Alternate interfaces may mediate homomeric and heteromeric assembly in the transmembrane domains of SNARE proteins

The fusion of a vesicle to a target membrane is mediated by temporally and spatially regulated interactions within a set of evolutionarily conserved proteins. Integral to proper fusion is the interaction between proteins originating on both vesicle and target membranes to form a protein bridge between the two membranes, known as the SNARE complex. This protein complex includes the single-pass transmembrane helix proteins: syntaxin and synaptobrevin. Experimental data and amino acid sequence analysis suggest that an interface of interaction is conserved between the transmembrane regions of the two proteins. However, conflicting reports have been presented on the role of the synaptobrevin transmembrane domain in mediating important protein-protein interactions. To address this question, a thermodynamic study was carried out to determine quantitatively the self-association propensities of the transmembrane domains of synaptobrevin and syntaxin. Our results show that the transmembrane domain of synaptobrevin has only a modest ability to self-associate, whereas the transmembrane domain of syntaxin is able to form stable homodimers. Nevertheless, by a single amino acid substitution, synaptobrevin can be driven to dimerize with the same affinity as syntaxin. Furthermore, crosslinking studies show that dimerization of synaptobrevin is promoted by oxidizing agents. Despite the presence of a conserved cysteine residue in the same location as in synaptobrevin, syntaxin dimerization is not promoted by oxidization. This analysis suggests that subtle yet distinct differences are present between the two transmembrane dimer interfaces. A syntaxin/synaptobrevin heterodimer is able to form under oxidizing conditions, and we propose that the interface of interaction for the heterodimer may resemble the homodimer interface formed by the synaptobrevin transmembrane domain. Computational analysis of the transmembrane sequences of syntaxin and synaptobrevin reveal structural models that correlate with the experimental data. These data may provide insight into the role of transmembrane segments in the mechanism of vesicle fusion.     

Mutational analysis of synaptobrevin transmembrane domain oligomerization

Synaptobrevin 2 is thought to facilitate fusion of synaptic vesicles with the presynaptic membrane through formation of a soluble NSF attachment protein receptor complex (SNARE) with syntaxin 1a and a synaptosomal associated protein of 25 kDa (SNAP-25). Previous reports have described a homodimer of synaptobrevin that is dependent on the transmembrane domain. However, these reports disagree about the magnitude of dimerization, which makes it difficult to assess the biological relevance of this interaction. We used SDS-PAGE and the TOXCAT genetic assay to reexamine the homodimerization of the synaptobrevin transmembrane domain in detergents and the Escherichia coli inner membrane, respectively. To gauge the magnitude of synaptobrevin homodimerization, we used the well-characterized glycophorin A homodimer as a positive standard. In contrast to previous studies, we found synaptobrevin homodimerization in E. coli is very weak when compared to glycophorin A. Recombinant synaptobrevin forms a small amount of dimer and higher order oligomers in detergents that are highly dependent on solublization conditions. We estimate a dissociation constant of 10 mM for synaptobrevin dimerization in detergent. Thus, the dimerization of synaptobrevin in membranes is very weak, questioning any possible functional role for this association in vivo.     

Action of complexin on SNARE complex

Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.     

Molecular mechanism of cholesterol- and polyphosphoinositide-mediated syntaxin clustering

The neuronal acceptor SNARE complex that functions as the receptor for synaptic vesicle docking and fusion at the presynaptic membrane is composed of the single-span transmembrane protein syntaxin-1A and the palmitoylated soluble protein SNAP-25. Previously, we explored interactions that promote the formation of syntaxin-1A clusters in membranes. Cholesterol activates clustering in native and model membranes, and its depletion in neuroendocrine cells results in a homogeneous distribution of the protein. However, as little as 1 mol % phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)) or 20 mol % phosphatidylserine was found to disperse syntaxin-1A clusters [Murray, D. H., and Tamm, L. K. (2009) Biochemistry 48, 4617-4625]. Strong evidence suggests that syntaxin-1A and its synaptic vesicle cognate synaptobrevin both interact directly with PI-4,5-P(2) and that this interaction activates fusion. However, the molecular details of this interaction and its relationship to the partial dispersion of syntaxin-1A clusters remain largely unexplored. Hence, we mutated the polybasic juxtamembrane motif of syntaxin-1A and found several residues that partially or fully abrogate the electrostatic interaction with PI-4,5-P(2). We further show that even in the presence of physiological concentrations of phosphatidylserine, the PI-4,5-P(2)-syntaxin interaction is sufficiently strong to disrupt syntaxin-1A clustering. The stereochemistry of PI-4,5-P(2) is not critical for this interaction as other polyphosphoinositides have similar effects. Forming an acceptor SNARE complex between syntaxin-1A and SNAP-25 weakens but does not abrogate cholesterol/PI-4,5-P(2)-controlled cluster formation. Potential consequences of these interactions with respect to synaptic vesicle fusion are discussed.     

