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Molecular Endocrinology 20(12):3042–3052
Copyright © 2006 by The Endocrine Society
doi: 10.1210/me.2005-0511
Structure of the Progesterone ReceptorDeoxyribonucleic Acid Complex: Novel Interactions
Required for Binding to Half-Site
Response Elements
Sarah C. Roemer, Douglas C. Donham, Lori Sherman, Vickie H. Pon, Dean P. Edwards, and
Mair E. A. Churchill
Program in Molecular Biology (S.C.R., D.P.E., M.E.A.C.), Department of Pharmacology (S.C.R.,
D.C.D., V.H.P., M.E.A.C.), and Department of Pathology (L.S., D.P.E.), University of Colorado at
Denver and Health Sciences Center, Aurora, Colorado 80045
The DNA binding domain (DBD) of nuclear hormone receptors contains a highly conserved globular domain and a less conserved carboxyl-terminal extension (CTE). Despite previous observations
that the CTEs of some classes of nuclear receptors
are structured and interact with DNA outside of the
hexanucleotide hormone response element (HRE),
there has been no evidence for such a CTE among
the steroid receptors. We have determined the
structure of the progesterone receptor (PR)-DBDCTE DNA complex at a resolution of 2.5 Å, which
revealed binding of the CTE to the minor groove
flanking the HREs. Alanine substitutions of the in-
teracting CTE residues reduced affinity for inverted
repeat HREs separated by three nucleotides, and
essentially abrogated binding to a single HRE. A
highly compressed minor groove of the trinucleotide spacer and a novel dimerization interface
were also observed. A PR binding site selection
experiment revealed sequence preferences in the
trinucleotide spacer and flanking DNA. These results, taken together, support the notion that sequences outside of the HREs influence the DNA
binding affinity and specificity of steroid receptors.
(Molecular Endocrinology 20: 3042–3052, 2006)
T
spacer (3N) between the half-sites (2–4). There are two
hexanucleotide HREs for the steroid receptors: AGGTCA
is preferred by estrogen receptor (ER), as well as by the
class II and orphan receptors, and AGAACA is preferred
by the glucocorticoid receptor (GR) subfamily, which
also includes androgen receptor (AR), mineralocorticoid
receptor (MR), and progesterone receptor (PR). Class II
receptors for the nonsteroidal hormones and dietary lipids such as retinoic acid receptor (RAR), thyroid hormone
receptor (TR), vitamin D3 receptor (VDR), and peroxisome proliferator-activated receptor (PPAR), bind primarily as heterodimers together with retinoic acid X receptor (RXR) to HREs arranged as direct repeats with
variable spacing between the half-sites (1). Orphan receptors, for which physiological ligands are unidentified,
interact through various configurations including monomer binding to extended HRE half-sites (1, 4–9).
The DNA binding regions of the nuclear receptors
share a core DBD, consisting of two ␣ helices and two
asymmetric zinc binding modules coordinated by eight
conserved cysteine residues, followed by a less conserved region termed the C-terminal extension (CTE).
The first helix of the core DBD inserts into the major
groove of the HRE, making base-specific contacts, and
helix 2 crosses over the top of helix 1 and the major
groove leading into the CTE. The dimerization domain (D
box) resides within the C-terminal zinc-binding module
and is important for mediating DNA binding-dependent
dimerization of receptors (8, 10–16).
HE NUCLEAR RECEPTOR superfamily is composed of ligand-dependent transcription factors
that regulate a variety of cellular processes including
metabolism, development, growth, differentiation, and
reproduction. Nuclear receptors are modular proteins
containing a conserved DNA binding domain (DBD), a
C-terminal ligand binding domain (LBD), and a diverse
amino-terminal region (1, 2). Transcriptional regulation
by nuclear receptors primarily occurs through direct
binding to specific hormone response elements (HREs)
of target genes. The nuclear receptor superfamily can be
subdivided based on distinct DNA binding mechanisms.
Class I receptors for steroid hormones interact optimally
as head-to-head homodimers with hexanucleotide
HREs arranged as inverted repeats with a trinucleotide
First Published Online August 24, 2006
Abbreviations: aa, Amino acids; AR, androgen receptor; CTE,
carboxyl-terminal extension; DBD, DNA binding domain; ER,
estrogen receptor; ERE, estrogen response element; ERR2,
estrogen-related receptor-2; GR, glucocorticoid receptor; GST,
glutathione-S-transferase; HRE, hexanucleotide hormone
response element; LBD, ligand binding domain; MR, mineralocorticoid receptor; 3N, trinucleotide spacer; NMR, nuclear magnetic resonance; PR, progesterone receptor; PRE, progesterone response element; RXR, retinoic acid X receptor; TR,
thyroid receptor; VDR, vitamin D receptor.
Molecular Endocrinology is published monthly by The
Endocrine Society (http://www.endo-society.org), the
foremost professional society serving the endocrine
community.
3042
Roemer et al. • Structure of the PR-DBD-DNA Complex
Unlike the core DBD, the CTE is not well conserved
in sequence or structure. Structural studies of class II
and orphan receptors have shown that the CTE, which
has a highly variable length of up to 40 amino acids
beyond the conserved Gly-Met sequence at the C
terminus of the core DBD (see Fig. 3D), provides additional contacts with the DNA minor groove outside of
the canonical HREs. The CTEs of TR and VDR form a
third ␣-helix that projects across the minor groove to
make extensive contacts with the phosphate backbone of spacer nucleotides. The CTEs of TR and VDR
are also important for DNA-dependent homo- and heterodimerization, as well as for proper spacing of receptor subunits between direct-repeat response-element half-sites (12, 17). The CTEs of the orphan
receptors [RevErb, nerve growth factor-I-␤, liver receptor homolog-1, and estrogen-related receptor-2
(ERR2)] form an extended loop that occupies the minor groove of DNA sites flanking the HRE (8, 9, 18, 19).
In addition to the extended loop the liver receptor
homolog-1 DBD contains a non-DNA binding ␣-helix
termed the Ftz-F1 motif (19). Conformational changes
in the CTE have been observed in response to DNA
binding. The RXR CTE is an alpha helix in solution but
forms an extended loop conformation when bound to
DNA, whereas the CTE of ERR2 is unstructured in
solution and becomes structured when bound to DNA
(9, 11, 13–15). Biochemical and mutagenesis studies
have also shown that the CTE is required for high
affinity DNA binding by class II and orphan nuclear
receptors (5, 11, 20–24). In particular, the CTE is important for the DNA binding of orphan receptors that
interact as monomers with a half-site HRE. The CTE
effectively extends the protein-DNA interface, increases the stability of monomer binding to HRE halfsites, and creates a preference for 5⬘ trinucleotide
sequences flanking half-site HREs.
In contrast to class II and orphan receptors, much
less is known about the steroid receptor CTEs. In
previous studies, we showed that the CTE of ER and
PR is required for interaction with the high mobility
group protein-1 and -2 (HMGB-1/-2) that function as
coregulatory proteins for all members of the steroid
class of receptors by facilitating receptor binding to
specific DNA target sites (25–32). Similar to the orphan
receptors, the CTE of ER and PR was also required for
efficient binding of ER and PR to HRE half-sites (31–
33). However, structural studies of steroid receptors
DBDs including GR, AR, and ER, either lacked sufficient CTE sequence in the protein expression constructs, or the CTE was disordered in the crystal structures (34–36). Furthermore, there are no reports of
structures of a PR-DBD/DNA complex.
