diff --git "a/annotation_IOB/test.tsv" "b/annotation_IOB/test.tsv"
new file mode 100644--- /dev/null
+++ "b/annotation_IOB/test.tsv"
@@ -0,0 +1,9956 @@
+In	O
+particular	O
+,	O
+the	O
+molecular	O
+basis	O
+of	O
+complex	B-chemical
+polysaccharide	I-chemical
+recognition	O
+,	O
+an	O
+essential	O
+prerequisite	O
+to	O
+hydrolysis	O
+by	O
+cell	O
+surface	O
+glycosidases	B-protein_type
+and	O
+subsequent	O
+metabolism	O
+,	O
+is	O
+generally	O
+poorly	O
+understood	O
+.	O
+
+The	O
+unique	O
+,	O
+tetra	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+structure	B-evidence
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+is	O
+comprised	O
+of	O
+tandem	B-structure_element
+Ig	I-structure_element
+-	I-structure_element
+like	I-structure_element
+folds	I-structure_element
+,	O
+with	O
+XyG	B-chemical
+binding	O
+mediated	O
+at	O
+the	O
+distal	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+.	O
+
+A	O
+remarkable	O
+feature	O
+of	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+is	O
+the	O
+packaging	O
+of	O
+genes	O
+for	O
+carbohydrate	O
+catabolism	O
+into	O
+discrete	O
+polysaccharide	B-gene
+utilization	I-gene
+loci	I-gene
+(	O
+PUL	B-gene
+),	O
+which	O
+are	O
+transcriptionally	O
+regulated	O
+by	O
+specific	O
+substrate	O
+signatures	O
+.	O
+
+The	O
+importance	O
+of	O
+PUL	B-gene
+as	O
+a	O
+successful	O
+evolutionary	O
+strategy	O
+is	O
+underscored	O
+by	O
+the	O
+observation	O
+that	O
+Bacteroidetes	B-taxonomy_domain
+such	O
+as	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+and	O
+Bacteroides	B-species
+ovatus	I-species
+devote	O
+~	O
+18	O
+%	O
+of	O
+their	O
+genomes	O
+to	O
+these	O
+systems	O
+.	O
+
+Xyloglucan	B-chemical
+and	O
+the	O
+Bacteroides	B-species
+ovatus	I-species
+xyloglucan	B-gene
+utilization	I-gene
+locus	I-gene
+(	O
+XyGUL	B-gene
+).	O
+(	O
+A	O
+)	O
+Representative	O
+structures	B-evidence
+of	O
+common	O
+xyloglucans	B-chemical
+using	O
+the	O
+Consortium	O
+for	O
+Functional	O
+Glycomics	O
+Symbol	O
+Nomenclature	O
+(	O
+http	O
+://	O
+www	O
+.	O
+functionalglycomics	O
+.	O
+org	O
+/	O
+static	O
+/	O
+consortium	O
+/	O
+Nomenclature	O
+.	O
+shtml	O
+).	O
+
+We	O
+describe	O
+here	O
+the	O
+detailed	O
+functional	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+the	O
+noncatalytic	B-protein_state
+SGBPs	B-protein_type
+encoded	O
+by	O
+Bacova_02651	B-gene
+and	O
+Bacova_02650	B-gene
+of	O
+the	O
+XyGUL	B-gene
+,	O
+here	O
+referred	O
+to	O
+as	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+to	O
+elucidate	O
+their	O
+molecular	O
+roles	O
+in	O
+carbohydrate	O
+acquisition	O
+in	O
+vivo	O
+.	O
+
+Here	O
+,	O
+the	O
+SGBPs	B-protein_type
+very	O
+likely	O
+work	O
+in	O
+concert	O
+with	O
+the	O
+cell	B-protein_type
+-	I-protein_type
+surface	I-protein_type
+-	I-protein_type
+localized	I-protein_type
+endo	I-protein_type
+-	I-protein_type
+xyloglucanase	I-protein_type
+B	B-species
+.	I-species
+ovatus	I-species
+GH5	B-protein
+(	O
+BoGH5	B-protein
+)	O
+to	O
+recruit	O
+and	O
+cleave	O
+XyG	B-chemical
+for	O
+subsequent	O
+periplasmic	O
+import	O
+via	O
+the	O
+SusC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+TBDT	I-protein_type
+of	O
+the	O
+XyGUL	B-gene
+(	O
+Fig	O
+.	O
+1B	O
+and	O
+C	O
+).	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+visualized	O
+by	O
+immunofluorescence	B-experimental_method
+.	O
+
+(	O
+D	O
+)	O
+FITC	B-evidence
+images	I-evidence
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+antibodies	O
+.	O
+
+All	O
+samples	O
+were	O
+loaded	O
+on	O
+the	O
+same	O
+gel	O
+next	O
+to	O
+the	O
+BSA	B-protein
+controls	O
+;	O
+thin	O
+black	O
+lines	O
+indicate	O
+where	O
+intervening	O
+lanes	O
+were	O
+removed	O
+from	O
+the	O
+final	O
+image	O
+for	O
+both	O
+space	O
+and	O
+clarity	O
+.	O
+
+ITC	B-experimental_method
+demonstrates	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+binds	O
+to	O
+XyG	B-chemical
+polysaccharide	B-chemical
+and	O
+XyGO2	B-chemical
+(	O
+based	O
+on	O
+a	O
+Glc8	B-structure_element
+backbone	I-structure_element
+)	O
+with	O
+essentially	O
+equal	O
+affinities	B-evidence
+,	O
+while	O
+no	O
+binding	O
+of	O
+XyGO1	B-chemical
+(	O
+Glc4	B-structure_element
+backbone	I-structure_element
+)	O
+was	O
+detectable	O
+(	O
+Table	O
+1	O
+;	O
+see	O
+Fig	O
+.	O
+S2	O
+and	O
+S3	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+Together	O
+,	O
+these	O
+data	O
+clearly	O
+suggest	O
+that	O
+polysaccharide	B-chemical
+binding	O
+of	O
+both	O
+SGBPs	B-protein_type
+is	O
+fulfilled	O
+by	O
+a	O
+dimer	B-oligomeric_state
+of	O
+the	O
+minimal	B-structure_element
+repeat	I-structure_element
+,	O
+corresponding	O
+to	O
+XyGO2	B-chemical
+(	O
+cf	O
+.	O
+
+Summary	O
+of	O
+thermodynamic	O
+parameters	O
+for	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+obtained	O
+by	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+at	O
+25	O
+°	O
+Ca	O
+
+Specifically	O
+,	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+overlays	B-experimental_method
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+SusD	B-protein
+(	O
+BtSusD	B-protein
+)	O
+with	O
+a	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+(	O
+RMSD	B-evidence
+)	O
+value	O
+of	O
+2	O
+.	O
+2	O
+Å	O
+for	O
+363	O
+Cα	O
+pairs	O
+,	O
+which	O
+is	O
+notable	O
+given	O
+the	O
+26	O
+%	O
+amino	O
+acid	O
+identity	O
+(	O
+40	O
+%	O
+similarity	O
+)	O
+between	O
+these	O
+homologs	O
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+
+The	O
+apo	B-protein_state
+structure	B-evidence
+is	O
+color	O
+ramped	O
+from	O
+blue	O
+to	O
+red	O
+.	O
+
+Binding	O
+thermodynamics	O
+are	O
+based	O
+on	O
+the	O
+concentration	O
+of	O
+the	O
+binding	O
+unit	O
+,	O
+XyGO2	B-chemical
+.	O
+
+The	O
+structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+alignment	I-experimental_method
+of	O
+these	O
+proteins	O
+reveals	O
+17	O
+%	O
+sequence	O
+identity	O
+,	O
+with	O
+a	O
+core	O
+RMSD	B-evidence
+of	O
+3	O
+.	O
+6	O
+Å	O
+for	O
+253	O
+aligned	O
+residues	O
+.	O
+
+While	O
+there	O
+is	O
+no	O
+substrate	O
+-	O
+complexed	O
+structure	O
+of	O
+Bacova_04391	B-protein
+available	O
+,	O
+the	O
+binding	B-site
+site	I-site
+is	O
+predicted	O
+to	O
+include	O
+W241	B-residue_name_number
+and	O
+Y404	B-residue_name_number
+,	O
+which	O
+are	O
+proximal	O
+to	O
+the	O
+XyGO	B-site
+binding	I-site
+site	I-site
+in	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+.	O
+However	O
+,	O
+the	O
+opposing	B-protein_state
+,	I-protein_state
+clamp	I-protein_state
+-	I-protein_state
+like	I-protein_state
+arrangement	I-protein_state
+of	O
+these	B-structure_element
+residues	I-structure_element
+in	O
+Bacova_04391	B-protein
+is	O
+clearly	O
+distinct	O
+from	O
+the	O
+planar	B-site
+surface	I-site
+arrangement	I-site
+of	O
+the	O
+residues	B-structure_element
+that	O
+interact	O
+with	O
+XyG	B-chemical
+in	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+described	O
+below	O
+).	O
+
+Inspection	O
+of	O
+the	O
+tertiary	O
+structure	B-evidence
+indicates	O
+that	O
+domains	O
+C	B-structure_element
+and	O
+D	B-structure_element
+are	O
+effectively	O
+inseparable	O
+,	O
+with	O
+a	O
+contact	O
+interface	O
+of	O
+396	O
+Å2	O
+.	O
+
+The	O
+backbone	O
+is	O
+flat	O
+,	O
+with	O
+less	O
+of	O
+the	O
+“	O
+twisted	O
+-	O
+ribbon	O
+”	O
+geometry	O
+observed	O
+in	O
+some	O
+cello	B-chemical
+-	I-chemical
+and	I-chemical
+xylogluco	I-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+.	O
+
+The	O
+aromatic	B-site
+platform	I-site
+created	O
+by	O
+W330	B-residue_name_number
+,	O
+W364	B-residue_name_number
+,	O
+and	O
+Y363	B-residue_name_number
+spans	O
+four	O
+glucosyl	B-chemical
+residues	O
+,	O
+compared	O
+to	O
+the	O
+longer	B-protein_state
+platform	B-site
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+,	O
+which	O
+supports	O
+six	O
+glucosyl	B-chemical
+residues	O
+(	O
+Fig	O
+.	O
+5E	O
+).	O
+
+Additional	O
+residues	B-structure_element
+surrounding	O
+the	O
+binding	B-site
+site	I-site
+,	O
+including	O
+Y369	B-residue_name_number
+and	O
+E412	B-residue_name_number
+,	O
+may	O
+contribute	O
+to	O
+the	O
+recognition	O
+of	O
+more	O
+highly	O
+decorated	O
+XyG	B-chemical
+,	O
+but	O
+precisely	O
+how	O
+this	O
+is	O
+mediated	O
+is	O
+presently	O
+unclear	O
+.	O
+
+While	O
+this	O
+may	O
+occur	O
+for	O
+a	O
+number	O
+of	O
+reasons	O
+in	O
+crystal	B-evidence
+structures	I-evidence
+,	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+poor	O
+ligand	O
+density	O
+even	O
+at	O
+higher	O
+resolution	O
+is	O
+due	O
+to	O
+movement	O
+or	O
+multiple	O
+orientations	O
+of	O
+the	O
+sugar	B-chemical
+averaged	O
+throughout	O
+the	O
+lattice	O
+.	O
+
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+have	O
+distinct	O
+,	O
+coordinated	O
+functions	O
+in	O
+vivo	O
+.	O
+
+To	O
+disentangle	O
+the	O
+functions	O
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+in	O
+XyG	B-chemical
+recognition	O
+and	O
+uptake	O
+,	O
+we	O
+created	O
+individual	O
+in	B-experimental_method
+-	I-experimental_method
+frame	I-experimental_method
+deletion	I-experimental_method
+and	I-experimental_method
+complementation	I-experimental_method
+mutant	I-experimental_method
+strains	O
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+.	O
+
+A	O
+strain	O
+in	O
+which	O
+the	O
+entire	O
+XyGUL	B-gene
+is	O
+deleted	B-experimental_method
+displays	O
+a	O
+lag	B-evidence
+of	O
+24	O
+.	O
+5	O
+h	O
+during	O
+growth	O
+on	O
+glucose	B-chemical
+compared	O
+to	O
+the	O
+isogenic	O
+parental	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+Δtdk	B-mutant
+strain	O
+,	O
+for	O
+which	O
+exponential	O
+growth	O
+lags	B-evidence
+for	O
+19	O
+.	O
+8	O
+h	O
+(	O
+see	O
+Fig	O
+.	O
+S8D	O
+).	O
+
+Complementation	B-experimental_method
+of	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+strain	O
+(	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+::	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+)	O
+restores	O
+growth	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+rates	O
+on	O
+xyloglucan	B-chemical
+and	O
+XyGO1	B-chemical
+,	O
+yet	O
+the	O
+calculated	O
+rate	O
+of	O
+the	O
+complemented	O
+strain	O
+is	O
+~	O
+72	O
+%	O
+that	O
+of	O
+the	O
+WT	B-protein_state
+Δtdk	B-mutant
+strain	O
+on	O
+XyGO2	B-chemical
+;	O
+similar	O
+results	O
+were	O
+obtained	O
+for	O
+the	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+complemented	O
+strain	O
+despite	O
+the	O
+fact	O
+that	O
+the	O
+growth	O
+curves	O
+do	O
+not	O
+appear	O
+much	O
+different	O
+(	O
+see	O
+Fig	O
+.	O
+S8C	O
+and	O
+F	O
+).	O
+
+This	O
+result	O
+mirrors	O
+our	O
+previous	O
+data	O
+for	O
+the	O
+canonical	O
+Sus	B-complex_assembly
+of	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+,	O
+which	O
+revealed	O
+that	O
+a	O
+homologous	O
+ΔsusD	B-mutant
+mutant	B-protein_state
+is	O
+unable	O
+to	O
+grow	O
+on	O
+starch	B-chemical
+or	O
+malto	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+,	O
+despite	O
+normal	O
+cell	O
+surface	O
+expression	O
+of	O
+all	O
+other	O
+PUL	B-gene
+-	O
+encoded	O
+proteins	O
+.	O
+
+More	O
+recently	O
+,	O
+we	O
+demonstrated	O
+that	O
+this	O
+phenotype	O
+is	O
+due	O
+to	O
+the	O
+loss	O
+of	O
+the	O
+physical	O
+presence	O
+of	O
+SusD	B-protein
+;	O
+complementation	B-experimental_method
+of	O
+ΔsusD	B-mutant
+with	O
+SusD	B-mutant
+*,	I-mutant
+a	O
+triple	B-protein_state
+site	I-protein_state
+-	I-protein_state
+directed	I-protein_state
+mutant	I-protein_state
+(	O
+W96A	B-mutant
+W320A	B-mutant
+Y296A	B-mutant
+)	O
+that	O
+ablates	B-protein_state
+glycan	I-protein_state
+binding	I-protein_state
+,	O
+restores	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+growth	O
+on	O
+malto	B-chemical
+-	I-chemical
+oligosaccharides	I-chemical
+and	O
+starch	B-chemical
+when	O
+sus	B-gene
+transcription	O
+is	O
+induced	O
+by	O
+maltose	B-chemical
+addition	O
+.	O
+
+The	O
+specific	O
+glycan	B-chemical
+signal	O
+that	O
+upregulates	O
+BoXyGUL	B-gene
+is	O
+currently	O
+unknown	O
+.	O
+
+The	O
+precise	O
+reason	O
+for	O
+this	O
+lag	B-evidence
+is	O
+unclear	O
+,	O
+but	O
+recapitulating	O
+our	O
+findings	O
+on	O
+the	O
+role	O
+of	O
+SusD	B-protein
+in	O
+malto	B-chemical
+-	I-chemical
+oligosaccharide	I-chemical
+sensing	O
+in	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+,	O
+this	O
+extended	O
+lag	B-evidence
+may	O
+be	O
+due	O
+to	O
+inefficient	O
+import	O
+and	O
+thus	O
+sensing	O
+of	O
+xyloglucan	B-chemical
+in	O
+the	O
+environment	O
+in	O
+the	O
+absence	O
+of	O
+glycan	B-chemical
+binding	O
+by	O
+essential	O
+SGBPs	B-protein_type
+.	O
+
+Our	O
+previous	O
+work	O
+demonstrates	O
+that	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+grown	O
+in	O
+minimal	O
+medium	O
+plus	O
+glucose	B-chemical
+express	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	B-gene
+transcript	O
+.	O
+
+Thus	O
+,	O
+in	O
+our	O
+experiments	O
+,	O
+we	O
+presume	O
+that	O
+each	O
+strain	O
+,	O
+initially	O
+grown	O
+in	O
+glucose	B-chemical
+,	O
+expresses	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	B-gene
+transcript	O
+and	O
+thus	O
+low	O
+levels	O
+of	O
+the	O
+XyGUL	B-gene
+-	O
+encoded	O
+surface	O
+proteins	O
+,	O
+including	O
+the	O
+vanguard	O
+GH5	B-protein
+.	O
+
+Presumably	O
+without	O
+glycan	B-chemical
+binding	O
+by	O
+the	O
+SGBPs	B-protein_type
+,	O
+the	O
+GH5	B-protein
+protein	O
+cannot	O
+efficiently	O
+process	O
+xyloglucan	B-chemical
+,	O
+and	O
+/	O
+or	O
+the	O
+lack	O
+of	O
+SGBP	B-protein_type
+function	O
+prevents	O
+efficient	O
+capture	O
+and	O
+import	O
+of	O
+the	O
+processed	O
+oligosaccharides	B-chemical
+.	O
+
+Likewise	O
+,	O
+such	O
+cognate	O
+interactions	O
+between	O
+homologous	O
+protein	O
+pairs	O
+such	O
+as	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+its	O
+TBDT	B-protein_type
+may	O
+underlie	O
+our	O
+observation	O
+that	O
+a	O
+ΔSGBP	B-mutant
+-	I-mutant
+A	I-mutant
+mutant	B-protein_state
+cannot	O
+grow	O
+on	O
