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.
authors.affiliation: The Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Arne
authors.initials: A
authors.lastname: Moeller
authors.forename: Chunxia
authors.initials: C
authors.lastname: Zhao
authors.forename: Michael G
authors.initials: MG
authors.lastname: Fried
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Wilson-Kubalek
authors.forename: Bridget
authors.initials: B
authors.lastname: Carragher
authors.forename: Sidney W
authors.initials: SW
authors.lastname: Whiteheart
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry*; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D008854:Microscopy, Electron; D008958:Models, Molecular; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D050600:SNARE Proteins / Q000378:metabolism*; D013499:Surface Properties; D014461:Ultracentrifugation
chemical_list: D000227:Adenine Nucleotides; D050600:SNARE Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins