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Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming

Abstract

UNC-13-Munc13s have a central function in synaptic-vesicle priming through their MUN domains. However, it is unclear whether this function arises from the ability of the MUN domain to mediate the transition from the Munc18-1–closed syntaxin-1 complex to the SNARE complex in vitro. The crystal structure of the rat Munc13-1 MUN domain now reveals an elongated, arch-shaped architecture formed by α-helical bundles, with a highly conserved hydrophobic pocket in the middle. Mutation of two residues (NF) in this pocket abolishes the stimulation caused by the Munc13-1 MUN domain on SNARE-complex assembly and on SNARE-dependent proteoliposome fusion in vitro. Moreover, the same mutation in UNC-13 abrogates synaptic-vesicle priming in Caenorhabditis elegans neuromuscular junctions. These results support the notion that orchestration of syntaxin-1 opening and SNARE-complex assembly underlies the central role of UNC-13-Munc13s in synaptic-vesicle priming.

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Figure 1: Domain organization of UNC-13-Munc13s.
Figure 2: Crystal structure of the Munc13-1 MUN domain.
Figure 3: A conserved hydrophobic pocket that mediates the stimulatory activity of MUN933 in the transition from the Munc18-1–syntaxin-1 complex to the SNARE complex.
Figure 4: The Munc13-1 NFAA mutation abolishes SNARE-mediated liposome fusion.
Figure 5: The UNC-13-Munc13 NFAA mutation abolishes synaptic-vesicle priming and exocytosis.
Figure 6: Working model for the mechanism underlying the activity of the MUN domain.

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Acknowledgements

We thank the BL19ID station at the Advanced Photon Source, Argonne National Laboratory, for helping with data collection. We thank J. Kaplan (Massachusetts General Hospital) for providing the unc-13(s69) strain and the Caenorhabditis Genetic Center for providing other strains used in this work. We thank Y. Li and X. Wang of the Huazhong University of Science and Technology (HUST) for initial efforts in constructing MUN-domain mutations and M. Zhang (Hong Kong University of Science and Technology) for insightful comments on the manuscript. This work was supported by grant 31370819 from the National Science Foundation of China and grant 2014CB910203 from the National Key Basic Research Program of China (both to C.M.), grants (31130065 and 91313301) from the National Science Foundation of China (both to T.X.) and grant NS37200 from the US National Institutes of Health (to J.R.).

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Contributions

C.M., M.Z., L.W. and R.Z. performed the structural-biology experiments; X.Y. and S.W. generated all mutants used in this study and performed in vitro experiments of SNARE-complex assembly and liposome fusion; Y.S., W.Z. and L.K. performed in vivo electrophysiology experiments; T.X. and C.M. conceived the experiments; and J.R., T.X. and C.M. wrote the paper.

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Correspondence to Tao Xu or Cong Ma.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Surface electrostatic potentials of MUN933 and hydrophobic region at the interface between the N- and C-terminal regions.

(a) The electrostatic potential calculated with the APBS tool and displayed by Pymol (www.pymol.org). The potential was scaled from -5kT/e to 5kT/e, with red and blue denoting negative and positive potential, respectively. A negative patch at one side of MUN933 subdomains A-B surface (see box 1) might be important for its weak binding with Munc18-1, or intramolecular binding with the N-terminal domains of Munc13-1, which are suggested to modulate the priming activity and synaptic plasticity, whereas a positive patch at one side of subdomains C-D interface (see box 2) suggests a possible interaction site for binding to membranes. (b) A hydrophobic patch at the midpoint of the structure between subdomains B and C (indicated by red arrow). Solvent accessible hydrophobic residues highlighted in orange, except for the isolated ones that do not have adjacent hydrophobic residues.

Supplementary Figure 2 Folding properties of MUN933, its fragments and the NF mutant, monitored by CD and gel filtration.

