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： 刺突糖蛋白（S-蛋白）是重症急性冠状病毒包膜上的病毒跨膜蛋白之一 呼吸综合征冠状病毒 2 (SARS-CoV-2)，它会导致 2019 年冠状病毒病 (COVID-19)。 蛋白质通过与人类结合，在介导病毒基因组首次进入宿主细胞中发挥着至关重要的作用 血管紧张素转换酶2（ACE2），然后诱导病毒包膜和细胞之间的融合 因此，S-蛋白是诊断和治疗分析（包括中和分析）的首选靶标。 单克隆抗体 (nAb) 迄今为止，S 蛋白的构象及其与 ACE2 的分子组装。 和/或 nAb 主要通过结构技术确定，包括晶体学和电子学 这些结构研究使我们能够了解病毒进入的分子基础。 并进一步开发针对 COVID-19 的治疗和预防疗法。然而，这些已解决的结构。 与生理条件下蛋白质的动态性质相比，它们是相当“静态的快照”。 技术难点，我们对S-蛋白的实时结构动力学及其实时性的了解 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功能性 的意义，还是很有限的。 在这个提案中，我的实验室将开发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（HS-AFM），用于实时观察S- 我们还将开发接近天然环境和各种条件下蛋白质的结构动力学。 定量表征包含 S-蛋白的分子组装体结构的新方法， ACE2 受体、nAb、宿主蛋白酶和酶以及生物膜，可介导 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 ACE2 结合期间， nAb 附着和 S 蛋白亚基的结构裂解我的实验室将进一步开发相关的研究。 荧光显微镜和 HS-AFM 来研究与 S 蛋白相关的这些动态事件 在我们提出的实验中获得的生物物理和生化信息将 提供对 S 蛋白构象状态、分子间结构的全面分子理解 S-蛋白和结合分子（ACE2和nAb）之间的相互作用，S-蛋白的构象变化 用于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 这里开发的方法可以进一步应用于其他受体介导的膜融合。 细胞进入系统。