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Spring-loaded unraveling of a single SNARE complex by NSF in one round of ATP turnover

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Science  27 Mar 2015:
Vol. 347, Issue 6229, pp. 1485-1489
DOI: 10.1126/science.aaa5267
  • Fig. 1 Reconstitution of NSF-mediated SNARE complex disassembly on a single-molecule fluorescence microscope.

    (A) Procedure of the experiment. (B) Exemplary TIR microscopy images after immobilization of acceptor vesicles (left), after SNARE complex formation (middle), and after disassembly reaction (right). Scale bar, 10 μm. (C) NSF imaged using negative stain electron microscopy (left). Scale bar, 100 nm. Representative two-dimensional class averages (right). Top views are shown. (D) Number of surface vesicles containing Cy3-sVAMP2 measured with various components of the disassembly reaction. (Left) Nonspecific binding of sVAMP2 in the absence of acceptor vesicles (dark gray). Disassembly (loss of fluorescence) is observable only when all components required for disassembly are present (red bar). ***P < 0.001, assessed using the paired t test, and all the errors are SD (n ≥ 20 TIR images) unless otherwise specified. (E) Procedure to allow for one-round ATP hydrolysis during the disassembly reaction. (F and G) Single-molecule immunolabeling assays for confirmation of α-SNAP-binding (F) and NSF-binding (G). Number of Alexa647 spots measured after incubation with the depicted components of the disassembly reaction. (H) Number of vesicles containing Cy3-sVAMP2 measured after SNARE complex formation (black), nonspecific binding of Cy3-sVAMP2 (gray), and after the experimental protocol in (E) with Mg2+ and ATP (blue) or with Mg2+ (red). Other bars indicate control experiments where, instead of Mg2+, either EDTA only, EDTA and ATP, or ADP (white) were injected during the ATP hydrolysis step. (I) Cumulative distributions of Δt for disassembly under conditions of one round of ATP hydrolysis and in the presence of excess NSF, ATP, and Mg2+. Δt was measured from t = 0 to the event of stepwise fluorescence decrease (fig. S2, F and G). Fitting of the distributions using a single exponential function gives time constants of 15.8 ± 1.1 s and 14.1 ± 1.9 s for disassembly with one-round ATP hydrolysis and free NSF, ATP, and Mg2+, respectively.

  • Fig. 2 Intermediates of NSF/α-SNAP disassembly of SNARE complex monitored by single-molecule FRET.

    (A) Labeling positions of Cy3 and Cy5 on either the N-terminal or the C-terminal end of the SNARE complex. According to the crystal structure (3), the expected distance between two dyes is less than 1.4 nm for both cases. (B) Experimental procedure. NSF, ATP, and EDTA were injected into the chamber containing α-SNAP-SNARE complexes. Movies were recorded starting at the same time as the injection of Mg2+. (C and D) Representative disassembly traces of C-terminal FRET pairs starting from low FRET (<0.3) (C) and high FRET (>0.65) (D). (E) Relative abundance of disassembly events classified as single burst (red) and other types (blue). Disassembly was carried out under one-round ATP hydrolysis condition (left) or with excess NSF, ATP, and Mg2+ (right). (F) Labeling positions of Cy3 and Cy5 on the C-terminal end of the SNARE complex with full-length VAMP2. (G) Relative abundance of disassembly events classified as single burst (red) and other types (blue) using C-terminal FRET of SNARE complex with full-length VAMP2.

  • Fig. 3 Observation of NSF-mediated SNARE-complex disassembly with single-molecule magnetic tweezers.

    (A to C) Representative real-time traces showing destabilization and disassembly of SNARE complexes driven by α-SNAP and NSF. The traces are categorized according to the disassembly types: complete disassembly within a temporal resolution of 16.7 ms (A), almost complete disassembly but with a few seconds delay before the final release (B), and repetitive unzipping of almost full SNARE complex before the final release (C). Pink dotted lines in (B) and (C) denote the fully unzipped state up to the seventh layer. (D) Extension distributions of the sequential stages colored as blue, red and green in the extension traces of (A) to (C) (n = 23 traces). (E) Structure diagram of the SNARE complex mapping the extension changes onto the corresponding positions in the structure. When a SNARE complex is unzipped to specific layers at 3.9 pN, the expected extension values are estimated with the wormlike chain model (shown at the bottom) (table S1). The positions of destabilizations (red for α-SNAP only and green for α-SNAP/NSF) and the position after the disassembly step (pink) are shown.

  • Fig. 4 Molecular model for disassembly of the SNARE complex mediated by NSF and α-SNAP.

    (A) Experimental design for sVAMP2 dissociation assay with Pi analogs. (B) Pi titration experiment. (C) Latency distributions of sVAMP2 dissociation via one-round ATP hydrolysis with Pi analogs. (D) Experimental design for SNAP-25 dissociation from the SNARE complex with full-length VAMP2 with Pi analogs. (E) Latency distributions of SNAP-25 dissociation with Pi analogs. (F) and (G) Mechanochemical model of the dwell-burst disassembly process for the power-stroke model (F) and the spring-loaded model (G).

Supplementary Materials

  • Spring-loaded unraveling of a single SNARE complex by NSF in one round of ATP turnover

    Je-Kyung Ryu, Duyoung Min, Sang-Hyun Rah, Soo Jin Kim, Yongsoo Park, Haesoo Kim, Changbong Hyeon, Ho Min Kim, Reinhard Jahn, Tae-Young Yoon

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S17
    • Table S1
    • References

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