DOI: 10.5061/dryad.mgqnk9978
title: A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release and Studies of Synaptotagmin-1 action by all-atom molecular dynamics simulations

description: 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 arginines
 to SNARE acidic residues at region II remains after disruption of region
 I, and that a mutation that impairs spontaneous and Ca2+-triggered
 neurotransmitter release enhances binding through region I. Moreover,
 fluorescence assays show that Ca2+ does not induce dissociation of
 synaptotagmin-1 from membrane-anchored SNARE complex but causes
 reorientation of the C2B domain. Based on these results and
 electrophysiological data described in Toulme et al.
 (https://doi.org/10.1073/pnas.2409636121), we propose that upon Ca2+
 binding the Synaptotagmin-1 C2B domain reorients on the membrane and
 dissociates from the SNAREs at region I but not region II, acting remotely
 as a lever that pulls the SNARE complex and facilitates linker zippering
 or other SNARE structural changes required for fast membrane fusion.
 Abstract 2: 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.
description: <strong>MD simulations. </strong>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 performed for the times indicated in Table
 S1 using 2 fs steps, isotropic Parrinello-Rahman pressure coupling and a
 1.1 nm cutoff for non-bonding interactions. Three different groups of
 atoms were used for Nose-Hoover temperature coupling: i) protein atoms;
 ii) lipid atoms; and iii) water and KCL ions. Periodic boundary conditions
 were imposed with Particle Mesh Ewald (PME) summation for long-range
 electrostatics. <strong>NMR spectroscopy.
 </strong>All NMR spectra were acquired at 25 °C on Agilent DD2
 spectrometers equipped with cold probes operating at 600 or 800 MHz.
 Titrations of WT and mutant
 <sup>2</sup>H,<sup>15</sup>N-labeled Syt1
 C<sub>2</sub>B domain specifically
 <sup>13</sup>CH<sub>3</sub>-labeled at the Ile d1
 and Met methyl groups (referred to as
 <sup>15</sup>N-C<sub>2</sub>B for simplicity) with
 SNARE complex four-helix bundle bound to a fragment spanning residues
 26-83 of complexin-1 (referred to as CpxSC) were performed as described in
 (3). Specific <sup>13</sup>CH<sub>3</sub> labeling
 was performed for acquisition of
 <sup>1</sup>H-<sup>13</sup>C heteronuclear
 multiple quantum (HMQC) spectra with these samples, although no such
 spectra are described here. Because the R322E/K325E mutation increases the
 stability of the C<sub>2</sub>B domain and slows down H/D
 exchange, some amide groups in the b-strands are not fully protonated
 after expression in D<sub>2</sub>O and purification in buffers
 containing H<sub>2</sub>O, resulting in signal loss for the
 corresponding peaks. All newly purified C<sub>2</sub>B mutants
 bearing the R322E/K325E mutation were incubated at RT for one week or at
 37 °C for 15 hours to facilitate full exchange, but some amide groups were
 still not fully exchanged after this procedure. The titrations were
 performed in NMR buffer containing 20 mM HEPES (pH 7.4), 100 mM KCl, 1 mM
 EDTA, 1 mM TCEP, 10% D<sub>2</sub>O and protease inhibitor
 cocktail [which contained 1 mM Antipain Dihydrochloride (Thermo Fischer
 Scientific: 50488492), 20 mM Leupeptin (Gold Bio: L01025) and 0.8 mM
 Aprotinin (Gold Bio: A655100)]. A
 <sup>1</sup>H-<sup>1</sup>5N TROSY-HSQC spectrum
 was acquired first for 32 μM isolated
 <sup>15</sup>N-C<sub>2</sub>B domain and
 additional <sup>1</sup>H-<sup>15</sup>N TROSY-HSQC
 spectra were acquired after adding increasing concentrations of CpxSC to
 the sample, resulting in gradual dilution of the
 <sup>15</sup>N-C<sub>2</sub>B domain. The protein
 concentrations of each titration step for each mutant are indicated in the
 legends of Fig. 4 and S15. Soluble SNARE complex was assembled as
 described in (3), concentrated at room temperature to a concentration
 above 250 μM using a 30 kDa centrifugation filter (Amicon) and exchanged
 into NMR buffer using Zeba Spin Desalting Columns, 7K MWCO, 10 mL (Thermo
 Fisher). Complexin-1 (26–83) was also concentrated above 250 μM using a 3
 kDa centrifugation filter (Amicon) and exchanged into NMR buffer.
