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 +1,21182 @@
+Biochemical	B-experimental_method
+analysis	I-experimental_method
+reveals	O
+that	O
+these	O
+outer	B-protein_type
+membrane	I-protein_type
+-	I-protein_type
+anchored	I-protein_type
+proteins	I-protein_type
+are	O
+in	O
+fact	O
+exquisitely	O
+specific	O
+for	O
+the	O
+highly	O
+branched	O
+xyloglucan	B-chemical
+(	O
+XyG	B-chemical
+)	O
+polysaccharide	B-chemical
+.	O
+
+Here	O
+,	O
+we	O
+provide	O
+the	O
+first	O
+biochemical	B-experimental_method
+,	I-experimental_method
+crystallographic	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+genetic	I-experimental_method
+insight	I-experimental_method
+into	O
+how	O
+two	O
+surface	B-protein_type
+glycan	I-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+from	O
+the	O
+complex	O
+Bacteroides	B-species
+ovatus	I-species
+xyloglucan	B-gene
+utilization	I-gene
+locus	I-gene
+(	O
+XyGUL	B-gene
+)	O
+enable	O
+recognition	O
+and	O
+uptake	O
+of	O
+this	O
+ubiquitous	O
+vegetable	B-taxonomy_domain
+polysaccharide	B-chemical
+.	O
+
+This	O
+microbial	B-taxonomy_domain
+community	O
+is	O
+largely	O
+bacterial	B-taxonomy_domain
+,	O
+with	O
+the	O
+Bacteroidetes	B-taxonomy_domain
+,	O
+Firmicutes	B-taxonomy_domain
+,	O
+and	O
+Actinobacteria	B-taxonomy_domain
+comprising	O
+the	O
+dominant	O
+phyla	O
+.	O
+
+In	O
+the	O
+archetypal	O
+starch	B-complex_assembly
+utilization	I-complex_assembly
+system	I-complex_assembly
+of	O
+B	B-species
+.	I-species
+thetaiotaomicron	I-species
+,	O
+starch	O
+binding	O
+to	O
+the	O
+cell	O
+surface	O
+is	O
+mediated	O
+at	O
+eight	O
+distinct	O
+starch	B-site
+-	I-site
+binding	I-site
+sites	I-site
+distributed	O
+among	O
+four	O
+surface	B-protein_type
+glycan	I-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+(	O
+SGBPs	B-protein_type
+):	O
+two	O
+within	O
+the	O
+amylase	B-protein_type
+SusG	B-protein
+,	O
+one	O
+within	O
+SusD	B-protein
+,	O
+two	O
+within	O
+SusE	B-protein
+,	O
+and	O
+three	O
+within	O
+SusF	B-protein
+.	O
+The	O
+functional	O
+redundancy	O
+of	O
+many	O
+of	O
+these	O
+sites	O
+is	O
+high	O
+:	O
+whereas	O
+SusD	B-protein
+is	O
+essential	O
+for	O
+growth	O
+on	O
+starch	B-chemical
+,	O
+combined	O
+mutations	O
+of	O
+the	O
+SusE	B-protein
+,	O
+SusF	B-protein
+,	O
+and	O
+SusG	B-protein
+binding	B-site
+sites	I-site
+are	O
+required	O
+to	O
+impair	O
+growth	O
+on	O
+the	O
+polysaccharide	B-chemical
+.	O
+
+Combined	O
+biochemical	B-experimental_method
+,	I-experimental_method
+structural	I-experimental_method
+,	I-experimental_method
+and	I-experimental_method
+reverse	I-experimental_method
+-	I-experimental_method
+genetic	I-experimental_method
+approaches	I-experimental_method
+clearly	O
+illuminate	O
+the	O
+distinct	O
+,	O
+yet	O
+complementary	O
+,	O
+functions	O
+that	O
+these	O
+two	O
+proteins	O
+play	O
+in	O
+XyG	B-chemical
+recognition	O
+as	O
+it	O
+impacts	O
+the	O
+physiology	O
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+.	O
+
+Hence	O
+,	O
+there	O
+is	O
+a	O
+critical	O
+need	O
+for	O
+the	O
+elucidation	O
+of	O
+detailed	O
+structure	O
+-	O
+function	O
+relationships	O
+among	O
+PUL	B-gene
+SGBPs	B-protein_type
+,	O
+in	O
+light	O
+of	O
+the	O
+manifold	O
+glycan	B-chemical
+structures	O
+in	O
+nature	O
+.	O
+
+Formalin	O
+-	O
+fixed	O
+,	O
+nonpermeabilized	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+were	O
+grown	O
+in	O
+minimal	O
+medium	O
+plus	O
+XyG	B-chemical
+,	O
+probed	O
+with	O
+custom	O
+rabbit	O
+antibodies	O
+to	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+or	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+,	O
+and	O
+then	O
+stained	O
+with	O
+Alexa	O
+Fluor	O
+488	O
+goat	O
+anti	O
+-	O
+rabbit	O
+IgG	O
+.	O
+(	O
+A	O
+)	O
+Overlay	B-experimental_method
+of	O
+bright	B-evidence
+-	I-evidence
+field	I-evidence
+and	I-evidence
+FITC	I-evidence
+images	I-evidence
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+A	O
+.	O
+(	O
+B	O
+)	O
+Overlay	B-experimental_method
+of	O
+bright	B-evidence
+-	I-evidence
+field	I-evidence
+and	I-evidence
+FITC	I-evidence
+images	I-evidence
+of	O
+B	B-species
+.	I-species
+ovatus	I-species
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+.	O
+(	O
+C	O
+)	O
+Bright	B-evidence
+-	I-evidence
+field	I-evidence
+image	I-evidence
+of	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+cells	O
+labeled	O
+with	O
+anti	O
+-	O
+SGBP	O
+-	O
+B	O
+antibodies	O
+.	O
+
+In	O
+our	O
+initial	O
+study	O
+focused	O
+on	O
+the	O
+functional	O
+characterization	O
+of	O
+the	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+of	O
+the	O
+XyGUL	B-gene
+,	O
+we	O
+reported	O
+preliminary	O
+affinity	B-experimental_method
+PAGE	I-experimental_method
+and	O
+isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+(	O
+ITC	B-experimental_method
+)	O
+data	O
+indicating	O
+that	O
+both	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+and	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+are	O
+competent	O
+xyloglucan	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+(	O
+affinity	B-evidence
+constant	I-evidence
+[	O
+Ka	B-evidence
+]	O
+values	O
+of	O
+3	O
+.	O
+74	O
+×	O
+105	O
+M	O
+−	O
+1	O
+and	O
+4	O
+.	O
+98	O
+×	O
+104	O
+M	O
+−	O
+1	O
+,	O
+respectively	O
+[	O
+23	O
+]).	O
+
+Cocrystallization	B-experimental_method
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+with	O
+XyGO2	B-chemical
+generated	O
+a	O
+substrate	B-complex_assembly
+complex	I-complex_assembly
+structure	B-evidence
+(	O
+2	O
+.	O
+3	O
+Å	O
+,	O
+Rwork	B-evidence
+=	O
+21	O
+.	O
+8	O
+%,	O
+Rfree	B-evidence
+=	O
+24	O
+.	O
+8	O
+%,	O
+residues	O
+36	B-residue_range
+to	I-residue_range
+546	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+4A	O
+and	O
+B	O
+;	O
+Table	O
+2	O
+)	O
+that	O
+revealed	O
+the	O
+distinct	O
+binding	B-site
+-	I-site
+site	I-site
+architecture	O
+of	O
+the	O
+XyG	B-protein_type
+binding	I-protein_type
+protein	I-protein_type
+.	O
+
+Molecular	O
+structure	B-evidence
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+Bacova_02651	B-gene
+).	O
+(	O
+A	O
+)	O
+Overlay	B-experimental_method
+of	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+from	O
+the	O
+apo	B-protein_state
+(	O
+rainbow	O
+)	O
+and	O
+XyGO2	B-chemical
+(	O
+gray	O
+)	O
+structures	B-evidence
+.	O
+
+The	O
+backbone	O
+glucose	B-chemical
+residues	O
+are	O
+numbered	O
+from	O
+the	O
+nonreducing	O
+end	O
+;	O
+xylose	B-chemical
+residues	O
+are	O
+labeled	O
+X1	B-residue_name_number
+and	O
+X2	B-residue_name_number
+.	O
+
+Most	O
+surprising	O
+in	O
+light	O
+of	O
+the	O
+saccharide	B-evidence
+-	I-evidence
+binding	I-evidence
+data	I-evidence
+,	O
+however	O
+,	O
+was	O
+a	O
+lack	O
+of	O
+extensive	O
+recognition	O
+of	O
+the	O
+XyG	B-chemical
+side	O
+chains	O
+;	O
+only	O
+Y84	B-residue_name_number
+appeared	O
+to	O
+provide	O
+a	O
+hydrophobic	B-site
+interface	I-site
+for	O
+a	O
+xylosyl	B-chemical
+residue	O
+(	O
+Xyl1	B-residue_name_number
+).	O
+
+Protein	O
+name	O
+Ka	B-evidence
+ΔG	O
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+ΔH	B-evidence
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+TΔS	B-evidence
+(	O
+kcal	O
+⋅	O
+mol	O
+−	O
+1	O
+)	O
+Fold	O
+changeb	O
+M	O
+−	O
+1	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+W82A	B-mutant
+W283A	B-mutant
+W306A	B-mutant
+)	O
+ND	O
+NB	O
+NB	O
+NB	O
+NB	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+W82A	B-mutant
+)	O
+c	O
+4	O
+.	O
+9	O
+9	O
+.	O
+1	O
+×	O
+104	O
+−	O
+6	O
+.	O
+8	O
+−	O
+6	O
+.	O
+3	O
+0	O
+.	O
+5	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+(	O
+W306	B-residue_name_number
+)	O
+ND	O
+NB	O
+NB	O
+NB	O
+NB	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+230	B-residue_range
+–	I-residue_range
+489	I-residue_range
+)	O
+0	O
+.	O
+7	O
+(	O
+8	O
+.	O
+6	O
+±	O
+0	O
+.	O
+20	O
+)	O
+×	O
+104	O
+−	O
+6	O
+.	O
+7	O
+−	O
+14	O
+.	O
+9	O
+±	O
+0	O
+.	O
+1	O
+−	O
+8	O
+.	O
+2	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+Y363A	B-mutant
+)	O
+19	O
+.	O
+7	O
+(	O
+2	O
+.	O
+9	O
+±	O
+0	O
+.	O
+10	O
+)	O
+×	O
+103	O
+−	O
+4	O
+.	O
+7	O
+−	O
+18	O
+.	O
+1	O
+±	O
+0	O
+.	O
+1	O
+−	O
+13	O
+.	O
+3	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+W364A	B-mutant
+)	O
+ND	O
+Weak	O
+Weak	O
+Weak	O
+Weak	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+(	O
+F414A	B-mutant
+)	O
+3	O
+.	O
+2	O
+(	O
+1	O
+.	O
+80	O
+±	O
+0	O
+.	O
+03	O
+)	O
+×	O
+104	O
+−	O
+5	O
+.	O
+8	O
+−	O
+11	O
+.	O
+4	O
+±	O
+0	O
+.	O
+1	O
+−	O
+5	O
+.	O
+6	O
+
+Weak	O
+binding	O
+represents	O
+a	O
+Ka	B-evidence
+of	O
+<	O
+500	O
+M	O
+−	O
+1	O
+.	O
+
+SGBP	B-protein
+-	I-protein
+B	I-protein
+has	O
+a	O
+multimodular	O
+structure	O
+with	O
+a	O
+single	O
+,	O
+C	O
+-	O
+terminal	O
+glycan	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+SGBP	B-protein
+-	I-protein
+B	I-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+XyGO2	B-chemical
+(	O
+2	O
+.	O
+37	O
+Å	O
+,	O
+Rwork	B-evidence
+=	O
+19	O
+.	O
+9	O
+%,	O
+Rfree	B-evidence
+=	O
+23	O
+.	O
+9	O
+%,	O
+residues	O
+34	B-residue_range
+to	I-residue_range
+489	I-residue_range
+)	O
+(	O
+Table	O
+2	O
+)	O
+revealed	O
+an	O
+extended	O
+structure	B-evidence
+composed	O
+of	O
+three	O
+tandem	B-structure_element
+immunoglobulin	I-structure_element
+(	I-structure_element
+Ig	I-structure_element
+)-	I-structure_element
+like	I-structure_element
+domains	I-structure_element
+(	O
+domains	O
+A	B-structure_element
+,	O
+B	B-structure_element
+,	O
+and	O
+C	B-structure_element
+)	O
+followed	O
+at	O
+the	O
+C	O
+terminus	O
+by	O
+a	O
+novel	O
+xyloglucan	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+domain	O
+D	B-structure_element
+)	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+Analogously	O
+,	O
+the	O
+outer	B-protein_state
+membrane	I-protein_state
+-	I-protein_state
+anchored	I-protein_state
+endo	B-protein_type
+-	I-protein_type
+xyloglucanase	I-protein_type
+BoGH5	B-protein
+of	O
+the	O
+XyGUL	B-gene
+contains	O
+a	O
+100	B-structure_element
+-	I-structure_element
+amino	I-structure_element
+-	I-structure_element
+acid	I-structure_element
+,	I-structure_element
+all	I-structure_element
+-	I-structure_element
+β	I-structure_element
+-	I-structure_element
+strand	I-structure_element
+,	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+module	I-structure_element
+and	O
+flexible	B-structure_element
+linker	I-structure_element
+that	O
+imparts	O
+conformational	O
+flexibility	O
+and	O
+distances	O
+the	O
+catalytic	B-structure_element
+module	I-structure_element
+from	O
+the	O
+cell	O
+surface	O
+.	O
+
+The	O
+Y363A	B-mutant
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutant	I-experimental_method
+of	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+displays	O
+a	O
+20	O
+-	O
+fold	O
+decrease	O
+in	O
+the	O
+Ka	B-evidence
+for	O
+XyG	B-chemical
+,	O
+while	O
+the	O
+W364A	B-mutant
+mutant	B-protein_state
+lacks	B-protein_state
+XyG	I-protein_state
+binding	I-protein_state
+(	O
+Table	O
+3	O
+;	O
+see	O
+Fig	O
+.	O
+S6	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+Hoping	O
+to	O
+achieve	O
+a	O
+higher	O
+-	O
+resolution	O
+view	O
+of	O
+the	O
+SGBP	B-protein
+-	I-protein
+B	I-protein
+–	O
+xyloglucan	B-chemical
+interaction	O
+,	O
+we	O
+solved	B-experimental_method
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+fused	B-mutant
+CD	I-mutant
+domains	I-mutant
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+XyGO2	B-chemical
+(	O
+1	O
+.	O
+57	O
+Å	O
+,	O
+Rwork	B-evidence
+=	O
+15	O
+.	O
+6	O
+%,	O
+Rfree	B-evidence
+=	O
+17	O
+.	O
+1	O
+%,	O
+residues	O
+230	B-residue_range
+to	I-residue_range
+489	I-residue_range
+)	O
+(	O
+Table	O
+2	O
+).	O
+
+Growth	O
+on	O
+glucose	B-chemical
+displayed	O
+the	O
+shortest	O
+lag	B-evidence
+time	I-evidence
+for	O
+each	O
+strain	O
+,	O
+and	O
+so	O
+lag	B-evidence
+times	I-evidence
+were	O
+normalized	O
+for	O
+each	O
+carbohydrate	B-chemical
+by	O
+subtracting	O
+the	O
+lag	B-evidence
+time	I-evidence
+of	O
+that	O
+strain	O
+in	O
+glucose	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+;	O
+see	O
+Fig	O
+.	O
+S8	O
+in	O
+the	O
+supplemental	O
+material	O
+).	O
+
+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
+
+Fig	O
+.	O
+1B	O
+)	O
+was	O
+completely	O
+incapable	O
+of	O
+growth	O
+on	O
+XyG	B-chemical
+,	O
+XyGO1	B-chemical
+,	O
+and	O
+XyGO2	B-chemical
+,	O
+indicating	O
+that	O
+SGBP	B-protein
+-	I-protein
+A	I-protein
+is	O
+essential	O
+for	O
+XyG	B-chemical
+utilization	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+Intriguingly	O
+,	O
+the	O
+ΔSGBP	B-mutant
+-	I-mutant
+B	I-mutant
+strain	O
+(	O
+ΔBacova_02650	B-mutant
+)	O
+(	O
+cf	O
+.	O
+
+In	O
+the	O
+BtSus	B-gene
+,	O
+SusD	B-protein
+and	O
+the	O
+TBDT	B-protein_type
+SusC	B-protein
+interact	O
+,	O
+and	O
+we	O
+speculate	O
+that	O
+this	O
+interaction	O
+is	O
+necessary	O
+for	O
+glycan	B-chemical
+uptake	O
+,	O
+as	O
+suggested	O
+by	O
+the	O
+fact	O
+that	O
+a	O
+ΔsusD	B-mutant
+mutant	B-protein_state
+cannot	O
+grow	O
+on	O
+starch	B-chemical
+,	O
+but	O
+a	O
+ΔsusD	B-mutant
+::	O
+SusD	B-mutant
+*	I-mutant
+strain	O
+regains	O
+this	O
+ability	O
+if	O
+a	O
+transcriptional	B-protein_type
+activator	I-protein_type
+of	O
+the	O
+sus	B-gene
+operon	I-gene
+is	O
+supplied	O
+.	O
+
+Recent	O
+work	O
+has	O
+elucidated	O
+that	O
+Bacteroidetes	B-taxonomy_domain
+cross	O
+-	O
+feed	O
+during	O
+growth	O
+on	O
+many	O
+glycans	B-chemical
+;	O
+the	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+expressed	O
+by	O
+one	O
+species	O
+liberate	O
+oligosaccharides	B-chemical
+for	O
+consumption	O
+by	O
+other	O
+members	O
+of	O
+the	O
+community	O
+.	O
+
+In	O
+this	O
+instance	O
+,	O
+coexpression	O
+of	O
+the	O
+susD	B-gene
+-	O
+like	O
+gene	O
+nanU	B-gene
+was	O
+not	O
+required	O
+,	O
+nor	O
+did	O
+the	O
+expression	O
+of	O
+the	O
+nanU	B-gene
+gene	O
+enhance	O
+growth	O
+kinetics	O
+.	O
+
+However	O
+,	O
+the	O
+natural	O
+diversity	O
+of	O
+these	O
+proteins	O
+represents	O
+a	O
+rich	O
+source	O
+for	O
+the	O
+discovery	O
+of	O
+unique	O
+carbohydrate	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+motifs	I-structure_element
+to	O
+both	O
+inform	O
+gut	O
+microbiology	O
+and	O
+generate	O
+new	O
+,	O
+specific	O
+carbohydrate	B-chemical
+analytical	O
+reagents	O
+.	O
+
+The	O
+ability	O
+of	O
+our	O
+resident	O
+gut	O
+bacteria	B-taxonomy_domain
+to	O
+recognize	O
+polysaccharides	B-chemical
+is	O
+the	O
+first	O
+committed	O
+step	O
+of	O
+glycan	B-chemical
+consumption	O
+by	O
+these	O
+organisms	O
+,	O
+a	O
+critical	O
+process	O
+that	O
+influences	O
+the	O
+community	O
+structure	O
+and	O
+thus	O
+the	O
+metabolic	O
+output	O
+(	O
+i	O
+.	O
+e	O
+.,	O
+short	O
+-	O
+chain	O
+fatty	O
+acid	O
+and	O
+metabolite	O
+profile	O
+)	O
+of	O
+these	O
+organisms	O
+.	O
+
+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
+
+Neisseria	B-protein
+adhesin	I-protein
+A	I-protein
+(	O
+NadA	B-protein
+)	O
+present	O
+on	O
+the	O
+meningococcal	B-taxonomy_domain
+surface	O
+can	O
+mediate	O
+binding	O
+to	O
+human	B-species
+cells	O
+and	O
+is	O
+one	O
+of	O
+the	O
+three	O
+MenB	B-species
+vaccine	O
+protein	O
+antigens	O
+.	O
+
+MarR	B-protein_type
+family	O
+proteins	O
+can	O
+promote	O
+bacterial	B-taxonomy_domain
+survival	O
+in	O
+the	O
+presence	O
+of	O
+antibiotics	O
+,	O
+toxic	O
+chemicals	O
+,	O
+organic	O
+solvents	O
+or	O
+reactive	O
+oxygen	O
+species	O
+and	O
+can	O
+regulate	O
+virulence	O
+factor	O
+expression	O
+.	O
+
+MarR	B-protein_type
+homologues	O
+can	O
+act	O
+either	O
+as	O
+transcriptional	O
+repressors	O
+or	O
+as	O
+activators	O
+.	O
+
+A	O
+potentially	O
+interesting	O
+exception	O
+comes	O
+from	O
+the	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+salicylate	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+forms	O
+of	O
+the	O
+Methanobacterium	B-species
+thermoautotrophicum	I-species
+protein	O
+MTH313	B-protein
+which	O
+revealed	O
+that	O
+two	O
+salicylate	B-chemical
+molecules	O
+bind	O
+to	O
+one	O
+MTH313	B-protein
+dimer	B-oligomeric_state
+and	O
+induce	O
+large	O
+conformational	O
+changes	O
+,	O
+apparently	O
+sufficient	O
+to	O
+prevent	O
+DNA	O
+binding	O
+.	O
+
+We	O
+obtained	O
+detailed	O
+new	O
+insights	O
+into	O
+ligand	O
+specificity	O
+,	O
+how	O
+the	O
+ligand	O
+allosterically	O
+influences	O
+the	O
+DNA	O
+-	O
+binding	O
+ability	O
+of	O
+NadR	B-protein
+,	O
+and	O
+the	O
+regulation	O
+of	O
+nadA	B-gene
+expression	O
+,	O
+thus	O
+also	O
+providing	O
+a	O
+deeper	O
+structural	O
+understanding	O
+of	O
+the	O
+ligand	O
+-	O
+responsive	O
+MarR	B-protein_type
+super	O
+-	O
+family	O
+.	O
+
+Since	O
+ligand	O
+-	O
+binding	O
+often	O
+increases	O
+protein	O
+stability	O
+,	O
+we	O
+also	O
+investigated	O
+the	O
+effect	O
+of	O
+various	O
+HPAs	B-chemical
+(	O
+Fig	O
+1A	O
+)	O
+on	O
+the	O
+melting	B-evidence
+temperature	I-evidence
+(	O
+Tm	B-evidence
+)	O
+of	O
+NadR	B-protein
+.	O
+As	O
+a	O
+control	O
+of	O
+specificity	O
+,	O
+we	O
+also	O
+tested	O
+salicylate	B-chemical
+,	O
+a	O
+known	O
+ligand	O
+of	O
+some	O
+MarR	B-protein_type
+proteins	O
+previously	O
+reported	O
+to	O
+increase	O
+the	O
+Tm	B-evidence
+of	O
+ST1710	B-protein
+and	O
+MTH313	B-protein
+.	O
+
+However	O
+,	O
+an	O
+increased	O
+thermal	O
+stability	O
+was	O
+induced	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+and	O
+,	O
+to	O
+a	O
+lesser	O
+extent	O
+,	O
+by	O
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+(	O
+A	O
+)	O
+Molecular	O
+structures	O
+of	O
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+MW	O
+152	O
+.	O
+2	O
+),	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+MW	O
+152	O
+.	O
+2	O
+),	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+MW	O
+186	O
+.	O
+6	O
+)	O
+and	O
+salicylic	B-chemical
+acid	I-chemical
+(	O
+MW	O
+160	O
+.	O
+1	O
+).	O
+(	O
+B	O
+)	O
+DSC	B-experimental_method
+profiles	B-evidence
+,	O
+colored	O
+as	O
+follows	O
+:	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+violet	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+salicylate	I-complex_assembly
+(	O
+red	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+3	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+green	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+blue	O
+),	O
+NadR	B-complex_assembly
++	I-complex_assembly
+3Cl	I-complex_assembly
+,	I-complex_assembly
+4	I-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+(	O
+pink	O
+).	O
+
+NadR	B-protein
+displays	O
+distinct	O
+binding	B-evidence
+affinities	I-evidence
+for	O
+hydroxyphenylacetate	B-chemical
+ligands	O
+
+To	O
+fully	O
+characterize	O
+the	O
+NadR	B-protein
+/	O
+HPA	B-chemical
+interactions	O
+,	O
+we	O
+sought	O
+to	O
+determine	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+NadR	B-protein
+in	O
+ligand	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+(	O
+holo	B-protein_state
+)	O
+and	O
+ligand	B-protein_state
+-	I-protein_state
+free	I-protein_state
+(	O
+apo	B-protein_state
+)	O
+forms	O
+.	O
+
+First	O
+,	O
+we	O
+crystallized	B-experimental_method
+NadR	B-protein
+(	O
+a	O
+selenomethionine	B-experimental_method
+-	I-experimental_method
+labelled	I-experimental_method
+derivative	I-experimental_method
+)	O
+in	O
+the	O
+presence	O
+of	O
+a	O
+200	O
+-	O
+fold	O
+molar	O
+excess	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+A	O
+single	O
+conserved	B-protein_state
+leucine	B-residue_name
+residue	O
+(	O
+L130	B-residue_name_number
+)	O
+is	O
+crucial	O
+for	O
+dimerization	O
+
+The	O
+NadR	B-protein
+dimer	B-site
+interface	I-site
+is	O
+formed	O
+by	O
+at	O
+least	O
+32	O
+residues	O
+,	O
+which	O
+establish	O
+numerous	O
+inter	O
+-	O
+chain	O
+salt	O
+bridges	O
+or	O
+hydrogen	O
+bonds	O
+,	O
+and	O
+many	O
+hydrophobic	O
+packing	O
+interactions	O
+(	O
+Fig	O
+3A	O
+and	O
+3B	O
+).	O
+
+Only	O
+the	O
+L130K	B-mutant
+mutation	O
+induced	O
+a	O
+notable	O
+change	O
+in	O
+the	O
+oligomeric	O
+state	O
+of	O
+NadR	B-protein
+(	O
+Fig	O
+3C	O
+).	O
+
+Chain	B-structure_element
+B	I-structure_element
+,	O
+grey	O
+surface	O
+,	O
+is	O
+marked	O
+blue	O
+to	O
+highlight	O
+residues	O
+probed	O
+by	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+(	O
+E136	B-residue_name_number
+only	O
+makes	O
+a	O
+salt	O
+bridge	O
+with	O
+K126	B-residue_name_number
+,	O
+therefore	O
+it	O
+was	O
+sufficient	O
+to	O
+make	O
+the	O
+K126A	B-mutant
+mutation	O
+to	O
+assess	O
+the	O
+importance	O
+of	O
+this	O
+ionic	O
+interaction	O
+;	O
+the	O
+H7	B-residue_name_number
+position	O
+is	O
+labelled	O
+for	O
+monomer	B-oligomeric_state
+A	B-structure_element
+,	O
+since	O
+electron	B-evidence
+density	I-evidence
+was	O
+lacking	O
+for	O
+monomer	B-oligomeric_state
+B	B-structure_element
+).	O
+(	O
+B	O
+)	O
+A	O
+zoom	O
+into	O
+the	O
+environment	O
+of	O
+helix	B-structure_element
+α6	B-structure_element
+to	O
+show	O
+how	O
+residue	O
+L130	B-residue_name_number
+chain	B-structure_element
+B	I-structure_element
+(	O
+blue	O
+side	O
+chain	O
+)	O
+is	O
+a	O
+focus	O
+of	O
+hydrophobic	O
+packing	O
+interactions	O
+with	O
+L130	B-residue_name_number
+,	O
+L133	B-residue_name_number
+,	O
+L134	B-residue_name_number
+and	O
+L137	B-residue_name_number
+of	O
+chain	B-structure_element
+A	I-structure_element
+(	O
+red	O
+side	O
+chains	O
+).	O
+
+*	O
+Bond	O
+distance	O
+between	O
+the	O
+ligand	O
+carboxylate	O
+group	O
+and	O
+the	O
+water	B-chemical
+molecule	O
+,	O
+which	O
+in	O
+turn	O
+makes	O
+H	O
+-	O
+bond	O
+to	O
+the	O
+SerA9	B-residue_name_number
+and	O
+AsnA11	B-residue_name_number
+side	O
+chains	O
+.	O
+
+Consequently	O
+,	O
+residues	O
+in	O
+the	O
+4	B-site
+-	I-site
+HPA	I-site
+binding	I-site
+pocket	I-site
+are	O
+mostly	O
+contributed	O
+by	O
+NadR	B-protein
+chain	B-structure_element
+B	I-structure_element
+,	O
+and	O
+effectively	O
+created	O
+a	O
+polar	O
+‘	O
+floor	O
+’	O
+and	O
+a	O
+hydrophobic	O
+‘	O
+ceiling	O
+’,	O
+which	O
+house	O
+the	O
+ligand	O
+.	O
+
+Collectively	O
+,	O
+this	O
+mixed	O
+network	O
+of	O
+polar	O
+and	O
+hydrophobic	O
+interactions	O
+endows	O
+NadR	B-protein
+with	O
+a	O
+strong	O
+recognition	O
+pattern	O
+for	O
+HPAs	B-chemical
+,	O
+with	O
+additional	O
+medium	O
+-	O
+range	O
+interactions	O
+potentially	O
+established	O
+with	O
+the	O
+hydroxyl	O
+group	O
+at	O
+the	O
+4	O
+-	O
+position	O
+.	O
+
+Structure	O
+-	O
+activity	O
+relationships	O
+:	O
+molecular	O
+basis	O
+of	O
+enhanced	O
+stabilization	O
+by	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+
+We	O
+modelled	B-experimental_method
+the	O
+binding	O
+of	O
+other	O
+HPAs	B-chemical
+by	O
+in	B-experimental_method
+silico	I-experimental_method
+superposition	I-experimental_method
+onto	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+the	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+structure	B-evidence
+,	O
+and	O
+thereby	O
+obtained	O
+molecular	O
+explanations	O
+for	O
+the	O
+binding	O
+specificities	O
+of	O
+diverse	O
+ligands	O
+.	O
+
+For	O
+example	O
+,	O
+similar	O
+to	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+the	O
+binding	O
+of	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+could	O
+involve	O
+multiple	O
+bonds	O
+towards	O
+the	O
+carboxylate	O
+group	O
+of	O
+the	O
+ligand	O
+and	O
+some	O
+to	O
+the	O
+4	O
+-	O
+hydroxyl	O
+group	O
+.	O
+
+Finally	O
+,	O
+salicylate	B-chemical
+is	O
+presumably	O
+unable	O
+to	O
+specifically	O
+bind	O
+NadR	B-protein
+due	O
+to	O
+the	O
+2	O
+-	O
+hydroxyl	O
+substitution	O
+and	O
+the	O
+shorter	O
+aliphatic	O
+chain	O
+connecting	O
+its	O
+carboxylate	O
+group	O
+(	O
+Fig	O
+1A	O
+):	O
+the	O
+compound	O
+simply	O
+seems	O
+too	O
+small	O
+to	O
+simultaneously	O
+establish	O
+the	O
+network	O
+of	O
+beneficial	O
+bonds	O
+observed	O
+in	O
+the	O
+NadR	B-protein
+/	O
+HPA	B-chemical
+interactions	O
+.	O
+
+The	O
+stoichiometry	O
+of	O
+the	O
+NadR	B-complex_assembly
+-	I-complex_assembly
+HPA	I-complex_assembly
+interactions	O
+was	O
+determined	O
+using	O
+Eq	O
+1	O
+(	O
+see	O
+Materials	O
+and	O
+Methods	O
+),	O
+and	O
+revealed	O
+stoichiometries	B-evidence
+of	O
+1	O
+.	O
+13	O
+for	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+1	O
+.	O
+02	O
+for	O
+3	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+and	O
+1	O
+.	O
+21	O
+for	O
+3Cl	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+strongly	O
+suggesting	O
+that	O
+one	O
+NadR	B-protein
+dimer	B-oligomeric_state
+bound	B-protein_state
+to	I-protein_state
+1	O
+HPA	B-chemical
+analyte	O
+molecule	O
+.	O
+
+The	O
+crystallographic	B-evidence
+data	I-evidence
+,	O
+supported	O
+by	O
+the	O
+SPR	B-experimental_method
+studies	O
+of	O
+binding	B-evidence
+stoichiometry	I-evidence
+,	O
+revealed	O
+the	O
+lack	O
+of	O
+a	O
+second	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+molecule	O
+in	O
+the	O
+homodimer	B-oligomeric_state
+,	O
+suggesting	O
+negative	O
+co	O
+-	O
+operativity	O
+,	O
+a	O
+phenomenon	O
+previously	O
+described	O
+for	O
+the	O
+MTH313	B-protein
+/	O
+salicylate	B-chemical
+interaction	O
+and	O
+for	O
+other	O
+MarR	B-protein_type
+family	O
+proteins	O
+.	O
+
+However	O
+,	O
+since	O
+residues	O
+of	O
+helix	B-structure_element
+α6	B-structure_element
+were	O
+not	O
+directly	O
+involved	O
+in	O
+ligand	O
+binding	O
+,	O
+an	O
+explanation	O
+for	O
+the	O
+lack	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+in	O
+monomer	B-oligomeric_state
+A	B-structure_element
+did	O
+not	O
+emerge	O
+by	O
+analyzing	O
+only	O
+these	O
+backbone	O
+atom	O
+positions	O
+,	O
+suggesting	O
+that	O
+a	O
+more	O
+complex	O
+series	O
+of	O
+allosteric	O
+events	O
+may	O
+occur	O
+.	O
+
+The	O
+broad	O
+spectral	O
+dispersion	O
+and	O
+the	O
+number	O
+of	O
+peaks	O
+observed	O
+,	O
+which	O
+is	O
+close	O
+to	O
+the	O
+number	O
+of	O
+expected	O
+backbone	O
+amide	O
+N	O
+-	O
+H	O
+groups	O
+for	O
+this	O
+polypeptide	O
+,	O
+confirmed	O
+that	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+is	O
+well	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+under	O
+these	O
+conditions	O
+and	O
+exhibits	O
+one	O
+conformation	O
+appreciable	O
+on	O
+the	O
+NMR	B-experimental_method
+timescale	O
+,	O
+i	O
+.	O
+e	O
+.	O
+in	O
+the	O
+NMR	B-experimental_method
+experiments	O
+at	O
+25	O
+°	O
+C	O
+,	O
+two	O
+or	O
+more	O
+distinct	O
+conformations	O
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+monomers	B-oligomeric_state
+were	O
+not	O
+readily	O
+apparent	O
+.	O
+
+(	O
+A	O
+)	O
+Superposition	B-experimental_method
+of	O
+two	O
+1H	B-experimental_method
+-	I-experimental_method
+15N	I-experimental_method
+TROSY	I-experimental_method
+-	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+recorded	O
+at	O
+25	O
+°	O
+C	O
+on	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+(	O
+cyan	O
+)	O
+and	O
+on	O
+NadR	B-protein
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+(	O
+red	O
+).	O
+
+Considering	O
+the	O
+small	O
+size	O
+,	O
+fast	O
+diffusion	O
+and	O
+relatively	O
+low	O
+binding	B-evidence
+affinity	I-evidence
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+it	O
+would	O
+not	O
+be	O
+surprising	O
+if	O
+the	O
+ligand	O
+associates	O
+and	O
+dissociates	O
+rapidly	O
+on	O
+the	O
+NMR	B-experimental_method
+time	O
+scale	O
+,	O
+resulting	O
+in	O
+only	O
+one	O
+set	O
+of	O
+peaks	O
+whose	O
+chemical	O
+shifts	O
+represent	O
+the	O
+average	O
+environment	O
+of	O
+the	O
+bound	B-protein_state
+and	O
+unbound	B-protein_state
+states	O
+.	O
+
+Overall	O
+apo	B-protein_state
+-	O
+and	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+structures	B-evidence
+are	O
+similar	O
+.	O
+
+To	O
+further	O
+investigate	O
+the	O
+conformational	O
+rearrangements	O
+of	O
+NadR	B-protein
+,	O
+we	O
+performed	O
+local	B-experimental_method
+structural	I-experimental_method
+alignments	I-experimental_method
+using	O
+only	O
+a	O
+subset	O
+of	O
+residues	O
+in	O
+the	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+helix	I-structure_element
+(	O
+α4	B-structure_element
+).	O
+
+By	O
+selecting	B-experimental_method
+and	O
+aligning	B-experimental_method
+residues	O
+Arg64	B-residue_range
