TIFAB regulates the TIFA–TRAF6 signaling pathway involved in innate immunity by forming a heterodimer complex with TIFA

Significance

The TIFA–TRAF6 signaling in nuclear factor κB (NF-κB) activation is involved in innate immunity. TIFAB, a homolog of TIFA, is a negative regulator of TIFA–TRAF6 signaling and is implicated in myeloid diseases; however, little is known about its biological function. We revealed a unique regulatory mechanism by which TIFAB suppresses TIFA–TRAF6 signaling by forming a heterodimer with TIFA. TIFAB binds to monomeric TIFA and blocks the formation of the TIFA homodimer, which is an intrinsic form of TIFA in NF-κB activation. The resultant TIFA/TIFAB complex is a pseudo-TIFA homodimer that suppresses TIFA–TRAF6 signaling. This study provides different insights into the regulation of NF-κB signaling and improves our understanding of the biological functions of TIFAB.

Abstract

Nuclear factor κB (NF-κB) is activated by various inflammatory and infectious molecules and is involved in immune responses. It has been elucidated that ADP-β-D-manno-heptose (ADP-Hep), a metabolite in gram-negative bacteria, activates NF-κB through alpha-kinase 1 (ALPK1)–TIFA–TRAF6 signaling. ADP-Hep stimulates the kinase activity of ALPK1 for TIFA phosphorylation. Complex formation between phosphorylation-dependent TIFA oligomer and TRAF6 promotes the polyubiquitination of TRAF6 for NF-κB activation. TIFAB, a TIFA homolog lacking a phosphorylation site and a TRAF6 binding motif, is a negative regulator of TIFA–TRAF6 signaling and is implicated in myeloid diseases. TIFAB is indicated to regulate TIFA–TRAF6 signaling through interactions with TIFA and TRAF6; however, little is known about its biological function. We demonstrated that TIFAB forms a complex not with the TIFA dimer, an intrinsic form of TIFA involved in NF-κB activation, but with monomeric TIFA. The structural analysis of the TIFA/TIFAB complex and the biochemical and cell-based analyses showed that TIFAB forms a stable heterodimer with TIFA, inhibits TIFA dimer formation, and suppresses TIFA–TRAF6 signaling. The resultant TIFA/TIFAB complex is a “pseudo-TIFA dimer” lacking the phosphorylation site and TRAF6 binding motif in TIFAB and cannot form the orderly structure as proposed for the phosphorylated TIFA oligomer involved in NF-κB activation. This study elucidated the molecular and structural basis for the regulation of TIFA–TRAF6 signaling by TIFAB.

Nuclear factor κB (NF-κB) is a crucial transcription factor involved in a wide range of immune responses; it is activated by various inflammatory and infectious molecules. TNF receptor-associated factor 6 (TRAF6) and TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA) were identified as signaling molecules in NF-κB activation (1–4). In TIFA–TRAF6 signaling, the complex formation between the phosphorylated TIFA (pTIFA) oligomer and TRAF6 promotes polyubiquitination of TRAF6, a crucial step in NF-κB activation (5, 6). In 2015, it was reported that D-glycero-D-manno-heptose-1,7-bisphosphate (HBP), a metabolite of gram-negative bacteria, triggers the phosphorylation-dependent oligomerization of TIFA and induces immune responses (7). ADP-β-D-manno-heptose (ADP-Hep), a derivative of HBP, has subsequently been identified as a pathogen-associated molecular pattern (PAMP) that binds and activates alpha-kinase 1 (ALPK1) for the phosphorylation of TIFA; it identifies ALPK1–TIFA–TRAF6 signaling pathway for modulating innate immunity (8–12).

