.	O
+
+A	O
+,	O
+a	O
+representative	O
+model	O
+of	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1	I-complex_assembly
+complex	O
+from	O
+the	O
+cluster	O
+closest	O
+to	O
+the	O
+lowest	O
+energy	O
+model	O
+produced	O
+using	O
+HADDOCK	B-experimental_method
+.	O
+
+Residues	O
+of	O
+Cdc42	B-protein
+that	O
+are	O
+affected	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+HR1	B-structure_element
+domain	O
+but	O
+are	O
+not	O
+in	O
+close	O
+proximity	O
+to	O
+it	O
+are	O
+colored	O
+in	O
+red	O
+and	O
+labeled	O
+.	O
+
+B	O
+,	O
+structure	B-evidence
+of	O
+Rac1	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+HR1b	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+(	O
+PDB	O
+code	O
+2RMK	O
+).	O
+
+C	O
+,	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+RhoA	B-protein
+,	O
+Cdc42	B-protein
+and	O
+Rac1	B-protein
+.	O
+
+Contact	O
+residues	O
+of	O
+RhoA	B-protein
+and	O
+Rac1	B-protein
+to	O
+PRK1	B-protein
+HR1a	B-structure_element
+and	O
+HR1b	B-structure_element
+,	O
+respectively	O
+,	O
+are	O
+colored	O
+cyan	O
+.	O
+
+Residues	O
+of	O
+Cdc42	B-protein
+that	O
+disappear	O
+or	O
+show	O
+chemical	O
+shift	O
+changes	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+TOCA1	B-protein
+are	O
+colored	O
+cyan	O
+if	O
+also	O
+identified	O
+as	O
+contacts	O
+in	O
+RhoA	B-protein
+and	O
+Rac1	B-protein
+and	O
+yellow	O
+if	O
+they	O
+are	O
+not	O
+.	O
+
+Residues	O
+equivalent	O
+to	O
+Rac1	B-protein
+and	O
+RhoA	B-protein
+contact	B-site
+sites	I-site
+but	O
+that	O
+are	O
+invisible	O
+in	O
+free	B-protein_state
+Cdc42	B-protein
+are	O
+gray	O
+.	O
+
+D	O
+,	O
+regions	O
+of	O
+interest	O
+of	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1	I-complex_assembly
+domain	O
+model	O
+.	O
+
+The	O
+four	O
+lowest	O
+energy	O
+structures	B-evidence
+in	O
+the	O
+chosen	O
+HADDOCK	B-experimental_method
+cluster	O
+are	O
+shown	O
+overlaid	O
+,	O
+with	O
+the	O
+residues	O
+of	O
+interest	O
+shown	O
+as	O
+sticks	O
+and	O
+labeled	O
+.	O
+
+Cdc42	O
+is	O
+shown	O
+in	O
+cyan	O
+,	O
+and	O
+TOCA1	B-protein
+is	O
+shown	O
+in	O
+purple	O
+.	O
+
+A	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+RhoA	B-protein
+,	O
+Cdc42	B-protein
+,	O
+and	O
+Rac1	B-protein
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+6C	O
+.	O
+
+The	O
+RhoA	B-protein
+and	O
+Rac1	B-protein
+contact	O
+residues	O
+in	O
+the	O
+switch	B-site
+regions	I-site
+are	O
+invisible	O
+in	O
+the	O
+spectra	B-evidence
+of	O
+Cdc42	B-protein
+,	O
+but	O
+they	O
+are	O
+generally	O
+conserved	B-protein_state
+between	O
+all	O
+three	O
+G	B-protein_type
+proteins	I-protein_type
+.	O
+
+Several	O
+Cdc42	B-protein
+residues	O
+identified	O
+by	O
+chemical	B-experimental_method
+shift	I-experimental_method
+mapping	I-experimental_method
+are	O
+not	O
+in	O
+close	O
+contact	O
+in	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+model	O
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+
+Some	O
+of	O
+these	O
+can	O
+be	O
+rationalized	O
+;	O
+for	O
+example	O
+,	O
+Thr	B-residue_name_number
+-	I-residue_name_number
+24Cdc42	I-residue_name_number
+,	O
+Leu	B-residue_name_number
+-	I-residue_name_number
+160Cdc42	I-residue_name_number
+,	O
+and	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+163Cdc42	I-residue_name_number
+all	O
+pack	O
+behind	O
+switch	B-site
+I	I-site
+and	O
+are	O
+likely	O
+to	O
+be	O
+affected	O
+by	O
+conformational	O
+changes	O
+within	O
+the	O
+switch	B-site
+,	O
+while	O
+Glu	B-residue_name_number
+-	I-residue_name_number
+95Cdc42	I-residue_name_number
+and	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+96Cdc42	I-residue_name_number
+are	O
+in	O
+the	O
+helix	B-structure_element
+behind	O
+switch	B-site
+II	I-site
+.	O
+
+Other	O
+residues	O
+that	O
+are	O
+affected	O
+in	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+complex	O
+but	O
+that	O
+do	O
+not	O
+correspond	O
+to	O
+contact	O
+residues	O
+of	O
+RhoA	B-protein
+or	O
+Rac1	B-protein
+(	O
+Fig	O
+.	O
+6C	O
+)	O
+include	O
+Gln	B-residue_name_number
+-	I-residue_name_number
+2Cdc42	I-residue_name_number
+,	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+16Cdc42	I-residue_name_number
+,	O
+Thr	B-residue_name_number
+-	I-residue_name_number
+52Cdc42	I-residue_name_number
+,	O
+and	O
+Arg	B-residue_name_number
+-	I-residue_name_number
+68Cdc42	I-residue_name_number
+.	O
+
+Lys	B-residue_name_number
+-	I-residue_name_number
+16Cdc42	I-residue_name_number
+is	O
+unlikely	O
+to	O
+be	O
+a	O
+contact	O
+residue	O
+because	O
+it	O
+is	O
+involved	O
+in	O
+nucleotide	O
+binding	O
+,	O
+but	O
+the	O
+others	O
+may	O
+represent	O
+specific	O
+Cdc42	B-complex_assembly
+-	I-complex_assembly
+TOCA1	I-complex_assembly
+contacts	O
+.	O
+
+Competition	O
+between	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+TOCA1	B-protein
+
+From	O
+the	O
+known	O
+interactions	O
+and	O
+effects	O
+of	O
+the	O
+proteins	O
+in	O
+biological	O
+systems	O
+,	O
+it	O
+has	O
+been	O
+suggested	O
+that	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+could	O
+bind	O
+Cdc42	B-protein
+simultaneously	O
+.	O
+
+Studies	O
+in	O
+CHO	O
+cells	O
+indicated	O
+that	O
+a	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+N	I-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+complex	O
+existed	O
+because	O
+FRET	B-evidence
+was	O
+observed	O
+between	O
+RFP	B-chemical
+-	O
+TOCA1	B-protein
+and	O
+GFP	B-chemical
+-	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+and	O
+the	O
+efficiency	O
+was	O
+decreased	O
+when	O
+an	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+mutant	B-protein_state
+was	O
+used	O
+that	O
+no	O
+longer	O
+binds	O
+Cdc42	B-protein
+.	O
+
+An	O
+overlay	B-experimental_method
+of	O
+the	O
+HADDOCK	B-experimental_method
+model	B-evidence
+of	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1TOCA1	I-complex_assembly
+complex	O
+and	O
+the	O
+structure	B-evidence
+of	O
+Cdc42	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+GBD	B-structure_element
+of	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+homologue	O
+,	O
+WASP	B-protein
+(	O
+PDB	O
+code	O
+1CEE	O
+),	O
+shows	O
+that	O
+the	O
+HR1	B-structure_element
+and	O
+GBD	B-site
+binding	I-site
+sites	I-site
+only	O
+partly	O
+overlap	O
+,	O
+and	O
+,	O
+therefore	O
+,	O
+a	O
+ternary	O
+complex	O
+remained	O
+possible	O
+(	O
+Fig	O
+.	O
+7A	O
+).	O
+
+Interestingly	O
+,	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+would	O
+not	O
+prevent	O
+the	O
+core	O
+CRIB	B-structure_element
+of	O
+WASP	B-protein
+from	O
+binding	O
+to	O
+Cdc42	B-protein
+,	O
+although	O
+the	O
+regions	O
+C	O
+-	O
+terminal	O
+to	O
+the	O
+CRIB	B-structure_element
+that	O
+are	O
+required	O
+for	O
+high	O
+affinity	O
+binding	O
+of	O
+WASP	B-protein
+would	O
+interfere	O
+sterically	O
+with	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+.	O
+
+A	O
+basic	O
+region	O
+in	O
+WASP	B-protein
+including	O
+three	O
+lysines	B-residue_name
+(	O
+residues	O
+230	B-residue_range
+–	I-residue_range
+232	I-residue_range
+),	O
+N	O
+-	O
+terminal	O
+to	O
+the	O
+core	O
+CRIB	B-structure_element
+,	O
+has	O
+been	O
+implicated	O
+in	O
+an	O
+electrostatic	O
+steering	O
+mechanism	O
+,	O
+and	O
+these	O
+residues	O
+would	O
+be	O
+free	O
+to	O
+bind	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+(	O
+Fig	O
+.	O
+7A	O
+).	O
+
+The	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+displaces	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+.	O
+
+A	O
+,	O
+the	O
+model	O
+of	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+HR1	B-structure_element
+domain	O
+complex	O
+overlaid	O
+with	O
+the	O
+Cdc42	B-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+structure	B-evidence
+.	O
+
+Cdc42	O
+is	O
+shown	O
+in	O
+green	O
+,	O
+and	O
+TOCA1	B-protein
+is	O
+shown	O
+in	O
+purple	O
+.	O
+
+The	O
+core	O
+CRIB	B-structure_element
+region	O
+of	O
+WASP	B-protein
+is	O
+shown	O
+in	O
+red	O
+,	O
+whereas	O
+its	O
+basic	O
+region	O
+is	O
+shown	O
+in	O
+orange	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+required	O
+for	O
+maximal	O
+affinity	O
+is	O
+shown	O
+in	O
+cyan	O
+.	O
+
+A	O
+semitransparent	O
+surface	O
+representation	O
+of	O
+Cdc42	B-protein
+and	O
+WASP	B-protein
+is	O
+shown	O
+overlaid	O
+with	O
+the	O
+schematic	O
+.	O
+
+B	O
+,	O
+competition	B-experimental_method
+SPA	I-experimental_method
+experiments	O
+carried	O
+out	O
+with	O
+indicated	O
+concentrations	O
+of	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+construct	O
+titrated	B-experimental_method
+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	B-structure_element
+and	O
+30	O
+nm	O
+Cdc42Δ7Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+.	O
+
+C	O
+,	O
+Selected	O
+regions	O
+of	O
+the	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+of	O
+145	O
+μm	O
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+with	O
+the	O
+indicated	O
+ratios	O
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+,	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+,	O
+or	O
+both	O
+,	O
+showing	O
+that	O
+the	O
+TOCA	B-protein
+HR1	B-structure_element
+domain	O
+does	O
+not	O
+displace	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+.	O
+
+D	O
+,	O
+selected	O
+regions	O
+of	O
+the	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+of	O
+600	O
+μm	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Cdc42	B-protein
+in	O
+the	O
+absence	B-protein_state
+and	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+,	O
+showing	O
+displacement	O
+of	O
+Cdc42	B-protein
+from	O
+the	O
+HR1	B-structure_element
+domain	O
+by	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+An	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+construct	O
+was	O
+produced	O
+,	O
+and	O
+its	O
+affinity	B-evidence
+for	O
+Cdc42	B-protein
+was	O
+measured	O
+by	O
+competition	B-experimental_method
+SPA	I-experimental_method
+(	O
+Fig	O
+.	O
+7B	O
+).	O
+
+The	O
+Kd	B-evidence
+that	O
+was	O
+determined	O
+(	O
+37	O
+nm	O
+)	O
+is	O
+consistent	O
+with	O
+the	O
+previously	O
+reported	O
+affinity	B-evidence
+.	O
+
+Unlabeled	B-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+was	O
+titrated	B-experimental_method
+into	O
+15N	B-chemical
+-	O
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+,	O
+and	O
+the	O
+backbone	O
+NH	O
+groups	O
+were	O
+monitored	O
+using	O
+HSQCs	B-experimental_method
+(	O
+Fig	O
+.	O
+7C	O
+).	O
+
+Unlabeled	B-protein_state
+HR1TOCA1	B-structure_element
+was	O
+then	O
+added	O
+to	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+N	I-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+complex	O
+,	O
+and	O
+no	O
+changes	O
+were	O
+seen	O
+,	O
+suggesting	O
+that	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+was	O
+not	O
+displaced	O
+even	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+a	O
+5	O
+-	O
+fold	O
+excess	O
+of	O
+HR1TOCA1	B-structure_element
+.	O
+
+These	O
+experiments	O
+were	O
+recorded	O
+at	O
+sufficiently	O
+high	O
+protein	O
+concentrations	O
+(	O
+145	O
+μm	O
+Cdc42	B-protein
+,	O
+145	O
+μm	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+,	O
+725	O
+μm	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+)	O
+to	O
+be	O
+far	O
+in	O
+excess	O
+of	O
+the	O
+Kd	B-evidence
+values	O
+of	O
+the	O
+individual	O
+interactions	O
+(	O
+TOCA1	B-protein
+Kd	B-evidence
+≈	O
+5	O
+μm	O
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+Kd	B-evidence
+=	O
+37	O
+nm	O
+).	O
+
+A	O
+comparison	O
+of	O
+the	O
+HSQC	B-experimental_method
+experiments	O
+recorded	O
+on	O
+15N	B-chemical
+-	O
+Cdc42	B-protein
+alone	B-protein_state
+,	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+,	O
+or	O
+both	O
+,	O
+shows	O
+that	O
+the	O
+spectra	B-evidence
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+both	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+TOCA1	B-protein
+HR1	B-structure_element
+are	O
+identical	O
+(	O
+Fig	O
+.	O
+7C	O
+).	O
+
+Furthermore	O
+,	O
+15N	B-chemical
+-	O
+TOCA1	B-protein
+HR1	B-structure_element
+was	O
+monitored	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+unlabeled	B-protein_state
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+(	O
+1	O
+:	O
+1	O
+)	O
+before	O
+and	O
+after	O
+the	O
+addition	O
+of	O
+0	O
+.	O
+25	O
+and	O
+1	O
+.	O
+0	O
+eq	O
+of	O
+unlabeled	B-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+.	O
+
+The	O
+spectrum	B-evidence
+when	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+TOCA1	B-protein
+were	O
+equimolar	O
+was	O
+identical	O
+to	O
+that	O
+of	O
+the	O
+free	B-protein_state
+HR1	B-structure_element
+domain	O
+,	O
+whereas	O
+the	O
+spectrum	B-evidence
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+0	O
+.	O
+25	O
+eq	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+was	O
+intermediate	O
+between	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+free	B-protein_state
+and	O
+complex	B-protein_state
+spectra	B-evidence
+(	O
+Fig	O
+.	O
+7D	O
+).	O
+
+When	O
+in	O
+fast	O
+exchange	O
+,	O
+the	O
+NMR	B-experimental_method
+signal	O
+represents	O
+a	O
+population	O
+-	O
+weighted	O
+average	O
+between	O
+free	B-protein_state
+and	O
+bound	B-protein_state
+states	O
+,	O
+so	O
+the	O
+intermediate	O
+spectrum	B-evidence
+indicates	O
+that	O
+the	O
+population	O
+comprises	O
+a	O
+mixture	O
+of	O
+free	B-protein_state
+and	O
+bound	B-protein_state
+HR1	B-structure_element
+domain	O
+.	O
+
+Again	O
+,	O
+the	O
+experiments	O
+were	O
+recorded	O
+on	O
+protein	O
+samples	O
+far	O
+in	O
+excess	O
+of	O
+the	O
+individual	O
+Kd	B-evidence
+values	O
+(	O
+600	O
+μm	O
+each	O
+protein	O
+).	O
+
+These	O
+data	O
+indicate	O
+that	O
+the	O
+HR1	B-structure_element
+domain	O
+is	O
+displaced	O
+from	O
+Cdc42	B-protein
+by	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+that	O
+a	O
+ternary	O
+complex	O
+comprising	O
+TOCA1	B-protein
+HR1	B-structure_element
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+,	O
+and	O
+Cdc42	B-protein
+is	O
+not	O
+formed	O
+.	O
+
+Taken	O
+together	O
+,	O
+the	O
+data	O
+in	O
+Fig	O
+.	O
+7	O
+,	O
+C	O
+and	O
+D	O
+,	O
+indicate	O
+unidirectional	O
+competition	O
+for	O
+Cdc42	B-protein
+binding	O
+in	O
+which	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+displaces	O
+TOCA1	B-protein
+HR1	B-structure_element
+but	O
+not	O
+vice	O
+versa	O
+.	O
+
+To	O
+extend	O
+these	O
+studies	O
+to	O
+a	O
+more	O
+complex	O
+system	O
+and	O
+to	O
+assess	O
+the	O
+ability	O
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+to	O
+compete	O
+with	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+pyrene	B-experimental_method
+actin	I-experimental_method
+assays	I-experimental_method
+were	O
+employed	O
+.	O
+
+These	O
+assays	O
+,	O
+described	O
+in	O
+detail	O
+elsewhere	O
+,	O
+were	O
+carried	O
+out	O
+using	O
+pyrene	B-chemical
+actin	I-chemical
+-	O
+supplemented	O
+Xenopus	B-taxonomy_domain
+extracts	O
+into	O
+which	O
+exogenous	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+or	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+was	O
+added	O
+,	O
+to	O
+assess	O
+their	O
+effects	O
+on	O
+actin	B-protein_type
+polymerization	O
+.	O
+
+Actin	B-protein_type
+polymerization	O
+in	O
+all	O
+cases	O
+was	O
+initiated	O
+by	O
+the	O
+addition	O
+of	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+-	O
+containing	O
+liposomes	O
+.	O
+
+Actin	B-protein_type
+polymerization	O
+triggered	O
+by	O
+the	O
+addition	O
+of	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+-	O
+containing	O
+liposomes	O
+has	O
+previously	O
+been	O
+shown	O
+to	O
+depend	O
+on	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+Endogenous	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+is	O
+present	O
+at	O
+∼	O
+100	O
+nm	O
+in	O
+Xenopus	B-taxonomy_domain
+extracts	O
+,	O
+whereas	O
+TOCA1	B-protein
+is	O
+present	O
+at	O
+a	O
+10	O
+-	O
+fold	O
+lower	O
+concentration	O
+than	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+The	O
+addition	B-experimental_method
+of	O
+the	O
+isolated	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+significantly	O
+inhibited	O
+the	O
+polymerization	O
+of	O
+actin	B-protein_type
+at	O
+concentrations	O
+as	O
+low	O
+as	O
+100	O
+nm	O
+and	O
+completely	O
+abolished	O
+polymerization	O
+at	O
+higher	O
+concentrations	O
+(	O
+Fig	O
+.	O
+8	O
+).	O
+
+The	O
+GBD	B-structure_element
+presumably	O
+acts	O
+as	O
+a	O
+dominant	O
+negative	O
+,	O
+sequestering	O
+endogenous	O
+Cdc42	B-protein
+and	O
+preventing	O
+endogenous	B-protein_state
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+from	O
+binding	O
+and	O
+becoming	O
+activated	O
+.	O
+
+The	O
+addition	B-experimental_method
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+to	O
+100	O
+μm	O
+had	O
+no	O
+significant	O
+effect	O
+on	O
+the	O
+rate	O
+of	O
+actin	B-protein_type
+polymerization	O
+or	O
+maximum	B-evidence
+fluorescence	I-evidence
+.	O
+
+This	O
+is	O
+consistent	O
+with	O
+endogenous	B-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+activated	O
+by	O
+other	O
+components	O
+of	O
+the	O
+assay	O
+,	O
+outcompeting	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+for	O
+Cdc42	B-protein
+binding	O
+.	O
+
+Actin	O
+polymerization	O
+downstream	O
+of	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+N	I-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+is	O
+inhibited	B-protein_state
+by	O
+excess	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+but	O
+not	O
+by	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+.	O
+
+Fluorescence	B-evidence
+curves	I-evidence
+show	O
+actin	O
+polymerization	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+increasing	B-experimental_method
+concentrations	I-experimental_method
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+or	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+as	O
+indicated	O
+.	O
+
+The	O
+Cdc42	B-protein
+-	O
+TOCA1	B-protein
+Interaction	O
+
+The	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+alone	B-protein_state
+is	O
+sufficient	O
+for	O
+Cdc42	B-protein
+binding	O
+in	O
+vitro	O
+,	O
+yet	O
+the	O
+affinity	B-evidence
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+for	O
+Cdc42	B-protein
+is	O
+remarkably	O
+low	O
+(	O
+Kd	B-evidence
+≈	O
+5	O
+μm	O
+).	O
+
+This	O
+is	O
+over	O
+100	O
+times	O
+lower	O
+than	O
+that	O
+of	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+(	O
+Kd	B-evidence
+=	O
+37	O
+nm	O
+)	O
+and	O
+considerably	O
+lower	O
+than	O
+other	O
+known	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+HR1	B-structure_element
+domain	O
+interactions	O
+.	O
+
+The	O
+polybasic	O
+tract	O
+within	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+Cdc42	B-protein
+does	O
+not	O
+appear	O
+to	O
+be	O
+required	O
+for	O
+binding	O
+to	O
+TOCA1	B-protein
+,	O
+which	O
+is	O
+in	O
+contrast	O
+to	O
+the	O
+interaction	O
+between	O
+Rac1	B-protein
+and	O
+the	O
+HR1b	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+but	O
+more	O
+similar	O
+to	O
+the	O
+PRK1	B-protein
+HR1a	B-structure_element
+-	O
+RhoA	B-protein
+interaction	O
+.	O
+
+A	O
+single	O
+binding	B-site
+interface	I-site
+on	O
+both	O
+the	O
+HR1	B-structure_element
+domain	O
+and	O
+Cdc42	B-protein
+can	O
+be	O
+concluded	O
+from	O
+the	O
+data	O
+presented	O
+here	O
+.	O
+
+Furthermore	O
+,	O
+the	O
+interfaces	B-site
+are	O
+comparable	O
+with	O
+those	O
+of	O
+other	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+HR1	B-structure_element
+interactions	O
+(	O
+Fig	O
+.	O
+4	O
+),	O
+and	O
+the	O
+lowest	O
+energy	O
+model	B-evidence
+produced	O
+in	O
+rigid	B-experimental_method
+body	I-experimental_method
+docking	I-experimental_method
+resembles	O
+previously	O
+studied	O
+G	B-complex_assembly
+protein	I-complex_assembly
+·	I-complex_assembly
+HR1	I-complex_assembly
+complexes	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+It	O
+seems	O
+,	O
+therefore	O
+,	O
+that	O
+the	O
+interaction	O
+,	O
+despite	O
+its	O
+relatively	O
+low	O
+affinity	O
+,	O
+is	O
+specific	O
+and	O
+sterically	O
+similar	O
+to	O
+other	O
+HR1	B-structure_element
+domain	O
+-	O
+G	B-protein_type
+protein	I-protein_type
+interactions	O
+.	O
+
+The	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+is	O
+a	O
+left	O
+-	O
+handed	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+comparable	O
+with	O
+other	O
+known	O
+HR1	B-structure_element
+domains	O
+.	O
+
+A	O
+short	O
+region	O
+N	O
+-	O
+terminal	O
+to	O
+the	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+exhibits	O
+a	O
+series	O
+of	O
+turns	O
+and	O
+contacts	O
+residues	O
+of	O
+both	O
+helices	O
+of	O
+the	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+(	O
+Fig	O
+.	O
+3	O
+).	O
+
+The	O
+corresponding	O
+sequence	O
+in	O
+CIP4	B-protein
+also	O
+includes	O
+a	O
+series	O
+of	O
+turns	O
+but	O
+is	O
+flexible	O
+,	O
+whereas	O
+in	O
+the	O
+HR1a	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+,	O
+the	O
+equivalent	O
+region	O
+adopts	O
+an	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+structure	I-structure_element
+that	O
+packs	O
+against	O
+the	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+.	O
+
+The	O
+contacts	O
+between	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+and	O
+the	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+are	O
+predominantly	O
+hydrophobic	O
+in	O
+both	O
+cases	O
+,	O
+but	O
+sequence	O
+-	O
+specific	O
+contacts	O
+do	O
+not	O
+appear	O
+to	O
+be	O
+conserved	O
+.	O
+
+This	O
+region	O
+is	O
+distant	O
+from	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+interface	I-site
+of	O
+the	O
+HR1	B-structure_element
+domains	O
+,	O
+so	O
+the	O
+structural	O
+differences	O
+may	O
+relate	O
+to	O
+the	O
+structure	O
+and	O
+regulation	O
+of	O
+these	O
+domains	O
+rather	O
+than	O
+their	O
+G	B-protein_type
+protein	I-protein_type
+interactions	O
+.	O
+
+The	O
+interhelical	B-structure_element
+loops	I-structure_element
+of	O
+TOCA1	B-protein
+and	O
+CIP4	B-protein
+differ	O
+from	O
+the	O
+same	O
+region	O
+in	O
+the	O
+HR1	B-structure_element
+domains	O
+of	O
+PRK1	B-protein
+in	O
+that	O
+they	O
+are	O
+longer	O
+and	O
+contain	O
+two	O
+short	O
+stretches	O
+of	O
+310	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+.	O
+
+This	O
+region	O
+lies	O
+within	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+surface	I-site
+of	O
+all	O
+of	O
+the	O
+HR1	B-structure_element
+domains	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+
+TOCA1	B-protein
+and	O
+CIP4	B-protein
+both	O
+bind	O
+weakly	O
+to	O
+Cdc42	B-protein
+,	O
+whereas	O
+the	O
+HR1a	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+binds	O
+tightly	O
+to	O
+RhoA	B-protein
+and	O
+Rac1	B-protein
+,	O
+and	O
+the	O
+HR1b	B-structure_element
+domain	O
+binds	O
+to	O
+Rac1	B-protein
+.	O
+
+The	O
+structural	O
+features	O
+shared	O
+by	O
+TOCA1	B-protein
+and	O
+CIP4	B-protein
+may	O
+therefore	O
+be	O
+related	O
+to	O
+Cdc42	B-protein
+binding	O
+specificity	O
+and	O
+the	O
+low	O
+affinities	O
+.	O
+
+In	O
+free	B-protein_state
+TOCA1	B-protein
+,	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+interhelical	B-structure_element
+region	I-structure_element
+make	O
+extensive	O
+contacts	O
+with	O
+residues	O
+in	O
+helix	B-structure_element
+1	I-structure_element
+.	O
+
+Many	O
+of	O
+these	O
+residues	O
+are	O
+significantly	O
+affected	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Cdc42	B-protein
+,	O
+so	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+conformation	O
+of	O
+this	O
+loop	B-structure_element
+is	O
+altered	O
+in	O
+the	O
+Cdc42	B-protein
+complex	O
+.	O
+
+These	O
+observations	O
+therefore	O
+provide	O
+a	O
+molecular	O
+mechanism	O
+whereby	O
+mutation	B-experimental_method
+of	O
+Met383	B-residue_name_number
+-	O
+Gly384	B-residue_name_number
+-	O
+Asp385	B-residue_name_number
+to	O
+Ile383	B-residue_name_number
+-	O
+Ser384	B-residue_name_number
+-	O
+Thr385	B-residue_name_number
+abolishes	O
+TOCA1	B-protein
+binding	O
+to	O
+Cdc42	B-protein
+.	O
+
+The	O
+lowest	O
+energy	O
+model	B-evidence
+produced	O
+by	O
+HADDOCK	B-experimental_method
+using	O
+ambiguous	O
+interaction	O
+restraints	O
+from	O
+the	O
+titration	B-evidence
+data	O
+resembled	O
+the	O
+NMR	B-experimental_method
+structures	B-evidence
+of	O
+RhoA	B-protein
+and	O
+Rac1	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+their	O
+HR1	B-structure_element
+domain	O
+partners	O
+.	O
+
+For	O
+example	O
+,	O
+Phe	B-residue_name_number
+-	I-residue_name_number
+56Cdc42	I-residue_name_number
+,	O
+which	O
+is	O
+not	O
+visible	O
+in	O
+free	B-protein_state
+Cdc42	B-protein
+or	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1TOCA1	I-complex_assembly
+,	O
+is	O
+close	O
+to	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+
+Phe	B-residue_name_number
+-	I-residue_name_number
+56Cdc42	I-residue_name_number
+,	O
+which	O
+is	O
+a	O
+Trp	B-residue_name
+in	O
+both	O
+Rac1	B-protein
+and	O
+RhoA	B-protein
+(	O
+Fig	O
+.	O
+6C	O
+),	O
+is	O
+thought	O
+to	O
+pack	O
+behind	O
+switch	B-site
+I	I-site
+when	O
+Cdc42	B-protein
+interacts	O
+with	O
+ACK	B-protein
+,	O
+maintaining	O
+the	O
+switch	O
+in	O
+a	O
+binding	O
+-	O
+competent	O
+orientation	O
+.	O
+
+This	O
+residue	O
+has	O
+also	O
+been	O
+identified	O
+as	O
+important	O
+for	O
+Cdc42	B-protein
+-	O
+WASP	B-protein
+binding	O
+.	O
+
+Phe	B-residue_name_number
+-	I-residue_name_number
+56Cdc42	I-residue_name_number
+is	O
+therefore	O
+likely	O
+to	O
+be	O
+involved	O
+in	O
+the	O
+Cdc42	B-protein
+-	O
+TOCA1	B-protein
+interaction	O
+,	O
+probably	O
+by	O
+stabilizing	O
+the	O
+position	O
+of	O
+switch	B-site
+I	I-site
+.	O
+
+Some	O
+residues	O
+that	O
+are	O
+affected	O
+in	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1TOCA1	I-complex_assembly
+complex	O
+but	O
+do	O
+not	O
+correspond	O
+to	O
+contact	O
+residues	O
+of	O
+RhoA	B-protein
+or	O
+Rac1	B-protein
+(	O
+Fig	O
+.	O
+6C	O
+)	O
+may	O
+contact	O
+HR1TOCA1	B-structure_element
+directly	O
+(	O
+Fig	O
+.	O
+6D	O
+).	O
+
+Gln	B-residue_name_number
+-	I-residue_name_number
+2Cdc42	I-residue_name_number
+,	O
+which	O
+has	O
+also	O
+been	O
+identified	O
+as	O
+a	O
+contact	O
+residue	O
+in	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+ACK	I-complex_assembly
+complex	O
+,	O
+contacts	O
+Val	B-residue_name_number
+-	I-residue_name_number
+376TOCA1	I-residue_name_number
+and	O
+Asn	B-residue_name_number
+-	I-residue_name_number
+380TOCA1	I-residue_name_number
+in	O
+the	O
+model	O
+and	O
+disrupts	O
+the	O
+contacts	O
+between	O
+the	O
+interhelical	B-structure_element
+loop	I-structure_element
+and	O
+the	O
+first	B-structure_element
+helix	I-structure_element
+of	O
+the	O
+TOCA1	B-protein
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+.	O
+
+Thr	B-residue_name_number
+-	I-residue_name_number
+52Cdc42	I-residue_name_number
+,	O
+which	O
+has	O
+also	O
+been	O
+identified	O
+as	O
+making	O
+minor	O
+contacts	O
+with	O
+ACK	B-protein
+,	O
+falls	O
+near	O
+the	O
+side	O
+chains	O
+of	O
+HR1TOCA1	B-structure_element
+helix	B-structure_element
+1	I-structure_element
+,	O
+particularly	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+372TOCA1	I-residue_name_number
+,	O
+whereas	O
+the	O
+equivalent	O
+position	O
+in	O
+Rac1	B-protein
+is	O
+Asn	B-residue_name_number
+-	I-residue_name_number
+52Rac1	I-residue_name_number
+.	O
+
+N52T	B-mutant
+is	O
+one	O
+of	O
+a	O
+combination	O
+of	O
+seven	O
+residues	O
+found	O
+to	O
+confer	O
+ACK	B-protein
+binding	O
+on	O
+Rac1	B-protein
+and	O
+so	O
+may	O
+represent	O
+a	O
+specific	O
+Cdc42	B-protein
+-	O
+effector	O
+contact	O
+residue	O
+.	O
+
+The	O
+position	O
+equivalent	O
+to	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+372TOCA1	I-residue_name_number
+in	O
+PRK1	B-protein
+is	O
+Glu	B-residue_name_number
+-	I-residue_name_number
+58HR1a	I-residue_name_number
+or	O
+Gln	B-residue_name_number
+-	I-residue_name_number
+151HR1b	I-residue_name_number
+.	O
+
+Thr	B-residue_name_number
+-	I-residue_name_number
+52Cdc42	I-residue_name_number
+-	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+372TOCA1	I-residue_name_number
+may	O
+therefore	O
+represent	O
+a	O
+specific	O
+Cdc42	B-protein
+-	O
+HR1TOCA1	B-structure_element
+contact	O
+.	O
+
+Arg	B-residue_name_number
+-	I-residue_name_number
+68Cdc42	I-residue_name_number
+of	O
+switch	B-site
+II	I-site
+is	O
+positioned	O
+close	O
+to	O
+Glu	B-residue_name_number
+-	I-residue_name_number
+395TOCA1	I-residue_name_number
+(	O
+Fig	O
+.	O
+6D	O
+),	O
+suggesting	O
+a	O
+direct	O
+electrostatic	O
+contact	O
+between	O
+switch	B-site
+II	I-site
+of	O
+Cdc42	B-protein
+and	O
+helix	B-structure_element
+2	I-structure_element
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+.	O
+
+The	O
+equivalent	O
+Arg	B-residue_name
+in	O
+Rac1	B-protein
+and	O
+RhoA	B-protein
+is	O
+pointing	O
+away	O
+from	O
+the	O
+HR1	B-structure_element
+domains	O
+of	O
+PRK1	B-protein
+.	O
+
+The	O
+importance	O
+of	O
+this	O
+residue	O
+in	O
+the	O
+Cdc42	B-protein
+-	O
+TOCA1	B-protein
+interaction	O
+remains	O
+unclear	O
+,	O
+although	O
+its	O
+mutation	B-experimental_method
+reduces	O
+binding	O
+to	O
+RhoGAP	B-protein
+,	O
+suggesting	O
+that	O
+it	O
+can	O
+be	O
+involved	O
+in	O
+Cdc42	B-protein
+interactions	O
+.	O
+
+The	O
+solution	B-evidence
+structure	I-evidence
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+presented	O
+here	O
+,	O
+along	O
+with	O
+the	O
+model	O
+of	O
+the	O
+HR1TOCA1	B-complex_assembly
+·	I-complex_assembly
+Cdc42	I-complex_assembly
+complex	O
+is	O
+consistent	O
+with	O
+a	O
+conserved	O
+mode	O
+of	O
+binding	O
+across	O
+the	O
+known	O
+HR1	B-structure_element
+domain	O
+-	O
+Rho	O
+family	O
+interactions	O
+,	O
+despite	O
+their	O
+differing	O
+affinities	O
+.	O
+
+The	O
+weak	O
+binding	O
+prevented	O
+detailed	O
+structural	B-experimental_method
+and	I-experimental_method
+thermodynamic	I-experimental_method
+studies	I-experimental_method
+of	O
+the	O
+complex	O
+.	O
+
+Nonetheless	O
+,	O
+structural	B-experimental_method
+studies	I-experimental_method
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+,	O
+combined	O
+with	O
+chemical	B-experimental_method
+shift	I-experimental_method
+mapping	I-experimental_method
+,	O
+have	O
+highlighted	O
+some	O
+potentially	O
+interesting	O
+differences	O
+between	O
+Cdc42	B-protein
+-	O
+HR1TOCA1	B-structure_element
+and	O
+RhoA	B-protein
+/	O
+Rac1	B-protein
+-	O
+HR1PRK1	B-structure_element
+binding	O
+.	O
+
+We	O
+have	O
+previously	O
+postulated	O
+that	O
+the	O
+inherent	O
+flexibility	O
+of	O
+HR1	B-structure_element
+domains	O
+contributes	O
+to	O
+their	O
+ability	O
+to	O
+bind	O
+to	O
+different	O
+Rho	B-protein_type
+family	I-protein_type
+G	I-protein_type
+proteins	I-protein_type
+,	O
+with	O
+Rho	O
+-	O
+binding	O
+HR1	B-structure_element
+domains	O
+displaying	O
+increased	O
+flexibility	O
+,	O
+reflected	O
+in	O
+their	O
+lower	O
+melting	B-evidence
+temperatures	I-evidence
+(	O
+Tm	B-evidence
+)	O
+and	O
+Rac	B-protein_type
+binders	O
+being	O
+more	O
+rigid	O
+.	O
+
+The	O
+Tm	B-evidence
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+is	O
+61	O
+.	O
+9	O
+°	O
+C	O
+(	O
+data	O
+not	O
+shown	O
+),	O
+which	O
+is	O
+the	O
+highest	O
+Tm	B-evidence
+that	O
+we	O
+have	O
+measured	O
+for	O
+an	O
+HR1	B-structure_element
+domain	O
+thus	O
+far	O
+.	O
+
+As	O
+such	O
+,	O
+the	O
+ability	O
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+to	O
+bind	O
+to	O
+Cdc42	B-protein
+(	O
+a	O
+close	O
+relative	O
+of	O
+Rac1	B-protein
+rather	O
+than	O
+RhoA	B-protein
+)	O
+fits	O
+this	O
+trend	O
+.	O
+
+An	O
+investigation	O
+into	O
+the	O
+local	O
+motions	O
+,	O
+particularly	O
+in	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+regions	I-site
+,	O
+may	O
+offer	O
+further	O
+insight	O
+into	O
+the	O
+differential	O
+specificities	O
+and	O
+affinities	O
+of	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+HR1	B-structure_element
+domain	O
+interactions	O
+.	O
+
+The	O
+low	O
+affinity	O
+of	O
+the	O
+Cdc42	B-protein
+-	O
+HR1TOCA1	B-structure_element
+interaction	O
+is	O
+consistent	O
+with	O
+a	O
+tightly	O
+spatially	O
+and	O
+temporally	O
+regulated	O
+pathway	O
+,	O
+requiring	O
+combinatorial	O
+signals	O
+leading	O
+to	O
+a	O
+series	O
+of	O
+coincident	O
+weak	O
+interactions	O
+that	O
+elicit	O
+full	O
+activation	O
+.	O
+
+The	O
+HR1	B-structure_element
+domains	O
+from	O
+other	O
+TOCA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+,	O
+CIP4	B-protein
+and	O
+FBP17	B-protein
+,	O
+also	O
+bind	O
+at	O
+low	O
+micromolar	O
+affinities	O
+to	O
+Cdc42	B-protein
+,	O
+so	O
+the	O
+low	O
+affinity	O
+interaction	O
+appears	O
+to	O
+be	O
+commonplace	O
+among	O
+this	O
+family	O
+of	O
+HR1	B-protein_type
+domain	I-protein_type
+proteins	I-protein_type
+,	O
+in	O
+contrast	O
+to	O
+the	O
+PRK	B-protein_type
+family	I-protein_type
+.	O
+
+The	O
+low	O
+affinity	O
+of	O
+the	O
+HR1TOCA1	B-structure_element
+-	O
+Cdc42	B-protein
+interaction	O
+in	O
+the	O
+context	O
+of	O
+the	O
+physiological	O
+concentration	O
+of	O
+TOCA1	B-protein
+in	O
+Xenopus	B-taxonomy_domain
+extracts	O
+(∼	O
+10	O
+nm	O
+)	O
+suggests	O
+that	O
+binding	O
+between	O
+TOCA1	B-protein
+and	O
+Cdc42	B-protein
+is	O
+likely	O
+to	O
+occur	O
+in	O
+vivo	O
+only	O
+when	O
+TOCA1	B-protein
+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	B-protein_state
+Cdc42	B-protein
+.	O
+
+Evidence	O
+suggests	O
+that	O
+the	O
+TOCA	B-protein_type
+family	I-protein_type
+of	O
+proteins	O
+are	O
+recruited	O
+to	O
+the	O
+membrane	O
+via	O
+an	O
+interaction	O
+between	O
+their	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+and	O
+specific	O
+signaling	O
+lipids	O
+.	O
+
+For	O
+example	O
+,	O
+electrostatic	O
+interactions	O
+between	O
+the	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+and	O
+the	O
+membrane	O
+are	O
+required	O
+for	O
+TOCA1	B-protein
+recruitment	O
+to	O
+membrane	O
+vesicles	O
+and	O
+tubules	O
+,	O
+and	O
+TOCA1	B-protein
+-	O
+dependent	O
+actin	O
+polymerization	O
+is	O
+known	O
+to	O
+depend	O
+specifically	O
+on	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+.	O
+
+Furthermore	O
+,	O
+the	O
+isolated	B-experimental_method
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+of	O
+FBP17	B-protein
+has	O
+been	O
+shown	O
+to	O
+induce	O
+membrane	O
+tubulation	O
+of	O
+brain	O
+liposomes	O
+and	O
+BAR	B-structure_element
+domain	O
+proteins	O
+that	O
+promote	O
+tubulation	O
+cluster	O
+on	O
+membranes	O
+at	O
+high	O
+densities	O
+.	O
+
+Once	O
+at	O
+the	O
+membrane	O
+,	O
+high	O
+local	O
+concentrations	O
+of	O
+TOCA1	B-protein
+could	O
+exceed	O
+the	O
+Kd	B-evidence
+of	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+dimerization	B-oligomeric_state
+(	O
+likely	O
+to	O
+be	O
+comparable	O
+with	O
+that	O
+of	O
+the	O
+FCHo2	B-protein
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+(	O
+2	O
+.	O
+5	O
+μm	O
+))	O
+and	O
+that	O
+of	O
+the	O
+Cdc42	B-protein
+-	O
+HR1TOCA1	B-structure_element
+interaction	O
+.	O
+
+Cdc42	B-protein
+-	O
+HR1TOCA1	B-structure_element
+binding	O
+would	O
+then	O
+be	O
+favorable	O
+,	O
+as	O
+long	O
+as	O
+coincident	O
+activation	O
+of	O
+Cdc42	B-protein
+had	O
+occurred	O
+,	O
+leading	O
+to	O
+stabilization	O
+of	O
+TOCA1	B-protein
+at	O
+the	O
+membrane	O
+and	O
+downstream	O
+activation	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+It	O
+has	O
+been	O
+postulated	O
+that	O
+WASP	B-protein_type
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+exist	O
+in	O
+equilibrium	O
+between	O
+folded	B-protein_state
+(	O
+inactive	B-protein_state
+)	O
+and	O
+unfolded	B-protein_state
+(	O
+active	B-protein_state
+)	O
+forms	O
+,	O
+and	O
+the	O
+affinity	B-evidence
+of	O
+Cdc42	B-protein
+for	O
+the	O
+unfolded	B-protein_state
+WASP	B-protein_type
+proteins	O
+is	O
+significantly	O
+enhanced	O
+.	O
+
+The	O
+unfolded	B-protein_state
+,	O
+high	O
+affinity	O
+state	O
+of	O
+WASP	B-protein_type
+is	O
+represented	O
+by	O
+a	O
+short	O
+peptide	B-chemical
+,	O
+the	O
+GBD	B-structure_element
+,	O
+which	O
+binds	O
+with	O
+a	O
+low	O
+nanomolar	O
+affinity	O
+to	O
+Cdc42	B-protein
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+best	O
+estimate	O
+of	O
+the	O
+affinity	B-evidence
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+WASP	B-protein_type
+for	O
+Cdc42	B-protein
+is	O
+low	O
+micromolar	O
+.	O
+
+In	O
+the	O
+inactive	B-protein_state
+state	O
+of	O
+WASP	B-protein_type
+,	O
+the	O
+actin	O
+-	O
+and	O
+Arp2	B-complex_assembly
+/	I-complex_assembly
+3	I-complex_assembly
+-	O
+binding	O
+VCA	B-structure_element
+domain	O
+contacts	O
+the	O
+GBD	B-structure_element
+,	O
+competing	O
+for	O
+Cdc42	B-protein
+binding	O
+.	O
+
+The	O
+high	O
+affinity	O
+of	O
+Cdc42	B-protein
+for	O
+the	O
+unfolded	B-protein_state
+,	O
+active	B-protein_state
+form	O
+pushes	O
+the	O
+equilibrium	O
+in	O
+favor	O
+of	O
+(	B-protein
+N	I-protein
+-)	I-protein
+WASP	I-protein
+activation	O
+.	O
+
+Binding	O
+of	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+to	O
+the	O
+basic	O
+region	O
+just	O
+N	O
+-	O
+terminal	O
+to	O
+the	O
+GBD	B-structure_element
+further	O
+favors	O
+the	O
+active	B-protein_state
+conformation	O
+.	O
+
+A	O
+substantial	O
+body	O
+of	O
+data	O
+has	O
+illuminated	O
+the	O
+complex	O
+regulation	O
+of	O
+WASP	B-protein_type
+/	I-protein_type
+N	I-protein_type
+-	I-protein_type
+WASP	I-protein_type
+proteins	I-protein_type
+,	O
+and	O
+current	O
+evidence	O
+suggests	O
+that	O
+these	O
+allosteric	O
+activation	O
+mechanisms	O
+and	O
+oligomerization	O
+combine	O
+to	O
+regulate	O
+WASP	B-protein_type
+activity	O
+,	O
+allowing	O
+the	O
+synchronization	O
+and	O
+integration	O
+of	O
+multiple	O
+potential	O
+activation	O
+signals	O
+(	O
+reviewed	O
+in	O
+Ref	O
+.).	O
+
+We	O
+envisage	O
+that	O
+TOCA1	B-protein
+is	O
+first	O
+recruited	O
+to	O
+the	O
+appropriate	O
+membrane	O
+in	O
+response	O
+to	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+via	O
+its	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+,	O
+where	O
+the	O
+local	O
+increase	O
+in	O
+concentration	O
+favors	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+-	O
+mediated	O
+dimerization	B-oligomeric_state
+of	O
+TOCA1	B-protein
+.	O
+
+Cdc42	B-protein
+is	O
+activated	O
+in	O
+response	O
+to	O
+co	O
+-	O
+incident	O
+signals	O
+and	O
+can	O
+then	O
+bind	O
+to	O
+TOCA1	B-protein
+,	O
+further	O
+stabilizing	O
+TOCA1	B-protein
+at	O
+the	O
+membrane	O
+.	O
+
+TOCA1	B-protein
+can	O
+then	O
+recruit	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+via	O
+an	O
+interaction	O
+between	O
+its	O
+SH3	B-structure_element
+domain	O
+and	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+proline	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+region	I-structure_element
+.	O
+
+The	O
+recruitment	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+alone	B-protein_state
+and	O
+of	O
+the	O
+N	B-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+WIP	I-complex_assembly
+complex	O
+by	O
+TOCA1	B-protein
+and	O
+FBP17	B-protein
+has	O
+been	O
+demonstrated	O
+.	O
+
+WIP	B-protein
+inhibits	O
+the	O
+activation	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+by	O
+Cdc42	B-protein
+,	O
+an	O
+effect	O
+that	O
+is	O
+reversed	O
+by	O
+TOCA1	B-protein
+.	O
+
+It	O
+may	O
+therefore	O
+be	O
+envisaged	O
+that	O
+WIP	B-protein
+and	O
+TOCA1	B-protein
+exert	O
+opposing	O
+allosteric	O
+effects	O
+on	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+with	O
+TOCA1	B-protein
+favoring	O
+the	O
+unfolded	B-protein_state
+,	O
+active	B-protein_state
+conformation	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+increasing	O
+its	O
+affinity	O
+for	O
+Cdc42	B-protein
+.	O
+
+TOCA1	B-protein
+may	O
+also	O
+activate	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+by	O
+effective	O
+oligomerization	O
+because	O
+clustering	O
+of	O
+TOCA1	B-protein
+at	O
+the	O
+membrane	O
+following	O
+coincident	O
+interactions	O
+with	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+and	O
+Cdc42	B-protein
+would	O
+in	O
+turn	O
+lead	O
+to	O
+clustering	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+in	O
+addition	O
+to	O
+pushing	O
+the	O
+equilibrium	O
+toward	O
+the	O
+unfolded	B-protein_state
+,	O
+active	B-protein_state
+state	O
+.	O
+
+In	O
+a	O
+cellular	O
+context	O
+,	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+are	O
+likely	O
+to	O
+have	O
+similar	O
+affinities	B-evidence
+for	O
+active	B-protein_state
+Cdc42	B-protein
+,	O
+but	O
+in	O
+the	O
+unfolded	B-protein_state
+,	O
+active	B-protein_state
+conformation	O
+,	O
+the	O
+affinity	B-evidence
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+for	O
+Cdc42	B-protein
+dramatically	O
+increases	O
+.	O
+
+Our	O
+binding	B-evidence
+data	I-evidence
+suggest	O
+that	O
+TOCA1	B-protein
+HR1	B-structure_element
+binding	O
+is	O
+not	O
+allosterically	O
+regulated	O
+,	O
+and	O
+our	O
+NMR	B-experimental_method
+data	O
+,	O
+along	O
+with	O
+the	O
+high	O
+stability	B-protein_state
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+,	O
+suggest	O
+that	O
+there	O
+is	O
+no	O
+widespread	O
+conformational	O
+change	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Cdc42	B-protein
+.	O
+
+As	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+TOCA1	B-protein
+and	O
+the	O
+isolated	B-protein_state
+HR1	B-structure_element
+domain	O
+bind	O
+Cdc42	B-protein
+with	O
+similar	O
+affinities	O
+,	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+-	O
+Cdc42	B-protein
+interaction	O
+will	O
+be	O
+favored	O
+because	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+can	O
+easily	O
+outcompete	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+for	O
+Cdc42	B-protein
+.	O
+
+A	O
+combination	O
+of	O
+allosteric	O
+activation	O
+by	O
+PI	B-chemical
+(	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+)	I-chemical
+P2	I-chemical
+,	O
+activated	B-protein_state
+Cdc42	B-protein
+and	O
+TOCA1	B-protein
+,	O
+and	O
+oligomeric	O
+activation	O
+implemented	O
+by	O
+TOCA1	B-protein
+would	O
+lead	O
+to	O
+full	B-protein_state
+activation	I-protein_state
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+downstream	O
+actin	O
+polymerization	O
+.	O
+
+In	O
+such	O
+an	O
+array	O
+of	O
+molecules	O
+localized	O
+to	O
+a	O
+discrete	O
+region	O
+of	O
+the	O
+membrane	O
+,	O
+it	O
+is	O
+plausible	O
+that	O
+WASP	B-protein
+could	O
+bind	O
+to	O
+a	O
+second	O
+Cdc42	B-protein
+molecule	O
+rather	O
+than	O
+displacing	O
+TOCA1	B-protein
+from	O
+its	O
+cognate	O
+Cdc42	B-protein
+.	O
+
+Our	O
+NMR	B-experimental_method
+and	O
+affinity	B-evidence
+data	I-evidence
+,	O
+however	O
+,	O
+are	O
+consistent	O
+with	O
+displacement	O
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+by	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+.	O
+
+Furthermore	O
+,	O
+TOCA1	B-protein
+is	O
+required	O
+for	O
+Cdc42	B-protein
+-	O
+mediated	O
+activation	O
+of	O
+N	B-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+WIP	I-complex_assembly
+,	O
+implying	O
+that	O
+it	O
+may	O
+not	O
+be	O
+possible	O
+for	O
+Cdc42	B-protein
+to	O
+bind	O
+and	O
+activate	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+prior	O
+to	O
+TOCA1	B-protein
+-	O
+Cdc42	B-protein
+binding	O
+.	O
+
+The	O
+commonly	O
+used	O
+MGD	B-mutant
+→	I-mutant
+IST	I-mutant
+(	O
+Cdc42	B-protein_state
+-	I-protein_state
+binding	I-protein_state
+deficient	I-protein_state
+)	O
+mutant	O
+of	O
+TOCA1	B-protein
+has	O
+a	O
+reduced	O
+ability	O
+to	O
+activate	O
+the	O
+N	B-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+WIP	I-complex_assembly
+complex	O
+,	O
+further	O
+indicating	O
+the	O
+importance	O
+of	O
+the	O
+Cdc42	B-protein
+-	O
+HR1TOCA1	B-structure_element
+interaction	O
+prior	O
+to	O
+downstream	O
+activation	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+In	O
+light	O
+of	O
+this	O
+,	O
+we	O
+favor	O
+an	O
+“	O
+effector	O
+handover	O
+”	O
+scheme	O
+whereby	O
+TOCA1	B-protein
+interacts	O
+with	O
+Cdc42	B-protein
+prior	O
+to	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+activation	O
+,	O
+after	O
+which	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+displaces	O
+TOCA1	B-protein
+from	O
+its	O
+bound	B-protein_state
+Cdc42	B-protein
+in	O
+order	O
+to	O
+be	O
+fully	B-protein_state
+activated	I-protein_state
+rather	O
+than	O
+binding	O
+a	O
+second	O
+Cdc42	B-protein
+molecule	O
+.	O
+
+Potentially	O
+,	O
+the	O
+TOCA1	B-protein
+-	O
+Cdc42	B-protein
+interaction	O
+functions	O
+to	O
+position	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+Cdc42	B-protein
+such	O
+that	O
+they	O
+are	O
+poised	O
+to	O
+interact	O
+with	O
+high	O
+affinity	O
+.	O
+
+The	O
+concomitant	O
+release	O
+of	O
+TOCA1	B-protein
+from	O
+Cdc42	B-protein
+while	O
+still	O
+bound	B-protein_state
+to	I-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+presumably	O
+enhances	O
+the	O
+ability	O
+of	O
+TOCA1	B-protein
+to	O
+further	O
+activate	O
+N	B-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+WIP	I-complex_assembly
+-	O
+induced	O
+actin	O
+polymerization	O
+.	O
+
+There	O
+is	O
+an	O
+advantage	O
+to	O
+such	O
+an	O
+effector	O
+handover	O
+,	O
+in	O
+that	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+would	O
+only	O
+be	O
+robustly	O
+recruited	O
+when	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domains	O
+are	O
+already	O
+present	O
+.	O
+
+Hence	O
+,	O
+actin	O
+polymerization	O
+cannot	O
+occur	O
+until	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domains	O
+are	O
+poised	O
+for	O
+membrane	O
+distortion	O
+.	O
+
+Our	O
+model	O
+of	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1TOCA1	I-complex_assembly
+complex	O
+indicates	O
+a	O
+mechanism	O
+by	O
+which	O
+such	O
+a	O
+handover	O
+could	O
+take	O
+place	O
+(	O
+Fig	O
+.	O
+9	O
+)	O
+because	O
+it	O
+shows	O
+that	O
+the	O
+effector	B-site
+binding	I-site
+sites	I-site
+only	O
+partially	O
+overlap	O
+on	O
+Cdc42	B-protein
+.	O
+
+The	O
+lysine	B-residue_name
+residues	O
+thought	O
+to	O
+be	O
+involved	O
+in	O
+an	O
+electrostatic	O
+steering	O
+mechanism	O
+in	O
+WASP	B-protein
+-	O
+Cdc42	B-protein
+binding	O
+are	O
+conserved	O
+in	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+would	O
+be	O
+able	O
+to	O
+interact	O
+with	O
+Cdc42	B-protein
+even	O
+when	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+is	O
+already	O
+bound	B-protein_state
+.	O
+
+It	O
+has	O
+been	O
+postulated	O
+that	O
+the	O
+initial	O
+interactions	O
+between	O
+this	O
+basic	O
+region	O
+and	O
+Cdc42	B-protein
+could	O
+stabilize	O
+the	O
+active	B-protein_state
+conformation	O
+of	O
+WASP	B-protein
+,	O
+leading	O
+to	O
+high	O
+affinity	O
+binding	O
+between	O
+the	O
+core	O
+CRIB	B-structure_element
+and	O
+Cdc42	B-protein
+.	O
+
+The	O
+region	O
+C	O
+-	O
+terminal	O
+to	O
+the	O
+core	O
+CRIB	B-structure_element
+,	O
+required	O
+for	O
+maximal	O
+affinity	O
+binding	O
+,	O
+would	O
+then	O
+fully	O
+displace	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+.	O
+
+A	O
+simplified	O
+model	O
+of	O
+the	O
+early	O
+stages	O
+of	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+N	I-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+-	O
+dependent	O
+actin	O
+polymerization	O
+.	O
+
+Step	O
+1	O
+,	O
+TOCA1	B-protein
+is	O
+recruited	O
+to	O
+the	O
+membrane	O
+via	O
+its	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+and	O
+/	O
+or	O
+Cdc42	B-protein
+interactions	O
+.	O
+
+F	O
+-	O
+BAR	O
+oligomerization	O
+is	O
+expected	O
+to	O
+occur	O
+following	O
+membrane	O
+binding	O
+,	O
+but	O
+a	O
+single	O
+monomer	B-oligomeric_state
+is	O
+shown	O
+for	O
+clarity	O
+.	O
+
+Step	O
+2	O
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+exists	O
+in	O
+an	O
+inactive	B-protein_state
+,	O
+folded	B-protein_state
+conformation	O
+.	O
+
+The	O
+TOCA1	B-protein
+SH3	B-structure_element
+domain	O
+interacts	O
+with	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+causing	O
+an	O
+activatory	O
+allosteric	O
+effect	O
+.	O
+
+The	O
+HR1TOCA1	B-structure_element
+-	O
+Cdc42	O
+and	O
+SH3TOCA1	B-structure_element
+-	O
+N	O
+-	O
+WASP	O
+interactions	O
+position	O
+Cdc42	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+for	O
+binding	O
+.	O
+
+Step	O
+3	O
+,	O
+electrostatic	O
+interactions	O
+between	O
+Cdc42	B-protein
+and	O
+the	O
+basic	O
+region	O
+upstream	O
+of	O
+the	O
+CRIB	B-structure_element
+initiate	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+N	I-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+binding	O
+.	O
+
+Step	O
+4	O
+,	O
+the	O
+core	O
+CRIB	B-structure_element
+binds	O
+with	O
+high	O
+affinity	O
+while	O
+the	O
+region	O
+C	O
+-	O
+terminal	O
+to	O
+the	O
+CRIB	B-structure_element
+displaces	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+and	O
+increases	O
+the	O
+affinity	O
+of	O
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+-	O
+Cdc42	O
+interaction	O
+further	O
+.	O
+
+The	O
+VCA	B-structure_element
+domain	O
+is	O
+released	O
+for	O
+downstream	O
+interactions	O
+,	O
+and	O
+actin	O
+polymerization	O
+proceeds	O
+.	O
+
+WH1	O
+,	O
+WASP	B-structure_element
+homology	I-structure_element
+1	I-structure_element
+domain	I-structure_element
+;	O
+PP	B-structure_element
+,	O
+proline	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+region	I-structure_element
+;	O
+VCA	B-structure_element
+,	O
+verprolin	B-structure_element
+homology	I-structure_element
+,	I-structure_element
+cofilin	I-structure_element
+homology	I-structure_element
+,	I-structure_element
+acidic	I-structure_element
+region	I-structure_element
+.	O
+
+In	O
+conclusion	O
+,	O
+the	O
+data	O
+presented	O
+here	O
+show	O
+that	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+is	O
+sufficient	O
+for	O
+Cdc42	B-protein
+binding	O
+in	O
+vitro	O
+and	O
+that	O
+the	O
+interaction	O
+is	O
+of	O
+micromolar	O
+affinity	O
+,	O
+lower	O
+than	O
+that	O
+of	O
+other	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+HR1	B-structure_element
+domain	O
+interactions	O
+.	O
+
+The	O
+analogous	O
+HR1	B-structure_element
+domains	O
+from	O
+other	O
+TOCA1	B-protein_type
+family	I-protein_type
+members	O
+,	O
+FBP17	B-protein
+and	O
+CIP4	B-protein
+,	O
+also	O
+exhibit	O
+micromolar	O
+affinity	O
+for	O
+Cdc42	B-protein
+.	O
+
+A	O
+role	O
+for	O
+the	O
+TOCA1	B-protein
+-,	O
+FBP17	B-protein
+-,	O
+and	O
+CIP4	B-protein
+-	O
+Cdc42	B-protein
+interactions	O
+in	O
+the	O
+recruitment	O
+of	O
+these	O
+proteins	O
+to	O
+the	O
+membrane	O
+therefore	O
+appears	O
+unlikely	O
+.	O
+
+Instead	O
+,	O
+our	O
+findings	O
+agree	O
+with	O
+earlier	O
+suggestions	O
+that	O
+the	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+domain	O
+is	O
+responsible	O
+for	O
+membrane	O
+recruitment	O
+.	O
+
+The	O
+role	O
+of	O
+the	O
+Cdc42	B-protein
+-	O
+TOCA1	B-protein
+interaction	O
+remains	O
+somewhat	O
+elusive	O
+,	O
+but	O
+it	O
+may	O
+serve	O
+to	O
+position	O
+activated	B-protein_state
+Cdc42	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+to	O
+allow	O
+full	B-protein_state
+activation	I-protein_state
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+as	O
+such	O
+serve	O
+to	O
+couple	O
+F	B-structure_element
+-	I-structure_element
+BAR	I-structure_element
+-	O
+mediated	O
+membrane	O
+deformation	O
+with	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+activation	O
+.	O
+
+We	O
+envisage	O
+a	O
+complex	O
+interplay	O
+of	O
+equilibria	O
+between	O
+free	B-protein_state
+and	O
+bound	B-protein_state
+,	O
+active	B-protein_state
+and	O
+inactive	B-protein_state
+Cdc42	B-protein
+,	O
+TOCA	B-protein_type
+family	I-protein_type
+,	O
+and	O
+WASP	B-protein_type
+family	O
+proteins	O
+,	O
+facilitating	O
+a	O
+tightly	O
+spatially	O
+and	O
+temporally	O
+regulated	O
+pathway	O
+requiring	O
+numerous	O
+simultaneous	O
+events	O
+in	O
+order	O
+to	O
+achieve	O
+appropriate	O
+and	O
+robust	O
+activation	O
+of	O
+the	O
+downstream	O
+pathway	O
+.	O
+
+Our	O
+data	O
+are	O
+therefore	O
+easily	O
+reconciled	O
+with	O
+the	O
+dynamic	O
+instability	O
+models	O
+described	O
+in	O
+relation	O
+to	O
+the	O
+formation	O
+of	O
+endocytic	O
+vesicles	O
+and	O
+with	O
+the	O
+current	O
+data	O
+pertaining	O
+to	O
+the	O
+complex	O
+activation	O
+of	O
+WASP	B-protein_type
+/	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+pathways	O
+by	O
+allosteric	O
+and	O
+oligomeric	O
+effects	O
+.	O
+
+It	O
+is	O
+clear	O
+from	O
+the	O
+data	O
+presented	O
+here	O
+that	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+do	O
+not	O
+bind	O
+Cdc42	B-protein
+simultaneously	O
+and	O
+that	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+is	O
+likely	O
+to	O
+outcompete	O
+TOCA1	B-protein
+for	O
+Cdc42	B-protein
+binding	O
+.	O
+
+We	O
+therefore	O
+postulate	O
+an	O
+effector	O
+handover	O
+mechanism	O
+based	O
+on	O
+current	O
+evidence	O
+surrounding	O
+WASP	B-protein
+/	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+activation	O
+and	O
+our	O
+model	O
+of	O
+the	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1TOCA1	I-complex_assembly
+complex	O
+.	O
+
+The	O
+displacement	O
+of	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+from	O
+Cdc42	B-protein
+by	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+may	O
+represent	O
+a	O
+unidirectional	O
+step	O
+in	O
+the	O
+pathway	O
+of	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+N	I-complex_assembly
+-	I-complex_assembly
+WASP	I-complex_assembly
+·	I-complex_assembly
+TOCA1	I-complex_assembly
+-	O
+dependent	O
+actin	O
+assembly	O
+.	O
+
+The	O
+dynamic	B-protein_state
+organization	O
+of	O
+fungal	B-taxonomy_domain
+acetyl	B-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylase	I-protein_type
+
+Acetyl	B-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylases	I-protein_type
+(	O
+ACCs	B-protein_type
+)	O
+catalyse	O
+the	O
+committed	O
+step	O
+in	O
+fatty	O
+-	O
+acid	O
+biosynthesis	O
+:	O
+the	O
+ATP	B-chemical
+-	O
+dependent	O
+carboxylation	O
+of	O
+acetyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+malonyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+They	O
+are	O
+important	O
+regulatory	O
+hubs	O
+for	O
+metabolic	O
+control	O
+and	O
+relevant	O
+drug	O
+targets	O
+for	O
+the	O
+treatment	O
+of	O
+the	O
+metabolic	O
+syndrome	O
+and	O
+cancer	O
+.	O
+
+Eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+are	O
+single	B-protein_type
+-	I-protein_type
+chain	I-protein_type
+multienzymes	I-protein_type
+characterized	O
+by	O
+a	O
+large	O
+,	O
+non	B-protein_state
+-	I-protein_state
+catalytic	I-protein_state
+central	B-structure_element
+domain	I-structure_element
+(	O
+CD	B-structure_element
+),	O
+whose	O
+role	O
+in	O
+ACC	B-protein_type
+regulation	O
+remains	O
+poorly	O
+characterized	O
+.	O
+
+Here	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+CD	B-structure_element
+,	O
+revealing	O
+a	O
+unique	O
+four	O
+-	O
+domain	O
+organization	O
+.	O
+
+A	O
+regulatory	B-structure_element
+loop	I-structure_element
+,	O
+which	O
+is	O
+phosphorylated	B-protein_state
+at	O
+the	O
+key	O
+functional	O
+phosphorylation	B-site
+site	I-site
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+wedges	O
+into	O
+a	O
+crevice	O
+between	O
+two	O
+domains	O
+of	O
+CD	B-structure_element
+.	O
+
+Combining	O
+the	O
+yeast	B-taxonomy_domain
+CD	B-structure_element
+structure	B-evidence
+with	O
+intermediate	O
+and	O
+low	O
+-	O
+resolution	O
+data	O
+of	O
+larger	B-mutant
+fragments	I-mutant
+up	O
+to	O
+intact	B-protein_state
+ACCs	B-protein_type
+provides	O
+a	O
+comprehensive	O
+characterization	O
+of	O
+the	O
+dynamic	B-protein_state
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+architecture	O
+.	O
+
+In	O
+contrast	O
+to	O
+related	O
+carboxylases	B-protein_type
+,	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	B-structure_element
+under	O
+phosphorylation	B-ptm
+control	O
+.	O
+
+Acetyl	B-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylases	I-protein_type
+are	O
+central	O
+regulatory	O
+hubs	O
+of	O
+fatty	O
+acid	O
+metabolism	O
+and	O
+are	O
+important	O
+targets	O
+for	O
+drug	O
+development	O
+in	O
+obesity	O
+and	O
+cancer	O
+.	O
+
+Here	O
+,	O
+the	O
+authors	O
+demonstrate	O
+that	O
+the	O
+regulation	O
+of	O
+these	O
+highly	B-protein_state
+dynamic	I-protein_state
+enzymes	B-protein_type
+in	O
+fungi	B-taxonomy_domain
+is	O
+governed	O
+by	O
+a	O
+mechanism	O
+based	O
+on	O
+phosphorylation	B-ptm
+-	O
+dependent	O
+conformational	O
+variability	O
+.	O
+
+Biotin	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+acetyl	I-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylases	I-protein_type
+(	O
+ACCs	B-protein_type
+)	O
+are	O
+essential	O
+enzymes	O
+that	O
+catalyse	O
+the	O
+ATP	B-chemical
+-	O
+dependent	O
+carboxylation	O
+of	O
+acetyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+malonyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+This	O
+reaction	O
+provides	O
+the	O
+committed	O
+activated	O
+substrate	O
+for	O
+the	O
+biosynthesis	O
+of	O
+fatty	B-chemical
+acids	I-chemical
+via	O
+fatty	B-protein_type
+-	I-protein_type
+acid	I-protein_type
+synthase	I-protein_type
+.	O
+
+By	O
+catalysing	O
+this	O
+rate	O
+-	O
+limiting	O
+step	O
+in	O
+fatty	O
+-	O
+acid	O
+biosynthesis	O
+,	O
+ACC	B-protein_type
+plays	O
+a	O
+key	O
+role	O
+in	O
+anabolic	O
+metabolism	O
+.	O
+
+ACC	B-experimental_method
+inhibition	I-experimental_method
+and	I-experimental_method
+knock	I-experimental_method
+-	I-experimental_method
+out	I-experimental_method
+studies	I-experimental_method
+show	O
+the	O
+potential	O
+of	O
+targeting	O
+ACC	B-protein_type
+for	O
+treatment	O
+of	O
+the	O
+metabolic	O
+syndrome	O
+.	O
+
+Furthermore	O
+,	O
+elevated	O
+ACC	B-protein_type
+activity	O
+is	O
+observed	O
+in	O
+malignant	O
+tumours	O
+.	O
+
+A	O
+direct	O
+link	O
+between	O
+ACC	B-protein_type
+and	O
+cancer	O
+is	O
+provided	O
+by	O
+cancer	O
+-	O
+associated	O
+mutations	B-mutant
+in	O
+the	O
+breast	B-protein
+cancer	I-protein
+susceptibility	I-protein
+gene	I-protein
+1	I-protein
+(	O
+BRCA1	B-protein
+),	O
+which	O
+relieve	O
+inhibitory	O
+interactions	O
+of	O
+BRCA1	B-protein
+with	O
+ACC	B-protein_type
+.	O
+
+Thus	O
+,	O
+ACC	B-protein_type
+is	O
+a	O
+relevant	O
+drug	O
+target	O
+for	O
+type	O
+2	O
+diabetes	O
+and	O
+cancer	O
+.	O
+
+Microbial	B-taxonomy_domain
+ACCs	B-protein_type
+are	O
+also	O
+the	O
+principal	O
+target	O
+of	O
+antifungal	O
+and	O
+antibiotic	O
+compounds	O
+,	O
+such	O
+as	O
+Soraphen	B-chemical
+A	I-chemical
+.	O
+
+The	O
+principal	O
+functional	O
+protein	O
+components	O
+of	O
+ACCs	B-protein_type
+have	O
+been	O
+described	O
+already	O
+in	O
+the	O
+late	O
+1960s	O
+for	O
+Escherichia	B-species
+coli	I-species
+(	O
+E	B-species
+.	I-species
+coli	I-species
+)	O
+ACC	B-protein_type
+:	O
+Biotin	B-protein_type
+carboxylase	I-protein_type
+(	O
+BC	B-protein_type
+)	O
+catalyses	O
+the	O
+ATP	B-chemical
+-	O
+dependent	O
+carboxylation	O
+of	O
+a	O
+biotin	B-chemical
+moiety	O
+,	O
+which	O
+is	O
+covalently	O
+linked	O
+to	O
+the	O
+biotin	B-protein_type
+carboxyl	I-protein_type
+carrier	I-protein_type
+protein	I-protein_type
+(	O
+BCCP	B-protein_type
+).	O
+
+Carboxyltransferase	B-protein_type
+(	O
+CT	B-protein_type
+)	O
+transfers	O
+the	O
+activated	O
+carboxyl	B-chemical
+group	O
+from	O
+carboxybiotin	B-chemical
+to	O
+acetyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+yield	O
+malonyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+Prokaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+are	O
+transient	B-protein_state
+assemblies	O
+of	O
+individual	O
+BC	B-protein_type
+,	O
+CT	B-protein_type
+and	O
+BCCP	B-protein_type
+subunits	O
+.	O
+
+Eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+,	O
+instead	O
+,	O
+are	O
+multienzymes	B-protein_type
+,	O
+which	O
+integrate	O
+all	O
+functional	O
+components	O
+into	O
+a	O
+single	O
+polypeptide	O
+chain	O
+of	O
+∼	O
+2	O
+,	O
+300	O
+amino	O
+acids	O
+.	O
+
+Human	B-species
+ACC	B-protein_type
+occurs	O
+in	O
+two	O
+closely	O
+related	O
+isoforms	B-protein_state
+,	O
+ACC1	B-protein
+and	O
+2	B-protein
+,	O
+located	O
+in	O
+the	O
+cytosol	O
+and	O
+at	O
+the	O
+outer	O
+mitochondrial	O
+membrane	O
+,	O
+respectively	O
+.	O
+
+In	O
+addition	O
+to	O
+the	O
+canonical	O
+ACC	B-structure_element
+components	I-structure_element
+,	O
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+contain	O
+two	O
+non	B-protein_state
+-	I-protein_state
+catalytic	I-protein_state
+regions	B-structure_element
+,	O
+the	O
+large	O
+central	B-structure_element
+domain	I-structure_element
+(	O
+CD	B-structure_element
+)	O
+and	O
+the	O
+BC	B-structure_element
+–	I-structure_element
+CT	I-structure_element
+interaction	I-structure_element
+domain	I-structure_element
+(	O
+BT	B-structure_element
+).	O
+
+The	O
+CD	B-structure_element
+comprises	O
+one	O
+-	O
+third	O
+of	O
+the	O
+protein	O
+and	O
+is	O
+a	O
+unique	B-protein_state
+feature	I-protein_state
+of	I-protein_state
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+without	O
+homologues	O
+in	O
+other	O
+proteins	O
+.	O
+
+The	O
+function	O
+of	O
+this	O
+domain	O
+remains	O
+poorly	O
+characterized	O
+,	O
+although	O
+phosphorylation	B-ptm
+of	O
+several	O
+serine	B-residue_name
+residues	O
+in	O
+the	O
+CD	B-structure_element
+regulates	O
+ACC	B-protein_type
+activity	O
+.	O
+
+The	O
+BT	B-structure_element
+domain	O
+has	O
+been	O
+visualized	O
+in	O
+bacterial	B-taxonomy_domain
+carboxylases	B-protein_type
+,	O
+where	O
+it	O
+mediates	O
+contacts	O
+between	O
+α	B-structure_element
+-	I-structure_element
+and	O
+β	B-structure_element
+-	I-structure_element
+subunits	I-structure_element
+.	O
+
+Structural	B-experimental_method
+studies	I-experimental_method
+on	O
+the	O
+functional	O
+architecture	O
+of	O
+intact	B-protein_state
+ACCs	B-protein_type
+have	O
+been	O
+hindered	O
+by	O
+their	O
+huge	O
+size	O
+and	O
+pronounced	O
+dynamics	O
+,	O
+as	O
+well	O
+as	O
+the	O
+transient	B-protein_state
+assembly	O
+mode	O
+of	O
+bacterial	B-taxonomy_domain
+ACCs	B-protein_type
+.	O
+
+However	O
+,	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+individual	O
+components	O
+or	O
+domains	O
+from	O
+prokaryotic	B-taxonomy_domain
+and	O
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+,	O
+respectively	O
+,	O
+have	O
+been	O
+solved	O
+.	O
+
+The	O
+structure	B-experimental_method
+determination	I-experimental_method
+of	O
+the	O
+holoenzymes	B-protein_state
+of	O
+bacterial	B-taxonomy_domain
+biotin	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+carboxylases	I-protein_type
+,	O
+which	O
+lack	B-protein_state
+the	O
+characteristic	O
+CD	B-structure_element
+,	O
+such	O
+as	O
+the	O
+pyruvate	B-protein_type
+carboxylase	I-protein_type
+(	O
+PC	B-protein_type
+),	O
+propionyl	B-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylase	I-protein_type
+,	O
+3	B-protein_type
+-	I-protein_type
+methyl	I-protein_type
+-	I-protein_type
+crotonyl	I-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylase	I-protein_type
+and	O
+a	O
+long	B-protein_type
+-	I-protein_type
+chain	I-protein_type
+acyl	I-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylase	I-protein_type
+revealed	O
+strikingly	O
+divergent	O
+architectures	O
+despite	O
+a	O
+general	O
+conservation	O
+of	O
+all	O
+functional	O
+components	O
+.	O
+
+In	O
+these	O
+structures	B-evidence
+,	O
+the	O
+BC	B-protein_type
+and	O
+CT	B-protein_type
+active	B-site
+sites	I-site
+are	O
+at	O
+distances	O
+between	O
+40	O
+and	O
+80	O
+Å	O
+,	O
+such	O
+that	O
+substrate	O
+transfer	O
+could	O
+be	O
+mediated	O
+solely	O
+by	O
+the	O
+mobility	O
+of	O
+the	O
+flexibly	B-protein_state
+tethered	I-protein_state
+BCCP	B-protein_type
+.	O
+
+Human	B-species
+ACC1	B-protein
+is	O
+regulated	B-protein_state
+allosterically	I-protein_state
+,	O
+via	O
+specific	O
+protein	O
+–	O
+protein	O
+interactions	O
+,	O
+and	O
+by	O
+reversible	O
+phosphorylation	B-ptm
+.	O
+
+Dynamic	O
+polymerization	O
+of	O
+human	B-species
+ACC1	B-protein
+is	O
+linked	O
+to	O
+increased	O
+activity	O
+and	O
+is	O
+regulated	B-protein_state
+allosterically	I-protein_state
+by	O
+the	O
+activator	O
+citrate	B-chemical
+and	O
+the	O
+inhibitor	O
+palmitate	B-chemical
+,	O
+or	O
+by	O
+binding	O
+of	O
+the	O
+small	O
+protein	O
+MIG	B-protein
+-	I-protein
+12	I-protein
+(	O
+ref	O
+.).	O
+
+Human	B-species
+ACC1	B-protein
+is	O
+further	O
+regulated	O
+by	O
+specific	O
+phosphorylation	B-ptm
+-	O
+dependent	O
+binding	O
+of	O
+BRCA1	B-protein
+to	O
+Ser1263	B-residue_name_number
+in	O
+the	O
+CD	B-structure_element
+.	O
+
+BRCA1	B-protein
+binds	O
+only	O
+to	O
+the	O
+phosphorylated	B-protein_state
+form	O
+of	O
+ACC1	B-protein
+and	O
+prevents	O
+ACC	B-protein_type
+activation	O
+by	O
+phosphatase	B-protein_type
+-	O
+mediated	O
+dephosphorylation	O
+.	O
+
+Furthermore	O
+,	O
+phosphorylation	B-ptm
+by	O
+AMP	B-protein
+-	I-protein
+activated	I-protein
+protein	I-protein
+kinase	I-protein
+(	O
+AMPK	B-protein
+)	O
+and	O
+cAMP	B-protein
+-	I-protein
+dependent	I-protein
+protein	I-protein
+kinase	I-protein
+(	O
+PKA	B-protein
+)	O
+leads	O
+to	O
+a	O
+decrease	O
+in	O
+ACC1	B-protein
+activity	O
+.	O
+
+AMPK	B-protein
+phosphorylates	O
+ACC1	B-protein
+in	O
+vitro	O
+at	O
+Ser80	B-residue_name_number
+,	O
+Ser1201	B-residue_name_number
+and	O
+Ser1216	B-residue_name_number
+and	O
+PKA	B-protein
+at	O
+Ser78	B-residue_name_number
+and	O
+Ser1201	B-residue_name_number
+.	O
+
+However	O
+,	O
+regulatory	O
+effects	O
+on	O
+ACC1	B-protein
+activity	O
+are	O
+mainly	O
+mediated	O
+by	O
+phosphorylation	B-ptm
+of	O
+Ser80	B-residue_name_number
+and	O
+Ser1201	B-residue_name_number
+(	O
+refs	O
+).	O
+
+Phosphorylated	B-protein_state
+Ser80	B-residue_name_number
+,	O
+which	O
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+only	O
+in	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+,	O
+presumably	O
+binds	O
+into	O
+the	O
+Soraphen	B-site
+A	I-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+The	O
+regulatory	O
+Ser1201	B-residue_name_number
+shows	O
+only	O
+moderate	B-protein_state
+conservation	I-protein_state
+across	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+,	O
+while	O
+the	O
+phosphorylated	B-protein_state
+Ser1216	B-residue_name_number
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+across	O
+all	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+However	O
+,	O
+no	O
+effect	O
+of	O
+Ser1216	B-residue_name_number
+phosphorylation	B-ptm
+on	O
+ACC	B-protein_type
+activity	O
+has	O
+been	O
+reported	O
+in	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+
+For	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+neither	O
+spontaneous	O
+nor	O
+inducible	O
+polymerization	O
+has	O
+been	O
+detected	O
+despite	O
+considerable	O
+sequence	O
+conservation	O
+to	O
+human	B-species
+ACC1	B-protein
+.	O
+
+The	O
+BRCA1	B-protein
+-	O
+interacting	O
+phosphoserine	B-residue_name
+position	O
+is	O
+not	B-protein_state
+conserved	I-protein_state
+in	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+and	O
+no	O
+other	O
+phospho	O
+-	O
+dependent	O
+protein	O
+–	O
+protein	O
+interactions	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+have	O
+been	O
+described	O
+.	O
+
+In	O
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+phosphorylation	B-site
+sites	I-site
+have	O
+been	O
+identified	O
+at	O
+Ser2	B-residue_name_number
+,	O
+Ser735	B-residue_name_number
+,	O
+Ser1148	B-residue_name_number
+,	O
+Ser1157	B-residue_name_number
+and	O
+Ser1162	B-residue_name_number
+(	O
+ref	O
+.).	O
+
+Of	O
+these	O
+,	O
+only	O
+Ser1157	B-residue_name_number
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+and	O
+aligns	B-experimental_method
+to	I-experimental_method
+Ser1216	B-residue_name_number
+in	O
+human	B-species
+ACC1	B-protein
+.	O
+
+Its	O
+phosphorylation	B-ptm
+by	O
+the	O
+AMPK	B-protein
+homologue	O
+SNF1	B-protein
+results	O
+in	O
+strongly	O
+reduced	O
+ACC	B-protein_type
+activity	O
+.	O
+
+Despite	O
+the	O
+outstanding	O
+relevance	O
+of	O
+ACC	B-protein_type
+in	O
+primary	O
+metabolism	O
+and	O
+disease	O
+,	O
+the	O
+dynamic	O
+organization	O
+and	O
+regulation	O
+of	O
+the	O
+giant	O
+eukaryotic	B-taxonomy_domain
+,	O
+and	O
+in	O
+particular	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+remain	O
+poorly	O
+characterized	O
+.	O
+
+Here	O
+we	O
+provide	O
+the	O
+structure	B-evidence
+of	O
+Saccharomyces	B-species
+cerevisiae	I-species
+(	O
+Sce	B-species
+)	O
+ACC	B-protein_type
+CD	B-structure_element
+,	O
+intermediate	O
+-	O
+and	O
+low	O
+-	O
+resolution	O
+structures	B-evidence
+of	O
+human	B-species
+(	O
+Hsa	B-species
+)	O
+ACC	B-protein_type
+CD	B-structure_element
+and	O
+larger	B-mutant
+fragments	I-mutant
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+from	O
+Chaetomium	B-species
+thermophilum	I-species
+(	O
+Cth	B-species
+;	O
+Fig	O
+.	O
+1a	O
+).	O
+
+Integrating	O
+these	O
+data	O
+with	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+(	O
+SAXS	B-experimental_method
+)	O
+and	O
+electron	B-experimental_method
+microscopy	I-experimental_method
+(	O
+EM	B-experimental_method
+)	O
+observations	O
+yield	O
+a	O
+comprehensive	O
+representation	O
+of	O
+the	O
+dynamic	O
+structure	O
+and	O
+regulation	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+The	O
+organization	O
+of	O
+the	O
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+CD	B-structure_element
+
+First	O
+,	O
+we	O
+focused	O
+on	O
+structure	B-experimental_method
+determination	I-experimental_method
+of	O
+the	O
+82	O
+-	O
+kDa	O
+CD	B-structure_element
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+CD	B-structure_element
+of	O
+SceACC	B-protein
+(	O
+SceCD	B-species
+)	O
+was	O
+determined	O
+at	O
+3	O
+.	O
+0	O
+Å	O
+resolution	O
+by	O
+experimental	B-experimental_method
+phasing	I-experimental_method
+and	O
+refined	B-experimental_method
+to	O
+Rwork	B-evidence
+/	O
+Rfree	B-evidence
+=	O
+0	O
+.	O
+20	O
+/	O
+0	O
+.	O
+24	O
+(	O
+Table	O
+1	O
+).	O
+
+The	O
+overall	O
+extent	O
+of	O
+the	O
+SceCD	B-species
+is	O
+70	O
+by	O
+75	O
+Å	O
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+,	O
+b	O
+),	O
+and	O
+the	O
+attachment	O
+points	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+26	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+linker	I-structure_element
+to	O
+the	O
+BCCP	B-structure_element
+domain	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+CT	B-structure_element
+domain	O
+are	O
+separated	O
+by	O
+46	O
+Å	O
+(	O
+the	O
+N	O
+-	O
+and	O
+C	O
+termini	O
+are	O
+indicated	O
+with	O
+spheres	O
+in	O
+Fig	O
+.	O
+1b	O
+).	O
+
+SceCD	B-species
+comprises	O
+four	O
+distinct	O
+domains	O
+,	O
+an	O
+N	O
+-	O
+terminal	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+domain	I-structure_element
+(	O
+CDN	B-structure_element
+),	O
+and	O
+a	O
+central	O
+four	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+linker	I-structure_element
+domain	I-structure_element
+(	O
+CDL	B-structure_element
+),	O
+followed	O
+by	O
+two	O
+α	B-structure_element
+–	I-structure_element
+β	I-structure_element
+-	I-structure_element
+fold	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domains	I-structure_element
+(	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+).	O
+
+CDN	B-structure_element
+adopts	O
+a	O
+letter	O
+C	B-protein_state
+shape	I-protein_state
+,	O
+where	O
+one	O
+of	O
+the	O
+ends	O
+is	O
+a	O
+regular	B-structure_element
+four	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+(	O
+Nα3	B-structure_element
+-	I-structure_element
+6	I-structure_element
+),	O
+the	O
+other	O
+end	O
+is	O
+a	O
+helical	B-structure_element
+hairpin	I-structure_element
+(	O
+Nα8	B-structure_element
+,	I-structure_element
+9	I-structure_element
+)	O
+and	O
+the	O
+bridging	B-structure_element
+region	I-structure_element
+comprises	O
+six	O
+helices	B-structure_element
+(	O
+Nα1	B-structure_element
+,	I-structure_element
+2	I-structure_element
+,	I-structure_element
+7	I-structure_element
+,	I-structure_element
+10	I-structure_element
+–	I-structure_element
+12	I-structure_element
+).	O
+
+CDL	B-structure_element
+is	O
+composed	O
+of	O
+a	O
+small	B-structure_element
+,	I-structure_element
+irregular	I-structure_element
+four	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+(	O
+Lα1	B-structure_element
+–	I-structure_element
+4	I-structure_element
+)	O
+and	O
+tightly	O
+interacts	O
+with	O
+the	O
+open	O
+face	O
+of	O
+CDC1	B-structure_element
+via	O
+an	O
+interface	B-site
+of	O
+1	O
+,	O
+300	O
+Å2	O
+involving	O
+helices	B-structure_element
+Lα3	B-structure_element
+and	O
+Lα4	B-structure_element
+.	O
+
+CDL	B-structure_element
+does	O
+not	O
+interact	O
+with	O
+CDN	B-structure_element
+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	B-structure_element
+via	O
+a	O
+loop	B-structure_element
+between	O
+Lα2	B-structure_element
+/	I-structure_element
+α3	I-structure_element
+and	O
+the	O
+N	O
+-	O
+terminal	O
+end	O
+of	O
+Lα1	B-structure_element
+,	O
+with	O
+an	O
+interface	B-site
+area	O
+of	O
+400	O
+Å2	O
+.	O
+
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+share	O
+a	O
+common	O
+fold	O
+;	O
+they	O
+are	O
+composed	O
+of	O
+six	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheets	I-structure_element
+flanked	O
+on	O
+one	O
+side	O
+by	O
+two	O
+long	B-structure_element
+,	I-structure_element
+bent	I-structure_element
+helices	I-structure_element
+inserted	O
+between	O
+strands	B-structure_element
+β3	B-structure_element
+/	I-structure_element
+β4	I-structure_element
+and	O
+β4	B-structure_element
+/	I-structure_element
+β5	I-structure_element
+.	O
+
+CDC2	B-structure_element
+is	O
+extended	B-protein_state
+at	O
+its	O
+C	O
+terminus	O
+by	O
+an	O
+additional	O
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+and	O
+an	O
+irregular	B-structure_element
+β	I-structure_element
+-	I-structure_element
+hairpin	I-structure_element
+.	O
+
+On	O
+the	O
+basis	O
+of	O
+a	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+of	O
+main	O
+chain	O
+atom	O
+positions	O
+of	O
+2	O
+.	O
+2	O
+Å	O
+,	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+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
+
+Close	O
+structural	O
+homologues	O
+could	O
+not	O
+be	O
+found	O
+for	O
+the	O
+CDN	B-structure_element
+or	O
+the	O
+CDC	B-structure_element
+domains	O
+.	O
+
+A	O
+regulatory	B-structure_element
+loop	I-structure_element
+mediates	O
+interdomain	O
+interactions	O
+
+To	O
+define	O
+the	O
+functional	O
+state	O
+of	O
+insect	B-experimental_method
+-	I-experimental_method
+cell	I-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+ACC	B-protein_type
+variants	O
+,	O
+we	O
+employed	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+(	O
+MS	B-experimental_method
+)	O
+for	O
+phosphorylation	B-experimental_method
+site	I-experimental_method
+detection	I-experimental_method
+.	O
+
+In	O
+insect	B-experimental_method
+-	I-experimental_method
+cell	I-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+SceACC	B-protein
+,	O
+the	O
+highly	B-protein_state
+conserved	I-protein_state
+Ser1157	B-residue_name_number
+is	O
+the	O
+only	O
+fully	B-protein_state
+occupied	I-protein_state
+phosphorylation	B-site
+site	I-site
+with	O
+functional	O
+relevance	O
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+.	O
+
+Additional	O
+phosphorylation	B-ptm
+was	O
+detected	O
+for	O
+Ser2101	B-residue_name_number
+and	O
+Tyr2179	B-residue_name_number
+;	O
+however	O
+,	O
+these	O
+sites	O
+are	O
+neither	B-protein_state
+conserved	I-protein_state
+across	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+nor	B-protein_state
+natively	I-protein_state
+phosphorylated	I-protein_state
+in	O
+yeast	B-taxonomy_domain
+.	O
+
+MS	B-experimental_method
+analysis	O
+of	O
+dissolved	B-experimental_method
+crystals	I-experimental_method
+confirmed	O
+the	O
+phosphorylated	B-protein_state
+state	O
+of	O
+Ser1157	B-residue_name_number
+also	O
+in	O
+SceCD	B-species
+crystals	B-evidence
+.	O
+
+The	O
+SceCD	B-species
+structure	B-evidence
+thus	O
+authentically	O
+represents	O
+the	O
+state	O
+of	O
+SceACC	B-protein
+,	O
+where	O
+the	O
+enzyme	B-protein
+is	O
+inhibited	B-protein_state
+by	O
+SNF1	B-ptm
+-	I-ptm
+dependent	I-ptm
+phosphorylation	I-ptm
+.	O
+
+In	O
+the	O
+SceCD	B-species
+crystal	B-evidence
+structure	I-evidence
+,	O
+the	O
+phosphorylated	B-protein_state
+Ser1157	B-residue_name_number
+resides	O
+in	O
+a	O
+regulatory	B-structure_element
+36	I-structure_element
+-	I-structure_element
+amino	I-structure_element
+-	I-structure_element
+acid	I-structure_element
+loop	I-structure_element
+between	O
+strands	B-structure_element
+β2	B-structure_element
+and	O
+β3	B-structure_element
+of	O
+CDC1	B-structure_element
+(	O
+Fig	O
+.	O
+1b	O
+,	O
+d	O
+),	O
+which	O
+contains	O
+two	O
+additional	O
+less	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+phosphorylation	B-site
+sites	I-site
+(	O
+Ser1148	B-residue_name_number
+and	O
+Ser1162	B-residue_name_number
+)	O
+confirmed	O
+in	O
+yeast	B-taxonomy_domain
+,	O
+but	O
+not	O
+occupied	O
+here	O
+.	O
+
+This	O
+regulatory	B-structure_element
+loop	I-structure_element
+wedges	O
+between	O
+the	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+domains	O
+and	O
+provides	O
+the	O
+largest	O
+contribution	O
+to	O
+the	O
+interdomain	B-site
+interface	I-site
+.	O
+
+The	O
+N	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+also	O
+directly	O
+contacts	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+CDC2	B-structure_element
+leading	O
+into	O
+CT	B-structure_element
+.	O
+
+Phosphoserine	B-residue_name_number
+1157	I-residue_name_number
+is	O
+tightly	O
+bound	O
+by	O
+two	O
+highly	B-protein_state
+conserved	I-protein_state
+arginines	B-residue_name
+(	O
+Arg1173	B-residue_name_number
+and	O
+Arg1260	B-residue_name_number
+)	O
+of	O
+CDC1	B-structure_element
+(	O
+Fig	O
+.	O
+1d	O
+).	O
+
+Already	O
+the	O
+binding	O
+of	O
+phosphorylated	B-protein_state
+Ser1157	B-residue_name_number
+apparently	O
+stabilizes	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+conformation	O
+;	O
+the	O
+accessory	O
+phosphorylation	B-site
+sites	I-site
+Ser1148	B-residue_name_number
+and	O
+Ser1162	B-residue_name_number
+in	O
+the	O
+same	B-structure_element
+loop	I-structure_element
+may	O
+further	O
+modulate	O
+the	O
+strength	O
+of	O
+interaction	O
+between	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+and	O
+the	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+domains	O
+.	O
+
+Phosphorylation	B-ptm
+of	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+thus	O
+determines	O
+interdomain	O
+interactions	O
+of	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+,	O
+suggesting	O
+that	O
+it	O
+may	O
+exert	O
+its	O
+regulatory	O
+function	O
+by	O
+modifying	O
+the	O
+overall	O
+structure	O
+and	O
+dynamics	O
+of	O
+the	O
+CD	B-structure_element
+.	O
+
+The	O
+functional	O
+role	O
+of	O
+Ser1157	B-residue_name_number
+was	O
+confirmed	O
+by	O
+an	O
+activity	B-experimental_method
+assay	I-experimental_method
+based	O
+on	O
+the	O
+incorporation	O
+of	O
+radioactive	O
+carbonate	O
+into	O
+acid	O
+non	O
+-	O
+volatile	O
+material	O
+.	O
+
+Phosphorylated	B-protein_state
+SceACC	B-protein
+shows	O
+only	O
+residual	O
+activity	O
+(	O
+kcat	B-evidence
+=	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	B-evidence
+=	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	B-protein_type
+protein	I-protein_type
+phosphatase	I-protein_type
+.	O
+
+The	O
+values	O
+obtained	O
+for	O
+dephosphorylated	B-protein_state
+SceACC	B-protein
+are	O
+comparable	O
+to	O
+earlier	O
+measurements	O
+of	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+expressed	B-experimental_method
+in	I-experimental_method
+E	B-species
+.	I-species
+coli	I-species
+.	O
+
+The	O
+variable	O
+CD	B-structure_element
+is	O
+conserved	B-protein_state
+between	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+
+To	O
+compare	O
+the	O
+organization	O
+of	O
+fungal	B-taxonomy_domain
+and	O
+human	B-species
+ACC	B-protein_type
+CD	B-structure_element
+,	O
+we	O
+determined	B-experimental_method
+the	I-experimental_method
+structure	I-experimental_method
+of	O
+a	O
+human	B-species
+ACC1	B-mutant
+fragment	I-mutant
+that	O
+comprises	O
+the	O
+BT	B-structure_element
+and	O
+CD	B-structure_element
+domains	O
+(	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+),	O
+but	O
+lacks	B-protein_state
+the	O
+mobile	O
+BCCP	B-structure_element
+in	O
+between	O
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+
+An	O
+experimentally	B-evidence
+phased	I-evidence
+map	I-evidence
+was	O
+obtained	O
+at	O
+3	O
+.	O
+7	O
+Å	O
+resolution	O
+for	O
+a	O
+cadmium	B-chemical
+-	O
+derivatized	O
+crystal	O
+and	O
+was	O
+interpreted	O
+by	O
+a	O
+poly	O
+-	O
+alanine	O
+model	O
+(	O
+Fig	O
+.	O
+1e	O
+and	O
+Table	O
+1	O
+).	O
+
+Each	O
+of	O
+the	O
+four	O
+CD	B-structure_element
+domains	O
+in	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+individually	O
+resembles	O
+the	O
+corresponding	O
+SceCD	B-species
+domain	O
+;	O
+however	O
+,	O
+human	B-species
+and	O
+yeast	B-taxonomy_domain
+CDs	B-structure_element
+exhibit	O
+distinct	O
+overall	O
+structures	B-evidence
+.	O
+
+In	O
+agreement	O
+with	O
+their	O
+tight	O
+interaction	O
+in	O
+SceCD	B-species
+,	O
+the	O
+relative	O
+spatial	O
+arrangement	O
+of	O
+CDL	B-structure_element
+and	O
+CDC1	B-structure_element
+is	O
+preserved	O
+in	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+,	O
+but	O
+the	O
+human	B-species
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+didomain	O
+is	O
+tilted	O
+by	O
+30	O
+°	O
+based	O
+on	O
+a	O
+superposition	B-experimental_method
+of	O
+human	B-species
+and	O
+yeast	B-taxonomy_domain
+CDC2	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+).	O
+
+As	O
+a	O
+result	O
+,	O
+the	O
+N	O
+terminus	O
+of	O
+CDL	B-structure_element
+at	O
+helix	B-structure_element
+Lα1	B-structure_element
+,	O
+which	O
+connects	O
+to	O
+CDN	B-structure_element
+,	O
+is	O
+shifted	O
+by	O
+12	O
+Å	O
+.	O
+Remarkably	O
+,	O
+CDN	B-structure_element
+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	B-species
+.	O
+
+With	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+superposed	B-experimental_method
+,	O
+CDN	B-structure_element
+in	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+is	O
+rotated	O
+by	O
+160	O
+°	O
+around	O
+a	O
+hinge	B-structure_element
+at	O
+the	O
+connection	O
+of	O
+CDN	B-structure_element
+/	O
+CDL	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+).	O
+
+This	O
+rotation	O
+displaces	O
+the	O
+N	O
+terminus	O
+of	O
+CDN	B-structure_element
+in	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+by	O
+51	O
+Å	O
+compared	O
+with	O
+SceCD	B-species
+,	O
+resulting	O
+in	O
+a	O
+separation	O
+of	O
+the	O
+attachment	O
+points	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+linker	B-structure_element
+to	O
+the	O
+BCCP	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+C	O
+-	O
+terminal	O
+CT	B-structure_element
+domain	O
+by	O
+67	O
+Å	O
+(	O
+the	O
+attachment	O
+points	O
+are	O
+indicated	O
+with	O
+spheres	O
+in	O
+Fig	O
+.	O
+1e	O
+).	O
+
+The	O
+BT	B-structure_element
+domain	O
+of	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+consists	O
+of	O
+a	O
+helix	B-structure_element
+that	O
+is	O
+surrounded	O
+at	O
+its	O
+N	O
+terminus	O
+by	O
+an	O
+antiparallel	B-structure_element
+eight	I-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+barrel	I-structure_element
+.	O
+
+It	O
+resembles	O
+the	O
+BT	B-structure_element
+of	O
+propionyl	B-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylase	I-protein_type
+;	O
+only	O
+the	O
+four	O
+C	O
+-	O
+terminal	O
+strands	B-structure_element
+of	I-structure_element
+the	I-structure_element
+β	I-structure_element
+-	I-structure_element
+barrel	I-structure_element
+are	O
+slightly	O
+tilted	O
+.	O
+
+On	O
+the	O
+basis	O
+of	O
+MS	B-experimental_method
+analysis	O
+of	O
+insect	B-experimental_method
+-	I-experimental_method
+cell	I-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+human	B-species
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+ACC	B-protein_type
+,	O
+Ser80	B-residue_name_number
+shows	O
+the	O
+highest	O
+degree	O
+of	O
+phosphorylation	B-ptm
+(	O
+90	O
+%).	O
+
+Ser29	B-residue_name_number
+and	O
+Ser1263	B-residue_name_number
+,	O
+implicated	O
+in	O
+insulin	B-ptm
+-	I-ptm
+dependent	I-ptm
+phosphorylation	I-ptm
+and	O
+BRCA1	B-protein
+binding	O
+,	O
+respectively	O
+,	O
+are	O
+phosphorylated	B-protein_state
+at	O
+intermediate	O
+levels	O
+(	O
+40	O
+%).	O
+
+The	O
+highly	B-protein_state
+conserved	I-protein_state
+Ser1216	B-residue_name_number
+(	O
+corresponding	O
+to	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+Ser1157	B-residue_name_number
+),	O
+as	O
+well	O
+as	O
+Ser1201	B-residue_name_number
+,	O
+both	O
+in	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+discussed	O
+above	O
+,	O
+are	O
+not	B-protein_state
+phosphorylated	I-protein_state
+.	O
+
+However	O
+,	O
+residual	O
+phosphorylation	B-ptm
+levels	O
+were	O
+detected	O
+for	O
+Ser1204	B-residue_name_number
+(	O
+7	O
+%)	O
+and	O
+Ser1218	B-residue_name_number
+(	O
+7	O
+%)	O
+in	O
+the	O
+same	B-structure_element
+loop	I-structure_element
+.	O
+
+MS	B-experimental_method
+analysis	O
+of	O
+the	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+crystallization	B-evidence
+sample	I-evidence
+reveals	O
+partial	O
+proteolytic	O
+digestion	O
+of	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+.	O
+
+Accordingly	O
+,	O
+most	O
+of	O
+this	B-structure_element
+loop	I-structure_element
+is	O
+not	O
+represented	O
+in	O
+the	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+The	O
+absence	B-protein_state
+of	I-protein_state
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+might	O
+be	O
+linked	O
+to	O
+the	O
+less	B-protein_state
+-	I-protein_state
+restrained	I-protein_state
+interface	B-site
+of	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+and	O
+altered	O
+relative	O
+orientations	O
+of	O
+these	O
+domains	B-structure_element
+.	O
+
+Besides	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+,	O
+also	O
+the	O
+phosphopeptide	B-site
+target	I-site
+region	I-site
+for	O
+BRCA1	B-protein
+interaction	O
+is	O
+not	O
+resolved	O
+presumably	O
+because	O
+of	O
+pronounced	O
+flexibility	O
+.	O
+
+At	O
+the	O
+level	O
+of	O
+isolated	B-experimental_method
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+CD	B-structure_element
+,	O
+the	O
+structural	B-experimental_method
+analysis	I-experimental_method
+indicates	O
+the	O
+presence	O
+of	O
+at	O
+least	O
+two	O
+hinges	B-structure_element
+,	O
+one	O
+with	O
+large	O
+-	O
+scale	O
+flexibility	O
+at	O
+the	O
+CDN	B-structure_element
+/	I-structure_element
+CDL	I-structure_element
+connection	I-structure_element
+,	O
+and	O
+one	O
+with	O
+tunable	O
+plasticity	O
+between	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+,	O
+plausibly	O
+affected	O
+by	O
+phosphorylation	B-ptm
+in	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+region	O
+.	O
+
+The	O
+integration	O
+of	O
+CD	B-structure_element
+into	O
+the	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+multienzyme	I-protein_type
+
+To	O
+further	O
+obtain	O
+insights	O
+into	O
+the	O
+functional	O
+architecture	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+we	O
+characterized	O
+larger	B-mutant
+multidomain	I-mutant
+fragments	I-mutant
+up	O
+to	O
+the	O
+intact	B-protein_state
+enzymes	B-protein
+.	O
+
+Using	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+based	O
+on	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+CD	B-structure_element
+and	O
+CT	B-structure_element
+models	O
+,	O
+we	O
+obtained	O
+structures	B-evidence
+of	O
+a	O
+variant	B-mutant
+comprising	O
+CthCT	B-species
+and	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+in	O
+two	B-evidence
+crystal	I-evidence
+forms	I-evidence
+at	O
+resolutions	O
+of	O
+3	O
+.	O
+6	O
+and	O
+4	O
+.	O
+5	O
+Å	O
+(	O
+CthCD	B-mutant
+-	I-mutant
+CTCter1	I-mutant
+/	I-mutant
+2	I-mutant
+),	O
+respectively	O
+,	O
+as	O
+well	O
+as	O
+of	O
+a	O
+CthCT	B-species
+linked	O
+to	O
+the	O
+entire	O
+CD	B-structure_element
+at	O
+7	O
+.	O
+2	O
+Å	O
+resolution	O
+(	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+;	O
+Figs	O
+1a	O
+and	O
+2	O
+,	O
+Table	O
+1	O
+).	O
+
+No	O
+crystals	O
+diffracting	O
+to	O
+sufficient	O
+resolution	O
+were	O
+obtained	O
+for	O
+larger	B-mutant
+BC	I-mutant
+-	I-mutant
+containing	I-mutant
+fragments	I-mutant
+,	O
+or	O
+for	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cth	B-species
+or	O
+SceACC	B-protein
+.	O
+
+To	O
+improve	B-experimental_method
+crystallizability	I-experimental_method
+,	O
+we	O
+generated	B-experimental_method
+ΔBCCP	B-mutant
+variants	I-mutant
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+ACC	B-protein_type
+,	O
+which	O
+,	O
+based	O
+on	O
+SAXS	B-experimental_method
+analysis	I-experimental_method
+,	O
+preserve	O
+properties	O
+of	O
+intact	B-protein_state
+ACC	B-protein_type
+(	O
+Supplementary	O
+Table	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+–	O
+c	O
+).	O
+
+For	O
+CthΔBCCP	B-mutant
+,	O
+crystals	B-evidence
+diffracting	O
+to	O
+8	O
+.	O
+4	O
+Å	O
+resolution	O
+were	O
+obtained	O
+.	O
+
+However	O
+,	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+did	O
+not	O
+reveal	O
+a	O
+unique	O
+positioning	O
+of	O
+the	O
+BC	B-structure_element
+domain	O
+.	O
+
+Owing	O
+to	O
+the	O
+limited	O
+resolution	O
+the	O
+discussion	O
+of	O
+structures	B-evidence
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+CthΔBCCP	B-mutant
+is	O
+restricted	O
+to	O
+the	O
+analysis	O
+of	O
+domain	O
+localization	O
+.	O
+
+Still	O
+,	O
+these	B-evidence
+structures	I-evidence
+contribute	O
+considerably	O
+to	O
+the	O
+visualization	O
+of	O
+an	O
+intrinsically	O
+dynamic	B-protein_state
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+In	O
+all	O
+these	O
+crystal	B-evidence
+structures	I-evidence
+,	O
+the	O
+CT	B-structure_element
+domains	O
+build	O
+a	O
+canonical	O
+head	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+tail	I-protein_state
+dimer	B-oligomeric_state
+,	O
+with	O
+active	B-site
+sites	I-site
+formed	O
+by	O
+contributions	O
+from	O
+both	O
+protomers	B-oligomeric_state
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+).	O
+
+The	O
+connection	B-structure_element
+of	O
+CD	B-structure_element
+and	O
+CT	B-structure_element
+is	O
+provided	O
+by	O
+a	O
+10	B-residue_range
+-	I-residue_range
+residue	I-residue_range
+peptide	I-residue_range
+stretch	I-residue_range
+,	O
+which	O
+links	O
+the	O
+N	O
+terminus	O
+of	O
+CT	B-structure_element
+to	O
+the	O
+irregular	B-structure_element
+β	I-structure_element
+-	I-structure_element
+hairpin	I-structure_element
+/	I-structure_element
+β	I-structure_element
+-	I-structure_element
+strand	I-structure_element
+extension	I-structure_element
+of	O
+CDC2	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+).	O
+
+The	O
+connecting	B-structure_element
+region	I-structure_element
+is	O
+remarkably	O
+similar	O
+in	O
+isolated	B-protein_state
+CD	B-structure_element
+and	O
+CthCD	B-mutant
+-	I-mutant
+CTCter	I-mutant
+structures	B-evidence
+,	O
+indicating	O
+inherent	O
+conformational	O
+stability	O
+.	O
+
+CD	B-structure_element
+/	O
+CT	B-structure_element
+contacts	O
+are	O
+only	O
+formed	O
+in	O
+direct	O
+vicinity	O
+of	O
+the	O
+covalent	O
+linkage	O
+and	O
+involve	O
+the	O
+β	B-structure_element
+-	I-structure_element
+hairpin	I-structure_element
+extension	I-structure_element
+of	O
+CDC2	B-structure_element
+as	O
+well	O
+as	O
+the	O
+loop	B-structure_element
+between	O
+strands	B-structure_element
+β2	I-structure_element
+/	I-structure_element
+β3	I-structure_element
+of	O
+the	O
+CT	B-structure_element
+N	I-structure_element
+-	I-structure_element
+lobe	I-structure_element
+,	O
+which	O
+contains	O
+a	O
+conserved	B-protein_state
+RxxGxN	B-structure_element
+motif	I-structure_element
+.	O
+
+The	O
+neighbouring	O
+loop	B-structure_element
+on	O
+the	O
+CT	B-structure_element
+side	O
+(	O
+between	O
+CT	B-structure_element
+β1	B-structure_element
+/	O
+β2	B-structure_element
+)	O
+is	O
+displaced	O
+by	O
+2	O
+.	O
+5	O
+Å	O
+compared	O
+to	O
+isolated	B-protein_state
+CT	B-structure_element
+structures	B-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+3c	O
+).	O
+
+On	O
+the	O
+basis	O
+of	O
+an	O
+interface	O
+area	O
+of	O
+∼	O
+600	O
+Å2	O
+and	O
+its	O
+edge	O
+-	O
+to	O
+-	O
+edge	O
+connection	O
+characteristics	O
+,	O
+the	O
+interface	B-site
+between	O
+CT	B-structure_element
+and	O
+CD	B-structure_element
+might	O
+be	O
+classified	O
+as	O
+conformationally	O
+variable	O
+.	O
+
+Indeed	O
+,	O
+the	O
+comparison	O
+of	O
+the	O
+positioning	O
+of	O
+eight	O
+instances	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+part	O
+of	O
+CD	B-structure_element
+relative	O
+to	O
+CT	B-structure_element
+in	O
+crystal	B-evidence
+structures	I-evidence
+determined	B-experimental_method
+here	O
+,	O
+reveals	O
+flexible	O
+interdomain	O
+linking	O
+(	O
+Fig	O
+.	O
+3a	O
+).	O
+
+The	O
+CDC2	B-site
+/	I-site
+CT	I-site
+interface	I-site
+acts	O
+as	O
+a	O
+true	B-structure_element
+hinge	I-structure_element
+with	O
+observed	O
+rotation	O
+up	O
+to	O
+16	O
+°,	O
+which	O
+results	O
+in	O
+a	O
+translocation	O
+of	O
+the	O
+distal	O
+end	O
+of	O
+CDC2	B-structure_element
+by	O
+8	O
+Å	O
+.	O
+
+The	O
+interface	B-site
+between	O
+CDC2	B-structure_element
+and	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+,	O
+which	O
+is	O
+mediated	O
+by	O
+the	O
+phosphorylated	B-protein_state
+regulatory	B-structure_element
+loop	I-structure_element
+in	O
+the	O
+SceCD	B-species
+structure	B-evidence
+,	O
+is	O
+less	O
+variable	O
+than	O
+the	O
+CD	B-structure_element
+–	I-structure_element
+CT	I-structure_element
+junction	I-structure_element
+,	O
+and	O
+permits	O
+only	O
+limited	O
+rotation	O
+and	O
+tilting	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+
+Analysis	O
+of	O
+the	O
+impact	O
+of	O
+phosphorylation	B-ptm
+on	O
+the	O
+interface	B-site
+between	O
+CDC2	B-structure_element
+and	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+in	O
+CthACC	B-mutant
+variant	I-mutant
+structures	B-evidence
+is	O
+precluded	O
+by	O
+the	O
+limited	O
+crystallographic	O
+resolution	O
+.	O
+
+However	O
+,	O
+MS	B-experimental_method
+analysis	O
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+CthΔBCCP	B-mutant
+constructs	O
+revealed	O
+between	O
+60	O
+and	O
+70	O
+%	O
+phosphorylation	B-ptm
+of	O
+Ser1170	B-residue_name_number
+(	O
+corresponding	O
+to	O
+SceACC	B-protein
+Ser1157	B-residue_name_number
+).	O
+
+The	O
+CDN	B-structure_element
+domain	O
+positioning	O
+relative	O
+to	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+is	O
+highly	O
+variable	O
+with	O
+three	O
+main	O
+orientations	O
+observed	O
+in	O
+the	O
+structures	B-evidence
+of	O
+SceCD	B-species
+and	O
+the	O
+larger	B-mutant
+CthACC	I-mutant
+fragments	I-mutant
+:	O
+CDN	B-structure_element
+tilts	O
+,	O
+resulting	O
+in	O
+a	O
+displacement	O
+of	O
+its	O
+N	O
+terminus	O
+by	O
+23	O
+Å	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+observed	O
+in	O
+both	O
+protomers	B-oligomeric_state
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+one	O
+protomer	B-oligomeric_state
+of	O
+CthΔBCCP	B-mutant
+,	O
+denoted	O
+as	O
+CthCD	B-mutant
+-	I-mutant
+CT1	I-mutant
+/	I-mutant
+2	I-mutant
+and	O
+CthΔBCCP1	B-mutant
+,	O
+respectively	O
+).	O
+
+In	O
+addition	O
+,	O
+CDN	B-structure_element
+can	O
+rotate	O
+around	O
+hinges	B-structure_element
+in	O
+the	O
+connection	O
+between	O
+CDN	B-structure_element
+/	O
+CDL	B-structure_element
+by	O
+70	O
+°	O
+(	O
+Fig	O
+.	O
+4b	O
+,	O
+observed	O
+in	O
+the	O
+second	O
+protomer	B-oligomeric_state
+of	O
+CthΔBCCP	B-mutant
+,	O
+denoted	O
+as	O
+CthΔBCCP2	B-mutant
+)	O
+and	O
+160	O
+°	O
+(	O
+Fig	O
+.	O
+4c	O
+,	O
+observed	O
+in	O
+SceCD	B-species
+)	O
+leading	O
+to	O
+displacement	O
+of	O
+the	O
+anchor	B-site
+site	I-site
+for	O
+the	O
+BCCP	B-structure_element
+linker	I-structure_element
+by	O
+up	O
+to	O
+33	O
+and	O
+40	O
+Å	O
+,	O
+respectively	O
+.	O
+
+Conformational	O
+variability	O
+in	O
+the	O
+CD	B-structure_element
+thus	O
+contributes	O
+considerably	O
+to	O
+variations	O
+in	O
+the	O
+spacing	O
+between	O
+the	O
+BC	B-structure_element
+and	O
+CT	B-structure_element
+domains	O
+,	O
+and	O
+may	O
+extend	O
+to	O
+distance	O
+variations	O
+beyond	O
+the	O
+mobility	O
+range	O
+of	O
+the	O
+flexibly	B-protein_state
+tethered	I-protein_state
+BCCP	B-structure_element
+.	O
+
+On	O
+the	O
+basis	O
+of	O
+the	O
+occurrence	O
+of	O
+related	O
+conformational	O
+changes	O
+between	O
+fungal	B-taxonomy_domain
+and	O
+human	B-species
+ACC	B-mutant
+fragments	I-mutant
+,	O
+the	O
+observed	O
+set	O
+of	O
+conformations	O
+may	O
+well	O
+represent	O
+general	O
+states	O
+present	O
+in	O
+all	O
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+.	O
+
+Large	O
+-	O
+scale	O
+conformational	O
+variability	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+
+To	O
+obtain	O
+a	O
+comprehensive	O
+view	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+dynamics	O
+in	B-protein_state
+solution	I-protein_state
+,	O
+we	O
+employed	O
+SAXS	B-experimental_method
+and	O
+EM	B-experimental_method
+.	O
+
+SAXS	B-experimental_method
+analysis	O
+of	O
+CthACC	B-protein
+agrees	O
+with	O
+a	O
+dimeric	B-oligomeric_state
+state	O
+and	O
+an	O
+elongated	B-protein_state
+shape	I-protein_state
+with	O
+a	O
+maximum	O
+extent	O
+of	O
+350	O
+Å	O
+(	O
+Supplementary	O
+Table	O
+1	O
+).	O
+
+The	O
+smooth	O
+appearance	O
+of	O
+scattering	B-evidence
+curves	I-evidence
+and	O
+derived	B-evidence
+distance	I-evidence
+distributions	I-evidence
+might	O
+indicate	O
+substantial	O
+interdomain	O
+flexibility	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+–	O
+c	O
+).	O
+
+Direct	O
+observation	O
+of	O
+individual	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+CthACC	B-protein