+-	I-residue_range
+Ala77	I-residue_range
+of	O
+one	O
+α4	B-structure_element
+helix	I-structure_element
+per	O
+dimer	B-oligomeric_state
+,	O
+superposition	B-experimental_method
+of	O
+the	O
+holo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+onto	O
+the	O
+two	O
+apo	B-protein_state
+-	O
+homodimers	B-oligomeric_state
+revealed	O
+differences	O
+in	O
+the	O
+monomer	B-oligomeric_state
+conformations	O
+of	O
+each	O
+structure	B-evidence
+.	O
+
+The	O
+three	O
+homodimers	B-oligomeric_state
+(	O
+chains	O
+AB	B-structure_element
+holo	B-protein_state
+,	O
+AB	B-structure_element
+apo	B-protein_state
+,	O
+and	O
+CD	B-structure_element
+apo	B-protein_state
+)	O
+were	O
+overlaid	B-experimental_method
+by	O
+structural	B-experimental_method
+alignment	I-experimental_method
+exclusively	O
+of	O
+all	O
+heavy	O
+atoms	O
+in	O
+residues	O
+R64	B-residue_range
+-	I-residue_range
+A77	I-residue_range
+(	O
+shown	O
+in	O
+red	O
+,	O
+with	O
+side	O
+chain	O
+sticks	O
+)	O
+of	O
+chains	O
+A	B-structure_element
+holo	B-protein_state
+,	O
+A	B-structure_element
+apo	B-protein_state
+,	O
+and	O
+C	B-structure_element
+apo	B-protein_state
+,	O
+belonging	O
+to	O
+helix	B-structure_element
+α4	B-structure_element
+(	O
+left	O
+).	O
+
+However	O
+,	O
+structural	B-experimental_method
+comparisons	I-experimental_method
+revealed	O
+that	O
+the	O
+shift	O
+of	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+helix	B-structure_element
+α4	B-structure_element
+induced	O
+by	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+was	O
+also	O
+accompanied	O
+by	O
+several	O
+changes	O
+at	O
+the	O
+holo	B-protein_state
+dimer	B-site
+interface	I-site
+,	O
+while	O
+such	O
+extensive	O
+structural	O
+differences	O
+were	O
+not	O
+observed	O
+in	O
+the	O
+apo	B-protein_state
+dimer	B-site
+interfaces	I-site
+,	O
+particularly	O
+notable	O
+when	O
+comparing	O
+the	O
+α6	B-structure_element
+helices	I-structure_element
+(	O
+S3	O
+Fig	O
+).	O
+
+Pairwise	B-experimental_method
+superpositions	I-experimental_method
+showed	O
+that	O
+the	O
+NadR	B-protein
+apo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+AB	B-structure_element
+was	O
+the	O
+most	O
+similar	O
+to	O
+OhrR	B-protein
+(	O
+rmsd	B-evidence
+2	O
+.	O
+6	O
+Å	O
+),	O
+while	O
+the	O
+holo	B-protein_state
+-	O
+homodimer	B-oligomeric_state
+was	O
+the	O
+most	O
+divergent	O
+(	O
+rmsd	B-evidence
+3	O
+.	O
+3	O
+Å	O
+)	O
+(	O
+Fig	O
+8C	O
+).	O
+
+Interestingly	O
+,	O
+and	O
+on	O
+the	O
+contrary	O
+,	O
+the	O
+nadR	B-gene
+N11A	B-mutant
+complemented	O
+strain	O
+showed	O
+hypo	O
+-	O
+repression	O
+(	O
+i	O
+.	O
+e	O
+.	O
+exhibited	O
+high	O
+expression	O
+of	O
+nadA	B-gene
+both	O
+in	O
+absence	O
+and	O
+presence	O
+of	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+).	O
+
+Western	B-experimental_method
+blot	I-experimental_method
+analyses	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+strain	O
+(	O
+lanes	O
+1	O
+–	O
+2	O
+)	O
+or	O
+isogenic	O
+nadR	B-gene
+knockout	O
+strains	O
+(	O
+ΔNadR	B-mutant
+)	O
+complemented	O
+to	O
+express	O
+the	O
+indicated	O
+NadR	B-protein
+WT	B-protein_state
+or	O
+mutant	B-protein_state
+proteins	O
+(	O
+lanes	O
+3	O
+–	O
+12	O
+)	O
+or	O
+not	O
+complemented	O
+(	O
+lanes	O
+13	O
+–	O
+14	O
+),	O
+grown	O
+in	O
+the	O
+presence	O
+(	O
+even	O
+lanes	O
+)	O
+or	O
+absence	O
+(	O
+odd	O
+lanes	O
+)	O
+of	O
+5mM	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+showing	O
+NadA	B-protein
+and	O
+NadR	B-protein
+expression	O
+.	O
+
+Complementation	O
+of	O
+ΔNadR	B-mutant
+with	O
+WT	B-protein_state
+NadR	B-protein
+enables	O
+induction	O
+of	O
+nadA	B-gene
+expression	O
+by	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+.	O
+
+Here	O
+,	O
+we	O
+determined	O
+the	O
+first	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+apo	B-protein_state
+-	O
+NadR	B-protein
+and	O
+holo	B-protein_state
+-	O
+NadR	B-protein
+.	O
+These	O
+experimentally	O
+-	O
+determined	O
+structures	B-evidence
+enabled	O
+a	O
+new	O
+detailed	O
+characterization	O
+of	O
+the	O
+ligand	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+Subsequently	O
+,	O
+we	O
+established	O
+the	O
+functional	O
+importance	O
+of	O
+His7	B-residue_name_number
+,	O
+Ser9	B-residue_name_number
+,	O
+Asn11	B-residue_name_number
+and	O
+Phe25	B-residue_name_number
+in	O
+the	O
+in	O
+vitro	O
+response	O
+of	O
+meningococcus	B-taxonomy_domain
+to	O
+4	B-chemical
+-	I-chemical
+HPA	I-chemical
+,	O
+via	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+.	O
+
+(	O
+B	O
+)	O
+A	O
+structural	B-experimental_method
+alignment	I-experimental_method
+of	O
+MTH313	B-protein
+chain	B-structure_element
+A	I-structure_element
+and	O
+ST1710	B-protein
+(	O
+pink	O
+)	O
+(	O
+Cα	O
+rmsd	B-evidence
+2	O
+.	O
+3Å	O
+),	O
+shows	O
+that	O
+they	O
+bind	O
+salicylate	B-chemical
+in	O
+equivalent	O
+sites	O
+(	O
+differing	O
+by	O
+only	O
+~	O
+3Å	O
+)	O
+and	O
+with	O
+the	O
+same	O
+orientation	O
+.	O
+
+In	O
+an	O
+alternative	O
+and	O
+less	O
+extensive	O
+manner	O
+,	O
+the	O
+binding	O
+of	O
+two	O
+salicylate	B-chemical
+molecules	O
+to	O
+the	O
+M	B-species
+.	I-species
+thermoautotrophicum	I-species
+protein	O
+MTH313	B-protein
+appeared	O
+to	O
+induce	O
+large	O
+changes	O
+in	O
+the	O
+wHTH	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+was	O
+associated	O
+with	O
+reduced	O
+DNA	O
+-	O
+binding	O
+activity	O
+.	O
+
+Here	O
+,	O
+we	O
+report	O
+two	O
+high	O
+-	O
+resolution	O
+PduL	B-protein_type
+crystal	B-evidence
+structures	I-evidence
+with	B-protein_state
+bound	I-protein_state
+substrates	I-protein_state
+.	O
+
+This	O
+reaction	O
+directly	O
+links	O
+an	O
+acyl	B-chemical
+-	I-chemical
+CoA	I-chemical
+with	O
+ATP	B-chemical
+generation	O
+via	O
+substrate	O
+-	O
+level	O
+phosphorylation	O
+,	O
+producing	O
+short	B-chemical
+-	I-chemical
+chain	I-chemical
+fatty	I-chemical
+acids	I-chemical
+(	O
+e	O
+.	O
+g	O
+.,	O
+acetate	B-chemical
+),	O
+and	O
+also	O
+provides	O
+a	O
+path	O
+for	O
+short	B-chemical
+-	I-chemical
+chain	I-chemical
+fatty	I-chemical
+acids	I-chemical
+to	O
+enter	O
+central	O
+metabolism	O
+.	O
+
+Not	O
+only	O
+does	O
+PduL	B-protein_type
+facilitate	O
+substrate	O
+level	O
+phosphorylation	O
+,	O
+but	O
+it	O
+also	O
+is	O
+critical	O
+for	O
+cofactor	O
+recycling	O
+within	O
+,	O
+and	O
+product	O
+efflux	O
+from	O
+,	O
+the	O
+organelle	O
+.	O
+
+More	O
+recently	O
+,	O
+bioinformatic	B-experimental_method
+studies	I-experimental_method
+have	O
+demonstrated	O
+the	O
+widespread	O
+distribution	O
+of	O
+BMCs	B-complex_assembly
+among	O
+diverse	O
+bacterial	B-taxonomy_domain
+phyla	I-taxonomy_domain
+and	O
+grouped	O
+them	O
+into	O
+23	O
+different	O
+functional	O
+types	O
+.	O
+
+They	O
+can	O
+also	O
+work	O
+in	O
+the	O
+reverse	O
+direction	O
+to	O
+activate	O
+acetate	B-chemical
+to	O
+the	O
+CoA	B-chemical
+-	I-chemical
+thioester	I-chemical
+.	O
+
+Another	O
+distinctive	O
+feature	O
+of	O
+BMC	B-protein_state
+-	I-protein_state
+associated	I-protein_state
+PduL	B-protein_type
+homologs	O
+is	O
+an	O
+N	O
+-	O
+terminal	O
+encapsulation	B-structure_element
+peptide	I-structure_element
+(	O
+EP	B-structure_element
+)	O
+that	O
+is	O
+thought	O
+to	O
+“	O
+target	O
+”	O
+proteins	O
+for	O
+encapsulation	O
+by	O
+the	O
+BMC	B-complex_assembly
+shell	B-structure_element
+.	O
+
+The	O
+primary	O
+structure	O
+of	O
+PduL	B-protein_type
+homologs	O
+is	O
+subdivided	O
+into	O
+two	O
+PF06130	B-structure_element
+domains	O
+,	O
+each	O
+roughly	O
+80	B-residue_range
+residues	I-residue_range
+in	I-residue_range
+length	I-residue_range
+.	O
+
+Structure	B-experimental_method
+Determination	I-experimental_method
+of	O
+PduL	B-protein_type
+
+While	O
+purifying	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+sPduL	B-protein
+,	O
+we	O
+observed	O
+a	O
+tendency	O
+to	O
+aggregation	O
+as	O
+described	O
+previously	O
+,	O
+with	O
+a	O
+large	O
+fraction	O
+of	O
+the	O
+expressed	O
+protein	O
+found	O
+in	O
+the	O
+insoluble	O
+fraction	O
+in	O
+a	O
+white	O
+,	O
+cake	O
+-	O
+like	O
+pellet	O
+.	O
+
+(	O
+a	O
+)	O
+Primary	O
+and	O
+secondary	O
+structure	O
+of	O
+rPduL	B-protein
+(	O
+tubes	O
+represent	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+,	O
+arrows	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+and	O
+dashed	O
+line	O
+residues	O
+disordered	O
+in	O
+the	O
+structure	B-evidence
+.	O
+
+The	O
+first	B-residue_range
+33	I-residue_range
+amino	I-residue_range
+acids	I-residue_range
+are	O
+present	O
+only	O
+in	O
+the	O
+wildtype	O
+construct	O
+and	O
+contains	O
+the	O
+predicted	O
+EP	B-structure_element
+alpha	B-structure_element
+helix	I-structure_element
+,	O
+α0	B-structure_element
+);	O
+the	O
+truncated	B-protein_state
+rPduLΔEP	B-mutant
+that	O
+was	O
+crystallized	B-experimental_method
+begins	O
+with	O
+M	B-residue_name
+-	O
+G	B-residue_name
+-	O
+V	B-residue_name
+.	O
+Coloring	O
+is	O
+according	O
+to	O
+structural	O
+domains	O
+(	O
+domain	B-structure_element
+1	I-structure_element
+D36	B-residue_range
+-	I-residue_range
+N46	I-residue_range
+/	O
+Q155	B-residue_range
+-	I-residue_range
+C224	I-residue_range
+,	O
+blue	O
+;	O
+loop	B-structure_element
+insertion	I-structure_element
+G61	B-residue_range
+-	I-residue_range
+E81	I-residue_range
+,	O
+grey	O
+;	O
+domain	B-structure_element
+2	I-structure_element
+R47	B-residue_range
+-	I-residue_range
+F60	I-residue_range
+/	O
+E82	B-residue_range
+-	I-residue_range
+A154	I-residue_range
+,	O
+red	O
+).	O
+
+Using	O
+a	O
+mercury	B-experimental_method
+-	I-experimental_method
+derivative	I-experimental_method
+crystal	I-experimental_method
+form	O
+diffracting	O
+to	O
+1	O
+.	O
+99	O
+Å	O
+(	O
+Table	O
+2	O
+),	O
+we	O
+obtained	O
+high	O
+quality	O
+electron	B-evidence
+density	I-evidence
+for	O
+model	O
+building	O
+and	O
+used	O
+the	O
+initial	O
+model	O
+to	O
+refine	O
+against	O
+the	O
+native	O
+data	O
+to	O
+Rwork	B-evidence
+/	O
+Rfree	B-evidence
+values	O
+of	O
+18	O
+.	O
+9	O
+/	O
+22	O
+.	O
+1	O
+%.	O
+
+There	O
+are	O
+two	O
+PduL	B-protein_type
+molecules	O
+in	O
+the	O
+asymmetric	O
+unit	O
+of	O
+the	O
+P212121	O
+unit	O
+cell	O
+.	O
+
+Structurally	O
+,	O
+PduL	B-protein_type
+consists	O
+of	O
+two	O
+domains	B-structure_element
+(	O
+Fig	O
+2	O
+,	O
+blue	O
+/	O
+red	O
+),	O
+each	O
+a	O
+beta	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+that	O
+is	O
+capped	O
+on	O
+both	O
+ends	O
+by	O
+short	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+.	O
+
+Consistent	O
+with	O
+this	O
+,	O
+results	O
+from	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+of	O
+rPduLΔEP	B-mutant
+suggest	O
+that	O
+it	O
+is	O
+a	O
+dimer	B-oligomeric_state
+in	O
+solution	O
+(	O
+Fig	O
+5e	O
+).	O
+
+The	O
+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
+
+CoA	B-chemical
+and	O
+the	O
+metal	O
+ions	O
+bind	O
+between	O
+the	O
+two	O
+domains	O
+,	O
+presumably	O
+in	O
+the	O
+active	B-site
+site	I-site
+(	O
+Figs	O
+2b	O
+and	O
+4a	O
+).	O
+
+The	O
+large	O
+differences	O
+between	O
+the	O
+anomalous	O
+signals	O
+confirm	O
+the	O
+presence	O
+of	O
+zinc	B-chemical
+at	O
+both	O
+metal	O
+sites	O
+(	O
+S3	O
+Fig	O
+).	O
+
+The	O
+first	O
+zinc	B-chemical
+ion	O
+(	O
+Zn1	B-chemical
+)	O
+is	O
+in	O
+a	O
+tetrahedral	O
+coordination	O
+state	O
+with	O
+His48	B-residue_name_number
+,	O
+His50	B-residue_name_number
+,	O
+Glu109	B-residue_name_number
+,	O
+and	O
+the	O
+CoA	B-chemical
+sulfur	B-chemical
+(	O
+Fig	O
+4a	O
+).	O
+
+Oligomeric	O
+States	O
+of	O
+PduL	B-protein_type
+Orthologs	O
+Are	O
+Influenced	O
+by	O
+the	O
+EP	B-structure_element
+
+In	O
+contrast	O
+,	O
+both	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+rPduL	B-protein
+and	O
+pPduL	B-protein
+appeared	O
+to	O
+exist	O
+in	O
+two	O
+distinct	O
+oligomeric	O
+states	O
+(	O
+Fig	O
+5b	O
+and	O
+5c	O
+respectively	O
+,	O
+orange	O
+curves	O
+),	O
+one	O
+form	O
+of	O
+the	O
+approximate	O
+size	O
+of	O
+a	O
+dimer	B-oligomeric_state
+and	O
+the	O
+second	O
+,	O
+a	O
+higher	O
+molecular	O
+weight	O
+oligomer	B-oligomeric_state
+(~	O
+150	O
+kDa	O
+).	O
+
+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
+
+Homologs	O
+of	O
+the	O
+predominant	O
+cofactor	O
+utilizer	O
+(	O
+aldehyde	B-protein_type
+dehydrogenase	I-protein_type
+)	O
+and	O
+NAD	B-chemical
++	I-chemical
+regenerator	O
+(	O
+alcohol	B-protein_type
+dehydrogenase	I-protein_type
+)	O
+have	O
+been	O
+structurally	O
+characterized	O
+,	O
+but	O
+until	O
+now	O
+structural	O
+information	O
+was	O
+lacking	O
+for	O
+PduL	B-protein_type
+,	O
+which	O
+recycles	O
+CoA	B-chemical
+in	O
+the	O
+organelle	O
+lumen	O
+.	O
+
+The	O
+PduL	B-protein_type
+signature	O
+primary	O
+structure	O
+,	O
+two	O
+PF06130	B-structure_element
+domains	O
+,	O
+occurs	O
+in	O
+some	O
+multidomain	O
+proteins	O
+,	O
+most	O
+of	O
+them	O
+annotated	O
+as	O
+Acks	B-protein_type
+,	O
+suggesting	O
+that	O
+PduL	B-protein_type
+may	O
+also	O
+replace	O
+Pta	B-protein_type
+in	O
+variants	O
+of	O
+the	O
+phosphotransacetylase	B-protein_type
+-	O
+Ack	B-protein_type
+pathway	O
+.	O
+
+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
+
+Structured	O
+aggregation	O
+of	O
+the	O
+core	O
+enzymes	O
+has	O
+been	O
+proposed	O
+to	O
+be	O
+the	O
+initial	O
+step	O
+in	O
+metabolosome	B-complex_assembly
+assembly	O
+and	O
+is	O
+known	O
+to	O
+be	O
+the	O
+first	O
+step	O
+of	O
+β	O
+-	O
+carboxysome	O
+biogenesis	O
+,	O
+where	O
+the	O
+core	O
+enzyme	O
+Ribulose	B-protein_type
+Bisphosphate	I-protein_type
+Carboxylase	I-protein_type
+/	I-protein_type
+Oxygenase	I-protein_type
+(	O
+RuBisCO	B-protein_type
+)	O
+is	O
+aggregated	O
+by	O
+the	O
+CcmM	B-protein_type
+protein	O
+.	O
+
+The	O
+close	O
+resemblance	O
+between	O
+the	O
+structures	O
+binding	O
+CoA	B-chemical
+and	O
+phosphate	B-chemical
+likely	O
+indicates	O
+that	O
+no	O
+large	O
+changes	O
+in	O
+protein	O
+conformation	O
+are	O
+involved	O
+in	O
+catalysis	O
+,	O
+and	O
+that	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+are	O
+representative	O
+of	O
+the	O
+active	B-protein_state
+form	O
+.	O
+
+This	O
+hypothesis	O
+is	O
+strengthened	O
+by	O
+the	O
+fact	O
+that	O
+the	O
+CoA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+crystals	B-evidence
+were	O
+obtained	O
+without	O
+added	O
+CoA	B-chemical
+,	O
+indicating	O
+that	O
+the	O
+protein	O
+bound	B-protein_state
+CoA	B-chemical
+from	O
+the	O
+E	B-species
+.	I-species
+coli	I-species
+expression	O
+strain	O
+and	O
+retained	O
+it	O
+throughout	O
+purification	O
+and	O
+crystallization	O
+.	O
+
+PduL	B-protein_type
+and	O
+Pta	B-protein_type
+are	O
+mechanistically	O
+and	O
+structurally	O
+distinct	O
+enzymes	O
+that	O
+catalyze	O
+the	O
+same	O
+reaction	O
+,	O
+a	O
+prime	O
+example	O
+of	O
+evolutionary	O
+convergence	O
+upon	O
+a	O
+function	O
+.	O
+
+This	O
+is	O
+not	O
+surprising	O
+,	O
+as	O
+β	B-protein_type
+-	I-protein_type
+lactamases	I-protein_type
+are	O
+not	O
+so	O
+widespread	O
+among	O
+bacteria	B-taxonomy_domain
+and	O
+therefore	O
+would	O
+be	O
+expected	O
+to	O
+have	O
+evolved	O
+independently	O
+several	O
+times	O
+as	O
+a	O
+defense	O
+mechanism	O
+against	O
+β	O
+-	O
+lactam	O
+antibiotics	O
+.	O
+
+These	O
+results	O
+suggest	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+RNase	B-protein_type
+activity	O
+is	O
+tightly	O
+controlled	O
+by	O
+both	O
+intramolecular	O
+(	O
+NTD	B-structure_element
+-	O
+PIN	B-structure_element
+)	O
+and	O
+intermolecular	O
+(	O
+PIN	B-structure_element
+-	O
+PIN	B-structure_element
+)	O
+interactions	O
+.	O
+
+The	O
+initial	O
+sensing	O
+of	O
+infection	O
+is	O
+mediated	O
+by	O
+a	O
+set	O
+of	O
+pattern	B-protein_type
+-	I-protein_type
+recognition	I-protein_type
+receptors	I-protein_type
+(	O
+PRRs	B-protein_type
+)	O
+such	O
+Toll	B-protein_type
+-	I-protein_type
+like	I-protein_type
+receptors	I-protein_type
+(	O
+TLRs	B-protein_type
+)	O
+and	O
+the	O
+intracellular	O
+signaling	O
+cascades	O
+triggered	O
+by	O
+TLRs	B-protein_type
+evoke	O
+transcriptional	O
+expression	O
+of	O
+inflammatory	O
+mediators	O
+that	O
+coordinate	O
+the	O
+elimination	O
+of	O
+pathogens	O
+and	O
+infected	O
+cells	O
+.	O
+
+Our	O
+data	O
+revealed	O
+that	O
+the	O
+catalytic	O
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+is	O
+regulated	O
+through	O
+both	O
+intra	O
+and	O
+intermolecular	O
+domain	O
+interactions	O
+in	O
+vitro	O
+.	O
+
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+was	O
+attempted	O
+for	O
+the	O
+fragment	O
+containing	O
+both	O
+the	O
+PIN	B-structure_element
+and	O
+ZF	B-structure_element
+domains	O
+,	O
+however	O
+,	O
+electron	B-evidence
+density	I-evidence
+was	O
+observed	O
+only	O
+for	O
+the	O
+PIN	B-structure_element
+domain	O
+(	O
+Fig	O
+.	O
+1c	O
+),	O
+consistent	O
+with	O
+a	O
+previous	O
+report	O
+on	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+derived	O
+from	O
+Homo	B-species
+sapiens	I-species
+.	O
+
+The	O
+domain	O
+structures	B-evidence
+of	O
+NTD	B-structure_element
+,	O
+ZF	B-structure_element
+,	O
+and	O
+CTD	B-structure_element
+were	O
+determined	O
+by	O
+NMR	B-experimental_method
+(	O
+Fig	O
+.	O
+1b	O
+,	O
+d	O
+,	O
+e	O
+).	O
+
+Although	O
+the	O
+PIN	B-structure_element
+domain	O
+is	O
+responsible	O
+for	O
+the	O
+catalytic	O
+activity	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+,	O
+the	O
+roles	O
+of	O
+the	O
+other	O
+domains	O
+are	O
+largely	O
+unknown	O
+.	O
+
+Upon	O
+addition	O
+of	O
+a	O
+larger	O
+amount	O
+of	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+,	O
+the	O
+fluorescence	B-evidence
+of	O
+free	B-protein_state
+RNA	B-chemical
+decreased	O
+,	O
+indicating	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+RNA	B-chemical
+.	O
+
+Direct	O
+binding	O
+of	O
+the	O
+ZF	B-structure_element
+domain	O
+and	O
+RNA	B-chemical
+were	O
+confirmed	O
+by	O
+NMR	B-experimental_method
+spectral	B-evidence
+changes	I-evidence
+.	O
+
+Dimer	B-oligomeric_state
+formation	O
+of	O
+the	O
+PIN	B-structure_element
+domains	O
+
+Mutation	B-experimental_method
+of	O
+Arg215	B-residue_name_number
+,	O
+whose	O
+side	O
+chain	O
+faces	O
+to	O
+the	O
+opposite	O
+side	O
+of	O
+the	O
+oligomeric	B-site
+surface	I-site
+,	O
+to	O
+Glu	B-residue_name
+preserved	O
+the	O
+monomer	B-oligomeric_state
+/	O
+dimer	B-oligomeric_state
+equilibrium	O
+,	O
+similar	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
+Based	O
+on	O
+the	O
+titration	B-evidence
+curve	I-evidence
+for	O
+the	O
+chemical	B-evidence
+shift	I-evidence
+changes	I-evidence
+of	O
+L58	B-residue_name_number
+,	O
+the	O
+apparent	O
+Kd	B-evidence
+between	O
+the	O
+isolated	O
+NTD	B-structure_element
+and	O
+PIN	B-structure_element
+was	O
+estimated	O
+to	O
+be	O
+110	O
+±	O
+5	O
+.	O
+8	O
+μM	O
+.	O
+Considering	O
+the	O
+fact	O
+that	O
+the	O
+NTD	B-structure_element
+and	O
+PIN	B-structure_element
+domains	O
+are	O
+attached	O
+by	O
+a	O
+linker	B-structure_element
+,	O
+the	O
+actual	O
+binding	B-evidence
+affinity	I-evidence
+is	O
+expected	O
+much	O
+higher	O
+in	O
+the	O
+native	B-protein_state
+protein	O
+.	O
+
+An	O
+in	B-experimental_method
+silico	I-experimental_method
+docking	I-experimental_method
+of	O
+the	O
+NTD	B-structure_element
+and	O
+PIN	B-structure_element
+domains	O
+using	O
+chemical	B-evidence
+shift	I-evidence
+restraints	I-evidence
+provided	O
+a	O
+model	O
+consistent	O
+with	O
+the	O
+NMR	B-experimental_method
+experiments	O
+(	O
+Fig	O
+.	O
+3c	O
+).	O
+
+When	O
+any	O
+members	O
+of	O
+the	O
+two	O
+groups	O
+are	O
+mixed	O
+,	O
+two	O
+kinds	O
+of	O
+heterodimers	B-oligomeric_state
+can	O
+be	O
+formed	O
+:	O
+one	O
+is	O
+composed	O
+of	O
+a	O
+DDNN	B-mutant
+primary	B-protein_state
+PIN	B-structure_element
+and	O
+a	O
+basic	O
+residue	O
+mutant	B-protein_state
+secondary	B-protein_state
+PIN	B-structure_element
+and	O
+is	O
+expected	O
+to	O
+exhibit	O
+no	O
+RNase	B-protein_type
+activity	O
+;	O
+the	O
+other	O
+is	O
+composed	O
+of	O
+a	O
+basic	O
+residue	O
+mutant	B-protein_state
+primary	B-protein_state
+PIN	B-structure_element
+and	O
+a	O
+DDNN	B-mutant
+secondary	B-protein_state
+PIN	B-structure_element
+and	O
+is	O
+predicted	O
+to	O
+rescue	O
+RNase	B-protein_type
+activity	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+
+When	O
+we	O
+compared	O
+the	O
+fluorescence	B-evidence
+intensity	I-evidence
+of	O
+uncleaved	B-protein_state
+IL	B-protein_type
+-	I-protein_type
+6	I-protein_type
+mRNA	B-chemical
+,	O
+basic	O
+residue	O
+mutants	B-protein_state
+W182A	B-mutant
+,	O
+K184A	B-mutant
+,	O
+R214A	B-mutant
+,	O
+and	O
+R220A	B-mutant
+were	O
+rescued	O
+upon	O
+addition	O
+of	O
+the	O
+DDNN	B-mutant
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+5b	O
+).	O
+
+Rescue	O
+of	O
+K184A	B-mutant
+and	O
+R214A	B-mutant
+by	O
+the	O
+DDNN	B-mutant
+mutant	B-protein_state
+was	O
+also	O
+confirmed	O
+by	O
+a	O
+significant	O
+increase	O
+in	O
+the	O
+cleaved	O
+products	O
+.	O
+
+R214	B-residue_name_number
+is	O
+an	O
+important	O
+residue	O
+for	O
+dimer	B-oligomeric_state
+formation	O
+as	O
+shown	O
+in	O
+Fig	O
+.	O
+2	O
+,	O
+therefore	O
+,	O
+R214A	B-mutant
+in	O
+the	O
+secondary	B-protein_state
+PIN	B-structure_element
+cannot	O
+dimerize	O
+.	O
+
+Due	O
+to	O
+this	O
+limitation	O
+,	O
+it	O
+is	O
+difficult	O
+to	O
+perform	O
+further	O
+structural	B-experimental_method
+analyses	I-experimental_method
+of	O
+mRNA	B-complex_assembly
+-	I-complex_assembly
+Regnase	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+complexes	O
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+or	O
+NMR	B-experimental_method
+.	O
+
+Moreover	O
+,	O
+our	O
+structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+mutational	I-experimental_method
+analyses	I-experimental_method
+showed	O
+these	O
+two	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+specific	O
+basic	O
+regions	O
+were	O
+essential	O
+for	O
+target	O
+mRNA	B-chemical
+cleavage	O
+in	O
+vitro	O
+.	O
+
+The	O
+affinity	B-evidence
+of	O
+the	O
+domain	O
+-	O
+domain	O
+interaction	O
+between	O
+two	O
+PIN	B-structure_element
+domains	O
+(	O
+Kd	B-evidence
+=	O
+~	O
+10	O
+−	O
+4	O
+M	O
+)	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+NTD	B-structure_element
+-	O
+PIN	B-structure_element
+(	O
+Kd	B-evidence
+=	O
+110	O
+±	O
+5	O
+.	O
+8	O
+μM	O
+)	O
+interactions	O
+;	O
+however	O
+,	O
+the	O
+covalent	O
+connection	O
+corresponding	O
+to	O
+residues	O
+90	B-residue_range
+–	I-residue_range
+133	I-residue_range
+between	O
+the	O
+NTD	B-structure_element
+and	O
+the	O
+primary	B-protein_state
+PIN	B-structure_element
+will	O
+greatly	O
+enhance	O
+the	O
+intramolecular	O
+domain	O
+interaction	O
+in	O
+the	O
+case	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Regnase	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+In	O
+this	O
+context	O
+,	O
+it	O
+is	O
+interesting	O
+that	O
+,	O
+in	O
+response	O
+to	O
+TCR	O
+stimulation	O
+,	O
+Malt1	B-protein
+cleaves	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+at	O
+R111	B-residue_name_number
+to	O
+control	O
+immune	O
+responses	O
+in	O
+vivo	O
+.	O
+
+Two	O
+PIN	B-structure_element
+molecules	O
+in	O
+the	O
+crystal	B-evidence
+were	O
+colored	O
+white	O
+and	O
+green	O
+,	O
+respectively	O
+.	O
+
+Eukaryotic	B-taxonomy_domain
+ribosome	O
+biogenesis	O
+is	O
+highly	O
+complex	O
+and	O
+requires	O
+a	O
+large	O
+number	O
+of	O
+non	O
+-	O
+ribosomal	O
+proteins	O
+and	O
+small	B-chemical
+non	I-chemical
+-	I-chemical
+coding	I-chemical
+RNAs	I-chemical
+in	O
+addition	O
+to	O
+ribosomal	B-chemical
+RNAs	I-chemical
+(	O
+rRNAs	B-chemical
+)	O
+and	O
+proteins	O
+.	O
+
+In	O
+addition	O
+,	O
+18S	B-chemical
+and	O
+25S	B-chemical
+(	O
+yeast	B-taxonomy_domain
+)/	O
+28S	B-chemical
+(	O
+humans	B-species
+)	O
+rRNAs	B-chemical
+contain	O
+several	O
+base	O
+modifications	O
+catalyzed	O
+by	O
+site	O
+-	O
+specific	O
+and	O
+snoRNA	B-chemical
+-	O
+independent	O
+enzymes	O
+.	O
+
+In	O
+Saccharomyces	B-species
+cerevisiae	I-species
+18S	B-chemical
+rRNA	I-chemical
+contains	O
+four	O
+base	O
+methylations	B-ptm
+,	O
+two	O
+acetylations	B-ptm
+and	O
+a	O
+single	O
+3	B-chemical
+-	I-chemical
+amino	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+carboxypropyl	I-chemical
+(	O
+acp	B-chemical
+)	O
+modification	O
+,	O
+whereas	O
+six	O
+base	O
+methylations	B-ptm
+are	O
+present	O
+in	O
+the	O
+25S	B-chemical
+rRNA	I-chemical
+.	O
+
+Defects	O
+of	O
+rRNA	B-chemical
+modification	O
+enzymes	O
+often	O
+lead	O
+to	O
+disturbed	O
+ribosome	O
+biogenesis	O
+or	O
+functionally	O
+impaired	O
+ribosomes	O
+,	O
+although	O
+the	O
+lack	O
+of	O
+individual	O
+rRNA	B-chemical
+modifications	O
+often	O
+has	O
+no	O
+or	O
+only	O
+a	O
+slight	O
+influence	O
+on	O
+the	O
+cell	O
+.	O
+
+Wild	B-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+and	O
+plasmid	O
+encoded	O
+18S	B-chemical
+rRNA	I-chemical
+(	O
+U1191U	B-mutant
+)	O
+show	O
+the	O
+14C	B-chemical
+-	I-chemical
+acp	I-chemical
+signal	O
+,	O
+whereas	O
+the	O
+14C	B-chemical
+-	I-chemical
+acp	I-chemical
+signal	O
+is	O
+missing	O
+in	O
+the	O
+U1191A	B-mutant
+mutant	B-protein_state
+plasmid	O
+encoded	O
+18S	B-chemical
+rRNA	I-chemical
+(	O
+U1191A	B-mutant
+)	O
+and	O
+Δtsr3	B-mutant
+mutants	O
+(	O
+Δtsr3	B-mutant
+).	O
+
+The	O
+primer	O
+extension	O
+arrest	O
+is	O
+reduced	O
+in	O
+HTC116	O
+cells	O
+transfected	O
+with	O
+siRNAs	B-chemical
+544	O
+and	O
+545	O
+.	O
+
+For	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+strain	O
+the	O
+HPLC	B-evidence
+elution	I-evidence
+profile	I-evidence
+of	O
+18S	B-chemical
+rRNA	I-chemical
+nucleosides	B-chemical
+(	O
+Figure	O
+1B	O
+)	O
+was	O
+very	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+pseudouridine	B-protein_type
+-	I-protein_type
+N1	I-protein_type
+methyltransferase	I-protein_type
+mutant	B-protein_state
+Δnep1	B-mutant
+,	O
+where	O
+a	O
+shoulder	O
+at	O
+∼	O
+7	O
+.	O
+4	O
+min	O
+elution	O
+time	O
+was	O
+missing	O
+in	O
+the	O
+elution	O
+profile	O
+.	O
+
+As	O
+previously	O
+reported	O
+this	O
+shoulder	O
+was	O
+identified	O
+by	O
+ESI	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+as	O
+corresponding	O
+to	O
+m1acp3Ψ	B-chemical
+.	O
+
+Similar	O
+to	O
+yeast	B-taxonomy_domain
+,	O
+siRNA	B-experimental_method
+-	I-experimental_method
+mediated	I-experimental_method
+depletion	I-experimental_method
+of	O
+the	O
+Ψ1248	B-protein_type
+N1	I-protein_type
+-	I-protein_type
+methyltransferase	I-protein_type
+Nep1	B-protein
+/	O
+Emg1	B-protein
+had	O
+no	O
+influence	O
+on	O
+the	O
+primer	B-evidence
+extension	I-evidence
+arrest	I-evidence
+(	O
+Figure	O
+1E	O
+).	O
+
+However	O
+,	O
+the	O
+Δtsr3	B-mutant
+deletion	O
+was	O
+synthetic	O
+sick	O
+with	O
+a	O
+Δsnr35	B-mutant
+deletion	O
+preventing	O
+pseudouridylation	B-ptm
+and	O
+Nep1	B-protein
+-	O
+catalyzed	O
+methylation	O
+of	O
+nucleotide	O
+1191	B-residue_number
+(	O
+Figure	O
+2A	O
+).	O
+
+Phenotypic	O
+characterization	O
+of	O
+yeast	B-taxonomy_domain
+TSR3	B-protein
+deletion	O
+(	O
+Δtrs3	B-mutant
+)	O
+and	O
+human	B-species
+TSR3	B-protein
+depletion	O
+(	O
+siRNAs	B-chemical
+544	O
+and	O
+545	O
+)	O
+and	O
+cellular	O
+localization	O
+of	O
+yeast	B-taxonomy_domain
+Tsr3	B-protein
+.	O
+(	O
+A	O
+)	O
+Growth	O
+of	O
+yeast	B-taxonomy_domain
+wild	B-protein_state
+type	I-protein_state
+,	O
+Δtsr3	B-mutant
+,	O
+Δsnr35	B-mutant
+and	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+segregants	O
+after	O
+meiosis	O
+and	O
+tetrad	O
+dissection	O
+of	O
+Δtsr3	B-mutant
+/	O
+TSR3	B-protein
+Δsnr35	B-mutant
+/	O
+SNR35	B-protein
+heterozygous	O
+diploids	O
+.	O
+
+Consistent	O
+with	O
+its	O
+role	O
+in	O
+late	O
+18S	B-chemical
+rRNA	I-chemical
+processing	O
+,	O
+TSR3	B-protein
+deletion	O
+leads	O
+to	O
+a	O
+ribosomal	O
+subunit	O
+imbalance	O
+with	O
+a	O
+reduced	O
+40S	B-complex_assembly
+to	O
+60S	B-complex_assembly
+ratio	O
+of	O
+0	O
+.	O
+81	O
+(	O
+σ	O
+=	O
+0	O
+.	O
+024	O
+)	O
+which	O
+was	O
+further	O
+increased	O
+in	O
+a	O
+Δtsr3	B-mutant
+Δsnr35	I-mutant
+recombinant	O
