Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol covalent attachment sites on estrogen receptor alpha.
Mass spectrometry was used to identify the sites of covalent attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). 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 ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 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 (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the covalent attachment sites of 17BAPE(2). 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 [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative covalent attachment sites of 17BAPE(2) 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-17BAPE(2) complex.
Mattras
Hélène
H
INSERM U 439, 70 rue de Navacelles, 34090 Montpellier, France.
Aliau
Sigrid
S
Richard
Eric
E
Bonnafous
Jean-Claude
JC
Jouin
Patrick
P
Borgna
Jean-Louis
JL
D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D004958:Estradiol / Q000819:agonists / Q000031:analogs & derivatives / Q000737:chemistry*; D047628:Estrogen Receptor alpha; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics; D010449:Peptide Mapping / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
C471207:17-bromoacetamidopropylestradiol; D000345:Affinity Labels; D002250:Carbon Radioisotopes; D047628:Estrogen Receptor alpha; D008024:Ligands; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D004958:Estradiol; D005982:Glutathione Transferase