Cardiolipin regulates free energy of folding and vesicle leakage of CM15 antimicrobial peptides in bacterial inner membranes

Globally rising antimicrobial resistance has led to antimicrobial peptides (AMPs) being explored as alternative therapeutics for bacterial infections. A better mechanistic understanding of the interaction of the peptide with membrane compositions can aid in targeting specific bacterial strains more effectively and lead to more resilient treatments. Using all-atom molecular dynamics simulations, we obtain the free-energy landscape of the cecropin-melittin hybrid peptide CM15 using a path-based method and investigate the effect of cardiolipin (CL), a four - tailed lipid found in bacterial membranes. Our analysis reveals that the transition of CM15 from an unfolded state in solution to a membrane-bound helical state proceeds through three main steps: membrane binding, insertion in an unfolded state, and subsequent folding beneath the lipid headgroups into an -helical conformation. In a phosphatidylethanolamine (PE)/phosphatidylglycerol (PG) membrane lacking CL, folding occurs spontaneously without appreciable free energy barriers. However, the addition of only 5% CL substantially alters the landscape, giving rise to two distinct transition pathways with barriers ranging from 2.5 to 8 k BT. This reflects the presence of a more rugged free-energy landscape associated with local heterogeneity in lipid composition. The emergence of these barriers is driven by strong interactions between CL and CM15, which promote CL sequestration around the peptide in regions of negative membrane curvature. Vesicle leakage experiments reveal the inhibitory influence of CL at lower peptide-to-lipid ratios with delayed kinetics as the CL content is increased. We attribute the reduced activity at low CM15 concentrations to the free-energy barriers associated with peptide folding and to strong CM15-CL interactions that stabilize a membrane bound peptide state, thereby hindering membrane disruption, pore formation, and leakage. Our study illustrates the putative role of CL in pore formation, providing molecular insights that can potentially aid the rational design of strain-specific synthetic AMPs.