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

脂质双层中流感 M2 蛋白的固态 NMR

• 批准号: 8894893
• 负责人: Mei Hong
• 金额: $ 28.54万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8894893
• 关键词: Amantadine; Amantadine resistance; Antiviral Agents; Binding; Binding Sites; Biological Models; C-terminal; Chemicals; Cholesterol; Cytoplasmic Tail; Dependence; Development; Diffusion; Drug Binding Site; Flu virus; Funding; Future; Gated Ion Channel; Hydrogen Bonding; Imidazole; Immune system; Influenza; Influenza A Virus, H1N1 Subtype; Ion Channel; Ion Transport; Knowledge; Lead; Length; Leukocytes; Lipid Bilayers; M2 protein; Magic; Measurement; Measures; Mediating; Membrane; Membrane Lipids; Modeling; Molecular; Molecular Conformation; Molecular Structure; Mutation; Pharmaceutical Preparations; Potassium Channel; Protein Dynamics; Proteins; Protons; Relaxation; Research Proposals; Resolution; Rimantadine; Structure; Techniques; Testing; Transmembrane Domain; Variant; Vertebral column; Virion; Virus; Virus Assembly; Virus Diseases; Work; abstracting; base; chelation; combat; deprotonation; dimer; flu; influenzavirus; inhibitor/antagonist; insight; pandemic disease; pandemic influenza; prevent; protein complex; protonation; research study; solid state nuclear magnetic resonance; three dimensional structure; voltage

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.

抽象的 流感M2蛋白形成pH激活的质子通道，这对于病毒至关重要 生命周期。抑制抗病毒药物类肿瘤类药物的H+通道活性已有 因M2跨膜结构域突变而无效。高分辨率结构 因此，M2的确定对于开发新的抗病毒药以靶向氨酰胺 - 抗性M2变体。这种小蛋白质包含pH激活所需的所有机械， H+选择性和门控，因此是理解较大的绝佳模型系统 电压门控的H+通道和pH门控离子通道。该研究建议资助的工作 以前已导致在M2中阐明了与药理相关的药物结合位点 并揭示了质子选择残基His37的pH依赖性动力学。但是，新的H+ 此后提出了传导模型，以及TRP41的通道门控的结构基础 尚未研究。该建议的目标1是阐明M2的H+传导机制 通过检查通道激活时在酸性pH时检查HIS37结构。 h键， 质子化/去质子化动力学以及抑制剂对HIS37结构的影响将是 测量。 amantadine和Cu2+均将用作抑制剂。目标2是阐明TRP41 通过测量化学位移和原子间距离，与HIS37的结构和相互作用。在 除H+通道活性外，M2通过引起膜曲率介导病毒萌芽 以胆固醇依赖的方式。我们将研究M2膜和M2-胆固醇 通过距离和放松NMR测量的相互作用。 AIM 3将衡量M2是否 优先结合膜的高度弯曲区域。 M2与矩阵相互作用 在病毒组装和萌芽过程中，蛋白M1通过其细胞质尾巴。没有结构 信息可用于细胞质结构域。在AIM 4中，我们将确定3D 使用多维MAS相关性脂质双层中全长M2的结构 实验。