Definition of the Surface in the Thyroid Hormone Receptor Ligand Binding Domain for Association as Homodimers and Heterodimers with Retinoid X Receptor

Abstract

Thyroid hormone receptors (TRs) bind as homodimers or heterodimers with retinoid X receptors (RXRs) to DNA elements with diverse orientations of AGGTCA half-sites. We performed a comprehensive x-ray crystal structure-guided mutation analysis of the TR ligand binding domain (TR LBD) surface to map the functional interface for TR homodimers and heterodimers with RXR in the absence and/or in the presence of DNA. We also identified the molecular contacts in TR LBDs crystallized as dimers. The results show that crystal dimer contacts differ from those found in the functional studies. We found that identical TR LBD residues found in helices 10 and 11 are involved in TR homodimerization and heterodimerization with RXR. Moreover, the same TR LBD surface is operative for dimerization with direct repeats spaced by 4 base pairs (DR-4) and with the inverted palindrome spaced by 6 base pairs (F2), but not with TREpal (unspaced palindrome), where homodimers appear to be simply two monomers binding independently to DNA. We also demonstrate that interactions between the TR and RXR DNA binding domains stabilize TR-RXR heterodimers on DR-4. The dimer interface can be functional in the cell, because disruption of key residues impairs transcriptional activity of TRs mediated through association with RXR LBD linked to GAL4 DNA-binding domain.Thyroid hormone receptors (TRs) 1 are members of the nuclear receptor superfamily, which includes receptors for steroid hormones, vitamins, retinoids, prostaglandins, fatty acids, and orphan receptors for which no ligands are known (1-4). These receptors are modular transcription factors that bind to specific sequences in promoters of target genes. They bind DNA as monomers, homodimers, or heterodimers through the DNA binding domain (DBD) (1, 5). Steroid receptors bind as homodimers to two half-sites of a DNA palindrome. In contrast, TRs, retinoic acid receptors (RARs), vitamin D receptors (VDRs), peroxisome proliferator-activated receptors (PPARs), and several orphan receptors bind as heterodimers with retinoid X receptors (RXRs) to direct repeats (DRs) of the AGGTCA half-site (1) but also bind to elements oriented as palindromes or inverted palindromes (1). Binding specificity of these receptors is determined by spacing between the half-sites, because PPARs, VDRs, TRs, and RARs bind preferentially to DRs spaced by 1, 3, 4, or 5 nucleotides, respectively (1). Unlike PPARs, VDRs, and RARs, TRs also bind DNA as monomers and as homodimers to DRs and to inverted palindromes spaced by 4 -6 nucleotides (F2) (1, 6, 7). Current concepts of the dimerization and heterodimerization interfaces for nuclear receptors are based on mutational and x-ray crystal structural data. The mutation data for LBDs are mostly limited to TR, RAR, VDR, and RXR. Little is known from mutation studies with the steroid receptors, because only few studies report single mutations in the LBDs that disrupt dimerization (8). This is due in part to the fact that the DBDs and amino-terminal fragments of these receptors contribute su...

Rearrangements in Thyroid Hormone Receptor Charge Clusters That StabilizeBound 3,5′,5-Triiodo-L-thyronine and Inhibit HomodimerFormation

“…Thus, it is thought that T 3 -dependent inhibition of homodimer formation relieves transcriptional repression by unliganded TRs. Nevertheless the mechanisms involved in coupling of T 3 binding to inhibition of DNA binding are not clear; TRs utilize the same surface at the junction of H10 and H11 in homodimer and heterodimer formation on DNA (27). The structural elements that render homodimers sensitive to T 3 are not known.…”

Multiple mutations contribute to repression by the v-Erb A oncoprotein

“…We mapped all four of these alterations in the dimerization properties of v-Erb A to the receptor I-box. This region represents an important site of receptor-receptor contact and mutations in the I-box are known to alter the dimerization properties of many nuclear receptors, including v-and c-Erb A (Forman and Samuels, 1990;Selmi and Samuels, 1991;Au-Fliegner et al, 1993;Glass, 1996;Perlmann et al, 1996;Reginato et al, 1996;Bauer et al, 1997;Shen and Subauste, 2000;Ribeiro et al, 2001). V-Erb A and (avian) c-Erb A differ in their sequence at four amino acids in the I-box region (Sap et al, 1986).…”

“…Analysis of these v-/c-Erb A chimeras localized the relevant sequences to a region bearing four amino-acid substitutions between c-and v-Erb A (Figure 3, compare the efficient RXR heterodimerization properties of the VVCV construct with the inefficient RXR heterodimerization properties of the CCVC construct). Notably, this region includes the 'I-box' previously implicated as a nuclear receptor dimerization interface (Forman and Samuels, 1990;Au-Fliegner et al, 1993;Glass, 1996;Perlmann et al, 1996;Reginato et al, 1996;Ribeiro et al, 2001).…”

The oligomeric state of thyroid receptor regulates hormone binding kinetics

“…Expression vectors for full-length TRa and TRb (pCMXTRb and pCMX-TRa) and TRb mutants (TRbP419R, L422R, M423R, and D355R) were previously described (Ribeiro et al 2001). TRb-LBD and DBD-LBD (amino acids 202-461 and 102-461) were expressed as fusion proteins containing an N-terminal poly-histidine tag.…”

“…RXR-TRs, TR monomers, and TR homodimers activate transcription from different TH response elements (TREs) in yeast and mammalian cells, indicating that each species is functionally important (Velasco et al 2007). Nevertheless, even though RXR-TR and TR-TR homodimer formation involves a common hydrophobic surface at the junction of H10 and H11 (Ribeiro et al 2001), fairly distant from the ligand binding pocket (LBP), T 3 selectively inhibits TR homodimer formation on DNA (Yen 2001, Togashi et al 2005b. Thus, basic thermodynamic principles predict that oligomerization should selectively affect T 3 interactions with receptor.…”