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Structure of the human lipid exporter ABCB4 in a lipid environment

Nature Structural & Molecular Biology volume 27, pages 62–70 (2020)Cite this article

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Abstract

ABCB4 is an ATP-binding cassette transporter that extrudes phosphatidylcholine into the bile canaliculi of the liver. Its dysfunction or inhibition by drugs can cause severe, chronic liver disease or drug-induced liver injury. We determined the cryo-EM structure of nanodisc-reconstituted human ABCB4 trapped in an ATP-bound state at a resolution of 3.2 Å. The nucleotide binding domains form a closed conformation containing two bound ATP molecules, but only one of the ATPase sites contains bound Mg2+. The transmembrane domains adopt a collapsed conformation at the level of the lipid bilayer, but we observed a large, hydrophilic and fully occluded cavity at the level of the cytoplasmic membrane boundary, with no ligand bound. This indicates a state following substrate release but prior to ATP hydrolysis. Our results rationalize disease-causing mutations in human ABCB4 and suggest an ‘alternating access’ mechanism of lipid extrusion, distinct from the ‘credit card swipe’ model of other lipid transporters.

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Fig. 1: Functional characterization of ABCB4.
Fig. 2: Structure of human ABCB4.
Fig. 3: Nucleotide binding to ABCB4.
Fig. 4: Functionally impairing mutants and chimeras of ABCB4.
Fig. 5: The structure of ABCB4 in the context of the predicted transport cycle, with states numbered.

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Data availability

Source data for Figs. 1b,d,e and 4d as well as Extended Data Fig. 2a are available with the online version of this paper. Coordinates for the deposited model have been deposited at the Protein Data Bank under ID 6S7P. The associated cryo-EM map has been deposited at the Electron Microscopy Data Bank under ID EMD-10111.

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Acknowledgements

This research was supported by the Swiss National Science Foundation (grant nos. 310030B_166672 and 310030_189111 to K.P.L.) and by The Danish Council for Independent Research (grant no. DFF-4181-00021 to J.A.O.). Cryo-EM data were collected at the electron microscopy facility at ETH Zürich (ScopeM) and the authors thank the ScopeM staff for technical support.

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  1. Jeppe A. Olsen

    Present address: Ichnos Sciences, Epalinges, Switzerland

  2. Amer Alam

    Present address: The Hormel Institute, University of Minnesota, Austin, MN, USA

Authors and Affiliations

  1. Department of Biology, ETH Zürich, Zürich, Switzerland

    Jeppe A. Olsen, Amer Alam, Julia Kowal & Kaspar P. Locher

  2. Department of Clinical Pharmacology and Toxicology, University Hospital Zürich, Zürich, Switzerland

    Bruno Stieger

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Contributions

J.A.O. performed experiments and prepared samples. J.A.O., A.A. and J.K. collected the cryo-EM data. J.A.O., A.A. and J.K. processed the cryo-EM data. J.A.O. and K.P.L. built and validated the model of nucleotide-bound ABCB4. J.A.O. and K.P.L. designed the experiments. J.A.O., B.S. and K.P.L. conceived the project. J.A.O. and K.P.L. wrote the manuscript, with all authors contributing to revisions.

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Correspondence to Kaspar P. Locher.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 SEC and FSEC profiles of ABCB4.

a,b, FSEC profiles of eYFP-labeled protein extracted by DDM:CHS from pellets of WT ABCB4 expressing Flp-In T-Rex 293 cells collected after harvesting of media for extrusion experiments. Black lines indicates signal from cells with expression induced by addition of tetracycline, red lines indicates signal from uninduced cells. Samples were loaded on a TSKgel G4000SWXL column at 0.4 ml −1min. a, Signals from cells expressing wt-ABCB4). b, Signals from cells expressing ABCB4[EQ]. c-f, SEC profiles of purified ABCB4 reconstituted into LPL:CHS nanodiscs and run on a a TSKgel G3000SWXL column at 0.4 ml −1min. c, ABCB4[EQ] d, Reinjection of the peak fraction from (c) at around 18 min, as also shown in Fig. 1c. e, wt-ABCB4 with reinjected peak fraction shown in (f), as also shown in Fig. 1c.

