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Structural basis of small-molecule inhibition of human multidrug transporter ABCG2

Nature Structural & Molecular Biology volume 25, pages 333–340 (2018)Cite this article

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Abstract

ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure–activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.

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Fig. 1: Functional characteristics of Ko143 derivatives.
Fig. 2: Structure of the ABCG2–MZ29–Fab complex.
Fig. 3: ABCG2 NBDs.
Fig. 4: Structure of MB136-bound ABCG2 revealing a central multidrug-binding site.
Fig. 5: ABCG2–lipid interactions.
Fig. 6: Proposed mechanism of inhibition.

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Acknowledgements

This research was supported by the Swiss National Science Foundation through the National Centre of Competence in Research (NCCR) TransCure and by a Swiss Federal Institute of Technology Zurich (ETH Zurich) research grant ETH-22-14-1. J.K. was also supported by the TransCure Young Investigator Award. N.M.I.T. was also supported by the University of Basel Research Fund for Junior Investigators. Cryo-EM data for the ABCG2–MZ29–Fab and ABCG2–MB136–Fab samples were collected at the electron microscopy facility at ETH Zurich (ScopeM); we thank P. Tittmann for technical support. Cryo-EM data for the ABCG2-MZ29 sample were collected at C-CINA, University of Basel; we thank K. Goldie, L. Kováčik and A. Fecteau-Lefebvre for technical support. We also thank J. Bloch for helpful discussions, F. Antoni, M. Scholler and D. Wifling (University of Regensburg) for technical assistance and helpful discussions and B. Sorrentino (St. Jude Children’s Research Hospital) for providing the 5D3-producing hybridoma cell line.

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Author notes

  1. Nicholas M. I. Taylor

    Present address: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

  2. These authors contributed equally: Scott M. Jackson, Ioannis Manolaridis, Julia Kowal, Melanie Zechner and Nicholas M. I. Taylor.

  3. Deceased: Armin Buschauer.

Authors and Affiliations

  1. Institute of Molecular Biology and Biophysics, Department of Biology, ETH Zurich, Zurich, Switzerland

    Scott M. Jackson, Ioannis Manolaridis, Julia Kowal & Kaspar P. Locher

  2. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland

    Melanie Zechner, Ruben Bartholomaeus & Karl-Heinz Altmann

  3. Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland

    Nicholas M. I. Taylor & Henning Stahlberg

  4. Faculty of Chemistry and Pharmacy, University of Regensburg, Regensburg, Germany

    Manuel Bause, Stefanie Bauer, Guenther Bernhardt, Burkhard Koenig & Armin Buschauer

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Contributions

I.M. expressed and purified ABCG2 and 5D3-Fab. I.M. and S.M.J. performed MST and thermostability experiments. S.M.J. reconstituted ABCG2 into liposomes and lipidic nanodiscs. J.K. prepared all cryo-grids and collected cryo-EM data for ABCG2–MZ29–Fab and ABCG2–MB136–Fab. I.M. and J.K. determined the structure of ABCG2–MZ29–Fab. J.K. determined the structure of ABCG2–MB136–Fab. N.M.I.T. and H.S. collected cryo-EM data and determined the structure of ABCG2–MZ29. I.M. and K.P.L. refined and validated the structures with the help of J.K. and N.M.I.T. M.Z. synthesized Ko143 and derivatives, and R.B. synthesized FKo143 and FKo132, under the supervision of K.-H.A. M.B. synthesized MB136. M.B., S.B., G.B., B.K. and A.B. designed MB136 and supervised and assisted in its synthesis. S.M.J. screened the compounds and performed all of the ATPase and transport assays. K.P.L., K.-H.A., S.M.J. and I.M. conceived the project. K.P.L., S.M.J. and I.M. planned the experiments. S.M.J., I.M. and K.P.L. wrote the manuscript; all authors contributed to revisions.

Corresponding authors

Correspondence to Karl-Heinz Altmann or Kaspar P. Locher.

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Integrated supplementary information

Supplementary Figure 1 Purification of ABCG2, SEC-TS and reconstitution of ABCG2–MZ29–Fab into nanodiscs.

a, Preparative SEC profile of detergent-purified ABCG2. The fraction used for SEC-TS and nanodisc preparation is indicated with an arrow. b, SEC-TS of ABCG2 before or after the addition of specified inhibitors at 10 µM concentration and E1S at 50 µM concentration. c, Preparative SEC profile of the nanodisc-reconstituted ABCG2-MZ29-Fab complex. The fraction used for cryo-EM grid preparation and SDS-PAGE analysis is indicated by ‘1’. d, Non-reducing SDS-PAGE of the nanodisc-reconstituted ABCG2-MZ29-Fab complex shown in c.

Source data

Supplementary Figure 2 Cryo-EM map generation and data processing flow chart of the ABCG2–MZ29–Fab complex.

a, An example micrograph (drift-corrected, dose-weighted, and low-pass filtered to 20 Å) of the nanodisc-reconstituted ABCG2–MZ29-Fab data set. White scale bar, 50 nm. b, Averages of 15 representative two-dimensional class averages of the final round of two-dimensional classification, sorted in decreasing order by the number of particles assigned to each class. c, The flow chart for the cryo-EM data processing and structure determination of the ABCG2-MZ29-Fab complex.

