Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
iThree Institute, University of Technology Sydney, Sydney, NSW 2007, Australia.
Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):12051-12056. doi: 10.1073/pnas.1713741114. Epub 2017 Oct 25.
Unlike their descendants, mitochondria and plastids, bacteria do not have dedicated protein import systems. However, paradoxically, import of protein bacteriocins, the mechanisms of which are poorly understood, underpins competition among pathogenic and commensal bacteria alike. Here, using X-ray crystallography, isothermal titration calorimetry, confocal fluorescence microscopy, and in vivo photoactivatable cross-linking of stalled translocation intermediates, we demonstrate how the iron transporter FpvAI in the opportunistic pathogen is hijacked to translocate the bacteriocin pyocin S2 (pyoS2) across the outer membrane (OM). FpvAI is a TonB-dependent transporter (TBDT) that actively imports the small siderophore ferripyoverdine (Fe-Pvd) by coupling to the proton motive force (PMF) via the inner membrane (IM) protein TonB1. The crystal structure of the N-terminal domain of pyoS2 (pyoS2) bound to FpvAI ( = 240 pM) reveals that the pyocin mimics Fe-Pvd, inducing the same conformational changes in the receptor. Mimicry leads to fluorescently labeled pyoS2 being imported into FpvAI-expressing cells by a process analogous to that used by bona fide TBDT ligands. PyoS2 induces unfolding by TonB1 of a force-labile portion of the plug domain that normally occludes the central channel of FpvAI. The pyocin is then dragged through this narrow channel following delivery of its own TonB1-binding epitope to the periplasm. Hence, energized nutrient transporters in bacteria also serve as rudimentary protein import systems, which, in the case of FpvAI, results in a protein antibiotic 60-fold bigger than the transporter's natural substrate being translocated across the OM.
与它们的后代线粒体和质体不同，细菌没有专门的蛋白质导入系统。然而，具有讽刺意味的是，细菌素的导入机制尚不清楚，但这是病原细菌和共生细菌竞争的基础。在这里，我们使用 X 射线晶体学、等温滴定量热法、共聚焦荧光显微镜和体内光活化停顿易位中间产物的交联，展示了机会性病原体 中的铁转运蛋白 FpvAI 如何被劫持来跨外膜（OM）转运细菌素 pyocin S2（pyoS2）。FpvAI 是一种 TonB 依赖性转运体（TBDT），通过与内膜（IM）蛋白 TonB1 耦合质子动力势（PMF），主动导入小分子铁载体 ferripyoverdine（Fe-Pvd）。pyoS2 的 N 端结构域与 FpvAI （=240pM）结合的晶体结构揭示了该噬菌体模拟了 Fe-Pvd，引起了受体的相同构象变化。模拟导致荧光标记的 pyoS2 通过与 bona fide TBDT 配体类似的过程被导入表达 FpvAI 的 细胞。TonB1 通过 TonB1 诱导插头结构域的力不稳定部分展开，该结构域通常阻塞 FpvAI 的中央通道。然后，在将自身的 TonB1 结合表位递送至周质后，将噬菌体拖过该狭窄通道。因此，细菌中的能量营养转运蛋白也充当基本的蛋白质导入系统，在 FpvAI 的情况下，导致比转运体的天然底物大 60 倍的蛋白质抗生素穿过 OM 转运。
