Solid-state NMR of influenza M2 protein in lipid bilayers

脂质双层中流感 M2 蛋白的固态 NMR

• 批准号: 7939909
• 负责人: Mei Hong
• 金额: $ 27.75万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7939909
• 关键词: Address; Amantadine; Amantadine resistance; Amines; Antiviral Agents; Binding; Binding Proteins; Binding Sites; Cells; Chemicals; Complex; Data; Detergents; Diffusion; Drug Binding Site; Drug resistance; Elements; Environment; Funding; Future; Goals; Heterogeneity; Influenza; Influenza A virus; Investigation; Lipid Bilayers; M2 protein; Magic; Measures; Membrane; Membrane Lipids; Methods; Molecular Conformation; Mutation; NMR Spectroscopy; Nuclear; Peptides; Pharmaceutical Preparations; Physiological; Proteins; Protons; Relaxation; Reporting; Resistance; Resolution; Rimantadine; Roentgen Rays; Sampling; Site; Solutions; Structure; Techniques; Temperature; Testing; Time; Transmembrane Domain; Vertebral column; Viral; Virus; Virus Diseases; Work; X-Ray Crystallography; base; cold temperature; design; flexibility; inhibitor/antagonist; mutant; pandemic influenza; peptide structure; prevent; research study; solid state nuclear magnetic resonance; viral RNA

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.

描述（由申请人提供）：流感A的M2蛋白病毒形成pH门控质子通道，对病毒感染和复制很重要。抗病毒药amantadine曾经有效阻止该通道，直到最近出现的M2跨膜结构域突变体S31N的出现使病毒完全抗性。最近通过X射线晶体学和溶液NMR确定了M2跨膜肽（M2TMP）的高分辨率结构。然而，这两个结构结束了截然不同的药物结合位点，明显不同的螺旋方向和侧chain构象。由于X射线和溶液NMR结构在洗涤剂中求解，因此在脂质双层的更生物学相关环境中，这些差异敦促进行高分辨率结构研究。阐明这种重要质子通道的原子水平结构将有助于开发新的抑制剂，以防止未来的流感流感。这项工作的广泛长期目标是使用固态NMR光谱法阐明其许多功能状态下脂质双层中M2蛋白的结构和动力学。我们希望了解M2如何导致质子，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。在第一个资金期间，我们将重点关注该蛋白质的跨膜结构域，并在封闭状态（高pH）中表征APO M2TMP，在封闭状态下的药物复杂性肽，以及在开放状态的APO肽（低pH）。我们还将研究主要抗肿瘤的突变体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的魔法旋转（MAS）固态NMR，它允许原子分辨率的结构信息从无调的水合脂质膜样品中获得。基于我们的初步数据，我们假设M2TMP的关键要素是其构象柔韧性，它表现为药物和pH诱导的骨架和侧chain构象变化，迁移率变化，膜诱导的螺旋方向的变化。我们将通过测量化学位移扰动，13C和15N线宽，核自旋松弛时间和螺旋取向来检验这一普遍的假设。生理温度下的实验将表征肽的动态构象波动，而低温实验将产生平均构象和构象分布。我们将使用定量的偶性回应实验和半定量自旋扩散技术，进一步确定药物与肽之间的分子间距离。我们的目标是获得具有骨架和侧链约束的双层结合的M2TMP的高分辨率结构，并阐明由于药物结合pH的变化和突变而引起的结构差异。公共卫生相关性：脂质双层中流感病毒M2蛋白的原子分辨率结构确定与治疗流感感染并防止美国和全球未来的流感大流传学直接相关。高分辨率的结构信息对于设计新抗病毒药物的设计至关重要，以靶向抗药性突变质子质子通道，S31N-M2在流感病毒中。