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 +1,10658 @@
+Biochemical	B-experimental_method
+analysis	I-experimental_method
+reveals	O
+that	O
+these	O
+outer	B-protein_type
+membrane	I-protein_type
+-	I-protein_type
+anchored	I-protein_type
+proteins	I-protein_type
+are	O
+in	O
+fact	O
+exquisitely	O
+specific	O
+for	O
+the	O
+highly	O
+branched	O
+xyloglucan	B-chemical
+(	O
+XyG	B-chemical
+)	O
+polysaccharide	B-chemical
+.	O
+
+However	O
+,	O
+there	O
+is	O
+a	O
+paucity	O
+of	O
+data	O
+regarding	O
+how	O
+the	O
+vast	O
+array	O
+of	O
+complex	B-chemical
+carbohydrate	I-chemical
+structures	O
+are	O
+selectively	O
+recognized	O
+and	O
+imported	O
+by	O
+members	O
+of	O
+the	O
+microbiota	B-taxonomy_domain
+,	O
+a	O
+critical	O
+process	O
+that	O
+enables	O
+these	O
+organisms	O
+to	O
+thrive	O
+in	O
+the	O
+competitive	O
+gut	O
+environment	O
+.	O
+
+The	O
+location	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+/	O
+B	B-protein
+is	O
+presented	O
+in	O
+this	O
+work	O
+;	O
+the	O
+location	O
+of	O
+GH5	B-protein
+has	O
+been	O
+empirically	O
+determined	O
+,	O
+and	O
+the	O
+enzymes	O
+have	O
+been	O
+placed	O
+based	O
+upon	O
+their	O
+predicted	O
+cellular	O
+location	O
+.	O
+
+These	O
+data	O
+extend	O
+our	O
+current	O
+understanding	O
+of	O
+the	O
+Sus	O
+-	O
+like	O
+glycan	B-chemical
+uptake	O
+paradigm	O
+within	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+and	O
+reveals	O
+how	O
+the	O
+complex	O
+dietary	O
+polysaccharide	B-chemical
+xyloglucan	B-chemical
+is	O
+recognized	O
+at	O
+the	O
+cell	O
+surface	O
+.	O
+
+In	O
+contrast	O
+,	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+encoded	O
+by	O
+locus	O
+tag	O
+Bacova_02650	B-gene
+,	O
+displays	O
+little	O
+sequence	O
+similarity	O
+to	O
+the	O
+products	O
+of	O
+similarly	O
+positioned	O
+genes	O
+in	O
+syntenic	O
+XyGUL	B-gene
+nor	O
+to	O
+any	O
+other	O
+gene	O
+product	O
+among	O
+the	O
+diversity	O
+of	O
+Bacteroidetes	B-taxonomy_domain
+PUL	B-gene
+.	O
+
+Formalin	O
+-	O
+fixed	O
+,	O
+nonpermeabilized	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+were	O
+grown	O
+in	O
+minimal	O
+medium	O
+plus	O
+XyG	B-chemical
+,	O
+probed	O
+with	O
+custom	O
+rabbit	O
+antibodies	O
+to	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+or	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+and	O
+then	O
+stained	O
+with	O
+Alexa	O
+Fluor	O
+488	O
+goat	O
+anti	O
+-	O
+rabbit	O
+IgG	O
+.	O
+(	O
+A	O
+)	O
+Overlay	B-experimental_method
+of	O
+bright	B-evidence
+-	I-evidence
+field	I-evidence
+and	I-evidence
+FITC	I-evidence
+images	I-evidence
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+A	O
+.	O
+(	O
+B	O
+)	O
+Overlay	B-experimental_method
+of	O
+bright	B-evidence
+-	I-evidence
+field	I-evidence
+and	I-evidence
+FITC	I-evidence
+images	I-evidence
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+.	O
+(	O
+C	O
+)	O
+Bright	B-evidence
+-	I-evidence
+field	I-evidence
+image	I-evidence
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+antibodies	O
+.	O
+
+The	O
+vanguard	O
+endo	B-protein_type
+-	I-protein_type
+xyloglucanase	I-protein_type
+of	O
+the	O
+XyGUL	B-gene
+,	O
+BoGH5	B-protein
+,	O
+preferentially	O
+cleaves	O
+the	O
+polysaccharide	B-chemical
+at	O
+unbranched	O
+glucosyl	B-chemical
+residues	O
+to	O
+generate	O
+xylogluco	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+(	O
+XyGOs	B-chemical
+)	O
+comprising	O
+a	O
+Glc4	B-structure_element
+backbone	I-structure_element
+with	O
+variable	B-structure_element
+side	I-structure_element
+-	I-structure_element
+chain	I-structure_element
+galactosylation	I-structure_element
+(	O
+XyGO1	B-chemical
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+;	O
+n	O
+=	O
+1	O
+)	O
+as	O
+the	O
+limit	O
+of	O
+digestion	O
+products	O
+in	O
+vitro	O
+;	O
+controlled	B-experimental_method
+digestion	I-experimental_method
+and	I-experimental_method
+fractionation	I-experimental_method
+by	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+allow	O
+the	O
+production	O
+of	O
+higher	O
+-	O
+order	O
+oligosaccharides	B-chemical
+(	O
+e	O
+.	O
+g	O
+.,	O
+XyGO2	B-chemical
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+;	O
+n	O
+=	O
+2	O
+).	O
+
+The	O
+approximate	O
+length	O
+of	O
+each	O
+glycan	B-site
+-	I-site
+binding	I-site
+site	I-site
+is	O
+displayed	O
+,	O
+colored	O
+to	O
+match	O
+the	O
+protein	B-evidence
+structures	I-evidence
+.	O
+(	O
+E	O
+)	O
+Stereo	O
+view	O
+of	O
+the	O
+xyloglucan	B-site
+-	I-site
+binding	I-site
+site	I-site
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+displaying	O
+all	O
+residues	O
+within	O
+4	O
+Å	O
+of	O
+the	O
+ligand	O
+.	O
+
+Most	O
+surprising	O
+in	O
+light	O
+of	O
+the	O
+saccharide	B-evidence
+-	I-evidence
+binding	I-evidence
+data	I-evidence
+,	O
+however	O
+,	O
+was	O
+a	O
+lack	O
+of	O
+extensive	O
+recognition	O
+of	O
+the	O
+XyG	B-chemical
+side	O
+chains	O
+;	O
+only	O
+Y84	B-residue_name_number
+appeared	O
+to	O
+provide	O
+a	O
+hydrophobic	B-site
+interface	I-site
+for	O
+a	O
+xylosyl	B-chemical
+residue	O
+(	O
+Xyl1	B-residue_name_number
+).	O
+
+Domains	O
+A	B-structure_element
+,	O
+B	B-structure_element
+,	O
+and	O
+C	B-structure_element
+display	O
+similar	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+folds	I-structure_element
+;	O
+domains	O
+B	B-structure_element
+(	O
+residues	O
+134	B-residue_range
+to	I-residue_range
+230	I-residue_range
+)	O
+and	O
+C	B-structure_element
+(	O
+residues	O
+231	B-residue_range
+to	I-residue_range
+313	I-residue_range
+)	O
+can	O
+be	O
+superimposed	B-experimental_method
+onto	O
+domain	O
+A	B-structure_element
+(	O
+residues	O
+34	B-residue_range
+to	I-residue_range
+133	I-residue_range
+)	O
+with	O
+RMSDs	B-evidence
+of	O
+1	O
+.	O
+1	O
+and	O
+1	O
+.	O
+2	O
+Å	O
+,	O
+respectively	O
+,	O
+for	O
+47	O
+atom	O
+pairs	O
+(	O
+23	O
+%	O
+and	O
+16	O
+%	O
+sequence	O
+identity	O
+,	O
+respectively	O
+).	O
+
+While	O
+neither	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+nor	O
+domain	O
+D	B-structure_element
+displays	O
+structural	O
+homology	O
+to	O
+known	O
+XyG	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+,	O
+the	O
+topology	O
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+resembles	O
+the	O
+xylan	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+Bacova_04391	B-protein
+(	O
+PDB	O
+3ORJ	O
+)	O
+encoded	O
+within	O
+a	O
+xylan	B-chemical
+-	O
+targeting	O
+PUL	B-gene
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+(	O
+Fig	O
+.	O
+5C	O
+).	O
+
+Six	O
+glucosyl	B-chemical
+residues	O
+,	O
+comprising	O
+the	O
+main	O
+chain	O
+,	O
+and	O
+three	O
+branching	O
+xylosyl	B-chemical
+residues	O
+of	O
+XyGO2	B-chemical
+can	O
+be	O
+modeled	O
+in	O
+the	O
+density	B-evidence
+(	O
+Fig	O
+.	O
+5D	O
+;	O
+cf	O
+.	O
+
+Complementation	B-experimental_method
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+with	O
+the	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*	I-mutant
+(	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+)	O
+variant	O
+,	O
+which	O
+does	O
+not	B-protein_state
+bind	I-protein_state
+XyG	B-chemical
+,	O
+supports	O
+growth	O
+on	O
+XyG	B-chemical
+and	O
+XyGOs	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+;	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-mutant
+-	I-mutant
+A	I-mutant
+*),	I-mutant
+with	O
+growth	O
+rates	O
+that	O
+are	O
+~	O
+70	O
+%	O
+that	O
+of	O
+the	O
+WT	B-protein_state
+.	O
+
+However	O
+,	O
+combined	B-experimental_method
+deletion	I-experimental_method
+of	I-experimental_method
+the	I-experimental_method
+genes	I-experimental_method
+encoding	I-experimental_method
+GH9	B-protein
+(	O
+encoded	O
+by	O
+Bacova_02649	B-gene
+)	O
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+does	O
+not	O
+exacerbate	O
+the	O
+growth	O
+defect	O
+on	O
+XyGO1	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+;	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+/	O
+ΔGH9	B-mutant
+).	O
+
+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
+
+Our	O
+observation	O
+here	O
+that	O
+the	O
+physical	O
+presence	O
+of	O
+the	O
+SusD	B-protein
+homolog	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+independent	O
+of	O
+XyG	B-chemical
+-	O
+binding	O
+ability	O
+,	O
+is	O
+both	O
+necessary	O
+and	O
+sufficient	O
+for	O
+XyG	B-chemical
+utilization	O
+further	O
+supports	O
+a	O
+model	O
+of	O
+glycan	B-chemical
+import	O
+whereby	O
+the	O
+SusC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+TBDTs	I-protein_type
+and	O
+the	O
+SusD	B-protein_type
+-	I-protein_type
+like	I-protein_type
+SGBPs	I-protein_type
+must	O
+be	O
+intimately	O
+associated	O
+to	O
+support	O
+glycan	B-chemical
+uptake	O
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+
+Because	O
+the	O
+intestinal	O
+ecosystem	O
+is	O
+a	O
+dense	O
+consortium	O
+of	O
+bacteria	B-taxonomy_domain
+that	O
+must	O
+compete	O
+for	O
+their	O
+nutrients	O
+,	O
+these	O
+multimodular	O
+SGBPs	B-protein_type
+may	O
+reflect	O
+ongoing	O
+evolutionary	O
+experiments	O
+to	O
+enhance	O
+glycan	B-chemical
+uptake	O
+efficiency	O
+.	O
+
+In	O
+conclusion	O
+,	O
+the	O
+present	O
+study	O
+further	O
+illuminates	O
+the	O
+essential	O
+role	O
+that	O
+surface	B-protein_type
+-	I-protein_type
+glycan	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+play	O
+in	O
+facilitating	O
+the	O
+catabolism	O
+of	O
+complex	O
+dietary	O
+carbohydrates	B-chemical
+by	O
+Bacteroidetes	B-taxonomy_domain
+.	O
+
+The	O
+PTAC	B-protein_type
+predominantly	O
+associated	O
+with	O
+metabolosomes	B-complex_assembly
+(	O
+PduL	B-protein_type
+)	O
+has	O
+no	O
+sequence	O
+homology	O
+to	O
+the	O
+PTAC	B-protein_type
+ubiquitous	O
+among	O
+fermentative	B-taxonomy_domain
+bacteria	I-taxonomy_domain
+(	O
+Pta	B-protein_type
+).	O
+
+Here	O
+,	O
+we	O
+report	O
+two	O
+high	O
+-	O
+resolution	O
+PduL	B-protein_type
+crystal	B-evidence
+structures	I-evidence
+with	B-protein_state
+bound	I-protein_state
+substrates	I-protein_state
+.	O
+
+Recently	O
+,	O
+a	O
+new	O
+type	O
+of	O
+phosphotransacylase	B-protein_type
+was	O
+described	O
+that	O
+shares	O
+no	O
+evolutionary	O
+relation	O
+to	O
+Pta	B-protein_type
+.	O
+
+Not	O
+only	O
+does	O
+PduL	B-protein_type
+facilitate	O
+substrate	O
+level	O
+phosphorylation	O
+,	O
+but	O
+it	O
+also	O
+is	O
+critical	O
+for	O
+cofactor	O
+recycling	O
+within	O
+,	O
+and	O
+product	O
+efflux	O
+from	O
+,	O
+the	O
+organelle	O
+.	O
+
+The	O
+reactions	O
+carried	O
+out	O
+in	O
+the	O
+majority	O
+of	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+(	O
+also	O
+known	O
+as	O
+metabolosomes	B-complex_assembly
+)	O
+fit	O
+a	O
+generalized	O
+biochemical	O
+paradigm	O
+for	O
+the	O
+oxidation	O
+of	O
+aldehydes	B-chemical
+(	O
+Fig	O
+1	O
+).	O
+
+Reaction	O
+1	O
+:	O
+acetyl	B-chemical
+-	I-chemical
+S	I-chemical
+-	I-chemical
+CoA	I-chemical
++	O
+Pi	B-chemical
+←→	O
+acetyl	B-chemical
+phosphate	I-chemical
++	O
+CoA	B-chemical
+-	I-chemical
+SH	I-chemical
+(	O
+PTAC	B-protein_type
+)	O
+
+The	O
+canonical	O
+PTAC	B-protein_type
+,	O
+Pta	B-protein_type
+,	O
+is	O
+an	O
+ancient	O
+enzyme	O
+found	O
+in	O
+some	O
+eukaryotes	B-taxonomy_domain
+and	O
+archaea	B-taxonomy_domain
+,	O
+and	O
+widespread	O
+among	O
+the	O
+bacteria	B-taxonomy_domain
+;	O
+90	O
+%	O
+of	O
+the	O
+bacterial	B-taxonomy_domain
+genomes	O
+in	O
+the	O
+Integrated	O
+Microbial	O
+Genomes	O
+database	O
+contain	O
+a	O
+gene	O
+encoding	O
+the	O
+PTA_PTB	B-protein_type
+phosphotransacylase	I-protein_type
+(	O
+Pfam	O
+domain	O
+PF01515	B-structure_element
+).	O
+
+This	O
+protein	O
+,	O
+PduL	B-protein_type
+(	O
+Pfam	O
+domain	O
+PF06130	B-structure_element
+),	O
+was	O
+shown	O
+to	O
+catalyze	O
+the	O
+conversion	O
+of	O
+propionyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+propionyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+and	O
+is	O
+associated	O
+with	O
+a	O
+BMC	B-complex_assembly
+involved	O
+in	O
+propanediol	O
+utilization	O
+,	O
+the	O
+PDU	B-complex_assembly
+BMC	I-complex_assembly
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+structure	B-evidence
+of	O
+PduL	B-protein_type
+,	O
+the	O
+PTAC	B-protein_type
+found	O
+in	O
+the	O
+vast	O
+majority	O
+of	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+,	O
+has	O
+not	O
+been	O
+determined	O
+.	O
+
+Here	O
+we	O
+report	O
+high	O
+-	O
+resolution	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+a	O
+PduL	B-protein_type
+-	I-protein_type
+type	I-protein_type
+PTAC	I-protein_type
+in	O
+both	O
+CoA	B-protein_state
+-	I-protein_state
+and	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+forms	O
+,	O
+completing	O
+our	O
+understanding	O
+of	O
+the	O
+structural	O
+basis	O
+of	O
+catalysis	O
+by	O
+the	O
+metabolosome	B-complex_assembly
+common	O
+core	O
+enzymes	O
+.	O
+
+Structure	B-experimental_method
+Determination	I-experimental_method
+of	O
+PduL	B-protein_type
+
+(	O
+a	O
+)	O
+Primary	O
+and	O
+secondary	O
+structure	O
+of	O
+rPduL	B-protein
+(	O
+tubes	O
+represent	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+,	O
+arrows	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+and	O
+dashed	O
+line	O
+residues	O
+disordered	O
+in	O
+the	O
+structure	B-evidence
+.	O
+
+Coenzyme	B-chemical
+A	I-chemical
+is	O
+shown	O
+in	O
+magenta	O
+sticks	O
+and	O
+Zinc	B-chemical
+(	O
+grey	O
+)	O
+as	O
+spheres	O
+.	O
+
+There	O
+are	O
+two	O
