Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

Pyrexia and acidosis act independently of neutrophil elastase reactive center loop cleavage to effect cortisol release from corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) transports cortisol and other steroids. High-affinity CBG (haCBG) undergoes proteolysis of the reactive center loop (RCL) by neutrophil elastase (NE) altering conformation to low-affinity CBG (laCBG). Elevated temperature reduces CBG:cortisol binding affinity. Surface plasmon resonance was used to determine binding profiles of 19 steroids to haCBG and laCBG at 25, 37, and 39°C mimicking pyrexia and pH 7.4 and 7.0 mimicking acidosis, pathophysiological conditions relevant to sepsis. An expected 4-8-fold reduction in affinity for cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, 17-hydroxyprogesterone, and prednisolone occurred with NE-mediated haCBG-to-laCBG conversion. CBG:cortisol binding affinity was further reduced 3.5-fold at 39°C relative to 37°C, binding affinity was also reduced by acidosis for both haCBG and laCBG. Using a conformational antibody generated against the RCL, we confirmed RCL antibody binding was eliminated by NE cleavage, but preserved in pyrexia and acidosis. Molecular modeling studies performed at 40°C confirmed a critical role for Trp371, positioned within the steroid-binding pocket, in ligand binding. These studies demonstrated CBG binding affinity to range of steroids is ligand specific and is reduced with NE-mediated haCBG-to-laCBG transition. Reduced CBG:cortisol binding occurs with increased temperature and in acidosis. Increased flexibility of the Trp371 side chain is proposed in the thermo-coupling mechanism of cortisol release. The synergy of NE cleavage, pyrexia, and acidosis on CBG:cortisol binding may serve to enhance cortisol delivery to the interstitial space in inflammation.

NewBG: A surrogate corticosteroid-binding globulin with an unprecedentedly high ligand release efficacy.

The introduction of ligand-binding sites into proteins and the engineering of molecular allosteric coupling pathways are topical issues in protein design. Here, we show that these issues can be addressed concurrently, using the serpin human α1-antichymotrypsin (ACT) as a model. We have introduced up to 15 amino acid substitutions into ACT, converting it into a surrogate corticosteroid-binding globulin (CBG), thereby creating a new binding globulin (NewBG). Human CBG and ACT share 46% sequence identity, and CBG served as the blue-print for our design, which was guided by side-chain-packing calculations, ITC measurements and crystal structure determinations. Upon transfer of ligand-interacting residues from CBG to ACT and mutation of specific second shell residues, a NewBG variant was obtained, which binds cortisol with 1.5 µM affinity. This novel serpin (NewBG-III) binds cortisol with a 33-fold lower affinity than CBG, but shares a similar ligand-binding profile and binding mode when probed with different steroid ligands and site-directed mutagenesis. An additional substitution, i.e. A349R, created NewBG-III-allo, which introduced an allosteric coupling between ligand binding and the serpin-like S-to-R transition in ACT. In NewBG-III-allo, the proteinase-triggered S-to-R transition leads to a greater than 200-fold reduction in ligand affinity, and crystal structures suggest that this is mediated by the L55V and A349R substitutions. This reduction significantly exceeds the 10-fold reduction in binding affinity observed in human CBG.

Neutrophil elastase-cleaved corticosteroid-binding globulin is absent in human plasma.

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.

Functional implications of corticosteroid-binding globulin N-glycosylation.

Corticosteroid-binding globulin (CBG) is a plasma carrier of glucocorticoids. Human and rat CBGs have six N-glycosylation sites. Glycosylation of human CBG influences its steroid-binding activity, and there are N-glycosylation sites in the reactive center loops (RCLs) of human and rat CBGs. Proteolysis of the RCL of human CBG causes a structural change that disrupts steroid binding. We now show that mutations of conserved N-glycosylation sites at N238 in human CBG and N230 in rat CBG disrupt steroid binding. Inhibiting glycosylation by tunicamycin also markedly reduced human and rat CBG steroid-binding activities. Deglycosylation of fully glycosylated human CBG or human CBG with only one N-glycan at N238 with Endo H-reduced steroid-binding affinity, while PNGase F-mediated deglycosylation does not, indicating that steroid binding is preserved by deamidation of N238 when its N-glycan is removed. When expressed in N-acetylglucosaminyltransferase-I-deficient Lec1 cells, human and rat CBGs, and a human CBG mutant with only one glycosylation site at N238, have higher (2-4 fold) steroid-binding affinities than when produced by sialylation-deficient Lec2 cells or glycosylation-competent CHO-S cells. Thus, the presence and composition of an N-glycan in this conserved position both appear to influence the steroid binding of CBG. We also demonstrate that neutrophil elastase cleaves the RCL of human CBG and reduces its steroid-binding capacity more efficiently than does chymotrypsin or the Pseudomonas aeruginosa protease LasB. Moreover, while glycosylation of N347 in the RCL limits these activities, N-glycans at other sites also appear to protect CBG from neutrophil elastase or chymotrypsin.