To understand the role of the CTE in PR-DNA binding, we determined the crystal structure of the PR DBD
including the CTE bound to consensus inverted-repeat progesterone response elements (PREs). Arg637
and Lys638 of the CTE contact the DNA minor groove
at sites flanking the PREs. Alanine substitutions of
Arg637 and Lys638 reduced binding affinity of PR to
Mol Endocrinol, December 2006, 20(12):3042–3052 3043
the inverted repeat PREs and abrogated binding of PR
to a half-site PRE. Other novel structural features of
the PR-DBD DNA complex included interactions of the
subunits with the PRE consensus sequence bound by
the opposite subunit, which coincided with a narrow
minor groove in the 3N spacer DNA. These studies
further our understanding of steroid receptor-DNA
recognition mechanism and suggest that minor groove
DNA interactions outside the HREs contribute to binding affinity and sequence selectivity.
RESULTS AND DISCUSSION
To examine the role of the CTE in PR-DNA binding, the
PR DBD including the sequence for the CTE was expressed, purified, and cocrystallized with PRE DNA. The
PR-DBD670 [amino acids (aa) 562–670] was designed to
have a CTE of the same length as TR in the crystal
structure of TR-DBD-CTE (12). However, purification of
the bacterially expressed PR-DBD670 polypeptide resulted in a mixture of two distinct species, the intact
PR-DBD670 and a C-terminal proteolytic degradation
product determined to be predominantly aa 562–648
(Fig. 1). Double-stranded oligonucleotides that were
tested in crystallization trials contained two inverted
repeat consensus PREs separated by a variable 3N
spacer sequence and variable 5⬘ and 3⬘ flanking sequences. The stoichiometric ratio, at which a maximum
amount of PR-DNA complex formed with a minimum of
free protein, was determined by stoichiometric EMSA
using concentrations of protein and DNA equivalent to
crystallization conditions. Crystals of the protein-DNA
complex were obtained at a molar protein/DNA ratio of
1.95:1 with the smaller PR DBD proteolytic product
and a double stranded DNA fragment of 18 nucleotides (designated PRE_2C7A), which contained a 3N
spacer derived from the mouse mammary tumor virus
promoter and a one base overhang on each side of the
PREs (Fig. 1).
The structure of PR-DBD-DNA complex was determined by x-ray crystallography to a resolution of 2.5 Å.
The structure was solved by molecular replacement
using a homology model of the PR-DNA complex
(Donham, D. C., and M. E. A. Churchill, unpublished
results) that was based on the ER DBD-estrogen response element (ERE) complex structure (35). Data
collection and refinement statistics are presented in
Table 1. The final model contains one homodimer of
PR-DBD (subunit A includes aa 562–640 and subunit B
includes aa 562–638), associated zinc ions and solvent molecules, and one DNA duplex, which forms a
pseudocontinuous helix with a base triple at the junction between adjacent asymmetric units. The base
triples create a discontinuous phosphate backbone
and slight inclination of the bases, which appears to
promote additional hydrogen bonding between adjacent base pairs and a narrowing of the minor groove at
the 5⬘ and 3⬘ ends of the DNA.
3044 Mol Endocrinol, December 2006, 20(12):3042–3052
Roemer et al. • Structure of the PR-DBD-DNA Complex
Fig. 1. PR DBD Polypeptide and DNA Used in the Structure
A, Schematic of the PR-DBD648 construct. Alpha helices are boxed. Direct dimer contacts (L602, C603, A604, R606, D608,
1610, and R615) are indicated by solid dots, and the water-mediated dimer contacts (K617 and N618) are marked with open dots.
DNA backbone interactions are marked according to whether they fall within or outside of the consensus PREs with solid (H578,
Y579, R616, K617, and R623) and open squares (C577 and R617), respectively. Base-specific interactions are denoted with
closed stars (K588, V589, R593). The CTE-DNA interaction (R637) is depicted with an open star. Water-mediated DNA contacts
are marked with triangles (D578, C577, Y579, G580, S586, and K617). N-terminal residues that are cloning artifacts are in lower
case (g and s). Residues that are disordered in the crystal (g, s, L562, K640, K641, F642, N643, K644, V645, R646, V647, and
V648) are marked with a dotted line. B, The DNA fragment named PRE_2C7A used in the crystals. The consensus PREs are
separated by 3N spacer nucleotides that are derived from mouse mammary tumor virus. Consensus inverted repeats are
highlighted in gray, and PRE numbering is indicated below the sequence. A and B denote the subunit of PR-DBD bound to the
hexanucleotide element.
Overall Structure
The overall structure of the PR-core DBD was similar
to that of other nuclear receptors. The PR-DBD binds
to the response element as a head-to-head dimer (Fig.
2), and the recognition helix (helix 1) of each monomer
Table 1. Data Collection and Refinement Statistics
Diffraction data
Space group unit cell (Å) P212121 39.53 ⫻ 107.85 ⫻ 111.96
Wave length (Å)
1.5418
Resolution (Å)
50–2.5
High-resolution shell (Å)
2.59–2.50
Unique reflections
16,986
Redundancy
2.9
Completeness (%)
98.4
25 (5.3)
Average I/␴ (last shell)
0.055 (.270)
Rmerge (last shell)
FOM (last shell)
0.846 (.759)
Crystallographic refinement
Resolution range
30–2.5
Reflections
14,739
RMS bond lengths
0.01
RMS bond angles
1.71
R value (%) (last shell)
19.8 (23)
24.6 (35)
Rfree (%) (last shell)
FOM, Figure of merit; RMS, root mean squared deviation
from ideality.
lies within the major groove of the PRE half-site where
it makes sequence-specific interactions. Helix 2
crosses over and is perpendicular to helix 1, and there
is also a short third helix just C-terminal to helix 2,
which we have termed helix 2⬘. Although the structures of the individual DBD subunits are similar, they
do not interact with the DNA equivalently. Solvent
accessibility and the atomic packing density at each
PR monomer-DNA interface, as determined by the
program FADE (37) and CCP4 (38), revealed that subunit A of the DBD dimer (shown in Fig. 2 as the PR DBD
subunit on the right) associates more closely with the
DNA than the B subunit. The same analysis applied to
the structures of other steroid receptor-DNA complexes showed that monomer binding of the GR subfamily is also not equivalent, with subunit A (as defined
in the PDB entries) associating more closely with the
DNA than the B subunits (34–36). This asymmetric
DNA interaction suggests that the GR subfamily members have a dominant monomer of the two involved in
binding to the inverted repeat HREs.
To more precisely determine DBD structural relationships, the best fit of the local protein backbone
atoms of each subunit of PR DBD (A and B) was
compared with each subunit of other steroid receptors
using root mean squared deviation analyses (55). PR
was most closely related to ER␣, and the most closely
related nonsteroid receptor was found to be class II
Roemer et al. • Structure of the PR-DBD-DNA Complex
Mol Endocrinol, December 2006, 20(12):3042–3052 3045
the most closely related subunit was not unexpected
considering that it is the subunit more loosely associated with the DNA and therefore under fewer structural
restraints.
