From the
The three P-box amino acids in the DNA recognition
The thyroid hormone receptor (T3R)
While it is likely that most receptors
utilize these conserved functions, individual receptors bind to, and
activate transcription from, distinct DNA sequences(4) .
Discrimination between DNA binding sites by the various receptors
occurs at three levels: 1) nucleotide preference of the third and
fourth base pairs of the core DNA element (9-11); 2) flanking
sequence preferences upstream and possibly downstream of the core DNA
element(12, 13, 14, 15) ; and 3)
orientation and spacing of core sequence half-sites for receptors which
bind to DNA as homodimers or
heterodimers(16, 17, 18, 19) . Crystal
structures and mutagenesis studies have indicated that recognition of
the third and fourth base pairs of the hexameric half-site are largely
determined by the identity of three amino acids within the DNA
recognition
In the
present study, we have tested the contextual nature of P-box function
in hT3R
The role of P-box amino acids in determining the unique DNA
binding specificity of hT3R
Perhaps the most
intriguing results were obtained using the everted repeat with AGTCCA
half-sites. Binding of hT3R
The P-box amino acids of the nuclear receptor family play a
crucial role in DNA binding site selection. This amino acid motif in
the recognition
DNA binding of the wild
type hT3R
For high affinity binding of variant hT3R
Compared
to the other two P-box positions, the third position can be substituted
with a variety of amino acids without compromising the binding of
hT3R
Of the everted
repeats with AGTNCA half-site sequences, only those with AGTTCA or
AGTCCA half-sites bound receptors with reasonably strong affinities.
These elements have a T-A base pair in the third position, which
correlates to the loss of the requirement for a glutamate in the first
P-box position for receptor binding. On the everted repeat with AGTTCA
half-sites, substitution of asparagine or aspartate for glutamate in
the first P-box position is compatible with high affinity binding of
variant hT3R
The wild type
hT3R
Of the 19
variant receptors with substitutions in the second P-box position, none
had detectable DNA binding affinity for the everted repeats with AGTTCA
or AGTCCA half-sites, and the wild type receptor was able to bind only
to the AGTTCA element. Substitution of the glycine at the third P-box
position with glutamine results in high affinity binding to the everted
repeat with AGTCCA half-site sequences. This variant receptor
(EGQ) also has an increased affinity for everted repeats with
AGTTCA or AGGCCA half-sites in comparison to the wild type (EGG)
receptor. These results suggest that the glutamine in the third P-box
position may make a base specific contact to these particular DNA
sequences, with the most probable site of interaction being the fourth
base pair of the half-site. An everted repeat with AGTCCA half-sites
requires a ``change in specificity'' mutation in the P-box of
hT3R
Taken
as a whole, the data suggest that one position of the P-box is involved
in making base contacts with the DNA, while amino acid substitution at
another position is subject to fairly stringent steric constraints, and
the final P-box position can accommodate a variety of amino acid
substitutions without affecting DNA binding affinity. Perhaps the most
striking feature of this observation is the fact that many of the P-box
variants which retain strong binding affinity to TREs with half-site
sequences commonly found in genomic DNA, have not been identified among
the known members of the superfamily of nuclear receptors. This
discrepancy suggests that either a variety of orphan receptors with
such P-box sequences remain to be identified, or that the natural
evolution of the P-box motif in these receptors has operated under
constraints in addition to DNA binding affinity.
Other features of
TREs besides half-site sequence dictate the binding affinity of
hT3R
We thank R. Evans for providing plasmid peA101, C.
Juricic for assistance in the construction of mutant receptor clones,
and J. Faris for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helix
of steroid/thyroid hormone receptors participate in the discrimination
of the central base pairs of the hexameric half-sites of receptor
response elements in DNA. A series of 57 variants of the
isoform
of the human thyroid hormone receptor were constructed in which the 19
possible amino acid substitutions were incorporated at each of the
three P-box positions. The effects of these substitutions on the
sequence specificity of the DNA binding activity of the receptor were
analyzed using 16 everted repeat elements which differed in sequence in
the two central base pairs of the hexameric half-sites. Only receptors
with glutamate or aspartate as the first P-box amino acid had
detectable DNA binding affinity on everted repeats with AGGNCA
half-sites. Only those receptors with alanine, glycine, serine, or
proline in the second P-box position were able to bind to this same
group of everted repeat elements. In contrast, many of the variant
receptors with substitutions at the third P-box position were capable
of binding to the AGGNCA group of repeat elements. The actual
substitutions at the third P-box position that were compatible with
binding depended upon the identity of the fourth base pair of the
AGGNCA half-sites. Of the remaining 12 everted repeat sequences, only
those with AGTTCA or AGTCCA half-sites were able to bind any of the
receptors. In addition to wild type receptor, several variant receptors
with amino acid substitutions in either the first or third P-box
position were able to bind to the everted repeat with AGTTCA
half-sites. The everted repeat with AGTCCA half-sites was bound by
receptors with a DGG, NGG, or EGQ P-box sequence, but not the wild type
receptor which has an EGG P-box sequence. These data demonstrate that
all three P-box positions of the thyroid hormone receptor function to
discriminate between half-sites that differ in sequence at the third
and fourth base pairs.
