Abstract
Human platelets contribute to hemostasis and thrombosis, the imbalance of which can cause cardiovascular diseases. The activation and accumulation of platelets can be induced by agonists or inhibited by antagonists. Thus, the human ABC transporter ABCC4, which pumps out platelet agonists and antagonists, might become a promising target for preventing cardiovascular diseases. Here we define five structures of human ABCC4: the apo and three complexed forms in the inward-facing conformation, in addition to an outward-facing occluded conformation upon ATP binding. Combined with biochemical assays, we structurally prove that U46619, a synthetic analog of the unstable agonist TXA2, and the antagonist aspirin are substrates of ABCC4. In addition, we found that the platelet antagonist dipyridamole is a strong competitive inhibitor against ABCC4. These complex structures also enable us to identify a transmembrane pocket in ABCC4 that provides a defined space for the rational design of specific platelet antagonists.
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Data availability
The cryo-EM structures of apo-form, U46619-bound, U46619-ATP-bound, dipyridamole-bound and aspirin-bound ABCC4 have been deposited at the Protein Data Bank under codes 8I4B, 8I4C, 8J3Z, 8I4A and 8J3W, respectively. The cryo-EM density maps of these structures have been deposited at the Electron Microscopy Data Bank under accession codes EMD-35168 for apo-form ABCC4, EMD-35169 for U46619-bound ABCC4, EMD-35968 for U46619-ATP-bound ABCC4, EMD-35167 for dipyridamole-bound ABCC4 and EMD-35967 for aspirin-bound ABCC4. All other data supporting the findings in this study are included in the main article and associated files.
References
Thomas, C. & Tampe, R. Structural and mechanistic principles of ABC transporters. Annu. Rev. Biochem. 89, 605–636 (2020).
Dean, M., Moitra, K. & Allikmets, R. The human ATP-binding cassette (ABC) transporter superfamily. Hum Mutat. 43, 1162–1182 (2022).
Gottesman, M. M., Fojo, T. & Bates, S. E. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat. Rev. Cancer 2, 48–58 (2002).
Schuetz, J. D. et al. MRP4: a previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nat. Med. 5, 1048–1051 (1999).
Lai, L. & Tan, T. M. Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues. Biochem. J 361, 497–503 (2002).
Evers, R. et al. Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export. Br. J. Cancer 83, 375–383 (2000).
Johnson, Z. L. & Chen, J. Structural basis of substrate recognition by the multidrug resistance protein MRP1. Cell 168, 1075–1085 (2017).
Rius, M., Nies, A. T., Hummel-Eisenbeiss, J., Jedlitschky, G. & Keppler, D. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38, 374–384 (2003).
Bortfeld, M. et al. Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system. Neuroscience 137, 1247–1257 (2006).
Chan, L. M., Lowes, S. & Hirst, B. H. The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. Eur. J. Pharm. Sci. 21, 25–51 (2004).
Imaoka, T. et al. Functional involvement of multidrug resistance-associated protein 4 (MRP4/ABCC4) in the renal elimination of the antiviral drugs adefovir and tenofovir. Mol. Pharmacol. 71, 619–627 (2007).
Kool, M. et al. Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res. 57, 3537–3547 (1997).
Hasegawa, M. et al. Multidrug resistance-associated protein 4 is involved in the urinary excretion of hydrochlorothiazide and furosemide. J. Am. Soc. Nephrol. 18, 37–45 (2007).
Ci, L. et al. Involvement of MRP4 (ABCC4) in the luminal efflux of ceftizoxime and cefazolin in the kidney. Mol. Pharmacol. 71, 1591–1597 (2007).
Fukuda, Y. et al. Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity. Mol. Pharmacol. 84, 361–371 (2013).
Adema, A. D. et al. Overexpression of MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the nucleoside analogs cytarabine and troxacitabine, but not gemcitabine. Springerplus 3, 732 (2014).
Tian, Q. et al. Topotecan is a substrate for multidrug resistance associated protein 4. Curr. Drug Metab. 7, 105–118 (2006).
Jedlitschky, G. et al. The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage. Blood 104, 3603–3610 (2004).
Rius, M., Thon, W. F., Keppler, D. & Nies, A. T. Prostanoid transport by multidrug resistance protein 4 (MRP4/ABCC4) localized in tissues of the human urogenital tract. J. Urol. 174, 2409–2414 (2005).
Wang, L. H., Tsai, A. L. & Hsu, P. Y. Substrate binding is the rate-limiting step in thromboxane synthase catalysis. J. Biol. Chem. 276, 14737–14743 (2001).
Hamberg, M., Svensson, J. & Samuelsson, B. Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc. Natl Acad. Sci. USA 72, 2994–2998 (1975).
Reid, G. et al. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc. Natl Acad. Sci. USA 100, 9244–9249 (2003).
Davì, G. & Patrono, C. Platelet activation and atherothrombosis. N. Engl. J. Med. 357, 2482–2494 (2007).
Jedlitschky, G., Greinacher, A. & Kroemer, H. K. Transporters in human platelets: physiologic function and impact for pharmacotherapy. Blood 119, 3394–3402 (2012).
Mattiello, T. et al. Aspirin extrusion from human platelets through multidrug resistance protein-4-mediated transport: evidence of a reduced drug action in patients after coronary artery bypass grafting. J. Am. Coll. Cardiol. 58, 752–761 (2011).
Kleindorfer, D. O. et al. 2021 guideline for the prevention of stroke in patients with stroke and transient ischemic attack: a guideline from the American Heart Association/American Stroke Association. Stroke 52, e364–e467 (2021).
Hwa, J. & Martin, K. in Basic & Clinical Pharmacology 14th edn (ed. Katzung, B. G.) Ch. 18 (McGraw-Hill Education, 2018).
Kalinski, P. Regulation of immune responses by prostaglandin E2. J. Immunol. 188, 21–28 (2012).
Hawkins, M. & Lipton, J. M. Analogs of endoperoxide precursors of prostaglandins: failure to affect body temperature when injected into primary and secondary central temperature controls. Prostaglandins 13, 209–218 (1977).
Sauna, Z. E., Nandigama, K. & Ambudkar, S. V. Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state. J. Biol. Chem. 279, 48855–48864 (2004).
Wang, L. et al. Structures of human bile acid exporter ABCB11 reveal a transport mechanism facilitated by two tandem substrate-binding pockets. Cell Res. 32, 501–504 (2022).
Chen, Z. P. et al. Structural basis of substrate recognition and translocation by human very long-chain fatty acid transporter ABCD1. Nat. Commun. 13, 3299 (2022).
Johnson, Z. L. & Chen, J. ATP binding enables substrate release from multidrug resistance protein 1. Cell 172, 81–89 (2018).
Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L. & Chen, J. Crystal structure of a catalytic intermediate of the maltose transporter. Nature 450, 515–521 (2007).
Ward, A., Reyes, C. L., Yu, J., Roth, C. B. & Chang, G. Flexibility in the ABC transporter MsbA: alternating access with a twist. Proc. Natl Acad. Sci. USA 104, 19005–19010 (2007).
Rivey, M. P., Alexander, M. R. & Taylor, J. W. Dipyridamole: a critical evaluation. Drug Intell. Clin. Pharm. 18, 869–880 (1984).
Cheepala, S. B. et al. The ABCC4 membrane transporter modulates platelet aggregation. Blood 126, 2307–2319 (2015).
Copsel, S. et al. Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation. J. Biol. Chem. 286, 6979–6988 (2011).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Acknowledgements
We thank Y.-X. Gao for technical support on cryo-EM data collection at the Cryo-EM Center at the University of Science and Technology of China (USTC). We also thank L. Sun at USTC for technical assistance with structure refinement. This work was supported by the Ministry of Science and Technology of China (grant no. 2019YFA0508500 to Yuxing Chen), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB37020202 to Yuxing Chen) and the Fundamental Research Funds for the Central Universities (grant no. YD9100002014 to Yuxing Chen).
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Yuxing Chen, Q.L. and C.-Z.Z. conceived the project and planned the experiments. Yu Chen and L.W. expressed and purified human ABCC4. Yu Chen and Z.-H.Z. performed cryo-EM data collection, structure determination and model refinement. Yu Chen, W.-T.H. and K.X. performed biochemical assays. Yuxing Chen, Q.L., C.-Z.Z. and Yu Chen wrote the paper. All authors discussed the data and read the paper.
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Nature Cardiovascular Research thanks Jochen Zimmer and John Hwa for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Structural formulas of ABCC4 substrates and inhibitor involved in this study.
a TXA2; b PGE2; c U46619; d TXB2; e dipyridamole and f aspirin. All the formulas were draw by software Kingdraw v5.0.
Extended Data Fig. 2 Cryo-EM analysis of apo-form ABCC4.
a Representative micrograph and 2D class averages. Bar: 50 nm. The micrograph is a representative of 2,892 cryo-EM images. b The flowchart for cryo-EM data processing. c The angular distribution plot of the final 3-D reconstruction of apo-form ABCC4. d Fourier shell correlation (FSC) curves for apo-form ABCC4. e The local resolution map of apo-form ABCC4. The color code for resolutions, shown with the unit Å, is calculated using CryoSPARC. f Cryo-EM maps for representative segments of apo-form ABCC4. Contour levels for TM1-12 and the lasso motif are set at 5σ, whereas those for the two NBDs are 3σ.
Extended Data Fig. 3 Cryo-EM analysis of U46619-bound ABCC4.
a Representative micrograph and 2D class averages. Bar: 50 nm. The micrograph is a representative of 2,211 cryo-EM images. b The flowchart for cryo-EM data processing. c The angular distribution plot of the final 3-D reconstruction of U46619-bound ABCC4. d FSC curves for U46619-bound ABCC4. e The local resolution map of U46619-bound ABCC4. The color code for resolutions, shown with the unit Å, is calculated using CryoSPARC. f Cryo-EM maps for representative segments of U46619-bound ABCC4. Contour levels for TM1-12 and the lasso motif are set at 5σ, whereas those for the U46619 molecule and coordinating TMs are 3σ.
