NMR solution structure of the precursor for carnobacteriocin B2, an antimicrobial peptide from Carnobacterium piscicola Implications of the a-helical leader section for export and inhibition of type IIa bacteriocin activity Tara Sprules 1 , Karen E. Kawulka 1 , Alan C. Gibbs 2 , David S. Wishart 2 and John C. Vederas 1 1 Department of Chemistry and 2 Faculty of Pharmacy, University of Alberta, Edmonton, AB, Canada Type IIa bacteriocins, which are isolated from lactic acid bacteria that are useful for food preservation, are potent antimicrobial peptides with considerable potential as therapeutic agents for gastrointestinal infections in mam- mals. They are ribosomally synthesized as precursors with an N-terminal leader, typically 18–24 amino acid residues in length, which is cleaved during export from the produ- cing cell. We have chemically synthesized the full precursor of carnobacteriocin B2, precarnobacteriocin (preCbnB2), which has a C-terminal amide rather than a carboxyl, and also produced preCbnB2(1–64), which is missing two amino acid residues at the C-terminus (Arg65 and Pro66), via expression in Escherichia coli as a maltose-binding protein fusion that is then cut with Factor Xa. PreC- bnB2(1–64) is readily labeled with 15 Nand 13 CforNMR studies using the latter approach. Multidimensional NMR analysis of preCbnB2(1–64) shows that, like the parent bacteriocin, it exists as a random coil in water but assumes a defined conformation in water/trifluoroethanol mixtures. In 70 : 30 trifluoroethanol/water, the 3D structure of the preCbnB2 section corresponding to the mature bacteriocin is essentially the same as reported previously by us for carnobacteriocin B2 (CbnB2). This structure maintains the highly conserved a-helix corresponding to residues 20–38 of CbnB2 that is believed to be responsible for interaction with a target receptor in sensitive cells, including Listeria monocytogenes. PreCbnB2 also has a second a-helix from residues 3–13 (i.e. )15 to )5 relative to CbnB2) in the leader section of the peptide. This helix appears to be conserved in related type IIa bacteriocin precursors based on sequence analysis. It is likely to be a key recognition element for export and processing, and is probably responsible for the considerably reduced antimicrobial activity of preCbnB2. The latter effect may assist the pro- ducing cell in avoiding the toxic effects of the bacteriocin. This is the first 3D structure determined for a prebacte- riocin from lactic acid bacteria. Keywords: antibacterials; bacteriocin; NMR structure; pep- tide synthesis; precarnobacteriocin B2. Bacteriocins are potent antimicrobial peptides secreted by bacteria. Those produced by lactic acid bacteria are the focus of extensive studies because of their potential application as nontoxic food preservatives, as well as their possible therapeutic uses in both human and veterinary medicine [1–5]. Nisin is approved in over 80 countries as a food additive [6]. Most bacteriocins from lactic acid bacteria are synthesized as prepeptides which undergo a variety of post-translational modifications, ranging from extensive formation of dehydro residues and lanthionine bridges in the case of lantibiotics (e.g. nisin A) to simple cleavage of a leader peptide and export across the cell membrane. These antimicrobial compounds are divided into classes according to their structural characteristics. Type IIa bacteriocins are single peptides characterized by a conserved YGNGVXC motif in the N-terminus, with the cysteine involved in a disulfide bridge, and are otherwise unmodified except for cleavage of the leader from their precursor (Table 1). They show potent activity against a number of potential Gram-positive food spoilage and pathogenic bacteria, e.g. Listeria monocyto- genes, but display no toxicity toward humans or other eukaryotes. We reported purification and the primary structure of the first member of this class, leucocin A [7], and in the meantime over 20 such compounds have been identified. These heat-stable, cationic peptides typically have 37–48 amino acid residues. The solution structures of