Structure and Function of a Phosphorylation Coupled Saccharide Transporter

磷酸化偶联糖转运蛋白的结构和功能

• 批准号: 8317627
• 负责人: Ming Zhou
• 金额: $ 23.2万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8317627
• 关键词: Acetylglucosamine; Active Biological Transport; Active Sites; Address; Affect; Bacillus cereus; Bacteria; Binding; Binding Sites; Biological Assay; Carbohydrates; Chemotaxis; Chitin; Cholera; Complex; Computer Simulation; Coupled; Coupling; Cysteine; Data; Diffusion; Disaccharides; Docking; Face; Family; Food; Glucose; Integral Membrane Protein; Life; Life Cycle Stages; Ligand Binding; Ligands; Light; Liposomes; Measures; Mediating; Membrane; Metabolism; Modeling; Modification; Molecular Conformation; Mutation; Nutrient; Organism; Organized by Structure Protein; Orthologous Gene; Pathogenicity; Phase; Phosphoenolpyruvate; Phosphorylation; Phosphotransferases; Phylogenetic Analysis; Play; Proteins; Reaction; Regulation; Resolution; Role; Side; Solvents; Source; Specificity; Structure; Substrate Specificity; System; Testing; Vibrio cholerae; Water; base; crosslink; design; inorganic phosphate; member; mutant; nutrition; pathogen; periplasm; prevent; protein B; protein structure; reconstitution; sugar; three dimensional structure; uptake

DESCRIPTION (provided by applicant): The phosphoenolpyruvate-dependent phosphotranferase system (PTS) is a multicomponent carbohydrate uptake system that is also involved in the regulation of metabolism, chemotaxis, and pathogenicity in bacteria. The PTS drives active transport of sugar by coupling the translocation of the ligand across the membrane with its concomitant covalent modification by phosphorylation to prevent efflux. The PTS has been the subject of extensive study for nearly half a century, but our understanding of the system has remained incomplete due to the lack of any structures for the integral membrane component EIIC responsible for the transport of the sugar across the inner membrane. The EIICs also confer specificity for a particular sugar to the PTS, and assist in the transfer of the phosphate from the cytoplasmic PTS protein EIIB to the sugar. We intend to address this gap in the mechanistic understanding of the PTS by combining structural and functional studies of ChbC, a member of the glucose EIIC superfamily that is specific for the uptake of N.N'- diacetylchitobiose. This sugar is produced by the breakdown of chitin, and as an important nutrient in the life cycle of pathogens such as Vibrio cholerae. To this end, we have solved the structure of a ChbC ortholog from Bacillus cereus (bcChbC), which has led us to propose hypotheses for how the transporter selectively binds sugar, translocates it across the membrane, and assists in coupling phosphorylation to transport. We will use this structure to understand the mechanism of EIIC function with three aims: (1) to determine the structural basis of bcChbC's substrate selectivity with binding and uptake assays, (2) to uncover the mechanism of phosphorylation by solving the structure of an bcChbC in complex with its partner EIIB, bcChbB, and (3) to reconstruct the conformational changes underlying the transport cycle by solving the structure of the outward- facing open state of bcChbC.

描述(申请人提供)：磷酸烯醇式丙酮酸依赖的磷酸转移酶系统(PTS)是一个多组分的碳水化合物摄取系统，也参与调节细菌的新陈代谢、趋化性和致病性。PTS通过偶联配体的跨膜转运和伴随的磷酸化共价修饰来防止糖的外流，从而驱动糖的主动转运。近半个世纪以来，PTS一直是广泛研究的对象，但由于缺乏负责糖跨内膜运输的完整膜组件EIIC的结构，我们对该系统的了解仍然不完整。EIICs还赋予特定糖对PTS的专一性，并帮助将磷酸盐从细胞质PTS蛋白EIIB转移到糖上。我们打算通过结合对ChbC的结构和功能的研究来解决对PTS机制理解上的这一差距。ChbC是葡萄糖EIIC超家族的成员，专用于摄取N，N‘-二乙酰基壳二糖。这种糖是由甲壳素分解产生的，是霍乱弧菌等病原体生命周期中的重要营养物质。为此，我们解决了蜡样芽孢杆菌ChbC同源物(BcChbC)的结构，这导致我们提出了假设，即转运蛋白如何选择性地与糖结合，将其跨膜转运，并协助偶联磷酸化进行转运。我们将利用这种结构来理解EIIC的作用机制，目的有三：(1)通过结合和摄取分析来确定bcChbC底物选择性的结构基础；(2)通过求解bcChbC与其伴侣EIIB(BcChbB)形成的复合体的结构来揭示磷酸化的机制；(3)通过求解bcChbC的外向开放状态的结构来重建运输循环背后的构象变化。