Structures of apo IRF-3 and IRF-7 DNA binding
domains: effect of loop L1 on DNA binding
Pablo De Ioannes1, Carlos R. Escalante2,* and Aneel K. Aggarwal1,*
1Department of Structural and Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison
Avenue, NY 10029 and2Department of Physiology and Biophysics, Virginia Commonwealth University,
School of Medicine, 1101 East Marshall Street, Box 980551, Richmond, VA 23298, USA
Received February 18, 2011; Revised April 20, 2011; Accepted April 21, 2011
Interferon regulatory factors IRF-3 and IRF-7 are
transcription factors essential in the activation of
interferon-b (IFN-b) gene in response to viral infec-
tions. Although, both proteins recognize the same
consensus IRF binding site AANNGAAA, they have
distinct DNA binding preferences for sites in vivo.
The X-ray structures of IRF-3 and IRF-7 DNA
binding domains (DBDs) bound to IFN-b promoter
elements revealed flexibility in the loops (L1–L3)
and the residues that make contacts with the
target sequence. To characterize the conformation-
al changes that occur on DNA binding and how they
differ between IRF family members, we have solved
the X-ray structures of IRF-3 and IRF-7 DBDs in the
absence of DNA. We found that loop L1, carrying the
conserved histidine that interacts with the DNA
minor groove, is disordered in apo IRF-3 but is
ordered in apo IRF-7. This is reflected in differences
in DNA binding affinities when the conserved histi-
dine in loop L1 is mutated to alanine in the two
proteins. The stability of loop L1 in IRF-7 derives
from a unique combination of hydrophobic residues
that pack against the protein core. Together, our data
show that differences in flexibility of loop L1 are an
The interferon regulatory factor (IRF) family of proteins
plays an essential role in the activation and regulation of
immune response genes implicated in both innate and
adaptive immunity (1–5). In addition, some members of
this family have critical roles in the differentiation and
development of hematopoietic cells and in the regulation
of apoptosis (6,7). In mammals, nine IRF proteins have
been identified so far and all share the same general archi-
tecture, with a highly conserved N-terminal domain of
approximately 120 residues, which is involved in DNA-
specific binding and a variable C-terminal domain or
IRF association domain (IAD) that mediates not only
homo or hetero-oligomerization among IRF factors, but
also mediates association with other transcription factors
and co-activators, like CBP/p300 (8,9). Despite the high
degree of structural similarity among the different IRF
DNA binding domains (DBDs), there are important dif-
ferences; for instance, IRF-4 contains an N-terminal ex-
tension of 20 amino acids that inhibit DNA binding (10)
and IRF-7 has a recognition helix a3 that is longer than
other members of the IRF family (11). Nevertheless,
all IRF proteins recognize a DNA element with a consen-
sus sequence AANNGAAA. This sequence is found in a
multitude of promoters from interferon response genes,
usually containing two or more of these elements
(12–14). The crystal structure of IRF-1 DBD bound to
DNA (15), revealed that the IRF DBD consists of a
modified version of the helix-turn-helix (HTH) motif
that includes a four-stranded antiparallel b-sheet and
three large loops (L1–L3) connecting the different sec-
ondary structural elements. Subsequent structures of
IRF-2 (16), IRF-4 (10), IRF-3 (17) and IRF-7 (11)
bound to DNA have confirmed that DNA recognition is
achieved by (i) conserved residues on helix a3 (Arg, Cys,
Asn) interacting with GAAA bases (AANNGAAA) in the
major groove and (ii) a conserved His on loop L1
protruding into the minor groove and making water-
mediated contacts with two consecutive A:T steps
upstream of the GAAA core sequence (AANNGAAA).
These sets of interactions are further stabilized through
a network of hydrogen bonds between the IRF proteins
and the DNA phosphate backbone. The recent structures
DNA element of the IFN-b enhancer and the structure
of IRF-3, IRF-7 and NF-kB DBDs bound to the
*To whom correspondence should be addressed. Tel: +1 804 628 1202; Fax: +1 804 828 6991; Email: email@example.com
Correspondence may also be addressed to Aneel K. Aggarwal. Tel: +1 212 659 8650; Fax: +1 212 849 2456; Email: firstname.lastname@example.org
Nucleic Acids Research, 2011, Vol. 39, No. 16Published online 19 May 2011
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
PRDII–PRDIII element have provided insights on the
basis of cooperativity when two or more IRF DBDs
bind to natural enhancers and promoters (11,18). From
the structures, due to the overlapping nature of the
individual sites, the binding of one IRF molecule affects
the binding of the second molecule primarily through
DNA bending rather than direct contacts with each
other. Also, IRF-3 appears to be capable of recogniz-
ing both consensus and non-consensus sites and IRF-7
can better accommodate sites with G:C base pairs
upstream of the AA element. In order to fully understand
the structural and thermodynamic determinants of IRF
DNA recognition, it is important to characterize the struc-
tural changes that occur upon DNA binding. To address
this issue, we have solved the structures of IRF-3 and
IRF-7 DBDs in the absence of DNA. Our data shows
that differences in the flexibility of loop L1 in IRF
proteins play an important role in DNA-specific binding.
MATERIALS AND METHODS
The cDNA fragments encoding human IRF-3 DBD
(residues 1–113) and mouse IRF-7 DBD (residues 1–134)
were generated by PCR and cloned in pET-15b vectors
(Novagene). All point mutations were introduced by the
Quick-change site-directed mutagenesis kit (Stratagene).
Proteins were purified as previously described (18). Briefly,
the proteins were expressed in the Escherichia coli strain
BL21(DE3) pLyS (Stratagene). Cells were grown in LB
medium supplemented with ampicillin (100mg/ml) at
37?C to OD6000.5 and induced with 0.4mM IPTG for
3h. Clarified lysates were applied to nickel affinity columns
(Qiagen) obtaining protein up to 90% pure as judged by
SDS–PAGE. The His-6 affinity tags were removed by
thrombin digestion (Sigma). The proteins were then
loaded onto a SP-Sepharose column (GE healthcare)
equilibrated with 25mM HEPES pH 7.0, 50mM NaCl,
1mM DTT and eluted using a linear gradient of 0–
1.0M NaCl in buffer. Further purification was carried
out using a SD75 gel filtration column (GE healthcare)
equilibrated with a buffer containing 25mM Tris–HCl
pH 7.5, 300mM NaCl and 1mM TCEP. The purified
proteins were concentrated and used for crystallization.
Crystallization and data collection
Selenomethionine (Semet) crystals of IRF-3 DBD were
obtained by mixing equal volumes of protein solution
(30mg/ml) and reservoir solution containing 6% PEG
1000, 100mM sodium acetate (pH=6.0), 300mM zinc
chloride and 8% cadaverine. Crystals grew at 20?C
within 2 days and belonged to space group P3221 with
unit cell dimensions of a=b=64.90 A˚, c=157.62 A˚.
Multiwavelength anomalous diffraction data (MAD)
were measured from a single frozen crystal at the
Advance Photon Source (APS, beamline 17ID) of
Argonne National laboratory. Data were measured at
(0.9796 A˚) and peak (0.9791 A˚) of the selenium edge ab-
sorption profile plus at remote point (0.9537 A˚). IRF-3
crystals diffracted to 2.1 A˚ resolution at APS. The data
were truncated to 2.3 A˚, due to the presence of an ice ring
in the 2.1–2.25 A˚resolution range.
IRF-7 DBD apo crystals were obtained mixing two
volumes of protein solution (46mg/ml) with one volume
of reservoir solution containing 18% PEG 1500. The
crystals grew over night and belonged to the trigonal space
group P31with unit cell dimension of a=b=68.59 A˚,
c=68.61 A˚. Crystals were flash frozen in liquid nitrogen
using mother liquor plus 20% glycerol as a cryoprotect-
Synchrotron Light Source (NSLS, beam lines X6A and
X29) at ?=1.0 A˚. The crystals diffracted to 1.3 A˚reso-
lution. The data were integrated and reduced using
HKL2000 package (19).
The structure of IRF-3 DBD was solved by molecular
replacement (MR) using one of the IRF monomers from
the IRF-3/PRDIII–PRDI complex (PDB id 2PI0) (18) as
a search model. The initial MR solution was obtained
using the program PHASER (20) and included only two
molecules in the asymmetric unit. The inspection of
density map calculated with this solution suggested the
presence of another IRF-3 molecule in the asymmetric
unit, which was found by the program MolRep (21). An
anomalous difference Fourier map was used to locate the
positions of selenium (and zinc) atoms. Structure refine-
ment was carried out by rounds of energy minimization
and B-factor refinement using the CNS package (22).