The role of corticosteroid-binding globulin in the evaluation of adrenal insufficiency.

Cortisol is a hydrophobic molecule that is largely bound to corticosteroid-binding globulin (CBG) in the circulation. In the assessment of adrenal insufficiency, many clinicians measure a total serum cortisol level, which assumes that CBG is present in normal concentrations and with a normal binding affinity for cortisol. CBG concentration and affinity are affected by a number of common factors including oral contraceptive pills (OCPs), fever and infection, as well as rare mutations in the serine protease inhibitor A6 (SERPINA6) gene, and as such, total cortisol levels might not be the ideal way to assess adrenal function in all clinical circumstances. This paper reviews the limitations of immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the measurement of total cortisol, the challenges of measuring free serum cortisol directly as well as the difficulties in calculating an estimated free cortisol from total cortisol, CBG and albumin concentrations. Newer approaches to the evaluation of adrenal insufficiency, including the measurement of cortisol and cortisone in the saliva, are discussed and a possible future role for these tests is proposed.

Endocrine disruption: In silico interactions between phthalate plasticizers and corticosteroid binding globulin.

Endocrine disruption is a phenomenon when a man-made or natural compound interferes with normal hormone function in human or animal body systems. Endocrine-disrupting compounds (EDCs) have assumed considerable importance as a result of industrial activity, mass production of synthetic chemicals and environmental pollution. Phthalate plasticizers are a group of chemicals used widely and diversely in industry especially in the plastic industry, and many of the phthalate compounds have endocrine-disrupting properties. Increasing evidence indicates that steroid nuclear receptors and steroid binding proteins are the main targets of endocrine disruption. Corticosteroid-binding globulin (CBG) is a steroid binding protein that binds and transports cortisol in the blood circulation and is a potential target for endocrine disruption. An imbalance of cortisol in the body leads to many health problems. Induced fit docking of nine important and environmentally relevant phthalate plasticizers (DMP, BBP, DBP, DIBP, DnHP, DEHP, DINP, DnOP, DIDP) showed interactions with 10-19 amino acid residues of CBG. Comparison of the interacting residues of CBG with phthalate ligands and cortisol showed an overlapping of the majority (53-82%) of residues for each phthalate. Five of nine phthalate compounds and cortisol shared a hydrogen bonding interaction with the Arg-252 residue of CBG. Long-chain phthalates, such as DEHP, DINP, DnOP and DIDP displayed a higher binding affinity and formed a number of interactions with CBG in comparison to short-chain phthalates. The similarity in structural binding characteristics of phthalate compounds and native ligand cortisol suggested potential competitive conflicts in CBG-cortisol binding function and possible disruption of cortisol and progesterone homeostasis.

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation.

Enhanced Activity of Topical Hydrocortisone by Competitive Binding of Corticosteroid-Binding Globulin.

Atopic dermatitis of sensitive areas such as the face, particularly in children, is a difficult disease to treat as the standard therapeutic, topical steroids, is contraindicated for this application in children. Hydrocortisone (HC) can be used in these instances because it has been shown to be safe, but is often ineffective as it is a relatively weak steroid, especially at over-the-counter concentrations. To enhance the local topical activity of HC, the terminal inactive metabolite of prednisolone, Δ(1)-cortienic acid (Δ(1)-CA), is added to HC, as Δ(1)-CA preferentially binds transcortin, liberating more HC to elicit its therapeutic effect. Skin blanching studies, which are used to evaluate the potency of topical steroids, were employed to assess the ability of Δ(1)-CA to enhance the activity of HC. The results demonstrate that Δ(1)-CA, when applied in combination with HC, does indeed potentiate the vasoconstriction effect of topically applied HC, while having no effect alone. Thus, addition of the inert prednisolone metabolite Δ(1)-CA can increase the therapeutic effect of over-the-counter concentrations of HC when applied topically.

