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：
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
项目类别：
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

项目摘要

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 pH 激活的质子通道，这对于病毒生命周期至关重要。 M2 跨膜结构域的突变使金刚烷胺类抗病毒药物对 H+ 通道活性的抑制变得无效。因此，M2 的高分辨率结构测定对于开发针对金刚烷胺耐药 M2 突变体的新型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、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出色的模型系统。该研究计划资助的工作已经 1) 阐明了 M2 中药理学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) 揭示了质子的新颖的 pH 依赖性动力学选择性残基，His37。然而，与此同时，新的替代H+传导模型已经被提出，并且Trp41通道门控的结构基础尚未被研究。该提案的首要目的是通过在弱酸性 pH 条件下首次激活通道时检查 His37 结构来阐明 M2 的 H+ 传导机制。将测量侧链氢键、质子化/去质子化动力学以及抑制剂对 His37 结构的影响。金刚烷胺和Cu2+都将被用作抑制剂，并且将探索Cu2+顺磁弛豫增强效应以用于结构测定。第二个目标是阐明 Trp41 结构以及与 His37 的相互作用作为 pH 函数的变化，以了解这两个残基如何协同作用以实现通道门控，同样是在双层环境中。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 测量来研究 M2-膜和 M2-胆固醇相互作用。 M2 优先定位于膜的高度弯曲区域的假设将得到检验。最后，在病毒组装和出芽过程中，M2 通过其细胞质尾部与基质蛋白 M1 相互作用。到目前为止，还没有细胞质结构域的结构信息。我们将使用多维魔角旋转固态核磁共振技术确定脂质双层中全长 M2 的三维结构，为未来研究对流感生命周期重要的 M2-M1 相互作用奠定基础。