Structural Characterization of a Minimal Functional Transactivation Domain from the Human Glucocorticoid Receptor

A 58-amino acid polypeptide containing the functional core region, the$\tau1$core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the$\tau1$core peptide fragment. Circular dichroism spectroscopy shows that the$\tau1$core and a larger fragment encompassing the intact$\tau1$domain are largely unstructured in water solution under a variety of pH conditions. The$\tau1$core, however, acquires a significant α-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for α-helical conformation. Two- and three-dimensional NMR spectroscopy of15N-labeled$\tau1$core, in the presence of trifluoroethanol, has allowed sequential assignment of1Hand15N resonances and identification of three protein segments with α-helical character. Potentially helix-breaking proline substitutions, in proposed α-helical regions, lead to reduced activity, suggesting that α-helices are important for transactivation in vivo.