Munc13-1 MUN Domain and Munc18-1 Cooperatively Chaperone SNARE Assembly Through a Tetrameric Complex

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 membrane (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 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.

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. Thus, Munc13-1 and Munc18-1 cooperatively the SNARE proteins in response to the pulling force and binding of chaperone SNARE assembly. the regulatory proteins were measured by the extension change of the protein-DNA tether with subnanometer and submillisecond resolution Results (39–41). MUN Domain Stabilizes the Template Complex. We modified our We first pulled a single SNARE complex in the absence of any previous experimental setup for pulling a single SNARE complex regulatory protein. The resultant force-extension curve (FEC, Fig. (Fig. 1B) (37). As before, syntaxin 1A and VAMP2 were cross- 1C, gray curve in FEC #1) indicated that a single SNARE complex linked at their N termini (between syntaxin R198C and VAMP2 unfolded in a stepwise manner (SI Appendix,Fig.S1): reversible C- N29C) and pulled from their C termini via 2 optically trapped beads. terminal domain (CTD) transition (Fig. 1B, oval region), irreversible Because the MUN domain is ∼16 nm long (23), we introduced a N-terminal domain (NTD) unfolding (gray arrow), and irreversible second DNA handle of ∼500 bp to isolate any MUN-SNARE t-SNARE unfolding and the accompanying SNAP-25B dissociation complex from bead surfaces and thereby minimize potential non- (green arrow) (11). Relaxing the remaining SNARE proteins did not specific interactions. The MUN domain and Munc18-1, either alone reveal any refolding events, suggesting minimum interactions be- or together, were added into the solution. To facilitate protein tween syntaxin 1 and VAMP2. However, the addition of 1 μM

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1914361117 Shu et al. Downloaded by guest on September 24, 2021 Munc18-1 MUN Energetics and Kinetics of MUN-Bound Template Complex. To further F=5.0 pN 10 nm 5 s characterize the MUN-bound template complex, we measured –+ 4 SNARE folding and unfolding transitions at constant mean force F=5.0 pN (42). We first compared the SNARE transitions at 5 pN in the –+ 6 presence of the MUN domain and/or Munc18-1 (Fig. 2). With 7 the MUN domain alone, no SNARE folding was observed, F=5.0 pN consistent with the pulling result described above. In the pres- ++ 9 ence of Munc18-1 alone, we detected fast transitions between F=6.8 pN 6 the template complex and partially closed syntaxin, with an ap- ++ 9 proximately equal probability of observing each state. With both Munc18-1 and the MUN domain, the template complex domi- Fig. 2. Extension-time trajectories at indicated constant mean forces in the nated (the third trace from the top), corroborating that the μ μ absence or presence of 1 M Munc18-1 or 1 M MUN domain. The equilib- MUN domain stabilized the template complex. Higher force rium 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 tem- induced unfolding of the MUN-bound template complex. Once plate complex. The red dashed lines indicate the average extensions of the unfolded, the SNAREs generally failed to refold for an extended indicated states (Fig. 1D and SI Appendix, Fig. S1). The trajectories were period of time (up to 10 min) at constant mean force, confirming obtained at constant trap separations, with the corresponding mean forces a large energy barrier to forming the MUN-bound template calculated as the means of 2 state forces (42). complex. Nevertheless, we observed a small number of trajectories with reversible template complex transitions that occurred at an average equilibrium force of 6.8 ± 0.1 pN (n = 6; Fig. 2, Munc18-1 induced 2 reversible transitions seen in the first relaxation bottom trace). As expected, the transition was slow, with an − curve (#2, black curve) and subsequent pulling and relaxation equilibrium rate of 0.5 ± 0.3 s 1 revealed by hidden-Markov curves (#3, cyan and black curves). The first one, at 8 to 14 pN with modeling (green trace), compared with an equilibrium rate of − 2 to 3 nm extension change, was caused by folding of the partially 3.2 ± 0.7 s 1 for the template complex in the absence of the closed syntaxin (Fig. 1D, state 6), while the second one, at 3 to 6 pN MUN domain (37). Based on the mechanical work required to with 5 to 6 nm extension change, resulted from formation of the reversibly unfold the MUN-bound template complex (Data template complex of Munc18-1:syntaxin 1:VAMP2 (state 7) (37). Analysis), we estimated unfolding energy as 7.3 ± 0.4 kBT for the All these observations are consistent with previous reports MUN-bound template complex. Given the smaller unfolding ± (11, 37, 39). energy, 5.2 0.2 kBT, of the template complex in the absence of BIOPHYSICS AND COMPUTATIONAL BIOLOGY The addition of both 1 μM MUN domain and 1 μM Munc18-1 the MUN domain (37), MUN domain binding significantly sta- led to marked hysteresis in the unfolding and refolding of the bilizes the template complex. template complex. In this case, the initial unfolding of the template complex during the pulling phase (Fig. 1C, #5 and 6, cyan MUN-Bound Template Complex Promotes SNAP-25B Binding and arrows) occurred at significantly higher force than its initial SNARE Assembly. We next tested whether the MUN-bound template complex supports SNAP-25B binding and SNARE as- refolding during the relaxation phase (#4 to 6, black arrows). We sembly. To this end, we added 131 nM SNAP-25B, 1 μM measured the unfolding and refolding forces and plotted their Munc18-1, and 1 μM MUN domain in the solution. Under our unimodal distributions (Fig. 1E) and cumulative distribution experimental conditions, spontaneous SNARE assembly is functions (SI Appendix, Fig. S2). The average unfolding and inhibited by Munc18-1, as previously shown (37). To prepare the ± ± refolding forces were 8.5 0.3 pN (mean SEM throughout the MUN-bound template complex, we first relaxed the unfolded = ± = text, n 95) and 4.9 0.1 pN (n 76), respectively. The dif- syntaxin-VAMP conjugate to form the template complex (Fig. 3, ference between the unfolding force and the refolding force #1, black arrow) and then pulled it to above 5 pN to confirm measured on the same pulling and relaxation cycle ranged from 1 MUN domain binding (#2, cyan curve). Next, the MUN-bound to 9 pN (Fig. 1F). The large force hysteresis suggested a higher- template complex was held at a constant mean force to await energy barrier for unfolding and refolding of the template SNAP-25B binding (#2, red region). Finally, the SNARE com- complex in the presence of the MUN domain. In comparison, plex was relaxed and pulled again to reset to the unfolded state, both transitions in the absence of the MUN domain reached after which we repeated the SNARE assembly cycle (#3 to 5). thermal equilibrium at 5.1 ± 0.1 pN (the equilibrium force) We found that SNAP-25B quickly associated with the MUN- without discernable hysteresis during pulling and relaxation (Fig. bound template complex (Fig. 3B), as indicated by a single 8- 1C, #2 and 3) (37). Thus, the MUN domain stabilized the to 9-nm extension drop (Fig. 3 A and C, red arrows). The re- template complex. However, MUN binding to the template sultant SNARE complex exhibited the same extension and complex barely changed its extension relative to the unfolded stepwise unfolding as the fully assembled SNARE complex (Fig. syntaxin-VAMP conjugate (Fig. 1C, compare #4 to 6 to #2 to 3; 3A, see the overlapping FECs in #1 to 3 and compare the gray Fig. 2), indicating that MUN binding likely did not significantly FEC in #1 and cyan FECs in #3 and #5), suggesting that SNAP- alter the conformation of the template complex. Consequently, 25B binding led to full SNARE assembly, likely by displacing the MUN domain and Munc18-1 from the 4-helix bundle (Fig. 3B). in our assay, MUN binding was mainly inferred from the enhanced In conclusion, the MUN-bound template complex supports mechanical stability of the MUN-bound template complex. Finally, SNARE assembly. the MUN domain alone did not affect the unfolding of the ternary Next, we examined how the MUN domain and Munc18-1 may SNARE complex (Fig. 1C, compare the pulling FECs in #1 and synergistically chaperone SNARE assembly. We initiated SNARE #4), nor did it induce folding of the disordered syntaxin-VAMP assembly by relaxing the unfolded syntaxin-VAMP conjugate in conjugate (compare the relaxation FEC in #1 and the overlapping the presence of 1 μM Munc18-1 and 40 nM SNAP-25B and then pulling and relaxation FECs in #7) under our experimental con- holding it at 5 pN constant mean force, the force that permitted ditions. These observations are consistent with negligible associa- both MUN-dependent and MUN-independent SNARE assembly. tions of 1 μM MUN domain with individual syntaxin and VAMP2 After a maximum of 100 s, the conjugate was relaxed, pulled to molecules estimated from their large dissociation constants (>40 μM) confirm its folding status, and reset to the unfolded state. Finally, (19, 30, 31). In contrast, the MUN domain readily binds to and sta- the procedure was repeated to detect the next round of SNARE bilizes the template complex (Fig. 1D, state 9). assembly. The probability of SNARE assembly per round was

