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.

Calcium-dependent exocytosis of synaptic vesicles mediates the release of neurotransmitters. Important proteins in this process have been identified such as the SNAREs, synaptotagmins, complexins, Munc18 and Munc13. Structural and functional studies have yielded a wealth of information about the physiological role of these proteins. However, it has been surprisingly difficult to arrive at a unified picture of the molecular sequence of events from vesicle docking to calcium-triggered membrane fusion. Using mainly a biochemical and biophysical perspective, we briefly survey the molecular mechanisms in an attempt to functionally integrate the key proteins into the emerging picture of the neuronal fusion machine.

SNARE [soluble NSF (N-ethylmaleimidesensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four ␣-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and RSNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.Intracellular membrane fusion involves conserved sets of membrane proteins that are commonly referred to as SNARE proteins [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor proteins] (1-4). SNARE proteins can be grouped into several small protein families with a growing number of members. The similarity between distant members of these protein families is rather limited, but it is thought that they all operate by means of a common mechanism. The variants functioning in neuronal exocytosis are among the best characterized; they include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the synaptic plasma membrane proteins SNAP-25 and syntaxin-1A. These proteins readily assemble into a stable ternary complex whose core structure has been recently solved by x-ray crystallography (5). The SNARE complex can be reversibly disassembled by the ATPase NSF in conjunction with soluble cofactors termed SNAPs (soluble NSF attachment proteins) (6, 7). The formation of the assembled complex is now believed to be a critical step leading to membrane fusion. Assembly proceeds spontaneously from less structured monomers and results in a stoichiometric and elongated complex with all membrane anchor domains located at one side of the rod-shaped particle (2,5,8,9). These findings led to a model that assembly juxtaposes membranes, thus overcoming the free energy barrier for fusion (2,5,9,10). However, it remains to be established whether more distantly related SNARE proteins form similar complexes and which of the structural features of the neuronal complex are generally relevant for SNARE protein function. METHODSSequence Analysis. Sequences were aligned by using the CLUSTALW software available at http:͞͞www2.ebi.ac.uk͞ clustalw (11). A nearest-neighbor dendrogram of the SNAP-25, syntaxin, and synaptobrevin fa...

Sec1/Munc18-like (SM) proteins functionally interact with SNARE proteins in vesicular fusion. Despite their high sequence conservation, structurally disparate binding modes for SM proteins with syntaxins have been observed. Several SM proteins appear to bind only to a short peptide present at the N terminus of syntaxin, designated the N-peptide, while Munc18a binds to a 'closed' conformation formed by the remaining portion of syntaxin 1a. Here, we show that the syntaxin 16 N-peptide binds to the SM protein Vps45, but the remainder of syntaxin 16 strongly enhances the affinity of the interaction. Likewise, the N-peptide of syntaxin 1a serves as a second binding site in the Munc18a/syntaxin 1a complex. When the syntaxin 1a N-peptide is bound to Munc18a, SNARE complex formation is blocked. Removal of the N-peptide enables binding of syntaxin 1a to its partner SNARE SNAP-25, while still bound to Munc18a. This suggests that Munc18a controls the accessibility of syntaxin 1a to its partners, a role that might be common to all SM proteins.