Research ArticleCELL BIOLOGY

Mechanistic insights into the SNARE complex disassembly

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Science Advances  10 Apr 2019:
Vol. 5, no. 4, eaau8164
DOI: 10.1126/sciadv.aau8164
  • Fig. 1 Structure of the α-SNAP–SNARE subcomplex.

    (A) The EM density map of the α-SNAP–SNARE subcomplex color-coded to show the local resolution as estimated by ResMap. The right panel shows a cut-through view of the interior of the map. (B) Cryo-EM density (mesh) of representative layers of the SNARE complex superimposed with respective atomic models (stick). Unsharpened map showing the extended density corresponding to the longer C terminus of VAMP is displayed in the inset. The red arrows indicate the extended density. (C) Interactions of each α-SNAP with the SNARE complex showing interacting residues. Eye symbols and arrowheads in the insets indicate view directions. Note that each α-SNAP molecule interacts with three of the four chains of the SNARE complex, and in each of the four α-SNAP molecules, almost an identical set of residues make contact with SNARE, but on the receiving side, different sets of residues are used by individual SNARE chains.

  • Fig. 2 Essential role of R116 and L197 of α-SNAP in the SNARE complex disassembly.

    (A) Effect of α-SNAP mutations on the α-SNAP–dependent binding of NSF to the SNARE complex and the SNARE complex disassembly. Values are normalized to wild-type (WT) α-SNAP and represent the mean ± SD. (B) Significance of α-SNAP R116 and L197 residues for the spontaneous neurotransmitter release. Left panel: representative traces of mEPSCs recorded from cultured WT hippocampal neurons alone (Ctrl), overexpressing WT α-SNAP (WT), and mutated α-SNAPs R116A and L197A. Middle and right panels: quantitative analysis of the frequency (middle) and amplitude (right) of mEPSCs. Shown are the mean ± SEM. The number of cells/independent cultures analyzed are 18, 22, 16, and 21 for Ctrl, WT, R116A, and L197A, respectively. Statistical assessment was performed by Student’s t test (*P < 0.05). (C) Interactions between SNAP R116 and the SNARE complex. Note that two R116 residues from α-SNAP-H and α-SNAP-E interact with two sites of VAMP, E62 and D68, respectively. (D) Interactions between SNAP L197 and the SNARE complex. Note that two L197 residues from α-SNAP-G and α-SNAP-H interact with three sites of VAMP, V48, V50, and L54, respectively. (E) Effect of mutations of individual SNARE proteins on the SNARE complex disassembly. Values are normalized to the WT SNARE complex and represent the mean ± SD.

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

    (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.

  • Fig. 4 Model of the SNARE complex disassembly.

    The rotation of the α-SNAP barrel induced by the rotation of the NSF-D1 ring upon ATP hydrolysis may generate mechanical force along the tangent direction of the cylindrical barrel, applied mainly on two sites of VAMP. These two sites correspond to their respective interacting residues on the α-SNAP, where one site interacts with the R116 residue and the other interacts with L197. Since the N terminus of the SNARE complex is directly anchored to the pore region of the NSF-D1, the right-handed torque would start to unwind and/or break the left-handed helical SNARE complex and thus disassemble the complex into individual proteins simultaneously or sequentially.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaau8164/DC1

    Fig. S1. Cryo-EM analysis of the whole 20S complex, the α-SNAP–SNARE subcomplex, and the NSF-D1D2 part.

    Fig. S2. The flowchart for EM data processing.

    Fig. S3. Representative raw gel images.

    Fig. S4. Analyses of the expression levels of the wild-type α-SNAP and the mutants in the neurons.

    Fig. S5. Sequence alignment of α-SNAP from different species.

    Fig. S6. Effects of multiple point mutations of VAMP on the α-SNAP–dependent binding of NSF to the SNARE complex.

    Fig. S7. Focused 3D classification of the α-SNAP–SNARE subcomplex together with the NSF N domain.

    Fig. S8. The N-terminally deleted SNARE proteins can form the 20S complex.

    Table S1. Cryo-EM data collection and refinement statistics.

    Table S2. CXMS analysis of the 20S complex formed with either wild-type SNARE proteins or the N-terminally truncated SNARE proteins.

    Table S3. The amino acid sequences of the NSF and SNARE proteins used in this study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Cryo-EM analysis of the whole 20S complex, the α-SNAP–SNARE subcomplex, and the NSF-D1D2 part.
    • Fig. S2. The flowchart for EM data processing.
    • Fig. S3. Representative raw gel images.
    • Fig. S4. Analyses of the expression levels of the wild-type α-SNAP and the mutants in the neurons.
    • Fig. S5. Sequence alignment of α-SNAP from different species.
    • Fig. S6. Effects of multiple point mutations of VAMP on the α-SNAP–dependent binding of NSF to the SNARE complex.
    • Fig. S7. Focused 3D classification of the α-SNAP–SNARE subcomplex together with the NSF N domain.
    • Fig. S8. The N-terminally deleted SNARE proteins can form the 20S complex.
    • Table S1. Cryo-EM data collection and refinement statistics.
    • Table S2. CXMS analysis of the 20S complex formed with either wild-type SNARE proteins or the N-terminally truncated SNARE proteins.
    • Table S3. The amino acid sequences of the NSF and SNARE proteins used in this study.

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