Altered Ligand Binding Properties and Enhanced Stability of a Constitutively Active Estrogen Receptor:  Evidence That an Open Pocket Conformation Is Required for Ligand Interaction

Abstract

To elucidate the ligand binding properties of the estrogen receptor (ER) and how ligand access to and release from the ligand binding pocket is affected by the conformational state of the receptor, we have measured the rates of estradiol association and dissociation, the equilibrium binding, and the stability of estradiol binding to denaturants, comparing wild-type human ER and a point mutant (Y537S ER) that shows full constitutive activity, i.e., the same full transcriptional activity in the absence or presence of estrogen. Ligand binding kinetics and affinity were measured with the full-length (1-595) ERs and with truncated forms of both receptors containing domains C through F (including the DNA binding, hinge, and ligand binding domains, amino acids 175-595) or domains E and F (the ligand binding domain; amino acids 304-595). With all ERs, the rates of ligand association and dissociation were considerably slower with the Y537S mutant ER than with wild-type ER (6-fold and 3-4-fold, respectively). These marked differences in ligand on and off rates for the wild-type and Y537S receptors result in a predicted (k-1/k+1) and measured Kd that is 2-fold lower for Y537S ER compared to wild-type ER. The binding of estradiol by wild-type ER was disrupted by high concentrations of urea (above 2 M), whereas the Y537S ER was distinctly more resistant to this disruption. These results are consistent with a model in which wild-type ER in the absence of ligand adopts a transcriptionally inactive collapsed pocket conformation, stabilized by specific interactions of Y537 with nearby regions of ER. When estradiol is bound, the wild-type ER adopts a transcriptionally active, closed pocket (ligand occupied) conformation. By contrast, the Y537S mutant ER favors the transcriptionally active closed pocket conformation, whether occupied by ligand or not, the latter state (closed pocket but unoccupied) accounting for its constitutive activity. Our findings suggest that the entry or exit of ligand from the binding pocket requires that ER adopt an open pocket conformation. The reduced rates of ligand association and dissociation in the constitutively active form of the ER, as well as its greater resistance to disruption of ligand binding by urea, support the supposition that the rate at which this open pocket conformation can be accessed from the unoccupied or ligand-occupied Y537S ER is slower than from the unoccupied or occupied forms of wild-type ER. Thus, the binding and release of ligand by ER require that the receptor access an open pocket state, and the ease with which this state can be accessed is affected by mutations that alter receptor conformation.

Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes

“…S9 and S10). Earlier comparative studies on the ERα-Y537S mutant established that it had somewhat elevated affinity for E 2 (36).…”

New insights into Vitamin D sterol-VDR proteolysis, allostery, structure–function from the perspective of a conformational ensemble model

“…In addition, apo-PPARγ NMR results indicate no single unliganded conformation exists in solution [46]. Furthermore, orphan NRs as well as single point mutations made in the estrogen receptor (ER), a NR that 'requires ligand' for activation, show high levels of basal and/or constitutive activity [10,47]. Collectively the results therefore suggest that mechanistically a conformational ensemble model may be useful in understanding NR/ VDR signaling.…”

“…It is inferred from such a model that the apo-NR molecule exists in a relatively rigid, opened conformation (Fig. 9, apo-VDR or c3) [10] where the C-terminal end of H11 must naturally move out of the way to expose the conserved H5 Arg residue (R274 of VDR, see Fig. 1C) and allow sterol to bind the G-pocket (Fig.…”

“…It is currently posited that H12 is opened in the absence of ligand (apo-NR) and is induced to close (e.g. activated by induced-fit) upon binding the ligand [9,10].…”

A Proteomic Microarray Approach for Exploring Ligand-initiated Nuclear Hormone Receptor Pharmacology, Receptor Selectivity, and Heterodimer Functionality

“…In addition to ER-LBDs, we have extended our NHR microarray approach to full-length ER and can also simultaneously monitor the activation state of different classes of NHRs, ER and TR, in the presence of estrogens and thyroid receptor ligands. Protein Preparations-The LBD of the human ER␣ (amino acids 304 -554) and the corresponding LBD of the human ER␤ (amino acids 256 -505) were expressed from a pET-15b vector in BL21(DE3)pLysS E. coli using methods reported previously (9). ER-LBD mutants with a single reactive cysteine were constructed as previously reported (10).…”