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A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease

Nature Structural Biology volume 10, pages 136–140 (2003)Cite this article

Abstract

Nuclear receptors are transcription factors that activate gene expression in response to ligands. The C-terminal helix (helix 12) of the ligand-binding domain plays a critical role in the activation mechanism. When bound to activating ligands, helix 12 adopts a conformation that promotes the binding of co-activator proteins. Helix 12 also adopts this 'active' position in several ligand-free structures, raising questions as to the exact role of helix 12. We proposed that the dynamic properties of helix 12 may be critical for the activation mechanism and, to test this, have used fluorescence anisotropy techniques to directly monitor the mobility of helix 12 in PPARγ. Our results suggest that helix 12 is significantly more mobile than the main body of the protein. Upon ligand binding, helix 12 shows reduced mobility, accounting for its role as a molecular switch. We also show that natural mutations in human PPARγ, associated with severe insulin resistance and diabetes mellitus, exhibit perturbations in the dynamic behavior of helix 12. Our findings provide the first direct observations of the mobility of helix 12 and suggest that the dynamic properties of this helix are key to the regulation of transcriptional activity.

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Figure 1: Specific labeling of PPARγ helix 12.
Figure 2: Steady-state fluorescence anisotropy.
Figure 3: Time-resolved fluorescence anisotropy decay.

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References

  1. Darimont, B.D. et al. Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev. 12, 3343–3356 (1998).

    Article  CAS  Google Scholar 

  2. Shiau, A.K. et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95, 927–937 (1998).

    Article  CAS  Google Scholar 

  3. Nolte, R. et al. Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor. Nature 395, 137–143 (1998).

    Article  CAS  Google Scholar 

  4. Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H. & Moras, D. Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-α. Nature 375, 377–382 (1995).

    Article  CAS  Google Scholar 

  5. Brzozowski, A.M. et al. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389, 753–758 (1997).

    Article  CAS  Google Scholar 

  6. Pike, A.C. et al. Structure of the ligand-binding domain of oestrogen receptor βε in the presence of a partial agonist and a full antagonist. EMBO J. 18, 4608–4618 (1999).

    Article  CAS  Google Scholar 

  7. Gampe, R.T. Jr. et al. Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix. Genes Dev. 14, 2229–2241 (2000).

    Article  CAS  Google Scholar 

  8. Bourguet, W. et al. Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains. Mol. Cell 5, 289–298 (2000).

    Article  CAS  Google Scholar 

  9. Pike, A.C. et al. Structural insights into the mode of action of a pure antiestrogen. Structure 9, 145–153 (2001).

    Article  CAS  Google Scholar 

  10. Watkins, R.E. et al. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science 292, 2329–2333 (2001).

    Article  CAS  Google Scholar 

  11. Clayton, G.M., Peak-Chew, S.Y., Evans, R.M. & Schwabe, J.W. The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation. Proc. Natl. Acad. Sci. USA 98, 1549–1554 (2001).

    Article  CAS  Google Scholar 

  12. Xu, H.E. et al. Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARα. Nature 415, 813–817 (2002).

    Article  CAS  Google Scholar 

  13. Hu, X. & Lazar, M.A. The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402, 93–96 (1999).

    Article  CAS  Google Scholar 

  14. Perissi, V. et al. Molecular determinants of nuclear receptor-corepressor interaction. Genes Dev. 13, 3198–3208 (1999).

    Article  CAS  Google Scholar 

  15. Nagy, L. et al. Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev. 13, 3209–3216 (1999).

    Article  CAS  Google Scholar 

  16. Webb, P. et al. The nuclear receptor corepressor (N-CoR) contains three isoleucine motifs (I/LXXII) that serve as receptor interaction domains (IDs). Mol. Endocrinol. 14, 1976–1985 (2000).

    Article  CAS  Google Scholar 

  17. Barroso, I. et al. Dominant negative mutations in human PPARγ associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 402, 880–883 (1999).

    Article  CAS  Google Scholar 

  18. Chong, S. et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271–281 (1997).

    Article  CAS  Google Scholar 

  19. Lehmann, J.M. et al. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ). J. Biol. Chem. 270, 12953–12956 (1995).

    Article  CAS  Google Scholar 

  20. Yguerabide, J., Epstein, H.F. & Stryer, L. Segmental flexibility in an antibody molecule. J. Mol. Biol. 51, 573–590 (1970).

