Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain

Abstract

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered
as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95 Å structure
of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response
element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD,
which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part
of the C-terminal extension (CTE) of the EcRDBD folds into an α-helix whose location in the minor groove does not match any
of the locations previously observed for nuclear receptors. Mutational analyses suggest that the α-helix is a component of
EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to
be a general feature of all known EcRs.

Full text

Published online 10 April 2007 Nucleic Acids Research, 2007, Vol. 35, No. 8 2705–2718
doi:10.1093/nar/gkm162
Novel DNA-binding element within the C-terminal
extension of the nuclear receptor DNA-binding
domain
Michał Jako
´b
1
, Robert Kołodziejczyk
1
, Marek Orłowski
1
, Szymon Krzywda
2
,
Agnieszka Kowalska
1
, Joanna Dutko-Gwo
´z
´dz
´
1
, Tomasz Gwo
´z
´dz
´
1
,
Marian Kochman
1
, Mariusz Jasko
´lski
2,3
and Andrzej O_
zyhar
1,
*
1
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrze_
ze Wyspian
´skiego
27, 50-370 Wrocław, Poland,
2
Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University,
Poznan
´, Poland and
3
Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy
of Sciences, Poznan
´, Poland
Received January 10, 2007; Revised March 2, 2007; Accepted March 5, 2007
ABSTRACT
The heterodimer of the ecdysone receptor (EcR)
and ultraspiracle (Usp), members of the nuclear
receptors superfamily, is considered as the func-
tional receptor for ecdysteroids initiating molting
and metamorphosis in insects. Here we report
the 1.95 A
˚structure of the complex formed by the
DNA-binding domains (DBDs) the EcR and the Usp,
bound to the natural pseudopalindromic response
element. Comparison of the structure with that
obtained previously, using an idealized response
element, shows how the EcRDBD, which has been
previously reported to possess extraordinary flex-
ibility, accommodates DNA-induced structural
changes. Part of the C-terminal extension (CTE) of
the EcRDBD folds into an a-helix whose location in
the minor groove does not match any of the
locations previously observed for nuclear receptors.
Mutational analyses suggest that the a-helix is a
component of EcR-box, a novel element indispens-
able for DNA-binding and located within the nuclear
receptor CTE. This element seems to be a general
feature of all known EcRs.
INTRODUCTION
Multicellular organisms require specific intercellular
communication to properly orchestrate the complex
body plan during embryogenesis and to maintain its
function during the entire lifespan. Classical signal
transduction cascades are initiated by ligand binding to
membrane-anchored receptors, eventually changing the
activity of specific nuclear transcription factors. In
contrast, members of the nuclear receptor superfamily
transduce their signals directly. The receptors have
evolved to combine the functions of signal responsiveness,
DNA-binding and transcriptional activation into one
protein composed of functionally separated modules/
domains (1). A typical nuclear receptor is composed of the
N-terminal AB region, the DNA-binding C domain, a
hinge D region, the ligand-binding E domain and the
C-terminal F region. The core DNA-binding domain
(DBD), which is a defining feature of the family (2), plays
a central role in the correct positioning of the receptors,
and complexes recruited by them, close to the genes whose
transcription is affected (3). To achieve this aim, the DBD
must overcome the challenge of finding small cognate
response elements within the entire genome. A long-
standing question is therefore, how this selection is
achieved, given that nuclear receptors employ a highly
conserved DBD and a set of response elements, which is
quite limited in both sequence and structure. To solve this
mystery on the molecular level, crystal structures of some
nuclear receptor DBDs in complex with DNA have been
analyzed. Unfortunately, most of the research in this field
has been carried out using idealized, highly symmetric
DNA duplexes, disregarding the fact that natural
response elements are characterized by high-sequence
degeneracy (3).
Representatives of the nuclear receptor superfamily
have been identified in almost all classes of metazoans and
the availability of complete genome sequences has
revealed some interesting data regarding the occurrence
of nuclear receptors. For example, the human genome
sequence reveals 48 members of the family with 21 genes
representing receptors with known ligands, usually small
lipophilic molecules, including steroids, the thyroid
*To whom correspondence should be addressed. Tel: þ48-71-320-6333; Fax: þ48-71-320-6337; Email: andrzej.ozyhar@pwr.wroc.pl
ß2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

