Interfacial activity and membrane interaction of Duttaphrynus melanostictus cathelicidin

Cathelicidin-DM is a host defence peptide isolated from the Asian common toad Duttaphrynus melanostictus. The peptide has been reported in the literature to exhibit antibacterial and wound-healing activities. The mechanism of antimicrobial activity, however, remains unclear. As most cathelicidins reported in the literature exhibit their antimicrobial activity through membrane perturbation, we investigated cathelicidin-DM's interfacial and membrane binding activities using molecular dynamics simulations and experimental methods that include surface activity at air/aqueous interface, binding with liposomes using fluorescence spectroscopy, binding with lipid monolayers using Langmuir set-up, and dye release assay. The molecular dynamics simulation of cathelicidin-DM in water/cyclohexane biphasic system reveals its localization at the water/cyclohexane interface. The experiment shows a saturation surface pressure of ∼10-11 mN/m at the air/aqueous interface, thereby establishing its interfacial activity. The simulation with the POPC/CHL bilayer results in extensive unfolding of the peptide, with the peptide showing transient interactions with the bilayer. With POPE/POPG bilayer, the peptide binds through its N-terminus and remains bound throughout the simulation. Lipid monolayer penetration assays with POPC/CHL (10:1) and POPE/POPG (7:3) monolayers exhibited critical insertion pressures of ∼33.2 and ∼46.7 mN/m, respectively. Preferential binding to negatively charged membranes is further established using tryptophan fluorescence assays. Counterintuitively, however, the peptide preferentially perturbs zwitterionic vesicles, sparing the negatively charged ones. Antimicrobial peptide molecules usually act cooperatively to bring about the membranolytic effects. Tight binding to membranes could, therefore, affect cooperativity, thereby negatively impacting activity.