Structure and Function of Colicin S4, a Colicin with a Duplicated Receptor-binding Domain
2008; Elsevier BV; Volume: 284; Issue: 10 Linguagem: Inglês
10.1074/jbc.m808504200
ISSN1083-351X
AutoresThomas Arnold, Kornelius Zeth, Dirk Linke,
Tópico(s)Bacteriophages and microbial interactions
ResumoColicins are plasmid-encoded toxic proteins produced by Escherichia coli strains to kill other E. coli strains that lack the corresponding immunity protein. Colicins intrude into the host cell by exploiting existing transport, diffusion, or efflux systems. We have traced the way colicin S4 takes to execute its function and show that it interacts specifically with OmpW, OmpF, and the Tol system before it inserts its pore-forming domain into the cytoplasmic membrane. The common structural architecture of colicins comprises a translocation, a receptor-binding, and an activity domain. We have solved the crystal structure of colicin S4 to a resolution of 2.5 Å, which shows a remarkably compact domain arrangement of four independent domains, including a unique domain duplication of the receptor-binding domain. Finally, we have determined the residues responsible for binding to the receptor OmpW by mutating exposed charged residues in one or both receptor-binding domains. Colicins are plasmid-encoded toxic proteins produced by Escherichia coli strains to kill other E. coli strains that lack the corresponding immunity protein. Colicins intrude into the host cell by exploiting existing transport, diffusion, or efflux systems. We have traced the way colicin S4 takes to execute its function and show that it interacts specifically with OmpW, OmpF, and the Tol system before it inserts its pore-forming domain into the cytoplasmic membrane. The common structural architecture of colicins comprises a translocation, a receptor-binding, and an activity domain. We have solved the crystal structure of colicin S4 to a resolution of 2.5 Å, which shows a remarkably compact domain arrangement of four independent domains, including a unique domain duplication of the receptor-binding domain. Finally, we have determined the residues responsible for binding to the receptor OmpW by mutating exposed charged residues in one or both receptor-binding domains. Colicins are plasmid-encoded toxic proteins produced by up to 50% of Escherichia coli strains in natural populations (1Riley M.A. Wertz J.E. Annu. Rev. Microbiol. 2002; 56: 117-137Crossref PubMed Scopus (837) Google Scholar). They are produced to kill competing E. coli strains under stress conditions and are regulated by the SOS response (2Cascales E. Buchanan S.K. Duche D. Kleanthous C. Lloubes R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (767) Google Scholar, 3Gillor O. Vriezen J.A. Riley M.A. Microbiology (Read.). 2008; 154: 1783-1792Crossref PubMed Scopus (58) Google Scholar). Among the toxic functions of colicins, pore formation in the cytoplasmic membrane followed by a breakdown of the electrochemical gradients is the most abundant killing mechanism (4Lakey J.H. Slatin S.L. Curr. Top. Microbiol. Immunol. 2001; 257: 131-161PubMed Google Scholar). Other colicins kill by exerting DNase, RNase, or phosphatase activity in the target cell (2Cascales E. Buchanan S.K. Duche D. Kleanthous C. Lloubes R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (767) Google Scholar). Colicin producer strains are protected from the toxic action by specific immunity proteins encoded on the same plasmid (5de Zamaroczy M. Buckingham R.H. Biochimie (Paris). 2002; 84: 423-432Crossref PubMed Scopus (32) Google Scholar, 6Duche D. Biochimie (Paris). 2002; 84: 455-464Crossref PubMed Scopus (10) Google Scholar, 7Kleanthous C. Hemmings A.M. Moore G.R. James R. Mol. Microbiol. 1998; 28: 227-233Crossref PubMed Scopus (57) Google Scholar). In some cases, an additional protein is encoded on the plasmid, which ensures an efficient lysis of dying producer cells and, as a consequence, the maximal release of colicin into the environment (2Cascales E. Buchanan S.K. Duche D. Kleanthous C. Lloubes R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (767) Google Scholar, 8Cavard D. J. Bacteriol. 