Article | Published:

Structure of the VP16 transactivator target in the Mediator

Nature Structural & Molecular Biology volume 18, pages 410415 (2011) | Download Citation

Abstract

The human Mediator coactivator complex interacts with many transcriptional activators and facilitates recruitment of RNA polymerase II to promote target gene transcription. The MED25 subunit is a critical target of the potent herpes simplex 1 viral transcriptional activator VP16. Here we determine the solution structure of the MED25 VP16-binding domain (VBD) and define its binding site for the N-terminal portion of the VP16 transactivation domain (TADn). A hydrophobic furrow, formed by a β-barrel and two α-helices in MED25 VBD, interacts tightly with VP16 TADn. Mutations in this furrow prevent binding of VP16 TAD to MED25 VBD and interfere with the ability of overexpressed MED25 VBD to inhibit VP16-dependent transcriptional activation in vivo. This detailed molecular understanding of transactivation by the benchmark activator VP16 could provide important insights into viral and cellular gene activation mechanisms.

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Primary accessions

Protein Data Bank

Referenced accessions

GenBank/EMBL/DDBJ

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Acknowledgements

We thank M. Oberer for initial NMR data collection, R. Rodriguez for critical advice on designing biochemical experiments, K. Edmonds for critical reading and Python script writing, S. Hiller and V. Orekhov for advice and assistance with nonlinear-sampling NMR data collection and processing, and H. Arthanari for advice on NMR data collection and manuscript editing. We are very grateful to M. Sattler and E. Vojnic of the Technical University of Munich for critical discussion of unpublished data. We are also thankful for H.C. Seou's assistance with DNA cloning procedures and A. Koglin for help with CNS calculations. A.G.M. was partially supported by a Deutscher Akademischer Austausch Dienst postdoctoral fellowship. P.S. was funded by the Human Frontier Science Program Organization long-term fellowship LT00686/2004-C. The work was supported by US National Institutes of Health grants CA127990 (G.W. and A.M.N.), GM47467 and EB002026 (G.W.) and GM071449 (A.M.N.).

Author information

Author notes

    • Koh Takeuchi
    •  & Philipp Selenko

    Present addresses: Biomedical Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan (K.T.), and Department of NMR-assisted Structural Biology, Leibniz Institute of Molecular Pharmacology, Berlin, Germany (P.S.).

    • Madhura Kulkarni
    •  & Tingfang Yi

    These authors contributed equally to this work.

Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Alexander G Milbradt
    • , Tingfang Yi
    • , Koh Takeuchi
    • , Zhen-Yu J Sun
    • , Rafael E Luna
    • , Philipp Selenko
    •  & Gerhard Wagner
  2. Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts, USA.

    • Madhura Kulkarni
    •  & Anders M Näär
  3. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Madhura Kulkarni
    •  & Anders M Näär

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Contributions

A.G.M. prepared protein samples, recorded and analyzed NMR data, calculated the structure, designed the MED25 VBD mutants and co-wrote the paper; M.K. generated point mutations, did pulldown assays and assisted with transcription assays; T.Y. did transcription assays; R.E.L. assisted with transcription assays, data interpretation and writing of the paper; K.T. and P.S. cloned the original construct, collected initial NMR data, obtained preliminary assignment, and assisted with editing and writing the paper; Z.-Y.J.S. assisted in recording NMR data, data analysis and structure calculation; A.M.N. and G.W. initiated the project, helped design experiments, advised on data collection and interpretation, and participated in writing and editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Anders M Näär or Gerhard Wagner.

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DOI

https://doi.org/10.1038/nsmb.1999

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