Mechanism of an ATP-coupled arsenical pump

ATP耦合砷泵的机制

• 批准号: 6797750
• 负责人: BARRY P. ROSEN
• 金额: $ 38.79万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6797750
• 关键词: Escherichia coli; adenosinetriphosphatase; antimony; arsenic; bacterial genetics; bacterial proteins; biological signal transduction; biological transport; chemical kinetics; drug resistance; enzyme induction /repression; fluorescence resonance energy transfer; intermolecular interaction; membrane reconstitution /synthesis; membrane transport proteins; molecular site; protein structure function; proteolysis; radiotracer; reporter genes; site directed mutagenesis; stoichiometry; stop flow technique; tryptophan; vanadium; western blottings

Resistance to arsenic and antimony is widely spread in both gram-positive and gram-negative bacteria. The best- characterized system encoded by the clinically isolated resistance factor plasmid R773 in E. coli confers resistance against oxyanions of arsenic (arsenite and arsenate) and antimony (antimonite). The ars operon codes for two regulatory (ArsR and ArsD) and three structural (ArsA, ArsB and ArsC) proteins. Resistance correlates with active extrusion of arsenite from the cell by a primary pump. The arsA gene product is an ATPase that serves as the catalytic subunit of the pump. ArsA is a 63-kDa peripheral membrane protein that catalyzes As(III)/Sb(III)- stimulated ATP hydrolysis. ArsA has two homologous halves, A1 and A2, connected by a short linker. Each has a consensus nucleotide binding site (NBS), and both NBS are required for activity. ArsA binds to ArsB, which s the ion-conducting subunit of the pump. ArsB is a 45-kDa integral membrane protein that spans the inner membrane 12 times. ArsB has a novel dual mode of energy coupling depending on its association with ArsA. Arsenic efflux bacteria can be catalyzed by either ArsB alone functioning as a secondary transporter or by the ArsAB complex, functioning as a transport ATPase. Our overall goal is elucidation of the molecular mechanism of the ArsAB pump. Analysis includes the following specific aims: 1. Structure and function of the ArsA ATPase: The function and properties of the metal binding site, nucleotide binding sites and signal transduction sites of ArsA will be examined. The composition and function of residues that form the interface between A1 and A2 will be determined. 2. Catalytic mechanism of the ArsA ATPase will be examined using single tryptophan ArsA mutants as spectroscopic probes, by isotope trapping and by vanadate trapping and cleavage experiments. 3. Structure of the ArsAB pump: Residues that form the sites of interaction of the ArsA and ArsB subunits will be identified. The stoichiometry of the ArsA and ArsB subunits in the pump will be determined.

对砷和锑的耐药性在革兰氏阳性和革兰氏阴性细菌中广泛传播。临床分离的耐药因子质粒R773在大肠杆菌中编码的特征最好的系统对砷(亚砷酸盐和砷酸盐)和锑(亚硫酸盐)的氧阴离子具有抵抗力。Ars操纵子编码两种调控蛋白(ArsR和ARSD)和三种结构蛋白(ARSA、ARSB和ArsC)。阻力与一次泵主动将亚砷酸盐从细胞中排出有关。ARSA基因产物是一种ATPase，作为泵的催化亚单位。ARSA是一种63 kDa的外周膜蛋白，催化As(III)/Sb(III)刺激的ATP水解酶。ARSA有两个同源的一半，A1和A2，由一个短接头连接。每个都有一个共同的核苷酸结合位点(NBS)，两个NBS都是活性所必需的。ArsA与Arsb结合，而Arsb是泵的离子传导亚单位S。ARSB是一种45 kDa的完整膜蛋白，跨内膜12次。ARSB有一种新的双重能量耦合模式，依赖于它与ARSA的结合。砷外排细菌既可以被ARSB单独作为二级转运体催化，也可以被ArsAB复合体作为转运体ATPase催化。我们的总体目标是阐明ArsAB泵的分子机制。研究内容包括以下几个方面：1.ARSA-ATPase的结构与功能：对ARSA的金属结合部位、核苷酸结合部位和信号转导部位的功能和性质进行研究。将确定形成A1和A2之间界面的残基的组成和功能。2.以单一色氨酸ARSA突变体为光谱探针，通过同位素捕获、钒酸捕获和裂解实验研究ARSA-ATPase的催化机理。3.ArsAB泵的结构：将确定形成ARSA和ARSB亚单位相互作用部位的残基。将测定泵中ARSA和ARSB亚基的化学计量比。