Crystal structure of human sex hormone‐binding globulin: steroid transport by a laminin G‐like domain

Human sex hormone‐binding globulin (SHBG) transports sex steroids in blood and regulates their access to target tissues. In biological fluids, SHBG exists as a homodimer and each monomer comprises two laminin G‐like domains (G domains). The crystal structure of the N‐terminal G domain of SHBG in complex with 5α‐dihydrotestosterone at 1.55 Å resolution reveals both the architecture of the steroid‐binding site and the quaternary structure of the dimer. We also show that G domains have jellyroll topology and are structurally related to pentraxin. In each SHBG monomer, the steroid intercalates into a hydrophobic pocket within the β‐sheet sandwich. The steroid and a 20 Å distant calcium ion are not located at the dimer interface. Instead, two separate steroid‐binding pockets and calcium‐binding sites exist per dimer. The structure displays intriguing disorder for loop segment Pro130–Arg135. In all other jellyroll proteins, this loop is well ordered. If modelled accordingly, it covers the steroid‐binding site and could thereby regulate access of ligands to the binding pocket.

[1]  J. Thornton,et al.  Depicting topology and handedness in jellyroll structures , 1992, FEBS letters.

[2]  R. Carrell,et al.  Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. , 1989, Biochemistry.

[3]  M. Parker.  Steroid hormone action , 1993 .

[4]  P. Pétra,et al.  Localization of the steroid‐binding site of the human sex steroid‐binding protein of plasma (SBP or SHBG) by site‐directed mutagenesis , 1992, FEBS letters.

[5]  P. A. Peterson,et al.  Studies on thyroid hormone-binding proteins. II. Binding of thyroid hormones, retinol-binding protein, and fluorescent probes to prealbumin and effects of thyroxine on prealbumin subunit self association. , 1975, The Journal of biological chemistry.

[6]  V S Lamzin,et al.  wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models. , 1997, Acta crystallographica. Section D, Biological crystallography.

[7]  J. Tesarik,et al.  Nongenomic actions of steroid hormones in reproductive tissues. , 1998, Endocrine reviews.

[8]  M. Hemler.  Dystroglycan Versatility , 1999, Cell.

[9]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[10]  T. Blundell,et al.  Structure of pentameric human serum amyloid P component , 1994, Nature.

[11]  Carolyn L. Smith,et al.  Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[12]  G. Hammond.  Determinants of steroid hormone bioavailability. , 1997, Biochemical Society transactions.

[13]  T. Gustafson,et al.  Complete amino acid sequence of human thyroxine-binding globulin deduced from cloned DNA: close homology to the serine antiproteases. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[14]  W. Rosner,et al.  The functions of corticosteroid-binding globulin and sex hormone-binding globulin: recent advances. , 1990, Endocrine reviews.

[15]  K. Titani,et al.  Amino acid sequence of the sex steroid binding protein of human blood plasma. , 1986, Biochemistry.

[16]  R. Timpl,et al.  The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin. , 1999, Molecular cell.

[17]  R. W. Kuhn,et al.  The serum transport of steroid hormones. , 1982, Recent progress in hormone research.

[18]  D. Carter,et al.  Atomic structure and chemistry of human serum albumin , 1992, Nature.

[19]  Peter J. Bryant,et al.  The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily , 1991, Cell.

[20]  D. C. Carter,et al.  Atomic structure and chemistry of human serum albumin , 1993, Nature.

[21]  J. Salzer,et al.  Neural Targeting of Mycobacterium leprae Mediated by the G Domain of the Laminin-α2 Chain , 1997, Cell.

[22]  T. A. Jones,et al.  Lipid-binding proteins: a family of fatty acid and retinoid transport proteins. , 1994, Advances in protein chemistry.

[23]  A. Skubitz,et al.  Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit , 1991, The Journal of cell biology.

[24]  W. Rosner,et al.  Estradiol causes the rapid accumulation of cAMP in human prostate. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[25]  G. Hammond,et al.  Sex hormone-binding globulin: gene organization and structure/function analyses. , 1996, Hormone research.

[26]  B. Oh,et al.  High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue. , 1997, Biochemistry.

[27]  P. Pétra,et al.  Observation and quantitation of metal-binding sites in the sex steroid-binding protein of human and rabbit sera using the luminescent probe terbium , 1985 .

[28]  J Engel,et al.  Structure and function of laminin: anatomy of a multidomain glycoprotein , 1990, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[29]  S. Jones,et al.  Principles of protein-protein interactions. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[30]  R. Frairia,et al.  MCF‐7 Cell Progesterone Receptor (PGR) Is Additionally Modulated by Sex Steroid Binding Protein (SBP) and Its Membrane Receptor (SBP‐R) through cAMP and PKA a , 1996, Annals of the New York Academy of Sciences.

[31]  P. Pétra,et al.  Direct evidence for the localization of the steroid‐binding site of the plasma sex steroid‐binding protein (SBP or SHBG) at the interface between the subunits , 1996, Protein science : a publication of the Protein Society.

[32]  S. Linse,et al.  Binding site for C4b-binding protein in vitamin K-dependent protein S fully contained in carboxy-terminal laminin-G-type repeats. A study using recombinant factor IX-protein S chimeras and surface plasmon resonance. , 1997, Biochemistry.

[33]  C. Cheng,et al.  There are two forms of androgen binding protein in human testes. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin. , 1985, The Journal of biological chemistry.

[34]  F. S. French,et al.  A high-affinity androgen-binding protein (ABP) in rat testis: evidence for secretion into efferent duct fluid and absorption by epididymis. , 1973, Endocrinology.

[35]  C. Blake,et al.  Protein–DNA and protein–hormone interactions in prealbumin: a model of the thyroid hormone nuclear receptor? , 1977, Nature.