+to	O
+0	O
+.	O
+73	O
+(	O
+σ	O
+=	O
+0	O
+.	O
+023	O
+)	O
+(	O
+Supplementary	O
+Figure	O
+S2F	O
+).	O
+
+N	O
+-	O
+terminal	O
+deletions	B-experimental_method
+of	O
+36	B-residue_range
+or	O
+45	B-residue_range
+amino	O
+acids	O
+and	O
+C	O
+-	O
+terminal	O
+deletions	B-experimental_method
+of	O
+43	B-residue_range
+or	O
+76	B-residue_range
+residues	O
+show	O
+a	O
+primer	B-evidence
+extension	I-evidence
+stop	I-evidence
+comparable	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
+Strong	O
+20S	O
+rRNA	O
+accumulation	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+Δtsr3	B-mutant
+deletion	B-experimental_method
+is	O
+observed	O
+for	O
+Tsr3	B-protein
+fragments	O
+37	B-residue_range
+–	I-residue_range
+223	I-residue_range
+or	O
+46	B-residue_range
+–	I-residue_range
+223	I-residue_range
+.	O
+
+Well	O
+diffracting	O
+crystals	B-evidence
+were	O
+obtained	O
+for	O
+Tsr3	B-protein
+homologs	O
+from	O
+the	O
+two	O
+crenarchaeal	B-taxonomy_domain
+species	O
+Vulcanisaeta	B-species
+distributa	I-species
+(	O
+VdTsr3	B-protein
+)	O
+and	O
+Sulfolobus	B-species
+solfataricus	I-species
+(	O
+SsTsr3	B-protein
+)	O
+which	O
+share	O
+36	O
+%	O
+(	O
+VdTsr3	B-protein
+)	O
+and	O
+38	O
+%	O
+(	O
+SsTsr3	B-protein
+)	O
+identity	O
+with	O
+the	O
+ScTsr3	B-protein
+core	B-structure_element
+region	I-structure_element
+(	O
+ScTsr3	B-protein
+aa	O
+46	B-residue_range
+–	I-residue_range
+223	I-residue_range
+).	O
+
+Crystals	B-evidence
+of	O
+VdTsr3	B-protein
+diffracted	O
+to	O
+a	O
+resolution	O
+of	O
+1	O
+.	O
+6	O
+Å	O
+whereas	O
+crystals	B-evidence
+of	O
+SsTsr3	B-protein
+diffracted	O
+to	O
+2	O
+.	O
+25	O
+Å	O
+.	O
+Serendipitously	O
+,	O
+VdTsr3	B-protein
+was	O
+purified	O
+and	O
+crystallized	B-experimental_method
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+endogenous	B-protein_state
+(	O
+E	B-species
+.	I-species
+coli	I-species
+)	O
+SAM	B-chemical
+(	O
+Supplementary	O
+Figure	O
+S4	O
+)	O
+while	O
+SsTsr3	B-protein
+crystals	B-evidence
+contained	O
+the	O
+protein	O
+in	O
+the	O
+apo	B-protein_state
+state	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+VdTsr3	B-protein
+can	O
+be	O
+divided	O
+into	O
+two	O
+domains	O
+(	O
+Figure	O
+4A	O
+).	O
+
+A	O
+red	O
+arrow	O
+marks	O
+the	O
+location	O
+of	O
+the	O
+topological	B-structure_element
+knot	I-structure_element
+in	O
+the	O
+structure	B-evidence
+.	O
+(	O
+B	O
+)	O
+Secondary	O
+structure	O
+representation	O
+of	O
+the	O
+VdTsr3	B-protein
+structure	B-evidence
+.	O
+
+Gel	B-experimental_method
+filtration	I-experimental_method
+experiments	O
+with	O
+both	O
+VdTsr3	B-protein
+and	O
+SsTsr3	B-protein
+(	O
+Figure	O
+4E	O
+)	O
+showed	O
+that	O
+both	O
+proteins	O
+are	O
+monomeric	B-oligomeric_state
+in	O
+solution	O
+thereby	O
+extending	O
+the	O
+structural	O
+similarities	O
+to	O
+Trm10	B-protein
+.	O
+
+This	O
+enzyme	O
+,	O
+Tyw2	B-protein
+,	O
+is	O
+part	O
+of	O
+the	O
+biosynthesis	O
+pathway	O
+of	O
+wybutosine	B-chemical
+nucleotides	I-chemical
+in	O
+tRNAs	B-chemical
+.	O
+
+SAM	B-chemical
+-	O
+binding	O
+by	O
+Tsr3	B-protein
+.	O
+
+3xHA	B-protein_state
+tagged	I-protein_state
+Tsr3	B-protein
+mutants	B-protein_state
+are	O
+expressed	O
+comparable	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+as	O
+shown	O
+by	O
+western	B-experimental_method
+blot	I-experimental_method
+(	O
+lower	O
+left	O
+).	O
+
+Accordingly	O
+,	O
+a	O
+W66A	B-mutant
+-	O
+mutation	B-experimental_method
+(	O
+W73	B-residue_name_number
+in	O
+VdTsr3	B-protein
+)	O
+of	O
+SsTsr3	B-protein
+significantly	O
+diminished	O
+SAM	B-evidence
+-	I-evidence
+binding	I-evidence
+in	O
+a	O
+filter	B-experimental_method
+binding	I-experimental_method
+assay	I-experimental_method
+compared	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+Figure	O
+5E	O
+).	O
+
+Furthermore	O
+,	O
+a	O
+W	B-experimental_method
+to	I-experimental_method
+A	I-experimental_method
+mutation	I-experimental_method
+at	O
+the	O
+equivalent	O
+position	O
+W114	B-residue_name_number
+in	O
+ScTsr3	B-protein
+strongly	O
+reduced	O
+the	O
+in	O
+vivo	O
+acp	B-protein_type
+transferase	I-protein_type
+activity	O
+(	O
+Figure	O
+5F	O
+).	O
+
+Furthermore	O
+,	O
+a	O
+negatively	O
+charged	O
+MES	B-chemical
+-	O
+ion	O
+is	O
+found	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+VdTsr3	B-protein
+complexed	B-protein_state
+to	I-protein_state
+the	O
+side	O
+chain	O
+of	O
+K22	B-residue_name_number
+in	O
+helix	B-structure_element
+α1	B-structure_element
+.	O
+
+In	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+,	O
+the	O
+surface	O
+exposed	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+α5	B-structure_element
+and	O
+α7	B-structure_element
+carry	O
+a	O
+significant	O
+amount	O
+of	O
+positively	O
+charged	O
+amino	O
+acids	O
+.	O
+
+For	O
+S	B-species
+.	I-species
+solfataricus	I-species
+the	O
+chemical	O
+identity	O
+of	O
+the	O
+hypermodified	B-protein_state
+nucleotide	B-chemical
+is	O
+not	O
+known	O
+but	O
+the	O
+existence	O
+of	O
+NEP1	B-protein
+and	O
+TSR3	B-protein
+homologs	O
+suggest	O
+that	O
+it	O
+is	O
+indeed	O
+N1	B-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+N3	I-chemical
+-	I-chemical
+acp	I-chemical
+-	I-chemical
+pseudouridine	I-chemical
+.	O
+
+5	O
+′-	O
+fluoresceine	B-chemical
+labeled	O
+RNA	B-chemical
+oligonucleotides	O
+corresponding	O
+either	O
+to	O
+the	O
+native	B-protein_state
+(	O
+20mer	B-oligomeric_state
+–	O
+see	O
+inset	O
+)	O
+or	O
+a	O
+stabilized	B-protein_state
+(	O
+20mer_GC	B-oligomeric_state
+-	O
+inset	O
+)	O
+helix	B-structure_element
+31	I-structure_element
+of	O
+the	O
+small	O
+ribosomal	O
+subunit	O
+rRNA	B-chemical
+from	O
+S	B-species
+.	I-species
+solfataricus	I-species
+were	O
+titrated	B-experimental_method
+with	I-experimental_method
+increasing	I-experimental_method
+amounts	I-experimental_method
+of	O
+SsTsr3	B-protein
+and	O
+the	O
+changes	O
+in	O
+the	O
+fluoresceine	B-chemical
+fluorescence	B-evidence
+anisotropy	I-evidence
+were	O
+measured	O
+and	O
+fitted	O
+to	O
+a	O
+binding	B-evidence
+curve	I-evidence
+(	O
+20mer	B-oligomeric_state
+–	O
+red	O
+,	O
+20mer_GC	B-oligomeric_state
+–	O
+blue	O
+).	O
+
+This	O
+suggests	O
+that	O
+Tsr3	B-protein
+is	O
+not	O
+stably	O
+incorporated	O
+into	O
+pre	B-complex_assembly
+-	I-complex_assembly
+ribosomal	I-complex_assembly
+particles	I-complex_assembly
+and	O
+that	O
+its	O
+binding	O
+to	O
+the	O
+nascent	O
+ribosomal	B-complex_assembly
+subunit	I-complex_assembly
+possibly	O
+requires	O
+additional	O
+interactions	O
+with	O
+other	O
+pre	O
+-	O
+ribosomal	O
+components	O
+.	O
+
+The	O
+cleavage	O
+step	O
+most	O
+likely	O
+acts	O
+as	O
+a	O
+quality	O
+control	O
+check	O
+that	O
+ensures	O
+the	O
+proper	O
+40S	B-complex_assembly
+subunit	I-complex_assembly
+assembly	O
+with	O
+only	O
+completely	O
+processed	O
+precursors	O
+.	O
+
+Finally	O
+,	O
+termination	B-protein_type
+factor	I-protein_type
+Rli1	B-protein
+,	O
+an	O
+ATPase	B-protein_type
+,	O
+promotes	O
+the	O
+dissociation	O
+of	O
+assembly	O
+factors	O
+and	O
+the	O
+80S	B-complex_assembly
+-	I-complex_assembly
+like	I-complex_assembly
+complex	I-complex_assembly
+dissociates	O
+and	O
+releases	O
+the	O
+mature	B-protein_state
+40S	B-complex_assembly
+subunit	I-complex_assembly
+.	O
+
+Thus	O
+,	O
+the	O
+acp	B-chemical
+transfer	O
+to	O
+m1Ψ1191	B-residue_name_number
+occurs	O
+during	O
+the	O
+step	O
+at	O
+which	O
+Rio2	B-protein
+leaves	O
+the	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+particle	I-complex_assembly
+.	O
+
+The	O
+current	O
+data	O
+together	O
+with	O
+the	O
+finding	O
+that	O
+acp	B-chemical
+modification	O
+takes	O
+place	O
+at	O
+the	O
+very	O
+last	O
+step	O
+in	O
+pre	B-complex_assembly
+-	I-complex_assembly
+40S	I-complex_assembly
+subunit	I-complex_assembly
+maturation	O
+indicate	O
+that	O
+the	O
+acp	B-chemical
+modification	O
+probably	O
+supports	O
+the	O
+formation	O
+of	O
+the	O
+decoding	B-site
+site	I-site
+and	O
+efficient	O
+20S	B-chemical
+pre	I-chemical
+-	I-chemical
+rRNA	I-chemical
+D	B-site
+-	I-site
+site	I-site
+cleavage	O
+.	O
+
+These	O
+studies	O
+also	O
+revealed	O
+a	O
+well	O
+ordered	O
+break	O
+in	O
+the	O
+polypeptide	O
+chain	O
+at	O
+Lys147	B-residue_name_number
+,	O
+resulting	O
+in	O
+a	O
+large	O
+conformational	O
+rearrangement	O
+close	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+PmC11	B-protein
+has	O
+an	O
+acidic	B-site
+binding	I-site
+pocket	I-site
+and	O
+a	O
+preference	O
+for	O
+basic	O
+substrates	O
+,	O
+and	O
+accepts	O
+substrates	O
+with	O
+Arg	B-residue_name
+and	O
+Lys	B-residue_name
+in	O
+P1	B-residue_number
+and	O
+does	O
+not	O
+require	O
+Ca2	B-chemical
++	I-chemical
+for	O
+activity	O
+.	O
+
+Clan	B-protein_type
+CD	I-protein_type
+families	I-protein_type
+are	O
+typically	O
+described	O
+using	O
+the	O
+name	O
+of	O
+their	O
+archetypal	O
+,	O
+or	O
+founding	O
+,	O
+member	O
+and	O
+also	O
+given	O
+an	O
+identification	O
+number	O
+preceded	O
+by	O
+a	O
+“	O
+C	O
+,”	O
+to	O
+denote	O
+cysteine	B-protein_type
+peptidase	I-protein_type
+.	O
+
+Although	O
+seven	O
+families	O
+(	O
+C14	O
+is	O
+additionally	O
+split	O
+into	O
+three	O
+subfamilies	O
+)	O
+have	O
+been	O
+described	O
+for	O
+this	O
+clan	O
+,	O
+crystal	B-evidence
+structures	I-evidence
+have	O
+only	O
+been	O
+determined	O
+from	O
+four	O
+:	O
+legumain	B-protein
+(	O
+C13	B-protein_type
+),	O
+caspase	B-protein
+(	O
+C14a	B-protein_type
+),	O
+paracaspase	B-protein
+(	O
+C14b	B-protein_type
+(	I-protein_type
+P	I-protein_type
+),	O
+metacaspase	B-protein
+(	O
+C14b	B-protein_type
+(	I-protein_type
+M	I-protein_type
+),	O
+gingipain	B-protein
+(	O
+C25	B-protein_type
+),	O
+and	O
+the	O
+cysteine	B-structure_element
+peptidase	I-structure_element
+domain	I-structure_element
+(	O
+CPD	B-structure_element
+)	O
+of	O
+various	O
+toxins	O
+(	O
+C80	B-protein_type
+).	O
+
+Clan	B-protein_type
+CD	I-protein_type
+enzymes	I-protein_type
+have	O
+a	O
+highly	B-protein_state
+conserved	I-protein_state
+His	B-site
+/	I-site
+Cys	I-site
+catalytic	I-site
+dyad	I-site
+and	O
+exhibit	O
+strict	O
+specificity	O
+for	O
+the	O
+P1	B-residue_number
+residue	O
+of	O
+their	O
+substrates	O
+.	O
+
+Structure	B-evidence
+of	O
+PmC11	B-protein
+
+The	O
+position	O
+of	O
+the	O
+catalytic	B-site
+dyad	I-site
+(	O
+H	B-residue_name
+,	O
+C	B-residue_name
+)	O
+and	O
+the	O
+processing	B-site
+site	I-site
+(	O
+Lys147	B-residue_name_number
+)	O
+are	O
+highlighted	O
+.	O
+
+Helices	O
+(	O
+1	O
+–	O
+14	O
+)	O
+and	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+(	O
+1	O
+–	O
+9	O
+and	O
+A	O
+-	O
+F	O
+)	O
+are	O
+numbered	O
+from	O
+the	O
+N	O
+terminus	O
+.	O
+
+The	O
+N	O
+and	O
+C	O
+termini	O
+(	O
+N	O
+and	O
+C	O
+)	O
+of	O
+PmC11	B-protein
+along	O
+with	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+(	O
+1	O
+–	O
+9	O
+),	O
+helix	B-structure_element
+α5	B-structure_element
+,	O
+and	O
+helices	B-structure_element
+α8	B-structure_element
+,	O
+α11	B-structure_element
+,	O
+and	O
+α13	B-structure_element
+from	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+,	O
+are	O
+all	O
+labeled	O
+.	O
+
+Of	O
+the	O
+interacting	O
+secondary	O
+structure	O
+elements	O
+,	O
+α5	B-structure_element
+is	O
+perhaps	O
+the	O
+most	O
+interesting	O
+.	O
+
+This	B-structure_element
+helix	I-structure_element
+makes	O
+a	O
+total	O
+of	O
+eight	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+CTD	B-structure_element
+,	O
+including	O
+one	O
+salt	O
+bridge	O
+(	O
+Arg191	B-residue_name_number
+-	O
+Asp255	B-residue_name_number
+)	O
+and	O
+is	O
+surrounded	O
+by	O
+the	O
+CTD	B-structure_element
+on	O
+one	O
+side	O
+and	O
+the	O
+main	B-structure_element
+core	I-structure_element
+of	O
+the	O
+enzyme	O
+on	O
+the	O
+other	O
+,	O
+acting	O
+like	O
+a	O
+linchpin	O
+holding	O
+both	O
+components	O
+together	O
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+
+The	O
+two	O
+ends	O
+of	O
+the	O
+autolytic	B-site
+cleavage	I-site
+site	I-site
+(	O
+Lys147	B-residue_name_number
+and	O
+Ala148	B-residue_name_number
+,	O
+green	O
+)	O
+are	O
+displaced	O
+by	O
+19	O
+.	O
+5	O
+Å	O
+(	O
+thin	O
+black	O
+line	O
+)	O
+from	O
+one	O
+another	O
+and	O
+residues	O
+in	O
+the	O
+potential	O
+substrate	B-site
+binding	I-site
+pocket	I-site
+are	O
+highlighted	O
+in	O
+blue	O
+.	O
+
+B	O
+,	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+of	O
+PmC11	B-protein
+.	O
+
+Elution	O
+fractions	O
+across	O
+the	O
+major	O
+peak	O
+(	O
+1	O
+–	O
+6	O
+)	O
+were	O
+analyzed	O
+by	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+on	O
+a	O
+4	O
+–	O
+12	O
+%	O
+gel	O
+in	O
+MES	O
+buffer	O
+.	O
+
+E	O
+,	O
+intermolecular	B-ptm
+processing	I-ptm
+of	O
+PmC11C179A	B-mutant
+by	O
+PmC11	B-protein
+.	O
+
+PmC11C179A	O
+(	O
+20	O
+μg	O
+)	O
+was	O
+incubated	O
+overnight	O
+at	O
+37	O
+°	O
+C	O
+with	O
+increasing	O
+amounts	O
+of	O
+processed	O
+PmC11	B-protein
+and	O
+analyzed	O
+on	O
+a	O
+10	O
+%	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+gel	O
+.	O
+
+A	O
+single	O
+lane	O
+of	O
+20	O
+μg	O
+of	O
+active	B-protein_state
+PmC11	B-protein
+(	O
+labeled	O
+20	O
+)	O
+is	O
+shown	O
+for	O
+comparison	O
+.	O
+
+The	O
+position	O
+of	O
+the	O
+catalytic	B-site
+dyad	I-site
+,	O
+one	O
+potential	O
+key	B-site
+substrate	I-site
+binding	I-site
+residue	I-site
+Asp177	B-residue_name_number
+,	O
+and	O
+the	O
+ends	O
+of	O
+the	O
+cleavage	B-site
+site	I-site
+Lys147	B-residue_name_number
+and	O
+Ala148	B-residue_name_number
+are	O
+indicated	O
+.	O
+
+To	O
+investigate	O
+this	O
+possibility	O
+,	O
+two	O
+mutant	O
+forms	O
+of	O
+the	O
+enzyme	O
+were	O
+created	O
+:	O
+PmC11C179A	B-mutant
+(	O
+a	O
+catalytically	B-protein_state
+inactive	I-protein_state
+mutant	I-protein_state
+)	O
+and	O
+PmC11K147A	B-mutant
+(	O
+a	O
+cleavage	B-protein_state
+-	I-protein_state
+site	I-protein_state
+mutant	I-protein_state
+).	O
+
+The	O
+PmC11K147A	B-mutant
+mutant	B-protein_state
+enzyme	O
+had	O
+a	O
+markedly	O
+different	O
+reaction	B-evidence
+rate	I-evidence
+(	O
+Vmax	B-evidence
+)	O
+compared	O
+with	O
+WT	B-protein_state
+,	O
+where	O
+the	O
+reaction	B-evidence
+velocity	I-evidence
+of	O
+PmC11	B-protein
+was	O
+10	O
+times	O
+greater	O
+than	O
+that	O
+of	O
+PmC11K147A	B-mutant
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+
+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
+
+Furthermore	O
+,	O
+Cu2	B-chemical
++,	I-chemical
+Fe2	B-chemical
++,	I-chemical
+and	O
+Zn2	B-chemical
++	I-chemical
+appear	O
+to	O
+inhibit	B-protein_state
+PmC11	B-protein
+.	O
+
+The	O
+structural	O
+similarity	O
+of	O
+PmC11	B-protein
+with	O
+its	O
+nearest	O
+structural	O
+neighbors	O
+in	O
+the	O
+PDB	O
+is	O
+decidedly	O
+low	O
+,	O
+overlaying	O
+better	O
+with	O
+six	O
+-	O
+stranded	O
+caspase	B-protein
+-	I-protein
+7	I-protein
+than	O
+any	O
+of	O
+the	O
+other	O
+larger	O
+members	O
+of	O
+the	O
+clan	O
+(	O
+Table	O
+2	O
+).	O
+
+PmC11	B-protein
+differs	O
+from	O
+clostripain	B-protein
+in	O
+that	O
+is	O
+does	O
+not	O
+appear	O
+to	O
+require	O
+divalent	O
+cations	O
+for	O
+activation	O
+.	O
+
+In	O
+addition	O
+,	O
+several	O
+members	O
+of	O
+clan	B-protein_type
+CD	I-protein_type
+exhibit	O
+self	O
+-	O
+inhibition	O
+,	O
+whereby	O
+regions	B-structure_element
+of	O
+the	O
+enzyme	O
+block	O
+access	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+Recently	O
+,	O
+we	O
+solved	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+YfiR	B-protein
+in	O
+both	O
+the	O
+non	B-protein_state
+-	I-protein_state
+oxidized	I-protein_state
+and	O
+the	O
+oxidized	B-protein_state
+states	O
+,	O
+revealing	O
+breakage	O
+/	O
+formation	O
+of	O
+one	O
+disulfide	B-ptm
+bond	I-ptm
+(	O
+Cys71	B-residue_name_number
+-	O
+Cys110	B-residue_name_number
+)	O
+and	O
+local	O
+conformational	O
+change	O
+around	O
+the	O
+other	O
+one	O
+(	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+),	O
+indicating	O
+that	O
+Cys145	B-residue_name_number
+-	O
+Cys152	B-residue_name_number
+plays	O
+an	O
+important	O
+role	O
+in	O
+maintaining	O
+the	O
+correct	O
+folding	O
+of	O
+YfiR	B-protein
+(	O
+Yang	O
+et	O
+al	O
+.,).	O
+
+In	O
+the	O
+present	O
+study	O
+,	O
+we	O
+solved	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+an	O
+N	O
+-	O
+terminal	O
+truncated	B-protein_state
+form	O
+of	O
+YfiB	B-protein
+(	O
+34	B-residue_range
+–	I-residue_range
+168	I-residue_range
+)	O
+and	O
+YfiR	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+an	O
+active	B-protein_state
+mutant	B-protein_state
+YfiBL43P	B-mutant
+.	O
+
+Compared	O
+with	O
+the	O
+reported	O
+complex	O
+structure	O
+,	O
+YfiBL43P	B-mutant
+in	O
+our	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+structure	B-evidence
+has	O
+additional	O
+visible	O
+N	O
+-	O
+terminal	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+58	I-residue_range
+that	O
+are	O
+shown	O
+to	O
+play	O
+essential	O
+roles	O
+in	O
+YfiB	B-protein
+activation	O
+and	O
+biofilm	O
+formation	O
+.	O
+
+In	O
+addition	O
+,	O
+there	O
+is	O
+a	O
+short	O
+helix	B-structure_element
+turn	I-structure_element
+connecting	O
+the	O
+β4	B-structure_element
+strand	I-structure_element
+and	O
+α4	B-structure_element
+helix	I-structure_element
+(	O
+Fig	O
+.	O
+1A	O
+and	O
+1B	O
+).	O
+
+The	O
+YfiR	B-protein
+molecules	O
+are	O
+shown	O
+in	O
+green	O
+and	O
+magenta	O
+.	O
+
+YfiBL43P	B-mutant
+and	O
+YfiR	B-protein
+are	O
+shown	O
+in	O
+cyan	O
+and	O
+green	O
+,	O
+respectively	O
+.	O
+(	O
+E	O
+and	O
+F	O
+)	O
+The	O
+conserved	B-site
+surface	I-site
+in	O
+YfiR	B-protein
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	B-protein
+.	O
+(	O
+G	O
+)	O
+The	O
+residues	B-structure_element
+of	O
+YfiR	B-protein
+responsible	O
+for	O
+interacting	O
+with	O
+YfiB	B-protein
+are	O
+shown	O
+in	O
+green	O
+sticks	O
+,	O
+and	O
+the	O
+proposed	O
+YfiN	B-site
+-	I-site
+interacting	I-site
+residues	I-site
+are	O
+shown	O
+in	O
+yellow	O
+sticks	O
+.	O
+
+The	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+is	O
+a	O
+2	O
+:	O
+2	O
+heterotetramer	B-oligomeric_state
+(	O
+Fig	O
+.	O
+3A	O
+)	O
+in	O
+which	O
+the	O
+YfiR	B-protein
+dimer	B-oligomeric_state
+is	O
+clamped	O
+by	O
+two	O
+separated	O
+YfiBL43P	B-mutant
+molecules	O
+with	O
+a	O
+total	O
+buried	O
+surface	O
+area	O
+of	O
+3161	O
+.	O
+2	O
+Å2	O
+.	O
+
+The	O
+observed	O
+changes	O
+in	O
+conformation	O
+of	O
+YfiB	B-protein
+and	O
+the	O
+results	O
+of	O
+mutagenesis	B-experimental_method
+suggest	O
+a	O
+mechanism	O
+by	O
+which	O
+YfiB	B-protein
+sequesters	O
+YfiR	B-protein
+.	O
+
+Region	B-structure_element
+I	I-structure_element
+is	O
+formed	O
+by	O
+numerous	O
+main	O
+-	O
+chain	O
+and	O
+side	O
+-	O
+chain	O
+hydrophilic	O
+interactions	O
+between	O
+residues	O
+E45	B-residue_name_number
+,	O
+G47	B-residue_name_number
+and	O
+E53	B-residue_name_number
+from	O
+the	O
+N	O
+-	O
+terminal	O
+extended	O
+loop	B-structure_element
+of	O
+YfiB	B-protein
+and	O
+residues	O
+S57	B-residue_name_number
+,	O
+R60	B-residue_name_number
+,	O
+A89	B-residue_name_number
+and	O
+H177	B-residue_name_number
+from	O
+YfiR	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+i	O
+)).	O
+
+Additionally	O
+,	O
+three	O
+hydrophobic	B-site
+anchoring	I-site
+sites	I-site
+exist	O
+in	O
+region	B-structure_element
+I	I-structure_element
+.	O
+The	O
+residues	O
+F48	B-residue_name_number
+and	O
+W55	B-residue_name_number
+of	O
+YfiB	B-protein
+are	O
+inserted	O
+into	O
+the	O
+hydrophobic	B-site
+cores	I-site
+mainly	O
+formed	O
+by	O
+the	O
+main	O
+chain	O
+and	O
+side	O
+chain	O
+carbon	O
+atoms	O
+of	O
+residues	O
+S57	B-residue_name_number
+/	O
+Q88	B-residue_name_number
+/	O
+A89	B-residue_name_number
+/	O
+N90	B-residue_name_number
+and	O
+R60	B-residue_name_number
+/	O
+R175	B-residue_name_number
+/	O
+H177	B-residue_name_number
+of	O
+YfiR	B-protein
+,	O
+respectively	O
+;	O
+and	O
+F57	B-residue_name_number
+of	O
+YfiB	B-protein
+is	O
+inserted	O
+into	O
+the	O
+hydrophobic	B-site
+pocket	I-site
+formed	O
+by	O
+L166	B-residue_name_number
+/	O
+I169	B-residue_name_number
+/	O
+V176	B-residue_name_number
+/	O
+P178	B-residue_name_number
+/	O
+L181	B-residue_name_number
+of	O
+YfiR	B-protein
+(	O
+Fig	O
+.	O
+3D	O
+-	O
+I	O
+(	O
+ii	O
+)).	O
+
+The	O
+results	O
+indicated	O
+that	O
+the	O
+PG	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+YfiBL43P	B-mutant
+is	O
+65	O
+.	O
+5	O
+μmol	O
+/	O
+L	O
+,	O
+which	O
+is	O
+about	O
+16	O
+-	O
+fold	O
+stronger	O
+than	O
+that	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+YfiB	B-protein
+(	O
+Kd	O
+=	O
+1	O
+.	O
+1	O
+mmol	O
+/	O
+L	O
+)	O
+(	O
+Fig	O
+.	O
+4E	O
+–	O
+F	O
+).	O
+
+As	O
+the	O
+experiment	O
+is	O
+performed	O
+in	B-protein_state
+the	I-protein_state
+absence	I-protein_state
+of	I-protein_state
+YfiR	B-protein
+,	O
+it	O
+suggests	O
+that	O
+an	O
+increase	O
+in	O
+the	O
+PG	B-evidence
+-	I-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+YfiB	B-protein
+is	O
+not	O
+a	O
+result	O
+of	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+interaction	O
+and	O
+is	O
+highly	O
+coupled	O
+to	O
+the	O
+activation	O
+of	O
+YfiB	B-protein
+characterized	O
+by	O
+a	O
+stretched	B-protein_state
+N	I-protein_state
+-	I-protein_state
+terminal	I-protein_state
+conformation	I-protein_state
+.	O
+
+Calculation	O
+using	O
+the	O
+ConSurf	B-experimental_method
+Server	I-experimental_method
+(	O
+http	O
+://	O
+consurf	O
+.	O
+tau	O
+.	O
+ac	O
+.	O
+il	O
+/),	O
+which	O
+estimates	O
+the	O
+evolutionary	B-evidence
+conservation	I-evidence
+of	O
+amino	O
+acid	O
+positions	O
+and	O
+visualizes	O
+information	O
+on	O
+the	O
+structure	B-site
+surface	I-site
+,	O
+revealed	O
+a	O
+conserved	B-site
+surface	I-site
+on	O
+YfiR	B-protein
+that	O
+contributes	O
+to	O
+the	O
+interaction	O
+with	O
+YfiB	B-protein
+(	O
+Fig	O
+.	O
+3E	O
+and	O
+3F	O
+).	O
+
+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
+
+YfiR	B-protein
+binds	O
+small	O
+molecules	O
+
+The	O
+results	O
+showed	O
+Kd	B-evidence
+values	O
+of	O
+1	O
+.	O
+4	O
+×	O
+10	O
+−	O
+7	O
+mol	O
+/	O
+L	O
+and	O
+5	O
+.	O
+3	O
+×	O
+10	O
+−	O
+7	O
+mol	O
+/	O
+L	O
+for	O
+YfiBL43P	B-mutant
+and	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+,	O
+respectively	O
+,	O
+revealing	O
+that	O
+the	O
+YfiBL43P	B-mutant
+/	O
+F57A	B-mutant
+mutant	B-protein_state
+caused	O
+a	O
+3	O
+.	O
+8	O
+-	O
+fold	O
+reduction	O
+in	O
+the	O
+binding	B-evidence
+affinity	I-evidence
+compared	O
+with	O
+the	O
+YfiBL43P	B-mutant
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+6F	O
+and	O
+6G	O
+).	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+YfiB	B-protein
+alone	B-protein_state
+and	O
+an	O
+active	B-protein_state
+mutant	B-protein_state
+YfiBL43P	B-mutant
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+YfiR	B-protein
+,	O
+indicating	O
+that	O
+YfiR	B-protein
+forms	O
+a	O
+2	O
+:	O
+2	O
+complex	B-protein_state
+with	I-protein_state
+YfiB	B-protein
+via	O
+a	O
+region	O
+composed	O
+of	O
+conserved	O
+residues	O
+.	O
+
+Thus	O
+,	O
+YfiB	B-protein
+alone	B-protein_state
+represents	O
+an	O
+inactive	B-protein_state
+form	O
+that	O
+may	O
+only	O
+partially	O
+insert	O
+into	O
+the	O
+PG	O
+matrix	O
+.	O
+
+The	O
+periplasmic	B-structure_element
+domain	I-structure_element
+of	O
+YfiB	B-protein
+and	O
+the	O
+YfiB	B-complex_assembly
+-	I-complex_assembly
+YfiR	I-complex_assembly
+complex	O
+are	O
+depicted	O
+according	O
+to	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+The	O
+lipid	O
+acceptor	O
+Cys26	B-residue_name_number
+is	O
+indicated	O
+as	O
+blue	O
+ball	O
+.	O
+
+These	O
+results	O
+,	O
+together	O
+with	O
+our	O
+observation	O
+that	O
+activated	B-protein_state
+YfiB	B-protein
+has	O
+a	O
+much	O
+higher	O
+cell	B-evidence
+wall	I-evidence
+binding	I-evidence
+affinity	I-evidence
+,	O
+and	O
+previous	O
+mutagenesis	O
+data	O
+showing	O
+that	O
+(	O
+1	O
+)	O
+both	O
+PG	B-chemical
+binding	O
+and	O
+membrane	O
+anchoring	O
+are	O
+required	O
+for	O
+YfiB	B-protein
+activity	O
+and	O
+(	O
+2	O
+)	O
+activating	O
+mutations	O
+possessing	O
+an	O
+altered	O
+N	O
+-	O
+terminal	O
+loop	B-structure_element
+length	O
+are	O
+dominant	O
+over	O
+the	O
+loss	O
+of	O
+PG	B-chemical
+binding	O
+(	O
+Malone	O
+et	O
+al	O
+.,),	O
+suggest	O
+an	O
+updated	O
+regulatory	O
+model	O
+of	O
+the	O
+YfiBNR	B-complex_assembly
+system	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+
+In	O
+this	O
+model	O
+,	O
+in	O
+response	O
+to	O
+a	O
+particular	O
+cell	O
+stress	O
+that	O
+is	O
+yet	O
+to	O
+be	O
+identified	O
+,	O
+the	O
+dimeric	B-oligomeric_state
+YfiB	B-protein
+is	O
+activated	B-protein_state
+from	O
+a	O
+compact	B-protein_state
+,	O
+inactive	B-protein_state
+conformation	B-protein_state
+to	O
+a	O
+stretched	B-protein_state
+conformation	I-protein_state
+,	O
+which	O
+possesses	O
+increased	O
+PG	B-chemical
+binding	O
+affinity	O
+.	O
+
+Ligands	O
+that	O
+regulate	O
+the	O
+dynamics	O
+and	O
+stability	O
+of	O
+the	O
+coactivator	B-site
+‐	I-site
+binding	I-site
+site	I-site
+in	O
+the	O
+C	O
+‐	O
+terminal	O
+ligand	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+,	O
+called	O
+activation	B-structure_element
+function	I-structure_element
+‐	I-structure_element
+2	I-structure_element
+(	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+),	O
+showed	O
+similar	O
+activity	O
+profiles	O
+in	O
+different	O
+cell	O
+types	O
+.	O
+
+For	O
+some	O
+ligand	O
+series	O
+,	O
+a	O
+single	O
+inter	B-evidence
+‐	I-evidence
+atomic	I-evidence
+distance	I-evidence
+in	O
+the	O
+ligand	B-structure_element
+‐	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+predicted	O
+their	O
+proliferative	O
+effects	O
+.	O
+
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+binds	O
+a	O
+separate	O
+surface	O
+on	O
+these	O
+coactivators	O
+(	O
+Webb	O
+et	O
+al	O
+,	O
+1998	O
+;	O
+Yi	O
+et	O
+al	O
+,	O
+2015	O
+).	O
+
+However	O
+,	O
+ERα	B-protein
+‐	O
+mediated	O
+proliferative	O
+responses	O
+vary	O
+in	O
+a	O
+ligand	O
+‐	O
+dependent	O
+manner	O
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+);	O
+thus	O
+,	O
+it	O
+is	O
+not	O
+known	O
+whether	O
+this	O
+canonical	O
+model	O
+is	O
+widely	O
+applicable	O
+across	O
+diverse	O
+ERα	B-protein
+ligands	O
+.	O
+
+Summary	O
+of	O
+ligand	B-experimental_method
+screening	I-experimental_method
+assays	I-experimental_method
+used	O
+to	O
+measure	O
+ER	O
+‐	O
+mediated	O
+activities	O
+.	O
+
+This	O
+wide	O
+variance	O
+enabled	O
+us	O
+to	O
+probe	O
+specific	O
+features	O
+of	O
+ERα	B-protein
+signaling	O
+using	O
+ligand	B-experimental_method
+class	I-experimental_method
+analyses	I-experimental_method
+,	O
+and	O
+identify	O
+signaling	O
+patterns	O
+shared	O
+by	O
+specific	O
+ligand	O
+series	O
+or	O
+scaffolds	O
+.	O
+
+The	O
+ERα	B-protein
+ligand	O
+library	O
+contains	O
+241	O
+ligands	O
+representing	O
+15	O
+indirect	O
+modulator	O
+scaffolds	O
+,	O
+plus	O
+4	O
+direct	O
+modulator	O
+scaffolds	O
+.	O
+
+Ligand	O
+‐	O
+specific	O
+signaling	O
+underlies	O
+ERα	B-protein
+‐	O
+mediated	O
+cell	O
+proliferation	O
+
+To	O
+test	O
+this	O
+idea	O
+,	O
+we	O
+compared	O
+the	O
+average	B-evidence
+L	I-evidence
+‐	I-evidence
+Luc	I-evidence
+activities	I-evidence
+of	O
+each	O
+scaffold	O
+in	O
+HepG2	O
+cells	O
+co	B-experimental_method
+‐	I-experimental_method
+transfected	I-experimental_method
+with	O
+wild	B-protein_state
+‐	I-protein_state
+type	I-protein_state
+ERα	B-protein
+or	O
+with	O
+ERα	B-protein
+lacking	B-protein_state
+the	I-protein_state
+AB	B-structure_element
+domain	O
+(	O
+Figs	O
+1B	O
+and	O
+EV1	O
+).	O
+
+Deletion	B-experimental_method
+of	I-experimental_method
+the	O
+AB	B-structure_element
+domain	O
+significantly	O
+reduced	O
+the	O
+average	B-evidence
+L	I-evidence
+‐	I-evidence
+Luc	I-evidence
+activities	I-evidence
+of	O
+14	O
+scaffolds	O
+(	O
+Student	B-experimental_method
+'	I-experimental_method
+s	I-experimental_method
+t	I-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+,	O
+P	B-evidence
+≤	O
+0	O
+.	O
+05	O
+)	O
+(	O
+Fig	O
+3B	O