Manual model rebuilding was performed with the program
COOT (23) using composite omit maps generated by
CNS. Final cycles of restrained refinement were per-
formed usingthe programs
PHENIX (25). The final model contains three monomers
in the asymmetric unit, named IRF-3A (amino acids 5–40
and 49–110), IRF-3B (amino acids 5–39 and 49–110),
IRF-3C (amino acids 5–39 and 49–110); 9 zinc ions; 3
sodium ions; 7 chloride ions and 151 water molecules
(Figure 1A). The model presents excellent geometry,
with the Ramachandran analysis indicating that 94.6%
of the residues are in the allowed regions and 5.4% are
in the preferred regions (Table 1).
The structure of mouse IRF-7 DBD apo form was
solved by MR using a homology search model from the
human IRF-7 DBD bound structure (PDB id 2O61) (11),
generated by the program CHAINSAW (26). The initial
MR solution was obtained with the program PHASER,
which suggested three molecules in the asymmetric unit.
After a round of rigid-body refinement, the Rfactand Rfree
values were 47 and 48%, respectively. A significant twin
fraction (a=0.36) was detected by the merohedral crystal
twinning server (http://nihserver.mbi.ucla.edu/twinning/)
Reflections for the free set were thus selected using the
highest lattice symmetry [P3(bar)m] in order to avoid a
bias in the calculation of Rfree, due to the pseudo-
symmetry generated by the twinning. In the final step of
refinement, twining was included, with a significant improve-
ment of the refinement statistics (final Rfree=20.0%).
Nucleic Acids Research, 2011,Vol.39, No. 167301
Iterative rounds of restrained refinement and building
were performed in the same manner as IRF3-DBD apo.
The final model contains three monomers in the asymmet-
ric unit, named IRF-7A (amino acids 9–130), IRF-7B
(amino acids 9–125) and IRF-7C (amino acids 8–125);
287 water molecules and 3 sodium ions. The model has
excellent geometry with 98.0% of the residues in the most
favored regions of the Ramachandran plot, and no
residues in the disallowed regions (Table 1). All figures
were made with the PyMol solftware (27).
Fluorescence anisotropy measurements
6-Carboxyfluorescein (6FAM) labeled (50-6FAM-GAGA
AGTGAAAGTGG-30) and unlabeled complementary
DNA oligonucleotides containing the PRDI consensus
Figure 1. Crystal structures of human IRF-3 DBD and mouse IRF-7 DBD. (A) Ribbon representation of three IRF-3 DBDs in the asymmetric unit.
IRF-3A is colored red, IRF-3B is colored blue, and IRF-3C is colored purple. Zinc atoms are colored green. Loop L1 is represented in dashed lines.
(B) Ribbon representation of IRF3-A. Chain was colored according to the secondary structure assignment in the alignment (see below). Missing
residues in loop L1 are represented in dashed lines. (C) Structural based sequence alignment of IRF DBDs. Missing residues in the apo IRF-3 DBD
structure are highlighted in red. The secondary structure elements in IRF-3 DBD are shown on top of the alignment and colored according to B.
Missing region in loop L1 is shown in dashed lines. Unique residues in L1 of IRF-7 are highlighted in green. (D) Ribbon representation of three apo
IRF-7 DBDs in the asymmetric unit. IRF-7A is colored green, IRF-7B is colored cyan, and IRF-7C is colored purple. (E) Ribbon representation of
IRF-7A. The chain was colored according to the secondary structure assignment in the alignment.
7302Nucleic Acids Research, 2011,Vol.39, No. 16
site were purchased form IDT DNA Technologies
(Coralville, IA, USA). Oligos were resuspended in
1?TE buffer supplemented with 100mM NaCl. DNA
duplex were formed by heating to 95?C a mixture of one
equivalent of the 6-FAM-labeled strand with one equiva-
lent of the complementary strand and permitting the
sample to cool down to room temperature. Each
reaction sample (volume 150ml) consisted of 5nM of
50-Fluorescein-labeled DNA and increasing concentra-
tions of IRF-3 DBD or IRF-7 DBD (0.5nM–300mM) in
a buffer containing 25mM HEPES pH 7.0, 50mM NaCl.