1991; 173: 191-196Crossref PubMed Google Scholar). The ubiquitous presence of non-colicinogenic strains in the same environments with colicin producers indicates that there has to be a narrow tradeoff between the costs and the benefits of colicin production (9Riley M.A. Mol. Biol. Evol. 1993; 10: 1048-1059PubMed Google Scholar).Colicins exhibit a modular structure, which suggests that their domains are frequently recombined to create new toxic functions in highly competitive environments (1Riley M.A. Wertz J.E. Annu. Rev. Microbiol. 2002; 56: 117-137Crossref PubMed Scopus (837) Google Scholar, 10Braun V. Patzer S.I. Hantke K. Biochimie (Paris). 2002; 84: 365-380Crossref PubMed Scopus (142) Google Scholar). The modules comprise the N-terminal translocation domain, followed by a specific receptor-binding domain and the activity domain at the C terminus. The most pronounced sequence conservation can be observed among the activity domains of pore-forming colicins. Accordingly, the structures of colicin pore-forming domains solved so far are highly similar (2Cascales E. Buchanan S.K. Duche D. Kleanthous C. Lloubes R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (767) Google Scholar, 11Zakharov S.D. Cramer W.A. Biochim. Biophys. Acta. 2002; 1565: 333-346Crossref PubMed Scopus (78) Google Scholar). The highest variability in sequence and structure can be seen in the central receptor-binding domains that are adapted to bind to very different receptors with high affinity (2Cascales E. Buchanan S.K. Duche D. Kleanthous C. Lloubes R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (767) Google Scholar, 12Cao Z. Klebba P.E. Biochimie (Paris). 2002; 84: 399-412Crossref PubMed Scopus (74) Google Scholar).The initial contact between colicin and the target cell is established between the receptor-binding domain and a specific receptor of the target cell, which in all cases is an outer membrane protein. Colicins recognize their receptor proteins with high specificity (12Cao Z. Klebba P.E. Biochimie (Paris). 2002; 84: 399-412Crossref PubMed Scopus (74) Google Scholar), and few point mutations in the receptor, or its entire deletion, can render the cell fully resistant to the colicin. This also explains the narrow range of strains that are sensitive to a certain colicin or groups of colicins.The N-terminal translocation domain is subsequently used either to penetrate through the receptor pore itself into the periplasm or to recruit an additional translocation pore, typically the general diffusion pore OmpF, to do so (13Housden N.G. Loftus S.R. Moore G.R. James R. Kleanthous C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13849-13854Crossref PubMed Scopus (77) Google Scholar, 14Zakharov S.D. Cramer W.A. Front. Biosci. 2004; 9: 1311-1317Crossref PubMed Scopus (38) Google Scholar). It is typically not resolved in crystal structures, and NMR spectroscopy indicates a high flexibility in this domain (15Deprez C. Blanchard L. Guerlesquin F. Gavioli M. Simorre J.P. Lazdunski C. Marion D. Lloubes R. Biochemistry. 2002; 41: 2589-2598Crossref PubMed Scopus (24) Google Scholar). For some colicins, it has been shown that a whole cascade of interactions between different periplasmic proteins of the target cell and the N terminus of the colicin is necessary for the successful translocation. Colicins have been classified into two groups according to the energized uptake system that they abuse; group A colicins exploit the Tol system for translocation, whereas group B colicins exploit the Ton system (2Cascales E. Buchanan S.K. Duche D. Kleanthous C. Lloubes R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (767) Google Scholar, 10Braun V. Patzer S.I. Hantke K. Biochimie (Paris). 2002; 84: 365-380Crossref PubMed Scopus (142) Google Scholar, 16Lazzaroni J.C. Dubuisson J.F. Vianney A. Biochimie (Paris). 2002; 84: 391-397Crossref PubMed Scopus (91) Google Scholar). The Tol system comprises the lipoprotein Pal, the β-propeller protein TolB, the inner membrane protein TolQ, and TolA and TolR, which span the cytoplasmic membrane but also have extended periplasmic domains. The function of the Tol system is still not fully understood (17Lloubes R. Cascales E. Walburger A. Bouveret E. Lazdunski C. Bernadac A. Journet L. Res. Microbiol. 