Structural Basis of Tetracycline Resistance by Efflux Pump TetL.

外排泵 TetL 的四环素耐药性的结构基础。

• 批准号: 8663548
• 负责人: DANENG WANG
• 金额: $ 10.17万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8663548
• 关键词: Amino Acids; Antibiotic Resistance; Antibiotics; Antibodies; Bacillus anthracis; Bacillus cereus; Bacillus subtilis; Bacteria; Belgium; Binding; Binding Sites; Biochemical; Biological Assay; Carrier Proteins; Characteristics; Clostridium; Collaborations; Complex; Crystallization; Detergents; Dimerization; Enterococcus; Enzyme Kinetics; Evaluation; Family member; Heavy Metals; Light; Lipids; Listeria; Magnesium; Mediating; Membrane; Membrane Transport Proteins; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Mutagenesis; Mutate; Pathway interactions; Phase; Play; Positioning Attribute; Proteins; Public Health; Resistance; Resolution; Ribosomes; Role; Solutions; Staphylococcus aureus; Streptococcus pneumoniae; Stress; Structure; Substrate Specificity; Tetanus Helper Peptide; Tetracycline Resistance; Tetracyclines; bacterial resistance; base; dimer; efflux pump; experience; improved; in vivo; molecular dynamics; monomer; mutant; nanobodies; pathogen; protein folding; proteoliposomes; reconstitution; research study; screening; thermostability

1. Summary of the original project Antibiotic resistance is a major threat to public health. An important mechanism of resistance to antibiotics such as tetracyclines is efflux mediated by membrane transporter proteins. The structural basis for substrate recognition by tetracycline efflux pumps, Tet proteins, is unknown. The efflux pump TetL from Bacillus subtilis exports tetracycline (Tc) in the form of a tetracycline-magnesium [Tc.Mg2+]+ complex, and is responsible for this bacterium’s resistance to the once widely efficacious antibiotic. TetL, with 14 transmembrane α-helices, is a member of the family of Tet efflux proteins in Gram-positive bacterial pathogens, including Bacillus anthracis, Bacillus cereus, Streptococcus pneumoniae, Staphylococcus aureus. Clostridium spp., Enterococcus spp. and Listeria spp. All Tet transporters belong to the major facilitator superfamily (MFS). No crystal structure is available for any Tet protein or any MFS protein with 14 helices. A crystal structure of TetL, in combination with biochemical and biophysical studies, will not only greatly advance our understanding of the molecular mechanism of efflux-mediated antibiotic resistance, it will also suggest new ways to modify tetracycline to reverse resistance. 2. Major setback due to Sandy We will use nanobodies to improve diffraction resolution. Our experience with antibodies allowed us to rapidly progress in the evaluation of nanobodies produced through a collaboration with the Steyaert Lab in Brussels, Belgium. We have generated a panel of 18 nanobodies that recognize purified TetL. These nanobodies can be readily made and purified from bacterial hosts, providing a continuous supply of nanobodies for structural and functional studies. Of these, 12 produce a stable complex that has been used in co-crystallization studies. Crystals have been grown for 9 of these, with the best resolution reaching 4-5 Å. These TetL/nanobody complexes are being further optimized to improve diffraction resolution. Additionally, the nanobodies are being combined to form heteromeric complexes to increase the available crystallization space in order to facilitate higher resolution diffraction. These nanobodies may also bind to preferential conformations of TetL, which may structurally shed light on the conformational changes during the tetracycline transport cycle.

1。原始项目的摘要 抗生素耐药性是对公共卫生的主要威胁。抵抗的重要机制 抗生素（例如四环素）是由膜转运蛋白介导的外排。这 四环素外排泵TET蛋白的底物识别的结构基础尚不清楚。 枯草芽孢杆菌的外排泵TETL以A的形式出口四环素（TC） 四环素 - 磁通量[TC.MG2+]+复合物，并负责该细菌对 曾经广泛有效的抗生素。 TETL，具有14个跨膜α-螺旋，是 革兰氏阳性细菌病原体中的TET外排蛋白家族，包括芽孢杆菌 炭疽，蜡状芽孢杆菌，肺炎链球菌，金黄色葡萄球菌。梭状芽胞杆菌。 肠球菌属。和李斯特菌。所有TET转运蛋白都属于主要的促进器 超家族（MFS）。任何TET蛋白或任何MFS蛋白都没有晶体结构 14个螺旋。 TETL的晶体结构，结合生化和生物物理研究， 我们不仅会大力提高我们对排出介导的分子机制的理解 抗生素耐药性，它还将提出新的方法来修改四环素以逆转电阻。 2。由于桑迪而引起的重大挫折 我们将使用纳米体来改善衍射分辨率。我们的抗体经验 使我们能够在评估通过协作产生的纳米体的评估中迅速进步 与比利时布鲁塞尔的Steyaert实验室一起。我们已经生成了一个18个纳米生物的面板 公认的纯化TETL。这些纳米生物可以很容易地从细菌中纯化和纯化 宿主，为结构和功能研究提供了连续的纳米构造。的 这些，12产生了一种稳定的复合物，该复合物已用于共结晶研究。晶体有 它们是在其中的9种生长，最好的分辨率达到4-5。这些TETL/纳米机构 复合物正在进一步优化以改善衍射分辨率。另外， 纳米体被合并以形成异源复合物以增加可用的 结晶空间以促进更高的分辨率衍射。这些纳米体可能 也与TETL的首选构象结合，该构象可能在结构上脱落在 四环素传输周期期间的构象变化。