hormone, retinoic acids and vitamin D (1). In contrast to
the complexity of the human hormone signaling pathways,
Drosophila melanogaster has only one lipophilic hormone
acting as a nuclear receptor ligand, the steroid hormone
20-hydroxyecdysone (20E), and its genome contains only
18 nuclear receptor genes (4). This makes D. melanogaster
an ideal system for studying nuclear-receptor function and
regulation. The 20E hormone, which is considered to be a
principal determinant of developmental timing, controls
diverse biological processes, including morphogenetic,
apoptotic, physiological, reproductive and behavioral
responses (5,6). Like vertebrate steroid hormones, 20E
exerts its effects via a member of the nuclear receptor
superfamily, a product of the EcR gene (7). Although
ecdysone receptor (EcR) can bind 20E on its own (8), the
binding is greatly stimulated (9) by its heterodimerization
partner, a product of the ultraspiracle gene (Usp), which is
another member of the nuclear receptor superfamily and
the D. melanogaster ortholog (10) of the mammalian
retinoid X receptors (RXRs). Since it has been also
observed that ligand binding stabilizes the EcR/Usp
complex and increases its affinity for 20E-response
elements, the EcR/Usp heterodimer is believed to be the
only functional form of the 20E receptor (11). Although
molecular studies of the EcR/Usp heterodimer are not as
advanced as those of vertebrate heterodimeric receptors, it
is already clear that the ecdysteroid receptor complex
holds an exceptional position within the nuclear receptor
family. It has been shown, for example, that the ligand-
binding domain (LBD) of EcR is characterized by unusual
flexibility and adaptability, which allows for molding of
this domain around different ligands (12). Although 20E
is thought to elicit most of the above-mentioned physio-
logical responses, mounting evidence indicates, that
alternate signaling pathways exist that are driven by
ecdysteroids other than 20E, some of which are present at
specific stages during development (13). According to a
recent hypothesis, ‘conformational compatibility’ between
the cognate receptor’s LBD and an ecdysteroid molecule
would determine the initiation of genomic versus
non-genomic response pathways (14). Interestingly, the
DBD of EcR appears to possess high plasticity as well
(15). Therefore, EcR could adopt different, although
ligand- and response-element-specific conformations
evoking numerous ecdysteroid-dependent effects (14).
Another feature distinguishing the EcR/Usp heterodimer
from its vertebrate counterparts, which tend to form
complexes on inherently asymmetric DNA-binding sites
composed of directly repeated half-sites, is its propensity
for response elements arranged as psudopalindromes with
a single intervening nucleotide (16). Our mutational
studies of the interaction of the Usp and EcR DBDs
(UspDBD and EcRDBD, respectively) with the pseudo-
palindromic response element from the hsp27 gene
promoter (hsp27pal), have demonstrated that natural
pseudopalindromic ecdysone response elements may act
as functionally asymmetric elements that fix the Usp/EcR
heterodimer in a specific orientation. In particular, it has
been shown that UspDBD, which preferentially
binds the 50half-site of hsp27pal, operates as a key
factor dictating the polarity of the heterocomplex
(50-UspDBD-EcRDBD-30) (16,17). Although this polarity
was verified by the crystal structures of the
UspDBD/EcRDBD and RXRDBD/EcRDBD
(RXRDBD/EcRDBD) complexes with an idealized
non-natural element organized as an inverted repeat of
the 50-AGGTCA-30sequence separated by 1 bp (IR-1)
(18), many important observations coming from earlier
biochemical studies could not be confirmed. In particular,
the previously reported importance of the EcRDBD
C-terminal extension (CTE) sequence for effective forma-
tion of the UspDBD/EcRDBD heterocomplexes (16)
could not be explained because the crystallographic data
allowed the visualization of only a few CTE residues,
mostly in the RXRDBD/EcRDBD-IR-1 complex, but not
in the UspDBD/EcRDBD-IR-1 complex.
To ultimately elucidate the molecular basis for DNA
target specificity of the UspDBD/EcRDBD heterodimer,
we have solved the crystal structure of both domains
bound to the natural response element from the hsp27
gene promoter. Our data reveal important molecular
details of the UspDBD/EcRDBD–DNA interaction,
which could not be observed previously, when unnatural
DNA response element was used for crystallization. Most
importantly, our crystallographic data demonstrate that
part of the CTE of EcRDBD folds into an a-helix whose
location in the minor groove does not match any of the
locations previously observed for other CTEs of other
nuclear receptors. Analysis of the crystallographic data,
together with mutational analyses, suggest that the a-helix
is a part of a novel DNA-binding supporting element,
previously unobserved for any of the nuclear receptors.
This element, which we refer to as EcR-box, seems to be
a characteristic feature of all known EcRs.
MATERIALS AND METHODS
Construction of expression vectors, site-directed mutagenesis,
overexpression and purification of the wild-type and
mutant proteins
The plasmid pGEX-2T (Amersham Biosciences, Freiburg,
Germany) containing the lacIq gene was used for the
expression of DBDs as fusion proteins with Schistosoma
japonicum glutathione-S-transferase (GST) in Escherichia
coli strain BL21(DE3)pLysS (Novagen, Germany).
The construction of the expression plasmids for the
wild-type D. melanogaster EcR, Usp GST-DBDs and
the wild-type Bombyx mori EcR GST-DBD has been
described previously (15,19). The expression plasmid
for the wild-type B. mori UspDBD (pGEX-
2T UspDBD
(104–202)
_Bm) was constructed using the
following primers: 50-gcccggggatccGCACCTCGACAG
CAAGAG-30(sense) and 50-gcccggggatccGTCTTCGAC
TGTGGTCGTA-30(antisense). Small letters in the above
sequences represent nucleotides added for cloning purpose
whereas the restriction sites are shown in italics. The
PCR-based megaprimer mutagenesis protocol (20) was
used to introduce new alanine codons within the cDNA
region encoding D. melanogaster EcRDBD A-box and
standard PCR to introduce deletion mutations within
the CTE of D. melanogaster and B. mori EcRDBD.
2706 Nucleic Acids Research, 2007, Vol. 35, No. 8

