structual characterization of protein import across bacterial outer membranes

E.大肠杆菌TonB依赖性转运蛋白，铁肠杆菌素转运蛋白（1）表明，这种铁转运蛋白使用一个22链β-桶跨越外膜，一个意外的插入结构域折叠到桶内部。栓结构域的功能是在膜的细胞外侧结合特异性铁螯合物，并在外膜的周质侧与能量转导蛋白TonB相互作用。在这种基态结构中，塞域完全闭塞的桶孔，揭示了一个意想不到的复杂性铁螯合物运输。最近，我们的研究小组主要关注来自病原菌的铁转运蛋白，假设这些蛋白质的结构可能导致新的抗生素和疫苗靶点，并有助于对细菌跨外膜转运的分子描述。2003年，我们发表了柠檬酸铁转运蛋白的三种结构，确定了特异性铁螯合物如何被识别以及配体结合如何跨外膜转导信号，为转运系统做好准备（2）。现在，我们正在扩展我们的研究，以了解这些相同的转运蛋白是如何被大的有毒蛋白质（大肠杆菌素）挪用而进入细胞的。 2007年完成了以下工作： 与其同源蛋白毒素复合的铁转运蛋白的结构测定： 大肠杆菌素Ia是一种69 kDa的蛋白质，可杀死敏感的大肠杆菌。通过结合特异性铁转运蛋白大肠杆菌素I受体（Cir; 70 kDa），随后将其通道形成结构域转位穿过周质空间，在周质空间中插入内膜并形成杀死细胞的电压门控离子通道，从而使大肠杆菌细胞死亡。我们解决了两个晶体结构的Cir，单独的和与大肠杆菌素Ia的受体结合结构域的复合物，分辨率分别为2.65 A和2.5 A。这是TonB依赖性大肠杆菌素/受体系统的第一个结构表征，我们观察到大肠杆菌素结合后受体中的大的和不寻常的构象变化。从这些结构中，我们能够模拟全长大肠杆菌素Ia与Cir结合时的相互作用。该模型提出的一个引人注目的问题是，如此大的蛋白质毒素如何通过相对较小的β-桶孔转移。这些结构的可用性现在允许我们询问有关大肠杆菌素输入的详细机制问题，以最终了解细菌毒素如何能够进入宿主细胞。 引用 1. Buchanan，S.K.，史密斯，B.S.，文卡特拉马尼湖Xia，D.，埃塞尔湖Palnitkar，M.，查克拉博蒂河货车德海尔姆，D. & Deisenhofer，J.大肠杆菌外膜活性转运蛋白FepA的晶体结构。Nat Struct Biol 6，56-63（1999）. 2. Yue，W.W.，Grizot，S. & Buchanan，S.K.无铁柠檬酸盐和柠檬酸铁与TonB依赖性外膜转运蛋白FecA结合的结构证据。J Mol Biol 332，353-68（2003）.

The first crystal structure of an E. coli TonB-dependent transporter, ferric enterobactin transporter(1), showed that this iron transporter uses a 22-stranded beta-barrel to span the outer membrane with an unanticipated plug domain folded into the barrel interior. The plug domain functions to bind a specific ferric chelate at the extracellular side of the membrane and to interact with an energy transducing protein, TonB, at the periplasmic side of the outer membrane. In this ground state structure, the plug domain completely occludes the barrel pore, revealing an unexpected complexity for ferric chelate transport. More recently our group has focused primarily on iron transporters from pathogenic bacteria with the hypothesis that structures of these proteins could lead to new antibiotics and vaccine targets, as well as contributing to a molecular description of bacterial transport across the outer membrane. In 2003, we published three structures of a ferric citrate transporter that established how specific ferric chelates are recognized and how ligand binding transduces a signal across the outer membrane, preparing the system for transport(2). Now we are extending our studies to ask how these same transporters are misappropriated by large, toxic proteins (colicins) for import into the cell. The following work was accomplished during 2007: Structure determination of an iron transporter in complex with its cognate protein toxin: Colicin Ia is a 69 kDa protein that kills susceptible E. coli cells by binding to a specific iron transporter, colicin I receptor (Cir; 70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-gated ion channel that kills the cell. We solved two crystal structures of Cir, alone and in complex with the receptor binding domain of colicin Ia, to resolutions of 2.65 A and 2.5 A, respectively. This is the first structural characterization of a TonB-dependent colicin/receptor system and we observed large and unusual conformational changes in the receptor upon colicin binding. From these structures, we were able to model the interaction with full-length colicin Ia when it binds to Cir. The striking question posed by the model is how such a large protein toxin can be translocated through a relatively small beta-barrel pore. The availability of these structures now allows us to ask detailed mechanistic questions about colicin import, to ultimately understand how bacterial toxins are able to enter host cells. References 1. Buchanan, S.K., Smith, B.S., Venkatramani, L., Xia, D., Esser, L., Palnitkar, M., Chakraborty, R., van der Helm, D. & Deisenhofer, J. Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat Struct Biol 6, 56-63 (1999). 2. Yue, W.W., Grizot, S. & Buchanan, S.K. Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. J Mol Biol 332, 353-68 (2003).