Structural Studies of a Tetracycline Efflux Pump

四环素外排泵的结构研究

• 批准号: 7811343
• 负责人: Bryan K. Czyzewski
• 金额: $ 4.14万
• 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: 2010-02-04 至 2012-02-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811343
• 关键词: Accounting; Antibiotic Resistance; Antibiotics; Bacillus (bacterium); Bacteremia; Bacterial Drug Resistance; Binding; Biochemical; Biological Assay; Cell membrane; Charge; Cholera; Collection; Communication; Complex; Crystallization; Data Set; Detergents; Development; Disease; Drug resistance; Dysentery; Effectiveness; Equilibrium; Family; Future; Genes; Goals; Gram-Positive Bacteria; Health; Homology Modeling; Human; Mediating; Microbial Antibiotic Resistance; Molecular; Multi-Drug Resistance; Play; Pneumonia; Preparation; Protein Binding; Proteins; Public Health; Pump; Research; Resistance; Resolution; Role; Sequence Alignment; Shigella dysenteriae; Staphylococcus aureus; Streptococcus pneumoniae; Structure; Substrate Specificity; System; Tetracycline Resistance; Tetracyclines; Vibrio cholerae; analog; antimicrobial; bacterial resistance; base; dimer; efflux pump; in vivo; milligram; pathogen; pathogenic bacteria; protein structure; proteoliposomes; public health relevance; reconstitution; research study

DESCRIPTION (provided by applicant): Antibiotics are arguably one of the most important discoveries impacting human health in the last century. Microbial resistance to antibiotics has steadily increased since they were first introduced. Tetracycline, once a widely prescribed broad specturum antimicrobial, has seen a sharp decline in effectiveness mainly due to the activity of resistance genes encoding multi-drug resistance pumps. TetL belongs to a family of tetracycline-specific transporters that account for the majority of resistance in most pathogenic bacteria responsible for diseases such as pneumonia, dysentery, cholera, and bacteremia. Detailed information about the molecular basis of interaction and mechanism of tetracycline binding and transport through TetL will be invaluable to the future development of antibiotics. The broad long term research objectives of this study are to use x-ray crystallagraphy to obtain the first complete picture of an efflux transporter-antibiotic complex and to characterize the mechanistic basis of this interaction. The specific aims for the proposed studies are as follows: Aim I. To understand the basis of efflux mediated tetracycline resistance, I propose to solve the structure of TetL, a tetracycline exporter from B.subtilis, alone and bound to an antibiotic. TetL has been crystallized and crystals currently diffract to 4.2 A. Improvement of these crystals to 3 A will allow for high resolution structural determination. Tetracycline analogs have been co-crystallized with TetL or soaked into TetL crystals, the first steps toward understanding the interaction of this multidrug resistance transporter with its substrate. AIM II. To understand TetL substrate specificity and the functional basis of transport of tetracycline, I propose to examine the role of residues predicted to be involved in substrate recognition and transport and the significance of TetL dimeric oligomerization. Charged residues involved in tetracycline binding and transport predicted from alignments, homology modeling, and the crystal structure of TetL, will be probed with fluorescent binding experiments, transport assays in reconstituted systems, and in vivo activity experiments. Allosteric communication within the dimer will be examined using equilibrium binding experiments. PUBLIC HEALTH RELEVANCE: Drug-resistant pathogens are global threats to public health. This research seeks to demonstrate the molecular basis of a tetracycline interaction with a bacterial drug resistance pump. Understanding the structural basis of drug resistance will open the field to future development of new antibiotics.

描述（由申请人提供）：抗生素可以说是上世纪影响人类健康的最重要的发现之一。自首次引入抗生素以来，微生物对抗生素的耐药性一直在稳步增加。四环素曾经是一种广泛使用的广谱抗菌药物，但其有效性急剧下降，主要是由于编码多药耐药泵的耐药基因的活性。 TetL 属于四环素特异性转运蛋白家族，在导致肺炎、痢疾、霍乱和菌血症等疾病的大多数病原菌中，该转运蛋白具有大部分耐药性。有关相互作用的分子基础以及四环素通过 TetL 结合和转运的机制的详细信息对于抗生素的未来发展将具有无价的价值。本研究的广泛长期研究目标是利用 X 射线晶体学获得外排转运蛋白-抗生素复合物的第一张完整图像，并表征这种相互作用的机制基础。拟议研究的具体目标如下： 目标 I. 为了了解外排介导的四环素耐药性的基础，我建议解析 TetL 的结构，TetL 是来自枯草芽孢杆菌的四环素输出蛋白，单独并与抗生素结合。 TetL 已经结晶，晶体目前的衍射强度达到 4.2 A。将这些晶体提高到 3 A 将能够实现高分辨率结构测定。四环素类似物已与 TetL 共结晶或浸入 TetL 晶体中，这是了解这种多药耐药性转运蛋白与其底物相互作用的第一步。目标二。为了了解 TetL 底物特异性和四环素转运的功能基础，我建议检查预测参与底物识别和转运的残基的作用以及 TetL 二聚寡聚化的意义。通过比对、同源建模和 TetL 晶体结构预测的参与四环素结合和转运的带电残基将通过荧光结合实验、重构系统中的转运测定和体内活性实验进行探测。将使用平衡结合实验检查二聚体内的变构通讯。 公共卫生相关性：耐药病原体是对公共卫生的全球威胁。这项研究旨在证明四环素与细菌耐药泵相互作用的分子基础。了解耐药性的结构基础将为新抗生素的未来开发开辟新领域。