The SNARE complex from yeast is partially unstructured on the membrane

Molecular recognition between cognate SNAREs leads to the formation of a four-helix bundle, which facilitates vesicle docking and membrane fusion. For a SNARE system involved in trafficking in yeast, target membrane (t-) SNARE Sso1p and vesicle associated (v-) SNARE Snc2p contribute one SNARE motif each, whereas another t-SNARE (Sec9) donates two N-terminal and C-terminal SNARE motifs (SN1 and SN2) to the helical bundle. By use of EPR, it is found that SN2 has a tendency to be uncoiled, leaving a significant population of the SNARE complexes to be partially unstructured on the membrane. In sharp contrast, SN2 is fully engaged in the four-helix bundle when removed from the membrane, showing that the membrane is the main destabilizing factor. Helix-breaking proline mutations in SN2 did not affect the rate of docking but reduced the rate of lipid mixing significantly, indicating that SN2 plays an essential role in activating the transition from docking to fusion.     

Topological arrangement of the intracellular membrane fusion machinery

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form a four-helix coiled-coil bundle that juxtaposes two bilayers and drives a basal level of membrane fusion. The Sec1/Munc18 (SM) protein binds to its cognate SNARE bundle and accelerates the basal fusion reaction. The question of how the topological arrangement of the SNARE helices affects the reactivity of the fusion proteins remains unanswered. Here we address the problem for the first time in a reconstituted system containing both SNAREs and SM proteins. We find that to be fusogenic a SNARE topology must support both basal fusion and SM stimulation. Certain topological combinations of exocytic SNAREs result in basal fusion but cannot support SM stimulation, whereas other topologies support SM stimulation without inducing basal fusion. It is striking that of all the possible topological combinations of exocytic SNARE helices, only one induces efficient fusion. Our results suggest that the intracellular membrane fusion complex is designed to fuse bilayers according to one genetically programmed topology.     

Accessory α-Helix of Complexin I Can Displace VAMP2 Locally in the Complexin-SNARE Quaternary Complex

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory α-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory α-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory α-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.     

SNARE assembly and disassembly exhibit a pronounced hysteresis.

SNARE proteins are essential for intracellular membrane fusion of eukaryotes. Their assembly into stable four-helix bundles bridges membranes and may provide the energy for initiating membrane fusion. In vitro, assembly of soluble SNARE fragments is accompanied by major structural rearrangements that can be described as a folding reaction. The pathways and the thermodynamics of SNARE protein interactions, however, are not known. Here we report that assembly and dissociation of two distantly related SNARE complexes exhibit a marked hysteresis. The assembled and disassembled native states are separated by a kinetic barrier and cannot equilibrate on biologically relevant timescales. We suggest that the hysteresis is a hallmark of all SNARE complexes and that complex assembly and disassembly follow different pathways that may be independently controlled.     

The neuronal t-SNARE complex is a parallel four-helix bundle

Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.     

SNAREs contribute to the specificity of membrane fusion

Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information for membrane compartment organization is not encoded in the inherent ability of SNAREs to form complexes. Here, we show that exocytosis of norepinephrine from PC12 cells is only inhibited or rescued by specific SNAREs. The data suggest that SNARE pairing does underlie vesicle trafficking fidelity, and that specific SNARE interactions with other proteins may facilitate the correct pairing.     

Cysteine-disulfide cross-linking to monitor SNARE complex assembly during endoplasmic reticulum-Golgi transport

Assembly of cognate SNARE proteins into SNARE complexes is required for many intracellular membrane fusion reactions. However, the mechanisms that govern SNARE complex assembly and disassembly during fusion are not well understood. We have devised a new in vitro cross-linking assay to monitor SNARE complex assembly during fusion of endoplasmic reticulum (ER)-derived vesicles with Golgi-acceptor membranes. In Saccharomyces cerevisiae, anterograde ER-Golgi transport requires four SNARE proteins: Sec22p, Bos1p, Bet1p, and Sed5p. After tethering of ER-derived vesicles to Golgi-acceptor membranes, SNARE proteins are thought to assemble into a four-helix coiled-coil bundle analogous to the structurally characterized neuronal and endosomal SNARE complexes. Molecular modeling was used to generate a structure of the four-helix ER-Golgi SNARE complex. Based on this structure, cysteine residues were introduced into adjacent SNARE proteins such that disulfide bonds would form if assembled into a SNARE complex. Our initial studies focused on disulfide bond formation between the SNARE motifs of Bet1p and Sec22p. Expression of SNARE cysteine derivatives in the same strain produced a cross-linked heterodimer of Bet1p and Sec22p under oxidizing conditions. Moreover, this Bet1p-Sec22p heterodimer formed during in vitro transport reactions when ER-derived vesicles containing the Bet1p derivative fused with Golgi membranes containing the Sec22p derivative. Using this disulfide cross-linking assay, we show that inhibition of transport with anti-Sly1p antibodies blocked formation of the Bet1p-Sec22p heterodimer. In contrast, chelation of divalent cations did not inhibit formation of the Bet1p-Sec22p heterodimer during in vitro transport but potently inhibited Golgi-specific carbohydrate modification of glyco-pro-alpha factor. This data suggests that Ca(2+) is not directly required for membrane fusion between ER-derived vesicles and Golgi-acceptor membranes.     

The membrane-dipped neuronal SNARE complex: a site-directed spin labeling electron paramagnetic resonance study

The formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is an essential process for membrane fusion and the neurotransmitter release in neurons. As an initial step toward the determination of the membrane topology of the SNARE complex, residues at the membrane-water interface were investigated with site-specific spin labeling electron paramagnetic resonance. EPR analysis revealed that the basic amino acid-rich interfacial region, which is universal for all transmembrane SNARE proteins, inserts into the membrane, eliminating the gap between the core complex and the membrane. The result raises the possibility that core complex formation directly leads to the apposition of two membranes, which could facilitate membrane fusion.     

SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved H_abc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the H_abc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin H_abc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Q_bc configuration. We found that neither the linker nor the Q_bc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.     

Insertion of the membrane-proximal region of the neuronal SNARE coiled coil into the membrane

In the neuron, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an alpha-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling.     

Self-association of the H3 region of syntaxin 1A. Implications for intermediates in SNARE complex assembly

Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion.     

Constitutive versus regulated SNARE assembly: a structural basis

SNARE complex formation is essential for intracellular membrane fusion. Vesicle-associated (v-) SNARE intertwines with target membrane (t-) SNARE to form a coiled coil that bridges two membranes and facilitates fusion. For the SNARE family involved in neuronal communications, complex formation is tightly regulated by the v-SNARE-membrane interactions. However, it was found using EPR that complex formation is spontaneous for a different SNARE family that is involved in protein trafficking in yeast. Further, reconstituted yeast SNAREs promoted membrane fusion, different from the inhibited fusion for reconstituted neuronal SNAREs. The EPR structural analysis showed that none of the coiled-coil residues of yeast v-SNARE is buried in the hydrophobic layer of the membrane, making the entire coiled-coil motif accessible, again different from the deep insertion of the membrane-proximal region of neuronal v-SNARE into the bilayer. Importantly, yeast membrane fusion is constitutively active, while synaptic membrane fusion is regulated, consistent with the present results for two SNARE families. Thus, the v-SNARE-membrane interaction may be a major molecular determinant for regulated versus constitutive membrane fusion in cells.     

Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexins in neuroexocytosis.

The Ca(2+)-triggered release of neurotransmitters is mediated by fusion of synaptic vesicles with the plasma membrane. The molecular machinery that translates the Ca(2+) signal into exocytosis is only beginning to emerge. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin, SNAP-25, and synaptobrevin are central components of the fusion apparatus. Assembly of a membrane-bridging ternary SNARE complex is thought to initiate membrane merger, but the roles of other factors are less understood. Complexins are two highly conserved proteins that modulate the Ca(2+) responsiveness of neurotransmitter release. In vitro, they bind in a 1:1 stoichiometry to the assembled synaptic SNARE complex, making complexins attractive candidates for controlling the exocytotic fusion apparatus. We have now performed a detailed structural, kinetic, and thermodynamic analysis of complexin binding to the SNARE complex. We found that no major conformational changes occur upon binding and that the complexin helix is aligned antiparallel to the four-helix bundle of the SNARE complex. Complexins bound rapidly (approximately 5 x 10(7) m(-1) s(-1)) and with high affinity (approximately 10 nm), making it one of the fastest protein-protein interactions characterized so far in membrane trafficking. Interestingly, neither affinity nor binding kinetics was substantially altered by Ca(2+) ions. No interaction of complexins was detectable either with individual SNARE proteins or with the binary syntaxin x SNAP-25 complex. Furthermore, complexin did not promote the formation of SNARE complex oligomers. Together, our data suggest that complexins modulate neuroexocytosis after assembly of membrane-bridging SNARE complexes.     

The structure of the yeast plasma membrane SNARE complex reveals destabilizing water-filled cavities

SNARE proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7A resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.     

A search for synthetic peptides that inhibit soluble N-ethylmaleimide sensitive-factor attachment receptor-mediated membrane fusion

Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.     

X-ray structure of a neuronal complexin-SNARE complex from squid

Nerve terminals release neurotransmitters from vesicles into the synaptic cleft upon transient increases in intracellular Ca(2+). This exocytotic process requires the formation of trans SNARE complexes and is regulated by accessory proteins including the complexins. Here we report the crystal structure of a squid core complexin-SNARE complex at 2.95-A resolution. A helical segment of complexin binds in anti-parallel fashion to the four-helix bundle of the core SNARE complex and interacts at its C terminus with syntaxin and synaptobrevin around the ionic zero layer of the SNARE complex. We propose that this structure is part of a multiprotein fusion machinery that regulates vesicle fusion at a late pre-fusion stage. Accordingly, Ca(2+) may initiate membrane fusion by acting directly or indirectly on complexin, thus allowing the conformational transitions of the trans SNARE complex that are thought to drive membrane fusion.     

An elaborate classification of SNARE proteins sheds light on the conservation of the eukaryotic endomembrane system

Proteins of the SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) family are essential for the fusion of transport vesicles with an acceptor membrane. Despite considerable sequence divergence, their mechanism of action is conserved: heterologous sets assemble into membrane-bridging SNARE complexes, in effect driving membrane fusion. Within the cell, distinct functional SNARE units are involved in different trafficking steps. These functional units are conserved across species and probably reflect the conservation of the particular transport step. Here, we have systematically analyzed SNARE sequences from 145 different species and have established a highly accurate classification for all SNARE proteins. Principally, all SNAREs split into four basic types, reflecting their position in the four-helix bundle complex. Among these four basic types, we established 20 SNARE subclasses that probably represent the original repertoire of a eukaryotic cenancestor. This repertoire has been modulated independently in different lines of organisms. Our data are in line with the notion that the ur-eukaryotic cell was already equipped with the various compartments found in contemporary cells. Possibly, the development of these compartments is closely intertwined with episodes of duplication and divergence of a prototypic SNARE unit.     

Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis

Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.     

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.     

Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~ 10 k_BT, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations.