Structural insights into the mode of action of the peptide antibiotic copsin

Defensins are a class of cysteine.rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Due to their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin.like peptide, previously identified in the mushroom Coprinopsis cinerea. It is active against a range of Gram positive bacteria through binding to the peptidoglycan precursor lipid II and prevention of a proper cell wall formation. In this study, we present a new workflow for the generation, production and activity-driven selection of copsin derivatives, based on the expression in Pichia pastoris. 152 single amino acid mutants and combinations thereof, allowed the identification of k.copsin, a peptide variant exhibiting a significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k.copsin, both in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47% and 35% of the total contacts for copsin and k.copsin, respectively. Mutated amino acids were located in loop-regions of k.copsin and showed to be crucial in perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed towards antibacterial therapies less prone to resistance issues.