Mechanisms of Fusion Protein Transmembrane Segments in Fusion Pore Formation

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are essential for membrane fusion, yet the mechanistic role of their transmembrane domains (TMDs) remains debated. Here, we use molecular dynamics simulations of a synaptic vesicle and a planar plasma membrane to elucidate how the aggregation state and copy number of SNAREs regulate fusion pore formation. We find that TMD aggregation markedly lowers the energetic barrier to pore initiation. Simulations with eight aggregated SNARE complexes rapidly generated a continuous, protein-lined aqueous channel connecting the vesicle lumen to the extracellular space. In contrast, systems with eight dispersed SNAREs failed to form a pore under identical conditions and required higher energy to achieve close membrane contact. The number of SNARE complexes also proved critical: while two or four complexes induced membrane deformation, only higher copy numbers, such as eight, consistently nucleated and stabilized a fusion pore. These results support a sequential proteolipid fusion model in which clustered TMDs first create a protein-rich conduit that subsequently incorporates lipids. Overall, our findings clarify how TMD aggregation and SNARE copy numbers cooperatively ensure efficient and robust synaptic vesicle exocytosis.