structual characterization of protein import across bacterial outer membranes

大肠杆菌TonB依赖的转运蛋白铁蛋白转运蛋白转运蛋白(1)的第一个晶体结构表明，这种铁转运蛋白转运蛋白使用一个22链的β-桶来跨越外膜，并将一个意想不到的塞区折叠到桶的内部。Plug结构域的功能是在膜的胞外侧结合特定的铁螯合物，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，塞状结构域完全封闭了桶状孔，揭示了铁螯合物传输的意想不到的复杂性。最近，我们的团队主要关注病原菌的铁转运蛋白，假设这些蛋白质的结构可能会导致新的抗生素和疫苗靶标，并对细菌跨外膜运输的分子描述做出贡献。2003年，我们发表了柠檬酸铁转运蛋白的三种结构，它们确定了特定的铁络合物如何被识别，以及配体结合如何通过外膜传递信号，为运输系统做好准备[2]。现在，我们正在扩大我们的研究范围，以了解这些转运蛋白是如何被大的有毒蛋白质(结肠素)挪用来输入细胞的。 2007年期间完成了以下工作： 铁转运蛋白及其同源蛋白毒素复合体的结构测定 Colicin Ia是一种69 kDa的蛋白质，它通过与特定的铁转运蛋白Colicin I受体(Cir；70 kDa)结合，然后将其通道形成域穿过周质空间，插入内膜，形成电压门控离子通道，从而杀死敏感的大肠杆菌细胞。我们解析了Cir的两个晶体结构，分别为2.65A和2.5A，分别是单独的和与结肠素Ia的受体结合域形成的复合体。这是TonB依赖的Colicin/受体系统的第一个结构特征，我们观察到Colicin结合时受体的大而不寻常的构象变化。从这些结构中，我们能够模拟当全长Colicin Ia与CIR结合时与它的相互作用。该模型提出的突出问题是，如此大的蛋白质毒素如何通过相对较小的贝塔桶孔转运。这些结构的可获得性现在使我们能够询问有关粘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。 参考文献 1.Buchanan，S.K.，Smith，B.S.，Venkatramani，L.，Xia，D.，Esser，L.，Palnitkar，M.，Chakraborty，R.，van der Helm，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.无铁柠檬酸和柠檬酸铁与TonB依赖的外膜转运蛋白FECA结合的结构证据。《摩尔生物杂志》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).