A Leu → His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol

Smith, Carolyn L.; Power, Stephanie; Hammond, Geoffrey L.

doi:10.1016/0960-0760(92)90107-t

Cited by 38 publications

(21 citation statements)

References 16 publications

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“…Moreover, like Met 276 , Lys 200 is distant from steroid-binding site or the site where the RCL insertion likely occurs upon proteolytic cleavage, and substitutions at these positions probably affect steroid binding only indirectly by altering the general stability of the protein. By contrast, the common S224A human CBG variant (Ser 116 in rat CBG) binds steroid normally (8), and this is understandable because its side chain points away from the protein surface and is not close to the steroid-binding pocket or the RCL insertion site.…”

Section: Resultsmentioning

confidence: 98%

Corticosteroid-binding Globulin, a Structural Basis for Steroid Transport and Proteinase-triggered Release

Klieber

Underhill

Hammond

et al. 2007

Journal of Biological Chemistry

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114

121

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Corticosteroid-binding globulin (CBG) is a serine proteinase inhibitor (serpin) family member that transports glucocorticoids in blood and regulates their access to target cells. The 1.9 Å crystal structure of rat CBG shows that its steroid-binding site resembles the thyroxin-binding site in the related serpin, thyroxin-binding globulin, and mutagenesis studies have confirmed the contributions of key residues that constitute the steroid-binding pocket. Unlike thyroxin-bound thyroxin-binding globulin, the cortisol-bound CBG displays an "active" serpin conformation with the proteinase-sensitive, reactive center loop (RCL) fully expelled from the regulatory ␤-sheet A. Moreover, the CBG structure allows us to predict that complete insertion of the proteolytically cleaved RCL into the serpin fold occurs in concert with a displacement and unwinding of helix D that would disrupt the steroid-binding site. This allosteric coupling between RCL positioning and occupancy of the CBG steroidbinding site, which resembles the ligand (glycosamino-glycan)-dependent activation of the thrombin inhibitory serpins heparin cofactor II and anti-thrombin RCLs, ensures both optimal recognition of CBG by target proteinases and efficient release of steroid to sites of action.Glucocorticoid hormones (cortisol and corticosterone) and progesterone are transported in the blood by a glycoprotein known as corticosteroid-binding globulin (CBG) 3 or transcortin (1). Plasma CBG also binds several synthetic glucocorticoids, such as prednisolone (2), and influences the half-life, distribution, and efficacy of these drugs in the same way as for natural steroids (3). The steroid binding characteristics of CBG from many species have been documented (1), and a benchmark set of CBG steroid binding characteristics has be used to develop three-dimensional quantitative structure activity relationship (3D-QSAR) computational methods (4, 5) aimed at predicting the binding affinities of ligands to target proteins. Although residues within CBG sequences critical for steroid binding have been identified through studies of naturally occurring variants (6 -10), photo-affinity labeling (11), and mutagenesis (12), a precise picture of its structure and steroidbinding site has been lacking.The primary structure of CBG defines it as a clade A member of the serine proteinase inhibitor (serpin) superfamily, together with the related transport protein for thyroxin, thyroxin-binding globulin (TBG), and the prototypical serpin members ␣1-antitrypsin (AAT) and ␣1-antichymotrypsin (ACT), among others (13,14). A hallmark of serpin structures is that they undergo conformational rearrangements as part of their biological function. The conformations they adopt are highly dependent on whether a surface-exposed loop, known as the reactive center loop (RCL) or "proteinase bait" domain, is intact. Cleavage of the RCL segment by proteinases usually causes a typical stressed to relaxed (S 3 R) transition in structure (15). This involves insertion of the entire N-terminal segment of t...

