Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex Tong Shua,b,c, Huaizhou Jina, James E. Rothmana,1, and Yongli Zhanga,1 aDepartment of Cell Biology, Yale University School of Medicine, New Haven, CT 06520; bIntegrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06511; and cDepartment of Physics, Yale University, New Haven, CT 06511 Edited by Randy Schekman, University of California, Berkeley, CA, and approved December 10, 2019 (received for review August 18, 2019) Munc13-1 is a large multifunctional protein essential for synaptic through its N- and C-terminal C2 and C1 domains (23, 26–29) and vesicle fusion and neurotransmitter release. Its dysfunction has been directly enhances SNARE assembly through the MUN domain linked to many neurological disorders. Evidence suggests that the (19, 25, 30–32). The latter activity has been recapitulated using the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate isolated MUN domain in vitro and requires its weak binding to SNARE assembly, thereby priming vesicles for fast calcium-triggered both syntaxin 1 and VAMP2 with affinities of 40 to 110 μM (19, 24, vesicle fusion. The underlying molecular mechanism, however, is 30–32). Recently, Munc13-1 was shown to cooperate with poorly understood. Recently, it was found that Munc18-1 catalyzes Munc18-1 to promote the accuracy of SNARE assembly (19). neuronal SNARE assembly through an obligate template complex Munc18-1 tightly associates with syntaxin 1 in a closed confor- intermediate containing Munc18-1 and 2 SNARE proteins—syntaxin 1 mation that inhibits syntaxin association with other SNAREs (33– and VAMP2. Here, using single-molecule force spectroscopy, we dis- 36). However, the closed syntaxin likely serves as a starting syn- covered that the MUN domain of Munc13-1 stabilizes the template taxin conformation in vivo and must be opened for SNARE as- complex by ∼2.1 kBT. The MUN-bound template complex enhances sembly (18, 25). Interestingly, with mutations that destabilize the SNAP-25 binding to the templated SNAREs and subsequent full closed conformation of syntaxin, Munc18-1 binds both syntaxin 1 SNARE assembly. Mutational studies suggest that the MUN-bound and VAMP2 to form a ternary template complex (37, 38). In the template complex is functionally important for SNARE assembly and complex, the N-terminal regions of the SNARE motifs of both neurotransmitter release. Taken together, our observations provide a SNAREs are aligned in helical conformations on the surface of BIOPHYSICS AND COMPUTATIONAL BIOLOGY potential molecular mechanism by which Munc13-1 and Munc18-1 Munc18-1, while the C-terminal regions are kept separated. The cooperatively chaperone SNARE folding and assembly, thereby reg- templated SNAREs nucleate SNAP-25B association and proper ulating synaptic vesicle fusion. SNARE assembly. Mutation experiments suggest that the stability of the template complex correlates with the rate of SNARE as- SNARE assembly | Munc13-1 | Munc18-1 | template complex | sembly or membrane fusion. Consistent with these observations, it optical tweezers has been hypothesized that Munc13-1 stabilizes the template complex (37). However, an experimental test of this hypothesis has eurotransmission relies on synaptic vesicle fusion and the been lacking. Ncorresponding release of neurotransmitters into the synaptic We investigated SNARE assembly in the presence of the junction (1, 2). Various proteins mediate and control the fusion MUN domain of both Munc13-1 and Munc18-1 using optical process with high precision (3, 4). Key proteins include 3 membrane- anchored SNARE proteins, syntaxin 1 and SNAP-25 on the plasma Significance membrane and VAMP2 (or synaptobrevin 2) on the vesicle mem- brane (5, 6), and at least 5 regulatory proteins, Munc13-1, Munc18-1, Neurons in the brain communicate with each other by release