Lipid Bilayer Remodeling and Protein Intermediates During Membrane Fusion

膜融合过程中的脂质双层重塑和蛋白质中间体

• 批准号: 10670375
• 负责人: Kelly Keisen Lee
• 金额: $ 58.95万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-25 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670375
• 关键词: 2019-nCoV; ACE2; Acute; Address; Adopted; Alphavirus; Architecture; Binding; Biological; Biological Process; CD4 Antigens; Cell fusion; Cells; Chikungunya virus; Chimeric Proteins; Complement; Coronavirus; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Deuterium; Development; Disease; Engineering; Event; Evolution; Fertilization; Freezing; Germ Cells; Goals; HIV; Hydrogen; Image; Individual; Influenza Hemagglutinin; Invaded; Investigation; Knowledge; Length; Lipid Bilayers; Maintenance; Mass Spectrum Analysis; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Methods; Mitochondria; Molecular; Molecular Conformation; Monitor; Mutation; Pathway interactions; Peptide Hydrolases; Physical Function; Placenta; Process; Proteins; Reaction; Research; Resolution; Sampling; Signal Transduction; Societies; Structure; Synapses; Synaptic Vesicles; System; TMPRSS2 gene; Testing; Time; Tissues; Vertebral column; Vesicle; Viral; Virion; Virus; Virus Diseases; Virus-like particle; Visualization; Work; density; dimer; experimental study; global health; influenzavirus; inhibitor; insight; molecular imaging; nanometer resolution; neutralizing antibody; novel; pandemic impact; prevent; programs; protein structure; receptor; receptor binding; targeted treatment; trafficking

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.

蛋白质介导的膜融合对于许多基本的生物过程是必不可少的。尽管 深入研究，目前我们对融合蛋白机制的了解有限 操纵脂膜以诱导其融合。这种知识的缺乏在以下方面尤为严重 膜中间体的结构，即它们的小叶弯曲或破裂成非双层的程度 结构，以及它们是如何由融合基因协调和重塑的。同样，就 融合蛋白本身，很少的结构信息可以用来描述它们如何随着 推动膜融合。这些过程是治疗药物的靶点，如融合抑制剂或 中和抗体在预防病毒感染的情况下，它们是一个可能会出错的过程 细胞融合原致病突变的结果。拟议的研究将扩大我们对这些问题的了解 基本过程和揭示发散融合机器所采用的一般原理。低温电子 显微镜和结构质谱学提供了强大的互补方法来直接成像和 探针膜融合，因为它们允许我们在自然条件下触发融合反应，然后捕获和 然后想象或分析反应过程中的中间状态。尤其是低温电子断层扫描 可以解析单个融合机和融合过程中捕获的薄膜小叶，并可以识别 当蛋白质和膜采用了非规范的中间结构时。氢/氢-- 交换质谱学补充了Cryo-EM，使我们能够在以下情况下监控本地主干动态 原生条件。这种方法在跟踪构象变化和比较时特别有效 不同状态下的蛋白质结构。在流感病毒工作的基础上，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 以及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 以及涉及受体启动之后的辅受体结合(Env)或蛋白水解性切割的触发 事件(S)。因此，这些系统提供了详细分析在 中间阶段，初级阶段。对于这些系统中的每一个，我们的目标都是描绘 膜重塑导致融合孔的形成并理解蛋白质 机械诱导两个分离的膜双层结合成一个。通过进行这样的分析，我们将 对I类蛋白介导的膜融合中的一般必备事件有了新的见解，同时还 揭示了特定于系统的机制。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，同时提供有价值的见解 探讨两种病毒入侵宿主的机制，这两种病毒已经引发了影响全球健康的主要流行病 和社会。

项目成果

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

• DOI: 10.1073/pnas.2220948120
• 发表时间: 2023-06-06
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Guenthoer, Jamie;Lilly, Michelle;Starr, Tyler N.;Dadonaite, Bernadeta;Lovendahl, Klaus N.;Croft, Jacob T.;Stoddard, Caitlin I.;Chohan, Vrasha;Ding, Shilei;Ruiz, Felicitas;Kopp, Mackenzie S.;Bloom, Jesse D.;Chu, Helen Y.;Lee, Kelly K.;Overbaugh, Julie
• 通讯作者: Overbaugh, Julie

De novo design of monomeric helical bundles for pH-controlled membrane lysis.

• DOI: 10.1002/pro.4769
• 发表时间: 2023-11
• 期刊: Protein science : a publication of the Protein Society