+particles	B-evidence
+,	O
+according	O
+to	O
+MS	B-experimental_method
+results	O
+predominantly	O
+in	O
+a	O
+phosphorylated	B-protein_state
+low	B-protein_state
+-	I-protein_state
+activity	I-protein_state
+state	I-protein_state
+,	O
+in	O
+negative	B-experimental_method
+stain	I-experimental_method
+EM	I-experimental_method
+reveals	O
+a	O
+large	O
+set	O
+of	O
+conformations	O
+from	O
+rod	B-protein_state
+-	I-protein_state
+like	I-protein_state
+extended	I-protein_state
+to	O
+U	B-protein_state
+-	I-protein_state
+shaped	I-protein_state
+particles	B-evidence
+.	O
+
+Class	B-evidence
+averages	I-evidence
+,	O
+obtained	O
+by	O
+maximum	B-experimental_method
+-	I-experimental_method
+likelihood	I-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+two	I-experimental_method
+-	I-experimental_method
+dimensional	I-experimental_method
+(	I-experimental_method
+2D	I-experimental_method
+)	I-experimental_method
+classification	I-experimental_method
+,	O
+are	O
+focused	O
+on	O
+the	O
+dimeric	B-oligomeric_state
+CT	B-structure_element
+domain	O
+and	O
+the	O
+full	B-protein_state
+BC	B-mutant
+–	I-mutant
+BCCP	I-mutant
+–	I-mutant
+CD	I-mutant
+domain	O
+of	O
+only	O
+one	O
+protomer	B-oligomeric_state
+,	O
+due	O
+to	O
+the	O
+non	O
+-	O
+coordinated	O
+motions	O
+of	O
+the	O
+lateral	O
+BC	B-structure_element
+/	O
+CD	B-structure_element
+regions	O
+relative	O
+to	O
+the	O
+CT	B-structure_element
+dimer	B-oligomeric_state
+.	O
+
+They	O
+identify	O
+the	O
+connections	O
+between	O
+CDN	B-structure_element
+/	O
+CDL	B-structure_element
+and	O
+between	O
+CDC2	B-structure_element
+/	O
+CT	B-structure_element
+as	O
+major	O
+contributors	O
+to	O
+conformational	O
+heterogeneity	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+
+The	O
+flexibility	O
+in	O
+the	O
+CDC2	B-structure_element
+/	I-structure_element
+CT	I-structure_element
+hinge	I-structure_element
+appears	O
+substantially	O
+larger	O
+than	O
+the	O
+variations	O
+observed	O
+in	O
+the	O
+set	O
+of	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+The	O
+BC	B-structure_element
+domain	O
+is	O
+not	O
+completely	O
+disordered	O
+,	O
+but	O
+laterally	O
+attached	O
+to	O
+BT	B-structure_element
+/	O
+CDN	B-structure_element
+in	O
+a	O
+generally	B-protein_state
+conserved	I-protein_state
+position	I-protein_state
+,	O
+albeit	O
+with	O
+increased	O
+flexibility	O
+.	O
+
+Surprisingly	O
+,	O
+in	O
+both	O
+the	O
+linear	B-protein_state
+and	I-protein_state
+U	I-protein_state
+-	I-protein_state
+shaped	I-protein_state
+conformations	I-protein_state
+,	O
+the	O
+approximate	O
+distances	O
+between	O
+the	O
+BC	B-structure_element
+and	O
+CT	B-structure_element
+active	B-site
+sites	I-site
+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	B-protein_state
+structures	B-evidence
+of	O
+any	O
+other	O
+related	O
+biotin	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+carboxylase	I-protein_type
+.	O
+
+Furthermore	O
+,	O
+based	O
+on	O
+an	O
+average	O
+length	O
+of	O
+the	O
+BCCP	B-structure_element
+–	I-structure_element
+CD	I-structure_element
+linker	I-structure_element
+in	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+of	O
+26	B-residue_range
+amino	I-residue_range
+acids	I-residue_range
+,	O
+mobility	O
+of	O
+the	O
+BCCP	B-structure_element
+alone	O
+would	O
+not	O
+be	O
+sufficient	O
+to	O
+bridge	O
+the	O
+active	B-site
+sites	I-site
+of	O
+BC	B-structure_element
+and	O
+CT	B-structure_element
+.	O
+
+The	O
+most	O
+relevant	O
+candidate	O
+site	O
+for	O
+mediating	O
+such	O
+additional	O
+flexibility	O
+and	O
+permitting	O
+an	O
+extended	O
+set	O
+of	O
+conformations	O
+is	O
+the	O
+CDC1	B-site
+/	I-site
+CDC2	I-site
+interface	I-site
+,	O
+which	O
+is	O
+rigidified	O
+by	O
+the	O
+Ser1157	B-residue_name_number
+-	O
+phosphorylated	B-protein_state
+regulatory	B-structure_element
+loop	I-structure_element
+,	O
+as	O
+depicted	O
+in	O
+the	O
+SceCD	B-species
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+Altogether	O
+,	O
+the	O
+architecture	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+is	O
+based	O
+on	O
+the	O
+central	O
+dimeric	B-oligomeric_state
+CT	B-structure_element
+domain	O
+(	O
+Fig	O
+.	O
+4d	O
+).	O
+
+The	O
+CD	B-structure_element
+consists	O
+of	O
+four	O
+distinct	O
+subdomains	B-structure_element
+and	O
+acts	O
+as	O
+a	O
+tether	O
+from	O
+the	O
+CT	B-structure_element
+to	O
+the	O
+mobile	B-protein_state
+BCCP	B-structure_element
+and	O
+an	O
+oriented	B-protein_state
+BC	B-structure_element
+domain	O
+.	O
+
+The	O
+CD	B-structure_element
+has	O
+no	O
+direct	O
+role	O
+in	O
+substrate	O
+recognition	O
+or	O
+catalysis	O
+but	O
+contributes	O
+to	O
+the	O
+regulation	O
+of	O
+all	O
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+.	O
+
+In	O
+higher	B-taxonomy_domain
+eukaryotic	I-taxonomy_domain
+ACCs	B-protein_type
+,	O
+regulation	O
+via	O
+phosphorylation	B-ptm
+is	O
+achieved	O
+by	O
+combining	O
+the	O
+effects	O
+of	O
+phosphorylation	B-ptm
+at	O
+Ser80	B-residue_name_number
+,	O
+Ser1201	B-residue_name_number
+and	O
+Ser1263	B-residue_name_number
+.	O
+
+In	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+however	O
+,	O
+Ser1157	B-residue_name_number
+in	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+of	O
+the	O
+CD	B-structure_element
+is	O
+the	O
+only	O
+phosphorylation	B-site
+site	I-site
+that	O
+has	O
+been	O
+demonstrated	O
+to	O
+be	O
+both	O
+phosphorylated	B-protein_state
+in	O
+vivo	O
+and	O
+involved	O
+in	O
+the	O
+regulation	O
+of	O
+ACC	B-protein_type
+activity	O
+.	O
+
+In	O
+its	O
+phosphorylated	B-protein_state
+state	O
+,	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+containing	O
+Ser1157	B-residue_name_number
+wedges	O
+between	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+and	O
+presumably	O
+limits	O
+the	O
+conformational	B-protein_state
+freedom	I-protein_state
+at	O
+this	O
+interdomain	B-site
+interface	I-site
+.	O
+
+However	O
+,	O
+flexibility	O
+at	O
+this	O
+hinge	B-structure_element
+may	O
+be	O
+required	O
+for	O
+full	B-protein_state
+ACC	I-protein_state
+activity	I-protein_state
+,	O
+as	O
+the	O
+distances	O
+between	O
+the	O
+BCCP	B-structure_element
+anchor	I-structure_element
+points	I-structure_element
+and	O
+the	O
+active	B-site
+sites	I-site
+of	O
+BC	B-structure_element
+and	O
+CT	B-structure_element
+observed	O
+here	O
+are	O
+such	O
+large	O
+that	O
+mobility	O
+of	O
+the	O
+BCCP	B-structure_element
+alone	O
+is	O
+not	O
+sufficient	O
+for	O
+substrate	O
+transfer	O
+.	O
+
+The	O
+current	O
+data	O
+thus	O
+suggest	O
+that	O
+regulation	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+is	O
+mediated	O
+by	O
+controlling	O
+the	O
+dynamics	O
+of	O
+the	O
+unique	B-protein_state
+CD	B-structure_element
+,	O
+rather	O
+than	O
+directly	O
+affecting	O
+catalytic	O
+turnover	O
+at	O
+the	O
+active	B-site
+sites	I-site
+of	O
+BC	B-structure_element
+and	O
+CT	B-structure_element
+.	O
+
+A	O
+comparison	O
+between	O
+fungal	B-taxonomy_domain
+and	O
+human	B-species
+ACC	B-protein_type
+will	O
+help	O
+to	O
+further	O
+discriminate	O
+mechanistic	O
+differences	O
+that	O
+contribute	O
+to	O
+the	O
+extended	O
+control	O
+and	O
+polymerization	O
+of	O
+human	B-species
+ACC	B-protein_type
+.	O
+
+Most	O
+recently	O
+,	O
+a	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+near	B-protein_state
+full	I-protein_state
+-	I-protein_state
+length	I-protein_state
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+ACC	B-protein_type
+from	O
+S	B-species
+.	I-species
+cerevisae	I-species
+(	O
+lacking	B-protein_state
+only	I-protein_state
+21	B-residue_range
+N	O
+-	O
+terminal	O
+amino	O
+acids	O
+,	O
+here	O
+denoted	O
+as	O
+flACC	B-mutant
+)	O
+was	O
+published	O
+by	O
+Wei	O
+and	O
+Tong	O
+.	O
+
+In	O
+flACC	B-mutant
+,	O
+the	O
+ACC	B-protein_type
+dimer	B-oligomeric_state
+obeys	O
+twofold	O
+symmetry	O
+and	O
+assembles	O
+in	O
+a	O
+triangular	B-protein_state
+architecture	I-protein_state
+with	O
+dimeric	B-oligomeric_state
+BC	B-structure_element
+domains	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+).	O
+
+In	O
+their	O
+study	O
+,	O
+mutational	B-experimental_method
+data	I-experimental_method
+indicate	O
+a	O
+requirement	O
+for	O
+BC	O
+dimerization	O
+for	O
+catalytic	O
+activity	O
+.	O
+
+The	O
+transition	O
+from	O
+the	O
+elongated	B-protein_state
+open	I-protein_state
+shape	I-protein_state
+,	O
+observed	O
+in	O
+our	O
+experiments	O
+,	O
+towards	O
+a	O
+compact	B-protein_state
+triangular	I-protein_state
+shape	I-protein_state
+is	O
+based	O
+on	O
+an	O
+intricate	O
+interplay	O
+of	O
+several	O
+hinge	O
+-	O
+bending	O
+motions	O
+in	O
+the	O
+CD	B-structure_element
+(	O
+Fig	O
+.	O
+4d	O
+).	O
+
+Comparison	B-experimental_method
+of	O
+flACC	B-mutant
+with	O
+our	O
+CthΔBCCP	B-mutant
+structure	B-evidence
+reveals	O
+the	O
+CDC2	B-structure_element
+/	I-structure_element
+CT	I-structure_element
+hinge	I-structure_element
+as	O
+a	O
+major	O
+contributor	O
+to	O
+conformational	O
+flexibility	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+
+In	O
+flACC	B-mutant
+,	O
+CDC2	B-structure_element
+rotates	O
+∼	O
+120	O
+°	O
+with	O
+respect	O
+to	O
+the	O
+CT	B-structure_element
+domain	O
+.	O
+
+A	O
+second	B-structure_element
+hinge	I-structure_element
+can	O
+be	O
+identified	O
+between	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+.	O
+
+On	O
+the	O
+basis	O
+of	O
+a	O
+superposition	B-experimental_method
+of	O
+CDC2	B-structure_element
+,	O
+CDC1	B-structure_element
+of	O
+the	O
+phosphorylated	B-protein_state
+SceCD	B-species
+is	O
+rotated	O
+by	O
+30	O
+°	O
+relative	O
+to	O
+CDC1	B-structure_element
+of	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+flACC	B-mutant
+(	O
+Supplementary	O
+Fig	O
+.	O
+5d	O
+),	O
+similar	O
+to	O
+what	O
+we	O
+have	O
+observed	O
+for	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+).	O
+
+When	O
+inspecting	B-experimental_method
+all	O
+individual	O
+protomer	B-oligomeric_state
+and	O
+fragment	B-mutant
+structures	B-evidence
+in	O
+their	O
+study	O
+,	O
+Wei	O
+and	O
+Tong	O
+also	O
+identify	O
+the	O
+CDN	B-structure_element
+/	I-structure_element
+CDC1	I-structure_element
+connection	I-structure_element
+as	O
+a	O
+highly	B-protein_state
+flexible	I-protein_state
+hinge	B-structure_element
+,	O
+in	O
+agreement	O
+with	O
+our	O
+observations	O
+.	O
+
+The	O
+only	O
+bona	O
+fide	O
+regulatory	B-protein_state
+phophorylation	B-site
+site	I-site
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+in	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+is	O
+directly	O
+participating	O
+in	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+domain	O
+interactions	O
+and	O
+thus	O
+stabilizes	O
+the	O
+hinge	B-structure_element
+conformation	I-structure_element
+.	O
+
+In	O
+flACC	B-mutant
+,	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+is	O
+mostly	B-protein_state
+disordered	I-protein_state
+,	O
+illustrating	O
+the	O
+increased	O
+flexibility	O
+due	O
+to	O
+the	O
+absence	O
+of	O
+the	O
+phosphoryl	B-chemical
+group	O
+.	O
+
+Only	O
+in	O
+three	O
+out	O
+of	O
+eight	O
+observed	O
+protomers	B-oligomeric_state
+a	O
+short	B-structure_element
+peptide	I-structure_element
+stretch	O
+(	O
+including	O
+Ser1157	B-residue_name_number
+)	O
+was	O
+modelled	B-evidence
+.	O
+
+In	O
+those	O
+instances	O
+the	O
+Ser1157	B-residue_name_number
+residue	O
+is	O
+located	O
+at	O
+a	O
+distance	O
+of	O
+14	O
+–	O
+20	O
+Å	O
+away	O
+from	O
+the	O
+location	O
+of	O
+the	O
+phosphorylated	B-protein_state
+serine	B-residue_name
+observed	O
+here	O
+,	O
+based	O
+on	O
+superposition	B-experimental_method
+of	O
+either	O
+CDC1	B-structure_element
+or	O
+CDC2	B-structure_element
+.	O
+
+Applying	B-experimental_method
+the	O
+conformation	O
+of	O
+the	O
+CDC1	B-structure_element
+/	I-structure_element
+CDC2	I-structure_element
+hinge	I-structure_element
+observed	O
+in	O
+SceCD	B-species
+on	O
+flACC	B-mutant
+leads	O
+to	O
+CDN	B-structure_element
+sterically	O
+clashing	O
+with	O
+CDC2	B-structure_element
+and	O
+BT	B-structure_element
+/	O
+CDN	B-structure_element
+clashing	O
+with	O
+CT	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+).	O
+
+Thus	O
+,	O
+in	O
+accordance	O
+with	O
+the	O
+results	O
+presented	O
+here	O
+,	O
+phosphorylation	B-ptm
+of	O
+Ser1157	B-residue_name_number
+in	O
+SceACC	B-protein
+most	O
+likely	O
+limits	O
+flexibility	O
+in	O
+the	O
+CDC1	B-structure_element
+/	I-structure_element
+CDC2	I-structure_element
+hinge	I-structure_element
+such	O
+that	O
+activation	O
+through	O
+BC	B-structure_element
+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
+
+In	O
+addition	O
+,	O
+EM	B-experimental_method
+micrographs	B-evidence
+of	O
+phosphorylated	B-protein_state
+and	O
+dephosphorylated	B-protein_state
+SceACC	B-protein
+display	O
+for	O
+both	O
+samples	O
+mainly	O
+elongated	B-protein_state
+and	I-protein_state
+U	I-protein_state
+-	I-protein_state
+shaped	I-protein_state
+conformations	I-protein_state
+and	O
+reveal	O
+no	O
+apparent	O
+differences	O
+in	O
+particle	B-evidence
+shape	I-evidence
+distributions	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+
+This	O
+implicates	O
+that	O
+the	O
+triangular	B-protein_state
+shape	I-protein_state
+with	O
+dimeric	B-oligomeric_state
+BC	B-structure_element
+domains	O
+has	O
+a	O
+low	O
+population	O
+also	O
+in	O
+the	O
+active	B-protein_state
+form	I-protein_state
+,	O
+even	O
+though	O
+a	O
+biasing	O
+influence	O
+of	O
+grid	O
+preparation	O
+cannot	O
+be	O
+excluded	O
+completely	O
+.	O
+
+Large	O
+-	O
+scale	O
+conformational	O
+variability	O
+has	O
+also	O
+been	O
+observed	O
+in	O
+most	O
+other	O
+carrier	B-protein_type
+protein	I-protein_type
+-	I-protein_type
+based	I-protein_type
+multienzymes	I-protein_type
+,	O
+including	O
+polyketide	B-protein_type
+and	I-protein_type
+fatty	I-protein_type
+-	I-protein_type
+acid	I-protein_type
+synthases	I-protein_type
+(	O
+with	O
+the	O
+exception	O
+of	O
+fungal	B-protein_type
+-	I-protein_type
+type	I-protein_type
+fatty	I-protein_type
+-	I-protein_type
+acid	I-protein_type
+synthases	I-protein_type
+),	O
+non	B-protein_type
+-	I-protein_type
+ribosomal	I-protein_type
+peptide	I-protein_type
+synthetases	I-protein_type
+and	O
+the	O
+pyruvate	B-protein_type
+dehydrogenase	I-protein_type
+complexes	I-protein_type
+,	O
+although	O
+based	O
+on	O
+completely	O
+different	O
+architectures	O
+.	O
+
+Together	O
+,	O
+this	O
+structural	B-evidence
+information	I-evidence
+suggests	O
+that	O
+variable	O
+carrier	O
+protein	O
+tethering	O
+is	O
+not	O
+sufficient	O
+for	O
+efficient	O
+substrate	O
+transfer	O
+and	O
+catalysis	O
+in	O
+any	O
+of	O
+these	O
+systems	O
+.	O
+
+The	O
+determination	B-experimental_method
+of	I-experimental_method
+a	I-experimental_method
+set	I-experimental_method
+of	I-experimental_method
+crystal	B-evidence
+structures	I-evidence
+of	O
+SceACC	B-protein
+in	O
+two	O
+states	O
+,	O
+unphosphorylated	B-protein_state
+and	O
+phosphorylated	B-protein_state
+at	O
+the	O
+major	B-site
+regulatory	I-site
+site	I-site
+Ser1157	B-residue_name_number
+,	O
+provides	O
+a	O
+unique	O
+depiction	O
+of	O
+multienzyme	O
+regulation	O
+by	O
+post	O
+-	O
+translational	O
+modification	O
+(	O
+Fig	O
+.	O
+4d	O
+).	O
+
+The	O
+phosphorylated	B-protein_state
+regulatory	B-structure_element
+loop	I-structure_element
+binds	O
+to	O
+an	O
+allosteric	B-site
+site	I-site
+at	O
+the	O
+interface	B-site
+of	O
+two	O
+non	B-protein_state
+-	I-protein_state
+catalytic	I-protein_state
+domains	O
+and	O
+restricts	O
+conformational	O
+freedom	O
+at	O
+several	O
+hinges	B-structure_element
+in	O
+the	O
+dynamic	B-protein_state
+ACC	B-protein_type
+.	O
+
+It	O
+disfavours	O
+the	O
+adoption	O
+of	O
+a	O
+rare	B-protein_state
+,	I-protein_state
+compact	I-protein_state
+conformation	I-protein_state
+,	O
+in	O
+which	O
+intramolecular	O
+dimerization	O
+of	O
+the	O
+BC	B-structure_element
+domains	O
+results	O
+in	O
+catalytic	O
+turnover	O
+.	O
+
+The	O
+regulation	O
+of	O
+activity	O
+thus	O
+results	O
+from	O
+restrained	O
+large	O
+-	O
+scale	O
+conformational	O
+dynamics	O
+rather	O
+than	O
+a	O
+direct	O
+or	O
+indirect	O
+influence	O
+on	O
+active	B-site
+site	I-site
+structure	I-site
+.	O
+
+To	O
+our	O
+best	O
+knowledge	O
+,	O
+ACC	B-protein_type
+is	O
+the	O
+first	O
+multienzyme	B-protein_type
+for	O
+which	O
+such	O
+a	O
+phosphorylation	B-ptm
+-	O
+dependent	O
+mechanical	O
+control	O
+mechanism	O
+has	O
+been	O
+visualized	O
+.	O
+
+However	O
+,	O
+the	O
+example	O
+of	O
+ACC	B-protein_type
+now	O
+demonstrates	O
+the	O
+possibility	O
+of	O
+regulating	O
+activity	O
+by	O
+controlled	O
+dynamics	O
+of	O
+non	B-structure_element
+-	I-structure_element
+enzymatic	I-structure_element
+linker	I-structure_element
+regions	I-structure_element
+also	O
+in	O
+other	O
+families	O
+of	O
+carrier	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+multienzymes	I-protein_type
+.	O
+
+The	O
+phosphorylated	B-protein_state
+central	B-structure_element
+domain	I-structure_element
+of	O
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+(	O
+a	O
+)	O
+Schematic	O
+overview	O
+of	O
+the	O
+domain	O
+organization	O
+of	O
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+.	O
+
+Crystallized	B-evidence
+constructs	I-evidence
+are	O
+indicated	O
+.	O
+
+(	O
+b	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+SceCD	B-species
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+CDN	B-structure_element
+is	O
+linked	O
+by	O
+a	O
+four	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+(	O
+CDL	B-structure_element
+)	O
+to	O
+two	B-structure_element
+α	I-structure_element
+–	I-structure_element
+β	I-structure_element
+-	I-structure_element
+fold	I-structure_element
+domains	I-structure_element
+(	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+).	O
+
+The	O
+regulatory	B-structure_element
+loop	I-structure_element
+is	O
+shown	O
+as	O
+bold	O
+cartoon	O
+,	O
+and	O
+the	O
+phosphorylated	B-protein_state
+Ser1157	B-residue_name_number
+is	O
+marked	O
+by	O
+a	O
+red	O
+triangle	O
+.	O
+
+(	O
+c	O
+)	O
+Superposition	B-experimental_method
+of	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+reveals	O
+highly	B-protein_state
+conserved	I-protein_state
+folds	B-structure_element
+.	O
+(	O
+d	O
+)	O
+The	O
+regulatory	B-structure_element
+loop	I-structure_element
+with	O
+the	O
+phosphorylated	B-protein_state
+Ser1157	B-residue_name_number
+is	O
+bound	O
+into	O
+a	O
+crevice	O
+between	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+,	O
+the	O
+conserved	B-protein_state
+residues	O
+Arg1173	B-residue_name_number
+and	O
+Arg1260	B-residue_name_number
+coordinate	O
+the	O
+phosphoryl	B-chemical
+-	O
+group	O
+.	O
+
+(	O
+e	O
+)	O
+Structural	O
+overview	O
+of	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+.	O
+
+The	O
+attachment	O
+points	O
+to	O
+the	O
+N	O
+-	O
+terminal	O
+BCCP	B-structure_element
+domain	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+CT	B-structure_element
+domain	O
+are	O
+indicated	O
+with	O
+spheres	O
+.	O
+
+Architecture	O
+of	O
+the	O
+CD	B-structure_element
+–	O
+CT	B-structure_element
+core	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+Cartoon	O
+representation	O
+of	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+multidomain	B-mutant
+constructs	I-mutant
+of	O
+CthACC	B-protein
+.	O
+
+One	O
+protomer	B-oligomeric_state
+is	O
+shown	O
+in	O
+colour	O
+and	O
+one	O
+in	O
+grey	O
+.	O
+
+Individual	O
+domains	O
+are	O
+labelled	O
+;	O
+the	O
+active	B-site
+site	I-site
+of	O
+CT	B-structure_element
+and	O
+the	O
+position	O
+of	O
+the	O
+conserved	B-protein_state
+regulatory	B-protein_state
+phosphoserine	B-site
+site	I-site
+based	O
+on	O
+SceCD	B-species
+are	O
+indicated	O
+by	O
+an	O
+asterisk	O
+and	O
+a	O
+triangle	O
+,	O
+respectively	O
+.	O
+
+Variability	O
+of	O
+the	O
+connections	O
+of	O
+CDC2	B-structure_element
+to	O
+CT	B-structure_element
+and	O
+CDC1	B-structure_element
+in	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+(	O
+a	O
+)	O
+Hinge	B-structure_element
+properties	O
+of	O
+the	O
+CDC2	B-structure_element
+–	I-structure_element
+CT	I-structure_element
+connection	I-structure_element
+analysed	O
+by	O
+a	O
+CT	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+superposition	I-experimental_method
+of	O
+eight	O
+instances	O
+of	O
+the	O
+CDC2	B-mutant
+-	I-mutant
+CT	I-mutant
+segment	I-mutant
+.	O
+
+For	O
+clarity	O
+,	O
+only	O
+one	O
+protomer	B-oligomeric_state
+of	O
+CthCD	B-mutant
+-	I-mutant
+CTCter1	I-mutant
+is	O
+shown	O
+in	O
+full	O
+colour	O
+as	O
+reference	O
+.	O
+
+For	O
+other	O
+instances	O
+,	O
+CDC2	B-structure_element
+domains	O
+are	O
+shown	O
+in	O
+transparent	O
+tube	O
+representation	O
+with	O
+only	O
+one	O
+helix	O
+each	O
+highlighted	O
+.	O
+
+The	O
+range	O
+of	O
+hinge	O
+bending	O
+is	O
+indicated	O
+and	O
+the	O
+connection	O
+points	O
+between	O
+CDC2	B-structure_element
+and	O
+CT	B-structure_element
+(	O
+blue	O
+)	O
+as	O
+well	O
+as	O
+between	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+(	O
+green	O
+and	O
+grey	O
+)	O
+are	O
+marked	O
+as	O
+spheres	O
+.	O
+
+(	O
+b	O
+)	O
+The	O
+interdomain	B-site
+interface	I-site
+of	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+exhibits	O
+only	O
+limited	O
+plasticity	O
+.	O
+
+Representation	O
+as	O
+in	O
+a	O
+,	O
+but	O
+the	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+are	O
+superposed	B-experimental_method
+based	O
+on	O
+CDC2	B-structure_element
+.	O
+
+One	O
+protomer	B-oligomeric_state
+of	O
+CthΔBCCP	B-mutant
+is	O
+shown	O
+in	O
+colour	O
+,	O
+the	O
+CDL	B-structure_element
+domains	O
+are	O
+omitted	O
+for	O
+clarity	O
+and	O
+the	O
+position	O
+of	O
+the	O
+phosphorylated	B-protein_state
+serine	B-residue_name
+based	O
+on	O
+SceCD	B-species
+is	O
+indicated	O
+with	O
+a	O
+red	O
+triangle	O
+.	O
+
+The	O
+connection	O
+points	O
+from	O
+CDC1	B-structure_element
+to	O
+CDC2	B-structure_element
+and	O
+to	O
+CDL	B-structure_element
+are	O
+represented	O
+by	O
+green	O
+spheres	O
+.	O
+
+The	O
+conformational	O
+dynamics	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+(	O
+a	O
+–	O
+c	O
+)	O
+Large	O
+-	O
+scale	O
+conformational	O
+variability	O
+of	O
+the	O
+CDN	B-structure_element
+domain	O
+relative	O
+to	O
+the	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+domain	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	B-experimental_method
+structures	I-experimental_method
+(	O
+as	O
+indicated	O
+,	O
+numbers	O
+after	O
+construct	O
+name	O
+differentiate	O
+between	O
+individual	O
+protomers	B-oligomeric_state
+)	O
+are	O
+shown	O
+in	O
+grey	O
+.	O
+
+Domains	O
+other	O
+than	O
+CDN	B-structure_element
+and	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+are	O
+omitted	O
+for	O
+clarity	O
+.	O
+
+The	O
+domains	O
+are	O
+labelled	O
+and	O
+the	O
+distances	O
+between	O
+the	O
+N	O
+termini	O
+of	O
+CDN	B-structure_element
+(	O
+spheres	O
+)	O
+in	O
+the	O
+compared	O
+structures	O
+are	O
+indicated	O
+.	O
+
+(	O
+d	O
+)	O
+Schematic	O
+model	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+showing	O
+the	O
+intrinsic	O
+,	O
+regulated	O
+flexibility	O
+of	O
+CD	B-structure_element
+in	O
+the	O
+phosphorylated	B-protein_state
+inhibited	B-protein_state
+or	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+activated	B-protein_state
+state	O
+.	O
+
+Flexibility	O
+of	O
+the	O
+CDC2	B-structure_element
+/	O
+CT	B-structure_element
+and	O
+CDN	B-structure_element
+/	O
+CDL	B-structure_element
+hinges	B-structure_element
+is	O
+illustrated	O
+by	O
+arrows	O
+.	O
+
+The	O
+Ser1157	B-residue_name_number
+phosphorylation	B-ptm
+site	O
+and	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+are	O
+schematically	O
+indicated	O
+in	O
+magenta	O
+.	O
+
+Structural	O
+insights	O
+into	O
+the	O
+Escherichia	B-species
+coli	I-species
+lysine	B-protein_type
+decarboxylases	I-protein_type
+and	O
+molecular	O
+determinants	O
+of	O
+interaction	O
+with	O
+the	O
+AAA	B-protein_type
++	I-protein_type
+ATPase	I-protein_type
+RavA	B-protein
+
+The	O
+inducible	B-protein_state
+lysine	B-protein_type
+decarboxylase	I-protein_type
+LdcI	B-protein
+is	O
+an	O
+important	O
+enterobacterial	B-taxonomy_domain
+acid	B-protein_type
+stress	I-protein_type
+response	I-protein_type
+enzyme	I-protein_type
+whereas	O
+LdcC	B-protein
+is	O
+its	O
+close	O
+paralogue	O
+thought	O
+to	O
+play	O
+mainly	O
+a	O
+metabolic	O
+role	O
+.	O
+
+A	O
+unique	O
+macromolecular	O
+cage	O
+formed	O
+by	O
+two	O
+decamers	B-oligomeric_state
+of	O
+the	O
+Escherichia	B-species
+coli	I-species
+LdcI	B-protein
+and	O
+five	O
+hexamers	B-oligomeric_state
+of	O
+the	O
+AAA	B-protein_type
++	I-protein_type
+ATPase	I-protein_type
+RavA	B-protein
+was	O
+shown	O
+to	O
+counteract	O
+acid	O
+stress	O
+under	O
+starvation	O
+.	O
+
+Previously	O
+,	O
+we	O
+proposed	O
+a	O
+pseudoatomic	B-evidence
+model	I-evidence
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+based	O
+on	O
+its	O
+cryo	B-experimental_method
+-	I-experimental_method
+electron	I-experimental_method
+microscopy	I-experimental_method
+map	B-evidence
+and	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+an	O
+inactive	B-protein_state
+LdcI	B-protein
+decamer	B-oligomeric_state
+and	O
+a	O
+RavA	B-protein
+monomer	B-oligomeric_state
+.	O
+
+We	O
+now	O
+present	O
+cryo	B-experimental_method
+-	I-experimental_method
+electron	I-experimental_method
+microscopy	I-experimental_method
+3D	B-evidence
+reconstructions	I-evidence
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+LdcI	B-protein
+and	O
+LdcC	B-protein
+,	O
+and	O
+an	O
+improved	B-evidence
+map	I-evidence
+of	O
+the	O
+LdcI	B-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+LARA	B-structure_element
+domain	I-structure_element
+of	O
+RavA	B-protein
+,	O
+at	O
+pH	B-protein_state
+optimal	I-protein_state
+for	O
+their	O
+enzymatic	O
+activity	O
+.	O
+
+Comparison	B-experimental_method
+with	O
+each	O
+other	O
+and	O
+with	O
+available	O
+structures	B-evidence
+uncovers	O
+differences	O
+between	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+explaining	O
+why	O
+only	O
+the	O
+acid	B-protein_type
+stress	I-protein_type
+response	I-protein_type
+enzyme	I-protein_type
+is	O
+capable	O
+of	O
+binding	O
+RavA	B-protein
+.	O
+We	O
+identify	O
+interdomain	O
+movements	O
+associated	O
+with	O
+the	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+with	O
+the	O
+RavA	B-protein
+binding	O
+.	O
+
+Multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+coupled	O
+to	O
+a	O
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+reveals	O
+that	O
+certain	O
+enterobacteria	B-taxonomy_domain
+exert	O
+evolutionary	O
+pressure	O
+on	O
+the	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+towards	O
+the	O
+cage	O
+-	O
+like	O
+assembly	O
+with	O
+RavA	B-protein
+,	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
+
+Enterobacterial	B-taxonomy_domain
+inducible	B-protein_state
+decarboxylases	B-protein_type
+of	O
+basic	B-protein_state
+amino	B-chemical
+acids	I-chemical
+lysine	B-residue_name
+,	O
+arginine	B-residue_name
+and	O
+ornithine	B-residue_name
+have	O
+a	O
+common	O
+evolutionary	O
+origin	O
+and	O
+belong	O
+to	O
+the	O
+α	B-protein_type
+-	I-protein_type
+family	I-protein_type
+of	O
+pyridoxal	B-chemical
+-	I-chemical
+5	I-chemical
+′-	I-chemical
+phosphate	I-chemical
+(	O
+PLP	B-chemical
+)-	O
+dependent	O
+enzymes	O
+.	O
+
+They	O
+counteract	O
+acid	O
+stress	O
+experienced	O
+by	O
+the	O
+bacterium	B-taxonomy_domain
+in	O
+the	O
+host	O
+digestive	O
+and	O
+urinary	O
+tract	O
+,	O
+and	O
+in	O
+particular	O
+in	O
+the	O
+extremely	O
+acidic	O
+stomach	O
+.	O
+
+Each	O
+decarboxylase	B-protein_type
+is	O
+induced	O
+by	O
+an	O
+excess	O
+of	O
+the	O
+target	O
+amino	B-chemical
+acid	I-chemical
+and	O
+a	O
+specific	O
+range	O
+of	O
+extracellular	O
+pH	O
+,	O
+and	O
+works	O
+in	O
+conjunction	O
+with	O
+a	O
+cognate	O
+inner	B-protein_type
+membrane	I-protein_type
+antiporter	I-protein_type
+.	O
+
+Decarboxylation	O
+of	O
+the	O
+amino	B-chemical
+acid	I-chemical
+into	O
+a	O
+polyamine	B-chemical
+is	O
+catalysed	O
+by	O
+a	O
+PLP	B-chemical
+cofactor	O
+in	O
+a	O
+multistep	O
+reaction	O
+that	O
+consumes	O
+a	O
+cytoplasmic	O
+proton	B-chemical
+and	O
+produces	O
+a	O
+CO2	B-chemical
+molecule	O
+passively	O
+diffusing	O
+out	O
+of	O
+the	O
+cell	O
+,	O
+while	O
+the	O
+polyamine	B-chemical
+is	O
+excreted	O
+by	O
+the	O
+antiporter	B-protein_type
+in	O
+exchange	O
+for	O
+a	O
+new	O
+amino	B-chemical
+acid	I-chemical
+substrate	O
+.	O
+
+Consequently	O
+,	O
+these	O
+enzymes	O
+buffer	O
+both	O
+the	O
+bacterial	B-taxonomy_domain
+cytoplasm	O
+and	O
+the	O
+local	O
+extracellular	O
+environment	O
+.	O
+
+These	O
+amino	B-protein_type
+acid	I-protein_type
+decarboxylases	I-protein_type
+are	O
+therefore	O
+called	O
+acid	O
+stress	O
+inducible	B-protein_state
+or	O
+biodegradative	B-protein_state
+to	O
+distinguish	O
+them	O
+from	O
+their	O
+biosynthetic	B-protein_state
+lysine	B-protein_type
+and	I-protein_type
+ornithine	I-protein_type
+decarboxylase	I-protein_type
+paralogs	O
+catalysing	O
+the	O
+same	O
+reaction	O
+but	O
+responsible	O
+for	O
+the	O
+polyamine	B-chemical
+production	O
+at	O
+neutral	B-protein_state
+pH	I-protein_state
+.	O
+
+Inducible	B-protein_state
+enterobacterial	B-taxonomy_domain
+amino	B-protein_type
+acid	I-protein_type
+decarboxylases	I-protein_type
+have	O
+been	O
+intensively	O
+studied	O
+since	O
+the	O
+early	O
+1940	O
+because	O
+the	O
+ability	O
+of	O
+bacteria	B-taxonomy_domain
+to	O
+withstand	O
+acid	O
+stress	O
+can	O
+be	O
+linked	O
+to	O
+their	O
+pathogenicity	O
+in	O
+humans	B-species
+.	O
+
+In	O
+particular	O
+,	O
+the	O
+inducible	B-protein_state
+lysine	B-protein_type
+decarboxylase	I-protein_type
+LdcI	B-protein
+(	O
+or	O
+CadA	B-protein
+)	O
+attracts	O
+attention	O
+due	O
+to	O
+its	O
+broad	B-protein_state
+pH	I-protein_state
+range	I-protein_state
+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	B-taxonomy_domain
+such	O
+as	O
+Salmonella	B-species
+enterica	I-species
+serovar	I-species
+Typhimurium	I-species
+,	O
+Vibrio	B-species
+cholerae	I-species
+and	O
+Vibrio	B-species
+vulnificus	I-species
+under	O
+acidic	O
+conditions	O
+.	O
+
+Furthermore	O
+,	O
+both	O
+LdcI	B-protein
+and	O
+the	O
+biosynthetic	B-protein_state
+lysine	B-protein_type
+decarboxylase	I-protein_type
+LdcC	B-protein
+of	O
+uropathogenic	B-species
+Escherichia	I-species
+coli	I-species
+(	O
+UPEC	B-species
+)	O
+appear	O
+to	O
+play	O
+an	O
+important	O
+role	O
+in	O
+increased	O
+resistance	O
+of	O
+this	O
+pathogen	O
+to	O
+nitrosative	O
+stress	O
+produced	O
+by	O
+nitric	B-chemical
+oxide	I-chemical
+and	O
+other	O
+damaging	O
+reactive	O
+nitrogen	O
+intermediates	O
+accumulating	O
+during	O
+the	O
+course	O
+of	O
+urinary	O
+tract	O
+infections	O
+(	O
+UTI	O
+).	O
+
+This	O
+effect	O
+is	O
+attributed	O
+to	O
+cadaverine	B-chemical
+,	O
+the	O
+diamine	O
+produced	O
+by	O
+decarboxylation	O
+of	O
+lysine	B-residue_name
+by	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+,	O
+that	O
+was	O
+shown	O
+to	O
+enhance	O
+UPEC	B-species
+colonisation	O
+of	O
+the	O
+bladder	O
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+biosynthetic	B-protein_state
+E	B-species
+.	I-species
+coli	I-species
+lysine	B-protein_type
+decarboxylase	I-protein_type
+LdcC	B-protein
+,	O
+long	O
+thought	O
+to	O
+be	O
+constitutively	O
+expressed	O
+in	O
+low	O
+amounts	O
+,	O
+was	O
+demonstrated	O
+to	O
+be	O
+strongly	O
+upregulated	O
+by	O
+fluoroquinolones	B-chemical
+via	O
+their	O
+induction	O
+of	O
+RpoS	B-protein
+.	O
+A	O
+direct	O
+correlation	O
+between	O
+the	O
+level	O
+of	O
+cadaverine	B-chemical
+and	O
+the	O
+resistance	O
+of	O
+E	B-species
+.	I-species
+coli	I-species
+to	O
+these	O
+antibiotics	O
+commonly	O
+used	O
+as	O
+a	O
+first	O
+-	O
+line	O
+treatment	O
+of	O
+UTI	O
+could	O
+be	O
+established	O
+.	O
+
+Both	O
+acid	B-protein_state
+pH	I-protein_state
+and	O
+cadaverine	B-chemical
+induce	O
+closure	O
+of	O
+outer	O
+membrane	O
+porins	B-protein_type
+thereby	O
+contributing	O
+to	O
+bacterial	B-taxonomy_domain
+protection	O
+from	O
+acid	O
+stress	O
+,	O
+but	O
+also	O
+from	O
+certain	O
+antibiotics	O
+,	O
+by	O
+reduction	O
+in	O
+membrane	O
+permeability	O
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+LdcI	B-protein
+as	O
+well	O
+as	O
+its	O
+low	O
+resolution	O
+characterisation	O
+by	O
+electron	B-experimental_method
+microscopy	I-experimental_method
+(	O
+EM	B-experimental_method
+)	O
+showed	O
+that	O
+it	O
+is	O
+a	O
+decamer	B-oligomeric_state
+made	O
+of	O
+two	O
+pentameric	B-oligomeric_state
+rings	B-structure_element
+.	O
+
+Each	O
+monomer	B-oligomeric_state
+is	O
+composed	O
+of	O
+three	O
+domains	O
+–	O
+an	O
+N	O
+-	O
+terminal	O
+wing	B-structure_element
+domain	I-structure_element
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+129	I-residue_range
+),	O
+a	O
+PLP	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+core	I-structure_element
+domain	I-structure_element
+(	O
+residues	O
+130	B-residue_range
+–	I-residue_range
+563	I-residue_range
+),	O
+and	O
+a	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+CTD	B-structure_element
+,	O
+residues	O
+564	B-residue_range
+–	I-residue_range
+715	I-residue_range
+).	O
+
+Monomers	B-oligomeric_state
+tightly	O
+associate	O
+via	O
+their	O
+core	B-structure_element
+domains	I-structure_element
+into	O
+2	B-protein_state
+-	I-protein_state
+fold	I-protein_state
+symmetrical	I-protein_state
+dimers	B-oligomeric_state
+with	O
+two	O
+complete	O
+active	B-site
+sites	I-site
+,	O
+and	O
+further	O
+build	O
+a	O
+toroidal	B-structure_element
+D5	I-structure_element
+-	I-structure_element
+symmetrical	I-structure_element
+structure	I-structure_element
+held	O
+by	O
+the	O
+wing	B-structure_element
+and	O
+core	B-structure_element
+domain	I-structure_element
+interactions	O
+around	O
+the	O
+central	B-structure_element
+pore	I-structure_element
+,	O
+with	O
+the	O
+CTDs	B-structure_element
+at	O
+the	O
+periphery	O
+.	O
+
+Ten	O
+years	O
+ago	O
+we	O
+showed	O
+that	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+AAA	B-protein_type
++	I-protein_type
+ATPase	I-protein_type
+RavA	B-protein
+,	O
+involved	O
+in	O
+multiple	O
+stress	O
+response	O
+pathways	O
+,	O
+tightly	O
+interacted	O
+with	O
+LdcI	B-protein
+but	O
+was	O
+not	O
+capable	O
+of	O
+binding	O
+to	O
+LdcC	B-protein
+.	O
+We	O
+described	O
+how	O
+two	O
+double	O
+pentameric	B-oligomeric_state
+rings	B-structure_element
+of	O
+the	O
+LdcI	B-protein
+tightly	O
+associate	O
+with	O
+five	O
+hexameric	B-oligomeric_state
+rings	B-structure_element
+of	O
+RavA	B-protein
+to	O
+form	O
+a	O
+unique	O
+cage	O
+-	O
+like	O
+architecture	O
+that	O
+enables	O
+the	O
+bacterium	B-taxonomy_domain
+to	O
+withstand	O
+acid	O
+stress	O
+even	O
+under	O
+conditions	O
+of	O
+nutrient	O
+deprivation	O
+eliciting	O
+stringent	O
+response	O
+.	O
+
+Furthermore	O
+,	O
+we	O
+recently	O
+solved	B-experimental_method
+the	I-experimental_method
+structure	I-experimental_method
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+complex	O
+by	O
+cryo	B-experimental_method
+-	I-experimental_method
+electron	I-experimental_method
+microscopy	I-experimental_method
+(	O
+cryoEM	B-experimental_method
+)	O
+and	O
+combined	O
+it	O
+with	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+individual	O
+proteins	O
+.	O
+
+This	O
+allowed	O
+us	O
+to	O
+make	O
+a	O
+pseudoatomic	B-evidence
+model	I-evidence
+of	O
+the	O
+whole	O
+assembly	O
+,	O
+underpinned	O
+by	O
+a	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+complex	O
+(	O
+with	O
+LARA	B-structure_element
+standing	O
+for	O
+LdcI	B-structure_element
+associating	I-structure_element
+domain	I-structure_element
+of	I-structure_element
+RavA	I-structure_element
+),	O
+and	O
+to	O
+identify	O
+conformational	O
+rearrangements	O
+and	O
+specific	O
+elements	O
+essential	O
+for	O
+complex	O
+formation	O
+.	O
+
+The	O
+main	O
+determinants	O
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+assembly	O
+appeared	O
+to	O
+be	O
+the	O
+N	O
+-	O
+terminal	O
+loop	B-structure_element
+of	O
+the	O
+LARA	B-structure_element
+domain	I-structure_element
+of	O
+RavA	B-protein
+and	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+LdcI	B-protein
+.	O
+
+In	O
+spite	O
+of	O
+this	O
+wealth	O
+of	O
+structural	B-evidence
+information	I-evidence
+,	O
+the	O
+fact	O
+that	O
+LdcC	B-protein
+does	O
+not	O
+interact	O
+with	O
+RavA	B-protein
+,	O
+although	O
+the	O
+two	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+are	O
+69	O
+%	O
+identical	O
+and	O
+84	O
+%	O
+similar	O
+,	O
+and	O
+the	O
+physiological	O
+significance	O
+of	O
+the	O
+absence	O
+of	O
+this	O
+interaction	O
+remained	O
+unexplored	O
+.	O
+
+To	O
+solve	O
+this	O
+discrepancy	O
+,	O
+in	O
+the	O
+present	O
+work	O
+we	O
+provided	O
+a	O
+three	O
+-	O
+dimensional	O
+(	O
+3D	O
+)	O
+cryoEM	B-experimental_method
+reconstruction	B-evidence
+of	O
+LdcC	B-protein
+and	O
+compared	O
+it	O
+with	O
+the	O
+available	O
+LdcI	B-protein
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+structures	B-evidence
+.	O
+
+Given	O
+that	O
+the	O
+LdcI	B-protein
+crystal	B-evidence
+structures	I-evidence
+were	O
+obtained	O
+at	O
+high	B-protein_state
+pH	I-protein_state
+where	O
+the	O
+enzyme	O
+is	O
+inactive	B-protein_state
+(	O
+LdcIi	B-protein
+,	O
+pH	B-protein_state
+8	I-protein_state
+.	I-protein_state
+5	I-protein_state
+),	O
+whereas	O
+the	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+of	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+were	O
+done	O
+at	O
+acidic	B-protein_state
+pH	I-protein_state
+optimal	I-protein_state
+for	O
+the	O
+enzymatic	O
+activity	O
+,	O
+for	O
+a	O
+meaningful	O
+comparison	O
+,	O
+we	O
+also	O
+produced	O
+a	O
+3D	B-evidence
+reconstruction	I-evidence
+of	O
+the	O
+LdcI	B-protein
+at	O
+active	B-protein_state
+pH	I-protein_state
+(	O
+LdcIa	B-protein
+,	O
+pH	B-protein_state
+6	I-protein_state
+.	I-protein_state
+2	I-protein_state
+).	O
+
+This	O
+comparison	O
+pinpointed	O
+differences	O
+between	O
+the	O
+biodegradative	B-protein_state
+and	O
+the	O
+biosynthetic	B-protein_state
+lysine	B-protein_type
+decarboxylases	I-protein_type
+and	O
+brought	O
+to	O
+light	O
+interdomain	O
+movements	O
+associated	O
+to	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+RavA	B-protein
+binding	O
+,	O
+notably	O
+at	O
+the	O
+predicted	O
+RavA	B-site
+binding	I-site
+site	I-site
+at	O
+the	O
+level	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+LdcI	B-protein
+.	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	B-experimental_method
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+in	O
+question	O
+.	O
+
+Finally	O
+,	O
+we	O
+performed	O
+multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+22	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+from	O
+Enterobacteriaceae	B-taxonomy_domain
+containing	O
+the	O
+ravA	B-gene
+-	I-gene
+viaA	I-gene
+operon	I-gene
+in	O
+their	O
+genome	O
+.	O
+
+Remarkably	O
+,	O
+this	O
+analysis	O
+revealed	O
+that	O
+several	O
+specific	B-structure_element
+residues	I-structure_element
+in	O
+the	O
+above	O
+-	O
+mentioned	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+,	O
+independently	O
+of	O
+the	O
+rest	O
+of	O
+the	O
+protein	O
+sequence	O
+,	O
+are	O
+sufficient	O
+to	O
+define	O
+if	O
+a	O
+particular	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+should	O
+be	O
+classified	O
+as	O
+an	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+or	O
+an	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”.	O
+
+This	O
+fascinating	O
+parallelism	O
+between	O
+the	O
+propensity	O
+for	O
+RavA	B-protein
+binding	O
+and	O
+the	O
+genetic	O
+environment	O
+of	O
+an	O
+enterobacterial	B-taxonomy_domain
+lysine	B-protein_type
+decarboxylase	I-protein_type
+,	O
+as	O
+well	O
+as	O
+the	O
+high	B-protein_state
+degree	I-protein_state
+of	I-protein_state
+conservation	I-protein_state
+of	O
+this	O
+small	B-structure_element
+structural	I-structure_element
+motif	I-structure_element
+,	O
+emphasize	O
+the	O
+functional	O
+importance	O
+of	O
+the	O
+interaction	O
+between	O
+biodegradative	B-protein_state
+enterobacterial	B-taxonomy_domain
+lysine	B-protein_type
+decarboxylases	I-protein_type
+and	O
+the	O
+AAA	B-protein_type
++	I-protein_type
+ATPase	I-protein_type
+RavA	B-protein
+.	O
+
+CryoEM	B-experimental_method
+3D	B-evidence
+reconstructions	I-evidence
+of	O
+LdcC	B-protein
+,	O
+LdcIa	B-protein
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+
+In	O
+the	O
+frame	O
+of	O
+this	O
+work	O
+,	O
+we	O
+produced	O
+two	O
+novel	O
+subnanometer	O
+resolution	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+lysine	B-protein_type
+decarboxylases	I-protein_type
+at	O
+pH	B-protein_state
+optimal	I-protein_state
+for	O
+their	O
+enzymatic	O
+activity	O
+–	O
+a	O
+5	O
+.	O
+5	O
+Å	O
+resolution	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcC	B-protein
+(	O
+pH	B-protein_state
+7	I-protein_state
+.	I-protein_state
+5	I-protein_state
+)	O
+for	O
+which	O
+no	O
+3D	O
+structural	O
+information	O
+has	O
+been	O
+previously	O
+available	O
+(	O
+Figs	O
+1A	O
+,	O
+B	O
+and	O
+S1	O
+),	O
+and	O
+a	O
+6	O
+.	O
+1	O
+Å	O
+resolution	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcIa	B-protein
+,	O
+(	O
+pH	B-protein_state
+6	I-protein_state
+.	I-protein_state
+2	I-protein_state
+)	O
+(	O
+Figs	O
+1C	O
+,	O
+D	O
+and	O
+S2	O
+).	O
+
+In	O
+addition	O
+,	O
+we	O
+improved	O
+our	O
+earlier	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+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
+
+Based	O
+on	O
+these	O
+reconstructions	B-evidence
+,	O
+reliable	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+the	O
+three	O
+assemblies	O
+were	O
+obtained	O
+by	O
+flexible	B-experimental_method
+fitting	I-experimental_method
+of	I-experimental_method
+either	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+LdcIi	B-protein
+or	O
+a	O
+derived	O
+structural	B-experimental_method
+homology	I-experimental_method
+model	I-experimental_method
+of	O
+LdcC	B-protein
+(	O
+Table	O
+S1	O
+).	O
+
+Significant	O
+differences	O
+between	O
+these	O
+pseudoatomic	B-evidence
+models	I-evidence
+can	O
+be	O
+interpreted	O
+as	O
+movements	O
+between	O
+specific	O
+biological	O
+states	O
+of	O
+the	O
+proteins	O
+as	O
+described	O
+below	O
+.	O
+
+The	O
+wing	B-structure_element
+domains	I-structure_element
+as	O
+a	O
+stable	O
+anchor	O
+at	O
+the	O
+center	O
+of	O
+the	O
+double	B-structure_element
+-	I-structure_element
+ring	I-structure_element
+
+As	O
+a	O
+first	O
+step	O
+of	O
+a	O
+comparative	O
+analysis	O
+,	O
+we	O
+superimposed	B-experimental_method
+the	O
+three	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+(	O
+LdcIa	B-protein
+,	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+and	O
+LdcC	B-protein
+)	O
+and	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+LdcIi	B-protein
+decamer	B-oligomeric_state
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Movie	O
+S1	O
+).	O
+
+This	O
+superposition	B-experimental_method
+reveals	O
+that	O
+the	O
+densities	B-evidence
+lining	O
+the	O
+central	B-structure_element
+hole	I-structure_element
+of	O
+the	O
+toroid	B-structure_element
+are	O
+roughly	O
+at	O
+the	O
+same	O
+location	O
+,	O
+while	O
+the	O
+rest	O
+of	O
+the	O
+structure	B-evidence
+exhibits	O
+noticeable	O
+changes	O
+.	O
+
+Specifically	O
+,	O
+at	O
+the	O
+center	O
+of	O
+the	O
+double	B-structure_element
+-	I-structure_element
+ring	I-structure_element
+the	O
+wing	B-structure_element
+domains	I-structure_element
+of	O
+the	O
+subunits	O
+provide	O
+the	O
+conserved	B-protein_state
+basis	O
+for	O
+the	O
+assembly	O
+with	O
+the	O
+lowest	B-evidence
+root	I-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+(	O
+RMSD	B-evidence
+)	O
+(	O
+between	O
+1	O
+.	O
+4	O
+and	O
+2	O
+Å	O
+for	O
+the	O
+Cα	O
+atoms	O
+only	O
+),	O
+whereas	O
+the	O
+peripheral	O
+CTDs	B-structure_element
+containing	O
+the	O
+RavA	B-site
+binding	I-site
+interface	I-site
+manifest	O
+the	O
+highest	O
+RMSD	B-evidence
+(	O
+up	O
+to	O
+4	O
+.	O
+2	O
+Å	O
+)	O
+(	O
+Table	O
+S2	O
+).	O
+
+In	O
+addition	O
+,	O
+the	O
+wing	B-structure_element
+domains	I-structure_element
+of	O
+all	O
+structures	B-evidence
+are	O
+very	O
+similar	O
+,	O
+with	O
+the	O
+RMSD	B-evidence
+after	O
+optimal	O
+rigid	O
+body	O
+alignment	O
+(	O
+RMSDmin	B-evidence
+)	O
+less	O
+than	O
+1	O
+.	O
+1	O
+Å	O
+.	O
+Thus	O
+,	O
+taking	O
+the	O
+limited	O
+resolution	O
+of	O
+the	O
+cryoEM	B-experimental_method
+maps	B-evidence
+into	O
+account	O
+,	O
+we	O
+consider	O
+that	O
+the	O
+wing	B-structure_element
+domains	I-structure_element
+of	O
+all	O
+the	O
+four	O
+structures	B-evidence
+are	O
+essentially	O
+identical	O
+and	O
+that	O
+in	O
+the	O
+present	O
+study	O
+the	O
+RMSD	B-evidence
+of	O
+less	O
+than	O
+2	O
+Å	O
+can	O
+serve	O
+as	O
+a	O
+baseline	O
+below	O
+which	O
+differences	O
+may	O
+be	O
+assumed	O
+as	O
+insignificant	O
+.	O
+
+This	O
+preservation	O
+of	O
+the	O
+central	B-structure_element
+part	I-structure_element
+of	O
+the	O
+double	O
+-	O
+ring	O
+assembly	O
+may	O
+help	O
+the	O
+enzymes	O
+to	O
+maintain	O
+their	O
+decameric	B-oligomeric_state
+state	O
+upon	O
+activation	O
+and	O
+incorporation	O
+into	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+.	O
+
+The	O
+core	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+active	B-site
+site	I-site
+rearrangements	O
+upon	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+LARA	O
+binding	O
+
+Both	O
+visual	B-experimental_method
+inspection	I-experimental_method
+(	O
+Fig	O
+.	O
+2	O
+)	O
+and	O
+RMSD	B-experimental_method
+calculations	I-experimental_method
+(	O
+Table	O
+S2	O
+)	O
+show	O
+that	O
+globally	O
+the	O
+three	O
+structures	B-evidence
+at	O
+active	B-protein_state
+pH	I-protein_state
+(	O
+LdcIa	B-protein
+,	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+and	O
+LdcC	B-protein
+)	O
+are	O
+more	O
+similar	O
+to	O
+each	O
+other	O
+than	O
+to	O
+the	O
+structure	O
+determined	O
+at	O
+high	B-protein_state
+pH	I-protein_state
+conditions	O
+(	O
+LdcIi	B-protein
+).	O
+
+The	O
+decameric	B-oligomeric_state
+enzyme	O
+is	O
+built	O
+of	O
+five	O
+dimers	B-oligomeric_state
+associating	O
+into	O
+a	O
+5	B-structure_element
+-	I-structure_element
+fold	I-structure_element
+symmetrical	I-structure_element
+double	I-structure_element
+-	I-structure_element
+ring	I-structure_element
+(	O
+two	O
+monomers	B-oligomeric_state
+making	O
+a	O
+dimer	B-oligomeric_state
+are	O
+delineated	O
+in	O
+Fig	O
+.	O
+1	O
+).	O
+
+As	O
+common	O
+for	O
+the	O
+α	B-protein_type
+family	I-protein_type
+of	O
+the	O
+PLP	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+decarboxylases	I-protein_type
+,	O
+dimerization	O
+is	O
+required	O
+for	O
+the	O
+enzymatic	O
+activity	O
+because	O
+the	O
+active	B-site
+site	I-site
+is	O
+buried	O
+in	O
+the	O
+dimer	B-site
+interface	I-site
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+
+This	O
+interface	B-site
+is	O
+formed	O
+essentially	O
+by	O
+the	O
+core	B-structure_element
+domains	I-structure_element
+with	O
+some	O
+contribution	O
+of	O
+the	O
+CTDs	B-structure_element
+.	O
+
+The	O
+core	B-structure_element
+domain	I-structure_element
+is	O
+built	O
+by	O
+the	O
+PLP	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+subdomain	I-structure_element
+(	O
+PLP	B-structure_element
+-	I-structure_element
+SD	I-structure_element
+,	O
+residues	O
+184	B-residue_range
+–	I-residue_range
+417	I-residue_range
+)	O
+flanked	O
+by	O
+two	O
+smaller	O
+subdomains	B-structure_element
+rich	O
+in	O
+partly	B-protein_state
+disordered	I-protein_state
+loops	B-structure_element
+–	O
+the	O
+linker	B-structure_element
+region	I-structure_element
+(	O
+residues	O
+130	B-residue_range
+–	I-residue_range
+183	I-residue_range
+)	O
+and	O
+the	O
+subdomain	B-structure_element
+4	I-structure_element
+(	O
+residues	O
+418	B-residue_range
+–	I-residue_range
+563	I-residue_range
+).	O
+
+Zooming	O
+in	O
+the	O
+variations	O
+in	O
+the	O
+PLP	B-structure_element
+-	I-structure_element
+SD	I-structure_element
+shows	O
+that	O
+most	O
+of	O
+the	O
+structural	O
+changes	O
+concern	O
+displacements	O
+in	O
+the	O
+active	B-site
+site	I-site
+(	O
+Fig	O
+.	O
+3C	O
+–	O
+F	O
+).	O
+
+The	O
+most	O
+conspicuous	O
+differences	O
+between	O
+the	O
+PLP	B-structure_element
+-	I-structure_element
+SDs	I-structure_element
+can	O
+be	O
+linked	O
+to	O
+the	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+activation	O
+of	O
+the	O
+enzymes	O
+.	O
+
+The	O
+resolution	O
+of	O
+the	O
+cryoEM	B-experimental_method
+maps	B-evidence
+does	O
+not	O
+allow	O
+modeling	O
+the	O
+position	O
+of	O
+the	O
+PLP	B-chemical
+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	B-chemical
+acids	I-chemical
+.	O
+
+In	O
+particular	O
+,	O
+transition	O
+from	O
+LdcIi	B-protein
+to	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+involves	O
+~	O
+3	O
+.	O
+5	O
+Å	O
+and	O
+~	O
+4	O
+.	O
+5	O
+Å	O
+shifts	O
+away	O
+from	O
+the	O
+5	O
+-	O
+fold	O
+axis	O
+in	O
+the	O
+active	B-site
+site	I-site
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+spanning	O
+residues	O
+218	B-residue_range
+–	I-residue_range
+232	I-residue_range
+and	O
+246	B-residue_range
+–	I-residue_range
+254	I-residue_range
+respectively	O
+(	O
+Fig	O
+.	O
+3C	O
+–	O
+E	O
+).	O
+
+Between	O
+these	O
+two	O
+extremes	O
+,	O
+the	O
+PLP	B-structure_element
+-	I-structure_element
+SDs	I-structure_element
+of	O
+LdcIa	B-protein
+and	O
+LdcC	B-protein
+are	O
+similar	O
+both	O
+in	O
+the	O
+context	O
+of	O
+the	O
+decamer	B-oligomeric_state
+(	O
+Fig	O
+.	O
+3F	O
+)	O
+and	O
+in	O
+terms	O
+of	O
+RMSDmin	B-evidence
+=	O
+0	O
+.	O
+9	O
+Å	O
+,	O
+which	O
+probably	O
+reflects	O
+the	O
+fact	O
+that	O
+,	O
+at	O
+the	O
+optimal	B-protein_state
+pH	I-protein_state
+,	O
+these	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+have	O
+a	O
+similar	O
+enzymatic	O
+activity	O
+.	O
+
+In	O
+addition	O
+,	O
+our	O
+earlier	O
+biochemical	B-experimental_method
+observation	I-experimental_method
+that	O
+the	O
+enzymatic	O
+activity	O
+of	O
+LdcIa	B-protein
+is	O
+unaffected	O
+by	O
+RavA	B-protein
+binding	O
+is	O
+consistent	O
+with	O
+the	O
+relatively	O
+small	O
+changes	O
+undergone	O
+by	O
+the	O
+active	B-site
+site	I-site
+upon	O
+transition	O
+from	O
+LdcIa	B-protein
+to	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+.	O
+
+Worthy	O
+of	O
+note	O
+,	O
+our	O
+previous	O
+comparison	O
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+LdcIi	B-protein
+with	O
+that	O
+of	O
+the	O
+inducible	B-protein_state
+arginine	B-protein_type
+decarboxylase	I-protein_type
+AdiA	B-protein
+revealed	O
+high	B-protein_state
+conservation	I-protein_state
+of	O
+the	O
+PLP	B-site
+-	I-site
+coordinating	I-site
+residues	I-site
+and	O
+identified	O
+a	O
+patch	B-site
+of	I-site
+negatively	I-site
+charged	I-site
+residues	I-site
+lining	O
+the	O
+active	B-site
+site	I-site
+channel	I-site
+as	O
+a	O
+potential	O
+binding	B-site
+site	I-site
+for	O
+the	O
+target	O
+amino	B-chemical
+acid	I-chemical
+substrate	O
+(	O
+Figs	O
+S3	O
+and	O
+S4	O
+in	O
+ref	O
+.).	O
+
+Rearrangements	O
+of	O
+the	O
+ppGpp	B-site
+binding	I-site
+pocket	I-site
+upon	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+LARA	B-structure_element
+binding	O
+
+An	O
+inhibitor	O
+of	O
+the	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+activity	O
+,	O
+the	O
+stringent	B-chemical
+response	I-chemical
+alarmone	I-chemical
+ppGpp	B-chemical
+,	O
+is	O
+known	O
+to	O
+bind	O
+at	O
+the	O
+interface	B-site
+between	O
+neighboring	O
+monomers	B-oligomeric_state
+within	O
+each	O
+ring	B-structure_element
+(	O
+Fig	O
+.	O
+S4	O
+).	O
+
+The	O
+ppGpp	B-site
+binding	I-site
+pocket	I-site
+is	O
+made	O
+up	O
+by	O
+residues	O
+from	O
+all	O
+domains	O
+and	O
+is	O
+located	O
+approximately	O
+30	O
+Å	O
+away	O
+from	O
+the	O
+PLP	B-chemical
+moiety	O
+.	O
+
+Whereas	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+ppGpp	B-complex_assembly
+-	I-complex_assembly
+LdcIi	I-complex_assembly
+was	O
+solved	B-experimental_method
+to	O
+2	O
+Å	O
+resolution	O
+,	O
+only	O
+a	O
+4	O
+.	O
+1	O
+Å	O
+resolution	O
+structure	B-evidence
+of	O
+the	O
+ppGpp	B-protein_state
+-	I-protein_state
+free	I-protein_state
+LdcIi	B-protein
+could	O
+be	O
+obtained	O
+.	O
+
+At	O
+this	O
+resolution	O
+,	O
+the	O
+apo	B-protein_state
+-	O
+LdcIi	B-protein
+and	O
+ppGpp	B-complex_assembly
+-	I-complex_assembly
+LdcIi	I-complex_assembly
+structures	B-evidence
+(	O
+both	O
+solved	O
+at	O
+pH	B-protein_state
+8	I-protein_state
+.	I-protein_state
+5	I-protein_state
+)	O
+appeared	O
+indistinguishable	O
+except	O
+for	O
+the	O
+presence	O
+of	O
+ppGpp	B-chemical
+(	O
+Fig	O
+.	O
+S11	O
+in	O
+ref	O
+.	O
+).	O
+
+Thus	O
+,	O
+we	O
+speculated	O
+that	O
+inhibition	O
+of	O
+LdcI	B-protein
+by	O
+ppGpp	B-chemical
+would	O
+be	O
+accompanied	O
+by	O
+a	O
+transduction	O
+of	O
+subtle	O
+structural	O
+changes	O
+at	O
+the	O
+level	O
+of	O
+individual	O
+amino	B-chemical
+acid	I-chemical
+side	O
+chains	O
+between	O
+the	O
+ppGpp	B-site
+binding	I-site
+pocket	I-site
+and	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+enzyme	O
+.	O
+
+All	O
+our	O
+current	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+of	O
+the	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+were	O
+obtained	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ppGpp	B-chemical
+in	O
+order	O
+to	O
+be	O
+closer	O
+to	O
+the	O
+active	B-protein_state
+state	O
+of	O
+the	O
+enzymes	O
+under	O
+study	O
+.	O
+
+While	O
+differences	O
+in	O
+the	O
+ppGpp	B-site
+binding	I-site
+site	I-site
+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
+
+The	O
+fact	O
+that	O
+interaction	O
+with	O
+RavA	B-protein
+reduces	O
+the	O
+ppGpp	B-chemical
+affinity	O
+for	O
+LdcI	B-protein
+despite	O
+the	O
+long	O
+distance	O
+of	O
+~	O
+30	O
+Å	O
+between	O
+the	O
+LARA	B-site
+domain	I-site
+binding	I-site
+site	I-site
+and	O
+the	O
+closest	O
+ppGpp	B-site
+binding	I-site
+pocket	I-site
+(	O
+Fig	O
+.	O
+S5	O
+)	O
+seems	O
+to	O
+favor	O
+an	O
+allosteric	O
+regulation	O
+mechanism	O
+.	O
+
+Interestingly	O
+,	O
+although	O
+a	O
+number	O
+of	O
+ppGpp	B-site
+binding	I-site
+residues	I-site
+are	O
+strictly	B-protein_state
+conserved	I-protein_state
+between	O
+LdcI	B-protein
+and	O
+AdiA	B-protein
+that	O
+also	O
+forms	O
+decamers	B-oligomeric_state
+at	O
+low	B-protein_state
+pH	I-protein_state
+optimal	I-protein_state
+for	O
+its	O
+arginine	B-protein_type
+decarboxylase	I-protein_type
+activity	O
+,	O
+no	O
+ppGpp	B-chemical
+regulation	O
+of	O
+AdiA	B-protein
+could	O
+be	O
+demonstrated	O
+.	O
+
+Swinging	O
+and	O
+stretching	O
+of	O
+the	O
+CTDs	B-structure_element
+upon	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+LdcI	B-protein
+activation	O
+and	O
+LARA	B-structure_element
+binding	O
+
+Inspection	O
+of	O
+the	O
+superimposed	B-experimental_method
+decameric	B-oligomeric_state
+structures	B-evidence
+(	O
+Figs	O
+2	O
+and	O
+S6	O
+)	O
+suggests	O
+a	O
+depiction	O
+of	O
+the	O
+wing	B-structure_element
+domains	I-structure_element
+as	O
+an	O
+anchor	O
+around	O
+which	O
+the	O
+peripheral	O
+CTDs	B-structure_element
+swing	O
+.	O
+
+This	O
+swinging	O
+movement	O
+seems	O
+to	O
+be	O
+mediated	O
+by	O
+the	O
+core	B-structure_element
+domains	I-structure_element
+and	O
+is	O
+accompanied	O
+by	O
+a	O
+stretching	O
+of	O
+the	O
+whole	O
+LdcI	B-protein
+subunits	B-structure_element
+attracted	O
+by	O
+the	O
+RavA	B-protein
+magnets	O
+.	O
+
+Indeed	O
+,	O
+all	O
+CTDs	B-structure_element
+have	O
+very	O
+similar	O
+structures	O
+(	O
+RMSDmin	B-evidence
+<	O
+1	O
+Å	O
+).	O
+
+Yet	O
+the	O
+superposition	B-experimental_method
+of	O
+the	O
+decamers	B-oligomeric_state
+lays	O
+bare	O
+a	O
+progressive	O
+movement	O
+of	O
+the	O
+CTD	B-structure_element
+as	O
+a	O
+whole	O
+upon	O
+enzyme	O
+activation	O
+by	O
+pH	O
+and	O
+the	O
+binding	O
+of	O
+LARA	B-structure_element
+.	O
+
+The	O
+LdcIi	B-protein
+monomer	B-oligomeric_state
+is	O
+the	O
+most	B-protein_state
+compact	I-protein_state
+,	O
+whereas	O
+LdcIa	B-protein
+and	O
+especially	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+gradually	B-protein_state
+extend	I-protein_state
+their	O
+CTDs	B-structure_element
+towards	O
+the	O
+LARA	B-structure_element
+domain	I-structure_element
+of	O
+RavA	B-protein
+(	O
+Figs	O
+2	O
+and	O
+4	O
+).	O
+
+These	O
+small	O
+but	O
+noticeable	O
+swinging	O
+and	O
+stretching	O
+(	O
+up	O
+to	O
+~	O
+4	O
+Å	O
+)	O
+may	O
+be	O
+related	O
+to	O
+the	O
+incorporation	O
+of	O
+the	O
+LdcI	B-protein
+decamer	B-oligomeric_state
+into	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+.	O
+
+The	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+a	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+as	O
+a	O
+major	O
+determinant	O
+of	O
+the	O
+interaction	O
+with	O
+RavA	B-protein
+
+In	O
+our	O
+previous	O
+contribution	O
+,	O
+based	O
+on	O
+the	O
+fit	O
+of	O
+the	O
+LdcIi	B-protein
+and	O
+the	O
+LARA	B-structure_element
+crystal	B-evidence
+structures	I-evidence
+into	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+cryoEM	B-experimental_method
+density	B-evidence
+,	O
+we	O
+predicted	O
+that	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+interaction	O
+should	O
+involve	O
+the	O
+C	O
+-	O
+terminal	O
+two	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+the	O
+LdcI	B-protein
+.	O
+Our	O
+present	O
+cryoEM	B-experimental_method
+maps	B-evidence
+and	O
+pseudoatomic	B-evidence
+models	I-evidence
+provide	O
+first	O
+structure	O
+-	O
+based	O
+insights	O
+into	O
+the	O
+differences	O
+between	O
+the	O
+inducible	B-protein_state
+and	O
+the	O
+constitutive	B-protein_state
+lysine	B-protein_type
+decarboxylases	I-protein_type
+.	O
+
+Therefore	O
+,	O
+we	O
+wanted	O
+to	O
+check	O
+the	O
+influence	O
+of	O
+the	O
+primary	O
+sequence	O
+of	O
+the	O
+two	O
+proteins	O
+in	O
+this	O
+region	O
+on	O
+their	O
+ability	O
+to	O
+interact	O
+with	O
+RavA	B-protein
+.	O
+To	O
+this	O
+end	O
+,	O
+we	O
+swapped	B-experimental_method
+the	O
+relevant	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+of	O
+the	O
+two	O
+proteins	O
+and	O
+produced	O
+their	O
+chimeras	B-mutant
+,	O
+namely	O
+LdcIC	B-mutant
+(	O
+i	O
+.	O
+e	O
+.	O
+LdcI	B-protein
+with	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+LdcC	B-protein
+)	O
+and	O
+LdcCI	B-mutant
+(	O
+i	O
+.	O
+e	O
+.	O
+LdcC	B-protein
+with	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+LdcI	B-protein
+)	O
+(	O
+Fig	O
+.	O
+5A	O
+–	O
+C	O
+).	O
+
+Both	B-mutant
+constructs	I-mutant
+could	O
+be	O
+purified	O
+and	O
+could	O
+form	O
+decamers	B-oligomeric_state
+visually	O
+indistinguishable	O
+from	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+proteins	O
+.	O
+
+As	O
+expected	O
+,	O
+binding	O
+of	O
+LdcI	B-protein
+to	O
+RavA	B-protein
+was	O
+completely	O
+abolished	O
+by	O
+this	O
+procedure	O
+and	O
+no	O
+LdcIC	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+complex	O
+could	O
+be	O
+detected	O
+.	O
+
+On	O
+the	O
+contrary	O
+,	O
+introduction	B-experimental_method
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+LdcI	B-protein
+into	O
+LdcC	B-protein
+led	O
+to	O
+an	O
+assembly	O
+of	O
+the	O
+LdcCI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+complex	O
+.	O
+
+On	O
+the	O
+negative	B-experimental_method
+stain	I-experimental_method
+EM	I-experimental_method
+grid	I-experimental_method
+,	O
+the	O
+chimeric	B-protein_state
+cages	O
+appeared	O
+less	O
+rigid	O
+than	O
+the	O
+native	B-protein_state
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+,	O
+which	O
+probably	O
+means	O
+that	O
+the	O
+environment	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+contributes	O
+to	O
+the	O
+efficiency	O
+of	O
+the	O
+interaction	O
+and	O
+the	O
+stability	O
+of	O
+the	O
+entire	O
+architecture	O
+(	O
+Fig	O
+.	O
+5D	O
+–	O
+F	O
+).	O
+
+The	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+a	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+is	O
+a	O
+highly	B-protein_state
+conserved	I-protein_state
+signature	O
+allowing	O
+to	O
+distinguish	O
+between	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+
+Alignment	B-experimental_method
+of	I-experimental_method
+the	I-experimental_method
+primary	I-experimental_method
+sequences	I-experimental_method
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+LdcI	B-protein
+and	O
+LdcC	B-protein
+shows	O
+that	O
+some	O
+amino	O
+acid	O
+residues	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+are	O
+the	O
+same	O
+in	O
+the	O
+two	O
+proteins	O
+,	O
+whereas	O
+others	O
+are	O
+notably	O
+different	O
+in	O
+chemical	O
+nature	O
+.	O
+
+Importantly	O
+,	O
+most	O
+of	O
+the	O
+amino	O
+acid	O
+differences	O
+between	O
+the	O
+two	O
+enzymes	O
+are	O
+located	O
+in	O
+this	O
+very	B-structure_element
+region	I-structure_element
+.	O
+
+Thus	O
+,	O
+to	O
+advance	O
+beyond	O
+our	O
+experimental	O
+confirmation	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+as	O
+a	O
+major	O
+determinant	O
+of	O
+the	O
+capacity	O
+of	O
+a	O
+particular	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+to	O
+form	O
+a	O
+cage	O
+with	O
+RavA	B-protein
+,	O
+we	O
+set	O
+out	O
+to	O
+investigate	O
+whether	O
+certain	B-structure_element
+residues	I-structure_element
+in	O
+this	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+are	O
+conserved	B-protein_state
+in	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+of	O
+different	O
+enterobacteria	B-taxonomy_domain
+that	O
+have	O
+the	O
+ravA	B-gene
+-	I-gene
+viaA	I-gene
+operon	I-gene
+in	O
+their	O
+genome	O
+.	O
+
+We	O
+inspected	B-experimental_method
+the	I-experimental_method
+genetic	I-experimental_method
+environment	I-experimental_method
+of	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+from	O
+22	O
+enterobacterial	B-taxonomy_domain
+species	O
+referenced	O
+in	O
+the	O
+NCBI	O
+database	O
+,	O
+corrected	O
+the	O
+gene	O
+annotation	O
+where	O
+necessary	O
+(	O
+Tables	O
+S3	O
+and	O
+S4	O
+),	O
+and	O
+performed	O
+multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+coupled	O
+to	O
+a	O
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+(	O
+see	O
+Methods	O
+).	O
+
+First	O
+of	O
+all	O
+,	O
+consensus	B-evidence
+sequence	I-evidence
+for	O
+the	O
+entire	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+family	O
+was	O
+derived	O
+.	O
+
+Second	O
+,	O
+the	O
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+clearly	O
+split	O
+the	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+into	O
+two	O
+groups	O
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+
+All	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+predicted	O
+to	O
+be	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+or	O
+biodegradable	B-protein_state
+based	O
+on	O
+their	O
+genetic	O
+environment	O
+,	O
+as	O
+for	O
+example	O
+their	O
+organization	O
+in	O
+an	O
+operon	O
+with	O
+a	O
+gene	O
+encoding	O
+the	O
+CadB	B-protein
+antiporter	B-protein_type
+(	O
+see	O
+Methods	O
+),	O
+were	O
+found	O
+in	O
+one	O
+group	O
+,	O
+whereas	O
+all	O
+enzymes	B-protein_type
+predicted	O
+as	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+or	O
+biosynthetic	B-protein_state
+partitioned	O
+into	O
+another	O
+group	O
+.	O
+
+Thus	O
+,	O
+consensus	B-evidence
+sequences	I-evidence
+could	O
+also	O
+be	O
+determined	O
+for	O
+each	O
+of	O
+the	O
+two	O
+groups	O
+(	O
+Figs	O
+6B	O
+,	O
+C	O
+and	O
+S7	O
+).	O
+
+Inspection	O
+of	O
+these	O
+consensus	B-evidence
+sequences	I-evidence
+revealed	O
+important	O
+differences	O
+between	O
+the	O
+groups	O
+regarding	O
+charge	O
+,	O
+size	O
+and	O
+hydrophobicity	O
+of	O
+several	O
+residues	O
+precisely	O
+at	O
+the	O
+level	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+that	O
+is	O
+responsible	O
+for	O
+the	O
+interaction	O
+with	O
+RavA	B-protein
+(	O
+Fig	O
+.	O
+6B	O
+–	O
+D	O
+).	O
+
+For	O
+example	O
+,	O
+in	O
+our	O
+previous	O
+study	O
+,	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutations	I-experimental_method
+identified	O
+Y697	B-residue_name_number
+as	O
+critically	O
+required	O
+for	O
+the	O
+RavA	B-protein
+binding	O
+.	O
+
+Our	O
+current	O
+analysis	O
+shows	O
+that	O
+Y697	B-residue_name_number
+is	O
+strictly	B-protein_state
+conserved	I-protein_state
+in	O
+the	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+group	O
+whereas	O
+the	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+enzymes	O
+always	B-protein_state
+have	I-protein_state
+a	O
+lysine	B-residue_name
+in	O
+this	O
+position	O
+;	O
+it	O
+also	O
+uncovers	O
+several	O
+other	O
+residues	O
+potentially	O
+essential	O
+for	O
+the	O
+interaction	O
+with	O
+RavA	B-protein
+which	O
+can	O
+now	O
+be	O
+addressed	O
+by	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+.	O
+
+The	O
+third	O
+and	O
+most	O
+remarkable	O
+finding	O
+was	O
+that	O
+exactly	O
+the	O
+same	O
+separation	O
+into	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+and	O
+“	O
+LdcC	B-protein_type
+”-	I-protein_type
+like	I-protein_type
+groups	O
+can	O
+be	O
+obtained	O
+based	O
+on	O
+a	O
+comparison	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+only	O
+,	O
+without	O
+taking	O
+the	O
+rest	O
+of	O
+the	O
+primary	O
+sequence	O
+into	O
+account	O
+.	O
+
+Therefore	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+emerges	O
+as	O
+being	O
+a	O
+highly	B-protein_state
+conserved	I-protein_state
+signature	B-structure_element
+sequence	I-structure_element
+,	O
+sufficient	O
+to	O
+unambiguously	O
+discriminate	O
+between	O
+the	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+and	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+enterobacterial	B-taxonomy_domain
+lysine	B-protein_type
+decarboxylases	I-protein_type
+independently	O
+of	O
+any	O
+other	O
+information	O
+(	O
+Figs	O
+6	O
+and	O
+S7	O
+).	O
+
+Our	O
+structures	B-evidence
+show	O
+that	O
+this	B-structure_element
+motif	I-structure_element
+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
+
+Thus	O
+,	O
+enterobacteria	B-taxonomy_domain
+identified	O
+here	O
+(	O
+Fig	O
+.	O
+6	O
+,	O
+Table	O
+S4	O
+)	O
+appear	O
+to	O
+exert	O
+evolutionary	O
+pressure	O
+on	O
+the	O
+biodegradative	B-protein_state
+lysine	B-protein_type
+decarboxylase	I-protein_type
+towards	O
+the	O
+RavA	B-protein
+binding	O
+.	O
+
+One	O
+of	O
+the	O
+elucidated	O
+roles	O
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+is	O
+to	O
+maintain	O
+LdcI	B-protein
+activity	O
+under	O
+conditions	O
+of	O
+enterobacterial	B-taxonomy_domain
+starvation	O
+by	O
+preventing	O
+LdcI	B-protein
+inhibition	O
+by	O
+the	O
+stringent	B-chemical
+response	I-chemical
+alarmone	I-chemical
+ppGpp	B-chemical
+.	O
+
+Furthermore	O
+,	O
+the	O
+recently	O
+documented	O
+interaction	O
+of	O
+both	O
+LdcI	B-protein
+and	O
+RavA	B-protein
+with	O
+specific	O
+subunits	B-structure_element
+of	O
+the	O
+respiratory	B-protein_type
+complex	I-protein_type
+I	I-protein_type
+,	O
+together	O
+with	O
+the	O
+unanticipated	O
+link	O
+between	O
+RavA	B-protein
+and	O
+maturation	O
+of	O
+numerous	O
+iron	B-protein_type
+-	I-protein_type
+sulfur	I-protein_type
+proteins	I-protein_type
+,	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
+
+The	O
+conformational	O
+rearrangements	O
+of	O
+LdcI	B-protein
+upon	O
+enzyme	O
+activation	O
+and	O
+RavA	B-protein
+binding	O
+revealed	O
+in	O
+this	O
+work	O
+,	O
+and	O
+our	O
+amazing	O
+finding	O
+that	O
+the	O
+molecular	O
+determinant	O
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+interaction	O
+is	O
+the	O
+one	O
+that	O
+straightforwardly	O
+determines	O
+if	O
+a	O
+particular	O
+enterobacterial	B-taxonomy_domain
+lysine	B-protein_type
+decarboxylase	I-protein_type
+belongs	O
+to	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+or	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+proteins	O
+,	O
+should	O
+give	O
+a	O
+new	O
+impetus	O
+to	O
+functional	O
+studies	O
+of	O
+the	O
+unique	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+.	O
+
+Besides	O
+,	O
+the	O
+structures	B-evidence
+and	O
+the	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+the	O
+active	B-protein_state
+ppGpp	B-protein_state
+-	I-protein_state
+free	I-protein_state
+states	O
+of	O
+both	O
+the	O
+biodegradative	B-protein_state
+and	O
+the	O
+biosynthetic	B-protein_state
+E	B-species
+.	I-species
+coli	I-species
+lysine	B-protein_type
+decarboxylases	I-protein_type
+offer	O
+an	O
+additional	O
+tool	O
+for	O
+analysis	O
+of	O
+their	O
+role	O
+in	O
+UPEC	B-species
+infectivity	O
+.	O
+
+Together	O
+with	O
+the	O
+apo	B-protein_state
+-	O
+LdcI	B-protein
+and	O
+ppGpp	B-complex_assembly
+-	I-complex_assembly
+LdcIi	I-complex_assembly
+crystal	B-evidence
+structures	I-evidence
+,	O
+our	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+provide	O
+a	O
+structural	O
+framework	O
+for	O
+future	O
+studies	O
+of	O
+structure	O
+-	O
+function	O
+relationships	O
+of	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+from	O
+other	O
+enterobacteria	B-taxonomy_domain
+and	O
+even	O
+of	O
+their	O
+homologues	O
+outside	O
+Enterobacteriaceae	B-taxonomy_domain
+.	O
+For	O
+example	O
+,	O
+the	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+of	O
+Eikenella	B-species
+corrodens	I-species
+is	O
+thought	O
+to	O
+play	O
+a	O
+major	O
+role	O
+in	O
+the	O
+periodontal	O
+disease	O
+and	O
+its	O
+inhibitors	O
+were	O
+shown	O
+to	O
+retard	O
+gingivitis	O
+development	O
+.	O
+
+Finally	O
+,	O
+cadaverine	B-chemical
+being	O
+an	O
+important	O
+platform	O
+chemical	O
+for	O
+the	O
+production	O
+of	O
+industrial	O
+polymers	O
+such	O
+as	O
+nylon	O
+,	O
+structural	O
+information	O
+is	O
+valuable	O
+for	O
+optimisation	O
+of	O
+bacterial	B-taxonomy_domain
+lysine	B-protein_type
+decarboxylases	I-protein_type
+used	O
+for	O
+its	O
+production	O
+in	O
+biotechnology	O
+.	O
+
+3D	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+of	O
+LdcC	B-protein
+,	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+and	O
+LdcIa	B-protein
+.	O
+
+(	O
+A	O
+,	O
+C	O
+,	O
+E	O
+)	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcC	B-protein
+(	O
+A	O
+),	O
+LdcIa	B-protein
+(	O
+C	O
+)	O
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+(	O
+E	O
+)	O
+decamers	B-oligomeric_state
+with	O
+one	O
+protomer	B-oligomeric_state
+in	O
+light	O
+grey	O
+.	O
+
+In	O
+the	O
+rest	O
+of	O
+the	O
+protomers	B-oligomeric_state
+,	O
+the	O
+wing	B-structure_element
+,	O
+core	B-structure_element
+and	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domains	I-structure_element
+are	O
+colored	O
+from	O
+light	O
+to	O
+dark	O
+in	O
+shades	O
+of	O
+green	O
+for	O
+LdcC	B-protein
+(	O
+A	O
+),	O
+pink	O
+for	O
+LdcIa	B-protein
+(	O
+C	O
+)	O
+and	O
+blue	O
+for	O
+LdcI	B-protein
+in	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+(	O
+E	O
+).	O
+
+In	O
+(	O
+E	O
+),	O
+the	O
+LARA	B-structure_element
+domain	I-structure_element
+density	O
+is	O
+shown	O
+in	O
+dark	O
+grey	O
+.	O
+
+Two	O
+monomers	B-oligomeric_state
+making	O
+a	O
+dimer	B-oligomeric_state
+are	O
+delineated	O
+.	O
+
+Scale	O
+bar	O
+50	O
+Å	O
+.	O
+(	O
+B	O
+,	O
+D	O
+,	O
+F	O
+)	O
+One	O
+protomer	B-oligomeric_state
+from	O
+the	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcC	B-protein
+(	O
+B	O
+),	O
+LdcIa	B-protein
+(	O
+D	O
+)	O
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+(	O
+F	O
+)	O
+in	O
+light	O
+grey	O
+with	O
+the	O
+pseudoatomic	B-evidence
+model	I-evidence
+represented	O
+as	O
+cartoons	O
+and	O
+colored	O
+as	O
+the	O
+densities	O
+in	O
+(	O
+A	O
+,	O
+C	O
+,	O
+E	O
+).	O
+
+Superposition	B-experimental_method
+of	O
+the	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+LdcC	B-protein
+,	O
+LdcI	B-protein
+from	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+and	O
+LdcIa	B-protein
+colored	O
+as	O
+in	O
+Fig	O
+.	O
+1	O
+,	O
+and	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+LdcIi	B-protein
+in	O
+shades	O
+of	O
+yellow	O
+.	O
+
+Only	O
+one	O
+of	O
+the	O
+two	O
+rings	B-structure_element
+of	O
+the	O
+double	B-structure_element
+toroid	I-structure_element
+is	O
+shown	O
+for	O
+clarity	O
+.	O
+
+The	O
+dashed	O
+circle	O
+indicates	O
+the	O
+central	O
+region	B-structure_element
+that	O
+remains	O
+virtually	O
+unchanged	O
+between	O
+all	O
+the	O
+structures	B-evidence
+,	O
+while	O
+the	O
+periphery	O
+undergoes	O
+visible	O
+movements	O
+.	O
+
+Conformational	O
+rearrangements	O
+in	O
+the	O
+enzyme	O
+active	B-site
+site	I-site
+.	O
+
+(	O
+A	O
+)	O
+LdcIi	B-protein
+crystal	B-evidence
+structure	I-evidence
+,	O
+with	O
+one	O
+ring	B-structure_element
+represented	O
+as	O
+a	O
+grey	O
+surface	O
+and	O
+the	O
+second	O
+as	O
+a	O
+cartoon	O
+.	O
+
+A	O
+monomer	B-oligomeric_state
+with	O
+its	O
+PLP	B-chemical
+cofactor	O
+is	O
+delineated	O
+.	O
+
+The	O
+PLP	B-chemical
+moieties	O
+of	O
+the	O
+cartoon	O
+ring	B-structure_element
+are	O
+shown	O
+in	O
+red	O
+.	O
+
+(	O
+B	O
+)	O
+The	O
+LdcIi	B-protein
+dimer	B-oligomeric_state
+extracted	O
+from	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+decamer	B-oligomeric_state
+.	O
+
+One	O
+monomer	B-oligomeric_state
+is	O
+colored	O
+in	O
+shades	O
+of	O
+yellow	O
+as	O
+in	O
+Figs	O
+1	O
+and	O
+2	O
+,	O
+while	O
+the	O
+monomer	B-oligomeric_state
+related	O
+by	O
+C2	O
+symmetry	O
+is	O
+grey	O
+.	O
+
+The	O
+PLP	B-chemical
+is	O
+red	O
+.	O
+
+The	O
+active	B-site
+site	I-site
+is	O
+boxed	O
+.	O
+
+Stretching	O
+of	O
+the	O
+LdcI	B-protein
+monomer	B-oligomeric_state
+upon	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+LARA	B-structure_element
+binding	O
+.	O
+
+(	O
+A	O
+–	O
+C	O
+)	O
+A	O
+slice	O
+through	O
+the	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+the	O
+LdcI	B-protein
+monomers	B-oligomeric_state
+extracted	O
+from	O
+the	O
+superimposed	B-experimental_method
+decamers	B-oligomeric_state
+(	O
+Fig	O
+.	O
+2	O
+)	O
+The	O
+rectangle	O
+indicates	O
+the	O
+regions	O
+enlarged	O
+in	O
+(	O
+D	O
+–	O
+F	O
+).	O
+
+(	O
+A	O
+)	O
+compares	O
+LdcIi	B-protein
+(	O
+yellow	O
+)	O
+and	O
+LdcIa	B-protein
+(	O
+pink	O
+),	O
+(	O
+B	O
+)	O
+compares	O
+LdcIa	B-protein
+(	O
+pink	O
+)	O
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+(	O
+blue	O
+),	O
+and	O
+(	O
+C	O
+)	O
+compares	O
+LdcIi	B-protein
+(	O
+yellow	O
+),	O
+LdcIa	B-protein
+(	O
+pink	O
+)	O
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+(	O
+blue	O
+)	O
+simultaneously	O
+in	O
+order	O
+to	O
+show	O
+the	O
+progressive	O
+stretching	O
+described	O
+in	O
+the	O
+text	O
+.	O
+
+The	O
+cryoEM	B-experimental_method
+density	B-evidence
+of	O
+the	O
+LARA	B-structure_element
+domain	I-structure_element
+is	O
+represented	O
+as	O
+a	O
+grey	O
+surface	O
+to	O
+show	O
+the	O
+position	O
+of	O
+the	O
+binding	B-site
+site	I-site
+and	O
+the	O
+direction	O
+of	O
+the	O
+movement	O
+.	O
+
+(	O
+D	O
+–	O
+F	O
+)	O
+Inserts	O
+zooming	O
+at	O
+the	O
+CTD	B-structure_element
+part	O
+in	O
+proximity	O
+of	O
+the	O
+LARA	B-site
+binding	I-site
+site	I-site
+.	O
+
+Analysis	O
+of	O
+the	O
+LdcIC	B-mutant
+and	O
+LdcCI	B-mutant
+chimeras	B-mutant
+.	O
+
+(	O
+A	O
+)	O
+A	O
+slice	O
+through	O
+the	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+the	O
+LdcIa	B-protein
+(	O
+purple	O
+)	O
+and	O
+LdcC	B-protein
+(	O
+green	O
+)	O
+monomers	B-oligomeric_state
+extracted	O
+from	O
+the	O
+superimposed	B-experimental_method
+decamers	B-oligomeric_state
+(	O
+Fig	O
+.	O
+2	O
+).	O
+(	O
+B	O
+)	O
+The	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+in	O
+LdcIa	B-protein
+and	O
+LdcC	B-protein
+enlarged	O
+from	O
+(	O
+A	O
+,	O
+C	O
+)	O
+Exchanged	O
+primary	O
+sequences	O
+(	O
+capital	O
+letters	O
+)	O
+and	O
+their	O
+immediate	O
+vicinity	O
+(	O
+lower	O
+case	O
+letters	O
+)	O
+colored	O
+as	O
+in	O
+(	O
+A	O
+,	O
+B	O
+),	O
+with	O
+the	O
+corresponding	O
+secondary	O
+structure	O
+elements	O
+and	O
+the	O
+amino	O
+acid	O
+numbering	O
+shown	O
+.	O
+
+(	O
+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	B-protein_state
+type	I-protein_state
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+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
+
+Sequence	B-experimental_method
+analysis	I-experimental_method
+of	O
+enterobacterial	B-taxonomy_domain
+lysine	B-protein_type
+decarboxylases	I-protein_type
+.	O
+
+(	O
+A	O
+)	O
+Maximum	B-evidence
+likelihood	I-evidence
+tree	I-evidence
+with	O
+the	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+and	O
+the	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+groups	O
+highlighted	O
+in	O
+green	O
+and	O
+pink	O
+,	O
+respectively	O
+.	O
+
+(	O
+B	O
+)	O
+Analysis	O
+of	O
+consensus	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+and	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+sequences	O
+around	O
+the	O
+first	O
+and	O
+second	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+.	O
+
+Numbering	O
+as	O
+in	O
+E	B-species
+.	I-species
+coli	I-species
+.	O
+
+(	O
+C	O
+)	O
+Signature	O
+sequences	O
+of	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+in	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+.	O
+
+Polarity	O
+differences	O
+are	O
+highlighted	O
+.	O
+(	O
+D	O
+)	O
+Position	O
+and	O
+nature	O
+of	O
+these	O
+differences	O
+at	O
+the	O
+surface	O
+of	O
+the	O
+respective	O
+cryoEM	B-experimental_method
+maps	B-evidence
+with	O
+the	O
+color	O
+code	O
+as	O
+in	O
+B	O
+.	O
+See	O
+also	O
+Fig	O
+.	O
+S7	O
+and	O
+Tables	O
+S3	O
+and	O
+S4	O
+.	O
+
+Crystal	B-evidence
+Structures	I-evidence
+of	O
+Putative	O
+Sugar	B-protein_type
+Kinases	I-protein_type
+from	O
+Synechococcus	B-species
+Elongatus	I-species
+PCC	I-species
+7942	I-species
+and	O
+Arabidopsis	B-species
+Thaliana	I-species
+
+The	O
+genome	O
+of	O
+the	O
+Synechococcus	B-species
+elongatus	I-species
+strain	I-species
+PCC	I-species
+7942	I-species
+encodes	O
+a	O
+putative	O
+sugar	B-protein_type
+kinase	I-protein_type
+(	O
+SePSK	B-protein
+),	O
+which	O
+shares	O
+44	O
+.	O
+9	O
+%	O
+sequence	O
+identity	O
+with	O
+the	O
+xylulose	B-protein
+kinase	I-protein
+-	I-protein
+1	I-protein
+(	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+)	O
+from	O
+Arabidopsis	B-species
+thaliana	I-species
+.	O
+
+Sequence	B-experimental_method
+alignment	I-experimental_method
+suggests	O
+that	O
+both	O
+kinases	B-protein_type
+belong	O
+to	O
+the	O
+ribulokinase	B-protein_type
+-	I-protein_type
+like	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+,	O
+a	O
+sub	O
+-	O
+family	O
+of	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+.	O
+
+Here	O
+we	O
+solved	B-experimental_method
+the	O
+structures	B-evidence
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+in	O
+both	O
+their	O
+apo	B-protein_state
+forms	O
+and	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+nucleotide	B-chemical
+substrates	O
+.	O
+
+The	O
+two	O
+kinases	O
+exhibit	O
+nearly	O
+identical	O
+overall	O
+architecture	O
+,	O
+with	O
+both	O
+kinases	B-protein_type
+possessing	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+substrates	I-protein_state
+.	O
+
+In	O
+addition	O
+,	O
+our	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+suggested	O
+that	O
+SePSK	B-protein
+has	O
+the	O
+capability	O
+to	O
+phosphorylate	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+.	O
+
+In	O
+order	O
+to	O
+understand	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	B-protein
+,	O
+we	O
+solved	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+SePSK	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+and	O
+found	O
+two	O
+potential	O
+substrate	B-site
+binding	I-site
+pockets	I-site
+in	O
+SePSK	B-protein
+.	O
+
+Using	O
+mutation	B-experimental_method
+and	I-experimental_method
+activity	I-experimental_method
+analysis	I-experimental_method
+,	O
+we	O
+further	O
+verified	O
+the	O
+key	O
+residues	O
+important	O
+for	O
+its	O
+catalytic	O
+activity	O
+.	O
+
+Moreover	O
+,	O
+our	O
+structural	B-experimental_method
+comparison	I-experimental_method
+with	O
+other	O
+family	O
+members	O
+suggests	O
+that	O
+there	O
+are	O
+major	O
+conformational	O
+changes	O
+in	O
+SePSK	B-protein
+upon	O
+substrate	O
+binding	O
+,	O
+facilitating	O
+the	O
+catalytic	O
+process	O
+.	O
+
+Together	O
+,	O
+these	O
+results	O
+provide	O
+important	O
+information	O
+for	O
+a	O
+more	O
+detailed	O
+understanding	O
+of	O
+the	O
+cofactor	O
+and	O
+substrate	O
+binding	O
+mode	O
+as	O
+well	O
+as	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	B-protein
+,	O
+and	O
+possible	O
+similarities	O
+with	O
+its	O
+plant	B-taxonomy_domain
+homologue	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+Carbohydrates	B-chemical
+are	O
+essential	O
+cellular	O
+compounds	O
+involved	O
+in	O
+the	O
+metabolic	O
+processes	O
+present	O
+in	O
+all	O
+organisms	O
+.	O
+
+Phosphorylation	B-ptm
+is	O
+one	O
+of	O
+the	O
+various	O
+pivotal	O
+modifications	O
+of	O
+carbohydrates	B-chemical
+,	O
+and	O
+is	O
+catalyzed	O
+by	O
+specific	O
+sugar	B-protein_type
+kinases	I-protein_type
+.	O
+
+These	O
+kinases	B-protein_type
+exhibit	O
+considerable	O
+differences	O
+in	O
+their	O
+folding	O
+pattern	O
+and	O
+substrate	O
+specificity	O
+.	O
+
+Based	O
+on	O
+sequence	B-experimental_method
+analysis	I-experimental_method
+,	O
+they	O
+can	O
+be	O
+divided	O
+into	O
+four	O
+families	O
+,	O
+namely	O
+HSP	B-protein_type
+70_NBD	I-protein_type
+family	I-protein_type
+,	O
+FGGY	B-protein_type
+family	I-protein_type
+,	O
+Mer_B	B-protein_type
+like	I-protein_type
+family	I-protein_type
+and	O
+Parm_like	B-protein_type
+family	I-protein_type
+.	O
+
+The	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+contain	O
+different	O
+types	O
+of	O
+sugar	B-protein_type
+kinases	I-protein_type
+,	O
+all	O
+of	O
+which	O
+possess	O
+different	O
+catalytic	O
+substrates	O
+with	O
+preferences	O
+for	O
+short	O
+-	O
+chained	O
+sugar	B-chemical
+substrates	O
+,	O
+ranging	O
+from	O
+triose	B-chemical
+to	O
+heptose	B-chemical
+.	O
+
+These	O
+sugar	B-chemical
+substrates	O
+include	O
+L	B-chemical
+-	I-chemical
+ribulose	I-chemical
+,	O
+erythritol	B-chemical
+,	O
+L	B-chemical
+-	I-chemical
+fuculose	I-chemical
+,	O
+D	B-chemical
+-	I-chemical
+glycerol	I-chemical
+,	O
+D	B-chemical
+-	I-chemical
+gluconate	I-chemical
+,	O
+L	B-chemical
+-	I-chemical
+xylulose	I-chemical
+,	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+,	O
+L	B-chemical
+-	I-chemical
+rhamnulose	I-chemical
+and	O
+D	B-chemical
+-	I-chemical
+xylulose	I-chemical
+.	O
+
+Structures	B-evidence
+reported	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+of	O
+the	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+exhibit	O
+a	O
+similar	O
+overall	O
+architecture	O
+containing	O
+two	O
+protein	O
+domains	O
+,	O
+one	O
+of	O
+which	O
+is	O
+responsible	O
+for	O
+the	O
+binding	O
+of	O
+substrate	O
+,	O
+while	O
+the	O
+second	O
+is	O
+used	O
+for	O
+binding	O
+cofactor	O
+ATP	B-chemical
+.	O
+
+While	O
+the	O
+binding	B-site
+pockets	I-site
+for	O
+substrates	O
+are	O
+at	O
+the	O
+same	O
+position	O
+,	O
+each	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+uses	O
+different	O
+substrate	B-site
+-	I-site
+binding	I-site
+residues	I-site
+,	O
+resulting	O
+in	O
+high	O
+substrate	O
+specificity	O
+.	O
+
+Synpcc7942_2462	B-gene
+from	O
+the	O
+cyanobacteria	B-taxonomy_domain
+Synechococcus	B-species
+elongatus	I-species
+PCC	I-species
+7942	I-species
+encodes	O
+a	O
+putative	O
+sugar	B-protein_type
+kinase	I-protein_type
+(	O
+SePSK	B-protein
+),	O
+and	O
+this	O
+kinase	B-protein_type
+contains	O
+426	B-residue_range
+amino	O
+acids	O
+.	O
+
+The	O
+At2g21370	B-gene
+gene	O
+product	O
+from	O
+Arabidopsis	B-species
+thaliana	I-species
+,	O
+xylulose	B-protein
+kinase	I-protein
+-	I-protein
+1	I-protein
+(	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+),	O
+whose	O
+mature	B-protein_state
+form	I-protein_state
+contains	O
+436	B-residue_range
+amino	O
+acids	O
+,	O
+is	O
+located	O
+in	O
+the	O
+chloroplast	O
+(	O
+ChloroP	O
+1	O
+.	O
+1	O
+Server	O
+).	O
+
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+display	O
+a	O
+sequence	O
+identity	O
+of	O
+44	O
+.	O
+9	O
+%,	O
+and	O
+belong	O
+to	O
+the	O
+ribulokinase	B-protein_type
+-	I-protein_type
+like	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+,	O
+a	O
+sub	O
+-	O
+family	O
+of	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+.	O
+
+Members	O
+of	O
+this	O
+sub	O
+-	O
+family	O
+are	O
+responsible	O
+for	O
+the	O
+phosphorylation	B-ptm
+of	O
+sugars	B-chemical
+similar	O
+to	O
+L	B-chemical
+-	I-chemical
+ribulose	I-chemical
+and	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+.	O
+
+The	O
+sequence	O
+and	O
+the	O
+substrate	O
+specificity	O
+of	O
+ribulokinase	B-protein_type
+-	I-protein_type
+like	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+are	O
+different	O
+,	O
+but	O
+they	O
+share	O
+the	O
+common	O
+folding	O
+feature	O
+with	O
+two	O
+domains	O
+.	O
+
+Domain	B-structure_element
+I	I-structure_element
+exhibits	O
+a	O
+ribonuclease	B-structure_element
+H	I-structure_element
+-	I-structure_element
+like	I-structure_element
+folding	I-structure_element
+pattern	I-structure_element
+,	O
+and	O
+is	O
+responsible	O
+for	O
+the	O
+substrate	O
+binding	O
+,	O
+while	O
+domain	B-structure_element
+II	I-structure_element
+possesses	O
+an	O
+actin	B-structure_element
+-	I-structure_element
+like	I-structure_element
+ATPase	I-structure_element
+domain	I-structure_element
+that	O
+binds	O
+cofactor	O
+ATP	B-chemical
+.	O
+
+Two	O
+possible	O
+xylulose	B-protein_type
+kinases	I-protein_type
+(	O
+xylulose	B-protein
+kinase	I-protein
+-	I-protein
+1	I-protein
+:	O
+XK	B-protein
+-	I-protein
+1	I-protein
+and	O
+xylulose	B-protein
+kinase	I-protein
+-	I-protein
+2	I-protein
+:	O
+XK	B-protein
+-	I-protein
+2	I-protein
+)	O
+from	O
+Arabidopsis	B-species
+thaliana	I-species
+were	O
+previously	O
+proposed	O
+.	O
+
+It	O
+was	O
+shown	O
+that	O
+XK	B-protein
+-	I-protein
+2	I-protein
+(	O
+At5g49650	B-gene
+)	O
+located	O
+in	O
+the	O
+cytosol	O
+is	O
+indeed	O
+xylulose	B-protein_type
+kinase	I-protein_type
+.	O
+
+However	O
+,	O
+the	O
+function	O
+of	O
+XK	B-protein
+-	I-protein
+1	I-protein
+(	O
+At2g21370	B-gene
+)	O
+inside	O
+the	O
+chloroplast	O
+stroma	O
+has	O
+remained	O
+unknown	O
+.	O
+
+SePSK	B-protein
+from	O
+Synechococcus	B-species
+elongatus	I-species
+strain	I-species
+PCC	I-species
+7942	I-species
+is	O
+the	O
+homolog	O
+of	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+,	O
+though	O
+its	O
+physiological	O
+function	O
+and	O
+substrates	O
+remain	O
+unclear	O
+.	O
+
+In	O
+order	O
+to	O
+obtain	O
+functional	O
+and	O
+structural	O
+information	O
+about	O
+these	O
+two	O
+proteins	O
+,	O
+here	O
+we	O
+reported	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+Our	O
+findings	O
+provide	O
+new	O
+details	O
+of	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	B-protein
+and	O
+lay	O
+the	O
+foundation	O
+for	O
+future	O
+studies	O
+into	O
+its	O
+homologs	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+Overall	O
+structures	B-evidence
+of	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+and	O
+apo	B-protein_state
+-	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+
+The	O
+attempt	O
+to	O
+solve	O
+the	O
+SePSK	B-protein
+structure	B-evidence
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+method	I-experimental_method
+failed	O
+with	O
+ribulokinase	B-protein
+from	O
+Bacillus	B-species
+halodurans	I-species
+(	O
+PDB	O
+code	O
+:	O
+3QDK	O
+,	O
+15	O
+.	O
+7	O
+%	O
+sequence	O
+identity	O
+)	O
+as	O
+an	O
+initial	O
+model	O
+.	O
+
+We	O
+therefore	O
+used	O
+single	B-experimental_method
+isomorphous	I-experimental_method
+replacement	I-experimental_method
+anomalous	I-experimental_method
+scattering	I-experimental_method
+method	I-experimental_method
+(	O
+SIRAS	B-experimental_method
+)	O
+for	O
+successful	O
+solution	O
+of	O
+the	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+structure	B-evidence
+at	O
+a	O
+resolution	O
+of	O
+2	O
+.	O
+3	O
+Å	O
+.	O
+Subsequently	O
+,	O
+the	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+structure	B-evidence
+was	O
+used	O
+as	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+model	I-experimental_method
+to	O
+solve	O
+all	O
+other	O
+structures	B-evidence
+identified	O
+in	O
+this	O
+study	O
+.	O
+
+Our	O
+structural	B-experimental_method
+analysis	I-experimental_method
+showed	O
+that	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+consists	O
+of	O
+one	O
+SePSK	B-protein
+protein	O
+molecule	O
+in	O
+an	O
+asymmetric	O
+unit	O
+.	O
+
+The	O
+amino	O
+-	O
+acid	O
+residues	O
+were	O
+traced	O
+from	O
+Val2	B-residue_name_number
+to	O
+His419	B-residue_name_number
+,	O
+except	O
+for	O
+the	O
+Met1	B-residue_name_number
+residue	O
+and	O
+the	O
+seven	O
+residues	O
+at	O
+the	O
+C	O
+-	O
+termini	O
+.	O
+
+Apo	B-protein_state
+-	O
+SePSK	B-protein
+contains	O
+two	O
+domains	O
+referred	O
+to	O
+further	O
+on	O
+as	O
+domain	B-structure_element
+I	I-structure_element
+and	O
+domain	B-structure_element
+II	I-structure_element
+(	O
+Fig	O
+1A	O
+).	O
+
+Domain	B-structure_element
+I	I-structure_element
+consists	O
+of	O
+non	O
+-	O
+contiguous	O
+portions	O
+of	O
+the	O
+polypeptide	O
+chains	O
+(	O
+aa	O
+.	O
+
+2	B-residue_range
+–	I-residue_range
+228	I-residue_range
+and	O
+aa	O
+.	O
+
+402	B-residue_range
+–	I-residue_range
+419	I-residue_range
+),	O
+exhibiting	O
+11	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+11	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+.	O
+
+Among	O
+all	O
+these	O
+structural	O
+elements	O
+,	O
+α4	B-structure_element
+/	O
+α5	B-structure_element
+/	O
+α11	B-structure_element
+/	O
+α18	B-structure_element
+,	O
+β3	B-structure_element
+/	O
+β2	B-structure_element
+/	O
+β1	B-structure_element
+/	O
+β6	B-structure_element
+/	O
+β19	B-structure_element
+/	O
+β20	B-structure_element
+/	O
+β17	B-structure_element
+and	O
+α21	B-structure_element
+/	O
+α32	B-structure_element
+form	O
+three	O
+patches	O
+,	O
+referred	O
+to	O
+as	O
+A1	B-structure_element
+,	O
+B1	B-structure_element
+and	O
+A2	B-structure_element
+,	O
+exhibiting	O
+the	O
+core	B-structure_element
+region	I-structure_element
+.	O
+
+In	O
+addition	O
+,	O
+four	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+(	O
+β7	B-structure_element
+,	O
+β10	B-structure_element
+,	O
+β12	B-structure_element
+and	O
+β16	B-structure_element
+)	O
+and	O
+five	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+α8	B-structure_element
+,	O
+α9	B-structure_element
+,	O
+α13	B-structure_element
+,	O
+α14	B-structure_element
+and	O
+α15	B-structure_element
+)	O
+flank	O
+the	O
+left	O
+side	O
+of	O
+the	O
+core	B-structure_element
+region	I-structure_element
+.	O
+
+Domain	B-structure_element
+II	I-structure_element
+is	O
+comprised	O
+of	O
+aa	O
+.	O
+
+229	B-residue_range
+–	I-residue_range
+401	I-residue_range
+and	O
+classified	O
+into	O
+B2	B-structure_element
+(	O
+β31	B-structure_element
+/	O
+β29	B-structure_element
+/	O
+β22	B-structure_element
+/	O
+β23	B-structure_element
+/	O
+β25	B-structure_element
+/	O
+β24	B-structure_element
+)	O
+and	O
+A3	B-structure_element
+(	O
+α26	B-structure_element
+/	O
+α27	B-structure_element
+/	O
+α28	B-structure_element
+/	O
+α30	B-structure_element
+)	O
+(	O
+Fig	O
+1A	O
+and	O
+S1	O
+Fig	O
+).	O
+
+In	O
+the	O
+SePSK	B-protein
+structure	B-evidence
+,	O
+B1	B-structure_element
+and	O
+B2	B-structure_element
+are	O
+sandwiched	O
+by	O
+A1	B-structure_element
+,	O
+A2	B-structure_element
+and	O
+A3	B-structure_element
+,	O
+and	O
+the	O
+whole	O
+structure	B-evidence
+shows	O
+the	O
+A1	B-structure_element
+/	O
+B1	B-structure_element
+/	O
+A2	B-structure_element
+/	O
+B2	B-structure_element
+/	O
+A3	B-structure_element
+(	O
+α	B-structure_element
+/	O
+β	B-structure_element
+/	O
+α	B-structure_element
+/	O
+β	B-structure_element
+/	O
+α	B-structure_element
+)	O
+folding	O
+pattern	O
+,	O
+which	O
+is	O
+in	O
+common	O
+with	O
+other	O
+members	O
+of	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+(	O
+S2	O
+Fig	O
+).	O
+
+The	O
+overall	O
+folding	O
+of	O
+SePSK	B-protein
+resembles	O
+a	O
+clip	O
+,	O
+with	O
+A2	B-structure_element
+of	O
+domain	B-structure_element
+I	I-structure_element
+acting	O
+as	O
+a	O
+hinge	B-structure_element
+region	I-structure_element
+.	O
+
+Overall	O
+structures	B-evidence
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+(	O
+A	O
+)	O
+Three	O
+-	O
+dimensional	O
+structure	B-evidence
+of	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+.	O
+
+The	O
+secondary	O
+structural	O
+elements	O
+are	O
+indicated	O
+(	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+:	O
+cyan	O
+,	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+:	O
+yellow	O
+).	O
+
+(	O
+B	O
+)	O
+Three	O
+-	O
+dimensional	O
+structure	B-evidence
+of	O
+apo	B-protein_state
+-	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+The	O
+secondary	O
+structural	O
+elements	O
+are	O
+indicated	O
+(	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+:	O
+green	O
+,	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+:	O
+wheat	O
+).	O
+
+Apo	B-protein_state
+-	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+exhibits	O
+a	O
+folding	O
+pattern	O
+similar	O
+to	O
+that	O
+of	O
+SePSK	B-protein
+in	O
+line	O
+with	O
+their	O
+high	O
+sequence	O
+identity	O
+(	O
+Fig	O
+1B	O
+and	O
+S1	O
+Fig	O
+).	O
+
+However	O
+,	O
+superposition	B-experimental_method
+of	O
+structures	B-evidence
+of	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+and	O
+SePSK	B-protein
+shows	O
+some	O
+differences	O
+,	O
+especially	O
+at	O
+the	O
+loop	B-structure_element
+regions	I-structure_element
+.	O
+
+A	O
+considerable	O
+difference	O
+is	O
+found	O
+in	O
+the	O
+loop3	B-structure_element
+linking	O
+β3	B-structure_element
+and	O
+α4	B-structure_element
+,	O
+which	O
+is	O
+stretched	O
+out	O
+in	O
+the	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+,	O
+while	O
+in	O
+the	O
+SePSK	B-protein
+structure	B-evidence
+,	O
+it	O
+is	O
+bent	O
+back	O
+towards	O
+the	O
+inner	O
+part	O
+.	O
+
+The	O
+corresponding	O
+residues	O
+between	O
+these	O
+two	O
+structures	B-evidence
+(	O
+SePSK	B-protein
+-	O
+Lys35	B-residue_name_number
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+-	O
+Lys48	B-residue_name_number
+)	O
+have	O
+a	O
+distance	O
+of	O
+15	O
+.	O
+4	O
+Å	O
+(	O
+S3	O
+Fig	O
+).	O
+
+Activity	B-experimental_method
+assays	I-experimental_method
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+
+In	O
+order	O
+to	O
+understand	O
+the	O
+function	O
+of	O
+these	O
+two	O
+kinases	O
+,	O
+we	O
+performed	O
+structural	B-experimental_method
+comparison	I-experimental_method
+using	O
+Dali	B-experimental_method
+server	I-experimental_method
+.	O
+
+The	O
+structures	B-evidence
+most	O
+closely	O
+related	O
+to	O
+SePSK	B-protein
+are	O
+xylulose	B-protein_type
+kinase	I-protein_type
+,	O
+glycerol	B-protein_type
+kinase	I-protein_type
+and	O
+ribulose	B-protein_type
+kinase	I-protein_type
+,	O
+implying	O
+that	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+might	O
+function	O
+similarly	O
+to	O
+these	O
+kinases	B-protein_type
+.	O
+
+We	O
+first	O
+tested	O
+whether	O
+both	O
+enzymes	O
+possessed	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+substrates	O
+.	O
+
+As	O
+shown	O
+in	O
+Fig	O
+2A	O
+,	O
+both	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+exhibited	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+.	O
+
+This	O
+finding	O
+is	O
+in	O
+agreement	O
+with	O
+a	O
+previous	O
+result	O
+showing	O
+that	O
+xylulose	B-protein_type
+kinase	I-protein_type
+(	O
+PDB	O
+code	O
+:	O
+2ITM	O
+)	O
+possessed	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+without	O
+adding	O
+substrate	O
+.	O
+
+To	O
+further	O
+identify	O
+the	O
+actual	O
+substrate	O
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+,	O
+five	O
+different	O
+sugar	O
+molecules	O
+,	O
+including	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+,	O
+L	B-chemical
+-	I-chemical
+ribulose	I-chemical
+,	O
+D	B-chemical
+-	I-chemical
+xylulose	I-chemical
+,	O
+L	B-chemical
+-	I-chemical
+xylulose	I-chemical
+and	O
+Glycerol	B-chemical
+,	O
+were	O
+used	O
+in	O
+enzymatic	B-experimental_method
+activity	I-experimental_method
+assays	I-experimental_method
+.	O
+
+As	O
+shown	O
+in	O
+Fig	O
+2B	O
+,	O
+the	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+of	O
+SePSK	B-protein
+greatly	O
+increased	O
+upon	O
+adding	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+than	O
+adding	O
+other	O
+potential	O
+substrates	O
+,	O
+suggesting	O
+that	O
+it	O
+has	O
+D	B-protein_type
+-	I-protein_type
+ribulose	I-protein_type
+kinase	I-protein_type
+activity	O
+.	O
+
+In	O
+contrary	O
+,	O
+limited	O
+increasing	O
+of	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+was	O
+detected	O
+for	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+upon	O
+addition	O
+of	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+(	O
+Fig	O
+2C	O
+),	O
+despite	O
+its	O
+structural	O
+similarity	O
+with	O
+SePSK	B-protein
+.	O
+
+The	O
+enzymatic	B-experimental_method
+activity	I-experimental_method
+assays	I-experimental_method
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+(	O
+A	O
+)	O
+The	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+Both	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+showed	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+substrate	O
+.	O
+
+While	O
+the	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+of	O
+SePSK	B-protein
+greatly	O
+increases	O
+upon	O
+addition	O
+of	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+(	O
+DR	B-chemical
+).	O
+
+(	O
+B	O
+)	O
+The	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+of	O
+SePSK	B-protein
+with	O
+addition	O
+of	O
+five	O
+different	O
