Loop-inserted and thermostabilized structure of P1-p1′ cleaved ovalbumin mutant R339T 1 1Edited by R. Huber

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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...

Section: Resultsmentioning

confidence: 99%

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

Klieber

Underhill

Hammond

et al. 2007

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121

“…Complementary studies on ovalbumin, a non-inhibitory serpin that has an arginine residue at P14 and lacks the loop insertion mechanism (29), indicate that when this P14 arginine (Arg-339) in ovalbumin is substituted with serine, the variant is characterized by an increased RCL insertion rate and spontaneous partial insertion of the cleaved RCL (30). Moreover, the crystal structure of the cleaved R339T ovalbumin variant clearly revealed that complete loop insertion can occur upon this single hinge mutation (31). These reports are all very much in line with our finding that substitution of the P14 valine (Val-336) in human CBG with arginine does not hinder cleavage of its RCL by elastase or alter its steroid binding properties after cleavage, presumably because the arginine at P14 prevents the reorientation and/or subsequent insertion of the cleaved 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

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...

“…demonstrated that cl-R339T has a T m much higher than that of int-R339T because it forms a relaxed serpin structure (27). In contrast, cl-OVA shows a T m slightly lower than that of int-OVA because it keeps a stressed serpin form slightly destabilized by the P1-P1Ј cleavage (41).…”

Section: Thermostability Of Refolded Cl-ova and Cl-r339t-our Previousmentioning

confidence: 98%

“…1), whereas typical inhibitory serpins have Thr or Ser. Indeed our crystallographic and biochemical evidence has demonstrated that the Arg 339 to Thr mutant of ovalbumin (R339T) is able to undergo RCL insertion (27,28). These characteristics imply that P1-P1Ј-cleaved ovalbumin (cl-OVA) may be capable of RCL insertion in a refolding intermediate state in which structures are not rigidly formed.…”

mentioning

confidence: 97%

Cleaved Serpin Refolds into the Relaxed State via a Stressed Conformer

Onda

Nakatani²,

Takehara

et al. 2008

Self Cite

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1 bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly 1 -Ala 352 ) and light (Ser 353 -Pro 385 ) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T m ‫؍‬ 68.4°C, whereas cl-R339T refolded into the relaxed form with T m ‫؍‬ 85.5°C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure. The serpins2 are believed to fold in vivo into a metastable "stressed form" as they undergo a conformational change to the thermostable "relaxed form" by insertion of the reactive center loop (RCL) between strands 3A and 5A (see Fig. 1) (1-3). The metastability of the native serpin is essential for the inhibition of serine proteinases, although this molecular flexibility also results in aberrant intermolecular linkage and polymerization that bring about conformational diseases such as dementia (4, 5), hepatocirrhosis (6), angioedema (7), and emphysema (8, 9). Therefore a central target of serpin studies is how the serpin polypeptide can fold into a metastable structure avoiding thermodynamically more stable conformations. Several serpin folding models have been suggested by various unfolding experiments, whereas refolding experiments from a fully unfolded state are limited (10 -14) because of serpin susceptibility to misfolding and aggregation.Ovalbumin is a major component of egg white proteins, and although it is not an apparent protease inhibitor its membership of the serpin family of protease inhibitors is clear from the close similarity of its primary and tertiary structure (15, 16). We have elucidated its refolding process from a fully denatured state as (10, 17, 18)