Hydrogen Exchange Differences between Chemoreceptor Signaling Complexes Localize to Functionally Important Subdomains
Hydrogen Exchange Differences between Chemoreceptor Signaling Complexes Localize to Functionally Important Subdomains
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DOI：
10.1021/bi500657v
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发表时间：
2014-12-16
期刊：
影响因子：
2.9
通讯作者：
Thompson, Lynmarie K.
中科院分区：
文献类型：
--
作者：
Koshy, Seena S.;Li, Xuni;Thompson, Lynmarie K.
跨膜信号是许多由膜蛋白介导的关键生命过程之一，理解跨膜信号机制的目标推动了对细菌趋化受体的大量研究。与受体的配基结合导致a螺旋在周质和跨膜区域的活塞运动，但目前尚不清楚信号是如何通过细胞质区域传播来控制相关的激酶CheA的活性。最近的建议表明，在细胞质区域的信号涉及到不同亚域的相反的动态变化。然而，很难测量功能系统中的动态，该系统由与另外两种蛋白质Chea和Chew的受体复合体的扩展阵列组成。我们将氢交换质谱学与受体胞浆结构域功能复合体的囊泡模板组装相结合，揭示了在交换过程中存在显著的信号相关变化，这些变化定位于参与兴奋和适应反应的受体的关键区域。甲基化亚域表现出复杂的变化，包括在激酶激活状态下复合体中氢交换较慢，这可能部分与该亚域在这种状态下稳定的说法一致。该信号亚域显示出显著的保护作用，使之不受处于激活状态的复合体中氢交换的影响，这表明在这种状态下与Chea和Chew有更紧密和/或更大的相互作用界面。这些对功能信号复合体内蛋白质亚域稳定性的首次测量表明，该方法在测量多蛋白质复合体的各种生理相关状态中的功能重要蛋白质动力学方面具有前景。
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 a 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.