Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Nov;15(11):1213-20.
doi: 10.1038/nsmb.1496. Epub 2008 Oct 5.

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

Weijun Chen  1 , Suvana S Lam, Hema Srinath, Zhaozhao Jiang, John J Correia, Celia A Schiffer, Katherine A Fitzgerald, Kai Lin, William E Royer Jr
Affiliations

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

Weijun Chen et al. Nat Struct Mol Biol. 2008 Nov.

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Ribbon diagrams of the C-terminal transactivation domain of IRF5 and IRF3. Monomers from three crystal structures have been aligned using 138 residues of the IAD (cyan). The rest of the IAD is in green, the C-terminal autoinhibitory region is in magenta and putative phosphorylation sites are shown as yellow balls. (a) IRF5 (222-267) S430D has a very extended C-terminal region that participates in dimer formation. This leaves most of the CBP binding site, formed by helices 3 and 4, exposed to solvent except for that covered by helix 1. (b) In the autoinhibited IRF3 (173-427) monomer, the autoinhibitory C-terminal region binds to and masks the CBP binding site . (c) In the complex of IRF3 (173-394) with CBP (2067-2112), CBP (gold) binds to helices 3 and 4, which were exposed by the removal of residues 395-427, and displaces the N-terminal helix 1 .
Figure 2
Figure 2
IRF5 dimer. (a) A ribbon diagram of the crystallographic IRF5 dimer is shown with one subunit in blue and one in green. Putative phosphorylation sites are shown as yellow balls. The C-terminal region lays across the surface of the second subunit making extensive contacts particularly involving helix 4, helix 5 and the interhelical region interacting with helix 2′ and loops L1′, L3′ and L5′. (Primes designate the second subunit.) (b) An expanded view of the major interacting region between subunits, rotated from a. Color code is the same as in a with key side chains shown, including those of putative phosphorylation sites with yellow carbon atoms. A hydrophobic patch on Helix 2′, formed from residues Y303′, L307′, V310′ and L403′ interacts with residues I431, L433 and I435 from the extended peptide between helices 4 and 5. Ionic interactions are contributed by residues in helix 5, including D442 with R353′, K441 with E354′ and K449 with D309′ and D312′; likely hydrogen bonds are shown as dashed lines. (c) Interface helix capping of helices 3 and 4. K401′ forms a likely hydrogen bond with the F372 carbonyl at the C-terminus of helix 3. Q373 forms a likely intrasubunit hydrogen bond with the F420 carbonyl at the C-terminus of helix 4, whereas R247′ forms a likely hydrogen bond with the main-chain carbonyl oxygen of S421. These interactions may stabilize the shorter length of helix 4 in dimeric IRF5 compared with monomeric IRF3. (d) Fo-Fc simulated annealing omit map showing the dimeric interactions of S436 with R328’ and D442 with R353′, whose atoms were removed from the atomic model for the simulated annealing process. Electron density is shown at the 3σ level. The interaction between R328′ and S436 is likely to be strengthened by phosphorylation of S436.
Figure 3
Figure 3
Structure guided mutagenesis of IRF5 and IRF3. (a) Gel exclusion chromatography on IRF5 dimeric interface mutants in the presence of CBP, all of which run as monomers in contrast to IRF5 S430D. (b) Gel exclusion chromatography on IRF3 (173-427) mutants in the background of the IRF3 S386D/S396D phosphomimetic, showing R285E and L362E disrupt dimer formation. (c) IFNβ luciferase activity measured from HEK293 cells transfected with the IFNβ reporter gene, TK-renilla, WT or mutants of IRF5 and increasing concentrations of MyD88 (20, 40, 80ng). (d) For IRF3 HEK assays, cells were transfected with the IFNβ reporter, TK-renilla and IRF3 as indicated and either uninfected (mock) or infected with NDV (8HAU/ml) for 16hrs prior to measuring luciferase activity. Western blot analysis of transfected proteins is shown in panel insets for both IRF5 (c) and IRF3 (d).(e) IRF3-deficient fibroblasts were transfected with WT or mutant versions of IRF3 and IFNβ reporter gene activity measured in uninfected cells (mock) or after NDV infection. In all cases, data are expressed as fold induction relative to the reporter-only control and are the mean ± SD.
Figure 4
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. Unphosphorylated IRF proteins generally remain in the cytoplasm in an autoinhibited state, with the CBP/p300 binding site masked. Phosphorylation induces an unfolding of the C-terminal region that triggers its assembly with another IRF to form a dimer and unmask the CBP binding site. Phosphorylated IRF dimers are translocated into the nucleus, where they interact with CBP/p300 and other transcription factors to form complexes that bind to the IFN-β enhancer region as well as other promoters. The DBDs bind to DNA in a tandem, rather than a two-fold, arrangement requiring that the two-fold symmetry of the IAD does not extend to the DBDs when bound to DNA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Honda K, Taniguchi T. IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol. 2006;6:644–58. - PubMed
    1. Paun A, Pitha PM. The IRF family, revisited. Biochimie. 2007;89:744–53. - PMC - PubMed
    1. Tamura T, Yanai H, Savitsky D, Taniguchi T. The IRF Family Transcription Factors in Immunity and Oncogenesis. Annu Rev Immunol. 2008;26:535–84. - PubMed
    1. Schoenemeyer A, et al. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J Biol Chem. 2005;280:17005–12. - PubMed
    1. 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. 2007;178:6876–85. - PubMed

Publication types

Associated data