Effects of phosphorylation, Mg2+, and conformation of the chemotaxis protein CheY on its binding to the flagellar switch protein FliM.
CheY is the response regulator of bacterial chemotaxis. Previously, we showed that CheY binds to the flagellar switch protein FliM and that this binding is increased upon phosphorylation of CheY [Welch, M., Oosawa, K., Aizawa, S.-I., & Eisenbach, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8787-8791]. Here, we demonstrate that it is the phosphorylated conformation of CheY, rather than the phosphate group itself, that is recognized and bound by FliM. We found that subsequent to the phosphorylation of CheY, Mg2+ was not required for the binding of CheY to FliM. However, phosphorylation of CheY did cause a change in the coordination properties of Mg2+ in the acid pocket of the protein. This change in the coordination of Mg2+ required the presence of the absolutely conserved residue Lys109. When Lys109 was substituted by arginine, the resulting CheY protein was unable to adopt an active conformation upon phosphorylation, and the protein was not bound by FliM. Surprisingly, the CheY13DK mutant protein, which is active in vivo but cannot be phosphorylated in vitro, exhibited only a low level of FliM binding activity, suggesting that its ability to cause clockwise rotation in the cell is not due to a constitutively high level of FliM binding. On the basis of these findings, we propose a mechanism for CheY activation by phosphorylation.
Welch
M
M
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Oosawa
K
K
Aizawa
S I
SI
Eisenbach
M
M
D001426:Bacterial Proteins / Q000378:metabolism*; D002633:Chemotaxis / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins; D005407:Flagella / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; C066821:FliM protein, Bacteria; D008274:Magnesium