Anand G S, Goudreau P N, Stock A M
Center for Advanced Biotechnology and Medicine, Howard Hughes Medical Institute, Rutgers University, New Brunswick, NJ, USA.
Biochemistry. 1998 Oct 6;37(40):14038-47. doi: 10.1021/bi980865d.
The response regulator CheB functions within the bacterial chemotaxis system together with the methyltransferase CheR to control the level of chemoreceptor methylation, influencing the signaling activities of the receptors. CheB catalyzes demethylation of specific methylglutamate residues introduced into the chemoreceptors by CheR. CheB has a two-domain architecture consisting of an N-terminal regulatory domain joined by a linker to a C-terminal effector domain. In the unphosphorylated state of the response regulator, the regulatory domain inhibits the methylesterase activity of the effector domain. Upon phosphorylation of a specific aspartate residue within the regulatory domain, the C-terminal methylesterase activity is stimulated, resulting in the subsequent demethylation of the chemoreceptors. We have investigated the mechanism of regulation of CheB activity by the N-terminal regulatory domain. First, we have found that phosphorylation of the N-terminal domain not only relieves inhibition of the C-terminal methylesterase activity but also provides an enhancement of this activity above that seen for the C-terminal effector domain alone. Second, we have identified mutations in CheB that show an enhancement of methylesterase activity in the absence of phosphorylation. Most of these single-site mutations are localized in the linker region joining the regulatory and effector domains. On the basis of these observations, we propose a model for activation of CheB in which phosphorylation of the regulatory domain results in a reorganization of the domain interface, allowing exposure of the active site to the receptor substrate and simultaneously stimulating methylesterase activity.
应答调节蛋白CheB在细菌趋化系统中与甲基转移酶CheR共同发挥作用，以控制化学感受器的甲基化水平，影响受体的信号传导活性。CheB催化由CheR引入化学感受器的特定甲基谷氨酸残基的去甲基化反应。CheB具有两个结构域，由一个N端调节结构域通过一个连接子与一个C端效应结构域相连。在应答调节蛋白的未磷酸化状态下，调节结构域会抑制效应结构域的甲酯酶活性。当调节结构域内的一个特定天冬氨酸残基发生磷酸化时，C端甲酯酶活性会被激活，导致随后化学感受器的去甲基化。我们研究了N端调节结构域对CheB活性的调节机制。首先，我们发现N端结构域的磷酸化不仅解除了对C端甲酯酶活性的抑制，还增强了该活性，使其高于单独C端效应结构域的活性。其次，我们在CheB中鉴定出了一些突变，这些突变在未磷酸化的情况下显示出甲酯酶活性增强。这些单点突变大多位于连接调节结构域和效应结构域的连接区域。基于这些观察结果，我们提出了一个CheB激活模型，其中调节结构域的磷酸化导致结构域界面的重组，使活性位点能够暴露于受体底物并同时刺激甲酯酶活性。