In addition to the core DBD, clear electron density
was visible for the first eight residues of the CTE projecting from the A subunit (aa 632–640) and up to six
residues (aa 632–638) from the subunit B (Figs. 1 and
3A). Thus, for the first time, the structure of a steroid
receptor CTE and its interaction with DNA has been
observed. The CTE interacts with the minor groove of
DNA flanking the two PRE half-sites (Fig. 2).
Core PR DBD Interactions with DNA
Fig. 2. Structure of PR-DBD⫹CTE-DNA Complex
The ribbon diagram of the structure shows the PR-DBD in
rainbow coloring from blue at the N terminus to red at the C
terminus. Zinc ions and the DNA are colored gray. DBD
subunits are labeled A and B, respectively. Helix 1 (H1), helix
2 (H2), and helix 2⬘ (H2⬘) are indicated on each DBD subunit.
The DNA ladder diagram details the contacts made between
the DBD subunit A and one hexanucleotide-half-site of the
PRE. The three-nucleotide spacer between the inverted response elements is shown in orange, and the consensus
sequence is colored green. The flanking sequence is red and
shows CTE interactions and the triple basepairs between the
inverted response elements. Nucleotides from the next asymmetric unit are boxed and the discontinuity in the phosphate
backbone is denoted by a break symbol. Predicted hydrogen
bonds are marked with black solid lines for major groove
interactions and red for minor groove interactions. The van
der Waals interactions occurring between T4 and Val589 are
indicated by a dashed line. Water molecules are named W,
and phosphate groups are dark teal. The number in superscript indicates nucleotide position within the response element. Protein DNA interface regions are indicated by the
brackets under the model.
receptor RXR (1.01 Å). Interestingly, all of the B subunits (as defined in the PDB entries) among the receptors were more closely related structurally than the A
subunits. The subunit B of PR is most structurally
comparable to the subunit B of ER␣ (0.81 Å) and AR
(0.94 Å), than GR (1.44 Å). The finding that subunit B is
Although there are slight differences in subunit orientation, the protein and DNA contacts are virtually indistinguishable in both subunits of the PR-DNA complex (Fig.
2). The sequence-specific interactions between residues
in helix 1 and sites in the major groove of the PRE are
nearly identical with those previously reported for GR
and AR (34, 36). The side-chain atoms Lys588-N␨ and
Arg593-NH1 and -NH2 make sequence-specific hydrogen bonds with Gua6-N7 and Gua3-N7 and -O6, respectively, and Val589 has significant van der Waals
interactions with Thy4-C5. These base-specific contacts
are further stabilized by numerous direct and watermediated phosphate backbone interactions summarized in the schematic (Fig. 1) and the DNA ladder diagram (Fig. 2; and the supplemental figure published as
supplemental data on The Endocrine Society’s Journals
Online web site at http://mend.endojournals.org) showing the A dimer subunit interactions with a single PRE, 3N
spacer, and flanking sequence.
CTE-DNA Interaction
The PR-CTE contacts the DNA outside of the canonical PRE, and effectively extends the protein-DNA interface made by the DBD (Figs. 2 and 3A). In both the
A and B PR DBD subunits, Arg637 of the CTE inserts
into the minor groove. There it forms hydrogen bonds
to Cyt8-O2 and -O4⬘, which are part of the base triple
at the junction of adjacent DNA molecules, and is
further stabilized by an apparent hydrogen bond between the NH1 and the phosphate of Gua8 in the
adjacent asymmetric unit. In subunit A, the carbonyl
oxygen of the next residue (Lys638) hydrogen bonds to
Arg637-NH1, which positions the side chain atom
Lys638-N␨ for a phosphate contact with Ade7. Phe639 is
the final CTE residue to have distinguishable electron
density for both the main chain and side-chain in the
structure of subunit A. Although, in this structure no
further side chains of the CTE were seen, the nuclear
magnetic resonance (NMR) structure of the ERR2-DNA
complex showed that a region of the ERR2 CTE, Cterminal to the minor groove interacting region, looped
back to interact with a hydrophobic pocket on the core
DBD (9). This region of the core PR DBD also has a
hydrophobic pocket (Val581 and Val633) in the same
3046 Mol Endocrinol, December 2006, 20(12):3042–3052
Roemer et al. • Structure of the PR-DBD-DNA Complex
position as the contact region of ERR2 (Val117 and
Leu169), as well as a unique surface patch of residues
(Val581, Leu634, and Met595) that have the potential for
additional interactions with hydrophobic residues of the
PR CTE (Fig. 3B).
To determine whether the CTE-DNA interactions
observed in the crystal structure were functionally important, DNA binding studies were conducted with
PR-DBD648 mutants. Arg637 and Lys638 were substituted with alanine either as single (R637A) or double
residue substitutions (R637A/K638A) in the context of
the PR-DBD648 construct. The wild-type and mutant
proteins were expressed and purified, and relative
DNA binding affinities for a DNA fragment containing
palindromic PREs were determined by EMSA (Fig. 3, B
and C). The single mutant (R637A) and wild type proteins had similar DNA binding affinities (Wt Kd ⫽ 104
nM and R637A Kd ⫽ 121 nM), whereas the double
mutant bound DNA with a 2.5-fold lower affinity (Kd ⫽
302 nM), and also had a decreased maximal binding at
saturation (0.7 vs. 0.9), indicating a weaker and more
labile interaction with the PRE_2C7A DNA.
This binding study also showed that a fraction of the
PR DBD bound to the DNA as a monomer, which was
detected as a faster mobility DNA complex than the
PR DBD dimer (Fig. 3B). Interestingly, no monomer
DNA binding was observed for the double mutant
R637A/K638A (Fig. 3B), which suggested an important
role for the CTE in binding to PRE half-sites. Therefore,
to examine this possibility more closely, we compared
Fig. 3. CTE-DNA Interaction
A, Interactions between CTE residues R637 and K638 and
the DNA. Red lines indicate predicted hydrogen bonds with
the minor groove. Carbon, oxygen, and nitrogen atoms are in
yellow, red, and blue, respectively. The DNA is colored gray
and CTE interacting nucleotides are labeled. Gua8 is boxed
to indicate that it is part of the adjacent asymmetric unit. The
electron density is a CCP4 FWT map with the contour set at
0.4 ␴. B and C, Quantitative EMSA with the palindromic PRE
(B) and half-site PRE (C). Increasing concentrations of wild
type PR DBD648, PR DBD R637A, and PR DBDR637A/
K638A were incubated with a single limiting concentration of
[32P] labeled PRE (0.6 nM). The gel insets are representative
experiments with DBD648 wild type, R637A, and R637K638A
and the resulting binding curves represent mean values
(⫾SEM) from replicate experiments (see Materials and Methods for details). D, Sequence alignment of steroid receptor
and ERR2 CTEs. Occurrences of the well-conserved RK sequence in steroid receptor CTEs are indicated by bold print.
