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:

Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5

Nature Structural & Molecular Biology volume 15, pages 1213–1220 (2008)Cite this article

Abstract

Interferon regulatory factors (IRFs) are essential in the innate immune response and other physiological processes. Activation of these proteins in the cytoplasm is triggered by phosphorylation of serine and threonine residues in a C-terminal autoinhibitory region, which stimulates dimerization, transport into the nucleus, assembly with the coactivator CBP/p300 and initiation of transcription. The crystal structure of the transactivation domain of pseudophosphorylated human IRF5 strikingly reveals a dimer in which the bulk of intersubunit interactions involve a highly extended C-terminal region. The corresponding region has previously been shown to block CBP/p300 binding to unphosphorylated IRF3. Mutation of key interface residues supports the observed dimer as the physiologically activated state of IRF5 and IRF3. Thus, phosphorylation is likely to activate IRF5 and other family members by triggering conformational rearrangements that switch the C-terminal segment from an autoinihibitory to a dimerization role.

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: Ribbon diagrams of the C-terminal transactivation domain of IRF5 and IRF3.
Figure 2: IRF5 dimer.
Figure 3: Structure-guided mutagenesis of IRF5 and IRF3.
Figure 4: Schematic diagram for the activation of IRF family members, in which the interferon association domain (IAD) is shown as a crescent and the DNA binding domain (DBD) is shown as an oval behind the IAD.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Honda, K. & Taniguchi, T. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 6, 644–658 (2006).

    Article  CAS  Google Scholar 

  2. Paun, A. & Pitha, P.M. The IRF family, revisited. Biochimie 89, 744–753 (2007).

    Article  CAS  Google Scholar 

  3. Tamura, T., Yanai, H., Savitsky, D. & Taniguchi, T. The IRF family transcription factors in immunity and oncogenesis. Annu. Rev. Immunol. 26, 535–584 (2008).

    Article  CAS  Google Scholar 

  4. Schoenemeyer, A. et al. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J. Biol. Chem. 280, 17005–17012 (2005).

    Article  CAS  Google Scholar 

  5. Yasuda, K. et al. Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production. J. Immunol. 178, 6876–6885 (2007).

    Article  CAS  Google Scholar 

  6. Takaoka, A. et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434, 243–249 (2005).

    Article  CAS  Google Scholar 

  7. Hu, G., Mancl, M.E. & Barnes, B.J. Signaling through IFN regulatory factor-5 sensitizes p53-deficient tumors to DNA damage-induced apoptosis and cell death. Cancer Res. 65, 7403–7412 (2005).

    Article  CAS  Google Scholar 

  8. Yanai, H. et al. Role of IFN regulatory factor 5 transcription factor in antiviral immunity and tumor suppression. Proc. Natl. Acad. Sci. USA 104, 3402–3407 (2007).

    Article  CAS  Google Scholar 

  9. Barnes, B.J., Moore, P.A. & Pitha, P.M. Virus-specific activation of a novel interferon regulatory factor, IRF-5, results in the induction of distinct interferon α genes. J. Biol. Chem. 276, 23382–23390 (2001).

    Article  CAS  Google Scholar 

  10. Barnes, B.J., Kellum, M.J., 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  Google Scholar 

  11. Mancl, M.E. et al. Two discrete promoters regulate the alternatively spliced human interferon regulatory factor-5 isoforms. Multiple isoforms with distinct cell type-specific expression, localization, regulation, and function. J. Biol. Chem. 280, 21078–21090 (2005).

    Article  CAS  Google Scholar 

  12. Mori, T. et al. Identification of the interferon regulatory factor 5 gene (IRF-5) as a direct target for p53. Oncogene 21, 2914–2918 (2002).

    Article  CAS  Google Scholar 

  13. Graham, R.R. et al. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat. Genet. 38, 550–555 (2006).

    Article  CAS  Google Scholar 

  14. Sigurdsson, S. et al. Polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associated with systemic lupus erythematosus. Am. J. Hum. Genet. 76, 528–537 (2005).

    Article  CAS  Google Scholar 

  15. Miceli-Richard, C. et al. Association of an IRF5 gene functional polymorphism with Sjogren's syndrome. Arthritis Rheum. 56, 3989–3994 (2007).

    Article  CAS  Google Scholar 

  16. Kristjansdottir, G. et al. Interferon regulatory factor 5 (IRF5) gene variants are associated with multiple sclerosis in three distinct populations. J. Med. Genet. 45, 362–369 (2008).

    Article  CAS  Google Scholar 

  17. Dideberg, V. et al. An insertion-deletion polymorphism in the interferon regulatory factor 5 (IRF5) gene confers risk of inflammatory bowel diseases. Hum. Mol. Genet. 16, 3008–3016 (2007).

    Article  CAS  Google Scholar 

  18. Hu, G. & Barnes, B.J. Interferon regulatory factor-5-regulated pathways as a target for colorectal cancer therapeutics. Expert Rev. Anticancer Ther. 6, 775–784 (2006).

    Article  CAS  Google Scholar 

  19. 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  Google Scholar 

  20. Panne, D., Maniatis, T. & Harrison, S.C. Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-β enhancer. EMBO J. 23, 4384–4393 (2004).

    Article  CAS  Google Scholar 

  21. Escalante, C.R., Nistal-Villan, E., Shen, L., Garcia-Sastre, A. & Aggarwal, A.K. Structure of IRF-3 bound to the PRDIII-I regulatory element of the human interferon-β enhancer. Mol. Cell 26, 703–716 (2007).

