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

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...

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Bibliographic Details
Published inJournal of structural biology Vol. 177; no. 2; pp. 335 - 343
Main Authors Moeller, Arne, Zhao, Chunxia, Fried, Michael G., Wilson-Kubalek, Elizabeth M., Carragher, Bridget, Whiteheart, Sidney W.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 01.02.2012
Subjects
AAA proteins
Adenine Nucleotides - chemistry
Amino Acid Substitution
Analytical centrifugation
Animals
CHO Cells
Cricetinae
Electron microscopy
Membrane trafficking
Microscopy, Electron
Models, Molecular
N-Ethylmaleimide-Sensitive Proteins - chemistry
N-Ethylmaleimide-Sensitive Proteins - genetics
Protein Binding
Protein Structure, Quaternary
Protein Structure, Tertiary
SNARE Proteins - metabolism
SNAREs
Surface Properties
Ultracentrifugation
NSF
AAA
VCP
EM
AAA proteins
SNAP
Electron microscopy
PDB
SNARE
AU
SNAREs
Membrane trafficking
Analytical centrifugation
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Summary: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/f0). 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.
ISSN:1047-8477
1095-8657
DOI:10.1016/j.jsb.2011.12.018