Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

  1. Ling Wang
  2. Zachary Lee Johnson
  3. Michael R Wasserman
  4. Jesper Levring
  5. Jue Chen  Is a corresponding author
  6. Shixin Liu  Is a corresponding author
  1. Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, United States
  2. Laboratory of Membrane Biology and Biophysics, The Rockefeller University, United States
  3. Howard Hughes Medical Institute, The Rockefeller University, United States
7 figures, 3 tables and 1 additional file

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
ConditiontIF (s)tOF (s)
WT, 5 mM ATP16.1 ± 2.228.8 ± 4.8
WT, 5 mM ATP + 10 µM LTC44.7 ± 0.830.8 ± 5.2
E1454Q, 5 mM ATP + 10 µM LTC47.7 ± 1.531.7 ± 5.5
  1. 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
MicroscopeTitan krios (FEI)
Voltage (kV)300
DetectorK2 Summit (Gatan)
Pixel size (Å)1.03
Defocus range (μm)0.7 to 2.4
Movies3604
Frames/movie50
Dose rate (electrons/pixel/s)8.0
Total dose (electrons/Å2)75
Number of particles1,143,729
Model composition
Non-hydrogen atoms9684
Protein residues1210
Lipids/Detergents/Ligands3 CHS/1 ATP/1 ADP/2 Mg2+
Refinement
Resolution (Å)3.23
Rwork0.265
Rfree0.276
RMS deviations
Bond lengths (Å)0.003
Bond angles (°)1.297
Validation
Molprobity score1.11
Clashscore, all atoms1.07
Favored rotamers (%)97.7
Ramachandran plot (%)
Favored95.7
Allowed4.3
Outliers0.0
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
AntibodyBiotinylated 6x-His Tag monoclonal antibodyInvitrogenCat# MA1-21315-BTINMolar ratio of 2:1 (antibody:bMRP1)
Cell lineSf9ATCCCRL-1711
Cell lineHEK293S GnTI-ATCCCRL-3022
Recombinant DNA reagentbovine MRP1 in pUC57 vectorBio BasicCodon-optimized
Recombinant DNA reagentbovine MRP1 in a modified pEG BacMam vectorJohnson and Chen, 2017Suitable for expression in mammalian cells
Recombinant DNA reagentbovine MRP1 with S6/A1 peptides for site-specific labelingThis paper
Recombinant DNA reagentbovine MRP1 E1454Q with S6/A1 peptides for site-specific labelingThis paper
Recombinant DNA reagentSfp pet29b C-terminal His Tag(Worthington and Burkart, 2006)Addgene Plasmid# 75015
Recombinant DNA reagentpET15b-ACPs (from S. pneumoniae)Gift from Michael JohnsonAddgene Plasmid# 63687
Chemical compound, drugCy3 maleimide mono-reactive dyeGE HealthcareCat# PA23031
Chemical compound, drugLD655 maleimide mono-reactive dyeLumidyne TechnologiesCat# LD655-MAL
Chemical compound, drugTroloxSigma-AldrichCat# 238813
Chemical compound, drug4-Nitrobenzyl alcohol (NBA)Sigma-AldrichCat# N12821
Chemical compound, drugCyclooctatetraene (COT)Sigma-AldrichCat# 138924
Chemical compound, drug3,4-Dihydroxybenzoic acid (PCA)Sigma-AldrichCat# 37580
Chemical compound, drugDigitoninSigma-AldrichCat# D141
Peptide, recombinant proteinProtocatechuate 3,4-Dioxygenase (PCD)Sigma-AldrichCat# P8279
Peptide, recombinant proteinLeukotriene C4Cayman ChemicalCat# 20210
Commercial assay, kitNHS-activated Sepharose 4 Fast Flow resinGE HealthcareCat# 17-0430-01
Commercial assay, kitSuperose 6, 10/300 GLGE HealthcareCat# 17-5172-01
Software, algorithmSPARTAN(Juette et al., 2016)https://www.scottcblanchardlab.com/software
Software, algorithmebFRET(van de Meent et al., 2014)http://ebfret.github.io
Software, algorithmMATLABMathWorkshttps://www.mathworks.com/products/matlab.html
Software, algorithmOriginOriginLabhttps://www.originlab.com
Software, algorithmGraphPad PrismGraphPadhttps://www.graphpad.com/scientific-software/prism/
Software, algorithmRELION 1.4(Scheres, 2012)https://www2.mrc-lmb.cam.ac.uk/relion
Software, algorithmFrealign(Grigorieff, 2016)https://grigoriefflab.janelia.org/frealign
Software, algorithmCoot(Emsley and Cowtan, 2004)https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot
Software, algorithmPHENIX(Adams et al., 2010)https://www.phenix-online.org
Software, algorithmREFMAC(Brown et al., 2015)https://www.ccp4.ac.uk/html/refmac5.html
Software, algorithmMolProbity(Chen et al., 2010)https://molprobity.biochem.duke.edu
Software, algorithmChimera(Pettersen et al., 2004)https://www.cgl.ucsf.edu/chimera
Software, algorithmPyMOLPyMOLhttps://www.pymol.org
Software, algorithmcryoSPARC(Punjani et al., 2017)https://cryosparc.com
OtherR1.2/1.3 400 mesh Au holey carbon gridsQuantifoilCat# 12106271 µg/mL

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