The plasmids: pGEX-2T EcRDBD
(256–364)
_Dm and
pGEX-2T EcRDBD
(199–307)
_Bm (15) were used as tem-
plates. The sequences of the recombinant cDNA frag-
ments were verified by dideoxy sequencing. Expression of
GST-DBDs and purification of wild-type and mutated
DBDs as GST-free proteins was performed using a
procedure described previously for UspDBD with deleted
C-terminal sequence (UspDBD
A/T
) (16). The concentra-
tion of the purified proteins was determined spectro-
photometrically at 280 nm using absorption coefficients
calculated according to Gill and von Hippel (21).
DNA-binding assays
The electrophoretic mobility shift assays (EMSA)
experiments (22) were performed under conditions
described previously (15) using
32
P-labeled ds oligo-
nucleotide 50-AGCGACAAGGGTTCAATGCACTTGT
CCAATGAA-30(only one strand is shown), based on
the natural 20E pseudopalindromic response element
(underlined) from the D. melanogaster hsp27 gene
promoter (23,24).
Crystallization and data collection
The protein–DNA complex was prepared using stoichio-
metric amounts of UspDBD, EcRDBD and duplex DNA
(for details see Figure 1) concentrated to 0.5 mM
(each species) in 15 mM MES buffer, pH 5.5, 50 mM
NaCl, 1 mM DTT, 5 mM ZnCl
2
, using AmiconÕUltra-4
Centrifugal Filters (Millipore, Poland). Best crystals
(0.6 0.1 0.015 mm) were grown at 208C in sitting
drops made of 1 ml of the protein–DNA complex solution
and an equivalent volume of precipitant solution, contain-
ing 21% PEG 3350, 100 mM MES buffer, pH 5.5, 100 mM
NaCl, 10 mM MgCl
2
, 1 mM DTT, 5 mM ZnCl
2
, LiCl or
10 mM urea.
For X-ray diffraction experiments the crystal was flash-
frozen to 100 K in nitrogen gas stream without additional
cryoprotection. Diffraction data extending to 1.95 A
˚
resolution were collected using a MAR Research CCD
detector at the EMBL beamline X11 of the DESY
synchrotron (Hamburg, Germany) operated at a wave-
length ¼0.8115 A
˚. The data were indexed, integrated
and scaled using the HKL package (25). The crystals
belong to the P2
1
space group with unit cell parameters
a¼46.7, b¼59.8, c¼65.2 A
˚,b¼106.728and contain one
complex molecule per asymmetric unit. Data collection
statistics are summarized in Table 1.
Structure solution and refinement
The structure was solved by molecular replacement using
the genetic algorithms EPMR program (26) and the
UspDBD/EcRDBD-IR-1 structure (18) (PDB ID 1R0O)
as a model. For the diffraction data between 15 and 4 A
˚,
the program located the expected single copy of the
complex in the asymmetric unit with a correlation
coefficient of 55%.
The model was manually rebuilt into electron
density maps during iterative cycles of modeling in
Quanta 2000 (Accelrys Inc., San Diego, CA, USA)
which alternated with maximum-likelihood restrained
refinement as implemented in the Refmac5 program
from the CCP4 suite (27). Two hundred and twenty-two
water molecules were added using the X-Solvate module
of Quanta 2000 at the final stages of the refinement.
The final model contains 165 amino acid residues
(78 UspDBD, 87 EcRDBD), 20 base-paired nucleotides,
four zinc cations and 222 water molecules. The refinement
converged with Rand R
free
factors of 0.180 and 0.217,
respectively.
Coordinates
The atomic coordinates and structure factors have been
deposited in the PDB with the accession code 2HAN.
RESULTS
Crystallization and structure determination
An 86-resiude fragment of D.melanogaster UspDBD
consisting of residues 10 to þ76 and a 109-residue
fragment of D. melanogaster EcRDBD, residues 8to
þ101 (Figure 1A, B), were expressed individually in E. coli
and purified to homogeneity. The primary structures of
the DBDs were the same as the structures of the DBDs
used previously in crystallization studies with an idealized
fully symmetric IR-1 element (18). Here, the purified
DBDs were co-crystallized with a DNA fragment contain-
ing the natural pseudopalindromic 20E response element
from the D. melanogaster hsp27 gene promoter (hsp27pal,
Figure 1C). The structure was solved by molecular
replacement using the PDB coordinates 1R0O of the
UspDBD/EcRDBD-IR-1 complex (18) as the search
model. The asymmetric unit of the crystal contains one
copy of the UspDBD/EcRDBD-DNA complex. The final
model, refined to 1.95 A
˚resolution, contains residues 3
to þ76 of UspDBD, residues 6toþ81 of EcRDBD, the
complete DNA and 222 water molecules. The refinement
is summarized in Table 1.
Four side chains of the protein components have clearly
defined alternative conformations (see further). Three of
them are located at crucial protein–protein and protein–
nucleic acid interfaces. All the multiple conformations in
the interface areas have unambiguous definition in
electron density maps. The course of the refinement and
the final model were validated using the R
free
test (28).
Overall architecture of the complex
In agreement with biochemical data (16) and with the
structure of UspDBD/EcRDBD bound to an idealized
IR-1 element (18), the UspDBD/EcRDBD heterodimer is
bound to the hsp27pal element with a defined polarity,
where the UspDBD occupies the 50-half-site of hsp27pal
and EcRDBD the 30-half-site. A superposition of the
structure of the UspDBD/EcRDBD-hsp27pal complex
with the UspDBD/EcRDBD-IR-1 structure is shown in
Figure 2 and is characterized by an r.m.s.d.-value of
0.54 A
˚calculated for the corresponding Caatoms.
Although the overall fold of the two heterodimers is
similar, the present structure of the UspDBD/EcRDBD
complex interacting with the natural element reveals in
Nucleic Acids Research, 2007, Vol. 35, No. 8 2707

Figure 1. The protein and response element constructs used in crystallization and their contacts. Sequences and interactions (legend is provided
within the figure) are shown for UspDBD (A) and EcRDBD (B), respectively. The numbering of the amino acid residues is relative to the first
conserved cysteine, with the authentic numbers (7,10) appearing in parentheses The sequences defined previously (16) as corresponding to T-box (42)
and A-box (33,34) are highlighted in green and red, respectively. In gray boxes the N- and C-terminal residues not visible in the electron density
maps are listed. Cloning artifacts from the pGEX-2T plasmid are indicated by lower case letters. The a-helices are boxed and the residues from
b-sheets are circled following the definition of DSSP (43). (C) The hsp27pal-based DNA used in cocrystallization. The symbols are as in (A) and (B).
2708 Nucleic Acids Research, 2007, Vol. 35, No. 8

