Dynamic formation of the protein-lipid prefusion 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 the PM upon Ca²⁺ binding; however, the mechanistic detail of this process is still debated. Syt1 interacts with the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex, a coiled-coil four-helical bundle that enables the SV-PM attachment. The SNARE-associated protein complexin (Cpx) promotes Ca²⁺-dependent fusion, possibly interacting with Syt1. We employed all-atom molecular dynamics to investigate the formation of the Syt1-SNARE-Cpx complex interacting with the lipid bilayers of the PM and SVs. Our simulations demonstrated that the PM-Syt1-SNARE-Cpx complex can transition to a "dead-end" state, wherein Syt1 attaches tightly to the PM but does not immerse into it, as opposed to a prefusion state, which has the tips of the Ca²⁺-bound C2 domains of Syt1 inserted into the PM. Our simulations unraveled the sequence of Syt1 conformational transitions, including the simultaneous docking of Syt1 to the SNARE-Cpx bundle and the PM, followed by Ca²⁺ chelation and the penetration of the tips of Syt1 domains into the PM, leading to the prefusion state of the protein-lipid complex. Importantly, we found that 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 prefusion 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.