DNA‐induced unfolding of the thyroid hormone receptor α A/B domain through allostery

The A/B domains of nuclear receptors such as thyroid receptor α (TRα) are considered to be conformationally flexible and can potentially adopt multiple structural conformations. We used intrinsic tryptophan fluorescence quenching and circular dichroism spectroscopy to characterize the unfolding of this A/B domain upon DNA binding to the contiguous DNA‐binding domain (DBD). We propose that this allosteric change in A/B domain conformation can allow it to make the multiple interactions with distinct molecular factors of the transcriptional preinitiation complex. We further suggest that by influencing the affinity of the DBD for DNA, A/B domain can fine‐tune the recognition of promotor DNA by TRα.

The A/B domains of nuclear receptors such as thyroid receptor a (TRa) are considered to be conformationally flexible and can potentially adopt multiple structural conformations. We used intrinsic tryptophan fluorescence quenching and circular dichroism spectroscopy to characterize the unfolding of this A/B domain upon DNA binding to the contiguous DNAbinding domain (DBD). We propose that this allosteric change in A/B domain conformation can allow it to make the multiple interactions with distinct molecular factors of the transcriptional preinitiation complex. We further suggest that by influencing the affinity of the DBD for DNA, A/B domain can fine-tune the recognition of promotor DNA by TRa.
The effects of the thyroid hormone (triiodothyronine, T3) are widespread in development, homeostasis and metabolism. The T3 receptors (thyroid hormone receptor, TR) are encoded by two closely related genes (a and b) [1]. The T3Ra genes in humans express the T3-binding isoform TRa1 [2]. The TRb gene expresses TRb1 and TRb2, which differ only in their N-terminal A/B regions, and are also distinct from the A/B region of TRa1 [3]. TRa is mostly expressed in the brain [4] and is associated with the development of the nervous system [5]. TRa is constitutively localized within the nucleus where it interacts with nucleosomal DNA [6,7]. In the absence of T3 ligand, TRa is observed to actively repress transcription through interactions with transcriptional corepressors such as SMRT and NCoR [8][9][10].
Thyroid hormone receptors are members of the nuclear receptor (NR) superfamily of ligand-mediated transcription factors [2]. NRs have common modular structural features that include an N-terminal domain (A/B domain, Fig. 1A). This A/B domain is of variable length and amino acid sequence and encompasses a ligand-independent transactivation function (AF1) domain that is critical for regulating transactivation [11,12]. Following the A/B domain is a highly conserved DNA-binding domain (DBD; C domain, Fig. 1A) that binds palindromic DNA sequences called hormone response elements (HRE). A short 'hinge' sequence (D domain) connects the DBD (C domain) to a C-terminal ligand-binding domain (LBD; E/F domain, Fig. 1A). Upon binding agonist-ligands, the LBD (E/F domain) undergoes conformational changes which results in the recruitment of coactivator molecules [13][14][15][16][17]. Antagonists and inverse agonists disrupt the 'active-state' LBD and the resulting LBD conformation functions as a docking site for corepressors [18][19][20]. Also, except for the A/B domains, the amino acid sequences of TRa and TRb are over 90% identical. Since TRs differ most significantly in the N-terminal A/B domain, it is suggested that this region plays a significant role in mediating the distinct roles of these receptors [21]. It has also been proposed that TRa-mediated transcriptional regulation can also occur through specific interactions of the A/B domain with the PIC, specifically with transcription factor IIB (TFIIB) [21][22][23][24]   domains have also been reported to modulate transactivation suggesting additional layers of regulation [32,44].
Here, we report a notable conformational change in the TRa A/B domain that is initiated through allostery through the TRa DBD by DNA. The shorter, 50amino acid A/B domain of TRa encompasses several of the structural motifs that have been identified in NRs with significantly larger A/B domains to be important for ligand-independent activity [24]. Of these, distinct variations of the KRKRK amino acid sequence motif are common to several NRs including TR, progesterone (PR) and the liver X receptor (LXR) [28]. We are able to observe that the TRa A/B domain can allosterically enhance the binding affinity of the receptor for direct repeat 4 (DR4) TRE DNA. Furthermore, using a combination of circular dichroism (CD) and intrinsic tryptophan fluorescence spectroscopy, we can report that the binding of DNA to the TRa DBD (C domain) induces unfolding within the flanking TRa A/B domain. Overall, these observations suggest a structural basis for intramolecular cooperativity within TRa that fine-tunes binding to specific DNA sites.