+substrates	O
+.	O
+
+The	O
+substrates	O
+are	O
+DR	B-chemical
+(	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+),	O
+LR	B-chemical
+(	O
+L	B-chemical
+-	I-chemical
+ribulose	I-chemical
+),	O
+DX	B-chemical
+(	O
+D	B-chemical
+-	I-chemical
+xylulose	I-chemical
+),	O
+LX	B-chemical
+(	O
+L	B-chemical
+-	I-chemical
+xylulose	I-chemical
+)	O
+and	O
+GLY	B-chemical
+(	O
+Glycerol	B-chemical
+).	O
+(	O
+C	O
+)	O
+The	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+with	O
+or	O
+without	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+.	O
+(	O
+D	O
+)	O
+The	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+and	O
+single	O
+-	O
+site	O
+mutants	O
+of	O
+SePSK	B-protein
+.	O
+
+Three	O
+single	O
+-	O
+site	O
+mutants	O
+of	O
+SePSK	B-protein
+are	O
+D8A	B-mutant
+-	O
+SePSK	B-protein
+,	O
+T11A	B-mutant
+-	O
+SePSK	B-protein
+and	O
+D221A	B-mutant
+-	O
+SePSK	B-protein
+.	O
+
+The	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+measured	O
+via	O
+luminescent	B-experimental_method
+ADP	I-experimental_method
+-	I-experimental_method
+Glo	I-experimental_method
+assay	I-experimental_method
+(	O
+Promega	O
+).	O
+
+To	O
+understand	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+SePSK	B-protein
+,	O
+we	O
+performed	O
+structural	B-experimental_method
+comparisons	I-experimental_method
+among	O
+xylulose	B-protein_type
+kinase	I-protein_type
+,	O
+glycerol	B-protein_type
+kinase	I-protein_type
+,	O
+ribulose	B-protein_type
+kinase	I-protein_type
+and	O
+SePSK	B-protein
+.	O
+
+Our	O
+results	O
+suggested	O
+that	O
+three	O
+conserved	O
+residues	O
+(	O
+D8	B-residue_name_number
+,	O
+T11	B-residue_name_number
+and	O
+D221	B-residue_name_number
+of	O
+SePSK	B-protein
+)	O
+play	O
+an	O
+important	O
+role	O
+in	O
+SePSK	B-protein
+function	O
+.	O
+
+Mutations	B-experimental_method
+of	O
+the	O
+corresponding	O
+residue	O
+in	O
+xylulose	B-protein_type
+kinase	I-protein_type
+and	O
+glycerol	B-protein_type
+kinase	I-protein_type
+from	O
+Escherichia	B-species
+coli	I-species
+greatly	O
+reduced	O
+their	O
+activity	O
+.	O
+
+To	O
+identify	O
+the	O
+function	O
+of	O
+these	O
+three	O
+residues	O
+of	O
+SePSK	B-protein
+,	O
+we	O
+constructed	O
+D8A	B-mutant
+,	O
+T11A	B-mutant
+and	O
+D221A	B-mutant
+mutants	B-protein_state
+.	O
+
+Using	O
+enzymatic	B-experimental_method
+activity	I-experimental_method
+assays	I-experimental_method
+,	O
+we	O
+found	O
+that	O
+all	O
+of	O
+these	O
+mutants	O
+exhibit	O
+much	O
+lower	O
+activity	O
+of	O
+ATP	B-chemical
+hydrolysis	O
+after	O
+adding	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+than	O
+that	O
+of	O
+wild	B-protein_state
+type	I-protein_state
+,	O
+indicating	O
+the	O
+possibility	O
+that	O
+these	O
+three	O
+residues	O
+are	O
+involved	O
+in	O
+the	O
+catalytic	O
+process	O
+of	O
+phosphorylation	B-ptm
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+and	O
+are	O
+vital	O
+for	O
+the	O
+function	O
+of	O
+SePSK	B-protein
+(	O
+Fig	O
+2D	O
+).	O
+
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+possess	O
+a	O
+similar	O
+ATP	B-site
+binding	I-site
+site	I-site
+
+To	O
+obtain	O
+more	O
+detailed	O
+information	O
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+ATP	B-chemical
+,	O
+we	O
+soaked	B-experimental_method
+the	O
+apo	B-protein_state
+-	O
+crystals	B-evidence
+in	O
+the	O
+reservoir	O
+adding	O
+cofactor	O
+ATP	B-chemical
+,	O
+and	O
+obtained	O
+the	O
+structures	B-evidence
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+bound	B-protein_state
+with	I-protein_state
+ATP	B-chemical
+at	O
+the	O
+resolution	O
+of	O
+2	O
+.	O
+3	O
+Å	O
+and	O
+1	O
+.	O
+8	O
+Å	O
+,	O
+respectively	O
+.	O
+
+In	O
+both	O
+structures	B-evidence
+,	O
+a	O
+strong	O
+electron	B-evidence
+density	I-evidence
+was	O
+found	O
+in	O
+the	O
+conserved	B-protein_state
+ATP	B-site
+binding	I-site
+pocket	I-site
+,	O
+but	O
+can	O
+only	O
+be	O
+fitted	O
+with	O
+an	O
+ADP	B-chemical
+molecule	O
+(	O
+S4	O
+Fig	O
+).	O
+
+Thus	O
+the	O
+two	O
+structures	B-evidence
+were	O
+named	O
+ADP	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+and	O
+ADP	B-complex_assembly
+-	I-complex_assembly
+AtXK	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+respectively	O
+.	O
+
+The	O
+extremely	O
+weak	O
+electron	B-evidence
+densities	I-evidence
+of	O
+ATP	O
+γ	O
+-	O
+phosphate	B-chemical
+in	O
+both	O
+structures	B-evidence
+suggest	O
+that	O
+the	O
+γ	O
+-	O
+phosphate	B-chemical
+group	O
+of	O
+ATP	B-chemical
+is	O
+either	O
+flexible	O
+or	O
+hydrolyzed	O
+by	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+This	O
+result	O
+was	O
+consistent	O
+with	O
+our	O
+enzymatic	B-experimental_method
+activity	I-experimental_method
+assays	I-experimental_method
+where	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+showed	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+without	O
+adding	O
+any	O
+substrates	O
+(	O
+Fig	O
+2A	O
+and	O
+2C	O
+).	O
+
+To	O
+avoid	O
+hydrolysis	O
+of	O
+ATP	B-chemical
+,	O
+we	O
+soaked	B-experimental_method
+the	O
+crystals	B-evidence
+of	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+and	O
+apo	B-protein_state
+-	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+into	O
+the	O
+reservoir	O
+adding	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+.	O
+
+However	O
+,	O
+we	O
+found	O
+that	O
+the	O
+electron	B-evidence
+densities	I-evidence
+of	O
+γ	O
+-	O
+phosphate	B-chemical
+group	O
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+(	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+γ	O
+-	O
+phosphate	B-chemical
+)	O
+are	O
+still	O
+weak	O
+in	O
+the	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+and	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+AtXK	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+structures	B-evidence
+,	O
+suggesting	O
+high	O
+flexibility	O
+of	O
+ATP	B-chemical
+-	O
+γ	O
+-	O
+phosphate	B-chemical
+.	O
+
+The	O
+γ	O
+-	O
+phosphate	B-chemical
+group	O
+of	O
+ATP	B-chemical
+is	O
+transferred	O
+to	O
+the	O
+sugar	B-chemical
+substrate	O
+during	O
+the	O
+reaction	O
+process	O
+,	O
+so	O
+this	O
+flexibility	O
+might	O
+be	O
+important	O
+for	O
+the	O
+ability	O
+of	O
+these	O
+kinases	B-protein_type
+.	O
+
+The	O
+overall	O
+structures	B-evidence
+as	O
+well	O
+as	O
+the	O
+coordination	O
+modes	O
+of	O
+ADP	B-chemical
+and	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+in	O
+the	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+AtXK	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+ADP	B-complex_assembly
+-	I-complex_assembly
+AtXK	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+ADP	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+and	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+structures	B-evidence
+are	O
+nearly	O
+identical	O
+(	O
+S5	O
+Fig	O
+),	O
+therefore	O
+the	O
+structure	B-evidence
+of	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+is	O
+used	O
+here	O
+to	O
+describe	O
+the	O
+structural	O
+details	O
+and	O
+to	O
+compare	O
+with	O
+those	O
+of	O
+other	O
+family	O
+members	O
+.	O
+
+As	O
+shown	O
+in	O
+Fig	O
+3A	O
+,	O
+one	O
+SePSK	B-protein
+protein	O
+molecule	O
+is	O
+in	O
+an	O
+asymmetric	O
+unit	O
+with	O
+one	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+molecule	O
+.	O
+
+The	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+is	O
+bound	O
+at	O
+the	O
+domain	B-structure_element
+II	I-structure_element
+,	O
+where	O
+it	O
+fits	O
+well	O
+inside	O
+a	O
+positively	B-site
+charged	I-site
+groove	I-site
+.	O
+
+The	O
+AMP	B-site
+-	I-site
+PNP	I-site
+binding	I-site
+pocket	I-site
+consists	O
+of	O
+four	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+α26	B-structure_element
+,	O
+α28	B-structure_element
+,	O
+α27	B-structure_element
+and	O
+α30	B-structure_element
+)	O
+and	O
+forms	O
+a	O
+shape	B-protein_state
+resembling	I-protein_state
+a	I-protein_state
+half	I-protein_state
+-	I-protein_state
+fist	I-protein_state
+(	O
+Fig	O
+3A	O
+and	O
+3B	O
+).	O
+
+The	O
+head	O
+group	O
+of	O
+the	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+is	O
+embedded	O
+in	O
+a	O
+pocket	B-site
+surrounded	O
+by	O
+Trp383	B-residue_name_number
+,	O
+Asn380	B-residue_name_number
+,	O
+Gly376	B-residue_name_number
+and	O
+Gly377	B-residue_name_number
+.	O
+
+The	O
+purine	O
+ring	O
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+is	O
+positioned	O
+in	O
+parallel	O
+to	O
+the	O
+indole	O
+ring	O
+of	O
+Trp383	B-residue_name_number
+.	O
+
+In	O
+addition	O
+,	O
+it	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+with	O
+the	O
+side	O
+chain	O
+amide	O
+of	O
+Asn380	B-residue_name_number
+(	O
+Fig	O
+3B	O
+).	O
+
+The	O
+tail	O
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+points	O
+to	O
+the	O
+hinge	B-structure_element
+region	I-structure_element
+of	O
+SePSK	B-protein
+,	O
+and	O
+its	O
+α	O
+-	O
+phosphate	B-chemical
+and	O
+β	O
+-	O
+phosphate	B-chemical
+groups	O
+are	O
+stabilized	O
+by	O
+Gly376	B-residue_name_number
+and	O
+Ser243	B-residue_name_number
+,	O
+respectively	O
+.	O
+
+Together	O
+,	O
+this	O
+structure	B-evidence
+clearly	O
+shows	O
+that	O
+the	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+-	O
+β	O
+-	O
+phosphate	B-chemical
+is	O
+sticking	O
+out	O
+of	O
+the	O
+ATP	B-site
+binding	I-site
+pocket	I-site
+,	O
+thus	O
+the	O
+γ	O
+-	O
+phosphate	B-chemical
+group	O
+is	O
+at	O
+the	O
+empty	O
+space	O
+between	O
+domain	B-structure_element
+I	I-structure_element
+and	O
+domain	B-structure_element
+II	I-structure_element
+and	O
+is	O
+unconstrained	O
+in	O
+its	O
+movement	O
+by	O
+the	O
+protein	O
+.	O
+
+Structure	B-evidence
+of	O
+SePSK	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+.	O
+
+(	O
+A	O
+)	O
+The	O
+electron	B-evidence
+density	I-evidence
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+.	O
+
+The	O
+SePSK	B-protein
+structure	B-evidence
+is	O
+shown	O
+in	O
+the	O
+electrostatic	O
+potential	O
+surface	O
+mode	O
+.	O
+
+The	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+is	O
+depicted	O
+as	O
+sticks	O
+with	O
+its	O
+ǀFoǀ	B-evidence
+-	I-evidence
+ǀFcǀ	I-evidence
+map	I-evidence
+contoured	O
+at	O
+3	O
+σ	O
+shown	O
+as	O
+cyan	O
+mesh	O
+.	O
+
+(	O
+B	O
+)	O
+The	O
+AMP	B-site
+-	I-site
+PNP	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+The	O
+head	O
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+is	O
+sandwiched	O
+by	O
+four	O
+residues	O
+(	O
+Leu293	B-residue_name_number
+,	O
+Gly376	B-residue_name_number
+,	O
+Gly377	B-residue_name_number
+and	O
+Trp383	B-residue_name_number
+).	O
+
+The	O
+four	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+α26	B-structure_element
+,	O
+α28	B-structure_element
+,	O
+α27	B-structure_element
+and	O
+α30	B-structure_element
+)	O
+are	O
+labeled	O
+in	O
+red	O
+.	O
+
+The	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+and	O
+coordinated	O
+residues	O
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+
+The	O
+potential	O
+substrate	B-site
+binding	I-site
+site	I-site
+in	O
+SePSK	B-protein
+
+The	O
+results	O
+from	O
+our	O
+activity	B-experimental_method
+assays	I-experimental_method
+suggested	O
+that	O
+SePSK	B-protein
+has	O
+D	B-protein_type
+-	I-protein_type
+ribulose	I-protein_type
+kinase	I-protein_type
+activity	O
+.	O
+
+To	O
+better	O
+understand	O
+the	O
+interaction	O
+pattern	O
+between	O
+SePSK	B-protein
+and	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+,	O
+the	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+crystals	B-experimental_method
+were	I-experimental_method
+soaked	I-experimental_method
+into	I-experimental_method
+the	O
+reservoir	B-experimental_method
+with	O
+10	O
+mM	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+(	O
+RBL	B-chemical
+)	O
+and	O
+the	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+structure	B-evidence
+was	O
+solved	B-experimental_method
+.	O
+
+As	O
+shown	O
+in	O
+S6	O
+Fig	O
+,	O
+two	O
+residual	O
+electron	B-evidence
+densities	I-evidence
+are	O
+visible	O
+in	O
+domain	B-structure_element
+I	I-structure_element
+,	O
+which	O
+can	O
+be	O
+interpreted	O
+as	O
+two	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+molecules	O
+with	O
+reasonable	O
+fit	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	B-chemical
+-	I-chemical
+ribulose	I-chemical
+molecules	O
+are	O
+approx	O
+.	O
+
+7	O
+.	O
+1	O
+Å	O
+(	O
+RBL1	B-residue_name_number
+-	O
+C4	O
+and	O
+RBL2	B-residue_name_number
+-	O
+C1	O
+).	O
+
+RBL1	B-residue_name_number
+is	O
+located	O
+in	O
+the	O
+pocket	B-site
+consisting	O
+of	O
+α21	B-structure_element
+and	O
+the	O
+loop	B-structure_element
+between	O
+β6	B-structure_element
+and	I-structure_element
+β7	I-structure_element
+.	O
+
+The	O
+O4	O
+and	O
+O5	O
+of	O
+RBL1	B-residue_name_number
+are	O
+coordinated	O
+with	O
+the	O
+side	O
+chain	O
+carboxyl	O
+group	O
+of	O
+Asp221	B-residue_name_number
+.	O
+
+Furthermore	O
+,	O
+the	O
+O2	O
+of	O
+RBL1	B-residue_name_number
+interacts	O
+with	O
+the	O
+main	O
+chain	O
+amide	O
+nitrogen	O
+of	O
+Ser72	B-residue_name_number
+(	O
+Fig	O
+4B	O
+).	O
+
+This	O
+pocket	B-site
+is	O
+at	O
+a	O
+similar	O
+position	O
+of	O
+substrate	B-site
+binding	I-site
+site	I-site
+of	O
+other	O
+sugar	B-protein_type
+kinase	I-protein_type
+,	O
+such	O
+as	O
+L	B-protein
+-	I-protein
+ribulokinase	I-protein
+(	O
+PDB	O
+code	O
+:	O
+3QDK	O
+)	O
+(	O
+S7	O
+Fig	O
+).	O
+
+However	O
+,	O
+structural	B-experimental_method
+comparison	I-experimental_method
+shows	O
+that	O
+the	O
+substrate	O
+ligating	O
+residues	O
+between	O
+the	O
+two	O
+structures	B-evidence
+are	O
+not	B-protein_state
+strictly	I-protein_state
+conserved	I-protein_state
+.	O
+
+Based	O
+on	O
+the	O
+structures	B-evidence
+,	O
+the	O
+ligating	O
+residues	O
+of	O
+RBL1	B-residue_name_number
+in	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+structure	B-evidence
+are	O
+Ser72	B-residue_name_number
+,	O
+Asp221	B-residue_name_number
+and	O
+Ser222	B-residue_name_number
+,	O
+and	O
+the	O
+interacting	O
+residues	O
+of	O
+L	B-chemical
+-	I-chemical
+ribulose	I-chemical
+with	O
+L	B-protein
+-	I-protein
+ribulokinase	I-protein
+are	O
+Ala96	B-residue_name_number
+,	O
+Lys208	B-residue_name_number
+,	O
+Asp274	B-residue_name_number
+and	O
+Glu329	B-residue_name_number
+(	O
+S7	O
+Fig	O
+).	O
+
+Glu329	B-residue_name_number
+in	O
+3QDK	O
+has	O
+no	O
+counterpart	O
+in	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+structure	B-evidence
+.	O
+
+In	O
+addition	O
+,	O
+although	O
+Lys208	B-residue_name_number
+of	O
+L	B-protein
+-	I-protein
+ribulokinase	I-protein
+has	O
+the	O
+corresponding	O
+residue	O
+(	O
+Lys163	B-residue_name_number
+)	O
+in	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+structure	B-evidence
+,	O
+the	O
+hydrogen	O
+bond	O
+of	O
+Lys163	B-residue_name_number
+is	O
+broken	O
+because	O
+of	O
+the	O
+conformational	O
+change	O
+of	O
+two	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+α9	B-structure_element
+and	O
+α13	B-structure_element
+)	O
+of	O
+SePSK	B-protein
+.	O
+
+The	O
+binding	O
+of	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+(	O
+RBL	B-chemical
+)	O
+with	O
+SePSK	B-protein
+.	O
+
+(	O
+A	O
+)	O
+The	O
+electrostatic	B-evidence
+potential	I-evidence
+surface	I-evidence
+map	I-evidence
+of	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+and	O
+a	O
+zoom	O
+-	O
+in	O
+view	O
+of	O
+RBL	B-site
+binding	I-site
+site	I-site
+.	O
+
+The	O
+RBL1	B-residue_name_number
+and	O
+RBL2	B-residue_name_number
+are	O
+depicted	O
+as	O
+sticks	O
+.	O
+(	O
+B	O
+)	O
+Interaction	O
+of	O
+two	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+molecules	O
+(	O
+RBL1	B-residue_name_number
+and	O
+RBL2	B-residue_name_number
+)	O
+with	O
+SePSK	B-protein
+.	O
+
+The	O
+RBL	B-chemical
+molecules	O
+(	O
+carbon	O
+atoms	O
+colored	O
+yellow	O
+)	O
+and	O
+amino	O
+acid	O
+residues	O
+of	O
+SePSK	B-protein
+(	O
+carbon	O
+atoms	O
+colored	O
+green	O
+)	O
+involved	O
+in	O
+RBL	B-chemical
+interaction	O
+are	O
+shown	O
+as	O
+sticks	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	B-experimental_method
+affinity	I-experimental_method
+assays	I-experimental_method
+of	O
+SePSK	B-protein
+with	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+.	O
+
+Single	B-experimental_method
+-	I-experimental_method
+cycle	I-experimental_method
+kinetic	I-experimental_method
+data	I-experimental_method
+are	O
+reflecting	O
+the	O
+interaction	O
+of	O
+SePSK	B-protein
+and	O
+D8A	B-mutant
+-	O
+SePSK	B-protein
+with	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+.	O
+
+It	O
+shows	O
+two	O
+experimental	O
+sensorgrams	B-evidence
+after	O
+minus	O
+the	O
+empty	O
+sensorgrams	B-evidence
+.	O
+
+The	O
+original	O
+data	O
+is	O
+shown	O
+as	O
+black	O
+curve	O
+,	O
+and	O
+the	O
+fitted	O
+data	O
+is	O
+shown	O
+as	O
+different	O
+color	O
+(	O
+wild	B-protein_state
+type	I-protein_state
+SePSK	B-protein
+:	O
+red	O
+curve	O
+,	O
+D8A	B-mutant
+-	O
+SePSK	B-protein
+:	O
+green	O
+curve	O
+).	O
+
+Dissociation	B-evidence
+rate	I-evidence
+constant	I-evidence
+of	O
+wild	B-protein_state
+type	I-protein_state
+and	O
+D8A	B-mutant
+-	O
+SePSK	B-protein
+are	O
+3	O
+ms	O
+-	O
+1	O
+and	O
+9	O
+ms	O
+-	O
+1	O
+,	O
+respectively	O
+.	O
+
+The	O
+binding	B-site
+pocket	I-site
+of	O
+RBL2	B-residue_name_number
+with	O
+relatively	O
+weak	O
+electron	B-evidence
+density	I-evidence
+is	O
+near	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+of	O
+SePSK	B-protein
+and	O
+is	O
+negatively	O
+charged	O
+.	O
+
+The	O
+side	O
+chain	O
+of	O
+Asp8	B-residue_name_number
+interacts	O
+strongly	O
+with	O
+O3	O
+and	O
+O4	O
+of	O
+RBL2	B-residue_name_number
+.	O
+
+The	O
+hydroxyl	O
+group	O
+of	O
+Ser12	B-residue_name_number
+coordinates	O
+with	O
+O2	O
+of	O
+RBL2	B-residue_name_number
+.	O
+
+The	O
+backbone	O
+amide	O
+nitrogens	O
+of	O
+Gly13	B-residue_name_number
+and	O
+Arg15	B-residue_name_number
+also	O
+keep	O
+hydrogen	O
+bonds	O
+with	O
+RBL2	B-residue_name_number
+(	O
+Fig	O
+4B	O
+).	O
+
+Structural	B-experimental_method
+comparison	I-experimental_method
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+showed	O
+that	O
+while	O
+the	O
+RBL1	B-site
+binding	I-site
+pocket	I-site
+is	O
+conserved	B-protein_state
+,	O
+the	O
+RBL2	B-site
+pocket	I-site
+is	O
+disrupted	O
+in	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+,	O
+despite	O
+the	O
+fact	O
+that	O
+the	O
+residues	O
+interacting	O
+with	O
+RBL2	B-residue_name_number
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+between	O
+the	O
+two	O
+proteins	O
+.	O
+
+In	O
+the	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+structure	B-evidence
+,	O
+a	O
+2	O
+.	O
+6	O
+Å	O
+hydrogen	O
+bond	O
+is	O
+present	O
+between	O
+RBL2	B-residue_name_number
+and	O
+Ser12	B-residue_name_number
+(	O
+Fig	O
+4B	O
+),	O
+while	O
+in	O
+the	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+this	O
+hydrogen	O
+bond	O
+with	O
+the	O
+corresponding	O
+residue	O
+(	O
+Ser22	B-residue_name_number
+)	O
+is	O
+broken	O
+.	O
+
+This	O
+break	O
+is	O
+probably	O
+induced	O
+by	O
+the	O
+conformational	O
+change	O
+of	O
+the	O
+two	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+(	O
+β1	B-structure_element
+and	O
+β2	B-structure_element
+),	O
+with	O
+the	O
+result	O
+that	O
+the	O
+linking	B-structure_element
+loop	I-structure_element
+(	O
+loop	B-structure_element
+1	I-structure_element
+)	O
+is	O
+located	O
+further	O
+away	O
+from	O
+the	O
+RBL2	B-site
+binding	I-site
+site	I-site
+.	O
+
+This	O
+change	O
+might	O
+be	O
+the	O
+reason	O
+that	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+only	O
+shows	O
+limited	O
+increasing	O
+in	O
+its	O
+ATP	B-chemical
+hydrolysis	O
+ability	O
+upon	O
+adding	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+as	O
+a	O
+substrate	O
+after	O
+comparing	O
+with	O
+SePSK	B-protein
+(	O
+Fig	O
+2C	O
+).	O
+
+Our	O
+SePSK	B-protein
+structure	B-evidence
+shows	O
+that	O
+the	O
+Asp8	B-residue_name_number
+residue	O
+forms	O
+strong	O
+hydrogen	O
+bond	O
+with	O
+RBL2	B-residue_name_number
+(	O
+Fig	O
+4B	O
+).	O
+
+In	O
+addition	O
+,	O
+our	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+indicated	O
+that	O
+Asp8	B-residue_name_number
+is	O
+important	O
+for	O
+the	O
+activity	O
+of	O
+SePSK	B-protein
+(	O
+Fig	O
+2D	O
+).	O
+
+To	O
+further	O
+verified	O
+this	O
+result	O
+,	O
+we	O
+measured	O
+the	O
+binding	B-evidence
+affinity	I-evidence
+for	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+of	O
+both	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+and	O
+D8A	B-mutant
+mutant	B-protein_state
+of	O
+SePSK	B-protein
+using	O
+a	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+method	I-experimental_method
+.	O
+
+The	O
+results	O
+showed	O
+that	O
+the	O
+affinity	B-evidence
+of	O
+D8A	B-mutant
+-	O
+SePSK	B-protein
+with	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+is	O
+weaker	O
+than	O
+that	O
+of	O
+WT	B-protein_state
+with	O
+a	O
+reduction	O
+of	O
+approx	O
+.	O
+
+Dissociation	B-evidence
+rate	I-evidence
+constant	I-evidence
+(	O
+Kd	B-evidence
+)	O
+of	O
+wild	B-protein_state
+type	I-protein_state
+and	O
+D8A	B-mutant
+-	O
+SePSK	B-protein
+are	O
+3	O
+ms	O
+-	O
+1	O
+and	O
+9	O
+ms	O
+-	O
+1	O
+,	O
+respectively	O
+.	O
+
+The	O
+results	O
+implied	O
+that	O
+the	O
+second	B-site
+RBL	I-site
+binding	I-site
+site	I-site
+plays	O
+a	O
+role	O
+in	O
+the	O
+D	B-protein_type
+-	I-protein_type
+ribulose	I-protein_type
+kinase	I-protein_type
+function	O
+of	O
+SePSK	B-protein
+.	O
+
+However	O
+,	O
+considering	O
+the	O
+high	O
+concentration	O
+of	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+used	O
+for	O
+crystal	B-experimental_method
+soaking	I-experimental_method
+,	O
+as	O
+well	O
+as	O
+the	O
+relatively	O
+weak	O
+electron	B-evidence
+density	I-evidence
+of	O
+RBL2	B-residue_name_number
+,	O
+it	O
+is	O
+also	O
+possible	O
+that	O
+the	O
+second	B-site
+binding	I-site
+site	I-site
+of	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+in	O
+SePSK	B-protein
+is	O
+an	O
+artifact	O
+.	O
+
+Simulated	O
+conformational	O
+change	O
+of	O
+SePSK	B-protein
+during	O
+the	O
+catalytic	O
+process	O
+
+It	O
+was	O
+reported	O
+earlier	O
+that	O
+the	O
+crossing	O
+angle	O
+between	O
+the	O
+domain	B-structure_element
+I	I-structure_element
+and	O
+domain	B-structure_element
+II	I-structure_element
+in	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+is	O
+different	O
+.	O
+
+In	O
+addition	O
+,	O
+this	O
+difference	O
+may	O
+be	O
+caused	O
+by	O
+the	O
+binding	O
+of	O
+substrates	O
+and	O
+/	O
+or	O
+ATP	B-chemical
+.	O
+
+As	O
+reported	O
+previously	O
+,	O
+members	O
+of	O
+the	O
+sugar	B-protein_type
+kinase	I-protein_type
+family	O
+undergo	O
+a	O
+conformational	O
+change	O
+to	O
+narrow	O
+the	O
+crossing	O
+angle	O
+between	O
+two	O
+domains	O
+and	O
+reduce	O
+the	O
+distance	O
+between	O
+substrate	O
+and	O
+ATP	B-chemical
+in	O
+order	O
+to	O
+facilitate	O
+the	O
+catalytic	O
+reaction	O
+of	O
+phosphorylation	B-ptm
+of	O
+sugar	O
+substrates	O
+.	O
+
+After	O
+comparing	O
+structures	B-evidence
+of	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+,	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+and	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+,	O
+we	O
+noticed	O
+that	O
+these	O
+structures	B-evidence
+presented	O
+here	O
+are	O
+similar	O
+.	O
+
+Superposing	B-experimental_method
+the	O
+structures	B-evidence
+of	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+and	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+,	O
+the	O
+results	O
+show	O
+that	O
+the	O
+nearest	O
+distance	O
+between	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+γ	O
+-	O
+phosphate	B-chemical
+and	O
+RBL1	B-residue_name_number
+/	O
+RBL2	B-residue_name_number
+is	O
+7	O
+.	O
+5	O
+Å	O
+(	O
+RBL1	B-residue_name_number
+-	O
+O5	O
+)/	O
+6	O
+.	O
+7	O
+Å	O
+(	O
+RBL2	B-residue_name_number
+-	O
+O1	O
+)	O
+(	O
+S8	O
+Fig	O
+).	O
+
+This	O
+distance	O
+is	O
+too	O
+long	O
+to	O
+transfer	O
+the	O
+γ	O
+-	O
+phosphate	B-chemical
+group	O
+from	O
+ATP	B-chemical
+to	O
+the	O
+substrate	O
+.	O
+
+Since	O
+the	O
+two	O
+domains	O
+of	O
+SePSK	B-protein
+are	O
+widely	O
+separated	O
+in	O
+this	O
+structure	B-evidence
+,	O
+we	O
+hypothesize	O
+that	O
+our	O
+structures	B-evidence
+of	O
+SePSK	B-protein
+represent	O
+its	O
+open	B-protein_state
+form	O
+,	O
+and	O
+that	O
+a	O
+conformational	O
+rearrangement	O
+must	O
+occur	O
+to	O
+switch	O
+to	O
+the	O
+closed	B-protein_state
+state	O
+in	O
+order	O
+to	O
+facilitate	O
+the	O
+catalytic	O
+process	O
+of	O
+phosphorylation	B-ptm
+of	O
+sugar	O
+substrates	O
+.	O
+
+For	O
+studying	O
+such	O
+potential	O
+conformational	O
+change	O
+,	O
+a	O
+simulation	B-experimental_method
+on	O
+the	O
+Hingeprot	B-experimental_method
+Server	I-experimental_method
+was	O
+performed	O
+to	O
+predict	O
+the	O
+movement	O
+of	O
+different	O
+SePSK	B-protein
+domains	O
+.	O
+
+The	O
+results	O
+showed	O
+that	O
+domain	B-structure_element
+I	I-structure_element
+and	O
+domain	B-structure_element
+II	I-structure_element
+are	O
+closer	O
+to	O
+each	O
+other	O
+with	O
+Ala228	B-residue_name_number
+and	O
+Thr401	B-residue_name_number
+in	O
+A2	B-structure_element
+as	O
+Hinge	B-structure_element
+-	I-structure_element
+residues	I-structure_element
+.	O
+
+Based	O
+on	O
+the	O
+above	O
+results	O
+,	O
+SePSK	B-protein
+is	O
+divided	O
+into	O
+two	O
+rigid	O
+parts	O
+.	O
+
+The	O
+domain	B-structure_element
+I	I-structure_element
+of	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+(	O
+aa	O
+.	O
+1	B-residue_range
+–	I-residue_range
+228	I-residue_range
+,	O
+aa	O
+.	O
+402	B-residue_range
+–	I-residue_range
+421	I-residue_range
+)	O
+and	O
+the	O
+domain	B-structure_element
+II	I-structure_element
+of	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+(	O
+aa	O
+.	O
+229	B-residue_range
+–	I-residue_range
+401	I-residue_range
+)	O
+were	O
+superposed	B-experimental_method
+with	O
+structures	B-evidence
+,	O
+including	O
+apo	B-protein_state
+-	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+,	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+,	O
+xylulose	B-protein_type
+kinase	I-protein_type
+from	O
+Lactobacillus	B-species
+acidophilus	I-species
+(	O
+PDB	O
+code	O
+:	O
+3LL3	O
+)	O
+and	O
+the	O
+S58W	B-mutant
+mutant	B-protein_state
+form	O
+of	O
+glycerol	B-protein_type
+kinase	I-protein_type
+from	O
+Escherichia	B-species
+coli	I-species
+(	O
+PDB	O
+code	O
+:	O
+1GLJ	O
+).	O
+
+The	O
+results	O
+of	O
+superposition	B-experimental_method
+displayed	O
+different	O
+crossing	O
+angle	O
+between	O
+these	O
+two	O
+domains	O
+.	O
+
+After	O
+superposition	B-experimental_method
+,	O
+the	O
+distances	O
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+γ	O
+-	O
+phosphate	B-chemical
+and	O
+the	O
+fifth	O
+hydroxyl	O
+group	O
+of	O
+RBL1	B-residue_name_number
+are	O
+7	O
+.	O
+9	O
+Å	O
+(	O
+superposed	B-experimental_method
+with	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+),	O
+7	O
+.	O
+4	O
+Å	O
+(	O
+superposed	B-experimental_method
+with	O
+SePSK	B-protein
+),	O
+6	O
+.	O
+6	O
+Å	O
+(	O
+superposed	B-experimental_method
+with	O
+3LL3	O
+)	O
+and	O
+6	O
+.	O
+1	O
+Å	O
+(	O
+superposed	B-experimental_method
+with	O
+1GLJ	O
+).	O
+
+Meanwhile	O
+,	O
+the	O
+distances	O
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+γ	O
+-	O
+phosphate	B-chemical
+and	O
+the	O
+first	O
+hydroxyl	O
+group	O
+of	O
+RBL2	B-residue_name_number
+are	O
+7	O
+.	O
+2	O
+Å	O
+(	O
+superposed	B-experimental_method
+with	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+),	O
+6	O
+.	O
+7	O
+Å	O
+(	O
+superposed	B-experimental_method
+with	O
+SePSK	B-protein
+),	O
+3	O
+.	O
+7	O
+Å	O
+(	O
+superposed	B-experimental_method
+with	O
+3LL3	O
+),	O
+until	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+γ	O
+-	O
+phosphate	B-chemical
+fully	O
+contacts	O
+RBL2	B-residue_name_number
+after	O
+superposition	B-experimental_method
+with	O
+1GLJ	O
+(	O
+Fig	O
+5	O
+).	O
+
+This	O
+distance	O
+between	O
+RBL2	B-residue_name_number
+and	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+-	O
+γ	O
+-	O
+phosphate	B-chemical
+is	O
+close	O
+enough	O
+to	O
+facilitate	O
+phosphate	B-chemical
+transferring	O
+.	O
+
+Together	O
+,	O
+our	O
+superposition	B-experimental_method
+results	O
+provided	O
+snapshots	O
+of	O
+the	O
+conformational	O
+changes	O
+at	O
+different	O
+catalytic	O
+stages	O
+of	O
+SePSK	B-protein
+and	O
+potentially	O
+revealed	O
+the	O
+closed	B-protein_state
+form	O
+of	O
+SePSK	B-protein
+.	O
+
+Simulated	O
+conformational	O
+change	O
+of	O
+SePSK	B-protein
+during	O
+the	O
+catalytic	O
+process	O
+.	O
+
+The	O
+structures	B-evidence
+are	O
+shown	O
+as	O
+cartoon	O
+and	O
+the	O
+ligands	O
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+
+Domain	B-structure_element
+I	I-structure_element
+from	O
+D	B-complex_assembly
+-	I-complex_assembly
+ribulose	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+(	O
+green	O
+)	O
+and	O
+Domain	B-structure_element
+II	I-structure_element
+from	O
+AMP	B-complex_assembly
+-	I-complex_assembly
+PNP	I-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+(	O
+cyan	O
+)	O
+are	O
+superposed	B-experimental_method
+with	O
+apo	B-protein_state
+-	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+(	O
+1st	O
+),	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+(	O
+2nd	O
+),	O
+3LL3	O
+(	O
+3rd	O
+)	O
+and	O
+1GLJ	O
+(	O
+4th	O
+),	O
+respectively	O
+.	O
+
+The	O
+numbers	O
+near	O
+the	O
+black	O
+dashed	O
+lines	O
+show	O
+the	O
+distances	O
+(	O
+Å	O
+)	O
+between	O
+two	O
+nearest	O
+atoms	O
+of	O
+RBL	B-chemical
+and	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+.	O
+
+In	O
+summary	O
+,	O
+our	O
+structural	B-experimental_method
+and	I-experimental_method
+enzymatic	I-experimental_method
+analyses	I-experimental_method
+provide	O
+evidence	O
+that	O
+SePSK	B-protein
+shows	O
+D	B-protein_type
+-	I-protein_type
+ribulose	I-protein_type
+kinase	I-protein_type
+activity	O
+,	O
+and	O
+exhibits	O
+the	O
+conserved	O
+features	O
+of	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+.	O
+
+Three	O
+conserved	B-site
+residues	O
+in	O
+SePSK	B-protein
+were	O
+identified	O
+to	O
+be	O
+essential	O
+for	O
+this	O
+function	O
+.	O
+
+Our	O
+results	O
+provide	O
+the	O
+detailed	O
+information	O
+about	O
+the	O
+interaction	O
+of	O
+SePSK	B-protein
+with	O
+ATP	B-chemical
+and	O
+substrates	O
+.	O
+
+Moreover	O
+,	O
+structural	B-experimental_method
+superposition	I-experimental_method
+results	O
+enable	O
+us	O
+to	O
+visualize	O
+the	O
+conformational	O
+change	O
+of	O
+SePSK	B-protein
+during	O
+the	O
+catalytic	O
+process	O
+.	O
+
+In	O
+conclusion	O
+,	O
+our	O
+results	O
+provide	O
+important	O
+information	O
+for	O
+a	O
+more	O
+detailed	O
+understanding	O
+of	O
+the	O
+mechanisms	O
+of	O
+SePSK	B-protein
+and	O
+other	O
+members	O
+of	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+.	O
+
+Structural	O
+basis	O
+for	O
+Mep2	B-protein_type
+ammonium	B-protein_type
+transceptor	I-protein_type
+activation	O
+by	O
+phosphorylation	B-ptm
+
+Mep2	B-protein_type
+proteins	I-protein_type
+are	O
+fungal	B-taxonomy_domain
+transceptors	B-protein_type
+that	O
+play	O
+an	O
+important	O
+role	O
+as	O
+ammonium	B-chemical
+sensors	O
+in	O
+fungal	B-taxonomy_domain
+development	O
+.	O
+
+Mep2	B-protein_type
+activity	O
+is	O
+tightly	O
+regulated	O
+by	O
+phosphorylation	B-ptm
+,	O
+but	O
+how	O
+this	O
+is	O
+achieved	O
+at	O
+the	O
+molecular	O
+level	O
+is	O
+not	O
+clear	O
+.	O
+
+Here	O
+we	O
+report	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+crystal	I-evidence
+structures	I-evidence
+of	O
+the	O
+Mep2	B-protein_type
+orthologues	O
+from	O
+Saccharomyces	B-species
+cerevisiae	I-species
+and	O
+Candida	B-species
+albicans	I-species
+and	O
+show	O
+that	O
+under	O
+nitrogen	O
+-	O
+sufficient	O
+conditions	O
+the	O
+transporters	B-protein_type
+are	O
+not	B-protein_state
+phosphorylated	I-protein_state
+and	O
+present	O
+in	O
+closed	B-protein_state
+,	O
+inactive	B-protein_state
+conformations	O
+.	O
+
+Relative	O
+to	O
+the	O
+open	B-protein_state
+bacterial	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+,	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+Mep2	B-protein_type
+exhibits	O
+shifts	O
+in	O
+cytoplasmic	B-structure_element
+loops	I-structure_element
+and	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+(	O
+CTR	B-structure_element
+)	O
+to	O
+occlude	O
+the	O
+cytoplasmic	O
+exit	B-site
+of	O
+the	O
+channel	B-site
+and	O
+to	O
+interact	O
+with	O
+His2	B-residue_name_number
+of	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+.	O
+
+The	O
+phosphorylation	B-site
+site	I-site
+in	O
+the	O
+CTR	B-structure_element
+is	O
+solvent	B-protein_state
+accessible	I-protein_state
+and	O
+located	O
+in	O
+a	O
+negatively	B-site
+charged	I-site
+pocket	I-site
+∼	O
+30	O
+Å	O
+away	O
+from	O
+the	O
+channel	B-site
+exit	I-site
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+phosphorylation	B-protein_state
+-	I-protein_state
+mimicking	I-protein_state
+Mep2	B-mutant
+variants	I-mutant
+from	O
+C	B-species
+.	I-species
+albicans	I-species
+show	O
+large	O
+conformational	O
+changes	O
+in	O
+a	O
+conserved	B-protein_state
+and	O
+functionally	O
+important	O
+region	O
+of	O
+the	O
+CTR	B-structure_element
+.	O
+
+The	O
+results	O
+allow	O
+us	O
+to	O
+propose	O
+a	O
+model	O
+for	O
+regulation	O
+of	O
+eukaryotic	B-taxonomy_domain
+ammonium	B-chemical
+transport	O
+by	O
+phosphorylation	B-ptm
+.	O
+
+Mep2	B-protein_type
+proteins	I-protein_type
+are	O
+tightly	O
+regulated	O
+fungal	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+.	O
+
+Here	O
+,	O
+the	O
+authors	O
+report	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+closed	B-protein_state
+states	O
+of	O
+Mep2	B-protein_type
+proteins	I-protein_type
+and	O
+propose	O
+a	O
+model	O
+for	O
+their	O
+regulation	O
+by	B-experimental_method
+comparing	I-experimental_method
+them	I-experimental_method
+with	I-experimental_method
+the	O
+open	B-protein_state
+ammonium	B-protein_type
+transporters	I-protein_type
+of	O
+bacteria	B-taxonomy_domain
+.	O
+
+Transceptors	B-protein_type
+are	O
+membrane	B-protein_type
+proteins	I-protein_type
+that	O
+function	O
+not	O
+only	O
+as	O
+transporters	O
+but	O
+also	O
+as	O
+receptors	O
+/	O
+sensors	O
+during	O
+nutrient	O
+sensing	O
+to	O
+activate	O
+downstream	O
+signalling	O
+pathways	O
+.	O
+
+A	O
+common	O
+feature	O
+of	O
+transceptors	B-protein_type
+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
+
+While	O
+most	O
+studies	O
+have	O
+focused	O
+on	O
+the	O
+Saccharomyces	B-species
+cerevisiae	I-species
+transceptors	B-protein_type
+for	O
+phosphate	B-chemical
+(	O
+Pho84	B-protein
+),	O
+amino	B-chemical
+acids	I-chemical
+(	O
+Gap1	B-protein
+)	O
+and	O
+ammonium	B-chemical
+(	O
+Mep2	B-protein
+),	O
+transceptors	B-protein_type
+are	O
+found	O
+in	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+as	O
+well	O
+(	O
+for	O
+example	O
+,	O
+the	O
+mammalian	B-taxonomy_domain
+SNAT2	B-protein
+amino	B-protein_type
+-	I-protein_type
+acid	I-protein_type
+transporter	I-protein_type
+and	O
+the	O
+GLUT2	B-protein
+glucose	B-protein_type
+transporter	I-protein_type
+).	O
+
+One	O
+of	O
+the	O
+most	O
+important	O
+unresolved	O
+questions	O
+in	O
+the	O
+field	O
+is	O
+how	O
+the	O
+transceptors	B-protein_type
+couple	O
+to	O
+downstream	O
+signalling	O
+pathways	O
+.	O
+
+One	O
+hypothesis	O
+is	O
+that	O
+downstream	O
+signalling	O
+is	O
+dependent	O
+on	O
+a	O
+specific	O
+conformation	O
+of	O
+the	O
+transporter	B-protein_type
+.	O
+
+Mep2	B-protein_type
+(	B-protein_type
+methylammonium	I-protein_type
+(	I-protein_type
+MA	I-protein_type
+)	I-protein_type
+permease	I-protein_type
+)	I-protein_type
+proteins	I-protein_type
+are	O
+ammonium	B-protein_type
+transceptors	I-protein_type
+that	O
+are	O
+ubiquitous	O
+in	O
+fungi	B-taxonomy_domain
+.	O
+
+They	O
+belong	O
+to	O
+the	O
+Amt	B-protein_type
+/	I-protein_type
+Mep	I-protein_type
+/	I-protein_type
+Rh	I-protein_type
+family	I-protein_type
+of	I-protein_type
+transporters	I-protein_type
+that	O
+are	O
+present	O
+in	O
+all	B-taxonomy_domain
+kingdoms	I-taxonomy_domain
+of	I-taxonomy_domain
+life	I-taxonomy_domain
+and	O
+they	O
+take	O
+up	O
+ammonium	B-chemical
+from	O
+the	O
+extracellular	O
+environment	O
+.	O
+
+Fungi	B-taxonomy_domain
+typically	O
+have	O
+more	O
+than	O
+one	O
+Mep	B-protein_type
+paralogue	O
+,	O
+for	O
+example	O
+,	O
+Mep1	B-protein
+-	I-protein
+3	I-protein
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+.	O
+
+Of	O
+these	O
+,	O
+only	O
+Mep2	B-protein_type
+proteins	I-protein_type
+function	O
+as	O
+ammonium	B-chemical
+receptors	O
+/	O
+sensors	O
+in	O
+fungal	B-taxonomy_domain
+development	O
+.	O
+
+Under	O
+conditions	O
+of	O
+nitrogen	O
+limitation	O
+,	O
+Mep2	B-protein
+initiates	O
+a	O
+signalling	O
+cascade	O
+that	O
+results	O
+in	O
+a	O
+switch	O
+from	O
+the	O
+yeast	O
+form	O
+to	O
+filamentous	O
+(	O
+pseudohyphal	O
+)	O
+growth	O
+that	O
+may	O
+be	O
+required	O
+for	O
+fungal	B-taxonomy_domain
+pathogenicity	O
+.	O
+
+As	O
+is	O
+the	O
+case	O
+for	O
+other	O
+transceptors	B-protein_type
+,	O
+it	O
+is	O
+not	O
+clear	O
+how	O
+Mep2	B-protein
+interacts	O
+with	O
+downstream	O
+signalling	O
+partners	O
+,	O
+but	O
+the	O
+protein	O
+kinase	O
+A	O
+and	O
+mitogen	O
+-	O
+activated	O
+protein	O
+kinase	O
+pathways	O
+have	O
+been	O
+proposed	O
+as	O
+downstream	O
+effectors	O
+of	O
+Mep2	B-protein
+(	O
+refs	O
+).	O
+
+Compared	O
+with	O
+Mep1	B-protein
+and	O
+Mep3	B-protein
+,	O
+Mep2	B-protein
+is	O
+highly	B-protein_state
+expressed	I-protein_state
+and	O
+functions	O
+as	O
+a	O
+low	O
+-	O
+capacity	O
+,	O
+high	O
+-	O
+affinity	O
+transporter	O
+in	O
+the	O
+uptake	O
+of	O
+MA	B-chemical
+.	O
+
+In	O
+addition	O
+,	O
+Mep2	B-protein
+is	O
+also	O
+important	O
+for	O
+uptake	O
+of	O
+ammonium	B-chemical
+produced	O
+by	O
+growth	O
+on	O
+other	O
+nitrogen	B-chemical
+sources	O
+.	O
+
+With	O
+the	O
+exception	O
+of	O
+the	O
+human	B-species
+RhCG	B-protein
+structure	B-evidence
+,	O
+no	O
+structural	O
+information	O
+is	O
+available	O
+for	O
+eukaryotic	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+.	O
+
+By	O
+contrast	O
+,	O
+several	O
+bacterial	B-taxonomy_domain
+Amt	B-protein_type
+orthologues	O
+have	O
+been	O
+characterized	O
+in	O
+detail	O
+via	O
+high	O
+-	O
+resolution	O
+crystal	B-evidence
+structures	I-evidence
+and	O
+a	O
+number	O
+of	O
+molecular	B-experimental_method
+dynamics	I-experimental_method
+(	O
+MD	B-experimental_method
+)	O
+studies	O
+.	O
+
+All	O
+the	O
+solved	O
+structures	B-evidence
+including	O
+that	O
+of	O
+RhCG	B-protein
+are	O
+very	O
+similar	O
+,	O
+establishing	O
+the	O
+basic	O
+architecture	O
+of	O
+ammonium	B-protein_type
+transporters	I-protein_type
+.	O
+
+The	O
+proteins	O
+form	O
+stable	B-protein_state
+trimers	B-oligomeric_state
+,	O
+with	O
+each	O
+monomer	B-oligomeric_state
+having	O
+11	O
+transmembrane	B-structure_element
+(	O
+TM	B-structure_element
+)	O
+helices	B-structure_element
+and	O
+a	O
+central	B-site
+channel	I-site
+for	O
+the	O
+transport	O
+of	O
+ammonium	B-chemical
+.	O
+
+All	O
+structures	B-evidence
+show	O
+the	O
+transporters	B-protein_type
+in	O
+open	B-protein_state
+conformations	O
+.	O
+
+Where	O
+earlier	O
+studies	O
+favoured	O
+the	O
+transport	O
+of	O
+ammonia	B-chemical
+gas	O
+,	O
+recent	O
+data	O
+and	O
+theoretical	O
+considerations	O
+suggest	O
+that	O
+Amt	B-protein_type
+/	I-protein_type
+Mep	I-protein_type
+proteins	I-protein_type
+are	O
+instead	O
+active	B-protein_state
+,	O
+electrogenic	B-protein_type
+transporters	I-protein_type
+of	O
+either	O
+NH4	B-chemical
++	I-chemical
+(	O
+uniport	O
+)	O
+or	O
+NH3	B-chemical
+/	O
+H	B-chemical
++	I-chemical
+(	O
+symport	O
+).	O
+
+A	O
+highly	B-protein_state
+conserved	I-protein_state
+pair	O
+of	O
+channel	B-site
+-	O
+lining	O
+histidine	B-residue_name
+residues	O
+dubbed	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+may	O
+serve	O
+as	O
+a	O
+proton	O
+relay	O
+system	O
+while	O
+NH3	B-chemical
+moves	O
+through	O
+the	O
+channel	B-site
+during	O
+NH3	B-chemical
+/	O
+H	B-chemical
++	I-chemical
+symport	O
+.	O
+
+Ammonium	B-chemical
+transport	O
+is	O
+tightly	O
+regulated	O
+.	O
+
+In	O
+animals	B-taxonomy_domain
+,	O
+this	O
+is	O
+due	O
+to	O
+toxicity	O
+of	O
+elevated	O
+intracellular	O
+ammonium	B-chemical
+levels	O
+,	O
+whereas	O
+for	O
+microorganisms	B-taxonomy_domain
+ammonium	B-chemical
+is	O
+a	O
+preferred	O
+nitrogen	O
+source	O
+.	O
+
+In	O
+bacteria	B-taxonomy_domain
+,	O
+amt	B-gene
+genes	O
+are	O
+present	O
+in	O
+an	O
+operon	O
+with	O
+glnK	B-gene
+,	O
+encoding	O
+a	O
+PII	B-protein_type
+-	I-protein_type
+like	I-protein_type
+signal	I-protein_type
+transduction	I-protein_type
+class	I-protein_type
+protein	I-protein_type
+.	O
+
+By	O
+binding	O
+tightly	O
+to	O
+Amt	B-protein_type
+proteins	I-protein_type
+without	O
+inducing	O
+a	O
+conformational	O
+change	O
+in	O
+the	O
+transporter	B-protein_type
+,	O
+GlnK	B-protein_type
+sterically	O
+blocks	O
+ammonium	B-chemical
+conductance	O
+when	O
+nitrogen	O
+levels	O
+are	O
+sufficient	O
+.	O
+
+Under	O
+conditions	O
+of	O
+nitrogen	B-chemical
+limitation	O
+,	O
+GlnK	B-protein_type
+becomes	O
+uridylated	B-protein_state
+,	O
+blocking	O
+its	O
+ability	O
+to	O
+bind	O
+and	O
+inhibit	O
+Amt	B-protein_type
+proteins	I-protein_type
+.	O
+
+Importantly	O
+,	O
+eukaryotes	B-taxonomy_domain
+do	O
+not	O
+have	O
+GlnK	B-protein_type
+orthologues	O
+and	O
+have	O
+a	O
+different	O
+mechanism	O
+for	O
+regulation	O
+of	O
+ammonium	B-chemical
+transport	O
+activity	O
+.	O
+
+In	O
+plants	B-taxonomy_domain
+,	O
+transporter	B-protein_type
+phosphorylation	B-ptm
+and	O
+dephosphorylation	B-ptm
+are	O
+known	O
+to	O
+regulate	O
+activity	O
+.	O
+
+In	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+,	O
+phosphorylation	B-ptm
+of	O
+Ser457	B-residue_name_number
+within	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+(	O
+CTR	B-structure_element
+)	O
+in	O
+the	O
+cytoplasm	O
+was	O
+recently	O
+proposed	O
+to	O
+cause	O
+Mep2	B-protein_type
+opening	O
+,	O
+possibly	O
+via	O
+inducing	O
+a	O
+conformational	O
+change	O
+.	O
+
+To	O
+elucidate	O
+the	O
+mechanism	O
+of	O
+Mep2	B-protein_type
+transport	O
+regulation	O
+,	O
+we	O
+present	O
+here	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+crystal	I-evidence
+structures	I-evidence
+of	O
+the	O
+Mep2	B-protein_type
+transceptors	I-protein_type
+from	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+and	O
+C	B-species
+.	I-species
+albicans	I-species
+.	O
+
+The	O
+structures	B-evidence
+are	O
+similar	O
+to	O
+each	O
+other	O
+but	O
+show	O
+considerable	O
+differences	O
+to	O
+all	O
+other	O
+ammonium	B-protein_type
+transporter	I-protein_type
+structures	B-evidence
+.	O
+
+The	O
+most	O
+striking	O
+difference	O
+is	O
+the	O
+fact	O
+that	O
+the	O
+Mep2	B-protein_type
+proteins	I-protein_type
+have	O
+closed	B-protein_state
+conformations	O
+.	O
+
+The	O
+putative	O
+phosphorylation	B-site
+site	I-site
+is	O
+solvent	B-protein_state
+accessible	I-protein_state
+and	O
+located	O
+in	O
+a	O
+negatively	B-site
+charged	I-site
+pocket	I-site
+∼	O
+30	O
+Å	O
+away	O
+from	O
+the	O
+channel	B-site
+exit	I-site
+.	O
+
+The	O
+channels	B-site
+of	O
+phosphorylation	B-protein_state
+-	I-protein_state
+mimicking	I-protein_state
+mutants	I-protein_state
+of	O
+C	B-species
+.	I-species
+albicans	I-species
+Mep2	B-protein
+are	O
+still	O
+closed	B-protein_state
+but	O
+show	O
+large	O
+conformational	O
+changes	O
+within	O
+a	O
+conserved	B-protein_state
+part	O
+of	O
+the	O
+CTR	B-structure_element
+.	O
+
+Together	O
+with	O
+a	O
+structure	B-evidence
+of	O
+a	O
+C	O
+-	O
+terminal	O
+Mep2	B-mutant
+variant	I-mutant
+lacking	B-protein_state
+the	O
+segment	B-structure_element
+containing	O
+the	O
+phosphorylation	B-site
+site	I-site
+,	O
+the	O
+results	O
+allow	O
+us	O
+to	O
+propose	O
+a	O
+structural	O
+model	O
+for	O
+phosphorylation	O
+-	O
+based	O
+regulation	O
+of	O
+eukaryotic	B-taxonomy_domain
+ammonium	B-chemical
+transport	O
+.	O
+
+General	O
+architecture	O
+of	O
+Mep2	B-protein_type
+ammonium	B-protein_type
+transceptors	I-protein_type
+
+The	O
+Mep2	B-protein
+protein	O
+of	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+(	O
+ScMep2	B-protein
+)	O
+was	O
+overexpressed	B-experimental_method
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+in	O
+high	O
+yields	O
+,	O
+enabling	O
+structure	B-experimental_method
+determination	I-experimental_method
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+using	O
+data	O
+to	O
+3	O
+.	O
+2	O
+Å	O
+resolution	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+(	O
+MR	B-experimental_method
+)	O
+with	O
+the	O
+archaebacterial	B-taxonomy_domain
+Amt	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+(	O
+see	O
+Methods	O
+section	O
+).	O
+
+Given	O
+that	O
+the	O
+modest	O
+resolution	O
+of	O
+the	O
+structure	B-evidence
+and	O
+the	O
+limited	O
+detergent	O
+stability	O
+of	O
+ScMep2	B-protein
+would	O
+likely	O
+complicate	O
+structure	B-experimental_method
+–	I-experimental_method
+function	I-experimental_method
+studies	I-experimental_method
+,	O
+several	O
+other	O
+fungal	B-taxonomy_domain
+Mep2	B-protein_type
+orthologues	O
+were	O
+subsequently	O
+overexpressed	B-experimental_method
+and	I-experimental_method
+screened	I-experimental_method
+for	I-experimental_method
+diffraction	O
+-	O
+quality	O
+crystals	B-evidence
+.	O
+
+Of	O
+these	O
+,	O
+Mep2	B-protein
+from	O
+C	B-species
+.	I-species
+albicans	I-species
+(	O
+CaMep2	B-protein
+)	O
+showed	O
+superior	O
+stability	O
+in	O
+relatively	O
+harsh	O
+detergents	O
+such	O
+as	O
+nonyl	O
+-	O
+glucoside	O
+,	O
+allowing	O
+structure	B-experimental_method
+determination	I-experimental_method
+in	O
+two	O
+different	O
+crystal	B-evidence
+forms	I-evidence
+to	O
+high	O
+resolution	O
+(	O
+up	O
+to	O
+1	O
+.	O
+5	O
+Å	O
+).	O
+
+Despite	O
+different	O
+crystal	O
+packing	O
+(	O
+Supplementary	O
+Table	O
+1	O
+),	O
+the	O
+two	O
+CaMep2	B-protein
+structures	B-evidence
+are	O
+identical	O
+to	O
+each	O
+other	O
+and	O
+very	O
+similar	O
+to	O
+ScMep2	B-protein
+(	O
+Cα	O
+r	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.	I-evidence
+
+(	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+)=	O
+0	O
+.	O
+7	O
+Å	O
+for	O
+434	O
+residues	O
+),	O
+with	O
+the	O
+main	O
+differences	O
+confined	O
+to	O
+the	O
+N	O
+terminus	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+Fig	O
+.	O
+1	O
+).	O
+
+Electron	B-evidence
+density	I-evidence
+is	O
+visible	O
+for	O
+the	O
+entire	O
+polypeptide	O
+chains	O
+,	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+43	B-residue_range
+(	O
+ScMep2	B-protein
+)	O
+and	O
+25	B-residue_range
+residues	O
+(	O
+CaMep2	B-protein
+),	O
+which	O
+are	O
+poorly	B-protein_state
+conserved	I-protein_state
+and	O
+presumably	O
+disordered	B-protein_state
+.	O
+
+Both	O
+Mep2	B-protein_type
+proteins	I-protein_type
+show	O
+the	O
+archetypal	O
+trimeric	B-oligomeric_state
+assemblies	O
+in	O
+which	O
+each	O
+monomer	B-oligomeric_state
+consists	O
+of	O
+11	O
+TM	B-structure_element
+helices	I-structure_element
+surrounding	O
+a	O
+central	B-structure_element
+pore	I-structure_element
+.	O
+
+Important	O
+functional	O
+features	O
+such	O
+as	O
+the	O
+extracellular	O
+ammonium	B-site
+binding	I-site
+site	I-site
+,	O
+the	O
+Phe	B-site
+gate	I-site
+and	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+within	O
+the	O
+hydrophobic	B-site
+channel	I-site
+are	O
+all	O
+very	O
+similar	O
+to	O
+those	O
+present	O
+in	O
+the	O
+bacterial	B-taxonomy_domain
+transporters	B-protein_type
+and	O
+RhCG	B-protein
+.	O
+
+In	O
+the	O
+remainder	O
+of	O
+the	O
+manuscript	O
+,	O
+we	O
+will	O
+specifically	O
+discuss	O
+CaMep2	B-protein
+due	O
+to	O
+the	O
+superior	O
+resolution	O
+of	O
+the	O
+structure	B-evidence
+.	O
+
+Unless	O
+specifically	O
+stated	O
+,	O
+the	O
+drawn	O
+conclusions	O
+also	O
+apply	O
+to	O
+ScMep2	B-protein
+.	O
+
+While	O
+the	O
+overall	O
+architecture	O
+of	O
+Mep2	B-protein
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+prokaryotic	B-taxonomy_domain
+transporters	B-protein_type
+(	O
+Cα	O
+r	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.	I-evidence
+with	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+=	O
+1	O
+.	O
+4	O
+Å	O
+for	O
+361	O
+residues	O
+),	O
+there	O
+are	O
+large	O
+differences	O
+within	O
+the	O
+N	O
+terminus	O
+,	O
+intracellular	B-structure_element
+loops	I-structure_element
+(	O
+ICLs	B-structure_element
+)	O
+ICL1	B-structure_element
+and	O
+ICL3	B-structure_element
+,	O
+and	O
+the	O
+CTR	B-structure_element
+.	O
+
+The	O
+N	O
+termini	O
+of	O
+the	O
+Mep2	B-protein_type
+proteins	I-protein_type
+are	O
+∼	O
+20	B-residue_range
+–	I-residue_range
+25	I-residue_range
+residues	O
+longer	O
+compared	O
+with	O
+their	O
+bacterial	B-taxonomy_domain
+counterparts	O
+(	O
+Figs	O
+1	O
+and	O
+2	O
+),	O
+substantially	O
+increasing	O
+the	O
+size	O
+of	O
+the	O
+extracellular	B-structure_element
+domain	I-structure_element
+.	O
+
+Moreover	O
+,	O
+the	O
+N	O
+terminus	O
+of	O
+one	O
+monomer	B-oligomeric_state
+interacts	O
+with	O
+the	O
+extended	O
+extracellular	B-structure_element
+loop	I-structure_element
+ECL5	B-structure_element
+of	O
+a	O
+neighbouring	O
+monomer	B-oligomeric_state
+.	O
+
+Together	O
+with	O
+additional	O
+,	O
+smaller	O
+differences	O
+in	O
+other	O
+extracellular	B-structure_element
+loops	I-structure_element
+,	O
+these	O
+changes	O
+generate	O
+a	O
+distinct	O
+vestibule	B-structure_element
+leading	O
+to	O
+the	O
+ammonium	B-site
+binding	I-site
+site	I-site
+that	O
+is	O
+much	O
+more	O
+pronounced	O
+than	O
+in	O
+the	O
+bacterial	B-taxonomy_domain
+proteins	O
+.	O
+
+The	O
+N	O
+-	O
+terminal	O
+vestibule	B-structure_element
+and	O
+the	O
+resulting	O
+inter	O
+-	O
+monomer	O
+interactions	O
+likely	O
+increase	O
+the	O
+stability	O
+of	O
+the	O
+Mep2	B-protein
+trimer	B-oligomeric_state
+,	O
+in	O
+support	O
+of	O
+data	O
+for	O
+plant	B-taxonomy_domain
+AMT	B-protein_type
+proteins	I-protein_type
+.	O
+
+However	O
+,	O
+given	O
+that	O
+an	O
+N	O
+-	O
+terminal	O
+deletion	B-protein_state
+mutant	I-protein_state
+(	O
+2	B-mutant
+-	I-mutant
+27Δ	I-mutant
+)	O
+grows	O
+as	O
+well	O
+as	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+Mep2	B-protein
+on	O
+minimal	O
+ammonium	B-chemical
+medium	O
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+),	O
+the	O
+importance	O
+of	O
+the	O
+N	O
+terminus	O
+for	O
+Mep2	B-protein
+activity	O
+is	O
+not	O
+clear	O
+.	O
+
+Mep2	B-protein
+channels	B-site
+are	O
+closed	B-protein_state
+by	O
+a	O
+two	O
+-	O
+tier	O
+channel	B-structure_element
+block	I-structure_element
+
+The	O
+largest	O
+differences	O
+between	O
+the	O
+Mep2	B-protein
+structures	B-evidence
+and	O
+the	O
+other	O
+known	O
+ammonium	B-protein_type
+transporter	I-protein_type
+structures	B-evidence
+are	O
+located	O
+on	O
+the	O
+intracellular	O
+side	O
+of	O
+the	O
+membrane	O
+.	O
+
+In	O
+the	O
+vicinity	O
+of	O
+the	O
+Mep2	B-protein
+channel	B-site
+exit	I-site
+,	O
+the	O
+cytoplasmic	O
+end	O
+of	O
+TM2	B-structure_element
+has	O
+unwound	O
+,	O
+generating	O
+a	O
+longer	O
+ICL1	B-structure_element
+even	O
+though	O
+there	O
+are	O
+no	O
+insertions	O
+in	O
+this	O
+region	O
+compared	O
+to	O
+the	O
+bacterial	B-taxonomy_domain
+proteins	O
+(	O
+Figs	O
+2	O
+and	O
+4	O
+).	O
+
+ICL1	B-structure_element
+has	O
+also	O
+moved	O
+inwards	O
+relative	O
+to	O
+its	O
+position	O
+in	O
+the	O
+bacterial	B-taxonomy_domain
+Amts	B-protein_type
+.	O
+
+The	O
+largest	O
+backbone	O
+movements	O
+of	O
+equivalent	O
+residues	O
+within	O
+ICL1	B-structure_element
+are	O
+∼	O
+10	O
+Å	O
+,	O
+markedly	O
+affecting	O
+the	O
+conserved	B-protein_state
+basic	B-protein_state
+RxK	B-structure_element
+motif	I-structure_element
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+The	O
+head	O
+group	O
+of	O
+Arg54	B-residue_name_number
+has	O
+moved	O
+∼	O
+11	O
+Å	O
+relative	O
+to	O
+that	O
+in	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+,	O
+whereas	O
+the	O
+shift	O
+of	O
+the	O
+head	O
+group	O
+of	O
+the	O
+variable	O
+Lys55	B-residue_name_number
+residue	O
+is	O
+almost	O
+20	O
+Å	O
+.	O
+The	O
+side	O
+chain	O
+of	O
+Lys56	B-residue_name_number
+in	O
+the	O
+basic	B-protein_state
+motif	B-structure_element
+points	O
+in	O
+an	O
+opposite	O
+direction	O
+in	O
+the	O
+Mep2	B-protein
+structures	B-evidence
+compared	O
+with	O
+that	O
+of	O
+,	O
+for	O
+example	O
+,	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+In	O
+addition	O
+to	O
+changing	O
+the	O
+RxK	B-structure_element
+motif	I-structure_element
+,	O
+the	O
+movement	O
+of	O
+ICL1	B-structure_element
+has	O
+another	O
+,	O
+crucial	O
+functional	O
+consequence	O
+.	O
+
+At	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+TM1	B-structure_element
+,	O
+the	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+group	O
+of	O
+the	O
+relatively	B-protein_state
+conserved	I-protein_state
+Tyr49	B-residue_name_number
+(	O
+Tyr53	B-residue_name_number
+in	O
+ScMep2	B-protein
+)	O
+makes	O
+a	O
+strong	O
+hydrogen	O
+bond	O
+with	O
+the	O
+ɛ2	O
+nitrogen	O
+atom	O
+of	O
+the	O
+absolutely	B-protein_state
+conserved	I-protein_state
+His342	B-residue_name_number
+of	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+(	O
+His348	B-residue_name_number
+in	O
+ScMep2	B-protein
+),	O
+closing	O
+the	O
+channel	B-site
+(	O
+Figs	O
+4	O
+and	O
+5	O
+).	O
+
+In	O
+bacterial	B-taxonomy_domain
+Amt	B-protein_type
+proteins	I-protein_type
+,	O
+this	O
+Tyr	B-residue_name
+side	O
+chain	O
+is	O
+rotated	O
+∼	O
+4	O
+Å	O
+away	O
+as	O
+a	O
+result	O
+of	O
+the	O
+different	O
+conformation	O
+of	O
+TM1	B-structure_element
+,	O
+leaving	O
+the	O
+channel	B-site
+open	B-protein_state
+and	O
+the	O
+histidine	B-residue_name
+available	O
+for	O
+its	O
+putative	O
+role	O
+in	O
+substrate	O
+transport	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+
+Compared	O
+with	O
+ICL1	B-structure_element
+,	O
+the	O
+backbone	O
+conformational	O
+changes	O
+observed	O
+for	O
+the	O
+neighbouring	O
+ICL2	B-structure_element
+are	O
+smaller	O
+,	O
+but	O
+large	O
+shifts	O
+are	O
+nevertheless	O
+observed	O
+for	O
+the	O
+conserved	B-protein_state
+residues	O
+Glu140	B-residue_name_number
+and	O
+Arg141	B-residue_name_number
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+Finally	O
+,	O
+the	O
+important	O
+ICL3	B-structure_element
+linking	O
+the	O
+pseudo	B-structure_element
+-	I-structure_element
+symmetrical	I-structure_element
+halves	I-structure_element
+(	O
+TM1	B-structure_element
+-	I-structure_element
+5	I-structure_element
+and	O
+TM6	B-structure_element
+-	I-structure_element
+10	I-structure_element
+)	O
+of	O
+the	O
+transporter	B-protein_type
+is	O
+also	O
+shifted	O
+up	O
+to	O
+∼	O
+10	O
+Å	O
+and	O
+forms	O
+an	O
+additional	O
+barrier	O
+that	O
+closes	O
+the	O
+channel	B-site
+on	O
+the	O
+cytoplasmic	O
+side	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+This	O
+two	O
+-	O
+tier	O
+channel	B-structure_element
+block	I-structure_element
+likely	O
+ensures	O
+that	O
+very	O
+little	O
+ammonium	B-chemical
+transport	O
+will	O
+take	O
+place	O
+under	O
+nitrogen	B-chemical
+-	O
+sufficient	O
+conditions	O
+.	O
+
+The	O
+closed	B-protein_state
+state	O
+of	O
+the	O
+channel	B-site
+might	O
+also	O
+explain	O
+why	O
+no	B-evidence
+density	I-evidence
+,	O
+which	O
+could	O
+correspond	O
+to	O
+ammonium	B-chemical
+(	O
+or	O
+water	B-chemical
+),	O
+is	O
+observed	O
+in	O
+the	O
+hydrophobic	O
+part	O
+of	O
+the	O
+Mep2	B-protein
+channel	B-site
+close	O
+to	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+.	O
+
+Significantly	O
+,	O
+this	O
+is	O
+also	O
+true	O
+for	O
+ScMep2	B-protein
+,	O
+which	O
+was	O
+crystallized	B-experimental_method
+in	O
+the	O
+presence	O
+of	O
+0	O
+.	O
+2	O
+M	O
+ammonium	B-chemical
+ions	O
+(	O
+see	O
+Methods	O
+section	O
+).	O
+
+The	O
+final	O
+region	O
+in	O
+Mep2	B-protein
+that	O
+shows	O
+large	O
+differences	O
+compared	O
+with	O
+the	O
+bacterial	B-taxonomy_domain
+transporters	B-protein_type
+is	O
+the	O
+CTR	B-structure_element
+.	O
+
+In	O
+Mep2	B-protein
+,	O
+the	O
+CTR	B-structure_element
+has	O
+moved	O
+away	O
+and	O
+makes	O
+relatively	O
+few	O
+contacts	O
+with	O
+the	O
+main	B-structure_element
+body	I-structure_element
+of	O
+the	O
+transporter	B-protein_type
+,	O
+generating	O
+a	O
+more	O
+elongated	B-protein_state
+protein	O
+(	O
+Figs	O
+1	O
+and	O
+4	O
+).	O
+
+By	O
+contrast	O
+,	O
+in	O
+the	O
+structures	B-evidence
+of	O
+bacterial	B-taxonomy_domain
+proteins	O
+,	O
+the	O
+CTR	B-structure_element
+is	O
+docked	O
+tightly	O
+onto	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+the	O
+transporters	B-protein_type
+(	O
+corresponding	O
+to	O
+TM1	B-structure_element
+-	I-structure_element
+5	I-structure_element
+),	O
+resulting	O
+in	O
+a	O
+more	O
+compact	B-protein_state
+structure	B-evidence
+.	O
+
+This	O
+is	O
+illustrated	O
+by	O
+the	O
+positions	O
+of	O
+the	O
+five	O
+universally	B-protein_state
+conserved	I-protein_state
+residues	O
+within	O
+the	O
+CTR	B-structure_element
+,	O
+that	O
+is	O
+,	O
+Arg415	B-residue_name_number
+(	O
+370	B-residue_number
+),	O
+Glu421	B-residue_name_number
+(	O
+376	B-residue_number
+),	O
+Gly424	B-residue_name_number
+(	O
+379	B-residue_number
+),	O
+Asp426	B-residue_name_number
+(	O
+381	B-residue_number
+)	O
+and	O
+Tyr	B-residue_name_number
+435	I-residue_name_number
+(	O
+390	B-residue_number
+)	O
+in	O
+CaMep2	B-protein
+(	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+)	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+
+These	O
+residues	O
+include	O
+those	O
+of	O
+the	O
+‘	B-structure_element
+ExxGxD	I-structure_element
+'	I-structure_element
+motif	I-structure_element
+,	O
+which	O
+when	O
+mutated	B-experimental_method
+generate	O
+inactive	B-protein_state
+transporters	B-protein_type
+.	O
+
+In	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+and	O
+other	O
+bacterial	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+,	O
+these	O
+CTR	B-structure_element
+residues	O
+interact	O
+with	O
+residues	O
+within	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+the	O
+protein	O
+.	O
+
+On	O
+one	O
+side	O
+,	O
+the	O
+Tyr390	B-residue_name_number
+hydroxyl	O
+in	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+is	O
+hydrogen	O
+bonded	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+the	O
+conserved	B-protein_state
+His185	B-residue_name_number
+at	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+loop	B-structure_element
+ICL3	B-structure_element
+.	O
+
+At	O
+the	O
+other	O
+end	O
+of	O
+ICL3	B-structure_element
+,	O
+the	O
+backbone	O
+carbonyl	O
+groups	O
+of	O
+Gly172	B-residue_name_number
+and	O
+Lys173	B-residue_name_number
+are	O
+hydrogen	O
+bonded	O
+to	O
+the	O
+side	O
+chain	O
+of	O
+Arg370	B-residue_name_number
+.	O
+
+Similar	O
+interactions	O
+were	O
+also	O
+modelled	B-experimental_method
+in	O
+the	O
+active	B-protein_state
+,	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+plant	B-taxonomy_domain
+AtAmt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+structure	B-evidence
+(	O
+for	O
+example	O
+,	O
+Y467	B-residue_name_number
+-	O
+H239	B-residue_name_number
+and	O
+D458	B-residue_name_number
+-	O
+K71	B-residue_name_number
+).	O
+
+The	O
+result	O
+of	O
+these	O
+interactions	O
+is	O
+that	O
+the	O
+CTR	B-structure_element
+‘	O
+hugs	O
+'	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+the	O
+transporters	B-protein_type
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+Also	O
+noteworthy	O
+is	O
+Asp381	B-residue_name_number
+,	O
+the	O
+side	O
+chain	O
+of	O
+which	O
+interacts	O
+strongly	O
+with	O
+the	O
+positive	O
+dipole	O
+on	O
+the	O
+N	O
+-	O
+terminal	O
+end	O
+of	O
+TM2	B-structure_element
+.	O
+
+This	O
+interaction	O
+in	O
+the	O
+centre	O
+of	O
+the	O
+protein	O
+may	O
+be	O
+particularly	O
+important	O
+to	O
+stabilize	O
+the	O
+open	B-protein_state
+conformations	O
+of	O
+ammonium	B-protein_type
+transporters	I-protein_type
+.	O
+
+In	O
+the	O
+Mep2	B-protein
+structures	B-evidence
+,	O
+none	O
+of	O
+the	O
+interactions	O
+mentioned	O
+above	O
+are	O
+present	O
+.	O
+
+Phosphorylation	B-site
+target	I-site
+site	I-site
+is	O
+at	O
+the	O
+periphery	O
+of	O
+Mep2	B-protein
+
+Recently	O
+Boeckstaens	O
+et	O
+al	O
+.	O
+provided	O
+evidence	O
+that	O
+Ser457	B-residue_name_number
+in	O
+ScMep2	B-protein
+(	O
+corresponding	O
+to	O
+Ser453	B-residue_name_number
+in	O
+CaMep2	B-protein
+)	O
+is	O
+phosphorylated	B-protein_state
+by	O
+the	O
+TORC1	B-protein_type
+effector	I-protein_type
+kinase	I-protein_type
+Npr1	B-protein
+under	O
+nitrogen	B-chemical
+-	O
+limiting	O
+conditions	O
+.	O
+
+In	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Npr1	B-protein
+,	O
+plasmid	B-experimental_method
+-	I-experimental_method
+encoded	I-experimental_method
+WT	B-protein_state
+Mep2	B-protein
+in	O
+a	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+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	B-chemical
+,	O
+suggesting	O
+that	O
+the	O
+transporter	B-protein_type
+is	O
+inactive	B-protein_state
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+
+Conversely	O
+,	O
+the	O
+phosphorylation	B-protein_state
+-	I-protein_state
+mimicking	I-protein_state
+S457D	B-mutant
+variant	O
+is	O
+active	B-protein_state
+both	O
+in	O
+the	O
+triple	B-mutant
+mepΔ	I-mutant
+background	O
+and	O
+in	O
+a	O
+triple	B-mutant
+mepΔ	I-mutant
+npr1Δ	I-mutant
+strain	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+
+Mutation	B-experimental_method
+of	O
+other	O
+potential	O
+phosphorylation	B-site
+sites	I-site
+in	O
+the	O
+CTR	B-structure_element
+did	O
+not	O
+support	O
+growth	O
+in	O
+the	O
+npr1Δ	B-mutant
+background	O
+.	O
+
+Collectively	O
+,	O
+these	O
+data	O
+suggest	O
+that	O
+phosphorylation	B-ptm
+of	O
+Ser457	B-residue_name_number
+opens	O
+the	O
+Mep2	B-protein
+channel	B-site
+to	O
+allow	O
+ammonium	B-chemical
+uptake	O
+.	O
+
+Ser457	B-residue_name_number
+is	O
+located	O
+in	O
+a	O
+part	O
+of	O
+the	O
+CTR	B-structure_element
+that	O
+is	O
+conserved	B-protein_state
+in	O
+a	O
+subgroup	O
+of	O
+Mep2	B-protein_type
+proteins	I-protein_type
+,	O
+but	O
+which	O
+is	O
+not	O
+present	O
+in	O
+bacterial	B-taxonomy_domain
+proteins	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+
+This	O
+segment	B-structure_element
+(	O
+residues	O
+450	B-residue_range
+–	I-residue_range
+457	I-residue_range
+in	O
+ScMep2	B-protein
+and	O
+446	B-residue_range
+–	I-residue_range
+453	I-residue_range
+in	O
+CaMep2	B-protein
+)	O
+was	O
+dubbed	O
+an	O
+autoinhibitory	B-structure_element
+(	I-structure_element
+AI	I-structure_element
+)	I-structure_element
+region	I-structure_element
+based	O
+on	O
+the	O
+fact	O
+that	O
+its	O
+removal	B-experimental_method
+generates	O
+an	O
+active	B-protein_state
+transporter	B-protein_type
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Npr1	B-protein
+(	O
+Fig	O
+.	O
+3	O
+).	O
+
+Where	O
+is	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+and	O
+the	O
+Npr1	B-protein
+phosphorylation	B-site
+site	I-site
+located	O
+?	O
+Our	O
+structures	B-evidence
+reveal	O
+that	O
+surprisingly	O
+,	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+is	O
+folded	O
+back	O
+onto	O
+the	O
+CTR	B-structure_element
+and	O
+is	O
+not	O
+located	O
+near	O
+the	O
+centre	O
+of	O
+the	O
+trimer	B-oligomeric_state
+as	O
+expected	O
+from	O
+the	O
+bacterial	B-taxonomy_domain
+structures	B-evidence
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+The	O
+AI	B-structure_element
+region	I-structure_element
+packs	O
+against	O
+the	O
+cytoplasmic	O
+ends	O
+of	O
+TM2	B-structure_element
+and	O
+TM4	B-structure_element
+,	O
+physically	O
+linking	O
+the	O
+main	B-structure_element
+body	I-structure_element
+of	O
+the	O
+transporter	B-protein_type
+with	O
+the	O
+CTR	B-structure_element
+via	O
+main	O
+chain	O
+interactions	O
+and	O
+side	O
+-	O
+chain	O
+interactions	O
+of	O
+Val447	B-residue_name_number
+,	O
+Asp449	B-residue_name_number
+,	O
+Pro450	B-residue_name_number
+and	O
+Arg452	B-residue_name_number
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+AI	B-structure_element
+regions	I-structure_element
+have	O
+very	O
+similar	O
+conformations	O
+in	O
+CaMep2	B-protein
+and	O
+ScMep2	B-protein
+,	O
+despite	O
+considerable	O
+differences	O
+in	O
+the	O
+rest	O
+of	O
+the	O
+CTR	B-structure_element
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+Strikingly	O
+,	O
+the	O
+Npr1	B-site
+target	I-site
+serine	I-site
+residue	O
+is	O
+located	O
+at	O
+the	O
+periphery	O
+of	O
+the	O
+trimer	B-oligomeric_state
+,	O
+far	O
+away	O
+(∼	O
+30	O
+Å	O
+)	O
+from	O
+any	O
+channel	B-site
+exit	I-site
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+Despite	O
+its	O
+location	O
+at	O
+the	O
+periphery	O
+of	O
+the	O
+trimer	B-oligomeric_state
+,	O
+the	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+serine	B-residue_name
+is	O
+well	O
+defined	O
+in	O
+both	O
+Mep2	B-protein
+structures	B-evidence
+and	O
+corresponds	O
+to	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+state	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+This	O
+makes	O
+sense	O
+since	O
+the	O
+proteins	O
+were	O
+expressed	O
+in	O
+rich	O
+medium	O
+and	O
+confirms	O
+the	O
+recent	O
+suggestion	O
+by	O
+Boeckstaens	O
+et	O
+al	O
+.	O
+that	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+form	O
+of	O
+Mep2	B-protein
+corresponds	O
+to	O
+the	O
+inactive	B-protein_state
+state	O
+.	O
+
+For	O
+ScMep2	B-protein
+,	O
+Ser457	B-residue_name_number
+is	O
+the	O
+most	O
+C	O
+-	O
+terminal	O
+residue	O
+for	O
+which	O
+electron	B-evidence
+density	I-evidence
+is	O
+visible	O
+,	O
+indicating	O
+that	O
+the	O
+region	O
+beyond	O
+Ser457	B-residue_name_number
+is	O
+disordered	B-protein_state
+.	O
+
+In	O
+CaMep2	B-protein
+,	O
+the	O
+visible	O
+part	O
+of	O
+the	O
+sequence	O
+extends	O
+for	O
+two	O
+residues	O
+beyond	O
+Ser453	B-residue_name_number
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+peripheral	O
+location	O
+and	O
+disorder	B-protein_state
+of	O
+the	O
+CTR	B-structure_element
+beyond	O
+the	O
+kinase	B-site
+target	I-site
+site	I-site
+should	O
+facilitate	O
+the	O
+phosphorylation	B-ptm
+by	O
+Npr1	B-protein
+.	O
+
+The	O
+disordered	B-protein_state
+part	O
+of	O
+the	O
+CTR	B-structure_element
+is	O
+not	B-protein_state
+conserved	I-protein_state
+in	O
+ammonium	B-protein_type
+transporters	I-protein_type
+(	O
+Fig	O
+.	O
+2	O
+),	O
+suggesting	O
+that	O
+it	O
+is	O
+not	O
+important	O
+for	O
+transport	O
+.	O
+
+Interestingly	O
+,	O
+a	O
+ScMep2	B-protein
+457Δ	B-mutant
+truncation	B-protein_state
+mutant	I-protein_state
+in	O
+which	O
+a	O
+His	O
+-	O
+tag	O
+directly	O
+follows	O
+Ser457	B-residue_name_number
+is	O
+highly	O
+expressed	O
+but	O
+has	O
+low	B-protein_state
+activity	I-protein_state
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+),	O
+suggesting	O
+that	O
+the	O
+His	O
+-	O
+tag	O
+interferes	O
+with	O
+phosphorylation	B-ptm
+by	O
+Npr1	B-protein
+.	O
+
+The	O
+same	O
+mutant	B-mutant
+lacking	B-protein_state
+the	I-protein_state
+His	I-protein_state
+-	I-protein_state
+tag	I-protein_state
+has	O
+WT	B-protein_state
+properties	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+),	O
+confirming	O
+that	O
+the	O
+region	O
+following	O
+the	O
+phosphorylation	B-site
+site	I-site
+is	O
+dispensable	O
+for	O
+function	O
+.	O
+
+Mep2	B-protein
+lacking	B-protein_state
+the	O
+AI	B-structure_element
+region	I-structure_element
+is	O
+conformationally	B-protein_state
+heterogeneous	I-protein_state
+
+Given	O
+that	O
+Ser457	B-residue_name_number
+/	O
+453	B-residue_number
+is	O
+far	O
+from	O
+any	O
+channel	B-site
+exit	I-site
+(	O
+Fig	O
+.	O
+6	O
+),	O
+the	O
+crucial	O
+question	O
+is	O
+how	O
+phosphorylation	B-ptm
+opens	O
+the	O
+Mep2	B-protein
+channel	B-site
+to	O
+generate	O
+an	O
+active	B-protein_state
+transporter	B-protein_type
+.	O
+
+Boeckstaens	O
+et	O
+al	O
+.	O
+proposed	O
+that	O
+phosphorylation	B-ptm
+does	O
+not	O
+affect	O
+channel	O
+activity	O
+directly	O
+,	O
+but	O
+instead	O
+relieves	O
+inhibition	O
+by	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+.	O
+
+The	O
+data	O
+behind	O
+this	O
+hypothesis	O
+is	O
+the	O
+observation	O
+that	O
+a	O
+ScMep2	B-protein
+449	B-mutant
+-	I-mutant
+485Δ	I-mutant
+deletion	B-protein_state
+mutant	I-protein_state
+lacking	B-protein_state
+the	O
+AI	B-structure_element
+region	I-structure_element
+is	O
+highly	B-protein_state
+active	I-protein_state
+in	O
+MA	B-chemical
+uptake	O
+both	O
+in	O
+the	O
+triple	B-mutant
+mepΔ	I-mutant
+and	O
+triple	B-mutant
+mepΔ	I-mutant
+npr1Δ	I-mutant
+backgrounds	O
+,	O
+implying	O
+that	O
+this	O
+Mep2	B-mutant
+variant	I-mutant
+has	O
+a	O
+constitutively	B-protein_state
+open	I-protein_state
+channel	B-site
+.	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	B-protein_state
+(	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	B-experimental_method
+and	I-experimental_method
+purified	I-experimental_method
+the	O
+analogous	O
+CaMep2	B-protein
+442Δ	B-mutant
+truncation	B-protein_state
+mutant	I-protein_state
+and	O
+determined	B-experimental_method
+the	O
+crystal	B-evidence
+structure	I-evidence
+using	O
+data	O
+to	O
+3	O
+.	O
+4	O
+Å	O
+resolution	O
+.	O
+
+The	O
+structure	B-evidence
+shows	O
+that	O
+removal	B-experimental_method
+of	I-experimental_method
+the	O
+AI	B-structure_element
+region	I-structure_element
+markedly	O
+increases	O
+the	O
+dynamics	O
+of	O
+the	O
+cytoplasmic	B-structure_element
+parts	I-structure_element
+of	O
+the	O
+transporter	B-protein_type
+.	O
+
+This	O
+is	O
+not	O
+unexpected	O
+given	O
+the	O
+fact	O
+that	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+bridges	O
+the	O
+CTR	B-structure_element
+and	O
+the	O
+main	B-structure_element
+body	I-structure_element
+of	O
+Mep2	B-protein
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+Density	B-evidence
+for	O
+ICL3	B-structure_element
+and	O
+the	O
+CTR	B-structure_element
+beyond	O
+residue	O
+Arg415	B-residue_name_number
+is	O
+missing	O
+in	O
+the	O
+442Δ	B-mutant
+mutant	B-protein_state
+,	O
+and	O
+the	O
+density	B-evidence
+for	O
+the	O
+other	O
+ICLs	B-structure_element
+including	O
+ICL1	B-structure_element
+is	O
+generally	O
+poor	O
+with	O
+visible	O
+parts	O
+of	O
+the	O
+structure	B-evidence
+having	O
+high	O
+B	O
+-	O
+factors	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+
+Interestingly	O
+,	O
+however	O
+,	O
+the	O
+Tyr49	B-residue_name_number
+-	O
+His342	B-residue_name_number
+hydrogen	O
+bond	O
+that	O
+closes	O
+the	O
+channel	O
+in	O
+the	O
+WT	B-protein_state
+protein	O
+is	O
+still	O
+present	O
+(	O
+Fig	O
+.	O
+7	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+
+Why	O
+then	O
+does	O
+this	O
+mutant	O
+appear	O
+to	O
+be	O
+constitutively	O
+active	B-protein_state
+?	O
+We	O
+propose	O
+two	O
+possibilities	O
+.	O
+
+The	O
+first	O
+one	O
+is	O
+that	O
+the	O
+open	B-protein_state
+state	O
+is	O
+disfavoured	O
+by	O
+crystallization	B-experimental_method
+because	O
+of	O
+lower	O
+stability	O
+or	O
+due	O
+to	O
+crystal	O
+packing	O
+constraints	O
+.	O
+
+The	O
+second	O
+possibility	O
+is	O
+that	O
+the	O
+Tyr	B-site
+–	I-site
+His	I-site
+hydrogen	I-site
+bond	I-site
+has	O
+to	O
+be	O
+disrupted	O
+by	O
+the	O
+incoming	O
+substrate	O
+to	O
+open	B-protein_state
+the	O
+channel	O
+.	O
+
+The	O
+latter	O
+model	O
+would	O
+fit	O
+well	O
+with	O
+the	O
+NH3	B-chemical
+/	O
+H	B-chemical
++	I-chemical
+symport	O
+model	O
+in	O
+which	O
+the	O
+proton	O
+is	O
+relayed	O
+by	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+.	O
+
+The	O
+importance	O
+of	O
+the	O
+Tyr	B-site
+–	I-site
+His	I-site
+hydrogen	I-site
+bond	I-site
+is	O
+underscored	O
+by	O
+the	O
+fact	O
+that	O
+its	O
+removal	B-experimental_method
+in	O
+the	O
+ScMep2	B-protein
+Y53A	B-mutant
+mutant	B-protein_state
+results	O
+in	O
+a	O
+constitutively	B-protein_state
+active	I-protein_state
+transporter	B-protein_type
+(	O
+Fig	O
+.	O
+3	O
+).	O
+
+Phosphorylation	B-ptm
+causes	O
+a	O
+conformational	O
+change	O
+in	O
+the	O
+CTR	B-structure_element
+
+Do	O
+the	O
+Mep2	B-protein
+structures	B-evidence
+provide	O
+any	O
+clues	O
+regarding	O
+the	O
+potential	O
+effect	O
+of	O
+phosphorylation	B-ptm
+?	O
+
+The	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+of	O
+Ser457	B-residue_name_number
+/	O
+453	B-residue_number
+is	O
+located	O
+in	O
+a	O
+well	O
+-	O
+defined	O
+electronegative	B-site
+pocket	I-site
+that	O
+is	O
+solvent	B-protein_state
+accessible	I-protein_state
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+closest	O
+atoms	O
+to	O
+the	O
+serine	B-residue_name
+hydroxyl	O
+group	O
+are	O
+the	O
+backbone	O
+carbonyl	O
+atoms	O
+of	O
+Asp419	B-residue_name_number
+,	O
+Glu420	B-residue_name_number
+and	O
+Glu421	B-residue_name_number
+,	O
+which	O
+are	O
+3	O
+–	O
+4	O
+Å	O
+away	O
+.	O
+
+We	O
+therefore	O
+predict	O
+that	O
+phosphorylation	B-ptm
+of	O
+Ser453	B-residue_name_number
+will	O
+result	O
+in	O
+steric	O
+clashes	O
+as	O
+well	O
+as	O
+electrostatic	O
+repulsion	O
+,	O
+which	O
+in	O
+turn	O
+might	O
+cause	O
+substantial	O
+conformational	O
+changes	O
+within	O
+the	O
+CTR	B-structure_element
+.	O
+
+To	O
+test	O
+this	O
+hypothesis	O
+,	O
+we	O
+determined	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+the	O
+phosphorylation	B-protein_state
+-	I-protein_state
+mimicking	I-protein_state
+R452D	B-mutant
+/	I-mutant
+S453D	I-mutant
+protein	O
+(	O
+hereafter	O
+termed	O
+‘	O
+DD	B-mutant
+mutant	I-mutant
+'),	O
+using	O
+data	O
+to	O
+a	O
+resolution	O
+of	O
+2	O
+.	O
+4	O
+Å	O
+.	O
+The	O
+additional	B-experimental_method
+mutation	I-experimental_method
+of	I-experimental_method
+the	O
+arginine	B-residue_name
+preceding	O
+the	O
+phosphorylation	B-site
+site	I-site
+was	O
+introduced	O
+(	O
+i	O
+)	O
+to	O
+increase	O
+the	O
+negative	O
+charge	O
+density	O
+and	O
+make	O
+it	O
+more	O
+comparable	O
+to	O
+a	O
+phosphate	B-chemical
+at	O
+neutral	O
+pH	O
+,	O
+and	O
+(	O
+ii	O
+)	O
+to	O
+further	O
+destabilize	O
+the	O
+interactions	O
+of	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+with	O
+the	O
+main	B-structure_element
+body	I-structure_element
+of	O
+the	O
+transporter	B-protein_type
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+ammonium	B-chemical
+uptake	O
+activity	O
+of	O
+the	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+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	B-protein_state
+Mep2	B-protein
+and	O
+the	O
+S453D	B-mutant
+mutant	B-protein_state
+,	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
+
+Unexpectedly	O
+,	O
+the	O
+AI	B-structure_element
+segment	I-structure_element
+containing	O
+the	O
+mutated	O
+residues	O
+has	O
+only	O
+undergone	O
+a	O
+slight	O
+shift	O
+compared	O
+with	O
+the	O
+WT	B-protein_state
+protein	O
+(	O
+Fig	O
+.	O
+8	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+
+By	O
+contrast	O
+,	O
+the	O
+conserved	B-protein_state
+part	O
+of	O
+the	O
+CTR	B-structure_element
+has	O
+undergone	O
+a	O
+large	O
+conformational	O
+change	O
+involving	O
+formation	O
+of	O
+a	O
+12	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+-	I-structure_element
+long	I-structure_element
+α	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+from	O
+Leu427	B-residue_range
+to	I-residue_range
+Asp438	I-residue_range
+.	O
+
+In	O
+addition	O
+,	O
+residues	O
+Glu420	B-residue_range
+-	I-residue_range
+Leu423	I-residue_range
+including	O
+Glu421	B-residue_name_number
+of	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+are	O
+now	O
+disordered	B-protein_state
+(	O
+Fig	O
+.	O
+8	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+
+This	O
+is	O
+the	O
+first	O
+time	O
+a	O
+large	O
+conformational	O
+change	O
+has	O
+been	O
+observed	O
+in	O
+an	O
+ammonium	B-protein_type
+transporter	I-protein_type
+as	O
+a	O
+result	O
+of	O
+a	O
+mutation	B-experimental_method
+,	O
+and	O
+confirms	O
+previous	O
+hypotheses	O
+that	O
+phosphorylation	B-ptm
+causes	O
+structural	O
+changes	O
+in	O
+the	O
+CTR	B-structure_element
+.	O
+
+To	O
+exclude	O
+the	O
+possibility	O
+that	O
+the	O
+additional	O
+R452D	B-mutant
+mutation	O
+is	O
+responsible	O
+for	O
+the	O
+observed	O
+changes	O
+,	O
+we	O
+also	O
+determined	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+the	O
+‘	O
+single	B-mutant
+D	I-mutant
+'	O
+S453D	B-mutant
+mutant	B-protein_state
+.	O
+
+As	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+,	O
+the	O
+consequence	O
+of	O
+the	O
+single	B-mutant
+D	I-mutant
+mutation	B-experimental_method
+is	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+DD	B-mutant
+substitution	I-mutant
+,	O
+with	O
+conformational	O
+changes	O
+and	O
+increased	O
+dynamics	O
+confined	O
+to	O
+the	O
+conserved	B-protein_state
+part	O
+of	O
+the	O
+CTR	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+
+To	O
+supplement	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+,	O
+we	O
+also	O
+performed	O
+modelling	B-experimental_method
+and	O
+MD	B-experimental_method
+studies	O
+of	O
+WT	B-protein_state
+CaMep2	B-protein
+,	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+and	O
+phosphorylated	B-protein_state
+protein	O
+(	O
+S453J	B-mutant
+).	O
+
+In	O
+the	O
+WT	B-protein_state
+structure	B-evidence
+,	O
+the	O
+acidic	O
+residues	O
+Asp419	B-residue_name_number
+,	O
+Glu420	B-residue_name_number
+and	O
+Glu421	B-residue_name_number
+are	O
+within	O
+hydrogen	O
+bonding	O
+distance	O
+of	O
+Ser453	B-residue_name_number
+.	O
+
+After	O
+200	O
+ns	O
+of	O
+MD	B-experimental_method
+simulation	B-experimental_method
+,	O
+the	O
+interactions	O
+between	O
+these	O
+residues	O
+and	O
+Ser453	B-residue_name_number
+remain	O
+intact	O
+.	O
+
+The	O
+protein	O
+backbone	O
+has	O
+an	O
+average	O
+r	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.	I-evidence
+of	O
+only	O
+∼	O
+3	O
+Å	O
+during	O
+the	O
+200	O
+-	O
+ns	O
+simulation	B-experimental_method
+,	O
+indicating	O
+that	O
+the	O
+protein	O
+is	O
+stable	B-protein_state
+.	O
+
+There	O
+is	O
+flexibility	O
+in	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+acidic	O
+residues	O
+so	O
+that	O
+they	O
+are	O
+able	O
+to	O
+form	O
+stable	B-protein_state
+hydrogen	O
+bonds	O
+with	O
+Ser453	B-residue_name_number
+.	O
+
+In	O
+particular	O
+,	O
+persistent	O
+hydrogen	O
+bonds	O
+are	O
+observed	O
+between	O
+the	O
+Ser453	B-residue_name_number
+hydroxyl	O
+group	O
+and	O
+the	O
+acidic	O
+group	O
+of	O
+Glu420	B-residue_name_number
+,	O
+and	O
+also	O
+between	O
+the	O
+amine	O
+group	O
+of	O
+Ser453	B-residue_name_number
+and	O
+the	O
+backbone	O
+carbonyl	O
+of	O
+Glu420	B-residue_name_number
+(	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+
+The	O
+DD	B-mutant
+mutant	I-mutant
+is	O
+also	O
+stable	B-protein_state
+during	O
+the	O
+simulations	B-experimental_method
+,	O
+but	O
+the	O
+average	O
+backbone	O
+r	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+of	O
+∼	O
+3	O
+.	O
+6	O
+Å	O
+suggests	O
+slightly	O
+more	O
+conformational	O
+flexibility	O
+than	O
+WT	B-protein_state
+.	O
+
+As	O
+the	O
+simulation	B-experimental_method
+proceeds	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+acidic	O
+residues	O
+move	O
+away	O
+from	O
+Asp452	B-residue_name_number
+and	O
+Asp453	B-residue_name_number
+,	O
+presumably	O
+to	O
+avoid	O
+electrostatic	O
+repulsion	O
+.	O
+
+For	O
+example	O
+,	O
+the	O
+distance	B-evidence
+between	O
+the	O
+Asp453	B-residue_name_number
+acidic	O
+oxygens	O
+and	O
+the	O
+Glu420	B-residue_name_number
+acidic	O
+oxygens	O
+increases	O
+from	O
+∼	O
+7	O
+to	O
+>	O
+22	O
+Å	O
+after	O
+200	O
+ns	O
+simulations	B-experimental_method
+,	O
+and	O
+thus	O
+these	O
+residues	O
+are	O
+not	O
+interacting	O
+.	O
+
+The	O
+protein	O
+is	O
+structurally	B-protein_state
+stable	I-protein_state
+throughout	O
+the	O
+simulation	B-experimental_method
+with	O
+little	O
+deviation	O
+in	O
+the	O
+other	O
+parts	O
+of	O
+the	O
+protein	O
+.	O
+
+Finally	O
+,	O
+the	O
+S453J	B-mutant
+mutant	B-protein_state
+is	O
+also	O
+stable	B-protein_state
+throughout	O
+the	O
+200	O
+-	O
+ns	O
+simulation	B-experimental_method
+and	O
+has	O
+an	O
+average	O
+backbone	O
+deviation	O
+of	O
+∼	O
+3	O
+.	O
+8	O
+Å	O
+,	O
+which	O
+is	O
+similar	O
+to	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+.	O
+
+The	O
+movement	O
+of	O
+the	O
+acidic	O
+residues	O
+away	O
+from	O
+Arg452	B-residue_name_number
+and	O
+Sep453	B-residue_name_number
+is	O
+more	O
+pronounced	O
+in	O
+this	O
+simulation	B-experimental_method
+in	O
+comparison	O
+with	O
+the	O
+movement	O
+away	O
+from	O
+Asp452	B-residue_name_number
+and	O
+Asp453	B-residue_name_number
+in	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+.	O
+
+The	O
+distance	B-evidence
+between	O
+the	O
+phosphate	B-chemical
+of	O
+Sep453	B-residue_name_number
+and	O
+the	O
+acidic	O
+oxygen	O
+atoms	O
+of	O
+Glu420	B-residue_name_number
+is	O
+initially	O
+∼	O
+11	O
+Å	O
+,	O
+but	O
+increases	O
+to	O
+>	O
+30	O
+Å	O
+after	O
+200	O
+ns	O
+.	O
+
+The	O
+short	B-structure_element
+helix	I-structure_element
+formed	O
+by	O
+residues	O
+Leu427	B-residue_range
+to	I-residue_range
+Asp438	I-residue_range
+unravels	O
+during	O
+the	O
+simulations	B-experimental_method
+to	O
+a	O
+disordered	B-protein_state
+state	O
+.	O
+
+Thus	O
+,	O
+the	O
+MD	B-experimental_method
+simulations	B-experimental_method
+support	O
+the	O
+notion	O
+from	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+that	O
+phosphorylation	B-ptm
+generates	O
+conformational	O
+changes	O
+in	O
+the	O
+conserved	B-protein_state
+part	O
+of	O
+the	O
+CTR	B-structure_element
+.	O
+
+However	O
+,	O
+the	O
+conformational	O
+changes	O
+for	O
+the	O
+phosphomimetic	B-mutant
+mutants	I-mutant
+in	O
+the	O
+crystals	B-evidence
+are	O
+confined	O
+to	O
+the	O
+CTR	B-structure_element
+(	O
+Fig	O
+.	O
+8	O
+),	O
+and	O
+the	O
+channels	B-site
+are	O
+still	O
+closed	B-protein_state
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+
+One	O
+possible	O
+explanation	O
+is	O
+that	O
+the	O
+mutants	B-mutant
+do	O
+not	O
+accurately	O
+mimic	O
+a	O
+phosphoserine	B-residue_name
+,	O
+but	O
+the	O
+observation	O
+that	O
+the	O
+S453D	B-mutant
+and	O
+DD	B-mutant
+mutants	I-mutant
+are	O
+fully	B-protein_state
+active	I-protein_state
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Npr1	B-protein
+suggests	O
+that	O
+the	O
+mutations	B-experimental_method
+do	O
+mimic	O
+the	O
+effect	O
+of	O
+phosphorylation	B-ptm
+(	O
+Fig	O
+.	O
+3	O
+).	O
+
+The	O
+fact	O
+that	O
+the	O
+S453D	B-mutant
+structure	B-evidence
+was	O
+obtained	O
+in	O
+the	O
+presence	O
+of	O
+10	O
+mM	O
+ammonium	B-chemical
+ions	O
+suggests	O
+that	O
+the	O
+crystallization	B-experimental_method
+process	O
+favours	O
+closed	B-protein_state
+states	O
+of	O
+the	O
+Mep2	B-protein
+channels	B-site
+.	O
+
+Knowledge	O
+about	O
+ammonium	B-protein_type
+transporter	I-protein_type
+structure	B-evidence
+has	O
+been	O
+obtained	O
+from	O
+experimental	O
+and	O
+theoretical	O
+studies	O
+on	O
+bacterial	B-taxonomy_domain
+family	O
+members	O
+.	O
+
+In	O
+addition	O
+,	O
+a	O
+number	O
+of	O
+biochemical	B-experimental_method
+and	I-experimental_method
+genetic	I-experimental_method
+studies	I-experimental_method
+are	O
+available	O
+for	O
+bacterial	B-taxonomy_domain
+,	O
+fungal	B-taxonomy_domain
+and	O
+plant	B-taxonomy_domain
+proteins	O
+.	O
+
+These	O
+efforts	O
+have	O
+advanced	O
+our	O
+knowledge	O
+considerably	O
+but	O
+have	O
+not	O
+yet	O
+yielded	O
+atomic	O
+-	O
+level	O
+answers	O
+to	O
+several	O
+important	O
+mechanistic	O
+questions	O
+,	O
+including	O
+how	O
+ammonium	B-chemical
+transport	O
+is	O
+regulated	O
+in	O
+eukaryotes	B-taxonomy_domain
+and	O
+the	O
+mechanism	O
+of	O
+ammonium	B-chemical
+signalling	O
+.	O
+
+In	O
+Arabidopsis	B-species
+thaliana	I-species
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+,	O
+phosphorylation	B-ptm
+of	O
+the	O
+CTR	B-structure_element
+residue	O
+T460	B-residue_name_number
+under	O
+conditions	O
+of	O
+high	O
+ammonium	B-chemical
+inhibits	O
+transport	O
+activity	O
+,	O
+that	O
+is	O
+,	O
+the	O
+default	O
+(	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+)	O
+state	O
+of	O
+the	O
+plant	B-taxonomy_domain
+transporter	B-protein_type
+is	O
+open	B-protein_state
+.	O
+
+Interestingly	O
+,	O
+phosphomimetic	B-mutant
+mutations	I-mutant
+introduced	O
+into	O
+one	O
+monomer	B-oligomeric_state
+inactivate	O
+the	O
+entire	O
+trimer	B-oligomeric_state
+,	O
+indicating	O
+that	O
+(	O
+i	O
+)	O
+heterotrimerization	O
+occurs	O
+and	O
+(	O
+ii	O
+)	O
+the	O
+CTR	B-structure_element
+mediates	O
+allosteric	O
+regulation	O
+of	O
+ammonium	B-chemical
+transport	O
+activity	O
+via	O
+phosphorylation	B-ptm
+.	O
+
+Owing	O
+to	O
+the	O
+lack	O
+of	O
+structural	O
+information	O
+for	O
+plant	B-taxonomy_domain
+AMTs	B-protein_type
+,	O
+the	O
+details	O
+of	O
+channel	B-site
+closure	O
+and	O
+inter	O
+-	O
+monomer	O
+crosstalk	O
+are	O
+not	O
+yet	O
+clear	O
+.	O
+
+Contrasting	O
+with	O
+the	O
+plant	B-taxonomy_domain
+transporters	B-protein_type
+,	O
+the	O
+inactive	B-protein_state
+states	O
+of	O
+Mep2	B-protein_type
+proteins	I-protein_type
+under	O
+conditions	O
+of	O
+high	O
+ammonium	B-chemical
+are	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+,	O
+with	O
+channels	B-site
+that	O
+are	O
+closed	B-protein_state
+on	O
+the	O
+cytoplasmic	O
+side	O
+.	O
+
+The	O
+reason	O
+why	O
+similar	O
+transporters	B-protein_type
+such	O
+as	O
+A	B-species
+.	I-species
+thaliana	I-species
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+and	O
+Mep2	B-protein
+are	O
+regulated	O
+in	O
+opposite	O
+ways	O
+by	O
+phosphorylation	B-ptm
+(	O
+inactivation	B-protein_state
+in	O
+plants	B-taxonomy_domain
+and	O
+activation	B-protein_state
+in	O
+fungi	B-taxonomy_domain
+)	O
+is	O
+not	O
+known	O
+.	O
+
+In	O
+fungi	B-taxonomy_domain
+,	O
+preventing	O
+ammonium	B-chemical
+entry	O
+via	O
+channel	O
+closure	O
+in	O
+ammonium	B-protein_type
+transporters	I-protein_type
+would	O
+be	O
+one	O
+way	O
+to	O
+alleviate	O
+ammonium	B-chemical
+toxicity	O
+,	O
+in	O
+addition	O
+to	O
+ammonium	B-chemical
+excretion	O
+via	O
+Ato	B-protein_type
+transporters	B-protein_type
+and	O
+amino	O
+-	O
+acid	O
+secretion	O
+.	O
+
+By	O
+determining	O
+the	O
+first	O
+structures	B-evidence
+of	O
+closed	B-protein_state
+ammonium	B-protein_type
+transporters	I-protein_type
+and	O
+comparing	B-experimental_method
+those	O
+structures	B-evidence
+with	O
+the	O
+permanently	B-protein_state
+open	I-protein_state
+bacterial	B-taxonomy_domain
+proteins	O
+,	O
+we	O
+demonstrate	O
+that	O
+Mep2	B-protein_type
+channel	B-site
+closure	O
+is	O
+likely	O
+due	O
+to	O
+movements	O
+of	O
+the	O
+CTR	B-structure_element
+and	O
+ICL1	B-structure_element
+and	O
+ICL3	B-structure_element
+.	O
+
+More	O
+specifically	O
+,	O
+the	O
+close	O
+interactions	O
+between	O
+the	O
+CTR	B-structure_element
+and	O
+ICL1	B-structure_element
+/	O
+ICL3	B-structure_element
+present	O
+in	O
+open	B-protein_state
+transporters	B-protein_type
+are	O
+disrupted	O
+,	O
+causing	O
+ICL3	B-structure_element
+to	O
+move	O
+outwards	O
+and	O
+block	O
+the	O
+channel	B-site
+(	O
+Figs	O
+4	O
+and	O
+9a	O
+).	O
+
+In	O
+addition	O
+,	O
+ICL1	B-structure_element
+has	O
+shifted	O
+inwards	O
+to	O
+contribute	O
+to	O
+the	O
+channel	B-site
+closure	O
+by	O
+engaging	O
+His2	B-residue_name_number
+from	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+via	O
+hydrogen	O
+bonding	O
+with	O
+a	O
+highly	B-protein_state
+conserved	I-protein_state
+tyrosine	B-residue_name
+hydroxyl	O
+group	O
+.	O
+
+Upon	O
+phosphorylation	B-ptm
+by	O
+the	O
+Npr1	B-protein
+kinase	B-protein_type
+in	O
+response	O
+to	O
+nitrogen	B-chemical
+limitation	O
+,	O
+the	O
+region	O
+around	O
+the	O
+conserved	B-protein_state
+ExxGxD	B-structure_element
+motif	I-structure_element
+undergoes	O
+a	O
+conformational	O
+change	O
+that	O
+opens	O
+the	O
+channel	B-site
+(	O
+Fig	O
+.	O
+9	O
+).	O
+
+Importantly	O
+,	O
+the	O
+structural	B-evidence
+similarities	I-evidence
+in	O
+the	O
+TM	B-structure_element
+parts	I-structure_element
+of	O
+Mep2	B-protein
+and	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+(	O
+Fig	O
+.	O
+5a	O
+)	O
+suggest	O
+that	O
+channel	B-site
+opening	O
+/	O
+closure	O
+does	O
+not	O
+require	O
+substantial	O
+changes	O
+in	O
+the	O
+residues	O
+lining	O
+the	O
+channel	B-site
+.	O
+
+How	O
+exactly	O
+the	O
+channel	B-site
+opens	O
+and	O
+whether	O
+opening	O
+is	O
+intra	O
+-	O
+monomeric	O
+are	O
+still	O
+open	B-protein_state
+questions	O
+;	O
+it	O
+is	O
+possible	O
+that	O
+the	O
+change	O
+in	O
+the	O
+CTR	B-structure_element
+may	O
+disrupt	O
+its	O
+interactions	O
+with	O
+ICL3	B-structure_element
+of	O
+the	O
+neighbouring	O
+monomer	B-oligomeric_state
+(	O
+Fig	O
+.	O
+9b	O
+),	O
+which	O
+could	O
+result	O
+in	O
+opening	O
+of	O
+the	O
+neighbouring	O
+channel	B-site
+via	O
+inward	O
+movement	O
+of	O
+its	O
+ICL3	B-structure_element
+.	O
+
+Owing	O
+to	O
+the	O
+crosstalk	O
+between	O
+monomers	B-oligomeric_state
+,	O
+a	O
+single	O
+phosphorylation	B-ptm
+event	O
+might	O
+lead	O
+to	O
+opening	O
+of	O
+the	O
+entire	O
+trimer	B-oligomeric_state
+,	O
+although	O
+this	O
+has	O
+not	O
+yet	O
+been	O
+tested	O
+(	O
+Fig	O
+.	O
+9b	O
+).	O
+
+Whether	O
+or	O
+not	O
+Mep2	B-protein_type
+channel	B-site
+opening	O
+requires	O
+,	O
+in	O
+addition	O
+to	O
+phosphorylation	B-ptm
+,	O
+disruption	O
+of	O
+the	O
+Tyr	B-site
+–	I-site
+His2	I-site
+interaction	I-site
+by	O
+the	O
+ammonium	B-chemical
+substrate	O
+is	O
+not	O
+yet	O
+clear	O
+.	O
+
+Is	O
+our	O
+model	O
+for	O
+opening	O
+and	O
+closing	O
+of	O
+Mep2	B-protein
+channels	B-site
+valid	O
+for	O
+other	O
+eukaryotic	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+?	O
+Our	O
+structural	B-evidence
+data	I-evidence
+support	O
+previous	O
+studies	O
+and	O
+clarify	O
+the	O
+central	O
+role	O
+of	O
+the	O
+CTR	B-structure_element
+and	O
+cytoplasmic	B-structure_element
+loops	I-structure_element
+in	O
+the	O
+transition	O
+between	O
+closed	B-protein_state
+and	O
+open	B-protein_state
+states	O
+.	O
+
+However	O
+,	O
+even	O
+the	O
+otherwise	O
+highly	O
+similar	O
+Mep2	B-protein_type
+proteins	I-protein_type
+of	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+and	O
+C	B-species
+.	I-species
+albicans	I-species
+have	O
+different	O
+structures	B-evidence
+for	O
+their	O
+CTRs	B-structure_element
+(	O
+Fig	O
+.	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+In	O
+addition	O
+,	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+of	O
+the	O
+CTR	B-structure_element
+containing	O
+the	O
+Npr1	B-site
+kinase	I-site
+site	I-site
+is	O
+conserved	B-protein_state
+in	O
+only	O
+a	O
+subset	O
+of	O
+fungal	B-taxonomy_domain
+transporters	B-protein_type
+,	O
+suggesting	O
+that	O
+the	O
+details	O
+of	O
+the	O
+structural	O
+changes	O
+underpinning	O
+regulation	O
+vary	O
+.	O
+
+Nevertheless	O
+,	O
+given	O
+the	O
+central	O
+role	O
+of	O
+absolutely	B-protein_state
+conserved	I-protein_state
+residues	O
+within	O
+the	O
+ICL1	B-site
+-	I-site
+ICL3	I-site
+-	I-site
+CTR	I-site
+interaction	I-site
+network	I-site
+(	O
+Fig	O
+.	O
+4	O
+),	O
+we	O
+propose	O
+that	O
+the	O
+structural	O
+basics	O
+of	O
+fungal	B-taxonomy_domain
+ammonium	B-chemical
+transporter	O
+activation	O
+are	O
+conserved	B-protein_state
+.	O
+
+The	O
+fact	O
+that	O
+Mep2	B-protein_type
+orthologues	O
+of	O
+distantly	O
+related	O
+fungi	B-taxonomy_domain
+are	O
+fully	O
+functional	O
+in	O
+ammonium	B-chemical
+transport	O
+and	O
+signalling	O
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+supports	O
+this	O
+notion	O
+.	O
+
+It	O
+should	O
+also	O
+be	O
+noted	O
+that	O
+the	O
+tyrosine	B-residue_name
+residue	O
+interacting	O
+with	O
+His2	B-residue_name_number
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+fungal	B-taxonomy_domain
+Mep2	B-protein_type
+orthologues	O
+,	O
+suggesting	O
+that	O
+the	O
+Tyr	B-site
+–	I-site
+His2	I-site
+hydrogen	I-site
+bond	I-site
+might	O
+be	O
+a	O
+general	O
+way	O
+to	O
+close	B-protein_state
+Mep2	B-protein_type
+proteins	I-protein_type
+.	O
+
+With	O
+regards	O
+to	O
+plant	B-taxonomy_domain
+AMTs	B-protein_type
+,	O
+it	O
+has	O
+been	O
+proposed	O
+that	O
+phosphorylation	B-ptm
+at	O
+T460	B-residue_name_number
+generates	O
+conformational	O
+changes	O
+that	O
+would	O
+close	O
+the	O
+neighbouring	O
+pore	B-site
+via	O
+the	O
+C	B-structure_element
+terminus	I-structure_element
+.	O
+
+This	O
+assumption	O
+was	O
+based	O
+partly	O
+on	O
+a	O
+homology	B-experimental_method
+model	I-experimental_method
+for	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+based	O
+on	O
+the	O
+(	O
+open	B-protein_state
+)	O
+archaebacterial	B-taxonomy_domain
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+,	O
+which	O
+suggested	O
+that	O
+the	O
+C	B-structure_element
+terminus	I-structure_element
+of	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+would	O
+extend	O
+further	O
+to	O
+the	O
+neighbouring	O
+monomer	B-oligomeric_state
+.	O
+
+Our	O
+Mep2	B-protein
+structures	B-evidence
+show	O
+that	O
+this	O
+assumption	O
+may	O
+not	O
+be	O
+correct	O
+(	O
+Fig	O
+.	O
+4	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+In	O
+addition	O
+,	O
+the	O
+considerable	O
+differences	O
+between	O
+structurally	O
+resolved	O
+CTR	B-structure_element
+domains	O
+means	O
+that	O
+the	O
+exact	O
+environment	O
+of	O
+T460	B-residue_name_number
+in	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+is	O
+also	O
+not	O
+known	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+Based	O
+on	O
+the	O
+available	O
+structural	B-evidence
+information	I-evidence
+,	O
+we	O
+consider	O
+it	O
+more	O
+likely	O
+that	O
+phosphorylation	O
+-	O
+mediated	O
+pore	O
+closure	O
+in	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+is	O
+intra	O
+-	O
+monomeric	O
+,	O
+via	O
+disruption	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+CTR	B-structure_element
+and	O
+ICL1	B-structure_element
+/	O
+ICL3	B-structure_element
+(	O
+for	O
+example	O
+,	O
+Y467	B-residue_name_number
+-	O
+H239	B-residue_name_number
+and	O
+D458	B-residue_name_number
+-	O
+K71	B-residue_name_number
+).	O
+
+There	O
+is	O
+generally	O
+no	O
+equivalent	O
+for	O
+CaMep2	B-protein
+Tyr49	B-residue_name_number
+in	O
+plant	B-taxonomy_domain
+AMTs	B-protein_type
+,	O
+indicating	O
+that	O
+a	O
+Tyr	B-site
+–	I-site
+His2	I-site
+hydrogen	I-site
+bond	I-site
+as	O
+observed	O
+in	O
+Mep2	B-protein
+may	O
+not	O
+contribute	O
+to	O
+the	O
+closed	B-protein_state
+state	O
+in	O
+plant	B-taxonomy_domain
+transporters	B-protein_type
+.	O
+
+We	O
+propose	O
+that	O
+intra	B-site
+-	I-site
+monomeric	I-site
+CTR	I-site
+-	I-site
+ICL1	I-site
+/	I-site
+ICL3	I-site
+interactions	I-site
+lie	O
+at	O
+the	O
+basis	O
+of	O
+regulation	O
+of	O
+both	O
+fungal	B-taxonomy_domain
+and	O
+plant	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+;	O
+close	O
+interactions	O
+generate	O
+open	B-protein_state
+channels	B-site
+,	O
+whereas	O
+the	O
+lack	B-protein_state
+of	I-protein_state
+‘	O
+intra	O
+-'	O
+interactions	O
+leads	O
+to	O
+inactive	B-protein_state
+states	O
+.	O
+
+The	O
+need	O
+to	O
+regulate	O
+in	O
+opposite	O
+ways	O
+may	O
+be	O
+the	O
+reason	O
+why	O
+the	O
+phosphorylation	B-site
+sites	I-site
+are	O
+in	O
+different	O
+parts	O
+of	O
+the	O
+CTR	B-structure_element
+,	O
+that	O
+is	O
+,	O
+centrally	O
+located	O
+close	O
+to	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+in	O
+AMTs	B-protein_type
+and	O
+peripherally	O
+in	O
+Mep2	B-protein
+.	O
+
+In	O
+this	O
+way	O
+,	O
+phosphorylation	B-ptm
+can	O
+either	O
+lead	O
+to	O
+channel	B-site
+closing	O
+(	O
+in	O
+the	O
+case	O
+of	O
+AMTs	B-protein_type
+)	O
+or	O
+channel	B-site
+opening	O
+in	O
+the	O
+case	O
+of	O
+Mep2	B-protein
+.	O
+
+Our	O
+model	O
+also	O
+provides	O
+an	O
+explanation	O
+for	O
+the	O
+observation	O
+that	O
+certain	B-mutant
+mutations	I-mutant
+within	O
+the	O
+CTR	B-structure_element
+completely	O
+abolish	O
+transport	O
+activity	O
+.	O
+
+An	O
+example	O
+of	O
+an	O
+inactivating	O
+residue	O
+is	O
+the	O
+glycine	B-residue_name
+of	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+of	O
+the	O
+CTR	B-structure_element
+.	O
+
+Mutation	B-experimental_method
+of	O
+this	O
+residue	O
+(	O
+G393	B-residue_name_number
+in	O
+EcAmtB	B-protein
+;	O
+G456	B-residue_name_number
+in	O
+AtAmt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+)	O
+inactivates	O
+transporters	B-protein_type
+as	O
+diverse	O
+as	O
+Escherichia	B-species
+coli	I-species
+AmtB	B-protein
+and	O
+A	B-species
+.	I-species
+thaliana	I-species
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+(	O
+refs	O
+).	O
+
+Such	O
+mutations	O
+likely	O
+cause	O
+structural	O
+changes	O
+in	O
+the	O
+CTR	B-structure_element
+that	O
+prevent	O
+close	O
+contacts	O
+between	O
+the	O
+CTR	B-structure_element
+and	O
+ICL1	B-structure_element
+/	O
+ICL3	B-structure_element
+,	O
+thereby	O
+stabilizing	O
+a	O
+closed	B-protein_state
+state	O
+that	O
+may	O
+be	O
+similar	O
+to	O
+that	O
+observed	O
+in	O
+Mep2	B-protein
+.	O
+
+Regulation	O
+and	O
+modulation	O
+of	O
+membrane	O
+transport	O
+by	O
+phosphorylation	B-ptm
+is	O
+known	O
+to	O
+occur	O
+in	O
+,	O
+for	O
+example	O
+,	O
+aquaporins	B-protein_type
+and	O
+urea	B-protein_type
+transporters	I-protein_type
+,	O
+and	O
+is	O
+likely	O
+to	O
+be	O
+a	O
+common	O
+theme	O
+for	O
+eukaryotic	B-taxonomy_domain
+channels	B-protein_type
+and	O
+transporters	B-protein_type
+.	O
+
+Recently	O
+,	O
+phosphorylation	B-ptm
+was	O
+also	O
+shown	O
+to	O
+modulate	O
+substrate	O
+affinity	O
+in	O
+nitrate	B-protein_type