Corticosteroid-Binding Globulin: A Review of Basic and Clinical Advances.

Corticosteroid-binding globulin (CBG, transcortin) is the primary cortisol binding protein. It is a non-inhibitory serine protease inhibitor, capable of conformational change from a high cortisol-binding affinity form to a low affinity form upon cleavage of its reactive centre loop by various proteases, such as neutrophil elastase. The burgeoning inflammatory role of CBG applies to acute, severe inflammation where depletion is associated with mortality, and to chronic inflammation where defects in cortisol delivery may perpetuate inflammation. Naturally occurring human mutations influence a wide range of CBG properties and point toward a role in hitherto unexplained chronic musculoskeletal pain and fatigue disorders as well as potentially affecting fertility outcomes including offspring gender. In vitro and knock-out animal models of CBG propose a role for CBG in cortisol transport to the brain, providing a foundation for understanding the human observations in those with CBG mutations and sex differences in stress-related mood and behaviour. Finally, CBG measurement has a practical role in the estimation of free cortisol, useful in clinical circumstances where CBG levels or cortisol binding affinity is reduced. Taken together, novel data suggest a role for cortisol in targeted cortisol delivery, with implications in acute and chronic inflammation, as well as roles in metabolism and neurocognitive function, implying that CBG is a multifaceted component in the mechanisms of hypothalamic-pituitary-adrenal axis related homeostasis.

Identification of Avian Corticosteroid-binding Globulin (SerpinA6) Reveals the Molecular Basis of Evolutionary Adaptations in SerpinA6 Structure and Function as a Steroid-binding Protein.

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.

Corticosteroid-binding globulin: modulating mechanisms of bioavailability of cortisol and its clinical implications.

Corticosteroid-binding globulin (CBG) is the principal transport protein of glucocorticoids. Approximately 80-90% of serum cortisol binds to CBG with high affinity and only about 5% of cortisol remain unbound and is considered biologically active. CBG seems to modulate and influence the bioavailability of cortisol to local tissues. In this review, we will discuss physicochemical properties of CBG and structure of CBG in the mechanisms of binding and release of cortisol. This review describes several factors affecting CBG functions, such as genetic factors or temperature. Furthermore, clinical implications of CBG abnormalities and the measurement of CBG and its use for assessment of free cortisol levels are described in this review.

Towards engineering hormone-binding globulins as drug delivery agents.

The treatment of many diseases such as cancer requires the use of drugs that can cause severe side effects. Off-target toxicity can often be reduced simply by directing the drugs specifically to sites of diseases. Amidst increasingly sophisticated methods of targeted drug delivery, we observed that Nature has already evolved elegant means of sending biological molecules to where they are needed. One such example is corticosteroid binding globulin (CBG), the major carrier of the anti-inflammatory hormone, cortisol. Targeted release of cortisol is triggered by cleavage of CBG's reactive centre loop by elastase, a protease released by neutrophils in inflamed tissues. This work aimed to establish the feasibility of exploiting this mechanism to carry therapeutic agents to defined locations. The reactive centre loop of CBG was altered with site-directed mutagenesis to favour cleavage by other proteases, to alter the sites at which it would release its cargo. Mutagenesis succeeded in making CBG a substrate for either prostate specific antigen (PSA), a prostate-specific serine protease, or thrombin, a key protease in the blood coagulation cascade. PSA is conspicuously overproduced in prostatic hyperplasia and is, therefore, a good way of targeting hyperplastic prostate tissues. Thrombin is released during clotting and consequently is ideal for conferring specificity to thrombotic sites. Using fluorescence-based titration assays, we also showed that CBG can be engineered to bind a new compound, thyroxine-6-carboxyfluorescein, instead of its physiological ligand, cortisol, thereby demonstrating that it is possible to tailor the hormone binding site to deliver a therapeutic drug. In addition, we proved that the efficiency with which CBG releases bound ligand can be increased by introducing some well-placed mutations. This proof-of-concept study has raised the prospect of a novel means of targeted drug delivery, using the serpin conformational change to combat the problem of off-target effects in the treatment of diseases.

