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• W2150167566 abstract "Transcriptional activation of interferon β (IFN-β), an antiviral cytokine, requires the assembly of IRF-3 and CBP/p300 at the promoter region of the IFN-β gene. The crystal structure of IRF-3 in complex with CBP reveals that CBP interacts with a hydrophobic surface on IRF-3, which in latent IRF-3 is covered by its autoinhibitory elements. This structural organization suggests that virus-induced phosphoactivation of IRF-3 triggers unfolding of the autoinhibitory elements and exposes the same hydrophobic surface for CBP interaction. The structure also reveals that the interacting CBP segment can exist in drastically different conformations, depending on the identity of the associating transcription cofactor. The finding suggests a possible regulatory mechanism in CBP/p300, by which the interacting transcription factor can specify the coactivator’s conformation and influence the transcriptional outcome. Transcriptional activation of interferon β (IFN-β), an antiviral cytokine, requires the assembly of IRF-3 and CBP/p300 at the promoter region of the IFN-β gene. The crystal structure of IRF-3 in complex with CBP reveals that CBP interacts with a hydrophobic surface on IRF-3, which in latent IRF-3 is covered by its autoinhibitory elements. This structural organization suggests that virus-induced phosphoactivation of IRF-3 triggers unfolding of the autoinhibitory elements and exposes the same hydrophobic surface for CBP interaction. The structure also reveals that the interacting CBP segment can exist in drastically different conformations, depending on the identity of the associating transcription cofactor. The finding suggests a possible regulatory mechanism in CBP/p300, by which the interacting transcription factor can specify the coactivator’s conformation and influence the transcriptional outcome. A key event in mounting the mammalian innate immune response upon virus infection is the transcriptional activation of the antiviral cytokine, interferon β (IFN-β) (Barnes et al., 2002Barnes B. Lubyova B. Pitha P.M. On the role of IRF in host defense.J. Interferon Cytokine Res. 2002; 22: 59-71Crossref PubMed Scopus (257) Google Scholar, Maniatis et al., 1998Maniatis T. Falvo J.V. Kim T.H. Kim T.K. Lin C.H. Parekh B.S. Wathelet M.G. Structure and function of the interferon-β enhanceosome.Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 609-620Crossref PubMed Scopus (305) Google Scholar, Taniguchi et al., 2001Taniguchi T. Ogasawara K. Takaoka A. Tanaka N. IRF family of transcription factors as regulators of host defense.Annu. Rev. Immunol. 2001; 19: 623-655Crossref PubMed Scopus (1218) Google Scholar, Taniguchi and Takaoka, 2002Taniguchi T. Takaoka A. The interferon-α/β system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors.Curr. Opin. Immunol. 2002; 14: 111-116Crossref PubMed Scopus (387) Google Scholar). The pathway leading to IFN-β expression is guarded by IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors (Hiscott et al., 1999Hiscott J. Pitha P. Genin P. Nguyen H. Heylbroeck C. Mamane Y. Algarte M. Lin R. Triggering the interferon response: the role of IRF-3 transcription factor.J. Interferon Cytokine Res. 1999; 19: 1-13Crossref PubMed Scopus (185) Google Scholar, Servant et al., 2002Servant M.J. Grandvaux N. Hiscott J. Multiple signaling pathways leading to the activation of interferon regulatory factor 3.Biochem. Pharmacol. 2002; 64: 985-992Crossref PubMed Scopus (128) Google Scholar, Yoneyama et al., 2002Yoneyama M. Suhara W. Fujita T. Control of IRF-3 activation by phosphorylation.J. Interferon Cytokine Res. 2002; 22: 73-76Crossref PubMed Scopus (129) Google Scholar). In the absence of virus, IRF-3 is localized in the cytoplasm in a latent form. Virus infection induces IRF-3 phosphorylation and translocation into the nucleus, where it forms a nucleoprotein complex with the closely related coactivators CREB binding protein (CBP) or p300 at the promoter region of the IFN-β gene to activate transcription (Lin et al., 1998Lin 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. 1998; 18: 2986-2996Crossref PubMed Scopus (712) Google Scholar, Parekh and Maniatis, 1999Parekh B.S. Maniatis T. Virus infection leads to localized hyperacetylation of histones H3 and H4 at the IFN-β promoter.Mol. Cell. 1999; 3: 125-129Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, Wathelet et al., 1998Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Virus infection induces the assembly of coordinately activated transcription factors on the IFN-β enhancer in vivo.Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, Weaver et al., 1998Weaver 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. 1998; 18: 1359-1368Crossref PubMed Scopus (292) Google Scholar, Yoneyama et al., 1998Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. 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. 1998; 17: 1087-1095Crossref PubMed Scopus (667) Google Scholar). IRF-3 consists of an N-terminal DNA binding domain and a C-terminal IRF association domain (IAD). The IAD mediates phosphorylation-dependent homooligomerization and heterooligomerization with other IRF members and interacts with CBP/p300. A functional analysis of IRF-3 revealed an autoinhibitory mechanism, mediated by the combined action of sequences flanking the IAD (Lin et al., 1999Lin R. Mamane Y. Hiscott J. Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains.Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (258) Google Scholar). The virus-induced phosphoactivation of IRF-3, thought to be mediated by IKKϵ and/or TBK1, occurs at Ser/Thr sites located C-terminal of the IAD (Fitzgerald et al., 2003Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. IKKϵ and TBK1 are essential components of the IRF3 signaling pathway.Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (1897) Google Scholar, Lin et al., 1998Lin 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. 1998; 18: 2986-2996Crossref PubMed Scopus (712) Google Scholar, Sharma et al., 2003Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Triggering the interferon antiviral response through an IKK-related pathway.Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1262) Google Scholar, Yoneyama et al., 1998Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. 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. 1998; 17: 1087-1095Crossref PubMed Scopus (667) Google Scholar). Virus-induced phosphorylation consequently triggers IRF-3 oligomerization, nuclear translocation, and interaction with CBP/p300 to activate IFN-β expression. Consistent with the results from functional studies, the crystal structure of latent IRF-3 defines a network of intramolecular interactions between N- and C-terminally flanking autoinhibitory elements, which together cover a hydrophobic surface on the IAD (Qin et al., 2003Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (160) Google Scholar). The structure suggests that phosphorylation results in unfolding of the autoinhibitory structures, thereby opening the hydrophobic surface for functional interactions. A parallel structural study (Takahasi et al., 2003Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. et al.X-ray crystal structure of IRF-3 and its functional implications.Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (117) Google Scholar) opposed the existence of an autoinhibitory mechanism and suggested that the phosphorylated residues of IRF-3 directly participate in subunit-subunit contacts within an active dimer without disturbing the structure. The same study also suggested that CBP/p300 interacts with a large acidic surface on IRF-3, formed as a result of subunit dimerization. The closely related CBP and p300 are general transcriptional coactivators that differentially and combinatorially interact with various transcription factors and comodulators to activate gene expression (Blobel, 2002Blobel G.A. CBP and p300: versatile coregulators with important roles in hematopoietic gene expression.J. Leukoc. Biol. 2002; 71: 545-556PubMed Google Scholar, Chan and La Thangue, 2001Chan H.M. La Thangue N.B. p300/CBP proteins: HATs for transcriptional bridges and scaffolds.J. Cell Sci. 2001; 114: 2363-2373Crossref PubMed Google Scholar, Goodman and Smolik, 2000Goodman R.H. Smolik S. CBP/p300 in cell growth, transformation, and development.Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar, Janknecht and Hunter, 1996Janknecht R. Hunter T. Versatile molecular glue. Transcriptional control.Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The numerous protein-protein interaction domains in CBP/p300 are all capable of recognizing multiple transcription factors with no apparent sequence or structural similarities. Activation of a specific gene depends on the formation of a nucleoprotein complex between CBP/p300 and a select set of transcription factors at the promoter region of the target gene. The coactivator function of CBP/p300 also depends on its intrinsic acetyltansferase activity and interaction with other acetyltransferases. Acetylation of histones and transcription factors leads to chromatin relaxation and transcript factor activation. The IRF-3 binding domain (IBiD) of CBP/p300 has been mapped to a 46 residue segment within the C-terminal glutamine-rich region (Lin et al., 2001aLin C.H. Hare B.J. Wagner G. Harrison S.C. Maniatis T. Fraenkel E. A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.Mol. Cell. 2001; 8: 581-590Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The IBiD can also interact with several other comodulators, such as Ets-2, the adenoviral oncoprotein E1A, the nuclear receptor coactivator protein ACTR, an IRF homolog encoded by the Kaposi’s sarcoma-associated herpesvirus, and p53 (Lin et al., 2001aLin C.H. Hare B.J. Wagner G. Harrison S.C. Maniatis T. Fraenkel E. A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.Mol. Cell. 2001; 8: 581-590Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, Lin et al., 2001bLin R. Genin P. Mamane Y. Sgarbanti M. Battistini A. Harrington Jr., W.J. Barber G.N. Hiscott J. HHV-8 encoded vIRF-1 represses the interferon antiviral response by blocking IRF-3 recruitment of the CBP/p300 coactivators.Oncogene. 2001; 20: 800-811Crossref PubMed Scopus (179) Google Scholar, Livengood et al., 2002Livengood J.A. Scoggin K.E. Van Orden K. McBryant S.J. Edayathumangalam R.S. Laybourn P.J. Nyborg J.K. p53 Transcriptional activity is mediated through the SRC1-interacting domain of CBP/p300.J. Biol. Chem. 2002; 277: 9054-9061Crossref PubMed Scopus (49) Google Scholar, Matsuda et al., 2004Matsuda S. Harries J.C. Viskaduraki M. Troke P.J. Kindle K.B. Ryan C. Heery D.M. A conserved α-helical motif mediates the binding of diverse nuclear proteins to the SRC1 interaction domain of CBP.J. Biol. Chem. 2004; 279: 14055-14064Crossref PubMed Scopus (27) Google Scholar). The NMR structures of the uncomplexed IBiD and a complex between the IBiD and ACTR have been determined (Demarest et al., 2002Demarest S.J. Martinez-Yamout M. Chung J. Chen H. Xu W. Dyson H.J. Evans R.M. Wright P.E. Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.Nature. 2002; 415: 549-553Crossref PubMed Scopus (337) Google Scholar, Lin et al., 2001aLin C.H. Hare B.J. Wagner G. Harrison S.C. Maniatis T. Fraenkel E. A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.Mol. Cell. 2001; 8: 581-590Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Both structures reveal a helical framework of the IBiD. However, the conformations of the IBiD in the two structures are different, suggesting that the IBiD undergoes a conformational transition upon interaction with a comodulator. The structural basis for the versatility of the IBiD in comodulator interactions is not known. Here, we report the crystal structure of a complex between the IBiD of CBP and the IAD of IRF-3 at 2.4 Å resolution. The structure reveals that the IBiD binds to a hydrophobic surface on IAD, which in latent IRF-3 is buried by the autoinhibitory elements. The structure further reveals a novel, to our knowledge, conformation of the IBiD that is markedly different from that of the free IBiD or the IBiD in the ACTR/IBiD complex. The result suggests a possible regulatory mechanism in CBP/p300, by which binding of a transcriptional comodulator at the IBiD can stabilize CBP/p300 in one of its multiple conformations to influence the transcriptional outcome. The C-terminal transactivation domain of IRF-3 consists of a central IAD flanked on both sides by autoinhibitory sequences (Lin et al., 1999Lin R. Mamane Y. Hiscott J. Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains.Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (258) Google Scholar, Qin et al., 2003Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (160) Google Scholar). To form a stable complex between IRF-3 and the IBiD, it was necessary to use an IRF-3 construct, IRF-3(173-394), without the C-terminal autoinhibitory sequence (data not shown). The crystal structure contains two copies of the IRF-3/IBiD complex in the asymmetric unit (Table 1). The two copies are related by a 2-fold axis, and they have essentially the same structure with a root mean square deviation of 0.1 Å, when superimposed over all atoms. Only one complex is described here since there is no evidence that the interactions between the two copies are physiologically relevant. Analytical ultracentrifugation analysis shows that the exact IRF-3/IBiD complex used in the structural study does not oligomerize in solution (Figure 1A and Figures S1 and S2; see the Supplemental Data available with this article