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Binding and Enhanced Binding between Key Immunity Proteins TRAF6 and
TIFA

Article  in  ChemBioChem · October 2018

DOI: 10.1002/cbic.201800436

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 DOI: 10.1002/cbic.201800436 Communications

Binding and Enhanced Binding between Key Immunity

Proteins TRAF6 and TIFA
Wei-Cheng Huang,[a] Jiahn-Haur Liao,[a] Tzu-Chun Hsiao,[a] Tong-You Wade Wei,[a]
Manuel Maestre-Reyna,[a] Yoshitaka Bessho,[a] and Ming-Daw Tsai*[a, b]

Human tumor necrosis factor receptor associated factor

(TRAF)-interacting protein, with a forkhead-associated domain
(TIFA), is a key regulator of NF-kB activation. It also plays a key
role in the activation of innate immunity in response to bacte-
rial infection, through heptose 1,7-bisphosphate (HBP); a me-
tabolite of lipopolysaccharide (LPS). However, the mechanism
of TIFA function is largely unexplored, except for the sugges-
tion of interaction with TRAF6. Herein, we provide evidence for
direct binding, albeit weak, between TIFA and the TRAF
domain of TRAF6, and it is shown that the binding is enhanced
for a rationally designed double mutant, TIFA S174Q/M179D.
Enhanced binding was also demonstrated for endogenous full-
Figure 1. A) Schematic illustration of the domain architecture of human
length TRAF6. Furthermore, the structures of the TRAF domain
TRAF6 and human TIFA. The dashed box designates the TRAF domain con-
complexes with the consensus TRAF-binding peptides from struct used in this study. B) Multiple sequence alignment based on the re-
the C terminus of wild-type and S174Q/M179D mutant TIFA, ported structures of the consensus TRAF-binding motifs bound to the TRAF
showing salt-bridge formation between residues 177–181 of domain of TRAF6. The peptides are from human CD40 (UniProtKB accession
number P25942),[14] human RANK (Q9Y6Q6),[14] human MAVS (Q7Z434),[17]
TIFA and the binding pocket residues of the TRAF domain,
and human TIFA (Q96CG3) and its double mutant in this work. The con-
were solved. Taken together, the results provide direct evi- served proline and glutamate residues are colored in red, and residues 174
dence and a structural basis for the TIFA–TRAF6 interaction, and 179 of TIFA are in green.
and show how this important biological function can be
modulated.

receptor signaling protein binding,[3] and a C-terminal TRAF

Cellular signal transduction involves a cascade of processes
mediated by protein–protein complexes. The tumor necrosis
factor receptor (TNFR) superfamily proteins recruit TNFR-associ-
ated factor (TRAF) proteins for activating downstream protein
kinases, such as IkB kinase (IkK) and c-Jun amino terminus
kinase (JNK), and transcription factors, such as nuclear factor-
kB (NF-kB) and activator protein 1 (AP-1), leading to a variety
of biological functions.[1] In addition, protein complexes in
TNFR, IL-1 receptor (IL-1R), and Toll-like receptor (TLR) signaling
pathways can cross talk with other molecules and transduce to
TRAF proteins, such as TRAF6.[2] As shown in Figure 1 A, human
TRAF6 consists of, from the N terminus, a RING domain, zinc
fingers, a coiled-coil region responsible for trimerization and
[a] Dr. W.-C. Huang, Dr. J.-H. Liao, T.-C. Hsiao, Dr. T.-Y. W. Wei,
Prof. M. Maestre-Reyna, Prof. Y. Bessho, Prof. M.-D. Tsai
Institute of Biological Chemistry, Academia Sinica
128 Academia Road Sec. 2, Nankang, Taipei, 115 (Taiwan)
E-mail: mdtsai@gate.sinica.edu.tw
[b] Prof. M.-D. Tsai
Institute of Biochemical Sciences, National (Taiwan) University
1, Roosevelt Road Sec. 4, Taipei 106 (Taiwan)
Supporting information and the ORCID identification numbers for the
authors of this article can be found under https://doi.org/10.1002/
cbic.201800436.
domain.[4]
The forkhead-associated (FHA) domain was demonstrated to
specifically recognize phosphothreonine (pThr).[5] The human
cytosolic TRAF-interacting protein with an FHA domain (TIFA)
contains an N-terminal Thr9 pThr site, a FHA domain, and a C-
terminal consensus TRAF-binding motif (Figure 1 A). Early stud-
ies suggested that overexpression of TIFA could activate NF-kB
and JNK.[6] Recent reports also suggested that TIFA sensed the
infecting pathogens through a TIFA-dependent cytosolic sur-
veillance pathway involving heptose 1,7-bisphosphate (HBP),
which is a metabolite of lipopolysaccharide (LPS), triggering
the NF-kB response; thus playing an important role in innate
immunity.[7] Along this line, TIFA is shown to be a crucial medi-
ator for nucleotide oligomerization domain-like receptor family
pyrin domain-containing protein 3 (NLRP3) inflammasome in
endothelial cells.[8] Furthermore, TIFA can also transduce DNA
damage-induced activation of NF-kB,[9] and support the pro-
gression of acute myeloid leukemia (AML) cells.[10] Importantly,
the interaction between phosphorylated Thr9 (pT9) of TIFA
and the FHA domain of TIFA is the key mechanism for TIFA-
mediated NF-kB activation,[11] presumably through interaction
with TRAF6 based on yeast two-hybrid screening,[6] glycerol-
gradient ultracentrifugation, and size-exclusion chromatogra-
phy.[12] However, direct interactions between TIFA and TRAF6

