Structural characterization of full-length NSF and 20S particles

Abstract

The 20S particle, which is composed of the N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs) and the SNAP receptor (SNARE) complex, has an essential role in intracellular vesicle fusion events. Using single-particle cryo-EM and negative stain EM, we reconstructed four related three-dimensional structures: Chinese hamster NSF hexamer in the ATPγS, ADP-AlFx and ADP states, and the 20S particle. These structures reveal a parallel arrangement between the D1 and D2 domains of the hexameric NSF and characterize the nucleotide-dependent conformational changes in NSF. The structure of the 20S particle shows that it holds the SNARE complex at two interaction interfaces around the C terminus and N-terminal half of the SNARE complex, respectively. These findings provide insight into the molecular mechanism underlying disassembly of the SNARE complex by NSF.

Cryo-EM structure of SNAP-SNARE assembly in 20S particle

“…The D1-D2 model segmented from the low-resolution reconstruction of the entire 20S particle shows very similar features to our previous 3D reconstruction of the NSF in the ATP state [17], allowing us to segment the upper part of each 20S particle image by subtracting the NSF D1-D2 ring density from the raw image and perform sin-npg www.cell-research.com | Cell Research gle-particle analysis of the resulted images (Supplementary information, Figures S3, S4 and Data S1) to overcome the flexibility difficulty. Indeed, this practice led to a much better defined 2D class averages of the upper part of the 20S particle with secondary structural elements clearly visible ( Figure 1C).…”

“…This indicates that the overall composition and architecture of the 20S particle is preserved in different means of reconstitution. 3D reconstruction from the negatively stained ND-20S particles generated a map with a similar overall shape to our previously reconstructed 20S particle [17], but having an addition cap density of the nanodisc on the top (Supplementary information, Figure S2D). …”

“…To understand the mechanism of SNARE disassembly in the 20S particle, we previously reconstituted 20S particles with a quality suitable for EM structural analysis [17]. The reconstituted 20S particles, in an NP40-containing buffer solution, had full SNARE assembly activity but showed partial aggregation with their SNARE ends attaching to detergent micelles as observed under EM (Supplementary information, Figure S1A-S1C).…”

“…D2 domain is responsible for maintaining NSF as a hexamer, while D1 domain is catalytically active and functions to hydrolyze ATP accompanied by a major conformational change of itself as well as the N-domain attached to it [16][17][18]. Previous studies indicated that the N-domain of NSF interacts with the C-terminal end of α-SNAP, which mediates the interaction of NSF and the SNARE complex [19][20][21][22][23].…”

“…The positively charged residues in the concave surface of the N-terminal sheet of α-SNAP were shown to be involved in the interaction with the SNARE complex [25]. Despite extensive studies of the structures of NSF, α-SNAP and the SNARE complex individually, how the SNARE complex is disassembled in the 20S particle, SNAP-SNARE assembly in 20S particle however, is still enigmatic due to the limited structural information for the entire 20S particle [17,[26][27][28].…”