Structural basis of tetracycline resistance by efflux pump TetL

外排泵TetL对四环素耐药的结构基础

• 批准号: 7887106
• 负责人: DANENG WANG
• 金额: $ 34.75万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7887106
• 关键词: Amino Acids; Antibiotic Resistance; Antibiotics; Bacillus anthracis; Bacillus cereus; Bacillus subtilis; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Carrier Proteins; Characteristics; Clostridium; Complex; Detergents; Dimerization; Doctor of Philosophy; Enterococcus; Enzyme Kinetics; Family; Family member; Heavy Metals; Lipids; Listeria; Magnesium; Mediating; Membrane; Membrane Transport Proteins; Molecular; Molecular Sieve Chromatography; Mutagenesis; Mutate; Names; Pathway interactions; Phase; Play; Positioning Attribute; Principal Investigator; Proteins; Public Health; Resistance; Resolution; Ribosomes; Role; Screening procedure; Site-Directed Mutagenesis; Solutions; Staphylococcus aureus; Streptococcus pneumoniae; Stress; Structure; Substrate Specificity; Tetanus Helper Peptide; Tetracycline Resistance; Tetracyclines; Work; bacterial resistance; base; dimer; efflux pump; improved; in vitro Assay; in vivo; molecular dynamics; monomer; mutant; pathogen; programs; protein folding; proteoliposomes; public health relevance; reconstitution; research study; thermostability

DESCRIPTION (provided by applicant): Antibiotic resistance is a major threat to public health and resistance to tetracyclines in particular has severely limited the use of this once efficacious, broad-spectrum family of antibiotics. A major mechanism of resistance to antibiotics like tetracyclines is mediated by membrane transporter proteins that catalyze efflux of tetracyclines in the form of tetracycline-magnesium complex. The structural basis for substrate recognition by such efflux proteins is poorly understood. The efflux pump TetL from Bacillus subtilis, with 14 transmembrane 1-helices, is a member of the family of antibiotic resistance efflux proteins (Tet) in Gram-positive bacterial pathogens, including Bacillus anthracis, Bacillus cereus, Streptococcus pneumoniae, and Staphylococcus aureus. Clostridium spp., Enterococcus spp. and Listeria spp. All Tet transporters belong to the major facilitator superfamily (MFS). The substrate binding sites of these Tet proteins are expected to be similar to homologous regions of the Gram-negative Tet proteins which have 12 transmembrane 1-helices. The proposed studies build upon recent preliminary structural work on TetL and on earlier structure-function studies using site- directed mutagenesis and in vitro assays. Specifically, our aims are: (I). To understand the molecular basis of efflux-mediated tetracycline resistance, we propose to determine the crystal structure of TetL. (II). To understand the structural basis of TetL's substrate specificity, we will characterize key tetracycline-binding residues using a combination of structural, computational, mutagenesis and biochemical approaches. (III). To investigate the role of TetL dimerization in substrate transport. PUBLIC HEALTH RELEVANCE: Antibiotic resistance is a major threat to public health. The major mechanism of resistance to antibiotics like tetracyclines is efflux mediated by membrane transporter proteins. The structural basis for substrate recognition by such efflux proteins is lacking. The efflux pump TetL from Bacillus subtilis exports tetracycline in the form of tetracycline-magnesium complex, and is responsible for this bacterium's resistance to the once widely efficacious antibiotic. A crystal structure of TetL, in combination with biochemical and biophysical studies, not only will greatly advance our understanding of the molecular mechanism of antibiotic resistance, it will also suggest new ways to modify tetracycline to reverse resistance.

描述（由申请人提供）：抗生素耐药性是对公共卫生的主要威胁，尤其是对四环素的抵抗，严重限制了这种曾经有效的，广泛的抗生素家族的使用。膜转运蛋白介导了对抗生素（如四环素）抗生素的主要机制，该膜转运蛋白以四环素 - 磁通剂的形式催化四环素的外排。这种外排蛋白识别底物识别的结构基础知之甚少。来自枯草芽孢杆菌的外排泵tetl，具有14个跨膜1螺旋，是革兰氏阴性细菌病原体中抗生素耐药性外排蛋白（TET）家族的成员，包括炭疽芽孢杆菌，脑杆菌，脑链球菌，肺炎链球菌，肺泡链球菌和孔氏菌coccus aufylococcus aurococcus aurococcus amococcus aucoccus aucoccus aucoccus aucoccus aucoccus aucoccus aucoccus aucoccus aucoccus aucrocus。梭状芽胞杆菌，肠球菌属。和李斯特菌。所有TET转运蛋白均属于主要促进剂超家族（MFS）。这些TET蛋白的底物结合位点有望与具有12个跨膜1螺旋的革兰氏阴性TET蛋白的同源区域相似。拟议的研究基于TETL的最新初步结构工作以及使用位置定向诱变和体外测定的早期结构功能研究。具体来说，我们的目标是：（i）。为了了解外排介导的四环素耐药的分子基础，我们建议确定TETL的晶体结构。 （ii）。为了了解TETL底物特异性的结构基础，我们将使用结构，计算，诱变和生化方法的组合来表征关键的四环素结合残基。 （iii）。研究TETL二聚化在底物转运中的作用。 公共卫生相关性：抗生素耐药性是对公共卫生的主要威胁。抗抗生素（如四环素）的耐药性的主要机制是由膜转运蛋白介导的外排。缺乏这种外排蛋白识别底物识别的结构基础。来自枯草芽孢杆菌的外排泵TETL以四环素 - 含量复合物的形式导出四环素，并负责该细菌对曾经广泛有效的抗生素的抗性。 TETL的晶体结构，结合生化和生物物理研究，不仅会极大地提高我们对抗生素耐药性分子机制的理解，还将提出新的方法来修改四环素以反向抗性。