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

基本信息

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
项目类别：
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

项目摘要

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，这对病毒至关重要生命周期。金刚烷胺类抗病毒药物对 H+ 通道活性的抑制 M2 跨膜结构域突变使其无效。高分辨率结构因此，M2 的测定对于开发针对金刚烷胺的新型抗病毒药物至关重要。抗性M2变体。这种小蛋白质包含 pH 激活所需的所有机制， H+ 选择性和门控，因此是了解更大的模型系统电压门控 H+ 通道和 pH 门控离子通道。本研究提案资助的工作之前已经阐明了 M2 中药理学相关的药物结合位点并揭示了质子选择性残基 His37 的 pH 依赖性动力学。然而，新的H+ 此后提出了传导模型，以及Trp41通道门控的结构基础还没有研究过。该提案的目标1是阐明M2的H+传导机制通过在通道激活时在酸性 pH 条件下检查 His37 结构。 H键，质子化/去质子化动力学，抑制剂对 His37 结构的影响将是测量。金刚烷胺和 Cu2+ 都将用作抑制剂。目标 2 是阐明 Trp41 通过测量化学位移和原子间距离来了解 His37 的结构和相互作用。在除了 H+ 通道活性外，M2 通过引起膜弯曲来介导病毒出芽以胆固醇依赖性方式。我们将研究 M2-膜和 M2-胆固醇通过距离和弛豫核磁共振测量的相互作用。目标 3 将测量 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出芽过程中，蛋白质 M1 通过其细胞质尾部发挥作用。无结构性细胞质结构域的信息尚不清楚。在目标 4 中，我们将确定 3D 使用多维 MAS 相关性研究脂质双层中全长 M2 的结构实验。