PR and ER␣ XGGR sequence (aa 634–637 642–645) that
closely resembles the VLRGGR (aa 642–646) sequence found
in the orphan nuclear receptor subclass III are underlined.
The ERR2 Tyr (aa 649), and potential residues for PR (F639,
F641, V644, V647, and V648), which may make hydrophobic
interactions with the core DBD, are marked with stars. Ribbon
diagrams show monomers of PR and ERR2 bound respectively to PRE half-site and an ERE half-site (9). The arginines
of the VXGGR sequences, which interact at comparable positions in the minor groove, are circled in black. Helix 1, 2, and
2⬘ are marked, the core DBD is colored blue and the CTE is
colored red. Amino acid numbering is indicated for PR.
Roemer et al. • Structure of the PR-DBD-DNA Complex
the binding affinities of wild-type and mutant PR DBDs
for a DNA fragment containing only a single PRE or
half-site (Fig. 3C). The single mutant R637A exhibited
a small 1.5-fold reduction in DNA binding affinity (Kd ⫽
205 nM) compared with wild-type DBD (Kd ⫽ 133 nM),
whereas the binding of the double mutant (R637A/
K638A) was essentially not detectable (Fig. 3C). These
findings confirmed, in solution without the influence of
any crystal packing contacts, that the CTE-DNA interactions observed in the crystal structure are important
for PR binding to a half-site PRE and potentially also to
imperfect inverted repeats.
CTE-DNA interactions are known to play an important role in class II and orphan nuclear receptor DNA
binding (4). The DNA interactions of the PR-CTE were
most comparable to those seen in the ERR2 orphan
receptor in the NMR structure of the ERR2-DNA complex (Fig. 3D) (9). ERR2 belongs to the type III group of
monomeric orphan receptors, which preferentially
bind extended half-site response elements with the
consensus sequence TNAAGGTCA (where TNA is the
preferred flanking sequence, and the consensus sequence is underlined). The CTE of ERR2 has a ValArg-Gly-Gly-Arg sequence (a Grip box-like sequence)
that interacts directly with the flanking TNA sequence
by inserting arginine residues into the minor groove to
make base-specific contacts (9, 18). The PR CTE sequence (Val-Leu-Gly-Gly-Arg-Lys) that inserts into the
minor groove is similar to that of ERR2, except that it
is positioned within the CTE closer to the GM boundary (Fig. 3D). A similar Gly-Gly-Arg sequence is also
present in the CTE of ER␣, and the ER␣ CTE was
shown previously by us and others to be important for
binding to ERE (estrogen response elements) halfsites (32, 33). In addition, all of the steroid class of
nuclear receptors except ER␤ have a conserved ArgLys sequence in a similar position in the CTE as the
Arg-Lys residues of PR that are seen interacting with
the minor groove (Fig. 3D), and mutations that disrupt
only this region of GR are functionally deficient (39).
These data, taken together, suggest that a motif in the
CTE for minor groove DNA interaction similar to that of
orphan receptors may extend to members of the steroid class of nuclear receptors.
Dimer Interface
The PR-DBD dimer interface consists of several direct
and water-mediated inter subunit interactions (Figs. 2
and 4A). The dimerization boxes (D-boxes, aa 604–
608) have interactions between Arg 606-Asp608 and
Ala604-Ile610, which are conserved with those seen in
the ER␣, GR, and AR structures (34–36). Outside of the
D-box an additional conserved interaction was observed between the main-chain carbonyl oxygen of
Leu602 and the side-chain atom N␧ of Arg615 in helix
2⬘ (aa 616–618), as well as a unique interaction between the main-chain carbonyl oxygen Cys603 and
the NH2 of Arg615. Helix 2⬘ also maintains watermediated contacts between opposing complementary
Mol Endocrinol, December 2006, 20(12):3042–3052 3047
Fig. 4. PR Dimer Interface in the PR-DBD Complex
A, Ribbon diagram showing the intersubunit interactions of
the PR dimer. Subunits are colored pink and green. Helix 1
(H1), helix 2 (H2), and helix 2⬘ (H2⬘) are indicated on each DBD
subunit, with subunit A on the left. Proposed hydrogen bonds
are marked with dashed red lines. Carbonyl and amine
groups involved in potential hydrogen bonding are colored
red and blue, respectively. B, Superpositioning of PR-DBD
(blue) and ER-DBD (red) (35) structures shows the different
positioning of Lys617 (K617). Amino acid numbering is for
PR. Proposed hydrogen bonds are marked with dashed red
lines and H1, H2, and H2⬘ are indicated.
Lys617 residues. Asymmetric interactions with water
molecules are coordinated by the A subunit Lys617
main-chain carbonyl oxygen and the B subunit side
chain amine of Lys617, and vice versa. A symmetric
interaction via a water molecule is coordinated by the
side-chain amine of Lys617 in both subunits. Solvent
accessibility and packing density between the subunits A and B were determined for all the steroid
receptor DBD-DNA structures by the programs FADE
and CCP4 (37, 38). PR-DBD was found to have a
looser packing density between the A and B subunits
than the other steroid receptor, suggesting that it has
a more relaxed dimer interface. The tighter association
between the PR-DBD subunits and the PRE (discussed in Overall Structure) potentially could compensate for a weaker inter-subunit interaction.
Several protein-DNA interactions are made by H2⬘
residues (Fig. 2). These include a direct phosphate
interaction between Arg616-N␧ and Gua3, a watermediated interaction with the phosphate of Thy4 via
the Arg616-NH2, and the carbonyl oxygen of Lys617
3048 Mol Endocrinol, December 2006, 20(12):3042–3052
also makes water-mediated contacts with the phosphate of Thy2. An interesting and unique interaction was
also made by the side chain of Lys617, which extends
across the 3N spacer to interact directly with the phosphate backbone (Thy2) of the PRE half-site bound by the
opposite PR DBD subunit. In other steroid receptor
structures, as shown here with ER-DBD as an example,
the side chain of the equivalent lysine to PR Lys617
bends back toward the DNA of the response element to
which the subunit is bound (Fig. 4B).
Narrow Minor Groove of the PRE in the
PR-DBD Complex
The PRE_2C7A DNA bound to PR (Table 2) is similar to
standard B-form DNA (40), with the exception of distortions at base pairs AT(⫺5), AT (0), TA (5), and
GC(⫺6) as well as the width of the minor groove. The
width of the minor groove, 3.6 Å, is well below the
mean value of 9.6 Å reported for B-DNA (Table 2 and
Fig. 5A). Comparison of the DNA in the steroid receptor DBD-DNA structures using CURVES (Fig. 5A)
showed that all of the complexes have compressed
minor grooves but revealed that the PR-DNA complex
had the most compressed minor groove on average
and in the 3N spacer region (41). The ERE in the ER
DBD complex also showed compression with an average minor groove width of 6.23 Å, whereas the AR
and GR structures with one subunit bound to a perfect
half-site and one associated with an incorrectly oriented half-site (designated AR and GR4S), had more
greatly compressed minor groove widths on the side
of the response element that contained the correct
consensus half-site. Even the minor groove width of
the DNA bound to GR, which had identical hexanucleotide HRE sequences and a similar crystal-packing
environment (designated GR3S) had a wider (4.8 Å)
minor groove than that of the PRE_2C7A DNA.
