Mutational activation of CheA, the protein kinase in the chemotaxis system of Escherichia coli.
In Escherichia coli and Salmonella typhimurium, appropriate changes of cell swimming patterns are mediated by CheA, an autophosphorylating histidine protein kinase whose activity is regulated by receptor/transducer proteins. The molecular mechanism underlying this regulation remains unelucidated but may involve CheA shifting between high-activity and low-activity conformations. We devised an in vivo screen to search for potential hyperkinase variants of CheA and used this screen to identify two cheA point mutations that cause the CheA protein to have elevated autokinase activity. Each point mutation resulted in alteration of proline 337. In vitro, CheA337PL and CheA337PS autophosphorylated significantly more rapidly than did wild-type CheA. This rate enhancement reflected the higher affinities of the mutant proteins for ATP and an increased rate constant for acquisition by CheA of the gamma-phosphoryl group of ATP within a kinetically defined CheA.ATP complex. In addition, the mutant proteins reacted with ADP more rapidly than did wild-type CheA. We considered the possibility that the mutations served to lock CheA into an activated signaling conformation; however, we found that both mutant proteins were regulated in a normal fashion by the transducer Tsr in the presence of CheW. We exploited the activated properties of one of these mutants to investigate whether the CheA subunits within a CheA dimer make equivalent contributions to the mechanism of trans phosphorylation. Our results indicate that CheA trans phosphorylation may involve active-site residues that are located both in cis and in trans to the autophosphorylation site and that the two protomers of a CheA dimer make nonequivalent contributions in determining the affinity of the ATP-binding site(s) of CheA.
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
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Stewart
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D002633:Chemotaxis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607604:tsr protein, E coli; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli