(267a) Elucidating the Mechanism of Melittin-Induced Pore Formation in Red Blood Cell Membranes with Molecular Dynamics Simulations

The antimicrobial resistance (AMR) crisis is a major global health threat where genetic changes in microbial populations over time, such as through mutations or natural selection of members that carry resistance genes, compromise the efficacy of clinically available drugs.¹ As a result, an emerging drug class that is being studied for the treatment of multi-drug resistant infections are antimicrobial peptides (AMPs), whose mode of action instead involves direct binding (through strong polar and electrostatic interactions with lipid head groups) and disruption (through either pore formation or permeabilization) of microbial cell membranes.² 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) have proved invaluable in elucidating the pore-formation mechanism of MEL in single-lipid zwitterionic membranes, however, an extension to mammalian-mimetic lipid bilayers such as RBC has been limited due to the inability of MD to capture long-timescale membrane restructuring events with atomistic detail dependent on lipid heterogeneity, leaflet asymmetry, and cholesterol content.

To address these challenges and access larger time scales for comparison with experiments, we utilized the coarse-grained MARTINI force field to model four lipid membranes of increasing complexity ranging from single-lipid POPC membranes to asymmetric RBC membranes containing cholesterol. Through the utilization of a nucleation collective variable (ξ) to create transmembrane pores and coarse-grained-to-atomistic backmapping strategy we developed previously³, we then studied MEL pore-lining affinity and free energy as a function of pore size 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 by increasing the ability of bilayers to resist lateral stress and lipid fluctuations, pore nucleation is more favorable in RBC versus single-lipid POPC membranes when CHOL is absent due to the enrichment of anionic POPS lipids near the pore that permit 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.

(1) Santos-Lopez, A.; Marshall, C. W.; Haas, A. L.; Turner, C.; Rasero, J.; Cooper, V. S. The roles of history, chance, and natural selection in the evolution of antibiotic resistance. Elife 2021, 10. DOI: 10.7554/eLife.70676 From NLM.

(2) Li, J.; Koh, J.-J.; Liu, S.; Lakshminarayanan, R.; Verma, C. S.; Beuerman, R. W. Membrane Active Antimicrobial Peptides: Translating Mechanistic Insights to Design. Frontiers in Neuroscience 2017, 11, Review.

(3) Richardson, J. D.; Van Lehn, R. C. Free Energy Analysis of Peptide-Induced Pore Formation in Lipid Membranes by Bridging Atomistic and Coarse-Grained Simulations. The Journal of Physical Chemistry B 2024, 128 (36), 8737-8752. DOI: 10.1021/acs.jpcb.4c03276.