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

摘要： S蛋白(S蛋白)是一种存在于重症急性冠状病毒囊膜上的跨膜蛋白。 导致2019年冠状病毒疾病的呼吸综合征冠状病毒2(新冠肺炎)。S- 蛋白质通过与人结合，在病毒基因组最初进入宿主细胞的过程中起着至关重要的作用 血管紧张素转换酶2(ACE2)诱导病毒被膜与细胞融合 薄膜。因此，S蛋白是包括中和在内的诊断和治疗分析的首选靶点 单抗(NAB)。迄今为止，S蛋白的构象及其与血管紧张素转换酶2的分子组装 和/或NaB主要由结构技术确定，包括结晶学和电子 显微方法。这些结构研究使我们能够理解病毒进入的分子基础。 并进一步开发新冠肺炎的治疗和预防疗法。然而，这些解析的结构 与生理条件下蛋白质的动态性质相比，它们更像是“静态快照”。由于 技术难点，我们对S蛋白质实时结构动力学及其实时结构动力学的认识 与宿主受体、NAB和其他相关生物分子的相互作用，这些生物分子可能具有 意义，仍然非常有限。 在这项提案中，我的实验室将开发一个仿生重建系统，并应用尖端技术 结构成像技术，高速原子力显微镜，实时观察S- 蛋白质在接近自然的环境和各种条件下的结构动力学。我们还将发展 定量表征S-蛋白质分子集合体结构的新方法 ACE2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。具体地说，我们将认定S的结构动力学-- 蛋白质处于不同的状态，并可视化状态转换是如何发生的，例如，在ACE2结合期间， NABS附着，S蛋白亚基的结构裂解。我的实验室将进一步发展相关的 用荧光显微镜和HS-AFM研究这些与S蛋白有关的动态事件 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化信息将 提供对S-蛋白质构象状态的全面分子理解，分子间 S-蛋白质与结合分子(ACE2和NaBS)的相互作用，S的构象变化- 启动病毒进入的膜融合过程的蛋白质，以及哺乳动物细胞表面如何影响 S-蛋白质。该方法可进一步应用于其他受体介导的膜融合。 用于单元格输入的系统。

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.