The 1.9 Å Resolution Crystal Structure of Phosphono-CheY, an Analogue of the Active Form of the Response Regulator, CheY^,

Abstract

To structurally characterize the activated state of the transiently phosphorylated signal transduction protein CheY, we have constructed an alpha-thiophosphonate derivative of the CheY D57C point mutant and determined its three-dimensional structure at 1.85 A 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 alpha-thiophosphonate substitution is clear and readily interpretable; omit refinement density at the phosphorus atom is greater than 10sigma. 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 alpha4 and alpha5 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.

Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

“…Similar interactions have been reported for many structures of the activated regulatory domains, such as of Spo0A, 45 FixJ 46 and CheY. 47,48 The area that differs most between the two structures is the β4-α4 loop, specifically residues Thr81, Gly82 and Arg83 ( Fig. 2(a)).…”

Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway

“…Previous genetic and nuclear magnetic resonance studies have indicated that it is the ␣4-␤5-␣5 face of CheY that binds to its target protein, FilM (4). When high-resolution structures of active and inactive CheY were compared, a conserved residue in this face, CheY Tyr106, was found to flip between a solvent-exposed inactive conformation and a buried active conformation (9). Similar conformation changes of the conserved Tyr (or Phe) were observed in other receiver domain structures (28).…”

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“…Recently, several structures of activated receiver domains were obtained by using a variety of means to overcome the problem of lability (6)(7)(8)(9)(10). Among these, formation of a complex with BeF 3 Ϫ , which yields an excellent aspartyl phosphate mimic, appears to be a simple general solution (11).…”