Shu et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 24, 2021 A + 1 μM Munc18-1 + 1 μM MUN + 131 nM SNAP-25B B – 1st pull – Subsequent pull – Relax MUN SNAP-25B Hold at constant force 9 8 #1-3 #1 #2 #3 #4 #5 25

5 5 5 20 Munc18-1

15 C a. F=7 pN SNAP-25B binding Force (pN) Force a 15 b 9 6 6 6 8

5 98 10 nm 98 b. F=5.5 pN 2 s 9 9 9 50 nm 9 0 Extension 8

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).

scored and compared for the experiments in the absence and Munc18-1 cooperatively chaperone SNARE assembly via the presence of 1 μM MUN domain. The addition of the MUN do- MUN-bound template complex intermediate. main increased the SNARE assembly probability from 0.41 to 0.71 (Fig. 4). Thus, the MUN domain significantly promotes Munc18- Conformation of the MUN-Bound Template Complex and Its Potential 1–chaperoned SNARE assembly. Physiological Relevance. To further explore the MUN-bound To demonstrate the role of the MUN-bound template com- template complex, we tested 2 modifications that are reported to perturb MUN–SNARE interactions, SNARE assembly, and plex in chaperoned SNARE assembly, we repeated the above neurotransmitter release (25, 30, 31). The first modification is experiment but at a reduced Munc18-1 concentration of 0.25 μM. the introduction of 2 alanine substitutions, N1128A and F1131A We found that the Munc18-1 concentration reduction decreased (NFAA mutation), at the center of the MUN domain (Figs. 5, the probability of SNARE assembly in the absence of the MUN Inset, and 1A) (25, 31). This modification impairs MUN domain domain to 0.18, with no significant change in the SNARE as- binding to the N-terminal linker region of syntaxin 1 between the sembly probability in the presence of the MUN domain (0.75). N-terminal regulatory domain (NRD) and the SNARE motif Taken together, our data confirm that the MUN domain and (Figs. 1B and 5). The second modification is truncation of the VAMP2 juxtamembrane linker domain (VAMP2 Δ85–96 or ΔLD) that was recently shown to bind one end of the MUN 1 domain (Fig. 1A) (30). To quantify the effects of these modifi- F=5 pN Munc18-1 cations on the MUN-bound template complex, we repeatedly Munc18-1 + MUN pulled and then relaxed the syntaxin-VAMP conjugate in the + 40 nM SNAP-25B μ μ 0.8 presence of 1 M Munc18-1 and 1 M MUN domain but in the absence of SNAP-25B. We found that both modifications dra- matically decreased the probability of MUN binding to the 0.6 template complex per relaxation (Fig. 5 and SI Appendix, Fig. 0.75 – 0.71 N=51 S3). This observation suggests that both MUN SNARE binding N=21 sites are crucial for the MUN domain to bind the template 0.4 complex. Given the reported functional significance of these binding sites, our data imply that the MUN-bound template complex is indispensable for SNARE assembly and synaptic 0.2 0.41 vesicle fusion in vivo. N=29 SNAP-25B binding probability 0.18 Discussion 0 N=28 Electrophysiological measurements and imaging by electron and 1 μM Munc18-1 0.25 μM Munc18-1 optical microscopy have demonstrated that synaptic vesicle fusion involves multiple intermediate states, including docking, 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 priming, fusion pore opening, and dilation (2, 43). How SNAREs concentrations of Munc18-1 and the MUN domain. The N value refers to the and regulatory proteins mediate these states remains a central total number of trials for SNAP-25B binding as described in the text, and the question in the field. Both Munc13-1 and Munc18-1 are involved error bar indicates the SEM. in vesicle docking and priming, but the underlying molecular

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1914361117 Shu et al. Downloaded by guest on September 24, 2021 1 16, 37, 46). Finally, further calcium-triggered SNARE zippering complete membrane fusion, leading to displacement of regulatory proteins from the SNARE 4-helix bundle. 0.8 MUN It remains unclear how Munc13-1 opens Munc18-1–bound NFAA closed syntaxin to allow SNARE assembly. Although no clear intermediates have been observed in Munc13-1–catalyzed 0.6 VAMP2 SNARE assembly starting from closed syntaxin (25, 30), it was ΔLD hypothesized that Munc13-1 induces opening of the closed syntaxin upon binding to the binary complex (31, 37, 47). To test this 0.4 0.65 hypothesis, we pulled single syntaxin along 2 directions in the N=147 presence of 1 μM Munc18-1 (48) and 1 μM MUN domain (SI Appendix, Fig. S4). We found that at this concentration, the 0.2