(
)belongs to a superfamily of ligand responsive
transcription factors which includes the steroid and retinoid
receptors, as well as a number of ``orphan'' receptors for
which regulatory ligands have not been
identified(1, 2, 3) . In general, these
receptors activate transcription in response to endocrine and metabolic
stimuli by binding to DNA elements found in the promoters of specific
genes. For most receptors studied to date the core DNA recognition
element is AGNNCA(4) . Structural analysis of the glucocorticoid
receptor and the estrogen receptor has shown that the DNA binding
domain consists of two interdependent zinc finger-like motifs with an
amphipathic
-helix at the C-terminal region of each
finger(5, 6, 7, 8) . The DNA recognition
-helix is positioned on the C-terminal side of the first zinc
finger and lies in the major groove of the core DNA sequence. Within
the DNA recognition
-helix conserved lysine and arginine residues
contact the conserved guanines at positions 2 and -5 of the core
DNA element(5, 8) . The
-helix following the second
finger folds over the DNA recognition
-helix to stabilize the
structure through hydrophobic interactions and provides phosphate
contacts to the DNA backbone.
-helix referred to as the ``P-box'' (Fig. 1). The glucocorticoid receptor contains a GSV P-box amino
acid sequence and binds with the highest affinity to an inverted repeat
with AGAACA half-site sequences. The valine forms a specific
interaction with the fourth base pair of this sequence (5). In
contrast, the estrogen receptor which has an EGA P-box sequence, binds
with the highest affinity to an AGGTCA half-site because of specific
interactions formed between the glutamate and the central base pairs of
this half-site(8) . The third P-box amino acid in the estrogen
receptor, unlike its counterpart in the glucocorticoid receptor, does
not contact any nucleotides. However, mutational studies of the
estrogen receptor, thyroid hormone receptor, and v-erbA
oncogene have shown that substitution of the third P-box amino acid has
profound effects on the sequence specific binding of these receptors to
DNA(20, 21, 22, 23) .
Figure 1:
Schematic representation of the DNA
binding domain of the hT3R and nucleotide contacts by nuclear
receptors. A, the hT3R
DNA binding domain. Shown in the
boxed regions are the predicted
-helices by comparison to the
structural analysis of the estrogen receptor and glucocorticoid
receptor (5, 8). The P-box amino acids within the DNA recognition
-helix are circled. B, three classes of response
elements bound by hT3R
, showing the differences in AGGTCA
half-site orientation and spacing between half-sites. C,
ladder diagram of specific nucleotide contacts demonstrated in the
crystal structures of the estrogen receptor and glucocorticoid receptor
bound to their respective DNA half-sites (5, 8) with numbering
according to the corresponding sequences in the
hT3R
.
The effects of
the natural variants of the P-box sequence within the thyroid hormone
receptor/estrogen receptor superfamily (EGG, EGA, EGS, ESG, and EAA)
have been studied in the context of hT3R(23) . This
receptor is able to interact with DNA elements that consist of two
AGGNCA half-sites oriented as a direct repeat, inverted repeat, or
everted repeat (Fig. 1). The results of studying this small
sample of P-box variants suggested that each amino acid in the P-box
functions within the context of the entire P-box sequence such that
substitution of an amino acid at one position may alter the
functionality of an amino acid at one of the other positions.
in greater detail. A total of 57 variant hT3R
receptors incorporating all possible amino acids at each of the three
P-box positions were constructed. The effects of these variant P-box
sequences on the DNA sequence specificity of the receptors was
investigated using a panel of 16 everted repeat elements which differ
in sequence at the central base pairs of the hexameric half-site. The
results of this study demonstrate that all three P-box amino acids in
hT3R
function to discriminate the nucleotide sequence of the third
and fourth base pairs of the half-sites in everted repeats. Several of
the variant receptors have a DNA binding specificity distinct from the
wild type receptor.
