Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution

Abstract

The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. Here we report the X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B. The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins. Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

“…Surprisingly, the phosphorylation sites in VAMP8 that we identified face the 3 other α‐helices of the SNARE complex (Fig 2A) (Fasshauer et al , 1998; Sutton et al , 1998; Diao et al , 2015) as part of the 16 conserved SNARE layers (Fasshauer et al , 1998) that are buried inside the SNARE complex (T47 at layer +3, S54 at layer +5, and T53 close to layer +5) (Figs 2B–D and EV2C). An additional site was listed as a putative phosphorylation site in the PhosphoSitePlus (Hornbeck et al , 2015) database (S61 at layer +7).…”

“…The soluble part of the SNARE complex consists of four α‐helices (SNARE domains) (Fasshauer et al , 1998; Sutton et al , 1998; Südhof & Rothman, 2009). Surprisingly, the phosphorylation sites in VAMP8 that we identified face the 3 other α‐helices of the SNARE complex (Fig 2A) (Fasshauer et al , 1998; Sutton et al , 1998; Diao et al , 2015) as part of the 16 conserved SNARE layers (Fasshauer et al , 1998) that are buried inside the SNARE complex (T47 at layer +3, S54 at layer +5, and T53 close to layer +5) (Figs 2B–D and EV2C).…”

“…While many SNARE‐associated proteins are known to influence vesicle fusion (Jahn & Fasshauer, 2012), less is known about posttranslational regulation of SNARE proteins in general and in particular about phosphorylation of SNARE domains which form α‐helices in the SNARE complex (Sutton et al , 1998). One phosphorylation site in the SNARE domain of SNAP25, S187, has been widely studied (Shimazaki et al , 1996).…”

“…One phosphorylation site in the SNARE domain of SNAP25, S187, has been widely studied (Shimazaki et al , 1996). Phosphorylation of S187 increases secretion (Shu et al , 2008) and the crystal structure of the SNARE complex reveals that S187 forms part of the outside surface area of the α‐helical bundle (Fasshauer et al , 1998; Sutton et al , 1998; Nagy et al , 2002). Additional SNARE domain phosphorylation sites have been reported: SNAP23 (S23, T24, and S160) (Polgár et al , 2003) and VAMP2 (T35 and S61) (Hirling & Scheller, 1996; Li et al , 2009), but their involvement in secretion has not been well defined.…”

Homozygous mutations in VAMP1 cause a presynaptic congenital myasthenic syndrome

“…Among these 6 variants, a homozygous missense mutation in VAMP1 (NM_014231: c.146G>C; p.Arg49Pro; see Fig 1D) emerged as the most likely explanation for the disease pathogenesis, as supported by protein function (the mutation affects a conserved amino acid within the active domain of the protein),14, 15 expression and role of this gene in the NMJ,12, 16 and the homozygous mutation identified in the patients from Family 1 presenting the same phenotype (see Fig 1C–G).…”