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

项目摘要 甲型和乙型流感病毒的M2蛋白(AM2和BM2)形成酸激活的 病毒进入的质子(H+)通道和介导膜断裂的胆固醇依赖型 病毒萌芽的时尚。AM2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突变株，没有抗病毒药物 目前还没有针对BM2的药物。因此，M2的结构和机制研究是 对于设计新的M2抑制剂以遏制季节性和大流行流感非常重要。由于它是模块化的 由于M2蛋白的性质及其小的尺寸，它也可以作为一个模型系统来理解 H+在离子通道中传输的结构原理和膜的作用机制 由蛋白质诱导的曲率。到目前为止，M2如何防止反向H+的结构性基础 从C端到N端的电流尚不清楚。N端胞外域是如何 而C-末端胞质尾巴通过调节药物敏感性H+传导 跨膜(TM)孔和诱导膜弯曲的机制还知之甚少。结构性 关于M2与胆固醇相互作用的信息很少。最后，流感的结构 BM2在脂质双分子层中的作用尚未被研究，而有关BM2如何 传导质子是稀疏的。我们建议使用固态核磁共振波谱来回答 流感病毒AM2和BM2在磷脂中的结构和机制问题 双层的。在目标1中，我们将研究H+传导动力学和药物结合平衡 包括胞外结构域和细胞质尾巴的全功能AM2。二维相关 同时检测TM和膜外残留物的实验和2H核磁共振实验 将进行药物定向和动力学探头。在目标2中，我们将调查 BM2的结构、动力学和H+传导机制。侧链构象和 将测量His和Trp在保守的HxxxW基序中的残基间接触，以及 通道残基的水化将通过1H-13C关联实验进行研究。在……里面 目的3，我们将研究AM2的门控缺陷突变体，以了解Trp41和Asp44如何 确保从N端到C端的H+单向流动。侧链构象， 动力学，以及关键功能残基之间的残基间距离将被测量。在……里面 目标4，我们将通过测量胆固醇取向和胆固醇来探讨M2与胆固醇的相互作用 动力学，胆固醇诱导的化学位移变化，以及分子间距离 抑制可能的M2-胆固醇复合体。

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.