Unstructured, disulfide-bridged C-terminus in helminth α-helical antimicrobial peptides enhances and modulates their activity.

Understanding antimicrobial peptide (AMP) structural determinants is crucial for clinical development. While most designed AMPs are short and helical, many natural ones have unstructured or cyclic C-terminal tails with poorly defined functions. We studied mesco-2 from the flatworm Mesocestoides corti, with an N-terminal α-helix kinked around a palindromic GRGIGRG motif and an unstructured C-terminal tail containing a disulfide-forming CLGRC motif, along with its disulfide-reduced analogue mesco-2 A. Similar CXXXC motifs are common in flatworm AMPs and typically occur in unstructured regions, as indicated by the sequence analysis. Molecular modelling revealed that the C-terminal disulfide loop modulates mesco-2 flexibility and oligomerization. Both peptides displayed strong antibacterial activity and low cytotoxicity. Differences appeared in their effect on bacterial growth kinetics at sub-bactericidal concentrations. Flow cytometry and fluorescence imaging confirmed membrane-related mechanisms, but for mesco-2 A the membrane-disruptive effect was slower. Atomic force microscopy confirmed their distinct membrane interaction modes, and circular dichroism in anionic liposomes revealed secondary-structure differences. Microscale thermophoresis confirmed distinct liposome binding, with mesco-2 A likely binding as monomers and mesco-2 forming assemblies, as also suggested by the modelling results. Overall, our findings show that the C-terminal cyclic tail is a tunable element for peptide engineering, enabling control over the speed, extent, and cooperativity of antimicrobial activity.