TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain

Abstract

A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.glucocorticoid receptor ͉ N-terminal activation function ͉ coregulatory protein ͉ protein folding T he glucocorticoid receptor (GR) is a ligand-activated transcription factor with the domain structural arrangement typical of the nuclear hormone receptors superfamily (1-4). The GR regulates transcription of target genes by binding DNA at specific hormone response elements and͞or by interacting with other transcription factors (5-8). Although the structural organization of steroid receptors into N-terminal domain (NTD), DNA binding domain (DBD), and ligand binding domain is well characterized (9-14), precisely how transcription is regulated by them is largely unknown. For all steroid receptors, this is in part due to the lack of information about their transcription activating domain AF1, located in the NTD (9, 10). The GR AF1 sequence resembles those of acidic transactivation domains of several transcription factors (15, 16). When expressed independently, the GR AF1 shows little structure and seems to exist as an ensemble of largely unstructured conformers (17, 18). The GR AF1 is known to interact with other transcription factors, and conditional folding has been suggested to be the key for these interactions (19)(20)(21)(22).It has been reported that in the presence of trifluoroethanol, the ''core'' of AF1 (AF1 c , amino acids 187-242), located toward the C-terminal end of AF1, adopts three helical segments (17). Independent experiments have shown that substitution of the ␣-helixbreaking amino acid proline for natural residues a...

Site-Specific Phosphorylation Induces Functionally Active Conformation in the Intrinsically Disordered N-Terminal Activation Function (AF1) Domain of the Glucocorticoid Receptor

“…Phosphorylationinduced conformational changes have also been proposed in the ID NTD of the estrogen receptor (8). ID activating regions of many TFs have been shown to function by recruiting the transcriptional apparatus to the promoter (24), and ID regions do indeed fold into a more ordered helical structure under physiological conditions (43). The characterization of phosphorylation-induced structure formation in facilitating protein-protein interactions should be of particular importance in understanding the mechanism by which kinase(s) regulate the transcriptional regulation of the target genes in the nucleus.…”

“…Site-specific phosphorylation represents an important regulatory mechanism in the activities of signaling proteins including steroid receptors (23,24,32). Since all of the known phosphorylation sites in the GR are located in the ID AF1/NTD, we hypothesize that site-specific phosphorylation of GR AF1 leads to changes in its conformations that are important for AF1's interaction with other critical coregulatory proteins and subsequent transcriptional activity.…”

Selective Regulation of Bone Cell Apoptosis by Translational Isoforms of the Glucocorticoid Receptor

“…It will be interesting to determine whether the N-terminal domains of GR isoforms have different molecular structures presenting different coregulator-interacting surfaces. While this notion is still speculative, recent evidence supports the hypothesis that after ligand and DNA binding, the GR N-terminal domain folds into an organized structure that promotes both intra-and intermolecular interactions and cofactor recruitment (14).…”

Regulation of the Structurally Dynamic N-terminal Domain of Progesterone Receptor by Protein-induced Folding

“…These protein interactions were initially thought to be a mechanism for receptor contact with basal transcriptional machinery required for activation. However, a core C-terminal domain of human TBP consisting of aa 159 -339 (TBP C ) was reported to interact with the NTDs of several SRs and to promote a more compact tertiary structure with increased ␣-helical content (14,(33)(34)(35)(36). In the case of glucocorticoid (GR) and mineralocorticoid receptors (MR), TBP C binding was also observed to be associated with an enhancement of NTD-dependent transcriptional activity (30,31,33).…”