SNARE assembly enlightened by cryo-EM structures of a synaptobrevin–Munc18-1–syntaxin-1 complex

Abstract

Munc18-1 forms a template to organize assembly of the neuronal SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its amino-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here, we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 amino-terminal region can help nucleate interactions between the amino termini of the syntaxin-1 and synaptobrevin SNARE motifs, while their carboxyl termini bind to distal sites of Munc18-1. These observations, together with mutagenesis, SNARE complex assembly experiments, and fusion assays with reconstituted proteoliposomes, support a model whereby these interactions are critical to initiate SNARE complex assembly and multiple energy barriers enable diverse mechanisms for exquisite regulation of neurotransmitter release.

Assembly-promoting protein Munc18c stimulates SNARE-dependent membrane fusion through its SNARE-like peptide

“…SLP has multiple binding modes during SNARE zippering. In addition to the t-SNARE complex, SLP and its adjacent regions can also recognize v-SNARE, facilitating the formation of the syntaxin-Munc18-VAMP template complex (16,22,47,(70)(71)(72). It was suggested that the physiological SNARE assemblies might start from Munc18-bound syntaxins (42,44,72).…”

“…It was suggested that the physiological SNARE assemblies might start from Munc18-bound syntaxins (42,44,72). Then, Munc18 interacts with v-SNARE to form a syntaxin-Munc18-VAMP template complex, which may be universally conserved for Munc18-chaperoned SNARE assembly (16,(70)(71)(72). The template complex guides the formation of a partially zippered trans-SNARE complex, whereas Munc18 SLP turns to interact with the CTDs of the intermediate t-SNARE complex.…”

A dynamic template complex mediates Munc18-chaperoned SNARE assembly

“…The tip of the Munc18-1 3a hairpin (a.a. 314-334) contains a loop (a.a. 314-326) that is disordered in all published Munc18-1 structures (Fig. 3A) (Misura et al, 2000;Stepien et al, 2022). The function of this loop is unclear.…”

“…In a major step toward understanding Munc18-1-chaperoned SNARE assembly, Stepien et al recently reported 3.5-3.7 Å-resolution cryo-EM structures of the synaptic template complex (Figure 1A) (Stepien et al, 2022). The structure confirms many features of the structural model derived from earlier single-molecule and structural studies: the N-terminal SNARE motifs of syntaxin-1 and VAMP2 are aligned as parallel helices, while the C-terminal halves are kept separated also in helical conformations (Baker et al, 2015; Jiao et al, 2018).…”

“…In addition, the syntaxin region N-terminal to the SNARE motif, including the N-terminal peptide and the Habc three-helix bundle, stabilizes the template complex (Jiao et al, 2018). Importantly, the cryo-EM structure reveals a small four-helix bundle formed by the N-terminal regions of the syntaxin-1 and VAMP2 SNARE motifs and the syntaxin linker region (Stepien et al, 2022). The syntaxin linker region connects the Habc domain to the SNARE motif and, in the cryo-EM structure, folds into two α-helices (Fig.…”