+transporters	I-protein_type
+.	O
+
+With	O
+respect	O
+to	O
+ammonium	B-chemical
+transport	O
+,	O
+phosphorylation	B-ptm
+has	O
+thus	O
+far	O
+only	O
+been	O
+shown	O
+for	O
+A	B-species
+.	I-species
+thaliana	I-species
+AMTs	B-protein_type
+and	O
+for	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+Mep2	B-protein
+(	O
+refs	O
+).	O
+
+However	O
+,	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+GlnK	B-protein_type
+proteins	I-protein_type
+in	O
+eukaryotes	B-taxonomy_domain
+suggests	O
+that	O
+phosphorylation	B-ptm
+-	O
+based	O
+regulation	O
+of	O
+ammonium	B-chemical
+transport	O
+may	O
+be	O
+widespread	O
+.	O
+
+With	O
+respect	O
+to	O
+Mep2	B-protein_type
+-	O
+mediated	O
+signalling	O
+to	O
+induce	O
+pseudohyphal	O
+growth	O
+,	O
+two	O
+models	O
+have	O
+been	O
+put	O
+forward	O
+as	O
+to	O
+how	O
+this	O
+occurs	O
+and	O
+why	O
+it	O
+is	O
+specific	O
+to	O
+Mep2	B-protein_type
+proteins	I-protein_type
+.	O
+
+In	O
+one	O
+model	O
+,	O
+signalling	O
+is	O
+proposed	O
+to	O
+depend	O
+on	O
+the	O
+nature	O
+of	O
+the	O
+transported	O
+substrate	O
+,	O
+which	O
+might	O
+be	O
+different	O
+in	O
+certain	O
+subfamilies	O
+of	O
+ammonium	B-protein_type
+transporters	I-protein_type
+(	O
+for	O
+example	O
+,	O
+Mep1	B-protein
+/	O
+Mep3	B-protein
+versus	O
+Mep2	B-protein
+).	O
+
+For	O
+example	O
+,	O
+NH3	B-chemical
+uniport	O
+or	O
+symport	O
+of	O
+NH3	B-chemical
+/	O
+H	B-chemical
++	I-chemical
+might	O
+result	O
+in	O
+changes	O
+in	O
+local	O
+pH	O
+,	O
+but	O
+NH4	B-chemical
++	I-chemical
+uniport	O
+might	O
+not	O
+,	O
+and	O
+this	O
+difference	O
+might	O
+determine	O
+signalling	O
+.	O
+
+In	O
+the	O
+other	O
+model	O
+,	O
+signalling	O
+is	O
+thought	O
+to	O
+require	O
+a	O
+distinct	O
+conformation	O
+of	O
+the	O
+Mep2	B-protein
+transporter	B-protein_type
+occurring	O
+during	O
+the	O
+transport	O
+cycle	O
+.	O
+
+While	O
+the	O
+current	O
+study	O
+does	O
+not	O
+specifically	O
+address	O
+the	O
+mechanism	O
+of	O
+signalling	O
+underlying	O
+pseudohyphal	O
+growth	O
+,	O
+our	O
+structures	B-evidence
+do	O
+show	O
+that	O
+Mep2	B-protein_type
+proteins	I-protein_type
+can	O
+assume	O
+different	O
+conformations	O
+.	O
+
+It	O
+is	O
+clear	O
+that	O
+ammonium	B-chemical
+transport	O
+across	O
+biomembranes	O
+remains	O
+a	O
+fascinating	O
+and	O
+challenging	O
+field	O
+in	O
+large	O
+part	O
+due	O
+to	O
+the	O
+unique	O
+properties	O
+of	O
+the	O
+substrate	O
+.	O
+
+Our	O
+Mep2	B-protein
+structural	O
+work	O
+now	O
+provides	O
+a	O
+foundation	O
+for	O
+future	O
+studies	O
+to	O
+uncover	O
+the	O
+details	O
+of	O
+the	O
+structural	O
+changes	O
+that	O
+occur	O
+during	O
+eukaryotic	B-taxonomy_domain
+ammonium	B-chemical
+transport	O
+and	O
+signaling	O
+,	O
+and	O
+to	O
+assess	O
+the	O
+possibility	O
+to	O
+utilize	O
+small	O
+molecules	O
+to	O
+shut	O
+down	O
+ammonium	B-chemical
+sensing	O
+and	O
+downstream	O
+signalling	O
+pathways	O
+in	O
+pathogenic	O
+fungi	B-taxonomy_domain
+.	O
+
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+crystal	I-evidence
+structures	I-evidence
+of	O
+Mep2	B-protein
+transceptors	B-protein_type
+.	O
+
+(	O
+a	O
+)	O
+Monomer	B-oligomeric_state
+cartoon	O
+models	O
+viewed	O
+from	O
+the	O
+side	O
+for	O
+(	O
+left	O
+)	O
+A	O
+.	O
+fulgidus	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+(	O
+PDB	O
+ID	O
+2B2H	O
+),	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+Mep2	B-protein
+(	O
+middle	O
+)	O
+and	O
+C	B-species
+.	I-species
+albicans	I-species
+Mep2	B-protein
+(	O
+right	O
+).	O
+
+The	O
+region	O
+showing	O
+ICL1	B-structure_element
+(	O
+blue	O
+),	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+)	O
+is	O
+boxed	O
+for	O
+comparison	O
+.	O
+
+(	O
+b	O
+)	O
+CaMep2	B-protein
+trimer	B-oligomeric_state
+viewed	O
+from	O
+the	O
+intracellular	O
+side	O
+(	O
+right	O
+).	O
+
+One	O
+monomer	B-oligomeric_state
+is	O
+coloured	O
+as	O
+in	O
+a	O
+and	O
+one	O
+monomer	B-oligomeric_state
+is	O
+coloured	O
+by	O
+B	O
+-	O
+factor	O
+(	O
+blue	O
+,	O
+low	O
+;	O
+red	O
+;	O
+high	O
+).	O
+
+The	O
+CTR	B-structure_element
+is	O
+boxed	O
+.	O
+
+(	O
+c	O
+)	O
+Overlay	B-experimental_method
+of	O
+ScMep2	B-protein
+(	O
+grey	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+rainbow	O
+),	O
+illustrating	O
+the	O
+differences	O
+in	O
+the	O
+CTRs	B-structure_element
+.	O
+
+Sequence	B-evidence
+conservation	I-evidence
+in	O
+ammonium	B-protein_type
+transporters	I-protein_type
+.	O
+
+ClustalW	B-experimental_method
+alignment	I-experimental_method
+of	O
+CaMep2	B-protein
+,	O
+ScMep2	B-protein
+,	O
+A	B-species
+.	I-species
+fulgidus	I-species
+Amt	B-protein
+-	I-protein
+1	I-protein
+,	O
+E	O
+.	O
+coli	O
+AmtB	B-protein
+and	O
+A	B-species
+.	I-species
+thaliana	I-species
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+.	O
+
+The	O
+secondary	O
+structure	O
+elements	O
+observed	O
+for	O
+CaMep2	B-protein
+are	O
+indicated	O
+,	O
+with	O
+the	O
+numbers	O
+corresponding	O
+to	O
+the	O
+centre	O
+of	O
+the	O
+TM	B-structure_element
+segment	I-structure_element
+.	O
+
+The	O
+conserved	B-protein_state
+RxK	B-structure_element
+motif	I-structure_element
+in	O
+ICL1	B-structure_element
+is	O
+boxed	O
+in	O
+blue	O
+,	O
+the	O
+ER	B-structure_element
+motif	I-structure_element
+in	O
+ICL2	B-structure_element
+in	O
+cyan	O
+,	O
+the	O
+conserved	B-protein_state
+ExxGxD	B-structure_element
+motif	I-structure_element
+of	O
+the	O
+CTR	B-structure_element
+in	O
+red	O
+and	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+in	O
+yellow	O
+.	O
+
+Coloured	O
+residues	O
+are	O
+functionally	O
+important	O
+and	O
+correspond	O
+to	O
+those	O
+of	O
+the	O
+Phe	B-site
+gate	I-site
+(	O
+blue	O
+),	O
+the	O
+binding	B-site
+site	I-site
+Trp	B-residue_name
+residue	O
+(	O
+magenta	O
+)	O
+and	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+(	O
+red	O
+).	O
+
+The	O
+Npr1	B-site
+kinase	I-site
+site	I-site
+in	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+is	O
+highlighted	O
+pink	O
+.	O
+
+The	O
+grey	O
+sequences	O
+at	O
+the	O
+C	O
+termini	O
+of	O
+CaMep2	B-protein
+and	O
+ScMep2	B-protein
+are	O
+not	O
+visible	O
+in	O
+the	O
+structures	B-evidence
+and	O
+are	O
+likely	B-protein_state
+disordered	I-protein_state
+.	O
+
+Growth	B-experimental_method
+of	O
+ScMep2	B-mutant
+variants	I-mutant
+on	O
+low	O
+ammonium	O
+medium	O
+.	O
+
+(	O
+a	O
+)	O
+The	O
+triple	B-mutant
+mepΔ	I-mutant
+strain	O
+(	O
+black	O
+)	O
+and	O
+triple	O
+mepΔ	O
+npr1Δ	O
+strain	O
+(	O
+grey	O
+)	O
+containing	O
+plasmids	O
+expressing	O
+WT	B-protein_state
+and	O
+variant	B-mutant
+ScMep2	I-mutant
+were	O
+grown	B-experimental_method
+on	I-experimental_method
+minimal	I-experimental_method
+medium	I-experimental_method
+containing	O
+1	O
+mM	O
+ammonium	B-chemical
+sulphate	I-chemical
+.	O
+
+The	O
+quantified	O
+cell	B-evidence
+density	I-evidence
+reflects	O
+logarithmic	O
+growth	O
+after	O
+24	O
+h	O
+.	O
+Error	O
+bars	O
+are	O
+the	O
+s	O
+.	O
+d	O
+.	O
+for	O
+three	O
+replicates	O
+of	O
+each	O
+strain	O
+(	O
+b	O
+)	O
+The	O
+strains	O
+used	O
+in	O
+a	O
+were	O
+also	O
+serially	O
+diluted	O
+and	O
+spotted	O
+onto	O
+minimal	O
+agar	O
+plates	O
+containing	O
+glutamate	B-chemical
+(	O
+0	O
+.	O
+1	O
+%)	O
+or	O
+ammonium	B-chemical
+sulphate	I-chemical
+(	O
+1	O
+mM	O
+),	O
+and	O
+grown	O
+for	O
+3	O
+days	O
+at	O
+30	O
+°	O
+C	O
+.	O
+
+Structural	O
+differences	O
+between	O
+Mep2	B-protein
+and	O
+bacterial	B-taxonomy_domain
+ammonium	O
+transporters	O
+.	O
+
+(	O
+a	O
+)	O
+ICL1	B-structure_element
+in	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+(	O
+light	O
+blue	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+dark	O
+blue	O
+),	O
+showing	O
+unwinding	O
+and	O
+inward	O
+movement	O
+in	O
+the	O
+fungal	B-taxonomy_domain
+protein	O
+.	O
+(	O
+b	O
+)	O
+Stereo	O
+diagram	O
+viewed	O
+from	O
+the	O
+cytosol	O
+of	O
+ICL1	B-structure_element
+,	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+)	O
+in	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+(	O
+light	O
+colours	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+dark	O
+colours	O
+).	O
+
+The	O
+side	O
+chains	O
+of	O
+residues	O
+in	O
+the	O
+RxK	B-structure_element
+motif	I-structure_element
+as	O
+well	O
+as	O
+those	O
+of	O
+Tyr49	B-residue_name_number
+and	O
+His342	B-residue_name_number
+are	O
+labelled	O
+.	O
+
+The	O
+numbering	O
+is	O
+for	O
+CaMep2	B-protein
+.	O
+
+(	O
+c	O
+)	O
+Conserved	B-protein_state
+residues	O
+in	O
+ICL1	B-structure_element
+-	I-structure_element
+3	I-structure_element
+and	O
+the	O
+CTR	B-structure_element
+.	O
+
+Views	O
+from	O
+the	O
+cytosol	O
+for	O
+CaMep2	B-protein
+(	O
+left	O
+)	O
+and	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+,	O
+highlighting	O
+the	O
+large	O
+differences	O
+in	O
+conformation	O
+of	O
+the	O
+conserved	B-protein_state
+residues	O
+in	O
+ICL1	B-structure_element
+(	O
+RxK	O
+motif	O
+;	O
+blue	O
+),	O
+ICL2	B-structure_element
+(	O
+ER	B-structure_element
+motif	I-structure_element
+;	O
+cyan	O
+),	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+).	O
+
+The	O
+labelled	O
+residues	O
+are	O
+analogous	O
+within	O
+both	O
+structures	B-evidence
+.	O
+
+In	O
+b	O
+and	O
+c	O
+,	O
+the	O
+centre	O
+of	O
+the	O
+trimer	B-oligomeric_state
+is	O
+at	O
+top	O
+.	O
+
+Channel	O
+closures	O
+in	O
+Mep2	B-protein
+.	O
+
+(	O
+a	O
+)	O
+Stereo	O
+superposition	B-experimental_method
+of	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+and	O
+CaMep2	B-protein
+showing	O
+the	O
+residues	O
+of	O
+the	O
+Phe	B-site
+gate	I-site
+,	O
+His2	B-residue_name_number
+of	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+and	O
+the	O
+tyrosine	B-residue_name
+residue	O
+Y49	B-residue_name_number
+in	O
+TM1	B-structure_element
+that	O
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+His2	B-residue_name_number
+in	O
+CaMep2	B-protein
+.	O
+(	O
+b	O
+)	O
+Surface	O
+views	O
+from	O
+the	O
+side	O
+in	O
+rainbow	O
+colouring	O
+,	O
+showing	O
+the	O
+two	O
+-	O
+tier	O
+channel	B-structure_element
+block	I-structure_element
+(	O
+indicated	O
+by	O
+the	O
+arrows	O
+)	O
+in	O
+CaMep2	B-protein
+.	O
+
+The	O
+Npr1	B-protein
+kinase	B-protein_type
+target	O
+Ser453	B-residue_name_number
+is	O
+dephosphorylated	B-protein_state
+and	O
+located	O
+in	O
+an	O
+electronegative	B-site
+pocket	I-site
+.	O
+
+(	O
+a	O
+)	O
+Stereoviews	O
+of	O
+CaMep2	B-protein
+showing	O
+2Fo	O
+–	O
+Fc	O
+electron	O
+density	O
+(	O
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+)	O
+for	O
+CTR	B-structure_element
+residues	O
+Asp419	B-residue_range
+-	I-residue_range
+Met422	I-residue_range
+and	O
+for	O
+Tyr446	B-residue_range
+-	I-residue_range
+Thr455	I-residue_range
+of	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+.	O
+
+The	O
+phosphorylation	B-ptm
+target	O
+residue	O
+Ser453	B-residue_name_number
+is	O
+labelled	O
+in	O
+bold	O
+.	O
+
+(	O
+b	O
+)	O
+Overlay	B-experimental_method
+of	O
+the	O
+CTRs	B-structure_element
+of	O
+ScMep2	B-protein
+(	O
+grey	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+green	O
+),	O
+showing	O
+the	O
+similar	O
+electronegative	O
+environment	O
+surrounding	O
+the	O
+phosphorylation	B-site
+site	I-site
+(	O
+P	O
+).	O
+
+The	O
+AI	B-structure_element
+regions	I-structure_element
+are	O
+coloured	O
+magenta	O
+.	O
+
+(	O
+c	O
+)	O
+Cytoplasmic	O
+view	O
+of	O
+the	O
+Mep2	B-protein
+trimer	B-oligomeric_state
+indicating	O
+the	O
+large	O
+distance	O
+between	O
+Ser453	B-residue_name_number
+and	O
+the	O
+channel	B-site
+exits	I-site
+(	O
+circles	O
+;	O
+Ile52	B-residue_name_number
+lining	O
+the	O
+channel	B-site
+exit	I-site
+is	O
+shown	O
+).	O
+
+Effect	O
+of	O
+removal	B-experimental_method
+of	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+on	O
+Mep2	B-protein
+structure	B-evidence
+.	O
+
+(	O
+a	O
+)	O
+Side	O
+views	O
+for	O
+WT	B-protein_state
+CaMep2	B-protein
+(	O
+left	O
+)	O
+and	O
+the	O
+truncation	B-protein_state
+mutant	I-protein_state
+442Δ	B-mutant
+(	O
+right	O
+).	O
+
+The	O
+latter	O
+is	O
+shown	O
+as	O
+a	O
+putty	O
+model	O
+according	O
+to	O
+B	O
+-	O
+factors	O
+to	O
+illustrate	O
+the	O
+disorder	B-protein_state
+in	O
+the	O
+protein	O
+on	O
+the	O
+cytoplasmic	O
+side	O
+.	O
+
+Missing	O
+regions	O
+are	O
+labelled	O
+.	O
+(	O
+b	O
+)	O
+Stereo	O
+superpositions	B-experimental_method
+of	O
+WT	B-protein_state
+CaMep2	B-protein
+and	O
+the	O
+truncation	B-protein_state
+mutant	I-protein_state
+.	O
+
+2Fo	O
+–	O
+Fc	O
+electron	O
+density	O
+(	O
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+)	O
+for	O
+residues	O
+Tyr49	B-residue_name_number
+and	O
+His342	B-residue_name_number
+is	O
+shown	O
+for	O
+the	O
+truncation	B-protein_state
+mutant	I-protein_state
+.	O
+
+Phosphorylation	B-ptm
+causes	O
+conformational	O
+changes	O
+in	O
+the	O
+CTR	B-structure_element
+.	O
+
+(	O
+a	O
+)	O
+Cytoplasmic	O
+view	O
+of	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+trimer	B-oligomeric_state
+,	O
+with	O
+WT	B-protein_state
+CaMep2	B-protein
+superposed	B-experimental_method
+in	O
+grey	O
+for	O
+one	O
+of	O
+the	O
+monomers	B-oligomeric_state
+.	O
+
+The	O
+arrow	O
+indicates	O
+the	O
+phosphorylation	B-site
+site	I-site
+.	O
+
+The	O
+AI	B-structure_element
+region	I-structure_element
+is	O
+coloured	O
+magenta	O
+.	O
+
+(	O
+b	O
+)	O
+Monomer	B-oligomeric_state
+side	O
+-	O
+view	O
+superposition	B-experimental_method
+of	O
+WT	B-protein_state
+CaMep2	B-protein
+and	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+,	O
+showing	O
+the	O
+conformational	O
+change	O
+and	O
+disorder	O
+around	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+.	O
+
+Side	O
+chains	O
+for	O
+residues	O
+452	B-residue_number
+and	O
+453	B-residue_number
+are	O
+shown	O
+as	O
+stick	O
+models	O
+.	O
+
+Schematic	O
+model	O
+for	O
+phosphorylation	O
+-	O
+based	O
+regulation	O
+of	O
+Mep2	B-protein
+ammonium	O
+transporters	O
+.	O
+
+(	O
+a	O
+)	O
+In	O
+the	O
+closed	B-protein_state
+,	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+state	O
+(	O
+i	O
+),	O
+the	O
+CTR	B-structure_element
+(	O
+magenta	O
+)	O
+and	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+are	O
+far	O
+apart	O
+with	O
+the	O
+latter	O
+blocking	O
+the	O
+intracellular	O
+channel	B-site
+exit	I-site
+(	O
+indicated	O
+with	O
+a	O
+hatched	O
+circle	O
+).	O
+
+Upon	O
+phosphorylation	B-ptm
+and	O
+mimicked	B-protein_state
+by	O
+the	O
+CaMep2	B-protein
+S453D	B-mutant
+and	O
+DD	B-mutant
+mutants	I-mutant
+(	O
+ii	O
+),	O
+the	O
+region	O
+around	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+undergoes	O
+a	O
+conformational	O
+change	O
+that	O
+results	O
+in	O
+the	O
+CTR	B-structure_element
+interacting	O
+with	O
+the	O
+inward	O
+-	O
+moving	O
+ICL3	B-structure_element
+,	O
+opening	O
+the	O
+channel	B-site
+(	O
+full	O
+circle	O
+)	O
+(	O
+iii	O
+).	O
+
+The	O
+open	B-protein_state
+-	O
+channel	B-site
+Mep2	B-protein
+structure	B-evidence
+is	O
+represented	O
+by	O
+archaebacterial	B-taxonomy_domain
+Amt	B-protein
+-	I-protein
+1	I-protein
+and	O
+shown	O
+in	O
+lighter	O
+colours	O
+consistent	O
+with	O
+Fig	O
+.	O
+4	O
+.	O
+
+As	O
+discussed	O
+in	O
+the	O
+text	O
+,	O
+similar	O
+structural	O
+arrangements	O
+may	O
+occur	O
+in	O
+plant	B-taxonomy_domain
+AMTs	B-protein_type
+.	O
+
+In	O
+this	O
+case	O
+however	O
+,	O
+the	O
+open	B-protein_state
+channel	B-site
+corresponds	O
+to	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+state	O
+;	O
+phosphorylation	B-ptm
+breaks	O
+the	O
+CTR	O
+–	O
+ICL3	O
+interactions	O
+leading	O
+to	O
+channel	B-site
+closure	O
+.	O
+(	O
+b	O
+)	O
+Model	O
+based	O
+on	O
+AMT	O
+transporter	O
+analogy	O
+showing	O
+how	O
+phosphorylation	B-ptm
+of	O
+a	O
+Mep2	B-protein
+monomer	B-oligomeric_state
+might	O
+allosterically	O
+open	B-protein_state
+channels	B-site
+in	O
+the	O
+entire	O
+trimer	B-oligomeric_state
+via	O
+disruption	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+CTR	B-structure_element
+and	O
+ICL3	B-structure_element
+of	O
+a	O
+neighbouring	O
+monomer	B-oligomeric_state
+(	O
+arrow	O
+).	O
+
+Visualizing	O
+chaperone	B-protein_type
+-	O
+assisted	O
+protein	O
+folding	O
+
+Challenges	O
+in	O
+determining	O
+the	O
+structures	B-evidence
+of	O
+heterogeneous	O
+and	O
+dynamic	O
+protein	O
+complexes	O
+have	O
+greatly	O
+hampered	O
+past	O
+efforts	O
+to	O
+obtain	O
+a	O
+mechanistic	O
+understanding	O
+of	O
+many	O
+important	O
+biological	O
+processes	O
+.	O
+
+One	O
+such	O
+process	O
+is	O
+chaperone	B-protein_type
+-	O
+assisted	O
+protein	O
+folding	O
+,	O
+where	O
+obtaining	O
+structural	O
+ensembles	O
+of	O
+chaperone	B-protein_type
+:	O
+substrate	O
+complexes	O
+would	O
+ultimately	O
+reveal	O
+how	O
+chaperones	B-protein_type
+help	O
+proteins	O
+fold	O
+into	O
+their	O
+native	O
+state	O
+.	O
+
+To	O
+address	O
+this	O
+problem	O
+,	O
+we	O
+devised	O
+a	O
+novel	O
+structural	O
+biology	O
+approach	O
+based	O
+on	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+,	O
+termed	O
+Residual	B-experimental_method
+Electron	I-experimental_method
+and	I-experimental_method
+Anomalous	I-experimental_method
+Density	I-experimental_method
+(	O
+READ	B-experimental_method
+).	O
+
+READ	B-experimental_method
+enabled	O
+us	O
+to	O
+visualize	O
+even	O
+sparsely	O
+populated	O
+conformations	O
+of	O
+the	O
+substrate	O
+protein	O
+immunity	B-protein
+protein	I-protein
+7	I-protein
+(	O
+Im7	B-protein
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+E	B-species
+.	I-species
+coli	I-species
+chaperone	B-protein_type
+Spy	B-protein
+.	O
+
+This	O
+study	O
+resulted	O
+in	O
+a	O
+series	O
+of	O
+snapshots	O
+depicting	O
+the	O
+various	O
+folding	O
+states	O
+of	O
+Im7	B-protein
+while	O
+bound	B-protein_state
+to	I-protein_state
+Spy	B-protein
+.	O
+
+The	O
+ensemble	O
+shows	O
+that	O
+Spy	B-protein_state
+-	I-protein_state
+associated	I-protein_state
+Im7	B-protein
+samples	O
+conformations	O
+ranging	O
+from	O
+unfolded	B-protein_state
+to	O
+partially	O
+folded	B-protein_state
+and	O
+native	B-protein_state
+-	O
+like	O
+states	O
+,	O
+and	O
+reveals	O
+how	O
+a	O
+substrate	O
+can	O
+explore	O
+its	O
+folding	O
+landscape	O
+while	O
+bound	B-protein_state
+to	I-protein_state
+a	O
+chaperone	B-protein_type
+.	O
+
+High	O
+-	O
+resolution	O
+structural	B-evidence
+models	I-evidence
+of	O
+protein	O
+-	O
+protein	O
+interactions	O
+are	O
+critical	O
+for	O
+obtaining	O
+mechanistic	O
+insights	O
+into	O
+biological	O
+processes	O
+.	O
+
+However	O
+,	O
+many	O
+protein	O
+-	O
+protein	O
+interactions	O
+are	O
+highly	B-protein_state
+dynamic	I-protein_state
+,	O
+making	O
+it	O
+difficult	O
+to	O
+obtain	O
+high	O
+-	O
+resolution	O
+data	O
+.	O
+
+Particularly	O
+challenging	O
+are	O
+interactions	O
+of	O
+intrinsically	B-protein_state
+or	I-protein_state
+conditionally	I-protein_state
+disordered	I-protein_state
+sections	O
+of	O
+proteins	O
+with	O
+their	O
+partner	O
+proteins	O
+.	O
+
+Recent	O
+advances	O
+in	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+and	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+continue	O
+to	O
+improve	O
+our	O
+ability	O
+to	O
+analyze	O
+biomolecules	O
+that	O
+exist	O
+in	O
+multiple	O
+conformations	O
+.	O
+
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+has	O
+historically	O
+provided	O
+valuable	O
+information	O
+on	O
+small	O
+-	O
+scale	O
+conformational	O
+changes	O
+,	O
+but	O
+observing	O
+large	O
+-	O
+amplitude	O
+heterogeneous	O
+conformational	O
+changes	O
+often	O
+falls	O
+beyond	O
+the	O
+reach	O
+of	O
+current	O
+crystallographic	O
+techniques	O
+.	O
+
+NMR	B-experimental_method
+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
+
+It	O
+is	O
+clear	O
+that	O
+molecular	O
+chaperones	B-protein_type
+aid	O
+in	O
+protein	O
+folding	O
+.	O
+
+Structural	O
+characterization	O
+of	O
+chaperone	B-protein_type
+-	O
+assisted	O
+protein	O
+folding	O
+likely	O
+would	O
+help	O
+bring	O
+clarity	O
+to	O
+this	O
+question	O
+.	O
+
+Structural	B-evidence
+models	I-evidence
+of	O
+chaperone	B-protein_type
+-	O
+substrate	O
+complexes	O
+have	O
+recently	O
+begun	O
+to	O
+provide	O
+information	O
+as	O
+to	O
+how	O
+a	O
+chaperone	B-protein_type
+can	O
+recognize	O
+its	O
+substrate	O
+.	O
+
+However	O
+,	O
+the	O
+impact	O
+that	O
+chaperones	B-protein_type
+have	O
+on	O
+their	O
+substrates	O
+,	O
+and	O
+how	O
+these	O
+interactions	O
+affect	O
+the	O
+folding	O
+process	O
+remain	O
+largely	O
+unknown	O
+.	O
+
+For	O
+most	O
+chaperones	B-protein_type
+,	O
+it	O
+is	O
+still	O
+unclear	O
+whether	O
+the	O
+chaperone	B-protein_type
+actively	O
+participates	O
+in	O
+and	O
+affects	O
+the	O
+folding	O
+of	O
+the	O
+substrate	O
+proteins	O
+,	O
+or	O
+merely	O
+provides	O
+a	O
+suitable	O
+microenvironment	O
+enabling	O
+the	O
+substrate	O
+to	O
+fold	O
+on	O
+its	O
+own	O
+.	O
+
+This	O
+is	O
+a	O
+truly	O
+fundamental	O
+question	O
+in	O
+the	O
+chaperone	B-protein_type
+field	O
+,	O
+and	O
+one	O
+that	O
+has	O
+eluded	O
+the	O
+community	O
+largely	O
+because	O
+of	O
+the	O
+highly	B-protein_state
+dynamic	I-protein_state
+nature	O
+of	O
+the	O
+chaperone	B-protein_type
+-	O
+substrate	O
+complexes	O
+.	O
+
+To	O
+address	O
+this	O
+question	O
+,	O
+we	O
+investigated	O
+the	O
+ATP	B-protein_state
+-	I-protein_state
+independent	I-protein_state
+Escherichia	B-species
+coli	I-species
+periplasmic	O
+chaperone	B-protein_type
+Spy	B-protein
+.	O
+
+Spy	B-protein
+prevents	O
+protein	O
+aggregation	O
+and	O
+aids	O
+in	O
+protein	O
+folding	O
+under	O
+various	O
+stress	O
+conditions	O
+,	O
+including	O
+treatment	O
+with	O
+tannin	B-chemical
+and	O
+butanol	B-chemical
+.	O
+
+We	O
+originally	O
+discovered	O
+Spy	B-protein
+by	O
+its	O
+ability	O
+to	O
+stabilize	O
+the	O
+protein	O
+-	O
+folding	O
+model	O
+Im7	B-protein
+in	O
+vivo	O
+and	O
+recently	O
+demonstrated	O
+that	O
+Im7	B-protein
+folds	O
+while	O
+associated	O
+with	O
+Spy	B-protein
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+Spy	B-protein
+revealed	O
+that	O
+it	O
+forms	O
+a	O
+thin	O
+α	O
+-	O
+helical	O
+homodimeric	B-oligomeric_state
+cradle	B-site
+.	O
+
+Crosslinking	B-experimental_method
+and	I-experimental_method
+genetic	I-experimental_method
+experiments	I-experimental_method
+suggested	O
+that	O
+Spy	B-protein
+interacts	O
+with	O
+substrates	O
+somewhere	O
+on	O
+its	O
+concave	O
+side	O
+.	O
+
+By	O
+using	O
+a	O
+novel	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+-	O
+based	O
+approach	O
+to	O
+model	O
+disorder	O
+in	O
+crystal	B-evidence
+structures	I-evidence
+,	O
+we	O
+have	O
+now	O
+determined	O
+the	O
+high	O
+-	O
+resolution	O
+ensemble	B-evidence
+of	O
+the	O
+dynamic	B-protein_state
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im7	I-complex_assembly
+complex	O
+.	O
+
+This	O
+work	O
+provides	O
+a	O
+detailed	O
+view	O
+of	O
+chaperone	B-protein_type
+-	O
+mediated	O
+protein	O
+folding	O
+and	O
+shows	O
+how	O
+substrates	O
+like	O
+Im7	B-protein
+find	O
+their	O
+native	O
+fold	O
+while	O
+bound	B-protein_state
+to	I-protein_state
+their	O
+chaperones	B-protein_type
+.	O
+
+Crystallizing	B-experimental_method
+the	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im7	I-complex_assembly
+complex	O
+
+We	O
+reasoned	O
+that	O
+to	O
+obtain	O
+crystals	B-evidence
+of	O
+complexes	O
+between	O
+Spy	B-protein
+(	O
+domain	O
+boundaries	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+)	O
+and	O
+its	O
+substrate	O
+proteins	O
+,	O
+our	O
+best	O
+approach	O
+was	O
+to	O
+identify	O
+crystallization	B-experimental_method
+conditions	I-experimental_method
+that	O
+yielded	O
+Spy	B-protein
+crystals	B-evidence
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+protein	O
+substrates	O
+but	O
+not	O
+in	O
+their	O
+absence	B-protein_state
+.	O
+
+We	O
+therefore	O
+screened	B-experimental_method
+crystallization	B-experimental_method
+conditions	I-experimental_method
+for	O
+Spy	B-protein
+with	O
+four	O
+different	O
+substrate	O
+proteins	O
+:	O
+a	O
+fragment	O
+of	O
+the	O
+largely	O
+unfolded	B-protein_state
+bovine	B-taxonomy_domain
+α	B-chemical
+-	I-chemical
+casein	I-chemical
+protein	O
+,	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+E	B-species
+.	I-species
+coli	I-species
+Im7	B-protein
+,	O
+an	O
+unfolded	B-protein_state
+variant	O
+of	O
+Im7	B-protein
+(	O
+L18A	B-mutant
+L19A	B-mutant
+L37A	B-mutant
+),	O
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+Im7	B-protein
+(	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+),	O
+which	O
+encompasses	O
+the	O
+entire	O
+Spy	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+portion	I-structure_element
+of	O
+Im7	B-protein
+.	O
+
+We	O
+found	O
+conditions	O
+in	O
+which	O
+all	O
+four	O
+substrates	O
+co	B-experimental_method
+-	I-experimental_method
+crystallized	I-experimental_method
+with	B-protein_state
+Spy	B-protein
+,	O
+but	O
+in	O
+which	O
+Spy	B-protein
+alone	B-protein_state
+did	O
+not	O
+yield	O
+crystals	B-evidence
+.	O
+
+Subsequent	O
+crystal	B-experimental_method
+washing	I-experimental_method
+and	I-experimental_method
+dissolution	I-experimental_method
+experiments	O
+confirmed	O
+the	O
+presence	O
+of	O
+the	O
+substrates	O
+in	O
+the	O
+co	B-experimental_method
+-	I-experimental_method
+crystals	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+
+The	O
+crystals	B-evidence
+diffracted	O
+to	O
+~	O
+1	O
+.	O
+8	O
+Å	O
+resolution	O
+.	O
+
+We	O
+used	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+selenomethionine	B-chemical
+crystals	B-evidence
+for	O
+phasing	O
+with	O
+single	B-experimental_method
+-	I-experimental_method
+wavelength	I-experimental_method
+anomalous	I-experimental_method
+diffraction	I-experimental_method
+(	O
+SAD	B-experimental_method
+)	O
+experiments	O
+,	O
+and	O
+used	O
+this	O
+solution	O
+to	O
+build	O
+the	O
+well	O
+-	O
+ordered	O
+Spy	B-protein
+portions	O
+of	O
+all	O
+four	O
+complexes	O
+.	O
+
+However	O
+,	O
+modeling	O
+of	O
+the	O
+substrate	O
+in	O
+the	O
+complex	O
+proved	O
+to	O
+be	O
+a	O
+substantial	O
+challenge	O
+,	O
+as	O
+the	O
+electron	B-evidence
+density	I-evidence
+of	O
+the	O
+substrate	O
+was	O
+discontinuous	O
+and	O
+fragmented	O
+.	O
+
+Even	O
+the	O
+minimal	B-structure_element
+binding	I-structure_element
+portion	I-structure_element
+of	O
+Im7	B-protein
+(	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+)	O
+showed	O
+highly	O
+dispersed	O
+electron	B-evidence
+density	I-evidence
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+
+We	O
+hypothesized	O
+that	O
+the	O
+fragmented	O
+density	B-evidence
+was	O
+due	O
+to	O
+multiple	O
+,	O
+partially	O
+occupied	O
+conformations	O
+of	O
+the	O
+substrate	O
+bound	O
+within	O
+the	O
+crystal	B-evidence
+.	O
+
+Such	O
+residual	O
+density	O
+is	O
+typically	O
+not	O
+considered	O
+usable	O
+by	O
+traditional	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+methods	O
+.	O
+
+Thus	O
+,	O
+we	O
+developed	O
+a	O
+new	O
+approach	O
+to	O
+interpret	O
+the	O
+chaperone	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+substrate	O
+in	O
+multiple	O
+conformations	O
+.	O
+
+READ	B-experimental_method
+:	O
+a	O
+strategy	O
+to	O
+visualize	O
+heterogeneous	O
+and	O
+dynamic	O
+biomolecules	O
+
+To	O
+determine	O
+the	O
+structure	B-evidence
+of	O
+the	O
+substrate	O
+portion	O
+of	O
+these	O
+Spy	B-protein
+:	O
+substrate	O
+complexes	O
+,	O
+we	O
+conceived	O
+of	O
+an	O
+approach	O
+that	O
+we	O
+term	O
+READ	B-experimental_method
+,	O
+for	O
+Residual	B-experimental_method
+Electron	I-experimental_method
+and	I-experimental_method
+Anomalous	I-experimental_method
+Density	I-experimental_method
+.	O
+
+We	O
+split	O
+this	O
+approach	O
+into	O
+five	O
+steps	O
+:	O
+(	O
+1	O
+)	O
+By	O
+using	O
+a	O
+well	O
+-	O
+diffracting	O
+Spy	B-protein
+:	O
+substrate	O
+co	B-evidence
+-	I-evidence
+crystal	I-evidence
+,	O
+we	O
+first	O
+determined	O
+the	O
+structure	B-evidence
+of	O
+the	O
+folded	B-protein_state
+domain	B-structure_element
+of	O
+Spy	B-protein
+and	O
+obtained	O
+high	O
+quality	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+within	O
+the	O
+dynamic	B-protein_state
+regions	O
+of	O
+the	O
+substrate	O
+.	O
+
+(	O
+2	O
+)	O
+We	O
+then	O
+labeled	O
+individual	O
+residues	O
+in	O
+the	O
+flexible	B-protein_state
+regions	O
+of	O
+the	O
+substrate	O
+with	O
+the	O
+strong	O
+anomalous	O
+scatterer	O
+iodine	B-chemical
+,	O
+which	O
+serves	O
+to	O
+locate	O
+these	O
+residues	O
+in	O
+three	O
+-	O
+dimensional	O
+space	O
+using	O
+their	O
+anomalous	B-evidence
+density	I-evidence
+.	O
+
+(	O
+3	O
+)	O
+We	O
+performed	O
+molecular	B-experimental_method
+dynamics	I-experimental_method
+(	O
+MD	B-experimental_method
+)	O
+simulations	B-experimental_method
+to	O
+generate	O
+a	O
+pool	O
+of	O
+energetically	O
+reasonable	O
+conformations	O
+of	O
+the	O
+dynamic	B-protein_state
+complex	O
+and	O
+(	O
+4	O
+)	O
+applied	O
+a	O
+sample	B-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+-	I-experimental_method
+select	I-experimental_method
+algorithm	I-experimental_method
+to	O
+determine	O
+the	O
+minimal	O
+set	O
+of	O
+substrate	O
+conformations	O
+that	O
+fit	O
+both	O
+the	O
+residual	B-evidence
+and	I-evidence
+anomalous	I-evidence
+density	I-evidence
+.	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	B-protein_state
+regions	O
+of	O
+the	O
+substrate	O
+with	O
+iodine	B-chemical
+,	O
+the	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+can	O
+provide	O
+spatial	O
+information	O
+on	O
+many	O
+of	O
+the	O
+other	O
+flexible	B-protein_state
+residues	O
+.	O
+
+The	O
+electron	B-evidence
+density	I-evidence
+then	O
+allowed	O
+us	O
+to	O
+connect	O
+the	O
+labeled	O
+residues	O
+of	O
+the	O
+substrate	O
+by	O
+confining	O
+the	O
+protein	O
+chain	O
+within	O
+regions	O
+of	O
+detectable	O
+density	B-evidence
+.	O
+
+In	O
+this	O
+way	O
+,	O
+the	O
+two	O
+forms	O
+of	O
+data	O
+together	O
+were	O
+able	O
+to	O
+describe	O
+multiple	O
+conformations	O
+of	O
+the	O
+substrate	O
+within	O
+the	O
+crystal	B-evidence
+.	O
+
+As	O
+described	O
+in	O
+detail	O
+below	O
+,	O
+we	O
+developed	O
+the	O
+READ	B-experimental_method
+method	O
+to	O
+uncover	O
+the	O
+ensemble	O
+of	O
+conformations	O
+that	O
+the	O
+Spy	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+of	O
+Im7	B-protein
+(	O
+i	O
+.	O
+e	O
+.,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+)	O
+adopts	O
+while	O
+bound	B-protein_state
+to	I-protein_state
+Spy	B-protein
+.	O
+
+However	O
+,	O
+we	O
+believe	O
+that	O
+READ	B-experimental_method
+will	O
+prove	O
+generally	O
+applicable	O
+to	O
+visualizing	O
+heterogeneous	O
+and	O
+dynamic	O
+complexes	O
+that	O
+have	O
+previously	O
+escaped	O
+detailed	O
+structural	O
+analysis	O
+.	O
+
+Collecting	O
+READ	B-experimental_method
+data	O
+for	O
+the	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+complex	O
+
+To	O
+apply	O
+the	O
+READ	B-experimental_method
+technique	I-experimental_method
+to	O
+the	O
+folding	O
+mechanism	O
+employed	O
+by	O
+the	O
+chaperone	B-protein_type
+Spy	B-protein
+,	O
+we	O
+selected	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+for	O
+further	O
+investigation	O
+because	O
+NMR	B-experimental_method
+data	O
+suggested	O
+that	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+could	O
+recapitulate	O
+unfolded	B-protein_state
+,	O
+partially	O
+folded	B-protein_state
+,	O
+and	O
+native	O
+-	O
+like	O
+states	O
+of	O
+Im7	B-protein
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+
+Moreover	O
+,	O
+binding	B-experimental_method
+experiments	I-experimental_method
+indicated	O
+that	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+comprises	O
+the	O
+entire	O
+Spy	B-site
+-	I-site
+binding	I-site
+region	I-site
+.	O
+
+To	O
+introduce	O
+the	O
+anomalous	O
+scatterer	O
+iodine	B-chemical
+,	O
+we	O
+replaced	B-experimental_method
+eight	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+residues	O
+with	O
+the	O
+non	O
+-	O
+canonical	O
+amino	O
+acid	O
+4	B-chemical
+-	I-chemical
+iodophenylalanine	I-chemical
+(	O
+pI	B-chemical
+-	I-chemical
+Phe	I-chemical
+).	O
+
+Its	O
+strong	O
+anomalous	B-evidence
+scattering	I-evidence
+allowed	O
+us	O
+to	O
+track	O
+the	O
+positions	O
+of	O
+these	O
+individual	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+residues	O
+one	O
+at	O
+a	O
+time	O
+,	O
+potentially	O
+even	O
+if	O
+the	O
+residue	O
+was	O
+found	O
+in	O
+several	O
+locations	O
+in	O
+the	O
+same	O
+crystal	B-evidence
+.	O
+
+We	O
+then	O
+co	B-experimental_method
+-	I-experimental_method
+crystallized	I-experimental_method
+Spy	B-protein
+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	B-chemical
+-	I-chemical
+Phe	I-chemical
+substitution	B-experimental_method
+at	O
+one	O
+distinct	O
+position	O
+,	O
+and	O
+collected	B-experimental_method
+anomalous	B-evidence
+data	I-evidence
+for	O
+all	O
+eight	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+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
+
+Consistent	O
+with	O
+our	O
+electron	B-evidence
+density	I-evidence
+map	I-evidence
+,	O
+we	O
+found	O
+that	O
+the	O
+majority	O
+of	O
+anomalous	B-evidence
+signals	I-evidence
+emerged	O
+in	O
+the	O
+cradle	B-site
+of	O
+Spy	B-protein
+,	O
+implying	O
+that	O
+this	O
+is	O
+the	O
+likely	O
+Im7	B-protein
+substrate	B-site
+binding	I-site
+site	I-site
+.	O
+
+Consistent	O
+with	O
+the	O
+fragmented	O
+density	B-evidence
+,	O
+however	O
+,	O
+we	O
+observed	O
+multiple	O
+iodine	B-chemical
+positions	O
+for	O
+seven	O
+of	O
+the	O
+eight	O
+substituted	O
+residues	O
+.	O
+
+Together	O
+,	O
+these	O
+results	O
+indicated	O
+that	O
+the	O
+Im7	B-protein
+substrate	O
+binds	O
+Spy	B-protein
+in	O
+multiple	O
+conformations	O
+.	O
+
+READ	B-experimental_method
+sample	B-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+-	I-experimental_method
+select	I-experimental_method
+procedure	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	B-protein_state
+to	I-protein_state
+Spy	B-protein
+,	O
+we	O
+combined	O
+the	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+and	O
+the	O
+anomalous	B-evidence
+signals	I-evidence
+from	O
+our	O
+pI	B-chemical
+-	I-chemical
+Phe	I-chemical
+substituted	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+complexes	O
+.	O
+
+To	O
+generate	O
+an	O
+accurate	O
+depiction	O
+of	O
+the	O
+chaperone	B-protein_type
+-	O
+substrate	O
+interactions	O
+,	O
+we	O
+devised	O
+a	O
+selection	O
+protocol	O
+based	O
+on	O
+a	O
+sample	B-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+-	I-experimental_method
+select	I-experimental_method
+procedure	O
+employed	O
+in	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+.	O
+
+During	O
+each	O
+round	O
+of	O
+the	O
+selection	O
+,	O
+a	O
+genetic	B-experimental_method
+algorithm	I-experimental_method
+alters	O
+the	O
+ensemble	O
+and	O
+its	O
+agreement	O
+to	O
+the	O
+experimental	O
+data	O
+is	O
+re	O
+-	O
+evaluated	O
+.	O
+
+If	O
+successful	O
+,	O
+the	O
+selection	O
+identifies	O
+the	O
+smallest	O
+group	O
+of	O
+specific	O
+conformations	O
+that	O
+best	O
+fits	O
+the	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+and	O
+anomalous	B-evidence
+signals	I-evidence
+.	O
+
+The	O
+READ	B-experimental_method
+sample	B-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+-	I-experimental_method
+select	I-experimental_method
+algorithm	I-experimental_method
+is	O
+diagrammed	O
+in	O
+Fig	O
+.	O
+2	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	B-protein_type
+-	O
+substrate	O
+complexes	O
+using	O
+coarse	B-experimental_method
+-	I-experimental_method
+grained	I-experimental_method
+MD	I-experimental_method
+simulations	I-experimental_method
+in	O
+a	O
+pseudo	B-experimental_method
+-	I-experimental_method
+crystal	I-experimental_method
+environment	I-experimental_method
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+
+The	O
+coarse	B-experimental_method
+-	I-experimental_method
+grained	I-experimental_method
+simulations	I-experimental_method
+are	O
+based	O
+on	O
+a	O
+single	O
+-	O
+residue	O
+resolution	O
+model	O
+for	O
+protein	O
+folding	O
+and	O
+were	O
+extended	O
+here	O
+to	O
+describe	O
+Spy	B-complex_assembly
+-	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+binding	O
+events	O
+(	O
+Online	O
+Methods	O
+).	O
+
+The	O
+initial	O
+conditions	O
+of	O
+the	O
+binding	B-experimental_method
+simulations	I-experimental_method
+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	B-protein_type
+-	O
+substrate	O
+interaction	O
+(	O
+Online	O
+Methods	O
+).	O
+
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+binds	O
+and	O
+unbinds	O
+to	O
+Spy	B-protein
+throughout	O
+the	O
+simulations	B-experimental_method
+.	O
+
+This	O
+strategy	O
+allows	O
+a	O
+wide	O
+range	O
+of	O
+substrate	O
+conformations	O
+to	O
+interact	O
+with	O
+the	O
+chaperone	B-protein_type
+.	O
+
+From	O
+the	O
+MD	B-experimental_method
+simulations	B-experimental_method
+,	O
+we	O
+extracted	O
+~	O
+10	O
+,	O
+000	O
+diverse	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+complexes	O
+to	O
+be	O
+used	O
+by	O
+the	O
+ensuing	O
+selection	O
+.	O
+
+Each	O
+complex	O
+within	O
+this	O
+pool	O
+comprises	O
+one	O
+Spy	B-protein
+dimer	B-oligomeric_state
+bound	B-protein_state
+to	I-protein_state
+a	O
+single	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+substrate	O
+.	O
+
+This	O
+pool	O
+was	O
+then	O
+used	O
+by	O
+the	O
+selection	O
+algorithm	O
+to	O
+identify	O
+the	O
+minimal	O
+ensemble	O
+that	O
+best	O
+satisfies	O
+both	O
+the	O
+residual	B-evidence
+electron	I-evidence
+and	I-evidence
+anomalous	I-evidence
+crystallographic	I-evidence
+data	I-evidence
+.	O
+
+The	O
+anomalous	B-evidence
+scattering	I-evidence
+portion	O
+of	O
+the	O
+selection	O
+uses	O
+our	O
+basic	O
+knowledge	O
+of	O
+pI	B-chemical
+-	I-chemical
+Phe	I-chemical
+geometry	O
+:	O
+the	O
+iodine	B-chemical
+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	B-chemical
+anomalous	B-evidence
+signals	I-evidence
+.	O
+
+Simultaneously	O
+,	O
+it	O
+uses	O
+the	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+to	O
+help	O
+choose	O
+ensembles	O
+.	O
+
+To	O
+make	O
+the	O
+electron	B-experimental_method
+density	I-experimental_method
+selection	I-experimental_method
+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	B-evidence
+density	I-evidence
+on	O
+-	O
+the	O
+-	O
+fly	O
+during	O
+the	O
+selection	O
+procedure	O
+.	O
+
+To	O
+accomplish	O
+this	O
+task	O
+,	O
+we	O
+generated	O
+a	O
+compressed	O
+version	O
+of	O
+the	O
+experimental	O
+2mFo	B-evidence
+−	I-evidence
+DFc	I-evidence
+electron	I-evidence
+density	I-evidence
+map	I-evidence
+for	O
+use	O
+in	O
+the	O
+selection	O
+.	O
+
+This	O
+process	O
+provided	O
+us	O
+with	O
+a	O
+target	O
+map	B-evidence
+that	O
+the	O
+ensuing	O
+selection	O
+tried	O
+to	O
+recapitulate	O
+.	O
+
+To	O
+reduce	O
+the	O
+extent	O
+of	O
+3D	O
+space	O
+to	O
+be	O
+explored	O
+,	O
+this	O
+compressed	O
+map	B-evidence
+was	O
+created	O
+by	O
+only	O
+using	O
+density	B-evidence
+from	O
+regions	O
+of	O
+space	O
+significantly	O
+sampled	O
+by	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+in	O
+the	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+MD	B-experimental_method
+simulations	B-experimental_method
+.	O
+
+For	O
+each	O
+of	O
+the	O
+~	O
+10	O
+,	O
+000	O
+complexes	O
+in	O
+the	O
+coarse	B-experimental_method
+-	I-experimental_method
+grained	I-experimental_method
+MD	B-experimental_method
+pool	O
+,	O
+the	O
+electron	B-evidence
+density	I-evidence
+at	O
+the	O
+Cα	O
+positions	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+was	O
+extracted	O
+and	O
+used	O
+to	O
+construct	O
+an	O
+electron	B-evidence
+density	I-evidence
+map	I-evidence
+(	O
+Online	O
+Methods	O
+).	O
+
+These	O
+individual	O
+electron	B-evidence
+density	I-evidence
+maps	I-evidence
+from	O
+the	O
+separate	O
+conformers	O
+could	O
+then	O
+be	O
+combined	O
+(	O
+Fig	O
+.	O
+2	O
+)	O
+and	O
+compared	O
+to	O
+the	O
+averaged	O
+experimental	O
+electron	B-evidence
+density	I-evidence
+map	I-evidence
+as	O
+part	O
+of	O
+the	O
+selection	O
+algorithm	O
+.	O
+
+This	O
+approach	O
+allowed	O
+us	O
+to	O
+simultaneously	O
+use	O
+both	O
+the	O
+iodine	B-chemical
+anomalous	B-evidence
+signals	I-evidence
+and	O
+the	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+in	O
+the	O
+selection	O
+procedure	O
+.	O
+
+The	O
+selection	O
+resulted	O
+in	O
+small	O
+ensembles	O
+from	O
+the	O
+MD	B-experimental_method
+pool	O
+that	O
+best	O
+fit	O
+the	O
+READ	B-experimental_method
+data	O
+(	O
+Fig	O
+.	O
+1c	O
+,	O
+d	O
+).	O
+
+Before	O
+analyzing	O
+the	O
+details	O
+of	O
+the	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+complex	O
+,	O
+we	O
+first	O
+engaged	O
+in	O
+a	O
+series	O
+of	O
+validation	O
+tests	O
+to	O
+verify	O
+the	O
+ensemble	O
+and	O
+selection	O
+procedure	O
+(	O
+Supplementary	O
+Note	O
+1	O
+,	O
+Figures	O
+1c	O
+,	O
+d	O
+,	O
+Supplemental	O
+Figures	O
+5	O
+-	O
+7	O
+).	O
+
+Of	O
+note	O
+,	O
+the	O
+final	O
+six	O
+-	O
+membered	O
+ensemble	O
+was	O
+the	O
+largest	O
+ensemble	O
+that	O
+could	O
+simultaneously	O
+decrease	O
+the	O
+RFree	B-evidence
+and	O
+pass	O
+the	O
+10	B-experimental_method
+-	I-experimental_method
+fold	I-experimental_method
+cross	I-experimental_method
+-	I-experimental_method
+validation	I-experimental_method
+test	I-experimental_method
+.	O
+
+This	O
+ensemble	O
+depicts	O
+the	O
+conformations	O
+that	O
+the	O
+substrate	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+adopts	O
+while	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+chaperone	B-protein_type
+Spy	B-protein
+(	O
+Fig	O
+.	O
+3	O
+Supplementary	O
+Movie	O
+1	O
+,	O
+and	O
+Table	O
+1	O
+).	O
+
+Folding	O
+and	O
+interactions	O
+of	O
+Im7	B-protein
+while	O
+bound	B-protein_state
+to	I-protein_state
+Spy	B-protein
+
+Our	O
+results	O
+showed	O
+that	O
+by	O
+using	O
+this	O
+novel	O
+READ	B-experimental_method
+approach	O
+,	O
+we	O
+were	O
+able	O
+to	O
+obtain	O
+structural	O
+information	O
+about	O
+the	O
+dynamic	O
+interaction	O
+of	O
+a	O
+chaperone	B-protein_type
+with	O
+its	O
+substrate	O
+protein	O
+.	O
+
+We	O
+were	O
+particularly	O
+interested	O
+in	O
+finding	O
+answers	O
+to	O
+one	O
+of	O
+the	O
+most	O
+fundamental	O
+questions	O
+in	O
+chaperone	B-protein_type
+biology	O
+—	O
+how	O
+does	O
+chaperone	B-protein_type
+binding	O
+affect	O
+substrate	O
+structure	O
+and	O
+vice	O
+versa	O
+.	O
+
+By	O
+analyzing	O
+the	O
+individual	O
+structures	B-evidence
+of	O
+the	O
+six	O
+-	O
+member	O
+ensemble	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+bound	B-protein_state
+to	I-protein_state
+Spy	B-protein
+,	O
+we	O
+observed	O
+that	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+takes	O
+on	O
+several	O
+different	O
+conformations	O
+while	O
+bound	B-protein_state
+.	O
+
+We	O
+found	O
+these	O
+conformations	O
+to	O
+be	O
+highly	O
+heterogeneous	O
+and	O
+to	O
+include	O
+unfolded	B-protein_state
+,	O
+partially	B-protein_state
+folded	I-protein_state
+,	O
+and	O
+native	B-protein_state
+-	I-protein_state
+like	I-protein_state
+states	O
+(	O
+Fig	O
+.	O
+3	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	B-protein
+cradle	B-site
+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	B-site
+(	O
+Figs	O
+.	O
+3	O
+,	O
+4a	O
+).	O
+
+We	O
+constructed	O
+a	O
+contact	B-evidence
+map	I-evidence
+of	O
+the	O
+complex	O
+,	O
+which	O
+shows	O
+the	O
+frequency	O
+of	O
+interactions	O
+for	O
+chaperone	B-protein_type
+-	O
+substrate	O
+residue	O
+pairs	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+We	O
+found	O
+that	O
+the	O
+primary	O
+interaction	B-site
+sites	I-site
+on	O
+Spy	B-protein
+reside	O
+at	O
+the	O
+N	O
+and	O
+C	O
+termini	O
+(	O
+Arg122	B-residue_name_number
+,	O
+Thr124	B-residue_name_number
+,	O
+and	O
+Phe29	B-residue_name_number
+)	O
+as	O
+well	O
+as	O
+on	O
+the	O
+concave	O
+face	O
+of	O
+the	O
+chaperone	B-protein_type
+(	O
+Arg61	B-residue_name_number
+,	O
+Arg43	B-residue_name_number
+,	O
+Lys47	B-residue_name_number
+,	O
+His96	B-residue_name_number
+,	O
+and	O
+Met46	B-residue_name_number
+).	O
+
+The	O
+Spy	B-site
+-	I-site
+contacting	I-site
+residues	I-site
+comprise	O
+a	O
+mixture	O
+of	O
+charged	O
+,	O
+polar	O
+,	O
+and	O
+hydrophobic	O
+residues	O
+.	O
+
+Surprisingly	O
+,	O
+we	O
+noted	O
+that	O
+in	O
+the	O
+ensemble	O
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+interacts	O
+with	O
+only	O
+38	O
+%	O
+of	O
+the	O
+hydrophobic	O
+residues	O
+in	O
+the	O
+Spy	B-protein
+cradle	B-site
+,	O
+but	O
+interacts	O
+with	O
+61	O
+%	O
+of	O
+the	O
+hydrophilic	O
+residues	O
+in	O
+the	O
+cradle	B-site
+.	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
+
+With	O
+respect	O
+to	O
+the	O
+substrate	O
+,	O
+we	O
+observed	O
+that	O
+nearly	O
+every	O
+residue	O
+in	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+is	O
+in	O
+contact	O
+with	O
+Spy	B-protein
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+
+However	O
+,	O
+we	O
+did	O
+notice	O
+that	O
+despite	O
+this	O
+uniformity	O
+,	O
+regions	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+preferentially	O
+interact	O
+with	O
+different	O
+regions	O
+in	O
+Spy	B-protein
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+
+For	O
+example	O
+,	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+binds	O
+more	O
+consistently	O
+in	O
+the	O
+Spy	B-protein
+cradle	B-site
+,	O
+whereas	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+predominantly	O
+binds	O
+to	O
+the	O
+outer	O
+edges	O
+of	O
+Spy	B-protein
+’	O
+s	O
+concave	B-site
+surface	I-site
+.	O
+
+Not	O
+unexpectedly	O
+,	O
+we	O
+found	O
+that	O
+as	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+progresses	O
+from	O
+the	O
+unfolded	B-protein_state
+to	O
+the	O
+native	B-protein_state
+state	O
+,	O
+its	O
+interactions	O
+with	O
+Spy	B-protein
+shift	O
+accordingly	O
+.	O
+
+Whereas	O
+the	O
+least	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+pose	O
+in	O
+the	O
+ensemble	O
+forms	O
+the	O
+most	O
+hydrophobic	O
+contacts	O
+with	O
+Spy	B-protein
+(	O
+Fig	O
+.	O
+3	O
+),	O
+the	O
+two	O
+most	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+conformations	O
+form	O
+the	O
+fewest	O
+hydrophobic	O
+contacts	O
+(	O
+Fig	O
+.	O
+3	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	B-site
+sites	I-site
+.	O
+
+The	O
+diversity	O
+of	O
+conformations	O
+and	O
+binding	B-site
+sites	I-site
+observed	O
+here	O
+emphasizes	O
+the	O
+dynamic	O
+and	O
+heterogeneous	O
+nature	O
+of	O
+the	O
+chaperone	B-protein_type
+-	O
+substrate	O
+ensemble	O
+.	O
+
+Although	O
+we	O
+do	O
+not	O
+yet	O
+have	O
+time	O
+resolution	O
+data	O
+of	O
+these	O
+various	O
+snapshots	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+,	O
+this	O
+ensemble	O
+illustrates	O
+how	O
+a	O
+substrate	O
+samples	O
+its	O
+folding	O
+landscape	O
+while	O
+bound	B-protein_state
+to	I-protein_state
+a	O
+chaperone	B-protein_type
+.	O
+
+Spy	B-protein
+changes	O
+conformation	O
+upon	O
+substrate	O
+binding	O
+
+Comparing	O
+the	O
+structure	B-evidence
+of	O
+Spy	B-protein
+in	O
+its	O
+substrate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+and	O
+apo	B-protein_state
+states	O
+revealed	O
+that	O
+the	O
+Spy	B-protein
+dimer	B-oligomeric_state
+also	O
+undergoes	O
+significant	O
+conformational	O
+changes	O
+upon	O
+substrate	O
+binding	O
+(	O
+Fig	O
+.	O
+5a	O
+and	O
+Supplementary	O
+Movie	O
+2	O
+).	O
+
+Upon	O
+substrate	O
+binding	O
+,	O
+the	O
+Spy	B-protein
+dimer	B-oligomeric_state
+twists	O
+9	O
+°	O
+about	O
+its	O
+center	O
+relative	O
+to	O
+its	O
+apo	B-protein_state
+form	O
+.	O
+
+This	O
+twist	O
+yields	O
+asymmetry	O
+and	O
+results	O
+in	O
+substantially	O
+different	O
+interaction	O
+patterns	O
+in	O
+the	O
+two	O
+Spy	B-protein
+monomers	B-oligomeric_state
+(	O
+Fig	O
+.	O
+4b	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	B-protein
+by	O
+providing	O
+more	O
+binding	O
+poses	O
+.	O
+
+Additionally	O
+,	O
+we	O
+observed	O
+that	O
+the	O
+linker	B-structure_element
+region	I-structure_element
+(	O
+residues	O
+47	B-residue_range
+–	I-residue_range
+57	I-residue_range
+)	O
+of	O
+Spy	B-protein
+,	O
+which	O
+participates	O
+in	O
+substrate	O
+interaction	O
+,	O
+becomes	O
+mostly	O
+disordered	B-protein_state
+upon	O
+binding	O
+the	O
+substrate	O
+.	O
+
+This	O
+increased	O
+disorder	O
+might	O
+explain	O
+how	O
+Spy	B-protein
+is	O
+able	O
+to	O
+recognize	O
+and	O
+bind	O
+different	O
+substrates	O
+and	O
+/	O
+or	O
+differing	O
+conformations	O
+of	O
+the	O
+same	O
+substrate	O
+.	O
+
+Importantly	O
+,	O
+we	O
+observed	O
+the	O
+same	O
+structural	O
+changes	O
+in	O
+Spy	B-protein
+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
+
+The	O
+RMSD	B-evidence
+between	O
+the	O
+well	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+sections	O
+of	O
+Spy	B-protein
+in	O
+the	O
+four	O
+chaperone	B-protein_type
+-	O
+substrate	O
+complexes	O
+was	O
+very	O
+small	O
+,	O
+less	O
+than	O
+0	O
+.	O
+3	O
+Å	O
+.	O
+Combined	O
+with	O
+competition	B-experimental_method
+experiments	I-experimental_method
+showing	O
+that	O
+the	O
+substrates	O
+compete	O
+in	O
+solution	O
+for	O
+Spy	B-protein
+binding	O
+(	O
+Fig	O
+.	O
+5c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+8	O
+),	O
+we	O
+conclude	O
+that	O
+all	O
+the	O
+tested	O
+substrates	O
+share	O
+the	O
+same	O
+overall	O
+Spy	B-site
+binding	I-site
+site	I-site
+.	O
+
+To	O
+shed	O
+light	O
+on	O
+how	O
+chaperones	B-protein_type
+interact	O
+with	O
+their	O
+substrates	O
+,	O
+we	O
+developed	O
+a	O
+novel	O
+structural	O
+biology	O
+method	O
+(	O
+READ	B-experimental_method
+)	O
+and	O
+applied	O
+it	O
+to	O
+determine	O
+a	O
+conformational	B-evidence
+ensemble	I-evidence
+of	O
+the	O
+chaperone	B-protein_type
+Spy	B-protein
+bound	B-protein_state
+to	I-protein_state
+substrate	I-protein_state
+.	O
+
+As	O
+a	O
+substrate	O
+,	O
+we	O
+used	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+,	O
+the	O
+chaperone	B-structure_element
+-	I-structure_element
+interacting	I-structure_element
+portion	I-structure_element
+of	O
+the	O
+protein	O
+-	O
+folding	O
+model	O
+protein	O
+Im7	B-protein
+.	O
+
+In	O
+the	O
+chaperone	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+ensemble	O
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+samples	O
+unfolded	B-protein_state
+,	O
+partially	O
+folded	B-protein_state
+,	O
+and	O
+native	B-protein_state
+-	O
+like	O
+states	O
+.	O
+
+The	O
+ensemble	O
+provides	O
+an	O
+unprecedented	O
+description	O
+of	O
+the	O
+conformations	O
+that	O
+a	O
+substrate	O
+assumes	O
+while	O
+exploring	O
+its	O
+chaperone	B-protein_type
+-	O
+associated	O
+folding	O
+landscape	O
+.	O
+
+This	O
+substrate	O
+-	O
+chaperone	B-protein_type
+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	B-protein_type
+aids	O
+protein	O
+folding	O
+.	O
+
+We	O
+recently	O
+showed	O
+that	O
+Im7	B-protein
+can	O
+fold	O
+while	O
+remaining	O
+continuously	B-protein_state
+bound	I-protein_state
+to	I-protein_state
+Spy	B-protein
+.	O
+
+The	O
+high	O
+-	O
+resolution	O
+ensemble	B-evidence
+obtained	O
+here	O
+now	O
+provides	O
+insight	O
+into	O
+exactly	O
+how	O
+this	O
+occurs	O
+.	O
+
+The	O
+structures	B-evidence
+of	O
+our	O
+ensemble	B-evidence
+agree	O
+well	O
+with	O
+lower	O
+-	O
+resolution	O
+crosslinking	O
+data	O
+,	O
+which	O
+indicate	O
+that	O
+chaperone	B-protein_type
+-	O
+substrate	O
+interactions	O
+primarily	O
+occur	O
+on	O
+the	O
+concave	B-site
+surface	I-site
+of	O
+Spy	B-protein
+.	O
+
+The	O
+ensemble	B-evidence
+suggests	O
+a	O
+model	O
+in	O
+which	O
+Spy	B-protein
+provides	O
+an	O
+amphipathic	B-site
+surface	I-site
+that	O
+allows	O
+substrate	O
+proteins	O
+to	O
+assume	O
+different	O
+conformations	O
+while	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+chaperone	B-protein_type
+.	O
+
+This	O
+model	O
+is	O
+consistent	O
+with	O
+previous	O
+studies	O
+postulating	O
+that	O
+the	O
+flexible	O
+binding	O
+of	O
+chaperones	B-protein_type
+allows	O
+for	O
+substrate	O
+protein	O
+folding	O
+.	O
+
+The	O
+amphipathic	O
+concave	B-site
+surface	I-site
+of	O
+Spy	B-protein
+likely	O
+facilitates	O
+this	O
+flexible	O
+binding	O
+and	O
+may	O
+be	O
+a	O
+crucial	O
+feature	O
+for	O
+Spy	B-protein
+and	O
+potentially	O
+other	O
+chaperones	B-protein_type
+,	O
+allowing	O
+them	O
+to	O
+bind	O
+multiple	O
+conformations	O
+of	O
+many	O
+different	O
+substrates	O
+.	O
+
+In	O
+contrast	O
+to	O
+Spy	B-protein
+’	O
+s	O
+binding	B-site
+hotspots	I-site
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+displays	O
+substantially	O
+less	O
+specificity	O
+in	O
+its	O
+binding	B-site
+sites	I-site
+.	O
+
+Nearly	O
+all	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+residues	O
+come	O
+in	O
+contact	O
+with	O
+Spy	B-protein
+.	O
+
+Unfolded	B-protein_state
+substrate	O
+conformers	O
+interact	O
+with	O
+Spy	B-protein
+through	O
+both	O
+hydrophobic	O
+and	O
+hydrophilic	O
+interactions	O
+,	O
+whereas	O
+the	O
+binding	O
+of	O
+native	B-protein_state
+-	I-protein_state
+like	I-protein_state
+states	O
+is	O
+mainly	O
+hydrophilic	O
+.	O
+
+This	O
+trend	O
+suggests	O
+that	O
+complex	O
+formation	O
+between	O
+an	O
+ATP	B-protein_state
+-	I-protein_state
+independent	I-protein_state
+chaperone	B-protein_type
+and	O
+its	O
+unfolded	B-protein_state
+substrate	O
+may	O
+initially	O
+involve	O
+hydrophobic	O
+interactions	O
+,	O
+effectively	O
+shielding	O
+the	O
+exposed	O
+aggregation	O
+-	O
+sensitive	O
+hydrophobic	B-site
+regions	I-site
+in	O
+the	O
+substrate	O
+.	O
+
+Once	O
+the	O
+substrate	O
+begins	O
+to	O
+fold	O
+within	O
+this	O
+protected	O
+environment	O
+,	O
+it	O
+progressively	O
+buries	O
+its	O
+own	O
+hydrophobic	O
+residues	O
+,	O
+and	O
+its	O
+interactions	O
+with	O
+the	O
+chaperone	B-protein_type
+shift	O
+towards	O
+becoming	O
+more	O
+electrostatic	O
+.	O
+
+Notably	O
+,	O
+the	O
+most	O
+frequent	O
+contacts	O
+between	O
+Spy	B-protein
+and	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+are	O
+charge	O
+-	O
+charge	O
+interactions	O
+.	O
+
+The	O
+negatively	O
+charged	O
+Im7	B-protein
+residues	O
+Glu21	B-residue_name_number
+,	O
+Asp32	B-residue_name_number
+,	O
+and	O
+Asp35	B-residue_name_number
+reside	O
+on	O
+the	O
+surface	O
+of	O
+Im7	B-protein
+and	O
+form	O
+interactions	O
+with	O
+Spy	B-protein
+’	O
+s	O
+positively	O
+charged	O
+cradle	B-site
+in	O
+both	O
+the	O
+unfolded	B-protein_state
+and	O
+native	B-protein_state
+-	I-protein_state
+like	I-protein_state
+states	O
+.	O
+
+Residues	O
+Asp32	B-residue_name_number
+and	O
+Asp35	B-residue_name_number
+are	O
+close	O
+to	O
+each	O
+other	O
+in	O
+the	O
+folded	B-protein_state
+state	O
+of	O
+Im7	B-protein
+.	O
+
+This	O
+proximity	O
+likely	O
+causes	O
+electrostatic	O
+repulsion	O
+that	O
+destabilizes	O
+Im7	B-protein
+’	O
+s	O
+native	B-protein_state
+state	O
+.	O
+
+Interaction	O
+with	O
+Spy	B-protein
+’	O
+s	O
+positively	O
+-	O
+charged	O
+residues	O
+likely	O
+relieves	O
+the	O
+charge	O
+repulsion	O
+between	O
+Asp32	B-residue_name_number
+and	O
+Asp35	B-residue_name_number
+,	O
+promoting	O
+their	O
+compaction	O
+into	O
+a	O
+helical	B-protein_state
+conformation	I-protein_state
+.	O
+
+As	O
+inter	O
+-	O
+molecular	O
+hydrophobic	O
+interactions	O
+between	O
+Spy	B-protein
+and	O
+the	O
+substrate	O
+become	O
+progressively	O
+replaced	O
+by	O
+intra	O
+-	O
+molecular	O
+interactions	O
+within	O
+the	O
+substrate	O
+,	O
+the	O
+affinity	O
+between	O
+chaperone	B-protein_type
+and	O
+substrates	O
+could	O
+decrease	O
+,	O
+eventually	O
+leading	O
+to	O
+release	O
+of	O
+the	O
+folded	B-protein_state
+client	O
+protein	O
+.	O
+
+Recently	O
+,	O
+we	O
+employed	O
+a	O
+genetic	B-experimental_method
+selection	I-experimental_method
+system	I-experimental_method
+to	O
+improve	O
+the	O
+chaperone	B-protein_type
+activity	O
+of	O
+Spy	B-protein
+.	O
+
+This	O
+selection	O
+resulted	O
+in	O
+“	O
+Super	O
+Spy	B-protein
+”	O
+variants	B-protein_state
+that	O
+were	O
+more	O
+effective	O
+at	O
+both	O
+preventing	O
+aggregation	O
+and	O
+promoting	O
+protein	O
+folding	O
+.	O
+
+In	O
+conjunction	O
+with	O
+our	O
+bound	B-protein_state
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+ensemble	B-evidence
+,	O
+these	O
+mutants	O
+now	O
+allowed	O
+us	O
+to	O
+investigate	O
+structural	O
+features	O
+important	O
+to	O
+chaperone	B-protein_type
+function	O
+.	O
+
+Previous	O
+analysis	O
+revealed	O
+that	O
+the	O
+Super	O
+Spy	B-protein
+variants	B-protein_state
+either	O
+bound	B-protein_state
+Im7	B-protein
+tighter	O
+than	O
+WT	B-protein_state
+Spy	B-protein
+,	O
+increased	O
+chaperone	B-protein_type
+flexibility	O
+as	O
+measured	O
+via	O
+H	B-experimental_method
+/	I-experimental_method
+D	I-experimental_method
+exchange	I-experimental_method
+,	O
+or	O
+both	O
+.	O
+
+Our	O
+ensemble	B-evidence
+revealed	O
+that	O
+two	O
+of	O
+the	O
+Super	O
+Spy	B-protein
+mutations	B-protein_state
+(	O
+H96L	B-mutant
+and	O
+Q100L	B-mutant
+)	O
+form	O
+part	O
+of	O
+the	O
+chaperone	B-site
+contact	I-site
+surface	I-site
+that	O
+binds	O
+to	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+
+Moreover	O
+,	O
+our	O
+co	B-evidence
+-	I-evidence
+structure	I-evidence
+suggests	O
+that	O
+the	O
+L32P	B-mutant
+substitution	O
+,	O
+which	O
+increases	O
+Spy	B-protein
+’	O
+s	O
+flexibility	O
+,	O
+could	O
+operate	O
+by	O
+unhinging	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+helix	I-structure_element
+and	O
+effectively	O
+expanding	O
+the	O
+size	O
+of	O
+the	O
+disordered	B-protein_state
+linker	B-structure_element
+.	O
+
+This	O
+possibility	O
+is	O
+supported	O
+by	O
+the	O
+Spy	B-protein
+:	O
+substrate	O
+structures	B-evidence
+,	O
+in	O
+which	O
+the	O
+linker	B-structure_element
+region	I-structure_element
+becomes	O
+more	O
+flexible	O
+compared	O
+to	O
+the	O
+apo	B-protein_state
+state	O
+(	O
+Fig	O
+.	O
+6a	O
+).	O
+
+By	O
+sampling	O
+multiple	O
+conformations	O
+,	O
+this	O
+linker	B-structure_element
+region	I-structure_element
+may	O
+allow	O
+diverse	O
+substrate	O
+conformations	O
+to	O
+be	O
+accommodated	O
+.	O
+
+Other	O
+Super	O
+Spy	B-protein
+mutations	B-protein_state
+(	O
+F115I	B-mutant
+and	O
+F115L	B-mutant
+)	O
+caused	O
+increased	O
+flexibility	O
+but	O
+not	O
+tighter	O
+substrate	O
+binding	O
+.	O
+
+This	O
+residue	O
+does	O
+not	O
+directly	O
+contact	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+in	O
+our	O
+READ	B-experimental_method
+-	O
+derived	O
+ensemble	B-evidence
+.	O
+
+Instead	O
+,	O
+when	O
+Spy	B-protein
+is	O
+bound	B-protein_state
+to	I-protein_state
+substrate	O
+,	O
+F115	B-residue_name_number
+engages	O
+in	O
+close	O
+CH	O
+⋯	O
+π	O
+hydrogen	O
+bonds	O
+with	O
+Tyr104	B-residue_name_number
+(	O
+Fig	O
+.	O
+6b	O
+).	O
+
+This	O
+interaction	O
+presumably	O
+reduces	O
+the	O
+mobility	O
+of	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+helix	I-structure_element
+.	O
+
+The	O
+F115I	B-mutant
+/	O
+L	B-mutant
+substitutions	O
+would	O
+replace	O
+these	O
+hydrogen	O
+bonds	O
+with	O
+hydrophobic	O
+interactions	O
+that	O
+have	O
+little	O
+angular	O
+dependence	O
+.	O
+
+As	O
+a	O
+result	O
+,	O
+the	O
+C	O
+-	O
+terminus	O
+,	O
+and	O
+possibly	O
+also	O
+the	O
+flexible	B-protein_state
+linker	B-structure_element
+,	O
+is	O
+likely	O
+to	O
+become	O
+more	O
+flexible	O
+and	O
+thus	O
+more	O
+accommodating	O
+of	O
+different	O
+conformations	O
+of	O
+substrates	O
+.	O
+
+Overall	O
+,	O
+comparison	O
+of	O
+our	O
+ensemble	B-evidence
+to	O
+the	O
+Super	O
+Spy	B-protein
+variants	B-protein_state
+provides	O
+specific	O
+examples	O
+to	O
+corroborate	O
+the	O
+importance	O
+of	O
+conformational	O
+flexibility	O
+in	O
+chaperone	B-protein_type
+-	O
+substrate	O
+interactions	O
+.	O
+
+Despite	O
+extensive	O
+studies	O
+,	O
+exactly	O
+how	O
+complex	O
+chaperone	B-protein_type
+machines	O
+help	O
+proteins	O
+fold	O
+remains	O
+controversial	O
+.	O
+
+Our	O
+study	O
+indicates	O
+that	O
+the	O
+chaperone	B-protein_type
+Spy	B-protein
+employs	O
+a	O
+simple	O
+surface	O
+binding	O
+approach	O
+that	O
+allows	O
+the	O
+substrate	O
+to	O
+explore	O
+various	O
+conformations	O
+and	O
+form	O
+transiently	O
+favorable	O
+interactions	O
+while	O
+being	O
+protected	O
+from	O
+aggregation	O
+.	O
+
+We	O
+speculate	O
+that	O
+many	O
+other	O
+chaperones	B-protein_type
+could	O
+utilize	O
+a	O
+similar	O
+strategy	O
+.	O
+
+ATP	B-chemical
+and	O
+co	O
+-	O
+chaperone	B-protein_type
+dependencies	O
+may	O
+have	O
+emerged	O
+later	O
+through	O
+evolution	O
+to	O
+better	O
+modulate	O
+and	O
+control	O
+chaperone	B-protein_type
+action	O
+.	O
+
+In	O
+addition	O
+to	O
+insights	O
+into	O
+chaperone	B-protein_type
+function	O
+,	O
+this	O
+work	O
+presents	O
+a	O
+new	O
+method	O
+for	O
+determining	O
+heterogeneous	O
+structural	O
+ensembles	O
+via	O
+a	O
+hybrid	O
+methodology	O
+of	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+and	O
+computational	B-experimental_method
+modeling	I-experimental_method
+.	O
+
+Heterogeneous	O
+dynamic	O
+complexes	O
+or	O
+disordered	B-protein_state
+regions	O
+of	O
+single	O
+proteins	O
+,	O
+once	O
+considered	O
+solely	O
+approachable	O
+by	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+,	O
+can	O
+now	O
+be	O
+visualized	O
+through	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+.	O
+
+Crystallographic	O
+data	O
+and	O
+ensemble	O
+selection	O
+.	O
+(	O
+a	O
+)	O
+2mFo	B-evidence
+−	I-evidence
+DFc	I-evidence
+omit	I-evidence
+map	I-evidence
+of	O
+residual	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+and	O
+flexible	B-structure_element
+linker	I-structure_element
+electron	B-evidence
+density	I-evidence
+contoured	O
+at	O
+0	O
+.	O
+5	O
+σ	O
+.	O
+
+This	O
+is	O
+the	O
+residual	O
+density	B-evidence
+that	O
+is	O
+used	O
+in	O
+the	O
+READ	B-experimental_method
+selection	O
+.	O
+
+(	O
+b	O
+)	O
+Composites	O
+of	O
+iodine	B-chemical
+positions	O
+detected	O
+from	O
+anomalous	B-evidence
+signals	I-evidence
+using	O
+pI	B-chemical
+-	I-chemical
+Phe	I-chemical
+substitutions	B-experimental_method
+,	O
+colored	O
+and	O
+numbered	O
+by	O
+sequence	O
+.	O
+
+Multiple	O
+iodine	B-chemical
+positions	O
+were	O
+detected	O
+for	O
+most	O
+residues	O
+.	O
+
+Agreement	O
+to	O
+the	O
+residual	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+electron	B-evidence
+density	I-evidence
+(	O
+c	O
+)	O
+and	O
+anomalous	B-evidence
+iodine	I-evidence
+signals	I-evidence
+(	O
+d	O
+)	O
+for	O
+ensembles	O
+of	O
+varying	O
+size	O
+generated	O
+by	O
+randomly	O
+choosing	O
+from	O
+the	O
+MD	B-experimental_method
+pool	O
+(	O
+blue	O
+)	O
+and	O
+from	O
+the	O
+selection	O
+procedure	O
+(	O
+black	O
+).	O
+
+The	O
+cost	B-evidence
+function	I-evidence
+,	O
+χ2	B-evidence
+,	O
+decreases	O
+as	O
+the	O
+agreement	O
+to	O
+the	O
+experimental	O
+data	O
+increases	O
+and	O
+is	O
+defined	O
+in	O
+the	O
+Online	O
+Methods	O
+.	O
+
+Flowchart	O
+of	O
+the	O
+READ	B-experimental_method
+sample	B-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+-	I-experimental_method
+select	I-experimental_method
+process	O
+.	O
+
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+ensemble	O
+,	O
+arranged	O
+by	O
+RMSD	B-evidence
+to	O
+native	B-protein_state
+state	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+.	O
+Although	O
+the	O
+six	O
+-	O
+membered	O
+ensemble	O
+from	O
+the	O
+READ	B-experimental_method
+selection	O
+should	O
+be	O
+considered	O
+only	O
+as	O
+an	O
+ensemble	O
+,	O
+for	O
+clarity	O
+,	O
+the	O
+individual	O
+conformers	O
+are	O
+shown	O
+separately	O
+here	O
+.	O
+
+Spy	B-protein
+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
+
+Atoms	O
+that	O
+were	O
+either	O
+not	O
+directly	O
+selected	O
+in	O
+the	O
+READ	B-experimental_method
+procedure	O
+,	O
+or	O
+whose	O
+position	O
+could	O
+not	O
+be	O
+justified	O
+based	O
+on	O
+agreement	O
+with	O
+the	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+were	O
+removed	O
+,	O
+leading	O
+to	O
+non	O
+-	O
+contiguous	O
+sections	O
+.	O
+
+Dashed	O
+lines	O
+connect	O
+non	O
+-	O
+contiguous	O
+segments	O
+of	O
+the	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+substrate	O
+.	O
+
+Residues	O
+of	O
+the	O
+Spy	B-protein
+flexible	O
+linker	B-structure_element
+region	I-structure_element
+that	O
+fit	O
+the	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+are	O
+shown	O
+as	O
+larger	O
+gray	O
+spheres	O
+.	O
+
+Shown	O
+below	O
+each	O
+ensemble	O
+member	O
+is	O
+the	O
+RMSD	B-evidence
+of	O
+each	O
+conformer	O
+to	O
+the	O
+native	B-protein_state
+state	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+,	O
+as	O
+well	O
+as	O
+the	O
+percentage	O
+of	O
+contacts	O
+between	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+and	O
+Spy	B-protein
+that	O
+are	O
+hydrophobic	O
+.	O
+
+Contact	B-evidence
+maps	I-evidence
+of	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+complex	O
+.	O
+
+(	O
+a	O
+)	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+contact	B-evidence
+map	I-evidence
+projected	O
+onto	O
+the	O
+bound	B-protein_state
+Spy	B-protein
+dimer	B-oligomeric_state
+(	O
+above	O
+)	O
+and	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+(	O
+below	O
+)	O
+structures	B-evidence
+.	O
+
+For	O
+clarity	O
+,	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+is	O
+represented	O
+with	O
+a	O
+single	O
+conformation	O
+.	O
+
+The	O
+frequency	O
+plotted	O
+is	O
+calculated	O
+as	O
+the	O
+average	O
+contact	B-evidence
+frequency	I-evidence
+from	O
+Spy	B-protein
+to	O
+every	O
+residue	O
+of	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+and	O
+vice	O
+-	O
+versa	O
+.	O
+
+As	O
+the	O
+residues	O
+involved	O
+in	O
+contacts	O
+are	O
+more	O
+evenly	O
+distributed	O
+in	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+compared	O
+to	O
+Spy	B-protein
+,	O
+its	O
+contact	B-evidence
+map	I-evidence
+was	O
+amplified	O
+.	O
+(	O
+b	O
+)	O
+Detailed	O
+contact	B-evidence
+maps	I-evidence
+of	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+.	O
+
+Contacts	O
+to	O
+the	O
+two	O
+Spy	B-protein
+monomers	B-oligomeric_state
+are	O
+depicted	O
+separately	O
+.	O
+
+Note	O
+that	O
+the	O
+flexible	B-protein_state
+linker	B-structure_element
+region	I-structure_element
+of	O
+Spy	B-protein
+(	O
+residues	O
+47	B-residue_range
+–	I-residue_range
+57	I-residue_range
+)	O
+is	O
+not	O
+represented	O
+in	O
+the	O
+2D	O
+contact	B-evidence
+maps	I-evidence
+.	O
+
+Spy	B-protein
+conformation	O
+changes	O
+upon	O
+substrate	O
+binding	O
+.	O
+
+(	O
+a	O
+)	O
+Overlay	B-experimental_method
+of	O
+apo	B-protein_state
+Spy	B-protein
+(	O
+PDB	O
+ID	O
+:	O
+3O39	O
+,	O
+gray	O
+)	O
+and	O
+bound	B-protein_state
+Spy	B-protein
+(	O
+green	O
+).	O
+(	O
+b	O
+)	O
+Overlay	B-experimental_method
+of	O
+WT	B-protein_state
+Spy	B-protein
+bound	B-protein_state
+to	I-protein_state
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+(	O
+green	O
+),	O
+H96L	B-mutant
+Spy	B-protein
+bound	B-protein_state
+to	I-protein_state
+Im7	B-protein
+L18A	B-mutant
+L19	B-mutant
+AL13A	I-mutant
+(	O
+blue	O
+),	O
+H96L	B-mutant
+Spy	B-protein
+bound	B-protein_state
+to	I-protein_state
+WT	B-protein_state
+Im7	B-protein
+(	O
+yellow	O
+),	O
+and	O
+WT	B-protein_state
+Spy	B-protein
+bound	B-protein_state
+to	I-protein_state
+casein	B-chemical
+(	O
+salmon	O
+).	O
+(	O
+c	O
+)	O
+Competition	B-experimental_method
+assay	I-experimental_method
+showing	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+competes	O
+with	O
+Im7	B-protein
+L18A	B-mutant
+L19A	B-mutant
+L37A	B-mutant
+H40W	B-mutant
+for	O
+the	O
+same	O
+binding	B-site
+site	I-site
+on	O
+Spy	B-protein
+(	O
+further	O
+substrate	B-experimental_method
+competition	I-experimental_method
+assays	I-experimental_method
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+8	O
+).	O
+
+Flexibility	O
+of	O
+Spy	B-protein
+linker	B-structure_element
+region	I-structure_element
+and	O
+effect	O
+of	O
+Super	O
+Spy	B-protein
+mutants	O
+.	O
+(	O
+a	O
+)	O
+The	O
+Spy	B-protein
+linker	B-structure_element
+region	I-structure_element
+adopts	O
+one	O
+dominant	O
+conformation	O
+in	O
+its	O
+apo	B-protein_state
+state	O
+(	O
+PDB	O
+ID	O
+3039	O
+,	O
+gray	O
+),	O
+but	O
+expands	O
+and	O
+adopts	O
+multiple	O
+conformations	O
+in	O
+bound	B-protein_state
+states	O
+(	O
+green	O
+).	O
+
+(	O
+b	O
+)	O
+F115	B-residue_name_number
+and	O
+L32	B-residue_name_number
+tether	O
+Spy	B-protein
+’	O
+s	O
+linker	B-structure_element
+region	I-structure_element
+to	O
+its	O
+cradle	B-site
+,	O
+decreasing	O
+Spy	B-protein
+activity	O
+by	O
+limiting	O
+linker	B-structure_element
+region	I-structure_element
+flexibility	O
+.	O
+
+The	O
+Super	O
+Spy	B-protein
+mutants	O
+F115L	B-mutant
+,	O
+F115I	B-mutant
+,	O
+and	O
+L32P	B-mutant
+are	O
+proposed	O
+to	O
+gain	O
+activity	O
+by	O
+increasing	O
+the	O
+flexibility	O
+or	O
+size	O
+of	O
+this	O
+linker	B-structure_element
+region	I-structure_element
+.	O
+
+L32	B-residue_name_number
+,	O
+F115	B-residue_name_number
+,	O
+and	O
+Y104	B-residue_name_number
+are	O
+rendered	O
+in	O
+purple	O
+to	O
+illustrate	O
+residues	O
+that	O
+are	O
+most	O
+affected	O
+by	O
+Super	O
+Spy	B-protein
+mutations	B-protein_state
+;	O
+CH	O
+⋯	O
+π	O
+hydrogen	O
+bonds	O
+are	O
+depicted	O
+by	O
+orange	O
+dashes	O
+.	O
+
+Structural	O
+diversity	O
+in	O
+a	O
+human	B-species
+antibody	B-protein_type
+germline	O
+library	O
+
+To	O
+support	O
+antibody	B-protein_type
+therapeutic	O
+development	O
+,	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+a	O
+set	O
+of	O
+16	O
+germline	O
+variants	O
+composed	O
+of	O
+4	O
+different	O
+kappa	B-structure_element
+light	I-structure_element
+chains	I-structure_element
+paired	O
+with	O
+4	O
+different	O
+heavy	B-structure_element
+chains	I-structure_element
+have	O
+been	O
+determined	O
+.	O
+
+All	O
+four	O
+heavy	B-structure_element
+chains	I-structure_element
+of	O
+the	O
+antigen	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+fragments	I-structure_element
+(	O
+Fabs	B-structure_element
+)	O
+have	O
+the	O
+same	O
+complementarity	B-structure_element
+-	I-structure_element
+determining	I-structure_element
+region	I-structure_element
+(	O
+CDR	B-structure_element
+)	O
+H3	B-structure_element
+that	O
+was	O
+reported	O
+in	O
+an	O
+earlier	O
+Fab	B-structure_element
+structure	B-evidence
+.	O
+
+The	O
+structure	B-experimental_method
+analyses	I-experimental_method
+include	O
+comparisons	O
+of	O
+the	O
+overall	O
+structures	B-evidence
+,	O
+canonical	O
+structures	B-evidence
+of	O
+the	O
+CDRs	B-structure_element
+and	O
+the	O
+VH	B-site
+:	I-site
+VL	I-site
+packing	I-site
+interactions	I-site
+.	O
+
+The	O
+CDR	B-structure_element
+conformations	O
+for	O
+the	O
+most	O
+part	O
+are	O
+tightly	O
+clustered	O
+,	O
+especially	O
+for	O
+the	O
+ones	O
+with	O
+shorter	O
+lengths	O
+.	O
+
+The	O
+longer	B-protein_state
+CDRs	B-structure_element
+with	O
+tandem	O
+glycines	B-residue_name
+or	O
+serines	B-residue_name
+have	O
+more	O
+conformational	O
+diversity	O
+than	O
+the	O
+others	O
+.	O
+
+CDR	B-structure_element
+H3	B-structure_element
+,	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
+
+About	O
+half	O
+of	O
+the	O
+structures	B-evidence
+have	O
+CDR	B-structure_element
+H3	B-structure_element
+conformations	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+parent	O
+;	O
+the	O
+others	O
+diverge	O
+significantly	O
+.	O
+
+One	O
+conclusion	O
+is	O
+that	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+conformations	O
+are	O
+influenced	O
+by	O
+both	O
+their	O
+amino	O
+acid	O
+sequence	O
+and	O
+their	O
+structural	O
+environment	O
+determined	O
+by	O
+the	O
+heavy	B-structure_element
+and	O
+light	B-structure_element
+chain	I-structure_element
+pairing	O
+.	O
+
+The	O
+stem	B-structure_element
+regions	I-structure_element
+of	O
+14	O
+of	O
+the	O
+variant	O
+pairs	O
+are	O
+in	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+,	O
+and	O
+only	O
+2	O
+are	O
+in	O
+the	O
+extended	B-protein_state
+conformation	O
+.	O
+
+The	O
+packing	O
+of	O
+the	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+domains	O
+is	O
+consistent	O
+with	O
+our	O
+knowledge	O
+of	O
+antibody	B-protein_type
+structure	B-evidence
+,	O
+and	O
+the	O
+tilt	B-evidence
+angles	I-evidence
+between	O
+these	O
+domains	O
+cover	O
+a	O
+range	O
+of	O
+11	O
+degrees	O
+.	O
+
+Two	O
+of	O
+16	O
+structures	B-evidence
+showed	O
+particularly	O
+large	O
+variations	O
+in	O
+the	O
+tilt	B-evidence
+angles	I-evidence
+when	O
+compared	O
+with	O
+the	O
+other	O
+pairings	O
+.	O
+
+The	O
+structures	B-evidence
+and	O
+their	O
+analyses	O
+provide	O
+a	O
+rich	O
+foundation	O
+for	O
+future	O
+antibody	B-protein_type
+modeling	O
+and	O
+engineering	O
+efforts	O
+.	O
+
+At	O
+present	O
+,	O
+therapeutic	O
+antibodies	B-protein_type
+are	O
+the	O
+largest	O
+class	O
+of	O
+biotherapeutic	O
+proteins	O
+that	O
+are	O
+in	O
+clinical	O
+trials	O
+.	O
+
+The	O
+use	O
+of	O
+monoclonal	O
+antibodies	B-protein_type
+as	O
+therapeutics	O
+began	O
+in	O
+the	O
+early	O
+1980s	O
+,	O
+and	O
+their	O
+composition	O
+has	O
+transitioned	O
+from	O
+murine	B-taxonomy_domain
+antibodies	B-protein_type
+to	O
+generally	O
+less	O
+immunogenic	O
+humanized	O
+and	O
+human	B-species
+antibodies	B-protein_type
+.	O
+
+The	O
+technologies	O
+currently	O
+used	O
+to	O
+obtain	O
+human	B-species
+antibodies	B-protein_type
+include	O
+transgenic	O
+mice	B-taxonomy_domain
+containing	O
+human	B-species
+antibody	B-protein_type
+repertoires	O
+,	O
+cloning	O
+directly	O
+from	O
+human	B-species
+B	O
+cells	O
+,	O
+and	O
+in	B-experimental_method
+vitro	I-experimental_method
+selection	I-experimental_method
+from	O
+antibody	B-experimental_method
+libraries	I-experimental_method
+using	O
+various	O
+display	O
+technologies	O
+.	O
+
+Once	O
+a	O
+candidate	O
+antibody	B-protein_type
+is	O
+identified	O
+,	O
+protein	B-experimental_method
+engineering	I-experimental_method
+is	O
+usually	O
+required	O
+to	O
+produce	O
+a	O
+molecule	O
+with	O
+the	O
+right	O
+biophysical	O
+and	O
+functional	O
+properties	O
+.	O
+
+All	O
+engineering	O
+efforts	O
+are	O
+guided	O
+by	O
+our	O
+understanding	O
+of	O
+the	O
+atomic	B-evidence
+structures	I-evidence
+of	O
+antibodies	B-protein_type
+.	O
+
+In	O
+such	O
+efforts	O
+,	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+specific	O
+antibody	B-protein_type
+may	O
+not	O
+be	O
+available	O
+,	O
+but	O
+modeling	O
+can	O
+be	O
+used	O
+to	O
+guide	O
+the	O
+engineering	O
+efforts	O
+.	O
+
+Today	O
+'	O
+s	O
+antibody	B-protein_type
+modeling	O
+approaches	O
+,	O
+which	O
+normally	O
+focus	O
+on	O
+the	O
+variable	B-structure_element
+region	I-structure_element
+,	O
+are	O
+being	O
+developed	O
+by	O
+the	O
+application	O
+of	O
+structural	O
+principles	O
+and	O
+insights	O
+that	O
+are	O
+evolving	O
+as	O
+our	O
+knowledge	O
+of	O
+antibody	B-protein_type
+structures	B-evidence
+continues	O
+to	O
+expand	O
+.	O
+
+Our	O
+current	O
+structural	O
+knowledge	O
+of	O
+antibodies	B-protein_type
+is	O
+based	O
+on	O
+a	O
+multitude	O
+of	O
+studies	O
+that	O
+used	O
+many	O
+techniques	O
+to	O
+gain	O
+insight	O
+into	O
+the	O
+functional	O
+and	O
+structural	O
+properties	O
+of	O
+this	O
+class	O
+of	O
+macromolecule	O
+.	O
+
+Five	O
+different	O
+antibody	B-protein_type
+isotypes	O
+occur	O
+,	O
+IgG	B-protein
+,	O
+IgD	B-protein
+,	O
+IgE	B-protein
+,	O
+IgA	B-protein
+and	O
+IgM	B-protein
+,	O
+and	O
+each	O
+isotype	O
+has	O
+a	O
+unique	O
+role	O
+in	O
+the	O
+adaptive	O
+immune	O
+system	O
+.	O
+
+IgG	B-protein
+,	O
+IgD	B-protein
+and	O
+IgE	B-protein
+isotypes	O
+are	O
+composed	O
+of	O
+2	O
+heavy	B-structure_element
+chains	I-structure_element
+(	O
+HCs	B-structure_element
+)	O
+and	O
+2	O
+light	B-structure_element
+chains	I-structure_element
+(	O
+LCs	B-structure_element
+)	O
+linked	O
+through	O
+disulfide	B-ptm
+bonds	I-ptm
+,	O
+while	O
+IgA	B-protein
+and	O
+IgM	B-protein
+are	O
+double	O
+and	O
+quintuple	O
+versions	O
+of	O
+antibodies	B-protein_type
+,	O
+respectively	O
+.	O
+
+Isotypes	O
+IgG	B-protein
+,	O
+IgD	B-protein
+and	O
+IgA	B-protein
+each	O
+have	O
+4	O
+domains	O
+,	O
+one	O
+variable	B-structure_element
+(	O
+V	B-structure_element
+)	O
+and	O
+3	O
+constant	B-structure_element
+(	O
+C	B-structure_element
+)	O
+domains	O
+,	O
+while	O
+IgE	B-protein
+and	O
+IgM	B-protein
+each	O
+have	O
+the	O
+same	O
+4	O
+domains	O
+along	O
+with	O
+an	O
+additional	O
+C	B-structure_element
+domain	I-structure_element
+.	O
+
+These	O
+multimeric	O
+forms	O
+are	O
+linked	O
+with	O
+an	O
+additional	O
+J	B-structure_element
+chain	O
+.	O
+
+The	O
+LCs	B-structure_element
+that	O
+associate	O
+with	O
+the	O
+HCs	B-structure_element
+are	O
+divided	O
+into	O
+2	O
+functionally	O
+indistinguishable	O
+classes	O
+,	O
+κ	B-structure_element
+and	O
+λ	B-structure_element
+.	O
+
+Both	O
+κ	B-structure_element
+and	O
+λ	B-structure_element
+polypeptide	O
+chains	O
+are	O
+composed	O
+of	O
+a	O
+single	O
+V	B-structure_element
+domain	I-structure_element
+and	O
+a	O
+single	O
+C	B-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+heavy	B-structure_element
+and	O
+light	B-structure_element
+chains	I-structure_element
+are	O
+composed	O
+of	O
+structural	B-structure_element
+domains	I-structure_element
+that	O
+have	O
+∼	B-residue_range
+110	I-residue_range
+amino	I-residue_range
+acid	I-residue_range
+residues	I-residue_range
+.	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	B-structure_element
+fold	I-structure_element
+,”	O
+formed	O
+by	O
+the	O
+packing	O
+together	O
+of	O
+2	O
+anti	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheets	I-structure_element
+.	O
+
+All	O
+immunoglobulin	B-protein_type
+chains	I-protein_type
+have	O
+an	O
+N	O
+-	O
+terminal	O
+V	B-structure_element
+domain	I-structure_element
+followed	O
+by	O
+1	O
+to	O
+4	O
+C	B-structure_element
+domains	I-structure_element
+,	O
+depending	O
+upon	O
+the	O
+chain	O
+type	O
+.	O
+
+In	O
+antibodies	B-protein_type
+,	O
+the	O
+heavy	B-structure_element
+and	I-structure_element
+light	I-structure_element
+chain	I-structure_element
+V	B-structure_element
+domains	I-structure_element
+pack	O
+together	O
+forming	O
+the	O
+antigen	B-site
+combining	I-site
+site	I-site
+.	O
+
+This	O
+site	O
+,	O
+which	O
+interacts	O
+with	O
+the	O
+antigen	O
+(	O
+or	O
+target	O
+),	O
+is	O
+the	O
+focus	O
+of	O
+current	O
+antibody	B-protein_type
+modeling	O
+efforts	O
+.	O
+
+This	O
+interaction	B-site
+site	I-site
+is	O
+composed	O
+of	O
+6	O
+complementarity	B-structure_element
+-	I-structure_element
+determining	I-structure_element
+regions	I-structure_element
+(	O
+CDRs	B-structure_element
+)	O
+that	O
+were	O
+identified	O
+in	O
+early	O
+antibody	B-experimental_method
+amino	I-experimental_method
+acid	I-experimental_method
+sequence	I-experimental_method
+analyses	I-experimental_method
+to	O
+be	O
+hypervariable	B-protein_state
+in	O
+nature	O
+,	O
+and	O
+thus	O
+are	O
+responsible	O
+for	O
+the	O
+sequence	O
+and	O
+structural	O
+diversity	O
+of	O
+our	O
+antibody	B-protein_type
+repertoire	O
+.	O
+
+The	O
+sequence	O
+diversity	O
+of	O
+the	O
+CDR	B-structure_element
+regions	I-structure_element
+presents	O
+a	O
+substantial	O
+challenge	O
+to	O
+antibody	B-protein_type
+modeling	O
+.	O
+
+However	O
+,	O
+an	O
+initial	O
+structural	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+combining	B-site
+sites	I-site
+of	O
+the	O
+small	O
+set	O
+of	O
+structures	B-evidence
+of	O
+immunoglobulin	O
+fragments	O
+available	O
+in	O
+the	O
+1980s	O
+found	O
+that	O
+5	O
+of	O
+the	O
+6	O
+hypervariable	B-structure_element
+loops	I-structure_element
+or	O
+CDRs	B-structure_element
+had	O
+canonical	O
+structures	O
+(	O
+a	O
+limited	O
+set	O
+of	O
+main	O
+-	O
+chain	O
+conformations	O
+).	O
+
+A	O
+CDR	B-structure_element
+canonical	O
+structure	O
+is	O
+defined	O
+by	O
+its	O
+length	O
+and	O
+conserved	O
+residues	O
+located	O
+in	O
+the	O
+hypervariable	B-structure_element
+loop	I-structure_element
+and	O
+framework	B-structure_element
+residues	I-structure_element
+(	O
+V	B-structure_element
+-	I-structure_element
+region	I-structure_element
+residues	O
+that	O
+are	O
+not	O
+part	O
+of	O
+the	O
+CDRs	B-structure_element
+).	O
+
+Furthermore	O
+,	O
+studies	O
+of	O
+antibody	B-protein_type
+sequences	O
+revealed	O
+that	O
+the	O
+total	O
+number	O
+of	O
+canonical	O
+structures	O
+are	O
+limited	O
+for	O
+each	O
+CDR	B-structure_element
+,	O
+indicating	O
+possibly	O
+that	O
+antigen	O
+recognition	O
+may	O
+be	O
+affected	O
+by	O
+structural	O
+restrictions	O
+at	O
+the	O
+antigen	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+
+Later	O
+studies	O
+found	O
+that	O
+the	O
+CDR	B-structure_element
+loop	I-structure_element
+length	O
+is	O
+the	O
+primary	O
+determining	O
+factor	O
+of	O
+antigen	B-site
+-	I-site
+binding	I-site
+site	I-site
+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
+
+Additional	O
+efforts	O
+have	O
+led	O
+to	O
+our	O
+current	O
+understanding	O
+that	O
+the	O
+LC	B-structure_element
+CDRs	B-structure_element
+L1	B-structure_element
+,	O
+L2	B-structure_element
+,	O
+and	O
+L3	B-structure_element
+have	O
+preferred	O
+sets	O
+of	O
+canonical	O
+structures	O
+based	O
+on	O
+length	O
+and	O
+amino	O
+acid	O
+sequence	O
+composition	O
+.	O
+
+This	O
+was	O
+also	O
+found	O
+to	O
+be	O
+the	O
+case	O
+for	O
+the	O
+H1	B-structure_element
+and	O
+H2	B-structure_element
+CDRs	B-structure_element
+.	O
+
+Classification	O
+schemes	O
+for	O
+the	O
+canonical	O
+structures	O
+of	O
+these	O
+5	O
+CDRs	B-structure_element
+have	O
+emerged	O
+and	O
+evolved	O
+as	O
+the	O
+number	O
+of	O
+depositions	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+of	O
+Fab	B-structure_element
+fragments	O
+of	O
+antibodies	B-protein_type
+grow	O
+.	O
+
+Recently	O
+,	O
+a	O
+comprehensive	O
+CDR	B-structure_element
+classification	O
+scheme	O
+was	O
+reported	O
+identifying	O
+72	O
+clusters	O
+of	O
+conformations	O
+observed	O
+in	O
+antibody	B-protein_type
+structures	B-evidence
+.	O
+
+The	O
+knowledge	O
+and	O
+predictability	O
+of	O
+these	O
+CDR	B-structure_element
+canonical	O
+structures	B-evidence
+have	O
+greatly	O
+advanced	O
+antibody	B-protein_type
+modeling	O
+efforts	O
+.	O
+
+In	O
+contrast	O
+to	O
+CDRs	B-structure_element
+L1	B-structure_element
+,	O
+L2	B-structure_element
+,	O
+L3	B-structure_element
+,	O
+H1	B-structure_element
+and	O
+H2	B-structure_element
+,	O
+no	O
+canonical	O
+structures	B-evidence
+have	O
+been	O
+observed	O
+for	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+which	O
+is	O
+the	O
+most	O
+variable	O
+in	O
+length	O
+and	O
+amino	O
+acid	O
+sequence	O
+.	O
+
+Some	O
+clustering	O
+of	O
+conformations	O
+was	O
+observed	O
+for	O
+the	O
+shortest	O
+lengths	O
+;	O
+however	O
+,	O
+for	O
+the	O
+longer	O
+loops	B-structure_element
+,	O
+only	O
+the	O
+portions	O
+nearest	O
+the	O
+framework	B-structure_element
+(	O
+torso	B-structure_element
+,	O
+stem	B-structure_element
+or	O
+anchor	B-structure_element
+region	I-structure_element
+)	O
+were	O
+found	O
+to	O
+have	O
+defined	O
+conformations	O
+.	O
+
+In	O
+the	O
+torso	B-structure_element
+region	I-structure_element
+,	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	B-structure_element
+region	I-structure_element
+.	O
+
+The	O
+“	O
+kinked	B-protein_state
+”	O
+or	O
+“	O
+bulged	B-protein_state
+”	O
+conformation	O
+is	O
+the	O
+most	O
+prevalent	O
+,	O
+but	O
+an	O
+“	O
+extended	B-protein_state
+”	O
+or	O
+“	O
+non	B-protein_state
+-	I-protein_state
+bulged	I-protein_state
+”	O
+conformation	O
+is	O
+also	O
+,	O
+but	O
+less	O
+frequently	O
+,	O
+observed	O
+.	O
+
+The	O
+cataloging	O
+and	O
+development	O
+of	O
+the	O
+rules	O
+for	O
+predicting	O
+the	O
+conformation	O
+of	O
+the	O
+anchor	B-structure_element
+region	I-structure_element
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+continue	O
+to	O
+be	O
+refined	O
+,	O
+producing	O
+new	O
+insight	O
+into	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+conformations	O
+and	O
+new	O
+tools	O
+for	O
+antibody	B-protein_type
+engineering	O
+.	O
+
+Current	O
+antibody	B-protein_type
+modeling	O
+approaches	O
+take	O
+advantage	O
+of	O
+the	O
+most	O
+recent	O
+advances	O
+in	O
+homology	B-experimental_method
+modeling	I-experimental_method
+,	O
+the	O
+evolving	O
+understanding	O
+of	O
+the	O
+CDR	B-structure_element
+canonical	O
+structures	B-evidence
+,	O
+the	O
+emerging	O
+rules	O
+for	O
+CDR	B-structure_element
+H3	B-structure_element
+modeling	O
+and	O
+the	O
+growing	O
+body	O
+of	O
+antibody	B-protein_type
+structural	O
+data	O
+available	O
+from	O
+the	O
+PDB	O
+.	O
+
+Recent	O
+antibody	B-experimental_method
+modeling	I-experimental_method
+assessments	I-experimental_method
+show	O
+continued	O
+improvement	O
+in	O
+the	O
+quality	O
+of	O
+the	O
+models	O
+being	O
+generated	O
+by	O
+a	O
+variety	O
+of	O
+modeling	O
+methods	O
+.	O
+
+Although	O
+antibody	B-protein_type
+modeling	O
+is	O
+improving	O
+,	O
+the	O
+latest	O
+assessment	O
+revealed	O
+a	O
+number	O
+of	O
+challenges	O
+that	O
+need	O
+to	O
+be	O
+overcome	O
+to	O
+provide	O
+accurate	O
+3	O
+-	O
+dimensional	O
+models	O
+of	O
+antibody	B-protein_type
+V	B-structure_element
+regions	I-structure_element
+,	O
+including	O
+accuracies	O
+in	O
+the	O
+modeling	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+.	O
+
+The	O
+need	O
+for	O
+improvement	O
+in	O
+this	O
+area	O
+was	O
+also	O
+highlighted	O
+in	O
+a	O
+recent	O
+study	O
+reporting	O
+an	O
+approach	O
+and	O
+results	O
+that	O
+may	O
+influence	O
+future	O
+antibody	B-protein_type
+modeling	O
+efforts	O
+.	O
+
+One	O
+important	O
+finding	O
+of	O
+the	O
+antibody	B-experimental_method
+modeling	I-experimental_method
+assessments	I-experimental_method
+was	O
+that	O
+errors	O
+in	O
+the	O
+structural	O
+templates	O
+that	O
+are	O
+used	O
+as	O
+the	O
+basis	O
+for	O
+homology	B-experimental_method
+models	I-experimental_method
+can	O
+propagate	O
+into	O
+the	O
+final	O
+models	O
+,	O
+producing	O
+inaccuracies	O
+that	O
+may	O
+negatively	O
+influence	O
+the	O
+predictive	O
+nature	O
+of	O
+the	O
+V	B-structure_element
+region	I-structure_element
+model	O
+.	O
+
+To	O
+support	O
+antibody	B-protein_type
+engineering	O
+and	O
+therapeutic	O
+development	O
+efforts	O
+,	O
+a	O
+phage	B-experimental_method
+library	I-experimental_method
+was	O
+designed	O
+and	O
+constructed	O
+based	O
+on	O
+a	O
+limited	O
+number	O
+of	O
+scaffolds	O
+built	O
+with	O
+frequently	O
+used	O
+human	B-species
+germ	O
+-	O
+line	O
+IGV	B-structure_element
+and	O
+IGJ	B-structure_element
+gene	O
+segments	O
+that	O
+encode	O
+antigen	B-site
+combining	I-site
+sites	I-site
+suitable	O
+for	O
+recognition	O
+of	O
+peptides	O
+and	O
+proteins	O
+.	O
+
+This	O
+Fab	B-structure_element
+library	O
+is	O
+composed	O
+of	O
+3	O
+HC	B-structure_element
+germlines	O
+,	O
+IGHV1	B-mutant
+-	I-mutant
+69	I-mutant
+(	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+),	O
+IGHV3	B-mutant
+-	I-mutant
+23	I-mutant
+(	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+)	O
+and	O
+IGHV5	B-mutant
+-	I-mutant
+51	I-mutant
+(	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+),	O
+and	O
+4	O
+LC	B-structure_element
+germlines	O
+(	O
+all	O
+κ	B-structure_element
+),	O
+IGKV1	B-mutant
+-	I-mutant
+39	I-mutant
+(	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+),	O
+IGKV3	B-mutant
+-	I-mutant
+11	I-mutant
+(	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+),	O
+IGKV3	B-mutant
+-	I-mutant
+20	I-mutant
+(	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+)	O
+and	O
+IGKV4	B-mutant
+-	I-mutant
+1	I-mutant
+(	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+).	O
+
+Selection	O
+of	O
+these	O
+genes	O
+was	O
+based	O
+on	O
+the	O
+high	O
+frequency	O
+of	O
+their	O
+use	O
+and	O
+their	O
+cognate	O
+canonical	O
+structures	B-evidence
+that	O
+were	O
+found	O
+binding	O
+to	O
+peptides	O
+and	O
+proteins	O
+,	O
+as	O
+well	O
+as	O
+their	O
+ability	O
+to	O
+be	O
+expressed	B-experimental_method
+in	I-experimental_method
+bacteria	I-experimental_method
+and	O
+displayed	B-experimental_method
+on	I-experimental_method
+filamentous	I-experimental_method
+phage	I-experimental_method
+.	O
+
+The	O
+implementation	O
+of	O
+the	O
+library	O
+involves	O
+the	O
+diversification	O
+of	O
+the	O
+human	B-species
+germline	O
+genes	O
+to	O
+mimic	O
+that	O
+found	O
+in	O
+natural	O
+human	B-species
+libraries	O
+.	O
+
+The	O
+crystal	B-experimental_method
+structure	I-experimental_method
+determinations	I-experimental_method
+and	O
+structural	B-experimental_method
+analyses	I-experimental_method
+of	O
+all	O
+germline	O
+Fabs	B-structure_element
+in	O
+the	O
+library	O
+described	O
+above	O
+along	O
+with	O
+the	O
+structures	B-evidence
+of	O
+a	O
+fourth	O
+HC	B-structure_element
+germline	O
+,	O
+IGHV3	B-mutant
+-	I-mutant
+53	I-mutant
+(	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+),	O
+paired	O
+with	O
+the	O
+4	O
+LCs	B-structure_element
+of	O
+the	O
+library	O
+have	O
+been	O
+carried	O
+out	O
+to	O
+support	O
+antibody	B-protein_type
+therapeutic	O
+development	O
+.	O
+
+All	O
+16	O
+HCs	B-structure_element
+of	O
+the	O
+Fabs	B-structure_element
+have	O
+the	O
+same	O
+CDR	B-structure_element
+H3	B-structure_element
+that	O
+was	O
+reported	O
+in	O
+an	O
+earlier	O
+Fab	B-structure_element
+structure	B-evidence
+.	O
+
+This	O
+is	O
+the	O
+first	O
+systematic	O
+study	O
+of	O
+the	O
+same	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+structures	B-evidence
+in	O
+the	O
+context	O
+of	O
+different	O
+pairings	O
+.	O
+
+The	O
+structure	O
+analyses	O
+include	O
+comparisons	O
+of	O
+the	O
+overall	O
+structures	B-evidence
+,	O
+canonical	O
+structures	B-evidence
+of	O
+the	O
+L1	B-structure_element
+,	O
+L2	B-structure_element
+,	O
+L3	B-structure_element
+,	O
+H1	B-structure_element
+and	O
+H2	B-structure_element
+CDRs	B-structure_element
+,	O
+the	O
+structures	B-evidence
+of	O
+all	O
+CDR	B-structure_element
+H3s	B-structure_element
+,	O
+and	O
+the	O
+VH	B-site
+:	I-site
+VL	I-site
+packing	I-site
+interactions	I-site
+.	O
+
+The	O
+structures	B-evidence
+and	O
+their	O
+analyses	O
+provide	O
+a	O
+foundation	O
+for	O
+future	O
+antibody	B-protein_type
+engineering	O
+and	O
+structure	O
+determination	O
+efforts	O
+.	O
+
+Crystal	B-evidence
+structures	I-evidence
+
+Crystal	B-evidence
+data	I-evidence
+,	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+,	O
+and	O
+refinement	B-evidence
+statistics	I-evidence
+.	O
+
+(	O
+Continued	O
+)	O
+Crystal	B-evidence
+data	I-evidence
+,	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+,	O
+and	O
+refinement	B-evidence
+statistics	I-evidence
+.	O
+
+The	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+a	O
+germline	B-experimental_method
+library	I-experimental_method
+composed	O
+of	O
+16	O
+Fabs	B-structure_element
+generated	O
+by	O
+combining	O
+4	O
+HCs	B-structure_element
+(	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+)	O
+and	O
+4	O
+LCs	B-structure_element
+(	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+)	O
+have	O
+been	O
+determined	O
+.	O
+
+The	O
+Fab	B-structure_element
+heavy	O
+and	O
+light	B-structure_element
+chain	I-structure_element
+sequences	O
+for	O
+the	O
+variants	O
+numbered	O
+according	O
+to	O
+Chothia	O
+are	O
+shown	O
+in	O
+Fig	O
+.	O
+S1	O
+.	O
+
+The	O
+four	O
+different	O
+HCs	B-structure_element
+all	O
+have	O
+the	O
+same	O
+CDR	B-structure_element
+H3	B-structure_element
+sequence	O
+,	O
+ARYDGIYGELDF	B-structure_element
+.	O
+
+Crystallization	B-experimental_method
+of	O
+the	O
+16	O
+Fabs	B-structure_element
+was	O
+previously	O
+reported	O
+.	O
+
+Three	O
+sets	O
+of	O
+the	O
+crystals	B-evidence
+were	O
+isomorphous	O
+with	O
+nearly	O
+identical	O
+unit	O
+cells	O
+(	O
+Table	O
+1	O
+).	O
+
+These	O
+include	O
+(	O
+1	O
+)	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+in	O
+P212121	O
+,	O
+(	O
+2	O
+)	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+in	O
+P6522	O
+,	O
+and	O
+(	O
+3	O
+)	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+in	O
+P212121	O
+.	O
+
+Variations	O
+occur	O
+in	O
+the	O
+pH	O
+(	O
+buffer	O
+)	O
+and	O
+the	O
+additives	O
+,	O
+and	O
+,	O
+in	O
+group	O
+3	O
+,	O
+PEG	B-chemical
+3350	I-chemical
+is	O
+the	O
+precipitant	O
+for	O
+one	O
+variants	O
+while	O
+ammonium	B-chemical
+sulfate	I-chemical
+is	O
+the	O
+precipitant	O
+for	O
+the	O
+other	O
+two	O
+.	O
+
+The	O
+similarity	O
+in	O
+the	O
+crystal	B-evidence
+forms	I-evidence
+is	O
+attributed	O
+in	O
+part	O
+to	O
+cross	O
+-	O
+seeding	O
+using	O
+the	O
+microseed	B-experimental_method
+matrix	I-experimental_method
+screening	I-experimental_method
+for	O
+groups	O
+2	O
+and	O
+3	O
+.	O
+
+The	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+16	O
+Fabs	B-structure_element
+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
+
+The	O
+number	O
+of	O
+Fab	B-structure_element
+molecules	O
+in	O
+the	O
+crystallographic	O
+asymmetric	O
+unit	O
+varies	O
+from	O
+1	O
+(	O
+for	O
+12	O
+Fabs	B-structure_element
+)	O
+to	O
+2	O
+(	O
+for	O
+4	O
+Fabs	B-structure_element
+).	O
+
+Overall	O
+the	O
+structures	B-evidence
+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	B-evidence
+are	O
+more	O
+complete	O
+than	O
+those	O
+for	O
+the	O
+lower	O
+resolution	O
+structures	B-evidence
+(	O
+Table	O
+S1	O
+).	O
+
+Invariably	O
+,	O
+the	O
+HCs	B-structure_element
+have	O
+more	O
+disorder	B-protein_state
+than	O
+the	O
+LCs	B-structure_element
+.	O
+
+For	O
+the	O
+LC	B-structure_element
+,	O
+the	O
+disorder	B-protein_state
+is	O
+observed	O
+at	O
+2	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+residues	O
+with	O
+few	O
+exceptions	O
+.	O
+
+Apart	O
+from	O
+the	O
+C	O
+-	O
+terminus	O
+,	O
+only	O
+a	O
+few	O
+surface	O
+residues	O
+in	O
+LC	B-structure_element
+are	O
+disordered	B-protein_state
+.	O
+
+The	O
+HCs	B-structure_element
+feature	O
+the	O
+largest	O
+number	O
+of	O
+disordered	B-protein_state
+residues	O
+,	O
+with	O
+the	O
+lower	O
+resolution	O
+structures	B-evidence
+having	O
+the	O
+most	O
+.	O
+
+The	O
+C	O
+-	O
+terminal	O
+residues	O
+including	O
+the	O
+6xHis	O
+tags	O
+are	O
+disordered	B-protein_state
+in	O
+all	O
+16	O
+structures	B-evidence
+.	O
+
+In	O
+addition	O
+to	O
+these	O
+,	O
+2	O
+primary	O
+disordered	O
+stretches	O
+of	O
+residues	O
+are	O
+observed	O
+in	O
+a	O
+number	O
+of	O
+structures	B-evidence
+(	O
+Table	O
+S1	O
+).	O
+
+One	O
+involves	O
+the	O
+loop	B-structure_element
+connecting	O
+the	O
+first	O
+2	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+of	O
+the	O
+constant	B-structure_element
+domain	I-structure_element
+(	O
+in	O
+all	O
+Fabs	B-structure_element
+except	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+).	O
+
+The	O
+other	O
+is	O
+located	O
+in	O
+CDR	B-structure_element
+H3	B-structure_element
+(	O
+in	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+,	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+in	O
+one	O
+of	O
+2	O
+copies	O
+of	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+).	O
+
+CDR	B-structure_element
+H1	B-structure_element
+and	O
+CDR	B-structure_element
+H2	B-structure_element
+also	O
+show	O
+some	O
+degree	O
+of	O
+disorder	B-protein_state
+,	O
+but	O
+to	O
+a	O
+lesser	O
+extent	O
+.	O
+
+CDR	B-structure_element
+canonical	O
+structures	B-evidence
+
+Several	O
+CDR	B-structure_element
+definitions	O
+have	O
+evolved	O
+over	O
+decades	O
+of	O
+antibody	B-protein_type
+research	O
+.	O
+
+Depending	O
+on	O
+the	O
+focus	O
+of	O
+the	O
+study	O
+,	O
+the	O
+CDR	B-structure_element
+boundaries	O
+differ	O
+slightly	O
+between	O
+various	O
+definitions	O
+.	O
+
+In	O
+this	O
+work	O
+,	O
+we	O
+use	O
+the	O
+CDR	B-structure_element
+definition	O
+of	O
+North	O
+et	O
+al	O
+.,	O
+which	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+Martin	O
+with	O
+the	O
+following	O
+exceptions	O
+:	O
+1	O
+)	O
+CDRs	B-structure_element
+H1	B-structure_element
+and	O
+H3	B-structure_element
+begin	O
+immediately	O
+after	O
+the	O
+Cys	B-residue_name
+;	O
+and	O
+2	O
+)	O
+CDR	B-structure_element
+L2	B-structure_element
+includes	O
+an	O
+additional	O
+residue	O
+at	O
+the	O
+N	O
+-	O
+terminal	O
+side	O
+,	O
+typically	O
+Tyr	B-residue_name
+.	O
+
+CDR	B-structure_element
+H1	B-structure_element
+
+The	O
+superposition	B-experimental_method
+of	O
+CDR	B-structure_element
+H1	B-structure_element
+backbones	O
+for	O
+all	O
+HC	B-complex_assembly
+:	I-complex_assembly
+LC	I-complex_assembly
+pairs	O
+with	O
+heavy	B-structure_element
+chains	I-structure_element
+:	O
+(	O
+A	O
+)	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+(	O
+B	O
+)	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+(	O
+C	O
+)	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+(	O
+D	O
+)	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+.	O
+
+CDRs	B-structure_element
+are	O
+defined	O
+using	O
+the	O
+Dunbrack	O
+convention	O
+[	O
+12	O
+].	O
+
+Assignments	O
+for	O
+2	O
+copies	O
+of	O
+the	O
+Fab	B-structure_element
+in	O
+the	O
+asymmetric	O
+unit	O
+are	O
+given	O
+for	O
+5	O
+structures	B-evidence
+.	O
+
+No	O
+assignment	O
+(	O
+NA	O
+)	O
+for	O
+CDRs	B-structure_element
+with	O
+missing	O
+residues	O
+.	O
+
+The	O
+four	O
+HCs	B-structure_element
+feature	O
+CDR	B-structure_element
+H1	B-structure_element
+of	O
+the	O
+same	O
+length	O
+,	O
+and	O
+their	O
+sequences	O
+are	O
+highly	O
+similar	O
+(	O
+Table	O
+2	O
+).	O
+
+The	O
+CDR	B-structure_element
+H1	B-structure_element
+backbone	O
+conformations	O
+for	O
+all	O
+variants	O
+for	O
+each	O
+of	O
+the	O
+HCs	B-structure_element
+are	O
+shown	O
+in	O
+Fig	O
+.	O
+1	O
+.	O
+
+Three	O
+of	O
+the	O
+HCs	B-structure_element
+,	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+,	O
+have	O
+the	O
+same	O
+canonical	O
+structure	O
+,	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+and	O
+the	O
+backbone	O
+conformations	O
+are	O
+tightly	O
