Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins
- Yale University School of Medicine, United States
- Yale University, United States
- UMR CNRS 8550 Associée aux Universités Paris 6 et Paris 7, Ecole Normale Supérieure, France
Figures
Figure 1 with 4 supplements
Chimeric SNARE construct and experimental setup used to study functional assembly of single SNARE complexes using dual-trap high-resolution optical tweezers.
(A) Modular parallel four-helix bundle structure of an assembled neuronal SNARE complex mediating membrane fusion. The SNARE complex contains different functional domains: an N-terminal domain …
Figure 1—figure supplement 1
Amino acid sequences of the chimeric SNARE protein constructs used for the single-molecule manipulation study of SNARE assembly.
The four sets of SNARE proteins are neuronal SNAREs of rat syntaxin (SX) 1A, rat VAMP2, and mouse SNAP-25B, GLUT4 SNAREs of rat syntaxin 4A, rat SNAP-23, and rat VAMP2, endosomal SNAREs of rat …
Figure 1—figure supplement 2
The chimeric neuronal SNARE protein correctly folds into an expected four-helix SNARE bundle.
Circular dichroism spectrum of the chimeric SNARE protein. The presence of two local minima at 208 nm and 222 nm indicates a high content of alpha-helical structure in the protein. Based on the CD …
Figure 1—figure supplement 3
The chimeric neuronal SNARE protein folds into a homogenous SNARE four-helix bundle with an expected molecular weight.
Gel filtration profile of the chimeric protein. The purified chimeric SNARE protein was eluted from the column (Superdex 200 10/300 GL) in one major peak corresponding to a molecular weight of 57 …
Figure 1—figure supplement 4
The chimeric t-SNARE protein supports lipid mixing between liposomes.
(A) Schematic representation of the chimeric t-SNARE and the v-SNARE used for the lipid-mixing assay. The chimeric t-SNARE protein is lipidated through a cysteine introduced to the C-terminus of …
Figure 2 with 4 supplements
Four representative SNARE complexes assemble or disassemble via common intermediates and pathways.
(A) Force-extension curves (FECs) of the neuronal, GLUT4, endosomal, and yeast SNARE complexes. FECs were obtained by pulling the complexes (black) or relaxing them (gray). The reversible C-terminal …
Figure 2—figure supplement 1
Distinct linker domain and C-terminal domain transitions.
Force-extension curves (FECs) of yeast SNARE complexes containing the v-SNARE Snc2 truncated in the linker domain (LD) region or in both the LD region and part of the C-terminal domain (CTD) region …
Figure 2—figure supplement 2
The neuronal t-SNARE complex as a transient unfolding intermediate of the half-zippered SNARE complex.
Force-extension curves (FECs) of a single neuronal SNARE complex corresponding to three pulling cycles. The inset shows close-up views of the unfolding process of the half-zippered SNARE complex in …
Figure 2—figure supplement 3
The GLUT4 t-SNARE complex as a transient unfolding intermediate of the half-zippered SNARE complex.
Force-extension curves (FECs) of the same GLUT4 SNARE complex corresponding to three pulling cycles. The overlapping FECs (‘All’) and individual FECs from successive pulling cycles (#1–#3) were …
Figure 2—figure supplement 4
The yeast t-SNARE complex is a stable unfolding intermediate of the half-zippered SNARE complex.
Force-extension curves (FECs) of a single yeast SNARE complex corresponding to two successive pulling cycles and their best fits by the worm-like chain model in the continuous phases. The …
Figure 3
Distinct transition kinetics and stabilities of SNARE C-terminal domain and N-terminal domain.
(A) Histogram distributions of the C-terminal domain (CTD) equilibrium force and the N-terminal domain (NTD) unzipping force for different SNARE complexes. The vertical axis shows the percentage of …
Figure 4
Overlapping force-extension curves obtained by repeatedly pulling a single neuronal or GLUT4 SNARE complex, revealing robust and common step-wise SNARE assembly and disassembly.
