Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation

Nature Structural & Molecular Biology volume 10, pages 913–921 (2003)Cite this article

Abstract

IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions as a molecular switch for antiviral activity. IRF-3 uses an autoinhibitory mechanism to suppress its transactivation potential in uninfected cells, and virus infection induces phosphorylation and activation of IRF-3 to initiate the antiviral responses. The crystal structure of the IRF-3 transactivation domain reveals a unique autoinhibitory mechanism, whereby the IRF association domain and the flanking autoinhibitory elements condense to form a hydrophobic core. The structure suggests that phosphorylation reorganizes the autoinhibitory elements, leading to unmasking of a hydrophobic active site and realignment of the DNA binding domain for transcriptional activation. IRF-3 exhibits marked structural and surface electrostatic potential similarity to the MH2 domain of the Smad protein family and the FHA domain, suggesting a common molecular mechanism of action among this superfamily of signaling mediators.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Crystal structure of the IRF-3 transactivation domain and its homology to the Smad MH2 domain.
Figure 2: Sequence alignment of the C-terminal transactivation domains of human IRF members that contain the IAD.
Figure 3: Structural basis of IRF-3 regulation by autoinhibition and virus-induced phosphoactivation.
Figure 4: Structural and surface electrostatic potential homology among the IAD, MH2 and FHA domains.
Figure 5: The basic and hydrophobic surfaces in IRF-3 are functional.
Figure 6: Model of regulation of IRF-3 activity by autoinhibition and phosphorylation.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Mamane, Y. et al. Interferon regulatory factors: the next generation. Gene 237, 1–14 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Barnes, B., Lubyova, B. & Pitha, P.M. On the role of IRF in host defense. J. Interferon Cytokine Res. 22, 59–71 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Taniguchi, T., Ogasawara, K., Takaoka, A. & Tanaka, N. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 19, 623–655 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Fujii, Y. et al. Crystal structure of an IRF-DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences. EMBO J. 18, 5028–5041 (1999).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Escalante, C.R., Yie, J., Thanos, D. & Aggarwal, A.K. Structure of IRF-1 with bound DNA reveals determinants of interferon regulation. Nature 391, 103–106 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Lin, R., Mamane, Y. & Hiscott, J. Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol. Cell Biol. 19, 2465–2474 (1999).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Lin, R., Mamane, Y. & Hiscott, J. Multiple regulatory domains control IRF-7 activity in response to virus infection. J. Biol. Chem. 275, 34320–34327 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Au, W.C., Yeow, W. & Pitha, P.M. Analysis of functional domains of interferon regulatory factor 7 and its association with IRF-3. Virology 280, 273–282 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Brass, A.L., Kehrli, E., Eisenbeis, C.F., Storb, U. & Singh, H. Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1. Genes Dev. 10, 2335–2347 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Barnes, B.J., Kellum, M., Field, A.E. & Pitha, P.M. Multiple regulatory domains of IRF-5 control activation, cellular localization, and induction of chemokines that mediate recruitment of T lymphocytes. Mol. Cell. Biol. 22, 5721–5740 (2002).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. tenOever, B.R., Servant, M.J., Grandvaux, N., Lin, R. & Hiscott, J. Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J. Virol. 76, 3659–3669 (2002).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Iwamura, T. et al. Induction of IRF-3/-7 kinase and NF-κB in response to double-stranded RNA and virus infection: common and unique pathways. Genes Cells 6, 375–388 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Sato, M. et al. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-α/β gene induction. Immunity 13, 539–548 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Wathelet, M.G. et al. Virus infection induces the assembly of coordinately activated transcription factors on the IFN-β enhancer in vivo. Mol. Cell 1, 507–518 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Yoneyama, M. et al. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J. 17, 1087–1095 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Lin, R., Heylbroeck, C., Pitha, P.M. & Hiscott, J. Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol. Cell Biol. 18, 2986–2996 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Servant, M.J. et al. Identification of distinct signaling pathways leading to the phosphorylation of interferon regulatory factor 3. J. Biol. Chem. 276, 355–363 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Sharma, S. et al. Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148–1151 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Fitzgerald, K.A. et al. IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491–496 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Au, W.C., Moore, P.A., LaFleur, D.W., Tombal, B. & Pitha, P.M. Characterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon A genes. J. Biol. Chem. 273, 29210–29217 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. Zhang, L. & Pagano, J.S. Structure and function of IRF-7. J. Interferon Cytokine Res. 1, 95–101 (2002).

    Article  Google Scholar 

  22. Levy, D.E., Marie, I., Smith, E. & Prakash, A. Enhancement and diversification of IFN induction by IRF-7-mediated positive feedback. J. Interferon Cytokine Res. 1, 87–93 (2002).

    Article  Google Scholar 

  23. Marie, I., Durbin, J.E. & Levy, D.E. Differential viral induction of distinct interferon-α genes by positive feedback through interferon regulatory factor-7. EMBO J. 17, 6660–6669 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Eroshkin, A. & Mushegian, A. Conserved transactivation domain shared by interferon regulatory factors and Smad morphogens. J. Mol. Med. 77, 403–405 (1999).

