title: Direct evidence for the localization of the steroid-binding site of the plasma sex steroid-binding protein (SBP or SHBG) at the interface between the subunits.
abstract: Complete dissociation of dimeric plasma sex steroid-binding protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca+2 or steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric steroid-binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds steroid can be considered meaningless; steroid-binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195, USA.
authors.forename: L M
authors.initials: LM
authors.lastname: Sui
authors.forename: W
authors.initials: W
authors.lastname: Hughes
authors.forename: A J
authors.initials: AJ
authors.lastname: Hoppe
authors.forename: P H
authors.initials: PH
authors.lastname: Pétra
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium; D019281:Dimerization; D006801:Humans; D011817:Rabbits; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000378:metabolism; D013256:Steroids / Q000378:metabolism*
chemical_list: D012738:Sex Hormone-Binding Globulin; D013256:Steroids; D002118:Calcium