Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association
- Yale University School of Medicine, United States
- Princeton University, United States
- University of California, San Diego, United States
Figures
Figure 1
Two potential pathways for Munc18-1-regulated neuronal SNARE assembly.
(i) Munc18-1 first serves as a syntaxin chaperone and binds syntaxin to inhibit its association with other SNAREs. (ii) Closed syntaxin is opened by Munc13-1, a large multifunctional protein that …
Figure 2 with 7 supplements
Single-molecule manipulation based on optical tweezers revealed a ternary template complex.
(A) Experimental setup and structural model of the template complex. Some key mutations tested in this study are indicated by dots: red (phosphomimetic mutations) or gray (others) for Munc18-1, …
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Figure 2—source data 1
MATLAB figure for the FECs shown in Figure 2C.
- https://doi.org/10.7554/eLife.41771.011
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Figure 2—source data 2
Complete time-dependent instantaneous force, extension, and trap separation obtained in a representative single-molecule experiment in the presence of WT 2 µM Munc18-1.
Data here correspond to Figure 2C (FECs #2–3) and Figure 2—figure supplement 2.
- https://doi.org/10.7554/eLife.41771.012
Figure 2—figure supplement 1
Sequences, domains, and crosslinking sites of the SNARE proteins used in this study.
The amino acids in hydrophobic layers (from −7 to +8) and the central ionic layer (0 layer) are colored yellow. The underlined sequences are added to facilitate crosslinking of Qa and R SNAREs, …
Figure 2—figure supplement 2
Time-dependent extension (top panel), force (middle panel), and trap separation (bottom panel) for a typical experiment to test template complex formation (Figure 2—source data 2).
Data here and FECs #2 and #3 in Figure 2C are acquired on the same Qa-R SNARE conjugate, with the same pulling round numbering. Close-up views of different time regions indicated by A-D are shown: (A…
Figure 2—figure supplement 3
Extension-time trajectories at two constant mean forces showing the opening-closing transition of the partially closed syntaxin molecule.
https://doi.org/10.7554/eLife.41771.006
Figure 2—figure supplement 4
Force-dependent syntaxin opening probabilities (top panel) and opening and closing rates (bottom panel) obtained by pulling syntaxin from the two N-terminal sites, R198C and I187C (Figure 2—figure supplement 1).
Curves are best model fits to derive the energies and kinetics at zero force associated with the transitions, with solid curves for unfolding and dashed curves for folding. The unfolding energies of …
Figure 2—figure supplement 5
FECs of Qa only (#1) or the Qa-R SNARE conjugate (other FECs) pulled from Site I187C in the presence of 2 μM Munc18-1 without (-) or with (+) 60 nM SNAP-25B.
The wide-type (‘WT’) syntaxin-1 here denotes syntaxin-1A (a.a. 1–265, I187C, C145S; see ‘SNARE protein constructs’), with additional mutations indicated. FECs in each bracket were obtained on the …
Figure 2—figure supplement 6
Extension-time trajectories at two constant mean forces showing the opening-closing transition of the syntaxin molecule pulled from the crosslinking site I187C (Figure 2—figure supplement 1).
The red curves are idealized state transitions derived from hidden-Markov modeling. State 6' represents the fully closed syntaxin (Figure 2B).
Figure 2—figure supplement 7
Extension-time trajectories showing conformational transitions of the template complex transition pulled from Site I187C in the absence (top trace) and presence (bottom) of 60 nM SNAP-25B.
The unfolding energy of the template complex crosslinked at I187C is estimated to be 4.8 ± 0.3 kBT, close to the unfolding energy of 5.2 ± 0.1 kBT of the template complex crosslinked at R198C. …
Figure 3 with 3 supplements
Stability, conformation, and folding kinetics of the template complex.
(A, D, E, I) Extension-time trajectories at constant mean forces with the WT template complex (A) or its variants containing indicated mutations in Munc18-1 (D), VAMP2 (E), or syntaxin (I). The red …
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Figure 3—source data 1
MATLAB figure corresponding to Figure 3A with an additional trace at force F = 5.0 pN.
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Figure 3—source data 2
MATLAB figure containing expanded traces shown in Figure 3D,E,I,H.
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Figure 3—source data 3
MATLAB figure for Figure 3C.
