RESOLUTION OF THE STEROID-BINDING AND DIMERIZATION DOMAINS OF HUMAN SEX HORMONE-BINDING GLOBULIN BY EXPRESSION IN ESCHERICHIA-COLI

To determine the minimal sequence requirements for steroid binding and dimerization of human sex hormone-binding globulin (SHBG), the SHBG polypeptide and various SHBG deletion mutants were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli: Fusion proteins containing the complete SHBG sequence, or the first 177 N-terminal residues of SHBG, bound steroids with high affinity and specificity. Further deletions from the C-terminus severely compromised steroid-binding activity, as did N-terminal deletions beyond residue 18 in the SHBG sequence. Thus, residues 18-177 in SHBG encompass a region required for its steroid-binding activity, and a disulfide bridge normally present between Cys-164 and Cys-188 in SHBG is not obviously essential for steroid binding. Most of the GST/SHBG fusion proteins undergo cleavage at 4 degrees C, releasing immunoreactive polypeptides that correspond approximately in size to their respective SHBG sequences. The 23-kDa immunoreactive cleavage product released from the fusion protein containing residues 1-205 in the SHBG sequence (SHBG 1-205) has a 50-fold greater steroid binding capacity but a 7.5-fold lower affinity than its parent fusion protein. In addition, the 22-kDa immunoreactive polypeptide released from SHBG(1-194) binds steroid, and its dimerization is promoted by steroid ligands that bind SHBG with high affinity. These data suggest that the N-terminal region of SHBG dimerizes readily in the absence of GST and in doing so acquires steroid-binding sites. In conclusion, the N-terminal half of SHBG contains all the essential elements for steroid binding and dimerization, and we suggest that the C-terminal region may stabilize the overall conformation of SHBG to enhance steroid-binding affinity and/or provide some other function.