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Structural basis of prostaglandin efflux by MRP4

Nature Structural & Molecular Biology volume 31, pages 621–632 (2024)Cite this article

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Abstract

Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.

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Fig. 1: Functional characterization of MRP4 and identification of three stimulating substrates.
Fig. 2: Structures of MRP4 in multiple conformations along the substrate transport cycle.
Fig. 3: Comparison of the inward-open, inward-narrow and outward-occluded conformations of MRP4.
Fig. 4: Structural asymmetry between NBD1 and NBD2.
Fig. 5: DHEA-S and prostaglandin binding to MRP4.
Fig. 6: Structural comparison between MRP4 and related ABCC family members.
Fig. 7: Proposed translocation cycle of MRP4.

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Data availability

All five three-dimensional cryo-EM density maps have been deposited to the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-40829 (apowide MRP4), EMD-40828 (aponarrow MRP4), EMD-40827 (PGE1-bound MRP4), EMD-40830 (PGE2-bound MRP4), EMD-40826 (DHEA-S-bound MRP4) and EMD-40821 (ATP-bound MRP4E1202Q). The coordinates for the atomic models have been deposited in the Protein Data Bank (PDB) under accession numbers 8SXA (apowide MRP4), 8SX9 (aponarrow MRP4), 8SX8 (PGE1-bound MRP4), 8SXB (PGE2-bound MRP4), 8SX7 (DHEA-S-bound MRP4) and 8SWN (ATP-bound MRP4E1202Q). See Table 1 for more details. Source data are provided with this paper.

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Acknowledgements

We thank P. Nguyen for assistance with virus production and D. Asarnow for discussions regarding purification and reconstitution of MRP4. This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (grants NIGMS GM24485 to R.M.S. and R35GM140847 to Y.C.). R.B. was supported in part by American Heart Association postdoctoral fellowship award 9POST34370101. Mass spectrometry was performed in the UCSF Mass Spectrometry facility supported by NIH grant P41 GM103481. We thank D. P. Bulkley, G. Gilbert and M. Harrington for support with cryo-EM data collection. All data were collected at the UCSF cryo-EM facility, which is supported by NIH grants S10OD020054, S10OD021741 and S10OD026881.

Author information

Author notes

  1. Sergei Pourmal

    Present address: Genentech, South San Francisco, CA, USA

  2. Evan Green

    Present address: Exelixis, Alameda, CA, USA

  3. Ilan E. Chemmama

    Present address: Brightseed, South San Francisco, CA, USA

  4. These authors contributed equally: Evan Green, Ruchika Bajaj.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA

    Sergei Pourmal, Evan Green, Meghna Gupta, Yifan Cheng & Robert M. Stroud

  2. Program in Chemistry and Chemical Biology, University of California, San Francisco, CA, USA

    Sergei Pourmal

  3. Biophysics Graduate Program, University of California, San Francisco, CA, USA

    Evan Green

  4. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA

    Ruchika Bajaj, Ilan E. Chemmama, Andrej Sali & Deanna L. Kroetz

  5. Quantitative Biosciences Institute, University of California, San Francisco, CA, USA

    Giselle M. Knudsen & Andrej Sali

  6. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA

    Andrej Sali & Charles S. Craik

  7. Howard Hughes Medical Institute, University of California, San Francisco, CA, USA

    Yifan Cheng

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Contributions

R.M.S., D.L.K., C.S.C., Y.C. and A.S. conceived this work. S.P., R.B. and G.M.K. expressed and functionally characterized proteins. S.P., E.G. and M.G. collected and processed cryo-EM data. S.P. and E.G. performed model building and refinement. S.P., R.B., E.G., I.E.C., R.M.S. and D.L.K. wrote the manuscript with contributions from all other authors.

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Extended data

Extended Data Fig. 1 Sequence alignment of MRP4 and MRP1 homologs.

Sequences of bovine and human MRP4 are aligned to reflect sequence conservation across evolution and similarity to the closely related MRP1. All references to MRP4 used throughout the text refer to the bovine sequence. The residue numbers for bovine MRP4 are indicated above the sequences. Red asterisks denote a residue involved in binding one of either DHEA-S, PGE1, or PGE2. Text boxes refer to conserved sequence motifs from NBD1 (pink) and NBD2 (cyan), NBD2’s inserted sequence (orange) and the C-terminal PDZ-binding motif (PDZ-BM, sand). Bovine MRP4 catalytic residue E1202 is highlighted in red.

