Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly

Arne Moeller; Chunxia Zhao; Michael G. Fried; Elizabeth M. Wilson-Kubalek; Bridget Carragher; Sidney W. Whiteheart

doi:10.1016/j.jsb.2011.12.018

Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly

Arne Moeller, Chunxia Zhao, Michael G. Fried, Elizabeth M. Wilson-Kubalek, Bridget Carragher, Sidney W. Whiteheart

Research output: Contribution to journal › Article › peer-review

20 Scopus citations

Abstract

Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f ₀). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.

Original language	English
Pages (from-to)	335-343
Number of pages	9
Journal	Journal of Structural Biology
Volume	177
Issue number	2
DOIs	https://doi.org/10.1016/j.jsb.2011.12.018
State	Published - Feb 2012

Bibliographical note

Funding Information:
The electron microscopy and image analysis presented here was conducted at the National Resource for Automated Molecular Microscopy which is supported by the National Institutes of Health though the National Center for Research Resources’ P41 program (RR017573). The work was also supported by Grants HL56652 and HL091893 from the National Institutes of Health to S.W.W. A.M. received support from the NIH Roadmap Award P50 GM073197 and E.W. and B.C. received support from GM095573 . We thank Dr. Lance Hellman for his help with the analytical ultracentrifugation experiments and Dr. Elena A. Matveeva for her insightful comments on the manuscript.

Keywords

AAA proteins
Analytical centrifugation
Electron microscopy
Membrane trafficking
SNAREs

ASJC Scopus subject areas

Structural Biology

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10.1016/j.jsb.2011.12.018

Cite this

@article{07da0f3040b545ec8c993aa3f62e1610,

title = "Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly",

abstract = "Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f 0). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.",

keywords = "AAA proteins, Analytical centrifugation, Electron microscopy, Membrane trafficking, SNAREs",

author = "Arne Moeller and Chunxia Zhao and Fried, {Michael G.} and Wilson-Kubalek, {Elizabeth M.} and Bridget Carragher and Whiteheart, {Sidney W.}",

note = "Funding Information: The electron microscopy and image analysis presented here was conducted at the National Resource for Automated Molecular Microscopy which is supported by the National Institutes of Health though the National Center for Research Resources{\textquoteright} P41 program (RR017573). The work was also supported by Grants HL56652 and HL091893 from the National Institutes of Health to S.W.W. A.M. received support from the NIH Roadmap Award P50 GM073197 and E.W. and B.C. received support from GM095573 . We thank Dr. Lance Hellman for his help with the analytical ultracentrifugation experiments and Dr. Elena A. Matveeva for her insightful comments on the manuscript. ",

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AU - Zhao, Chunxia

AU - Fried, Michael G.

AU - Wilson-Kubalek, Elizabeth M.

AU - Carragher, Bridget

AU - Whiteheart, Sidney W.

N1 - Funding Information: The electron microscopy and image analysis presented here was conducted at the National Resource for Automated Molecular Microscopy which is supported by the National Institutes of Health though the National Center for Research Resources’ P41 program (RR017573). The work was also supported by Grants HL56652 and HL091893 from the National Institutes of Health to S.W.W. A.M. received support from the NIH Roadmap Award P50 GM073197 and E.W. and B.C. received support from GM095573 . We thank Dr. Lance Hellman for his help with the analytical ultracentrifugation experiments and Dr. Elena A. Matveeva for her insightful comments on the manuscript.

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N2 - Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f 0). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.

AB - Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f 0). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.

KW - AAA proteins

KW - Analytical centrifugation

KW - Electron microscopy

KW - Membrane trafficking

KW - SNAREs

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Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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