Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Nature volume 424, pages 464–468 (2003)Cite this article

Abstract

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants1. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use2. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug–drug interactions.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

Buy

All prices are NET prices.

Figure 1: Structure of P450 CYP2C9.
Figure 2: Stereo view of the binding site of S-warfarin in CYP2C9.
Figure 3: The surface of the active site of CYP2C9.
Figure 4: View of the region of the active site of CYP2C9 that remains available to accommodate additional ligand(s) after S-warfarin.

References

  1. Anzenbacher, P. & Anzenbacherova, E. Cytochromes P450 and metabolism of xenobiotics. Cell. Mol. Life Sci. 58, 737–747 (2001)

    CAS  Article  PubMed  Google Scholar 

  2. Hodgson, J. ADMET–turning chemicals into drugs. Nature Biotechnol. 19, 722–726 (2001)

    CAS  Article  Google Scholar 

  3. Li, H. & Poulos, T. L. conformational dynamics in cytochromeP450-substrate interactions. Biochimie 78, 695–699 (1996)

    CAS  Article  PubMed  Google Scholar 

  4. Li, H. & Poulos, T. L. The structure of the cytochrome P450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nature Struct. Biol. 4, 140–146 (1997)

    CAS  Article  PubMed  Google Scholar 

  5. Schlichting, I., Jung, C. & Schulze, H. Crystal structure of cytochrome P-450cam complexed with the (1S)-camphor enantiomer. FEBS Lett. 415, 253–257 (1997)

    CAS  Article  PubMed  Google Scholar 

  6. Podust, L. M., Poulos, T. L. & Waterman, M. R. Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proc. Natl Acad. Sci. USA 98, 3068–3073 (2001)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F. & McRee, D. E. Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol. Cell 5, 121–131 (2000)

    CAS  Article  PubMed  Google Scholar 

  8. Ibeanu, G. C. et al. Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity. J. Biol. Chem. 271, 12496–12501 (1996)

    CAS  Article  PubMed  Google Scholar 

  9. Melet, A. et al. Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid. Arch. Biochem. Biophys. 409, 80–91 (2003)

    CAS  Article  PubMed  Google Scholar 

  10. Haines, D. C., Tomchick, D. R., Machius, M. & Peterson, J. A. Pivotal role of water in the mechanism of P450BM-3. Biochemistry 40, 13456–13465 (2001)

    CAS  Article  PubMed  Google Scholar 

  11. Ridderstrom, M. et al. Arginines 97 and 108 in CYP2C9 are important determinants of the catalytic function. Biochem. Biophys. Res. Commun. 270, 983–987 (2000)

    CAS  Article  PubMed  Google Scholar 

  12. de Groot, M. J., Alex, A. A. & Jones, B. C. Development of a combined protein and pharmacophore nodel for cytochrome P450 2C9. J. Med. Chem. 45, 1983–1993 (2002)

    CAS  Article  PubMed  Google Scholar 

  13. Mancy, A., Broto, P., Dijols, S., Dansette, P. M. & Mansuy, D. The substrate binding site of human liver cytochrome P450 2C9: an approach using designed tienilic acid derivatives and molecular modeling. Biochemistry 34, 10365–10375 (1995)

    CAS  Article  PubMed  Google Scholar 

  14. Jones, B. C. et al. Putative active site template model for cytochrome P4502C9 (tolbutamide hydroxylase). Drug Metab. Dispos. 24, 260–266 (1996)

    CAS  PubMed  Google Scholar 

  15. Lewis, D. F. On the recognition of mammalian microsomal cytochrome P450 substrates and their characteristics: towards the prediction of human p450 substrate specificity and metabolism. Biochem. Pharmacol. 60, 293–306 (2000)

    CAS  Article  PubMed  Google Scholar 

  16. Takahashi, H. & Echizen, H. Pharmacogenetics of warfarin elimination and its clinical implications. Clin. Pharmacokinet. 40, 587–603 (2001)

    CAS  Article  PubMed  Google Scholar 

  17. Kaminsky, L. S. & Zhang, Z. Y. Human P450 metabolism of warfarin. Pharmacol. Ther. 73, 67–74 (1997)

    CAS  Article  PubMed  Google Scholar 

  18. Haining, R. L. et al. Enzymatic determinants of the substrate specificity of CYP2C9: role of B′-C loop residues in providing the pi-stacking anchor site for warfarin binding. Biochemistry 38, 3285–3292 (1999)

