CheY's acetylation sites responsible for generating clockwise flagellar rotation in Escherichia coli.
Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high-resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate-dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is nonmodified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations, we show that K91 repression is manifested in the conformational dynamics of the β4α4 loop, shifted toward an active state upon mutation. Removal of β4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination, we further suggest that K109 acetylation is actively involved in generating clockwise rotation.
Department of Biological Chemistry, The Weizmann Institute of Science, 7610001, Rehovot, Israel.
Milana
M
Fraiberg
Oshri
O
Afanzar
C Keith
CK
Cassidy
Alexandra
A
Gabashvili
Klaus
K
Schulten
Yishai
Y
Levin
Michael
M
Eisenbach
D000085:Acetates / Q000378:metabolism; D000107:Acetylation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002633:Chemotaxis; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D056004:Molecular Dynamics Simulation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D020124:Thermotoga maritima / Q000378:metabolism
D000085:Acetates; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; D008239:Lysine