Whether these differences in minor groove width represent intrinsic abilities of different steroid receptors to
compress the minor groove or reflect differential flexibility or curvature of DNA due to the sequence of the
3N spacer and/or flanking region cannot be determined from crystallographic analysis.
PR DBD Preference for 3N Spacer and
Sequences Flanking the PREs
The observed novel interactions of the PR-DBD-CTE
with the minor groove outside of the HREs raised the
Roemer et al. • Structure of the PR-DBD-DNA Complex
question of whether there is an intrinsic sequence preference in the 3N spacer and/or sequences flanking the
hexanucleotide HREs for optimal PR binding. To address
this question, we conducted a binding site selection
experiment. The selectable DNA contained fixed canonical PRE sequences with a randomized 3N spacer and
four randomized nucleotides immediately flanking either
side of the PREs (N4AGAACAN3TGTTCTN4). Purified PR
DBD was bound to the randomized DNA, complexes
were immunoprecipitated, the purified DNA was amplified, and after five rounds of selection, the selected
DNA was subcloned and sequenced (42).
A slight strand bias and nonrandom distribution of
bases in the 3N spacer and flanking DNA was detected using the MEME program (Fig. 5B) (43). The
occurrence of a 3N spacer itself generates asymmetry
in an otherwise palindromic binding site, which the
alignment programs are sensitive to. However, when
these sequences were aligned by MEME, an orientation was selected where position 0 was predominantly
a pyrimidine, and the bases at ⫺1 and ⫹1 appeared to
have a greater propensity also to be pyrimidines. This
also corresponded to the A and B subunit orientation
seen in the PR-DNA crystal structure. Interestingly, the
PRE_2C7A DNA has a T-track in the 3N spacer, which
is a sequence consistent with a narrow minor groove
(reviewed in Ref. 44). The predicted curvature for the
3N spacer of the selected elements, as determined by
the program BendIt (45) was increased across the 3N
spacer similar to that predicted for the PRE_2C7A
DNA used in the structure (Fig. 5A). Furthermore, it has
been observed by several investigators that the steroid
receptors have higher affinity for HREs with certain
nucleotides within the 3N spacer (46–49).
The selection experiments also revealed a preference for sequences flanking the PRE, with stronger
selection for the bases flanking half-site B, which is the
subunit with the looser interactions with DNA. The
sequence preference seen in the bases flanking the
HREs is difficult to explain unless the CTE, which
binds to these sites, actually contributes to sequence
selectivity. Arg637 forms hydrogen bonds directly with
the bases in the minor groove, and this may result in
slight sequence bias in these regions, similar to what
has been observed for the CTEs of the class II and
orphan receptors (9, 18). Taken together, these results
support the model that receptors have a dominant
subunit (A), which selects for a pyrimidine-rich 3N
spacer 3⬘, and which creates the opportunity for the
Table 2. DNA Parameters for PR-DBD Complex
Shiftb (Å)
Average
B-DNA
SD
0.39
0.02
0.5
Slideb (Å)
⫺0.77
⫺0.07
0.7
Riseb (Å)
3.26
3.4
0.21
B-form DNA values reported from Ref. 40.
Tilt (°)
4.14
0.2
3.4
Rolla (°)
⫺1.40
0.9
2.5
Twista (°)
35.89
35.9
6.2
Major Groove
Minor Groove
Sugar Pucker
Width (Å)
Depth (Å)
Width (Å)
Depth (Å)
12.76
13.6
0.9
4.37
5.8
1.7
3.65
9.6
1.6
5.38
6.7
0.5
C2⬘-endo
C2⬘-endo
Roemer et al. • Structure of the PR-DBD-DNA Complex
Mol Endocrinol, December 2006, 20(12):3042–3052 3049
PR binding to PRE half-sites. This role for the CTE may
be biologically relevant because many natural target
genes for PR contain multiple weak half-sites or palindromic PREs that deviate from consensus sequences, and may require the extended protein-DNA
interface provided by the CTE to assemble a stable
PR-DNA complex needed for gene activation (48). The
unique cross PRE half-site interactions correlated with
a particularly narrow minor groove width, and the CTE
interactions flanking the PREs are consistent with the
observed subtle preference of PR for sequences outside of the consensus PREs. In particular, different 3N
spacer sequence may have propensities to conform to
different DNA structural features, and the CTE interaction may also confer some sequence selectivity to
the PRE-flanking DNA. It is well known that the GR
subgroup of steroid receptors can recognize the same
consensus HRE, yet each receptor clearly regulates
different sets of target genes in vivo (48). Although
coregulatory proteins no doubt play a role in target site
selection, the studies herein suggest that compression
of the minor groove and sequence preferences in the
three-nucleotide spacer and flanking DNA could also
contribute to the differential ability of closely related
receptors in the GR subgroup to bind and activate
specific target genes.
MATERIALS AND METHODS
Protein Expression and Purification
Fig. 5. Analysis of the PRE_2C7A DNA in the PR-DBD Complex
A, Graph of minor groove width (measured by the program
CURVES) vs. nucleotide position of the DNA response elements. The protein names of the oligonucleotide used in the
text, and the PDB IDs (R4R, 1R4O, 1HCQ, 1R4I) are given for
each complex along with the sequence of the DNA fragment
from steroid receptor DBD-DNA crystal structures. HREs are
indicated by gray boxes. B, Analysis of the sequences selected by PR-DBD-648 by MEME. The sequence of the inverted repeat PREs and the preferred sequences in the selection are shown on the x-axis, and the information content
of the alignment for each position is shown on the y-axis.
for weaker half-site (B) to be more greatly influenced
by the flanking sequence and the binding of the CTE.
Conclusions
The crystal structure of PR-DBD bound to DNA has
provided insight into steroid receptor DNA interaction.
The observed CTE in the structure demonstrates for
the first time that a steroid receptor can make additional DNA contacts outside of the consensus HREs.
These CTE interactions, analogous to the CTE interactions of class II and orphan receptors, are crucial for
The expression vector used to produce the for the PR-DBD670
glutathione-S-transferase (GST)-fusion protein for crystallization
was previously described (31). Based on the polypeptide observed in the structure, a similar, but truncated, DBD termed
PR-DBD648 (aa 562–648) was subcloned into the pGEX2T expression vector (Amersham Biosciences, Piscataway, NJ) via
BamH1 and EcoR1 restriction enzyme sites that were introduced using standard PCR protocols. The N-terminal primer
was previously described for PR-DBD670 the C-terminal primer
is 5⬘-GATCGAATTCACTACACAACTCTGACTTTATTGAAC-3⬘.
