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Geoffrey L Hammond - One of the best experts on this subject based on the ideXlab platform.
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Identification of Avian CorticoSteroid-Binding Globulin (SerpinA6) Reveals the Molecular Basis of Evolutionary Adaptations in SerpinA6 Structure and Function as a Steroid-Binding Protein.
Journal of Biological Chemistry, 2016Co-Authors: Ganna Vashchenko, Samir Das, Kyung-mee Moon, Jason C. Rogalski, Matthew D. Taves, Kiran K. Soma, Filip Van Petegem, Leonard J. Foster, Geoffrey L HammondAbstract:CorticoSteroid-Binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG Steroid-Binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only ∼40% identity, and their Steroid-Binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG Steroid-Binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its Steroid-Binding affinity, but we present evidence that it may participate in protein folding and Steroid-Binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct Steroid-Binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts Steroid Binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex Steroids in these species.
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Residues in the Human CorticoSteroid-Binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage
Journal of Biological Chemistry, 2008Co-Authors: Hai-yan Lin, Yves A. Muller, Caroline Underhill, Bernd R. Gardill, Geoffrey L HammondAbstract:Abstract CorticoSteroid-Binding globulin (CBG) is a non-inhibitory serine proteinase inhibitor (serpin) that transports cortisol and progesterone in blood. Crystal structures of rat CBG and a thrombin-cleaved human CBG:anti-trypsin (Pittsburgh) chimera show how structural transitions after proteolytic cleavage of the CBG reactive center loop (RCL) could disrupt Steroid Binding. This ligand release mechanism is assumed to involve insertion of the cleaved RCL into the β-sheet A of the serpin structure. We have, therefore, examined how amino acid substitutions in the human CBG RCL influence Steroid Binding before and after its cleavage by neutrophil elastase. Elastase-cleaved wild-type CBG or variants with substitutions at P15 and/or P16 (E334G/G335N or E334A) lost Steroid Binding completely, whereas deletion of Glu-334 resulted in no loss of Steroid Binding after RCL cleavage, presumably because this prevents its insertion into β-sheet A. Similarly, the Steroid Binding properties of CBG variants with substitutions at P15 (G335P), P14 (V336R), or P12 (T338P) in the RCL hinge were largely unaffected after elastase cleavage, most likely because the re-orientation and/or insertion of the cleaved RCL was blocked. Substitutions at P10 (G340P, G340S) or P8 (T342P, T342N) resulted in a partial loss of Steroid Binding after proteolysis which we attribute to incomplete insertion of the cleaved RCL. Remarkably, several substitutions (E334A, V336R, G340S, and T342P) increased the Steroid Binding affinities of human CBG even before elastase cleavage, consistent with the concept that CBG normally toggles between a high affinity ligand Binding state where the RCL is fully exposed and a lower affinity state in which the RCL is partly inserted into β-sheet A.
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Resolution of a Disordered Region at the Entrance of the Human Sex Hormone-Binding Globulin Steroid-Binding Site
Journal of Molecular Biology, 2002Co-Authors: Irina Grishkovskaya, George V. Avvakumov, Geoffrey L Hammond, Yves A. MullerAbstract:Abstract The crystal structure of human sex hormone-Binding globulin (SHBG) has revealed how 5α-dihydrotestosterone intercalates between the two seven-stranded β-sheets of its amino-terminal laminin G-like domain. However, a region of disorder (residues 130 to 135 of SHBG) was identified together with a zinc-Binding site in immediate proximity to the Steroid. It has been important to resolve the structure of this region because previous studies have suggested that these residues may contribute to Steroid Binding directly. Here, we present the 2.35 A and 1.7 A crystal structures of the amino-terminal LG domain of SHBG obtained from a tetragonal crystal form and by EDTA-soaking of a trigonal crystal form, respectively. In both of these new structures, residues Pro130 to Arg135 are now clearly visible. Substitution of the two residues (Leu131Gly and Lys134Ala) pointing towards the Steroid has shown that only Leu131 contributes significantly to Steroid Binding. Rather than covering the Steroid-Binding pocket in an extended conformation, a 3 10 helical turn is formed by residues Leu131 to Lys134 in this segment. Unfolding of this secondary structure element can either facilitate the entry of the Steroids into the Binding site or modulate the important contribution that Leu131 makes to Steroid Binding. A comparison with previous structures supports the concept that zinc Binding re-orients the side-chain of His136, and this residue serves as a lever causing disorder within the loop structure between Pro130 and Arg135.
