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Abstract
The estrogen receptor dimerizes and exhibits cooperative ligand binding as part of its normal functioning. Interaction of the estrogen receptor with its ligands is mediated by a C-terminal hormone-binding domain (HBD), and residues within the HBD are thought to contribute to dimerization. To examine dimer interactions in the isolated HBD, a human estrogen receptor HBD fragment was expressed in high yield as a cleavable fusion protein in Escherichia coli. The isolated HBD peptide exhibited affinity for estradiol, ligand discrimination, and cooperative estradiol binding (Hill coefficient approximately 1.6) similar to the full-length protein. Circular dichroism spectroscopy suggests that the HBD contains significant amounts of alpha-helix ( approximately 60%) and some beta-strand ( approximately 7%) and that ligand binding induces little change in secondary structure. HBD dimer dissociation, measured using size exclusion chromatography, exhibited a half-life of approximately 1.2 h, which ligand binding increased approximately 3-fold (estradiol) to approximately 4-fold (4-hydroxytamoxifen). These results suggest that the isolated estrogen receptor HBD dimerizes and undergoes conformational changes associated with cooperative ligand binding in a manner comparable to the full-length protein, and that one effect of ligand binding is to alter the receptor dimer dissociation kinetics.
Highlights
Protein, (ii) a highly conserved central domain of ϳ80 amino acids involved in DNA binding, and (iii) a less well conserved C-terminal domain of ϳ250 amino acids that is involved in ligand binding
The C-terminal hormone-binding domain (HBD)1 of the receptor is of particular interest because, in addition to its ligand binding activity, it appears to contain many of the regulatory functions of the protein
It is not known how much of the interprotein interaction involved in dimer formation is contained within the HBD and whether the isolated HBD is fully capable of cooperative ligand binding
Summary
Protein, (ii) a highly conserved central domain of ϳ80 amino acids involved in DNA binding, and (iii) a less well conserved C-terminal domain of ϳ250 amino acids that is involved in ligand binding. Chimeric constructs containing fusions of fragments of the estrogen receptor with unrelated proteins such as the myc oncogene product, for example, display hormonal regulation of the activity of the fused gene products [3] This suggests that, even when removed from its normal environment, the HBD is capable of specific ligand binding, but may retain the capacity to undergo the conformational changes that normally regulate the function of the receptor. The isolated estrogen receptor DNA-binding domain has been shown to dimerize in the presence of DNA, suggesting that some of the dimerization interface resides within this portion of the protein; the isolated DNA-binding domain, is monomeric in solution [5, 6] It is not known how much of the interprotein interaction involved in dimer formation is contained within the HBD and whether the isolated HBD is fully capable of cooperative ligand binding. The HBD peptide is produced as a fusion protein with the E. coli
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