Construction and Expression of hT3R
P-box amino acid substitution mutants of hT3R
Mutants
were
created by a site-directed mutagenesis strategy described
previously(23) . DNA sequencing was used to verify that only the
desired mutation was introduced into the hT3R
cDNA. Proteins were
prepared by in vitro transcription/translation using a T7 RNA
polymerase coupled-rabbit reticulocyte translation system (Promega) and
DNA templates that had been linearized at a HindIII site
downstream of the hT3R
cDNA. Protein yield in each reaction was
quantified by monitoring the conversion of
S-labeled
methionine into trichloroacetic acid-precipitated form, and by
verifying the production of full-length receptor by SDS-polyacrylamide
gel electrophoresis. Translated proteins were diluted to identical
concentrations using unprogrammed lysate and stored frozen at -70
°C in 3-µl aliquots.
Construction of Mutant TREs
A series of
complementary oligonucleotides were synthesized with the core sequence
of the upper strand being TGACCTctgcagAGGTCA flanked by HindIII compatible overhangs. In the complete series of
everted repeat elements, the nucleotide positions in bold in the
AGGTCA half-site on each strand were replaced with GA, GG, GC,
AT, AA, AG, AC, CT, CA, CG, CC, TT, TA, TG, or TC. Each everted repeat
was cloned into the HindIII site of pUC19. DNA fragments
containing the everted repeats were excised from pUC19 by digestion
with HindIII, labeled with P, and purified by gel
electrophoresis as described previously(23) .
DNA Binding Analysis
Aliquots of in vitro translated proteins (3 µl) were preincubated on ice for 10 min
in a 10-µl volume containing 50 mM KCl, 20 mM HEPES, pH 7.8, 1 mM -mercaptoethanol, 5% glycerol,
and 2 µg of poly(dI-dC) (Pharmacia). Labeled DNA (10,000 cpm, about
2 fmol) in a 2-µl volume was added and incubation was continued on
ice for another 10 min. Under these conditions, the wild type hT3R
protein bound approximately 20-30% of the labeled DNA when the
everted repeat element had the wild type AGGTCA half-site sequences
flanked by 5`-CAG(23) . The binding of mutant receptors with
less than 1% of the activity of the wild type receptor could be
detected under these binding conditions when the time for
autoradiography was extended from 16 to 96 h(24) . Reactions
were loaded onto a 5% polyacrylamide gel (55:1
acrylamide:bisacrylamide) containing 0.3
TBE and 0.1% Nonidet
P-40. Gels were run for 2 h at 15 volts/cm at approximately 15 °C
in 0.3
TBE. Gels were dried and autoradiographed at -70
°C using Kodak X-AR film. Experiments were repeated a minimum of
three times using different preparations of protein.
was investigated by creating 19-amino
acid substitution mutants at each of the three P-box positions (Fig. 1A). These mutants in conjunction with the wild
type receptor were then assayed for DNA binding specificity using 16
different everted repeat elements that varied in the identity of the
third and fourth base pairs of the hexameric half-site. The everted
configuration of half-sites was chosen because hT3R
homodimers
have a high DNA binding affinity for this orientation and spacing of
half-sites (16, 17) and because the symmetrical nature
of the everted repeat elements provided identical flanking sequences
for the two half-sites (Fig. 1B).
Effects of Amino Acid Substitutions at the First P-box Position
on the Interaction of hT3R
The effects on DNA binding specificity of the
20-amino acid variants in the first P-box position were tested using
everted repeat elements containing AGGNCA half-sites. The results of
the gel shift analysis demonstrated that binding of hT3R with TREs Containing AGGNCA
Half-sites
receptors
to these four DNA elements was largely restricted to those containing
the wild type EGG P-box, with varying degrees of binding
observed for the mutant receptor with a DGG P-box depending upon
the identity of the fourth nucleotide of the half-site (Fig. 2).
