Real-time structural and functional studies of SARS-CoV-2 spike proteins

摘要： 刺突糖蛋白（S-蛋白）是重症急性呼吸道综合征（SARS）病毒包膜上的一种跨膜蛋白。 呼吸道综合征冠状病毒2（SARS-CoV-2），导致2019冠状病毒病（COVID-19）。S-的 蛋白质在介导病毒基因组通过与人细胞结合而初始进入宿主细胞中起关键作用。 血管紧张素转换酶2（ACE 2），然后诱导病毒包膜与细胞之间的融合 膜的因此，S蛋白是诊断和治疗测定的选择靶，包括中和 单克隆抗体（nAbs）。迄今为止，S蛋白及其与ACE 2的分子组装体的构象 和/或nAb主要通过结构技术来确定，包括晶体学和电子学技术。 显微镜方法这些结构研究使我们能够了解病毒进入的分子基础 并进一步开发COVID-19的治疗和预防疗法。然而，这些分解结构 与蛋白质在生理条件下的动态性质相比，它们是“静态快照”。由于 技术上的困难，我们对S-蛋白质的实时结构动力学及其实时 与宿主受体、nAb和其他相关生物分子的相互作用，其可能具有功能性 意义，仍然非常有限。 在这个提案中，我的实验室将开发一种生物模拟重建系统，并应用最先进的 结构成像技术，高速原子力显微镜（HS-AFM），用于实时观察S- 蛋白质在接近天然环境和各种条件下的结构动力学。我们还将开发 定量表征包含S蛋白的分子组装体结构的新方法， ACE 2受体，nAb，宿主蛋白酶和酶，以及生物膜，它们可以介导 膜融合和病毒进入过程。具体来说，我们将确定S的“实时”结构动力学， 不同状态的蛋白质，并可视化状态转换如何发生，例如在ACE 2结合期间， nAbs附着，以及S蛋白亚基的结构裂解。我的实验室将进一步研究 荧光显微镜和HS-AFM来研究这些与S-蛋白相关的动态事件。 哺乳动物细胞表面。在我们提议的实验中获得的生物物理和生物化学信息将 提供了一个全面的分子理解的构象状态的S-蛋白，分子间 S蛋白和结合分子（ACE 2和nAb）之间的相互作用，S蛋白中的构象变化， 蛋白质启动膜融合过程的病毒进入，以及哺乳动物细胞表面如何影响 S蛋白该方法可进一步应用于其他受体介导的膜融合 细胞进入系统。

Abstract: Spike glycoprotein (S-protein) is one of the viral transmembrane proteins on the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19). S- protein plays a crucial role in mediating the initial entry of viral genome into the host cell by binding to the human angiotensin-converting enzyme 2 (ACE2) and then inducing fusion between the virus envelope and cell membrane. Thus, S-protein is a target of choice for diagnostic and therapeutic assays, including neutralizing monoclonal antibodies (nAbs). To date, the conformations of S-protein and its molecular assemblies with ACE2 and/or nAbs have been mainly determined by structural techniques, including crystallographic and electron microscopic methods. These structural studies allow us to understand the molecular basis underlying viral entry and to further develop treatment and preventive therapeutics for COVID-19. However, these resolved structures are rather “static snapshots” compared to the dynamic nature of proteins in physiological conditions. Due to the technical difficulties, our knowledge about the real-time structural dynamics of S-protein and its real-time interactions with host receptors, nAbs, and the other relevant biomolecules, which may have functional significance, is still very limited. In this proposal, my lab will develop a bio-mimicking reconstitution system and apply a cutting-edge structural imaging technique, high-speed atomic force microscopy (HS-AFM), for real-time observations of S- protein’s structural dynamics in close-to-native environments and under various conditions. We will also develop novel methods to quantitatively characterize the architecture of molecular assemblies comprising S-protein, ACE2 receptor, nAbs, host proteases and enzymes, and biological membranes, which can mediate the membrane fusion and viral entry processes. Specifically, we will identify the “real-time” structural dynamics of S- protein in different states and visualize how the state transitions happen, for example, during ACE2 binding, nAbs attachment, and the structural cleavages in S-protein subunits. My lab will further develop correlated fluorescence microscopy and HS-AFM to study these dynamic events associated with S-protein on the mammalian cell surface. The biophysical and biochemical information acquired in our proposed experiments will provide a comprehensive molecular understanding of the conformational states of S-protein, intermolecular interactions between S-protein and binding molecules (ACE2 and nAbs), the conformational changes in S- protein for initiating membrane fusion processes for viral entry, and how the mammalian cell surface impacts the S-protein. The developed methods here can further apply to the other receptor-mediated membrane fusion systems for cell entry.