Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses
- Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, United States
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, United States
- Howard Hughes Medical Institute, The Rockefeller University, United States
Figures
Figure 1 with 1 supplement
Structure-guided smFRET design for probing MRP1 dynamics.
(A) Structures of bMRP1 captured in ligand-free (left), LTC4-bound (middle), and ATP-bound (right) conformations (PDB accession numbers: 5UJ9, 5UJA, and 6BHU). The positions of tag insertions for …
Figure 1—figure supplement 1
Site-specific labeling and purification of MRP1.
(A) Schematic of the surface immobilization and single-molecule imaging strategy. Labeled bMRP1 molecules were immobilized on a glass surface and imaged at room temperature with …
Figure 2 with 2 supplements
Conformational landscapes of MRP1 revealed by smFRET.
(A) Contour plots (top) and histograms (bottom) of FRET distributions obtained with WT bMRP1 in the following conditions (from left to right): apo, + LTC4 (10 µM), + ATP (5 mM), + ATP/LTC4 (5 mM/10 …
Figure 2—figure supplement 1
Determination of model parameters for idealizing smFRET trajectories.
(A) Evidence lower bound determined by ebFRET, which was used to determine the number of non-zero FRET states and provide initial estimates of model parameters. (B–F) Models with two (B), three (C), …
Figure 2—figure supplement 2
Dependence of the conformational distribution of MRP1 on substrate and ATP.
(A) FRET contour plots (top) and histograms (bottom) obtained at varying concentrations of LTC4. n denotes the number of molecules analyzed for each condition. (B) FRET contour plots and histograms …
Figure 3 with 2 supplements
Real-time dynamics of MRP1 under steady state.
(A) Representative single-molecule donor (green) and acceptor (red) fluorescence trajectories, and the corresponding FRET trajectories (blue) obtained at a frame rate of 25 ms. Idealized FRET states …
Figure 3—figure supplement 1
Additional representative fluorescence and FRET trajectories obtained at 25 ms time resolution for the indicated conditions.
Idealized FRET states are overlaid in orange.
Figure 3—figure supplement 2
Transition density plots displaying the distributions of FRET values before (x-axis) and after (y-axis) each transition identified in the idealized FRET traces.
Dashed lines represent the mean values for the five discrete FRET states.
Figure 4 with 2 supplements
Transitions between IF and OF states from perturbation experiments.
(A) Representative smFRET trajectories of active molecules from the perturbation experiments obtained at a frame rate of 300 ms. A limiting (10 µM) or saturating (5 mM) concentration of ATP with or …
Figure 4—figure supplement 1
Differentiation between inactive and active molecules.
(A) Representative fluorescence and FRET trajectories of the inactive molecules. These molecules transitioned among distinct IF states but never visited the OF state before photobleaching. (B) …
Figure 4—figure supplement 2
Characteristics of IF-to-OF transitions under different conditions.
(A) Contour plots (top) and corresponding transition density plots (bottom) of smFRET trajectories aligned at the appearance of the first OF state from the perturbation experiments in which 10 µM …
Figure 5 with 1 supplement
OF state lifetime under different conditions.
(A) Example smFRET trajectories for apo and LTC4-only conditions showing spontaneous transitions into the OF state independent of ATP. (B) Average lifetime of the OF states under different …
Figure 5—figure supplement 1
Histograms of OF state lifetimes for different conditions and their fit to single-exponential decay functions.
The corresponding time constants (τOF) are reported.
Figure 6 with 2 supplements
Cryo-EM structure of wild-type MRP1 in its post-hydrolytic state.
(A) The structure of WT bMRP1 in the presence of saturating ATP and LTC4 shown in blue, overlaid with the structure of the non-hydrolyzing bMRP1-E1454Q mutant in the presence of saturating ATP and …
Figure 6—figure supplement 1
Cryo-EM data processing workflow.
The number of particles used in each step is indicated next to the arrow associated with that step. After initial classification and refinement in RELION, two separate routes were taken to generate …
Figure 6—figure supplement 2
Quality of the cryo-EM maps.
(A) Cryo-EM density in the degenerate ATPase site from the Frealign map using all particles, displayed in the same way as Figure 5C. (B) Cryo-EM density in the consensus ATPase site from the …
Figure 7 with 1 supplement
Kinetic model for the transport cycle of MRP1.
MRP1 is intrinsically dynamic, transitioning between multiple IF conformations both in the absence and presence of ATP. Under physiological conditions, ATP rapidly binds to the IF state, promoting …
Figure 7—figure supplement 1
Perturbation experiments with E1454Q mutant MRP1.
(A) FRET contour plot for the perturbation experiment in which 5 mM ATP and 10 µM LTC4 were delivered to bMRP1-E1454Q at 10 s. (B) Representative smFRET trajectories for the above experiment. (C) …
Tables
Table 1
Kinetics of the transitions between IF and OF conformations
| Condition | tIF (s) | tOF (s) |
|---|---|---|
| WT, 5 mM ATP | 16.1 ± 2.2 | 28.8 ± 4.8 |
| WT, 5 mM ATP + 10 µM LTC4 | 4.7 ± 0.8 | 30.8 ± 5.2 |
| E1454Q, 5 mM ATP + 10 µM LTC4 | 7.7 ± 1.5 | 31.7 ± 5.5 |
-
Shown are the average lifetimes (mean ± SEM) of the composite IF state and the OF state for WT and E1454Q MRP1 with indicated ATP and substrate concentrations. The effect of dye photobleaching on the apparent IF/OF lifetime has been corrected for.
