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Letters Crystal Structure of the Endosomal SNARE Complex letters sent an evolutionarily conserved superfamily of membrane-asso- Crystal structure of the ciated proteins that are differentially distributed among intra- endosomal SNARE complex cellular membranes1,2. SNAREs are characterized by heptad repeats of ∼60 amino acids that are referred to as SNARE motifs. reveals common structural Much of our knowledge is based upon the SNAREs that func- tion in neuronal exocytosis. They include the vesicle protein principles of all SNAREs synaptobrevin 2 (also referred to as VAMP-2), and the plasma membrane proteins syntaxin 1 and SNAP-25. Detailed investiga- Wolfram Antonin1, Dirk Fasshauer1, Stefan Becker2, tions of their conformational changes and their assembly and Reinhard Jahn1 and Thomas R. Schneider3 disassembly have resulted in a coherent, although still contro- versial, concept for their role in membrane fusion3. As 1Department of Neurobiology and 2NMR-based Structural Biology, Max- monomers, the SNARE motifs of these SNAREs are largely Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 unstructured. Upon assembly, they roll up into a highly stable Göttingen, Germany. 3University of Göttingen, Department of Structural helical bundle, with the membrane anchor domains extending Chemistry, Tammannstrasse 4, 37077 Göttingen, Germany. from one end of the bundle. This SNARE complex can be reversibly disassembled by the ATPase NSF and α-SNAP (solu- Published online: 14 January 2002, DOI: 10.1038/nsb746 ble NSF attachment protein). When complementary sets of SNAREs are anchored in opposed membranes, assembly would SNARE proteins are crucial for intracellular membrane tightly connect these membranes, thus largely overcoming the fusion in all eukaryotes. These proteins assemble into tight repulsive forces that prevent fusion4. Indeed, incorporation of complexes that connect membranes and may induce fusion. SNAREs into liposomes causes fusion that is dependent on the The crystal structure of the neuronal core complex is repre- ability of the SNAREs to form such complexes5. sented by an unusually long bundle of four ␣-helices con- Major support for this hypothesis was provided by the crystal nected by 16 layers of mostly hydrophobic amino acids. Here structure of the neuronal SNARE complex6, a bundle of four we report the 1.9 Å resolution crystal structure of an endo- α-helices that are aligned in parallel. Syntaxin 1 and synapto- somal SNARE core complex containing four SNAREs: syn- brevin 2 each contribute one helix, whereas SNAP-25 con- taxin 7, syntaxin 8, vti1b and endobrevin/VAMP-8. Despite tributes two helices to the bundle. In the interior, the helices limited sequence homology, the helix alignment and the form 16 layers of interacting amino acid side chains that are layer structure of the endosomal complex are remarkably mostly hydrophobic and arranged perpendicular to the axis of similar to those of the neuronal complex. However, subtle the helix bundle. In the middle of the bundle, an unusual variations are evident that characterize different SNARE sub- hydrophilic layer (0-layer) was discovered that consists of three families. We conclude that the structure of the SNARE core Gln residues, contributed by syntaxin 1 and SNAP-25, and one © http://structbio.nature.com Group 2002 Nature Publishing complex is an evolutionarily conserved hallmark of all Arg residue, contributed by synaptobrevin 2. These amino acids SNARE complexes and is intimately associated with the gen- are conserved throughout the SNARE superfamily, which led to eral role of SNAREs in membrane fusion. the classification of these proteins into Q- and R-SNAREs, Controlled fusion of biological membranes is fundamental to respectively7. During assembly, the 16 interacting layers may intracellular compartmentalization and, thus, essential for form sequentially in a zipper-like fashion, beginning at the growth and differentiation of eukaryotic cells. Although phos- N- and progressing towards the C-terminus4. pholipid bilayers can be induced to fuse by a variety of agents and Much less is known about SNARE complexes that function in treatments, cellular membranes require specific proteins for this other cellular fusion reactions. Comparisons of SNARE task. The best candidates for forming the core of such fusion sequences suggest that the major hallmarks of the neuronal machinery are the SNARE (soluble N-ethylmaleimide-sensitive SNARE complex are conserved. These include the overall com- factor (NSF) attachment protein receptor) proteins. They repre- position and length, as well as the positions of some interacting Fig. 1 Sequence alignment of the fragments in the neuronal SNARE complex (top lines) and the endosomal SNARE complex (bottom lines). Amino acids participating in layer formation are shown in red, and numbers of the layers are indicated on the top. Residues present in the crystal