Homotetrameric structure of the SNAP-23 N-terminal coiled-coil domain

SNARE proteins mediate intracellular membrane fusion by forming a coiled-coil complex to merge opposing membranes. A "fusion-active" neuronal SNARE complex is a parallel four-helix bundle containing two coiled-coil domains from SNAP-25 and one coiled-coil domain each from syntaxin-1a and VAMP-2. "Prefusion" assembly intermediate complexes can also form from these SNAREs. We studied the N-terminal coiled-coil domain of SNAP-23 (SNAP-23N), a non-neuronal homologue of SNAP-25, and its interaction with other coiled-coil domains. SNAP-23N can assemble spontaneously with the coiled-coil domains from SNAP-23C, syntaxin-4, and VAMP-3 to form a heterotetrameric complex. Unexpectedly, pure SNAP-23N crystallizes as a coiled-coil homotetrameric complex. The four helices have a parallel orientation and are symmetrical about the long axis. The complex is stabilized through the interaction of conserved hydrophobic residues comprising the a and d positions of the coiled-coil heptad repeats. In addition, a central, highly conserved glutamine residue (Gln-48) is buried within the interface by hydrogen bonding between glutamine side chains derived from adjacent subunits and to solvent molecules. A comparison of the SNAP-23N structure to other SNARE complex structures reveals how a simple coiled-coil motif can form diverse SNARE complexes.     

The polybasic juxtamembrane region of Sso1p is required for SNARE function in vivo

Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the "SNARE domain" or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.     

The length of the flexible SNAREpin juxtamembrane region is a critical determinant of SNARE-dependent fusion.

The topology of a SNARE complex bridging two docked vesicles could act as a mechanical couple to do work on the lipid bilayer resulting in fusion. To test this, we prepared a series of modified SNARE proteins and determined their effects on SNARE-dependent membrane fusion. When two helix-breaking proline residues are introduced into the juxtamembrane region of VAMP, there is little or no effect on fusion, and the same change in syntaxin 1A only reduced the extent and rate of fusion by half. The insertion of a flexible linker between the transmembrane domain and the conserved coiled-coil domain only moderately affected fusion; however, fusion efficiency systematically decreased with increasing length of the linker. Together, these results rule out a requirement for helical continuity and suggest that distance is a critical factor for membrane fusion.     

Functional analysis of conserved structural elements in yeast syntaxin Vam3p

Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion. Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion. Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion. As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present. Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.     

Lipidic antagonists to SNARE-mediated fusion

SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.     

Two Coiled-Coil Domains of Chlamydia trachomatis IncA Affect Membrane Fusion Events during Infection

Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA’s fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.     

The syntaxins

Summary  

The SNARE hypothesis predicts that a family of SNAP receptors are localized to and function in diverse intracellular membrane compartments where membrane fusion processes take place. Syntaxins, the prototype family of SNARE proteins, have a carboxy-terminal tail-anchor and multiple coiled-coil domains. There are 15 members of the syntaxin family in the human genome and 7 syntaxin-like genes in the yeast Saccharomyces cerevisiae. In conjunction with other SNAREs and with the cytoplasmic NSF and SNAP proteins, syntaxins mediate vesicle fusion in diverse vesicular transport processes along the exocytic and the endocytic pathway. They are crucial components that both drive and provide specificity to the myriad vesicular fusion processes that characterize the eukaryotic cell.     

Specific interaction between SNAREs and epsin N-terminal homology (ENTH) domains of epsin-related proteins in trans-Golgi network to endosome transport

SNARE proteins on transport vesicles and target membranes have important roles in vesicle targeting and fusion. Therefore, localization and activity of SNAREs have to be tightly controlled. Regulatory proteins bind to N-terminal domains of some SNAREs. vti1b is a mammalian SNARE that functions in late endosomal fusion. To investigate the role of the N terminus of vti1b we performed a yeast two-hybrid screen. The N terminus of vti1b interacted specifically with the epsin N-terminal homology (ENTH) domain of enthoprotin/CLINT/epsinR. The interaction was confirmed using in vitro binding assays. This complex formation between a SNARE and an ENTH domain was conserved between mammals and yeast. Yeast Vti1p interacted with the ENTH domain of Ent3p. ENTH proteins are involved in the formation of clathrin-coated vesicles. Both epsinR and Ent3p bind adaptor proteins at the trans-Golgi network. Vti1p is required for multiple transport steps in the endosomal system. Genetic interactions between VTI1 and ENT3 were investigated. Synthetic defects suggested that Vti1p and Ent3p cooperate in transport from the trans-Golgi network to the prevacuolar endosome. Our experiments identified the first cytoplasmic protein binding to specific ENTH domains. These results point toward a novel function of the ENTH domain and a connection between proteins that function either in vesicle formation or in vesicle fusion.     

