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.
Petra
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PH
Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA. hue@u.washington.edu
Adman
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Orr
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Woodcock
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Groff
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Sui
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D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D020033:Protein Isoforms; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D020033:Protein Isoforms; D012738:Sex Hormone-Binding Globulin; D007532:Isoleucine; D004958:Estradiol; D001120:Arginine