+PduL	B-protein_type
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+of	O
+the	O
+P212121	O
+unit	O
+cell	O
+.	O
+
+Structurally	O
+,	O
+PduL	B-protein_type
+consists	O
+of	O
+two	O
+domains	B-structure_element
+(	O
+Fig	O
+2	O
+,	O
+blue	O
+/	O
+red	O
+),	O
+each	O
+a	O
+beta	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+that	O
+is	O
+capped	O
+on	O
+both	O
+ends	O
+by	O
+short	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+.	O
+
+Residues	O
+in	O
+this	O
+region	O
+(	O
+Gln42	B-residue_name_number
+,	O
+Pro43	B-residue_name_number
+,	O
+Gly44	B-residue_name_number
+),	O
+covering	O
+the	O
+active	B-site
+site	I-site
+,	O
+are	O
+strongly	B-protein_state
+conserved	I-protein_state
+(	O
+Fig	O
+3	O
+).	O
+
+The	O
+position	O
+numbers	O
+shown	O
+correspond	O
+to	O
+the	O
+residue	O
+numbering	O
+of	O
+rPduL	B-protein
+;	O
+note	O
+that	O
+some	O
+positions	O
+in	O
+the	O
+logo	O
+represent	O
+gaps	O
+in	O
+the	O
+rPduL	B-protein
+sequence	O
+.	O
+
+The	O
+folds	O
+of	O
+the	O
+two	O
+chains	O
+in	O
+the	O
+asymmetric	O
+unit	O
+are	O
+very	O
+similar	O
+,	O
+superimposing	B-experimental_method
+with	O
+a	O
+rmsd	B-evidence
+of	O
+0	O
+.	O
+16	O
+Å	O
+over	O
+2	O
+,	O
+306	O
+aligned	O
+atom	O
+pairs	O
+.	O
+
+(	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
+large	O
+differences	O
+between	O
+the	O
+anomalous	O
+signals	O
+confirm	O
+the	O
+presence	O
+of	O
+zinc	B-chemical
+at	O
+both	O
+metal	O
+sites	O
+(	O
+S3	O
+Fig	O
+).	O
+
+Oligomeric	O
+States	O
+of	O
+PduL	B-protein_type
+Orthologs	O
+Are	O
+Influenced	O
+by	O
+the	O
+EP	B-structure_element
+
+It	O
+has	O
+been	O
+proposed	O
+that	O
+the	O
+catabolic	B-protein_state
+BMCs	B-complex_assembly
+may	O
+assemble	O
+in	O
+a	O
+core	O
+-	O
+first	O
+manner	O
+,	O
+with	O
+the	O
+luminal	O
+enzymes	O
+(	O
+signature	O
+enzyme	O
+,	O
+aldehyde	B-protein_type
+,	I-protein_type
+and	I-protein_type
+alcohol	I-protein_type
+dehydrogenases	I-protein_type
+and	O
+the	O
+BMC	B-complex_assembly
+PTAC	B-protein_type
+)	O
+forming	O
+an	O
+initial	O
+bolus	O
+,	O
+or	O
+prometabolosome	O
+,	O
+around	O
+which	O
+a	O
+shell	B-structure_element
+assembles	O
+.	O
+
+However	O
+,	O
+when	O
+the	O
+putative	O
+EP	B-structure_element
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+27	I-residue_range
+)	O
+was	O
+removed	B-experimental_method
+(	O
+sPduL	B-mutant
+ΔEP	I-mutant
+),	O
+the	O
+truncated	B-protein_state
+protein	O
+was	O
+stable	O
+and	O
+eluted	O
+as	O
+a	O
+single	O
+peak	O
+(	O
+Fig	O
+5a	O
+)	O
+consistent	O
+with	O
+the	O
+size	O
+of	O
+a	O
+monomer	B-oligomeric_state
+(	O
+Fig	O
+5d	O
+,	O
+blue	O
+curve	O
+).	O
+
+rPduL	B-protein
+full	B-protein_state
+length	I-protein_state
+runs	O
+as	O
+Mw	B-evidence
+=	O
+140	O
+.	O
+3	O
+kDa	O
++/−	O
+1	O
+.	O
+2	O
+%	O
+and	O
+Mn	B-evidence
+=	O
+140	O
+.	O
+5	O
+kDa	O
++/−	O
+1	O
+.	O
+2	O
+%.	O
+
+Our	O
+structure	B-evidence
+reveals	O
+that	O
+the	O
+two	O
+assigned	O
+PF06130	B-structure_element
+domains	O
+(	O
+Fig	O
+3	O
+)	O
+do	O
+not	O
+form	O
+structurally	O
+discrete	O
+units	O
+;	O
+this	O
+reduces	O
+the	O
+apparent	O
+sequence	O
+conservation	O
+at	O
+the	O
+level	O
+of	O
+primary	O
+structure	O
+.	O
+
+The	O
+closest	O
+structural	O
+homolog	O
+of	O
+the	O
+PduL	B-protein_type
+barrel	B-structure_element
+domain	I-structure_element
+is	O
+a	O
+subdomain	O
+of	O
+a	O
+multienzyme	O
+complex	O
+,	O
+the	O
+alpha	B-structure_element
+subunit	I-structure_element
+of	O
+ethylbenzene	B-protein_type
+dehydrogenase	I-protein_type
+(	O
+S5b	O
+Fig	O
+,	O
+rmsd	B-evidence
+of	O
+2	O
+.	O
+26	O
+Å	O
+over	O
+226	O
+aligned	O
+atoms	O
+consisting	O
+of	O
+one	O
+beta	B-structure_element
+barrel	I-structure_element
+and	O
+one	O
+capping	B-structure_element
+helix	I-structure_element
+).	O
+
+The	O
+PduL	B-protein_type
+signature	O
+primary	O
+structure	O
+,	O
+two	O
+PF06130	B-structure_element
+domains	O
+,	O
+occurs	O
+in	O
+some	O
+multidomain	O
+proteins	O
+,	O
+most	O
+of	O
+them	O
+annotated	O
+as	O
+Acks	B-protein_type
+,	O
+suggesting	O
+that	O
+PduL	B-protein_type
+may	O
+also	O
+replace	O
+Pta	B-protein_type
+in	O
+variants	O
+of	O
+the	O
+phosphotransacetylase	B-protein_type
+-	O
+Ack	B-protein_type
+pathway	O
+.	O
+
+These	O
+PduL	B-protein_type
+homologs	O
+lack	B-protein_state
+EPs	B-structure_element
+,	O
+and	O
+their	B-protein_type
+fusion	O
+to	O
+Ack	B-protein_type
+may	O
+have	O
+evolved	O
+as	O
+a	O
+way	O
+to	O
+facilitate	O
+substrate	O
+channeling	O
+between	O
+the	O
+two	O
+enzymes	O
+.	O
+
+sPduL	B-protein
+has	O
+also	O
+previously	O
+been	O
+reported	O
+to	O
+localize	O
+to	O
+inclusion	O
+bodies	O
+when	O
+overexpressed	B-experimental_method
+;	O
+we	O
+show	O
+here	O
+that	O
+this	O
+is	O
+dependent	O
+on	O
+the	O
+presence	O
+of	O
+the	O
+EP	B-structure_element
+.	O
+
+Structured	O
+aggregation	O
+of	O
+the	O
+core	O
+enzymes	O
+has	O
+been	O
+proposed	O
+to	O
+be	O
+the	O
+initial	O
+step	O
+in	O
+metabolosome	B-complex_assembly
+assembly	O
+and	O
+is	O
+known	O
+to	O
+be	O
+the	O
+first	O
+step	O
+of	O
+β	O
+-	O
+carboxysome	O
+biogenesis	O
+,	O
+where	O
+the	O
+core	O
+enzyme	O
+Ribulose	B-protein_type
+Bisphosphate	I-protein_type
+Carboxylase	I-protein_type
+/	I-protein_type
+Oxygenase	I-protein_type
+(	O
+RuBisCO	B-protein_type
+)	O
+is	O
+aggregated	O
+by	O
+the	O
+CcmM	B-protein_type
+protein	O
+.	O
+
+As	O
+expected	O
+,	O
+the	O
+amino	O
+acid	O
+sequence	O
+conservation	O
+is	O
+highest	O
+in	O
+the	O
+region	O
+around	O
+the	O
+proposed	O
+active	B-site
+site	I-site
+(	O
+Fig	O
+4d	O
+);	O
+highly	B-protein_state
+conserved	I-protein_state
+residues	O
+are	O
+also	O
+involved	O
+in	O
+CoA	B-chemical
+binding	O
+(	O
+Figs	O
+2a	O
+and	O
+3	O
+,	O
+residues	O
+Ser45	B-residue_name_number
+,	O
+Lys70	B-residue_name_number
+,	O
+Arg97	B-residue_name_number
+,	O
+Leu99	B-residue_name_number
+,	O
+His204	B-residue_name_number
+,	O
+Asn211	B-residue_name_number
+).	O
+
+In	O
+contrast	O
+,	O
+in	O
+the	O
+rPduL	B-protein
+structure	B-evidence
+,	O
+there	O
+are	O
+no	O
+conserved	O
+aspartate	B-residue_name
+residues	O
+in	O
+or	O
+around	O
+the	O
+active	B-site
+site	I-site
+,	O
+and	O
+the	O
+only	O
+well	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+glutamate	B-residue_name
+residue	O
+in	O
+the	O
+active	B-site
+site	I-site
+is	O
+involved	O
+in	O
+coordinating	O
+one	O
+of	O
+the	O
+metal	O
+ions	O
+.	O
+
+These	O
+observations	O
+strongly	O
+suggest	O
+that	O
+an	O
+acidic	B-protein_state
+residue	B-structure_element
+is	O
+not	O
+directly	O
+involved	O
+in	O
+catalysis	O
+by	O
+PduL	B-protein_type
+.	O
+Instead	O
+,	O
+the	O
+dimetal	B-site
+active	I-site
+site	I-site
+of	O
+PduL	B-protein_type
+may	O
+create	O
+a	O
+nucleophile	O
+from	O
+one	O
+of	O
+the	O
+hydroxyl	O
+groups	O
+on	O
+free	O
+phosphate	B-chemical
+to	O
+attack	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+the	O
+thioester	O
+bond	O
+of	O
+an	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+.	O
+In	O
+the	O
+reverse	O
+direction	O
+,	O
+the	O
+metal	O
+ion	O
+(	O
+s	O
+)	O
+could	O
+stabilize	O
+the	O
+thiolate	O
+anion	O
+that	O
+would	O
+attack	O
+the	O
+carbonyl	O
+carbon	O
+of	O
+an	O
+acyl	B-chemical
+-	I-chemical
+phosphate	I-chemical
+;	O
+a	O
+similar	O
+mechanism	O
+has	O
+been	O
+described	O
+for	O
+phosphatases	B-protein_type
+where	O
+hydroxyl	O
+groups	O
+or	O
+hydroxide	O
+ions	O
+can	O
+act	O
+as	O
+a	O
+base	O
+when	O
+coordinated	O
+by	O
+a	O
+dimetal	B-site
+active	I-site
+site	I-site
+.	O
+
+Our	O
+structures	B-evidence
+provide	O
+the	O
+foundation	O
+for	O
+studies	O
+to	O
+elucidate	O
+the	O
+details	O
+of	O
+the	O
+catalytic	O
+mechanism	O
+of	O
+PduL	B-protein_type
+.	O
+Conserved	B-protein_state
+residues	O
+in	O
+the	O
+active	B-site
+site	I-site
+that	O
+may	O
+contribute	O
+to	O
+substrate	O
+binding	O
+and	O
+/	O
+or	O
+transition	O
+state	O
+stabilization	O
+include	O
+Ser127	B-residue_name_number
+,	O
+Arg103	B-residue_name_number
+,	O
+Arg194	B-residue_name_number
+,	O
+Gln107	B-residue_name_number
+,	O
+Gln74	B-residue_name_number
+,	O
+and	O
+Gln	B-residue_name_number
+/	O
+Glu77	B-residue_name_number
+.	O
+
+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
+
+PduL	B-protein_type
+and	O
+Pta	B-protein_type
+are	O
+mechanistically	O
+and	O
+structurally	O
+distinct	O
+enzymes	O
+that	O
+catalyze	O
+the	O
+same	O
+reaction	O
+,	O
+a	O
+prime	O
+example	O
+of	O
+evolutionary	O
+convergence	O
+upon	O
+a	O
+function	O
+.	O
+
+However	O
+,	O
+apparently	O
+less	O
+frequent	O
+is	O
+functional	O
+convergence	O
+that	O
+is	O
+supported	O
+by	O
+distinctly	O
+different	O
+active	B-site
+sites	I-site
+and	O
+accordingly	O
+catalytic	O
+mechanism	O
+,	O
+as	O
+revealed	O
+by	O
+comparison	O
+of	O
+the	O
+structures	O
+of	O
+Pta	B-protein_type
+and	O
+PduL	B-protein_type
+.	O
+One	O
+well	O
+-	O
+studied	O
+example	O
+of	O
+this	O
+is	O
+the	O
+β	B-protein_type
+-	I-protein_type
+lactamase	I-protein_type
+family	O
+of	O
+enzymes	O
+,	O
+in	O
+which	O
+the	O
+active	B-site
+site	I-site
+of	O
+Class	O
+A	O
+and	O
+Class	O
+C	O
+enzymes	O
+involve	O
+serine	O
+-	O
+based	O
+catalysis	O
+,	O
+but	O
+Class	O
+B	O
+enzymes	O
+are	O
+metalloproteins	B-protein_type
+.	O
+
+This	O
+is	O
+not	O
+surprising	O
+,	O
+as	O
+β	B-protein_type
+-	I-protein_type
+lactamases	I-protein_type
+are	O
+not	O
+so	O
+widespread	O
+among	O
+bacteria	B-taxonomy_domain
+and	O
+therefore	O
+would	O
+be	O
+expected	O
+to	O
+have	O
+evolved	O
+independently	O
+several	O
+times	O
+as	O
+a	O
+defense	O
+mechanism	O
+against	O
+β	O
+-	O
+lactam	O
+antibiotics	O
+.	O
+
+Cysteine	B-protein_type
+peptidases	I-protein_type
+play	O
+crucial	O
+roles	O
+in	O
+the	O
+virulence	O
+of	O
+bacterial	B-taxonomy_domain
+and	O
+other	O
+eukaryotic	B-taxonomy_domain
+pathogens	O
+.	O
+
+Clostripain	B-protein
+has	O
+been	O
+described	O
+as	O
+an	O
+arginine	B-protein_type
+-	I-protein_type
+specific	I-protein_type
+peptidase	I-protein_type
+with	O
+a	O
+requirement	O
+for	O
+Ca2	B-chemical
++	I-chemical
+and	O
+loss	O
+of	O
+an	O
+internal	B-structure_element
+nonapeptide	I-structure_element
+for	O
+full	B-protein_state
+activation	I-protein_state
+;	O
+lack	O
+of	O
+structural	O
+information	O
+on	O
+the	O
+family	O
+appears	O
+to	O
+have	O
+prohibited	O
+further	O
+investigation	O
+.	O
+
+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
+
+Six	O
+of	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+in	O
+PmC11	B-protein
+(	O
+β1	B-structure_element
+–	I-structure_element
+β2	I-structure_element
+and	O
+β5	B-structure_element
+–	I-structure_element
+β8	I-structure_element
+)	O
+share	O
+the	O
+same	O
+topology	O
+as	O
+the	O
+six	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+found	O
+in	O
+caspases	B-protein_type
+,	O
+with	O
+strands	B-structure_element
+β3	B-structure_element
+,	O
+β4	B-structure_element
+,	O
+and	O
+β9	B-structure_element
+located	O
+on	O
+the	O
+outside	O
+of	O
+this	O
+core	B-structure_element
+structure	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+box	O
+).	O
+
+Summary	O
+of	O
+PDBeFOLD	B-experimental_method
+superposition	I-experimental_method
+of	O
+structures	O
+found	O
+to	O
+be	O
+most	O
+similar	O
+to	O
+PmC11	B-protein
+in	O
+the	O
+PBD	O
+based	O
+on	O
+DaliLite	B-experimental_method
+
+B	O
+,	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+of	O
+PmC11	B-protein
+.	O
+
+PmC11C179A	O
+(	O
+20	O
+μg	O
+)	O
+was	O
+incubated	O
+overnight	O
+at	O
+37	O
+°	O
+C	O
+with	O
+increasing	O
+amounts	O
+of	O
+processed	O
+PmC11	B-protein
+and	O
+analyzed	O
+on	O
+a	O
+10	O
+%	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+gel	O
+.	O
+
+Inactive	O
+PmC11C179A	B-mutant
+was	O
+not	O
+processed	O
+to	O
+a	O
+major	O
+extent	O
+by	O
+active	B-protein_state
+PmC11	B-protein
+until	O
+around	O
+a	O
+ratio	O
+of	O
+1	O
+:	O
+4	O
+(	O
+5	O
+μg	O
+of	O
+active	B-protein_state
+PmC11	B-protein
+).	O
+
+Incubation	B-experimental_method
+of	O
+PmC11	B-protein
+at	O
+37	O
+°	O
+C	O
+for	O
+16	O
+h	O
+,	O
+resulted	O
+in	O
+a	O
+fully	B-protein_state
+processed	I-protein_state
+enzyme	O
+that	O
+remained	O
+as	O
+an	O
+intact	B-protein_state
+monomer	B-oligomeric_state
+when	O
+applied	O
+to	O
+a	O
+size	O
+-	O
+exclusion	O
+column	O
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+
+The	O
+single	O
+cleavage	B-site
+site	I-site
+of	O
+PmC11	B-protein
+at	O
+Lys147	B-residue_name_number
+is	O
+found	O
+immediately	O
+after	O
+α3	B-structure_element
+,	O
+in	O
+loop	B-structure_element
+L5	B-structure_element
+within	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+Figs	O
+.	O
+1	O
+,	O
+A	O
+and	O
+B	O
+,	O
+and	O
+2A	O
+).	O
+
+The	O
+two	O
+ends	O
+of	O
+the	O
+cleavage	B-site
+site	I-site
+are	O
+remarkably	O
+well	O
+ordered	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+and	O
+displaced	O
+from	O
+one	O
+another	O
+by	O
+19	O
+.	O
+5	O
+Å	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+Initial	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+and	O
+Western	B-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+both	O
+mutants	O
+revealed	O
+no	O
+discernible	O
+processing	O
+occurred	O
+as	O
+compared	O
+with	O
+active	B-protein_state
+PmC11	B-protein
+(	O
+Fig	O
+.	O
+2C	O
+).	O
+
+C	O
+,	O
+divalent	O
+cations	O
+do	O
+not	O
+increase	O
+the	O
+activity	O
