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.
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
S K
SK
Drake
R B
RB
Bourret
L A
LA
Luck
M I
MI
Simon
J J
JJ
Falke
D001426:Bacterial Proteins; D001665:Binding Sites; D002633:Chemotaxis; D029968:Escherichia coli Proteins; D005461:Fluorine; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D010755:Organophosphates / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D015202:Protein Engineering
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010755:Organophosphates; C000607606:cheY protein, E coli; D005461:Fluorine; C011632:acetyl phosphate; D008274:Magnesium