ALTERED LIGAND-BINDING PROPERTIES AND ENHANCED STABILITY OF A CONSTITUTIVELY ACTIVE ESTROGEN-RECEPTOR - EVIDENCE THAT AN OPEN POCKET CONFORMATION IS REQUIRED FOR LIGAND INTERACTION

TO elucidate the ligand binding properties of the estrogen receptor (E R) and how ligand access to and release from the ligand binding pocket is affected by the conformational state of the receptor, we have meas ured the rates of estradiol association and dissociation, the equilibr ium binding, and the stability of estradiol binding to denaturants, co mparing wild-type human ER and a point mutant (Y537S ER) that shows fu ll constitutive activity, i.e., the same full transcriptional activity in the absence or presence of estrogen. Ligand binding kinetics and a ffinity were measured with the full-length (1-595) ERs and with trunca ted forms of both receptors containing domains C through F (including the DNA binding, hinge, and ligand binding domains, amino acids 175-59 5) 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 pre dicted (k(-1)/k(+1)) and measured K-d that is 2-fold lower for Y537S E R compared to wild-type ER. The binding of estradiol by wild-type ER w as disrupted by high concentrations of urea (above 2 M), whereas the Y 537S ER was distinctly more resistant to this disruption. These result s are consistent with a model in which wild-type ER in the absence of ligand adopts a transcriptionally inactive collapsed pocket conformati on, stabilized by specific interactions of Y537 with nearby regions of ER. When estradiol is bound, the wild-type ER adopts a transcriptiona lly 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 (cl osed 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 reduce d rates of ligand association and dissociation in the constitutively a ctive form of the ER, as well as its greater resistance to disruption of ligand binding by urea, support the supposition that the rate at wh ich this open pocket conformation can be accessed from the unoccupied or ligand-occupied Y537S ER is slower than from the unoccupied or occu pied 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 eas e with which this state can be accessed is affected by mutations that alter receptor conformation.