The reactive centre loop of corticosteroid-binding globulin (CBG) is a protease target for cortisol release.

Corticosteroid-binding globulin (CBG) binds more than 90% of circulating cortisol and is a non-inhibitory member of the family of serine protease inhibitors (SERPINS) with an exposed elastase sensitive reactive centre loop (RCL). At sites of inflammation neutrophil activation can release elastase which may cleave the RCL and result in cortisol release from CBG. The RCL sequence also has two theoretical chymotrypsin cleavage sites and we used a monoclonal antibody with specificity for the RCL to investigate chymotrypsin cleavage of CBG. Here we show, for the first time, rapid chymotrypsin cleavage of the RCL of CBG, resulting in undetectable levels of intact CBG, whereas total CBG levels were unchanged. Coincident with both chymotrypsin and elastase cleavage there was an increase in the free cortisol fraction of serum to levels similar to when CBG had been inactivated by heat indicating total cortisol release from CBG. These findings demonstrate a new mechanism for cortisol release from its binding globulin.

3-O-methoxyimino group inhibits interactions between progestins and blood transcortin.

The interactions between E- and Z-isomers of 3-O-methoxyimino-pregn-4-ene-20-one and its 17α-hydroxy derivative and transcortin from human blood were investigated. The substitution of the progesterone 3-oxo group for a 3-O-methoxyimino group was shown to diminish the affinity of the steroid for transcortin by approximately one order of magnitude irrespective of the substituent's orientation. The data suggests that progesterone derivatives substituted thereby must have higher bioavailability compared to progesterone and must not significantly affect the biodynamics of glucocorticoid in vivo.

Intact or "active" corticosteroid-binding globulin (CBG) and total CBG in plasma: determination by parallel ELISAs using monoclonal antibodies.

The predominant carrier of cortisol in circulation is corticosteroid-binding globulin (CBG) which is a non-functional member of the family of serine protease inhibitors. Corticosteroid-binding globulin possesses an exposed elastase sensitive loop and upon cleavage it adopts a "relaxed" conformation promoting the delivery of cortisol to sites of inflammation. Recently we have developed monoclonal antibodies which recognise only the intact exposed elastase loop, including an N-glycosylation site, which, in concert with another monoclonal antibody to CBG, offered the potential for the determination of intact and total CBG which may both be present in circulation. Here we validate these parallel ELISAs and show that like total CBG there is little diurnal variation of intact plasma CBG. Furthermore in a normal reference population the majority of CBG is in the intact or active form but a significant level of apparently cleaved CBG is evident. In some subjects there is gross discordance between total CBG and intact CBG implying a predominance of apparently cleaved CBG in circulation and this significantly affects calculated free cortisol levels. Gross differences in total and intact CBG levels may not be due to differences in N-glycosylation affecting antibody binding as CBG levels are unaffected by PNGase F treatment.

High frequency of SERPINA6 polymorphisms that reduce plasma corticosteroid-binding globulin activity in Chinese subjects.

CONTEXT: Cortisol is transported by corticosteroid-binding globulin (CBG) in blood. Single nucleotide polymorphisms (SNP) in the human CBG (SERPINA6) gene that disrupt CBG production or steroid binding are considered rare. OBJECTIVE: The objective of the study was to identify and determine the frequency of SNP in SERPINA6 that influence the production or cortisol-binding properties of CBG in Chinese subjects. PARTICIPANTS AND DESIGN: Blood samples from 2287 anonymous Chinese workers undergoing routine health tests were screened for the SERPINA6 coding sequence polymorphisms. MAIN OUTCOME MEASURES AND RESULTS: In a pilot study of 108 Chinese women, two nonsynonymous SNP were identified within SERPINA6 exon 2 encoding CBG A51V (n = 3) and CBG E102G (n = 1) variants. Sequence analysis of SERPINA6 exon 2 in a further 137 Chinese women revealed two other individuals with nonsynonymous SNP encoding CBGs R64Q and R64W as well as another CBG A51V carrier. The surprisingly high frequency of heterozygous CBG A51V carriers was confirmed in 1011 Chinese men (1:35) and 1031 other women (1:37). Individuals homozygous for these SNP were not identified. When expressed in Chinese hamster ovary cells, CBG A51V bound steroid normally, but its production/secretion was severely impaired; CBG E102G was produced normally, but its cortisol-binding capacity was abnormally low, whereas CBG R64Q and R64W were produced and bound cortisol normally. CONCLUSIONS: Defects in CBG A51V production explain why plasma CBG levels in individuals heterozygous for this variant are approximately 50% lower than normal. The high frequency of CBG A51V will allow clinical consequences of CBG deficiencies to be assessed for the first time in large patient populations.

