Solid-state NMR of the influenza M2 protein in lipid bilayers

摘要 流感M2蛋白形成了一个pH激活的质子通道，这是病毒所必需的 生命周期金刚烷胺类抗病毒药物抑制H+通道活性， M2跨膜结构域的突变使其无效。高分辨率结构 因此，M2的测定对于开发靶向金刚烷胺的新抗病毒药物至关重要- M2变种。这种小蛋白质含有pH激活所需的所有机制， H+选择性和门控，因此是一个很好的模型系统，用于了解更大的 电压门控H+通道和pH门控离子通道。本研究计划资助的工作 先前已导致M2中与药物相关的药物结合位点的阐明 并揭示了质子选择性残基His 37的pH依赖性动力学。新H+ 此后提出了传导模型，并且Trp 41的沟道门控的结构基础 尚未被研究。目的1是阐明M2的H+传导机制 通过检查His 37结构在酸性pH值时，通道被激活。H键， 质子化/去质子化动力学，以及抑制剂对His 37结构的影响将被 测定了金刚烷胺和Cu 2+均将用作抑制剂。目的二是阐明Trp 41的分子结构 通过测量化学位移和原子间距离来分析结构和与His 37的相互作用。在 除了H+通道活性外，M2还通过引起膜弯曲介导病毒出芽 以胆固醇依赖的方式。我们将研究M2膜和M2胆固醇 通过距离和弛豫NMR测量的相互作用。目标3将衡量M2是否 优先结合到膜的高度弯曲区域。M2与基质相互作用 蛋白M1在病毒装配和出芽过程中通过其胞质尾区。没有结构 胞质结构域的信息仍然可用。在目标4中，我们将确定 利用多维MAS相关性研究脂双层中全长M2结构 实验

Abstract The influenza M2 protein forms a pH-activated proton channel that is essential for the virus lifecycle. Inhibition of the H+ channel activity by the amantadine class of antiviral drugs has been made ineffective by mutations in the M2 transmembrane domain. High-resolution structure determination of M2 is thus paramount for developing new antiviral drugs to target amantadine- resistant M2 variants. This small protein contains all the machinery necessary for pH activation, H+ selectivity, and gating, and is thus an excellent model system for understanding larger voltage-gated H+ channels and pH-gated ion channels. Work funded by this research proposal has previously led to the elucidation of the pharmacologically relevant drug-binding site in M2 and revealed pH-dependent dynamics of the proton-selective residue, His37. However, new H+ conduction models have since been proposed, and the structure basis for channel gating by Trp41 has not been studied. Aim 1 of this proposal is to elucidate the H+ conduction mechanism of M2 by examining His37 structure at acidic pH when the channel is activated. H-bonding, protonation/deprotonation dynamics, and the effects of inhibitors on His37 structure will be measured. Both amantadine and Cu2+ will be used as inhibitors. Aim 2 is to elucidate Trp41 structure and interaction with His37 by measuring chemical shifts and inter-atomic distances. In addition to the H+ channel activity, M2 mediates virus budding by causing membrane curvature in a cholesterol-dependent fashion. We will investigate M2-membrane and M2-cholesterol interactions by distance and relaxation NMR measurements. Aim 3 will measure whether M2 preferentially binds to highly curved regions of the membrane. M2 interacts with the matrix protein M1 through its cytoplasmic tail during virus assembly and budding. No structural information is yet available for the cytoplasmic domain. In aim 4 we will determine the 3D structure of full-length M2 in lipid bilayers using multidimensional MAS correlation experiments.