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Homodimeric DNA-Binding and Ligand-Binding Interactions of Human Thyroid Hormone Receptor

Justin Taft '13, Andrew Gipson '13, and Patrick Mershon '14


I. Introduction

    The organization of a complex multicellular organism requires a sophisticated system of chemical communication to coordinate the cooperation of various tissues. Depending on stage of development, dietary intake, or even mood, the profile of signaling molecules in an animal will change as a response to these conditions. These responses are regulated by a group of chemical-synthesizing organs known collectively as the endocrine system.
    The thyroid is an endocrine gland responsible for regulating metabolism and growth. Its primary mode of effect is the production of the thyroid hormone thyroxine ( ), which is converted to triiodothyronine ( )
in a tissue-specific manner. These hormones both act as transcriptional activators, binding the nuclear receptor known as thyroid hormone receptor (TR), a transcription factor constitutively bound to hormone response elements (HREs). Like most nuclear receptors, TR does not bind to DNA alone; rather, it most frequently forms a heterodimer with the retinoid X receptor (RXR), another nuclear receptor. The heterodimer acts as a transcriptional activator. Occasionally it forms a homodimer with another TR molecule and acts as a repressor until a hormone ligand arrives and causes it to dissociate.
    TR proteins are encoded by two genes, TR alpha and TR beta, with multiple isoforms for each gene. Although their central role as a transcription factor is the same in all forms, they exhibit differential expression at different times in development and in different tissues. Despite these differences, the TRs all share a domain scheme common to the nuclear receptors. The A/B domain at the amino terminus is involved in regulation of tertiary structure. Moving towards the carboxy terminus, the DNA binding domain (DBD) binds to the HREs with sequence specificity. The hinge domain is a flexible linker that connects the DBD to the ligand binding domain (LBD). The LBD mediates dimerization interactions and binds a wide variety of ligands, including endogenous hormones and drugs that affect its function as a transcriptional activator or repressor.

II. Comparison of TR-β LBD with T3 Vs. T4

    TR binds the active form of thyroid hormone triiodothyronine (T3) with high affinity, but also binds its precursor thyroxine (T4) with a roughly 30-fold lower affinity. TR-T4 complexes are less stable than TR-T3 complexes, and dissociate more readily. While the protein adopts a different conformation with each ligand, T4 can also act as an agonist in the cell. T4’s agonist activity is only about 10% that of T3, but 4- to 6-fold higher T4 levels mean that T4 likely plays an active role in TR activation. 

    When bound to T3 or T4, the ligand binding domain (LBD) undergoes a conformational change that packs the C-terminal helix 12 against the LBD, burying the hormone in the ligand-binding pocket. The T3-bound LBD has a smaller binding pocket than the T4-bound structure; the pocket expands to accommodate the bulky 5’ iodine group not present in T3. In order to accommodate this group, backbone shifts occur in Helix 12, the H11-H12 loop and the wall of the binding pocket near the 5’ iodine . Other backbone shifts involve residues 199-212, Helix 2 residues 234-243, regions of beta sheet (residues 318-321 and 325-339), the H2-H3 loop (232-236), and regions of Helix 3 (248-267)

    The conformational change in helix 12 that occurs in either T3 or T4 binding is sufficient to cause folding into the active conformation. This causes the region of the LBD involved in dimerization to assume the proper positioning. Both ligands therefore stimulate the association of coactivators, the release of corepressors, and the dissociation of TR homodimers (although not TR-RXR heterodimers). 

III. TR-β LBD + HPPE (Inhibitor)

    There are several diseases associated with nuclear receptor activity, but some of the more dangerous involve excess production of thyroid hormone, the most severe cases of which are called thyroid storms. Many drugs used in the treatment of these conditions resemble agonists of TR and so bind to the LBD to prevent activation of TR by those agonists, but they lack specificity and will bind the LBD of other nuclear receptors, causing unwanted side effects. This problem can be circumvented by targeting other regions more specific to TR to inhibit functions such as DNA or coactivator binding. This latter approach is the method by which the surface-interacting drug DHPPA and its active form HPPE act on TRβ

    TRβ is normally activated by dimerizing with the steroid receptor coactivator 2 (SRC2) after hormone binding induces a conformational change in the collection of proximate residues called the ligand-dependent transactivation function AF-2 , which then recruits SRC2. The addition of DHPPA prevents this dimerization and thus inhibits the activating function of TRβ. However, DHPPA is not the molecule contributing directly to this effect; rather, a derived form called HPPE (Inhibitor Structures) is the compound responsible for forming a covalent bond at the AF-2 site and occluding SRC2. DHPPA rapidly converts to HPPE at the AF-2 surface to form the intermediate structure shown here , and it may be that the protein itself catalyzes this reaction by an as yet undetermined mechanism.  

