The aspartate chemoreceptor Tar is effectively methylated by binding to the methyltransferase mainly through hydrophobic interaction.
In the chemotaxis of Escherichia coli, adaptation requires the methylation and demethylation of transmembrane receptors, which are catalysed by the methyltransferase CheR and the methylesterase CheB respectively. CheR binds to major chemoreceptors through their C-terminal motif NWETF, which is distinct from the methylation sites. In this study, we carried out a systematic mutagenesis of the pentapeptide sequence of Tar. Receptor methylation and adaptation were severely impaired by the alanine substitution of residue W550 and, to a lesser extent, by that of F553. Substitution of residues N549, E551 and T552 had only a slight or little effect. The defects of the W550A and F553A mutations were suppressed by high- and low-level overproduction of CheR respectively. Expression of a fusion protein containing the NWETF sequence, but not its W550A and F553A versions, inhibited chemotaxis of the Che+ strain. In an in vitro assay, CheR bound to the wild-type version but not to the mutant versions. These results and further mutagenesis suggest that the hydrophobicity and the size of residues W550 and F553 are critical in the interaction with CheR, a conclusion that is consistent with the crystal structure of a CheR-NWETF complex. On the other hand, the negatively charged side chain of E551 and the polar side chains of N549 and T552 may not be strictly required, although the presence of a salt bridge and hydrogen bonds between these residues and residues from CheR has been noted in the co-crystal.
Shiomi
D
D
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Okumura
H
H
Homma
M
M
Kawagishi
I
I
D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002628:Chemoreceptor Cells; D002633:Chemotaxis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; D001224:Aspartic Acid; D008780:Methyltransferases; C038706:chemotaxis methyltransferase