Mechanism of CheA protein kinase activation in receptor signaling complexes.
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.
Levit
M N
MN
Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
Liu
Y
Y
Stock
J B
JB
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
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