4.5 Article

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

Journal

MOLECULAR MICROBIOLOGY
Volume 95, Issue 2, Pages 231-244

Publisher

WILEY
DOI: 10.1111/mmi.12858

Keywords

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Funding

  1. Israel Science Foundation [534/10]
  2. Minerva Foundation
  3. Federal German Ministry for Education and Research
  4. National Institutes of Health [9P41GM104601]
  5. National Science Foundation [PHY-1430124, ACI-1440026]
  6. Direct For Mathematical & Physical Scien
  7. Division Of Physics [1430124] Funding Source: National Science Foundation
  8. Office of Advanced Cyberinfrastructure (OAC)
  9. Direct For Computer & Info Scie & Enginr [1440026] Funding Source: National Science Foundation

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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 44 loop, shifted toward an active state upon mutation. Removal of 44 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.

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