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and their structural basis remain to be demonstrated, and is
the main focus of this study.
The structure of human TIFA (residues 1–150, with truncated
C-terminal flexible region 151–184) was solved as an intrinsic
dimer recently. In addition, the structure of the complex of this
truncated TIFA with its binding peptide from the N terminus of
TIFA, including pT9, was also obtained, leading to a proposed
model of pT9–TIFA oligomers.[13] However, the binding motif of
TRAF6 has been shown to lie in the C terminus, including
Glu178.[6, 11] Therefore, the full-length TIFA and a C-terminal
peptide of TIFA are used in the present TIFA–TRAF6 binding
study.
Structural studies of human TRAF6 were first reported by
using the construct from residues 346 to 504 of TRAF6, which
existed as a monomer in solution.[14] In this study, we used a
similar construct with a minor shortening of the flexible region
(residues 350 to 501; Figure 1 A), which also existed in a stable
monomeric form in solution.
We first performed chemical crosslinking experiments for
the full-length TIFA and TRAF domain of TRAF6 by using the
BS3 crosslinker (bis(sulfosuccinimidyl)suberate), which is known
to probe protein–protein interactions within a distance of
11.4  by linking the amine groups of two lysine side chains.[15]
Although several crosslinked peptide sets of the wild-type
TIFA/TRAF6 mixture were found to meet the crosslinking crite-
ria in each experiment, they were not consistently repeatable
(and thus, not reported herein); thus suggesting that wild-type
TIFA does interact with TRAF6, but the interaction is weak. This
is not unreasonable because protein–protein interactions in
vivo often involve additional partners, which can enhance
binding synergistically. However, for the purpose of structural
characterization of the TIFA–TRAF interaction in vitro, it is nec-
essary to obtain a more stable complex.
Because it has been reported that CD40 binding with the
TRAF family proteins can be modulated by mutagenesis,[16] we
set out to design mutants of TIFA with enhanced binding affin-
ity for TRAF6. The consensus binding motif for the TRAF
domain of TRAF6 has been reported as -P-x-E-x-x-[Ar/Ac]-, in
which Ar is an aromatic residue and Ac is an acidic residue.[14]
The consensus TRAF-binding motif of the human wild-type
TIFA was reported to be 174-SSPTEMDE-181.[6] We analyzed the
reported TRAF domain structures (from TRAF6) in complex
with the peptides of the consensus TRAF-binding motif from
wild-type human CD40 and its N237D mutant,[14] human
RANK,[14] and human MAVS.[17] Based on structural and se-
quence analyses (Figure 1 B), we predicted and selected a
double mutant, S174Q/M179D TIFA, for this study.
As expected, the double mutant S174Q/M179D TIFA showed
more stable interactions with the TRAF domain, since two
pairs of crosslinked lysine residues were repeatedly observed,
between TIFA Lys76 and TRAF6 Lys388 (Figure 2 A left and
Figure S1 in the Supporting Information), and between TIFA
Lys108 and TRAF6 Lys489 (Figures 2 A right and S2). These re-
sults support that the double mutant S174Q/M179D of TIFA
stabilizes the interaction between TIFA and TRAF6.
Consistent with the results of chemical crosslinking, the re-
sults of size-exclusion chromatography also showed that the
& ChemBioChem 2018, 19, 1 – 8 www.chembiochem.org
Communications
S174Q/M179D double mutant TIFA, relative to the wild-type
TIFA, could form a more stable complex with the TRAF domain
of TRAF6. As shown in Figure 2 B left, the chromatogram of the
1:1 mixture of TIFA/TRAF domain consists of two peaks only
slightly shifted from the peaks of TIFA alone (intrinsic dimer,
44 kDa)[13] and TRAF domain alone (18 kDa), and the results of
SDS-PAGE showed only a trace amount of the TRAF domain in
the fractions containing TIFA. On the other hand, the chroma-
togram of the 1:1 mixture of the TIFA double mutant and the
TRAF domain (Figure 2 B right) consists of a major peak at the
position near 70 kDa (along with a minor peak near the posi-
tion of the TRAF domain); thus indicating the formation of a
complex between the TIFA mutant dimer and the TRAF
domain. This was further supported by SDS-PAGE analysis.
The weak binding of wild-type TIFA with the TRAF domain,
and the enhanced binding of the S174Q/M179D double
mutant TIFA, were further supported by analytical ultracentrifu-
