Structural Studies of a Tetracycline Efflux Pump

描述（由申请人提供）：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稳步增加。四环素曾是一种广泛使用的广谱抗菌素，但其有效性急剧下降，主要原因是编码多重耐药泵的耐药基因的活性。tel属于四环素特异性转运蛋白家族，该家族是导致肺炎、痢疾、霍乱和菌血症等疾病的大多数致病菌产生耐药性的主要原因。详细了解四环素相互作用的分子基础以及四环素通过tel结合和转运的机制对未来抗生素的开发具有重要意义。本研究的长期研究目标是利用x射线结晶学获得外排转运体-抗生素复合物的第一张完整图片，并表征这种相互作用的机制基础。拟开展研究的具体目的如下：目的1 .为了了解外排介导的四环素耐药性的基础，我提出解决来自枯草芽孢杆菌的四环素出口蛋白TetL的结构，该蛋白单独与抗生素结合。tel已经结晶，晶体目前衍射到4.2 A。这些晶体的改进到3a将允许高分辨率的结构测定。四环素类似物已经与TetL共结晶或浸泡在TetL晶体中，这是了解这种多药耐药转运体与其底物相互作用的第一步。目的二世。为了了解TetL底物特异性和四环素运输的功能基础，我建议研究预测参与底物识别和运输的残基的作用以及TetL二聚体寡聚化的意义。通过定位、同源性建模和TetL的晶体结构预测四环素结合和运输的带电残基将通过荧光结合实验、重组系统中的运输分析和体内活性实验进行探测。二聚体内的变构通信将通过平衡结合实验进行检验。

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.