SUMMARY Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.

Pediocin-like antimicrobial peptides (AMPs) form a group of lactic acid bacteria produced, cationic membrane-permeabilizing peptides with 37 to 48 residues. Upon exposure to membrane-mimicking entities, their hydrophilic, cationic, and highly conserved N-terminal region forms a three-stranded antiparallel beta-sheet supported by a conserved disulfide bridge. This N-terminal beta-sheet region is followed by a central amphiphilic alpha-helix and this in most (if not all) of these peptides is followed by a rather extended C-terminal tail that folds back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal half. There is a flexible hinge between the beta-sheet N-terminal region and the hairpin C-terminal region and one thus obtains two domains that may move relative to each other. The cationic N-terminal beta-sheet domain mediates binding of the pediocin-like AMPs to the target-cell surface through electrostatic interactions, while the more hydrophobic and amphiphilic C-terminal hairpin domain penetrates into the hydrophobic part of the target-cell membrane, thereby mediating leakage through the membrane. The hinge provides the structural flexibility that enables the C-terminal hairpin domain to dip into the hydrophobic part of the membrane. Despite extensive sequence similarities, these AMPs differ markedly in their target-cell specificity, and results obtained with hybrid AMPs indicate that the membrane-penetrating hairpin-like C-terminal domain is the major specificity determinant. Bacteria that produce pediocin-like AMPs also produce a 11-kDa cognate immunity protein that protects the producer. The immunity proteins are well-structured, 4-helix bundle cytosolic proteins. They show a high degree of specificity in that they largely recognize and confer immunity only to their cognate AMP and in some cases to a few AMPs that are closely related to their cognate AMP. The C-terminal half of the immunity proteins contains a domain that is involved in specific recognition of the C-terminal membrane-penetrating specificity-determining hairpin domain of the cognate AMP.

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.

The three-dimensional structures in dodecylphosphocholine (DPC) micelles and in trifluoroethanol (TFE) of the pediocin-like antimicrobial peptide sakacin P and an engineered variant of sakacin P (termed sakP[N24C+44C]) have been determined by use of nuclear magnetic resonance spectroscopy. SakP[N24C+44C] has an inserted non-native activity- and structure-stabilizing C-terminal disulfide bridge that ties the C-terminus to the middle part of the peptide. In the presence of DPC, the cationic N-terminal region (residues 1-17) of both peptides has an S-shaped conformation that is reminiscent of a three-stranded antiparallel beta-sheet and that is more pronounced when the peptide was dissolved in TFE instead of DPC. The four positively charged residues located in the N-terminal part are found pointing to the same direction. For both peptides, the N-terminal region is followed by a well-defined central amphiphilic alpha-helix (residues 18-33), and this in turn is followed by the C-terminal tail (residues 34-43 for sakacin P and 34-44 for sakP[N24C+44C]) that lacks any apparent common secondary structural motif. In the presence of DPC, the C-terminal tails in both peptides fold back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal halves. The lack of long-range NOEs between the beta-sheet Nu-terminal region and the hairpin-like C-terminal half indicates that there is a flexible hinge between these regions. We discuss which implications such a structural arrangement has on the interaction with the target cell membrane.