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Figure 3—figure supplement 1
FECs obtained in the presence of 2 µM Munc18-1.
Dashed blue ovals mark template complex transitions. Note that the partially closed syntaxin state was abrogated by modifications that are known to destabilize the closed syntaxin, including …
Figure 3—figure supplement 2
Circular Dichroism (CD) spectra show that the mutations tested in our experiments do not significantly alter Munc18-1 folding.
The CD spectra of Munc18-1 mutants that abolished or weakened the template complex are shown, including Munc18-1 F-pocket mutations L247R, T248G, L247A/T248G, disease-related mutations L341P and …
Figure 3—figure supplement 3
Snapshots of the extension-time trajectories at constant mean forces showing sporadic folding of the template complex in the absence of syntaxin NRD.
https://doi.org/10.7554/eLife.41771.018
Figure 4
Stability of the template complex correlates with SNARE-mediated membrane fusion and neurotransmitter release.
(A) Unfolding energy of the WT and mutant template complexes; see also Table 1. Unfolding energy that is less than our detection limit (1.5 kBT) is plotted as zero. The unfolding energy is derived …
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Figure 4—source data 1
Data summary table for the results shown in Figure 4.
- https://doi.org/10.7554/eLife.41771.023
Figure 5
Spontaneous SNARE assembly in the absence of Munc18-1 is inefficient.
(A) Representative FECs obtained by consecutively pulling and relaxing a single Qa-R SNARE conjugate in the presence of 60 nM SNAP-25B (Figure 5—source data 1). No SNARE assembly is observed. (B) …
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Figure 5—source data 1
MATLAB figure corresponding to Figure 5A.
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Figure 5—source data 2
MATLAB figure corresponding to Figure 5B (FEC#1–4).
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Figure 5—source data 3
MATLAB figure corresponding to Figure 5C.
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Figure 6
Comparison of SNARE assembly in the absence and presence of Munc18-1.
Bars indicate probabilities of Munc18-1-independent or spontaneous SNARE assembly (blue), Munc18-1-chaperoned SNARE assembly (red), and SNARE misassembly (black). See also Figure 6—source data 1.
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Figure 6—source data 1
Data summary table for the results shown in Figure 6.
Dataset 1. This dataset contains 18 MATLAB figures corresponding to Figures 2–10, as are listed below. Each plot or curve in a MATLAB figure has its associated data embedded. The data values for all data points in a plot can be obtained using MATLAB command get(plot_handle,’xdata’) or get(plot_handle,’ydata’), where plot_handle is a unique identifier of the plot.
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Figure 7 with 2 supplements
Template complex facilitates SNARE assembly.
(A) Representative FECs obtained in the presence of 60 nM SNAP-25B and 2 µM WT Munc18-1 (#1–5 ) or Munc18-1 mutants P335A (#6) or T248G (#7). FECs #1–5 represent consecutive rounds of manipulation …
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Figure 7—source data 1
MATLAB figure corresponding to Figure 7A (FECs #1–5).
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Figure 7—source data 2
MATLAB figure corresponding to Figure 7B.
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Figure 7—figure supplement 1
FECs obtained in the presence of 2 µM Munc18-1 and 60 nM SNAP-25B in the solution.
Red arrows mark events of SNAP-25B binding and SNARE assembly. Note that syntaxin +2 layer mutation I233G/E234G/Y235G significantly weakens CTD zippering.
Figure 7—figure supplement 2
Extension-time trajectories at constant mean forces.
The trajectories exhibit reversible folding and unfolding transitions of the mutant template complexes and irreversible SNAP-25 binding (indicated by red arrows).
Figure 8
Munc18-1 does not significantly accelerate zippering between t- and v-SNAREs.
(A) FECs obtained by pulling single WT SNARE complexes in 2 µM soluble VAMP2 in the absence or presence of 2 µM Munc18-1. Magenta arrows mark binding of the VAMP2 molecules in the solution to the …
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Figure 8—source data 1
MATLAB figure corresponding to Figure 8A.
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Figure 8—source data 2
MATLAB figure corresponding to Figure 8B.
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Figure 9
Munc18-1 phosphomimetic and disease mutations altered chaperoned SNARE assembly.
