1. Microbiology and Infectious Disease
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NSF-mediated disassembly of on- and off-pathway SNARE complexes and inhibition by complexin

  1. Ucheor B Choi
  2. Minglei Zhao
  3. K Ian White
  4. Richard A Pfuetzner
  5. Luis Esquivies
  6. Qiangjun Zhou
  7. Axel T Brunger  Is a corresponding author
  1. Stanford University, United States
  2. Howard Hughes Medical Institute, Stanford University, United States
  3. University of Chicago, United States
Research Article
Cite this article as: eLife 2018;7:e36497 doi: 10.7554/eLife.36497
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Structure of the L-20S (NSF/αSNAP/L-SNARE) complex.

(A) Domain diagram of the L-20S complex consisting of NSF (orange, light blue, pink), αSNAP (yellow), and the L-SNARE complex composed of the cytoplasmic domain of syntaxin-1A (red, amino acid range 181–262), full-length SNAP-25A (green), and the cytoplasmic domain of synaptobrevin-2 (blue, amino acid range 25–96) fused by the synthetic linkers Sp1 and Sp2 and a biotinylation sequence (Avi-tag) at the C-terminus (Materials and methods). (B) Two orthogonal side views of the cryo-EM map of the L-20S complex filtered to a resolution of 7.0 Å and sharpened with a B-factor of −225 Å2 (Table 1). The color code remains the same as in panel A. (C) Two orthogonal views of the three-dimensional model of the L-20S complex.

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Figure 1—figure supplement 1
Single particle cryo-EM analysis.

(A) Representative electron micrograph of the L-20S (NSF/αSNAP/L-SNARE) complex. (B) Selected 2D class averages. (C) Plots of the angular distribution of particle projections. The radius of the sphere at each projection direction is proportional to the number of particle images assigned to it. Two orthogonal views are shown. (D) FSC curve for the 3D density map after RELION post-processing. The resolution is estimated to be 7.0 Å by the FSC = 0.143 criterion.

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Figure 1—figure supplement 2
Protein quantification of the 20S complex using a gel-based assay.

(A) Domain diagram of the 20S complex consisting of NSF (orange, light blue, pink), αSNAP (yellow), and the SNARE complex composed of the cytoplasmic domain of syntaxin-1A (red, amino acid range 180–262), two SNARE domains of SNAP-25A (green, amino acid range 1–85 and 120–206) or full-length SNAP-25A (green, amino acid range 1–206), and the cytoplasmic domain of synaptobrevin-2 (blue, amino acid range 1–96) (Materials and methods). (B) SDS-PAGE gel of the 20S complex formed in the absence and presence of the native linker of SNAP-25A that connects its two SNARE domains. (C) The intensities of the protein bands in the SDS-PAGE gel were quantified using ImageJ (Schneider et al., 2012). Shown are mean values ± SD for protein band intensities of αSNAP divided by the protein band intensities of NSF from two independent gels prepared by the same samples. The protein band intensities from the boiled samples were used since the molecular weight of the SNARE complex without the S25 linker was only slightly higher than that of αSNAP, interfering with densitometry. Additionally, all protein intensities were background-subtracted using the intensity of a nearby selected rectangular area.

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Figure 2 with 1 supplement
NSF-mediated disassembly and reassembly of single SNARE complexes.