Identification of SNAP-47, a novel Qbc-SNARE with ubiquitous expression

The SNARE proteins are essential components of the intracellular fusion machinery. It is thought that they form a tight four-helix complex between membranes, in effect initiating fusion. Most SNAREs contain a single coiled-coil region, referred to as the SNARE motif, directly adjacent to a single transmembrane domain. The neuronal SNARE SNAP-25 defines a subfamily of SNARE proteins with two SNARE helices connected by a longer linker, comprising also the proteins SNAP-23 and SNAP-29. We now report the initial characterization of a novel vertebrate homologue termed SNAP-47. Northern blot and immunoblot analysis revealed ubiquitous tissue distribution, with particularly high levels in nervous tissue. In neurons, SNAP-47 shows a widespread distribution on intracellular membranes and is also enriched in synaptic vesicle fractions. In vitro, SNAP-47 substituted for SNAP-25 in SNARE complex formation with the neuronal SNAREs syntaxin 1a and synaptobrevin 2, and it also substituted for SNAP-25 in proteoliposome fusion. However, neither complex assembly nor fusion was as efficient as with SNAP-25.     

Coiled-coil domains enhance the membrane association of Salmonella type III effectors

Coiled-coil domains in eukaryotic and prokaryotic proteins contribute to diverse structural and regulatory functions. Here we have used in silico analysis to predict which proteins in the proteome of the enteric pathogen, Salmonella enterica serovar Typhimurium, harbour coiled-coil domains. We found that coiled-coil domains are especially prevalent in virulence-associated proteins, including type III effectors. Using SopB as a model coiled-coil domain type III effector, we have investigated the role of this motif in various aspects of effector function including chaperone binding, secretion and translocation, protein stability, localization and biological activity. Compared with wild-type SopB, SopB coiled-coil mutants were unstable, both inside bacteria and after translocation into host cells. In addition, the putative coiled-coil domain was required for the efficient membrane association of SopB in host cells. Since many other Salmonella effectors were predicted to contain coiled-coil domains, we also investigated the role of this motif in their intracellular targeting in mammalian cells. Mutation of the predicted coiled-coil domains in PipB2, SseJ and SopD2 also eliminated their membrane localization in mammalian cells. These findings suggest that coiled-coil domains represent a common membrane-targeting determinant for Salmonella type III effectors.     

Engineering botulinum neurotoxin domains for activation by toxin light chain

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.     

The transmembrane domain of Vam3 affects the composition of cis- and trans-SNARE complexes to promote homotypic vacuole fusion

It is presently not clear how the function of SNARE proteins is affected by their transmembrane domains. Here, we analyzed the role of the transmembrane domain of the vacuolar SNARE Vam3 by replacing it by a lipid anchor. Vacuoles with mutant Vam3 fuse poorly and have increased amounts of cis-SNARE complexes, indicating that they are more stable. As a consequence efficient cis-SNARE complex disassembly that occurs at priming as a prerequisite of fusion requires addition of exogenous Sec18. trans-SNARE complexes in this mutant accumulate up to 4-fold over wild type, suggesting that the transmembrane domain of Vam3 is required to transit through this step. Finally, palmitoylation of Vac8, a reaction that also occurs early during priming is reduced by almost one-half. Since palmitoylated Vac8 is required beyond trans-SNARE complex formation, this may partially explain the fusion deficiency.     

Distinct domains of complexins bind SNARE complexes and clamp fusion in vitro

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca(2+)) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.     

Sec22p export from the endoplasmic reticulum is independent of SNARE pairing

Molecularly distinct sets of SNARE proteins localize to specific intracellular compartments and catalyze membrane fusion events. Although their central role in membrane fusion is appreciated, little is known about the mechanisms by which individual SNARE proteins are targeted to specific organelles. Here we investigated functional domains in Sec22p that direct this SNARE protein to the endoplasmic reticulum (ER), to Golgi membranes, and into SNARE complexes with Bet1p, Bos1p, and Sed5p. A series of Sec22p deletion mutants were monitored in COPII budding assays, subcellular fractionation gradients, and SNARE complex immunoprecipitations. We found that the N-terminal "profilin-like" domain of Sec22p was required but not sufficient for COPII-dependent export of Sec22p from the ER. Interestingly, versions of Sec22p that lacked the N-terminal domain were assembled into ER/Golgi SNARE complexes. Analyses of Sec22p SNARE domain mutants revealed a second signal within the SNARE motif (between layers -4 and -1) that was required for efficient ER export. Other SNARE domain mutants that contained this signal were efficiently packaged into COPII vesicles but failed to assemble into SNARE complexes. Together these results indicated that SNARE complex formation is neither required nor sufficient for Sec22p packaging into COPII transport vesicles and subsequent targeting to the Golgi complex. We propose that the COPII budding machinery has a preference for unassembled ER/Golgi SNARE proteins.     