Preparation of DR4 TRE DNA adduct
19-mer DNA oligos containing the thyroid hormone response element (TRE) consensus site (DR4: 5 0 -CCAGGTCATTTCAGGTCAG-3 0 , where the underlined sequence is the NR binding site) were commercially obtained (Life Technologies Inc.) as single-stranded oligomers [45]. Double-stranded DR4 TRE was prepared by mixing the complementary strands in equimolar ratios to a final concentration of 2 mM, followed by heat denaturation at 95 ᵒC for 5 min and annealing by gradual cooling to room temperature.

Isothermal titration calorimetry (ITC)
Thyroid receptor a (A/B + DBD) and TRa (DBD), purified by SEC, were used for isothermal titration calorimetry (ITC) measurements using VP-ITC MicroCal TM (MicroCal Inc., Northampton, MA, USA). Protein and ligand were prepared in 50 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM MgCl 2 and 1 mM TCEP. For titration experiments, protein concentration ranged from 30 to 45 lM and ligand DR4 TRE: 5 0 -CCAGGTCATTTCAGGTCAG-3 0 concentration ranged from 300 to 400 lM. Both protein and ligand were degassed for 5-10 min. The experiments were initiated by injecting 28 9 10 lL aliquots of DR4 TRE from the syringe into the calorimetric cell containing 1.5 mL of protein solution. All the titrations were performed at 25°C and the buffer (pH adjusted to 7.5 at 25°C). The change in thermal power as a function of each injection was automatically recorded using MICROCAL ORIGIN software and the raw data were further processed to yield binding isotherms of heat released per injection as a function of molar ratio of DR4 TRE to TRa (A/B + DBD) or TRa (C domain). The data were acquired and processed using the MICROCAL ORIGIN (MicroCal Inc.) software. Data were collected in triplicate.

Fluorescence spectroscopy
Fluorescence emission spectra of purified TRa (A/B + DBD) in 50 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM MgCl 2 , 1 mM TCEP were recorded at various concentrations of DR4 TRE. A total of 2 mL protein (2 lM) was used to which 2 lL of DR4 TRE (0-9.4 lM) was added for each scan. To monitor the effect of sample dilution due to DR4 TRE titrations into protein, equal volumes of buffer were titrated into 2 mL protein (2 lM). The spectra were monitored using a PerkinElmer-LS 55 Fluorescence Spectrometer at excitation wavelength of 295 nm at 300 nmÁmin À1 .
Emission wavelength range was set at 310 nm to 450 nm, with slit width of 5.0 nm; 1 cm path length rectangular cuvettes were used to take all measurements at room temperature. The final fluorescence intensity change curve was a result of three averaged curves from individual experiments. The contribution of DR4-TRE to the TRa (A/ B + DBD) + DR4 TRE spectrum was corrected by subtracting the spectrum of TRa (A/B + DBD) + buffer. Since multiple studies have shown that two molecules of TR bind a single TRE DNA [32,46], titration data curves were fitted to a two-site binding, nonlinear regression fitting model by PRISM7

Circular dichroism (CD)
Circular dichroism spectra of TRa (A/B + DBD) and TRa (DBD; in 50 mM sodium phosphate buffer, pH = 7.5-8.0, 80 mM NaCl, and 5 mM MgCl 2 , 1 mM TCEP) in the presence and absence of DR4 TRE DNA were recorded using a JASCO J-815 CD spectrometer. Protein to DNA ratio was 1: 1.1 for all experiments. All spectra were collected at 100 nmÁmin À1 scan rate in 2 mm cuvettes maintained at 4°C. The band width was 4 nm with data pitch 1 nm. CD spectra of buffer and DR4 TRE (4-5 lM) were also recorded separately as controls. Each spectrum shown is the result of 30 spectra accumulations, averaged and smoothed. All the spectra were corrected for the contributions of the buffer and TRE DR4 [47]. Mean residue ellipticity ([h], (deg cm 2 dmol À1 ) was calculated using the CAPITO software [48].