MUN binding probability 0.29 MUN domain alone did not significantly affect the conformation N=104 0.24 of the closed syntaxin and its unfolding transition. Therefore, the N=92 MUN domain does not appear to directly induce opening of the 0 closed syntaxin under our experimental conditions. We thus SNARE WT VAMP2 ΔLD WT WT WT NFAA propose that Munc13-1 collaborates with VAMP2 to open closed MUN syntaxin by forming the stabilized template complex. Consistent Fig. 5. Probability of MUN binding to the template complex observed per with this view, we found that the MUN-bound template complex round of pulling and relaxation for the WT and altered MUN domain or is as stable as closed syntaxin (7.3 ± 0.4 kBT vs. 7.2 ± 0.2 kBT) VAMP2. (Inset) A structural model of the MUN-bound template complex and (37). Therefore, the template complex may thermodynamically the 2 modifications tested. The probability of MUN binding was calculated as compete with closed syntaxin to allow SNARE assembly. How- the ratio of the total occurrence number of MUN-stabilized template com- ever, our data do not rule out the possibility that at a higher plexes to that of all template complexes measured in all pulling rounds. The concentration, the MUN domain binds the Munc18-1–syntaxin N value refers to the total round of pulling and relaxation, and the error bar complex to slightly change the conformation of the closed syn- indicates the SEM. taxin (31), because our assay is not sensitive to such minor conformational change (SI Appendix, Fig. S4). mechanism is unclear (1, 20, 21, 27, 44). It remains challenging to In summary, the tetrameric complex identified by us may serve study interactions between SNAREs and their regulatory pro- as a crucial intermediate to regulate SNARE assembly. Future BIOPHYSICS AND COMPUTATIONAL BIOLOGY teins, partly because these interactions are generally weak and work will examine how other regulatory proteins target the highly dynamic (19, 30–32). Using optical tweezers, we found complex and cooperatively control synaptic vesicle fusion. that the MUN domain of Munc13-1, Munc18-1, syntaxin 1, and Materials and Methods VAMP2 associate into a tetrameric complex to initiate SNARE assembly. Combined with previous studies, our finding implies Protein Constructs and Purification. The cytoplasmic domains of rat syntaxin- that the complex may participate in vesicle docking and priming. 1A (amino acids [a.a.] 1 to 265, R198C) and VAMP2 (a.a. 1 to 96, N29C), rat Munc18-1, and the MUN domain of rat Munc13-1 (a.a. 859 to 1407 and 1453 Our data also shed light on the structure of the tetrameric to 1531, with the loop region 1408 to 1452 replaced by EF residues) were complex. Using single-molecule force spectroscopy, we recently described elsewhere in detail and were purified accordingly (19, 25, 30, 31, 37). identified the template complex consisting of Munc18-1, syn- Briefly, the MUN gene was cloned into the pGEX vector encoding a GST tag taxin, and VAMP2 that acts as an essential intermediate for and a thrombin cleavage site N-terminal to the MUN sequence. The MUN Munc18-1–chaperoned SNARE assembly (37). In this template protein was expressed in BL21 Escherichia coli cells and purified using complex, Munc18-1 aligns the N-terminal regions of the SNARE glutathione-agarose beads followed by GST tag removal. Syntaxin 1A, VAMP2, motifs of syntaxin 1 and VAMP2 while keeping their C-terminal and Munc18-1 genes were cloned into the pET-SUMO vector encoding 6 his- regions separated on the Munc18-1 surface. The NRD of syntaxin tidine followed by a SUMO tag at the N termini. The full-length rat SNAP-25B stabilizes the template complex. In contrast, the N-terminal linker was cloned into pET-15b vector encoding 6 histidine tag at the N terminus. These SNAREs and Munc18-1 were expressed and purified from BL21 E. coli region of syntaxin between the NRD and the SNARE motif and the cells using Ni-NTA-agarose beads. Syntaxin-1A was biotinylated at its C- C-terminal linker domain of VAMP2 do not appear to bind terminal Avi-tag with the biotin ligase as previously described (49). Munc18-1. Our data here show that the MUN domain binds both regions to stabilize the template complex, and the MUN binding SNARE Complex Formation and Cross-Linking. Ternary SNARE complexes were does not appear to significantly alter the extension of the template prepared and cross-linked with DNA handles as was previously described (11, 37, complex. The distance between the 2 regions inferred from the 49). Briefly, ternary SNARE complexes were assembled by mixing the purified structural model of the template complex is consistent with the syntaxin-1A, SNAP-25B, and VAMP2 at molar ratio 0.8:1:1.2 and incubating at distance of the cognate binding sites on the MUN domain (Fig. 5, 4 °C overnight. Assembled SNARE complexes were purified by binding to Ni- Inset) (23, 25, 30, 37). Thus, the MUN domain binds both SNARE NTA-agarose through the His-tag on SNAP-25B. The SNARE complexes were cross-linked with DTDP (2,2′-dithiodipyridine disulfide) treated DNA handles with proteins at sites left accessible upon formation of the template a molar ratio of 50:1 in 100 mM phosphate buffer, 500 mM NaCl, pH 8.5. complex and may stabilize the template complex by clamping the templated SNAREs in a half-zippered state. This binding mode Single-Molecule Manipulation Experiments. Dual-trap optical tweezers and exposes the SNARE motifs for SNAP-25 binding and subsequent basic protocols for single-molecule experiments have been described in detail SNARE assembly. The VAMP2 linker domain contains many elsewhere (40, 41). Briefly, the 2 optical traps were formed by focusing 2 positively charged and hydrophobic residues that are shown to orthogonally polarized beams by a water-immersed 60× objective with a strongly interact with membranes (45). Thus, the MUN domain 1.2 numerical aperture (Olympus). The 2 beams were split from a single may compete with the membrane to bind the linker domain of 1,064-nm laser beam generated by a solid-state laser (Spectra-Physics). One membrane-anchored VAMP2. In addition, transmembrane binding of the 2 beams is deflected by a mirror mounted on a piezoelectrically of Munc13-1 via its terminal C1 and C2 domains (27) may affect its controlled stage that can tilt along 2 orthogonal axes (Mad City Labs), which was used to move one trap relative to the other. The outgoing laser beams association with the template complex. Synaptotagmin and com- were collimated by a second water immersion objective, split, and projected plexin, as well as SNAP-25, may bind the tetrameric complex to onto 2 position-sensitive detectors (Pacific Silicon Sensor). Bead displacements form a partially zipperedSNAREcomplextoassist vesicle priming were detected by back-focal plane interferometry. Aliquots of the 2 DNA han- and resist premature SNARE disassembly by NSF/SNAP (4, 11, 14, dles, one cross-linked with the SNARE complex and the other bound by a

