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。对四环素等抗生素耐药的主要机制是由膜转运蛋白介导的，该转运蛋白催化四环素以四环素-镁复合物的形式外排。这种外排蛋白识别底物的结构基础尚不清楚。枯草芽孢杆菌的外排泵TetL是革兰氏阳性细菌病原体（包括炭疽芽孢杆菌、蜡样芽孢杆菌、肺炎链球菌和金黄色葡萄球菌）中抗生素耐药外排蛋白（Tet）家族的一员，具有14个跨膜1螺旋。梭状芽孢杆菌、肠球菌和李斯特菌都是Tet转运体，它们都属于主要的促进体超家族（MFS）。这些Tet蛋白的底物结合位点预计与革兰氏阴性Tet蛋白的同源区域相似，后者具有12个跨膜1-螺旋。拟议的研究建立在最近对tel的初步结构工作和早期使用定点诱变和体外测定的结构功能研究的基础上。具体来说，我们的目标是：(1)。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，我们将使用结构、计算、诱变和生化方法的组合来表征关键的四环素结合残基。(3)。探讨tel二聚化在底物转运中的作用。

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.