structual characterization of protein import across bacterial outer membranes

Diabetes, Digestive, Kidney Diseases

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Abstract

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.[unreadable] The following work was accomplished during 2007:[unreadable] [unreadable] Structure determination of an iron transporter in complex with its cognate protein toxin:[unreadable] [unreadable] 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.[unreadable] [unreadable] References[unreadable] [unreadable] 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).[unreadable] [unreadable] 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).