2001; 152: 523-529Crossref PubMed Scopus (137) Google Scholar). The Ton system comprises TonB in complex with ExbB and ExbD; this complex uses the protonmotive force to interact with outer membrane transporters to energize the import of bound substrates, e.g. iron or cobalamin, and of colicins (18Braun V. ACS Chem. Biol. 2006; 1: 352-354Crossref PubMed Scopus (19) Google Scholar, 19Postle K. Kadner R.J. Mol. Microbiol. 2003; 49: 869-882Crossref PubMed Scopus (247) Google Scholar). In the final step of colicin import, the C-terminal activity domain is translocated through the outer membrane to reach its place of action, which is the cytoplasmic membrane for pore-forming colicins, the periplasm for colicin phosphatases, or the cytosol for colicins with DNase or RNase activity.In contrast to the common domain architecture, the three-dimensional structures known so far differ remarkably in their shape. The first colicin structures (e.g. colicins Ia and E3) solved by x-ray crystallography have been summarized as elongated, mainly α-helical molecules (20Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (123) Google Scholar, 21Wiener M. Freymann D. Ghosh P. Stroud R.M. Nature. 1997; 385: 461-464Crossref PubMed Scopus (224) Google Scholar). Compact structures are observed in colicins M, N, and B (22Hilsenbeck J.L. Park H. Chen G. Youn B. Postle K. Kang C. Mol. Microbiol. 2004; 51: 711-720Crossref PubMed Scopus (63) Google Scholar, 23Vetter I.R. Parker M.W. Tucker A.D. Lakey J.H. Pattus F. Tsernoglou D. Structure. 1998; 6: 863-874Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 24Zeth K. Römer C. Patzer S.I. Braun V. J. Biol. Chem. 2008; 283: 25324-25331Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Intrigued by the unusual receptor-binding domain duplication in colicin S4 and by its binding to the small outer membrane protein OmpW (25Pilsl H. Smajs D. Braun V. J. Bacteriol. 1999; 181: 3578-3581Crossref PubMed Google Scholar), we set out to investigate the pathway that colicin S4 takes through the outer membrane and the periplasm to insert into the cytoplasmic membrane. To this end, we have tested a series of E. coli knock-out strains with deletions of outer membrane proteins and proteins of the Tol and Ton systems for their resistance to colicin S4 and show that it is a Tol-dependent group A colicin. Using single channel conductance measurements, we were able to characterize the pore-forming activity. We have solved the structure of colicin S4 using x-ray crystallography, which displays an unusual, Y-shaped structure with two almost identical receptor-binding domains that form the arms. The sequence similarity of the two receptor-binding domains and their arrangement in the colicin structure raised the question whether colicin S4 would indeed need two functional receptor-binding domains to bind to target cells. On the basis of the structure, we have mapped the crucial charged residues for receptor binding and mutated them to alanines in one or both domains.EXPERIMENTAL PROCEDURESStrains, Constructs, and Primers-All strains and constructs used in this study are listed in supplemental Table SI, and all primers used can be found in supplemental Table SII.Sequence Analysis-Homology searches were done using PSI-BLAST and the non-redundant data base (26Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59166) Google Scholar). Alignments were computed with ClustalW (27Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55203) Google Scholar) and were further edited manually. Secondary structure elements in the pore alignment were assigned using Protein Data Bank codes 2i88 (colicin E1) and 1cii (colicin Ia). All relevant GenBank™ identifiers are listed in supplemental Table SIII.Construction of the 5KΔompW Strain-The ompW deletion strain was produced using the pKO3 vector for homologous recombination (28Link A.J. Phillips D. Church G.M. J. Bacteriol. 1997; 179: 6228-6237Crossref PubMed Scopus (744) Google Scholar); to be able to compare it with the spontaneous ompW mutation generated in a mutagenesis screen (25Pilsl H. Smajs D. Braun V. J. Bacteriol. 