ACTIVATION OF THE PHOSPHOSIGNALING PROTEIN CHEY .2. ANALYSIS OF ACTIVATED MUTANTS BY F-19 NMR AND PROTEIN ENGINEERING

The Escherichia coli CheY protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mecha nism of activation, an extensive collection of mutant CheY proteins wa s analyzed by behavioral assays, in vitro phosphorylation, and F-19 NM R chemical shift measurements. Substitution of a positively charged re sidue (Arg or Lys) in place of Asp13 in the CheY activation site resul ts in activation, even for mutants which cannot be phosphorylated. Thu s phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational ''switch'' in th e activation site, is required for activation of CheY by either phosph orylation or mutation. The F-19 NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simo n, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081-13088) was again used to monitor six phenylalanine positions in CheY, including o ne position which probed the vicinity of Lys109. Mutations which activ ate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformati onal change. Two striking contrasts were observed between activation b y mutation and phosphorylation. (i) Each activating mutation generates a relatively localized perturbation in the activation site region, wh ereas phosphorylation triggers a global structural change. (ii) The pe rturbation of the Lys109 region observed for activating mutations is n ot detected in the phosphorylated protein. These results are consisten t with a two-step model of activated CheY docking to the flagellar swi tch.