Conformational coupling in the chemotaxis response regulator CheY

CheY, a response regulator protein in bacterial chemotaxis, serves as a pro totype for the analysis of response regulator function in two-component sig nal transduction. Phosphorylation of a conserved aspartate at the active si te mediates a conformational change at a distal signaling surface that modu lates interactions with the flagellar motor component FliM, the sensor kina se CheA, and the phosphatase CheZ. The objective of th is study was to prob e the conformational coupling between the phosphorylation site and the sign aling surface of CheY in the reverse direction by quantifying phosphorylati on activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatical ly impacted autophosphorylation of CheY by small molecule phosphodonors, wh ich is indicative of reverse signal propagation in CheY. Autodephosphorylat ion and substrate affinity, however, were not significantly affected. Kinet ic characterization of several CheY mutants suggested that conserved residu es Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, ar e not essential for conformational coupling. These findings provide structu ral and conceptual insights into the mechanism of CheY activation. Our resu lts are consistent with a multistate thermodynamic model of response regula tor activation.