total eleven additional amino acid residues: three at the
N-terminus of EcRDBD (R-6, V-5, Q-4), six in the CTE of
EcRDBD (Q76, C77, A78, M79, K80, R81), i.e., in the
N-terminal region corresponding to the so-called A-box,
and one residue at each end of UspDBD (K-3, R75)
(Figure 1A, B). The N-terminal fragment of EcRDBD is
directed away from the DNA molecule into the solvent
and does not exhibit any specific interactions with the
DNA molecule. A remarkable feature of the UspDBD/
EcRDBD-hsp27pal complex is the presence of an a-helix
at the C-terminus of the EcRDBD molecule, involved in
interactions with the DNA. This fragment was disordered
in EcRDBD complexed with UspDBD on the non-natural
IR-1 element (18), and therefore its involvement in
binding the DNA response element is reported here for
the first time. Furthermore, this a-helix may be a part of a
unique structural element indispensable for effective
DNA-binding, previously unobserved for any of the
nuclear receptors (see subsequently).
According to recently published fluorescence resonance
energy transfer data, binding to the UspDBD/EcRDBD
heterodimer induces a significant bend of the hsp27pal
element. Steady-state data indicate a bend of about
23 38whereas a value of 21 48could be estimated
using fluorescence decay measurements. These observa-
tions were reinforced by gel retardation experiments where
the apparent bend was estimated as 20.98(29). To examine
if any distortion of the hsp27pal element could be observed
in the crystal structure, we analyzed the coordinate file
using the 3DNA software (30). The local helical para-
meters obtained from the 3DNA software were used as
input to the Madbend program for calculation of the bend
magnitude and global roll of the DNA molecules (31).
Calculations using Madbend with a reference plane in the
middle of the hsp27pal molecule indicated that this
element is bent toward the minor groove by 20.98
(Figure 3A). Devarakonda et al. (18) reported that in
the UspDBD/EcRDBD-IR-1 complex there was no
significant distortion of the IR-1 element, except for the
spacer. In a re-evaluation of those data, using the same
3DNA/Madbend procedure and the coordinate file
deposited for the UspDBD/EcRDBD-IR-1 complexes in
the PDB (accession code 1R0O) the bend angle could be
calculated as 24.18(Figure 3A). Therefore, we conclude
that formation of UspDBD/EcRDBD complexes on
different response elements is accompanied by well-defined
deformation of the DNA architecture (Figure 3B).
The basis for the recognition of the hsp27pal sequence by the
Usp and EcR core DBDs
As shown in Figure 3A the sequence of the naturally
occurring hsp27pal element is highly degenerated. Five of
the fifteen base pairs making up the two heptameric half-
site sequences deviate from perfect palindromic sequence.
In contrast, the idealized IR-1 is a fully symmetric
palindrome (Figure 3A), which according to gel shift
studies can bind the UspDBD/EcRDBD heterocomplex,
and also the complex of the full-length Usp and EcR, with
higher affinity than hsp27pal (16,32). A side-by-side
comparison of the UspDBD/EcRDBD-hsp27pal structure
solved here with the previously published crystallo-
graphic data of the UspDBD/EcRDBD-IR-1 complex
(18), demonstrates that complexes with natural and
idealized response elements differ significantly in the
mode of protein–DNA and protein–protein interactions.
Table 1. Crystallographic data. The values in parentheses represent the
values for the last resolution shell.
Data collection
Radiation source Beamline X11, EMBL Hamburg
Wavelength (A
˚) 0.8115
Temperature (K) 100
Space group P2
1
Unit-cell parameters (A
˚,8)a¼46.7, b¼59.8, c¼65.2, b¼106.7
Resolution (A
˚) 30–1.95
Number of observed reflections 242 671
Number of unique reflections 25 368
Redundancy 9.6
Completeness (%) 99.9 (100)
R
merge
0.068 (0.549)
5I/(I)424.2 (3.4)
Refinement statistics
Resolution range (A
˚) 26.92–1.95
Number of all/test reflections 23 899/1272
R/R
free
0.180/0.217
5B4(A
˚
2
)/number of atoms
Protein 36.0/1348
DNA 40.9/812
Zn
2þ
28.4/4
Solvent 38.1/222
R.m.s. deviations from ideal
Bond lengths (A
˚) 0.016
Bond angles (8) 2.03
Ramachandran statistics (%)
Most favored/additionally
allowed regions
84.4/14.3
Figure 2. Superposition of the UspDBD/EcRDBD-hsp27pal and
UspDBD/EcRDBD-IR-1 structures. Shown are UspDBD (red) and
EcRDBD (blue) bound to hsp27pal oligonucleotide (gold), as well as
UspDBD (yellow) and EcRDBD (green) bound to the IR-1 element
(gray). The UspDBD/EcRDBD-IR-1 structure (18) is based on the
coordinate file deposited in the PDB (accession code 1R0O).
Nucleic Acids Research, 2007, Vol. 35, No. 8 2709

Figure 3. Evaluation of the crystallographic data indicates bending in hsp27pal and IR-1 elements. (A) The ds oligonucleotides used in the
crystallographic analysis of the UspDBD/EcRDBD heterodimer complexed with the IR-1 and hsp27pal response elements. The sequences of the
response elements are shown in frames, and ovals represent localization of the respective DBDs. Models and parameters of the DNA molecules were
generated using the 3DNA software (30), data from this study and the atomic coordinate file of the UspDBD/EcRDBD-IR-1 structure (1R0O) (18).
DNA base pairs are shown as rectangular blocks, and the idealized helical axis based on the axis computed by the 3DNA software (dotted line) is
shown in black. (B) The minor and major grove widths of the hsp27pal (solid line) and IR-1 (broken line) ds oligonucleotides. Only the heptameric
half-sites are shown. The values were derived using the 3DNA software. The solid black line represent standard values for B-DNA.
2710 Nucleic Acids Research, 2007, Vol. 35, No. 8

