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

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 流感病毒。这种蛋白质的主要功能是传导质子，使细胞内部酸化。 病毒，用于从衣壳释放病毒RNA以复制和传播感染性颗粒（11- 14）。跨膜结构域构成了M2蛋白的功能核心，对于M2蛋白的功能至关重要。 四聚化和质子传导（15），而N-和C-末端结构域是必要的病毒 萌芽（13，14，16，17）。从生物物理的角度来看，M2也是一个有趣的系统，以探测质子 扩散和pH依赖的构象变化，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途径。因为这 蛋白质对于病毒的生命周期至关重要，详细了解M2蛋白质的结构及其 质子传导的机制不仅涉及质子传导的基本过程， 和稳定膜蛋白，但也将有助于靶向M2通道阻滞剂的开发。 本文提出的研究策略旨在阐明AM 2和AM3中的质子传导机制。 BM 2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。 蛋白质在传染性流感病毒复制和传播中的作用。在这项研究中，我们建议： 1.将AM 2的质子耦合构象变化与质子传导动力学联系起来，并阐明 孔隙衬里残留物和沃茨的氢键网络，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AM 2与其相互作用配偶体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BM 2的高分辨率结构， 通过蛋白质的传导途径，并进行结构的比较分析， AM 2和BM 2的质子传导机制。

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.