Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor alpha.

A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the covalent attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile covalent attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The covalent attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the covalent attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).