Structural Analysis of Membrane Tethering and Fusion Proteins

膜束缚和融合蛋白的结构分析

• 批准号: 10210474
• 负责人: FREDERICK M HUGHSON
• 金额: $ 37.53万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10210474
• 关键词: Architecture; Attention; Awareness; Binding; Biological Process; Carrier Proteins; Cell membrane; Cells; Chimeric Proteins; Closure by clamp; Coated vesicle; Collaborations; Complex; Coupled; Cryoelectron Microscopy; Disease; Eukaryotic Cell; Exocytosis; Foundations; Future; Goals; In Vitro; Infection; Lasso; Lipids; Lysosomes; Mediating; Membrane; Membrane Fusion; Modification; Molecular Chaperones; Mutation; Neurons; Organelles; Pathway interactions; Pattern; Process; Protein Sortings; Proteins; Proteomics; Reaction; Reporting; Research; Role; SNAP receptor; Sorting - Cell Movement; Structure; Surface; Synapses; System; Therapeutic Intervention; Thermodynamics; Vacuole; Vesicle; Work; X-Ray Crystallography; Yeasts; base; biochemical tools; design; experimental study; falls; flexibility; human disease; improved; in vivo; laser tweezer; late endosome; membrane assembly; nanomachine; neurotransmitter release; programs; protein function; reconstitution; single molecule; syntaxin binding protein 1; trafficking; vesicle transport; virtual

PROJECT SUMMARY / ABSTRACT The traffic patterns established by transport vesicles are of fundamental importance for protein localization, modification, and function within eukaryotic cells. Cargo transported by these vesicles is delivered through the fusion of the vesicle with the membrane of a target organelle or, in the case of exocytosis, the plasma membrane. Membrane fusion is executed by SNARE complexes that bridge the vesicle and target membranes. The formation of these complexes requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. We hypothesize that SNARE complex assembly reactions in the cell are orchestrated by `topologically aware' chaperones called multisubunit tethering complexes (MTCs). We furthermore propose that the key task of catalyzing four-helix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with—and sometimes as integral subunits of—the MTCs. Therefore, the overarching goal of this proposal is to achieve an improved structural and mechanistic understanding of MTC and SM function, especially as they relate to one another, in the assembly of membrane fusogenic SNARE complexes. Aim 1 is focused on SM proteins with the goal of characterizing their precise catalytic role in SNARE complex assembly. Principally through the use of X-ray crystallography and complementary single-molecule optical tweezers experiments, we will determine the structures and thermodynamic stabilities of SM-bound SNARE assembly intermediates. In Aims 2 and 3, we broaden our focus to include MTCs. In Aim 2, we will investigate the simplest known MTC, the yeast Dsl1 complex, and its interactions with SNAREs and the SM protein Sly1. Cryo-EM studies of arrested SNARE assembly intermediates in complex with both the Dsl1 complex and Sly1 are designed to reveal how the Dsl1 complex and Sly1 collaborate. In Aim 3, we will turn our attention to the homotypic fusion and vacuole protein sorting (HOPS) complex, a well-studied MTC that is required for fusion at late endosomes and lysosomes/vacuoles. Importantly, HOPS contains an SM protein as an integral subunit, making it an ideal system for studying MTC–SM collaboration. In order to elucidate how HOPS organizes SNAREs for assembly, we will expand our ongoing cryo-EM studies of HOPS to include bound SNAREs and SNARE assembly intermediates. Overall, this research program has the potential to revolutionize our mechanistic understanding of chaperoned SNARE complex assembly, with potentially profound implications for elucidating diverse biological processes and their subversion during infection and disease. While the proposed work is more fundamental than applied, it will lay a foundation for efforts to manipulate trafficking and other processes entailing membrane fusion, with potential future applications to therapeutic intervention.

项目概要/摘要 运输囊泡建立的运输模式对于蛋白质定位至关重要， 真核细胞内的修饰和功能。这些囊泡运输的货物通过 囊泡与目标细胞器膜的融合，或者在胞吐作用的情况下，与血浆融合 膜。膜融合由桥接囊泡和靶标的 SNARE 复合体执行 膜。这些复合物的形成需要四种不同的 SNARE 蛋白，锚定在两个 不同的膜，经历耦合的折叠和组装反应，在此期间 SNARE 图案拉链 形成平行的四螺旋束。这个复杂的过程在体外效率很低，而且肯定是均匀的 在体内更具挑战性，它必须与各种非同源和旁路的形成竞争 圈套复合体。我们假设细胞中的 SNARE 复杂组装反应是由 “拓扑感知”分子伴侣称为多亚基束缚复合物（MTC）。我们还建议 催化四螺旋束形成的关键任务落在 Sec1/Munc18 (SM) 蛋白身上， 与 MTC 一起（有时作为 MTC 的组成部分）。因此，本次活动的总体目标是 建议是为了更好地理解 MTC 和 SM 功能的结构和机制， 特别是当它们在膜融合 SNARE 复合体的组装中彼此相关时。目标 1 是 专注于 SM 蛋白，目标是表征它们在 SNARE 复合体中的精确催化作用 集会。主要通过使用 X 射线晶体学和互补单分子光学 镊子实验，我们将确定 SM 结合 SNARE 的结构和热力学稳定性 组装中间体。在目标 2 和 3 中，我们将重点扩大到包括 MTC。在目标 2 中，我们将调查 已知最简单的 MTC，即酵母 Dsl1 复合体，及其与 SNARE 和 SM 蛋白 Sly1 的相互作用。 与 Dsl1 复合物和 Sly1 复合物中捕获的 SNARE 组装中间体的冷冻电镜研究 旨在揭示 Dsl1 复合体和 Sly1 如何协作。在目标 3 中，我们将把注意力转向 同型融合和液泡蛋白分选 (HOPS) 复合体，这是一种经过充分研究的 MTC，是融合所需的 晚期内体和溶酶体/液泡。重要的是，HOPS 含有 SM 蛋白作为完整的亚基， 使其成为研究 MTC-SM 协作的理想系统。为了阐明 HOPS 如何组织 用于组装的 SNARE，我们将扩展我们正在进行的 HOPS 冷冻电镜研究，以包括结合的 SNARE 和 SNARE 组装中间体。总体而言，该研究计划有可能彻底改变我们的 对伴随 SNARE 复杂组装的机械理解，对以下领域具有潜在的深远影响： 阐明感染和疾病期间的不同生物过程及其颠覆。虽然提议的 工作比应用更为基础，它将为操纵贩运和其他行为的努力奠定基础 涉及膜融合的过程，未来可能应用于治疗干预。