Fig. 10: An expanded model for NSF action across cellular and atomic scales.

A A model for disassembling non-fusogenic SNARE species by NSF and ɑ-SNAP at the presynapse and progression to priming and fusion. Syntaxin nanodomains comprised at least in part of tetrameric syntaxin SNARE complexes serve as a reservoir for the liberation of individual, fusogenic syntaxin molecules by NSF upon disassembly. These syntaxin molecules may also enter non-productive binary complexes with SNAP-25, with 2:1 or 2:2 stoichiometry; such complexes would be disassembled by NSF to ensure trans-SNARE complex formation with synaptobrevin on incoming synaptic vesicles in concert with other factors, such as Munc18, Munc13, complexin, and synaptotagmin. Where possible, full-length structures (structures reported here and PDB IDs 1N7S, 4JEU, 5W5C) were supplemented with AlphaFold2⁹⁴ structures to provide additional context, and ISOLDE⁸⁵ was used with secondary structure restraints to reposition flexible regions. B A sequential hydrolysis model for SNARE complex disassembly by NSF is inferred from comparing different classes from non-hydrolyzing and hydrolyzing conditions. Following hydrolysis-independent substrate side loading, the D1 ring is found in a pre-hydrolysis state with ATP in protomer E of D1. Protomer F nucleotide exchange then enables ATP exchange and rebinding to both SNARE substrate and the formation of the interprotomer interface with protomer E, triggering ATP hydrolysis in protomer E and substrate translocation. This is accompanied by P_i and Mg²⁺ release from down-ring protomers; in many structures, the release occurs between protomers C and B. The cycle then begins anew. Blue corresponds to ADP-bound, red to ATP-bound, orange to ATP+Mg²⁺ bound, purple to ADP•P_i bound, and gray to nucleotide-free. See Movie S1 for an animated rendition.