Arginine-140 and isoleucine-141 determine the 17beta-estradiol-binding specificity of the sex-steroid-binding protein (SBP, or SHBG) of human plasma.

Arginine-140 and isoleucine-141 were identified as key determinants of 17beta-estradiol (E(2)) binding affinity of the sex-steroid-binding protein (SBP, or SHBG) of human plasma. Amino acid residues that differ between human and rabbit SBP sequences were replaced in the human protein and the products tested for lowered E(2)binding activity as are seen in the rabbit protein. Only mutants containing either R140K or I141L replacements display an E(2) equilibrium dissociation constant (Kd) higher than the wild type, reaching a value of 30 nM when both were present. The 5alpha-dihydrotestosterone (DHT) equilibrium dissociation constant of these mutants was unaffected. The quadruple mutant M107I/I138V/R140K/I141L yielded an E(2) Kd of 65 nM, significantly closer to the 80 nM rabbit SBP E(2) Kd value. Although mutants containing the M107I and I138V replacements in the absence of R140K and I141L had normal E(2) Kds, the presence of the M107I replacement in the quadruple mutant was necessary to obtain an accurate E(2) Kd value by competitive Scatchard analysis. Molecular modeling using coordinates for the recently determined N-terminal domain of human SBP revealed a significant shift of the F56 phenyl ring away from ring A of E(2) in mutant models containing the R140K and I141L replacements. We conclude that R140 and I141 are required for sustaining the right proximity of the phenyl ring of F56 to ring A of 17beta-estradiol, thus optimizing the E(2)-binding affinity of human SBP.

The sex steroid binding protein (SBP or SHBG) of human plasma: identification of Tyr-57 and Met-107 in the steroid binding site.

Tyrosine-57 (Y57) and methionine-107 (M107) have been identified in the binding site of the sex steroid binding protein (SBP) (or sex hormone binding globulin) of human plasma by replacing the two amino acids with a number of residues of varying structure. Replacement of Y57 with phenylalanine resulted in a fourfold increase in the K(d) of 5 alpha-dihydrotestosterone but left the K(d) of 17 beta-estradiol unchanged. Except in two cases, no further loss in binding took place when replacing Y57 with other residues, suggesting that the phenolic group of Y57 may form a hydrogen bond with the ligand. Replacement of M107 with isoleucine increased the 5 alpha-dihydrotestosterone K(d) fourfold to a value equal to that of rabbit SBP, which contains isoleucine at the corresponding position; however, the K(d) of 17 beta-estradiol remained unchanged. Replacement of M107 with threonine resulted in a tenfold decrease in 5 alpha-dihydrotestosterone binding affinity, whereas replacement with leucine left the K(d) unchanged. These data indicate that substitutions on the beta-carbon of the amino acid side-chain at position 107 causes significant loss of binding affinity but, as in the case of Y57, the activity was not totally eliminated. We conclude that Y57 and M107 form part of a structural motif within the steroid binding site and specifically contribute binding energy to ring A of 5 alpha-dihydrotestosterone but not to ring A of 17 beta-estradiol. We also propose that the integrated contribution of several side chains may be required to optimize the ligand affinity of the steroid binding site. This proposal may fit a 'lock and key' model where little movement of the side chains occurs during binding as might be expected for a rigid structure like the steroid nucleus.