Activating and inhibitory mutations in the regulatory domain of CheB, the methylesterase in bacterial chemotaxis.
In the chemotaxis system of Escherichia coli, CheB promotes sensory adaptation by interacting with the chemotaxis receptor-transducer proteins to catalyze removal of their gamma-glutamyl methyl ester groups. CheB is comprised of two functional domains; the C-terminal domain contains the methylesterase active site, and the N-terminal domain regulates the activity of this active site. The chemotaxis system controls CheB methylesterase activity via a mechanism involving phosphorylation of the CheB regulatory domain by the chemotaxis protein kinase CheA. To further explore the communication between the regulatory and methylesterase domains of CheB, I generated mutations in the CheB regulatory domain that affect methylesterase activity in vitro. Three of these mutations (D11K, E58K, and E91K) caused increased methylesterase activity in the absence of phosphorylation, and several other mutations (R42H, R73H, and K107R) caused decreased methylesterase activity in the purified proteins. Several of these mutations (D10N, D11K, R42H, E58K, and K107R) also affected the phosphorylation biochemistry of CheB by reducing the rate of CheA-mediated phosphorylation of CheB and/or by decreasing the autodephosphorylation rate of CheB. In addition, all of these mutations diminished the ability of excess CheA to inhibit CheB methylesterase activity. The locations of these mutations in the deduced three-dimensional structure of the CheB N-terminal domain indicate that the region of the protein surrounding the putative phosphorylation site plays important roles in its interaction with the CheB C-terminal domain as well as in its interactions with CheA.
Stewart
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RC
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002630:Chemotactic Factors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002630:Chemotactic Factors; C069656:CheB protein, Bacteria; D000251:Adenosine Triphosphatases