+of	O
+PmC11	B-protein
+.	O
+
+Furthermore	O
+,	O
+Cu2	B-chemical
++,	I-chemical
+Fe2	B-chemical
++,	I-chemical
+and	O
+Zn2	B-chemical
++	I-chemical
+appear	O
+to	O
+inhibit	B-protein_state
+PmC11	B-protein
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+predicted	O
+primary	O
+S1	B-site
+-	I-site
+binding	I-site
+residue	I-site
+in	O
+PmC11	B-protein
+Asp177	B-residue_name_number
+also	O
+overlays	B-experimental_method
+with	O
+the	O
+residue	O
+predicted	O
+to	O
+be	O
+the	O
+P1	B-site
+specificity	I-site
+determining	I-site
+residue	I-site
+in	O
+clostripain	B-protein
+(	O
+Asp229	B-residue_name_number
+,	O
+Fig	O
+.	O
+1A	O
+).	O
+
+Surprisingly	O
+,	O
+Ca2	B-chemical
++	I-chemical
+did	O
+not	O
+enhance	O
+PmC11	B-protein
+activity	O
+and	O
+,	O
+furthermore	O
+,	O
+other	O
+divalent	O
+cations	O
+,	O
+Mg2	B-chemical
++,	I-chemical
+Mn2	B-chemical
++,	I-chemical
+Co2	B-chemical
++,	I-chemical
+Fe2	B-chemical
++,	I-chemical
+Zn2	B-chemical
++,	I-chemical
+and	O
+Cu2	B-chemical
++,	I-chemical
+were	O
+not	O
+necessary	O
+for	O
+PmC11	B-protein
+activity	O
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+
+In	O
+addition	O
+,	O
+the	O
+structure	B-evidence
+suggested	O
+a	O
+mechanism	O
+of	O
+self	O
+-	O
+inhibition	O
+in	O
+both	O
+PmC11	B-protein
+and	O
+clostripain	B-protein
+and	O
+an	O
+activation	O
+mechanism	O
+that	O
+requires	O
+autoprocessing	B-ptm
+.	O
+
+All	O
+other	O
+clan	B-protein_type
+CD	I-protein_type
+members	I-protein_type
+requiring	O
+cleavage	B-ptm
+for	O
+full	B-protein_state
+activation	I-protein_state
+do	O
+so	O
+at	O
+sites	B-site
+external	O
+to	O
+their	O
+central	O
+sheets	B-structure_element
+.	O
+
+The	O
+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
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+YfiB	B-protein
+alone	B-protein_state
+and	O
+of	O
+an	O
+active	B-protein_state
+mutant	B-protein_state
+YfiBL43P	B-mutant
+complexed	B-protein_state
+with	I-protein_state
+YfiR	B-protein
+with	O
+2	O
+:	O
+2	O
+stoichiometry	O
+.	O
+
+Bis	B-chemical
+-(	I-chemical
+3	I-chemical
+’-	I-chemical
+5	I-chemical
+’)-	I-chemical
+cyclic	I-chemical
+dimeric	I-chemical
+GMP	I-chemical
+(	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+)	O
+is	O
+a	O
+ubiquitous	O
+second	O
+messenger	O
+that	O
+bacteria	B-taxonomy_domain
+use	O
+to	O
+facilitate	O
+behavioral	O
+adaptations	O
+to	O
+their	O
+ever	O
+-	O
+changing	O
+environment	O
+.	O
+
+The	O
+YfiBNR	B-complex_assembly
+system	O
+contains	O
+three	O
+protein	O
+members	O
+and	O
+modulates	O
+intracellular	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+levels	O
+in	O
+response	O
+to	O
+signals	O
+received	O
+in	O
+the	O
+periplasm	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+It	O
+has	O
+been	O
+reported	O
+that	O
+the	O
+activation	O
+of	O
+YfiN	B-protein
+may	O
+be	O
+induced	O
+by	O
+redox	O
+-	O
+driven	O
+misfolding	O
+of	O
+YfiR	B-protein
+(	O
+Giardina	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+It	O
+is	O
+also	O
+proposed	O
+that	O
+the	O
+sequestration	O
+of	O
+YfiR	B-protein
+by	O
+YfiB	B-protein
+can	O
+be	O
+induced	O
+by	O
+certain	O
+YfiB	B-protein
+-	O
+mediated	O
+cell	O
+wall	O
+stress	O
+,	O
+and	O
+mutagenesis	B-experimental_method
+studies	I-experimental_method
+revealed	O
+a	O
+number	O
+of	O
+activation	B-structure_element
+residues	I-structure_element
+of	O
+YfiB	B-protein
+that	O
+were	O
+located	O
+in	O
+close	O
+proximity	O
+to	O
+the	O
+predicted	B-protein_state
+first	B-structure_element
+helix	I-structure_element
+of	O
+the	O
+periplasmic	B-structure_element
+domain	I-structure_element
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+In	O
+the	O
+present	O
+study	O
+,	O
+we	O
+solved	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+an	O
+N	O
+-	O
+terminal	O
+truncated	B-protein_state
+form	O
+of	O
+YfiB	B-protein
+(	O
+34	B-residue_range
+–	I-residue_range
+168	I-residue_range
+)	O
+and	O
+YfiR	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+an	O
+active	B-protein_state
+mutant	B-protein_state
+YfiBL43P	B-mutant
+.	O
+
+Compared	O
+with	O
+the	O
+reported	O
+complex	O
+structure	O
+,	O
+YfiBL43P	B-mutant
+in	O
+our	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+structure	B-evidence
+has	O
+additional	O
+visible	O
+N	O
+-	O
+terminal	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+58	I-residue_range
+that	O
+are	O
+shown	O
+to	O
+play	O
+essential	O
+roles	O
+in	O
+YfiB	B-protein
+activation	O
+and	O
+biofilm	O
+formation	O
+.	O
+
+In	O
+addition	O
+,	O
+we	O
+found	O
+that	O
+the	O
+YfiBL43P	B-mutant
+shows	O
+a	O
+much	O
+higher	O
+PG	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+than	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+,	O
+most	O
+likely	O
+due	O
+to	O
+its	O
+more	O
+compact	O
+PG	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+Overall	O
+structure	B-evidence
+of	O
+YfiB	B-protein
+
+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
+
+As	O
+expected	O
+,	O
+both	O
+mutants	O
+form	O
+a	O
+stable	B-protein_state
+complex	B-protein_state
+with	I-protein_state
+YfiR	B-protein
+.	O
+Finally	O
+,	O
+we	O
+crystalized	B-experimental_method
+YfiR	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+YfiBL43P	B-mutant
+mutant	B-protein_state
+and	O
+solved	O
+the	O
+structure	B-evidence
+at	O
+1	O
+.	O
+78	O
+Å	O
+resolution	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+using	O
+YfiR	B-protein
+and	O
+YfiB	B-protein
+as	O
+models	O
+.	O
+
+The	O
+YfiB	B-site
+-	I-site
+YfiR	I-site
+interface	I-site
+can	O
+be	O
+divided	O
+into	O
+two	O
+regions	O
+(	O
+Fig	O
+.	O
+3A	O
+and	O
+3D	O
+).	O
+
+(	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
+
+Apo	B-protein_state
+YfiB	B-protein
+is	O
+shown	O
+in	O
+yellow	O
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+in	O
+cyan	O
+.	O
+(	O
+E	O
+and	O
+F	O
+)	O
+MST	B-experimental_method
+data	O
+and	O
+analysis	O
+for	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+(	O
+E	O
+)	O
+YfiB	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+(	O
+F	O
+)	O
+YfiBL43P	B-mutant
+with	O
+PG	B-chemical
+.	O
+(	O
+G	O
+)	O
+The	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+and	O
+E	B-species
+.	I-species
+coli	I-species
+sources	O
+of	O
+YfiB	B-protein
+,	O
+Pal	B-protein_type
+and	O
+the	O
+periplasmic	B-structure_element
+domain	I-structure_element
+of	O
+OmpA	B-protein_type
+
+In	O
+parallel	O
+,	O
+to	O
+better	O
+understand	O
+the	O
+putative	O
+functional	O
+role	O
+of	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+,	O
+yfiB	B-gene
+was	O
+deleted	B-experimental_method
+in	O
+a	O
+PAO1	B-species
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+strain	O
+,	O
+and	O
+a	O
+construct	B-experimental_method
+expressing	I-experimental_method
+the	O
+YfiBL43P	B-mutant
+mutant	B-protein_state
+was	O
+transformed	B-experimental_method
+into	I-experimental_method
+the	O
+PAO1	B-species
+ΔyfiB	B-mutant
+strain	O
+to	O
+trigger	O
+YfiBL43P	B-mutant
+-	O
+induced	O
+biofilm	O
+formation	O
+.	O
+
+To	O
+answer	O
+the	O
+question	O
+whether	O
+competition	O
+of	O
+VB6	B-chemical
+or	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+for	O
+the	O
+YfiB	B-protein
+F57	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+of	O
+YfiR	B-protein
+plays	O
+an	O
+essential	O
+role	O
+in	O
+inhibiting	O
+biofilm	O
+formation	O
+,	O
+we	O
+measured	O
+the	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+for	O
+YfiR	B-protein
+via	O
+BIAcore	B-experimental_method
+experiments	O
+.	O
+
+In	O
+addition	O
+to	O
+the	O
+preceding	B-residue_range
+8	I-residue_range
+aa	I-residue_range
+loop	B-structure_element
+(	O
+from	O
+the	O
+lipid	O
+acceptor	O
+Cys26	B-residue_range
+to	I-residue_range
+Gly34	I-residue_range
+),	O
+the	O
+full	B-protein_state
+length	I-protein_state
+of	O
+the	O
+periplasmic	O
+portion	O
+of	O
+apo	B-protein_state
+YfiB	B-protein
+can	O
+reach	O
+approximately	O
+60	O
+Å	O
+.	O
+It	O
+was	O
+reported	O
+that	O
+the	O
+distance	O
+between	O
+the	O
+outer	O
+membrane	O
+and	O
+the	O
+cell	O
+wall	O
+is	O
+approximately	O
+50	O
+Å	O
+and	O
+that	O
+the	O
+thickness	O
+of	O
+the	O
+PG	O
+layer	O
+is	O
+approximately	O
+70	O
+Å	O
+(	O
+Matias	O
+et	O
+al	O
+.,).	O
+
+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
+
+Regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	B-complex_assembly
+tripartite	B-protein_state
+system	O
+.	O
+
+The	O
+lipid	O
+acceptor	O
+Cys26	B-residue_name_number
+is	O
+indicated	O
+as	O
+blue	O
+ball	O
+.	O
+
+The	O
+loop	B-structure_element
+connecting	O
+Cys26	B-residue_name_number
+and	O
+Gly34	B-residue_name_number
+of	O
+YfiB	B-protein
+is	O
+modeled	O
+.	O
+
+Once	O
+activated	B-protein_state
+by	O
+certain	O
+cell	O
+stress	O
+,	O
+the	O
+dimeric	B-oligomeric_state
+YfiB	B-protein
+transforms	O
+from	O
+a	O
+compact	B-protein_state
+conformation	I-protein_state
+to	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+,	O
+allowing	O
+the	O
+periplasmic	B-structure_element
+domain	I-structure_element
+of	O
+the	O
+membrane	B-protein_state
+-	I-protein_state
+anchored	I-protein_state
+YfiB	B-protein
+to	O
+penetrate	O
+the	O
+cell	O
+wall	O
+and	O
+sequester	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+,	O
+thus	O
+relieving	O
+the	O
+repression	O
+of	O
+YfiN	B-protein
+
+These	O
+results	O
+,	O
+together	O
+with	O
+our	O
+observation	O
+that	O
+activated	B-protein_state
+YfiB	B-protein
+has	O
+a	O
+much	O
+higher	O
+cell	B-evidence
+wall	I-evidence
+binding	I-evidence
+affinity	I-evidence
+,	O
+and	O
+previous	O
+mutagenesis	O
+data	O
+showing	O
+that	O
+(	O
+1	O
+)	O
+both	O
+PG	B-chemical
+binding	O
+and	O
+membrane	O
+anchoring	O
+are	O
+required	O
+for	O
+YfiB	B-protein
+activity	O
+and	O
+(	O
+2	O
+)	O
+activating	O
+mutations	O
+possessing	O
+an	O
+altered	O
+N	O
+-	O
+terminal	O
+loop	B-structure_element
+length	O
+are	O
+dominant	O
+over	O
+the	O
+loss	O
+of	O
+PG	B-chemical
+binding	O
+(	O
+Malone	O
+et	O
+al	O
+.,),	O
+suggest	O
+an	O
+updated	O
+regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	B-complex_assembly
+system	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+
+In	O
+this	O
+model	O
+,	O
+in	O
+response	O
+to	O
+a	O
+particular	O
+cell	O
+stress	O
+that	O
+is	O
+yet	O
+to	O
+be	O
+identified	O
+,	O
+the	O
+dimeric	B-oligomeric_state
+YfiB	B-protein
+is	O
+activated	B-protein_state
+from	O
+a	O
+compact	B-protein_state
+,	O
+inactive	B-protein_state
+conformation	B-protein_state
+to	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+,	O
+which	O
+possesses	O
+increased	O
+PG	B-chemical
+binding	O
+affinity	O
+.	O
+
+A	O
+regulatory	B-structure_element
+loop	I-structure_element
+,	O
+which	O
+is	O
+phosphorylated	B-protein_state
+at	O
+the	O
+key	O
+functional	O
+phosphorylation	B-site
+site	I-site
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+wedges	O
+into	O
+a	O
+crevice	O
+between	O
+two	O
+domains	O
+of	O
+CD	B-structure_element
+.	O
+
+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
+
+By	O
+catalysing	O
+this	O
+rate	O
+-	O
+limiting	O
+step	O
+in	O
+fatty	O
+-	O
+acid	O
+biosynthesis	O
+,	O
+ACC	B-protein_type
+plays	O
+a	O
+key	O
+role	O
+in	O
+anabolic	O
+metabolism	O
+.	O
+
+The	O
+principal	O
+functional	O
+protein	O
+components	O
+of	O
+ACCs	B-protein_type
+have	O
+been	O
+described	O
+already	O
+in	O
+the	O
+late	O
+1960s	O
+for	O
+Escherichia	B-species
+coli	I-species
+(	O
+E	B-species
+.	I-species
+coli	I-species
+)	O
+ACC	B-protein_type
+:	O
+Biotin	B-protein_type
+carboxylase	I-protein_type
+(	O
+BC	B-protein_type
+)	O
+catalyses	O
+the	O
+ATP	B-chemical
+-	O
+dependent	O
+carboxylation	O
+of	O
+a	O
+biotin	B-chemical
+moiety	O
+,	O
+which	O
+is	O
+covalently	O
+linked	O
+to	O
+the	O
+biotin	B-protein_type
+carboxyl	I-protein_type
+carrier	I-protein_type
+protein	I-protein_type
+(	O
+BCCP	B-protein_type
+).	O
+
+Human	B-species
+ACC	B-protein_type
+occurs	O
+in	O
+two	O
+closely	O
+related	O
+isoforms	B-protein_state
+,	O
+ACC1	B-protein
+and	O
+2	B-protein
+,	O
+located	O
+in	O
+the	O
+cytosol	O
+and	O
+at	O
+the	O
+outer	O
+mitochondrial	O
+membrane	O
+,	O
+respectively	O
+.	O
+
+However	O
+,	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+individual	O
+components	O
+or	O
+domains	O
+from	O
+prokaryotic	B-taxonomy_domain
+and	O
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+,	O
+respectively	O
+,	O
+have	O
+been	O
+solved	O
+.	O
+
+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
+
+Despite	O
+the	O
+outstanding	O
+relevance	O
+of	O
+ACC	B-protein_type
+in	O
+primary	O
+metabolism	O
+and	O
+disease	O
+,	O
+the	O
+dynamic	O
+organization	O
+and	O
+regulation	O
+of	O
+the	O
+giant	O
+eukaryotic	B-taxonomy_domain
+,	O
+and	O
+in	O
+particular	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+remain	O
+poorly	O
+characterized	O
+.	O
+
+First	O
+,	O
+we	O
+focused	O
+on	O
+structure	B-experimental_method
+determination	I-experimental_method
+of	O
+the	O
+82	O
+-	O
+kDa	O
+CD	B-structure_element
+.	O
+
+The	O
+overall	O
+extent	O
+of	O
+the	O
+SceCD	B-species
+is	O
+70	O
+by	O
+75	O
+Å	O
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+,	O
+b	O
+),	O
+and	O
+the	O
+attachment	O
+points	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+26	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+linker	I-structure_element
+to	O
+the	O
+BCCP	B-structure_element
+domain	O
+and	O
+the	O
+C	O
+-	O
+terminal	O
+CT	B-structure_element
+domain	O
+are	O
+separated	O
+by	O
+46	O
+Å	O
+(	O
+the	O
+N	O
+-	O
+and	O
+C	O
+termini	O
+are	O
+indicated	O
+with	O
+spheres	O
+in	O
+Fig	O
+.	O
+1b	O
+).	O
+
+On	O
+the	O
+basis	O
+of	O
+a	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+of	O
+main	O
+chain	O
+atom	O
+positions	O
+of	O
+2	O
+.	O
+2	O
+Å	O
+,	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+are	O
+structurally	O
+more	O
+closely	O
+related	O
+to	O
+each	O
+other	O
+than	O
+to	O
+any	O
+other	O
+protein	O
+(	O
+Fig	O
+.	O
+1c	O
+);	O
+they	O
+may	O
+thus	O
+have	O
+evolved	O
+by	O
+duplication	O
+.	O
+
+Additional	O
+phosphorylation	B-ptm
+was	O
+detected	O
+for	O
+Ser2101	B-residue_name_number
+and	O
+Tyr2179	B-residue_name_number
+;	O
+however	O
+,	O
+these	O
+sites	O
+are	O
+neither	B-protein_state
+conserved	I-protein_state
+across	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+nor	B-protein_state
+natively	I-protein_state
+phosphorylated	I-protein_state
+in	O
+yeast	B-taxonomy_domain
+.	O
+
+MS	B-experimental_method
+analysis	O
+of	O
+dissolved	B-experimental_method
+crystals	I-experimental_method
+confirmed	O
+the	O
+phosphorylated	B-protein_state
+state	O
+of	O
+Ser1157	B-residue_name_number
+also	O
+in	O
+SceCD	B-species
+crystals	B-evidence
+.	O
+
+Owing	O
+to	O
+the	O
+limited	O
+resolution	O
+the	O
+discussion	O
+of	O
+structures	B-evidence
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+CthΔBCCP	B-mutant
+is	O
+restricted	O
+to	O
+the	O
+analysis	O
+of	O
+domain	O
+localization	O
+.	O
+
+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
+
+The	O
+CDN	B-structure_element
+domain	O
+positioning	O
+relative	O
+to	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+is	O
+highly	O
+variable	O
+with	O
+three	O
+main	O
+orientations	O
+observed	O
+in	O
+the	O
+structures	B-evidence
+of	O
+SceCD	B-species
+and	O
+the	O
+larger	B-mutant
+CthACC	I-mutant
+fragments	I-mutant
+:	O
+CDN	B-structure_element
+tilts	O
+,	O
+resulting	O
+in	O
+a	O
+displacement	O
+of	O
+its	O
+N	O
+terminus	O
+by	O
+23	O
+Å	O
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+observed	O
+in	O
+both	O
+protomers	B-oligomeric_state
+of	O
+CthCD	B-mutant
+-	I-mutant
+CT	I-mutant
+and	O
+one	O
+protomer	B-oligomeric_state
+of	O
+CthΔBCCP	B-mutant
+,	O
+denoted	O
+as	O
+CthCD	B-mutant
+-	I-mutant
+CT1	I-mutant
+/	I-mutant
+2	I-mutant
+and	O
+CthΔBCCP1	B-mutant
+,	O
+respectively	O
+).	O
+
+Most	O
+recently	O
+,	O
+a	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+near	B-protein_state
+full	I-protein_state
+-	I-protein_state
+length	I-protein_state
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+ACC	B-protein_type
+from	O
+S	B-species
+.	I-species
+cerevisae	I-species
+(	O
+lacking	B-protein_state
+only	I-protein_state
+21	B-residue_range
+N	O
+-	O
+terminal	O
+amino	O
+acids	O
+,	O
+here	O
+denoted	O
+as	O
+flACC	B-mutant
+)	O
+was	O
+published	O
+by	O
+Wei	O
+and	O
+Tong	O
+.	O
+
+In	O
+flACC	B-mutant
+,	O
+the	O
+ACC	B-protein_type
+dimer	B-oligomeric_state
+obeys	O
+twofold	O
+symmetry	O
+and	O
+assembles	O
+in	O
+a	O
+triangular	B-protein_state
+architecture	I-protein_state
+with	O
+dimeric	B-oligomeric_state
+BC	B-structure_element
+domains	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+).	O
+
+Comparison	B-experimental_method
+of	O
+flACC	B-mutant
+with	O
+our	O
+CthΔBCCP	B-mutant
+structure	B-evidence
+reveals	O
+the	O
+CDC2	B-structure_element
+/	I-structure_element
+CT	I-structure_element
+hinge	I-structure_element
+as	O
+a	O
+major	O
+contributor	O
+to	O
+conformational	O
+flexibility	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+
+In	O
+flACC	B-mutant
+,	O
+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
+
+Applying	B-experimental_method
+the	O
+conformation	O
+of	O
+the	O
+CDC1	B-structure_element
+/	I-structure_element
+CDC2	I-structure_element
+hinge	I-structure_element
+observed	O
+in	O
+SceCD	B-species
+on	O
+flACC	B-mutant
+leads	O
+to	O
+CDN	B-structure_element
+sterically	O
+clashing	O
+with	O
+CDC2	B-structure_element
+and	O
+BT	B-structure_element
+/	O
+CDN	B-structure_element
+clashing	O
+with	O
+CT	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+).	O
+
+In	O
+addition	O
+,	O
+EM	B-experimental_method
+micrographs	B-evidence
+of	O
+phosphorylated	B-protein_state
+and	O
+dephosphorylated	B-protein_state
+SceACC	B-protein
+display	O
+for	O
+both	O
+samples	O
+mainly	O
+elongated	B-protein_state
+and	I-protein_state
+U	I-protein_state
+-	I-protein_state
+shaped	I-protein_state
+conformations	I-protein_state
+and	O
+reveal	O
+no	O
+apparent	O
+differences	O
+in	O
+particle	B-evidence
+shape	I-evidence
+distributions	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+
+It	O
+disfavours	O
+the	O
+adoption	O
+of	O
+a	O
+rare	B-protein_state
+,	I-protein_state
+compact	I-protein_state
+conformation	I-protein_state
+,	O
+in	O
+which	O
+intramolecular	O
+dimerization	O
+of	O
+the	O
+BC	B-structure_element
+domains	O
+results	O
+in	O
+catalytic	O
+turnover	O
+.	O
+
+Cartoon	O
+representation	O
+of	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+multidomain	B-mutant
+constructs	I-mutant
+of	O
+CthACC	B-protein
+.	O
+
+(	O
+a	O
+)	O
+Hinge	B-structure_element
+properties	O
+of	O
+the	O
+CDC2	B-structure_element
+–	I-structure_element
+CT	I-structure_element
+connection	I-structure_element
+analysed	O
+by	O
+a	O
+CT	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+superposition	I-experimental_method
+of	O
+eight	O
+instances	O
+of	O
+the	O
+CDC2	B-mutant
+-	I-mutant
+CT	I-mutant
+segment	I-mutant
+.	O
+
+The	O
+range	O
+of	O
+hinge	O
+bending	O
+is	O
+indicated	O
+and	O
+the	O
+connection	O
+points	O
+between	O
+CDC2	B-structure_element
+and	O
+CT	B-structure_element
+(	O
+blue	O
+)	O
+as	O
+well	O
+as	O
+between	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+(	O
+green	O
+and	O
+grey	O
+)	O
+are	O
+marked	O
+as	O
+spheres	O
+.	O
+
+The	O
+conformational	O
+dynamics	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+Comparison	B-experimental_method
+with	O
+each	O
+other	O
+and	O
+with	O
+available	O
+structures	B-evidence
+uncovers	O
+differences	O
+between	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+explaining	O
+why	O
+only	O
+the	O
+acid	B-protein_type
+stress	I-protein_type
+response	I-protein_type
+enzyme	I-protein_type
+is	O
+capable	O
+of	O
+binding	O
+RavA	B-protein
+.	O
+We	O
+identify	O
+interdomain	O
+movements	O
+associated	O
+with	O
+the	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+with	O
+the	O
+RavA	B-protein
+binding	O
+.	O
+
+Enterobacterial	B-taxonomy_domain
+inducible	B-protein_state
+decarboxylases	B-protein_type
+of	O
+basic	B-protein_state
+amino	B-chemical
+acids	I-chemical
+lysine	B-residue_name
+,	O
+arginine	B-residue_name
+and	O
+ornithine	B-residue_name
+have	O
+a	O
+common	O
+evolutionary	O
+origin	O
+and	O
+belong	O
+to	O
+the	O
+α	B-protein_type
+-	I-protein_type
+family	I-protein_type
+of	O
+pyridoxal	B-chemical
+-	I-chemical
+5	I-chemical
+′-	I-chemical
+phosphate	I-chemical
+(	O
+PLP	B-chemical
+)-	O
+dependent	O
+enzymes	O
+.	O
+
+Each	O
+decarboxylase	B-protein_type
+is	O
+induced	O
+by	O
+an	O
+excess	O
+of	O
+the	O
+target	O
+amino	B-chemical
+acid	I-chemical
+and	O
+a	O
+specific	O
+range	O
+of	O
+extracellular	O
+pH	O
+,	O
+and	O
+works	O
+in	O
+conjunction	O
+with	O
+a	O
+cognate	O
+inner	B-protein_type
+membrane	I-protein_type
+antiporter	I-protein_type
+.	O
+
+In	O
+particular	O
+,	O
+the	O
+inducible	B-protein_state
+lysine	B-protein_type
+decarboxylase	I-protein_type
+LdcI	B-protein
+(	O
+or	O
+CadA	B-protein
+)	O
+attracts	O
+attention	O
+due	O
+to	O
+its	O
+broad	B-protein_state
+pH	I-protein_state
+range	I-protein_state
+of	O
+activity	O
+and	O
+its	O
+capacity	O
+to	O
+promote	O
+survival	O
+and	O
+growth	O
+of	O
+pathogenic	O
+enterobacteria	B-taxonomy_domain
+such	O
+as	O
+Salmonella	B-species
+enterica	I-species
+serovar	I-species
+Typhimurium	I-species
+,	O
+Vibrio	B-species
+cholerae	I-species
+and	O
+Vibrio	B-species
+vulnificus	I-species
+under	O
+acidic	O
+conditions	O
+.	O
+
+This	O
+effect	O
+is	O
+attributed	O
+to	O
+cadaverine	B-chemical
+,	O
+the	O
+diamine	O
+produced	O
+by	O
+decarboxylation	O
+of	O
+lysine	B-residue_name
+by	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+,	O
+that	O
+was	O
+shown	O
+to	O
+enhance	O
+UPEC	B-species
+colonisation	O
+of	O
+the	O
+bladder	O
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+LdcI	B-protein
+as	O
+well	O
+as	O
+its	O
+low	O
+resolution	O
+characterisation	O
+by	O
+electron	B-experimental_method
+microscopy	I-experimental_method
+(	O
+EM	B-experimental_method
+)	O
+showed	O
+that	O
+it	O
+is	O
+a	O
+decamer	B-oligomeric_state
+made	O
+of	O
+two	O
+pentameric	B-oligomeric_state
+rings	B-structure_element
+.	O
+
+Each	O
+monomer	B-oligomeric_state
+is	O
+composed	O
+of	O
+three	O
+domains	O
+–	O
+an	O
+N	O
+-	O
+terminal	O
+wing	B-structure_element
+domain	I-structure_element
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+129	I-residue_range
+),	O
+a	O
+PLP	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+core	I-structure_element
+domain	I-structure_element
+(	O
+residues	O
+130	B-residue_range
+–	I-residue_range
+563	I-residue_range
+),	O
+and	O
+a	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+CTD	B-structure_element
+,	O
+residues	O
+564	B-residue_range
+–	I-residue_range
+715	I-residue_range
+).	O
+
+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
+
+Based	O
+on	O
+these	O
+reconstructions	B-evidence
+,	O
+reliable	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+the	O
+three	O
+assemblies	O
+were	O
+obtained	O
+by	O
+flexible	B-experimental_method
+fitting	I-experimental_method
+of	I-experimental_method
+either	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+LdcIi	B-protein
+or	O
+a	O
+derived	O
+structural	B-experimental_method
+homology	I-experimental_method
+model	I-experimental_method
+of	O
+LdcC	B-protein
+(	O
+Table	O
+S1	O
+).	O
+
+As	O
+a	O
+first	O
+step	O
+of	O
+a	O
+comparative	O
+analysis	O
+,	O
+we	O
+superimposed	B-experimental_method
+the	O
+three	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+(	O
+LdcIa	B-protein
+,	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+and	O
+LdcC	B-protein
+)	O
+and	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+LdcIi	B-protein
+decamer	B-oligomeric_state
+(	O
+Fig	O
+.	O
+2	O
+and	O
+Movie	O
+S1	O
+).	O
+
+This	O
+superposition	B-experimental_method
+reveals	O
+that	O
+the	O
+densities	B-evidence
+lining	O
+the	O
+central	B-structure_element
+hole	I-structure_element
+of	O
+the	O
+toroid	B-structure_element
+are	O
+roughly	O
+at	O
+the	O
+same	O
+location	O
+,	O
+while	O
+the	O
+rest	O
+of	O
+the	O
+structure	B-evidence
+exhibits	O
+noticeable	O
+changes	O
+.	O
+
+In	O
+addition	O
+,	O
+our	O
+earlier	O
+biochemical	B-experimental_method
+observation	I-experimental_method
+that	O
+the	O
+enzymatic	O
+activity	O
+of	O
+LdcIa	B-protein
+is	O
+unaffected	O
+by	O
+RavA	B-protein
+binding	O
+is	O
+consistent	O
+with	O
+the	O
+relatively	O
+small	O
+changes	O
+undergone	O
+by	O
+the	O
+active	B-site
+site	I-site
+upon	O
+transition	O
+from	O
+LdcIa	B-protein
+to	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+.	O
+
+Rearrangements	O
+of	O
+the	O
+ppGpp	B-site
+binding	I-site
+pocket	I-site
+upon	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+LARA	B-structure_element
+binding	O
+
+Indeed	O
+,	O
+all	O
+CTDs	B-structure_element
+have	O
+very	O
+similar	O
+structures	O
+(	O
+RMSDmin	B-evidence
+<	O
+1	O
+Å	O
+).	O
+
+In	O
+our	O
+previous	O
+contribution	O
+,	O
+based	O
+on	O
+the	O
+fit	O
+of	O
+the	O
+LdcIi	B-protein
+and	O
+the	O
+LARA	B-structure_element
+crystal	B-evidence
+structures	I-evidence
+into	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+cryoEM	B-experimental_method
+density	B-evidence
+,	O
+we	O
+predicted	O
+that	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+interaction	O
+should	O
+involve	O
+the	O
+C	O
+-	O
+terminal	O
+two	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+the	O
+LdcI	B-protein
+.	O
+Our	O
+present	O
+cryoEM	B-experimental_method
+maps	B-evidence
+and	O
+pseudoatomic	B-evidence
+models	I-evidence
+provide	O
+first	O
+structure	O
+-	O
+based	O
+insights	O
+into	O
+the	O
+differences	O
+between	O
+the	O
+inducible	B-protein_state
+and	O
+the	O
+constitutive	B-protein_state
+lysine	B-protein_type
+decarboxylases	I-protein_type
+.	O
+
+Our	O
+current	O
+analysis	O
+shows	O
+that	O
+Y697	B-residue_name_number
+is	O
+strictly	B-protein_state
+conserved	I-protein_state
+in	O
+the	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+group	O
+whereas	O
+the	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+enzymes	O
+always	B-protein_state
+have	I-protein_state
+a	O
+lysine	B-residue_name
+in	O
+this	O
+position	O
+;	O
+it	O
+also	O
+uncovers	O
+several	O
+other	O
+residues	O
+potentially	O
+essential	O
+for	O
+the	O
+interaction	O
+with	O
+RavA	B-protein
+which	O
+can	O
+now	O
+be	O
+addressed	O
+by	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+.	O
+
+The	O
+conformational	O
+rearrangements	O
+of	O
+LdcI	B-protein
+upon	O
+enzyme	O
+activation	O
+and	O
+RavA	B-protein
+binding	O
+revealed	O
+in	O
+this	O
+work	O
+,	O
+and	O
+our	O
+amazing	O
+finding	O
+that	O
+the	O
+molecular	O
+determinant	O
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+interaction	O
+is	O
+the	O
+one	O
+that	O
+straightforwardly	O
+determines	O
+if	O
+a	O
+particular	O
+enterobacterial	B-taxonomy_domain
+lysine	B-protein_type
+decarboxylase	I-protein_type
+belongs	O
+to	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+or	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+proteins	O
+,	O
+should	O
+give	O
+a	O
+new	O
+impetus	O
+to	O
+functional	O
+studies	O
+of	O
+the	O
+unique	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+.	O
+
+3D	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+of	O
+LdcC	B-protein
+,	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+and	O
+LdcIa	B-protein
+.	O
+
+Only	O
+one	O
+of	O
+the	O
+two	O
+rings	B-structure_element
+of	O
+the	O
+double	B-structure_element
+toroid	I-structure_element
+is	O
+shown	O
+for	O
+clarity	O
+.	O
+
+The	O
+PLP	B-chemical
+moieties	O
+of	O
+the	O
+cartoon	O
+ring	B-structure_element
+are	O
+shown	O
+in	O
+red	O
+.	O
+
+Stretching	O
+of	O
+the	O
+LdcI	B-protein
+monomer	B-oligomeric_state
+upon	O
+pH	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+enzyme	O
+activation	O
+and	O
+LARA	B-structure_element
+binding	O
+.	O
+
+(	O
+D	O
+–	O
+F	O
+)	O
+Inserts	O
+zooming	O
+at	O
+the	O
+CTD	B-structure_element
+part	O
+in	O
+proximity	O
+of	O
+the	O
+LARA	B-site
+binding	I-site
+site	I-site
+.	O
+
+(	O
+A	O
+)	O
+Maximum	B-evidence
+likelihood	I-evidence
+tree	I-evidence
+with	O
+the	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+and	O
+the	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+groups	O
+highlighted	O
+in	O
+green	O
+and	O
+pink	O
+,	O
+respectively	O
+.	O
+
+Numbering	O
+as	O
+in	O
+E	B-species
+.	I-species
+coli	I-species
+.	O
+
+(	O
+C	O
+)	O
+Signature	O
+sequences	O
+of	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+in	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+.	O
+
+Polarity	O
+differences	O
+are	O
+highlighted	O
+.	O
+(	O
+D	O
+)	O
+Position	O
+and	O
+nature	O
+of	O
+these	O
+differences	O
+at	O
+the	O
+surface	O
+of	O
+the	O
+respective	O
+cryoEM	B-experimental_method
+maps	B-evidence
+with	O
+the	O
+color	O
+code	O
+as	O
+in	O
+B	O
+.	O
+See	O
+also	O
+Fig	O
+.	O
+S7	O
+and	O
+Tables	O
+S3	O
+and	O
+S4	O
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+phosphorylation	B-protein_state
+-	I-protein_state
+mimicking	I-protein_state
+Mep2	B-mutant
+variants	I-mutant
+from	O
+C	B-species
+.	I-species
+albicans	I-species
+show	O
+large	O
+conformational	O
+changes	O
+in	O
+a	O
+conserved	B-protein_state
+and	O
+functionally	O
+important	O
+region	O
+of	O
+the	O
+CTR	B-structure_element
+.	O
+
+One	O
+of	O
+the	O
+most	O
+important	O
+unresolved	O
+questions	O
+in	O
+the	O
+field	O
+is	O
+how	O
+the	O
+transceptors	B-protein_type
+couple	O
+to	O
+downstream	O
+signalling	O
+pathways	O
+.	O
+
+One	O
+hypothesis	O
+is	O
+that	O
+downstream	O
+signalling	O
+is	O
+dependent	O
+on	O
+a	O
+specific	O
+conformation	O
+of	O
+the	O
+transporter	B-protein_type
+.	O
+
+Mep2	B-protein_type
+(	B-protein_type
+methylammonium	I-protein_type
+(	I-protein_type
+MA	I-protein_type
+)	I-protein_type
+permease	I-protein_type
+)	I-protein_type
+proteins	I-protein_type
+are	O
+ammonium	B-protein_type
+transceptors	I-protein_type
+that	O
+are	O
+ubiquitous	O
+in	O
+fungi	B-taxonomy_domain
+.	O
+
+By	O
+contrast	O
+,	O
+several	O
+bacterial	B-taxonomy_domain
+Amt	B-protein_type
+orthologues	O
+have	O
+been	O
+characterized	O
+in	O
+detail	O
+via	O
+high	O
+-	O
+resolution	O
+crystal	B-evidence
+structures	I-evidence
+and	O
+a	O
+number	O
+of	O
+molecular	B-experimental_method
+dynamics	I-experimental_method
+(	O
+MD	B-experimental_method
+)	O
+studies	O
+.	O
+
+The	O
+structures	B-evidence
+are	O
+similar	O
+to	O
+each	O
+other	O
+but	O
+show	O
+considerable	O
+differences	O
+to	O
+all	O
+other	O
+ammonium	B-protein_type
+transporter	I-protein_type
+structures	B-evidence
+.	O
+
+The	O
+putative	O
+phosphorylation	B-site
+site	I-site
+is	O
+solvent	B-protein_state
+accessible	I-protein_state
+and	O
+located	O
+in	O
+a	O
+negatively	B-site
+charged	I-site
+pocket	I-site
+∼	O
+30	O
+Å	O
+away	O
+from	O
+the	O
+channel	B-site
+exit	I-site
+.	O
+
+In	O
+the	O
+remainder	O
+of	O
+the	O
+manuscript	O
+,	O
+we	O
+will	O
+specifically	O
+discuss	O
+CaMep2	B-protein
+due	O
+to	O
+the	O
+superior	O
+resolution	O
+of	O
+the	O
+structure	B-evidence
+.	O
+
+Moreover	O
+,	O
+the	O
+N	O
+terminus	O
+of	O
+one	O
+monomer	B-oligomeric_state
+interacts	O
+with	O
+the	O
+extended	O
+extracellular	B-structure_element
+loop	I-structure_element
+ECL5	B-structure_element
+of	O
+a	O
+neighbouring	O
+monomer	B-oligomeric_state
+.	O
+
+Together	O
+with	O
+additional	O
+,	O
+smaller	O
+differences	O
+in	O
+other	O
+extracellular	B-structure_element
+loops	I-structure_element
+,	O
+these	O
+changes	O
+generate	O
+a	O
+distinct	O
+vestibule	B-structure_element
+leading	O
+to	O
+the	O
+ammonium	B-site
+binding	I-site
+site	I-site
+that	O
+is	O
+much	O
+more	O
+pronounced	O
+than	O
+in	O
+the	O
+bacterial	B-taxonomy_domain
+proteins	O
+.	O
+
+However	O
+,	O
+given	O
+that	O
+an	O
+N	O
+-	O
+terminal	O
+deletion	B-protein_state
+mutant	I-protein_state
+(	O
+2	B-mutant
+-	I-mutant
+27Δ	I-mutant
+)	O
+grows	O
+as	O
+well	O
+as	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+Mep2	B-protein
+on	O
+minimal	O
+ammonium	B-chemical
+medium	O
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+),	O
+the	O
+importance	O
+of	O
+the	O
+N	O
+terminus	O
+for	O
+Mep2	B-protein
+activity	O
+is	O
+not	O
+clear	O
+.	O
+
+In	O
+the	O
+vicinity	O
+of	O
+the	O
+Mep2	B-protein
+channel	B-site
+exit	I-site
+,	O
+the	O
+cytoplasmic	O
+end	O
+of	O
+TM2	B-structure_element
+has	O
+unwound	O
+,	O
+generating	O
+a	O
+longer	O
+ICL1	B-structure_element
+even	O
+though	O
+there	O
+are	O
+no	O
+insertions	O
+in	O
+this	O
+region	O
+compared	O
+to	O
+the	O
+bacterial	B-taxonomy_domain
+proteins	O
+(	O
+Figs	O
+2	O
+and	O
+4	O
+).	O
+
+At	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+TM1	B-structure_element
+,	O
+the	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+group	O
+of	O
+the	O
+relatively	B-protein_state
+conserved	I-protein_state
+Tyr49	B-residue_name_number
+(	O
+Tyr53	B-residue_name_number
+in	O
+ScMep2	B-protein
+)	O
+makes	O
+a	O
+strong	O
+hydrogen	O
+bond	O
+with	O
+the	O
+ɛ2	O
+nitrogen	O
+atom	O
+of	O
+the	O
+absolutely	B-protein_state
+conserved	I-protein_state
+His342	B-residue_name_number
+of	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+(	O
+His348	B-residue_name_number
+in	O
+ScMep2	B-protein
+),	O
+closing	O
+the	O
+channel	B-site
+(	O
+Figs	O
+4	O
+and	O
+5	O
+).	O
+
+Compared	O
+with	O
+ICL1	B-structure_element
+,	O
+the	O
+backbone	O
+conformational	O
+changes	O
+observed	O
+for	O
+the	O
+neighbouring	O
+ICL2	B-structure_element
+are	O
+smaller	O
+,	O
+but	O
+large	O
+shifts	O
+are	O
+nevertheless	O
+observed	O
+for	O
+the	O
+conserved	B-protein_state
+residues	O
+Glu140	B-residue_name_number
+and	O
+Arg141	B-residue_name_number
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+The	O
+closed	B-protein_state
+state	O
+of	O
+the	O
+channel	B-site
+might	O
+also	O
+explain	O
+why	O
+no	B-evidence
+density	I-evidence
+,	O
+which	O
+could	O
+correspond	O
+to	O
+ammonium	B-chemical
+(	O
+or	O
+water	B-chemical
+),	O
+is	O
+observed	O
+in	O
+the	O
+hydrophobic	O
+part	O
+of	O
+the	O
+Mep2	B-protein
+channel	B-site
+close	O
+to	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+.	O
+
+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
+
+Despite	O
+its	O
+location	O
+at	O
+the	O
+periphery	O
+of	O
+the	O
+trimer	B-oligomeric_state
+,	O
+the	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+serine	B-residue_name
+is	O
+well	O
+defined	O
+in	O
+both	O
+Mep2	B-protein
+structures	B-evidence
+and	O
+corresponds	O
+to	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+state	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+data	O
+behind	O
+this	O
+hypothesis	O
+is	O
+the	O
+observation	O
+that	O
+a	O
+ScMep2	B-protein
+449	B-mutant
+-	I-mutant
+485Δ	I-mutant
+deletion	B-protein_state
+mutant	I-protein_state
+lacking	B-protein_state
+the	O
+AI	B-structure_element
+region	I-structure_element
+is	O
+highly	B-protein_state
+active	I-protein_state
+in	O
+MA	B-chemical
+uptake	O
+both	O
+in	O
+the	O
+triple	B-mutant
+mepΔ	I-mutant
+and	O
+triple	B-mutant
+mepΔ	I-mutant
+npr1Δ	I-mutant
+backgrounds	O
+,	O
+implying	O
+that	O
+this	O
+Mep2	B-mutant
+variant	I-mutant
+has	O
+a	O
+constitutively	B-protein_state
+open	I-protein_state
+channel	B-site
+.	O
+
+The	O
+latter	O
+model	O
+would	O
+fit	O
+well	O
+with	O
+the	O
+NH3	B-chemical
+/	O
+H	B-chemical
++	I-chemical
+symport	O
+model	O
+in	O
+which	O
+the	O
+proton	O
+is	O
+relayed	O
+by	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+.	O
+
+The	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+of	O
+Ser457	B-residue_name_number
+/	O
+453	B-residue_number
+is	O
+located	O
+in	O
+a	O
+well	O
+-	O
+defined	O
+electronegative	B-site
+pocket	I-site
+that	O
+is	O
+solvent	B-protein_state
+accessible	I-protein_state
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+closest	O
+atoms	O
+to	O
+the	O
+serine	B-residue_name
+hydroxyl	O
+group	O
+are	O
+the	O
+backbone	O
+carbonyl	O
+atoms	O
+of	O
+Asp419	B-residue_name_number
+,	O
+Glu420	B-residue_name_number
+and	O
+Glu421	B-residue_name_number
+,	O
+which	O
+are	O
+3	O
+–	O
+4	O
+Å	O
+away	O
+.	O
+
+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
+
+By	O
+contrast	O
+,	O
+the	O
+conserved	B-protein_state
+part	O
+of	O
+the	O
+CTR	B-structure_element
+has	O
+undergone	O
+a	O
+large	O
+conformational	O
+change	O
+involving	O
+formation	O
+of	O
+a	O
+12	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+-	I-structure_element
+long	I-structure_element
+α	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+from	O
+Leu427	B-residue_range
+to	I-residue_range
+Asp438	I-residue_range
+.	O
+
+As	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+,	O
+the	O
+consequence	O
+of	O
+the	O
+single	B-mutant
+D	I-mutant
+mutation	B-experimental_method
+is	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+DD	B-mutant
+substitution	I-mutant
+,	O
+with	O
+conformational	O
+changes	O
+and	O
+increased	O
+dynamics	O
+confined	O
+to	O
+the	O
+conserved	B-protein_state
+part	O
+of	O
+the	O
+CTR	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+
+As	O
+the	O
+simulation	B-experimental_method
+proceeds	O
+,	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+acidic	O
+residues	O
+move	O
+away	O
+from	O
+Asp452	B-residue_name_number
+and	O
+Asp453	B-residue_name_number
+,	O
+presumably	O
+to	O
+avoid	O
+electrostatic	O
+repulsion	O
+.	O
+
+The	O
+short	B-structure_element
+helix	I-structure_element
+formed	O
+by	O
+residues	O
+Leu427	B-residue_range
+to	I-residue_range
+Asp438	I-residue_range
+unravels	O
+during	O
+the	O
+simulations	B-experimental_method
+to	O
+a	O
+disordered	B-protein_state
+state	O
+.	O
+
+One	O
+possible	O
+explanation	O
+is	O
+that	O
+the	O
+mutants	B-mutant
+do	O
+not	O
+accurately	O
+mimic	O
+a	O
+phosphoserine	B-residue_name
+,	O
+but	O
+the	O
+observation	O
+that	O
+the	O
+S453D	B-mutant
+and	O
+DD	B-mutant
+mutants	I-mutant
+are	O
+fully	B-protein_state
+active	I-protein_state
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Npr1	B-protein
+suggests	O
+that	O
+the	O
+mutations	B-experimental_method
+do	O
+mimic	O
+the	O
+effect	O
+of	O
+phosphorylation	B-ptm
+(	O
+Fig	O
+.	O
+3	O
+).	O
+
+Interestingly	O
+,	O
+phosphomimetic	B-mutant
+mutations	I-mutant
+introduced	O
+into	O
+one	O
+monomer	B-oligomeric_state
+inactivate	O
+the	O
+entire	O
+trimer	B-oligomeric_state
+,	O
+indicating	O
+that	O
+(	O
+i	O
+)	O
+heterotrimerization	O
+occurs	O
+and	O
+(	O
+ii	O
+)	O
+the	O
+CTR	B-structure_element
+mediates	O
+allosteric	O
+regulation	O
+of	O
+ammonium	B-chemical
+transport	O
+activity	O
+via	O
+phosphorylation	B-ptm
+.	O
+
+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
+
+However	O
+,	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+GlnK	B-protein_type
+proteins	I-protein_type
+in	O
+eukaryotes	B-taxonomy_domain
+suggests	O
+that	O
+phosphorylation	B-ptm
+-	O
+based	O
+regulation	O
+of	O
+ammonium	B-chemical
+transport	O
+may	O
+be	O
+widespread	O
+.	O
+
+The	O
+conserved	B-protein_state
+RxK	B-structure_element
+motif	I-structure_element
+in	O
+ICL1	B-structure_element
+is	O
+boxed	O
+in	O
+blue	O
+,	O
+the	O
+ER	B-structure_element
+motif	I-structure_element
+in	O
+ICL2	B-structure_element
+in	O
+cyan	O
+,	O
+the	O
+conserved	B-protein_state
+ExxGxD	B-structure_element
+motif	I-structure_element
+of	O
+the	O
+CTR	B-structure_element
+in	O
+red	O
+and	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+in	O
+yellow	O
+.	O
+
+Coloured	O
+residues	O
+are	O
+functionally	O
+important	O
+and	O
+correspond	O
+to	O
+those	O
+of	O
+the	O
+Phe	B-site
+gate	I-site
+(	O
+blue	O
+),	O
+the	O
+binding	B-site
+site	I-site
+Trp	B-residue_name
+residue	O
+(	O
+magenta	O
+)	O
+and	O
+the	O
+twin	O
+-	O
+His	O
+motif	O
+(	O
+red	O
+).	O
+
+(	O
+a	O
+)	O
+The	O
+triple	B-mutant
+mepΔ	I-mutant
+strain	O
+(	O
+black	O
+)	O
+and	O
+triple	O
+mepΔ	O
+npr1Δ	O
+strain	O
+(	O
+grey	O
+)	O
+containing	O
+plasmids	O