Results
Here, we present data from studies on a 154-amino acid, two-domain molecular construct that encompasses the contiguous A/B (N terminus) and the C domains (DBD) of TRa (Fig. 1A). The TRa A/B domain comprises approximately 50 amino acids with an evolutionary conserved KRKRK motif (Fig. 1B) consisting of multiple charged residues [21,24]. Additionally, this construct contains a single tryptophan residue that is conveniently located within the A/B domain ( 19 Trp) and adjacent to the KRKRK motif which has enabled us to monitor the local changes in conformation with steady-state intrinsic tryptophan fluorescence spectroscopy. In summary, we present data on the structural conformation of the TRa A/B domain, the conformational changes in this domain that are transmitted by allostery when the DBD (C domain) binds DNA, and the effect of the A/B domain on DNA recognition and binding.

TRE binding to the DBD can influence specific local conformation of the A/B domain
Our studies above indicate that there is an allosteric pathway that links the DNA-binding site within the TRa DBD to the N-terminal TRa A/B domain (Fig. 2). Here, we sought to determine if the DNA-  where n is the stoichiometry parameter, K a is the association constant = 1/K d and M tot is the concentration of the macromolecule, TRa) range from 6.5 to 9, which is within the ideal range for determining binding constants by ITC [73]. Data obtained are summarized in Table 1. due to allostery [16]. We monitored the dose-dependent changes in intrinsic steady-state tryptophan fluorescence, accompanied by an approximately 5 nm red-shift in fluorescence maxima, within TRa (A/ B + DBD) in the presence of DR4 TRE (Fig. 3A). The measurable decrease in fluorescence suggests a specific change in the 19 Trp conformation, and furthermore, the conformational changes within the 19 Trp sidechain are more likely from a progressive decrease in its local hydrophobic environment, presumably from an increased exposure to the surrounding buffer [16]. These titrations were also analysed to provide a quantitative measure of binding affinity:

TRE binding to the DBD results in unfolding of the TRa A/B domain
The spectroscopic analyses above suggest an allosteric conformational change within the TRa A/B domain upon binding DNA at the TRa(DBD). To determine the specific DNA-dependent changes in structure within the TRa A/B domain, we utilized CD spectroscopy. Given that minor changes in the secondary structure of proteins can be detected in the raw CD spectra (h in rad cm À1 vs. wavelength in nm) in the far-UV (k = 190-260 nm) range, we compared the CD spectra of the TRa (A/B + DBD) domains with TRa (DBD) in the absence and when complexed with DR4 TRE (Fig. 3B). For the TRa (DBD), there is a prominent change in the minima at 208 nm and 222 nm of the CD spectrum in the presence of DNA, which suggests a significant increase in a-helical structure of the TR(DBD) upon binding DNA (Fig. 3C). Such conformational changes in NR DBDs have been previously observed using NMR spectroscopy confirming a dosage-dependent stabilization of the NR DBD upon binding DNA [51][52][53][54][55]. In this study, the CD spectra of TRa (A/B + DBD) indicates that while the TRa segment is predominantly a-helical, the complexation of TRa (A/B + DBD) with DNA results in a markedly smaller change in secondary structure from the DNAfree protein when compared with the corresponding structural changes within the TRa DBD-only (Fig. 3C).  (Fig. 3D). Additionally, we do not detect significant secondary structure changes to the isolated TRa (A/B domain) in the presence of DR4 TRE (Fig. 3D inset). Taken together, these results suggest that the TRa (A/B domain) has partial a-helical secondary structure within the 'DNA-free' TRa (A/ B + DBD). Upon binding DNA, the contiguous A/B domain and the DBD undergo contrasting conformational changeswhile the A/B domain appears to convert from a more structured to a conformationally less-rigid state, the DBD becomes conformationally more stable. Overall, this a-helical-to-random coil unfolding of the TRa A/B domain appears to counteract the propensity for greater a-helicity within the TRa(DBD) upon binding DR4 TRE. This may explain, in part, the smaller overall change in TRa (A/ B + DBD) in comparison with the TRa(DBD), upon binding DR4 TRE.

Discussion
Multiple lines of evidence suggest that the NR A/B domains are flexible and can adopt distinct conformations through allostery initiated by DNA:DBD interactions [12,34,43,49,50,[56][57][58][59]. A common observation is that the A/B domains in all NRs studied to date, the DNA-initiated allostery elicits an increase in secondary structure (mostly a-helicity) of this domain. Multiple attempts to determine the structures of full-length NRs have failed to identify the conformation of their N-terminal domains [60]. Yet, all these structures have indicated that there is no apparent direct interaction between the A/B domain and the DBD. Our observations suggest that DNA-dependent conformational changes within the TRa A/B domain are distinct from the corresponding changes within the other NR A/B domains listed above. The implications for the unique mode of TRa A/B domain ↔ DBD allostery are broad. For instance, the TRa A/B domain is reported to interact with several cellular cofactors including TFIIB [21-24] and TBP [25]. Similar interactions have been observed between NRs and the PIC, such as the androgen (AR) [61,62], COUP-TF [63], oestrogen (ER) [63,64], GR [65], mineralocorticoid (MR) [66] and PR [34,63] receptors, among others. In each of these NRs, and distinct from TRa as reported here, the A/B domain is constrained to a more folded conformation by DNAallostery. This more-structurally constrained A/B domain is observed to enhance the NR↔cofactor interaction.
In TRa, the sequence of basic residues 23 KRKR 27 K has been identified to make specific interactions with TFIIB ( Fig. 1B) [24]). Adjacent to this basic motif is 19 Trp, which we show here by DR4 TRE DNA dosedependent fluorescence quenching to undergo conformational changes to a more exposed environment and this would be expected with the unfolding of this region of the TRa A/B domain. From truncation and associated binding studies, the corresponding TRainteracting domain of TFIIB is identified to be contained within residues 178-201 of an amphipathic ahelix [24]. Curiously, this TRa-interacting TFIIB ahelix has also separately been identified as integral to the binding interface between TFIIB and DNA [67]. Together, these studies suggest that the formation of the TRa:TFIIB and the TFIIB:DNA complexes are mutually exclusive and that binding to TRa can disrupt the TFIIB-DNA complex. In the absence of direct structural data, it is tempting to speculate that the DNA-induced unfolding of the TRa A/B domain plays a role in inserting itself into the TFIIB-DNA complex and the newly created TRa:TFIIB is stabilized by both interactions made by the charged 23 KRKR 27 K and through the exposed apolar backbone of the TRa A/B domain. Indeed, such DNAinduced unfolding events are less commonly reported in the literature and the Ets-1 transcription factor is a singular prior example of an analogous DNA-induced unfolding within a flanking domain through allostery [68,69]. In Ets-1, this induced unfolding is proposed to ameliorate inhibitory intramolecular interactions and encourage intermolecular interactions that promote gene transcription.
Additionally, this study reinforces the observation that DNA recognition is finely tuned by the domains flanking the NR DBD. In both DNA-bound TRa: RXR heterodimeric [45] and the TRb monomeric [46] structures, the conformation of the TR DBD is virtually identical, suggesting a generic mechanism for DNA recognition and binding. Yet, using DBD and DBD-LBD constructs of TRa, we have earlier established that the affinity of the DBD for DNA can be modulated through intramolecular allostery [32]. Moreover, even subtle changes within these flanking domains (A/B or E/F domains) such as mutations [70] and interactions with cellular factors [32] or smallmolecule ligands [71] can affect DNA binding. Given the distinct unfolding process of the TRa A/B domain, the mechanism by which this domain can allosterically influence DBD↔DNA interactions is likely to be different from those of AR [35] and PR [72].
In summary, our data here suggest a distinct consequence of allostery within TRa. The data from CD spectroscopy show that conformational changes induced within the TR(DBD) are transmitted 'upstream' to the flanking A/B domain. The resultant conformation of the TRa A/B domain is less ordered within the intact, DNA-bound TRa (A/B + DBD) than in the absence of DNA. This unfolding results in the repositioning of 19 Trp observed from the quenching of tryptophan fluorescence. The unusual feature of DNA-induced, allosterically driven conformational changes within the TRa A/B domain is the overall loss in secondary structure, quantified as a decrease in its a-helicity. Finally, this study showcases the diversity in the structural response to allostery within the NR superfamily. We are drawn to hypothesize that such structural responses have been evolutionarily selected to optimize the specific behaviour of individual members of these NR transcription factors.