+xyloglucan	B-chemical
+.	O
+
+The	O
+ability	O
+of	O
+gut	O
+-	O
+adapted	O
+microorganisms	B-taxonomy_domain
+to	O
+thrive	O
+in	O
+the	O
+gastrointestinal	O
+tract	O
+is	O
+critically	O
+dependent	O
+upon	O
+their	O
+ability	O
+to	O
+efficiently	O
+recognize	O
+,	O
+cleave	O
+,	O
+and	O
+import	O
+glycans	B-chemical
+.	O
+
+PUL	B-gene
+-	O
+encoded	O
+TBDTs	B-protein_type
+in	O
+Bacteroidetes	B-taxonomy_domain
+are	O
+larger	O
+than	O
+the	O
+well	O
+-	O
+characterized	O
+iron	B-protein_type
+-	I-protein_type
+targeting	I-protein_type
+TBDTs	I-protein_type
+from	O
+many	O
+Proteobacteria	B-taxonomy_domain
+and	O
+are	O
+further	O
+distinguished	O
+as	O
+the	O
+only	O
+known	O
+glycan	B-protein_type
+-	I-protein_type
+importing	I-protein_type
+TBDTs	I-protein_type
+coexpressed	O
+with	O
+an	O
+SGBP	B-protein_type
+.	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+there	O
+is	O
+clear	O
+evidence	O
+for	O
+independent	O
+TBDTs	B-protein_type
+in	O
+Bacteroidetes	B-taxonomy_domain
+that	O
+do	O
+not	O
+require	O
+SGBP	B-protein_type
+association	O
+for	O
+activity	O
+.	O
+
+Furthermore	O
+,	O
+considering	O
+the	O
+broader	O
+distribution	O
+of	O
+TBDTs	B-protein_type
+in	O
+PUL	B-gene
+lacking	O
+SGBPs	B-protein_type
+(	O
+sometimes	O
+known	O
+as	O
+carbohydrate	B-gene
+utilization	I-gene
+containing	I-gene
+TBDT	I-gene
+[	I-gene
+CUT	I-gene
+]	I-gene
+loci	I-gene
+;	O
+see	O
+reference	O
+and	O
+reviewed	O
+in	O
+reference	O
+)	O
+across	O
+bacterial	B-taxonomy_domain
+phyla	O
+,	O
+it	O
+appears	O
+that	O
+the	O
+intimate	O
+biophysical	O
+association	O
+of	O
+these	O
+substrate	O
+-	O
+transport	O
+and	O
+-	O
+binding	O
+proteins	O
+is	O
+the	O
+result	O
+of	O
+specific	O
+evolution	O
+within	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+.	O
+
+Such	O
+is	O
+the	O
+case	O
+for	O
+XyGUL	B-gene
+from	O
+related	O
+Bacteroides	B-taxonomy_domain
+species	O
+,	O
+which	O
+may	O
+encode	O
+either	O
+one	O
+or	O
+two	O
+of	O
+these	O
+predicted	O
+SGBPs	B-protein_type
+,	O
+and	O
+these	O
+proteins	O
+vary	O
+considerably	O
+in	O
+length	O
+.	O
+
+Mucosal	O
+glycan	B-chemical
+foraging	O
+enhances	O
+fitness	O
+and	O
+transmission	O
+of	O
+a	O
+saccharolytic	O
+human	O
+gut	O
+bacterial	O
+symbiont	O
+
+The	O
+PduL	B-protein_type
+fold	B-structure_element
+is	O
+unrelated	O
+to	O
+that	B-structure_element
+of	O
+Pta	B-protein_type
+;	O
+it	O
+contains	O
+a	O
+dimetal	B-site
+active	I-site
+site	I-site
+involved	O
+in	O
+a	O
+catalytic	O
+mechanism	O
+distinct	O
+from	O
+that	O
+of	O
+the	O
+housekeeping	B-protein_state
+PTAC	B-protein_type
+.	O
+
+Substrates	O
+and	O
+cofactors	O
+involving	O
+the	O
+PTAC	B-protein_type
+reaction	O
+are	O
+shown	O
+in	O
+red	O
+;	O
+other	O
+substrates	O
+and	O
+enzymes	O
+are	O
+shown	O
+in	O
+black	O
+,	O
+and	O
+other	O
+cofactors	O
+are	O
+shown	O
+in	O
+gray	O
+.	O
+
+Both	O
+enzymes	O
+are	O
+,	O
+however	O
+,	O
+not	O
+restricted	O
+to	O
+fermentative	B-taxonomy_domain
+organisms	I-taxonomy_domain
+.	O
+
+Remarkably	O
+,	O
+after	O
+removing	B-experimental_method
+the	O
+N	O
+-	O
+terminal	O
+putative	O
+EP	B-structure_element
+(	O
+27	B-residue_range
+amino	I-residue_range
+acids	I-residue_range
+),	O
+most	O
+of	O
+the	O
+sPduLΔEP	B-mutant
+protein	O
+was	O
+in	O
+the	O
+soluble	O
+fraction	O
+upon	O
+cell	O
+lysis	O
+.	O
+
+Similar	O
+differences	O
+in	O
+solubility	O
+were	O
+observed	O
+for	O
+pPduL	B-protein
+and	O
+rPduL	B-protein
+when	O
+comparing	O
+EP	B-protein_state
+-	I-protein_state
+truncated	I-protein_state
+forms	O
+to	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+,	O
+but	O
+none	O
+were	O
+quite	O
+as	O
+dramatic	O
+as	O
+for	O
+sPduL	B-protein
+.	O
+We	O
+confirmed	O
+that	O
+all	O
+homologs	O
+were	O
+active	B-protein_state
+(	O
+S1a	O
+and	O
+S1b	O
+Fig	O
+).	O
+
+Structural	O
+overview	O
+of	O
+R	B-species
+.	I-species
+palustris	I-species
+PduL	B-protein_type
+from	O
+the	O
+grm3	B-gene
+locus	I-gene
+.	O
+
+Metal	B-site
+coordination	I-site
+residues	I-site
+are	O
+highlighted	O
+in	O
+light	O
+blue	O
+and	O
+CoA	B-site
+contacting	I-site
+residues	I-site
+in	O
+magenta	O
+,	O
+residues	O
+contacting	O
+the	O
+CoA	B-chemical
+of	O
+the	O
+other	O
+chain	O
+are	O
+also	O
+outlined	O
+.	O
+
+(	O
+b	O
+)	O
+Cartoon	O
+representation	O
+of	O
+the	O
+structure	B-evidence
+colored	O
+by	O
+domains	O
+and	O
+including	O
+secondary	O
+structure	B-evidence
+numbering	O
+.	O
+
+Residues	O
+100	O
+%	O
+conserved	O
+across	O
+all	O
+PduL	B-protein_type
+homologs	O
+in	O
+our	O
+dataset	O
+are	O
+noted	O
+with	O
+an	O
+asterisk	O
+,	O
+and	O
+residues	O
+conserved	O
+in	O
+over	O
+90	O
+%	O
+of	O
+sequences	O
+are	O
+noted	O
+with	O
+a	O
+colon	O
+.	O
+
+The	O
+sequences	O
+aligning	O
+to	O
+the	O
+PF06130	B-structure_element
+domain	O
+(	O
+determined	O
+by	O
+BLAST	O
+)	O
+are	O
+highlighted	O
+in	O
+red	O
+and	O
+blue	O
+.	O
+
+Distances	O
+between	O
+atom	O
+centers	O
+are	O
+indicated	O
+in	O
+Å	O
+.	O
+(	O
+a	O
+)	O
+Coenzyme	B-chemical
+A	I-chemical
+containing	O
+,	O
+(	O
+b	O
+)	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+.	O
+
+The	O
+asterisk	O
+and	O
+double	O
+arrow	O
+marks	O
+the	O
+location	O
+of	O
+the	O
+π	O
+–	O
+π	O
+interaction	O
+between	O
+F116	B-residue_name_number
+and	O
+the	O
+CoA	B-chemical
+base	O
+of	O
+the	O
+other	O
+dimer	B-oligomeric_state
+chain	O
+.	O
+
+The	O
+first	O
+zinc	B-chemical
+ion	O
+(	O
+Zn1	B-chemical
+)	O
+is	O
+in	O
+a	O
+tetrahedral	O
+coordination	O
+state	O
+with	O
+His48	B-residue_name_number
+,	O
+His50	B-residue_name_number
+,	O
+Glu109	B-residue_name_number
+,	O
+and	O
+the	O
+CoA	B-chemical
+sulfur	B-chemical
+(	O
+Fig	O
+4a	O
+).	O
+
+The	O
+phosphate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+aligns	B-experimental_method
+well	O
+with	O
+the	O
+CoA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+(	O
+0	O
+.	O
+43	O
+Å	O
+rmsd	B-evidence
+over	O
+2	O
+,	O
+361	O
+atoms	O
+for	O
+the	O
+monomer	B-oligomeric_state
+,	O
+0	O
+.	O
+83	O
+Å	O
+over	O
+5	O
+,	O
+259	O
+aligned	O
+atoms	O
+for	O
+the	O
+dimer	B-oligomeric_state
+).	O
+
+Upon	O
+deletion	B-experimental_method
+of	O
+the	O
+putative	O
+EP	B-structure_element
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+47	I-residue_range
+for	O
+rPduL	B-protein
+,	O
+and	O
+1	B-residue_range
+–	I-residue_range
+20	I-residue_range
+for	O
+pPduL	B-protein
+),	O
+there	O
+was	O
+a	O
+distinct	O
+change	O
+in	O
+the	O
+elution	O
+profiles	O
+(	O
+Fig	O
+5b	O
+and	O
+5c	O
+respectively	O
+,	O
+blue	O
+curves	O
+).	O
+
+In	O
+contrast	O
+,	O
+rPduLΔEP	B-mutant
+eluted	O
+as	O
+one	O
+smaller	O
+oligomer	O
+,	O
+possibly	O
+a	O
+dimer	B-oligomeric_state
+.	O
+
+Curiously	O
+,	O
+while	O
+the	O
+housekeeping	B-protein_state
+Pta	B-protein_type
+could	O
+provide	O
+this	O
+function	O
+,	O
+and	O
+indeed	O
+does	O
+so	O
+in	O
+the	O
+case	O
+of	O
+one	O
+type	O
+of	O
+ethanolamine	B-complex_assembly
+-	I-complex_assembly
+utilizing	I-complex_assembly
+(	I-complex_assembly
+EUT	I-complex_assembly
+)	I-complex_assembly
+BMC	I-complex_assembly
+,	O
+the	O
+evolutionarily	O
+unrelated	O
+PduL	B-protein_type
+fulfills	O
+this	O
+function	O
+for	O
+the	O
+majority	O
+of	O
+metabolosomes	B-complex_assembly
+using	O
+a	O
+novel	O
+structure	B-evidence
+and	O
+active	B-site
+site	I-site
+for	O
+convergent	O
+evolution	O
+of	O
+function	O
+.	O
+
+Refined	O
+domain	O
+assignment	O
+based	O
+on	O
+our	O
+structure	B-evidence
+should	O
+be	O
+able	O
+to	O
+predict	O
+domains	O
+of	O
+PF06130	B-structure_element
+homologs	O
+much	O
+more	O
+accurately	O
+.	O
+
+For	O
+BMC	B-complex_assembly
+-	O
+encapsulated	O
+proteins	O
+to	O
+properly	O
+function	O
+together	O
+,	O
+they	O
+must	O
+be	O
+targeted	O
+to	O
+the	O
+lumen	O
+and	O
+assemble	O
+into	O
+an	O
+organization	O
+that	O
+facilitates	O
+substrate	O
+/	O
+product	O
+channeling	O
+among	O
+the	O
+different	O
+catalytic	B-site
+sites	I-site
+of	O
+the	O
+signature	O
+and	O
+core	O
+enzymes	O
+.	O
+
+All	O
+of	O
+the	O
+metal	B-site
+-	I-site
+coordinating	I-site
+residues	I-site
+(	O
+Fig	O
+2a	O
+)	O
+are	O
+absolutely	B-protein_state
+conserved	I-protein_state
+,	O
+implicating	O
+them	O
+in	O
+catalysis	O
+or	O
+the	O
+correct	O
+spatial	O
+orientation	O
+of	O
+the	O
+substrates	O
+.	O
+
+However	O
+,	O
+for	O
+the	O
+clostripain	B-protein_type
+family	I-protein_type
+(	O
+denoted	O
+C11	B-protein_type
+),	O
+little	O
+is	O
+currently	O
+known	O
+.	O
+
+The	O
+structure	B-experimental_method
+was	I-experimental_method
+analyzed	I-experimental_method
+,	O
+and	O
+the	O
+enzyme	O
+was	O
+biochemically	B-experimental_method
+characterized	I-experimental_method
+to	O
+provide	O
+the	O
+first	O
+structure	O
+/	O
+function	O
+correlation	O
+for	O
+a	O
+C11	B-protein_type
+peptidase	I-protein_type
+.	O
+
+A	O
+single	B-ptm
+cleavage	I-ptm
+was	O
+observed	O
+in	O
+the	O
+polypeptide	O
+chain	O
+at	O
+Lys147	B-residue_name_number
+(	O
+Fig	O
+.	O
+1	O
+,	O
+A	O
+and	O
+B	O
+),	O
+where	O
+both	O
+ends	O
+of	O
+the	O
+cleavage	B-site
+site	I-site
+are	O
+fully	O
+visible	O
+and	O
+well	O
+ordered	O
+in	O
+the	O
+electron	B-evidence
+density	I-evidence
+.	O
+
+The	O
+secondary	O
+structure	O
+of	O
+PmC11	B-protein
+from	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+is	O
+mapped	O
+onto	O
+its	O
+sequence	O
+with	O
+the	O
+position	O
+of	O
+the	O
+PmC11	B-protein
+catalytic	B-site
+dyad	I-site
+,	O
+autocatalytic	B-site
+cleavage	I-site
+site	I-site
+(	O
+Lys147	B-residue_name_number
+),	O
+and	O
+S1	B-site
+binding	I-site
+pocket	I-site
+Asp	B-residue_name
+(	O
+Asp177	B-residue_name_number
+)	O
+highlighted	O
+by	O
+a	O
+red	O
+star	O
+,	O
+a	O
+red	O
+downturned	O
+triangle	O
+,	O
+and	O
+a	O
+red	O
+upturned	O
+triangle	O
+,	O
+respectively	O
+.	O
+
+Sequences	O
+around	O
+the	O
+catalytic	B-site
+site	I-site
+of	O
+clostripain	B-protein
+and	O
+PmC11	B-protein
+align	O
+well	O
+.	O
+
+The	O
+CTD	B-structure_element
+of	O
+PmC11	B-protein
+is	O
+composed	O
+of	O
+a	O
+tight	B-structure_element
+helical	I-structure_element
+bundle	I-structure_element
+formed	O
+from	O
+helices	B-structure_element
+α8	B-structure_element
+–	I-structure_element
+α14	I-structure_element
+and	O
+includes	O
+strands	B-structure_element
+βC	B-structure_element
+and	O
+βF	B-structure_element
+,	O
+and	O
+β	B-structure_element
+-	I-structure_element
+hairpin	I-structure_element
+βD	B-structure_element
+–	I-structure_element
+βE	I-structure_element
+.	O
+The	O
+CTD	B-structure_element
+sits	O
+entirely	O
+on	O
+one	O
+side	O
+of	O
+the	O
+enzyme	O
+interacting	O
+only	O
+with	O
+α3	B-structure_element
+,	O
+α5	B-structure_element
+,	O
+β9	B-structure_element
+,	O
+and	O
+the	O
+loops	B-structure_element
+surrounding	O
+β8	B-structure_element
+.	O
+
+Biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+PmC11	B-protein
+.	O
+
+A	O
+,	O
+ribbon	O
+representation	O
+of	O
+the	O
+overall	O
+structure	O
+of	O
+PmC11	B-protein
+illustrating	O
+the	O
+catalytic	B-site
+site	I-site
+,	O
+cleavage	O
+site	O
+displacement	O
+,	O
+and	O
+potential	O
+S1	B-site
+binding	I-site
+site	I-site
+.	O
+
+Km	O
+and	O
+Vmax	B-evidence
+of	O
+PmC11	B-protein
+and	O
+K147A	B-mutant
+mutant	O
+were	O
+determined	O
+by	O
+monitoring	O
+change	O
+in	O
+the	O
+fluorescence	O
+corresponding	O
+to	O
+AMC	O
+release	O
+from	O
+Bz	B-chemical
+-	I-chemical
+R	I-chemical
+-	I-chemical
+AMC	I-chemical
+.	O
+
+Five	O
+of	O
+the	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+surrounding	O
+the	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+of	O
+PmC11	B-protein
+(	O
+α1	B-structure_element
+,	O
+α2	B-structure_element
+,	O
+α4	B-structure_element
+,	O
+α6	B-structure_element
+,	O
+and	O
+α7	B-structure_element
+)	O
+are	O
+found	O
+in	O
+similar	O
+positions	O
+to	O
+the	O
+five	O
+structurally	B-protein_state
+conserved	I-protein_state
+helices	B-structure_element
+in	O
+caspases	B-protein_type
+and	O
+other	O
+members	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+,	O
+apart	O
+from	O
+family	O
+C80	B-protein_type
+.	O
+
+Autoprocessing	B-ptm
+of	O
+PmC11	B-protein
+
+Substrate	O
+Specificity	O
+of	O
+PmC11	B-protein
+
+The	O
+autocatalytic	B-ptm
+cleavage	I-ptm
+of	O
+PmC11	B-protein
+at	O
+Lys147	B-residue_name_number
+(	O
+sequence	O
+KLK	O
+∧	O
+A	O
+)	O
+demonstrates	O
+that	O
+the	O
+enzyme	O
+accepts	O
+substrates	O
+with	O
+Lys	B-residue_name
+in	O
+the	O
+P1	B-residue_number
+position	O
+.	O
+
+This	O
+pocket	B-site
+is	O
+lined	O
+with	O
+the	O
+potential	O
+functional	O
+side	O
+chains	O
+of	O
+Asn50	B-residue_name_number
+,	O
+Asp177	B-residue_name_number
+,	O
+and	O
+Thr204	B-residue_name_number
+with	O
+Gly134	B-residue_name_number
+,	O
+Asp207	B-residue_name_number
+,	O
+and	O
+Met205	B-residue_name_number
+also	O
+contributing	O
+to	O
+the	O
+pocket	B-site
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+Thus	O
+,	O
+Asn50	B-residue_name_number
+,	O
+Asp177	B-residue_name_number
+,	O
+and	O
+Asp207	B-residue_name_number
+are	O
+most	O
+likely	O
+responsible	O
+for	O
+the	O
+substrate	O
+specificity	O
+of	O
+PmC11	B-protein
+.	O
+
+Cleavage	O
+of	O
+Bz	B-chemical
+-	I-chemical
+R	I-chemical
+-	I-chemical
+AMC	I-chemical
+by	O
+PmC11	B-protein
+was	O
+measured	O
+in	O
+a	O
+fluorometric	B-experimental_method
+activity	I-experimental_method
+assay	I-experimental_method
+with	O
+(+,	O
+purple	O
+)	O
+and	O
+without	O
+(−,	O
+red	O
+)	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+.	O
+
+A	O
+three	B-experimental_method
+-	I-experimental_method
+dimensional	I-experimental_method
+structural	I-experimental_method
+overlay	I-experimental_method
+of	O
+Z	B-chemical
+-	I-chemical
+VRPR	I-chemical
+-	I-chemical
+FMK	I-chemical
+from	O
+the	O
+MALT1	B-protein
+-	I-protein
+P	I-protein
+complex	O
+onto	O
+PmC11	B-protein
+.	O
+
+Comparison	O
+with	O
+Clostripain	B-protein
+
+In	O
+support	O
+of	O
+these	O