+).	O
+
+For	O
+example	O
+,	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+,	O
+furan	B-chemical
+,	O
+and	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+2	I-chemical
+drove	O
+positively	O
+correlated	O
+GREB1	B-protein
+levels	O
+and	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+but	O
+not	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+activity	O
+(	O
+Fig	O
+3C	O
+lanes	O
+5	O
+–	O
+7	O
+).	O
+
+This	O
+is	O
+demonstrated	O
+by	O
+directly	O
+comparing	O
+the	O
+signaling	O
+specificities	O
+of	O
+matched	O
+OBHS	B-chemical
+(	O
+indirect	O
+modulator	O
+,	O
+cluster	O
+1	O
+)	O
+and	O
+OBHS	B-chemical
+‐	I-chemical
+BSC	I-chemical
+analogs	O
+(	O
+direct	O
+modulator	O
+,	O
+cluster	O
+3	O
+),	O
+which	O
+differ	O
+only	O
+in	O
+the	O
+basic	O
+side	O
+chain	O
+(	O
+Fig	O
+2E	O
+).	O
+
+Thus	O
+,	O
+examining	O
+the	O
+correlated	O
+patterns	O
+of	O
+ERα	B-protein
+activity	O
+within	O
+each	O
+scaffold	O
+demonstrates	O
+that	O
+an	O
+extended	O
+side	O
+chain	O
+is	O
+not	O
+required	O
+for	O
+cell	O
+‐	O
+specific	O
+signaling	O
+.	O
+
+Similarly	O
+,	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+F	B-structure_element
+domain	O
+did	O
+not	O
+abolish	O
+correlations	O
+between	O
+the	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+and	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+or	O
+GREB1	B-protein
+levels	O
+induced	O
+by	O
+OBHS	B-chemical
+analogs	O
+(	O
+Fig	O
+EV3F	O
+).	O
+
+Thus	O
+,	O
+ligands	O
+in	O
+cluster	O
+2	O
+rely	O
+on	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+both	O
+activity	O
+(	O
+Fig	O
+3B	O
+)	O
+and	O
+signaling	O
+specificity	O
+(	O
+Fig	O
+3D	O
+).	O
+
+Direct	O
+modulators	O
+showed	O
+low	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+(	O
+Fig	O
+EV2F	O
+–	O
+H	O
+),	O
+but	O
+only	O
+OBHS	B-chemical
+‐	I-chemical
+ASC	I-chemical
+analogs	O
+had	O
+NCOA2	B-protein
+recruitment	O
+profiles	O
+that	O
+predicted	O
+a	O
+full	O
+range	O
+of	O
+effects	O
+on	O
+GREB1	B-protein
+levels	O
+(	O
+Figs	O
+3E	O
+lanes	O
+9	O
+,	O
+11	O
+,	O
+18	O
+–	O
+19	O
+,	O
+and	O
+EV2A	O
+).	O
+
+Out	O
+of	O
+11	O
+indirect	O
+modulator	O
+series	O
+in	O
+cluster	O
+2	O
+or	O
+3	O
+,	O
+only	O
+the	O
+S	B-chemical
+‐	I-chemical
+OBHS	I-chemical
+‐	I-chemical
+3	I-chemical
+class	O
+had	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+profiles	O
+that	O
+predicted	O
+GREB1	B-protein
+levels	O
+(	O
+Fig	O
+3E	O
+lane	O
+12	O
+).	O
+
+The	O
+significant	O
+correlations	O
+with	O
+GREB1	B-protein
+expression	O
+and	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+observed	O
+in	O
+this	O
+cluster	O
+are	O
+consistent	O
+with	O
+the	O
+canonical	O
+signaling	O
+model	O
+(	O
+Fig	O
+1D	O
+),	O
+where	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+recruitment	O
+determines	O
+GREB1	B-protein
+expression	O
+,	O
+which	O
+then	O
+drives	O
+proliferation	O
+.	O
+
+Therefore	O
+,	O
+we	O
+first	O
+performed	O
+a	O
+time	B-experimental_method
+‐	I-experimental_method
+course	I-experimental_method
+study	I-experimental_method
+,	O
+and	O
+found	O
+that	O
+E2	B-chemical
+and	O
+the	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analog	O
+,	O
+AAPII	B-chemical
+‐	I-chemical
+151	I-chemical
+‐	I-chemical
+4	I-chemical
+,	O
+induced	O
+recruitment	O
+of	O
+NCOA3	B-protein
+to	O
+the	O
+GREB1	B-protein
+promoter	O
+in	O
+a	O
+temporal	O
+cycle	O
+that	O
+peaked	O
+after	O
+45	O
+min	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+(	O
+Fig	O
+4A	O
+).	O
+
+Kinetic	B-experimental_method
+ChIP	I-experimental_method
+assay	I-experimental_method
+examining	O
+recruitment	O
+of	O
+NCOA3	B-protein
+to	O
+the	O
+GREB1	B-protein
+gene	O
+in	O
+MCF	O
+‐	O
+7	O
+cells	O
+stimulated	O
+with	O
+E2	B-chemical
+or	O
+the	O
+indicated	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analog	O
+.	O
+
+The	O
+M2H	B-experimental_method
+assay	I-experimental_method
+for	O
+NCOA3	B-protein
+recruitment	O
+broadly	O
+correlated	O
+with	O
+the	O
+other	O
+assays	O
+,	O
+and	O
+was	O
+predictive	O
+for	O
+GREB1	B-protein
+expression	O
+and	O
+cell	O
+proliferation	O
+(	O
+Fig	O
+3E	O
+).	O
+
+However	O
+,	O
+the	O
+ChIP	B-experimental_method
+assays	I-experimental_method
+for	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+‐	O
+induced	O
+recruitment	O
+of	O
+NCOA3	B-protein
+to	O
+the	O
+GREB1	B-protein
+promoter	O
+did	O
+not	O
+correlate	O
+with	O
+any	O
+of	O
+the	O
+other	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+activity	O
+profiles	O
+(	O
+Fig	O
+4D	O
+),	O
+although	O
+the	O
+positive	O
+correlation	O
+between	O
+ChIP	B-experimental_method
+assays	I-experimental_method
+and	O
+NCOA3	B-protein
+recruitment	O
+via	O
+M2H	B-experimental_method
+assay	I-experimental_method
+showed	O
+a	O
+trend	O
+toward	O
+significance	O
+with	O
+r	B-evidence
+2	I-evidence
+=	O
+0	O
+.	O
+36	O
+and	O
+P	B-evidence
+=	O
+0	O
+.	O
+09	O
+(	O
+F	B-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+for	O
+nonzero	O
+slope	O
+).	O
+
+Nevertheless	O
+,	O
+the	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activities	O
+of	O
+both	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+cyclofenil	B-chemical
+analogs	O
+were	O
+better	O
+predicted	O
+by	O
+their	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERα	B-protein
+‐	O
+WT	B-protein_state
+than	O
+L	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+ERβ	B-protein
+activities	O
+(	O
+Fig	O
+EV4A	O
+and	O
+B	O
+).	O
+
+ERα	B-protein
+activity	O
+of	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+cyclofenil	B-chemical
+analogs	O
+correlates	O
+with	O
+E	B-experimental_method
+‐	I-experimental_method
+Luc	I-experimental_method
+activity	O
+.	O
+
+Based	O
+on	O
+our	O
+original	O
+OBHS	B-chemical
+structure	B-evidence
+,	O
+the	O
+OBHS	B-chemical
+,	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+,	O
+and	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+compounds	O
+were	O
+modified	O
+with	O
+h11	B-structure_element
+‐	O
+directed	O
+pendant	O
+groups	O
+(	O
+Zheng	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Zhu	O
+et	O
+al	O
+,	O
+2012	O
+;	O
+Liao	O
+et	O
+al	O
+,	O
+2014	O
+).	O
+
+For	O
+the	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+,	O
+the	O
+displacement	O
+of	O
+h11	B-structure_element
+was	O
+in	O
+a	O
+perpendicular	O
+direction	O
+,	O
+away	O
+from	O
+Ile424	B-residue_name_number
+in	O
+h8	B-structure_element
+and	O
+toward	O
+h12	B-structure_element
+.	O
+
+Structure	B-experimental_method
+‐	I-experimental_method
+class	I-experimental_method
+analysis	I-experimental_method
+of	O
+triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+.	O
+
+Triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+superposed	B-experimental_method
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+are	O
+shown	O
+.	O
+
+Triaryl	B-chemical
+‐	I-chemical
+ethylene	I-chemical
+analogs	O
+induce	O
+variance	O
+of	O
+ERα	B-protein
+conformations	O
+at	O
+the	O
+C	O
+‐	O
+terminal	O
+region	O
+of	O
+h11	B-structure_element
+.	O
+
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+side	O
+groups	O
+subtly	O
+nudge	O
+h12	B-structure_element
+Leu540	B-residue_name_number
+.	O
+
+Structure	B-experimental_method
+‐	I-experimental_method
+class	I-experimental_method
+analysis	I-experimental_method
+of	O
+indirect	O
+modulators	O
+in	O
+cluster	O
+1	O
+.	O
+
+Crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+ERα	B-protein
+LBD	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+OBHS	B-chemical
+and	O
+OBHS	B-chemical
+‐	I-chemical
+N	I-chemical
+analogs	O
+were	O
+superposed	B-experimental_method
+.	O
+
+As	O
+visualized	O
+in	O
+four	O
+LBD	B-structure_element
+structures	B-evidence
+(	O
+Srinivasan	O
+et	O
+al	O
+,	O
+2013	O
+),	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+analogs	O
+were	O
+designed	O
+with	O
+small	O
+substitutions	O
+that	O
+slightly	O
+nudge	O
+h12	B-structure_element
+Leu540	B-residue_name_number
+,	O
+without	O
+exiting	O
+the	O
+ligand	B-site
+‐	I-site
+binding	I-site
+pocket	I-site
+(	O
+Fig	O
+5G	O
+and	O
+H	O
+).	O
+
+This	O
+difference	O
+in	O
+ligand	O
+positioning	O
+altered	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+via	O
+a	O
+shift	O
+in	O
+the	O
+N	O
+‐	O
+terminus	O
+of	O
+h12	B-structure_element
+,	O
+which	O
+directly	O
+contacts	O
+the	O
+coactivator	O
+.	O
+
+Crystal	B-evidence
+structures	I-evidence
+show	O
+that	O
+a	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+analog	O
+shifts	O
+h3	B-structure_element
+(	O
+F	B-structure_element
+)	O
+and	O
+the	O
+NCOA2	B-protein
+(	O
+G	O
+)	O
+peptide	O
+compared	O
+to	O
+an	O
+A	B-chemical
+‐	I-chemical
+CD	I-chemical
+‐	O
+ring	O
+estrogen	B-chemical
+(	O
+PDB	O
+4PPS	O
+,	O
+5DTV	O
+).	O
+
+The	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+showed	O
+perturbation	O
+of	O
+h11	B-structure_element
+,	O
+as	O
+well	O
+as	O
+h3	B-structure_element
+,	O
+which	O
+forms	O
+part	O
+of	O
+the	O
+AF	B-site
+‐	I-site
+2	I-site
+surface	I-site
+.	O
+
+We	O
+observed	O
+a	O
+difference	O
+of	O
+0	O
+.	O
+4	O
+Å	O
+that	O
+was	O
+significant	O
+(	O
+two	O
+‐	O
+tailed	O
+Student	B-experimental_method
+'	I-experimental_method
+s	I-experimental_method
+t	I-experimental_method
+‐	I-experimental_method
+test	I-experimental_method
+,	O
+P	B-evidence
+=	O
+0	O
+.	O
+002	O
+)	O
+due	O
+to	O
+the	O
+very	O
+tight	O
+clustering	O
+of	O
+the	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+‐	O
+induced	O
+LBD	B-structure_element
+conformation	O
+.	O
+
+The	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTPD	I-chemical
+analogs	O
+also	O
+induced	O
+a	O
+shift	O
+in	O
+h3	B-structure_element
+positioning	O
+,	O
+which	O
+translated	O
+again	O
+into	O
+a	O
+shift	O
+in	O
+the	O
+bound	O
+coactivator	O
+peptide	O
+(	O
+Fig	O
+6F	O
+).	O
+
+Despite	O
+the	O
+similar	O
+average	O
+activities	O
+of	O
+these	O
+ligand	O
+classes	O
+(	O
+Fig	O
+3A	O
+and	O
+B	O
+),	O
+2	B-chemical
+,	I-chemical
+5	I-chemical
+‐	I-chemical
+DTP	I-chemical
+and	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+‐	I-chemical
+DTP	I-chemical
+analogs	O
+displayed	O
+remarkably	O
+different	O
+peptide	O
+recruitment	O
+patterns	O
+(	O
+Fig	O
+6H	O
+),	O
+consistent	O
+with	O
+the	O
+structural	B-experimental_method
+analyses	I-experimental_method
+.	O
+
+This	O
+finding	O
+can	O
+be	O
+explained	O
+by	O
+the	O
+fact	O
+that	O
+NCOA1	B-protein
+/	I-protein
+2	I-protein
+/	I-protein
+3	I-protein
+contain	O
+distinct	O
+binding	B-site
+sites	I-site
+for	O
+interaction	O
+with	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+and	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+(	O
+McInerney	O
+et	O
+al	O
+,	O
+1996	O
+;	O
+Webb	O
+et	O
+al	O
+,	O
+1998	O
+),	O
+which	O
+allows	O
+ligands	O
+to	O
+nucleate	O
+ERα	B-complex_assembly
+–	I-complex_assembly
+NCOA1	I-complex_assembly
+/	I-complex_assembly
+2	I-complex_assembly
+/	I-complex_assembly
+3	I-complex_assembly
+interaction	O
+through	O
+AF	B-structure_element
+‐	I-structure_element
+2	I-structure_element
+,	O
+and	O
+reinforce	O
+this	O
+interaction	O
+with	O
+additional	O
+binding	O
+to	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+.	O
+
+Also	O
+,	O
+we	O
+have	O
+used	O
+siRNA	B-experimental_method
+screening	I-experimental_method
+to	O
+identify	O
+a	O
+number	O
+of	O
+coregulators	O
+required	O
+for	O
+ERα	B-protein
+‐	O
+mediated	O
+repression	O
+of	O
+the	O
+IL	O
+‐	O
+6	O
+gene	O
+(	O
+Nwachukwu	O
+et	O
+al	O
+,	O
+2014	O
+).	O
+
+Secondly	O
+,	O
+our	O
+finding	O
+that	O
+WAY	B-chemical
+‐	I-chemical
+C	I-chemical
+compounds	O
+do	O
+not	O
+rely	O
+of	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+signaling	O
+efficacy	O
+may	O
+derive	O
+from	O
+the	O
+slight	O
+contacts	O
+with	O
+h12	B-structure_element
+observed	O
+in	O
+crystal	B-evidence
+structures	I-evidence
+(	O
+Figs	O
+3B	O
+and	O
+5H	O
+),	O
+unlike	O
+other	O
+compounds	O
+in	O
+cluster	O
+1	O
+that	O
+dislocate	O
+h11	B-structure_element
+and	O
+rely	O
+on	O
+AF	B-structure_element
+‐	I-structure_element
+1	I-structure_element
+for	O
+signaling	O
+efficacy	O
+(	O
+Figs	O
+3B	O
+and	O
+5C	O
+,	O
+and	O
+EV5B	O
+).	O
+
+We	O
+have	O
+also	O
+investigated	O
+the	O
+binding	O
+of	O
+the	O
+TOCA	B-protein
+HR1	B-structure_element
+domain	O
+to	O
+Cdc42	B-protein
+and	O
+the	O
+potential	O
+ternary	O
+complex	O
+between	O
+Cdc42	B-protein
+and	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+regions	I-site
+of	O
+TOCA1	B-protein
+and	O
+a	O
+member	O
+of	O
+the	O
+Wiskott	B-protein_type
+-	I-protein_type
+Aldrich	I-protein_type
+syndrome	I-protein_type
+protein	I-protein_type
+family	I-protein_type
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+The	O
+guanine	B-protein_type
+nucleotide	I-protein_type
+exchange	I-protein_type
+factors	I-protein_type
+mediate	O
+formation	O
+of	O
+the	O
+active	B-protein_state
+state	O
+by	O
+promoting	O
+the	O
+dissociation	O
+of	O
+GDP	B-chemical
+,	O
+allowing	O
+GTP	B-chemical
+to	O
+bind	O
+.	O
+
+The	O
+Rho	B-protein_type
+family	I-protein_type
+comprises	O
+20	O
+members	O
+,	O
+of	O
+which	O
+three	O
+,	O
+RhoA	B-protein
+,	O
+Rac1	B-protein
+,	O
+and	O
+Cdc42	B-protein
+,	O
+have	O
+been	O
+relatively	O
+well	O
+studied	O
+.	O
+
+Following	O
+their	O
+release	O
+,	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+regions	I-structure_element
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+are	O
+free	O
+to	O
+interact	O
+with	O
+G	B-protein_type
+-	I-protein_type
+actin	I-protein_type
+and	O
+a	O
+known	O
+nucleator	O
+of	O
+actin	O
+assembly	O
+,	O
+the	O
+Arp2	B-complex_assembly
+/	I-complex_assembly
+3	I-complex_assembly
+complex	O
+.	O
+
+The	O
+structures	B-evidence
+of	O
+the	O
+PRK1	B-protein
+HR1a	B-structure_element
+domain	O
+in	O
+complex	B-protein_state
+with	I-protein_state
+RhoA	B-protein
+and	O
+the	O
+HR1b	B-structure_element
+domain	O
+in	O
+complex	B-protein_state
+with	I-protein_state
+Rac1	B-protein
+show	O
+that	O
+the	O
+HR1	B-structure_element
+domain	O
+comprises	O
+an	O
+anti	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+coiled	I-structure_element
+-	I-structure_element
+coil	I-structure_element
+that	O
+interacts	O
+with	O
+its	O
+G	B-protein_type
+protein	I-protein_type
+binding	O
+partner	O
+via	O
+both	O
+helices	B-structure_element
+.	O
+
+Both	O
+of	O
+the	O
+G	B-site
+protein	I-site
+switch	I-site
+regions	I-site
+are	O
+involved	O
+in	O
+the	O
+interaction	O
+.	O
+
+The	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+fold	I-structure_element
+is	O
+shared	O
+by	O
+the	O
+HR1	B-structure_element
+domain	O
+of	O
+the	O
+TOCA	B-protein_type
+family	I-protein_type
+protein	I-protein_type
+,	O
+CIP4	B-protein
+,	O
+and	O
+,	O
+based	O
+on	O
+sequence	O
+homology	O
+,	O
+by	O
+TOCA1	B-protein
+itself	O
+.	O
+
+How	O
+different	O
+HR1	B-structure_element
+domain	O
+proteins	O
+distinguish	O
+their	O
+specific	O
+G	B-protein_type
+protein	I-protein_type
+partners	O
+remains	O
+only	O
+partially	O
+understood	O
+,	O
+and	O
+structural	O
+characterization	O
+of	O
+a	O
+novel	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+HR1	B-structure_element
+domain	O
+interaction	O
+would	O
+add	O
+to	O
+the	O
+growing	O
+body	O
+of	O
+information	O
+pertaining	O
+to	O
+these	O
+protein	O
+complexes	O
+.	O
+
+The	O
+interactions	O
+of	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+with	O
+Cdc42	B-protein
+as	O
+well	O
+as	O
+with	O
+each	O
+other	O
+have	O
+raised	O
+questions	O
+as	O
+to	O
+whether	O
+the	O
+two	O
+Cdc42	B-protein
+effectors	O
+can	O
+interact	O
+with	O
+a	O
+single	O
+molecule	O
+of	O
+Cdc42	B-protein
+simultaneously	O
+.	O
+
+We	O
+also	O
+present	O
+data	O
+pertaining	O
+to	O
+binding	O
+of	O
+the	O
+TOCA	B-protein_type
+HR1	B-structure_element
+domain	O
+to	O
+Cdc42	B-protein
+,	O
+which	O
+is	O
+the	O
+first	O
+biophysical	O
+description	O
+of	O
+an	O
+HR1	B-structure_element
+domain	O
+binding	O
+this	O
+particular	O
+Rho	B-protein_type
+family	I-protein_type
+small	I-protein_type
+G	I-protein_type
+protein	I-protein_type
+.	O
+
+Finally	O
+,	O
+we	O
+investigate	O
+the	O
+potential	O
+ternary	O
+complex	O
+between	O
+Cdc42	B-protein
+and	O
+the	O
+G	B-site
+protein	I-site
+-	I-site
+binding	I-site
+regions	I-site
+of	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+contributing	O
+to	O
+our	O
+understanding	O
+of	O
+G	B-protein_type
+protein	I-protein_type
+-	O
+effector	O
+interactions	O
+as	O
+well	O
+as	O
+the	O
+roles	O
+of	O
+Cdc42	B-protein
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+,	O
+and	O
+TOCA1	B-protein
+in	O
+the	O
+pathways	O
+that	O
+govern	O
+actin	O
+dynamics	O
+.	O
+
+TOCA1	B-protein
+was	O
+identified	O
+in	O
+Xenopus	B-taxonomy_domain
+extracts	O
+as	O
+a	O
+protein	O
+necessary	O
+for	O
+Cdc42	B-protein
+-	O
+dependent	O
+actin	O
+assembly	O
+and	O
+was	O
+shown	O
+to	O
+bind	O
+to	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+GTPγS	I-complex_assembly
+but	O
+not	O
+to	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+GDP	I-complex_assembly
+or	O
+to	O
+Rac1	B-protein
+and	O
+RhoA	B-protein
+.	O
+Given	O
+its	O
+homology	O
+to	O
+other	O
+Rho	B-site
+family	I-site
+binding	I-site
+modules	I-site
+,	O
+it	O
+is	O
+likely	O
+that	O
+the	O
+HR1	B-structure_element
+domain	O
+of	O
+TOCA1	B-protein
+is	O
+sufficient	O
+to	O
+bind	O
+Cdc42	B-protein
+.	O
+
+The	O
+binding	O
+of	O
+TOCA1	B-protein
+HR1	B-structure_element
+to	O
+Cdc42	B-protein
+was	O
+unexpectedly	O
+weak	O
+,	O
+with	O
+a	O
+Kd	B-evidence
+of	O
+>	O
+1	O
+μm	O
+.	O
+
+A	O
+,	O
+curves	O
+derived	O
+from	O
+direct	B-experimental_method
+binding	I-experimental_method
+assays	I-experimental_method
+in	O
+which	O
+the	O
+indicated	O
+concentrations	O
+of	O
+Cdc42Δ7Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+were	O
+incubated	B-experimental_method
+with	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+PAK	I-mutant
+or	O
+HR1	B-mutant
+-	I-mutant
+His6	I-mutant
+in	O
+SPAs	B-experimental_method
+.	O
+
+The	O
+data	O
+were	O
+fitted	O
+to	O
+a	O
+binding	B-evidence
+isotherm	I-evidence
+to	O
+give	O
+an	O
+apparent	O
+Kd	B-evidence
+and	O
+are	O
+expressed	O
+as	O
+a	O
+percentage	O
+of	O
+the	O
+maximum	O
+signal	O
+;	O
+B	O
+and	O
+C	O
+,	O
+competition	B-experimental_method
+SPA	I-experimental_method
+experiments	O
+were	O
+carried	O
+out	O
+with	O
+the	O
+indicated	O
+concentrations	O
+of	O
+ACK	B-protein
+GBD	B-structure_element
+(	O
+B	O
+)	O
+or	O
+HR1	B-structure_element
+domain	O
+(	O
+C	O
+)	O
+titrated	B-experimental_method
+into	O
+30	O
+nm	O
+GST	B-mutant
+-	I-mutant
+ACK	I-mutant
+and	O
+either	O
+30	O
+nm	O
+Cdc42Δ7Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+or	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42Q61L	B-complex_assembly
+·[	I-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+.	O
+
+Isothermal	B-experimental_method
+titration	I-experimental_method
+calorimetry	I-experimental_method
+was	O
+carried	O
+out	O
+,	O
+but	O
+no	O
+heat	O
+changes	O
+were	O
+observed	O
+at	O
+a	O
+range	O
+of	O
+concentrations	O
+and	O
+temperatures	O
+(	O
+data	O
+not	O
+shown	O
+),	O
+suggesting	O
+that	O
+the	O
+interaction	O
+is	O
+predominantly	O
+entropically	O
+driven	O
+.	O
+
+The	O
+binding	B-experimental_method
+experiments	I-experimental_method
+were	O
+repeated	O
+with	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+[	B-complex_assembly
+3H	I-complex_assembly
+]	I-complex_assembly
+GTP	I-complex_assembly
+·	I-complex_assembly
+Cdc42	I-complex_assembly
+,	O
+but	O
+the	O
+affinity	B-evidence
+of	O
+the	O
+HR1	B-structure_element
+domain	O
+for	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Cdc42	B-protein
+was	O
+similar	O
+to	O
+its	O
+affinity	B-evidence
+for	O
+truncated	B-protein_state
+Cdc42	B-protein
+(	O
+Kd	B-evidence
+≈	O
+5	O
+μm	O
+;	O
+Fig	O
+.	O
+1C	O
+).	O
+
+Full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+TOCA1	B-protein
+and	O
+ΔSH3	B-mutant
+TOCA1	B-protein
+bound	B-protein_state
+with	O
+micromolar	O
+affinity	O
+(	O
+Fig	O
+.	O
+2B	O
+),	O
+in	O
+a	O
+similar	O
+manner	O
+to	O
+the	O
+isolated	O
+HR1	B-structure_element
+domain	O
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+
+There	O
+were	O
+1	O
+,	O
+845	O
+unambiguous	O
+NOEs	B-evidence
+and	O
+757	O
+ambiguous	O
+NOEs	B-evidence
+after	O
+eight	O
+iterations	O
+.	O
+
+a	O
+<	O
+SA	O
+>,	O
+the	O
+average	B-evidence
+root	I-evidence
+mean	I-evidence
+square	I-evidence
+deviations	I-evidence
+for	O
+the	O
+ensemble	O
+±	O
+S	O
+.	O
+D	O
+.	O
+
+B	O
+,	O
+a	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+HR1	B-structure_element
+domains	O
+from	O
+TOCA1	B-protein
+,	O
+CIP4	B-protein
+,	O
+and	O
+PRK1	B-protein
+.	O
+
+A	O
+series	O
+of	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+experiments	O
+was	O
+recorded	O
+on	O
+15N	B-chemical
+-	O
+labeled	B-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+increasing	B-experimental_method
+concentrations	I-experimental_method
+of	O
+unlabeled	B-protein_state
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+to	O
+map	O
+the	O
+Cdc42	B-site
+-	I-site
+binding	I-site
+surface	I-site
+.	O
+
+Residues	O
+with	O
+significantly	O
+affected	O
+backbone	O
+or	O
+side	O
+chain	O
+chemical	O
+shifts	O
+when	O
+Cdc42	B-protein_state
+bound	I-protein_state
+and	O
+that	O
+are	O
+buried	O
+are	O
+colored	O
+dark	O
+blue	O
+,	O
+whereas	O
+those	O
+that	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+accessible	I-protein_state
+are	O
+colored	O
+yellow	O
+.	O
+
+Therefore	O
+,	O
+13C	B-experimental_method
+HSQC	I-experimental_method
+and	O
+methyl	B-experimental_method
+-	I-experimental_method
+selective	I-experimental_method
+SOFAST	I-experimental_method
+-	I-experimental_method
+HMQC	I-experimental_method
+experiments	O
+were	O
+also	O
+recorded	O
+on	O
+15N	B-chemical
+,	O
+13C	B-chemical
+-	O
+labeled	B-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+to	O
+yield	O
+more	O
+information	O
+on	O
+side	O
+chain	O
+involvement	O
+.	O
+
+As	O
+was	O
+the	O
+case	O
+when	O
+labeled	B-protein_state
+HR1	B-structure_element
+was	O
+observed	O
+,	O
+several	O
+peaks	O
+were	O
+shifted	O
+in	O
+the	O
+complex	O
+,	O
+but	O
+many	O
+disappeared	O
+,	O
+indicating	O
+exchange	O
+on	O
+an	O
+unfavorable	O
+,	O
+millisecond	O
+time	O
+scale	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+B	O
+,	O
+CSPs	B-experimental_method
+are	O
+shown	O
+for	O
+backbone	O
+NH	O
+groups	O
+.	O
+
+C	O
+,	O
+the	O
+residues	O
+with	O
+significantly	O
+affected	O
+backbone	O
+and	O
+side	O
+chain	O
+groups	O
+are	O
+highlighted	O
+on	O
+an	O
+NMR	B-experimental_method
+structure	B-evidence
+of	O
+free	B-protein_state
+Cdc42Δ7Q61L	B-complex_assembly
+·	I-complex_assembly
+GMPPNP	I-complex_assembly
+;	O
+those	O
+that	O
+are	O
+buried	O
+are	O
+colored	O
+dark	O
+blue	O
+,	O
+whereas	O
+those	O
+that	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+accessible	I-protein_state
+are	O
+colored	O
+red	O
+.	O
+
+Although	O
+the	O
+binding	B-site
+interface	I-site
+may	O
+be	O
+overestimated	O
+,	O
+this	O
+suggests	O
+that	O
+the	O
+switch	B-site
+regions	I-site
+are	O
+involved	O
+in	O
+binding	O
+to	O
+TOCA1	B-protein
+.	O
+
+HADDOCK	B-experimental_method
+was	O
+therefore	O
+used	O
+to	O
+perform	O
+rigid	O
+body	B-experimental_method
+docking	I-experimental_method
+based	O
+on	O
+the	O
+structures	B-evidence
+of	O
+free	B-protein_state
+HR1	B-structure_element
+domain	O
+and	O
+Cdc42	B-protein
+and	O
+ambiguous	O
+interaction	O
+restraints	O
+derived	O
+from	O
+the	O
+titration	B-experimental_method
+experiments	I-experimental_method
+described	O
+above	O
+.	O
+
+The	O
+cluster	O
+with	O
+the	O
+lowest	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+from	O
+the	O
+lowest	O
+energy	O
+structure	B-evidence
+is	O
+assumed	O
+to	O
+be	O
+the	O
+best	O
+model	O
+.	O
+
+Residues	O
+of	O
+Cdc42	B-protein
+that	O
+are	O
+affected	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+HR1	B-structure_element
+domain	O
+but	O
+are	O
+not	O
+in	O
+close	O
+proximity	O
+to	O
+it	O
+are	O
+colored	O
+in	O
+red	O
+and	O
+labeled	O
+.	O
+
+B	O
+,	O
+structure	B-evidence
+of	O
+Rac1	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+HR1b	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+(	O
+PDB	O
+code	O
+2RMK	O
+).	O
+
+Cdc42	O
+is	O
+shown	O
+in	O
+cyan	O
+,	O
+and	O
+TOCA1	B-protein
+is	O
+shown	O
+in	O
+purple	O
+.	O
+
+D	O
+,	O
+selected	O
+regions	O
+of	O
+the	O
+15N	B-experimental_method
+HSQC	I-experimental_method
+of	O
+600	O
+μm	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Cdc42	B-protein
+in	O
+the	O
+absence	B-protein_state
+and	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+,	O
+showing	O
+displacement	O
+of	O
+Cdc42	B-protein
+from	O
+the	O
+HR1	B-structure_element
+domain	O
+by	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+.	O
+
+The	O
+Kd	B-evidence
+that	O
+was	O
+determined	O
+(	O
+37	O
+nm	O
+)	O
+is	O
+consistent	O
+with	O
+the	O
+previously	O
+reported	O
+affinity	B-evidence
+.	O
+
+A	O
+comparison	O
+of	O
+the	O
+HSQC	B-experimental_method
+experiments	O
+recorded	O
+on	O
+15N	B-chemical
+-	O
+Cdc42	B-protein
+alone	B-protein_state
+,	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+TOCA1	B-protein
+HR1	B-structure_element
+,	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+,	O
+or	O
+both	O
+,	O
+shows	O
+that	O
+the	O
+spectra	B-evidence
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+both	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+and	O
+TOCA1	B-protein
+HR1	B-structure_element
+are	O
+identical	O
+(	O
+Fig	O
+.	O
+7C	O
+).	O
+
+These	O
+assays	O
+,	O
+described	O
+in	O
+detail	O
+elsewhere	O
+,	O
+were	O
+carried	O
+out	O
+using	O
+pyrene	B-chemical
+actin	I-chemical
+-	O
+supplemented	O
+Xenopus	B-taxonomy_domain
+extracts	O
+into	O
+which	O
+exogenous	O
+TOCA1	B-protein
+HR1	B-structure_element
+domain	O
+or	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+GBD	B-structure_element
+was	O
+added	O
+,	O
+to	O
+assess	O
+their	O
+effects	O
+on	O
+actin	B-protein_type
+polymerization	O
+.	O
+
+The	O
+corresponding	O
+sequence	O
+in	O
+CIP4	B-protein
+also	O
+includes	O
+a	O
+series	O
+of	O
+turns	O
+but	O
+is	O
+flexible	O
+,	O
+whereas	O
+in	O
+the	O
+HR1a	B-structure_element
+domain	O
+of	O
+PRK1	B-protein
+,	O
+the	O
+equivalent	O
+region	O
+adopts	O
+an	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+structure	I-structure_element
+that	O
+packs	O
+against	O
+the	O
+coiled	B-structure_element
+-	I-structure_element
+coil	I-structure_element
+.	O
+
+The	O
+lowest	O
+energy	O
+model	B-evidence
+produced	O
+by	O
+HADDOCK	B-experimental_method
+using	O
+ambiguous	O
+interaction	O
+restraints	O
+from	O
+the	O
+titration	B-evidence
+data	O
+resembled	O
+the	O
+NMR	B-experimental_method
+structures	B-evidence
+of	O
+RhoA	B-protein
+and	O
+Rac1	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+their	O
+HR1	B-structure_element
+domain	O
+partners	O
+.	O
+
+For	O
+example	O
+,	O
+Phe	B-residue_name_number
+-	I-residue_name_number
+56Cdc42	I-residue_name_number
+,	O
+which	O
+is	O
+not	O
+visible	O
+in	O
+free	B-protein_state
+Cdc42	B-protein
+or	O
+Cdc42	B-complex_assembly
+·	I-complex_assembly
+HR1TOCA1	I-complex_assembly
+,	O
+is	O
+close	O
+to	O
+the	O
+TOCA1	B-protein
+HR1	B-structure_element
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+
+Phe	B-residue_name_number
+-	I-residue_name_number
+56Cdc42	I-residue_name_number
+is	O
+therefore	O
+likely	O
+to	O
+be	O
+involved	O
+in	O
+the	O
+Cdc42	B-protein
+-	O
+TOCA1	B-protein
+interaction	O
+,	O
+probably	O
+by	O
+stabilizing	O
+the	O
+position	O
+of	O
+switch	B-site
+I	I-site
+.	O
+
+Thr	B-residue_name_number
+-	I-residue_name_number
+52Cdc42	I-residue_name_number
+,	O
+which	O
+has	O
+also	O
+been	O
+identified	O
+as	O
+making	O
+minor	O
+contacts	O
+with	O
+ACK	B-protein
+,	O
+falls	O
+near	O
+the	O
+side	O
+chains	O
+of	O
+HR1TOCA1	B-structure_element
+helix	B-structure_element
