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Structures of fusion-machinery components
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Pictures in cell biology Structures of fusion-machinery components Earlier this year, there was a spate of papers describing the structures of two of the major components of the intracellular membrane-fusion machinery: the SNARE complex1 and the D2 domain of N-ethylmaleimide-sensitive factor (NSF)2,3. These structures have resolved some uncertainties about the fusion machinery but also leave some questions unanswered. SNAREs (SNAP receptors; SNAPs are soluble NSF-attachment proteins) are small conserved proteins involved in vesicle fusion within the secretory pathway4. A current model for this process involves the formation of a complex between SNAREs on the two fusing membranes, which promotes fusion. The SNARE complex crystallized in this study contains syntaxin 1A, synaptobrevin-II and SNAP-25B – all SNAREs involved in synaptic vesicle exocytosis. This complex contains four a-helical domains – two from SNAP-25B and one each from synaptobrevin-II and syntaxin 1A, which form a stable coiled-coil region. Sutton et al. crystallized this coiled-coil domain and found, against expectation, that all four of the helices are in a parallel orientation (Fig. 1). Within the coiled-coil, there is an unusual but conserved salt bridge involving all four helices. The authors propose that this acts as a ‘register-check’, which could ensure correct alignment of the helices and enforce 1 : 1 : 1 stoichiometry. Another study also came to the conclusion that the four helices within this
SNARE complex are in a parallel orientation5. Rather than solving the crystal structure, Poirier et al. used spin-labelling electro-paramagnetic resonance spectroscopy. A third group looked at the question in yet another way – using deep-etch electron microscopy6. They studied mainly the yeast exocytic SNARE complex, containing Sso1p, Snc2p and the SNAP-25 homologue Sec9p. Using epitope tags, antibodies and globular protein markers, Katz et al. also concluded that the four helices are parallel. Similar studies on the synaptic SNARE complex came to the same conclusion6. This confirmation of a parallel helix arrangement rationalizes a point about SNARE complexes that was previously unclear. SNAP-25B is unusual among SNAREs in containing two a-helical regions and no transmembrane domain – most SNAREs are membrane proteins with only one a-helix. This, combined with other information, led to the prediction that other SNARE complexes contain four molecules, each contributing one a-helix7. However, it seemed likely that the helices of SNAP-25B were in opposite orientations since the intervening sequence between them is quite short, whereas in a four-member complex all the helices would be parallel. The demonstration that the SNAP-25B helices are in fact parallel removes this potential disparity, and it now seems likely that other SNARE complexes are indeed analogous in structure to the crystallized one.
Acknowledgements With thanks to Axel Brunger for providing images and consultation, and Axel Brunger and Patrick Brennwald for communicating results prior to publication.
502
FIGURE 1 Hypothetical model of the synaptic fusion complex joining two membranes. The crystallized helices of SNAP-25B are in green, synaptobrevin II in blue and syntaxin 1A in red. Synaptobrevin II and syntaxin 1A have been extended to include their transmembrane domains (yellow) and the inter-helical loop of SNAP-25B is also added (pink). Adapted, with permission, from Fig. 5 of Ref. 1.
0962-8924/98/$ – see front matter © 1998 Elsevier Science. All rights reserved. PII: S0962-8924(98)01404-4
A major remaining question is whether SNARE complex formation is sufficient to induce membrane fusion8. The stability and ease of assembly of the SNARE complex suggests that its formation is an energetically favourable process, which could be a driving force for fusion. One study suggested that it might be sufficient9, but the fusion observed was very inefficient, leaving room for the involvement of accessory factors in vivo. Another recent paper10 suggests that such accessory factors might be required to overcome the inherent inhibition of complex formation by certain SNARE protein domains. The stability of the SNARE complex also relates to the role of the other crystallized protein, NSF. As described in this issue11, NSF acts as an ATP-dependent chaperone to dissociate SNARE complexes and thus recycle SNAREs. The NSF crystallization is not quite as instructive as that of the SNAREs because it only involves one part of the protein – the D2 or oligomerization domain2,3. This domain binds to ATP, but its hydrolysis rate is low and, instead, hydrolysis by another domain, D1, is crucial for disassembly of the SNARE complex. This structure does, however, confirm that the D2 domain assembles as a hexamer, with sixfold radial symmetry. It also shows the structure of the AAA domain, within which NSF shares homology with a number of other AAA proteins12, and thus might provide a paradigm for such domains. The homology between the D1 and D2 domains allows predictions about the ATPase mechanism of the D1 domain and why the two domains differ in activity. However, a clear understanding of NSF activity will really require analysis of the N-terminal and D1 domains of NSF as well; with this, we should have a much better idea of how NSF works. References 1 Sutton, R. B. et al. (1998) Nature 395, 347–353 2 Lenzen, C. U. et al. (1998) Cell 94, 525–536 3 Yu, R. C. et al. (1998) Nat. Struct. Biol. 5, 803–811 4 Götte, M. and Fischer von Mollard, G. (1998) Trends Cell Biol. 8, 215–218 5 Poirier, M. et al. (1998) Nat. Struct. Biol. 5, 765–769 6 Katz, L. et al. EMBO J. (in press) 7 Weimbs, T. et al. (1998) Trends Cell Biol. 8, 260–262 8 Hanson, P. I., Heuser, J. and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315 9 Weber, T. et al. (1998) Cell 92, 759–772 10 Nicholson, K. L. (1998) Nat. Struct. Biol. 5, 793–802 11 Haas, A. (1998) Trends Cell Biol. 8, 471–473 12 Patel, S. and Latterich, M. (1998) Trends Cell Biol. 8, 65–71
