Molecular Microbiology (1988) 2(6). 807-811 Notes Functional domains of colicin A D. Baty,^* M. Frenette,^ R. Lloubes,^ V. Geti,^ S. P. Hovt^ard,^ F. Pattus^ and C. LazdunskP 'Centre de Biochimie et de Biologie Moleculaire du C.N.R.S., 31 Chemin Joseph Aiguier, BP71, 13402 Marseitte Cedex 9, France. ^European Motecular Biology Laboratory, Postfach 10.2209. Meyerhofstrasse 1, 6900 t-teidetberg, FRG. Summary A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar Iipid bilayers) was assayed. The receptor-binding properties of each protein were also analysed. From these results, we suggest that the NH2-terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor-binding domain. The COOHterminal domain (residues 389 to 592) carries the pore-forming activity. Introduction Colicins are bactericidal proteins produced by and active against Escherichia coli and closely related bacteria. The molecular mechanisms involved in the binding of colicins to their specific receptors and their penetration across the cell envelope to bring them to their biochemical targets are not yet understood. The mode of action of colicins involves several steps of transfer across membranes. They are first produced in the cytoplasm (Varenne et at., 1981) and then subsequently released to the extracellular medium by a mechanism that does not involve any specific region of the polypeptide chain (Baty et at., 1987b). The mode of action of coiicins appears to involve 3 steps: (i) binding to a specific receptor located in the outer membrane; (ii) translocation across the membrane(s); (Iii) biochemical interaction of the colicin with its target in the cell. Evidence has been put forward that there is a linear organization of three distinct domains along the poiypeptide chain of several colicins: A (Martinez ef a/., 1983; Received 12 April, 1988; revised 22 June, 1988. 'For correspondence. Crozel etat., 1984; Baty etat., 1987a), E1 (Ohno-lwashita and Imahori, 1982; Cleveland e( a/., 1983; Brunden ef a/.. 1984; Liu ef ai, 1986), E2 (Ohno-lwashita and Imahori. 1980; de Graaf and Oudega, 1986), E3 (Suzuki etat., 1980; Ohno et at., 1980; Ohno-lwashita and Imahori, 1980; de Graaf and Oudega, 1986), la and lb (Mankovlch ef at., 1986), N (Pugsley, 1988) and cloacin DF13 (Gaastra etat., 1978; Neville and Hudson, 1986). To each of these domains, a specific function has been assigned. The central domain of colicins appears to be involved in receptor binding, the NH2-terminai domain seems to be required for transiocation across the outer membrane, and the COOH-terminal domain carries the lethal activity whether it carries an enzymatic (colicins E2, E3, Cloacin DF13) or an ionophoric activity (coiicins A, E l , la, lb, N). Most results obtained so far have been obtained by using limited digestion of colicin polypeptide chains. For colicin A for example, we have demonstrated that partial digestion with bromelain or thermolysin allows the isolation of a 20 kD fragment endowed with ionophoric activity (Martinez ef at., 1983; Tucker ef at.. 1986). In the present study, we have constructed many proteins derived from coiicin A which lack various regions of the polypeptide chain. These proteins have been purified and their ability to bind to the receptor (constituted by the two proteins OmpF and BtuB (Cavard and Lazdunski, 1981) and to form ion channels has been tested. Results Construction of colicin A derivatives tactiing various regions of the potypeptide chain The restriction sites in the colicin A gene (caa) were determined from the nucleotide sequence of the gene (Morion ef at., 1983). In addition, a new restriction site (Mtu\) was created within the gene by site-directed mutagenesis (Baty ef a/., 1987a). Using these sites, deletions which preserved the reading frame were introduced within caa as indicated in Fig. 1. All of the constructed proteins were released to the medium and were sufficiently stable to be purified. The anaiysis by poiyacrylamide gei electrophoresis is shown in Fig. 2. These proteins were then assayed for their in vivo and in vitro ionophoric activity and for their ability to bind to the colicin A receptor. 