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

Abstract

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.

Keywords: A/B domain; interdomain allostery; intrinsically disordered protein domain; nuclear receptor; protein–DNA interactions; thyroid receptor.

Figure 1

(A) NR domain topology displaying single‐letter domain assignments. The region circled in green (above) is the focus of this study and the structural topology shows the relative orientation of the domains with DR4 TRE DNA (below). (B) The amino acid sequence of the TRα A/B + C domain molecular construct is colour coded (A/B domain in red and C domain in green). The single tryptophan is shown in blue. (C and D) Results from the Escherichia coli overexpression and purification of the TRα (A/B + DBD) and TRα (DBD) molecular constructs, respectively. The molecular weight standards are on lane 6 (the positions of the 75 kDa and 25 kDa standards are labelled) and the purified proteins are in lane 7.

Figure 2

ITC measurements were performed to measure heat changes upon titrating DR4 TRE DNA into (A). TRα (A/B + DBD) and (B). TRα (DBD). For all titrations, the c values (c = nK _a M _tot, where n is the stoichiometry parameter, K _a is the association constant = 1/K _d and M _tot is the concentration of the macromolecule, TRα) 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.

Figure 3

Conformational changes determined by Fluorescence and CD spectroscopy. (A) Change in intrinsic tryptophan fluorescence of TRα (A/B + DBD) is monitored in response to increasing levels of DR4 TRE DNA. The data above are obtained after subtracting buffer and DR4 TRE DNA contributions. In addition, no static quenching of molecular Trp was observed by DR4 TRE DNA. (B and C) Raw CD spectra of TRα (A/B + DBD) and TRα (DBD), respectively, ± DR4 TRE DNA. (D) The CD ([θ], (deg cm² dmol⁻¹) vs. wavelength, nm) spectra of the TRα (A/B domain) was calculated by individually subtracting the [θ] values for TRα (DBD) from TRα (A/B + DBD), for each corresponding wavelength, ± DR4 TRE DNA, respectively. The assumption made is that the conformations of the TRα (C domain), ± DR4 TRE DNA, are the same in both TRα (A/B + DBD) and TRα (DBD). Inset, CD spectra of TRα (A/B domain) measured directly ± DR4 TRE.