1999; 181: 3578-3581Crossref PubMed Google Scholar), the same background strain (E. coli 5K) was chosen. The genome regions upstream and downstream of the OmpW coding region were amplified by PCR and fused in a second PCR using primers KOvfwd, KOvrev, KOhfwd, and KOhrev, which contain compatible ends. The resulting PCR product was subcloned into pCR-BLUNT (Invitrogen), digested with BamHI, and ligated into the pKO3 vector. Selection for deletion clones was done as described (28Link A.J. Phillips D. Church G.M. J. Bacteriol. 1997; 179: 6228-6237Crossref PubMed Scopus (744) Google Scholar) and was verified by PCR of the genomic DNA.Cloning, Expression, and Purification of OmpW-OmpW was cloned, expressed, and purified as described (29Albrecht R. Zeth K. Soding J. Lupas A. Linke D. Acta Crystallogr. F Struct. Biol. Crystalliz. Comm. 2006; 62: 415-418Crossref PubMed Scopus (32) Google Scholar).Cloning and Expression of Colicins S4 and S4His-The DNA sequence was amplified from the pColS4 plasmid using primers S4-fwd and S4-rev or S4-His-rev, respectively. The PCR product was digested with Eco91I and cloned into an Eco91I-digested pASK-IBA33+ vector (IBA GmbH, Göttingen, Germany). The E. coli strain 5KΔompW was transformed with the resulting plasmids, p33-S4 and p33-S4His. Expression of colicin S4 or S4His was induced by adding 0.2 μg/ml anhydrotetracycline (IBA GmbH).Generation of Mutant Colicin Variants-To introduce mutations into the receptor-binding domain, the sequence was first divided between the receptor-binding domains to avoid misannealing of primers in the almost identical sequence parts. The first part was engineered by fusing the PCR product obtained from the wild-type colicin S4 sequence with primers S4-fwd-all and Colmut-2-rev with the PCR product obtained by fusion of primers Colmut-3-fwd, Colmut-4-rev, Colmut-5-fwd, and Colmut-6-rev. To generate the first part without amino acid mutation in receptor-binding domain R1, the primer Colmut-4wt-rev was used instead of Colmut-4-rev. The second part was generated from the wild-type colicin S4 sequence using primers S4-fwd-R2P and ColS4-rev-long. The mutations in the second part were introduced by fusing the PCR products obtained with primers S4-fwd-R2P and S4mut-R2rev and primers Colmut-R2fwd and ColS4-rev-long using the previously generated second part fragment as a template. The two parts were fused by adding primers S4-fwd-all and ColS4-rev-long using parts with and without amino acid changes in every combination to yield S4m1, S4m2, and S4m12, respectively. The PCR products were digested with Eco91I and cloned into Eco91I-digested pASK-IBA33+ vector (IBA GmbH).Purification of Colicin S4 and Its Mutant Variants-4 h post-induction, cells were pelleted at 7000 × g. After resuspension of the pellet in buffer containing 20 mm Tris (pH 8.5), 1 mm MgSO4, 1 mm MnSO4, and a pinch of DNase, cells were passed three times through a French pressure cell. The lysate was centrifuged in an ultracentrifuge at 60,000 × g for 30 min. The supernatant was applied to a Mono Q column (GE Healthcare). The protein was eluted with a gradient to 1 m NaCl. The fractions containing colicin S4 were pooled, concentrated using an Amicon concentrator (Millipore), and applied to a preparative Superdex 200 column (GE Healthcare) equilibrated with 150 mm NaCl and 20 mm MOPS 3The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid. (pH 7.0).Purification of Colicin S4His-Cell lysis was performed as described for colicin S4. After centrifugation in an ultracentrifuge for 30 min at 60,000 × g, the supernatant was applied to a nickel-nitrilotriacetic acid column (GE Healthcare). The protein was eluted with a gradient to 0.5 m imidazole. The colicin S4-containing fractions were pooled, concentrated using an Amicon concentrator, and applied to a preparative Superdex 200 column equilibrated with 150 mm NaCl and 20 mm MOPS (pH 7.0).Colicin S4 Sensitivity Assay-The E. coli strains used in this assay (supplemental Table SI) were obtained from the Keio Collection (30Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Systems Biol. 2006; 22006.0008Crossref PubMed Scopus (5271) Google Scholar). 