Colicin N binds to the periphery of its receptor and translocator, outer membrane protein F

Colicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.     

Colicin pore-forming domains bind to Escherichia coli trimeric porins

Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain. It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA. Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II. Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain). No binding was seen with the monomeric porin OmpA. In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive. Pull-down assays in solution show that porin-P-domain complexes also form in the presence of outer membrane lipopolysaccharide. This indicates that an additional colicin-porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain. This may help to explain the role of porins and TolA-II in the later stages of colicin translocation.     

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.     

Minimum length requirement of the flexible N-terminal translocation subdomain of colicin E3

The 315-residue N-terminal T domain of colicin E3 functions in translocation of the colicin across the outer membrane through its interaction with outer membrane proteins including the OmpF porin. The first 83 residues of the T domain are known from structure studies to be disordered. This flexible translocation subdomain contains the TolB box (residues 34 to 46) that must cross the outer membrane in an early translocation event, allowing the colicin to bind to the TolB protein in the periplasm. In the present study, it was found that cytotoxicity of the colicin requires a minimum length of 19 to 23 residues between the C terminus (residue 46) of the TolB box and the end of the flexible subdomain (residue 83). Colicin E3 molecules of sufficient length display normal binding to TolB and occlusion of OmpF channels in vitro. The length of the N-terminal subdomain is critical because it allows the TolB box to cross the outer membrane and interact with TolB. It is proposed that the length constraint is a consequence of ordered structure in the downstream segment of the T domain (residues 84 to 315) that prevents its insertion through the outer membrane via a translocation pore that includes OmpF.     

Daring to be different: colicin N finds another way

The mechanisms by which colicins, protein toxins produced by Escherichia coli, kill other E. coli, have become much better understood in recent years. Most colicins initially bind to an outer membrane protein receptor, and then search for a separate nearby outer membrane protein translocator that serves as a pathway into target cells. Many colicins use the outer membrane porin, OmpF, as that translocator, while using a different primary receptor. Colicin N is unique among known colicins in that only OmpF had been identified as being required for uptake of the colicin and it was presumed to somehow serve as both receptor and translocator. Genetic screens also identified a number of genes required for lipopolysaccharide (LPS) synthesis as uniquely required for killing by colicin N, but not by other colicins. Johnson et al. show that the receptor‐binding domain of colicin N binds to LPS, and does not require OmpF for that binding. LPS of a minimal length is required for binding, explaining the requirement for specific elements of the LPS biosynthetic pathway. For colicin N, the receptor‐binding domain does not recognize a protein, but rather the most abundant component of the outer membrane itself, LPS.     

Colicin import and pore formation: a system for studying protein transport across membranes?

Pore-forming colicins are a family of protein toxins (M_r40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient-uptake systems and the so-called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage-gated channels). The Tol-dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α-helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin-resistant mutant of OmpF. The X-ray crystal structure of the channel-forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site-directed muta-genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N-terminal domains of colicins interact with the lumen of the OmpF pore and then with the C-terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C-terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.     

Structure of the complex of the colicin E2 R-domain and its BtuB receptor. The outer membrane colicin translocon

The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-A crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg(452) at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.     

Stable self-assembly of a protein engineering scaffold on gold surfaces

The outer membrane protein OmpF from Escherichia coli is a member of a large family of beta-barrel membrane proteins. Some, like OmpF, are pore-forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor-binding domain (R-domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF-E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF-E183C binds directly to gold surfaces and creates high-density protein layers by self-assembly from detergent solution. When the gold surface is pretreated with beta-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its beta-rich structure. Furthermore, we could also measure R-domain binding by SPR and IS, confirming the functional reconstitution of a self-assembled membrane protein monolayer at the gold surface. Because these beta-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.     

Involvement of OmpF during reception and translocation steps of colicin N entry

[125I]-colicin N binds to OmpF receptor sites (70,000 per cell) with an average Kassoc of 3.2 x 10(6) M-1 at 23 degrees C. Monoclonal antibody directed against a cell-surface-exposed epitope of OmpF is able to complete with the binding of the colicin in vitro and also to protect against colicin N in vivo. OmpF is an absolute requirement for colicin N uptake. OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A. Colicin N does not cross-react with various monoclonal antibodies directed against colicin A.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.     

