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. 原项目概要 抗生素耐药性是对公众健康的主要威胁。重要的抵抗机制 四环素等抗生素是由膜转运蛋白介导的外排。这 四环素外排泵（Tet 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 以四环素 (Tc) 的形式输出 四环素-镁 [Tc.Mg2+]+ 复合物，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（包括芽孢杆菌）中的 Tet 外排蛋白家族 炭疽杆菌、蜡样芽孢杆菌、肺炎链球菌、金黄色葡萄球菌。梭菌属， 肠球菌属和李斯特菌属。所有 Tet 转运蛋白都属于主要促进者 超家族（MFS）。任何 Tet 蛋白或任何 MFS 蛋白都没有可用的晶体结构 14 个螺旋。 TetL的晶体结构，结合生化和生物物理研究， 不仅将极大地促进我们对外排介导的分子机制的理解 抗生素耐药性，它还将提出修改四环素以逆转耐药性的新方法。 2.桑迪造成的重大挫折 我们将使用纳米抗体来提高衍射分辨率。我们在抗体方面的经验 使我们能够在评估通过合作生产的纳米抗体方面取得快速进展 与比利时布鲁塞尔的 Steyaert 实验室合作。我们已经生成了一个由 18 个纳米体组成的面板 识别纯化的 TetL。这些纳米抗体可以很容易地从细菌中制备和纯化 宿主，为结构和功能研究提供持续的纳米抗体供应。的 其中 12 种产生了稳定的复合物，已用于共结晶研究。水晶有 其中 9 个已生长，最佳分辨率达到 4-5 Å。这些TetL/纳米抗体 配合物正在进一步优化以提高衍射分辨率。此外， 纳米抗体被组合形成异聚复合物，以增加可用的 结晶空间，以利于更高分辨率的衍射。这些纳米抗体可以 也与 TetL 的优先构象结合，这可能在结构上揭示 四环素转运周期中的构象变化。