ACTIVATION OF THE PHOSPHOSIGNALING PROTEIN CHEY .1. ANALYSIS OF THE PHOSPHORYLATED CONFORMATION BY F-19 NMR AND PROTEIN ENGINEERING

CheY, the 14-kDa response regulator protein of the Escherichia coli ch emotaxis pathway, is activated by phosphorylation of Asp57. In order t o probe the structural changes associated with activation, an approach which combines F-19 NMR, protein engineering, and the known crystal s tructure of one conformer has been utilized. This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conf ormation of CheY. The molecule was selectively labeled at its six phen ylalanine positions by incorporation of 4-fluorophenylalanine, which y ielded no significant effect on activity. One of these F-19 probe posi tions monitored the vicinity of Lys109, which forms a salt bridge to A sp57 in the apoprotein and has been proposed to act as a structural '' switch'' in activation. F-19 NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered lo cal structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with ph osphorylation were then examined, utilizing acetyl phosphate to chemic ally generate phospho-CheY during NMR acquisition. Phosphorylation tri ggered a long-range conformational change extending from the activatio n site to a cluster of 4 phenylalanine residues at the other end of th e molecule. However, phosphorylation did not perturb the probe of Lys1 09. The observed phosphorylated conformer is proposed to be the first step in the activation of CheY; later steps appear to perturb Lys109, as evidenced in the following paper. Together these results may give i nsight into the activation of other prokaryotic response regulators.