IDENTIFICATION OF THE CYSTEINE IN THE STEROID-BINDING SITE OF HUMAN CORTICOSTEROID-BINDING GLOBULIN BY SITE-DIRECTED MUTAGENESIS AND SITE-SPECIFIC CHEMICAL MODIFICATION

Binding of corticosteroids by human corticosteroid binding globulin (hCBG) is thought to involve interaction with one of its two cysteine residues (Cys(60) and Cys(228)). To identify which of the two cysteine residues mediates steroid binding, we have produced mutant hCBGs containing serine or alanine in place of Cys(228) by site-directed mutagenesis. Alteration of Cys(228) to serine or alanine does not change the steroid binding affinity of hCBG, demonstrating that Cys(228) is not involved in the binding interaction. This finding strongly suggests that Cys(60) 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 Cys(60) is present at the steroid binding site, and that it may be directly involved in steroid binding. This result also identifies Cys(60) as the accessible cysteine reported in previous studies.