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Structural basis for the recognition of hydroxyproline in HIF-1α by pVHL

Abstract

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability1. HIF-1α is the oxygen-regulated subunit of HIF-1, an αβ heterodimeric complex1. HIF-1α is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel–Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex2,3. Capture of HIF-1α by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1α4,5,6,7. The crystal structure of a hydroxylated HIF-1α peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

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Figure 1: Electron density for the bound CODD peptide.
Figure 2: Structure of VCB–CODD complex, and sequence alignments of HIF ODD regions and pVHL.
Figure 3: CODD–pVHL interactions.

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References

  1. Wang, G. L., Jiang, B. H., Rue, E. A. & Semenza, G. L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl Acad. Sci. USA 92, 5510–5514 (1995)

    Article  ADS  CAS  Google Scholar 

  2. Maxwell, P. H. et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 (1999)

    Article  ADS  CAS  Google Scholar 

  3. Ohh, M. et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel-Lindau protein. Nature Cell Biol. 2, 423–427 (2000)

    Article  CAS  Google Scholar 

  4. Ivan, M. et al. HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464–468 (2001)

    Article  ADS  CAS  Google Scholar 

  5. Jaakkola, P. et al. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468–472 (2001)

    Article  ADS  CAS  Google Scholar 

  6. Yu, F., White, S. B., Zhao, Q. & Lee, F. S. HIF-1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc. Natl Acad. Sci. USA 98, 9630–9635 (2001)

    Article  ADS  CAS  Google Scholar 

  7. Masson, N., Willam, C., Maxwell, P. H., Pugh, C. W. & Ratcliffe, P. J. Independent function of two destruction domains in hypoxia-inducible factor-α chains activated by prolyl hydroxylation. EMBO J. 20, 5197–5206 (2001)

    Article  CAS  Google Scholar 

  8. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998)

    Article  CAS  Google Scholar 

  9. Tyers, M. & Jorgensen, P. Proteolysis and the cell cycle: with this RING I do thee destroy. Curr. Opin. Genet. Dev. 10, 54–64 (2000)

    Article  CAS  Google Scholar 

  10. Kamura, T. et al. Rbx1, a component of the VHL tumour suppressor complex and SCF ubiquitin ligase. Science 284, 657–661 (1999)

    Article  ADS  CAS  Google Scholar 

  11. Bruick, R. K. & McKnight, S. L. A conserved family of prolyl-4-hydroxylases that modify HIF. Science 294, 1337–1340 (2001)

    Article  ADS  CAS  Google Scholar 

  12. Epstein, A. C. et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107, 43–54 (2001)

    Article  CAS  Google Scholar 

  13. Pugh, C. W., O'Rourke, J. F., Nagao, M., Gleadle, J. M. & Ratcliffe, P. J. Activation of hypoxia-inducible factor-1; definition of regulatory domains within the α subunit. J. Biol. Chem. 272, 11205–11214 (1997)

    Article  CAS  Google Scholar 

  14. Huang, L. E., Gu, J., Schau, M. & Bunn, H. F. Regulation of hypoxia-inducible factor 1α is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl Acad. Sci. USA 95, 7987–7992 (1998)

    Article  ADS  CAS  Google Scholar 

  15. Stebbins, C. E., Kaelin, W. G. Jr & Pavletich, N. P. Structure of the VHL–ElonginC–ElonginB complex: implications for VHL tumour suppressor function. Science 284, 455–461 (1999)

    Article  ADS  CAS  Google Scholar 

  16. Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993)

    Article  CAS  Google Scholar 

  17. Beroud, C. et al. Software and database for the analysis of mutations in the VHL gene. Nucleic Acids Res. 26, 256–258 (1998)

    Article  CAS  Google Scholar 

  18. Fersht, A. R. et al. Hydrogen bonding and biological specificity analysed by protein engineering. Nature 314, 235–238 (1985)

    Article  ADS  CAS  Google Scholar 

  19. Semenza, G. L. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell Dev. Biol. 15, 551–578 (1999)

    Article  CAS  Google Scholar 

  20. Elson, D. A. et al. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1α. Genes Dev. 15, 2520–2532 (2001)

    Article  CAS  Google Scholar 

  21. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326

  22. Kissinger, C. R., Gehlhaar, D. K. & Fogel, D. B. Rapid automated molecular replacement by evolutionary search. Acta Crystallogr. D 55, 484–491 (1999)

    Article  CAS  Google Scholar 

  23. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  24. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  25. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996)

    Article  CAS  Google Scholar 

  26. Hubbard, S. J. & Thornton, J. M. ‘NACCESS’, Computer Program Version 2.1.1 (Department of Biochemistry and Molecular Biology, Univ. Coll. London, London, 1996)

    Google Scholar 

  27. Kleywegt, G. J. Use of non-crystallographic symmetry in protein structure refinement. Acta Crystallogr. D 52, 842–857 (1996)

    Article  CAS  Google Scholar 

  28. CCP4 The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  29. Myszka, D. G. Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. Curr. Opin. Biotechnol. 8, 50–57 (1997)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the staff at beamline ID14-2 of the ESRF, France. We are grateful to A. van der Merwe and A. Kearney for advice and assistance with the surface plasmon resonance experiments. L. M. McNeil assisted with the NMR analysis. This work was supported by Cancer Research UK and the Wellcome Trust. W.-C.H. and M.I.W. were recipients of a Human Frontiers long-term fellowship and a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowship, respectively. The Medical Research Council supports C.W.P. and D.I.S., and Cancer Research UK supports E.Y.J.

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Correspondence to E. Yvonne Jones.

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Hon, WC., Wilson, M., Harlos, K. et al. Structural basis for the recognition of hydroxyproline in HIF-1α by pVHL. Nature 417, 975–978 (2002). https://doi.org/10.1038/nature00767

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