three type IIa bacteriocins have been determined by NMR methods: leucocin A [8], carnobacteriocin B2 (CbnB2) [9], and, very recently, sakacin P [10]. Interest- ingly, the high sequence homology of the N-terminal portion does not lead to conserved 3D structures for this section of the molecules because of polarity differences of the few varying residues [9]. However, in contrast, the much more variable sequences of the C-terminal halves of Correspondence to J. C. Vederas, Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada. Fax: + 1 780 492 8231, Tel.: + 1 780 492 5475, E-mail: john.vederas@ualberta.ca Abbreviations: ABC, ATP-binding cassette; BHI, brain heart infusion; CbnB2, carnobacteriocin B2; DMF, N,N-dimethylformamide; HSQC, heteronuclear single quantum coherence; MBP, maltose- binding protein; preCbnB2, precarnobacteriocin B2. (Received 7 January 2004, revised 24 February 2004, accepted 11 March 2004) Eur. J. Biochem. 271, 1748–1756 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04085.x these bacteriocins maintain a highly conserved a-helical structure which determines antimicrobial specificity [9,11] and is likely to be responsible for recognition of a cell surface protein receptor in the target bacterial cells. Disruptions of this helix through mutations that change amino acid polarity generally abolish antimicrobial activ- ity [12–14]. The leader peptides of type IIa bacteriocins are also homologous [5,15] (Table 1). They range in length from 18 to 24 amino acids, terminating in two glycine residues. Hydrophobic residues are found at positions )4, )7, )12 and )15, and hydrophilic residues are found at positions )5, )6, )8, )9, )11, )13 and )14. Along with genes that encode bacteriocin and immunity proteins, which protect the producer strain from attack by its own antimicrobial peptides, genes that encode ATP-binding cassette (ABC) transporter proteins are usually found in these bacteriocin operons [5]. ABC transporters are a family of large transmembrane proteins responsible for the ATP-driven transport of a variety of compounds ranging from ions to oligosaccharides and proteins. The ABC proteins associ- ated with type IIa bacteriocin production have an extra N-terminal cysteine protease domain, which is responsible for cleavage of the leader peptide after the double glycine motif [16,17]. This cleavage occurs on the cytoplasmic side of the membrane during export of the bioactive peptide [18]. The leader peptide is undoubtedly recognized by the ABC protein responsible for export and processing. In the case of carnobacteriocin B2 (CbnB2), a cationic thermo- stable type IIa bacteriocin produced by Carnobacterium piscicola LV17B [19], the additional 18 amino acids in the leader peptide of precarnobacteriocin B2 (preCbnB2) also render it  125 times less active than the mature peptide [14]. To help determine the structural basis of the inhibition of the antimicrobial activity of CbnB2 by the leader and to assist future analysis of the interaction of preCbnB2 with its ABC transporter protease, it is essential to establish the preferred geometry of the precursor. We now report the chemical synthesis, bio- chemical production, and solution structure of preCbnB2, and compare it with the structure of the mature bacteriocin, CbnB2. Materials and methods Chemical synthesis of preCbnB2 with a C-terminal amide Stepwise synthesis of preCbnB2 (but with a C-terminal amide instead of carboxyl) was achieved manually on a 0.3 mmol scale of Rink amide resin using standard Fmoc solid-phase peptide chemistry. Fmoc-protected L -amino acids (Sigma) were used with the following orthogonal protection: Arg(Pmc), Asn(Trt), Asp(tBu), Cys(Acm), Cys(Trt), Glu(tBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc), Tyr(tBu). Initially, Fmoc-Pro was coupled to Rink resin using N,N-dicyclohexyl-carbodiimide as the activating agent in the presence of a catalytic amount of N-hydroxybenzotriazole in N,N-dimethylformamide (DMF). The peptide chain was assembled in a stepwise manner with deprotection, activation and coupling cycles. All steps were followed by washing sequentially with DMF, dichloromethane, propan-2-ol, dichloromethane and DMF, to remove excess reagents and protecting groups. Fmoc deprotection was catalyzed by 20% piperdine in DMF. Amino acid activation was achieved with O-benzotriazole- N,N,N¢,N¢-tetramethyluronium hexafluorophosphate in DMF, and a fourfold excess of the Fmoc-protected amino acid was used to maximize yield of the N-hydroxybenzo- triazole-amino acid ester. The extent of peptide elongation, or coupling, was monitored by using a ninhydrin assay to check for residual free a-amine. To facilitate coupling of difficult residues, combinations of the following condi- tions were used: elevated temperatures of up to 50 °C, O-7-azabenzotriazole-1-yl-N,N,N¢,N¢-tetramethyluronium hexafluorophosphate as activation agent, and N-methyl- pyrrolidinone as solvent. Test cleavages were performed after every five-residue coupling, and the desired product was confirmed by MALDI-TOF MS. Peptide was cleaved from the resin with a mixture of 87.5% trifluoroacetic acid, 5% phenol, 5% water, 2.5% dithiothreitol, and 2.5% anisole for  90 min at 20 °C. The filtrate from the cleavage reactions was collected, combined with trifluoroacetic acid washes (3 · 2 min, 1 mL), and concentrated in vacuo.Cold diethyl ether ( 15 mL) was added to precipitate the crude cleaved peptide. Disulfide bond formation was achieved by Table 1. Comparison of the amino acid sequence of selected type IIa bacteriocins. Conserved hydrophobic residues within the leader peptide are italicized, and hydrophilic residues are underlined. The YGNGVXC motif is highlighted in bold text. Bacteriocin Leader peptide Mature peptide Reference PreCbnB2 MNSVKELNVKEMKQLHGG VNYGNGVSCSKTKCSVNWGQAFQERYTAGINSFVSGVASGAGS IGRRP [19] Sakacin G MKNAKSLTIQEMKSITGG KYYGNGVSCNSHGCSVNWGQAWTCGVNHLANGGHGVC [20] Plantaricin 423 MMKKIEKLTEKEMANIIGG KYYGNGVTCGKHSCSVNWGQAFSCSVSHLANFGHGKC [21] Piscicolin 126 MKTVKELSVKEMQLTTGG KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTGGAAGWNKG [22] CbnBM1 MKSVKELNKKEMQQINGG AISYGNGVYCNKEKCWVNKAENKQAITGIVIGGWASSLAGMGH [19] Leucocin A MMNMKPTESYEQLDNSALEQVVGG KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW [7] Pediocin MKKIEKLTEKEMANIIGG KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATGGHQGNHKC [23,24] Mesentericin Y105 MTNMKSVEAYQQLDNQNLKKVVGG KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW [25] Sakacin A MNNVKELSMTELQTITGG ARSYGNGVYCNNKKCWVNRGEATQSIIGGMISGWASGLAGM [26] Divercin V41 MKNLKEGSYTAVNTDELKSINGG TKYYGNGVYCNSKKCWVDWGQASGCIGQTVVGGWLGGAIPGKC [27] Sakacin P MEKFIELSLKEVTAITGG KYYGNGVHCGKHSCTVDWGTAIGNIGNNAAANWATGGNAGWNK [28] Ó FEBS 2004 Solution structure of precarnobacteriocin B2 (Eur. J. Biochem. 271) 1749 stirring the reaction mixture with elemental iodine (0.1 M in methanol) under argon for 2 h. The oxidation was quenched by addition of aqueous ascorbic acid (1 M ), and the crude product was purified by RP-HPLC. The fraction showing the correct mass spectrum (mass 6990.8) was collected and lyophilized to give 10 mg peptide with >90% purity. Expression of labeled MBP-PreCbnB2 fusion protein, cleavage and purification of PreCbnB2(1–64) Escherichia coli BL21 (DE3) cells transformed with the plasmid pLQP, expressing preCbnB2 as a maltose-binding protein (MBP) fusion, was grown with shaking at 37 °C in M9 minimal medium as described previously [14]. For labeling experiments, [ 15 NH 4 ] 2 SO 4 , or alternatively [ 15 NH 4 ] 2 SO 4 and D -[U- 13 C]glucose (99% isotopic purity; Cambridge Isotope Laboratories, Woburn, MA, USA), were used as sole nitrogen and carbon sources. Recom- binant protein production was induced with 0.3 m M isopropyl thio-b- D -galactoside when A 600 of the cell culture reached 0.5. The culture was incubated for a further 3 h at 37 °C, and the cells were harvested by centrifugation. The cell pellet was resuspended in column buffer (20 m M Tris/ HCl, 200 m M NaCl, 1 m M EDTA, 1 m M NaN 3 and 1 m M dithiothreitol; 20 mLÆL )1 cell culture), and lysozyme (0.1 mgÆmL )1 ) was added. The cells were subjected to three freeze-thaw cycles and sonicated for 2 min. The cells were then centrifuged, applied to a column