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: 8508272
• 负责人: Mei Hong
• 金额: $ 28.24万
• 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 2014-07-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8508272
• 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.

描述（由申请人提供）：流感M2蛋白形成对病毒生命周期必不可少的pH激活质子通道。通过M2跨膜结构域突变使抗病毒药物类肿瘤类别的H+通道活性抑制。因此，对于开发新的抗病毒药物以靶向抗肿瘤的M2突变体，M2的高分辨率结构测定至关重要。小型M2蛋白包含pH激活，H+选择性和门控所需的所有机械，因此还提供了一个出色的模型系统，用于了解更大，更复杂的电压门控H+通道和其他pH门控离子通道。该研究建议资助的工作已经已经1）导致M2中的药理相关药物结合位点阐明了脂质双层中的药物相关的药物结合位点，以及2）揭示了质子选择性残留物的新型pH依赖性动力学，HIS37。但是，在此期间已经提出了新的替代H+传导模型，并且尚未研究TRP41通道门控的结构基础。该提案的第一个目的是通过在首次激活通道时检查中等酸性pH的HIS37结构来阐明M2的H+传导机制。 Sidechain H键，质子化/去质子化动力学以及抑制剂对HIS37结构的影响。 Amantadine和Cu2+都将用作抑制剂，将探索Cu2+顺磁性弛豫增强效应以进行结构测定。第二个目的是阐明TRP41结构并与HIS37作为pH的函数相互作用，以了解这两个残基如何在双层环境中再次起作用以实现通道门控。除了H+通道活性外，M2还通过以胆固醇依赖性的方式引起膜曲率来介导病毒萌芽。我们将通过距离和松弛NMR测量来研究M2膜和M2-胆固醇的相互作用。将测试M2优先定位于膜的高度弯曲区域的假设。最后，在病毒组装和萌芽过程中，M2通过其细胞质尾巴与基质蛋白M1相互作用。到目前为止，对于细胞质结构域尚无结构信息。我们将使用多维魔术旋转固态NMR技术来确定脂质双层中全长M2的三维结构，以为对流感生命周期重要的M2-M1相互作用的未来研究奠定基础。