Mechanism of CheA protein kinase activation in receptor signaling complexes

Mikhail N. Levit, Yi Liu, Jeffry B. Stock

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Abstract

The chemotaxis receptor for aspartate, Tar, generates responses by regulating the activity of an associated histidine kinase, CheA. Tar is composed of an extracellular sensory domain connected by a transmembrane sequence to a cytoplasmic signaling domain. The cytoplasmic domain fused to a leucine zipper dimerization domain forms soluble active ternary complexes with CheA and an adapter protein, CheW. The kinetics of kinase activity within these complexes compared to CheA alone indicate approximately a 50% decrease in the K(M) for ATP and a 100-fold increase in the V(max). A truncated CheA construct that lacks the phosphoaccepting H-domain and the CheY/CheB-binding domain forms an activated ternary complex that is similar to the one formed by the full-length CheA protein. The V(max) of H-domain phosphorylation by this complex is enhanced approximately 60-fold, the K(M) for ATP decreased to 50%, and the K(M) for H-domain decreased to 20% of the values obtained with the same CheA construct in the absence of receptor and CheW. The kinetic data support a mechanism of CheA regulation that involves perturbation of an equilibrium between an inactive form where the H-domain is loosely bound and an active form where the H-domain is tightly associated with the CheA active site and properly positioned for phosphotransfer. The data are consistent with an asymmetric mechanism of CheA activation [Levit, M., Liu, I., Surette, M. G., and Stock, J. B. (1996) J. Biol. Chem. 271, 32057-32063] wherein only one phosphoaccepting domain of CheA at a time can interact with an active center within a CheA dimer.

Original languageEnglish (US)
Pages (from-to)6651-6658
Number of pages8
JournalBiochemistry
Volume38
Issue number20
DOIs
StatePublished - May 18 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry

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