+expressing	O
+WT	B-protein_state
+and	O
+variant	B-mutant
+ScMep2	I-mutant
+were	O
+grown	B-experimental_method
+on	I-experimental_method
+minimal	I-experimental_method
+medium	I-experimental_method
+containing	O
+1	O
+mM	O
+ammonium	B-chemical
+sulphate	I-chemical
+.	O
+
+Channel	O
+closures	O
+in	O
+Mep2	B-protein
+.	O
+
+(	O
+c	O
+)	O
+Cytoplasmic	O
+view	O
+of	O
+the	O
+Mep2	B-protein
+trimer	B-oligomeric_state
+indicating	O
+the	O
+large	O
+distance	O
+between	O
+Ser453	B-residue_name_number
+and	O
+the	O
+channel	B-site
+exits	I-site
+(	O
+circles	O
+;	O
+Ile52	B-residue_name_number
+lining	O
+the	O
+channel	B-site
+exit	I-site
+is	O
+shown	O
+).	O
+
+Side	O
+chains	O
+for	O
+residues	O
+452	B-residue_number
+and	O
+453	B-residue_number
+are	O
+shown	O
+as	O
+stick	O
+models	O
+.	O
+
+(	O
+a	O
+)	O
+In	O
+the	O
+closed	B-protein_state
+,	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+state	O
+(	O
+i	O
+),	O
+the	O
+CTR	B-structure_element
+(	O
+magenta	O
+)	O
+and	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+are	O
+far	O
+apart	O
+with	O
+the	O
+latter	O
+blocking	O
+the	O
+intracellular	O
+channel	B-site
+exit	I-site
+(	O
+indicated	O
+with	O
+a	O
+hatched	O
+circle	O
+).	O
+
+We	O
+determined	B-experimental_method
+four	I-experimental_method
+structures	I-experimental_method
+of	O
+an	O
+extended	B-protein_state
+U2AF65	B-structure_element
+–	I-structure_element
+RNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+bound	B-protein_state
+to	I-protein_state
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+oligonucleotides	I-chemical
+at	O
+resolutions	O
+between	O
+2	O
+.	O
+0	O
+and	O
+1	O
+.	O
+5	O
+Å	O
+.	O
+These	O
+structures	B-evidence
+together	O
+with	O
+RNA	B-experimental_method
+binding	I-experimental_method
+and	I-experimental_method
+splicing	I-experimental_method
+assays	I-experimental_method
+reveal	O
+unforeseen	O
+roles	O
+for	O
+U2AF65	B-protein
+inter	B-site
+-	I-site
+domain	I-site
+residues	I-site
+in	O
+recognizing	O
+a	O
+contiguous	B-structure_element
+,	O
+nine	O
+-	O
+nucleotide	B-chemical
+Py	B-chemical
+tract	I-chemical
+.	O
+
+In	O
+turn	O
+,	O
+the	O
+ternary	B-complex_assembly
+complex	I-complex_assembly
+of	O
+U2AF65	B-protein
+with	O
+SF1	B-protein
+and	O
+U2AF35	B-protein
+identifies	O
+the	O
+surrounding	O
+BPS	B-site
+and	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+junctions	O
+.	O
+
+Biochemical	B-experimental_method
+characterizations	I-experimental_method
+of	O
+U2AF65	B-protein
+demonstrated	O
+that	O
+tandem	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+)	O
+recognize	O
+the	O
+Py	B-chemical
+tract	I-chemical
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+
+Milestone	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+core	B-protein_state
+U2AF65	B-protein
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+connected	O
+by	O
+a	O
+shortened	B-protein_state
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+(	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+)	O
+detailed	O
+a	O
+subset	O
+of	O
+nucleotide	O
+interactions	O
+with	O
+the	O
+individual	O
+U2AF65	B-protein
+RRMs	B-structure_element
+.	O
+
+The	O
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+minimal	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+domain	O
+comprising	O
+the	O
+core	B-protein_state
+RRM1	B-structure_element
+–	O
+RRM2	B-structure_element
+folds	B-structure_element
+(	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+,	O
+residues	O
+148	B-residue_range
+–	I-residue_range
+336	I-residue_range
+)	O
+is	O
+relatively	O
+weak	O
+compared	O
+with	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF65	B-protein
+(	O
+Fig	O
+.	O
+1a	O
+,	O
+b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+
+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
+
+Based	O
+on	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	B-evidence
+,	O
+we	O
+originally	O
+hypothesized	O
+that	O
+the	O
+U2AF65	B-protein
+RRMs	B-structure_element
+would	O
+bind	O
+the	O
+minimal	B-protein_state
+seven	O
+nucleotides	B-chemical
+observed	O
+in	O
+these	O
+structures	B-evidence
+.	O
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+characterize	O
+ribose	B-chemical
+(	O
+r	B-chemical
+)	O
+nucleotides	B-chemical
+at	O
+all	O
+of	O
+the	O
+binding	B-site
+sites	I-site
+except	O
+the	O
+seventh	B-residue_number
+and	O
+eighth	B-residue_number
+deoxy	B-chemical
+-(	I-chemical
+d	I-chemical
+)	I-chemical
+U	I-chemical
+,	O
+which	O
+are	O
+likely	O
+to	O
+lack	O
+2	O
+′-	O
+hydroxyl	O
+contacts	O
+based	O
+on	O
+the	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structure	B-evidence
+.	O
+
+The	O
+rU6	B-residue_name_number
+base	O
+edge	O
+is	O
+relatively	O
+solvent	B-protein_state
+exposed	I-protein_state
+;	O
+accordingly	O
+,	O
+the	O
+rU6	B-residue_name_number
+hydrogen	O
+bonds	O
+with	O
+U2AF65	B-protein
+are	O
+water	B-chemical
+mediated	O
+apart	O
+from	O
+a	O
+single	O
+direct	O
+interaction	O
+by	O
+the	O
+RRM1	B-structure_element
+-	O
+N196	B-residue_name_number
+side	O
+chain	O
+.	O
+
+The	O
+energetic	O
+penalties	O
+due	O
+to	O
+these	O
+mutations	O
+(	O
+ΔΔG	B-evidence
+0	O
+.	O
+8	O
+–	O
+0	O
+.	O
+9	O
+kcal	O
+mol	O
+−	O
+1	O
+)	O
+are	O
+consistent	O
+with	O
+the	O
+loss	O
+of	O
+each	O
+hydrogen	O
+bond	O
+with	O
+the	O
+rU5	B-residue_name_number
+base	O
+and	O
+support	O
+the	O
+relevance	O
+of	O
+the	O
+central	O
+nucleotide	O
+interactions	O
+observed	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+.	O
+
+Despite	O
+12	B-experimental_method
+concurrent	I-experimental_method
+mutations	I-experimental_method
+,	O
+the	O
+AdML	B-gene
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+12Gly	I-mutant
+variant	B-protein_state
+was	O
+reduced	O
+by	O
+only	O
+three	O
+-	O
+fold	O
+relative	O
+to	O
+the	O
+unmodified	B-protein_state
+protein	B-protein
+(	O
+Fig	O
+.	O
+4b	O
+),	O
+which	O
+is	O
+less	O
+than	O
+the	O
+penalty	O
+of	O
+the	O
+V254P	B-mutant
+mutation	O
+that	O
+disrupts	O
+the	O
+rU5	B-residue_name_number
+hydrogen	O
+bond	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+i	O
+).	O
+
+To	O
+test	O
+the	O
+interplay	O
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+with	O
+its	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+RRM	B-structure_element
+extensions	I-structure_element
+,	O
+we	O
+constructed	B-experimental_method
+an	O
+internal	O
+linker	B-experimental_method
+deletion	I-experimental_method
+of	O
+20	B-residue_range
+-	I-residue_range
+residues	I-residue_range
+within	O
+the	O
+extended	B-protein_state
+RNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+).	O
+
+Notably	O
+,	O
+the	O
+Q147A	B-mutant
+/	O
+V254P	B-mutant
+/	O
+R227A	B-mutant
+mutation	B-experimental_method
+reduced	O
+the	O
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+3Mut	I-mutant
+protein	O
+by	O
+30	O
+-	O
+fold	O
+more	O
+than	O
+would	O
+be	O
+expected	O
+based	O
+on	O
+simple	O
+addition	O
+of	O
+the	O
+ΔΔG	B-evidence
+'	O
+s	O
+for	O
+the	O
+single	O
+mutations	O
+.	O
+
+This	O
+difference	O
+indicates	O
+that	O
+the	O
+linearly	B-protein_state
+distant	I-protein_state
+regions	B-structure_element
+of	O
+the	O
+U2AF65	B-protein
+primary	O
+sequence	O
+,	O
+including	O
+Q147	B-residue_name_number
+in	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	B-structure_element
+extension	I-structure_element
+and	O
+R227	B-residue_name_number
+/	O
+V254	B-residue_name_number
+in	O
+the	O
+N	O
+-/	O
+C	O
+-	O
+terminal	O
+linker	B-structure_element
+regions	I-structure_element
+at	O
+the	O
+fifth	B-site
+nucleotide	I-site
+site	I-site
+,	O
+cooperatively	O
+recognize	O
+the	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+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
+
+The	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+dynamics	O
+of	O
+U2AF65	B-protein
+were	O
+followed	O
+using	O
+FRET	B-experimental_method
+between	O
+fluorophores	B-chemical
+attached	O
+to	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+(	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+,	O
+Methods	O
+).	O
+
+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
+
+Double	O
+-	O
+cysteine	B-residue_name
+variant	B-protein_state
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+was	O
+modified	B-experimental_method
+with	O
+equimolar	O
+amount	O
+of	O
+Cy3	B-chemical
+and	O
+Cy5	B-chemical
+.	O
+
+We	O
+first	O
+characterized	O
+the	O
+conformational	O
+dynamics	O
+spectrum	O
+of	O
+U2AF65	B-protein
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+6c	O
+,	O
+d	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+,	O
+b	O
+).	O
+
+The	O
+FRET	B-evidence
+distribution	I-evidence
+histogram	I-evidence
+built	O
+from	O
+more	O
+than	O
+a	O
+thousand	O
+traces	B-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ligand	B-chemical
+showed	O
+an	O
+extremely	O
+broad	O
+distribution	O
+centred	O
+at	O
+a	O
+FRET	B-evidence
+efficiency	I-evidence
+of	O
+∼	O
+0	O
+.	O
+4	O
+(	O
+Fig	O
+.	O
+6d	O
+).	O
+
+Despite	O
+the	O
+large	O
+width	O
+of	O
+the	O
+FRET	B-evidence
+-	I-evidence
+distribution	I-evidence
+histogram	I-evidence
+,	O
+the	O
+majority	O
+(	O
+80	O
+%)	O
+of	O
+traces	B-evidence
+that	O
+showed	O
+fluctuations	O
+sampled	O
+only	O
+two	O
+distinct	O
+FRET	B-evidence
+states	I-evidence
+(	O
+for	O
+example	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+
+We	O
+introduced	B-experimental_method
+an	O
+rArA	B-chemical
+purine	B-chemical
+dinucleotide	I-chemical
+within	O
+a	O
+variant	O
+of	O
+the	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+(	O
+detailed	O
+in	O
+Methods	O
+).	O
+
+Nevertheless	O
+,	O
+the	O
+predominant	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+state	I-evidence
+in	O
+the	O
+presence	O
+of	O
+RNA	B-chemical
+agrees	O
+with	O
+the	O
+Py	B-protein_state
+-	I-protein_state
+tract	I-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystal	B-evidence
+structure	I-evidence
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+.	O
+
+Notably	O
+,	O
+a	O
+triple	B-protein_state
+mutation	I-protein_state
+of	O
+three	O
+residues	O
+(	O
+V254P	B-mutant
+,	O
+Q147A	B-mutant
+and	O
+R227A	B-mutant
+)	O
+in	O
+the	O
+respective	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+,	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extensions	I-structure_element
+non	O
+-	O
+additively	O
+reduce	O
+RNA	B-evidence
+binding	I-evidence
+by	O
+150	O
+-	O
+fold	O
+.	O
+
+Altogether	O
+,	O
+these	O
+data	O
+indicate	O
+that	O
+interactions	O
+among	O
+the	O
+U2AF65	B-protein
+RRM1	B-structure_element
+/	O
+RRM2	B-structure_element
+,	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+,	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extensions	I-structure_element
+are	O
+mutually	O
+inter	O
+-	O
+dependent	O
+for	O
+cognate	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+.	O
+
+These	O
+transitions	O
+could	O
+correspond	O
+to	O
+rearrangement	O
+from	O
+the	O
+‘	O
+closed	B-protein_state
+'	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+-	O
+based	O
+U2AF65	B-protein
+conformation	O
+in	O
+which	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+surface	I-site
+of	O
+only	O
+a	O
+single	B-protein_state
+RRM	B-structure_element
+is	O
+exposed	O
+and	O
+available	O
+for	O
+RNA	O
+binding	O
+,	O
+to	O
+the	O
+structural	O
+state	O
+seen	O
+in	O
+the	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+,	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+The	O
+regions	O
+of	O
+RRM1	B-structure_element
+,	O
+RRM2	B-structure_element
+and	O
+linker	B-structure_element
+contacts	O
+are	O
+indicated	O
+above	O
+by	O
+respective	O
+black	O
+and	O
+blue	O
+arrows	O
+from	O
+N	O
+-	O
+to	O
+C	O
+-	O
+terminus	O
+.	O
+
+Crystallographic	O
+statistics	O
+are	O
+given	O
+in	O
+Table	O
+1	O
+and	O
+the	O
+overall	O
+conformations	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+/	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+structures	B-evidence
+are	O
+compared	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+.	O
+
+Residues	O
+V249	B-residue_name_number
+,	O
+V250	B-residue_name_number
+,	O
+V254	B-residue_name_number
+(	O
+yellow	O
+)	O
+are	O
+mutated	B-experimental_method
+to	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+V254G	B-mutant
+in	O
+the	O
+3Gly	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+P255	B-residue_name_number
+(	O
+red	O
+)	O
+along	O
+with	O
+V254	B-residue_name_number
+are	O
+mutated	B-experimental_method
+to	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+5Gly	B-mutant
+mutant	I-mutant
+or	O
+to	O
+S251N	B-mutant
+/	O
+T252L	B-mutant
+/	O
+V253A	B-mutant
+/	O
+V254L	B-mutant
+/	O
+P255A	B-mutant
+in	O
+the	O
+NLALA	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+M144	B-residue_name_number
+,	O
+L235	B-residue_name_number
+,	O
+M238	B-residue_name_number
+,	O
+V244	B-residue_name_number
+,	O
+V246	B-residue_name_number
+(	O
+orange	O
+)	O
+along	O
+with	O
+V249	B-residue_name_number
+,	O
+V250	B-residue_name_number
+,	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+V254	B-residue_name_number
+,	O
+P255	B-residue_name_number
+are	O
+mutated	B-experimental_method
+to	O
+M144G	B-mutant
+/	O
+L235G	B-mutant
+/	O
+M238G	B-mutant
+/	O
+V244G	B-mutant
+/	O
+V246G	B-mutant
+/	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+12Gly	B-mutant