Reactions were incubated at 20?C for 15min prior to
measurement. Fluorescence anisotropy intensity data
were collected on a Beacon 2000 fluorescence polarization
system (Invitrogen) by setting excitation filter at 490nm
and emission filter at 520nm. The fraction of DNA bound
(B) was calculated using the following equation:
where [A]xequals the anisotropy measured at protein con-
centration X, [A]DNA is the anisotropy in absence of
protein and [A]finalis the anisotropy value at saturation.
Each data point was calculated from an average of five
anisotropy measurements. DNA fraction bound values
were then plotted versus domain concentration and the
data were fitted by hyperbolic regression, using Origin
6.1 (OriginLab), to the following equation:
where B is the fraction of DNA duplex bound, D0is the
total concentration of the DBD, and Kdis the dissociation
constant of the complex.
As in previous structures of IRF DBD/DNA complexes,
the apo forms of IRF-3 and IRF-7 DBD’s retain an a/b
architecture consisting of three a helices (a1–a3) flanked
by a four-stranded antiparallel b-sheet (b1–b4), a variant
of the helix-turn-helix (HTH) motif found commonly in
transcription factors (Figure 1B and E) (28). As is char-
acteristic for this family, three large loops (L1– L3)
connect different parts of the secondary structure. Loop
L1 connects b2 and a2, loop L2 connects a2 and a3, and
loop L3 connects b3 and b4. The electron density for
apoIRF-3 DBD is well defined with the exception of 4
residuesat the N-terminus
residues from loop L1. In the case of IRF-7 DBD, only
the eight residues at the N-terminus are not defined. The
apo IRF-3 and IRF-7 DBD structures superimpose with
an RMSD of 1.39A˚, but if the superposition is performed
with only the three a-helices and the b-sheet, the RMSD
decreases to 1.19A˚. The largest deviations between the
two structures occur in loops L1 and L2.
IRF-3 DBD apo form
The three IRF-3 DBD molecules in the crystallographic
asymmetric unit are arranged with their loops (L1–L3)
and helix a3 exposed to the solvent while the rest of the
molecules pack against each other. Interestingly, the apo
IRF-3 DBD crystals grow only in the presence of ZnCl2.
We identified nine Zn+2ions via their anomalous signal
and together these ions mediate intermolecular inter-
actions between the three symmetry-related molecules
by coordinating a set of Asp, Glu and His residues
(Figure 1A), as well as chloride ions. The coordination is
typically tetrahedral with an average Zn2+-ligand distance
of 2.1A˚(29). A superposition of the three molecules yields
a low RMSD value of 0.5 A˚2for 98 Ca’s. The largest
deviation occurs in the linker between helix a3 and
strand b3 (RMSD of 1.47A˚) of molecule C and molecules
A/B. A notable feature of the structure is the absence of
density for most of loop L1 (amino acids Gly41–Asn48) in
all three molecules of the asymmetric unit (Figure 1A).
In the previous IRF-3-DNA complex structures, loop
L1 interacts with the DNA minor groove, with residues
Trp38, Gly41, Leu42 and Gln44 contacting the DNA
backbone and His40 making water-mediated contacts
with the two contiguous A:T base pairs upstream of the
GAAA core sequence (AANNGAAA) (11,17,18). The
loop is somewhat flexible in the DNA complexes (average
B-factor of ?70A˚2) but, as we show here, in the absence
Table 1. Data collection and refinement statistics
Data collectionhIRF3-DBD mIRF7-DBD
Molecules in AU
Cell parameters (A˚)
Data coverage (%)
Average B factors (A˚2)
RMSD bonds (A˚)
RMSD angles (?)
Ramachandran quality plot
Most favored (%)
aHigh-resolution shell (2.2–2.1 A˚).
bHigh-resolution shell (1.35–1.30 A˚).
Rsym¼ 100 ?PjI ? hIij
observed and calculated structure factors, respectively (Fo>0s). Rfree
was determined from ?5% of the data
Rfact¼ 100 ?PjjF0j ? jFcjjÞ=PjF0jðÞ,
Nucleic Acids Research, 2011,Vol.39, No. 16 7303
of DNA, it is largely disordered (Figures 1B and 2A).
