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Structure and VP16 binding of the Mediator Med25 activator interaction domain

Nature Structural & Molecular Biology volume 18, pages 404–409 (2011)Cite this article

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Abstract

Eukaryotic transcription is regulated by interactions between gene-specific activators and the coactivator complex Mediator. Here we report the NMR structure of the Mediator subunit Med25 (also called Arc92) activator interaction domain (ACID) and analyze the structural and functional interaction of ACID with the archetypical acidic transcription activator VP16. Unlike other known activator targets, ACID forms a seven-stranded β-barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in a in vitro system. The activator-binding ACID faces are functionally required and conserved among higher eukaryotes. Comparison with published activator structures reveals that the VP16 activation domain uses distinct interaction modes to adapt to unrelated target surfaces and folds that evolved for activator binding.

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Figure 1: Solution structure of Med25 ACID.
Figure 2: VP16–ACID interaction.
Figure 3: VP16-binding interface of ACID.
Figure 4: Functional ACID-VP16 interaction.
Figure 5: Model of an activated transcription initiation complex.
Figure 6: Comparison of ACID with known activator–target complexes.

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References

  1. Brent, R. & Ptashne, M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43, 729–736 (1985).

    Article  CAS  Google Scholar 

  2. Keegan, L., Gill, G. & Ptashne, M. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science 231, 699–704 (1986).

    Article  CAS  Google Scholar 

  3. Mitchell, P.J. & Tjian, R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371–378 (1989).

    Article  CAS  Google Scholar 

  4. Gerster, T. & Roeder, R.G. A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins. Proc. Natl. Acad. Sci. USA 85, 6347–6351 (1988).

    Article  CAS  Google Scholar 

  5. Katan, M., Haigh, A., Verrijzer, C.P., van der Vliet, P.C. & O'Hare, P. Characterization of a cellular factor which interacts functionally with Oct-1 in the assembly of a multicomponent transcription complex. Nucleic Acids Res. 18, 6871–6880 (1990).

    Article  CAS  Google Scholar 

  6. Stern, S., Tanaka, M. & Herr, W. The Oct-1 homoeodomain directs formation of a multiprotein-DNA complex with the HSV transactivator VP16. Nature 341, 624–630 (1989).

    Article  CAS  Google Scholar 

  7. Wilson, A.C., LaMarco, K., Peterson, M.G. & Herr, W. The VP16 accessory protein HCF is a family of polypeptides processed from a large precursor protein. Cell 74, 115–125 (1993).

    Article  CAS  Google Scholar 

  8. Wysocka, J. & Herr, W. The herpes simplex virus VP16-induced complex: the makings of a regulatory switch. Trends Biochem. Sci. 28, 294–304 (2003).

    Article  CAS  Google Scholar 

  9. Greaves, R. & O'Hare, P. Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65. J. Virol. 63, 1641–1650 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Greaves, R.F. & O'Hare, P. Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences. J. Virol. 64, 2716–2724 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Lai, J.S. & Herr, W. Interdigitated residues within a small region of VP16 interact with Oct-1, HCF, and DNA. Mol. Cell. Biol. 17, 3937–3946 (1997).

    Article  CAS  Google Scholar 

  12. Triezenberg, S.J., Kingsbury, R.C. & McKnight, S.L. Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 2, 718–729 (1988a).

    Article  CAS  Google Scholar 

  13. Triezenberg, S.J., LaMarco, K.L. & McKnight, S.L. Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16. Genes Dev. 2, 730–742 (1988b).

    Article  CAS  Google Scholar 

  14. Sadowski, I., Ma, J., Triezenberg, S. & Ptashne, M. GAL4–VP16 is an unusually potent transcriptional activator. Nature 335, 563–564 (1988).

    Article  CAS  Google Scholar 

  15. Cousens, D.J., Greaves, R., Goding, C.R. & O'Hare, P. The C-terminal 79 amino acids of the herpes simplex virus regulatory protein, Vmw65, efficiently activate transcription in yeast and mammalian cells in chimeric DNA-binding proteins. EMBO J. 8, 2337–2342 (1989).

    Article  CAS  Google Scholar 

  16. Berger, S.L., Cress, W.D., Cress, A., Triezenberg, S.J. & Guarente, L. Selective inhibition of activated but not basal transcription by the acidic activation domain of VP16: evidence for transcriptional adaptors. Cell 61, 1199–1208 (1990).

    Article  CAS  Google Scholar 

  17. Chasman, D.I., Leatherwood, J., Carey, M., Ptashne, M. & Kornberg, R.D. Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro. Mol. Cell. Biol. 9, 4746–4749 (1989).

    Article  CAS  Google Scholar 

  18. Kobayashi, N., Boyer, T.G. & Berk, A.J. A class of activation domains interacts directly with TFIIA and stimulates TFIIA-TFIID-promoter complex assembly. Mol. Cell. Biol. 15, 6465–6473 (1995).

    Article  CAS  Google Scholar 

  19. Kobayashi, N. et al. DA-complex assembly activity required for VP16C transcriptional activation. Mol. Cell. Biol. 18, 4023–4031 (1998).