The protein–DNA interactions for both complexes are
depicted schematically in Figure 4. In agreement with
previous structural characterizations of nuclear receptor
DBD–DNA complexes (3), the key sequence-specific base
contacts in the major groove are maintained in both
complexes mostly by residues from the so-called DNA-
recognition a-helix (for definition see Figure 1A, B).
However, as shown in Figure 4, equivalent amino
acid residues generate significantly different interaction
patterns. Surprisingly, this is also true for residues
interacting with identical DNA sequences, for example
base pairs from positions 3to1. In total, ten
interactions with the hsp27pal bases could be defined for
the UspDBD molecule, three direct (involving E19, K22,
R27) and seven water-mediated (involving K22, E19, K26,
R27, H12). In comparison, for the UspDBD interacting
with the IR-1 element only eight interactions with bases
could be observed in total, two direct ones (E19, R27)
Figure 4. Comparison of protein–DNA contacts observed for the hsp27pal and IR-1 ds oligonucleotides bound by the UspDBD/EcRDBD
heterocomplex. The crystallographic data obtained here for the UspDBD/EcRDBD-hsp27pal complex and published previously for the UspDBD/
EcRDBD-IR-1 complex (PDB accession code 1R0O) (18) were analyzed in order to identify protein–DNA interactions. The DNA double helices are
unwrapped to show schematically the base pairs and the respective contacts in the upstream (beige) and downstream (gold) half-sites of the hsp27pal
and IR-1 elements. Gray circles represent phosphodiester groups, black circles are ordered water molecules that mediate protein-DNA contacts.
Amino acid residues that contact the DNA using side chains are shown as red (EcRDBD) or blue (UspDBD) ovals. Asterisks denote residues which
assume two well-defined conformations. Red solid lines indicate direct or water-mediated specific hydrogen bonding to base pairs. Blue dashed lines
indicate hydrogen bonds to sugar-phosphate backbone.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2711

and six water-mediated (K22, E19, K26, R27, H12).
EcRDBD on the other hand makes seven contacts with
the hsp27pal 30half-site and eight with the 30half-site of
the IR-1. However, the impact of direct or water-mediated
contacts differs significantly for both elements. In parti-
cular, for the hsp27pal sequence there are two direct
(K22, E19) and five water-mediated contacts (E19, K22,
R27, R51) whereas for IR-1 four direct (E19, K22, R27,
R26) and four water-mediated (Y13, K22, R26) con-
tacts exist. Interestingly, only one residue, K22, of the
EcRDBD exhibits the same sequence-specific interaction
pattern in the UspDBD/EcRDBD-hsp27pal and
UspDBD/EcRDBD-IR-1 structures.
A novel feature observed in the UspDBD/EcRDBD
complex bound to the hsp27pal element is the ability of
several amino acid residues of the EcRDBD to assume
two well-defined conformations, which differ functionally.
The first of these residues is R26 from the DNA-
recognition a-helix. One of the R26 conformers makes
contact with the phosphate backbone, whereas the other
one, corresponding to that observed in EcRDBD com-
plexed to the IR-1 element, is not involved in any
interactions (Figure 5A, B). Similar observations could
be made for Q54 (data not shown). Another residue
assuming two conformations is R51, which belongs to a
group of residues forming the subunit interface. Here, one
of the conformers interacts with N51 of the UspDBD
(Figure 5C), similarly as in the EcRDBD in the UspDBD/
EcRDBD-IR-1 complex (not shown). Additionally, this
conformer forms a novel hsp27pal-specific direct
contact with the spacer A/T base-pair (Figure 5D).
The second R51 conformer forms a set of direct
and/or water-mediated contacts with UspDBD residues
(Figure 5C, D). Dual conformation was also observed for
R67, which is located on the surface of the EcRDBD
molecule (not shown).
An a-helix of the CTE segment of EcRDBD is indispensable
for efficient interaction with DNA
Earlier mutational and crystallographic studies of nuclear
receptors, bound to asymmetric response elements orga-
nized as direct repeats, have emphasized the functional
importance of the so-called CTE sequence of the core
DBD. It has been shown that the CTE, consisting of the
T-box and the adjacent a-helix (A-box), plays an
important role in response element recognition, especially
by vertebrate heterodimeric DBD complexes and by
DBDs, which bind their cognate sequences as monomers
(3). While the A-box residues are mainly involved in
specific contacts with the response element, the T-box
performs different functions, including specific base-pair
recognition, formation of the dimer interface and support
of the proper orientation of the A-box a-helix. In contrast
to the high-sequence conservation of the core DBD region
within the nuclear receptor superfamily, the CTE
sequences are not preserved. Consequently it has been
suggested that the CTE, as a DBD-characteristic element,
would play an important role in response element
discrimination by the DBDs interacting as hetero- and
homodimers with the inherently asymmetrical directly
repeated elements (33). The first experimental evidence
suggesting the CTE would be also essential for the
interaction of the DBDs with the palindromic response
elements, was published by Niedziela-Majka et al. (16)
who showed that deletion of the D. melanogaster
EcRDBD CTE fragment encompassing the A-box
sequence disrupted EcRDBD homo- and heterodimeriza-
tion with UspDBD on hsp27pal. Moreover, detailed
biochemical analyses demonstrated that the T-box of
EcRDBD plays an important role in binding to hsp27pal
(15). Unfortunately, these important functions of the CTE
fragment could not be fully explained by the crystal
structure of the UspDBD/EcRDBD heterocomplex
bound to the idealized IR-1 element (18) where none of
the A-box residues were included in the model. In
contrast, in the present structure obtained for the natural
hsp27pal element, seven residues of the EcRDBD A-box
(N75-R81) are clearly seen in electron density. As shown
in Figure 6, the residues form an a-helix, which definitely
rests in the minor groove suggesting that this part of the
EcRDBD may be involved in some interactions with
the DNA. An analysis of the electron density suggests that
the last two residues of the a-helix, K80 and R81, would
be the prime candidates for such interactions (Figure 6).
To validate this supposition we have obtained EcRDBD
derivatives where K80 or R81 were substituted by alanine
(Figure 7A). The binding affinities of the K80A and
R81A mutants were determined by EMSA using a
double-stranded oligonucleotide containing the original
hsp27pal sequence. As hsp27pal had been shown pre-
viously to bind specifically and in a cooperative manner
both the EcRDBD homo- and UspDBD/EcRDBD
heterodimer, and since biochemical experiments indicate
that A-box is necessary for these interactions (16), we
tested the putative influence of the alanine substitutions on
both homo- and heterodimer interactions. The binding of
DNA by EcRDBD homodimers was clearly reduced
by alanine substitutions at positions K80 and R81
(Figure 7B, C). The analyses presented in Figure 7D, E
reveal that substitution of K80 and R81 by alanine is also
detrimental to the effective formation of the UspDBD/
EcRDBD-hsp27pal complex. Importantly, as demon-
strated by EMSA results obtained for the respective
alanine mutants (Figure 7A), this is also true for some
other residues from the EcRDBD A-box, including K84,
K85, Q87, K88 and K90 (Figure 7B–E), which are not
visualized in the present structure. This observation is
further supported by the EMSAs results obtained for
EcRDBD CTE deletion mutants. As shown in Figure 8,
deletion of the CTE up to the residue K92 does not change
the affinity of the EcRDBD homo- and heterodimers
(Figure 8B–E). In contrast, more extensive deletions
encompassing residues identified here by means of crystal-
lography and/or directed mutagenesis, reduced (deletion
up to residue K85) or completely abolished (deletion up to
N75) the binding of the homo- and/or heterodimers
(Figure 8B, C and D, E). Together, the above results
clearly indicate that some of the EcRDBD CTE residues,
including K80 and R81, build up within the A-box
a discrete functional entity, which is indispensable
for the efficient interaction of the EcRDBD with DNA.
2712 Nucleic Acids Research, 2007, Vol. 35, No. 8