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Resolution of the Human Sex Hormone-Binding Globulin Dimer Interface and Evidence for Two Steroid-Binding Sites per Homodimer
Journal of Biological Chemistry, 2001Co-Authors: George V. Avvakumov, Irina Grishkovskaya, Yves A. Muller, Geoffrey L HammondAbstract:Human sex hormone-Binding globulin (SHBG) transports sex Steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its Steroid Binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single Steroid-Binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and Steroid-Binding sites are distinct and that both monomers within a homodimeric complex are capable of Binding Steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a Steroid-Binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical Steroid Binding capacities. These data indicate that both subunits of the SHBG homodimer bind Steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold.
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potential functions of plasma Steroid Binding proteins
Trends in Endocrinology and Metabolism, 1995Co-Authors: Geoffrey L HammondAbstract:The plasma Steroid-Binding proteins, sex hormone-Binding globulin (SHBG) and corticoSteroid-Binding globulin (CBG), transport Steroid hormones in the blood and regulate their access to target tissues. Recent biochemical and molecular analyses of these proteins and their genes, and studies of their biosynthesis and localization in the liver and other tissues during development, have led to the realization that CBG and SHBG function in much more sophisticated ways. In particular, the presence of plasma membrane Binding sites for both CBG and SHBG on Steroid target cells, and evidence for interactions between CBG and specific proteinases at sites of inflammation or tissue remodeling, suggest that these proteins control Steroid hormone bioavailability and/or action in a highly selective or targeted fashion. This new information should not only serve to extend our understanding of the basis of Steroid-hormone dependent diseases, but may influence the design of Steroid hormone agonists and antagonist of therapeutic potential.
George V. Avvakumov - One of the best experts on this subject based on the ideXlab platform.
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Resolution of a Disordered Region at the Entrance of the Human Sex Hormone-Binding Globulin Steroid-Binding Site
Journal of Molecular Biology, 2002Co-Authors: Irina Grishkovskaya, George V. Avvakumov, Geoffrey L Hammond, Yves A. MullerAbstract:Abstract The crystal structure of human sex hormone-Binding globulin (SHBG) has revealed how 5α-dihydrotestosterone intercalates between the two seven-stranded β-sheets of its amino-terminal laminin G-like domain. However, a region of disorder (residues 130 to 135 of SHBG) was identified together with a zinc-Binding site in immediate proximity to the Steroid. It has been important to resolve the structure of this region because previous studies have suggested that these residues may contribute to Steroid Binding directly. Here, we present the 2.35 A and 1.7 A crystal structures of the amino-terminal LG domain of SHBG obtained from a tetragonal crystal form and by EDTA-soaking of a trigonal crystal form, respectively. In both of these new structures, residues Pro130 to Arg135 are now clearly visible. Substitution of the two residues (Leu131Gly and Lys134Ala) pointing towards the Steroid has shown that only Leu131 contributes significantly to Steroid Binding. Rather than covering the Steroid-Binding pocket in an extended conformation, a 3 10 helical turn is formed by residues Leu131 to Lys134 in this segment. Unfolding of this secondary structure element can either facilitate the entry of the Steroids into the Binding site or modulate the important contribution that Leu131 makes to Steroid Binding. A comparison with previous structures supports the concept that zinc Binding re-orients the side-chain of His136, and this residue serves as a lever causing disorder within the loop structure between Pro130 and Arg135.
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Resolution of the Human Sex Hormone-Binding Globulin Dimer Interface and Evidence for Two Steroid-Binding Sites per Homodimer
Journal of Biological Chemistry, 2001Co-Authors: George V. Avvakumov, Irina Grishkovskaya, Yves A. Muller, Geoffrey L HammondAbstract:Human sex hormone-Binding globulin (SHBG) transports sex Steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its Steroid Binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single Steroid-Binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and Steroid-Binding sites are distinct and that both monomers within a homodimeric complex are capable of Binding Steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a Steroid-Binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical Steroid Binding capacities. These data indicate that both subunits of the SHBG homodimer bind Steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold.
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Glycosylation of human corticoSteroid-Binding globulin at aspargine 238 is necessary for Steroid Binding.
