17R-(Haloacetamidoalkyl)estradiols Alkylate the Human Estrogen Receptor at Cysteine Residues 417 and 530 † Sigrid Aliau, ‡ Driss El Garrouj, ‡ Abdelaziz Yasri, § Benita S. Katzenellenbogen, | and Jean-Louis Borgna* ,‡ INSERM Unite ´ 439, 70 rue de NaVacelles, 34090 Montpellier, France, Syntem, Parc Scientifique Georges Besse, 30000 Nimes, France, and Department of Molecular and IntegratiVe Physiology, UniVersity of Illinois, Urbana, Illinois 61801 ReceiVed December 18, 1996; ReVised Manuscript ReceiVed February 19, 1997 X ABSTRACT: Results obtained in a previous study suggested that cysteine residues in the estrogen receptor were covalent attachment sites for four 17R-(haloacetamidoalkyl)estradiols (halo, bromo or iodo; alkyl, methyl, ethyl, or propyl). To identify the putative concerned cysteines, we expressed wild-type and various cysteine f alanine mutants of the human estrogen receptor in COS cells and determined their ability to be alkylated by the four electrophiles. The quadruple mutant, in which all the cysteines (residues 381, 417, 447, and 530) of the hormone-binding site were changed to alanines, showed very little electrophile labeling, whereas the four single mutants (C381A, C417A, C447A, and C530A) were alkylated as efficiently as the wild-type receptor. These results (i) demonstrate that cysteine residues were covalent attachment sites of electrophiles and (ii) indicate that more than one cysteine residue could be alkylated. Analysis of three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) provided strong evidence that only C417 and C530 were sites for electrophile covalent attachment. Since C530 was also alkylated by tamoxifen aziridine, a nonsteroidal affinity-labeling agent, we propose a selective mode of superimposition of tamoxifen-class antiestrogens with estradiol, which could account for the relative positioning of the two types of ligands in the receptor hormone-binding pocket. According to the structure of the hormone-binding pocket of nuclear receptors, as inferred from crystallographic studies and general sequence alignment of hormone-binding domains, C417 and C530 appear to be (1) located at the extreme border or in structural elements involved in delineation of the hormone-binding pocket, (2) spatially in close proximity to each other, and (3) in positions highly homologous to those of glucocorticoid receptor sites alkylated by affinity- and photoaffinity-labeling agents, respectively. The nuclear receptor superfamily (Evans, 1988; Mangels- dorf et al., 1995b) presently includes more than 150 related intracellular proteins which act as transcriptional regulators and are involved in development, differentiation, and ho- meostasis. This superfamily is made up of a group of established receptors, which are ligand-modulated transcrip- tional factors that include receptors for steroid and thyroid hormones, vitamins D, retinoids, and related compounds, and of a group of orphan receptors, including proteins without ligands and possibly proteins whose cognate ligands are still unknown (Mangelsdorf & Evans, 1995a). Nuclear receptors are modular proteins with evolutionarily conserved DNA- and ligand-binding domains (Krust et al., 1986). In the steroid hormone receptor subgroup, hormone binding, through a receptor conformation change, induces receptor dimeriza- tion, interaction of the dimer with target DNA sequences, usually located in the vicinity of hormone-regulated genes, and interaction with transcription factors, thus allowing modulation of transcription of these genes (Tsai et al., 1994). The DNA-binding domain of nuclear receptors includes about 70 amino acids; its structure, with two perpendicular helices, was established in the early 1990s, through NMR (Hard et al., 1990; Schwabe et al., 1990) and crystallographic studies (Luisi et al., 1991). The nuclear receptor hormone- binding domain includes about 250 amino acids; in addition to the hormone-binding function, this domain is involved in binding of 90 kDa heat-shock proteins (Pratt, 1993), the receptor dimerization process (Glass, 1994), and activation of transcription (Tora et al., 1989). The overall structure of this domain was unknown until recent reports involving crystallographic studies of the hormone-binding domain from unliganded human retinoic X receptor R (RXRR) 1 (Bourguet et al., 1995), liganded retinoic acid receptor γ (RARγ) (Renaud et al., 1995), and rat thyroid hormone receptor R 1 (TRR 1 ) (Wagner et al., 1995). The hormone-binding domain of these receptors appears to comprise 11 (or 12) R-helices and 2 (or 4) -strands. The helices are organized in a three- layer structure with 5 (or 4) internal helices sandwiched between the most external helices. The ligand-binding pocket is predominantly formed by hydrophobic residues located in 8 (or 10) structural elements (Renaud et al., 1995; Wagner et al., 1995), some identical and others differing with respect to RARγ and TRR 1 . On the basis of a comparison of structures determined for unfilled and hormone-filled receptors, Wurtz et al. (1996) proposed a mechanism whereby a ligand-induced confor- † This work was supported by the Institut National de la Sante ´ et de la Recherche Me ´dicale (J.-L.B.), the Association pour la Recherche sur le Cancer (J.-L.B.), and the National Institutes of Health (CA 18119 to B.S.K.). * To whom correspondence should be addressed. ‡ INSERM Unite ´ 439. § Syntem. | University of Illinois. X Abstract published in AdVance ACS Abstracts, April 15, 1997. 1 Abbreviations: RXRR, retinoic X receptor R; RAR γ, retinoic acid receptor γ; TRR1, thyroid hormone receptor R1; DME medium, Dulbecco’s modified Eagle’s tissue culture medium; PBS, phosphate- buffered saline; T20, 20 mM Tris-HCl buffer at pH 8.5. 5861 Biochemistry 1997, 36, 5861-5867 S0006-2960(96)03111-X CCC: $14.00 © 1997 American Chemical Society