The region preceding the C-terminal NWETF pentapeptide modulates baseline activity and aspartate inhibition of Escherichia coli Tar.
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The Tar chemoreceptor-CheA-CheW ternary complex of Escherichia coli is a transmembrane allosteric enzyme in which binding of ligands to the periplasmic domain modulates the activity of CheA kinase. Kinase activity is also affected by reversible methylation of four glutamyl residues in the cytoplasmic domain of the receptor. E. coli Tar contains 553 residues. Residues 549-553 comprise the NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. The crystal structure of the similar Tsr chemoreceptor predicts that residues 263-289 and 490-515 of Tar form the most membrane-proximal portion of the extended CD1-CD2 four-helix bundle of the cytoplasmic domain. The last methylation site, Glu-491, is in the C19 heptad, and the N22-19 and C22-19 heptads are present in all classes of bacterial transmembrane chemoreceptors. Residues 516-548 probably serve as a flexible tether for the NWETF pentapeptide. Here, we present a mutational analysis of residues 505-548. The more of this region that is deleted, the less sensitive Tar is to inhibition by aspartate. Tar deleted from residue 505 through the NWETF sequence stimulates CheA in vitro but is not inhibited by aspartate. Thus, interaction of the last two heptads (C21 and C22) of CD2 with the first two heptads (N22 and N21) of CD1 must be important for transmitting an inhibitory signal from the HAMP domain to the four-helix bundle. The R514A, K523A, R529A, R540A, and R542A substitutions, singly or together, increase the level of activation of CheA in vitro, whereas the R505A substitution decreases the level of CheA stimulation by 40% and lowers the aspartate K(i) 7-fold. The R505E substitution completely abolishes stimulation of CheA in vitro. Glu-505 may interact electrostatically with Asp-273 to destabilize the "on" signaling state by loosening the four-helix bundle.
author list (cited authors)
Lai, R., Bormans, A. F., Draheim, R. R., Wright, G. A., & Manson, M. D.
complete list of authors
Lai, Run-Zhi||Bormans, Arjan F||Draheim, Roger R||Wright, Gus A||Manson, Michael D