Shu et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 24, 2021 streptavidin molecule, were separately bound to anti-digoxigenin antibody unfolding equilibrium constant, and ΔGs is the entropic energy to stretch the μ coated polystyrene beads of 2.1 m in diameter (Spherotech). The 2 kinds of unfolded syntaxin-VAMP conjugate to force F. At the equilibrium force F1/2 beads were injected into a microfluidic channel and trapped. The 2 beads are with Ku = 1 measured under our experimental conditions, ΔGu = F1/2 × Δx − brought close to allow a single SNARE complex to be tethered between them. ΔGs, where the extension change Δx at the equilibrium force was de- All manipulation experiments were carried out in PBS buffer supplemented with termined based on the measured state extensions at constant trap separa-

the oxygen scavenging system. All single molecules were pulled and relaxed tions, as is shown in SI Appendix, Fig. S7, in ref. 11. The entropic energy ΔGs by increasing and decreasing, respectively, the trap separation at a speed of was calculated based on the worm-like chain model for the unfolded poly- 10 nm/s or held at constant mean forces by keeping the trap separation peptide, as is shown in eq. 6 in ref. 11, with a persistence length of 0.6 nm constant (42). and a contour length of 0.365 nm per amino acid.

Data Analysis. Our methods were described in detail elsewhere (11, 42, 50). Data Availability. The MATLAB codes for our data analysis have been pub- Briefly, the extension trajectories were analyzed by 2-state hidden-Markov lished elsewhere (11, 37). modeling (HMM), which yielded the probability, extension, force, and life- time for each state (50). The unfolding energy of the MUN-bound template ACKNOWLEDGMENTS. We thank Josep Rizo for providing the plasmid for Δ complex Gu was determined based on the Boltzmann distribution under MUN domain purification and Fred Hughson for reading the manuscript. constant force, i.e., ΔGu = F × Δx − kBT × lnKu − ΔGs, where F is the force, Δx This work is supported by the NIH grants GM120193, GM131714, and is the extension increase associated with the equilibrium transition, Ku is the GM093341 to Y.Z., and DK027044 to J.E.R.

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