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

项目摘要 甲型和B型流感病毒的M2蛋白（AM 2和BM 2）形成酸活化的 质子（H+）通道的病毒进入和介导膜断裂的胆固醇依赖性 病毒萌芽的时尚。AM 2被金刚烷胺类抗病毒药物抑制，直到 最近在流行的流感病毒中出现了耐药的M2突变体， 目前还没有针对BM 2的药物。因此，M2的结构和机制研究是 这对于设计新的M2抑制剂以抑制季节性流感和大流行流感非常重要。由于其模块化 M2蛋白质的性质和它的小尺寸，也可以作为一个模型系统，用于了解 离子通道中H+转运的结构原理和膜- 蛋白质的曲率诱导。到目前为止，M2如何防止反向H+的结构基础 从C-末端到N-末端的电流尚不清楚。N端胞外域 和C-末端胞质尾调节药物敏感的H+传导通过 跨膜（TM）孔和诱导膜弯曲知之甚少。结构 关于M2与胆固醇相互作用的信息很少。最后，流感的结构 尚未研究脂质双层中的BM 2，并且关于BM 2 传导质子的很少。我们建议采用固态核磁共振光谱来回答 磷脂中关于流感病毒AM 2和BM 2的结构和机制问题 双层。在目标1中，我们将研究H+传导动力学和药物结合平衡 包含胞外域和胞质尾区的全功能AM 2。2D相关 检测TM和膜外残留物的实验和 将执行探针药物定向和动力学。在目标2中，我们将研究 结构、动力学和H+传导机制。侧链构象和 将测量保守的HxxxW基序中His和Trp的残基间接触，并且 将使用1H-13 C相关实验研究通道残留物的水合作用。在 目的3，我们将研究AM 2的门控缺陷突变体，以了解Trp 41和Asp 44 确保H+从N-末端到C-末端的单向流动。侧链构象， 动力学和关键功能残基之间的残基间距离将被测量。在 目的4，我们将通过测量胆固醇取向来探测M2-胆固醇相互作用， 动力学，胆固醇诱导的化学位移变化，和分子间距离， 限制了假定的M2-胆固醇复合物。

Project Summary The M2 protein of influenza A and B viruses (AM2 and BM2) forms an acid-activated proton (H+) channel for virus entry and mediates membrane scission in a cholesterol-dependent fashion for virus budding. AM2 is inhibited by the amantadine class of antiviral drugs until the recent emergence of drug-resistant M2 mutants among circulating flu viruses, and no antiviral drugs are yet available against BM2. Thus, structural and mechanistic studies of M2 are important for designing new M2 inhibitors to curb seasonal and pandemic flu. Due to its modular nature and its small size, the M2 protein also serves as a model system for understanding the structural principles governing H+ transport in ion channels and the mechanism of membrane- curvature induction by proteins. So far, the structural basis for how M2 prevents reverse H+ current from the C-terminus to the N-terminus is not yet known. How the N-terminal ectodomain and the C-terminal cytoplasmic tail modulate drug-sensitive H+ conduction through the transmembrane (TM) pore and induce membrane curvature is poorly understood. Structural information about M2 interaction with cholesterol is scarce. Finally, the structure of influenza BM2 in lipid bilayers has not been investigated, and mechanistic information about how BM2 conducts protons is sparse. We propose to employ solid-state NMR spectroscopy to answer these structural and mechanistic questions about influenza AM2 and BM2 in phospholipid bilayers. In Aim 1, we will investigate the H+ conduction dynamics and drug binding equilibrium of fully functional AM2 containing the ectodomain and the cytoplasmic tail. 2D correlation experiments that detect both TM and extra-membrane residues and 2H NMR experiments that probes drug orientation and dynamics will be performed. In Aim 2, we will investigate the structure, dynamics and H+ conduction mechanism of BM2. The sidechain conformation and inter-residue contacts of His and Trp in the conserved HxxxW motif will be measured, and hydration of the channel residues will be investigated using 1H-13C correlation experiments. In Aim 3, we will study gating-deficient mutants of AM2 to understand how Trp41 and Asp44 ensure unidirectional H+ flow from the N-terminus to the C-terminus. Sidechain conformation, dynamics, and inter-residue distances among the key functional residues will be measured. In Aim 4, we will probe M2-cholesterol interactions by measuring cholesterol orientation and dynamics, cholesterol-induced chemical shift changes, and intermolecular distances to constrain the putative M2-cholesterol complex.