Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, California 93106, USA.
J Am Chem Soc. 2012 Oct 3;134(39):16107-10. doi: 10.1021/ja3056694. Epub 2012 Sep 21.
A two-component signal transduction pathway underlies the phenomenon of bacterial chemotaxis that allows bacteria to modulate their swimming behavior in response to environmental stimuli. The dimeric five-domain histidine kinase, CheA, plays a central role in the pathway, converting sensory signals to a chemical signal via trans-autophosphorylation between the P1 and P4 domains. This autophosphorylation is regulated via the networked interactions among the P5 domain of CheA, CheW, and chemoreceptors. Despite a wealth of structural information about these components and their interactions, the key question of how the kinase activity of the catalytic P4 domain is regulated by the signal received from the regulatory P5 domain remains poorly understood. We performed replica exchange molecular dynamics simulations on the CheA kinase core and found that while individual domains maintained their structural fold, these domains exhibited a variety of interdomain orientations due to two interdomain linkers. A partially populated conformation that adopts an interdomain arrangement is suitable for building a functional ternary complex. An allosteric network derived from this structural model implies critical roles for two linkers in CheA's activity. The biochemical and biological functions of these linkers were assigned via a series of biochemical and genetic assays that show the P4-P5 linker controls the activation of CheA and the P3-P4 linker controls both the basal autophosphorylation activity and the activation of CheA. These results reveal the functional dependence between the two linkers and the essential role of the linkers in passing signal information from one domain to another.
细菌趋化性现象的背后存在着双组分信号转导途径，使细菌能够根据环境刺激来调节其游动行为。二聚体五结构域组氨酸激酶 CheA 在该途径中起着核心作用，通过 P1 和 P4 结构域之间的转磷酸化将感觉信号转化为化学信号。这种自磷酸化通过 CheA 的 P5 结构域、CheW 和趋化受体之间的网络相互作用进行调节。尽管有大量关于这些成分及其相互作用的结构信息，但激酶活性如何被来自调节 P5 结构域的信号调节的关键问题仍未得到很好的理解。我们对 CheA 激酶核心进行了复制交换分子动力学模拟，发现尽管各个结构域保持其结构折叠，但由于两个结构域间接头的存在，这些结构域表现出各种结构域取向。一种部分占据的构象采用结构域间排列，适合构建功能三元复合物。从该结构模型得出的变构网络表明，两个接头在 CheA 的活性中起着关键作用。通过一系列生化和遗传实验分配了这些接头的生化和生物学功能，表明 P4-P5 接头控制 CheA 的激活，而 P3-P4 接头控制 CheA 的基础自磷酸化活性和激活。这些结果揭示了两个接头之间的功能依赖性，以及接头在将信号信息从一个结构域传递到另一个结构域中的重要作用。
