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 orients the Ca²⁺-binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C₂ domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C₂B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca²⁺ causes reorientation of the C₂B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and electrophysiological data described in the accompanying paper, we propose that upon Ca²⁺ binding Synaptotagmin-1 reorients and acts as a lever that pulls the SNARE complex, facilitating linker zippering or other SNARE structural changes that lead to fast membrane fusion.synaptotagmin, SNAREs, neurotransmitter release, membrane fusion, molecular dynamics simulation, synaptic vesicle fusion

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 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.

NMR spectroscopy. 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 ²H,¹⁵N-labeled Syt1 C₂B domain specifically ¹³CH₃-labeled at the Ile d1 and Met methyl groups (referred to as ¹⁵N-C₂B 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 ¹³CH₃ labeling was performed for acquisition of ¹H-¹³C 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 C₂B domain and slows down H/D exchange, some amide groups in the b-strands are not fully protonated after expression in D₂O and purification in buffers containing H₂O, resulting in signal loss for the corresponding peaks. All newly purified C₂B 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% D₂O 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 ¹H-¹5N TROSY-HSQC spectrum was acquired first for 32 μM isolated ¹⁵N-C₂B domain and additional ¹H-¹⁵N TROSY-HSQC spectra were acquired after adding increasing concentrations of CpxSC to the sample, resulting in gradual dilution of the ¹⁵N-C₂B 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 ¹⁵N-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.

Bimane fluorescence quenching assay. 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% PIP₂. The experiments were performed in 25 mM HEPES pH 7.4 100 mM KCl 0.1 mM TCEP 2.5 mM MgCl₂, 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 C₂A-T285W without or with 2.0 mM CaCl₂ (1.0 mM free Ca²⁺).

FRET assays. 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 Ca²⁺-induced changes in FRET, spectra were acquired for separate samples prepared under identical conditions before and after addition of 2.0 mM CaCl₂ (1.0 mM free Ca²⁺). Because the Syt1 C₂AB 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 MgCl₂, 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 C₂AB-SNARE-complexes get reconstituted inside the liposomes and hence are inaccessible to the disassembly machinery.

References

(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. Elife 11, 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. Proc Natl Acad Sci U S A 121, 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. Elife 9, e57154 (2020).