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Mechanistic insights into the recycling machine of the SNARE complex.

Zhao M, Wu S, Zhou Q, Vivona S, Cipriano DJ, Cheng Y, Brunger AT - Nature (2015)

Bottom Line: The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex.SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism.The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.

ABSTRACT
Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

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Detrimental effect of imposing C6 symmetry on the EM reconstruction of NSF and C3symmetry on the EM reconstruction of the 20S supercomplexa, For the ADP-bound NSF maps, in order to visualize densities ofthe N domains, an unsharpened map is displayed (translucent surface, C1: 1.2 σ,C6: 0.6 σ) together with the sharpened map using no symmetry (C1) or C6 symmetryduring reconstruction (colored surface, C1: 5.9 σ, C6: 7.0 σ). For thereconstruction that uses C6 symmetry, symmetric densities for the N domains at top andside positions appear in the unsharpened map, however, these densities cannot be matchedto the crystal structure of the N domain. Likewise, the D1 domains appear compressed andcannot be fit well using the structure of the D1 domain that we obtained by asymmetricreconstruction. b, Reconstruction of state I of the 20S supercomplexwithout symmetry (C1) or with C3 symmetry. Maps are shown in colored surfaces similar toFig. 3 (C1: 4.7 σ, C3: 4.9 σ).The C3 averaging causes the D1 domains to display alternating up and down positions. Thedensity for the SNARE complex is a featureless rod without the characteristicleft-handed twist of the four α-helix bundle. Densities for only three SNAPsemerge, but without any interpretable features (e.g. grooves betweenhelices), preventing a match with the crystal structure of the known homolog ofαSNAP, Sec17. The N domain densities are weak and none of them exhibit theexpected kidney shape.
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Figure 13: Detrimental effect of imposing C6 symmetry on the EM reconstruction of NSF and C3symmetry on the EM reconstruction of the 20S supercomplexa, For the ADP-bound NSF maps, in order to visualize densities ofthe N domains, an unsharpened map is displayed (translucent surface, C1: 1.2 σ,C6: 0.6 σ) together with the sharpened map using no symmetry (C1) or C6 symmetryduring reconstruction (colored surface, C1: 5.9 σ, C6: 7.0 σ). For thereconstruction that uses C6 symmetry, symmetric densities for the N domains at top andside positions appear in the unsharpened map, however, these densities cannot be matchedto the crystal structure of the N domain. Likewise, the D1 domains appear compressed andcannot be fit well using the structure of the D1 domain that we obtained by asymmetricreconstruction. b, Reconstruction of state I of the 20S supercomplexwithout symmetry (C1) or with C3 symmetry. Maps are shown in colored surfaces similar toFig. 3 (C1: 4.7 σ, C3: 4.9 σ).The C3 averaging causes the D1 domains to display alternating up and down positions. Thedensity for the SNARE complex is a featureless rod without the characteristicleft-handed twist of the four α-helix bundle. Densities for only three SNAPsemerge, but without any interpretable features (e.g. grooves betweenhelices), preventing a match with the crystal structure of the known homolog ofαSNAP, Sec17. The N domain densities are weak and none of them exhibit theexpected kidney shape.

Mentions: The cryo-EM datasets of the NSF particles used in this study were of sufficientquality to determine and refine 3D reconstructions to high resolution without imposing anysymmetry, which turned out to be critical (Extended Data Fig.5, see SupplementaryInformation for a detailed discussion).


Mechanistic insights into the recycling machine of the SNARE complex.

Zhao M, Wu S, Zhou Q, Vivona S, Cipriano DJ, Cheng Y, Brunger AT - Nature (2015)

Detrimental effect of imposing C6 symmetry on the EM reconstruction of NSF and C3symmetry on the EM reconstruction of the 20S supercomplexa, For the ADP-bound NSF maps, in order to visualize densities ofthe N domains, an unsharpened map is displayed (translucent surface, C1: 1.2 σ,C6: 0.6 σ) together with the sharpened map using no symmetry (C1) or C6 symmetryduring reconstruction (colored surface, C1: 5.9 σ, C6: 7.0 σ). For thereconstruction that uses C6 symmetry, symmetric densities for the N domains at top andside positions appear in the unsharpened map, however, these densities cannot be matchedto the crystal structure of the N domain. Likewise, the D1 domains appear compressed andcannot be fit well using the structure of the D1 domain that we obtained by asymmetricreconstruction. b, Reconstruction of state I of the 20S supercomplexwithout symmetry (C1) or with C3 symmetry. Maps are shown in colored surfaces similar toFig. 3 (C1: 4.7 σ, C3: 4.9 σ).The C3 averaging causes the D1 domains to display alternating up and down positions. Thedensity for the SNARE complex is a featureless rod without the characteristicleft-handed twist of the four α-helix bundle. Densities for only three SNAPsemerge, but without any interpretable features (e.g. grooves betweenhelices), preventing a match with the crystal structure of the known homolog ofαSNAP, Sec17. The N domain densities are weak and none of them exhibit theexpected kidney shape.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4320033&req=5

Figure 13: Detrimental effect of imposing C6 symmetry on the EM reconstruction of NSF and C3symmetry on the EM reconstruction of the 20S supercomplexa, For the ADP-bound NSF maps, in order to visualize densities ofthe N domains, an unsharpened map is displayed (translucent surface, C1: 1.2 σ,C6: 0.6 σ) together with the sharpened map using no symmetry (C1) or C6 symmetryduring reconstruction (colored surface, C1: 5.9 σ, C6: 7.0 σ). For thereconstruction that uses C6 symmetry, symmetric densities for the N domains at top andside positions appear in the unsharpened map, however, these densities cannot be matchedto the crystal structure of the N domain. Likewise, the D1 domains appear compressed andcannot be fit well using the structure of the D1 domain that we obtained by asymmetricreconstruction. b, Reconstruction of state I of the 20S supercomplexwithout symmetry (C1) or with C3 symmetry. Maps are shown in colored surfaces similar toFig. 3 (C1: 4.7 σ, C3: 4.9 σ).The C3 averaging causes the D1 domains to display alternating up and down positions. Thedensity for the SNARE complex is a featureless rod without the characteristicleft-handed twist of the four α-helix bundle. Densities for only three SNAPsemerge, but without any interpretable features (e.g. grooves betweenhelices), preventing a match with the crystal structure of the known homolog ofαSNAP, Sec17. The N domain densities are weak and none of them exhibit theexpected kidney shape.
Mentions: The cryo-EM datasets of the NSF particles used in this study were of sufficientquality to determine and refine 3D reconstructions to high resolution without imposing anysymmetry, which turned out to be critical (Extended Data Fig.5, see SupplementaryInformation for a detailed discussion).

Bottom Line: The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex.SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism.The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.

ABSTRACT
Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

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