Center for Computational Biology, The University of Kansas, Lawrence, United States.
Department of Molecular Biosciences, The University of Kansas, Lawrence, United States.
Elife. 2022 Feb 24;11:e73297. doi: 10.7554/eLife.73297.
The double membrane architecture of Gram-negative bacteria forms a barrier that is impermeable to most extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, that hijacks the outer membrane proteins (OMPs) TolC and BtuB to enter the cell. Here, we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryo-electron microscopy to obtain the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore-an architectural rearrangement from colicin E1's unbound conformation. This binding is stable in live cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plug the channel, inhibiting its native efflux function as an antibiotic efflux pump, and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other OMP functions.
革兰氏阴性菌的双层膜结构形成了一道屏障，使大多数细胞外威胁无法穿透。细菌素蛋白进化到可以利用外膜中嵌入的可及的、表面暴露的蛋白质来输送细胞毒性货物。肠毒素 E1 是一种由革兰氏阴性菌产生的细菌素，对革兰氏阴性菌有毒性，它劫持外膜蛋白（OMPs）TolC 和 BtuB 进入细胞。在这里，我们通过高分辨率冷冻电镜捕获了位于其膜受体 TolC 内的肠毒素 E1 转运结构域，从而获得了第一个报道的与 TolC 结合的细菌素结构。肠毒素 E1 以打开的铰链形式稳定地结合 TolC 通过 TolC 孔-从肠毒素 E1 的未结合构象的结构重排。如单分子荧光显微镜所示，这种结合在活细胞中是稳定的。最后，结合到 TolC 上的肠毒素 E1 片段堵塞了通道，抑制了其作为抗生素外排泵的天然外排功能，从而增加了对三类抗生素的敏感性。除了证明这些蛋白质片段是开发新型抗生素增效剂的有用起点之外，这种方法还可以扩展到其他肠毒素，以抑制其他 OMP 功能。