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Section: Resultsmentioning

confidence: 98%

Corticosteroid-binding Globulin, a Structural Basis for Steroid Transport and Proteinase-triggered Release

Klieber

Underhill

Hammond

et al. 2007

Journal of Biological Chemistry

Self Cite

114

121

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“…However, this effect was significant in two of three transfection assays. Genetic variants of CBG have been reported in rat (Smith and Hammond 1991), in mouse (Orava et al 1994), and in humans (Van Baelen et al 1982;Smith et al 1992;Emptoz-Bonneton et al 2000;Brunner et al 2003). The alignment of pig and rat amino acid sequences shows that the pig Arg307 amino acid is located at two residues of the substitution Met276Ile in the rat that is implicated in reduced steroid binding affinity of the BioBreeding rat (Smith and Hammond 1991).…”

Section: Discussionmentioning

confidence: 99%

Functional Implication of an Arg307Gly Substitution in Corticosteroid-Binding Globulin, a Candidate Gene for a Quantitative Trait Locus Associated With Cortisol Variability and Obesity in Pig

Guyonnet‐Duperat¹,

Geverink²,

Plastow³

et al. 2006

Genetics

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We previously reported that corticosteroid-binding globulin gene (Cbg) may be the causal gene of a quantitative trait locus associated with cortisol levels, fat deposition, and muscle content in a pig intercross. Sequence analysis of parental animals allowed us to identify four amino-acid substitutions. Here we have examined if any of these single amino acid substitutions could be responsible for the difference in CBG binding and affinity for cortisol between the parental breeds, using in vitro assays of Cbg variants after transfection of mammalian cells. Additionally, the Cbg coding region was analyzed in samples from a synthetic pig line to study association between polymorphism and CBG biochemical properties, carcass composition, and meat quality. Both in vitro transfection assays and the association studies suggest a role of the Arg307Gly mutation in increasing CBG capacity (by .70%) and decreasing CBG affinity for cortisol (by 30%). The Ile265Val substitution may also have an effect on decreasing CBG affinity for cortisol by 25%. The mutations Ser15Ile and Thr257Met do not seem to have an effect on CBG parameters. The Arg307Gly substitution was the only mutation associated with a parameter of meat quality and no mutation was linked to carcass composition.

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“…As mentioned above, amino acid substitutions in the RCL hinge regions of some serpins interfere with their inhibitory activity, and naturally occurring mutations within serpin RCLs have been linked to disease, such as at P10 of anti-thrombin III (35)(36)(37) as well as at P10 (38), P12 (39), and P14 (40) of C1 inhibitor. The rat CBG crystal structure (13) has revealed how several naturally occurring CBG variants could account for defects in steroid binding (13), including human CBG variants L93H (41,42) located in helix D and D367N (43) located in strand s4B as well as the rat CBG variant M276I (44) in strand s6A and the mouse CBG variant K201E (45) in strand s2C. Almost all of these naturally occurring CBG mutations were identified initially because they had low steroid binding affinities, and our data raise the interesting possibility that other CBG variants exist with abnormally high affinity steroid-binding sites that retain their activities after elastase cleavage of the RCL.…”

Section: Discussionmentioning

confidence: 99%

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Lin

Underhill

Gardill

et al. 2009

Journal of Biological Chemistry

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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 ␤-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 ␤-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 ␤-sheet A. Corticosteroid-binding globulin (CBG)2 is the major carrier protein for natural glucocorticoids (cortisol and corticosterone) and progesterone in blood (1), and it regulates the bioavailability of steroids that control numerous physiological processes including reproduction, inflammation, stress responses, and tissue development (2). Because CBG binds up to 90% of the glucocorticoids in blood plasma, it serves as a reservoir of anti-inflammatory steroids that can be released at their sites of action (3). The latter concept was proposed when it was discovered that human CBG exhibits remarkable sequence identity with the archetypal serine proteinase inhibitor, ␣1-anti-trypsin (AAT), and was based on the realization that CBG might also be a target of specific classes of proteinases (4).The close structural relationship between CBG and AAT defines it as a clade A serine proteinase inhibitor (serpin) family member (5). Many of these serpin A family members, including CBG, are encoded by genes in the human 14q21.1 chromosome cluster and are thought to have arisen by gene duplication to produce serpins with similar properties and physiological functions (6). Unlike most clade A serpins, CBG is not known to act as a proteinase inhibitor but appears to be a suicide substrate of specific protei...

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A Leu → His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol

Cited by 38 publications

References 16 publications

Corticosteroid-binding Globulin, a Structural Basis for Steroid Transport and Proteinase-triggered Release

Corticosteroid-binding Globulin, a Structural Basis for Steroid Transport and Proteinase-triggered Release

Functional Implication of an Arg307Gly Substitution in Corticosteroid-Binding Globulin, a Candidate Gene for a Quantitative Trait Locus Associated With Cortisol Variability and Obesity in Pig

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

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