SIGNAL-TRANSDUCTION IN CHEMOTAXIS - A PROPAGATING CONFORMATION CHANGEUPON PHOSPHORYLATION OF CHEY

The CheY protein from Escherichia coli and Salmonella typhimurium are among the best characterized proteins of the receiver domain family of two component signal transduction systems in bacteria. Phosphorylatio n of CheY plays a central role in bacterial chemo taxis. However, it i s not entirely clear how its state of phosphorylation contributes to i ts function. Genetic evidence suggests that CheY changes its conformat ion upon phosphorylation. We present evidence for this con formation c hange by comparing the NMR N-15 - H-1 correlation spectra of CheY.Mg2 complex and phospho-CheY in the presence of magnesium. Large changes in chemical shift are used as indicators of chemical changes and proba ble structural changes in the protein backbone. Our observations sugge st that significant structural changes occur in CheY upon phosphorylat ion and that these changes are distinct from the changes produced by m agnesium ion binding. In addition to residues Asn-59 and Gly-65 that a re immediately adjacent to the site of phosphorylation at Asp-57, a la rge number of other residues show significant chemical shift changes a s a result of phosphorylation. These include Met-17, Val-21, Asn-23, G ly-39, Met-60, Met-63, Asp-64, Leu-66, Glu-67, Leu-68, Leu-69, Met-85, Val-86, Thr-87, Ala-88, Asn-94, Val-107, Lys-109, Thr-112, Ala-113, A la-114, and Asn-121. These results appear inconsistent with the recent suggestion that phosphorylation produces the same structural changes as magnesium binding (Bell-solell, L., Prieto, J., Serrano, L., and Ce ll, M. (1994) J. Mel. Biol. 238, 489-495). We find that some regions c hange overlap with a genetically defined motor binding face. We theref ore propose that the conformation switch modulates the interaction of CheY with its target, the flagellar motor. Other regions also change, possibly reflecting the many different functions of CheY homologues.