The innate immune system is the first line of defense against bacterial and viral infections. The recognition of pathogen-associated molecular patterns by the RIG-I–like receptors, TLRs, and cGAS leads to the induction of IFN-I by activating the transcription factor IRF-3. Although the mechanism of IRF-3 activation has been extensively studied, the structural basis of IRF-3 activation upon phosphorylation is not fully understood. In this study, we determined the crystal structures of phosphorylated human and mouse IRF-3 bound to CREB-binding protein (CBP), which reveal that phosphorylated IRF-3 forms a dimer via pSer386 (pSer379 in mouse IRF-3) and a downstream pLxIS motif. Size-exclusion chromatography and cell-based studies show that mutations of key residues interacting with pSer386 severely impair IRF-3 activation and IFN-β induction. By contrast, phosphorylation of Ser396 within the pLxIS motif of human IRF-3 only plays a moderate role in IRF-3 activation. The mouse IRF-3/CBP complex structure reveals that the mechanism of mouse IRF-3 activation is similar but distinct from human IRF-3. These structural and functional studies reveal the detailed mechanism of IRF-3 activation upon phosphorylation.

The innate immune system detects the pathogen-associated molecular patterns, such as nucleic acids, LPS through pattern recognition receptors, which trigger the induction of a variety of cytokines, including IFN-I, to initiate host defense against pathogens (17). Viral or bacterial DNA in cytosol are recognized by cyclic GMP-AMP synthase (cGAS), which catalyzes the synthesis of a cyclic dinucleotide cyclic GMP-AMP (cGAMP). cGAMP binds to the adaptor stimulator of IFN genes (STING) and mediates the recruitment and activation of TANK-binding kinase 1 (TBK1) and IFN regulatory factor (IRF) 3 (5, 8). Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene with other transcription factors, such as NF-κB (7, 913). dsRNA and LPS can be recognized by retinoic acid-inducible protein 1 (RIG-I)–like receptors (RLRs) and TLRs, respectively, to induce the expression of IFN-Is through the adaptors mitochondrial antiviral signaling (MAVS) and TIR domain-containing adaptor-inducing IFN-β (TRIF). Interestingly, all three of these signaling pathways converge at the recruitment of IRF-3 via a conserved pLxIS motif (p, hydrophilic residue, x, any residue, S, phosphorylation site) within the adaptor proteins (913).

The IRF family transcription factors contain nine members (IRF-1 through IRF-9). These transcription factors contain a highly conserved N-terminal DNA-binding domain and a relatively divergent C-terminal regulatory domain, suggesting that most members function nonredundantly (14). IRF-3 is a key transcription factor that regulates the expression of IFN-I genes. Under resting conditions, IRF-3 adopts an autoinhibited conformation and is ubiquitously accumulated in the cytoplasm. Bacterial or viral infection triggers the activation of IRF-3 through various innate immune sensing pathways. Phosphorylated IRF-3 binds to p300/CREB-binding protein (CBP), translocates to the nucleus, and initiates the transcription of IFN-I genes (1416). Interestingly, IRF-7, which is closely related to IRF-3 in terms of the conserved regulatory domain, also plays an important role in regulating the expression of IFN-Is (17).

The mechanism of IRF-3 activation has been extensively studied, showing that IRF-3 is activated through phosphorylation of the C-terminal serine-rich repeat (16). Previous studies by the Fujita laboratory (1820) showed that the phosphorylation site 1 (residues Ser385 and Ser386 of human IRF-3 [hIRF-3]) plays a key role in IRF-3 activation. The phosphorylation of Ser386 induced by viral infection has been detected by a specific Ab. They also observed that the dimerization of IRF-3 was abolished by the mutation of Ser386. In addition, the Hiscott laboratory (21, 22) observed that the phosphorylation site 2, which includes residues Ser396, Ser398, Ser402, Thr404, and Ser405 of hIRF-3, plays a critical role in IRF-3 activation. The phosphomimetic mutation of these residues (IRF-3 5D) results in a constitutively active phenotype. Moreover, they observed that the S396D mutation alone induces IFN-I expression, suggesting that Ser396 also plays a critical role in IRF-3 activation. Another study by the Harrison laboratory (23) proposed a two-step phosphorylation and activation model, which suggests that phosphorylation at site 2 leads to the alleviation of IRF-3 autoinhibition that facilitates the phosphorylation at site 1 and eventually leads to the activation of IRF-3. Later on, studies by the Lin group (24) showed that IRF-3 mutant S386D/S396D bound to CBP forms a stable oligomer, suggesting that the phosphorylation of both Ser386 and Ser396 is essential for hIRF-3 activation. Based on these studies, we mutated Ser386 and Ser396 to glutamic acid in a truncated form of IRF-3 (residues 189–398) and determined the structure of the phosphomimetic IRF-3 in complex with CBP, revealing that the phosphomimetic IRF-3 mutant forms a dimer (12). However, the higher affinity between phosphorylated hIRF-3/CBP (phIRF-3/CBP) complex compared to that of the S386/396E mutant indicates that the phosphomimetic mutation does not fully recapitulate the interactions between phosphorylated IRF-3.

To elucidate the exact mechanism of IRF-3 activation upon phosphorylation by TBK1, we coexpressed truncated forms of hIRF-3 and mouse IRF-3 (mIRF-3) C-terminal domains with a CBP fragment, phosphorylated these complexes with TBK1, and determined the crystal structures of the phosphorylated IRF-3/CBP dimers. These structures reveal the molecular basis of IRF-3 activation upon phosphorylation. Biochemical and functional studies based on the structures show that mutations of the key residues mediating IRF-3 dimerization upon phosphorylation dramatically impair IRF-3 dimerization and IRF-3–mediated signaling.

The cDNA encoding hIRF-3 (residues 189–398) and mIRF-3 (residues 184–390) dimerization domains were cloned into a modified pET-28a (+) vector containing an N-terminal His6-SUMO tag with appropriate primers obtained from Integrated DNA Technologies. SUMO fusion of human CBP (residues 2065–2111) was cloned into the pET-22b (+) vector using appropriate primers from Integrated DNA Technologies. Sequences of all the constructs were confirmed by DNA sequencing. The plasmid containing hIRF-3 or mIRF-3 dimerization domain was cotransformed with CBP plasmid into Escherichia coli BL21 (DE3) cells. The cells were grown on Luria–Bertani agar plates containing both kanamycin and ampicillin. On the next day, the cell colonies from the plates were transferred to 6 liters of Luria–Bertani liquid medium in flasks with kanamycin and ampicillin in an incubator shaker at 37°C under 225 rpm. When OD600 reached ∼1.2, BL21 cells were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside overnight at 16°C. The cells were harvested by centrifugation at 4000 rpm for 10 min and then suspended in a 200-ml lysis buffer containing 300 mM NaCl and 50 mM Tris-HCl (pH 8). The cells were lysed by sonication for 10 min with a 0.5-s pulse and a 0.5-s rest, and the cell lysate was centrifuged at 16,000 rpm for 30 min. The supernatant was loaded onto a Ni2+-NTA column (QIAGEN). Then, 200 ml of washing buffer containing 500 mM NaCl, 20 mM Tris-HCl, 25 mM imidazole (pH 7.5) was used to wash nonspecific binding proteins off the Ni2+-NTA column. The target proteins were then eluted with 75 ml of elution buffer containing 150 mM NaCl, 20 mM Tris-HCl, and 250 mM imidazole (pH 7.5). The His6-SUMO tag was cleaved with SUMO protease at a concentration of 10 μg/ml at 4°C overnight and removed using an Ni2+-NTA column. The target proteins in the flow-through were centrifuged to ∼2 ml and further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 Column (GE Healthcare) equilibrated with a running buffer containing 150 mM NaCl and 20 mM Tris-HCl (pH 7.5). All the mutations were introduced into a full-length hIRF-3 pET-28a (+) plasmid using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) using proper primers. Sequences of all the plasmids were confirmed by DNA sequencing. The IRF-3 mutants were expressed and purified in the same way as the IRF-3 dimerization domain. Mouse TBK1 (mTBK1) was cloned into the pAcGHLTc vector with an N-terminal GST tag and a His6 tag. The 2-μg plasmid was transfected together with 2.5 μl of BaculoGold Bright Linearized baculovirus DNA (BD Biosciences) into Spodoptera frugiperda (sf9) insect cells to generate recombinant baculovirus. The recombinant viruses were amplified for at least two rounds (4–6 d/round) before the large-scale protein expression. The insect cells at a density of 2.5 × 106 cells/ml were infected with the TBK1 recombinant baculovirus, cultured at 27°C, and harvested 72 h postinfection by centrifugation at 3000 rpm for 10 min. The cells were lysed in a buffer containing 150 mM NaCl, 0.2 M Tris-HCl, 1% NP-40, and 1 mM PMSF (pH 8) in a shaker at 4°C for 2 h. The cell lysate was centrifuged at 16,000 rpm for 30 min. The GST-TBK1 protein in the supernatant was mixed with 6 ml Ni2+-NTA beads and incubated in a shaker at 4°C for 2 h. The beads were then spun down and washed three times using a buffer containing 500 mM NaCl, 20 mM Tris-HCl, and 25 mM imidazole (pH 7.5). The target protein was eluted with a buffer containing 150 mM NaCl, 20 mM Tris-HCl, and 250 mM imidazole (pH 7.5). The eluted protein was further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 200 column.

