Elucidating the Impact of Red Blood Cell Membrane Components on Melittin-Induced Pore Formation with Molecular Dynamics Simulations.

Understanding the membrane-disrupting mode of action of antimicrobial peptides (AMPs) in complex biological membranes is critical for the design of therapeutically viable AMPs that are both active against microbial pathogens and nontoxic to human cells. To assess human toxicity in AMP design studies, melittin (MEL) (a highly charged 26-amino acid AMP sourced from bee venom) is often used as the positive control in experimental human red blood cell (RBC) hemolysis assays. Molecular dynamics (MD) simulations have proven invaluable in elucidating the pore-formation mechanism of MEL in single-lipid zwitterionic membranes. However, modeling pore formation in lipid bilayers containing multiple lipid species, like RBC membranes, has been limited due to the challenges of using atomistic MD simulations to capture long-time-scale membrane restructuring events that depend on lipid heterogeneity, leaflet asymmetry, and cholesterol content. To address these challenges and access larger time scales, in this work, we utilize the coarse-grained MARTINI force field to model four lipid membranes of increasing complexity, ranging from single-lipid POPC membranes to asymmetric RBC-mimetic membranes containing cholesterol. Through the application of a nucleation collective variable (ξ) to create transmembrane pores and a coarse-grained-to-atomistic backmapping strategy, we studied MEL pore-lining affinity and pore nucleation free energies to assess the effect of lipid complexity and cholesterol on MEL pore formation. We find that although cholesterol strongly inhibits MEL-induced pore formation regardless of lipid content, pore nucleation is more favorable in RBC versus single-lipid POPC membranes when cholesterol is absent due to the enrichment of anionic POPS lipids near the pore that permits increased conformational flexibility for MEL. These results provide new physical insight into factors that affect pore formation in compositionally complex membranes and are a step toward understanding how AMPs can be designed to selectively induce pores in membranes with different compositions.