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

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 ␤-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...

Effect of mutations of the human serpin protein corticosteroid-binding globulin on cortisol-binding, thermal and protease sensitivity

“…In contrast to human TBG rat CBG displays an active serpin conformation similar to thrombin inhibitory serpins [19]. Lin et al analyzed in detail the effects of amino acid mutations of the hCBG RCL on ligand binding in the context of HNE cleavage [27].…”

Ecophysiology of avian migration in the face of current global hazards

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Single nucleotide polymorphisms in the human corticosteroid-binding globulin promoter alter transcriptional activity

“…The release of ligands from CBG at their sites of action is controlled in a targeted manner, as illustrated by crystal structure studies [1]. The best example for this is at sites of inflammation neutrophil elastase cleaves CBG at a single site in a surface exposed reactive center loop, which causes a significant rearrangement in the tertiary structure of CBG that disrupts steroid-binding, thus providing a highly efficient mechanism for the local release of cortisol [11][12][13]. As there is evidence that CBG is the target of other proteinases [9], CBG accumulated at the maternal-fetal interface [6] can be cleaved by placenta-specific proteinases and thus involved in regulating local progesterone bioavailability and action.…”