Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

Abstract

SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors ␣-, ␤-, and ␥-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant ␣-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in ␣-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.Neurons release their neurotransmitters by the Ca 2ϩ -dependent exocytosis of synaptic vesicles. In recent years, several membrane proteins have been identified which are required for exocytotic membrane fusion. These proteins include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) 1 and the synaptic membrane proteins syntaxin and SNAP-25, collectively referred to as SNAREs. Synaptobrevin and syntaxin both contain a single transmembrane domain at the C terminus (1, 2). SNAP-25 does not contain a transmembrane domain but carries palmitoyl side chains attached to cysteine residues in the middle of the sequence (3, 4). Homologues of these proteins have been identified in many eukaryotic cells including yeast, suggesting that fusion of trafficking vesicles with their respective target membranes is mediated by a conserved mechanism (2,(5)(6)(7)(8).While the evidence linking synaptobrevin, SNAP-25, and syntaxin to exocytosis is compelling, their precise role is unknown. In detergent extracts of brain membranes, the three proteins form a tight complex (9, 10). A ternary complex with properties similar to the native complex can be formed using recombinant proteins lacking their transmembrane anchors (11). Both native and recombinant complexes can be disassembled by the concerted action of the ATPase NSF and the protein ␣-SNAP (9, 10, 12, 13). The latter two proteins are soluble, abundant, and highly conserved through evolution. They are essential for the fusion of trafficking vesicles with their target membranes (9, 10).The assembly and disassembly of the SNARE proteins has not yet been integrated into a coherent picture of exocytosis. However, any interference with these reactions seriously inhibits membrane fusion (6 -8). To further understand these essential reac...

The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

“…X-ray crystallographic studies reveal that the N-termini of the helices lie in parallel, with two of the four helices contributed by SNAP-25 and one each by syntaxin and VAMP (Sutton et al, 1998). Studies of the proteins in solution are consistent with this structure (Fasshauer et al, 1997;Lin and Scheller, 1997), which may reflect a configuration of the SNAREs during or after fusion. Physiological interactions of SNAP-25 and syntaxin in the plasma membrane probably occur with a 1:1 ratio (Fasshauer, 2003;Rickman et al, 2004), although in vitro studies of soluble SNARE motifs also demonstrate a 2:1 binding stoichiometry (Fasshauer et al, 1997;Xiao et al, 2001).…”

“…Studies of the proteins in solution are consistent with this structure (Fasshauer et al, 1997;Lin and Scheller, 1997), which may reflect a configuration of the SNAREs during or after fusion. Physiological interactions of SNAP-25 and syntaxin in the plasma membrane probably occur with a 1:1 ratio (Fasshauer, 2003;Rickman et al, 2004), although in vitro studies of soluble SNARE motifs also demonstrate a 2:1 binding stoichiometry (Fasshauer et al, 1997;Xiao et al, 2001). Little is known about the interactions of membranebound SNAREs in living cells and the relationships of the interactions to secretion.…”

Regulation of Synaptic Transmission by RAB-3 and RAB-27 inCaenorhabditis elegans

“…rab-27 was amplified from cDNA using oligonucleotide 5Ј-CATGGCCATGGGTGACTACGACTATCTC-3Ј and 5Ј-TCGGGTCGACGCATAGGAAGAAGCGGCCG-3Ј, digested with NcoI and SalI, and inserted into NcoI-SalI pHO2d (Fasshauer et al, 1997). The final construct contained the complete rab-27 coding sequences minus the last four amino acids fused to a linker sequence [ASTSLNSG] and ending in a His 6 -tag.…”

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

“…Alternative models proposed the involvement of accessory proteins, such as synaptotagmin (Littleton et al, 2001) or complexin (Tokumaru et al, 2001), or the transmembrane domains of syb2 and syntaxin 1A (Laage et al, 2000), in synaptic SNARE complex multimerization. However, SNARE complexes assembled from recombinant coils and lacking transmembrane domains are able to associate with each other (Fasshauer et al, 1997;Fasshauer et al, 1998;Margittai et al, 2001;Ernst and Brü nger, 2003), arguing that at least some of the interactions that support multimerization may require neither accessory proteins nor transmembrane domains. The precise multimeric nature and configuration of these recombinant cytosolic SNARE complexes is ambiguous.…”