synaptotagmin, complexin, and NSF (4, 7, 8). SNARE proteins of neurotransmitters. Neurotransmitter release is mediated by consist of characteristic SNARE motifs of ∼60 amino acids, which 3 membrane-anchored SNARE proteins and various regulatory are intrinsically disordered in solution. Coupled folding and assembly proteins, including Munc13-1 and Munc18-1. SNAREs couple of the 4 SNARE motifs in the 3 SNAREs into a 4-helix bundle draw their folding and assembly to membrane fusion in a regulatory their associated membranes into proximity, inducing membrane fu- protein-dependent manner. The physiological pathway of the sion (9–11). Synaptotagmin and complexin suspend the assembly of regulated SNARE assembly, however, is unclear. We found that the membrane-bridging trans-SNARE complex midway but promote the MUN domain of Munc13-1, Munc18-1, and 2 SNAREs— its full assembly and membrane fusion when triggered by calcium syntaxin 1 and VAMP2—associate into a weak tetrameric upon the arrival of an action potential (3, 7, 12–14). After fusion, complex. The third SNARE protein, SNAP-25B, rapidly binds the NSF and its adaptor protein SNAP disassemble the fully assembled 2 SNAREs in the complex to form a ternary SNARE complex cis-SNARE complex in an ATP-dependent manner for next round likely with a displacement of the regulatory proteins. There- of SNARE assembly (15, 16). Despite decades of research, it re- fore, Munc13-1 and Munc18-1 cooperatively chaperone SNARE mains unclear how SNAREs and regulatory proteins collaborate to assembly, a process required for neurotransmitter release. drive membrane fusion. Both Munc13-1 and Munc18-1 initiate SNARE assembly and Author contributions: T.S., J.E.R., and Y.Z. designed research; T.S., H.J., and Y.Z. performed + help prime synaptic vesicles for subsequent Ca2 -triggered fusion research; T.S. analyzed data; and T.S., J.E.R., and Y.Z. wrote the paper. (17–21). Evolutionarily unrelated, Munc13-1 and Munc18-1 were The authors declare no competing interest. first identified as mammalian homologs of Unc13 and Unc18, This article is a PNAS Direct Submission. respectively, Caenorhabditis elegans mutations that cause un- Published under the PNAS license. coordinated motion (22). Munc13-1 is a large multifunctional rod- 1To whom correspondence may be addressed. Email: [email protected] or yongli. like protein containing N-terminal C2A, C1, and C2Bdomains;a [email protected]. central MUN domain; and a C-terminal C2C domain (Fig. 1A) This article contains supporting information online at https://www.pnas.org/lookup/suppl/ (23–25). Munc13-1 has been shown to promote membrane fusion doi:10.1073/pnas.1914361117/-/DCSupplemental. by 2 means: it tethers synaptic vesicles to the plasma membrane www.pnas.org/cgi/doi/10.1073/pnas.1914361117 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 ACN1128/F1131 D1358 C A C C B MUN C C __ __ + + 164567 859 1531 1735 MUN Munc18-1 __ + + __ B Syntaxin 1 VAMP2 SNAP-25B 25 NRD Dig. – 1st pull – Subsequent pull – Relax S-S #1 #2 #3 #4 #5 #6 #7 Munc18-1 Biotin 20 Streptavidin 55 5 554 NTD MUN 15 3 CTD 2 4 10 Force (pN) Force 96 DNA handle DNA handle 6 4 1 6 6 96 (500 bp) (2260 bp) 5 Extension 7 7 50 nm 9 0 MUN Extension D 9 E F 6 0.6 Unfolding 0.3 Refolding 0.4 N=76 7 N=95 0.2 N=76 0.2 Probabiltiy Probability 0.1 Munc18-1 4 5 0 0 246810 12 14 16 1234567 8910 Syntaxin 1 VAMP2 Force (pN) 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 (+)orabsence(−)of1μM 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 distri- bution 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. tweezers. We found that the MUN domain indeed stabilizes the preparation, we used the recombinant MUN domain as in previous template complex and significantly promotes SNAP-25 binding and experiments (19, 25, 30, 31). The folding and unfolding transitions of SNARE assembly.