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.

摘要