Structure and Conformational Changes in NSF and Its Membrane Receptor Complexes Visualized by Quick-Freeze/Deep-Etch Electron Microscopy

TLDR

The images suggest that NSF may dissociate the SNARE complex, linking its association/dissociation to membrane fusion. Quick‑freeze/deep‑etch EM shows NSF as a hollow 10 × 16 nm cylinder whose nucleotide‑dependent conformation changes to a splayed form, while the SNARE complex is a 4 × 14 nm rod with syntaxin and synaptobrevin aligned parallel, and the rod binds α‑SNAP to one end of NSF to form an asymmetric 20S complex.

Abstract

Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 × 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 × 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with α-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.