Activation of the phosphosignaling protein CheY. I. Analysis of the phosphorylated conformation by 19F NMR and protein engineering
CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57. In order to probe the structural changes associated with activation, an approach which combines 19F NMR, protein engineering, and the known crystal structure of one conformer has been utilized. This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conformation of CheY. The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity. One of these 19F probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural "switch" in activation. 19F NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phsopho-CheY during NMR acquisition. Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule. However, phosphorylation did not perturb the probe of Lys109. 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 insight into the activation of other prokaryotic response regulators.
Additional InformationCopyright © 1993 by the American Society for Biochemistry and Molecular Biology. (Received for publication, November 30, 1992, and in revised form, March 4, 1993) We are indebted to Dr. Karl Volz (University of Illinois) for the generous gift of CheY coordinates prior to publication; to Drs. Rick Dahlquist, Phil Matsumura, Karl Volz, and Ann Stock for helpful discussions; and to Stephen Chervitz for comments on the manuscript. This work was supported by National Institutes of Health Grants GM40731 (to J.J.F.) and A119296 (to M.I.S.), and by National Research Service Award Fellowship A107798 (to R.B.B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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