Mechanical and energetic insights into pore formation in asymmetric lipid bilayers

Compositional asymmetry is a hallmark of biological membranes and plays a crucial role in pore formation. However, the molecular mechanism by which leaflet asymmetry influences pore formation remains unclear. Here, we employed molecular dynamics simulations of asymmetric lipid bilayers composed of zwitterionic phosphatidylcholine (POPC) and anionic phosphatidylserine (POPS) to elucidate this process. The calculated free-energy barrier for pore nucleation in asymmetric bilayers was higher than that in symmetric ones. In the asymmetric system, water defects initiating pore formation preferentially appeared on the POPC leaflet, whereas the subsequent pore growth involved rearrangements in the POPS leaflet. During this process, hydrogen bonds between POPS molecules were disrupted and partially replaced by hydrogen bonds between POPS and water molecules, accompanied by increased headgroup cohesion and structural ordering within POPS-rich regions, thereby increasing the energetic cost of pore nucleation. In contrast, POPC molecules lacked intermolecular hydrogen bonding, resulting in weaker headgroup cohesion. We then used a continuum-based theoretical model to rationalize the preferential water entry on one leaflet observed in asymmetric membranes. Finally, using an erythrocyte-mimetic multicomponent membrane model, we show that leaflet-biased water entry and the accompanying reorganization of headgroup interaction networks persist in a multicomponent asymmetric membrane. Overall, leaflet-resolved headgroup chemistry and membrane mechanics jointly shape the free-energy landscape of pore nucleation in asymmetric membranes. Lipid-lipid headgroup hydrogen bonding, together with membrane thickness and elastic response, can modulate pore nucleation and early pore stabilization, and may be relevant to asymmetry-dependent processes such as apoptosis and transmembrane signaling.