Action at a Distance: Amino Acid Substitutions That Affect Binding of the Phosphorylated CheY Response Regulator and Catalysis of Dephosphorylation Can Be Far from the CheZ Phosphatase Active Site
| Autor: | Beth M. Mole, Ashalla M. Freeman, Ruth E. Silversmith, Robert B. Bourret |
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| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular Phosphatase Methyl-Accepting Chemotaxis Proteins Cooperativity Plasma protein binding Biology Microbiology Phosphates Dephosphorylation Suppression Genetic Bacterial Proteins Catalytic Domain Escherichia coli Binding site Protein Structure Quaternary Molecular Biology Binding Sites Methyl-accepting chemotaxis protein Escherichia coli Proteins Membrane Proteins Active site Phosphoric Monoester Hydrolases Amino Acid Substitution Biochemistry biology.protein Phosphorylation Locomotion Protein Binding Signal Transduction |
| Zdroj: | Journal of Bacteriology. 193:4709-4718 |
| ISSN: | 1098-5530 0021-9193 |
| Popis: | Two-component regulatory systems, in which phosphorylation controls the activity of a response regulator protein, provide signal transduction in bacteria. For example, the phosphorylated CheY response regulator (CheYp) controls swimming behavior. In Escherichia coli , the chemotaxis phosphatase CheZ stimulates the dephosphorylation of CheYp. CheYp apparently binds first to the C terminus of CheZ and then binds to the active site where dephosphorylation occurs. The phosphatase activity of the CheZ 2 dimer exhibits a positively cooperative dependence on CheYp concentration, apparently because the binding of the first CheYp to CheZ 2 is inhibited compared to the binding of the second CheYp. Thus, CheZ phosphatase activity is reduced at low CheYp concentrations. The CheZ21IT gain-of-function substitution, located far from either the CheZ active site or C-terminal CheY binding site, enhances CheYp binding and abolishes cooperativity. To further explore mechanisms regulating CheZ activity, we isolated 10 intragenic suppressor mutations of cheZ21IT that restored chemotaxis. The suppressor substitutions were located along the central portion of CheZ and were not allele specific. Five suppressor mutants tested biochemically diminished the binding of CheYp and/or the catalysis of dephosphorylation, even when the suppressor substitutions were distant from the active site. One suppressor mutant also restored cooperativity to CheZ21IT. Consideration of results from this and previous studies suggests that the binding of CheYp to the CheZ active site (not to the C terminus) is rate limiting and leads to cooperative phosphatase activity. Furthermore, amino acid substitutions distant from the active site can affect CheZ catalytic activity and CheYp binding, perhaps via the propagation of structural or dynamic perturbations through a helical bundle. |
| Databáze: | OpenAIRE |
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