Purified full-length hIRF-3 proteins were mixed with GST-mTBK1 in a ratio of 10:1 (w/w) in a 1-ml reaction buffer with 20 mM HEPES (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 5 mM ATP, 0.1 mM Na3VO4, 5 mM NaF, and 5 mM DTT at 27°C for ∼24 h. The final concentration of the proteins was ∼1 mg/ml. After ∼24 h of incubation, the phosphorylated IRF-3 proteins were analyzed using a Superdex 200 (10/300 GL) column eluted with a buffer containing 20 mM Tris-HCl and 150 mM NaCl (pH 7.5).

Full-length wild-type (WT) IRF-3 and all the mutant proteins were purified using an Ni2+-NTA column followed by gel filtration chromatography, as described above. Ten micrograms of each sample was mixed with 5× loading dye of 250 mM Tris-HCl (pH 6.8), 10% SDS, 30% (v/v) glycerol, 10 mM DTT, and 0.05% (w/v) bromophenol blue and then resolved on 4–20% gradient gels in a buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS (pH 8.4) at 100 V for 1 h. The gel was stained with Coomassie blue for 1 h and destained with a solution containing H2O, methanol, and acetic acid in a ratio of 50:40:10 (v/v/v) until the bands were clearly seen. The gel image was taken using a Bio-Rad imager. For native gel electrophoresis, the purified proteins were phosphorylated with GST-mTBK1 using the method described above, and each of the phosphorylated proteins with nonphosphorylated WT was resolved on 10% native gels running in a buffer containing 25 mM Tris and 192 mM glycine (pH 8.4) at 4°C at 100 V for 30 min. The gels were stained and destained the same way as the gradient gels.

Purified hIRF-3 (residues 189–398) and mIRF3 (residues 184–390) bound to the CBP fragment (residues 2065–2111) were phosphorylated by GST-mTBK1. After 24 h of incubation, the proteins were purified using a HiLoad 16/60 Superdex 75 column eluted with 20 mM Tris-HCl and 150 mM NaCl (pH 7.5). The purified phosphorylated proteins were concentrated to a final concentration of ∼5 mg/ml. The crystallization screen was performed by hanging drop vapor diffusion technique at 4°C using Index, Crystal Screen and Crystal Screen 2 reagent kits from Hampton Research. Crystals of hIRF-3 bound to CBP were grown in 0.1 M sodium acetate (pH 5), 0.2 M MgCl2, and ∼5% polyethylene glycol 3350. Crystals of mIRF-3 in complex with CBP were grown in 0.2 M ammonium citrate tribasic (pH 7) with ∼12% polyethylene glycol 3350. The crystals were flash frozen in liquid nitrogen in the reservoir solution containing 25% (v/v) glycerol. Diffraction data were collected at the Advanced Light Source beamlines 5.0.1, using a Quantum 315r CCD detector. The diffraction data were indexed and integrated with iMosflm and merged with Aimless in the CCP4 package (25). The structures of the pIRF-3/CBP complex were determined by molecular replacement using the structure of our phosphomimetic IRF-3/CBP complex (Protein Data Bank [PDB] identification code: 5JEM) as the search model using Phaser in the Phenix package (26). The structures were manually rebuilt using Coot and refined with Phenix. Details of data quality and structure refinement are summarized in Table I. The structural figures were generated with PyMOL (https://www.pymol.org).

The cDNA encoding WT hIRF-3 was cloned into a pcDNA3.1(−) vector using appropriate primers. Mutants of hIRF-3 were generated using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). Sequences of the mutants were confirmed by DNA sequencing. HEK293T cells were plated in Costar White 96-Well Plates at 4 × 104 cells per well, and each well contained 100 μl DMEM (1×) plus GlutaMAX medium (Life Technologies) supplemented with 10% FBS (Life Technologies). After ∼24 h of incubation at 37°C, the cells were transfected with the IRF-3 plasmids (10 ng per transfection) using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM (Life Technologies) together with a constant amount of IFN-β firefly luciferase reporter plasmids (20 ng per transfection), phRL-TK–Renilla luciferase plasmids (2 ng per transfection) (Promega), and human STING plasmids (0.2 ng per transfection). Transfections with empty pcDNA3.1(−) and WT hIRF-3 with no STING plasmid were used as controls. The cells were incubated for another 24 h to allow for expression of the genes. The half of the cells in the plates were treated with 30 μg/ml cGAMP dissolved in DMEM (1×) plus GlutaMAX medium, and the other half were treated with the medium only. After ∼16 h of incubation, the cells were analyzed using the Dual-Glo Luciferase Reporter Assay kit (Promega). Luminescence was quantified with the BioTek Synergy HTX Multi-Mode Microplate Reader. The relative firefly luciferase activity was normalized by the Renilla luciferase activity. The relative IFN-β reporter fold of induction represents the ratio normalized to control plasmid values with the same treatment.

HEK293T cells were transfected with empty pcDNA3.1(−), WT IRF-3 or IRF-3 mutants together with human STING plasmid. The cells were stimulated by 30 μg/ml cGAMP added to the culture media 24 h posttransfection. After ∼16 h of incubation, the cells were washed and suspended in PBS and then lysed in 150 mM NaCl, 200 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1% Nonidet P-40 supplemented with one cOmplete Protease Inhibitor Cocktail EDTA-free Tablet (Roche) and one PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche) for each 10 ml of lysis buffer. The proteins were resolved on 10% SDS-PAGE at 100 V for 1.5 h and transferred to a PVDF membrane in a transfer buffer containing 1× Tris-glycine plus 20% methanol for another 1.5 h. Then, 5% milk in 1× PBS with Tween 20 (PBST) solution was used to block the membrane for 1 h, followed by three rinses with PBST. Next, the membrane was incubated with primary Abs dissolved in 1× PBST with 5% BSA overnight at 4°C. The membrane was washed three times using PBST on the next day and further incubated with the corresponding HRP-conjugated secondary Abs dissolved in PBST. The following Abs were used in the Western blot experiment: anti–IRF-3 (1:1000; sc-9082; Santa Cruz Biotechnology) and anti-actin (1: 4000; HHF35; Pierce). The proteins were visualized using the Western Lightening Plus-ECL (PerkinElmer) according to the manufacturer’s protocol. For the detection of phosphorylated IRF-3, half of the cells were transfected with empty pcDNA3.1(−), WT IRF-3 were stimulated by 30 μg/ml cGAMP, and the other half were treated with the medium only. Anti–IRF-3 p-Ser386 (1:2500; ab76493; Abcam) and anti–IRF-3 p-Ser396 (1:1000; 4947S; Cell Signaling Technology) were used.

