Structural analysis of inner membrane platform in the type 2 secretion system

2型分泌系统内膜平台的结构分析

• 批准号: 10308683
• 负责人: Wei Mi
• 金额: $ 22.41万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308683
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Architecture; Bacterial Infections; Binding; Biochemical; Bioinformatics; Cell surface; Cholera Toxin; Complex; Core Protein; Cryoelectron Microscopy; Cytoplasm; Enterotoxins; Evolution; Family; Fluorescence; Future; Gram-Negative Bacteria; Green Fluorescent Proteins; Image; Individual; Interdisciplinary Study; Intervention; Ion Channel; Knowledge; Membrane; Membrane Proteins; Modeling; Molecular Sieve Chromatography; Monitor; Mutagenesis; Negative Staining; Nomenclature; Pathway interactions; Periplasmic Binding Proteins; Pharmacology; Pilum; Positioning Attribute; Proteins; Research; Resolution; Screening Result; Secretin; Structure; System; Therapeutic; Therapeutic Intervention; Toxin; Type II Secretion System Pathway; Vibrio cholerae; Virulence; Work; base; cell envelope; crosslink; enterotoxigenic Escherichia coli; extracellular; membrane assembly; overexpression; particle; pathogen; pathogenic bacteria; periplasm; prevent; protein complex; protein folding; screening; tool; two-dimensional; unnatural amino acids; vacuolar H+-ATPase

ABSTRACT The type II secretion system (T2SS) exists widely in gram-negative bacteria and exports a variety of folded protein substrates, such as cholera toxin (CT) from Vibrio cholerae and heat- labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC). The T2SS machinery spans the entire cell envelope and consists of three subassemblies: the mysterious inner membrane platform (IMP), the dynamic pseudopilus, and the channel-forming outer membrane complex. In current mechanism models, protein substrate binding to the T2SS in the periplasm triggers the cytoplasmic ATPase (GspE) to hydrolyze ATP, which energizes the incorporation of the major pseudopilin subunit (GspG) into a pilus-like structure. This growing pseudopilus acts as a piston or Archimedes screw, pushing the folded protein substrates through the outer membrane channel to the cell surface or extracellular milieu. Despite decades of research, the structure of IMP is not yet available; mainly for this reason, it is still unknown how the inner membrane assembly platform converts energy from ATP hydrolysis in the cytoplasm to extend the pseudopilus and push the substrates across the outer membrane. It is also unknown how the inner membrane proteins regulate the GspE ATPase activity. Our proposal exploits fluorescence size exclusion chromatography and coevolution analysis to identify interactions among components of IMP. We will use an assistant-multimer strategy and unnatural amino acid crosslinking to purify stable inner membrane protein complexes, followed by single particle cryo-electron microscopy to determine complex structures. We will analysis the ATPase activity of GspE with different inner membrane protein complexes. This proposal will substantially increase the fundamental scientific knowledge about the architecture and mechanisms of the T2SS, and thereby provide a new basis for developing future therapeutic interventions against a broad range of bacterial infections.

抽象的 II型分泌系统（T2SS）广泛存在于革兰氏阴性细菌中，并导出A 多种折叠蛋白底物，例如霍乱和热的霍乱毒素（CT） 肠毒素大肠杆菌（ETEC）的不稳定肠毒素（LT）。 T2SS机械 跨越整个细胞信封，由三个子组件组成：神秘的内心 膜平台（IMP），动态假核心和频道形成的外膜 复杂的。在当前的机理模型中，蛋白质底物与pariplasm的T2SS结合 触发细胞质ATPase（GSPE）以水解ATP，该ATP充满活力 主要的假肽亚基（GSPG）成类似菌毛的结构。这种增长的假核行为 作为活塞或阿基梅德螺钉，将折叠的蛋白质底物推到外部 膜通道通向细胞表面或细胞外环境。尽管进行了数十年的研究，但 IMP的结构尚不可用；主要是由于这个原因，它仍然未知内部 膜组件平台转换来自细胞质中ATP水解的能量，以延伸 假核心并将底物推到外膜上。这也未知 内膜蛋白调节GSPE ATPase活性。我们的建议利用 荧光尺寸排除色谱和协同进化分析以识别相互作用 在Imp的组成部分中。我们将使用助理 - 培训策略和不自然的氨基 酸交联以纯化稳定的内膜蛋白复合物，然后是单个颗粒 冷冻电子显微镜确定复杂的结构。我们将分析ATPase活动 具有不同内膜蛋白复合物的GSPE。该提议将大大 增加有关建筑和机制的基本科学知识 T2SS，因此为开发未来的治疗干预措施提供了新的基础 广泛的细菌感染。