Molecular Dynamics-Enhanced Sampling Reveals Electrofusion Mechanisms and Pathways

While electroporation mechanisms in biomembranes are well-established, the molecular basis of electrofusion remains unclear due to sampling limitations and artificial dismissal of prestalk membrane leakage. By integrating molecular dynamics simulations with enhanced sampling, employing coarse-grained (extended sampling for conformational exploration, requiring rationalized polarizable water settings) and atomistic force fields (high spatial resolution to capture details, necessitating overcoming prevalent sampling artifacts), we elucidate the field-strength-dependent free energy landscape of electrofusion (focusing on stalk formation) and noncanonical fusion pathways. A critical electric field threshold (Ec), validated by water dipole orientation, mass density, and transmembrane potential profiles, governs distinct regimes: for E < Ec, discontinuous aqueous defects emerge on noncontact monolayers, causing sluggish membrane deformation and attenuation of stalk formation energy under increasing field. For E > Ec, prestalk single bilayer leakage (resembling peptide-induced π-shaped pores, hence named) redistributes local fields, triggering cooperative rupture on opposing leaflets to form 2π-shaped fusion pores and inducing a precipitous drop in stalk formation energy with heightened field sensitivity. This threshold mechanism may unify kinetic disparities in synaptic transmission/viral fusion, partly attributable to bias variations imposed by fusion proteins. In summary, our work advances understanding of electrofusion mechanisms and pathways.