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

摘要 流感M2蛋白形成一个pH激活的质子通道，这对病毒是必不可少的 生命周期。金刚烷胺类抗病毒药物对H+通道活性的抑制作用 由于M2跨膜区的突变而变得无效。高分辨率结构 因此，M2的测定对于开发针对金刚烷胺的新的抗病毒药物至关重要。 抗性M2变种。这种小蛋白质包含了激活pH所需的所有机制， H+选择性和门控，因此是了解更大 电压门控H+通道和pH门控离子通道。这项研究计划资助的工作 曾导致阐明M2中与药物相关的药物结合部位 揭示了质子选择性残基His37的pH依赖动力学。然而，新的H+ 此后，人们提出了传导模型，Trp41提出了通道选通的结构基础 还没有被研究过。这项建议的目的1是阐明M2的H+传导机制 通过检测通道被激活时在酸性pH下的His37结构。氢键， 质子化/去质子化动力学，以及抑制剂对His37结构的影响 量过了。金刚烷胺和Cu2+都将被用作抑制剂。目的2是阐明Trp41 通过测量化学位移和原子间距离，研究His37的结构和相互作用。在……里面 除了H+通道活性外，M2还通过引起膜弯曲来介导病毒的萌发 以依赖胆固醇的方式。我们将研究M2膜和M2-胆固醇 通过距离和松弛核磁共振测量的相互作用。目标3将衡量M2 优先结合到膜的高度弯曲区域。M2与矩阵相互作用 在病毒组装和萌发过程中，蛋白质M1通过其细胞质尾巴。无结构 关于细胞质区域的信息仍然可用。在目标4中，我们将确定3D 多维MAS关联研究脂质双层中全长M2的结构 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.