Koshy Seena S, Li Xuni, Eyles Stephen J, Weis Robert M, Thompson Lynmarie K
Department of Chemistry, ‡Department of Biochemistry and Molecular Biology, and §Program in Molecular and Cellular Biology, University of Massachusetts , Amherst, Massachusetts 01003, United States.
Biochemistry. 2014 Dec 16;53(49):7755-64. doi: 10.1021/bi500657v. Epub 2014 Dec 3.
The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes.
理解跨膜信号传导机制是膜蛋白介导的众多关键生命过程之一，这一目标推动了对细菌趋化性受体的大量研究。配体与受体结合会导致周质和跨膜结构域中α螺旋的活塞式运动，但尚不清楚信号随后如何通过细胞质结构域传播以控制相关激酶CheA的活性。最近的提议表明，细胞质结构域中的信号传导涉及不同亚结构域动力学的相反变化。然而，在由受体复合物与另外两种蛋白质CheA和CheW组成的扩展阵列构成的功能系统中测量动力学一直很困难。我们将氢交换质谱与受体细胞质结构域功能复合物的囊泡模板组装相结合，以揭示交换中存在与信号传导相关的显著变化，并且这些变化定位于受体中参与兴奋和适应反应的关键区域。甲基化亚结构域表现出复杂的变化，包括处于激酶激活状态的复合物中氢交换较慢，这可能部分与该亚结构域在这种状态下稳定的提议一致。信号传导亚结构域在处于激酶激活状态的复合物中表现出对氢交换的显著保护，表明在这种状态下与CheA和CheW的相互作用界面更紧密和/或更大。这些对功能信号复合物中蛋白质亚结构域稳定性的首次测量证明了这种方法在测量多蛋白复合物各种生理相关状态下功能重要的蛋白质动力学方面的前景。
