Data from: Cryo-EM and NMR reveal a membrane-gated Doc2B–Munc13-1 complex and a helical Doc2B–SNARE-binding interface
Abstract
Doc2s modulate neurotransmitter release through interactions with Munc13-1, SNAREs, lipids and Ca2+, which involve the Doc2 C2 domains and Munc13-interacting domain (MID). However, it is unclear how these interactions are coupled, how Doc2s bind to Munc13-1 and how they bind to the SNAREs. Here we determined the cryo-electron microscopy structure of a Doc2B-Munc13-1 complex aided by an AI-designed Munc13-1 binder, revealing how Doc2B MID binds to a Munc13-1 hydrophobic pocket essential for opening syntaxin-1 and explaining why Doc2B inhibits SNARE assembly. NMR and fluorescence spectroscopy show that Doc2B MID binds lipids and that MID-lipid and MID-Munc13-1 interactions are mutually exclusive, suggesting that membrane binding helps release the inhibition of Munc13-1. NMR shows that Doc2B binds to the SNARE complex via two helices of C2B. Based on these results and liposome fusion assays, we propose a working model whereby: 1. Doc2B MID-Munc13-1 interactions facilitate synergistic binding of both proteins to the plasma membrane, with the MID inhibiting Munc13-1. 2. Membrane binding facilitates MID-lipid interactions that transiently relieve the inhibition of Munc13-1, allowing SNARE complex assembly. 3. Doc2B binding to the SNARE complex inhibits neurotransmitter release but generates a primed state that mediates neurotransmitter release by an as yet unknown mechanism.
Rastegar C.T., Powell J., Wosztyl A., David G., Jaczynska K., Bai X.-C., Han Y., Chen Z., Rizo J. In press.
Overview
This dataset consists of the source data for the figures of the manuscript titled "Cryo-EM and NMR reveal a membrane-gated Doc2B–Munc13-1 complex and a helical Doc2B–SNARE-binding interface". The data is organized with one folder for each figure, with the folder name corresponding to the figure number. Every folder has its own README detailing the folder contents, experimental details, and how the figures were generated from the source data. To improve readability, in instances where a figure consists of data from different types of experiments, a separate README is provided for each set of experiments.
File: Dryad.tar
Folders
| Folder | Contents |
|---|---|
Figure_2 |
FRET-based binding of Doc2B to liposomes |
Figure_3 |
NMR spectra of the Doc2B IDR with either the MUN domain or with liposomes (panels A,B); FRET-monitored displacement of Doc2B from liposomes by MUN (panels D,E) |
Figure_4 |
FRET-based binding of Doc2B C2AB to liposome-anchored SNARE complex |
Figure_5 |
NMR titration of Doc2B C2AB K176Q,K179Q,K316Q,K319Q with soluble SNARE complex (panel A); FRET-based binding and competition experiments (panels C–E) |
Figure_6 |
Content mixing from liposome fusion assays |
Figure_S2 |
ITC titrations of the de novo designed Munc13-1 binder and the binder–Doc2B IDR fusion with the MUN domain |
Figure_S5 |
ITC titrations of full-length Doc2B WT and F37A variant with the MUN domain |
Figure_S6 |
FRET-based competition for Doc2B IDR binding to fluorescent liposomes (panels A–C) or fluorescent MUN domain (panels E,F) |
Figure_S7 |
Ca2+ dependence of Doc2B C2AB binding to liposome-anchored SNARE complex (in three subfolders by panel) |
Figure_S8 |
NMR titration of Doc2B C2AB K316Q,K319Q with soluble SNARE complex |
Figure_S9 |
NMR titration of the Doc2B C2A domain with soluble SNARE complex (panel A) |
Figure_S10 |
NMR titration of the Doc2B C2B K316Q,K319Q domain with soluble SNARE complex |
Figure_S11 |
FRET-based binding of Doc2B C2AB polybasic mutants to liposomes |
Figure_S12 |
NMR titration of the Doc2B C2A K176Q,K179Q domain with soluble SNARE complex |
Figure_S13 |
README only. The source data used in this figure are also used in Figure_5 and are not duplicated here |
Figure_S15 |
Sequence alignments of the two C2B domain helices |
Figure_S17 |
NMR spectra and the calculated cross-peak intensities of Doc2B C2AB K176Q,K179Q,K316Q,K319Q with soluble SNARE complex and/or Complexin |
Figure_S18 |
Lipid mixing from liposome fusion assays |
Related data depositions
- Cryo-EM movies: EMPIAR accession code EMPIAR-13857
- Cryo-EM maps: EMDB accession codes EMD-73826, EMD-73827 and EMD-78200
- Atomic coordinates: PDB accession code 37IC
- Resonance assignments: NMR data used to obtain resonance assignments (raw time-domain data, processed .nv files, and peak lists) of the Doc2B C2A and C2B K316Q,K319Q domains (Figures S9B and S14) are deposited in the Biological Magnetic Resonance Data Bank (BMRB accession codes C2A: 54032; C2B K316Q,K319Q: 54031)
Figures with no corresponding Dryad folder present either:
- Cryo-EM data deposited in the aforementioned repositories (Figures 1, S1, S3, S4)
- Structural models and schematics with no tabular source data (Figures 7, S16)
- Resonance assignments deposited in BMRB (Figure S14).
File types and data conventions
.xlsx— spreadsheets containing the values used to generate each panel. Each sheet
is described in the README in the same folder. Prepared in Microsoft Excel with curve
fitting and statistics performed in GraphPad Prism..itc— raw isothermal titration calorimetry data generated with a MicroCal
PEAQ-ITC instrument..nvand.par— NMR spectra and their associated parameter files, in NMRViewJ format..fiddirectories — raw NMR time-domain data from Varian 600 or 800 MHz spectrometers, provided
with the associated NMRPipe conversion and processing scripts used to generate the.nvfiles..ipynb— Jupyter notebooks consisting of the analysis pipeline for the Ca2+ titration
data (Figure S7), provided along with the raw instrument exports used in the analysis..aln— Clustal-format sequence alignments.
Two abbreviations pertaining to the NMR experiments recur in the file names and
sample descriptions across several folders:
- MCC is the soluble SNARE complex described in the methods section for the NMR experiments
- 1X JRR is the protease inhibitor cocktail described in the supporting information
Figure-specific abbreviations are defined in their corresponding README.
Contact
In case of any questions, please do not hesitate to contact Josep Rizo.
Name: Josep Rizo
Email: Jose.Rizo-Rey@UTSouthwestern.edu
Institution: Department of Biophysics, UT Southwestern Medical Center Dallas
Address: 6001 Forest Park Road, Dallas, TX 75390-8816