TIFA–TRAF6 signaling is complexly regulated by the molecular assembly of dimeric TIFA and trimeric TRAF6 through TIFA phosphorylation. TIFA is an adaptor protein of TRAF6; it has an FHA domain [a phosphothreonine (pThr)-binding domain], an N-terminal phosphorylation site (Thr9) for oligomerization, and a C-terminal TRAF6 binding motif [PXEXX(Ar/Ac); X, any; Ar, aromatic; Ac, acidic] for interaction with the C-terminal domain of TRAF6 (TRAF6-C) (SI Appendix, Fig. S1) (3, 6). TRAF6 is an E3 ligase that consists of an N-terminal RING and zinc finger domain, a central coiled-coil domain, and a C-terminal TRAF6-C domain; it forms a trimer via the coiled-coil domain (1, 2). The crystal structures of human TIFA revealed an intrinsic dimeric form (in the absence of phosphorylation) and recognition of pThr9 through the FHA domain, suggesting head-to-tail oligomerization of the TIFA dimer (13). Subsequent structural analysis of mouse TIFA suggested a TIFA–TRAF6 oligomeric complex, based on the head-to-tail oligomerization of dimeric TIFA and the interaction between the TRAF6 binding motif of TIFA and TRAF6-C (14, 15). The RING domain of TRAF6 forms a dimer and catalyzes the ubiquitination of TRAF6 (16–18). The TIFA–TRAF6 complex, consisting of the phosphorylated TIFA oligomer and trimeric TRAF6, potentially strengthens the alignment of the RING and zinc finger domain of TRAF6 for effective ubiquitination (14, 16).

TIFAB was identified as a negative regulator of TIFA–TRAF6 signaling (19, 20). TIFAB shares 22% amino acid identity with TIFA but has no phosphorylation site at the N terminus; however, it has a partially conserved TRAF6 binding motif (AEETDE in human TIFAB; underlined characters indicate conserved residues) at the C terminus (SI Appendix, Fig. S1). The overexpression and immunoprecipitation of TIFAB suggested that TIFAB suppresses TIFA–TRAF6 signaling by interacting with TIFA and TRAF6 (19). TIFAB is expressed in B cells, dendritic cells, and macrophages, where it regulates TIFA–TRAF6 signaling (20). In addition, TIFAB is implicated in myelodysplastic syndrome and acute myeloid leukemia (21–23), and hematopoiesis is influenced by the balance between TIFA and TIFAB expression (24). However, little is known about the biological function of TIFAB, especially the molecular and structural basis of the regulation of TIFA–TRAF6 signaling. Here, we investigated the regulatory mechanism of TIFA–TRAF6 signaling by TIFAB using structural, biochemical, biophysical, and cell-based analyses and revealed that TIFAB regulates TIFA–TRAF6 signaling by forming a heterodimer complex with TIFA.

Results

TIFAB Forms a Heterodimer with TIFA during Protein Expression.

Previous immunoprecipitation experiments indicated that TIFAB coprecipitates with TIFA and TRAF6 (19). However, size-exclusion chromatography using a solution containing purified TIFA and TIFAB showed two separate peaks for the TIFA dimer and TIFAB monomer, indicating that TIFAB is expressed in a monomeric form and cannot interact with the TIFA dimer (Fig. 1). Considering this result and the amino acid sequence similarity between TIFA and TIFAB, we hypothesized that TIFAB and TIFA form a complex before the dimer formation of TIFA. Coexpression of TIFA and TIFAB in Escherichia coli enabled the purification of a complex form of TIFA and TIFAB with a single peak using size-exclusion chromatography (Fig. 1). The estimated molecular weight of the TIFA/TIFAB complex and the ratio of the band intensities of TIFA and TIFAB clearly showed that TIFA and TIFAB form a heterodimer (Fig. 1).

Fig. 1.

Complex formation between TIFA and TIFAB. Size-exclusion chromatography experiments using a mixture of separately purified TIFA dimer and TIFAB (TIFA + TIFAB, black) and coexpressed TIFA/TIFAB (pink), and SDS-PAGE analysis.

Structure of TIFA/TIFAB Heterodimer.

