Chemical and Biochemical Issues Related to X-ray Crystallography of the Ligand-Binding Domain of Estrogen Receptor α
| Autor: | Kieran F. Geoghegan, Steven W. Goldstein, Jon Bordner, Lise R. Hoth |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Pyrrolidines
Tetrahydronaphthalenes Iodoacetic acid Stereochemistry Molecular Sequence Data Biomedical Engineering Pharmaceutical Science Bioengineering Crystallography X-Ray Ligands Peptide Mapping High-performance liquid chromatography Chromatography Affinity Mass Spectrometry chemistry.chemical_compound Affinity chromatography medicine Humans Amino Acid Sequence Receptor Pharmacology Binding Sites Chromatography Molecular Structure Chemistry Carbocysteine Organic Chemistry Estrogen Antagonists Estrogen Receptor alpha Diastereomer Trypsin Protein Structure Tertiary Receptors Estrogen Estrogen receptor alpha Biotechnology Cysteine medicine.drug |
| Zdroj: | Bioconjugate Chemistry. 12:406-413 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc000127d |
| Popis: | Careful attention to technical issues preceded successful crystallography of the ligand-binding domain of estrogen receptor alpha (ERalpha) complexed with CP-336156, a nonsteroidal estrogen agonist/antagonist. An affinity column based on immobilized estradiol was prepared according to the scheme of Greene et al. (Greene, G. L., Nolan, C., Engler, J. P., and Jensen, E. V. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5115-5119). It was shown by X-ray crystallography that the major and less polar isomer of the affinity column precursor was 17alpha-((S)-2',3'-epoxyprop-1'-yl)estra-1,3,5(10)-triene-3,17beta-diol. This diastereomer was coupled to Thiopropyl Sepharose, with coupling monitored by observing loss of the phenolic absorption band of estradiol from the reaction supernatant, and gave an affinity matrix containing about 9 micromol of estradiol per milliliter of wet gel. Recombinant ERalpha ligand binding domain was selectively removed from E. coli cell lysate by binding to the column and was partly S-carboxymethylated by treatment with iodoacetic acid while bound to the column as described by previous workers. After being eluted from the column as a complex with drug, the receptor fragment was shown by mass spectrometry to be a mixture of differently modified forms. It was further S-carboxymethylated in solution, after which anion-exchange chromatography was used to isolate protein in which two of the four cysteine residues were S-carboxymethylated. This material, which afforded diffraction-quality crystals, was subjected to digestion with trypsin and peptide mapping analysis by HPLC coupled with mass spectrometry. For this experiment, the two previously unmodified cysteines were alkylated with 4-vinylpyridine to allow definitive identification. It was shown that Cys-417 and Cys-530 were S-carboxymethylated in the crystallized protein, while Cys-381 and Cys-447 remained unmodified. Close attention to such technical issues may be important in structural studies of other nuclear receptors, a very important class of potential drug targets. |
| Databáze: | OpenAIRE |
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