A Coarse Grained Model for a Lipid Membrane with Physiological Composition and Leaflet Asymmetry

The resemblance of lipid membrane models to physiological membranes determines how well molecular dynamics (MD) simulations imitate the dynamic behavior of cell membranes and membrane proteins. Physiological lipid membranes are composed of multiple types of phospholipids, and the leaflet compositions are generally asymmetric. Here we describe an approach for self-assembly of a Coarse-Grained (CG) membrane model with physiological composition and leaflet asymmetry using the MARTINI force field. An initial set-up of two boxes with different types of lipids according to the leaflet asymmetry of mammalian cell membranes stacked with 0.5 nm overlap, reliably resulted in the self-assembly of bilayer membranes with leaflet asymmetry resembling that of physiological mammalian cell membranes. Self-assembly in the presence of a fragment of the plasma membrane protein syntaxin 1A led to spontaneous specific positioning of phosphatidylionositol(4,5)bisphosphate at a positively charged stretch of syntaxin consistent with experimental data. An analogous approach choosing an initial set-up with two concentric shells filled with different lipid types results in successful assembly of a spherical vesicle with asymmetric leaflet composition. Self-assembly of the vesicle in the presence of the synaptic vesicle protein synaptobrevin 2 revealed the correct position of the synaptobrevin transmembrane domain. This is the first CG MD method to form a membrane with physiological lipid composition as well as leaflet asymmetry by self-assembly and will enable unbiased studies of the incorporation and dynamics of membrane proteins in more realistic CG membrane models.     

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain.

A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.     

UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.     

Targeting of the Synaptic Vesicle Protein Synaptobrevin in the Axon of Cultured Hippocampal Neurons: Evidence for Two Distinct Sorting Steps

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10– amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61–70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.     

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.     

The C-terminal transmembrane region of synaptobrevin binds synaptophysin from adult synaptic vesicles

Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal synaptobrevin cleavage product. In addition, the His-tagged C-terminal synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding synaptobrevin, synaptophysin may positively modulate neurotransmission.     

Role of the Vam3p transmembrane segment in homodimerization and SNARE complex formation

Intracellular membrane fusion in eukaryotic cells is mediated by SNARE (soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor) proteins and is known to involve assembly of cognate subunits to heterooligomeric complexes. For synaptic SNAREs, it has previously been shown that the transmembrane segments drive homotypic and support heterotypic interactions. Here, we demonstrate that a significant fraction of the yeast vacuolar SNARE Vam3p is a homodimer in detergent extracts of vacuolar membranes. This homodimer exists in parallel to the heterooligomeric SNARE complex. A Vam3p homodimer also formed from the isolated recombinant protein. Interestingly, homodimerization depended on the transmembrane segment. In contrast, formation of the quaternary SNARE complex from recombinant Vam3p, Nyv1p, Vti1p, and Vam7p subunits did not depend on the transmembrane segment of Vam3p nor on the transmembrane segments of its partner proteins. We conclude that Vam3p homodimerization, but not quaternary SNARE complex formation, is promoted by TMS-TMS interaction. As the transmembrane segments of Vam3p and other SNARE homologues were previously shown to be critical for membrane fusion downstream of membrane apposition, our results may shed light on the functional significance of SNARE TMS-TMS interactions.     

NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin

Synaptobrevin is a synaptic vesicle protein that has an essential role in exocytosis and forms the SNARE complex with syntaxin and SNAP-25. We have analyzed the structure of isolated synaptobrevin and its binary interaction with syntaxin using NMR spectroscopy. Our results demonstrate that isolated synaptobrevin is largely unfolded in solution. The entire SNARE motif of synaptobrevin is capable of interacting with the isolated C-terminal SNARE motif of syntaxin but only a few residues bind to the full-length cytoplasmic region of syntaxin. This result suggests an interaction between the N- and C-terminal regions of syntaxin that competes with core complex assembly.     

