An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

Abstract

The interaction of coactivators with the ligand-binding domain of nuclear receptors (NRs) is mediated by amphipathic ␣-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.

Alternative Mechanisms by Which Mediator Subunit MED1/TRAP220 Regulates Peroxisome Proliferator-Activated Receptor γ-Stimulated Adipogenesis and Target Gene Expression

“…Biochemical and genetic evidence strongly suggest a mechanism in which different (promoter-bound) activators recruit Mediator through interactions with distinct Mediator subunits (4,21,26). Relevant to the present study, many nuclear receptors have been shown to interact, in a ligand-dependent manner, with the Mediator MED1/TRAP220 subunit, through interactions between the liganded AF2 receptor domain and the MED1/TRAP220 LXXLL motifs (8,20,30,32,33,38,39,43,44), and studies of mutated Mediator complexes have established a key role for MED1/TRAP220 and resident LXXLL domains in strong interactions between TR␣ and the complete Mediator complex (25).…”

Quantitative Proteomics of the Thyroid Hormone Receptor-Coregulator Interactions

“…1B). Previous screens with coactivator peptides established amino acid residues at ϩ6 to ϩ12 as critical for binding (38,42). To capture these specificity determinants, peptides of 20-amino acid length were generated.…”

Transcriptional activation by estrogen receptor (ERalpha) and steroid receptor coactivator (SRC1) involves distinct mechanisms in yeast and mammalian cells

“…In contrast, Y537 (the human equivalent of mouse Y541) does not appear to be in close contact with the LXXLL core motif sequence. However, sequences flanking the core motifs are known to be important for differential interactions of coactivators with steroid receptors and other NRs (Needham et al 2000, Coulthard et al 2003, although such sequences have yet to be observed in LBD/coactivator peptide crystal structures. Thus, it remains to be established how replacement of Y541 with alanine or acidic residues enhances ligand-independent binding of coactivators in mammalian cells.…”