©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Relationship between P-box Amino Acid Sequence and DNA Binding Specificity of the Thyroid Hormone Receptor
THE EFFECTS OF HALF-SITE SEQUENCE IN EVERTED REPEATS (*)

Colleen C. Nelson (§) , Stephen C. Hendy , Paul J. Romaniuk (¶)

From the (1)Department of Biochemistry and Microbiology, P. O. Box 3055, University of Victoria, Victoria, British Columbia V8W 3P6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The three P-box amino acids in the DNA recognition -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.


INTRODUCTION

The thyroid hormone receptor (T3R)()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.

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 -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 the present study, we have tested the contextual nature of P-box function in hT3R 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.


EXPERIMENTAL PROCEDURES

Construction and Expression of hT3R Mutants

P-box amino acid substitution mutants of hT3R 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.


RESULTS

The role of P-box amino acids in determining the unique DNA binding specificity of hT3R 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 with TREs Containing AGGNCA Half-sites

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 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 with TREs Containing AGGNCA Half-sites

The effects of amino acid substitutions at the second P-box position of hT3R 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 with TREs Containing AGGNCA Half-sites

Amino acid substitutions at the third P-box position of hT3R 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).

Perhaps the most intriguing results were obtained using the everted repeat with AGTCCA half-sites. Binding of hT3R 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.


DISCUSSION

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 -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.

DNA binding of the wild type hT3R 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.

For high affinity binding of variant hT3R 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) .

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 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.

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 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) .

The wild type hT3R 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.

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 from the wild type EGG to the variant EGQ sequence.

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. 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.


FOOTNOTES

*
This work was supported in part by a grant (to P. J. R.) from the National Cancer Institute of Canada with funds from the Canadian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a National Cancer Institute of Canada postdoctoral fellowship.

To whom correspondence should be addressed. Tel.: 604-721-7088; Fax: 604-721-6227.

The abbreviations used are: T3R, thyroid hormone receptor; TRE, thyroid hormone response element.


ACKNOWLEDGEMENTS

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.


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