Extended Data Fig. 2 Cryo-EM data processing of vanadate-trapped wt-ABCB4.

a, ATPase activity of vanadate-trapped wt-ABCB4 (black squares and curve) shown relative to the activity without vanadate (dashed blue line, curve from Fig. 1d). Data points indicate means of three independent measurements, error bars represent S.E.M. b, Representative motion-corrected micrograph of ABCB4 at a nominal magnification of 165kx and a defocus of 2.7 µm. c, Representative 2D classes from cryoSPARC. d, cryoSPARC processing workflow. Data for graph in panel a are available as.

Source data

Extended Data Fig. 3 Cryo-EM structure determination of nucleotide-bound ABCB4.

a, Representative motion-corrected micrograph of ABCB4 at nominal magnification of 165 kx and a defocus of 2.2 µm. Representative 2D classes from RELION are shown to the left. b, RELION processing workflow. c, Local resolution calculated with ResMap in RELION and presented on the sharpened EM map, color coded according to the scale bar next to the density. d, The 3.2 Å map used for model building (cyan) is displayed inside the unsharpened map contoured at lower level (grey). e, Fourier shell correlation curves from RELION. f, Distribution of orientations of particles going into the final class.

Extended Data Fig. 4 Cryo-EM map and model.

The model of nucleotide-bound ABCB4 built according to density. Contour level is set at σ = 5 and density is carved at a distance of 2 Å. a, Density contours from b-factor sharpened map is shown for all six helix pairs as well as the NBDs. b, Density contours shown around central slices of the NBDs, for the same views as shown in (a). Nucleotides are shown in red, Magnesium in yellow.

Extended Data Fig. 5 Stereo representations of disease causing mutations mapped onto the closed conformation of ABCB4.

Stereo representations of disease causing mutations mapped onto the closed conformation of ABCB4. a, Mutations affecting function. b, For clarity, panel (a) of main Fig. 4 is shown with residue identities indicated. c, Mutations affecting expression or trafficking. d, Panel (b) of main Fig. 4 is shown with residue identities labelled.

Extended Data Fig. 6 ABCB4 and ABCB1 chimeras reveal important residues.

a, The different levels of protein extracted with DDM:CHS from pellets of experiments in Fig. 4d are indicated by luminescence units relative to wt-ABCB4. Main peak heights (peak around 20 min) were used to normalize results of the extrusion assays. b, The three positions mutated in the 3 M construct (V985M, H989Q and A990V) are shown in a model of the ATP-Mg2+ bound ABCB4 in stick representation, and residues within 4 Å are shown in line representation. c, Residues V985, H989 and A990 and nearby residues on TMH12, from the model of ABCB4 in the closed conformation, are shown with associated density contoured at a level of σ = 6.5 and carved at a distance of 2 Å from residues. d, Residue positions corresponding to those in (a) are shown on the previously published model of human-mouse chimeric ABCB1 bound to the inhibitor zosuquidar44, used for homology modelling, with density from the associated map contoured at a level of σ = 6.5 and carved at a distance of 2 Å from residues.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Table 1.

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Source data

Source Data Fig. 1

Raw data for Figs. 1b, 1d and 1e.

Source Data Fig. 4

Raw data for Fig. 4d.

Source Data Extended Data Fig. 2

Raw data for Extended Data Fig. 2a.

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Olsen, J.A., Alam, A., Kowal, J. et al. Structure of the human lipid exporter ABCB4 in a lipid environment. Nat Struct Mol Biol 27, 62–70 (2020). https://doi.org/10.1038/s41594-019-0354-3

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