The Journal of biological chemistry, 1993Co-Authors: George V. Avvakumov, S Warmels-rodenhiser, Geoffrey L HammondAbstract:Abstract Human corticoSteroid Binding-globulin (CBG) is a plasma glycoprotein that binds and regulates the biological activity of glucocorticoids and progesterone. Carbohydrates comprise approximately 25% of its molecular mass being represented by bi- and triantennary N-linked oligosaccharides of the N-acetyllactosamine type. To assess the impact of these carbohydrate chains on CBG production and Steroid Binding, we mutated a human CBG cDNA so that the six consensus sites for N-glycosylation in the CBG polypeptide were eliminated individually and in various combinations. Expression of the mutant cDNAs in Chinese hamster ovary cells showed that all consensus sites may be utilized during the CBG biosynthesis and that the immunochemical properties of the recombinant glycoproteins are similar to those of CBG isolated from human serum. Removal of sugar chains generally led to a reduction in the secretion of recombinant CBG, but complete removal of N-glycosylation sites did not prevent production or secretion of the protein. Our data indicate that an oligosaccharide linked to Asn238 is essential for Steroid Binding, and we suggest that an interaction between this sugar chain and the polypeptide may be essential for the creation of a high affinity Steroid-Binding site. In addition, concanavalin A chromatography of mutants containing only one N-glycosylation site at either Asn74 or Asn238 indicated that processing of the oligosaccharides at these positions is site-specific.
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Specific Steroid-Binding glycoproteins of human blood plasma: novel data on their structure and function.
Journal of Steroid Biochemistry, 1990Co-Authors: Oleg A. Strel'chyonok, George V. AvvakumovAbstract:Abstract In this review, the modern data on the polypeptide and carbohydrate structures of human corticoSteroid-Binding globulin (CBG) and sex hormone-Binding globulin (SHBG) as well as on the biochemical properties and biological functions of these Steroid-Binding glycoproteins are discussed.
Philip H. Petra - One of the best experts on this subject based on the ideXlab platform.
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The sex Steroid Binding protein (SBP or SHBG) of human plasma: identification of Tyr-57 and Met-107 in the Steroid Binding site.
The Journal of Steroid Biochemistry and Molecular Biology, 2000Co-Authors: Philip H. Petra, Katherine T. Woodcock, William R. Orr, Dat Nguyen, Li-ming SuiAbstract:Abstract 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 5α-dihydrotestosterone but left the K d of 17β-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 5α-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 17β-estradiol remained unchanged. Replacement of M107 with threonine resulted in a tenfold decrease in 5α-dihydrotestosterone Binding affinity, whereas replacement with leucine left the K d unchanged. These data indicate that substitutions on the β-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 5α-dihydrotestosterone but not to ring A of 17β-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.
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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
European Journal of Biochemistry, 1997Co-Authors: Konrad Beck, Li-ming Sui, Tanja M. Gruber, Catherine C. Ridgway, William Hughes, Philip H. PetraAbstract:We have analyzed the secondary structure, shape and dimensions of plasma sex Steroid-Binding protein (SBP) by CD, size-exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for beta-sheet structures. Analysis with different algorithms indicates 15% alpha-helix, 43% beta-sheet and 10-16% beta-turn structures. An irreversible structural change is observed upon heating above 60 degrees C, which correlates with the loss of Steroid-Binding activity. As the SBP sequence shows similarity with domains of several multidomain proteins, including laminins, we evaluated the structure of domain G of laminin-1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolution results in 13% alpha-helix, 32% beta-sheet and 15% beta-turn structures. Steroid-Binding assays indicate that laminin and fragments thereof have no activity. Size-exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the Steroid-Binding site located in the center of the rod-like particle.
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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.
Protein Science, 1996Co-Authors: Li-ming Sui, William Hughes, Agnes J. Hoppe, Philip H. PetraAbstract:Complete dissociation of dimeric plasma sex Steroid-Binding protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca+2 or Steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric Steroid-Binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for Binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds Steroid can be considered meaningless; Steroid-Binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.
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Localization of the Steroid-Binding site of the human sex Steroid-Binding protein of plasma (SBP or SHBG) by site-directed mutagenesis
FEBS Letters, 1992Co-Authors: Li-ming Sui, Audrey W.c. Cheung, Pearl C. Namkung, Philip H. PetraAbstract:The amino-terminal region of the human sex Steroid-Binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the Steroid-Binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M 139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in Steroid Binding. The fully active H235S mutant reveals that H235 is not involved in the Steroid-Binding process.