    Article  CAS  Google Scholar 

  20. Hall, D.B. & Struhl, K. The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo. J. Biol. Chem. 277, 46043–46050 (2002).

    Article  CAS  Google Scholar 

  21. Langlois, C. et al. NMR structure of the complex between the Tfb1 subunit of TFIIH and the activation domain of VP16: structural similarities between VP16 and p53. J. Am. Chem. Soc. 130, 10596–10604 (2008).

    Article  CAS  Google Scholar 

  22. Xiao, H. et al. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol. Cell. Biol. 14, 7013–7024 (1994).

    Article  CAS  Google Scholar 

  23. Uesugi, M., Nyanguile, O., Lu, H., Levine, A.J. & Verdine, G.L. Induced alpha helix in the VP16 activation domain upon binding to a human TAF. Science 277, 1310–1313 (1997).

    Article  CAS  Google Scholar 

  24. Mittler, G. et al. A novel docking site on Mediator is critical for activation by VP16 in mammalian cells. EMBO J. 22, 6494–6504 (2003).

    Article  CAS  Google Scholar 

  25. Bourbon, H.M. et al. A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol. Cell 14, 553–557 (2004).

    Article  CAS  Google Scholar 

  26. Näär, A.M. et al. Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828–832 (1999).

    Article  Google Scholar 

  27. Yang, F., DeBeaumont, R., Zhou, S. & Naar, A.M. The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator. Proc. Natl. Acad. Sci. USA 101, 2339–2344 (2004).

    Article  CAS  Google Scholar 

  28. Kornberg, R.D. Mediator and the mechanism of transcriptional activation. Trends Biochem. Sci. 30, 235–239 (2005).

    Article  CAS  Google Scholar 

  29. Malik, S. & Roeder, R.G. Dynamic regulation of pol II transcription by the mammalian Mediator complex. Trends Biochem. Sci. 30, 256–263 (2005).

    Article  CAS  Google Scholar 

  30. Thakur, J.K. et al. A nuclear receptor-like pathway regulating multidrug resistance in fungi. Nature 452, 604–609 (2008).

    Article  CAS  Google Scholar 

  31. Yang, F. et al. An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis. Nature 442, 700–704 (2006).

    Article  CAS  Google Scholar 

  32. Grossmann, J.G., Sharff, A.J., O′Hare, P. & Luisi, B. Molecular shapes of transcription factors TFIIB and VP16 in solution: implications for recognition. Biochemistry 40, 6267–6274 (2001).

    Article  CAS  Google Scholar 

  33. Kim, D.H. et al. Multiple hTAF(II)31-binding motifs in the intrinsically unfolded transcriptional activation domain of VP16. BMB Rep. 42, 411–417 (2009).

    Article  CAS  Google Scholar 

  34. Liu, Y., Gong, W., Huang, C.C., Herr, W. & Cheng, X. Crystal structure of the conserved core of the herpes simplex virus transcriptional regulatory protein VP16. Genes Dev. 13, 1692–1703 (1999).

    Article  CAS  Google Scholar 

  35. Ikeda, K., Stuehler, T. & Meisterernst, M. The H1 and H2 regions of the activation domain of herpes simplex virion protein 16 stimulate transcription through distinct molecular mechanisms. Genes Cells 7, 49–58 (2002).

    Article  CAS  Google Scholar 

  36. Walker, S., Greaves, R. & O'Hare, P. Transcriptional activation by the acidic domain of Vmw65 requires the integrity of the domain and involves additional determinants distinct from those necessary for TFIIB binding. Mol. Cell. Biol. 13, 5233–5244 (1993).

    Article  CAS  Google Scholar 

  37. Cress, W.D. & Triezenberg, S.J. Critical structural elements of the VP16 transcriptional activation domain. Science 251, 87–90 (1991).

    Article  CAS  Google Scholar 

  38. Regier, J.L., Shen, F. & Triezenberg, S.J. Pattern of aromatic and hydrophobic amino acids critical for one of two subdomains of the VP16 transcriptional activator. Proc. Natl. Acad. Sci. USA 90, 883–887 (1993).

    Article  CAS  Google Scholar 

  39. Sullivan, S.M. et al. Mutational analysis of a transcriptional activation region of the VP16 protein of herpes simplex virus. Nucleic Acids Res. 26, 4487–4496 (1998).

    Article  CAS  Google Scholar 

  40. Jonker, H.R., Wechselberger, R.W., Boelens, R., Folkers, G.E. & Kaptein, R. Structural properties of the promiscuous VP16 activation domain. Biochemistry 44, 827–839 (2005).

    Article  CAS  Google Scholar 

  41. Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995).

    Article  CAS  Google Scholar 

  42. Ariyoshi, M. & Schwabe, J.W. A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling. Genes Dev. 17, 1909–1920 (2003).

    Article  CAS  Google Scholar 

  43. Walker, J.R., Corpina, R.A. & Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614 (2001).

    Article  CAS  Google Scholar 

  44. Ranish, J.A. & Hahn, S. The yeast general transcription factor TFIIA is composed of two polypeptide subunits. J. Biol. Chem. 266, 19320–19327 (1991).

