Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by super(19)F NMR and protein engineering

The Escherichia coli CheY protein is activate 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 super(19)F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp super(13) 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. Lys super(109), 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 super(19) F NMR chemical shift assay was used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys super(109). Mutations which activate CheY were observed to perturb the Lys super(109) probe, providing further evidence that Lys super(109) 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 Lys super(109) 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.