The 1.9 angstrom resolution crystal structure of phosphono-CheY, an analogue of the active form of the response regulator, CheY

To structurally characterize the activated state of the transiently phospho rylated signal transduction protein CheY, we have constructed an alpha-thio phosphonate derivative of the CheY D57C point mutant and determined its thr ee-dimensional structure at 1.85 A resolution. We have also characterized t his analogue with high-resolution NMR and studied its binding to a peptide derived from FliM, CheY's target component of the flagellar motor. The chem ically modified derivative, phosphono-CheY, exhibits many of the chemical p roperties of phosphorylated wild-type CheY, except that it is indefinitely stable. Electron density for the alpha-thiophosphonate substitution is clea r and readily interpretable; omit refinement density at the phosphorus atom is greater than 10 sigma. The molecule shows a number of localized conform ational changes that are believed to constitute the postphosphorylation act ivation events. The most obvious of these changes include movement of the s ide chain of the active site base, Lys 109, and a predominately buried conf ormation of the side chain of Tyr 106. In addition, there are a number of m ore subtle changes more distant from the active site involving the alpha 14 and alpha 5 helices. These results are consistent with our previous struct ural interpretations of other CheY activation mutants, and with our earlier hypotheses concerning CheY activation through propagation of structural ch anges away from the active site.