(A) FECs for Munc18-1 mutations with 0 nM (#3) or 60 nM (others) SNAP-25B. See also Figure 9—source data 1. (B) Extension-time trajectories at the indicated constant mean forces, some of which (e …
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Figure 9—source data 1
MATLAB figure corresponding to Figure 9A (FECs #3–7).
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Figure 9—source data 2
MATLAB figure corresponding to Figure 9B.
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Figure 10 with 3 supplements
Munc18-3 and Vps33 catalyze SNARE assembly via template complexes.
(A) FECs of the Munc18-3 or Vps33 cognate Qa-R SNARE conjugate in the presence of the indicated protein(s). See also Figure 10—source datas 1 and 2. (B–C) Extension-time trajectories at the …
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Figure 10—source data 1
MATLAB figure corresponding to Figure 10A (FECs #1–3).
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Figure 10—source data 2
MATLAB figure corresponding to Figure 10A (FECs #4–7).
- https://doi.org/10.7554/eLife.41771.047
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Figure 10—source data 3
MATLAB figure corresponding to Figure 10B (traces a-e).
- https://doi.org/10.7554/eLife.41771.048
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Figure 10—source data 4
MATLAB figure corresponding to Figure 10C (traces g-i).
- https://doi.org/10.7554/eLife.41771.049
Figure 10—figure supplement 1
FECs obtained by pulling and relaxing a single syntaxin-4-VAMP2 conjugate (#1–4) or Vam3-Nyv1 conjugate (#5–8) in the presence of the indicated protein or proteins.
SNARE CTD transitions and template complex transitions are marked by gray and blue ovals, respectively. Gray arrows indicate SNARE unzipping. Vps33(Δ354–376) is analogous to Munc18-1 Δ324–339 and is …
Figure 10—figure supplement 2
FECs displaying Vps33-catalyzed vacuolar SNARE assembly, marked by red arrows.
Gray arrows indicate SNARE unzipping. Template complex transitions are marked by blue ovals.
Figure 10—figure supplement 3
Probabilities of vacuolar SNARE assembly per relaxation under different conditions.
The insert shows the pulling direction and the region of the Vps33 truncation (yellow).
Videos
Video 1
SNARE complex unfolding and subsequent template complex formation as inferred from single-molecule measurements.
The proposed state transitions associated with FEC #2 in Figure 2C or Figure 2—figure supplement 2 are simulated.
Video 2
Template complex facilitates SNAP-25B binding and SNARE assembly.
The extension at a constant mean force of 6.0 pN corresponding to trace b in Figure 7B and its associated state transition are simulated. For simplicity, only the right bead was simulated to move in …
Tables
Table 1
Properties of the neuronal template complex.
https://doi.org/10.7554/eLife.41771.014| SNARE or SM | Mutation or truncation | Unfolding energy (kBT) | Equilibrium force* (pN) | Folding rate (s−1) | Unfolding rate (s−1) | Partially closed syntaxin† | Template formation | SNAP-25 binding | |||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Prob. | Prob.‡ | N§ | Prob.¶ | N** | |||||||