On the element with AGGTCA half-sites, the receptor with a EGG
P-box bound with the highest affinity, the receptor with the DGG
P-box bound with lower affinity, and there was faint, but detectable
binding by the mutant receptor with a NGG P-box. Receptor
binding on the element containing AGGACA half-sites demonstrated that
the EGG P-box directs high affinity binding, while the DGG P-box directs very low affinity binding. On AGGGCA and
AGGCCA half-sites, the wild type receptor with an EGG P-box
bound with a slightly higher affinity than the mutant receptor with the DGG P-box. Thus the ratio of the affinities of these two
receptors on these two elements is distinctly different from the
relative binding affinities of these proteins on the elements with
AGGTCA and AGGACA half-sites, where EGG was strongly preferred
over DGG.
Figure 2:
Gel
mobility shift analysis of amino acid substitution mutants in the first
P-box position of hT3R. DNA-protein interactions were analyzed by
gel mobility shift analysis on DNA elements composed of everted
half-sites of the sequence AGGNCA, where N was T, A, G, or C as
indicated. The P-box amino acid sequence of the receptor protein is
indicated by the amino acid one letter code, where the amino acid in
bold is the one varied within this set of mutants. Shown are strips of
the autoradio-graphs containing the homodimer-DNA
complexes.
The identity of the fourth nucleotide in the
half-site alters the overall affinity for the binding of hT3R
receptors. This difference in receptor binding affinity between the
four elements is not apparent in Fig. 2because autoradiographic
exposure times were adjusted to identify the P-box variants of
hT3R
that would bind to a particular DNA element. DNA binding
affinity for this set of P-box variants was highest for the consensus
AGGTCA half-sites, weaker for AGGACA half-sites, and considerably
weaker for AGGCCA and AGGGCA half-sites.
Effects of Amino Acid Substitutions at the Second P-box
Position on the Interaction of hT3R
The effects of amino acid substitutions at the second
P-box position of hT3R with TREs Containing AGGNCA
Half-sites
were tested using everted repeats with
AGGNCA half-sites (Fig. 3). On AGGTCA half-sites, DNA binding was
observed for the wild type hT3R
(EGG P-box) and the mutant
receptor with an EAG P-box. A minimal amount of binding to this
element was also observed for the mutant receptor with an ESG
P-box. On everted repeats with either AGGGCA or AGGCCA half-sites, only
those receptors with EGG and EAG P-box sequences were
able to bind with high affinity. The everted repeat consisting of
AGGACA half-sites was able to bind a greater variety of mutant
hT3R
receptors: in addition to receptors with EGG and
EAG P-box sequences, mutant receptors with ESG and
EPG P-box sequences also bound to this element. It is
interesting to note that the mutant receptor with an EAG P-box
sequence bound with a substantially higher affinity than the wild type
receptor with the EGG P-box to the AGGACA half-sites, whereas
these two receptors had roughly equal affinity for binding to the other
three half-site sequences in this series (Fig. 3).
Figure 3:
Gel
mobility shift analysis of amino acid substitution mutants in the
second P-box position of hT3R. DNA-protein interactions were
analyzed by gel mobility shift analysis on DNA elements composed of
everted half-sites of the sequence AGGNCA, where N was T, A, G, or C as
indicated. The P-box amino acid sequence of the receptor protein is
indicated by the amino acid one letter code, where the amino acid in
bold is the one varied within this set of mutants. Shown are strips of
the autoradio-graphs containing the homodimer-DNA
complexes.
Effects of Amino Acid Substitutions at the Third P-box
Position on the Interaction of hT3R
Amino acid substitutions at the third P-box position
of hT3R with TREs Containing AGGNCA
Half-sites
had far fewer deleterious effects on the binding of the
receptor to everted repeats with AGGNCA half-sites (Fig. 4).
Mutant hT3R
receptors with EGA, EGV, EGL,
EGM, EGG, EGS, and EGT P-box sequences
bound with the highest affinities to the element with AGGTCA
half-sites. The mutant receptor with an EGC P-box bound to this
everted repeat with a strong affinity both as a monomer and a dimer. In
comparison, receptors with EGI, EGN, and EGQ P-box
sequences bound to the AGGTCA element with a somewhat lower affinity
and binding of the mutant receptor with the EGK P-box had the
lowest affinity. The binding of this panel of hT3R
receptors to
the everted repeat with AGGACA half-sites differed significantly: only
the wild type receptor with an EGG P-box had a strong affinity
for this element, while mutant receptors with the P-box sequences
EGA, EGV, EGL, EGI, EGM, EGT,
and EGN bound with very low affinities. A third distinct pattern
of binding was observed for the everted repeat with AGGGCA half-sites.