+findings	O
+,	O
+EGTA	B-chemical
+did	O
+not	O
+inhibit	O
+PmC11	B-protein
+suggesting	O
+that	O
+,	O
+unlike	O
+clostripain	B-protein
+,	O
+PmC11	B-protein
+does	O
+not	O
+require	O
+Ca2	B-chemical
++	I-chemical
+or	O
+other	O
+divalent	O
+cations	O
+,	O
+for	O
+activity	O
+.	O
+
+Several	O
+other	O
+members	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+require	O
+processing	B-ptm
+for	O
+full	B-protein_state
+activation	I-protein_state
+including	O
+legumain	B-protein
+,	O
+gingipain	B-protein
+-	I-protein
+R	I-protein
+,	O
+MARTX	B-protein
+-	I-protein
+CPD	I-protein
+,	O
+and	O
+the	O
+effector	B-protein_type
+caspases	I-protein_type
+,	O
+e	O
+.	O
+g	O
+.	O
+caspase	B-protein
+-	I-protein
+7	I-protein
+.	O
+
+Structural	O
+insights	O
+into	O
+the	O
+regulatory	O
+mechanism	O
+of	O
+the	O
+Pseudomonas	B-species
+aeruginosa	I-species
+YfiBNR	B-complex_assembly
+system	O
+
+In	O
+response	O
+to	O
+cell	O
+stress	O
+,	O
+YfiB	B-protein
+in	O
+the	O
+outer	O
+membrane	O
+can	O
+sequester	O
+the	O
+periplasmic	O
+protein	O
+YfiR	B-protein
+,	O
+releasing	O
+its	O
+inhibition	O
+of	O
+YfiN	B-protein
+on	O
+the	O
+inner	O
+membrane	O
+and	O
+thus	O
+provoking	O
+the	O
+diguanylate	O
+cyclase	O
+activity	O
+of	O
+YfiN	B-protein
+to	O
+induce	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+production	O
+.	O
+
+Biofilm	O
+formation	O
+protects	O
+pathogenic	O
+bacteria	B-taxonomy_domain
+from	O
+antibiotic	O
+treatment	O
+,	O
+and	O
+c	O
+-	O
+di	O
+-	O
+GMP	O
+-	O
+regulated	O
+biofilm	O
+formation	O
+has	O
+been	O
+extensively	O
+studied	O
+in	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+(	O
+Evans	O
+,;	O
+Kirisits	O
+et	O
+al	O
+.,;	O
+Malone	O
+,;	O
+Reinhardt	O
+et	O
+al	O
+.,).	O
+
+Recently	O
+,	O
+Malone	O
+and	O
+coworkers	O
+identified	O
+the	O
+tripartite	B-protein_state
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+signaling	O
+module	O
+system	O
+YfiBNR	B-complex_assembly
+(	O
+also	O
+known	O
+as	O
+AwsXRO	B-complex_assembly
+(	O
+Beaumont	O
+et	O
+al	O
+.,;	O
+Giddens	O
+et	O
+al	O
+.,)	O
+or	O
+Tbp	B-complex_assembly
+(	O
+Ueda	O
+and	O
+Wood	O
+,))	O
+by	O
+genetic	B-experimental_method
+screening	I-experimental_method
+for	O
+mutants	O
+that	O
+displayed	O
+SCV	O
+phenotypes	O
+in	O
+P	B-species
+.	I-species
+aeruginosa	I-species
+PAO1	I-species
+(	O
+Malone	O
+et	O
+al	O
+.,;	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+More	O
+recently	O
+,	O
+this	O
+system	O
+was	O
+also	O
+reported	O
+in	O
+other	O
+Gram	B-taxonomy_domain
+-	I-taxonomy_domain
+negative	I-taxonomy_domain
+bacteria	I-taxonomy_domain
+,	O
+such	O
+as	O
+Escherichia	B-species
+coli	I-species
+(	O
+Hufnagel	O
+et	O
+al	O
+.,;	O
+Raterman	O
+et	O
+al	O
+.,;	O
+Sanchez	O
+-	O
+Torres	O
+et	O
+al	O
+.,),	O
+Klebsiella	B-species
+pneumonia	I-species
+(	O
+Huertas	O
+et	O
+al	O
+.,)	O
+and	O
+Yersinia	B-species
+pestis	I-species
+(	O
+Ren	O
+et	O
+al	O
+.,).	O
+
+After	O
+the	O
+sequestration	O
+of	O
+YfiR	B-protein
+by	O
+YfiB	B-protein
+,	O
+the	O
+c	B-chemical
+-	I-chemical
+di	I-chemical
+-	I-chemical
+GMP	I-chemical
+produced	O
+by	O
+activated	B-protein_state
+YfiN	B-protein
+increases	O
+the	O
+biosynthesis	O
+of	O
+the	O
+Pel	B-chemical
+and	O
+Psl	B-chemical
+EPSs	B-chemical
+,	O
+resulting	O
+in	O
+the	O
+appearance	O
+of	O
+the	O
+SCV	O
+phenotype	O
+,	O
+which	O
+indicates	O
+enhanced	O
+biofilm	O
+formation	O
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+Recently	O
+,	O
+we	O
+solved	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+YfiR	B-protein
+in	O
+both	O
+the	O
+non	B-protein_state
+-	I-protein_state
+oxidized	I-protein_state
+and	O
+the	O
+oxidized	B-protein_state
+states	O
+,	O
+revealing	O
+breakage	O
+/	O
+formation	O
+of	O
+one	O
+disulfide	B-ptm
+bond	I-ptm
+(	O
+Cys71	B-residue_name_number
+-	O
+Cys110	B-residue_name_number
+)	O
+and	O
+local	O
+conformational	O
+change	O
+around	O
+the	O
+other	O
+one	O
+(	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+),	O
+indicating	O
+that	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+plays	O
+an	O
+important	O
+role	O
+in	O
+maintaining	O
+the	O
+correct	O
+folding	O
+of	O
+YfiR	B-protein
+(	O
+Yang	O
+et	O
+al	O
+.,).	O
+
+We	O
+obtained	O
+two	O
+crystal	B-evidence
+forms	I-evidence
+of	O
+YfiB	B-protein
+(	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+168	I-residue_range
+,	O
+lacking	B-protein_state
+the	O
+signal	B-structure_element
+peptide	I-structure_element
+from	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+26	I-residue_range
+and	O
+periplasmic	O
+residues	O
+27	B-residue_range
+–	I-residue_range
+33	I-residue_range
+),	O
+crystal	O
+forms	O
+I	O
+and	O
+II	O
+,	O
+belonging	O
+to	O
+space	O
+groups	O
+P21	O
+and	O
+P41	O
+,	O
+respectively	O
+.	O
+
+The	O
+“	O
+back	B-protein_state
+to	I-protein_state
+back	I-protein_state
+”	O
+dimer	B-oligomeric_state
+presents	O
+a	O
+Y	B-protein_state
+shape	I-protein_state
+.	O
+
+Therefore	O
+,	O
+we	O
+constructed	B-experimental_method
+two	I-experimental_method
+such	I-experimental_method
+single	I-experimental_method
+mutants	I-experimental_method
+of	O
+YfiB	B-protein
+(	O
+YfiBL43P	B-mutant
+and	O
+YfiBF48S	B-mutant
+).	O
+
+The	O
+N	O
+-	O
+terminal	O
+structural	O
+conformation	O
+of	O
+YfiBL43P	B-mutant
+,	O
+from	O
+the	O
+foremost	O
+N	O
+-	O
+terminus	O
+to	O
+residue	O
+D70	B-residue_name_number
+,	O
+is	O
+significantly	O
+altered	O
+compared	O
+with	O
+that	O
+of	O
+the	O
+apo	B-protein_state
+YfiB	B-protein
+.	O
+The	O
+majority	O
+of	O
+the	O
+α1	B-structure_element
+helix	I-structure_element
+(	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+43	I-residue_range
+)	O
+is	O
+invisible	O
+on	O
+the	O
+electron	B-evidence
+density	I-evidence
+map	I-evidence
+,	O
+and	O
+the	O
+α2	B-structure_element
+helix	I-structure_element
+and	O
+β1	B-structure_element
+and	O
+β2	B-structure_element
+strands	I-structure_element
+are	O
+rearranged	O
+to	O
+form	O
+a	O
+long	O
+loop	B-structure_element
+containing	O
+two	O
+short	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+turns	I-structure_element
+(	O
+Fig	O
+.	O
+3B	O
+and	O
+3C	O
+),	O
+thus	O
+embracing	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+.	O
+
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+is	O
+shown	O
+in	O
+cyan	O
+;	O
+the	O
+sulfate	B-chemical
+ion	O
+,	O
+in	O
+green	O
+;	O
+and	O
+the	O
+water	B-chemical
+molecule	O
+,	O
+in	O
+yellow	O
+.	O
+(	O
+D	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+PG	B-site
+-	I-site
+binding	I-site
+sites	I-site
+of	O
+apo	B-protein_state
+YfiB	B-protein
+and	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+,	O
+the	O
+key	O
+residues	O
+are	O
+shown	O
+in	O
+stick	O
+.	O
+
+Similarly	O
+,	O
+in	O
+the	O
+YfiR	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+YfiBL43P	B-mutant
+structure	B-evidence
+,	O
+the	O
+sulfate	B-chemical
+ion	O
+interacts	O
+with	O
+the	O
+side	O
+-	O
+chain	O
+atoms	O
+of	O
+D102	B-residue_name_number
+(	O
+corresponding	O
+to	O
+D71	B-residue_name_number
+in	O
+Pal	B-protein_type
+)	O
+and	O
+R117	B-residue_name_number
+(	O
+corresponding	O
+to	O
+R86	B-residue_name_number
+in	O
+Pal	B-protein_type
+)	O
+and	O
+the	O
+main	O
+-	O
+chain	O
+amide	O
+of	O
+N68	B-residue_name_number
+(	O
+corresponding	O
+to	O
+D37	B-residue_name_number
+in	O
+Pal	B-protein_type
+).	O
+
+Therefore	O
+,	O
+we	O
+proposed	O
+that	O
+the	O
+PG	B-chemical
+-	O
+binding	O
+ability	O
+of	O
+inactive	B-protein_state
+YfiB	B-protein
+might	O
+be	O
+weaker	O
+than	O
+that	O
+of	O
+active	B-protein_state
+YfiB	B-protein
+.	O
+To	O
+validate	O
+this	O
+,	O
+we	O
+performed	O
+a	O
+microscale	B-experimental_method
+thermophoresis	I-experimental_method
+(	O
+MST	B-experimental_method
+)	O
+assay	O
+to	O
+measure	O
+the	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+PG	B-chemical
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+and	O
+YfiBL43P	B-mutant
+,	O
+respectively	O
+.	O
+
+As	O
+the	O
+experiment	O
+is	O
+performed	O
+in	B-protein_state
+the	I-protein_state
+absence	I-protein_state
+of	I-protein_state
+YfiR	B-protein
+,	O
+it	O
+suggests	O
+that	O
+an	O
+increase	O
+in	O
+the	O
+PG	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+YfiB	B-protein
+is	O
+not	O
+a	O
+result	O
+of	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+and	O
+is	O
+highly	O
+coupled	O
+to	O
+the	O
+activation	O
+of	O
+YfiB	B-protein
+characterized	O
+by	O
+a	O
+stretched	B-protein_state
+N	I-protein_state
+-	I-protein_state
+terminal	I-protein_state
+conformation	I-protein_state
+.	O
+
+Malone	O
+JG	O
+et	O
+al	O
+.	O
+have	O
+reported	O
+that	O
+F151	B-residue_name_number
+,	O
+E163	B-residue_name_number
+,	O
+I169	B-residue_name_number
+and	O
+Q187	B-residue_name_number
+,	O
+located	O
+near	O
+the	O
+C	O
+-	O
+terminus	O
+of	O
+YfiR	B-protein
+,	O
+comprise	O
+a	O
+putative	O
+YfiN	B-site
+binding	I-site
+site	I-site
+(	O
+Malone	O
+et	O
+al	O
+.,).	O
+
+Interestingly	O
+,	O
+these	O
+residues	O
+are	O
+part	O
+of	O
+the	O
+conserved	B-site
+surface	I-site
+of	O
+YfiR	B-protein
+(	O
+Fig	O
+.	O
+3G	O
+).	O
+
+F151	B-residue_name_number
+,	O
+E163	B-residue_name_number
+and	O
+I169	B-residue_name_number
+form	O
+a	O
+hydrophobic	B-site
+core	I-site
+while	O
+,	O
+Q187	B-residue_name_number
+is	O
+located	O
+at	O
+the	O
+end	O
+of	O
+the	O
+α6	B-structure_element
+helix	I-structure_element
+.	O
+
+E163	B-residue_name_number
+and	O
+I169	B-residue_name_number
+are	O
+YfiB	B-site
+-	I-site
+interacting	I-site
+residues	I-site
+of	O
+YfiR	B-protein
+,	O
+in	O
+which	O
+E163	B-residue_name_number
+forms	O
+a	O
+hydrogen	O
+bond	O
+with	O
+R96	B-residue_name_number
+of	O
+YfiB	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+II	O
+)	O
+and	O
+I169	B-residue_name_number
+is	O
+involved	O
+in	O
+forming	O
+the	O
+L166	B-residue_name_number
+/	O
+I169	B-residue_name_number
+/	O
+V176	B-residue_name_number
+/	O
+P178	B-residue_name_number
+/	O
+L181	B-residue_name_number
+hydrophobic	B-site
+core	I-site
+for	O
+anchoring	O
+F57	B-residue_name_number
+of	O
+YfiB	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+ii	O
+)).	O
+
+(	O
+C	O
+and	O
+D	O
+)	O
+BIAcore	B-experimental_method
+data	O
+and	O
+analysis	O
+for	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+(	O
+C	O
+)	O
+VB6	B-chemical
+and	O
+(	O
+D	O
+)	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+with	O
+YfiR	B-protein
+.	O
+(	O
+E	O
+–	O
+G	O
+)	O
+ITC	B-experimental_method
+data	O
+and	O
+analysis	O
+for	O
+titration	B-experimental_method
+of	O
+(	O
+E	O
+)	O
+YfiB	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+,	O
+(	O
+F	O
+)	O
+YfiBL43P	O
+,	O
+and	O
+(	O
+G	O
+)	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+into	O
+YfiR	B-protein
+
+Structural	B-experimental_method
+analyses	I-experimental_method
+revealed	O
+that	O
+the	O
+VB6	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+Trp	I-chemical
+molecules	O
+are	O
+bound	B-protein_state
+at	I-protein_state
+the	O
+periphery	O
+of	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+,	O
+but	O
+not	O
+at	O
+the	O
+dimer	B-site
+interface	I-site
+.	O
+
+To	O
+evaluate	O
+the	O
+importance	O
+of	O
+F57	B-residue_name_number
+in	O
+YfiBL43P	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+,	O
+the	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+YfiBL43P	B-mutant
+and	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+for	O
+YfiR	B-protein
+were	O
+measured	O
+by	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+ITC	B-experimental_method
+).	O
+
+Provided	O
+that	O
+the	O
+diameter	O
+of	O
+the	O
+widest	O
+part	O
+of	O
+the	O
+YfiB	B-protein
+dimer	B-oligomeric_state
+is	O
+approximately	O
+64	O
+Å	O
+,	O
+which	O
+is	O
+slightly	O
+smaller	O
+than	O
+the	O
+smallest	O
+diameter	O
+of	O
+the	O
+PG	O
+pore	O
+(	O
+70	O
+Å	O
+)	O
+(	O
+Meroueh	O
+et	O
+al	O
+.,),	O
+the	O
+YfiB	B-protein
+dimer	B-oligomeric_state
+should	O
+be	O
+able	O
+to	O
+penetrate	O
+the	O
+PG	O
+layer	O
+.	O
+
+A	O
+direct	O
+link	O
+between	O
+ACC	B-protein_type
+and	O
+cancer	O
+is	O
+provided	O
+by	O
+cancer	O
+-	O
+associated	O
+mutations	B-mutant
+in	O
+the	O
+breast	B-protein
+cancer	I-protein
+susceptibility	I-protein
+gene	I-protein
+1	I-protein
+(	O
+BRCA1	B-protein
+),	O
+which	O
+relieve	O
+inhibitory	O
+interactions	O
+of	O
+BRCA1	B-protein
+with	O
+ACC	B-protein_type
+.	O
+
+Structural	B-experimental_method
+studies	I-experimental_method
+on	O
+the	O
+functional	O
+architecture	O
+of	O
+intact	B-protein_state
+ACCs	B-protein_type
+have	O
+been	O
+hindered	O
+by	O
+their	O
+huge	O
+size	O
+and	O
+pronounced	O
+dynamics	O
+,	O
+as	O
+well	O
+as	O
+the	O
+transient	B-protein_state
+assembly	O
+mode	O
+of	O
+bacterial	B-taxonomy_domain
+ACCs	B-protein_type
+.	O
+
+Phosphorylated	B-protein_state
+Ser80	B-residue_name_number
+,	O
+which	O
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+only	O
+in	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+,	O
+presumably	O
+binds	O
+into	O
+the	O
+Soraphen	B-site
+A	I-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+The	O
+regulatory	O
+Ser1201	B-residue_name_number
+shows	O
+only	O
+moderate	B-protein_state
+conservation	I-protein_state
+across	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+,	O
+while	O
+the	O
+phosphorylated	B-protein_state
+Ser1216	B-residue_name_number
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+across	O
+all	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+In	O
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+phosphorylation	B-site
+sites	I-site
+have	O
+been	O
+identified	O
+at	O
+Ser2	B-residue_name_number
+,	O
+Ser735	B-residue_name_number
+,	O
+Ser1148	B-residue_name_number
+,	O
+Ser1157	B-residue_name_number
+and	O
+Ser1162	B-residue_name_number
+(	O
+ref	O
+.).	O
+
+CDL	B-structure_element
+is	O
+composed	O
+of	O
+a	O
+small	B-structure_element
+,	I-structure_element
+irregular	I-structure_element
+four	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+(	O
+Lα1	B-structure_element
+–	I-structure_element
+4	I-structure_element
+)	O
+and	O
+tightly	O
+interacts	O
+with	O
+the	O
+open	O
+face	O
+of	O
+CDC1	B-structure_element
+via	O
+an	O
+interface	B-site
+of	O
+1	O
+,	O
+300	O
+Å2	O
+involving	O
+helices	B-structure_element
+Lα3	B-structure_element
+and	O
+Lα4	B-structure_element
+.	O
+
+To	O
+define	O
+the	O
+functional	O
+state	O
+of	O
+insect	B-experimental_method
+-	I-experimental_method
+cell	I-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+ACC	B-protein_type
+variants	O
+,	O
+we	O
+employed	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+(	O
+MS	B-experimental_method
+)	O
+for	O
+phosphorylation	B-experimental_method
+site	I-experimental_method
+detection	I-experimental_method
+.	O
+
+The	O
+N	O
+-	O
+terminal	O
+region	O
+of	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+also	O
+directly	O
+contacts	O
+the	O
+C	O
+-	O
+terminal	O
+region	O
+of	O
+CDC2	B-structure_element
+leading	O
+into	O
+CT	B-structure_element
+.	O
+
+Phosphoserine	B-residue_name_number
+1157	I-residue_name_number
+is	O
+tightly	O
+bound	O
+by	O
+two	O
+highly	B-protein_state
+conserved	I-protein_state
+arginines	B-residue_name
+(	O
+Arg1173	B-residue_name_number
+and	O
+Arg1260	B-residue_name_number
+)	O
+of	O
+CDC1	B-structure_element
+(	O
+Fig	O
+.	O
+1d	O
+).	O
+
+The	O
+values	O
+obtained	O
+for	O
+dephosphorylated	B-protein_state
+SceACC	B-protein
+are	O
+comparable	O
+to	O
+earlier	O
+measurements	O
+of	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+expressed	B-experimental_method
+in	I-experimental_method
+E	B-species
+.	I-species
+coli	I-species
+.	O
+
+To	O
+compare	O
+the	O
+organization	O
+of	O
+fungal	B-taxonomy_domain
+and	O
+human	B-species
+ACC	B-protein_type
+CD	B-structure_element
+,	O
+we	O
+determined	B-experimental_method
+the	I-experimental_method
+structure	I-experimental_method
+of	O
+a	O
+human	B-species
+ACC1	B-mutant
+fragment	I-mutant
+that	O
+comprises	O
+the	O
+BT	B-structure_element
+and	O
+CD	B-structure_element
+domains	O
+(	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+),	O
+but	O
+lacks	B-protein_state
+the	O
+mobile	O
+BCCP	B-structure_element
+in	O
+between	O
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+
+An	O
+experimentally	B-evidence
+phased	I-evidence
+map	I-evidence
+was	O
+obtained	O
+at	O
+3	O
+.	O
+7	O
+Å	O
+resolution	O
+for	O
+a	O
+cadmium	B-chemical
+-	O
+derivatized	O
+crystal	O
+and	O
+was	O
+interpreted	O
+by	O
+a	O
+poly	O
+-	O
+alanine	O
+model	O
+(	O
+Fig	O
+.	O
+1e	O
+and	O
+Table	O
+1	O
+).	O
+
+Each	O
+of	O
+the	O
+four	O
+CD	B-structure_element
+domains	O
+in	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+individually	O
+resembles	O
+the	O
+corresponding	O
+SceCD	B-species
+domain	O
+;	O
+however	O
+,	O
+human	B-species
+and	O
+yeast	B-taxonomy_domain
+CDs	B-structure_element
+exhibit	O
+distinct	O
+overall	O
+structures	B-evidence
+.	O
+
+The	O
+BT	B-structure_element
+domain	O
+of	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+consists	O
+of	O
+a	O
+helix	B-structure_element
+that	O
+is	O
+surrounded	O
+at	O
+its	O
+N	O
+terminus	O
+by	O
+an	O
+antiparallel	B-structure_element
+eight	I-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+barrel	I-structure_element
+.	O
+
+It	O
+resembles	O
+the	O
+BT	B-structure_element
+of	O
+propionyl	B-protein_type
+-	I-protein_type
+CoA	I-protein_type
+carboxylase	I-protein_type
+;	O
+only	O
+the	O
+four	O
+C	O
+-	O
+terminal	O
+strands	B-structure_element
+of	I-structure_element
+the	I-structure_element
+β	I-structure_element
+-	I-structure_element
+barrel	I-structure_element
+are	O
+slightly	O
+tilted	O
+.	O
+
+On	O
+the	O
+basis	O
+of	O
+MS	B-experimental_method
+analysis	O
+of	O
+insect	B-experimental_method
+-	I-experimental_method
+cell	I-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+human	B-species
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+ACC	B-protein_type
+,	O
+Ser80	B-residue_name_number
+shows	O
+the	O
+highest	O
+degree	O
+of	O
+phosphorylation	B-ptm
+(	O
+90	O
+%).	O
+
+However	O
+,	O
+residual	O
+phosphorylation	B-ptm
+levels	O
+were	O
+detected	O
+for	O
+Ser1204	B-residue_name_number
+(	O
+7	O
+%)	O
+and	O
+Ser1218	B-residue_name_number
+(	O
+7	O
+%)	O
+in	O
+the	O
+same	B-structure_element
+loop	I-structure_element
+.	O
+
+Besides	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+,	O
+also	O
+the	O
+phosphopeptide	B-site
+target	I-site
+region	I-site
+for	O
+BRCA1	B-protein
+interaction	O
+is	O
+not	O
+resolved	O
+presumably	O
+because	O
+of	O
+pronounced	O
+flexibility	O
+.	O
+
+To	O
+improve	B-experimental_method
+crystallizability	I-experimental_method
+,	O
+we	O
+generated	B-experimental_method
+ΔBCCP	B-mutant
+variants	I-mutant
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+ACC	B-protein_type
+,	O
+which	O
+,	O
+based	O
+on	O
+SAXS	B-experimental_method
+analysis	I-experimental_method
+,	O
+preserve	O
+properties	O
+of	O
+intact	B-protein_state
+ACC	B-protein_type
+(	O
+Supplementary	O
+Table	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+–	O
+c	O
+).	O
+
+For	O
+CthΔBCCP	B-mutant
+,	O
+crystals	B-evidence
+diffracting	O
+to	O
+8	O
+.	O
+4	O
+Å	O
+resolution	O
+were	O
+obtained	O
+.	O
+
+On	O
+the	O
+basis	O
+of	O
+the	O
+occurrence	O
+of	O
+related	O
+conformational	O
+changes	O
+between	O
+fungal	B-taxonomy_domain
+and	O
+human	B-species
+ACC	B-mutant
+fragments	I-mutant
+,	O
+the	O
+observed	O
+set	O
+of	O
+conformations	O
+may	O
+well	O
+represent	O
+general	O
+states	O
+present	O
+in	O
+all	O
+eukaryotic	B-taxonomy_domain
+ACCs	B-protein_type
+.	O
+
+To	O
+obtain	O
+a	O
+comprehensive	O
+view	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+dynamics	O
+in	B-protein_state
+solution	I-protein_state
+,	O
+we	O
+employed	O
+SAXS	B-experimental_method
+and	O
+EM	B-experimental_method
+.	O
+
+They	O
+identify	O
+the	O
+connections	O
+between	O
+CDN	B-structure_element
+/	O
+CDL	B-structure_element
+and	O
+between	O
+CDC2	B-structure_element
+/	O
+CT	B-structure_element
+as	O
+major	O
+contributors	O
+to	O
+conformational	O
+heterogeneity	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+
+Furthermore	O
+,	O
+based	O
+on	O
+an	O
+average	O
+length	O
+of	O
+the	O
+BCCP	B-structure_element
+–	I-structure_element
+CD	I-structure_element
+linker	I-structure_element
+in	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+of	O
+26	B-residue_range
+amino	I-residue_range
+acids	I-residue_range
+,	O
+mobility	O
+of	O
+the	O
+BCCP	B-structure_element
+alone	O
+would	O
+not	O
+be	O
+sufficient	O
+to	O
+bridge	O
+the	O
+active	B-site
+sites	I-site
+of	O
+BC	B-structure_element
+and	O
+CT	B-structure_element
+.	O
+
+The	O
+CD	B-structure_element
+consists	O
+of	O
+four	O
+distinct	O
+subdomains	B-structure_element
+and	O
+acts	O
+as	O
+a	O
+tether	O
+from	O
+the	O
+CT	B-structure_element
+to	O
+the	O
+mobile	B-protein_state
+BCCP	B-structure_element
+and	O
+an	O
+oriented	B-protein_state
+BC	B-structure_element
+domain	O
+.	O
+
+In	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+,	O
+however	O
+,	O
+Ser1157	B-residue_name_number
+in	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+of	O
+the	O
+CD	B-structure_element
+is	O
+the	O
+only	O
+phosphorylation	B-site
+site	I-site
+that	O
+has	O
+been	O
+demonstrated	O
+to	O
+be	O
+both	O
+phosphorylated	B-protein_state
+in	O
+vivo	O
+and	O
+involved	O
+in	O
+the	O
+regulation	O
+of	O
+ACC	B-protein_type
+activity	O
+.	O
+
+A	O
+comparison	O
+between	O
+fungal	B-taxonomy_domain
+and	O
+human	B-species
+ACC	B-protein_type
+will	O
+help	O
+to	O
+further	O
+discriminate	O
+mechanistic	O
+differences	O
+that	O
+contribute	O
+to	O
+the	O
+extended	O
+control	O
+and	O
+polymerization	O
+of	O
+human	B-species
+ACC	B-protein_type
+.	O
+
+In	O
+their	O
+study	O
+,	O
+mutational	B-experimental_method
+data	I-experimental_method
+indicate	O
+a	O
+requirement	O
+for	O
+BC	O
+dimerization	O
+for	O
+catalytic	O
+activity	O
+.	O
+
+In	O
+flACC	B-mutant
+,	O
+CDC2	B-structure_element
+rotates	O
+∼	O
+120	O
+°	O
+with	O
+respect	O
+to	O
+the	O
+CT	B-structure_element
+domain	O
+.	O
+
+A	O
+second	B-structure_element
+hinge	I-structure_element
+can	O
+be	O
+identified	O
+between	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+.	O
+
+In	O
+those	O
+instances	O
+the	O
+Ser1157	B-residue_name_number
+residue	O
+is	O
+located	O
+at	O
+a	O
+distance	O
+of	O
+14	O
+–	O
+20	O
+Å	O
+away	O
+from	O
+the	O
+location	O
+of	O
+the	O
+phosphorylated	B-protein_state
+serine	B-residue_name
+observed	O
+here	O
+,	O
+based	O
+on	O
+superposition	B-experimental_method
+of	O
+either	O
+CDC1	B-structure_element
+or	O
+CDC2	B-structure_element
+.	O
+
+The	O
+phosphorylated	B-protein_state
+central	B-structure_element
+domain	I-structure_element
+of	O
+yeast	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+Architecture	O
+of	O
+the	O
+CD	B-structure_element
+–	O
+CT	B-structure_element
+core	O
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+.	O
+
+Flexibility	O
+of	O
+the	O
+CDC2	B-structure_element
+/	O
+CT	B-structure_element
+and	O
+CDN	B-structure_element
+/	O
+CDL	B-structure_element
+hinges	B-structure_element
+is	O
+illustrated	O
+by	O
+arrows	O
+.	O
+
+The	O
+inducible	B-protein_state
+lysine	B-protein_type
+decarboxylase	I-protein_type
+LdcI	B-protein
+is	O
+an	O
+important	O
+enterobacterial	B-taxonomy_domain
+acid	B-protein_type
+stress	I-protein_type
+response	I-protein_type
+enzyme	I-protein_type
+whereas	O
+LdcC	B-protein
+is	O
+its	O
+close	O
+paralogue	O
+thought	O
+to	O
+play	O
+mainly	O
+a	O
+metabolic	O
+role	O
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+biosynthetic	B-protein_state
+E	B-species
+.	I-species
+coli	I-species
+lysine	B-protein_type
+decarboxylase	I-protein_type
+LdcC	B-protein
+,	O
+long	O
+thought	O
+to	O
+be	O
+constitutively	O
+expressed	O
+in	O
+low	O
+amounts	O
+,	O
+was	O
+demonstrated	O
+to	O
+be	O
+strongly	O
+upregulated	O
+by	O
+fluoroquinolones	B-chemical
+via	O
+their	O
+induction	O
+of	O
+RpoS	B-protein
+.	O
+A	O
+direct	O
+correlation	O
+between	O
+the	O
+level	O
+of	O
+cadaverine	B-chemical
+and	O
+the	O
+resistance	O
+of	O
+E	B-species
+.	I-species
+coli	I-species
+to	O
+these	O
+antibiotics	O
+commonly	O
+used	O
+as	O
+a	O
+first	O
+-	O
+line	O
+treatment	O
+of	O
+UTI	O
+could	O
+be	O
+established	O
+.	O
+
+Furthermore	O
+,	O
+we	O
+recently	O
+solved	B-experimental_method
+the	I-experimental_method
+structure	I-experimental_method
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+complex	O
+by	O
+cryo	B-experimental_method
+-	I-experimental_method
+electron	I-experimental_method
+microscopy	I-experimental_method
+(	O
+cryoEM	B-experimental_method
+)	O
+and	O
+combined	O
+it	O
+with	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+individual	O
+proteins	O
+.	O
+
+CryoEM	B-experimental_method
+3D	B-evidence
+reconstructions	I-evidence
+of	O
+LdcC	B-protein
+,	O
+LdcIa	B-protein
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+
+In	O
+the	O
+frame	O
+of	O
+this	O
+work	O
+,	O
+we	O
+produced	O
+two	O
+novel	O
+subnanometer	O
+resolution	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+of	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+lysine	B-protein_type
+decarboxylases	I-protein_type
+at	O
+pH	B-protein_state
+optimal	I-protein_state
+for	O
+their	O
+enzymatic	O
+activity	O
+–	O
+a	O
+5	O
+.	O
+5	O
+Å	O
+resolution	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcC	B-protein
+(	O
+pH	B-protein_state
+7	I-protein_state
+.	I-protein_state
+5	I-protein_state
+)	O
+for	O
+which	O
+no	O
+3D	O
+structural	O
+information	O
+has	O
+been	O
+previously	O
+available	O
+(	O
+Figs	O
+1A	O
+,	O
+B	O
+and	O
+S1	O
+),	O
+and	O
+a	O
+6	O
+.	O
+1	O
+Å	O
+resolution	O
+cryoEM	B-experimental_method
+map	B-evidence
+of	O
+the	O
+LdcIa	B-protein
+,	O
+(	O
+pH	B-protein_state
+6	I-protein_state
+.	I-protein_state
+2	I-protein_state
+)	O
+(	O
+Figs	O
+1C	O
+,	O
+D	O
+and	O
+S2	O
+).	O
+
+Zooming	O
+in	O
+the	O
+variations	O
+in	O
+the	O
+PLP	B-structure_element
+-	I-structure_element
+SD	I-structure_element
+shows	O
+that	O
+most	O
+of	O
+the	O
+structural	O
+changes	O
+concern	O
+displacements	O
+in	O
+the	O
+active	B-site
+site	I-site
+(	O
+Fig	O
+.	O
+3C	O
+–	O
+F	O
+).	O
+
+An	O
+inhibitor	O
+of	O
+the	O
+LdcI	B-protein
+and	O
+LdcC	B-protein
+activity	O
+,	O
+the	O
+stringent	B-chemical
+response	I-chemical
+alarmone	I-chemical
+ppGpp	B-chemical
+,	O
+is	O
+known	O
+to	O
+bind	O
+at	O
+the	O
+interface	B-site
+between	O
+neighboring	O
+monomers	B-oligomeric_state
+within	O
+each	O
+ring	B-structure_element
+(	O
+Fig	O
+.	O
+S4	O
+).	O
+
+Thus	O
+,	O
+to	O
+advance	O
+beyond	O
+our	O
+experimental	O
+confirmation	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+as	O
+a	O
+major	O
+determinant	O
+of	O
+the	O
+capacity	O
+of	O
+a	O
+particular	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+to	O
+form	O
+a	O
+cage	O
+with	O
+RavA	B-protein
+,	O
+we	O
+set	O
+out	O
+to	O
+investigate	O
+whether	O
+certain	B-structure_element
+residues	I-structure_element
+in	O
+this	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+are	O
+conserved	B-protein_state
+in	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+of	O
+different	O
+enterobacteria	B-taxonomy_domain
+that	O
+have	O
+the	O
+ravA	B-gene
+-	I-gene
+viaA	I-gene
+operon	I-gene
+in	O
+their	O
+genome	O
+.	O
+
+The	O
+third	O
+and	O
+most	O
+remarkable	O
+finding	O
+was	O
+that	O
+exactly	O
+the	O
+same	O
+separation	O
+into	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+and	O
+“	O
+LdcC	B-protein_type
+”-	I-protein_type
+like	I-protein_type
+groups	O
+can	O
+be	O
+obtained	O
+based	O
+on	O
+a	O
+comparison	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+only	O
+,	O
+without	O
+taking	O
+the	O
+rest	O
+of	O
