The Ca²⁺ sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving ‘polybasic’, ‘primary’ and ‘tripartite’ interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca²⁺-free synaptotagmin-1 binds to SNARE complexes anchored on PIP₂-containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca²⁺ induces tight binding of synaptotagmin-1 to PIP₂-containing nanodiscs, releasing synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca²⁺-dependent synaptotagmin-1-PIP₂-membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca²⁺ releases this interaction, inducing PIP₂/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release.

The dataset consists of ¹H ¹⁵N TROSY HSQC and ¹H¹³ HMQC spectra acquired to investigate interactions of Synaptotagmin-1 with Complexin-1(26-83)-SNARE complex in solution.

All NMR spectra in the data set were acquired at 25°C on Agilent DD2 spectrometers operating at 600 or 800 MHz and equipped with cold probes. Detailed sample preparation is described in the methods section of Voleti et al, 2020. NMR data were processed with NMRPipe (Delaglio et al., 1995) and analyzed with NMRView (Johnson and Blevins, 1994).

The data sets are organized in main directories that correspond to the figures of Voleti et al, 2020, i.e. all the files that were used for generating Figure 1 are included in main directory “Fig1”. 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 extension .fid and their name specifies the experimental conditions (including mutations and protein concentration). Each of these directories includes files that contain the raw NMR data (fid), the acquisition parameters (procpar), a log file (log), a text file with a more detailed 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. Below is a summary of the main directories and the directories corresponding to each titration.

NMR figures list:

Fig1

TRHS_201803_SL-IM_C2B_Syt1_WT_mcc-cpx26-83_titration_EDTA

 

Fig1-S2

TRHS_201803_SL-IM_C2B_Syt1_WT_mcc-cpx26-83_titration_CaCl2

 

Fig2

TRHS_201805_SL-IM_C2B_Syt1_RRQQ_mcc-cpx26-83_titration_EDTA

TRHS_20190505_SL-IM_C2B_Syt1_RKEE_mcc-cpx26-83_titration_EDTA

TRHS_201907_SL-IM_C2B_Syt1_RKRREEQQ_mcc-cpx26-83_titration_EDTA

 

Fig2-S1

TRHS_201806_SL-IM_C2B_Syt1_R399Q_mcc-cpx26-83_titration_EDTA

TRHS_201806_SL-IM_C2B_Syt1_R398Q_mcc-cpx26-83_titration_EDTA

 

Fig3

TRHS_201805_2H15N_Cpx2683mcc_S1C2B_WT_titration_EDTA

TRHS_201904_2H15N_Cpx2683mcc_S1C2B_RKRREEQQ_titration_EDTA

 

Fig3-S1

201805_SL-IM_C2B_Syt1_LLQQ_mcc-cpx2683_titration_EDTA

 

Fig4

TRHS_201906_SL-IM_C2B_Syt1_EYAW_mcc-cpx26-83_titration_EDTA

 

Fig4-S1

HMQC_201803_SL-IM_C2B_Syt1_WT_mcc-cpx26-83_titration_EDTA

HMQC_201905_SL-IM_C2B_Syt1_RKEE_mcc-cpx26-83_titration_EDTA

HMQC_201906_SL-IM_C2B_Syt1_EYAW_mcc-cpx26-83_titration_EDTA

HMQC_201805_SL-IM_C2B_Syt1_RRQQ_mcc-cpx26-83_titration_EDTA

HMQC_201907_SL-IM_C2B_Syt1_RKRREEQQ_mcc-cpx26-83_titration_EDTA

To view the files, please download the file Voleti_Jaczynska_NMR_data.tar.gz and use the following commands:

gunzip Voleti_Jaczynska_NMR_data.tar.gz

tar xvf Voleti_Jaczynska_NMR_data.tar

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 the authors.