+clustered	O
+for	O
+each	O
+set	O
+of	O
+Fab	B-structure_element
+structures	B-evidence
+as	O
+reflected	O
+in	O
+the	O
+rmsd	B-evidence
+values	I-evidence
+(	O
+Fig	O
+.	O
+1B	O
+-	O
+D	O
+).	O
+
+Some	O
+deviation	O
+is	O
+observed	O
+for	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+,	O
+mostly	O
+due	O
+to	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+which	O
+exhibits	O
+a	O
+significant	O
+degree	O
+of	O
+disorder	O
+in	O
+CDR	B-structure_element
+H1	B-structure_element
+.	O
+
+The	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+backbone	O
+is	O
+weak	O
+and	O
+discontinuous	O
+,	O
+and	O
+completely	O
+missing	O
+for	O
+several	O
+side	O
+chains	O
+.	O
+
+The	O
+CDR	B-structure_element
+H1	B-structure_element
+structures	B-evidence
+with	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+shown	O
+in	O
+Fig	O
+.	O
+1A	O
+are	O
+quite	O
+variable	O
+,	O
+both	O
+for	O
+the	O
+structures	B-evidence
+with	O
+different	O
+LCs	B-structure_element
+and	O
+for	O
+the	O
+copies	O
+of	O
+the	O
+same	O
+Fab	B-structure_element
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+and	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+.	O
+
+In	O
+total	O
+,	O
+6	O
+independent	O
+Fab	B-structure_element
+structures	B-evidence
+produce	O
+5	O
+different	O
+canonical	O
+structures	B-evidence
+,	O
+namely	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+3	I-mutant
+,	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+4	I-mutant
+,	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+6	I-mutant
+and	O
+H1	B-mutant
+-	I-mutant
+13	I-mutant
+-	I-mutant
+10	I-mutant
+.	O
+
+A	O
+major	O
+difference	O
+of	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+from	O
+the	O
+other	O
+germlines	O
+in	O
+the	O
+experimental	O
+data	O
+set	O
+is	O
+the	O
+presence	O
+of	O
+Gly	B-residue_name
+instead	O
+of	O
+Phe	B-residue_name
+or	O
+Tyr	B-residue_name
+at	O
+position	O
+27	B-residue_number
+(	O
+residue	O
+5	O
+of	O
+13	O
+in	O
+CDR	B-structure_element
+H1	B-structure_element
+).	O
+
+Glycine	B-residue_name
+introduces	O
+the	O
+possibility	O
+of	O
+a	O
+higher	O
+degree	O
+of	O
+conformational	O
+flexibility	O
+that	O
+undoubtedly	O
+translates	O
+to	O
+the	O
+differences	O
+observed	O
+,	O
+and	O
+contributes	O
+to	O
+the	O
+elevated	O
+thermal	O
+parameters	O
+for	O
+the	O
+atoms	O
+in	O
+the	O
+amino	O
+acid	O
+residues	O
+in	O
+this	O
+region	O
+.	O
+
+CDR	B-structure_element
+H2	B-structure_element
+
+The	O
+superposition	B-experimental_method
+of	O
+CDR	B-structure_element
+H2	B-structure_element
+backbones	O
+for	O
+all	O
+HC	B-complex_assembly
+:	I-complex_assembly
+LC	I-complex_assembly
+pairs	O
+with	O
+heavy	B-structure_element
+chains	I-structure_element
+:	O
+(	O
+A	O
+)	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+(	O
+B	O
+)	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+(	O
+C	O
+)	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+(	O
+D	O
+)	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+.	O
+
+The	O
+canonical	O
+structures	O
+of	O
+CDR	B-structure_element
+H2	B-structure_element
+have	O
+fairly	O
+consistent	O
+conformations	O
+(	O
+Table	O
+2	O
+,	O
+Fig	O
+.	O
+2	O
+).	O
+
+Each	O
+of	O
+the	O
+4	O
+HCs	B-structure_element
+adopts	O
+only	O
+one	O
+canonical	O
+structure	O
+regardless	O
+of	O
+the	O
+pairing	O
+LC	B-structure_element
+.	O
+
+Germlines	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+have	O
+the	O
+same	O
+canonical	O
+structure	O
+assignment	O
+H2	B-mutant
+-	I-mutant
+10	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+has	O
+H2	B-mutant
+-	I-mutant
+10	I-mutant
+-	I-mutant
+2	I-mutant
+,	O
+and	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+has	O
+H2	B-mutant
+-	I-mutant
+9	I-mutant
+-	I-mutant
+3	I-mutant
+.	O
+
+The	O
+conformations	O
+for	O
+all	O
+of	O
+these	O
+CDR	B-structure_element
+H2s	B-structure_element
+are	O
+tightly	O
+clustered	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+
+In	O
+one	O
+case	O
+,	O
+in	O
+the	O
+second	O
+Fab	B-structure_element
+of	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+CDR	B-structure_element
+H2	B-structure_element
+is	O
+partially	B-protein_state
+disordered	I-protein_state
+(	O
+Δ55	B-mutant
+-	I-mutant
+60	I-mutant
+).	O
+
+Although	O
+three	O
+of	O
+the	O
+germlines	O
+have	O
+CDR	B-structure_element
+H2	B-structure_element
+of	O
+the	O
+same	O
+length	O
+,	O
+10	B-residue_range
+residues	I-residue_range
+,	O
+they	O
+adopt	O
+2	O
+distinctively	O
+different	O
+conformations	O
+depending	O
+mostly	O
+on	O
+the	O
+residue	O
+at	O
+position	O
+71	B-residue_number
+from	O
+the	O
+so	O
+-	O
+called	O
+CDR	B-structure_element
+H4	B-structure_element
+.	O
+
+Arg71	B-residue_name_number
+in	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+fills	O
+the	O
+space	O
+between	O
+CDRs	B-structure_element
+H2	B-structure_element
+and	O
+H4	B-structure_element
+,	O
+and	O
+defines	O
+the	O
+conformation	O
+of	O
+the	O
+tip	O
+of	O
+CDR	B-structure_element
+H2	B-structure_element
+so	O
+that	O
+residue	O
+54	B-residue_number
+points	O
+away	O
+from	O
+the	O
+antigen	B-site
+binding	I-site
+site	I-site
+.	O
+
+Germlines	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+are	O
+unique	O
+in	O
+the	O
+human	B-species
+repertoire	O
+in	O
+having	O
+an	O
+Ala	B-residue_name
+at	O
+position	O
+71	B-residue_number
+that	O
+leaves	O
+enough	O
+space	O
+for	O
+H	B-structure_element
+-	O
+Pro52a	B-residue_name_number
+to	O
+pack	O
+deeper	O
+against	O
+CDR	B-structure_element
+H4	B-structure_element
+so	O
+that	O
+the	O
+following	O
+residues	O
+53	B-residue_number
+and	O
+54	B-residue_number
+point	O
+toward	O
+the	O
+putative	O
+antigen	O
+.	O
+
+Conformations	O
+of	O
+CDR	B-structure_element
+H2	B-structure_element
+in	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+,	O
+both	O
+of	O
+which	O
+have	O
+canonical	O
+structure	O
+H2	B-mutant
+-	I-mutant
+10	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+show	O
+little	O
+deviation	O
+within	O
+each	O
+set	O
+of	O
+4	O
+structures	B-evidence
+.	O
+
+However	O
+,	O
+there	O
+is	O
+a	O
+significant	O
+shift	O
+of	O
+the	O
+CDR	B-structure_element
+as	O
+a	O
+rigid	O
+body	O
+when	O
+the	O
+2	O
+sets	O
+are	O
+superimposed	B-experimental_method
+.	O
+
+Most	O
+likely	O
+this	O
+is	O
+the	O
+result	O
+of	O
+interaction	O
+of	O
+CDR	B-structure_element
+H2	B-structure_element
+with	O
+CDR	B-structure_element
+H1	B-structure_element
+,	O
+namely	O
+with	O
+the	O
+residue	O
+at	O
+position	O
+33	B-residue_number
+(	O
+residue	O
+11	O
+of	O
+13	O
+in	O
+CDR	B-structure_element
+H1	B-structure_element
+).	O
+
+Germline	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+has	O
+Ala	B-residue_name
+at	O
+position	O
+33	B-residue_number
+whereas	O
+in	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+position	O
+33	B-residue_number
+is	O
+occupied	O
+by	O
+a	O
+bulky	O
+Trp	B-residue_name
+,	O
+which	O
+stacks	O
+against	O
+H	B-structure_element
+-	O
+Tyr52	B-residue_name_number
+and	O
+drives	O
+CDR	B-structure_element
+H2	B-structure_element
+away	O
+from	O
+the	O
+center	O
+.	O
+
+CDR	B-structure_element
+L1	B-structure_element
+
+The	O
+superposition	B-experimental_method
+of	O
+CDR	B-structure_element
+L1	B-structure_element
+backbones	O
+for	O
+all	O
+HC	B-complex_assembly
+:	I-complex_assembly
+LC	I-complex_assembly
+pairs	O
+with	O
+light	B-structure_element
+chains	I-structure_element
+:	O
+(	O
+A	O
+)	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+(	O
+B	O
+)	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+(	O
+C	O
+)	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+(	O
+D	O
+)	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+.	O
+
+The	O
+four	O
+LC	B-structure_element
+CDRs	B-structure_element
+L1	B-structure_element
+feature	O
+3	O
+different	O
+lengths	O
+(	O
+11	B-residue_range
+,	O
+12	B-residue_range
+and	O
+17	B-residue_range
+residues	O
+)	O
+having	O
+a	O
+total	O
+of	O
+4	O
+different	O
+canonical	O
+structure	O
+assignments	O
+.	O
+
+Of	O
+these	O
+LCs	B-structure_element
+,	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+and	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+have	O
+the	O
+same	O
+canonical	O
+structure	O
+,	O
+L1	B-mutant
+-	I-mutant
+11	I-mutant
+-	I-mutant
+1	I-mutant
+,	O
+and	O
+superimpose	B-experimental_method
+very	O
+well	O
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+
+For	O
+the	O
+remaining	O
+2	O
+,	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+has	O
+2	O
+different	O
+assignments	O
+,	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+1	I-mutant
+and	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+2	I-mutant
+,	O
+while	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+has	O
+a	O
+single	O
+assignment	O
+,	O
+L1	B-mutant
+-	I-mutant
+17	I-mutant
+-	I-mutant
+1	I-mutant
+.	O
+
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+has	O
+the	O
+longest	O
+CDR	B-structure_element
+L1	B-structure_element
+,	O
+composed	O
+of	O
+17	B-residue_range
+amino	I-residue_range
+acid	I-residue_range
+residues	I-residue_range
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+
+Despite	O
+this	O
+,	O
+the	O
+conformations	O
+are	O
+tightly	O
+clustered	O
+(	O
+rmsd	B-evidence
+is	O
+0	O
+.	O
+20	O
+Å	O
+).	O
+
+The	O
+backbone	O
+conformations	O
+of	O
+the	O
+stem	B-structure_element
+regions	I-structure_element
+superimpose	O
+well	O
+.	O
+
+Some	O
+changes	O
+in	O
+conformation	O
+occur	O
+between	O
+residues	O
+30a	B-residue_number
+and	O
+30f	B-residue_number
+(	O
+residues	O
+8	B-residue_number
+and	O
+13	B-residue_number
+of	O
+17	B-residue_number
+in	O
+CDR	B-structure_element
+L1	B-structure_element
+).	O
+
+This	O
+is	O
+the	O
+tip	O
+of	O
+the	O
+loop	B-structure_element
+region	I-structure_element
+,	O
+which	O
+appears	O
+to	O
+have	O
+similar	O
+conformations	O
+that	O
+fan	O
+out	O
+the	O
+structures	B-evidence
+because	O
+of	O
+the	O
+slight	O
+differences	O
+in	O
+torsion	O
+angles	O
+in	O
+the	O
+backbone	O
+near	O
+Tyr30a	B-residue_name_number
+and	O
+Lys30f	B-residue_name_number
+.	O
+
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+is	O
+the	O
+most	O
+variable	O
+in	O
+CDR	B-structure_element
+L1	B-structure_element
+among	O
+the	O
+4	O
+germlines	O
+as	O
+indicated	O
+by	O
+an	O
+rmsd	B-evidence
+of	O
+0	O
+.	O
+54	O
+Å	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+Two	O
+structures	B-evidence
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+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
+
+The	O
+third	O
+structure	O
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+has	O
+CDR	B-structure_element
+L1	B-structure_element
+as	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+2	I-mutant
+,	O
+which	O
+deviates	O
+from	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+1	I-mutant
+at	O
+residues	O
+29	B-residue_range
+-	I-residue_range
+32	I-residue_range
+,	O
+i	O
+.	O
+e	O
+.,	O
+at	O
+the	O
+site	O
+of	O
+insertion	O
+with	O
+respect	O
+to	O
+the	O
+11	B-residue_range
+-	I-residue_range
+residue	I-residue_range
+CDR	B-structure_element
+.	O
+
+The	O
+fourth	O
+member	O
+of	O
+the	O
+set	O
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+was	O
+crystallized	B-experimental_method
+with	O
+2	O
+Fabs	B-structure_element
+in	O
+the	O
+asymmetric	O
+unit	O
+.	O
+
+The	O
+conformation	O
+of	O
+CDR	B-structure_element
+L1	B-structure_element
+in	O
+these	O
+2	O
+Fabs	B-structure_element
+is	O
+slightly	O
+different	O
+,	O
+and	O
+both	O
+conformations	O
+fall	O
+somewhere	O
+between	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+1	I-mutant
+and	O
+L1	B-mutant
+-	I-mutant
+12	I-mutant
+-	I-mutant
+2	I-mutant
+.	O
+
+This	O
+reflects	O
+the	O
+lack	O
+of	O
+accuracy	O
+in	O
+the	O
+structure	B-evidence
+due	O
+to	O
+low	O
+resolution	O
+of	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+(	O
+3	O
+.	O
+3	O
+Å	O
+).	O
+
+CDR	B-structure_element
+L2	B-structure_element
+
+The	O
+superposition	B-experimental_method
+of	O
+CDR	B-structure_element
+L2	B-structure_element
+backbones	O
+for	O
+all	O
+HC	B-complex_assembly
+:	I-complex_assembly
+LC	I-complex_assembly
+pairs	O
+with	O
+light	B-structure_element
+chains	I-structure_element
+:	O
+(	O
+A	O
+)	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+(	O
+B	O
+)	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+(	O
+C	O
+)	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+(	O
+D	O
+)	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+.	O
+
+All	O
+four	O
+LCs	B-structure_element
+have	O
+CDR	B-structure_element
+L2	B-structure_element
+of	O
+the	O
+same	O
+length	O
+and	O
+canonical	O
+structure	O
+,	O
+L2	B-mutant
+-	I-mutant
+8	I-mutant
+-	I-mutant
+1	I-mutant
+(	O
+Table	O
+2	O
+).	O
+
+The	O
+CDR	B-structure_element
+L2	B-structure_element
+conformations	O
+for	O
+each	O
+of	O
+the	O
+LCs	B-structure_element
+paired	O
+with	O
+the	O
+4	O
+HCs	B-structure_element
+are	O
+clustered	O
+more	O
+tightly	O
+than	O
+any	O
+of	O
+the	O
+other	O
+CDRs	B-structure_element
+(	O
+rmsd	B-evidence
+values	O
+are	O
+in	O
+the	O
+range	O
+0	O
+.	O
+09	O
+-	O
+0	O
+.	O
+16	O
+Å	O
+),	O
+and	O
+all	O
+4	O
+sets	O
+have	O
+virtually	O
+the	O
+same	O
+conformation	O
+despite	O
+the	O
+sequence	O
+diversity	O
+of	O
+the	O
+loop	B-structure_element
+.	O
+
+CDR	B-structure_element
+L3	B-structure_element
+
+The	O
+superposition	B-experimental_method
+of	O
+CDR	B-structure_element
+L3	B-structure_element
+backbones	O
+for	O
+all	O
+HC	B-complex_assembly
+:	I-complex_assembly
+LC	I-complex_assembly
+pairs	O
+with	O
+light	B-structure_element
+chains	I-structure_element
+:	O
+(	O
+A	O
+)	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+(	O
+B	O
+)	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+(	O
+C	O
+)	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+(	O
+D	O
+)	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+.	O
+
+As	O
+with	O
+CDR	B-structure_element
+L2	B-structure_element
+,	O
+all	O
+4	O
+LCs	B-structure_element
+have	O
+CDR	B-structure_element
+L3	B-structure_element
+of	O
+the	O
+same	O
+length	O
+and	O
+canonical	O
+structure	B-evidence
+,	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
+
+The	O
+conformations	O
+of	O
+CDR	B-structure_element
+L3	B-structure_element
+for	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+and	O
+particularly	O
+for	O
+L320	O
+,	O
+are	O
+not	O
+as	O
+tightly	O
+clustered	O
+as	O
+those	O
+of	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+The	O
+slight	O
+conformational	O
+variability	O
+occurs	O
+in	O
+the	O
+region	O
+of	O
+amino	O
+acid	O
+residues	O
+90	B-residue_range
+-	I-residue_range
+92	I-residue_range
+,	O
+which	O
+is	O
+in	O
+contact	O
+with	O
+CDR	B-structure_element
+H3	B-structure_element
+.	O
+
+CDR	B-structure_element
+H3	B-structure_element
+conformational	O
+diversity	O
+
+As	O
+mentioned	O
+earlier	O
+,	O
+all	O
+16	O
+Fabs	B-structure_element
+have	O
+the	O
+same	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+for	O
+which	O
+the	O
+amino	O
+acid	O
+sequence	O
+is	O
+derived	O
+from	O
+the	O
+anti	O
+-	O
+CCL2	O
+antibody	B-protein_type
+CNTO	B-chemical
+888	I-chemical
+.	O
+
+The	O
+loop	B-structure_element
+and	O
+the	O
+2	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+of	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+in	O
+this	O
+‘	O
+parent	O
+’	O
+structure	B-evidence
+are	O
+stabilized	O
+by	O
+H	O
+-	O
+bonds	O
+between	O
+the	O
+carbonyl	O
+oxygen	O
+and	O
+peptide	O
+nitrogen	O
+atoms	O
+in	O
+the	O
+2	O
+strands	O
+.	O
+
+An	O
+interesting	O
+feature	O
+of	O
+these	O
+CDR	B-structure_element
+H3	B-structure_element
+structures	B-evidence
+is	O
+the	O
+presence	O
+of	O
+a	O
+water	B-chemical
+molecule	O
+that	O
+interacts	O
+with	O
+the	O
+peptide	O
+nitrogens	O
+and	O
+carbonyl	O
+oxygens	O
+near	O
+the	O
+bridging	O
+loop	B-structure_element
+connecting	O
+the	O
+2	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+.	O
+
+This	O
+water	B-chemical
+is	O
+present	O
+in	O
+both	O
+the	O
+bound	B-protein_state
+(	O
+4DN4	O
+)	O
+and	O
+unbound	B-protein_state
+(	O
+4DN3	O
+)	O
+forms	O
+of	O
+CNTO	B-chemical
+888	I-chemical
+.	O
+
+The	O
+stem	B-structure_element
+region	I-structure_element
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+in	O
+the	O
+parental	O
+Fab	B-structure_element
+is	O
+in	O
+a	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+,	O
+in	O
+which	O
+the	O
+indole	O
+nitrogen	O
+of	O
+Trp103	B-residue_name_number
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+the	O
+carbonyl	O
+oxygen	O
+of	O
+Leu100b	B-residue_name_number
+.	O
+
+The	O
+carboxyl	O
+group	O
+of	O
+Asp101	B-residue_name_number
+forms	O
+a	O
+salt	O
+bridge	O
+with	O
+Arg94	B-residue_name_number
+.	O
+
+Ribbon	O
+representations	O
+of	O
+(	O
+A	O
+)	O
+the	O
+superposition	B-experimental_method
+of	O
+all	O
+CDR	B-structure_element
+H3s	B-structure_element
+of	O
+the	O
+structures	B-evidence
+with	O
+complete	O
+backbone	O
+traces	O
+.	O
+(	O
+B	O
+)	O
+The	O
+CDR	B-structure_element
+H3s	B-structure_element
+rotated	O
+90	O
+°	O
+about	O
+the	O
+y	O
+axis	O
+of	O
+the	O
+page	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+each	O
+CDR	B-structure_element
+H3	B-structure_element
+is	O
+represented	O
+with	O
+a	O
+different	O
+color	O
+.	O
+
+Despite	O
+having	O
+the	O
+same	O
+amino	O
+acid	O
+sequence	O
+in	O
+all	O
+variants	O
+,	O
+CDR	B-structure_element
+H3	B-structure_element
+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	B-structure_element
+in	O
+the	O
+experimental	O
+set	O
+.	O
+
+Three	O
+of	O
+the	O
+21	O
+Fab	B-structure_element
+structures	B-evidence
+(	O
+including	O
+multiple	O
+copies	O
+in	O
+the	O
+asymmetric	O
+unit	O
+),	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+,	O
+H551	B-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+(	O
+one	O
+of	O
+the	O
+2	O
+Fabs	B-structure_element
+),	O
+have	O
+missing	B-protein_state
+(	O
+disordered	B-protein_state
+)	O
+residues	O
+at	O
+the	O
+apex	O
+of	O
+the	O
+CDR	B-structure_element
+loop	I-structure_element
+.	O
+
+Another	O
+four	O
+of	O
+the	O
+Fabs	B-structure_element
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+have	O
+missing	O
+side	O
+-	O
+chain	O
+atoms	O
+.	O
+
+The	O
+variations	O
+in	O
+CDR	B-structure_element
+H3	B-structure_element
+conformation	O
+are	O
+illustrated	O
+in	O
+Fig	O
+.	O
+6	O
+for	O
+the	O
+18	O
+Fab	B-structure_element
+structures	B-evidence
+that	O
+have	O
+ordered	O
+backbone	O
+atoms	O
+.	O
+
+A	O
+comparison	O
+of	O
+representatives	O
+of	O
+the	O
+“	O
+kinked	B-protein_state
+”	O
+and	O
+“	O
+extended	B-protein_state
+”	O
+structures	B-evidence
+.	O
+
+(	O
+A	O
+)	O
+The	O
+“	O
+kinked	B-protein_state
+”	O
+CDR	B-structure_element
+H3	B-structure_element
+of	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+with	O
+purple	O
+carbon	O
+atoms	O
+and	O
+yellow	O
+dashed	O
+lines	O
+connecting	O
+the	O
+H	O
+-	O
+bond	O
+pairs	O
+for	O
+Leu100b	B-residue_name_number
+O	O
+and	O
+Trp103	B-residue_name_number
+NE1	O
+,	O
+Arg94	B-residue_name_number
+NE	O
+and	O
+Asp101	B-residue_name_number
+OD1	O
+,	O
+and	O
+Arg94	B-residue_name_number
+NH2	O
+and	O
+Asp101	B-residue_name_number
+OD2	O
+.	O
+
+(	O
+B	O
+)	O
+The	O
+“	O
+extended	B-protein_state
+”	O
+CDR	B-structure_element
+H3	B-structure_element
+of	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+with	O
+green	O
+carbon	O
+atoms	O
+and	O
+yellow	O
+dashed	O
+lines	O
+connecting	O
+the	O
+H	O
+-	O
+bond	O
+pairs	O
+for	O
+Asp101	B-residue_name_number
+OD1	O
+and	O
+OD2	O
+and	O
+Trp103	B-residue_name_number
+NE1	O
+.	O
+
+In	O
+10	O
+of	O
+the	O
+18	O
+Fab	B-structure_element
+structures	B-evidence
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+(	O
+2	O
+Fabs	B-structure_element
+),	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+(	O
+2	O
+Fabs	B-structure_element
+),	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+the	O
+CDRs	B-structure_element
+have	O
+similar	O
+conformations	O
+to	O
+that	O
+found	O
+in	O
+4DN3	O
+.	O
+
+The	O
+bases	O
+of	O
+these	O
+structures	B-evidence
+have	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+with	O
+the	O
+H	O
+-	O
+bond	O
+between	O
+Trp103	B-residue_name_number
+and	O
+Leu100b	B-residue_name_number
+.	O
+
+A	O
+representative	O
+CDR	B-structure_element
+H3	B-structure_element
+structure	B-evidence
+for	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+illustrating	O
+this	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+7A	O
+.	O
+
+The	O
+largest	O
+backbone	O
+conformational	O
+deviation	O
+for	O
+the	O
+set	O
+is	O
+at	O
+Tyr99	B-residue_name_number
+,	O
+where	O
+the	O
+C	O
+=	O
+O	O
+is	O
+rotated	O
+by	O
+90	O
+°	O
+relative	O
+to	O
+that	O
+observed	O
+in	O
+4DN3	O
+.	O
+
+Also	O
+,	O
+it	O
+is	O
+worth	O
+noting	O
+that	O
+only	O
+one	O
+of	O
+these	O
+structures	B-evidence
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+has	O
+the	O
+conserved	B-protein_state
+water	B-chemical
+molecule	O
+in	O
+CDR	B-structure_element
+H3	B-structure_element
+observed	O
+in	O
+the	O
+4DN3	O
+and	O
+4DN4	O
+structures	B-evidence
+.	O
+
+In	O
+fact	O
+,	O
+it	O
+is	O
+the	O
+only	O
+Fab	B-structure_element
+in	O
+the	O
+set	O
+that	O
+has	O
+a	O
+water	B-chemical
+molecule	O
+present	O
+at	O
+this	O
+site	O
+.	O
+
+The	O
+CDR	B-structure_element
+H3	B-structure_element
+for	O
+this	O
+structure	B-evidence
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+S3	O
+.	O
+
+The	O
+remaining	O
+8	O
+Fabs	B-structure_element
+can	O
+be	O
+grouped	O
+into	O
+5	O
+different	O
+conformational	O
+classes	O
+.	O
+
+Three	O
+of	O
+the	O
+Fabs	B-structure_element
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+,	O
+have	O
+distinctive	O
+conformations	O
+.	O
+
+The	O
+stem	B-structure_element
+regions	I-structure_element
+in	O
+these	O
+3	O
+cases	O
+are	O
+in	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+consistent	O
+with	O
+that	O
+observed	O
+for	O
+4DN3	O
+.	O
+
+The	O
+five	O
+remaining	O
+Fabs	B-structure_element
+,	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+(	O
+2	O
+copies	O
+),	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+(	O
+2	O
+copies	O
+)	O
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+have	O
+3	O
+different	O
+CDR	B-structure_element
+H3	B-structure_element
+conformations	O
+(	O
+Fig	O
+.	O
+S4	O
+).	O
+
+The	O
+stem	B-structure_element
+regions	I-structure_element
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+for	O
+the	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+Fabs	B-structure_element
+are	O
+in	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+while	O
+,	O
+surprisingly	O
+,	O
+those	O
+of	O
+the	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+pair	O
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+are	O
+in	O
+the	O
+‘	O
+extended	B-protein_state
+’	O
+conformation	O
+(	O
+Fig	O
+.	O
+7B	O
+).	O
+
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+domain	O
+packing	O
+
+The	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+domains	O
+have	O
+a	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+structure	I-structure_element
+(	O
+also	O
+often	O
+referred	O
+as	O
+a	O
+Greek	B-structure_element
+key	I-structure_element
+motif	I-structure_element
+)	O
+and	O
+each	O
+is	O
+composed	O
+of	O
+a	O
+4	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+and	I-structure_element
+a	I-structure_element
+5	I-structure_element
+-	I-structure_element
+stranded	I-structure_element
+antiparallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheets	I-structure_element
+.	O
+
+The	O
+two	O
+domains	O
+pack	O
+together	O
+such	O
+that	O
+the	O
+5	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheets	I-structure_element
+,	O
+which	O
+have	O
+hydrophobic	O
+surfaces	O
+,	O
+interact	O
+with	O
+each	O
+other	O
+bringing	O
+the	O
+CDRs	B-structure_element
+from	O
+both	O
+the	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+domains	O
+into	O
+close	O
+proximity	O
+.	O
+
+The	O
+domain	O
+packing	O
+of	O
+the	O
+variants	O
+was	O
+assessed	O
+by	O
+computing	O
+the	O
+domain	B-site
+interface	I-site
+interactions	O
+,	O
+the	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+tilt	B-evidence
+angles	I-evidence
+,	O
+the	O
+buried	O
+surface	O
+area	O
+and	O
+surface	O
+complementarity	O
+.	O
+
+VH	B-site
+:	I-site
+VL	I-site
+interface	I-site
+amino	O
+acid	O
+residue	O
+interactions	O
+
+The	O
+conserved	B-protein_state
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+interactions	O
+as	O
+viewed	O
+along	O
+the	O
+VH	B-structure_element
+/	O
+VL	B-structure_element
+axis	O
+.	O
+
+The	O
+VH	B-structure_element
+residues	O
+are	O
+in	O
+blue	O
+,	O
+the	O
+VL	B-structure_element
+residues	O
+are	O
+in	O
+orange	O
+.	O
+
+The	O
+VH	B-site
+:	I-site
+VL	I-site
+interface	I-site
+is	O
+pseudosymmetric	B-protein_state
+,	O
+and	O
+involves	O
+2	O
+stretches	O
+of	O
+the	O
+polypeptide	O
+chain	O
+from	O
+each	O
+domain	O
+,	O
+namely	O
+CDR3	B-structure_element
+and	O
+the	O
+framework	B-structure_element
+region	I-structure_element
+between	O
+CDRs	B-structure_element
+1	I-structure_element
+and	I-structure_element
+2	I-structure_element
+.	O
+
+These	O
+stretches	O
+form	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+hairpins	I-structure_element
+within	O
+the	O
+internal	O
+5	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+.	O
+
+There	O
+are	O
+a	O
+few	O
+principal	O
+inter	O
+-	O
+domain	O
+interactions	O
+that	O
+are	O
+conserved	O
+not	O
+only	O
+in	O
+the	O
+experimental	O
+set	O
+of	O
+16	O
+Fabs	B-structure_element
+,	O
+but	O
+in	O
+all	O
+human	B-species
+antibodies	B-protein_type
+.	O
+
+They	O
+include	O
+:	O
+1	O
+)	O
+a	O
+bidentate	O
+hydrogen	O
+bond	O
+between	O
+L	B-structure_element
+-	O
+Gln38	B-residue_name_number
+and	O
+H	B-structure_element
+-	O
+Gln39	B-residue_name_number
+;	O
+2	O
+)	O
+H	B-structure_element
+-	O
+Leu45	B-residue_name_number
+in	O
+a	O
+hydrophobic	B-site
+pocket	I-site
+between	O
+L	B-structure_element
+-	O
+Phe98	B-residue_name_number
+,	O
+L	B-structure_element
+-	O
+Tyr87	B-residue_name_number
+and	O
+L	B-structure_element
+-	O
+Pro44	B-residue_name_number
+;	O
+3	O
+)	O
+L	B-structure_element
+-	O
+Pro44	B-residue_name_number
+stacked	O
+against	O
+H	B-structure_element
+-	O
+Trp103	B-residue_name_number
+;	O
+and	O
+4	O
+)	O
+L	B-structure_element
+-	O
+Ala43	B-residue_name_number
+opposite	O
+the	O
+face	O
+of	O
+H	B-structure_element
+-	O
+Tyr91	B-residue_name_number
+(	O
+Fig	O
+.	O
+8	O
+).	O
+
+With	O
+the	O
+exception	O
+of	O
+L	B-structure_element
+-	O
+Ala43	B-residue_name_number
+,	O
+all	O
+other	O
+residues	O
+are	O
+conserved	O
+in	O
+human	B-species
+germlines	O
+.	O
+
+Position	O
+43	B-residue_number
+may	O
+be	O
+alternatively	O
+occupied	O
+by	O
+Ser	B-residue_name
+,	O
+Val	B-residue_name
+or	O
+Pro	B-residue_name
+(	O
+as	O
+in	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+),	O
+but	O
+the	O
+hydrophobic	O
+interaction	O
+with	O
+H	B-structure_element
+-	O
+Tyr91	B-residue_name_number
+is	O
+preserved	O
+.	O
+
+These	O
+core	O
+interactions	O
+provide	O
+enough	O
+stability	O
+to	O
+the	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+dimer	B-oligomeric_state
+so	O
+that	O
+additional	O
+VH	B-site
+-	I-site
+VL	I-site
+contacts	I-site
+can	O
+tolerate	O
+amino	O
+acid	O
+sequence	O
+variations	O
+in	O
+CDRs	B-structure_element
+H3	B-structure_element
+and	O
+L3	B-structure_element
+that	O
+form	O
+part	O
+of	O
+the	O
+VH	B-site
+:	I-site
+VL	I-site
+interface	I-site
+.	O
+
+In	O
+total	O
+,	O
+about	O
+20	B-residue_range
+residues	I-residue_range
+are	O
+involved	O
+in	O
+the	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+interactions	O
+on	O
+each	O
+side	O
+(	O
+Fig	O
+.	O
+S5	O
+).	O
+
+Half	O
+of	O
+them	O
+are	O
+in	O
+the	O
+framework	B-structure_element
+regions	I-structure_element
+and	O
+those	O
+residues	O
+(	O
+except	O
+residue	O
+61	B-residue_number
+in	O
+HC	B-structure_element
+,	O
+which	O
+is	O
+actually	O
+in	O
+CDR2	B-structure_element
+in	O
+Kabat	O
+'	O
+s	O
+definition	O
+)	O
+are	O
+conserved	O
+in	O
+the	O
+set	O
+of	O
+16	O
+Fabs	B-structure_element
+.	O
+
+One	O
+notable	O
+exception	O
+is	O
+H	B-structure_element
+-	O
+Trp47	B-residue_name_number
+,	O
+which	O
+exhibits	O
+2	O
+conformations	O
+of	O
+the	O
+indole	O
+ring	O
+.	O
+
+In	O
+most	O
+of	O
+the	O
+structures	B-evidence
+,	O
+it	O
+has	O
+the	O
+χ2	B-evidence
+angle	O
+of	O
+∼	O
+80	O
+°,	O
+while	O
+the	O
+ring	O
+is	O
+flipped	O
+over	O
+(	O
+χ2	B-evidence
+=	O
+−	O
+100	O
+°)	O
+in	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+:	I-complex_assembly
+11	I-complex_assembly
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+.	O
+
+Interestingly	O
+,	O
+these	O
+are	O
+the	O
+only	O
+2	O
+structures	B-evidence
+with	O
+residues	O
+missing	B-protein_state
+in	O
+CDR	B-structure_element
+H3	B-structure_element
+because	O
+of	O
+disorder	O
+,	O
+although	O
+both	O
+structures	B-evidence
+are	O
+determined	O
+at	O
+high	O
+resolution	O
+and	O
+the	O
+rest	O
+of	O
+the	O
+structure	B-evidence
+is	O
+well	O
+defined	O
+.	O
+
+Apparently	O
+,	O
+residues	O
+flanking	O
+CDR	B-structure_element
+H3	B-structure_element
+in	O
+the	O
+2	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+pairings	O
+are	O
+inconsistent	O
+with	O
+any	O
+stable	B-protein_state
+conformation	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+which	O
+translates	O
+into	O
+a	O
+less	O
+restricted	O
+conformational	O
+space	O
+for	O
+some	O
+of	O
+them	O
+,	O
+including	O
+H	B-structure_element
+-	O
+Trp47	B-residue_name_number
+.	O
+
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+tilt	B-evidence
+angles	I-evidence
+
+The	O
+relative	O
+orientation	O
+of	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+has	O
+been	O
+measured	O
+in	O
+a	O
+number	O
+of	O
+different	O
+ways	O
+.	O
+
+The	O
+first	O
+approach	O
+uses	O
+ABangles	B-experimental_method
+,	O
+the	O
+results	O
+of	O
+which	O
+are	O
+shown	O
+in	O
+Table	O
+S2	O
+.	O
+
+The	O
+four	O
+LCs	B-structure_element
+all	O
+are	O
+classified	O
+as	O
+Type	O
+A	O
+because	O
+they	O
+have	O
+a	O
+proline	B-residue_name
+at	O
+position	O
+44	B-residue_number
+,	O
+and	O
+the	O
+results	O
+for	O
+each	O
+orientation	B-evidence
+parameter	I-evidence
+are	O
+within	O
+the	O
+range	O
+of	O
+values	O
+of	O
+this	O
+type	O
+reported	O
+by	O
+Dunbar	O
+and	O
+co	O
+-	O
+workers	O
+.	O
+
+In	O
+fact	O
+,	O
+the	O
+parameter	O
+values	O
+for	O
+the	O
+set	O
+of	O
+16	O
+Fabs	B-structure_element
+are	O
+in	O
+the	O
+middle	O
+of	O
+the	O
+distribution	O
+observed	O
+for	O
+351	O
+non	O
+-	O
+redundant	O
+antibody	B-protein_type
+structures	B-evidence
+determined	O
+at	O
+3	O
+.	O
+0	O
+Å	O
+resolution	O
+or	O
+better	O
+.	O
+
+The	O
+only	O
+exception	O
+is	O
+HC1	B-structure_element
+,	O
+which	O
+is	O
+shifted	O
+toward	O
+smaller	O
+angles	O
+with	O
+the	O
+mean	O
+value	O
+of	O
+70	O
+.	O
+8	O
+°	O
+as	O
+compared	O
+to	O
+the	O
+distribution	O
+centered	O
+at	O
+72	O
+°	O
+for	O
+the	O
+entire	O
+PDB	O
+.	O
+
+This	O
+probably	O
+reflects	O
+the	O
+invariance	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+in	O
+the	O
+current	O
+set	O
+as	O
+opposed	O
+to	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+diversity	O
+in	O
+the	O
+PDB	O
+.	O
+
+The	O
+second	O
+approach	O
+used	O
+for	O
+comparing	O
+tilt	B-evidence
+angles	I-evidence
+involved	O
+computing	O
+the	O
+difference	B-evidence
+in	O
+the	O
+tilt	B-evidence
+angles	I-evidence
+between	O
+all	O
+pairs	O
+of	O
+structures	B-evidence
+.	O
+
+For	O
+structures	B-evidence
+with	O
+2	O
+copies	O
+of	O
+the	O
+Fab	B-structure_element
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+only	O
+one	O
+structure	B-evidence
+was	O
+used	O
+.	O
+
+The	O
+differences	O
+between	O
+independent	O
+Fabs	B-structure_element
+in	O
+the	O
+same	O
+structure	B-evidence
+are	O
+4	O
+.	O
+9	O
+°	O
+for	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+1	O
+.	O
+6	O
+°	O
+for	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+,	O
+1	O
+.	O
+4	O
+°	O
+for	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+3	O
+.	O
+3	O
+°	O
+for	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+,	O
+and	O
+2	O
+.	O
+5	O
+°	O
+for	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+.	O
+
+With	O
+the	O
+exception	O
+of	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+the	O
+angles	O
+are	O
+within	O
+the	O
+range	O
+of	O
+2	O
+-	O
+3	O
+°	O
+as	O
+are	O
+observed	O
+in	O
+the	O
+identical	O
+structures	B-evidence
+in	O
+the	O
+PDB	O
+.	O
+
+In	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+one	O
+of	O
+the	O
+Fabs	B-structure_element
+is	O
+substantially	O
+disordered	B-protein_state
+so	O
+that	O
+part	O
+of	O
+CDR	B-structure_element
+H2	B-structure_element
+(	O
+the	O
+outer	O
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+,	O
+residues	O
+55	B-residue_range
+-	I-residue_range
+60	I-residue_range
+)	O
+is	O
+completely	O
+missing	O
+.	O
+
+This	O
+kind	O
+of	O
+disorder	O
+may	O
+compromise	O
+the	O
+integrity	O
+of	O
+the	O
+VH	B-structure_element
+domain	O
+and	O
+its	O
+interaction	O
+with	O
+the	O
+VL	B-structure_element
+.	O
+
+Indeed	O
+,	O
+this	O
+Fab	B-structure_element
+has	O
+the	O
+largest	O
+twist	B-evidence
+angle	I-evidence
+HC2	B-structure_element
+within	O
+the	O
+experimental	O
+set	O
+that	O
+exceeds	O
+the	O
+mean	O
+value	O
+by	O
+2	O
+.	O
+5	O
+standard	O
+deviations	O
+(	O
+Table	O
+S2	O
+).	O
+
+An	O
+illustration	O
+of	O
+the	O
+difference	O
+in	O
+tilt	O
+angle	O
+for	O
+2	O
+pairs	O
+of	O
+variants	O
+by	O
+the	O
+superposition	B-experimental_method
+of	O
+the	O
+VH	B-structure_element
+domains	O
+of	O
+(	O
+A	O
+)	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+on	O
+that	O
+of	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+(	O
+the	O
+VL	B-structure_element
+domain	O
+is	O
+off	O
+by	O
+a	O
+rigid	O
+-	O
+body	O
+roatation	O
+of	O
+10	O
+.	O
+5	O
+°)	O
+and	O
+(	O
+B	O
+)	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+on	O
+that	O
+of	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+(	O
+the	O
+VL	B-structure_element
+domain	O
+is	O
+off	O
+by	O
+a	O
+rigid	O
+-	O
+body	O
+roatation	O
+of	O
+1	O
+.	O
+6	O
+°).	O
+
+Differences	O
+in	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+tilt	B-evidence
+angles	I-evidence
+.	O
+
+The	O
+differences	B-evidence
+in	O
+the	O
+tilt	B-evidence
+angle	I-evidence
+are	O
+shown	O
+for	O
+all	O
+pairs	O
+of	O
+V	B-structure_element
+regions	I-structure_element
+in	O
+Table	O
+3	O
+.	O
+
+The	O
+smallest	O
+differences	O
+in	O
+the	O
+tilt	B-evidence
+angle	I-evidence
+are	O
+between	O
+the	O
+Fabs	B-structure_element
+in	O
+isomorphous	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+
+The	O
+largest	O
+deviations	O
+in	O
+the	O
+tilt	B-evidence
+angle	I-evidence
+,	O
+up	O
+to	O
+11	O
+.	O
+0	O
+°,	O
+are	O
+found	O
+for	O
+2	O
+structures	B-evidence
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+that	O
+stand	O
+out	O
+from	O
+the	O
+other	O
+Fabs	B-structure_element
+.	O
+
+One	O
+of	O
+the	O
+2	O
+structures	B-evidence
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+has	O
+its	O
+CDR	B-structure_element
+H3	B-structure_element
+in	O
+the	O
+‘	O
+extended	B-protein_state
+’	O
+conformation	O
+;	O
+the	O
+other	O
+structure	O
+has	O
+it	O
+in	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	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	B-evidence
+angles	I-evidence
+are	O
+shown	O
+in	O
+Fig	O
+.	O
+9	O
+.	O
+
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+buried	O
+surface	O
+area	O
+and	O
+complementarity	O
+
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+surface	O
+areas	O
+and	O
+surface	O
+complementarity	O
+.	O
+
+Some	O
+side	O
+chain	O
+atoms	O
+in	O
+CDR	B-structure_element
+H3	B-structure_element
+are	O
+missing	O
+.	O
+
+Residues	O
+in	O
+CDR	B-structure_element
+H3	B-structure_element
+are	O
+missing	O
+:	O
+YGE	B-structure_element
+in	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+,	O
+GIY	B-structure_element
+in	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+.	O
+
+The	O
+results	O
+of	O
+the	O
+PISA	B-experimental_method
+contact	B-experimental_method
+surface	I-experimental_method
+calculation	I-experimental_method
+and	O
+surface	B-experimental_method
+complementarity	I-experimental_method
+calculation	I-experimental_method
+are	O
+shown	O
+in	O
+Table	O
+4	O
+.	O
+
+The	O
+interface	B-site
+areas	O
+are	O
+calculated	O
+as	O
+the	O
+average	O
+of	O
+the	O
+VH	B-site
+and	I-site
+VL	I-site
+contact	I-site
+surfaces	I-site
+.	O
+
+Six	O
+of	O
+the	O
+16	O
+structures	B-evidence
+have	O
+CDR	B-structure_element
+H3	B-structure_element
+side	O
+chains	O
+or	O
+complete	O
+residues	O
+missing	B-protein_state
+,	O
+and	O
+therefore	O
+their	O
+interfaces	B-site
+are	O
+much	O
+smaller	O
+than	O
+in	O
+the	O
+other	O
+10	O
+structures	B-evidence
+with	O
+complete	B-protein_state
+CDRs	B-structure_element
+(	O
+the	O
+results	O
+are	O
+provided	O
+for	O
+all	O
+Fabs	B-structure_element
+for	O
+completeness	O
+).	O
+
+Among	O
+the	O
+complete	B-protein_state
+structures	B-evidence
+,	O
+the	O
+interface	B-site
+areas	O
+range	O
+from	O
+684	O
+to	O
+836	O
+Å2	O
+.	O
+
+Interestingly	O
+,	O
+the	O
+2	O
+structures	B-evidence
+that	O
+have	O
+the	O
+largest	O
+tilt	B-evidence
+angle	I-evidence
+differences	I-evidence
+with	O
+the	O
+other	O
+variants	O
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+have	O
+the	O
+smallest	O
+VH	B-site
+:	I-site
+VL	I-site
+interfaces	I-site
+,	O
+684	O
+and	O
+725	O
+Å2	O
+,	O
+respectively	O
+.	O
+
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+is	O
+also	O
+unique	O
+in	O
+that	O
+it	O
+has	O
+the	O
+lowest	O
+value	O
+(	O
+0	O
+.	O
+676	O
+)	O
+of	O
+surface	B-evidence
+complementarity	I-evidence
+.	O
+
+Melting	B-evidence
+temperatures	I-evidence
+for	O
+the	O
+16	O
+Fabs	B-structure_element
+.	O
+
+Colors	O
+:	O
+blue	O
+(	O
+Tm	B-evidence
+<	O
+70	O
+°	O
+C	O
+),	O
+green	O
+(	O
+70	O
+°	O
+C	O
+<	O
+Tm	B-evidence
+<	O
+73	O
+°	O
+C	O
+),	O
+yellow	O
+(	O
+73	O
+°	O
+C	O
+<	O
+Tm	B-evidence
+<	O
+78	O
+°	O
+C	O
+),	O
+orange	O
+(	O
+Tm	B-evidence
+>	O
+78	O
+°	O
+C	O
+).	O
+
+Melting	B-evidence
+temperatures	I-evidence
+(	O
+Tm	B-evidence
+)	O
+were	O
+measured	O
+for	O
+all	O
+Fabs	B-structure_element
+using	O
+differential	B-experimental_method
+scanning	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+Table	O
+5	O
+).	O
+
+It	O
+appears	O
+that	O
+for	O
+each	O
+given	O
+LC	B-structure_element
+,	O
+the	O
+Fabs	B-structure_element
+with	O
+germlines	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+are	O
+substantially	O
+more	O
+stable	B-protein_state
+than	O
+those	O
+with	O
+germlines	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+.	O
+
+In	O
+addition	O
+,	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+provides	O
+a	O
+much	O
+higher	O
+degree	O
+of	O
+stabilization	O
+than	O
+the	O
+other	O
+3	O
+LC	B-structure_element
+germlines	O
+when	O
+combined	O
+with	O
+any	O
+of	O
+the	O
+HCs	B-structure_element
+.	O
+
+As	O
+a	O
+result	O
+,	O
+the	O
+Tm	B-evidence
+for	O
+pairs	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+is	O
+12	O
+-	O
+13	O
+°	O
+higher	O
+than	O
+for	O
+pairs	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+.	O
+
+These	O
+findings	O
+correlate	O
+well	O
+with	O
+the	O
+degree	O
+of	O
+conformational	O
+disorder	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Parts	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+main	O
+chain	O
+are	O
+completely	O
+disordered	B-protein_state
+,	O
+and	O
+were	O
+not	O
+modeled	O
+in	O
+Fabs	B-structure_element
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+that	O
+have	O
+the	O
+lowest	O
+Tms	B-evidence
+in	O
+the	O
+set	O
+.	O
+
+No	O
+electron	B-evidence
+density	I-evidence
+is	O
+observed	O
+for	O
+a	O
+number	O
+of	O
+side	O
+chains	O
+in	O
+CDRs	B-structure_element
+H3	B-structure_element
+and	O
+L3	B-structure_element
+in	O
+all	O
+Fabs	B-structure_element
+with	O
+germline	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+,	O
+which	O
+indicates	O
+loose	O
+packing	O
+of	O
+the	O
+variable	B-structure_element
+domains	I-structure_element
+.	O
+
+All	O
+those	O
+molecules	O
+are	O
+relatively	O
+unstable	O
+,	O
+as	O
+is	O
+reflected	O
+in	O
+their	O
+low	O
+Tms	B-evidence
+.	O
+
+This	O
+is	O
+the	O
+first	O
+report	O
+of	O
+a	O
+systematic	B-experimental_method
+structural	I-experimental_method
+investigation	I-experimental_method
+of	O
+a	O
+phage	B-experimental_method
+germline	I-experimental_method
+library	I-experimental_method
+.	O
+
+The	O
+16	O
+Fab	B-structure_element
+structures	B-evidence
+offer	O
+a	O
+unique	O
+look	O
+at	O
+all	O
+pairings	O
+of	O
+4	O
+different	O
+HCs	B-structure_element
+(	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+,	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+,	O
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+)	O
+and	O
+4	O
+different	O
+LCs	B-structure_element
+(	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+),	O
+all	O
+with	O
+the	O
+same	O
+CDR	B-structure_element
+H3	B-structure_element
+.	O
+
+The	O
+structural	B-evidence
+data	I-evidence
+set	O
+taken	O
+as	O
+a	O
+whole	O
+provides	O
+insight	O
+into	O
+how	O
+the	O
+backbone	O
+conformations	O
+of	O
+the	O
+CDRs	B-structure_element
+of	O
+a	O
+specific	O
+heavy	O
+or	O
+light	B-structure_element
+chain	I-structure_element
+vary	O
+when	O
+it	O
+is	O
+paired	O
+with	O
+4	O
+different	O
+light	O
+or	O
+heavy	B-structure_element
+chains	I-structure_element
+,	O
+respectively	O
+.	O
+
+A	O
+large	O
+variability	O
+in	O
+the	O
+CDR	B-structure_element
+conformations	O
+for	O
+the	O
+sets	O
+of	O
+HCs	B-structure_element
+and	O
+LCs	B-structure_element
+is	O
+observed	O
+.	O
+
+In	O
+some	O
+cases	O
+the	O
+CDR	B-structure_element
+conformations	O
+for	O
+all	O
+members	O
+of	O
+a	O
+set	O
+are	O
+virtually	O
+identical	O
+,	O
+for	O
+others	O
+subtle	O
+changes	O
+occur	O
+in	O
+a	O
+few	O
+members	O
+of	O
+a	O
+set	O
+,	O
+and	O
+in	O
+some	O
+cases	O
+larger	O
+deviations	O
+are	O
+observed	O
+within	O
+a	O
+set	O
+.	O
+
+The	O
+five	O
+variants	O
+that	O
+crystallized	B-experimental_method
+with	O
+2	O
+copies	O
+of	O
+the	O
+Fab	B-structure_element
+in	O
+the	O
+asymmetric	O
+unit	O
+serve	O
+somewhat	O
+as	O
+controls	O
+for	O
+the	O
+influence	O
+of	O
+crystal	O
+packing	O
+on	O
+the	O
+conformations	O
+of	O
+the	O
+CDRs	B-structure_element
+.	O
+
+In	O
+four	O
+of	O
+the	O
+5	O
+structures	B-evidence
+the	O
+CDR	B-structure_element
+conformations	O
+are	O
+consistent	O
+.	O
+
+In	O
+only	O
+one	O
+case	O
+,	O
+that	O
+of	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+(	O
+the	O
+lowest	O
+resolution	O
+structure	B-evidence
+),	O
+do	O
+we	O
+see	O
+differences	O
+in	O
+the	O
+conformations	O
+of	O
+the	O
+2	O
+copies	O
+of	O
+CDRs	B-structure_element
+H1	B-structure_element
+and	O
+L1	B-structure_element
+.	O
+
+This	O
+variability	O
+is	O
+likely	O
+a	O
+result	O
+of	O
+2	O
+factors	O
+,	O
+crystal	O
+packing	O
+interactions	O
+and	O
+internal	O
+instability	O
+of	O
+the	O
+variable	B-structure_element
+domain	I-structure_element
+.	O
+
+For	O
+the	O
+CDRs	B-structure_element
+with	O
+canonical	O
+structures	O
+,	O
+the	O
+largest	O
+changes	O
+in	O
+conformation	O
+occur	O
+for	O
+CDR	B-structure_element
+H1	B-structure_element
+of	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+.	O
+
+The	O
+other	O
+2	O
+HCs	B-structure_element
+,	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+,	O
+have	O
+canonical	O
+structures	O
+that	O
+are	O
+remarkably	B-protein_state
+well	I-protein_state
+conserved	I-protein_state
+(	O
+Fig	O
+.	O
+1	O
+).	O
+
+Of	O
+the	O
+4	O
+HCs	B-structure_element
+,	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+has	O
+the	O
+greatest	O
+number	O
+of	O
+canonical	O
+structure	O
+assignments	O
+(	O
+Table	O
+2	O
+).	O
+
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+is	O
+unique	O
+in	O
+having	O
+a	O
+pair	O
+of	O
+glycine	B-residue_name
+residues	O
+at	O
+positions	O
+26	B-residue_number
+and	O
+27	B-residue_number
+,	O
+which	O
+provide	O
+more	O
+conformational	B-protein_state
+freedom	I-protein_state
+in	O
+CDR	B-structure_element
+H1	B-structure_element
+.	O
+
+Besides	O
+IGHV1	B-mutant
+-	I-mutant
+69	I-mutant
+,	O
+only	O
+the	O
+germlines	O
+of	O
+the	O
+VH4	B-structure_element
+family	O
+possess	O
+double	O
+glycines	B-residue_name
+in	O
+CDR	B-structure_element
+H1	B-structure_element
+,	O
+and	O
+it	O
+will	O
+be	O
+interesting	O
+to	O
+see	O
+if	O
+they	O
+are	O
+also	O
+conformationally	B-protein_state
+unstable	I-protein_state
+.	O
+
+Having	O
+all	O
+16	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+pairs	O
+with	O
+the	O
+same	O
+CDR	B-structure_element
+H3	B-structure_element
+provides	O
+some	O
+insights	O
+into	O
+why	O
+molecular	O
+modeling	O
+efforts	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+have	O
+proven	O
+so	O
+difficult	O
+.	O
+
+As	O
+mentioned	O
+in	O
+the	O
+Results	O
+section	O
+,	O
+this	O
+data	O
+set	O
+is	O
+composed	O
+of	O
+21	O
+Fabs	B-structure_element
+,	O
+since	O
+5	O
+of	O
+the	O
+16	O
+variants	O
+have	O
+2	O
+Fab	B-structure_element
+copies	O
+in	O
+the	O
+asymmetric	O
+unit	O
+.	O
+
+For	O
+the	O
+18	O
+Fabs	B-structure_element
+with	O
+complete	O
+backbone	O
+atoms	O
+for	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+10	O
+have	O
+conformations	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+parent	O
+,	O
+while	O
+the	O
+others	O
+have	O
+significantly	O
+different	O
+conformations	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+Thus	O
+,	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+conformation	O
+is	O
+dependent	O
+upon	O
+2	O
+dominating	O
+factors	O
+:	O
+1	O
+)	O
+amino	O
+acid	O
+sequence	O
+;	O
+and	O
+2	O
+)	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+context	O
+.	O
+
+More	O
+than	O
+half	O
+of	O
+the	O
+variants	O
+retain	O
+the	O
+conformation	O
+of	O
+the	O
+parent	O
+despite	O
+having	O
+differences	O
+in	O
+the	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+pairing	O
+.	O
+
+This	O
+subset	O
+includes	O
+2	O
+structures	B-evidence
+with	O
+2	O
+copies	O
+of	O
+the	O
+Fab	B-structure_element
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+all	O
+of	O
+which	O
+are	O
+nearly	O
+identical	O
+in	O
+conformation	O
+.	O
+
+The	O
+remaining	O
+8	O
+structures	B-evidence
+exhibit	O
+“	O
+non	O
+-	O
+parental	O
+”	O
+conformations	O
+,	O
+indicating	O
+that	O
+the	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+context	O
+can	O
+also	O
+be	O
+a	O
+dominating	O
+factor	O
+influencing	O
+CDR	B-structure_element
+H3	B-structure_element
+.	O
+
+This	O
+subset	O
+also	O
+has	O
+2	O
+structures	B-evidence
+with	O
+2	O
+Fab	B-structure_element
+copies	O
+in	O
+the	O
+asymmetric	O
+unit	O
+.	O
+
+Interestingly	O
+,	O
+as	O
+described	O
+earlier	O
+,	O
+these	O
+2	O
+pairs	O
+differ	O
+in	O
+the	O
+stem	B-structure_element
+regions	I-structure_element
+with	O
+the	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+pair	O
+in	O
+the	O
+‘	O
+extended	B-protein_state
+’	O
+conformation	O
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+pair	O
+in	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+.	O
+
+The	O
+CDR	B-structure_element
+H3	B-structure_element
+conformational	B-experimental_method
+analysis	I-experimental_method
+shows	O
+that	O
+,	O
+for	O
+each	O
+set	O
+of	O
+variants	O
+of	O
+one	O
+HC	B-structure_element
+paired	O
+with	O
+the	O
+4	O
+different	O
+LCs	B-structure_element
+,	O
+both	O
+“	O
+parental	O
+”	O
+and	O
+“	O
+non	O
+-	O
+parental	O
+”	O
+conformations	O
+are	O
+observed	O
+.	O
+
+The	O
+same	O
+variability	O
+is	O
+observed	O
+for	O
+the	O
+sets	O
+of	O
+variants	O
+composed	O
+of	O
+one	O
+LC	B-structure_element
+paired	O
+with	O
+each	O
+of	O
+the	O
+4	O
+HCs	B-structure_element
+.	O
+
+Thus	O
+,	O
+no	O
+patterns	O
+of	O
+conformational	O
+preference	O
+for	O
+a	O
+particular	O
+HC	B-structure_element
+or	O
+LC	B-structure_element
+emerge	O
+to	O
+shed	O
+any	O
+direct	O
+light	O
+on	O
+what	O
+drives	O
+the	O
+conformational	O
+differences	O
+.	O
+
+This	O
+finding	O
+supports	O
+the	O
+hypothesis	O
+of	O
+Weitzner	O
+et	O
+al	O
+.	O
+that	O
+the	O
+H3	B-structure_element
+conformation	O
+is	O
+controlled	O
+both	O
+by	O
+its	O
+sequence	O
+and	O
+its	O
+environment	O
+.	O
+
+In	O
+looking	O
+at	O
+a	O
+possible	O
+correlation	O
+between	O
+the	O
+tilt	B-evidence
+angle	I-evidence
+and	O
+the	O
+conformation	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+no	O
+clear	O
+trends	O
+are	O
+observed	O
+.	O
+
+Two	O
+variants	O
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+have	O
+the	O
+largest	O
+differences	O
+in	O
+the	O
+tilt	O
+angles	O
+compared	O
+to	O
+other	O
+variants	O
+as	O
+seen	O
+in	O
+Table	O
+3	O
+.	O
+
+The	O
+absolute	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+orientation	B-evidence
+parameters	I-evidence
+for	O
+the	O
+2	O
+Fabs	B-structure_element
+(	O
+Table	O
+S2	O
+)	O
+show	O
+significant	O
+deviation	B-evidence
+in	O
+HL	B-structure_element
+,	O
+LC1	B-structure_element
+and	O
+HC2	B-structure_element
+values	O
+(	O
+2	O
+-	O
+3	O
+standard	O
+deviations	O
+from	O
+the	O
+mean	O
+).	O
+
+One	O
+of	O
+the	O
+variants	O
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+has	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+conformation	O
+similar	O
+to	O
+the	O
+parent	O
+,	O
+but	O
+the	O
+other	O
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+is	O
+different	O
+.	O
+
+As	O
+noted	O
+in	O
+the	O
+Results	O
+section	O
+,	O
+the	O
+2	O
+variants	O
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+are	O
+outliers	O
+in	O
+terms	O
+of	O
+the	O
+tilt	B-evidence
+angle	I-evidence
+;	O
+at	O
+the	O
+same	O
+time	O
+,	O
+both	O
+have	O
+the	O
+smallest	O
+VH	B-site
+:	I-site
+VL	I-site
+interface	I-site
+.	O
+
+These	O
+smaller	O
+interfaces	B-site
+may	O
+perhaps	O
+translate	O
+to	O
+a	O
+significant	O
+deviation	O
+in	O
+how	O
+VH	B-structure_element
+is	O
+oriented	O
+relative	O
+to	O
+VL	B-structure_element
+than	O
+the	O
+other	O
+variants	O
+.	O
+
+These	O
+deviations	O
+from	O
+the	O
+other	O
+variants	O
+can	O
+also	O
+be	O
+seen	O
+to	O
+some	O
+extent	O
+in	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+orientation	O
+parameters	O
+in	O
+Table	O
+S2	O
+,	O
+as	O
+well	O
+as	O
+in	O
+the	O
+smaller	O
+number	O
+of	O
+residues	O
+involved	O
+in	O
+the	O
+VH	B-site
+:	I-site
+VL	I-site
+interfaces	I-site
+of	O
+these	O
+2	O
+variants	O
+(	O
+Fig	O
+.	O
+S5	O
+).	O
+
+These	O
+differences	O
+undoubtedly	O
+influence	O
+the	O
+conformation	O
+of	O
+the	O
+CDRs	B-structure_element
+,	O
+in	O
+particular	O
+CDR	B-structure_element
+H1	B-structure_element
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+and	O
+CDR	B-structure_element
+L1	B-structure_element
+(	O
+Fig	O
+.	O
+3C	O
+),	O
+especially	O
+with	O
+the	O
+tandem	O
+glycines	B-residue_name
+and	O
+multiple	O
+serines	B-residue_name
+present	O
+,	O
+respectively	O
+.	O
+
+Pairing	O
+of	O
+different	O
+germlines	O
+yields	O
+antibodies	B-protein_type
+with	O
+various	O
+degrees	O
+of	O
+stability	O
+.	O
+
+As	O
+indicated	O
+by	O
+the	O
+melting	B-evidence
+temperatures	I-evidence
+,	O
+germlines	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+for	O
+HC	B-structure_element
+and	O
+germline	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+for	O
+LC	B-structure_element
+produce	O
+more	O
+stable	B-protein_state
+Fabs	B-structure_element
+compared	O
+to	O
+the	O
+other	O
+germlines	O
+in	O
+the	O
+experimental	O
+set	O
+.	O
+
+One	O
+possible	O
+explanation	O
+of	O
+the	O
+clear	O
+preference	O
+of	O
+LC	B-structure_element
+germline	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+is	O
+that	O
+CDR	B-structure_element
+L3	B-structure_element
+has	O
+smaller	O
+residues	O
+at	O
+positions	O
+91	B-residue_number
+and	O
+94	B-residue_number
+,	O
+allowing	O
+for	O
+more	O
+room	O
+to	O
+accommodate	O
+CDR	B-structure_element
+H3	B-structure_element
+.	O
+
+Other	O
+germlines	O
+have	O
+bulky	O
+residues	O
+,	O
+Tyr	B-residue_name
+,	O
+Arg	B-residue_name
+and	O
+Trp	B-residue_name
+,	O
+at	O
+these	O
+positions	O
+,	O
+whereas	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+has	O
+Ser	B-residue_name
+and	O
+Thr	B-residue_name
+.	O
+
+Various	O
+combinations	O
+of	O
+germline	O
+sequences	O
+for	O
+VL	B-structure_element
+and	O
+VH	B-structure_element
+impose	O
+certain	O
+constraints	O
+on	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+which	O
+has	O
+to	O
+adapt	O
+to	O
+the	O
+environment	O
+.	O
+
+A	O
+more	O
+compact	B-protein_state
+CDR	B-structure_element
+L3	B-structure_element
+may	O
+be	O
+beneficial	O
+in	O
+this	O
+situation	O
+.	O
+
+At	O
+the	O
+other	O
+end	O
+of	O
+the	O
+stability	O
+range	O
+is	O
+LC	B-structure_element
+germline	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+,	O
+which	O
+yields	O
+antibodies	B-protein_type
+with	O
+the	O
+lowest	O
+Tms	B-evidence
+.	O
+
+While	O
+pairings	O
+with	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+may	O
+be	O
+safely	O
+called	O
+a	O
+mismatch	O
+,	O
+those	O
+with	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+have	O
+Tms	B-evidence
+about	O
+5	O
+-	O
+6	O
+°	O
+higher	O
+.	O
+
+Curiously	O
+,	O
+the	O
+2	O
+Fabs	B-structure_element
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+deviate	O
+markedly	O
+in	O
+their	O
+tilt	B-evidence
+angles	I-evidence
+from	O
+the	O
+rest	O
+of	O
+the	O
+panel	O
+.	O
+
+It	O
+is	O
+possible	O
+that	O
+by	O
+adopting	O
+extreme	O
+tilt	B-evidence
+angles	I-evidence
+the	O
+structure	B-evidence
+modulates	O
+CDR	B-structure_element
+H3	B-structure_element
+and	O
+its	O
+environment	O
+,	O
+which	O
+apparently	O
+cannot	O
+be	O
+achieved	O
+solely	O
+by	O
+conformational	O
+rearrangement	O
+of	O
+the	O
+CDR	B-structure_element
+.	O
+
+Note	O
+that	O
+most	O
+of	O
+the	O
+VH	B-site
+:	I-site
+VL	I-site
+interface	I-site
+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
+
+Yet	O
+,	O
+for	O
+the	O
+2	O
+antibodies	B-protein_type
+,	O
+the	O
+total	O
+gain	O
+in	O
+stability	O
+merits	O
+the	O
+domain	O
+repacking	O
+.	O
+
+Overall	O
+,	O
+the	O
+stability	O
+of	O
+the	O
+Fab	B-structure_element
+,	O
+as	O
+measured	O
+by	O
+Tm	B-evidence
+,	O
+is	O
+a	O
+result	O
+of	O
+the	O
+mutual	O
+adjustment	O
+of	O
+the	O
+HC	B-structure_element
+and	O
+LC	B-structure_element
+variable	B-structure_element
+domains	I-structure_element
+and	O
+adjustment	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+to	O
+the	O
+VH	B-site
+:	I-site
+VL	I-site
+cleft	I-site
+.	O
+
+The	O
+final	O
+conformation	O
+represents	O
+an	O
+energetic	O
+minimum	O
+;	O
+however	O
+,	O
+in	O
+most	O
+cases	O
+it	O
+is	O
+very	O
+shallow	O
+,	O
+so	O
+that	O
+a	O
+single	O
+mutation	O
+can	O
+cause	O
+a	O
+dramatic	O
+rearrangement	O
+of	O
+the	O
+structure	B-evidence
+.	O
+
+In	O
+summary	O
+,	O
+the	O
+analysis	O
+of	O
+this	O
+structural	B-experimental_method
+library	I-experimental_method
+of	O
+germline	O
+variants	O
+composed	O
+of	O
+all	O
+pairs	O
+of	O
+4	O
+HCs	B-structure_element
+and	O
+4LCs	O
+,	O
+all	O
+with	O
+the	O
+same	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+offers	O
+some	O
+unique	O
+insights	O
+into	O
+antibody	B-protein_type
+structure	B-evidence
+and	O
+how	O
+pairing	O
+and	O
+sequence	O
+may	O
+influence	O
+,	O
+or	O
+not	O
+,	O
+the	O
+canonical	O
+structures	O
+of	O
+the	O
+L1	B-structure_element
+,	O
+L2	B-structure_element
+,	O
+L3	B-structure_element
+,	O
+H1	B-structure_element
+and	O
+H2	B-structure_element
+CDRs	B-structure_element
+.	O
+
+Comparison	O
+of	O
+the	O
+CDR	B-structure_element
+H3s	B-structure_element
+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	B-structure_element
+H3	B-structure_element
+conformation	O
+.	O
+
+Quite	O
+unexpectedly	O
+,	O
+2	O
+of	O
+the	O
+variants	O
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+,	O
+have	O
+the	O
+‘	O
+extended	B-protein_state
+’	O
+stem	B-structure_element
+region	I-structure_element
+differing	O
+from	O
+the	O
+other	O
+14	O
+that	O
+have	O
+a	O
+‘	O
+kinked	B-protein_state
+’	O
+stem	B-structure_element
+region	I-structure_element
+.	O
+
+These	O
+data	O
+reveal	O
+the	O
+difficulty	O
+of	O
+modeling	O
+CDR	B-structure_element
+H3	B-structure_element
+accurately	O
+,	O
+as	O
+shown	O
+again	O
+in	O
+Antibody	O
+Modeling	O
+Assessment	O
+II	O
+.	O
+
+Furthermore	O
+,	O
+antibody	B-protein_type
+CDRs	B-structure_element
+,	O
+H3	B-structure_element
+in	O
+particular	O
+,	O
+may	O
+go	O
+through	O
+conformational	O
+changes	O
+upon	O
+binding	O
+their	O
+targets	O
+,	O
+making	O
+structural	O
+prediction	O
+for	O
+docking	O
+purposes	O
+an	O
+even	O
+more	O
+difficult	O
+task	O
+.	O
+
+Fortunately	O
+,	O
+for	O
+most	O
+applications	O
+of	O
+antibody	B-protein_type
+modeling	O
+,	O
+such	O
+as	O
+engineering	O
+affinity	O
+and	O
+biophysical	O
+properties	O
+,	O
+an	O
+accurate	O
+CDR	B-structure_element
+H3	B-structure_element
+structure	B-evidence
+is	O
+not	O
+always	O
+necessary	O
+.	O
+
+For	O
+those	O
+applications	O
+where	O
+accurate	O
+CDR	B-structure_element
+structures	B-evidence
+are	O
+essential	O
+,	O
+such	O
+as	O
+docking	O
+,	O
+the	O
+results	O
+in	O
+this	O
+work	O
+demonstrate	O
+the	O
+importance	O
+of	O
+experimental	O
+structures	B-evidence
+.	O
+
+With	O
+the	O
+recent	O
+advances	O
+in	O
+expression	B-experimental_method
+and	I-experimental_method
+crystallization	I-experimental_method
+methods	I-experimental_method
+,	O
+Fab	B-structure_element
+structures	B-evidence
+can	O
+be	O
+obtained	O
+rapidly	O
+.	O
+
+The	O
+set	O
+of	O
+16	O
+germline	O
+Fab	B-structure_element
+structures	B-evidence
+offers	O
+a	O
+unique	O
+dataset	O
+to	O
+facilitate	O
+software	O
+development	O
+for	O
+antibody	B-protein_type
+modeling	O
+.	O
+
+The	O
+results	O
+essentially	O
+support	O
+the	O
+underlying	O
+idea	O
+of	O
+canonical	O
+structures	B-evidence
+,	O
+indicating	O
+that	O
+most	O
+CDRs	B-structure_element
+with	O
+germline	O
+sequences	O
+tend	O
+to	O
+adopt	O
+predefined	O
+conformations	O
+.	O
+
+From	O
+this	O
+point	O
+of	O
+view	O
+,	O
+a	O
+novel	O
+approach	O
+to	O
+design	O
+combinatorial	O
+antibody	B-protein_type
+libraries	O
+would	O
+be	O
+to	O
+cover	O
+the	O
+range	O
+of	O
+CDR	B-structure_element
+conformations	O
+that	O
+may	O
+not	O
+necessarily	O
+coincide	O
+with	O
+the	O
+germline	O
+usage	O
+in	O
+the	O
+human	B-species
+repertoire	O
+.	O
+
+This	O
+would	O
+insure	O
+more	O
+structural	O
+diversity	O
+,	O
+leading	O
+to	O
+a	O
+more	O
+diverse	O
+panel	O
+of	O
+antibodies	B-protein_type
+that	O
+would	O
+bind	O
+to	O
+a	O
+broad	O
+spectrum	O
+of	O
+targets	O
+.	O
+
+An	O
+extended	B-protein_state
+U2AF65	B-structure_element
+–	I-structure_element
+RNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+recognizes	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+signal	O
+
+How	O
+the	O
+essential	O
+pre	B-protein_type
+-	I-protein_type
+mRNA	I-protein_type
+splicing	I-protein_type
+factor	I-protein_type
+U2AF65	B-protein
+recognizes	O
+the	O
+polypyrimidine	B-chemical
+(	O
+Py	B-chemical
+)	O
+signals	O
+of	O
+the	O
+major	O
+class	O
+of	O
+3	B-site
+′	I-site
+splice	I-site
+sites	I-site
+in	O
+human	B-species
+gene	O
+transcripts	O
+remains	O
+incompletely	O
+understood	O
+.	O
+
+We	O
+determined	B-experimental_method
+four	I-experimental_method
+structures	I-experimental_method
+of	O
+an	O
+extended	B-protein_state
+U2AF65	B-structure_element
+–	I-structure_element
+RNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+bound	B-protein_state
+to	I-protein_state
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+oligonucleotides	I-chemical
+at	O
+resolutions	O
+between	O
+2	O
+.	O
+0	O
+and	O
+1	O
+.	O
+5	O
+Å	O
+.	O
+These	O
+structures	B-evidence
+together	O
+with	O
+RNA	B-experimental_method
+binding	I-experimental_method
+and	I-experimental_method
+splicing	I-experimental_method
+assays	I-experimental_method
+reveal	O
+unforeseen	O
+roles	O
+for	O
+U2AF65	B-protein
+inter	B-site
+-	I-site
+domain	I-site
+residues	I-site
+in	O
+recognizing	O
+a	O
+contiguous	B-structure_element
+,	O
+nine	O
+-	O
+nucleotide	B-chemical
+Py	B-chemical
+tract	I-chemical
+.	O
+
+The	O
+U2AF65	B-protein
+linker	B-structure_element
+residues	O
+between	O
+the	O
+dual	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRMs	B-structure_element
+)	O
+recognize	O
+the	O
+central	O
+nucleotide	B-chemical
+,	O
+whereas	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+RRM	B-structure_element
+extensions	I-structure_element
+recognize	O
+the	O
+3	B-site
+′	I-site
+terminus	I-site
+and	O
+third	B-residue_number
+nucleotide	B-chemical
+.	O
+
+Single	B-experimental_method
+-	I-experimental_method
+molecule	I-experimental_method
+FRET	I-experimental_method
+experiments	O
+suggest	O
+that	O
+conformational	O
+selection	O
+and	O
+induced	O
+fit	O
+of	O
+the	O
+U2AF65	B-protein
+RRMs	B-structure_element
+are	O
+complementary	O
+mechanisms	O
+for	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+association	O
+.	O
+
+Altogether	O
+,	O
+these	O
+results	O
+advance	O
+the	O
+mechanistic	O
+understanding	O
+of	O
+molecular	O
+recognition	O
+for	O
+a	O
+major	O
+class	O
+of	O
+splice	B-site
+site	I-site
+signals	O
+.	O
+
+The	O
+pre	B-protein_type
+-	I-protein_type
+mRNA	I-protein_type
+splicing	I-protein_type
+factor	I-protein_type
+U2AF65	B-protein
+recognizes	O
+3	B-site
+′	I-site
+splice	I-site
+sites	I-site
+in	O
+human	B-species
+gene	O
+transcripts	O
+,	O
+but	O
+the	O
+details	O
+are	O
+not	O
+fully	O
+understood	O
+.	O
+
+Here	O
+,	O
+the	O
+authors	O
+report	O
+U2AF65	B-protein
+structures	B-evidence
+and	O
+single	B-experimental_method
+molecule	I-experimental_method
+FRET	I-experimental_method
+that	O
+reveal	O
+mechanistic	O
+insights	O
+into	O
+splice	B-site
+site	I-site
+recognition	O
+.	O
+
+The	O
+differential	O
+skipping	O
+or	O
+inclusion	O
+of	O
+alternatively	O
+spliced	O
+pre	B-structure_element
+-	I-structure_element
+mRNA	I-structure_element
+regions	I-structure_element
+is	O
+a	O
+major	O
+source	O
+of	O
+diversity	O
+for	O
+nearly	O
+all	O
+human	B-species
+gene	O
+transcripts	O
+.	O
+
+The	O
+splice	B-site
+sites	I-site
+are	O
+marked	O
+by	O
+relatively	O
+short	B-structure_element
+consensus	I-structure_element
+sequences	I-structure_element
+and	O
+are	O
+regulated	O
+by	O
+additional	O
+pre	B-structure_element
+-	I-structure_element
+mRNA	I-structure_element
+motifs	I-structure_element
+(	O
+reviewed	O
+in	O
+ref	O
+.).	O
+
+At	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+of	O
+the	O
+major	O
+intron	O
+class	O
+,	O
+these	O
+include	O
+a	O
+polypyrimidine	B-chemical
+(	I-chemical
+Py	I-chemical
+)	I-chemical
+tract	I-chemical
+comprising	O
+primarily	O
+Us	B-residue_name
+or	O
+Cs	B-residue_name
+,	O
+which	O
+is	O
+preceded	O
+by	O
+a	O
+branch	B-site
+point	I-site
+sequence	I-site
+(	O
+BPS	B-site
+)	O
+that	O
+ultimately	O
+serves	O
+as	O
+the	O
+nucleophile	O
+in	O
+the	O
+splicing	O
+reaction	O
+and	O
+an	O
+AG	B-chemical
+-	I-chemical
+dinucleotide	I-chemical
+at	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+junction	O
+.	O
+
+Disease	O
+-	O
+causing	O
+mutations	O
+often	O
+compromise	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+splicing	O
+(	O
+reviewed	O
+in	O
+refs	O
+),	O
+yet	O
+a	O
+priori	O
+predictions	O
+of	O
+splice	B-site
+sites	I-site
+and	O
+the	O
+consequences	O
+of	O
+their	O
+mutations	O
+are	O
+challenged	O
+by	O
+the	O
+brevity	O
+and	O
+degeneracy	O
+of	O
+known	O
+splice	B-site
+site	I-site
+sequences	O
+.	O
+
+High	O
+-	O
+resolution	O
+structures	B-evidence
+of	O
+intact	B-protein_state
+splicing	B-complex_assembly
+factor	I-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complexes	O
+would	O
+offer	O
+key	O
+insights	O
+regarding	O
+the	O
+juxtaposition	O
+of	O
+the	O
+distinct	O
+splice	B-site
+site	I-site
+consensus	O
+sequences	O
+and	O
+their	O
+relationship	O
+to	O
+disease	O
+-	O
+causing	O
+point	O
+mutations	O
+.	O
+
+The	O
+early	O
+-	O
+stage	O
+pre	B-protein_type
+-	I-protein_type
+mRNA	I-protein_type
+splicing	I-protein_type
+factor	I-protein_type
+U2AF65	B-protein
+is	O
+essential	O
+for	O
+viability	O
+in	O
+vertebrates	B-taxonomy_domain
+and	O
+other	O
+model	O
+organisms	O
+(	O
+for	O
+example	O
+,	O
+ref	O
+.).	O
+
+A	O
+tightly	O
+controlled	O
+assembly	B-complex_assembly
+among	O
+U2AF65	B-protein
+,	O
+the	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+,	O
+and	O
+partner	O
+proteins	O
+sequentially	O
+identifies	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+and	O
+promotes	O
+association	O
+of	O
+the	O
+spliceosome	B-complex_assembly
+,	O
+which	O
+ultimately	O
+accomplishes	O
+the	O
+task	O
+of	O
+splicing	O
+.	O
+
+Initially	O
+U2AF65	B-protein
+recognizes	O
+the	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+splice	B-site
+site	I-site
+signal	O
+.	O
+
+In	O
+turn	O
+,	O
+the	O
+ternary	B-complex_assembly
+complex	I-complex_assembly
+of	O
+U2AF65	B-protein
+with	O
+SF1	B-protein
+and	O
+U2AF35	B-protein
+identifies	O
+the	O
+surrounding	O
+BPS	B-site
+and	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+junctions	O
+.	O
+
+Subsequently	O
+U2AF65	B-protein
+recruits	O
+the	O
+U2	B-complex_assembly
+small	I-complex_assembly
+nuclear	I-complex_assembly
+ribonucleoprotein	I-complex_assembly
+particle	I-complex_assembly
+(	O
+snRNP	B-complex_assembly
+)	O
+and	O
+ultimately	O
+dissociates	O
+from	O
+the	O
+active	B-protein_state
+spliceosome	B-complex_assembly
+.	O
+
+Biochemical	B-experimental_method
+characterizations	I-experimental_method
+of	O
+U2AF65	B-protein
+demonstrated	O
+that	O
+tandem	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+)	O
+recognize	O
+the	O
+Py	B-chemical
+tract	I-chemical
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+
+Milestone	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+core	B-protein_state
+U2AF65	B-protein
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+connected	O
+by	O
+a	O
+shortened	B-protein_state
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+(	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+)	O
+detailed	O
+a	O
+subset	O
+of	O
+nucleotide	O
+interactions	O
+with	O
+the	O
+individual	O
+U2AF65	B-protein
+RRMs	B-structure_element
+.	O
+
+A	O
+subsequent	O
+NMR	B-experimental_method
+structure	B-evidence
+characterized	O
+the	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+arrangement	O
+of	O
+the	O
+minimal	B-protein_state
+U2AF65	B-protein
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+connected	O
+by	O
+a	O
+linker	B-structure_element
+of	O
+natural	B-protein_state
+length	I-protein_state
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+),	O
+yet	O
+depended	O
+on	O
+the	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+crystal	B-evidence
+structures	I-evidence
+for	O
+RNA	B-chemical
+interactions	O
+and	O
+an	O
+ab	O
+initio	O
+model	O
+for	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+conformation	O
+.	O
+
+As	O
+such	O
+,	O
+the	O
+molecular	O
+mechanisms	O
+for	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+by	O
+the	O
+intact	B-protein_state
+U2AF65	B-structure_element
+–	I-structure_element
+RNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+remained	O
+unknown	O
+.	O
+
+Here	O
+,	O
+we	O
+use	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+and	O
+biochemical	B-experimental_method
+studies	I-experimental_method
+to	O
+reveal	O
+new	O
+roles	O
+in	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+for	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+and	O
+key	O
+residues	O
+surrounding	O
+the	O
+core	B-protein_state
+U2AF65	B-protein
+RRMs	B-structure_element
+.	O
+
+We	O
+use	O
+single	B-experimental_method
+-	I-experimental_method
+molecule	I-experimental_method
+Förster	I-experimental_method
+resonance	I-experimental_method
+energy	I-experimental_method
+transfer	I-experimental_method
+(	O
+smFRET	B-experimental_method
+)	O
+to	O
+characterize	O
+the	O
+conformational	B-evidence
+dynamics	I-evidence
+of	O
+this	O
+extended	B-protein_state
+U2AF65	B-structure_element
+–	I-structure_element
+RNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+during	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+.	O
+
+Cognate	O
+U2AF65	B-protein
+–	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+requires	O
+RRM	B-structure_element
+extensions	I-structure_element
+
+The	O
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+minimal	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+domain	O
+comprising	O
+the	O
+core	B-protein_state
+RRM1	B-structure_element
+–	O
+RRM2	B-structure_element
+folds	B-structure_element
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+,	O
+residues	O
+148	B-residue_range
+–	I-residue_range
+336	I-residue_range
+)	O
+is	O
+relatively	O
+weak	O
+compared	O
+with	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF65	B-protein
+(	O
+Fig	O
+.	O
+1a	O
+,	O
+b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+
+Historically	O
+,	O
+this	O
+difference	O
+was	O
+attributed	O
+to	O
+the	O
+U2AF65	B-protein
+arginine	B-structure_element
+–	I-structure_element
+serine	I-structure_element
+rich	I-structure_element
+domain	I-structure_element
+,	O
+which	O
+contacts	O
+pre	B-complex_assembly
+-	I-complex_assembly
+mRNA	I-complex_assembly
+–	I-complex_assembly
+U2	I-complex_assembly
+snRNA	I-complex_assembly
+duplexes	I-complex_assembly
+outside	O
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+We	O
+noticed	O
+that	O
+the	O
+RNA	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+domain	O
+was	O
+greatly	O
+enhanced	O
+by	O
+the	O
+addition	B-experimental_method
+of	I-experimental_method
+seven	I-experimental_method
+and	I-experimental_method
+six	I-experimental_method
+residues	I-experimental_method
+at	O
+the	O
+respective	O
+N	O
+and	O
+C	O
+termini	O
+of	O
+the	O
+minimal	B-protein_state
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+residues	O
+141	B-residue_range
+–	I-residue_range
+342	I-residue_range
+;	O
+Fig	O
+.	O
+1a	O
+).	O
+
+In	O
+a	O
+fluorescence	B-experimental_method
+anisotropy	I-experimental_method
+assay	I-experimental_method
+for	O
+binding	O
+a	O
+representative	O
+Py	B-chemical
+tract	I-chemical
+derived	O
+from	O
+the	O
+well	O
+-	O
+characterized	O
+splice	B-site
+site	I-site
+of	O
+the	O
+adenovirus	B-gene
+major	I-gene
+late	I-gene
+promoter	I-gene
+(	O
+AdML	B-gene
+),	O
+the	O
+RNA	B-evidence
+affinity	I-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+increased	O
+by	O
+100	O
+-	O
+fold	O
+relative	O
+to	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+to	O
+comparable	O
+levels	O
+as	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF65	B-protein
+(	O
+Fig	O
+.	O
+1b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+–	O
+d	O
+).	O
+
+Likewise	O
+,	O
+both	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF65	B-protein
+showed	O
+similar	O
+sequence	B-evidence
+specificity	I-evidence
+for	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+stretches	I-structure_element
+in	O
+the	O
+5	B-site
+′-	I-site
+region	I-site
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+and	O
+promiscuity	O
+for	O
+C	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+regions	I-structure_element
+in	O
+the	O
+3	B-site
+′-	I-site
+region	I-site
+(	O
+Fig	O
+.	O
+1c	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+1e	O
+–	O
+h	O
+).	O
+
+U2AF65	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+Py	B-chemical
+tract	I-chemical
+comprises	O
+nine	O
+contiguous	B-structure_element
+nucleotides	B-chemical
+
+To	O
+investigate	O
+the	O
+structural	O
+basis	O
+for	O
+cognate	O
+U2AF65	B-protein
+recognition	O
+of	O
+a	O
+contiguous	B-structure_element
+Py	B-chemical
+tract	I-chemical
+,	O
+we	O
+determined	B-experimental_method
+four	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+bound	B-protein_state
+to	I-protein_state
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+oligonucleotides	I-chemical
+(	O
+Fig	O
+.	O
+2a	O
+;	O
+Table	O
+1	O
+).	O
+
+By	O
+sequential	B-experimental_method
+boot	I-experimental_method
+strapping	I-experimental_method
+(	O
+Methods	O
+),	O
+we	O
+optimized	O
+the	O
+oligonucleotide	B-chemical
+length	O
+,	O
+the	O
+position	O
+of	O
+a	O
+Br	B-chemical
+-	I-chemical
+dU	I-chemical
+,	O
+and	O
+the	O
+identity	O
+of	O
+the	O
+terminal	O
+nucleotide	B-chemical
+(	O
+rU	B-residue_name
+,	O
+dU	B-residue_name
+and	O
+rC	B-residue_name
+)	O
+to	O
+achieve	O
+full	O
+views	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+bound	B-protein_state
+to	I-protein_state
+contiguous	B-structure_element
+Py	B-chemical
+tracts	I-chemical
+at	O
+up	O
+to	O
+1	O
+.	O
+5	O
+Å	O
+resolution	O
+.	O
+
+The	O
+protein	O
+and	O
+oligonucleotide	B-chemical
+conformations	O
+are	O
+nearly	O
+identical	O
+among	O
+the	O
+four	O
+new	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+).	O
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+associate	O
+with	O
+the	O
+Py	B-chemical
+tract	I-chemical
+in	O
+a	O
+parallel	B-protein_state
+,	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+arrangement	O
+(	O
+shown	O
+for	O
+representative	O
+structure	O
+iv	O
+in	O
+Fig	O
+.	O
+2b	O
+,	O
+c	O
+;	O
+Supplementary	O
+Movie	O
+1	O
+).	O
+
+An	O
+extended	B-protein_state
+conformation	I-protein_state
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+traverses	O
+across	O
+the	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+surface	I-structure_element
+of	O
+RRM1	B-structure_element
+and	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+of	O
+RRM2	B-structure_element
+and	O
+is	O
+well	O
+defined	O
+in	O
+the	O
+electron	B-evidence
+density	I-evidence
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+
+The	O
+extensions	B-structure_element
+at	O
+the	O
+N	O
+terminus	O
+of	O
+RRM1	B-structure_element
+and	O
+C	O
+terminus	O
+of	O
+RRM2	B-structure_element
+adopt	O
+well	O
+-	O
+ordered	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+.	O
+
+Both	O
+RRM1	B-structure_element
+/	O
+RRM2	B-structure_element
+extensions	B-structure_element
+and	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+directly	O
+recognize	O
+the	O
+bound	B-protein_state
+oligonucleotide	B-chemical
+.	O
+
+We	O
+compare	O
+the	O
+global	O
+conformation	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+with	O
+the	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+crystal	B-evidence
+structure	I-evidence
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+NMR	B-experimental_method
+structure	B-evidence
+in	O
+the	O
+Supplementary	O
+Discussion	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+.	O
+
+The	O
+discovery	O
+of	O
+nine	O
+U2AF65	B-site
+-	I-site
+binding	I-site
+sites	I-site
+for	O
+contiguous	B-structure_element
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+nucleotides	I-chemical
+was	O
+unexpected	O
+.	O
+
+Based	O
+on	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	B-evidence
+,	O
+we	O
+originally	O
+hypothesized	O
+that	O
+the	O
+U2AF65	B-protein
+RRMs	B-structure_element
+would	O
+bind	O
+the	O
+minimal	B-protein_state
+seven	O
+nucleotides	B-chemical
+observed	O
+in	O
+these	O
+structures	B-evidence
+.	O
+
+Surprisingly	O
+,	O
+the	O
+RRM2	B-structure_element
+extension	I-structure_element
+/	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+contribute	O
+new	O
+central	O
+nucleotide	B-site
+-	I-site
+binding	I-site
+sites	I-site
+near	O
+the	O
+RRM1	B-site
+/	I-site
+RRM2	I-site
+junction	I-site
+and	O
+the	O
+RRM1	B-structure_element
+extension	I-structure_element
+recognizes	O
+the	O
+3	O
+′-	O
+terminal	O
+nucleotide	B-chemical
+(	O
+Fig	O
+.	O
+2c	O
+;	O
+Supplementary	O
+Movie	O
+1	O
+).	O
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+characterize	O
+ribose	B-chemical
+(	O
+r	B-chemical
+)	O
+nucleotides	B-chemical
+at	O
+all	O
+of	O
+the	O
+binding	B-site
+sites	I-site
+except	O
+the	O
+seventh	B-residue_number
+and	O
+eighth	B-residue_number
+deoxy	B-chemical
+-(	I-chemical
+d	I-chemical
+)	I-chemical
+U	I-chemical
+,	O
+which	O
+are	O
+likely	O
+to	O
+lack	O
+2	O
+′-	O
+hydroxyl	O
+contacts	O
+based	O
+on	O
+the	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structure	B-evidence
+.	O
+
+Qualitatively	O
+,	O
+a	O
+subset	O
+of	O
+the	O
+U2AF651	B-site
+,	I-site
+2L	I-site
+-	I-site
+nucleotide	I-site
+-	I-site
+binding	I-site
+sites	I-site
+(	O
+sites	B-site
+1	I-site
+–	I-site
+3	I-site
+and	O
+7	B-site
+–	I-site
+9	I-site
+)	O
+share	O
+similar	O
+locations	O
+to	O
+those	O
+of	O
+the	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	B-evidence
+(	O
+Supplementary	O
+Figs	O
+2c	O
+,	O
+d	O
+and	O
+3	O
+).	O
+
+Yet	O
+,	O
+only	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+interactions	O
+at	O
+sites	B-site
+1	I-site
+and	I-site
+7	I-site
+are	O
+nearly	O
+identical	O
+to	O
+those	O
+of	O
+the	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	B-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+,	O
+f	O
+).	O
+
+In	O
+striking	O
+departures	O
+from	O
+prior	O
+partial	O
+views	O
+,	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+reveal	O
+three	O
+unanticipated	O
+nucleotide	B-site
+-	I-site
+binding	I-site
+sites	I-site
+at	O
+the	O
+centre	O
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+,	O
+as	O
+well	O
+as	O
+numerous	O
+new	O
+interactions	O
+that	O
+underlie	O
+cognate	O
+recognition	O
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+(	O
+Fig	O
+.	O
+3a	O
+–	O
+h	O
+).	O
+
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+interacts	O
+with	O
+the	O
+Py	B-chemical
+tract	I-chemical
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM2	B-structure_element
+,	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+and	O
+RRM1	B-structure_element
+concomitantly	O
+recognize	O
+the	O
+three	O
+central	O
+nucleotides	B-chemical
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+,	O
+which	O
+are	O
+likely	O
+to	O
+coordinate	O
+the	O
+conformational	O
+arrangement	O
+of	O
+these	O
+disparate	O
+portions	O
+of	O
+the	O
+protein	O
+.	O
+
+Residues	O
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+comprise	O
+a	O
+centrally	O
+located	O
+binding	B-site
+site	I-site
+for	O
+the	O
+fifth	B-residue_number
+nucleotide	B-chemical
+on	O
+the	O
+RRM2	B-site
+surface	I-site
+and	O
+abutting	O
+the	O
+RRM1	B-site
+/	I-site
+RRM2	I-site
+interface	I-site
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+The	O
+backbone	O
+amide	O
+of	O
+the	O
+linker	B-structure_element
+V254	B-residue_name_number
+and	O
+the	O
+carbonyl	O
+of	O
+T252	B-residue_name_number
+engage	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+rU5	B-residue_name_number
+-	O
+O4	O
+and	O
+-	O
+N3H	O
+atoms	O
+.	O
+
+In	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+of	O
+RRM1	B-structure_element
+,	O
+the	O
+side	O
+chains	O
+of	O
+K225	B-residue_name_number
+and	O
+R227	B-residue_name_number
+donate	O
+additional	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+rU5	B-residue_name_number
+-	O
+O2	O
+lone	O
+pair	O
+electrons	O
+.	O
+
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+also	O
+participates	O
+in	O
+the	O
+preceding	O
+rU4	B-site
+-	I-site
+binding	I-site
+site	I-site
+,	O
+where	O
+the	O
+V254	B-residue_name_number
+backbone	O
+carbonyl	O
+and	O
+D256	B-residue_name_number
+carboxylate	O
+position	O
+the	O
+K260	B-residue_name_number
+side	O
+chain	O
+to	O
+hydrogen	O
+bond	O
+with	O
+the	O
+rU4	B-residue_name_number
+-	O
+O4	O
+(	O
+Fig	O
+.	O
+3c	O
+).	O
+
+Otherwise	O
+,	O
+the	O
+rU4	B-residue_name_number
+nucleotide	B-chemical
+packs	O
+against	O
+F304	B-residue_name_number
+in	O
+the	O
+signature	O
+ribonucleoprotein	B-structure_element
+consensus	I-structure_element
+motif	I-structure_element
+(	I-structure_element
+RNP	I-structure_element
+)-	I-structure_element
+2	I-structure_element
+of	O
+RRM2	B-structure_element
+.	O
+
+At	O
+the	O
+opposite	O
+side	O
+of	O
+the	O
+central	O
+fifth	B-residue_number
+nucleotide	B-chemical
+,	O
+the	O
+sixth	B-residue_number
+rU6	B-residue_name_number
+nucleotide	B-chemical
+is	O
+located	O
+at	O
+the	O
+inter	B-site
+-	I-site
+RRM1	I-site
+/	I-site
+RRM2	I-site
+interface	I-site
+(	O
+Fig	O
+.	O
+3e	O
+;	O
+Supplementary	O
+Movie	O
+1	O
+).	O
+
+This	O
+nucleotide	B-chemical
+twists	O
+to	O
+face	O
+away	O
+from	O
+the	O
+U2AF65	B-protein
+linker	B-structure_element
+and	O
+instead	O
+inserts	O
+the	O
+rU6	B-residue_name_number
+-	O
+uracil	B-residue_name
+into	O
+a	O
+sandwich	O
+between	O
+the	O
+β2	B-structure_element
+/	I-structure_element
+β3	I-structure_element
+loops	I-structure_element
+of	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+.	O
+
+The	O
+rU6	B-residue_name_number
+base	O
+edge	O
+is	O
+relatively	O
+solvent	B-protein_state
+exposed	I-protein_state
+;	O
+accordingly	O
+,	O
+the	O
+rU6	B-residue_name_number
+hydrogen	O
+bonds	O
+with	O
+U2AF65	B-protein
+are	O
+water	B-chemical
+mediated	O
+apart	O
+from	O
+a	O
+single	O
+direct	O
+interaction	O
+by	O
+the	O
+RRM1	B-structure_element
+-	O
+N196	B-residue_name_number
+side	O
+chain	O
+.	O
+
+We	O
+tested	B-experimental_method
+the	I-experimental_method
+contribution	I-experimental_method
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+interactions	O
+with	O
+the	O
+new	O
+central	O
+nucleotide	B-chemical
+to	O
+Py	B-evidence
+-	I-evidence
+tract	I-evidence
+affinity	I-evidence
+(	O
+Fig	O
+.	O
+3i	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+
+Mutagenesis	B-experimental_method
+of	O
+either	O
+V254	B-residue_name_number
+in	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+to	O
+proline	B-residue_name
+or	O
+RRM1	B-structure_element
+–	O
+R227	B-residue_name_number
+to	O
+alanine	B-residue_name
+,	O
+which	O
+remove	O
+the	O
+hydrogen	O
+bond	O
+with	O
+the	O
+fifth	B-residue_number
+uracil	B-residue_name
+-	O
+O4	O
+or	O
+-	O
+O2	O
+,	O
+reduced	O
+the	O
+affinities	B-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+for	O
+the	O
+representative	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+by	O
+four	O
+-	O
+or	O
+five	O
+-	O
+fold	O
+,	O
+respectively	O
+.	O
+
+The	O
+energetic	O
+penalties	O
+due	O
+to	O
+these	O
+mutations	O
+(	O
+ΔΔG	B-evidence
+0	O
+.	O
+8	O
+–	O
+0	O
+.	O
+9	O
+kcal	O
+mol	O
+−	O
+1	O
+)	O
+are	O
+consistent	O
+with	O
+the	O
+loss	O
+of	O
+each	O
+hydrogen	O
+bond	O
+with	O
+the	O
+rU5	B-residue_name_number
+base	O
+and	O
+support	O
+the	O
+relevance	O
+of	O
+the	O
+central	O
+nucleotide	O
+interactions	O
+observed	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+.	O
+
+U2AF65	B-protein
+RRM	B-structure_element
+extensions	I-structure_element
+interact	O
+with	O
+the	O
+Py	B-chemical
+tract	I-chemical
+
+The	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extensions	I-structure_element
+of	O
+the	O
+U2AF65	B-protein
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+directly	O
+contact	O
+the	O
+bound	B-protein_state
+Py	B-chemical
+tract	I-chemical
+.	O
+
+Rather	O
+than	O
+interacting	O
+with	O
+a	O
+new	O
+5	O
+′-	O
+terminal	O
+nucleotide	B-chemical
+as	O
+we	O
+had	O
+hypothesized	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+of	O
+RRM2	B-structure_element
+instead	O
+folds	O
+across	O
+one	O
+surface	O
+of	O
+rU3	B-residue_name_number
+in	O
+the	O
+third	B-site
+binding	I-site
+site	I-site
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+
+There	O
+,	O
+a	O
+salt	O
+bridge	O
+between	O
+the	O
+K340	B-residue_name_number
+side	O
+chain	O
+and	O
+nucleotide	B-chemical
+phosphate	O
+,	O
+as	O
+well	O
+as	O
+G338	B-residue_name_number
+-	O
+base	O
+stacking	O
+and	O
+a	O
+hydrogen	O
+bond	O
+between	O
+the	O
+backbone	O
+amide	O
+of	O
+G338	B-residue_name_number
+and	O
+the	O
+rU3	B-residue_name_number
+-	O
+O4	O
+,	O
+secure	O
+the	O
+RRM2	B-structure_element
+extension	I-structure_element
+.	O
+
+Indirectly	O
+,	O
+the	O
+additional	O
+contacts	O
+with	O
+the	O
+third	B-residue_number
+nucleotide	B-chemical
+shift	O
+the	O
+rU2	B-residue_name_number
+nucleotide	B-chemical
+in	O
+the	O
+second	B-site
+binding	I-site
+site	I-site
+closer	O
+to	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+of	O
+RRM2	B-structure_element
+.	O
+
+Consequently	O
+,	O
+the	O
+U2AF651	B-protein_state
+,	I-protein_state
+2L	I-protein_state
+-	I-protein_state
+bound	I-protein_state
+rU2	B-residue_name_number
+-	O
+O4	O
+and	O
+-	O
+N3H	O
+form	O
+dual	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+K329	B-residue_name_number
+backbone	O
+atoms	O
+(	O
+Fig	O
+.	O
+3a	O
+),	O
+rather	O
+than	O
+a	O
+single	O
+hydrogen	O
+bond	O
+with	O
+the	O
+K329	B-residue_name_number
+side	O
+chain	O
+as	O
+in	O
+the	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structure	B-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+).	O
+
+At	O
+the	O
+N	O
+terminus	O
+,	O
+the	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+extension	I-structure_element
+of	O
+U2AF65	B-protein
+RRM1	B-structure_element
+positions	O
+the	O
+Q147	B-residue_name_number
+side	O
+chain	O
+to	O
+bridge	O
+the	O
+eighth	B-residue_number
+and	O
+ninth	B-residue_number
+nucleotides	B-chemical
+at	O
+the	O
+3	B-site
+′	I-site
+terminus	I-site
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+(	O
+Fig	O
+.	O
+3f	O
+–	O
+h	O
+).	O
+
+The	O
+Q147	B-residue_name_number
+residue	O
+participates	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+-	O
+N3H	O
+of	O
+the	O
+eighth	B-residue_number
+uracil	B-residue_name
+and	O
+-	O
+O2	O
+of	O
+the	O
+ninth	B-residue_number
+pyrimidine	B-chemical
+.	O
+
+The	O
+adjacent	O
+R146	B-residue_name_number
+guanidinium	O
+group	O
+donates	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+3	O
+′-	O
+terminal	O
+ribose	B-chemical
+-	O
+O2	O
+′	O
+and	O
+O3	O
+′	O
+atoms	O
+,	O
+where	O
+it	O
+could	O
+form	O
+a	O
+salt	O
+bridge	O
+with	O
+a	O
+phospho	O
+-	O
+diester	O
+group	O
+in	O
+the	O
+context	O
+of	O
+a	O
+longer	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+.	O
+
+Consistent	O
+with	O
+loss	O
+of	O
+a	O
+hydrogen	O
+bond	O
+with	O
+the	O
+ninth	B-residue_number
+pyrimidine	B-chemical
+-	O
+O2	O
+(	O
+ΔΔG	B-evidence
+1	O
+.	O
+0	O
+kcal	O
+mol	O
+−	O
+1	O
+),	O
+mutation	B-experimental_method
+of	O
+the	O
+Q147	B-residue_name_number
+to	O
+an	O
+alanine	B-residue_name
+reduced	O
+U2AF651	B-evidence
+,	I-evidence
+2L	I-evidence
+affinity	I-evidence
+for	O
+the	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+by	O
+five	O
+-	O
+fold	O
+(	O
+Fig	O
+.	O
+3i	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4c	O
+).	O
+
+We	O
+compare	B-experimental_method
+U2AF65	B-protein
+interactions	O
+with	O
+uracil	B-residue_name
+relative	O
+to	O
+cytosine	B-residue_name
+pyrimidines	B-chemical
+at	O
+the	O
+ninth	B-site
+binding	I-site
+site	I-site
+in	O
+Fig	O
+.	O
+3g	O
+,	O
+h	O
+and	O
+the	O
+Supplementary	O
+Discussion	O
+.	O
+
+Versatile	O
+primary	O
+sequence	O
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+reveal	O
+that	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+mediates	O
+an	O
+extensive	B-site
+interface	I-site
+with	O
+the	O
+second	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+of	O
+RRM1	B-structure_element
+,	O
+the	O
+β2	B-structure_element
+/	I-structure_element
+β3	I-structure_element
+strands	I-structure_element
+of	O
+RRM2	B-structure_element
+and	O
+the	O
+N	O
+-	O
+terminal	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+extension	I-structure_element
+of	O
+RRM1	B-structure_element
+.	O
+
+Altogether	O
+,	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+residues	O
+(	O
+R228	B-residue_range
+–	I-residue_range
+K260	I-residue_range
+)	O
+bury	O
+2	O
+,	O
+800	O
+Å2	O
+of	O
+surface	O
+area	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+holo	B-protein_state
+-	I-protein_state
+protein	I-protein_state
+,	O
+suggestive	O
+of	O
+a	O
+cognate	B-site
+interface	I-site
+compared	O
+with	O
+1	O
+,	O
+900	O
+Å2	O
+for	O
+a	O
+typical	O
+protein	O
+–	O
+protein	O
+complex	O
+.	O
+
+The	O
+path	O
+of	O
+the	O
+linker	B-structure_element
+initiates	O
+at	O
+P229	B-residue_name_number
+following	O
+the	O
+core	B-protein_state
+RRM1	B-structure_element
+β	B-structure_element
+-	I-structure_element
+strand	I-structure_element
+,	O
+in	O
+a	O
+kink	B-structure_element
+that	O
+is	O
+positioned	O
+by	O
+intra	O
+-	O
+molecular	O
+stacking	O
+among	O
+the	O
+consecutive	O
+R228	B-residue_name_number
+,	O
+Y232	B-residue_name_number
+and	O
+P234	B-residue_name_number
+side	O
+chains	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+lower	O
+right	O
+).	O
+
+A	O
+second	B-structure_element
+kink	I-structure_element
+at	O
+P236	B-residue_name_number
+,	O
+coupled	O
+with	O
+respective	O
+packing	O
+of	O
+the	O
+L235	B-residue_name_number
+and	O
+M238	B-residue_name_number
+side	O
+chains	O
+on	O
+the	O
+N	O
+-	O
+terminal	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+RRM1	I-structure_element
+extension	I-structure_element
+and	O
+the	O
+core	B-protein_state
+RRM1	B-structure_element
+α2	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+,	O
+reverses	O
+the	O
+direction	O
+of	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+towards	O
+the	O
+RRM1	B-site
+/	I-site
+RRM2	I-site
+interface	I-site
+and	O
+away	O
+from	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+
+In	O
+the	O
+neighbouring	O
+apical	O
+region	O
+of	O
+the	O
+linker	B-structure_element
+,	O
+the	O
+V244	B-residue_name_number
+and	O
+V246	B-residue_name_number
+side	O
+chains	O
+pack	O
+in	O
+a	O
+hydrophobic	B-site
+pocket	I-site
+between	O
+two	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+of	O
+the	O
+core	B-protein_state
+RRM1	B-structure_element
+.	O
+
+The	O
+adjacent	O
+V249	B-residue_name_number
+and	O
+V250	B-residue_name_number
+are	O
+notable	O
+for	O
+their	O
+respective	O
+interactions	O
+that	O
+connect	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+at	O
+this	O
+distal	O
+interface	B-site
+from	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+site	I-site
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+top	O
+).	O
+
+A	O
+third	B-structure_element
+kink	I-structure_element
+stacks	O
+P247	B-residue_name_number
+and	O
+G248	B-residue_name_number
+with	O
+Y245	B-residue_name_number
+and	O
+re	O
+-	O
+orients	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+linker	B-structure_element
+towards	O
+the	O
+RRM2	B-structure_element
+and	O
+bound	B-protein_state
+RNA	B-chemical
+.	O
+
+At	O
+the	O
+RNA	B-chemical
+surface	O
+,	O
+the	O
+key	O
+V254	B-residue_name_number
+that	O
+recognizes	O
+the	O
+fifth	B-residue_number
+uracil	B-residue_name
+is	O
+secured	O
+via	O
+hydrophobic	O
+contacts	O
+between	O
+its	O
+side	O
+chain	O
+and	O
+the	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+surface	I-structure_element
+of	O
+RRM2	B-structure_element
+,	O
+chiefly	O
+the	O
+consensus	O
+RNP1	B-structure_element
+-	O
+F304	B-residue_name_number
+residue	O
+that	O
+stacks	O
+with	O
+the	O
+fourth	B-residue_number
+uracil	B-residue_name
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+lower	O
+left	O
+).	O
+
+Few	O
+direct	O
+contacts	O
+are	O
+made	O
+between	O
+the	O
+remaining	O
+residues	O
+of	O
+the	O
+linker	B-structure_element
+and	O
+the	O
+U2AF65	B-protein
+RRM2	B-structure_element
+;	O
+instead	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+conformation	O
+of	O
+the	O
+linker	B-structure_element
+appears	O
+primarily	O
+RNA	B-chemical
+mediated	O
+(	O
+Fig	O
+.	O
+3c	O
+,	O
+d	O
+).	O
+
+We	O
+investigated	O
+whether	O
+the	O
+observed	O
+contacts	O
+between	O
+the	O
+RRMs	B-structure_element
+and	O
+linker	B-structure_element
+were	O
+critical	O
+for	O
+RNA	O
+binding	O
+by	O
+structure	B-experimental_method
+-	I-experimental_method
+guided	I-experimental_method
+mutagenesis	I-experimental_method
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+
+We	O
+titrated	B-experimental_method
+these	O
+mutant	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+proteins	O
+into	O
+fluorescein	B-chemical
+-	O
+labelled	O
+AdML	B-gene
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+RNA	I-chemical
+and	O
+fit	O
+the	O
+fluorescence	B-evidence
+anisotropy	I-evidence
+changes	I-evidence
+to	O
+obtain	O
+the	O
+apparent	O
+equilibrium	B-evidence
+affinities	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+–	O
+h	O
+).	O
+
+We	O
+introduced	O
+glycine	B-residue_name
+substitutions	B-experimental_method
+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
+
+First	O
+,	O
+we	O
+replaced	B-experimental_method
+V249	B-residue_name_number
+and	O
+V250	B-residue_name_number
+at	O
+the	O
+RRM1	B-site
+/	I-site
+RRM2	I-site
+interface	I-site
+and	O
+V254	B-residue_name_number
+at	O
+the	O
+bound	B-protein_state
+RNA	B-chemical
+site	O
+with	O
+glycine	B-residue_name
+(	O
+3Gly	B-mutant
+).	O
+
+However	O
+,	O
+the	O
+resulting	O
+decrease	O
+in	O
+the	O
+AdML	B-gene
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+3Gly	I-mutant
+mutant	B-protein_state
+relative	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+protein	B-protein
+was	O
+not	O
+significant	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+
+In	O
+parallel	O
+,	O
+we	O
+replaced	B-experimental_method
+five	O
+linker	B-structure_element
+residues	I-structure_element
+(	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+V254	B-residue_name_number
+and	O
+P255	B-residue_name_number
+)	O
+at	O
+the	O
+fifth	B-site
+nucleotide	I-site
+-	I-site
+binding	I-site
+site	I-site
+with	O
+glycines	B-residue_name
+(	O
+5Gly	B-mutant
+)	O
+and	O
+also	O
+found	O
+that	O
+the	O
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+5Gly	I-mutant
+mutant	B-protein_state
+likewise	O
+decreased	O
+only	O
+slightly	O
+relative	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+protein	B-protein
+.	O
+
+A	O
+more	O
+conservative	B-experimental_method
+substitution	I-experimental_method
+of	O
+these	O
+five	O
+residues	O
+(	O
+251	B-residue_range
+–	I-residue_range
+255	I-residue_range
+)	O
+with	O
+an	O
+unrelated	O
+sequence	O
+capable	O
+of	O
+backbone	O
+-	O
+mediated	O
+hydrogen	O
+bonds	O
+(	O
+STVVP	B-mutant
+>	I-mutant
+NLALA	I-mutant
+)	O
+confirmed	O
+the	O
+subtle	O
+impact	O
+of	O
+this	O
+versatile	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+sequence	I-structure_element
+on	O
+affinity	B-evidence
+for	O
+the	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+.	O
+
+Finally	O
+,	O
+to	O
+ensure	O
+that	O
+these	O
+selective	O
+mutations	O
+were	O
+sufficient	O
+to	O
+disrupt	O
+the	O
+linker	B-structure_element
+/	O
+RRM	B-structure_element
+contacts	O
+,	O
+we	O
+substituted	B-experimental_method
+glycine	B-residue_name
+for	O
+the	O
+majority	O
+of	O
+buried	O
+hydrophobic	O
+residues	O
+in	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+(	O
+including	O
+M144	B-residue_name_number
+,	O
+L235	B-residue_name_number
+,	O
+M238	B-residue_name_number
+,	O
+V244	B-residue_name_number
+,	O
+V246	B-residue_name_number
+,	O
+V249	B-residue_name_number
+,	O
+V250	B-residue_name_number
+,	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+V254	B-residue_name_number
+,	O
+P255	B-residue_name_number
+;	O
+called	O
+12Gly	B-mutant
+).	O
+
+Despite	O
+12	B-experimental_method
+concurrent	I-experimental_method
+mutations	I-experimental_method
+,	O
+the	O
+AdML	B-gene
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+12Gly	I-mutant
+variant	B-protein_state
+was	O
+reduced	O
+by	O
+only	O
+three	O
+-	O
+fold	O
+relative	O
+to	O
+the	O
+unmodified	B-protein_state
+protein	B-protein
+(	O
+Fig	O
+.	O
+4b	O
+),	O
+which	O
+is	O
+less	O
+than	O
+the	O
+penalty	O
+of	O
+the	O
+V254P	B-mutant
+mutation	O
+that	O
+disrupts	O
+the	O
+rU5	B-residue_name_number
+hydrogen	O
+bond	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+i	O
+).	O
+
+To	O
+test	O
+the	O
+interplay	O
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+with	O
+its	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+RRM	B-structure_element
+extensions	I-structure_element
+,	O
+we	O
+constructed	B-experimental_method
+an	O
+internal	O
+linker	B-experimental_method
+deletion	I-experimental_method
+of	O
+20	B-residue_range
+-	I-residue_range
+residues	I-residue_range
+within	O
+the	O
+extended	B-protein_state
+RNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+).	O
+
+We	O
+found	O
+that	O
+the	O
+affinity	B-evidence
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+for	O
+the	O
+AdML	B-gene
+RNA	B-chemical
+was	O
+significantly	O
+reduced	O
+relative	O
+to	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+(	O
+four	O
+-	O
+fold	O
+,	O
+Figs	O
+1b	O
+and	O
+4b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4i	O
+).	O
+
+Yet	O
+,	O
+it	O
+is	O
+well	O
+known	O
+that	O
+the	O
+linker	B-experimental_method
+deletion	I-experimental_method
+in	O
+the	O
+context	O
+of	O
+the	O
+minimal	B-protein_state
+RRM1	B-structure_element
+–	O
+RRM2	B-structure_element
+boundaries	O
+has	O
+no	O
+detectable	O
+effect	O
+on	O
+the	O
+RNA	B-evidence
+affinities	I-evidence
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+compared	O
+with	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+(	O
+refs	O
+;	O
+Figs	O
+1b	O
+and	O
+4b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4j	O
+).	O
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+suggest	O
+that	O
+an	O
+extended	B-protein_state
+conformation	I-protein_state
+of	O
+the	O
+truncated	B-protein_state
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+would	O
+suffice	O
+to	O
+connect	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM1	B-structure_element
+C	O
+terminus	O
+to	O
+the	O
+N	O
+terminus	O