The overlapping force-extension curves (FECs) (designated by ‘All’) are shifted along the x-axis to reveal individual FECs corresponding to different pulling cycles (numbered). The cooperative …
Figure 5
Comparison of the two-state C-terminal domain transitions of four SNARE complexes.
(A) Force-dependent extension-time trajectories under approximately constant forces (f) revealing the unfolding probability (p) of C-terminal domain (CTD) as indicated. The idealized two-state …
Figure 6 with 2 supplements
Comparison of the two-state linker domain transitions of four SNARE complexes.
(A) Extension-time trajectories (black lines) and their best hidden Markov model (HMM) fits (red lines) showing fast binary transitions of linker domains (LDs) under constant forces. The force (f) …
Figure 6—figure supplement 1
Folding energy and kinetics of SNARE linker domains.
Unfolding probabilities (top panel) and folding rates (open symbols in bottom panel) or unfolding rates (solid symbols) of linker domains (LDs) in the yeast, endosomal, and neuronal SNARE complexes. …
Figure 6—figure supplement 2
Minor effect of the spacer sequences in the chimeric SNARE proteins on the folding energy of SNARE complexes.
Predicted structures of the folded and unfolded states in linker domain (LD) transition and the accompanying extension change of the spacer sequence connecting syntaxin and SNAP-25 (red dashed …
Figure 7 with 1 supplement
Energy landscape of SNARE zippering perfectly meets the needs of membrane fusion.
(A) Cartoons of different assembly states of the trans-SNARE complex corresponding to the points indicated in B. (B) Free energy of membrane fusion per SNARE complex (red line), a single loaded …
Figure 7—figure supplement 1
Estimation of the average forces generated by zippering of the N-terminal and C-terminal CTD of neuronal SNARE complex.
We estimated the C-terminal domain (CTD) zippering forces based on a simplified energy landscape model for a two-state process. The energy landscape is characterized by the experimentally measured …
Tables
Table 1
Average equilibrium force, extension change, folding energy, and folding energy barrier and position associated with C-terminal domain and linker domain transitions of the four different SNARE …
| SNARE complex | C-terminal domain | Linker domain | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Force (pN) | Extension change (nm) | Folding energy (kBT) | Transition state energy* (kBT) | Transition state position† (a.a.) | Force (pN) | Extension change (nm) | Folding energy (kBT) | Transition state energy* (kBT) | Transition state position† (a.a.) | |
| Neuron | 16.2 (0.9) | 7.2 (1.2) | −27 (4.7) | −5.5 (1.5) | 17 (3) | 8 (1) | 4.7 (0.5) | −9.7 (1.6) | 5.5 (1.5) | 31 (1) |
| GLUT4 | 18.5 (1.8) | 6.0 (0.9) | −23 (4.1) | −0.8 (1.0) | 11 (2) | 8.6 (0.9) | 5.6 (1.1) | −12 (2.7) | 2 (1.0) | 30 (1) |
| Endosome | 11.9 (0.9) | 6.9 (0.4) | −16 (1.5) | 2.1 (1.4) | 12 (2) | 6.3 (1.2) | 5.1 (1.8) | −6.1 (2.4) | 4.9 (1.5) | 32 (2) |
| Yeast | 10.1 (1.4) | 5.8 (0.8) | −13 (2.5) | 3.2 (1.5) | 13 (2) | 6.0 (1.6) | 5.1 (1.2) | −5.7 (2.0) | 3.6 (2.0) | 32 (2) |
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*
Here, negative energy indicates downhill protein folding (Yang and Gruebele, 2003).
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†
The number of the amino acids in the R SNARE C-terminal to the ionic layer (chosen as 0).
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The equilibrium force and extension change were determined at an unfolding probability of 0.5 for the two-state processes. The standard deviations of the averages are shown in parenthesis. The equilibrium force distribution, the number of transitions, and the number of single molecules scored for C-terminal domain (CTD) transitions are shown in Figure 3. For parameters related to linker domain (LD) transitions, a total of 18, 35, 11, and 24 LD transitions in single neuronal, GLUT4, endosomal, and yeast SNARE complexes were scored, respectively.