 SNARE-Complexin-1 (26–83) complex was preassembled with 20% excess
 Complexin-1 (26–83) before mixing with <sup>15</sup>N-C2B
 domain. Total acquisition times ranged from 3.5 to 87.5 hr, depending on
 the sensitivity of the spectra. All NMR data were processed with NMRPipe
 and analyzed with NMRViewJ. <strong>Bimane
 fluorescence quenching assay. </strong>All fluorescence emission
 scans were collected on a PTI Quantamaster 400 spectrofluorometer
 (T-format) at room temperature with slits set to 1.25 mm. For
 tryptophan-induced bimane fluorescence quenching assays, we used SNARE
 complexes anchored on nanodiscs as described (3) and labeled with bimane
 at position R59C of SNAP-25_N. The lipid composition of nanodiscs was 84%
 POPC, 15% DOPS, 1% PIP<sub>2</sub>. The experiments were
 performed in 25 mM HEPES pH 7.4 100 mM KCl 0.1 mM TCEP 2.5 mM
 MgCl<sub>2</sub>, 2 mM ATP 1 mM EGTA containing 1.5 μM BSA to
 prevent sample binding to the cuvette.  Fluorescence emission spectra
 (excitation at 380 nm) were acquired for samples containing 1 mM
 SC-59-bimane-nanodiscs alone or in the presence of 4 mM
 C<sub>2</sub>A-T285W without or with 2.0 mM
 CaCl<sub>2</sub> (1.0 mM free
 Ca<sup>2+</sup>). <strong>FRET
 assays. </strong>All fluorescence emission scans were collected on a
 PTI Quantamaster 400 spectrofluorometer (T-format) at room temperature
 with excitation at 550 nm and slits set to 1.25 mm. Each sample contained
 0.125 μM C2AB-SNARE-complex-liposomes in 25 mM HEPES pH 7.4 100 mM KCl
 buffer, 1 mM EGTA, 2.5 mM MgCl2, 2 mM ATP, 1.5 μM BSA, and 0.15 μM
 full-length Complexin-1. Mg-ATP and complexin-1 were included to hinder
 non-specific interactions. To examine the reproducibility of
 Ca<sup>2+</sup>-induced changes in FRET, spectra were acquired
 for separate samples prepared under identical conditions before and after
 addition of 2.0 mM CaCl<sub>2</sub> (1.0 mM free
 Ca<sup>2+</sup>). Because the Syt1 C<sub>2</sub>AB
 fragment and SNARE complex were fused covalently via the 37 aa linker and
 the SNARE complex is resistant to SDS, control spectra to measure the
 maximum donor fluorescence observable without FRET were acquired after
 incubation for 5 minutes at 37 oC with 0.4 µM NSF and 2 µM aSNAP (to
 disassemble the SNARE complex) in 25 mM HEPES pH 7.4 100 mM KCl buffer
 containing 1 mM EGTA, 2.5 mM MgCl<sub>2</sub>, 2 mM ATP, 1.5
 μM BSA and 1% BOG. The presence of detergent proved to be necessary to
 recover maximum signal because a subset of the
 C<sub>2</sub>AB-SNARE-complexes get reconstituted inside the
 liposomes and hence are inaccessible to the disassembly
 machinery.
 <strong>References</strong> (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) J.
 Rizo, L. Sari, K. Jaczynska, C. Rosenmund, M. M. Lin, Molecular mechanism
 underlying SNARE-mediated membrane fusion enlightened by all-atom
 molecular dynamics simulations. <em>Proc Natl Acad Sci U S
 A</em> <strong>121</strong>, e2321447121
 (2024). (3) R. Voleti, K. Jaczynska, J. Rizo,
 Ca(2+)-dependent release of Synaptotagmin-1 from the SNARE complex on
 phosphatidylinositol 4,5-bisphosphate-containing membranes.
