diff --git "a/annotation_IOB/dev.tsv" "b/annotation_IOB/dev.tsv"
new file mode 100644--- /dev/null
+++ "b/annotation_IOB/dev.tsv"
@@ -0,0 +1,20355 @@
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+Stability	O
+of	O
+NadR	B-protein
+is	O
+increased	O
+by	O
+small	O
+molecule	O
+ligands	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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+Primary	O
+structure	O
+conservation	O
+of	O
+the	O
+PduL	B-protein_type
+protein	O
+family	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
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+Catalytic	B-protein_state
+Asp	B-residue_name
+residues	O
+were	O
+shown	O
+in	O
+sticks	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
+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
+
+Catalytic	B-site
+residues	I-site
+and	O
+mutated	O
+residues	O
+were	O
+shown	O
+in	O
+sticks	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
+
+(	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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+C	O
+)	O
+14C	B-chemical
+-	I-chemical
+acp	I-chemical
+labeling	O
+of	O
+18S	B-chemical
+rRNAs	I-chemical
+.	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+The	O
+“	O
+back	B-protein_state
+to	I-protein_state
+back	I-protein_state
+”	O
+dimer	B-oligomeric_state
+.	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
+
+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
+
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+−,	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
+
+Identifying	O
+cell	O
+‐	O
+specific	O
+signaling	O
+clusters	O
+in	O
+ERα	B-protein
+ligand	O
+classes	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+affinity	B-evidence
+was	O
+therefore	O
+determined	O
+using	O
+competition	B-experimental_method
+SPAs	I-experimental_method
+.	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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+The	O
+organization	O
+of	O
+the	O
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+CD	B-structure_element
+
+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
+
+A	O
+regulatory	B-structure_element
+loop	I-structure_element
+mediates	O
+interdomain	O
+interactions	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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+The	O
+active	B-site
+site	I-site
+is	O
+boxed	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+All	O
+structures	B-evidence
+show	O
+the	O
+transporters	B-protein_type
+in	O
+open	B-protein_state
+conformations	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
+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
+
+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
+
+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
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+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
+
+Growth	B-experimental_method
+of	O
+ScMep2	B-mutant
+variants	I-mutant
+on	O
+low	O
+ammonium	O
+medium	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
+
+(	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
+
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+These	O
+multimeric	O
+forms	O
+are	O
+linked	O
+with	O
+an	O
+additional	O
+J	B-structure_element
+chain	O
+.	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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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-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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+Schematic	O
+models	O
+of	O
+U2AF65	B-protein
+recognizing	O
+the	O
+Py	B-chemical
+tract	I-chemical
+.	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+RNA	B-chemical
+binding	O
+reduces	O
+radiation	O
+-	O
+induced	O
+disorder	O
+on	O
+the	O
+atomic	O
+scale	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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+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
+
+(	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
+
+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
+