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Helical extension of the neuronal SNARE complex into the membrane.

Stein A, Weber G, Wahl MC, Jahn R - Nature (2009)

Bottom Line: Here, we report the X-ray structure of the neuronal SNARE complex, consisting of rat syntaxin 1A, SNAP-25 and synaptobrevin 2, with the carboxy-terminal linkers and transmembrane regions at 3.4 A resolution.The structure shows that assembly proceeds beyond the already known core SNARE complex, resulting in a continuous helical bundle that is further stabilized by side-chain interactions in the linker region.Our results suggest that the final phase of SNARE assembly is directly coupled to membrane merger.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

ABSTRACT
Neurotransmission relies on synaptic vesicles fusing with the membrane of nerve cells to release their neurotransmitter content into the synaptic cleft, a process requiring the assembly of several members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family. SNAREs represent an evolutionarily conserved protein family that mediates membrane fusion in the secretory and endocytic pathways of eukaryotic cells. On membrane contact, these proteins assemble in trans between the membranes as a bundle of four alpha-helices, with the energy released during assembly being thought to drive fusion. However, it is unclear how the energy is transferred to the membranes and whether assembly is conformationally linked to fusion. Here, we report the X-ray structure of the neuronal SNARE complex, consisting of rat syntaxin 1A, SNAP-25 and synaptobrevin 2, with the carboxy-terminal linkers and transmembrane regions at 3.4 A resolution. The structure shows that assembly proceeds beyond the already known core SNARE complex, resulting in a continuous helical bundle that is further stabilized by side-chain interactions in the linker region. Our results suggest that the final phase of SNARE assembly is directly coupled to membrane merger.

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Synaptobrevin 2 and syntaxin 1A form continuous helices.a, Ribbon plot of the synaptic SNARE complex including linkers and TMRs (colour coding as in Fig. 1a). Sulfate ions and two glycylglycylglycine molecules are depicted as spheres and black sticks, respectively.b, Surface plot showing the electrostatic potential of the synaptic SNARE complex (blue, positive charge; red, negative charge). The electrostatic surface was contoured between – 16 kT/e and + 16 kT/e.c, Ribbon plot showing linkers and TMRs of synaptobrevin 2 and syntaxin 1A. Side chains are shown as sticks, with carbons coloured as the corresponding backbone; oxygen, red; nitrogen, blue. The hydrogen-bond between Asn92 in synaptobrevin 2 and Lys264 in syntaxin 1A is depicted as a black dashed line. The right panel is rotated by 90° about the vertical axis as indicated.d, Amino acids forming interactions between the two linkers and TMRs. Amino acids that are close to each other are connected by black lines, those further away but facing towards each other by gray lines. The last two amino acids in syntaxin 1A were not resolved in the structure. +8 indicates the C-terminal layer of the four helix bundle 49. Note that this layer is not completely resolved as electron density for the last four amino acids of the second helix of SNAP-25 (including the layer-forming amino acid Met202) is absent.
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Figure 2: Synaptobrevin 2 and syntaxin 1A form continuous helices.a, Ribbon plot of the synaptic SNARE complex including linkers and TMRs (colour coding as in Fig. 1a). Sulfate ions and two glycylglycylglycine molecules are depicted as spheres and black sticks, respectively.b, Surface plot showing the electrostatic potential of the synaptic SNARE complex (blue, positive charge; red, negative charge). The electrostatic surface was contoured between – 16 kT/e and + 16 kT/e.c, Ribbon plot showing linkers and TMRs of synaptobrevin 2 and syntaxin 1A. Side chains are shown as sticks, with carbons coloured as the corresponding backbone; oxygen, red; nitrogen, blue. The hydrogen-bond between Asn92 in synaptobrevin 2 and Lys264 in syntaxin 1A is depicted as a black dashed line. The right panel is rotated by 90° about the vertical axis as indicated.d, Amino acids forming interactions between the two linkers and TMRs. Amino acids that are close to each other are connected by black lines, those further away but facing towards each other by gray lines. The last two amino acids in syntaxin 1A were not resolved in the structure. +8 indicates the C-terminal layer of the four helix bundle 49. Note that this layer is not completely resolved as electron density for the last four amino acids of the second helix of SNAP-25 (including the layer-forming amino acid Met202) is absent.

Mentions: As previously seen in the structure of the core complex 7, the four SNARE motifs form a four-helix bundle. The most striking feature is that both syntaxin 1A and synaptobrevin 2 form continuous helices throughout their SNARE motifs, linker regions and TMRs (see figure 2a for an overview, 2b for an electrostatic surface plot, 2c for a close-up view on the linkers and TMRs, and 2d for a schematic representation of the interacting amino acids). In the crystal, hydrophobic contacts between the TMRs of synaptobrevin 2 and syntaxin 1A mediate the association of four complexes via their TMRs, forming an X-shaped assembly. Four synaptobrevin 2 TMRs build the core of this complex, surrounded by four TMRs from syntaxin 1A (supplementary figure S3a).


Helical extension of the neuronal SNARE complex into the membrane.

Stein A, Weber G, Wahl MC, Jahn R - Nature (2009)

Synaptobrevin 2 and syntaxin 1A form continuous helices.a, Ribbon plot of the synaptic SNARE complex including linkers and TMRs (colour coding as in Fig. 1a). Sulfate ions and two glycylglycylglycine molecules are depicted as spheres and black sticks, respectively.b, Surface plot showing the electrostatic potential of the synaptic SNARE complex (blue, positive charge; red, negative charge). The electrostatic surface was contoured between – 16 kT/e and + 16 kT/e.c, Ribbon plot showing linkers and TMRs of synaptobrevin 2 and syntaxin 1A. Side chains are shown as sticks, with carbons coloured as the corresponding backbone; oxygen, red; nitrogen, blue. The hydrogen-bond between Asn92 in synaptobrevin 2 and Lys264 in syntaxin 1A is depicted as a black dashed line. The right panel is rotated by 90° about the vertical axis as indicated.d, Amino acids forming interactions between the two linkers and TMRs. Amino acids that are close to each other are connected by black lines, those further away but facing towards each other by gray lines. The last two amino acids in syntaxin 1A were not resolved in the structure. +8 indicates the C-terminal layer of the four helix bundle 49. Note that this layer is not completely resolved as electron density for the last four amino acids of the second helix of SNAP-25 (including the layer-forming amino acid Met202) is absent.
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Figure 2: Synaptobrevin 2 and syntaxin 1A form continuous helices.a, Ribbon plot of the synaptic SNARE complex including linkers and TMRs (colour coding as in Fig. 1a). Sulfate ions and two glycylglycylglycine molecules are depicted as spheres and black sticks, respectively.b, Surface plot showing the electrostatic potential of the synaptic SNARE complex (blue, positive charge; red, negative charge). The electrostatic surface was contoured between – 16 kT/e and + 16 kT/e.c, Ribbon plot showing linkers and TMRs of synaptobrevin 2 and syntaxin 1A. Side chains are shown as sticks, with carbons coloured as the corresponding backbone; oxygen, red; nitrogen, blue. The hydrogen-bond between Asn92 in synaptobrevin 2 and Lys264 in syntaxin 1A is depicted as a black dashed line. The right panel is rotated by 90° about the vertical axis as indicated.d, Amino acids forming interactions between the two linkers and TMRs. Amino acids that are close to each other are connected by black lines, those further away but facing towards each other by gray lines. The last two amino acids in syntaxin 1A were not resolved in the structure. +8 indicates the C-terminal layer of the four helix bundle 49. Note that this layer is not completely resolved as electron density for the last four amino acids of the second helix of SNAP-25 (including the layer-forming amino acid Met202) is absent.
Mentions: As previously seen in the structure of the core complex 7, the four SNARE motifs form a four-helix bundle. The most striking feature is that both syntaxin 1A and synaptobrevin 2 form continuous helices throughout their SNARE motifs, linker regions and TMRs (see figure 2a for an overview, 2b for an electrostatic surface plot, 2c for a close-up view on the linkers and TMRs, and 2d for a schematic representation of the interacting amino acids). In the crystal, hydrophobic contacts between the TMRs of synaptobrevin 2 and syntaxin 1A mediate the association of four complexes via their TMRs, forming an X-shaped assembly. Four synaptobrevin 2 TMRs build the core of this complex, surrounded by four TMRs from syntaxin 1A (supplementary figure S3a).

Bottom Line: Here, we report the X-ray structure of the neuronal SNARE complex, consisting of rat syntaxin 1A, SNAP-25 and synaptobrevin 2, with the carboxy-terminal linkers and transmembrane regions at 3.4 A resolution.The structure shows that assembly proceeds beyond the already known core SNARE complex, resulting in a continuous helical bundle that is further stabilized by side-chain interactions in the linker region.Our results suggest that the final phase of SNARE assembly is directly coupled to membrane merger.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

ABSTRACT
Neurotransmission relies on synaptic vesicles fusing with the membrane of nerve cells to release their neurotransmitter content into the synaptic cleft, a process requiring the assembly of several members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family. SNAREs represent an evolutionarily conserved protein family that mediates membrane fusion in the secretory and endocytic pathways of eukaryotic cells. On membrane contact, these proteins assemble in trans between the membranes as a bundle of four alpha-helices, with the energy released during assembly being thought to drive fusion. However, it is unclear how the energy is transferred to the membranes and whether assembly is conformationally linked to fusion. Here, we report the X-ray structure of the neuronal SNARE complex, consisting of rat syntaxin 1A, SNAP-25 and synaptobrevin 2, with the carboxy-terminal linkers and transmembrane regions at 3.4 A resolution. The structure shows that assembly proceeds beyond the already known core SNARE complex, resulting in a continuous helical bundle that is further stabilized by side-chain interactions in the linker region. Our results suggest that the final phase of SNARE assembly is directly coupled to membrane merger.

Show MeSH
Related in: MedlinePlus