Evaluation of synaptotagmin-1 action models by all-atom molecular dynamics simulations

Neurotransmitter release is triggered in microseconds by the two C₂ domains of the Ca²⁺ sensor synaptotagmin-1 and by SNARE complexes, which form four-helix bundles that bridge the vesicle and plasma membranes. The synaptotagmin-1 C₂B domain binds to the SNARE complex via a 'primary interface', but the mechanism that couples Ca²⁺-sensing to membrane fusion is unknown. Widespread models postulate that the synaptotagmin-1 Ca²⁺-binding loops accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but these models do not seem compatible with SNARE binding through the primary interface, which orients the Ca²⁺-binding loops away from the fusion site. To test these models, we performed molecular dynamics simulations of SNARE complexes bridging a vesicle and a flat bilayer, including the synaptotagmin-1 C₂ domains in various configurations. Our data do not support the notion that insertion of the synaptotagmin-1 Ca²⁺-binding loops causes substantial membrane curvature or major perturbations of the lipid bilayers that could facilitate membrane fusion. We observed membrane bridging by the synaptotagmin-1 C₂ domains, but such bridging or the presence of the C₂ domains near the site of fusion hindered the action of the SNAREs in bringing the membranes together. These results argue against models predicting that synaptotagmin-1 triggers neurotransmitter release by inducing membrane curvature, perturbing bilayers or bridging membranes. Instead, our data support the hypothesis that binding via the primary interface keeps the synaptotagmin-1 C₂ domains away from the site of fusion, orienting them such that they trigger release through a remote action.