+the	O
+primary	O
+sequence	O
+into	O
+account	O
+.	O
+
+Together	O
+with	O
+the	O
+apo	B-protein_state
+-	O
+LdcI	B-protein
+and	O
+ppGpp	B-complex_assembly
+-	I-complex_assembly
+LdcIi	I-complex_assembly
+crystal	B-evidence
+structures	I-evidence
+,	O
+our	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+provide	O
+a	O
+structural	O
+framework	O
+for	O
+future	O
+studies	O
+of	O
+structure	O
+-	O
+function	O
+relationships	O
+of	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+from	O
+other	O
+enterobacteria	B-taxonomy_domain
+and	O
+even	O
+of	O
+their	O
+homologues	O
+outside	O
+Enterobacteriaceae	B-taxonomy_domain
+.	O
+For	O
+example	O
+,	O
+the	O
+lysine	B-protein_type
+decarboxylase	I-protein_type
+of	O
+Eikenella	B-species
+corrodens	I-species
+is	O
+thought	O
+to	O
+play	O
+a	O
+major	O
+role	O
+in	O
+the	O
+periodontal	O
+disease	O
+and	O
+its	O
+inhibitors	O
+were	O
+shown	O
+to	O
+retard	O
+gingivitis	O
+development	O
+.	O
+
+The	O
+dashed	O
+circle	O
+indicates	O
+the	O
+central	O
+region	B-structure_element
+that	O
+remains	O
+virtually	O
+unchanged	O
+between	O
+all	O
+the	O
+structures	B-evidence
+,	O
+while	O
+the	O
+periphery	O
+undergoes	O
+visible	O
+movements	O
+.	O
+
+(	O
+A	O
+)	O
+LdcIi	B-protein
+crystal	B-evidence
+structure	I-evidence
+,	O
+with	O
+one	O
+ring	B-structure_element
+represented	O
+as	O
+a	O
+grey	O
+surface	O
+and	O
+the	O
+second	O
+as	O
+a	O
+cartoon	O
+.	O
+
+Analysis	O
+of	O
+the	O
+LdcIC	B-mutant
+and	O
+LdcCI	B-mutant
+chimeras	B-mutant
+.	O
+
+With	O
+the	O
+exception	O
+of	O
+the	O
+human	B-species
+RhCG	B-protein
+structure	B-evidence
+,	O
+no	O
+structural	O
+information	O
+is	O
+available	O
+for	O
+eukaryotic	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+.	O
+
+Ammonium	B-chemical
+transport	O
+is	O
+tightly	O
+regulated	O
+.	O
+
+By	O
+binding	O
+tightly	O
+to	O
+Amt	B-protein_type
+proteins	I-protein_type
+without	O
+inducing	O
+a	O
+conformational	O
+change	O
+in	O
+the	O
+transporter	B-protein_type
+,	O
+GlnK	B-protein_type
+sterically	O
+blocks	O
+ammonium	B-chemical
+conductance	O
+when	O
+nitrogen	O
+levels	O
+are	O
+sufficient	O
+.	O
+
+Under	O
+conditions	O
+of	O
+nitrogen	B-chemical
+limitation	O
+,	O
+GlnK	B-protein_type
+becomes	O
+uridylated	B-protein_state
+,	O
+blocking	O
+its	O
+ability	O
+to	O
+bind	O
+and	O
+inhibit	O
+Amt	B-protein_type
+proteins	I-protein_type
+.	O
+
+General	O
+architecture	O
+of	O
+Mep2	B-protein_type
+ammonium	B-protein_type
+transceptors	I-protein_type
+
+The	O
+Mep2	B-protein
+protein	O
+of	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+(	O
+ScMep2	B-protein
+)	O
+was	O
+overexpressed	B-experimental_method
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+in	O
+high	O
+yields	O
+,	O
+enabling	O
+structure	B-experimental_method
+determination	I-experimental_method
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+using	O
+data	O
+to	O
+3	O
+.	O
+2	O
+Å	O
+resolution	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+(	O
+MR	B-experimental_method
+)	O
+with	O
+the	O
+archaebacterial	B-taxonomy_domain
+Amt	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+(	O
+see	O
+Methods	O
+section	O
+).	O
+
+Unless	O
+specifically	O
+stated	O
+,	O
+the	O
+drawn	O
+conclusions	O
+also	O
+apply	O
+to	O
+ScMep2	B-protein
+.	O
+
+In	O
+addition	O
+to	O
+changing	O
+the	O
+RxK	B-structure_element
+motif	I-structure_element
+,	O
+the	O
+movement	O
+of	O
+ICL1	B-structure_element
+has	O
+another	O
+,	O
+crucial	O
+functional	O
+consequence	O
+.	O
+
+Finally	O
+,	O
+the	O
+important	O
+ICL3	B-structure_element
+linking	O
+the	O
+pseudo	B-structure_element
+-	I-structure_element
+symmetrical	I-structure_element
+halves	I-structure_element
+(	O
+TM1	B-structure_element
+-	I-structure_element
+5	I-structure_element
+and	O
+TM6	B-structure_element
+-	I-structure_element
+10	I-structure_element
+)	O
+of	O
+the	O
+transporter	B-protein_type
+is	O
+also	O
+shifted	O
+up	O
+to	O
+∼	O
+10	O
+Å	O
+and	O
+forms	O
+an	O
+additional	O
+barrier	O
+that	O
+closes	O
+the	O
+channel	B-site
+on	O
+the	O
+cytoplasmic	O
+side	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+In	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+and	O
+other	O
+bacterial	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+,	O
+these	O
+CTR	B-structure_element
+residues	O
+interact	O
+with	O
+residues	O
+within	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+the	O
+protein	O
+.	O
+
+For	O
+ScMep2	B-protein
+,	O
+Ser457	B-residue_name_number
+is	O
+the	O
+most	O
+C	O
+-	O
+terminal	O
+residue	O
+for	O
+which	O
+electron	B-evidence
+density	I-evidence
+is	O
+visible	O
+,	O
+indicating	O
+that	O
+the	O
+region	O
+beyond	O
+Ser457	B-residue_name_number
+is	O
+disordered	B-protein_state
+.	O
+
+In	O
+CaMep2	B-protein
+,	O
+the	O
+visible	O
+part	O
+of	O
+the	O
+sequence	O
+extends	O
+for	O
+two	O
+residues	O
+beyond	O
+Ser453	B-residue_name_number
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+Density	B-evidence
+for	O
+ICL3	B-structure_element
+and	O
+the	O
+CTR	B-structure_element
+beyond	O
+residue	O
+Arg415	B-residue_name_number
+is	O
+missing	O
+in	O
+the	O
+442Δ	B-mutant
+mutant	B-protein_state
+,	O
+and	O
+the	O
+density	B-evidence
+for	O
+the	O
+other	O
+ICLs	B-structure_element
+including	O
+ICL1	B-structure_element
+is	O
+generally	O
+poor	O
+with	O
+visible	O
+parts	O
+of	O
+the	O
+structure	B-evidence
+having	O
+high	O
+B	O
+-	O
+factors	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+
+We	O
+therefore	O
+predict	O
+that	O
+phosphorylation	B-ptm
+of	O
+Ser453	B-residue_name_number
+will	O
+result	O
+in	O
+steric	O
+clashes	O
+as	O
+well	O
+as	O
+electrostatic	O
+repulsion	O
+,	O
+which	O
+in	O
+turn	O
+might	O
+cause	O
+substantial	O
+conformational	O
+changes	O
+within	O
+the	O
+CTR	B-structure_element
+.	O
+
+To	O
+supplement	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+,	O
+we	O
+also	O
+performed	O
+modelling	B-experimental_method
+and	O
+MD	B-experimental_method
+studies	O
+of	O
+WT	B-protein_state
+CaMep2	B-protein
+,	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+and	O
+phosphorylated	B-protein_state
+protein	O
+(	O
+S453J	B-mutant
+).	O
+
+The	O
+protein	O
+is	O
+structurally	B-protein_state
+stable	I-protein_state
+throughout	O
+the	O
+simulation	B-experimental_method
+with	O
+little	O
+deviation	O
+in	O
+the	O
+other	O
+parts	O
+of	O
+the	O
+protein	O
+.	O
+
+Finally	O
+,	O
+the	O
+S453J	B-mutant
+mutant	B-protein_state
+is	O
+also	O
+stable	B-protein_state
+throughout	O
+the	O
+200	O
+-	O
+ns	O
+simulation	B-experimental_method
+and	O
+has	O
+an	O
+average	O
+backbone	O
+deviation	O
+of	O
+∼	O
+3	O
+.	O
+8	O
+Å	O
+,	O
+which	O
+is	O
+similar	O
+to	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+.	O
+
+The	O
+distance	B-evidence
+between	O
+the	O
+phosphate	B-chemical
+of	O
+Sep453	B-residue_name_number
+and	O
+the	O
+acidic	O
+oxygen	O
+atoms	O
+of	O
+Glu420	B-residue_name_number
+is	O
+initially	O
+∼	O
+11	O
+Å	O
+,	O
+but	O
+increases	O
+to	O
+>	O
+30	O
+Å	O
+after	O
+200	O
+ns	O
+.	O
+
+In	O
+Arabidopsis	B-species
+thaliana	I-species
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+,	O
+phosphorylation	B-ptm
+of	O
+the	O
+CTR	B-structure_element
+residue	O
+T460	B-residue_name_number
+under	O
+conditions	O
+of	O
+high	O
+ammonium	B-chemical
+inhibits	O
+transport	O
+activity	O
+,	O
+that	O
+is	O
+,	O
+the	O
+default	O
+(	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+)	O
+state	O
+of	O
+the	O
+plant	B-taxonomy_domain
+transporter	B-protein_type
+is	O
+open	B-protein_state
+.	O
+
+In	O
+addition	O
+,	O
+ICL1	B-structure_element
+has	O
+shifted	O
+inwards	O
+to	O
+contribute	O
+to	O
+the	O
+channel	B-site
+closure	O
+by	O
+engaging	O
+His2	B-residue_name_number
+from	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+via	O
+hydrogen	O
+bonding	O
+with	O
+a	O
+highly	B-protein_state
+conserved	I-protein_state
+tyrosine	B-residue_name
+hydroxyl	O
+group	O
+.	O
+
+However	O
+,	O
+even	O
+the	O
+otherwise	O
+highly	O
+similar	O
+Mep2	B-protein_type
+proteins	I-protein_type
+of	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+and	O
+C	B-species
+.	I-species
+albicans	I-species
+have	O
+different	O
+structures	B-evidence
+for	O
+their	O
+CTRs	B-structure_element
+(	O
+Fig	O
+.	O
+1	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+With	O
+regards	O
+to	O
+plant	B-taxonomy_domain
+AMTs	B-protein_type
+,	O
+it	O
+has	O
+been	O
+proposed	O
+that	O
+phosphorylation	B-ptm
+at	O
+T460	B-residue_name_number
+generates	O
+conformational	O
+changes	O
+that	O
+would	O
+close	O
+the	O
+neighbouring	O
+pore	B-site
+via	O
+the	O
+C	B-structure_element
+terminus	I-structure_element
+.	O
+
+This	O
+assumption	O
+was	O
+based	O
+partly	O
+on	O
+a	O
+homology	B-experimental_method
+model	I-experimental_method
+for	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+based	O
+on	O
+the	O
+(	O
+open	B-protein_state
+)	O
+archaebacterial	B-taxonomy_domain
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+,	O
+which	O
+suggested	O
+that	O
+the	O
+C	B-structure_element
+terminus	I-structure_element
+of	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+would	O
+extend	O
+further	O
+to	O
+the	O
+neighbouring	O
+monomer	B-oligomeric_state
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+considerable	O
+differences	O
+between	O
+structurally	O
+resolved	O
+CTR	B-structure_element
+domains	O
+means	O
+that	O
+the	O
+exact	O
+environment	O
+of	O
+T460	B-residue_name_number
+in	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+is	O
+also	O
+not	O
+known	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+We	O
+propose	O
+that	O
+intra	B-site
+-	I-site
+monomeric	I-site
+CTR	I-site
+-	I-site
+ICL1	I-site
+/	I-site
+ICL3	I-site
+interactions	I-site
+lie	O
+at	O
+the	O
+basis	O
+of	O
+regulation	O
+of	O
+both	O
+fungal	B-taxonomy_domain
+and	O
+plant	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+;	O
+close	O
+interactions	O
+generate	O
+open	B-protein_state
+channels	B-site
+,	O
+whereas	O
+the	O
+lack	B-protein_state
+of	I-protein_state
+‘	O
+intra	O
+-'	O
+interactions	O
+leads	O
+to	O
+inactive	B-protein_state
+states	O
+.	O
+
+The	O
+need	O
+to	O
+regulate	O
+in	O
+opposite	O
+ways	O
+may	O
+be	O
+the	O
+reason	O
+why	O
+the	O
+phosphorylation	B-site
+sites	I-site
+are	O
+in	O
+different	O
+parts	O
+of	O
+the	O
+CTR	B-structure_element
+,	O
+that	O
+is	O
+,	O
+centrally	O
+located	O
+close	O
+to	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+in	O
+AMTs	B-protein_type
+and	O
+peripherally	O
+in	O
+Mep2	B-protein
+.	O
+
+With	O
+respect	O
+to	O
+ammonium	B-chemical
+transport	O
+,	O
+phosphorylation	B-ptm
+has	O
+thus	O
+far	O
+only	O
+been	O
+shown	O
+for	O
+A	B-species
+.	I-species
+thaliana	I-species
+AMTs	B-protein_type
+and	O
+for	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+Mep2	B-protein
+(	O
+refs	O
+).	O
+
+The	O
+region	O
+showing	O
+ICL1	B-structure_element
+(	O
+blue	O
+),	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+)	O
+is	O
+boxed	O
+for	O
+comparison	O
+.	O
+
+The	O
+grey	O
+sequences	O
+at	O
+the	O
+C	O
+termini	O
+of	O
+CaMep2	B-protein
+and	O
+ScMep2	B-protein
+are	O
+not	O
+visible	O
+in	O
+the	O
+structures	B-evidence
+and	O
+are	O
+likely	B-protein_state
+disordered	I-protein_state
+.	O
+
+(	O
+a	O
+)	O
+ICL1	B-structure_element
+in	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+(	O
+light	O
+blue	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+dark	O
+blue	O
+),	O
+showing	O
+unwinding	O
+and	O
+inward	O
+movement	O
+in	O
+the	O
+fungal	B-taxonomy_domain
+protein	O
+.	O
+(	O
+b	O
+)	O
+Stereo	O
+diagram	O
+viewed	O
+from	O
+the	O
+cytosol	O
+of	O
+ICL1	B-structure_element
+,	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+)	O
+in	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+(	O
+light	O
+colours	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+dark	O
+colours	O
+).	O
+
+Views	O
+from	O
+the	O
+cytosol	O
+for	O
+CaMep2	B-protein
+(	O
+left	O
+)	O
+and	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+,	O
+highlighting	O
+the	O
+large	O
+differences	O
+in	O
+conformation	O
+of	O
+the	O
+conserved	B-protein_state
+residues	O
+in	O
+ICL1	B-structure_element
+(	O
+RxK	O
+motif	O
+;	O
+blue	O
+),	O
+ICL2	B-structure_element
+(	O
+ER	B-structure_element
+motif	I-structure_element
+;	O
+cyan	O
+),	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+).	O
+
+The	O
+labelled	O
+residues	O
+are	O
+analogous	O
+within	O
+both	O
+structures	B-evidence
+.	O
+
+The	O
+Npr1	B-protein
+kinase	B-protein_type
+target	O
+Ser453	B-residue_name_number
+is	O
+dephosphorylated	B-protein_state
+and	O
+located	O
+in	O
+an	O
+electronegative	B-site
+pocket	I-site
+.	O
+
+Missing	O
+regions	O
+are	O
+labelled	O
+.	O
+(	O
+b	O
+)	O
+Stereo	O
+superpositions	B-experimental_method
+of	O
+WT	B-protein_state
+CaMep2	B-protein
+and	O
+the	O
+truncation	B-protein_state
+mutant	I-protein_state
+.	O
+
+(	O
+a	O
+)	O
+Cytoplasmic	O
+view	O
+of	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+trimer	B-oligomeric_state
+,	O
+with	O
+WT	B-protein_state
+CaMep2	B-protein
+superposed	B-experimental_method
+in	O
+grey	O
+for	O
+one	O
+of	O
+the	O
+monomers	B-oligomeric_state
+.	O
+
+The	O
+arrow	O
+indicates	O
+the	O
+phosphorylation	B-site
+site	I-site
+.	O
+
+(	O
+b	O
+)	O
+Monomer	B-oligomeric_state
+side	O
+-	O
+view	O
+superposition	B-experimental_method
+of	O
+WT	B-protein_state
+CaMep2	B-protein
+and	O
+the	O
+DD	B-mutant
+mutant	I-mutant
+,	O
+showing	O
+the	O
+conformational	O
+change	O
+and	O
+disorder	O
+around	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+.	O
+
+Schematic	O
+model	O
+for	O
+phosphorylation	O
+-	O
+based	O
+regulation	O
+of	O
+Mep2	B-protein
+ammonium	O
+transporters	O
+.	O
+
+Upon	O
+phosphorylation	B-ptm
+and	O
+mimicked	B-protein_state
+by	O
+the	O
+CaMep2	B-protein
+S453D	B-mutant
