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Crystal Structure of a Colicin N Fragment Suggests a Model for Toxicity

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Crystal Structure of a Colicin N Fragment Suggests a Model for Toxicity Research Article 863 Crystal structure of a colicin N fragment suggests a model for toxicity Ingrid R Vetter†*, Michael W Parker‡*, Alec D Tucker§, Jeremy H Lakey#, Franc Pattus¶ and Demetrius Tsernoglou¥ Background: Pore-forming colicins are water-soluble bacteriocins capable of Address: European Molecular Biology Laboratory, binding to and translocating through the Escherichia coli cell envelope. They Postfach 10.2209, D-6900 Heidelberg, Germany. then undergo a transition to a transmembrane ion channel in the cytoplasmic Present addresses: †Max-Planck-Institute for membrane leading to bacterial death. Colicin N is the smallest pore-forming Molecular Physiology, Rheinlanddamm 201, 44139 colicin known to date (40 kDa instead of the more usual 60 kDa) and the crystal Dortmund, Germany, ‡St. Vincent’s Institute of structure of its membrane receptor, the porin OmpF, is already known. Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia, §Pfizer Central Research, Structural knowledge of colicin N is therefore important for a molecular Molecular Sciences Department, Sandwich, Kent, understanding of colicin toxicity and is relevant to toxic mechanisms in general. CT13 9NJ, UK, #Department of Biochemistry and Genetics, The Medical School, The University of Results: The crystal structure of colicin N reveals a novel receptor-binding Newcastle-upon-Tyne, NE2 4HH, UK, ¶Département Récepteurs et Protéines Membranaires, UPR 9050, domain containing a six-stranded antiparallel β sheet wrapped around the CNRS, ESBS, Rue Sébastien Brant, F-67400 α 63 Å long N-terminal helix of the pore-forming domain. The pore-forming Illkirch, France and ¥Department of Biochemical domain adopts a ten α-helix bundle that has been observed previously in the Sciences, Università di Roma, ‘La Sapienza’, pore-forming domains of colicin A, Ia and E1. The translocation domain, Piazzale Aldo Moro 5, I-00185 Rome, Italy. however, does not appear to adopt any regular structure. Models for receptor *Corresponding authors. binding and translocation through the outer membrane are proposed based E-mail: [email protected] on the structure and biochemical data. [email protected] Conclusions: The colicin N–ompF system is now the structurally best-defined Key words: colicins, membrane translocation, porin, receptor binding, toxin structure translocation pathway. Knowledge of the colicin N structure, coupled with the structure of its receptor, OmpF, and previously published biochemical data, Received: 9 February 1998 limits the numerous possibilities of translocation and leads to a model in which Revisions requested: 25 March 1998 the translocation domain inserts itself through the porin pore, the Revisions received: 28 April 1998 Accepted: 27 May 1998 receptor-binding domain stays outside and the pore-forming domain translocates along the outer wall of the trimeric porin channel. Structure 15 July 1998, 6:863–874 http://biomednet.com/elecref/0969212600600863 © Current Biology Ltd ISSN 0969-2126 Introduction energy-dependent uptake of vitamin B12 and iron sidero- Colicins are plasmid-encoded bacteriocins, produced by phores [6,7]. The Tol system consists of six proteins: Escherichia coli, that kill sensitive E. coli strains and related TolA, TolB, TolQ, TolR, PAL and Orf2 [8,9]. Group B bacteria (for recent reviews see [1–5]). The cell envelope colicins (B, D, Ia, Ib, M, 5 and 10) are taken up via the Ton of gram negative bacteria, with its two membranes, pre- pathway, whereas the translocation of group A colicins (A, sents a serious obstacle for the uptake of macromolecules E1–9, K, N and cloacin DF13) involves interaction with of the size of colicins (40–60 kDa). To cross this barrier TolA and different combinations of the proteins from the and reach their target, colicins must first bind to their Tol family [10–12]. Additional proteins are also essential cognate receptor on the cell surface and then translocate for the translocation of some colicins: the porin OmpF for across the outer membrane. The pore-forming colicins (E1, colicins A, K and N; TolC for colicins E1 and 10; and A, B, N, Ia, Ib, K, 10 and 5) and colicin M, an inhibitor of OmpA for colicin K [13]. murein biosynthesis, insert into the inner membrane, whereas colicins with nuclease activity (E2–E9 and cloacin Colicins consist of distinctive protein modules, which pre- DF13) must enter the cytoplasm. Uptake of colicins sumably originated from the genetic recombination of through the outer membrane follows two alternative path- DNA fragments that encoded for different specialized func- ways mediated by the Ton or Tol systems. These two tions. The N-terminal domain specifies the translocation systems are also involved in the transport of bacteriophage route (Ton or Tol pathways), a central domain specifies the DNA into the cells. The Ton system consists of the pro- receptor, and a C-terminal domain carries the killing activ- teins TonB, ExbB and ExbD and is also involved in the ity. Colicins E1 and 10 contain a fourth domain, the TolC 864 Structure 1998, Vol 6 No 7 domain, located between the translocation and the receptor recently solved tripartite structure of colicin Ia at 3.0 Å domains [14]. TonB-dependent colicins contain a homolo- resolution [26] is compatible with such organization. The gous sequence of five amino acids, called the TonB box, in molecule is 210 Å long with the translocation and pore- their N-terminal translocation domain, which is also present forming domains separated from the receptor-binding in TonB-dependent receptors [15]. The Tol-dependent col- domain by a pair of helices, each 160 Å long. icins possess a glycine-rich and proline-rich region in their N-terminal translocation domains. The existence of a TolA Colicin N, which belongs to the group A colicins, possesses box has been proposed with the following motif: Asp–Gly– similar properties and functions to other pore-forming col- (Ser,Thr)–Gly–Trp [14]. But mutation of serine in this icins, but has a much shorter polypeptide chain (385 amino motif to phenylalanine in colicin E3 inhibits TolB binding acids) and a single receptor. It thus provides a simpler instead [12] and the motif is present in only one subset of model system than other colicins for pursuing structure– Tol-dependent colicins. Another TolA box with the motif function studies. Here, we present the crystal structure, at Glu–Gly–Gly–Gly–Ser has been proposed for protein III of 3.1 Å resolution, of a large fragment of colicin N (missing phage f1 [16]. There is experimental evidence that the the first 66 residues) that contains the pore-forming and N-terminal domains of both group A and group B colicins the receptor-binding domains. The pore-forming domain interact with components of the translocation system on the adopts the canonical ten helix bundle seen in other colicins inner side of the outer membrane; translocation through the [24–26], whereas the receptor-binding domain adopts a outer membrane therefore occurs during colicin uptake. novel fold. The crystal structure of the colicin N receptor, Mutations in the TonB box of colicins B and M impaired the protein ompF, which is one of the major unspecific uptake, but this was partially restored by a mutation in porins from E. coli, has been solved previously at 2.4 Å TonB [17]. Expression of the N-terminal domain of either resolution [37]. The structures have enabled us to look at colicin B or colicin E3 in the periplasm make E. coli strains possible interactions between the colicin receptor-binding tolerant to the corresponding colicins [12]. domain and its receptor, leading to new insights into the translocation mechanism of colicins. Pore-forming colicins induce voltage-dependent potass- ium and phosphate efflux across the plasma membrane of Results the target bacterium [18] and form voltage-gated ion chan- The pore-forming domain nels in artificial membranes [19–23]. The pore activity is The structure of colicin N is shown in Figure 1 and the carried by the C-terminal domain [20,23]. Near the car- corresponding topology diagram is presented in Figure 2. boxyl end of the pore-forming domain is a hydrophobic The pore-forming domain of colicin N consists of a bundle segment present in all the pore-forming colicins. The of ten α helices arranged in three layers forming the so- crystal structure of the pore-forming domain of colicin A, called ‘globin-like’ topology [38,39], also found in the pore- solved to 2.4 Å resolution, revealed that this hydrophobic forming apoptosis regulatory protein Bcl-xL [40]. This topol- segment forms a buried α-helical hairpin, surrounded by ogy is identical to that observed in the pore-forming an outer bundle of eight amphipathic helices [24]. A similar domains of other colicins (Figure 2; [24–26]). A character- fold has been identified recently in the crystal structures istic feature of the pore-forming domains of colicins is of the pore-forming domain of colicin E1 and the full- their hydrophobic core consisting of a buried helical hairpin, length colicin Ia [25,26]. A large amount of data have been formed by α helices 8 and 9, and surrounded by a cluster accumulated concerning the mechanism of membrane inser- of aromatic residues. Although the hairpin is present in all tion and pore formation by this domain [1–4,27–31]. A uni- pore-forming colicins, some colicins exhibit extensive dele- fying model has been proposed recently that reconciles tions in this region, for example colicins E1 and Ia. various proposals on the orientation of the hairpin with Colicin N has a deletion of one amino acid in helix 8 com- respect to the membrane [32] and segments of the poly- pared to colicin A (corresponding to Leu165 in the colicin A peptide chain have been identified that move across the pore-forming domain; see Figures 3 and 4).
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