Dynamic Formation of the Protein-Lipid Pre-fusion Complex

Abstract Synaptic vesicles (SVs) fuse with the presynaptic membrane (PM) to release neuronal transmitters. 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 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 Ca 2+ -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 Ca 2+ -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 Ca 2+ 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. Statement of Significance Neurons communicate by releasing transmitter molecules. Transmitters are packed in synaptic vesicles (SVs) and released by the fusion of SVs with the presynaptic membrane (PM). This process is regulated by a dynamic complex of fusion proteins, including the coil-coiled SNARE bundle that attaches SV to PM, Synaptotagmin that serves as a Ca 2+ sensor for the release process, and Complexin that attaches to the SNARE bundle and promotes the Ca 2+ -dependent release. To understand how these proteins interact dynamically with the lipid bilayers of SV and PM, we employed molecular dynamics, a computational approach that enables simulating the behavior of proteins and lipids at the atomistic resolution. Our simulations enabled us to delineate the stages of the formation of prefusion protein-lipid complex.