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 kBT. 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.

Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

“…In this context, a recent report suggested that interactions of the Munc13‐1 MUN domain with the juxtamembrane region of synaptobrevin contribute to its activity in stimulating SNARE complex assembly and described a crystal structure of a peptide corresponding to this region bound to the MUN domain . The functional importance of this interaction was supported by mutagenesis, and the synaptobrevin juxtamembrane region was reported to contribute to the binding of the MUN domain to the syntaxin‐1‐Munc18‐1‐synaptobrevin template . However, as explained above, this region of synaptobrevin is known to bind also to Munc18‐1 and to membranes, and its high promiscuity likely arises because of the presence of three aromatic and multiple basic residues in its sequence.…”

“…Such recruitment is expected to be facilitated by binding of the Munc13‐1 C 2 C domain to the vesicle membrane and requires unfurling of the Munc18‐1 loop that covers the synaptobrevin binding site . Since the Munc13‐1 MUN domain binds weakly to Munc18‐1, and also binds to the template complex formed by Munc18‐1, syntaxin‐1, and synaptobrevin, an intriguing possibility is that the MUN domain helps to unfurl the Munc18‐1 loop. Once the template complex is formed, SNAP‐25 needs to be incorporated and the interactions of the synaptobrevin and syntaxin‐1 SNARE motifs with Munc18‐1 need to be released to form the SNARE four‐helix bundle.…”

Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly

“…This activator function is attributed to Munc18-1's ability to bind and sequester partially-assembled SNARE complexes, termed SNAREpins (Shen et al 2007). Indeed, recent structural and biophysical studies suggest that Munc18-1 likely serves as a template for the formation of an early SNARE assembly intermediate (termed a 'template complex') wherein Syntaxin-1 and VAMP2 are properly oriented and aligned for nucleation into a four helix bundle (Baker et al 2015;Shu et al 2020;Jiao et al 2018;Sitarska et al 2017). Thus, the dual-binding modes of Munc18-1 to monomeric Syntaxin-1 and SNARE complexes allow it to both negatively-regulate and positively-assist SNARE complex assembly.…”

“…This catalytic function has been mapped to the large central module within Munc13 called the MUN domain (Yang et al 2015;Basu et al 2005), but the precise mechanism is not known. Independent of Munc18-1, Munc13-1 has been shown to promote the proper alignment of Syntaxin-1 and VAMP2 (Lai et al 2017) thereby accelerating and stabilizing the Munc18/Syntaxin-1/VAMP2 template complex (Shu et al 2020). The mechanism involved was recently clarified with the findings that Munc13-1 independently binds both VAMP2 (Wang et al 2019) and Syntaxin-1 (Wang et al 2017;Ma et al 2011) at distinct sites of its MUN domain potentially enabling it to simultaneously interact with and orient these two SNAREs in molecular proximity to Munc18-1.…”

“…Recently, a single-molecule force spectroscopy study revealed that the Munc13-1 MUN domain enhances the probability of SNAP25 binding to the Syntaxin-1/VAMP2/Munc18 template complex and subsequently complete assembly of the ternary SNARE complex (Shu et al 2020). This suggested that Munc13-1 may play a role in recruiting SNAP25 to initiate SNARE complex formation.…”