Regulation of the Amino-Terminal Transcription Activation Domain of Progesterone Receptor by a Cofactor-Induced Protein Folding Mechanism

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

“…The NTDs of SRs are predicted by computational analysis to contain a high percentage of random coil, and solution phase biophysical analyses have directly confirmed the IDP characteristics of isolated NTDs of several SRs (22)(23)(24)(25)(26)(27)(28). NTDs of selected SRs have also been observed to undergo an increase in overall secondary and tertiary structure in the presence of natural osmolytes such as trimethylamine-N-oxide (TMAO) and trehalose (10,12,14,29).…”

“…Amino acid substitutions were introduced into the PR NTD fragments using the Stratagene QuikChange Lightning site-directed mutagenesis kit. Mammalian cell expression plasmids under the control of the Rous sarcoma virus promoter for full-length PR and domains of receptor have been described previously, including phPR-B and a two-domain PR-B NTD/DBD fragment (aa 1-650) (28). DNA sequencing of all plasmids was performed to verify correct sequences and point mutations.…”

“…Plasmids and Antibodies-Recombinant baculovirus vectors expressing different domains of human progesterone receptor (PR) containing polyhistidine tags at the N terminus have been previously described (28). Transfer plasmids used for construction of baculoviruses were pBlueBacHis (Invitrogen) and encode the following domains and sequence regions of PR (PR-A, aa 165-933; PRB-NTD, aa 1-534; PR-A NTD, aa 165-534; hinge LBD, aa 634 -933).…”

“…Recombinant Protein Expression and Purification-PR was expressed from baculovirus vectors in Sf9 insect cells as described previously (28). Full-length PR (A or B isoform) was bound to the synthetic progestin R5020 during expression in Sf9 cells, and cells were lysed in 20 mM sodium phosphate buffer, pH 7.4, containing 350 mM NaCl, 10 mM imidazole, 10% glycerol, 15 mM ␤ mercaptoethanol, 50 mM sodium fluoride, 1 M urea, protease inhibitors (leupeptin, aprotinin, bacitracin, pepstatin A, and PMSF) and 1.2 units/ml benzonase nuclease.…”

“…Cells were also transfected with a GRE/PRE-dependent SEAP reporter gene, and reporter gene activity was measured as described previously (33). As anticipated, the two-domain PR-B NTD-DBD construct exhibited constitutive transcriptional activity in the absence of hormone (28). Co-transfection with TBP increased the transcriptional activity mediated by PR-B NTD-DBD (Fig.…”