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CLASSIC STRUCTURES

SNARE complex structure

Axel Brunger

Introduction

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Vesicular trafficking in eukaryotic cells is essential for diverse cellular processes, including maintenance of distinct subcellular compartments, protein and hormone secretion, egg fertilization, and neurotransmitter release. The life cycle of a vesicle generally consists of three stages: endocytosis or formation of the vesicle from specific cellular membranes, exocytosis or fusion of the vesicle with its target membrane, and recycling of the components of the protein machinery after exocytosis. Essential to the process of vesicular exocytosis are the SNARE (Soluble NSF Attachment protein REceptor; NSF is N-ethylmaleimide-sensitive fusion protein) proteins (3717; 290; 4252). These proteins are part of a machinery that is conserved from yeast to man. Structural analysis of the SNARE complex suggests how its assembly contributes to vesicular exocytosis.

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Background

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The synaptic SNARE complex consists of three proteins:

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  • Synaptobrevin (also referred to as VAMP, for Vesicle Associated Membrane Protein): a 12 kDa protein with an N-terminal domain with unknown function, a SNARE binding domain (i.e., the region that interacts with other SNAREs to form the ternary SNARE complex), and a single-spanning transmembrane domain.
  • Syntaxin: a 35 kDa protein with an N-terminal three-helix bundle regulatory domain, a SNARE binding domain, and a single-spanning transmembrane domain.
  • SNAP-25 [SyNaptosome-Associated Protein]: a 25 kDa protein, with two SNARE binding domains and a linker of approximately 45 amino acids, that connects the two SNARE domains. SNAP-25 is targeted to the plasma membrane by its association with syntaxin and via palmitoylation (covalent attachment of fatty acids to cysteine residues) in the linker.

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Figure 1  
Vesicle membrane fusion requires formation of the SNARE complex: synaptobrevin, syntaxin, and SNAP-25. After vesicular exocytosis, the ATPase NSF and α-SNAP takes apart the SNARE complex, allowing the proteins to be recycled.

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Figure 1 illustrates the process of exocytosis in the presynaptic neuron, and the involvement of the SNARE proteins. During the priming/docking step, synaptobrevin on the vesicle makes contact with syntaxin and SNAP-25 on the target membrane. Fusion of the vesicle with the target membrane occurs upon an increase in calcium concentration due to neuronal depolarization, and subsequent formation of the complete SNARE complex. Finally, the SNARE complex is disassembled, with the help of the ATPase NSF (N-ethylmaleimide sensitive factor) and its cofactor α-SNAP.

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Before the structure of the SNARE complex was known, the proteins were considered to be fundamental to exocytosis because site-specific cleavage of SNAREs by the botulinum and tetanus neurotoxins (both from the Clostridium bacteria) inhibits neurotransmission (4248). More recent experiments both in vitro and in vivo have demonstrated that the SNARE proteins are essential in membrane fusion (786; 4238; 4258) (for more information on the SNARE complex, see The SNARE complex and its role in the specificity of membrane fusion and SNARES are responsible for membrane fusion). However, the precise function of the SNAREs during exocytosis is still a topic of some debate.

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Of the uncomplexed SNARE proteins, only syntaxin possesses some structure; the other two proteins are unstructured in the absence of binding partners (4240; 4244). However, the SNARE binding domains of the SNARE proteins spontaneously assemble into a four-helical bundle in vitro (4244; 4240). SNAP-25 contributes two helices; synaptobrevin and syntaxin contribute one helix each. Thus, complex formation dramatically induces structure in syntaxin, SNAP-25, and synaptobrevin.

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We undertook structural analysis of the SNARE complex in part to see the configuration of the individual SNARE domains (i.e., parallel vs. anti-parallel) within the bundle. A parallel configuration would promote fusion by placing the transmembrane domains close to each other whereas an anti-parallel configuration would position the membranes 100 Å apart. Therefore, knowledge of the arrangement of the different domains may provide insight into the role of SNARE complex assembly in membrane fusion.

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Structure and function

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Figure 2  
Ribbon representation of the neuronal SNARE complex, adapted from 3742. The PDB identifier of the SNARE complex is 1SFC.

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The spontaneous assembly of the SNARE binding domains produces a mixture of both parallel and anti-parallel configurations in vitro (4255). The most stable configuration consists of a parallel four-helical bundle which lends itself to crystallization. We obtained a 2.4 Å crystal structure of the "core" of the synaptic complex (3742). As shown in Figure 2, the structure of the synaptic SNARE complex reveals a 120 Å-long and 20 Å-wide parallel four-helix bundle. The surface consists of many charged residues, in particular a large negatively charged center region, and a highly positively charged membrane proximal end.