To obtain structural insights into the complex formation of TIFA and TIFAB, the crystal structure of TIFA/TIFAB was determined at 1.79 Å resolution (SI Appendix, Table S1 and Fig. 2A). C-terminal-truncated mouse TIFA (residues 1 to 150) and TIFAB (residues 1 to 139) were used for crystallization (SI Appendix, Fig. S1). Cys36 in mouse TIFA was replaced with Ser to prevent the formation of the disulfide bond observed in the crystals of the mouse TIFA dimer (14).

Fig. 2.

Crystal structure of a TIFA/TIFAB heterodimer. (A) Overall structure of TIFA/TIFAB. TIFA and TIFAB are shown in cyan and pink, respectively. N and C indicate the N and C terminus, respectively. (B) Superposition of TIFA and TIFAB monomers. The regions with low sequence identity between TIFA and TIFAB are highlighted in blue and red, respectively. (C) Superposition of TIFA/TIFAB and the TIFA dimer (PDB ID: 6L9U) (14). TIFA and TIFAB of the heterodimer are shown in blue and red, respectively. TIFA dimer is shown in gray.

TIFAB and TIFA form a heterodimer, as predicted by size-exclusion chromatography (Fig. 2A). The overall structure of TIFAB is similar to that of TIFA except the region from Thr16 to Val38 of TIFAB (α-1, β-2, and β-3), which shows a low sequence identity with TIFA (Fig. 2B and SI Appendix, Fig. S1). This region (from Thr16 to Val38 in TIFAB) does not strongly contribute to the interactions with TIFA (discussed in the next section). The RMSD is 0.97 Å for the corresponding 105 Cα atoms between TIFA and TIFAB without this region. TIFAB dimerizes with TIFA through the molecular interface of TIFA that is also involved in TIFA dimer formation (Fig. 2C). However, detailed interactions between TIFAB and TIFA differ from those in the TIFA dimer (discussed in the next section). The differences in the interactions between TIFA/TIFAB and the TIFA dimer rotate the TIFA/TIFAB dimer interface by approximately 10° relative to that of the TIFA dimer (indicated by red arrows in Fig. 2C).

Interactions between TIFA and TIFAB.

TIFAB interacts with TIFA through 19 hydrogen bond/salt bridges (Fig. 3A). Glu61, Gln96, and Lys66 in TIFAB, which are non-conserved residues (corresponding to Gln73, Lys108, and Phe78 in TIFA, respectively), contribute to a network of hydrogen bonds or salt bridges with TIFA, which are different from those observed in the TIFA dimer (Fig. 3B) (14). Glu61 in TIFAB forms hydrogen bonds/salt bridges with Gln73, Lys88, and Lys108 in TIFA. Gln96 and Lys66 in TIFAB interact with Glu86 and Glu102 (main chain) in TIFA, respectively. Cys74 in TIFAB is located at the center of the dimer interface of TIFA/TIFAB and corresponds to Glu86 in TIFA (SI Appendix, Figs. S1 and S2). Glu86 in TIFA plays a significant role in the network of hydrogen bonds/salt bridges in TIFA dimer formation (Fig. 3B), whereas Cys74 in TIFAB is not involved in the interaction with TIFA (SI Appendix, Fig. S2). The different contributions of Glu61, Gln96, and Cys74 of TIFAB in such interactions influence the orientation of the main chain of TIFA/TIFAB, resulting in a rotation of the TIFA/TIFAB dimer interface (indicated by red arrows in Fig. 2C and SI Appendix, Fig. S2). Arg35 in TIFAB forms new hydrogen bonds with the main chain of Ile44 in TIFA; this interaction is unique to mouse TIFA/TIFAB, because Arg35 is not conserved in human TIFAB (Pro35 in human). The conserved or functionally conserved residues in TIFAB, Lys76, Arg80, Tyr93, and Glu123 (corresponding to Lys88, Lys92, Tyr105, and Glu135, respectively), contribute to interactions with TIFA that are similar to those observed in the TIFA dimer (Fig. 3 A and B).