[36]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[37]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[38]  A. D. de Vos,et al.  Hematopoietic receptor complexes. , 1996, Annual review of biochemistry.

[39]  P. Fernlund,et al.  SHBG region of the anticoagulant cofactor protein S: Secondary structure prediction, circular dichroism spectroscopy, and analysis of naturally occurring mutations , 1997, Proteins.

[40]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[41]  M Carlquist,et al.  Structure of the ligand‐binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist , 1999, The EMBO journal.

[42]  G. Hammond,et al.  Steroid-binding and dimerization domains of human sex hormone-binding globulin partially overlap: steroids and Ca2+ stabilize dimer formation. , 1994, Biochemistry.

[43]  S. Nilsson.  Studies on thyroid hormone-binding proteins , 1973 .

[44]  P. Bork,et al.  Merging extracellular domains: fold prediction for laminin G-like and amino-terminal thrombospondin-like modules based on homology to pentraxins. , 1998, Journal of molecular biology.

[45]  K. Walsh,et al.  Identification of lysine 134 in the steroid-binding site of the sex steroid-binding protein of human plasma. , 1990, The Journal of biological chemistry.

[46]  K. Umesono,et al.  The nuclear receptor superfamily: The second decade , 1995, Cell.

[47]  M. Mark,et al.  Characterization of Gas6, a Member of the Superfamily of G Domain-containing Proteins, as a Ligand for Rse and Axl (*) , 1996, The Journal of Biological Chemistry.

[48]  S. Goff,et al.  Tyro-3 family receptors are essential regulators of mammalian spermatogenesis , 1999, Nature.

[49]  David Eisenberg,et al.  Generalized method of determining heavy-atom positions using the difference Patterson function , 1987 .

[50]  D. R. Joseph,et al.  Structure-function relationships of rat androgen--binding protein/human sex-hormone binding globulin: the effect of mutagenesis on steroid-binding parameters. , 1994, Endocrinology.

[51]  J. Deisenhofer,et al.  The structure of the ligand-binding domain of neurexin Ibeta: regulation of LNS domain function by alternative splicing. , 1999, Cell.

[52]  Y. Muller,et al.  Crystallization of the N-terminal domain of human sex hormone-binding globulin, the major sex steroid carrier in blood. , 1999, Acta crystallographica. Section D, Biological crystallography.

[53]  K. D. Hardman,et al.  Structure of concanavalin A at 2.4-A resolution. , 1972, Biochemistry.

[54]  G. Hammond,et al.  Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli. , 1995, Biochemistry.

[55]  S. Carbonetto,et al.  Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin. , 1993, The Journal of biological chemistry.

[56]  D. R. Joseph,et al.  Structure, function, and regulation of androgen-binding protein/sex hormone-binding globulin. , 1994, Vitamins and hormones.

[57]  C. Mendel,et al.  The free hormone hypothesis. Distinction from the free hormone transport hypothesis. , 1992, Journal of andrology.

[58]  J. Schlessinger,et al.  Regulation of signal transduction and signal diversity by receptor oligomerization. , 1994, Trends in biochemical sciences.

[59]  D. F. Senear,et al.  Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins. , 1986, Journal of steroid biochemistry.

[60]  E. Baulieu,et al.  Hormonal and immunological aspects of the phylogeny of sex steroid binding plasma protein. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[61]  G. Hammond,et al.  Sex hormone-binding globulin/androgen-binding protein: Steroid-binding and dimerization domains , 1995, The Journal of Steroid Biochemistry and Molecular Biology.

[62]  Wolfgang Kabsch,et al.  Evaluation of Single-Crystal X-ray Diffraction Data from a Position-Sensitive Detector , 1988 .

[63]  A. Wilson,et al.  The probability distribution of X-ray intensities , 1949 .

[64]  Karl D. Hardman,et al.  Structure of concanavalin A at 2.4-Ang resolution , 1972 .

[65]  U. Westphal.  Steroid-Protein Interactions II , 1986, Monographs on Endocrinology.

[66]  J. Deisenhofer,et al.  Regulation of LNS Domain Function by Alternative Splicing: The Structure of the Ligand-Binding Domain of Neurexin Iβ , 1999, Cell.

[67]  G. Hammond,et al.  Structure/function analyses of human sex hormone‐binding globulin by site‐directed mutagenesis , 1992, FEBS letters.

[68]  P B Sigler,et al.  Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[69]  M. S. Khan,et al.  Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin. , 1990, The Journal of biological chemistry.

[70]  T. Blachère,et al.  Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents. , 1992, Biochemistry.

[71]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[72]  M. Baker,et al.  Sex hormone‐binding globulin, androgen‐binding protein, and vitamin K‐dependent protein S are homologous to laminin A, merosin, and Drosophila crumbs protein , 1992, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[73]  C. Porto,et al.  Receptors for androgen-binding proteins: Internalization and intracellular signalling , 1995, The Journal of Steroid Biochemistry and Molecular Biology.

[74]  D. R. Joseph.  Sequence and functional relationships between androgen-binding protein/sex hormone-binding globulin and its homologs protein S, Gas6, laminin, and agrin , 1997, Steroids.

[75]  C. Chambon,et al.  Photoaffinity labeling of homologous Met-133 and Met-139 amino acids of rabbit and sheep sex hormone-binding globulins with the unsubstituted Delta 6-testosterone photoreagent. , 1998, Biochemistry.

[76]  T. Gruber,et al.  Secondary structure and shape of plasma sex steroid-binding protein--comparison with domain G of laminin results in a structural model of plasma sex steroid-binding protein. , 1997, European journal of biochemistry.

[77]  Ulrich Westphal,et al.  Steroid-Protein Interactions , 1971, Monographs on Endocrinology.