Structural requirements for high affinity ligand binding by estrogen receptors: A comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells

Cynthia Harris Wooge, Gisela M. Nilsson, Agneta Heierson, Donald P. McDonnell, Benita S. Katzenellenbogen

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In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast. yeast, as well as the bacterially expressed ER, were as stable as the full length MCF-7 ER, with minimal loss of the initial binding capacity of the unoccupied receptor even after 10 h at 25 C. In contrast, there was no binding of either reversibly (estradiol) or covalently attaching (ketononestrol aziridine, tamoxifen aziridine) ligands to a 42-amino acid synthetic peptide (human ER amino acids 510-551) comprising a portion of the hormone binding domain considered essential for ligand binding and which encompasses Cys-530, shown previously to be the amino acid covalently labeled with ER affinity labeling ligands. These studies demonstrate that the hormone binding domain of the ER alone (amino acids 302-595) is sufficient to bind ligand with good affinity (ca. 30% that of full length ER) and with appropriate ligand structural specificity. Such expressed truncated proteins should be valuable in further studies to characterize the three-dimensional structure of the ligand binding pocket of the receptor.

Original languageEnglish (US)
Pages (from-to)861-869
Number of pages9
JournalMolecular Endocrinology
Issue number6
StatePublished - Jun 1 1992


ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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