Molecular determinants of the interactions between LXR/RXR heterodimers and TRAP220

Dimerization-induced activation of LXR is a unique allosteric mechanism described only for LXR/RXR heterodimers. Previously, we demonstrated that RXR functions as an allosteric activator of LXR binding to ASC-2 coactivator rather than as a direct interaction partner. Here, we investigated the molecular basis of the interaction between LXR/RXR and TRAP220 fragment (TN1/2) harboring two NR boxes. We found that either LXR binding to NR box-2 or RXR binding to NR box-1 was sufficient for optimal LXR/RXR binding to TN1/2, indicating that both receptors contribute equally in this interaction. Notably, the AF2 deletion of either receptor completely abolished LXR/RXR-TN1/2 interaction, suggesting dual roles for both AF2 domains in direct interaction with target NR boxes as well as in allosteric activation of partner receptors. We also found specific residues within NR box-2 required for LXR binding using one- plus two-hybrid system and identified Pro⁶⁴³ residue as a major determinant for NR specificity.     

Protein-protein interactions: structural motifs and molecular recognition.

The past year has brought new insights into common structural motifs used for protein-protein interactions by DNA-binding proteins. In addition, there have been significant advances in our understanding of antibody-protein complexes.     

An extended LXXLL motif sequence determines the nuclear receptor binding specificity of TRAP220

The interaction of coactivators with the ligand-binding domain of nuclear receptors (NRs) is mediated by amphipathic alpha-helices containing the signature motif LXXLL. TRAP220 contains two LXXLL motifs (LXM1 and LXM2) that are required for its interaction with NRs. Here we show that the nuclear receptor interaction domain (NID) of TRAP220 interacts weakly with Class I NRs. In contrast, SRC1 NID binds strongly to both Class I and Class II NRs. Interaction assays using nine amino acid LXXLL core motifs derived from SRC1 and TRAP220 revealed no discriminatory NR binding preferences. However, an extended LXM1 sequence containing amino acids -4 to +9, (where the first conserved leucine is +1) showed selective binding to thyroid hormone receptor and reduced binding to estrogen receptor. Replacement of either TRAP220 LXXLL motif with the corresponding 13 amino acids of SRC1 LXM2 strongly enhanced the interaction of the TRAP220 NID with the estrogen receptor. Mutational analysis revealed combinatorial effects of the LXM1 core and flanking sequences in the determination of the NR binding specificity of the TRAP220 NID. In contrast, a mutation that increased the spacing between TRAP220 LXM1 and LXM2 had little effect on the binding properties of this domain. Thus, a 13-amino acid sequence comprising an extended LXXLL motif acts as the key determinant of the NR binding specificity of TRAP220. Finally, we show that the NR binding specificity of full-length TRAP220 can be altered by swapping extended LXM sequences.     

Antibody against nuclear thyroid hormone receptors

Rat liver nuclear thyroid hormone receptor was purified to 700-1600 pmol T3 binding capacity/mg protein by sequentially using hydroxylapatite column, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column, DEAE-Sephadex A-50 column, and heparin-Sepharose column. Serum from a mouse immunized using this purified receptor preparation caused a shift of [125I]T3-receptor peak on glycerol density gradient sedimentation from 3.4 S to approximately 7 S. [125I]T3-receptor complex was immunoprecipitated using this serum and goat anti-mouse IgG. The serum showed reduced ability to immunoprecipitate the globular T3 binding fragment with Stokes radius of 22 A produced by trypsin digestion, a receptor fragment which has core histone and hormone binding but not DNA binding activity. These data indicate the production of anti-nuclear thyroid hormone receptor antibody which mainly recognized epitopes unrelated to hormone and core histone binding domain.     

