Cryo-Electron and Biochemical Anaysis of Native Paramyxovirus Fusion Complexes

描述（由申请人提供）：蛋白质介导的膜融合对于维持真核细胞组织和主要人类病毒的繁殖至关重要。许多临床相关的副粘病毒家族成员依靠两种包膜糖蛋白（附着蛋白和融合蛋白）的协同作用，将其包膜与靶细胞质膜融合，从而进入细胞。然而，尽管它们具有临床重要性，但控制天然副粘病毒融合复合物的组织和功能的基本机制原理尚不清楚。为了阐明这些原理，本项目将在三个基本问题上探讨这个问题：在感染颗粒表面显示的天然亚稳构象中，包膜糖蛋白异聚物复合物的空间组织是什么？受体结合如何影响附着蛋白的组织？是什么分子机制将受体结合与融合蛋白重新折叠成热动力学稳定的融合后构象？随着不同副粘病毒糖蛋白分离外结构域的晶体结构的掌握，拟议的研究将集中在麻疹病毒包膜糖蛋白上，以一种综合的、跨学科的方法，结合创新的成像、生化、功能和计算实验策略来解决这些问题。冷冻电子断层扫描结合包膜糖蛋白工程将阐明在病毒颗粒表面显示的水合融合复合物的整体空间组织，单独和经过可溶性受体处理（目的1）。天然凝胶电泳、H低聚物稳定和不稳定修饰以及H双分子互补将通过评估受体结合对附着蛋白组织的影响以及表征启动融合蛋白重折叠信号的分子性质来提取功能信息（目的2）。分子模型引导的诱变、生化接触域定位、肽结合和质谱将交叉检验、扩展和功能表征在初步研究中发现的候选分子间接触，从而鉴定出糖蛋白中的离散微域，这些微域排列在异寡聚物界面上，并控制天然副粘病毒融合复合体的结构完整性（目标3）。

DESCRIPTION (provided by applicant): Protein-mediated membrane fusion is essential for maintaining eukaryotic cell organization and propagation of major human viruses. Many clinically relevant members of the paramyxovirus family rely on the concerted action of two envelope glycoproteins, the attachment and fusion protein, to fuse their envelope with the target cell plasma membrane for cell entry. However, despite their clinical importance, fundamental mechanistic principles that govern the organization and function of native paramyxovirus fusion complexes are not understood. Towards the overarching goal of elucidating these principles, this project will pursue the problem in three basic questions: What is the spatial organization of the envelope glycoprotein hetero-oligomer complexes in the native, metastable conformation displayed on the surface of infectious particles? How does receptor binding affect attachment protein organization? What is the molecular mechanism that links receptor binding with fusion protein refolding into the thermodynamically stable postfusion conformation? With crystal structures of isolated ectodomains of different paramyxovirus glycoproteins at hand, the proposed studies will focus on measles virus envelope glycoproteins to address these questions in a comprehensive, interdisciplinary approach that interfaces innovative imaging, biochemical, functional, and computational experimental strategies. Cryo-electron tomography combined with envelope glycoprotein engineering will elucidate the overall spatial organization of hydrated fusion complexes displayed on the surface of viral particles, alone and after treatment with soluble receptor (aim 1). Native gel electrophoresis, H oligomer stabilizing and destabilizing modifications, and H bimolecular complementation will extract functional information by assessing the effect of receptor binding on attachment protein organization and characterizing the molecular nature of the signal that initiates fusion protein refolding (aim 2). Molecular modeling-guided mutagenesis, biochemical contact domain mapping, peptide binding and mass spectrometry will cross-examine, expand and functionally characterize candidate intermolecular contacts found in pilot studies, resulting in the identification of discrete microdomains in either glycoprotein that line the hetero-oligomer interface and control the structural integrity of a native paramyxovirus fusion complex (aim 3).