Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex

Abstract

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 k_BT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.

Keywords: Munc13-1; Munc18-1; SNARE assembly; optical tweezers; template complex.

Fig. 1.

Optical tweezers reveal a template complex stabilized by the MUN domain. (A) Different functional domains of Munc13-1 with their borders labeled by a.a. numbers. Amino acids in 2 distinct SNARE binding sites (N1128/F1131 and D1358) are indicated. (B) Schematic diagram of the experimental setup. A single SNARE complex (Protein Data Bank [PDB] ID 1SFC) was pulled from the C termini of syntaxin 1A (red) and VAMP2 (blue) via 2 DNA handles attached to 2 optically trapped beads. The N termini of both SNARE proteins were cross-linked via a disulfide bond. Munc18-1 (gray; derived from PDB ID 3C98) and the MUN domain of Munc13-1 (yellow; PDB ID 5UE8) were added in the solution. The syntaxin 1A molecule contains the N-terminal regulatory domain (NRD). (C) Representative FECs obtained in the presence (+) or absence (−) of 1 µM MUN domain or 1 µM Munc18-1. The syntaxin–VAMP conjugate was pulled or relaxed by changing the separation between 2 optical traps at a speed of 10 nm/s. Throughout the figures, all FECs are color coded in the same fashion: gray for pulling the initial purified SNARE complex, cyan for subsequent pulls, and black for relaxations. FECs obtained from consecutive pulling/relaxation rounds (e.g., #4 to 6) are offset along the x axis and indicated by the same lines above the FECs. States associated with different FEC regions (indicated by red dashed lines if necessary) are indicated by the corresponding state numbers (see D; SI Appendix, Fig. S1; and video 1 in ref. 37). (D) Schematic diagrams of different SNARE folding and protein binding states: 4, fully unfolded SNARE motifs; 5, unfolded SNARE motifs with Munc18-1 bound; 6, partially closed syntaxin; 7, template complex; and 9, MUN-bound template complex (11, 37). Other states are depicted in SI Appendix, Fig. S1. (E) Histogram distributions of the unfolding and refolding forces of all MUN-bound template complexes. The corresponding cumulative distribution functions are shown in SI Appendix, Fig. S2. (F) Histogram distribution of the difference between the unfolding force and the refolding of the MUN-bound template complexes.

Fig. 2.

Extension-time trajectories at indicated constant mean forces in the absence or presence of 1 µM Munc18-1 or 1 µM MUN domain. The equilibrium force in the presence of the MUN domain (∼6.8 pN) is higher than that in its absence (∼5 pN), indicating that the MUN domain stabilizes the template complex. The red dashed lines indicate the average extensions of the indicated states (Fig. 1D and SI Appendix, Fig. S1). The trajectories were obtained at constant trap separations, with the corresponding mean forces calculated as the means of 2 state forces (42).

Fig. 3.

Mun-bound template complex supports efficient SNAP-25B binding and SNARE assembly. (A) FECs obtained by consecutively pulling and relaxing a single syntaxin-VAMP conjugate for 5 rounds in the presence of MUN, Munc18-1, and SNAP-25B with their concentrations indicated. During relaxation, the SNAREs were held at constant mean forces to allow SNAP-25B biding (red regions). The corresponding time-dependent extension trajectories are shown in C. Binding by the MUN domain and SNAP-25B are indicated by black arrows and red arrows, respectively. (B) Schematic diagram of SNARE assembly mediated by the MUN-bound template complex. (C) Extension-time trajectories at indicated constant mean forces showing SNAP-25B binding to the MUN-bound template complex. The red dashed lines indicate the average extensions of the corresponding states labeled with their state numbers in red (Fig. 1D).

Fig. 4.

Probability of chaperoned SNARE assembly observed within 100 s at 5 pN constant mean force in the presence of 40 nM SNAP-25B and different concentrations of Munc18-1 and the MUN domain. The N value refers to the total number of trials for SNAP-25B binding as described in the text, and the error bar indicates the SEM.

Fig. 5.

Probability of MUN binding to the template complex observed per round of pulling and relaxation for the WT and altered MUN domain or VAMP2. (Inset) A structural model of the MUN-bound template complex and the 2 modifications tested. The probability of MUN binding was calculated as the ratio of the total occurrence number of MUN-stabilized template complexes to that of all template complexes measured in all pulling rounds. The N value refers to the total round of pulling and relaxation, and the error bar indicates the SEM.