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Review
. 2021 Jun 20:90:581-603.
doi: 10.1146/annurev-biochem-081820-103615. Epub 2021 Apr 6.

Chaperoning SNARE Folding and Assembly

Yongli Zhang  1 , Frederick M Hughson  2
Affiliations
Review

Chaperoning SNARE Folding and Assembly

Yongli Zhang et al. Annu Rev Biochem. .

Abstract

SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.

Keywords: Munc18-1; SM proteins; SNARE assembly; membrane fusion; optical tweezers; template complex.

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Figures

Figure 1
Figure 1
Stepwise SNARE folding and assembly drives membrane fusion. (a) Schematic diagram of chaperoned SNARE assembly and membrane fusion. (b) Typical experimental setup to pull a single synaptic SNARE complex using dual-trap optical tweezers (17, 28). The cartoon of the fully assembled cytoplasmic SNARE complex incorporates the X-ray structure of the four-helix SNARE motif bundle (PDB ID: 3HD7) and the nuclear magnetic resonance structure of the syntaxin-1 Habc domain (PDB ID: 1BR0). A 2,260-base-pair DNA handle is used to attach a single SNARE complex to the surfaces of two polystyrene beads held in optical traps, whose separation is accurately controlled to change the pulling force applied to the SNARE complex. Red numbers indicate the hydrophobic layers and the ionic zero layer. (c) Energetics of uncatalyzed synaptic SNARE assembly as measured using the experimental setup in panel b. The free energies of states ❶–❹ relative to the fully assembled SNARE complex (state ❺) are shown below the state diagrams. (d) Representative time-dependent extension at constant force. The red dashed lines represent states ❸ and ❹ (as shown in panel c), and the transitions between them represent the rapid, reversible folding and unfolding of the SNARE CTD. Panel d modified [with permission] from Reference . Abbreviations: CTD, C-terminal domain; LD, juxtamembrane linker domain; Munc, mammalian homolog of unc; NTD, N-terminal domain; PDB ID, Protein Data Bank identifier; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor.
Figure 2
Figure 2
Structures of SM–Qa-SNARE complexes. (a) The SM protein Munc18-1 bound to the Qa-SNARE syntaxin-1 (PDB ID: 3C98). Syntaxin-1 adopts a closed conformation in which the SNARE motif (red) interacts with the NRD (pink). The helical hairpin (gold) of Munc18-1 (gray surface) adopts a furled conformation, hiding the R-SNARE binding site. (b) The SM protein Vps45 bound to the Qa-SNARE Tlg2 (PDB ID: 6XM1). Tlg2 adopts an open conformation, with only minimal interactions between the SNARE motif and the NRD. In addition, the linker connecting the three-helical Habc domain (pink) to the SNARE motif (red) is disordered, unlike in the Munc18-1–syntaxin-1 structure. The helical hairpin is unfurled, exposing the presumptive R-SNARE binding site. (c) A composite model combining two SM–SNARE motif crystal structures: Vps33 with the Qa-SNARE Vam3 (PDB ID: 5BUZ) and Vps33 with the R-SNARE Nyv1 (PDB ID: 5BV0). In both the Munc18-1–syntaxin-1 and Vps45–Tlg2 structures, the N-peptide-binding site is at the left, behind the SM protein; Vps33 does not possess an N-peptide-binding site. Abbreviations: NRD, N-terminal regulatory domain; PDB ID, Protein Data Bank identifier; SM, Sec1/Munc18.
Figure 3
Figure 3
Schematic diagrams of key intermediates and pathways of SNARE assembly chaperoned by Munc18-1 and the MUN domain of Munc13-1. Open syntaxin-1 can be generated by local unfolding of the syntaxin-1 linker region, through either thermal fluctuation, perturbation by mutation, or binding of Munc13-1. VAMP2 binding to the opened Munc18-1–syntaxin-1 complex forms the template complex, which in turn can be stabilized by the MUN domain of Munc13-1. SNAP-25 binding concludes SNARE assembly and displaces both chaperones from the four-helix bundle. Stars (outlined in yellow) indicate SNARE-binding sites on the MUN domain; the binding site for SNAP-25 has not been mapped and is therefore arbitrary. Abbreviation: NRD, N-terminal regulatory domain.
Figure 4
Figure 4
Model for Munc18-1- and Munc13-1-chaperoned SNARE assembly and synaptic vesicle fusion. Munc13-1 first tethers the synaptic vesicle to the plasma membrane by simultaneously binding to both membranes through its terminal C2 and C1 domains and associates with VAMP2, SNAP-25, and Munc18-1-bound syntaxin. The latter association opens closed syntaxin. The open syntaxin then binds VAMP2 to form the template complex that is stabilized by Munc13-1. SNAP-25 binds to the templated SNAREs, generating a half-zippered SNARE complex in the presence of other SNARE chaperones like synaptotagmin and complexin. Finally, calcium triggers full SNARE assembly to induce membrane fusion and neurotransmitter release. (Top middle) The domain structure of Munc13-1 inferred from the crystal structure of the Munc13-1 fragment containing the C1, C2B, and MUN domains (122), with the N-terminal C2A domain omitted.
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