of amylose resin (New England Biolabs), washed overnight with column buffer, andelutedwith10m M maltose, according to the manufac- turer’s protocol. The fractions were analyzed by MALDI- TOF MS and also by SDS/PAGE (12%) to determine if they contained the expected MBP-PreCbnB2 fusion protein. The appropriate fractions were combined and dialyzed extensively against distilled, deionized water and lyophi- lized. Typical yield was 70–100 mg fusion protein per litre of culture. The target peptide was cleaved from MBP with Factor Xa (Borean Biologics Aps, Aarhus, Denmark) in 20 m M Tris/HCl/100 m M NaCl/1 m M CaCl 2 [0.01 mgÆ(mg fusion protein) )1 ] overnight at room temperature. The resulting peptide was separated from MBP on a C18 column (Waters PrepPak), with a 20 minute gradient of 20–60% acetonitrile in water with 0.1% trifluoroacetic acid. The target peptide was eluted at 28% acetonitrile. Fractions containing this peptide were combined and lyophilized. Typical yield is 4–6 mg per litre of initial fermentation. MS analysis (see below) of the MBP fusion protein was in agreement with that expected (mass 48 300 Da) for unlabe- led protein. However, the MS of the target peptide showed that the two C-terminal amino acid residues (Arg65 and Pro66) were absent [observed 6739.2 ± 0.8 for unlabeled peptide; calculated 6738.5 for preCbnB2(1–64) missing the two C-terminal amino acids; calculated 6991.8 for preC- bnB2 having all 66 residues]. The mass spectra of 15 N-labeled preCbnB2(1–64) derived from [ 15 NH 4 ] 2 SO 4 showed a predominant mass peak at 6825.8 ± 0.8, corres- ponding to complete 15 N-labeling of all 87 nitrogens (backbone and side chain). PreCbnB2(1–64) showed CD spectra and biological activity that were indistinguishable from mature preCbnB2. As the peptide samples were susceptible to degradation when stored in solid form for a prolonged period, they were dissolved and stored in 70 : 30 trifluoroethanol-d 3 (99.9%)/H 2 O or 70 : 30 trifluoroetha- nol-d 3 (99.9%)/D 2 O (100%) at a concentration of  1m M . Mass spectrometry MS analysis used MALDI-TOF on an Applied Biosystems Voyager Elite instrument in the positive ion mode with an acceleration voltage of 20 kV using a nitrogen laser (k ¼ 337 nm). Samples were prepared using a-cyano-4- hydroxycinnamic acid (Aldrich) or sinapinic acid (Aldrich) as a matrix, and fixed to a gold or stainless-steel target before analysis. The instrument was calibrated daily before each experiment using apomyoglobin [MH + ¼ 16 952.56] and trypsinogen [MH + ¼ 23 981.9] as standards for MBP fusion proteins and insulin [MH + ¼ 5734.59] and insulin chain B [MH + ¼ 3496.96] for peptides. CD spectroscopy All CD measurements were performed by R. Luty (Depart- ment of Biochemistry, University of Alberta) on a Jasco J-720 spectrophotometer equipped with JASCO J 700 soft- ware. A thermally controlled quartz cell with a 0.02 cm path length over 180–250 nm was used. CD spectra of preCbnB2 at different concentrations and varying the solvent from 0% to 70% trifluoroethanol in water were collected at 25 °C. Data were collected every 0.05 nm and were the average of eight scans. The bandwidth was set at 1.0 nm and the sensitivity at 50 mdegrees, and the response time was 0.25 s. In all cases, baseline scans of aqueous buffer were subtracted from the experimental readings. Results are expressed in units of molar ellipticity per residue (degreesÆcm 2 Ædmol )1 ) and plotted against wavelength. Antibacterial activity Antibacterial activity was determined by the spot-on-lawn test essentially as previously reported [14]. The indicator organism, L. monocytogenes LI0502, was grown overnight in 7.5 mL tryptic soy broth with yeast extract or brain heart infusion (BHI) broth without shaking at 30 °C. Serial twofold dilutions of preCbnB2 (in Luria–Bertani broth) werespotted(20mL)ontoaBHIhardagarplate,allowed to dry, and overlaid with 7.5 mL of a 1% solution of the indicator organism in BHI soft agar. Zones of inhibition were measured after 16 h incubation at 37 °C. NMR spectroscopy NMR experiments were recorded on a Varian INOVA-600 spectrometer. Unless otherwise stated, all experiments were performed on labeled preCbnB2(1–64), the biological activity and CD spectra of which were indistinguishable from the parent 66-amino acid peptide, preCbnB2. 