Multi-target QSAR and docking study of steroids binding to corticosteroid-binding globulin and sex hormone-binding globulin.

The QSAR and docking studies were performed on fifty seven steroids with binding affinities for corticosteroid-binding globulin (CBG) and eighty four steroids with binding affinities for sex hormone-binding globulin (SHBG). Since the steroidal compounds have binding affinity for both CBG and SHBG, multi-target QSAR approach was employed to establish a unique QSAR method for simultaneous evaluation of the CBG and SHBG binding affinities. The constitutional, geometrical, physico-chemical and electronic descriptors were computed for the examined structures by use of the Chem3D Ultra 7.0.0, the Dragon 6.0, the MOPAC2009, and the Chemical Descriptors Library (CDL) program. Partial least squares regression (PLSR) has been applied for selection of the most relevant molecular descriptors and QSAR models building. The QSAR (SHGB) model, QSAR model (CBG), and multi-target QSAR model (CBG, SHBG) were created. The multi-target QSAR model (CBG and SHBG) was found to be more effective in describing the CBG and SHBG affinity of steroids in comparison to the one target models (QSAR (SHGB) model, QSAR model (CBG)). The multi-target QSAR study indicated the importance of the electronic descriptor (Mor16v), steric/symmetry descriptors (Eig06_EA(ed)), 2D autocorrelation descriptor (GATS4m), distance distribution descriptor (RDF045m), and atom type fingerprint descriptor (CDL-ATFP 253) in describing the CBG and SHBG affinity of steroidal compounds. Results of the created multi-target QSAR model were in accordance with the performed docking studies. The theoretical study defined physicochemical, electronic and structural requirements for selective and effective binding of steroids to the CBG and SHBG active sites.

Corticosteroid-binding globulin: structure-function implications from species differences.

Corticosteroid-binding globulin (CBG) transports glucocorticoids and progesterone in the blood and thereby modulates the tissue availability of these hormones. As a member of the serine protease inhibitor (SERPIN) family, CBG displays a reactive center loop (RCL) that is targeted by proteinases. Cleavage of the RCL is thought to trigger a SERPIN-typical stressed-to-relaxed (S-to-R) transition that leads to marked structural rearrangements and a reduced steroid-binding affinity. To characterize structure-function relationships in CBG we studied various conformational states of E. coli-produced rat and human CBG. In the 2.5 Å crystal structure of human CBG in complex with progesterone, the RCL is cleaved at a novel site that differs from the known human neutrophil elastase recognition site. Although the cleaved RCL segment is five residues longer than anticipated, it becomes an integral part of ß-sheet A as a result of the S-to-R transition. The atomic interactions observed between progesterone and CBG explain the lower affinity of progesterone in comparison to corticosteroids. Surprisingly, CD measurements in combination with thermal unfolding experiments show that rat CBG fails to undergo an S-to-R transition upon proteolytic cleavage of the RCL hinting that the S-to-R transition observed in human CBG is not a prerequisite for CBG function in rat. This observation cautions against drawing general conclusions about molecular mechanisms by comparing and merging structural data from different species.

Serpins as hormone carriers: modulation of release.

The hormone-carrying serpins, thyroxine- and corticosteroid-binding globulins, TBG and CBG, provide a clear example of the way the serpin conformational mechanism can be adapted not only to give an irreversible switching-off of function but also more significantly to allow a constant dynamic modulation of activity. This is illustrated here with the demonstration that hormone release from both TBG and CBG is responsive to changes in ambient temperature and specifically to changes in body temperature. An exception to this adaptation of the serpin mechanism is seen with another family member, angiotensinogen, in which hormone release is modulated by a redox switch and is apparently independent of changes in the serpin framework.