A structural and biophysical study of the matrix proteins in influenza A/B viruses: Mechanisms of proton conduction and roles of protein-protein interactions

甲型/乙型流感病毒基质蛋白的结构和生物物理学研究：质子传导机制和蛋白质-蛋白质相互作用的作用

• 批准号: 9767794
• 负责人: Huong Tran Kratochvil
• 金额: $ 6.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767794
• 关键词: Address; Advanced Development; Amantadine; Antigens; Binding; Biophysics; C-terminal; Capsid; Cells; Complex; Coupled; Data; Dependence; Development; Diffuse; Diffusion; Electrophysiology (science); Endosomes; Golgi Apparatus; Hemagglutinin; Human; Hydrogen Bonding; Immunoprecipitation; Influenza; Influenza A virus; Influenza B Virus; Investigation; Kinetics; Length; Life Cycle Stages; Link; M2 protein; Measures; Mediating; Membrane; Membrane Proteins; Methods; Modeling; Molecular Conformation; Motion; Movement; N-terminal; Nature; Pathway interactions; Peptides; Pharmaceutical Preparations; Play; Process; Proteins; Protons; Pump; Research; Resistance; Resolution; Roentgen Rays; Role; Specificity; Spectrum Analysis; Structural Protein; Structure; System; Time; Transmembrane Domain; Viral; Virus; Virus Replication; Water; Work; X-Ray Crystallography; anti-influenza; base; biophysical analysis; channel blockers; comparative; design; hydroxyl group; influenzavirus; insight; particle; premature; prevent; protein function; protein protein interaction; protein structure; single molecule; stem; stoichiometry; stop flow technique; targeted treatment; transmission process; viral RNA

Project Summary/Abstract The matrix protein M2 is a 97-residue homotetrameric protein that performs many important functions for the influenza virus. The primary function of this protein is to conduct protons to acidify the interior of the virus for the release of viral RNA from the capsid for replication and the transmission of infectious particles (11- 14). The transmembrane domain constitutes the functional core of the M2 protein, essential for the tetramerization and proton conductance (15), while the N- and C-terminal domains are necessary for viral budding (13, 14, 16, 17). From a biophysical perspective, M2 is also an interesting system to probe proton diffusion and pH-dependent conformational change as many of these structural and functional studies would yield insight into the proton conduction pathways of more complex proton channels and pumps. Because this protein is crucial for the viral life cycle, a detailed understanding of the M2 protein structure and its mechanisms of proton conduction will not only address the very fundamental processes of proton conduction and stabilization in membrane proteins, but will also aid in the development of targeted M2 channel blockers. The proposed research strategy herein seeks to elucidate the proton conduction mechanism in both AM2 and BM2 as well as probe its interactions with another matrix protein M1 to better understand the roles of these proteins in the replication and transmission of infectious influenza viruses. In this research, we propose to: 1. Link the proton-coupled conformational changes of AM2 to the kinetics of proton conduction and elucidate the hydrogen-bonding network of pore-lining residues and waters that stabilize the proton as it diffuses to the His tetrad to obtain a detailed picture of the conduction pathway through this protein. 2. Confirm the specificity and stoichiometry of binding of full-length AM2 to its interaction partner M1 and obtain a high-resolution structure of the resulting complex. 3. Determine the high-resolution structure of the functional core and of full-length BM2 to establish the conduction pathway through the protein and carry out a comparative analysis of the structures and mechanisms of proton conduction of AM2 and BM2.

项目摘要/摘要 基质蛋白M2是一种97个残基的同源四聚体蛋白，具有许多重要功能 流感病毒。这种蛋白质的主要功能是传导质子，使其内部酸化。 用于从衣壳中释放病毒RNA以进行复制和传播感染颗粒的病毒(11- 14)。跨膜结构域构成M2蛋白的功能核心，对 四聚和质子电导(15)，而N-末端和C-末端结构域是病毒所必需的 萌芽(13、14、16、17)。从生物物理的角度来看，M2也是一个有趣的探测质子的系统 扩散和pH依赖的构象变化，就像许多这样的结构和功能研究 深入了解更复杂的质子通道和泵的质子传导路径。因为这件事 蛋白质对于病毒的生命周期至关重要，详细了解M2蛋白质的结构和它的 质子传导机制不仅涉及质子传导的最基本过程 并稳定在膜蛋白，但也将有助于开发靶向M2通道阻滞剂。 本文提出的研究策略试图阐明AM2和AM2中的质子传导机制。 以及探索其与另一种基质蛋白M1的相互作用，以更好地了解这些蛋白的作用 与传染性流感病毒的复制和传播有关的蛋白质。在这项研究中，我们建议： 1.将AM2的质子偶联构象变化与质子传导动力学联系起来，并阐明 由孔衬残渣和水组成的氢键网络，当质子扩散到 His四分体，以获得通过该蛋白质的传导途径的详细图像。 2.确认全长AM2与其相互作用伙伴M1和 获得所得到的复合体的高分辨率结构。 3.确定功能核和全长BM2的高分辨率结构，以建立 并对蛋白质的传导途径和结构进行了比较分析 AM2和BM2的质子传导机制。