Structural Studies of a Tetracycline Efflux Pump

描述(申请人提供)：抗生素可以说是上个世纪影响人类健康的最重要的发现之一。自抗生素首次引入以来，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，但由于编码多药耐药泵的耐药基因的活性，其有效性已急剧下降。TetL属于四环素特异性转运蛋白家族，在肺炎、痢疾、霍乱和菌血症等疾病的大多数病原菌中，TetL占大多数耐药性。关于四环素相互作用的分子基础和四环素通过TetL的结合和转运机制的详细信息将对未来抗生素的发展具有非常重要的意义。这项研究广泛的长期研究目标是使用X射线结晶学获得外排转运体-抗生素复合体的第一张完整图片，并表征这种相互作用的机制基础。本研究的具体目的如下：目的I.为了了解外排介导的四环素耐药性的基础，我建议解决枯草杆菌四环素输出物TetL的结构，并与抗生素结合。TetL已经结晶，晶体目前的衍射率为4.2A。将这些晶体的衍射率提高到3A将使高分辨率结构确定成为可能。四环素类似物已经与TetL共结晶或渗透到TetL晶体中，这是了解这种多药耐药转运蛋白与其底物相互作用的第一步。目的II.为了了解TetL底物的特异性和四环素转运的功能基础，我建议研究参与底物识别和转运的残基的作用以及TetL二聚体齐聚的意义。从比对、同源模拟和TetL的晶体结构预测的四环素结合和运输所涉及的带电残基，将通过荧光结合实验、重组体系中的运输分析和体内活性实验来探索。二聚体内的变构通讯将通过平衡结合实验来检验。 公共卫生相关性：抗药性病原体是对公共卫生的全球性威胁。这项研究试图证明四环素与细菌耐药泵相互作用的分子基础。了解耐药的结构基础将为未来新抗生素的开发开辟新的领域。

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.