Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA+ ATPase N-Ethylmaleimide-Sensitive Factor (NSF)

Abstract

N-ethylmaleimide-sensitive factor (NSF), first discovered in 1988, is a key factor for eukaryotic trafficking, including protein and hormone secretion and neurotransmitter release. It is a member of the AAA+ family (ATPases associated with diverse cellular activities). NSF disassembles soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes in conjunction with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP). Structural studies of NSF and its complex with SNAREs and SNAPs (known as 20S supercomplex) started about 20years ago. Crystal structures of individual N and D2 domains of NSF and low-resolution electron microscopy structures of full-length NSF and 20S supercomplex have been reported over the years. Nevertheless, the molecular architecture of the 20S supercomplex and the molecular mechanism of NSF-mediated SNARE complex disassembly remained unclear until recently. Here we review recent atomic-resolution or near-atomic resolution structures of NSF and of the 20S supercomplex, as well as recent insights into the molecular mechanism and energy requirements of NSF. We also compare NSF with other known AAA+ family members.

Keywords: ATPase; NSF; SNAREs; eukaryotic trafficking; protein disassembly.

Fig. 1. Domain architecture of NSF

Crystal structures of the N domain (PDB accession code: 1qcs), and the D2 domain (PDB accession code: 1nsf) are shown. The structure of the D1 domain is from the cryo-EM structure of NSF in the ATP state (PDB accession code: 3j94). The structures are shown in scale, with subdomains marked. D1 and D2 domains are aligned in a similar orientation with all the conserved secondary structure elements of the AAA+ domain (defined in [22]) labeled. Compared to the D2 domain, the D1 domain has a characteristic bent α2 helix.

Fig. 2. A history of EM studies on 20S supercomplex

Representative images or reconstructions are shown on the time axis. They are adapted from Figure 6 of Hanson et al., 1997 (panel a), Figure 2 of Hohl et al., 1998 (panel b), and Figure 6 of Chang et al, 2012 (panel d), respectively. In addition, the maps of Furst et al., 2003 (panel c) and Zhao et al., 2015 (EMD-6206, state I, panel e) are recolored in the same scheme as in Figures 3 and 4.

Fig. 3. EM structure of the 20S supercomplex (state I) at 7.6 Å resolution in the presence of AMPPNP

Structures of individual proteins domains had been previously determined by X-ray crystallography or cryo-EM at higher resolution as indicated by references in the figure, and were fit to the EM density map of the 20S supercomplex.

Fig. 4. Cryo-EM structures of ATP- and ADP-bound full-length NSF

The cryo-EM structures are derived from PDB entries 3j94 and 3j95 with the N domains docked into the density of unsharpened maps of ATP- and ADP-bound NSF respectively as rigid bodies, using the crystal structure of the N domain (PDB accession code: 1qcs). (a), Side views. The six chains are colored red, orange, yellow, green, blue, and purple counter-clockwise as viewed from the top. The red chain is always the one with the lowest α2 helix in the D1 domain. (b), Top views of the N domains. The D1 rings are colored white. (c), Top views of the D1 domains. (d), Top views of the D2 domains. Conformational changes upon ATP hydrolysis are highlighted in the ATP-bound structures on the left in panels (b–d). All structures are shown in scale.

Fig. 5. Cryo-EM structures of the 20S supercomplex

NSF is colored the same as in Fig. 2. αSNAP is colored white and black. SNARE complex is colored dark red. (a) 20S supercomplex with truncated neuronal SNARE complex (state I, PDB accession code: 3j96). (b) V7-20S supercomplex that involves the Vamp-7 SNARE complex. The particular construct of the SNARE complex is shown at the bottom right of each panel. The dark red shades correspond to the actual segments included in the structure.

Fig. 6. Different states of the 20S supercomplex

Cartoon representations of the top views are shown. (a) There are nine asymmetric configurations with four SNAP proteins. (b) The actual pattern observed for the V7-20S supercomplex that involves the Vamp-7 SNARE complex. There are two SNAP proteins, and four resolved N domains in the structure (see Fig. 3b). The black circles indicate the configurations that have been observed [35].

Fig. 7. Comparison of NSF and VCP/p97 structures

(a) Overall structures of ATP-bound NSF (PDB accession code 3j94), ADP-bound NSF (PDB accession code 3j95), and ADP-bound VCP/p97 (PDB accession code 3cf3). Only ATPase rings are shown. The structures have been aligned and are on the same scale. (b) Same view as in (a) but only showing the α2 helices. Dashed lines are in the same positions as the top two lines in (a) to help visualization. (c)-(f) Superposition of AAA+ domains of NSF and VCP/p97. Root mean square deviations (RMSD) of the main chains are provided. α2 helices are highlighted in dashed boxes. The following PDB models are used: NSF D1 (accession code 3j94), NSF D2 (accession code 1nsf), VCP/p97 D1 (accession code 1e32), and VCP/p97 D2 (accession code 3cf0).