    CAS  PubMed  Google Scholar 

  45. Radhakrishnan, I. et al. Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions. Cell 91, 741–752 (1997).

    Article  CAS  Google Scholar 

  46. Razeto, A. et al. Structure of the NCoA-1/SRC-1 PAS-B domain bound to the LXXLL motif of the STAT6 transactivation domain. J. Mol. Biol. 336, 319–329 (2004).

    Article  CAS  Google Scholar 

  47. Kussie, P.H. et al. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 274, 948–953 (1996).

    Article  CAS  Google Scholar 

  48. Benedit, P. et al. PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks. Oncogene 20, 1455–1464 (2001).

    Article  CAS  Google Scholar 

  49. Santamaría, A. et al. PTOV1 enables the nuclear translocation and mitogenic activity of flotillin-1, a major protein of lipid rafts. Mol. Cell. Biol. 25, 1900–1911 (2005).

    Article  Google Scholar 

  50. McEwan, I.J., Dahlman-Wright, K., Ford, J. & Wright, A.P. Functional interaction of the c-Myc transactivation domain with the TATA binding protein: evidence for an induced fit model of transactivation domain folding. Biochemistry 35, 9584–9593 (1996).

    Article  CAS  Google Scholar 

  51. D'Alessio, J.A., Wright, K.J. & Tjian, R. Shifting players and paradigms in cell-specific transcription. Mol. Cell 36, 924–931 (2009).

    Article  CAS  Google Scholar 

  52. Levine, M. & Tjian, R. Transcription regulation and animal diversity. Nature 424, 147–151 (2003).

    Article  CAS  Google Scholar 

  53. Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996).

    Article  CAS  Google Scholar 

  54. Chen, Z.A. et al. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 29, 717–726 (2010).

    Article  CAS  Google Scholar 

  55. Kostrewa, D. et al. RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature 462, 323–330 (2009).

    Article  CAS  Google Scholar 

  56. Linge, J.P., Williams, M.A., Spronk, C.A., Bonvin, A.M. & Nilges, M. Refinement of protein structures in explicit solvent. Proteins 50, 496–506 (2003).

    Article  CAS  Google Scholar 

  57. 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 

  58. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009).

    Article  CAS  Google Scholar 

  59. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

  60. Johnson, B.A. & Blevins, R.A. NMRView: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    Article  CAS  Google Scholar 

  61. Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999).

    Article  CAS  Google Scholar 

  62. Farrow, N.A. et al. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994).

    Article  CAS  Google Scholar 

  63. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    Google Scholar 

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

    Article  Google Scholar 

  65. Hooft, R.W., Vriend, G., Sander, C. & Abola, E.E. Errors in protein structures. Nature 381, 272 (1996).

    Article  CAS  Google Scholar 

  66. Larivière, L. et al. Structure-system correlation identifies a gene regulatory Mediator submodule. Genes Dev. 22, 872–877 (2008).

    Article  Google Scholar 

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Acknowledgements

We thank D. Kostrewa and K. Leike for help, and T. Fröhlich for help with MALDI and ESI experiments. M. Seizl was supported by the Boehringer Ingelheim Fonds and Elitenetzwerk Bayern. A.M. was supported by a Ph.D. fellowship (SFRH/BD/22323/2005) from the Portuguese Foundation for Science and Technology (FCT). M. Sattler acknowledges support by the Deutsche Forschungsgemeinschaft, the European Commission (3D Repertoire LSHG-CT-2005-512028) and the Bavarian NMR Center. P.C. was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich SFB646, the SFB Transregio 5, the Jung-Stiftung and the Fonds der chemischen Industrie.

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  1. Sonja Baumli

    Present address: Present address: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK.,

Authors and Affiliations

  1. Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany

    Erika Vojnic, Martin Seizl, Larissa Wenzeck, Laurent Larivière, Sonja Baumli & Patrick Cramer

  2. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany

    André Mourão & Michael Sattler

  3. Department Chemie, Biomolecular NMR and Center for Integrated Protein Science Munich (CIPSM), Garching, Germany

    André Mourão & Michael Sattler

  4. European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    André Mourão & Bernd Simon

  5. Institute of Molecular Tumor Biology, University of Muenster, Muenster, Germany

    Karen Baumgart & Michael Meisterernst

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Contributions

E.V., A.M. and B.S., NMR data acquisition and analysis; E.V., M. Seizl, L.W., L.L. and S.B., sample preparation and functional assays; K.B. and M.M., mammalian transcription assays; M.M., M. Sattler and P.C., project design and supervision; E.V., M. Sattler and P.C., manuscript preparation.

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Correspondence to Michael Sattler or Patrick Cramer.

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Vojnic, E., Mourão, A., Seizl, M. et al. Structure and VP16 binding of the Mediator Med25 activator interaction domain. Nat Struct Mol Biol 18, 404–409 (2011). https://doi.org/10.1038/nsmb.1997

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