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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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Comparison of ATP- and ADP-bound NSF structures (a–c), and ATPase domainsof ATP-bound NSF and 20S supercomplex (d–f)a–c, Surface representations of the D2, D1 and N domainsof ATP- and ADP-bound NSF (looking down from the N side of the NSF hexamer). The maximumdiameters of the D2 and D1 rings, and the interface areas (calculated by PISA66) between ATPase domains are indicated.Each protomer chain is colored as in Fig. 2. The D1ring is also shown in panel c and colored white to help with visualization.d–f, The ATPase domains of the structure of the 20S supercomplex(state I) were superposed on the ATP-bound NSF using the D1 ring as the reference forthe fit. Six protomer chains from ATP-bound NSF are rainbow colored counterclockwisefrom the top based on the relative positions of the D1 domains to the D2 ring. TheATPase domains of the 20S supercomplex are colored in white and grey. Note that thedensity of Chain F in the EM reconstruction of ATP-bound NSF alone is poorly resolved(Fig. 1b), whereas in the 20S reconstruction itis well defined, although the overall resolution of the 20S reconstruction is lower.d, Side views. e, Top view of the D2 rings. Each individualD2 domain is labeled. Percentages of interface area change (from NSF to 20S) between theD2 domains are provided in the figure. The interface areas between the D2 domains aresimilar in the NSF and 20S structures, except for a significant increase (12%)between Chains D and E for 20S compared to NSF alone. f, Top view of the D1rings. Each D1 domain is labeled, with the split between Chains A and F indicated by ablack arrow. The translation of the α7 helix in α subdomain of Chain Ais illustrated in the inset. Percentages of interface area change (from NSF to 20S)between the D1 domains are shown. Three stay the same; the one between Chains A and Bdecreases, whereas those between Chains E and F, and Chains F and A increasesignificantly.
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Figure 15: Comparison of ATP- and ADP-bound NSF structures (a–c), and ATPase domainsof ATP-bound NSF and 20S supercomplex (d–f)a–c, Surface representations of the D2, D1 and N domainsof ATP- and ADP-bound NSF (looking down from the N side of the NSF hexamer). The maximumdiameters of the D2 and D1 rings, and the interface areas (calculated by PISA66) between ATPase domains are indicated.Each protomer chain is colored as in Fig. 2. The D1ring is also shown in panel c and colored white to help with visualization.d–f, The ATPase domains of the structure of the 20S supercomplex(state I) were superposed on the ATP-bound NSF using the D1 ring as the reference forthe fit. Six protomer chains from ATP-bound NSF are rainbow colored counterclockwisefrom the top based on the relative positions of the D1 domains to the D2 ring. TheATPase domains of the 20S supercomplex are colored in white and grey. Note that thedensity of Chain F in the EM reconstruction of ATP-bound NSF alone is poorly resolved(Fig. 1b), whereas in the 20S reconstruction itis well defined, although the overall resolution of the 20S reconstruction is lower.d, Side views. e, Top view of the D2 rings. Each individualD2 domain is labeled. Percentages of interface area change (from NSF to 20S) between theD2 domains are provided in the figure. The interface areas between the D2 domains aresimilar in the NSF and 20S structures, except for a significant increase (12%)between Chains D and E for 20S compared to NSF alone. f, Top view of the D1rings. Each D1 domain is labeled, with the split between Chains A and F indicated by ablack arrow. The translation of the α7 helix in α subdomain of Chain Ais illustrated in the inset. Percentages of interface area change (from NSF to 20S)between the D1 domains are shown. Three stay the same; the one between Chains A and Bdecreases, whereas those between Chains E and F, and Chains F and A increasesignificantly.

Mentions: Both the ATP- and ADP-bound structures of NSF are organized into three layers: tworings consisting of six D2 domains and six D1 domains, respectively, and a layer of six(four) N domains for ATP (ADP)-bound NSF (Figs. 1c, e,and 2a, b). For ADP-bound NSF, the remaining two Ndomains are flipped along the sides of the ATPase rings with well resolved densitiescompared to the N domains atop the D1 ring, leaving little doubt as regards the identity ofthese two densities (Fig. 1e and Extended Data Figs. 4c, e and 7c).


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)

Comparison of ATP- and ADP-bound NSF structures (a–c), and ATPase domainsof ATP-bound NSF and 20S supercomplex (d–f)a–c, Surface representations of the D2, D1 and N domainsof ATP- and ADP-bound NSF (looking down from the N side of the NSF hexamer). The maximumdiameters of the D2 and D1 rings, and the interface areas (calculated by PISA66) between ATPase domains are indicated.Each protomer chain is colored as in Fig. 2. The D1ring is also shown in panel c and colored white to help with visualization.d–f, The ATPase domains of the structure of the 20S supercomplex(state I) were superposed on the ATP-bound NSF using the D1 ring as the reference forthe fit. Six protomer chains from ATP-bound NSF are rainbow colored counterclockwisefrom the top based on the relative positions of the D1 domains to the D2 ring. TheATPase domains of the 20S supercomplex are colored in white and grey. Note that thedensity of Chain F in the EM reconstruction of ATP-bound NSF alone is poorly resolved(Fig. 1b), whereas in the 20S reconstruction itis well defined, although the overall resolution of the 20S reconstruction is lower.d, Side views. e, Top view of the D2 rings. Each individualD2 domain is labeled. Percentages of interface area change (from NSF to 20S) between theD2 domains are provided in the figure. The interface areas between the D2 domains aresimilar in the NSF and 20S structures, except for a significant increase (12%)between Chains D and E for 20S compared to NSF alone. f, Top view of the D1rings. Each D1 domain is labeled, with the split between Chains A and F indicated by ablack arrow. The translation of the α7 helix in α subdomain of Chain Ais illustrated in the inset. Percentages of interface area change (from NSF to 20S)between the D1 domains are shown. Three stay the same; the one between Chains A and Bdecreases, whereas those between Chains E and F, and Chains F and A increasesignificantly.
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Figure 15: Comparison of ATP- and ADP-bound NSF structures (a–c), and ATPase domainsof ATP-bound NSF and 20S supercomplex (d–f)a–c, Surface representations of the D2, D1 and N domainsof ATP- and ADP-bound NSF (looking down from the N side of the NSF hexamer). The maximumdiameters of the D2 and D1 rings, and the interface areas (calculated by PISA66) between ATPase domains are indicated.Each protomer chain is colored as in Fig. 2. The D1ring is also shown in panel c and colored white to help with visualization.d–f, The ATPase domains of the structure of the 20S supercomplex(state I) were superposed on the ATP-bound NSF using the D1 ring as the reference forthe fit. Six protomer chains from ATP-bound NSF are rainbow colored counterclockwisefrom the top based on the relative positions of the D1 domains to the D2 ring. TheATPase domains of the 20S supercomplex are colored in white and grey. Note that thedensity of Chain F in the EM reconstruction of ATP-bound NSF alone is poorly resolved(Fig. 1b), whereas in the 20S reconstruction itis well defined, although the overall resolution of the 20S reconstruction is lower.d, Side views. e, Top view of the D2 rings. Each individualD2 domain is labeled. Percentages of interface area change (from NSF to 20S) between theD2 domains are provided in the figure. The interface areas between the D2 domains aresimilar in the NSF and 20S structures, except for a significant increase (12%)between Chains D and E for 20S compared to NSF alone. f, Top view of the D1rings. Each D1 domain is labeled, with the split between Chains A and F indicated by ablack arrow. The translation of the α7 helix in α subdomain of Chain Ais illustrated in the inset. Percentages of interface area change (from NSF to 20S)between the D1 domains are shown. Three stay the same; the one between Chains A and Bdecreases, whereas those between Chains E and F, and Chains F and A increasesignificantly.
Mentions: Both the ATP- and ADP-bound structures of NSF are organized into three layers: tworings consisting of six D2 domains and six D1 domains, respectively, and a layer of six(four) N domains for ATP (ADP)-bound NSF (Figs. 1c, e,and 2a, b). For ADP-bound NSF, the remaining two Ndomains are flipped along the sides of the ATPase rings with well resolved densitiescompared to the N domains atop the D1 ring, leaving little doubt as regards the identity ofthese two densities (Fig. 1e and Extended Data Figs. 4c, e and 7c).

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.

Show MeSH
Related in: MedlinePlus