15 N HSQC [29], 15 N HSQC-TOCSY [30], 15 NHSQC-NOESY [30], 13 CHSQC, 13 C HSQC-NOESY [31] and a 2D NOESY spectrum were recorded at 35 °C. 15 NHSQC, 15 N HSQC-NOESY, HNHA [32–34], 13 CHSQCand 13 C HSQC-NOESY spectra were recorded at 20 °C. 15 N HSQC-NOESY mixing times were 200 ms; the 13 CHSQC- NOESY experiments were recorded with 150 ms mixing 1750 T. Sprules et al.(Eur. J. Biochem. 271) Ó FEBS 2004 times. The TOCSY experiment was recorded with a 60-ms spinlock. Chemical shifts were referenced to an internal standard of 2,2-dimethyl-2-silapentane-5-sulfonic acid [35]. Data were processed with NMRpipe [36], and data analysis was performed with NMRView [37]. Data were multiplied by a 90°-shifted sine-bell-squared function in all dimensions. Indirect dimensions were doubled by linear prediction and zero-filled to the nearest power of 2, before Fourier transformation. Structure calculations A total of 649 NOE restraints were obtained from 15 Nand 13 C edited HSQC-NOESY experiments on labeled preC- bnB2(1–64), and classified as strong, medium and weak, corresponding to distance restraints of 1.8–2.8 A ˚ , 1.8–3.4 A ˚ and 1.8–5.0 A ˚ , respectively. Thirty-three backbone / angles were obtained from analysis of the diagonal-peak to cross- peak intensity ratio in the HNHA experiment, with torsion angles calculated from the Karplus equation [34] and assigned a variance of ± 15°. Fifty-eight backbone w angle restraints were obtained from analysis of d Na /d aN ratios [38]. The w angle restraint was set to )30° ±110° for d Na / d aN ratios less than 1, and to 120° ±100° for d Na /d aN ratios greater than 1. Initially 100 structures were calculated with CNS 1.1 [39] using 327 intraresidue, 201 sequential, 115 medium-range and six long-range NOEs. The resulting structures were subjected to a second round of simulated annealing with the addition of 22 hydrogen bonds and 91 dihedral angles. The 20 lowest energy accepted structures (no NOE violations >0.5 A ˚ , no dihedral angle violations >5°)withnoresiduesinthedisallowedregionofthe Ramachandran plot ( PROCHECK [40] analysis) were chosen to represent the structure. The co-ordinates of preCbnB2(1– 64) have been deposited in the Protein Data Bank as 1RY3. Results and discussion Production of target peptides Like other bacteriocins, CbnB2 is ribosomally synthesized as a prepeptide, PreCbnB2, by C. piscicola [19]. PreCbnB2 consists of the mature CbnB2 sequence (48 amino acids) preceded at the N-terminus by an 18-amino acid leader peptide, which is cleaved at the Gly–Gly site during the maturation process to liberate the active peptide. The goal of this study was determination of the 3D solution structure of preCbnB2 to assist in obtaining a molecular level understanding of the reduced activity of preCbnB2 (relative to the mature bacteriocin) and the recognition events required for export and cleavage by the ABC transporter. Initially it appeared that the structure of preCbnB2 could be obtained by modern protein NMR techniques using the compound without isotopic labels. Hence, we chemically synthesized this 66-amino acid peptide as its C-terminal amide using standard solid-phase methods with Fmoc chemistry and acid-labile protecting groups where necessary on the side chains. This provided sufficient material (10 mg) suitable for a variety of biological (e.g. antimicrobial activity) and spectroscopic (e.g. MS, CD) studies. However, it soon became evident that preCbnB2 has limited solubility as well as a strong tendency to self-associate and precipitate as insoluble aggregates from aqueous solutions at concen- trations required for effective NMR analysis. PreCbnB2 also displays a tendency to decompose or denature in the absence of solvent. Thus, the enhanced sensitivity in NMR studies afforded by isotopic labeling with 15 Nand 13 Cof more dilute samples proved essential. A practical route to isotopic labeling involves the previously reported [14] expression of a fusion of MBP and preCbnB2 in E. coli. This system allows use of ( 15 NH 4 ) 2 SO 4 , or alternatively ( 15 NH 4 ) 2 SO 4 and D -[U- 13 C]glucose, as sole nitrogen and carbon sources. Purification of substantial quantities (70–100 mg per litre of fermentation) of fusion protein is easy, but large-scale proteolytic cleavage of the MBP portion by Factor Xa also results in removal of two C-terminal amino acids of preCbnB2, namely Arg65 and Pro66, to yield the truncated preCbnB2(1–64) as the major product. However, this material displays CD spectra and antimicrobial properties indistinguishable from the complete preCbnB2. The two missing hydrophilic residues are at the C-terminus of the peptide, which is unstructured in the mature CbnB2 [9], and distant from the N-terminal leader portion. The C-terminal section is also random coil in preCbnB2(1–64) (see below). Significantly, substitution of Arg46, corresponding to Arg64 in preCbnB2, with glycine in CbnB2 has no effect on its activity [14]. Hence no significant conformational differ- ences are expected between preCbnB2 and preCbnB2(1–64). Structure of preCbnB2 The structures of all mature type IIa bacteriocins examined thus far are primarily random coil in water, but in lipophilic solvents, such as trifluoroethanol or dodecylphosphocholine micelles, assume a highly conserved amphipathic a-helix which begins at about residue 18 and continues for several turns toward the C-terminus [8–10]. As these bacteriocins are well known to interact with membrane lipids and recognize a chiral membrane-bound receptor molecule [2,11], the a-helical portion of the structure of mature type IIa bacteriocins is critical for their biological activity [8–10]. CD experiments (data not shown) demonstrate that, like the mature bacteriocin, preCbnB2 is unstructured in pure water but contains one or more a-helical sections in more lipophilic trifluoroethanol/water mixtures. To directly compare the structure of PreCbnB2(1–64) with that reported by us for mature CbnB2 [9], the prepeptide was initially dissolved in a 90 : 10 trifluoroethanol/water. However, preCbnB2 is very sparingly soluble in this solvent system, possibly because of the increase in the number of charged and hydrophilic residues in the leader peptide region. Increasing the propor- tion of water to 30% results in the best combination of peptide solubility and spectral dispersion of the amide protons, which indicates the maintenance of organized structural elements (Fig. 1). As described below, the use of a higher proportion of water does not significantly alter the 3D structure of the section corresponding to the mature peptide. Complete proton, nitrogen and carbon assignments were obtained for PreCbnB2(1–64) using a combination of 15 N HSQC-TOCSY, 15 N HSQC-NOESY and 13 CHSQC spectra. Sequential assignments were made by following the pattern of d N,N±1 NOEs. Analysis of 3 J HNHa coupling constants, Ha,Ca and Cb chemical shifts, and NOEs Ó FEBS 2004 Solution structure of precarnobacteriocin B2 (Eur. J. Biochem. 271) 1751 indicated the presence of two a-helices: one running from residues )15 to )5 in the leader peptide, and a second from residues 20–38 in the C-terminal portion of the molecule. No other regular secondary-structure elements were identified. The solution structure of PreCbnB2(1–64) was calculated based on 649 NOEs, 91 dihedral angles, and 22 hydrogen bonds. The two a-helices are separated by a stretch of relatively unstructured peptide (Fig. 2). The rmsd for helix 1, from residues )15 to )5 is 0.6 ± 0.2 A ˚ , and for helix 2 0.8 ± 0.3 A ˚ . In contrast with mature CbnB2, where no NOEs were observed indicative of a disulfide bridge [9], at 35 °CHa-Ha and Ha-Hb NOEs are seen across the disulfide bridge, although at lower temperature (20 °C) line broadening precludes their observation. Cysteine b carbon chemical shifts have been found to be quite sensitive to oxidation state [41]. The Cb chemical shifts for oxidized cysteine is 40.7 ± 3.8 p.p.m. and those of reduced cysteine fall in the range of 28.4 ± 2.4 p.p.m. The Cb chemical shifts for both cysteines in PreCbnB2(1–64) fall clearly in the oxidized region (44.1 and 43.9 p.p.m.), confirming that a disulfide bridge is present. Because of the absence of NOE and 13 C chemical shift data indicative of disulfide bond formation in mature CbnB2, MS and chemical modification experiments were used to determine that the disulfide bridge was present [9]. The structures of the region common to both mature CbnB2 and its precursor are nearly identical: both possess a poorly defined reverse turn and relatively unstructured N-terminus, where only sequential NOEs are observed, followed by the highly conserved amphipathic helix from residues 20–38. The a-helix in PreCbnB2(1–64) is very slightly less well defined at its N-terminus. This may be due to fewer hydrogen bond and dihedral angle restraints observed because of overlap of Ha and HN chemical shifts. The slightly increased proportion of water (30% vs. 10% for previous experiments with mature CbnB2 [9]) may also Fig. 1. 15 N HSQC of preCbnB2. Spectra recorded in 70 : 30 trifluoroethanol/H 2 Oat 35 °C and 600 MHz. Fig. 2. Solution structure of preCbnB2 in 70% trifluoroethanol. The positions of the N-terminus and C-terminus are indicated, as are the residues at the start and finish of each a-helix. The position of the double glycine motif where cleavage occurs during maturation of the bacteriocin is indicated by an arrow. 1752 T. Sprules et al.(Eur. J. Biochem. 271) Ó FEBS 2004 account for this very minor difference. The side chain chemical shifts are very similar across the protein (within ± 0.1 p.p.m.), with the most variance observed at the N-terminus, next to the junction with the leader peptide. Similar patterns of NOEs are observed as well. Thus, the critical a-helix in the Ômature portionÕ of the bacteriocin is conserved in lipophilic environments, not only in all type IIa bacteriocins examined to date [8–10], but also in the precursor, preCbnB2. Mutations that disrupt the helix of mature CbnB2, for example replacement of Phe33 by serine, render the bacteriocin completely inactive and greatly alter its retention time on RP-HPLC [14]. Many additional experiments support the importance of this helix for membrane-bound receptor recognition and consequent antimicrobial activity [2,10]. For example, Fimland et al. [42] have shown that interchange of large domains of different type IIa bacterio- cins, such as pediocin PA-1, sakacin P, and curvacin A, gives chimeric peptides, the antimicrobial specificity of which for target organisms corresponds to the C-terminal region. It has also been demonstrated that a C-terminal fragment of pediocin (residues 20–34) specifically inhibits pediocin PA-1 activity [43]. Interestingly, this short peptide is not antimicrobially active and does not significantly inhibit closely related bacteriocins such as leucocin A, sakacin P, and curvacin A. The receptor for type IIa bacteriocins may involve an extracellular domain present in the MptD subunit of EII Man t of the mannose phospho- transferase system [44]. Although these types of experiments have not yet been performed with prebacteriocins, the present work shows that the leader peptide does not interfere with formation of the key C-terminal a-helix, and as a result, all of the precursors are likely to possess significant antimicrobial activity with specificity similar to the mature type IIa bacteriocin. The leader peptide, which consists of the first 18 amino acids of PreCbnB2(1–64), forms a 10-amino acid a-helix, from Val15 to Gln5, as we had previously predicted [19]. Although it is not as clearly defined as the amphipathic a-helix in the C-terminus of the polypeptide, the leader peptide helix presents both a charged and hydrophobic face (Fig. 3). The region between the two a-helices is relatively unstructured, permitting them to approach one another (Fig. 4), thereby making contact between the hydrophobic faces of the two helices possible to give a closed ÔjackknifeÕ structure. This could interfere to some extent with recog- nition of the mature bacteriocin a-helix by the putative receptor in membranes of target bacteria and