Also, residues that flank the loop deviate substantially in
conformation from that observed in complex with DNA.
For example, the Ca of Phe51 is shifted by ?10A˚and the
Ca of His40 is displaced by ?3.7A˚. Also, side chain
density of His40 is defined only in one of the three mol-
ecules in the asymmetric unit (Figure 2A). With the excep-
tion of loop L1 (and the flanking residues), the rest of the
apo structure (including loops L2 and L3) superimposes
well with the DNA complex, with an RMSD of ?1.06A˚
for 98 Ca’s (Figure 3A). Most of the conformational
changes are limited to the local rearrangement of side
chains. Most notably, Lys77, Arg78, Arg81 and Arg86
(on helix a3) that make in contacts with the DNA in the
complex adopt different (or multiple) conformations in
the absence of DNA (Figure 3A) (18).
IRF-7 DBD apo form
Although the IRF-7 DBD crystallizes in a different space
group than the IRF-3 DBD there are again three mol-
ecules in the asymmetric unit (Figure 1B). The three mol-
ecules superimpose with a maximum RMSD of ?0.5A˚for
Figure 3. Conformational changes in IRF-3 DBD and IRF-7 DBD
upon DNA binding. (A) Structural comparison between apo and
DNA-bound forms of IRF3-DBD. Apo IRF-3 DBD superimposes
with an RMSD of 1.04 A˚ on the DNA bound form (PDB ID=2PI0
chain B). Note the disordered region in loop L1, represented by a
dashed line in apo IRF-3 DBD. Side chains of His40 and Leu42 on
loop L1 and Lys77, Arg78, Arg81 and Arg86 on Helix a3 are repre-
sented by sticks. DNA molecule from the complex is colored in grey,
where the core recognition sequence (AANNGAAA) is highlighted in
yellow. (B) Structural comparison between apo and DNA-bound
forms of and IRF-7 DBD. Apo mouse IRF-7 DBD superimposes
with an RMSD of 1.41 A˚ on the DNA-bound form of human IRF-7
DBD (PDB ID 2O61). Side chains of His44 and Phe45 on loop L1
and Lys92, Thr93 and His100 on helix a3 of mouse IRF-7 (and
counterparts on human IRF-7) are represented by stick. Note that
helix a2 is extended and all three loops (L1–L3) are ordered in apo
Figure 2. Structure of loop L1 in IRF-3 DBD and IRF-7 DBD. Stereo
view of the 2Fo–Fcelectron density map for loop L1 region of IRF-3A
DBD (A, red) and IRF-7A DBD (B, green), contoured at 1s. For
clarity L1 is colored in yellow in both structures. IRF-7C DBD is
colored cyan (B). Note that loop L1 in IRF-3 DBD lacks density
form residues 41–48, whereas in IRF-7 DBD there is good definition
for the density.
7304Nucleic Acids Research, 2011,Vol.39, No. 16
117 Ca’s. In contrast to the apo IRF-3 DBD, loop L1 is
well defined (Figure 1E and 2B). This ordering of loop L1
in IRF-7 does not appear to be a crystallization artifact,
as the conformation of the loop is almost identical in
all three molecules in the asymmetric unit despite their
different crystal packing environments. Loop L1 in
IRF-7 differs from that in other IRF family members in
containing a phenylalanine (Phe45 instead of an alanine)
after the conserved histidine (His44) (Figure 1C). Phe45
packs against Leu50 (also unique to IRF-7) in loop L1
and Phe58 in helix a2, as part of the hydrophobic core
of the IRF-7 DBD (Figure 2B). Together, these hydropho-
bic interactions appear to confer a more restrictive con-
formation to loop L1, which is reflected in lower average
B-factors for this region (?25 A˚2) compared with the same
region in IRF-3.
Loop L2 is much longer in the IRF-7 DBD than in
other IRFs and has been proposed to interact with the
p50 subunit of NF-kB when the two transcription factors
bind to adjacent sites on the interferon-b enhancer (10).
Because of its length and the abundance of proline
and glycine residues, it is the most flexible segment of
the IRF-7 DBD structure. Indeed, in the structure of
IRF-7 bound to DNA, loop L2 is partially unstructured
(11). In the apo structure, loop L2 is well defined but
has the highest average B-factors (?29 A˚2) (Figure 3B).
