Identification of covalent attachment site of antiestrogenic estradiol 11β-derivatives on human estrogen receptor α ligand-binding domain

Abstract Affinity labeling of human estrogen receptor α (ERα) by high affinity and antiestrogenic estradiol (E 2 ) 11β-derivatives, 11β-bromoacetamidoethoxyphenylE 2 (11BAEOPE 2 ) and 11β-bromoacetamidopentoxyphenylE 2 (11BAPOPE 2 ) was studied using glutathione- S -transferase (GST) fused to the ligand-binding domain (LBD) of human ERα. To identify and quantify the electrophile covalent attachment sites on LBD, [ 14 C]11BAEOPE 2 - and [ 14 C]11BAPOPE 2 -alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE 2 and 11BAPOPE 2 , respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys 530 Lys 531 LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the covalent attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD–ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys 530 could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common attachment to Cys 530 of estrogenic E 2 17α-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor α, J. Steroid Biochem. Mol. Biol. 98 (4–5), in press] and antiestrogenic E 2 11β-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.