808 D.Baty eial 2 3 3 Js 3 Ik- II + + I— C UJ o ^ 1 •5 --a 0) S 3 1^ "MS .E (D £ M sa T3 •= 2 1 P 9E J CQ ^' S o 1 ^ S^ 2 5 •a •o ^ < •I CO Q CQ UJ Pi Q ea U Q ll Functionat domains of coticin A A BD2 ARl BEI P449 DR2 3S1 DQ16 DTI BRJ BC7 BQ2 DV8 T.F. 809 Locatization of the receptor-binding domain - 68 — 43 — 25.7 — 18.4 Fig. 2. Deletion derivatives of colicin A. The purified derivatives of colicin A and the thermolysin fragment (T.F.) were analysed by electrophoresis in NaDodSOV12.5% PAGE. Pore-forming activity of the partially deleted colicin A derivatives Coiicin A derivatives can kill sensitive ceils provided that they are abie to bind to the receptor, to be translocated across the outer membrane and to depolarize the inner membrane. In contrast, even if they cannot fulfil these requirements, they should be able to form channels in planar Iipid bilayers provided that they carry the poreforming region. Only protein DQ16, which lacks the region between residues 336 to 372, was as active in vivo as the wild-type colicin A (Fig. 1). This result suggests that this region is not implicated in any of the three steps of the mode of action of colicin A. The other proteins were not active at all in vivo, which suggests that one or more steps were affected. For this reason, it was necessary to study each step individually. All of the colicin A derivatives carrying at least residues 389 to 592 exhibited ionophoric activity in phospholipid planar bilayers (Fig. 1), as did the thermolysin fragment (Tucker etat., 1986). However, most of them (BD2, A R l , BEI, P449, DR2and DS1) were unable to kill sensitive cells in vivo because one or both of the two first steps of the mode of action were affected. Various regions from residues 1 to 388 have been deleted in these proteins. These deletions did not change the single channel conductance (i.e. the size of the pore was the same as that formed by the wild-type colicin A). Proteins lacking the COOH-terminal domain (BC7, BR3, DV8, BQ2 and DTI) were active neither in vivo nor in vitro. These results are in agreement with the fact that the COOH-terminal domain (residues 389 to 592) of colicin A contains the poreforming activity (Martinez ef a/., 1983; Tucker ef a/., 1986). The rest of the molecule has an effect on the voltage dependence of the pore (Martinez ef a/., 1983; Baty ef a/., 1987a; Collarini etal., 1987). Proteins that carry the receptor-binding domain but which cannot kill sensitive cetls should protect these cells from the action of wild-type colicin A by competing for binding to the receptor. Using the test described in Experimentat procedures, we observed that some of the deletion derivatives fulfilled this criterion (Fig. 1). Colicin A at 50ng is able to kill 90% of sensitive cells (see the colicin A curve in Fig. 3). The proteins BD2, AR1, BR3, DV8, BQ2 and DTI couid provide full protection against this colicin A concentration, whereas BEI, P449, DR2, DS1, BC7 and the thermolysin fragment could not. Two representative examples (BD2 and DS1) are shown in Fig. 3. The other proteins classified as 'protecting' or 'non-protecting' produced comparable curves corresponding to one of the two examples. From these results, it can be deduced that the receptor-binding domain must be located between residues 173 and 336. The absence of this domain in DS1 and DR2 was verified in bypass experiments in which we found that both were able to kill btuB mutant cells (H. Benedetti, unpublished results). | /'•' n, Localization of the translocation domain The localization of the translocation domain can be inferred from the results presented above. Proteins BD2 and ARl having both receptor-binding and ionophoric activity (in vitro) were unable to kill sensitive cells in vivo. This suggested that the region between residues 1 and 172 is responsible for the translocation of colicin A to the 80- BD2 60< > Pi 40 - 20- 0.5 5 50 500 PROTEIN (ng) Fig. 3. Assay of receptor binding for partially deleted colicin A proteins. The percentage survival of sensitive cells was plotted as a function of the amount of protein added. For colicin A, the sensitive cells were incubated for 20 min at 37°C . then colicin A was added at the indicated concentrations for 20 min at 37°C . For BD2 and DS1, the proteins were incubated with the sensitive cells at Z1''Z for 20 min at the indicated concentrations and then 50 ng of colicin A was added and incubation was continued for a further 20 min. 810 D.Baty e\a\. inner membrane, although because of the size of the deletions the results must be interpreted carefully. Discussion The results presented in this work al!ow us to