| WT | - | 5.2 (0.1) | 5.1 (0.1) | 132 | 0.7 | 0.4 | 0.5 | 346 | 0.7 | 50 | |
| Munc18-1 | L247R | 1.6 (0.3) | 2.3 (0.1) | - | - | 0.3 | 0.3 | 99 | 0.7 | 6 | |
| T248G | 2.9 (0.2) | 3.1 (0.1) | - | - | 0 | 0.3 | 155 | 0.3 | 16 | ||
| L247A/ T248G | <1.5*** | - | - | - | 0 | 0 | 241 | - | - | ||
| S306D¶¶ | 5.8 (0.1) | 5.6 (0.1) | 184 | 0.6 | 0.4 | 0.9 | 123 | 0.9 | 53 | ||
| L307R | 4.1 (0.2) | 4.6 (0.1) | 0.07 | 0.43 | 114 | 0.58 | 19 | ||||
| S313D¶¶ | 6.1 (0.2) | 5.7 (0.1) | 568 | 1.5 | 0.4 | 1 | 162 | 0.8 | 70 | ||
| Δ324– 339††,‡‡ | <1.5*** | - | - | 0 | 0 | 105 | 0 | 0 | |||
| D326K¶¶ | 6.5 (0.2) | 5.7 (0.1) | 420 | 0.6 | 0.03 | 0.9 | 103 | 1 | 27 | ||
| P335A§§ | 6.0 (0.3) | 5.9 (0.1) | 258 | 0.5 | 0.02 | 0.7 | 155 | 0.9 | 11 | ||
| P335L§§ | 4.3 (0.1) | 4.8 (0.1) | 17 | 0.2 | 0.4 | 0.3 | 224 | 0.8 | 36 | ||
| L341P§§ | <1.5*** | - | - | 0.06 | 0.04 | 176 | 0.5 | 4 | |||
| L348R††,‡‡ | <1.5*** | - | - | 0.02 | 0.04 | 222 | 0.7 | 6 | |||
| Y473D‡‡ | 4.0 (0.1) | 4.3 (0.2) | - | - | 0 | 0.1 | 395 | 0.5 | 24 | ||
| VAMP2 | L32G/Q33G | 3.4 (0.2) | 3.9 (0.1) | 310 | 10 | 0.4 | 0.6 | 170 | 0.06 | 33 | |
| V39D | 3.8 (0.4) | 3.9 (0.2) | 90 | 2 | 0.3 | 0.1 | 175 | 0.8 | 13 | ||
| M46A | 5.2 (0.4) | 5.1 (0.2) | 130 | 0.7 | 0.3 | 0.5 | 52 | 0.8 | 13 | ||
| E62T†† | 4.1 (0.2) | 4.8 (0.2) | 107 | 5 | 0.4 | 0.5 | 104 | 0.4 | 23 | ||
| S61D/ E62T†† | 3.6 (0.2) | 4.1 (0.1) | 0.4 | 0.7 | 56 | 0.2 | 12 | ||||
| Q76A†† | 4.7 (0.2) | 4.8 (0.1) | 166 | 2 | 0.4 | 0.6 | 62 | 0.3 | 12 | ||
| F77A‡‡ | 1.5 (0.3) | 2.3 | - | - | 0.5 | 0.1 | 121 | 0.5 | 6 | ||
| A81G/A82G | 5.0 (0.3) | 4.9 (0.2) | 130 | 0.8 | 0.4 | 0.5 | 149 | 0.4 | 42 | ||
| Δ85–94 | 5.1 (0.2) | 5.0 (0.1) | 120 | 0.7 | 0.4 | 0.5 | 87 | 0.7 | 29 | ||
| Syntaxin-1 | ΔNRD††,‡‡ | <1.5*** | - | - | - | 0 | 0.08 | 105 | 0.2 | 12 | |
| ΔN- peptide††,‡‡ | 3.2 (0.2) | 4.6 (0.1) | 42 | 2 | 0.03 | 0.5 | 328 | 0.4 | 46 | ||
| ΔHabc‡‡ | <1.5*** | - | - | - | 0 | 0.06 | 140 | 0.5 | 4 | ||
| L165A/E166A (LE)¶¶ | 6.7 (0.2) | 6.1 (0.1) | 406 | 0.5 | 0.07 | 0.7 | 83 | 0.9 | 26 | ||
| LE/E76K | 6.4 (0.2) | 6.0 (0.2) | 123 | 0.2 | 0.07 | 0.9 | 81 | 0.7 | 30 | ||
| I202G/I203G | 3.0 (0.3) | 3.8 (0.1) | 240 | 12 | 0.4 | 0.5 | 177 | 0.4 | 33 | ||
| F216A | 3.7 (0.1) | 5.1 (0.1) | 82 | 2 | 0 | 0.6 | 155 | 0.9 | 32 | ||
| I230G/D231G/ R232G††† | 3.6 (0.2) | 4.3 (0.1) | - | - | 0 | 0.5 | 111 | 0.4 | 7 | ||
| I233G/E234G/ Y235G††† | 3.0 (0.2) | 4.1 (0.1) | - | - | 0 | 0.6 | 122 | 0.7 | 30 | ||
| V237G/E238G/ H239G | 5.2 (0.2) | 4.9 (0.1) | 124 | 0.7 | 0.01 | 0.3 | 182 | 0.4 | 14 | ||
| T251G/K252G | 5.2 (0.1) | 4.9 (0.1) | 126 | 0.7 | 0.5 | 0.8 | 197 | 0.7 | 47 | ||
| Δ255–264 | 5.4 (0.2) | 5.1 (0.1) | 140 | 0.6 | 0.5 | 0.5 | 134 | 0.7 | 29 | ||
| Syntaxin-1 | L165A/E166A | 6.6 (0.2) | 6.2 (0.1) | 72 | 0.1 | 0.2 | 0.9 | 85 | 0.2 | 11 | |
| Munc18-1 | D326K¶¶ | ||||||||||
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* Mean of two average forces for the unfolded and folded states when the two states are equally populated (Rebane et al., 2016). The equilibrium force of the template complex generally correlates with its unfolding energy. The number in parentheses is the standard error of the mean.