10 μl of an overnight culture was mixed with 3 ml of melted LB top agar and poured on an LB agar plate. After cooling to room temperature, round filter platelets (Schleicher & Schüll) were placed on the plates. The filter platelets were subsequently soaked with colicin S4 dilutions, with total colicin S4 amounts ranging from 10 to 10-7 μg. The so-treated LB agar plates were incubated overnight at 37 °C. Colicin S4 sensitivities of the different strains were evaluated by checking for clear inhibition zones around the filter platelets.Single Channel Conductance Measurements-Single channel conductance values were recorded using a BLM workstation (Warner Instruments, Hamden, CT) with a BC-535 amplifier and an LPF-8 Bessel filter connected to an Axxon Digidata 1440A digitizer. Data were recorded and evaluated using pCLAMP 10.0 software (Molecular Devices, Sunnyvale, CA) supplied with the digitizer. First of all, 3 μl of a 1% (w/v) solution of ultrapure hen egg phosphatidylcholine (a gift from Lipoid GmbH, Ludwigshafen, Germany) in 1:1 (v/v) methanol/chloroform was applied to a 150-μm aperture in a 4-ml polysulfone cuvette (Warner Instruments). After evaporation of the solving agents, the cuvette was filled with the measurement buffer, which was 1 m KCl and 20 mm MES (pH 6.0). 3 μl of a 1% (w/v) solution of ultrapure hen egg phosphatidylcholine in 9:1 (v/v) n-decane/butanol was painted onto the 150-μm aperture of the cuvette. 1 μl colicin of S4 solution (3-4 mg/ml) was added to the cuvette, which contained the ground electrode of the setup. Colicin S4 inserted readily into the membrane at a potential of approximately -100 mV.Protein Crystallization-Crystals of colicin S4 were obtained at 293 K by the vapor diffusion hanging drop method against 200 μl of a reservoir solution. Crystal drops were prepared by mixing 2.5 μl of protein at 30 mg/ml concentration with 2.5 μl of reservoir solution. Crystals were obtained with 0.16 m MgCl2, 24% polyethylene glycol 4000, and 20% glycerol in 80 mm Tris-HCl (pH 8.5) at a size of up to 1000 × 100 × 100 μm. Platinum derivatives were obtained by soaking the crystal in the mother liquid containing 2 mm of K2PtCl4, (NH4)2PtCl4, K2Pt(NO2)4, or K2Pt(CN)4.X-ray Data Collection-Single crystals were flash-frozen in their mother liquid, and data collection was performed at 100 K. The crystal system is R32 with cell constants of a = b = 240.34 Å, c = 80.03 Å, α = β = 90°, and γ = 120°. The crystals contained one monomer in the asymmetric unit, diffracted to a resolution limit of 2.45 Å, and showed a solvent content of 70%. All data sets were collected at Swiss Light Source beamline PXII on a MarCCD225 detector at 90 K. Data were indexed, integrated, and scaled with the XDS program package (31Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3214) Google Scholar).Structure Determination and Refinement-Data sets were collected at the theoretical platinum edge + 10 eV, and 180 images (1°/image) were recorded. All data of native and derivative crystals were processed identically and were transferred to the SHARP program package (32Vonrhein C. Blanc E. Roversi P. Bricogne G. Methods Mol. Biol. 2007; 364: 215-230PubMed Google Scholar). The electron density provided by SHARP and DM was used in Buccaneer for initial model building (33Cowtan K. Acta Crystallogr. 2006; 62: 1002-1011Crossref PubMed Scopus (1435) Google Scholar, 34Wang B.C. Methods Enzymol. 1985; 115: 90-112Crossref PubMed Scopus (942) Google Scholar). This initial model was partially improved by using the pore-forming domain structure of colicin A (Protein Data Bank code 1col) together with model bits derived from Buccaneer. This model was then refined in REF-MAC5 (35Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar), and both maps including experimental and model phases were used for model building. To improve this model, Coot (36Emsley P. Cowtan K. Acta Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22815) Google Scholar) and iterative REFMAC rebuilding were used to rebuild side chains and to add missing residues. A random set of 5% of the data was neglected during the refinement process and marked as the test set for cross-validation. Atoms were refined and TLS parameters determined using the TLS server and REF-MAC (37Winn M.D. Murshudov G.N. Papiz M.Z. Methods Enzymol. 