Cooperativity and Dimerization of Recombinant Human Estrogen Receptor Hormone-binding Domain

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 ϳ1.6) similar to the full-length protein. Circular dichroism spectroscopy suggests that the HBD contains significant amounts of ␣-helix (ϳ60%) and some ␤-strand (ϳ7%) and that ligand binding induces little change in secondary structure. HBD dimer dissociation, measured using size exclusion chromatography, exhibited a halflife of ϳ1.2 h, which ligand binding increased ϳ3-fold (estradiol) to ϳ4-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.The estrogen receptor is a member of a superfamily of nuclear proteins that includes the receptors for the steroid hormones, for vitamins A and D, and for thyroid hormone (1, 2). The binding of ligands to these receptors is the initial step in a complex series of events culminating in an interaction of the ligand-bound receptor with the transcription machinery and modulation of gene expression. These receptor proteins exhibit four distinct properties required to exert their actions: hormone binding, multimeric complex formation, sequence specific DNA binding, and transcriptional modulation. The currently proposed schematic structure of these receptor proteins (shown in Fig. 1 for the estrogen receptor), based on sequence similarities and deletion analyses (summarized in Ref.2), suggests that these proteins fold into at least three separate structural and functional domains: (i) an N-terminal domain having a highly variable length and amino acid sequence and believed to mediate much of the transcriptional enhancement activity of the 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. 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 i...

Oscillations and patterns in spatially discrete models for developmental intercellular signalling

“…The nonlinear terms can then be neglected to give α ≈ −a 3 (K)/a 2 (K), and a good approximation to K corresponding to the largest possible α is given by K c in (40). In contrast to earlier work by Collier et al [4], Wearing et al demonstrated that the juxtacrine model (1) can generate a wide range of pattern wavelengths.…”

“…In both the string and two-dimensional square arrays, K(λ) is bounded between ±1 as λ varies. However, K(λ) is reduced to [0,1] Substituting into the linearized model and dividing through by exp(αt + iλ · x), the condition for non-trivial solutions gives the cubic characteristic…”

“…Each nicotinic receptor has two binding sites for acetylcholine, one of a group of biochemicals known as neurotransmitters that carry nerve impulses from one neuron to another. The estrogen receptor dimerizes and exhibits cooperative ligand binding as part of its normal functioning [1]. Another important co-operative behaviour is when the ligand is multivalent, such as platelet derived growth factor (PDGF) which can cross-link and bind to two or more receptors [9,11].…”

Structural Basis for Ca2+-induced Activation and Dimerization of Estrogen Receptor α by Calmodulin

“…The 1:2 stoichiometry suggests that CaM may facilitate functional dimerization of ER-␣, which could help place two ER-␣ DNA binding domains in contact with tandem repeat sequences in ERE (36,37). The ER-␣ LBD (residues 305-552) forms a dimer in the x-ray crystal structure of the E2-bound state (12), whereas the E2-free apo-LBD has weaker dimerization affinity (38,39). Thus, ER-␣ dimerization stabilized by E2 binding may be important for activating transcription (3).…”

Interaction of Transcriptional Intermediary Factor 2 Nuclear Receptor Box Peptides with the Coactivator Binding Site of Estrogen Receptor α

“…However, in reality the actual binding model could be more complicated because liganded ER is typically dimeric and therefore capable of binding two peptide motifs (one for each LBD in an ER homodimer). Based on the reported equilibrium constants for ER dimerization (37,38), we have assumed that, at the concentrations of ER and ER␣LBD used in this study (Ն16 nM), the receptor exists as a homodimer. To further simplify the system, we also immobilized limited amounts of each peptide to the chip surfaces so as to make it impossible for two peptides to bind simultaneously to a single ER homodimer.…”