Fig. 3.

Interactions at the interface of TIFA/TIFAB and the TIFA dimer. (A) Dimer interface of TIFA/TIFAB. Non-conserved residues of TIFAB (TIFAB vs. TIFA) are highlighted in red. Hydrogen bonds/salt bridges are shown as dashed lines. (B) Dimer interface of TIFA dimer (PDB ID: 6L9U).

TIFA/TIFAB Exhibits Higher Structural Stability than the TIFA Dimer.

The crystal structure of TIFA/TIFAB suggests that TIFAB inhibits TIFA dimer formation by forming a heterodimeric complex with TIFA. Further, structural differences between TIFA/TIFAB and the TIFA dimer were analyzed (Fig. 4A). No significant difference in the electrostatic surface potentials at the dimer interface was observed between TIFA/TIFAB and the TIFA dimer. Analysis using PISA (25) and SC (26) showed that TIFAB binds to TIFA with a smaller buried surface area (970 Ų of TIFA/TIFAB vs. 1,050 Ų of TIFA dimer), larger shape complementarity (0.74 of TIFA/TIFAB vs. 0.69 of TIFA dimer), and similar number of hydrogen bonds/salt bridges (19 of TIFA/TIFAB vs. 18 of TIFA dimer) compared to those in the TIFA dimer (Fig. 4A). These results indicate that TIFA/TIFAB exhibits structural stability similar to that of the TIFA dimer. To further investigate the molecular mechanism underlying the inhibition of TIFA dimer formation by TIFAB, the thermostabilities of TIFA/TIFAB, the TIFA dimer, and TIFAB monomer were examined using differential scanning fluorometry (DSF) (Fig. 4B). The melting temperatures (T_m) of TIFA/TIFAB and the TIFA dimer are 67.6 and 62.4 °C, respectively, indicating that TIFA/TIFAB has a higher thermostability than the TIFA dimer. However, TIFAB monomer has incredibly low thermostability with a T_m value of 36.6 °C. The much lower thermostability of TIFAB than that of TIFA/TIFAB suggests that monomeric TIFAB, which is unstable after the process of translation, immediately forms a TIFA/TIFAB complex with higher structural stability than that of the TIFA dimer; this blocks the formation of the TIFA dimer, which is an essential building block for the pTIFA–TRAF6 oligomeric complex (6, 13, 14).

Fig. 4.

Comparison of dimerization of TIFA/TIFAB and the TIFA dimer. (A) Electrostatic surface potentials of the dimer interface of TIFA/TIFAB and the TIFA dimer (PDB ID: 6L9U). The electrostatic surface potentials are shown in the range from +5 kT/e (blue) to −5 kT/e (red). Buried surface area, shape complementarity, and number of hydrogen bonds/salt bridges were calculated using the programs, PISA and SC. (B) DSF curves of TIFA/TIFAB, the TIFA dimer, and TIFAB monomer are shown in pink, blue, and green, respectively.

Complex Formation of TIFA/TIFAB Suppresses TIFA–TRAF6 Signaling Pathway and Inhibits NF-κB Activation.