Supplementary Figure 3 Atomic model refinement and local resolution of the ABCG2–MZ29–Fab complex.

a, FSC from the RELION auto-refine procedure of the unmasked half-maps (blue), the random-phase corrected half-maps (brown), the half-maps after masking (cyan), and the half-maps after masking and correction for the influence of the mask (pink). A horizontal line (blue) is drawn for the FSC = 0.143 criterion. For both the unmasked and the corrected FSC curves, their intersection with the FSC = 0.143 line is indicated by an arrow, and the resolution at this point is indicated. b, FSC curve of the final 3.1 Å refined model versus the map it was refined against (FSCfull, black line). FSC curve of the final model with introduced shifts (mean value of 0.3 Å) refined against the first of two independent half-maps (half-map 1) (to which it was refined against; FSCwork red line) or the same refined model versus the second independent half-map (to which is was not refined; FSChalf2, green line). c, Full view of the RELION local-resolution-filtered map of ABCG2-MZ29-Fab colored by local resolution as calculated by ResMap with the clipping plane in the middle of the molecule. ABCG2, Fab and nanodiscs are labeled. d, Resolution distribution histogram generated in ResMap. e, Angular distribution plot for the final reconstruction.

Supplementary Figure 4 Fit of the model to the density of the ABCG2–MZ29–Fab complex.

a, Fit of the TM helices of the final model of the ABCG2 TMD to the post-processed and masked map from RELION. A region of up to 2 Å around the atoms is shown. b, Same as a but showing the intramolecular disulfide (C592-C608), the intermolecular disulfide (C603-C603’) and N596 decorated with two GlcNAcs. c, Same as a but showing the Walker A motif with selected residues numbered. d, Same as a but showing the Walker B motif and the D loop. e, Same as a but showing the α-helix containing Q141 with selected residues numbered. f, Same as a but showing the fit of MZ29 and surrounding residues.

Supplementary Figure 5 Cryo-EM map generation and data processing flow chart of the ABCG2–MB136–Fab complex.

a, An example micrograph (drift-corrected, dose-weighted, and low-pass filtered to 20 Å) of the nanodisc-reconstituted ABCG2–MB136-Fab data set. White scale bar, 50 nm. b, Averages of 15 representative two-dimensional class averages of the final round of two-dimensional classification, sorted in decreasing order by the number of particles assigned to each class. c, The flow chart for the cryo-EM data processing and structure determination of the ABCG2-MB136-Fab complex.

Supplementary Figure 6 Atomic model refinement and local resolution of the ABCG2–MB136–Fab complex.

a, FSC from the RELION auto-refine procedure of the unmasked half-maps (blue), the random-phase corrected half-maps (brown), the half-maps after masking (cyan), and the half-maps after masking and correction for the influence of the mask (pink). A horizontal line (blue) is drawn for the FSC = 0.143 criterion. For both the unmasked and the corrected FSC curves, their intersection with the FSC = 0.143 line is indicated by an arrow, and the resolution at this point is indicated. b, Angular distribution plot for the final reconstruction. c, Full view of the RELION local-resolution-filtered map of ABCG2-MB136-Fab colored by local resolution as calculated by ResMap with the clipping plane in the middle of the molecule. ABCG2, Fab and nanodiscs are labeled. d, Resolution distribution histogram generated in ResMap. e, Fit of one MB136 molecule into the EM density of the ABCG2-MB136-Fab structure processed with C2 symmetry. f, Fit of one MB136 molecule into the EM density of the ABCG2-MB136-Fab structure processed with C1 symmetry.

Supplementary Figure 7 Cryo-EM map generation and data processing flow chart of the ABCG2–MZ29 complex.

a, An example micrograph (drift-corrected, dose-weighted, and low-pass filtered to 20 Å) of the nanodisc-reconstituted ABCG2–MZ29 data set. White scale bar, 50 nm. b, Averages of 15 representative two-dimensional class averages of the final round of two-dimensional classification, sorted in decreasing order by the number of particles assigned to each class. c, The flow chart for the cryo-EM data processing and structure determination of the ABCG2-MZ29 complex.

Supplementary Figure 8 Atomic model refinement and local resolution of the ABCG2–MZ29 complex.

a, FSC from the RELION auto-refine procedure of the unmasked half-maps (blue), the random-phase corrected half-maps (brown), the half-maps after masking (cyan), and the half-maps after masking and correction for the influence of the mask (pink). A horizontal line (blue) is drawn for the FSC = 0.143 criterion. For both the unmasked and the corrected FSC curves, their intersection with the FSC = 0.143 line is indicated by an arrow, and the resolution at this point is indicated. b, FSC curve of the final 3.56 Å refined model versus the map it was refined against (FSCfull, black line). FSC curve of the final model with introduced shifts (mean value of 0.3 Å) refined against the first of two independent half-maps (half-map1) (to which it was refined against; FSCwork red line) or the same refined model versus the second independent half-map (to which is was not refined; FSChalf2, green line). c, Full view of the RELION local resolution filtered map of ABCG2-MZ29 colored by local resolution as calculated by ResMap with the clipping plane in the middle of the molecule. d, Resolution distribution histogram generated in ResMap. e, Angular distribution plot for the final reconstruction. f, Superposition of the ABCG2-MZ29-Fab structure with the Fabs removed (blue) and the ABCG2-MZ29 structure (red). The insert shows the superposition of the bound MZ29 molecules.

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Supplementary Figures 1–8, Supplementary Tables 1–5 and Supplementary Note

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Supplementary Dataset 1

Combined source data for Figs. 1a–c, 4b and 6a and Supplementary Fig. 1b

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Jackson, S.M., Manolaridis, I., Kowal, J. et al. Structural basis of small-molecule inhibition of human multidrug transporter ABCG2. Nat Struct Mol Biol 25, 333–340 (2018). https://doi.org/10.1038/s41594-018-0049-1

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