+and	O
+DD	B-mutant
+mutants	I-mutant
+(	O
+ii	O
+),	O
+the	O
+region	O
+around	O
+the	O
+ExxGxD	B-structure_element
+motif	I-structure_element
+undergoes	O
+a	O
+conformational	O
+change	O
+that	O
+results	O
+in	O
+the	O
+CTR	B-structure_element
+interacting	O
+with	O
+the	O
+inward	O
+-	O
+moving	O
+ICL3	B-structure_element
+,	O
+opening	O
+the	O
+channel	B-site
+(	O
+full	O
+circle	O
+)	O
+(	O
+iii	O
+).	O
+
+The	O
+open	B-protein_state
+-	O
+channel	B-site
+Mep2	B-protein
+structure	B-evidence
+is	O
+represented	O
+by	O
+archaebacterial	B-taxonomy_domain
+Amt	B-protein
+-	I-protein
+1	I-protein
+and	O
+shown	O
+in	O
+lighter	O
+colours	O
+consistent	O
+with	O
+Fig	O
+.	O
+4	O
+.	O
+
+An	O
+extended	B-protein_state
+U2AF65	B-structure_element
+–	I-structure_element
+RNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+recognizes	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+signal	O
+
+The	O
+U2AF65	B-protein
+linker	B-structure_element
+residues	O
+between	O
+the	O
+dual	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRMs	B-structure_element
+)	O
+recognize	O
+the	O
+central	O
+nucleotide	B-chemical
+,	O
+whereas	O
+the	O
+N	O
+-	O
+and	O
+C	O
+-	O
+terminal	O
+RRM	B-structure_element
+extensions	I-structure_element
+recognize	O
+the	O
+3	B-site
+′	I-site
+terminus	I-site
+and	O
+third	B-residue_number
+nucleotide	B-chemical
+.	O
+
+The	O
+differential	O
+skipping	O
+or	O
+inclusion	O
+of	O
+alternatively	O
+spliced	O
+pre	B-structure_element
+-	I-structure_element
+mRNA	I-structure_element
+regions	I-structure_element
+is	O
+a	O
+major	O
+source	O
+of	O
+diversity	O
+for	O
+nearly	O
+all	O
+human	B-species
+gene	O
+transcripts	O
+.	O
+
+High	O
+-	O
+resolution	O
+structures	B-evidence
+of	O
+intact	B-protein_state
+splicing	B-complex_assembly
+factor	I-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complexes	O
+would	O
+offer	O
+key	O
+insights	O
+regarding	O
+the	O
+juxtaposition	O
+of	O
+the	O
+distinct	O
+splice	B-site
+site	I-site
+consensus	O
+sequences	O
+and	O
+their	O
+relationship	O
+to	O
+disease	O
+-	O
+causing	O
+point	O
+mutations	O
+.	O
+
+Likewise	O
+,	O
+both	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+and	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+U2AF65	B-protein
+showed	O
+similar	O
+sequence	B-evidence
+specificity	I-evidence
+for	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+stretches	I-structure_element
+in	O
+the	O
+5	B-site
+′-	I-site
+region	I-site
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+and	O
+promiscuity	O
+for	O
+C	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+regions	I-structure_element
+in	O
+the	O
+3	B-site
+′-	I-site
+region	I-site
+(	O
+Fig	O
+.	O
+1c	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+1e	O
+–	O
+h	O
+).	O
+
+U2AF65	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+Py	B-chemical
+tract	I-chemical
+comprises	O
+nine	O
+contiguous	B-structure_element
+nucleotides	B-chemical
+
+An	O
+extended	B-protein_state
+conformation	I-protein_state
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+traverses	O
+across	O
+the	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+surface	I-structure_element
+of	O
+RRM1	B-structure_element
+and	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+of	O
+RRM2	B-structure_element
+and	O
+is	O
+well	O
+defined	O
+in	O
+the	O
+electron	B-evidence
+density	I-evidence
+(	O
+Fig	O
+.	O
+2b	O
+).	O
+
+We	O
+compare	O
+the	O
+global	O
+conformation	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+with	O
+the	O
+prior	O
+dU2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+crystal	B-evidence
+structure	I-evidence
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+NMR	B-experimental_method
+structure	B-evidence
+in	O
+the	O
+Supplementary	O
+Discussion	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+.	O
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRM2	B-structure_element
+,	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+and	O
+RRM1	B-structure_element
+concomitantly	O
+recognize	O
+the	O
+three	O
+central	O
+nucleotides	B-chemical
+of	O
+the	O
+Py	B-chemical
+tract	I-chemical
+,	O
+which	O
+are	O
+likely	O
+to	O
+coordinate	O
+the	O
+conformational	O
+arrangement	O
+of	O
+these	O
+disparate	O
+portions	O
+of	O
+the	O
+protein	O
+.	O
+
+Residues	O
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+comprise	O
+a	O
+centrally	O
+located	O
+binding	B-site
+site	I-site
+for	O
+the	O
+fifth	B-residue_number
+nucleotide	B-chemical
+on	O
+the	O
+RRM2	B-site
+surface	I-site
+and	O
+abutting	O
+the	O
+RRM1	B-site
+/	I-site
+RRM2	I-site
+interface	I-site
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+Otherwise	O
+,	O
+the	O
+rU4	B-residue_name_number
+nucleotide	B-chemical
+packs	O
+against	O
+F304	B-residue_name_number
+in	O
+the	O
+signature	O
+ribonucleoprotein	B-structure_element
+consensus	I-structure_element
+motif	I-structure_element
+(	I-structure_element
+RNP	I-structure_element
+)-	I-structure_element
+2	I-structure_element
+of	O
+RRM2	B-structure_element
+.	O
+
+This	O
+nucleotide	B-chemical
+twists	O
+to	O
+face	O
+away	O
+from	O
+the	O
+U2AF65	B-protein
+linker	B-structure_element
+and	O
+instead	O
+inserts	O
+the	O
+rU6	B-residue_name_number
+-	O
+uracil	B-residue_name
+into	O
+a	O
+sandwich	O
+between	O
+the	O
+β2	B-structure_element
+/	I-structure_element
+β3	I-structure_element
+loops	I-structure_element
+of	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+.	O
+
+The	O
+Q147	B-residue_name_number
+residue	O
+participates	O
+in	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+-	O
+N3H	O
+of	O
+the	O
+eighth	B-residue_number
+uracil	B-residue_name
+and	O
+-	O
+O2	O
+of	O
+the	O
+ninth	B-residue_number
+pyrimidine	B-chemical
+.	O
+
+Consistent	O
+with	O
+loss	O
+of	O
+a	O
+hydrogen	O
+bond	O
+with	O
+the	O
+ninth	B-residue_number
+pyrimidine	B-chemical
+-	O
+O2	O
+(	O
+ΔΔG	B-evidence
+1	O
+.	O
+0	O
+kcal	O
+mol	O
+−	O
+1	O
+),	O
+mutation	B-experimental_method
+of	O
+the	O
+Q147	B-residue_name_number
+to	O
+an	O
+alanine	B-residue_name
+reduced	O
+U2AF651	B-evidence
+,	I-evidence
+2L	I-evidence
+affinity	I-evidence
+for	O
+the	O
+AdML	B-gene
+Py	B-chemical
+tract	I-chemical
+by	O
+five	O
+-	O
+fold	O
+(	O
+Fig	O
+.	O
+3i	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4c	O
+).	O
+
+We	O
+compare	B-experimental_method
+U2AF65	B-protein
+interactions	O
+with	O
+uracil	B-residue_name
+relative	O
+to	O
+cytosine	B-residue_name
+pyrimidines	B-chemical
+at	O
+the	O
+ninth	B-site
+binding	I-site
+site	I-site
+in	O
+Fig	O
+.	O
+3g	O
+,	O
+h	O
+and	O
+the	O
+Supplementary	O
+Discussion	O
+.	O
+
+Versatile	O
+primary	O
+sequence	O
+of	O
+the	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+reveal	O
+that	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+mediates	O
+an	O
+extensive	B-site
+interface	I-site
+with	O
+the	O
+second	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+of	O
+RRM1	B-structure_element
+,	O
+the	O
+β2	B-structure_element
+/	I-structure_element
+β3	I-structure_element
+strands	I-structure_element
+of	O
+RRM2	B-structure_element
+and	O
+the	O
+N	O
+-	O
+terminal	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+extension	I-structure_element
+of	O
+RRM1	B-structure_element
+.	O
+
+The	O
+adjacent	O
+V249	B-residue_name_number
+and	O
+V250	B-residue_name_number
+are	O
+notable	O
+for	O
+their	O
+respective	O
+interactions	O
+that	O
+connect	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+at	O
+this	O
+distal	O
+interface	B-site
+from	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+site	I-site
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+top	O
+).	O
+
+Few	O
+direct	O
+contacts	O
+are	O
+made	O
+between	O
+the	O
+remaining	O
+residues	O
+of	O
+the	O
+linker	B-structure_element
+and	O
+the	O
+U2AF65	B-protein
+RRM2	B-structure_element
+;	O
+instead	O
+,	O
+the	O
+C	O
+-	O
+terminal	O
+conformation	O
+of	O
+the	O
+linker	B-structure_element
+appears	O
+primarily	O
+RNA	B-chemical
+mediated	O
+(	O
+Fig	O
+.	O
+3c	O
+,	O
+d	O
+).	O
+
+We	O
+introduced	O
+glycine	B-residue_name
+substitutions	B-experimental_method
+to	O
+maximally	O
+reduce	O
+the	O
+buried	O
+surface	O
+area	O
+without	O
+directly	O
+interfering	O
+with	O
+its	O
+hydrogen	O
+bonds	O
+between	O
+backbone	O
+atoms	O
+and	O
+the	O
+base	O
+.	O
+
+However	O
+,	O
+the	O
+resulting	O
+decrease	O
+in	O
+the	O
+AdML	B-gene
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+3Gly	I-mutant
+mutant	B-protein_state
+relative	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+protein	B-protein
+was	O
+not	O
+significant	O
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+
+We	O
+found	O
+that	O
+the	O
+affinity	B-evidence
+of	O
+dU2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+for	O
+the	O
+AdML	B-gene
+RNA	B-chemical
+was	O
+significantly	O
+reduced	O
+relative	O
+to	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+(	O
+four	O
+-	O
+fold	O
+,	O
+Figs	O
+1b	O
+and	O
+4b	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+4i	O
+).	O
+
+Paramagnetic	B-experimental_method
+resonance	I-experimental_method
+enhancement	I-experimental_method
+(	O
+PRE	B-experimental_method
+)	O
+measurements	O
+previously	O
+had	O
+suggested	O
+a	O
+predominant	O
+back	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+back	I-protein_state
+,	O
+or	O
+‘	O
+closed	B-protein_state
+'	O
+conformation	O
+of	O
+the	O
+apo	B-protein_state
+-	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+in	O
+equilibrium	O
+with	O
+a	O
+minor	O
+‘	O
+open	B-protein_state
+'	O
+conformation	O
+resembling	O
+the	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+arrangement	O
+.	O
+
+Addition	O
+of	O
+the	O
+AdML	B-gene
+RNA	B-chemical
+to	O
+tethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+selectively	O
+increases	O
+a	O
+fraction	O
+of	O
+molecules	O
+showing	O
+an	O
+∼	O
+0	O
+.	O
+45	O
+apparent	O
+FRET	B-evidence
+efficiency	I-evidence
+,	O
+suggesting	O
+that	O
+RNA	O
+binding	O
+stabilizes	O
+a	O
+single	O
+conformation	O
+,	O
+which	O
+corresponds	O
+to	O
+the	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+state	I-evidence
+(	O
+Fig	O
+.	O
+6e	O
+,	O
+f	O
+).	O
+
+To	O
+assess	O
+the	O
+possible	O
+contributions	O
+of	O
+RNA	B-protein_state
+-	I-protein_state
+free	I-protein_state
+conformations	O
+of	O
+U2AF65	B-protein
+and	O
+/	O
+or	O
+structural	O
+heterogeneity	O
+introduced	O
+by	O
+tethering	B-experimental_method
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+to	O
+the	O
+slide	O
+to	O
+the	O
+observed	O
+distribution	B-evidence
+of	I-evidence
+FRET	I-evidence
+values	I-evidence
+,	O
+we	O
+reversed	B-experimental_method
+the	I-experimental_method
+immobilization	I-experimental_method
+scheme	I-experimental_method
+.	O
+
+We	O
+tethered	B-protein_state
+the	O
+AdML	B-gene
+RNA	B-chemical
+to	O
+the	O
+slide	O
+via	O
+a	O
+biotinylated	B-chemical
+oligonucleotide	I-chemical
+DNA	I-chemical
+handle	O
+and	O
+added	B-experimental_method
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+biotin	B-chemical
+-	I-chemical
+NTA	I-chemical
+resin	I-chemical
+(	O
+Fig	O
+.	O
+6g	O
+,	O
+h	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+
+Nevertheless	O
+,	O
+in	O
+the	O
+presence	O
+of	O
+saturating	O
+concentrations	O
+of	O
+rArA	B-chemical
+-	O
+interrupted	O
+RNA	B-chemical
+slide	B-protein_state
+-	I-protein_state
+tethered	I-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+showed	O
+a	O
+prevalent	O
+∼	O
+0	O
+.	O
+45	O
+apparent	O
+FRET	B-evidence
+value	I-evidence
+(	O
+Fig	O
+.	O
+6i	O
+,	O
+j	O
+),	O
+which	O
+was	O
+also	O
+predominant	O
+in	O
+the	O
+presence	O
+of	O
+continuous	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+Therefore	O
+,	O
+RRM1	B-structure_element
+-	O
+to	O
+-	O
+RRM2	B-structure_element
+distance	O
+remains	O
+similar	O
+regardless	O
+of	O
+whether	O
+U2AF65	B-protein
+is	O
+bound	B-protein_state
+to	I-protein_state
+interrupted	O
+or	O
+continuous	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+The	O
+majority	O
+of	O
+traces	B-evidence
+that	O
+show	O
+fluctuations	O
+began	O
+at	O
+high	O
+(	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+)	O
+FRET	B-evidence
+value	I-evidence
+and	O
+transitioned	O
+to	O
+a	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+).	O
+
+Thus	O
+,	O
+the	O
+sequence	O
+of	O
+structural	O
+rearrangements	O
+of	O
+U2AF65	B-protein
+observed	O
+in	O
+smFRET	B-experimental_method
+traces	B-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7c	O
+–	O
+g	O
+)	O
+suggests	O
+that	O
+a	O
+‘	O
+conformational	O
+selection	O
+'	O
+mechanism	O
+of	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+(	O
+that	O
+is	O
+,	O
+RNA	O
+ligand	O
+stabilization	O
+of	O
+a	O
+pre	B-protein_state
+-	I-protein_state
+configured	I-protein_state
+U2AF65	B-protein
+conformation	O
+)	O
+is	O
+complemented	O
+by	O
+‘	O
+induced	O
+fit	O
+'	O
+(	O
+that	O
+is	O
+,	O
+RNA	O
+-	O
+induced	O
+rearrangement	O
+of	O
+the	O
+U2AF65	B-protein
+RRMs	B-structure_element
+to	O
+achieve	O
+the	O
+final	O
+‘	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+'	O
+conformation	O
+),	O
+as	O
+discussed	O
+below	O
+.	O
+
+Likewise	O
+,	O
+deletion	B-experimental_method
+of	O
+20	B-residue_range
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+linker	I-structure_element
+residues	I-structure_element
+significantly	O
+reduces	O
+U2AF65	B-protein
+–	O
+RNA	B-chemical
+binding	O
+only	O
+when	O
+introduced	O
+in	O
+the	O
+context	O
+of	O
+the	O
+longer	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+construct	O
+comprising	O
+the	O
+RRM	B-structure_element
+extensions	I-structure_element
+,	O
+which	O
+in	O
+turn	O
+position	O
+the	O
+linker	B-structure_element
+for	O
+RNA	B-chemical
+interactions	O
+.	O
+
+The	O
+lesser	O
+0	O
+.	O
+65	O
+–	O
+0	O
+.	O
+8	O
+and	O
+0	O
+.	O
+2	O
+–	O
+0	O
+.	O
+3	O
+FRET	B-evidence
