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

The signal transduction protein CheY displays an alpha/beta -parallel polyp eptide folding, including a highly unstable helix alpha4 and a strongly cha rged active site. Helix alpha4 has been shown to adopt various positions an d conformations in different crystal structures, suggesting that it is a mo bile segment. Furthermore, the instability of this helix is believed to hav e functional significance because it is involved in protein-protein contact s with the transmitter protein kinase CheA, the target protein FliM and the phosphatase CheZ. The active site of CheY comprises a cluster of three asp artic acid residues and a lysine residue, all of which participate in the b inding of the Mg2+ needed for the protein activation. Two steps were follow ed to study the activation mechanism of CheY upon phosphorylation: first, w e independently substituted the three aspartic acid residues in the active site with alanine; second, several mutations were designed in helix alpha4, both to increase its level of stability and to improve its packing against the protein core. The structural and thermodynamic analysis of these mutan t proteins provides further evidence of the connection between the active-s ite area and helix alpha4, and helps to understand how small movements at t he active site are transmitted and amplified to the protein surface. (C) 20 00 Academic Press.