Structural Basis of Tetracycline Resistance by Efflux Pump TetL.

1.原项目总结 抗生素耐药性是对公共健康的主要威胁。一种重要的抗病机制 四环素类抗生素的外排是由膜转运蛋白介导的。这个 四环素外排泵识别底物的结构基础，Tet蛋白，尚不清楚。 枯草芽孢杆菌外排泵TetL以a形式输出四环素(TC) 四环素-镁[Tc.Mg~(2+)]+络合物，是导致该菌对 曾经广泛有效的抗生素。TetL具有14个跨膜α-螺旋，是 包括芽孢杆菌在内的革兰氏阳性细菌中的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(Clostridium spp.) 肠球菌属和李斯特氏菌(Listeria spp.)所有的TET运输者都属于主要的促进者 超级家族(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 在抗生素耐药性方面，它还将提出修改四环素以逆转耐药性的新方法。 2.桑迪带来的重大挫折 我们将使用纳米体来提高衍射分辨率。我们使用抗体的经验 使我们能够在评估通过合作产生的纳米实体方面取得快速进展 位于比利时布鲁塞尔的斯泰厄特实验室。我们已经生成了一个由18个纳米实体组成的面板 识别纯化的TetL。这些纳米小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体。的 这12种化合物产生了一种稳定的络合物，已用于共结晶研究。水晶有 已经生长了其中的9个，最好的分辨率达到了4-5ä。这些TetL/纳米体 络合物正在进一步优化，以提高衍射分辨率。此外， 纳米体正在被结合起来形成异构体络合物，以增加可用的 结晶空间，以便于更高分辨率的衍射。这些纳米体可能 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。

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.