(a) CD spectra of samples of MUN933, MUN933 NFAA, MUN-AB, MUN-BC, MUN-ABC and MUN-BCD acquired from 200 nm to 250 nm on CD spectrometer. Red, magenta, green, orange, blue and cyan traces correspond to MUN-BC, MUN-AB, MUN-BCD, MUN-ABC, MUN933 and MUN933 NFAA respectively. (b) Unfolding curves of MUN933 (WT) and MUN933 NFAA (NFAA) from room temperature (25°C) to 95°C recording from the CD signal at 222 nm. Data were normalized to calculate the fraction of unfolded protein (from 0 to 1) using Origin 8. (c) Gel filtration profiles on Superdex-200 of MUN933 (WT) and MUN933 NFAA (NFAA).

Supplementary Figure 3 Screening of residues that are key for MUN933 activity by a new native gel assay.

(a) Ribbon diagrams showing the residues screened in SNARE complex assembly assays. Residues screened are labeled in red in the MUN933 structure. All residues were selected on the surface of the structure and mutated to Ala, and their folding properties were similar to MUN933. (b) Native gel assays detecting the activity of MUN933 in the transition from the Munc18-1–syntaxin-12–253 complex (M18–Syx) to the SNARE complex. The M18–Syx and the SNARE complex show strong and clear bands in native gel (indicated by asterisks). In the presence of MUN933, the band of M18–Syx is weakened, whereas the band of SNARE complex is intensified, showing that our native gel assays can detect the activity of MUN933 in the M18–Syx to SNARE complex transition, and can be used for screening key residues responsible for this activity. (c) Native gel experiments showing the effects of the mutations (see a) in the screened residues on MUN933 activity. Bands of M18–Syx were displayed below each bar respectively. Bands were scanned using Image J for gray level. The intensity in the negative control (i.e. without MUN933) was used for normalization to 0 and the intensity upon addition of MUN933 (i.e. WT in the figure) was used for normalization to 100% activity. Mutations of N1128A and F1131A strikingly impair the activity of MUN933 in "opening" the M18–Syx and stimulating SNARE complex assembly. (data represent mean values, ± s.d.; n = 3 technical replicates).

Supplementary Figure 4 The UNC-13-Munc13 NF sequence is vital for the locomotion behavior of C. elegans.

The locomotion trajectories (a) and average speed (b) are shown for the indicated genotypes. Compared to UNC-13S rescue, UNC-13S NFAA failed to rescue the locomotion rate defect of unc-13(s69). Scale bar: 1 mm. Values that differ significantly from controls are indicated (***p < 0.001, n = 10 for each genotype indicated). Error bars represent s.e.m.

Supplementary Figure 5 The NF sequence mediates the binding between the MUN domain and the Munc18-1–syntaxin-1 liposomes, as detected by liposome coflotation experiments.

Liposomes were prepared as indicated in Online Methods. Proteins loaded before co-flotation are indicated in the left panel, proteins floated with liposomes after co-flotation are indicated in the right panel. The MUN domain bound to liposomes containing reconstituted Munc18-1–syntaxin-1 complex but not liposomes containing isolated syntaxin-1 or syntaxin-1–SNAP-25 complex. The NFAA mutation disrupted binding of the MUN domain to the Munc18-1–syntaxin-1 liposomes. MUN933 or MUN NFAA bound to the Munc18-1–syntaxin-1 liposomes are indicated by asterisk.

Supplementary Figure 6 Key residues responsible for forming the NF pocket of UNC-13-Munc13s are not shared by other tethering proteins.

Structure-based sequence alignments were performed by PROMALS3D [Pei, J., Kim, B.H., Grishin, N.V. PROMALS3D: a tool for multiple sequence and structure alignment. Nucleic Acids Res. 36, 2295–2300 (2008)] and ESPript [Gouet, P., Robert, X., & Courcelle, E. ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res. 31, 3320–3323 (2003)]. The NF sequence is colored in red, similar residues (consensus level >70%) are bold and shaded in yellow box. Residues N-1128 and F-1131 colored in red are not conserved in other tethering proteins (Sec6p, Exo70 and Tip20). Conserved hydrophobic residues in the "pocket" are in cyan box. Secondary structure information (derived by MUN933) is indicated on the top of the sequences.

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Yang, X., Wang, S., Sheng, Y. et al. Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming. Nat Struct Mol Biol 22, 547–554 (2015). https://doi.org/10.1038/nsmb.3038

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