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Surprisingly, the crystal structure revealed a largely conserved core of interacting residues, strongly suggesting that SNAREs cannot be the sole determinants for vesicle targeting specificity. The original SNARE hypothesis (290) proposed that membrane trafficking specificity is mediated by preferential interactions between particular v (vesicle membrane) and t (target membrane)-SNARE combinations. Since most interactions in the core of the four-helix bundle are highly conserved (4241), perhaps interactions with other proteins or factors facilitate the correct pairing between donor and acceptor membranes. More recent experiments in PC12 cells support this hypothesis, although the interactions remain to be identified (4251).

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The set of conserved interacting residues includes a conserved "ionic" layer at the center of the bundle consisting of an arginine and three glutamine residues. The function of the ionic layer is still being investigated (4254). Most likely it plays a role during NSF-driven disassembly of the SNARE complex since mutations of the central layer can disrupt this process (4250).

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Directed assembly of the SNARE complex drives membrane fusion

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The parallel orientation of the helices and charged regions of the complex suggest a "zipper" mechanism for membrane fusion. In the zipper model, complex formation proceeds in a directed fashion starting at the membrane-distal end (N-terminus) of the complex towards the membrane-proximal C-terminus (4246). The directed assembly process would bring membranes into close proximity, thus overcoming the free-energy barrier for membrane fusion.

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Figure 3  
The model is adapted from 3742, with hypothetical placement of the transmembrane domains (gray) of syntaxin (red) and synaptobrevin (blue), and the loop that connects the two fragments of SNAP-25 (green). The information button reveals the cleavage sites of the tetanus toxin (TeNT) and types A-F of botulinum toxin (BoNT/A-F, respectively).

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Consistent with the zipper model is the presumed existence of the SNARE complex in a partially assembled state (Figure 1, first panel). Although this state has not been directly observed, there is indirect evidence for such an intermediate state. Figure 3 shows that the cleavage sites of all clostridial neurotoxin proteases are located in the C-terminal (membrane-proximal) half of the core complex (4248). Since SNAREs are protected against proteolysis in the fully assembled complex, the vulnerability of the complex to proteases suggests that SNAREs must exist in partly assembled or "loose" states for significant periods of time.

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More recent experiments indicate the existence of loose and tight SNARE complex states. The C-terminus, but not the N-terminus, of synaptobrevin is sensitive to toxins in the docked state (4247). Kinetic studies of chromaffin cell exocytosis revealed a fusion-competent state that is sensitive to the attack of clostridial neurotoxins (4256). Inhibition of SNARE complex assembly by antibody-binding differentially affected kinetic components of exocytosis (4257). Taken together, these experiments provide additional evidence for the zipper mechanism of SNARE complex assembly.

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Legacy

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While the elegance of the zipper model is appealing, it is too simplistic. We recently observed the existence of anti-parallel configurations of the SNARE complex in addition to the highly stable parallel configuration in vitro (4255). Thus, chaperones or the membrane environment are required in vivo to promote directed assembly into the parallel configuration. Clearly, a physiological role of the less stable anti-parallel configurations, if any, needs to be tested.

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Figure 4  
Arg-56 can assume different conformations in the ionic layer of the SNARE complex. Here, analogous residues are compared from the neuronal SNARE complex solved at 2.4 Å (cyan), the neuronal SNARE complex solved at 1.4 Å (yellow), and the endosomal SNARE complex (gray). Figure adapted from 4239.

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We also determined the SNARE complex structure at higher resolution (1.4 Å), which provided additional insights into the role of the ionic central layer (4239; PDB code 1N7S at RCSB). In particular, we found that the arginine residue at the ionic central layer exhibited an alternate conformation, shown in Figure 4. The conformational variability of the central layer would allow unraveling or unwinding of the complex starting at the arginine. This result further supports a possible role for this layer in NSF-mediated disassembly of the SNARE complex.

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>Structural analysis of the SNARE complex not only helps elucidate the process of vesicular exocytosis, but it also aids our understanding of membrane fusion in general. For example, the high thermal and chemical stability of the SNARE complex makes it similar to the proteins involved in viral fusion (4116). Both systems involve α-helical bundles although there are differences in oligomeric state and configuration. In addition, the orientation of the helices places the membrane-associated regions at one end of the helical bundle. These similarities indicate a possible common ancestral mechanism for both fusion systems.

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The author

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Dr. Brunger is Investigator in the Howard Hughes Medical Institute. He is also Professor of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and the Stanford Synchrotron Radiation Laboratory at Stanford University. He received his Ph.D. degree from the Technical University of Munich. He held a NATO postdoctoral fellowship and subsequently became a research associate in the Harvard University Department of Chemistry before joining the faculty at Yale University, where he was an HHMI investigator. Dr. Brunger is corecipient of the 2003 Gregori Aminoff Prize of the Royal Swedish Academy of Sciences.

Photograph by L.A. Cicero of the Stanford Report.