(A) Schematic of NSF-mediated disassembly of a single L-SNARE complex (colored as described in Figure 1A). Residues on syntaxin-1A and synaptobrevin-2 were stochastically labeled with fluorescent dyes at residues 249 and 82, respectively (indicated by stars) (referred to as L-SNARE-CC). The labeled L-SNARE-CC complex was surface tethered to a passivated surface and disassembly was subsequently initiated by adding the disassembly factors (Materials and methods). (B) Representative single-molecule fluorescence intensity time traces in the absence (top left panel) and presence (top right panel) of disassembly factors. The fluorescence intensities were converted to FRET efficiency time traces (bottom panels, Materials and methods). (C) Point-wise FRET efficiency histogram of all smFRET efficiency time traces of L-SNARE-CC molecules with transitions in the presence of disassembly factors. (D) Fraction of L-SNARE-CC molecules without transitions (mean values ± SD, Materials and methods, Figure 2—source data 1). (E) Expanded view of a smFRET efficiency time trace (black line) in the presence of disassembly factors in the selected time range (20–75 s), and a fit by HMM (red line, Materials and methods). (F) Probability distribution histogram of smFRET efficiencies (black line, left axis) and the corresponding dwell times (red dots, right axis) obtained from HMM. (G–H) smFRET efficiency dwell time histograms obtained from HMM on both linear and logarithmic timescales. Dashed red lines separate the short- and long-lived states (0.56 s for high FRET dwell times and 0.32 s for low FRET dwell times). Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Materials and methods, Figure 2—source data 2).

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Figure 2—source data 1

Data summary table for the results shown in Figure 2D.

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Figure 2—source data 2

Data summary table for the results shown in Figure 2G-H.

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Figure 2—figure supplement 1
Representative smFRET efficiency time traces of L-SNARE-CC.

Single-molecule fluorescence time traces were converted to FRET efficiency time traces and divided into three sections (I, II, III) (Materials and methods). Only section II was used for data analysis.

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Increasing ionic strength or decreasing αSNAP concentration reduces NSF-mediated disassembly of L-SNARE-CC.

(A) Representative smFRET efficiency time traces. (B) Corresponding point-wise FRET efficiency histograms using all observed time traces vs. NaCl concentrations. (C) Fraction of molecules without transitions (mean values ± SD, Figure 3—source data 1). (D–E) High and low smFRET efficiency dwell time histograms obtained from HMM. Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 3—source data 2). (F) Representative smFRET efficiency time traces of NSF-mediated disassembly of L-SNARE-CC. (G) Corresponding point-wise FRET efficiency histograms using all smFRET efficiency time trace of molecules with transitions. (H) Fraction of molecules without transitions (mean values ± SD, Figure 3—source data 3). (I–J) smFRET efficiency dwell time histograms obtained from HMM. Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 3—source data 4).

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Figure 3—source data 1

Data summary table for the results shown in Figure 3C.

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Figure 3—source data 2

Data summary table for the results shown in Figure 3D-E.

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Figure 3—source data 3

Data summary table for the results shown in Figure 3H.

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Figure 3—source data 4

Data summary table for the results shown in Figure 3I-J.

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αSNAP mutants reduce NSF-mediated disassembly of L-SNARE-CC.

(A) Structural schematic showing the locations of αSNAP mutations. (B) Representative smFRET efficiency time traces of L-SNARE-CC disassembly in the presence 10 µM wild type and mutant αSNAP. (C) Corresponding point-wise smFRET efficiency histograms using all smFRET efficiency time trace of molecules with transitions. (D) Fraction of molecules without transition (mean values ± SD, Figure 4—source data 1). (E–F) smFRET efficiency dwell time histograms obtained from HMM. Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 4—source data 2).

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Figure 4—source data 1

Data summary table for the results shown in Figure 4D.

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Figure 4—source data 2

Data summary table for the results shown in Figure 4E-F.

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Effect of the N-terminal Habc domain of syntaxin-1A on disassembly.

(A) Schematic of NSF-mediated disassembly of a single L-SNAREfull-CC complex labeled with fluorescent dyes (indicated by stars) at residue 249 of syntaxin-1A and residue 82 of synaptobvrevin-2. (B) Representative smFRET efficiency time traces for NSF-mediated L-SNAREfull-CC disassembly. (C) Corresponding point-wise smFRET efficiency histograms using all smFRET efficiency time trace of molecules with transitions. (D) Fraction of molecules without transitions (mean values ± SD, Figure 5—source data 1). (E–F) smFRET efficiency dwell time histograms obtained from HMM. Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 5—source data 2).

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Figure 5—source data 1

Data summary table for the results shown in Figure 5D.

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Figure 5—source data 2

Data summary table for the results shown in Figure 5E-F.

