The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY.
A key event in signal transduction during chemotaxis of Salmonella typhimurium and related bacterial species is the interaction between the phosphorylated form of the response regulator CheY (CheY approximately P) and the switch of the flagellar motor, located at its base. The consequence of this interaction is a shift in the direction of flagellar rotation from the default, counterclockwise, to clockwise. The docking site of CheY approximately P at the switch is the protein FliM. The purpose of this study was to identify the CheY-binding domain of FliM. We cloned 17 fliM mutants, each defective in switching and having a point mutation at a different location, and then overexpressed and purified their products. The CheY-binding ability of each of the FliM mutant proteins was determined by chemical crosslinking. All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM10EG, bound CheY approximately P to a much lesser extent than did wild-type FliM. CheY approximately P-binding of the other mutant proteins was similar to wild-type FliM. To investigate whether the FliM domain that includes these three mutations is indeed the CheY-binding domain, we synthesized a peptide composed of the first 16 amino acid residues of FliM, including a highly conserved region of FliM (residues 6 to 15). The peptide bound CheY and, to a larger extent, CheY approximately P. It also competed with full-length FliM on CheY approximately P. These results indicate that the CheY-binding domain of FliM is located at the N terminus, within residues 1 to 16, and suggest that FliM monomers can form a complete site for CheY binding.
Bren
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A
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel.
Eisenbach
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M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002633:Chemotaxis; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005407:Flagella / Q000502:physiology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000502:physiology*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011994:Recombinant Proteins; C000607606:cheY protein, E coli; C066821:FliM protein, Bacteria