Interestingly, residues 71–78 in L2 are unique to IRF-7
and adopt an extended conformation characteristic
of poly glycine and poly proline chains (Figure 3B).
This extended conformation appears to be important
in allowing Arg67 on loop L2 to contact Asn75 on
Intriguingly, the recognition helix a3 in the apo struc-
ture is longer at the N-terminus by three amino acids
when compared to IRF-7 DBD in the presence of DNA
(Figure 3B). Among these amino acids is Arg89, which
in the DNA complex makes contacts with the DNA
backbone but in the absence of DNA is directed toward
the interior of the protein and makes contacts with struc-
tural water in the protein cavity. Interestingly, Arg89
in apo IRF-7 adopts a similar conformation as Lys92 in
DNA complex, pointing to a switch in position between
the two residues upon DNA binding. One feature of
helix a3 that is conserved in the apo and DNA bound
forms of IRF-7 is a kink in the helix at Gly90, indicating
that this bending is not a consequence of the DNA
We also identify a putative metal ion between helix a3
and strand b3 in the apo structure that is coordinated with
carboxylic groups of Leu99, Gly103 and Phe105. The
metal (possibly Na+from the crystallization mix) appears
to compensate for the large dipole moment of long helix
a3. A similar metal binding site is located in the DNA
bound structure of IRF-2 (16).
Mutations in loop L1
The structures of all IRF/DNA complexes have shown
that a conserved histidine on loop L1 (His40 in IRF-3
and His44 in IRF-7) partially enters the DNA minor
groove and makes water mediated contacts with A:T
base pairs upstream of the GAAA core sequence
(AANNGAAA). The observed difference in flexibility of
loop L1 in IRF-3 and IRF-7 DBDs raises the question of
the effect of mutating residues in loop L1 on DNA
binding. To address this, we constructed two mutants:
(i) IRF3-H40A, in which His40 in IRF-3 DBD is
mutated to alanine, (ii) IRF7-H44A, in which His44 in
IRF-7 DBD is also mutated to alanine. The wild-type
(WT) and mutant proteins were tested for DNA binding
by fluorescence anisotropy. Figure 5 shows the result
obtained using the PRDI DNA element with sequence G
AGAAGTGAAAGT. Our experiments show that IRF-3
DBDwt binds with a dissociation constant of 486nM,
while IRF-7 DBDwtbinds with a slightly lower affinity
of 630nM. These values are consistent with previously
reported affinities of IRFs proteins for the PRD-I
element (30). However, the affinity of IRF-3-H40A de-
creases by ?3.6-fold (Kd=1706nM), IRF-7-H44A binds
with almost the same affinity (Kd=532nM) as the WT
We present here the first crystal structures of IRF-3 and
IRF-7 DBDs in the absence of DNA. Superposition of
apo and DNA bound IRF-3 DBDs reveals two primary
N-terminus of IRF-3 undergoes a disorder to order tran-
sition, with Lys5 making contacts with DNA along
the minor groove, (ii) loop L1 becomes ordered and ap-
proximately 10 residues that are not visible in the apo
IRF3 structure are well defined in the DNA-bound struc-
ture. In contrast to IRF-3, the L1 loop is ordered in the
apo IRF-7 DBD structure and the major change is a re-
arrangement of the loop as a rigid body around the minor
groove. The inherent flexibility of loop L1 is consistent
with the apo NMR structure of IRF-2 DBD, wherein
L1 displays a large number of conformations (31). A
conserved PWKH motif in loop L1 appears to be import-
ant in positioning L1 for its interaction with DNA. That
is, the proline (Pro37 in IRF-3) fixes the adjoining trypto-
phan (Trp38) in an outward conformation for interaction
with the DNA backbone. The indole ring of this trypto-
phan moves by about 1.6 A˚ in order to interact with a
DNA phosphate group. At the same time, the main chain
carbonyl group of Lys39 is positioned to make a hydrogen
bond with Lys77 (on the recognition helix a3), providing
stability to the overall loop conformation. Notably, the
imidazole ring of His40 swings by almost 6 A˚ into the
DNA minor groove in order to make water-mediated
contacts with A:T base pairs, while the main chain
carboxyl group of His40 interacts with the main chain
amide group of Arg43 to further stabilize the loop
