Using multiscale molecular dynamics simulations to explore the fusion machinery underlying neurotransmitter release.

Neurotransmitter release via synaptic vesicle fusion with the plasma membrane is driven by SNARE proteins (Synaptobrevin, Syntaxin, and SNAP-25) and accessory proteins (Synaptotagmin, Complexin, Munc13, and Munc18). While extensively studied experimentally, the precise mechanisms and dynamics remain elusive due to spatiotemporal limitations. Molecular dynamics (MD) simulations-both all-atom (AA) and coarse-grained (CG)-bridge these gaps by capturing fusion dynamics beyond experimental resolution. This review explores the use of these simulations in understanding SNARE-mediated membrane fusion and its regulation by Synaptotagmin and Complexin. We first examine two competing hypotheses regarding the driving force of fusion: (1) SNARE zippering transducing energy through rigid juxtamembrane domains (JMDs) and (2) SNAREs generating entropic forces via flexible JMDs. Despite different origins of forces, the conserved fusion pathway - from membrane adhesion to stalk and fusion pore (FP) formation - emerges across models. We also highlight the critical role of SNARE transmembrane domains (TMDs) and their regulation by post-translational modifications like palmitoylation in fast fusion. Further, we review Ca²⁺-dependent interactions of Synaptotagmin's C2 domains with lipids and SNAREs at the primary and tripartite interfaces, and how these interactions regulate fusion timing. Complexin's role in clamping spontaneous fusion while facilitating evoked release via its central and accessory helices is also discussed. We present a case study leveraging AA and CG simulations to investigate ion selectivity in FPs, balancing timescale and accuracy. We conclude with the limitations in current simulations and using AI tools to construct complete fusion machinery and explore isoform-specific functions in fusion machinery.