gation (AUC) analyses. The TIFA peak was shifted only slightly
to a higher sedimentation coefficient in the 1:1 mixture of
wild-type TIFA and the TRAF domain of TRAF6 (Figure 2 C left),
but substantially in the corresponding mixture with the TIFA
double mutant (Figure 2 C right). The latter was further probed
by varying the ratio of the two proteins, which led to a pattern
of shifts in the sedimentation coefficient supporting rapid
chemical interconversion between the complex and the two
proteins (Figure 2 D left).[18] Fitting of the data with the Hill
equation[19] gave a Kd value of (16.5  0.7) mm and a coopera-
tive Hill coefficient n = 1.9  0.1 (Figure 2 D right), based on the
two-binding-site model for the dimeric TIFA protein (Figure 2 D
bottom).[13] The corresponding Kd value for wild-type TIFA was
not measurable by this method due to weak binding.
To provide support that enhanced binding of the S174Q/
M179D TIFA mutant, relative to wild-type TIFA, was applicable
not only to the TRAF domain, but also to the full-length
TRAF6, we used a TRAF6-specific antibody on Dynabeads to
pull down endogenous TRAF6 from 293T cells, which was, in
turn, incubated with wild-type or mutant TIFA. Western blot
analysis of washed protein–bead complex showed that full-
length TRAF6 bound more recombinant mutant TIFA than re-
combinant wild-type TIFA, as shown in Figure 2 E.
Structures of TRAF domain complexes with interacting part-
ners have been reported only with peptides, not with full-
length proteins.[14, 17] We also have the challenge of obtaining
crystals of the complex between full-length TIFA and the TRAF
domain. As an alternative approach, we successfully cocrystal-
lized the TRAF domain of TRAF6 with a C-terminal peptide of
TIFA (residues 170–184) containing the consensus TRAF-bind-
ing motif, for both wild-type TIFA and the double mutant (with
peptide sequences shown in Figure 1 B). The structures of the
two complexes were solved by using molecular replacement at
a resolution of 2.1 and 2.6 , respectively. Detailed structural
statistics are shown in Table 1.
In comparison with other reported structures of the com-
plexes of the TRAF domain of TRAF6 with the consensus TRAF-
binding peptides from CD40,[14] RANK,[14] and MAVS,[17] all
TRAF-binding peptides are bound in very similar orientations
(Figure 3 A top). In particular, two key consensus residues,
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Figure 2. Analyses of complex formation between human TIFA and human TRAF6. A) Two triplicate sets of the BS3 crosslinked peptide from the complex of
the S174Q/M179D mutant TIFA and the TRAF domain of TRAF6. In the crosslinked peptide of the first pair, crosslinking was also observed between TIFA resi-
dues Lys77 and Lys108. B) Size-exclusion chromatographic analysis on a Superdex 75 10/300 GL analytical gel filtration column for complex formation of wild-
type (left) or mutant (right) TIFA with the TRAF domain of TRAF6. TIFA alone is in green, the TRAF domain alone in gray, and the mixture (1:1) in blue. The
concentrations were 80:80 mm for wild-type/mutant TIFA:TRAF domain in Tris·HCl buffer (50 mm, pH 8.0), containing 180 mm NaCl. The collected fractions of
the complex were analyzed by 4–15 % SDS-PAGE. C) Plots of continuous sedimentation coefficient distribution c(S) versus sedimentation coefficient (S) for
TIFA alone (green), TRAF domain alone (gray), and 1:1 mixture (blue) for wild-type (left) and mutant (right) TIFA. The concentrations were 27 mm for all pro-
teins. The buffer conditions are the same as those given in B). D) AUC analysis for complex formation between the S174Q/M179D mutant TIFA (10 mm) and
the TRAF domain (0–50 mm). Left: Plots of c(S) versus S as a representative set from three repeats. An additional set of plots with different protein concentra-
tion ratios are shown in Figure S3. Right: Plots of the difference of the weighted-average sedimentation coefficients (DSw) versus TRAF6 concentration, and
fitting of the data by using the Hill equation. The standard error came from the fitting program. Bottom: The two-binding site model for the TIFA dimer. E) Ex
vivo association of full-length TRAF6, immunoprecipitated from 293T cells, with recombinant wild-type and mutant TIFA proteins. Lane 1 is a control without
TIFA. The numbers below the gel indicate the intensity ratios of [TIFA]/[TRAF6]. Representative of experiments performed in triplicate.

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Table 1. Statistics of data collection and refinement for the two complexes. High resolution statistics are shown in parentheses.

Crystal TRAF6 with wild-type TIFA TRAF6 with double mutant TIFA S174Q/M179D
PDB ID 6A33 5ZUJ
TRAF6 residues 350–501 residues 350–501
TIFA peptide sequence 170-SSQSSSPTEMDENES-184 170-SSQSQSPTEDDENES-184
l [] 1 1
Data collection and processing statistics
resolution [] 41.4–2.1 (2.175–2.1) 44.62–2.6 (2.693–2.6)
space group P61 2 2 P 61 2 2
unit cell
a, b, c [] 51.43, 51.43, 336.57 51.518, 51.518, 335.679
a, b, g [8] 90, 90, 120 90, 90, 120
total reflns 32 830 (3156) 17 691 (1689)
unique reflns 16 418 (1576) 9019 (863)
multiplicity 2.0 (2.0) 2.0 (2.0)
completeness [%] 98.40 (97.40) 99.81 (99.88)
mean I/s 16.43 (2.02) 10.57 (2.17)
Wilson B factor 40.85 49.3
Rmerge 0.0198 (0.3512) 0.05055 (0.4142)
Rmeas 0.028 (0.4967) 0.07149 (0.5858)
Rpim 0.0198 (0.3512) 0.05055 (0.4142)
CC1/2 1 (0.827) 0.997 (0.777)
CC* 1 (0.952) 0.999 (0.935)
Refinement statistics
reflns used in refinement 16 399 (1576) 9015 (863)
reflns used for Rfree 820 (78) 443 (47)
Rwork 0.1937 (0.2648) 0.1929 (0.2735)
Rfree 0.2467 (0.3222) 0.2420 (0.3171)
CC(work) 0.955 (0.863) 0.954 (0.902)
CC(free) 0.939 (0.795) 0.962 (0.696)
number of non-H atoms 1427 1390
macromolecules 1303 1327
solvent 124 63
protein residues 163 165
RMS[a] bonds [] 0.007 0.008
RMS[a] angles [8] 0.85 1.01
Ramachandran favored [%] 97.48 94.41
Ramachandran allowed [%] 2.52 5.59
Ramachandran outliers [%] 0 0
rotamer outliers [%] 0 0.69
Clash score 8.2 5.35
average B factors 48.52 46.20
protein 46.04 45.23
peptide substrate 77.22 57.50
solvent 55.91 48.92

[a] RMS: root mean square.