+of	O
+RRM2	B-structure_element
+(	O
+24	O
+Å	O
+distance	O
+between	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+R227	B-residue_name_number
+-	O
+Cα	O
+–	O
+H259	B-residue_name_number
+-	O
+Cα	O
+atoms	O
+),	O
+which	O
+agrees	O
+with	O
+the	O
+greater	O
+RNA	B-evidence
+affinities	I-evidence
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+dual	B-protein_state
+RRMs	B-structure_element
+compared	O
+with	O
+the	O
+individual	B-protein_state
+U2AF65	B-protein
+RRMs	B-structure_element
+.	O
+
+However	O
+,	O
+stretching	O
+of	O
+the	O
+truncated	B-protein_state
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+linker	B-structure_element
+to	O
+connect	O
+the	O
+RRM	B-structure_element
+termini	I-structure_element
+is	O
+expected	O
+to	O
+disrupt	O
+its	O
+nucleotide	O
+interactions	O
+.	O
+
+Likewise	O
+,	O
+deletion	B-experimental_method
+of	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	B-structure_element
+extension	I-structure_element
+in	O
+the	O
+shortened	B-protein_state
+constructs	O
+would	O
+remove	O
+packing	O
+interactions	O
+that	O
+position	O
+the	O
+linker	B-structure_element
+in	O
+a	O
+kinked	B-structure_element
+turn	I-structure_element
+following	O
+P229	B-residue_name_number
+(	O
+Fig	O
+.	O
+4a	O
+),	O
+consistent	O
+with	O
+the	O
+lower	O
+RNA	B-evidence
+affinities	I-evidence
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+compared	O
+with	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+.	O
+
+To	O
+further	O
+test	O
+cooperation	O
+among	O
+the	O
+U2AF65	B-protein
+RRM	B-structure_element
+extensions	I-structure_element
+and	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+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	B-experimental_method
+(	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
+
+Notably	O
+,	O
+the	O
+Q147A	B-mutant
+/	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+mutation	B-experimental_method
+reduced	O
+the	O
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+3Mut	I-mutant
+protein	O
+by	O
+30	O
+-	O
+fold	O
+more	O
+than	O
+would	O
+be	O
+expected	O
+based	O
+on	O
+simple	O
+addition	O
+of	O
+the	O
+ΔΔG	B-evidence
+'	O
+s	O
+for	O
+the	O
+single	O
+mutations	O
+.	O
+
+This	O
+difference	O
+indicates	O
+that	O
+the	O
+linearly	B-protein_state
+distant	I-protein_state
+regions	B-structure_element
+of	O
+the	O
+U2AF65	B-protein
+primary	O
+sequence	O
+,	O
+including	O
+Q147	B-residue_name_number
+in	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	B-structure_element
+extension	I-structure_element
+and	O
+R227	B-residue_name_number
+/	O
+V254	B-residue_name_number
+in	O
+the	O
+N	O
+-/	O
+C	O
+-	O
+terminal	O
+linker	B-structure_element
+regions	I-structure_element
+at	O
+the	O
+fifth	B-site
+nucleotide	I-site
+site	I-site
+,	O
+cooperatively	O
+recognize	O
+the	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+Altogether	O
+,	O
+we	O
+conclude	O
+that	O
+the	O
+conformation	O
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+is	O
+key	O
+for	O
+recognizing	O
+RNA	B-chemical
+and	O
+is	O
+positioned	O
+by	O
+the	O
+RRM	B-structure_element
+extension	I-structure_element
+but	O
+otherwise	O
+relatively	O
+independent	O
+of	O
+the	O
+side	O
+chain	O
+composition	O
+.	O
+
+The	O
+non	O
+-	O
+additive	O
+effects	O
+of	O
+the	O
+Q147A	B-mutant
+/	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+triple	B-experimental_method
+mutation	I-experimental_method
+,	O
+coupled	O
+with	O
+the	O
+context	O
+-	O
+dependent	O
+penalties	O
+of	O
+an	O
+internal	O
+U2AF65	B-protein
+linker	B-experimental_method
+deletion	I-experimental_method
+,	O
+highlights	O
+the	O
+importance	O
+of	O
+the	O
+structural	O
+interplay	O
+among	O
+the	O
+U2AF65	B-protein
+linker	B-structure_element
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extensions	I-structure_element
+flanking	O
+the	O
+core	B-protein_state
+RRMs	B-structure_element
+.	O
+
+Importance	O
+of	O
+U2AF65	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+contacts	O
+for	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+splicing	O
+
+We	O
+proceeded	O
+to	O
+test	O
+the	O
+importance	O
+of	O
+new	O
+U2AF65	B-complex_assembly
+–	I-complex_assembly
+Py	I-complex_assembly
+-	I-complex_assembly
+tract	I-complex_assembly
+interactions	O
+for	O
+splicing	O
+of	O
+a	O
+model	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+substrate	O
+in	O
+a	O
+human	B-species
+cell	O
+line	O
+(	O
+Fig	O
+.	O
+5	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+
+As	O
+a	O
+representative	O
+splicing	O
+substrate	O
+,	O
+we	O
+utilized	O
+a	O
+well	O
+-	O
+characterized	O
+minigene	B-chemical
+splicing	I-chemical
+reporter	I-chemical
+(	O
+called	O
+pyPY	B-chemical
+)	O
+comprising	O
+a	O
+weak	O
+(	O
+that	O
+is	O
+,	O
+degenerate	O
+,	O
+py	B-chemical
+)	O
+and	O
+strong	O
+(	O
+that	O
+is	O
+,	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+,	O
+PY	B-chemical
+)	O
+polypyrimidine	B-chemical
+tracts	I-chemical
+preceding	O
+two	O
+alternative	O
+splice	B-site
+sites	I-site
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+
+When	O
+transfected	B-experimental_method
+into	O
+HEK293T	O
+cells	O
+containing	O
+only	O
+endogenous	B-protein_state
+U2AF65	B-protein
+,	O
+the	O
+PY	B-site
+splice	I-site
+site	I-site
+is	O
+used	O
+and	O
+the	O
+remaining	O
+transcript	O
+remains	O
+unspliced	O
+.	O
+
+When	O
+co	B-experimental_method
+-	I-experimental_method
+transfected	I-experimental_method
+with	O
+an	O
+expression	B-experimental_method
+plasmid	I-experimental_method
+for	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+U2AF65	B-protein
+,	O
+use	O
+of	O
+the	O
+py	B-site
+splice	I-site
+site	I-site
+significantly	O
+increases	O
+(	O
+by	O
+more	O
+than	O
+five	O
+-	O
+fold	O
+)	O
+and	O
+as	O
+documented	O
+converts	O
+a	O
+fraction	O
+of	O
+the	O
+unspliced	O
+to	O
+spliced	O
+transcript	O
+.	O
+
+The	O
+strong	O
+PY	B-site
+splice	I-site
+site	I-site
+is	O
+insensitive	O
+to	O
+added	O
+U2AF65	B-protein
+,	O
+suggesting	O
+that	O
+endogenous	B-protein_state
+U2AF65	B-protein
+levels	O
+are	O
+sufficient	O
+to	O
+saturate	O
+this	O
+site	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+).	O
+
+We	O
+introduced	O
+the	O
+triple	B-experimental_method
+mutation	I-experimental_method
+(	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+/	O
+Q147A	B-mutant
+)	O
+that	O
+significantly	O
+reduced	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+association	O
+with	O
+the	O
+Py	B-chemical
+tract	I-chemical
+(	O
+Fig	O
+.	O
+4b	O
+)	O
+in	O
+the	O
+context	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF65	B-protein
+(	O
+U2AF65	B-mutant
+-	I-mutant
+3Mut	I-mutant
+).	O
+
+Co	B-experimental_method
+-	I-experimental_method
+transfection	I-experimental_method
+of	O
+the	O
+U2AF65	B-mutant
+-	I-mutant
+3Mut	I-mutant
+with	O
+the	O
+pyPY	B-chemical
+splicing	O
+substrate	O
+significantly	O
+reduced	O
+splicing	O
+of	O
+the	O
+weak	O
+‘	B-site
+py	I-site
+'	I-site
+splice	I-site
+site	I-site
+relative	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+U2AF65	B-protein
+(	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+
+We	O
+conclude	O
+that	O
+the	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+interactions	O
+with	O
+these	O
+residues	O
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+and	O
+RRM	B-structure_element
+extensions	I-structure_element
+are	O
+important	O
+for	O
+splicing	O
+as	O
+well	O
+as	O
+for	O
+binding	O
+a	O
+representative	O
+of	O
+the	O
+major	B-structure_element
+U2	I-structure_element
+-	I-structure_element
+class	I-structure_element
+of	I-structure_element
+splice	I-structure_element
+sites	I-structure_element
+.	O
+
+Sparse	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+contacts	O
+underlie	O
+apo	B-protein_state
+-	O
+U2AF65	B-protein
+dynamics	O
+
+The	O
+direct	O
+interface	B-site
+between	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+is	O
+minor	O
+,	O
+burying	O
+265	O
+Å2	O
+of	O
+solvent	O
+accessible	O
+surface	O
+area	O
+compared	O
+with	O
+570	O
+Å2	O
+on	O
+average	O
+for	O
+a	O
+crystal	O
+packing	O
+interface	O
+.	O
+
+A	O
+handful	O
+of	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+hydrogen	O
+bonds	O
+are	O
+apparent	O
+between	O
+the	O
+side	O
+chains	O
+of	O
+RRM1	B-structure_element
+-	O
+N155	B-residue_name_number
+and	O
+RRM2	B-structure_element
+-	O
+K292	B-residue_name_number
+,	O
+RRM1	B-structure_element
+-	O
+N155	B-residue_name_number
+and	O
+RRM2	B-structure_element
+-	O
+D272	B-residue_name_number
+as	O
+well	O
+as	O
+the	O
+backbone	O
+atoms	O
+of	O
+RRM1	B-structure_element
+-	O
+G221	B-residue_name_number
+and	O
+RRM2	B-structure_element
+-	O
+D273	B-residue_name_number
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+
+This	O
+minor	O
+U2AF65	B-protein
+RRM1	B-site
+/	I-site
+RRM2	I-site
+interface	I-site
+,	O
+coupled	O
+with	O
+the	O
+versatile	O
+sequence	O
+of	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+,	O
+highlighted	O
+the	O
+potential	O
+role	O
+for	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+conformational	O
+dynamics	O
+in	O
+U2AF65	B-protein
+-	O
+splice	O
+site	O
+recognition	O
+.	O
+
+Paramagnetic	B-experimental_method
+resonance	I-experimental_method
+enhancement	I-experimental_method
+(	O
+PRE	B-experimental_method
+)	O
+measurements	O
+previously	O
+had	O
+suggested	O
+a	O
+predominant	O
+back	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+back	I-protein_state
+,	O
+or	O
+‘	O
+closed	B-protein_state
+'	O
+conformation	O
+of	O
+the	O
+apo	B-protein_state
+-	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+in	O
+equilibrium	O
+with	O
+a	O
+minor	O
+‘	O
+open	B-protein_state
+'	O
+conformation	O
+resembling	O
+the	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+arrangement	O
+.	O
+
+Yet	O
+,	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+(	O
+SAXS	B-experimental_method
+)	O
+data	O
+indicated	O
+that	O
+both	O
+the	O
+minimal	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+longer	O
+constructs	O
+comprise	O
+a	O
+highly	B-protein_state
+diverse	I-protein_state
+continuum	I-protein_state
+of	I-protein_state
+conformations	I-protein_state
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+RNA	B-chemical
+that	O
+includes	O
+the	O
+‘	O
+closed	B-protein_state
+'	O
+and	O
+‘	O
+open	B-protein_state
+'	O
+conformations	O
+.	O
+
+To	O
+complement	O
+the	O
+static	O
+portraits	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	B-evidence
+that	O
+we	O
+had	O
+determined	O
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+,	O
+we	O
+used	O
+smFRET	B-experimental_method
+to	O
+characterize	O
+the	O
+probability	B-evidence
+distribution	I-evidence
+functions	I-evidence
+and	O
+time	O
+dependence	O
+of	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+conformational	O
+dynamics	O
+in	O
+solution	O
+.	O
+
+The	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+dynamics	O
+of	O
+U2AF65	B-protein
+were	O
+followed	O
+using	O
+FRET	B-experimental_method
+between	O
+fluorophores	B-chemical
+attached	O
+to	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+(	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+,	O
+Methods	O
+).	O
+
+The	O
+positions	O
+of	O
+single	O
+cysteine	B-residue_name
+mutations	B-experimental_method
+for	O
+fluorophore	B-chemical
+attachment	O
+(	O
+A181C	B-mutant
+in	O
+RRM1	B-structure_element
+and	O
+Q324C	B-mutant
+in	O
+RRM2	B-structure_element
+)	O
+were	O
+chosen	O
+based	O
+on	O
+inspection	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+and	O
+the	O
+‘	O
+closed	B-protein_state
+'	O
+model	O
+of	O
+apo	B-protein_state
+-	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+.	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	B-complex_assembly
+/	I-complex_assembly
+RNA	I-complex_assembly
+or	O
+inter	B-site
+-	I-site
+RRM	I-site
+interfaces	I-site
+,	O
+(	O
+ii	O
+)	O
+inter	O
+-	O
+dye	O
+distances	O
+(	O
+50	O
+Å	O
+for	O
+U2AF651	B-complex_assembly
+,	I-complex_assembly
+2L	I-complex_assembly
+–	I-complex_assembly
+Py	I-complex_assembly
+tract	I-complex_assembly
+and	O
+30	O
+Å	O
+for	O
+the	O
+closed	B-protein_state
+apo	B-protein_state
+-	O
+model	O
+)	O
+that	O
+are	O
+expected	O
+to	O
+be	O
+near	O
+the	O
+Förster	B-experimental_method
+radius	I-experimental_method
+(	I-experimental_method
+Ro	I-experimental_method
+)	I-experimental_method
+for	O
+the	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+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	B-evidence
+efficiencies	I-evidence
+that	O
+are	O
+calculated	O
+to	O
+be	O
+significantly	O
+greater	O
+for	O
+the	O
+‘	O
+closed	B-protein_state
+'	O
+apo	B-protein_state
+-	O
+model	O
+as	O
+opposed	O
+to	O
+the	O
+‘	O
+open	B-protein_state
+'	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structures	B-evidence
+(	O
+by	O
+∼	O
+30	O
+%).	O
+
+The	O
+FRET	B-evidence
+efficiencies	I-evidence
+of	O
+either	O
+of	O
+these	O
+structurally	O
+characterized	O
+conformations	O
+also	O
+are	O
+expected	O
+to	O
+be	O
+significantly	O
+greater	O
+than	O
+elongated	B-protein_state
+U2AF65	B-protein
+conformations	O
+that	O
+lack	B-protein_state
+inter	O
+-	O
+RRM	B-structure_element
+contacts	O
+.	O
+
+Double	O
+-	O
+cysteine	B-residue_name
+variant	B-protein_state
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+was	O
+modified	B-experimental_method
+with	O
+equimolar	O
+amount	O
+of	O
+Cy3	B-chemical
+and	O
+Cy5	B-chemical
+.	O
+
+Only	O
+traces	B-evidence
+that	O
+showed	O
+single	O
+photobleaching	O
+events	O
+for	O
+both	O
+donor	O
+and	O
+acceptor	O
+dyes	O
+and	O
+anti	O
+-	O
+correlated	O
+changes	O
+in	O
+acceptor	O
+and	O
+donor	O
+fluorescence	O
+were	O
+included	O
+in	O
+smFRET	B-experimental_method
+data	O
+analysis	O
+.	O
+
+We	O
+first	O
+characterized	O
+the	O
+conformational	O
+dynamics	O
+spectrum	O
+of	O
+U2AF65	B-protein
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+6c	O
+,	O
+d	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+,	O
+b	O
+).	O
+
+The	O
+double	O
+-	O
+labelled	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+protein	O
+was	O
+tethered	B-protein_state
+to	O
+a	O
+slide	O
+via	O
+biotin	B-chemical
+-	I-chemical
+NTA	I-chemical
+/	I-chemical
+Ni	I-chemical
++	I-chemical
+2	I-chemical
+resin	I-chemical
+.	O
+
+Virtually	O
+no	O
+fluorescent	O
+molecules	O
+were	O
+detected	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+biotin	B-chemical
+-	I-chemical
+NTA	I-chemical
+/	I-chemical
+Ni	I-chemical
++	I-chemical
+2	I-chemical
+,	O
+which	O
+demonstrates	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+detectable	O
+non	O
+-	O
+specific	O
+binding	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+to	O
+the	O
+slide	O
+.	O
+
+The	O
+FRET	B-evidence
+distribution	I-evidence
+histogram	I-evidence
+built	O
+from	O
+more	O
+than	O
+a	O
+thousand	O
+traces	B-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ligand	B-chemical
+showed	O
+an	O
+extremely	O
+broad	O
+distribution	O
+centred	O
+at	O
+a	O
+FRET	B-evidence
+efficiency	I-evidence
+of	O
+∼	O
+0	O
+.	O
+4	O
+(	O
+Fig	O
+.	O
+6d	O
+).	O
+
+Approximately	O
+40	O
+%	O
+of	O
+the	O
+smFRET	B-experimental_method
+traces	B-evidence
+showed	O
+apparent	O
+transitions	O
+between	O
+multiple	O
+FRET	B-evidence
+values	I-evidence
+(	O
+for	O
+example	O
+,	O
+Fig	O
+.	O
+6c	O
+).	O
+
+Despite	O
+the	O
+large	O
+width	O
+of	O
+the	O
+FRET	B-evidence
+-	I-evidence
+distribution	I-evidence
+histogram	I-evidence
+,	O
+the	O
+majority	O
+(	O
+80	O
+%)	O
+of	O
+traces	B-evidence
+that	O
+showed	O
+fluctuations	O
+sampled	O
+only	O
+two	O
+distinct	O
+FRET	B-evidence
+states	I-evidence
+(	O
+for	O
+example	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+
+Approximately	O
+70	O
+%	O
+of	O
+observed	O
+fluctuations	O
+were	O
+interchanges	O
+between	O
+the	O
+∼	O
+0	O
+.	O
+65	O
+and	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+values	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7b	O
+).	O
+
+We	O
+cannot	O
+exclude	O
+a	O
+possibility	O
+that	O
+tethering	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+to	O
+the	O
+microscope	O
+slide	O
+introduces	O
+structural	O
+heterogeneity	O
+into	O
+the	O
+protein	O
+and	O
+,	O
+thus	O
+,	O
+contributes	O
+to	O
+the	O
+breadth	O
+of	O
+the	O
+FRET	B-evidence
+distribution	I-evidence
+histogram	I-evidence
+.	O
+
+However	O
+,	O
+the	O
+presence	O
+of	O
+repetitive	O
+fluctuations	O
+between	O
+particular	O
+FRET	B-evidence
+values	I-evidence
+supports	O
+the	O
+hypothesis	O
+that	O
+RNA	B-protein_state
+-	I-protein_state
+free	I-protein_state
+U2AF65	B-protein
+samples	O
+several	O
+distinct	O
+conformations	O
+.	O
+
+This	O
+result	O
+is	O
+consistent	O
+with	O
+the	O
+broad	O
+ensembles	O
+of	O
+extended	B-protein_state
+solution	O
+conformations	O
+that	O
+best	O
+fit	O
+the	O
+SAXS	B-experimental_method
+data	O
+collected	O
+for	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+as	O
+well	O
+as	O
+for	O
+a	O
+longer	O
+construct	O
+(	O
+residues	O
+136	B-residue_range
+–	I-residue_range
+347	I-residue_range
+).	O
+
+We	O
+conclude	O
+that	O
+weak	O
+contacts	O
+between	O
+the	O
+U2AF65	B-protein
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+permit	O
+dissociation	O
+of	O
+these	O
+RRMs	B-structure_element
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+RNA	B-chemical
+.	O
+
+U2AF65	B-protein
+conformational	O
+selection	O
+and	O
+induced	O
+fit	O
+by	O
+bound	B-protein_state
+RNA	B-chemical
+
+We	O
+next	O
+used	O
+smFRET	B-experimental_method
+to	O
+probe	O
+the	O
+conformational	O
+selection	O
+of	O
+distinct	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+arrangements	O
+following	O
+association	O
+of	O
+U2AF65	B-protein
+with	O
+the	O
+AdML	B-gene
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+prototype	O
+.	O
+
+Addition	O
+of	O
+the	O
+AdML	B-gene
+RNA	B-chemical
+to	O
+tethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+selectively	O
+increases	O
+a	O
+fraction	O
+of	O
+molecules	O
+showing	O
+an	O
+∼	O
+0	O
+.	O
+45	O
+apparent	O
+FRET	B-evidence
+efficiency	I-evidence
+,	O
+suggesting	O
+that	O
+RNA	O
+binding	O
+stabilizes	O
+a	O
+single	O
+conformation	O
+,	O
+which	O
+corresponds	O
+to	O
+the	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+state	I-evidence
+(	O
+Fig	O
+.	O
+6e	O
+,	O
+f	O
+).	O
+
+To	O
+assess	O
+the	O
+possible	O
+contributions	O
+of	O
+RNA	B-protein_state
+-	I-protein_state
+free	I-protein_state
+conformations	O
+of	O
+U2AF65	B-protein
+and	O
+/	O
+or	O
+structural	O
+heterogeneity	O
+introduced	O
+by	O
+tethering	B-experimental_method
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+to	O
+the	O
+slide	O
+to	O
+the	O
+observed	O
+distribution	B-evidence
+of	I-evidence
+FRET	I-evidence
+values	I-evidence
+,	O
+we	O
+reversed	B-experimental_method
+the	I-experimental_method
+immobilization	I-experimental_method
+scheme	I-experimental_method
+.	O
+
+We	O
+tethered	B-protein_state
+the	O
+AdML	B-gene
+RNA	B-chemical
+to	O
+the	O
+slide	O
+via	O
+a	O
+biotinylated	B-chemical
+oligonucleotide	I-chemical
+DNA	I-chemical
+handle	O
+and	O
+added	B-experimental_method
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+biotin	B-chemical
+-	I-chemical
+NTA	I-chemical
+resin	I-chemical
+(	O
+Fig	O
+.	O
+6g	O
+,	O
+h	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+
+A	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+was	O
+again	O
+predominant	O
+,	O
+indicating	O
+a	O
+similar	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+conformation	O
+and	O
+structural	O
+dynamics	O
+for	O
+the	O
+untethered	B-protein_state
+and	O
+tethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+).	O
+
+We	O
+examined	O
+the	O
+effect	O
+on	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+conformations	O
+of	O
+purine	B-experimental_method
+interruptions	I-experimental_method
+that	O
+often	O
+occur	O
+in	O
+relatively	O
+degenerate	O
+human	B-species
+Py	B-chemical
+tracts	I-chemical
+.	O
+
+We	O
+introduced	B-experimental_method
+an	O
+rArA	B-chemical
+purine	B-chemical
+dinucleotide	I-chemical
+within	O
+a	O
+variant	O
+of	O
+the	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+(	O
+detailed	O
+in	O
+Methods	O
+).	O
+
+Insertion	B-experimental_method
+of	O
+adenine	B-chemical
+nucleotides	I-chemical
+decreased	O
+binding	B-evidence
+affinity	I-evidence
+of	O
+U2AF65	B-protein
+to	O
+RNA	B-chemical
+by	O
+approximately	O
+five	O
+-	O
+fold	O
+.	O
+
+Nevertheless	O
+,	O
+in	O
+the	O
+presence	O
+of	O
+saturating	O
+concentrations	O
+of	O
+rArA	B-chemical
+-	O
+interrupted	O
+RNA	B-chemical
+slide	B-protein_state
+-	I-protein_state
+tethered	I-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+showed	O
+a	O
+prevalent	O
+∼	O
+0	O
+.	O
+45	O
+apparent	O
+FRET	B-evidence
+value	I-evidence
+(	O
+Fig	O
+.	O
+6i	O
+,	O
+j	O
+),	O
+which	O
+was	O
+also	O
+predominant	O
+in	O
+the	O
+presence	O
+of	O
+continuous	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+Therefore	O
+,	O
+RRM1	B-structure_element
+-	O
+to	O
+-	O
+RRM2	B-structure_element
+distance	O
+remains	O
+similar	O
+regardless	O
+of	O
+whether	O
+U2AF65	B-protein
+is	O
+bound	B-protein_state
+to	I-protein_state
+interrupted	O
+or	O
+continuous	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+The	O
+inter	B-evidence
+-	I-evidence
+fluorophore	I-evidence
+distances	I-evidence
+derived	O
+from	O
+the	O
+observed	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+state	I-evidence
+agree	O
+with	O
+the	O
+distances	O
+between	O
+the	O
+α	O
+-	O
+carbon	O
+atoms	O
+of	O
+the	O
+respective	O
+residues	O
+in	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+bound	B-protein_state
+to	I-protein_state
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+oligonucleotides	I-chemical
+.	O
+
+It	O
+should	O
+be	O
+noted	O
+that	O
+inferring	O
+distances	O
+from	O
+FRET	B-evidence
+values	I-evidence
+is	O
+prone	O
+to	O
+significant	O
+error	O
+because	O
+of	O
+uncertainties	O
+in	O
+the	O
+determination	O
+of	O
+fluorophore	O
+orientation	O
+factor	O
+κ2	O
+and	O
+Förster	O
+radius	O
+R0	O
+,	O
+the	O
+parameters	O
+used	O
+in	O
+distance	O
+calculations	O
+.	O
+
+Nevertheless	O
+,	O
+the	O
+predominant	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+state	I-evidence
+in	O
+the	O
+presence	O
+of	O
+RNA	B-chemical
+agrees	O
+with	O
+the	O
+Py	B-protein_state
+-	I-protein_state
+tract	I-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystal	B-evidence
+structure	I-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+.	O
+
+Importantly	O
+,	O
+the	O
+majority	O
+of	O
+traces	B-evidence
+(∼	O
+70	O
+%)	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+slide	O
+-	O
+tethered	O
+RNA	B-chemical
+lacked	O
+FRET	O
+fluctuations	O
+and	O
+predominately	O
+exhibited	O
+a	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+(	O
+for	O
+example	O
+,	O
+Fig	O
+.	O
+6g	O
+).	O
+
+The	O
+remaining	O
+∼	O
+30	O
+%	O
+of	O
+traces	B-evidence
+for	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+slide	O
+-	O
+tethered	O
+RNA	B-chemical
+showed	O
+fluctuations	O
+between	O
+distinct	O
+FRET	B-evidence
+values	I-evidence
+.	O
+
+The	O
+majority	O
+of	O
+traces	B-evidence
+that	O
+show	O
+fluctuations	O
+began	O
+at	O
+high	O
+(	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+)	O
+FRET	B-evidence
+value	I-evidence
+and	O
+transitioned	O
+to	O
+a	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+
+Hidden	B-experimental_method
+Markov	I-experimental_method
+modelling	I-experimental_method
+analysis	I-experimental_method
+of	O
+smFRET	B-experimental_method
+traces	B-evidence
+suggests	O
+that	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+can	O
+sample	O
+at	O
+least	O
+two	O
+other	O
+conformations	O
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+7	O
+–	O
+0	O
+.	O
+8	O
+and	O
+∼	O
+0	O
+.	O
+3	O
+FRET	B-evidence
+values	I-evidence
+in	O
+addition	O
+to	O
+the	O
+predominant	O
+conformation	O
+corresponding	O
+to	O
+the	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+state	I-evidence
+.	O
+
+Although	O
+a	O
+compact	O
+conformation	O
+(	O
+or	O
+multiple	O
+conformations	O
+)	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+7	O
+–	O
+0	O
+.	O
+8	O
+FRET	B-evidence
+values	I-evidence
+can	O
+bind	O
+RNA	B-chemical
+,	O
+on	O
+RNA	B-chemical
+binding	O
+,	O
+these	O
+compact	B-protein_state
+conformations	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+transition	O
+into	O
+a	O
+more	O
+stable	O
+structural	O
+state	O
+that	O
+corresponds	O
+to	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+and	O
+is	O
+likely	O
+similar	O
+to	O
+the	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+-	O
+arrangement	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Thus	O
+,	O
+the	O
+sequence	O
+of	O
+structural	O
+rearrangements	O
+of	O
+U2AF65	B-protein
+observed	O
+in	O
+smFRET	B-experimental_method
+traces	B-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+)	O
+suggests	O
+that	O
+a	O
+‘	O
+conformational	O
+selection	O
+'	O
+mechanism	O
+of	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+(	O
+that	O
+is	O
+,	O
+RNA	O
+ligand	O
+stabilization	O
+of	O
+a	O
+pre	B-protein_state
+-	I-protein_state
+configured	I-protein_state
+U2AF65	B-protein
+conformation	O
+)	O
+is	O
+complemented	O
+by	O
+‘	O
+induced	O
+fit	O
+'	O
+(	O
+that	O
+is	O
+,	O
+RNA	O
+-	O
+induced	O
+rearrangement	O
+of	O
+the	O
+U2AF65	B-protein
+RRMs	B-structure_element
+to	O
+achieve	O
+the	O
+final	O
+‘	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+'	O
+conformation	O
+),	O
+as	O
+discussed	O
+below	O
+.	O
+
+The	O
+U2AF65	B-protein
+structures	B-evidence
+and	O
+analyses	B-evidence
+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	B-site
+′	I-site
+splice	I-site
+site	I-site
+.	O
+
+Several	O
+observations	O
+indicate	O
+that	O
+the	O
+numerous	O
+intramolecular	O
+contacts	O
+,	O
+here	O
+revealed	O
+among	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+and	O
+RRM1	B-structure_element
+,	O
+RRM2	B-structure_element
+,	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	B-structure_element
+extension	I-structure_element
+,	O
+synergistically	O
+coordinate	O
+U2AF65	B-protein
+–	O
+Py	O
+-	O
+tract	O
+recognition	O
+.	O
+
+Truncation	B-experimental_method
+of	O
+U2AF65	B-protein
+to	O
+the	O
+core	B-protein_state
+RRM1	B-structure_element
+–	I-structure_element
+RRM2	I-structure_element
+region	I-structure_element
+reduces	O
+its	O
+RNA	B-evidence
+affinity	I-evidence
+by	O
+100	O
+-	O
+fold	O
+.	O
+
+Likewise	O
+,	O
+deletion	B-experimental_method
+of	O
+20	B-residue_range
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+residues	I-structure_element
+significantly	O
+reduces	O
+U2AF65	B-protein
+–	O
+RNA	B-chemical
+binding	O
+only	O
+when	O
+introduced	O
+in	O
+the	O
+context	O
+of	O
+the	O
+longer	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+construct	O
+comprising	O
+the	O
+RRM	B-structure_element
+extensions	I-structure_element
+,	O
+which	O
+in	O
+turn	O
+position	O
+the	O
+linker	B-structure_element
+for	O
+RNA	B-chemical
+interactions	O
+.	O
+
+Notably	O
+,	O
+a	O
+triple	B-protein_state
+mutation	I-protein_state
+of	O
+three	O
+residues	O
+(	O
+V254P	B-mutant
+,	O
+Q147A	B-mutant
+and	O
+R227A	B-mutant
+)	O
+in	O
+the	O
+respective	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+,	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extensions	I-structure_element
+non	O
+-	O
+additively	O
+reduce	O
+RNA	B-evidence
+binding	I-evidence
+by	O
+150	O
+-	O
+fold	O
+.	O
+
+Altogether	O
+,	O
+these	O
+data	O
+indicate	O
+that	O
+interactions	O
+among	O
+the	O
+U2AF65	B-protein
+RRM1	B-structure_element
+/	O
+RRM2	B-structure_element
+,	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+,	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extensions	I-structure_element
+are	O
+mutually	O
+inter	O
+-	O
+dependent	O
+for	O
+cognate	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+.	O
+
+The	O
+implications	O
+of	O
+this	O
+finding	O
+for	O
+U2AF65	B-protein
+conservation	O
+and	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+are	O
+detailed	O
+in	O
+the	O
+Supplementary	O
+Discussion	O
+.	O
+
+Recently	O
+,	O
+high	B-experimental_method
+-	I-experimental_method
+throughput	I-experimental_method
+sequencing	I-experimental_method
+studies	I-experimental_method
+have	O
+shown	O
+that	O
+somatic	O
+mutations	O
+in	O
+pre	B-protein_type
+-	I-protein_type
+mRNA	I-protein_type
+splicing	I-protein_type
+factors	I-protein_type
+occur	O
+in	O
+the	O
+majority	O
+of	O
+patients	O
+with	O
+myelodysplastic	O
+syndrome	O
+(	O
+MDS	O
+).	O
+
+MDS	O
+-	O
+relevant	O
+mutations	O
+are	O
+common	O
+in	O
+the	O
+small	B-protein_state
+U2AF	B-protein_type
+subunit	I-protein_type
+(	O
+U2AF35	B-protein
+,	O
+or	O
+U2AF1	B-protein
+),	O
+yet	O
+such	O
+mutations	O
+are	O
+rare	O
+in	O
+the	O
+large	B-protein_state
+U2AF65	B-protein
+subunit	O
+(	O
+also	O
+called	O
+U2AF2	B-protein
+)—	O
+possibly	O
+due	O
+to	O
+the	O
+selective	O
+versus	O
+nearly	O
+universal	O
+requirements	O
+of	O
+these	O
+factors	O
+for	O
+splicing	O
+.	O
+
+A	O
+confirmed	O
+somatic	O
+mutation	O
+of	O
+U2AF65	B-protein
+in	O
+patients	O
+with	O
+MDS	O
+,	O
+L187V	B-mutant
+,	O
+is	O
+located	O
+on	O
+a	O
+solvent	B-site
+-	I-site
+exposed	I-site
+surface	I-site
+of	O
+RRM1	B-structure_element
+that	O
+is	O
+distinct	O
+from	O
+the	O
+RNA	B-site
+interface	I-site
+(	O
+Fig	O
+.	O
+7a	O
+).	O
+
+This	O
+L187	B-residue_name_number
+surface	O
+is	O
+oriented	O
+towards	O
+the	O
+N	O
+terminus	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+construct	O
+,	O
+where	O
+it	O
+is	O
+expected	O
+to	O
+abut	O
+the	O
+U2AF35	B-site
+-	I-site
+binding	I-site
+site	I-site
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF	B-protein
+heterodimer	B-oligomeric_state
+.	O
+
+Likewise	O
+,	O
+an	O
+unconfirmed	O
+M144I	B-mutant
+mutation	O
+reported	O
+by	O
+the	O
+same	O
+group	O
+corresponds	O
+to	O
+the	O
+N	O
+-	O
+terminal	O
+residue	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+which	O
+is	O
+separated	O
+by	O
+only	O
+∼	O
+20	O
+residues	O
+from	O
+the	O
+U2AF35	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+
+As	O
+such	O
+,	O
+we	O
+suggest	O
+that	O
+the	O
+MDS	O
+-	O
+relevant	O
+U2AF65	B-protein
+mutations	O
+contribute	O
+to	O
+MDS	O
+progression	O
+indirectly	O
+,	O
+by	O
+destabilizing	O
+a	O
+relevant	O
+conformation	O
+of	O
+the	O
+conjoined	O
+U2AF35	B-protein
+subunit	O
+rather	O
+than	O
+affecting	O
+U2AF65	B-protein
+functions	O
+in	O
+RNA	B-chemical
+binding	O
+or	O
+spliceosome	B-complex_assembly
+recruitment	O
+per	O
+se	O
+.	O
+
+Our	O
+smFRET	B-experimental_method
+results	O
+agree	O
+with	O
+prior	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+evidence	O
+for	O
+multi	O
+-	O
+domain	O
+conformational	O
+selection	O
+as	O
+one	O
+mechanistic	O
+basis	O
+for	O
+U2AF65	B-protein
+–	O
+RNA	B-chemical
+association	O
+(	O
+Fig	O
+.	O
+7b	O
+).	O
+
+An	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+is	O
+likely	O
+to	O
+correspond	O
+to	O
+the	O
+U2AF65	B-protein
+conformation	O
+visualized	O
+in	O
+our	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+crystal	B-evidence
+structures	I-evidence
+,	O
+in	O
+which	O
+the	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+bind	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+to	O
+the	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+oligonucleotide	I-chemical
+.	O
+
+The	O
+lesser	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+and	O
+0	O
+.	O
+2	O
+–	O
+0	O
+.	O
+3	O
+FRET	B-evidence
+values	I-evidence
+in	O
+the	O
+untethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+experiment	O
+could	O
+correspond	O
+to	O
+respective	O
+variants	O
+of	O
+the	O
+‘	O
+closed	B-protein_state
+',	O
+back	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+back	I-protein_state
+U2AF65	B-protein
+conformations	O
+characterized	O
+by	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+data	O
+,	O
+or	O
+to	O
+extended	B-protein_state
+U2AF65	B-protein
+conformations	O
+,	O
+in	O
+which	O
+the	O
+intramolecular	O
+RRM1	B-structure_element
+/	O
+RRM2	B-structure_element
+interactions	O
+have	O
+dissociated	O
+the	O
+protein	B-protein
+is	O
+bound	B-protein_state
+to	I-protein_state
+RNA	B-chemical
+via	O
+single	B-protein_state
+RRMs	B-structure_element
+.	O
+
+An	O
+increased	O
+prevalence	O
+of	O
+the	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+following	O
+U2AF65	B-protein
+–	O
+RNA	B-chemical
+binding	O
+,	O
+coupled	O
+with	O
+the	O
+apparent	O
+absence	B-protein_state
+of	I-protein_state
+transitions	O
+in	O
+many	O
+∼	O
+0	O
+.	O
+45	O
+-	O
+value	O
+single	O
+molecule	O
+traces	B-evidence
+(	O
+for	O
+example	O
+,	O
+Fig	O
+.	O
+6e	O
+),	O
+suggests	O
+a	O
+population	O
+shift	O
+in	O
+which	O
+RNA	B-chemical
+binds	O
+to	O
+(	O
+and	O
+draws	O
+the	O
+equilibrium	O
+towards	O
+)	O
+a	O
+pre	B-protein_state
+-	I-protein_state
+configured	I-protein_state
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+proximity	O
+that	O
+most	O
+often	O
+corresponds	O
+to	O
+the	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+.	O
+
+Notably	O
+,	O
+our	O
+smFRET	B-experimental_method
+results	O
+reveal	O
+that	O
+U2AF65	B-protein
+–	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+can	O
+be	O
+characterized	O
+by	O
+an	O
+‘	O
+extended	O
+conformational	O
+selection	O
+'	O
+model	O
+(	O
+Fig	O
+.	O
+7b	O
+).	O
+
+Examples	O
+of	O
+‘	O
+extended	B-protein_state
+conformational	O
+selection	O
+'	O
+during	O
+ligand	O
+binding	O
+have	O
+been	O
+characterized	O
+for	O
+a	O
+growing	O
+number	O
+of	O
+macromolecules	O
+(	O
+for	O
+example	O
+,	O
+adenylate	B-protein_type
+kinase	I-protein_type
+,	O
+LAO	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+,	O
+poly	B-protein_type
+-	I-protein_type
+ubiquitin	I-protein_type
+,	O
+maltose	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+and	O
+the	O
+preQ1	B-protein_type
+riboswitch	I-protein_type
+,	O
+among	O
+others	O
+).	O
+
+Here	O
+,	O
+the	O
+majority	O
+of	O
+changes	O
+in	O
+smFRET	B-experimental_method
+traces	B-evidence
+for	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+bound	B-protein_state
+to	I-protein_state
+slide	O
+-	O
+tethered	O
+RNA	B-chemical
+began	O
+at	O
+high	O
+(	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+)	O
+FRET	B-evidence
+value	I-evidence
+and	O
+transition	O
+to	O
+the	O
+predominant	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+
+These	O
+transitions	O
+could	O
+correspond	O
+to	O
+rearrangement	O
+from	O
+the	O
+‘	O
+closed	B-protein_state
+'	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+-	O
+based	O
+U2AF65	B-protein
+conformation	O
+in	O
+which	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+surface	I-site
+of	O
+only	O
+a	O
+single	B-protein_state
+RRM	B-structure_element
+is	O
+exposed	O
+and	O
+available	O
+for	O
+RNA	O
+binding	O
+,	O
+to	O
+the	O
+structural	O
+state	O
+seen	O
+in	O
+the	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+,	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+As	O
+such	O
+,	O
+the	O
+smFRET	B-experimental_method
+approach	O
+reconciles	O
+prior	O
+inconsistencies	O
+between	O
+two	O
+major	O
+conformations	O
+that	O
+were	O
+detected	O
+by	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+experiments	O
+and	O
+a	O
+broad	O
+ensemble	O
+of	O
+diverse	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+arrangements	O
+that	O
+fit	O
+the	O
+SAXS	B-experimental_method
+data	O
+for	O
+the	O
+apo	B-protein_state
+-	O
+protein	B-protein
+.	O
+
+Similar	O
+interdisciplinary	O
+structural	O
+approaches	O
+are	O
+likely	O
+to	O
+illuminate	O
+whether	O
+similar	O
+mechanistic	O
+bases	O
+for	O
+RNA	O
+binding	O
+are	O
+widespread	O
+among	O
+other	O
+members	O
+of	O
+the	O
+vast	O
+multi	O
+-	O
+RRM	B-structure_element
+family	O
+.	O
+
+The	O
+finding	O
+that	O
+U2AF65	B-protein
+recognizes	O
+a	O
+nine	O
+base	O
+pair	O
+Py	B-chemical
+tract	I-chemical
+contributes	O
+to	O
+an	O
+elusive	O
+‘	O
+code	O
+'	O
+for	O
+predicting	O
+splicing	O
+patterns	O
+from	O
+primary	O
+sequences	O
+in	O
+the	O
+post	O
+-	O
+genomic	O
+era	O
+(	O
+reviewed	O
+in	O
+ref	O
+.).	O
+
+Based	O
+on	O
+(	O
+i	O
+)	O
+similar	O
+RNA	B-evidence
+affinities	I-evidence
+of	O
+U2AF65	B-protein
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+(	O
+ii	O
+)	O
+indistinguishable	O
+conformations	O
+among	O
+four	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+in	O
+two	O
+different	O
+crystal	O
+packing	O
+arrangements	O
+and	O
+(	O
+iii	O
+)	O
+penalties	B-evidence
+of	O
+structure	B-experimental_method
+-	I-experimental_method
+guided	I-experimental_method
+mutations	I-experimental_method
+in	O
+RNA	B-experimental_method
+binding	I-experimental_method
+and	I-experimental_method
+splicing	I-experimental_method
+assays	I-experimental_method
+,	O
+we	O
+suggest	O
+that	O
+the	O
+extended	B-protein_state
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+regions	I-structure_element
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+underlie	O
+cognate	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+by	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF65	B-protein
+protein	O
+.	O
+
+Further	O
+research	O
+will	O
+be	O
+needed	O
+to	O
+understand	O
+the	O
+roles	O
+of	O
+SF1	B-protein
+and	O
+U2AF35	B-protein
+subunits	O
+in	O
+the	O
+conformational	O
+equilibria	O
+underlying	O
+U2AF65	B-protein
+association	O
+with	O
+Py	B-chemical
+tracts	I-chemical
+.	O
+
+Moreover	O
+,	O
+structural	O
+differences	O
+among	O
+U2AF65	B-protein
+homologues	O
+and	O
+paralogues	O
+may	O
+regulate	O
+splice	B-site
+site	I-site
+selection	O
+.	O
+
+Ultimately	O
+,	O
+these	O
+guidelines	O
+will	O
+assist	O
+the	O
+identification	O
+of	O
+3	B-site
+′	I-site
+splice	I-site
+sites	I-site
+and	O
+the	O
+relationship	O
+of	O
+disease	O
+-	O
+causing	O
+mutations	O
+to	O
+penalties	O
+for	O
+U2AF65	B-protein
+association	O
+.	O
+
+The	O
+intact	B-protein_state
+U2AF65	B-protein
+RRM1	B-structure_element
+/	O
+RRM2	B-structure_element
+-	O
+containing	O
+domain	O
+and	O
+flanking	O
+residues	O
+are	O
+required	O
+for	O
+binding	O
+contiguous	B-structure_element
+Py	B-chemical
+tracts	I-chemical
+.	O
+
+(	O
+a	O
+)	O
+Domain	O
+organization	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+(	O
+fl	B-protein_state
+)	O
+U2AF65	B-protein
+and	O
+constructs	O
+used	O
+for	O
+RNA	B-chemical
+binding	O
+and	O
+structural	O
+experiments	O
+.	O
+
+An	O
+internal	O
+deletion	O
+(	O
+d	B-mutant
+,	O
+Δ	B-mutant
+)	O
+of	O
+residues	O
+238	B-residue_range
+–	I-residue_range
+257	I-residue_range
+removes	O
+a	O
+portion	O
+of	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+from	O
+the	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+and	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+constructs	O
+.	O
+
+(	O
+b	O
+)	O
+Comparison	O
+of	O
+the	O
+apparent	O
+equilibrium	B-evidence
+affinities	I-evidence
+of	O
+various	O
+U2AF65	B-protein
+constructs	O
+for	O
+binding	O
+the	O
+prototypical	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+(	O
+5	B-chemical
+′-	I-chemical
+CCCUUUUUUUUCC	I-chemical
+-	I-chemical
+3	I-chemical
+′).	I-chemical
+
+The	O
+flU2AF65	B-protein
+protein	O
+includes	O
+a	O
+heterodimerization	B-structure_element
+domain	I-structure_element
+of	O
+the	O
+U2AF35	B-protein
+subunit	O
+to	O
+promote	O
+solubility	O
+and	O
+folding	O
+.	O
+
+The	O
+apparent	O
+equilibrium	B-evidence
+dissociation	I-evidence
+constants	I-evidence
+(	O
+KD	B-evidence
+)	O
+for	O
+binding	O
+the	O
+AdML	B-gene
+13mer	O
+are	O
+as	O
+follows	O
+:	O
+flU2AF65	B-protein
+,	O
+30	O
+±	O
+3	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+35	O
+±	O
+6	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+,	O
+3	O
+,	O
+600	O
+±	O
+300	O
+nM	O
+.	O
+(	O
+c	O
+)	O
+Comparison	O
+of	O
+the	O
+RNA	B-evidence
+sequence	I-evidence
+specificities	I-evidence
+of	O
+flU2AF65	B-protein
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+constructs	O
+binding	O
+C	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+Py	B-chemical
+tracts	I-chemical
+with	O
+4U	O
+'	O
+s	O
+embedded	O
+in	O
+either	O
+the	O
+5	O
+′-	O
+(	O
+light	O
+grey	O
+fill	O
+)	O
+or	O
+3	O
+′-	O
+(	O
+dark	O
+grey	O
+fill	O
+)	O
+regions	O
+.	O
+
+The	O
+KD	B-evidence
+'	O
+s	O
+for	O
+binding	O
+5	B-chemical
+′-	I-chemical
+CCUUUUCCCCCCC	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+are	O
+:	O
+flU2AF65	B-protein
+,	O
+41	O
+±	O
+2	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+31	O
+±	O
+3	O
+nM	O
+.	O
+The	O
+KD	B-evidence
+'	O
+s	O
+for	O
+binding	O
+5	B-chemical
+′-	I-chemical
+CCCCCCCUUUUCC	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+are	O
+:	O
+flU2AF65	B-protein
+,	O
+414	O
+±	O
+12	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+417	O
+±	O
+10	O
+nM	O
+.	O
+Bar	O
+graphs	O
+are	O
+hatched	O
+to	O
+match	O
+the	O
+constructs	O
+shown	O
+in	O
+a	O
+.	O
+The	O
+average	B-evidence
+apparent	I-evidence
+equilibrium	I-evidence
+affinity	I-evidence
+(	O
+KA	B-evidence
+)	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
+
+The	O
+purified	O
+protein	O
+and	O
+average	B-evidence
+fitted	I-evidence
+fluorescence	I-evidence
+anisotropy	I-evidence
+RNA	I-evidence
+-	I-evidence
+binding	I-evidence
+curves	I-evidence
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+.	O
+
+RRM	B-structure_element
+,	O
+RNA	B-structure_element
+recognition	I-structure_element
+motif	I-structure_element
+;	O
+RS	B-structure_element
+,	O
+arginine	B-structure_element
+-	I-structure_element
+serine	I-structure_element
+rich	I-structure_element
+;	O
+UHM	B-structure_element
+,	O
+U2AF	B-structure_element
+homology	I-structure_element
+motif	I-structure_element
+;	O
+ULM	B-structure_element
+,	O
+U2AF	B-structure_element
+ligand	I-structure_element
+motif	I-structure_element
+.	O
+
+Structures	B-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+recognizing	O
+a	O
+contiguous	B-structure_element
+Py	B-chemical
+tract	I-chemical
+.	O
+
+(	O
+a	O
+)	O
+Alignment	B-experimental_method
+of	O
+oligonucleotide	B-chemical
+sequences	O
+that	O
+were	O
+co	B-experimental_method
+-	I-experimental_method
+crystallized	I-experimental_method
+in	O
+the	O
+indicated	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+.	O
+
+The	O
+regions	O
+of	O
+RRM1	B-structure_element
+,	O
+RRM2	B-structure_element
+and	O
+linker	B-structure_element
+contacts	O
+are	O
+indicated	O
+above	O
+by	O
+respective	O
+black	O
+and	O
+blue	O
+arrows	O
+from	O
+N	O
+-	O
+to	O
+C	O
+-	O
+terminus	O
+.	O
+
+For	O
+clarity	O
+,	O
+we	O
+consistently	O
+number	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+nucleotide	B-site
+-	I-site
+binding	I-site
+sites	I-site
+from	O
+one	O
+to	O
+nine	O
+,	O
+although	O
+in	O
+some	O
+cases	O
+the	O
+co	B-experimental_method
+-	I-experimental_method
+crystallized	I-experimental_method
+oligonucleotide	B-chemical
+comprises	O
+eight	O
+nucleotides	B-chemical
+and	O
+as	O
+such	O
+leaves	O
+the	O
+first	B-site
+binding	I-site
+site	I-site
+empty	O
+.	O
+
+The	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+nucleotide	B-site
+-	I-site
+binding	I-site
+sites	I-site
+are	O
+given	O
+in	O
+parentheses	O
+(	O
+site	O
+4	O
+'	O
+interacts	O
+with	O
+dU2AF65	B-mutant
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+by	O
+crystallographic	O
+symmetry	O
+).	O
+
+(	O
+b	O
+)	O
+Stereo	O
+views	O
+of	O
+a	O
+‘	O
+kicked	O
+'	O
+2	B-evidence
+|	I-evidence
+Fo	I-evidence
+|−|	I-evidence
+Fc	I-evidence
+|	I-evidence
+electron	I-evidence
+density	I-evidence
+map	I-evidence
+contoured	O
+at	O
+1σ	O
+for	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+,	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+residues	O
+(	O
+blue	O
+)	O
+or	O
+bound	O
+oligonucleotide	B-chemical
+of	O
+a	O
+representative	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	O
+(	O
+structure	O
+iv	O
+,	O
+bound	B-protein_state
+to	I-protein_state
+5	O
+′-(	O
+P	O
+)	O
+rUrUrUdUrUrU	O
+(	O
+BrdU	O
+)	O
+dUrC	O
+)	O
+(	O
+magenta	O
+).	O
+
+Crystallographic	O
+statistics	O
+are	O
+given	O
+in	O
+Table	O
+1	O
+and	O
+the	O
+overall	O
+conformations	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+/	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	B-evidence
+are	O
+compared	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+.	O
+
+BrdU	B-chemical
+,	O
+5	B-chemical
+-	I-chemical
+bromo	I-chemical
+-	I-chemical
+deoxy	I-chemical
+-	I-chemical
+uridine	I-chemical
+;	O
+d	B-chemical
+,	O
+deoxy	B-chemical
+-	I-chemical
+ribose	I-chemical
+;	O
+P	B-chemical
+-,	I-chemical
+5	B-chemical
+′-	I-chemical
+phosphorylation	I-chemical
+;	O
+r	B-chemical
+,	O
+ribose	B-chemical
+.	O
+
+Representative	O
+views	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+interactions	O
+with	O
+each	O
+new	O
+nucleotide	B-chemical
+of	O
+the	O
+bound	B-protein_state
+Py	B-chemical
+tract	I-chemical
+.	O
+
+New	O
+residues	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+are	O
+coloured	O
+a	O
+darker	O
+shade	O
+of	O
+blue	O
+,	O
+apart	O
+from	O
+residues	O
+that	O
+were	O
+tested	O
+by	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+,	O
+which	O
+are	O
+coloured	O
+yellow	O
+.	O
+
+The	O
+nucleotide	B-site
+-	I-site
+binding	I-site
+sites	I-site
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structure	B-evidence
+are	O
+compared	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+–	O
+h	O
+.	O
+The	O
+first	B-site
+and	I-site
+seventh	I-site
+U2AF651	I-site
+,	I-site
+2L	I-site
+-	I-site
+binding	I-site
+sites	I-site
+are	O
+unchanged	O
+from	O
+the	O
+prior	O
+dU2AF651	B-complex_assembly
+,	I-complex_assembly
+2	I-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+structure	B-evidence
+and	O
+are	O
+portrayed	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+,	O
+f	O
+.	O
+The	O
+four	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+are	O
+similar	O
+with	O
+the	O
+exception	O
+of	O
+pH	O
+-	O
+dependent	O
+variations	O
+at	O
+the	O
+ninth	B-site
+site	I-site
+that	O
+are	O
+detailed	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+3i	O
+,	O
+j	O
+.	O
+The	O
+representative	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	B-evidence
+shown	O
+has	O
+the	O
+highest	O
+resolution	O
+and	O
+/	O
+or	O
+ribose	B-chemical
+nucleotide	I-chemical
+at	O
+the	O
+given	O
+site	O
+:	O
+(	O
+a	O
+)	O
+rU2	B-residue_name_number
+of	O
+structure	O
+iv	O
+;	O
+(	O
+b	O
+)	O
+rU3	B-residue_name_number
+of	O
+structure	O
+iii	O
+;	O
+(	O
+c	O
+)	O
+rU4	B-residue_name_number
+of	O
+structure	O
+i	O
+;	O
+(	O
+d	O
+)	O
+rU5	B-residue_name_number
+of	O
+structure	O
+iii	O
+;	O
+(	O
+e	O
+)	O
+rU6	B-residue_name_number
+of	O
+structure	O
+ii	O
+;	O
+(	O
+f	O
+)	O
+dU8	B-residue_name_number
+of	O
+structure	O
+iii	O
+;	O
+(	O
+g	O
+)	O
+dU9	B-residue_name_number
+of	O
+structure	O
+iii	O
+;	O
+(	O
+h	O
+)	O
+rC9	B-residue_name_number
+of	O
+structure	O
+iv	O
+.	O
+
+(	O
+i	O
+)	O
+Bar	O
+graph	O
+of	O
+apparent	O
+equilibrium	B-evidence
+affinities	I-evidence
+(	O
+KA	B-evidence
+)	O
+of	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+blue	O
+)	O
+and	O
+the	O
+indicated	O
+mutant	B-protein_state
+(	O
+yellow	O
+)	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+proteins	O
+binding	O
+the	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+(	O
+5	B-chemical
+′-	I-chemical
+CCCUUUUUUUUCC	I-chemical
+-	I-chemical
+3	I-chemical
+′).	I-chemical
+
+The	O
+apparent	O
+equilibrium	B-evidence
+dissociation	I-evidence
+constants	I-evidence
+(	O
+KD	B-evidence
+)	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+mutant	B-protein_state
+proteins	O
+are	O
+:	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+),	O
+35	O
+±	O
+6	O
+nM	O
+;	O
+R227A	B-mutant
+,	O
+166	O
+±	O
+2	O
+nM	O
+;	O
+V254P	B-mutant
+,	O
+137	O
+±	O
+10	O
+nM	O
+;	O
+Q147A	B-mutant
+,	O
+171	O
+±	O
+21	O
+nM	O
+.	O
+The	O
+average	O
+KA	B-evidence
+and	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+for	O
+three	O
+independent	O
+titrations	O
+are	O
+plotted	O
+.	O
+
+The	O
+average	O
+fitted	O
+fluorescence	O
+anisotropy	O
+RNA	B-evidence
+-	I-evidence
+binding	I-evidence
+curves	I-evidence
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+–	O
+c	O
+.	O
+
+The	O
+U2AF65	B-protein
+linker	B-structure_element
+/	O
+RRM	B-structure_element
+and	O
+inter	O
+-	O
+RRM	B-structure_element
+interactions	O
+.	O
+
+(	O
+a	O
+)	O
+Contacts	O
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+with	O
+the	O
+RRMs	B-structure_element
+.	O
+
+A	O
+semi	O
+-	O
+transparent	O
+space	O
+-	O
+filling	O
+surface	O
+is	O
+shown	O
+for	O
+the	O
+RRM1	B-structure_element
+(	O
+green	O
+)	O
+and	O
+RRM2	B-structure_element
+(	O
+light	O
+blue	O
+).	O
+
+Residues	O
+V249	B-residue_name_number
+,	O
+V250	B-residue_name_number
+,	O
+V254	B-residue_name_number
+(	O
+yellow	O
+)	O
+are	O
+mutated	B-experimental_method
+to	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+V254G	B-mutant
+in	O
+the	O
+3Gly	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+P255	B-residue_name_number
+(	O
+red	O
+)	O
+along	O
+with	O
+V254	B-residue_name_number
+are	O
+mutated	B-experimental_method
+to	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+5Gly	B-mutant
+mutant	I-mutant
+or	O
+to	O
+S251N	B-mutant
+/	O
+T252L	B-mutant
+/	O
+V253A	B-mutant
+/	O
+V254L	B-mutant
+/	O
+P255A	B-mutant
+in	O
+the	O
+NLALA	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+M144	B-residue_name_number
+,	O
+L235	B-residue_name_number
+,	O
+M238	B-residue_name_number
+,	O
+V244	B-residue_name_number
+,	O
+V246	B-residue_name_number
+(	O
+orange	O
+)	O
+along	O
+with	O
+V249	B-residue_name_number
+,	O
+V250	B-residue_name_number
+,	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+V254	B-residue_name_number
+,	O
+P255	B-residue_name_number
+are	O
+mutated	B-experimental_method
+to	O
+M144G	B-mutant
+/	O
+L235G	B-mutant
+/	O
+M238G	B-mutant
+/	O
+V244G	B-mutant
+/	O
+V246G	B-mutant
+/	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+12Gly	B-mutant
+mutant	I-mutant
+.	O
+
+Other	O
+linker	B-structure_element
+residues	O
+are	O
+coloured	O
+either	O
+dark	O
+blue	O
+for	O
+new	O
+residues	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	O
+or	O
+light	O
+blue	O
+for	O
+the	O
+remaining	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+residues	O
+.	O
+
+The	O
+central	O
+panel	O
+shows	O
+an	O
+overall	O
+view	O
+with	O
+stick	O
+diagrams	O
+for	O
+mutated	O
+residues	O
+;	O
+boxed	O
+regions	O
+are	O
+expanded	O
+to	O
+show	O
+the	O
+C	O
+-	O
+terminal	O
+(	O
+bottom	O
+left	O
+)	O
+and	O
+central	B-structure_element
+linker	I-structure_element
+regions	I-structure_element
+(	O
+top	O
+)	O
+at	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+interfaces	I-structure_element
+,	O
+and	O
+N	O
+-	O
+terminal	O
+linker	O
+region	O
+contacts	O
+with	O
+RRM1	B-structure_element
+(	O
+bottom	O
+right	O
+).	O
+
+(	O
+b	O
+)	O
+Bar	O
+graph	O
+of	O
+apparent	O
+equilibrium	B-evidence
+affinities	I-evidence
+(	O
+KA	B-evidence
+)	O
+for	O
+the	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+(	O
+5	B-chemical
+′-	I-chemical
+CCCUUUUUUUUCC	I-chemical
+-	I-chemical
+3	I-chemical
+′)	I-chemical
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	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	B-experimental_method
+deletions	I-experimental_method
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+in	O
+the	O
+minimal	B-protein_state
+RRM1	B-structure_element
+–	I-structure_element
+RRM2	I-structure_element
+region	I-structure_element
+(	O
+residues	O
+148	B-residue_range
+–	I-residue_range
+237	I-residue_range
+,	O
+258	B-residue_range
+–	I-residue_range
+336	I-residue_range
+)	O
+or	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+(	O
+residues	O
+141	B-residue_range
+–	I-residue_range
+237	I-residue_range
+,	O
+258	B-residue_range
+–	I-residue_range
+342	I-residue_range
+).	O
+
+The	O
+apparent	O
+equilibrium	B-evidence
+dissociation	I-evidence
+constants	I-evidence
+(	O
+KD	B-evidence
+)	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+mutant	B-protein_state
+proteins	O
+are	O
+:	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+),	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	B-evidence
+and	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+for	O
+three	O
+independent	O
+titrations	O
+are	O
+plotted	O
+.	O
+
+The	O
+fitted	O
+fluorescence	O
+anisotropy	O
+RNA	B-evidence
+-	I-evidence
+binding	I-evidence
+curves	I-evidence
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+–	O
+j	O
+.	O
+(	O
+c	O
+)	O
+Close	O
+view	O
+of	O
+the	O
+U2AF65	B-protein
+RRM1	B-site
+/	I-site
+RRM2	I-site
+interface	I-site
+following	O
+a	O
+two	O
+-	O
+fold	O
+rotation	O
+about	O
+the	O
+x	O
+-	O
+axis	O
+relative	O
+to	O
+a	O
+.	O
+
+U2AF65	B-protein
+inter	O
+-	O
+domain	O
+residues	O
+are	O
+important	O
+for	O
+splicing	O
+a	O
+representative	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+substrate	O
+in	O
+human	B-species
+cells	O
+.	O
+
+(	O
+a	O
+)	O
+Schematic	O
+diagram	O
+of	O
+the	O
+pyPY	B-chemical
+reporter	O
+minigene	O
+construct	O
+comprising	O
+two	O
+alternative	O
+splice	B-site
+sites	I-site
+preceded	O
+by	O
+either	O
+the	O
+weak	O
+IgM	O
+Py	B-chemical
+tract	I-chemical
+(	O
+py	B-chemical
+)	O
+or	O
+the	O
+strong	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+(	O
+PY	B-chemical
+)	O
+(	O
+sequences	O
+inset	O
+).	O
+
+(	O
+b	O
+)	O
+Representative	O
+RT	B-experimental_method
+-	I-experimental_method
+PCR	I-experimental_method
+of	O
+pyPY	B-chemical
+transcripts	O
+from	O
+HEK293T	O
+cells	O
+co	B-experimental_method
+-	I-experimental_method
+transfected	I-experimental_method
+with	O
+constructs	O
+encoding	O
+the	O
+pyPY	B-chemical
+minigene	O
+and	O
+either	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+U2AF65	B-protein
+or	O
+a	O
+triple	O
+U2AF65	B-protein
+mutant	B-protein_state
+(	O
+3Mut	B-mutant
+)	O
+of	O
+Q147A	B-mutant
+,	O
+R227A	B-mutant
+and	O
+V254P	B-mutant
+residues	O
+.	O
+(	O
+c	O
+)	O
+A	O
+bar	O
+graph	O
+of	O
+the	O
+average	O
+percentage	O
+of	O
+the	O
+py	B-chemical
+-	O
+spliced	O
+mRNA	B-chemical
+relative	O
+to	O
+total	O
+detected	O
+pyPY	B-chemical
+transcripts	O
+(	O
+spliced	O
+and	O
+unspliced	O
+)	O
+for	O
+the	O
+corresponding	O
+gel	O
+lanes	O
+(	O
+black	O
+,	O
+no	O
+U2AF65	B-protein
+added	O
+;	O
+white	O
+,	O
+WT	B-protein_state
+U2AF65	B-protein
+;	O
+grey	O
+,	O
+3Mut	B-mutant
+U2AF65	B-protein
+).	O
+
+Protein	B-experimental_method
+overexpression	I-experimental_method
+and	O
+qRT	B-experimental_method
+-	I-experimental_method
+PCR	I-experimental_method
+results	O
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+.	O
+
+RNA	O
+binding	O
+stabilizes	O
+the	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+conformation	O
+of	O
+U2AF65	B-protein
+RRMs	B-structure_element
+.	O
+
+(	O
+a	O
+,	O
+b	O
+)	O
+Views	O
+of	O
+FRET	B-experimental_method
+pairs	O
+chosen	O
+to	O
+follow	O
+the	O
+relative	O
+movement	O
+of	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+on	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+‘	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+'	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRMs	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+a	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+oligonucleotide	I-chemical
+(	O
+a	O
+,	O
+representative	O
+structure	O
+iv	O
+)	O
+or	O
+‘	O
+closed	B-protein_state
+'	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+-	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	B-structure_element
+.	O
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+proteins	O
+were	O
+doubly	O
+labelled	O
+at	O
+A181C	B-mutant
+/	O
+Q324C	B-mutant
+such	O
+that	O
+a	O
+mixture	O
+of	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+fluorophores	B-chemical
+are	O
+expected	O
+to	O
+be	O
+present	O
+at	O
+each	O
+site	O
+.	O
+
+(	O
+c	O
+–	O
+f	O
+,	O
+i	O
+,	O
+j	O
+)	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+protein	O
+was	O
+immobilized	O
+on	O
+the	O
+microscope	O
+slide	O
+via	O
+biotin	B-chemical
+-	I-chemical
+NTA	I-chemical
+/	I-chemical
+Ni	I-chemical
++	I-chemical
+2	I-chemical
+(	O
+orange	O
+line	O
+)	O
+on	O
+a	O
+neutravidin	O
+(	O
+black	O
+X	O
+)-	O
+biotin	O
+-	O
+PEG	O
+(	O
+orange	O
+triangle	O
+)-	O
+treated	O
+surface	O
+and	O
+imaged	O
+either	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ligands	B-chemical
+(	O
+c	O
+,	O
+d	O
+),	O
+in	O
+the	O
+presence	O
+of	O
+5	O
+μM	O
+AdML	B-gene
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+RNA	I-chemical
+(	O
+5	B-chemical
+′-	I-chemical
+CCUUUUUUUUCC	I-chemical
+-	I-chemical
+3	I-chemical
+′)	I-chemical
+(	O
+e	O
+,	O
+f	O
+),	O
+or	O
+in	O
+the	O
+presence	O
+of	O
+10	O
+μM	O
+adenosine	B-residue_name
+-	O
+interrupted	O
+variant	O
+RNA	B-chemical
+(	O
+5	B-chemical
+′-	I-chemical
+CUUUUUAAUUUCCA	I-chemical
+-	I-chemical
+3	I-chemical
+′)	I-chemical
+(	O
+i	O
+,	O
+j	O
+).	O
+
+The	O
+untethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+protein	O
+(	O
+1	O
+nM	O
+)	O
+was	O
+added	O
+to	O
+AdML	B-gene
+RNA	B-chemical
+–	I-chemical
+polyethylene	I-chemical
+-	I-chemical
+glycol	I-chemical
+-	I-chemical
+linker	I-chemical
+–	I-chemical
+DNA	I-chemical
+oligonucleotide	I-chemical
+(	O
+10	O
+nM	O
+),	O
+which	O
+was	O
+immobilized	O
+on	O
+the	O
+microscope	O
+slide	O
+by	O
+annealing	O
+with	O
+a	O
+complementary	O
+biotinyl	B-chemical
+-	I-chemical
+DNA	I-chemical
+oligonucleotide	I-chemical
+(	O
+black	O
+vertical	O
+line	O
+).	O
+
+Typical	O
+single	B-experimental_method
+-	I-experimental_method
+molecule	I-experimental_method
+FRET	I-experimental_method
+traces	B-evidence
+(	O
+c	O
+,	O
+e	O
+,	O
+g	O
+,	O
+i	O
+)	O
+show	O
+fluorescence	O
+intensities	O
+from	O
+Cy3	B-chemical
+(	O
+green	O
+)	O
+and	O
+Cy5	B-chemical
+(	O
+red	O
+)	O
+and	O
+the	O
+calculated	B-evidence
+apparent	I-evidence
+FRET	I-evidence
+efficiency	I-evidence
+(	O
+blue	O
+).	O
+
+Additional	O
+traces	B-evidence
+for	O
+untethered	B-protein_state
+,	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+,	O
+d	O
+.	O
+Histograms	B-evidence
+(	O
+d	O
+,	O
+f	O
+,	O
+h	O
+,	O
+j	O
+)	O
+show	O
+the	O
+distribution	B-evidence
+of	I-evidence
+FRET	I-evidence
+values	I-evidence
+in	O
+RNA	B-protein_state
+-	I-protein_state
+free	I-protein_state
+,	O
+slide	B-protein_state
+-	I-protein_state
+tethered	I-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+(	O
+d	O
+);	O
+AdML	B-gene
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+,	O
+slide	B-protein_state
+-	I-protein_state
+tethered	I-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+(	O
+f	O
+);	O
+AdML	B-gene
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+,	O
+untethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+(	O
+h	O
+)	O
+and	O
+adenosine	O
+-	O
+interrupted	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+,	O
+slide	B-protein_state
+-	I-protein_state
+tethered	I-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+(	O
+j	O
+).	O
+
+N	O
+is	O
+the	O
+number	O
+of	O
+single	O
+-	O
+molecule	O
+traces	B-evidence
+compiled	O
+for	O
+each	O
+histogram	B-evidence
+.	O
+
+Schematic	O
+models	O
+of	O
+U2AF65	B-protein
+recognizing	O
+the	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+(	O
+a	O
+)	O
+Diagram	O
+of	O
+the	O
+U2AF65	B-protein
+,	O
+SF1	B-protein
+and	O
+U2AF35	B-protein
+splicing	O
+factors	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+consensus	O
+elements	O
+of	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+.	O
+
+A	O
+surface	O
+representation	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+is	O
+shown	O
+bound	B-protein_state
+to	I-protein_state
+nine	O
+nucleotides	B-chemical
+(	O
+nt	O
+);	O
+the	O
+relative	O
+distances	O
+and	O
+juxtaposition	O
+of	O
+the	O
+branch	B-site
+point	I-site
+sequence	I-site
+(	O
+BPS	B-site
+)	O
+and	O
+consensus	O
+AG	B-chemical
+dinucleotide	I-chemical
+at	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+are	O
+unknown	O
+.	O
+
+MDS	O
+-	O
+relevant	O
+mutated	O
+residues	O
+of	O
+U2AF65	B-protein
+are	O
+shown	O
+as	O
+yellow	O
+spheres	O
+(	O
+L187	B-residue_name_number
+and	O
+M144	B-residue_name_number
+).	O
+
+(	O
+b	O
+)	O
+Following	O
+binding	O
+to	O
+the	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+RNA	I-chemical
+,	O
+a	O
+conformation	O
+corresponding	O
+to	O
+high	B-evidence
+FRET	I-evidence
+and	O
+consistent	O
+with	O
+the	O
+‘	O
+closed	B-protein_state
+',	O
+back	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+back	I-protein_state
+apo	B-protein_state
+-	O
+U2AF65	B-protein
+model	O
+resulting	O
+from	O
+PRE	B-experimental_method
+/	O
+NMR	B-experimental_method
+characterization	O
+(	O
+PDB	O
+ID	O
+2YH0	O
+)	O
+often	O
+transitions	O
+to	O
+a	O
+conformation	O
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+,	O
+which	O
+is	O
+consistent	O
+with	O
+‘	O
+open	B-protein_state
+',	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+RRMs	B-structure_element
+such	O
+as	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Alternatively	O
+,	O
+a	O
+conformation	O
+of	O
+U2AF65	B-protein
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+can	O
+directly	O
+bind	O
+to	O
+RNA	B-chemical
+;	O
+RNA	B-chemical
+binding	O
+stabilizes	O
+the	O
+‘	O
+open	B-protein_state
+',	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+conformation	O
+and	O
+thus	O
+shifts	O
+the	O
+U2AF65	B-protein
+population	O
+towards	O
+the	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+.	O
+
+RRM1	B-structure_element
+,	O
+green	O
+;	O
+RRM2	B-structure_element
+,	O
+pale	O
+blue	O
+;	O
+RRM	B-structure_element
+extensions	I-structure_element
+/	O
+linker	B-structure_element
+,	O
+blue	O
+.	O
+
+RNA	B-chemical
+protects	O
+a	O
+nucleoprotein	B-complex_assembly
+complex	O
+against	O
+radiation	O
+damage	O
+
+Systematic	O
+analysis	O
+of	O
+radiation	O
+damage	O
+within	O
+a	O
+protein	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+MGy	O
+)	O
+reveals	O
+significant	O
+differential	O
+susceptibility	O
+of	O
+RNA	B-chemical
+and	O
+protein	O
+.	O
+
+A	O
+new	O
+method	O
+of	O
+difference	B-experimental_method
+electron	I-experimental_method
+-	I-experimental_method
+density	I-experimental_method
+quantification	I-experimental_method
+is	O
+presented	O
+.	O
+
+Radiation	O
+damage	O
+during	O
+macromolecular	B-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallographic	I-experimental_method
+data	I-experimental_method
+collection	I-experimental_method
+is	O
+still	O
+the	O
+main	O
+impediment	O
+for	O
+many	O
+macromolecular	B-experimental_method
+structure	I-experimental_method
+determinations	I-experimental_method
+.	O
+
+Although	O
+this	O
+has	O
+been	O
+well	O
+characterized	O
+within	O
+protein	O
+crystals	B-evidence
+,	O
+far	O
+less	O
+is	O
+known	O
+about	O
+specific	O
+damage	O
+effects	O
+within	O
+the	O
+larger	O
+class	O
+of	O
+nucleoprotein	O
+complexes	O
+.	O
+
+Here	O
+,	O
+a	O
+methodology	O
+has	O
+been	O
+developed	O
+whereby	O
+per	B-evidence
+-	I-evidence
+atom	I-evidence
+density	I-evidence
+changes	I-evidence
+could	O
+be	O
+quantified	O
+with	O
+increasing	O
+dose	O
+over	O
+a	O
+wide	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+.	O
+0	O
+MGy	O
+)	O
+range	O
+and	O
+at	O
+higher	O
+resolution	O
+(	O
+1	O
+.	O
+98	O
+Å	O
+)	O
+than	O
+the	O
+previous	O
+systematic	O
+specific	O
+damage	O
+study	O
+on	O
+a	O
+protein	O
+–	O
+DNA	B-chemical
+complex	O
+.	O
+
+Specific	O
+damage	O
+manifestations	O
+were	O
+determined	O
+within	O
+the	O
+large	O
+trp	B-protein_type
+RNA	I-protein_type
+-	I-protein_type
+binding	I-protein_type
+attenuation	I-protein_type
+protein	I-protein_type
+(	O
+TRAP	B-complex_assembly
+)	O
+bound	B-protein_state
+to	I-protein_state
+a	O
+single	O
+-	O
+stranded	O
+RNA	B-chemical
+that	O
+forms	O
+a	O
+belt	O
+around	O
+the	O
+protein	O
+.	O
+
+Over	O
+a	O
+large	O
+dose	O
+range	O
+,	O
+the	O
+RNA	B-chemical
+was	O
+found	O
+to	O
+be	O
+far	O
+less	O
+susceptible	O
+to	O
+radiation	O
+-	O
+induced	O
+chemical	O
+changes	O
+than	O
+the	O
+protein	O
+.	O
+
+The	O
+availability	O
+of	O
+two	O
+TRAP	B-complex_assembly
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+,	O
+of	O
+which	O
+only	O
+one	O
+contained	O
+bound	B-protein_state
+RNA	B-chemical
+,	O
+allowed	O
+a	O
+controlled	O
+investigation	O
+into	O
+the	O
+exact	O
+role	O
+of	O
+RNA	B-chemical
+binding	O
+in	O
+protein	O
+specific	O
+damage	O
+susceptibility	O
+.	O
+
+The	O
+11	O
+-	O
+fold	O
+symmetry	O
+within	O
+each	O
+TRAP	B-complex_assembly
+ring	B-structure_element
+permitted	O
+statistically	O
+significant	O
+analysis	O
+of	O
+the	O
+Glu	B-residue_name
+and	O
+Asp	B-residue_name
+damage	O
+patterns	O
+,	O
+with	O
+RNA	B-chemical
+binding	O
+unexpectedly	O
+being	O
+observed	O
+to	O
+protect	O
+these	O
+otherwise	O
+highly	O
+sensitive	O
+residues	O
+within	O
+the	O
+11	O
+RNA	B-site
+-	I-site
+binding	I-site
+pockets	I-site
+distributed	O
+around	O
+the	O
+outside	O
+of	O
+the	O
+protein	O
+molecule	O
+.	O
+
+Additionally	O
+,	O
+the	O
+method	O
+enabled	O
+a	O
+quantification	O
+of	O
+the	O
+reduction	O
+in	O
+radiation	O
+-	O
+induced	O
+Lys	B-residue_name
+and	O
+Phe	B-residue_name
+disordering	O
+upon	O
+RNA	B-chemical
+binding	O
+directly	O
+from	O
+the	O
+electron	B-evidence
+density	I-evidence
+.	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	B-experimental_method
+determination	I-experimental_method
+using	O
+macromolecular	B-experimental_method
+crystallography	I-experimental_method
+(	O
+MX	B-experimental_method
+)	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
+
+Significant	O
+progress	O
+has	O
+been	O
+made	O
+in	O
+recent	O
+years	O
+in	O
+understanding	O
+the	O
+inevitable	O
+manifestations	O
+of	O
+X	O
+-	O
+ray	O
+-	O
+induced	O
+RD	O
+within	O
+protein	O
+crystals	B-evidence
+,	O
+and	O
+there	O
+is	O
+now	O
+a	O
+body	O
+of	O
+literature	O
+on	O
+possible	O
+strategies	O
+to	O
+mitigate	O
+the	O
+effects	O
+of	O
+RD	O
+(	O
+e	O
+.	O
+g	O
+.	O
+Zeldin	O
+,	O
+Brockhauser	O
+et	O
+al	O
+.,	O
+2013	O
+;	O
+Bourenkov	O
+&	O
+Popov	O
+,	O
+2010	O
+).	O
+
+However	O
+,	O
+there	O
+is	O
+still	O
+no	O
+general	O
+consensus	O
+within	O
+the	O
+field	O
+on	O
+how	O
+to	O
+minimize	O
+RD	O
+during	O
+MX	B-experimental_method
+data	O
+collection	O
+,	O
+and	O
+debates	O
+on	O
+the	O
+dependence	O
+of	O
+RD	O
+progression	O
+on	O
+incident	O
+X	O
+-	O
+ray	O
+energy	O
+(	O
+Shimizu	O
+et	O
+al	O
+.,	O
+2007	O
+;	O
+Liebschner	O
+et	O
+al	O
+.,	O
+2015	O
+)	O
+and	O
+the	O
+efficacy	O
+of	O
+radical	O
+scavengers	O
+(	O
+Allan	O
+et	O
+al	O
+.,	O
+2013	O
+)	O
+have	O
+yet	O
+to	O
+be	O
+resolved	O
+.	O
+
+Global	O
+radiation	O
+damage	O
+is	O
+observed	O
+within	O
+reciprocal	O
+space	O
+as	O
+the	O
+overall	O
+decay	O
+of	O
+the	O
+summed	O
+intensity	O
+of	O
+reflections	O
+detected	O
+within	O
+the	O
+diffraction	B-evidence
+pattern	I-evidence
+as	O
+dose	O
+increases	O
+(	O
+Garman	O
+,	O
+2010	O
+;	O
+Murray	O
+&	O
+Garman	O
+,	O
+2002	O
+).	O
+
+Dose	O
+is	O
+defined	O
+as	O
+the	O
+absorbed	O
+energy	O
+per	O
+unit	O
+mass	O
+of	O
+crystal	O
+in	O
+grays	O
+(	O
+Gy	O
+;	O
+1	O
+Gy	O
+=	O
+1	O
+J	O
+kg	O
+−	O
+1	O
+),	O
+and	O
+is	O
+the	O
+metric	O
+against	O
+which	O
+damage	O
+progression	O
+should	O
+be	O
+monitored	O
+during	O
+MX	B-experimental_method
+data	O
+collection	O
+,	O
+as	O
+opposed	O
+to	O
+time	O
+.	O
+
+At	O
+100	O
+K	O
+,	O
+an	O
+experimental	O
+dose	O
+limit	O
+of	O
+30	O
+MGy	O
+has	O
+been	O
+recommended	O
+as	O
+an	O
+upper	O
+limit	O
+beyond	O
+which	O
+the	O
+biological	O
+information	O
+derived	O
+from	O
+any	O
+macromolecular	O
+crystal	B-evidence
+may	O
+be	O
+compromised	O
+(	O
+Owen	O
+et	O
+al	O
+.,	O
+2006	O
+).	O
+
+Specific	B-experimental_method
+radiation	I-experimental_method
+damage	I-experimental_method
+(	O
+SRD	B-experimental_method
+)	O
+is	O
+observed	O
+in	O
+the	O
+real	B-evidence
+-	I-evidence
+space	I-evidence
+electron	I-evidence
+density	I-evidence
+,	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
+
+Indeed	O
+,	O
+the	O
+C	O
+—	O
+Se	B-chemical
+bond	O
+in	O
+selenomethionine	B-chemical
+,	O
+the	O
+stability	O
+of	O
+which	O
+is	O
+key	O
+for	O
+the	O
+success	O
+of	O
+experimental	O
+phasing	O
+methods	O
+,	O
+can	O
+be	O
+cleaved	O
+at	O
+a	O
+dose	O
+as	O
+low	O
+as	O
+2	O
+MGy	O
+for	O
+a	O
+crystal	B-evidence
+maintained	O
+at	O
+100	O
+K	O
+(	O
+Holton	O
+,	O
+2007	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	B-ptm
+-	I-ptm
+bond	I-ptm
+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
+
+There	O
+are	O
+a	O
+number	O
+of	O
+cases	O
+where	O
+SRD	O
+manifestations	O
+have	O
+compromised	O
+the	O
+biological	O
+information	O
+extracted	O
+from	O
+MX	B-experimental_method
+-	I-experimental_method
+determined	I-experimental_method
+structures	B-evidence
+at	O
+much	O
+lower	O
+doses	O
+than	O
+the	O
+recommended	O
+30	O
+MGy	O
+limit	O
+,	O
+leading	O
+to	O
+false	O
+structural	O
+interpretations	O
+of	O
+protein	O
+mechanisms	O
+.	O
+
+Active	B-site
+-	I-site
+site	I-site
+residues	I-site
+appear	O
+to	O
+be	O
+particularly	O
+susceptible	O
+,	O
+particularly	O
+for	O
+photosensitive	O
+proteins	O
+and	O
+in	O
+instances	O
+where	O
+chemical	O
+strain	O
+is	O
+an	O
+intrinsic	O
+feature	O
+of	O
+the	O
+reaction	O
+mechanism	O
+.	O
+
+For	O
+instance	O
+,	O
+structure	B-experimental_method
+determination	I-experimental_method
+of	O
+the	O
+purple	O
+membrane	O
+protein	O
+bacterio	B-protein_type
+­	I-protein_type
+rhodopsin	I-protein_type
+required	O
+careful	O
+corrections	O
+for	O
+radiation	O
+-	O
+induced	O
+structural	O
+changes	O
+before	O
+the	O
+correct	O
+photosensitive	O
+intermediate	O
+states	O
+could	O
+be	O
+isolated	O
+(	O
+Matsui	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+
+The	O
+significant	O
+chemical	O
+strain	O
+required	O
+for	O
+catalysis	O
+within	O
+the	O
+active	B-site
+site	I-site
+of	O
+phosphoserine	B-protein_type
+aminotransferase	I-protein_type
+has	O
+been	O
+observed	O
+to	O
+diminish	O
+during	O
+X	O
+-	O
+ray	O
+exposure	O
+(	O
+Dubnovitsky	O
+et	O
+al	O
+.,	O
+2005	O
+).	O
+
+Since	O
+the	O
+majority	O
+of	O
+SRD	B-experimental_method
+studies	I-experimental_method
+to	O
+date	O
+have	O
+focused	O
+on	O
+proteins	O
+,	O
+much	O
+less	O
+is	O
+known	O
+about	O
+the	O
+effects	O
+of	O
+X	O
+-	O
+ray	O
+irradiation	O
+on	O
+the	O
+wider	O
+class	O
+of	O
+crystalline	O
+nucleoprotein	B-complex_assembly
+complexes	O
+or	O
+how	O
+to	O
+correct	O
+for	O
+such	O
+radiation	O
+-	O
+induced	O
+structural	O
+changes	O
+.	O
+
+Understanding	O
+RD	O
+to	O
+such	O
+complexes	O
+is	O
+crucial	O
+,	O
+since	O
+DNA	B-chemical
+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	B-chemical
+repair	O
+.	O
+
+As	O
+of	O
+early	O
+2016	O
+,	O
+>	O
+5400	O
+nucleoprotein	B-complex_assembly
+complex	O
+structures	B-evidence
+have	O
+been	O
+deposited	O
+within	O
+the	O
+PDB	O
+,	O
+with	O
+91	O
+%	O
+solved	O
+by	O
+MX	B-experimental_method
+.	O
+
+It	O
+is	O
+essential	O
+to	O
+understand	O
+how	O
+these	O
+increasingly	O
+complex	O
+macromolecular	O
+structures	B-evidence
+are	O
+affected	O
+by	O
+the	O
+radiation	O
+used	O
+to	O
+solve	O
+them	O
+.	O
+
+Nucleoproteins	B-complex_assembly
+also	O
+represent	O
+one	O
+of	O
+the	O
+main	O
+targets	O
+of	O
+radiotherapy	O
+,	O
+and	O
+an	O
+insight	O
+into	O
+the	O
+damage	O
+mechanisms	O
+induced	O
+by	O
+X	O
+-	O
+ray	O
+irradiation	O
+could	O
+inform	O
+innovative	O
+treatments	O
+.	O
+
+Investigations	O
+on	O
+sub	O
+-	O
+ionization	O
+-	O
+level	O
+LEEs	O
+(	O
+0	O
+–	O
+15	O
+eV	O
+)	O
+interacting	O
+with	O
+both	O
+dried	O
+and	O
+aqueous	O
+oligonucleotides	O
+(	O
+Alizadeh	O
+&	O
+Sanche	O
+,	O
+2014	O
+;	O
+Simons	O
+,	O
+2006	O
+)	O
+concluded	O
+that	O
+resonant	O
+electron	O
+attachment	O
+to	O
+DNA	B-chemical
+bases	O
+and	O
+the	O
+sugar	O
+-	O
+phosphate	O
+backbone	O
+could	O
+lead	O
+to	O
+the	O
+preferential	O
+cleavage	O
+of	O
+strong	O
+(∼	O
+4	O
+eV	O
+,	O
+385	O
+kJ	O
+mol	O
+−	O
+1	O
+)	O
+sugar	O
+-	O
+phosphate	O
+C	O
+—	O
+O	O
+covalent	O
+bonds	O
+within	O
+the	O
+DNA	B-chemical
+backbone	O
+and	O
+then	O
+base	O
+-	O
+sugar	O
+N1	O
+—	O
+C	O
+bonds	O
+,	O
+eventually	O
+leading	O
+to	O
+single	O
+-	O
+strand	O
+breakages	O
+(	O
+SSBs	O
+;	O
+Ptasińska	O
+&	O
+Sanche	O
+,	O
+2007	O
+).	O
+
+Electrons	O
+have	O
+been	O
+shown	O
+to	O
+be	O
+mobile	O
+at	O
+77	O
+K	O
+by	O
+electron	B-experimental_method
+spin	I-experimental_method
+resonance	I-experimental_method
+spectroscopy	I-experimental_method
+studies	O
+(	O
+Symons	O
+,	O
+1997	O
+;	O
+Jones	O
+et	O
+al	O
+.,	O
+1987	O
+),	O
+with	O
+rapid	O
+electron	O
+quantum	O
+tunnelling	O
+and	O
+positive	O
+hole	O
+migration	O
+along	O
+the	O
+protein	O
+backbone	O
+and	O
+through	O
+stacked	O
+DNA	B-chemical
+bases	O
+indicated	O
+as	O
+a	O
+dominant	O
+mechanism	O
+by	O
+which	O
+oxidative	O
+and	O
+reductive	O
+damage	O
+localizes	O
+at	O
+distances	O
+from	O
+initial	O
+ionization	B-site
+sites	I-site
+at	O
+100	O
+K	O
+(	O
+O	O
+’	O
+Neill	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+
+The	O
+investigation	O
+of	O
+naturally	O
+forming	O
+nucleoprotein	O
+complexes	O
+circumvents	O
+the	O
+inherent	O
+challenges	O
+in	O
+making	O
+controlled	O
+comparisons	O
+of	O
+damage	O
+mechanisms	O
+between	O
+protein	O
+and	O
+nucleic	O
+acids	O
+crystallized	B-experimental_method
+separately	O
+.	O
+Recently	O
+,	O
+for	O
+a	O
+well	O
+characterized	O
+bacterial	B-taxonomy_domain
+protein	O
+–	O
+DNA	B-chemical
+complex	O
+(	O
+C	B-complex_assembly
+.	I-complex_assembly
+Esp1396I	I-complex_assembly
+;	O
+PDB	O
+entry	O
+3clc	O
+;	O
+resolution	O
+2	O
+.	O
+8	O
+Å	O
+;	O
+McGeehan	O
+et	O
+al	O
+.,	O
+2008	O
+)	O
+it	O
+was	O
+concluded	O
+that	O
+over	O
+a	O
+wide	O
+dose	O
+range	O
+(	O
+2	O
+.	O
+1	O
+–	O
+44	O
+.	O
+6	O
+MGy	O
+)	O
+the	O
+protein	O
+was	O
+far	O
+more	O
+susceptible	O
+to	O
+SRD	O
+than	O
+the	O
+DNA	B-chemical
+within	O
+the	O
+crystal	B-evidence
+(	O
+Bury	O
+et	O
+al	O
+.,	O
+2015	O
+).	O
+
+Only	O
+at	O
+doses	O
+above	O
+20	O
+MGy	O
+were	O
+precursors	O
+of	O
+phosphodiester	O
+-	O
+bond	O
+cleavage	O
+observed	O
+within	O
+AT	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+regions	I-structure_element
+of	O
+the	O
+35	O
+-	O
+mer	O
+DNA	B-chemical
+.	O
+
+For	O
+crystalline	O
+complexes	O
+such	O
+as	O
+C	B-complex_assembly
+.	I-complex_assembly
+Esp1396I	I-complex_assembly
+,	O
+whether	O
+the	O
+protein	O
+is	O
+intrinsically	O
+more	O
+susceptible	O
+to	O
+X	O
+-	O
+ray	O
+-	O
+induced	O
+damage	O
+or	O
+whether	O
+the	O
+protein	O
+scavenges	O
+electrons	O
+to	O
+protect	O
+the	O
+DNA	B-chemical
+remains	O
+unclear	O
+in	O
+the	O
+absence	O
+of	O
+a	O
+non	O
+-	O
+nucleic	O
+acid	O
+-	O
+bound	O
+protein	O
+control	O
+obtained	O
+under	O
+exactly	O
+the	O
+same	O
+crystallization	O
+and	O
+data	O
+-	O
+collection	O
+conditions	O
+.	O
+
+To	O
+monitor	O
+the	O
+effects	O
+of	O
+nucleic	O
+acid	O
+binding	O
+on	O
+protein	O
+damage	O
+susceptibility	O
+,	O
+a	O
+crystal	B-evidence
+containing	O
+two	O
+protein	O
+molecules	O
+per	O
+asymmetric	O
+unit	O
+,	O
+only	O
+one	O
+of	O
+which	O
+was	O
+bound	B-protein_state
+to	I-protein_state
+RNA	B-chemical
+,	O
+is	O
+reported	O
+here	O
+(	O
+Fig	O
+.	O
+1	O
+▸).	O
+
+Using	O
+newly	O
+developed	O
+methodology	O
+,	O
+we	O
+present	O
+a	O
+controlled	B-experimental_method
+SRD	I-experimental_method
+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	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+:	O
+trp	B-protein_type
+RNA	I-protein_type
+-	I-protein_type
+binding	I-protein_type
+attenuation	I-protein_type
+protein	I-protein_type
+(	O
+TRAP	B-complex_assembly
+)	O
+bound	B-protein_state
+to	I-protein_state
+a	O
+53	O
+bp	O
+RNA	B-chemical
+sequence	O
+(	B-chemical
+GAGUU	I-chemical
+)	I-chemical
+10GAG	I-chemical
+(	O
+PDB	O
+entry	O
+1gtf	O
+;	O
+Hopcroft	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+
+TRAP	B-complex_assembly
+consists	O
+of	O
+11	O
+identical	O
+subunits	B-structure_element
+assembled	O
+into	O
+a	O
+ring	B-structure_element
+with	O
+11	O
+-	O
+fold	O
+rotational	O
+symmetry	O
+.	O
+
+It	O
+binds	O
+with	O
+high	O
+affinity	O
+(	O
+K	B-evidence
+d	I-evidence
+≃	O
+1	O
+.	O
+0	O
+nM	O
+)	O
+to	O
+RNA	B-chemical
+segments	O
+containing	O
+11	O
+GAG	B-structure_element
+/	I-structure_element
+UAG	I-structure_element
+triplets	I-structure_element
+separated	O
+by	O
+two	O
+or	O
+three	O
+spacer	B-structure_element
+nucleotides	I-structure_element
+(	O
+Elliott	O
+et	O
+al	O
+.,	O
+2001	O
+)	O
+to	O
+regulate	O
+the	O
+transcription	O
+of	O
+tryptophan	B-chemical
+biosynthetic	O
+genes	O
+in	O
+Bacillus	B-species
+subtilis	I-species
+(	O
+Antson	O
+et	O
+al	O
+.,	O
+1999	O
+).	O
+
+In	O
+this	O
+structure	B-evidence
+,	O
+the	O
+bases	O
+of	O
+the	O
+G1	B-chemical
+-	I-chemical
+A2	I-chemical
+-	I-chemical
+G3	I-chemical
+nucleotides	O
+form	O
+direct	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+protein	O
+,	O
+unlike	O
+the	O
+U4	B-chemical
+-	I-chemical
+U5	I-chemical
+nucleotides	O
+,	O
+which	O
+appear	O
+to	O
+be	O
+more	O
+flexible	O
+.	O
+
+Ten	O
+successive	O
+1	O
+.	O
+98	O
+Å	O
+resolution	O
+MX	B-experimental_method
+data	O
+sets	O
+were	O
+collected	O
+from	O
+the	O
+same	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+crystal	B-evidence
+to	O
+analyse	O
+X	O
+-	O
+ray	O
+-	O
+induced	O
+structural	O
+changes	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+d	O
+1	O
+=	O
+1	O
+.	O
+3	O
+MGy	O
+to	O
+d	O
+10	O
+=	O
+25	O
+.	O
+0	O
+MGy	O
+).	O
+
+To	O
+avoid	O
+the	O
+previous	O
+necessity	O
+for	O
+visual	O
+inspection	O
+of	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+maps	I-evidence
+to	O
+detect	O
+SRD	B-site
+sites	I-site
+,	O
+a	O
+computational	O
+approach	O
+was	O
+designed	O
+to	O
+quantify	O
+the	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+change	I-evidence
+for	O
+each	O
+refined	O
+atom	O
+with	O
+increasing	O
+dose	O
+,	O
+thus	O
+providing	O
+a	O
+rapid	O
+systematic	O
+method	O
+for	O
+SRD	O
+study	O
+on	O
+such	O
+large	O
+multimeric	O
+complexes	O
+.	O
+
+By	O
+employing	O
+the	O
+high	O
+11	O
+-	O
+fold	O
+structural	O
+symmetry	O
+within	O
+each	O
+TRAP	B-complex_assembly
+macromolecule	O
+,	O
+this	O
+approach	O
+permitted	O
+a	O
+thorough	O
+statistical	O
+quantification	O
+of	O
+the	O
+RD	O
+effects	O
+of	O
+RNA	B-chemical
+binding	O
+to	O
+TRAP	B-complex_assembly
+.	O
+
+Per	B-experimental_method
+-	I-experimental_method
+atom	I-experimental_method
+quantification	I-experimental_method
+of	I-experimental_method
+electron	I-experimental_method
+density	I-experimental_method
+
+To	O
+quantify	O
+the	O
+exact	O
+effects	O
+of	O
+nucleic	O
+acid	O
+binding	O
+to	O
+a	O
+protein	O
+on	O
+SRD	O
+susceptibility	O
+,	O
+a	O
+high	O
+-	O
+throughput	O
+and	O
+automated	O
+pipeline	O
+was	O
+created	O
+to	O
+systematically	O
+calculate	O
+the	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+change	I-evidence
+for	O
+every	O
+refined	O
+atom	O
+within	O
+the	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+structure	B-evidence
+as	O
+a	O
+function	O
+of	O
+dose	O
+.	O
+
+This	O
+provides	O
+an	O
+atom	O
+-	O
+specific	O
+quantification	O
+of	O
+density	B-evidence
+–	I-evidence
+dose	I-evidence
+dynamics	I-evidence
+,	O
+which	O
+was	O
+previously	O
+lacking	O
+within	O
+the	O
+field	O
+.	O
+
+Previous	O
+studies	O
+have	O
+characterized	O
+SRD	B-site
+sites	I-site
+by	O
+reporting	O
+magnitudes	O
+of	O
+F	B-evidence
+obs	I-evidence
+(	I-evidence
+d	I-evidence
+n	I-evidence
+)	I-evidence
+−	I-evidence
+F	I-evidence
+obs	I-evidence
+(	I-evidence
+d	I-evidence
+1	I-evidence
+)	I-evidence
+Fourier	I-evidence
+difference	I-evidence
+map	I-evidence
+peaks	I-evidence
+in	O
+terms	O
+of	O
+the	O
+sigma	B-evidence
+(	O
+σ	B-evidence
+)	O
+contour	O
+level	O
+(	O
+the	O
+number	O
+of	O
+standard	B-evidence
+deviations	I-evidence
+from	O
+the	O
+mean	B-evidence
+map	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+value	I-evidence
+)	O
+at	O
+which	O
+peaks	O
+become	O
+visible	O
+.	O
+
+However	O
+,	O
+these	O
+σ	B-evidence
+levels	O
+depend	O
+on	O
+the	O
+standard	B-evidence
+deviation	I-evidence
+values	O
+of	O
+the	O
+map	B-evidence
+,	O
+which	O
+can	O
+deviate	O
+between	O
+data	O
+sets	O
+,	O
+and	O
+are	O
+thus	O
+unsuitable	O
+for	O
+quantitative	O
+comparison	O
+of	O
+density	B-evidence
+between	O
+different	O
+dose	O
+data	O
+sets	O
+.	O
+
+Instead	O
+,	O
+we	O
+use	O
+here	O
+a	O
+maximum	B-evidence
+density	I-evidence
+-	I-evidence
+loss	I-evidence
+metric	I-evidence
+(	O
+D	B-evidence
+loss	I-evidence
+),	O
+which	O
+is	O
+the	O
+per	O
+-	O
+atom	O
+equivalent	O
+of	O
+the	O
+magnitude	O
+of	O
+these	O
+negative	B-evidence
+Fourier	I-evidence
+difference	I-evidence
+map	I-evidence
+peaks	I-evidence
+in	O
+units	O
+of	O
+e	O
+Å	O
+−	O
+3	O
+.	O
+
+Large	O
+positive	O
+D	B-evidence
+loss	I-evidence
+values	O
+indicate	O
+radiation	O
+-	O
+induced	O
+atomic	O
+disordering	O
+reproducibly	O
+throughout	O
+the	O
+unit	O
+cells	O
+with	O
+respect	O
+to	O
+the	O
+initial	O
+low	O
+-	O
+dose	O
+data	O
+set	O
+.	O
+
+For	O
+each	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+data	O
+set	O
+,	O
+the	O
+D	B-evidence
+loss	I-evidence
+metric	I-evidence
+successfully	O
+identified	O
+the	O
+recognized	O
+forms	O
+of	O
+protein	O
+SRD	B-experimental_method
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+a	O
+),	O
+with	O
+clear	O
+Glu	B-residue_name
+and	O
+Asp	B-residue_name
+side	O
+-	O
+chain	O
+decarboxylation	O
+even	O
+in	O
+the	O
+first	O
+difference	B-evidence
+map	I-evidence
+calculated	O
+(	O
+3	O
+.	O
+9	O
+MGy	O
+;	O
+Fig	O
+.	O
+3	O
+▸	O
+a	O
+).	O
+
+The	O
+main	O
+sequence	O
+of	O
+TRAP	B-complex_assembly
+does	O
+not	O
+contain	O
+any	O
+Trp	B-residue_name
+and	O
+Cys	B-residue_name
+residues	O
+(	O
+and	O
+thus	O
+contains	O
+no	O
+disulfide	O
+bonds	O
+).	O
+
+The	O
+substrate	O
+Trp	B-chemical
+amino	O
+-	O
+acid	O
+ligands	O
+also	O
+exhibited	O
+disordering	O
+of	O
+the	O
+free	O
+terminal	O
+carboxyl	O
+groups	O
+at	O
+higher	O
+doses	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+a	O
+);	O
+however	O
+,	O
+no	O
+clear	O
+Fourier	B-evidence
+difference	I-evidence
+peaks	I-evidence
+could	O
+be	O
+observed	O
+visually	O
+.	O
+
+Even	O
+for	O
+radiation	O
+-	O
+insensitive	O
+residues	O
+(	O
+e	O
+.	O
+g	O
+.	O
+Gly	B-residue_name
+)	O
+the	O
+average	O
+D	B-evidence
+loss	I-evidence
+increases	O
+with	O
+dose	O
+:	O
+this	O
+is	O
+the	O
+effect	O
+of	O
+global	O
+radiation	O
+damage	O
+,	O
+since	O
+as	O
+dose	O
+increases	O
+the	O
+electron	B-evidence
+density	I-evidence
+associated	O
+with	O
+each	O
+refined	O
+atom	O
+becomes	O
+weaker	O
+as	O
+the	O
+atomic	O
+occupancy	O
+decreases	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+b	O
+).	O
+
+Only	O
+Glu	B-residue_name
+and	O
+Asp	B-residue_name
+residues	O
+exhibit	O
+a	O
+rate	O
+of	O
+D	B-evidence
+loss	I-evidence
+increase	O
+that	O
+consistently	O
+exceeds	O
+the	O
+average	O
+decay	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+b	O
+,	O
+dashed	O
+line	O
+)	O
+at	O
+each	O
+dose	O
+.	O
+
+The	O
+rate	O
+of	O
+D	B-evidence
+loss	I-evidence
+(	O
+attributed	O
+to	O
+side	O
+-	O
+chain	O
+decarboxylation	O
+)	O
+was	O
+consistently	O
+larger	O
+for	O
+Glu	B-residue_name
+compared	O
+with	O
+Asp	B-residue_name
+residues	O
+over	O
+the	O
+large	O
+dose	O
+range	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+S3	O
+);	O
+this	O
+observation	O
+is	O
+consistent	O
+with	O
+our	O
+calculations	O
+on	O
+model	O
+systems	O
+(	O
+see	O
+above	O
+)	O
+that	O
+suggest	O
+that	O
+,	O
+without	O
+considering	O
+differential	O
+hydrogen	O
+-	O
+bonding	O
+environments	O
+,	O
+CO2	B-chemical
+loss	O
+is	O
+more	O
+exothermic	O
+by	O
+around	O
+8	O
+kJ	O
+mol	O
+−	O
+1	O
+from	O
+oxidized	B-protein_state
+Glu	B-residue_name
+residues	O
+than	O
+from	O
+their	O
+Asp	B-residue_name
+counterparts	O
+.	O
+
+RNA	B-chemical
+is	O
+less	O
+susceptible	O
+to	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+loss	O
+than	O
+protein	O
+within	O
+the	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+
+Visual	B-experimental_method
+inspection	I-experimental_method
+of	I-experimental_method
+Fourier	B-evidence
+difference	I-evidence
+maps	I-evidence
+illustrated	O
+the	O
+clear	O
+lack	O
+of	O
+RNA	B-chemical
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+degradation	I-evidence
+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
+
+Only	O
+at	O
+the	O
+highest	O
+doses	O
+investigated	O
+(>	O
+20	O
+MGy	O
+)	O
+was	O
+density	O
+loss	O
+observed	O
+at	O
+the	O
+RNA	B-chemical
+phosphate	O
+and	O
+C	O
+—	O
+O	O
+bonds	O
+of	O
+the	O
+phosphodiester	O
+backbone	O
+.	O
+
+However	O
+,	O
+the	O
+median	O
+D	B-evidence
+loss	I-evidence
+was	O
+lower	O
+by	O
+a	O
+factor	O
+of	O
+>	O
+2	O
+for	O
+RNA	B-chemical
+P	O
+atoms	O
+than	O
+for	O
+Glu	B-residue_name
+and	O
+Asp	B-residue_name
+side	O
+-	O
+chain	O
+groups	O
+at	O
+25	O
+.	O
+0	O
+MGy	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+S4	O
+),	O
+and	O
+furthermore	O
+could	O
+not	O
+be	O
+numerically	O
+distinguished	O
+from	O
+Gly	B-residue_name
+Cα	O
+atoms	O
+within	O
+TRAP	B-complex_assembly
+,	O
+which	O
+are	O
+not	O
+radiation	O
+-	O
+sensitive	O
+at	O
+the	O
+doses	O
+tested	O
+here	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+S3	O
+).	O
+
+RNA	B-chemical
+binding	O
+protects	O
+radiation	O
+-	O
+sensitive	O
+residues	O
+
+For	O
+the	O
+large	O
+number	O
+of	O
+acidic	O
+residues	O
+per	O
+TRAP	B-complex_assembly
+ring	B-structure_element
+(	O
+four	O
+Asp	B-residue_name
+and	O
+six	O
+Glu	B-residue_name
+residues	O
+per	O
+protein	O
+monomer	B-oligomeric_state
+),	O
+a	O
+strong	O
+dependence	O
+of	O
+decarboxylation	O
+susceptibility	O
+on	O
+local	O
+environment	O
+was	O
+observed	O
+(	O
+Fig	O
+.	O
+4	O
+▸).	O
+
+For	O
+each	O
+Glu	B-residue_name
+Cδ	O
+or	O
+Asp	B-residue_name
+Cγ	O
+atom	O
+,	O
+D	B-evidence
+loss	I-evidence
+provided	O
+a	O
+direct	O
+measure	O
+of	O
+the	O
+rate	O
+of	O
+side	O
+-	O
+chain	O
+carboxyl	O
+-	O
+group	O
+disordering	O
+and	O
+subsequent	O
+decarboxylation	O
+.	O
+
+For	O
+acidic	O
+residues	O
+with	O
+no	O
+differing	O
+interactions	O
+between	O
+nonbound	B-protein_state
+and	O
+bound	B-protein_state
+TRAP	B-complex_assembly
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+a	O
+),	O
+similar	O
+damage	O
+was	O
+apparent	O
+between	O
+the	O
+two	O
+rings	O
+within	O
+the	O
+asymmetric	O
+unit	O
+,	O
+as	O
+expected	O
+.	O
+
+However	O
+,	O
+TRAP	B-complex_assembly
+residues	O
+directly	O
+on	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+exhibited	O
+greater	O
+damage	O
+accumulation	O
+in	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+b	O
+),	O
+and	O
+for	O
+residues	O
+at	O
+the	O
+ring	B-site
+–	I-site
+ring	I-site
+interfaces	I-site
+(	O
+where	O
+crystal	O
+contacts	O
+were	O
+detected	O
+)	O
+bound	B-protein_state
+TRAP	B-complex_assembly
+exhibited	O
+enhanced	O
+SRD	O
+accumulation	O
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+c	O
+).	O
+
+Three	O
+acidic	O
+residues	O
+(	O
+Glu36	B-residue_name_number
+,	O
+Asp39	B-residue_name_number
+and	O
+Glu42	B-residue_name_number
+)	O
+are	O
+involved	O
+in	O
+RNA	B-chemical
+interactions	O
+within	O
+each	O
+of	O
+the	O
+11	O
+TRAP	B-complex_assembly
+ring	B-structure_element
+subunits	B-structure_element
+,	O
+and	O
+Fig	O
+.	O
+5	O
+▸	O
+shows	O
+their	O
+density	B-evidence
+changes	I-evidence
+with	O
+increasing	O
+dose	O
+.	O
+
+Hotelling	B-experimental_method
+’	I-experimental_method
+s	I-experimental_method
+T	I-experimental_method
+-	I-experimental_method
+squared	I-experimental_method
+test	I-experimental_method
+(	O
+the	O
+multivariate	O
+counterpart	O
+of	O
+Student	B-experimental_method
+’	I-experimental_method
+s	I-experimental_method
+t	I-experimental_method
+-	I-experimental_method
+test	I-experimental_method
+)	O
+was	O
+used	O
+to	O
+reject	O
+the	O
+null	O
+hypothesis	O
+that	O
+the	O
+means	O
+of	O
+the	O
+D	B-evidence
+loss	I-evidence
+metric	I-evidence
+were	O
+equal	O
+for	O
+the	O
+bound	B-protein_state
+and	O
+nonbound	B-protein_state
+groups	O
+in	O
+Fig	O
+.	O
+5	O
+▸.	O
+
+A	O
+significant	O
+reduction	O
+in	O
+D	B-evidence
+loss	I-evidence
+is	O
+seen	O
+for	O
+Glu36	B-residue_name_number
+in	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+compared	O
+with	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+,	O
+indicative	O
+of	O
+a	O
+lower	O
+rate	O
+of	O
+side	O
+-	O
+chain	O
+decarboxylation	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+a	O
+;	O
+p	O
+=	O
+6	O
+.	O
+06	O
+×	O
+10	O
+−	O
+5	O
+).	O
+
+For	O
+each	O
+TRAP	B-complex_assembly
+ring	B-structure_element
+subunit	B-structure_element
+,	O
+the	O
+Glu36	B-residue_name_number
+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	B-residue_name_number
+RNA	B-chemical
+base	O
+.	O
+
+In	O
+our	O
+analysis	O
+,	O
+Asp39	B-residue_name_number
+in	O
+the	O
+TRAP	B-complex_assembly
+–(	I-complex_assembly
+GAGUU	I-complex_assembly
+)	I-complex_assembly
+10GAG	I-complex_assembly
+structure	B-evidence
+appears	O
+to	O
+exhibit	O
+two	O
+distinct	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+G1	B-residue_name_number
+base	O
+within	O
+each	O
+of	O
+the	O
+11	O
+TRAP	B-site
+–	I-site
+RNA	I-site
+interfaces	I-site
+,	O
+as	O
+does	O
+Glu36	B-residue_name_number
+to	O
+G3	B-residue_name_number
+;	O
+however	O
+,	O
+the	O
+reduction	O
+in	O
+density	B-evidence
+disordering	O
+upon	O
+RNA	B-chemical
+binding	O
+is	O
+far	O
+less	O
+significant	O
+for	O
+Asp39	B-residue_name_number
+than	O
+for	O
+Glu36	B-residue_name_number
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+b	O
+,	O
+p	O
+=	O
+0	O
+.	O
+0925	O
+).	O
+
+RNA	B-chemical
+binding	O
+reduces	O
+radiation	O
+-	O
+induced	O
+disorder	O
+on	O
+the	O
+atomic	O
+scale	O
+
+One	O
+oxygen	O
+(	O
+O	O
+∊	O
+1	O
+)	O
+of	O
+Glu42	B-residue_name_number
+appears	O
+to	O
+form	O
+a	O
+hydrogen	O
+bond	O
+to	O
+a	O
+nearby	O
+water	B-chemical
+within	O
+each	O
+TRAP	B-site
+RNA	I-site
+-	I-site
+binding	I-site
+pocket	I-site
+,	O
+with	O
+the	O
+other	O
+(	O
+O	O
+∊	O
+2	O
+)	O
+being	O
+involved	O
+in	O
+a	O
+salt	O
+-	O
+bridge	O
+interaction	O
+with	O
+Arg58	B-residue_name_number
+(	O
+Hopcroft	O
+et	O
+al	O
+.,	O
+2002	O
+;	O
+Antson	O
+et	O
+al	O
+.,	O
+1999	O
+).	O
+
+Salt	O
+-	O
+bridge	O
+interactions	O
+have	O
+previously	O
+been	O
+suggested	O
+to	O
+reduce	O
+the	O
+glutamate	B-residue_name
+decarboxylation	O
+rate	O
+within	O
+the	O
+large	O
+(∼	O
+62	O
+.	O
+4	O
+kDa	O
+)	O
+myrosinase	B-protein_type
+protein	O
+structure	B-evidence
+(	O
+Burmeister	O
+,	O
+2000	O
+).	O
+
+A	O
+significant	O
+difference	O
+was	O
+observed	O
+between	O
+the	O
+D	B-evidence
+loss	I-evidence
+dynamics	I-evidence
+for	O
+the	O
+nonbound	B-protein_state
+/	O
+bound	B-protein_state
+Glu42	B-residue_name_number
+O	O
+∊	O
+1	O
+atoms	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+c	O
+;	O
+p	O
+=	O
+0	O
+.	O
+007	O
+)	O
+but	O
+not	O
+for	O
+the	O
+Glu42	B-residue_name_number
+O	O
+∊	O
+2	O
+atoms	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+d	O
+;	O
+p	O
+=	O
+0	O
+.	O
+239	O
+),	O
+indicating	O
+that	O
+the	O
+stabilizing	O
+strength	O
+of	O
+this	O
+salt	O
+-	O
+bridge	O
+interaction	O
+was	O
+conserved	O
+upon	O
+RNA	B-chemical
+binding	O
+and	O
+that	O
+the	O
+water	B-chemical
+-	O
+mediated	O
+hydrogen	O
+bond	O
+had	O
+a	O
+greater	O
+relative	O
+susceptibility	O
+to	O
+atomic	O
+disordering	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+RNA	B-chemical
+.	O
+
+The	O
+density	B-evidence
+-	I-evidence
+change	I-evidence
+dynamics	I-evidence
+were	O
+statistically	O
+indistinguishable	O
+between	O
+bound	B-protein_state
+and	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+for	O
+each	O
+Glu42	B-residue_name_number
+carboxyl	O
+group	O
+Cδ	O
+atom	O
+(	O
+p	O
+=	O
+0	O
+.	O
+435	O
+),	O
+indicating	O
+that	O
+upon	O
+RNA	B-chemical
+binding	O
+the	O
+conserved	O
+salt	O
+-	O
+bridge	O
+interaction	O
+ultimately	O
+dictated	O
+the	O
+overall	O
+Glu42	B-residue_name_number
+decarboxylation	O
+rate	O
+.	O
+
+The	O
+RNA	B-chemical
+-	O
+stabilizing	O
+effect	O
+was	O
+not	O
+restricted	O
+to	O
+radiation	O
+-	O
+sensitive	O
+acidic	O
+residues	O
+.	O
+
+The	O
+side	O
+chain	O
+of	O
+Phe32	B-residue_name_number
+stacks	O
+against	O
+the	O
+G3	B-residue_name_number
+base	O
+within	O
+the	O
+11	O
+TRAP	B-site
+RNA	I-site
+-	I-site
+binding	I-site
+interfaces	I-site
+(	O
+Antson	O
+et	O
+al	O
+.,	O
+1999	O
+).	O
+
+With	O
+increasing	O
+dose	O
+,	O
+the	O
+D	B-evidence
+loss	I-evidence
+associated	O
+with	O
+the	O
+Phe32	B-residue_name_number
+side	O
+chain	O
+was	O
+significantly	O
+reduced	O
+upon	O
+RNA	B-chemical
+binding	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+e	O
+;	O
+Phe32	B-residue_name_number
+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	B-residue_name_number
+had	O
+been	O
+reduced	O
+.	O
+
+The	O
+extended	O
+aliphatic	O
+Lys37	B-residue_name_number
+side	O
+chain	O
+stacks	O
+against	O
+the	O
+nearby	O
+G1	B-residue_name_number
+base	O
+,	O
+making	O
+a	O
+series	O
+of	O
+nonpolar	O
+contacts	O
+within	O
+each	O
+RNA	B-site
+-	I-site
+binding	I-site
+interface	I-site
+.	O
+
+The	O
+D	B-evidence
+loss	I-evidence
+for	O
+Lys37	B-residue_name_number
+side	O
+-	O
+chain	O
+atoms	O
+was	O
+also	O
+reduced	O
+when	O
+stacked	O
+against	O
+the	O
+G1	B-residue_name_number
+base	O
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+f	O
+;	O
+p	O
+=	O
+0	O
+.	O
+0243	O
+for	O
+Lys37	B-residue_name_number
+C	O
+∊	O
+atoms	O
+).	O
+
+Representative	O
+Phe32	B-residue_name_number
+and	O
+Lys37	B-residue_name_number
+atoms	O
+were	O
+selected	O
+to	O
+illustrate	O
+these	O
+trends	O
+.	O
+
+Here	O
+,	O
+MX	B-experimental_method
+radiation	O
+-	O
+induced	O
+specific	O
+structural	O
+changes	O
+within	O
+the	O
+large	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+assembly	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+.	O
+0	O
+MGy	O
+)	O
+have	O
+been	O
+analysed	O
+using	O
+a	O
+high	O
+-	O
+throughput	O
+quantitative	O
+approach	O
+,	O
+providing	O
+a	O
+measure	O
+of	O
+the	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+distribution	I-evidence
+for	O
+each	O
+refined	O
+atom	O
+with	O
+increasing	O
+dose	O
+,	O
+D	B-evidence
+loss	I-evidence
+.	O
+
+Compared	O
+with	O
+previous	O
+studies	O
+,	O
+the	O
+results	O
+provide	O
+a	O
+further	O
+step	O
+in	O
+the	O
+detailed	O
+characterization	O
+of	O
+SRD	O
+effects	O
+in	O
+MX	B-experimental_method
+.	O
+
+Our	O
+method	O
+­	O
+ology	O
+,	O
+which	O
+eliminated	O
+tedious	O
+and	O
+error	O
+-	O
+prone	O
+visual	O
+inspection	O
+,	O
+permitted	O
+the	O
+determination	O
+on	O
+a	O
+per	O
+-	O
+atom	O
+basis	O
+of	O
+the	O
+most	O
+damaged	O
+sites	O
+,	O
+as	O
+characterized	O
+by	O
+F	B-evidence
+obs	I-evidence
+(	I-evidence
+d	I-evidence
+n	I-evidence
+)	I-evidence
+−	I-evidence
+F	I-evidence
+obs	I-evidence
+(	I-evidence
+d	I-evidence
+1	I-evidence
+)	I-evidence
+Fourier	I-evidence
+difference	I-evidence
+map	I-evidence
+peaks	I-evidence
+between	O
+successive	O
+data	O
+sets	O
+collected	O
+from	O
+the	O
+same	O
+crystal	B-evidence
+.	O
+
+Here	O
+,	O
+it	O
+provided	O
+the	O
+precision	O
+required	O
+to	O
+quantify	O
+the	O
+role	O
+of	O
+RNA	B-chemical
+in	O
+the	O
+damage	O
+susceptibilities	O
+of	O
+equivalent	O
+atoms	O
+between	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+and	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+,	O
+but	O
+it	O
+is	O
+applicable	O
+to	O
+any	O
+MX	B-experimental_method
+SRD	O
+study	O
+.	O
+
+The	O
+RNA	B-chemical
+was	O
+found	O
+to	O
+be	O
+substantially	O
+more	O
+radiation	B-protein_state
+-	I-protein_state
+resistant	I-protein_state
+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	B-experimental_method
+investigation	I-experimental_method
+of	O
+the	O
+C	B-complex_assembly
+.	I-complex_assembly
+Esp1396I	I-complex_assembly
+protein	O
+–	O
+DNA	B-chemical
+complex	O
+(	O
+Bury	O
+et	O
+al	O
+.,	O
+2015	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	B-evidence
+obs	I-evidence
+(	I-evidence
+d	I-evidence
+n	I-evidence
+)	I-evidence
+−	I-evidence
+F	I-evidence
+obs	I-evidence
+(	I-evidence
+d	I-evidence
+1	I-evidence
+)	I-evidence
+map	I-evidence
+density	I-evidence
+was	O
+detected	O
+around	O
+P	O
+,	O
+O3	O
+′	O
+and	O
+O5	O
+′	O
+atoms	O
+of	O
+the	O
+RNA	B-chemical
+backbone	O
+,	O
+with	O
+no	O
+significant	O
+difference	B-evidence
+density	I-evidence
+localized	O
+to	O
+RNA	B-chemical
+ribose	O
+and	O
+basic	O
+subunits	B-structure_element
+.	O
+
+RNA	B-chemical
+backbone	O
+disordering	O
+thus	O
+appears	O
+to	O
+be	O
+the	O
+main	O
+radiation	O
+-	O
+induced	O
+effect	O
+in	O
+RNA	B-chemical
+,	O
+with	O
+the	O
+protein	O
+–	O
+base	O
+interactions	O
+maintained	O
+even	O
+at	O
+high	O
+doses	O
+(>	O
+20	O
+MGy	O
+).	O
+
+The	O
+U4	B-residue_name_number
+phosphate	B-chemical
+exhibited	O
+marginally	O
+larger	O
+D	B-evidence
+loss	I-evidence
+values	O
+above	O
+20	O
+MGy	O
+than	O
+G1	B-residue_name_number
+,	O
+A2	B-residue_name_number
+and	O
+G3	B-residue_name_number
+(	O
+Supplementary	O
+Fig	O
+.	O
+S4	O
+).	O
+
+Since	O
+U4	B-residue_name_number
+is	O
+the	O
+only	O
+refined	O
+nucleotide	O
+not	O
+to	O
+exhibit	O
+significant	O
+base	O
+–	O
+protein	O
+interactions	O
+around	O
+TRAP	B-complex_assembly
+(	O
+with	O
+a	O
+water	B-chemical
+-	O
+mediated	O
+hydrogen	O
+bond	O
+detected	O
+in	O
+only	O
+three	O
+of	O
+the	O
+11	O
+subunits	B-structure_element
+and	O
+a	O
+single	O
+Arg58	B-residue_name_number
+hydrogen	O
+bond	O
+suggested	O
+in	O
+a	O
+further	O
+four	O
+subunits	B-structure_element
+),	O
+this	O
+increased	O
+U4	B-residue_name_number