To confirm whether the complex formation of TIFA/TIFAB observed in the crystal structure inhibits NF-κB activation, GST pull-down assay and cell-based luciferase assay were performed. Human TIFA and TIFAB were used in the experiments. Mutations were introduced at the dimer interface of TIFAB. Glu61, Tyr93, Leu94, and Gln96, which are conserved between human and mouse TIFAB and are important for TIFA/TIFAB dimer formation, were mutated to alanine. Glu61, Tyr93, and Gln96 are involved in hydrogen bond/salt bridges (Fig. 3A), and Leu94 interacts with TIFA through van der Waals interactions (SI Appendix, Fig. S3). Three TIFAB mutants, E61A/Q96A, Y93A/L94A/Q96A, and E61A/Y93A/L94A/Q96A, were prepared and referred to as the M1, M2, and M3 mutants, respectively. In the GST pull-down assay, GST-TIFA (wild-type) and His-TIFAB (wild-type, M1, M2, or M3) were coexpressed in E. coli, and each cell lysate was collected (input). GST-TIFA and His-TIFAB in the lysate were bound to the glutathione Sepharose resin, and the coeluted GST-TIFA and His-TIFAB (pull-down) were analyzed using SDS-PAGE and Western blotting (Fig. 5A). Complex formation between TIFA and TIFAB was observed with a combination of GST-TIFA and wild-type TIFAB. In contrast, the band intensities of the coeluted M1, M2, and M3 mutants of TIFAB were weaker than that of the wild-type TIFAB, showing that the M1, M2, and M3 mutants have a reduced ability to form a complex with TIFA. These results confirm that TIFA and TIFAB form a heterodimer complex through the interaction interface identified in the crystal structure.

Fig. 5.

TIFAB forms a heterodimer with TIFA and suppresses TIFA–TRAF6 signaling. (A) GST pull-down assay. GST-TIFA and His-TIFAB [wild-type (WT), M1, M2, or M3] were coexpressed in E. coli cells. The cell lysates were analyzed using SDS-PAGE (Input, lanes 1 to 4). The eluted fractions of cell lysates obtained using the glutathione Sepharose resin were analyzed using SDS-PAGE (Pull-down, lanes 1 to 4). His-TIFAB was detected by Western blotting using anti-His-tag antibody. (B) Luciferase assay. HEK293T cells were cotransfected using pME-Myc-TIFAB (WT, M1, M2, or M3), 3κB-tk-Luc, and pRL-Luc. The cells were treated with 0.1 μg/mL ADP-Hep for 20 h and then subjected to luciferase assays. The data represent the mean ± SE of three independent experiments. n.s.: not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 vs. indicated group. Expression of Myc-TIFAB (WT, M1, M2, or M3) was detected by Western blotting using anti-Myc-tag antibody. (C) Phosphorylation and degradation of IκBα in NF-κB signaling. HEK293T cells were transfected using pME-Myc-TIFAB (WT, M1, M2, or M3). The cells were treated with 10 μg/mL ADP-Hep for 30 min and then subjected to Western blotting. Phospho-IκBα (p-IκBα), IκBα, and Myc-TIFAB were detected, respectively. Expression levels of IκBα were quantified using the band intensities of IκBα and GAPDH.

We carried out cell-based luciferase assay to further examine whether the complex formation of TIFA/TIFAB is essential to inhibit NF-κB activation through the ALPK1–TIFA–TRAF6 signaling induced by ADP-Hep. TIFAB (wild-type, M1, M2, or M3) was expressed in HEK293T cells, and the activation of NF-κB induced by ADP-Hep was quantified using a luciferase assay (Fig. 5B). Treatment with ADP-Hep activates NF-κB, but the expression of the wild-type TIFAB suppresses NF-κB activation to a level similar to that in the untreated control. In contrast, expression of the TIFAB mutant (M1, M2, or M3) fails to inhibit NF-κB activation. Inhibitory effects against NF-κB activation by the TIFAB mutants (M1, M2, and M3) are consistent with the results of the GST pull-down assay where M1, M2, and M3 fail to form a stable complex with TIFA. In addition, we examined the effects of TIFAB on the phosphorylation and degradation of IκBα in NF-κB signaling (Fig. 5C). Wild-type TIFAB inhibits the IκBα phosphorylation and degradation induced by ADP-Hep, whereas M1, M2, and M3 mutants lose the inhibitory effect (Fig. 5C), supporting the results of the luciferase assay (Fig. 5B). These experiments clearly show that TIFA/TIFAB complex formation suppresses the ALPK1–TIFA–TRAF6 signaling pathway and inhibits NF-κB activation.

TIFA/TIFAB Interacts with TRAF6 only through the TRAF6 Binding Motif of TIFA.

