Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering.
The Escherichia coli CheY protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection of mutant CheY proteins was analyzed by behavioral assays, in vitro phosphorylation, and 19F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp13 in the CheY activation site results in activation, even for mutants which cannot be phosphorylated. Thus phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational "switch" in the activation site, is required for activation of CheY by either phosphorylation or mutation. The 19F NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081-13088) was again used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys109. Mutations which activate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformational change. Two striking contrasts were observed between activation by mutation and phosphorylation. (i) Each activating mutation generates a relatively localized perturbation in the activation site region, whereas phosphorylation triggers a global structural change. (ii) The perturbation of the Lys109 region observed for activating mutations is not detected in the phosphorylated protein. These results are consistent with a two-step model of activated CheY docking to the flagellar switch.
Bourret
R B
RB
Division of Biology, California Institute of Technology, Pasadena 91125.
Drake
S K
SK
Chervitz
S A
SA
Simon
M I
MI
Falke
J J
JJ
D001426:Bacterial Proteins; D002633:Chemotaxis; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005461:Fluorine; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D015202:Protein Engineering
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; D005461:Fluorine