Solid-state NMR of influenza M2 protein in lipid bilayers

描述（由申请人提供）：甲型流感病毒M2蛋白形成ph门控质子通道，对病毒感染和复制很重要。抗病毒药物金刚烷胺过去可以有效阻断这一通道，直到最近出现的M2跨膜结构域的突变体S31N使病毒完全具有耐药性。最近用x射线晶体学和溶液核磁共振测定了M2跨膜肽（M2TMP）的高分辨率结构。然而，这两种结构的药物结合位点明显不同，螺旋取向和侧链构象也明显不同。由于在洗涤剂中解决了x射线和溶液核磁共振结构，这些差异迫切需要在更具有生物学相关性的脂质双分子层环境中进行高分辨率结构研究。阐明这一重要质子通道的原子水平结构将有助于开发新的抑制剂，以预防未来的流感大流行。这项工作的长期目标是利用固态核磁共振波谱技术阐明脂质双分子层中M2蛋白在许多功能状态下的结构和动力学。我们希望了解M2如何传导质子，药物分子如何阻断通道，以及位点特异性突变如何改变结构以逃避药物结合。在第一期资助期内，我们将重点研究该蛋白的跨膜结构域，对封闭状态（高pH）的载脂蛋白M2TMP、封闭状态的药物络合肽和开放状态（低pH）的载脂蛋白肽进行表征。我们还将研究主要的金刚烷胺抗性突变体S31N-M2TMP的结构，并将其与野生型肽的结构进行比较。我们的主要方法是高分辨率魔角旋转（MAS）固态核磁共振，它允许从无取向水合脂膜样品中获得原子分辨率的结构信息。根据我们的初步数据，我们假设M2TMP的一个关键因素是其构象灵活性，表现为药物和ph诱导的主链和侧链构象改变、迁移率改变和膜诱导的螺旋取向改变。我们将通过测量化学位移扰动、13C和15N线宽、核自旋弛豫时间和肽的各种状态下的螺旋取向来测试这一一般假设。生理温度下的实验将表征肽的动态构象波动，低温下的实验将得到平均构象和构象分布。我们将进一步利用定量偶极重偶联实验和半定量自旋扩散技术确定药物和肽之间的分子间距离。我们的目标是获得具有主链和侧链约束的双层结合M2TMP的高分辨率结构，并阐明由于药物结合pH变化和突变导致的结构差异。公共卫生相关性：甲型流感病毒脂质双层中M2蛋白的原子分辨率结构测定与治疗流感感染和预防未来在美国和世界范围内的流感大流行直接相关。高分辨率的结构信息对于设计针对甲型流感病毒中耐药突变质子通道S31N-M2的新型抗病毒药物至关重要。

DESCRIPTION (provided by applicant): The M2 protein of influenza A virus forms a pH-gated proton channel that is important for viral infection and replication. The antiviral drug amantadine used to be effective in blocking this channel, until the recent emergence of a mutant, S31N, of the M2 transmembrane domain rendered the viruses completely resistant. The high- resolution structure of the M2 transmembrane peptide (M2TMP) is recently determined by X-ray crystallography and solution NMR. However, the two structures concluded dramatically different drug binding sites and noticeably different helix orientations and sidechain conformations. Since the X-ray and solution NMR structures were solved in detergents, these differences urge for high-resolution structural investigations in the more biologically relevant environment of lipid bilayers. Elucidation of the atomic-level structure of this important proton channel will help to develop new inhibitors to prevent future influenza pandemics. The broad, long-term objective of this work is to elucidate the structure and dynamics of the M2 protein in lipid bilayers in many of its functional states using solid- state NMR spectroscopy. We wish to understand how M2 conducts protons, how drug molecules block the channel, and how site-specific mutations alter the structure to evade drug binding. In the first funding period, we will focus on the transmembrane domain of the protein, and characterize the apo M2TMP in the closed state (high pH), the drug-complexed peptide in the closed state, and the apo peptide in the open state (low pH). We will also investigate the structure of the main amantadine-resistant mutant, S31N-M2TMP, and compare it with the structure of the wild-type peptide. Our main method is high-resolution magic-angle spinning (MAS) solid-state NMR, which allows atomic-resolution structural information to be obtained from unoriented hydrated lipid membrane samples. Based on our preliminary data, we hypothesize that a key element of M2TMP is its conformational flexibility, which is manifested as drug- and pH-induced backbone and sidechain conformational changes, mobility changes, and membrane-induced helix orientation changes. We will test this general hypothesis by measuring chemical shift perturbations, 13C and 15N linewidths, nuclear spin relaxation times, and the helix orientation in various states of the peptide. Experiments at physiological temperature will characterize the dynamic conformational fluctuations of the peptide, while low-temperature experiments will yield the average conformation and conformational distribution. We will further determine intermolecular distances between the drug and the peptide using both quantitative dipolar recoupling experiments and semi-quantitative spin diffusion techniques. Our goal is to obtain a high-resolution structure of bilayer-bound M2TMP with both backbone and sidechain constraints, and to elucidate the structural differences due to drug binding pH change, and mutation. PUBLIC HEALTH RELEVANCE: Atomic-resolution structure determination of the M2 protein of influenza A viruses in lipid bilayers is directly relevant to treating influenza infection and preventing future flu pandemics in the US and worldwide. The high-resolution structural information will be crucial for the design of new antiviral drugs to target the drug-resistant mutant proton channel, S31N-M2 in influenza A viruses.