title: Identification of the cysteine in the steroid-binding site of human corticosteroid binding globulin by site-directed mutagenesis and site-specific chemical modification.
abstract: Binding of corticosteroids by human corticosteroid binding globulin (hCBG) is thought to involve interaction with one of its two cysteine residues (Cys60 and Cys228). To identify which of the two cysteine residues mediates steroid binding, we have produced mutant hCBGs containing serine or alanine in place of Cys228 by site-directed mutagenesis. Alteration of Cys228 to serine or alanine does not change the steroid binding affinity of hCBG, demonstrating that Cys228 is not involved in the binding interaction. This finding strongly suggests that Cys60 is the functionally important cysteine. By modifying the wild-type and mutant hCBGs with the sulfhydryl-specific reagents N-ethylmaleimide, iodoacetamide, and sodium tetrathionate, we have demonstrated that Cys60 is present at the steroid binding site, and that it may be directly involved in steroid binding. This result also identifies Cys60 as the accessible cysteine reported in previous studies.
authors.affiliation: Department of Biochemistry, Mount Sinai School of Medicine, New York, NY 10029-6574.
authors.forename: J
authors.initials: J
authors.lastname: Ghose-Dastidar
authors.forename: R
authors.initials: R
authors.lastname: Green
authors.forename: J B
authors.initials: JB
authors.lastname: Ross
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013256:Steroids / Q000378:metabolism*; D013439:Sulfhydryl Reagents; D014156:Transcortin / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D013256:Steroids; D013439:Sulfhydryl Reagents; D014156:Transcortin; D003545:Cysteine