Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor.

Abstract

A 58-amino acid polypeptide containing the functional core region, the x1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-701% ofthe activity ofthe intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the T1 core peptide fragment. Circular dichroism spectroscopy shows that the T1 core and a larger fragment encompassing the intact Tl domain are largely unstructured in water solution under a variety of pH conditions. The T1 core, however, acquires a significant a-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for a-helical conformation.Two-and three-dimensional NMR spectroscopy of 1-N-labeled T1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 'H and 15N resonances and identification of three protein segments with a-helical character.

Analysis of the architecture of the transcription factor σN (σ54) and its domains by circular dichroism

“…The amino-terminal activation Region I appears to contribute little to the backbone and tertiary structure of N . In isolation, the Region I peptide was structured with respect to helix and sheet, in contrast to observations consistently made for activation domains of eukaryotic transcription factors (for example Dahlman-Wright et al, 1995). A lack of intrinsic conformational preference of the Region I peptide would indicate that a particular Region I secondary structure within N could be directed by tertiary interactions between Region I and other parts of N .…”

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

“…Independent experiments have shown that substitution of the ␣-helixbreaking amino acid proline for natural residues at critical positions in these putative helices significantly reduces the transcriptional potency of the GR (17). Other experiments have shown that mutations of the hydrophobic amino acids in AF1 reduce both its interactions with other coregulatory proteins and its transactivation function (21).…”

“…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 at critical positions in these putative helices significantly reduces the transcriptional potency of the GR (17).…”

“…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).…”

Role of Regulatory F-domain in Hepatocyte Nuclear Factor-4α Ligand Specificity

“…Mutants: Circular Dichroism-TFE is known to induce helical secondary structure in proteins that contain unordered structures and have potential for developing helixes under a hydrophobic environment (41,42). Since the long F-domain in HNF-4␣ molecule is random coil-proline-rich with very low helix content (22,38), the potential of the three forms of HNF-4␣ to form helix structure in the presence of TFE and the influence of F-domain were examined (Fig.…”