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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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Linkers and transmembrane regions add stability to SNARE complexes.a, Protein fragments used in this study. The complex used for crystallization contained all colored segments. For CD measurements, complexes were formed with the same syntaxin 1A fragment but with full-length synaptobrevin 2 and SNAP-25a (all cysteines replaced by serines).b–e, Unfolding of SNARE complexes, monitored by CD spectroscopy at 222nm.b, Thermal unfolding of synaptic SNARE complexes in which the TMRs were either present or in which one or both of the TMRs were lacking. Note that the complex used for crystallization unfolded at approximately 97 °C (see table 1). Sx1a, syntaxin 1A; Syb2, synaptobrevin 2.c, Unfolding of the same complexes at increasing concentrations of guanidine hydrochloride (GdnHCl).d, Thermal unfolding of endosomal SNARE complexes consisting of syntaxin 7, vti1b, syntaxin 8, and endobrevin, either containing or lacking its four TMRs.e, Unfolding of the endosomal complexes at increasing concentrations of GdnHCl.
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Figure 1: Linkers and transmembrane regions add stability to SNARE complexes.a, Protein fragments used in this study. The complex used for crystallization contained all colored segments. For CD measurements, complexes were formed with the same syntaxin 1A fragment but with full-length synaptobrevin 2 and SNAP-25a (all cysteines replaced by serines).b–e, Unfolding of SNARE complexes, monitored by CD spectroscopy at 222nm.b, Thermal unfolding of synaptic SNARE complexes in which the TMRs were either present or in which one or both of the TMRs were lacking. Note that the complex used for crystallization unfolded at approximately 97 °C (see table 1). Sx1a, syntaxin 1A; Syb2, synaptobrevin 2.c, Unfolding of the same complexes at increasing concentrations of guanidine hydrochloride (GdnHCl).d, Thermal unfolding of endosomal SNARE complexes consisting of syntaxin 7, vti1b, syntaxin 8, and endobrevin, either containing or lacking its four TMRs.e, Unfolding of the endosomal complexes at increasing concentrations of GdnHCl.

Mentions: SNAREs are characterized by conserved stretches of 60–70 amino acids termed SNARE motifs 2,8,9. In syntaxin 1A and synaptobrevin 2, the SNARE motifs are connected by short linkers to C-terminal transmembrane regions (TMRs) (Fig. 1a). SNAP-25 is anchored to the plasma membrane via palmitoyl chains bound to cysteine residues in a loop region connecting its two SNARE motifs. SNARE motifs are largely unstructured but spontaneously assemble into helical SNARE complexes 10,11. The X-ray structure of the synaptic core complex, which is paradigmatic for all other SNARE complexes studied so far, revealed four intertwined, parallel α-helices, with each helix being provided by a different SNARE motif. The centre of the bundle contains 16 stacked layers of interacting, mostly hydrophobic side chains 7.


Helical extension of the neuronal SNARE complex into the membrane.

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

Linkers and transmembrane regions add stability to SNARE complexes.a, Protein fragments used in this study. The complex used for crystallization contained all colored segments. For CD measurements, complexes were formed with the same syntaxin 1A fragment but with full-length synaptobrevin 2 and SNAP-25a (all cysteines replaced by serines).b–e, Unfolding of SNARE complexes, monitored by CD spectroscopy at 222nm.b, Thermal unfolding of synaptic SNARE complexes in which the TMRs were either present or in which one or both of the TMRs were lacking. Note that the complex used for crystallization unfolded at approximately 97 °C (see table 1). Sx1a, syntaxin 1A; Syb2, synaptobrevin 2.c, Unfolding of the same complexes at increasing concentrations of guanidine hydrochloride (GdnHCl).d, Thermal unfolding of endosomal SNARE complexes consisting of syntaxin 7, vti1b, syntaxin 8, and endobrevin, either containing or lacking its four TMRs.e, Unfolding of the endosomal complexes at increasing concentrations of GdnHCl.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3108252&req=5

Figure 1: Linkers and transmembrane regions add stability to SNARE complexes.a, Protein fragments used in this study. The complex used for crystallization contained all colored segments. For CD measurements, complexes were formed with the same syntaxin 1A fragment but with full-length synaptobrevin 2 and SNAP-25a (all cysteines replaced by serines).b–e, Unfolding of SNARE complexes, monitored by CD spectroscopy at 222nm.b, Thermal unfolding of synaptic SNARE complexes in which the TMRs were either present or in which one or both of the TMRs were lacking. Note that the complex used for crystallization unfolded at approximately 97 °C (see table 1). Sx1a, syntaxin 1A; Syb2, synaptobrevin 2.c, Unfolding of the same complexes at increasing concentrations of guanidine hydrochloride (GdnHCl).d, Thermal unfolding of endosomal SNARE complexes consisting of syntaxin 7, vti1b, syntaxin 8, and endobrevin, either containing or lacking its four TMRs.e, Unfolding of the endosomal complexes at increasing concentrations of GdnHCl.
Mentions: SNAREs are characterized by conserved stretches of 60–70 amino acids termed SNARE motifs 2,8,9. In syntaxin 1A and synaptobrevin 2, the SNARE motifs are connected by short linkers to C-terminal transmembrane regions (TMRs) (Fig. 1a). SNAP-25 is anchored to the plasma membrane via palmitoyl chains bound to cysteine residues in a loop region connecting its two SNARE motifs. SNARE motifs are largely unstructured but spontaneously assemble into helical SNARE complexes 10,11. The X-ray structure of the synaptic core complex, which is paradigmatic for all other SNARE complexes studied so far, revealed four intertwined, parallel α-helices, with each helix being provided by a different SNARE motif. The centre of the bundle contains 16 stacked layers of interacting, mostly hydrophobic side chains 7.

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