+values	I-evidence
+in	O
+the	O
+untethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+experiment	O
+could	O
+correspond	O
+to	O
+respective	O
+variants	O
+of	O
+the	O
+‘	O
+closed	B-protein_state
+',	O
+back	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+back	I-protein_state
+U2AF65	B-protein
+conformations	O
+characterized	O
+by	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+data	O
+,	O
+or	O
+to	O
+extended	B-protein_state
+U2AF65	B-protein
+conformations	O
+,	O
+in	O
+which	O
+the	O
+intramolecular	O
+RRM1	B-structure_element
+/	O
+RRM2	B-structure_element
+interactions	O
+have	O
+dissociated	O
+the	O
+protein	B-protein
+is	O
+bound	B-protein_state
+to	I-protein_state
+RNA	B-chemical
+via	O
+single	B-protein_state
+RRMs	B-structure_element
+.	O
+
+Examples	O
+of	O
+‘	O
+extended	B-protein_state
+conformational	O
+selection	O
+'	O
+during	O
+ligand	O
+binding	O
+have	O
+been	O
+characterized	O
+for	O
+a	O
+growing	O
+number	O
+of	O
+macromolecules	O
+(	O
+for	O
+example	O
+,	O
+adenylate	B-protein_type
+kinase	I-protein_type
+,	O
+LAO	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+,	O
+poly	B-protein_type
+-	I-protein_type
+ubiquitin	I-protein_type
+,	O
+maltose	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+and	O
+the	O
+preQ1	B-protein_type
+riboswitch	I-protein_type
+,	O
+among	O
+others	O
+).	O
+
+Similar	O
+interdisciplinary	O
+structural	O
+approaches	O
+are	O
+likely	O
+to	O
+illuminate	O
+whether	O
+similar	O
+mechanistic	O
+bases	O
+for	O
+RNA	O
+binding	O
+are	O
+widespread	O
+among	O
+other	O
+members	O
+of	O
+the	O
+vast	O
+multi	O
+-	O
+RRM	B-structure_element
+family	O
+.	O
+
+Further	O
+research	O
+will	O
+be	O
+needed	O
+to	O
+understand	O
+the	O
+roles	O
+of	O
+SF1	B-protein
+and	O
+U2AF35	B-protein
+subunits	O
+in	O
+the	O
+conformational	O
+equilibria	O
+underlying	O
+U2AF65	B-protein
+association	O
+with	O
+Py	B-chemical
+tracts	I-chemical
+.	O
+
+The	O
+apparent	O
+equilibrium	B-evidence
+dissociation	I-evidence
+constants	I-evidence
+(	O
+KD	B-evidence
+)	O
+for	O
+binding	O
+the	O
+AdML	B-gene
+13mer	O
+are	O
+as	O
+follows	O
+:	O
+flU2AF65	B-protein
+,	O
+30	O
+±	O
+3	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+35	O
+±	O
+6	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+,	O
+3	O
+,	O
+600	O
+±	O
+300	O
+nM	O
+.	O
+(	O
+c	O
+)	O
+Comparison	O
+of	O
+the	O
+RNA	B-evidence
+sequence	I-evidence
+specificities	I-evidence
+of	O
+flU2AF65	B-protein
+and	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+constructs	O
+binding	O
+C	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+Py	B-chemical
+tracts	I-chemical
+with	O
+4U	O
+'	O
+s	O
+embedded	O
+in	O
+either	O
+the	O
+5	O
+′-	O
+(	O
+light	O
+grey	O
+fill	O
+)	O
+or	O
+3	O
+′-	O
+(	O
+dark	O
+grey	O
+fill	O
+)	O
+regions	O
+.	O
+
+The	O
+KD	B-evidence
+'	O
+s	O
+for	O
+binding	O
+5	B-chemical
+′-	I-chemical
+CCUUUUCCCCCCC	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+are	O
+:	O
+flU2AF65	B-protein
+,	O
+41	O
+±	O
+2	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+31	O
+±	O
+3	O
+nM	O
+.	O
+The	O
+KD	B-evidence
+'	O
+s	O
+for	O
+binding	O
+5	B-chemical
+′-	I-chemical
+CCCCCCCUUUUCC	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+are	O
+:	O
+flU2AF65	B-protein
+,	O
+414	O
+±	O
+12	O
+nM	O
+;	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+,	O
+417	O
+±	O
+10	O
+nM	O
+.	O
+Bar	O
+graphs	O
+are	O
+hatched	O
+to	O
+match	O
+the	O
+constructs	O
+shown	O
+in	O
+a	O
+.	O
+The	O
+average	B-evidence
+apparent	I-evidence
+equilibrium	I-evidence
+affinity	I-evidence
+(	O
+KA	B-evidence
+)	O
+and	O
+s	O
+.	O
+e	O
+.	O
+m	O
+.	O
+for	O
+three	O
+independent	O
+titrations	O
+are	O
+plotted	O
+.	O
+
+Representative	O
+views	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+interactions	O
+with	O
+each	O
+new	O
+nucleotide	B-chemical
+of	O
+the	O
+bound	B-protein_state
+Py	B-chemical
+tract	I-chemical
+.	O
+
+The	O
+average	O
+fitted	O
+fluorescence	O
+anisotropy	O
+RNA	B-evidence
+-	I-evidence
+binding	I-evidence
+curves	I-evidence
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4a	O
+–	O
+c	O
+.	O
+
+RNA	O
+binding	O
+stabilizes	O
+the	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+conformation	O
+of	O
+U2AF65	B-protein
+RRMs	B-structure_element
+.	O
+
+A	O
+surface	O
+representation	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+is	O
+shown	O
+bound	B-protein_state
+to	I-protein_state
+nine	O
+nucleotides	B-chemical
+(	O
+nt	O
+);	O
+the	O
+relative	O
+distances	O
+and	O
+juxtaposition	O
+of	O
+the	O
+branch	B-site
+point	I-site
+sequence	I-site
+(	O
+BPS	B-site
+)	O
+and	O
+consensus	O
+AG	B-chemical
+dinucleotide	I-chemical
+at	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+are	O
+unknown	O
+.	O
+
+This	O
+sequence	O
+pattern	O
+is	O
+conserved	B-protein_state
+among	O
+diverse	O
+plant	B-taxonomy_domain
+peptides	B-chemical
+,	O
+suggesting	O
+that	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormone	I-protein_type
+receptors	I-protein_type
+may	O
+share	O
+a	O
+common	O
+ligand	O
+binding	O
+mode	O
+and	O
+activation	O
+mechanism	O
+.	O
+
+Plants	B-taxonomy_domain
+can	O
+shed	O
+their	O
+leaves	O
+,	O
+flowers	O
+or	O
+other	O
+organs	O
+when	O
+they	O
+no	O
+longer	O
+need	O
+them	O
+.	O
+But	O
+how	O
+does	O
+a	O
+leaf	O
+or	O
+a	O
+flower	O
+know	O
+when	O
+to	O
+let	O
+go	O
+?	O
+A	O
+receptor	B-protein_type
+protein	I-protein_type
+called	O
+HAESA	B-protein
+is	O
+found	O
+on	O
+the	O
+surface	O
+of	O
+the	O
+cells	O
+that	O
+surround	O
+a	O
+future	O
+break	O
+point	O
+on	O
+the	O
+plant	O
+.	O
+When	O
+its	O
+time	O
+to	O
+shed	O
+an	O
+organ	O
+,	O
+a	O
+hormone	B-chemical
+called	O
+IDA	B-protein
+instructs	O
+HAESA	B-protein
+to	O
+trigger	O
+the	O
+shedding	O
+process	O
+.	O
+
+The	O
+experiments	O
+show	O
+that	O
+IDA	B-protein
+binds	B-protein_state
+directly	I-protein_state
+to	I-protein_state
+a	O
+canyon	B-protein_state
+shaped	I-protein_state
+pocket	B-site
+in	O
+HAESA	B-protein
+that	O
+extends	O
+out	O
+from	O
+the	O
+surface	O
+of	O
+the	O
+cell	O
+.	O
+
+Hydrophobic	O
+contacts	O
+and	O
+a	O
+hydrogen	B-site
+-	I-site
+bond	I-site
+network	I-site
+mediate	O
+the	O
+interaction	O
+between	O
+HAESA	B-protein
+and	O
+the	O
+peptide	B-protein_type
+hormone	I-protein_type
+IDA	B-protein
+.	O
+
+HAESA	B-site
+interface	I-site
+residues	I-site
+are	O
+shown	O
+as	O
+sticks	O
+,	O
+selected	O
+hydrogen	O
+bond	O
+interactions	O
+are	O
+denoted	O
+as	O
+dotted	O
+lines	O
+(	O
+in	O
+magenta	O
+).	O
+(	O
+B	O
+)	O
+View	O
+of	O
+the	O
+complete	O
+IDA	B-protein
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+yellow	O
+)	O
+binding	B-site
+pocket	I-site
+in	O
+HAESA	B-protein
+(	O
+surface	O
+view	O
+,	O
+in	O
+blue	O
+).	O
+
+The	O
+IDA	B-complex_assembly
+-	I-complex_assembly
+HAESA	I-complex_assembly
+and	O
+SERK1	B-complex_assembly
+-	I-complex_assembly
+HAESA	I-complex_assembly
+complex	O
+interfaces	B-site
+are	O
+conserved	B-protein_state
+among	O
+HAESA	B-protein
+and	O
+HAESA	B-protein_type
+-	I-protein_type
+like	I-protein_type
+proteins	I-protein_type
+from	O
+different	O
+plant	B-taxonomy_domain
+species	O
+.	O
+
+Details	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+central	O
+Hyp	B-structure_element
+anchor	I-structure_element
+in	O
+IDA	B-protein
+and	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+with	O
+HAESA	B-protein
+are	O
+highlighted	O
+in	O
+(	O
+E	O
+)	O
+and	O
+(	O
+F	O
+),	O
+respectively	O
+.	O
+
+Close	O
+-	O
+up	O
+views	O
+of	O
+(	O
+A	O
+)	O
+IDA	B-protein
+,	O
+(	O
+B	O
+)	O
+the	O
+N	B-protein_state
+-	I-protein_state
+terminally	I-protein_state
+extended	I-protein_state
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+and	O
+(	O
+C	O
+)	O
+IDL1	B-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+HAESA	B-protein
+hormone	B-site
+binding	I-site
+pocket	I-site
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+yellow	O
+)	O
+and	O
+including	O
+simulated	B-experimental_method
+annealing	I-experimental_method
+2Fo	B-evidence
+–	I-evidence
+Fc	I-evidence
+omit	I-evidence
+electron	I-evidence
+density	I-evidence
+maps	I-evidence
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+.	O
+
+Petal	O
+break	O
+-	O
+strength	O
+was	O
+found	O
+significantly	O
+increased	O
+in	O
+almost	O
+all	O
+positions	O
+(	O
+indicated	O
+with	O
+a	O
+*)	O
+for	O
+haesa	B-gene
+/	O
+hsl2	B-gene
+and	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+mutant	B-protein_state
+plants	B-taxonomy_domain
+with	O
+respect	O
+to	O
+the	O
+Col	O
+-	O
+0	O
+control	O
+.	O
+
+A	O
+SDS	B-experimental_method
+PAGE	I-experimental_method
+of	O
+the	O
+peak	O
+fractions	O
+is	O
+shown	O
+alongside	O
+.	O
+
+Transphosphorylation	O
+activity	O
+from	O
+the	O
+active	B-protein_state
+kinase	O
+to	O
+the	O
+mutated	B-protein_state
+form	O
+can	O
+be	O
+observed	O
+in	O
+both	O
+directions	O
+(	O
+lanes	O
+5	O
++	O
+6	O
+).	O
+
+A	O
+Hyp	B-protein_state
+-	I-protein_state
+modified	I-protein_state
+dodecamer	B-structure_element
+comprising	O
+the	O
+highly	B-protein_state
+conserved	I-protein_state
+PIP	B-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+(	O
+Figure	O
+1A	O
+)	O
+interacts	O
+with	O
+HAESA	B-protein
+with	O
+1	O
+:	O
+1	O
+stoichiometry	O
+(	O
+N	O
+)	O
+and	O
+with	O
+a	O
+dissociation	B-evidence
+constant	I-evidence
+(	O
+Kd	B-evidence
+)	O
+of	O
+~	O
+20	O
+μM	O
+(	O
+Figure	O
+1B	O
+).	O
+
+Consistently	O
+,	O
+PKGV	B-mutant
+-	I-mutant
+IDA	I-mutant
+and	O
+IDA	B-protein
+have	O
+similar	O
+binding	B-evidence
+affinities	I-evidence
+in	O
+our	O
+ITC	B-experimental_method
+assays	I-experimental_method
+,	O
+further	O
+indicating	O
+that	O
+HAESA	B-protein
+senses	O
+a	O
+dodecamer	B-structure_element
+peptide	B-chemical
+comprising	O
+residues	O
+58	B-residue_range
+-	I-residue_range
+69IDA	I-residue_range
+(	O
+Figure	O
+2D	O
+).	O
+
+IDL1	B-protein
+,	O
+which	O
+can	O
+rescue	O
+IDA	B-protein
+loss	O
+-	O
+of	O
+-	O
+function	O
+mutants	O
+when	O
+introduced	O
+in	O
+abscission	O
+zone	O
+cells	O
+,	O
+can	O
+also	O
+be	O
+sensed	O
+by	O
+HAESA	B-protein
+,	O
+albeit	O
+with	O
+lower	O
+affinity	B-evidence
+(	O
+Figure	O
+2D	O
+).	O
+
+The	O
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+kinase	I-protein_type
+SERK1	B-protein
+allows	O
+for	O
+high	O
+-	O
+affinity	O
+IDA	O
+sensing	O
+
+As	O
+all	O
+five	O
+SERK	B-protein_type
+family	I-protein_type
+members	I-protein_type
+appear	O
+to	O
+be	O
+expressed	O
+in	O
+the	O
+Arabidopsis	B-taxonomy_domain
+abscission	O
+zone	O
+,	O
+we	O
+quantified	O
+their	O
+relative	O
+contribution	O
+to	O
+floral	O
+abscission	O
+in	O
+Arabidopsis	B-taxonomy_domain
+using	O
+a	O
+petal	B-experimental_method
+break	I-experimental_method
+-	I-experimental_method
+strength	I-experimental_method
+assay	I-experimental_method
+.	O
+
+Importantly	O
+,	O
+hydroxyprolination	B-ptm
+of	O
+IDA	B-protein
+is	O
+critical	O
+for	O
+HAESA	B-complex_assembly
+-	I-complex_assembly
+IDA	I-complex_assembly
+-	I-complex_assembly
+SERK1	I-complex_assembly
+complex	O
+formation	O
+(	O
+Figure	O
+3C	O
+,	O
+D	O
+).	O
+
+Our	O
+calorimetry	B-experimental_method
+experiments	O
+now	O
+reveal	O
+that	O
+SERKs	B-protein_type
+may	O
+render	O
+HAESA	B-protein
+,	O
+and	O
+potentially	O
+other	O
+receptor	B-protein_type
+kinases	I-protein_type
+,	O
+competent	O
+for	O
+high	O
+-	O
+affinity	O
+sensing	O
+of	O
+their	O
+cognate	O
+ligands	O
+.	O
+
+(	O
+A	O
+)	O
+Overview	O
+of	O
+the	O
+ternary	O
+complex	O
+with	O
+HAESA	B-protein
+in	O
+blue	O
+(	O
+surface	O
+representation	O
+),	O
+IDA	B-protein
+in	O
+yellow	O
+(	O
+bonds	O
+representation	O
+)	O
+and	O
+SERK1	B-protein
+in	O
+orange	O
+(	O
+surface	O
+view	O
+).	O
+(	O
+B	O
+)	O
+The	O
+HAESA	B-protein
+ectodomain	B-structure_element
+undergoes	O
+a	O
+conformational	O
+change	O
+upon	O
+SERK1	B-protein
+co	O
+-	O
+receptor	O
+binding	O
+.	O
+
+SERK1	B-protein
+loop	B-structure_element
+residues	O
+establish	O
+multiple	O
+hydrophobic	O
+and	O
+polar	O
+contacts	O
+with	O
+Lys66IDA	B-residue_name_number
+and	O
+the	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+(	O
+Figure	O
+4C	O
+).	O
+
+Deletion	B-experimental_method
+of	O
+the	O
+C	O
+-	O
+terminal	O
+Asn69IDA	B-residue_name_number
+completely	O
+inhibits	B-protein_state
+complex	O
+formation	O
+.	O
+
+(	O
+C	O
+)	O
+Quantitative	O
+petal	B-experimental_method
+break	I-experimental_method
+-	I-experimental_method
+strength	I-experimental_method
+assay	I-experimental_method
+for	O
+Col	O
+-	O
+0	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+flowers	O
+and	O
+35S	B-gene
+::	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+35S	B-gene
+::	O
+IDA	B-mutant
+K66A	I-mutant
+/	I-mutant
+R67A	I-mutant
+mutant	B-protein_state
+flowers	O
+.	O
+
+We	O
+thus	O
+assessed	O
+their	O
+contribution	O
+to	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+SERK1	I-complex_assembly
+complex	O
+formation	O
+.	O
+
+In	O
+contrast	O
+,	O
+over	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+the	O
+IDA	B-mutant
+Lys66IDA	I-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+double	B-protein_state
+mutant	I-protein_state
+significantly	O
+delays	O
+floral	O
+abscission	O
+when	O
+compared	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+control	O
+plants	B-taxonomy_domain
+,	O
+suggesting	O
+that	O
+the	O
+mutant	B-protein_state
+IDA	B-chemical
+peptide	I-chemical
+has	O
+reduced	O
+activity	O
+in	O
+planta	B-taxonomy_domain
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+
+Comparison	O
+of	O
+35S	B-gene
+::	O
+IDA	B-protein
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+plants	B-taxonomy_domain
+further	O
+indicates	O
+that	O
+mutation	B-experimental_method
+of	O
+Lys66IDA	B-mutant
+/	I-mutant
+Arg67IDA	I-mutant
+→	I-mutant
+Ala	I-mutant
+may	O
+cause	O
+a	O
+weak	O
+dominant	O
+negative	O
+effect	O
