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Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between α-soluble NSF attachment protein (SNAP) and SNARE complex.

Vivona S, Cipriano DJ, O'Leary S, Li YH, Fenn TD, Brunger AT - J. Biol. Chem. (2013)

Bottom Line: By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering.We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding.Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

View Article: PubMed Central - PubMed

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

ABSTRACT
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

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Conservation of the electrostatic potential surface of ternary SNARE complexes.Upper, electrostatic potential surface maps of (from left to right) the VAMP2-syntaxin1-SNAP25, VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP8-syntaxin4-SNAP23 core complexes (i.e. without the respective N-terminal domains). Center, 120° counterclockwise rotation of the top view. Lower, 120° counterclockwise rotation of the center view. The electrostatic potential surface maps were calculated in vacuum with PyMOL and contoured with the ABPS module from −10 kT/e (red) to +10 kT/e (blue). The SNARE core complexes of VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP7-syntaxin4-SNAP23 were modeled based on the crystal structure of VAMP2-syntaxin1-SNAP25 (Protein Data Bank code 1SFC).
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Figure 4: Conservation of the electrostatic potential surface of ternary SNARE complexes.Upper, electrostatic potential surface maps of (from left to right) the VAMP2-syntaxin1-SNAP25, VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP8-syntaxin4-SNAP23 core complexes (i.e. without the respective N-terminal domains). Center, 120° counterclockwise rotation of the top view. Lower, 120° counterclockwise rotation of the center view. The electrostatic potential surface maps were calculated in vacuum with PyMOL and contoured with the ABPS module from −10 kT/e (red) to +10 kT/e (blue). The SNARE core complexes of VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP7-syntaxin4-SNAP23 were modeled based on the crystal structure of VAMP2-syntaxin1-SNAP25 (Protein Data Bank code 1SFC).

Mentions: Our kinetic and binding data suggest a conserved mechanism for NSF-driven disassembly of SNARE complexes, mediated by a conserved interaction between α-SNAP and both binary and ternary SNARE complexes. These common features may arise from a conserved electrostatic potential surface of the SNARE complex, based on the observation that the SNARE complex interacts with α-SNAP mostly through its acidic residues (17). Indeed, modeling the SNARE core bundles of VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP8-syntaxin4-SNAP23 based on the crystal structure of the neuronal SNARE complex (VAMP2-syntaxin1-SNAP25; Protein Data Bank Code 1SFC (4)) revealed a conserved electrostatic potential surface distribution (Fig. 4), suggesting that α-SNAP recognizes different SNARE complexes via conserved features of their electrostatic potential surfaces.


Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between α-soluble NSF attachment protein (SNAP) and SNARE complex.

Vivona S, Cipriano DJ, O'Leary S, Li YH, Fenn TD, Brunger AT - J. Biol. Chem. (2013)

Conservation of the electrostatic potential surface of ternary SNARE complexes.Upper, electrostatic potential surface maps of (from left to right) the VAMP2-syntaxin1-SNAP25, VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP8-syntaxin4-SNAP23 core complexes (i.e. without the respective N-terminal domains). Center, 120° counterclockwise rotation of the top view. Lower, 120° counterclockwise rotation of the center view. The electrostatic potential surface maps were calculated in vacuum with PyMOL and contoured with the ABPS module from −10 kT/e (red) to +10 kT/e (blue). The SNARE core complexes of VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP7-syntaxin4-SNAP23 were modeled based on the crystal structure of VAMP2-syntaxin1-SNAP25 (Protein Data Bank code 1SFC).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750193&req=5

Figure 4: Conservation of the electrostatic potential surface of ternary SNARE complexes.Upper, electrostatic potential surface maps of (from left to right) the VAMP2-syntaxin1-SNAP25, VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP8-syntaxin4-SNAP23 core complexes (i.e. without the respective N-terminal domains). Center, 120° counterclockwise rotation of the top view. Lower, 120° counterclockwise rotation of the center view. The electrostatic potential surface maps were calculated in vacuum with PyMOL and contoured with the ABPS module from −10 kT/e (red) to +10 kT/e (blue). The SNARE core complexes of VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP7-syntaxin4-SNAP23 were modeled based on the crystal structure of VAMP2-syntaxin1-SNAP25 (Protein Data Bank code 1SFC).
Mentions: Our kinetic and binding data suggest a conserved mechanism for NSF-driven disassembly of SNARE complexes, mediated by a conserved interaction between α-SNAP and both binary and ternary SNARE complexes. These common features may arise from a conserved electrostatic potential surface of the SNARE complex, based on the observation that the SNARE complex interacts with α-SNAP mostly through its acidic residues (17). Indeed, modeling the SNARE core bundles of VAMP7-syntaxin1-SNAP25, VAMP7-syntaxin4-SNAP23, and VAMP8-syntaxin4-SNAP23 based on the crystal structure of the neuronal SNARE complex (VAMP2-syntaxin1-SNAP25; Protein Data Bank Code 1SFC (4)) revealed a conserved electrostatic potential surface distribution (Fig. 4), suggesting that α-SNAP recognizes different SNARE complexes via conserved features of their electrostatic potential surfaces.

Bottom Line: By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering.We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding.Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

View Article: PubMed Central - PubMed

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

ABSTRACT
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

Show MeSH
Related in: MedlinePlus