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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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An aromatic layer appears to be crucial for linker contact. Stereo view of the aromatic residues in the linker regions viewed from the N-terminus. Carbon atoms in the side chains of aromatic amino acids are shown in black; otherwise, the same colour-code as in figure 2 is used.
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Figure 3: An aromatic layer appears to be crucial for linker contact. Stereo view of the aromatic residues in the linker regions viewed from the N-terminus. Carbon atoms in the side chains of aromatic amino acids are shown in black; otherwise, the same colour-code as in figure 2 is used.

Mentions: The two complexes in an asymmetric unit are very similar (rmsd = 0.6 Å). The structures of the four-helical core of the complex closely resemble the previously published structures (rmsd = 0.90 and 0.82 Å compared to the structure in ref. 20, except of the last layer (+ 8) that is not completely resolved). The linker region contains a collar of aromatic residues that is surrounded predominantly by basic residues (Fig. 3). Tyr257 is part of this layer and is deeply buried in a pocket formed by three flanking lysine residues of syntaxin 1A (Lys253, Lys256, and Lys260) and four residues of synaptobrevin 2 (Lys85, Arg86, Trp89, and Asn92). In addition, Lys264 of syntaxin 1A engages in a hydrogen bond with Asn92 of synaptobrevin 2. To investigate the importance of amino acid contacts in the linker region, we mutated several residues of syntaxin 1A to alanines (which are readily accommodated in α-helices) and subjected the corresponding complexes to thermal unfolding. Only substitution of Tyr257 resulted in reduced thermal stability while the effects of the other mutations on the melting transition temperature were not significant (table 1). Apparently, intermolecular side chain contacts in the aromatic layer are crucial for the stability and may stiffen the linker region.


Helical extension of the neuronal SNARE complex into the membrane.

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

An aromatic layer appears to be crucial for linker contact. Stereo view of the aromatic residues in the linker regions viewed from the N-terminus. Carbon atoms in the side chains of aromatic amino acids are shown in black; otherwise, the same colour-code as in figure 2 is used.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: An aromatic layer appears to be crucial for linker contact. Stereo view of the aromatic residues in the linker regions viewed from the N-terminus. Carbon atoms in the side chains of aromatic amino acids are shown in black; otherwise, the same colour-code as in figure 2 is used.
Mentions: The two complexes in an asymmetric unit are very similar (rmsd = 0.6 Å). The structures of the four-helical core of the complex closely resemble the previously published structures (rmsd = 0.90 and 0.82 Å compared to the structure in ref. 20, except of the last layer (+ 8) that is not completely resolved). The linker region contains a collar of aromatic residues that is surrounded predominantly by basic residues (Fig. 3). Tyr257 is part of this layer and is deeply buried in a pocket formed by three flanking lysine residues of syntaxin 1A (Lys253, Lys256, and Lys260) and four residues of synaptobrevin 2 (Lys85, Arg86, Trp89, and Asn92). In addition, Lys264 of syntaxin 1A engages in a hydrogen bond with Asn92 of synaptobrevin 2. To investigate the importance of amino acid contacts in the linker region, we mutated several residues of syntaxin 1A to alanines (which are readily accommodated in α-helices) and subjected the corresponding complexes to thermal unfolding. Only substitution of Tyr257 resulted in reduced thermal stability while the effects of the other mutations on the melting transition temperature were not significant (table 1). Apparently, intermolecular side chain contacts in the aromatic layer are crucial for the stability and may stiffen the linker region.

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