somewhat diminish the activity of the prebacteriocin. Although no Fig. 3. Amphipathic a-helical structure in PreCbnB2. (A) Leader pep- tide a-helix. (B) C-terminal a-helix. The charged residues are coloured blue, hydrophilic residues white, and hydrophobic residues purple. Fig. 4. Superimposition of preCbnB2 structures. (A) Superimposition of the backbone of the leader peptide a-helix for 20 structures. (B) Superimposition of the backbone of helix 2. Both helix domains are highly conserved in each case, but the flexible linker region displaces their relative positions. Ó FEBS 2004 Solution structure of precarnobacteriocin B2 (Eur. J. Biochem. 271) 1753 long-range NOEs were observed between side chains in the two a-helices under the conditions used in this study, this may reflect a relatively weak or short-lived interaction. The hydrophilic positively charged face of the leader helix, which contains three lysines, could potentially also interact with the negatively charged membrane of Gram-positive bac- teria, thereby hindering the ability of the prebacteriocin to fully interact with the target receptor. These observations are consistent with the maintenance of significant antibac- terial activity for the prebacteriocin, but at a greatly reduced level ( 125 fold). The amphipathic nature of the leader peptide is likely to be critical for its export and processing. Analysis of the sequences of leader peptides for type IIa bacteriocins (Table 1) indicates that an a-helix as seen in this section with preCbnB2(1–64) (Fig. 5) is likely to be present in all of the structures of the prepeptides. The sakacins and carno- bacteriocin BM1 (CbnBM1), which have short leader peptides, are especially closely related in the arrangement of hydrophobic and hydrophilic residues in this portion. The ABC transporter protein must recognize this leader portion not only for export, but also for processing by its cysteine proteinase domain [17]. In the absence of 3D structures of these transporter proteases, at this stage it is impossible to ascertain the molecular details of these recognition events. However, it is likely that interactions of the hydrophobic faces of the leader helix with the ABC transporter play a key role in recognition, as the bacteriocin must pass through the membrane, a helix-inducing envi- ronment, during export and processing. In summary, this work describes the chemical synthesis and properties of the 66-amino acid bacteriocin precursor preCbnB2, and the biochemical production and isotopic labeling of preCbnB2(1–64), along with its detailed NMR analysis and 3D structure. The sequence homology with other type IIa prebacteriocins indicates that the structural motif of two amphipathic a-helices connected in the cleavage region by a flexible hinge will be present in all such peptides. Knowledge of such structures provides a basis for understanding the protein–protein interactions involved in transport, processing and antimicrobial activity. Acknowledgements We thank Robert Luty (Department Biochemistry, University of Alberta) for performing all CD experiments. Albin Otter (Department of Chemistry) and Ryan T. McKay (NANUC, University of Alberta) are gratefully acknowledged for assistance with NMR experiments. 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Supplementary material The following material is available from http:// blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4085/EJB4085sm.htm Fig. S1. Helical wheel representation of leader peptide residues –4 to –15 for selected type IIa bacteriocins. Fig. S2. CD spectra of preCbnB2(1–64) (0–60% aqueous trifluoroethanol). Table S1. Nitrogen and carbon chemical shifts. Table S2. Proton chemical shifts. Table S3. Structure statistics. 1756 T. Sprules et al.(Eur. J. Biochem. 271) Ó FEBS 2004 . NMR solution structure of the precursor for carnobacteriocin B2, an antimicrobial peptide from Carnobacterium piscicola Implications of the a-helical leader. basis of the inhibition of the antimicrobial activity of CbnB2 by the leader and to assist future analysis of the interaction of preCbnB2 with its ABC transporter