Nucleotide binding is the critical regulator of ABCG2 conformational transitions

  1. Zsuzsanna Gyöngy
  2. Gábor Mocsár
  3. Éva Hegedűs
  4. Thomas Stockner
  5. Zsuzsanna Ritter
  6. László Homolya
  7. Anita Schamberger
  8. Tamás I Orbán
  9. Judit Remenyik
  10. Gergely Szakacs
  11. Katalin Goda  Is a corresponding author
  1. Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary
  2. Doctoral School of Molecular Cell and Immune Biology, University of Debrecen, Hungary
  3. Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria
  4. Institute of Enzymology, Research Centre for Natural Sciences, Hungary
  5. Institute of Food Technology, Faculty of Agricultural and Food Sciences and Environmental Management, University of Debrecen, Hungary
  6. Institute of Cancer Research, Medical University of Vienna, Austria
8 figures, 2 tables and 1 additional file


The IF and OF conformers of ABCG2.

A slice through IF (A) and OF (B) ABCG2 shows the lower and upper cavity and reveal their volume changes induced by the transition. Two rotationally symmetric 5D3 epitopes are formed in the IF conformation (C, E) and are absent in the OF conformation of ABCG2 (D, F). Panels (C) and (D) show an extracellular view of the 5D3-bound IF (PDB ID: 6ETI), and the OF (PDB ID: 6HBU) ABCG2 conformations, respectively. A side view of the IF (E) and the OF (F) ABCG2 is also shown. The two protomers of ABCG2 are colored in pink and green in cartoon backbone and transparent surface rendering. The two epitopes are highlighted in blue and orange, and include all residues that are in direct contact with the 5D3 Fab (white) in the 6ETI cryo-EM structure. The membrane boundaries are indicated by dashed lines. cryo-EM, cryogenic electron microscopy; IF, inward-facing; OF, outward-facing.

Functional characterization of human ABCG2 and ABCG2-GFP.

Western blot showing comparable expression of ABCG2 and ABCG2-GFP in MDCK II cells using the BXP-21 anti-ABCG2 mAb (A). ABCG2 and ABCG2-GFP expressing cells show decreased mitoxantrone (MX) accumulation, which is reversed to the level of the ABCG2-negative cells by 2 µM Ko143 (B–D). 2 µM Ko143 pre-treatment causes an increase in the 5D3-reactivity of ABCG2 (F) and ABCG2-GFP (G) expressing cells. ABCG2-negative cells show only a negligible 5D3 binding that is not affected by Ko143 (E).

Nucleotide dependence of 5D3-reactivity.

MDCK-ABCG2 cells were permeabilized to allow the titration of intracellular nucleotide concentrations. Dose-response curves of 5D3 binding with increasing concentrations of ATP/Mg2+ (panels (A), and (B)), ATP in the presence of EDTA (panels (C) and (D)), AMP-PNP/Mg2+ (panels (G) and (H)), or ADP/Mg2+ (panels (I) and (J)) were obtained in the presence or absence of Vi (left panels) or BeFx (right panels). Samples were pre-treated with nucleotides at 37°C for 10 min, then further incubated with 5 µg/ml 5D3-A647 at 37°C for 20 min except for (panels (E) and (F)), where all the treatments were carried out on ice. In case of nucleotide trapping, permeabilized cells were co-treated with nucleotides and BeFx or Vi at 37°C for 30 min, then un-trapped nucleotides were washed out and 5D3 labeling was carried out on ice for 45 min. Representative curves are shown from three to five independent experiments. The small inserts in panel (A) depict the IF and OF conformations of ABCG2. IF, inward-facing; OF, outward-facing.

Effect of transported substrates on the kinetics of the formation of the Vi- and BeFx-trapped complexes.

Permeabilized MDCK-ABCG2 cells were pre-treated or not with 10 µM quercetin or E3S for 10 min at 37°C and then further incubated with 0.5 mM ATP/Mg2+ or ADP/Mg2+ in the presence of Vi or BeFx. Samples taken at different time points were stained with 5 µg/ml 5D3-A647 on ice for 45 min. Panel (A) shows a representative Vi-trapping experiment in the absence or presence of E3S, while panel (B) summarizes the t1/2 values calculated from the exponential fit of the kinetic curves (see Materials and methods). Mean ± SD of 3–5 independent experiments is shown. Significant differences compared to substrate untreated samples are shown by ***: p < 0.001.

Effects of substrates and nucleotides on the kinetics of 5D3 dissociation when substrates were added simultaneously with nucleotides (A, B), or prior to nucleotides (C).

Permeabilized MDCK-ABCG2 cells were pre-labeled with 5D3-A647 in the absence (panels (A) and (B)) or presence (panel (C)) of substrates (10 µM quercetin or 10 µM E3S) for 20 min at 37°C. After removal of the unbound antibody, cells were further incubated with 3 mM ATP/Mg2+ or AMP-PNP/Mg2+ in the absence or presence of substrates. Panel (A) shows representative 5D3 dissociation curves in the indicated conditions, while panels (B) and (C) summarizes the t1/2 values calculated from the exponential fit of the dissociation curves (see Materials and methods). Means ± SD of 3–5 independent experiments are shown. Significant differences compared to untreated samples (first column) are shown by ###: p < 0.001 or ##: p < 0.01. Significant differences compared to only nucleotide-treated samples are indicated by ***: p < 0.001.

Cellular distribution of MX (red) in MDCK cells expressing ABCG2-GFP (green) in response to ATP depletion and/or Ko143 or Vi treatment (A).

All treatments increased the intracellular MX fluorescence intensity (B), while the ABCG2-GFP fluorescence intensity in the plasma membrane remained unchanged (C). Pearson’s correlation coefficients between the intensity distributions of MX and ABCG2-GFP in the plasma membrane pixels reveal the stabilization of a high-affinity substrate binding ABCG2 conformation in energy-deprived cells (D). ATP depletion was induced by 15  min pre-treatment of cells with 10  mM sodium azide and 8  mM 2-deoxy-D-glucose. Ko143, a non-fluorescent, competitive ABCG2 inhibitor, was added 10  min before MX staining. In panels (B) and (C), bars represent mean ± SD values, while panel (D) shows box and whisker plots. For each treatment group, 150–200 cells were analyzed from three to five independent experiments. Significant differences compared to the untreated control are shown by ***: p < 0.001.

Fluorescence autocorrelation functions (A) and diffusion constants (B) of ABCG2-GFP (green) and MX (orange) in the plasma membrane of intact MDCK cells.

ABCG2-GFP expressing cells without pre-treatments or following ATP depletion or Ko143 pre-treatment were stained with 100 nM MX for 15 min at 37°C. Each bar shows the mean ± SD for n = 50–100 cells from at least three independent measurements. Significant differences compared to the diffusion constant of ABCG2-GFP (green bar) in the plasma membrane of untreated cells are shown by ***: p < 0.001.

Substrate affinity changes of ABCG2 in permeabilized cells.

SLO-permeabilized ABCG2-GFP expressing cells were pre-treated or not with 5 mM AMP-PNP/Mg2+ for 15 min and then stained with 500 nM MX for 15 min at 37°C. Permeabilized cells were identified by PI staining. Box and whisker plots show Pearson’s correlation coefficients of n > 150 cells from three independent experiments. ***p < 0.001 by Kolmogorov-Smirnov test. MX, mitoxantrone; PI, propidium iodide; SLO, streptolysin-O.


