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/f0). Hydrodynamic analyses of NSF mutants 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.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 SWW. AM received support from the NIH Roadmap Award P50 GM073197.