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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hildebrand
C
C
MRC Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Canada.
Bocchinfuso
W P
WP
Dales
D
D
Hammond
G L
GL
D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007120:Immunochemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D012738:Sex Hormone-Binding Globulin; D013256:Steroids