A previous report suggested that TIFAB interacts with TIFA and TRAF6 (19). TIFAB has no TRAF6 binding motif but has a partially conserved motif (AE/QETDE in human/mouse) at the C terminus (SI Appendix, Fig. S1). To understand the complex formation between TIFA/TIFAB and TRAF6, size-exclusion chromatography was performed using mouse TIFA/TIFAB and TRAF6-C. Full-length TIFA/TIFAB (TIFA/TIFAB), TIFA without the TRAF6 binding motif/full-length TIFAB [TIFA(1−150)/TIFAB], and trimeric TRAF6-C (14) were prepared. Size-exclusion chromatography showed that TIFA/TIFAB and TRAF6-C were coeluted at higher molecular weight range compared to TIFA/TIFAB alone and TRAF6-C alone (Fig. 6A). In contrast, TIFA(1−150)/TIFAB and TRAF6-C were eluted separately (Fig. 6B). Therefore, the partially conserved TRAF6 binding motif of TIFAB has no binding ability to TRAF6. This result indicates that the TIFA/TIFAB heterodimer is a “pseudo-TIFA dimer” lacking the TRAF6 binding motif as well as the phosphorylation site in TIFAB.

Fig. 6.

Complex formation between TIFA/TIFAB and TRAF6. Size-exclusion chromatography experiments using TIFA/TIFAB and TRAF6. (A) Chromatograms of a mixture of TIFA/TIFAB and TRAF6-C (TIFA/TIFAB+TRAF6-C, green), TIFA/TIFAB (orange), and TRAF6-C (black), and SDS-PAGE analysis. (B) Chromatograms of a mixture of TIFA(1−150)/TIFAB and TRAF6-C [TIFA(1−150)/TIFAB+TRAF6-C, red], TIFA(1−150)/TIFAB (cyan), and TRAF6-C (black), and SDS-PAGE analysis.

Discussion

We determined the crystal structure of TIFA/TIFAB and demonstrated that TIFAB interacts with TIFA through a molecular interface for TIFA dimer formation. The structure proposes that TIFAB blocks TIFA dimer formation by forming a heterodimer complex with TIFA; this inhibits the activation of NF-κB. DSF analysis indicates that TIFAB, which is expressed as an unstable monomer, forms a heterodimer with TIFA, which has a higher thermostability than the TIFA dimer. In addition, the interactions between TIFA and TIFAB observed in the crystal structure are essential for TIFA/TIFAB formation, leading to the inhibition of NF-κB activation.

Based on these results, we propose a molecular and structural basis for the regulation of TIFA–TRAF6 signaling by TIFAB (Fig. 7). TIFA forms a homodimer and each monomer has the N-terminal phosphorylation site (Thr9) and the C-terminal TRAF6 binding motif. Through the stimulation of ADP-Hep, ALPK1 phosphorylates Thr9 of TIFA, leading to oligomerization of pTIFA and further to the complex formation between the pTIFA oligomer and TRAF6 for the activation of NF-κB (indicated by a blue arrow in Fig. 7) (14, 16). In contrast, TIFAB has no phosphorylation site and no TRAF6 binding motif and is expressed as an unstable monomer. TIFAB forms a heterodimer with monomeric TIFA during protein expression (indicated by a red arrow in Fig. 7) and blocks TIFA dimer formation; this reduces the amount of TIFA dimers and suppresses the subsequent oligomerization of pTIFA. The resultant TIFA/TIFAB, a pseudo-TIFA dimer owing to the lack of the phosphorylation site and TRAF6 binding motif in TIFAB, cannot form the orderly structure as proposed for the pTIFA oligomer and pTIFA/TRAF6 complex. Through this mechanism, TIFAB expressed in B cells, dendritic cells, and macrophages would block the accidental activation of TIFA–TRAF6 signaling (20). In the absence of gram-negative bacteria, TIFA–TRAF6 signaling is suppressed by TIFAB by forming an inhibitory complex with TIFA. However, during bacterial infection, the expression of TIFAB in these cells is down-regulated by bacterial LPS (20), the formation of TIFA dimers is promoted, and the inhibition of TIFA–TRAF6 signaling by TIFAB is lifted. Subsequently, ADP-Hep, also released from bacteria, promotes the formation of the pTIFA/TRAF6 complex and activates NF-κB. This study revealed the regulatory mechanism of TIFA–TRAF6 signaling by TIFAB and provided fundamental insights into the biological functions of TIFAB.

