Article | Published:

Structure of the human multidrug transporter ABCG2

Nature volume 546, pages 504509 (22 June 2017) | Download Citation

Abstract

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.

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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 Zürich (ETH Zürich) research grant ETH-22-14-1. We thank the staff of the X06SA beamline of the Swiss Light Source for support during data collection. We thank K. Goldie, A. Fecteau-LeFebre, and R. McLeod for support during electron microscope use, and R. Adaixo, L. Muckenfuss, N. Biyani, and R. Righetto for support and discussions during EM data analysis. We thank L. Lancien and B. Prinz for help with protein expression and cell culture work. We thank B. Sorrentino for providing the 5D3-producing hybridoma cell line, B. Stieger for discussions about transport assays, and J.-I. Kim for initial ABCG2 expression and purification experiments.

Author information

Author notes

    • Nicholas M. I. Taylor
    • , Ioannis Manolaridis
    •  & Scott M. Jackson

    These authors contributed equally to this work.

Affiliations

  1. Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Mattenstrasse 26, 4058 Basel, Switzerland

    • Nicholas M. I. Taylor
    •  & Henning Stahlberg
  2. Institute of Molecular Biology and Biophysics, ETH Zürich, Otto-Stern-Weg 5, 8093 Zürich, Switzerland

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

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Contributions

I.M. expressed human and rat ABCG2. I.M. and S.M.J. purified and reconstituted ABCG2 in liposomes and nanodiscs. S.M.J. performed functional assays. J.K. performed initial negative-stain and cryo-EM analyses and prepared all grids. N.M.I.T. and H.S. performed cryo-EM data collection. N.M.I.T. performed ABCG2 structure determination and built the ABCG2 model. I.M., K.P.L. and N.M.I.T. revised the model, and N.M.I.T. performed model refinement. I.M. determined the 5D3-Fab crystal structure. K.P.L., I.M., S.M.J., and H.S. conceived the project and planned the experiments. I.M., S.M.J. and K.P.L. wrote the manuscript; all authors contributed to its revision.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Henning Stahlberg or Kaspar P. Locher.

Reviewer Information Nature thanks A. Ward and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature22345

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