Increase in the Stability and Helical Content of Estrogen Receptor R in the Presence of the Estrogen Response Element: Analysis by Circular Dichroism Spectroscopy † Norma Greenfield, ‡ Veena Vijayanathan, § T. J. Thomas, § Michael A. Gallo, | and Thresia Thomas* ,| Departments of Neuroscience and Cell Biology, Medicine, and EnVironmental and Community Medicine, EnVironmental and Occupational Health Sciences Institute, and The Cancer Institute of New Jersey, UniVersity of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 ReceiVed December 15, 2000; ReVised Manuscript ReceiVed March 29, 2001 ABSTRACT: Ligand-dependent stabilization of the estrogen receptor (ER) is often postulated, with limited support from experimental data. We studied the thermal unfolding of recombinant ERR by circular dichroism (CD) spectroscopy. The T M of unfolding of ERR was 38 ( 2.4 °C, and the van’t Hoff enthalpy of unfolding was 31.7 ( 3.4 kcal/mol in the absence of ligands. Addition of estradiol (E 2 ) increased the T M to 43.6 ( 2.3 °C, while addition of E 2 and an oligonucleotide harboring the estrogen response element (ERE) increased the T M to 47.9 ( 1.6 °C. Addition of the antiestrogen 4-hydroxytamoxifen (HT) alone did not increase the T M ; however, a combination of HT and the ERE increased the T M to 48.9 ( 1.0 °C. The ERE alone increased the T M to 46.1 ( 0.9 °C. Addition of E 2 alone had no effect on the apparent enthalpy of unfolding; however, the ERE alone increased the apparent enthalpy from 31.7 to 36.1 kcal/ mol. ERR samples containing the ERE also exhibited an increase in the negative ellipticity at 208 and 222 nm, relative to that of ligand-free ERR, suggesting a stabilization of the R-helix. CD data analysis further showed that the presence of the ERE caused a large increase in R-helical content of ERR in both the presence and absence of the ligands. This increase in R-helical content of ERR was not observed in the presence of a nonspecific oligonucleotide. These results show that the ERE can increase the thermal stability of ERR, enhance its R-helical content, and facilitate the cooperativity of the folding transition. Estrogen receptors (ERR and ER) 1 are ligand-activated transcription factors that mediate the actions of the female sex hormone, estradiol, its metabolites, and synthetic mimics (1-4). Estradiol and other ligands are believed to alter the conformation of ER so that it acquires a high affinity for DNA and binds to specific sequences, the ERE, present in the promoter/enhancer region of estrogen-responsive genes. This binding and the associated changes in the chromatin structure initiate transcriptional activation of estrogen- responsive genes. Activation of a cell- and tissue-specific network of genes enables estradiol and ERs to exert multiple functions, not only on the reproductive system but also on several other organ systems (5, 6). Recent X-ray crystallographic studies on the ligand binding domain of ER (R and ) have identified the positioning of the ligand in a well-defined hydrophobic pocket of the protein (7-9). Similar studies on the DNA binding domain have indicated that the protein binds as a symmetrical dimer to the palindromic consensus sequence (10, 11). There has been no crystallographic or NMR study on the whole protein to assess ligand-induced changes in the total ER protein structure. A conventional avenue for exploring ligand-induced con- formational changes has been to examine changes in protease sensitivity due to ligand or DNA binding. Studies on protease sensitivity of the whole protein have produced contradictory results. Fritch et al. (12) did not observe any difference in the proteolytic pattern of unbound ER, or estrogen/anties- trogen-bound ER. Emmus et al. (13) also did not find any difference in the sensitivity of ER bound to estrogens or antiestrogens to trypsin, chymotrypsin, papain, or elastase. However, Attardi and Happe (14) reported differences in the sensitivity to proteolysis of the nuclear receptor bound to estrogen and antiestrogens. More recent studies by Kraichely et al. (15) demonstrated distinct differences in the protease digestion pattern of ER (R and ) bound to agonistic or antagonistic ligands. Studies on the DNA binding domain of ERR also showed differences in chymotrypsin sensitivity when bound to classical and altered EREs (16). However, direct measurements of ligand-induced conformational changes in intact ER have not been possible. † This work was supported by Public Health Service Grants CA42439, CA73058, and CA80163 from the National Cancer Institute and Grant ES05022 from the National Institute of Environmental Health Sciences. * To whom correspondence should be addressed: 125 Paterson St., Clinical Academic Building, Room 7092, New Brunswick, NJ 08903. Phone: (732) 235-8458. Fax: (732) 235-8473. E-mail: thomasth@ umdnj.edu. ‡ Department of Neuroscience and Cell Biology. § Department of Medicine. | Department of Environmental and Community Medicine. 1 Abbreviations: ER, estrogen receptor; CD, circular dichroism; ERE, estrogen response element; E2, estradiol; HT, 4-hydroxytamoxifen; HPLC, high-performance liquid chromatography; PAGE, polyacryla- mide gel electrophoresis; ODN, oligodeoxyribonucleotide; DTT, dithio- threitol; DCC, dextran-coated charcoal; EMSA, electrophoretic mobility shift assay; TEGD, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, and 10% glycerol. 6646 Biochemistry 2001, 40, 6646-6652 10.1021/bi002846l CCC: $20.00 © 2001 American Chemical Society Published on Web 05/10/2001