trends in CELL BIOLOGY (Vol. 8) November 1998
Pictures in cell biology Structures of fusion-machinery components Earlier this year, there was a spate of papers describing the structures of two of the major components of the intracellular membrane-fusion machinery: the SNARE complex1 and the D2 domain of N-ethylmaleimide-sensitive factor (NSF)2,3. These structures have resolved some uncertainties about the fusion machinery but also leave some questions unanswered. SNAREs (SNAP receptors; SNAPs are soluble NSF-attachment proteins) are small conserved proteins involved in vesicle fusion within the secretory pathway4. A current model for this process involves the formation of a complex between SNAREs on the two fusing membranes, which promotes fusion. The SNARE complex crystallized in this study contains syntaxin 1A, synaptobrevin-II and SNAP-25B – all SNAREs involved in synaptic vesicle exocytosis. This complex contains four a-helical domains – two from SNAP-25B and one each from synaptobrevin-II and syntaxin 1A, which form a stable coiled-coil region. Sutton et al. crystallized this coiled-coil domain and found, against expectation, that all four of the helices are in a parallel orientation (Fig. 1). Within the coiled-coil, there is an unusual but conserved salt bridge involving all four helices. The authors propose that this acts as a ‘register-check’, which could ensure correct alignment of the helices and enforce 1 : 1 : 1 stoichiometry. Another study also came to the conclusion that the four helices within this
SNARE complex are in a parallel orientation5. Rather than solving the crystal structure, Poirier et al. used spin-labelling electro-paramagnetic resonance spectroscopy. A third group looked at the question in yet another way – using deep-etch electron microscopy6. They studied mainly the yeast exocytic SNARE complex, containing Sso1p, Snc2p and the SNAP-25 homologue Sec9p. Using epitope tags, antibodies and globular protein markers, Katz et al. also concluded that the four helices are parallel. Similar studies on the synaptic SNARE complex came to the same conclusion6. This confirmation of a parallel helix arrangement rationalizes a point about SNARE complexes that was previously unclear. SNAP-25B is unusual among SNAREs in containing two a-helical regions and no transmembrane domain – most SNAREs are membrane proteins with only one a-helix. This, combined with other information, led to the prediction that other SNARE complexes contain four molecules, each contributing one a-helix7. However, it seemed likely that the helices of SNAP-25B were in opposite orientations since the intervening sequence between them is quite short, whereas in a four-member complex all the helices would be parallel. The demonstration that the SNAP-25B helices are in fact parallel removes this potential disparity, and it now seems likely that other SNARE complexes are indeed analogous in structure to the crystallized one.
Acknowledgements With thanks to Axel Brunger for providing images and consultation, and Axel Brunger and Patrick Brennwald for communicating results prior to publication.
502
FIGURE 1 Hypothetical model of the synaptic fusion complex joining two membranes. The crystallized helices of SNAP-25B are in green, synaptobrevin II in blue and syntaxin 1A in red. Synaptobrevin II and syntaxin 1A have been extended to include their transmembrane domains (yellow) and the inter-helical loop of SNAP-25B is also added (pink). Adapted, with permission, from Fig. 5 of Ref. 1.
0962-8924/98/$ – see front matter © 1998 Elsevier Science. All rights reserved. PII: S0962-8924(98)01404-4
A major remaining question is whether SNARE complex formation is sufficient to induce membrane fusion8. The stability and ease of assembly of the SNARE complex suggests that its formation is an energetically favourable process, which could be a driving force for fusion. One study suggested that it might be sufficient9, but the fusion observed was very inefficient, leaving room for the involvement of accessory factors in vivo. Another recent paper10 suggests that such accessory factors might be required to overcome the inherent inhibition of complex formation by certain SNARE protein domains. The stability of the SNARE complex also relates to the role of the other crystallized protein, NSF. As described in this issue11, NSF acts as an ATP-dependent chaperone to dissociate SNARE complexes and thus recycle SNAREs. The NSF crystallization is not quite as instructive as that of the SNAREs because it only involves one part of the protein – the D2 or oligomerization domain2,3. This domain binds to ATP, but its hydrolysis rate is low and, instead, hydrolysis by another domain, D1, is crucial for disassembly of the SNARE complex. This structure does, however, confirm that the D2 domain assembles as a hexamer, with sixfold radial symmetry. It also shows the structure of the AAA domain, within which NSF shares homology with a number of other AAA proteins12, and thus might provide a paradigm for such domains. The homology between the D1 and D2 domains allows predictions about the ATPase mechanism of the D1 domain and why the two domains differ in activity. However, a clear understanding of NSF activity will really require analysis of the N-terminal and D1 domains of NSF as well; with this, we should have a much better idea of how NSF works. References 1 Sutton, R. B. et al. (1998) Nature 395, 347–353 2 Lenzen, C. U. et al. (1998) Cell 94, 525–536 3 Yu, R. C. et al. (1998) Nat. Struct. Biol. 5, 803–811 4 Götte, M. and Fischer von Mollard, G. (1998) Trends Cell Biol. 8, 215–218 5 Poirier, M. et al. (1998) Nat. Struct. Biol. 5, 765–769 6 Katz, L. et al. EMBO J. (in press) 7 Weimbs, T. et al. (1998) Trends Cell Biol. 8, 260–262 8 Hanson, P. I., Heuser, J. and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315 9 Weber, T. et al. (1998) Cell 92, 759–772 10 Nicholson, K. L. (1998) Nat. Struct. Biol. 5, 793–802 11 Haas, A. (1998) Trends Cell Biol. 8, 471–473 12 Patel, S. and Latterich, M. (1998) Trends Cell Biol. 8, 65–71
trends in CELL BIOLOGY (Vol. 8) November 1998