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The SNAREs syntaxin (yellow and orange in the figures), SNAP-25 (green) and synaptobrevin (red) play a crucial role in neurotransmitter release by forming a tight four helix bundle through their SNARE motifs [1, 2] (on the right in the image above). The SNARE complex brings the synaptic vesicle and plasma membranes together, which is key for membrane fusion [3, 4], and is disassembled after membrane fusion by NSF and alphaSNAP (no relation to SNAP-25) [5]. A widespread textbook model assumes that assembly of the SNARE complex involves formation of a syntaxin-SNAP-25 heterodimer on the plasma membrane that binds to synaptobrevin. Our research showed that this non-regulated assembly pathway is not productive because alphaSNAP binds to syntaxin-SNAP-25 heterodimers, preventing synaptobrevin binding, and to trans-SNARE complexes, preventing membrane fusion [6]. The resulting ‘dead-end’ alphaSNAP/SNARE complexes are disassembled by NSF [5, 7-9]. Thus, while many studies reported that the SNAREs and synaptotagmin are sufficient to induce efficient fusion between reconstituted proteoliposomes, such fusion is abolished by NSF-alphaSNAP [7]. We further demonstrated that, in the presence of NSF-alphaSNAP, liposome fusion strictly requires Munc18 and Munc13 (panel A below) because these two proteins orchestrate SNARE complex assembly via a pathway that is resistant to NSF-alphaSNAP [6-9]. These assays reconstituted synaptic vesicle fusion with the most central components of the release machinery and provided a compelling explanation for the total abrogation of neurotransmitter release observed in the absence of Munc18 or Munc13 [10-12].

Initial key findings that led us to delineate this pathway were : i) the identification of an N-terminal domain in syntaxin that forms a three-helix bundle (the Habc domain, orange) [13]; and ii) the discovery that the Habc domain binds intramolecularly to the SNARE motif to form a self-inhibited ‘closed’ conformation that binds tightly to Munc18-1 [14], as shown also by the crystal structure solved in the lab of Bill Weis [15] (on the left in the image above, with Munc18 in violet). Subsequent crystal structures of the yeast vacuolar Munc18 homologue Vps33 bound to yeast homologues of syntaxin and synaptobrevin (in the center of the image above), which were solved in the lab of Fred Hughson, showed that these interactions place the two SNARES close to each other, leading to a model whereby Munc18 and its homologues form a template for SNARE complex assembly [16]. We and others provided strong evidence that neuronal Munc18 also binds to synaptobrevin and forms such a template [16-18]. Thus, Munc18 hinders SNARE complex assembly by binding to closed syntaxin [19] but facilitates assembly in downstream events (panel B).

In parallel research, we identified a large domain in Munc13 called the MUN domain [20]  and showed that this domain facilitates opening of syntaxin, accelerating the transition from the syntaxin-Munc18 complex to the SNARE complex [19, 21, 22]. Moreover, we found that Munc13 also plays a key role in facilitating SNARE complex assembly by bridging the vesicle and plasma membranes [8, 23] through its highly elongated structure [24] (panel B, with Munc13 in cyan; see page on plasticity for further details).

The physiological relevance of this mechanism was supported my multiple evidence, including the observation that a so-called LE mutation that we designed to open syntaxin [14] leads to an increase in neurotransmitter release probability [25] and partially rescues neurotransmitter release in the absence of Unc13, the invertebrate homologue of Munc13, in C. elegans [26]. In fact, the LE mutation rescues defects in neurotransmitter release observed in a wide variety of genetic backgrounds [27], showing the critical functional importance of opening syntaxin. We also showed that the furled conformation of a Munc18-1 loop hinders binding to synaptobrevin and that a mutation that unfurls the loop leads to a strong gain of function in Munc18-1 [18]. Moreover, another mutation that results in a similar gain of function also leads to a partial rescue of release in Unc13 nulls in C. elegans [28].


All these findings show that SNARE complex formation is hindered by multiple energy barriers that render neurotransmitter release highly dependent on Munc13, which acts as a master regulator of release and mediates multiple forms of presynaptic plasticity (see page on plasticity for more details). Thus, we established that assembly of the SNARE complex through the Munc18-Munc13 pathway is critical for the exquisite regulation of release, but fundamental questions remain about the molecular mechanisms underlying how Munc13 opens syntaxin to help forming the template complex of Munc18 with synaptobrevin and syntaxin, and how this template complex transits to the SNARE complex. We are addressing these questions using a combination of cryo-EM, X-ray crystallography, NMR spectroscopy, FRET and reconstitution assays. 



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