Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290, USA.
J Bacteriol. 2011 Sep;193(18):4709-18. doi: 10.1128/JB.00070-11. Epub 2011 Jul 15.
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.
双组分调控系统中，磷酸化控制响应调节蛋白的活性，为细菌提供信号转导。例如，磷酸化 CheY 响应调节剂（CheYp）控制游动行为。在大肠杆菌中，趋化作用磷酸酶 CheZ 刺激 CheYp 的去磷酸化。CheYp 显然首先与 CheZ 的 C 末端结合，然后结合到发生去磷酸化的活性部位。CheZ(2)二聚体的磷酸酶活性对 CheYp 浓度表现出正协同依赖性，显然是因为与第二个 CheYp 的结合相比，第一个 CheYp 与 CheZ(2)的结合受到抑制。因此，CheZ 磷酸酶活性在 CheYp 浓度低时降低。CheZ21IT 功能获得性取代位于 CheZ 活性位点或 C 末端 CheY 结合位点都很远的位置，增强了 CheYp 的结合并消除了协同性。为了进一步探索调节 CheZ 活性的机制，我们分离了 cheZ21IT 的 10 个基因内抑制突变，这些突变恢复了趋化作用。抑制突变位于 CheZ 的中央部分，并且不是等位基因特异性的。五种经生化测试的抑制突变体降低了 CheYp 的结合和/或去磷酸化的催化作用，即使抑制突变远离活性位点也是如此。一个抑制突变体也恢复了 CheZ21IT 的协同性。考虑到这项和以前的研究结果表明，CheYp 与 CheZ 活性位点（而不是 C 末端）的结合是限速的，并且导致协同磷酸酶活性。此外，远离活性位点的氨基酸取代可以影响 CheZ 催化活性和 CheYp 结合，可能是通过结构或动态扰动通过螺旋束传播。
