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

SARS-CoV-2 刺突蛋白的实时结构和功能研究

• 批准号: 10715467
• 负责人: Yi-Chih Lin
• 金额: $ 38.98万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715467
• 关键词: 2019-nCoV; ACE2; Architecture; Atomic Force Microscopy; Binding; Biochemical; Biological; Biological Assay; Biophysics; COVID-19; COVID-19 therapeutics; Cell membrane; Cell surface; Cells; Crystallography; Diagnostic; Electrons; Environment; Enzymes; Event; Fluorescence Microscopy; Glycoproteins; Human; Imaging Techniques; Integral Membrane Protein; Knowledge; Mammalian Cell; Mediating; Membrane; Membrane Fusion; Methods; Microscopic; Molecular; Molecular Conformation; Nature; Peptide Hydrolases; Physiological; Play; Preventive; Process; Proteins; Role; SARS-CoV-2 spike protein; Speed; Structure; System; Techniques; Therapeutic; Time; Viral; Viral Genome; Virus; Visualization; experimental study; intermolecular interaction; molecular assembly/self assembly; neutralizing monoclonal antibodies; novel; receptor; reconstitution; structural imaging; virus envelope

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.

抽象的： 尖峰糖蛋白（S蛋白）是严重急性信封上的病毒跨膜蛋白之一 呼吸综合征冠状病毒2（SARS-COV-2），导致2019年冠状病毒病（COVID-19）。 s- 蛋白质在介导病毒基因组进入宿主细胞的初始进入中起着至关重要的作用 血管紧张素转换酶2（ACE2），然后诱导病毒包膜与细胞之间的融合 膜。那是S蛋白是诊断和治疗测定的首选目标，包括中和 单克隆抗体（NABS）。迄今为止，S蛋白及其分子组件与ACE2的构象 和/或NABS主要由结构技术确定，包括晶体学和电子 微观方法。这些结构研究使我们能够了解分子基础的基础病毒进入 并进一步开发治疗和预防性治疗COVID-19。但是，这些解决的结构 与物理条件下蛋白质的动态性质相比，相当“静态快照”。由于 技术困难，我们对S蛋白的实时结构动态及其实时的了解 与宿主接收器，NAB和其他相关生物分子的相互作用，它们可能具有功能性 意义，仍然非常有限。 在此提案中，我的实验室将开发一个模仿生物的重建系统，并应用尖端 结构成像技术，高速原子力显微镜（HS-AFM），用于实时观察S- 蛋白质的结构动力在近乎本地的环境和各种条件下。我们还将发展 定量表征完成S-蛋白质的分子组件结构的新方法， ACE2受体，NABS，宿主蛋白酶和酶以及生物膜，可以介导 膜融合和病毒进入过程。具体而言，我们将确定S-的“实时”结构动力学 在不同状态下的蛋白质，并可视化状态转变的发生方式，例如在ACE2结合期间， NABS附着以及S蛋白亚基中的结构分裂。我的实验室将进一步发展相关 荧光显微镜和HS-AFM研究与S蛋白有关的这些动态事件 哺乳动物细胞表面。我们提出的实验中获得的生物物理和生化信息将 对S蛋白，分子间的构象状态提供全面的分子理解 S蛋白与结合分子（ACE2和NABS）之间的相互作用，S-的构象变化 用于发射病毒进入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。这里开发的方法可以进一步应用于其他接收器介导的膜融合 细胞进入系统。