† Detected as the syntaxin- and Munc18-1-dependent transition in the force range of 10–15 pN.
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‡ Probability per relaxation or pulling measured in the absence of SNAP-25B.
§ Total number of pulling or relaxation FECs acquired, in which transitions of the template complex or syntaxin are scored, including their average equilibrium forces and extension changes.
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¶ Probability of SNAP-25B binding and SNARE assembly per relaxation upon formation of the template complex.
** Total number of relaxation FECs containing the template complex transition.
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†† Mutation that reduces membrane fusion in vitro (Parisotto et al., 2014; Shen et al., 2010; Shen et al., 2007).
‡‡ Mutation that impairs exocytosis or neurotransmitter release in vivo (Meijer et al., 2018; Munch et al., 2016; Walter et al., 2010).
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§§ Mutation associated with epilepsy (Stamberger et al., 2016).
¶¶ Mutation that enhances membrane fusion in vitro or neurotransmitter release in the cell (Genc et al., 2014; Gerber et al., 2008; Lai et al., 2017; Munch et al., 2016; Parisotto et al., 2014; Richmond et al., 2001).
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*** Unfolding energy below the detection limit of our method, estimated to be 1.5 kBT, or not available due to no, infrequent, or heterogeneous template complex transition.
††† In the observed template complex transition, the template complex frequently dwelled in the unfolded state for an unusually long time (Figure 3—figure supplement 1). Thus, the transition is no longer two-state.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (species) | BL21 Gold (DE3) competent cells | Agilent echnologies | Cat#230132 | |
| Commercial assay or kit | BirA-500: BirA biotin-protein ligase standard reaction kit | Avidity | Cat#BirA500 | |
| Chemical compound, drug | dNTP mix (10 mM) | Invitrogen | Cat#18427013 | |
| Chemical compound, drug | 2,2'-dithiodipyridine disulfide (DTDP) | Sigma-Aldrich | CAS#2127-03-9 | |
| Chemical compound, drug | Protease inhibitor cocktail tablet, EDTA free | Roche | Cat#05892791001 | |
| Peptide, recombinant protein | Catalase from bovine liver | Sigma-Aldrich | CAS#9001-05-2 | |
| Peptide, recombinant protein | Glucose Oxidase from Aspergillus niger | Sigma-Aldrich | CAS#9001-37-0 | |
| Software, algorithm | LabVIEW VIs | (Gao et al., 2012) | instrument control, data acquisition, and data analysis | |
| Software, algorithm | MATLAB scripts | (Gao et al., 2012) (Rebane et al., 2016) | data analysis | |
| Software, algorithm | Geneious | Geneious | DNA primer design | |
| Software, algorithm | GraphPad Prism7 | GraphPad Software | ||
| Other | Micro Bio- spin six columns | Bio-RAD | Cat#732–6221 | |
| Other | Ni Sepharose 6 Fast Flow | GE healthcare Lifesciences | Cat#17531801 | |
| Other | Anti-digoxigenin antibody coated polystyrene particles | Spherotech | Cat#DIGP-20–2 | 2.1 µm, called DIG beads |
| Other | Streptavidin-coated polystyrene particles | Spherotech | Cat#SVP-15–5 | 1.8 µm |
| Other | Customized glass tubing: bead dispenser tubes with 100 µm outer diameter (OD) and 25 µm inner diameter (ID) | King Precision Glass, Inc | ||
| Other | Polyethylene tubing PE50 | Becton Dickinson | Cat# 22–270835 | |
| Other | Dual optical trap setup | (Gao et al., 2012) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.41771.050