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+
+This	O
+observation	O
+is	O
+consistent	O
+with	O
+our	O
+complex	O
+structure	B-evidence
+in	O
+which	O
+receptor	O
+and	O
+co	O
+-	O
+receptor	O
+together	O
+form	O
+the	O
+IDA	B-site
+binding	I-site
+pocket	I-site
+.	O
+
+In	O
+both	O
+cases	O
+however	O
+,	O
+the	O
+co	O
+-	O
+receptor	O
+completes	O
+the	O
+hormone	B-site
+binding	I-site
+pocket	I-site
+.	O
+
+Diverse	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormones	I-protein_type
+may	O
+thus	O
+also	O
+bind	O
+their	O
+LRR	B-protein_type
+-	I-protein_type
+RK	I-protein_type
+receptors	I-protein_type
+in	O
+an	O
+extended	B-protein_state
+conformation	I-protein_state
+along	O
+the	O
+inner	O
+surface	O
+of	O
+the	O
+LRR	B-structure_element
+domain	I-structure_element
+and	O
+may	O
+also	O
+use	O
+small	B-protein_state
+,	O
+shape	B-protein_state
+-	I-protein_state
+complementary	I-protein_state
+co	B-protein_type
+-	I-protein_type
+receptors	I-protein_type
+for	O
+high	O
+-	O
+affinity	O
+ligand	O
+binding	O
+and	O
+receptor	O
+activation	O
+.	O
+
+Biogenesis	O
+of	O
+the	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+is	O
+tightly	O
+regulated	O
+.	O
+
+Substitution	B-experimental_method
+of	O
+Thr1	B-residue_name_number
+by	O
+Cys	B-residue_name
+disrupts	O
+the	O
+interaction	O
+with	O
+Lys33	B-residue_name_number
+and	O
+inactivates	B-protein_state
+the	O
+proteasome	B-complex_assembly
+.	O
+
+Although	O
+a	O
+Thr1Ser	B-mutant
+mutant	B-protein_state
+is	O
+active	B-protein_state
+,	O
+it	O
+is	O
+less	O
+efficient	O
+compared	O
+with	O
+wild	B-protein_state
+type	I-protein_state
+because	O
+of	O
+the	O
+unfavourable	O
+orientation	O
+of	O
+Ser1	B-residue_name_number
+towards	O
+incoming	O
+substrates	O
+.	O
+
+This	O
+work	O
+provides	O
+insights	O
+into	O
+the	O
+basic	O
+mechanism	O
+of	O
+proteolysis	O
+and	O
+propeptide	B-ptm
+autolysis	I-ptm
+,	O
+as	O
+well	O
+as	O
+the	O
+evolutionary	O
+pressures	O
+that	O
+drove	O
+the	O
+proteasome	B-complex_assembly
+to	O
+become	O
+a	O
+threonine	B-protein_type
+protease	I-protein_type
+.	O
+
+While	O
+the	O
+inactive	B-protein_state
+α	B-protein
+subunits	I-protein
+build	O
+the	O
+two	O
+outer	O
+rings	B-structure_element
+,	O
+the	O
+β	B-protein
+subunits	I-protein
+form	O
+the	O
+inner	O
+rings	B-structure_element
+.	O
+
+Data	O
+from	O
+biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+analyses	I-experimental_method
+of	O
+proteasome	O
+variants	O
+with	O
+mutations	O
+in	O
+the	O
+β5	B-protein
+propeptide	B-structure_element
+and	O
+the	O
+active	B-site
+site	I-site
+strongly	O
+support	O
+the	O
+model	O
+and	O
+deliver	O
+novel	O
+insights	O
+into	O
+the	O
+structural	O
+constraints	O
+required	O
+for	O
+the	O
+autocatalytic	B-ptm
+activation	I-ptm
+of	O
+the	O
+proteasome	B-complex_assembly
+.	O
+
+By	O
+contrast	O
+,	O
+the	O
+T1A	B-mutant
+mutation	O
+in	O
+subunit	O
+β5	B-protein
+has	O
+been	O
+reported	O
+to	O
+be	O
+lethal	O
+or	O
+nearly	O
+so	O
+.	O
+
+Our	O
+present	O
+crystallographic	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+mutant	B-protein_state
+demonstrates	O
+that	O
+the	O
+mutation	B-experimental_method
+per	O
+se	O
+does	O
+not	O
+structurally	O
+alter	O
+the	O
+catalytic	B-site
+active	I-site
+site	I-site
+and	O
+that	O
+the	O
+trans	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+β5	B-protein
+propeptide	B-structure_element
+is	O
+not	B-protein_state
+bound	I-protein_state
+in	O
+the	O
+β5	B-protein
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+(	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+).	O
+
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+positions	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+O	O
+via	O
+hydrogen	O
+bonding	O
+(∼	O
+2	O
+.	O
+8	O
+Å	O
+)	O
+in	O
+a	O
+perfect	O
+trajectory	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+by	O
+Thr1Oγ	B-residue_name_number
+(	O
+Fig	O
+.	O
+1b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+
+Surprisingly	O
+,	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+is	O
+completely	O
+extended	O
+and	O
+forces	O
+the	O
+histidine	B-residue_name
+side	O
+chain	O
+at	O
+position	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+to	O
+occupy	O
+the	O
+S2	B-site
+instead	O
+of	O
+the	O
+S1	B-site
+pocket	I-site
+,	O
+thereby	O
+disrupting	O
+the	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+.	O
+
+Remarkably	O
+,	O
+eukaryotic	B-taxonomy_domain
+proteasomal	O
+β5	B-protein
+subunits	O
+bear	O
+a	O
+His	B-residue_name
+residue	O
+in	O
+position	O
+(-	B-residue_number
+2	I-residue_number
+)	I-residue_number
+of	O
+the	O
+propeptide	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+).	O
+
+As	O
+proven	O
+by	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+crystal	B-evidence
+structures	I-evidence
+,	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+hydrogen	O
+bonds	O
+to	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+O	O
+.	O
+Although	O
+this	O
+interaction	O
+was	O
+not	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+H	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+T	I-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+2c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4c	O
+,	O
+i	O
+),	O
+exchange	B-experimental_method
+of	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+by	O
+Val	B-residue_name
+in	O
+β2	B-protein
+,	O
+a	O
+conservative	O
+mutation	O
+regarding	O
+size	O
+but	O
+drastic	O
+with	O
+respect	O
+to	O
+polarity	O
+,	O
+was	O
+found	O
+to	O
+inhibit	O
+maturation	O
+of	O
+this	O
+subunit	O
+(	O
+Fig	O
+.	O
+2d	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4e	O
+,	O
+j	O
+).	O
+
+Instead	O
+,	O
+Lys33NH2	B-residue_name_number
+,	O
+which	O
+is	O
+in	O
+hydrogen	O
+-	O
+bonding	O
+distance	O
+to	O
+Thr1Oγ	B-residue_name_number
+(	O
+2	O
+.	O
+7	O
+Å	O
+)	O
+in	O
+all	O
+catalytically	B-protein_state
+active	I-protein_state
+β	B-protein
+subunits	I-protein
+(	O
+Fig	O
+.	O
+3a	O
+,	O
+b	O
+),	O
+was	O
+proposed	O
+to	O
+serve	O
+as	O
+the	O
+proton	O
+acceptor	O
+.	O
+
+A	O
+proposed	O
+catalytic	B-site
+tetrad	I-site
+model	O
+involving	O
+Thr1OH	B-residue_name_number
+,	O
+Thr1NH2	B-residue_name_number
+,	O
+Lys33NH2	B-residue_name_number
+and	O
+Asp17Oδ	B-residue_name_number
+,	O
+as	O
+well	O
+as	O
+a	O
+nucleophilic	O
+water	B-chemical
+molecule	O
+as	O
+the	O
+proton	O
+shuttle	O
+appeared	O
+to	O
+accommodate	O
+all	O
+possible	O
+views	O
+of	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+.	O
+
+The	O
+phenotype	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+mutant	B-protein_state
+was	O
+however	O
+less	O
+pronounced	O
+than	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+yeast	B-taxonomy_domain
+(	O
+Fig	O
+.	O
+4a	O
+).	O
+
+Since	O
+no	O
+acetylation	B-ptm
+of	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+was	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+K33A	I-mutant
+pp	B-chemical
+trans	B-protein_state
+apo	B-protein_state
+crystal	B-evidence
+structure	I-evidence
+,	O
+the	O
+reduced	O
+reactivity	O
+towards	O
+substrates	O
+and	O
+inhibitors	O
+indicates	O
+that	O
+Lys33NH2	B-residue_name_number
+,	O
+rather	O
+than	O
+Thr1NH2	B-residue_name_number
+,	O
+deprotonates	O
+and	O
+activates	O
+Thr1OH	B-residue_name_number
+.	O
+
+Autolysis	B-ptm
+and	O
+residual	O
+catalytic	O
+activity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+mutants	O
+may	O
+originate	O
+from	O
+the	O
+carbonyl	O
+group	O
+of	O
+Asn17	B-residue_name_number
+,	O
+which	O
+albeit	O
+to	O
+a	O
+lower	O
+degree	O
+still	O
+can	O
+polarize	O
+Lys33	B-residue_name_number
+for	O
+the	O
+activation	O
+of	O
+Thr1	B-residue_name_number
+.	O
+
+Together	O
+,	O
+these	O
+observations	O
+suggest	O
+that	O
+efficient	O
+peptide	O
+-	O
+bond	O
+hydrolysis	O
+requires	O
+that	O
+Lys33NH2	B-residue_name_number
+hydrogen	O
+bonds	O
+to	O
+the	O
+active	O
+site	O
+nucleophile	O
+.	O
+
+Crystal	B-evidence
+structure	I-evidence
+analysis	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+confirmed	O
+precursor	B-ptm
+processing	I-ptm
+(	O
+Fig	O
+.	O
+4g	O
+),	O
+and	O
+ligand	B-complex_assembly
+-	I-complex_assembly
+complex	I-complex_assembly
+structures	B-evidence
+with	O
+bortezomib	B-chemical
+and	O
+carfilzomib	B-chemical
+unambiguously	O
+corroborated	O
+the	O
+reactivity	O
+of	O
+Ser1	B-residue_name_number
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+In	O
+vitro	O
+,	O
+the	O
+mutant	B-protein_state
+proteasome	B-complex_assembly
+is	O
+less	O
+susceptible	O
+to	O
+proteasome	B-complex_assembly
+inhibition	O
+by	O
+bortezomib	B-chemical
+(	O
+3	O
+.	O
+7	O
+-	O
+fold	O
+)	O
+and	O
+carfilzomib	B-chemical
+(	O
+1	O
+.	O
+8	O
+-	O
+fold	O
+;	O
+Fig	O
+.	O
+5	O
+).	O
+
+Hence	O
+,	O
+the	O
+mean	B-evidence
+residence	I-evidence
+time	I-evidence
+of	O
+carfilzomib	B-chemical
+at	O
+the	O
+active	B-site
+site	I-site
+is	O
+prolonged	O
+and	O
+the	O
+probability	O
+to	O
+covalently	O
+react	O
+with	O
+Ser1	B-residue_name_number
+is	O
+increased	O
+.	O
+
+In	O
+eukaryotes	B-taxonomy_domain
+,	O
+deletion	O
+of	O
+or	O
+failure	O
+to	O
+cleave	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+propeptides	B-structure_element
+is	O
+well	O
+tolerated	O
+.	O
+
+From	O
+these	O
+data	O
+we	O
+conclude	O
+that	O
+only	O
+the	O
+positioning	O
+of	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+and	O
+Thr1	B-residue_name_number
+as	O
+well	O
+as	O
+the	O
+integrity	O
+of	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+are	O
+required	O
+for	O
+autolysis	B-ptm
+.	O
+
+We	O
+propose	O
+a	O
+catalytic	B-site
+triad	I-site
+for	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+CP	B-complex_assembly
+consisting	O
+of	O
+residues	O
+Thr1	B-residue_name_number
+,	O
+Lys33	B-residue_name_number
+and	O
+Asp	B-residue_name
+/	O
+Glu17	B-residue_name_number
+,	O
+which	O
+are	O
+conserved	O
+among	O
+all	O
+proteolytically	O
+active	O
+eukaryotic	B-taxonomy_domain
+,	O
+bacterial	B-taxonomy_domain
+and	O
+archaeal	B-taxonomy_domain
+proteasome	B-complex_assembly
+subunits	O
+.	O
+
+Analogously	O
+,	O
+Thr1NH3	B-residue_name_number
++	O
+might	O
+promote	O
+the	O
+bivalent	O
+reaction	O
+mode	O
+of	O
+epoxyketone	O
+inhibitors	O
+by	O
+protonating	O
+the	O
+epoxide	O
+moiety	O
+to	O
+create	O
+a	O
+positively	O
+charged	O
+trivalent	O
+oxygen	O
+atom	O
+that	O
+is	O
+subsequently	O
+nucleophilically	O
+attacked	O
+by	O
+Thr1NH2	B-residue_name_number
+.	O
+
+The	O
+residues	O
+Ser129	B-residue_name_number
+and	O
+Asp166	B-residue_name_number
+are	O
+expected	O
+to	O
+increase	O
+the	O
+pKa	O
+value	O
+of	O
+Thr1N	B-residue_name_number
+,	O
+thereby	O
+favouring	O
+its	O
+charged	O
+state	O
+.	O
+
+In	O
+accord	O
+with	O
+the	O
+proposed	O
+Thr1	B-residue_name_number
+–	O
+Lys33	B-residue_name_number
+–	O
+Asp17	B-residue_name_number
+catalytic	B-site
+triad	I-site
+,	O
+crystallographic	B-evidence
+data	I-evidence
+on	O
+the	O
+proteolytically	B-protein_state
+inactive	I-protein_state
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+mutant	B-protein_state
+demonstrate	O
+that	O
+the	O
+interaction	O
+of	O
+Lys33NH2	B-residue_name_number
+and	O
+Cys1	B-residue_name_number
+is	O
+broken	O
+.	O
+
+Notably	O
+,	O
+in	O
+the	O
+Ntn	B-protein_type
+hydrolase	I-protein_type
+penicillin	B-protein_type
+acylase	I-protein_type
+,	O
+substitution	B-experimental_method
+of	O
+the	O
+catalytic	B-protein_state
+N	O
+-	O
+terminal	O
+Ser	B-residue_name
+residue	O
+by	O
+Cys	B-residue_name
+also	O
+inactivates	B-protein_state
+the	O
+enzyme	B-protein_type
+but	O
+still	O
+enables	O
+precursor	B-ptm
+processing	I-ptm
+.	O
+
+Notably	O
+,	O
+proteolytically	B-protein_state
+active	I-protein_state
+proteasome	B-complex_assembly
+subunits	O
+from	O
+archaea	B-taxonomy_domain
+,	O
+yeast	B-taxonomy_domain
+and	O
+mammals	B-taxonomy_domain
+,	O
+including	O
+constitutive	O
+,	O
+immuno	O
+-	O
+and	O
+thymoproteasome	O
+subunits	O
+,	O
+either	O
+encode	O
+Thr	B-residue_name
+or	O
+Ile	B-residue_name
+at	O
+position	O
+3	B-residue_number
+,	O
+indicating	O
+the	O
+importance	O
+of	O
+the	O
+Cγ	O
+for	O
+fixing	O
+the	O
+position	O
+of	O
+the	O
+nucleophilic	O
+Thr1	B-residue_name_number
+.	O
+
+In	O
+contrast	O
+to	O
+Thr1	B-residue_name_number
+,	O
+the	O
+hydroxyl	O
+group	O
+of	O
+Ser1	B-residue_name_number
+occupies	O
+the	O
+position	O
+of	O
+the	O
+Thr1	B-residue_name_number
+methyl	O
+side	O
+chain	O
+in	O
+the	O
+WT	B-protein_state
+enzyme	B-complex_assembly
+,	O
+which	O
+requires	O
+its	O
+reorientation	O
+relative	O
+to	O
+the	O
+substrate	O
+to	O
+allow	O
+cleavage	O
+(	O
+Fig	O
+.	O
+4g	O
+,	O
+h	O
+).	O
+
+The	O
+resulting	O
+deprotonated	O
+Thr1NH2	B-residue_name_number
+finally	O
+activates	O
+a	O
+water	B-chemical
+molecule	O
+for	O
+hydrolysis	O
+of	O
+the	O
+acyl	O
+-	O
+enzyme	O
+.	O
+
+The	O
+prosegment	B-structure_element
+is	O
+cleaved	B-protein_state
+but	O
+still	B-protein_state
+bound	I-protein_state
+in	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+channel	I-site
+.	O
+
+(	O
+h	O
+)	O
+The	O
+methyl	O
+group	O
+of	O
+Thr1	B-residue_name_number
+is	O
+anchored	O
+by	O
+hydrophobic	O
+interactions	O
+with	O
+Ala46Cβ	B-residue_name_number
+and	O
+Thr3Cγ	B-residue_name_number
+.	O
+
+Note	O
+that	O
+IC50	B-evidence
+values	I-evidence
+depend	O
+on	O
+time	O
+and	O
+enzyme	O
+concentration	O
+.	O
+
+The	O
+WT	B-protein_state
+proteasome	B-complex_assembly
+:	I-complex_assembly
+inhibitor	I-complex_assembly
+complex	I-complex_assembly
+structures	B-evidence
+(	O
+inhibitor	O
+in	O
+grey	O
+;	O
+Thr1	B-residue_name_number
+in	O
+black	O
+)	O
+are	O
+superimposed	B-experimental_method
+and	O
+demonstrate	O
+that	O
+mutation	B-experimental_method
+of	O
+Thr1	B-residue_name_number
+to	O
+Ser	B-residue_name
+does	O
+not	O
+affect	O
+the	O
+binding	O
+mode	O
+of	O
+bortezomib	B-chemical
+or	O
+carfilzomib	B-chemical
+.	O
+