Fig. 7.

Molecular and structural basis for the regulation of TIFA–TRAF6 signaling by TIFAB. TIFAB is expressed as an unstable monomer; it forms a heterodimer with monomeric TIFA; this blocks the formation of TIFA dimer. The resulting TIFA/TIFAB complex cannot form the orderly structure as proposed for the pTIFA oligomer and pTIFA/TRAF6 complex.

Materials and Methods

Protein Preparation for X-Ray Crystallography, DSF, and Size-Exclusion Chromatography.

Mouse TIFA and TIFAB were prepared for X-ray crystallography, DSF, and size-exclusion chromatography. The TIFA/TIFAB heterodimer was coexpressed in BL21(DE3) cells transformed with the pET30b(+) vector encoding TIFA and the pGEX-6P-1 vector encoding the N-terminal GST fusion TIFAB. TIFA/TIFAB was purified using glutathione Sepharose, cation exchange, and gel filtration columns. The GST-tag of TIFAB was removed using the HRV 3C protease, prior to cation-exchange column chromatography. Three types of TIFA/TIFAB were prepared as described above: TIFA/TIFAB (full-length TIFA/TIFAB), TIFA(1−150/C36S)/TIFAB(1−139) (residues 1−150 and C36S mutant of TIFA/residues 1−139 of TIFAB), and TIFA(1−150)/TIFAB (residues 1−150 of TIFA/full-length TIFAB). The TIFAB monomer was expressed as an N-terminal His-tag fusion protein using pET28b(+) and BL21(DE3) cells and was purified using Ni Sepharose, cation exchange, and gel filtration columns. Mouse TIFA dimer and trimeric TRAF6-C were prepared as described previously (14).

X-Ray Crystallography.

Crystallization conditions were screened using mouse TIFA/TIFAB proteins [TIFA/TIFAB, TIFA(1−150/C36S)/TIFAB(1−139), and TIFA(1−150)/TIFAB], which were concentrated to 10 mg/mL in a buffer solution containing 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 1 mM β-mercaptoethanol. Only TIFA(1−150/C36S)/TIFAB(1−139) was crystallized. The crystals were obtained using a reservoir containing 0.1 M HEPES (pH 7.0), 17% PEG2000, and 5% 2,5-hexanediol. The crystals were cryoprotected using 20% glycerol and frozen in liquid nitrogen. X-ray diffraction data were collected at 100 K on beamlines BL5A, BL17A, and AR-NE3A at the Photon Factory and processed using XDS (27). The phases were determined by molecular replacement using MOLREP (28) in the CCP4 program suite (29), with the coordinates of the TIFA dimer (PDB ID:6L9U) (14). The structure of TIFA/TIFAB was built and refined using COOT (30) and PHENIX (31). The electrostatic surface potentials were calculated using APBS and PDB2PQR (32). The data collection and refinement statistics are presented in SI Appendix, Table S1. Molecular graphics were generated using PyMOL (Schrödinger, LLC.).

DSF.

The DSF was measured using CFX Connect (Bio-Rad). The protein solution for DSF contained 10 μM protein (mouse TIFA dimer, TIFA/TIFAB, or TIFAB monomer), 20 mM Tris-HCl (pH 8.0), 250 mM NaCl, and 0.5 mM β-mercaptoethanol. Before measurement, SYPRO Orange Protein Gel Stain (Thermo Fisher Scientific) was added (1,000 times dilution) to the protein solutions.

GST Pull-Down Assay.