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Figure 6 with 1 supplement
Cpx interferes with NSF-mediated disassembly of the SNARE complex.

(A) Schematic of NSF-mediated disassembly of a single L-SNARE-CC complex labeled with fluorescent dyes (indicated by stars) at residue 249 of syntaxin-1A and residue 82 of synaptobvrevin-2 in the presence of Cpx. (B) Representative smFRET efficiency time traces corresponding to SNARE-CC disassembly in the absence or presence of 1 µM or 10 µM wildtype Cpx or 10 µM 4M mutant of Cpx. Concentrations of 1 µM and 10 µM Cpx correspond to a 10:1 and 1:1 molar ratio of αSNAP:Cpx, respectively. (C) Corresponding point-wise smFRET efficiency histograms using all smFRET efficiency time traces of molecules with transitions. (D) Fraction of molecules without transitions (mean values ± SD, Figure 6—source data 1). (E–F) smFRET efficiency dwell time histograms obtained from HMM . Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 6—source data 2). (G) Superposition of the structure of the αSNAP/L-SNARE subcomplex (from the L-20S EM structure) with the crystal structure of the Cpx/SNARE complex (PDB ID: 1KIL). Cpx is shown as a surface and colored pink. The two αSNAPs are colored yellow and brown.

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Figure 6—source data 1

Data summary table for the results shown in Figure 6D.

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Figure 6—source data 2

Data summary table for the results shown in Figure 6E-F.

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Figure 6—figure supplement 1
Competition of αSNAP and Cpx binding to the L-SNARE complex.

A single-molecule competition experiment. Alexa 647 labeled L-SNARE complex was surface-tethered through biotin-streptavidin linkage on a PEG coated surface of the microscope slide. Alexa 555-labeled αSNAP was concurrently mixed with 0.1 µM, 1.0 µM, and 10 µM Cpx (corresponding to a 1:10, 1:100, and 1:1000 molar ratio of αSNAP:Cpx, respectively) and incubated for 5 min to the surface-tethered L-SNARE complex. The Cpx 4M mutant that blocks the binding to the SNARE complex was used as control. Proteins that did not bind to the surface-tethered L-SNARE complex were washed away before smFRET efficiency measurements. Shown are mean values ± SD for the number of Alexa 555-labeled αSNAP divided by the number of Alexa 647 labeled L-SNARE complexes observed in three fields of view.

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Figure 7 with 1 supplement
NSF-mediated disassembly of the binary t-SNARE complex.

(A) Schematic of NSF-mediated disassembly of a single linked binary t-SNARE complex composed of syntaxin-1A and SNAP-25A. L-SNAREbinary-CC1 and L-SNAREbinary-CC2 were labeled (indicated by stars) at residue 249 of syntaxin-1A and at either residue 76 or 197 of SNAP-25A, respectively. (B) Representative smFRET efficiency time traces of L-SNAREbinary-CC1 (left panel) and L-SNAREbinary-CC2 (right panel). Red arrows indicate intermediate FRET efficiency states. (C–D) Corresponding point-wise smFRET efficiency histograms using all smFRET efficiency time traces of molecules with transitions. A sum of three Gaussian functions was fit to the histogram representing the distribution of the individual states (black: observed point-wise FRET; red: Gaussian components; green: sum of Gaussian fits). (E) Fraction of molecules without transitions (mean values ± SD, Figure 7—source data 1). (F–G) Probability distribution histogram of smFRET efficiencies (black line, left axis) and the corresponding dwell times (red dots, right axis) obtained from HMM. (H–I) smFRET efficiency dwell time histograms obtained from HMM. Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 7—source data 2).

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Figure 7—source data 1

Data summary table for the results shown in Figure 7E.

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Figure 7—source data 2

Data summary table for the results shown in Figure 7H–I.

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Figure 7—figure supplement 1
Configurations of isolated linked binary t-SNARE complexes.