HEK293T cells were grown on poly-l-lysine–coated coverslips, placed in 12-well plates for 24 h, then cotransfected with 20 ng of WT IRF-3–HA and 1 ng of STING plasmids or 20 ng of mutant IRF-3–HA and 1 ng of STING, respectively, using Lipofectamine 2000 reagent mixed with Opti-MEM (Life Technologies). Twenty-four hours posttransfection, the medium was replaced with fresh DMEM (1×) plus GlutaMAX medium with or without 30 μg/ml cGAMP. After 12 h of incubation, cells were washed using PBS, then fixed by 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with PBST containing 0.5% Triton X-100 in PBS. Cells were washed and blocked with 5% FBS in PBST and then incubated with anti–HA tag primary Ab (3724; 1:100; Cell Signaling Technology) overnight. The cells were washed three times with PBS and incubated with Alexa Fluor 488 Goat anti-Rabbit IgG (A11034, 1:1000; Thermo Fisher Scientific) at room temperature for 1 h. The coverslips were washed with PBS, mounted on slides with ProLong Gold Antifade Reagent with DAPI, then imaged under an Olympus FV1000 fluorescence microscope. The scale bars in the images correspond to 20 μm in length. To quantify the amount of nuclear translocation, 12 IRF-3–HA highly expressed cells or cell clusters were randomly selected in each field. The region of interest of nuclear or total IRF-3–HA fluorescence in the same cell or cell cluster was manually drawn. The area of integrated fluorescence intensity was calculated by ImageJ (Version 1.51n).

Molecular mass of WT IRF-3 and phosphorylated IRF-3 were determined by MALDI-TOF mass spectrometry (MS) using a Bruker UltrafleXtreme TOF/TOF mass spectrometer (Protein Chemistry Laboratory, Texas A&M University). The samples were solid-phase extracted using Protea C4 LithTips and analyzed using α-cyanohydroxycinnamic acid as a matrix and using the dried-droplet method. The mass spectrometer was operated in reflector mode and calibrated with angiotensin II, fibrinopeptide, renin substrate, and adrenocorticotropic hormone (18–39 fragment).

HEK293T cells (American Type Culture Collection, CRL-3216) were cultured in DMEM (1×) plus GlutaMAX medium (Life Technologies) supplemented with 10% FBS (Life Technologies), streptomycin (100 μg/ml), and penicillin (100 U/ml) at 37°C in a humidified atmosphere containing 5% CO2.

Statistical analyses for the luciferase reporter assay and quantification of fluorescence were carried out by Microsoft Excel and Prism, respectively. All of the data are presented as mean ± SEM. Two-way ANOVA with a Tukey multiple comparisons test was used to compare different groups. The statistical significance between the indicated samples and the control is designated as *p < 0.05, **p < 0.01, ***p < 0.001, or NS (p > 0.05).

The atomic coordinates and structural factors of the phosphorylated human and mouse IRF-3/CBP complexes have been deposited in the Worldwide Protein Data Bank, www.wwpdb.org (PDB: 7JFL and 7JFM, respectively).

To investigate how phosphorylation activates IRF-3, we expressed and purified a truncated hIRF-3 (residues 189–398) in complex with a CBP fragment (residues 2065–2111) and phosphorylated the complex with TBK1. MS analysis showed that only one residue of IRF-3 is phosphorylated (Fig. 1A). Next, we crystalized the phIRF-3/CBP and determined the structure at 1.8 Å resolution (Table I). Consistent with the MS analysis, the IRF-3/CBP structure showed that only Ser386 was phosphorylated (Fig. 1B–E). Overall, phIRF-3/CBP forms a dimer through phosphorylated Ser386 and the downstream DLHIS sequence (known as pLxIS motif) (Fig. 1B, 1C). Superposition of phIRF-3/CBP complex and autoinhibited IRF-3 (PDB indentification code: 1QWT) structures reveal a dramatic conformational change of the C-terminal tail upon phosphorylation (Supplemental Fig. 1A, 1B). In autoinhibited IRF-3, the C-terminal tail is folded and blocks the binding of CBP. In the phIRF-3/CBP complex, the C-terminal tail of IRF-3 unfolds and interacts with another IRF-3 molecule through extensive hydrogen bonds, hydrophobic interaction, and electrostatic interaction (Fig. 1E, Supplemental Fig. 1C, 1D, 1F). In addition, the IRF-3 dimer is also stabilized via hydrophobic interactions and hydrogen bonds in the central core region of the dimer (Supplemental Fig. 1E).

FIGURE 1.

Crystal structure of the phosphorylated hIRF-3 bound to CBP. (A) MS analyses of hIRF-3 (residues 189–398) before and after TBK1 phosphorylation. (B) Structure of phosphorylated hIRF-3 (residues 189–398) bound to CBP. Phosphorylated Ser386 and residues of the pLxIS motif are indicated by ball-and-stick models. IRF-3 are shown as green and cyan ribbons. CBP are shown by magenta and blue ribbons. (C) Structure of IRF-3 C-terminal region containing phosphorylated Ser386 and the pLxIS motif. One IRF-3 molecule is shown by green ball-and-stick model. The other IRF-3 in the IRF-3 dimer is shown by the surface representation with positively charged and negatively charged surfaces in blue and red, respectively. (D) 2Fo–Fc map showing pSer386 and its interacting residues contoured at 2.0 σ. (E) Interactions between pSer386 and surrounding residues. (F) Interactions between Glu386 and surrounding residues in the phosphomimetic S386/396E IRF-3 dimer (PDB identification code: 5JEM). (G) Superposition of the phIRF-3/CBP dimer and pSTING/IRF-3 complex structures (PDB identification code: 5JEJ). The green and cyan colored ribbons represent IRF-3 in the IRF-3/CBP dimer. The magenta ribbon represents phosphorylated STING and the pink colored ribbon indicates IRF-3 in pSTING/IRF-3 complex. (H) Native gel electrophoresis showing the dimerization state of WT IRF-3 and its S386A and S396A mutants upon phosphorylation by TBK1. (I) SEC showing how mutation S386A affects the dimerization of phosphorylated IRF-3 as compared with WT IRF-3. (J) SEC showing how mutation S396A affects the dimerization of phosphorylated IRF-3. (K) IFN-β luciferase reporter assays showing the effects of S386A and S396A mutations on IRF-3–mediated signaling. The data are mean ± SEM and representative of three independent assays. ***p < 0.001 values were calculated by comparisons of signals in cells transfected with S386A and S396A mutants and those transfected with WT IRF-3.

FIGURE 1.

Crystal structure of the phosphorylated hIRF-3 bound to CBP. (A) MS analyses of hIRF-3 (residues 189–398) before and after TBK1 phosphorylation. (B) Structure of phosphorylated hIRF-3 (residues 189–398) bound to CBP. Phosphorylated Ser386 and residues of the pLxIS motif are indicated by ball-and-stick models. IRF-3 are shown as green and cyan ribbons. CBP are shown by magenta and blue ribbons. (C) Structure of IRF-3 C-terminal region containing phosphorylated Ser386 and the pLxIS motif. One IRF-3 molecule is shown by green ball-and-stick model. The other IRF-3 in the IRF-3 dimer is shown by the surface representation with positively charged and negatively charged surfaces in blue and red, respectively. (D) 2Fo–Fc map showing pSer386 and its interacting residues contoured at 2.0 σ. (E) Interactions between pSer386 and surrounding residues. (F) Interactions between Glu386 and surrounding residues in the phosphomimetic S386/396E IRF-3 dimer (PDB identification code: 5JEM). (G) Superposition of the phIRF-3/CBP dimer and pSTING/IRF-3 complex structures (PDB identification code: 5JEJ). The green and cyan colored ribbons represent IRF-3 in the IRF-3/CBP dimer. The magenta ribbon represents phosphorylated STING and the pink colored ribbon indicates IRF-3 in pSTING/IRF-3 complex. (H) Native gel electrophoresis showing the dimerization state of WT IRF-3 and its S386A and S396A mutants upon phosphorylation by TBK1. (I) SEC showing how mutation S386A affects the dimerization of phosphorylated IRF-3 as compared with WT IRF-3. (J) SEC showing how mutation S396A affects the dimerization of phosphorylated IRF-3. (K) IFN-β luciferase reporter assays showing the effects of S386A and S396A mutations on IRF-3–mediated signaling. The data are mean ± SEM and representative of three independent assays. ***p < 0.001 values were calculated by comparisons of signals in cells transfected with S386A and S396A mutants and those transfected with WT IRF-3.

