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. 2019 Apr 10;5(4):eaau8164. doi: 10.1126/sciadv.aau8164

Fig. 3. Direct interaction between the N terminus of the SNARE complex and NSF-D1.

Fig. 3

(A) Different positions of the SNARE complexes relative to the D1 ring in the six states of the 20S complex. The identified six states were aligned with respect to the D1D2 rings. The SNARE complex is represented as a cylinder. The volume in gray at the bottom of the SNARE complexes represented the distribution of the residue E12 from SNAP25-N. (B) Top view of (A) showing the relative positions between the SNARE complexes and the D1 ring. (C) Unsharpened density map of the SNARE complex superimposed with the model. The red arrow indicates the residue located at the very N terminus of the SNARE complex. (D) Top view of (A) with the SNARE cylinders omitted. Note that the volume (gray) representing the distribution of the residue E12 from SNAP25-N overlaps with the D1 pore loop (red) of chain E (red arrow). (E) Volume representing the distribution of the residue E12 from SNAP25-N is close to the YVG motif (red spheres). NSF K193 and E297 (blue) were found cross-linked to the N terminus of the SNARE complex from 20S treated with chemical cross-linkers. (F) Cryo-EM density (mesh) of the D1 pore loops superimposed with the atomic model (stick) showing the quality of the EM map at the D1 pore loops. (G) Summary of the chemical cross-linking results. Circular plot showing the distribution of the identified cross-linked residue pairs (tables S2 and S3) mapped to protein sequences. VAMP, Stx, 25N, and 25C are the WT VAMP, Syntaxin, SNAP25-N, and SNAP25-C proteins, respectively. VAMP-D, Stx-D, 25N-D, and 25C-D are the N-terminally deleted VAMP, Syntaxin, SNAP25-N, and SNAP25-C proteins, respectively. Note that only DSS (and not EDC) was able to cross-link the N terminus of the SNARE complex with the pore loop region of NSF-D1 in the mutated 20S particle formed with four N-terminally deleted SNARE chains. (H) Effects of the deletion of the N termini of the four SNARE chains (del) on the α-SNAP–dependent binding of NSF to the SNARE complex and the SNARE complex disassembly. Values are normalized to the WT SNARE complex (WT) and represent the mean ± SD.