Fig. 3: NSF and ɑ-SNAP engage a 2:1 binary SNARE complex of syntaxin H3 and SNAP-25 SN1 and SN2 SNARE domains under non-hydrolyzing conditions.

A representative 21bin20S model (class #5, 3.39 Å) and ɑ-SNAP—2:1 binary SNARE subcomplex model (class #13, 3.74 Å). A Hexameric NSF engages the ɑ-SNAP—2:1 binary SNARE subcomplex via multiple N-domains (salmon), which engage three ɑ-SNAP molecules (gold) surrounding the 2:1 binary SNARE complex composed of two syntaxin H3 domains (dark red) and the SN1 and SN2 SNARE domains of a single SNAP-25 molecule (green). A linker N-terminal to one of the syntaxin H3 domains is found within the catalytic D1 ring of NSF (light blue). The D2 ring of NSF (purple) does not engage substrate. A hypothetical membrane is illustrated in gray. In the protein domain legend, ordered syntaxin and SNAP-25 domains are shaded and those visible in the reconstruction of the SNARE complex are denoted with an asterisk; syntaxin Habc domains were not observed. B NSF (light blue) engages one of the two syntaxin molecules in the 2:1 binary SNARE complex (dark red) via pore loop interactions involving Y294 (protomers A–F, indicated). A sharpened map is shown, contoured at 5 σ. Of the eight 21bin20S classes with the 2:1 binary SNARE complex from the dataset collected under non-hydrolyzing conditions, six engage syntaxin and two engage SNAP-25 within the pore of the D1 ring. C The ɑ-SNAP—2:1 binary SNARE subcomplex model. Three ɑ-SNAP molecules bind the 2:1 binary SNARE complex; a flexible linker between SNAP-25 SN1 and SN2 blocks access by a fourth. The 2:1 binary SNARE complex forms a twisted, parallel ɑ-helical bundle with a series of characteristic SNARE complex layers preserved. This configuration also places the N-terminal portion of the linker, bearing cysteines often palmitoylated in vivo, near the membrane⁹⁷. D Layer slices through the ɑ-SNAP—2:1 binary SNARE subcomplex model and sharpened map. Each layer is characterized by nearly planar interactions between four side chains within the helical bundle. The primary interactions between ɑ-SNAP and the syntaxin H3 complex are found near the -2 and -3 layers, where several functionally essential⁶¹ ɑ-SNAP side chains (L197, Y200) are positioned by a loop that buries them within the bundle groove.