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: 8669013
• 负责人: Ming Zhou
• 金额: $ 29.74万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8669013
• 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（由申请人提供）：磷酸烯醇化酸依赖性磷酸转移酶系统（PTS）是一种多组分碳水化合物摄取系统，也参与细菌代谢、趋化性和致病性的调节。PTS通过偶联配体跨膜的易位及其伴随的通过磷酸化的共价修饰以防止流出来驱动糖的主动转运。近半个世纪以来，PTS一直是广泛研究的主题，但由于缺乏负责糖穿过内膜运输的完整膜组分EIIC的任何结构，我们对该系统的理解仍然不完整。EIIC还赋予PTS对特定糖的特异性，并帮助磷酸盐从细胞质PTS蛋白EIIB转移到糖。我们打算通过结合ChbC的结构和功能研究来解决PTS的机制理解中的这一差距，ChbC是葡萄糖EIIC超家族的成员，其特异性用于摄取N，N '-二乙酰壳二糖。这种糖是由几丁质分解产生的，并且是病原体如霍乱弧菌生命周期中的重要营养素。为此，我们已经解决了从蜡状芽孢杆菌（bcChbC），这使我们提出的转运蛋白如何选择性地结合糖，跨膜转运，并协助耦合磷酸化运输的直向同源物的结构。我们将使用这个结构来理解EIIC功能的机制，有三个目标：（1）用结合和摄取测定确定bcChbC底物选择性的结构基础，（2）通过解析bcChbC与其配偶体EIIB，bcChbB复合的结构来揭示磷酸化的机制，（3）通过求解bcChbC外向开放态的结构，重建转运循环中的构象变化。