+D	B-evidence
+loss	I-evidence
+can	O
+be	O
+explained	O
+owing	O
+to	O
+its	O
+greater	O
+flexibility	O
+.	O
+
+At	O
+25	O
+.	O
+0	O
+MGy	O
+,	O
+the	O
+magnitude	O
+of	O
+the	O
+RNA	B-chemical
+backbone	O
+D	B-evidence
+loss	I-evidence
+was	O
+of	O
+the	O
+same	O
+order	O
+as	O
+for	O
+the	O
+radiation	O
+-	O
+insensitive	O
+Gly	B-residue_name
+Cα	O
+atoms	O
+and	O
+on	O
+average	O
+less	O
+than	O
+half	O
+that	O
+of	O
+the	O
+acidic	O
+residues	O
+of	O
+the	O
+protein	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+S3	O
+).	O
+
+Consequently	O
+,	O
+no	O
+clear	O
+single	O
+-	O
+strand	O
+breaks	O
+could	O
+be	O
+located	O
+,	O
+and	O
+since	O
+RNA	B-chemical
+-	O
+binding	O
+within	O
+the	O
+current	O
+TRAP	B-complex_assembly
+–(	I-complex_assembly
+GAGUU	I-complex_assembly
+)	I-complex_assembly
+10GAG	I-complex_assembly
+complex	O
+is	O
+mediated	O
+predominantly	O
+through	O
+base	O
+–	O
+protein	O
+interactions	O
+,	O
+the	O
+biological	O
+integrity	O
+of	O
+the	O
+RNA	B-chemical
+complex	O
+was	O
+dictated	O
+by	O
+the	O
+rate	O
+at	O
+which	O
+protein	O
+decarboxylation	O
+occurred	O
+.	O
+
+RNA	B-chemical
+interacting	O
+with	O
+TRAP	B-complex_assembly
+was	O
+shown	O
+to	O
+offer	O
+significant	O
+protection	O
+against	O
+radiation	O
+-	O
+induced	O
+structural	O
+changes	O
+.	O
+
+Both	O
+Glu36	B-residue_name_number
+and	O
+Asp39	B-residue_name_number
+bind	O
+directly	O
+to	O
+RNA	B-chemical
+,	O
+each	O
+through	O
+two	O
+hydrogen	O
+bonds	O
+to	O
+guanine	B-chemical
+bases	O
+(	O
+G3	B-residue_name_number
+and	O
+G1	B-residue_name_number
+,	O
+respectively	O
+).	O
+
+However	O
+,	O
+compared	O
+with	O
+Asp39	B-residue_name_number
+,	O
+Glu36	B-residue_name_number
+is	O
+strikingly	O
+less	O
+decarboxylated	O
+when	O
+bound	B-protein_state
+to	I-protein_state
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+4	O
+▸).	O
+
+This	O
+is	O
+in	O
+good	O
+agreement	O
+with	O
+previous	O
+mutagenesis	B-experimental_method
+and	I-experimental_method
+nucleoside	I-experimental_method
+analogue	I-experimental_method
+studies	I-experimental_method
+(	O
+Elliott	O
+et	O
+al	O
+.,	O
+2001	O
+),	O
+which	O
+indicated	O
+that	O
+the	O
+G1	B-residue_name_number
+nucleotide	O
+does	O
+not	O
+bind	O
+to	O
+TRAP	B-complex_assembly
+as	O
+strongly	O
+as	O
+do	O
+A2	B-residue_name_number
+and	O
+G3	B-residue_name_number
+,	O
+and	O
+plays	O
+little	O
+role	O
+in	O
+the	O
+high	O
+RNA	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+TRAP	B-complex_assembly
+(	O
+K	B-evidence
+d	I-evidence
+≃	O
+1	O
+.	O
+1	O
+±	O
+0	O
+.	O
+4	O
+nM	O
+).	O
+
+For	O
+Glu36	B-residue_name_number
+and	O
+Asp39	B-residue_name_number
+,	O
+no	O
+direct	O
+quantitative	O
+correlation	O
+could	O
+be	O
+established	O
+between	O
+hydrogen	O
+-	O
+bond	O
+length	O
+and	O
+D	B-evidence
+loss	I-evidence
+(	O
+linear	B-evidence
+R	I-evidence
+2	I-evidence
+of	O
+<	O
+0	O
+.	O
+23	O
+for	O
+all	O
+doses	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+S5	O
+).	O
+
+Thus	O
+,	O
+another	O
+factor	O
+must	O
+be	O
+responsible	O
+for	O
+this	O
+clear	O
+reduction	O
+in	O
+Glu36	B-residue_name_number
+CO2	O
+decarboxyl	O
+­	O
+ation	O
+in	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+TRAP	B-complex_assembly
+.	O
+
+The	O
+Glu36	B-residue_name_number
+carboxyl	O
+side	O
+chain	O
+also	O
+potentially	O
+forms	O
+hydrogen	O
+bonds	O
+to	O
+His34	B-residue_name_number
+and	O
+Lys56	B-residue_name_number
+,	O
+but	O
+since	O
+these	O
+interactions	O
+are	O
+conserved	B-protein_state
+irrespective	O
+of	O
+G3	B-residue_name_number
+nucleotide	O
+binding	O
+,	O
+this	O
+cannot	O
+directly	O
+account	O
+for	O
+the	O
+stabilization	O
+effect	O
+on	O
+Glu36	B-residue_name_number
+in	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+TRAP	B-complex_assembly
+.	O
+
+When	O
+bound	B-protein_state
+to	I-protein_state
+RNA	B-chemical
+,	O
+the	O
+average	O
+solvent	O
+-	O
+accessible	O
+area	O
+of	O
+the	O
+Glu36	B-residue_name_number
+side	O
+-	O
+chain	O
+O	O
+atoms	O
+is	O
+reduced	O
+from	O
+∼	O
+15	O
+to	O
+0	O
+Å2	O
+.	O
+
+We	O
+propose	O
+that	O
+with	O
+no	O
+solvent	O
+accessibility	O
+Glu36	B-residue_name_number
+decarboxylation	O
+is	O
+inhibited	O
+,	O
+since	O
+the	O
+CO2	B-evidence
+-	I-evidence
+formation	I-evidence
+rate	I-evidence
+K	I-evidence
+2	I-evidence
+is	O
+greatly	O
+reduced	O
+,	O
+and	O
+suggest	O
+that	O
+steric	O
+hindrance	O
+prevents	O
+each	O
+radicalized	O
+Glu36	B-residue_name_number
+CO2	O
+group	O
+from	O
+achieving	O
+the	O
+planar	O
+conformation	O
+required	O
+for	O
+complete	O
+dissociation	O
+from	O
+TRAP	B-complex_assembly
+.	O
+
+The	O
+electron	B-evidence
+-	I-evidence
+recombination	I-evidence
+rate	I-evidence
+K	I-evidence
+−	I-evidence
+1	I-evidence
+remains	O
+high	O
+,	O
+however	O
+,	O
+owing	O
+to	O
+rapid	O
+electron	O
+migration	O
+through	O
+the	O
+protein	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+to	O
+refill	O
+the	O
+Glu36	B-residue_name_number
+positive	B-site
+hole	I-site
+(	O
+the	O
+precursor	O
+for	O
+Glu	B-residue_name
+decarboxylation	O
+).	O
+
+Upon	O
+RNA	B-chemical
+binding	O
+,	O
+the	O
+Asp39	B-residue_name_number
+side	O
+-	O
+chain	O
+carboxyl	O
+group	O
+solvent	O
+-	O
+accessible	O
+area	O
+changes	O
+from	O
+∼	O
+75	O
+to	O
+35	O
+Å2	O
+,	O
+still	O
+allowing	O
+a	O
+high	O
+CO2	B-chemical
+-	O
+formation	O
+rate	B-evidence
+K	I-evidence
+2	I-evidence
+.	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	B-evidence
+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
+
+By	O
+comparing	O
+equivalent	O
+acidic	O
+residues	O
+with	B-protein_state
+and	O
+without	B-protein_state
+RNA	B-chemical
+,	O
+we	O
+have	O
+now	O
+deconvoluted	O
+the	O
+role	O
+of	O
+solvent	O
+accessibility	O
+from	O
+other	O
+local	O
+protein	O
+environment	O
+factors	O
+,	O
+and	O
+thus	O
+propose	O
+a	O
+suitable	O
+mechanism	O
+by	O
+which	O
+exceptionally	O
+low	O
+solvent	O
+accessibility	O
+can	O
+reduce	O
+the	O
+rate	O
+of	O
+decarboxylation	O
+.	O
+
+Apart	O
+from	O
+these	O
+RNA	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+,	O
+RNA	B-chemical
+binding	O
+was	O
+seen	O
+to	O
+enhance	O
+decarboxylation	O
+for	O
+residues	O
+Glu50	B-residue_name_number
+,	O
+Glu71	B-residue_name_number
+and	O
+Glu73	B-residue_name_number
+,	O
+all	O
+of	O
+which	O
+are	O
+involved	O
+in	O
+crystal	O
+contacts	O
+between	O
+TRAP	B-complex_assembly
+rings	B-structure_element
+(	O
+Fig	O
+.	O
+4	O
+▸	O
+c	O
+).	O
+
+However	O
+,	O
+for	O
+each	O
+of	O
+these	O
+residues	O
+the	O
+exact	O
+crystal	O
+contacts	O
+are	O
+not	O
+preserved	O
+between	O
+bound	B-protein_state
+and	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+or	O
+even	O
+between	O
+monomers	O
+within	O
+one	O
+TRAP	B-complex_assembly
+ring	B-structure_element
+.	O
+
+For	O
+example	O
+,	O
+in	O
+bound	B-protein_state
+TRAP	B-complex_assembly
+,	O
+Glu73	B-residue_name_number
+hydrogen	O
+-	O
+bonds	O
+to	O
+a	O
+nearby	O
+lysine	B-residue_name
+on	O
+each	O
+of	O
+the	O
+11	O
+subunits	B-structure_element
+,	O
+whereas	O
+in	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+no	O
+such	O
+interaction	O
+exists	O
+and	O
+Glu73	B-residue_name_number
+interacts	O
+with	O
+a	O
+variable	O
+number	O
+of	O
+refined	O
+waters	B-chemical
+in	O
+each	O
+subunit	B-structure_element
+.	O
+
+Radiation	O
+-	O
+induced	O
+side	O
+-	O
+chain	O
+conformational	O
+changes	O
+have	O
+been	O
+poorly	O
+characterized	O
+in	O
+previous	O
+SRD	B-experimental_method
+investigations	I-experimental_method
+owing	O
+to	O
+their	O
+strong	O
+dependence	O
+on	O
+packing	O
+density	O
+and	O
+geometric	O
+strain	O
+.	O
+
+Such	O
+structural	O
+changes	O
+are	O
+known	O
+to	O
+have	O
+significant	O
+roles	O
+within	O
+enzymatic	O
+pathways	O
+,	O
+and	O
+experimenters	O
+must	O
+be	O
+aware	O
+of	O
+these	O
+possible	O
+confounding	O
+factors	O
+when	O
+assigning	O
+true	O
+functional	O
+mechanisms	O
+using	O
+MX	B-experimental_method
+.	O
+
+Our	O
+results	O
+show	O
+that	O
+RNA	B-chemical
+binding	O
+to	O
+TRAP	B-complex_assembly
+physically	O
+stabilizes	O
+non	O
+-	O
+acidic	O
+residues	O
+within	O
+the	O
+TRAP	B-complex_assembly
+macromolecule	O
+,	O
+most	O
+notably	O
+Lys37	B-residue_name_number
+and	O
+Phe32	B-residue_name_number
+,	O
+which	O
+stack	O
+against	O
+the	O
+G1	B-residue_name_number
+and	O
+G3	B-residue_name_number
+bases	O
+,	O
+respectively	O
+.	O
+
+It	O
+has	O
+been	O
+suggested	O
+(	O
+Burmeister	O
+,	O
+2000	O
+)	O
+that	O
+Tyr	B-residue_name
+residues	O
+can	O
+lose	O
+their	O
+aromatic	O
+–	O
+OH	O
+group	O
+owing	O
+to	O
+radiation	O
+-	O
+induced	O
+effects	O
+;	O
+however	O
+,	O
+no	O
+energetically	O
+favourable	O
+pathway	O
+for	O
+–	O
+OH	O
+cleavage	O
+exists	O
+and	O
+this	O
+has	O
+not	O
+been	O
+detected	O
+in	O
+aqueous	O
+radiation	O
+-	O
+chemistry	O
+studies	O
+.	O
+
+In	O
+TRAP	B-complex_assembly
+,	O
+D	B-evidence
+loss	I-evidence
+increased	O
+at	O
+a	O
+similar	O
+rate	O
+for	O
+both	O
+the	O
+Tyr	B-residue_name
+O	O
+atoms	O
+and	O
+aromatic	O
+ring	B-structure_element
+atoms	O
+,	O
+suggesting	O
+that	O
+full	O
+ring	B-structure_element
+conformational	O
+disordering	O
+is	O
+more	O
+likely	O
+.	O
+
+Indeed	O
+,	O
+no	O
+convincing	O
+reproducible	O
+Fourier	B-evidence
+difference	I-evidence
+peaks	I-evidence
+above	O
+the	O
+background	O
+map	B-evidence
+noise	O
+were	O
+observed	O
+around	O
+any	O
+Tyr	B-residue_name
+terminal	O
+–	O
+OH	O
+groups	O
+.	O
+
+The	O
+RNA	B-chemical
+-	O
+stabilization	O
+effects	O
+on	O
+protein	O
+are	O
+observed	O
+at	O
+short	O
+ranges	O
+and	O
+are	O
+restricted	O
+to	O
+within	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+around	O
+the	O
+TRAP	B-complex_assembly
+ring	B-structure_element
+.	O
+
+For	O
+example	O
+,	O
+Asp17	B-residue_name_number
+is	O
+located	O
+∼	O
+6	O
+.	O
+8	O
+Å	O
+from	O
+the	O
+G1	B-residue_name_number
+base	O
+,	O
+outside	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+,	O
+and	O
+has	O
+indistinguishable	O
+Cγ	O
+atom	O
+D	O
+loss	B-evidence
+dose	I-evidence
+-	I-evidence
+dynamics	I-evidence
+between	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+and	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+(	O
+p	O
+>	O
+0	O
+.	O
+9	O
+).	O
+
+An	O
+increase	O
+in	O
+the	O
+dose	O
+at	O
+which	O
+functionally	O
+important	O
+residues	O
+remain	O
+intact	O
+has	O
+biological	O
+ramifications	O
+for	O
+understanding	O
+the	O
+mechanisms	O
+at	O
+which	O
+ionizing	O
+radiation	O
+damage	O
+is	O
+mitigated	O
+within	O
+naturally	O
+forming	O
+DNA	B-complex_assembly
+–	I-complex_assembly
+protein	I-complex_assembly
+and	O
+RNA	B-complex_assembly
+–	I-complex_assembly
+protein	I-complex_assembly
+complexes	O
+.	O
+
+Observations	O
+of	O
+lower	O
+protein	O
+radiation	O
+-	O
+sensitivity	O
+in	O
+DNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+forms	O
+have	O
+been	O
+recorded	O
+in	O
+solution	O
+at	O
+RT	O
+at	O
+much	O
+lower	O
+doses	O
+(∼	O
+1	O
+kGy	O
+)	O
+than	O
+those	O
+used	O
+for	O
+typical	O
+MX	B-experimental_method
+experiments	O
+[	O
+e	O
+.	O
+g	O
+.	O
+an	O
+oestrogen	O
+response	O
+element	O
+–	O
+receptor	O
+complex	O
+(	O
+Stísová	O
+et	O
+al	O
+.,	O
+2006	O
+)	O
+and	O
+a	O
+DNA	B-protein_type
+glycosylase	I-protein_type
+and	O
+its	O
+abasic	B-site
+DNA	I-site
+target	I-site
+site	I-site
+(	O
+Gillard	O
+et	O
+al	O
+.,	O
+2004	O
+)].	O
+
+In	O
+these	O
+studies	O
+,	O
+the	O
+main	O
+damaging	O
+species	O
+is	O
+predicted	O
+to	O
+be	O
+the	O
+oxidizing	O
+hydroxyl	O
+radical	O
+produced	O
+through	O
+solvent	O
+irradiation	O
+,	O
+which	O
+is	O
+known	O
+to	O
+add	O
+to	O
+double	O
+covalent	O
+bonds	O
+within	O
+both	O
+DNA	B-chemical
+and	O
+RNA	B-chemical
+bases	O
+to	O
+induce	O
+strand	O
+breaks	O
+and	O
+base	O
+modification	O
+(	O
+Spotheim	O
+-	O
+Maurizot	O
+&	O
+Davídková	O
+,	O
+2011	O
+;	O
+Chance	O
+et	O
+al	O
+.,	O
+1997	O
+).	O
+
+It	O
+was	O
+suggested	O
+that	O
+physical	O
+screening	O
+of	O
+DNA	B-chemical
+by	O
+protein	O
+shielded	O
+the	O
+DNA	B-site
+–	I-site
+protein	I-site
+interaction	I-site
+sites	I-site
+from	O
+radical	O
+damage	O
+,	O
+yielding	O
+an	O
+extended	O
+life	O
+-	O
+dose	O
+for	O
+the	O
+nucleoprotein	O
+complex	O
+compared	O
+with	O
+separate	O
+protein	O
+and	O
+DNA	B-chemical
+constituents	O
+at	O
+RT	O
+.	O
+
+However	O
+,	O
+in	O
+the	O
+current	O
+MX	B-experimental_method
+study	O
+at	O
+100	O
+K	O
+,	O
+the	O
+main	O
+damaging	O
+species	O
+are	O
+believed	O
+to	O
+be	O
+migrating	O
+LEEs	O
+and	O
+holes	O
+produced	O
+directly	O
+within	O
+the	O
+protein	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+components	O
+or	O
+in	O
+closely	O
+associated	O
+solvent	O
+.	O
+
+The	O
+results	O
+presented	O
+here	O
+suggest	O
+that	O
+biologically	O
+relevant	O
+nucleoprotein	B-complex_assembly
+complexes	O
+also	O
+exhibit	O
+prolonged	O
+life	O
+-	O
+doses	O
+under	O
+the	O
+effect	O
+of	O
+LEE	O
+-	O
+induced	O
+structural	O
+changes	O
+,	O
+involving	O
+direct	O
+physical	O
+protection	O
+of	O
+key	O
+RNA	B-site
+-	I-site
+binding	I-site
+residues	I-site
+.	O
+
+Such	O
+reduced	O
+radiation	O
+-	O
+sensitivity	O
+in	O
+this	O
+case	O
+ensures	O
+that	O
+the	O
+interacting	O
+protein	O
+remains	O
+bound	B-protein_state
+long	O
+enough	O
+to	O
+the	O
+RNA	B-chemical
+to	O
+complete	O
+its	O
+function	O
+,	O
+even	O
+whilst	O
+exposed	O
+to	O
+ionizing	O
+radiation	O
+.	O
+
+Within	O
+the	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+macromolecule	O
+,	O
+the	O
+acidic	O
+residues	O
+within	O
+the	O
+unoccupied	O
+RNA	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+(	O
+Asp39	B-residue_name_number
+,	O
+Glu36	B-residue_name_number
+,	O
+Glu42	B-residue_name_number
+)	O
+are	O
+notably	O
+amongst	O
+the	O
+most	O
+susceptible	O
+residues	O
+within	O
+the	O
+asymmetric	O
+unit	O
+(	O
+Fig	O
+.	O
+4	O
+▸).	O
+
+When	O
+exposed	O
+to	O
+X	O
+-	O
+rays	O
+,	O
+these	O
+residues	O
+will	O
+be	O
+preferentially	O
+damaged	O
+by	O
+X	O
+-	O
+rays	O
+and	O
+subsequently	O
+reduce	O
+the	O
+affinity	O
+with	O
+which	O
+TRAP	B-complex_assembly
+binds	O
+to	O
+RNA	B-chemical
+.	O
+
+Within	O
+the	O
+cellular	O
+environment	O
+,	O
+this	O
+mechanism	O
+could	O
+reduce	O
+the	O
+risk	O
+that	O
+radiation	O
+-	O
+damaged	O
+proteins	O
+might	O
+bind	O
+to	O
+RNA	B-chemical
+,	O
+thus	O
+avoiding	O
+the	O
+detrimental	O
+introduction	O
+of	O
+incorrect	O
+DNA	B-chemical
+-	O
+repair	O
+,	O
+transcriptional	O
+and	O
+base	O
+-	O
+modification	O
+pathways	O
+.	O
+
+The	O
+TRAP	B-complex_assembly
+–(	I-complex_assembly
+GAGUU	I-complex_assembly
+)	I-complex_assembly
+10GAG	I-complex_assembly
+complex	O
+asymmetric	O
+unit	O
+(	O
+PDB	O
+entry	O
+1gtf	O
+;	O
+Hopcroft	O
+et	O
+al	O
+.,	O
+2002	O
+).	O
+
+Bound	B-protein_state
+tryptophan	B-chemical
+ligands	O
+are	O
+represented	O
+as	O
+coloured	O
+spheres	O
+.	O
+
+RNA	B-chemical
+is	O
+shown	O
+is	O
+yellow	O
+.	O
+
+(	O
+a	O
+)	O
+Electron	O
+-	O
+density	O
+loss	O
+sites	O
+as	O
+indicated	O
+by	O
+D	O
+loss	O
+in	O
+the	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+crystal	B-evidence
+by	O
+residue	O
+/	O
+nucleotide	O
+type	O
+for	O
+five	O
+doses	O
+[	O
+sites	O
+determined	O
+above	O
+the	O
+4	O
+×	O
+average	O
+D	O
+loss	O
+threshold	O
+,	O
+calculated	O
+over	O
+the	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+structure	B-evidence
+for	O
+the	O
+first	O
+difference	B-evidence
+map	I-evidence
+:	O
+F	O
+obs	O
+(	O
+d	O
+2	O
+)	O
+−	O
+F	O
+obs	O
+(	O
+d	O
+1	O
+)].	O
+
+(	O
+b	O
+)	O
+Average	O
+D	O
+loss	O
+for	O
+each	O
+residue	O
+/	O
+nucleotide	O
+type	O
+with	O
+respect	O
+to	O
+the	O
+DWD	B-evidence
+(	O
+diffraction	B-evidence
+-	I-evidence
+weighted	I-evidence
+dose	I-evidence
+;	O
+Zeldin	O
+,	O
+Brock	O
+­	O
+hauser	O
+et	O
+al	O
+.,	O
+2013	O
+).	O
+
+Only	O
+a	O
+subset	O
+of	O
+key	O
+TRAP	B-complex_assembly
+residue	O
+types	O
+are	O
+included	O
+.	O
+
+The	O
+average	O
+D	O
+loss	O
+(	O
+calculated	O
+over	O
+the	O
+whole	O
+TRAP	B-complex_assembly
+asymmetric	O
+unit	O
+)	O
+is	O
+shown	O
+at	O
+each	O
+dose	O
+(	O
+dashed	O
+line	O
+).	O
+
+In	O
+(	O
+a	O
+)	O
+clear	O
+difference	B-evidence
+density	I-evidence
+is	O
+observed	O
+around	O
+the	O
+Glu42	B-residue_name_number
+carboxyl	O
+side	O
+chain	O
+in	O
+chain	O
+H	O
+,	O
+within	O
+the	O
+lowest	B-evidence
+dose	I-evidence
+difference	I-evidence
+map	I-evidence
+at	O
+d	O
+2	O
+=	O
+3	O
+.	O
+9	O
+MGy	O
+.	O
+
+Radiation	O
+-	O
+induced	O
+protein	O
+disordering	O
+is	O
+evident	O
+across	O
+the	O
+large	O
+dose	O
+range	O
+(	O
+b	O
+,	O
+c	O
+);	O
+in	O
+comparison	O
+,	O
+no	O
+clear	O
+deterioration	O
+of	O
+the	O
+RNA	B-chemical
+density	B-evidence
+was	O
+observed	O
+.	O
+
+D	O
+loss	O
+calculated	O
+for	O
+all	O
+side	O
+-	O
+chain	O
+carboxyl	O
+group	O
+Glu	B-residue_name
+Cδ	O
+and	O
+Asp	B-residue_name
+Cγ	O
+atoms	O
+within	O
+the	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+for	O
+a	O
+dose	O
+of	O
+19	O
+.	O
+3	O
+MGy	O
+(	O
+d	O
+8	O
+).	O
+
+Residues	O
+have	O
+been	O
+grouped	O
+by	O
+amino	O
+-	O
+acid	O
+number	O
+,	O
+and	O
+split	O
+into	O
+bound	B-protein_state
+and	O
+nonbound	B-protein_state
+groupings	O
+,	O
+with	O
+each	O
+bar	O
+representing	O
+the	O
+mean	O
+calculated	O
+over	O
+11	O
+equivalent	O
+atoms	O
+around	O
+a	O
+TRAP	B-complex_assembly
+ring	B-structure_element
+.	O
+
+D	O
+loss	O
+against	O
+dose	O
+for	O
+(	O
+a	O
+)	O
+Glu36	B-residue_name_number
+Cδ	O
+,	O
+(	O
+b	O
+)	O
+Asp39	B-residue_name_number
+Cγ	O
+,	O
+(	O
+c	O
+)	O
+Glu42	B-residue_name_number
+O	O
+∊	O
+1	O
+,	O
+(	O
+d	O
+)	O
+Glu42	B-residue_name_number
+O	O
+∊	O
+2	O
+,	O
+(	O
+e	O
+)	O
+Phe32	B-residue_name_number
+Cζ	O
+and	O
+(	O
+f	O
+)	O
+Lys37	B-residue_name_number
+C	O
+∊	O
+atoms	O
+.	O
+
+95	O
+%	O
+CI	O
+are	O
+included	O
+for	O
+each	O
+set	O
+of	O
+11	O
+equivalent	O
+atoms	O
+grouped	O
+as	O
+bound	B-protein_state
+/	O
+nonbound	B-protein_state
+.	O
+
+RNA	B-site
+-	I-site
+binding	I-site
+interface	I-site
+interactions	O
+are	O
+shown	O
+for	O
+TRAP	B-complex_assembly
+chain	O
+N	O
+,	O
+with	O
+the	O
+F	O
+obs	O
+(	O
+d	O
+7	O
+)	O
+−	O
+F	O
+obs	O
+(	O
+d	O
+1	O
+)	O
+Fourier	O
+difference	O
+map	O
+(	O
+dose	O
+16	O
+.	O
+7	O
+MGy	O
+)	O
+overlaid	O
+and	O
+contoured	O
+at	O
+a	O
+±	O
+4σ	O
+level	O
+.	O
+
+Mechanistic	O
+insight	O
+into	O
+a	O
+peptide	B-protein_type
+hormone	I-protein_type
+signaling	O
+complex	O
+mediating	O
+floral	O
+organ	O
+abscission	O
+
+Plants	B-taxonomy_domain
+constantly	O
+renew	O
+during	O
+their	O
+life	O
+cycle	O
+and	O
+thus	O
+require	O
+to	O
+shed	O
+senescent	O
+and	O
+damaged	O
+organs	O
+.	O
+
+Floral	O
+abscission	O
+is	O
+controlled	O
+by	O
+the	O
+leucine	B-protein_type
+-	I-protein_type
+rich	I-protein_type
+repeat	I-protein_type
+receptor	I-protein_type
+kinase	I-protein_type
+(	O
+LRR	B-protein_type
+-	I-protein_type
+RK	I-protein_type
+)	O
+HAESA	B-protein
+and	O
+the	O
+peptide	B-protein_type
+hormone	I-protein_type
+IDA	B-protein
+.	O
+
+It	O
+is	O
+unknown	O
+how	O
+expression	O
+of	O
+IDA	B-protein
+in	O
+the	O
+abscission	O
+zone	O
+leads	O
+to	O
+HAESA	B-protein
+activation	O
+.	O
+
+Here	O
+we	O
+show	O
+that	O
+IDA	B-protein
+is	O
+sensed	O
+directly	O
+by	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+
+Crystal	B-evidence
+structures	I-evidence
+of	O
+HAESA	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+IDA	B-protein
+reveal	O
+a	O
+hormone	B-site
+binding	I-site
+pocket	I-site
+that	O
+accommodates	O
+an	O
+active	B-protein_state
+dodecamer	B-structure_element
+peptide	B-chemical
+.	O
+
+A	O
+central	O
+hydroxyproline	B-residue_name
+residue	O
+anchors	O
+IDA	B-protein
+to	O
+the	O
+receptor	O
+.	O
+
+The	O
+HAESA	B-protein
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+SERK1	B-protein
+,	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	B-protein_type
+hormone	I-protein_type
+by	O
+binding	O
+to	O
+an	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+.	O
+
+This	O
+sequence	O
+pattern	O
+is	O
+conserved	B-protein_state
+among	O
+diverse	O
+plant	B-taxonomy_domain
+peptides	B-chemical
+,	O
+suggesting	O
+that	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormone	I-protein_type
+receptors	I-protein_type
+may	O
+share	O
+a	O
+common	O
+ligand	O
+binding	O
+mode	O
+and	O
+activation	O
+mechanism	O
+.	O
+
+Plants	B-taxonomy_domain
+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	B-protein_type
+protein	I-protein_type
+called	O
+HAESA	B-protein
+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	B-chemical
+called	O
+IDA	B-protein
+instructs	O
+HAESA	B-protein
+to	O
+trigger	O
+the	O
+shedding	O
+process	O
+.	O
+
+However	O
+,	O
+the	O
+molecular	O
+details	O
+of	O
+how	O
+IDA	B-protein
+triggers	O
+organ	O
+shedding	O
+are	O
+not	O
+clear	O
+.	O
+
+The	O
+shedding	O
+of	O
+floral	O
+organs	O
+(	O
+or	O
+leaves	O
+)	O
+can	O
+be	O
+easily	O
+studied	O
+in	O
+a	O
+model	O
+plant	B-taxonomy_domain
+called	O
+Arabidopsis	B-taxonomy_domain
+.	O
+
+Santiago	O
+et	O
+al	O
+.	O
+used	O
+protein	B-experimental_method
+biochemistry	I-experimental_method
+,	O
+structural	B-experimental_method
+biology	I-experimental_method
+and	O
+genetics	B-experimental_method
+to	O
+uncover	O
+how	O
+the	O
+IDA	B-protein
+hormone	B-chemical
+activates	O
+HAESA	B-protein
+.	O
+
+The	O
+experiments	O
+show	O
+that	O
+IDA	B-protein
+binds	B-protein_state
+directly	I-protein_state
+to	I-protein_state
+a	O
+canyon	B-protein_state
+shaped	I-protein_state
+pocket	B-site
+in	O
+HAESA	B-protein
+that	O
+extends	O
+out	O
+from	O
+the	O
+surface	O
+of	O
+the	O
+cell	O
+.	O
+
+IDA	B-protein
+binding	O
+to	O
+HAESA	B-protein
+allows	O
+another	O
+receptor	B-protein_type
+protein	I-protein_type
+called	O
+SERK1	B-protein
+to	B-protein_state
+bind	I-protein_state
+to	I-protein_state
+HAESA	B-protein
+,	O
+which	O
+results	O
+in	O
+the	O
+release	O
+of	O
+signals	O
+inside	O
+the	O
+cell	O
+that	O
+trigger	O
+the	O
+shedding	O
+of	O
+organs	O
+.	O
+
+The	O
+next	O
+step	O
+following	O
+on	O
+from	O
+this	O
+work	O
+is	O
+to	O
+understand	O
+what	O
+signals	O
+are	O
+produced	O
+when	O
+IDA	B-protein
+activates	O
+HAESA	B-protein
+.	O
+
+Another	O
+challenge	O
+will	O
+be	O
+to	O
+find	O
+out	O
+where	O
+IDA	B-protein
+is	O
+produced	O
+in	O
+the	O
+plant	B-taxonomy_domain
+and	O
+what	O
+causes	O
+it	O
+to	O
+accumulate	O
+in	O
+specific	O
+places	O
+in	O
+preparation	O
+for	O
+organ	O
+shedding	O
+.	O
+
+The	O
+HAESA	B-protein
+ectodomain	B-structure_element
+folds	O
+into	O
+a	O
+superhelical	B-structure_element
+assembly	I-structure_element
+of	O
+21	O
+leucine	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+repeats	I-structure_element
+.	O
+
+(	O
+A	O
+)	O
+SDS	B-experimental_method
+PAGE	I-experimental_method
+analysis	O
+of	O
+the	O
+purified	O
+Arabidopsis	B-species
+thaliana	I-species
+HAESA	B-protein
+ectodomain	B-structure_element
+(	O
+residues	O
+20	B-residue_range
+–	I-residue_range
+620	I-residue_range
+)	O
+obtained	O
+by	O
+secreted	B-experimental_method
+expression	I-experimental_method
+in	I-experimental_method
+insect	I-experimental_method
+cells	I-experimental_method
+.	O
+
+The	O
+calculated	O
+molecular	O
+mass	O
+is	O
+65	O
+.	O
+7	O
+kDa	O
+,	O
+the	O
+actual	O
+molecular	O
+mass	O
+obtained	O
+by	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+is	O
+74	O
+,	O
+896	O
+Da	O
+,	O
+accounting	O
+for	O
+the	O
+N	B-chemical
+-	I-chemical
+glycans	I-chemical
+.	O
+(	O
+B	O
+)	O
+Ribbon	O
+diagrams	O
+showing	O
+front	O
+(	O
+left	O
+panel	O
+)	O
+and	O
+side	O
+views	O
+(	O
+right	O
+panel	O
+)	O
+of	O
+the	O
+isolated	O
+HAESA	B-protein
+LRR	B-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+N	O
+-	O
+(	O
+residues	O
+20	B-residue_range
+–	I-residue_range
+88	I-residue_range
+)	O
+and	O
+C	O
+-	O
+terminal	O
+(	O
+residues	O
+593	B-residue_range
+–	I-residue_range
+615	I-residue_range
+)	O
+capping	B-structure_element
+domains	I-structure_element
+are	O
+shown	O
+in	O
+yellow	O
+,	O
+the	O
+central	O
+21	O
+LRR	B-structure_element
+motifs	I-structure_element
+are	O
+in	O
+blue	O
+and	O
+disulphide	B-ptm
+bonds	I-ptm
+are	O
+highlighted	O
+in	O
+green	O
+(	O
+in	O
+bonds	O
+representation	O
+).	O
+(	O
+C	O
+)	O
+Structure	B-experimental_method
+based	I-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+21	O
+leucine	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+repeats	I-structure_element
+in	O
+HAESA	B-protein
+with	O
+the	O
+plant	B-taxonomy_domain
+LRR	B-structure_element
+consensus	O
+sequence	O
+shown	O
+for	O
+comparison	O
+.	O
+
+Conserved	B-protein_state
+hydrophobic	B-protein_state
+residues	B-structure_element
+are	O
+shaded	O
+in	O
+gray	O
+,	O
+N	B-site
+-	I-site
+glycosylation	I-site
+sites	I-site
+visible	O
+in	O
+our	O
+structures	B-evidence
+are	O
+highlighted	O
+in	O
+blue	O
+,	O
+cysteine	B-residue_name
+residues	O
+involved	O
+in	O
+disulphide	B-ptm
+bridge	I-ptm
+formation	O
+in	O
+green	O
+.	O
+(	O
+D	O
+)	O
+Asn	B-ptm
+-	I-ptm
+linked	I-ptm
+glycans	I-ptm
+mask	O
+the	O
+N	O
+-	O
+terminal	O
+portion	O
+of	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+
+Oligomannose	B-chemical
+core	O
+structures	O
+(	O
+containing	O
+two	O
+N	B-chemical
+-	I-chemical
+actylglucosamines	I-chemical
+and	O
+three	O
+terminal	O
+mannose	B-chemical
+units	O
+)	O
+as	O
+found	O
+in	O
+Trichoplusia	B-species
+ni	I-species
+cells	O
+and	O
+in	O
+plants	B-taxonomy_domain
+were	O
+modeled	O
+onto	O
+the	O
+seven	O
+glycosylation	B-site
+sites	I-site
+observed	O
+in	O
+our	O
+HAESA	B-protein
+structures	B-evidence
+,	O
+to	O
+visualize	O
+the	O
+surface	O
+areas	O
+potentially	O
+not	O
+masked	O
+by	O
+carbohydrate	B-chemical
+.	O
+
+The	O
+HAESA	B-protein
+ectodomain	B-structure_element
+is	O
+shown	O
+in	O
+blue	O
+(	O
+in	O
+surface	O
+representation	O
+),	O
+the	O
+glycan	B-chemical
+structures	O
+are	O
+shown	O
+in	O
+yellow	O
+.	O
+
+Hydrophobic	O
+contacts	O
+and	O
+a	O
+hydrogen	B-site
+-	I-site
+bond	I-site
+network	I-site
+mediate	O
+the	O
+interaction	O
+between	O
+HAESA	B-protein
+and	O
+the	O
+peptide	B-protein_type
+hormone	I-protein_type
+IDA	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Details	O
+of	O
+the	O
+IDA	B-site
+binding	I-site
+pocket	I-site
+.	O
+
+HAESA	B-protein
+is	O
+shown	O
+in	O
+blue	O
+(	O
+ribbon	O
+diagram	O
+),	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+(	O
+left	O
+panel	O
+),	O
+the	O
+central	O
+Hyp	B-structure_element
+anchor	I-structure_element
+(	O
+center	O
+)	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+Pro	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+(	O
+right	O
+panel	O
+)	O
+are	O
+shown	O
+in	O
+yellow	O
+(	O
+in	O
+bonds	O
+representation	O
+).	O
+
+HAESA	B-site
+interface	I-site
+residues	I-site
+are	O
+shown	O
+as	O
+sticks	O
+,	O
+selected	O
+hydrogen	O
+bond	O
+interactions	O
+are	O
+denoted	O
+as	O
+dotted	O
+lines	O
+(	O
+in	O
+magenta	O
+).	O
+(	O
+B	O
+)	O
+View	O
+of	O
+the	O
+complete	O
+IDA	B-protein
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+yellow	O
+)	O
+binding	B-site
+pocket	I-site
+in	O
+HAESA	B-protein
+(	O
+surface	O
+view	O
+,	O
+in	O
+blue	O
+).	O
+
+Orientation	O
+as	O
+in	O
+(	O
+A	O
+).	O
+(	O
+C	O
+)	O
+Structure	B-experimental_method
+based	I-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+leucine	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+repeats	I-structure_element
+in	O
+HAESA	B-protein
+with	O
+the	O
+plant	B-taxonomy_domain
+LRR	B-structure_element
+consensus	B-evidence
+sequence	I-evidence
+shown	O
+for	O
+comparison	O
+.	O
+
+Residues	O
+mediating	O
+hydrophobic	O
+interactions	O
+with	O
+the	O
+IDA	B-chemical
+peptide	I-chemical
+are	O
+highlighted	O
+in	O
+blue	O
+,	O
+residues	O
+contributing	O
+to	O
+hydrogen	O
+bond	O
+interactions	O
+and	O
+/	O
+or	O
+salt	O
+bridges	O
+are	O
+shown	O
+in	O
+red	O
+.	O
+
+The	O
+IDA	B-site
+binding	I-site
+pocket	I-site
+covers	O
+LRRs	B-structure_element
+2	I-structure_element
+–	I-structure_element
+14	I-structure_element
+and	O
+all	O
+residues	O
+originate	O
+from	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+HAESA	B-protein
+superhelix	B-structure_element
+.	O
+
+The	O
+IDA	B-complex_assembly
+-	I-complex_assembly
+HAESA	I-complex_assembly
+and	O
+SERK1	B-complex_assembly
+-	I-complex_assembly
+HAESA	I-complex_assembly
+complex	O
+interfaces	B-site
+are	O
+conserved	B-protein_state
+among	O
+HAESA	B-protein
+and	O
+HAESA	B-protein_type
+-	I-protein_type
+like	I-protein_type
+proteins	I-protein_type
+from	O
+different	O
+plant	B-taxonomy_domain
+species	O
+.	O
+
+Structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+HAESA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+:	O
+Arabidopsis	B-species
+thaliana	I-species
+HAESA	B-protein
+(	O
+Uniprot	O
+(	O
+http	O
+://	O
+www	O
+.	O
+uniprot	O
+.	O
+org	O
+)	O
+ID	O
+P47735	O
+),	O
+Arabidopsis	B-species
+thaliana	I-species
+HSL2	B-protein
+(	O
+Uniprot	O
+ID	O
+C0LGX3	O
+),	O
+Capsella	B-species
+rubella	I-species
+HAESA	B-protein
+(	O
+Uniprot	O
+ID	O
+R0F2U6	O
+),	O
+Citrus	B-species
+clementina	I-species
+HSL2	B-protein
+(	O
+Uniprot	O
+ID	O
+V4U227	O
+),	O
+Vitis	B-species
+vinifera	I-species
+HAESA	B-protein
+(	O
+Uniprot	O
+ID	O
+F6HM39	O
+).	O
+
+The	O
+alignment	O
+includes	O
+a	O
+secondary	O
+structure	O
+assignment	O
+calculated	O
+with	O
+the	O
+program	O
+DSSP	O
+and	O
+colored	O
+according	O
+to	O
+Figure	O
+1	O
+,	O
+with	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+caps	B-structure_element
+and	O
+the	O
+21	O
+LRR	B-structure_element
+motifs	I-structure_element
+indicated	O
+in	O
+orange	O
+and	O
+blue	O
+,	O
+respectively	O
+.	O
+
+Cysteine	B-residue_name
+residues	O
+engaged	O
+in	O
+disulphide	B-ptm
+bonds	I-ptm
+are	O
+depicted	O
+in	O
+green	O
+.	O
+
+HAESA	B-protein
+residues	O
+interacting	O
+with	O
+the	O
+IDA	B-chemical
+peptide	I-chemical
+and	O
+/	O
+or	O
+the	O
+SERK1	B-protein
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+kinase	I-protein_type
+ectodomain	B-structure_element
+are	O
+highlighted	O
+in	O
+blue	O
+and	O
+orange	O
+,	O
+respectively	O
+.	O
+
+The	O
+peptide	B-protein_type
+hormone	I-protein_type
+IDA	B-protein
+binds	O
+to	O
+the	O
+HAESA	B-protein
+LRR	B-structure_element
+ectodomain	I-structure_element
+.	O
+
+(	O
+A	O
+)	O
+Multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+selected	O
+IDA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+.	O
+
+The	O
+conserved	B-protein_state
+PIP	B-structure_element
+motif	I-structure_element
+is	O
+highlighted	O
+in	O
+yellow	O
+,	O
+the	O
+central	O
+Hyp	B-residue_name
+in	O
+blue	O
+.	O
+
+The	O
+PKGV	B-structure_element
+motif	I-structure_element
+present	O
+in	O
+our	O
+N	B-protein_state
+-	I-protein_state
+terminally	I-protein_state
+extended	I-protein_state
+IDA	B-chemical
+peptide	I-chemical
+is	O
+highlighted	O
+in	O
+red	O
+.	O
+(	O
+B	O
+)	O
+Isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+of	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+vs	O
+.	O
+IDA	B-protein
+and	O
+including	O
+the	O
+synthetic	B-protein_state
+peptide	B-chemical
+sequence	O
+.	O
+
+(	O
+C	O
+)	O
+Structure	O
+of	O
+the	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+IDA	I-complex_assembly
+complex	O
+with	O
+HAESA	B-protein
+shown	O
+in	O
+blue	O
+(	O
+ribbon	O
+diagram	O
+).	O
+
+IDA	B-protein
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+surface	O
+view	O
+included	O
+)	O
+is	O
+depicted	O
+in	O
+yellow	O
+.	O
+
+The	O
+peptide	B-site
+binding	I-site
+pocket	I-site
+covers	O
+HAESA	B-protein
+LRRs	B-structure_element
+2	I-structure_element
+–	I-structure_element
+14	I-structure_element
+.	O
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+entire	O
+IDA	B-protein
+(	O
+in	O
+yellow	O
+)	O
+peptide	B-site
+binding	I-site
+site	I-site
+in	O
+HAESA	B-protein
+(	O
+in	O
+blue	O
+).	O
+
+Details	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+central	O
+Hyp	B-structure_element
+anchor	I-structure_element
+in	O
+IDA	B-protein
+and	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+with	O
+HAESA	B-protein
+are	O
+highlighted	O
+in	O
+(	O
+E	O
+)	O
+and	O
+(	O
+F	O
+),	O
+respectively	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	B-chemical
+molecule	O
+is	O
+shown	O
+as	O
+a	O
+red	O
+sphere	O
+.	O
+
+During	O
+their	O
+growth	O
+,	O
+development	O
+and	O
+reproduction	O
+plants	B-taxonomy_domain
+use	O
+cell	O
+separation	O
+processes	O
+to	O
+detach	O
+no	O
+-	O
+longer	O
+required	O
+,	O
+damaged	O
+or	O
+senescent	O
+organs	O
+.	O
+
+Abscission	O
+of	O
+floral	O
+organs	O
+in	O
+Arabidopsis	B-taxonomy_domain
+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
+
+The	O
+LRR	B-structure_element
+-	I-structure_element
+RKs	I-structure_element
+HAESA	B-protein
+(	O
+greek	O
+:	O
+to	O
+adhere	O
+to	O
+)	O
+and	O
+HAESA	B-protein
+-	I-protein
+LIKE	I-protein
+2	I-protein
+(	O
+HSL2	B-protein
+)	O
+redundantly	O
+control	O
+floral	O
+abscission	O
+.	O
+
+Loss	O
+-	O
+of	O
+-	O
+function	O
+of	O
+the	O
+secreted	O
+small	O
+protein	O
+INFLORESCENCE	B-protein
+DEFICIENT	I-protein
+IN	I-protein
+ABSCISSION	I-protein
+(	O
+IDA	B-protein
+)	O
+causes	O
+floral	O
+organs	O
+to	O
+remain	O
+attached	O
+while	O
+its	O
+over	O
+-	O
+expression	O
+leads	O
+to	O
+premature	O
+shedding	O
+.	O
+
+Full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+IDA	B-protein
+is	O
+proteolytically	B-ptm
+processed	I-ptm
+and	O
+a	O
+conserved	B-protein_state
+stretch	B-residue_range
+of	I-residue_range
+20	I-residue_range
+amino	I-residue_range
+-	I-residue_range
+acids	I-residue_range
+(	O
+termed	O
+EPIP	B-structure_element
+)	O
+can	O
+rescue	O
+the	O
+IDA	B-protein
+loss	O
+-	O
+of	O
+-	O
+function	O
+phenotype	O
+(	O
+Figure	O
+1A	O
+).	O
+
+It	O
+has	O
+been	O
+demonstrated	O
+that	O
+a	O
+dodecamer	B-structure_element
+peptide	B-chemical
+within	O
+EPIP	B-structure_element
+is	O
+able	O
+to	O
+activate	O
+HAESA	B-protein
+and	O
+HSL2	B-protein
+in	O
+transient	B-experimental_method
+assays	I-experimental_method
+in	O
+tobacco	B-taxonomy_domain
+cells	O
+.	O
+
+This	B-structure_element
+sequence	I-structure_element
+motif	I-structure_element
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+among	O
+IDA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+(	O
+IDA	B-protein_type
+-	I-protein_type
+LIKE	I-protein_type
+PROTEINS	I-protein_type
+,	O
+IDLs	B-protein_type
+)	O
+and	O
+contains	O
+a	O
+central	O
+Pro	B-residue_name
+residue	O
+,	O
+presumed	O
+to	O
+be	O
+post	B-protein_state
+-	I-protein_state
+translationally	I-protein_state
+modified	I-protein_state
+to	O
+hydroxyproline	B-residue_name
+(	O
+Hyp	B-residue_name
+;	O
+Figure	O
+1A	O
+).	O
+
+The	O
+available	O
+genetic	O
+and	O
+biochemical	O
+evidence	O
+suggests	O
+that	O
+IDA	B-protein
+and	O
+HAESA	B-protein
+together	O
+control	O
+floral	O
+abscission	O
+,	O
+but	O
+it	O
+is	O
+poorly	O
+understood	O
+if	O
+IDA	B-protein
+is	O
+directly	O
+sensed	O
+by	O
+the	O
+receptor	B-protein_type
+kinase	I-protein_type
+HAESA	B-protein
+and	O
+how	O
+IDA	B-protein
+binding	O
+at	O
+the	O
+cell	O
+surface	O
+would	O
+activate	O
+the	O
+receptor	O
+.	O
+
+IDA	B-protein
+directly	O
+binds	O
+to	O
+the	O
+LRR	B-structure_element
+domain	I-structure_element
+of	O
+HAESA	B-protein
+
+Active	B-protein_state
+IDA	B-protein_type
+-	I-protein_type
+family	I-protein_type
+peptide	I-protein_type
+hormones	I-protein_type
+are	O
+hydroxyprolinated	B-protein_state
+dodecamers	B-structure_element
+.	O
+
+Close	O
+-	O
+up	O
+views	O
+of	O
+(	O
+A	O
+)	O
+IDA	B-protein
+,	O
+(	O
+B	O
+)	O
+the	O
+N	B-protein_state
+-	I-protein_state
+terminally	I-protein_state
+extended	I-protein_state
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+and	O
+(	O
+C	O
+)	O
+IDL1	B-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+HAESA	B-protein
+hormone	B-site
+binding	I-site
+pocket	I-site
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+yellow	O
+)	O
+and	O
+including	O
+simulated	B-experimental_method
+annealing	I-experimental_method
+2Fo	B-evidence
+–	I-evidence
+Fc	I-evidence
+omit	I-evidence
+electron	I-evidence
+density	I-evidence
+maps	I-evidence
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+.	O
+
+Note	O
+that	O
+Pro58IDA	B-residue_name_number
+and	O
+Leu67IDA	B-residue_name_number
+are	O
+the	O
+first	O
+residues	O
+defined	O
+by	O
+electron	B-evidence
+density	I-evidence
+when	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+(	O
+D	O
+)	O
+Table	O
+summaries	O
+for	O
+equilibrium	B-evidence
+dissociation	I-evidence
+constants	I-evidence
+(	O
+Kd	B-evidence
+),	O
+binding	B-evidence
+enthalpies	I-evidence
+(	O
+ΔH	B-evidence
+),	O
+binding	B-evidence
+entropies	I-evidence
+(	O
+ΔS	B-evidence
+)	O
+and	O
+stoichoimetries	O
+(	O
+N	O
+)	O
+for	O
+different	O
+IDA	B-chemical
+peptides	I-chemical
+binding	O
+to	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+(	O
+±	O
+fitting	O
+errors	O
+;	O
+n	O
+.	O
+d	O
+.	O
+
+no	O
+detectable	O
+binding	O
+).	O
+(	O
+E	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+active	B-protein_state
+IDA	B-protein
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+gray	O
+)	O
+and	O
+IDL1	B-chemical
+peptide	I-chemical
+(	O
+in	O
+yellow	O
+)	O
+hormones	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+
+Root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+(	O
+r	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.)	I-evidence
+is	O
+1	O
+.	O
+0	O
+Å	O
+comparing	O
+100	O
+corresponding	O
+atoms	O
+.	O
+
+The	O
+receptor	B-protein_type
+kinase	I-protein_type
+SERK1	B-protein
+acts	O
+as	O
+a	O
+HAESA	B-protein_type
+co	I-protein_type
+-	I-protein_type
+receptor	I-protein_type
+and	O
+promotes	O
+high	O
+-	O
+affinity	O
+IDA	B-protein
+sensing	O
+.	O
+
+(	O
+A	O
+)	O
+Petal	B-experimental_method
+break	I-experimental_method
+-	I-experimental_method
+strength	I-experimental_method
+assays	I-experimental_method
+measure	O
+the	O
+force	O
+(	O
+expressed	O
+in	O
+gram	O
+equivalents	O
+)	O
+required	O
+to	O
+remove	O
+the	O
+petals	O
+from	O
+the	O
+flower	O
+of	O
+serk	B-gene
+mutant	B-protein_state
+plants	B-taxonomy_domain
+compared	O
+to	O
+haesa	B-gene
+/	O
+hsl2	B-gene
+mutant	B-protein_state
+and	O
+Col	O
+-	O
+0	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+flowers	O
+.	O
+
+Petal	O
+break	O
+-	O
+strength	O
+was	O
+found	O
+significantly	O
+increased	O
+in	O
+almost	O
+all	O
+positions	O
+(	O
+indicated	O
+with	O
+a	O
+*)	O
+for	O
+haesa	B-gene
+/	O
+hsl2	B-gene
+and	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+mutant	B-protein_state
+plants	B-taxonomy_domain
+with	O
+respect	O
+to	O
+the	O
+Col	O
+-	O
+0	O
+control	O
+.	O
+
+(	O
+B	O
+)	O
+Analytical	B-experimental_method
+size	I-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+.	O
+
+The	O
+HAESA	B-protein
+LRR	B-structure_element
+domain	I-structure_element
+elutes	O
+as	O
+a	O
+monomer	B-oligomeric_state
+(	O
+black	O
+dotted	O
+line	O
+),	O
+as	O
+does	O
+the	O
+isolated	O
+SERK1	B-protein
+ectodomain	B-structure_element
+(	O
+blue	O
+dotted	O
+line	O
+).	O
+
+A	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+IDA	I-complex_assembly
+–	I-complex_assembly
+SERK1	I-complex_assembly
+complex	O
+elutes	O
+as	O
+an	O
+apparent	O
+heterodimer	B-oligomeric_state
+(	O
+red	O
+line	O
+),	O
+while	O
+a	O
+mixture	O
+of	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+yields	O
+two	O
+isolated	O
+peaks	O
+that	O
+correspond	O
+to	O
+monomeric	B-oligomeric_state
+HAESA	B-protein
+and	O
+SERK1	B-protein
+,	O
+respectively	O
+(	O
+black	O
+line	O
+).	O
+
+Void	O
+(	O
+V0	O
+)	O
+volume	O
+and	O
+total	O
+volume	O
+(	O
+Vt	O
+)	O
+are	O
+shown	O
+,	O
+together	O
+with	O
+elution	O
+volumes	O
+for	O
+molecular	O
+mass	O
+standards	O
+(	O
+A	O
+,	O
+Thyroglobulin	B-protein
+,	O
+669	O
+,	O
+000	O
+Da	O
+;	O
+B	O
+,	O
+Ferritin	B-protein
+,	O
+440	O
+,	O
+00	O
+Da	O
+,	O
+C	O
+,	O
+Aldolase	B-protein
+,	O
+158	O
+,	O
+000	O
+Da	O
+;	O
+D	O
+,	O
+Conalbumin	B-protein
+,	O
+75	O
+,	O
+000	O
+Da	O
+;	O
+E	O
+,	O
+Ovalbumin	B-protein
+,	O
+44	O
+,	O
+000	O
+Da	O
+;	O
+F	O
+,	O
+Carbonic	B-protein
+anhydrase	I-protein
+,	O
+29	O
+,	O
+000	O
+Da	O
+).	O
+
+A	O
+SDS	B-experimental_method
+PAGE	I-experimental_method
+of	O
+the	O
+peak	O
+fractions	O
+is	O
+shown	O
+alongside	O
+.	O
+
+Purified	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+are	O
+~	O
+75	O
+and	O
+~	O
+28	O
+kDa	O
+,	O
+respectively	O
+.	O
+(	O
+C	O
+)	O
+Isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+Hyp64	B-mutant
+→	I-mutant
+Pro	I-mutant
+IDA	I-mutant
+versus	O
+the	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+ectodomains	B-structure_element
+.	O
+
+The	O
+titration	B-experimental_method
+of	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+versus	O
+the	O
+isolated	O
+HAESA	B-protein
+ectodomain	B-structure_element
+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
+
+no	O
+detectable	O
+binding	O
+)	O
+(	O
+D	O
+)	O
+Analytical	B-experimental_method
+size	I-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+IDA	B-mutant
+Hyp64	I-mutant
+→	I-mutant
+Pro	I-mutant
+mutant	B-protein_state
+peptide	B-chemical
+reveals	O
+no	O
+complex	O
+formation	O
+between	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+ectodomains	B-structure_element
+.	O
+
+(	O
+E	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+kinase	I-experimental_method
+assays	I-experimental_method
+of	O
+the	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+kinase	B-structure_element
+domains	I-structure_element
+.	O
+
+Wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+HAESA	B-protein
+and	O
+SERK1	B-protein
+kinase	B-structure_element
+domains	I-structure_element
+(	O
+KDs	B-structure_element
+)	O
+exhibit	O
+auto	O
+-	O
+phosphorylation	O
+activities	O
+(	O
+lanes	O
+1	O
++	O
+3	O
+).	O
+
+Mutant	B-protein_state
+(	O
+m	O
+)	O
+versions	O
+,	O
+which	O
+carry	O
+point	B-experimental_method
+mutations	I-experimental_method
+in	O
+their	O
+active	B-site
+sites	I-site
+(	O
+Asp837HAESA	B-mutant
+→	I-mutant
+Asn	I-mutant
+,	O
+Asp447SERK1	B-mutant
+→	I-mutant
+Asn	I-mutant
+)	O
+possess	O
+no	O
+autophosphorylation	O
+activity	O
+(	O
+lanes	O
+2	O
++	O
+4	O
+).	O
+
+Transphosphorylation	O
+activity	O
+from	O
+the	O
+active	B-protein_state
+kinase	O
+to	O
+the	O
+mutated	B-protein_state
+form	O
+can	O
+be	O
+observed	O
+in	O
+both	O
+directions	O
+(	O
+lanes	O
+5	O
++	O
+6	O
+).	O
+
+We	O
+purified	B-experimental_method
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+(	O
+residues	O
+20	B-residue_range
+–	I-residue_range
+620	I-residue_range
+)	O
+from	O
+baculovirus	B-experimental_method
+-	I-experimental_method
+infected	I-experimental_method
+insect	I-experimental_method
+cells	I-experimental_method
+(	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	B-protein_type
+with	O
+synthetic	B-protein_state
+IDA	B-chemical
+peptides	I-chemical
+using	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+ITC	B-experimental_method
+).	O
+
+A	O
+Hyp	B-protein_state
+-	I-protein_state
+modified	I-protein_state
+dodecamer	B-structure_element
+comprising	O
+the	O
+highly	B-protein_state
+conserved	I-protein_state
+PIP	B-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+(	O
+Figure	O
+1A	O
+)	O
+interacts	O
+with	O
+HAESA	B-protein
+with	O
+1	O
+:	O
+1	O
+stoichiometry	O
+(	O
+N	O
+)	O
+and	O
+with	O
+a	O
+dissociation	B-evidence
+constant	I-evidence
+(	O
+Kd	B-evidence
+)	O
+of	O
+~	O
+20	O
+μM	O
+(	O
+Figure	O
+1B	O
+).	O
+
+We	O
+next	O
+determined	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+apo	B-protein_state
+HAESA	B-protein
+ectodomain	B-structure_element
+and	O
+of	O
+a	O
+HAESA	B-complex_assembly
+-	I-complex_assembly
+IDA	I-complex_assembly
+complex	O
+,	O
+at	O
+1	O
+.	O
+74	O
+and	O
+1	O
+.	O
+86	O
+Å	O
+resolution	O
+,	O
+respectively	O
+(	O
+Figure	O
+1C	O
+;	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1B	O
+–	O
+D	O
+;	O
+Tables	O
+1	O
+,	O
+2	O
+).	O
+
+IDA	B-protein
+binds	O
+in	O
+a	O
+completely	B-protein_state
+extended	I-protein_state
+conformation	I-protein_state
+along	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+,	O
+covering	O
+LRRs	B-structure_element
+2	I-structure_element
+–	I-structure_element
+14	I-structure_element
+(	O
+Figure	O
+1C	O
+,	O
+D	O
+,	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+
+The	O
+central	O
+Hyp64IDA	B-residue_name_number
+is	O
+buried	O
+in	O
+a	O
+specific	O
+pocket	B-site
+formed	O
+by	O
+HAESA	B-protein
+LRRs	B-structure_element
+8	I-structure_element
+–	I-structure_element
+10	I-structure_element
+,	O
+with	O
+its	O
+hydroxyl	O
+group	O
+establishing	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+strictly	B-protein_state
+conserved	I-protein_state
+Glu266HAESA	B-residue_name_number
+and	O
+with	O
+a	O
+water	B-chemical
+molecule	O
+,	O
+which	O
+in	O
+turn	O
+is	O
+coordinated	O
+by	O
+the	O
+main	O
+chain	O
+oxygens	O
+of	O
+Phe289HAESA	B-residue_name_number
+and	O
+Ser311HAESA	B-residue_name_number
+(	O
+Figure	O
+1E	O
+;	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+).	O
+
+The	O
+restricted	O
+size	O
+of	O
+the	O
+Hyp	B-site
+pocket	I-site
+suggests	O
+that	O
+IDA	B-protein
+does	O
+not	O
+require	O
+arabinosylation	B-ptm
+of	O
+Hyp64IDA	B-residue_name_number
+for	O
+activity	O
+in	O
+vivo	O
+,	O
+a	O
+modification	O
+that	O
+has	O
+been	O
+reported	O
+for	O
+Hyp	B-residue_name
+residues	O
+in	O
+plant	B-taxonomy_domain
+CLE	B-protein_type
+peptide	I-protein_type
+hormones	I-protein_type
+.	O
+
+The	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+maps	O
+to	O
+a	O
+cavity	B-site
+formed	O
+by	O
+HAESA	B-protein
+LRRs	B-structure_element
+11	I-structure_element
+–	I-structure_element
+14	I-structure_element
+(	O
+Figure	O
+1D	O
+,	O
+F	O
+).	O
+
+The	O
+COO	O
+-	O
+group	O
+of	O
+Asn69IDA	B-residue_name_number
+is	O
+in	O
+direct	O
+contact	O
+with	O
+Arg407HAESA	B-residue_name_number
+and	O
+Arg409HAESA	B-residue_name_number
+and	O
+HAESA	B-protein
+cannot	O
+bind	O
+a	O
+C	B-protein_state
+-	I-protein_state
+terminally	I-protein_state
+extended	I-protein_state
+IDA	B-mutant
+-	I-mutant
+SFVN	I-mutant
+peptide	O
+(	O
+Figures	O
+1D	O
+,	O
+F	O
+,	O
+2D	O
+).	O
+
+This	O
+suggests	O
+that	O
+the	O
+conserved	B-protein_state
+Asn69IDA	B-residue_name_number
+may	O
+constitute	O
+the	O
+very	O
+C	O
+-	O
+terminus	O
+of	O
+the	O
+mature	B-protein_state
+IDA	B-chemical
+peptide	I-chemical
+in	O
+planta	B-taxonomy_domain
+and	O
+that	O
+active	B-protein_state
+IDA	B-protein
+is	O
+generated	O
+by	O
+proteolytic	O
+processing	O
+from	O
+a	O
+longer	O
+pre	O
+-	O
+protein	O
+.	O
+
+Mutation	B-experimental_method
+of	O
+Arg417HSL2	B-residue_name_number
+(	O
+which	O
+corresponds	O
+to	O
+Arg409HAESA	B-residue_name_number
+)	O
+causes	O
+a	O
+loss	O
+-	O
+of	O
+-	O
+function	O
+phenotype	O
+in	O
+HSL2	B-protein
+,	O
+which	O
+indicates	O
+that	O
+the	O
+peptide	B-site
+binding	I-site
+pockets	I-site
+in	O
+different	O
+HAESA	B-protein_type
+receptors	I-protein_type
+have	O
+common	O
+structural	O
+and	O
+sequence	O
+features	O
+.	O
+
+Indeed	O
+,	O
+we	O
+find	O
+many	O
+of	O
+the	O
+residues	O
+contributing	O
+to	O
+the	O
+formation	O
+of	O
+the	O
+IDA	B-site
+binding	I-site
+surface	I-site
+in	O
+HAESA	B-protein
+to	O
+be	O
+conserved	B-protein_state
+in	O
+HSL2	B-protein
+and	O
+in	O
+other	O
+HAESA	B-protein_type
+-	I-protein_type
+type	I-protein_type
+receptors	I-protein_type
+in	O
+different	O
+plant	B-taxonomy_domain
+species	O
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+).	O
+
+A	O
+N	O
+-	O
+terminal	O
+Pro	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+makes	O
+contacts	O
+with	O
+LRRs	B-structure_element
+2	I-structure_element
+–	I-structure_element
+6	I-structure_element
+of	O
+the	O
+receptor	O
+(	O
+Figure	O
+1D	O
+,	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2A	O
+–	O
+C	O
+).	O
+
+Other	O
+hydrophobic	O
+and	O
+polar	O
+interactions	O
+are	O
+mediated	O
+by	O
+Ser62IDA	B-residue_name_number
+,	O
+Ser65IDA	B-residue_name_number
+and	O
+by	O
+backbone	O
+atoms	O
+along	O
+the	O
+IDA	B-chemical
+peptide	I-chemical
+(	O
+Figure	O
+1D	O
+,	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+2A	O
+–	O
+C	O
+).	O
+
+HAESA	B-protein
+specifically	O
+senses	O
+IDA	B-protein_type
+-	I-protein_type
+family	I-protein_type
+dodecamer	B-structure_element
+peptides	B-chemical
+
+We	O
+next	O
+investigated	O
+whether	O
+HAESA	B-protein
+binds	O
+N	B-protein_state
+-	I-protein_state
+terminally	I-protein_state
+extended	I-protein_state
+versions	O
+of	O
+IDA	B-protein
+.	O
+
+We	O
+obtained	O
+a	O
+structure	B-evidence
+of	O
+HAESA	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+peptide	B-chemical
+at	O
+1	O
+.	O
+94	O
+Å	O
+resolution	O
+(	O
+Table	O
+2	O
+).	O
+
+In	O
+this	O
+structure	B-evidence
+,	O
+no	O
+additional	O
+electron	B-evidence
+density	I-evidence
+accounts	O
+for	O
+the	O
+PKGV	B-structure_element
+motif	I-structure_element
+at	O
+the	O
+IDA	B-protein
+N	O
+-	O
+terminus	O
+(	O
+Figure	O
+2A	O
+,	O
+B	O
+).	O
+
+Consistently	O
+,	O
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+and	O
+IDA	B-protein
+have	O
+similar	O
+binding	B-evidence
+affinities	I-evidence
+in	O
+our	O
+ITC	B-experimental_method
+assays	I-experimental_method
+,	O
+further	O
+indicating	O
+that	O
+HAESA	B-protein
+senses	O
+a	O
+dodecamer	B-structure_element
+peptide	B-chemical
+comprising	O
+residues	O
+58	B-residue_range
+-	I-residue_range
+69IDA	I-residue_range
+(	O
+Figure	O
+2D	O
+).	O
+
+We	O
+next	O
+tested	O
+if	O
+HAESA	B-protein
+binds	O
+other	O
+IDA	B-chemical
+peptide	I-chemical
+family	I-chemical
+members	I-chemical
+.	O
+
+IDL1	B-protein
+,	O
+which	O
+can	O
+rescue	O
+IDA	B-protein
+loss	O
+-	O
+of	O
+-	O
+function	O
+mutants	O
+when	O
+introduced	O
+in	O
+abscission	O
+zone	O
+cells	O
+,	O
+can	O
+also	O
+be	O
+sensed	O
+by	O
+HAESA	B-protein
+,	O
+albeit	O
+with	O
+lower	O
+affinity	B-evidence
+(	O
+Figure	O
+2D	O
+).	O
+
+A	O
+2	O
+.	O
+56	O
+Å	O
+co	B-evidence
+-	I-evidence
+crystal	I-evidence
+structure	I-evidence
+with	O
+IDL1	B-protein
+reveals	O
+that	O
+different	O
+IDA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+use	O
+a	O
+common	O
+binding	O
+mode	O
+to	O
+interact	O
+with	O
+HAESA	B-protein_type
+-	I-protein_type
+type	I-protein_type
+receptors	I-protein_type
+(	O
+Figure	O
+2A	O
+–	O
+C	O
+,	O
+E	O
+,	O
+Table	O
+2	O
+).	O
+
+We	O
+do	O
+not	O
+detect	O
+interaction	O
+between	O
+HAESA	B-protein
+and	O
+a	O
+synthetic	B-protein_state
+peptide	B-chemical
+missing	B-protein_state
+the	I-protein_state
+C	I-protein_state
+-	I-protein_state
+terminal	I-protein_state
+Asn69IDA	B-residue_name_number
+(	O
+ΔN69	B-mutant
+),	O
+highlighting	O
+the	O
+importance	O
+of	O
+the	O
+polar	O
+interactions	O
+between	O
+the	O
+IDA	B-protein
+carboxy	O
+-	O
+terminus	O
+and	O
+Arg407HAESA	B-residue_name_number
+/	O
+Arg409HAESA	B-residue_name_number
+(	O
+Figures	O
+1F	O
+,	O
+2D	O
+).	O
+
+Replacing	B-experimental_method
+Hyp64IDA	B-residue_name_number
+,	O
+which	O
+is	O
+common	O
+to	O
+all	O
+IDLs	B-protein_type
+,	O
+with	O
+proline	B-residue_name
+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	B-protein_state
+-	I-protein_state
+mutant	I-protein_state
+discussed	O
+below	O
+(	O
+Figure	O
+1A	O
+,	O
+2D	O
+).	O
+
+Notably	O
+,	O
+HAESA	B-protein
+can	O
+discriminate	O
+between	O
+IDLs	B-protein_type
+and	O
+functionally	B-protein_state
+unrelated	I-protein_state
+dodecamer	B-structure_element
+peptides	B-chemical
+with	O
+Hyp	B-ptm
+modifications	I-ptm
+,	O
+such	O
+as	O
+CLV3	B-protein
+(	O
+Figures	O
+2D	O
+,	O
+7	O
+).	O
+
+The	O
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+kinase	I-protein_type
+SERK1	B-protein
+allows	O
+for	O
+high	O
+-	O
+affinity	O
+IDA	O
+sensing	O
+
+Our	O
+binding	B-experimental_method
+assays	I-experimental_method
+reveal	O
+that	O
+IDA	B-chemical
+family	I-chemical
+peptides	I-chemical
+are	O
+sensed	O
+by	O
+the	O
+isolated	B-protein_state
+HAESA	B-protein
+ectodomain	B-structure_element
+with	O
+relatively	O
+weak	O
+binding	B-evidence
+affinities	I-evidence
+(	O
+Figures	O
+1B	O
+,	O
+2A	O
+–	O
+D	O
+).	O
+
+It	O
+has	O
+been	O
+recently	O
+reported	O
+that	O
+SOMATIC	B-protein_type
+EMBRYOGENESIS	I-protein_type
+RECEPTOR	I-protein_type
+KINASES	I-protein_type
+(	O
+SERKs	B-protein_type
+)	O
+are	O
+positive	O
+regulators	O
+of	O
+floral	O
+abscission	O
+and	O
+can	O
+interact	O
+with	O
+HAESA	B-protein
+and	O
+HSL2	B-protein
+in	O
+an	O
+IDA	O
+-	O
+dependent	O
+manner	O
+.	O
+
+As	O
+all	O
+five	O
+SERK	B-protein_type
+family	I-protein_type
+members	I-protein_type
+appear	O
+to	O
+be	O
+expressed	O
+in	O
+the	O
+Arabidopsis	B-taxonomy_domain
+abscission	O
+zone	O
+,	O
+we	O
+quantified	O
+their	O
+relative	O
+contribution	O
+to	O
+floral	O
+abscission	O
+in	O
+Arabidopsis	B-taxonomy_domain
+using	O
+a	O
+petal	B-experimental_method
+break	I-experimental_method
+-	I-experimental_method
+strength	I-experimental_method
+assay	I-experimental_method
+.	O
+
+Our	O
+experiments	O
+suggest	O
+that	O
+among	O
+the	O
+SERK	B-protein_type
+family	I-protein_type
+members	I-protein_type
+,	O
+SERK1	B-protein
+is	O
+a	O
+positive	O
+regulator	O
+of	O
+floral	O
+abscission	O
+.	O
+
+We	O
+found	O
+that	O
+the	O
+force	O
+required	O
+to	O
+remove	O
+the	O
+petals	O
+of	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+mutants	B-protein_state
+is	O
+significantly	O
+higher	O
+than	O
+that	O
+needed	O
+for	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+plants	B-taxonomy_domain
+,	O
+as	O
+previously	O
+observed	O
+for	O
+haesa	B-gene
+/	O
+hsl2	B-gene
+mutants	B-protein_state
+,	O
+and	O
+that	O
+floral	O
+abscission	O
+is	O
+delayed	O
+in	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+(	O
+Figure	O
+3A	O
+).	O
+
+The	O
+serk2	B-gene
+-	I-gene
+2	I-gene
+,	O
+serk3	B-gene
+-	I-gene
+1	I-gene
+,	O
+serk4	B-gene
+-	I-gene
+1	I-gene
+and	O
+serk5	B-gene
+-	I-gene
+1	I-gene
+mutant	B-protein_state
+lines	O
+showed	O
+a	O
+petal	O
+break	O
+-	O
+strength	O
+profile	O
+not	O
+significantly	O
+different	O
+from	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+plants	B-taxonomy_domain
+.	O
+
+Possibly	O
+because	O
+SERKs	B-protein_type
+have	O
+additional	O
+roles	O
+in	O
+plant	O
+development	O
+such	O
+as	O
+in	O
+pollen	O
+formation	O
+and	O
+brassinosteroid	O
+signaling	O
+,	O
+we	O
+found	O
+that	O
+higher	O
+-	O
+order	O
+SERK	O
+mutants	O
+exhibit	O
+pleiotropic	O
+phenotypes	O
+in	O
+the	O
+flower	O
+,	O
+rendering	O
+their	O
+analysis	O
+and	O
+comparison	O
+by	O
+quantitative	B-experimental_method
+petal	I-experimental_method
+break	I-experimental_method
+-	I-experimental_method
+strength	I-experimental_method
+assays	I-experimental_method
+difficult	O
+.	O
+
+We	O
+thus	O
+focused	O
+on	O
+analyzing	O
+the	O
+contribution	O
+of	O
+SERK1	B-protein
+to	O
+HAESA	B-protein
+ligand	O
+sensing	O
+and	O
+receptor	O
+activation	O
+.	O
+
+In	O
+vitro	O
+,	O
+the	O
+LRR	B-structure_element
+ectodomain	I-structure_element
+of	O
+SERK1	B-protein
+(	O
+residues	O
+24	B-residue_range
+–	I-residue_range
+213	I-residue_range
+)	O
+forms	O
+stable	B-protein_state
+,	O
+IDA	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+heterodimeric	B-oligomeric_state
+complexes	B-protein_state
+with	I-protein_state
+HAESA	B-protein
+in	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+experiments	O
+(	O
+Figure	O
+3B	O
+).	O
+
+We	O
+next	O
+quantified	O
+the	O
+contribution	O
+of	O
+SERK1	B-protein
+to	O
+IDA	B-protein
+recognition	O
+by	O
+HAESA	B-protein
+.	O
+
+We	O
+found	O
+that	O
+HAESA	B-protein
+senses	O
+IDA	B-protein
+with	O
+a	O
+~	O
+60	O
+fold	O
+higher	O
+binding	B-evidence
+affinity	I-evidence
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+SERK1	B-protein
+,	O
+suggesting	O
+that	O
+SERK1	B-protein
+is	O
+involved	O
+in	O
+the	O
+specific	O
+recognition	O
+of	O
+the	O
+peptide	B-protein_type
+hormone	I-protein_type
+(	O
+Figure	O
+3C	O
+).	O
+
+We	O
+next	O
+titrated	B-experimental_method
+SERK1	B-protein
+into	O
+a	O
+solution	O
+containing	O
+only	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+
+In	O
+this	O
+case	O
+,	O
+there	O
+was	O
+no	O
+detectable	O
+interaction	O
+between	O
+receptor	O
+and	O
+co	O
+-	O
+receptor	O
+,	O
+while	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+IDA	B-protein
+,	O
+SERK1	B-protein
+strongly	O
+binds	O
+HAESA	B-protein
+with	O
+a	O
+dissociation	B-evidence
+constant	I-evidence
+in	O
+the	O
+mid	O
+-	O
+nanomolar	O
+range	O
+(	O
+Figure	O
+3C	O
+).	O
+
+This	O
+suggests	O
+that	O
+IDA	B-protein
+itself	O
+promotes	O
+receptor	O
+–	O
+co	O
+-	O
+receptor	O
+association	O
+,	O
+as	O
+previously	O
+described	O
+for	O
+the	O
+steroid	B-chemical
+hormone	I-chemical
+brassinolide	B-chemical
+and	O
+for	O
+other	O
+LRR	B-complex_assembly
+-	I-complex_assembly
+RK	I-complex_assembly
+complexes	O
+.	O
+
+Importantly	O
+,	O
+hydroxyprolination	B-ptm
+of	O
+IDA	B-protein
+is	O
+critical	O
+for	O
+HAESA	B-complex_assembly
+-	I-complex_assembly
+IDA	I-complex_assembly
+-	I-complex_assembly
+SERK1	I-complex_assembly
+complex	O
+formation	O
+(	O
+Figure	O
+3C	O
+,	O
+D	O
+).	O
+
+Our	O
+calorimetry	B-experimental_method
+experiments	O
+now	O
+reveal	O
+that	O
+SERKs	B-protein_type
+may	O
+render	O
+HAESA	B-protein
+,	O
+and	O
+potentially	O
+other	O
+receptor	B-protein_type
+kinases	I-protein_type
+,	O
+competent	O
+for	O
+high	O
+-	O
+affinity	O
+sensing	O
+of	O
+their	O
+cognate	O
+ligands	O
+.	O
+
+Upon	O
+IDA	B-protein
+binding	O
+at	O
+the	O
+cell	O
+surface	O
+,	O
+the	O
+kinase	B-structure_element
+domains	I-structure_element
+of	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+,	O
+which	O
+have	O
+been	O
+shown	O
+to	O
+be	O
+active	B-protein_state
+protein	B-protein_type
+kinases	I-protein_type
+,	O
+may	O
+interact	O
+in	O
+the	O
+cytoplasm	O
+to	O
+activate	O
+each	O
+other	O
+.	O
+
+Consistently	O
+,	O
+the	O
+HAESA	B-protein
+kinase	B-structure_element
+domain	I-structure_element
+can	O
+transphosphorylate	O
+SERK1	B-protein
+and	O
+vice	O
+versa	O
+in	O
+in	O
+vitro	O
+transphosphorylation	B-experimental_method
+assays	I-experimental_method
+(	O
+Figure	O
+3E	O
+).	O
+
+Together	O
+,	O
+our	O
+genetic	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+experiments	I-experimental_method
+implicate	O
+SERK1	B-protein
+as	O
+a	O
+HAESA	B-protein_type
+co	I-protein_type
+-	I-protein_type
+receptor	I-protein_type
+in	O
+the	O
+Arabidopsis	B-taxonomy_domain
+abscission	O
+zone	O
+.	O
+
+SERK1	B-protein
+senses	O
+a	O
+conserved	B-protein_state
+motif	B-structure_element
+in	O
+IDA	B-chemical
+family	I-chemical
+peptides	I-chemical
+
+Crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+IDA	I-complex_assembly
+–	I-complex_assembly
+SERK1	I-complex_assembly
+signaling	O
+complex	O
+.	O
+
+(	O
+A	O
+)	O
+Overview	O
+of	O
+the	O
+ternary	O
+complex	O
+with	O
+HAESA	B-protein
+in	O
+blue	O
+(	O
+surface	O
+representation	O
+),	O
+IDA	B-protein
+in	O
+yellow	O
+(	O
+bonds	O
+representation	O
+)	O
+and	O
+SERK1	B-protein
+in	O
+orange	O
+(	O
+surface	O
+view	O
+).	O
+(	O
+B	O
+)	O
+The	O
+HAESA	B-protein
+ectodomain	B-structure_element
+undergoes	O
+a	O
+conformational	O
+change	O
+upon	O
+SERK1	B-protein
+co	O
+-	O
+receptor	O
+binding	O
+.	O
+
+Shown	O
+are	O
+Cα	O
+traces	O
+of	O
+a	O
+structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+unbound	B-protein_state
+(	O
+yellow	O
+)	O
+and	O
+SERK1	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+(	O
+blue	O
+)	O
+HAESA	B-protein
+ectodomains	B-structure_element
+(	O
+r	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.	I-evidence
+is	O
+1	O
+.	O
+5	O
+Å	O
+between	O
+572	O
+corresponding	O
+Cα	O
+atoms	O
+).	O
+
+SERK1	B-protein
+(	O
+in	O
+orange	O
+)	O
+and	O
+IDA	B-protein
+(	O
+in	O
+red	O
+)	O
+are	O
+shown	O
+alongside	O
+.	O
+
+The	O
+conformational	O
+change	O
+in	O
+the	O
+C	O
+-	O
+terminal	O
+LRRs	B-structure_element
+and	O
+capping	B-structure_element
+domain	I-structure_element
+is	O
+indicated	O
+by	O
+an	O
+arrow	O
+.	O
+(	O
+C	O
+)	O
+SERK1	B-protein
+forms	O
+an	O
+integral	O
+part	O
+of	O
+the	O
+receptor	O
+'	O
+s	O
+peptide	B-site
+binding	I-site
+pocket	I-site
+.	O
+
+The	O
+N	O
+-	O
+terminal	O
+capping	B-structure_element
+domain	I-structure_element
+of	O
+SERK1	B-protein
+(	O
+in	O
+orange	O
+)	O
+directly	O
+contacts	O
+the	O
+C	O
+-	O
+terminal	O
+part	O
+of	O
+IDA	B-protein
+(	O
+in	O
+yellow	O
+,	O
+in	O
+bonds	O
+representation	O
+)	O
+and	O
+the	O
+receptor	B-protein_type
+HAESA	B-protein
+(	O
+in	O
+blue	O
+).	O
+
+Polar	O
+contacts	O
+of	O
+SERK1	B-protein
+with	O
+IDA	B-protein
+are	O
+shown	O
+in	O
+magenta	O
+,	O
+with	O
+the	O
+HAESA	B-protein
+LRR	B-structure_element
+domain	I-structure_element
+in	O
+gray	O
+.	O
+(	O
+D	O
+)	O
+Details	O
+of	O
+the	O
+zipper	B-structure_element
+-	I-structure_element
+like	I-structure_element
+SERK1	B-site
+-	I-site
+HAESA	I-site
+interface	I-site
+.	O
+
+Ribbon	O
+diagrams	O
+of	O
+HAESA	B-protein
+(	O
+in	O
+blue	O
+)	O
+and	O
+SERK1	B-protein
+(	O
+in	O
+orange	O
+)	O
+are	O
+shown	O
+with	O
+selected	O
+interface	B-site
+residues	I-site
+(	O
+in	O
+bonds	O
+representation	O
+).	O
+
+To	O
+understand	O
+in	O
+molecular	O
+terms	O
+how	O
+SERK1	B-protein
+contributes	O
+to	O
+high	O
+-	O
+affinity	O
+IDA	B-protein
+recognition	O
+,	O
+we	O
+solved	O
+a	O
+2	O
+.	O
+43	O
+Å	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+ternary	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+IDA	I-complex_assembly
+–	I-complex_assembly
+SERK1	I-complex_assembly
+complex	O
+(	O
+Figure	O
+4A	O
+,	O
+Table	O
+2	O
+).	O
+
+HAESA	B-protein
+LRRs	B-structure_element
+16	I-structure_element
+–	I-structure_element
+21	I-structure_element
+and	O
+its	O
+C	O
+-	O
+terminal	O
+capping	B-structure_element
+domain	I-structure_element
+undergo	O
+a	O
+conformational	O
+change	O
+upon	O
+SERK1	B-protein
+binding	O
+(	O
+Figure	O
+4B	O
+).	O
+
+The	O
+SERK1	B-protein
+ectodomain	B-structure_element
+interacts	O
+with	O
+the	O
+IDA	B-site
+peptide	I-site
+binding	I-site
+site	I-site
+using	O
+a	O
+loop	B-structure_element
+region	I-structure_element
+(	O
+residues	O
+51	B-residue_range
+-	I-residue_range
+59SERK1	I-residue_range
+)	O
+from	O
+its	O
+N	O
+-	O
+terminal	O
+cap	B-structure_element
+(	O
+Figure	O
+4A	O
+,	O
+C	O
+).	O
+
+SERK1	B-protein
+loop	B-structure_element
+residues	O
+establish	O
+multiple	O
+hydrophobic	O
+and	O
+polar	O
+contacts	O
+with	O
+Lys66IDA	B-residue_name_number
+and	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+(	O
+Figure	O
+4C	O
+).	O
+
+SERK1	B-protein
+LRRs	B-structure_element
+1	I-structure_element
+–	I-structure_element
+5	I-structure_element
+and	O
+its	O
+C	O
+-	O
+terminal	O
+capping	B-structure_element
+domain	I-structure_element
+form	O
+an	O
+additional	O
+zipper	B-structure_element
+-	I-structure_element
+like	I-structure_element
+interface	B-site
+with	O
+residues	O
+originating	O
+from	O
+HAESA	B-protein
+LRRs	B-structure_element
+15	I-structure_element
+–	I-structure_element
+21	I-structure_element
+and	O
+from	O
+the	O
+HAESA	B-protein
+C	O
+-	O
+terminal	O
+cap	B-structure_element
+(	O
+Figure	O
+4D	O
+).	O
+
+SERK1	B-protein
+binds	O
+HAESA	B-protein
+using	O
+these	O
+two	O
+distinct	O
+interaction	B-site
+surfaces	I-site
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+),	O
+with	O
+the	O
+N	B-structure_element
+-	I-structure_element
+cap	I-structure_element
+of	O
+the	O
+SERK1	B-protein
+LRR	B-structure_element
+domain	I-structure_element
+partially	O
+covering	O
+the	O
+IDA	B-site
+peptide	I-site
+binding	I-site
+cleft	I-site
+.	O
+
+The	O
+IDA	B-protein
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+motif	I-structure_element
+is	O
+required	O
+for	O
+HAESA	B-complex_assembly
+-	I-complex_assembly
+SERK1	I-complex_assembly
+complex	O
+formation	O
+and	O
+for	O
+IDA	O
+bioactivity	O
+.	O
+
+(	O
+A	O
+)	O
+Size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+experiments	O
+similar	O
+to	O
+Figure	O
+3B	O
+,	O
+D	O
+reveal	O
+that	O
+IDA	B-protein
+mutant	B-protein_state
+peptides	B-chemical
+targeting	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+motif	I-structure_element
+do	O
+not	O
+form	O
+biochemically	B-protein_state
+stable	I-protein_state
+HAESA	B-complex_assembly
+-	I-complex_assembly
+IDA	I-complex_assembly
+-	I-complex_assembly
+SERK1	I-complex_assembly
+complexes	O
+.	O
+
+Deletion	B-experimental_method
+of	O
+the	O
+C	O
+-	O
+terminal	O
+Asn69IDA	B-residue_name_number
+completely	O
+inhibits	B-protein_state
+complex	O
+formation	O
+.	O
+
+Purified	B-experimental_method
+HAESA	B-protein
+and	O
+SERK1	B-protein
+are	O
+~	O
+75	O
+and	O
+~	O
+28	O
+kDa	O
+,	O
+respectively	O
+.	O
+
+Left	O
+panel	O
+:	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+;	O
+center	O
+:	O
+IDA	B-mutant
+ΔN69	I-mutant
+,	O
+right	O
+panel	O
+:	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+of	O
+peak	O
+fractions	O
+.	O
+
+Note	O
+that	O
+the	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+input	O
+lanes	O
+have	O
+already	O
+been	O
+shown	O
+in	O
+Figure	O
+3D	O
+.	O
+(	O
+B	O
+)	O
+Isothermal	B-evidence
+titration	I-evidence
+thermographs	I-evidence
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+IDA	B-chemical
+peptides	I-chemical
+titrated	B-experimental_method
+into	O
+a	O
+HAESA	B-protein
+-	O
+SERK1	B-protein
+mixture	O
+in	O
+the	O
+cell	O
+.	O
+
+Table	O
+summaries	O
+for	O
+calorimetric	B-evidence
+binding	I-evidence
+constants	I-evidence
+and	O
+stoichoimetries	O
+for	O
+different	O
+IDA	B-chemical
+peptides	I-chemical
+binding	O
+to	O
+the	O
+HAESA	B-protein
+–	O
+SERK1	B-protein
+ectodomain	B-structure_element
+mixture	O
+(	O
+±	O
+fitting	O
+errors	O
+;	O
+n	O
+.	O
+d	O
+.	O
+
+(	O
+C	O
+)	O
+Quantitative	O
+petal	B-experimental_method
+break	I-experimental_method
+-	I-experimental_method
+strength	I-experimental_method
+assay	I-experimental_method
+for	O
+Col	O
+-	O
+0	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+flowers	O
+and	O
+35S	B-gene
+::	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+mutant	B-protein_state
+flowers	O
+.	O
+
+35S	B-gene
+::	O
+IDA	B-protein
+plants	B-taxonomy_domain
+showed	O
+significantly	O
+increased	O
+abscission	O
+compared	O
+to	O
+Col	O
+-	O
+0	O
+controls	O
+in	O
+inflorescence	O
+positions	O
+2	O
+and	O
+3	O
+(	O
+a	O
+).	O
+
+Up	O
+to	O
+inflorescence	O
+position	O
+4	O
+,	O
+petal	O
+break	O
+in	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+mutant	B-protein_state
+plants	B-taxonomy_domain
+was	O
+significantly	O
+increased	O
+compared	O
+to	O
+both	O
+Col	O
+-	O
+0	O
+control	O
+plants	B-taxonomy_domain
+(	O
+b	O
+)	O
+and	O
+35S	B-gene
+::	O
+IDA	B-protein
+plants	B-taxonomy_domain
+(	O
+c	O
+)	O
+(	O
+D	O
+)	O
+Normalized	O
+expression	O
+levels	O
+(	O
+relative	O
+expression	O
+±	O
+standard	O
+error	O
+;	O
+ida	O
+:	O
+-	O
+0	O
+.	O
+02	O
+±	O
+0	O
+.	O
+001	O
+;	O
+Col	O
+-	O
+0	O
+:	O
+1	O
+±	O
+0	O
+.	O
+11	O
+;	O
+35S	B-gene
+::	O
+IDA	B-protein
+124	O
+±	O
+0	O
+.	O
+75	O
+;	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+:	O
+159	O
+±	O
+0	O
+.	O
+58	O
+)	O
+of	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+transcripts	O
+in	O
+the	O
+35S	B-experimental_method
+promoter	I-experimental_method
+over	I-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+lines	I-experimental_method
+analyzed	O
+in	O
+(	O
+C	O
+).	O
+(	O
+E	O
+)	O
+Magnified	O
+view	O
+of	O
+representative	O
+abscission	O
+zones	O
+from	O
+35S	B-gene
+::	O
+IDA	B-protein
+,	O
+Col	O
+-	O
+0	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+double	B-protein_state
+-	I-protein_state
+mutant	I-protein_state
+T3	B-experimental_method
+transgenic	I-experimental_method
+lines	I-experimental_method
+.	O
+
+15	O
+out	O
+of	O
+15	O
+35S	B-gene
+::	O
+IDA	B-protein
+plants	B-taxonomy_domain
+,	O
+0	O
+out	O
+of	O
+15	O
+Col	O
+-	O
+0	O
+plants	B-taxonomy_domain
+and	O
+0	O
+out	O
+of	O
+15	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+double	B-protein_state
+-	I-protein_state
+mutant	I-protein_state
+plants	B-taxonomy_domain
+,	O
+showed	O
+an	O
+enlarged	O
+abscission	O
+zone	O
+,	O
+respectively	O
+(	O
+3	O
+independent	O
+lines	O
+were	O
+analyzed	O
+).	O
+
+The	O
+four	O
+C	O
+-	O
+terminal	O
+residues	O
+in	O
+IDA	B-protein
+(	O
+Lys66IDA	B-residue_range
+-	I-residue_range
+Asn69IDA	I-residue_range
+)	O
+are	O
+conserved	B-protein_state
+among	O
+IDA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+and	O
+are	O
+in	O
+direct	O
+contact	O
+with	O
+SERK1	B-protein
+(	O
+Figures	O
+1A	O
+,	O
+4C	O
+).	O
+
+We	O
+thus	O
+assessed	O
+their	O
+contribution	O
+to	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+SERK1	I-complex_assembly
+complex	O
+formation	O
+.	O
+
+Deletion	B-experimental_method
+of	O
+the	O
+buried	O
+Asn69IDA	B-residue_name_number
+completely	B-protein_state
+inhibits	I-protein_state
+receptor	O
+–	O
+co	O
+-	O
+receptor	O
+complex	O
+formation	O
+and	O
+HSL2	O
+activation	O
+(	O
+Figure	O
+5A	O
+,	O
+B	O
+).	O
+
+A	O
+synthetic	B-protein_state
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+mutant	B-protein_state
+peptide	B-chemical
+(	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R66A	I-mutant
+)	O
+showed	O
+a	O
+10	O
+fold	O
+reduced	O
+binding	B-evidence
+affinity	I-evidence
+when	O
+titrated	B-experimental_method
+in	O
+a	O
+HAESA	B-protein
+/	O
+SERK1	B-protein
+protein	O
+solution	O
+(	O
+Figures	O
+5A	O
+,	O
+B	O
+,	O
+2D	O
+).	O
+
+We	O
+over	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+IDA	B-protein
+or	O
+this	O
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+double	B-protein_state
+-	I-protein_state
+mutant	I-protein_state
+to	O
+similar	O
+levels	O
+in	O
+Col	O
+-	O
+0	O
+Arabidopsis	B-taxonomy_domain
+plants	B-taxonomy_domain
+(	O
+Figure	O
+5D	O
+).	O
+
+We	O
+found	O
+that	O
+over	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+IDA	B-protein
+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
+
+In	O
+contrast	O
+,	O
+over	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+the	O
+IDA	B-mutant
+Lys66IDA	I-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+double	B-protein_state
+mutant	I-protein_state
+significantly	O
+delays	O
+floral	O
+abscission	O
+when	O
+compared	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+control	O
+plants	B-taxonomy_domain
+,	O
+suggesting	O
+that	O
+the	O
+mutant	B-protein_state
+IDA	B-chemical
+peptide	I-chemical
+has	O
+reduced	O
+activity	O
+in	O
+planta	B-taxonomy_domain
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+
+Comparison	O
+of	O
+35S	B-gene
+::	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+plants	B-taxonomy_domain
+further	O
+indicates	O
+that	O
+mutation	B-experimental_method
+of	O
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+may	O
+cause	O
+a	O
+weak	O
+dominant	O
+negative	O
+effect	O
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+
+In	O
+agreement	O
+with	O
+our	O
+structures	B-evidence
+and	O
+biochemical	B-experimental_method
+assays	I-experimental_method
+,	O
+this	O
+experiment	O
+suggests	O
+a	O
+role	O
+of	O
+the	O
+conserved	B-protein_state
+IDA	B-protein
+C	O
+-	O
+terminus	O
+in	O
+the	O
+control	O
+of	O
+floral	O
+abscission	O
+.	O
+
+In	O
+contrast	O
+to	O
+animal	B-taxonomy_domain
+LRR	B-protein_type
+receptors	I-protein_type
+,	O
+plant	B-taxonomy_domain
+LRR	B-structure_element
+-	I-structure_element
+RKs	I-structure_element
+harbor	O
+spiral	B-protein_state
+-	I-protein_state
+shaped	I-protein_state
+ectodomains	B-structure_element
+and	O
+thus	O
+they	O
+require	O
+shape	B-protein_state
+-	I-protein_state
+complementary	I-protein_state
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+proteins	I-protein_type
+for	O
+receptor	O
+activation	O
+.	O
+
+For	O
+a	O
+rapidly	O
+growing	O
+number	O
+of	O
+plant	B-taxonomy_domain
+signaling	O
+pathways	O
+,	O
+SERK	B-protein_type
+proteins	I-protein_type
+act	O
+as	O
+these	O
+essential	O
+co	B-protein_type
+-	I-protein_type
+receptors	I-protein_type
+(;	O
+).	O
+
+SERK1	O
+has	O
+been	O
+previously	O
+reported	O
+as	O
+a	O
+positive	O
+regulator	O
+in	O
+plant	B-taxonomy_domain
+embryogenesis	O
+,	O
+male	O
+sporogenesis	O
+,	O
+brassinosteroid	O
+signaling	O
+and	O
+in	O
+phytosulfokine	O
+perception	O
+.	O
+
+Recent	O
+findings	O
+by	O
+and	O
+our	O
+mechanistic	O
+studies	O
+now	O
+also	O
+support	O
+a	O
+positive	O
+role	O
+for	O
+SERK1	B-protein
+in	O
+floral	O
+abscission	O
+.	O
+
+As	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+mutant	B-protein_state
+plants	B-taxonomy_domain
+show	O
+intermediate	O
+abscission	O
+phenotypes	O
+when	O
+compared	O
+to	O
+haesa	B-gene
+/	O
+hsl2	O
+mutants	B-protein_state
+,	O
+SERK1	B-protein
+likely	O
+acts	O
+redundantly	O
+with	O
+other	O
+SERKs	B-protein_type
+in	O
+the	O
+abscission	O
+zone	O
+(	O
+Figure	O
+3A	O
+).	O
+
+It	O
+has	O
+been	O
+previously	O
+suggested	O
+that	O
+SERK1	B-protein
+can	O
+inhibit	O
+cell	O
+separation	O
+.	O
+
+However	O
+our	O
+results	O
+show	O
+that	O
+SERK1	B-protein
+also	O
+can	O
+activate	O
+this	O
+process	O
+upon	O
+IDA	B-protein
+sensing	O
+,	O
+indicating	O
+that	O
+SERKs	B-protein_type
+may	O
+fulfill	O
+several	O
+different	O
+functions	O
+in	O
+the	O
+course	O
+of	O
+the	O
+abscission	O
+process	O
+.	O
+
+While	O
+the	O
+sequence	O
+of	O
+the	O
+mature	B-protein_state
+IDA	B-chemical
+peptide	I-chemical
+has	O
+not	O
+been	O
+experimentally	O
+determined	O
+in	O
+planta	B-taxonomy_domain
+,	O
+our	O
+HAESA	B-complex_assembly
+-	I-complex_assembly
+IDA	I-complex_assembly
+complex	O
+structures	B-evidence
+and	O
+calorimetry	B-evidence
+assays	I-evidence
+suggest	O
+that	O
+active	B-protein_state
+IDLs	B-protein_type
+are	O
+hydroxyprolinated	B-protein_state
+dodecamers	B-structure_element
+.	O
+
+It	O
+will	O
+be	O
+thus	O
+interesting	O
+to	O
+see	O
+if	O
+proteolytic	O
+processing	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+IDA	B-protein
+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
+
+The	O
+central	O
+Hyp	B-residue_name
+residue	O
+in	O
+IDA	B-protein
+is	O
+found	O
+buried	O
+in	O
+the	O
+HAESA	B-protein
+peptide	B-site
+binding	I-site
+surface	I-site
+and	O
+thus	O
+this	O
+post	O
+-	O
+translational	O
+modification	O
+may	O
+regulate	O
+IDA	B-protein
+bioactivity	O
+.	O
+
+Our	O
+comparative	B-experimental_method
+structural	I-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+analysis	I-experimental_method
+further	O
+suggests	O
+that	O
+IDLs	B-protein_type
+share	O
+a	O
+common	O
+receptor	O
+binding	O
+mode	O
+,	O
+but	O
+may	O
+preferably	O
+bind	O
+to	O
+HAESA	B-protein
+,	O
+HSL1	B-protein
+or	O
+HSL2	B-protein
+in	O
+different	O
+plant	B-taxonomy_domain
+tissues	O
+and	O
+organs	O
+.	O
+
+In	O
+our	O
+quantitative	B-experimental_method
+biochemical	I-experimental_method
+assays	I-experimental_method
+,	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+SERK1	B-protein
+dramatically	O
+increases	O
+the	O
+HAESA	B-protein
+binding	O
+specificity	O
+and	O
+affinity	O
+for	O
+IDA	B-protein
+.	O
+
+This	O
+observation	O
+is	O
+consistent	O
+with	O
+our	O
+complex	O
+structure	B-evidence
+in	O
+which	O
+receptor	O
+and	O
+co	O
+-	O
+receptor	O
+together	O
+form	O
+the	O
+IDA	B-site
+binding	I-site
+pocket	I-site
+.	O
+
+The	O
+fact	O
+that	O
+SERK1	B-protein
+specifically	O
+interacts	O
+with	O
+the	O
+very	O
+C	O
+-	O
+terminus	O
+of	O
+IDLs	B-protein_type
+may	O
+allow	O
+for	O
+the	O
+rational	O
+design	O
+of	O
+peptide	B-chemical
+hormone	I-chemical
+antagonists	I-chemical
+,	O
+as	O
+previously	O
+demonstrated	O
+for	O
+the	O
+brassinosteroid	O
+pathway	O
+.	O
+
+Importantly	O
+,	O
+our	O
+calorimetry	B-experimental_method
+assays	I-experimental_method
+reveal	O
+that	O
+the	O
+SERK1	B-protein
+ectodomain	B-structure_element
+binds	B-protein_state
+HAESA	B-protein
+with	O
+nanomolar	O
+affinity	O
+,	O
+but	O
+only	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+IDA	B-protein
+(	O
+Figure	O
+3C	O
+).	O
+
+This	O
+ligand	O
+-	O
+induced	O
+formation	O
+of	O
+a	O
+receptor	O
+–	O
+co	O
+-	O
+receptor	O
+complex	O
+may	O
+allow	O
+the	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+kinase	B-structure_element
+domains	I-structure_element
+to	O
+efficiently	O
+trans	O
+-	O
+phosphorylate	O
+and	O
+activate	O
+each	O
+other	O
+in	O
+the	O
+cytoplasm	O
+.	O
+
+It	O
+is	O
+of	O
+note	O
+that	O
+our	O
+reported	O
+binding	B-evidence
+affinities	I-evidence
+for	O
+IDA	B-protein
+and	O
+SERK1	B-protein
+have	O
+been	O
+measured	O
+using	O
+synthetic	B-protein_state
+peptides	B-chemical
+and	O
+the	O
+isolated	B-experimental_method
+HAESA	B-protein
+and	O
+SERK1	B-protein
+ectodomains	B-structure_element
+,	O
+and	O
+thus	O
+might	O
+differ	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+,	O
+membrane	B-protein_state
+-	I-protein_state
+embedded	I-protein_state
+signaling	O
+complex	O
+.	O
+
+SERK1	B-protein
+uses	O
+partially	O
+overlapping	O
+surface	O
+areas	O
+to	O
+activate	O
+different	O
+plant	B-taxonomy_domain
+signaling	B-protein_type
+receptors	I-protein_type
+.	O
+
+(	O
+A	O
+)	O
+Structural	B-experimental_method
+comparison	I-experimental_method
+of	O
+plant	B-taxonomy_domain
+steroid	B-chemical
+and	O
+peptide	B-protein_type
+hormone	I-protein_type
+membrane	B-protein_type
+signaling	I-protein_type
+complexes	I-protein_type
+.	O
+
+Left	O
+panel	O
+:	O
+Ribbon	O
+diagram	O
+of	O
+HAESA	B-protein
+(	O
+in	O
+blue	O
+),	O
+SERK1	B-protein
+(	O
+in	O
+orange	O
+)	O
+and	O
+IDA	B-protein
+(	O
+in	O
+bonds	O
+and	O
+surface	O
+represention	O
+).	O
+
+Right	O
+panel	O
+:	O
+Ribbon	O
+diagram	O
+of	O
+the	O
+plant	B-taxonomy_domain
+steroid	B-protein_type
+receptor	I-protein_type
+BRI1	B-protein
+(	O
+in	O
+blue	O
+)	O
+bound	B-protein_state
+to	I-protein_state
+brassinolide	B-chemical
+(	O
+in	O
+gray	O
+,	O
+in	O
+bonds	O
+representation	O
+)	O
+and	O
+to	O
+SERK1	B-protein
+,	O
+shown	O
+in	O
+the	O
+same	O
+orientation	O
+(	O
+PDB	O
+-	O
+ID	O
+.	O
+4lsx	O
+).	O
+
+(	O
+B	O
+)	O
+View	O
+of	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+SERK1	B-protein
+LRR	B-structure_element
+domain	I-structure_element
+(	O
+PDB	O
+-	O
+ID	O
+4lsc	O
+,	O
+surface	O
+representation	O
+,	O
+in	O
+gray	O
+).	O
+
+A	O
+ribbon	O
+diagram	O
+of	O
+SERK1	B-protein
+in	O
+the	O
+same	O
+orientation	O
+is	O
+shown	O
+alongside	O
+.	O
+
+Residues	O
+interacting	O
+with	O
+the	O
+HAESA	B-protein
+or	O
+BRI1	B-protein
+LRR	B-structure_element
+domains	I-structure_element
+are	O
+shown	O
+in	O
+orange	O
+or	O
+magenta	O
+,	O
+respectively	O
+.	O
+
+Comparison	B-experimental_method
+of	O
+our	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+IDA	I-complex_assembly
+–	I-complex_assembly
+SERK1	I-complex_assembly
+structure	B-evidence
+with	O
+the	O
+brassinosteroid	O
+receptor	O
+signaling	O
+complex	O
+,	O
+where	O
+SERK1	B-protein
+also	O
+acts	O
+as	O
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+,	O
+reveals	O
+an	O
+overall	O
+conserved	B-protein_state
+mode	O
+of	O
+SERK1	B-protein
+binding	O
+,	O
+while	O
+the	O
+ligand	B-site
+binding	I-site
+pockets	I-site
+map	O
+to	O
+very	O
+different	O
+areas	O
+in	O
+the	O
+corresponding	O
+receptors	O
+(	O
+LRRs	B-structure_element
+2	I-structure_element
+–	I-structure_element
+14	I-structure_element
+;	O
+HAESA	B-protein
+;	O
+LRRs	B-structure_element
+21	I-structure_element
+–	I-structure_element
+25	I-structure_element
+,	O
+BRI1	B-protein
+)	O
+and	O
+may	O
+involve	O
+an	O
+island	O
+domain	O
+(	O
+BRI1	B-protein
+)	O
+or	O
+not	O
+(	O
+HAESA	B-protein
+)	O
+(	O
+Figure	O
+6A	O
+).	O
+
+Several	O
+residues	O
+in	O
+the	O
+SERK1	B-protein
+N	O
+-	O
+terminal	O
+capping	B-structure_element
+domain	I-structure_element
+(	O
+Thr59SERK1	B-residue_name_number
+,	O
+Phe61SERK1	B-residue_name_number
+)	O
+and	O
+the	O
+LRR	B-site
+inner	I-site
+surface	I-site
+(	O
+Asp75SERK1	B-residue_name_number
+,	O
+Tyr101SERK1	B-residue_name_number
+,	O
+SER121SERK1	B-residue_name_number
+,	O
+Phe145SERK1	B-residue_name_number
+)	O
+contribute	O
+to	O
+the	O
+formation	O
+of	O
+both	O
+complexes	O
+(	O
+Figures	O
+4C	O
+,	O
+D	O
+,	O
+6B	O
+).	O
+
+In	O
+addition	O
+,	O
+residues	O
+53	B-residue_range
+-	I-residue_range
+55SERK1	I-residue_range
+from	O
+the	O
+SERK1	B-protein
+N	O
+-	O
+terminal	O
+cap	B-structure_element
+mediate	O
+specific	O
+interactions	O
+with	O
+the	O
+IDA	B-chemical
+peptide	I-chemical
+(	O
+Figures	O
+4C	O
+,	O
+6B	O
+).	O
+
+These	O
+residues	O
+are	O
+not	O
+involved	O
+in	O
+the	O
+sensing	O
+of	O
+the	O
+steroid	B-chemical
+hormone	I-chemical
+brassinolide	B-chemical
+.	O
+
+In	O
+both	O
+cases	O
+however	O
+,	O
+the	O
+co	O
+-	O
+receptor	O
+completes	O
+the	O
+hormone	B-site
+binding	I-site
+pocket	I-site
+.	O
+
+This	O
+fact	O
+together	O
+with	O
+the	O
+largely	O
+overlapping	O
+SERK1	B-site
+binding	I-site
+surfaces	I-site
+in	O
+HAESA	B-protein
+and	O
+BRI1	B-protein
+allows	O
+us	O
+to	O
+speculate	O
+that	O
+SERK1	B-protein
+may	O
+promote	O
+high	O
+-	O
+affinity	O
+peptide	B-protein_type
+hormone	I-protein_type
+and	O
+brassinosteroid	O
+sensing	O
+by	O
+simply	O
+slowing	O
+down	O
+dissociation	O
+of	O
+the	O
+ligand	O
+from	O
+its	O
+cognate	O
+receptor	O
+.	O
+
+Different	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormone	I-protein_type
+families	I-protein_type
+contain	O
+a	O
+C	O
+-	O
+terminal	O
+(	B-structure_element
+Arg	I-structure_element
+)-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+,	O
+which	O
+in	O
+IDA	B-protein
+represents	O
+the	O
+co	B-site
+-	I-site
+receptor	I-site
+recognition	I-site
+site	I-site
+.	O
+
+Structure	B-experimental_method
+-	I-experimental_method
+guided	I-experimental_method
+multiple	I-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+IDA	B-protein
+and	O
+IDA	B-chemical
+-	I-chemical
+like	I-chemical
+peptides	I-chemical
+with	O
+other	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormone	I-protein_type
+families	I-protein_type
+,	O
+including	O
+CLAVATA3	B-protein_type
+–	I-protein_type
+EMBRYO	I-protein_type
+SURROUNDING	I-protein_type
+REGION	I-protein_type
+-	I-protein_type
+RELATED	I-protein_type
+(	O
+CLV3	B-protein_type
+/	I-protein_type
+CLE	I-protein_type
+),	O
+ROOT	B-protein_type
+GROWTH	I-protein_type
+FACTOR	I-protein_type
+–	I-protein_type
+GOLVEN	I-protein_type
+(	O
+RGF	B-protein_type
+/	I-protein_type
+GLV	I-protein_type
+),	O
+PRECURSOR	B-protein_type
+GENE	I-protein_type
+PROPEP1	I-protein_type
+(	O
+PEP1	B-protein_type
+)	O
+from	O
+Arabidopsis	B-species
+thaliana	I-species
+.	O
+
+The	O
+conserved	B-protein_state
+(	B-structure_element
+Arg	I-structure_element
+)-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+is	O
+highlighted	O
+in	O
+red	O
+,	O
+the	O
+central	O
+Hyp	B-residue_name
+residue	O
+in	O
+IDLs	B-protein_type
+and	O
+CLEs	B-protein_type
+is	O
+marked	O
+in	O
+blue	O
+.	O
+
+Our	O
+experiments	O
+reveal	O
+that	O
+SERK1	B-protein
+recognizes	O
+a	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+.	O
+
+Importantly	O
+,	O
+this	B-structure_element
+motif	I-structure_element
+can	O
+also	O
+be	O
+found	O
+in	O
+other	O
+peptide	B-protein_type
+hormone	I-protein_type
+families	I-protein_type
+(	O
+Figure	O
+7	O
+).	O
+
+Among	O
+these	O
+are	O
+the	O
+CLE	B-chemical
+peptides	I-chemical
+regulating	O
+stem	O
+cell	O
+maintenance	O
+in	O
+the	O
+shoot	O
+and	O
+the	O
+root	O
+.	O
+
+It	O
+is	O
+interesting	O
+to	O
+note	O
+,	O
+that	O
+CLEs	B-protein_type
+in	O
+their	O
+mature	B-protein_state
+form	I-protein_state
+are	O
+also	O
+hydroxyprolinated	B-protein_state
+dodecamers	B-structure_element
+,	O
+which	O
+bind	O
+to	O
+a	O
+surface	B-site
+area	I-site
+in	O
+the	O
+BARELY	B-protein_type
+ANY	I-protein_type
+MERISTEM	I-protein_type
+1	I-protein_type
+receptor	I-protein_type
+that	O
+would	O
+correspond	O
+to	O
+part	O
+of	O
+the	O
+IDA	B-site
+binding	I-site
+cleft	I-site
+in	O
+HAESA	B-protein
+.	O
+
+Diverse	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormones	I-protein_type
+may	O
+thus	O
+also	O
+bind	O
+their	O
+LRR	B-protein_type
+-	I-protein_type
+RK	I-protein_type
+receptors	I-protein_type
+in	O
+an	O
+extended	B-protein_state
+conformation	I-protein_state
+along	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+LRR	B-structure_element
+domain	I-structure_element
+and	O
+may	O
+also	O
+use	O
+small	B-protein_state
+,	O
+shape	B-protein_state
+-	I-protein_state
+complementary	I-protein_state
+co	B-protein_type
+-	I-protein_type
+receptors	I-protein_type
+for	O
+high	O
+-	O
+affinity	O
+ligand	O
+binding	O
+and	O
+receptor	O
+activation	O
+.	O
+
+The	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+is	O
+a	O
+reader	O
+of	O
+histone	B-protein_type
+crotonylation	B-ptm
+
+The	O
+discovery	O
+of	O
+new	O
+histone	B-protein_type
+modifications	O
+is	O
+unfolding	O
+at	O
+startling	O
+rates	O
+,	O
+however	O
+,	O
+the	O
+identification	O
+of	O
+effectors	O
+capable	O
+of	O
+interpreting	O
+these	O
+modifications	O
+has	O
+lagged	O
+behind	O
+.	O
+
+Here	O
+we	O
+report	O
+the	O
+YEATS	B-structure_element
+domain	I-structure_element
+as	O
+an	O
+effective	O
+reader	O
+of	O
+histone	B-protein_type
+lysine	B-residue_name
+crotonylation	B-ptm
+–	O
+an	O
+epigenetic	O
+signature	O
+associated	O
+with	O
+active	O
+transcription	O
+.	O
+
+We	O
+show	O
+that	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+engages	O
+crotonyllysine	B-residue_name
+via	O
+a	O
+unique	O
+π	O
+-	O
+π	O
+-	O
+π	O
+-	O
+stacking	O
+mechanism	O
+and	O
+that	O
+other	O
+YEATS	B-structure_element
+domains	I-structure_element
+have	O
+crotonyllysine	B-residue_name
+binding	O
+activity	O
+.	O
+
+Crotonylation	B-ptm
+of	O
+lysine	B-residue_name
+residues	O
+(	O
+crotonyllysine	B-residue_name
+,	O
+Kcr	B-residue_name
+)	O
+has	O
+emerged	O
+as	O
+one	O
+of	O
+the	O
+fundamental	O
+histone	B-protein_type
+post	O
+-	O
+translational	O
+modifications	O
+(	O
+PTMs	O
+)	O
+found	O
+in	O
+mammalian	B-taxonomy_domain
+chromatin	O
+.	O
+
+The	O
+crotonyllysine	B-residue_name
+mark	O
+on	O
+histone	B-protein_type
+H3K18	B-protein_type
+is	O
+produced	O
+by	O
+p300	B-protein
+,	O
+a	O
+histone	B-protein_type
+acetyltransferase	I-protein_type
+also	O
+responsible	O
+for	O
+acetylation	B-ptm
+of	O
+histones	O
+.	O
+
+Owing	O
+to	O
+some	O
+differences	O
+in	O
+their	O
+genomic	O
+distribution	O
+,	O
+the	O
+crotonyllysine	B-residue_name
+and	O
+acetyllysine	B-residue_name
+(	O
+Kac	B-residue_name
+)	O
+modifications	O
+have	O
+been	O
+linked	O
+to	O
+distinct	O
+functional	O
+outcomes	O
+.	O
+
+p300	B-protein
+-	O
+catalyzed	O
+histone	B-protein_type
+crotonylation	B-ptm
+,	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	B-protein
+-	O
+catalyzed	O
+acetylation	B-ptm
+.	O
+
+The	O
+discovery	O
+of	O
+individual	O
+biological	O
+roles	O
+for	O
+the	O
+crotonyllysine	B-residue_name
+and	O
+acetyllysine	B-residue_name
+marks	O
+suggests	O
+that	O
+these	O
+PTMs	O
+can	O
+be	O
+read	O
+by	O
+distinct	O
+readers	O
+.	O
+
+While	O
+a	O
+number	O
+of	O
+acetyllysine	B-residue_name
+readers	O
+have	O
+been	O
+identified	O
+and	O
+characterized	O
+,	O
+a	O
+specific	O
+reader	O
+of	O
+the	O
+crotonyllysine	B-residue_name
+mark	O
+remains	O
+unknown	O
+(	O
+reviewed	O
+in	O
+).	O
+
+A	O
+recent	O
+survey	O
+of	O
+bromodomains	B-structure_element
+(	O
+BDs	B-structure_element
+)	O
+demonstrates	O
+that	O
+only	O
+one	O
+BD	B-structure_element
+associates	O
+very	O
+weakly	O
+with	O
+a	O
+crotonylated	B-protein_state
+peptide	O
+,	O
+however	O
+it	O
+binds	O
+more	O
+tightly	O
+to	O
+acetylated	B-protein_state
+peptides	O
+,	O
+inferring	O
+that	O
+bromodomains	B-structure_element
+do	O
+not	O
+possess	O
+physiologically	O
+relevant	O
+crotonyllysine	B-residue_name
+binding	O
+activity	O
+.	O
+
+The	O
+family	O
+of	O
+acetyllysine	B-residue_name
+readers	O
+has	O
+been	O
+expanded	O
+with	O
+the	O
+discovery	O
+that	O
+the	O
+YEATS	B-structure_element
+(	O
+Yaf9	B-protein
+,	O
+ENL	B-protein
+,	O
+AF9	B-protein
+,	O
+Taf14	B-protein
+,	O
+Sas5	B-protein
+)	O
+domains	O
+of	O
+human	B-species
+AF9	B-protein
+and	O
+yeast	B-taxonomy_domain
+Taf14	B-protein
+are	O
+capable	O
+of	O
+recognizing	O
+the	O
+histone	B-protein_type
+mark	O
+H3K9ac	B-protein_type
+.	O
+
+The	O
+acetyllysine	B-residue_name
+binding	O
+function	O
+of	O
+the	O
+AF9	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+is	O
+essential	O
+for	O
+the	O
+recruitment	O
+of	O
+the	O
+histone	B-protein_type
+methyltransferase	I-protein_type
+DOT1L	B-protein
+to	O
+H3K9ac	B-protein_type
+-	O
+containing	O
+chromatin	O
+and	O
+for	O
+DOT1L	B-protein
+-	O
+mediated	O
+H3K79	B-protein_type
+methylation	B-ptm
+and	O
+transcription	O
+.	O
+
+Similarly	O
+,	O
+activation	O
+of	O
+a	O
+subset	O
+of	O
+genes	O
+and	O
+DNA	O
+damage	O
+repair	O
+in	O
+yeast	B-taxonomy_domain
+require	O
+the	O
+acetyllysine	B-residue_name
+binding	O
+activity	O
+of	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+.	O
+
+Consistent	O
+with	O
+its	O
+role	O
+in	O
+gene	O
+regulation	O
+,	O
+Taf14	B-protein
+was	O
+identified	O
+as	O
+a	O
+core	O
+component	O
+of	O
+the	O
+transcription	O
+factor	O
+complexes	O
+TFIID	B-complex_assembly
+and	O
+TFIIF	B-complex_assembly
+.	O
+
+However	O
+,	O
+Taf14	B-protein
+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	B-complex_assembly
+,	O
+SWI	B-complex_assembly
+/	I-complex_assembly
+SNF	I-complex_assembly
+and	O
+RSC	B-complex_assembly
+)	O
+and	O
+the	O
+histone	B-protein_type
+acetyltransferase	I-protein_type
+complex	O
+NuA3	B-complex_assembly
+,	O
+indicating	O
+a	O
+multifaceted	O
+role	O
+of	O
+Taf14	B-protein
+in	O
+transcriptional	O
+regulation	O
+and	O
+chromatin	O
+biology	O
+.	O
+
+In	O
+this	O
+study	O
+,	O
+we	O
+identified	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+as	O
+a	O
+reader	O
+of	O
+crotonyllysine	B-residue_name
+that	O
+binds	O
+to	O
+histone	B-protein_type
+H3	B-protein_type
+crotonylated	B-protein_state
+at	O
+lysine	B-residue_name_number
+9	I-residue_name_number
+(	O
+H3K9cr	B-protein_type
+)	O
+via	O
+a	O
+distinctive	O
+binding	O
+mechanism	O
+.	O
+
+We	O
+found	O
+that	O
+H3K9cr	B-protein_type
+is	O
+present	O
+in	O
+yeast	B-taxonomy_domain
+and	O
+is	O
+dynamically	O
+regulated	O
+.	O
+
+To	O
+elucidate	O
+the	O
+molecular	O
+basis	O
+for	O
+recognition	O
+of	O
+the	O
+H3K9cr	B-protein_type
+mark	O
+,	O
+we	O
+obtained	O
+a	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+H3K9cr5	B-chemical
+-	I-chemical
+13	I-chemical
+(	O
+residues	O
+5	B-residue_range
+–	I-residue_range
+13	I-residue_range
+of	O
+H3	B-protein_type
+)	O
+peptide	O
+(	O
+Fig	O
+.	O
+1	O
+,	O
+Supplementary	O
+Results	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+and	O
+Supplementary	O
+Table	O
+1	O
+).	O
+
+The	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+adopts	O
+an	O
+immunoglobin	B-structure_element
+-	I-structure_element
+like	I-structure_element
+β	I-structure_element
+sandwich	I-structure_element
+fold	I-structure_element
+containing	O
+eight	O
+anti	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+strands	I-structure_element
+linked	O
+by	O
+short	O
+loops	B-structure_element
+that	O
+form	O
+a	O
+binding	B-site
+site	I-site
+for	O
+H3K9cr	B-protein_type
+(	O
+Fig	O
+.	O
+1b	O
+).	O
+
+The	O
+H3K9cr	B-protein_type
+peptide	O
+lays	O
+in	O
+an	O
+extended	B-protein_state
+conformation	I-protein_state
+in	O
+an	O
+orientation	O
+orthogonal	O
+to	O
+the	O
+β	B-structure_element
+strands	I-structure_element
+and	O
+is	O
+stabilized	O
+through	O
+an	O
+extensive	O
+network	O
+of	O
+direct	O
+and	O
+water	O
+-	O
+mediated	O
+hydrogen	O
+bonds	O
+and	O
+a	O
+salt	O
+bridge	O
+(	O
+Fig	O
+.	O
+1c	O
+).	O
+
+The	O
+most	O
+striking	O
+feature	O
+of	O
+the	O
+crotonyllysine	B-residue_name
+recognition	O
+mechanism	O
+is	O
+the	O
+unique	O
+coordination	O
+of	O
+crotonylated	B-protein_state
+lysine	B-residue_name
+residue	O
+.	O
+
+The	O
+fully	O
+extended	O
+side	O
+chain	O
+of	O
+K9cr	B-residue_name_number
+transverses	O
+the	O
+narrow	O
+tunnel	O
+,	O
+crossing	O
+the	O
+β	B-structure_element
+sandwich	I-structure_element
+at	O
+right	O
+angle	O
+in	O
+a	O
+corkscrew	O
+-	O
+like	O
+manner	O
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Figure	O
+1b	O
+).	O
+
+The	O
+planar	O
+crotonyl	O
+group	O
+is	O
+inserted	O
+between	O
+Trp81	B-residue_name_number
+and	O
+Phe62	B-residue_name_number
+of	O
+the	O
+protein	O
+,	O
+the	O
+aromatic	O
+rings	O
+of	O
+which	O
+are	O
+positioned	O
+strictly	O
+parallel	O
+to	O
+each	O
+other	O
+and	O
+at	O
+equal	O
+distance	O
+from	O
+the	O
+crotonyl	O
+group	O
+,	O
+yielding	O
+a	O
+novel	O
+aromatic	O
+-	O
+amide	O
+/	O
+aliphatic	O
+-	O
+aromatic	O
+π	O
+-	O
+π	O
+-	O
+π	O
+-	O
+stacking	O
+system	O
+that	O
+,	O
+to	O
+our	O
+knowledge	O
+,	O
+has	O
+not	O
+been	O
+reported	O
+previously	O
+for	O
+any	O
+protein	O
+-	O
+protein	O
+interaction	O
+(	O
+Fig	O
+.	O
+1d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+).	O
+
+The	O
+side	O
+chain	O
+of	O
+Trp81	B-residue_name_number
+appears	O
+to	O
+adopt	O
+two	O
+conformations	O
+,	O
+one	O
+of	O
+which	O
+provides	O
+maximum	O
+π	O
+-	O
+stacking	O
+with	O
+the	O
+alkene	O
+functional	O
+group	O
+while	O
+the	O
+other	O
+rotamer	O
+affords	O
+maximum	O
+π	O
+-	O
+stacking	O
+with	O
+the	O
+amide	O
+π	O
+electrons	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+).	O
+
+The	O
+dual	O
+conformation	O
+of	O
+Trp81	B-residue_name_number
+is	O
+likely	O
+due	O
+to	O
+the	O
+conjugated	O
+nature	O
+of	O
+the	O
+C	O
+=	O
+C	O
+and	O
+C	O
+=	O
+O	O
+π	O
+-	O
+orbitals	O
+within	O
+the	O
+crotonyl	O
+functional	O
+group	O
+.	O
+
+This	O
+provides	O
+the	O
+capability	O
+for	O
+the	O
+alkene	O
+moiety	O
+to	O
+form	O
+electrostatic	O
+contacts	O
+,	O
+as	O
+Cα	O
+and	O
+Cβ	O
+lay	O
+within	O
+electrostatic	O
+interaction	O
+distances	O
+of	O
+the	O
+carbonyl	O
+oxygen	O
+of	O
+Gln79	B-residue_name_number
+and	O
+of	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Thr61	B-residue_name_number
+,	O
+respectively	O
+.	O
+
+The	O
+hydroxyl	O
+group	O
+of	O
+Thr61	B-residue_name_number
+also	O
+participates	O
+in	O
+a	O
+hydrogen	O
+bond	O
+with	O
+the	O
+amide	O
+nitrogen	O
+of	O
+the	O
+K9cr	B-residue_name_number
+side	O
+chain	O
+(	O
+Fig	O
+.	O
+1d	O
+).	O
+
+The	O
+fixed	O
+position	O
+of	O
+the	O
+Thr61	B-residue_name_number
+hydroxyl	O
+group	O
+,	O
+which	O
+facilitates	O
+interactions	O
+with	O
+both	O
+the	O
+amide	O
+and	O
+Cα	O
+of	O
+K9cr	B-residue_name_number
+,	O
+is	O
+achieved	O
+through	O
+a	O
+hydrogen	O
+bond	O
+with	O
+imidazole	O
+ring	O
+of	O
+His59	B-residue_name_number
+.	O
+
+Extra	O
+stabilization	O
+of	O
+K9cr	B-residue_name_number
+is	O
+attained	O
+by	O
+a	O
+hydrogen	O
+bond	O
+formed	O
+between	O
+its	O
+carbonyl	O
+oxygen	O
+and	O
+the	O
+backbone	O
+nitrogen	O
+of	O
+Trp81	B-residue_name_number
+,	O
+as	O
+well	O
+as	O
+a	O
+water	B-chemical
+-	O
+mediated	O
+hydrogen	O
+bond	O
+with	O
+the	O
+backbone	O
+carbonyl	O
+group	O
+of	O
+Gly82	B-residue_name_number
+(	O
+Fig	O
+1d	O
+).	O
+
+This	O
+distinctive	O
+mechanism	O
+was	O
+corroborated	O
+through	O
+mapping	O
+the	O
+Taf14	B-protein
+YEATS	B-site
+-	I-site
+H3K9cr	I-site
+binding	I-site
+interface	I-site
+in	O
+solution	O
+using	O
+NMR	B-experimental_method
+chemical	I-experimental_method
+shift	I-experimental_method
+perturbation	I-experimental_method
+analysis	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+).	O
+
+Binding	O
+of	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+to	O
+H3K9cr	B-protein_type
+is	O
+robust	O
+.	O
+
+The	O
+dissociation	B-evidence
+constant	I-evidence
+(	O
+Kd	B-evidence
+)	O
+for	O
+the	O
+Taf14	B-complex_assembly
+YEATS	I-complex_assembly
+-	I-complex_assembly
+H3K9cr5	I-complex_assembly
+-	I-complex_assembly
+13	I-complex_assembly
+complex	O
+was	O
+found	O
+to	O
+be	O
+9	O
+.	O
+5	O
+μM	O
+,	O
+as	O
+measured	O
+by	O
+fluorescence	B-experimental_method
+spectroscopy	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+).	O
+
+This	O
+value	O
+is	O
+in	O
+the	O
+range	O
+of	O
+binding	B-evidence
+affinities	I-evidence
+exhibited	O
+by	O
+the	O
+majority	O
+of	O
+histone	O
+readers	O
+,	O
+thus	O
+attesting	O
+to	O
+the	O
+physiological	O
+relevance	O
+of	O
+the	O
+H3K9cr	B-protein_type
+recognition	O
+by	O
+Taf14	B-protein
+.	O
+
+To	O
+determine	O
+whether	O
+H3K9cr	B-protein_type
+is	O
+present	O
+in	O
+yeast	B-taxonomy_domain
+,	O
+we	O
+generated	O
+whole	B-experimental_method
+cell	I-experimental_method
+extracts	I-experimental_method
+from	O
+logarithmically	O
+growing	O
+yeast	B-taxonomy_domain
+cells	O
+and	O
+subjected	O
+them	O
+to	O
+Western	B-experimental_method
+blot	I-experimental_method
+analysis	I-experimental_method
+using	O
+antibodies	O
+directed	O
+towards	O
+H3K9cr	B-protein_type
+,	O
+H3K9ac	B-protein_type
+and	O
+H3	B-protein_type
+(	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
+
+Both	O
+H3K9cr	B-protein_type
+and	O
+H3K9ac	B-protein_type
+were	O
+detected	O
+in	O
+yeast	B-taxonomy_domain
+histones	B-protein_type
+;	O
+to	O
+our	O
+knowledge	O
+,	O
+this	O
+is	O
+the	O
+first	O
+report	O
+of	O
+H3K9cr	B-protein_type
+occurring	O
+in	O
+yeast	B-taxonomy_domain
+.	O
+
+We	O
+next	O
+asked	O
+if	O
+H3K9cr	B-protein_type
+is	O
+regulated	O
+by	O
+the	O
+actions	O
+of	O
+histone	B-protein_type
+acetyltransferases	I-protein_type
+(	O
+HATs	B-protein_type
+)	O
+and	O
+histone	B-protein_type
+deacetylases	I-protein_type
+(	O
+HDACs	B-protein_type
+).	O
+
+Towards	O
+this	O
+end	O
+,	O
+we	O
+probed	O
+extracts	O
+derived	O
+from	O
+yeast	B-taxonomy_domain
+cells	O
+in	O
+which	O
+major	O
+yeast	B-taxonomy_domain
+HATs	B-protein_type
+(	O
+HAT1	B-protein
+,	O
+Gcn5	B-protein
+,	O
+and	O
+Rtt109	B-protein
+)	O
+or	O
+HDACs	B-protein_type
+(	O
+Rpd3	B-protein
+,	O
+Hos1	B-protein
+,	O
+and	O
+Hos2	B-protein
+)	O
+were	O
+deleted	B-experimental_method
+.	O
+
+As	O
+shown	O
+in	O
+Figure	O
+2a	O
