Identification of two cysteines closely positioned in the ligand-binding pocket of the human estrogen receptor: Roles in ligand binding and transcriptional activation

We have previously identified cysteine 530 in the human estrogen receptor (ER) as the major site of attachment for covalently binding affinity ligands and have shown that when this cysteine is mutated to alanine (C530A mutant), the affinity ligand [tamoxifen aziridine (TAZ)] can still bind covalently to the ER, presumably by interaction with a different cysteine(s) in the hormone-binding domain (HBD). Using site-directed mutagenesis, we have determined the alternative ligand attachment site and the functional importance of the cysteines (residues 381, 417, 447, and 530) in the HBD of the ER to the hormone-binding and transcriptional responses to estrogens and antiestrogens. Cysteine 530 plus one or more of these other cysteines were mutated to alanines. Analysis of these mutant ERs expressed in Chinese hamster ovary cells provides strong evidence that cysteine 381 is the residue that is preferentially covalently labeled by TAZ in the C530A mutant. Hence, portions of the HBD that are far apart in the linear receptor sequence, namely regions near C381 and C530, are probably closely positioned in the ligand-binding pocket, with the cysteine thiols being 1.1 nm or less apart. The affinity of estradiol binding to receptors was reduced only 2- and 5-fold, respectively, in the double and quadruple Cys to Ala mutants, and estradiol was an effective stimulator of transcription from an estrogen-responsive reporter gene [(ERE)2-TATA-CAT]. While reversibly binding antiestrogens were able to suppress estradiol-stimulated transcriptional activity evoked by all of the mutant receptors, the effectiveness of TAZ as an estrogen antagonist was reduced in the C530A mutant relative to wild-type ER and was essentially eliminated in all three of the double Cys mutants (i.e. C381A,C530A; C417,C530A; and C447,C530A) and in the quadruple Cys to Ala mutant (C381A,C417A,C447A,C530A). Hence, TAZ is less effective as an estrogen antagonist when covalent attachment is at C381 (in the C530A ERs) instead of C530 (in wild-type ER). Our study shows that while the four Cys residues in the HBD are not essential for the binding or transactivation activity of estradiol or other reversibly binding estrogens and antiestrogens, they do affect the potency of a covalently attaching antiestrogen.