Helix 12 Dynamics and Thyroid Hormone Receptor Activity: Experimental and Molecular Dynamics Studies of Ile280 Mutants

We would like to express our deepest gratitude to our late friend and colleague Ralff Ribeiro and dedicate this work to his beloved memory.
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Abstract

Nuclear hormone receptors (NRs) form a family of transcription factors that mediate cellular responses initiated by hormone binding. It is generally recognized that the structure and dynamics of the C-terminal helix 12 (H12) of NRs' ligand binding domain (LBD) are fundamental to the recognition of coactivators and corepressors that modulate receptor function. Here we study the role of three mutations in the I280 residue of H12 of thyroid hormone receptors using site-directed mutagenesis, functional assays, and molecular dynamics simulations. Although residues at position 280 do not interact with coactivators or with the ligand, we show that its mutations can selectively block coactivator and corepressor binding, and affect hormone binding affinity differently. Molecular dynamics simulations suggest that ligand affinity is reduced by indirectly displacing the ligand in the binding pocket, facilitating water penetration and ligand destabilization. Mutations I280R and I280K link H12 to the LBD by forming salt bridges with E457 in H12, stabilizing H12 in a conformation that blocks both corepressor and coactivator recruitment. The I280M mutation, in turn, blocks corepressor binding, but appears to enhance coactivator affinity, suggesting stabilization of H12 in agonist conformation.

Research Highlights

► Mutations in NR's H12 impair transcriptional activity usually by destabilizing H12–LBD interactions. Here we show that mutants can selectively impair the affinities for ligands and coregulators. Ile280 mutants stabilize inactive H12–LBD interfaces, whereas ligand affinity is reduced by facilitating water penetration in the binding site.

Introduction

Thyroid hormone receptors (TRs) are transcription factors modulated by thyroid hormone binding.1, 2, 3 They belong to the nuclear hormone receptor (NR) superfamily, one of the major targets of pharmaceuticals comprising receptors for estrogens and its analogs, corticosteroids, and retinoic acid and derivatives, to mention a few. NRs contain three domains: a variable N-terminal domain with unknown structure, a DNA binding domain that recognizes DNA response elements, and a ligand binding domain (LBD) that selectively recognizes hormones and contains interfaces for dimerization and cofactor recruitment.4, 5, 6, 7, 8, 9, 10

The structure and dynamics of the LBD are essential for transcription regulation. It is currently accepted that in the absence of ligand, the C-terminal helix [helix 12 (H12)] of the LBD is positioned such that it exposes an interface for corepressor binding. In positively regulated genes, the NR inhibits gene transcription while bound to the corepressor. Ligand binding perturbs the dynamic equilibrium of H12, which adopts a novel preferential orientation that favors coactivator—instead of corepressor—recruitment. Dissociation of corepressor and binding of coactivator initiate transcription.8, 9, 10 Thus, H12 conformation and dynamics are key factors that modulate ligand-dependent transcription regulation.

The dynamics of H12 was initially believed to involve its detachment from the body of the LBD, as exemplified by apo retinoid X receptor (RXR) and holo retinoic acid receptor (RAR) crystallographic structures.11, 12 The recruitment of corepressors and coactivators with specific H12 conformations suggests that the movements of H12 are more subtle, as shown in Fig. 1, and are mostly determined by preferential orientations H12 assumes while docked to the surface of the LBD. Ligand entry and exit may occur through subtle movements of H12 or other structural elements. Other crystal structures of apo-LBDs,13, 14, 15, 16, 17 the constitutive activity of receptors,18, 19, 20, 21 molecular dynamics (MD) computer simulation studies,22, 23, 24, 25, 26, 27 and hydrogen–deuterium exchange experiments28, 29, 30, 31, 32, 33, 34 support the view of a dynamic but compact LBD in which H12 can assume both corepressor-favorable and coactivator-favorable conformations in the presence or in the absence of ligand, but with different populations in each case.

In TRs, corepressor and coactivator interfaces overlap and are formed by residues V284, K288, I302, and K306 from helices 3, 5, and 6 (residue numbering according to TRβ isoform). The corepressor binding surface is further complemented by residues T277, I280, T281, V283, and C309, which also belong to helices 3, 5, and 6 but are spatially closer to H12 in holo-TR, whereas the coactivators require residues L454 and E457 from H12 to interact with TR.35, 36, 37

H12 is docked over residues I280, V283, and C309 in holo-TR structures, so that corepressor binding requires a conformational shift of H12 from this position. The role of these three residues (I280, V283, and C309) in coactivator and corepressor binding is essential for the comprehension of H12 conformational equilibrium and dynamics. As coactivator—but not corepressor—binding is dependent on direct interactions with H12, deletion of H12 blocks coactivator interactions but increases corepressor association by exposing its interaction surface.37 Some mutants in this region are also linked to resistance-to-thyroid-hormone syndrome,38, 39, 40 which is usually associated with reduced transcriptional activity and reduced hormone affinity for the receptor.41

Here, we report an experimental and computational study of the effects of mutations I280M, I280R, and I280K on the association of coactivators and corepressors, heterodimerization, and ligand affinity. We show that different mutations at position 280 affect each of these functional characteristics of the receptors differently, and MD simulations provide the structural basis for such differential effects.

Section snippets

Mutants impair transcriptional activity

Reporter gene assays were used to probe the transcriptional activities of native and mutant I280M, I280R, I280K, F451X, and I280K/F451X. Only mutant I280M preserved significant levels of transcriptional activity relative to wild-type TRβ, as shown in Fig. 2a–c. The relative transcriptional activity is response-element-dependent for each mutant: I280M preserved about 82% of native activity in DR4, 42% of native activity in F2, and 63% of native activity in TREpal. Activation promoted by mutants

Conclusions

The conformation and mobility of NRs' H12 are key structural factors affecting NR transcriptional activity. H12 is mobile and assumes different preferential conformations in the presence and in the absence of ligand. However, the few structural models of apo-LBDs and limited direct experimental information on H12 and LBD dynamics result in the incomplete comprehension of relationships between NR dynamics and function. Here, we address the effects of the I280 mutants of the LBD of TR to further

Plasmid vectors

The construction of plasmids used for the synthesis of TRβ1 (pCMX-hTRβ1) had been previously described.35, 55, 56 Vectors encoding TRβ1 with an exchange of isoleucine 280 with lysine or methionine (pCMX-I280K and pCMX-I280M) were donated by Professor Brian West and have been already described.37 The vectors with mutation of isoleucine 280 to arginine (pCMX-I280R) and with deletion of the last 10 amino acids of TRβ1 (pCMX-F451X) were generated by site-directed mutagenesis (QuickChange

Acknowledgements

The authors thank the following Brazilian agencies for financial support: Fundação de Amparo a Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grants 476895/2008-1 and 620195/2008), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

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