The Ig cell adhesion molecule Basigin controls compartmentalization and vesicle release at Drosophila melanogaster synapses

Synapses can undergo rapid changes in size as well as in their vesicle release function during both plasticity processes and development. This fundamental property of neuronal cells requires the coordinated rearrangement of synaptic membranes and their associated cytoskeleton, yet remarkably little is known of how this coupling is achieved. In a GFP exon-trap screen, we identified Drosophila melanogaster Basigin (Bsg) as an immunoglobulin domain-containing transmembrane protein accumulating at periactive zones of neuromuscular junctions. Bsg is required pre- and postsynaptically to restrict synaptic bouton size, its juxtamembrane cytoplasmic residues being important for that function. Bsg controls different aspects of synaptic structure, including distribution of synaptic vesicles and organization of the presynaptic cortical actin cytoskeleton. Strikingly, bsg function is also required specifically within the presynaptic terminal to inhibit nonsynchronized evoked vesicle release. We thus propose that Bsg is part of a transsynaptic complex regulating synaptic compartmentalization and strength, and coordinating plasma membrane and cortical organization.     

Determinants of synaptobrevin regulation in membranes

Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion.     

Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca(2+)-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca(2+)-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca(2+) signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.     

Polyunsaturated fatty acids influence synaptojanin localization to regulate synaptic vesicle recycling

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.     

A conserved membrane-spanning amino acid motif drives homomeric and supports heteromeric assembly of presynaptic SNARE proteins

Assembly of the SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 to binary and ternary complexes is important for docking and/or fusion of presynaptic vesicles to the neuronal plasma membrane prior to regulated neurotransmitter release. Despite the well characterized structure of their cytoplasmic assembly domains, little is known about the role of the transmembrane segments in SNARE protein assembly and function. Here, we identified conserved amino acid motifs within the transmembrane segments that are required for homodimerization of synaptobrevin II and syntaxin 1A. Minimal motifs of 6-8 residues grafted onto an otherwise monomeric oligoalanine host sequence were sufficient for self-interaction of both transmembrane segments in detergent solution or membranes. These motifs constitute contiguous areas of interfacial residues assuming alpha-helical secondary structures. Since the motifs are conserved, they also contributed to heterodimerization of synaptobrevin II and syntaxin 1A and therefore appear to constitute interaction domains independent of the cytoplasmic coiled coil regions. Interactions between the transmembrane segments may stabilize the SNARE complex, cause its multimerization to previously observed multimeric superstructures, and/or be required for the fusogenic activity of SNARE proteins.     

In vivo imaging of retrogradely transported synaptic vesicle proteins in Caenorhabditis elegans neurons

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.     

SNAP-25 is targeted to the plasma membrane through a novel membrane-binding domain.

SNAP-25, syntaxin, and synaptobrevin are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. Membrane attachment of syntaxin and synaptobrevin is achieved through a C-terminal hydrophobic tail, whereas SNAP-25 association with membranes appears to depend upon palmitoylation of cysteine residues located in the center of the molecule. This process requires an intact secretory pathway and is inhibited by brefeldin A. Here we show that the minimal plasma membrane-targeting domain of SNAP-25 maps to residues 85-120. This sequence is both necessary and sufficient to target a heterologous protein to the plasma membrane. Palmitoylation of this domain is sensitive to brefeldin A, suggesting that it uses the same membrane-targeting mechanism as the full-length protein. As expected, the palmitoylated cysteine cluster is present within this domain, but surprisingly, membrane anchoring requires an additional five-amino acid sequence that is highly conserved among SNAP-25 family members. Significantly, the membrane-targeting module coincides with the protease-sensitive stretch (residues 83-120) that connects the two alpha-helices that SNAP-25 contributes to the four-helix bundle of the synaptic SNARE complex. Our results demonstrate that residues 85-120 of SNAP-25 represent a protein module that is physically and functionally separable from the SNARE complex-forming domains.     

Interaction of the Complexin Accessory Helix with the C-Terminus of the SNARE Complex: Molecular-Dynamics Model of the Fusion Clamp

SNARE complexes form between the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 to drive membrane fusion. A cytosolic protein, complexin (Cpx), binds to the SNARE bundle, and its accessory helix (AH) functions to clamp synaptic vesicle fusion. We performed molecular-dynamics simulations of the SNARE/Cpx complex and discovered that at equilibrium the Cpx AH forms tight links with both synaptobrevin and SNAP25. To simulate the effect of electrostatic repulsion between vesicle and membrane on the SNARE complex, we calculated the electrostatic force and performed simulations with an external force applied to synaptobrevin. We found that the partially unzipped state of the SNARE bundle can be stabilized by interactions with the Cpx AH, suggesting a simple mechanistic explanation for the role of Cpx in fusion clamping. To test this model, we performed experimental and computational characterizations of the syx^3-69Drosophila mutant, which has a point mutation in syntaxin that causes increased spontaneous fusion. We found that this mutation disrupts the interaction of the Cpx AH with synaptobrevin, partially imitating the cpx null phenotype. Our results support a model in which the Cpx AH clamps fusion by binding to the synaptobrevin C-terminus, thus preventing full SNARE zippering.