structure of the endosomal complex are boxed. Italic characters denote the remaining residues of the affinity tag. nature structural biology • volume 9 number 2 • february 2002 107 letters a Fig. 2 Overall structure of the endosomal core complex. a, Schematic view of the endosomal SNARE core complex (endobrevin in blue, syntaxin 7 in red, vti1b in light green and syntaxin 8 in dark green; N and C indicate the N- and C-termini of the core complex). Side chains partici- pating in layer formation are shown as sticks. b, Superposition of the three neuronal complexes (light red) and the endosomal complex (dark red and blue) based on the 83 Cα atoms (blue) of the endosomal com- plex that can be accommodated in the conformational ensemble of the neuronal complex. The side chains of the 0-layer of the endosomal com- b plex are shown for orientation. The orientation of (a) and (b) are identi- cal. c, B-factors of Cα atoms in the endosomal complex. d, Mean r.m.s. deviations for the layer-wise superposition of the Cα atoms of the three models for the neuronal complex onto the endosomal complex. c were formed from segments that included only the regions cor- 45.0 responding to the layers –8 to 8 of the neuronal complex (Fig. 1), 40.0 endobrevin syntaxin 7 crystals were obtained that diffracted to better than 2 Å resolu- 35.0 vti1b tion on a laboratory X-ray source. The crystal structure of this ) syntaxin 8 2 30.0 Å complex was solved by molecular replacement using the neu- 25.0 ronal SNARE complex as a search model and refined to an 20.0 B-value ( R-value of 17.6% at a resolution of 1.9 Å (Table 1). The model 15.0 contains all residues of the protein fragments, with the exception 10.0 of four nongeneric N-terminal amino acids (present due to 5.0 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 affinity tagging) and some additional N- and C-terminal Layer number residues (Fig. 1). d 0.8 Overall structure ) Å The overall structure of the endosomal core complex (Fig. 2a) 0.6 lends support to the hypothesis that SNARE complexes are gen- 0.4 erally formed by four-helix bundles, with the helices being arranged in parallel and connected by hydrophobic layers of 0.2 interaction7. As predicted, endobrevin occupies the position of R.m.s. deviations ( 0.0 synaptobrevin 2 in the neuronal complex (a-helix), syntaxin 7 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 that of syntaxin 1 (b-helix), and vti1b (c-helix) and syntaxin 8 Layer number © http://structbio.nature.com Group 2002 Nature Publishing (d-helix) that of the N- and C-terminal domains, respectively, of layers. Also, the biochemical properties of some of these com- SNAP-25 (ref. 15). plexes appear to be similar. For instance, individual components Most important, the structure is remarkably similar to that of of the neuronal complex can be replaced by corresponding the neuronal SNARE complex. Least-squares superposition of family members in vitro8,9. The overall sequence identities all Cα atoms (228 atoms) of the present structure with the corre- between distant family members, however, are rather limited. sponding Cα atoms in the three crystallographically indepen- Furthermore, only appropriate sets of SNAREs have been pro- dent models of the neuronal SNARE complex gives root mean posed to form fusion-competent complexes, implying that there square (r.m.s.) deviations of 1.22, 1.29 and 1.10 Å. The superpo- are significant structural differences in the interacting surfaces10. sition of only the conformationally invariant Cα atoms (83 Moreover, other compositions of SNARE complexes were atoms, marked in blue in Fig. 2b) of the endosomal core com- reported, such as complexes containing two or more identical plex gives r.m.s. deviations of 0.78, 0.68 and 0.63 Å, respectively, SNAREs11 or five instead of four SNARE motifs12. Finally, based which is within the experimental uncertainty (see Methods). on the in vitro study of yeast vacuole fusion, the idea that SNAREs The latter superposition, together with increasing B-values function as general fusion catalysts has been challenged13,14. Thus, towards both termini (Fig. 2c), suggests that the conformation of determining whether all SNARE complexes are formed by a bun- SNARE complexes is less conserved towards their C-terminal dle of four helices, whether features of the neuronal SNAREs are ends, which also exhibit a higher sequence diversity7. general hallmarks of all SNAREs and which of them reflect specializations that may have evolved to fulfill the highly sophisti- Structure of individual layers cated needs of synaptic vesicle exocytosis is essential. The structures of the individual layers are very similar to the lay- Here we report the 1.9 Å crystal structure of an endosomal ers of the neuronal complex. Only small variations are seen in core complex containing four SNAREs: syntaxin 7, syntaxin 8, the relative positions of the Cα atoms, with mean r.m.s.
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