Interaction of Transcriptional Intermediary Factor 2 Nuclear Receptor Box Peptides with the Coactivator Binding Site of Estrogen Receptor α

Abstract

The activation function 2/ligand-dependent interaction between nuclear receptors and their coregulators is mediated by a short consensus motif, the so-called nuclear receptor (NR) box. Nuclear receptors exhibit distinct preferences for such motifs depending both on the bound ligand and on the NR box sequence. To better understand the structural basis of motif recognition, we characterized the interaction between estrogen receptor ␣ and the NR box regions of the p160 coactivator TIF2. We have determined the crystal structures of complexes between the ligand-binding domain of estrogen receptor ␣ and 12-mer peptides from the Box 2 and Box 3 regions of TIF2. Surprisingly, the Box 3 module displays an unexpected binding mode that is distinct from the canonical LXXLL interaction observed in other ligand-binding domain/NR box crystal structures. The peptide is shifted along the coactivator binding site in such a way that the interaction motif becomes LXXYL rather than the classical LXXLL. However, analysis of the binding properties of wild type NR box peptides, as well as mutant peptides designed to probe the Box 3 orientation, suggests that the Box 3 peptide primarily adopts the "classical" LXXLL orientation in solution. These results highlight the potential difficulties in interpretation of protein-protein interactions based on cocrystal structures using short peptide motifs. The estrogen receptor ␣ (ER␣)1 is a ligand-activated transcription factor that mediates the biological effects of the steroid hormone estrogen. Like other nuclear receptors (NRs), ER␣ exhibits a characteristic modular domain organization that includes two autonomous transcriptional activation functions (AF1 and AF2) that regulates transcription through interactions with NR coregulators (1-3). AF1, which resides in the N-terminal region of ER␣, is constitutively active and regulated by growth factors (4 -6). In contrast, AF2, which is located in the C-terminal ligand-binding domain (LBD) of ER, is entirely dependent on ligand for its activity.In recent years, structural and functional studies of both the AF2 domain of ER␣ and associated coregulators have greatly enhanced our knowledge of ligand-dependent, ER-mediated transcriptional activation. A large number of coactivators have been isolated that primarily target the LBD of the receptor in a ligand-and AF2-dependent manner (7,8). The most widely studied group of AF2 coactivators includes the p160 family of proteins (steroid receptor coactivator 1, TIF2/glucocorticoid receptor-interacting protein 1, and steroid receptor coactivator 3/AIB1) (9 -13) and the p300/cAMP-responsive element-binding protein-binding protein (14, 15). These factors possess intrinsic histone acetyltransferase activity and/or function in complexes with other acetyltransferases such as p300/cAMPresponsive element-binding protein-binding protein-associated factor (16, 17). They act to remodel chromatin through the regulation of histone acetylation status (18) and are therefore believed to influence promoter accessibility. The...

Multiple Binding Modes between HNF4α and the LXXLL Motifs of PGC-1α Lead to Full Activation

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