For mutagenesis, complementary synthetic oligonucleotides
(Macromolecular Resources, Ft. Collins, CO) of PR-DBD648,
containing a StyI site and the codon change for R637A (5⬘-GATCCCTTGGAGGTgccAAATTTAAAAAGTTCAATAAAGTCAGAGTTGTGTAGTGAATTCCGATCA-3⬘ and 5⬘-GATCGGAATTCACTACACAACTCTGACTTTATTGAACTTTTTAAATTTggcACC
TCCAAGGGATCA-3⬘), or the double mutant R637A/K638A
underlined (5⬘-GATCCCTTGGAGGTgccgccTTTAAAAAGTTCAATAAAGTCAGAGTTGTGTAGTGAATTCCGATCA-3⬘ and 5⬘GATCGGAATTCACTACACAACTCTGACTTTATTGAACTTTTTAAAggcggcACCTCCAAGGGATCA-3⬘). Annealed oligonucleotides were inserted into the pGEX2T vector containing PR-DBD670 via the StyI and EcoRI sites yielding the PR-DBD648,
which contained either the point mutant R637A or the double
mutant R637A/K638A. The PR coding regions of plasmids were
confirmed by DNA sequencing.
GST-tagged PR-DBD670, PR-DBD648, and PR-DBD648
mutants were overexpressed in Escherichia coli strain BL21
(DE3) and purified by GST affinity chromatography (Amersham Biosciences). The proteins were cleaved from the glutathione-Sepharose resin with thrombin (Sigma, St. Louis,
MO) at room temperature for 12–16 h. PR-DBD670 was further purified using size-exclusion chromatography in 20 mM
Tris (pH 7.5), 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1
3050 Mol Endocrinol, December 2006, 20(12):3042–3052
mM dithiothreitol (Superdex 30; Amersham Biosciences) and
concentrated. PR-DBD648 and mutant proteins cleaved from
the GST resin were further purified with a cation exchange
column (Source 15S; Amersham Biosciences). The final concentration of the proteins was determined by UV absorbance
spectroscopy (␧ ⫽ 2580 at 280 nm), and the homogeneity of the
DBD was examined using SDS-PAGE, native gel analysis, and
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (Tufts Protein Chemistry Facility, Boston, MA).
DNA Purification
Oligonucleotides (Macromolecular Resources) for crystallography (dCCAGAACAAACTGTTCTG and dCCAGAACAGTTTGTTCTG) were purified by HPLC using an ion-pair reverse-phase
chromatography with XTERRA (Waters, Milford, MA) oligonucleotide column (50). Fractions were pooled, lyophilized, and
resuspended in water. The concentration of each of the DNA
strands was determined by UV absorbance spectroscopy, so
that equal amounts of complementary strands were annealed
by boiling and slow cooling. Correct annealing was assessed
by non-denaturing PAGE, followed by staining with Stainsall
(Sigma) or Vistra Green (Amersham Biosciences).
EMSAs
The stoichiometric gel shifts were carried out with high concentrations of protein (0–45 ␮M) and DNA (10–15 ␮M) to
simulate crystallization conditions. Increasing concentrations
of PR-DBD were incubated with PRE in 0.5⫻ TAE and 30%
glycerol on ice. Reactions were electrophoresed on a cooled
6% nondenaturing polyacrylamide gel (running buffer 0.5⫻
TAE) and stained with Vistra Green (Amersham Biosciences)
for visualization via phosphorimaging (GE Healthcare, Piscataway, NJ). The ratio of DBD to PRE, at which 100% complex
formation was observed, was decreased by 2.5% in crystallization trials, providing a slight excess of DNA needed for
maximum crystallization.
The quantitative EMSA was carried out as described
previously (31–33). PR-DBD648 or PR-DBD648 mutants
were incubated with 10 mM Tris-HCl (pH 7.5), 5 mM NaCl,
5% glycerol, 2 mM MgCl2, 1 mM EDTA, and 5 mM dithiothreitol in the presence of 0.1 ␮g polydA-polydT and 1 ␮g
ovalbumin as a carrier protein, in 25 ␮l total volume, for
30 min. The DNA used for the palindromic PRE and halfsite PRE were (5⬘-GATCTTTGAGAACAAACTGTTCTTAAAACGAGGATC-3⬘) and (5⬘-GATCAAGTTTATAAAAGAACATGCTCAATTT-3⬘), respectively, with the half-sites underlined. The 32P end-labeled DNA was added to reactions
to a final concentration of 0.6 nM and incubated for a
further 30 min on ice. Reactions were electrophoresed on
6% nondenaturing polyacrylamide gels (37.5:1 acrylamide/
bisacrylamide ratio) in 0.5⫻ TAE buffer [0.02 M Tris-acetate
(pH 8.0), and 0.5 mM EDTA] at 4 C. Gels were dried under
vacuum at 80 C and the fraction of bound to free 32Plabeled probe was determined with a series 400 Molecular
Dynamics PhosphorImager. The fraction of DNA bound as
a function of protein concentration was determined from
the average of at least three independent experiments and
graphed as a best-fit binding curves using the equation y ⫽
(x/Kd)/1⫹(x/Kd), where y is the fraction of PRE bound and
x is the concentration of DBD, and the error bars indicate
the standard error.
Binding Site Selection
The binding site selection method described in Pollock and
Treisman (42) was modified to identify PR sequence preferences outside of the hexanucleotide response elements. A
polyclonal antiserum against PR-DBD was used to immunoprecipitate complexes of PR-DBD648 bound to DNA frag-
Roemer et al. • Structure of the PR-DBD-DNA Complex
ments containing a single inverted repeat PRE with a randomized 3N spacer and four randomized nucleotides on
either side of the response element and flanked by fixed ends
for PCR priming and subsequent subcloning. Complex formation was carried out as described earlier. The immunoprecipitated DNA was washed with 10 mM Tris-HCl (pH 7.8), 1 M
NaCl, 5% glycerol, 1 mM EDTA, 2 mM MgCl, 1 ␮M ZnCl, 0.1%
Nonidet P-40, amplified using PCR, and purified using QIAquick Nucleotide Removal Kit (QIAGEN, Valencia, CA). This
process was repeated five times using a decreasing concentration of PR-DBD648 (50, 25, 10, 5, and 2.5 nM), and we
noted that the amount of DNA precipitated during each cycle
increased steadily. After the fifth amplification, the selected
DNA was electrophoresed in the presence of 2.5 nM PRDBD648 on a 8% polyacrylamide gel (37.5:1 acrylamide:bisacrylamide) for 90 min at 20 mA in 0.5⫻ TAE (Tris-acetateEDTA). The shifted DNA was visualized by Vistra Greenstaining (Amersham Biosciences), gel-purified using the
crush and soak procedure (51), subcloned into the BamHIEcoRI site of a pUC18 vector and sequenced using standard
methods. The sequences were aligned using MEME (43). The
alignment used 27 sequences with the option to select the
best alignment of between 15 and 23 nucleotides in length.
The program selected 18 as the optimal length of the alignment. Preference for the sequences shown is indicated by
the value of the information content, where a value of 0.21–
0.42 represents a bias of the sequences such that approximately 70% of the selected sequences are the bases shown.
Crystallization and Data Collection
PR-DBD654 (140 ␮M) and PRE_2C7A DNA (71.75 ␮M) were
incubated together on ice for 20 min to form the complex.