+1	I-structure_element
+,	O
+particularly	O
+Lys	B-residue_name_number
+-	I-residue_name_number
+372TOCA1	I-residue_name_number
+,	O
+whereas	O
+the	O
+equivalent	O
+position	O
+in	O
+Rac1	B-protein
+is	O
+Asn	B-residue_name_number
+-	I-residue_name_number
+52Rac1	I-residue_name_number
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+best	O
+estimate	O
+of	O
+the	O
+affinity	B-evidence
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+WASP	B-protein_type
+for	O
+Cdc42	B-protein
+is	O
+low	O
+micromolar	O
+.	O
+
+WIP	B-protein
+inhibits	O
+the	O
+activation	O
+of	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+by	O
+Cdc42	B-protein
+,	O
+an	O
+effect	O
+that	O
+is	O
+reversed	O
+by	O
+TOCA1	B-protein
+.	O
+
+It	O
+has	O
+been	O
+postulated	O
+that	O
+the	O
+initial	O
+interactions	O
+between	O
+this	O
+basic	O
+region	O
+and	O
+Cdc42	B-protein
+could	O
+stabilize	O
+the	O
+active	B-protein_state
+conformation	O
+of	O
+WASP	B-protein
+,	O
+leading	O
+to	O
+high	O
+affinity	O
+binding	O
+between	O
+the	O
+core	O
+CRIB	B-structure_element
+and	O
+Cdc42	B-protein
+.	O
+
+We	O
+envisage	O
+a	O
+complex	O
+interplay	O
+of	O
+equilibria	O
+between	O
+free	B-protein_state
+and	O
+bound	B-protein_state
+,	O
+active	B-protein_state
+and	O
+inactive	B-protein_state
+Cdc42	B-protein
+,	O
+TOCA	B-protein_type
+family	I-protein_type
+,	O
+and	O
+WASP	B-protein_type
+family	O
+proteins	O
+,	O
+facilitating	O
+a	O
+tightly	O
+spatially	O
+and	O
+temporally	O
+regulated	O
+pathway	O
+requiring	O
+numerous	O
+simultaneous	O
+events	O
+in	O
+order	O
+to	O
+achieve	O
+appropriate	O
+and	O
+robust	O
+activation	O
+of	O
+the	O
+downstream	O
+pathway	O
+.	O
+
+It	O
+is	O
+clear	O
+from	O
+the	O
+data	O
+presented	O
+here	O
+that	O
+TOCA1	B-protein
+and	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+do	O
+not	O
+bind	O
+Cdc42	B-protein
+simultaneously	O
+and	O
+that	O
+N	B-protein
+-	I-protein
+WASP	I-protein
+is	O
+likely	O
+to	O
+outcompete	O
+TOCA1	B-protein
+for	O
+Cdc42	B-protein
+binding	O
+.	O
+
+Furthermore	O
+,	O
+elevated	O
+ACC	B-protein_type
+activity	O
+is	O
+observed	O
+in	O
+malignant	O
+tumours	O
+.	O
+
+The	O
+principal	O
+functional	O
+protein	O
+components	O
+of	O
+ACCs	B-protein_type
+have	O
+been	O
+described	O
+already	O
+in	O
+the	O
+late	O
+1960s	O
+for	O
+Escherichia	B-species
+coli	I-species
+(	O
+E	B-species
+.	I-species
+coli	I-species
+)	O
+ACC	B-protein_type
+:	O
+Biotin	B-protein_type
+carboxylase	I-protein_type
+(	O
+BC	B-protein_type
+)	O
+catalyses	O
+the	O
+ATP	B-chemical
+-	O
+dependent	O
+carboxylation	O
+of	O
+a	O
+biotin	B-chemical
+moiety	O
+,	O
+which	O
+is	O
+covalently	O
+linked	O
+to	O
+the	O
+biotin	B-protein_type
+carboxyl	I-protein_type
+carrier	I-protein_type
+protein	I-protein_type
+(	O
+BCCP	B-protein_type
+).	O
+
+Human	B-species
+ACC1	B-protein
+is	O
+further	O
+regulated	O
+by	O
+specific	O
+phosphorylation	B-ptm
+-	O
+dependent	O
+binding	O
+of	O
+BRCA1	B-protein
+to	O
+Ser1263	B-residue_name_number
+in	O
+the	O
+CD	B-structure_element
+.	O
+
+Furthermore	O
+,	O
+phosphorylation	B-ptm
+by	O
+AMP	B-protein
+-	I-protein
+activated	I-protein
+protein	I-protein
+kinase	I-protein
+(	O
+AMPK	B-protein
+)	O
+and	O
+cAMP	B-protein
+-	I-protein
+dependent	I-protein
+protein	I-protein
+kinase	I-protein
+(	O
+PKA	B-protein
+)	O
+leads	O
+to	O
+a	O
+decrease	O
+in	O
+ACC1	B-protein
+activity	O
+.	O
+
+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
+
+SceCD	B-species
+comprises	O
+four	O
+distinct	O
+domains	O
+,	O
+an	O
+N	O
+-	O
+terminal	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+domain	I-structure_element
+(	O
+CDN	B-structure_element
+),	O
+and	O
+a	O
+central	O
+four	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+linker	I-structure_element
+domain	I-structure_element
+(	O
+CDL	B-structure_element
+),	O
+followed	O
+by	O
+two	O
+α	B-structure_element
+–	I-structure_element
+β	I-structure_element
+-	I-structure_element
+fold	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domains	I-structure_element
+(	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+).	O
+
+In	O
+insect	B-experimental_method
+-	I-experimental_method
+cell	I-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+SceACC	B-protein
+,	O
+the	O
+highly	B-protein_state
+conserved	I-protein_state
+Ser1157	B-residue_name_number
+is	O
+the	O
+only	O
+fully	B-protein_state
+occupied	I-protein_state
+phosphorylation	B-site
+site	I-site
+with	O
+functional	O
+relevance	O
+in	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+.	O
+
+Additional	O
+phosphorylation	B-ptm
+was	O
+detected	O
+for	O
+Ser2101	B-residue_name_number
+and	O
+Tyr2179	B-residue_name_number
+;	O
+however	O
+,	O
+these	O
+sites	O
+are	O
+neither	B-protein_state
+conserved	I-protein_state
+across	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+nor	B-protein_state
+natively	I-protein_state
+phosphorylated	I-protein_state
+in	O
+yeast	B-taxonomy_domain
+.	O
+
+Already	O
+the	O
+binding	O
+of	O
+phosphorylated	B-protein_state
+Ser1157	B-residue_name_number
+apparently	O
+stabilizes	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+conformation	O
+;	O
+the	O
+accessory	O
+phosphorylation	B-site
+sites	I-site
+Ser1148	B-residue_name_number
+and	O
+Ser1162	B-residue_name_number
+in	O
+the	O
+same	B-structure_element
+loop	I-structure_element
+may	O
+further	O
+modulate	O
+the	O
+strength	O
+of	O
+interaction	O
+between	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+and	O
+the	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+domains	O
+.	O
+
+The	O
+variable	O
+CD	B-structure_element
+is	O
+conserved	B-protein_state
+between	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+
+To	O
+compare	O
+the	O
+organization	O
+of	O
+fungal	B-taxonomy_domain
+and	O
+human	B-species
+ACC	B-protein_type
+CD	B-structure_element
+,	O
+we	O
+determined	B-experimental_method
+the	I-experimental_method
+structure	I-experimental_method
+of	O
+a	O
+human	B-species
+ACC1	B-mutant
+fragment	I-mutant
+that	O
+comprises	O
+the	O
+BT	B-structure_element
+and	O
+CD	B-structure_element
+domains	O
+(	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+),	O
+but	O
+lacks	B-protein_state
+the	O
+mobile	O
+BCCP	B-structure_element
+in	O
+between	O
+(	O
+Fig	O
+.	O
+1a	O
+).	O
+
+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
+
+The	O
+neighbouring	O
+loop	B-structure_element
+on	O
+the	O
+CT	B-structure_element
+side	O
+(	O
+between	O
+CT	B-structure_element
+β1	B-structure_element
+/	O
+β2	B-structure_element
+)	O
+is	O
+displaced	O
+by	O
+2	O
+.	O
+5	O
+Å	O
+compared	O
+to	O
+isolated	B-protein_state
+CT	B-structure_element
+structures	B-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+3c	O
+).	O
+
+The	O
+interface	B-site
+between	O
+CDC2	B-structure_element
+and	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+,	O
+which	O
+is	O
+mediated	O
+by	O
+the	O
+phosphorylated	B-protein_state
+regulatory	B-structure_element
+loop	I-structure_element
+in	O
+the	O
+SceCD	B-species
+structure	B-evidence
+,	O
+is	O
+less	O
+variable	O
+than	O
+the	O
+CD	B-structure_element
+–	I-structure_element
+CT	I-structure_element
+junction	I-structure_element
+,	O
+and	O
+permits	O
+only	O
+limited	O
+rotation	O
+and	O
+tilting	O
+(	O
+Fig	O
+.	O
+3b	O
+).	O
+
+The	O
+BC	B-structure_element
+domain	O
+is	O
+not	O
+completely	O
+disordered	O
+,	O
+but	O
+laterally	O
+attached	O
+to	O
+BT	B-structure_element
+/	O
+CDN	B-structure_element
+in	O
+a	O
+generally	B-protein_state
+conserved	I-protein_state
+position	I-protein_state
+,	O
+albeit	O
+with	O
+increased	O
+flexibility	O
+.	O
+
+The	O
+most	O
+relevant	O
+candidate	O
+site	O
+for	O
+mediating	O
+such	O
+additional	O
+flexibility	O
+and	O
+permitting	O
+an	O
+extended	O
+set	O
+of	O
+conformations	O
+is	O
+the	O
+CDC1	B-site
+/	I-site
+CDC2	I-site
+interface	I-site
+,	O
+which	O
+is	O
+rigidified	O
+by	O
+the	O
+Ser1157	B-residue_name_number
+-	O
+phosphorylated	B-protein_state
+regulatory	B-structure_element
+loop	I-structure_element
+,	O
+as	O
+depicted	O
+in	O
+the	O
+SceCD	B-species
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+In	O
+flACC	B-mutant
+,	O
+the	O
+ACC	B-protein_type
+dimer	B-oligomeric_state
+obeys	O
+twofold	O
+symmetry	O
+and	O
+assembles	O
+in	O
+a	O
+triangular	B-protein_state
+architecture	I-protein_state
+with	O
+dimeric	B-oligomeric_state
+BC	B-structure_element
+domains	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+).	O
+
+On	O
+the	O
+basis	O
+of	O
+a	O
+superposition	B-experimental_method
+of	O
+CDC2	B-structure_element
+,	O
+CDC1	B-structure_element
+of	O
+the	O
+phosphorylated	B-protein_state
+SceCD	B-species
+is	O
+rotated	O
+by	O
+30	O
+°	O
+relative	O
+to	O
+CDC1	B-structure_element
+of	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+flACC	B-mutant
+(	O
+Supplementary	O
+Fig	O
+.	O
+5d	O
+),	O
+similar	O
+to	O
+what	O
+we	O
+have	O
+observed	O
+for	O
+the	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+(	O
+Supplementary	O
+Fig	O
+.	O
+1d	O
+).	O
+
+When	O
+inspecting	B-experimental_method
+all	O
+individual	O
+protomer	B-oligomeric_state
+and	O
+fragment	B-mutant
+structures	B-evidence
+in	O
+their	O
+study	O
+,	O
+Wei	O
+and	O
+Tong	O
+also	O
+identify	O
+the	O
+CDN	B-structure_element
+/	I-structure_element
+CDC1	I-structure_element
+connection	I-structure_element
+as	O
+a	O
+highly	B-protein_state
+flexible	I-protein_state
+hinge	B-structure_element
+,	O
+in	O
+agreement	O
+with	O
+our	O
+observations	O
+.	O
+
+The	O
+only	O
+bona	O
+fide	O
+regulatory	B-protein_state
+phophorylation	B-site
+site	I-site
+of	O
+fungal	B-taxonomy_domain
+ACC	B-protein_type
+in	O
+the	O
+regulatory	B-structure_element
+loop	I-structure_element
+is	O
+directly	O
+participating	O
+in	O
+CDC1	B-structure_element
+/	O
+CDC2	B-structure_element
+domain	O
+interactions	O
+and	O
+thus	O
+stabilizes	O
+the	O
+hinge	B-structure_element
+conformation	I-structure_element
+.	O
+
+The	O
+phosphorylated	B-protein_state
+regulatory	B-structure_element
+loop	I-structure_element
+binds	O
+to	O
+an	O
+allosteric	B-site
+site	I-site
+at	O
+the	O
+interface	B-site
+of	O
+two	O
+non	B-protein_state
+-	I-protein_state
+catalytic	I-protein_state
+domains	O
+and	O
+restricts	O
+conformational	O
+freedom	O
+at	O
+several	O
+hinges	B-structure_element
+in	O
+the	O
+dynamic	B-protein_state
+ACC	B-protein_type
+.	O
+
+However	O
+,	O
+the	O
+example	O
+of	O
+ACC	B-protein_type
+now	O
+demonstrates	O
+the	O
+possibility	O
+of	O
+regulating	O
+activity	O
+by	O
+controlled	O
+dynamics	O
+of	O
+non	B-structure_element
+-	I-structure_element
+enzymatic	I-structure_element
+linker	I-structure_element
+regions	I-structure_element
+also	O
+in	O
+other	O
+families	O
+of	O
+carrier	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+multienzymes	I-protein_type
+.	O
+
+CDN	B-structure_element
+is	O
+linked	O
+by	O
+a	O
+four	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+(	O
+CDL	B-structure_element
+)	O
+to	O
+two	B-structure_element
+α	I-structure_element
+–	I-structure_element
+β	I-structure_element
+-	I-structure_element
+fold	I-structure_element
+domains	I-structure_element
+(	O
+CDC1	B-structure_element
+and	O
+CDC2	B-structure_element
+).	O
+
+(	O
+e	O
+)	O
+Structural	O
+overview	O
+of	O
+HsaBT	B-mutant
+-	I-mutant
+CD	I-mutant
+.	O
+
+(	O
+a	O
+–	O
+c	O
+)	O
+Large	O
+-	O
+scale	O
+conformational	O
+variability	O
+of	O
+the	O
+CDN	B-structure_element
+domain	O
+relative	O
+to	O
+the	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+domain	O
+.	O
+
+Domains	O
+other	O
+than	O
+CDN	B-structure_element
+and	O
+CDL	B-structure_element
+/	O
+CDC1	B-structure_element
+are	O
+omitted	O
+for	O
+clarity	O
+.	O
+
+Structural	O
+insights	O
+into	O
+the	O
+Escherichia	B-species
+coli	I-species
+lysine	B-protein_type
+decarboxylases	I-protein_type
+and	O
+molecular	O
+determinants	O
+of	O
+interaction	O
+with	O
+the	O
+AAA	B-protein_type
++	I-protein_type
+ATPase	I-protein_type
+RavA	B-protein
+
+Previously	O
+,	O
+we	O
+proposed	O
+a	O
+pseudoatomic	B-evidence
+model	I-evidence
+of	O
+the	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+cage	O
+based	O
+on	O
+its	O
+cryo	B-experimental_method
+-	I-experimental_method
+electron	I-experimental_method
+microscopy	I-experimental_method
+map	B-evidence
+and	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+an	O
+inactive	B-protein_state
+LdcI	B-protein
+decamer	B-oligomeric_state
+and	O
+a	O
+RavA	B-protein
+monomer	B-oligomeric_state
+.	O
+
+Enterobacterial	B-taxonomy_domain
+inducible	B-protein_state
+decarboxylases	B-protein_type
+of	O
+basic	B-protein_state
+amino	B-chemical
+acids	I-chemical
+lysine	B-residue_name
+,	O
+arginine	B-residue_name
+and	O
+ornithine	B-residue_name
+have	O
+a	O
+common	O
+evolutionary	O
+origin	O
+and	O
+belong	O
+to	O
+the	O
+α	B-protein_type
+-	I-protein_type
+family	I-protein_type
+of	O
+pyridoxal	B-chemical
+-	I-chemical
+5	I-chemical
+′-	I-chemical
+phosphate	I-chemical
+(	O
+PLP	B-chemical
+)-	O
+dependent	O
+enzymes	O
+.	O
+
+Inducible	B-protein_state
+enterobacterial	B-taxonomy_domain
+amino	B-protein_type
+acid	I-protein_type
+decarboxylases	I-protein_type
+have	O
+been	O
+intensively	O
+studied	O
+since	O
+the	O
+early	O
+1940	O
+because	O
+the	O
+ability	O
+of	O
+bacteria	B-taxonomy_domain
+to	O
+withstand	O
+acid	O
+stress	O
+can	O
+be	O
+linked	O
+to	O
+their	O
+pathogenicity	O
+in	O
+humans	B-species
+.	O
+
+Each	O
+monomer	B-oligomeric_state
+is	O
+composed	O
+of	O
+three	O
+domains	O
+–	O
+an	O
+N	O
+-	O
+terminal	O
+wing	B-structure_element
+domain	I-structure_element
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+129	I-residue_range
+),	O
+a	O
+PLP	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+core	I-structure_element
+domain	I-structure_element
+(	O
+residues	O
+130	B-residue_range
+–	I-residue_range
+563	I-residue_range
+),	O
+and	O
+a	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+(	O
+CTD	B-structure_element
+,	O
+residues	O
+564	B-residue_range
+–	I-residue_range
+715	I-residue_range
+).	O
+
+Monomers	B-oligomeric_state
+tightly	O
+associate	O
+via	O
+their	O
+core	B-structure_element
+domains	I-structure_element
+into	O
+2	B-protein_state
+-	I-protein_state
+fold	I-protein_state
+symmetrical	I-protein_state
+dimers	B-oligomeric_state
+with	O
+two	O
+complete	O
+active	B-site
+sites	I-site
+,	O
+and	O
+further	O
+build	O
+a	O
+toroidal	B-structure_element
+D5	I-structure_element
+-	I-structure_element
+symmetrical	I-structure_element
+structure	I-structure_element
+held	O
+by	O
+the	O
+wing	B-structure_element
+and	O
+core	B-structure_element
+domain	I-structure_element
+interactions	O
+around	O
+the	O
+central	B-structure_element
+pore	I-structure_element
+,	O
+with	O
+the	O
+CTDs	B-structure_element
+at	O
+the	O
+periphery	O
+.	O
+
+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
+
+To	O
+solve	O
+this	O
+discrepancy	O
+,	O
+in	O
+the	O
+present	O
+work	O
+we	O
+provided	O
+a	O
+three	O
+-	O
+dimensional	O
+(	O
+3D	O
+)	O
+cryoEM	B-experimental_method
+reconstruction	B-evidence
+of	O
+LdcC	B-protein
+and	O
+compared	O
+it	O
+with	O
+the	O
+available	O
+LdcI	B-protein
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+structures	B-evidence
+.	O
+
+Given	O
+that	O
+the	O
+LdcI	B-protein
+crystal	B-evidence
+structures	I-evidence
+were	O
+obtained	O
+at	O
+high	B-protein_state
+pH	I-protein_state
+where	O
+the	O
+enzyme	O
+is	O
+inactive	B-protein_state
+(	O
+LdcIi	B-protein
+,	O
+pH	B-protein_state
+8	I-protein_state
+.	I-protein_state
+5	I-protein_state
+),	O
+whereas	O
+the	O
+cryoEM	B-experimental_method
+reconstructions	B-evidence
+of	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+RavA	I-complex_assembly
+and	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+were	O
+done	O
+at	O
+acidic	B-protein_state
+pH	I-protein_state
+optimal	I-protein_state
+for	O
+the	O
+enzymatic	O
+activity	O
+,	O
+for	O
+a	O
+meaningful	O
+comparison	O
+,	O
+we	O
+also	O
+produced	O
+a	O
+3D	B-evidence
+reconstruction	I-evidence
+of	O
+the	O
+LdcI	B-protein
+at	O
+active	B-protein_state
+pH	I-protein_state
+(	O
+LdcIa	B-protein
+,	O
+pH	B-protein_state
+6	I-protein_state
+.	I-protein_state
+2	I-protein_state
+).	O
+
+As	O
+common	O
+for	O
+the	O
+α	B-protein_type
+family	I-protein_type
+of	O
+the	O
+PLP	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+decarboxylases	I-protein_type
+,	O
+dimerization	O
+is	O
+required	O
+for	O
+the	O
+enzymatic	O
+activity	O
+because	O
+the	O
+active	B-site
+site	I-site
+is	O
+buried	O
+in	O
+the	O
+dimer	B-site
+interface	I-site
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+
+In	O
+addition	O
+,	O
+our	O
+earlier	O
+biochemical	B-experimental_method
+observation	I-experimental_method
+that	O
+the	O
+enzymatic	O
+activity	O
+of	O
+LdcIa	B-protein
+is	O
+unaffected	O
+by	O
+RavA	B-protein
+binding	O
+is	O
+consistent	O
+with	O
+the	O
+relatively	O
+small	O
+changes	O
+undergone	O
+by	O
+the	O
+active	B-site
+site	I-site
+upon	O
+transition	O
+from	O
+LdcIa	B-protein
+to	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+.	O
+
+Second	O
+,	O
+the	O
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+clearly	O
+split	O
+the	O
+lysine	B-protein_type
+decarboxylases	I-protein_type
+into	O
+two	O
+groups	O
+(	O
+Fig	O
+.	O
+6A	O
+).	O
+
+Inspection	O
+of	O
+these	O
+consensus	B-evidence
+sequences	I-evidence
+revealed	O
+important	O
+differences	O
+between	O
+the	O
+groups	O
+regarding	O
+charge	O
+,	O
+size	O
+and	O
+hydrophobicity	O
+of	O
+several	O
+residues	O
+precisely	O
+at	O
+the	O
+level	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+that	O
+is	O
+responsible	O
+for	O
+the	O
+interaction	O
+with	O
+RavA	B-protein
+(	O
+Fig	O
+.	O
+6B	O
+–	O
+D	O
+).	O
+
+For	O
+example	O
+,	O
+in	O
+our	O
+previous	O
+study	O
+,	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutations	I-experimental_method
+identified	O
+Y697	B-residue_name_number
+as	O
+critically	O
+required	O
+for	O
+the	O
+RavA	B-protein
+binding	O
+.	O
+
+Our	O
+current	O
+analysis	O
+shows	O
+that	O
+Y697	B-residue_name_number
+is	O
+strictly	B-protein_state
+conserved	I-protein_state
+in	O
+the	O
+“	O
+LdcI	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+group	O
+whereas	O
+the	O
+“	O
+LdcC	B-protein_type
+-	I-protein_type
+like	I-protein_type
+”	O
+enzymes	O
+always	B-protein_state
+have	I-protein_state
+a	O
+lysine	B-residue_name
+in	O
+this	O
+position	O
+;	O
+it	O
+also	O
+uncovers	O
+several	O
+other	O
+residues	O
+potentially	O
+essential	O
+for	O
+the	O
+interaction	O
+with	O
+RavA	B-protein
+which	O
+can	O
+now	O
+be	O
+addressed	O
+by	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+.	O
+
+Superposition	B-experimental_method
+of	O
+the	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+LdcC	B-protein
+,	O
+LdcI	B-protein
+from	O
+LdcI	B-complex_assembly
+-	I-complex_assembly
+LARA	I-complex_assembly
+and	O
+LdcIa	B-protein
+colored	O
+as	O
+in	O
+Fig	O
+.	O
+1	O
+,	O
+and	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+LdcIi	B-protein
+in	O
+shades	O
+of	O
+yellow	O
+.	O
+
+(	O
+B	O
+)	O
+The	O
+LdcIi	B-protein
+dimer	B-oligomeric_state
+extracted	O
+from	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+decamer	B-oligomeric_state
+.	O
+
+(	O
+A	O
+)	O
+A	O
+slice	O
+through	O
+the	O
+pseudoatomic	B-evidence
+models	I-evidence
+of	O
+the	O
+LdcIa	B-protein
+(	O
+purple	O
+)	O
+and	O
+LdcC	B-protein
+(	O
+green	O
+)	O
+monomers	B-oligomeric_state
+extracted	O
+from	O
+the	O
+superimposed	B-experimental_method
+decamers	B-oligomeric_state
+(	O
+Fig	O
+.	O
+2	O
+).	O
+(	O
+B	O
+)	O
+The	O
+C	O
+-	O
+terminal	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+in	O
+LdcIa	B-protein
+and	O
+LdcC	B-protein
+enlarged	O
+from	O
+(	O
+A	O
+,	O
+C	O
+)	O
+Exchanged	O
+primary	O
+sequences	O
+(	O
+capital	O
+letters	O
+)	O
+and	O
+their	O
+immediate	O
+vicinity	O
+(	O
+lower	O
+case	O
+letters	O
+)	O
+colored	O
+as	O
+in	O
+(	O
+A	O
+,	O
+B	O
+),	O
+with	O
+the	O
+corresponding	O
+secondary	O
+structure	O
+elements	O
+and	O
+the	O
+amino	O
+acid	O
+numbering	O
+shown	O
+.	O
+
+Sequence	B-experimental_method
+alignment	I-experimental_method
+suggests	O
+that	O
+both	O
+kinases	B-protein_type
+belong	O
+to	O
+the	O
+ribulokinase	B-protein_type
+-	I-protein_type
+like	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+,	O
+a	O
+sub	O
+-	O
+family	O
+of	O
+FGGY	B-protein_type
+family	I-protein_type
+carbohydrate	I-protein_type
+kinases	I-protein_type
+.	O
+
+In	O
+addition	O
+,	O
+our	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+suggested	O
+that	O
+SePSK	B-protein
+has	O
+the	O
+capability	O
+to	O
+phosphorylate	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+.	O
+
+These	O
+kinases	B-protein_type
+exhibit	O
+considerable	O
+differences	O
+in	O
+their	O
+folding	O
+pattern	O
+and	O
+substrate	O
+specificity	O
+.	O
+
+Domain	B-structure_element
+I	I-structure_element
+exhibits	O
+a	O
+ribonuclease	B-structure_element
+H	I-structure_element
+-	I-structure_element
+like	I-structure_element
+folding	I-structure_element
+pattern	I-structure_element
+,	O
+and	O
+is	O
+responsible	O
+for	O
+the	O
+substrate	O
+binding	O
+,	O
+while	O
+domain	B-structure_element
+II	I-structure_element
+possesses	O
+an	O
+actin	B-structure_element
+-	I-structure_element
+like	I-structure_element
+ATPase	I-structure_element
+domain	I-structure_element
+that	O
+binds	O
+cofactor	O
+ATP	B-chemical
+.	O
+
+2	B-residue_range
+–	I-residue_range
+228	I-residue_range
+and	O
+aa	O
+.	O
+
+Domain	B-structure_element
+II	I-structure_element
+is	O
+comprised	O
+of	O
+aa	O
+.	O
+
+229	B-residue_range
+–	I-residue_range
+401	I-residue_range
+and	O
+classified	O
+into	O
+B2	B-structure_element
+(	O
+β31	B-structure_element
+/	O
+β29	B-structure_element
+/	O
+β22	B-structure_element
+/	O
+β23	B-structure_element
+/	O
+β25	B-structure_element
+/	O
+β24	B-structure_element
+)	O
+and	O
+A3	B-structure_element
+(	O
+α26	B-structure_element
+/	O
+α27	B-structure_element
+/	O
+α28	B-structure_element
+/	O
+α30	B-structure_element
+)	O
+(	O
+Fig	O
+1A	O
+and	O
+S1	O
+Fig	O
+).	O
+
+(	O
+A	O
+)	O
+Three	O
+-	O
+dimensional	O
+structure	B-evidence
+of	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+.	O
+
+(	O
+B	O
+)	O
+Three	O
+-	O
+dimensional	O
+structure	B-evidence
+of	O
+apo	B-protein_state
+-	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+In	O
+order	O
+to	O
+understand	O
+the	O
+function	O
+of	O
+these	O
+two	O
+kinases	O
+,	O
+we	O
+performed	O
+structural	B-experimental_method
+comparison	I-experimental_method
+using	O
+Dali	B-experimental_method
+server	I-experimental_method
+.	O
+
+Both	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+showed	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+substrate	O
+.	O
+
+This	O
+result	O
+was	O
+consistent	O
+with	O
+our	O
+enzymatic	B-experimental_method
+activity	I-experimental_method
+assays	I-experimental_method
+where	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+showed	O
+ATP	B-chemical
+hydrolysis	O
+activity	O
+without	O
+adding	O
+any	O
+substrates	O
+(	O
+Fig	O
+2A	O
+and	O
+2C	O
+).	O
+
+(	O
+A	O
+)	O
+The	O
+electron	B-evidence
+density	I-evidence
+of	O
+AMP	B-chemical
+-	I-chemical
+PNP	I-chemical
+.	O
+
+To	O
+better	O
+understand	O
+the	O
+interaction	O
+pattern	O
+between	O
+SePSK	B-protein
+and	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+,	O
+the	O
+apo	B-protein_state
+-	O
+SePSK	B-protein
+crystals	B-experimental_method
+were	I-experimental_method
+soaked	I-experimental_method
+into	I-experimental_method
+the	O
+reservoir	B-experimental_method
+with	O
+10	O
+mM	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+(	O
+RBL	B-chemical
+)	O
+and	O
+the	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+structure	B-evidence
+was	O
+solved	B-experimental_method
+.	O
+
+As	O
+shown	O
+in	O
+Fig	O
+4A	O
+,	O
+the	O
+nearest	O
+distance	O
+between	O
+the	O
+carbon	O
+skeleton	O
+of	O
+two	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+molecules	O
+are	O
+approx	O
+.	O
+
+Furthermore	O
+,	O
+the	O
+O2	O
+of	O
+RBL1	B-residue_name_number
+interacts	O
+with	O
+the	O
+main	O
+chain	O
+amide	O
+nitrogen	O
+of	O
+Ser72	B-residue_name_number
+(	O
+Fig	O
+4B	O
+).	O
+
+(	O
+A	O
+)	O
+The	O
+electrostatic	B-evidence
+potential	I-evidence
+surface	I-evidence
+map	I-evidence
+of	O
+RBL	B-complex_assembly
+-	I-complex_assembly
+SePSK	I-complex_assembly
+and	O
+a	O
+zoom	O
+-	O
+in	O
+view	O
+of	O
+RBL	B-site
+binding	I-site
+site	I-site
+.	O
+
+The	O
+RBL	B-chemical
+molecules	O
+(	O
+carbon	O
+atoms	O
+colored	O
+yellow	O
+)	O
+and	O
+amino	O
+acid	O
+residues	O
+of	O
+SePSK	B-protein
+(	O
+carbon	O
+atoms	O
+colored	O
+green	O
+)	O
+involved	O
+in	O
+RBL	B-chemical
+interaction	O
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+
+Structural	B-experimental_method
+comparison	I-experimental_method
+of	O
+SePSK	B-protein
+and	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+showed	O
+that	O
+while	O
+the	O
+RBL1	B-site
+binding	I-site
+pocket	I-site
+is	O
+conserved	B-protein_state
+,	O
+the	O
+RBL2	B-site
+pocket	I-site
+is	O
+disrupted	O
+in	O
+AtXK	B-protein
+-	I-protein
+1	I-protein
+structure	B-evidence
+,	O
+despite	O
+the	O
+fact	O
+that	O
+the	O
+residues	O
+interacting	O
+with	O
+RBL2	B-residue_name_number
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+between	O
+the	O
+two	O
+proteins	O
+.	O
+
+To	O
+further	O
+verified	O
+this	O
+result	O
+,	O
+we	O
+measured	O
+the	O
+binding	B-evidence
+affinity	I-evidence
+for	O
+D	B-chemical
+-	I-chemical
+ribulose	I-chemical
+of	O
+both	O
+wild	B-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+and	O
+D8A	B-mutant
+mutant	B-protein_state
+of	O
+SePSK	B-protein
+using	O
+a	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+method	I-experimental_method
+.	O
+
+Dissociation	B-evidence
+rate	I-evidence
+constant	I-evidence
+(	O
+Kd	B-evidence
+)	O
+of	O
+wild	B-protein_state
+type	I-protein_state
+and	O
+D8A	B-mutant
+-	O
+SePSK	B-protein
+are	O
+3	O
+ms	O
+-	O
+1	O
+and	O
+9	O
+ms	O
+-	O
+1	O
+,	O
+respectively	O
+.	O
+
+Simulated	O
+conformational	O
+change	O
+of	O
+SePSK	B-protein
+during	O
+the	O
+catalytic	O
+process	O
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+phosphorylation	B-protein_state
+-	I-protein_state
+mimicking	I-protein_state
+Mep2	B-mutant
+variants	I-mutant
+from	O
+C	B-species
+.	I-species
+albicans	I-species
+show	O
+large	O
+conformational	O
+changes	O
+in	O
+a	O
+conserved	B-protein_state
+and	O
+functionally	O
+important	O
+region	O
+of	O
+the	O
+CTR	B-structure_element
+.	O
+
+Mep2	B-protein_type
+proteins	I-protein_type
+are	O
+tightly	O
+regulated	O
+fungal	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+.	O
+
+While	O
+most	O
+studies	O
+have	O
+focused	O
+on	O
+the	O
+Saccharomyces	B-species
+cerevisiae	I-species
+transceptors	B-protein_type
+for	O
+phosphate	B-chemical
+(	O
+Pho84	B-protein
+),	O
+amino	B-chemical
+acids	I-chemical
+(	O
+Gap1	B-protein
+)	O
+and	O
+ammonium	B-chemical
+(	O
+Mep2	B-protein
+),	O
+transceptors	B-protein_type
+are	O
+found	O
+in	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+as	O
+well	O
+(	O
+for	O
+example	O
+,	O
+the	O
+mammalian	B-taxonomy_domain
+SNAT2	B-protein
+amino	B-protein_type
+-	I-protein_type
+acid	I-protein_type
+transporter	I-protein_type
+and	O
+the	O
+GLUT2	B-protein
+glucose	B-protein_type
+transporter	I-protein_type
+).	O
+
+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
+
+In	O
+bacteria	B-taxonomy_domain
+,	O
+amt	B-gene
+genes	O
+are	O
+present	O
+in	O
+an	O
+operon	O
+with	O
+glnK	B-gene
+,	O
+encoding	O
+a	O
+PII	B-protein_type
+-	I-protein_type
+like	I-protein_type
+signal	I-protein_type
+transduction	I-protein_type
+class	I-protein_type
+protein	I-protein_type
+.	O
+
+In	O
+plants	B-taxonomy_domain
+,	O
+transporter	B-protein_type
+phosphorylation	B-ptm
+and	O
+dephosphorylation	B-ptm
+are	O
+known	O
+to	O
+regulate	O
+activity	O
+.	O
+
+In	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+,	O
+phosphorylation	B-ptm
+of	O
+Ser457	B-residue_name_number
+within	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+(	O
+CTR	B-structure_element
+)	O
+in	O
+the	O
+cytoplasm	O
+was	O
+recently	O
+proposed	O
+to	O
+cause	O
+Mep2	B-protein_type
+opening	O
+,	O
+possibly	O
+via	O
+inducing	O
+a	O
+conformational	O
+change	O
+.	O
+
+The	O
+most	O
+striking	O
+difference	O
+is	O
+the	O
+fact	O
+that	O
+the	O
+Mep2	B-protein_type
+proteins	I-protein_type
+have	O
+closed	B-protein_state
+conformations	O
+.	O
+
+Electron	B-evidence
+density	I-evidence
+is	O
+visible	O
+for	O
+the	O
+entire	O
+polypeptide	O
+chains	O
+,	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+43	B-residue_range
+(	O
+ScMep2	B-protein
+)	O
+and	O
+25	B-residue_range
+residues	O
+(	O
+CaMep2	B-protein
+),	O
+which	O
+are	O
+poorly	B-protein_state
+conserved	I-protein_state
+and	O
+presumably	O
+disordered	B-protein_state
+.	O
+
+Together	O
+with	O
+additional	O
+,	O
+smaller	O
+differences	O
+in	O
+other	O
+extracellular	B-structure_element
+loops	I-structure_element
+,	O
+these	O
+changes	O
+generate	O
+a	O
+distinct	O
+vestibule	B-structure_element
+leading	O
+to	O
+the	O
+ammonium	B-site
+binding	I-site
+site	I-site
+that	O
+is	O
+much	O
+more	O
+pronounced	O
+than	O
+in	O
+the	O
+bacterial	B-taxonomy_domain
+proteins	O
+.	O
+
+The	O
+largest	O
+backbone	O
+movements	O
+of	O
+equivalent	O
+residues	O
+within	O
+ICL1	B-structure_element
+are	O
+∼	O
+10	O
+Å	O
+,	O
+markedly	O
+affecting	O
+the	O
+conserved	B-protein_state
+basic	B-protein_state
+RxK	B-structure_element
+motif	I-structure_element
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+The	O
+head	O
+group	O
+of	O
+Arg54	B-residue_name_number
+has	O
+moved	O
+∼	O
+11	O
+Å	O
+relative	O
+to	O
+that	O
+in	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+,	O
+whereas	O
+the	O
+shift	O
+of	O
+the	O
+head	O
+group	O
+of	O
+the	O
+variable	O
+Lys55	B-residue_name_number
+residue	O
+is	O
+almost	O
+20	O
+Å	O
+.	O
+The	O
+side	O
+chain	O
+of	O
+Lys56	B-residue_name_number
+in	O
+the	O
+basic	B-protein_state
+motif	B-structure_element
+points	O
+in	O
+an	O
+opposite	O
+direction	O
+in	O
+the	O
+Mep2	B-protein
+structures	B-evidence
+compared	O
+with	O
+that	O
+of	O
+,	O
+for	O
+example	O
+,	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+Compared	O
+with	O
+ICL1	B-structure_element
+,	O
+the	O
+backbone	O
+conformational	O
+changes	O
+observed	O
+for	O
+the	O
+neighbouring	O
+ICL2	B-structure_element
+are	O
+smaller	O
+,	O
+but	O
+large	O
+shifts	O
+are	O
+nevertheless	O
+observed	O
+for	O
+the	O
+conserved	B-protein_state
+residues	O
+Glu140	B-residue_name_number
+and	O
+Arg141	B-residue_name_number
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+The	O
+closed	B-protein_state
+state	O
+of	O
+the	O
+channel	B-site
+might	O
+also	O
+explain	O
+why	O
+no	B-evidence
+density	I-evidence
+,	O
+which	O
+could	O
+correspond	O
+to	O
+ammonium	B-chemical
+(	O
+or	O
+water	B-chemical
+),	O
+is	O
+observed	O
+in	O
+the	O
+hydrophobic	O
+part	O
+of	O
+the	O
+Mep2	B-protein
+channel	B-site
+close	O
+to	O
+the	O
+twin	B-structure_element
+-	I-structure_element
+His	I-structure_element
+motif	I-structure_element
+.	O
+
+The	O
+final	O
+region	O
+in	O
+Mep2	B-protein
+that	O
+shows	O
+large	O
+differences	O
+compared	O
+with	O
+the	O
+bacterial	B-taxonomy_domain
+transporters	B-protein_type
+is	O
+the	O
+CTR	B-structure_element
+.	O
+
+In	O
+Mep2	B-protein
+,	O
+the	O
+CTR	B-structure_element
+has	O
+moved	O
+away	O
+and	O
+makes	O
+relatively	O
+few	O
+contacts	O
+with	O
+the	O
+main	B-structure_element
+body	I-structure_element
+of	O
+the	O
+transporter	B-protein_type
+,	O
+generating	O
+a	O
+more	O
+elongated	B-protein_state
+protein	O
+(	O
+Figs	O
+1	O
+and	O
+4	O
+).	O
+
+This	O
+is	O
+illustrated	O
+by	O
+the	O
+positions	O
+of	O
+the	O
+five	O
+universally	B-protein_state
+conserved	I-protein_state
+residues	O
+within	O
+the	O
+CTR	B-structure_element
+,	O
+that	O
+is	O
+,	O
+Arg415	B-residue_name_number
+(	O
+370	B-residue_number
+),	O
+Glu421	B-residue_name_number
+(	O
+376	B-residue_number
+),	O
+Gly424	B-residue_name_number
+(	O
+379	B-residue_number
+),	O
+Asp426	B-residue_name_number
+(	O
+381	B-residue_number
+)	O
+and	O
+Tyr	B-residue_name_number
+435	I-residue_name_number
+(	O
+390	B-residue_number
+)	O
+in	O
+CaMep2	B-protein
+(	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+)	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+
+On	O
+one	O
+side	O
+,	O
+the	O
+Tyr390	B-residue_name_number
+hydroxyl	O
+in	O
+Amt	B-protein
+-	I-protein
+1	I-protein
+is	O
+hydrogen	O
+bonded	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+the	O
+conserved	B-protein_state
+His185	B-residue_name_number
+at	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+loop	B-structure_element
+ICL3	B-structure_element
+.	O
+
+In	O
+the	O
+Mep2	B-protein
+structures	B-evidence
+,	O
+none	O
+of	O
+the	O
+interactions	O
+mentioned	O
+above	O
+are	O
+present	O
+.	O
+
+In	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Npr1	B-protein
+,	O
+plasmid	B-experimental_method
+-	I-experimental_method
+encoded	I-experimental_method
+WT	B-protein_state
+Mep2	B-protein
+in	O
+a	O
+S	B-species
+.	I-species
+cerevisiae	I-species
+mep1	B-mutant
+-	I-mutant
+3Δ	I-mutant
+strain	O
+(	O
+triple	B-mutant
+mepΔ	I-mutant
+)	O
+does	O
+not	O
+allow	O
+growth	O
+on	O
+low	O
+concentrations	O
+of	O
+ammonium	B-chemical
+,	O
+suggesting	O
+that	O
+the	O
+transporter	B-protein_type
+is	O
+inactive	B-protein_state
+(	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+
+Conversely	O
+,	O
+the	O
+phosphorylation	B-protein_state
+-	I-protein_state
+mimicking	I-protein_state
+S457D	B-mutant
+variant	O
+is	O
+active	B-protein_state
+both	O
+in	O
+the	O
+triple	B-mutant
+mepΔ	I-mutant
+background	O
+and	O
+in	O
+a	O
+triple	B-mutant
+mepΔ	I-mutant
+npr1Δ	I-mutant
+strain	O
+(	O
+Fig	O
+.	O
+3	O
+).	O
+
+Mutation	B-experimental_method
+of	O
+other	O
+potential	O
+phosphorylation	B-site
+sites	I-site
+in	O
+the	O
+CTR	B-structure_element
+did	O
+not	O
+support	O
+growth	O
+in	O
+the	O
+npr1Δ	B-mutant
+background	O
+.	O
+
+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
+
+Boeckstaens	O
+et	O
+al	O
+.	O
+proposed	O
+that	O
+phosphorylation	B-ptm
+does	O
+not	O
+affect	O
+channel	O
+activity	O
+directly	O
+,	O
+but	O
+instead	O
+relieves	O
+inhibition	O
+by	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+.	O
+
+The	O
+first	O
+one	O
+is	O
+that	O
+the	O
+open	B-protein_state
+state	O
+is	O
+disfavoured	O
+by	O
+crystallization	B-experimental_method
+because	O
+of	O
+lower	O
+stability	O
+or	O
+due	O
+to	O
+crystal	O
+packing	O
+constraints	O
+.	O
+
+The	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+of	O
+Ser457	B-residue_name_number
+/	O
+453	B-residue_number
+is	O
+located	O
+in	O
+a	O
+well	O
+-	O
+defined	O
+electronegative	B-site
+pocket	I-site
+that	O
+is	O
+solvent	B-protein_state
+accessible	I-protein_state
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+In	O
+the	O
+WT	B-protein_state
+structure	B-evidence
+,	O
+the	O
+acidic	O
+residues	O
+Asp419	B-residue_name_number
+,	O
+Glu420	B-residue_name_number
+and	O
+Glu421	B-residue_name_number
+are	O
+within	O
+hydrogen	O
+bonding	O
+distance	O
+of	O
+Ser453	B-residue_name_number
+.	O
+
+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
+
+These	O
+efforts	O
+have	O
+advanced	O
+our	O
+knowledge	O
+considerably	O
+but	O
+have	O
+not	O
+yet	O
+yielded	O
+atomic	O
+-	O
+level	O
+answers	O
+to	O
+several	O
+important	O
+mechanistic	O
+questions	O
+,	O
+including	O
+how	O
+ammonium	B-chemical
+transport	O
+is	O
+regulated	O
+in	O
+eukaryotes	B-taxonomy_domain
+and	O
+the	O
+mechanism	O
+of	O
+ammonium	B-chemical
+signalling	O
+.	O
+
+In	O
+Arabidopsis	B-species
+thaliana	I-species
+Amt	B-protein
+-	I-protein
+1	I-protein
+;	I-protein
+1	I-protein
+,	O
+phosphorylation	B-ptm
+of	O
+the	O
+CTR	B-structure_element
+residue	O
+T460	B-residue_name_number
+under	O
+conditions	O
+of	O
+high	O
+ammonium	B-chemical
+inhibits	O
+transport	O
+activity	O
+,	O
+that	O
+is	O
+,	O
+the	O
+default	O
+(	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+)	O
+state	O
+of	O
+the	O
+plant	B-taxonomy_domain
+transporter	B-protein_type
+is	O
+open	B-protein_state
+.	O
+
+Interestingly	O
+,	O
+phosphomimetic	B-mutant
+mutations	I-mutant
+introduced	O
+into	O
+one	O
+monomer	B-oligomeric_state
+inactivate	O
+the	O
+entire	O
+trimer	B-oligomeric_state
+,	O
+indicating	O
+that	O
+(	O
+i	O
+)	O
+heterotrimerization	O
+occurs	O
+and	O
+(	O
+ii	O
+)	O
+the	O
+CTR	B-structure_element
+mediates	O
+allosteric	O
+regulation	O
+of	O
+ammonium	B-chemical
+transport	O
+activity	O
+via	O
+phosphorylation	B-ptm
+.	O
+
+More	O
+specifically	O
+,	O
+the	O
+close	O
+interactions	O
+between	O
+the	O
+CTR	B-structure_element
+and	O
+ICL1	B-structure_element
+/	O
+ICL3	B-structure_element
+present	O
+in	O
+open	B-protein_state
+transporters	B-protein_type
+are	O
+disrupted	O
+,	O
+causing	O
+ICL3	B-structure_element
+to	O
+move	O
+outwards	O
+and	O
+block	O
+the	O
+channel	B-site
+(	O
+Figs	O
+4	O
+and	O
+9a	O
+).	O
+
+How	O
+exactly	O
+the	O
+channel	B-site
+opens	O
+and	O
+whether	O
+opening	O
+is	O
+intra	O
+-	O
+monomeric	O
+are	O
+still	O
+open	B-protein_state
+questions	O
+;	O
+it	O
+is	O
+possible	O
+that	O
+the	O
+change	O
+in	O
+the	O
+CTR	B-structure_element
+may	O
+disrupt	O
+its	O
+interactions	O
+with	O
+ICL3	B-structure_element
+of	O
+the	O
+neighbouring	O
+monomer	B-oligomeric_state
+(	O
+Fig	O
+.	O
+9b	O
+),	O
+which	O
+could	O
+result	O
+in	O
+opening	O
+of	O
+the	O
+neighbouring	O
+channel	B-site
+via	O
+inward	O
+movement	O
+of	O
+its	O
+ICL3	B-structure_element
+.	O
+
+Is	O
+our	O
+model	O
+for	O
+opening	O
+and	O
+closing	O
+of	O
+Mep2	B-protein
+channels	B-site
+valid	O
+for	O
+other	O
+eukaryotic	B-taxonomy_domain
+ammonium	B-protein_type
+transporters	I-protein_type
+?	O
+Our	O
+structural	B-evidence
+data	I-evidence
+support	O
+previous	O
+studies	O
+and	O
+clarify	O
+the	O
+central	O
+role	O
+of	O
+the	O
+CTR	B-structure_element
+and	O
+cytoplasmic	B-structure_element
+loops	I-structure_element
+in	O
+the	O
+transition	O
+between	O
+closed	B-protein_state
+and	O
+open	B-protein_state
+states	O
+.	O
+
+There	O
+is	O
+generally	O
+no	O
+equivalent	O
+for	O
+CaMep2	B-protein
+Tyr49	B-residue_name_number
+in	O
+plant	B-taxonomy_domain
+AMTs	B-protein_type
+,	O
+indicating	O
+that	O
+a	O
+Tyr	B-site
+–	I-site
+His2	I-site
+hydrogen	I-site
+bond	I-site
+as	O
+observed	O
+in	O
+Mep2	B-protein
+may	O
+not	O
+contribute	O
+to	O
+the	O
+closed	B-protein_state
+state	O
+in	O
+plant	B-taxonomy_domain
+transporters	B-protein_type
+.	O
+
+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
+
+In	O
+one	O
+model	O
+,	O
+signalling	O
+is	O
+proposed	O
+to	O
+depend	O
+on	O
+the	O
+nature	O
+of	O
+the	O
+transported	O
+substrate	O
+,	O
+which	O
+might	O
+be	O
+different	O
+in	O
+certain	O
+subfamilies	O
+of	O
+ammonium	B-protein_type
+transporters	I-protein_type
+(	O
+for	O
+example	O
+,	O
+Mep1	B-protein
+/	O
+Mep3	B-protein
+versus	O
+Mep2	B-protein
+).	O
+
+In	O
+the	O
+other	O
+model	O
+,	O
+signalling	O
+is	O
+thought	O
+to	O
+require	O
+a	O
+distinct	O
+conformation	O
+of	O
+the	O
+Mep2	B-protein
+transporter	B-protein_type
+occurring	O
+during	O
+the	O
+transport	O
+cycle	O
+.	O
+
+The	O
+region	O
+showing	O
+ICL1	B-structure_element
+(	O
+blue	O
+),	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+)	O
+is	O
+boxed	O
+for	O
+comparison	O
+.	O
+
+(	O
+a	O
+)	O
+ICL1	B-structure_element
+in	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+(	O
+light	O
+blue	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+dark	O
+blue	O
+),	O
+showing	O
+unwinding	O
+and	O
+inward	O
+movement	O
+in	O
+the	O
+fungal	B-taxonomy_domain
+protein	O
+.	O
+(	O
+b	O
+)	O
+Stereo	O
+diagram	O
+viewed	O
+from	O
+the	O
+cytosol	O
+of	O
+ICL1	B-structure_element
+,	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+and	O
+the	O
+CTR	B-structure_element
+(	O
+red	O
+)	O
+in	O
+AfAmt	B-protein
+-	I-protein
+1	I-protein
+(	O
+light	O
+colours	O
+)	O
+and	O
+CaMep2	B-protein
+(	O
+dark	O
+colours	O
+).	O
+
+The	O
+labelled	O
+residues	O
+are	O
+analogous	O
+within	O
+both	O
+structures	B-evidence
+.	O
+
+Channel	O
+closures	O
+in	O
+Mep2	B-protein
+.	O
+
+The	O
+Npr1	B-protein
+kinase	B-protein_type
+target	O
+Ser453	B-residue_name_number
+is	O
+dephosphorylated	B-protein_state
+and	O
+located	O
+in	O
+an	O
+electronegative	B-site
+pocket	I-site
+.	O
+
+(	O
+a	O
+)	O
+Stereoviews	O
+of	O
+CaMep2	B-protein
+showing	O
+2Fo	O
+–	O
+Fc	O
+electron	O
+density	O
+(	O
+contoured	O
+at	O
+1	O
+.	O
+0	O
+σ	O
+)	O
+for	O
+CTR	B-structure_element
+residues	O
+Asp419	B-residue_range
+-	I-residue_range
+Met422	I-residue_range
+and	O
+for	O
+Tyr446	B-residue_range
+-	I-residue_range
+Thr455	I-residue_range
+of	O
+the	O
+AI	B-structure_element
+region	I-structure_element
+.	O
+
+Phosphorylation	B-ptm
+causes	O
+conformational	O
+changes	O
+in	O
+the	O
+CTR	B-structure_element
+.	O
+
+(	O
+a	O
+)	O
+In	O
+the	O
+closed	B-protein_state
+,	O
+non	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+state	O
+(	O
+i	O
+),	O
+the	O
+CTR	B-structure_element
+(	O
+magenta	O
+)	O
+and	O
+ICL3	B-structure_element
+(	O
+green	O
+)	O
+are	O
+far	O
+apart	O
+with	O
+the	O
+latter	O
+blocking	O
+the	O
+intracellular	O
+channel	B-site
+exit	I-site
+(	O
+indicated	O
+with	O
+a	O
+hatched	O
+circle	O
+).	O
+
+Visualizing	O
+chaperone	B-protein_type
+-	O
+assisted	O
+protein	O
+folding	O
+
+READ	B-experimental_method
+enabled	O
+us	O
+to	O
+visualize	O
+even	O
+sparsely	O
+populated	O
+conformations	O
+of	O
+the	O
+substrate	O
+protein	O
+immunity	B-protein
+protein	I-protein
+7	I-protein
+(	O
+Im7	B-protein
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+E	B-species
+.	I-species
+coli	I-species
+chaperone	B-protein_type
+Spy	B-protein
+.	O
+
+It	O
+is	O
+clear	O
+that	O
+molecular	O
+chaperones	B-protein_type
+aid	O
+in	O
+protein	O
+folding	O
+.	O
+
+Structural	B-evidence
+models	I-evidence
+of	O
+chaperone	B-protein_type
+-	O
+substrate	O
+complexes	O
+have	O
+recently	O
+begun	O
+to	O
+provide	O
+information	O
+as	O
+to	O
+how	O
+a	O
+chaperone	B-protein_type
+can	O
+recognize	O
+its	O
+substrate	O
+.	O
+
+For	O
+most	O
+chaperones	B-protein_type
+,	O
+it	O
+is	O
+still	O
+unclear	O
+whether	O
+the	O
+chaperone	B-protein_type
+actively	O
+participates	O
+in	O
+and	O
+affects	O
+the	O
+folding	O
+of	O
+the	O
+substrate	O
+proteins	O
+,	O
+or	O
+merely	O
+provides	O
+a	O
+suitable	O
+microenvironment	O
+enabling	O
+the	O
+substrate	O
+to	O
+fold	O
+on	O
+its	O
+own	O
+.	O
+
+We	O
+therefore	O
+screened	B-experimental_method
+crystallization	B-experimental_method
+conditions	I-experimental_method
+for	O
+Spy	B-protein
+with	O
+four	O
+different	O
+substrate	O
+proteins	O
+:	O
+a	O
+fragment	O
+of	O
+the	O
+largely	O
+unfolded	B-protein_state
+bovine	B-taxonomy_domain
+α	B-chemical
+-	I-chemical
+casein	I-chemical
+protein	O
+,	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+E	B-species
+.	I-species
+coli	I-species
+Im7	B-protein
+,	O
+an	O
+unfolded	B-protein_state
+variant	O
+of	O
+Im7	B-protein
+(	O
+L18A	B-mutant
+L19A	B-mutant
+L37A	B-mutant
+),	O
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+half	I-structure_element
+of	O
+Im7	B-protein
+(	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+),	O
+which	O
+encompasses	O
+the	O
+entire	O
+Spy	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+portion	I-structure_element
+of	O
+Im7	B-protein
+.	O
+
+Subsequent	O
+crystal	B-experimental_method
+washing	I-experimental_method
+and	I-experimental_method
+dissolution	I-experimental_method
+experiments	O
+confirmed	O
+the	O
+presence	O
+of	O
+the	O
+substrates	O
+in	O
+the	O
+co	B-experimental_method
+-	I-experimental_method
+crystals	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+
+We	O
+split	O
+this	O
+approach	O
+into	O
+five	O
+steps	O
+:	O
+(	O
+1	O
+)	O
+By	O
+using	O
+a	O
+well	O
+-	O
+diffracting	O
+Spy	B-protein
+:	O
+substrate	O
+co	B-evidence
+-	I-evidence
+crystal	I-evidence
+,	O
+we	O
+first	O
+determined	O
+the	O
+structure	B-evidence
+of	O
+the	O
+folded	B-protein_state
+domain	B-structure_element
+of	O
+Spy	B-protein
+and	O
+obtained	O
+high	O
+quality	O
+residual	B-evidence
+electron	I-evidence
+density	I-evidence
+within	O
+the	O
+dynamic	B-protein_state
+regions	O
+of	O
+the	O
+substrate	O
+.	O
+
+The	O
+READ	B-experimental_method
+sample	B-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+-	I-experimental_method
+select	I-experimental_method
+algorithm	I-experimental_method
+is	O
+diagrammed	O
+in	O
+Fig	O
+.	O
+2	O
+.	O
+
+To	O
+make	O
+the	O
+electron	B-experimental_method
+density	I-experimental_method
+selection	I-experimental_method
+practical	O
+,	O
+we	O
+needed	O
+to	O
+develop	O
+a	O
+method	O
+to	O
+rapidly	O
+evaluate	O
+the	O
+agreement	O
+between	O
+the	O
+selected	O
+sub	O
+-	O
+ensembles	O
+and	O
+the	O
+experimental	O
+electron	B-evidence
+density	I-evidence
+on	O
+-	O
+the	O
+-	O
+fly	O
+during	O
+the	O
+selection	O
+procedure	O
+.	O
+
+To	O
+reduce	O
+the	O
+extent	O
+of	O
+3D	O
+space	O
+to	O
+be	O
+explored	O
+,	O
+this	O
+compressed	O
+map	B-evidence
+was	O
+created	O
+by	O
+only	O
+using	O
+density	B-evidence
+from	O
+regions	O
+of	O
+space	O
+significantly	O
+sampled	O
+by	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+in	O
+the	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+MD	B-experimental_method
+simulations	B-experimental_method
+.	O
+
+We	O
+were	O
+particularly	O
+interested	O
+in	O
+finding	O
+answers	O
+to	O
+one	O
+of	O
+the	O
+most	O
+fundamental	O
+questions	O
+in	O
+chaperone	B-protein_type
+biology	O
+—	O
+how	O
+does	O
+chaperone	B-protein_type
+binding	O
+affect	O
+substrate	O
+structure	O
+and	O
+vice	O
+versa	O
+.	O
+
+We	O
+constructed	O
+a	O
+contact	B-evidence
+map	I-evidence
+of	O
+the	O
+complex	O
+,	O
+which	O
+shows	O
+the	O
+frequency	O
+of	O
+interactions	O
+for	O
+chaperone	B-protein_type
+-	O
+substrate	O
+residue	O
+pairs	O
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+This	O
+twist	O
+yields	O
+asymmetry	O
+and	O
+results	O
+in	O
+substantially	O
+different	O
+interaction	O
+patterns	O
+in	O
+the	O
+two	O
+Spy	B-protein
+monomers	B-oligomeric_state
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+
+Additionally	O
+,	O
+we	O
+observed	O
+that	O
+the	O
+linker	B-structure_element
+region	I-structure_element
+(	O
+residues	O
+47	B-residue_range
+–	I-residue_range
+57	I-residue_range
+)	O
+of	O
+Spy	B-protein
+,	O
+which	O
+participates	O
+in	O
+substrate	O
+interaction	O
+,	O
+becomes	O
+mostly	O
+disordered	B-protein_state
+upon	O
+binding	O
+the	O
+substrate	O
+.	O
+
+Importantly	O
+,	O
+we	O
+observed	O
+the	O
+same	O
+structural	O
+changes	O
+in	O
+Spy	B-protein
+regardless	O
+of	O
+which	O
+of	O
+the	O
+four	O
+substrates	O
+was	O
+bound	O
+(	O
+Fig	O
+.	O
+5b	O
+,	O
+Table	O
+1	O
+).	O
+
+We	O
+recently	O
+showed	O
+that	O
+Im7	B-protein
+can	O
+fold	O
+while	O
+remaining	O
+continuously	B-protein_state
+bound	I-protein_state
+to	I-protein_state
+Spy	B-protein
+.	O
+
+This	O
+model	O
+is	O
+consistent	O
+with	O
+previous	O
+studies	O
+postulating	O
+that	O
+the	O
+flexible	O
+binding	O
+of	O
+chaperones	B-protein_type
+allows	O
+for	O
+substrate	O
+protein	O
+folding	O
+.	O
+
+The	O
+amphipathic	O
+concave	B-site
+surface	I-site
+of	O
+Spy	B-protein
+likely	O
+facilitates	O
+this	O
+flexible	O
+binding	O
+and	O
+may	O
+be	O
+a	O
+crucial	O
+feature	O
+for	O
+Spy	B-protein
+and	O
+potentially	O
+other	O
+chaperones	B-protein_type
+,	O
+allowing	O
+them	O
+to	O
+bind	O
+multiple	O
+conformations	O
+of	O
+many	O
+different	O
+substrates	O
+.	O
+
+The	O
+negatively	O
+charged	O
+Im7	B-protein
+residues	O
+Glu21	B-residue_name_number
+,	O
+Asp32	B-residue_name_number
+,	O
+and	O
+Asp35	B-residue_name_number
+reside	O
+on	O
+the	O
+surface	O
+of	O
+Im7	B-protein
+and	O
+form	O
+interactions	O
+with	O
+Spy	B-protein
+’	O
+s	O
+positively	O
+charged	O
+cradle	B-site
+in	O
+both	O
+the	O
+unfolded	B-protein_state
+and	O
+native	B-protein_state
+-	I-protein_state
+like	I-protein_state
+states	O
+.	O
+
+This	O
+selection	O
+resulted	O
+in	O
+“	O
+Super	O
+Spy	B-protein
+”	O
+variants	B-protein_state
+that	O
+were	O
+more	O
+effective	O
+at	O
+both	O
+preventing	O
+aggregation	O
+and	O
+promoting	O
+protein	O
+folding	O
+.	O
+
+By	O
+sampling	O
+multiple	O
+conformations	O
+,	O
+this	O
+linker	B-structure_element
+region	I-structure_element
+may	O
+allow	O
+diverse	O
+substrate	O
+conformations	O
+to	O
+be	O
+accommodated	O
+.	O
+
+Overall	O
+,	O
+comparison	O
+of	O
+our	O
+ensemble	B-evidence
+to	O
+the	O
+Super	O
+Spy	B-protein
+variants	B-protein_state
+provides	O
+specific	O
+examples	O
+to	O
+corroborate	O
+the	O
+importance	O
+of	O
+conformational	O
+flexibility	O
+in	O
+chaperone	B-protein_type
+-	O
+substrate	O
+interactions	O
+.	O
+
+ATP	B-chemical
+and	O
+co	O
+-	O
+chaperone	B-protein_type
+dependencies	O
+may	O
+have	O
+emerged	O
+later	O
+through	O
+evolution	O
+to	O
+better	O
+modulate	O
+and	O
+control	O
+chaperone	B-protein_type
+action	O
+.	O
+
+As	O
+the	O
+residues	O
+involved	O
+in	O
+contacts	O
+are	O
+more	O
+evenly	O
+distributed	O
+in	O
+Im76	B-mutant
+-	I-mutant
+45	I-mutant
+compared	O
+to	O
+Spy	B-protein
+,	O
+its	O
+contact	B-evidence
+map	I-evidence
+was	O
+amplified	O
+.	O
+(	O
+b	O
+)	O
+Detailed	O
+contact	B-evidence
+maps	I-evidence
+of	O
+Spy	B-complex_assembly
+:	I-complex_assembly
+Im76	I-complex_assembly
+-	I-complex_assembly
+45	I-complex_assembly
+.	O
+
+The	O
+Super	O
+Spy	B-protein
+mutants	O
+F115L	B-mutant
+,	O
+F115I	B-mutant
+,	O
+and	O
+L32P	B-mutant
+are	O
+proposed	O
+to	O
+gain	O
+activity	O
+by	O
+increasing	O
+the	O
+flexibility	O
+or	O
+size	O
+of	O
+this	O
+linker	B-structure_element
+region	I-structure_element
+.	O
+
+To	O
+support	O
+antibody	B-protein_type
+therapeutic	O
+development	O
+,	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+a	O
+set	O
+of	O
+16	O
+germline	O
+variants	O
+composed	O
+of	O
+4	O
+different	O
+kappa	B-structure_element
+light	I-structure_element
+chains	I-structure_element
+paired	O
+with	O
+4	O
+different	O
+heavy	B-structure_element
+chains	I-structure_element
+have	O
+been	O
+determined	O
+.	O
+
+The	O
+longer	B-protein_state
+CDRs	B-structure_element
+with	O
+tandem	O
+glycines	B-residue_name
+or	O
+serines	B-residue_name
+have	O
+more	O
+conformational	O
+diversity	O
+than	O
+the	O
+others	O
+.	O
+
+Two	O
+of	O
+16	O
+structures	B-evidence
+showed	O
+particularly	O
+large	O
+variations	O
+in	O
+the	O
+tilt	B-evidence
+angles	I-evidence
+when	O
+compared	O
+with	O
+the	O
+other	O
+pairings	O
+.	O
+
+These	O
+domains	O
+have	O
+a	O
+common	O
+folding	O
+pattern	O
+often	O
+referred	O
+to	O
+as	O
+the	O
+“	O
+immunoglobulin	B-structure_element
+fold	I-structure_element
+,”	O
+formed	O
+by	O
+the	O
+packing	O
+together	O
+of	O
+2	O
+anti	B-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheets	I-structure_element
+.	O
+
+All	O
+immunoglobulin	B-protein_type
+chains	I-protein_type
+have	O
+an	O
+N	O
+-	O
+terminal	O
+V	B-structure_element
+domain	I-structure_element
+followed	O
+by	O
+1	O
+to	O
+4	O
+C	B-structure_element
+domains	I-structure_element
+,	O
+depending	O
+upon	O
+the	O
+chain	O
+type	O
+.	O
+
+The	O
+cataloging	O
+and	O
+development	O
+of	O
+the	O
+rules	O
+for	O
+predicting	O
+the	O
+conformation	O
+of	O
+the	O
+anchor	B-structure_element
+region	I-structure_element
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+continue	O
+to	O
+be	O
+refined	O
+,	O
+producing	O
+new	O
+insight	O
+into	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+conformations	O
+and	O
+new	O
+tools	O
+for	O
+antibody	B-protein_type
+engineering	O
+.	O
+
+One	O
+important	O
+finding	O
+of	O
+the	O
+antibody	B-experimental_method
+modeling	I-experimental_method
+assessments	I-experimental_method
+was	O
+that	O
+errors	O
+in	O
+the	O
+structural	O
+templates	O
+that	O
+are	O
+used	O
+as	O
+the	O
+basis	O
+for	O
+homology	B-experimental_method
+models	I-experimental_method
+can	O
+propagate	O
+into	O
+the	O
+final	O
+models	O
+,	O
+producing	O
+inaccuracies	O
+that	O
+may	O
+negatively	O
+influence	O
+the	O
+predictive	O
+nature	O
+of	O
+the	O
+V	B-structure_element
+region	I-structure_element
+model	O
+.	O
+
+The	O
+structures	B-evidence
+and	O
+their	O
+analyses	O
+provide	O
+a	O
+foundation	O
+for	O
+future	O
+antibody	B-protein_type
+engineering	O
+and	O
+structure	O
+determination	O
+efforts	O
+.	O
+
+The	O
+similarity	O
+in	O
+the	O
+crystal	B-evidence
+forms	I-evidence
+is	O
+attributed	O
+in	O
+part	O
+to	O
+cross	O
+-	O
+seeding	O
+using	O
+the	O
+microseed	B-experimental_method
+matrix	I-experimental_method
+screening	I-experimental_method
+for	O
+groups	O
+2	O
+and	O
+3	O
+.	O
+
+The	O
+number	O
+of	O
+Fab	B-structure_element
+molecules	O
+in	O
+the	O
+crystallographic	O
+asymmetric	O
+unit	O
+varies	O
+from	O
+1	O
+(	O
+for	O
+12	O
+Fabs	B-structure_element
+)	O
+to	O
+2	O
+(	O
+for	O
+4	O
+Fabs	B-structure_element
+).	O
+
+For	O
+the	O
+LC	B-structure_element
+,	O
+the	O
+disorder	B-protein_state
+is	O
+observed	O
+at	O
+2	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+residues	O
+with	O
+few	O
+exceptions	O
+.	O
+
+CDR	B-structure_element
+H1	B-structure_element
+
+The	O
+canonical	O
+structures	O
+of	O
+CDR	B-structure_element
+H2	B-structure_element
+have	O
+fairly	O
+consistent	O
+conformations	O
+(	O
+Table	O
+2	O
+,	O
+Fig	O
+.	O
+2	O
+).	O
+
+In	O
+one	O
+case	O
+,	O
+in	O
+the	O
+second	O
+Fab	B-structure_element
+of	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+CDR	B-structure_element
+H2	B-structure_element
+is	O
+partially	B-protein_state
+disordered	I-protein_state
+(	O
+Δ55	B-mutant
+-	I-mutant
+60	I-mutant
+).	O
+
+Despite	O
+this	O
+,	O
+the	O
+conformations	O
+are	O
+tightly	O
+clustered	O
+(	O
+rmsd	B-evidence
+is	O
+0	O
+.	O
+20	O
+Å	O
+).	O
+
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+is	O
+the	O
+most	O
+variable	O
+in	O
+CDR	B-structure_element
+L1	B-structure_element
+among	O
+the	O
+4	O
+germlines	O
+as	O
+indicated	O
+by	O
+an	O
+rmsd	B-evidence
+of	O
+0	O
+.	O
+54	O
+Å	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+The	O
+CDR	B-structure_element
+L2	B-structure_element
+conformations	O
+for	O
+each	O
+of	O
+the	O
+LCs	B-structure_element
+paired	O
+with	O
+the	O
+4	O
+HCs	B-structure_element
+are	O
+clustered	O
+more	O
+tightly	O
+than	O
+any	O
+of	O
+the	O
+other	O
+CDRs	B-structure_element
+(	O
+rmsd	B-evidence
+values	O
+are	O
+in	O
+the	O
+range	O
+0	O
+.	O
+09	O
+-	O
+0	O
+.	O
+16	O
+Å	O
+),	O
+and	O
+all	O
+4	O
+sets	O
+have	O
+virtually	O
+the	O
+same	O
+conformation	O
+despite	O
+the	O
+sequence	O
+diversity	O
+of	O
+the	O
+loop	B-structure_element
+.	O
+
+The	O
+superposition	B-experimental_method
+of	O
+CDR	B-structure_element
+L3	B-structure_element
+backbones	O
+for	O
+all	O
+HC	B-complex_assembly
+:	I-complex_assembly
+LC	I-complex_assembly
+pairs	O
+with	O
+light	B-structure_element
+chains	I-structure_element
+:	O
+(	O
+A	O
+)	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+,	O
+(	O
+B	O
+)	O
+L3	B-mutant
+-	I-mutant
+11	I-mutant
+,	O
+(	O
+C	O
+)	O
+L3	B-mutant
+-	I-mutant
+20	I-mutant
+and	O
+(	O
+D	O
+)	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+.	O
+
+An	O
+interesting	O
+feature	O
+of	O
+these	O
+CDR	B-structure_element
+H3	B-structure_element
+structures	B-evidence
+is	O
+the	O
+presence	O
+of	O
+a	O
+water	B-chemical
+molecule	O
+that	O
+interacts	O
+with	O
+the	O
+peptide	O
+nitrogens	O
+and	O
+carbonyl	O
+oxygens	O
+near	O
+the	O
+bridging	O
+loop	B-structure_element
+connecting	O
+the	O
+2	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+.	O
+
+A	O
+representative	O
+CDR	B-structure_element
+H3	B-structure_element
+structure	B-evidence
+for	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L1	I-complex_assembly
