Cysteine 530 of the human estrogen receptor alpha is the main covalent attachment site of 11 beta-(aziridinylalkoxyphenyl)estradiols

The efficiency of 11 beta-[p(aziridinylethoxy)phenyl]estradiol 1 and 11 bet a-[p(aziridinylpentoxy)phenyl]estradiol 2 affinity labeling of the estrogen receptor alpha (ER alpha) was evaluated on the basis of their capacity to inhibit [H-3]estradiol binding to lamb and human ER alpha s. Relative to RU 39 411 (11 beta-[p(dimethylaminoethoxy)phenyl]estradiol) the most closely related and chemically inert analogue of 1, the two electrophiles irreversi bly inhibited [H-3]estradiol binding to the lamb ER alpha. The fact that th e compound effects were prevented (i) when the ER alpha hormone-binding sit e was occupied by estradiol and (ii) when the ER alpha-containing extracts were pretreated with methyl methanethiosulfonate (an SH-specific reagent) s uggested that the compounds specifically alkylated ER alpha at cysteine res idues. Wild-type human ER alpha was alkylated as efficiently as lamb ER, wh ereas the quadruple cysteine --> alanine mutant, in which all cysteines of the hormone-binding domain (residues 381, 417, 447, and 530) were changed t o alanines, showed no significant electrophile labeling. The single C530A m utant was much less sensitive to the action of the electrophiles than the t hree other single mutants (C381A, C417A, and C447A). Moreover, analysis of the three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) showed that only the C381A/C530A mutant was less susceptible to electrophile labe ling than the single C530A mutant. We concluded that in the hormone-binding pocket C530 was the main covalent attachment site of aziridines 1 and 2, w hereas C381 could be a secondary site. These results agreed with the crysta l structure of the hormone-binding domain of the human ER alpha bound to es trogen or antiestrogen, since C381 and C530 appeared to be (i) located in s tructural elements involved in delineating the hormone-binding pocket and ( ii) in spatial proximity to each other, which was closer in the crystal str ucture of the ER:antiestrogen complex than in that of the ER:estrogen compl ex. Since C530 and C381 were also the main and secondary covalent attachmen t sites of tamoxifen aziridine (a nonsteroidal affinity-labeling agent), we propose a selective mode of superimposition of tamoxifen-class antiestroge ns with RU 39 411-class antiestrogens, which could account for the relative positioning of the two types of ligands in the ER alpha hormone-binding po cket.