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
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. Full length and truncated receptors expressed in 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.
Wooge, CH; Nilsson, GM; Heierson, A; McDonnell, DP; Katzenellenbogen, BS
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