Human TIFA and TIFAB were used in the GST pull-down assay. The assay was performed using N-terminal GST fusion TIFA (GST-TIFA) and N-terminal His-tag fusion TIFAB (His-TIFAB). GST-TIFA and His-TIFAB (encoded by pGEX-6P-1 and pET28b(+), respectively) were coexpressed in BL21(DE3) cells. Plasmid vectors of the three His-TIFAB mutants (E61A/Q96A, Y93A/L94A/Q96A, and E61A/Y93A/L94A/Q96A) were prepared using the KOD-Plus-Mutagenesis kit (Toyobo). The cells were disrupted, and the supernatants containing GST-TIFA and His-TIFAB (input) were loaded onto a glutathione Sepharose resin. The proteins bound to the resin were eluted using a buffer containing 40 mM glutathione (reduced form). The supernatants (input) and eluted fractions (pull-down) were analyzed using SDS-PAGE. His-TIFAB was detected by Western blotting using Anti-His-tag mAB-HRP-DirecT (Medical & Biological Laboratories Co.).

Luciferase Assay.

Human HEK293T cells (ATCC) were cultured in DMEM (Fujifilm Wako) supplemented with 10% heat-inactivated FBS (Selborne). The cells were cotransfected using Lipofectamine 3000 (Thermo Fisher Scientific) with three vectors [pME-Myc-TIFAB (encoding N-terminal Myc-tag fusion human TIFAB) (19), 3κB-tk-Luc (with a triple κB sequence that binds to NF-κB joined to a downstream firefly luciferase gene, provided by Dr. Shigeki Miyamoto at The University of Wisconsin), and a control vector pRL-Luc (with a β-actin promoter joined to a downstream renilla luciferase gene) (33)]. The pME-Myc-TIFAB vectors of the E61A/Q96A, Y93A/L94A/Q96A, and E61A/Y93A/L94A/Q96A mutants were prepared using the KOD-Plus-Mutagenesis kit. After transfection, the cells were incubated for 24 h and then treated with 0.1 μg/mL ADP-heptose (InvivoGen) for 20 h. The cell lysate was analyzed using the Dual-luciferase reporter assay system (Promega). The values of luminescence for NF-κB activation were normalized using the ratio of luminescence derived from firefly and renilla luciferases. The expression level of TIFAB in the cell lysates was analyzed by Western blotting. Myc-TIFAB and β-actin were detected using anti-Myc-tag-mAb (Medical & Biological Laboratories Co.) and anti-β-actin clone AC-15 (Sigma-Aldrich), respectively.

Western Blotting.

Human HEK293T cells transfected with pME-Myc-TIFAB (WT, E61A/Q96A, Y93A/L94A/Q96A, and E61A/Y93A/L94A/Q96A) were treated with 10 μg/mL ADP-heptose for 30 min. The cell lysates were analyzed by Western blotting. Phospho-IκBα (Ser32/36) (5A5) Mouse mAb (Cell Signaling), IκBα Antibody (Cell Signaling), anti-Myc-tag-mAb, and anti-GAPDH antibody 0411 (Santa Cruz) were used for Western blotting. Band intensity was quantified using ImageJ software 2.0.0 (NIH).

Size-Exclusion Chromatography.

Complex formation between mouse TIFA dimer and TIFAB and between mouse TIFA/TIFAB and TRAF6-C was analyzed using Superdex 75 10/300 GL or Superdex 200 10/300 GL (Cytiva). The TIFA dimer and TIFAB were mixed at a molar ratio of 1:1 (monomeric TIFA:monomeric TIFAB). TIFA/TIFAB and TRAF6-C were mixed at a molar ratio of 1:1 (TIFA/TIFAB complex:monomeric TRAF6-C). The buffer contained 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 1 mM β-mercaptoethanol.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

Atomic coordinates and structure factors of mouse TIFA/TIFAB have been deposited in the Protein Data Bank under an accession ID 8WWY (34).