A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release and Studies of Synaptotagmin-1 action by all-atom molecular dynamics simulations

Abstract 1:
Neurotransmitter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2-domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a ‘primary interface’ comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding through the primary interface orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. To clarify this paradox, we have used NMR and fluorescence spectroscopy. NMR experiments reveal that binding of C2B domain ar..., MD simulations. All-atom MD simulations were performed using Gromacs with the CHARMM36 force field. Solvation and ion addition for system setup were performed at the BioHPC supercomputing facility of UT Southwestern. Minimizations, equilibration steps and production molecular dynamics (MD) simulations were carried out on Frontera at the Texas Advanced Computing Center (TACC). Pymol (Schrödinger, LLC) was used for system design, manual manipulation and system visualization. The methodology used to set up the systems and run MD simulations was analogous to that described previously (1, 2). Systems were energy minimized using double precision, whereas the default mixed precision was used in all MD simulations. The systems were heated to the desired temperature running a 1 ns simulation in the NVT ensemble with 1 fs steps, and then equilibrated to 1 atm for 1 ns in the NPT ensemble with isotropic Parrinello-Rahman pressure coupling and 2 fs steps. NPT production MD simulations were performe..., , # A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release

[https://doi.org/10.5061/dryad.mgqnk9978](https://doi.org/10.5061/dryad.mgqnk9978)

Change log: This deposition contains files corresponding to molecular dynamics (MD) simulations, bimane fluorescence quenching assays, nuclear magnetic resonance (NMR) spectra and fluorescence resonance energy transfer (FRET) experiments described in a paper that was submitted to Proceedings of the National Academy of Sciences USA (PNAS). During peer review of this paper, we decided to remove five of the MD simulations from the PNAS submission because they convey a separate, complementary message from the main message of the paper (the lever hypothesis of synaptotagmin-1 action and the data supporting this hypothesis). This paper has been accepted for publication and is referred to below as the PNAS paper. The final version of the paper describes additional bimane fluorescence and FRET experiments that are included in this updat...