Coarse Grain Simulations Reveal Movement of Synaptobrevin C Terminus in Response to Piconewton Forces Suggesting a Novel Fusion Pore Mechanism

Abstract

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C terminal transmembrane (TM) helix. In coarse grain molecular dynamics simulations self-assembly of the membrane occurred with the syb2 TM helix inserted as expected from experimental data. The free energy profile for the position of the TM domain in the membrane was determined applying harmonic potentials to the peptide in its unbiased position, pulling it towards new biased equilibrium positions. The energy profile determined in this way predicts the energy landscapes for pulling syb2 towards the extravesicular side as expected for SNARE complex zippering. Applying a constant pulling force of 160 pN detaches the synaptobrevin C terminus from the vesicle's inner leaflet lipid head groups within ∼100ns and pulls the C terminus deeper into the membrane. This C terminal movement should occur on the physiological millisecond time scale at ∼120 pN force. It is facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. These results suggest a mechanism where fusion pore formation is induced by movement of the charged syb2 C terminus into the hydrophobic core of the membrane in response to the force generated by C terminal zippering of the SNARE complex. This displacement of the charged C terminus is expected to destabilize the membrane providing a plausible pathway to fusion pore formation. Supported by NIH grants R01GM085808, R21NS072577, EDICT Project grant 201924, an MRC fellowship and the Wellcome Trust.