 <em>Elife</em> <strong>9</strong>, e57154
 (2020).
description: # 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 updated Dryad dataset. The five MD simulations that were removed from the PNAS paper emphasize the need to reconsider widespread models of how synaptotagmin-1 functions and are described in a separate paper that has been submitted to FEBS Open Bio (below referred to as FEBS Open Bio paper). ## **Description of the data and file structure** The deposition has two tar files: * dryadMDSyt1action1.tar.gz contains the MD simulation data * dryadMDSyt1action1c.tar contains the bimane, NMR and FRET data To extract the files, use the following commands in Linux: `gunzip dryadMDSyt1action1.tar.gz` `tar xvf dryadMDSyt1action1.tar` `tar xvf dryadMDSyt1action1c.tar` ### **Content of dryadMDSyt1action1.tar.gz** 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 in more detail in the references associated with this deposition (see the results parts, figure legends and Tables of these references). The FEBS Open Bio paper described the following simulations: cac2absc: four trans-SNARE complexes zippered to distinct extents at the C-terminus, bridging a vesicle and a flat bilayer, and including four synaptotagmin-1 C2AB molecules bound to five Ca2+ ions each and interspersed between the SNARE complexes to investigate whether the synaptotagmin-1 C2 domains spontaneously insert into the bilayers, perturb or bridge the bilayers, and perhaps even initiate membrane fusion fusiong: similar systems as cac2absc but with all SNARE four-helix bundles almost fully assembled and with the C2B domain Ca2+-binding loops oriented to the center of the membrane-membrane interface, to further explore the possibility that the C2 domains might cooperate with the SNAREs and increase the probability of observing fusion. nosytfusion: control system analogous to fusiong but without C2AB molecules to examine the possibility that the C2AB molecules might actually hinder SNARE action in this system. sytfusion2: similar system as fusiong but with two C2AB molecules instead of four; the C2 domains were located between the two membranes with the Ca2+-binding loops oriented toward the center of the membrane-membrane interface such that one of the loops can bind to the vesicle and the other to the flat bilayer, to explore the possibility that Ca2+-binding might favor movement of lipids toward the Ca2+-binding sites to destabilize the bilayers and initiate fusion. sytfusion3: similar system as fusiong but with the four trans-SNARE complexes closer to the center of the membrane-membrane interface and with four C2AB molecules in positions in which the two C2 domains were poised to bridge the two membranes, to investigate whether , the C2 domains might act as wedges that prevent the membranes from coming closer while the SNARE complexes pull the membranes together in the center, resulting in torque forces that help to bend the membranes to initiate fusion. The PNAS paper describes the following simulation: s1action2: four almost fully assembled SNAREs complexes bridging a vesicle and a flat bilayer, and bound to one C2AB molecule each through the primary interface to investigate the interactions between C2B domain arginines and SNARE acidic residues at this interface. For each simulation, filenames have a common beginning (e.g. cac2absc for the simulation in the cac2absc 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) ### **Content of dryadMDSyt1action1c.tar** This tar file includes the following files, corresponding to data described in the PNAS paper: Fig1Bimanedata.xlsx: Excel file with the bimane fluorescence data of Fig. 1 of the PNAS paper, including the replicates used for the statistics of Fig. 1D. Fig5FRETdata.xlsx: Excel file with the FRET data of Fig. 5 of the PNAS paper, including the replicates used for the statistics of Fig. 5E,H. Fig2andS1toS3 and Fig3andS4toS9: Directories including the NMR data sets corresponding to Figs. 2, 3 and S1-S9 of the PNAS paper. Within each of these directories there is one directory for each titration included in the corresponding figure and within each titration directory there is one directory for each spectrum acquired during the titration. These directories have the extension .fid and their name specifies the synaptotagmin-1 C2B domain version uses for the experiments [wild type (WT) or mutant]. Each of these directories includes files that contain the raw NMR data in Agilent format (fid), the acquisition parameters (procpar), a log file (log), a text file with a description of experimental conditions (text), macros to convert from Agilent format to NMRPipe format (fid.com) and to process the data with NMRPipe (jrtrgood2c.com), an intermediate processing file (tr.ft), the processed NMR spectrum in NMRPipe format (tr.ft2) and the same spectrum converted to NMR view format (same name as the directory but with extension .nv). For each titration, there is also a directory called nvs that contains all the .nv files. To increase the signal to noise ratio, some of the experiments were acquired multiple times and added. For added experiments, an additional directory was created (directory name includes \_add) and those nv files were used for further analysis. The spectra included in the addition are indicated by the numbers at the end of the filename. Processed files with extension .nv or .ft2 are ready to be viewed in NMRViewJ or NMRDraw, respectively. In case of any questions, please do not hesitate to contact Josep Rizo. 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)