Contact details

Axel Brunger
The Howard Hughes Medical Institute and
Department of Molecular and Cellular Physiology
Neurology and Neurological Sciences
Stanford Synchrotron Radiation Laboratory
Stanford University
Stanford, CA
Phone: 650 736 1031
Fax: 650 745 1463
E-mail: axel.brunger@stanford.edu

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Last Revised on September 10, 2004

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reviews
  • 290 Rothman, J. E. (1994).  Mechanisms of intracellular protein transport.  Nature 372, 55-68.  PubMed   Journal
  • 3717 Ferro-Novick, S. and Jahn, R. (1994).  Vesicle fusion from yeast to man.  Nature 370, 191-193.  PubMed   Journal
  • 4116 Skehel, J. J. and Wiley, D. C. (1998).  Coiled coils in both intracellular vesicle and viral membrane fusion.  Cell 95, 871-874.  PubMed   Journal
  • 4246 Hanson, P. I., Heuser, J. E., and Jahn, R. (1997).  Neurotransmitter release - four years of SNARE complexes.  Curr. Opin. Neurobiol. 7, 310-315.  PubMed  
  • 4248 Jahn, R. and Niemann, H. (1994).  Molecular mechanisms of clostridial neurotoxins.  Ann. N Y Acad. Sci. 733, 245-255.  PubMed  
  • 4252 Südhof, T. C. (1995).  The synaptic vesicle cycle: a cascade of protein-protein interactions.  Nature 375, 645-653.  PubMed   Journal
Last Revised on September 10, 2004

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reviews
  • 786 Weber, T., Zemelman, B., McNew, J., Westermann, B., Gmachl, M., Parlati, F., Sollner, T.H., Rothman, J.E. (1998).  SNAREpins: minimal machinery for membrane fusion.  Cell 92, 759-772.  PubMed   Journal
  • 3742 Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998).  Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution.  Nature 395, 347-353.  PubMed   Journal
  • 4238 Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y. C., and Scheller, R. H. (1999).  SNARE complex formation is triggered by Ca2+ and drives membrane fusion.  Cell 97, 165-174.  PubMed   Journal
  • 4239 Ernst, J. A. and Brunger, A. T. (2003).  High resolution structure, stability, and synaptotagmin binding of a truncated neuronal SNARE complex.  J. Biol. Chem. 278, 8630-8636.  PubMed   Journal
  • 4240 Fasshauer, D., Otto, H., Eliason, W.K., Jahn, R., and Brunger, A.T. (1997).  Structural changes are associated with SNARE-complex formation.  J. Biol. Chem. 242, 28036-28041..
  • 4241 Fasshauer, D., Sutton, R. B., Brunger, A. T., and Jahn, R. (1998).  Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs.  Proc. Natl. Acad. Sci. USA 95, 15781-15786.  PubMed   Journal
  • 4244 Fiebig, K. M., Rice, L. M., Pollock, E., and Brunger, A. T. (1999).  Folding intermediates of SNARE complex assembly.  Nat. Struct. Biol. 6, 117-123.  PubMed   Journal
  • 4247 Hua, S. Y. and Charlton, M. P. (1999).  Activity-dependent changes in partial VAMP complexes during neurotransmitter release.  Nat Neurosci 2, 1078-1083.  PubMed   Journal
  • 4250 Scales, S. J., Yoo, B. Y., and Scheller, R. H. (2001).  The ionic layer is required for efficient dissociation of the SNARE complex by alpha-SNAP and NSF.  Proc. Natl. Acad. Sci. USA 98, 14262-14267.  PubMed   Journal
  • 4251 Scales, S.J., Chen, Y.A., Yoo, B.Y., Patel, S.M., Doung, Y.-C., and Scheller, R.H. (2000).  SNAREs contribute to the specificity of membrane fusion.  Neuron 26, 457-464..  PubMed  
  • 4254 Wei, S., Xu, T., Ashery, U., Kollewe, A., Matti, U., Antonin, W., Rettig, J., and Neher, E. (2000).  Exocytotic mechanism studied by truncated and zero layer mutants of the C-terminus of SNAP-25.  EMBO J. 19, 1279-1289.  PubMed   Journal
  • 4255 Weninger, K., Bowen, M.E., Chu, S., Brunger, and A.T., (2003).  Single molecule studies of SNARE complex assembly reveal parallel and anti-parallel configurations.  Proc. Natl. Acad. Sci. USA  100, 14800-14805.  PubMed   Journal
  • 4256 Xu, T., Binz, T., Niemann, H., and Neher, E. (1998).  Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity.  Nat Neurosci 1, 192-200.  PubMed   Journal
  • 4257 Xu, T., Rammner, B., Margittai, M., Artalejo, A. R., Neher, E., and Jahn, R. (1999).  Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis.  Cell 99, 713-722.  PubMed   Journal
  • 4258 Schoch, S., Deák, F., Königstorfer, A., Mozhayeva, M., Sara, Y., Südhof, T. C., and Kavalali, E. T. (2001).  SNARE function analyzed in synaptobrevin/VAMP knockout mice.  Science 294, 1117-1122.  PubMed   Journal

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