CheY's acetylation sites responsible for generating clockwise flagellar rotation in E scherichia coli

• Published in: Molecular microbiology 2015-01, Vol.95 (2), p.231-244
• Main Authors: Fraiberg, Milana, Afanzar, Oshri, Cassidy, C. Keith, Gabashvili, Alexandra, Schulten, Klaus, Levin, Yishai, Eisenbach, Michael
• Format: Article
• Language: English
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.12858

Stimulation of E scherichia 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 K 91 and K 109 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 K 91 and K 109 are also the sites mainly responsible for acetate‐dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K 91 is nonmodified, as evidenced by an increased ability of CheY to generate clockwise rotation when K 91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations, we show that K 91 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 T hermotoga maritima . By way of elimination, we further suggest that K 109 acetylation is actively involved in generating clockwise rotation.