+mutant	I-mutant
+.	O
+
+Other	O
+linker	B-structure_element
+residues	O
+are	O
+coloured	O
+either	O
+dark	O
+blue	O
+for	O
+new	O
+residues	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	O
+or	O
+light	O
+blue	O
+for	O
+the	O
+remaining	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+residues	O
+.	O
+
+The	O
+central	O
+panel	O
+shows	O
+an	O
+overall	O
+view	O
+with	O
+stick	O
+diagrams	O
+for	O
+mutated	O
+residues	O
+;	O
+boxed	O
+regions	O
+are	O
+expanded	O
+to	O
+show	O
+the	O
+C	O
+-	O
+terminal	O
+(	O
+bottom	O
+left	O
+)	O
+and	O
+central	B-structure_element
+linker	I-structure_element
+regions	I-structure_element
+(	O
+top	O
+)	O
+at	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+interfaces	I-structure_element
+,	O
+and	O
+N	O
+-	O
+terminal	O
+linker	O
+region	O
+contacts	O
+with	O
+RRM1	B-structure_element
+(	O
+bottom	O
+right	O
+).	O
+
+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
+
+Schematic	O
+models	O
+of	O
+U2AF65	B-protein
+recognizing	O
+the	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+(	O
+b	O
+)	O
+Following	O
+binding	O
+to	O
+the	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+RNA	I-chemical
+,	O
+a	O
+conformation	O
+corresponding	O
+to	O
+high	B-evidence
+FRET	I-evidence
+and	O
+consistent	O
+with	O
+the	O
+‘	O
+closed	B-protein_state
+',	O
+back	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+back	I-protein_state
+apo	B-protein_state
+-	O
+U2AF65	B-protein
+model	O
+resulting	O
+from	O
+PRE	B-experimental_method
+/	O
+NMR	B-experimental_method
+characterization	O
+(	O
+PDB	O
+ID	O
+2YH0	O
+)	O
+often	O
+transitions	O
+to	O
+a	O
+conformation	O
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+,	O
+which	O
+is	O
+consistent	O
+with	O
+‘	O
+open	B-protein_state
+',	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+RRMs	B-structure_element
+such	O
+as	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Floral	O
+abscission	O
+is	O
+controlled	O
+by	O
+the	O
+leucine	B-protein_type
+-	I-protein_type
+rich	I-protein_type
+repeat	I-protein_type
+receptor	I-protein_type
+kinase	I-protein_type
+(	O
+LRR	B-protein_type
+-	I-protein_type
+RK	I-protein_type
+)	O
+HAESA	B-protein
+and	O
+the	O
+peptide	B-protein_type
+hormone	I-protein_type
+IDA	B-protein
+.	O
+
+It	O
+is	O
+unknown	O
+how	O
+expression	O
+of	O
+IDA	B-protein
+in	O
+the	O
+abscission	O
+zone	O
+leads	O
+to	O
+HAESA	B-protein
+activation	O
+.	O
+
+Here	O
+we	O
+show	O
+that	O
+IDA	B-protein
+is	O
+sensed	O
+directly	O
+by	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+
+However	O
+,	O
+the	O
+molecular	O
+details	O
+of	O
+how	O
+IDA	B-protein
+triggers	O
+organ	O
+shedding	O
+are	O
+not	O
+clear	O
+.	O
+
+Santiago	O
+et	O
+al	O
+.	O
+used	O
+protein	B-experimental_method
+biochemistry	I-experimental_method
+,	O
+structural	B-experimental_method
+biology	I-experimental_method
+and	O
+genetics	B-experimental_method
+to	O
+uncover	O
+how	O
+the	O
+IDA	B-protein
+hormone	B-chemical
+activates	O
+HAESA	B-protein
+.	O
+
+HAESA	B-protein
+is	O
+shown	O
+in	O
+blue	O
+(	O
+ribbon	O
+diagram	O
+),	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+(	O
+left	O
+panel	O
+),	O
+the	O
+central	O
+Hyp	B-structure_element
+anchor	I-structure_element
+(	O
+center	O
+)	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+Pro	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+(	O
+right	O
+panel	O
+)	O
+are	O
+shown	O
+in	O
+yellow	O
+(	O
+in	O
+bonds	O
+representation	O
+).	O
+
+Structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+HAESA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+:	O
+Arabidopsis	B-species
+thaliana	I-species
+HAESA	B-protein
+(	O
+Uniprot	O
+(	O
+http	O
+://	O
+www	O
+.	O
+uniprot	O
+.	O
+org	O
+)	O
+ID	O
+P47735	O
+),	O
+Arabidopsis	B-species
+thaliana	I-species
+HSL2	B-protein
+(	O
+Uniprot	O
+ID	O
+C0LGX3	O
+),	O
+Capsella	B-species
+rubella	I-species
+HAESA	B-protein
+(	O
+Uniprot	O
+ID	O
+R0F2U6	O
+),	O
+Citrus	B-species
+clementina	I-species
+HSL2	B-protein
+(	O
+Uniprot	O
+ID	O
+V4U227	O
+),	O
+Vitis	B-species
+vinifera	I-species
+HAESA	B-protein
+(	O
+Uniprot	O
+ID	O
+F6HM39	O
+).	O
+
+The	O
+alignment	O
+includes	O
+a	O
+secondary	O
+structure	O
+assignment	O
+calculated	O
+with	O
+the	O
+program	O
+DSSP	O
+and	O
+colored	O
+according	O
+to	O
+Figure	O
+1	O
+,	O
+with	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+caps	B-structure_element
+and	O
+the	O
+21	O
+LRR	B-structure_element
+motifs	I-structure_element
+indicated	O
+in	O
+orange	O
+and	O
+blue	O
+,	O
+respectively	O
+.	O
+
+HAESA	B-protein
+residues	O
+interacting	O
+with	O
+the	O
+IDA	B-chemical
+peptide	I-chemical
+and	O
+/	O
+or	O
+the	O
+SERK1	B-protein
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+kinase	I-protein_type
+ectodomain	B-structure_element
+are	O
+highlighted	O
+in	O
+blue	O
+and	O
+orange	O
+,	O
+respectively	O
+.	O
+
+The	O
+PKGV	B-structure_element
+motif	I-structure_element
+present	O
+in	O
+our	O
+N	B-protein_state
+-	I-protein_state
+terminally	I-protein_state
+extended	I-protein_state
+IDA	B-chemical
+peptide	I-chemical
+is	O
+highlighted	O
+in	O
+red	O
+.	O
+(	O
+B	O
+)	O
+Isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+of	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+vs	O
+.	O
+IDA	B-protein
+and	O
+including	O
+the	O
+synthetic	B-protein_state
+peptide	B-chemical
+sequence	O
+.	O
+
+IDA	B-protein
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+surface	O
+view	O
+included	O
+)	O
+is	O
+depicted	O
+in	O
+yellow	O
+.	O
+
+Active	B-protein_state
+IDA	B-protein_type
+-	I-protein_type
+family	I-protein_type
+peptide	I-protein_type
+hormones	I-protein_type
+are	O
+hydroxyprolinated	B-protein_state
+dodecamers	B-structure_element
+.	O
+
+Void	O
+(	O
+V0	O
+)	O
+volume	O
+and	O
+total	O
+volume	O
+(	O
+Vt	O
+)	O
+are	O
+shown	O
+,	O
+together	O
+with	O
+elution	O
+volumes	O
+for	O
+molecular	O
+mass	O
+standards	O
+(	O
+A	O
+,	O
+Thyroglobulin	B-protein
+,	O
+669	O
+,	O
+000	O
+Da	O
+;	O
+B	O
+,	O
+Ferritin	B-protein
+,	O
+440	O
+,	O
+00	O
+Da	O
+,	O
+C	O
+,	O
+Aldolase	B-protein
+,	O
+158	O
+,	O
+000	O
+Da	O
+;	O
+D	O
+,	O
+Conalbumin	B-protein
+,	O
+75	O
+,	O
+000	O
+Da	O
+;	O
+E	O
+,	O
+Ovalbumin	B-protein
+,	O
+44	O
+,	O
+000	O
+Da	O
+;	O
+F	O
+,	O
+Carbonic	B-protein
+anhydrase	I-protein
+,	O
+29	O
+,	O
+000	O
+Da	O
+).	O
+
+The	O
+titration	B-experimental_method
+of	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+versus	O
+the	O
+isolated	O
+HAESA	B-protein
+ectodomain	B-structure_element
+from	O
+Figure	O
+1B	O
+is	O
+shown	O
+for	O
+comparison	O
+(	O
+red	O
+line	O
+;	O
+n	O
+.	O
+d	O
+.	O
+
+We	O
+next	O
+determined	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+apo	B-protein_state
+HAESA	B-protein
+ectodomain	B-structure_element
+and	O
+of	O
+a	O
+HAESA	B-complex_assembly
+-	I-complex_assembly
+IDA	I-complex_assembly
+complex	O
+,	O
+at	O
+1	O
+.	O
+74	O
+and	O
+1	O
+.	O
+86	O
+Å	O
+resolution	O
+,	O
+respectively	O
+(	O
+Figure	O
+1C	O
+;	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1B	O
+–	O
+D	O
+;	O
+Tables	O
+1	O
+,	O
+2	O
+).	O
+
+The	O
+COO	O
+-	O
+group	O
+of	O
+Asn69IDA	B-residue_name_number
+is	O
+in	O
+direct	O
+contact	O
+with	O
+Arg407HAESA	B-residue_name_number
+and	O
+Arg409HAESA	B-residue_name_number
+and	O
+HAESA	B-protein
+cannot	O
+bind	O
+a	O
+C	B-protein_state
+-	I-protein_state
+terminally	I-protein_state
+extended	I-protein_state
+IDA	B-mutant
+-	I-mutant
+SFVN	I-mutant
+peptide	O
+(	O
+Figures	O
+1D	O
+,	O
+F	O
+,	O
+2D	O
+).	O
+
+A	O
+2	O
+.	O
+56	O
+Å	O
+co	B-evidence
+-	I-evidence
+crystal	I-evidence
+structure	I-evidence
+with	O
+IDL1	B-protein
+reveals	O
+that	O
+different	O
+IDA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+use	O
+a	O
+common	O
+binding	O
+mode	O
+to	O
+interact	O
+with	O
+HAESA	B-protein_type
+-	I-protein_type
+type	I-protein_type
+receptors	I-protein_type
+(	O
+Figure	O
+2A	O
+–	O
+C	O
+,	O
+E	O
+,	O
+Table	O
+2	O
+).	O
+
+The	O
+N	O
+-	O
+terminal	O
+capping	B-structure_element
+domain	I-structure_element
+of	O
+SERK1	B-protein
+(	O
+in	O
+orange	O
+)	O
+directly	O
+contacts	O
+the	O
+C	O
+-	O
+terminal	O
+part	O
+of	O
+IDA	B-protein
+(	O
+in	O
+yellow	O
+,	O
+in	O
+bonds	O
+representation	O
+)	O
+and	O
+the	O
+receptor	B-protein_type
+HAESA	B-protein
+(	O
+in	O
+blue	O
+).	O
+
+The	O
+SERK1	B-protein
+ectodomain	B-structure_element
+interacts	O
+with	O
+the	O
+IDA	B-site
+peptide	I-site
+binding	I-site
+site	I-site
+using	O
+a	O
+loop	B-structure_element
+region	I-structure_element
+(	O
+residues	O
+51	B-residue_range
+-	I-residue_range
+59SERK1	I-residue_range
+)	O
+from	O
+its	O
+N	O
+-	O
+terminal	O
+cap	B-structure_element
+(	O
+Figure	O
+4A	O
+,	O
+C	O
+).	O
+
+SERK1	B-protein
+binds	O
+HAESA	B-protein
+using	O
+these	O
+two	O
+distinct	O
+interaction	B-site
+surfaces	I-site
+(	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+),	O
+with	O
+the	O
+N	B-structure_element
+-	I-structure_element
+cap	I-structure_element
+of	O
+the	O
+SERK1	B-protein
+LRR	B-structure_element
+domain	I-structure_element
+partially	O
+covering	O
+the	O
+IDA	B-site
+peptide	I-site
+binding	I-site
+cleft	I-site
+.	O
+
+(	O
+A	O
+)	O
+Size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+experiments	O
+similar	O
+to	O
+Figure	O
+3B	O
+,	O
+D	O
+reveal	O
+that	O
+IDA	B-protein
+mutant	B-protein_state
+peptides	B-chemical
+targeting	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+motif	I-structure_element
+do	O
+not	O
+form	O
+biochemically	B-protein_state
+stable	I-protein_state
+HAESA	B-complex_assembly
+-	I-complex_assembly
+IDA	I-complex_assembly
+-	I-complex_assembly
+SERK1	I-complex_assembly
+complexes	O
+.	O
+
+Purified	B-experimental_method
+HAESA	B-protein
+and	O
+SERK1	B-protein
+are	O
+~	O
+75	O
+and	O
+~	O
+28	O
+kDa	O
+,	O
+respectively	O
+.	O
+
+Note	O
+that	O
+the	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+input	O
+lanes	O
+have	O
+already	O
+been	O
+shown	O
+in	O
+Figure	O
+3D	O
+.	O
+(	O
+B	O
+)	O
+Isothermal	B-evidence
+titration	I-evidence
+thermographs	I-evidence
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+IDA	B-chemical
+peptides	I-chemical
+titrated	B-experimental_method
+into	O
+a	O
+HAESA	B-protein
+-	O
+SERK1	B-protein
+mixture	O
+in	O
+the	O
+cell	O
+.	O
+
+Table	O
+summaries	O
+for	O
+calorimetric	B-evidence
+binding	I-evidence
+constants	I-evidence
+and	O
+stoichoimetries	O
+for	O
+different	O
+IDA	B-chemical
+peptides	I-chemical
+binding	O
+to	O
+the	O
+HAESA	B-protein
+–	O
+SERK1	B-protein
+ectodomain	B-structure_element
+mixture	O
+(	O
+±	O
+fitting	O
+errors	O
+;	O
+n	O
+.	O
+d	O
+.	O
+
+Up	O
+to	O
+inflorescence	O
+position	O
+4	O
+,	O
+petal	O
+break	O
+in	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+mutant	B-protein_state
+plants	B-taxonomy_domain
+was	O
+significantly	O
+increased	O
+compared	O
+to	O
+both	O
+Col	O
+-	O
+0	O
+control	O
+plants	B-taxonomy_domain
+(	O
+b	O
+)	O
+and	O
+35S	B-gene
+::	O
+IDA	B-protein
+plants	B-taxonomy_domain
+(	O
+c	O
+)	O
+(	O
+D	O
+)	O
+Normalized	O
+expression	O
+levels	O
+(	O
+relative	O
+expression	O
+±	O
+standard	O
+error	O
+;	O
+ida	O
+:	O
+-	O
+0	O
+.	O
+02	O
+±	O
+0	O
+.	O
+001	O
+;	O
+Col	O
+-	O
+0	O
+:	O
+1	O
+±	O
+0	O
+.	O
+11	O
+;	O
+35S	B-gene
+::	O
+IDA	B-protein
+124	O
+±	O
+0	O
+.	O
+75	O
+;	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+:	O
+159	O
+±	O
+0	O
+.	O
+58	O
+)	O
+of	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+transcripts	O
+in	O
+the	O
+35S	B-experimental_method
+promoter	I-experimental_method
+over	I-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+lines	I-experimental_method
+analyzed	O
+in	O
+(	O
+C	O
+).	O
+(	O
+E	O
+)	O
+Magnified	O
+view	O
+of	O
+representative	O
+abscission	O
+zones	O
+from	O
+35S	B-gene
+::	O
+IDA	B-protein
+,	O
+Col	O
+-	O
+0	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+double	B-protein_state
+-	I-protein_state
+mutant	I-protein_state
+T3	B-experimental_method
+transgenic	I-experimental_method
+lines	I-experimental_method
+.	O
+
+The	O
+four	O
+C	O
+-	O
+terminal	O
+residues	O
+in	O
+IDA	B-protein
+(	O
+Lys66IDA	B-residue_range
+-	I-residue_range
+Asn69IDA	I-residue_range
+)	O
+are	O
+conserved	B-protein_state
+among	O
+IDA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+and	O
+are	O
+in	O
+direct	O
+contact	O
+with	O
+SERK1	B-protein
+(	O
+Figures	O
+1A	O
+,	O
+4C	O
+).	O
+
+We	O
+found	O
+that	O
+over	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+IDA	B-protein
+leads	O
+to	O
+early	O
+floral	O
+abscission	O
+and	O
+an	O
+enlargement	O
+of	O
+the	O
+abscission	O
+zone	O
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+
+SERK1	O
+has	O
+been	O
+previously	O
+reported	O
+as	O
+a	O
+positive	O
+regulator	O
+in	O
+plant	B-taxonomy_domain
+embryogenesis	O
+,	O
+male	O
+sporogenesis	O
+,	O
+brassinosteroid	O
+signaling	O
+and	O
+in	O
+phytosulfokine	O
+perception	O
+.	O
+
+The	O
+fact	O
+that	O
+SERK1	B-protein
+specifically	O
+interacts	O
+with	O
+the	O
+very	O
+C	O
+-	O
+terminus	O
+of	O
+IDLs	B-protein_type
+may	O
+allow	O
+for	O
+the	O
+rational	O
+design	O
+of	O
+peptide	B-chemical
+hormone	I-chemical
+antagonists	I-chemical
+,	O
+as	O
+previously	O
+demonstrated	O
+for	O
+the	O
+brassinosteroid	O
+pathway	O
+.	O
+
+These	O
+residues	O
+are	O
+not	O
+involved	O
+in	O
+the	O
+sensing	O
