Molecular mechanism of a protein disassembly machinery

Overview.  In concert with the adaptor protein, SNAP, the ATPase NSF disassembles the SNARE complex into individual proteins upon ATP hydrolysis (Cipriano et al., 2013)(Vivona et al., 2013). In addition, NSF is capable of disassembling off-pathway (including anti-parallel) SNARE complexes, suggesting a role in quality control (Choi et al., 2018).

Figure 1. Disassembly of the SNARE complex by NSF and SNAP. Binding constants are from (Cipriano et al., 2013) and (Vivina et al., 2013).

NSF is a member of the AAA+ family consisting of two ATPase rings (known as Type II AAA+), and an N-terminal domain. In 2015, we determined EM structures of NSF at 4.2 Å and of the 20S complex at 7.6 Å resolution without imposing symmetry (Zhao et al., 2015). Two key factors led to these higher-resolution EM reconstructions: (1) much improved sample homogeneity and (2) advances in cryo-EM technology, especially the availability of direct electron detectors. We subsequently obtained an even higher-resolution reconstruction of the 20S particle at 4.5 Å resolution (White et al., 2018). This reconstruction showed unprecedented detail about the interaction of SNAREs with both NSF and SNAPs. Two αSNAP molecules bind to the ternary SNARE complex primarily involving a large, negatively charged region at the center of the SNARE complex (Figure 1a). Although the αSNAP stoichiometry is different for the two reconstructions of the 20S complex (related to the absence of the SNAP-25 linker in the previous reconstruction), the interactions of the common two αSNAP molecules are nearly identical for all structures determined to date. The interacting regions on the SNARE complex and the two αSNAP molecules have complementary electrostatic features that may be a key contributor to this preferred binding mode. Taken together, these structures suggest that the 2:1 αSNAP:SNARE binding mode is likely conserved in all 20S complexes.


Figure 2. Cryo-EM structure of the SNARE/SNAP/NSF complex at 4.5 Å resolution (White et al., 2018), composed of one hexameric NSF molecule, two αSNAP molecules, one ternary SNARE complex (consisting of the cytoplasmic domains of synaptobrevin-2 and syntaxin-1A, and full-length SNAP-25) in the presence of ATP. (a) Sharpened EM map. Synaptobrevin-2, blue; SNAP-25, green; syntaxin-1A, red; αSNAP, yellow; NSF N domains, gold; NSF D1 domains, light blue; NSF D2 domains, magenta. (b) Close-up view of interactions between the N-terminal residues of SNAP-25 (green) and pore residues (tyrosine residues shown as sticks) of five of the six D1 ATPase domains of NSF (labelled A-E).

Key insights. The N-terminus of SNAP25 interacts with the D1 ring pore of NSF and suggests a ratchet-like threading mechanism prior to disassembly (Figure 1b) (White et al., 2018). This configuration is reminiscent of other recent structures of Type II AAA+ proteins in complex with peptides, suggesting a common, conserved mechanism for substrate unfolding. The substrate is recognized by two αSNAP molecules that, in turn, guide a part of the substrate (i.e., the N-terminal residues of SNAP-25) into the D1 ATPase ring of NSF and unfold this part. More broadly, this suggests that different parts of AAA ATPases and their adapters are responsible for substrate recognition and initiation of the unfolding or disassembly process.

Figure 3: Model of NSF/SNAP-mediated disassembly of the SNARE complex.