title: Mutational analysis of the role of charged residues in target-cell binding, potency and specificity of the pediocin-like bacteriocin sakacin P.
abstract: The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P. Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction. All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2-15-fold reduction in potency. The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T). This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance. Somewhat more deleterious was the removal of positive charges at the N- and C-terminal ends (K1T, K43T). Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins. Mutated peptides in which the net positive charge was increased by one were also constructed. Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e. an extra positive charge at the C-terminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e. an extra positive charge at the N-terminus) had reduced potency. Sakacin P contains only one negatively charged residue (Asp17). This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious. Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic. Thus, charged residues in the N-terminal half may - apparently via the initial electrostatic binding of the bacteriocin to target cells - influence the target-cell specificity.
authors.affiliation: Department of Biochemistry, University of Oslo, Post Box 1041, Blindern, 0316 Oslo, Norway1.
authors.forename: Maja
authors.initials: M
authors.lastname: Kazazic
authors.affiliation: Department of Biochemistry, University of Oslo, Post Box 1041, Blindern, 0316 Oslo, Norway1.
authors.forename: Jon
authors.initials: J
authors.lastname: Nissen-Meyer
authors.affiliation: Department of Biochemistry, University of Oslo, Post Box 1041, Blindern, 0316 Oslo, Norway1.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Fimland
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000187:drug effects; D001430:Bacteriocins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007778:Lactobacillus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D001430:Bacteriocins; C087217:sakacin P protein, Lactobacillus