To structurally characterize the activated state of the transiently phosphorylated signal transduction protein CheY, we have constructed an α- thiophosphonate derivative of the CheY D57C point mutant and determined its three-dimensional structure at 1.85 Å resolution. We have also characterized this analogue with high-resolution NMR and studied its binding to a peptide derived from FliM, CheY's target component of the flagellar motor. The chemically modified derivative, phosphono-CheY, exhibits many of the chemical properties of phosphorylated wild-type CheY, except that it is indefinitely stable. Electron density for the α-thiophosphonate substitution is clear and readily interpretable; omit refinement density at the phosphorus atom is greater than 10σ. The molecule shows a number of localized conformational changes that are believed to constitute the postphosphorylation activation events. The most obvious of these changes include movement of the side chain of the active site base, Lys 109, and a predominately buried conformation of the side chain of Tyr 106. In addition, there are a number of more subtle changes more distant from the active site involving the α4 and α5 helices. These results are consistent with our previous structural interpretations of other CheY activation mutants, and with our earlier hypotheses concerning CheY activation through propagation of structural changes away from the active site.
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