define roughly the domains involved in the various steps of colicin A action on sensitive cells. The NHg-terminal domain, involved in translocation, may comprise residues 1 to 173. The central domain, involved in receptor binding, appears to contain residues 173 to 336, and the ionophoric activity is contained in a region encompassing residues 389 to 592. However, it should be pointed out that these are maximum estimates, and the receptor-binding and ionophoric domains may be shorter. It is also difficult to give a definite size for the translocation domain, which may be shorter or may contain a stretch downstream from residue 173. These uncertainties are demonstrated by the example of colicin E l , for which the ionophoric domain was first reported to contain 152 amino acid residues (Cleveland et ai, 1983) while more recent work has demonstrated that it is composed of 88 residues (Liu et al., 1986). This also appears to hold true for colicin A since the ionophoric domain has been found to be located between residues 457 and 592 (D. Baty, unpublished results). The COOH-terminal region of colicin N (residues 187 to 387) shows a high degree of sequence homology with that of colicin A, suggesting that it is the ionophoric domain (Pugsley, 1988). The fact that colicin N, containing only 387 residues, has the properties of larger colicins like A, la and Ib also supports the contention that specific domains may be shorter than those defined in this work. Indeed, we have deleted 36 amino acid residues of the protein to produce DQ16 without any detectable effect on colicin A activity (Fig. 1). This deletion is interesting since it is located precisely in a central region sharing a high degree of homotogy with colicins E1, E2, E3andcloacinDF13(de Graaf and Oudega, 1986). Such a feature may have suggested an important function for this region but this does not appear to be the case. While the concept and demonstration of domains responsible for receptor-binding and ionophoric activity are rather straightforward, the existence of a domain required for translocation cannot be easily demonstrated at present. One of the main features of the NH^-terminal domain is that it is always rich in glycine and proline residues for colicins A, E l , E2, E3, N and cloacin DF13 (Pugsiey, 1988). As a result, it is not highly structured and cannot be isolated by limited proteolytic digestion. The possible role of the glycine-rich region for translocation has been challenged by results from Pugsley demonstrating that a LacZ-ColN hybrid having a significantly reduced gtycine content (the 16 first residues of the colicin were deleted) was still active (Pugsley, 1988). On the other hand, a LacZ-ColN hybrid in which the 44 first residues of the colicin N were deleted was not active. Similar results were obtained here with protein BD2. which lacked only 14 residues (residues 16 to 29) in the NH2-terminal domain and yet had lost its activity. This suggests that some 'targeting' sequence involved in recognition of envelope proteins in the sensitive cells may be contained in this domain. With regard to this point, it is of interest that some point mutations in the NH2-terminal domain of colicin E3 (Escuyer and Mock, 1987) and cloacin DF13 (Verschoor et ai. 1988) reduced the translocation of these proteins into susceptible cells. Finally, it is notable that none of the colicins isolated so far contains a disulphide bridge. This may allow more flexibility for the partial unfolding and refolding that may occur during uptake into sensitive cells. Some of our partially deleted colicin A proteins are very interesting because they correspond to delimited domains. For example, the proteins BC7, DV8 and BE1 correspond, respectively, to the translocation, the receptor-binding and the pore-forming domains. Thus, these protein domains can now be prepared without using limited proteolytic digestions, which often results in heterologous protein products. Using Bal31 and Exolll exonucleases, we are now trying to restrict the various domains of colicin A to their minimal sizes and studies are currently being carried out in attempts to understand the interplay between these domains during colicin action. Experimental procedures Bacterial strains, growth conditions and plasmids The bacterial host strain W3110 and plasmid ColA9 (colicin A wild type) have been described previously (Lloubes etai. 1986). The W3110 strain containing the various plasmids was grown in LB medium (Miller. 