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

被引:1
作者
Mattras, H
Aliau, S
Demey, E
Poncet, JL
Borgna, JL
机构
[1] INSERM, U540, F-34090 Montpellier 5, France
[2] INSERM, U554, F-34090 Montpellier 5, France
[3] CNRS, UPR 2580, F-34094 Montpellier 5, France
关键词
estrogen receptor; ligand-binding domain; estradiol; 17; alpha-derivatives; affinity labeling; Mass spectrometry;
D O I
10.1016/j.jsbmb.2005.10.006
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A purified preparation of human estrogen receptor alpha (hER alpha) ligand-binding domain (LBD) involving mainly the Ser(309) Ala(569) (similar to 30%) and Ser(309)Ala(571) (similar to 63%) ER portions Was used to identify the covalent attachment sites of two closely related estrogenic ER affinity labels 17 alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17 alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantity the electrophile covalent attachment sites, [C-14]17BAP(2)- and [C-14]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,. 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 17 alpha-derivatives differ from the Structure of crystallized LBD bound to E-2. (C) 2006 Elsevier Ltd. All rights reserved.
引用
收藏
页码:236 / 247
页数:12
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