title: Mechanism of CheA protein kinase activation in receptor signaling complexes.
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 KM for ATP and a 100-fold increase in the Vmax. 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 Vmax of H-domain phosphorylation by this complex is enhanced approximately 60-fold, the KM for ATP decreased to 50%, and the KM 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.
authors.affiliation: Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
authors.forename: M N
authors.initials: MN
authors.lastname: Levit
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: J B
authors.initials: JB
authors.lastname: Stock
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002384:Catalysis; D002628:Chemoreceptor Cells; D002633:Chemotaxis / Q000502:physiology*; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008956:Models, Chemical; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D012486:Salmonella typhimurium; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; C489103:CheW protein, E coli; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; C000607606:cheY protein, E coli; C051236:CheW protein, Bacteria; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli