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Published June 25, 1993 | Published
Journal Article Open

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.

Additional Information

Copyright © 1993 by the American Society for Biochemistry and Molecular Biology. (Received for publication, November 30, 1992, and in revised form, March 4, 1993) We are indebted to Karl Volz for access to the crystal structure of E. coli CheY prior to publication. We thank Nancy Kleckner, Steve Roman, and Alan Wolfe for providing bacterial strains and plasmids, and Lisa Alex, Stephan Schuster, and Ron Swanson for comments on the manuscript. This work was supported by National Research Service Award Fellowship AI07798 (to R.B.B.) and National Institutes of Health Grants AI19296 (to M.I.S.) and GM40731 (to J.J.F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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