Resolution of the human sex hormone-binding globulin dimer interface and evidence for two steroid-binding sites per homodimer

Human sex hormone-binding globulin (SHBG) transports sex steroids in the bl ood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its steroid binding stoichiomet ry is controversial. The prevailing assumption is that each homodimeric SHB G molecule contains a single steroid-binding site at the dimer interface. H owever, crystallographic analysis of the aminoterminal Iaminin G-like domai n of human SHBG has shown that the dimerization and steroid-binding sites a re distinct and that both monomers within a homodimeric complex are capable of binding steroid. To validate our crystallographic model of the SHBG hom odimer, we have used site-directed mutagenesis to create SHBG variants in w hich single amino acid substitutions (V89E and L122E) were introduced to pr oduce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a stero id-binding site with an affinity and specificity indistinguishable from wil dtype SHBG. Moreover, when equalized in terms of their monomeric subunit co ntent, dimerization-deficient and wild-type SHBGs have essentially identica l steroid binding capacities. These data indicate that both subunits of the SHBG homodimer bind steroid and that measurements of the molar concentrati on of SHBG homodimer in serum samples have been overestimated by 2-fold.