Table 2
Summary of EM data and structure refinement statistics
| Data collection | ||
|---|---|---|
| Microscope | Titan krios (FEI) | |
| Voltage (kV) | 300 | |
| Detector | K2 Summit (Gatan) | |
| Pixel size (Å) | 1.03 | |
| Defocus range (μm) | 0.7 to 2.4 | |
| Movies | 3604 | |
| Frames/movie | 50 | |
| Dose rate (electrons/pixel/s) | 8.0 | |
| Total dose (electrons/Å2) | 75 | |
| Number of particles | 1,143,729 | |
| Model composition | ||
| Non-hydrogen atoms | 9684 | |
| Protein residues | 1210 | |
| Lipids/Detergents/Ligands | 3 CHS/1 ATP/1 ADP/2 Mg2+ | |
| Refinement | ||
| Resolution (Å) | 3.23 | |
| Rwork | 0.265 | |
| Rfree | 0.276 | |
| RMS deviations | ||
| Bond lengths (Å) | 0.003 | |
| Bond angles (°) | 1.297 | |
| Validation | ||
| Molprobity score | 1.11 | |
| Clashscore, all atoms | 1.07 | |
| Favored rotamers (%) | 97.7 | |
| Ramachandran plot (%) | ||
| Favored | 95.7 | |
| Allowed | 4.3 | |
| Outliers | 0.0 | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Biotinylated 6x-His Tag monoclonal antibody | Invitrogen | Cat# MA1-21315-BTIN | Molar ratio of 2:1 (antibody:bMRP1) |
| Cell line | Sf9 | ATCC | CRL-1711 | |
| Cell line | HEK293S GnTI- | ATCC | CRL-3022 | |
| Recombinant DNA reagent | bovine MRP1 in pUC57 vector | Bio Basic | Codon-optimized | |
| Recombinant DNA reagent | bovine MRP1 in a modified pEG BacMam vector | Johnson and Chen, 2017 | Suitable for expression in mammalian cells | |
| Recombinant DNA reagent | bovine MRP1 with S6/A1 peptides for site-specific labeling | This paper | ||
| Recombinant DNA reagent | bovine MRP1 E1454Q with S6/A1 peptides for site-specific labeling | This paper | ||
| Recombinant DNA reagent | Sfp pet29b C-terminal His Tag | (Worthington and Burkart, 2006) | Addgene Plasmid# 75015 | |
| Recombinant DNA reagent | pET15b-ACPs (from S. pneumoniae) | Gift from Michael Johnson | Addgene Plasmid# 63687 | |
| Chemical compound, drug | Cy3 maleimide mono-reactive dye | GE Healthcare | Cat# PA23031 | |
| Chemical compound, drug | LD655 maleimide mono-reactive dye | Lumidyne Technologies | Cat# LD655-MAL | |
| Chemical compound, drug | Trolox | Sigma-Aldrich | Cat# 238813 | |
| Chemical compound, drug | 4-Nitrobenzyl alcohol (NBA) | Sigma-Aldrich | Cat# N12821 | |
| Chemical compound, drug | Cyclooctatetraene (COT) | Sigma-Aldrich | Cat# 138924 | |
| Chemical compound, drug | 3,4-Dihydroxybenzoic acid (PCA) | Sigma-Aldrich | Cat# 37580 | |
| Chemical compound, drug | Digitonin | Sigma-Aldrich | Cat# D141 | |
| Peptide, recombinant protein | Protocatechuate 3,4-Dioxygenase (PCD) | Sigma-Aldrich | Cat# P8279 | |
| Peptide, recombinant protein | Leukotriene C4 | Cayman Chemical | Cat# 20210 | |
| Commercial assay, kit | NHS-activated Sepharose 4 Fast Flow resin | GE Healthcare | Cat# 17-0430-01 | |
| Commercial assay, kit | Superose 6, 10/300 GL | GE Healthcare | Cat# 17-5172-01 | |
| Software, algorithm | SPARTAN | (Juette et al., 2016) | https://www.scottcblanchardlab.com/software | |
| Software, algorithm | ebFRET | (van de Meent et al., 2014) | http://ebfret.github.io | |
| Software, algorithm | MATLAB | MathWorks | https://www.mathworks.com/products/matlab.html | |
| Software, algorithm | Origin | OriginLab | https://www.originlab.com | |
| Software, algorithm | GraphPad Prism | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | RELION 1.4 | (Scheres, 2012) | https://www2.mrc-lmb.cam.ac.uk/relion | |
| Software, algorithm | Frealign | (Grigorieff, 2016) | https://grigoriefflab.janelia.org/frealign | |
| Software, algorithm | Coot | (Emsley and Cowtan, 2004) | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot | |
| Software, algorithm | PHENIX | (Adams et al., 2010) | https://www.phenix-online.org | |
| Software, algorithm | REFMAC | (Brown et al., 2015) | https://www.ccp4.ac.uk/html/refmac5.html | |
| Software, algorithm | MolProbity | (Chen et al., 2010) | https://molprobity.biochem.duke.edu | |
| Software, algorithm | Chimera | (Pettersen et al., 2004) | https://www.cgl.ucsf.edu/chimera | |
| Software, algorithm | PyMOL | PyMOL | https://www.pymol.org | |
| Software, algorithm | cryoSPARC | (Punjani et al., 2017) | https://cryosparc.com | |
| Other | R1.2/1.3 400 mesh Au holey carbon grids | Quantifoil | Cat# 1210627 | 1 µg/mL |
