Dynamic Formation of the Protein-Lipid Pre-fusion Complex

Synaptic vesicles (SVs) fuse with the presynaptic membrane (PM) to release neuronal transmitters. The SV protein Synaptotagmin 1 (Syt1) serves as a Ca²⁺ sensor for evoked fusion. Syt1 is thought to trigger fusion by penetrating into PM upon Ca²⁺ binding, however the mechanistic detail of this process is still debated. Syt1 interacts with the SNARE complex, a coiled-coil four-helical bundle that enables the SV-PM attachment. The SNARE-associated protein Complexin (Cpx) promotes the Ca²⁺-dependent fusion, possibly interacting with Syt1. We employed all-atom molecular dynamics (MD) to investigate the formation of the Syt1-SNARE-Cpx complex interacting with the lipid bilayers of PM and SV. Our simulations demonstrated that the PM-Syt1-SNARE-Cpx complex can transition to a "dead-end" state, wherein Syt1 attaches tightly to PM but does not immerse into it, as opposed to a pre-fusion state, which has the tips of the Ca²⁺-bound C2 domains of Syt1 inserted into PM. Our simulations unraveled the sequence of Syt1 conformational transitions, including the simultaneous Syt1 docking to the SNARE-Cpx bundle and PM, followed by the Ca²⁺ chelation and the penetration of the tips of Syt1 domains into PM, leading to the pre-fusion state of the protein-lipid complex. Importantly, we found that the direct Syt1-Cpx interactions are required to promote these transitions. Thus, we developed the all-atom dynamic model of the conformational transitions that lead to the formation of the pre-fusion PM-Syt1-SNARE-Cpx complex. Our simulations also revealed an alternative "dead-end" state of the protein-lipid complex that can be formed if this pathway is disrupted.