+,	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+3e	O
+,	O
+H3K9cr	B-protein_type
+levels	O
+were	O
+abolished	O
+or	O
+reduced	O
+considerably	O
+in	O
+the	O
+HAT	B-protein_type
+deletion	B-experimental_method
+strains	O
+,	O
+whereas	O
+they	O
+were	O
+dramatically	O
+increased	O
+in	O
+the	O
+HDAC	B-protein_type
+deletion	B-experimental_method
+strains	O
+.	O
+
+Furthermore	O
+,	O
+fluctuations	O
+in	O
+the	O
+H3K9cr	B-protein_type
+levels	O
+were	O
+more	O
+substantial	O
+than	O
+fluctuations	O
+in	O
+the	O
+corresponding	O
+H3K9ac	B-protein_type
+levels	O
+.	O
+
+Together	O
+,	O
+these	O
+results	O
+reveal	O
+that	O
+H3K9cr	B-protein_type
+is	O
+a	O
+dynamic	O
+mark	O
+of	O
+chromatin	O
+in	O
+yeast	B-taxonomy_domain
+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	B-protein_type
+and	O
+HDACs	B-protein_type
+.	O
+
+We	O
+have	O
+previously	O
+shown	O
+that	O
+among	O
+acetylated	B-protein_state
+histone	B-protein_type
+marks	O
+,	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+prefers	O
+acetylated	B-protein_state
+H3K9	B-protein_type
+(	O
+also	O
+see	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+),	O
+however	O
+it	O
+binds	O
+to	O
+H3K9cr	B-protein_type
+tighter	O
+.	O
+
+The	O
+selectivity	O
+of	O
+Taf14	B-protein
+towards	O
+crotonyllysine	B-residue_name
+was	O
+substantiated	O
+by	O
+1H	B-experimental_method
+,	I-experimental_method
+15N	I-experimental_method
+HSQC	I-experimental_method
+experiments	O
+,	O
+in	O
+which	O
+either	O
+H3K9cr5	B-chemical
+-	I-chemical
+13	I-chemical
+or	O
+H3K9ac5	B-chemical
+-	I-chemical
+13	I-chemical
+peptide	O
+was	O
+titrated	B-experimental_method
+into	O
+the	O
+15N	B-protein_state
+-	I-protein_state
+labeled	I-protein_state
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+.	O
+2c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+
+Binding	O
+of	O
+H3K9cr	B-protein_type
+induced	O
+resonance	B-evidence
+changes	I-evidence
+in	O
+slow	O
+exchange	O
+regime	O
+on	O
+the	O
+NMR	B-experimental_method
+time	O
+scale	O
+,	O
+indicative	O
+of	O
+strong	O
+interaction	O
+.	O
+
+In	O
+contrast	O
+,	O
+binding	O
+of	O
+H3K9ac	B-protein_type
+resulted	O
+in	O
+an	O
+intermediate	O
+exchange	O
+,	O
+which	O
+is	O
+characteristic	O
+of	O
+a	O
+weaker	O
+association	O
+.	O
+
+Furthermore	O
+,	O
+crosspeaks	B-evidence
+of	O
+Gly80	B-residue_name_number
+and	O
+Trp81	B-residue_name_number
+of	O
+the	O
+YEATS	B-structure_element
+domain	I-structure_element
+were	O
+uniquely	O
+perturbed	O
+by	O
+H3K9cr	B-protein_type
+and	O
+H3K9ac	B-protein_type
+,	O
+indicating	O
+a	O
+different	O
+chemical	O
+environment	O
+in	O
+the	O
+respective	O
+crotonyllysine	B-site
+and	I-site
+acetyllysine	I-site
+binding	I-site
+pockets	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+).	O
+
+These	O
+differences	O
+support	O
+our	O
+model	O
+that	O
+Trp81	B-residue_name_number
+adopts	O
+two	O
+conformations	O
+upon	O
+complex	O
+formation	O
+with	O
+the	O
+H3K9cr	B-protein_type
+mark	O
+as	O
+compared	O
+to	O
+H3K9ac	B-protein_type
+(	O
+Supplementary	O
+Figs	O
+.	O
+1c	O
+,	O
+d	O
+and	O
+4c	O
+).	O
+
+One	O
+of	O
+the	O
+conformations	O
+,	O
+characterized	O
+by	O
+the	O
+π	O
+stacking	O
+involving	O
+two	O
+aromatic	O
+residues	O
+and	O
+the	O
+alkene	O
+group	O
+,	O
+is	O
+observed	O
+only	O
+in	O
+the	O
+YEATS	B-complex_assembly
+-	I-complex_assembly
+H3K9cr	I-complex_assembly
+complex	O
+.	O
+
+To	O
+establish	O
+whether	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+is	O
+able	O
+to	O
+recognize	O
+other	O
+recently	O
+identified	O
+acyllysine	B-residue_name
+marks	O
+,	O
+we	O
+performed	O
+solution	B-experimental_method
+pull	I-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assays	I-experimental_method
+using	O
+H3	B-protein_type
+peptides	O
+acetylated	B-protein_state
+,	O
+propionylated	B-protein_state
+,	O
+butyrylated	B-protein_state
+,	O
+and	O
+crotonylated	B-protein_state
+at	O
+lysine	B-residue_name_number
+9	I-residue_name_number
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+20	I-residue_range
+of	O
+H3	B-protein_type
+).	O
+
+As	O
+shown	O
+in	O
+Figure	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+,	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+binds	O
+more	O
+strongly	O
+to	O
+H3K9cr1	B-chemical
+-	I-chemical
+20	I-chemical
+,	O
+as	O
+compared	O
+to	O
+other	O
+acylated	B-protein_state
+histone	O
+peptides	O
+.	O
+
+The	O
+preference	O
+for	O
+H3K9cr	B-protein_type
+over	O
+H3K9ac	B-protein_type
+,	O
+H3K9pr	B-protein_type
+and	O
+H3K9bu	B-protein_type
+was	O
+supported	O
+by	O
+1H	B-experimental_method
+,	I-experimental_method
+15N	I-experimental_method
+HSQC	I-experimental_method
+titration	I-experimental_method
+experiments	I-experimental_method
+.	O
+
+Addition	O
+of	O
+H3K9ac1	B-chemical
+-	I-chemical
+20	I-chemical
+,	O
+H3K9pr1	B-chemical
+-	I-chemical
+20	I-chemical
+,	O
+and	O
+H3K9bu1	B-chemical
+-	I-chemical
+20	I-chemical
+peptides	O
+caused	O
+chemical	B-evidence
+shift	I-evidence
+perturbations	I-evidence
+in	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+in	O
+intermediate	O
+exchange	O
+regime	O
+,	O
+implying	O
+that	O
+these	O
+interactions	O
+are	O
+weaker	O
+compared	O
+to	O
+the	O
+interaction	O
+with	O
+the	O
+H3K9cr1	B-chemical
+-	I-chemical
+20	I-chemical
+peptide	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+).	O
+
+We	O
+concluded	O
+that	O
+H3K9cr	B-protein_type
+is	O
+the	O
+preferred	O
+target	O
+of	O
+this	O
+domain	O
+.	O
+
+From	O
+comparative	B-experimental_method
+structural	I-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+YEATS	O
+complexes	O
+,	O
+Gly80	B-residue_name_number
+emerged	O
+as	O
+candidate	O
+residue	O
+potentially	O
+responsible	O
+for	O
+the	O
+preference	O
+for	O
+crotonyllysine	B-residue_name
+.	O
+
+In	O
+attempt	O
+to	O
+generate	O
+a	O
+mutant	O
+capable	O
+of	O
+accommodating	O
+a	O
+short	O
+acetyl	O
+moiety	O
+but	O
+discriminating	O
+against	O
+a	O
+longer	O
+,	O
+planar	O
+crotonyl	O
+moiety	O
+,	O
+we	O
+mutated	B-protein_state
+Gly80	B-residue_name_number
+to	O
+more	O
+bulky	O
+residues	O
+,	O
+however	O
+all	O
+mutants	B-protein_state
+of	I-protein_state
+Gly80	B-residue_name_number
+lost	O
+their	O
+binding	O
+activities	O
+towards	O
+either	O
+acylated	B-protein_state
+peptide	O
+,	O
+suggesting	O
+that	O
+Gly80	B-residue_name_number
+is	O
+absolutely	O
+required	O
+for	O
+the	O
+interaction	O
+.	O
+
+In	O
+contrast	O
+,	O
+mutation	B-experimental_method
+of	O
+Val24	B-residue_name_number
+,	O
+a	O
+residue	O
+located	O
+on	O
+another	O
+side	O
+of	O
+Trp81	B-residue_name_number
+,	O
+had	O
+no	O
+effect	O
+on	O
+binding	O
+(	O
+Fig	O
+.	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+,	O
+c	O
+).	O
+
+To	O
+determine	O
+if	O
+the	O
+binding	O
+to	O
+crotonyllysine	B-residue_name
+is	O
+conserved	B-protein_state
+,	O
+we	O
+tested	O
+human	B-species
+YEATS	B-structure_element
+domains	I-structure_element
+by	O
+pull	B-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+experiments	I-experimental_method
+using	O
+singly	O
+and	O
+multiply	O
+acetylated	B-protein_state
+,	O
+propionylated	B-protein_state
+,	O
+butyrylated	B-protein_state
+,	O
+and	O
+crotonylated	B-protein_state
+histone	B-protein_type
+peptides	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+We	O
+found	O
+that	O
+all	O
+YEATS	B-structure_element
+domains	I-structure_element
+tested	O
+are	O
+capable	O
+of	O
+binding	O
+to	O
+crotonyllysine	B-residue_name
+peptides	O
+,	O
+though	O
+they	O
+display	O
+variable	O
+preferences	O
+for	O
+the	O
+acyl	O
+moieties	O
+.	O
+
+While	O
+YEATS2	B-protein
+and	O
+ENL	B-protein
+showed	O
+selectivity	O
+for	O
+the	O
+crotonylated	B-protein_state
+peptides	O
+,	O
+GAS41	B-protein
+and	O
+AF9	B-protein
+bound	O
+acylated	B-protein_state
+peptides	O
+almost	O
+equally	O
+well	O
+.	O
+
+Unlike	O
+the	O
+YEATS	B-structure_element
+domain	I-structure_element
+,	O
+a	O
+known	O
+acetyllysine	B-protein_type
+reader	I-protein_type
+,	O
+bromodomain	B-structure_element
+,	O
+does	O
+not	O
+recognize	O
+crotonyllysine	B-residue_name
+.	O
+
+We	O
+assayed	O
+a	O
+large	O
+set	O
+of	O
+BDs	B-structure_element
+in	O
+pull	B-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+experiments	I-experimental_method
+and	O
+found	O
+that	O
+this	O
+module	O
+is	O
+highly	O
+specific	O
+for	O
+acetyllysine	B-residue_name
+and	O
+propionyllysine	B-residue_name
+containing	O
+peptides	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+
+However	O
+,	O
+bromodomains	B-structure_element
+did	O
+not	O
+interact	O
+(	O
+or	O
+associated	O
+very	O
+weakly	O
+)	O
+with	O
+longer	O
+acyl	O
+modifications	O
+,	O
+including	O
+crotonyllysine	B-residue_name
+,	O
+as	O
+in	O
+the	O
+case	O
+of	O
+BDs	B-structure_element
+of	O
+TAF1	B-protein
+and	O
+BRD2	B-protein
+,	O
+supporting	O
+recent	O
+reports	O
+.	O
+
+These	O
+results	O
+demonstrate	O
+that	O
+the	O
+YEATS	B-structure_element
+domain	I-structure_element
+is	O
+currently	O
+the	O
+sole	O
+reader	O
+of	O
+crotonyllysine	B-residue_name
+.	O
+
+In	O
+conclusion	O
+,	O
+we	O
+have	O
+identified	O
+the	O
+YEATS	B-structure_element
+domain	I-structure_element
+of	O
+Taf14	B-protein
+as	O
+the	O
+first	O
+reader	O
+of	O
+histone	B-protein_type
+crotonylation	B-ptm
+.	O
+
+We	O
+further	O
+demonstrate	O
+that	O
+H3K9cr	B-protein_type
+exists	O
+in	O
+yeast	B-taxonomy_domain
+and	O
+is	O
+dynamically	O
+regulated	O
+by	O
+HATs	B-protein_type
+and	O
+HDACs	B-protein_type
+.	O
+
+As	O
+we	O
+previously	O
+showed	O
+the	O
+importance	O
+of	O
+acyllysine	B-residue_name
+binding	O
+by	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+for	O
+the	O
+DNA	O
+damage	O
+response	O
+and	O
+gene	O
+transcription	O
+,	O
+it	O
+will	O
+be	O
+essential	O
+in	O
+the	O
+future	O
+to	O
+define	O
+the	O
+physiological	O
+role	O
+of	O
+crotonyllysine	B-residue_name
+recognition	O
+and	O
+to	O
+differentiate	O
+the	O
+activities	O
+of	O
+Taf14	B-protein
+that	O
+are	O
+due	O
+to	O
+binding	O
+to	O
+crotonyllysine	B-residue_name
+and	O
+acetyllysine	B-residue_name
+modifications	O
+.	O
+
+Furthermore	O
+,	O
+the	O
+functional	O
+significance	O
+of	O
+crotonyllysine	B-residue_name
+recognition	O
+by	O
+other	O
+YEATS	B-protein_type
+proteins	O
+will	O
+be	O
+of	O
+great	O
+importance	O
+to	O
+elucidate	O
+and	O
+compare	O
+.	O
+
+The	O
+structural	O
+mechanism	O
+for	O
+the	O
+recognition	O
+of	O
+H3K9cr	B-protein_type
+
+(	O
+a	O
+)	O
+Chemical	O
+structure	O
+of	O
+crotonyllysine	B-residue_name
+.	O
+(	O
+b	O
+)	O
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+(	O
+wheat	O
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+H3K9cr5	B-chemical
+-	I-chemical
+13	I-chemical
+peptide	O
+(	O
+green	O
+).	O
+(	O
+c	O
+)	O
+H3K9cr	B-protein_type
+is	O
+stabilized	O
+via	O
+an	O
+extensive	O
+network	O
+of	O
+intermolecular	O
+electrostatic	O
+and	O
+polar	O
+interactions	O
+with	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+.	O
+
+(	O
+d	O
+)	O
+The	O
+π	O
+-	O
+π	O
+-	O
+π	O
+stacking	O
+mechanism	O
+involving	O
+the	O
+alkene	O
+moiety	O
+of	O
+crotonyllysine	B-residue_name
+.	O
+
+H3K9cr	B-protein_type
+is	O
+a	O
+selective	O
+target	O
+of	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+
+(	O
+a	O
+,	O
+b	O
+)	O
+Western	B-experimental_method
+blot	I-experimental_method
+analysis	O
+comparing	O
+the	O
+levels	O
+of	O
+H3K9cr	B-protein_type
+and	O
+H3K9ac	B-protein_type
+in	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+),	O
+HAT	O
+deletion	O
+,	O
+or	O
+HDAC	B-protein_type
+deletion	B-experimental_method
+yeast	B-taxonomy_domain
+strains	O
+.	O
+
+Total	O
+H3	B-protein_type
+was	O
+used	O
+as	O
+a	O
+loading	O
+control	O
+.	O
+
+(	O
+c	O
+)	O
+Superimposed	O
+1H	B-experimental_method
+,	I-experimental_method
+15N	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+of	O
+Taf14	B-protein
+YEATS	B-structure_element
+recorded	O
+as	O
+H3K9cr5	B-chemical
+-	I-chemical
+13	I-chemical
+and	O
+H3K9ac5	B-chemical
+-	I-chemical
+13	I-chemical
+peptides	O
+were	O
+titrated	B-experimental_method
+in	O
+.	O
+
+Spectra	B-evidence
+are	O
+color	O
+coded	O
+according	O
+to	O
+the	O
+protein	O
+:	O
+peptide	O
+molar	O
+ratio	O
+.	O
+
+(	O
+d	O
+)	O
+Western	B-experimental_method
+blot	I-experimental_method
+analyses	O
+of	O
+peptide	B-experimental_method
+pull	I-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assays	I-experimental_method
+using	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutated	B-protein_state
+Taf14	B-protein
+YEATS	B-structure_element
+domains	I-structure_element
+and	O
+indicated	O
+peptides	O
+.	O
+
+A	O
+unified	O
+mechanism	O
+for	O
+proteolysis	O
+and	O
+autocatalytic	B-ptm
+activation	I-ptm
+in	O
+the	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+
+Biogenesis	O
+of	O
+the	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+is	O
+tightly	O
+regulated	O
+.	O
+
+The	O
+N	O
+-	O
+terminal	O
+propeptides	B-structure_element
+protecting	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+threonines	B-residue_name
+are	O
+autocatalytically	B-ptm
+released	O
+only	O
+on	O
+completion	O
+of	O
+assembly	O
+.	O
+
+However	O
+,	O
+the	O
+trigger	O
+for	O
+the	O
+self	O
+-	O
+activation	O
+and	O
+the	O
+reason	O
+for	O
+the	O
+strict	B-protein_state
+conservation	I-protein_state
+of	O
+threonine	B-residue_name
+as	O
+the	O
+active	O
+site	O
+nucleophile	O
+remain	O
+enigmatic	O
+.	O
+
+Here	O
+we	O
+use	O
+mutagenesis	B-experimental_method
+,	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+and	O
+biochemical	B-experimental_method
+assays	I-experimental_method
+to	O
+suggest	O
+that	O
+Lys33	B-residue_name_number
+initiates	O
+nucleophilic	O
+attack	O
+of	O
+the	O
+propeptide	B-structure_element
+by	O
+deprotonating	O
+the	O
+Thr1	B-residue_name_number
+hydroxyl	O
+group	O
+and	O
+that	O
+both	O
+residues	O
+together	O
+with	O
+Asp17	B-residue_name_number
+are	O
+part	O
+of	O
+a	O
+catalytic	B-site
+triad	I-site
+.	O
+
+Substitution	B-experimental_method
+of	O
+Thr1	B-residue_name_number
+by	O
+Cys	B-residue_name
+disrupts	O
+the	O
+interaction	O
+with	O
+Lys33	B-residue_name_number
+and	O
+inactivates	B-protein_state
+the	O
+proteasome	B-complex_assembly
+.	O
+
+Although	O
+a	O
+Thr1Ser	B-mutant
+mutant	B-protein_state
+is	O
+active	B-protein_state
+,	O
+it	O
+is	O
+less	O
+efficient	O
+compared	O
+with	O
+wild	B-protein_state
+type	I-protein_state
+because	O
+of	O
+the	O
+unfavourable	O
+orientation	O
+of	O
+Ser1	B-residue_name_number
+towards	O
+incoming	O
+substrates	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	B-ptm
+autolysis	I-ptm
+,	O
+as	O
+well	O
+as	O
+the	O
+evolutionary	O
+pressures	O
+that	O
+drove	O
+the	O
+proteasome	B-complex_assembly
+to	O
+become	O
+a	O
+threonine	B-protein_type
+protease	I-protein_type
+.	O
+
+The	O
+proteasome	B-complex_assembly
+,	O
+an	O
+essential	O
+molecular	O
+machine	O
+,	O
+is	O
+a	O
+threonine	B-protein_type
+protease	I-protein_type
+,	O
+but	O
+the	O
+evolution	O
+and	O
+the	O
+components	O
+of	O
+its	O
+proteolytic	O
+centre	O
+are	O
+unclear	O
+.	O
+
+Here	O
+,	O
+the	O
+authors	O
+use	O
+structural	O
+biology	O
+and	O
+biochemistry	O
+to	O
+investigate	O
+the	O
+role	O
+of	O
+proteasome	B-complex_assembly
+active	B-site
+site	I-site
+residues	O
+on	O
+maturation	O
+and	O
+activity	O
+.	O
+
+The	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+core	I-complex_assembly
+particle	I-complex_assembly
+(	O
+CP	B-complex_assembly
+)	O
+is	O
+the	O
+key	O
+non	B-protein_type
+-	I-protein_type
+lysosomal	I-protein_type
+protease	I-protein_type
+of	O
+eukaryotic	B-taxonomy_domain
+cells	O
+.	O
+
+Its	O
+seven	O
+different	O
+α	B-protein
+and	O
+seven	O
+different	O
+β	B-protein
+subunits	I-protein
+assemble	O
+into	O
+four	O
+heptameric	B-oligomeric_state
+rings	B-structure_element
+that	O
+are	O
+stacked	O
+on	O
+each	O
+other	O
+to	O
+form	O
+a	O
+hollow	B-structure_element
+cylinder	I-structure_element
+.	O
+
+While	O
+the	O
+inactive	B-protein_state
+α	B-protein
+subunits	I-protein
+build	O
+the	O
+two	O
+outer	O
+rings	B-structure_element
+,	O
+the	O
+β	B-protein
+subunits	I-protein
+form	O
+the	O
+inner	O
+rings	B-structure_element
+.	O
+
+Only	O
+three	O
+out	O
+of	O
+the	O
+seven	O
+different	O
+β	B-protein
+subunits	I-protein
+,	O
+namely	O
+β1	B-protein
+,	O
+β2	B-protein
+and	O
+β5	B-protein
+,	O
+bear	O
+N	O
+-	O
+terminal	O
+proteolytic	B-site
+active	I-site
+centres	I-site
+,	O
+and	O
+before	O
+CP	B-complex_assembly
+maturation	O
+these	O
+are	O
+protected	O
+by	O
+propeptides	B-structure_element
+.	O
+
+In	O
+the	O
+last	O
+stage	O
+of	O
+CP	B-complex_assembly
+biogenesis	O
+,	O
+the	O
+prosegments	B-structure_element
+are	O
+autocatalytically	B-ptm
+removed	I-ptm
+through	O
+nucleophilic	O
+attack	O
+by	O
+the	O
+active	B-site
+site	I-site
+residue	I-site
+Thr1	B-residue_name_number
+on	O
+the	O
+preceding	O
+peptide	O
+bond	O
+involving	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+).	I-residue_name_number
+
+Release	O
+of	O
+the	O
+propeptides	B-structure_element
+creates	O
+a	O
+functionally	O
+active	B-protein_state
+CP	B-complex_assembly
+that	O
+cleaves	O
+proteins	O
+into	O
+short	O
+peptides	O
+.	O
+
+Although	O
+the	O
+chemical	O
+nature	O
+of	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+and	O
+hence	O
+substrate	O
+preferences	O
+are	O
+unique	O
+to	O
+each	O
+of	O
+the	O
+distinct	O
+active	B-protein_state
+β	B-protein
+subunits	I-protein
+,	O
+all	O
+active	B-site
+sites	I-site
+employ	O
+an	O
+identical	O
+reaction	O
+mechanism	O
+to	O
+hydrolyse	O
+peptide	O
+bonds	O
+.	O
+
+Nucleophilic	O
+attack	O
+of	O
+Thr1Oγ	B-residue_name_number
+on	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+the	O
+scissile	O
+peptide	O
+bond	O
+creates	O
+a	O
+first	O
+cleavage	O
+product	O
+and	O
+a	O
+covalent	O
+acyl	O
+-	O
+enzyme	O
+intermediate	O
+.	O
+
+Hydrolysis	O
+of	O
+this	O
+complex	B-complex_assembly
+by	O
+the	O
+addition	O
+of	O
+a	O
+nucleophilic	O
+water	B-chemical
+molecule	O
+regenerates	O
+the	O
+enzyme	B-complex_assembly
+and	O
+releases	O
+the	O
+second	O
+peptide	B-chemical
+fragment	O
+.	O
+
+The	O
+proteasome	B-complex_assembly
+belongs	O
+to	O
+the	O
+family	O
+of	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+nucleophilic	I-protein_type
+(	I-protein_type
+Ntn	I-protein_type
+)	I-protein_type
+hydrolases	I-protein_type
+,	O
+and	O
+the	O
+free	B-protein_state
+N	O
+-	O
+terminal	O
+amine	O
+group	O
+of	O
+Thr1	B-residue_name_number
+was	O
+proposed	O
+to	O
+deprotonate	O
+the	O
+Thr1	B-residue_name_number
+hydroxyl	O
+group	O
+to	O
+generate	O
+a	O
+nucleophilic	O
+Thr1Oγ	B-residue_name_number
+for	O
+peptide	O
+-	O
+bond	O
+cleavage	O
+.	O
+
+This	O
+mechanism	O
+,	O
+however	O
+,	O
+cannot	O
+explain	O
+autocatalytic	B-ptm
+precursor	I-ptm
+processing	I-ptm
+because	O
+in	O
+the	O
+immature	B-protein_state
+active	B-site
+sites	I-site
+,	O
+Thr1N	B-residue_name_number
+is	O
+part	O
+of	O
+the	O
+peptide	O
+bond	O
+with	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+),	I-residue_name_number
+the	O
+bond	O
+that	O
+needs	O
+to	O
+be	O
+hydrolysed	O
+.	O
+
+An	O
+alternative	O
+candidate	O
+for	O
+deprotonating	O
+the	O
+Thr1	B-residue_name_number
+hydroxyl	O
+group	O
+is	O
+the	O
+side	O
+chain	O
+of	O
+Lys33	B-residue_name_number
+as	O
+it	O
+is	O
+within	O
+hydrogen	O
+-	O
+bonding	O
+distance	O
+to	O
+Thr1OH	B-residue_name_number
+(	O
+2	O
+.	O
+7	O
+Å	O
+).	O
+
+In	O
+principle	O
+it	O
+could	O
+function	O
+as	O
+the	O
+general	O
+base	O
+during	O
+both	O
+autocatalytic	B-ptm
+removal	I-ptm
+of	O
+the	O
+propeptide	B-structure_element
+and	O
+protein	O
+substrate	O
+cleavage	O
+.	O
+
+Here	O
+we	O
+provide	O
+experimental	O
+evidences	O
+for	O
+this	O
+distinct	O
+view	O
+of	O
+the	O
+proteasome	B-complex_assembly
+active	B-site
+-	I-site
+site	I-site
+mechanism	O
+.	O
+
+Data	O
+from	O
+biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+analyses	I-experimental_method
+of	O
+proteasome	O
+variants	O
+with	O
+mutations	O
+in	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+and	O
+the	O
+active	B-site
+site	I-site
+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	B-ptm
+activation	I-ptm
+of	O
+the	O
+proteasome	B-complex_assembly
+.	O
+
+Furthermore	O
+,	O
+we	O
+determine	O
+the	O
+advantages	O
+of	O
+Thr	B-residue_name
+over	O
+Cys	B-residue_name
+or	O
+Ser	B-residue_name
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+using	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+together	O
+with	O
+activity	B-experimental_method
+and	I-experimental_method
+inhibition	I-experimental_method
+assays	I-experimental_method
+.	O
+
+Inactivation	O
+of	O
+proteasome	B-complex_assembly
+subunits	B-protein
+by	O
+T1A	B-mutant
+mutations	B-experimental_method
+
+Proteasome	B-complex_assembly
+-	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	B-taxonomy_domain
+cells	O
+.	O
+
+Inactivation	O
+of	O
+the	O
+active	B-site
+site	I-site
+Thr1	B-residue_name_number
+by	O
+mutation	B-experimental_method
+to	I-experimental_method
+Ala	B-residue_name
+has	O
+been	O
+used	O
+to	O
+study	O
+substrate	O
+specificity	O
+and	O
+the	O
+hierarchy	O
+of	O
+the	O
+proteasome	B-complex_assembly
+active	B-site
+sites	I-site
+.	O
+
+Yeast	B-taxonomy_domain
+strains	O
+carrying	O
+the	O
+single	O
+mutations	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+or	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+or	O
+both	O
+,	O
+are	O
+viable	O
+,	O
+even	O
+though	O
+one	O
+or	O
+two	O
+of	O
+the	O
+three	O
+distinct	O
+catalytic	B-protein_state
+β	B-protein
+subunits	I-protein
+are	O
+disabled	B-protein_state
+and	O
+carry	B-protein_state
+remnants	I-protein_state
+of	I-protein_state
+their	O
+N	O
+-	O
+terminal	O
+propeptides	B-structure_element
+(	O
+Table	O
+1	O
+).	O
+
+These	O
+results	O
+indicate	O
+that	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+proteolytic	O
+activities	O
+are	O
+not	O
+essential	O
+for	O
+cell	O
+survival	O
+.	O
+
+By	O
+contrast	O
+,	O
+the	O
+T1A	B-mutant
+mutation	O
+in	O
+subunit	O
+β5	B-protein
+has	O
+been	O
+reported	O
+to	O
+be	O
+lethal	O
+or	O
+nearly	O
+so	O
+.	O
+
+Viability	O
+is	O
+restored	O
+if	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+subunit	O
+has	O
+its	O
+propeptide	B-structure_element
+(	O
+pp	B-chemical
+)	O
+deleted	B-experimental_method
+but	I-experimental_method
+expressed	I-experimental_method
+separately	I-experimental_method
+in	O
+trans	B-protein_state
+(	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+),	O
+although	O
+substantial	O
+phenotypic	O
+impairment	O
+remains	O
+(	O
+Table	O
+1	O
+).	O
+
+Our	O
+present	O
+crystallographic	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+mutant	B-protein_state
+demonstrates	O
+that	O
+the	O
+mutation	B-experimental_method
+per	O
+se	O
+does	O
+not	O
+structurally	O
+alter	O
+the	O
+catalytic	B-site
+active	I-site
+site	I-site
+and	O
+that	O
+the	O
+trans	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+β5	B-protein
+propeptide	B-structure_element
+is	O
+not	B-protein_state
+bound	I-protein_state
+in	O
+the	O
+β5	B-protein
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+).	O
+
+The	O
+extremely	O
+weak	O
+growth	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+pp	B-chemical
+cis	B-protein_state
+described	O
+by	O
+Chen	O
+and	O
+Hochstrasser	O
+compared	O
+with	O
+the	O
+inviability	O
+reported	O
+by	O
+Heinemeyer	O
+et	O
+al	O
+.	O
+prompted	O
+us	O
+to	O
+analyse	O
+this	O
+discrepancy	O
+.	O
+
+Sequencing	B-experimental_method
+of	I-experimental_method
+the	I-experimental_method
+plasmids	I-experimental_method
+,	O
+testing	O
+them	O
+in	O
+both	O
+published	O
+yeast	B-taxonomy_domain
+strain	O
+backgrounds	O
+and	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+revealed	O
+that	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+pp	B-chemical
+cis	B-protein_state
+is	O
+viable	O
+,	O
+but	O
+suffers	O
+from	O
+a	O
+marked	O
+growth	O
+defect	O
+that	O
+requires	O
+extended	O
+incubation	O
+of	O
+4	O
+–	O
+5	O
+days	O
+for	O
+initial	O
+colony	O
+formation	O
+(	O
+Table	O
+1	O
+and	O
+Supplementary	O
+Methods	O
+).	O
+
+We	O
+also	O
+identified	O
+an	O
+additional	O
+point	O
+mutation	O
+K81R	B-mutant
+in	O
+subunit	O
+β5	B-protein
+that	O
+was	O
+present	O
+in	O
+the	O
+allele	O
+used	O
+in	O
+ref	O
+..	O
+This	B-experimental_method
+single	I-experimental_method
+amino	I-experimental_method
+-	I-experimental_method
+acid	I-experimental_method
+exchange	I-experimental_method
+is	O
+located	O
+at	O
+the	O
+interface	B-site
+of	O
+the	O
+subunits	O
+α4	B-protein
+,	O
+β4	B-protein
+and	O
+β5	B-protein
+(	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+)	O
+and	O
+might	O
+weakly	O
+promote	O
+CP	B-complex_assembly
+assembly	O
+by	O
+enhancing	O
+inter	O
+-	O
+subunit	O
+contacts	O
+.	O
+
+The	O
+slightly	O
+better	O
+growth	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	B-protein_state
+allowed	O
+us	O
+to	O
+solve	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+yeast	B-taxonomy_domain
+proteasome	B-complex_assembly
+(	O
+yCP	B-complex_assembly
+)	O
+with	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutation	O
+,	O
+which	O
+is	O
+discussed	O
+in	O
+the	O
+following	O
+section	O
+(	O
+for	O
+details	O
+see	O
+Supplementary	O
+Note	O
+1	O
+).	O
+
+Propeptide	B-structure_element
+conformation	O
+and	O
+triggering	O
+of	O
+autolysis	B-ptm
+
+In	O
+the	O
+final	O
+steps	O
+of	O
+proteasome	B-complex_assembly
+biogenesis	O
+,	O
+the	O
+propeptides	B-structure_element
+are	O
+autocatalytically	B-ptm
+cleaved	I-ptm
+from	O
+the	O
+mature	B-protein_state
+β	B-protein
+-	I-protein
+subunit	I-protein
+domains	I-protein
+.	O
+
+For	O
+subunit	O
+β1	B-protein
+,	O
+this	O
+process	O
+was	O
+previously	O
+inferred	O
+to	O
+require	O
+that	O
+the	O
+propeptide	B-structure_element
+residue	O
+at	O
+position	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+of	O
+the	O
+subunit	O
+precursor	O
+occupies	O
+the	O
+S1	B-site
+specificity	I-site
+pocket	I-site
+of	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+formed	O
+by	O
+amino	O
+acid	O
+45	B-residue_number
+(	O
+for	O
+details	O
+see	O
+Supplementary	O
+Note	O
+2	O
+).	O
+
+Furthermore	O
+,	O
+it	O
+was	O
+observed	O
+that	O
+the	O
+prosegment	B-structure_element
+forms	O
+an	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+in	O
+the	O
+active	B-site
+site	I-site
+,	O
+and	O
+that	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+adopts	O
+a	O
+γ	B-structure_element
+-	I-structure_element
+turn	I-structure_element
+conformation	I-structure_element
+,	O
+which	O
+by	O
+definition	O
+is	O
+characterized	O
+by	O
+a	O
+hydrogen	O
+bond	O
+between	O
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+O	O
+and	O
+Thr1NH	B-residue_name_number
+(	O
+ref	O
+.).	O
+
+Here	O
+we	O
+again	O
+analysed	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+crystallographically	B-experimental_method
+but	O
+in	O
+addition	O
+determined	O
+the	O
+structures	B-evidence
+of	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+single	O
+and	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+β2	I-mutant
+-	I-mutant
+T1A	I-mutant
+double	O
+mutants	O
+(	O
+Protein	O
+Data	O
+Bank	O
+(	O
+PDB	O
+)	O
+entry	O
+codes	O
+are	O
+provided	O
+in	O
+Supplementary	O
+Table	O
+1	O
+).	O
+
+In	O
+subunit	O
+β1	B-protein
+,	O
+we	O
+found	O
+that	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+indeed	O
+forms	O
+a	O
+sharp	B-structure_element
+turn	I-structure_element
+,	O
+which	O
+relaxes	O
+on	O
+prosegment	B-ptm
+cleavage	I-ptm
+(	O
+Fig	O
+.	O
+1a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+).	O
+
+However	O
+,	O
+the	O
+γ	B-structure_element
+-	I-structure_element
+turn	I-structure_element
+conformation	I-structure_element
+and	O
+the	O
+associated	O
+hydrogen	O
+bond	O
+initially	O
+proposed	O
+is	O
+for	O
+geometric	O
+and	O
+chemical	O
+reasons	O
+inappropriate	O
+and	O
+would	O
+not	O
+perfectly	O
+position	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+for	O
+nucleophilic	O
+attack	O
+by	O
+Thr1	B-residue_name_number
+.	O
+
+Regarding	O
+the	O
+β2	B-protein
+propeptide	B-structure_element
+,	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+occupies	O
+the	O
+S1	B-site
+pocket	I-site
+but	O
+is	O
+less	O
+deeply	O
+anchored	O
+compared	O
+with	O
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+β1	B-protein
+,	O
+which	O
+might	O
+be	O
+due	O
+to	O
+the	O
+rather	O
+large	O
+β2	B-protein
+-	O
+S1	B-site
+pocket	I-site
+created	O
+by	O
+Gly45	B-residue_name_number
+.	O
+
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+positions	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+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	B-residue_name_number
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+
+Next	O
+,	O
+we	O
+examined	O
+the	O
+position	O
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+in	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	B-protein_state
+.	O
+
+Surprisingly	O
+,	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+is	O
+completely	O
+extended	O
+and	O
+forces	O
+the	O
+histidine	B-residue_name
+side	O
+chain	O
+at	O
+position	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+to	O
+occupy	O
+the	O
+S2	B-site
+instead	O
+of	O
+the	O
+S1	B-site
+pocket	I-site
+,	O
+thereby	O
+disrupting	O
+the	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+.	O
+
+Nonetheless	O
+,	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+would	O
+be	O
+ideally	O
+placed	O
+for	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	B-residue_name_number
+(	O
+Fig	O
+.	O
+1c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+,	O
+d	O
+).	O
+
+As	O
+the	O
+K81R	B-mutant
+mutation	O
+is	O
+located	O
+far	O
+from	O
+the	O
+active	B-site
+site	I-site
+(	O
+Thr1Cα	B-residue_name_number
+–	O
+Arg81Cα	B-residue_name_number
+:	O
+24	O
+Å	O
+),	O
+any	O
+influence	O
+on	O
+propeptide	B-structure_element
+conformation	O
+can	O
+be	O
+excluded	O
+.	O
+
+Instead	O
+,	O
+the	O
+plasticity	O
+of	O
+the	O
+β5	B-protein
+S1	B-site
+pocket	I-site
+caused	O
+by	O
+the	O
+rotational	O
+flexibility	O
+of	O
+Met45	B-residue_name_number
+might	O
+prevent	O
+stable	O
+accommodation	O
+of	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+the	O
+S1	B-site
+site	I-site
+and	O
+thus	O
+also	O
+promote	O
+its	O
+immediate	O
+release	O
+after	O
+autolysis	B-ptm
+.	O
+
+Processing	O
+of	O
+β	O
+-	O
+subunit	O
+precursors	O
+requires	O
+deprotonation	O
+of	O
+Thr1OH	B-residue_name_number
+;	O
+however	O
+,	O
+the	O
+general	O
+base	O
+initiating	O
+autolysis	B-ptm
+is	O
+unknown	O
+.	O
+
+Remarkably	O
+,	O
+eukaryotic	B-taxonomy_domain
+proteasomal	O
+β5	B-protein
+subunits	O
+bear	O
+a	O
+His	B-residue_name
+residue	O
+in	O
+position	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+of	O
+the	O
+propeptide	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+).	O
+
+As	O
+histidine	B-residue_name
+commonly	O
+functions	O
+as	O
+a	O
+proton	O
+shuttle	O
+in	O
+the	O
+catalytic	B-site
+triads	I-site
+of	O
+serine	B-protein_type
+proteases	I-protein_type
+,	O
+we	O
+investigated	O
+the	O
+role	O
+of	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+processing	O
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+by	O
+exchanging	B-experimental_method
+it	I-experimental_method
+for	I-experimental_method
+Asn	B-residue_name
+,	O
+Lys	B-residue_name
+,	O
+Phe	B-residue_name
+and	O
+Ala	B-residue_name
+.	O
+All	O
+yeast	B-taxonomy_domain
+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
+
+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	B-protein_state
+yCPs	B-complex_assembly
+was	O
+impaired	O
+in	O
+situ	O
+and	O
+in	O
+vitro	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3c	O
+).	O
+
+Structural	B-experimental_method
+analyses	I-experimental_method
+revealed	O
+that	O
+the	O
+propeptides	B-structure_element
+of	O
+all	O
+mutant	B-protein_state
+yCPs	B-complex_assembly
+shared	O
+residual	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+densities	I-evidence
+.	O
+
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+and	O
+Phe	B-residue_name
+/	O
+Lys	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+were	O
+visualized	O
+at	O
+low	O
+occupancy	O
+,	O
+while	O
+Ala	B-residue_name
+/	O
+Asn	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+could	O
+not	O
+be	O
+assigned	O
+.	O
+
+This	O
+observation	O
+indicates	O
+a	O
+mixture	O
+of	O
+processed	B-protein_state
+and	O
+unprocessed	B-protein_state
+β5	B-protein
+subunits	O
+and	O
+partially	O
+impaired	O
+autolysis	B-ptm
+,	O
+thereby	O
+excluding	O
+any	O
+essential	O
+role	O
+of	O
+residue	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+as	O
+the	O
+general	O
+base	O
+.	O
+
+Next	O
+,	O
+we	O
+examined	O
+the	O
+effect	O
+of	O
+residue	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+on	O
+the	O
+orientation	O
+of	O
+the	O
+propeptide	B-structure_element
+by	O
+creating	B-experimental_method
+mutants	I-experimental_method
+that	I-experimental_method
+combine	I-experimental_method
+the	O
+T1A	B-mutant
+(	O
+K81R	B-mutant
+)	O
+mutation	B-experimental_method
+(	I-experimental_method
+s	I-experimental_method
+)	I-experimental_method
+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	B-experimental_method
+.	O
+
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+is	O
+encoded	O
+in	O
+the	O
+yeast	B-taxonomy_domain
+β1	B-protein
+subunit	O
+precursor	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+);	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+is	O
+generally	O
+part	O
+of	O
+β2	B-protein
+-	O
+propeptides	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+);	O
+and	O
+Ala	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+was	O
+expected	O
+to	O
+fit	O
+the	O
+β5	B-protein
+-	O
+S1	B-site
+pocket	I-site
+without	O
+inducing	O
+conformational	O
+changes	O
+of	O
+Met45	B-residue_name_number
+,	O
+allowing	O
+it	O
+to	O
+accommodate	O
+‘	O
+β1	O
+-	O
+like	O
+'	O
+propeptide	O
+positioning	O
+.	O
+
+As	O
+expected	O
+from	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutants	O
+,	O
+the	O
+yeasts	B-taxonomy_domain
+show	O
+severe	O
+growth	O
+phenotypes	O
+,	O
+with	O
+minor	O
+variations	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+and	O
+Table	O
+1	O
+).	O
+
+We	O
+determined	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+A	I-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutants	O
+(	O
+Supplementary	O
+Table	O
+1	O
+).	O
+
+For	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+A	I-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+variant	O
+,	O
+only	O
+the	O
+residues	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+and	O
+Ala	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+could	O
+be	O
+visualized	O
+,	O
+indicating	O
+that	O
+Ala	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+leads	O
+to	O
+insufficient	O
+stabilization	O
+of	O
+the	O
+propeptide	B-structure_element
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+).	O
+
+By	O
+contrast	O
+,	O
+the	O
+prosegments	B-structure_element
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutants	O
+were	O
+significantly	O
+better	O
+resolved	O
+in	O
+the	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+maps	I-evidence
+yet	O
+not	O
+at	O
+full	O
+occupancy	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4b	O
+,	O
+c	O
+and	O
+Supplementary	O
+Table	O
+1	O
+),	O
+suggesting	O
+that	O
+the	O
+natural	O
+propeptide	B-structure_element
+bearing	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+is	O
+most	O
+favourable	O
+.	O
+
+Nevertheless	O
+,	O
+both	O
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+and	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+were	O
+found	O
+to	O
+occupy	O
+the	O
+S1	B-site
+specificity	I-site
+pocket	I-site
+formed	O
+by	O
+Met45	B-residue_name_number
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4f	O
+–	O
+h	O
+).	O
+
+This	O
+result	O
+proves	O
+that	O
+the	O
+naturally	O
+occurring	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+does	O
+not	O
+stably	O
+fit	O
+into	O
+the	O
+S1	B-site
+site	I-site
+.	O
+
+Since	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+adopts	O
+the	O
+same	O
+position	O
+in	O
+both	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+and	O
+mutant	B-protein_state
+β5	B-protein
+propeptides	B-structure_element
+,	O
+and	O
+since	O
+in	O
+all	O
+cases	O
+its	O
+carbonyl	O
+carbon	O
+is	O
+perfectly	O
+placed	O
+for	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	B-residue_name_number
+(	O
+Fig	O
+.	O
+2b	O
+),	O
+we	O
+propose	O
+that	O
+neither	O
+binding	O
+of	O
+residue	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+to	O
+the	O
+S1	B-site
+pocket	I-site
+nor	O
+formation	O
+of	O
+the	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+is	O
+essential	O
+for	O
+autolysis	B-ptm
+of	O
+the	O
+propeptide	B-structure_element
+.	O
+
+Next	O
+,	O
+we	O
+determined	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+chimeric	B-protein_state
+yCP	B-complex_assembly
+having	O
+the	O
+yeast	B-taxonomy_domain
+β1	B-protein
+-	O
+propeptide	B-structure_element
+replaced	B-experimental_method
+by	I-experimental_method
+its	O
+β5	B-protein
+counterpart	B-structure_element
+.	O
+
+Although	O
+we	O
+observed	O
+fragments	O
+of	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+density	I-evidence
+in	O
+the	O
+β1	B-protein
+active	B-site
+site	I-site
+,	O
+the	O
+data	O
+were	O
+not	O
+interpretable	O
+.	O
+
+Bearing	O
+in	O
+mind	O
+that	O
+in	O
+contrast	O
+to	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+β2	B-protein
+,	O
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+subunit	O
+β1	B-protein
+is	O
+not	B-protein_state
+conserved	I-protein_state
+among	O
+species	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+),	O
+we	O
+created	B-experimental_method
+a	O
+β2	B-mutant
+-	I-mutant
+T	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+V	I-mutant
+proteasome	B-complex_assembly
+mutant	B-protein_state
+.	O
+
+As	O
+proven	O
+by	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+crystal	B-evidence
+structures	I-evidence
+,	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+hydrogen	O
+bonds	O
+to	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+O	O
+.	O
+Although	O
+this	O
+interaction	O
+was	O
+not	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+2c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4c	O
+,	O
+i	O
+),	O
+exchange	B-experimental_method
+of	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+by	O
+Val	B-residue_name
+in	O
+β2	B-protein
+,	O
+a	O
+conservative	O
+mutation	O
+regarding	O
+size	O
+but	O
+drastic	O
+with	O
+respect	O
+to	O
+polarity	O
+,	O
+was	O
+found	O
+to	O
+inhibit	O
+maturation	O
+of	O
+this	O
+subunit	O
+(	O
+Fig	O
+.	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4e	O
+,	O
+j	O
+).	O
+
+Notably	O
+,	O
+the	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+map	I-evidence
+displays	O
+a	O
+different	O
+orientation	O
+for	O
+the	O
+β2	B-protein
+propeptide	B-structure_element
+than	O
+has	O
+been	O
+observed	O
+for	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+proteasome	B-complex_assembly
+.	O
+
+In	O
+particular	O
+,	O
+Val	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+is	O
+displaced	O
+from	O
+the	O
+S1	B-site
+site	I-site
+and	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+is	O
+severely	O
+shifted	O
+(	O
+movement	O
+of	O
+the	O
+carbonyl	O
+oxygen	O
+atom	O
+of	O
+3	O
+.	O
+8	O
+Å	O
+),	O
+thereby	O
+preventing	O
+nucleophilic	O
+attack	O
+of	O
+Thr1	B-residue_name_number
+(	O
+Fig	O
+.	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4j	O
+,	O
+k	O
+).	O
+
+These	O
+results	O
+further	O
+confirm	O
+that	O
+correct	O
+positioning	O
+of	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+residues	I-site
+and	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+is	O
+decisive	O
+for	O
+the	O
+maturation	O
+of	O
+the	O
+proteasome	B-complex_assembly
+.	O
+
+The	O
+active	B-site
+site	I-site
+of	O
+the	O
+proteasome	B-complex_assembly
+
+Proton	O
+shuttling	O
+from	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+Thr1OH	B-residue_name_number
+to	O
+Thr1NH2	B-residue_name_number
+via	O
+a	O
+nucleophilic	O
+water	B-chemical
+molecule	O
+was	O
+suggested	O
+to	O
+initiate	O
+peptide	O
+-	O
+bond	O
+hydrolysis	O
+.	O
+
+However	O
+,	O
+in	O
+the	O
+immature	B-protein_state
+particle	B-complex_assembly
+Thr1NH2	B-residue_name_number
+is	O
+blocked	O
+by	O
+the	O
+propeptide	B-structure_element
+and	O
+cannot	O
+activate	O
+Thr1Oγ	B-residue_name_number
+.	O
+
+Instead	O
+,	O
+Lys33NH2	B-residue_name_number
+,	O
+which	O
+is	O
+in	O
+hydrogen	O
+-	O
+bonding	O
+distance	O
+to	O
+Thr1Oγ	B-residue_name_number
+(	O
+2	O
+.	O
+7	O
+Å	O
+)	O
+in	O
+all	O
+catalytically	B-protein_state
+active	I-protein_state
+β	B-protein
+subunits	I-protein
+(	O
+Fig	O
+.	O
+3a	O
+,	O
+b	O
+),	O
+was	O
+proposed	O
+to	O
+serve	O
+as	O
+the	O
+proton	O
+acceptor	O
+.	O
+
+A	O
+proposed	O
+catalytic	B-site
+tetrad	I-site
+model	O
+involving	O
+Thr1OH	B-residue_name_number
+,	O
+Thr1NH2	B-residue_name_number
+,	O
+Lys33NH2	B-residue_name_number
+and	O
+Asp17Oδ	B-residue_name_number
+,	O
+as	O
+well	O
+as	O
+a	O
+nucleophilic	O
+water	B-chemical
+molecule	O
+as	O
+the	O
+proton	O
+shuttle	O
+appeared	O
+to	O
+accommodate	O
+all	O
+possible	O
+views	O
+of	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+.	O
+
+Twenty	O
+years	O
+later	O
+,	O
+with	O
+a	O
+plethora	O
+of	O
+yCP	B-complex_assembly
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structures	I-evidence
+in	O
+hand	O
+,	O
+we	O
+decided	O
+to	O
+re	O
+-	O
+analyse	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+proteasome	B-complex_assembly
+and	O
+to	O
+resolve	O
+the	O
+uncertainty	O
+regarding	O
+the	O
+nature	O
+of	O
+the	O
+general	O
+base	O
+.	O
+
+Mutation	B-experimental_method
+of	O
+β5	B-protein
+-	O
+Lys33	B-residue_name_number
+to	O
+Ala	B-residue_name
+causes	O
+a	O
+strongly	O
+deleterious	O
+phenotype	O
+,	O
+and	O
+previous	O
+structural	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+analyses	I-experimental_method
+confirmed	O
+that	O
+this	O
+is	O
+caused	O
+by	O
+failure	O
+of	O
+propeptide	B-ptm
+cleavage	I-ptm
+,	O
+and	O
+consequently	O
+,	O
+lack	O
+of	O
+ChT	O
+-	O
+L	O
+activity	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+and	O
+Table	O
+1	O
+;	O
+for	O
+details	O
+see	O
+Supplementary	O
+Note	O
+1	O
+).	O
+
+The	O
+phenotype	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+mutant	B-protein_state
+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	B-taxonomy_domain
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+
+This	O
+discrepancy	O
+in	O
+growth	O
+was	O
+traced	O
+to	O
+an	O
+additional	O
+point	O
+mutation	O
+L	B-mutant
+(-	I-mutant
+49	I-mutant
+)	I-mutant
+S	I-mutant
+in	O
+the	O
+β5	B-protein
+-	O
+propeptide	B-structure_element
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+mutant	B-protein_state
+(	O
+see	O
+also	O
+Supplementary	O
+Note	O
+1	O
+).	O
+
+Structural	B-experimental_method
+comparison	I-experimental_method
+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	B-site
+sites	I-site
+revealed	O
+that	O
+mutation	B-experimental_method
+of	O
+Lys33	B-residue_name_number
+to	O
+Ala	B-residue_name
+creates	O
+a	O
+cavity	O
+that	O
+is	O
+filled	O
+with	O
+Thr1	B-residue_name_number
+and	O
+the	O
+remnant	O
+propeptide	B-structure_element
+.	O
+
+This	O
+structural	O
+alteration	O
+destroys	O
+active	B-site
+-	I-site
+site	I-site
+integrity	O
+and	O
+abolishes	O
+catalytic	O
+activity	O
+of	O
+the	O
+β5	B-protein
+active	B-site
+site	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+).	O
+
+Additional	O
+proof	O
+for	O
+the	O
+key	O
+function	O
+of	O
+Lys33	B-residue_name_number
+was	O
+obtained	O
+from	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+mutant	B-protein_state
+,	O
+with	O
+the	O
+propeptide	B-structure_element
+expressed	B-experimental_method
+separately	I-experimental_method
+from	O
+the	O
+main	O
+subunit	O
+(	O
+pp	B-chemical
+trans	B-protein_state
+).	O
+
+The	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+of	O
+this	O
+mutant	B-protein_state
+is	O
+not	O
+blocked	O
+by	O
+the	O
+propeptide	B-structure_element
+,	O
+yet	O
+its	O
+catalytic	O
+activity	O
+is	O
+reduced	O
+by	O
+∼	O
+83	O
+%	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6b	O
+).	O
+
+Consistent	O
+with	O
+this	O
+,	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+mutant	B-protein_state
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+carfilzomib	B-chemical
+only	O
+showed	O
+partial	O
+occupancy	O
+of	O
+the	O
+ligand	O
+at	O
+the	O
+β5	B-protein
+active	B-site
+sites	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+and	O
+Supplementary	O
+Table	O
+1	O
+).	O
+
+Since	O
+no	O
+acetylation	B-ptm
+of	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+was	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+apo	B-protein_state
+crystal	B-evidence
+structure	I-evidence
+,	O
+the	O
+reduced	O
+reactivity	O
+towards	O
+substrates	O
+and	O
+inhibitors	O
+indicates	O
+that	O
+Lys33NH2	B-residue_name_number
+,	O
+rather	O
+than	O
+Thr1NH2	B-residue_name_number
+,	O
+deprotonates	O
+and	O
+activates	O
+Thr1OH	B-residue_name_number
+.	O
+
+Furthermore	O
+,	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+mutant	B-protein_state
+without	B-protein_state
+inhibitor	I-protein_state
+revealed	O
+that	O
+Thr1Oγ	B-residue_name_number
+strongly	O
+coordinates	O
+a	O
+well	O
+-	O
+defined	O
+water	B-chemical
+molecule	O
+(∼	O
+2	O
+Å	O
+;	O
+Fig	O
+.	O
+3c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+5c	O
+,	O
+d	O
+).	O
+
+This	O
+water	B-chemical
+hydrogen	O
+bonds	O
+also	O
+to	O
+Arg19O	B-residue_name_number
+(∼	O
+3	O
+.	O
+0	O
+Å	O
+)	O
+and	O
+Asp17Oδ	B-residue_name_number
+(∼	O
+3	O
+.	O
+0	O
+Å	O
+),	O
+and	O
+thereby	O
+presumably	O
+enables	O
+residual	O
+activity	O
+of	O
+the	O
+mutant	B-protein_state
+.	O
+
+Remarkably	O
+,	O
+the	O
+solvent	O
+molecule	O
+occupies	O
+the	O
+position	O
+normally	O
+taken	O
+by	O
+Lys33NH2	B-residue_name_number
+in	O
+the	O
+WT	B-protein_state
+proteasome	B-complex_assembly
+structure	B-evidence
+(	O
+Fig	O
+.	O
+3c	O
+),	O
+further	O
+corroborating	O
+the	O
+essential	O
+role	O
+of	O
+Lys33	B-residue_name_number
+as	O
+the	O
+general	O
+base	O
+for	O
+autolysis	B-ptm
+and	O
+proteolysis	O
+.	O
+
+Conservative	B-experimental_method
+substitution	I-experimental_method
+of	O
+Lys33	B-residue_name_number
+by	O
+Arg	B-residue_name
+delays	O
+autolysis	B-ptm
+of	O
+the	O
+β5	B-protein
+precursor	O
+and	O
+impairs	O
+yeast	B-taxonomy_domain
+growth	O
+(	O
+for	O
+details	O
+see	O
+Supplementary	O
+Note	O
+1	O
+).	O
+
+While	O
+Thr1	B-residue_name_number
+occupies	O
+the	O
+same	O
+position	O
+as	O
+in	O
+WT	B-protein_state
+yCPs	B-complex_assembly
+,	O
+Arg33	B-residue_name_number
+is	O
+unable	O
+to	O
+hydrogen	O
+bond	O
+to	O
+Asp17	B-residue_name_number
+,	O
+thereby	O
+inactivating	O
+the	O
+β5	B-protein
+active	B-site
+site	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+5e	O
+).	O
+
+The	O
+conservative	B-experimental_method
+mutation	I-experimental_method
+of	O
+Asp17	B-residue_name_number
+to	O
+Asn	B-residue_name
+in	O
+subunit	O
+β5	B-protein
+of	O
+the	O
+yCP	B-complex_assembly
+also	O
+provokes	O
+a	O
+severe	O
+growth	O
+defect	O
+(	O
+Supplementary	O
+Note	O
+1	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+and	O
+Table	O
+1	O
+).	O
+
+Notably	O
+,	O
+only	O
+with	O
+the	O
+additional	O
+point	O
+mutation	O
+L	B-mutant
+(-	I-mutant
+49	I-mutant
+)	I-mutant
+S	I-mutant
+present	O
+in	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+could	O
+we	O
+purify	O
+a	O
+small	O
+amount	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+mutant	B-protein_state
+yCP	B-complex_assembly
+.	O
+
+As	O
+determined	O
+by	O
+crystallographic	B-experimental_method
+analysis	I-experimental_method
+,	O
+this	O
+mutant	B-protein_state
+β5	B-protein
+subunit	O
+was	O
+partially	B-protein_state
+processed	I-protein_state
+(	O
+Table	O
+1	O
+)	O
+but	O
+displayed	O
+impaired	O
+reactivity	O
+towards	O
+the	O
+proteasome	B-complex_assembly
+inhibitor	O
+carfilzomib	B-chemical
+compared	O
+with	O
+the	O
+subunits	O
+β1	B-protein
+and	O
+β2	B-protein
+,	O
+and	O
+with	O
+WT	B-protein_state
+β5	B-protein
+(	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+
+In	O
+contrast	O
+to	O
+the	O
+cis	B-protein_state
+-	O
+construct	O
+,	O
+expression	B-experimental_method
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+in	O
+trans	B-protein_state
+allowed	O
+straightforward	O
+isolation	B-experimental_method
+and	O
+crystallization	B-experimental_method
+of	O
+the	O
+D17N	B-mutant
+mutant	B-protein_state
+proteasome	B-complex_assembly
+.	O
+
+The	O
+ChT	O
+-	O
+L	O
+activity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+pp	B-chemical
+in	O
+trans	B-protein_state
+CP	B-complex_assembly
+towards	O
+the	O
+canonical	O
+β5	B-protein
+model	O
+substrates	O
+N	B-chemical
+-	I-chemical
+succinyl	I-chemical
+-	I-chemical
+Leu	I-chemical
+-	I-chemical
+Leu	I-chemical
+-	I-chemical
+Val	I-chemical
+-	I-chemical
+Tyr	I-chemical
+-	I-chemical
+7	I-chemical
+-	I-chemical
+amino	I-chemical
+-	I-chemical
+4	I-chemical
+-	I-chemical
+methylcoumarin	I-chemical
+(	O
+Suc	B-chemical
+-	I-chemical
+LLVY	I-chemical
+-	I-chemical
+AMC	I-chemical
+)	O
+and	O
+carboxybenzyl	B-chemical
+-	I-chemical
+Gly	I-chemical
+-	I-chemical
+Gly	I-chemical
+-	I-chemical
+Leu	I-chemical
+-	I-chemical
+para	I-chemical
+-	I-chemical
+nitroanilide	I-chemical
+(	O
+Z	B-chemical
+-	I-chemical
+GGL	I-chemical
+-	I-chemical
+pNA	I-chemical
+)	O
+was	O
+severely	O
+reduced	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6b	O
+),	O
+confirming	O
+that	O
+Asp17	B-residue_name_number
+is	O
+of	O
+fundamental	O
+importance	O
+for	O
+the	O
+catalytic	O
+activity	O
+of	O
+the	O
+mature	B-protein_state
+proteasome	B-complex_assembly
+.	O
+
+Even	O
+though	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+pp	B-chemical
+trans	B-protein_state
+yCP	B-complex_assembly
+crystal	B-evidence
+structure	I-evidence
+appeared	O
+identical	O
+to	O
+the	O
+WT	B-protein_state
+yCP	B-complex_assembly
+(	O
+Supplementary	O
+Fig	O
+.	O
+7b	O
+),	O
+the	O
+co	B-evidence
+-	I-evidence
+crystal	I-evidence
+structure	I-evidence
+with	O
+the	O
+α	B-chemical
+′,	I-chemical
+β	I-chemical
+′	I-chemical
+epoxyketone	I-chemical
+inhibitor	O
+carfilzomib	B-chemical
+visualized	O
+only	O
+partial	O
+occupancy	O
+of	O
+the	O
+ligand	O
+in	O
+the	O
+β5	B-protein
+active	B-site
+site	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+
+This	O
+observation	O
+is	O
+consistent	O
+with	O
+a	O
+strongly	O
+reduced	O
+reactivity	O
+of	O
+β5	B-protein
+-	O
+Thr1	B-residue_name_number
+and	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+pp	B-chemical
+cis	B-protein_state
+mutant	B-protein_state
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+carfilzomib	B-chemical
+.	O
+
+Autolysis	B-ptm
+and	O
+residual	O
+catalytic	O
+activity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+mutants	O
+may	O
+originate	O
+from	O
+the	O
+carbonyl	O
+group	O
+of	O
+Asn17	B-residue_name_number
+,	O
+which	O
+albeit	O
+to	O
+a	O
+lower	O
+degree	O
+still	O
+can	O
+polarize	O
+Lys33	B-residue_name_number
+for	O
+the	O
+activation	O
+of	O
+Thr1	B-residue_name_number
+.	O
+
+In	O
+agreement	O
+,	O
+an	O
+E17A	B-mutant
+mutant	B-protein_state
+in	O
+the	O
+proteasomal	O
+β	B-protein
+-	I-protein
+subunit	I-protein
+of	O
+the	O
+archaeon	B-taxonomy_domain
+Thermoplasma	B-species
+acidophilum	I-species
+prevents	O
+autolysis	B-ptm
+and	O
+catalysis	O
+.	O
+
+Strikingly	O
+,	O
+although	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+on	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+mutant	B-protein_state
+with	O
+the	O
+propeptide	B-structure_element
+expressed	B-experimental_method
+in	O
+cis	B-protein_state
+and	O
+in	O
+trans	B-protein_state
+looked	O
+similar	O
+,	O
+there	O
+was	O
+a	O
+pronounced	O
+difference	O
+in	O
+their	O
+growth	O
+phenotypes	O
+observed	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7b	O
+).	O
+
+On	O
+the	O
+basis	O
+of	O
+these	O
+results	O
+,	O
+we	O
+propose	O
+that	O
+CPs	B-complex_assembly
+from	O
+all	O
+domains	O
+of	O
+life	O
+use	O
+a	O
+catalytic	B-site
+triad	I-site
+consisting	O
+of	O
+Thr1	B-residue_name_number
+,	O
+Lys33	B-residue_name_number
+and	O
+Asp	B-residue_name
+/	O
+Glu17	B-residue_name_number
+for	O
+both	O
+autocatalytic	B-ptm
+precursor	I-ptm
+processing	I-ptm
+and	O
+proteolysis	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+This	O
+model	O
+is	O
+also	O
+consistent	O
+with	O
+the	O
+fact	O
+that	O
+no	O
+defined	O
+water	B-chemical
+molecule	O
+is	O
+observed	O
+in	O
+the	O
+mature	B-protein_state
+WT	B-protein_state
+proteasomal	O
+active	B-site
+site	I-site
+that	O
+could	O
+shuttle	O
+the	O
+proton	O
+from	O
+Thr1Oγ	B-residue_name_number
+to	O
+Thr1NH2	B-residue_name_number
+.	O
+
+To	O
+explore	O
+this	O
+active	B-site
+-	I-site
+site	I-site
+model	O
+further	O
+,	O
+we	O
+exchanged	B-experimental_method
+the	I-experimental_method
+conserved	I-experimental_method
+Asp166	B-residue_name_number
+residue	O
+for	O
+Asn	B-residue_name
+in	O
+the	O
+yeast	B-taxonomy_domain
+β5	B-protein
+subunit	O
+.	O
+
+Asp166Oδ	B-residue_name_number
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Thr1NH2	B-residue_name_number
+via	O
+Ser129OH	B-residue_name_number
+and	O
+Ser169OH	B-residue_name_number
+,	O
+and	O
+therefore	O
+was	O
+proposed	O
+to	O
+be	O
+involved	O
+in	O
+catalysis	O
+.	O
+
+The	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+pp	B-chemical
+cis	B-protein_state
+yeast	B-taxonomy_domain
+mutant	B-protein_state
+is	O
+significantly	O
+impaired	O
+in	O
+growth	O
+and	O
+its	O
+ChT	O
+-	O
+L	O
+activity	O
+is	O
+drastically	O
+reduced	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+and	O
+Table	O
+1	O
+).	O
+
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+on	O
+the	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+mutant	B-protein_state
+indicate	O
+that	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+is	O
+hydrolysed	O
+,	O
+but	O
+due	O
+to	O
+reorientation	O
+of	O
+Ser129OH	B-residue_name_number
+,	O
+the	O
+interaction	O
+with	O
+Asn166Oδ	B-residue_name_number
+is	O
+disrupted	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8a	O
+).	O
+
+Instead	O
+,	O
+a	O
+water	B-chemical
+molecule	O
+is	O
+bound	B-protein_state
+to	I-protein_state
+Ser129OH	B-residue_name_number
+and	O
+Thr1NH2	B-residue_name_number
+(	O
+Supplementary	O
+Fig	O
+.	O
+8b	O
+),	O
+which	O
+may	O
+enable	O
+precursor	B-ptm
+processing	I-ptm
+.	O
+
+The	O
+hydrogen	O
+bonds	O
+involving	O
+Ser169OH	B-residue_name_number
+are	O
+intact	O
+and	O
+may	O
+account	O
+for	O
+residual	O
+substrate	O
+turnover	O
+.	O
+
+Soaking	B-experimental_method
+the	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+crystals	B-experimental_method
+with	O
+carfilzomib	B-chemical
+and	O
+MG132	B-chemical
+resulted	O
+in	O
+covalent	O
+modification	O
+of	O
+Thr1	B-residue_name_number
+at	O
+high	O
+occupancy	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+
+In	O
+the	O
+carfilzomib	B-complex_assembly
+complex	I-complex_assembly
+structure	B-evidence
+,	O
+Thr1Oγ	B-residue_name_number
+and	O
+Thr1N	B-residue_name_number
+incorporate	O
+into	O
+a	O
+morpholine	O
+ring	O
+structure	O
+and	O
+Ser129	B-residue_name_number
+adopts	O
+its	O
+WT	B-protein_state
+-	O
+like	O
+orientation	O
+.	O
+
+In	O
+the	O
+MG132	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+state	I-protein_state
+,	O
+Thr1N	B-residue_name_number
+is	O
+unmodified	B-protein_state
+,	O
+and	O
+we	O
+again	O
+observe	O
+that	O
+Ser129	B-residue_name_number
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+a	O
+water	B-chemical
+molecule	O
+instead	O
+of	O
+Asn166	B-residue_name_number
+.	O
+
+Whereas	O
+Asn	B-residue_name
+can	O
+to	O
+some	O
+degree	O
+replace	O
+Asp166	B-residue_name_number
+due	O
+to	O
+its	O
+carbonyl	O
+group	O
+in	O
+the	O
+side	O
+chain	O
+,	O
+Ala	B-residue_name
+at	O
+this	O
+position	O
+was	O
+found	O
+to	O
+prevent	O
+both	O
+autolysis	B-ptm
+and	O
+catalysis	O
+.	O
+
+These	O
+results	O
+suggest	O
+that	O
+Asp166	B-residue_name_number
+and	O
+Ser129	B-residue_name_number
+function	O
+as	O
+a	O
+proton	O
+shuttle	O
+and	O
+affect	O
+the	O
+protonation	O
+state	O
+of	O
+Thr1N	B-residue_name_number
+during	O
+autolysis	B-ptm
+and	O
+catalysis	O
+.	O
+
+Substitution	B-experimental_method
+of	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+Thr1	B-residue_name_number
+by	O
+Cys	B-residue_name
+
+Mutation	B-experimental_method
+of	O
+Thr1	B-residue_name_number
+to	O
+Cys	B-residue_name
+inactivates	O
+the	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+from	O
+the	O
+archaeon	B-taxonomy_domain
+T	B-species
+.	I-species
+acidophilum	I-species
+.	O
+
+In	O
+yeast	B-taxonomy_domain
+,	O
+this	O
+mutation	B-experimental_method
+causes	O
+a	O
+strong	O
+growth	O
+defect	O
+(	O
+Fig	O
+.	O
+4a	O
+and	O
+Table	O
+1	O
+),	O
+although	O
+the	O
+propeptide	B-structure_element
+is	O
+hydrolysed	O
+,	O
+as	O
+shown	O
+here	O
+by	O
+its	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structure	I-evidence
+.	O
+
+In	O
+one	O
+of	O
+the	O
+two	O
+β5	B-protein
+subunits	O
+,	O
+however	O
+,	O
+we	O
+found	O
+the	O
+cleaved	B-protein_state
+propeptide	B-structure_element
+still	B-protein_state
+bound	I-protein_state
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+occupies	O
+the	O
+S2	B-site
+pocket	I-site
+like	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	B-protein_state
+,	O
+but	O
+in	O
+contrast	O
+to	O
+the	O
+latter	O
+,	O
+the	O
+propeptide	B-structure_element
+in	O
+the	O
+T1C	B-mutant
+mutant	B-protein_state
+adopts	O
+an	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+conformation	O
+as	O
+known	O
+from	O
+inhibitors	O
+like	O
+MG132	B-chemical
+(	O
+Fig	O
+.	O
+4c	O
+–	O
+e	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9b	O
+).	O
+
+On	O
+the	O
+basis	O
+of	O
+the	O
+phenotype	O
+of	O
+the	O
+T1C	B-mutant
+mutant	B-protein_state
+and	O
+the	O
+propeptide	B-structure_element
+remnant	O
+identified	O
+in	O
+its	O
+active	B-site
+site	I-site
+,	O
+we	O
+suppose	O
+that	O
+autolysis	B-ptm
+is	O
+retarded	O
+and	O
+may	O
+not	O
+have	O
+been	O
+completed	O
+before	O
+crystallization	B-experimental_method
+.	O
+
+Owing	O
+to	O
+the	O
+unequal	O
+positions	O
+of	O
+the	O
+two	O
+β5	B-protein
+subunits	O
+within	O
+the	O
+CP	B-complex_assembly
+in	O
+the	O
+crystal	O
+lattice	O
+,	O
+maturation	O
+and	O
+propeptide	B-structure_element
+displacement	O
+may	O
+occur	O
+at	O
+different	O
+timescales	O
+in	O
+the	O
+two	O
+subunits	O
+.	O
+
+Despite	O
+propeptide	B-ptm
+hydrolysis	I-ptm
+,	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+active	B-site
+site	I-site
+is	O
+catalytically	B-protein_state
+inactive	I-protein_state
+(	O
+Fig	O
+.	O
+4b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9a	O
+).	O
+
+In	O
+agreement	O
+,	O
+soaking	B-experimental_method
+crystals	I-experimental_method
+with	O
+the	O
+CP	B-complex_assembly
+inhibitors	O
+bortezomib	B-chemical
+or	O
+carfilzomib	B-chemical
+modifies	O
+only	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+active	B-site
+sites	I-site
+,	O
+while	O
+leaving	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+proteolytic	B-site
+centres	I-site
+unmodified	B-protein_state
+even	O
+though	O
+they	O
+are	O
+only	O
+partially	O
+occupied	O
+by	O
+the	O
+cleaved	B-protein_state
+propeptide	B-structure_element
+remnant	O
+.	O
+
+Moreover	O
+,	O
+the	O
+structural	B-evidence
+data	I-evidence
+reveal	O
+that	O
+the	O
+thiol	O
+group	O
+of	O
+Cys1	B-residue_name_number
+is	O
+rotated	O
+by	O
+74	O
+°	O
+with	O
+respect	O
+to	O
+the	O
+hydroxyl	O
+side	O
+chain	O
+of	O
+Thr1	B-residue_name_number
+(	O
+Fig	O
+.	O
+4f	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9b	O
+).	O
+
+Consequently	O
+,	O
+the	O
+hydrogen	O
+bond	O
+bridging	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+and	O
+Lys33	B-residue_name_number
+in	O
+WT	B-protein_state
+CPs	B-complex_assembly
+is	O
+broken	O
+with	O
+Cys1	B-residue_name_number
+.	O
+
+Notably	O
+,	O
+the	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+map	I-evidence
+of	O
+the	O
+T1C	B-mutant
+mutant	B-protein_state
+also	O
+indicates	O
+that	O
+Lys33NH2	B-residue_name_number
+is	O
+disordered	B-protein_state
+.	O
+
+Together	O
+,	O
+these	O
+observations	O
+suggest	O
+that	O
+efficient	O
+peptide	O
+-	O
+bond	O
+hydrolysis	O
+requires	O
+that	O
+Lys33NH2	B-residue_name_number
+hydrogen	O
+bonds	O
+to	O
+the	O
+active	O
+site	O
+nucleophile	O
+.	O
+
+The	O
+benefit	O
+of	O
+Thr	B-residue_name
+over	O
+Ser	B-residue_name
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+
+All	O
+proteasomes	B-complex_assembly
+strictly	B-protein_state
+employ	I-protein_state
+threonine	B-residue_name
+as	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+residue	I-site
+instead	O
+of	O
+serine	B-residue_name
+.	O
+
+To	O
+investigate	O
+the	O
+reason	O
+for	O
+this	O
+singularity	O
+,	O
+we	O
+analysed	O
+a	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+,	O
+which	O
+is	O
+viable	O
+but	O
+suffers	O
+from	O
+growth	O
+defects	O
+(	O
+Fig	O
+.	O
+4a	O
+and	O
+Table	O
+1	O
+).	O
+
+Activity	B-experimental_method
+assays	I-experimental_method
+with	O
+the	O
+β5	B-protein
+-	O
+specific	O
+substrate	O
+Suc	B-chemical
+-	I-chemical
+LLVY	I-chemical
+-	I-chemical
+AMC	I-chemical
+demonstrated	O
+that	O
+the	O
+ChT	O
+-	O
+L	O
+activity	O
+of	O
+the	O
+T1S	B-mutant
+mutant	B-protein_state
+is	O
+reduced	O
+by	O
+40	O
+–	O
+45	O
+%	O
+compared	O
+with	O
+WT	B-protein_state
+proteasomes	B-complex_assembly
+depending	O
+on	O
+the	O
+incubation	O
+temperature	O
+(	O
+Fig	O
+.	O
+4b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9c	O
+).	O
+
+By	O
+contrast	O
+,	O
+turnover	O
+of	O
+the	O
+substrate	O
+Z	B-chemical
+-	I-chemical
+GGL	I-chemical
+-	I-chemical
+pNA	I-chemical
+,	O
+used	O
+to	O
+monitor	O
+ChT	O
+-	O
+L	O
+activity	O
+in	O
+situ	O
+but	O
+in	O
+a	O
+less	O
+quantitative	O
+fashion	O
+,	O
+is	O
+not	O
+detectably	O
+impaired	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+9a	O
+).	O
+
+Crystal	B-evidence
+structure	I-evidence
+analysis	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+confirmed	O
+precursor	B-ptm
+processing	I-ptm
+(	O
+Fig	O
+.	O
+4g	O
+),	O
+and	O
+ligand	B-complex_assembly
+-	I-complex_assembly
+complex	I-complex_assembly
+structures	B-evidence
+with	O
+bortezomib	B-chemical
+and	O
+carfilzomib	B-chemical
+unambiguously	O
+corroborated	O
+the	O
+reactivity	O
+of	O
+Ser1	B-residue_name_number
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+However	O
+,	O
+the	O
+apo	B-protein_state
+crystal	B-evidence
+structure	I-evidence
+revealed	O
+that	O
+Ser1Oγ	B-residue_name_number
+is	O
+turned	O
+away	O
+from	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+(	O
+Fig	O
+.	O
+4g	O
+).	O
+
+Compared	O
+with	O
+Thr1Oγ	B-residue_name_number
+in	O
+WT	B-protein_state
+CP	B-complex_assembly
+structures	B-evidence
+,	O
+Ser1Oγ	B-residue_name_number
+is	O
+rotated	O
+by	O
+60	O
+°.	O
+
+Because	O
+both	O
+conformations	O
+of	O
+Ser1Oγ	B-residue_name_number
+are	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Lys33NH2	B-residue_name_number
+(	O
+Fig	O
+.	O
+4h	O
+),	O
+the	O
+relay	O
+system	O
+is	O
+capable	O
+of	O
+hydrolysing	O
+peptide	O
+substrates	O
+,	O
+albeit	O
+at	O
+lower	O
+rates	O
+compared	O
+with	O
+Thr1	B-residue_name_number
+.	O
+
+The	O
+active	B-site
+-	I-site
+site	I-site
+residue	I-site
+Thr1	B-residue_name_number
+is	O
+fixed	O
+in	O
+its	O
+position	O
+,	O
+as	O
+its	O
+methyl	O
+group	O
+is	O
+engaged	O
+in	O
+hydrophobic	O
+interactions	O
+with	O
+Thr3	B-residue_name_number
+and	O
+Ala46	B-residue_name_number
+(	O
+Fig	O
+.	O
+4h	O
+).	O
+
+Consequently	O
+,	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Thr1	B-residue_name_number
+requires	O
+no	O
+reorientation	O
+before	O
+substrate	O
+cleavage	O
+and	O
+is	O
+thus	O
+more	O
+catalytically	O
+efficient	O
+than	O
+Ser1	B-residue_name_number
+.	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	B-protein_state
+is	O
+unable	O
+to	O
+grow	O
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+
+In	O
+vitro	O
+,	O
+the	O
+mutant	B-protein_state
+proteasome	B-complex_assembly
+is	O
+less	O
+susceptible	O
+to	O
+proteasome	B-complex_assembly
+inhibition	O
+by	O
+bortezomib	B-chemical
+(	O
+3	O
+.	O
+7	O
+-	O
+fold	O
+)	O
+and	O
+carfilzomib	B-chemical
+(	O
+1	O
+.	O
+8	O
+-	O
+fold	O
+;	O
+Fig	O
+.	O
+5	O
+).	O
+
+Nevertheless	O
+,	O
+inhibitor	B-complex_assembly
+complex	I-complex_assembly
+structures	B-evidence
+indicate	O
+identical	O
+binding	O
+modes	O
+compared	O
+with	O
+the	O
+WT	B-protein_state
+yCP	B-complex_assembly
+structures	B-evidence
+,	O
+with	B-protein_state
+the	I-protein_state
+same	I-protein_state
+inhibitors	I-protein_state
+.	O
+
+Notably	O
+,	O
+the	O
+affinity	B-evidence
+of	O
+the	O
+tetrapeptide	O
+carfilzomib	B-chemical
+is	O
+less	O
+impaired	O
+,	O
+as	O
+it	O
+is	O
+better	O
+stabilized	O
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+than	O
+the	O
+dipeptide	O
+bortezomib	B-chemical
+,	O
+which	O
+lacks	O
+a	O
+defined	O
+P3	O
+site	O
+and	O
+has	O
+only	O
+a	O
+few	O
+interactions	O
+with	O
+the	O
+surrounding	O
+protein	O
+.	O
+
+Hence	O
+,	O
+the	O
+mean	B-evidence
+residence	I-evidence
+time	I-evidence
+of	O
+carfilzomib	B-chemical
+at	O
+the	O
+active	B-site
+site	I-site
+is	O
+prolonged	O
+and	O
+the	O
+probability	O
+to	O
+covalently	O
+react	O
+with	O
+Ser1	B-residue_name_number
+is	O
+increased	O
+.	O
+
+Considered	O
+together	O
+,	O
+these	O
+results	O
+provide	O
+a	O
+plausible	O
+explanation	O
+for	O
+the	O
+invariance	O
+of	O
+threonine	B-residue_name
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+in	O
+proteasomes	B-complex_assembly
+in	O
+all	O
+three	O
+domains	O
+of	O
+life	O
+,	O
+as	O
+well	O
+as	O
+in	O
+proteasome	B-protein_type
+-	I-protein_type
+like	I-protein_type
+proteases	I-protein_type
+such	O
+as	O
+HslV	B-protein
+(	O
+ref	O
+.).	O
+
+The	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+CP	B-complex_assembly
+is	O
+the	O
+major	O
+non	B-protein_type
+-	I-protein_type
+lysosomal	I-protein_type
+protease	I-protein_type
+in	O
+eukaryotic	B-taxonomy_domain
+cells	O
+,	O
+and	O
+its	O
+assembly	O
+is	O
+highly	O
+organized	O
+.	O
+
+The	O
+β	B-protein
+-	I-protein
+subunit	I-protein
+propeptides	B-structure_element
+,	O
+particularly	O
+that	O
+of	O
+β5	B-protein
+,	O
+are	O
+key	O
+factors	O
+that	O
+help	O
+drive	O
+proper	O
+assembly	O
+of	O
+the	O
+CP	B-complex_assembly
+complex	O
+.	O
+
+In	O
+addition	O
+,	O
+they	O
+prevent	O
+irreversible	O
+inactivation	O
+of	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+by	O
+N	B-ptm
+-	I-ptm
+acetylation	I-ptm
+.	O
+
+By	O
+contrast	O
+,	O
+the	O
+prosegments	B-structure_element
+of	O
+β	B-protein
+subunits	I-protein
+are	O
+dispensable	O
+for	O
+archaeal	B-taxonomy_domain
+proteasome	B-complex_assembly
+assembly	O
+,	O
+at	O
+least	O
+when	O
+heterologously	B-experimental_method
+expressed	I-experimental_method
+in	O
+Escherichia	B-species
+coli	I-species
+.	O
+
+In	O
+eukaryotes	B-taxonomy_domain
+,	O
+deletion	O
+of	O
+or	O
+failure	O
+to	O
+cleave	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+propeptides	B-structure_element
+is	O
+well	O
+tolerated	O
+.	O
+
+However	O
+,	O
+removal	B-experimental_method
+of	I-experimental_method
+the	O
+β5	B-protein
+prosegment	B-structure_element
+or	O
+any	O
+interference	O
+with	O
+its	O
+cleavage	O
+causes	O
+severe	O
+phenotypic	O
+defects	O
+.	O
+
+These	O
+observations	O
+highlight	O
+the	O
+unique	O
+function	O
+and	O
+importance	O
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+as	O
+well	O
+as	O
+the	O
+β5	B-protein
+active	B-site
+site	I-site
+for	O
+maturation	O
+and	O
+function	O
+of	O
+the	O
+eukaryotic	B-taxonomy_domain
+CP	B-complex_assembly
+.	O
+
+Here	O
+we	O
+have	O
+described	O
+the	O
+atomic	B-evidence
+structures	I-evidence
+of	O
+various	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutants	O
+,	O
+which	O
+allowed	O
+for	O
+the	O
+first	O
+time	O
+visualization	O
+of	O
+the	O
+residual	O
+β5	B-protein
+propeptide	B-structure_element
+.	O
+
+Depending	O
+on	O
+the	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+residue	O
+we	O
+observed	O
+various	O
+propeptide	B-structure_element
+conformations	O
+,	O
+but	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+is	O
+in	O
+all	O
+structures	B-evidence
+perfectly	O
+located	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	B-residue_name_number
+,	O
+although	O
+it	O
+does	O
+not	O
+adopt	O
+the	O
+tight	B-structure_element
+turn	I-structure_element
+observed	O
+for	O
+the	O
+prosegment	B-structure_element
+of	O
+subunit	O
+β1	B-protein
+.	O
+
+From	O
+these	O
+data	O
+we	O
+conclude	O
+that	O
+only	O
+the	O
+positioning	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+and	O
+Thr1	B-residue_name_number
+as	O
+well	O
+as	O
+the	O
+integrity	O
+of	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+are	O
+required	O
+for	O
+autolysis	B-ptm
+.	O
+
+In	O
+this	O
+regard	O
+,	O
+inappropriate	O
+N	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+cannot	O
+be	O
+removed	O
+by	O
+Thr1Oγ	B-residue_name_number
+due	O
+to	O
+the	O
+rotational	O
+freedom	O
+and	O
+flexibility	O
+of	O
+the	O
+acetyl	O
+group	O
+.	O
+
+The	O
+propeptide	B-structure_element
+needs	O
+some	O
+anchoring	O
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+to	O
+properly	O
+position	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+),	I-residue_name_number
+but	O
+this	O
+seems	O
+to	O
+be	O
+independent	O
+of	O
+the	O
+orientation	O
+of	O
+residue	O
+(-	B-residue_number
+2	I-residue_number
+).	I-residue_number
+
+Autolytic	O
+activation	O
+of	O
+the	O
+CP	B-complex_assembly
+constitutes	O
+one	O
+of	O
+the	O
+final	O
+steps	O
+of	O
+proteasome	O
+biogenesis	O
+,	O
+but	O
+the	O
+trigger	O
+for	O
+propeptide	B-ptm
+cleavage	I-ptm
+had	O
+remained	O
+enigmatic	O
+.	O
+
+On	O
+the	O
+basis	O
+of	O
+the	O
+numerous	O
+CP	B-complex_assembly
+:	I-complex_assembly
+ligand	I-complex_assembly
+complexes	O
+solved	O
+during	O
+the	O
+past	O
+18	O
+years	O
+and	O
+in	O
+the	O
+current	O
+study	O
+,	O
+we	O
+provide	O
+a	O
+revised	O
+interpretation	O
+of	O
+proteasome	B-complex_assembly
+active	B-site
+-	I-site
+site	I-site
+architecture	I-site
+.	O
+
+We	O
+propose	O
+a	O
+catalytic	B-site
+triad	I-site
+for	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+CP	B-complex_assembly
+consisting	O
+of	O
+residues	O
+Thr1	B-residue_name_number
+,	O
+Lys33	B-residue_name_number
+and	O
+Asp	B-residue_name
+/	O
+Glu17	B-residue_name_number
+,	O
+which	O
+are	O
+conserved	O
+among	O
+all	O
+proteolytically	O
+active	O
+eukaryotic	B-taxonomy_domain
+,	O
+bacterial	B-taxonomy_domain
+and	O
+archaeal	B-taxonomy_domain
+proteasome	B-complex_assembly
+subunits	O
+.	O
+
+Lys33NH2	B-residue_name_number
+is	O
+expected	O
+to	O
+act	O
+as	O
+the	O
+proton	O
+acceptor	O
+during	O
+autocatalytic	B-ptm
+removal	I-ptm
+of	O
+the	O
+propeptides	B-structure_element
+,	O
+as	O
+well	O
+as	O
+during	O
+substrate	O
+proteolysis	O
+,	O
+while	O
+Asp17Oδ	B-residue_name_number
+orients	O
+Lys33NH2	B-residue_name_number
+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	B-residue_name_number
++–	O
+Asp17Oδ	B-residue_name_number
+:	O
+2	O
+.	O
+9	O
+Å	O
+).	O
+
+Analogously	O
+to	O
+the	O
+proteasome	B-complex_assembly
+,	O
+a	O
+Thr	B-site
+–	I-site
+Lys	I-site
+–	I-site
+Asp	I-site
+triad	I-site
+is	O
+also	O
+found	O
+in	O
+L	B-protein_type
+-	I-protein_type
+asparaginase	I-protein_type
+.	O
+
+Thus	O
+,	O
+specific	O
+protein	O
+surroundings	O
+can	O
+significantly	O
+alter	O
+the	O
+chemical	O
+properties	O
+of	O
+amino	O
+acids	O
+such	O
+as	O
+Lys	B-residue_name
+to	O
+function	O
+as	O
+an	O
+acid	O
+–	O
+base	O
+catalyst	O
+.	O
+
+In	O
+this	O
+new	O
+view	O
+of	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+,	O
+the	O
+positively	O
+charged	O
+Thr1NH3	B-residue_name_number
++-	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	B-ptm
+cleavage	I-ptm
+by	O
+Thr1Oγ	B-residue_name_number
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+Consistent	O
+with	O
+this	O
+model	O
+,	O
+the	O
+positively	O
+charged	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+is	O
+engaged	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+inhibitory	O
+compounds	O
+like	O
+fellutamide	B-chemical
+B	I-chemical
+(	O
+ref	O
+.),	O
+α	B-chemical
+-	I-chemical
+ketoamides	I-chemical
+,	O
+homobelactosin	B-chemical
+C	I-chemical
+(	O
+ref	O
+.)	O
+and	O
+salinosporamide	B-chemical
+A	I-chemical
+(	O
+ref	O
+.).	O
+
+Furthermore	O
+,	O
+opening	O
+of	O
+the	O
+β	O
+-	O
+lactone	O
+compound	O
+omuralide	B-chemical
+by	O
+Thr1	B-residue_name_number
+creates	O
+a	O
+C3	O
+-	O
+hydroxyl	O
+group	O
+,	O
+whose	O
+proton	O
+originates	O
+from	O
+Thr1NH3	B-residue_name_number
++.	O
+
+The	O
+resulting	O
+uncharged	O
+Thr1NH2	B-residue_name_number
+is	O
+hydrogen	O
+-	O
+bridged	O
+to	O
+the	O
+C3	O
+-	O
+OH	O
+group	O
+.	O
+
+In	O
+agreement	O
+,	O
+acetylation	B-ptm
+of	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+irreversibly	O
+blocks	O
+hydrolytic	O
+activity	O
+,	O
+and	O
+binding	O
+of	O
+substrates	O
+is	O
+prevented	O
+for	O
+steric	O
+reasons	O
+.	O
+
+By	O
+acting	O
+as	O
+a	O
+proton	O
+donor	O
+during	O
+catalysis	O
+,	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+may	O
+also	O
+favour	O
+cleavage	O
+of	O
+substrate	O
+peptide	O
+bonds	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+Cleavage	O
+of	O
+the	O
+scissile	O
+peptide	O
+bond	O
+requires	O
+protonation	O
+of	O
+the	O
+emerging	O
+free	O
+amine	O
+,	O
+and	O
+in	O
+the	O
+proteasome	B-complex_assembly
+,	O
+the	O
+Thr1	B-residue_name_number
+amine	O
+group	O
+is	O
+likely	O
+to	O
+assume	O
+this	O
+function	O
+.	O
+
+Analogously	O
+,	O
+Thr1NH3	B-residue_name_number
++	O
+might	O
+promote	O
+the	O
+bivalent	O
+reaction	O
+mode	O
+of	O
+epoxyketone	O
+inhibitors	O
+by	O
+protonating	O
+the	O
+epoxide	O
+moiety	O
+to	O
+create	O
+a	O
+positively	O
+charged	O
+trivalent	O
+oxygen	O
+atom	O
+that	O
+is	O
+subsequently	O
+nucleophilically	O
+attacked	O
+by	O
+Thr1NH2	B-residue_name_number
+.	O
+
+During	O
+autolysis	B-ptm
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+is	O
+engaged	O
+in	O
+a	O
+hydroxyoxazolidine	O
+ring	O
+intermediate	O
+(	O
+Fig	O
+.	O
+3d	O
+),	O
+which	O
+is	O
+unstable	O
+and	O
+short	O
+-	O
+lived	O
+.	O
+
+Breakdown	O
+of	O
+this	O
+tetrahedral	O
+transition	O
+state	O
+releases	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+that	O
+is	O
+protonated	O
+by	O
+aspartic	B-residue_name_number
+acid	I-residue_name_number
+166	I-residue_name_number
+via	O
+Ser129OH	B-residue_name_number
+to	O
+yield	O
+Thr1NH3	B-residue_name_number
++.	O
+
+The	O
+residues	O
+Ser129	B-residue_name_number
+and	O
+Asp166	B-residue_name_number
+are	O
+expected	O
+to	O
+increase	O
+the	O
+pKa	O
+value	O
+of	O
+Thr1N	B-residue_name_number
+,	O
+thereby	O
+favouring	O
+its	O
+charged	O
+state	O
+.	O
+
+Consistent	O
+with	O
+playing	O
+an	O
+essential	O
+role	O
+in	O
+proton	O
+shuttling	O
+,	O
+the	O
+mutation	B-experimental_method
+D166A	B-mutant
+prevents	O
+autolysis	B-ptm
+of	O
+the	O
+archaeal	B-taxonomy_domain
+CP	B-complex_assembly
+and	O
+the	O
+exchange	B-experimental_method
+D166N	B-mutant
+impairs	O
+catalytic	O
+activity	O
+of	O
+the	O
+yeast	B-taxonomy_domain
+CP	B-complex_assembly
+about	O
+60	O
+%.	O
+
+The	O
+mutation	B-experimental_method
+D166N	B-mutant
+lowers	O
+the	O
+pKa	O
+of	O
+Thr1N	B-residue_name_number
+,	O
+which	O
+is	O
+thus	O
+more	O
+likely	O
+to	O
+exist	O
+in	O
+the	O
+uncharged	O
+deprotonated	O
+state	O
+(	O
+Thr1NH2	B-residue_name_number
+).	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	B-protein_state
+on	O
+the	O
+one	O
+hand	O
+,	O
+and	O
+the	O
+ability	O
+to	O
+react	O
+readily	O
+with	O
+carfilzomib	B-chemical
+on	O
+the	O
+other	O
+.	O
+
+Hence	O
+,	O
+the	O
+proteasome	B-complex_assembly
+can	O
+be	O
+viewed	O
+as	O
+having	O
+a	O
+second	B-site
+triad	I-site
+that	O
+is	O
+essential	O
+for	O
+efficient	O
+proteolysis	O
+.	O
+
+While	O
+Lys33NH2	B-residue_name_number
+and	O
+Asp17Oδ	B-residue_name_number
+are	O
+required	O
+to	O
+deprotonate	O
+the	O
+Thr1	B-residue_name_number
+hydroxyl	O
+side	O
+chain	O
+,	O
+Ser129OH	B-residue_name_number
+and	O
+Asp166OH	B-residue_name_number
+serve	O
+to	O
+protonate	O
+the	O
+N	O
+-	O
+terminal	O
+amine	O
+group	O
+of	O
+Thr1	B-residue_name_number
+.	O
+
+In	O
+accord	O
+with	O
+the	O
+proposed	O
+Thr1	B-residue_name_number
+–	O
+Lys33	B-residue_name_number
+–	O
+Asp17	B-residue_name_number
+catalytic	B-site
+triad	I-site
+,	O
+crystallographic	B-evidence
+data	I-evidence
+on	O
+the	O
+proteolytically	B-protein_state
+inactive	I-protein_state
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+mutant	B-protein_state
+demonstrate	O
+that	O
+the	O
+interaction	O
+of	O
+Lys33NH2	B-residue_name_number
+and	O
+Cys1	B-residue_name_number
+is	O
+broken	O
+.	O
+
+However	O
+,	O
+owing	O
+to	O
+Cys	B-residue_name
+being	O
+a	O
+strong	O
+nucleophile	O
+,	O
+the	O
+propeptide	B-structure_element
+can	O
+still	O
+be	O
+cleaved	B-protein_state
+off	O
+over	O
+time	O
+.	O
+
+While	O
+only	O
+one	O
+single	O
+turnover	O
+is	O
+necessary	O
+for	O
+autolysis	B-ptm
+,	O
+continuous	O
+enzymatic	O
+activity	O
+is	O
+required	O
+for	O
+significant	O
+and	O
+detectable	O
+substrate	O
+hydrolysis	O
+.	O
+
+Notably	O
+,	O
+in	O
+the	O
+Ntn	B-protein_type
+hydrolase	I-protein_type
+penicillin	B-protein_type
+acylase	I-protein_type
+,	O
+substitution	B-experimental_method
+of	O
+the	O
+catalytic	B-protein_state
+N	O
+-	O
+terminal	O
+Ser	B-residue_name
+residue	O
+by	O
+Cys	B-residue_name
+also	O
+inactivates	B-protein_state
+the	O
+enzyme	B-protein_type
+but	O
+still	O
+enables	O
+precursor	B-ptm
+processing	I-ptm
+.	O
+
+To	O
+investigate	O
+why	O
+the	O
+CP	B-complex_assembly
+specifically	O
+employs	O
+threonine	B-residue_name
+as	O
+its	O
+active	B-site
+-	I-site
+site	I-site
+residue	I-site
+,	O
+we	O
+used	O
+a	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+of	O
+the	O
+yCP	B-complex_assembly
+and	O
+characterized	O
+it	O
+biochemically	B-experimental_method
+and	I-experimental_method
+structurally	I-experimental_method
+.	O
+
+Activity	B-experimental_method
+assays	I-experimental_method
+with	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+revealed	O
+reduced	O
+turnover	O
+of	O
+Suc	B-chemical
+-	I-chemical
+LLVY	I-chemical
+-	I-chemical
+AMC	I-chemical
+.	O
+
+We	O
+also	O
+observed	O
+slightly	O
+lower	O
+affinity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+yCP	B-complex_assembly
+for	O
+the	O
+Food	O
+and	O
+Drug	O
+Administration	O
+-	O
+approved	O
+proteasome	B-complex_assembly
+inhibitors	O
+bortezomib	B-chemical
+and	O
+carfilzomib	B-chemical
+.	O
+
+Structural	B-evidence
+analyses	I-evidence
+support	O
+these	O
+findings	O
+with	O
+the	O
+T1S	B-mutant
+mutant	B-protein_state
+and	O
+provide	O
+an	O
+explanation	O
+for	O
+the	O
+strict	B-protein_state
+use	I-protein_state
+of	I-protein_state
+Thr	B-residue_name
+residues	O
+in	O
+proteasomes	B-complex_assembly
+.	O
+
+Thr1	B-residue_name_number
+is	O
+well	O
+anchored	O
+in	O
+the	O
+active	B-site
+site	I-site
+by	O
+hydrophobic	O
+interactions	O
+of	O
+its	O
+Cγ	O
+methyl	O
+group	O
+with	O
+Ala46	B-residue_name_number
+(	O
+Cβ	O
+),	O
+Lys33	B-residue_name_number
+(	O
+carbon	O
+side	O
+chain	O
+)	O
+and	O
+Thr3	B-residue_name_number
+(	O
+Cγ	O
+).	O
+
+Notably	O
+,	O
+proteolytically	B-protein_state
+active	I-protein_state
+proteasome	B-complex_assembly
+subunits	O
+from	O
+archaea	B-taxonomy_domain
+,	O
+yeast	B-taxonomy_domain
+and	O
+mammals	B-taxonomy_domain
+,	O
+including	O
+constitutive	O
+,	O
+immuno	O
+-	O
+and	O
+thymoproteasome	O
+subunits	O
+,	O
+either	O
+encode	O
+Thr	B-residue_name
+or	O
+Ile	B-residue_name
+at	O
+position	O
+3	B-residue_number
+,	O
+indicating	O
+the	O
+importance	O
+of	O
+the	O
+Cγ	O
+for	O
+fixing	O
+the	O
+position	O
+of	O
+the	O
+nucleophilic	O
+Thr1	B-residue_name_number
+.	O
+
+In	O
+contrast	O
+to	O
+Thr1	B-residue_name_number
+,	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Ser1	B-residue_name_number
+occupies	O
+the	O
+position	O
+of	O
+the	O
+Thr1	B-residue_name_number
+methyl	O
+side	O
+chain	O
+in	O
+the	O
+WT	B-protein_state
+enzyme	B-complex_assembly
+,	O
+which	O
+requires	O
+its	O
+reorientation	O
+relative	O
+to	O
+the	O
+substrate	O
+to	O
+allow	O
+cleavage	O
+(	O
+Fig	O
+.	O
+4g	O
+,	O
+h	O
+).	O
+
+Notably	O
+,	O
+in	O
+the	O
+threonine	B-protein_type
+aspartase	I-protein_type
+Taspase1	B-protein
+,	O
+mutation	B-experimental_method
+of	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+Thr234	B-residue_name_number
+to	O
+Ser	B-residue_name
+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	B-residue_name_number
+in	O
+the	O
+WT	B-protein_state
+,	O
+thereby	O
+reducing	O
+catalytic	O
+activity	O
+.	O
+
+Similarly	O
+,	O
+although	O
+the	O
+serine	B-residue_name
+mutant	B-protein_state
+is	O
+active	B-protein_state
+,	O
+threonine	B-residue_name
+is	O
+more	O
+efficient	O
+in	O
+the	O
+context	O
+of	O
+the	O
+proteasome	B-complex_assembly
+active	B-site
+site	I-site
+.	O
+
+The	O
+greater	O
+suitability	O
+of	O
+threonine	B-residue_name
+for	O
+the	O
+proteasome	B-complex_assembly
+active	B-site
+site	I-site
+,	O
+which	O
+has	O
+been	O
+noted	O
+in	O
+biochemical	O
+as	O
+well	O
+as	O
+in	O
+kinetic	O
+studies	O
+,	O
+constitutes	O
+a	O
+likely	O
+reason	O
+for	O
+the	O
+conservation	B-protein_state
+of	O
+the	O
+Thr1	B-residue_name_number
+residue	O
+in	O
+all	O
+proteasomes	B-complex_assembly
+from	O
+bacteria	B-taxonomy_domain
+to	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+Conformation	O
+of	O
+proteasomal	O
+propeptides	B-structure_element
+.	O
+
+(	O
+a	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+and	O
+the	O
+matured	B-protein_state
+WT	B-protein_state
+β1	B-protein
+active	B-site
+-	I-site
+site	I-site
+Thr1	B-residue_name_number
+.	O
+
+Only	O
+the	O
+residues	O
+(-	B-residue_range
+5	I-residue_range
+)	I-residue_range
+to	I-residue_range
+(-	I-residue_range
+1	I-residue_range
+)	I-residue_range
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+are	O
+displayed	O
+.	O
+
+The	O
+major	O
+determinant	O
+of	O
+the	O
+S1	B-site
+specificity	I-site
+pocket	I-site
+,	O
+residue	O
+45	B-residue_number
+,	O
+is	O
+depicted	O
+.	O
+
+Note	O
+the	O
+tight	O
+conformation	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+and	O
+Ala1	B-residue_name_number
+before	O
+propeptide	B-structure_element
+removal	O
+(	O
+G	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+turn	O
+;	O
+cyan	O
+double	O
+arrow	O
+)	O
+compared	O
+with	O
+the	O
+relaxed	O
+,	O
+processed	B-protein_state
+WT	B-protein_state
+active	B-site
+-	I-site
+site	I-site
+Thr1	B-residue_name_number
+(	O
+red	O
+double	O
+arrow	O
+).	O
+
+The	O
+black	O
+arrow	O
+indicates	O
+the	O
+attack	O
+of	O
+Thr1Oγ	B-residue_name_number
+onto	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+).	I-residue_name_number
+
+(	O
+b	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+and	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+highlights	O
+subtle	O
+differences	O
+in	O
+their	O
+conformations	O
+,	O
+but	O
+illustrates	O
+that	O
+Ala1	B-residue_name_number
+and	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+match	O
+well	O
+.	O
+
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+OH	O
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+O	O
+(∼	O
+2	O
+.	O
+8	O
+Å	O
+;	O
+black	O
+dashed	O
+line	O
+).	O
+
+(	O
+c	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+propeptide	B-structure_element
+remnants	O
+depict	O
+their	O
+differences	O
+in	O
+conformation	O
+.	O
+
+While	O
+residue	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+of	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+prosegments	B-structure_element
+fit	O
+the	O
+S1	B-site
+pocket	I-site
+,	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+occupies	O
+the	O
+S2	B-site
+pocket	I-site
+.	O
+
+Nonetheless	O
+,	O
+in	O
+all	O
+mutants	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+is	O
+ideally	O
+placed	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	B-residue_name_number
+.	O
+
+The	O
+hydrogen	O
+bond	O
+between	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+OH	O
+and	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+O	O
+(∼	O
+2	O
+.	O
+8	O
+Å	O
+)	O
+is	O
+indicated	O
+by	O
+a	O
+black	O
+dashed	O
+line	O
+.	O
+
+Mutations	B-experimental_method
+of	O
+residue	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+and	O
+their	O
+influence	O
+on	O
+propeptide	B-structure_element
+conformation	O
+and	O
+autolysis	B-ptm
+.	O
+
+(	O
+a	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+propeptide	B-structure_element
+.	O
+
+The	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+residues	O
+of	O
+both	O
+prosegments	B-structure_element
+point	O
+into	O
+the	O
+S1	B-site
+pocket	I-site
+.	O
+
+(	O
+b	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β5	B-protein
+propeptides	B-structure_element
+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	B-protein_state
+proteasomes	B-complex_assembly
+.	O
+
+While	O
+the	O
+residues	O
+(-	B-residue_range
+2	I-residue_range
+)	I-residue_range
+to	I-residue_range
+(-	I-residue_range
+4	I-residue_range
+)	I-residue_range
+vary	O
+in	O
+their	O
+conformation	O
+,	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+and	O
+Ala1	B-residue_name_number
+are	O
+located	O
+in	O
+all	O
+structures	B-evidence
+at	O
+the	O
+same	O
+positions	O
+.	O
+
+(	O
+c	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+propeptide	B-structure_element
+.	O
+
+The	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+residues	O
+of	O
+both	O
+prosegments	B-structure_element
+point	O
+into	O
+the	O
+S1	B-site
+pocket	I-site
+,	O
+but	O
+only	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+OH	O
+of	O
+β2	B-protein
+forms	O
+a	O
+hydrogen	O
+bridge	O
+to	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+O	O
+(	O
+black	O
+dashed	O
+line	O
+).	O
+
+(	O
+d	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+matured	B-protein_state
+β2	B-protein
+active	B-site
+site	I-site
+,	O
+the	O
+WT	B-protein_state
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+and	O
+the	O
+β2	B-mutant
+-	I-mutant
+T	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+V	I-mutant
+mutant	B-protein_state
+propeptide	B-structure_element
+.	O
+
+Notably	O
+,	O
+Val	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+of	O
+the	O
+latter	O
+does	O
+not	O
+occupy	O
+the	O
+S1	B-site
+pocket	I-site
+,	O
+thereby	O
+changing	O
+the	O
+orientation	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+and	O
+preventing	O
+nucleophilic	O
+attack	O
+of	O
+Thr1Oγ	B-residue_name_number
+on	O
+the	O
+carbonyl	O
+carbon	O
+atom	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+).	I-residue_name_number
+
+Architecture	O
+and	O
+proposed	O
+reaction	O
+mechanism	O
+of	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+.	O
+
+(	O
+a	O
+)	O
+Hydrogen	B-site
+-	I-site
+bonding	I-site
+network	I-site
+at	O
+the	O
+mature	B-protein_state
+WT	B-protein_state
+β5	B-protein
+proteasomal	O
+active	B-site
+site	I-site
+(	O
+dotted	O
+lines	O
+).	O
+
+Thr1OH	B-residue_name_number
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Lys33NH2	B-residue_name_number
+(	O
+2	O
+.	O
+7	O
+Å	O
+),	O
+which	O
+in	O
+turn	O
+interacts	O
+with	O
+Asp17Oδ	B-residue_name_number
+.	O
+
+The	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+is	O
+engaged	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+Ser129Oγ	B-residue_name_number
+,	O
+the	O
+carbonyl	O
+oxygen	O
+of	O
+residue	O
+168	B-residue_number
+,	O
+Ser169Oγ	B-residue_name_number
+and	O
+Asp166Oδ	B-residue_name_number
+.	O
+(	O
+b	O
+)	O
+The	O
+orientations	O
+of	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+residues	I-site
+involved	O
+in	O
+hydrogen	O
+bonding	O
+are	O
+strictly	B-protein_state
+conserved	I-protein_state
+in	O
+each	O
+proteolytic	B-site
+centre	I-site
+,	O
+as	O
+shown	O
+by	O
+superposition	B-experimental_method
+of	O
+the	O
+β	B-protein
+subunits	I-protein
+.	O
+
+(	O
+c	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+WT	B-protein_state
+β5	B-protein
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+mutant	B-protein_state
+active	B-site
+site	I-site
+.	O
+
+In	O
+the	O
+latter	O
+,	O
+a	O
+water	B-chemical
+molecule	O
+(	O
+red	O
+sphere	O
+)	O
+is	O
+found	O
+at	O
+the	O
+position	O
+where	O
+in	O
+the	O
+WT	B-protein_state
+structure	O
+the	O
+side	O
+chain	O
+amine	O
+group	O
+of	O
+Lys33	B-residue_name_number
+is	O
+located	O
+.	O
+
+Similarly	O
+to	O
+Lys33	B-residue_name_number
+,	O
+the	O
+water	B-chemical
+molecule	O
+hydrogen	O
+bonds	O
+to	O
+Arg19O	B-residue_name_number
+,	O
+Asp17Oδ	B-residue_name_number
+and	O
+Thr1OH	B-residue_name_number
+.	O
+
+Note	O
+,	O
+the	O
+strong	O
+interaction	O
+with	O
+the	O
+water	B-chemical
+molecule	O
+causes	O
+a	O
+minor	O
+shift	O
+of	O
+Thr1	B-residue_name_number
+,	O
+while	O
+all	O
+other	O
+active	B-site
+-	I-site
+site	I-site
+residues	I-site
+remain	O
+in	O
+place	O
+.	O
+
+(	O
+d	O
+)	O
+Proposed	O
+chemical	O
+reaction	O
+mechanism	O
+for	O
+autocatalytic	B-ptm
+precursor	I-ptm
+processing	I-ptm
+and	O
+proteolysis	O
+in	O
+the	O
+proteasome	B-complex_assembly
+.	O
+
+The	O
+active	B-site
+-	I-site
+site	I-site
+Thr1	B-residue_name_number
+is	O
+depicted	O
+in	O
+blue	O
+,	O
+the	O
+propeptide	B-structure_element
+segment	O
+and	O
+the	O
+peptide	O
+substrate	O
+are	O
+coloured	O
+in	O
+green	O
+,	O
+whereas	O
+the	O
+scissile	O
+peptide	O
+bond	O
+is	O
+highlighted	O
+in	O
+red	O
+.	O
+
+Autolysis	B-ptm
+(	O
+left	O
+set	O
+of	O
+structures	O
+)	O
+is	O
+initiated	O
+by	O
+deprotonation	O
+of	O
+Thr1OH	B-residue_name_number
+via	O
+Lys33NH2	B-residue_name_number
+and	O
+the	O
+formation	O
+of	O
+a	O
+tetrahedral	O
+transition	O
+state	O
+.	O
+
+The	O
+strictly	B-protein_state
+conserved	I-protein_state
+oxyanion	O
+hole	O
+Gly47NH	B-residue_name_number
+stabilizing	O
+the	O
+negatively	O
+charged	O
+intermediate	O
+is	O
+illustrated	O
+as	O
+a	O
+semicircle	O
+.	O
+
+Collapse	O
+of	O
+the	O
+transition	O
+state	O
+frees	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+(	O
+by	O
+completing	O
+an	O
+N	O
+-	O
+to	O
+-	O
+O	O
+acyl	O
+shift	O
+of	O
+the	O
+propeptide	B-structure_element
+),	O
+which	O
+is	O
+subsequently	O
+protonated	O
+by	O
+Asp166OH	B-residue_name_number
+via	O
+Ser129OH	B-residue_name_number
+.	O
+
+Next	O
+,	O
+Thr1NH2	B-residue_name_number
+polarizes	O
+a	O
+water	B-chemical
+molecule	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+of	O
+the	O
+acyl	O
+-	O
+enzyme	O
+intermediate	O
+.	O
+
+On	O
+hydrolysis	O
+of	O
+the	O
+latter	O
+,	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+Thr1	B-residue_name_number
+is	O
+ready	O
+for	O
+catalysis	O
+(	O
+right	O
+set	O
+of	O
+structures	O
+).	O
+
+The	O
+charged	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+may	O
+engage	O
+in	O
+the	O
+orientation	O
+of	O
+the	O
+amide	O
+moiety	O
+and	O
+donate	O
+a	O
+proton	O
+to	O
+the	O
+emerging	O
+N	O
+terminus	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+cleavage	O
+product	O
+.	O
+
+The	O
+resulting	O
+deprotonated	O
+Thr1NH2	B-residue_name_number
+finally	O
+activates	O
+a	O
+water	B-chemical
+molecule	O
+for	O
+hydrolysis	O
+of	O
+the	O
+acyl	O
+-	O
+enzyme	O
+.	O
+
+The	O
+proteasome	B-complex_assembly
+favours	O
+threonine	B-residue_name
+as	O
+the	O
+active	O
+-	O
+site	O
+nucleophile	O
+.	O
+
+(	O
+a	O
+)	O
+Growth	B-experimental_method
+tests	I-experimental_method
+by	I-experimental_method
+serial	I-experimental_method
+dilution	I-experimental_method
+of	O
+WT	B-protein_state
+and	O
+pre2	O
+(	O
+β5	B-protein
+)	O
+mutant	B-protein_state
+yeast	B-taxonomy_domain
+cultures	O
+reveal	O
+growth	O
+defects	O
+of	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+mutants	B-experimental_method
+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
+b	O
+)	O
+Purified	O
+WT	B-protein_state
+and	O
+mutant	B-protein_state
+proteasomes	B-complex_assembly
+were	O
+tested	O
+for	O
+their	O
+chymotrypsin	O
+-	O
+like	O
+activity	O
+(	O
+β5	B-protein
+)	O
+using	O
+the	O
+substrate	O
+Suc	B-chemical
+-	I-chemical
+LLVY	I-chemical
+-	I-chemical
+AMC	I-chemical
+.	O
+
+(	O
+c	O
+)	O
+Illustration	O
+of	O
+the	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+map	I-evidence
+(	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	B-structure_element
+fragment	O
+.	O
+
+The	O
+prosegment	B-structure_element
+is	O
+cleaved	B-protein_state
+but	O
+still	B-protein_state
+bound	I-protein_state
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+.	O
+
+Notably	O
+,	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+does	O
+not	O
+occupy	O
+the	O
+S1	B-site
+pocket	I-site
+formed	O
+by	O
+Met45	B-residue_name_number
+,	O
+similar	O
+to	O
+what	O
+was	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	B-protein_state
+.	O
+
+(	O
+d	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+mutant	B-protein_state
+subunits	O
+onto	O
+the	O
+WT	B-protein_state
+β5	B-protein
+subunit	O
+.	O
+(	O
+e	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+propeptide	B-structure_element
+onto	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+active	B-site
+site	I-site
+(	O
+blue	O
+)	O
+and	O
+the	O
+WT	B-protein_state
+β5	B-protein
+active	B-site
+site	I-site
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+proteasome	B-complex_assembly
+inhibitor	O
+MG132	B-chemical
+(	O
+ref	O
+.).	O
+
+The	O
+inhibitor	B-chemical
+as	O
+well	O
+as	O
+the	O
+propeptides	B-structure_element
+adopt	O
+similar	O
+conformations	O
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+.	O
+
+(	O
+f	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+WT	B-protein_state
+β5	B-protein
+and	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+mutant	B-protein_state
+active	B-site
+sites	I-site
+illustrates	O
+the	O
+different	O
+orientations	O
+of	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Thr1	B-residue_name_number
+and	O
+the	O
+thiol	O
+side	O
+chain	O
+of	O
+Cys1	B-residue_name_number
+.	O
+
+(	O
+g	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+WT	B-protein_state
+β5	B-protein
+and	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+active	B-site
+sites	I-site
+reveals	O
+different	O
+orientations	O
+of	O
+the	O
+hydroxyl	O
+groups	O
+of	O
+Thr1	B-residue_name_number
+and	O
+Ser1	B-residue_name_number
+,	O
+respectively	O
+.	O
+
+The	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+map	I-evidence
+for	O
+Ser1	B-residue_name_number
+(	O
+blue	O
+mesh	O
+contoured	O
+at	O
+1σ	O
+)	O
+is	O
+illustrated	O
+.	O
+
+(	O
+h	O
+)	O
+The	O
+methyl	O
+group	O
+of	O
+Thr1	B-residue_name_number
+is	O
+anchored	O
+by	O
+hydrophobic	O
+interactions	O
+with	O
+Ala46Cβ	B-residue_name_number
+and	O
+Thr3Cγ	B-residue_name_number
+.	O
+
+Ser1	B-residue_name_number
+lacks	B-protein_state
+this	O
+stabilization	O
+and	O
+is	O
+therefore	O
+rotated	O
+by	O
+60	O
+°.	O
+
+Inhibition	O
+of	O
+WT	B-protein_state
+and	O
+mutant	B-protein_state
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+proteasomes	B-complex_assembly
+by	O
+bortezomib	B-chemical
+and	O
+carfilzomib	B-chemical
+.	O
+
+Inhibition	B-experimental_method
+assays	I-experimental_method
+(	O
+left	O
+panel	O
+).	O
+
+Purified	O
+yeast	B-taxonomy_domain
+proteasomes	B-complex_assembly
+were	O
+tested	O
+for	O
+the	O
+susceptibility	O
+of	O
+their	O
+ChT	O
+-	O
+L	O
+(	O
+β5	B-protein
+)	O
+activity	O
+to	O
+inhibition	O
+by	O
+bortezomib	B-chemical
+and	O
+carfilzomib	B-chemical
+using	O
+the	O
+substrate	O
+Suc	B-chemical
+-	I-chemical
+LLVY	I-chemical
+-	I-chemical
+AMC	I-chemical
+.	O
+
+IC50	B-evidence
+values	I-evidence
+were	O
+determined	O
+in	O
+triplicate	O
+;	O
+s	O
+.	O
+d	O
+.'	O
+s	O
+are	O
+indicated	O
+by	O
+error	O
+bars	O
+.	O
+
+Note	O
+that	O
+IC50	B-evidence
+values	I-evidence
+depend	O
+on	O
+time	O
+and	O
+enzyme	O
+concentration	O
+.	O
+
+Proteasomes	B-complex_assembly
+(	O
+final	O
+concentration	O
+:	O
+66	O
+nM	O
+)	O
+were	O
+incubated	O
+with	O
+inhibitor	O
+for	O
+45	O
+min	O
+before	O
+substrate	O
+addition	O
+(	O
+final	O
+concentration	O
+:	O
+200	O
+μM	O
+).	O
+
+Structures	B-evidence
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+in	O
+complex	B-complex_assembly
+with	I-complex_assembly
+both	I-complex_assembly
+ligands	I-complex_assembly
+(	O
+green	O
+)	O
+prove	O
+the	O
+reactivity	O
+of	O
+Ser1	B-residue_name_number
+(	O
+right	O
+panel	O
+).	O
+
+The	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+maps	I-evidence
+(	O
+blue	O
+mesh	O
+)	O
+for	O
+Ser1	B-residue_name_number
+(	O
+brown	O
+)	O
+and	O
+the	O
+covalently	O
+bound	O
+ligands	O
+(	O
+green	O
+;	O
+only	O
+the	O
+P1	B-site
+site	I-site
+(	O
+Leu1	B-residue_name_number
+)	O
+is	O
+shown	O
+)	O
+are	O
+contoured	O
+at	O
+1σ	O
+.	O
+
+The	O
+WT	B-protein_state
+proteasome	B-complex_assembly
+:	I-complex_assembly
+inhibitor	I-complex_assembly
+complex	I-complex_assembly
+structures	B-evidence
+(	O
+inhibitor	O
+in	O
+grey	O
+;	O
+Thr1	B-residue_name_number
+in	O
+black	O
+)	O
+are	O
+superimposed	B-experimental_method
+and	O
+demonstrate	O
+that	O
+mutation	B-experimental_method
+of	O
+Thr1	B-residue_name_number
+to	O
+Ser	B-residue_name
+does	O
+not	O
+affect	O
+the	O
+binding	O
+mode	O
+of	O
+bortezomib	B-chemical
+or	O
+carfilzomib	B-chemical
+.	O
+