Structural basis of tetracycline resistance by efflux pump TetL

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，特别是对四环素类抗生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。对抗生素如四环素类的耐药性的主要机制是由膜转运蛋白介导的，该膜转运蛋白以四环素-镁复合物的形式催化四环素类的外排。这种外排蛋白的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，是革兰氏阳性细菌病原体（包括炭疽芽孢杆菌、蜡状芽孢杆菌、肺炎链球菌和金黄色葡萄球菌）中的抗生素抗性外排蛋白（泰特）家族的成员。梭菌属，肠球菌属和李斯特菌属（Listeria spp.）所有的泰特转运蛋白都属于主要易化蛋白超家族（MFS）。预期这些泰特蛋白的底物结合位点与具有12个跨膜1-螺旋的革兰氏阴性泰特蛋白的同源区域相似。拟议的研究建立在最近的初步结构工作TetL和早期的结构功能研究，使用定点诱变和体外试验。具体而言，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的底物特异性的结构基础，我们将使用结构，计算，诱变和生物化学方法的组合来表征关键的四环素结合残基。（三）。探讨TetL二聚化在底物转运中的作用。 公共卫生相关性：抗生素耐药性是公共卫生的主要威胁。四环素类抗生素耐药的主要机制是膜转运蛋白介导的外排。缺乏这种外排蛋白的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-镁复合物的形式输出四环素，并且负责该细菌对曾经广泛有效的抗生素的抗性。TetL的晶体结构与生物化学和生物物理学研究相结合，不仅将大大推进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饰四环素以逆转耐药性。

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.