+-	I-complex_assembly
+39	I-complex_assembly
+illustrating	O
+this	O
+is	O
+shown	O
+in	O
+Fig	O
+.	O
+7A	O
+.	O
+
+The	O
+stem	B-structure_element
+regions	I-structure_element
+in	O
+these	O
+3	O
+cases	O
+are	O
+in	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+consistent	O
+with	O
+that	O
+observed	O
+for	O
+4DN3	O
+.	O
+
+The	O
+stem	B-structure_element
+regions	I-structure_element
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+for	O
+the	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+Fabs	B-structure_element
+are	O
+in	O
+the	O
+‘	O
+kinked	B-protein_state
+’	O
+conformation	O
+while	O
+,	O
+surprisingly	O
+,	O
+those	O
+of	O
+the	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+pair	O
+and	O
+H3	B-complex_assembly
+-	I-complex_assembly
+53	I-complex_assembly
+:	I-complex_assembly
+L4	I-complex_assembly
+-	I-complex_assembly
+1	I-complex_assembly
+are	O
+in	O
+the	O
+‘	O
+extended	B-protein_state
+’	O
+conformation	O
+(	O
+Fig	O
+.	O
+7B	O
+).	O
+
+The	O
+two	O
+domains	O
+pack	O
+together	O
+such	O
+that	O
+the	O
+5	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+β	I-structure_element
+-	I-structure_element
+sheets	I-structure_element
+,	O
+which	O
+have	O
+hydrophobic	O
+surfaces	O
+,	O
+interact	O
+with	O
+each	O
+other	O
+bringing	O
+the	O
+CDRs	B-structure_element
+from	O
+both	O
+the	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+domains	O
+into	O
+close	O
+proximity	O
+.	O
+
+VH	B-site
+:	I-site
+VL	I-site
+interface	I-site
+amino	O
+acid	O
+residue	O
+interactions	O
+
+Position	O
+43	B-residue_number
+may	O
+be	O
+alternatively	O
+occupied	O
+by	O
+Ser	B-residue_name
+,	O
+Val	B-residue_name
+or	O
+Pro	B-residue_name
+(	O
+as	O
+in	O
+L4	B-mutant
+-	I-mutant
+1	I-mutant
+),	O
+but	O
+the	O
+hydrophobic	O
+interaction	O
+with	O
+H	B-structure_element
+-	O
+Tyr91	B-residue_name_number
+is	O
+preserved	O
+.	O
+
+These	O
+core	O
+interactions	O
+provide	O
+enough	O
+stability	O
+to	O
+the	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+dimer	B-oligomeric_state
+so	O
+that	O
+additional	O
+VH	B-site
+-	I-site
+VL	I-site
+contacts	I-site
+can	O
+tolerate	O
+amino	O
+acid	O
+sequence	O
+variations	O
+in	O
+CDRs	B-structure_element
+H3	B-structure_element
+and	O
+L3	B-structure_element
+that	O
+form	O
+part	O
+of	O
+the	O
+VH	B-site
+:	I-site
+VL	I-site
+interface	I-site
+.	O
+
+One	O
+notable	O
+exception	O
+is	O
+H	B-structure_element
+-	O
+Trp47	B-residue_name_number
+,	O
+which	O
+exhibits	O
+2	O
+conformations	O
+of	O
+the	O
+indole	O
+ring	O
+.	O
+
+Apparently	O
+,	O
+residues	O
+flanking	O
+CDR	B-structure_element
+H3	B-structure_element
+in	O
+the	O
+2	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+pairings	O
+are	O
+inconsistent	O
+with	O
+any	O
+stable	B-protein_state
+conformation	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+,	O
+which	O
+translates	O
+into	O
+a	O
+less	O
+restricted	O
+conformational	O
+space	O
+for	O
+some	O
+of	O
+them	O
+,	O
+including	O
+H	B-structure_element
+-	O
+Trp47	B-residue_name_number
+.	O
+
+Differences	O
+in	O
+VH	B-complex_assembly
+:	I-complex_assembly
+VL	I-complex_assembly
+tilt	B-evidence
+angles	I-evidence
+.	O
+
+The	O
+differences	B-evidence
+in	O
+the	O
+tilt	B-evidence
+angle	I-evidence
+are	O
+shown	O
+for	O
+all	O
+pairs	O
+of	O
+V	B-structure_element
+regions	I-structure_element
+in	O
+Table	O
+3	O
+.	O
+
+Residues	O
+in	O
+CDR	B-structure_element
+H3	B-structure_element
+are	O
+missing	O
+:	O
+YGE	B-structure_element
+in	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+,	O
+GIY	B-structure_element
+in	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+.	O
+
+Interestingly	O
+,	O
+the	O
+2	O
+structures	B-evidence
+that	O
+have	O
+the	O
+largest	O
+tilt	B-evidence
+angle	I-evidence
+differences	I-evidence
+with	O
+the	O
+other	O
+variants	O
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+have	O
+the	O
+smallest	O
+VH	B-site
+:	I-site
+VL	I-site
+interfaces	I-site
+,	O
+684	O
+and	O
+725	O
+Å2	O
+,	O
+respectively	O
+.	O
+
+It	O
+appears	O
+that	O
+for	O
+each	O
+given	O
+LC	B-structure_element
+,	O
+the	O
+Fabs	B-structure_element
+with	O
+germlines	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+and	O
+H3	B-mutant
+-	I-mutant
+23	I-mutant
+are	O
+substantially	O
+more	O
+stable	B-protein_state
+than	O
+those	O
+with	O
+germlines	O
+H3	B-mutant
+-	I-mutant
+53	I-mutant
+and	O
+H5	B-mutant
+-	I-mutant
+51	I-mutant
+.	O
+
+Parts	O
+of	O
+CDR	B-structure_element
+H3	B-structure_element
+main	O
+chain	O
+are	O
+completely	O
+disordered	B-protein_state
+,	O
+and	O
+were	O
+not	O
+modeled	O
+in	O
+Fabs	B-structure_element
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+and	O
+H5	B-complex_assembly
+-	I-complex_assembly
+51	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+11	I-complex_assembly
+that	O
+have	O
+the	O
+lowest	O
+Tms	B-evidence
+in	O
+the	O
+set	O
+.	O
+
+All	O
+those	O
+molecules	O
+are	O
+relatively	O
+unstable	O
+,	O
+as	O
+is	O
+reflected	O
+in	O
+their	O
+low	O
+Tms	B-evidence
+.	O
+
+Of	O
+the	O
+4	O
+HCs	B-structure_element
+,	O
+H1	B-mutant
+-	I-mutant
+69	I-mutant
+has	O
+the	O
+greatest	O
+number	O
+of	O
+canonical	O
+structure	O
+assignments	O
+(	O
+Table	O
+2	O
+).	O
+
+The	O
+remaining	O
+8	O
+structures	B-evidence
+exhibit	O
+“	O
+non	O
+-	O
+parental	O
+”	O
+conformations	O
+,	O
+indicating	O
+that	O
+the	O
+VH	B-structure_element
+and	O
+VL	B-structure_element
+context	O
+can	O
+also	O
+be	O
+a	O
+dominating	O
+factor	O
+influencing	O
+CDR	B-structure_element
+H3	B-structure_element
+.	O
+
+Thus	O
+,	O
+no	O
+patterns	O
+of	O
+conformational	O
+preference	O
+for	O
+a	O
+particular	O
+HC	B-structure_element
+or	O
+LC	B-structure_element
+emerge	O
+to	O
+shed	O
+any	O
+direct	O
+light	O
+on	O
+what	O
+drives	O
+the	O
+conformational	O
+differences	O
+.	O
+
+One	O
+of	O
+the	O
+variants	O
+,	O
+H3	B-complex_assembly
+-	I-complex_assembly
+23	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+has	O
+the	O
+CDR	B-structure_element
+H3	B-structure_element
+conformation	O
+similar	O
+to	O
+the	O
+parent	O
+,	O
+but	O
+the	O
+other	O
+,	O
+H1	B-complex_assembly
+-	I-complex_assembly
+69	I-complex_assembly
+:	I-complex_assembly
+L3	I-complex_assembly
+-	I-complex_assembly
+20	I-complex_assembly
+,	O
+is	O
+different	O
+.	O
+
+These	O
+differences	O
+undoubtedly	O
+influence	O
+the	O
+conformation	O
+of	O
+the	O
+CDRs	B-structure_element
+,	O
+in	O
+particular	O
+CDR	B-structure_element
+H1	B-structure_element
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+and	O
+CDR	B-structure_element
+L1	B-structure_element
+(	O
+Fig	O
+.	O
+3C	O
+),	O
+especially	O
+with	O
+the	O
+tandem	O
+glycines	B-residue_name
+and	O
+multiple	O
+serines	B-residue_name
+present	O
+,	O
+respectively	O
+.	O
+
+Pairing	O
+of	O
+different	O
+germlines	O
+yields	O
+antibodies	B-protein_type
+with	O
+various	O
+degrees	O
+of	O
+stability	O
+.	O
+
+Other	O
+germlines	O
+have	O
+bulky	O
+residues	O
+,	O
+Tyr	B-residue_name
+,	O
+Arg	B-residue_name
+and	O
+Trp	B-residue_name
+,	O
+at	O
+these	O
+positions	O
+,	O
+whereas	O
+L1	B-mutant
+-	I-mutant
+39	I-mutant
+has	O
+Ser	B-residue_name
+and	O
+Thr	B-residue_name
+.	O
+
+The	O
+set	O
+of	O
+16	O
+germline	O
+Fab	B-structure_element
+structures	B-evidence
+offers	O
+a	O
+unique	O
+dataset	O
+to	O
+facilitate	O
+software	O
+development	O
+for	O
+antibody	B-protein_type
+modeling	O
+.	O
+
+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
+splice	B-site
+sites	I-site
+are	O
+marked	O
+by	O
+relatively	O
+short	B-structure_element
+consensus	I-structure_element
+sequences	I-structure_element
+and	O
+are	O
+regulated	O
+by	O
+additional	O
+pre	B-structure_element
+-	I-structure_element
+mRNA	I-structure_element
+motifs	I-structure_element
+(	O
+reviewed	O
+in	O
+ref	O
+.).	O
+
+The	O
+early	O
+-	O
+stage	O
+pre	B-protein_type
+-	I-protein_type
+mRNA	I-protein_type
+splicing	I-protein_type
+factor	I-protein_type
+U2AF65	B-protein
+is	O
+essential	O
+for	O
+viability	O
+in	O
+vertebrates	B-taxonomy_domain
+and	O
+other	O
+model	O
+organisms	O
+(	O
+for	O
+example	O
+,	O
+ref	O
+.).	O
+
+A	O
+tightly	O
+controlled	O
+assembly	B-complex_assembly
+among	O
+U2AF65	B-protein
+,	O
+the	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+,	O
+and	O
+partner	O
+proteins	O
+sequentially	O
+identifies	O
+the	O
+3	B-site
+′	I-site
+splice	I-site
+site	I-site
+and	O
+promotes	O
+association	O
+of	O
+the	O
+spliceosome	B-complex_assembly
+,	O
+which	O
+ultimately	O
+accomplishes	O
+the	O
+task	O
+of	O
+splicing	O
+.	O
+
+We	O
+use	O
+single	B-experimental_method
+-	I-experimental_method
+molecule	I-experimental_method
+Förster	I-experimental_method
+resonance	I-experimental_method
+energy	I-experimental_method
+transfer	I-experimental_method
+(	O
+smFRET	B-experimental_method
+)	O
+to	O
+characterize	O
+the	O
+conformational	B-evidence
+dynamics	I-evidence
+of	O
+this	O
+extended	B-protein_state
+U2AF65	B-structure_element
+–	I-structure_element
+RNA	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+during	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+recognition	O
+.	O
+
+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
+
+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
+
+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
+rU6	B-residue_name_number
+base	O
+edge	O
+is	O
+relatively	O
+solvent	B-protein_state
+exposed	I-protein_state
+;	O
+accordingly	O
+,	O
+the	O
+rU6	B-residue_name_number
+hydrogen	O
+bonds	O
+with	O
+U2AF65	B-protein
+are	O
+water	B-chemical
+mediated	O
+apart	O
+from	O
+a	O
+single	O
+direct	O
+interaction	O
+by	O
+the	O
+RRM1	B-structure_element
+-	O
+N196	B-residue_name_number
+side	O
+chain	O
+.	O
+
+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
+
+Despite	O
+12	B-experimental_method
+concurrent	I-experimental_method
+mutations	I-experimental_method
+,	O
+the	O
+AdML	B-gene
+RNA	B-evidence
+affinity	I-evidence
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+-	I-mutant
+12Gly	I-mutant
+variant	B-protein_state
+was	O
+reduced	O
+by	O
+only	O
+three	O
+-	O
+fold	O
+relative	O
+to	O
+the	O
+unmodified	B-protein_state
+protein	B-protein
+(	O
+Fig	O
+.	O
+4b	O
+),	O
+which	O
+is	O
+less	O
+than	O
+the	O
+penalty	O
+of	O
+the	O
+V254P	B-mutant
+mutation	O
+that	O
+disrupts	O
+the	O
+rU5	B-residue_name_number
+hydrogen	O
+bond	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+i	O
+).	O
+
+This	O
+difference	O
+indicates	O
+that	O
+the	O
+linearly	B-protein_state
+distant	I-protein_state
+regions	B-structure_element
+of	O
+the	O
+U2AF65	B-protein
+primary	O
+sequence	O
+,	O
+including	O
+Q147	B-residue_name_number
+in	O
+the	O
+N	O
+-	O
+terminal	O
+RRM1	B-structure_element
+extension	I-structure_element
+and	O
+R227	B-residue_name_number
+/	O
+V254	B-residue_name_number
+in	O
+the	O
+N	O
+-/	O
+C	O
+-	O
+terminal	O
+linker	B-structure_element
+regions	I-structure_element
+at	O
+the	O
+fifth	B-site
+nucleotide	I-site
+site	I-site
+,	O
+cooperatively	O
+recognize	O
+the	O
+Py	B-chemical
+tract	I-chemical
+.	O
+
+When	O
+transfected	B-experimental_method
+into	O
+HEK293T	O
+cells	O
+containing	O
+only	O
+endogenous	B-protein_state
+U2AF65	B-protein
+,	O
+the	O
+PY	B-site
+splice	I-site
+site	I-site
+is	O
+used	O
+and	O
+the	O
+remaining	O
+transcript	O
+remains	O
+unspliced	O
+.	O
+
+Co	B-experimental_method
+-	I-experimental_method
+transfection	I-experimental_method
+of	O
+the	O
+U2AF65	B-mutant
+-	I-mutant
+3Mut	I-mutant
+with	O
+the	O
+pyPY	B-chemical
+splicing	O
+substrate	O
+significantly	O
+reduced	O
+splicing	O
+of	O
+the	O
+weak	O
+‘	B-site
+py	I-site
+'	I-site
+splice	I-site
+site	I-site
+relative	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+U2AF65	B-protein
+(	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+
+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
+
+To	O
+complement	O
+the	O
+static	O
+portraits	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	B-evidence
+that	O
+we	O
+had	O
+determined	O
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+,	O
+we	O
+used	O
+smFRET	B-experimental_method
+to	O
+characterize	O
+the	O
+probability	B-evidence
+distribution	I-evidence
+functions	I-evidence
+and	O
+time	O
+dependence	O
+of	O
+U2AF65	B-protein
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+conformational	O
+dynamics	O
+in	O
+solution	O
+.	O
+
+The	O
+positions	O
+of	O
+single	O
+cysteine	B-residue_name
+mutations	B-experimental_method
+for	O
+fluorophore	B-chemical
+attachment	O
+(	O
+A181C	B-mutant
+in	O
+RRM1	B-structure_element
+and	O
+Q324C	B-mutant
+in	O
+RRM2	B-structure_element
+)	O
+were	O
+chosen	O
+based	O
+on	O
+inspection	O
+of	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structures	B-evidence
+and	O
+the	O
+‘	O
+closed	B-protein_state
+'	O
+model	O
+of	O
+apo	B-protein_state
+-	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+.	O
+
+Approximately	O
+70	O
+%	O
+of	O
+observed	O
+fluctuations	O
+were	O
+interchanges	O
+between	O
+the	O
+∼	O
+0	O
+.	O
+65	O
+and	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+values	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+7b	O
+).	O
+
+However	O
+,	O
+the	O
+presence	O
+of	O
+repetitive	O
+fluctuations	O
+between	O
+particular	O
+FRET	B-evidence
+values	I-evidence
+supports	O
+the	O
+hypothesis	O
+that	O
+RNA	B-protein_state
+-	I-protein_state
+free	I-protein_state
+U2AF65	B-protein
+samples	O
+several	O
+distinct	O
+conformations	O
+.	O
+
+U2AF65	B-protein
+conformational	O
+selection	O
+and	O
+induced	O
+fit	O
+by	O
+bound	B-protein_state
+RNA	B-chemical
+
+Insertion	B-experimental_method
+of	O
+adenine	B-chemical
+nucleotides	I-chemical
+decreased	O
+binding	B-evidence
+affinity	I-evidence
+of	O
+U2AF65	B-protein
+to	O
+RNA	B-chemical
+by	O
+approximately	O
+five	O
+-	O
+fold	O
+.	O
+
+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
+
+Residues	O
+V249	B-residue_name_number
+,	O
+V250	B-residue_name_number
+,	O
+V254	B-residue_name_number
+(	O
+yellow	O
+)	O
+are	O
+mutated	B-experimental_method
+to	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+V254G	B-mutant
+in	O
+the	O
+3Gly	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+P255	B-residue_name_number
+(	O
+red	O
+)	O
+along	O
+with	O
+V254	B-residue_name_number
+are	O
+mutated	B-experimental_method
+to	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+5Gly	B-mutant
+mutant	I-mutant
+or	O
+to	O
+S251N	B-mutant
+/	O
+T252L	B-mutant
+/	O
+V253A	B-mutant
+/	O
+V254L	B-mutant
+/	O
+P255A	B-mutant
+in	O
+the	O
+NLALA	B-mutant
+mutant	I-mutant
+;	O
+residues	O
+M144	B-residue_name_number
+,	O
+L235	B-residue_name_number
+,	O
+M238	B-residue_name_number
+,	O
+V244	B-residue_name_number
+,	O
+V246	B-residue_name_number
+(	O
+orange	O
+)	O
+along	O
+with	O
+V249	B-residue_name_number
+,	O
+V250	B-residue_name_number
+,	O
+S251	B-residue_name_number
+,	O
+T252	B-residue_name_number
+,	O
+V253	B-residue_name_number
+,	O
+V254	B-residue_name_number
+,	O
+P255	B-residue_name_number
+are	O
+mutated	B-experimental_method
+to	O
+M144G	B-mutant
+/	O
+L235G	B-mutant
+/	O
+M238G	B-mutant
+/	O
+V244G	B-mutant
+/	O
+V246G	B-mutant
+/	O
+V249G	B-mutant
+/	O
+V250G	B-mutant
+/	O
+S251G	B-mutant
+/	O
+T252G	B-mutant
+/	O
+V253G	B-mutant
+/	O
+V254G	B-mutant
+/	O
+P255G	B-mutant
+in	O
+the	O
+12Gly	B-mutant
+mutant	I-mutant
+.	O
+
+Other	O
+linker	B-structure_element
+residues	O
+are	O
+coloured	O
+either	O
+dark	O
+blue	O
+for	O
+new	O
+residues	O
+in	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+structure	O
+or	O
+light	O
+blue	O
+for	O
+the	O
+remaining	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+residues	O
+.	O
+
+The	O
+central	O
+panel	O
+shows	O
+an	O
+overall	O
+view	O
+with	O
+stick	O
+diagrams	O
+for	O
+mutated	O
+residues	O
+;	O
+boxed	O
+regions	O
+are	O
+expanded	O
+to	O
+show	O
+the	O
+C	O
+-	O
+terminal	O
+(	O
+bottom	O
+left	O
+)	O
+and	O
+central	B-structure_element
+linker	I-structure_element
+regions	I-structure_element
+(	O
+top	O
+)	O
+at	O
+the	O
+inter	B-structure_element
+-	I-structure_element
+RRM	I-structure_element
+interfaces	I-structure_element
+,	O
+and	O
+N	O
+-	O
+terminal	O
+linker	O
+region	O
+contacts	O
+with	O
+RRM1	B-structure_element
+(	O
+bottom	O
+right	O
+).	O
+
+The	O
+fitted	O
+fluorescence	O
+anisotropy	O
+RNA	B-evidence
+-	I-evidence
+binding	I-evidence
+curves	I-evidence
+are	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4d	O
+–	O
+j	O
+.	O
+(	O
+c	O
+)	O
+Close	O
+view	O
+of	O
+the	O
+U2AF65	B-protein
+RRM1	B-site
+/	I-site
+RRM2	I-site
+interface	I-site
+following	O
+a	O
+two	O
+-	O
+fold	O
+rotation	O
+about	O
+the	O
+x	O
+-	O
+axis	O
+relative	O
+to	O
+a	O
+.	O
+
+(	O
+a	O
+,	O
+b	O
+)	O
+Views	O
+of	O
+FRET	B-experimental_method
+pairs	O
+chosen	O
+to	O
+follow	O
+the	O
+relative	O
+movement	O
+of	O
+RRM1	B-structure_element
+and	O
+RRM2	B-structure_element
+on	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+‘	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+'	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+RRMs	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+a	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+oligonucleotide	I-chemical
+(	O
+a	O
+,	O
+representative	O
+structure	O
+iv	O
+)	O
+or	O
+‘	O
+closed	B-protein_state
+'	O
+NMR	B-experimental_method
+/	O
+PRE	B-experimental_method
+-	O
+based	O
+model	O
+of	O
+U2AF651	B-mutant
+,	I-mutant
+2	I-mutant
+(	O
+b	O
+,	O
+PDB	O
+ID	O
+2YH0	O
+)	O
+in	O
+identical	O
+orientations	O
+of	O
+RRM2	B-structure_element
+.	O
+
+(	O
+c	O
+–	O
+f	O
+,	O
+i	O
+,	O
+j	O
+)	O
+The	O
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+protein	O
+was	O
+immobilized	O
+on	O
+the	O
+microscope	O
+slide	O
+via	O
+biotin	B-chemical
+-	I-chemical
+NTA	I-chemical
+/	I-chemical
+Ni	I-chemical
++	I-chemical
+2	I-chemical
+(	O
+orange	O
+line	O
+)	O
+on	O
+a	O
+neutravidin	O
+(	O
+black	O
+X	O
+)-	O
+biotin	O
+-	O
+PEG	O
+(	O
+orange	O
+triangle	O
+)-	O
+treated	O
+surface	O
+and	O
+imaged	O
+either	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ligands	B-chemical
+(	O
+c	O
+,	O
+d	O
+),	O
+in	O
+the	O
+presence	O
+of	O
+5	O
+μM	O
+AdML	B-gene
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+RNA	I-chemical
+(	O
+5	B-chemical
+′-	I-chemical
+CCUUUUUUUUCC	I-chemical
+-	I-chemical
+3	I-chemical
+′)	I-chemical
+(	O
+e	O
+,	O
+f	O
+),	O
+or	O
+in	O
+the	O
+presence	O
+of	O
+10	O
+μM	O
+adenosine	B-residue_name
+-	O
+interrupted	O
+variant	O
+RNA	B-chemical
+(	O
+5	B-chemical
+′-	I-chemical
+CUUUUUAAUUUCCA	I-chemical
+-	I-chemical
+3	I-chemical
+′)	I-chemical
+(	O
+i	O
+,	O
+j	O
+).	O
+
+The	O
+untethered	B-protein_state
+U2AF651	B-mutant
+,	I-mutant
+2LFRET	I-mutant
+(	O
+Cy3	B-chemical
+/	O
+Cy5	B-chemical
+)	O
+protein	O
+(	O
+1	O
+nM	O
+)	O
+was	O
+added	O
+to	O
+AdML	B-gene
+RNA	B-chemical
+–	I-chemical
+polyethylene	I-chemical
+-	I-chemical
+glycol	I-chemical
+-	I-chemical
+linker	I-chemical
+–	I-chemical
+DNA	I-chemical
+oligonucleotide	I-chemical
+(	O
+10	O
+nM	O
+),	O
+which	O
+was	O
+immobilized	O
+on	O
+the	O
+microscope	O
+slide	O
+by	O
+annealing	O
+with	O
+a	O
+complementary	O
+biotinyl	B-chemical
+-	I-chemical
+DNA	I-chemical
+oligonucleotide	I-chemical
+(	O
+black	O
+vertical	O
+line	O
+).	O
+
+(	O
+b	O
+)	O
+Following	O
+binding	O
+to	O
+the	O
+Py	B-chemical
+-	I-chemical
+tract	I-chemical
+RNA	I-chemical
+,	O
+a	O
+conformation	O
+corresponding	O
+to	O
+high	B-evidence
+FRET	I-evidence
+and	O
+consistent	O
+with	O
+the	O
+‘	O
+closed	B-protein_state
+',	O
+back	B-protein_state
+-	I-protein_state
+to	I-protein_state
+-	I-protein_state
+back	I-protein_state
+apo	B-protein_state
+-	O
+U2AF65	B-protein
+model	O
+resulting	O
+from	O
+PRE	B-experimental_method
+/	O
+NMR	B-experimental_method
+characterization	O
+(	O
+PDB	O
+ID	O
+2YH0	O
+)	O
+often	O
+transitions	O
+to	O
+a	O
+conformation	O
+corresponding	O
+to	O
+∼	O
+0	O
+.	O
+45	O
+FRET	B-evidence
+value	I-evidence
+,	O
+which	O
+is	O
+consistent	O
+with	O
+‘	O
+open	B-protein_state
+',	O
+side	B-protein_state
+-	I-protein_state
+by	I-protein_state
+-	I-protein_state
+side	I-protein_state
+RRMs	B-structure_element
+such	O
+as	O
+the	O
+U2AF651	B-mutant
+,	I-mutant
+2L	I-mutant
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+RRM1	B-structure_element
+,	O
+green	O
+;	O
+RRM2	B-structure_element
+,	O
+pale	O
+blue	O
+;	O
+RRM	B-structure_element
+extensions	I-structure_element
+/	O
+linker	B-structure_element
+,	O
+blue	O
+.	O
+
+Systematic	O
+analysis	O
+of	O
+radiation	O
+damage	O
+within	O
+a	O
+protein	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+MGy	O
+)	O
+reveals	O
+significant	O
+differential	O
+susceptibility	O
+of	O
+RNA	B-chemical
+and	O
+protein	O
+.	O
+
+With	O
+the	O
+wide	O
+use	O
+of	O
+high	O
+-	O
+flux	O
+third	O
+-	O
+generation	O
+synchrotron	O
+sources	O
+,	O
+radiation	O
+damage	O
+(	O
+RD	O
+)	O
+has	O
+once	O
+again	O
+become	O
+a	O
+dominant	O
+reason	O
+for	O
+the	O
+failure	O
+of	O
+structure	B-experimental_method
+determination	I-experimental_method
+using	O
+macromolecular	B-experimental_method
+crystallography	I-experimental_method
+(	O
+MX	B-experimental_method
+)	O
+in	O
+experiments	O
+conducted	O
+both	O
+at	O
+room	O
+temperature	O
+and	O
+under	O
+cryocooled	O
+conditions	O
+(	O
+100	O
+K	O
+).	O
+
+Significant	O
+progress	O
+has	O
+been	O
+made	O
+in	O
+recent	O
+years	O
+in	O
+understanding	O
+the	O
+inevitable	O
+manifestations	O
+of	O
+X	O
+-	O
+ray	O
+-	O
+induced	O
+RD	O
+within	O
+protein	O
+crystals	B-evidence
+,	O
+and	O
+there	O
+is	O
+now	O
+a	O
+body	O
+of	O
+literature	O
+on	O
+possible	O
+strategies	O
+to	O
+mitigate	O
+the	O
+effects	O
+of	O
+RD	O
+(	O
+e	O
+.	O
+g	O
+.	O
+Zeldin	O
+,	O
+Brockhauser	O
+et	O
+al	O
+.,	O
+2013	O
+;	O
+Bourenkov	O
+&	O
+Popov	O
+,	O
+2010	O
+).	O
+
+There	O
+are	O
+a	O
+number	O
+of	O
+cases	O
+where	O
+SRD	O
+manifestations	O
+have	O
+compromised	O
+the	O
+biological	O
+information	O
+extracted	O
+from	O
+MX	B-experimental_method
+-	I-experimental_method
+determined	I-experimental_method
+structures	B-evidence
+at	O
+much	O
+lower	O
+doses	O
+than	O
+the	O
+recommended	O
+30	O
+MGy	O
+limit	O
+,	O
+leading	O
+to	O
+false	O
+structural	O
+interpretations	O
+of	O
+protein	O
+mechanisms	O
+.	O
+
+Understanding	O
+RD	O
+to	O
+such	O
+complexes	O
+is	O
+crucial	O
+,	O
+since	O
+DNA	B-chemical
+is	O
+rarely	O
+naked	O
+within	O
+a	O
+cell	O
+,	O
+instead	O
+dynamically	O
+interacting	O
+with	O
+proteins	O
+,	O
+facilitating	O
+replication	O
+,	O
+transcription	O
+,	O
+modification	O
+and	O
+DNA	B-chemical
+repair	O
+.	O
+
+Nucleoproteins	B-complex_assembly
+also	O
+represent	O
+one	O
+of	O
+the	O
+main	O
+targets	O
+of	O
+radiotherapy	O
+,	O
+and	O
+an	O
+insight	O
+into	O
+the	O
+damage	O
+mechanisms	O
+induced	O
+by	O
+X	O
+-	O
+ray	O
+irradiation	O
+could	O
+inform	O
+innovative	O
+treatments	O
+.	O
+
+Investigations	O
+on	O
+sub	O
+-	O
+ionization	O
+-	O
+level	O
+LEEs	O
+(	O
+0	O
+–	O
+15	O
+eV	O
+)	O
+interacting	O
+with	O
+both	O
+dried	O
+and	O
+aqueous	O
+oligonucleotides	O
+(	O
+Alizadeh	O
+&	O
+Sanche	O
+,	O
+2014	O
+;	O
+Simons	O
+,	O
+2006	O
+)	O
+concluded	O
+that	O
+resonant	O
+electron	O
+attachment	O
+to	O
+DNA	B-chemical
+bases	O
+and	O
+the	O
+sugar	O
+-	O
+phosphate	O
+backbone	O
+could	O
+lead	O
+to	O
+the	O
+preferential	O
+cleavage	O
+of	O
+strong	O
+(∼	O
+4	O
+eV	O
+,	O
+385	O
+kJ	O
+mol	O
+−	O
+1	O
+)	O
+sugar	O
+-	O
+phosphate	O
+C	O
+—	O
+O	O
+covalent	O
+bonds	O
+within	O
+the	O
+DNA	B-chemical
+backbone	O
+and	O
+then	O
+base	O
+-	O
+sugar	O
+N1	O
+—	O
+C	O
+bonds	O
+,	O
+eventually	O
+leading	O
+to	O
+single	O
+-	O
+strand	O
+breakages	O
+(	O
+SSBs	O
+;	O
+Ptasińska	O
+&	O
+Sanche	O
+,	O
+2007	O
+).	O
+
+To	O
+avoid	O
+the	O
+previous	O
+necessity	O
+for	O
+visual	O
+inspection	O
+of	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+maps	I-evidence
+to	O
+detect	O
+SRD	B-site
+sites	I-site
+,	O
+a	O
+computational	O
+approach	O
+was	O
+designed	O
+to	O
+quantify	O
+the	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+change	I-evidence
+for	O
+each	O
+refined	O
+atom	O
+with	O
+increasing	O
+dose	O
+,	O
+thus	O
+providing	O
+a	O
+rapid	O
+systematic	O
+method	O
+for	O
+SRD	O
+study	O
+on	O
+such	O
+large	O
+multimeric	O
+complexes	O
+.	O
+
+To	O
+quantify	O
+the	O
+exact	O
+effects	O
+of	O
+nucleic	O
+acid	O
+binding	O
+to	O
+a	O
+protein	O
+on	O
+SRD	O
+susceptibility	O
+,	O
+a	O
+high	O
+-	O
+throughput	O
+and	O
+automated	O
+pipeline	O
+was	O
+created	O
+to	O
+systematically	O
+calculate	O
+the	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+change	I-evidence
+for	O
+every	O
+refined	O
+atom	O
+within	O
+the	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+structure	B-evidence
+as	O
+a	O
+function	O
+of	O
+dose	O
+.	O
+
+This	O
+provides	O
+an	O
+atom	O
+-	O
+specific	O
+quantification	O
+of	O
+density	B-evidence
+–	I-evidence
+dose	I-evidence
+dynamics	I-evidence
+,	O
+which	O
+was	O
+previously	O
+lacking	O
+within	O
+the	O
+field	O
+.	O
+
+However	O
+,	O
+these	O
+σ	B-evidence
+levels	O
+depend	O
+on	O
+the	O
+standard	B-evidence
+deviation	I-evidence
+values	O
+of	O
+the	O
+map	B-evidence
+,	O
+which	O
+can	O
+deviate	O
+between	O
+data	O
+sets	O
+,	O
+and	O
+are	O
+thus	O
+unsuitable	O
+for	O
+quantitative	O
+comparison	O
+of	O
+density	B-evidence
+between	O
+different	O
+dose	O
+data	O
+sets	O
+.	O
+
+The	O
+rate	O
+of	O
+D	B-evidence
+loss	I-evidence
+(	O
+attributed	O
+to	O
+side	O
+-	O
+chain	O
+decarboxylation	O
+)	O
+was	O
+consistently	O
+larger	O
+for	O
+Glu	B-residue_name
+compared	O
+with	O
+Asp	B-residue_name
+residues	O
+over	O
+the	O
+large	O
+dose	O
+range	O
+(	O
+Fig	O
+.	O
+2	O
+▸	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+S3	O
+);	O
+this	O
+observation	O
+is	O
+consistent	O
+with	O
+our	O
+calculations	O
+on	O
+model	O
+systems	O
+(	O
+see	O
+above	O
+)	O
+that	O
+suggest	O
+that	O
+,	O
+without	O
+considering	O
+differential	O
+hydrogen	O
+-	O
+bonding	O
+environments	O
+,	O
+CO2	B-chemical
+loss	O
+is	O
+more	O
+exothermic	O
+by	O
+around	O
+8	O
+kJ	O
+mol	O
+−	O
+1	O
+from	O
+oxidized	B-protein_state
+Glu	B-residue_name
+residues	O
+than	O
+from	O
+their	O
+Asp	B-residue_name
+counterparts	O
+.	O
+
+In	O
+our	O
+analysis	O
+,	O
+Asp39	B-residue_name_number
+in	O
+the	O
+TRAP	B-complex_assembly
+–(	I-complex_assembly
+GAGUU	I-complex_assembly
+)	I-complex_assembly
+10GAG	I-complex_assembly
+structure	B-evidence
+appears	O
+to	O
+exhibit	O
+two	O
+distinct	O
+hydrogen	O
+bonds	O
+to	O
+the	O
+G1	B-residue_name_number
+base	O
+within	O
+each	O
+of	O
+the	O
+11	O
+TRAP	B-site
+–	I-site
+RNA	I-site
+interfaces	I-site
+,	O
+as	O
+does	O
+Glu36	B-residue_name_number
+to	O
+G3	B-residue_name_number
+;	O
+however	O
+,	O
+the	O
+reduction	O
+in	O
+density	B-evidence
+disordering	O
+upon	O
+RNA	B-chemical
+binding	O
+is	O
+far	O
+less	O
+significant	O
+for	O
+Asp39	B-residue_name_number
+than	O
+for	O
+Glu36	B-residue_name_number
+(	O
+Fig	O
+.	O
+5	O
+▸	O
+b	O
+,	O
+p	O
+=	O
+0	O
+.	O
+0925	O
+).	O
+
+One	O
+oxygen	O
+(	O
+O	O
+∊	O
+1	O
+)	O
+of	O
+Glu42	B-residue_name_number
+appears	O
+to	O
+form	O
+a	O
+hydrogen	O
+bond	O
+to	O
+a	O
+nearby	O
+water	B-chemical
+within	O
+each	O
+TRAP	B-site
+RNA	I-site
+-	I-site
+binding	I-site
+pocket	I-site
+,	O
+with	O
+the	O
+other	O
+(	O
+O	O
+∊	O
+2	O
+)	O
+being	O
+involved	O
+in	O
+a	O
+salt	O
+-	O
+bridge	O
+interaction	O
+with	O
+Arg58	B-residue_name_number
+(	O
+Hopcroft	O
+et	O
+al	O
+.,	O
+2002	O
+;	O
+Antson	O
+et	O
+al	O
+.,	O
+1999	O
+).	O
+
+The	O
+density	B-evidence
+-	I-evidence
+change	I-evidence
+dynamics	I-evidence
+were	O
+statistically	O
+indistinguishable	O
+between	O
+bound	B-protein_state
+and	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+for	O
+each	O
+Glu42	B-residue_name_number
+carboxyl	O
+group	O
+Cδ	O
+atom	O
+(	O
+p	O
+=	O
+0	O
+.	O
+435	O
+),	O
+indicating	O
+that	O
+upon	O
+RNA	B-chemical
+binding	O
+the	O
+conserved	O
+salt	O
+-	O
+bridge	O
+interaction	O
+ultimately	O