Figure 5. Four amino acids (R26, Q54, R51 and R67) of the EcRDBD assume two well-defined conformations in the UspDBD/EcRDBD complex
bound to the hsp27pal element. The R26 residue is from the DNA-recognition a-helix. One of the R26 conformers makes contact with the phosphate
backbone, whereas the second conformer, related to that observed in the IR-1 DNA complex, is not involved in any interactions (A, B). R51 is one
of the residues forming the DBDs interface (C, D). One R51 conformer interacts with residue N51 of the UspDBD (C) and simultaneously forms a
direct contact with the A/T base-pair (D). The second R51 conformer forms direct and water-mediated contacts with UspDBD (C, D). Hydrogen
bonds in the stereodiagrams (A-D) are indicated as black dotted lines. Water molecules are shown as red spheres. For more details concerning other
residues see text. The 2Fo-Fc electron density maps shown for selected side chains of the present UspDBD/EcRDBD-hsp27pal complex have been
contoured at the 1.0 level.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2713

The mutational analyses presented in Figures 7 and 8
indicate that this conclusion is also true for the EcRDBD
from B. mori.
DISCUSSION
The crystallographic data presented here for the
UspDBD/EcRDBD heterodimer in complex with the
natural pseudopalindromic response element from
the hsp27 gene promoter, and especially their comparison
with the data published previously for the UspDBD/
EcRDBD bound to the idealized IR-1 element (18),
demonstrate the basis of the molecular rearrangements
within both DBDs that permit them to adapt to different
DNA sequences. Most importantly, the use of the natural
element reveals some unexpected molecular details, which
could not be observed in the structure published
previously. Although the key sequence-specific base
contacts are maintained in both structures essentially by
the same amino acid residues from the DNA-recognition
a-helices of the UspDBD and EcRDBD, the details of
these interactions differ significantly and only one residue
(K22 from EcRDBD) exhibits the same sequence-specific
interaction pattern. At the same time, the UspDBD
seems to form more sequence-specific contacts with the
50half of the hsp27pal than with the corresponding part
of the IR-1. A total of ten and eight interactions could
be identified, respectively. On the other hand, EcRDBD
forms seven contacts with the hsp27pal sequence and
eight with the IR-1 element. Furthermore, the impact of
water-mediated contacts is significantly higher when
the EcRDBD interacts with the natural element. In
particular, five of seven contacts belong to this category
whereas water mediates four of eight contacts with the
IR-1 element. We emphasize that these significant
hsp27pal-specific changes in the interaction pattern
uncover unusual, previously not observed, molecular
characteristics of the EcRDBD, such as dual conforma-
tion of the side chains of four amino acid residues. Only
one of them, R67, situated at the surface of the domain, is
apparently not involved in any protein–protein or
protein–DNA interactions. The rotamers of two residues
(R26 and Q54) are involved in hsp27pal-specific protein–
DNA interactions. Finally, the R51 residue, located at the
subunit interface, uses two conformations to create the
direct and the water-mediated contacts with the UspDBD
and with the hsp27pal sequence. We speculate that the
alternate side chain conformations indicate that the
EcRDBD molecule, functioning as part of the hetero-
complex bound to the natural element but not to the
unnatural symmetric element, retains some extraordinary
structural flexibility reported previously for the isolated
(i.e. not interacting with DNA and with UspDBD)
domain (15). It has been also suggested that due to this
property EcRDBD could easily accommodate DNA-
induced changes in the secondary and tertiary structure
(15). Indeed, part of the CTE of the EcRDBD bound in
complex with the UspDBD on the hsp27pal folds into a
novel a-helix not observed in the structure of the
UspDBD/EcRDBD-IR-1 complex (18). The EMSA
experiments presented here clearly demonstrate that the
a-helix seems to be a component of a well-defined
functional element, which is absolutely necessary for the
effective formation of the UspDBD/EcRDBD-hsp27pal
complex. The element extends from N75 to K92 within the
CTE part previously defined (16) as corresponding to the
so-called A-box (34). As indicated by the previously
published alignment of the EcRDBD sequences (15),
amino acid residues of the EcR CTE fragment exhibit a
remarkable conservation although this fragment is not
present in other nuclear receptors. As noted before, the
sequence of B.mori EcR exhibits some puzzling differ-
ences (15). Nevertheless, as demonstrated by the EMSA
results presented here, the CTE of the B. mori contains a
well-defined fragment (P75-G89), which is critical for the
formation of the UspDBD/EcRDBD-hsp27pal complex as
well. Thus, the presence within the CTE of an element
supporting DNA-binding, which we refer to as the
EcR-box, seems to be a general feature of the EcR
proteins. Taking into account our crystallographic data,
which show that at least the N-terminal part of the
D. melanogaster EcR-box (residues N75–R81) forms an
a-helix, as well as secondary structure predictions done for
the D. melanogaster and B. mori EcRDBDs (Figure 9A),
we speculate that the entire EcR-box could fold into an
a-helix (see subsequently).
As discussed earlier, in addition to the core DBD, the
variable CTE region has been implicated in DNA
response recognition and discrimination by the particular
receptor. In contrast to the core DBD, which has the same
structure in all receptor DBDs solved to date (3), the
structure of each CTE fragment determined thus far has
been unique. Moreover, distinct functions have been
ascribed to the established CTEs. The crystal structure
of the RXR/thyroid receptor DBDs heterodimer
(RXRDBD/TRDBD) on its response element was the
first to reveal the a-helical structure of the TR CTE.
Figure 6. The A-box residues of the EcRDBD form an a-helical
structure that interacts with the minor groove of the hsp27pal element.
Comparison of the structures of the CTE fragments of the EcRDBD
from the UspDBD/EcRDBD heterocomplex bound to the hsp27pal
(blue) and IR-1 (green) elements. Dotted lines indicate hydrogen bonds
formed by the K81 and R81 residues.
2714 Nucleic Acids Research, 2007, Vol. 35, No. 8

It makes no tertiary contacts with the rest of the DBD but
instead projects across the minor grove, where it makes an
extensive interface with DNA (33) (Figure 9B). Although
the CTE of the vitamin D receptor (VDR) DBD bears
striking structural resemblance to the CTE of the TR
(Figure 9B), it makes quantitatively different interactions
with its cognate response element. It has been suggested
that the primary role of the VDR CTE is to mediate
response element discrimination, and not to provide
additional DNA affinity (35). For other nuclear receptor
DBDs, including nerve growth factor-inducible factor B
(NGFI-B) (36), human liver receptor homologue-1 (LRH-
1) (37) and RevErba(38) where the CTEs do not fold into
a defined a-helix (Figure 9B), it has been observed that
they trace over one of the phosphate backbones and
descend into the major groove contacting base pairs
located 50to the response element half-site. The EcR-box
fragment observed here on the hsp27pal element appears
to have a novel and unique structural and possibly also
multifunctional characteristic. First, being an a-helix the
fragment does not project across the minor groove, as it
was observed for TR and VDR CTEs, but descends into
the minor groove, similarly as it was observed for non-
helical CTEs (Figure 9B). However, due to the g-turn
formed by the P73, E74 and N75 residues, the orientation
of this fragment, and possibly of the entire EcR-box, does
Figure 7. Effects of amino acids substitutions in the A-box of D. melanogaster EcRDBD on the interaction with the hsp27pal element. Sixteen
individual residues from the A-box of the EcRDBD (N75–K92) (16) (A) were substituted with alanine. EMSA experiments were carried out with an
hsp27pal-containing ds oligonucleotide and with the indicated homogenous EcRDBD (B) or equimolar mixture of the respective EcRDBD and the
wild-type UspDBD (D). Panels (C) and (E) represent quantitative analysis of the EMSA data presented in panel (B) (lanes 2–18) and panel (D) (lanes
2–18), respectively. The columns indicate mean values of three independent experiments and error bars indicate SD-values. The designations of the
respective mutant EcRDBDs are based on the amino acid single-letter code. The respective complexes formed by one DBD molecule are indicated by
CI, and those originating from homo- or heterodimers are indicated by CII; F, free probe. For clarity, the wild-type complexes formed by EcRDBD
are denoted as E and by UspDBD as U. The protein concentrations were: (B), lanes 2–18, 240 nM of the indicated EcRDBD; lane 19, the same
amount of the wild-type UspDBD; lane 20, 120 nM of each wild-type DBD; (E), lanes 2–18, 120 nM of wild-type UspDBD and 120 nM of the
indicated EcRDBD; lane 19, 240 nM of wild-type UspDBD; lane 20, 240 nM of wild-type EcRDBD.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2715

