Department of Chemistry, University of Michigan, Ann Arbor, MI 48109.
Department of Chemistry and Biochemistry, California State University, San Marcos, CA 92096.
Proc Natl Acad Sci U S A. 2020 Feb 18;117(7):3583-3591. doi: 10.1073/pnas.1913385117. Epub 2020 Feb 3.
The matrix-2 (M2) protein from influenza A virus is a tetrameric, integral transmembrane (TM) protein that plays a vital role in viral replication by proton flux into the virus. The His37 tetrad is a pH sensor in the center of the M2 TM helix that activates the channel in response to the low endosomal pH. M2 consists of different regions that are believed to be involved in membrane targeting, packaging, nucleocapsid binding, and proton transport. Although M2 has been the target of many experimental and theoretical studies that have led to significant insights into its structure and function under differing conditions, the main mechanism of proton transport, its conformational dynamics, and the role of the amphipathic helices (AHs) on proton conductance remain elusive. To this end, we have applied explicit solvent constant pH molecular dynamics using the multisite λ-dynamics approach (CpHMD) to investigate the buried ionizable residues comprehensively and to elucidate their effect on the conformational transition. Our model recapitulates the pH-dependent conformational transition of M2 from closed to open state when the AH domain is included in the M2 construct, revealing the role of the amphipathic helices on this transition and shedding light on the proton-transport mechanism. This work demonstrates the importance of including the amphipathic helices in future experimental and theoretical studies of ion channels. Finally, our work shows that explicit solvent CpHMD provides a realistic pH-dependent model for membrane proteins.
甲型流感病毒的基质蛋白 2（M2）是一个四聚体、完整的跨膜（TM）蛋白，通过质子流入病毒中在病毒复制中起着至关重要的作用。His37 四联体是 M2 TM 螺旋中心的 pH 传感器，它响应低内体 pH 激活通道。M2 由不同的区域组成，这些区域被认为参与膜靶向、包装、核衣壳结合和质子转运。尽管 M2 已经成为许多实验和理论研究的目标，这些研究对其在不同条件下的结构和功能有了重要的了解，但质子转运的主要机制、其构象动力学以及两性螺旋（AHs）在质子传导中的作用仍然难以捉摸。为此，我们应用了包含 pH 不变性的溶剂的分子动力学模拟，并采用多位点 λ 动力学方法（CpHMD）来全面研究埋藏的可离子化残基及其对构象转变的影响。我们的模型再现了 M2 从关闭到开放状态的 pH 依赖性构象转变，当 AH 结构域包含在 M2 结构中时，揭示了两性螺旋对这种转变的作用，并阐明了质子转运机制。这项工作表明，在未来对离子通道的实验和理论研究中，包括两性螺旋是很重要的。最后，我们的工作表明，包含 pH 不变性溶剂的分子动力学模拟为膜蛋白提供了一个现实的 pH 依赖性模型。