In contrast to IRF-3, the L1 loop is ordered in the apo
IRF-7 DBD structure and stabilized in part by two hydro-
phobic residues (Phe45 and Leu50) that fold back into the
core of the protein. Upon DNA binding, the loop
undergoes a rigid body transition of ?2 A˚
around two points at both ends of the loop. At the
DNAbinding: (i) the
Nucleic Acids Research, 2011,Vol.39, No. 167305
N-terminal end, the Ca of the conserved residue Trp42
moves by about 1 A˚ in order to interact with the DNA
backbone. Moreover, the displacement of Trp42 pushes
against the other pivot site in residue Phe60. Similarly to
IRF-3 DBD, the imidazole ring of the conserved Histidine
residue (His44) rotates around the chi1 bond by ?25?to
position itself to interact with the minor groove. The dif-
ference in the inherent flexibility of loop L1 between
IRF-3 and IRF-7 seems to be reflected on the effect of
His mutations on DNA binding. Thus, while mutation
of His40 to alanine significantly decreases DNA binding
of IRF-3 by ?3.6-fold, the equivalent mutation in IRF-7
shows no significant change in binding (Figure 5). The
decrease in binding with the IRF-3 H40A mutation
likely represents the loss of interactions that stabilize
loop L1 upon DNA binding, including the interactions
between His40 and the DNA minor groove. However,
because loop L1 is already ordered in apo IRF-7 and
His44 is involved in interactions with Ser112 and several
water molecules, it is conceivable that there is less of a gain
in enthalpy when His44 (compared to His40 in IRF-3)
interacts with DNA. Our data suggest that differences in
the inherent flexibility of loop L1 between IRF-3 and
IRF-7 have a direct effect on DNA binding and may
play a role in the distinct DNA binding specificities
observed between the two proteins. Indeed, there is some
indication from biochemical studies that IRF-3 and IRF-7
interact differently with DNA. IRF-7, for example, is
more tolerant than IRF-3 to changes in the AA
sequence upstream of the GAAA core (AANNGAAA)
suggesting a somewhat looser interaction with DNA
(9,31). To more fully explore the differences between
IRF-3 and IRF-7 in DNA selection will require additional
mutations and a more detailed thermodynamic analysis on
several DNA sites.
Taken together, we show here that the structures apo
IRF-3 and IRF-7 DBD’s are generally similar to the DNA
bound forms, including a kink in the recognition helix a3
and ordered loops L2 and L3. The primary difference
is in loop L1, which undergoes a disorder to order transi-
tion in the IRF-3 DBD and a conformational change
in the IRF-7 DBD. The varying intrinsic flexibility of
loops and tails in the IRF family may serve as a mechan-
ism to modulate the binding specificity of its members and
Figure 4. Structural comparison of Loop L1 between apo and DNA bound form of IRF DBDs. (A) Close up view of Loop L1 of IRF-3 DBD apo
(magenta) and bound (green) forms. Conserved histidine residue (His40) is shown in stick representation. (B) Close up view of loop L1 of IRF-7
DBD apo (blue) and bound (orange) forms. Conserved histidines (His46 on human and His44 on mouse) are shown in stick representation. For
clarity, DNA is not shown. Loop L1 and the conserved histidine adopt similar conformations when bound to DNA, but adopt different (IRF-7) or
disordered (IRF-3) conformations in the absence of DNA.
Figure 5. Effect of mutations in loop L1 on DNA binding. Isothermal
binding curves over the PRDI consensus DNA site for IRF-3 DBD
wild type (black squares), IRF-3 DBD H40A (red dots), IRF-7 DBD
wild type (green triangles) and IRF-7 DBD H44A (blue inverted tri-
angles). The calculated Kd’s are shown in the table.
7306Nucleic Acids Research, 2011,Vol.39, No. 16
to respond to a larger population of diverse promoter
Coordinates have been submitted to the RCSB Protein
Data Bank with accession codes: 3QU6 for IRF-3 and
3QU3 for IRF-7
We thank the staff at APS beamline 17ID and NSLS
beamline X6A for help with data collection.
US National Institutes of Health (R01 AI41706 to A.K.A.
and R01 GM092854 to C.R.E.). Funding for open access
Conflict of interest statement. None declared.
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