Extended Data Fig. 4 Structural comparisons.
a Superposition of U46619-bound ABCC4 (limon) against apo-form ABCC4 (gray). b Superposition of U46619-bound ABCC4 (limon) against LTC4-bound bMRP1 (gray, PDB code:5UJA). c Superposition of key residues in the U46619-binding pocket (limon) against those in apo-form ABCC4 (gray). The binding residues are shown as sticks and labeled.
Extended Data Fig. 5 Multiple-sequence alignment of human ABCC4 and homologs.
Black triangles indicate the binding residues in the substrate-binding pocket of ABCC4. Notably, residues Leu321, Leu363 and Met992 involved in the interaction with dipyridamole are labeled with red triangles, whereas residues His152, Phe156 interacting with U46619 in ATP-bound ABCC4 and Thr846 interacting with aspirin are labeled with blue triangles. All the primary sequences were provided as a Source Data file.
Extended Data Fig. 6 Cryo-EM analysis of U46619-ATP-bound ABCC4.
a Representative micrograph and 2D class averages. Bar: 50 nm. The micrograph is a representative of 4,768 cryo-EM images. b The flowchart for cryo-EM data processing. c The angular distribution plot of the final 3-D reconstruction of U46619-ATP-bound ABCC4. d FSC curves for U46619-ATP-bound ABCC4. e The local resolution map of U46619-ATP-bound ABCC4. The color code for resolutions, shown with the unit Å, is calculated using CryoSPARC. f Cryo-EM maps for representative segments of U46619-ATP-bound ABCC4. Contour levels for TM1-12, the lasso motif, the U46619 molecule and coordinating TMs are set at 5σ.
Extended Data Fig. 7 Structural comparison of outward-facing occluded ABCC4 against inward-facing ABCC4.
a, b Superposition of a NBD1-linking bundle and b NBD2-linking bundle in outward-facing against those in inward-facing conformation, respectively. The NBDs and TMs of outward-facing occluded ABCC4 are colored as overall structure in Fig. 3d, whereas those of inward-facing ABCC4 are colored in gray. c, d Superposition of overall structures of outward-facing occluded and inward-facing ABCC4 in c side view and d top view. The NBD1-linking bundle is colored in yellow, whereas the NBD2-linking bundle is colored in magenta.
Extended Data Fig. 8 Cryo-EM analysis of dipyridamole-bound ABCC4.
a Representative micrograph and 2D class averages. Bar: 50 nm. The micrograph is a representative of 2,219 cryo-EM images. b The flowchart for cryo-EM data processing. c The angular distribution plot of the final 3-D reconstruction of dipyridamole-bound ABCC4. d FSC curves for dipyridamole-bound ABCC4. e The local resolution map of dipyridamole-bound ABCC4. The color code for resolutions, shown with the unit Å, is calculated using CryoSPARC. f Cryo-EM maps for representative segments of dipyridamole-bound ABCC4. Contour levels for TM1-12 and the lasso motif are set at 5σ, whereas those for the dipyridamole molecule and coordinating TMs are 4σ.
Extended Data Fig. 9 Structural comparisons.
Superposition of dipyridamole-bound ABCC4 (yellow) against a apo-form ABCC4 (gray) and b U46619-bound ABCC4 (gray). c Superposition of the key residues in the dipyridamole-binding pocket against those in the apo-form ABCC4. The interacting residues are shown as sticks and labeled. d Superposition of aspirin-bound ABCC4 (cyan) against dipyridamole-bound ABCC4 (gray).
Extended Data Fig. 10 Cryo-EM analysis of aspirin-bound ABCC4.
a Representative micrograph and 2D class averages. Bar: 50 nm. The micrograph is a representative of 8,712 cryo-EM images. b The flowchart for cryo-EM data processing. c The angular distribution plot of the final 3-D reconstruction of aspirin-bound ABCC4. d FSC curves for aspirin-bound ABCC4. e The local resolution map of aspirin-bound ABCC4. The color code for resolutions, shown with the unit Å, is calculated using CryoSPARC. f Cryo-EM maps for representative segments of aspirin-bound ABCC4. Contour levels for TM1-12, the lasso motif, the aspirin molecule and coordinating TMs are set at 5σ.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2, Tables 1 and 2 and Source Data for Supplementary Fig. 1.
Source data
Source Data Fig. 1
Statistical source data for ATPase activity and SPR measurements.
Source Data Fig. 3
Statistical source data for ATPase activity measurements.
Source Data Extended Data Fig. 5
Primary sequences of ABCC4 and its homologs.
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Chen, Y., Wang, L., Hou, WT. et al. Structural insights into human ABCC4-mediated transport of platelet agonist and antagonist. Nat Cardiovasc Res 2, 693–701 (2023). https://doi.org/10.1038/s44161-023-00289-9
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DOI: https://doi.org/10.1038/s44161-023-00289-9
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