Close modal
Table I.
Data collection and refinement statistics for phIRF-3/CBP and pmIRF-3/CBP complexes
phIRF-3/CBPpmIRF-3/CBP
Data collection   
 Space group C 2 P 62 
 Molecules per asymmetric unit 2 phIRF-3, 2 CBP 2 pmIRF-3, 2 CBP 
 Cell dimensions   
  a, b, c (Å) 124.01, 68.03, 55.92 118.80, 118.80, 72.17 
  α, β, γ (°) 90.0, 106.24, 90.0 90.0, 90.0, 120.0 
 Resolution (Å) 1.68 (1.7–1.68)a 2.23 (2.30–2.23) 
 Rmerge 10.9% (124.3%) 8.9% (292%) 
 Rpim 6.8% (79.0%) 2.7% (86.8%) 
 CC(1/2) (%) 99.2 (35.2) 99.9 (38.2) 
 Unique reflections 48,116 28,422 
I/σI 6.5 (1.0) 12.6 (1.0) 
 Completeness (%) 94.7 (90.6) 99.9 (100.0) 
 Redundancy 3.4 (3.1) 12.1 (12.3) 
Refinement   
 Resolution (Å) 46.93–1.68 51.44–2.23 
 Number of reflections (F > 0) 48,093 28,389 
 Rwork/Rfree 19.1% (22.3%) 23.1% (25.2%) 
 Number of atoms   
  Protein 3,670 3,702 
  Water 252 14 
 B-factors (Å2  
  Protein 24.2 91.3 
  Water 27.8 67.9 
 Root mean square deviations   
  Bond lengths (Å) 0.006 0.001 
  Bond angles (°) 0.771 0.420 
 Ramachandran plot favored (%) 97.98 95.78 
 Ramachandran plot outlier (%) 0.45 0.0 
phIRF-3/CBPpmIRF-3/CBP
Data collection   
 Space group C 2 P 62 
 Molecules per asymmetric unit 2 phIRF-3, 2 CBP 2 pmIRF-3, 2 CBP 
 Cell dimensions   
  a, b, c (Å) 124.01, 68.03, 55.92 118.80, 118.80, 72.17 
  α, β, γ (°) 90.0, 106.24, 90.0 90.0, 90.0, 120.0 
 Resolution (Å) 1.68 (1.7–1.68)a 2.23 (2.30–2.23) 
 Rmerge 10.9% (124.3%) 8.9% (292%) 
 Rpim 6.8% (79.0%) 2.7% (86.8%) 
 CC(1/2) (%) 99.2 (35.2) 99.9 (38.2) 
 Unique reflections 48,116 28,422 
I/σI 6.5 (1.0) 12.6 (1.0) 
 Completeness (%) 94.7 (90.6) 99.9 (100.0) 
 Redundancy 3.4 (3.1) 12.1 (12.3) 
Refinement   
 Resolution (Å) 46.93–1.68 51.44–2.23 
 Number of reflections (F > 0) 48,093 28,389 
 Rwork/Rfree 19.1% (22.3%) 23.1% (25.2%) 
 Number of atoms   
  Protein 3,670 3,702 
  Water 252 14 
 B-factors (Å2  
  Protein 24.2 91.3 
  Water 27.8 67.9 
 Root mean square deviations   
  Bond lengths (Å) 0.006 0.001 
  Bond angles (°) 0.771 0.420 
 Ramachandran plot favored (%) 97.98 95.78 
 Ramachandran plot outlier (%) 0.45 0.0 
a

One crystal was used to collect each of the dataset. Values in parentheses are for highest-resolution shell.

The phIRF-3/CBP complex structure reveals that pSer386 reaches into a highly positively charged pocket surrounded by residues Arg211, Arg380, Arg341, and Lys360 and interacts with these residues via electrostatic interactions and an extensive network of hydrogen bonds (Fig. 1E). Specifically, pSer386 interacts with Arg211 from another IRF-3 molecule through a network of three hydrogen bonds. In addition, Arg211 also interacts with Lys360 and Glu388 through hydrogen bonds, thus making a critical contribution to the formation of IRF-3 dimer. Arg380 forms a hydrogen bond with pSer386 within the same IRF-3 molecule via its side chain guanidinium group. A water molecule forms a network of three hydrogen bonds with Arg380, pSer386, and Asp254, making additional contributions to the interactions between pSer386 and Arg380. In addition, Asp254 of the other IRF-3 molecule interacts with Ser385 upstream of pSer386 via two hydrogen bonds. Arg341 is within 4.0 Å from pSer386 and interacts with pSer386 through electrostatic interactions. Moreover, Arg341 also interacts with the phosphate group of pSer386 via a solvent-mediated hydrogen bond. Similarly, the sidechain of Lys360 from the other IRF-3 in the dimer is within 4.0 Å from the phosphate group and interacts with pSer386 via electrostatic interaction and a solvent-mediated hydrogen bond. In addition, Ser339 forms a hydrogen bond with pSer386 through its side chain hydroxyl group. Ser339 also interacts with the phosphate group of pSer386 via a solvent-mediated hydrogen bond through its main chain amine group. Based on these structural analyses, it is obvious that Arg380, Arg211, Ser339, and Asp254 play key roles in promoting IRF-3 dimerization by interacting with pSer386 through electrostatic interactions and a network of hydrogen bonds (Fig. 1E). Strikingly, every polar atom of pSer386 contributes to one or more hydrogen bonds in the phosphorylated IRF-3 dimer.

Although the overall structure of the phosphomimetic S386/396E IRF-3/CBP dimer is similar to the phIRF-3/CBP dimer (Supplemental Fig. 2A), Glu386, which mimics pSer386, contributes much less significantly to IRF-3 dimerization (Fig. 1F). Similar to the phIRF-3/CBP dimer, Arg380 is <4 Å from Glu386, and they likely interact with each other via electrostatic interactions. By contrast, the closest distance between the side chains of Arg211 and Glu386 is over 5 Å, and Arg211 forms no hydrogen bonds directly with Glu386. Instead, Arg211 stabilizes the phosphomimetic IRF-3 dimer mainly through its interaction with the side chain of Glu388. In addition, Arg341 and Lys360 are farther away from Glu386 and do not interact with Glu386 directly. Moreover, Lys360, Gln356, and Ser351 are not involved in the interaction with any residues downstream of Glu386 in the phosphomimetic dimer (Supplemental Fig. 2B). Based on this structural comparison, the phosphomimetic dimer does not fully recapitulate the extensive intermolecular interactions observed in the phIRF-3/CBP dimer, explaining why the phosphorylated IRF-3 dimer is more stable compared with the phosphomimetic dimer.

In addition to Ser386, Ser396 within the pLxIS motif is also involved in IRF-3 activation and can be phosphorylated by TBK1 (6, 21, 23, 24, 2729). Western blot showed that both Ser386 and Ser396 are phosphorylated upon cGAMP stimulation in HEK293T cells transfected with WT IRF-3 (Supplemental Fig. 2C). In the phIRF-3/CBP complex structure, the electron density for Ser396 and surrounding residues was well defined. However, we did not observe the phosphorylation of Ser396 in the structure, likely because of the truncation at residue Ser398 that prevents the phosphorylation of Ser396 by TBK1. To investigate how phosphorylation of Ser396 contributes to IRF-3 activation, we superimposed the structure of pSTING/IRF-3 (PDB identification code: 5JEJ) complex over the phIRF-3/CBP complex structure (Fig. 1G). The pLxIS motif of the phosphorylated STING is well aligned with the pIRF-3 pLxIS motif (Fig. 1G). Thus, it is likely that phosphorylated Ser396 could reach into the positively charged pocket surrounded by Arg285, His288, His290, and Lys313 and interact with them through electrostatic interactions in a similar fashion as pSer366 of pSTING (Fig. 1G).

To distinguish the roles of Ser386 and Ser396 in IRF-3 activation, we expressed and purified both S386A and S396A mutants of hIRF-3, phosphorylated them by TBK1, and analyzed them by native PAGE. Interestingly, the phosphorylated S386A mutant showed a single lower band, which is indicative of a monomer, whereas the phosphorylated S396A mutant exhibited two bands, indicating a mixture of both monomer and dimer (Fig. 1H). Consistent with the native gel result, size-exclusion chromatography (SEC) showed that phosphorylated S386A mutant was eluted at the same position as unphosphorylated S386A and WT IRF-3, whereas the phosphorylated S396A mutant showed two peaks, which correspond to a mixture of IRF-3 monomer and dimer (Fig. 1I, 1J). These results demonstrate that both Ser386 and Ser396 are involved in IRF-3 dimerization but Ser386 plays a more important role in IRF-3 activation compared with Ser396. To further explore how these two residues affect IRF-3–mediated signaling, we conducted IFN-β luciferase reporter assays in cells transfected with STING and IRF-3. We observed that the S386A mutation blocked the IFN-β reporter activation and the S396A mutation reduced the reporter signal by ∼50%, demonstrating that both Ser386 and Ser396 are involved in IRF–mediated signaling but that Ser386 is more crucial (Fig. 1K). Altogether, these extensive structural and functional studies provide critical insights into the detailed mechanism of IRF-3 activation upon phosphorylation.

Based on the structure of the phIRF-3/CBP complex, the C-terminal tail of IRF-3 mediates the IRF-3 dimerization upon phosphorylation. We wondered whether a similar mechanism is involved in IRF-3 activation in other mammalian species. First, we aligned the C-terminal sequences of IRF-3 across different species and observed that Ser386 and the pLxIS motif containing Ser396 of hIRF-3 are highly conserved in other species (Fig. 2A). However, Lys381 of mIRF-3 and Lys383 of rat IRF-3 replace Glu388 and Asn389 of hIRF-3 (Fig. 2A). To elucidate the mechanism of mIRF-3 activation, we phosphorylated the mIRF-3 (residues 184–390)/CBP complex using TBK1 and determined the crystal structure of the phosphorylated mIRF-3/CBP (pmIRF-3/CBP) complex (Supplemental Fig. 3A, Table I). Similar to hIRF-3, MS analysis showed that only one residue of mIRF-3 is phosphorylated (Supplemental Fig. 3B). The overall structures of the pmIRF-3/CBP and phIRF-3/CBP complexes are similar (root mean square deviation 1.3 Å, Fig. 2B). We observed that Ser379, which corresponds to Ser386 of hIRF-3, is phosphorylated in the structure (Fig. 2B, 2C). Because of the replacement of Glu388 and Asn389 of hIRF-3 by Lys381, this region of pmIRF-3 is restructured. Unlike Glu388 in human IRF-3, which contributes to hIRF-3 activation by interacting with Arg211, Lys360, and Gln356, Lys381 of mIRF-3 flips into the solvent and does not interact with any residues nearby (Fig. 2C). However, structures of pSer379 and the pLxIS motif downstream are well preserved. Arg373 is structurally conserved and interacts with pSer379 in a similar fashion as Arg380 in phIRF-3 (Fig. 2C). The side chain of Arg205 adopts a slightly different conformation compared with Arg211 of phIRF-3 and interacts with pSer379 through electrostatic interaction and hydrogen bonds (Fig. 2C). The interactions between Asp247, pSer379, and Ser378 are also well preserved in both mouse and hIRF-3 (Fig. 2C). By contrast, the side chains of Arg334 and Lys353 move away from pSer379 and do not interact with pSer379 directly (Fig. 2C). These structural comparisons demonstrate that Arg373 and Arg205 of mIRF-3, which correspond to Arg211 and Arg380 of hIRF-3, are critical for the interactions with pSer379 and contribute to the dimerization of mIRF-3 upon phosphorylation.

FIGURE 2.

Comparison of structures of phosphorylated mouse and hIRF-3 bound to CBP. (A) Sequence alignment of C-terminal tail of IRF-3 across different species showing the conserved phosphorylation sites and the pLxIS motif in red. Residues corresponding to Glu388 and Asn389 of hIRF-3 are highlighted in blue. Other potential phosphorylation sites are indicated by the asterisks. (B) Comparison of structures of phosphorylated mouse and hIRF-3/CBP complexes. pmIRF-3/CBP is shown by the orange ribbon. hIRF-3 dimer is colored in green and cyan with CBP bound to hIRF-3 in magenta and blue. Phosphorylated Ser379 of mIRF-3 and Ser386 of hIRF-3 are shown by the ball-and-stick models. (C) Distinct interactions between phosphorylated mouse and hIRF-3. Key residues mediating human and mIRF-3 dimerization are colored in green and orange, respectively. Residues interacting with Glu388 of hIRF-3 are in cyan. (D) IFN-β luciferase reporter assays showing the effect of E388A mutation on the signaling mediated by hIRF-3. The data are mean ± SEM and representative of three independent assays. **p < 0.01, ***p < 0.001 values were calculated by comparisons of signals in cells transfected with E388A mutant and those transfected with WT IRF-3. (E) Native gel electrophoresis showing the dimerization of WT hIRF-3 and its E388A mutant upon phosphorylation. (F) SEC showing the effect of E388A mutation on the dimerization of hIRF-3 upon phosphorylation as compared with WT IRF-3.

FIGURE 2.

Comparison of structures of phosphorylated mouse and hIRF-3 bound to CBP. (A) Sequence alignment of C-terminal tail of IRF-3 across different species showing the conserved phosphorylation sites and the pLxIS motif in red. Residues corresponding to Glu388 and Asn389 of hIRF-3 are highlighted in blue. Other potential phosphorylation sites are indicated by the asterisks. (B) Comparison of structures of phosphorylated mouse and hIRF-3/CBP complexes. pmIRF-3/CBP is shown by the orange ribbon. hIRF-3 dimer is colored in green and cyan with CBP bound to hIRF-3 in magenta and blue. Phosphorylated Ser379 of mIRF-3 and Ser386 of hIRF-3 are shown by the ball-and-stick models. (C) Distinct interactions between phosphorylated mouse and hIRF-3. Key residues mediating human and mIRF-3 dimerization are colored in green and orange, respectively. Residues interacting with Glu388 of hIRF-3 are in cyan. (D) IFN-β luciferase reporter assays showing the effect of E388A mutation on the signaling mediated by hIRF-3. The data are mean ± SEM and representative of three independent assays. **p < 0.01, ***p < 0.001 values were calculated by comparisons of signals in cells transfected with E388A mutant and those transfected with WT IRF-3. (E) Native gel electrophoresis showing the dimerization of WT hIRF-3 and its E388A mutant upon phosphorylation. (F) SEC showing the effect of E388A mutation on the dimerization of hIRF-3 upon phosphorylation as compared with WT IRF-3.