Receptors with the highest affinities for this element had EGI and EGT P-box sequences. Strong binding was also observed
for mutant receptors with EGA, EGM, EGS, and
EGK P-box sequences. In comparison, mutant receptors with the
P-box sequences EGV and EGY bound with reduced affinity
to the everted repeat with AGGGCA half-sites and there was barely
detectable binding observed for the receptors with EGL,
EGF, EGG, and EGN P-box sequences. On the element
containing AGGCCA half-sites the highest binding affinity was observed
for those mutant receptors with EGN, EGQ, and EGK P-box sequences, whereas a lower binding affinity was observed for
those receptors with EGA, EGG, and EGS P-box
sequences. Weak DNA binding was detected on this element for mutant
receptors with EGI, EGM, and EGT P-box sequences.
Figure 4:
Gel
mobility shift analysis of amino acid substitution mutants in the third
P-box position of hT3R. DNA-protein interactions were analyzed by
gel mobility shift analysis on DNA elements composed of everted
half-sites of the sequence AGGNCA, where N was T, A, G, or C as
indicated. The P-box amino acid sequence of the receptor protein is
indicated by the amino acid one letter code, with the amino acid in
bold being analyzed. Shown are strips of the autoradiographs containing
the monomer-DNA complexes (M) and the homodimer-DNA complexes (D).
Some P-box Substitution Mutants Also Bind to AGTNCA
Half-sites
Some of the P-box variants of hT3R were able to
bind to elements with half-sites that had a thymidine in the third
nucleotide position (AGTNCA). Receptor binding on the everted repeat
with AGTTCA half-sites demonstrated a unique pattern of amino acid
compatibility at all three P-box positions (Fig. 5). On this
element amino acid variants in the first P-box position of hT3R
that retained strong DNA binding affinity included NGG, DGG, and EGG; detectable DNA binding was observed for
receptors with P-box sequences of AGG, GGG, and SGG. With the exception of the wild type EGG P-box, none
of the mutant receptors with an amino acid substitution at the second
P-box position were able to bind to the AGTTCA half-sites. Of the
substitution mutations at the third P-box position, the highest DNA
binding affinities were achieved by receptors with EGQ and
EGA P-box sequences, while those receptors with EGL,
EGG, EGS, and EGN P-box sequences bound to this
element with lower affinities. It is interesting to note that the
AGTTCA half-site sequence is commonly found in naturally occurring
retinoic acid and vitamin D
response elements, and that the
corresponding receptors have a higher affinity for this motif than
AGGTCA half-sites.
Figure 5:
P-box mutants of hT3R were analyzed
for DNA binding on AGTTCA half-sites. DNA-protein interactions were
analyzed by gel mobility shift analyses on an everted repeat of AGTTCA
half-sites. The position of the P-box substitution is indicated at the
left-hand column by the letter ``X,'' with the
identity of X being shown in the top row by the amino acid one
letter code. Shown are autoradiograph strips of the homodimer-DNA
complexes.
The everted repeat with AGTACA half-sites had a
very weak affinity for the DGG P-box variant receptor, and even
less affinity for mutant receptors with NGG, EAG, and
EGA P-box sequences. The wild type hT3R receptor with a EGG
P-box did not bind to this element. On an everted repeat with AGTGCA
half-sites, very weak binding was observed for receptors with EGG, DGG, and NGG P-box sequences, and barely
detectable binding was observed for receptors with EGA and
EGS P-box sequences (data not shown).
