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

The efficiency of 11β-[p(aziridinylethoxy)phenyl]estradiol 1 and 11β-[p(aziridinylpentoxy)phenyl]estradiol 2 affinity labeling of the estrogen receptor α (ERα) was evaluated on the basis of their capacity to inhibit [3H]estradiol binding to lamb and human ERαs. Relative to RU 39 411 (11β-[p(dimethylaminoethoxy)phenyl]estradiol), the most closely related and chemically inert analogue of 1, the two electrophiles irreversibly inhibited [3H]estradiol binding to the lamb ERα. The fact that the compound effects were prevented (i) when the ERα hormone-binding site was occupied by estradiol and (ii) when the ERα-containing extracts were pretreated with methyl methanethiosulfonate (an SH-specific reagent) suggested that the compounds specifically alkylated ERα at cysteine residues. Wild-type human ERα was alkylated as efficiently as lamb ER, whereas the quadruple cysteine → alanine mutant, in which all cysteines of the hormone-binding domain (residues 381, 417, 447, and 530) were changed to alanines, showed no sig...