Mechanism of an ATP-coupled arsenical pump

ATP耦合砷泵的机制

• 批准号: 6525901
• 负责人: BARRY P. ROSEN
• 金额: $ 36.73万
• 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/6525901
• 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.

对砷和锑的抗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表征最好的系统具有对砷（亚砷酸盐和砷酸盐）和锑（锑矿）氧离子的抗性。ars操纵子编码两个调节蛋白（ArsR和ArsD）和三个结构蛋白（ArsA、ArsB和ArsC）。电阻与主泵从电池中主动挤出亚砷酸盐有关。arsA基因产物是一种atp酶，作为泵的催化亚基。ArsA是一种63 kda的外周膜蛋白，可催化As(III)/Sb（III）刺激的ATP水解。ArsA有两个同源半部分，A1和A2，由一个短连接体连接。每个都有一个一致的核苷酸结合位点（NBS），两个NBS都是活性所必需的。ArsA与ArsB结合，ArsB是泵的离子传导亚基。ArsB是一种45 kda的完整膜蛋白，横跨细胞膜12次。ArsB与ArsA之间存在一种新型的双能量耦合模式。砷外排细菌既可以被作为二级转运体的ArsB单独催化，也可以被作为转运atp酶的ArsAB复合物催化。我们的总体目标是阐明ArsAB泵的分子机制。分析包括以下具体目的：1。ArsA atp酶的结构和功能：研究ArsA的金属结合位点、核苷酸结合位点和信号转导位点的功能和性质。形成A1和A2之间界面的残基的组成和功能将被确定。2. 利用单一色氨酸ArsA突变体作为光谱探针，通过同位素捕获和钒酸盐捕获和裂解实验来研究ArsA atp酶的催化机制。3. ArsAB泵的结构：形成ArsA和ArsB亚基相互作用位点的残基将被识别。将确定泵中ArsA和ArsB亚基的化学计量。