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.
Budimir et al. (2026) studied this question.