Signal transduction by the chemotaxis response regulator CheY

The response regulator protein CheY in bacterial chemotaxis serves as a prototype for the analysis of response regulator function in prokaryotic signal transduction. Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ. This work addresses the nature of the interaction between CheY and FliM, and the conformational coupling between the active site and the signaling surface of CheY. The genetic, biochemical, and crystallographic characterization of CheY mutants with single site substitutions allowed us to define a role for a conserved isoleucine in the interaction with FliM and to establish a direct correlation between FliM binding and motor response. Conformational coupling in CheY was probed by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatically impacted phosphorylation activity, indicating that signal propagation in CheY also occurs in a reverse direction. Characterization of CheY mutants suggested that conserved residues implicated in the activation mechanism are not essential for conformational coupling. Our results are consistent with a multi-state thermodynamic model of response regulator function.