SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex

Abstract

Munc18-1 forms a template to organize assembly of the neuronal SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its amino-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here, we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 amino-terminal region can help nucleate interactions between the amino termini of the syntaxin-1 and synaptobrevin SNARE motifs, while their carboxyl termini bind to distal sites of Munc18-1. These observations, together with mutagenesis, SNARE complex assembly experiments, and fusion assays with reconstituted proteoliposomes, support a model whereby these interactions are critical to initiate SNARE complex assembly and multiple energy barriers enable diverse mechanisms for exquisite regulation of neurotransmitter release.

Fig. 1.. Two cryo-EM structures of the template complex.

(A) Domain diagrams of syntaxin-1 and synaptobrevin, and summary of the fragments used to prepare SyxLE/Syb. N-pep, N-peptide; SNARE, SNARE motif. (B and C) 3D reconstructions of two structures of the template complex, class1 (A) and class2 (B), and corresponding ribbon diagrams fitted into the cryo-EM maps at 3.7 and 3.5 Å, respectively. (D to F) Comparison of the crystal structure of the syntaxin-1–Munc18-1 complex (SyxM18) (PDB code 3C98) (8, 34) (D) with the two cryo-EM structures of the template complex, class1 (E) and class2 (F). The surface of Munc18-1 is shown in blue, and the SNAREs are represented by ribbon diagrams with synaptobrevin (Syb) in red and syntaxin-1 in orange (N-peptide and H_abc domain), pink (linker), and yellow (SNARE motif). The domains of Munc18-1 (D1, D2, D3a, and D3b) are labeled. The helices formed by syntaxin-1 (named Ha-Hg) are indicated. The N and C termini of Syb, as well as the C terminus of SyxLE, are labeled. (G) Summary of the locations of the helices observed in SyxM18, class1, and class2. The helices are represented by cylinders, and they are shown below a domain diagram of syntaxin-1 with selected residue numbers above to indicate domain boundaries. The helix formed by the N-peptide at the very N terminus is named N-pep, and subsequent helices of class1 and class2 are named Ha to Hg. To facilitate comparisons, the same nomenclature is used for SyxM18 helices in the same or similar positions as those observed in class1 and class2, but not that there is no He helix in SyxM18.

Fig. 2.. Structural changes in Munc18-1 that lead to synaptobrevin binding and template complex formation.

(A to C) Close-up views of the area where the Munc18-1 loop unfurls to allow synaptobrevin binding in the syntaxin-1–Munc18-1 complex (SyxM18) before unfurling (A) and in class1 (B) and class2 (C), where the loop is unfurled and synaptobrevin is bound. Munc18-1 is colored in blue, synaptobrevin (Syb) in red, and syntaxin-1 in orange (N-peptide and H_abc domain), pink (linker), and yellow (SNARE motif). P335 is shown as a stick model, with carbon atoms in cyan. The positions of the furled loop and D326 in the Munc18-1–syntaxin-1 complex, as well as of synaptobrevin (Syb), P335, a nearby β-hairpin, and selected helices of Munc18-1 and syntaxin-1 are indicated.

Fig. 3.. Conformational changes in syntaxin-1 that lead to template complex formation.

(A to C) Close-up views of the region where the syntaxin-1 SNARE motif interacts with the H_abc domain and the linker in SyxM18 (A) as well as with synaptobrevin in class1 (B) and class2 (C). (D to F) Superpositions among SyxM18 (gray), class1 (green), and class2 (purple) in the same close-up views shown in (A) to (C). The structures were superimposed using the atoms of the H_abc domain. (G) Superposition of class1 and class2 in a similar close-up view but superimposing the atoms of the residues of the syntaxin-1 and synaptobrevin SNARE motifs (residues 206–219 and 37–50, respectively) that form the small four-helix bundle with helices Hd and He of the syntaxin-1 linker, showing that the small four-helix bundle has a similar structure in class1 and class2. Munc18-1 is not shown for simplicity in (A) to (G). (H to K) Close-up views of the small four-helix bundle in two different orientations. The views of (J) and (K) are rotated 90° with respect to those of (H) and (I). The N-terminal residues of the synaptobrevin SNARE motif and helix Hf formed by the SNARE motif of syntaxin-1, as well as the N- and C-terminal residues of helix He formed by the syntaxin-1 linker, are indicated. The sequence of the linker forming helix Hd could not be assigned, but we speculate that it corresponds to the same sequence that forms helix Hd in the Munc18-1–closed syntaxin-1 complex. The position of residue 166 resulting from this assumption is indicated. In (H) to (K), Munc18-1, the H_abc domain, and the N-peptide are not shown for simplicity.

