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: 8211383
• 负责人: Mei Hong
• 金额: $ 27.35万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8211383
• 关键词: Amantadine; Amantadine resistance; Antiviral Agents; Bacteria; Binding Sites; Biological Models; C-terminal; Cholesterol; Complex; Cytoplasmic Tail; Dependence; Development; Diffusion; Drug Binding Site; Environment; Flu virus; Funding; Future; Gated Ion Channel; Hydrogen Bonding; Imidazole; Influenza; Influenza A Virus, H1N1 Subtype; Investigation; Ion Channel; Ion Transport; Knowledge; Lead; Length; Leukocytes; Life Cycle Stages; 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; base; cell killing; chelation; combat; deprotonation; dimer; flu; influenzavirus; inhibitor/antagonist; insight; mutant; novel; pandemic disease; pandemic influenza; prevent; protein complex; protonation; research study; solid state nuclear magnetic resonance; three dimensional structure; voltage

DESCRIPTION (provided by applicant): 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 mutants. The small M2 protein contains all the machinery necessary for pH activation, H+ selectivity, and gating, and thus also provides an excellent model system for understanding larger and more complex voltage-gated H+ channels and other pH-gated ion channels. Work funded by this research proposal has already 1) led to the elucidation of the pharmacologically relevant drug binding site in M2 and the drug-complexed high-resolution structure in the lipid bilayer, and 2) revealed novel pH-dependent dynamics of the proton-selective residue, His37. However, new alternative H+ conduction models have been proposed in the meantime, and the structure basis for channel gating by Trp41 has not been studied. The first aim of this proposal is to elucidate the H+ conduction mechanism of M2 by examining His37 structure at mildly acidic pH when the channel is first activated. Sidechain 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, and Cu2+ paramagnetic relaxation enhancement effects will be explored for structure determination. The second aim is to elucidate Trp41 structure and interaction with His37 as a function of pH, to understand how these two residues act in unison to achieve channel gating, again in a bilayer environment. In addition to the H+ channel activity, M2 also 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. The hypothesis that M2 preferentially localizes to highly curved regions of the membrane will be tested. Finally, M2 interacts with matrix protein M1 through its cytoplasmic tail during virus assembly and budding. No structural information is available so far for the cytoplasmic domain. We will determine the three-dimensional structure of full-length M2 in lipid bilayers using multidimensional magic-angle-spinning solid-state NMR techniques, to lay the ground for future investigations of the M2-M1 interactions important for the influenza life cycle. PUBLIC HEALTH RELEVANCE: The influenza virus M2 protein is the target of an antiviral drug but has recently evolved to evade it. This project seeks to elucidate the atomic structure of the M2 protein in the hope that this knowledge will lead to new antiviral drugs to combat future flu pandemics. Elucidating the molecular basis for the proton-channel activity of the M2 protein may also give insights into the inner workings of proton channels of white blood cells that kill bacteria.

描述（由申请方提供）：流感病毒M2蛋白形成了一个pH激活的质子通道，这对病毒的生命周期至关重要。金刚烷胺类抗病毒药物对H+通道活性的抑制由于M2跨膜结构域的突变而变得无效。因此，M2的高分辨率结构测定对于开发针对金刚烷胺抗性M2突变体的新的抗病毒药物至关重要。小M2蛋白包含pH激活、H+选择性和门控所需的所有机制，因此也为理解更大和更复杂的电压门控H+通道和其他pH门控离子通道提供了一个极好的模型系统。这项研究计划资助的工作已经：1）阐明了M2中与药物相关的药物结合位点和脂质双层中药物复合的高分辨率结构，2）揭示了质子选择性残基His 37的新pH依赖性动力学。然而，新的替代H+导电模型已被提出的同时，和Trp 41的通道门控的结构基础还没有被研究。该建议的第一个目的是通过检查His 37结构在弱酸性pH值时，通道首次激活，以阐明H+传导机制的M2。侧链氢键，质子化/去质子化动力学，和抑制剂对His 37结构的影响将被测量。金刚烷胺和Cu 2+将被用作抑制剂，并且Cu 2+顺磁弛豫增强效应将被探索用于结构测定。第二个目的是阐明Trp 41的结构和与His 37的相互作用作为pH的函数，以了解这两个残基如何一致地作用以实现通道门控，再次在双层环境中。除了H+通道活性外，M2还通过以胆固醇依赖性方式引起膜弯曲来介导病毒出芽。我们将通过距离和弛豫NMR测量研究M2-膜和M2-胆固醇的相互作用。将检验M2优先定位于膜的高度弯曲区域的假设。最后，在病毒装配和出芽过程中，M2通过其胞质尾区与基质蛋白M1相互作用。到目前为止，还没有关于胞质结构域的结构信息。我们将使用多维魔角旋转固态NMR技术确定脂质双层中全长M2的三维结构，为未来研究流感生命周期中重要的M2-M1相互作用奠定基础。 公共卫生关系：流感病毒M2蛋白是一种抗病毒药物的靶点，但最近已经进化到逃避它。该项目旨在阐明M2蛋白的原子结构，希望这一知识将导致新的抗病毒药物来对抗未来的流感大流行。阐明M2蛋白质质子通道活性的分子基础也可能使我们深入了解杀死细菌的白色血细胞质子通道的内部工作原理。