    Article  CAS  PubMed  Google Scholar 

  25. Zhang, Y., Feng, X.H., Wu, R.Y. & Derynck, R. Receptor-associated Mad homologues synergize as effectors of the TGF-β response. Nature 383, 168–172 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Kawabata, M., Inoue, H., Hanyu, A., Imamura, T. & Miyazono, K. Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors. EMBO J. 17, 4056–4065 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Chacko, B.M. et al. The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization. Nat. Struct. Biol. 8, 248–253 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Qin, B.Y. et al. Structural basis of Smad1 activation by receptor kinase phosphorylation. Mol. Cell 8, 1303–1312 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Durocher, D. et al. The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. Mol. Cell 6, 1169–1182 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Liao, H., Byeon, I.J. & Tsai, M.D. Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53. J. Mol. Biol. 294, 1041–1049 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Meraro, D. et al. Protein-protein and DNA-protein interactions affect the activity of lymphoid-specific IFN regulatory factors. J. Immunol. 163, 6468–6478 (1999).

    CAS  PubMed  Google Scholar 

  32. Levi, B.Z., Hashmueli, S., Gleit-Kielmanowicz, M., Azriel, A. & Meraro, D. ICSBP/IRF-8 transactivation: a tale of protein-protein interaction. J. Interferon Cytokine Res. 22, 153–160 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Maniatis, T. et al. Structure and function of the interferon-β enhanceosome. Cold Spring Harb. Symp. Quant. Biol. 63, 609–620 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Servant, M.J. et al. Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA. J. Biol. Chem. 278, 9441–9447 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Yang, H. et al. Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein-protein interactions. Eur. J. Biochem. 269, 6142–6151 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Huse, M. et al. The TGF-β receptor activation process: an inhibitor- to substrate-binding switch. Mol. Cell 8, 671–682 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Wu, J.W. et al. Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-β signaling. Mol. Cell 8, 1277–1289 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Tsukazaki, T., Chiang, T.A., Davison, A.F., Attisano, L. & Wrana, J.L. SARA, a FYVE domain protein that recruits smad2 to the TGF-β receptor. Cell 95, 779–791 (1998).

    Article  CAS  PubMed  Google Scholar 

  39. Wu, G. et al. Structural basis of smad2 recognition by the smad anchor for receptor activation. Science 287, 92–97 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Qin, B.Y., Lam, S.S., Correia, J.J. & Lin, K. Smad3 allostery links TGF-β receptor kinase activation to transcriptional control. Genes Dev. 16, 1950–1963 (2002).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Luo, K. et al. The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signaling. Genes Dev. 13, 196–206 (1999).

    Article  Google Scholar 

  42. Bell, D.W. et al. Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome. Science 286, 2528–2531 (1999).

    Article  CAS  PubMed  Google Scholar 

  43. Weaver, B.K., Kumar, K.P. & Reich, N.C. Interferon regulatory factor 3 and CREB-binding protein/p300 are subunits of double-stranded RNA-activated transcription factor DRAF1. Mol. Cell Biol. 18, 1359–1368 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Takahasi, K. et al. X-ray crystal structure of IRF-3 and its functional implications. Nat. Struct. Biol. advance online publication, 12 October 2003 (doi:10.1038/nsb1001).

  45. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation model. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  46. 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  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  48. Zhang, Y. & Derynck, R. Transcriptional regulation of the transforming growth factor-β-inducible mouse germ line Ig α constant region gene by functional cooperation of Smad, CREB, and AML family members. J. Biol. Chem. 275, 16979–16985 (2000).

    Article  CAS  PubMed  Google Scholar 

  49. Feng, X.H., Filvaroff, E.H. & Derynck, R. Transforming growth factor-β (TGF-β)-induced down-regulation of cyclin A expression requires a functional TGF-β receptor complex. Characterization of chimeric and truncated type I and type II receptors. J. Biol. Chem. 270, 24237–24245 (1995).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank J. Qing for generating the expression plasmid for GST-fused IRF-3 and B. Chacko for critical reading of the manuscript. We thank the staff members of Advanced Light Source for assistance with the data collection of IRF-3 crystals. This research was supported by grants from the US National Institutes of Health to K.L and R.D.

Author information

Author notes

  1. Bin Y Qin and Cheng Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, 01605, Massachusetts, USA

    Bin Y Qin, Suvana S Lam, Hema Srinath & Kai Lin

  2. Departments of Growth and Development, and Anatomy, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, 94143-0640, California, USA

    Cheng Liu, Rachel Delston & Rik Derynck

  3. Department of Biochemistry, University of Mississippi Medical Center, Jackson, 39216, Mississippi, USA

    John J Correia

Authors
  1. Bin Y Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cheng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suvana S Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hema Srinath
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rachel Delston
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John J Correia
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rik Derynck
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kai Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Rik Derynck or Kai Lin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Qin, B., Liu, C., Lam, S. et al. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation. Nat Struct Mol Biol 10, 913–921 (2003). https://doi.org/10.1038/nsb1002

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb1002

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing