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

• Published in: Protein science Vol. 10; no. 9; pp. 1811 - 1821
• Main Authors: Petra, Philip H., Adman, Elinor T., Orr, William R., Woodcock, Katherine T., Groff, Christine, Sui, Li‐Ming
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.09.2001
• Subjects: 17β‐estradiol; ABP, androgen binding protein; DHT, 5α‐dihydrotestosterone; dihydrotestosterone; E2, 17β‐estradiol; hSBP, human SBP; I101, isoleucine‐101; K134, lysine‐134; M107, methionine‐107; M107A, replacement of M107 by alanine at position 107; M107I, replacement of M107 by isoleucine at position 107; M107L, replacement of M107 by leucine at position 107; M107T, replacement of M107 by threonine at position 107; M139, methionine‐139; rSBP, rabbit SBP; SBP, plasma sex steroid‐binding protein; sex‐hormone‐binding globulin, SHBG; Sex‐steroid‐binding protein, SBP; SHBG, sex hormone binding globulin; steroid‐binding site; T, testosterone; testosterone; Y57, tyrosine‐57; Y57A, replacement of Y57 by alanine at position 57; Y57F, replacement of Y57 by phenylalanine at position 57; Y57G, replacement of Y57 by glycine at position 57; Y57L, replacement of Y57 by leucine at position 57; Y57S, replacement of Y57 by serine at position 57; Y57T, replacement of Y57 by threonine at position 57
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.02301

Arginine‐140 and isoleucine‐141 were identified as key determinants of 17β‐estradiol (E2) 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 E2binding activity as are seen in the rabbit protein. Only mutants containing either R140K or I141L replacements display an E2 equilibrium dissociation constant (Kd) higher than the wild type, reaching a value of 30 nM when both were present. The 5α‐dihydrotestosterone (DHT) equilibrium dissociation constant of these mutants was unaffected. The quadruple mutant M107I/I138V/R140K/I141L yielded an E2 Kd of 65 nM, significantly closer to the 80 nM rabbit SBP E2 Kd value. Although mutants containing the M107I and I138V replacements in the absence of R140K and I141L had normal E2 Kds, the presence of the M107I replacement in the quadruple mutant was necessary to obtain an accurate E2 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 E2 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 17β‐estradiol, thus optimizing the E2‐binding affinity of human SBP.