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Complete enzymatic deglycosylation of native sex Steroid-Binding protein (SBP or SHBG) of human and rabbit plasma: effect on the Steroid-Binding activity.
Protein Science, 1992Co-Authors: Philip H. Petra, Wei Zhang, Patrick R. Griffin, John R. Yates, Katherine H. MooreAbstract:An enzymatic procedure for the complete removal of the N-linked and O-linked oligosaccharide side chains of the sex Steroid-Binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N-glycanase, neuraminidase, and O-glycanase and was monitored by SDS-PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with N-glycanase generated a major 39,777-Da protein and two minor ones of 39,389 and 39,545 Da. The molecular weight of the major protein agrees with the molecular weight calculated from the sequence of the sugar-free polypeptide monomer (39,769 Da: Griffin, P.R., Kumar, S., Shabanowitz, J., Charbonneau, H., Namkung, P.C., Walsh, K.A., Hunt, D.F., & Petra, P.H., 1989, J. Biol. Chem. 264, 19066-19075), whereas the other two are deglycosylated proteolytic cleavage products lacking the TQR and TQ sequences at the amino-terminus. The N- and O-linked side chains of human SBP were removed by sequential digestion with N-glycanase and neuraminidase/O-glycanase. A 38,771-Da protein was generated, which agrees well with the molecular weight of the sugar-free polypeptide monomer (Walsh, K.A., Titani, K., Kumar, S., Hayes, R., & Petra, P.H., 1986, Biochemistry 25, 7584-7590). N-deglycosylation of human and rabbit SBP has no effect on the Steroid-Binding activity, but removal of the O-linked side chains of N-deglycosylated human SBP results in an apparent 50% loss of Steroid-Binding activity and an increase in the Kd for the Binding of 5 alpha-dihydrotestosterone from 0.3 mM to 0.9 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
Yves A. Muller - One of the best experts on this subject based on the ideXlab platform.
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Residues in the Human CorticoSteroid-Binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage
Journal of Biological Chemistry, 2008Co-Authors: Hai-yan Lin, Yves A. Muller, Caroline Underhill, Bernd R. Gardill, Geoffrey L HammondAbstract:Abstract CorticoSteroid-Binding globulin (CBG) is a non-inhibitory serine proteinase inhibitor (serpin) that transports cortisol and progesterone in blood. Crystal structures of rat CBG and a thrombin-cleaved human CBG:anti-trypsin (Pittsburgh) chimera show how structural transitions after proteolytic cleavage of the CBG reactive center loop (RCL) could disrupt Steroid Binding. This ligand release mechanism is assumed to involve insertion of the cleaved RCL into the β-sheet A of the serpin structure. We have, therefore, examined how amino acid substitutions in the human CBG RCL influence Steroid Binding before and after its cleavage by neutrophil elastase. Elastase-cleaved wild-type CBG or variants with substitutions at P15 and/or P16 (E334G/G335N or E334A) lost Steroid Binding completely, whereas deletion of Glu-334 resulted in no loss of Steroid Binding after RCL cleavage, presumably because this prevents its insertion into β-sheet A. Similarly, the Steroid Binding properties of CBG variants with substitutions at P15 (G335P), P14 (V336R), or P12 (T338P) in the RCL hinge were largely unaffected after elastase cleavage, most likely because the re-orientation and/or insertion of the cleaved RCL was blocked. Substitutions at P10 (G340P, G340S) or P8 (T342P, T342N) resulted in a partial loss of Steroid Binding after proteolysis which we attribute to incomplete insertion of the cleaved RCL. Remarkably, several substitutions (E334A, V336R, G340S, and T342P) increased the Steroid Binding affinities of human CBG even before elastase cleavage, consistent with the concept that CBG normally toggles between a high affinity ligand Binding state where the RCL is fully exposed and a lower affinity state in which the RCL is partly inserted into β-sheet A.