1972). The synthesis of colicin A or colicin A derivatives was induced by adding 300ng ml ' of mitomycin C (MTC) to cultures at an ODBOO of 1.0. Construction of recombinant plasmids All enzymes were purchased from Boehringer-Mannheim and New England Biolabs Inc. The recombinant plasmids were constructed from pColA9 as described by Baty et al. (1987b). The enzyme restriction sites used to delete regions in the coding sequence of the colicin A are indicated in Fig. 1. The junctions of the partially deleted recombinants were sequenced using the Maxam and Gilbert (1980) method to verify the reading frame. Colicin purification and assay Colicin purification and assay were carried out as described previously (Baty et ai, 1987a; Cavard and Lazdunski. 1979). Functionat domains of coticin A Assay of cett survival in ceil protection experiments The experimental protocol indicated above was used. Briefly, 0.1ml of the mutant colicin was incubated for 20 min with sensitive cells before the addition of 0.1ml of colicin A (500ng ml '); SDS was added 20 min later, and the absorbance measured after a further 10 min of incubation. Formation of voitage-dependent pores in ptanar tipid biiayers Formation of planar bilayers from two monolayers was performed according to Pattus et al. (1983). Conductance measurements were performed for the different colicin A derivatives as described previously (Baty ef a/. 1987a). Acknowledgements This work was supported by the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Fondation pour la Recherche Medicale, and the Direction des Recherches, Etudes et Techniques. M.F. is the recipient of a post-doctoral fellowship from the Fonds de la Recherche en Sante du Quebec and S.P.H. is the recipient of a post-doctoral fellowship from N.S.E.R.C. References Baty, D.. Knibiehler, M., Verheij, H., Pattus, F., Shire, D., Bernadac, A., and Lazdunski, C. (1987a) Site-directed mutagenesis of the COOH-terminal region of colicin A: effect on secretion and voltage-dependent channel activity. Proc Natt Acad Sci USA 84: 1152-1156. Baty, D., Lloubes, R., Geli, V., Lazdunski, C , and Howard, S.P. (1987b) Extracellular release of coiicin A is non-specific. EMBO J 6: 2463-2468. Brunden, K.R., Cramer. W.A., and Cohen, F.S. 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Varenne, S., Chartier, M., and Lazdunski, C (1983) Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate. J Mot Biot 170:271-285. Neville, D.M.. and Hudson, T.H. (1986) Transmembrane transport of diphtheria toxin, related toxins, and colicins. Ann Rev S/oc/jem 55:195-224. Ohno, Y., Saito, K., Suzuki, K., and Imahori. K. (1980) The effects of carboxypeptidase digestion on the function of colicin E3. J Biochem 87: 989-992. Ohno-lwashita. Y., and Imahori, K. (1980) Assignment of the functional loci in colicin El and E3 molecules by the characterization of their proteolytic fragments. Biochemistry 19: 652659. Ohno-lwashita, Y.. and Imahori, K. (1982) Assignment of the functional loci in the colicin El molecule by characterization of its proteoiytic fragments. J Biot Chem 257: 6446-6451. Pattus, F., Cavard, D.. Verger, R., Lazdunski, C , Rosenbuch, J., and Schindler, H. (1983) Formation of voltage-dependent pores in planar bilayers by colicin A. In Physical Chemistry of Transmembrane tons Motions. Spach, G. (ed.). Amsterdam: Elsevier Biomedical Press, pp. 407-413. Pugsley, A.P. (1988) Nucleotide sequencing of the structural gene for colicin N reveals homology between the catalytic C-terminal domains of colicins A and N. Mol Microbiol 1: 317-325. Suzuki, K., Ohno, S., and Imahori, K. (1980) Studies on the physicochemical structure and stability of an active fragment fT2A) of colicin E3. J Biochem 87: 761-769. Tucker, A.D.. Pattus, F., and Tsernoglou, D. (1986) Crystallization of the C-terminal domain of colicin A carrying the voltagedependent pore activity of the protein. J/Wo/S/o/190: 133-134. Varenne, S., Cavard, D., and Lazdunski. C (1981) Biosynthesis and export of colicin A in Citrobacter ireundii CA31. Eur J Biochem 116: 615-620. Verschoor, E.J., Luirink, J., de Graaf, F.K., and Oudega, B. (1988) Characterization of a mutation in the cloacin structural gene causing a reduced uptake of cloacin DF13 by susceptible cells. FEMS Microbiol Letts 49:403-409.