Crystals were grown by sitting drop vapor diffusion in 96-well
plates at 4 C with the addition of 1 ␮l of 70 ␮M DBD654-PRE
complex to an equal volume of reservoir solution [0.1 M
2-(N-morpholino)ethanesulfonic acid (pH 6.0), 10 mM NaCl,
2.5 mM spermine, and 5% hexanediol]. In 1 wk, the crystals
grew to approximately 300 ␮m ⫻ 50 ␮m ⫻ 20 ␮m in space
group P212121. Cryo-preservation was achieved with the addition of 20% glycerol before flash freezing in liquid nitrogen
for data collection at ⫺180 C. Images were collected for 30
min each at 0.5° oscillation over a range of 74°, at the University of Colorado at Denver and Health Sciences Center
Biomolecular X-Ray Crystallography Center, using a Rigaku/
MSC (The Woodlands, TX) Ru-H3R generator (␭⫽1.5418 Å)
equipped with an Raxis IV⫹⫹ area detector, confocal optics
and XTREAM system. Data were indexed and scaled with
HKL2000 (HKL Research, Inc., Charlottesville, VA).
Structure Determination, Refinement, and Analysis
A PR-DNA homology model, based on the ER-ERE complex
structure (35), was used to obtain a molecular replacement
solution using AmoRe within the Crystallography and NMR
Software suite (CNS) (52). Model building was performed with
O (53). Initial refinement was performed with CNS and completed with the program Refmac 5 from CCP4 (38). The
program CONTACT (CCP4: Supported Program (38) was
used to determine interatomic distances and hydrogen
bonds within the structure. GETAREA (54), a web-based
server, was used to determine solvent accessible surface
area with a 1.4 Å probe. DNA conformation and groove width
was determined using the program CURVES (41). Images in
the figures were made using the program Pymol (DeLano
Scientific LLC, San Carlos, CA). The coordinates have the
PDB ID 2C7A.
Acknowledgments
Received December 13, 2005. Accepted August 16, 2006.
Roemer et al. • Structure of the PR-DBD-DNA Complex
Address all correspondence and requests for reprints to:
Mair E. A. Churchill, Department of Pharmacology, MS
8303, P.O. Box 6511, Aurora, Colorado 80045. E-mail:
[email protected].
We acknowledge the support of the Howard Hughes Medical Institute, University of Colorado Cancer Center (Core
Grant P30-CA46934) for the University of Colorado Health
Sciences Center Biomolecular X-ray Crystallography Center.
This work was also supported by grants from the National
Institutes of Health (CA46938 to D.P.E. and GM56881 to
M.E.A.C.).
Current address for D.P.E.: Baylor College of Medicine,
Department of Molecular and Cellular Biology and Pathology,
Houston, Texas 77030. E-mail: [email protected].
Disclosure Statement: The authors have nothing to disclose.
Mol Endocrinol, December 2006, 20(12):3042–3052 3051
15.
16.
17.
18.
19.
REFERENCES
20.
1. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P,
Schutz G, Umesono K, Blumberg B, Kastner P, Mark M,
Chambon P, Evans RM 1995 The nuclear receptor
superfamily: the second decade. Cell 83:835–839
2. Beato M, Klug J 2000 Steroid hormone receptors: an
update. Hum Reprod Update 6:225–236
3. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895
4. Khorasanizadeh S, Rastinejad F 2001 Nuclear-receptor
interactions on DNA-response elements. Trends Biochem Sci 26:384–390
5. Wilson TE, Fahrner TJ, Milbrandt J 1993 The orphan
receptors NGFI-B and steroidogenic factor 1 establish
monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol Cell Biol 13:5794–5804
6. Giguere V, Tini M, Flock G, Ong E, Evans RM, Otulakowski G 1994 Isoform-specific amino-terminal domains
dictate DNA-binding properties of ROR ␣, a novel family
of orphan hormone nuclear receptors. Genes Dev
8:538–553
7. Yan ZH, Medvedev A, Hirose T, Gotoh H, Jetten AM 1997
Characterization of the response element and DNA binding properties of the nuclear orphan receptor germ cell
nuclear factor/retinoid receptor-related testis-associated
receptor. J Biol Chem 272:10565–10572
8. Meinke G, Sigler PB 1999 DNA-binding mechanism of
the monomeric orphan nuclear receptor NGFI-B. Nat
Struct Biol 6:471–477
9. Gearhart MD, Holmbeck SM, Evans RM, Dyson HJ,
Wright PE 2003 Monomeric complex of human orphan
estrogen related receptor-2 with DNA: a pseudo-dimer
interface mediates extended half-site recognition. J Mol
Biol 327:819–832
10. Zilliacus J, Wright AP, Carlstedt-Duke J, Gustafsson JA
1995 Structural determinants of DNA-binding specificity
by steroid receptors. Mol Endocrinol 9:389–400
11. Lee MS, Kliewer SA, Provencal J, Wright PE, Evans RM
1993 Structure of the retinoid X receptor ␣ DNA binding
domain: a helix required for homodimeric DNA binding.
Science 260:1117–1121
12. Rastinejad F, Perlmann T, Evans RM, Sigler PB 1995
Structural determinants of nuclear receptor assembly on
DNA direct repeats. Nature 375:203–211
13. Sem DS, Casimiro DR, Kliewer SA, Provencal J, Evans
RM, Wright PE 1997 NMR spectroscopic studies of the
DNA-binding domain of the monomer-binding nuclear
orphan receptor, human estrogen related receptor-2.
The carboxyl-terminal extension to the zinc-finger region
is unstructured in the free form of the protein. J Biol
Chem 272:18038–18043
14. Rastinejad F, Wagner T, Zhao Q, Khorasanizadeh S 2000
Structure of the RXR-RAR DNA-binding complex on the
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
retinoic acid response element DR1. EMBO J 19:
1045–1054
Zhao Q, Chasse SA, Devarakonda S, Sierk ML, Ahvazi B,
Rastinejad F 2000 Structural basis of RXR-DNA interactions. J Mol Biol 296:509–520
Freedman LP 1992 Anatomy of the steroid receptor zinc
finger region. Endocr Rev 13:129–145
Shaffer PL, Gewirth DT 2002 Structural basis of VDRDNA interactions on direct repeat response elements.
EMBO J 21:2242–2252
Zhao Q, Khorasanizadeh S, Miyoshi Y, Lazar MA, Rastinejad F 1998 Structural elements of an orphan nuclear
receptor-DNA complex. Mol Cell 1:849–861
Solomon IH, Hager JM, Safi R, McDonnell DP, Redinbo
MR, Ortlund EA 2005 Crystal structure of the human
LRH-1 DBD-DNA complex reveals Ftz-F1 domain positioning is required for receptor activity. J Mol Biol 354:
1091–1102
Wilson TE, Paulsen RE, Padgett KA, Milbrandt J 1992
Participation of non-zinc finger residues in DNA binding
by two nuclear orphan receptors. Science 256:107–110
Zechel C, Shen XQ, Chambon P, Gronemeyer H 1994
Dimerization interfaces formed between the DNA binding
domains determine the cooperative binding of RXR/RAR
and RXR/TR heterodimers to DR5 and DR4 elements.
