Structural basis of tetracycline resistance by efflux pump TetL

描述(申请人提供)：抗生素耐药性是对公众健康的主要威胁，尤其是对四环素的耐药性严重限制了这个曾经有效的广谱抗生素家族的使用。四环素类抗生素耐药的一个主要机制是通过膜转运蛋白以四环素-镁复合体的形式催化四环素的外排。这种外排蛋白识别底物的结构基础还不是很清楚。枯草杆菌外排泵TetL由14个跨膜1螺旋组成，是炭疽杆菌、蜡状芽孢杆菌、肺炎链球菌和金黄色葡萄球菌等革兰氏阳性细菌耐药外排蛋白家族的成员。梭状芽胞杆菌、肠球菌属。和李斯特氏菌(Listeria spp.)所有的TET转运蛋白都属于主要的促进剂超家族(MFS)。这些Tet蛋白的底物结合部位预计与革兰氏阴性Tet蛋白的同源区域相似，后者有12个跨膜1-螺旋。拟议的研究建立在最近关于TetL的初步结构工作和早期使用定点突变和体外分析进行的结构-功能研究的基础上。具体来说，我们的目标是：(I)。为了了解外排介导的四环素耐药的分子基础，我们建议确定TetL的晶体结构。(Ii)。为了了解TetL底物特异性的结构基础，我们将结合结构、计算、突变和生化方法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。

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.