Extended Data Fig. 2 Biochemical characterization of MRP4.

Fold change in ATPase activity relative to basal levels with the addition of increasing concentrations of a, PGE1, PGE2, b, DHEAS, c, cAMP, cGMP, d, LTC4, e, tenofovir, f, E217G, g, folic acid, h, S1P. Data in each panel were obtained from separate preparations of MRP4 in MSP lipid nanodisc. The basal ATPase specific activity across the samples used in panels a-f was 13.6 ± 4.5 nmol min-1 mg-1 from 10 biological replicates. PGE1, prostaglandin E1; PGE2, prostaglandin E2; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; LTC4, leukotriene C4; DHEA-S, dehydroepiandrosterone sulfate; E217G, β-estradiol-17β-D-glucuronide; S1P, sphingosine-1-phosphate.

Source data

Extended Data Fig. 3 cAMP does not elicit a conformational change in MRP4 in cryo-EM.

a Final density of MRP4 in the presence of 1 mM cAMP from cryoSPARC. Sharpened output volume in gray, lowpass filtered volume showing the nanodisc and NBDs as black silhouette. b Rigid body fitting of apo MRP4 into density obtained in the presence of 1 mM cAMP. Density in gray, MRP4 domains colored as before. c View of substrate binding residues of the rigid-body fit apo MRP4 model in the density obtained for MRP4 in the presence of 1 mM cAMP. Map contoured at low threshold, revealing non-protein density similar to our apo MRP4 refinement. Density in purple mesh, MRP4 domains colored as before.

Extended Data Fig. 4 The domains of MRP4 move as rigid bodies throughout the substrate transport cycle.

Superposition of a, bundle 1 (TMs 1, 2, 3, 6, 10, 11) b, bundle 2 (TMs 4, 5, 7, 8, 9, 12) c, NBD1 and d, NBD2 across all five structures. Apowide MRP4 in salmon, aponarrow MRP4 in green, DHEA-S-bound MRP4 in cyan, PGE1-bound MRP4 in grey, PGE2-bound MRP4 in pink, ATP-Mg2+-bound MRP4 in yellow. e, Superposition of the three substrate-bound structures reveals them to share an inward-open, narrow conformation. RMSD between all Cα of DHEA-S-bound MRP4 and PGE1-bound MRP4 is 1.2 Å; between DHEA-S-bound and PGE2-bound is 0.87 Å; between PGE1-bound and PGE2-bound is 0.90 Å. Structures are colored as in a-d.

Extended Data Fig. 5 The outward-facing occluded state of MRP4 closely resembles the structure of SUR1 bound to ADP/ATP.

a Front view, b side view, and c top view of MRP4E1202Q bound to ATP-Mg2+ and SUR1 bound to ADP-ATP (shown as single chain from PDB ID:6C3O). TMD0 domain in SUR1 is hidden for clarity in a and c. Cα RMSD between our ATP-bound structure and SUR1 with TMD0 domain deleted is 4.36 Å. MRP4E1202Q bound to ATP-Mg2+ in pink, SUR1 bound to ADP-ATP in cyan, TMD0 domain in light gray.

Extended Data Table 1 Kinetic parameters for ATPase activity of MRP4
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Supplementary information

Source data

Source Data Fig. 1

SEC A280 source data for Fig. 1b; Spectromax A340 source data for Fig. 1c,d,e.

Source Data Fig. 2

Uncropped gel image for Fig. 1b. Coomassie brilliant blue stained gel. Alternating lanes of ladders and serial dilutions of the peak MRP4–nanodisc fraction from SEC.

Source Data Extended Data Fig. 2

Spectromax A340 source data for Extended Fig. 2.

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Pourmal, S., Green, E., Bajaj, R. et al. Structural basis of prostaglandin efflux by MRP4. Nat Struct Mol Biol 31, 621–632 (2024). https://doi.org/10.1038/s41594-023-01176-4

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