online).Table 1Summary of Crystal Analysis for the IRF3/IBiD ComplexParameterIRF-3/IBiDCrystal Parameters and Crystallographic DataSpace groupP6(1)Unit cell dimensionsa = b = 81.7, c = 242.1Diffraction limit (Å)aValues in brackets are for the highest-resolution shell.2.37 (2.41–2.37)Total reflections122,107Unique reflections35,879Completeness (%)96.0 (98.4)Intensity/σ14.4 (2.0)Rmerge (%)bRmerge = ∑ |Ihkl − <Ihkl>|/∑ Ihkl.7.8 (44.7)Refinement StatisticsProtein atoms3,720Water molecules418R factor (%)cR factor = ∑hkl ||Fobs| − |Fcalc||/∑hkl|Fobs| for all data.21.5Rfree (%)dRfree = ∑hkl ||Fobs| − |Fcalc||/∑hkl|Fobs| for 10% of the data not used in refinement.22.6Rmsd from ideal Bond (Å)0.007 Bond (°)1.3Ramachandran Core (%)89.1 Allow (%)10.9B factor average (Å2) Chain A (IRF-3)63.6 Chain B (IRF-3)63.5 Chain C (the IBiD)69.0 Chain D (the IBiD)68.7a Values in brackets are for the highest-resolution shell.b Rmerge = ∑ |Ihkl − <Ihkl>|/∑ Ihkl.c R factor = ∑hkl ||Fobs| − |Fcalc||/∑hkl|Fobs| for all data.d Rfree = ∑hkl ||Fobs| − |Fcalc||/∑hkl|Fobs| for 10% of the data not used in refinement. Open table in a new tab The IAD segment of IRF-3 and the IBiD segment of CBP form a 1:1 complex through interactions primarily between α helices (Figure 1B). The structure of IRF-3 consists of a β sandwich core capped by α helices and loops. Helices H3 and H4 of IRF-3, located on one end of the structural core, form the major interaction surface for the IBiD. The IBiD consists of three α helices (H1, H2, and H3) and a loop (L1), forming an approximately rectangular framework, which interacts with IRF-3 via a flat surface. In the crystal structure of latent IRF-3, the H3 and H4 helices are covered by concerted intramolecular interactions of the autoinhibitory elements, consisting of the N-terminal H1 helix and the C-terminal β12-L6-β13-H5 structure (Figure 1C) (Qin et al., 2003Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (160) Google Scholar). In the IRF-3/IBiD complex, the IBiD covers the same surface as the autoinhibitory elements in the latent IRF-3 structure (compare Figures 1B and 1C). These observations indicate that the autoinhibitory conformation of latent IRF-3 and the interaction of IRF-3 with the IBiD are mutually exclusive. The association between IRF-3 and the IBiD covers a hydrophobic interface of approximately 1800 Å2. The interacting hydrophobic residues of IRF-3 involve primarily Ile326, Leu329, and Ile330 in the H3 helix, as well as Cys371, Leu372, Leu375, Met378, and Val381 in the H4 helix (Figure 1D, right). In addition, Trp202 on the β1 strand also contributes to the buried hydrophobic interface. The interacting hydrophobic residues of the IBiD are distributed over three helices, involving primarily Leu2071 and Leu2075 in the H1 helix; Val2087, Ile2090, and Leu2091in the H2 helix; as well as Leu2097 and Phe2101 in the H3 helix (Figure 1D, left). In addition to these core hydrophobic contacts, the complex is also stabilized by a limited set of H bond interactions on the outskirts of the interface (Figure 1D). The Glu203 side chain of IRF-3 forms an H bond with the Ser2080 side chain of CBP, and the Ser221 side chain of IRF-3 forms an H bond with the Gln2086 side chain of CBP. The Glu201 side chain of IRF-3, although not involved in H bonding interactions, forms a nice van der Waals contact with the Gln2086 side chain of CBP. To verify that the peptide conformation of the IBiD observed in the crystal structure corresponds to its interaction with IRF-3 in vivo, we performed coimmunoprecipitations of endogenous CBP with ectopically expressed full-length wild-type or mutant IRF-3 in the absence or presence of Sendai viral infection (Figure 2A ). Virus infection results in IRF-3 activation and consequent stabilization of the IRF-3/CBP interaction. The H3A and H4A mutants contained mutations of the hydrophobic residues in the H3 and H4 helices, respectively, of IRF-3 and were designed to disrupt the hydrophobic interaction with CBP. The SA and EA mutants of IRF-3 were designed to disrupt the polar contacts with CBP, whereas the SA mutant had Ser221 mutated to alanine, and the EA mutant had both Glu201 and Glu203 mutated to alanine. While Myc-tagged, wild-type IRF-3 exhibited some interaction with CBP, which was substantially enhanced by viral infection, the mutants, with the exception of IRF-3(SA), did not detectably interact with CBP in the presence or absence of virus infection. The IRF-3(SA) mutant showed a low-level interaction with CBP in the presence of virus, but no detectable