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DNA binding alters coactivator interaction surfaces of the intact VDR–RXR complex

Abstract

The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators.

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Figure 1: The interactions along the dimer interface of the RXR–VDR heterodimer.
Figure 2: Ligand-induced domain-domain interactions within the RXR–VDR heterodimer complex.
Figure 3: The interactions between RXR and VDR when they are bound to DNA.
Figure 4: Ligand dependency of SRC1 RID binding to the RXR–VDR heterodimer complex.

References

  1. Gennari, L., Merlotti, D., De Paola, V., Martini, G. & Nuti, R. Update on the pharmacogenetics of the vitamin D receptor and osteoporosis. Pharmacogenomics 10, 417–433 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Narvaez, C.J., Matthews, D., Broun, E., Chan, M. & Welsh, J. Lean phenotype and resistance to diet-induced obesity in vitamin D receptor knockout mice correlates with induction of uncoupling protein-1 in white adipose tissue. Endocrinology 150, 651–661 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Marshall, T.G., Lee, R.E. & Marshall, F.E. Common angiotensin receptor blockers may directly modulate the immune system via VDR, PPAR and CCR2b. Theor. Biol. Med. Model. 3, 1 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Krishnan, A.V. et al. Tissue-selective regulation of aromatase expression by calcitriol: implications for breast cancer therapy. Endocrinology 151, 32–42 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Köstner, K. et al. The relevance of vitamin D receptor (VDR) gene polymorphisms for cancer: a review of the literature. Anticancer Res. 29, 3511–3536 (2009).

    PubMed  Google Scholar 

  6. Dilworth, F.J. & Chambon, P. Nuclear receptors coordinate the activities of chromatin remodeling complexes and coactivators to facilitate initiation of transcription. Oncogene 20, 3047–3054 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Chandra, V. et al. Structure of the intact PPAR-γ–RXR-α nuclear receptor complex on DNA. Nature 456, 350–356 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Iacob, R.E. et al. Conformational disturbance in Abl kinase upon mutation and deregulation. Proc. Natl. Acad. Sci. USA 106, 1386–1391 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hamuro, Y. et al. Hydrogen/deuterium-exchange (H/D-Ex) of PPARγ LBD in the presence of various modulators. Protein Sci. 15, 1883–1892 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hsu, Y.H., Burke, J.E., Li, S., Woods, V.L. Jr. & Dennis, E.A. Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry. J. Biol. Chem. 284, 23652–23661 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bruning, J.B. et al. Partial agonists activate PPARγ using a helix 12 independent mechanism. Structure 15, 1258–1271 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Hamuro, Y. et al. Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry. J. Biomol. Tech. 14, 171–182 (2003).

    PubMed  PubMed Central  Google Scholar 

  13. Yan, X., Broderick, D., Leid, M.E., Schimerlik, M.I. & Deinzer, M.L. Dynamics and ligand-induced solvent accessibility changes in human retinoid X receptor homodimer determined by hydrogen deuterium exchange and mass spectrometry. Biochemistry 43, 909–917 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Yan, X . et al. Deuterium exchange and mass spectrometry reveal the interaction differences of two synthetic modulators of RXRα LBD. Protein Sci. 16, 2491–2501 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chalmers, M.J., Busby, S.A., Pascal, B.D., Southern, M.R. & Griffin, P.R. A two-stage differential hydrogen deuterium exchange method for the rapid characterization of protein/ligand interactions. J. Biomol. Tech. 18, 194–204 (2007).

    PubMed  PubMed Central  Google Scholar 

  16. Dai, S.Y. et al. Prediction of the tissue-specificity of selective estrogen receptor modulators by using a single biochemical method. Proc. Natl. Acad. Sci. USA 105, 7171–7176 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Dai, S.Y. et al. Unique ligand binding patterns between estrogen receptor α and β revealed by hydrogen-deuterium exchange. Biochemistry 48, 9668–9676 (2009).

    Article  CAS  PubMed  Google Scholar 

  18. Choi, J.H. et al. Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARγ by Cdk5. Nature 466, 451–456 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang, J. et al. Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer. Structure 18, 1332–1341 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  21. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Rastinejad, F., Perlmann, T., Evans, R.M. & Sigler, P.B. Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 375, 203–211 (1995).

    Article  CAS  PubMed  Google Scholar 

  23. Gampe, R.T. Jr. et al. Asymmetry in the PPARγ/RXRα crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. Mol. Cell 5, 545–555 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. 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  PubMed  Google Scholar 

  25. Rochel, N., Wurtz, J.M., Mitschler, A., Klaholz, B. & Moras, D. The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol. Cell 5, 173–179 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Carlberg, C. & Polly, P. Gene regulation by vitamin D3. Crit. Rev. Eukaryot. Gene Expr. 8, 19–42 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Schulman, I.G., Li, C., Schwabe, J.W. & Evans, R.M. The phantom ligand effect: allosteric control of transcription by the retinoid X receptor. Genes Dev. 11, 299–308 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Willy, P.J. & Mangelsdorf, D.J. Unique requirements for retinoid-dependent transcriptional activation by the orphan receptor LXR. Genes Dev. 11, 289–298 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Egea, P.F. et al. Crystal structure of the human RXRα ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J. 19, 2592–2601 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  31. Zhang, J., Simisky, J., Tsai, F.T. & Geller, D.S. A critical role of helix 3-helix 5 interaction in steroid hormone receptor function. Proc. Natl. Acad. Sci. USA 102, 2707–2712 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lemon, B.D. & Freedman, L.P. Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated gene activation in vivo. Mol. Cell. Biol. 16, 1006–1016 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhao, Q., Khorasanizadeh, S., Miyoshi, Y., Lazar, M.A. & Rastinejad, F. Structural elements of an orphan nuclear receptor–DNA complex. Mol. Cell 1, 849–861 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Miyamoto, T. et al. The role of hinge domain in heterodimerization and specific DNA recognition by nuclear receptors. Mol. Cell. Endocrinol. 181, 229–238 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Rachez, C. & Freedman, L.P. Mechanisms of gene regulation by vitamin D3 receptor: a network of coactivator interactions. Gene 246, 9–21 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Heery, D.M., Kalkhoven, E., Hoare, S. & Parker, M.G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387, 733–736 (1997).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  38. Glass, C.K. & Rosenfeld, M.G. The co-regulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 14, 121–141 (2000).

    CAS  PubMed  Google Scholar 

  39. Pogenberg, V. et al. Characterization of the interaction between retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers and transcriptional coactivators through structural and fluorescence anisotropy studies. J. Biol. Chem. 280, 1625–1633 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Kersten, S., Dong, D., Lee, W., Reczek, P.R. & Noy, N. Auto-silencing by the retinoid X receptor. J. Mol. Biol. 284, 21–32 (1998).

    Article  CAS  PubMed  Google Scholar 

  41. Sánchez-Martínez, R., Castillo, A.I., Steinmeyer, A. & Aranda, A. The retinoid X receptor ligand restores defective signalling by the vitamin D receptor. EMBO Rep. 7, 1030–1034 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Meijsing, S.H. et al. DNA binding site sequence directs glucocorticoid receptor structure and activity. Science 324, 407–410 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Shoemaker, B.A., Portman, J.J. & Wolynes, P.G. Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. Proc. Natl. Acad. Sci. USA 97, 8868–8873 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Levy, Y., Onuchic, J.N. & Wolynes, P.G. Fly-casting in protein-DNA binding: frustration between protein folding and electrostatics facilitates target recognition. J. Am. Chem. Soc. 129, 738–739 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. von Hippel, P.H. Biochemistry. Completing the view of transcriptional regulation. Science 305, 350–352 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Leung, T.H., Hoffmann, A. & Baltimore, D. One nucleotide in a κB site can determine cofactor specificity for NF-κB dimers. Cell 118, 453–464 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Lefstin, J.A. & Yamamoto, K.R. Allosteric effects of DNA on transcriptional regulators. Nature 392, 885–888 (1998).

    Article  CAS  PubMed  Google Scholar 

  48. Chalmers, M.J. et al. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Anal. Chem. 78, 1005–1014 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Pascal, B.D., Chalmers, M.J., Busby, S.A. & Griffin, P.R. HD desktop: an integrated platform for the analysis and visualization of H/D exchange data. J. Am. Soc. Mass Spectrom. 20, 601–610 (2009).

    Article  CAS  PubMed  Google Scholar 

  50. Zhang, Z. & Smith, D.L. Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci. 2, 522–531 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ericsson, U.B., Hallberg, B.M., Detitta, G.T., Dekker, N. & Nordlund, P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem. 357, 289–298 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Matulis, D., Kranz, J.K., Salemme, F.R. & Todd, M.J. Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258–5266 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Marky, L.A. & Breslauer, K.J. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26, 1601–1620 (1987).

    Article  CAS  PubMed  Google Scholar 

  54. Pantoliano, M.W. et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J. Biomol. Screen. 6, 429–440 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Bruylants, G., Wouters, J. & Michaux, C. Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design. Curr. Med. Chem. 12, 2011–2020 (2005).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful for support from M. Southern and S. Willis for software analyzing the HDX data. This work was supported in part by the Intramural Research Program of the US National Institutes of Health (NIH), National Institute of Mental Health (Grant U54-MH074404 to H. Rosen), the National Institute of General Medical Sciences (R01-GM084041 to P.R.G.), the National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK080201to T.P.B.) and the James and Esther King Biomedical Research Program, Florida Department of Health (10KN-09 to D.J.K.).

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J.Z., M.J.C., K.R.S., J.A.D. and P.R.G. conceived of the project and designed the research; J.Z., L.L.B., Y.W., S.A.B., B.D.P., R.D.G.-O., J.B.B., M.A.I. and D.J.K. conducted the research; J.Z., M.J.C., K.R.S., L.L.B., Y.W., S.A.B., B.D.P., R.D.G.-O., J.B.B., M.A.I., D.J.K., T.P.B., J.A.D. and P.R.G. analyzed the data; and J.Z., K.R.S., T.P.B., S.A.B. and P.R.G. wrote the paper, with contributions from all authors.

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Correspondence to Patrick R Griffin.

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Zhang, J., Chalmers, M., Stayrook, K. et al. DNA binding alters coactivator interaction surfaces of the intact VDR–RXR complex. Nat Struct Mol Biol 18, 556–563 (2011). https://doi.org/10.1038/nsmb.2046

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