    Article  CAS  Google Scholar 

  21. Vergani, B. et al. Backbone dynamics of Tet repressor α8–α9 loop. Biochemistry 39, 2759–2768 (2000).

    Article  CAS  Google Scholar 

  22. Gangal, M. et al. Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations. Biochemistry 37, 13728–13735 (1998).

    Article  CAS  Google Scholar 

  23. Mielke, T., Alexiev, U., Glasel, M., Otto, H. & Heyn, M.P. Light-induced changes in the structure and accessibility of the cytoplasmic loops of rhodopsin in the activated MII state. Biochemistry 41, 7875–7884 (2002).

    Article  CAS  Google Scholar 

  24. Clore, G.M., Driscoll, P.C., Wingfield, P.T. & Gronenborn, A.M. Analysis of the backbone dynamics of interleukin-1β using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry 29, 7387–7401 (1990).

    Article  CAS  Google Scholar 

  25. Johnson, B.A. et al. Ligand-induced stabilization of PPARγ monitored by NMR spectroscopy: implications for nuclear receptor activation. J. Mol. Biol. 298, 187–194 (2000).

    Article  CAS  Google Scholar 

  26. Cronet, P. et al. Structure of the PPARα and -γ ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family. Structure 9, 699–706 (2001).

    Article  CAS  Google Scholar 

  27. Gangloff, M. et al. Crystal structure of a mutant hERα ligand-binding domain reveals key structural features for the mechanism of partial agonism. J. Biol. Chem. 276, 15059–15065 (2001).

    Article  CAS  Google Scholar 

  28. Schulman, I.G., Juguilon, H. & Evans, R.M. Activation and repression by nuclear hormone receptors: hormone modulates an equilibrium between active and repressive states. Mol. Cell. Biol. 16, 3807–3813 (1996).

    Article  CAS  Google Scholar 

  29. Tanenbaum, D.M., Wang, Y., Williams, S.P. & Sigler, P.B. Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains. Proc. Natl. Acad. Sci. USA 95, 5998–6003 (1998).

    Article  CAS  Google Scholar 

  30. Love, J.D. et al. The structural basis for the specificity of retinoid-X-receptor selective agonists: new insights into the role of helix H12. J. Biol. Chem. 277, 11385–11391 (2002).

    Article  CAS  Google Scholar 

  31. Gregory, C., Hayes, M., Jones, G. & Pantos, E. FLUOR — a program to analyse fluorescence data. in Daresbury Laboratory Technical Memorandum (Daresbury Laboratory, Warrington; 1994).

    Google Scholar 

  32. Brown, K.K. et al. A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-γ reverses the diabetic phenotype of the Zucker diabetic fatty rat. Diabetes 48, 1415–1424 (1999).

    Article  CAS  Google Scholar 

  33. Li, F. et al. Evidence for an internal entropy contribution to phosphoryl transfer: a study of domain closure, backbone flexibility, and the catalytic cycle of cAMP-dependent protein kinase. J. Mol. Biol. 315, 459–469 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to D. Veprintsev for assistance with the steady state measurements, T. Willson and Glaxo-SmithKline for the gift of PPARγ ligands, D. Owen for synthesis of fluorophore and co-activator peptide, D. Shaw and M. Martin-Fernandez for assistance at the SRS beamline and T. Mielke, P. Evans, D. Neuhaus and D. Rhodes for helpful discussions. The work was supported in part by HFSP and EU-RTN grants.

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Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Bettina C. Kallenberger, James D. Love & John W. R. Schwabe

  2. Department of Medicine, University of Cambridge, Cambridge, CB2 2QQ, UK

    Bettina C. Kallenberger & V. Krishna K. Chatterjee

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  1. Bettina C. Kallenberger
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Correspondence to John W. R. Schwabe.

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Kallenberger, B., Love, J., Chatterjee, V. et al. A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease. Nat Struct Mol Biol 10, 136–140 (2003). https://doi.org/10.1038/nsb892

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