EMBO J 13:1414–1424
Hsieh JC, Jurutka PW, Selznick SH, Reeder MC, Haussler CA, Whitfield GK, Haussler MR 1995 The T-box near
the zinc fingers of the human vitamin D receptor is required for heterodimeric DNA binding and transactivation. Biochem Biophys Res Commun 215:1–7
Hsieh JC, Whitfield GK, Oza AK, Dang HT, Price JN, Galligan MA, Jurutka PW, Thompson PD, Haussler CA, Haussler MR 1999 Characterization of unique DNA-binding and
transcriptional-activation functions in the carboxyl-terminal
extension of the zinc finger region in the human vitamin D
receptor. Biochemistry 38:16347–16358
Quack M, Szafranski K, Rouvinen J, Carlberg C 1998 The
role of the T-box for the function of the vitamin D receptor
on different types of response elements. Nucleic Acids
Res 26:5372–5378
Verrier CS, Roodi N, Yee CJ, Bailey LR, Jensen RA,
Bustin M, Parl FF 1997 High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor
TAF(II)30 affect estrogen receptor-mediated transcriptional activation. Mol Endocrinol 11:1009–1019
Boonyaratanakornkit V, Melvin V, Prendergast P, Altmann M, Ronfani L, Bianchi ME, Taraseviciene L, Nordeen SK, Allegretto EA, Edwards DP 1998 High-mobility
group chromatin proteins 1 and 2 functionally interact
with steroid hormone receptors to enhance their DNA
binding in vitro and transcriptional activity in mammalian
cells. Mol Cell Biol 18:4471–4487
Romine LE, Wood JR, Lamia LA, Prendergast P, Edwards DP, Nardulli AM 1998 The high mobility group
protein 1 enhances binding of the estrogen receptor DNA
binding domain to the estrogen response element. Mol
Endocrinol 12:664–674
Zhang CC, Krieg S, Shapiro DJ 1999 HMG-1 stimulates
estrogen response element binding by estrogen receptor
from stably transfected HeLa cells. Mol Endocrinol 13:
632–643
Verrijdt G, Haelens A, Schoenmakers E, Rombauts W,
Claessens F 2002 Comparative analysis of the influence
of the high-mobility group box 1 protein on DNA binding
and transcriptional activation by the androgen, glucocorticoid, progesterone and mineralocorticoid receptors.
Biochem J 361:97–103
Melvin VS, Edwards DP 1999 Coregulatory proteins in
steroid hormone receptor action: the role of chromatin
high mobility group proteins HMG-1 and -2. Steroids
64:576–586
3052 Mol Endocrinol, December 2006, 20(12):3042–3052
31. Melvin VS, Roemer SC, Churchill ME, Edwards DP 2002
The C-terminal extension (CTE) of the nuclear hormone
receptor DNA binding domain determines interactions
and functional response to the HMGB-1/-2 co-regulatory
proteins. J Biol Chem 277:25115–25124
32. Melvin VS, Harrell C, Adelman JS, Kraus WL, Churchill
ME, Edwards DP 2004 The role of the C-terminal extension (CTE) of the estrogen receptor ␣ and ␤ DNA binding
domain in DNA binding and interaction with HMGB.
J Biol Chem 279:14763–14771
33. Das D, Peterson RC, Scovell WM 2004 High mobility group
B proteins facilitate strong estrogen receptor binding to
classical and half-site estrogen response elements and relax binding selectivity. Mol Endocrinol 18:2616–2632
34. Luisi BF, Xu WX, Otwinowski Z, Freedman LP,
Yamamoto KR, Sigler PB 1991 Crystallographic analysis
of the interaction of the glucocorticoid receptor with
DNA. Nature 352:497–505
35. Schwabe JW, Chapman L, Finch JT, Rhodes D 1993 The
crystal structure of the estrogen receptor DNA-binding
domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567–578
36. Shaffer PL, Jivan A, Dollins DE, Claessens F, Gewirth DT
2004 Structural basis of androgen receptor binding to
selective androgen response elements. Proc Natl Acad
Sci USA 101:4758–4763
37. Mitchell JC, Kerr R, Ten Eyck LF 2001 Rapid atomic
density methods for molecular shape characterization.
J Mol Graph Model 19:325–330:388–390
38. Collaborative Computational Project Number 4 1994 The
CCP4 suite: programs for protein crystallography. Acta
Crystallogr D Biol Crystallogr 50:760–763
39. Giguere V, Hollenberg SM, Rosenfeld MG, Evans RM
1986 Functional domains of the glucocorticoid receptor.
Cell 46:645–652
40. Jones S, van Heyningen P, Berman HM, Thornton JM 1999
Protein-DNA interactions: a structural analysis. J Mol Biol
287:877–896
41. Lavery R, Sklenar H 1988 The definition of generalized
helicoidal parameters and of axis curvature for irregular
nucleic acids. J Biomol Struct Dyn 6:63–91
42. Pollock R, Treisman R 1990 A sensitive method for the
determination of protein-DNA binding specificities. Nucleic Acids Res 18:6197–6204
43. Bailey TL, Elkan C 1994 Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2:28–36
Roemer et al. • Structure of the PR-DBD-DNA Complex
44. Travers AA 2004 The structural basis of DNA flexibility.
Philos Transact A Math Phys Eng Sci 362:1423–1438
45. Vlahovicek K, Kajan L, Pongor S 2003 DNA analysis
servers: plot.it, bend.it, model.it and IS. Nucleic Acids
Res 31:3686–3687
46. Klinge CM 2001 Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res 29:
2905–2919
47. Nelson CC, Hendy SC, Shukin RJ, Cheng H, Bruchovsky
N, Koop BF, Rennie PS 1999 Determinants of DNA sequence specificity of the androgen, progesterone, and
glucocorticoid receptors: evidence for differential steroid
receptor response elements. Mol Endocrinol 13:
2090–2107
48. Lieberman BA, Bona BJ, Edwards DP, Nordeen SK 1993
The constitution of a progesterone response element.
Mol Endocrinol 7:515–527
49. Nordeen SK, Suh BJ, Kuhnel B, Hutchison CD 1990
Structural determinants of a glucocorticoid receptor recognition element. Mol Endocrinol 4:1866–1873
50. Gilar M, Fountain KJ, Budman Y, Neue UD, Yardley KR,
Rainville PD, Russell 2nd RJ, Gebler JC 2002 Ion-pair
reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction.
J Chromatogr A 958:167–182
51. Maxam AM, Gilbert W 1980 Sequencing end-labeled
DNA with base-specific chemical cleavages. Methods
Enzymol 65:499–560
52. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P,
Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M,
Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL
1998 Crystallography, NMR system: a new software suite
for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54(Pt 5):905–921
53. Jones TA, Zou JY, Cowan SW, Kjeldgaard M 1991 Improved methods for building protein models in electron
density maps and the location of errors in these models.
Acta Crystallogr A 47(Pt 2):110–119
54. Fraczkiewicz R, Braun W 1998 Exact and efficient analytical calculation of the accessible surface areas and
their gradients for macromolecules. J Comp Chem 19:
319–333
55. Maiti R, Van Domselaar GH, Zhang H, Wishart DS 2004
Super Pose: a simple server for sophisticated structural
super position. Nucleic Acids Res 32:W590–W594
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