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众健康的主要威胁，特别是对四环素的耐药性严重限制了这种曾经有效的广谱抗生素家族的使用。对四环素等抗生素的耐药性的主要机制是由膜转运蛋白介导的，膜转运蛋白催化四环素以四环素-镁复合物的形式流​​出。这种外排蛋白识别底物的结构基础尚不清楚。来自枯草芽孢杆菌的外排泵 TetL 具有 14 个跨膜 1 螺旋，是革兰氏阳性细菌病原体（包括炭疽杆菌、蜡样芽孢杆菌、肺炎链球菌和金黄色葡萄球菌）中抗生素抗性外排蛋白 (Tet) 家族的成员。梭菌属、肠球菌属和李斯特菌属。所有 Tet 转运蛋白均属于主要促进子超家族 (MFS)。这些 Tet 蛋白的底物结合位点预计与具有 12 个跨膜 1-螺旋的革兰氏阴性 Tet 蛋白的同源区域相似。拟议的研究建立在最近对 TetL 的初步结构工作以及使用定点诱变和体外测定的早期结构功能研究的基础上。具体来说，我们的目标是：（一）．为了了解外排介导的四环素耐药性的分子基础，我们建议确定 TetL 的晶体结构。 （二）。为了了解 TetL 底物特异性的结构基础，我们将结合结构、计算、诱变和生化方法来表征关键的四环素结合残基。 （三）。研究 TetL 二聚化在底物运输中的作用。 公共卫生相关性：抗生素耐药性是对公共卫生的主要威胁。对四环素等抗生素耐药的主要机制是膜转运蛋白介导的外排。缺乏此类外排蛋白识别底物的结构基础。枯草芽孢杆菌的外排泵 TetL 以四环素-镁复合物的形式输出四环素，并导致该细菌对曾经广泛有效的抗生素产生耐药性。 TetL的晶体结构与生物化学和生物物理研究相结合，不仅将极大地促进我们对抗生素耐药性分子机制的理解，还将提出修饰四环素以逆转耐药性的新方法。