Towards understanding a molecular switch mechanism: thermodynamic and crystallographic studies of the signal transduction protein CheY

Abstract

The signal transduction protein CheY displays an α/β-parallel polypeptide folding, including a highly unstable helix α4 and a strongly charged active site. Helix α4 has been shown to adopt various positions and conformations in different crystal structures, suggesting that it is a mobile segment. Furthermore, the instability of this helix is believed to have functional significance because it is involved in protein-protein contacts with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ. The active site of CheY comprises a cluster of three aspartic acid residues and a lysine residue, all of which participate in the binding of the Mg 2+ needed for the protein activation. Two steps were followed to study the activation mechanism of CheY upon phosphorylation: first, we independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix α4, both to increase its level of stability and to improve its packing against the protein core. The structural and thermodynamic analysis of these mutant proteins provides further evidence of the connection between the active-site area and helix α4, and helps to understand how small movements at the active site are transmitted and amplified to the protein surface.