not match any of the previously observed orientations of
the CTEs (Figure 9B, C), including that recently reported
(39) for the homodimeric complex of the progesterone
receptor DBDs (not shown). Our crystallographic data
along with the EMSA experiments suggest that this
orientation of the EcR-box would allow interactions of
some amino acid residues in the minor groove. For the
other above-mentioned proteins, which insert the CTE
into the minor groove, numerous hydrogen-bonding
contacts, including sequence-specific base contacts, have
been observed. Our crystallographic data allow the
identification of two residues involved in contacts with
the sugar-phosphate backbone. Other key residues,
indicated by the EMSA experiments, could not be
modeled in the electron density maps, possibly indicating
that they are located in an EcR-box fragment with
increased dynamic properties. Apparently, interaction
with the hsp27pal element is not sufficient to bring about
a disorder-to-order transition of this EcR-box section and
other factors are needed to achieve this. According to
recent suggestions the predominant molecular function of
disordered protein segments appears to involve molecular
recognition in eukaryotes. The flexibility of the disordered
fragments would allow them to be targets for multiple
binding partners and post-translational modifications and
thus enhance their ability to participate in multiple
Figure 8. DNA-binding activities of CTE-truncated EcRDBDs of D. melanogaster and B. mori. Schematic diagram of the primary structure of the
wild-type D. melanogaster and B. mori EcRDBD CTEs and their truncated derivatives, in which fragments from the region corresponding to the
A-box (15,16) have been deleted (A). The numbering is relative to the first Zn
2þ
-coordinating cysteine of the DBDs. In order to analyze the effect of
the deletion on the EcRDBD activity, the EMSA experiments were carried out with an hsp27pal-containing ds oligonuleotide and with the indicated
EcRDBD derivative or wild-type EcRDBD (B), or with an equimolar mixture of the respective EcRDBD and wild-type UspDBD (D). Panels (C)
and (E) represent quantitative analysis of the EMSA data presented in panel B (lanes 2–12) and panel D (lanes 2–10), respectively. For clarity, the
wild-type full-length EcRDBDs were denoted by E
D
for EcRDBD from D. melanogaster and by E
B
for EcRDBD from B. mori. Similarly, the
full-length UspDBDs from D. melanogaster and B. mori were designated as U
D
and U
B
, respectively. Other details are as in the legend to Figure 7.
The protein concentrations were: (B), lanes 2–5 and 8–10, 600 nM of the indicated EcRDBD; lanes 6 and 11, 600 nM of the indicated wild-type
UspDBD; lanes 7 and 12, 300 nM of each indicated wild-type DBD; (D), lanes 2–10, 300 nM of the indicated wild-type UspDBD and 300 nM of the
indicated EcRDBD.
2716 Nucleic Acids Research, 2007, Vol. 35, No. 8

signaling pathways (40,41). For some nuclear receptors it
has been demonstrated that the CTE can serve as a
recruitment site for co-activators and as a target for post-
translational modification (3). Although the molecular
mechanism linking these regulatory events with control of
the activity of the receptors remains elusive, it is reason-
able to assume that also the EcR-box could be involved in
similar processes. However, the questions of possible EcR-
box interaction partners and of its post-translational
modifications are still open.
SUPPLEMENTARY DATA
Supplementary Data is available at NAR Online.
ACKNOWLEDGEMENTS
We are grateful to Professor Jacek Otlewski (Institute of
Biochemistry and Molecular Biology, University of
Wroclaw) for giving us the opportunity to perform
crystallization experiments. We thank Professor Kostas
Iatrou and Dr Luc Swevers (Institute of Biology, National
Center for Scientific Research ‘Demokritos,’ Athens,
Greece) for the kind gift of the cDNA clone encoding
the full length of B. mori EcR, Dr George Tzertzinis (New
England Biolabs, Beverly, MA, USA) for plasmid encod-
ing the full length of B. mori Usp. In addition, we thank
Professor Olaf Pongs and Dr Dirk Isbrandt (Center for
Molecular Neurobiology, Hamburg, Germany) for their
generous support. Supported by a grant number 3 T09A
040 28 from the State Committee for Scientific Research
and by a subsidy from the Foundation for Polish Science
to M.J. Some of the calculations were carried out in the
Poznan
´Metropolitan Supercomputing and Networking
Center. Funding to pay the Open Access publication
charges for this article was provided by the State
Committee for Scientific Research (grant number 3
T09A 040 28).
Conflict of interest statement. None declared.
Figure 9. CTE of the EcRDBD folds into an a-helix which is part of
the EcR-specific element supporting DNA-binding. (A) Secondary
structure predictions of D. melanogaster and B. mori A-box segments.
The predictions were performed using the GORIV, PROF and
PSIPRED algorithms available at the ExPASy Proteomics Server
(http://au.expasy.org/). The predicted a-helices are depicted as cylin-
ders. The seven-residue segment forming the short a-helix in the CTE
A-box of the D. melanogaster EcRDBD, observed in the present crystal
structure, is boxed with broken line. The average confidence score
values of the GORIV algorithm for the residues forming a potential
a-helix within the D. melanogaster and B. mori A-box segments are 736
and 661, respectively. The PROF algorithm gives a prediction and a
confidence value between 0 and 1 for each position in the amino acid
sequence. The average confidence values for the D. melanogaster and
B. mori sequences are 0.68 and 0.53, respectively. The PSIPRED
algorithm gives a confidence value between 0 and 9. The average
confidence scores for the D. melanogaster and B. mori are 5.1 and 4.8,
respectively. (B) Superposition of the corresponding Caatoms of
the TR (PDB accession code 2NLL) (33), VDR (PDB accession
code 1KB4) (35), LRH-1 (PDB accession code 2A66) (37), Rev-ERBa
(PDB accession code 1A6Y) (38), NGFI-B (PDB accession code 1CIT)
(36) and EcRDBD (from the present UspDBD/EcRDBD-hsp27pal
complex) proteins complexed to DNA. (C) A detailed view of the CTEs
and DNA shown in (B). C and N denote C- and N-termini of
respective DBDs.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2717

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