Identification by MALDI-TOF Mass Spectrometry of 17α-Bromoacetamidopropylestradiol Covalent Attachment Sites on Estrogen Receptor α

• Published in: Biochemistry (Easton) 2002-12, Vol.41 (52), p.15713-15727
• Main Authors: Mattras, Hélène, Aliau, Sigrid, Richard, Eric, Bonnafous, Jean-Claude, Jouin, Patrick, Borgna, Jean-Louis
• Format: Article
• Language: English
• Subjects: Affinity Labels; Affinity Labels - chemistry; Amino Acid Sequence; Animals; Binding Sites; Biochemistry, Molecular Biology; Carbon Radioisotopes; Estradiol; Estradiol - agonists; Estradiol - analogs & derivatives; Estradiol - chemistry; Estrogen Receptor alpha; Glutathione Transferase; Glutathione Transferase - chemistry; Glutathione Transferase - genetics; Humans; Life Sciences; Ligands; Mice; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Peptide Fragments - analysis; Peptide Fragments - genetics; Peptide Mapping; Peptide Mapping - methods; Protein Structure, Tertiary; Protein Structure, Tertiary - genetics; Receptors, Estrogen; Receptors, Estrogen - biosynthesis; Receptors, Estrogen - chemistry; Receptors, Estrogen - genetics; Receptors, Estrogen - isolation & purification; Recombinant Fusion Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0205092

Mass spectrometry was used to identify the sites of covalent attachment of [14C]-17α-bromoacetamidopropylestradiol ([14C]17BAPE2, an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor α (ERα). A glutathione S-transferase (GST)−LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERα (Ser313−Ile599), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [14C]17BAPE2 covalent labeling experiments were carried out on the GST−LBD chimera immobilized on glutathione−Sepharose. After thrombin cleavage of the chimeric LBD, two major [14C]17BAPE2-labeled species of 34 (∼75%) and 30 kDa (∼25%) were detected by SDS−PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS):  two main signals were consistent with the mass of the full-length (Ser313−Ile599) and truncated LBD (Ser313−Ala573), both comprising the extra 14 N-terminal amino acids and covalently bound [14C]17BAPE2 (via HBr elimination). A purified 14C-labeled LBD preparation was trypsinized to identify the covalent attachment sites of 17BAPE2. HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [14C]17BAPE2-alkylated Cys534Lys535 and Cys421−Arg438 peptides, respectively. Hydrolysis of the second 14C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys421−Glu423 tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative covalent attachment sites of 17BAPE2 on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol−LBD and raloxifene− or 4-hydroxytamoxifen−LBD complexes. The covalent attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD−17BAPE2 complex.