Fig. 4.. Analysis of Munc18-1–SNARE interactions by mass photometry.

(A to H) Normalized histograms of mass distributions observed for samples containing the indicated concentrations of WT or mutant Munc18-1 (M18) plus WT or mutant syntaxin-1 (2–253) (Syx) (A to D), or mutant Munc18-1 plus SyxLE/Syb with or without the M183A or D184P mutations (E to H). Gaussian fits (solid lines) were used to calculate the populations of free and bound Munc18-1 and derive dissociation constants (K_D’s). Binding of Munc18-1 D326K to SyxLE M183A/Syb and SyxLE D184P/Syb was too weak to derive reliable K_D’s. (I and J) Bar diagrams illustrating the average K_D’s (table S3) obtained from six independent experiments for samples containing WT or mutant Munc18-1 plus WT or mutant syntaxin-1 (2–253) (I), or WT or mutant Munc18-1 plus SyxLE/Syb with or without the M183A or D184P mutations (J). Error bars represent SDs. Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with the Holm-Sidak test (***P < 0.001).

Fig. 5.. Effects of mutations in Munc18-1 on SNARE complex assembly rates.

(A) Diagram summarizing the assays used to monitor SNARE complex assembly in solution starting with Munc18-1 (blue) bound to the syntaxin-1 cytoplasmic region (H_abc domain, orange; linker, pink; SNARE motif, yellow) alone (Syx) or covalently linked to the Munc13-1 MUN domain (purple) (SyxMUN). Synaptobrevin (red) was labeled with a donor fluorescent probe (green dot), and SNAP-25 (green) was labeled with an acceptor probe (red dot). SNARE complex assembly was monitored by the development of FRET between the probes. (B to D) Decrease in relative donor fluorescence (normalized to the first time point) as a function of time in SNARE complex assembly reactions where synaptobrevin and SNAP-25 were added to Syx or SyxMUN free or pre-bound to WT Munc18-1 (M18) (B), to SyxMUN bound to WT or mutant Munc18-1 (C), or to Syx bound to WT or mutant Munc18-1 (as indicated) (D). (E) Diagram summarizing trans-SNARE complex assembly assays between V-liposomes containing synaptobrevin labeled with a donor fluorescent probe (green dot) and S-liposomes containing syntaxin-1 labeled with an acceptor probe (red dot) in the presence of Munc13C, a SNAP-25 mutant (SNAP-25m), and WT or mutant Munc18-1. (F) Decrease in relative donor fluorescence (normalized to the first time point) as a function of time in trans-SNARE complex assembly assays performed with WT or mutant Munc18-1 as indicated. Reactions were initiated in the presence of 100 μM EGTA, and Ca²⁺ (600 μM) was added at the time indicated by the arrow.

Fig. 6.. Effects of Munc18-1 and syntaxin-1 mutations on liposome fusion.

(A) Diagram summarizing the content mixing between T-liposomes containing syntaxin-1 and SNAP-25, which were preincubated with Munc18-1, NSF, and αSNAP, and V-liposomes containing synaptobrevin in the presence of Munc13C. V-liposomes contain trapped Cy5-strepatavidin, and T-liposomes contain trapped PhycoE-biotin. (B to D) Content mixing between V-liposomes and T-liposomes was monitored from the increase in the fluorescence signal of Cy5-streptavidin caused by FRET with PhycoE-biotin. Assays were performed with WT or mutant Munc18-1 (B), or WT or mutant syntaxin-1 and WT (C) or D326K mutant (D) Munc18-1, as indicated. Experiments were started in the presence of 100 μM EGTA, and Ca²⁺ (600 μM) was added at 300 s.

Fig. 7.. Model of SNARE complex assembly templated by Munc18-1 and the syntaxin-1 N-terminal region.

The model postulates that assembly starts with Munc18-1 bound to closed syntaxin-1 (A) (PDB code 3C98) and is initiated when the Munc18-1 loop unfurls to allow synaptobrevin binding (B) (model built manually). (C and D) Conformational changes in syntaxin-1 that are stimulated by Munc13-1 (not shown) lead to template complex configurations such as those of class1 and class2, where syntaxin-1 gradually opens and the syntaxin-1 linker nucleates interactions between the syntaxin-1 and synaptobrevin SNARE motifs. The color code is the same as in Fig. 1. (E) Binding to SNAP-25 (green) eventually leads to formation of the SNARE complex (PDB codes: H_abc domain 1BR0; SNARE four-helix bundle 1SFC). The N and C termini of the SNAREs are indicated in the relevant panels.