interaction in the absence of virus. These results are consistent with the structure and support that the hydrophobic contacts mediated by the H3 and H4 helices, and the polar contacts mediated by Ser221 and Glu203 of IRF-3, are involved in the interaction of activated IRF-3 with CBP in vivo. The reported substantial decrease in the transactivation activity of IRF-3 resulting from mutations in the H3 and H4 helices (Qin et al., 2003Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (160) Google Scholar) may therefore be caused by disruption of the interaction between IRF-3 and CBP/p300. To further verify that IRF-3(173-394) used in the structural study faithfully mimics the interaction of full-length IRF-3 with CBP, we performed a competition experiment to assess whether IRF-3(173-394) can compete with Sendai virus-activated, full-length IRF-3 for CBP interaction. As shown in Figure 2B, increasing amounts of IRF-3(173-394) decreased the virus-induced association of full-length IRF-3 with endogenous CBP, strongly suggesting that IRF-3(173-394) exhibits the same mode of binding to endogenous CBP as activated full-length IRF-3. Since IRF-3(173-394) cannot bind to the promoter DNA due to the absence of its DNA binding domain, its interaction with CBP does not confer transcription activity (data not shown) and should allow for competitive interference with IRF-3-driven transcription through CBP/p300. Accordingly, coexpression of IRF-3(173-394) decreased the virus-induced transcription activity of IRF-3 from an IRF-3 binding promoter (Figure 2C), demonstrating that IRF-3(173-394) functionally competes with activated IRF-3 for CBP interaction. Finally, the effects of subunit interface mutations on the interaction between IRF-3(173-394) and CBP were tested (Figure 2D). The same mutations were generated in IRF-3(173-394) as in full-length IRF-3 (Figure 2A); however, as the inhibitory domain was removed in this construct, no viral activation was required for the interaction of IRF-3(173-394). Coimmunoprecipitation experiments showed that IRF-3(173-394) interacted well with endogenous CBP, and that this interaction was abolished by the interface mutations. As with full-size IRF-3(SA) following viral activation (Figure 2A), IRF-3(173-394, SA) showed a low-level interaction with CBP (Figure 2D). All of these functional studies indicate that the structure of the IRF-3/IBiD complex is physiologically relevant. There is a remarkable similarity between the interface of the IRF-3/IBiD complex and the interface buried by the autoinhibitory elements in latent IRF-3. This results from a similar spatial display of the interacting side chains in the IBiD and the IRF-3 autoinhibitory elements, despite their distinct secondary structural arrangements (Figure 1D, left). Specifically, T2074 in the H1 helix of the IBiD occupies the same space as N389 in the autoinhibitory structure; Q2083, V2087, I2090, L2091, and N2094 in the H2 helix of the IBiD occupy the same space as H394, L393, L195, L192, and L415, respectively, in the autoinhibitory structure; L2097 and F2101 in the H3 helix of the IBiD occupy the same space as L412 and Y408, respectively, in the autoinhibitory structure. In addition to the above-mentioned pairs of matching residues, L2071 in the IBiD and V391 in the autoinhibitory structure occupy a distinct, nonoverlapping space, suggesting that a minor structural rearrangement on the complementary interface of IRF-3 is required in the transition from the autoinhibited to the IBiD bound state. Indeed, the side chain of M378 in the H4 helix of IRF-3 needs to undergo a rotameric rearrangement to form a complex with the IBiD (Figure 1D, right). The mutually exclusive nature of the IRF-3 autoinhibitory conformation and the IRF-3/IBiD interaction suggests that the autoinhibitory structures of IRF-3 need to be displaced upon activation. Upon virus infection, the IKK-related kinases, IKKϵ and/or TBK1, activate latent IRF-3 through phosphorylation of defined Ser/Thr residues within the C-terminal autoinhibitory elements (Fitzgerald et al., 2003Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. IKKϵ and TBK1 are essential components of the IRF3 signaling pathway.Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (1897) Google Scholar, Sharma et al., 2003Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Triggering the interferon antiviral response through an IKK-related pathway.Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1262) Google Scholar). The crystal structure of latent IRF-3 revealed that these phosphorylation sites are partially buried in a hydrophobic environment (Figure 1C) (Qin et al., 2003Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (160) Google Scholar). A plausible activation mechanism, consistent with the structures, is that the repulsive force between the phosphorylated residues and their hydrophobic environment leads to unfolding of the autoinhibitory elements and consequent exposure of the H3 and H4 helices for interaction with the IBiD. The autoinhibitory elements of IRF-3 are not observed in the IRF-3/IBiD complex, due to exclusion of the C-terminal autoinhibitory elements in the expression construct, and the structural disordering of the N-terminal autoinhibitory element. The L5 loop of IRF-3 in complex with the IBiD assumes a different structural arrangement when compared to that in the autoinhibited IRF-3 (compare Figures 1B and 1C). Although a definitive function of the L5 loop has not been assigned, mutation of the basic residues K360 and R361 within the loop disables the virus-induced IRF-3 oligomerization and transcriptional activation, suggesting that the L5 loop is important for activation (Qin et al., 2003Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (160) Google Scholar). It is not obvious whether the observed structural change of the L5 loop has functional implication since the L5 loop also forms part of the crystal packing contacts in the IRF-3/IBiD complex. The structure of the uncomplexed IBiD has been investigated by NMR. In one study, the free IBiD forms a small globular domain with three interacting helices (Lin et al., 2001aLin C.H. Hare B.J. Wagner G. Harrison S.C. Maniatis T. Fraenkel E. A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.Mol. Cell. 2001; 8: 581-590Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, another study suggested that the free IBiD contains a helical secondary structure, but no fixed tertiary structure, characteristic of a molten globule (Demarest et al., 2004Demarest S.J. Deechongkit S. Dyson H.J. Evans R.M. Wright P.E. Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein.Protein Sci. 2004; 13: 203-210Crossref PubMed Scopus (55) Google Scholar). Thus, the reported structure of the free IBiD, which is used for comparison here, may represent the more stable form among the many possible conformations that can exist in solution. The IBiD in the IRF-3/IBiD complex has a different conformation when compared to the uncomplexed IBiD structure (Figure 3). The H2 helix in the IRF-3/IBiD complex is longer, as a consequence of a disorder-to-order transition of a glutamine-rich segment from residue 2079 to residue 2085 upon complex formation. The ordering of this region correlates with its direct contact with IRF-3. There is also a significant change in the relative positions of the H1 and H2 helices. When viewed through the plane formed by the H2 and H3 helices, the H1 helix in the IRF-3/IBiD complex points toward the opposite side of the plane as compared to that in the uncomplexed structure (Figure 3, right). These structural rearrangements of the IBiD make available a cluster of the above-mentioned hydrophobic side chains that are important for interaction with IRF-3 but are otherwise buried in an intramolecular hydrophobic core in the uncomplexed structure. As mentioned, the IBiD can interact with diverse transcriptional partners with no apparent sequence or structural homology. In addition to the IRF-3/IBiD complex structure described here, an NMR structure of a complex between the IBiD and the steroid hormone receptor coactivator ACTR has been reported (Demarest et al., 2002Demarest S.J. Martinez-Yamout M. Chung J. Chen H. Xu W. Dyson H.J. Evans R.M. Wright P.E. Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators.Nature. 2002; 415: 549-553Crossref PubMed Scopus (337) Google Scholar). Comparison of both the IBiD complex structures demonstrates different IBiD conformations dictated by the interacting transcriptional partner. Unlike the contacts of the IBiD with IRF-3 through a planar interface, the IBiD forms a V-shaped pocket to embrace ACTR, with the H3 helix lining up one side of the pocket and the H1-L1-H2 structure capping the other side (Figure 4). There is a marked difference in the relative position of the N and C termini between the two drastically different conformations of the IBiD. Since the IBiD is an internal segment of CBP, these observations suggest that the differences in the IBiD structures, depending upo" @default.
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