2003; 374: 300-321Crossref PubMed Scopus (673) Google Scholar). ARP/wARP was used to build the solvent structure (38Cohen S.X. Ben Jelloul M. Long F. Vagin A. Knipscheer P. Lebbink J. Sixma T.K. Lamzin V.S. Murshudov G.N. Perrakis A. Acta Crystallogr. Sect. D. 2008; 64: 49-60Crossref PubMed Scopus (131) Google Scholar). Together, this procedure returned a final model consisting of 3530 non-hydrogen atoms and 199 water molecules (corresponding to Val68-His499). Together with the hydrogen atoms generated for all amino acid residues, a crystallographic R/Rfree factor of 0.22/0.25 was achieved.Secondary structure elements were defined according to DSSP criteria (molbio.info.nih.gov/structbio/basic.html). Figures were prepared using the programs DINO, RasMol, the Swiss-PdbViewer (39Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9472) Google Scholar), and POV-Ray. The atomic coordinates of colicin S4 have been deposited in the Protein Data Bank (code 3few).RESULTSColicin S4 Sequence Features and Classification of Pore-forming Domains-The colicin S4 sequence shares regions of high homology with other colicins but also comprises sequence parts that are unique (25Pilsl H. Smajs D. Braun V. J. Bacteriol. 1999; 181: 3578-3581Crossref PubMed Google Scholar). As in other colicins, the N-terminal translocation domain of colicin S4 (residues 1-118) is very rich in glycines (22%). The N-terminal 43 residues of colicins K and S4 are 98% identical, but a canonical TolA or TolB box (2Cascales E. Buchanan S.K. Duche D. Kleanthous C. Lloubes R. Postle K. Riley M. Slatin S. Cavard D. Microbiol. Mol. Biol. Rev. 2007; 71: 158-229Crossref PubMed Scopus (767) Google Scholar) is missing. The receptor-binding domain of colicin S4 is unique in two ways: it has no sequence homology to other colicin domains (presumably because it is the only colicin that binds to OmpW), and it arose from a recent domain duplication event (25Pilsl H. Smajs D. Braun V. J. Bacteriol. 1999; 181: 3578-3581Crossref PubMed Google Scholar). PSI-BLAST searches using only the sequence of this domain against the non-redundant data base did not yield significant hits. The two 86-residue repeats display a sequence identity of 61% (25Pilsl H. Smajs D. Braun V. J. Bacteriol. 1999; 181: 3578-3581Crossref PubMed Google Scholar). Colicin S4 belongs to the group of pore-forming colicins, as the C-terminal 201 residues of colicin S4 (residues 299-499) and colicin A (residues 392-592) are 77% identical (see alignment in Fig. 1C) (25Pilsl H. Smajs D. Braun V. J. Bacteriol. 1999; 181: 3578-3581Crossref PubMed Google Scholar).Based on the sequence alignment, the pore-forming domains of colicins can be divided into three groups. These differences can also be observed on the structural level (Fig. 1A). Based on these observations, we decided to term the groups (see alignment in Fig. 1C). Colicin S4 is more similar to colicins A, B, U, Y, and N (group PI) than to colicins E1, 5, 10, and K (group PIIa) and colicins Ia and Ib, pyocin S5 from Pseudomonas aeruginosa, and alveicin B from Hafnia alvei (group PIIb). The main difference between the group PI pore-forming domains and the other two groups is in the different length of helices Pα1, Pα3, Pα4, and Pα8; in an insertion in the turn region of the hydrophobic hairpin between helices Pα8 and Pα9; and in the angle between Pα1 and Pα2. The differences between groups PIIa and PIIb are more subtle and are based mostly on point mutations and on four short β-strands visible only in group PIIb.Colicin S4 Expression and Purification-The DNA sequence of colicin S4 was obtained from plasmid pColS4 (25Pilsl H. Smajs D. Braun V. J. Bacteriol. 1999; 181: 3578-3581Crossref PubMed Google Scholar). To express the protein, the gene was cloned in a tet-inducible expression vector (pASK-IBA33+). Upon induction, colicin S4 accumulated in the cytosol of the expressing bacteria. However, the presence of the receptor protein OmpW rendered the cells sensitive to colicin S4, which was released into the growth medium at low concentrations, most likely after lysis of single cells. This led to the death of all cells shortly after induction, and the protein yield remained low. To overcome this problem, we used a ΔompW knock-out strain based on E. coli 5K, which was resistant to colicin S4.Colicin S4 can be easily obtained in vast amounts and high purity using either ion exchange or, for His-tagged constructs, nickel affinity chromatography and preparative gel-sizing chromatography for polishing. In solution, colicin S4 is a monomer. The protein can be concentrated up to 80 mg/ml using spin concentrators. The protein remains active (i.e. kills wild-type E. coli cells and displays pore-forming activity in single channel conductance measurements) for several weeks when stored on ice.Colicin S4 Forms Pores in Artificial Membranes-To show that the killing activity of colicin S4 is due to pore formation in the same fashion as in other colicins, we performed single channel conductance measurements. Black lipid membranes were produced from hen egg phosphatidylcholine, and colicin S4 was added to the bilayer setup on the ground side of the membrane. In the first assays, colicin S4 with a C-terminal His tag was used. This protein inserted spontaneously into the membrane but only with very low frequency and at high negative voltages (less than -100 mV). When colicin S4 without the His tag was used, the insertion frequency was much higher, and the voltage needed for insertion was lower (less than -80 mV). It seems that the C-terminal His tag hinders membrane insertion, but once the pore is formed, it does influence the conductance compared with the non-tagged protein (data not shown). Intriguingly, when very small amounts of purified OmpW were added to the bilayer setup with the His-tagged colicin, the insertion frequency increased dramatically. We assume that OmpW recruits the colicin to the proximity of the bilayer and thus increases the chances of insertion. The effect of OmpW on the insertion frequency seemed much less pronounced for the non-tagged colicin, albeit this is difficult to quantify. After these observations, we exclusively used non-tagged colicin (without addition of OmpW) in all single channel conductance measurements.Colicin S4 inserted spontaneously into the lipid bilayer in 1 m KCl buffers at pH <7. At higher pH, no insertion events were observed. The recordings shown in Fig. 2 were taken in 20 mm MES-KOH (pH 6) and 1 m KCl. In Fig. 2A, numerous successive insertion events are visible. Fig. 2B shows a close-up of the same recording, where the signal of an individual colicin channel can be seen. As with other colicins, the single channel characteristics are very noisy, and the channels have different conductance states that make a quantitative evaluation of the conductance difficult (40Davidson V.L. Brunden K.R. Cramer W.A. Cohen F.S. J. Membr. Biol. 1984; 79: 105-118Crossref PubMed Scopus (50) Google Scholar). But the behavior of colicin S4 in these experiments seems similar to that of colicin A (41Collarini M. Amblard G. Lazdunski C. Pattus F. Eur. Biophys. J. 1987; 14: 147-153Crossref PubMed Scopus (54) Google Scholar) as expected from the sequence alignments (Fig. 1C).FIGURE 2Single channel conductance measurements. A shows the insertion of numerous channels just after injection of the protein. B shows the action of a single channel flickering at a high frequency, prior to permanent opening. The trace was recorded at -100 mV. The buffer conditions were 1 m KCl and 20 mm MES (pH 6.0).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Colicin S4 Exploits OmpW as a Receptor and OmpF and Parts of the Tol System for Translocation-To examine the interaction partners of colicin S4 in the target cell, we performed a sensitivity screening using single knock-out strains of genes that were previously shown to be involved in colicin import and function, obtained from the Keio Collection (30Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Systems Biol. 2006; 22006.0008Crossref PubMed Scopus (5271) Google Scholar). The putat
Referência(s)