    CAS  Article  PubMed  Google Scholar 

  19. Modi, S., Sutcliffe, M. J., Primrose, W. U., Lian, L. Y. & Roberts, G. C. The catalytic mechanism of cytochrome P450 BM3 involves a 6 Å movement of the bound substrate on reduction. Nature Struct. Biol. 3, 414–417 (1996)

    CAS  Article  PubMed  Google Scholar 

  20. Modi, S. et al. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase. Biochemistry 36, 4461–4470 (1997)

    CAS  Article  PubMed  Google Scholar 

  21. Nave, C. Radiation damage in protein crystallography. Radiat. Phys. Chem. 45, 483–490 (1995)

    ADS  CAS  Article  Google Scholar 

  22. Hlavica, P. & Lewis, D. F. Allosteric phenomena in cytochrome P450-catalyzed monooxygenations. European Journal of Biochemistry 268, 4817–4832 (2001)

    CAS  Article  PubMed  Google Scholar 

  23. Tang, W. & Stearns, R. A. Heterotropic cooperativity of cytochrome P450 3A4 and potential drug-drug interactions. Curr. Drug Metab. 2, 185–198 (2001)

    CAS  Article  PubMed  Google Scholar 

  24. Hutzler, J. M., Hauer, M. J. & Tracy, T. S. Dapsone activation of CYP2C9-mediated metabolism: evidence for activation of multiple substrates and a two-site model. Drug Metab. Dispos. 29, 1029–1034 (2001)

    CAS  PubMed  Google Scholar 

  25. Hutzler, J. M., Wienkers, L. C., Wahhlstrom, J. L., Carlson, T. J. & Tracy, T. S. Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations. Arch. Biochem. Biophys. 410, 16–24 (2003)

    Article  PubMed  Google Scholar 

  26. Stresser, D. M., Ackermann, J. M., Miller, V. P. & Crespi, C. L. Fluorometric cytochromes P450 2C8, 2C9 and 2C19 inhibition assays 〈http://www.gentest.com/〉.

  27. Hemeryck, A., De Vriendt, C. & Belpaire, F. M. Inhibition of CYP2C9 by selective serotonin reuptake inhibitors: in vitro studies with tolbutamide and (S)-warfarin using human liver microsomes. Eur. J. Clin. Pharmacol. 54 (12), 947–951 (1999)

    CAS  Article  PubMed  Google Scholar 

  28. Leslie, A. G. W. MOSFLM. Joint CCP4 and EESF-EACMB Newsletter on Protein Crystallography 26 (SERC Daresbury Laboratory, Warrington, 1992)

    Google Scholar 

  29. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994)

    Article  Google Scholar 

  30. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 50, 760–763 (1997)

    Google Scholar 

Download references

Acknowledgements

We thank R. Mallender, L. Vuillard, S. Rich and S. Kirton for assistance with protein production, crystallization, characterization, analysis and modelling, and R. Carr, D. Cross, C. Murray and G. Williams for valuable discussions. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and would like to thank the industrial user support group for assistance. The authors are grateful to F. W. Dahlquist for provision of the pCWOri + expression vector.

Author information

Author notes

  1. Alison Ward

    Present address: AstraZeneca, R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, UK

  2. Harren Jhoti: Coordinates have been deposited in the Protein Data Bank under accession codes 1OG2 and 1OG5.

  3. Pamela A. Williams and Jose Cosme: These authors contributed equally to this work

Authors and Affiliations

  1. Astex Technology, 436 Cambridge Science Park, Milton Road, CB4 0QA, Cambridge, UK

    Pamela A. Williams, Jose Cosme, Alison Ward, Hayley C. Angove, Dijana Matak Vinković & Harren Jhoti

Authors
  1. Pamela A. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jose Cosme
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alison Ward
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hayley C. Angove
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dijana Matak Vinković
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Harren Jhoti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Harren Jhoti.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Williams, P., Cosme, J., Ward, A. et al. Crystal structure of human cytochrome P450 2C9 with bound warfarin. Nature 424, 464–468 (2003). https://doi.org/10.1038/nature01862

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature01862

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing