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Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine.

Cipriano DJ, Jung J, Vivona S, Fenn TD, Brunger AT, Bryant Z - J. Biol. Chem. (2013)

Bottom Line: NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism.We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF.We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.

ABSTRACT
SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

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Possible models for the action of NSF in SNARE disassembly. For simplicity, the model only shows the core SNARE helices (and respective transmembrane domains), which are drawn as cylinders. Three classes of models are shown. Distributive unwinding involves multiple NSF binding and rebinding events, whereas processive and global unwinding involve the binding and action of a single NSF hexamer. Shown are specific examples of a global model (the socket-wrench model) and a processive model (the threading/helicase-like model) that have been proposed in the literature (16). In the socket-wrench model, conformational changes in NSF are transmitted through α-SNAP to apply a torque or force on the SNAREs that results in the global unwinding of the SNARE core. In the threading/helicase-like model, NSF unwinds the SNARES by the ATP-powered processive threading of one of the SNARE proteins through the pore of the hexamer. Note that the stoichiometry of the 20S complex is still uncertain; initial work suggested that three α-SNAP bound to one SNARE complex and one NSF (56). However, recent work (see Footnote 4) indicates that the initial α-SNAP-SNARE complex has a stoichiometry of 1:1, although this finding does not rule out the involvement of additional α-SNAPs in the 20S complex and during the disassembly reaction. For simplicity, the graphics in Fig. 1 are shown with three α-SNAPs.
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Figure 1: Possible models for the action of NSF in SNARE disassembly. For simplicity, the model only shows the core SNARE helices (and respective transmembrane domains), which are drawn as cylinders. Three classes of models are shown. Distributive unwinding involves multiple NSF binding and rebinding events, whereas processive and global unwinding involve the binding and action of a single NSF hexamer. Shown are specific examples of a global model (the socket-wrench model) and a processive model (the threading/helicase-like model) that have been proposed in the literature (16). In the socket-wrench model, conformational changes in NSF are transmitted through α-SNAP to apply a torque or force on the SNAREs that results in the global unwinding of the SNARE core. In the threading/helicase-like model, NSF unwinds the SNARES by the ATP-powered processive threading of one of the SNARE proteins through the pore of the hexamer. Note that the stoichiometry of the 20S complex is still uncertain; initial work suggested that three α-SNAP bound to one SNARE complex and one NSF (56). However, recent work (see Footnote 4) indicates that the initial α-SNAP-SNARE complex has a stoichiometry of 1:1, although this finding does not rule out the involvement of additional α-SNAPs in the 20S complex and during the disassembly reaction. For simplicity, the graphics in Fig. 1 are shown with three α-SNAPs.

Mentions: In this work, we endeavor to distinguish between three broad classes of models for the action of NSF on the SNARE complex (Fig. 1). In a distributive model, complete unwinding of the SNARE complex requires many NSF binding and release events; a single binding event leads to only partial unwinding of the SNARE core, and the partially unwound complex then requires additional NSF binding events to complete the disassembly process. In a global unwinding model, NSF acts to destabilize the entire SNARE complex, whereas in a processive unwinding model, NSF advances stepwise from one end of the complex, progressively increasing the number of unwound residues until the entire complex is disassembled. The previously proposed “socket-wrench model” (10, 16, 17) is an example of global unwinding, and the “threading/helicase-like model” (16, 17) is an example of processive unwinding.


Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine.

Cipriano DJ, Jung J, Vivona S, Fenn TD, Brunger AT, Bryant Z - J. Biol. Chem. (2013)

Possible models for the action of NSF in SNARE disassembly. For simplicity, the model only shows the core SNARE helices (and respective transmembrane domains), which are drawn as cylinders. Three classes of models are shown. Distributive unwinding involves multiple NSF binding and rebinding events, whereas processive and global unwinding involve the binding and action of a single NSF hexamer. Shown are specific examples of a global model (the socket-wrench model) and a processive model (the threading/helicase-like model) that have been proposed in the literature (16). In the socket-wrench model, conformational changes in NSF are transmitted through α-SNAP to apply a torque or force on the SNAREs that results in the global unwinding of the SNARE core. In the threading/helicase-like model, NSF unwinds the SNARES by the ATP-powered processive threading of one of the SNARE proteins through the pore of the hexamer. Note that the stoichiometry of the 20S complex is still uncertain; initial work suggested that three α-SNAP bound to one SNARE complex and one NSF (56). However, recent work (see Footnote 4) indicates that the initial α-SNAP-SNARE complex has a stoichiometry of 1:1, although this finding does not rule out the involvement of additional α-SNAPs in the 20S complex and during the disassembly reaction. For simplicity, the graphics in Fig. 1 are shown with three α-SNAPs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520572&req=5

Figure 1: Possible models for the action of NSF in SNARE disassembly. For simplicity, the model only shows the core SNARE helices (and respective transmembrane domains), which are drawn as cylinders. Three classes of models are shown. Distributive unwinding involves multiple NSF binding and rebinding events, whereas processive and global unwinding involve the binding and action of a single NSF hexamer. Shown are specific examples of a global model (the socket-wrench model) and a processive model (the threading/helicase-like model) that have been proposed in the literature (16). In the socket-wrench model, conformational changes in NSF are transmitted through α-SNAP to apply a torque or force on the SNAREs that results in the global unwinding of the SNARE core. In the threading/helicase-like model, NSF unwinds the SNARES by the ATP-powered processive threading of one of the SNARE proteins through the pore of the hexamer. Note that the stoichiometry of the 20S complex is still uncertain; initial work suggested that three α-SNAP bound to one SNARE complex and one NSF (56). However, recent work (see Footnote 4) indicates that the initial α-SNAP-SNARE complex has a stoichiometry of 1:1, although this finding does not rule out the involvement of additional α-SNAPs in the 20S complex and during the disassembly reaction. For simplicity, the graphics in Fig. 1 are shown with three α-SNAPs.
Mentions: In this work, we endeavor to distinguish between three broad classes of models for the action of NSF on the SNARE complex (Fig. 1). In a distributive model, complete unwinding of the SNARE complex requires many NSF binding and release events; a single binding event leads to only partial unwinding of the SNARE core, and the partially unwound complex then requires additional NSF binding events to complete the disassembly process. In a global unwinding model, NSF acts to destabilize the entire SNARE complex, whereas in a processive unwinding model, NSF advances stepwise from one end of the complex, progressively increasing the number of unwound residues until the entire complex is disassembled. The previously proposed “socket-wrench model” (10, 16, 17) is an example of global unwinding, and the “threading/helicase-like model” (16, 17) is an example of processive unwinding.

Bottom Line: NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism.We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF.We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.

ABSTRACT
SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

Show MeSH
Related in: MedlinePlus