Table 1
Apparent nucleotide affinities (KA) determined in 5D3-reactivity experiments.
TreatmentConditionsKA (mM ± SD)nStatistical comparisons to ATP/Mg2+ (1st row)Figure
ATP37°C+4.15 ± 1.0811Figure 3A
ATP+Vi37°C+0.34 ± 0.2511p < 0.001Figure 3A
ATP+BeFx37°C+0.40 ± 0.063p < 0.001Figure 3B
ATP37°C3.69 ± 0.497nsFigure 3C
ATP+Vi37°C3.77 ± 0.384nsFigure 3C
ATP+BeFx37°C3.20 ± 0.153nsFigure 3D
ATP4°C+4.38 ± 0.246nsFigure 3E
ATP+Vi4°C+4.01 ± 0.233nsFigure 3E
ATP+BeFx4°C+4.06 ± 0.463nsFigure 3F
AMP-PNP37°C+5.29 ± 0.222nsFigure 3G
AMP-PNP+Vi37°C+4.911ndFigure 3G
AMP-PNP+BeFx37°C+5.881ndFigure 3H
ADP37°C+7.37 ± 2.316p < 0.001Figure 3I
ADP+Vi37°C+0.44 ± 0.103p < 0.001Figure 3I
ADP+BeFx37°C+0.57 ± 0.323p < 0.001Figure 3J
Key resources table
Reagent type or resourceDesignationSource or referenceIdentifiersAdditional information
Antibody5D3 (mouse monoclonal)Hybridoma was donated by Brian P. SorrentinoPrepared from hybridoma supernatant by affinity chromatography
AntibodyBXP-21 (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-58222(1:2500 dilution)
AntibodyC-2 (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-8432(1:2500 dilution)
AntibodyGoat anti-mouse IgG-HRP (polyclonal)Santa Cruz BiotechnologyCat# sc-2005(1:2500 dilution)
Cell line (Canis familiaris, dog)MDCK II, (epithelial-like cells from kidney distal tubule)Obtained from Prof. Gerrit van MeerECACC 00062107Mycoplasma-free
Cell line (Mus musculus, mouse)5D3 hybridomaDonated by Brian P. SorrentinoMycoplasma-free
Chemical compound, drugMitoxantroneSigma-AldrichCat# M6545
Chemical compound, drugKo143Sigma-AldrichCat# K2145
Chemical compound, drugQuercetinSigma-AldrichCat# Q4951
Chemical compound, drugEstrone-3-sulfateSigma-AldrichCat# E9145
Chemical compound, drugADPSigma-AldrichCat# A2754
Chemical compound, drugAMP-PNPSigma-AldrichCat# A2647
Chemical compound, drugATPSigma-AldrichCat# A2383
Chemical compound, drugSodium-orthovanadateSigma-AldrichCat# S6508
Chemical compound, drugProtease Inhibitor CocktailSigma-AldrichCat# P2714
Chemical compound, drugDL-DithiothreitolSigma-AldrichCat# D0632
Software, algorithmFlowing softwareTurku Centre for Biotechnology
Software, algorithmMATLABMathworks Inc.
Software, algorithmQuickFit 3.0
Software, algorithmSigmaPlotSystat Software Inc.
OtherEight-well chambered coverslip plateibidi GmbHCat# 80826-90Imaging chamber for microscopy
OtherStreptolysin-O (SLO) from Streptococcus pyogenesSigma-AldrichCat# S5265(250 U/ml)
OtherAlexa 647 succinimidyl esterLife Technologies Inc.Cat# A20006Fluorescent dye
OtherPropidium iodideSigma-AldrichCat# P4170Fluorescent dye
OtherSuperSignal West Pico PLUS Chemiluminescent SubstrateThermo Fisher ScientificCat# 34579Reagent for protein detection in western blot analysis

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  1. Zsuzsanna Gyöngy
  2. Gábor Mocsár
  3. Éva Hegedűs
  4. Thomas Stockner
  5. Zsuzsanna Ritter
  6. László Homolya
  7. Anita Schamberger
  8. Tamás I Orbán
  9. Judit Remenyik
  10. Gergely Szakacs
  11. Katalin Goda
Nucleotide binding is the critical regulator of ABCG2 conformational transitions
eLife 12:e83976.