+of	O
+the	O
+steroid	B-chemical
+hormone	I-chemical
+brassinolide	B-chemical
+.	O
+
+Structure	B-experimental_method
+-	I-experimental_method
+guided	I-experimental_method
+multiple	I-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+IDA	B-protein
+and	O
+IDA	B-chemical
+-	I-chemical
+like	I-chemical
+peptides	I-chemical
+with	O
+other	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormone	I-protein_type
+families	I-protein_type
+,	O
+including	O
+CLAVATA3	B-protein_type
+–	I-protein_type
+EMBRYO	I-protein_type
+SURROUNDING	I-protein_type
+REGION	I-protein_type
+-	I-protein_type
+RELATED	I-protein_type
+(	O
+CLV3	B-protein_type
+/	I-protein_type
+CLE	I-protein_type
+),	O
+ROOT	B-protein_type
+GROWTH	I-protein_type
+FACTOR	I-protein_type
+–	I-protein_type
+GOLVEN	I-protein_type
+(	O
+RGF	B-protein_type
+/	I-protein_type
+GLV	I-protein_type
+),	O
+PRECURSOR	B-protein_type
+GENE	I-protein_type
+PROPEP1	I-protein_type
+(	O
+PEP1	B-protein_type
+)	O
+from	O
+Arabidopsis	B-species
+thaliana	I-species
+.	O
+
+Our	O
+experiments	O
+reveal	O
+that	O
+SERK1	B-protein
+recognizes	O
+a	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+.	O
+
+A	O
+unified	O
+mechanism	O
+for	O
+proteolysis	O
+and	O
+autocatalytic	B-ptm
+activation	I-ptm
+in	O
+the	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+
+Here	O
+we	O
+use	O
+mutagenesis	B-experimental_method
+,	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+and	O
+biochemical	B-experimental_method
+assays	I-experimental_method
+to	O
+suggest	O
+that	O
+Lys33	B-residue_name_number
+initiates	O
+nucleophilic	O
+attack	O
+of	O
+the	O
+propeptide	B-structure_element
+by	O
+deprotonating	O
+the	O
+Thr1	B-residue_name_number
+hydroxyl	O
+group	O
+and	O
+that	O
+both	O
+residues	O
+together	O
+with	O
+Asp17	B-residue_name_number
+are	O
+part	O
+of	O
+a	O
+catalytic	B-site
+triad	I-site
+.	O
+
+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
+
+In	O
+the	O
+last	O
+stage	O
+of	O
+CP	B-complex_assembly
+biogenesis	O
+,	O
+the	O
+prosegments	B-structure_element
+are	O
+autocatalytically	B-ptm
+removed	I-ptm
+through	O
+nucleophilic	O
+attack	O
+by	O
+the	O
+active	B-site
+site	I-site
+residue	I-site
+Thr1	B-residue_name_number
+on	O
+the	O
+preceding	O
+peptide	O
+bond	O
+involving	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+).	I-residue_name_number
+
+Release	O
+of	O
+the	O
+propeptides	B-structure_element
+creates	O
+a	O
+functionally	O
+active	B-protein_state
+CP	B-complex_assembly
+that	O
+cleaves	O
+proteins	O
+into	O
+short	O
+peptides	O
+.	O
+
+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
+
+In	O
+the	O
+final	O
+steps	O
+of	O
+proteasome	B-complex_assembly
+biogenesis	O
+,	O
+the	O
+propeptides	B-structure_element
+are	O
+autocatalytically	B-ptm
+cleaved	I-ptm
+from	O
+the	O
+mature	B-protein_state
+β	B-protein
+-	I-protein
+subunit	I-protein
+domains	I-protein
+.	O
+
+Instead	O
+,	O
+the	O
+plasticity	O
+of	O
+the	O
+β5	B-protein
+S1	B-site
+pocket	I-site
+caused	O
+by	O
+the	O
+rotational	O
+flexibility	O
+of	O
+Met45	B-residue_name_number
+might	O
+prevent	O
+stable	O
+accommodation	O
+of	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+the	O
+S1	B-site
+site	I-site
+and	O
+thus	O
+also	O
+promote	O
+its	O
+immediate	O
+release	O
+after	O
+autolysis	B-ptm
+.	O
+
+As	O
+histidine	B-residue_name
+commonly	O
+functions	O
+as	O
+a	O
+proton	O
+shuttle	O
+in	O
+the	O
+catalytic	B-site
+triads	I-site
+of	O
+serine	B-protein_type
+proteases	I-protein_type
+,	O
+we	O
+investigated	O
+the	O
+role	O
+of	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+processing	O
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+by	O
+exchanging	B-experimental_method
+it	I-experimental_method
+for	I-experimental_method
+Asn	B-residue_name
+,	O
+Lys	B-residue_name
+,	O
+Phe	B-residue_name
+and	O
+Ala	B-residue_name
+.	O
+All	O
+yeast	B-taxonomy_domain
+mutants	O
+were	O
+viable	O
+at	O
+30	O
+°	O
+C	O
+,	O
+but	O
+suffered	O
+from	O
+growth	O
+defects	O
+at	O
+37	O
+°	O
+C	O
+with	O
+the	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+N	I-mutant
+and	O
+H	B-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+F	I-mutant
+mutants	O
+being	O
+most	O
+affected	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3b	O
+and	O
+Table	O
+1	O
+).	O
+
+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
+
+The	O
+active	B-site
+site	I-site
+of	O
+the	O
+proteasome	B-complex_assembly
+
+Twenty	O
+years	O
+later	O
+,	O
+with	O
+a	O
+plethora	O
+of	O
+yCP	B-complex_assembly
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structures	I-evidence
+in	O
+hand	O
+,	O
+we	O
+decided	O
+to	O
+re	O
+-	O
+analyse	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+proteasome	B-complex_assembly
+and	O
+to	O
+resolve	O
+the	O
+uncertainty	O
+regarding	O
+the	O
+nature	O
+of	O
+the	O
+general	O
+base	O
+.	O
+
+As	O
+determined	O
+by	O
+crystallographic	B-experimental_method
+analysis	I-experimental_method
+,	O
+this	O
+mutant	B-protein_state
+β5	B-protein
+subunit	O
+was	O
+partially	B-protein_state
+processed	I-protein_state
+(	O
+Table	O
+1	O
+)	O
+but	O
+displayed	O
+impaired	O
+reactivity	O
+towards	O
+the	O
+proteasome	B-complex_assembly
+inhibitor	O
+carfilzomib	B-chemical
+compared	O
+with	O
+the	O
+subunits	O
+β1	B-protein
+and	O
+β2	B-protein
+,	O
+and	O
+with	O
+WT	B-protein_state
+β5	B-protein
+(	O
+Supplementary	O
+Fig	O
+.	O
+7a	O
+).	O
+
+This	O
+observation	O
+is	O
+consistent	O
+with	O
+a	O
+strongly	O
+reduced	O
+reactivity	O
+of	O
+β5	B-protein
+-	O
+Thr1	B-residue_name_number
+and	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+pp	B-chemical
+cis	B-protein_state
+mutant	B-protein_state
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+carfilzomib	B-chemical
+.	O
+
+In	O
+agreement	O
+,	O
+an	O
+E17A	B-mutant
+mutant	B-protein_state
+in	O
+the	O
+proteasomal	O
+β	B-protein
+-	I-protein
+subunit	I-protein
+of	O
+the	O
+archaeon	B-taxonomy_domain
+Thermoplasma	B-species
+acidophilum	I-species
+prevents	O
+autolysis	B-ptm
+and	O
+catalysis	O
+.	O
+
+This	O
+model	O
+is	O
+also	O
+consistent	O
+with	O
+the	O
+fact	O
+that	O
+no	O
+defined	O
+water	B-chemical
+molecule	O
+is	O
+observed	O
+in	O
+the	O
+mature	B-protein_state
+WT	B-protein_state
+proteasomal	O
+active	B-site
+site	I-site
+that	O
+could	O
+shuttle	O
+the	O
+proton	O
+from	O
+Thr1Oγ	B-residue_name_number
+to	O
+Thr1NH2	B-residue_name_number
+.	O
+
+The	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+pp	B-chemical
+cis	B-protein_state
+yeast	B-taxonomy_domain
+mutant	B-protein_state
+is	O
+significantly	O
+impaired	O
+in	O
+growth	O
+and	O
+its	O
+ChT	O
+-	O
+L	O
+activity	O
+is	O
+drastically	O
+reduced	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+,	O
+b	O
+and	O
+Table	O
+1	O
+).	O
+
+Instead	O
+,	O
+a	O
+water	B-chemical
+molecule	O
+is	O
+bound	B-protein_state
+to	I-protein_state
+Ser129OH	B-residue_name_number
+and	O
+Thr1NH2	B-residue_name_number
+(	O
+Supplementary	O
+Fig	O
+.	O
+8b	O
+),	O
+which	O
+may	O
+enable	O
+precursor	B-ptm
+processing	I-ptm
+.	O
+
+In	O
+one	O
+of	O
+the	O
+two	O
+β5	B-protein
+subunits	O
+,	O
+however	O
+,	O
+we	O
+found	O
+the	O
+cleaved	B-protein_state
+propeptide	B-structure_element
+still	B-protein_state
+bound	I-protein_state
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+(	O
+Fig	O
+.	O
+4c	O
+).	O
+
+In	O
+agreement	O
+,	O
+soaking	B-experimental_method
+crystals	I-experimental_method
+with	O
+the	O
+CP	B-complex_assembly
+inhibitors	O
+bortezomib	B-chemical
+or	O
+carfilzomib	B-chemical
+modifies	O
+only	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+active	B-site
+sites	I-site
+,	O
+while	O
+leaving	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+proteolytic	B-site
+centres	I-site
+unmodified	B-protein_state
+even	O
+though	O
+they	O
+are	O
+only	O
+partially	O
+occupied	O
+by	O
+the	O
+cleaved	B-protein_state
+propeptide	B-structure_element
+remnant	O
+.	O
+
+However	O
+,	O
+the	O
+apo	B-protein_state
+crystal	B-evidence
+structure	I-evidence
+revealed	O
+that	O
+Ser1Oγ	B-residue_name_number
+is	O
+turned	O
+away	O
+from	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+(	O
+Fig	O
+.	O
+4g	O
+).	O
+
+Lys33NH2	B-residue_name_number
+is	O
+expected	O
+to	O
+act	O
+as	O
+the	O
+proton	O
+acceptor	O
+during	O
+autocatalytic	B-ptm
+removal	I-ptm
+of	O
+the	O
+propeptides	B-structure_element
+,	O
+as	O
+well	O
+as	O
+during	O
+substrate	O
+proteolysis	O
+,	O
+while	O
+Asp17Oδ	B-residue_name_number
+orients	O
+Lys33NH2	B-residue_name_number
+and	O
+makes	O
+it	O
+more	O
+prone	O
+to	O
+protonation	O
+by	O
+raising	O
+its	O
+pKa	O
+(	O
+hydrogen	O
+bond	O
+distance	O
+:	O
+Lys33NH3	B-residue_name_number
++–	O
+Asp17Oδ	B-residue_name_number
+:	O
+2	O
+.	O
+9	O
+Å	O
+).	O
+
+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
+
+The	O
+resulting	O
+uncharged	O
+Thr1NH2	B-residue_name_number
+is	O
+hydrogen	O
+-	O
+bridged	O
+to	O
+the	O
+C3	O
+-	O
+OH	O
+group	O
+.	O
+
+The	O
+greater	O
+suitability	O
+of	O
+threonine	B-residue_name
+for	O
+the	O
+proteasome	B-complex_assembly
+active	B-site
+site	I-site
+,	O
+which	O
+has	O
+been	O
+noted	O
+in	O
+biochemical	O
+as	O
+well	O
+as	O
+in	O
+kinetic	O
+studies	O
+,	O
+constitutes	O
+a	O
+likely	O
+reason	O
+for	O
+the	O
+conservation	B-protein_state
+of	O
+the	O
+Thr1	B-residue_name_number
+residue	O
+in	O
+all	O
+proteasomes	B-complex_assembly
+from	O
+bacteria	B-taxonomy_domain
+to	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+(	O
+c	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+propeptide	B-structure_element
+remnants	O
+depict	O
+their	O
+differences	O
+in	O
+conformation	O
+.	O
+
+While	O
+residue	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+of	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+prosegments	B-structure_element
+fit	O
+the	O
+S1	B-site
+pocket	I-site
+,	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+of	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+occupies	O
+the	O
+S2	B-site
+pocket	I-site
+.	O
+
+(	O
+a	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β1	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+and	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+propeptide	B-structure_element
+.	O
+
+(	O
+b	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+β5	B-protein
+propeptides	B-structure_element
+in	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+L	I-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+,	O
+β5	B-mutant
+-(	I-mutant
+H	I-mutant
+-	I-mutant
+2	I-mutant
+)	I-mutant
+A	I-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+and	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	B-protein_state
+proteasomes	B-complex_assembly
+.	O
+
+(	O
+d	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+matured	B-protein_state
+β2	B-protein
+active	B-site
+site	I-site
+,	O
+the	O
+WT	B-protein_state
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+propeptide	B-structure_element
+and	O
+the	O
+β2	B-mutant
+-	I-mutant
+T	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+V	I-mutant
+mutant	B-protein_state
+propeptide	B-structure_element
+.	O
+
+(	O
+a	O
+)	O
+Hydrogen	B-site
+-	I-site
+bonding	I-site
+network	I-site
+at	O
+the	O
+mature	B-protein_state
+WT	B-protein_state
+β5	B-protein
+proteasomal	O
+active	B-site
+site	I-site
+(	O
+dotted	O
+lines	O
+).	O
+
+The	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+is	O
+engaged	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+Ser129Oγ	B-residue_name_number
+,	O
+the	O
+carbonyl	O
+oxygen	O
+of	O
+residue	O
+168	B-residue_number
+,	O
+Ser169Oγ	B-residue_name_number
+and	O
+Asp166Oδ	B-residue_name_number
+.	O
+(	O
+b	O
+)	O
+The	O
+orientations	O
+of	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+residues	I-site
+involved	O
+in	O
+hydrogen	O
+bonding	O
+are	O
+strictly	B-protein_state
+conserved	I-protein_state
+in	O
+each	O
+proteolytic	B-site
+centre	I-site
+,	O
+as	O
+shown	O
+by	O
+superposition	B-experimental_method
+of	O
+the	O
+β	B-protein
+subunits	I-protein
+.	O
+
+The	O
+strictly	B-protein_state
+conserved	I-protein_state
+oxyanion	O
+hole	O
+Gly47NH	B-residue_name_number
+stabilizing	O
+the	O
+negatively	O
+charged	O
+intermediate	O
+is	O
+illustrated	O
+as	O
+a	O
+semicircle	O
+.	O
+
+On	O
+hydrolysis	O
+of	O
+the	O
+latter	O
+,	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+Thr1	B-residue_name_number
+is	O
+ready	O
+for	O
+catalysis	O
+(	O
+right	O
+set	O
+of	O
+structures	O
+).	O
+
+(	O
+a	O
+)	O
+Growth	B-experimental_method
+tests	I-experimental_method
+by	I-experimental_method
+serial	I-experimental_method
+dilution	I-experimental_method
+of	O
+WT	B-protein_state
+and	O
+pre2	O
+(	O
+β5	B-protein
+)	O
+mutant	B-protein_state
+yeast	B-taxonomy_domain
+cultures	O
+reveal	O
+growth	O
+defects	O
+of	O
+the	O
+active	B-site
+-	I-site
+site	I-site
+mutants	B-experimental_method
+under	O
+the	O
+indicated	O
+conditions	O
+after	O
+2	O
+days	O
+(	O
+2	O
+d	O
+)	O
+of	O
+incubation	O
+.	O
+
+Notably	O
+,	O
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+does	O
+not	O
+occupy	O
+the	O
+S1	B-site
+pocket	I-site
+formed	O
+by	O
+Met45	B-residue_name_number
+,	O
+similar	O
+to	O
+what	O
+was	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	B-protein_state
+.	O
+
+(	O
+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
+