A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli

Inducible hybrid genes encoding two large domains, a periplasmic domain consisting of the PhoS sequence and an outer membrane domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesized hybrid polypeptides are correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivity toward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogold labelling on ultrathin cryosections. Insertion of a hydrophobic sequence between PhoS and OmpF improves the trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimeric conformation which is recognized by specific antibodies. Moreover, the deletion of the C-terminal region impairs the functional insertion of the OmpF domain; this indicates that the last membrane-spanning region of OmpF is necessary for the correct folding and orientation of the protein in the outer membrane.     

The colicin E3 outer membrane translocon: immunity protein release allows interaction of the cytotoxic domain with OmpF porin

The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.     

Localization and assembly into the Escherichia coli envelope of a protein required for entry of colicin A.

Mutations in tolQ, previously designated fii, render cells tolerant to high concentrations of colicin A. In addition, a short deletion in the amino-terminal region of colicin A (amino acid residues 16 to 29) prevents its lethal action, although this protein can still bind the receptor and forms channels in planar lipid bilayers in vitro. These defects in translocation across the outer membrane in the tolQ cells or the colicin A mutant cannot be bypassed by osmotic shock. The TolQ protein, which is constitutively expressed at a low level, was studied in recombinant plasmid constructs allowing the expression of various TolQ fusion proteins under the control of the inducible caa promoter. The TolQ protein was thus "tagged" with an epitope from the colicin A protein for which a monoclonal antibody is available. A fusion protein containing the entire TolQ protein plus the 30 N-terminal residues of colicin A was shown to complement the tolQ mutation. Pulse-chase labeling followed by gradient fractionation indicated that the bulk of the overproduced fusion protein was rapidly incorporated into the inner membrane, with small amounts localized to regions corresponding to the attachment sites between inner and outer membranes and to the outer membrane itself. However, most of the protein was rapidly degraded, leaving only that localized to the attachment sites and the outer membrane remaining at very late times of chase.     

The unstructured domain of colicin N kills Escherichia coli

Bacteria often produce toxins which kill competing bacteria. Colicins, produced by and toxic to Escherichia coli bacteria are three‐domain proteins so efficient that one molecule can kill a cell. The C‐terminal domain carries the lethal activity and the central domain is required for surface receptor binding. The N‐terminal domain, required for translocation across the outer membrane, is always intrinsically unstructured. It has always been assumed therefore that the C‐terminal cytotoxic domain is required for the bactericidal activity. Here we report the unexpected finding that in isolation, the 90‐residue unstructured N‐terminal domain of colicin N is cytotoxic. Furthermore it causes ion leakage from cells but, unlike known antimicrobial peptides (AMPs) with this property, shows no membrane binding behaviour. Finally, its activity remains strictly dependent upon the same receptor proteins (OmpF and TolA) used by full‐length colicin N. This mechanism of rapid membrane disruption, via receptor mediated binding of a soluble peptide, may reveal a new target for the development of highly specific antibacterials.     

Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1

Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.     

Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.     

Assembly of colicin A in the outer membrane of producing Escherichia coli cells requires both phospholipase A and one porin,but phospholipase A is sufficient for secretion

Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.     

Discovery of critical Tol A-binding residues in the bactericidal toxin colicin N: a biophysical approach

Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal regions. Finally, the toxic C-terminal region is inserted into or across the cytoplasmic membrane. We have measured the binding of colicin N to TolA by isothermal titration microcalorimetry (ITC) and tryptophan fluorescence. The isolated N-terminal domain exhibits a higher affinity for TolA ( K _d = 1 μM) than does the whole colicin (18 μM), and similar behaviour has been observed when the N-terminal domain of the g3p protein of the bacteriophage fd, which also binds TolA, is examined in isolation and in situ . This may indicate a similar mechanism in which a cryptic TolA binding site is revealed after primary receptor binding. The isolated colicin N N-terminal domain appears to be unstructured in circular dichroism and fluorescence studies. We have used mutagenesis and ITC to characterize the TolA binding site and have shown it to be of a different sequence and much further from the N-terminus than previously thought.