    Article  CAS  Google Scholar 

  22. Panne, D., Maniatis, T. & Harrison, S.C. An atomic model of the interferon-β enhanceosome. Cell 129, 1111–1123 (2007).

    Article  CAS  Google Scholar 

  23. 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  Google Scholar 

  24. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Sato, M., Tanaka, N., Hata, N., Oda, E. & Taniguchi, T. Involvement of the IRF family transcription factor IRF-3 in virus-induced activation of the IFN-β gene. FEBS Lett. 425, 112–116 (1998).

    Article  CAS  Google Scholar 

  28. 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  Google Scholar 

  29. 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  Google Scholar 

  30. Takahasi, K. et al. X-ray crystal structure of IRF-3 and its functional implications. Nat. Struct. Biol. 10, 922–927 (2003).

    Article  CAS  Google Scholar 

  31. Qin, B.Y. et al. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation. Nat. Struct. Biol. 10, 913–921 (2003).

    Article  CAS  Google Scholar 

  32. Qin, B.Y. et al. Crystal structure of IRF-3 in complex with CBP. Structure 13, 1269–1277 (2005).

    Article  CAS  Google Scholar 

  33. Cheng, T.F. et al. Differential activation of IFN regulatory factor (IRF)-3 and IRF-5 transcription factors during viral infection. J. Immunol. 176, 7462–7470 (2006).

    Article  CAS  Google Scholar 

  34. Wu, Y., Vadrevu, R., Kathuria, S., Yang, X. & Matthews, C.R. A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the α subunit of tryptophan synthase, a TIM barrel protein. J. Mol. Biol. 366, 1624–1638 (2007).

    Article  CAS  Google Scholar 

  35. Aurora, R. & Rose, G.D. Helix capping. Protein Sci. 7, 21–38 (1998).

    Article  CAS  Google Scholar 

  36. 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  Google Scholar 

  37. Chen, W. et al. Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3. J. Mol. Biol. 379, 251–260 (2008).

    Article  CAS  Google Scholar 

  38. Panne, D., McWhirter, S.M., Maniatis, T. & Harrison, S.C. Interferon regulatory factor 3 is regulated by a dual phosphorylation-dependent switch. J. Biol. Chem. 282, 22816–22822 (2007).

    Article  CAS  Google Scholar 

  39. Mori, M. et al. Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation. J. Biol. Chem. 279, 9698–9702 (2004).

    Article  CAS  Google Scholar 

  40. 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  Google Scholar 

  41. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J. Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin α. Cell 94, 193–204 (1998).

    Article  CAS  Google Scholar 

  42. Cutress, M.L., Whitaker, H.C., Mills, I.G., Stewart, M. & Neal, D.E. Structural basis for the nuclear import of the human androgen receptor. J. Cell Sci. 121, 957–968 (2008).

    Article  CAS  Google Scholar 

  43. Stewart, M. Molecular mechanism of the nuclear protein import cycle. Nat. Rev. Mol. Cell Biol. 8, 195–208 (2007).

    Article  CAS  Google Scholar 

  44. Wiseman, T., Williston, S., Brandts, J.F. & Lin, L.N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137 (1989).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  46. Terwilliger, T.C. SOLVE and RESOLVE: automated structure solution and density modification. Methods Enzymol. 374, 22–37 (2003).

    Article  CAS  Google Scholar 

  47. Brunger, 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  CAS  Google Scholar 

  48. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  49. Winn, M.D., Isupov, M.N. & Murshudov, G.N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57, 122–133 (2001).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

Download references

Acknowledgements

This paper is dedicated to the memory of K. Lin, whose guidance initiated our investigations. We thank B. Hilbert for technical assistance and R. Gilmore, M. Munson and M. Moore for helpful comments on the manuscript. This work was supported by US National Institutes of Health (NIH) grants AI056080 (W.E.R. and C.A.S.) and AI067497 (K.A.F.). We thank the staff of National Synchrotron Light Source beamline X29. Financial support for this facility comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources of the NIH. All analytical ultracentrifugation runs were done in the University of Mississippi Medical Center Analytical Ultracentrifugation Facility.

Author information

Author notes

  1. Kai Lin: Deceased.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, 364 Plantation Street, Worcester, 01605, Massachusetts, USA

    Weijun Chen, Suvana S Lam, Hema Srinath, Celia A Schiffer, Kai Lin & William E Royer Jr

  2. Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, 01605, Massachusetts, USA

    Zhaozhao Jiang & Katherine A Fitzgerald

  3. Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, 39216, Mississippi, USA

    John J Correia

Authors
  1. Weijun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suvana S Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hema Srinath
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhaozhao Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John J Correia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Celia A Schiffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Katherine A Fitzgerald
    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

  9. William E Royer Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.L., W.C., W.E.R., C.A.S., J.J.C. and K.A.F. designed the experiments; W.C., S.S.L. and H.S. prepared, characterized and crystallized the protein; W.C. and C.A.S. carried out and analyzed the ITC experiments; W.C. and W.E.R. performed the crystallographic analysis; Z.J. and K.A.F. performed and analyzed the cell-based assays; J.J.C. carried out and analyzed the sedimentation experiments; W.E.R., W.C., K.A.F., C.A.S. and J.J.C. prepared the manuscript.

Corresponding author

Correspondence to William E Royer Jr.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Methods (PDF 421 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, W., Lam, S., Srinath, H. et al. Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5. Nat Struct Mol Biol 15, 1213–1220 (2008). https://doi.org/10.1038/nsmb.1496

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1496

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