Revised Manuscript Received June 14, 1994® abstract: Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with a single steroid-binding site for biologically active sex steroids, and a methionine at position 139 (Ml39) interacts with the photoaffinity ligand, A6-testosterone. We have introduced amino acid substitutions into this and other locations in the SHBG molecule and have examined their impact on steroid binding and dimerization. As a result, substitutions at residues 134-139 generate alterations in steroid-binding specificity. In particular, substitutions at residues 134-138 were characterized by altered binding affinities for estradiol relative to 5a-dihydrotestosterone (DHT), and one of them (R135L) also showed a 2-fold increase in affinity for C19 steroids with a 3/3-hydroxy group. Unlike all of the other mutants we have examined, the M139W mutant has a 5-fold lower affinity for DHT, and itsaffinities for testosterone, 5a-androstane-3/3, 17/?-diol, and estradiol also appear to be reduced to a similar extent. By contrast, M139W appears to bind androst-5-ene-3/3, 17/3-diol with only 2-fold less affinity than wild-type SHBG, while its affinity for 19-nortestosterone remains unaffected. Substitutions at other positions, including those immediately C-terminal to Ml39, had no effect on steroid-binding affinity and/or specificity. These data provide evidence that residues 134-139 influence the recognition of specific A/B ring conformations of steroid ligands and may constitute part of the steroid-binding domain. We have also foundthat substitutions at residues 138-148impair dimerization and that this defect may be abrogated by occupancy of the steroid-binding site. The removal of divalent cations also destabilizes dimer formation of mutants with substitutions at residues 140-148, and Ca2+ or Zn2+, but not Mg2+, can restore their ability to form dimers. Steroid ligands and divalent cations also appear to act independently to stabilize dimer formation. These data lead us to conclude that the steroid-binding and dimerization domains of human SHBG partially overlap and that steroid ligands and divalent cations influence the dimerization interface to enhance subunit association.

Plasma sex hormone-binding globulin (SHBG1) and testicular androgen-binding protein (ABP) are differentially glycosylated products of a gene located on the short arm of human chromosome 17 (Hammond, 1993). They function as homodimers with a single binding site for androgens and 17/3-estradiol (Hammond et al., 1986; Danzoetal., 1989), and the requirements for ligand binding to SHBG include a planar steroid molecule with an electronegative moiety at C3 and a/3-hydroxyl group at C17 (Cunningham etal., 1981). Details about the orientation of ligands in the binding site and its topography are limited, but studies involving affinity labeling (Grenot et al., 1992) and site-directed mutagenesis (Bocchinfuso et al., 1992; Sui et al., 1992) have indicated that a methionine at position 139 (Ml39) is probably important for interaction with the steroid B ring. In addition, the steroidbinding characteristics of a human SHBG/rat ABP chimera suggest that residues important for high-affinity steroid binding are situated within the N-terminal 205 amino acids of human SHBG (Bocchinfuso et al., 1992).