Close modal

In contrast to mIRF-3, Glu388 of hIRF-3 interacts with Arg211, Lys360, and Gln356 through electrostatic interactions and the solvent-mediated hydrogen bond and likely plays additional roles in hIRF-3 activation (Fig. 2C). Indeed, the luciferase reporter assay showed that mutating Glu388 to alanine in hIRF-3 reduced the IFN-β reporter signal by ∼45% (Fig. 2D). Furthermore, the phosphorylated E388A mutant showed two bands on native gel compared with phosphorylated WT IRF-3 (Fig. 2E). In agreement with these results, SEC shows that the E388A mutant of phIRF-3 elutes as two peaks, indicating the dimerization of hIRF-3 was compromised by this mutation (Fig. 2F). Taken together, these structural analyses reveal that mIRF-3 is activated in a similar but distinct manner compared with hIRF-3.

To investigate how the residues interacting with pSer386 and pSer396 contribute to IRF-3 activation, we generated 12 mutants of full-length hIRF-3 that include mutations R211A, R380A, S339A, R211A/R380A, and R211A/R380A/S339A of residues interacting with pSer386, and mutations R285A, H288A, H290A, K313A, R285A/K313A, H290A/K313A, and H288A/H290A/K313A of residues that are likely to interact with pSer396. Each of these mutants was expressed and purified for in vitro phosphorylation (Supplemental Fig. 3C, 3D). MS analyses showed that WT full-length IRF-3 can be efficiently phosphorylated by TBK1 (Fig. 3A). Each of the IRF-3 mutants was phosphorylated and analyzed by SEC (Fig. 3B–N). Strikingly, the R211A, R211A/R380A, and R211A/R380A/S339A mutants failed to form dimers upon phosphorylation compared with the WT control (Fig. 3B, 3C, 3F, 3G). In addition, mutation R380A severely impaired the dimerization of IRF-3 upon phosphorylation (Fig. 3D). By contrast, mutations S339A, R285A, R285A/K313A, H290A/K313A, and H288A/H290A/K313A have moderate effects on IRF-3 dimer formation (Fig. 3E, 3H, 3L–N). Moreover, mutations H288A, H290A, and K313A have little effect on IRF-3 dimerization (Fig. 3I–K). To further investigate the effect of these mutations on IRF-3 activation, we analyzed these IRF-3 mutants by native gel electrophoresis (Fig. 3O, 3P). We observed that phosphorylated R211A, R211A/R380A, and R211A/R380A/S339A mutants only showed a single band similar to unphosphorylated IRF-3, suggesting the activation of IRF-3 was disrupted by these mutations. Phosphorylated R380A mutant appeared as two bands, and only a small fraction of this mutant formed dimers, indicating that the activation of IRF-3 was dramatically impaired by this mutation. By contrast, mutations S339A, R285A, R285A/K313A, H290A/K313A, and H288A/H290A/K313A moderately affected the dimerization of IRF-3 upon phosphorylation. Three other mutations H288A, H290A, and K313A, do not individually affect the dimerization of IRF-3 (Fig. 3O, 3P). These results are consistent with the SEC analyses of phosphorylated IRF-3 mutants. Taken together, these studies show that mutations of key residues interacting with pSer386 significantly impair the activation of IRF-3. By contrast, mutations of residues that interact with pSer396 have moderate effects on IRF-3 activation, demonstrating that phosphorylation of Ser386 is more critical for the activation of IRF-3.

FIGURE 3.

Mutations of key residues interacting with pSer386 and pSer396 of hIRF-3 affect IRF-3 dimerization upon phosphorylation. (A) MS analyses of full-length hIRF-3 before and after TBK1 phosphorylation. (BN) SEC analyses showing how mutations of key residues interacting with pSer386 and pSer396 affect IRF-3 dimerization upon phosphorylation. (O and P) Native gel electrophoreses showing how IRF-3 mutations affect its dimerization upon phosphorylation.

FIGURE 3.

Mutations of key residues interacting with pSer386 and pSer396 of hIRF-3 affect IRF-3 dimerization upon phosphorylation. (A) MS analyses of full-length hIRF-3 before and after TBK1 phosphorylation. (BN) SEC analyses showing how mutations of key residues interacting with pSer386 and pSer396 affect IRF-3 dimerization upon phosphorylation. (O and P) Native gel electrophoreses showing how IRF-3 mutations affect its dimerization upon phosphorylation.

Close modal

It has been reported that IRF-3 dimerizes in the cytosol upon phosphorylation and then translocates to the nucleus to initiate the transcription of the IFN-β gene (14, 3033). To investigate how IRF-3–mediated signaling is affected by mutations of residues involved in IRF-3 dimerization, we conducted IFN-β luciferase reporter assay in cells transfected with full-length IRF-3 mutants. As is shown in Fig. 4A, mutations R211A, R380A, R211A/R380A, and R211A/K380A/S339A blocked the induction of the IFN-β reporter, whereas mutation S339A reduced the IFN-β reporter signal by ∼50%. As controls, the cells transfected with pcDNA3.1(−) or WT IRF-3 plasmid in the absence of STING plasmid showed almost no signals. The cells cotransfected with pcDNA3.1(−) and STING plasmids also showed very little signal (Fig. 4A). Western blot showed that the expression level of the IRF-3 mutants was similar, whereas only a very faint band was observed in the control vector, which corresponds to endogenous IRF-3 (Supplemental Fig. 3E). Mutations of residues surrounding pSer396, such as mutations R285A, R285A/K313A, H290A/K313A, and H288A/H290A/K313A reduced the reporter signal by 60–70%. By contrast, mutations H288A, H290A, and K313A, individually, barely impacted the activation of the reporter (Fig. 4B). Similarly, Western blot of cells transfected with WT IRF-3 and mutants indicated that these IRF-3 mutants are expressed at similar levels (Supplemental Fig. 3F). These results demonstrate that the mutations of key residues interacting with pSer386 abrogated IFN-β reporter activation, whereas the mutations of residues interacting with pSer396 only partially inhibited IFN-β reporter activation, suggesting that residues mediating IRF-3 dimerization are critical in IRF-3–mediated signaling.

FIGURE 4.

Mutations of residues interacting with pSer386 and pSer396 of hIRF-3 affect IRF-3–mediated signaling and nuclear localization. (A and B) IFN-β luciferase reporter assays showing mutations of residues interacting with pSer386 and pSer396 affect IRF-3–mediated signaling. The data are mean ± SEM and representative of three independent assays. **p < 0.01, ***p < 0.001, NS, when p > 0.05, values were calculated by comparisons of signals in cells transfected with IRF-3 mutants and those transfected with WT IRF-3. (C) Confocal microscopy of HEK293T cells transfected with IRF-3 mutants and STING upon cGAMP stimulation. Scale bar, 20 μm.

FIGURE 4.

Mutations of residues interacting with pSer386 and pSer396 of hIRF-3 affect IRF-3–mediated signaling and nuclear localization. (A and B) IFN-β luciferase reporter assays showing mutations of residues interacting with pSer386 and pSer396 affect IRF-3–mediated signaling. The data are mean ± SEM and representative of three independent assays. **p < 0.01, ***p < 0.001, NS, when p > 0.05, values were calculated by comparisons of signals in cells transfected with IRF-3 mutants and those transfected with WT IRF-3. (C) Confocal microscopy of HEK293T cells transfected with IRF-3 mutants and STING upon cGAMP stimulation. Scale bar, 20 μm.

Close modal

To investigate how these mutations affect the subcellular localization of IRF-3, we conducted confocal microscopy analyses of cells transfected with the IRF-3 mutants (Fig. 4C, Supplemental Fig. 4A). We observed that WT IRF-3 efficiently translocated to the nuclei after cGAMP treatment, whereas the R211A/R380A, R211A/R380A/S339A, R211A, and R380A mutants are mostly localized in the cytosol. The S339A, R285A, R285A/K313A, H290A/K313A, and H288A/H290A/K313A mutants partially entered the nuclei. By contrast, the H288A, H290A, and K313A mutants behaved similar to WT IRF-3 (Fig. 4C, Supplemental Fig. 4A). We used ImageJ to quantify the amount of nuclear translocation of IRF-3 in the cells and observed that mutations R211A, R380A, R211A/R380A, and R211A/R380A/S339A greatly impaired the amount of IRF-3 translocated to the nucleus after cGAMP treatment, whereas mutations H288A, H290A, and K313A did not individually affect IRF-3 nuclear translocation (Supplemental Fig. 4B). These results demonstrate that the mutations that affect IRF-3 dimerization impair IRF-3–mediated signaling and nuclear translocation of IRF-3.