Pro176 (position 0) and Glu178 (position 2), are anchored at
the same position as that in all other TRAF-binding peptides
by the interaction between the side chain of TIFA Glu178 and
the backbone of TRAF6 Leu457 and Ala458 (Figure 3 A top).
The backbone of the residue in position 3 also interacts with
the backbone of TRAF6 Gly470 (Figure 3 A top). In addition,
the corresponding residues in position 3 of RANK and MAVS
are Asp and Asn, respectively, which form additional salt
bridges with the side chain of TRAF6 Arg392 (Figure 3 A top).
This was taken into account in the rational design of the
double mutant TIFA. Despite similar numbers of salt bridges
between the consensus TRAF-binding peptides and the TRAF
domain (nine for RANK, nine for MAVS, ten for CD40, ten for
TIFA), a unique salt bridge was conferred between Glu181 of
TIFA and R466 of TRAF6 (Figure 3 A bottom). The correspond-
ing residue at position 5 of the CD40 peptide is Phe, which
interacts with Phe410 of TRAF6 through a perpendicular, non-
stacking p–p interaction. Meanwhile, in RANK or MAVS, posi-
tion 5 is occupied by Tyr, which lacks any close contact with
TRAF6.
A comparison of the wild type and S174Q/M179D mutant in
the complexes with the TRAF domain is shown in Figure 3 B. In
the structure of the bound wild-type peptide, the Met179 side
chain (position 3) was not well defined in the electron density,
and therefore, was not built in the refined structural model. By
comparison, the structure of the bound S174Q/M179D peptide
is very similar to that of the wild-type peptide, except that the
M179D mutation results in a salt bridge between its side chain
and Arg392 of TRAF6 within a distance of about 3 , which
was also observed in the reported RANK peptide/TRAF6 com-