Sequence-specific conformational flexibility of SNARE transmembrane helices probed by hydrogen/deuterium exchange

SNARE proteins mediate fusion of intracellular eukaryotic membranes and their α-helical transmembrane domains are known to contribute to lipid bilayer mixing. Synthetic transmembrane domain peptides were previously shown to mimic the function of SNARE proteins in that they trigger liposome fusion in a sequence-specific fashion. Here, we performed a detailed investigation of the conformational dynamics of the transmembrane helices of the presynaptic SNAREs synaptobrevin II and syntaxin 1a. To this end, we recorded deuterium/hydrogen-exchange kinetics in isotropic solution as well as in the membrane-embedded state. In solution, the exchange kinetics of each peptide can be described by three different classes of amide deuteriums that exchange with different rate constants. These are likely to originate from exchange at different domains of the helices. Interestingly, the rate constants of each class vary with the TMD sequence. Thus, the exchange rate is position-specific and sequence-specific. Further, the rate constants correlate with the previously determined membrane fusogenicities. In membranes, exchange is retarded and a significant proportion of amide hydrogens are protected from exchange. We conclude that the conformational dynamics of SNARE TMD helices is mechanistically linked to their ability to drive lipid mixing.     

Structure and function of SNARE and SNARE-interacting proteins

This review focuses on the so-called SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins that are involved in exocytosis at the pre-synpatic plasma membrane. SNAREs play a role in docking and fusion of synaptic vesicles to the active zone, as well as in the Ca2+-triggering step itself, most likely in combination with the Ca2+ sensor synaptotagmin. Different SNARE domains are involved in different processes, such as regulation, docking, and fusion. SNAREs exhibit multiple configurational, conformational, and oliogomeric states. These different states allow SNAREs to interact with their matching SNARE partners, auxiliary proteins, or with other SNARE domains, often in a mutually exclusive fashion. SNARE core domains undergo progressive disorder to order transitions upon interactions with other proteins, culminating with the fully folded post-fusion (cis) SNARE complex. Physiological concentrations of neuronal SNAREs can juxtapose membranes, and promote fusion in vitro under certain conditions. However, significantly more work will be required to reconstitute an in vitro system that faithfully mimics the Ca2+-triggered fusion of a synaptic vesicle at the active zone.     

Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane-related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum-related transport pathways throughout eukaryotic evolution.     

Conserved conformational dynamics of membrane fusion protein transmembrane domains and flanking regions indicated by sequence statistics

SNARE proteins and fusogenic viral membrane proteins represent the major classes of integral membrane proteins that mediate fusion of eukaryotic lipid bilayers. Although both classes have different primary structures, they share a number of basic architectural features. There is ample evidence that the fusogenic function of representative fusion proteins is influenced by the primary structure of the single transmembrane domain (TMD) and the region linking it to the soluble assembly domains. Here, we used comprehensive non-redundant datasets to examine potential over- and underrepresentation of amino acid types in the TMDs and flanking regions relative to control proteins that share similar biosynthetic origins. Our results reveal conserved overall and/or site-specific enrichment of β-branched residues and Gly within the TMDs, underrepresentation of Gly and Pro in regions flanking the TMD N-terminus, and overrepresentation of the same residue types in C-terminal flanks of SNAREs and viral fusion proteins. Furthermore, the basic Lys and Arg are enriched within SNARE N-terminal flanking regions. These results suggest evolutionary conservation of key structural features of fusion proteins and are discussed in light of experimental findings that link these features to the fusogenic function of these proteins.     

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.     

The N-terminal domains of syntaxin 7 and vti1b form three-helix bundles that differ in their ability to regulate SNARE complex assembly

The SNAREs syntaxin 7, syntaxin 8, vti1b, and endobrevin/VAMP8 function in the fusion of late endosomes. Although the core complex formed by these SNAREs is very similar to the neuronal SNARE complex, it differs from the neuronal complex in that three of the four SNAREs contain extended N-terminal regions of unknown structure and function. Here we show that the N-terminal regions of syntaxin 7, syntaxin 8, and vti1b contain well folded alpha-helical domains. Multidimensional NMR spectroscopy revealed that in syntaxin 7 and vti1b, the domains form three-helix bundles resembling those of syntaxin 1, Sso1p, and Vam3p. The three-helix bundle domain of vti1b is the first of its kind identified in a SNARE outside the syntaxin family. Only syntaxin 7 adopts a closed conformation, whereas in vti1b and syntaxin 8, the N-terminal domains do not interact with the adjacent SNARE motifs. Accordingly, the rate of SNARE complex assembly is retarded about 7-fold when syntaxin 7 contains its N-terminal domain, whereas the N-terminal domains of vti1b and syntaxin 8 have no influence on assembly kinetics. We conclude that three-helix bundles represent a common fold for SNARE N-terminal domains, not restricted to the syntaxin family. However, they differ in their ability to adopt closed conformations and thus to regulate the assembly of SNARE complexes.