+dictated	O
+the	O
+overall	O
+Glu42	B-residue_name_number
+decarboxylation	O
+rate	O
+.	O
+
+The	O
+RNA	B-chemical
+-	O
+stabilizing	O
+effect	O
+was	O
+not	O
+restricted	O
+to	O
+radiation	O
+-	O
+sensitive	O
+acidic	O
+residues	O
+.	O
+
+Here	O
+,	O
+MX	B-experimental_method
+radiation	O
+-	O
+induced	O
+specific	O
+structural	O
+changes	O
+within	O
+the	O
+large	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+assembly	O
+over	O
+a	O
+large	O
+dose	O
+range	O
+(	O
+1	O
+.	O
+3	O
+–	O
+25	O
+.	O
+0	O
+MGy	O
+)	O
+have	O
+been	O
+analysed	O
+using	O
+a	O
+high	O
+-	O
+throughput	O
+quantitative	O
+approach	O
+,	O
+providing	O
+a	O
+measure	O
+of	O
+the	O
+electron	B-evidence
+-	I-evidence
+density	I-evidence
+distribution	I-evidence
+for	O
+each	O
+refined	O
+atom	O
+with	O
+increasing	O
+dose	O
+,	O
+D	B-evidence
+loss	I-evidence
+.	O
+
+The	O
+RNA	B-chemical
+was	O
+found	O
+to	O
+be	O
+substantially	O
+more	O
+radiation	B-protein_state
+-	I-protein_state
+resistant	I-protein_state
+than	O
+the	O
+protein	O
+,	O
+even	O
+at	O
+the	O
+highest	O
+doses	O
+investigated	O
+(∼	O
+25	O
+.	O
+0	O
+MGy	O
+),	O
+which	O
+is	O
+in	O
+strong	O
+concurrence	O
+with	O
+our	O
+previous	O
+SRD	B-experimental_method
+investigation	I-experimental_method
+of	O
+the	O
+C	B-complex_assembly
+.	I-complex_assembly
+Esp1396I	I-complex_assembly
+protein	O
+–	O
+DNA	B-chemical
+complex	O
+(	O
+Bury	O
+et	O
+al	O
+.,	O
+2015	O
+).	O
+
+For	O
+example	O
+,	O
+Asp17	B-residue_name_number
+is	O
+located	O
+∼	O
+6	O
+.	O
+8	O
+Å	O
+from	O
+the	O
+G1	B-residue_name_number
+base	O
+,	O
+outside	O
+the	O
+RNA	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+,	O
+and	O
+has	O
+indistinguishable	O
+Cγ	O
+atom	O
+D	O
+loss	B-evidence
+dose	I-evidence
+-	I-evidence
+dynamics	I-evidence
+between	O
+RNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+and	O
+nonbound	B-protein_state
+TRAP	B-complex_assembly
+(	O
+p	O
+>	O
+0	O
+.	O
+9	O
+).	O
+
+However	O
+,	O
+in	O
+the	O
+current	O
+MX	B-experimental_method
+study	O
+at	O
+100	O
+K	O
+,	O
+the	O
+main	O
+damaging	O
+species	O
+are	O
+believed	O
+to	O
+be	O
+migrating	O
+LEEs	O
+and	O
+holes	O
+produced	O
+directly	O
+within	O
+the	O
+protein	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+components	O
+or	O
+in	O
+closely	O
+associated	O
+solvent	O
+.	O
+
+The	O
+results	O
+presented	O
+here	O
+suggest	O
+that	O
+biologically	O
+relevant	O
+nucleoprotein	B-complex_assembly
+complexes	O
+also	O
+exhibit	O
+prolonged	O
+life	O
+-	O
+doses	O
+under	O
+the	O
+effect	O
+of	O
+LEE	O
+-	O
+induced	O
+structural	O
+changes	O
+,	O
+involving	O
+direct	O
+physical	O
+protection	O
+of	O
+key	O
+RNA	B-site
+-	I-site
+binding	I-site
+residues	I-site
+.	O
+
+Such	O
+reduced	O
+radiation	O
+-	O
+sensitivity	O
+in	O
+this	O
+case	O
+ensures	O
+that	O
+the	O
+interacting	O
+protein	O
+remains	O
+bound	B-protein_state
+long	O
+enough	O
+to	O
+the	O
+RNA	B-chemical
+to	O
+complete	O
+its	O
+function	O
+,	O
+even	O
+whilst	O
+exposed	O
+to	O
+ionizing	O
+radiation	O
+.	O
+
+RNA	B-chemical
+is	O
+shown	O
+is	O
+yellow	O
+.	O
+
+Only	O
+a	O
+subset	O
+of	O
+key	O
+TRAP	B-complex_assembly
+residue	O
+types	O
+are	O
+included	O
+.	O
+
+D	O
+loss	O
+calculated	O
+for	O
+all	O
+side	O
+-	O
+chain	O
+carboxyl	O
+group	O
+Glu	B-residue_name
+Cδ	O
+and	O
+Asp	B-residue_name
+Cγ	O
+atoms	O
+within	O
+the	O
+TRAP	B-complex_assembly
+–	I-complex_assembly
+RNA	I-complex_assembly
+complex	O
+for	O
+a	O
+dose	O
+of	O
+19	O
+.	O
+3	O
+MGy	O
+(	O
+d	O
+8	O
+).	O
+
+Another	O
+challenge	O
+will	O
+be	O
+to	O
+find	O
+out	O
+where	O
+IDA	B-protein
+is	O
+produced	O
+in	O
+the	O
+plant	B-taxonomy_domain
+and	O
+what	O
+causes	O
+it	O
+to	O
+accumulate	O
+in	O
+specific	O
+places	O
+in	O
+preparation	O
+for	O
+organ	O
+shedding	O
+.	O
+
+The	O
+HAESA	B-protein
+ectodomain	B-structure_element
+folds	O
+into	O
+a	O
+superhelical	B-structure_element
+assembly	I-structure_element
+of	O
+21	O
+leucine	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+repeats	I-structure_element
+.	O
+
+(	O
+A	O
+)	O
+Details	O
+of	O
+the	O
+IDA	B-site
+binding	I-site
+pocket	I-site
+.	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
+
+The	O
+peptide	B-site
+binding	I-site
+pocket	I-site
+covers	O
+HAESA	B-protein
+LRRs	B-structure_element
+2	I-structure_element
+–	I-structure_element
+14	I-structure_element
+.	O
+(	O
+D	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+entire	O
+IDA	B-protein
+(	O
+in	O
+yellow	O
+)	O
+peptide	B-site
+binding	I-site
+site	I-site
+in	O
+HAESA	B-protein
+(	O
+in	O
+blue	O
+).	O
+
+Hydrogren	O
+bonds	O
+are	O
+depicted	O
+as	O
+dotted	O
+lines	O
+(	O
+in	O
+magenta	O
+),	O
+a	O
+water	B-chemical
+molecule	O
+is	O
+shown	O
+as	O
+a	O
+red	O
+sphere	O
+.	O
+
+This	B-structure_element
+sequence	I-structure_element
+motif	I-structure_element
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+among	O
+IDA	B-protein_type
+family	I-protein_type
+members	I-protein_type
+(	O
+IDA	B-protein_type
+-	I-protein_type
+LIKE	I-protein_type
+PROTEINS	I-protein_type
+,	O
+IDLs	B-protein_type
+)	O
+and	O
+contains	O
+a	O
+central	O
+Pro	B-residue_name
+residue	O
+,	O
+presumed	O
+to	O
+be	O
+post	B-protein_state
+-	I-protein_state
+translationally	I-protein_state
+modified	I-protein_state
+to	O
+hydroxyproline	B-residue_name
+(	O
+Hyp	B-residue_name
+;	O
+Figure	O
+1A	O
+).	O
+
+Active	B-protein_state
+IDA	B-protein_type
+-	I-protein_type
+family	I-protein_type
+peptide	I-protein_type
+hormones	I-protein_type
+are	O
+hydroxyprolinated	B-protein_state
+dodecamers	B-structure_element
+.	O
+
+no	O
+detectable	O
+binding	O
+).	O
+(	O
+E	O
+)	O
+Structural	B-experimental_method
+superposition	I-experimental_method
+of	O
+the	O
+active	B-protein_state
+IDA	B-protein
+(	O
+in	O
+bonds	O
+representation	O
+,	O
+in	O
+gray	O
+)	O
+and	O
+IDL1	B-chemical
+peptide	I-chemical
+(	O
+in	O
+yellow	O
+)	O
+hormones	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+
+Petal	O
+break	O
+-	O
+strength	O
+was	O
+found	O
+significantly	O
+increased	O
+in	O
+almost	O
+all	O
+positions	O
+(	O
+indicated	O
+with	O
+a	O
+*)	O
+for	O
+haesa	B-gene
+/	O
+hsl2	B-gene
+and	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+mutant	B-protein_state
+plants	B-taxonomy_domain
+with	O
+respect	O
+to	O
+the	O
+Col	O
+-	O
+0	O
+control	O
+.	O
+
+(	O
+B	O
+)	O
+Analytical	B-experimental_method
+size	I-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+.	O
+
+The	O
+central	O
+Hyp64IDA	B-residue_name_number
+is	O
+buried	O
+in	O
+a	O
+specific	O
+pocket	B-site
+formed	O
+by	O
+HAESA	B-protein
+LRRs	B-structure_element
+8	I-structure_element
+–	I-structure_element
+10	I-structure_element
+,	O
+with	O
+its	O
+hydroxyl	O
+group	O
+establishing	O
+hydrogen	O
+bonds	O
+with	O
+the	O
+strictly	B-protein_state
+conserved	I-protein_state
+Glu266HAESA	B-residue_name_number
+and	O
+with	O
+a	O
+water	B-chemical
+molecule	O
+,	O
+which	O
+in	O
+turn	O
+is	O
+coordinated	O
+by	O
+the	O
+main	O
+chain	O
+oxygens	O
+of	O
+Phe289HAESA	B-residue_name_number
+and	O
+Ser311HAESA	B-residue_name_number
+(	O
+Figure	O
+1E	O
+;	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+3	O
+).	O
+
+In	O
+this	O
+structure	B-evidence
+,	O
+no	O
+additional	O
+electron	B-evidence
+density	I-evidence
+accounts	O
+for	O
+the	O
+PKGV	B-structure_element
+motif	I-structure_element
+at	O
+the	O
+IDA	B-protein
+N	O
+-	O
+terminus	O
+(	O
+Figure	O
+2A	O
+,	O
+B	O
+).	O
+
+We	O
+do	O
+not	O
+detect	O
+interaction	O
+between	O
+HAESA	B-protein
+and	O
+a	O
+synthetic	B-protein_state
+peptide	B-chemical
+missing	B-protein_state
+the	I-protein_state
+C	I-protein_state
+-	I-protein_state
+terminal	I-protein_state
+Asn69IDA	B-residue_name_number
+(	O
+ΔN69	B-mutant
+),	O
+highlighting	O
+the	O
+importance	O
+of	O
+the	O
+polar	O
+interactions	O
+between	O
+the	O
+IDA	B-protein
+carboxy	O
+-	O
+terminus	O
+and	O
+Arg407HAESA	B-residue_name_number
+/	O
+Arg409HAESA	B-residue_name_number
+(	O
+Figures	O
+1F	O
+,	O
+2D	O
+).	O
+
+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
+
+We	O
+found	O
+that	O
+the	O
+force	O
+required	O
+to	O
+remove	O
+the	O
+petals	O
+of	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+mutants	B-protein_state
+is	O
+significantly	O
+higher	O
+than	O
+that	O
+needed	O
+for	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+plants	B-taxonomy_domain
+,	O
+as	O
+previously	O
+observed	O
+for	O
+haesa	B-gene
+/	O
+hsl2	B-gene
+mutants	B-protein_state
+,	O
+and	O
+that	O
+floral	O
+abscission	O
+is	O
+delayed	O
+in	O
+serk1	B-gene
+-	I-gene
+1	I-gene
+(	O
+Figure	O
+3A	O
+).	O
+
+In	O
+vitro	O
+,	O
+the	O
+LRR	B-structure_element
+ectodomain	I-structure_element
+of	O
+SERK1	B-protein
+(	O
+residues	O
+24	B-residue_range
+–	I-residue_range
+213	I-residue_range
+)	O
+forms	O
+stable	B-protein_state
+,	O
+IDA	B-protein_state
+-	I-protein_state
+dependent	I-protein_state
+heterodimeric	B-oligomeric_state
+complexes	B-protein_state
+with	I-protein_state
+HAESA	B-protein
+in	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+experiments	O
+(	O
+Figure	O
+3B	O
+).	O
+
+We	O
+next	O
+titrated	B-experimental_method
+SERK1	B-protein
+into	O
+a	O
+solution	O
+containing	O
+only	O
+the	O
+HAESA	B-protein
+ectodomain	B-structure_element
+.	O
+
+In	O
+this	O
+case	O
+,	O
+there	O
+was	O
+no	O
+detectable	O
+interaction	O
+between	O
+receptor	O
+and	O
+co	O
+-	O
+receptor	O
+,	O
+while	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+IDA	B-protein
+,	O
+SERK1	B-protein
+strongly	O
+binds	O
+HAESA	B-protein
+with	O
+a	O
+dissociation	B-evidence
+constant	I-evidence
+in	O
+the	O
+mid	O
+-	O
+nanomolar	O
+range	O
+(	O
+Figure	O
+3C	O
+).	O
+
+Upon	O
+IDA	B-protein
+binding	O
+at	O
+the	O
+cell	O
+surface	O
+,	O
+the	O
+kinase	B-structure_element
+domains	I-structure_element
+of	O
+HAESA	B-protein
+and	O
+SERK1	B-protein
+,	O
+which	O
+have	O
+been	O
+shown	O
+to	O
+be	O
+active	B-protein_state
+protein	B-protein_type
+kinases	I-protein_type
+,	O
+may	O
+interact	O
+in	O
+the	O
+cytoplasm	O
+to	O
+activate	O
+each	O
+other	O
+.	O
+
+Consistently	O
+,	O
+the	O
+HAESA	B-protein
+kinase	B-structure_element
+domain	I-structure_element
+can	O
+transphosphorylate	O
+SERK1	B-protein
+and	O
+vice	O
+versa	O
+in	O
+in	O
+vitro	O
+transphosphorylation	B-experimental_method
+assays	I-experimental_method
+(	O
+Figure	O
+3E	O
+).	O
+
+Crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+HAESA	B-complex_assembly
+–	I-complex_assembly
+IDA	I-complex_assembly
+–	I-complex_assembly
+SERK1	I-complex_assembly
+signaling	O
+complex	O
+.	O
+
+(	O
+A	O
+)	O
+Overview	O
+of	O
+the	O
+ternary	O
+complex	O
+with	O
+HAESA	B-protein
+in	O
+blue	O
+(	O
+surface	O
+representation	O
+),	O
+IDA	B-protein
+in	O
+yellow	O
+(	O
+bonds	O
+representation	O
+)	O
+and	O
+SERK1	B-protein
+in	O
+orange	O
+(	O
+surface	O
+view	O
+).	O
+(	O
+B	O
+)	O
+The	O
+HAESA	B-protein
+ectodomain	B-structure_element
+undergoes	O
+a	O
+conformational	O
+change	O
+upon	O
+SERK1	B-protein
+co	O
+-	O
+receptor	O
+binding	O
+.	O
+
+Polar	O
+contacts	O
+of	O
+SERK1	B-protein
+with	O
+IDA	B-protein
+are	O
+shown	O
+in	O
+magenta	O
+,	O
+with	O
+the	O
+HAESA	B-protein
+LRR	B-structure_element
+domain	I-structure_element
+in	O
+gray	O
+.	O
+(	O
+D	O
+)	O
+Details	O
+of	O
+the	O
+zipper	B-structure_element
+-	I-structure_element
+like	I-structure_element
+SERK1	B-site
+-	I-site
+HAESA	I-site
+interface	I-site
+.	O
+
+The	O
+SERK1	B-protein
+ectodomain	B-structure_element
+interacts	O
+with	O
+the	O
+IDA	B-site
+peptide	I-site
+binding	I-site
+site	I-site
+using	O
+a	O
+loop	B-structure_element
+region	I-structure_element
+(	O
+residues	O
+51	B-residue_range
+-	I-residue_range
+59SERK1	I-residue_range
+)	O
+from	O
+its	O
+N	O
+-	O
+terminal	O
+cap	B-structure_element
+(	O
+Figure	O
+4A	O
+,	O
+C	O
+).	O
+
+We	O
+found	O
+that	O
+over	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+IDA	B-protein
+leads	O
+to	O
+early	O
+floral	O
+abscission	O
+and	O
+an	O
+enlargement	O
+of	O
+the	O
+abscission	O
+zone	O
+(	O
+Figure	O
+5C	O
+–	O
+E	O
+).	O
+
+In	O
+contrast	O
+to	O
+animal	B-taxonomy_domain
+LRR	B-protein_type
+receptors	I-protein_type
+,	O
+plant	B-taxonomy_domain
+LRR	B-structure_element
+-	I-structure_element
+RKs	I-structure_element
+harbor	O
+spiral	B-protein_state
+-	I-protein_state
+shaped	I-protein_state
+ectodomains	B-structure_element
+and	O
+thus	O
+they	O
+require	O
+shape	B-protein_state
+-	I-protein_state
+complementary	I-protein_state
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+proteins	I-protein_type
+for	O
+receptor	O
+activation	O
+.	O
+
+However	O
+our	O
+results	O
+show	O
+that	O
+SERK1	B-protein
+also	O
+can	O
+activate	O
+this	O
+process	O
+upon	O
+IDA	B-protein
+sensing	O
+,	O
+indicating	O
+that	O
+SERKs	B-protein_type
+may	O
+fulfill	O
+several	O
+different	O
+functions	O
+in	O
+the	O
+course	O
+of	O
+the	O
+abscission	O
+process	O
+.	O
+
+The	O
+central	O
+Hyp	B-residue_name
+residue	O
+in	O
+IDA	B-protein
+is	O
+found	O
+buried	O
+in	O
+the	O
+HAESA	B-protein
+peptide	B-site
+binding	I-site
+surface	I-site
+and	O
+thus	O
+this	O
+post	O
+-	O
+translational	O
+modification	O
+may	O
+regulate	O
+IDA	B-protein
+bioactivity	O
+.	O
+
+In	O
+our	O
+quantitative	B-experimental_method
+biochemical	I-experimental_method
+assays	I-experimental_method
+,	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+SERK1	B-protein
+dramatically	O
+increases	O
+the	O
+HAESA	B-protein
+binding	O
+specificity	O
+and	O
+affinity	O
+for	O
+IDA	B-protein
+.	O
+
+A	O
+ribbon	O
+diagram	O
+of	O
+SERK1	B-protein
+in	O
+the	O
+same	O
+orientation	O
+is	O
+shown	O
+alongside	O
+.	O
+
+These	O
+residues	O
+are	O
+not	O
+involved	O
+in	O
+the	O
+sensing	O
+of	O
+the	O
+steroid	B-chemical
+hormone	I-chemical
+brassinolide	B-chemical
+.	O
+
+Different	O
+plant	B-taxonomy_domain
+peptide	B-protein_type
+hormone	I-protein_type
+families	I-protein_type
+contain	O
+a	O
+C	O
+-	O
+terminal	O
+(	B-structure_element
+Arg	I-structure_element
+)-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+,	O
+which	O
+in	O
+IDA	B-protein
+represents	O
+the	O
+co	B-site
+-	I-site
+receptor	I-site
+recognition	I-site
+site	I-site
+.	O
+
+Our	O
+experiments	O
+reveal	O
+that	O
+SERK1	B-protein
+recognizes	O
+a	O
+C	O
+-	O
+terminal	O
+Arg	B-structure_element
+-	I-structure_element
+His	I-structure_element
+-	I-structure_element
+Asn	I-structure_element
+motif	I-structure_element
+in	O
+IDA	B-protein
+.	O
+
+Among	O
+these	O
+are	O
+the	O
+CLE	B-chemical
+peptides	I-chemical
+regulating	O
+stem	O
+cell	O
+maintenance	O
+in	O
+the	O
+shoot	O
+and	O
+the	O
+root	O
+.	O
+
+The	O
+crotonyllysine	B-residue_name
+mark	O
+on	O
+histone	B-protein_type
+H3K18	B-protein_type
+is	O
+produced	O
+by	O
+p300	B-protein
+,	O
+a	O
+histone	B-protein_type
+acetyltransferase	I-protein_type
+also	O
+responsible	O
+for	O
+acetylation	B-ptm
+of	O
+histones	O
+.	O
+
+The	O
+family	O
+of	O
+acetyllysine	B-residue_name
+readers	O
+has	O
+been	O
+expanded	O
+with	O
+the	O
+discovery	O
+that	O
+the	O
+YEATS	B-structure_element
+(	O
+Yaf9	B-protein
+,	O
+ENL	B-protein
+,	O
+AF9	B-protein
+,	O
+Taf14	B-protein
+,	O
+Sas5	B-protein
+)	O
+domains	O
+of	O
+human	B-species
+AF9	B-protein
+and	O
+yeast	B-taxonomy_domain
+Taf14	B-protein
+are	O
+capable	O
+of	O
+recognizing	O
+the	O
+histone	B-protein_type
+mark	O
+H3K9ac	B-protein_type
+.	O
+
+Similarly	O
+,	O
+activation	O
+of	O
+a	O
+subset	O
+of	O
+genes	O
+and	O
+DNA	O
+damage	O
+repair	O
+in	O
+yeast	B-taxonomy_domain
+require	O
+the	O
+acetyllysine	B-residue_name
+binding	O
+activity	O
+of	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+.	O
+
+However	O
+,	O
+Taf14	B-protein
+is	O
+also	O
+found	O
+in	O
+a	O
+number	O
+of	O
+chromatin	O
+-	O
+remodeling	O
+complexes	O
+(	O
+i	O
+.	O
+e	O
+.,	O
+INO80	B-complex_assembly
+,	O
+SWI	B-complex_assembly
+/	I-complex_assembly
+SNF	I-complex_assembly
+and	O
+RSC	B-complex_assembly
+)	O
+and	O
+the	O
+histone	B-protein_type
+acetyltransferase	I-protein_type
+complex	O
+NuA3	B-complex_assembly
+,	O
+indicating	O
+a	O
+multifaceted	O
+role	O
+of	O
+Taf14	B-protein
+in	O
+transcriptional	O
+regulation	O
+and	O
+chromatin	O
+biology	O
+.	O
+
+We	O
+found	O
+that	O
+H3K9cr	B-protein_type
+is	O
+present	O
+in	O
+yeast	B-taxonomy_domain
+and	O
+is	O
+dynamically	O
+regulated	O
+.	O
+
+This	O
+distinctive	O
+mechanism	O
+was	O
+corroborated	O
+through	O
+mapping	O
+the	O
+Taf14	B-protein
+YEATS	B-site
+-	I-site
+H3K9cr	I-site
+binding	I-site
+interface	I-site
+in	O
+solution	O
+using	O
+NMR	B-experimental_method
+chemical	I-experimental_method
+shift	I-experimental_method
+perturbation	I-experimental_method
+analysis	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+).	O
+
+Binding	O
+of	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+to	O
+H3K9cr	B-protein_type
+is	O
+robust	O
+.	O
+
+We	O
+concluded	O
+that	O
+H3K9cr	B-protein_type
+is	O
+the	O
+preferred	O
+target	O
+of	O
+this	O
+domain	O
+.	O
+
+However	O
+,	O
+bromodomains	B-structure_element
+did	O
+not	O
+interact	O
+(	O
+or	O
+associated	O
+very	O
+weakly	O
+)	O
+with	O
+longer	O
+acyl	O
+modifications	O
+,	O
+including	O
+crotonyllysine	B-residue_name
+,	O
+as	O
+in	O
+the	O
+case	O
+of	O
+BDs	B-structure_element
+of	O
+TAF1	B-protein
+and	O
+BRD2	B-protein
+,	O
+supporting	O
+recent	O
+reports	O
+.	O
+
+As	O
+we	O
+previously	O
+showed	O
+the	O
+importance	O
+of	O
+acyllysine	B-residue_name
+binding	O
+by	O
+the	O
+Taf14	B-protein
+YEATS	B-structure_element
+domain	I-structure_element
+for	O
+the	O
+DNA	O
+damage	O
+response	O
+and	O
+gene	O
+transcription	O
+,	O
+it	O
+will	O
+be	O
+essential	O
+in	O
+the	O
+future	O
+to	O
+define	O
+the	O
+physiological	O
+role	O
+of	O
+crotonyllysine	B-residue_name
+recognition	O
+and	O
+to	O
+differentiate	O
+the	O
+activities	O
+of	O
+Taf14	B-protein
+that	O
+are	O
+due	O
+to	O
+binding	O
+to	O
+crotonyllysine	B-residue_name
+and	O
+acetyllysine	B-residue_name
+modifications	O
+.	O
+
+Furthermore	O
+,	O
+the	O
+functional	O
+significance	O
+of	O
+crotonyllysine	B-residue_name
+recognition	O
+by	O
+other	O
+YEATS	B-protein_type
+proteins	O
+will	O
+be	O
+of	O
+great	O
+importance	O
+to	O
+elucidate	O
+and	O
+compare	O
+.	O
+
+The	O
+structural	O
+mechanism	O
+for	O
+the	O
+recognition	O
+of	O
+H3K9cr	B-protein_type
+
+Total	O
+H3	B-protein_type
+was	O
+used	O
+as	O
+a	O
+loading	O
+control	O
+.	O
+
+(	O
+c	O
+)	O
+Superimposed	O
+1H	B-experimental_method
+,	I-experimental_method
+15N	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+of	O
+Taf14	B-protein
+YEATS	B-structure_element
+recorded	O
+as	O
+H3K9cr5	B-chemical
+-	I-chemical
+13	I-chemical
+and	O
+H3K9ac5	B-chemical
+-	I-chemical
+13	I-chemical
+peptides	O
+were	O
+titrated	B-experimental_method
+in	O
+.	O
+
+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
+
+The	O
+proteasome	B-complex_assembly
+,	O
+an	O
+essential	O
+molecular	O
+machine	O
+,	O
+is	O
+a	O
+threonine	B-protein_type
+protease	I-protein_type
+,	O
+but	O
+the	O
+evolution	O
+and	O
+the	O
+components	O
+of	O
+its	O
+proteolytic	O
+centre	O
+are	O
+unclear	O
+.	O
+
+In	O
+the	O
+last	O
+stage	O
+of	O
+CP	B-complex_assembly
+biogenesis	O
+,	O
+the	O
+prosegments	B-structure_element
+are	O
+autocatalytically	B-ptm
+removed	I-ptm
+through	O
+nucleophilic	O
+attack	O
+by	O
+the	O
+active	B-site
+site	I-site
+residue	I-site
+Thr1	B-residue_name_number
+on	O
+the	O
+preceding	O
+peptide	O
+bond	O
+involving	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+).	I-residue_name_number
+
+These	O
+results	O
+indicate	O
+that	O
+the	O
+β1	B-protein
+and	O
+β2	B-protein
+proteolytic	O
+activities	O
+are	O
+not	O
+essential	O
+for	O
+cell	O
+survival	O
+.	O
+
+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
+
+Sequencing	B-experimental_method
+of	I-experimental_method
+the	I-experimental_method
+plasmids	I-experimental_method
+,	O
+testing	O
+them	O
+in	O
+both	O
+published	O
+yeast	B-taxonomy_domain
+strain	O
+backgrounds	O
+and	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+revealed	O
+that	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+mutant	B-protein_state
+pp	B-chemical
+cis	B-protein_state
+is	O
+viable	O
+,	O
+but	O
+suffers	O
+from	O
+a	O
+marked	O
+growth	O
+defect	O
+that	O
+requires	O
+extended	O
+incubation	O
+of	O
+4	O
+–	O
+5	O
+days	O
+for	O
+initial	O
+colony	O
+formation	O
+(	O
+Table	O
+1	O
+and	O
+Supplementary	O
+Methods	O
+).	O
+
+In	O
+subunit	O
+β1	B-protein
+,	O
+we	O
+found	O
+that	O
+Gly	B-residue_name_number
+(-	I-residue_name_number
+1	I-residue_name_number
+)	I-residue_name_number
+indeed	O
+forms	O
+a	O
+sharp	B-structure_element
+turn	I-structure_element
+,	O
+which	O
+relaxes	O
+on	O
+prosegment	B-ptm
+cleavage	I-ptm
+(	O
+Fig	O
+.	O
+1a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+).	O
+
+Regarding	O
+the	O
+β2	B-protein
+propeptide	B-structure_element
+,	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+occupies	O
+the	O
+S1	B-site
+pocket	I-site
+but	O
+is	O
+less	O
+deeply	O
+anchored	O
+compared	O
+with	O
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+β1	B-protein
+,	O
+which	O
+might	O
+be	O
+due	O
+to	O
+the	O
+rather	O
+large	O
+β2	B-protein
+-	O
+S1	B-site
+pocket	I-site
+created	O
+by	O
+Gly45	B-residue_name_number
+.	O
+
+Nevertheless	O
+,	O
+both	O
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+and	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+were	O
+found	O
+to	O
+occupy	O
+the	O
+S1	B-site
+specificity	I-site
+pocket	I-site
+formed	O
+by	O
+Met45	B-residue_name_number
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4f	O
+–	O
+h	O
+).	O
+
+Bearing	O
+in	O
+mind	O
+that	O
+in	O
+contrast	O
+to	O
+Thr	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+β2	B-protein
+,	O
+Leu	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+in	O
+subunit	O
+β1	B-protein
+is	O
+not	B-protein_state
+conserved	I-protein_state
+among	O
+species	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3a	O
+),	O
+we	O
+created	B-experimental_method
+a	O
+β2	B-mutant
+-	I-mutant
+T	I-mutant
+(-	I-mutant
+2	I-mutant
+)	I-mutant
+V	I-mutant
+proteasome	B-complex_assembly
+mutant	B-protein_state
+.	O
+
+Notably	O
+,	O
+the	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+map	I-evidence
+displays	O
+a	O
+different	O
+orientation	O
+for	O
+the	O
+β2	B-protein
+propeptide	B-structure_element
+than	O
+has	O
+been	O
+observed	O
+for	O
+the	O
+β2	B-mutant
+-	I-mutant
+T1A	I-mutant
+proteasome	B-complex_assembly
+.	O
+
+The	O
+active	B-site
+site	I-site
+of	O
+the	O
+proteasome	B-complex_assembly
+
+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
+
+In	O
+agreement	O
+,	O
+an	O
+E17A	B-mutant
+mutant	B-protein_state
+in	O
+the	O
+proteasomal	O
+β	B-protein
+-	I-protein
+subunit	I-protein
+of	O
+the	O
+archaeon	B-taxonomy_domain
+Thermoplasma	B-species
+acidophilum	I-species
+prevents	O
+autolysis	B-ptm
+and	O
+catalysis	O
+.	O
+
+Strikingly	O
+,	O
+although	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+on	O
+the	O
+β5	B-mutant
+-	I-mutant
+D17N	I-mutant
+mutant	B-protein_state
+with	O
+the	O
+propeptide	B-structure_element
+expressed	B-experimental_method
+in	O
+cis	B-protein_state
+and	O
+in	O
+trans	B-protein_state
+looked	O
+similar	O
+,	O
+there	O
+was	O
+a	O
+pronounced	O
+difference	O
+in	O
+their	O
+growth	O
+phenotypes	O
+observed	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7b	O
+).	O
+
+Whereas	O
+Asn	B-residue_name
+can	O
+to	O
+some	O
+degree	O
+replace	O
+Asp166	B-residue_name_number
+due	O
+to	O
+its	O
+carbonyl	O
+group	O
+in	O
+the	O
+side	O
+chain	O
+,	O
+Ala	B-residue_name
+at	O
+this	O
+position	O
+was	O
+found	O
+to	O
+prevent	O
+both	O
+autolysis	B-ptm
+and	O
+catalysis	O
+.	O
+
+His	B-residue_name_number
+(-	I-residue_name_number
+2	I-residue_name_number
+)	I-residue_name_number
+occupies	O
+the	O
+S2	B-site
+pocket	I-site
+like	O
+observed	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1A	I-mutant
+-	I-mutant
+K81R	I-mutant
+mutant	B-protein_state
+,	O
+but	O
+in	O
+contrast	O
+to	O
+the	O
+latter	O
+,	O
+the	O
+propeptide	B-structure_element
+in	O
+the	O
+T1C	B-mutant
+mutant	B-protein_state
+adopts	O
+an	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+sheet	I-structure_element
+conformation	O
+as	O
+known	O
+from	O
+inhibitors	O
+like	O
+MG132	B-chemical
+(	O
+Fig	O
+.	O
+4c	O
+–	O
+e	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9b	O
+).	O
+
+Activity	B-experimental_method
+assays	I-experimental_method
+with	O
+the	O
+β5	B-protein
+-	O
+specific	O
+substrate	O
+Suc	B-chemical
+-	I-chemical
+LLVY	I-chemical
+-	I-chemical
+AMC	I-chemical
+demonstrated	O
+that	O
+the	O
+ChT	O
+-	O
+L	O
+activity	O
+of	O
+the	O
+T1S	B-mutant
+mutant	B-protein_state
+is	O
+reduced	O
+by	O
+40	O
+–	O
+45	O
+%	O
+compared	O
+with	O
+WT	B-protein_state
+proteasomes	B-complex_assembly
+depending	O
+on	O
+the	O
+incubation	O
+temperature	O
+(	O
+Fig	O
+.	O
+4b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+9c	O
+).	O
+
+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
+
+The	O
+20S	B-complex_assembly
+proteasome	I-complex_assembly
+CP	B-complex_assembly
+is	O
+the	O
+major	O
+non	B-protein_type
+-	I-protein_type
+lysosomal	I-protein_type
+protease	I-protein_type
+in	O
+eukaryotic	B-taxonomy_domain
+cells	O
+,	O
+and	O
+its	O
+assembly	O
+is	O
+highly	O
+organized	O
+.	O
+
+The	O
+β	B-protein
+-	I-protein
+subunit	I-protein
+propeptides	B-structure_element
+,	O
+particularly	O
+that	O
+of	O
+β5	B-protein
+,	O
+are	O
+key	O
+factors	O
+that	O
+help	O
+drive	O
+proper	O
+assembly	O
+of	O
+the	O
+CP	B-complex_assembly
+complex	O
+.	O
+
+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
+
+The	O
+resulting	O
+uncharged	O
+Thr1NH2	B-residue_name_number
+is	O
+hydrogen	O
+-	O
+bridged	O
+to	O
+the	O
+C3	O
+-	O
+OH	O
+group	O
+.	O
+
+In	O
+agreement	O
+,	O
+acetylation	B-ptm
+of	O
+the	O
+Thr1	B-residue_name_number
+N	O
+terminus	O
+irreversibly	O
+blocks	O
+hydrolytic	O
+activity	O
+,	O
+and	O
+binding	O
+of	O
+substrates	O
+is	O
+prevented	O
+for	O
+steric	O
+reasons	O
+.	O
+
+This	O
+interpretation	O
+agrees	O
+with	O
+the	O
+strongly	O
+reduced	O
+catalytic	O
+activity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+D166N	I-mutant
+mutant	B-protein_state
+on	O
+the	O
+one	O
+hand	O
+,	O
+and	O
+the	O
+ability	O
+to	O
+react	O
+readily	O
+with	O
+carfilzomib	B-chemical
+on	O
+the	O
+other	O
+.	O
+
+We	O
+also	O
+observed	O
+slightly	O
+lower	O
+affinity	O
+of	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1S	I-mutant
+mutant	B-protein_state
+yCP	B-complex_assembly
+for	O
+the	O
+Food	O
+and	O
+Drug	O
+Administration	O
+-	O
+approved	O
+proteasome	B-complex_assembly
+inhibitors	O
+bortezomib	B-chemical
+and	O
+carfilzomib	B-chemical
+.	O
+
+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
+
+Architecture	O
+and	O
+proposed	O
+reaction	O
+mechanism	O
+of	O
+the	O
+proteasomal	O
+active	B-site
+site	I-site
+.	O
+
+Thr1OH	B-residue_name_number
+is	O
+hydrogen	O
+-	O
+bonded	O
+to	O
+Lys33NH2	B-residue_name_number
+(	O
+2	O
+.	O
+7	O
+Å	O
+),	O
+which	O
+in	O
+turn	O
+interacts	O
+with	O
+Asp17Oδ	B-residue_name_number
+.	O
+
+Autolysis	B-ptm
+(	O
+left	O
+set	O
+of	O
+structures	O
+)	O
+is	O
+initiated	O
+by	O
+deprotonation	O
+of	O
+Thr1OH	B-residue_name_number
+via	O
+Lys33NH2	B-residue_name_number
+and	O
+the	O
+formation	O
+of	O
+a	O
+tetrahedral	O
+transition	O
+state	O
+.	O
+
+Next	O
+,	O
+Thr1NH2	B-residue_name_number
+polarizes	O
+a	O
+water	B-chemical
+molecule	O
+for	O
+the	O
+nucleophilic	O
+attack	O
+of	O
+the	O
+acyl	O
+-	O
+enzyme	O
+intermediate	O
+.	O
+
+(	O
+c	O
+)	O
+Illustration	O
+of	O
+the	O
+2FO	B-evidence
+–	I-evidence
+FC	I-evidence
+electron	I-evidence
+-	I-evidence
+density	I-evidence
+map	I-evidence
+(	O
+blue	O
+mesh	O
+contoured	O
+at	O
+1σ	O
+)	O
+for	O
+the	O
+β5	B-mutant
+-	I-mutant
+T1C	I-mutant
+propeptide	B-structure_element
+fragment	O
+.	O
+
+(	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
+