Lipid Bilayer Remodeling and Protein Intermediates During Membrane Fusion

蛋白质介导的膜融合是许多基本生物过程所必需的。尽管 通过深入研究，目前我们对融合蛋白机器如何运作的机制了解有限。 操纵脂质膜以诱导它们的融合。这种知识的缺乏在以下方面尤其严重： 膜中间体的结构，其小叶弯曲或破坏成非双层的程度 结构，以及它们如何被融合体协调和重塑。同样，在结构方面， 融合蛋白本身，很少有结构信息可以描述它们在融合过程中如何变化 驱动膜融合。这些是治疗剂如融合抑制剂或 中和抗体在预防病毒感染的情况下，它们是可能出错的过程， 细胞融合体的疾病突变的结果。拟议的研究将扩大我们对这些问题的理解， 基本过程，并揭示了一般原则所采用的发散聚变机。低温电子 显微镜和结构质谱法提供了强大的互补方法来直接成像， 探针膜融合，因为它们允许我们在天然条件下触发融合反应，然后捕获， 然后成像或分析反应过程中的中间状态。尤其是冷冻电子断层扫描 可以分辨融合过程中捕获的单个融合器和膜小叶， 当蛋白质和膜采用非规范的中间结构时。氢/氘- 交换质谱补充冷冻EM，使我们能够监测当地骨干动力学下， 原生条件。这种方法对于跟踪构象变化和比较 不同状态下的蛋白质结构。基于我们对流感病毒的研究，我们将把这些方法应用于 研究两种I类病毒融合系统中的膜融合途径：HIV使用的Env融合蛋白 和SARS-CoV-2使用的S刺突蛋白。这些融合机器采用顺序激活模式 触发涉及受体引发，随后是辅受体结合（Env）或蛋白水解裂解 事件（S）。因此，这些系统提供了详细分析在一个特定的温度下停止的聚变系统的机会。 中间，启动阶段。对于这些系统中的每一个，我们的目标是对 膜重塑导致融合孔的形成，并了解蛋白质 机械诱导两个分离的膜双层结合成一个。通过分析，我们将 获得新的洞察一般，强制性的事件，在I类蛋白质介导的膜融合，同时也 揭示系统特定的机制。因此，我们的研究应该推进我们的结构和机制， 了解生物膜融合的基本过程，同时也提供有价值的见解 研究两种病毒入侵宿主的机制，这两种病毒引发了影响全球健康的重大流行病 和社会

Protein-mediated membrane fusion is essential for a multitude of fundamental biological processes. Despite intensive study, at present we have a limited mechanistic understanding of how fusion protein machinery manipulates lipid membranes in order to induce their fusion. This lack of knowledge is particularly acute regarding the structure of membrane intermediates, the extent to which their leaflets are bent or disrupted into nonbilayer structures, and how they are coordinated and remodeled by fusogens. Similarly, in terms of the structure of the fusion proteins themselves, very little structural information is available to describe how they change as they drive membrane fusion. These are processes that are targeted by therapeutics such as fusion inhibitors or neutralizing antibodies in the case of preventing virus infection, and they are processes that can go awry as a result of disease mutations for cellular fusogens. The proposed studies will expand our understanding of these fundamental processes and reveal general principles employed by divergent fusion machines. Cryo-electron microscopy and structural mass spectrometry provide powerful complementary methods to directly image and probe membrane fusion because they allow us to trigger a fusion reaction under native conditions then trap and then image or analyze intermediate states over the course of the reaction. Cryo-electron tomography in particular can resolve individual fusion machines and membrane leaflets captured in the process of fusing and can discern when the proteins and membranes have adopted non-canonical intermediate structures. Hydrogen/deuterium- exchange mass spectrometry complements cryo-EM by enabling us to monitor local backbone dynamics under native conditions. This approach is particularly effective for tracking conformational changes and for comparing protein structure in different states. Building on our work with influenza virus, we will apply these methods to investigate pathways of membrane fusion in two Class I viral fusion systems: the Env fusion protein used by HIV and the S spike protein used by SARS-CoV-2. These fusion machines employ sequential modes of activation and triggering involving receptor priming followed by either coreceptor binding (Env) or a proteolytic cleavage event (S). These systems thus offer the opportunity to analyze in detail the fusion system arrested at an intermediate, primed stage. For each of these systems, our goal is to image the architecture and progression of membrane remodeling leading to formation of fusion pores and to understand the means by which the protein machinery induces two separate membrane bilayers to join into one. By performing such an analysis, we will gain novel insight into general, obligatory events in Class I protein-mediated membrane fusion, while also revealing system-specific mechanisms. Our study should thus advance our structural and mechanistic understanding of the fundamental process of biological membrane fusion while also providing valuable insight into the mechanism of host invasion by two viruses that have ignited major pandemics impacting global health and society.