DOI: 10.5061/dryad.9kd51c5rc
title: Molecular mechanism underlying SNARE-mediated membrane fusion enlightened by all-atom molecular dynamics simulations

description: The SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin mediate
 neurotransmitter release by forming tight SNARE complexes that fuse
 synaptic vesicles with the plasma membranes in microseconds. Membrane
 fusion is generally explained by the action of proteins on macroscopic
 membrane properties such as curvature, elastic modulus and tension, and a
 widespread model envisions that the SNARE motifs, juxtamembrane linkers
 and C-terminal transmembrane regions of synaptobrevin and syntaxin-1 form
 continuous helices that act mechanically as semi-rigid rods, squeezing the
 membranes together as they assemble (‘zipper’) from the N- to the
 C-termini. However, the mechanism underlying fast SNARE-induced membrane
 fusion remains unknown. We have used all-atom molecular dynamics
 simulations to investigate this mechanism. Our results need to be
 interpreted with caution because of the limited number and length of the
 simulations, but they suggest a model of membrane fusion that has a
 natural physicochemical basis, emphasizes local molecular events over
 general membrane properties, and explains extensive experimental data. In
 this model, the central event that initiates fast (microsecond scale)
 membrane fusion occurs when the SNARE helices zipper into the
 juxtamembrane linkers which, together with the adjacent transmembrane
 regions, promote encounters of acyl chains from both bilayers at the polar
 interface. The resulting hydrophobic nucleus rapidly expands into
 stalk-like structures that gradually progress to form a fusion pore, aided
 by the SNARE transmembrane regions and without clearly discernible
 intermediates. The propensity of polyunsaturated lipids to participate in
 encounters that initiate fusion suggests that these lipids may be
 important for the high speed of neurotransmitter release.
description: The methodology used in system setups, equilibration and
 production MD simulations was analogous to that described previously (1).
 The vesicle generated previously (1) was adapted for each system by moving
 lipids manually to accommodate different positions of the SNARE TM regions
 or their absence. A flat square bilayer of 30.5 nm x 30.5 nm was built at
 the CHARMM-GUI website (2) (https://charmm-gui.org/) for the fusion2g system and was adapted for all other systems (also moving lipids manually to accommodate SNARE TM regions) except for the snfreet350 system, which used a smaller bilayer adapted from the qscv simulation of ref. (1). The lipid compositions of the original vesicle and flat bilayers are described in Table 1 of the manuscript, and those of each system were only slightly altered by adding or removing a few lipids as needed. The number of atoms, box dimensions and simulation temperatures of each system are also listed in Table 1 of the manuscript. All systems were solvated with explicit water molecules (TIP3P model), adding potassium and chloride ions as needed to reach a concentration of 145 mM and make the system neutral.                All systems were energy minimized using double precision, heated to the desired temperature over the course of a 1 ns MD simulation in the NVT ensemble with 1 fs steps, and equilibrated to 1 atm for 1 ns in the NPT ensemble using isotropic Parrinello-Rahman pressure coupling and 2 fs steps (3). NPT production level MD simulations were performed for the times indicated in Table 1 using 2 fs steps, isotropic Parrinello-Rahman pressure coupling and a 1.1 nm cutoff for non-bonding interactions. Nose-Hoover temperature coupling (4) was used separately for three groups: i) protein atoms; ii) lipid atoms; and iii) water and KCL. Periodic boundary conditions were imposed with Particle Mesh Ewald (PME) (5) summation for long-range electrostatics. The default mixed precision was used in all MD simulations. The speeds of the production simulations ran on Frontera at TACC were typically about 28 ns/day for a typical system of about 5.2 million atoms using 40 nodes. (1) J. Rizo, L. Sari, Y. Qi, W. Im, M. M. Lin, All-atom molecular dynamics simulations of Synaptotagmin-SNARE-complexin complexes bridging a vesicle and a flat lipid bilayer. <em>Elife</em> <strong>11</strong>, e76356 (2022). (2) S. Jo, T. Kim, V. G. Iyer, W. Im, CHARMM-GUI: a web-based graphical user interface for CHARMM. <em>J Comput Chem</em> <strong>29</strong>, 1859-1865 (2008). (3) Parrinello M, Rahman A. 1981. Polymorphic transitions in single crystals: A new molecular dynamics method. Journal of Applied Physics 52:7182–7190 (4) Hoover T. 1985. Canonical dynamics: Equilibrium phase-space distributions. Physical Review. A, General Physics 31:1695–1697. (5) Darden T, York D, Pedersen L. 1993. Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. The Journal of Chemical Physics 98:10089–10092.
description: These data sets contain files associated with all-atom molecular dynamics
 simulations of the neurotransmitter release machinery between a flat
 bilayer and a vesicle. The simulations were performed with gromacs. Each
 directory corresponds to one simulation as described briefly below and
 with more detail in the reference associated with this deposition (see the
 results part, figure legends and Table 1 of the reference). fusion2g:
 simulation of four almost completely helical trans-SNARE complexes
 bridging a vesicle and a flat bilayer and close to the center of the
 interface at 310 K. t350f2: continuation of the fusion2g simulation but at
 350 K. t350f2e: continuation of the fusion2g simulation but at 350 K after
 re-centering the system and increasing the box size. t350f4link:
 simulation of four trans-SNARE complexes bridging a vesicle and a flat
 bilayer, with the linkers zippered but no pulling force to keep them
 zippered at 350 K. pull4link: simulation of four trans-SNARE complexes
 bridging a vesicle and a flat bilayer, with the linkers zippered and a
 pulling force to keep them zippered at 350 K. snfree: simulation of a flat
 bilayer and a vesicle bridged by a splayed lipid. nscpull4l: simulation of
 a flat bilayer bridged to a vesicle by a few lipids that form a small
 hydrophobic core at the interface, generated from the 475 ns frame of the
 pullf4link simulation. For each simulation, filenames have a common
 beginning (e.g. t350f2 for the simulation in the t350f2 directory)
 followed by a few letters that indicate the nature or order of the
 simulation: nvt for temperature equilibration runs; par for pressure
 equilibration runs and mdx for production simulations, where x is a number
 that denotes the order of a particular run within the chain of
 concatenated simulations. The filename extensions follow the common
 gromacs nomenclature. Here is a list of the types of files included in the
 deposition. Filenamea.sh or Filenameb.sh: text files with the SLURM
 instructions to run the simulation. These files show which files were used
 to run the particular simulation. Filename.top: topology file that
 includes calls to the force field and to other topology files and/or
 restraint files Filename.itp: topology or restraint file Filename.ndx:
 index file Filename.mdp: file containing the molecular dynamics parameters
 Filename.log: log file for the simulation Filename.gro: file with the
 coordinates and velocities at the end of the simulation Filename.cpt:
 checkpoint file containing the final coordinates and velocities with high
 precision to continue the simulation Filaname.edr: energy file In
 addition, each simulation directory contains force field, restraint and/or
 topology directories. The filename.xtc trajectory files that contain
 coordinates and velocities of frames saved during the simulations are not
 included because of their large size, which would render the deposition
 very expensive. These files are available from Josep Rizo upon reasonable
 request. This is the contact information: Name: Josep Rizo Institution:
 Department of Biophysics, UT Southwestern Medical Center Dallas Address:
 6001 Forest Park Road, Dallas, TX 75390-8816 Email:
 [Jose.Rizo-Rey@UTSouhtwestern.edu](mailto:Jose.Rizo-Rey@UTSouhtwestern.edu)