All-atom molecular dynamics simulations of synaptic vesicle fusion I: a glimpse at the primed Synaptotagmin-SNARE-complexin complex

Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca2+-binding to Syt1. This state likely includes trans-SNARE complexes between the vesicle and plasma membranes that are bound to Syt1 and complexins. However, the nature of this state and the steps leading to membrane fusion are unclear, in part because of the difficulty of studying this dynamic process experimentally. To shed light into these questions, we performed all-atom molecular dynamics simulations of systems containing trans-SNARE complexes between two flat bilayers or a vesicle and a flat bilayer with or without fragments of Syt1 and/or complexin-1. Our results need to be interpreted with caution because of the limited simulation times and the absence of key components, but suggest mechanistic features that may control release and help visualize potential states of the primed Syt1-SNARE-complexin-1 complex. In particular, the simulations suggest that SNAREs alone induce formation of extended membrane-membrane contact interfaces that may fuse slowly, and that the primed state contains macromolecular assemblies of trans-SNARE complexes bound to the Syt1 C2B domain and complexin-1 in a spring-loaded configuration that prevents premature membrane merger and formation of extended interfaces but keeps the system ready for fast fusion upon Ca2+ influx.

Methods
After energy minimization, all systems were heated to 310 K over the course of a 1 ns MD simulation in the NVT ensemble and equilibrated for 1 ns in the NPT ensemble using isotropic Parrinello-Rahman pressure coupling. NPT production MD simulations were performed for the times indicated in Table 1 for each system using 2 fs steps, isotropic Parrinello-Rahman pressure coupling and a 1.1 nm cutoff for non-bonding interactions. All simulations were performed at 310 K except one simulation with the qscff system, which was performed at 325 K after a 310 K simulation. Nose-Hoover temperature coupling was used separately for three groups: i) protein atoms plus Ca2+ ions if present; ii) lipid atoms; and ii) water and KCL. Periodic boundary conditions were imposed with Particle Mesh Ewald (PME) summation for long-range electrostatics.