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Figure 3. A) Superimposed stereostructures of the consensus TRAF-binding
peptides (sticks) bound to the TRAF domain (gray ribbons), highlighting po-
sitions 0, 2, and 3 (top), and position 5 (bottom). The peptide from TIFA is
colored in blue, MAVS in pink, CD40 in cyan, and RANK in green. B) A com-
parison of the TIFA–TRAF interaction between the peptides of wild-type
TIFA (blue) and the S174Q/M179D mutant (green).
plex.[14] In addition, the Gln174 residue forms two more salt
bridges, with Glu448 and Thr475 of TRAF6 (Figure 3 B). Interest-
ingly, in spite of a lower resolution for the mutant versus wild-
type crystal structure, the overall peptide B factor for the
mutant was about 25 % lower than that of the wild-type
(Table 1). Furthermore, the weaker diffracting mutant cocrystal
resulted in higher quality peptide electron density, which
allowed us to model one additional peptide residue (Ser173).
Overall, we suggest that the stabilization afforded by the
Gln174 interaction network, relative to the wild-type complex,
underlies these surprising features.
The present study is an important step toward elucidating
the structure of the complex between full-length TIFA and full-
length TRAF6. This would be a major challenge because TRAF6
is a large trimeric protein;[14] TIFA also forms oligomers in vivo
upon phosphorylation,[11] and together they form large aggre-
gates/speckles in vivo, along with other proteins.[11, 20] However,
considering examples in which TIFA is a potential therapeutic
target for AML,[10] whereas TRAF6 is a potential therapeutic
target for multiple myeloma,[21] structural information about
the TIFA–TRAF6 interaction should be useful for the future
development of therapeutics against cancer and possibly infec-
tious diseases as well. Along with the results of the double
mutant with enhanced binding, this study also suggests that it
will be possible to modulate inflammatory and immunity
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Communications
responses by attenuating or enhancing the TIFA–TRAF6 inter-
action.
Experimental Section
Protein expression and purification: Expression vector pET43.1a
was constructed with the C terminal His-tag TRAF domain of
TRAF6 (350–501) at multiple cloning sites by using NdeI and XhoI
and amplified in the Escherichia coli expression strain BL21(DE3).
The culture containing the desired target expression gene was
grown at 37 8C to an OD600 of 0.8. The cell culture was induced by
addition of isopropyl b-d-1-thiogalactopyranoside (IPTG) to a final
concentration of 0.3 mm at 16 8C for 16 h. The cell pellets were har-
vested and resuspended in the buffer containing Tris·HCl (50 mm,
pH 8.0), NaCl (200 mm), and imidazole (10 mm). The proteins were
initially purified by using a self-packed column carrying Ni-NTA
resin (Chelating Sepharose Fast Flow, GE Healthcare). The Ni-NTA
resin was equilibrated in the binding buffer before the sample was
loaded onto the column. The column was washed with the wash-
ing buffer (50 mm Tris·HCl, pH 8.0), 200 mm NaCl, and 80 mm imi-
dazole) and then eluted with the elution buffer (50 mm Tris·HCl,
pH 8.0, 200 mm NaCl, and 400 mm imidazole). The eluent was con-
centrated and further purified with a HiLoad 16/600 Superdex 75
column (GE Healthcare) in a buffer of Tris·HCl (50 mm, pH 8.0, and
300 mm NaCl. The separated fractions were analyzed by means of
4–15 % SDS-PAGE. The TIFA proteins with an N-terminal His tag
were purified as previously described.[13]
Analytical ultracentrifugation: Procedures for AUC were based on
reported sedimentation velocity AUC methods[22] with minor modi-
fications. The samples were dissolved in Tris·HCl (50 mm, pH 8.0),
and NaCl (180 mm) and analyzed by using a Beckman-Coulter XL-A
analytical ultracentrifuge. The samples were loaded into 12 mm
standard double-sector Epon charcoal-filled centerpieces mounted
in an An-50 Ti rotor for ultracentrifugation at 163 004 g at 4 8C and
monitored at l = 235 nm. The continuous sedimentation coefficient
distribution, c(S), values were obtained by using Sedfit software.[23]
The Gilbert Sw fast isotherm was determined by integration of the
c(S) peaks of the fast boundary component in c(S).[23a, 24] The disso-
ciation constant was obtained from an isothermal plot generated
by plotting the difference of the weighted-average sedimentation
coefficients (DSw) against the concentration of the TRAF domain
by using the Hill equation analysis[19, 25] in the OriginPro v8.5 pro-
gram.
Chemical crosslinking: The sample consisted of TIFA protein
(2 mm) and TRAF domain (2 mm) in the crosslinking buffer (50 mm
boric acid, 100 mm NaCl, pH 8.0). The crosslinker BS3 (0.5 mm;
Thermo Fisher Scientific) was added to the sample mixture. After
the mixture had reacted in the dark at room temperature for
60 min, ammonium bicarbonate (100 mm) was added to stop the
reaction, and the sample mixture was incubated in the dark at
room temperature for a further 30 min. The reaction mixture was
then concentrated and washed with the crosslinking buffer by
using Amicon Ultra 3K 0.5 mL centrifugal filters to remove the
crosslinker, followed by SDS-PAGE analysis. The crosslinked bands
were excised for in-gel digestion by chymotrypsin. The peptide
samples were analyzed by Orbitrap Fusion mass spectrometry, as
previously reported.[5c] The crosslinked peptides were analyzed by
using the MassMatrix software.[26] The MS raw data have been de-
posited to the PRIDE proteomics data repository (The European
Bioinformatics Institute, Cambridge, UK http://www.ebi.ac.uk/pride/
archive/) with access number PXD010473.
5  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &

Ex vivo binding assay: The 293T cells were lysed with CHAPS lysis
buffer, as described previously.[10] TRAF6 antibody (3 mg, GeneTex)
was precoated with M-280 Dynabeads (100 mL; Invitrogen) over-
night at 4 8C. Cell extract (1 mg) was incubated with antibody-
coated Dynabeads (25 mL) at 4 8C overnight. The protein–bead
complex was then washed (3 ) with 1  Tris-buffered saline Tween
20 (TBST) buffer and subjected to coincubation with recombinant
His-tagged wild-type or S174Q/M179D double mutant TIFA (1 mg)
in the binding buffer containing 1  TBS with 1 % bovine serum al-
bumin (BSA) supplemented with protease inhibitor cocktail (Roche)
and phosphatase inhibitor (Sigma) for 1 h at 25 8C. The protein–
bead complex was then washed (3 ) with 1  TBST buffer and sub-
jected to western blot analysis. The experiments were repeated
three times.
Crystallization and structural determination: The TRAF domain of
TRAF6 was exchanged into the Tris·HCl (20 mm, pH 8.0) buffer con-
taining NaCl (180 mm). The TRAF-binding peptides were synthe-
sized by Peptide Synthesis Laboratory, IBC, Academia Sinica. The
protein and peptide were mixed at a 1:10 molar ratio (150 mm
TRAF6/1.5 mm peptide). The crystals were obtained in crystalliza-
tion buffer containing HEPES sodium (0.1 m, pH 7.5), sodium citrate
tribasic dihydrate (1 m), and tris(2-carboxyethyl)phosphine (TCEP;
10 mm) at 4 8C. XRD data were collected at beamline BL05A of the
National Synchrotron Radiation Research Center (Hsinchu, Taiwan)
and beamline BL32XU of SPring-8 (Hyogo, Japan). Data sets were
collected at 100 K by using wavelengths of 1 . Diffraction intensi-
ties were integrated with the XDS package. The structures were
determined by molecular replacement by using the previously re-
ported CD40-TRAF6 complex as a searching model.[14] Refinement
used PHENIX[27] and CCP4i.[28] Both wild-type TIFA–TRAF6 and
double mutant TIFA–TRAF6 structures were deposited in the Pro-
tein Data Bank (PDB) with the accession numbers 6A33 and 5ZUJ,
respectively.
Acknowledgements
We acknowledge support from the National Synchrotron Radia-
tion Research Center (NSRRC) and the SPring-8 Synchrotron
Center for use of the protein crystal beamlines (TPS05A and
BL32XU). Synchrotron radiation experiments at the BL32XU
beamline of SPring-8 were with the approval of the Japan Syn-
chrotron Radiation Research Institute (JASRI; proposals
2017A2576, 2017B2576, and 2018A2514). We thank Dr. Yoshiaki
Kawano for helpful support during BL32XU experiments. We also
thank Academia Sinica for supporting crystallization facilities,
mass spectrometry facilities, and biophysics facilities; Dr. Meng-
Ru Ho and Dr. Shu-Chuan Jao for technical support; and Dr.
Wen-Jin Winston Wu for useful discussions. This work was sup-
ported by the Taiwan Protein Project (grant no. AS-KPQ-105-TPP).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: biological activity · mutagenesis · noncovalent
interactions · protein–protein interactions · structure
elucidation
& ChemBioChem 2018, 19, 1 – 8 www.chembiochem.org
Communications
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Manuscript received: July 30, 2018
Revised manuscript received: October 30, 2018
Accepted manuscript online: October 31, 2018
Version of record online: && &&, 0000
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 COMMUNICATIONS
W.-C. Huang, J.-H. Liao, T.-C. Hsiao, Bridging the gap: Human tumor ne-
T.-Y. W. Wei, M. Maestre-Reyna, Y. Bessho, crosis factor receptor associated factor
M.-D. Tsai* (TRAF)-interacting protein, with a fork-
head-associated domain (TIFA), is a key
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regulator of NF-kB activation and plays
Binding and Enhanced Binding a key role in activating the immune re-
between Key Immunity Proteins sponse to bacterial infection. An impor-
TRAF6 and TIFA tant signal transduction protein–protein
complex of human TIFA and TRAF6 pro-
vides direct evidence and a structural
basis for their interaction.

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