    HPPE is positioned in the AF-2 pocket through electrostatic interactions between the carbonyl oxygen of HPPE and residues K306 and E457, whereas the alkyl chain and aromatic ring of HPPE are held in place by hydrophobic interactions with residues L454, V284, and I302 . The beta carbon of the enone group of HPPE is positioned 6.5 angstroms from . Surprisingly, though, it is C298 that is covalently bound to HPPE . The proximity of the residue to the carbonyl group of HPPE would suggest that the sulfhydryl group of the cysteine attacks the carbonyl directly to form the bond; however, the inactivity of the HPPE analog HPPA, which lacks the alpha-beta unsaturated moiety (see Inhibitor Structures), gives evidence that the sulfhydryl undergoes 1,4-nucleophilic addition with the beta carbon of HPPE. How the molecule is oriented to fit this predicted bonding configuration is unclear, but the distance to C298 is only 10.0 angstroms; this implies that it would not be necessary for the entire molecule to shift in order to accommodate the bond.  

    Regardless of the exact position of covalently bound HPPE, its presence in the AF-2 pocket occludes the binding of SRC2 through steric interference. Short domains of the coactivator that have the consensus sequence L-X-X-L-L normally interact with the six hydrophobic residues of AF-2 . Similar to drugs that act on the ligand-binding pocket, the bulky hydrophobic features of HPPE act as an insurmountable barrier to coactivator binding .

IV. Homodimeric DNA Binding

TR, like all other nuclear receptors, interacts with DNA in a sequence specific manner, binding to thyroid responsive elements (TREs) located near the promoter region of the target gene. Specifically, a monomer of the TR DNA binding domain (DBD) recognizes a highly conserved core hexameric sequence of the TRE (5’-AGGTCA-3’), often referred to as a half-site, that is the generic sequence of all steroidal and non-steroidal nuclear receptors. This consensus sequence often exists as two repeats in the genome that can be arranged in multiple ways, and this arrangement appears to dictate the inter-subunit interactions of the DBD. Thus, these differing TRE conformations provide an additional means of gene regulation beyond ligand-binding. For example, the TRβ homodimer selectively binds a less common palindromic repeat separated by 6 bp, while the more common TR/RXR heterodimer preferentially binds a direct repeat separated by 4 bp. Mutational experiments have shown that insertions and deletions in the DNA separating the consensus sequences disrupt the optimal spatial arrangement that allows inter-subunit contacts by the C-terminal extension (CTE) that can be vital for dimerization.

In this instance, each TRβ homodimer subunit binds a palindromic half-site of DNA separated by a 6 bp insert . The TR-DBD is composed of two nonequivalent zinc finger motifs, characteristic of nuclear receptors , and contains three main helices. The N-terminal helix (Cys 18 - Lys31) or recognition helix, inserts into the major groove and  is responsible for the vast majority of DNA contacts. Base-Protein Interactions in the recognition helix occur between  Glu19,  Arg26, Arg27 and G4, A5, C6 on the 5’ to 3’ strand, and between Lys22, Arg26 and G16, G17 on the 3’ to 5’ strand . Helix 3 (Cys55 - Val66) lies perpendicular to helix one and participates in stabilizing hydrophobic interactions with aromatic residues (Phe24, Phe25, Phe50, Ile29 and Leu64) from both the recognition helix and helix 2(Leu33 - Ser36) . The T-box region (69 - 76) is located at the 5’ end of the CTE and acts as a hinge between the CTE and the DBD via the helical turn (Thr 70 - Leu72) . The hinge action of the helical turn allows the CTE to interact with the phosphate backbone as well as insert into the minor grove and make direct base contacts outside of the consensus repeat sequence.

V. References

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Schultz, Steve C., George C. Shields, and Thomas A. Steitz. 1991. Crystal Structure of a CAP-DNA complex: The DNA Is Bent by 90 degrees Science 253: 1001-1007.

Vaney, Marie Christine, Gary L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry 28:4568-4574.

Weber, Irene T., Gary L. Gilliland, James G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a Cyclic AMP-independent Mutant of Catabolite Activator Protein. The Journal of Biological Chemistry 262:5630-5636.

Zhou, Yuhong, Ziaoping Zhang, and Richard H. Ebright. 1993. Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation. Proceedings of the National Academy of Sciences of the United States of America 90:6081-6085.

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