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Resolution of a Disordered Region at the Entrance of the Human Sex Hormone-Binding Globulin Steroid-Binding Site
Journal of Molecular Biology, 2002Co-Authors: Irina Grishkovskaya, George V. Avvakumov, Geoffrey L Hammond, Yves A. MullerAbstract:Abstract The crystal structure of human sex hormone-Binding globulin (SHBG) has revealed how 5α-dihydrotestosterone intercalates between the two seven-stranded β-sheets of its amino-terminal laminin G-like domain. However, a region of disorder (residues 130 to 135 of SHBG) was identified together with a zinc-Binding site in immediate proximity to the Steroid. It has been important to resolve the structure of this region because previous studies have suggested that these residues may contribute to Steroid Binding directly. Here, we present the 2.35 A and 1.7 A crystal structures of the amino-terminal LG domain of SHBG obtained from a tetragonal crystal form and by EDTA-soaking of a trigonal crystal form, respectively. In both of these new structures, residues Pro130 to Arg135 are now clearly visible. Substitution of the two residues (Leu131Gly and Lys134Ala) pointing towards the Steroid has shown that only Leu131 contributes significantly to Steroid Binding. Rather than covering the Steroid-Binding pocket in an extended conformation, a 3 10 helical turn is formed by residues Leu131 to Lys134 in this segment. Unfolding of this secondary structure element can either facilitate the entry of the Steroids into the Binding site or modulate the important contribution that Leu131 makes to Steroid Binding. A comparison with previous structures supports the concept that zinc Binding re-orients the side-chain of His136, and this residue serves as a lever causing disorder within the loop structure between Pro130 and Arg135.
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Resolution of the Human Sex Hormone-Binding Globulin Dimer Interface and Evidence for Two Steroid-Binding Sites per Homodimer
Journal of Biological Chemistry, 2001Co-Authors: George V. Avvakumov, Irina Grishkovskaya, Yves A. Muller, Geoffrey L HammondAbstract:Human sex hormone-Binding globulin (SHBG) transports sex Steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its Steroid Binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single Steroid-Binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and Steroid-Binding sites are distinct and that both monomers within a homodimeric complex are capable of Binding Steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a Steroid-Binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical Steroid Binding capacities. These data indicate that both subunits of the SHBG homodimer bind Steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold.
Li-ming Sui - One of the best experts on this subject based on the ideXlab platform.
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The sex Steroid Binding protein (SBP or SHBG) of human plasma: identification of Tyr-57 and Met-107 in the Steroid Binding site.
The Journal of Steroid Biochemistry and Molecular Biology, 2000Co-Authors: Philip H. Petra, Katherine T. Woodcock, William R. Orr, Dat Nguyen, Li-ming SuiAbstract:Abstract 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 5α-dihydrotestosterone but left the K d of 17β-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 5α-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 17β-estradiol remained unchanged. Replacement of M107 with threonine resulted in a tenfold decrease in 5α-dihydrotestosterone Binding affinity, whereas replacement with leucine left the K d unchanged. These data indicate that substitutions on the β-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 5α-dihydrotestosterone but not to ring A of 17β-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.
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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
European Journal of Biochemistry, 1997Co-Authors: Konrad Beck, Li-ming Sui, Tanja M. Gruber, Catherine C. Ridgway, William Hughes, Philip H. PetraAbstract:We have analyzed the secondary structure, shape and dimensions of plasma sex Steroid-Binding protein (SBP) by CD, size-exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for beta-sheet structures. Analysis with different algorithms indicates 15% alpha-helix, 43% beta-sheet and 10-16% beta-turn structures. An irreversible structural change is observed upon heating above 60 degrees C, which correlates with the loss of Steroid-Binding activity. As the SBP sequence shows similarity with domains of several multidomain proteins, including laminins, we evaluated the structure of domain G of laminin-1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolution results in 13% alpha-helix, 32% beta-sheet and 15% beta-turn structures. Steroid-Binding assays indicate that laminin and fragments thereof have no activity. Size-exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the Steroid-Binding site located in the center of the rod-like particle.
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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.
Protein Science, 1996Co-Authors: Li-ming Sui, William Hughes, Agnes J. Hoppe, Philip H. PetraAbstract:Complete dissociation of dimeric plasma sex Steroid-Binding protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca+2 or Steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric Steroid-Binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for Binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds Steroid can be considered meaningless; Steroid-Binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.
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Localization of the Steroid-Binding site of the human sex Steroid-Binding protein of plasma (SBP or SHBG) by site-directed mutagenesis
FEBS Letters, 1992Co-Authors: Li-ming Sui, Audrey W.c. Cheung, Pearl C. Namkung, Philip H. PetraAbstract:The amino-terminal region of the human sex Steroid-Binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the Steroid-Binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M 139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in Steroid Binding. The fully active H235S mutant reveals that H235 is not involved in the Steroid-Binding process.