(A) Schematic of configurations of the linked binary t-SNARE complexes, L-SNAREbinary-CC1 and L-SNAREbinary-CC2, with labels (indicated by stars) at residue 249 of syntaxin-1A and residue 76 or residue 197 of SNAP-25A, respectively. (B) Representative single-molecule fluorescence intensity time traces of L-SNAREbinary-CC1 in the absence of disassembly factors (top panels). The fluorescence intensities were converted to FRET efficiency time traces (bottom panels). (C) Corresponding point-wise FRET efficiency histograms using all observed traces.

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Different labeling combinations produce similar kinetics.

(A) Schematic of NSF-mediated disassembly of a single L-SNARE-CC complex (labels at residue 249 of syntaxin-1A and residue 82 of synaptobrevin-2), L-SNARE-NN (labels at residue 193 of syntaxin-1A and residue 28 of synaptobrevin-2), L-SNAREternary-CC1 (labels at residue 249 of syntaxin-1A and residue 76 of SNAP-25A), and L-SNAREternary-CC2 (labels at residue 249 of syntaxin-1A and residue 197 of SNAP-25A). Stars indicate fluorescent dyes. (B) Representative smFRET efficiency time traces of NSF-mediated disassembly of L-SNARE-CC, L-SNARE-NN, L-SNAREternary-CC1, and L-SNAREternary-CC2. (C) Corresponding point-wise FRET efficiency histograms using all observed traces. (D) Fraction of molecules without transitions (mean values ± SD, Figure 8—source data 1). (E–F) smFRET efficiency dwell time histograms obtained from the HMM. Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 8—source data 2).

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Figure 8—source data 1

Data summary table for the results shown in Figure 8D.

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Figure 8—source data 2

Data summary table for the results shown in Figure 8E–F.

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Figure 9 with 1 supplement
NSF disassembles anti-parallel L-SNARE complexes.

(A) Schematic illustrating disassembly of the anti-parallel reporting L-SNARE-CN construct that was labeled with fluorescent dyes indicated by stars at residue 249 of syntaxin-1A and residue 28 of synaptobrevin-2. (B) Representative smFRET efficiency time traces of L-SNARE-CN. (C) Corresponding point-wise FRET efficiency histogram using all observed time traces. (D) Fraction of molecules without transitions (mean values ± SD, Figure 9—source data 1). (E) Probability distribution histogram of smFRET efficiency (black line, left axis) and the corresponding dwell times (red dots, right axis) obtained from the HMM. (F–G) smFRET efficiency dwell time histograms obtained from HMM. Right subpanels show the populations (mean values ± SD) of the dwell times in the short- and long-lived states (Figure 9—source data 2).

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Figure 9—source data 1

Data summary table for the results shown in Figure 9D.

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Figure 9—source data 2

Data summary table for the results shown in Figure 9F-G.

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Figure 9—figure supplement 1
A 7.5 M urea wash prevents improperly assembled L-SNARE complexes.

(A) FRET efficiency histogram corresponding to disassembly of surface tethered L-SNARE-CN with an anti-parallel-reporting FRET labeling pair when purified with and without a supplemental 7.5 M urea wash prior to tethering. Note that NSF and αSNAP were not included in these experiments. (B) Representative single-molecule fluorescence intensity time traces of L-SNARE-CN in anti-parallel configurations in the absence of disassembly factors and without urea wash (top panels). The fluorescence intensities were converted to FRET efficiency time traces (bottom panels). (C) Representative single-molecule fluorescence intensity time traces of L-SNARE-CN (prepared with a urea wash step during purification) in the presence of disassembly factors (top panels). The fluorescence intensities were converted to FRET efficiency time traces (bottom panels).

https://doi.org/10.7554/eLife.36497.033

Tables

Table 1
Data collection and processing statistics of the L-20S complex.
https://doi.org/10.7554/eLife.36497.005
Electron microscopeTitan Krios
Accelerating voltage (kV)300
Defocus range (μm)−1.5 — −3.0
Electron dose (e-2)78
Pixel size (Å)1.01
Initial particles (No.)220,554
Final particles (No.)45,194
Map resolution (Å)7.0
Map sharpening B-factor (Å2)−225

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