IRF-3 is the key transcription factor regulating the expression of IFN-Is in response to various pathogens. In this study, we determined the crystal structures of phosphorylated human and mIRF-3/CBP complexes, which reveal that IRF-3 forms a dimer upon phosphorylation. Compared with autoinhibited IRF-3, the C-terminal tail of IRF-3 undergoes a dramatic conformational change upon phosphorylation, extending to the binding surface on another IRF-3 molecule, and mediates the dimerization of IRF-3. Phosphorylated Ser386 interacts with several residues in a positively charged pocket through extensive electrostatic interaction and hydrogen bonds. Cell-based studies combined with in vitro phosphorylation assays demonstrate that mutations of Ser386 and the residues surrounding pSer386 abrogate IRF-3 dimerization, block its translocation to the nuclei and abolish IRF-3–mediated signaling. By contrast, phosphorylation of Ser396 within the pLxIS motif likely plays a moderate role in IRF-3 activation. Mutations of Ser396 or residues that may interact with pSer396 only partially impair IRF-3 activation and signaling. Moreover, the structural analyses reveal that Glu388 plays additional roles in the activation of hIRF-3. These structural and functional studies established the molecular basis of IRF-3 activation upon phosphorylation.

In previous studies, Ser386 and the adjacent Ser385 (Ser378 and Ser379 in mouse) were considered as two important phosphorylation sites and were identified as critical residues for IRF-3–mediated signaling (18, 19, 34). However, the structures of the human and mIRF-3 dimer clearly show that only Ser386 is phosphorylated. The phIRF-3/CBP structure reveals that Ser385 interacts with Asp254 via hydrogen bonds to stabilize the IRF-3 dimer. Phosphorylated Ser386 reaches into a large positively charged pocket formed between two molecules of IRF-3 and contributes significantly to IRF-3 dimerization. Mutations of Ser386 and residues interacting with pSer386 dramatically impair IRF-3 dimerization, nuclear translocation, and signaling. These results suggest that phosphorylation of Ser386 but not Ser385 is essential for IRF-3–mediated signaling. Consistent with these results, two previous studies showed that mutation of Ser385 to aspartic acid impairs IRF-3 dimerization, and no phosphorylated Ser385 was detected with specific Abs (20, 24).

Although Ser396 (Ser388 in mIRF-3) within the pLxIS motif is not phosphorylated in the human and mIRF-3 dimer structures presented in this study, these residues can be phosphorylated in vivo (Supplemental Fig. 2C). Mutations of Ser396 and key residues that may interact with pSer396 impair IRF-3 dimerization and its functions, suggesting that phosphorylation of Ser396 also plays an important role in IRF-3 activation. Our previous studies demonstrated that the adaptors STING, MAVS, and TRIF employ the conserved pLxIS motif to recruit IRF-3 upon phosphorylation (6, 12). Interestingly, the phosphorylated pLxIS motifs from the adaptors or IRF-3 itself bind to the same surface on IRF-3, suggesting that the pLxIS motif of IRF-3 itself has two functions. First, the pLxIS motif of pIRF-3 occupies the pLxIS motif-binding surface of another IRF-3 molecule, displacing the adaptors and allowing pIRF-3 to dissociate from the adaptors. Second, the phosphorylated pLxIS motif works together with the phosphorylated Ser386 to facilitate IRF-3 dimerization, thus promoting IRF-3 activation.

The structures of the phosphorylated IRF-3 dimer and the adaptors bound to IRF-3 reveal that Arg285 (Arg278 in mouse) is a key residue interacting with the phosphorylated serine residue within the pLxIS motif. Mutation of Arg285 to alanine moderately impairs IRF-3 activation. These results explain why the R285Q mutation impaired IFN responses to HSV-1 infection and the R285E mutation in either HEK293 cells or fibroblasts of IRF-3–deficient mice showed much less activity upon Newcastle disease virus infection compared with WT IRF-3 (35, 36). Besides Ser386 and the pLxIS motif, Glu388 of hIRF-3 also plays a role in hIRF-3 activation. SEC and cell-based studies confirm the contribution of Glu388 in IRF-3 dimerization and IRF-3–mediated signaling. Interestingly, Glu388 is highly conserved in various species, except for mouse and rat. The replacement of the two residues Glu388 and Asn389 in human by Lys381 in mIRF-3 restructures this region and likely reduces the intermolecular interactions of mIRF-3 upon phosphorylation that may affect the kinetics of IFN-I induction by mice. In summary, these extensive structural and functional studies provide critical insights into the molecular basis of IRF-3 activation upon phosphorylation.

We thank The Berkeley Center for Structural Biology for helping to collect x-ray diffraction data. We would also like to acknowledge the members of the Li laboratory for help with revising the manuscript and critical feedback.

This work was supported in part by the Welch Foundation (Grant A-1931 to P.L.) and the National Institutes of Health, National Institute of Allergy and Infectious Diseases (Grant R01 AI-145287 to P.L.).

The atomic coordinates and structural factors presented in this article have been submitted to the Worldwide Protein Data Bank (http://www.wwpdb.org) under identifiers 7JFL and 7JFM.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • CBP

    CREB-binding protein

  •  
  • cGAMP

    cyclic GMP-AMP

  •  
  • hIRF-3

    human IRF-3

  •  
  • IRF

    IFN regulatory factor

  •  
  • mIRF-3

    mouse IRF-3

  •  
  • MS

    mass spectrometry

  •  
  • mTBK1

    mouse TBK1

  •  
  • PBST

    PBS with Tween 20

  •  
  • PDB

    Protein Data Bank

  •  
  • phIRF-3/CBP

    phosphorylated hIRF-3/CBP

  •  
  • pmIRF-3/CBP

    phosphorylated mIRF-3/CBP

  •  
  • SEC

    size-exclusion chromatography

  •  
  • STING

    stimulator of IFN genes

  •  
  • TRIF

    TIR domain-containing adaptor-inducing IFN-β

  •  
  • WT

    wild-type.