Intrinsic disorder in nuclear hormone receptors

Many proteins possess intrinsic disorder (ID) and lack a rigid three-dimensional structure in at least part of their sequence. ID has been hypothesized to influence protein-protein and protein-ligand interactions. We calculated ID for nearly 400 vertebrate and invertebrate members of the biomedically important nuclear hormone receptor (NHR) superfamily, including all 48 known human NHRs. The predictions correctly identified regions in 20 of the 23 NHRs suggested as disordered based on published X-ray and NMR structures. Of the four major NHR domains (N-terminal domain, DNA-binding domain, D-domain, and ligand-binding domain), we found ID to be highest in the D-domain, a region of NHRs critical in DNA recognition and heterodimerization, coactivator/corepressor interactions and protein-protein interactions. ID in the D-domain and LBD was significantly higher in "hub" human NHRs that have 10 or more downstream proteins in their interaction networks compared to "non-hub" NHRs that interact with fewer than 10 downstream proteins. ID in the D-domain and LBD was also higher in classic, ligand-activated NHRs than in orphan, ligand-independent NHRs in human. The correlation between ID in human and mouse NHRs was high. Less correlation was found for ID between mammalian and non-mammalian vertebrate NHRs. For some invertebrate species, particularly sea squirts ( Ciona), marked differences were observed in ID between invertebrate NHRs and their vertebrate orthologs. Our results indicate that variability of ID within NHRs, particularly in the D-domain and LBD, is likely an important evolutionary force in shaping protein-protein interactions and NHR function. This information enables further understanding of these therapeutic targets.     

Comparative analysis of docking motifs in MAP-kinases and nuclear receptors

Nuclear receptor (NR) agonists induce activation of mitogen-activated protein kinases (MAPK) through an yet unknown rapid non-genomic mechanism. Vice versa, NR are targets for phosphorylation by MAPK. By multiple alignment of the amino acid sequences and comparative analysis of the secondary and tertiary structures we identified four peptides in MAPK with similarity to bona fide protein-protein-interaction motifs in NR. In both molecule species, these motifs mediate selective docking to dimerization partners, coregulators or phosphoacceptors. We therefore propose that similar motifs may direct the site-specific association of NR with MAPK. Based on mutual allosteric interactions within a kinase-receptor complex, we discuss a novel principle how NR-agonists may regulate kinase activity and thus expression of hormone-dependent genes.     

Nuclear cytoplasmic shuttling by thyroid hormone receptors. multiple protein interactions are required for nuclear retention

In this report, we have studied the intracellular dynamics and distribution of the thyroid hormone receptor-beta (TRbeta) in living cells, utilizing fusions to the green fluorescent protein. Wild-type TRbeta was mostly nuclear in both the absence and presence of triiodothyronine; however, triiodothyronine induced a nuclear reorganization of TRbeta. By mutating defined regions of TRbeta, we found that both nuclear corepressor and retinoid X receptor are involved in maintaining the unliganded receptor within the nucleus. A TRbeta mutant defective in DNA binding had only a slightly altered nuclear/cytoplasmic distribution compared with wild-type TRbeta; thus, site-specific DNA binding is not essential for maintaining TRbeta within the nucleus. Both ATP depletion studies and heterokaryon analysis demonstrated that TRbeta rapidly shuttles between the nuclear and the cytoplasmic compartments. Cotransfection of nuclear corepressor and retinoid X receptor markedly decreased the shuttling by maintaining unliganded TRbeta within the nucleus. In summary, our findings demonstrate that TRbeta rapidly shuttles between the nucleus and the cytoplasm and that protein-protein interactions of TRbeta with various cofactors, rather than specific DNA interactions, play the predominant role in determining the intracellular distribution of the receptor.     

On the mechanism of DNA binding by nuclear hormone receptors: A structural and functional perspective

The nuclear hormone receptor DNA-binding domain consists of two zinc finger-like modules whose amino acids are highly conserved among the members of the receptor superfamily. In this review, we describe the various genetic, biochemical, and structural experiments that have been carried out primarily for the DNA-binding domains of the glucocorticoid and estrogen receptors. We describe how the structural and functional information have permitted us to predict properties of the DNA-binding domains of other nuclear receptors. We postulate how receptors discriminate closely related response elements through sequence-specific contacts and distinguish symmetry of target sites through protein-protein interactions. This mechanism explains in part how the receptors regulate diverse sets of genes from a limited repertoire of core response elements. Lastly, we describe the stereochemical basis of nuclear receptor dysfunction in certain clinical disorders. © 1993 Wiley-Liss, Inc.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.