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.

项目概要/摘要 基质蛋白 M2 是一种具有 97 个残基的同源四聚体蛋白，具有许多重要功能 对于流感病毒。该蛋白质的主要功能是传导质子以酸化细胞内部 病毒从衣壳中释放病毒RNA以进行复制和传播感染性颗粒（11- 14）。跨膜结构域构成 M2 蛋白的功能核心，对于 四聚化和质子电导 (15)，而 N 端和 C 端结构域是病毒所必需的 正在萌芽（13、14、16、17）。从生物物理学的角度来看，M2也是一个有趣的探测质子的系统 扩散和 pH 依赖性构象变化，因为许多这些结构和功能研究将 深入了解更复杂的质子通道和泵的质子传导路径。因为这个 蛋白质对于病毒生命周期至关重要，详细了解 M2 蛋白质结构及其作用 质子传导机制不仅会解决质子传导的基本过程 和膜蛋白的稳定性，但也有助于开发靶向 M2 通道阻滞剂。 本文提出的研究策略旨在阐明 AM2 和 AM2 中的质子传导机制。 BM2 以及探测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 传染性流感病毒复制和传播中的蛋白质。在这项研究中，我们建议： 1. 将AM2的质子耦合构象变化与质子传导动力学联系起来并加以阐明 孔隙衬里残留物和水的氢键网络，在质子扩散到 His 四分体以获得通过该蛋白质的传导途径的详细图片。 2. 确认全长 AM2 与其相互作用伙伴 M1 结合的特异性和化学计量 获得所得复合物的高分辨率结构。 3. 确定功能核心和全长BM2的高分辨率结构，以建立 通过蛋白质的传导途径并进行结构和比较分析 AM2 和 BM2 的质子传导机制。