1
Paludan
,
S. R.
,
A. G.
Bowie
.
2013
.
Immune sensing of DNA.
Immunity
38
:
870
880
.
2
Bhat
,
N.
,
K. A.
Fitzgerald
.
2014
.
Recognition of cytosolic DNA by cGAS and other STING-dependent sensors.
Eur. J. Immunol.
44
:
634
640
.
3
Burdette
,
D. L.
,
R. E.
Vance
.
2013
.
STING and the innate immune response to nucleic acids in the cytosol.
Nat. Immunol.
14
:
19
26
.
4
Barbalat
,
R.
,
S. E.
Ewald
,
M. L.
Mouchess
,
G. M.
Barton
.
2011
.
Nucleic acid recognition by the innate immune system.
Annu. Rev. Immunol.
29
:
185
214
.
5
Shu
,
C.
,
X.
Li
,
P.
Li
.
2014
.
The mechanism of double-stranded DNA sensing through the cGAS-STING pathway.
Cytokine Growth Factor Rev.
25
:
641
648
.
6
Liu
,
S.
,
X.
Cai
,
J.
Wu
,
Q.
Cong
,
X.
Chen
,
T.
Li
,
F.
Du
,
J.
Ren
,
Y. T.
Wu
,
N. V.
Grishin
,
Z. J.
Chen
.
2015
.
Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.
Science
347
: aaa2630.
7
Takahasi
,
K.
,
N. N.
Suzuki
,
M.
Horiuchi
,
M.
Mori
,
W.
Suhara
,
Y.
Okabe
,
Y.
Fukuhara
,
H.
Terasawa
,
S.
Akira
,
T.
Fujita
,
F.
Inagaki
.
2003
.
X-ray crystal structure of IRF-3 and its functional implications.
Nat. Struct. Biol.
10
:
922
927
.
8
Shu
,
C.
,
G.
Yi
,
T.
Watts
,
C. C.
Kao
,
P.
Li
.
2012
.
Structure of STING bound to cyclic di-GMP reveals the mechanism of cyclic dinucleotide recognition by the immune system.
Nat. Struct. Mol. Biol.
19
:
722
724
.
9
Sun
,
L.
,
J.
Wu
,
F.
Du
,
X.
Chen
,
Z. J.
Chen
.
2013
.
Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.
Science
339
:
786
791
.
10
Xiao
,
T. S.
,
K. A.
Fitzgerald
.
2013
.
The cGAS-STING pathway for DNA sensing.
Mol. Cell
51
:
135
139
.
11
Hiscott
,
J.
,
R.
Lin
.
2005
.
IRF-3 releases its inhibitions.
Structure
13
:
1235
1236
.
12
Zhao
,
B.
,
C.
Shu
,
X.
Gao
,
B.
Sankaran
,
F.
Du
,
C. L.
Shelton
,
A. B.
Herr
,
J. Y.
Ji
,
P.
Li
.
2016
.
Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins.
Proc. Natl. Acad. Sci. USA
113
:
E3403
E3412
.
13
Li
,
X.
,
C.
Shu
,
G.
Yi
,
C. T.
Chaton
,
C. L.
Shelton
,
J.
Diao
,
X.
Zuo
,
C. C.
Kao
,
A. B.
Herr
,
P.
Li
.
2013
.
Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.
Immunity
39
:
1019
1031
.
14
Yanai
,
H.
,
H.
Negishi
,
T.
Taniguchi
.
2012
.
The IRF family of transcription factors: inception, impact and implications in oncogenesis.
OncoImmunology
1
:
1376
1386
.
15
Qin
,
B. Y.
,
C.
Liu
,
H.
Srinath
,
S. S.
Lam
,
J. J.
Correia
,
R.
Derynck
,
K.
Lin
.
2005
.
Crystal structure of IRF-3 in complex with CBP.
Structure
13
:
1269
1277
.
16
Qin
,
B. Y.
,
C.
Liu
,
S. S.
Lam
,
H.
Srinath
,
R.
Delston
,
J. J.
Correia
,
R.
Derynck
,
K.
Lin
.
2003
.
Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation.
Nat. Struct. Biol.
10
:
913
921
.
17
Ning
,
S.
,
J. S.
Pagano
,
G. N.
Barber
.
2011
.
IRF7: activation, regulation, modification and function.
Genes Immun.
12
:
399
414
.
18
Yoneyama
,
M.
,
W.
Suhara
,
T.
Fujita
.
2002
.
Control of IRF-3 activation by phosphorylation.
J. Interferon Cytokine Res.
22
:
73
76
.
19
Yoneyama
,
M.
,
W.
Suhara
,
Y.
Fukuhara
,
M.
Fukuda
,
E.
Nishida
,
T.
Fujita
.
1998
.
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
.
20
Mori
,
M.
,
M.
Yoneyama
,
T.
Ito
,
K.
Takahashi
,
F.
Inagaki
,
T.
Fujita
.
2004
.
Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation.
J. Biol. Chem.
279
:
9698
9702
.
21
Servant
,
M. J.
,
N.
Grandvaux
,
B. R.
tenOever
,
D.
Duguay
,
R.
Lin
,
J.
Hiscott
.
2003
.
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
.
22
Lin
,
R.
,
Y.
Mamane
,
J.
Hiscott
.
1999
.
Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains.
Mol. Cell. Biol.
19
:
2465
2474
.
23
Panne
,
D.
,
S. M.
McWhirter
,
T.
Maniatis
,
S. C.
Harrison
.
2007
.
Interferon regulatory factor 3 is regulated by a dual phosphorylation-dependent switch.
J. Biol. Chem.
282
:
22816
22822
.
24
Chen
,
W.
,
H.
Srinath
,
S. S.
Lam
,
C. A.
Schiffer
,
W. E.
Royer
Jr.
,
K.
Lin
.
2008
.
Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.
J. Mol. Biol.
379
:
251
260
.
25
Powell
,
H. R.
,
T. G. G.
Battye
,
L.
Kontogiannis
,
O.
Johnson
,
A. G. W.
Leslie
.
2017
.
Integrating macromolecular X-ray diffraction data with the graphical user interface iMosflm.
Nat. Protoc.
12
:
1310
1325
.
26
Adams
,
P. D.
,
P. V.
Afonine
,
G.
Bunkóczi
,
V. B.
Chen
,
I. W.
Davis
,
N.
Echols
,
J. J.
Headd
,
L. W.
Hung
,
G. J.
Kapral
,
R. W.
Grosse-Kunstleve
, et al
.
2010
.
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
Acta Crystallogr. D Biol. Crystallogr.
66
:
213
221
.
27
Tanaka
,
Y.
,
Z. J.
Chen
.
2012
.
STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway.
Sci. Signal.
5
:
ra20
.
28
Wu
,
J.
,
Z. J.
Chen
.
2014
.
Innate immune sensing and signaling of cytosolic nucleic acids.
Annu. Rev. Immunol.
32
:
461
488
.
29
Lin
,
R.
,
C.
Heylbroeck
,
P. M.
Pitha
,
J.
Hiscott
.
1998
.
Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation.
Mol. Cell. Biol.
18
:
2986
2996
.
30
Hiscott
,
J.
,
P.
Pitha
,
P.
Genin
,
H.
Nguyen
,
C.
Heylbroeck
,
Y.
Mamane
,
M.
Algarte
,
R.
Lin
.
1999
.
Triggering the interferon response: the role of IRF-3 transcription factor.
J. Interferon Cytokine Res.
19
:
1
13
.
31
Servant
,
M. J.
,
N.
Grandvaux
,
J.
Hiscott
.
2002
.
Multiple signaling pathways leading to the activation of interferon regulatory factor 3.
Biochem. Pharmacol.
64
:
985
992
.
32
Taniguchi
,
T.
,
K.
Ogasawara
,
A.
Takaoka
,
N.
Tanaka
.
2001
.
IRF family of transcription factors as regulators of host defense.
Annu. Rev. Immunol.
19
:
623
655
.
33
Tamura
,
T.
,
H.
Yanai
,
D.
Savitsky
,
T.
Taniguchi
.
2008
.
The IRF family transcription factors in immunity and oncogenesis.
Annu. Rev. Immunol.
26
:
535
584
.
34
Suhara
,
W.
,
M.
Yoneyama
,
T.
Iwamura
,
S.
Yoshimura
,
K.
Tamura
,
H.
Namiki
,
S.
Aimoto
,
T.
Fujita
.
2000
.
Analyses of virus-induced homomeric and heteromeric protein associations between IRF-3 and coactivator CBP/p300.
J. Biochem.
128
:
301
307
.
35
Andersen
,
L. L.
,
N.
Mørk
,
L. S.
Reinert
,
E.
Kofod-Olsen
,
R.
Narita
,
S. E.
Jørgensen
,
K. A.
Skipper
,
K.
Höning
,
H. H.
Gad
,
L.
Østergaard
, et al
.
2015
.
Functional IRF3 deficiency in a patient with herpes simplex encephalitis.
J. Exp. Med.
212
:
1371
1379
.
36
Chen
,
W.
,
S. S.
Lam
,
H.
Srinath
,
Z.
Jiang
,
J. J.
Correia
,
C. A.
Schiffer
,
K. A.
Fitzgerald
,
K.
Lin
,
W. E.
Royer
Jr
.
2008
.
Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5.
Nat. Struct. Mol. Biol.
15
:
1213
1220
.

The authors have no financial conflicts of interest.

Supplementary data