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The Pediocin PA-1 Accessory Protein Ensures Correct Disulfide Bond Formation in the Antimicrobial Peptide Pediocin PA-1
- Oppegård, Camilla, Fimland, Gunnar, Anonsen, Jan Haug, Nissen-Meyer, Jon
- Biochemistry 2015 v.54 no.19 pp. 2967-2974
- ABC transporters, active sites, antimicrobial peptides, bacteriocins, disulfide bonds, mutation, proteolysis, secretion
- Peptides, in contrast to proteins, are generally not large enough to form stable and well-defined three-dimensional structures. However, peptides are still able to form correct disulfide bonds. Using pediocin-like bacteriocins, we have examined how this may be achieved. Some pediocin-like bacteriocins, such as pediocin PA-1 and sakacin P[N24C+44C], have four cysteines. There are three possible ways by which the four cysteines may combine to form two disulfide bonds, and the three variants are expected to be produced in approximately equal amounts if their formation is random. Pediocin PA-1 and sakacin P[N24C+44C] with correct disulfide bonds were the main products when they were secreted by the pediocin PA-1 ABC transporter and accessory protein, but when they were secreted by the corresponding secretion machinery for sakacin A, a pediocin-like bacteriocin with one disulfide bond (two cysteines), peptides with all three possible disulfide bonds were produced in approximately equal amounts. All five cysteines in the pediocin PA-1 ABC transporter and the two cysteines (that form a CxxC motif) in the accessory protein were individually replaced with serines to examine their involvement in disulfide bond formation in pediocin PA-1. The Cys86Ser mutation in the accessory protein caused a 2-fold decrease in the amount of pediocin PA-1 with correct disulfide bonds, while the Cys83Ser mutation nearly abolished the production of pediocin PA-1 and resulted in the production of all three disufide bond variants in equal amounts. The Cys19Ser mutation in the ABC transporter completely abolished secretion of pediocin PA-1, suggesting that Cys19 is in the proteolytic active site and involved in cleaving the prebacteriocin. Replacing the other four cysteines in the ABC transporter with serines caused a slight reduction in the overall amount of secreted pediocin PA-1, but the relative amount with the correct disulfide bonds remained large. These results indicate that the pediocin PA-1 accessory protein has a chaperone-like activity in that it ensures the formation of the correct disulfide bond in pediocin PA-1.