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的磷酸酶系统（PTS）是一种多组分碳水化合物摄取系统，也参与了细菌中代谢，趋化性和致病性的调节。 PTS通过将配体在整个膜上的易位及其伴随磷酸化的共价修饰来驱动糖的主动运输，以防止外排。近半个世纪以来，PT一直是广泛研究的主题，但是由于缺乏负责糖在整个膜上运输的整体膜成分的任何结构，我们对系统的理解仍然不完整。 EIIC还将特定糖的特异性赋予PTS，并有助于将磷酸盐从细胞质PTS蛋白EIIB转移到糖中。我们打算通过结合CHBC的结构和功能研究来解决PTS的机械理解，这是CHBC的结构和功能研究，这是葡萄糖EIIC超级家族的成员，该研究特定于摄取N.N'-二乙酰基鸡蛋白。这种糖是由几丁质分解产生的，并且是病原体（例如弧菌霍乱）生命周期中的重要营养。为此，我们解决了从蜡状芽孢杆菌（BCCHBC）的CHBC直系同源物的结构，这使我们提出了转运蛋白如何选择性结合糖，将其跨膜易位的假设，并有助于将磷酸化偶联到运输。我们将使用该结构以三个目的来理解EIIC功能的机制：（1）确定BCCHBC的底物选择性的结构基础，使用结合和吸收测定，（2）通过与其伴侣的结构进行循环的结构来解决磷酸化的机制，从而解决了BCCHBC的结构，以解决其伙伴EIIB，BCCHBB和BCCHBB和（3）的结构。 BCCHBC的向外面向开放状态。 公共卫生相关性：N，N'-DiActelyChitobiose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弧菌霍乱，它在其在其水上造期间的微质量宿主的壳中生存。由于N'-DiactelyChitobiose的摄取系统是细菌独有的，因此它是破坏这种毁灭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'-DiActelyChitobiose摄取系统的机制。