variant receptors to this element
demonstrated a clear ``change in specificity.'' In
particular, the wild type receptor was unable to bind to this everted
repeat, while several mutant receptors bound with high affinity (Fig. 6). Of the variant receptors with an amino acid
substitution in the first P-box position, the receptor with a DGG P-box bound to the AGTCCA everted repeat with highest
affinity. Strong DNA binding affinity was also observed for the
receptor with an NGG P-box and lower binding affinities were
observed for the receptors with AGG, LGG, PGG, GGG, SGG, and VGG P-box sequences. The
incompatibility of a glutamate in the first P-box position of hT3R
with binding to AGTCCA half-sites could not be overcome by any amino
acid substitution in the second P-box position. However, amino acid
substitution in the third P-box position of hT3R
resulted in high
affinity binding to the everted repeat with AGTCCA half-sites by the
receptor with a EGQ P-box sequence and barely detectable binding
of receptors with EGA and EGS P-box sequences. The effect
of the glutamine for glycine substitution in the third P-box position
is somewhat different for the binding of the receptor to everted
repeats that have only the thymidine substitution in the third
nucleotide of the half-site (AGTTCA; Fig. 5) or only the cytidine
substitution in the fourth nucleotide of the half-site (AGGCCA; Fig. 4). In each of these cases, the presence of a glutamine in
the third P-box position (EGQ) provides an increase in DNA
binding affinity in comparison to the wild type receptor (EGG)
which has a reasonable affinity for these two elements. However, as the
data in Fig. 6show, if the half-sites of the everted repeat
contain both of these nucleotide substitutions (AGTCCA) the receptor
with the EGQ P-box sequence binds with high affinity whereas the
wild type receptor is unable to bind at all.
Figure 6:
P-box mutants of hT3R were analyzed
for DNA binding on AGTCCA half-sites. DNA-protein interactions were
analyzed by gel mobility shift analyses on an everted repeat of AGTCCA
half-sites. The position of the P-box substitution is indicated at the
left-hand column by the letter ``X,'' with the
identity of X being shown in the top row by the amino acid one
letter code. Shown are autoradiograph strips of the homodimer-DNA
complexes.
Adenine or Cytosine in the Third Nucleotide of the TRE
Half-site Sequence Block Receptor Binding
No binding was
detected for either wild type hT3R or the 57 P-box variant
receptors on the eight DNA elements that had either an adenosine
(AGANCA) or cytidine (AGCNCA) in the third nucleotide position of the
half-site (data not shown). This result indicates that there is a
strict requirement for a guanosine or thymidine in the third nucleotide
position of the TRE half-site for binding of this series of related
receptors. The simplest explanations for this observation would be that
either a critical contact is formed between the receptor and the AGGNCA
and AGTNCA half-sites that is lost when the other two nucleotides are
substituted into the third position, or that substitution of an A or C
at that position of the half-site sterically hinders the recognition
-helix of the receptor from interacting with the DNA. It seems
likely that there are other P-box combinations in the complete set of
8000 possible P-box sequences that would interact with TREs containing
AGANCA or AGCNCA half-site sequences.
-helix of these proteins determines the two
central base pairs of the hexameric half-site for a cognate
receptor-DNA interaction(9, 10, 11) . Other
amino acids in the same
-helix interact with the base pairs that
are common to the consensus binding sites of this family of
receptors(5, 8) . In the present study we have used
site-directed mutagenesis of both the P-box amino acids of hT3R
and the central base pairs of an everted repeat element to probe the
interaction of these species in more detail.
and certain of the 57 P-box variant receptors that were
constructed was restricted to two sets of related everted repeats:
those with half-site sequences in the series AGGNCA and those with
half-site sequences in the series AGTNCA. On the AGGNCA set of
elements, the first P-box position of hT3R
must contain either the
wild type glutamate or an aspartate for binding of the receptor to DNA.
This result can be best understood by drawing analogy to the crystal
structure of the estrogen receptor-DNA complex, since both wild type
receptors have EG(G or A) P-box sequences. In the estrogen receptor-DNA
complex, the glutamate in the first P-box position makes hydrogen
bonding contacts to the cytidine that is based paired to the guanosine
at the third nucleotide position of the half-site. In the consensus
AGGTCA half-site that is common to thyroid hormone response elements
and estrogen response elements, the glutamate also forms hydrogen bonds
to the N6 and N7 of the adenosine that is base paired to the thymidine
at the fourth position(8) . Our data indicates that the wild
type hT3R
receptor is capable of binding with appreciable affinity
to all four half-site sequences in the series AGGNCA, which suggests
that the glutamate contacts to this adenosine are not critical for DNA
binding.
receptors to
the AGGNCA elements, the second P-box position must be either the wild
type glycine or an alanine. This requirement for aliphatic amino acids
with small side chains suggests that there is some type of steric
constraint. This explanation is consistent with the placement of the
second P-box amino acid in the estrogen receptor-DNA complex, where the
backbone amide group of the glycine forms a hydrogen bond to the
phosphate adjacent to the fourth nucleotide of the
half-site(8) . Lower affinity binding to the everted repeat with
AGGACA half-sites was also observed for variant hT3R
proteins that
have either a proline or serine in the second P-box position. This
result suggests that in the complex formed between hT3R
and the
DNA with AGGACA half-sites, the pocket that accommodates the side chain
of the second P-box amino acid is slightly larger than the same pocket
in the wild type receptor-DNA complex. This compatibility of additional
substitutions in the second P-box position with binding of the receptor
to the AGGACA half-site is intriguing because amino acid substitutions
at either the first or third P-box position of hT3R
are not
compatible with high affinity binding to this half-site sequence. A
peptide containing the DNA binding domain of the glucocorticoid
receptor with a variant ESG P-box sequence is also able to bind with
high affinity to AGGACA half-sites(25, 26) .
to everted repeats with AGGNCA half-site sequences. The
results suggest that the identity of the fourth base pair of the
half-site dictates which amino acids in the third P-box position are
compatible with hT3R
binding. For instance, the element with wild
type AGGTCA half-sites binds variant hT3R
proteins with small
polar and non-polar aliphatic amino acids in the third P-box position.
On the everted repeat with AGGACA half-sites only the wild type
receptor with glycine in the third P-box position binds with reasonable
affinity, suggesting that there is some type of steric constraint
operating. It is particularly interesting that there are a number of
hT3R
variants that bind with considerably higher affinity than the
wild type receptor to everted repeats with AGGGCA or AGGCCA half-sites.
The serine found in the third P-box position of the v-erbA
oncogene enhances the affinity of this protein for binding to TREs with
these same half-site sequences(21) . Of the hT3R
variants
studied here, the three which have much higher affinity than the wild
type receptor for binding to the everted repeat with AGGCCA half-sites
have amino acids in the third P-box position which could form hydrogen
bonds with the DNA. High affinity receptor binding to the element with
AGGGCA half-sites required either an aliphatic amino acid with a
relatively small side chain or a polar amino acid with a hydroxyl
group. The variant receptor with a lysine in the third P-box position
also bound with a higher affinity than the wild type receptor to this
everted repeat. The reduced binding affinity of the wild type hT3R
to the AGGGCA element is consistent with the loss of hydrogen bonding
contacts presumably formed between the glutamate in the first P-box
position and the T-A base pair found at the fourth position of the wild
type TRE. The increased binding affinity to the everted repeat that has
AGGGCA half-sites that results from substituting either a small
aliphatic amino acid, a polar amino acid, or lysine suggests that these
variant receptors are able to form different, fortuitous contacts to
the DNA that increase the free energy of binding.
proteins to the DNA. However, weaker binding to the
everted repeats with AGTTCA and AGTCCA half-sites by receptors with AGG, LGG, GGG, and SGG P-box sequences may
indicate that an amino acid outside the P-box also contacts the DNA and
helps to stabilize the variant receptor-DNA complex. In the estrogen
receptor-DNA complex, the third base pair of the half-site is contacted
by a lysine residue at the C-terminal end of the DNA recognition
-helix(8) . Thus it seems possible that the arginine in the
comparable position of hT3R
(R127) may serve a similar function.
It is interesting to note that other receptors that bind with high
affinity to an AGTTCA half-site also have an arginine at this position
in the recognition
-helix. For example, both the vitamin D
receptor and the retinoic acid receptor bind with higher affinity
to an AGTTCA half-site than they do to an AGGTCA half-site, and
consequently a number of naturally occurring response elements for
these receptors have AGTTCA
half-sites(17, 27, 28) .
does not bind to the everted repeat with AGTCCA half-sites,
whereas the variant receptors with either a DGG or NGG
P-box sequence bind with high affinity. If the P-box glutamate of
hT3R
acts analogously to the same amino acid in the estrogen
receptor, then the 2-base pair replacement in the AGTCCA would result
in the loss of all hydrogen bonding contacts normally formed between
this glutamate and the DNA. The side chains of asparagine and aspartate
must provide hydrogen bonding contacts to the central base pairs of the
AGTCCA half-sites to allow for the high affinity binding of the variant
receptors with DGG and NGG P-box sequences.
from the wild type EGG to the variant EGQ sequence.
. These features include half-site orientation, spacing, and
the identity of the base pairs flanking the half-sites. In order to
understand these effects on a systematic basis, and to relate them to
constraints on P-box sequences in the receptors, we report in the
accompanying article (29) on the effects that flanking sequences
in everted repeat TREs have on the binding affinities of this series of
variant hT3R
proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.