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: 8160526
• 负责人: Ming Zhou
• 金额: $ 30.4万
• 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 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8160526
• 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. PUBLIC HEALTH RELEVANCE: N,N'-diactelychitobiose, a degradation product of chitin, is a vital food source for many bacteria, including the cholera-causing pathogen Vibrio cholerae, which survives on the chitin in the shells of its microcrustacean hosts during its water-bourne phase. Because the uptake system for N,N'-diactelychitobiose is unique to bacteria, it is a potential target for disrupting the lifecycle of this devastating pathogen. Our studies of the ChbC transporter will help shed light on the mechanism of the N,N'-diactelychitobiose uptake system.

描述（由申请人提供）：磷酸烯醇丙酮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 通过耦合配体跨膜易位及其伴随的通过磷酸化进行的共价修饰来驱动糖的主动转运，以防止外流。近半个世纪以来，PTS 一直是广泛研究的主题，但由于缺乏负责跨内膜转运糖的完整膜组件 EIIC 的任何结构，我们对该系统的了解仍然不完整。 EIIC 还赋予 PTS 对特定糖的特异性，并协助磷酸盐从细胞质 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 机制理解中的这一差距，ChbC 是葡萄糖 EIIC 超家族的成员，专门用于 N.N'-二乙酰壳二糖的摄取。这种糖是由几丁质分解产生的，是霍乱弧菌等病原体生命周期中的重要营养素。为此，我们解析了来自蜡样芽孢杆菌 (bcChbC) 的 ChbC 直系同源物的结构，这使我们提出了关于转运蛋白如何选择性结合糖、将其跨膜转运并协助磷酸化与转运偶联的假设。我们将使用该结构来了解 EIIC 功能的机制，以实现三个目标：(1) 通过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 与其伙伴 EIIB、bcChbB 复合物的结构来揭示磷酸化机制，以及 (3) 通过解析 bcChbC 的结构来重建转运循环背后的构象变化 bcChbC 的向外打开状态。 公众健康相关性：N,N'-双乳壳二糖是几丁质的降解产物，是许多细菌的重要食物来源，包括引起霍乱的病原体霍乱弧菌，它在水相阶段依靠微甲壳动物宿主壳中的几丁质生存。由于 N,N'-双乳壳二糖的摄取系统是细菌所独有的，因此它是破坏这种破坏性病原体生命周期的潜在目标。我们对 ChbC 转运蛋白的研究将有助于阐明 N,N'-双乳糖壳二糖摄取系统的机制。