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

Marie Togashi,Phuong Nguyen,Robert Fletterick,John D Baxter,Paul Webb

doi:10.1074/jbc.m501615200

Abstract

In this study, we investigated how thyroid hormone (3,5',5-triiodo-l-thyronine, T3) inhibits binding of thyroid hormone receptor (TR) homodimers, but not TR-retinoid X receptor heterodimers, to thyroid hormone response elements. Specifically we asked why a small subset of TRbeta mutations that arise in resistance to thyroid hormone syndrome inhibit both T3 binding and formation of TRbeta homodimers on thyroid hormone response elements. We reasoned that these mutations may affect structural elements involved in the coupling of T3 binding to inhibition of TR DNA binding activity. Analysis of TR x-ray structures revealed that each of these resistance to thyroid hormone syndrome mutations affects a cluster of charged amino acids with potential for ionic bond formation between oppositely charged partners. Two clusters (1 and 2) are adjacent to the dimer surface at the junction of helices 10 and 11. Targeted mutagenesis of residues in Cluster 1 (Arg338, Lys342, Asp351, and Asp355) and Cluster 2 (Arg429, Arg383, and Glu311) confirmed that the clusters are required for stable T3 binding and for optimal TR homodimer formation on DNA but also revealed that different arrangements of charged residues are needed for these effects. We propose that the charge clusters are homodimer-specific extensions of the dimer surface and further that T3 binding promotes specific rearrangements of these surfaces that simultaneously block homodimer formation on DNA and stabilize the bound hormone. Our data yield insight into the way that T3 regulates TR DNA binding activity and also highlight hitherto unsuspected T3-dependent conformational changes in the receptor ligand binding domain.

Highlights

Thyroid hormone receptors (TR␣ and thyroid hormone receptor (TR)␤)1 are conditional transcription factors that play important roles in development
We examined how TR DNA binding activity is regulated by its ligand binding domains (LBDs) and by ligand
To begin to understand this issue, we asked why some resistance to thyroid hormone syndrome (RTH) mutations (R316H, R338W, K342I, and R429Q) that reduce the affinity of TR␤ for T3 inhibit binding of TR homodimers, but not heterodimers, to thyroid hormone response elements (TREs) [30, 31]

Summary

Introduction

Thyroid hormone receptors (TR␣ and TR␤) are conditional transcription factors that play important roles in development,. Drugs that reverse actions of unliganded TRs could be useful for treating hypothyroidism and would avoid risk of thyroid hormone excess [7]. TRs utilize their DNA binding domain to recognize specific thyroid hormone response elements (TREs) comprised of AGGTCA repeats and bind these elements either as heterodimers with the closely related retinoid X receptor (RXR) or as homodimers and monomers. It is thought that T3-dependent inhibition of homodimer formation relieves transcriptional repression by unliganded TRs. the mechanisms involved in coupling of T3 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 T3 are not known

Methods

Results

Conclusion