Dynamic Formation of the Protein-Lipid Pre-fusion State.

Synaptic vesicles (SVs) release neuronal transmitters by the fusion with the presynaptic membrane (PM), and the SV protein Synaptotagmin 1 (Syt1) serves as a Ca 2+ sensor for evoked fusion. Syt1 is thought to trigger fusion by penetrating into PM upon Ca 2+ binding, however the mechanistic detail of this process are still debated. Syt1 interacts with the SNARE complex, a coiled-coil four-helical bundle that mediates the SV-PM attachment, and the protein Complexin (Cpx) attaches to the SNARE bundle and promotes Ca 2+ -dependent fusion. We employed all-atom molecular dynamics (MD) to investigate the formation of the Syt1-SNARE-Cpx complex interacting with 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 calcium-bound C2 domains of Syt1 inserted into PM. We simulated the sequence of Syt1 conformational transitions, including Syt1 docking to the SNARE bundle and PM, as well as Ca 2+ chelation, and found that the direct Syt1-Cpx interaction is required to promote these transitions. We developed the all-atom dynamic model of the Cpx-mediated sequence of conformational transitions that lead to the formation of the pre-fusion state of the PM-Syt1-SNARE-Cpx complex. We also show that an alternative "dead-end" complex can be formed if this pathway is disrupted.